The Novel Methoxetamine Analogs N-Ethylnorketamine

Psychopharmacology (2019) 236:2201–2210 https://doi.org/10.1007/s00213-019-05219-x

ORIGINAL INVESTIGATION

The novel methoxetamine analogs N-ethylnorketamine hydrochloride (NENK), 2-MeO-N-ethylketamine hydrochloride (2-MeO-NEK), and 4-MeO-N-ethylketamine hydrochloride (4-MeO-NEK) elicit rapid antidepressant effects via activation of AMPA and 5-HT2 receptors

Leandro Val Sayson1 & Chrislean Jun Botanas1 & Raly James Perez Custodio1 & Arvie Abiero1 & Mikyung Kim1 & Hyun Jun Lee1 & Hee Jin Kim1 & Sung Yeun Yoo2 & Kun Won Lee2 & Hye Won Ryu2 & Srijan Acharya3 & Kyeong-Man Kim3 & Yong Sup Lee2 & Jae Hoon Cheong1

Received: 5 November 2018 /Accepted: 1 March 2019 /Published online: 19 March 2019 # Springer-Verlag GmbH Germany, part of Springer Nature 2019

Abstract Rationale Depressive syndrome or depression is a debilitating brain disorder affecting numerous people worldwide. Although readily available, current antidepressants have low remission rates and late onset times. Recently, N-methyl-D-aspartate (NMDA) receptor antagonists, like ketamine and methoxetamine (MXE), were found to elicit rapid antidepressant effects. As the search for glutamatergic-based antidepressants is increasing, we synthesized three novel MXE analogs, N-ethylnorketamine hydrochloride (NENK), 2-MeO-N-ethylketamine hydrochloride (2-MeO-NEK), and 4-MeO-N-ethylketamine hydrochloride (4-MeO-NEK). Objectives To determine whether the three novel MXE analogs induce antidepressant effects and explore their mechanistic correlation. Methods We examined their affinity for NMDA receptors through a radioligand binding assay. Mice were treated with each drug (2.5, 5, and 10 mg/kg), and their behavior was assessed 30 min later in the forced swimming test (FST), tail suspension test (TST), elevated plus-maze (EPM) test, and open-field test (OFT). Another group of mice were pretreated with 2,3-dihydroxy-6-nitro-7- sulfamoyl-benzo(f)quinoxaline-2,3-dione (NBQX), an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, or ketanserin (KS), a 5-HT2 receptor antagonist, during the FST. We also measured mRNA levels of the AMPA receptor subunits GluA1 and GluA2, brain-derived neurotrophic factor (BDNF), and mammalian target of rapamycin (mTOR) in the hippocampus and prefrontal cortex. Results The MXE analogs showed affinity to NMDA receptors and decreased immobility time during the FST and TST. NBQX and KS blocked their effects in the FST. The compounds did not induce behavioral alteration during the EPM and OFT. The compounds altered GluA1, GluA2, and BDNF mRNA levels. Conclusion These results suggest that the novel MXE analogs induce antidepressant effects, which is likely via AMPA and 5-

HT2 receptor activation.

Keywords Depressive syndrome . NMDA receptor antagonists . Methoxetamine analogs . AMPA receptors . 5-HT2 receptors

Leandro Val Sayson and Chrislean Jun Botanas contributed equally to this work.

* Yong Sup Lee 2 Medicinal Chemistry Laboratory, Department of Pharmacy & [email protected] Department of Life and Nanopharmaceutical Sciences, College of Pharmacy, Kyung Hee University, 26 Kyungheedae-ro, Seoul 02447, * Jae Hoon Cheong Republic of Korea [email protected] 3 College of Pharmacy, Chonnam National University, 77 1 Uimyung Research Institute for Neuroscience, Department of Yongbong-ro, Yongbong-dong, Buk-gu, Gwangju, Republic of Pharmacy, Sahmyook University, 815 Hwarangro, Nowon-gu, Korea Seoul 01795, Republic of Korea 2202 Psychopharmacology (2019) 236:2201–2210

