Design, synthesis and biological evaluation of tricyclic diterpene derivatives as novel neuroprotective agents against ischemic brain injury

European Journal of Medicinal Chemistry 103 (2015) 396e408

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Design, synthesis and biological evaluation of tricyclic diterpene derivatives as novel neuroprotective agents against ischemic brain injury Ying-Ying Wang a, 1, Yuan-Xue Gao b, 1, Wei Gao a, Yuan Xu b, Ya-Zhou Xu b, Yun-Jie Wang b, Sai Chang b, Li-Gang Yu a, Lu-Yong Zhang b, Hong Liao b, Lian-Fang Yang a, Tao Pang b, **, Wen-Wei Qiu a, * a b

Department of Chemistry, School of Chemistry and Molecular Engineering, East China Normal University, 500 Dongchuan Road, Shanghai 200241, China Jiangsu Key Laboratory of Drug Screening, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 May 2015 Received in revised form 27 August 2015 Accepted 28 August 2015 Available online 10 September 2015

Lead compound 7 has neuroprotective effects, and it was discovered by screening a small synthetic natural product-like (NPL) library. Based on the lead, a series of tricyclic diterpene derivatives was designed and synthesized, and their neuroprotective effects were further evaluated against glutamate-, oxygen and glucose deprivation (OGD)- and nutrient deprivation-induced neuronal injury using cellbased assays. To our delight, most of these synthetic compounds exhibited increased neuroprotective effects and bloodebrain barrier (BBB) permeability without cellular toxicity. The most potent compound, compound 30, showed significantly improved neuroprotection against neuronal injury in primary neurons. Furthermore, compound 30 exhibited remarkable neuroprotection in transient middle cerebral artery occlusion (tMCAO) rats by reducing their infarct sizes and neurological deficit scores. A mechanistic exploration using in vitro and in vivo experiments showed that the neuroprotection of these compounds was at least partly mediated by improving the levels of glutathione (GSH), superoxide dismutase (SOD) and heme oxygenase-1 (HO-1) protein. Therefore, these tricyclic diterpene derivatives could be used as promising leads for the development of a new type of neuroprotective agents against ischemic brain injury. © 2015 Published by Elsevier Masson SAS.

Keywords: Diterpenoids Neuroprotection Glutamate Oxygeneglucose deprivation Nutrient deprivation Ischemic stroke

1. Introduction Ischemic stroke, which results from a transient or permanent reduction in cerebral blood flow, accounts for more than 80% of all strokes and is a leading cause of human morbidity and mortality throughout the world [1]. In general, brain ischemia is characterized by excess glutamate release, insufficient nutrient supply, and oxygen and glucose deprivation (OGD), which result in neuronal death. Glutamate is a major excitatory neurotransmitter that plays an important role in the mammalian brain [2]. However, excessive release of this amino acid can induce excitotoxicity, which is a

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (T. Pang), [email protected] (W.-W. Qiu). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ejmech.2015.08.057 0223-5234/© 2015 Published by Elsevier Masson SAS.

major factor in neuronal injury that is associated with many acute and chronic brain disorders, such as neurodegenerative diseases (for example, amyotrophic lateral sclerosis, Parkinson's, Alzheimer's and Huntington's diseases) [3,4], traumatic brain injury [5], and especially brain ischemic stroke [6]. An excessive level of the excitatory neurotransmitter glutamate is not only a key factor in ischemia-induced neuronal injury, but it also produces reactive oxygen species (ROS) and further leads to the inhibition of antioxidants such as superoxide dismutase (SOD) and glutathione (GSH) synthesis [7]. Oxidative stress is also considered to be one of the major mechanisms that triggers the pathogenic actions of ischemic stroke [8], which is produced when ROS surpass the endogenous antioxidant system, leading to the injury of essential components in neural cells [9]. Therefore, the reduction of ROS has been considered a promising remedy to attenuate neuronal damage from ischemia [10]. This reduction includes elements such as the antioxidative enzyme heme oxygenase-1 (HO-1), which

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protects neurons from the deleterious effects of ROS [11]. To date, the only drug that is currently approved by the FDA to treat ischemic strokes is tissue plasminogen activator (t-PA), which has a very short therapeutic window (3 h) and is used in less than 5% of stroke patients, thus significantly limiting the percentage of treatable subjects [12,13]. This deficiency indicates an urgent need to search for novel agents with neuroprotective effects to provide treatments for brain ischemic strokes. Diterpenoids are a large family of natural products that exhibit a wide range of biological activities, such as anti-inflammatory, antiHIV, anti-tumor, anti-diabetic, antibacterial and especially neuroprotective activity [14e16]. For instance (Fig. 1), carnosic acid, a component of rosemary, strongly promotes neurite outgrowth by activating antioxidant responsive element (ARE)-mediated transcription through the activation of Nrf2 [17]; serofendic acid, a sulfur-containing diterpenoid derived from fetal calf serum, exhibits potent neuroprotective actions in neurons against the cytotoxicity of glutamate and nitric oxide [18,19]; triptolide, the major active component of Tripterygium extracts, exerts both neuroprotective and neurotrophic activities in Parkinson's disease models [20,21]. Natural products have provided a rich resource for drug discovery in recent years [22]. However, the scarcity and poor activities of these products frequently limit their development. Recently, the production of modified, natural-based compounds and synthetic natural analogs has been shown to be an effective strategy in drug discovery. In our search for novel and potent anti-ischemic agents, we report the discovery of neuroprotective leads that were uncovered by screening our small synthetic natural product-like (NPL) library against glutamate-induced neuronal injury. This novel small NPL library contains approximately 200 tricyclic diterpene analogs, and it was constructed based on the cyclization reaction [23,24]. Compound 7 (Scheme 1) of the NPL library was chosen as a novel neuroprotective lead, and its neuroprotective percentage against glutamate-induced neuronal injury was 53.7% (Table 1), respectively. A series of tricyclic diterpene analogs was synthesized based on the lead compound. These newly synthesized analogs were evaluated for their neuroprotective effects against glutamate-, OGD- and nutrient deprivation-induced injury in cell-based assays. The most potent analog was further evaluated for its neuroprotective effects in a rat cerebral ischemia model. 2. Chemistry The lead compound and a series of tricyclic diterpene derivatives (methylol, esters, acyls and amides) was synthesized according to the pathways described in Schemes 1e3. The synthesis of lead compound and diterpenoid methylol and esters is outlined in Scheme 1. Compound 6 was synthesized according to our previously reported procedure [25]. The details were as follows. The coupling reaction of 6,7-epoxygeranyl acetate with 4-methoxybenzylmagnesium chloride in the presence of Li2CuCl4 yielded compound 1. Key intermediate 2 was obtained by the cyclization of 1 under the Lewis acid Et2AlCl. The bromination of

Fig. 1. Chemical structures of neuroprotective diterpenoids.

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compound 2 with Br2 in CH2Cl2 furnished intermediate 3. Compound 4 was produced by the protection of the 3-hydroxyl group of 3 with TBSCl. The treatment of 4 with n-butyllithium, and then with dry CO2 afforded compound 5. The deprotection of the TBS group under boron trifluoride etherate afforded compound 6. According to the Stork-Eschenmoser hypothesis [26,27], compound 2 is a racemate, thus all the synthesized diterpenoids herein belong to racemates. The esterification of 6 with corresponding alcohols in the presence of a catalytic amount of H2SO4 gave lead compound 7 and compounds 8 and 9. The methylol 11 was obtained by the reduction of 5 with LiAlH4 and then the deprotection of the TBS group under boron trifluoride etherate. The synthesis of diterpenoid acyls is outlined in Scheme 2. Compounds 13e17 were furnished by the esterification of compound 2 with acetic anhydride under DMAP, followed by FriedeleCrafts acylation with various acyl chlorides respectively. The hydrolysis of 13e17 with NaOH in MeOH produced compounds 18e22. Treatment of 18 with BBr3 produced compound 23. The oxidation of 18 with 2-iodoxybenzoic acid (IBX) formed compound 24. Compound 25 was prepared by the esterification of 18 with trifluoroacetic anhydride. The synthesis of diterpenoid amides is outlined in Scheme 3. Amide derivatives 26e32 were synthesized by the condensation of 6 with amines or their hydrochlorides under 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDCI) and 1hydroxybenzotriazole (HOBt) in CH2Cl2. The hydrolysis of 29 with LiOH in THF afforded 33. 3. Result and discussion 3.1. Neuroprotective effects against neuronal damage induced by glutamate Glutamate is the primary excitatory amino acid in the mammalian brain. The glutamate-induced over-excitation of neurons plays a pivotal role in the pathogenesis of neurodegeneration [28]. For instance, the ischemic stroke is related to a dramatic increase in excitatory glutamate in the extracellular space. The neuroprotective effects of the lead compound 7 and their synthetic derivatives 6 and 8e33 were evaluated against glutamate-induced injury in primary rat cerebellar granule neuronal cells. The results are shown in Table 1. Compared with lead compound 7 (53.7%, neuroprotection), most of these derivatives showed increased or equivalent cell viability against glutamate-induced neuronal injury. Compounds 13, 18, 23, 25e27 and 30e32 exhibited more neuroprotective abilities than the leads. Compounds 18 and 30 in particular possessed much more neuroprotective potency than 7. For acyl-substituted compounds, the small acetyl group (18) showed much more potent activity than the relatively large propionyl (19), 3-methoxypropionyl (20), isobutyryl (21) and octanoyl (22) groups. Notably, 21 and 22 showed almost no activity against glutamate-induced neuronal injury. If the methoxy group on the benzene ring was hydrolyzed to a hydroxyl group (Scheme 2), the activity was clearly decreased, and a similar situation occurred as the 3-hydroxyl group was oxidized to carbonyl (18 vs 23 and 24). For amide substituted compounds, the butyramide group (30) was better than other amide substituents (26e28 and 31e33). Compound 30 (10 mM) showed the most potent neuroprotective activity against neuronal damage induced by glutamate, and was more effective than positive control edaravone (50 mM). For ester-substituted compounds, regardless of the ethyl and butyl esters (8 and 9), they had no obvious improving effects in terms of neuroprotection compared with 7, and a similar situation

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Scheme 1. Synthesis of lead compound 7 and diterpenoid esters (8 and 9) and methylol (11). Reagents and conditions: (a) 4-methoxybenzylmagnesium chloride, Li2CuCl4 (0.1 M in THF), THF, 0
C, 67%; (b) Et2AlCl (2.0 M in hexane), CH2Cl2, 78
C, 68%; (c) Br2, CH2Cl2, 0
C, 96%; (d) TBSCl, imidazole, DMF, rt, 95%; (e) n-BuLi, CO2, THF, 78
C, 81%; (f) boron trifluoride etherate, CHCl3, rt, 79% for 6 and 87% for 11; (g) H2SO4, alcohols, reflux, 81e92%; (h) LiAlH4, THF, rt, 85%.

