Novel Nitric Oxide-Releasing Derivatives of Brusatol as Anti

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Novel Nitric Oxide-Releasing Derivatives of Brusatol as AntiInflammatory Agents: Design, Synthesis, Biological Evaluation, and Nitric Oxide Release Studies Weibin Tang,† Jianlin Xie,† Song Xu, Haining Lv, Mingbao Lin, Shaopeng Yuan, Jinye Bai, Qi Hou,* and Shishan Yu* State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 1 Xian Nong Tan Street, Beijing 100050, China S Supporting Information *

ABSTRACT: Brusatol, a biologically active natural product, was modified in four distinct positions through the covalent attachment of a furoxan moiety, which acts as a nitric oxide (NO) donor. Forty derivatives were synthesized and evaluated for their inhibitory effects on excess NO biosynthesis in activated macrophages. Among them, compound 75 demonstrated inhibition (IC50 = 0.067 μM) comparable to that of brusatol but were less cytotoxic. More importantly, even at very low doses (2 μmol/kg/day), compound 75 also showed substantial inhibitory efficacy against chronic obstructive pulmonary disease (COPD)-like inflammation in the mouse model induced by cigarette smoke (CS) and lipopolysaccharide (LPS). Particularly, this compound was over 100-fold less toxic (LD50 > 3852 μmol/ kg) than brusatol and could be a promising lead for further studies. Notably, the improved properties of this derivative are associated with its NO-releasing capability.

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INTRODUCTION The natural product brusatol (Figure 1) is a biologically active degraded triterpenoid compound and is especially prevalent in

Although efforts have been made to modify the original structure of brusatol, no derivatives with improved anti-inflammatory activity and reduced toxicity have been reported to date.2 Thus, there is an urgent need to develop a new strategy to modify the structure of brusatol. Nitric oxide (NO) is a physiological messenger that triggers a variety of actions in several systems.3 It can relax vascular smooth muscle, inhibit platelet aggregation, reduce neutrophil adherence and activation, suppress inducible NO synthase (iNOS) overexpression, mitigate cell death, and promote cell proliferation.4−11 Because of its anti-inflammatory properties and cytoprotective effects, adjunctive NO has been considered a plausible means for improving the anti-inflammatory activity and safety of drugs. Structurally diverse NO-releasing nonsteroid anti-inflammatory drugs (NSAIDs), including the NO-donating derivatives of aspirin, naproxen, indomethacin, diclofenac, and ibuprofen, have been synthesized and validated in laboratory and clinical research.12−16 Several NO-donor NSAIDs exhibit greater anti-inflammatory activity than their respective parent NSAIDs but with less gastrointestinal and cardiorenal toxicity; these NOreleasing NSAIDs may even promote ulcer healing. In particular, the promising naproxcinod (Nitronaproxen, AZD-3582, HCT3012) demonstrates improved activity and gastrointestinal safety

Figure 1. Structure of brusatol.

plants belonging to the Brucea genus. Brusatol and its acylated derivatives demonstrate a wide range of pharmacological activities, including antitumor, antimalaria, anti-inflammatory, antiviral, and insecticide activity.1 In a previous study by Hall et al., brusatol was reported to be a more potent inhibitor of induced rat paw inflammation and arthritis than indomethacin. Preliminary studies have indicated that although brusatol is not a corticosteroid, it can also stabilize lysosomal membranes, reducing the release of hydrolytic enzymes that cause damage to surrounding tissues. Additionally, brusatol’s inhibition of protein synthesis may serve an important role in antiinflammatory activity.2 However, the high toxicity of brusatol has severely limited its use as an anti-inflammatory agent. © XXXX American Chemical Society

Received: May 15, 2014

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dx.doi.org/10.1021/jm5007534 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Scheme 1. Synthesis of NO Donors 9−19a

Reagents and conditions: (i) succinic anhydride or phthalic anhydride, DMAP, CH2Cl2, 40 °C; (ii) PPh3, imidazole, I2, CH2Cl2, rt; (iii) PBr3, CH2Cl2, 0 °C, rt.

a

Scheme 2. Synthesis of NO Donors 24−32a,b

a Reagents and conditions: (i) succinic anhydride or phthalic anhydride, DMAP, CH2Cl2, 40 °C; (ii) PBr3, CH2Cl2, 0 °C, rt. bThe symbol “/ ”represents no atoms or atom groups.

Scheme 3. Synthesis of Brusatol Derivatives 33−47 Modified at the 3-Positiona,b

a b

Reagents and conditions: (i) compounds 9−17 and 24−28, EDCI, DMAP, CH2Cl2, 0 °C, rt. or 40 °C; (ii) compound 29, K2CO3, CH3CN, reflux. The symbol “/ ”represents no atoms or atom groups.

and has successfully completed advanced-phase clinical trials.13 Therefore, these studies prove that the linkage of an NO-

releasing moiety to an anti-inflammatory agent may reduce the toxicity and improve the activity of the original compound. B



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a Reagents and conditions: (i) t-BDMS-Cl, imidazole, DMAP, DMF, rt; (ii) 1 N KOMe-MeOH, 0 °C; (iii) Dowex 50-X2 (H+) resin; (iv) compounds 9−17 and 24−28, EDCI, DMAP, CH2Cl2, 0 °C, rt or 40 °C; (v) Bu4NF, HOAc, THF, rt. bThe symbol “/ ”represents no atoms or atom groups.


a Reagents and conditions: (i) LiI, pyridine, 100 °C; (ii) Bu4NF, HOAc, THF, rt; (iii) compounds 1−2 and 7−8, EDCI, DMAP, CH2Cl2, rt; (iv) compounds 18−19 and 29−32, K2CO3, CH3CN, reflux; (v) Bu4NF, HOAc, THF, rt. bThe symbol “/ ”represents no atoms or atom groups.

On the basis of the above-mentioned studies and our own previous work,17 we designed 40 novel NO-donating derivatives, which were derived through the conjugation of brusatol to NOdonor furoxan substructures and were endowed with various NO-releasing capabilities. Here, we report the synthesis, antiinflammatory properties, acute toxicities, potential structure− activity relationships (SARs), and nitric oxide release data for these structures. Compared with brusatol analogues without

NO-donating properties, the NO-releasing furoxan hybrids exhibit additional biological activities that could enhance the overall pharmacological profile of the parent compound.

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RESULTS AND DISCUSSION Chemistry. Brusatol has an enolic hydroxyl group at C-3, a secondary and slightly congested hydroxyl group at C-12, an esterified hydroxyl group at C-15, and an esterified carboxyl

C



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Scheme 6. Synthesis of Brusatol Derivatives 74−76a

a

Reagents and conditions: (i) compound 10, EDCI, DMAP, CH2Cl2, 0 °C, rt.

compounds 68 and 70−73. Because these bromides were reasonably active, the yields were acceptable. The target compounds 74−76, each of which possesses a free carboxyl group at the 21-position, were prepared from 21-Odesmethyl brusatol (65) using a similar esterification method to that described above (Scheme 6). This resulted in different principal products and byproducts depending on the ratios of the reagents. When 10 and 65 were reacted at a 1:1 ratio, the monoesters 74 and 76 were obtained as principal products. The yield of diester 75 was improved by increasing the equivalents of 10 used in the reaction. When the ratio of 10 to 65 was greater than 2, compound 75 was the principal product. In Vitro Inhibition of LPS-Induced Excess NO Production in Macrophages. The brusatol derivatives were evaluated for their inhibition of excessive NO production in murine peritoneal macrophages stimulated by LPS. NO possesses a “dual personality”: it displays a variety of beneficial effects at endogenous concentrations but is detrimental at superphysiological concentrations. In inflammatory diseases, large amounts of NO are produced through the inducible nitric oxide synthase (iNOS) pathway and amplify the inflammatory response.20 Compounds that inhibit excess NO production are thus considered to possess potential anti-inflammatory activities. Interestingly, small amounts of NO (provided by NO-releasing drugs) could not only protect cells but also inhibit iNOS expression through feedback mechanisms to abrogate the upregulation of NO biosynthesis in inflammation.21 This is also the reason that NO-releasing nonsteroid anti-inflammatory drugs (NSAIDs) exhibit lower toxicity and higher antiinflammatory activity than traditional NSAIDs. Therefore, our NO-releasing derivatives may display properties superior to those of their corresponding parent compounds. First, all brusatol derivatives were screened and assayed for the inhibition of excessive NO production at a concentration of 10 μM, using the parent compound brusatol and 65 as controls. Because NO is very reactive and can be quickly converted to the stable metabolite nitrite, nitrite determined using the Griess assay, was used as a proxy for NO produced by cells in our studies. Meanwhile, the cytotoxicities of all derivatives were determined by the MTT assay. The preliminary results (Supporting Information, Table S1) indicated that nearly all derivatives as well as brusatol and dexamethasone sodium phosphate (DEX) dramatically decreased the amount of NO produced by active macrophages. However, all of the effective derivatives still demonstrated obvious toxicities to cells, with the exception of compounds 74−76, the parent compound of which was 21-O-desmethyl brusatol (65). Additionally, compared with the parent compound 65, the hybrids 74−76 exhibited