Introduction we examined these compounds for their antidepressant effects. Depressive syndrome or depression (major depressive To ascertain whether the novel compounds have af- disorder) is a chronic and debilitating brain disorder that finity for NMDA receptors, we conducted a radioligand affects over 350 million people worldwide (Cipriani binding assay and confirmed that NENK, 2-MeO-NEK, et al. 2018; Coppola and Mondola 2012). Current phar- and 4-MeO-NEK all have an affinity for NMDA recep- macotherapies include monoaminergic-based antidepres- tors. We then determined whether these compounds ex- sants (Hashimoto 2011; Hillhouse and Porter 2015), ert antidepressant effects by submitting treated mice to such as selective serotonin reuptake inhibitors (SSRI), the forced swimming test (FST), tail suspension test tricyclic antidepressants (TCA), serotonin– (TST), elevated plus-maze (EPM) test, and open-field norepinephrine reuptake inhibitors (SNRI), noradrenergic test (OFT). We also examined the involvement of the and specific serotonergic antidepressants (NaSSA), and glutamatergic system by pretreating mice with 2,3-dihy- norepinephrine–dopamine reuptake inhibitors (NDRI). droxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline-2,3-dione However, these drugs are associated with limited clini- (NBQX), an AMPA receptor antagonist, in the FST, and cal efficacy, low remission rates, and delayed onset of performing quantitative real-time polymerase chain reac- therapeutic effects (Dwyer et al. 2012; Monteggia and tion (qRT-PCR) to measure the mRNA expression levels Zarate Jr 2015;Rushetal.2006), highlighting the need of the AMPA receptor subunits GluA1 and GluA2, for better antidepressants. Recent studies have shown BDNF, and mTOR in the hippocampus and prefrontal that the N-methyl-D-aspartate (NMDA) antagonist keta- cortex, regions of the brain that are implicated in the mine (KET) engenders rapid and robust antidepressant pathophysiology of depressive syndrome (Liu et al. effects in both humans and rodents (Murrough 2012; 2017). The aforementioned genes were selected as they Skolnick et al. 2015). Remarkably, these effects are ob- have been profoundly implicated in the mechanism of served within hours of a single treatment and may last action of the antidepressant effects of KET (Zanos et al. up to a week (Murrough 2012; Zarate et al. 2006). It is 2016; Aleksandrova et al. 2017; Pochwat et al. 2014). suggested that activation of α-amino-3-hydroxy-5-meth- In addition, we pretreated mice with ketanserin (KS), a yl-4-isoxazolepropionic acid (AMPA) receptors, along 5-HT2 receptor antagonist, during the FST to determine with increased brain-derived neurotrophic factor the involvement of 5-HT2 receptors in the antidepressant (BDNF) levels and mammalian target of rapamycin effects of the compounds. Parallel experiments were (mTOR) signaling stimulation, is responsible for the an- conducted with KET. NENK, 2-MeO-NEK, and 4- tidepressant effects of KET (Zhou et al. 2014;Yang MeO-NEK exhibited antidepressant effects in the FST et al. 2013). These robust antidepressant effects of and TST, but these effects were inhibited by NBQX KET can overcome the limitations of current antidepres- and KS in the FST. Lastly, the novel compounds altered sants and provide an opportunity for the development of the levels of GluA1, GluA2, and BDNF mRNA in the new and better glutamate-based antidepressants. hippocampus and prefrontal cortex. We have previously shown that methoxetamine (MXE), a KET analog, also elicits rapid and sustained antidepressant effects in mice (Botanas et al. 2017), and the mechanism of action is attributable to the capacity of MXE to affect the Materials and methods glutamatergic and serotonergic systems. Consequently, we hy- pothesized that a novel substance with a chemical structure Animals similar to MXE or KET may also exhibit antidepressant properties. In this study, we synthesized three novel substances We used six-week-old male ICR mice weighing 25–30 g, ac- derived from MXE:N-ethylnorketamine hydrochloride quired from the Hanlim Animal Laboratory Co. (Hwasung, (NENK), 2-MeO-N-ethylketamine hydrochloride (2-MeO- Korea). They were housed 8 to 10 per cage and kept in a room NEK), and 4-MeO-N-ethylketamine hydrochloride (4-MeO- with controlled temperature (22 ± 2 °C) and humidity (55 ± NEK). As shown in Fig. 1,comparedtoMXE,NENKhasa2- 5%), with a 12-h light/12-h dark schedule (07:00 to 19:00 chloro group (R2) instead of a methoxy group (R3) in the light). Food and water were available ad libitum. All animals phenyl ring, whereas 2-MeO-NEK and 4-MeO-NEK have a were habituated to the laboratory setting for 5 days before the methoxy group at R2 and R4, respectively, instead of R3. experiments. Different cohorts of mice were used for each Compared to KET, NENK, 2-MeO-NEK, and 4-MeO-NEK experiment. All tests were performed in accordance with the present an N-ethyl group instead of an N-methyl group, in Principles of Laboratory Animal Care (NIH Publication No. addition to the methoxy group at R2 and R4 presented by 2- 85-23, revised 1985) and the Animal Care and Use Guidelines MeO-NEK and 4-MeO-NEK on the phenyl ring. Thereafter, of Sahmyook University, Korea. Psychopharmacology (2019) 236:2201–2210 2203

