The discovery of 071031B, a novel serotonin and noradrenaline reuptake inhibitor

Neuroscience Letters 544 (2013) 68–73

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The discovery of 071031B, a novel serotonin and noradrenaline reuptake inhibitor Rui Xue a , Yan-Ping Zhang b , Zeng-Liang Jin a , Li Yuan a , Xin-Hua He b , Nan Zhao a , Hong-Xia Chen a , Li-Ming Zhang a , Shi-Yong Fan b , Bo-Hua Zhong b,∗ , You-Zhi Zhang a,∗ , Yun-Feng Li a a b

Department of New Drug Evaluation, Beijing Institute of Pharmacology and Toxicology, Beijing, PR China Department of Drug Synthesis, Beijing Institute of Pharmacology and Toxicology, Beijing, PR China

h i g h l i g h t s • • • •

We establish a system to screen monoamine reuptake inhibitors fast and accurately. Using this system, we discover a potential antidepressant 071031B. 071031B has robust antidepressant effect in vivo and transporter affinity in vitro. 071031B is expected to be a novel serotonin and noradrenaline reuptake inhibitor.

a r t i c l e

i n f o

Article history: Received 7 January 2013 Received in revised form 19 February 2013 Accepted 24 February 2013 Keywords: Antidepressants Dual reuptake inhibitors Behavioral despair models Monoamine transporters Integrated evaluation system

a b s t r a c t Depression is a severe mood disorder with increasing morbidity and suicidality, while the current therapy is not satisfactory. Serotonin and noradrenaline reuptake inhibitors (SNRIs) have been reported to have higher efficacy and/or faster acting rate than commonly used antidepressants. The present study was designed to screen the potential SNRIs, using in vitro radioligand receptor binding assays and in vivo animal tests, and introduced the discovery of 071031B. In the tail suspension test and forced swimming test in mice, six compounds (071017S, 071026W, 071031A, 071031B, 080307A and 080307B) showed robust antidepressant activity, without stimulant effect on the locomotor activity or other side effects, and the minimal effective dose of 071017S, 071026W, 071031A and 071031B was less than that of duloxetine; in vitro binding tests indicated that 071031B had high affinity to both serotonin transporter and noradrenaline transporter with similar inhibitory rates to duloxetine at 1 and 100 nM; acute toxicity test indicated that the LD50 value of 071031B was similar to that of duloxetine. These findings demonstrated that this integrated system, combining high throughput screening technology and in vivo animal tests, is effective to screen potential monoamine reuptake inhibitors fast and accurately; 071031B is expected to be a novel serotonin and noradrenaline reuptake inhibitor for its robust antidepressant activity and transporter affinity. © 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Depression is the most common mood disorder, with increasing mortality, suicidality, and heavy burden to society [8].

Abbreviations: TCAs, tricyclic antidepressants; SSRIs, selective serotonin reuptake inhibitors; NRIs, norepinephrine reuptake inhibitors; SNRIs, serotonin and norepinephrine reuptake inhibitors; NDRIs, norepinephrine and dopamine reuptake inhibitors; MAOI’s, monoamine oxidase inhibitors; 5-HT, serotonin; R&D, Research & Development; SERTs, serotonin transporters; NETs, norepinephrine transporters; TST, tail suspension test; FST, forced swimming test; LD50 , median lethal dosage; ANOVA, one-way analysis of variance; CNS, central nervous system. ∗ Corresponding authors at: Department of New Drug Evaluation, Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Beijing 100850, PR China. Tel.: +86 10 66874606; fax: +86 10 68211656. E-mail addresses: [email protected] (B.-H. Zhong), [email protected] (Y.-Z. Zhang). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.02.076

Currently, pharmacotherapy is the most effective strategy to treat depression, and most available antidepressants exert therapeutic activity by activating monoaminergic neural transmission. According to their characteristics and mechanism of action, commercially available antidepressants are classified as: (1) monoamine reuptake inhibitors, including tricyclic antidepressants (TCAs), selective serotonin reuptake inhibitors (SSRIs), selective norepinephrine reuptake inhibitors (NRIs), serotonin and norepinephrine reuptake inhibitors (SNRIs), and norepinephrine and dopamine reuptake inhibitors (NDRIs); (2) monoamine oxidase inhibitors (MAOIs), including irreversible nonselective MAOIs and reversible selective inhibitor of monoamine oxidase-A (e.g. moclobemide); (3) other antidepressants with multiple targets, e.g. Mirtazapine [10], with ␣2 -adrenoceptor/5HT2 /5-HT3 receptor antagonistic activity; vilazodone [6], with selective serotonin reuptake and 5-HT1A receptor partial agonistic activity; agomelatine [7], with melatonergic agonistic (at