Table 1 Neuroprotective effects of target compounds against glutamate-induced neuronal injury at 10 mM. Compd

% Cell viability

6 7 8 9 11 13 18 19 20 21 22

54.0 72.4 73.1 71.0 64.4 80.4 85.2 63.2 71.5 38.8 40.5

± ± ± ± ± ± ± ± ± ± ±

0.8 2.6 5.4 1.8 6.2 4.9 6.7 5.4 4.4 3.2 3.0

% Neuroprotection

Compd

% Cell viability

22.8 53.7 54.9 51.3 40.3 67.1 75.2 38.3 52.2 e e

23 24 25 26 27 28 30 31 32 33 Edaravonea

77.1 69.7 81.0 77.3 78.2 70.7 94.1 81.7 79.1 57.1 86.7

± ± ± ± ± ± ± ± ± ± ±

6.7 5.4 3.4 4.2 5.2 7.2 4.9 0.7 3.4 4.1 5.6

% Neuroprotection 61.6 49.2 68.1 61.9 63.4 50.8 90.1 69.3 64.9 28.0 77.7

The cell viability obtained with the control was considered as 100%, and the basal percentage of viable neurons after treatment with 100 mM glutamate was 40.4%. The percentages of neuroprotection are determined from the basal values. The data are expressed as the mean ± SD, n ¼ 3. a Edaravone was 50 mM.

was found as the methyl ester (7) was replaced by methylol (11) or free carboxylic acid (6). In addition, the compounds that possessed a neuroprotective potency of more than 65 percent of neuroprotection were selected for activity testing at different doses. The results showed that these compounds exerted a well-defined dose-dependent response

against glutamate-induced neuronal damage (Fig. 2).

3.2. Neuronal protection effects against neuronal damage induced by OGD OGD can cause long-lasting damage to the central nervous

Scheme 2. Synthesis of diterpenoid Acyls (18e25). Reagents and conditions: (a) acetic anhydride, DMAP, CH2Cl2, rt, 88%; (b) acyl chlorides, AlCl3, CH2Cl2, 10
C, 75e97%; (c) 1 M NaOH, MeOH, reflux, 57e97%; (d) AlCl3, CH2Cl2, reflux, 81%; (e) IBX, THF, DMSO, rt, 85%; (f) trifluoroacetic anhydride, DMAP, CH2Cl2, rt, 86%.

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Scheme 3. Synthesis of diterpenoid amides (26e28 and 30e33). Reagents and conditions: (a) amines or their hydrochlorides, EDCI, HOBt, DMAP, CH2Cl2, rt, 77e98%; (b) LiOH, THF, H2O, rt, 84%.

Fig. 2. Neuroprotective effects of the parent compound and target compounds against glutamate-induced injury in primary rat cerebellar granule neuronal cells at 0.1, 1, and 10 mM. The neurons were pretreated with the test compounds for 24 h, and then the cells were treated with 100 mM glutamate (Glu) for another 24 h. Cell viability was determined by MTT assay. The viability of untreated cells (Control) is defined as 100%. The data are expressed as the mean ± SD. ***p < 0.001, compared with Control; #p < 0.05, ##p < 0.01, ###p < 0.001, compared with the Glu group.

system (CNS) and cell death. Lead compound 7 and compounds 13, 18, 25, 30 and 31 possessed neuroprotective potencies of more than 65% (Table 1) against glutamate-induced neuronal damage, and they were further selected to examine their protective effects against OGD-induced neuronal death. Edaravone was used as a positive control. The results showed that the effects of the protection against OGD-induced damage were not exactly the same with glutamate-induced neuronal damage (Table 2). Most selected compounds showed decreased activity compared with 7, except 18 and 30. When the compound concentrations were increased from 1 mM to 10 mM, the cell viability percentages increased. The neuroprotection percentage of acyl substituted compound 18 was 11.9%

more potent than that of 7. The most potent amide-substituted compound, which was compound 30, exhibited 75.4% neuroprotection against OGD-induced injury at 10 mM, which was almost 2-fold more potent than 7. 3.3. Neuronal protection effects against neuronal damage induced by serum deprivation The differentiated neurons in the central nervous system (CNS) require nutrients, including serum, to survive and exert their functions [29]. In several brain diseases, such as stroke and traumatic brain injury (TBI), there are insufficient nutrients to support

Table 2 Neuroprotective effects of the tested compounds against oxygeneglucose deprivation (OGD)-induced injury in primary rat cerebellar granule neuronal cells. Compd

% Cell viability 1 mM

7 13 18 25 30 31 Edaravonea

43.9 50.0 51.1 48.2 58.9 59.7

± ± ± ± ± ±

% Neuroprotection 10 mM

4.7 3.1 3.6 4.7 3.9 3.2

68.4 60.0 74.5 60.5 87.4 67.8 85.8

± ± ± ± ± ± ±

10 mM 5.6 2.0 2.1 2.4 6.0 1.2 5.1

38.4 22.0 50.3 23.0 75.4 37.2 72.3

The neuroprotective effects of the tested compounds against OGD-induced injury in cerebellar granule neurons. The cell viability obtained with the control was considered as 100%, the basal percentage of viable neurons after OGD exposure was 48.7%. The percentages of neuroprotection are determined from the basal values. The data are expressed as the mean ± SD, n ¼ 3. a Edaravone was 50 mM.

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neuron survival, resulting in neuronal death. The cultured neurons can survive in the presence of the nutrient B27, a serum substitute. If the B27 is removed, the majority of the neurons will die [30]. Lead compound 7 and compounds 13, 18, 25, 30 and 31 were selected to evaluate their activity against nutrient deprivation-induced damage in neuronal cells. N-acetylcysteine (NAC) was used as a positive control. As shown in Table 3, most tested compounds showed obvious effects against damage induced by serum deprivation (-B27), and three compounds (18, 30 and 31) possessed more potent activity than 7 at 10 mM. The best active compound, which was compound 30, exhibited 25.9% neuroprotection at 10 mM. 3.4. Detection of related protein expression in neuroprotection HO-1 expression is induced by oxidative stress, which may be compromised by cerebral ischemia, and in animal models, increasing the HO-1 expression is neuroprotective [31]. SODs are enzymes that alternately catalyze the dismutation of the toxic superoxide radical (O 2 ) into either ordinary molecular oxygen (O2) or hydrogen peroxide (H2O2) [32], which serves an important antioxidant role in neuronal injury. GSH is a key neuroprotective molecule in the brain. The strategy for increasing the neuronal GSH level is a promising approach to the treatment of neurodegenerative diseases [33]. These neuroprotective factors play key roles in the prevention of neurological damage, and their expression levels in vitro under the action of compounds 18 and 30 are displayed in Figs. 3 and 4. The results showed that these two compounds induced HO-1 protein expression effectively in a dose-dependent manner and increased the SOD and GSH levels in primary neurons at 10 mM. These findings indicated that the increases in HO-1, SOD and GSH expression levels were important ways for the neuroprotection of our synthetic tricyclic diterpene compounds. 3.5. In vitro bloodebrain barrier permeation assay The ability to penetrate the bloodebrain barrier (BBB) is important for the effectiveness of CNS drugs. To explore whether these synthetic ditepenoids could enter the brain, we used a parallel artificial membrane permeation assay for the bloodebrain barrier (PAMPA-BBB) [34]. The in vitro permeability (Pe) of the tested compounds 7, 13, 18, 25, 30 and 31 and the control drugs verapamil, clonidine and atenolol through a lipid extract of porcine brain was determined using PBS as the solvent. According to the results, we established that compounds with a Pe above 4.0  106 cm S1could penetrate into the CNS by passive diffusion (CNSþ), whereas compounds with Pe below 2.0  106 cm S1could not enter (CNS-). Given a Pe from 4.0 to 2.0  106 cm S1, the CNS permeation was considered to be uncertain (CNS±). From the results in Table 4 (except compounds 13 which was not determined