Fig. 1 Comparison of the chemical structures of (a) methoxetamine (MXE), ring, whereas 2-MeO-NEK and 4-MeO-NEK show a methoxy group at R2 (b) ketamine (KET), (c) N-ethylnorketamine hydrochloride (NENK), (d)2- and R4, respectively, instead at R3. Compared to KET, NENK, 2-MeO-NEK, MeO-N-ethylketamine hydrochloride (2-MeO-NEK), and (e)4-MeO-N- and 4-MeO-NEK present an N-ethyl instead of an N-methyl group. In addi- ethylketamine hydrochloride (4-MeO-NEK). Compared to MXE, NENK tion, 2-MeO-NEK and 4-MeO-NEK display a methoxy group at R2 and R4 has a 2-chloro group (R2) instead of a methoxy group (R3) in the phenyl in the phenyl ring

Drugs 118.41, 112.49, 70.43, 54.99, 38.43, 37.59, 35.57, 29.24, + + 21.21, 10.73; HR-MS calculated for C15H22NO2 ([M-Cl] ): N-ethylnorketamine hydrochloride 248.1645, found 248.1673.

NENK was synthesized from cyclopentylmagnesium 4-MeO-N-ethylketamine hydrochloride bromide as described previously (Hays et al. 2012). Cyclopentylmagnesium bromide was reacted with 2- 4-MeO-NEK was synthesized from cyclopentylmagnesium chlorobenzonitrile to form 2-chlorophenyl cyclopentyl bromide as described previously (Hays et al. 2012). methanone, which was then brominated alpha to the ketone. Cyclopentylmagnesium bromide was reacted with 4- The alpha-bromo ketone was converted to a Schiff’sbasewith methoxybenzonitrile to form 4-methoxyphenyl cyclopentyl ethyl amine, which was then heated to form NENK. The methanone, which was then brominated alpha to the ketone. resulting compound was treated with HCl to produce NENK The alpha-bromo ketone was converted to a Schiff’sbasewith HCl. The structure was confirmed by the following spectro- ethyl amine, which was then heated to form 4-MeO-NEK. The 1 δ scopic analyses: H NMR (400 MHz, D2O) 7.83 (1H, m), resulting compound was treated with HCl to form 4-MeO- 7.56–7.60 (3H, m), 3.31 (1H, m), 2.82 (2H, q, J = 7.2 Hz), NEK HCl. Its structure was confirmed by the following spec- – 1 2.60 2.64 (2H, m, H-6), 2.08 (1H, m), 1.96 (1H, m) 1.83 (1H, troscopic analyses: H NMR (400 MHz, D2O) δ 7.33 (d, J = 13 m), 1.70–1.72 (2H, m), 1.15 (3H, t, J = 7.2 Hz); CNMR 8.0 Hz, 2H), 7.10 (d, J = 8.0 Hz, 2H), 3.82 (s, 3H), 3.13 (m, δ (100 MHz, DMSO-d6) 208.74, 138.70, 133.65, 131.15, 1H), 2.82 (m, 1H), 2.56–2.42 (m, 3H), 1.87–2.00 (m, 3H), 129.17, 128.56, 126.64, 69.95, 39.45, 39.02, 36.83, 27.65, 1.65–1.71 (m, 2H), 1.10 (t, J = 7.2 Hz, 3H); 13CNMR + 21.86, 15.75; HR-MS calculated for C14H19ClNO ([M- (100 MHz, D2O) δ 209.38, 160.48, 129.93(2C), 121.75, + Cl] ): 252.1150, found 252.1155. 115.30(2C),71.88,55.56,38.84,37.00,32.52,27.42,21.09, + + 10.67; HR-MS calculated for C15H22NO2 ([M-Cl] ): 2-MeO-N-ethylketamine hydrochloride 248.1645, found 248.1664. KET was purchased from Bayer Animal Health Co. 2-MeO-NEK was synthesized from cyclopentylmagnesium (Suwon, Korea). NBQX and KS were obtained from Sigma- bromide as described previously (Hays et al. 2012). Aldrich (South Korea). All drugs were diluted in normal saline Cyclopentylmagnesium bromide was reacted with 2- (0.9% w/v NaCl) and administered intraperitoneally (IP). methoxybenzonitrile to form 2-methoxyphenyl cyclopentyl methanone, which was then brominated alpha to the ketone. Receptor binding assay The alpha-bromo ketone was converted to a Schiff’sbasewith ethyl amine, which was then heated to form 2-MeO-NEK. The HEK-293 cells were transfected with the expression con- resulting compound was treated with HCl to produce 2-MeO- structs containing the NR1 and NR2B subunits of the NEK HCl. Its structure was confirmed by the following spec- NMDA receptor, which were purchased from Addgene 1 troscopic analyses: H NMR (400 MHz, D2O) δ 7.61 (d, J = (Cambridge, MA, USA). After 24 h, the cells were split into 7.8 Hz, 1H), 7.52 (t, J = 7.9 Hz, 1H), 7.15 (t, J = 7.6 Hz, 1H), a 24-well plate. On the next day, the cells were incubated with 7.09 (d, J = 8.4 Hz, 1H), 3.70 (s, 3H), 3.13 (m, 1H), 2.69 (q, 10 nM [3H]-TCP (40 Ci/mmol), purchased from PerkinElmer J = 7.3 Hz, 2H), 2.35–2.39 (m, 2H), 1.96 (m, 1H), 1.87–1.75 (Waltham, MA, USA), for 1 h at room temperature. Cells were (m, 2H), 1.58–1.61 (m, 2H), 1.06 (t, J = 7.3 Hz); 13CNMR washed three times with ice-cold serum-free media, dissolved