R. Xue et al. / Neuroscience Letters 544 (2013) 68–73

both MT1 and MT2 receptors) and 5-HT2C antagonistic properties. As we look back 60 years to the discovery of the first antidepressant, Research & Development (R&D) strategies for novel antidepressants experienced several phases: from by chance discovery strategy, to single target discovery strategy, then to multitarget discovery strategy. In 1950s, TCAs and nonselective MAOIs, the first generation of antidepressants, were discovered absolutely by chance [5,9]. However, besides therapeutic targets, i.e. serotonin transporter (SERT) and noradrenaline transporter (NET), TCAs also had high affinity for adrenergic, muscarinic, and other receptors, which led to severe side effects. Then, most drug discovery projects aimed to discover an antidepressant that was highly selective for a single target, and SERT was chosen as the most commonly studied target due to the close relationship between serotonin and depression. We must admit that fluoxetine, the first SSRI to be marketed, represent an important milestone in the history of antidepressants [20]. However, the efficacy and onset of SSRIs were still not satisfactory. How to accelerate onset of action and increase response rate has been a crucial point for antidepressant R&D. As is wellknown, complex diseases, such as depression, are not caused by a single gene, transmitter, or system. Accordingly, by modulating multiple targets simultaneously, drugs may have the potential to provide superior efficacy. SNRIs, activating serotonergic and noradrenergic neurotransmission simultaneously, have been reported to have higher efficacy and/or faster acting rate than commonly used antidepressants [23]. SNRIs have been first-line antidepressants, and the economic revenue is attractive to pharmaceutical companies. Taking duloxetine as the leading compound, we synthesized lots of compounds with novel structures. The present study was designed to screen potential SNRIs using the integrated evaluation system established by our group [24], which combined in vitro high throughput screening technology, and in vivo animal tests, and find novel SNRI candidate with high efficacy and independent intellectual property. This study, by in vivo and in vitro screening and comparison, led to the discovery of 071031B, a novel serotonin and noradrenaline reuptake inhibitor. 2. Materials and methods 2.1. Animals Male or female ICR mice weighing 18–20 g and male SD rats weighing 180–200 g were purchased from Beijing Vital River Laboratory Animal Technology Company (Beijing, China). Animals were group housed in rooms maintained at 22 ± 2 ◦ C, with humidity of 40–60% and 12 h:12 h-light/dark cycle (lights on at 8:00 am). Experiments were conducted in compliance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH publication No. 86-23, revised 1996). 2.2. Drugs and reagents Fourteen compounds with novel structure were synthesized in Beijing Institute of Pharmacology and Toxicology (Beijing, China). Duloxetine hydrochloride was purchased from Beijing Furenkang biopharmaceutical corporation, Ltd (Beijing, China). [3 H]citalopram and [3 H]nisoxetine were purchased from PerkinElmer Life Sciences (NEN, Boston, MA, USA). Fluoxetine and desipramine were purchased from Sigma (St. Louis, MO, USA). 2.3. Tail suspension test in mice The tail suspension test (TST) was performed according to that described previously [18,25]. Mice were treated with various doses of novel compounds (intraperitoneal injection, i.p.)