group at C-21 that could be modified by coupling with NO donors (Figure 1). In our study, brusatol was isolated from the seeds of Brucea javanica, and the NO donors, including eight phenylsulfonyl-substituted furoxans (1−8) and four phenylsubstituted furoxans (20−23), were synthesized as described in previous publications (Schemes 1 and 2).18,19 Subsequently, as shown in Scheme 1, compounds 1−8 were reacted with succinic anhydride or phthalic anhydride in the presence of 4-N,Ndimethylaminopyridine (DMAP) to produce the corresponding acids 9−17 with good yields. Additionally, the halogenated compound 18 was prepared by the iodination of 1 with iodine (I2), and 19 was obtained by the bromination of 7 with phosphorus tribromide (PBr3). Following the same procedures described above, the acids 24−28 and the bromides 29−32 were prepared from the corresponding alcohols 20−23 as shown in Scheme 2. The preparation of brusatol derivatives functionalized at the 3position is illustrated in Scheme 3. Compounds 33−46 were directly obtained from brusatol through the addition of the corresponding phenylsulfonyl- or phenyl-substituted furoxans (9−17 or 24−28) in the presence of 1-ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and DMAP. The target compound 47 was prepared by treating the exocyclic hydroxyl group at C-3 of brusatol with the corresponding bromide 29 and K2CO3. Prior to modifying the esterified 15-hydroxyl group, the key intermediate compound 49 for the synthesis of the target compounds 50−63 was produced by silylation of the 3-hydroxyl group with dimethyl-t-butylsilyl chloride (t-BDMS-Cl), followed by selective removal of the ester group at C-15 with potassium methoxide, as shown in Scheme 4. The subsequent esterification of the 15-hydroxyl group and removal of the protecting group of the 3-hydroxyl group yielded the desired derivatives. To synthesize compounds 66−73, which are brusatol analogues bearing different furoxan fragments at C-21, compound 48 was treated with lithium iodide in pyridine at 100 °C for 24 h to yield the key carboxylic acid 64, as shown in Scheme 5. Compounds 66−69 were obtained from 64 by the deprotection of the siloxane moiety. Furthermore, the esterification of the 21-carboxyl group with appropriate alcohols (9−10 or 15−16) yielded the desired derivatives. The method above was simple but offered poor yields, perhaps due to the large steric hindrance of the ester group at C-15 and weak acidity of the carboxyl group. Compound 66 could also be prepared with slightly better yields in two steps by treating compound 64 with the corresponding iodide and K2CO3 followed by desilylation (Scheme 5). Similarly, compound 64 was reacted with the corresponding bromides and then desilylated to generate target D



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RPMI-1640 supplemented with 5% (v/v) newborn calf serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. Then they were exposed to each test compound (10 μM) for 24 h. The levels of nitrite produced in the supernatant of the medium were characterized using the Griess assay. The results are presented in Figure 2.

significantly improved inhibitory activities and retained low toxicities. This finding implies that NO donors play a specific role in the inhibition of excess NO biosynthesis in inflammatory cells. To comprehensively evaluate this class of derivatives, the promising compounds 74−76 and eight other structurally typical compounds were selected for IC50 determination and tested at least twice in triplicate. As seen from the results summarized in Table 1, general inhibition was observed for the 11 brusatol Table 1. Cytotoxicity and Inhibitory Activities against LPSInduced NO Production in Macrophages of Select Compounds inhibition of NO production (A)

a

compd

IC50 (μM)

brusatol 34 41 42 47 51 59 65 67 70 74 75 76

0.050 0.089 0.69 0.047 2.1 0.21 0.38 >10 1.1 2.2 11 0.067 1.3

95% confidence interval (μM) 0.032−0.078 0.066−0.12 0.49−0.97 0.035−0.064 1.4−3.2 0.14−0.32 0.22−0.64 0.91−1.4 1.7−2.8 6.6−18 0.034−0.13 0.86−2.1

cytotoxicity (B)

IC50 (μM) 0.14 0.79 1.3 0.15 >10 1.2 0.82 >10 3.0 3.9 >10 >10 >10

95% confidence interval (μM) 0.12−0.17 0.50−1.3 1.0−1.6 0.12−0.18

SIa (B/A)

0.97−1.6 0.59−1.1

2.8 8.9 1.9 3.2 >4.7 5.7 2.2

2.4−3.7 3.2−4.8

2.7 1.8

Figure 2. Intrinsic NO-release capabilities of certain compounds. Macrophage cells without stimulation by LPS were treated with each compound at 10 μM, and the content of nitrite in each supernatant was determined using the Griess assay. No treatment or treatment with vehicle or LPS (1 μg/mL) was used as the control group. The individual values were determined by measuring the absorbance at 570 nm and were calculated according to the standard curve. The data presented are the mean values ± SEMs of individual compounds at 24 h posttreatment from three independent experiments. Statistical analysis was performed using Student’s t test: (*) P < 0.05 vs vehicle, (**) P < 0.01 vs vehicle.

>149 >7.6

Selectivity index.

As expected, treatment with compound 42 (phenylsubstituted furoxan hybrid) as well as the parent compounds brusatol and 65 did not result in more nitrite in the medium than the background (diluent-treated wells). In contrast, treatment with compounds 34 and 74−76 (phenylsulfonyl-substituted furoxan hybrids) promoted detectable levels of nitrite. In particular, treatment using compound 75, which had two phenylsulfonyl-substituted furoxan groups, generated the greatest amounts of nitrite. However, their quantities were far less than those produced by the LPS group. By comparison with the parent compound brusatol or 65, these findings indicate that phenylsulfonyl-substituted furoxan hybrids, rather than phenylsubstituted furoxan hybrids, possess obvious and intrinsic NOgenerating abilities. Interestingly, the furoxan substituent group has been shown to influence NO release as reported.23 The electron withdrawing moiety phenylsulfonyl as a substituent group might be beneficial to the NO-release ability of furoxan. This inference was also further validated by testing the NOrelease abilities of several additional compounds (Supporting Information, Table S2). An Analysis of the Relationships between the Activity/ Toxicity and NO-Release Capability of Compounds In Vitro. In light of the above findings, the NO-release capabilities of the compounds are related to the activities and toxicities exhibited in vitro. Because of the intrinsic NO-release capabilities, hybrids such as 34 and 74−76 demonstrated lower toxicity and/or superior activity compared with the corresponding parent compound. The reason for these results may be that the phenylsulfonyl-substituted furoxan hybrids (unlike phenylsubstituted furoxan hybrids) can obviously release a small amount of NO. The low levels of NO released by the compounds could not only protect the cells but could also inhibit the

derivatives. In particular, compound 75, with two identical phenylsulfonylfuroxan NO-releasing groups, demonstrated outstanding inhibition (IC50 = 0.067 μM) comparable to that of brusatol (IC50 = 0.050 μM) but with low cytotoxicity (IC50 > 10 μM). This property of 75 may reflect the synergistic effect of NO and the parent compound 65. Excluding compound 75, the 3position derivatives 34 and 42 exhibited the best biological effects while still possessing high toxicities. Substantial NO production inhibition by the three compounds was also confirmed by measuring the amount of intracellular NO using a fluorescent method (Supporting Information, Figure S1).22 Perhaps because of coupling with the same phenylsulfonylfuroxan NO-releasing group, 34 displayed reduced cytotoxicity (over 5-fold reduction) and an improved selectivity index (cytotoxicity IC50/NO inhibition IC50) compared with the parent compound brusatol and 42. Additionally, 51 and 67 also exhibited superior selectivity indices compared with 59 and 70, respectively, which were modified with phenylfuroxan. On the basis of these results, the high inhibition and reduced toxicity of these compounds appear to be associated with the intrinsic NOrelease capability of phenylsulfonylfuroxan. In Vitro Intrinsic NO-Release Capability of Compounds. To investigate the relationship between intrinsic NO release capability and activity/toxicity of compounds, NO release was evaluated in 34, 42, and 74−76 using their corresponding parent compounds brusatol and 21-O-desmethyl brusatol (65) as controls. The test conditions were similar to those used in the previous section, In Vitro Inhibition of LPS-Induced Excess NO Production in Macrophages, but without macrophage stimulation by LPS. In brief, macrophage cells were cultured for 2 h in E



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validate that the improved properties of our derivatives are associated with their NO-release capabilities. In Vivo Anti-Inflammatory Activity. Compounds 34, 42, and 75, exhibiting the highest activities in vitro, were selected to evaluate their anti-inflammatory effects by studying croton oilinduced ear edema in mice, using brusatol as the reference standard. The mice were randomized and treated with 5, 10, or 20 μmol/kg of test compound or vehicle alone prior to the application of croton oil. As shown in Table 2, oral treatment

overexpression of iNOS through feedback regulation, as previously reported.21 In fact, the introduction of NO-releasing groups is a wellknown approach to reduce toxicities of compounds, which was also evidenced by comparing the toxicity of brusatol, 34 and 42. To validate the feedback regulation mechanism proposed above, determining whether our hybrids with obvious NO-release capabilities exhibited greater inhibitory effect on iNOS overexpression than the corresponding parent compound was necessary. Thus, to eliminate the toxicity effect, hybrids 74−76 and their parent compound 65, which possess lower cytotoxicities, were selected to determine their effect on iNOS expression in stimulated macrophages by immunoblotting assays. As observed in Figure 3, iNOS expression was not clearly

Table 2. Effects of 34, 42, and 75 (po) on Ear Edema Induced by Croton Oil in Mice compd vehicle brusatol brusatol brusatol 34 34 34 42 42 42 75 75 75

dose po (μmol/kg)

edema degreea (mg)