because of the undetectable optical density of the reference solution), the majority of the target compounds would be able to cross the BBB, and compounds 7, 18 and 31 in particular exhibited greater permeation. 3.6. In vitro cytotoxicity study One of the major hindrances in developing compounds into drugs that provide effective neuroprotection is their toxicity to normal cells. Thus, it is important to measure the cytotoxicity of novel neuroprotective agents in drug discovery. Compounds 7, 13, 18, 25, 30 and 31 were tested for their cytotoxic activity in primary rat cerebellar granule neuronal cells by MTT assay. The cytotoxicity results of these compounds are summarized in Fig. 5. As shown in the figure, these tested compounds showed no obvious cytotoxicity at concentrations blow 10 mM, and compounds 13, 18, 30 and 31 showed a little cytotoxicity at high concentrations (50 and 100 mM), especially compounds 30 and 31. However, there was no obvious toxicity observed in tMCAO rats treated with compound 30 (10 mg/ kg dose). 3.7. In vivo anti-ischemic activity Owing to its good cellular activity, compound 30 was chosen for in vivo evaluation in the animal brain ischemic model. The widely accepted tMCAO stroke model was used in this study [35]. To mimic the clinical situation of patients with acute strokes, we used a postischemia administration by employing a 10 mg/kg dose, as described in the Experimental section. When the rats were first treated at 2 h post-ischemia induction, followed by the administration of 4 h and 6 h post-ischemia induction, as shown in Fig. 6, compound 30 significantly reduced the infarct volume by 44% and ameliorated the neurological deficit, similar to the positive control Edaravone. There was no significant difference in the infarct size, and the neurological deficit between vehicle-treated and salinetreated rats (data not shown) ruled out the protective effect of the vehicle. 3.8. In vivo detection of GSH and SOD Reactive oxygen species production is usually involved in the pathological process of ischemia and ischemia-induced brain neuronal injury. The antioxidant system in the brain contains the oxidant scavenger GSH and the antioxidant enzyme SOD. At 24 h of MCAO, the GSH concentration and SOD activity in the ipsilateral cortex of rats were also measured to investigate the effects of compound 30 on the anti-oxidant ability in ischemic rats. As shown in Fig. 7, the brain ischemia markedly decreased the GSH concentration and SOD activity in the ipsilateral cortex, and compound 30

Table 3 Neuroprotective effects of the tested compounds against nutrient deprivation-induced injury in primary rat cerebellar granule neuronal cells. Compd

% Cell viability 1 mM

7 13 18 25 30 31 NACa

49.0 46.2 49.0 44.3 49.1 48.3

± ± ± ± ± ±

% Neuroprotection 10 mM

3.1 3.9 1.0 2.5 0.6 4.6

51.7 47.2 54.1 46.9 57.4 53.6 53.7

± ± ± ± ± ± ±

10 mM 3.4 4.0 5.7 3.9 3.8 3.5 1.2

13.6 6.0 19.5 5.0 25.9 17.0 17.2

The neuroprotective effects of the tested compounds against nutrient deprivation (B27, serum substitute deprivation)-induced injury in cerebellar granule neurons. The cell viability obtained with the control was considered as 100%, and the basal percentage of viable neurons after nutrient deprivation (-B27) was 44.1%. The data are expressed as the mean ± SD, n ¼ 3. a N-acetylcysteine (NAC) was 10 mM.

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Fig. 3. Test compounds exhibit neuroprotection by inducing HO-1 protein expression in primary neurons. The cells were treated with different concentrations of compounds 18 and 30 for 24 h, and the HO-1 protein expression was determined by western blot analysis. b-actin was used as an internal control. Panel A shows representative blots. Panel B illustrates the results of densitometric analysis. The data are expressed as the mean ± SD, n ¼ 3. *p < 0.05, **p < 0.01, ***p < 0.001, compared with Control (0 mM).

Fig. 4. Test compounds prevent glutamate-induced neuronal death by inducing SOD and GSH levels in primary neurons. The cells were pretreated with 10 mM compounds 18 and 30 for 24 h and then glutamate (Glu) was added. After 24 h of glutamate incubation, the cellular SOD (A) and GSH levels (B) were determined. The data are expressed as the mean ± SD, n ¼ 3. ***p < 0.001, compared with Control; #p < 0.05, ##p < 0.01, compared with the Glu group.

significantly increased the GSH content and SOD activity, which was similar to the action of Edaravone. These findings were consistent with the results of the previous in vitro studies in neurons.

These findings imply that these synthetic novel tricyclic diterpene derivatives may protect neurons against oxidative stress that was induced by brain ischemia through the induction of endogenous antioxidant systems. The key proteins regulating cellular

Table 4 Permeability results of target compounds and control drugs from the PAMPA-BBB assay. Compd 7 13 18 25 30

Pe (106 cm S1) a

Equi NDb Equi 7.31 ± 0.45 11.26 ± 0.34

Prediction

Compd

Pe (106 cm S1)

Prediction

CNSþ ND CNSþ CNSþ CNSþ

31 Verapamil Clonidine Atenolol

Equi 13.4 ± 0.98 5.26 ± 0.49 0.64 ± 0.43

CNSþ CNSþ CNSþ CNSe

The data are expressed as the mean ± SD, n ¼ 3. PBS was used as the solvent. a Equi: equilibrated concentration of the compound in donor and acceptor compartment and represents high permeable compound. b ND: not determined due to undetectable optical density of the reference solution with tested compound.

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Fig. 5. Cytotoxicity of the tested compounds in primary rat cerebellar granule neuronal cells. The cells were treated with different concentrations of compounds for 72 h, and cell viability was determined by MTT assay. The data are expressed as the mean ± SD, n ¼ 3. *p < 0.05, ***p < 0.001, compared with Control.

Fig. 6. Effect of compound 30 on acute cerebral ischemia in rats at 24 h after MCAO. Rats were intravenously treated with compound 30 (dissolved in DMSO, 10 mg/kg) or Edaravone (dissolved in saline, 3 mg/kg) at 2 h, 4 h and 6 h after ischemia onset. Compound 30 ameliorated the ischemia-induced infarct volume and the neurological deficits, similar to the positive control Edaravone. Panel A shows representative images of coronal brain sections stained with TTC. The infarct area and the neurological deficit scores are illustrated in panels B and C, respectively. The data are expressed as the mean ± SD, n ¼ 8e12. *p < 0.05, ***p < 0.001, compared with Vehicle (MCAO group).

Fig. 7. Effects of compound 30 on GSH concentration and SOD activity in the ipsilateral cortex of rats with MCAO. The rats were treated with 10 mg/kg 30 or 3 mg/kg Edaravone during MCAO. The GSH concentration (A) and SOD activity (B) were determined at 24 h after MCAO. The data are expressed as the mean ± SD, n ¼ 3e5. *p < 0.05, ***p < 0.001, compared with the Sham group; #p < 0.05, compared with Vehicle (the MCAO group).

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antioxidant systems, such as Nrf2, HO-1, may be the targets for our tested compounds, and whether these compounds antagonize glutamate receptors to exhibit neuroprotective effects, needs to be investigated. Further in vitro and in vivo studies are needed to clarify the molecular mechanisms of their neuroprotective roles in detail. All the in vivo studies with compound 30 indicated that these tricyclic diterpene derivatives that were used as novel neuroprotective agents can penetrate the BBB effectively and may have beneficial effects when treating for brain ischemic stroke related to oxidative damage. 4. Conclusion We synthesized over 20 new tricyclic diterpene derivatives and evaluated their neuroprotective effects against glutamate-, OGDand nutrient deprivation-induced injury in primary neurons. These derivatives exhibited good neuroprotective effects and no cellular toxicity. Among them, compounds 18 and 30 exhibited potent neuroprotective activity. The in vitro detection of related protein expression in terms of neuroprotection showed that the levels of HO-1, SOD and GSH expression were promoted by these two compounds. According to the PAMPA-BBB assay, most of these derivatives would be able to cross the BBB with great permeability. The most promising compound, which was compound 30, was chosen for the in vivo evaluation of anti-ischemic activity and exhibited neuroprotection in rats with MCAO by reducing the infarct size and neurological deficit scores. In addition, further mechanistic studies with 30 showed that during the pathological process of ischemia-induced brain neuronal injury, this compound improved the expression levels of SOD and GSH in the cortex of rats with MCAO, which was consistent with the in vitro experiments. In conclusion, we are the first to report on the use of synthetic tricyclic diterpene derivatives as a series of new chemical entities with a potential neuroprotective role. In particular, compound 30, which exhibited potent neuroprotection in vitro and in vivo, could be used as a promising lead for the development of a new class of neuroprotective agents against ischemic brain injury. 5. Experimental section 5.1. General All reagents and chemicals were purchased from commercial suppliers and used without further purification unless otherwise stated. When needed, the reactions were carried out in flame or oven-dried glassware under a positive pressure of dry N2. Column chromatography was performed on silica gel (QinDao, 200e300 mesh) using the indicated eluents. Thin-layer chromatography was carried out on silica gel plates (QinDao) with a layer thickness of 0.25 mm. Melting points were determined using the MEL-TEMP 3.0 apparatus and uncorrected. 1H (300, 400 and 500 MHz) and 13C (100 and 125 MHz) NMR spectra were recorded on Varian Mercury300, Bruker AM-400 and Bruker AV-500 spectrometer with CDCl3 or DMSO-d6 as solvent and tetramethylsilane (TMS) as the internal standard. All chemical shift values were reported in units of d (ppm). The following abbreviations were used to indicate the peak multiplicity: s ¼ singlet; d ¼ doublet; t ¼ triplet; m ¼ multiplet; br ¼ broad. High-resolution mass data were obtained on a BrukermicroOTOF-Q II spectrometer. Purity of all final tricyclic diterpene derivatives for biological testing was confirmed to be >95% as determined by HPLC analysis (for data, see Supporting Information). HPLC analysis was conducted according to the following method with the retention time expressed in min at UV detection of 254 nm. For HPLC method, an Agilent 1200 series HPLC instrument was used, with chromatography performed on a

