Discovery and Structure-Based Optimization of 6-Bromotryptamine Derivatives as Potential 5-HT2A Receptor Antagonists

5-Hydroxytryptamine type 2A (5-HT2A) receptor is an important target for developing innovative antipsychotic agents in neuropsychiatric disorder therapies. To search for 5-HT2A receptor antagonists, a new indole alkaloid termed 6-bromo-N-propionyltryptamine (1), together with one known homologue 6-bromo-N-acetyltryptamine (2) were isolated and identified from a marine bacterium Pseudoalteromonas rubra QD1-2. Compound 1 with an N-propionyl side chain exhibited stronger 5-HT2A receptor antagonist activity than that of N-acetyl derivative (2), indicating that 6-bromotryptamine analogues with a longer chain acyl group perhaps displayed a more potent capacity to the target. Therefore, a series of new 6-bromotryptamine analogues (3–7) with different chain length of the acyl group (C4–C8) were prepared and evaluated activity against 5-HT2A receptor. Remarkably, 6-bromo-N-hexanoyltryptamine (5) displayed the most effective inhibitory activity, which was 5-fold stronger than that of the parent compound 1 and showed 70% efficacy of the positive control (ketanserin tartrate).


Introduction
Serotonin or 5-hydroxytryptamine (5-HT) is a monoamine neurotransmitter that modulates a wide range of physiological and behavioral functions of the central nervous system (CNS), including cognition, mood, mating, feeding, and sleeping [1][2][3]. 5-HT receptors are members of the G-protein-coupled receptor superfamily, which are categorized into seven major families, 5-HT1−7. Among them, the 5-HT2 receptor subfamily contains three members, namely 5-HT2A, 5-HT2B, and 5-HT2C. Extensive studies have revealed that the 5-HT2A receptor played a critical role in the regulation of neuropsychiatric disorders associated with depression, Parkinson's and Alzheimer's disease [4][5][6][7]. Hence, a 5-HT2A receptor antagonist is therapeutically relevant for neurological disorders. For instance, M100907 with the 5-HT2A antagonist activity has entered onto the phase II clinical trial for the treatment of schizophrenia [8].
In our continuing program on the discovery of 5-HT2A receptor antagonists from marine bacterial culture broths, the EtOAc extract from Pseudoalteromonas rubra QD1-2 displayed promising activity. Bioassay-guided isolation procedures led to the obtaining of 6-bromo-N-propionyltryptamine (1) and 6-bromo-N-acetyltryptamine (2) (Figure 1). Intriguingly, the new compound (1) with N-propionyl side chain showed a higher inhibitory activity than that of N-acetyl derivative (2), thus revealing that a longer chain acyl group attached to 6-bromotryptamine probably possessed a better inhibition capacity to 5-HT2A receptor. In order to test this hypothesis, a new series of 6-bromotryptamine derivatives with a longer chain length than that of compounds 1 and 2, were synthesized (3-7, Scheme 1) and evaluated the preliminary structure-activity relationships (SAR). Consequently, compound 5 with N-hexanoyl side chain was identified as the most potent one in the series of 6-bromotryptamine scaffold. Herein, we report the isolation, identification, synthesis, and SAR of 6-bromotryptamine derivatives.