5 10 20 5 10 20 5 10 20 5 10 20

27.7 ± 1.9 21.0 ± 1.9* 20.2 ± 1.9* 14.7 ± 2.3** 19.6 ± 1.1** 16.6 ± 1.7** 12.8 ± 1.3** 28.5 ± 1.6 27.7 ± 2.1 27.5 ± 1.8 27.5 ± 1.3 25.9 ± 1.4 25.6 ± 1.8

inhibition rate (%) 24.3 27.1 46.8 29.4 39.9 53.9

0.9 6.4 7.8

Data are shown as the means ± SEMs (n = 10). Statistical analysis was performed using Student’s t test: (*) P < 0.05 vs vehicle-treated mice, (**) P < 0.01 vs vehicle-treated mice. a

using hybrid 34, with the NO-release property, could dosedependently reduce croton oil-induced ear swelling in mice compared with the vehicle alone. At a 20 μmol/kg dose, the inhibition rate of 34 was greater than 50%. Moreover, hybrid 34 also provided better swelling inhibition than the parent compound brusatol at the same dose. However, compound 42, without obvious NO-release capability, exhibited no activity at multiple doses. This finding further indicates that the NO-release capabilities of the compounds are associated with activity improvement. Additionally, compound 75, with NO-release capability, the parent compound of which was 21-O-desmethyl brusatol (65), did not offer significant inhibition at doses below 20 μmol/kg. Nevertheless, as shown in Table 3, subcutaneous treatment with 75 at high doses (77, 154, and 308 μmol/kg) yielded significant and dose-dependent anti-inflammatory activity (P < 0.05 vs vehicle control). At a 154 μmol/kg dose, the inhibition rate of 75 was greater than 60%. These results suggest that the small

Figure 3. Effects of select compounds on iNOS expression in macrophages stimulated by LPS. Control group, macrophages with no treatment; LPS group, macrophages with stimulation by LPS for 24 h; 1400W group, macrophages pretreated with 75 μM selective iNOS inhibitor N-[3-(aminomethyl)benzyl]acetamidine dihydrochloride (1400W)24 for 1 h, followed by challenging with LPS for 24 h. Other groups: macrophages pretreated with 1 μM selected compounds for 1 h, followed by challenging with LPS for 24 h. (A) A representative blot. A representative image from three independent experiments is shown. (B) Quantitative analyses. The relative levels of iNOS to β-actin control were determined through densitometric scanning. The data represent the mean values ± SEMs from three independent experiments. Statistical analysis was performed using Student’s t test: (*) P < 0.05 vs LPS, (**) P < 0.01 vs LPS, (#) P < 0.05 vs 65.

Table 3. Effect of 75 (sc) on Ear Edema Induced by Croton Oil in Mice

detected from macrophages without stimulation. However, macrophages stimulated with LPS showed significant induction of iNOS expression. As expected, iNOS expression was suppressed upon treating the stimulated cells with compounds 65 and 74−76. Through a simple comparison, hybrids 74−76 showed higher inhibition than their parent compound 65, especially 75, having the most obvious NO-release capability. The significantly higher inhibition by hybrid 75 on iNOS expression suggests that the low level of NO released by 75 may enhance the inhibitory activity of the parent compound 65 on iNOS expression and consequent NO production in stimulated macrophages. This result provided support for the feedback regulation. Additionally, the following in vivo results further

compd vehicle 75 75 75

dose sc (μmol/kg)

edema degreea (mg)

inhibition rate (%)

77b 154c 308d

20.4 ± 1.0 14.0 ± 2.1* 7.9 ± 0.7** 4.8 ± 0.5**

31.4 61.0 76.5

Data are shown as the means ± SEMs (n = 8−9). The statistical analysis was performed using Student’s t test: (*) P < 0.05 vs vehicletreated mice, (**) P < 0.01 vs vehicle-treated mice. bThe equivalent dose is 100 mg/kg. cThe equivalent dose is 200 mg/kg. dThe equivalent dose is 400 mg/kg. a

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Figure 4. Effects of select compounds on COPD-like lung inflammation. Control group, air-exposed mice; model group, CS-exposed mice; DEX group, CS-exposed mice with DEX treatment (1 μmol/kg per day for 10 days). Other groups: CS-exposed mice treated with brusatol or one of its derivatives (2 μmol/kg per day for 35 days). (A) Effects of brusatol and brusatol derivatives on the numbers of white blood cells (WBC), neutrophils (NEU), and eosinophils (EOS). (B) Effects of brusatol and brusatol derivatives on TNF-α. (C) Effects of brusatol and brusatol derivatives on IL-1β. (D) Effects of brusatol and brusatol derivatives on IL-6. (E) Effects of brusatol and brusatol derivatives on IL-8. (F) Effects of brusatol and brusatol derivatives on IL17. The concentrations of the inflammatory factors were measured using ELISA kits. The data are shown as the means ± SEMs (n = 8−11). Statistical analysis was performed using Student’s t test: (*) P < 0.05 vs the model group, (**) P < 0.01 vs the model group, (#) P < 0.05 vs the control group, (##) P < 0.01 vs the control group.

determined (Figure 4). Compared with the vehicle-treated model group, compounds 34 and 75, with NO-release properties, could significantly reduce the numbers of white blood cells, neutrophils, and eosinophils and suppress the levels of various inflammatory factors in the BAL fluid. In particular, compound 34 demonstrated very high inhibition of neutrophils (inhibition rate = 85% ± 5%). Although the parent compound of 75 was not brusatol, 75 also significantly reduced the IL-8 level by 48 ± 4% compared with that of the model group. The inhibitory effect of 75 was comparable to that of brusatol (46 ± 7%). Overall, these data clearly demonstrate that even at very low doses (2 μmol/kg per day), both 34 and 75 exhibited high inhibitory efficacy against COPD-like lung inflammation in mice. Conversely, compound 42, without obvious NO-release capability, again exhibited no significant activity in vivo. Thus, the NO-release properties of the compounds appear to affect their inhibitory effects on COPD-like lung inflammation in the mouse model. Acute Toxicity Studies. To evaluate the safety of 34 and 75, groups of KM mice (half male and half female) were administered either a single dose or the vehicle control by gavage. The survival of the mice was monitored and recorded over 14 days (Supporting Information, Tables S3 and S4). The

amounts of NO released by 75 might play a synergistic role in anti-inflammation. In Vivo Activity against Chronic Obstructive Pulmonary Disease (COPD)-Like Inflammation. The positive results obtained for the in vitro and in vivo tests prompted us to evaluate the inhibitory effects of compounds 34 and 75 on COPD-like lung inflammation in the mouse model induced by cigarette smoke (CS) and LPS, using brusatol, DEX, and 42 as controls. A variety of leukocytes are involved in the occurrence and progression of COPD, and the numbers of these leukocytes are often increased. Neutrophils in particular play a major role in the pathological progress of COPD; the number of neutrophils is most significantly increased.25 The secretion levels of inflammatory factors, particularly the chemoattractant for neutrophils interleukin-8 (IL-8), are also dramatically increased.25 Excessive neutrophils and inflammatory factors will aggravate the patients’ condition. Therefore, the number of neutrophils and the level of inflammatory factors can serve as the basis for judging the state of the illness and evaluating the therapeutic effects of drugs. Thus, in our study, the total and differential leukocyte counts as well as the levels of tumor necrosis factor-α (TNF-α), IL-1β, IL-6, IL-8, and IL-17 in the bronchoalveolar lavage (BAL) fluid were G



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LD50 values of the tested compounds are presented in Table 4, which also includes the results for brusatol. Although 34

a

compd

LD50 (μmol/kg)

95% confidence interval (μmol/kg)