403

ZORBAX 150 mm  4.6 mm, 5 mm C18 column with mobile phase gradient of 10e60% H2O in MeOH or 10e60% H2O in MeOH (contain 0.5% CF3COOH), with a flow rate of 1.5 mL/min. 5.2. General procedure for the synthesis of compounds 7e9 To a solution of 6 (60 mg, 0.2 mmol) in alcohols (10 mL) was added H2SO4 (10 drops) under N2. The reaction mixture was heated under reflux for 3 h. After cooling, the reaction mixture was poured into saturated NaHCO3 (20 mL) and extracted with EtOAc (10 mL  3). The organic layer was washed with brine, dried with anhydrous Na2SO4, and concentrated. The residue was purified by silica gel chromatography (petroleum ether/EtOAc, 3/1 v/v) to afford the desired product. 5.2.1. Methyl 7-hydroxy-3-methoxy-4b,8,8-trimethyl4b,5,6,7,8,8a,9,10-octahydrophenanthrene-2-carboxylate (7) White solid (92%), mp 145e147
C. 1H NMR (400 MHz, CDCl3) d 7.49 (s, 1H), 6.82 (s, 1H), 3.85 (s, 6H), 3.30 (dd, J ¼ 11.1, 5.0 Hz, 1H), 2.96e2.86 (m, 1H), 2.82e2.71 (m, 1H), 2.28 (dt, J ¼ 12.9, 3.4 Hz, 1H), 1.63e1.47 (m, 2H), 1.29 (dd, J ¼ 12.3, 2.1 Hz, 1H), 1.19 (s, 3H), 1.07 (s, 3H), 0.89 (s, 3H), 13C NMR (100 MHz, CDCl3) d 166.79, 157.49, 155.33, 132.37, 127.15, 117.59, 108.43, 78.57, 56.28, 51.99, 49.55, 39.17, 38.36, 36.93, 29.67, 28.24, 27.94, 24.73, 18.87, 15.54. HRMS (ESI): calcd for C20H28NaO4 [MþNa]þ, 355.1880, found 355.1902. 5.2.2. Ethyl 7-hydroxy-3-methoxy-4b,8,8-trimethyl4b,5,6,7,8,8a,9,10-octahydrophenanthrene-2-carboxylate (8) Colorless oil (89%). 1H NMR (400 MHz, DMSO-d6) d 7.28 (s, 1H), 6.91 (s, 1H), 4.45 (d, J ¼ 5.0 Hz, 1H), 4.20 (q, J ¼ 7.1 Hz, 2H), 3.76 (s, 3H), 3.09 (dd, J ¼ 13.6, 7.3 Hz, 1H), 2.89e2.77 (m, 1H), 2.75e2.61 (m, 1H), 2.32 (d, J ¼ 13.0 Hz, 1H), 1.84e1.75 (m, 1H), 1.47e1.33 (m, 1H), 1.25 (t, J ¼ 7.1 Hz, 3H), 1.18 (d, J ¼ 11.5 Hz, 1H), 1.13 (s, 3H), 0.98 (s, 3H), 0.79 (s, 3H). 13C NMR (100 MHz, CDCl3) d 166.28, 157.46, 155.10, 132.12, 127.09, 118.02, 108.48, 78.55, 60.73, 56.30, 49.55, 39.15, 38.32, 36.91, 29.69, 28.24, 27.92, 24.73, 18.86, 15.55, 14.45. HRMS (ESI): calcd for C21H30NaO4 [MþNa]þ, 369.2036, found 369.2074. 5.2.3. Butyl 7-hydroxy-3-methoxy-4b,8,8-trimethyl4b,5,6,7,8,8a,9,10-octahydrophenanthrene-2-carboxylate (9) Colorless oil (81%). 1H NMR (400 MHz, CDCl3) d 7.47 (s, 1H), 6.82 (s, 1H), 4.27 (t, J ¼ 6.6 Hz, 2H), 3.85 (s, 3H), 3.31 (dd, J ¼ 11.2, 4.9 Hz, 1H), 2.98e2.86 (m, 1H), 2.85e2.71 (m, 1H), 2.34e2.24 (m, 1H), 1.62e1.56 (m, 1H), 1.51e1.41 (m, 3H), 1.33e1.24 (m, 3H), 1.20 (s, 3H), 1.08 (s, 3H), 0.96 (t, J ¼ 7.4 Hz, 3H), 0.90 (s, 3H). 13C NMR (100 MHz, CDCl3) d 166.44, 157.48, 155.08, 132.15, 127.09, 118.10, 108.48, 78.58, 64.64, 56.27, 49.56, 39.16, 38.34, 36.93, 30.90, 29.71, 28.25, 27.94, 24.74, 19.37, 18.88, 15.55, 13.89. HRMS (ESI): calcd for C23H34NaO4 [MþNa]þ, 397.2349, found 397.2320. 5.3. Synthesis of ((8aR)-7-(tert-butyldimethylsilyloxy)-3-methoxy4b,8,8-trimethyl-4b,5,6,7,8,8a,9, 10-octahydrophenanthren-2-yl) methanol (10) To a solution of 5 (200 mg, 0.5 mmol) in THF (10 mL) was added LiAlH4 (70 mg, 1.9 mmol) under N2 at 0
C. The reaction mixture was stirred at room temperature for 3 h and then added H2O (0.07 mL), NaOH solution (15%, 0.07 mL), H2O (0.21 mL) in sequence and filtered. The filtrate was concentrated and the residue was purified by silica gel chromatography (petroleum ether/AcOEt, 3/1 v/v) to give 10 (165 mg, 85%) as a white solid. 1H NMR (400 MHz, CDCl3) d 6.92 (s, 1H), 6.74 (s, 1H), 4.61 (s, 2H), 3.83 (s, 3H), 3.26 (dd, J ¼ 11.3, 4.6 Hz, 1H), 2.92e2.82 (m, 1H), 2.81e2.71 (m, 1H), 2.28e2.18 (m, 2H), 1.91e1.64 (m, 4H), 1.56e1.47 (m, 1H), 1.29e1.24 (m, 1H), 1.20 (s, 3H), 0.98 (s, 3H), 0.91 (s, 9H), 0.86 (s, 3H), 0.06 (s,

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3H), 0.05 (s, 3H). 5.4. Synthesis of (10aR)-7-(hydroxymethyl)-6-methoxy-1,1,4atrimethyl-1,2,3,4,4a,9,10,10a-octahydrophenanthren-2-ol (11) By a similar procedure described for 6, 11 was obtained as a white solid; mp 132e134
C. 1H NMR (400 MHz, CDCl3) d 6.94 (s, 1H), 6.74 (s, 1H), 4.61 (s, 2H), 3.83 (s, 3H), 3.29 (dd, J ¼ 11.1, 5.1 Hz, 1H), 2.94e2.85 (m, 1H), 2.83e2.69 (m, 1H), 2.31e2.25 (m, 1H), 1.92e1.67 (m, 4H), 1.57e1.51 (m, 1H), 1.32e1.27 (m, 1H), 1.21 (s, 3H), 1.07 (s, 3H), 0.90 (s, 3H). 13C NMR (125 MHz, CDCl3) d 155.82, 150.04, 129.54, 127.18, 126.70, 106.44, 78.77, 62.12, 55.43, 49.97, 39.15, 38.03, 37.20, 29.98, 28.31, 28.08, 24.93, 19.00, 15.54. HRMS (ESI): calcd for C19H28NaO3 [MþNa]þ, 327.1931, found 327.1936. 5.5. Synthesis of (10aR)-6-methoxy-1,1,4a-trimethyl1,2,3,4,4a,9,10,10a-octahydrophenanthren-2-yl acetate (12) To a solution of 2 (600 mg, 2.2 mmol) in CH2Cl2 (10 mL) was added DMAP (53 mg, 0.4 mmol) and Ac2O (0.6 mL, 6.6 mmol) under N2. The reaction mixture was stirred for 12 h at room temperature, and then poured into saturated aqueous NaHCO3 (20 mL) and extracted with CH2Cl2 (20 mL  3). The organic layer was washed with brine, dried with anhydrous Na2SO4, and concentrated. The residue was purified by silica gel chromatography (petroleum ether/AcOEt, 8/1 v/v) to give 12 (610 mg, 88%) as a white solid. 1H NMR (400 MHz, CDCl3) d 6.97 (d, J ¼ 8.4 Hz, 1H), 6.77 (d, J ¼ 2.6 Hz, 1H), 6.67 (dd, J ¼ 8.4, 2.6 Hz, 1H), 4.56 (dd, J ¼ 11.5, 4.8 Hz, 1H), 3.77 (s, 3H), 2.94e2.86 (m, 1H), 2.84e2.73 (m, 1H), 2.27 (dt, J ¼ 13.1, 3.4 Hz, 1H), 2.08 (s, 3H), 1.90e1.68 (m, 4H), 1.66e1.58 (m, 1H), 1.40 (dd, J ¼ 12.1, 2.3 Hz, 1H), 1.22 (s, 3H), 0.97 (s, 3H), 0.96 (s, 3H). 5.6. General procedure for the synthesis of compounds 13e17 To a solution of 12 (100 mg, 0.3 mmol) in CH2Cl2 (10 mL) was added AlCl3 (126 mg, 1.0 mmol) and acyl chlorides (1.0 mmol) under N2 at 10
C. The reaction mixture was stirred for 4 h at this temperature and then poured into water (30 mL) and extracted with CH2Cl2 (15 mL  3). The organic layer was washed with brine, dried with anhydrous Na2SO4, and concentrated. The residue was purified by silica gel chromatography (petroleum ether/EtOAc, 7/1 v/v) to afford the desired product. 5.6.1. (10aR)-7-Acetyl-6-methoxy-1,1,4a-trimethyl1,2,3,4,4a,9,10,10a-octahydrophenanthren-2-yl acetate (13) White solid (89%); mp 157e159
C. 1H NMR (400 MHz, CDCl3) d 7.44 (s, 1H), 6.79 (s, 1H), 4.55 (dd, J ¼ 11.5, 4.6 Hz, 1H), 3.86 (s, 3H), 2.98e2.87 (m, 1H), 2.84e2.71 (m, 1H), 2.57 (s, 3H), 2.29 (dt, J ¼ 13.0, 3.4 Hz, 1H), 2.07 (s, 3H), 1.38 (dd, J ¼ 12.2, 2.3 Hz, 1H), 1.23 (s, 3H), 0.97 (s, 3H), 0.96 (s, 3H). 13C NMR (100 MHz, CDCl3) d 199.59, 171.03, 157.43, 155.24, 131.05, 127.33, 126.01, 107.61, 80.44, 55.63, 49.68, 38.28, 38.11, 36.62, 31.91, 29.48, 28.24, 24.75, 24.36, 21.42, 18.79, 16.70. HRMS (ESI): calcd for C22H30NaO4 [MþNa]þ, 381.2036, found 381.2055. 5.6.2. (10aR)-6-methoxy-1,1,4a-trimethyl-7-propionyl1,2,3,4,4a,9,10,10a-octahydrophenanthren-2-yl acetate (14) White solid (82%); 1H NMR (400 MHz, CDCl3) d 7.39 (s, 1H), 6.78 (s, 1H), 4.55 (dd, J ¼ 11.2, 4.7 Hz, 1H), 3.85 (s, 3H), 3.04e2.86 (m, 3H), 2.85e2.69 (m, 1H), 2.29 (d, J ¼ 12.8 Hz, 1H), 2.08 (s, 3H), 1.38 (d, J ¼ 12.0 Hz, 1H), 1.23 (s, 3H), 1.14 (t, J ¼ 7.3 Hz, 3H), 0.97 (d, J ¼ 4.6 Hz, 6H).