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0.5 h before TST; then, mice were suspended 5 cm above the bottom of apparatus. The duration of immobility during the last 4 min of the total 6 min was recorded. Mice were judged to be immobile when they hung passively without moving. Inhibition rate was calculated by the following formula: Inhibition rate (%) = (immobility time of vehicle group − immobility time of compound group) × 100/immobility time of vehicle group. 2.4. Forced swimming test in mice The forced swimming test (FST) in mice was performed according to that described previously [13,25]. Thirty minutes after intraperitoneal injection of various doses of novel compounds (2.5–20 or 5–30 mg/kg), mice were individually placed in glass cylinders (diameter 10 cm, height 20 cm) containing 10 cm of water maintained at 25 ◦ C. The duration of immobility during the last 4 min of the total 6 min was measured. Mice were considered to be immobile when they floated motionless, making only the movement necessary to keep their heads above the water. Inhibition rate was calculated by the same formula as mentioned above. 2.5. Locomotor activity test in mice Compounds were injected intraperitoneally 30 min prior to tests, then mice were placed in the corner of a plastic box and allowed to habituate for 5 min. As for 071031A, locomotor activity was determined using VIDEOMEX-V image analytic system (Columbus Instruments, USA), and traveling distance in 5 min was recorded. As for 071017S, 071026W, and 071031B, locomotor activity was determined using visual method, and parameters including the number of crossing and number of rearing were recorded in 5 min. 2.6. Binding affinity for rat SERTs or NETs The affinity of compounds for rat SERTs or NETs was determined by competition of [3 H]citalopram or [3 H]nisoxetine, according to that described previously [1,21]. Membrane protein was prepared from rat frontal cortex. Competitive binding assays were performed in reaction buffer containing 20 ␮g membrane protein, 1.25 nM [3 H]citalopram or 1.0 nM [3 H]nisoxetine, and various concentration of compounds (1 and 100 nM) at 25 ◦ C (SERTs) or 4 ◦ C for 60 min (NETs). Nonspecific binding was determined using10 ␮M fluoxetine or 10 ␮M desipramine. Inhibition rate was calculated by the following formula: Inhibition rate = (total binding − drug binding) × 100%/(total binding − nonspecific binding). All data are presented as mean values of three dependent assays. 2.7. Acute toxicity test Acute toxicity test was conducted with 071031B in mice using intraperitoneal administration. Male or female mice were treated with various doses of 071031B (98.0, 89.1, 81.0, or 73.6 mg/kg), and observed for 2 weeks. Toxic symptoms and mortality rates were recorded, and the median lethal dosage (LD50 ) and 95% confidence interval values were calculated. 2.8. Statistical analysis Statistical analysis was performed using GraphPad Prism (GraphPad Prism 5.0, version 2.0; GraphPad Software Inc., San Diego, CA). Data from the TST, FST, and locomotor activity test were showed as means ± SD, and were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. The transporter binding data were analyzed using one-site nonlinear regression of concentration–effect curve. The LD50 and 95% confidence interval

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Table 1 Effects of compounds on immobility time in the tail suspension test and forced swimming test in mice. Group

Dose (mg/kg)

TST

FST

Immobility time (s) Duloxetinea

2007091701

2007091702

2007091703

2007091704

2007091705

2007091706

2007091707

071017S

071026W

070919A

071031A

071031B

080307A

0 2.5 5 10 20 40 0 5 10 20 30 0 5 10 20 30 0 5 10 20 30 0 2.5 5 10 20 0 2.5 5 10 20 0 2.5 5 10 20 0 2.5 5 10 20 0 2.5 5 10 20 0 2.5 5 10 20 0 2.5 5 10 20 0 2.5 5 10 20 0 2.5 5 10 20 0 10 20

113.3 85.6 55.5 57.6 19.3 17.9 100.1 164.3 140.6 119.7 104.3 107.4 54.8 12.7 0 5.2 111.6 58.6 97.2 73.9 71.2 87.3 111.5 72.5 47.6 35.9 108.0 98.2 101.4 89.9 99.5 88.7 78.1 61.4 77.4 31.3 81.3 87.3 92.4 73.5 26.7 80.6 38.2 20.2 6.2 2.0 108.7 51.2 53.9 22.5 11.9 113.1 64.7 76.3 79.5 55.2 95.0 30.7 21.9 33.3 0.3 94.2 35.0 20.8 9.0 2.5 95.7 50.7 17.4

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

24.6 35.7 37.7*** 36.4*** 14.6*** 21.4*** 40.5 35.5 45.0 42.5 47.8 34.5 48.7*** 20.3*** 0*** 11.2*** 38.3 24.5* 65.0 38.3 38.1 38.6 49.8 31.4 38.2 45.4* 34.1 38.3 43.1 55.2 45.1 40.8 61.4 45.7 57.8 43.3* 40.8 31.1 46.5 28.8 26.9** 32.8 32.5** 17.1*** 7.4*** 6.3*** 25.7 34.3*** 46.9** 28.1*** 16.8*** 27.8 51.3 45.1 48.4 37.7* 28.7 44.1*** 21.5*** 31.2*** 0.9*** 49.8 30.0*** 32.1*** 4.0*** 4.1*** 47.5 36.0* 27.0***