31.33 41.08 >3852a

23.38−38.23 34.56−49.42

EXPERIMENTAL SECTION

Chemistry. All commercial chemicals were used as supplied unless otherwise indicated. All reactions were performed in oven-dried glassware. All yields reported refer to the yields of the isolated compounds. 1H NMR and 13C NMR spectra were recorded using a Varian VNS-600 spectrometer (600 MHz), a Bruker AV III-500 spectrometer (500 MHz), an Agilent DD2-500 spectrometer (500 MHz), a Varian Mercury-400 spectrometer (400 MHz), and a Varian Mercury-300 spectrometer (300 MHz). High-resolution mass spectra were obtained using an Agilent 6520 Accurate-Mass Q-TOF LC/MS. ESI-MS was performed using an Agilent 1100 series LC-MSD-Trap-SL mass spectrometer. Silica gel TLC plates (Qing Dao Marine Chemical Factory, Qingdao, China) were used to monitor the progression of the reactions. Column chromatography was performed using silica gel (200−300 mesh size, Qing Dao Marine Chemical Factory, Qingdao, China), Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Sweden), or ODS (50 μm, Merck, Germany). The purity of the compounds was determined by HPLC analysis using an Agilent 1100 series instrument equipped with a DAD detector and a Waters xTerra C18 column (250 mm × 4.6 mm, 5 μm). HPLC conditions were the following: solvent A = water or water with 0.05% trifluoroacetic acid (v/v), solvent B = MeCN; flow rate = 1.0 mL/min; column temperature 35 °C; gradient of 20− 95% B (0−30 min). Purities of all final compounds were ≥95%. Preparative HPLC was performed on a Shimadzu instrument equipped with an LC-6AD pump and an SPD-10A detector using a YMC-PACK ODS-A column (250 mm × 20 mm, 5 μm). Known compounds 1− 8,15b,18 9−14,26,27 17,27 20−23,15c,18d,19 27,28 48−49,29 and 6430were synthesized as previously described. General Procedures for the Preparation of 15−16, 24−26, and 28. To a solution of the appropriate alcohol (1.0 mmol) in dry CH2Cl2 (20 mL), succinic anhydride or phthalic anhydride (2.0 mmol) and DMAP (49 mg, 0.40 mmol) were added. The mixture was stirred for 24 h at 40 °C. Then, the reaction mixture was washed with water, dried over Na2SO4, and concentrated. The residue was chromatographed on silica gel (CHCl3/MeOH = 40:1) to obtain the product. Compound 15. The title compound was obtained starting from 7 and succinic anhydride. White powder, 90.3% yield. 1H NMR (300 MHz, CD3COCD3) δ 8.08 (2H, dd, J = 7.8, 0.9 Hz, ArH), 7.90 (1H, t, J = 7.5 Hz, ArH), 7.76 (2H, t, J = 7.8 Hz, ArH), 7.52 (2H, d, J = 9.0 Hz, ArH), 7.42 (2H, d, J = 9.0 Hz, ArH), 5.17 (2H, s, OCH2), 2.65 (4H, m, COCH2CH2CO). HRESIMS m/z = 447.0500 [M − H]− (calcd for C19H15N2O9S, 447.0504). The yields and spectroscopic data for compounds 16, 24−26, and 28 are included in the Supporting Information. Compound 18. I2 (76 mg, 0.3 mmol) was added to a solution of 1 (85 mg, 0.28 mmol), triphenyl phosphine (79 mg, 0.3 mmol) and imidazole (38 mg, 0.56 mmol) in dry CH2Cl2 (10 mL). The mixture was stirred for 5 h in the dark at room temperature. Then, the reaction mixture was sequentially washed with saturated NaHCO3 solution and 10% aqueous Na2S2O3 solution and then dried over Na2SO4. After filtration, the filtrate was evaporated to dryness in vacuo, and the crude product was purified by silica gel column chromatography (PE/EtOAc = 4:1) to yield the title compound as a white solid powder (100 mg, 86.2% yield). 1H NMR (500 MHz, CDCl3) δ 8.06 (2H, dd, J = 8.0, 1.0 Hz, ArH), 7.77 (1H, t, J = 7.5 Hz, ArH), 7.64 (2H, t, J = 8.0 Hz, ArH), 4.53 (2H, t, J = 6.0 Hz, OCH2), 3.36 (2H, t, J = 6.5 Hz, CH2I), 2.35 (2H, m, CH2). HRESIMS m/z = 410.9510 [M + H]+ (calcd for C11H12IN2O5S, 410.9506). General Procedures for the Preparation of 19 and 29−32. To a solution of the appropriate alcohol (0.3 mmol) in CH2Cl2 (5 mL) at 0 °C, PBr3 (81.2 mg, 0.3 mmol) was added. After stirring for 10 min at 0 °C, the reaction mixture was warmed to room temperature, stirred for 1 h. and quenched with H2O (5 mL). The water layer was extracted with CH2Cl2. The combined organic layers were dried over anhydrous Na2SO4 and concentrated. The residue was purified by silica gel column chromatography (petroleum/EtOAc = 10:1) to yield the product. Compound 19. The title compound was obtained starting from 7 and PBr3. White powder, 91.8% yield. 1H NMR (500 MHz, CD3COCD3) δ 8.07 (2H, dd, J = 8.0, 1.0 Hz, ArH), 7.79 (1H, t, J =

Table 4. Median Lethal Dose (LD50) Values of the Tested Compounds brusatol 34 75

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The equivalent dose is 5 g/kg.

exhibited reduced toxicity compared with brusatol, it remains unsuitable as a candidate drug. By contrast, 75 exhibited over 100-fold less toxicity than brusatol. Even at a very high dose (3852 μmol/kg), oral administration of 75 did not cause any abnormality in the mice throughout the observation period as in the case of the vehicle alone. This finding suggests that compound 75 is worthy of further study. Structure−Activity Relationships (SARs). Some SARs among these compounds were revealed through a simple analysis. First, phenylsulfonylfuroxan was crucial to the performance of the hybrids. For instance, the phenylsulfonylfuroxansubstituted derivative 34 displayed greater activity in vivo than the phenylfuroxan-substituted derivative 42. Furthermore, the presence of a free C-21 carboxyl group had a considerable impact on biological activity and toxicity: compounds 65 and 76 were less active and less cytotoxic than brusatol and 34, respectively. Moreover, the coupling site of brusatol with the furoxans was also of importance to the compounds’ activities and toxicities. The 3position derivatives exhibited better biological activities and higher toxicities than the 15-position and 21-position derivatives, as observed by comparing 34 with 51 and 67. Additionally, the type of linker that connected the NO-donor moiety to the 3position of brusatol also affected the biological results. For example, compound 34, with succinic anhydride as the linker, was more active than the corresponding compound 41, which used phthalic anhydride as the linker. Compound 47, with the ether linker, exhibited reduced inhibition and toxicity compared with compound 42, with the ester linker. In accordance with the SARs described above, compound 75 exhibited high activity and low toxicity both in vitro and in vivo. The success of compound 75 further validates these SARs. However, the precise SARs of these derivatives still require further investigation.

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CONCLUSIONS We synthesized a series of brusatol derivatives containing NOdonor furoxan moieties. As an anti-inflammatory agent, compound 75 exhibited substantial inhibitory efficacy on COPD-like lung inflammation in the mouse model induced by CS and LPS. Additionally, compound 75 was also over 100-fold less toxic (LD50 > 3,852 μmol/kg) than brusatol. The superior properties of this hybrid may be attributed to the introduction of phenylsulfonyl-substituted furoxan NO-releasing groups. Compound 75 could be a promising candidate for intensive study. Additionally, our SAR study can serve as a guide for further research. Overall, the NO-donor analogues of brusatol described here represent a new and interesting class of polyvalent drugs potentially useful in the treatment of a number of inflammationrelated diseases. H