5.6.3. (10aR)-7-(3-Chloropropanoyl)-6-methoxy-1,1,4a-trimethyl1,2,3,4,4a,9,10,10a-octahydrophenanthren-2-yl acetate (15) White solid (75%); 1H NMR (400 MHz, CDCl3) d 7.48 (s, 1H), 6.79 (s, 1H), 4.55 (dd, J ¼ 11.5, 4.6 Hz, 1H), 3.88 (s, 3H), 3.87e3.82 (m, 2H), 3.48e3.41 (m, 2H), 2.98e2.89 (m, 1H), 2.84e2.73 (m, 1H), 2.29 (dt, J ¼ 12.9, 3.3 Hz, 1H), 2.08 (s, 3H), 1.38 (dd, J ¼ 12.2, 2.3 Hz, 1H), 1.23 (s, 3H), 0.98 (s, 3H), 0.96 (s, 3H). 5.6.4. 1 (10aR)-6-methoxy-1,1,4a-trimethyl-7-octanoyl1,2,3,4,4a,9,10,10a-octahydrophenanthren-2-yl acetate (16) White solid (97%); 1H NMR (400 MHz, CDCl3) d 7.36 (s, 1H), 6.77 (s, 1H), 4.55 (dd, J ¼ 11.5, 4.6 Hz, 1H), 3.84 (s, 3H), 3.01e2.69 (m, 4H), 2.38e2.20 (m, 1H), 2.08 (s, 3H), 1.95e1.70 (m, 4H), 1.42e1.24 (m, 9H), 1.22 (s, 3H), 0.96 (d, J ¼ 6.2 Hz, 6H), 0.87 (t, J ¼ 6.7 Hz, 3H). 5.6.5. (10aR)-7-isobutyryl-6-methoxy-1,1,4a-trimethyl1,2,3,4,4a,9,10,10a-octahydrophenanthren-2-yl acetate (17) White solid (79%); 1H NMR (400 MHz, CDCl3) d 7.24 (s, 1H), 6.76 (s, 1H), 4.55 (dd, J ¼ 11.5, 4.6 Hz, 1H), 3.83 (s, 3H), 3.53e2.43 (m, 1H), 2.97e2.87 (m, 1H), 2.84e2.73 (m, 1H), 2.33e2.24 (m, 1H), 2.08 (s, 3H), 1.39 (dd, J ¼ 12.2, 2.1 Hz, 1H), 1.22 (s, 3H), 1.15e1.07 (m, 6H), 0.97 (s, 3H), 0.96 (s, 3H). 5.7. General procedure for the synthesis of compounds 18e22 One of compounds 13e17 (0.3 mmol) was dissolved in MeOH (10 mL) and NaOH (1.0 mL, 1 M) was added. The reaction mixture was heated under reflux for 3 h. After cooling, the reaction mixture was poured into HCl (20 mL, 1 M) and extracted with AcOEt (30 mL  3). The organic layer was washed with brine, dried with anhydrous Na2SO4, and concentrated. The residue was purified by silica gel chromatography (petroleum ether/AcOEt, 1.5/1 v/v) to afford the desired product. 5.7.1. 1-((8aR)-7-hydroxy-3-methoxy-4b,8,8-trimethyl4b,5,6,7,8,8a,9,10-octahydrophenanthren-2-yl)ethanone (18) White solid (95%); mp 120e122
C. 1H NMR (400 MHz, CDCl3) d 7.44 (s, 1H), 6.80 (s, 1H), 3.86 (s, 3H), 3.30 (dd, J ¼ 11.1, 5.0 Hz, 1H), 2.97e2.88 (m, 1H), 2.83e2.70 (m, 1H), 2.57 (s, 3H), 2.31e2.23 (m, 1H), 1.93e1.53 (m, 6H), 1.29 (dd, J ¼ 12.3, 2.2 Hz, 1H), 1.21 (s, 3H), 1.07 (s, 3H), 0.90 (s, 3H). 13C NMR (100 MHz, CDCl3) d 199.63, 157.39, 155.62, 131.03, 127.47, 125.90, 107.66, 78.59, 55.61, 49.58, 39.19, 38.40, 36.99, 31.90, 29.66, 28.26, 27.95, 24.71, 18.89, 15.55. HRMS (ESI): calcd for C20H28NaO3 [MþNa]þ, 339.1931, found 339.1934. 5.7.2. 1-((8aR)-7-hydroxy-3-methoxy-4b,8,8-trimethyl4b,5,6,7,8,8a,9,10-octahydrophenanthren-2-yl)propan-1-one (19) White solid (97%); mp 139e141
C. 1H NMR (400 MHz, CDCl3) d 7.39 (s, 1H), 6.79 (s, 1H), 3.85 (s, 3H), 3.30 (dd, J ¼ 11.1, 4.9 Hz, 1H), 3.00e2.78 (m, 3H), 2.83e2.69 (m, 1H), 2.29 (d, J ¼ 12.9 Hz, 1H), 1.92e1.56 (m, 6H), 1.29 (d, J ¼ 12.3 Hz, 1H), 1.20 (s, 3H), 1.14 (t, J ¼ 7.3 Hz, 3H), 1.07 (s, 3H), 0.90 (s, 3H). 13C NMR (100 MHz, CDCl3) d 203.22, 156.95, 155.01, 130.90, 127.49, 126.13, 107.64, 78.62, 55.63, 49.62, 39.19, 38.35, 37.00, 37.00, 29.70, 28.26, 27.96, 24.73, 18.90, 15.55, 8.62. HRMS (ESI): calcd for C21H30NaO3 [MþNa]þ, 353.2087, found 353.2079. 5.7.3. 1-((8aR)-7-hydroxy-3-methoxy-4b,8,8-trimethyl4b,5,6,7,8,8a,9,10-octahydrophenanthren-2-yl)-3-methoxypropan1-one (20) White solid (80%); mp 101e103
C. 1H NMR (400 MHz, CDCl3) d 7.43 (s, 1H), 6.79 (s, 1H), 3.85 (s, 3H), 3.75 (t, J ¼ 6.7 Hz, 2H), 3.35 (s, 3H), 3.32e3.27 (m, 1H), 3.25 (t, J ¼ 6.7 Hz, 2H), 2.97e2.86 (m, 1H), 2.83e2.70 (m, 1H), 2.28 (dt, J ¼ 12.9, 3.3 Hz, 1H), 1.92e1.65 (m, 4H), 1.30e1.25 (m, 1H), 1.20 (s, 3H), 1.06 (s, 3H), 0.89 (s, 3H). 13C NMR