Inhibition rate (%) – 24 51 49 83 84 – −64 −40 −20 −4 – 49 88 100 95 – 47 13 34 36 – −28 17 45 59 – 9 6 17 8 – 12 31 13 65 – −7 −13 10 67 – 53 75 92 98 – 53 50 79 89 – 43 33 20 51 – 67 77 65 99 – 63 78 98 98 – 47 82

Immobility time (s)

Inhibition rate (%)

120.5 ± 54.5 115.5 ± 51.7 129.9 ± 31.6 107.5 ± 49.4 88.3 ± 54.2 60.4 ± 31.6* 154.8 ± 48.4 112.2 ± 48.2 149.0 ± 46.4 140.8 ± 53.2 102.7 ± 34.9 138.2 ± 53.8 110.5 ± 46.9 95.5 ± 40.6 42.7 ± 54.8*** 56.2 ± 37.7** 167.0 ± 37.0 118.4 ± 45.4 143.7 ± 67.0 153.7 ± 57.3 128.4 ± 37.3 150.4 ± 36.0 109.1 ± 63.0 113.3 ± 36.5 98.0 ± 47.0 127.9 ± 47.4 138.1 ± 42.8 122.9 ± 49.5 124.2 ± 46.9 113.5 ± 66.2 117.7 ± 33.0 137.5 ± 41.1 119.0 ± 49.0 138.5 ± 60.3 135.9 ± 50.5 78.7 ± 50.8* 115.5 ± 65.9 81.5 ± 37.6 96.4 ± 52.4 87.8 ± 46.9 58.7 ± 49.9 150.2 ± 29.3 148.7 ± 40.3 86.7 ± 44.7** 54.6 ± 44.0*** 18.6 ± 15.7*** 115.4 ± 46.3 92.4 ± 39.2 96.5 ± 43.3 108.2 ± 46.7 61.2 ± 44.5* 146.7 ± 36.5 110.5 ± 59.1 132.5 ± 59.6 138.1 ± 40.4 96.2 ± 45.7 146.7 ± 36.5 110.5 ± 59.1 132.5 ± 59.6* 138.1 ± 40.4** 96.2 ± 45.7*** 131.2 ± 51.5 113.6 ± 47.4 119.4 ± 47.0 79.2 ± 36.4* 43.1 ± 37.1*** 122.7 ± 56.0 80.6 ± 50.0 44.1 ± 59.4**

– 4 −8 11 27 50 – 28 4 9 34 – 20 31 69 59 – 25 14 8 23 – 27 25 35 15 – 11 10 18 15 – 13 −1 1 43 – 29 17 24 49 – 1 42 64 88 – 20 16 6 47 – 25 10 6 34 – 32 43 49 79 – 13 9 40 67 – 34 64

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Table 1 (Continued) Group

Dose (mg/kg)

080307B

0 2.5 5 10 20

TST

FST

Immobility time (s) 106.0 98.2 72.8 62.4 47.2

± ± ± ± ±

31.3 47.6 33.3 26.1* 32.3**

Inhibition rate (%)

Immobility time (s)

– 7 31 41 55

122.7 129.9 96.1 95.0 27.4

± ± ± ± ±

Inhibition rate (%)

56.0 30.5 53.4 43.6 25.5***

– −6 22 23 78

Values are expressed as mean ± SD. * P < 0.05 versus vehicle, one-way ANOVA followed by Dunnett’s t-test. n = 10. ** P < 0.01 versus vehicle, one-way ANOVA followed by Dunnett’s t-test. n = 10. *** P < 0.001 versus vehicle, one-way ANOVA followed by Dunnett’s t-test. n = 10. a Data from Xue et al. [22].

values in the acute toxicity test were calculated using the method described by Bliss [1]. 3. Results

Table 2 Effects of 071017S, 071026W, 071031B, and 071031A on the locomotor activity in mice. Group

Dose (mg/kg)

