Accepted Manuscript Synthesis and biological evaluation of 4-nitroindole derivatives as 5-HT2A receptor antagonists Faisal Hayat, Ambily Nath Indu Viswanath, Ae Nim Pae, Hyewhon Rhim, WooKyu Park, Hea-Young Park Choo PII: DOI: Reference:

S0968-0896(15)00048-6 http://dx.doi.org/10.1016/j.bmc.2015.01.032 BMC 12035

To appear in:

Bioorganic & Medicinal Chemistry

Received Date: Revised Date: Accepted Date:

17 November 2014 18 January 2015 19 January 2015

Please cite this article as: Hayat, F., Nath Indu Viswanath, A., Nim Pae, A., Rhim, H., Park, W-K., Park Choo, HY., Synthesis and biological evaluation of 4-nitroindole derivatives as 5-HT2A receptor antagonists, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.01.032

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Synthesis and biological evaluation of 4-nitroindole derivatives as 5-HT2A receptor antagonists Faisal Hayat a, Ambily Nath Indu Viswanath b, d, Ae Nim Pae b, d, Hyewhon Rhim b, Woo-Kyu Parkc, Hea-Young Park Choo a,* a

College of Pharmacy & Division of Life & Pharmaceutical Sciences, Ewha Womans University, Seoul 120-750, Republic of Korea b Center for Neuroscience, Korea Institute of Science & Technology, PO Box 131, Cheongryang, Seoul 130-650, Republic of Korea c

Pharmaceutical Screening Research Team, Korea Research Institute of Chemical Technology, Daejeon 305-343, Republic of Korea d

Department of Biological Chemistry, School of Science, Korea University of Science and Technology, 52 Eoeun Dong, Yuseong-Gu, 305-333 Daejeon, Republic of Korea

Corresponding author: Hea-Young Park Choo, Tel: +82-2-3277-3042;

Fax: +82- 2 -3277- 2821; e-mail: [email protected]

Abstract: A

novel

series

of

4-nitroindole

sulfonamides

containing

a

methyleneamino-N,N-

dimethylformamidine were prepared. The binding of these compounds to 5-HT2A and 5-HT2C was evaluated, and most of the compounds showed IC50 values of less than 1µM, and exhibited high selectivity for the 5-HT2C receptor. However, little selectivity was observed in the functional assay for 5-HT6 receptors. The computational modeling studies further validated the biological results and also demonstrated a reasonable correlation between the activity of compounds and the mode of superimposition with specified pharmacophoric features.

Key-words: 4-nitroindole, sulfonamides, methyleneamino-N,N-dimethylformamidine group, 5 HT2A, 5 HT2C, 5 HT6 receptor

1. Introduction Serotonin 5-HT2 subfamily are coupled to Gq/G11, increase the activity of phospholipase C and/or phospholipase, activate the second messenger cascade and mediate excitatory neurotransmission.1 5-HT2 receptor subtypes are involved in the regulation of brain function, therefore they are therapeutic targets for obesity, sleep, memory, addiction or psychiatric disorders such as schizophrenia, anxiety and depression. 2-5 Among 5-HT2 receptor subtypes (5HT2A, 5-HT2B, and 5-HT2C), 5-HT2A is the main excitatory receptor subtype among the GPCRs for 5-HT, although 5-HT2A may also have an inhibitory effect on certain areas such as the orbitofrontal cortex and the visual cortex.6 5-HT2A receptors are mainly expressed in pyramidal glutamatergic neurons at the frontal cortex,