(100 MHz, D2O) δ 210.68, 157.24, 132.68, 129.58, 121.68, with 1% SDS, mixed with a liquid scintillation cocktail, and 2204 Psychopharmacology (2019) 236:2201–2210 counted with a Wallac 1450 MicroBeta® TriLux liquid scin- (10 mg/kg), or saline 30 min previously were placed at the tillation counter (PerkinElmer). Binding of the remaining center, facing one of the open arms. An entry into an arm [3H]-TCP incubated with 10 μM MK-801 was defined as required all four paws of the animal to be placed in the non-specific. The IC50 values were determined with predefined area. The time spent in the open arms was recorded GraphPad Prism 7.0, using non-linear regression with log throughout the 5-min duration of the test. The percentage of concentration plotted against percent-specific binding. Ki entries into the open arm (100 × open/total entries) was values were calculated using the equation described previous- calculated. 3 ly (Cheng and Prusoff 1974). The Kd value for [ H]-TCP, 54.3 nM, was based on previous studies (Mitrovic et al. 1991). Open-field test

Forced swimming test This protocol was derived from a previous study (Botanas et al. 2017). Thirty minutes after NENK, 2-MeO-NEK, 4- This test was based on those described previously (Botanas et al. MeO-NEK (2.5, 5, and 10 mg/kg), KET (10 mg/kg), or saline 2017; Zanos et al. 2016). Mice (10 per group) treated with treatment, mice (10 per group) were put into a square black NENK, 2-MeO-NEK, 4-MeO-NEK (2.5, 5, and 10 mg/kg), Plexiglass container with an open field (42 × 42 × 42 cm) for KET (10 mg/kg), or saline 30 min previously were subjected to 12 min. The first 2 min of recording was excluded from the a 6-min swim session in clear Plexiglass cylinders (30 cm analysis to eliminate the effects of animal handling. The dis- height × 20 cm diameter) filled with 15 cm tap water (23 ± tance moved was measured using a video tracking system 1 °C). The test was performed under normal lighting conditions (Ethovision, Noldus, Netherlands). (100 lx) (Vollenweider et al. 2011). Each session was recorded using a digital video camera, and immobility time was measured Tissue collection, RNA preparation, and qRT-PCR by two trained observers (naïve of the groupings) during the last 4 min of the 6-min test. Mice were deemed immobile when they This procedure was patterned after previous studies (Kim et al. inactively floated with no further activity, other than that neces- 2018; Botanas et al. 2017; Custodio et al. 2017; Lecointre sary to keep their head above the water. Another group of mice (8 et al. 2015), with some modifications. We used 5 mice per per group) were treated with NBQX (10 mg/kg) or KS group in this experiment (Kong et al. 2015;Hébertetal. (0.5 mg/kg) 15 min before administration of the analogs. 2010). Mice treated with NENK, 2-MeO-NEK, 4-MeO- NEK, KET (10 mg/kg), or saline were euthanized and decap- Tail suspension test itated for brain extraction 30 min after treatment. The 10 mg/kg dose was chosen because it induced the lowest This test was performed as described previously (Botanas immobility time in the FST and TST. Brains were extracted et al. 2017; Koike et al. 2011a) with some modifications. and placed in ice-cold saline. The hippocampus and prefrontal Mice were suspended by the tail from a metal rod (45 cm cortex were isolated and immediately frozen at − 80 °C for above the