Structure Elucidation of 6-Bromotryptamine Derivatives 1-2
Compound 1 was obtained as a yellowish powder. The molecular formula was established as C13H15BrN2O by high-resolution electrospray ionization mass spectroscopy (HRESIMS), which gave equal-intensity ions at m/z 317.0269/319.0254 [M + Na] + , suggesting 1 contained one bromine atom.  (Table 1). Furthermore, these HMBC connectivities together with the COSY correlation of H-4/H-5, showed the location of the bromine atom at C-6, which was finally determined by comparison of the observed chemical shifts with those reported for 5 or 6-bromoindole-containing compounds [9,10]. In addition, the COSY correlation of H-10 (δH 2.91, t, J = 7.2 Hz)/H-11 (δH 3.44, t, J = 7.3 Hz) for 1 disclosed the presence of a pair of coupled methylene groups, which was attached to the 6-bromoindole moiety at C-3 due to the HMBC correlations of H-10/C-2 and C-3 and H-11/C-3, thus forming a 6-bromotryptamine moiety ( Figure 2). The remaining spectroscopic features of 1 indicated the presence of a propionyl group based on the COSY correlation of H-14 (δH 2.16, q, J = 7.6 Hz)/H-15 (δH 1.09, t, J = 7.7 Hz) and HMBC correlations of H-14 and H-15/C-13 ( Figure 2). Finally, the propionyl group was connected to 6-bromotryptamine moiety through an amide bond by the HMBC correlation of H-11/C-13. According to the aforementioned information, the structure of 1 was unambiguously assigned as 6-bromo-N-propionyltryptamine.
Compound 2 was a known synthetic compound but now isolated for the first time as a natural product. Its structure was determined as 6-bromo-N-acetyltryptamine, which was confirmed by comparing their spectral data with the reference reported [11].

Syntheses of 6-Bromotryptamine Derivatives 3-7
The synthetic route of 3-7 is depicted in Scheme 1, which was carried out by the condensation of 6-bromotryptamine and butyric acid, pentanoic acid, hexanoic acid, heptanoic acid and octanoic acid, respectively, using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and hydroxybenzotriazole (HOBt) in dichloromethane (DCM) solution at room temperature for 12 h [12][13][14]. Compounds 3-7 were synthesized for the first time in yields ranging from 79% to 85%, the structures of which were identified and characterized by ESI-MS and NMR spectroscopy .This method was proved to provide a rapid, and high yielding procedure to afford acyl chain derivatives of 6-bromotryptamine.

Structure-Activity Relationships
The 6-bromotryptamine derivatives (1-7) were evaluated at a concentration of 10 μM for their antagonist activity on 5-HT2A receptor detectable by calcium flux assay. The results (Figure 3) highlighted the importance of the length of the acyl chain in the 6-bromotryptamine derivatives for the antagonist activity. Interestingly, we observed that the activity gradually improved in response to increasing chain length (C2-C6), but a reverse tendency occurred when the chain length was C7 or C8. 6-Bromo-Nhexanoyltryptamine (5) thus exhibited an approximately 70% inhibition activity compared with the positive control, which was the most potency in the series. Notably, compound 5 displayed a roughly 4-fold higher inhibitory activity than the parent analogue 1. These observations might be explained by two mechanisms. A longer acyl side chain related to a higher lipophilicity, has the positive consequence in membrane permeation, probably eliciting more effective inhibitory potency for the target. However, the bulk chain (when > C6) caused an acyl group-dependent decrease in the binding affinity, and likely reduced the inhibitory potency for 5-HT2A receptor [15].

General Experimental Procedures
Preparative HPLC equipment was carried out on a Waters 600 pump equipped with a Waters model 2996 diode array detector and Waters Empower System (Waters Co., Milford, CT, USA). HRESIMS analysis was recorded using a triple quadrupole coupled with time-of-flight mass spectrometry with an ESI interface (Waters Co.). ESI-MS analysis was analyzed using a triple quadrupoles mass spectrometer (Thermo, San Jose, CA, USA). NMR experiments were performed using Bruker AVANCE 400 and 500 MHz NMR spectrometers (Bruker, Karlsruhe, Germany). A Sun-Fire C18 column (20 × 250 mm, ID. 5 μm) was used for reverse-phase separations. All solvents were HPLC grade (Shanghai ANPEL Scientific Instrument, Shanghai, China). 6-Bromotryptamine was purchased from Lianchuang Company (Anhui Lianchuang Pharmceutical Chemistry Co., Ltd., Hefei, China). All aliphatic acids, N- (3-(dimethylamino) propyl)-N-ethylcarbodiimide hydrochloride (EDC·HCl), and hydroxybenzotriazole hydrate (HOBt) were purchased from Aldrich Chemicals Company (Milwaukee, WI, USA). Calcium flux assay was performed on Fluorescence Image Plate Reader (Molecular Devices, San Francisco, CA, USA) with a FLIPR calcium assay kit. Ketanserin tartrate was purchased from Tocris Cookson Inc. (Ellisville, MO, USA). CHO-K1 (Chinese hamster ovary) was purchased from GenScript Corporation (Piscataway, NJ, USA). . All values are expressed as means ± SD (n = 3) and * indicates significant at p < 0.05 relative to compound 2. Ketanserin tartrate was used as positive control.