hippocampus, and inhibitory GABAnergic

interneuron, all of which play an important role in schizophrenia.7-9 Most of antipsychotics, such as clozapine, risperidone, and arypiprazole, are strong blocking agents for 5-HT2A receptor. 5HT2A receptor is also related to the mood control. Antidepressants such as trazodone and nefazodone work as antidepressant by 5-HT2A antagonism.10 The 5-HT2C receptor is present in the choroid plexus, hypothalalmus, basal ganglia and parts of the limbic system.11,12 The 5-HT2C receptor has similar characteristics to the 5-HT2A receptor with respect to pharmacology and signal transduction. Antagonism of 5-HT2C receptors is also useful for improving cortical function. Generally the drugs with a high affinity for 5-HT2A also show a relatively high affinity for 5-HT2C. Therefore, it would be quite reasonable to anticipate that antipsychotic drugs acting on both 5-HT2A and 5-HT2C receptors be more effective than drugs acting on only one receptor. Recently, we have reported a novel series of 5-HT2A ligands that contain a (phenylpiperazinylpropyl)arylsulfonamide skeleton.13,14 For example, N-(Cyclohexylmethyl)-N-(3-(4-phenyl piperazin-1-yl)propyl)naphthalene-2-sulfonamide showed good affinity at 5-HT2A (IC50 = 0.7 nM ) and good selectivity over 5-HT2C (ca. 100 times) and 5-HT7 (ca. 1800 times). Also, we have previously reported three different series of 5-HT6 receptor antagonists in which the central aromatic core are either the benzoisoxazole or benzothiazole ring structures. 15-17 The compounds include N,N-dimethylformimiamide or piperazine as the positive ionizable groups. Compounds such as (2-Methyl-1-(naphthalensulfonyl)-1H-indol-3-yl)-N-(4-methylpiperazin-1-

yl)methanimine (IC50 value of 2.04 µM), 2-(4-Methylpiperazin-1-yl)-6-(1-naphthalenyl)sulfon amidobenzo[d]thiazol (IC50 value of 3.9 µM), N,N-dimethyl-N’-(5-(4-methylphenylsulfonamido) benzo-[d]isothiazol-3-yl)formimidamide (IC50 value of 0.36 µM) showed good 5-HT6 inhibitory activity as shown in Fig 1. As a part of our continuous effort to find 5-HT ligands, here we have synthesized a series of 4-nitroindole sulfonamides and evaluated their affinity to 5-HT2A, 5-HT2C, and 5-HT6 receptors. Prompted by the previously reported works, we synthesized a novel series of antagonists that have an indole central aromatic core and a methyleneamino-N, Ndimethylformamidine group .

Figure 1.

2. Result and Discussion 2.1. Chemistry The synthesis of a novel series of 5-HT receptor antagonists was performed in a manner as outlined in Scheme 1. Initially, the coupling of 2-methylindole-3-carboxaldehyde (1 mmol) and hydrazine hydrate (1.5 mmol) was chosen as a model reaction to establish the optimized reaction conditions. All reactions were performed in the presence of various solvents using two to three drops of acetic acid as a catalyst at different temperature ranges under anhydrous conditions. The desired product was isolated in 70% yields when the reaction was carried out using anhydrous ethanol at reflux temperature for 2 h. In the second step, the desired product (1E)-N'-((4-nitro1H-indol-3-yl) methyleneamino)-N, N-dimethylformamidine was synthesized from (Z)-1-((4nitro-1H-indol-3-yl)methylene) hydrazine according to the reported procedure18,19 in good yield. For the final product synthesis, three test reactions were performed in three different solvents (DCM, THF and DMF) over a range of temperatures by using Et3 N (1.5–3.0 mmol) or NaH (1.2–1.5 mmol) as bases. After 24 h of stirring at room temperature, TLC did not progress in the first test reaction using DCM as a solvent and Et3N as a base. In two other test reactions with DMF and THF as the solvents and NaH as the base, TLC showed little progress in the reactions. Heating the reaction mixture up to the reflux temperature failed to provide the desired products.

Finally the reaction was successful when the reaction was run in the anhydrous THF solvent employing KH as a base. The reactions were completed within 13–15 h. Each synthesized compound was characterized by 1H NMR and HRMS.

Scheme1.