table) using adhesive tape and positioned at least subsequent analysis. Total RNA was isolated with TRIzol® 20 cm away from the nearest object. Each session was record- reagent (Invitrogen, Carlsbad, CA, USA) according to the ed for 6 min, and immobility time was determined by two manufacturer’s instructions. The RNA was further purified trained observers (naïve of the groupings). Mice were consid- using the Hybrid-R™ kit (Geneall Biotechnology, Seoul, ered immobile when they hung inactively and unmoving. Like Korea). The RNA concentrations were measured using a in the FST, mice (10 per group) were administered NENK, 2- Colibri Microvolume Spectrometer (Titertek-Berthold, MeO-NEK, 4-MeO-NEK (2.5, 5, and 10 mg/kg), KET Pforzheim, Germany). (10 mg/kg), or saline 30 min before the experiment. We used qRT-PCR to determine the mRNA expressions levels of the GluA1 and GluA2 AMPA receptor subunits, Elevated plus-maze test BDNF, and mTOR in the hippocampus and prefrontal cortex. Briefly, 1 μg of total RNA was reverse-transcribed using The experiment was designed according to a previous study AccuPower® CycleScript RT PreMix (Bioneer, Seoul, (Botanas et al. 2017). The plus-maze consisted of two open Korea), following the manufacturer’s protocol. The cDNA arms and two closed arms, all measuring 30 × 6 cm, with a 6 × was amplified using a set of custom sequence-specific primers 6 cm area in the center. The closed arms had enclosing 20-cm (Cosmogenetech, Seoul, Korea) and detected with SYBR® high walls. The plus-maze was raised 50 cm above the floor. Green (Solgent, Korea). The input concentration for cDNA The test was conducted with indirect lighting (12 lx) to pre- synthesis was 2.6 μg/μL. The cycling conditions were as fol- vent shadowed areas that could become a place of preference lows: 94 °C for 1 min (denaturing step), followed by anneal- for the mice. Mice (10 per group) administered with NENK, ing at primer-specific temperature for 1 min, and then 72 °C 2-MeO-NEK, 4-MeO-NEK (2.5, 5, and 10 mg/kg), KET for 45 s. The primer sequences used were as follows: GluA1: Psychopharmacology (2019) 236:2201–2210 2205 forward: 5′-TCC CCA ACA ATA TCC AGA TAG GG-3′, NENK, 2-MeO-NEK, and 4-MeO-NEK elicit rapid reverse: 5′-AAG CCG CAT GTT CCT GTG ATT-3′; antidepressant effects GluA2: forward: 5′-GCC GAG GCG AAA CGA ATG A-3′, reverse: 5′-CAC TCT CGA TGC CAT ATA CGT TG-3′; AsshowninFig.3, NENK, 2-MeO-NEK, 4-MeO-NEK, BDNF: forward: 5′-TCA TAC TTC GGT TGC ATG AAG and KET decreased the immobility time of the mice in G-3′, reverse: 5′-AGA CCT CTC GAA CCT GCC C-3′; the FST (F (10, 99) = 3.518, p < 0.001). Similarly, the mTOR: forward: 5′-ACC GGC ACA CAT TTG AAG three analogs and KET also reduced the immobility time AAG-3′,reverse:5′-CTC GTT GAG GAT CAG CAA GG- of mice during the TST (F (10, 99) = 3.754, p <0.001). 3′;GAPDH:forward:5′-AGG TCG GTG TGA ACG GAT In contrast, NENK, 2-MeO-NEK, 4-MeO-NEK, and TTG-3′,reverse:5′-TGT AGA CCA TGT AGT TGA GGT KET did not significantly affect the percentage of time CA-3′. All qRT-PCR analyses were done in triplicate. Values spent in the open arms in the EPM (F (10, 99) = 0.8481, were normalized to the relative amount of GAPDH mRNA. p > 0.05) or the distance moved during the OFT (F (10, Each result is shown as a relative expression level calculated 99) = 0.9969, p >0.05). using the 2−ΔΔCT method (VanGuilder et al. 2008).