071017S

0 2.5 5 10 20 0 2.5 5 10 20 0 2.5 5 10 20

Locomotor activity No. of crossing

3.1. Effects of compounds in the TST and FST in mice Table 1 illustrated the effects of compounds on the immobility time in the TST and FST in mice. In the TST, 12 compounds, including 2007091702 (5–30 mg/kg), 2007091703 (5 mg/kg), 2007091704 (20 mg/kg), 2007091706 (20 mg/kg), 2007091707 (20 mg/kg), 071017S (2.5–20 mg/kg), 071026W (2.5–20 mg/kg), 071019A (20 mg/kg), 071031A (2.5–20 mg/kg), 071031B (2.5–20 mg/kg), 080307A (10, 20 mg/kg) and 080307B (10, 20 mg/kg), significantly reduced the immobility time. In the FST, 8 compounds, including 2007091702 (20, 30 mg/kg), 2007091706 (20 mg/kg), 071017S (5–20 mg/kg), 071026W (20 mg/kg), 071031A (5–20 mg/kg), 071031B (10, 20 mg/kg), 080307A (20 mg/kg) and 080307B (20 mg/kg), decreased the duration of immobility time. 2007091701 (2.5–30 mg/kg) and 2007091705 (2.5–20 mg/kg) showed no antidepressant effect in the TST and FST.

071026W

071031B

3.3. Binding affinity of compounds for rat SERTs and NETs As 071017S, 071026W, 071031A, and 071031B showed superior antidepressant activity in behavioral despair tests in mice, in vitro competitive binding experiments were conducted to evaluate the affinity of these four compounds for SERTs and NETs. Table 3 illustrated inhibition of [3 H]citalopram and [3 H]nisoxetine binding to rat SERTs and NETs by 071017S, 071026W, 071031A, 071031B, and duloxetine at 1 nM and 100 nM. 071017S potently inhibited the binding of [3 H]citalopram to SERT at 100 nM with inhibitory rate of 67%, while the inhibitory effect on binding of [3 H]nisoxetine to NET was weaker. 071026W inhibited the binding of [3 H]nisoxetine to NET at 100 nM with inhibitory rate of 50%, while the inhibitory rate on binding of [3 H]citalopram to SERT was only 10%. 071031A had weak affinity to both rat SERT and NET, as the inhibitory rate was less than 50% at 100 nM. 071031B dose-dependently inhibited binding of [3 H]citalopram to SERT and [3 H]nisoxetine to NET, with

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

No. of rearing

25.8 24.2* 24.5 37.0 40.1* 20.5 13.6 22.2 25.4 28.3* 18.8 16.4 29.0 9.0 40.3*

33.5 17.2 27.5 15.0 13.4 38.6 42.1 27.6 26.0 11.9 31.0 26.8 24.5 22.8 3.3

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

19.6 9.9 13.6 16.0 22.1* 12.3 12.9 16.5 20.1 13.2** 5.7 11.1 17.8 13.4 7.4***

Group

Dose (mg/kg)

Locomotor activity Distance (mm)

071031A

0 2.5 5 10 20

685.1 479.2 479.9 473.6 305.1

3.2. Effects of compounds on the locomotor activity in mice As 071017S, 071026W, 071031A, and 071031B showed superior antidepressant activity in behavioral despair tests in mice, locomotor activity tests were performed to eliminate the possibility of false positive results. Table 2 illustrated the effects of these four compounds on the locomotor activity in mice. Compared with vehicle group, 071017S (2.5, 20 mg/kg), 071026W(20 mg/kg), and 071031B (20 mg/kg) significantly decreased the number of crossings and rearings; 071031A (2.5–20 mg/kg) also decreased the distance in 5 min.

83.6 44.0 74.1 62.8 47.6 85.4 74.4 61.3 63.8 53.6 74.0 68.0 73.3 72.3 38.9

± ± ± ± ±

136.4 128.0** 163.2** 130.0** 138.0**

Values are expressed as mean ± SD. * P < 0.05 versus vehicle, one-way ANOVA followed by Dunnett’s t-test. n = 8–10. ** P < 0.01 versus vehicle, one-way ANOVA followed by Dunnett’s t-test. n = 8–10. *** P < 0.001 versus vehicle, one-way ANOVA followed by Dunnett’s t-test. n = 8–10.