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7.5 Hz, ArH), 7.65 (2H, t, J = 8.0 Hz, ArH), 7.47 (2H, d, J = 8.5 Hz, ArH), 7.28 (2H, d, J = 8.5 Hz, ArH), 4.50 (2H, s, CH2Br). HRESIMS m/ z = 432.9474 [M + Na]+ (calcd for C15H11BrN2NaO5S, 432.9464). The yields and spectroscopic data for compounds 29−32 are included in the Supporting Information. General Procedures for the Preparation of 33−46. A solution of brusatol (90 mg, 0.17 mmol), the appropriate acid (0.34 mmol), EDCI (78 mg, 0.41 mmol), and DMAP (42 mg, 0.34 mmol) in dry CH2Cl2 (10 mL) was stirred at 0 °C. After stirring for 2 h at 0 °C, the reaction mixture was warmed to room temperature or to 40 °C and stirred for 24−48 h. The solution was diluted with CH2Cl2, washed sequentially with diluted HCl, saturated NaHCO3 solution, and brine, dried over anhydrous Na2SO4, and concentrated. The crude product was purified by preparative HPLC (CH3CN/H2O = 55:45, flow rate = 6 mL/min). Compound 34. The title compound was obtained starting from brusatol and 10. White powder, 65.5% yield. 1H NMR (400 MHz, CDCl3) δ 8.05 (2H, br d, J = 7.6 Hz, ArH), 7.76 (1H, t, J = 7.6 Hz, ArH), 7.62 (2H, t, J = 7.6 Hz, ArH), 6.24 (1H, br s, H-15), 5.62 (1H, s, H-2′), 4.79 (1H, s, H-7), 4.72 (1H, d, J = 7.6 Hz, Ha-20), 4.44 (2H, t, J = 6.0 Hz, OCH2), 4.24 (1H, d, J = 2.8 Hz, H-11), 4.20 (2H, t, J = 6.0 Hz, OCH2), 4.19 (1H, s, H-12), 3.79 (1H, d, J = 7.6 Hz, Hb-20), 3.78 (3H, s, CH3O21), 3.14 (1H, br s, H-14), 3.04 (1H, d, J = 12.4 Hz, H-5), 2.95 (1H, d, J = 16.0 Hz, Hβ-1), 2.88 (2H, t, J = 6.4 Hz, COCH2), 2.72 (2H, m, COCH2), 2.39 (1H, d, J = 16.0 Hz, Hα-1), 2.38 (1H, d, J = 14.0 Hz, Hα6), 2.19 (3H, s, H-5′), 2.09 (1H, br s, H-9), 1.97 (2H, m, CH2), 1.93 (3H, s, H-4′), 1.84 (2H, m, CH2), 1.84 (1H, overlapped with CH2, Hβ6), 1.81 (3H, s, H-18), 1.47 (3H, s, H-19). 13C NMR (100 MHz, CDCl3) δ 188.9, 172.0, 171.9, 170.2, 166.9, 164.4, 161.0, 158.9, 145.7, 142.1, 138.0, 135.6, 129.7, 128.5, 114.0, 110.5, 82.1, 81.2, 75.8, 73.9, 71.0, 70.8, 65.7, 63.9, 53.0, 51.6, 50.1, 45.3, 42.8, 41.7, 40.7, 29.0, 28.8, 28.5, 27.7, 25.1, 25.0, 20.6, 15.4, 14.5. HRESIMS m/z = 939.2450 [M + Na]+ (calcd for C42H48N2NaO19S, 939.2464). Compound 42. The title compound was obtained starting from brusatol and 24. White powder, 30.7% yield. 1H NMR (500 MHz, CD3COCD3) δ 7.88 (2H, dd, J = 8.5, 1.5 Hz, ArH), 7.61 (3H, m, ArH), 7.36 (2H, d, J = 8.5 Hz, ArH), 7.03 (2H, d, J = 8.5 Hz, ArH), 6.10 (1H, br s, H-15), 5.63 (1H, s, H-2′), 5.27 (2H, s, OCH2), 5.08 (2H, s, OCH2), 4.96 (1H, s, H-7), 4.73 (1H, d, J = 7.5 Hz, Ha-20), 4.27 (1H, d, J = 4.5 Hz, H-11), 4.26 (1H, s, H-12), 3.78 (1H, d, J = 7.5 Hz, Hb-20), 3.70 (3H, s, CH3O-21), 3.32 (1H, br s, H-14), 3.24 (1H, d, J = 12.5 Hz, H-5), 2.84 (2H, m, COCH2), 2.81 (1H, d, J = 15.5 Hz, Hβ-1), 2.72 (2H, t, J = 6.0 Hz, COCH2), 2.70 (1H, overlapped with COCH2, Hα-1), 2.40 (1H, br s, H-9), 2.27 (1H, dt, J = 14.5, 2.0 Hz, Hα-6), 2.13 (3H, s, H-5′), 1.97 (1H, ddd, J = 14.5, 12.5, 2.0 Hz, Hβ-6), 1.91 (3H, s, H-4′), 1.77 (3H, d, J = 1.0 Hz, H-18), 1.50 (3H, s, H-19). 13C NMR (125 MHz, CD3COCD3) δ 189.9, 172.4, 171.8, 170.7, 167.6, 164.9, 159.0, 158.3, 158.1, 146.8, 142.7, 132.2, 131.1, 130.8, 130.2, 128.6, 127.3, 115.8, 115.8, 113.1, 83.2, 82.3, 76.4, 74.0, 72.7, 67.7, 66.3, 59.6, 52.7, 51.0, 50.4, 46.1, 43.2, 41.6, 41.5, 29.4, 29.1, 27.2, 20.2, 15.8, 14.5. HRESIMS m/z = 923.2844 [M + Na]+ (calcd for C46H48N2NaO17, 923.2845). The yields and spectroscopic data for compounds 33, 35−41, and 43−46 are included in the Supporting Information. Compound 47. Potassium carbonate (64 mg, 0.46 mmol) and the corresponding bromide 29 (126 mg, 0.35 mmol) were added to a solution of brusatol (120 mg, 0.23 mmol) in acetonitrile (20 mL), and the mixture was stirred at 50 °C under an argon atmosphere for 5 h. The solvent was removed in vacuo. The residue was dissolved in chloroform (20 mL), and the insoluble matter was removed by filtration. The filtrate was concentrated in vacuo, and the crude product was purified by silica gel column chromatography using CH2Cl2/CH3OH (50:1) to yield the final compound as a white solid powder (100 mg, 54.2% yield). 1H NMR (500 MHz, CD3COCD3) δ 7.88 (2H, dd, J = 8.0, 1.5 Hz, ArH), 7.60 (3H, m, ArH), 7.38 (2H, d, J = 8.5 Hz, ArH), 7.01 (2H, d, J = 8.5 Hz, ArH), 6.10 (1H, br s, H-15), 5.63 (1H, s, H-2′), 5.27 (2H, s, OCH2), 4.93 (1H, s, H-7), 4.84, 4.78 (each 1H, d, J = 11.0 Hz, OCH2), 4.71 (1H, d, J = 7.5 Hz, Ha-20), 4.26 (1H, d, J = 5.0 Hz, H-11), 4.25 (1H, s, H-12), 3.75 (1H, d, J = 7.5 Hz, Hb-20), 3.70 (3H, s, CH3O-21), 3.30 (1H, br s, H-14), 3.09 (1H, d, J = 12.5 Hz, H-5), 2.78 (1H, d, J = 16.0 Hz, Hβ-1), 2.63 (1H, d, J = 16.0 Hz, Hα-1), 2.31 (1H, br s, H-9), 2.22 (1H, dt, J =

14.5, 2.0 Hz, Hα-6), 2.13 (3H, s, H-5′), 1.91 (3H, s, H-4′), 1.84 (1H, ddd, J = 14.5, 12.5, 2.0 Hz, Hβ-6), 1.76 (3H, d, J = 1.5 Hz, H-18), 1.32 (3H, s, H-19). 13C NMR (125 MHz, CD3COCD3) δ 193.4, 171.7, 167.6, 165.0, 159.0, 158.2, 158.0, 148.1, 144.9, 132.7, 132.2, 131.2, 130.2, 128.5, 127.2, 115.8, 115.7, 113.1, 83.4, 82.3, 76.4, 74.1, 73.3, 72.6, 67.6, 59.6, 52.7, 51.6, 51.2, 46.1, 43.2, 41.8, 41.2, 27.2, 20.2, 15.7, 14.4. HRESIMS m/z = 801.2867 [M + H]+ (calcd for C42H45N2O14, 801.2865). General Procedures for the Preparation of 50−63. A solution of 49 (100 mg, 0.18 mmol) and the appropriate acid (0.36 mmol) in dry CH2Cl2 (20 mL) was treated with EDCI (86 mg, 0.44 mmol) and DMAP (44 mg, 0.36 mmol). The resulting reaction mixture was stirred in an ice bath for 2 h and then reacted at room temperature or 40 °C for 24−48 h. The mixture was diluted with CH2Cl2, washed sequentially with diluted HCl, saturated NaHCO3 solution, and brine, dried over Na2SO4, and filtered. The solvent was removed in vacuo to obtain the residue, which was then dissolved in tetrahydrofuran (THF), and 2−3 drops of acetic acid and excess tetrabutylammonium fluoride solution (1.0 M solution in THF) were added. The mixture was stirred at room temperature for 12 h. Water was added to the mixture, and the entire mixture was extracted with CH2Cl2. The organic layer was washed with brine, dried over Na2SO4, and concentrated. The residue was purified by preparative HPLC (CH3CN/H2O = 50:50, flow rate = 6 mL/min) to obtain the product. Compound 51. The title compound was obtained starting from 49 and 10. White powder, 58.0% yield. 1H NMR (300 MHz, CDCl3) δ 8.06 (2H, br d, J = 7.8 Hz, ArH), 7.77 (1H, t, J = 7.5 Hz, ArH), 7.63 (2H, t, J = 7.5 Hz, ArH), 6.38 (1H, d, J = 12.9 Hz, H-15), 6.08 (1H, s, 3-OH), 4.74 (1H, s, H-7), 4.73 (1H, d, J = 7.5 Hz, Ha-20), 4.46 (2H, t, J = 6.0 Hz, OCH2), 4.25 (1H, m, H-11), 4.22 (1H, s, H-12), 4.19 (2H, overlapped with H-12, OCH2), 3.88 (3H, s, CH3O-21), 3.81 (1H, d, J = 7.5 Hz, Hb20), 3.06 (1H, d, J = 13.2 Hz, H-14), 2.98 (1H, J = 16.2 Hz, Hβ-1), 2.93 (1H, d, J = 12.9 Hz, H-5), 2.54−2.81 (4H, m, COCH2CH2CO), 2.40 (1H, J = 16.5 Hz, Hα-1), 2.37 (1H, d, J = 14.7 Hz, Hα-6), 2.18 (1H, d, J = 3.3 Hz, H-9), 1.95 (2H, m, CH2), 1.84 (2H, m, CH2), 1.84 (3H, s, H18), 1.77 (1H, ddd, J = 14.7, 12.9, 2.4 Hz, Hβ-6), 1.39 (3H, s, H-19). 13C NMR (75 MHz, CDCl3) δ 192.1, 172.3, 171.6, 170.6, 166.7, 158.9, 144.0, 137.9, 135.6, 129.6, 128.5, 127.8, 110.4, 82.8, 81.4, 75.5, 73.9, 70.9, 66.9, 64.1, 53.1, 51.5, 48.4, 45.6, 41.9, 41.8, 41.0, 28.9, 28.7, 28.3, 25.1, 24.8, 15.3, 13.2. HRESIMS m/z = 857.2050 [M + Na]+ (calcd for C37H42N2NaO18S, 857.2046). Compound 59. The title compound was obtained starting from 49 and 24. White powder, 56.4% yield. 1H NMR (300 MHz, CDCl3) δ 7.83 (2H, dd, J = 7.5, 1.2 Hz, ArH), 7.53 (3H, m, ArH), 7.31 (2H, d, J = 7.8 Hz, ArH), 6.97 (2H, d, J = 7.8 Hz, ArH), 6.36 (1H, d, J = 12.9 Hz, H-15), 6.12 (1H, s, 3-OH), 5.09 (2H, s, OCH2), 5.07 (2H, s, OCH2), 4.74 (1H, s, H-7), 4.72 (1H, d, J = 8.1 Hz, Ha-20), 4.25 (1H, d, J = 3.9 Hz, H-11), 4.22 (1H, s, H-12), 3.84 (3H, s, CH3O-21), 3.79 (1H, d, J = 8.1 Hz, Hb20), 3.06 (1H, d, J = 13.2 Hz, H-14), 2.96 (1H, J = 16.2 Hz, Hβ-1), 2.91 (1H, overlapped with Hβ-1, H-5), 2.55−2.81 (4H, m, COCH2CH2CO), 2.41(1H, J = 16.2 Hz, Hα-1), 2.35 (1H, d, J = 14.1 Hz, Hα-6), 2.18 (1H, d, J = 3.0 Hz, H-9), 1.83 (3H, s, H-18), 1.76 (1H, ddd, J = 14.1, 12.6, 1.5 Hz, Hβ-6), 1.38 (3H, s, H-19). 13C NMR (75 MHz, CDCl3) δ 192.1, 172.2, 171.6, 170.6, 166.8, 157.0, 156.9, 144.0, 131.4, 130.3, 129.5, 129.3, 127.7, 127.6, 126.0, 114.9, 112.0, 82.8, 81.4, 75.5, 74.0, 70.9, 66.9, 66.3, 58.3, 53.1, 51.6, 48.4, 45.7, 42.0, 41.9, 41.0, 29.0, 28.8, 28.3, 15.3, 13.3. HRESIMS m/z = 841.2436 [M + Na]+ (calcd for C41H42N2NaO16, 841.2427). The yields and spectroscopic data for compounds 50, 52−58, and 60−63 are included in the Supporting Information. Compound 65. To a solution of 64 (100 mg, 0.16 mmol) in tetrahydrofuran (0.5 mL), a tetrabutylammonium fluoride solution (1.0 M solution in tetrahydrofuran, 0.5 mL, 0.5 mmol) was added. The mixture was stirred at room temperature for 3 h. Then the solvent was evaporated to yield a residue, which was chromatographed on ODS using MeOH/H2O/CF3COOH (75:25:0.01) as an eluent to obtain the product as a colorless amorphous solid (70 mg, 85.8% yield). 1H NMR (500 MHz, CD3OD) δ 6.17 (1H, br s, H-15), 5.68 (1H, s, H-2′), 4.91 (1H, s, H-7), 4.71 (1H, d, J = 7.5 Hz, Ha-20), 4.17 (1H, d, J = 4.0 Hz, H11), 4.14 (1H, s, H-12), 3.74 (1H, d, J = 7.5 Hz, Hb-20), 3.24 (1H, m, H14), 2.97 (1H, d, J = 13.0 Hz, H-5), 2.84 (1H, d, J = 16.0 Hz, Hβ-1), 2.53 I