The antidepressant effects of NENK, 2-MeO-NEK, Data analysis and 4-MeO-NEK involve AMPA receptor activation All data are presented as mean ± standard error of the mean NENK, 2-MeO-NEK, 4-MeO-NEK, and KET reduced (S.E.M.) and were analyzed using GraphPad Prism 7.0 soft- the immobility time of mice (F (9, 70) = 11.84, ware (San Diego, CA, USA). Comparisons were made using p < 0.001) in the FST (Fig. 4). However, NBQX treat- one-way analysis of variance (ANOVA) followed by ment significantly increased the immobility time com- Dunnett’s or Bonferroni’s post-test to determine the effects pared to NENK, 2-MeO-NEK, 4-MeO-NEK, and KET. of NENK, 2-MeO-NEK, 4-MeO-NEK, and KET in the FST, AsshowninFig.5, NENK and 4-MeO-NEK in- TST, EPM, OFT, and qRT-PCR. A p valuelessthan0.05was creased the expression levels of GluA1 in the hippo- considered significant. campus (F (4, 20) = 6.217, p < 0.01) but not in the prefrontal cortex (F (4, 35) = 0.2921, p >0.05). 2-MeO- NEK and KET also upregulated the expression of the Results GluA2 gene in the hippocampus (F (4, 20) = 18.94, p < 0.001), whereas only 2-MeO-NEK increased GluA2 NENK, 2-MeO-NEK, and 4-MeO-NEK exhibit affinity expression in the prefrontal cortex (F (4, 20) = 7.417, for NMDA receptors p < 0.001). NENK, 2-MeO-NEK, 4-MeO-NEK, and KET elicited higher expression levels of BDNF in the Figure 2ashowsthe(+)-[3H]-TCP displacement curves with hippocampus (F (4, 20) = 5.834, p < 0.01). In the pre- concentrations of KET, MXE, NENK, 2-MeO-NEK, and 4- frontal cortex, NENK and KET treatment resulted in a MeO-NEK at 10 and 100 nM and at 1, 5, and 10 μM. As significant increase in the expression of BDNF mRNA shown in Fig. 2b, 2-MeO-NEK and 4-MeO-NEK showed an (F (4, 20) = 11.81, p < 0.001). Only KET treatment led affinity with calculated Ki values of 0.40 and 0.10 μM, respec- to enhanced mTOR gene expression in both the hippo- tively, higher than KET (0.78 μM) and MXE (4.30 μM). campus (F (4, 20) = 6.141, p < 0.01) and prefrontal cor-

NENK exhibited lower affinity with 6.27 μM Ki value. tex (F (4, 20) = 5.633, p <0.01).

3 Fig. 2 Competitive binding of [ H]-TCP with NENK, 2-MeO-NEK, and MeO-NEK, and NENK. (b) The table shows the calculated IC50 and Ki 4-MeO-NEK to the NMDA receptors. (a) The displacement curves show values for KET, MXE, NENK, 2-MeO-NEK, and 4-MeO-NEK the concentration–response relationship of KET, MXE, 2-MeO-NEK, 4- 2206 Psychopharmacology (2019) 236:2201–2210

Fig. 3 Effects of NENK, 2-MeO-NEK, 4-MeO-NEK, or KET treatment altered the behavior of the mice in the EPM and OFT. Values are mean ± in mice 30 min prior to the FST, TST, EPM, and OFT; n = 10 animals per S.E.M. *p <0.05,**p < 0.01, and ***p < 0.001 are significantly different group. NENK, 2-MeO-NEK, 4-MeO-NEK, and KET reduced the to the saline treatment (Dunnett’s post-test) immobility time of mice in the FST and TST. None of the compounds

A possible role for 5-HT2 receptor activation NENK, 2-MeO-NEK, and 4-MeO-NEK. However, KS did in the antidepressant effects of NENK, 2-MeO-NEK, not inhibit the effects of KET. and 4-MeO-NEK

As shown in Fig. 6, NENK, 2-MeO-NEK, 4-MeO-NEK, and Discussion KET decreased the immobility time of the mice in the FST (F (9, 70) = 10.23, p < 0.001). KS pretreatment blocked these In the present study, we examined the affinity of NENK, 2- effects, increasing the immobility time of mice treated with MeO-NEK, and 4-MeO-NEK for the NMDA receptors. We