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(100 MHz, CDCl3) d 199.76, 157.14, 155.58, 131.07, 127.53, 125.57, 107.51, 78.53, 68.25, 58.94, 55.54, 49.52, 43.97, 39.16, 38.36, 36.94, 29.65, 28.24, 27.91, 24.72, 18.85, 15.55. HRMS (ESI): calcd for C22H32NaO4 [MþNa]þ, 383.2193, found 383.2217. 5.7.4. 1-((8aR)-7-hydroxy-3-methoxy-4b,8,8-trimethyl4b,5,6,7,8,8a,9,10-octahydrophenanthren-2-yl)-2-methylpropan-1one (21) White solid (84%); mp 115e117
C. 1H NMR (400 MHz, CDCl3) d 7.23 (s, 1H), 6.77 (s, 1H), 3.82 (s, 3H), 3.55e3.37 (m, 1H), 3.28 (dd, J ¼ 10.9, 5.1 Hz, 1H), 2.99e2.86 (m, 1H), 2.83e2.67 (m, 1H), 2.32e2.21 (m, 1H), 1.92e1.64 (m, 5H),1.61e1.49 (m, 1H), 1.28 (dd, J ¼ 12.2, 1.6 Hz, 1H), 1.19 (s, 3H), 1.14e1.08 (m, 6H), 1.06 (s, 3H), 0.89 (s, 3H). 13C NMR (100 MHz, CDCl3) d 207.83, 156.23, 154.44, 130.76, 127.48, 126.46, 107.50, 78.54, 55.66, 49.61, 39.94, 39.14, 38.27, 36.99, 29.69, 28.23, 27.93, 24.71, 18.87, 18.84, 18.69, 15.53. HRMS (ESI): calcd for C22H32NaO3 [MþNa]þ, 367.2244, found 367.2252. 5.7.5. 1-((8aR)-7-hydroxy-3-methoxy-4b,8,8-trimethyl4b,5,6,7,8,8a,9,10-octahydrophenanthren-2-yl)octan-1-one (22) White solid (94%); mp 47e49
C. 1H NMR (400 MHz, CDCl3) d 7.36 (s, 1H), 6.79 (s, 1H), 3.85 (s, 3H), 3.31 (dd, J ¼ 11.0, 4.7 Hz, 1H), 2.99e2.84 (m, 3H), 2.83e2.71 (m, 1H), 2.33e2.24 (m, 1H), 1.21 (s, 3H), 1.07 (s, 3H), 0.93e0.82 (m, 6H). 13C NMR (100 MHz, CDCl3) d 203.02, 156.75, 154.95, 130.76, 127.40, 126.29, 107.54, 78.42, 55.53, 49.56, 43.74, 39.09, 38.25, 36.94, 31.77, 29.63, 29.44, 29.20, 28.18, 27.86, 24.64, 24.53, 22.66, 18.82, 15.49, 14.13. HRMS(ESI): calcd for C26H40NaO3 [MþNa]þ, 423.2870, found 423.2869. 5.8. Synthesis of 1-((8aR)-3,7-dihydroxy-4b,8,8-trimethyl4b,5,6,7,8,8a,9,10-octahydrophenanthren-2-yl)ethanone (23) To a solution of 18 (100 mg, 0.3 mmol) in CH2Cl2 (10 mL), AlCl3 (126 mg, 1.0 mmol) was added. The reaction mixture was heated under reflux for 6 h under N2. After cooling, the reaction mixture was poured into H2O (30 mL) and extracted with CH2Cl2 (15 mL  3). The organic layer was washed with brine, dried with anhydrous Na2SO4, and concentrated. The residue was purified by silica gel chromatography (petroleum ether/AcOEt, 1.5/1 v/v) to give 23 (78 mg, 81%) as a light yellow solid; mp 171e173
C. 1H NMR (400 MHz, CDCl3) d 11.89 (s, 1H), 7.40 (s, 1H), 6.85 (s, 1H), 3.30 (dd, J ¼ 11.3, 4.8 Hz, 1H), 3.00e2.89 (m, 1H), 2.88e2.73 (m, 1H), 2.58 (s, 3H), 2.30e2.21 (m, 1H), 1.97e1.66 (m, 4H), 1.31e1.23 (m, 2H), 1.19 (s, 3H), 1.08 (s, 3H), 0.90 (s, 3H). 13C NMR (100 MHz, DMSO-d6) d 204.05, 158.84, 158.73, 131.33, 125.78, 118.09, 112.75, 76.38, 48.86, 38.73, 37.90, 36.18, 29.06, 28.18, 27.64, 27.18, 24.12, 18.40, 15.82. HRMS (ESI): calcd for C19H26NaO3 [MþNa]þ, 325.1774, found 325.1779. 5.9. Synthesis of (10aR)-7-acetyl-6-methoxy-1,1,4a-trimethyl4,4a,10,10a-tetrahydrophenanthren-2(1H,3H,9H)-one (24) To a solution of 18 (100 mg, 0.3 mmol) in THF (10 mL) and DMSO (10 mL), IBX (114 mg, 0.6 mmol) was added. The reaction mixture was stirred for 12 h at room temperature and then poured into H2O (30 mL) and extracted with CH2Cl2 (20 mL  3). The organic layer was washed with brine, dried with anhydrous Na2SO4, and concentrated. The residue was purified by silica gel chromatography (petroleum ether/AcOEt, 5/1 v/v) to give 24 (86 mg, 85%) as a white solid; mp 112e114
C. 1H NMR (400 MHz, CDCl3) d 7.46 (s, 1H), 6.80 (s, 1H), 3.88 (s, 3H), 2.99e2.90 (m, 1H), 2.85e2.66 (m, 2H), 2.66e2.60 (m, 1H), 2.58 (s, 3H), 2.52e2.42 (m, 1H), 2.05e1.95 (m, 1H), 1.93e1.87 (m, 1H), 1.87e1.68 (m, 2H), 1.31 (s, 3H), 1.17 (s, 3H), 1.14 (s, 3H). 13C NMR (100 MHz, CDCl3) d 216.77, 199.53, 157.45, 153.42, 131.12, 127.34, 126.38, 108.46, 55.66, 50.39, 47.48, 38.11,

405

37.49, 34.54, 31.89, 29.84, 26.99, 24.56, 21.22, 20.25. HRMS (ESI): calcd for C20H26NaO3 [MþNa]þ, 337.1774, found 337.1764. 5.10. Synthesis of (10aR)-7-acetyl-6-methoxy-1,1,4a-trimethyl1,2,3,4,4a,9,10,10a-octahydrophenanthren-2-yl 2,2,2trifluoroacetate (25) To a solution of 18 (100 mg, 0.3 mmol) in CH2Cl2 (10 mL), DMAP (193 mg, 1.6 mmol) and trifluoroacetic anhydride (0.1 mL, 0.9 mmol) was added under N2. The reaction mixture was stirred for 12 h at room temperature and then poured into H2O (30 mL) and extracted with CH2Cl2 (20 mL  3). The organic layer was washed with brine, dried with anhydrous Na2SO4, and concentrated. The residue was purified by silica gel chromatography (petroleum ether/AcOEt, 8/1 v/v) to give 25 (115 mg, 86%) as a white solid; mp 114e116
C. 1H NMR (400 MHz, CDCl3) d 7.45 (s, 1H), 6.78 (s, 1H), 4.80e4.66 (m, 1H), 3.87 (s, 3H), 3.00e2.87 (m, 1H), 2.85e2.76 (m, 1H), 2.58 (s, 3H), 2.43e2.32 (m, 1H), 2.03e1.87 (m, 3H), 1.82e1.65 (m, 2H), 1.42 (d, J ¼ 12.1 Hz, 1H), 1.26 (s, 3H), 1.04 (s, 3H), 1.01 (s, 3H). 13 C NMR (100 MHz, CDCl3) d 199.59, 157.45 (q, J ¼ 41.9 Hz),157.44, 154.56, 131.14, 127.11, 126.11, 114.77 (q, J ¼ 286.1 Hz), 107.45, 85.62, 55.62, 49.55, 38.34, 38.17, 36.38, 31.96, 29.35, 28.06, 24.69, 23.92, 18.68, 16.43. HRMS (ESI): calcd for C22H27F3NaO4 [MþNa]þ, 435.1754, found 435.1773. 5.11. General procedure for the synthesis of compounds 26e32 To a solution of 6 (100 mg, 0.3 mmol) in CH2Cl2 (10 mL), EDC$HCl (126 mg, 0.6 mmol), HOBt (86 mg, 0.6 mmol), DMAP (227 mg, 1.9 mmol) and amines or their hydrochlorides (0.9 mmol) were added under N2. The reaction mixture was stirred for 12 h at room temperature and then poured into water and extracted with CH2Cl2 (10 mL  3). The organic layer was washed with brine, dried with anhydrous Na2SO4, and concentrated. The residue was purified by silica gel chromatography (petroleum ether/AcOEt, 1/1 v/v) to afford the desired product. 5.11.1. (8aR)-7-hydroxy-3-methoxy-N,4b,8,8-tetramethyl4b,5,6,7,8,8a,9,10-octahydrophenanthrene-2-carboxamide (26) White solid (98%); mp 200e201
C. 1H NMR (400 MHz, CDCl3) d 7.85 (s, 1H), 7.82 (s, 1H), 6.78 (s, 1H), 3.89 (s, 3H), 3.28 (dd, J ¼ 10.8, 5.0 Hz, 1H), 3.04e2.87 (m, 4H), 2.86e2.71 (m, 1H), 2.30e2.20 (m, 2H), 1.60e1.49 (m, 1H), 1.27 (d, J ¼ 11.8 Hz, 1H), 1.18 (s, 3H), 1.05 (s, 3H), 0.88 (s, 3H). 13C NMR (100 MHz, CDCl3) d 166.26, 155.62, 154.25, 132.69, 128.14, 118.95, 107.30, 78.51, 55.96, 49.57, 39.13, 38.21, 37.01, 29.68, 28.21, 27.88, 26.59, 24.76, 18.84, 15.52. HRMS (ESI): calcd for C20H29NNaO3 [MþNa]þ, 354.2040, found 354.2036. 5.11.2. (8aR)-N-ethyl-7-Hydroxy-3-methoxy-4b,8,8-trimethyl4b,5,6,7,8,8a,9,10-octahydrophenanthrene-2-carboxamide (27) White solid (93%); mp 78e80
C. 1H NMR (400 MHz, CDCl3) d 7.85 (s, 1H), 7.81 (s, 1H), 6.79 (s, 1H), 3.90 (s, 3H), 3.52e3.38 (m, 2H), 3.29 (dd, J ¼ 11.0, 5.1 Hz, 1H), 3.00e2.87 (m, 1H), 2.86e2.72 (m, 1H), 2.29e2.19 (m, 1H), 1.55 (td, J ¼ 12.7, 4.7 Hz, 1H), 1.28 (dd, J ¼ 12.3, 2.1 Hz, 1H), 1.24e1.16 (m, 6H), 1.06 (s, 3H), 0.88 (s, 3H). 13C NMR (100 MHz, CDCl3) d 165.35, 155.65, 154.18, 132.72, 128.19, 119.22, 107.43, 78.54, 56.01, 49.60, 39.15, 38.22, 37.03, 34.57, 29.71, 28.23, 27.92, 24.79, 18.87, 15.54, 15.03. HRMS (ESI): calcd for C21H31NNaO3 [MþNa]þ, 368.2196, found 368.2213. 5.11.3. (8aR)-7-hydroxy-3-methoxy-N,N,4b,8,8-pentamethyl4b,5,6,7,8,8a,9,10-octahydrophenanthrene-2-carboxamide (28) White solid (77%); mp 209e211
C. 1H NMR (400 MHz, CDCl3) d 6.89 (s, 1H), 6.72 (s, 1H), 3.77 (s, 3H), 3.08 (s, 3H), 2.85 (s, 3H), 2.79e2.69 (m, 1H), 2.23 (d, J ¼ 10.9 Hz, 1H), 1.17 (s, 3H), 1.01 (s, 3H),