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(1H, d, J = 16.0 Hz, Hα-1), 2.30 (1H, dt, J = 14.5, 2.0 Hz, Hα-6), 2.21 (1H, s, H-9), 2.15 (3H, s, H-5′), 1.92 (3H, s, H-4′), 1.88 (1H, J = 14.5, 13.0, 2.0 Hz, Hβ-6), 1.84 (3H, d, J = 1.5 Hz, H-18), 1.37 (3H, s, H-19). 13 C NMR (125 MHz, CD3OD) δ 194.4, 178.4, 169.7, 166.5, 159.6, 145.8, 130.4, 116.4, 84.8, 83.4, 78.6, 74.5, 70.9, 66.9, 51.4, 49.8, 46.2, 43.1, 42.7, 42.2, 30.1, 27.5, 20.5, 16.0, 13.4. HRESIMS m/z = 505.1706 [M − H]−(calcd for C25H29O11, 505.1715). General Procedures for the Preparation of 66, 68, and 70−73. To a solution of 64 (20 mg, 0.032 mmol) in acetonitrile (1 mL) were added potassium carbonate (22 mg, 0.16 mmol) and the corresponding iodide or bromide (0.048 mmol). Then, the mixture was stirred at 50 °C under an argon atmosphere for 5 h. The mixture was diluted with CH2Cl2 (20 mL), and the insoluble matter was removed by filtration. The filtrate was concentrated in vacuo, and the residue was purified by column chromatography on silica gel (CH2Cl2/CH3OH = 50:1) to remove the excess iodide or bromide. Then, the crude intermediate was dissolved in THF and CH2Cl2, and 2−3 drops of acetic acid and excess tetrabutylammonium fluoride solution (1.0 M solution in THF) were added. The mixture was stirred at room temperature for 12 h. Water was added to the mixture, and the entire mixture was extracted with CH2Cl2. The organic layer was washed with brine, dried over Na2SO4, and concentrated. The residue was purified by preparative HPLC (CH3CN/ H2O = 55:45, flow rate = 6 mL/min) to produce the compound. Compound 66. The title compound was obtained starting from 64 and 18. White powder, 17.5% yield. 1H NMR (400 MHz, CDCl3) δ 8.05 (2H, br d, J = 7.6 Hz, ArH), 7.77 (1H, t, J = 7.6 Hz, ArH), 7.63 (2H, t, J = 7.6 Hz, ArH), 6.32 (1H, br s, H-15), 6.08 (1H, s, OH-3), 5.64 (1H, s, H2′), 4.80 (1H, s, H-7), 4.71 (1H, d, J = 7.6 Hz, Ha-20), 4.55 (2H, t, J = 6.0 Hz, OCH2), 4.49, 4.33 (each 1H, m, OCH2), 4.25 (1H, d, J = 2.8 Hz, H11), 4.22 (1H, s, H-12), 3.78 (1H, d, J = 7.6 Hz, Hb-20), 3.14 (1H, d, J = 11.6 Hz, H-14), 2.98 (1H, d, J = 16.0 Hz, Hβ-1), 2.95 (1H, overlapped with Hβ-1, H-5), 2.39 (1H, d, J = 16.0 Hz, Hα-1), 2.39 (1H, overlapped with Hα-1, Hα-6), 2.30 (2H, m, CH2), 2.19 (3H, s, H-5′), 2.12 (1H, br s, H-9), 1.93 (3H, s, H-4′), 1.84 (3H, d, J = 1.2 Hz, H-18), 1.76 (1H, ddd, J = 14.8, 12.8, 2.0 Hz, Hβ-6), 1.40 (3H, s, H-19). 13C NMR (100 MHz, CDCl3) δ 191.9, 171.7, 167.0, 164.4, 161.4, 158.8, 144.0, 137.7, 135.8, 129.7, 128.6, 127.6, 113.9, 110.5, 82.3, 81.3, 75.8, 74.1, 71.0, 68.0, 65.7, 62.6, 51.7, 48.6, 45.5, 42.0, 41.8, 41.1, 29.1, 27.7, 27.7, 20.7, 15.4, 13.3. HRESIMS m/z = 811.1979 [M + Na]+ (calcd for C36H40N2NaO16S, 811.1991). Compound 70. The title compound was obtained starting from 64 and 29. White powder, 60.9% yield. 1H NMR (400 MHz, CDCl3) δ 7.86 (2H, dd, J = 8.0, 1.2 Hz, ArH), 7.56 (3H, m, ArH), 7.32 (2H, d, J = 8.4 Hz, ArH), 7.02 (2H, d, J = 8.4 Hz, ArH), 6.32 (1H, d, br s, H-15), 6.07 (1H, s, OH-3), 5.54 (1H, s, H-2′), 5.16, 5.11 (each 1H, d, J = 12.4 Hz, OCH2), 5.13 (2H, s, OCH2), 4.78 (1H, s, H-7), 4.70 (1H, d, J = 8.0 Hz, Ha-20), 4.25 (1H, d, J = 3.2 Hz, H-11), 4.20 (1H, s, H-12), 3.79 (1H, d, J = 8.0 Hz, Hb-20), 3.10 (1H, d, J = 12.8 Hz, H-14), 2.98 (1H, d, J = 16.0 Hz, Hβ-1), 2.96 (1H, d, J = 12.8 Hz, H-5), 2.39 (1H, d, J = 16.0 Hz, Hα1), 2.39 (1H, overlapped with Hα-1, Hα-6), 2.17 (3H, s, H-5′), 2.13 (1H, br s, H-9), 1.91 (3H, s, H-4′), 1.85 (3H, d, J = 1.2 Hz, H-18), 1.76 (1H, ddd, J = 14.0, 12.8, 1.2 Hz, Hβ-6), 1.40 (3H, s, H-19). 13C NMR (100 MHz, CDCl3) δ 192.1, 171.4, 167.1, 164.4, 161.1, 157.2, 157.0, 144.0, 131.5, 130.2, 129.4, 128.5, 128.0, 127.6, 126.0, 115.1, 114.0, 112.0, 82.3, 81.3, 75.8, 73.9, 71.0, 67.4, 65.7, 58.4, 51.5, 48.5, 45.5, 41.8, 41.8, 41.1, 29.0, 27.7, 20.6, 15.4, 13.3. HRESIMS m/z = 787.2714 [M + H]+ (calcd for C41H43N2O14, 787.2709). The yields and spectroscopic data for compounds 68 and 71−73 are included in the Supporting Information. General Procedures for the Preparation of 67 and 69. A solution of 65 (24 mg, 0.047 mmol) and 2 or 8 (0.094 mmol) in dry CH2Cl2 (20 mL) was treated with EDCI (18 mg, 0.094 mmol) and DMAP (17 mg, 0.141 mmol). Then, the mixture was stirred at room temperature. After 24 h, the solution was washed sequentially with water and saturated NaCl solution, dried over anhydrous Na2SO4, and concentrated in vacuo. The crude product was purified by preparative HPLC (CH3CN/H2O = 55:45, flow rate = 6 mL/min). Compound 67. The title compound was obtained starting from 65 and 2. White powder, 23.7% yield. 1H NMR (400 MHz, CDCl3) δ 8.05 (2H, br d, J = 7.6 Hz, ArH), 7.77 (1H, t, J = 7.6 Hz, ArH), 7.63 (2H, t, J =