inhibitory rate of 83% and 73% at 100 nM or 33% and 56% at 1 nM, respectively. 3.4. Acute toxicity test As 071031B showed superior antidepressant activity in vivo and transporter binding characteristics in vitro, acute toxicity test was applied to get the preliminary safety information. 071031B dose-dependently increased the mortality rate of mice, and the LD50 value was 85.6 mg/kg (Table 4). The primary toxicological Table 3 Inhibition of binding to SERT and NET in vitro by 071017S, 071026W, 071031B, and 071031A and duloxetine. Each concentration was conducted in triplicate. Group

071017S 071026W 071031A 071031B Duloxetine

Binding to SERT

Binding to NET

I% (100 nM)

I% (1 nM)

I% (100 nM)

I% (1 nM)

67 10 36 83 98

20 4 0 33 32

33 50 16 73 72

10 23 0 56 32

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Table 4 Median lethal dosage (LD50 ) and 95% confidence interval values of acute intraperitoneal administration of 071031B and duloxetine in mice. Group

071031B Duloxetinea

Dose (mg/kg)

Mortality rate (%)

98.0 89.1 81.0 73.6 –

80 70 40 30 –

LD50 (mg/kg)

95% confidence interval (mg/kg)

85.6

69.9–91.2

75.5

62.8–80.9

LD50 and 95% confidence interval values were calculated using the method described by Bliss [1]. n = 10 (5 per group for each sex). a Data from Xue et al. [22].

symptoms included tremor, convulsion, salivation, and hyperresponsiveness. 4. Discussion Currently, only three SNRIs, venlafaxine (Effexor® ), desvenlafaxine (Pristiq® ), and duloxetine (Cymbalta® ), were approved for depression therapy in the United State. Milnacipran (Ixel® ), another SNRI, was only available in some countries in European and Asia. Venlafaxine, approved by FDA in 1994, was the first SNRI to be marketed. Unfortunately, Redrobe et al. reported that venlafaxine, only at higher doses, enhanced 5-HT and NE concentration simultaneously [15]. In addition, venlafaxine can cause severe cardiovascular toxicity in some patients [12]. In 2008, desvenlafaxine, the active metabolite of venlafaxine [3], was approved to be marketed in the United State, which filled the market vacancy of venlafaxine. Duloxetine was deemed as the first real SNRI for its robust and balanced affinity for SERT and NET. Both preclinical and clinical evidence demonstrates that, compared with TCAs and MAOIs, duloxetine are better tolerated; compared with SSRIs, duloxetine provide greater therapeutic response. Furthermore, duloxetine also produces strong relief on painful physical symptoms associated with depression, which may contribute to their therapeutic efficacy [2]. In 2011, worldwide sales of Cymbalta® have reached $4.16 billion [4]. Given the prospective future of SNRIs R&D, we designed and synthesized a series of compounds with novel structure. Firstly, we performed screening for their antidepressant activity in vivo using the TST and FST; then, we conducted locomotor activity tests to eliminate the possibility of false positive results; furthermore, we determined the affinity of novel compounds for rat SERTs and NETs; finally, in order to reduce the risk of new drug R&D, acute toxicity test was conducted with the drug candidate. The TST and FST, also known as behavioral despair tests, are the earliest developed and most widely used animal tests for antidepressant screening. The TST and FST are based on principles that forcing mice at inescapable stress, i.e. swimming in narrow space or being suspended by tail, will develop “despair state” [11]. Our previous study demonstrated that minimal effective doses of duloxetine (i.p.) in the TST and FST were 5 mg/kg and 40 mg/kg, respectively [22]. Results in the present study indicated that 8 compounds, including 2007091702, 2007091706, 071017S, 071026W, 071031A, 071031B, 080307A, and 080307B showed robust antidepressant activity in both the TST and FST. Notably, the minimal effective dose of 071017S, 071026W, 071031A and 071031B was less than 2.5 mg/kg or 10 mg/kg in the TST or FST, respectively, which was lower than that of duloxetine. Unfortunately, although 2007091702, 2007091706, and 2007091707 showed antidepressant effect in the behavioral tests, these compounds produced side effects under effective doses, such as tremor, salivation, and irritation. 2007091701 and 2007091705 showed no antidepressant effect in the TST and FST. As 071017S, 071026W, 071031A, and 071031B showed superior antidepressant activity in behavioral despair tests in mice, locomotor activity tests and in vitro competitive binding experiments were conducted.