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0.86 (s, 3H). 13C NMR (100 MHz, CDCl3) d 169.87, 153.58, 151.63, 128.38, 127.68, 124.07, 107.14, 77.48, 55.76, 49.90, 39.07, 38.48, 38.13, 37.16, 34.86, 29.94, 28.26, 28.02, 24.90, 18.90, 15.55. HRMS (ESI): calcd for C21H31NNaO3 [MþNa]þ, 368.2196, found 368.2203. 5.11.4. Methyl 2-((10aR)-2-hydroxy-6-methoxy-1,1,4a-trimethyl1,2,3,4,4a,9,10,10a-octahydrophenanthrene-7-carboxamido)acetate (29) White solid (78%); 1H NMR (400 MHz, CDCl3) d 8.45 (t, J ¼ 4.9 Hz, 1H), 7.85 (s, 1H), 6.80 (s, 1H), 4.23 (d, J ¼ 5.1 Hz, 2H), 3.93 (s, 3H), 3.76 (s, 3H), 3.27 (dd, J ¼ 10.9, 5.1 Hz, 1H), 2.98e2.88 (m, 1H), 2.83e2.71 (m, 1H), 2.30e2.20 (m, 1H), 1.58e1.49 (m, 1H), 1.18 (s, 3H), 1.05 (s, 3H), 0.87 (s, 3H). 5.11.5. (8aR)-N-butyl-7-hydroxy-3-methoxy-4b,8,8-trimethyl4b,5,6,7,8,8a,9,10-octahydrophenanthrene-2-carboxamide (30) White solid (82%); mp 124e126
C. 1H NMR (400 MHz, CDCl3) d 7.87 (s, 1H), 7.84 (s, 1H), 6.79 (s, 1H), 3.90 (s, 3H), 3.47e3.39 (m, 2H), 3.30 (dd, J ¼ 11.0, 4.9 Hz, 1H), 3.05e2.89 (m, 1H), 2.85e2.72 (m, 1H), 2.27 (d, J ¼ 12.9 Hz, 1H), 1.61e1.51 (m, 3H), 1.39 (dt, J ¼ 14.5, 7.4 Hz, 2H), 1.31e1.26 (m, 1H), 1.19 (s, 3H), 1.06 (s, 3H), 0.94 (t, J ¼ 7.3 Hz, 3H), 0.89 (s, 3H). 13C NMR (100 MHz, CDCl3) d 165.36, 155.63, 154.10, 132.77, 128.20, 119.30, 107.41, 78.57, 56.05, 49.59, 39.45, 39.16, 38.22, 37.03, 31.82, 29.72, 28.24, 27.94, 24.82, 20.35, 18.88, 15.55, 13.93. HRMS (ESI): calcd for C23H36NO3 [MþH]þ, 374.2690, found 374.2697. 5.11.6. (8aR)-7-hydroxy-3-methoxy-4b,8,8-trimethyl-N-(prop-2ynyl)-4b,5,6,7,8,8a,9,10-octahydrophenanthrene-2-carboxamide (31) White solid (93%); mp 159e161
C. 1H NMR (400 MHz, CDCl3) d 8.03 (s, 1H), 7.88 (s, 1H), 6.81 (s, 1H), 4.24 (dd, J ¼ 5.1, 2.5 Hz, 2H), 3.93 (s, 3H), 3.32 (dd, J ¼ 11.1, 5.0 Hz, 1H), 3.02e2.91 (m, 1H), 2.86e2.74 (m, 1H), 2.33e2.24 (m, 1H), 2.23 (t, J ¼ 2.5 Hz, 1H), 1.64e1.57 (m, 1H), 1.21 (s, 3H), 1.08 (s, 3H), 0.91 (s, 3H). 13C NMR (100 MHz, CDCl3) d 165.19, 155.77, 154.77, 132.90, 128.28, 118.29, 107.40, 80.31, 78.56, 71.17, 56.05, 49.52, 39.16, 38.28, 37.00, 29.68, 29.42, 28.23, 27.91, 24.80, 18.84, 15.55. HRMS (ESI): calcd for C22H29NNaO3 [MþNa]þ, 378.2040, found 378.1993. 5.11.7. ((8aR)-7-hydroxy-3-methoxy-4b,8,8-trimethyl4b,5,6,7,8,8a,9,10-octahydrophenanthren-2-yl) (piperidin-1-yl) methanone (32) White solid (85%); mp 184e186
C. 1H NMR (500 MHz, DMSOd6) d 6.84 (s, 1H), 6.75 (s, 1H), 4.49 (dd, J ¼ 15.6, 4.9 Hz, 1H), 3.72 (s, 3H), 3.62e3.51 (m, 1H), 3.50e3.41 (m, 1H), 3.14e3.00 (m, 3H), 2.84e2.75 (m, 1H), 2.72e2.61 (m, 1H), 2.31 (d, J ¼ 12.9 Hz, 1H), 1.82e1.73 (m, 1H), 1.70e1.59 (m, 3H), 1.58e1.53 (m, 2H), 1.52e1.45 (m, 2H), 1.13 (d, J ¼ 7.1 Hz, 3H), 0.97 (s, 3H), 0.78 (s, 3H). 13C NMR (100 MHz, DMSO-d6) d 166.41, 153.02, 151.06, 127.55, 126.79, 123.80, 107.10, 76.55, 55.40, 49.38, 47.17, 41.64, 38.69, 37.65, 36.41, 29.33, 28.23, 27.76, 25.93, 25.34, 24.48, 24.10, 18.43, 15.82. HRMS (ESI): calcd for C24H35NNaO3 [MþNa]þ, 408.2509, found 408.2524. 5.12. Synthesis of 2-((10aR)-2-hydroxy-6-methoxy-1,1,4atrimethyl-1,2,3,4,4a,9,10,10a-octahydrophenanthrene-7carboxamido)acetic acid (33) To a solution of 29 (135 mg, 0.4 mmol) in THF (5 mL) and H2O (5 mL) LiOH$H2O (44 mg, 1.0 mmol) was added. The reaction mixture was stirred for 12 h at room temperature and then poured into HCl (20 mL, 1 M) and extracted with CH2Cl2 (15 mL  3). The organic layer was washed with brine, dried with anhydrous Na2SO4, and concentrated. The residue was purified by silica gel chromatography (CH2Cl2/MeOH, 30/1 v/v) to give 33 (110 mg, 84%) as a