7.6 Hz, ArH), 6.28 (1H, br s, H-15), 6.08 (1H, s, OH-3), 5.62 (1H, s, H2′), 4.80 (1H, s, H-7), 4.69 (1H, d, J = 8.0 Hz, Ha-20), 4.49 (2H, t, J = 4.8 Hz, OCH2), 4.36, 4.24 (each 1H, m, OCH2), 4.24 (1H, br s, H-11), 4.22 (1H, s, H-12), 3.78 (1H, d, J = 8.0 Hz, Hb-20), 3.14 (1H, d, J = 11.6 Hz, H-14), 2.98 (1H, d, J = 16.0 Hz, Hβ-1), 2.94 (1H, overlapped with Hβ-1, H-5), 2.38 (1H, d, J = 16.0 Hz, Hα-1), 2.37 (1H, d, J = 14.4 Hz, Hα-6), 2.19 (3H, s, H-5′), 2.11 (1H, br s, H-9), 2.00 (2H, m, CH2), 1.93 (2H, m, CH2), 1.92 (3H, s, H-4′), 1.84 (3H, s, H-18), 1.76 (1H, ddd, J = 14.4, 12.8, 2.0 Hz, Hβ-6), 1.39 (3H, s, H-19). 13C NMR (100 MHz, CDCl3) δ 192.0, 171.6, 167.0, 164.4, 161.1, 159.0, 144.0, 137.8, 135.7, 129.7, 128.6, 127.7, 114.0, 110.6, 82.3, 81.2, 75.8, 74.0, 71.0, 70.8, 65.8, 65.7, 51.6, 48.6, 45.5, 41.9, 41.8, 41.1, 29.1, 27.7, 25.4, 24.8, 20.7, 15.4, 13.3. HRESIMS m/z = 803.2337 [M + H]+ (calcd for C37H43N2O16S, 803.2328). The yield and spectroscopic data for compound 69 are included in the Supporting Information. General Procedures for the Preparation of 74 and 76. A solution of 10 (315 mg, 0.76 mmol), EDCI (146 mg, 0.76 mmol), and DMAP (185 mg, 1.52 mmol) in dry CH2Cl2 (20 mL) was stirred at 0 °C for 3 h. Then 65 (385 mg, 0.76 mmol) was added to the solution and the mixture was stirred at room temperature for 3 h. The solution was washed with diluted HCl, dried over Na2SO4, and concentrated in vacuo. The product was purified by preparative HPLC (CH3CN/H2O/ CF3COOH = 50:50:0.03, flow rate = 6 mL/min) to obtain the final compound. Compound 74. White powder, 32.3% yield. 1H NMR (500 MHz, CDCl3) δ 8.05 (2H, br d, J = 8.0 Hz, ArH), 7.76 (1H, t, J = 7.5 Hz, ArH), 7.63 (2H, t, J = 8.0 Hz, ArH), 6.08 (1H, br s, H-15), 6.08 (1H, overlapped with H-15, OH-3), 5.68 (1H, s, H-2′), 5.26 (1H, s, H-12), 4.82 (1H, s, H-7), 4.78 (1H, d, J = 6.5 Hz, Ha-20), 4.45 (2H, t, J = 6.0 Hz, OCH2), 4.19 (1H, br s, H-11), 4.15 (2H, m, OCH2), 3.80 (1H, d, J = 6.0 Hz, Hb-20), 3.25 (1H, br s, H-14), 2.97 (1H, d, J = 13.0 Hz, H-5), 2.92 (1H, d, J = 17.0 Hz, Hβ-1), 2.67 (1H, d, J = 17.0 Hz, Hα-1), 2.62 (1H, overlapped with Hα-1, Hα-6), 2.34−2.60 (4H, m, COCH2CH2CO), 2.16 (3H, s, H-5′), 2.16 (1H, overlapped with H-5′, H-9), 1.95 (2H, m, CH2), 1.90 (3H, s, H-4′), 1.84 (3H, s, H-18), 1.81 (2H, m, CH2), 1.78 (1H, overlapped with CH2, Hβ-6), 1.37 (3H, s, H-19). 13C NMR (125 MHz, CDCl3) δ 192.3, 172.4, 171.5, 170.9, 167.1, 164.7, 160.8, 158.9, 144.0, 137.9, 135.7, 129.7, 128.6, 128.4, 114.2, 110.5, 82.6, 80.1, 74.7, 74.0, 71.0, 68.9, 65.5, 64.3, 51.3, 48.4, 45.2, 42.0, 41.8, 41.0, 29.3, 29.0, 28.8, 27.6, 25.2, 24.9, 20.5, 15.5, 13.4. HRESIMS m/z = 925.2316 [M + Na]+ (calcd for C41H46N2NaO19S, 925.2308). Compound 76. White powder, 23.1% yield. 1H NMR (500 MHz, CDCl3) δ 8.04 (2H, br d, J = 7.5 Hz, ArH), 7.76 (1H, t, J = 7.5 Hz, ArH), 7.62 (2H, t, J = 7.5 Hz, ArH), 6.30 (1H, br s, H-15), 5.66 (1H, s, H-2′), 4.82 (1H, s, H-7), 4.69 (1H, br s, Ha-20), 4.44 (2H, t, J = 6.0 Hz, OCH2), 4.19 (2H, t, J = 6.0 Hz, OCH2), 4.19 (1H, overlapped with OCH2, H11), 4.11 (1H, s, H-12), 3.77 (1H, br s, Hb-20), 3.07 (1H, d, J = 10.5 Hz, H-14), 3.07 (1H, overlapped with H-14, H-5), 2.91 (1H, overlapped with COCH2, Hβ-1), 2.88 (2H, t, J = 6.0 Hz, COCH2), 2.71 (2H, t, J = 6.0 Hz, COCH2), 2.49 (1H, d, J = 14.0 Hz, Hα-1), 2.34 (1H, d, J = 12.0 Hz, Hα-6), 2.19 (1H, br s, H-9), 2.12 (3H, s, H-5′), 1.94 (2H, m, CH2), 1.88 (3H, s, H-4′), 1.82 (2H, m, CH2), 1.80 (1H, overlapped with CH2, Hβ-6), 1.80 (3H, s, H-18), 1.41 (3H, s, H-19). 13C NMR (125 MHz, CDCl3) δ 190.2, 172.7, 172.2, 170.6, 167.3, 165.0, 161.0, 158.9, 147.3, 141.9, 137.9, 135.7, 129.7, 128.5, 114.2, 110.5, 82.3, 80.9, 75.7, 73.7, 71.0, 70.7, 65.8, 64.2, 51.2, 49.7, 45.3, 42.8, 41.2, 40.7, 29.0, 28.7, 28.5, 27.6, 25.2, 24.9, 20.7, 15.5, 14.6. HRESIMS m/z = 925.2266 [M + Na]+ (calcd for C41H46N2NaO19S, 925.2308). Compound 75. A solution of 10 (630 mg, 1.52 mmol), EDCI (292 mg, 1.52 mmol), and DMAP (556 mg, 4.56 mmol) in dry CH2Cl2 (20 mL) was stirred at 0 °C for 3 h. Then 65 (385 mg, 0.76 mmol) was added to the solution and the mixture was stirred at room temperature for 24 h. To improve the yield of 75, 10 (110 mg, 0.27 mmol), EDCI (52 mg, 0.27 mmol), and DMAP (50 mg, 0.41 mmol) were added to the reaction mixture. The solution was stirred for a further 24 h and concentrated to obtain the residue, which was chromatographed on Sephadex LH-20 (MeOH/H2O = 1:1) to obtain the product as a white powder (504 mg, 51.0% yield). 1H NMR (500 MHz, CDCl3) δ 8.04 (4H, br d, J = 7.5 Hz, ArH), 7.75 (2H, t, J = 7.5 Hz, ArH), 7.62 (4H, t, J = 7.5 Hz, ArH), 6.08 J