Bacterial Material and Fermentation
The strain P. rubra QD1-2 was isolated from a seawater sample collected from Qingdao Sea area in China, which was stored in China General Microbiological Culture Collection Center (CGMCC, No. 6555). The fermentation was implemented in two steps: seed growth and scale-up fermentation. For the seed growth stage, P. rubra QD1-2 from a plate culture was inoculated into 100 mL of the basic seed medium (peptone 1 g/L, yeast extract 2.5 g/L, glucose 0.1 g/L dissolved in seawater, pH 7.0) in a 500 mL Erlenmeyer flask and grown in a shaker incubator at 25 °C and 150 rpm for 24 h. Then, seed cultures were inoculated into 1 L Erlenmeyer flasks containing 400 mL of fermentation medium (peptone 1 g/L, yeast extract 2.5 g/L, glucose 0.1 g/L dissolved in seawater, pH 7.0). The strain was fermented on rotary shakers at 25 °C with 150 rpm for 7 days.

Extraction and Isolation
A total of the 20 L fermentation broth was harvested and filtered by centrifuge and extracted with EtOAc three times. The extract was concentrated under reduced pressure on a rotary evaporator at 37 °C, which was combined to obtain 1.1 g of crude extract. The extract was directly purified by preparative-HPLC with an isocratic elution of 65% MeOH/H2O (Flow 8 mL/min, UV detection at 280 nm) to afford compounds 1 (12 mg) and 2 (14 mg).

Antagonist Activity
All the tested compounds were prepared as follows and stored at −20 °C. Compounds 1-7 and the positive control were tested at 10 μM in HBSS buffer (with 20 mM HEPES buffer, pH 7.4) with 0.1% DMSO. Ketanserin tartrate was employed as positive control. According to the previous method, the inhibitory activity was performed in CHO-K1 (Chinese hamster ovary) cells where recombinant human 5-HT2A receptor was expressed, measured by the calcium flux assay [16,17]. The experiments were carried out in triplicates.

Conclusions
In this paper, two 6-bromotryptamine derivatives with the 5-HT2A receptor antagonist activity, including a new 6-bromo-N-propionyltryptamine (1) and a known 6-bromo-N-acetyltryptamine (2) were isolated and characterized from the culture broth of a marine-derived P. rubra QD1-2. Moreover, we synthesized a series of new 6-bromotryptamine congeners (3-7) with different chain lengths (C4-C8) and evaluated their antagonist activity for 5-HT2A receptor. Notably, the preliminary SAR studies indicated that the length of the acyl chain dramatically influenced the antagonist activity of 6-bromotryptamine derivatives. Their antagonist activity increased with elongation of the side chain acyl group (C2-C6), while the activity of compounds 6 and 7 with the side chain longer than that of 6-bromo-Nhexanoyltryptamine (C6) significantly declined or disappeared. As the most potent one in this series, 6-bromo-N-hexanoyltryptamine (5) displayed roughly 4-fold activity enhancement over the parent analogue 1, which possessed an approximately 70% inhibition activity comparable to the positive control. Overall, 6-bromo-N-hexanoyltryptamine (5) with a sound antagonist activity for 5-HT2A receptor was identified and our work provides an insight into the further structural optimization of 6-bromotryptamine derivatives for developing potential therapeutic agents to treat neurological disorders.