Fig. 4 Effect of NBQX treatment (10 mg/kg) 15 min prior to S.E.M. *p < 0.05, **p < 0.01, and ***p < 0.001 are significant to the administration of NENK, 2-MeO-NEK, 4-MeO-NEK, or KET saline treatment, and #p <0.05, ##p <0.01,and ###p < 0.001 are (10 mg/kg) in mice during the FST. Treatment with NBQX inhibited significantly different to the saline + drug treatment (Bonferroni’spost- the reduction in immobility time induced by NENK, 2-MeO-NEK, 4- test) MeO-NEK, and KET; n = 8 animals per group. Values are mean ± Psychopharmacology (2019) 236:2201–2210 2207

Fig. 5 mRNA expression levels of (a, e)GluA1,(b, f)GluA2,(c, g) Significant differences in mRNA expression levels were observed after brain-derived neurotrophic factor (BDNF), and (d, h)mammaliantarget administration of the compounds; n = 5 animals per group. Values are of rapamycin (mTOR) in the mouse hippocampus and prefrontal cortex mean ± S.E.M. *p < 0.05, **p < 0.01, and ***p < 0.001 are significantly 30 min after NENK, 2-MeO-NEK, 4-MeO-NEK, or KET treatment. different to the saline treatment (Dunnett’s post-test) also determined the capacity of these novel compounds to MeO-NEK show considerably high affinity for the NMDA induce antidepressant effects in mice and explored their mech- receptors, while NENK displays lower affinity compared to anistic correlates. Here, we found that 2-MeO-NEK and 4- the earlier compounds.

Fig. 6 Effect on mice of ketanserin (KS) treatment (0.5 mg/kg) 15 min but not for KET; n = 8 animals per group. Values are mean ± S.E.M. prior to treatment with NENK, 2-MeO-NEK, 4-MeO-NEK, or KET *p <0.05,**p < 0.01, and ***p < 0.001 are significantly different from (10 mg/kg) during the FST. Treatment with KS inhibited the reduction saline treatment, and ##p <0.01and###p < 0.001 are significantly differ- in immobility time induced by NENK, 2-MeO-NEK, and 4-MeO-NEK, ent to the saline + drug treatment (Bonferroni’s post-test) 2208 Psychopharmacology (2019) 236:2201–2210

As shown in Fig. 3, NENK (5 and 10 mg/kg), 2-MeO-NEK inhibitor rapamycin blocks the antidepressant effects of KET (2.5, 5, and 10 mg/kg), and 4-MeO-NEK (2.5, 5, and (Zanos and Gould 2018; Duman et al. 2016;Lietal.2010). A 10 mg/kg) reduced the immobility time of mice in the FST single antidepressant dose of KET induces mTOR phosphor- and TST, similar to KET (10 mg/kg). This behavioral effect ylation in the prefrontal cortex and hippocampus in animals suggests that the novel MXE analogs exert rapid (Pazini et al. 2016; Jernigan et al. 2011). Furthermore, the antidepressant-like effects, in agreement with the results for KET-induced increases in mTOR and BDNF levels can be MXE and other NMDA receptor antagonists (Autry et al. attenuated by the AMPA receptor antagonist NBQX (Zhou 2011; Botanas et al. 2017). Furthermore, the compounds did et al. 2014). This information suggests that mTOR activation not alter the locomotor activity of mice, suggesting that the plays an important role in the AMPA receptor-mediated anti- observed antidepressant effects were not due to altered loco- depressant effects of KET. The present results show that motor activity (Browne and Lucki 2013). However, none of NENK, 2-MeO-NEK, 4-MeO-NEK, and KET increased the compounds altered the behavior of mice during the EPM, BDNF mRNA expression in the hippocampus, whereas only which was surprising considering these drugs are analogs of NENK and KET increased BDNF expression in the prefrontal MXE, which has been shown to induce anxiolytic-like effects cortex. Although KET treatment resulted in increased levels (Botanas et al. 2017). The reason for these results is unclear of mTOR mRNA in the hippocampus and prefrontal cortex, and requires further investigation. Nevertheless, the results are treatment with the three novel compounds did not. The reason consistent with the findings that KET did not evoke for this result is unclear, however, a possible explanation may anxiolytic-like effects in the EPM (Autry et al. 2011). Taken be that the compounds exert their antidepressant effects together, our findings suggest that NENK, 2-MeO-NEK, and through post-transcriptional modifications rather than chang- 4-MeO-NEK elicit fast-acting, antidepressant-like properties. ing gene expression. This is supported by previous findings Increasing evidence suggests that the stimulation of AMPA wherein KET did not alter mTOR gene expression levels but receptors is responsible for the rapid antidepressant effects of enhanced protein expression (du Jardin et al. 2016;Zhouetal. drugs targeting the glutamatergic system, such as NMDA re- 2014;Yangetal.2013). The inconsistent results between our ceptor antagonists (Botanas et al. 2017; Koike et al. 2011b). study and of du Jardin et al. (2016) regarding mTOR mRNA AMPA receptor potentiators are reportedly effective in behav- levels could be attributed to methodological differences ioral despair models of depression (Coppola and Mondola among studies (i.e., strain and euthanization time). 2012). Previous studies have shown that the antidepressant- Additional experiments are needed to examine the effects of like effects of KET are inhibited by NBQX in different animal NENK, 2-MeO-NEK, and 4-MeO-NEK on mTOR protein models of depression (e.g., FST, TST, and learned helpless- expression, as well as other genes or proteins (e.g., eEF2 and ness test) (Zanos et al. 2016; Koike et al. 2011a; Maeng et al. Trkß) that may have contributed to the antidepressant effects 2008). Consistent with this finding, our results show that of the compounds. Nevertheless, treatment with these novel NBQX treatment blocked the antidepressant-like effects of compounds resulted in increased levels of BDNF mRNA.