Notably, stimulants, like amphetamine and caffeine, can also decrease the immobility time [13,18]. It is crucial to exclude the false positive results by determining locomotor activity. Results from locomotor activity tests indicated that 071017S, 071026W, 071031A, and 071031B did not induce stimulatory effects on central nervous system (CNS). Actually, 071017S, 071026W, and 071031B induced suppressive effects on locomotor activity at high dose, while 071031A showed suppressive effects at lower doses. The suppressive effect on CNS was similar to the characteristics of many antidepressants, such as fluoxetine and duloxetine [15,16]. Currently, most antidepressants under research were based on monoamine hypothesis, which proposed that depression was caused by deficit of 5-HT and NE [14,17]. As most drugs were synthetized with target orientation, it is the first step for antidepressant screening to identify the target, i.e. SERT and NET. In vitro competitive binding experiments were conducted to evaluate the affinity of 071017S, 071026W, 071031A, and 071031B for SERTs and NETs, and the results indicated that only 071031B showed high affinity to both SERT and NET with similar inhibitory rates to duloxetine at 1 and 100 nM. Interestingly, despite the robust activity in behavioral tests, 071031A showed weak affinity for SERT and NET, indicating that 071031A may exert antidepressant effects by other mechanisms. In vivo and in vitro tests indicated that 070131B showed robust antidepressant effects and high transporter affinity. However, in addition to therapeutic benefit, the toxicology profile also plays an important role in new drug R&D. Accordingly, we conducted acute toxicity test to disclose the preliminary toxicological profile of 071031B. Results indicated that the toxic symptoms were related to the CNS reaction, which were similar to that of duloxetine, and the LD50 value was slightly higher than that of duloxetine [22]. As is well known, new drug R&D need huge investment and is accompanied by high risk. So it is necessary and important to perform screening for potential monoamine inhibitors faster. Following long term practice, our team established an integrated system for antidepressant screening and discovery of monoamine reuptake inhibitors, combining high throughput

Fig. 1. Chemical structure of 071031B.

R. Xue et al. / Neuroscience Letters 544 (2013) 68–73

screening technology and in vivo animal tests together [24]. This system can screen novel compounds fast, and was helpful in discovering side effects on CNS effectively in early phase. Using this system, we discovered a potential SNRI 071031B with independent intellectual property [(7)-3-(benzo[d] [1,3]dioxol4-yloxy)-N-methyl-3-(thiophen-2-yl) propan-1-amine; Fig. 1]. Notably, 071031B may undergo hydrolysis under acidic conditions, and the product of hydrolysis may be seasamol, which has definite hepatoprotective effect [19]. Therefore, this structure design may improve the hepatoxicology effects of duloxetine [2]. However, the hepatoprotective effects of 071031B needs further study. 5. Conclusion Our study demonstrated that the integrated system established by our group, combining high throughput screening technology and in vivo animal tests together, is effective to screen potential monoamine reuptake inhibitors fast and accurately, and 071031B is expected to be a novel serotonin and noradrenaline reuptake inhibitor for its robust antidepressant activity and transporter affinity. Contributors Author Xue contributed to the performance of in vivo behavioral tests, research design, data analysis, and manuscript writing. Authors Zhang (Yan-Ping Zhang), He, and Fan contributed to the synthesis of novel compounds. Author Jin contributed to the performance of in vitro binding assays and data analysis. Authors Yuan, Zhao, Chen, and Zhang (Li-Ming Zhang) participated in behavioral tests. Authors Zhang (You-Zhi Zhang), Zhong and Li contributed to research design, data analysis, and manuscript revision. Conflict of interest All authors declare that they have no conflicts of interest. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 30973516 and 81274117), and the National Key New Drug Creation Program (No. 2012ZX09102101-004). References [1] C.I. Bliss, Statistics in Biology, New York, McGraw-Hill Book Company, 1967. [2] F.P. Bymaster, T.C. Lee, M.P. Knadler, M.J. Detke, S. Iyengar, The dual transporter inhibitor duloxetine: a review of its preclinical pharmacology, pharmacokinetic profile, and clinical results in depression, Curr. Pharm. Des. 11 (2005) 1475–1493.

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