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(1H, br s, H-15), 5.67 (1H, s, H-2′), 5.25 (1H, s, H-12), 4.83 (1H, s, H7), 4.76 (1H, d, J = 7.5 Hz, Ha-20), 4.45 (4H, m, OCH2 × 2), 4.19 (1H, overlapped with OCH2, H-11), 4.19 (2H, t, J = 6.5 Hz, OCH2), 4.14 (2H, m, OCH2), 3.79 (1H, d, J = 7.5 Hz, Hb-20), 3.26 (1H, br s, H-14), 3.06 (1H, d, J = 13.0 Hz, H-5), 2.89 (1H, d, J = 17.5 Hz, Hβ-1), 2.87 (2H, t, J = 6.5 Hz, COCH2), 2.71 (2H, m, COCH2), 2.65 (1H, overlapped with Hα-6, Hα-1), 2.64 (1H, overlapped with Hα-1, Hα-6), 2.35−2.60 (4H, m, COCH2CH2CO), 2.15 (3H, s, H-5′), 2.15 (1H, overlapped with H-5′, H-9), 1.94 (4H, m, CH2 × 2), 1.90 (3H, s, H-4′), 1.82 (4H, m, CH2 × 2), 1.80 (3H, s, H-18), 1.78 (1H, overlapped with H-18, Hβ-6), 1.44 (3H, s, H-19). 13C NMR (125 MHz, CDCl3) δ 189.3, 172.5, 172.0, 171.4, 170.5, 170.3, 167.1, 164.7, 160.9, 158.9, 158.9, 146.2, 142.0, 138.0, 137.9, 135.7, 135.7, 129.7, 129.7, 128.5, 114.2, 110.5, 110.4, 82.3, 80.1, 74.6, 73.7, 71.0, 68.8, 65.5, 64.3, 64.0, 51.2, 49.8, 45.0, 42.8, 41.9, 40.7, 29.3, 29.0, 28.8, 28.5, 27.6, 25.2, 24.9, 24.9, 20.5, 15.5, 14.5. HRESIMS m/z = 1297.2962 [M − H]− (calcd for C57H61N4O27S2, 1297.2970). Biological Assays/Pharmacology. Lipopolysaccharide (LPS), dimethyl sulfoxide (DMSO), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT), and dexamethasone sodium phosphate (DEX) were obtained from Sigma Co. (St. Louis, MO, USA). The TNFα and IL family ELISA kits were purchased from BioLegend Inc. (San Diego, CA, USA). Rabbit polyclonal antibody to iNOS and rabbit polyclonal antibody to β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). Antirabbit IgG secondary antibody was purchased from Zhong Shan Golden Bridge Biotechnology (Beijing, China). The enhanced chemiluminescence detection kit was obtained from Tiangen Biotech (Beijing, China). In Vitro Inhibitory Activity on Excess NO Production. Cell Culture, Stimulation and Measurement of Nitrite Production. C57BL/6J male mice (20−22 g) were purchased from the Animal Center of Military Medical Science (Beijing, China). Three days after intraperitoneal injection (ip) of brewer thioglycollate medium (5 mL/ 100 g body weight), the C57BL/6J male mice were ethically sacrificed and submerged in 75% ethanol for 5 min. Then under sterile conditions, 5−8 mL of phosphate buffered saline (PBS) was injected into the peritoneal cavity. After needle withdrawal, the abdomen was kneaded several times. The needle was then inserted into the upper part of the abdomen, and peritoneal fluids were collected. This procedure was repeated three times as mentioned above. The resultant cell suspensions were centrifuged at 1000 rpm for 5 min. Then, the macrophages obtained were seeded in 96-well plates (Costar, USA) at a cell density of 1 × 105 cell/well in RPMI-1640 supplemented with 5% (v/v) newborn calf serum (Gibco, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin. After being cultured for 2 h, the cells were washed to remove any nonadherent cells, and the medium was replaced with fresh medium. The cells were then treated with or without various concentrations of test compounds. After incubation for another 1 h, the cells were challenged with 1 μg/mL of LPS, followed by additional culturing. After 24 h, the supernatants of the culture media were mixed with an equivalent volume of Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride in 5% phosphoric acid), and the absorbances at 570 nm were then measured. The cells treated with diluent were used as controls for the background levels of nitrite production, and sodium nitrate at various concentrations was used as the positive control for the standard curve. All incubation procedures were performed under 5% CO2 in humidified air at 37 °C. Cell Viability Assay. Cell viabilities were measured using the MTT assay. Briefly, peritoneal macrophages from C57BL/6 male mice were seeded in 96-well plates at concentrations of 1 × 105 cells per well. After incubation for 2 h, the cells were incubated with compounds for 24 h and then washed with PBS three times. Following the washing step, 20 μL of MTT solution (5 mg/mL in PBS) was added to each well, and the cells were incubated at 37 °C for another 4 h. Finally, the culture medium was removed, and the formazan crystal was dissolved by adding 150 μL of DMSO. Absorbances at 570 nm were measured using a microplate reader (BioTek, Winooski, VT, USA). Western Blot Analyses of iNOS Protein Expression in Murine Peritoneal Macrophages. Peritoneal macrophages from C57BL/6 male mice were pretreated with each test compound for 1 h and stimulated with 1 μg/mL of LPS for an additional 24 h. Cells were

washed with PBS and lysed with lysis buffer. Lysates were centrifuged (12000g) at 4 °C for 20 min. Then, equal amounts of cell protein extract were separated on 10% SDS-polyacrylamide gel and transferred onto PVDF membranes. After blocking with 5% nonfat milk for 2 h, the membranes were incubated with a rabbit polyclonal antibody to iNOS (1:1000) and a rabbit polyclonal antibody to β-actin (1:1000) overnight at 4 °C. After three washes, the membranes were incubated with antirabbit IgG secondary antibodies (1:2000) at the appropriate dilutions for 1 h at room temperature. The labeled protein bands were visualized using an enhanced chemiluminescence detection kit. The relative levels of iNOS to control β-actin were determined by densitometric scanning. In Vivo Anti-Inflammatory Activities. Animal Model and Treatments. Kunming male mice (18−20 g) were purchased from the Animal Center of Military Medical Science (Beijing, China). The animal experiments were performed in accordance with the Institutional Guidelines for Animal Care and Use of the Chinese Academy of Medical Sciences and Peking Union Medical College. Ear edema was induced in the left ear of each Kunming male mouse through the topical application of croton oil at a concentration of 0.4 mg/ear. Various doses of brusatol and its derivatives were administered 1 h prior to croton oil application. A control group of mice was treated with an equal volume of vehicle. The mice were then euthanized under sodium pentobarbital anesthesia, and ear samples (8 mm diameter punches of tissue) were collected 4 h after the application of croton oil. The difference between the weights of the left and right ear patches was measured to determine anti-inflammatory activity. The inhibitory rates were determined from the weight differences between the groups treated with test compounds and the vehicle groups. In Vivo Inhibitory Effects on COPD-Like Lung Inflammation. Animal Model and Treatments. Specific pathogen-free (SPF) male BALB/c mice (18−20 g) from Beijing HFK Bioscience Co, Ltd. (Beijing, China) were exposed to either CS or filtered air under identical conditions. The mice were exposed to CS in a 4 L homemade Plexiglas container for 5 min per exposure twice a day for 33 days (day 2−day 13, day 15−day 35) and were infused with 20 μg of LPS in 30 μL of normal saline once a day for 2 days (day 1 and day 14). At 1 h prior to the first CS exposure or injection of LPS, the mice were treated with brusatol, a brusatol derivative or dexamethasone sodium phosphate (DEX), depending on the group to which they were assigned. The oral dose for all groups was 2 μmol/kg for 35 days, with the exception of DEX, for which the dose was 1 μmol/kg for day 26−day 35. Approximately 24 h after the final administration of the test compounds, the mice were euthanized for tissue harvesting. The study consisted of 7 groups: a control group (air-exposed mice), a model group (CS-exposed mice), and five treatment groups (CS-exposed mice with treatment). The procedures for the care and use of the animals were in accordance with the institutional guidelines of the Experimental Animal Center of the Institute of Materia Medica, Beijing, China. BAL Fluid Analysis. In brief, the lungs were lavaged three times through an intratracheal cannula with 0.8 mL of ice-cold saline. Then, the inflammatory-cell differential and inflammatory cytokines in the BAL fluid were assessed. Measurements were acquired as described in a previous publication.31 The BAL samples were centrifuged, and the supernatant was stored at −20 °C for further analysis. The cell pellets were resuspended in 200 μL of PBS and counted using a hemocytometer (Beckman Coulter LH750, USA). The levels of TNF-α and the IL family (IL-1β, IL-6, IL-8, and IL-17) in the supernatant were measured using ELISA kits in accordance with the manufacturer’s instructions. Acute Toxicity Assay. SPF Kunming mice (half male and half female) were purchased from the Animal Center of Military Medical Science (Beijing, China). After 16 h of fasting (food but not water was withheld), the mice were weighed and randomly divided into groups (10 mice/group). Test compounds were administered by gavage (0.1 mL/ 10 g) at various doses, whereas the control groups received solvent only. After the compound was administered, food was withheld for 1 h. Behavioral changes, signs of toxicity or death and the latency of death were observed over a span of 14 days. Because of the lower water solubility of the compounds, 0.5% sodium carboxymethyl cellulose (CMC-Na) was chosen as the solvent. K



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ASSOCIATED CONTENT

S Supporting Information *

Yields and spectroscopic data for new compounds, NMR (1H and 13C) spectra of all target compounds, HPLC results, NO production inhibitory data, measurement of intracellular NO, and acute toxicity data. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*For S.Y.: phone, +86-10-63165326; fax, 86-10-63017757; Email, [email protected]. *For Q.H.: phone, +86-10-63165191; fax, 86-10-63017757; Email, [email protected]. Author Contributions †

The authors W.T. and J.X. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by National Natural Science Foundation of China (grant no. 21132009) and the National Science and Technology Project of China (grant no. 2012ZX09301002-002).

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ABBREVIATIONS USED iNOS, inducible NO synthase; NSAIDs, nonsteroid antiinflammatory drugs; LPS, lipopolysaccharide; COPD, chronic obstructive pulmonary diseases; EDCI, 1-ethyl-3-(3dimethyllaminopropyl)carbodiimide hydrochloride; t-BDMSCl, dimethyl-t-butylsilyl chloride; DEX, dexamethasone sodium phosphate; SAR, structure−activity relationship; SPF, specific pathogen free; CS, cigarette smoke; BAL, bronchoalveolar lavage; IL-1β, interleukin-1β; IL-6, interleukin-6; IL-8, interleukin-8; IL-17, interleukin-17; CMC-Na, sodium carboxymethyl cellulose

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REFERENCES

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