NENK, 2-MeO-NEK, and 4-MeO-NEK in the FST. It has been suggested that the 5-HT2 receptors are as- Furthermore, NENK and 4-MeO-NEK increased the expres- sociated with the pathophysiology of depressive syn- sion levels of GluA1 mRNA in the hippocampus, whereas 2- drome (Meyer et al. 2003), and activation of these recep- MeO-NEK increased GluA2 mRNA levels both in the hippo- tors has been implicated in the modulation of mood dis- campus and prefrontal cortex. This indicates that AMPA re- orders. Previous studies (Zomkowski et al. 2004) showed ceptor stimulation is necessary to elicit the antidepressant-like that DOI, a 5-HT2 agonist, enhanced the antidepressant effects of NENK, 2-MeO-NEK, and 4-MeO-NEK. effects of some compounds in the FST. In addition, KS,

BDNF is a protein vital for neuronal survival, morphogen- a5-HT2 antagonist, blocks the antidepressant effects of esis, and plasticity. Previous studies have suggested that MXE and other compounds like ferulic acid and berberine AMPA receptor activation induces the release of BDNF from (Botanas et al. 2017;Zenietal.2012; Fan et al. 2017). synaptic vesicles, increasing its level in the brain (Duman and Our results show that the antidepressant-like effects in- Vo le ti 2012). BDNF has been implicated in depressive syn- duced by NENK, 2-MeO-NEK, and 4-MeO-NEK in the drome, that is, reduced brain BDNF levels predispose to the FST were inhibited by KS pretreatment. This finding in- disorder, whereas increased brain BDNF levels induce an an- dicates the possible involvement of 5-HT2 receptor acti- tidepressant effect (Zhang et al. 2010). BDNF also activates vation in the antidepressant-like effects of NENK, 2- the mTOR signaling pathway, a serine/threonine protein ki- MeO-NEK, and 4-MeO-NEK, in addition to AMPA re- nase that acts as a central regulator of cell growth and survival ceptors. Consistent with our previous results (Botanas in response to stress signals (Yang et al. 2013). Activation of et al. 2017), KS did not block the antidepressant effects the mTOR pathway has been reported to contribute to the of KET in the FST, suggesting that the antidepressant antidepressant activity of KET (Zhou et al. 2014). effects of KET are independent of 5-HT2 receptor activa- Particularly, pre-administration with the selective mTOR tion. Taken together, in addition to the AMPA receptors, Psychopharmacology (2019) 236:2201–2210 2209 the antidepressant effects of NENK, 2-MeO-NEK, and 4- JH (2017) Evaluation of the abuse potential of novel amphetamine MeO-NEK are associated with the stimulation of 5-HT derivatives with modifications on the amine (NBNA) and phenyl 2 (EDA, PMEA, 2-APN) sites. Biomol Ther 25:578–585 receptors. du Jardin KG, Müller HK, Sanchez C, Wegener G, Elfving B (2016) A In conclusion, NENK, 2-MeO-NEK, and 4-MeO-NEK in- single dose of vortioxetine, but not ketamine or fluoxetine, increases duce antidepressant effects, similar to MXE and KET. The plasticity-related gene expression in the rat frontal cortex. 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