white solid; mp 156e158
C. 1H NMR (400 MHz, DMSO-d6) d 8.45 (t, J ¼ 5.6 Hz, 1H), 7.54 (s, 1H), 6.94 (s, 1H), 4.45 (s, 1H), 3.96 (d, J ¼ 5.7 Hz, 2H), 3.88 (s, 3H), 3.13e3.06 (m, 1H), 2.90e2.82 (m, 1H), 2.76e2.66 (m, 1H), 2.38e2.28 (m, 1H), 1.85e1.76 (m, 1H), 1.71e1.56 (m, 3H), 1.46e1.34 (m, 1H), 1.19 (dd, J ¼ 12.7, 2.2 Hz, 1H), 1.14 (s, 3H), 0.98 (s, 3H), 0.79 (s, 3H). 13C NMR (100 MHz, DMSO-d6) d 171.33, 164.61, 155.52, 154.30, 131.30, 126.94, 118.72, 108.05, 76.51, 55.96, 49.28, 41.45, 38.72, 37.82, 36.36, 29.28, 28.23, 27.71, 24.33, 18.42, 15.84. HRMS (ESI): calcd for C21H29NNaO5 [MþNa]þ, 398.1938, found 398.1928. 5.13. Biological assay Cell culture medium and supplements were obtained from Invitrogen (Carlsbad, CA, USA). Glutamate was purchased from SigmaeAldrich (St. Louis, MO, USA). Primary antibodies used for Western blot analysis were: rabbit anti-HO-1 (1:500), mouse antib-actin (1:1000), all from Santa Cruz Biotechnology Company (Danvers, MA, USA). Secondary antibodies for Western blot analysis were: goat anti-rabbit IgG (1:5000, AmershamBioSciences, Piscataway, NJ, USA); goat anti-mouse IgG (1:5,000, Jackson ImmunoResearch, West Grove, PA, USA). SuperSignal West Dura Substrate for chemiluminescent detection was purchased from Thermo Fisher Scientific (Pittsburg, PA, USA). The cell lysis buffer, protease inhibitor cocktail, BCA-based protein quantification kit, the SOD and GSH assay kit were obtained from Beyotime Institute of Biotechnology (Shanghai, China). All other chemicals were obtained from SigmaeAldrich unless otherwise stated. 5.13.1. Primary rat cerebellar granule neuron cultures Cerebellar granule neuronal cells (CGCs) were isolated from 8day old Sprague Dawley rat pups as described previously [36]. Cerebella were collected and placed in ice-cold Hank's balanced salt solution (HBSS) (Invitrogen). After removal of the meninges, the cerebellas were dispersed into the same buffer containing 0.25% trypsin (Invitrogen) and digested for 15 min at 37
C. Trypsin digestion was stopped by adding a two-fold volume of DMEM (Invitrogen), supplemented with 10% FBS (Invitrogen) and 0.1 mg/ ml DNase I (SigmaeAldrich). After gentle trituration, digested tissues were centrifuged at 1000 rpm for 5 min. The cell pellets were resuspended in the complete Neurobasal culture medium (Invitrogen) supplemented with 2% B27 (Invitrogen) and 0.5 mMGlutaMax (Invitrogen). After filtration through 70 mm cell strainer (BD Falcon, Vernon Hills, IL, USA), cells were plated at a density of 1  106 cells/ml onto poly-L-lysine coated 96-well or 6-well plates (Becton Dickinson and Company, Franklin Lakes, NJ, USA). Cultures were incubated in a humidified atmosphere of 5% CO2-95% air at 37
C. Cytosine arabinofuranoside (10 mM) was added to the cultures 24 h after plating to arrest the growth of glia cells. Cultures 6e8 days in vitro were used in this study. Immunocytochemical validation with anti-MAP2 antibody and DAPI revealed that more than 95% of the cells in our cultures system were neurons at the time of experiment. 5.13.2. Oxygeneglucose deprivation (OGD) When CGCs were cultured at DIV 6, the complete medium was removed and the cells were washed with a glucose-free Earle's balanced salt solution (EBSS, pH 7.4). Then the cultures were placed in fresh glucose-free EBSS and transferred to a hypoxia chamber containing a mixture of 95% N2 and 5% CO2 at 37
C for 6 h. After OGD, the glucose was added to attain a final concentration of 4.5 mg/ml, followed by incubation for additional 24 h in normal conditions with 5% CO2 and 95% air at 37
C. Compounds at indicated concentrations were added into the cultures 24 h before OGD treatment. Control cells without OGD and reoxygenation were

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maintained in the incubator under normal conditions and used for viability experiment. 5.13.3. Cell viability assay by MTT method The CGCs were pretreated with compounds at indicated concentrations for 24 h and then incubated with 100 mM glutamate or without nutrient B27 for another 24 h, or under OGD condition. All compounds were dissolved in DMSO. DMSO was present in all samples at a final 0.1% concentration in the culture medium, which had no neuroprotective effect in above biological assays. Cell viability was evaluated by incubating with 0.5 mg/ml 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) for 4 h under 5% CO2/95% air at 37
C. Active mitochondrial dehydrogenases of living cells cause cleavage and reduction of soluble yellow MTT dye to the insoluble purple formazan, which was extracted in dimethyl sulfoxide (DMSO). Media were replaced with 100 mL DMSO, and the optical density was measured at 570 nm in a BD plate-reader. 5.13.4. Western blotting To determine HO-1 blots, primary neurons or brain cortex samples were lysed in RIPA lysis buffer (Vazyme, Jiangsu, China). Cell homogenates were centrifuged at 13,200 rpm for 20 min and tissue samples homogenates were centrifuged at 10,000 rpm for 5 min. Total protein concentration of the supernatants was assessed by BCA kit (Thermo, Rockford, IL). The equal amounts of the extracted proteins were separated by electrophoresis on 15% SDSPAGE gels, and then transferred onto PVDF membranes. The membranes were blocked with 5% BSA for 1 h and then incubated overnight at 4
C with the primary antibody followed by washing and exposure to the secondary antibody for 30 min at room temperature. b-actin was used as a loading control for all samples. The membranes were exposed to SuperSignal West Dura Substrate kit for chemiluminescent detection. 5.13.5. Measurements of SOD and GSH The CGCs were pretreated with compounds at indicated concentrations for 24 h and then incubated with 100 mM glutamate. After 24 h of glutamate incubation, the cells were lysed by a lysis buffer consisting of a protease inhibitor cocktail. After 24 h of ischemia, the rat brain cortices were lysed by a RIPA lysis buffer. The extracted protein from cell lysates or brain cortex homogenates was quantified by BCA method, and then subjected to the measurement of SOD and GSH, respectively. The SOD activity and the GSH concentration were determined using the SOD assay kit and the GSH assay kit according to the manufacturer's instructions, respectively. 5.13.6. In vitro bloodebrain barrier permeability assay The ability to cross the BBB was predicted and evaluated using PAMPA [37]. Tested compounds were dissolved in DMSO at 5 mg/ml as stock solutions. A 10 mL sample of stock solution was diluted in PBS to make a secondary stock solution (final concentration 25 mg/ ml). These solutions were filtered. A 300 mL sample of secondary stock solution was added to the donor well. The porcine polar brain lipid was dissolved in dodecane at 20 mg/ml. The filter membrane was coated with 4 mL of porcine polar brain lipid solution, and the acceptor well was filled with 150 mL of PBS. The acceptor filter plate was carefully put on the donor plate to form a “sandwich” which was composed of the donor with tested compounds on the bottom, artificial lipid membrane in the middle, and the acceptor on the top. The sandwich was incubated undisturbed at room temperature for 18 h. The donor plate was removed after incubation. The concentrations of tested compounds in the acceptor and reference solutions were determined by a UV plate reader. Reference solutions were prepared by diluting the sample secondary stock solution to

407

the same concentration as that with no membrane barrier. Every sample was analyzed at three wavelengths, in three wells, and in three independent runs. 5.13.7. Animals Adult male SpragueeDawley (SD) rats weighing 260e280 g were purchased from Zhejiang Laboratory Animals Center (Hangzhou, China) and kept under standard housing conditions at a temperature between 20
C and 23
C, with a 12 h lightedark cycle and a relative humidity of 50%. Animal housing and handling were carried out in accordance with the US National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals published by the US National Academy of Sciences (http://oacu.od.nih.gov/ regs/index.htm). All animal tests and experimental procedures were approved by the Administration Committee of Experimental Animals in Jiangsu Province and the Ethics Committee of China Pharmaceutical University. 5.13.8. Focal cerebral ischemia procedure Transient middle cerebral artery occlusion (tMCAO) was performed as reported previously [35]. The external carotid artery was isolated and coagulated, a monofilament nylon suture (diameter of approximately 0.26 mm) with a round tip was inserted into the internal carotid artery through the external carotid artery stump, occluding the middle cerebral artery for 2 h. After occlusion, the animals were reanesthetized and the filament was withdrawn to restore blood flow. Body temperature was regulated at 37
C with a temperature control system. All animals had free access to food and water. 5.13.9. Drug delivery paradigm Animals were randomly divided into five groups, such as Sham, Vehicle (DMSO or saline), compound 30 and Edaravone as a positive control groups, respectively. In order to mimic clinical situations, rats were intravenously treated with compound 30 (dissolved in DMSO, 10 mg/kg) or Edaravone (dissolved in saline, 3 mg/kg) at 2 h, 4 h and 6 h after ischemia onset [38]. Rats intravenously injected with DMSO or saline were taken as a control (Vehicle group), showing similar ischemic effects. 5.13.10. Assessment of cerebral infarct volume and neurological deficit Animals were carried with neurological scoring at 24 h after MCAO followed by euthanization, then brains were harvested and sectioned into 2 mm coronal sections. Sections were inspected for the presence of macroscopic intracranial hemorrhage, then stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC) for 20 min at 37
C, fixed with 10% formalin, and evaluated for infarct size by morphometric analysis (image-pro plus) as described [35]. Total infarct volume was expressed as a percentage of the volume of the all sectional area to the lesion. The neurological scores were evaluated 24 h after the MCAO using a scoring system as previously reported [34]. 5.13.11. Statistical analysis Statistical significance was determined using GraphPad Prism 5 Software (GraphPad Software, San Diego, CA). Multiple group comparisons were performed by one-way ANOVA followed by Bonferroni posttest. Differences were considered statistically significant at p < 0.05. Values are expressed as the mean ± SD. Acknowledgment This work was supported by Shanghai Science and Technology Council (Grant 12ZR1408500), the National Natural Science

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Foundation of China (21402241), the Program for Jiangsu Province “Shuang Chuang” Team, the Natural Science Foundation of Jiangsu Province (BK20130653), the Fundamental Research Funds for the Central Universities (JKZD2013006), and the Open Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University (No. SKLNMKF201407).

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