2.2 Biological evaluation All the synthesized compounds were evaluated in vitro against the human recombinant 5-HT2A serotonin receptor in a stable CHO cell line.20 [3H]Ketanserin binding assay results are shown in Table 1. Most compounds showed good binding affinity to the 5-HT2A receptor with IC50 values ranging from 0.1 – 1.6 µM. The most active compound 14 had an IC50 value of 100 nM, which has the 2,3-dihydro-1,4-benzodioxinyl group on the sulfonyl moiety. The methoxypheny sulfonamides (compounds 9 and 10) showed good activity, with the IC50 values of 0.205 and 0.479 µM, respectively. The synthesized compounds were further evaluated for their selectivity on the 5-HT2A receptor over the 5-HT2C receptor (Table 1). Only compounds 13, 7 and 9 showed affinity to 5-HT2C, with IC50 values of 0.463, 0.480 and 1.040 µM, respectively. In vitro evaluation of the synthesized compounds against the human recombinant 5-HT6 serotonin receptor was also performed.21 The functional efficacy of each compound was evaluated by measuring the 5-HT-induced Ca2+ increases using a HeLa cell line expressing the cloned human 5-HT6 receptor (Table 2). Most of the synthesized compounds significantly inhibited the 5-HT-induced Ca2+ increases (9; IC50 = 0.74 µM, 10; IC50 = 1.40 µM, and 14; IC50= 0.17 µΜ) compared to other derivatives, indicating that compounds 9, 10 and 14 are potent 5-HT6 receptor antagonists, too. 2.3. Pharmacophore Mapping The statistical validation of our compounds has been carried out by the generation and subsequent mapping of a common feature pharmacophore model with active and inactive compounds. The mapping study derived satisfactory correlation between the fit value and

activity of compounds. The qualitative model generated from 5-HT2A ligands (termed as Hypo1; Figure 3A) consisted of two aromatic rings (AR), one positive ionizable (PI), and one hydrophobic (HYD) features. The feature disposition in the pharmacophore was nearly equal to the proposed model with specified distance constraints.22 The mapping of Hypo1 with the most active compound 14 and the inactive compound 8 showed fit values of 2.84 and 2.45, respectively. In 14 (Figure 3B), the benzene ring of the indole occupied one AR feature and the phenyl group of dihydrobenzodioxine superimposed with the second AR and HYD features. The PI element, although omitted from mapping, was positioned close to the nitrogen atom of the methyleneamino group. Compound 8 (Figure 3C) showed good fitting of HYD and AR features with the indole and butyl phenyl groups. The orientation of PI was on the opposite side of methyleneamino-N, N-dimethylformamidine, and also excluded from mapping. Since the biological evaluation has revealed the inhibitory potential of 4-nitroindole compounds towards 5-HT6 receptor, here we discussed the mapping pattern of chemical features in 14 and 8 with the pharmacophoric groups in a 5-HT6-based 3D model. Composed of two AR, two HBA, one PI and one HYD features, the six-point hypothesis (referred to as Hypo2) (Figure 4A) derived from 5-HT6 compounds, showed reasonable mapping with 14 and 8, resulting in fit values of 3.83 and 2.25, respectively. In 14 (Figure 4B), the dihydrobenzodioxine scaffold fitted with AR and HYD and the phenyl ring in indole superimposed over the second AR. The two HBA features found to locate on the sulfonyl group. Whereas in 8 (Figure 4C), HYD, AR and HBA showed poor merging with butyl phenyl, indole, and sulfonyl groups. Similar to 5-HT2A, here too, the omitted feature was PI.

Table 1. Table 2.

2.4. Discussion The 5-HT2A, 5-HT2B, and 5-HT2C receptors are involved in the regulation of a variety of physiological brain functions.23 Therefore, they are therapeutic targets for obesity, sleep,

memory, addiction and psychiatric disorders such as schizophrenia, anxiety, and depression.24-27 Pharmacogenetic studies in human have reported that one of the most investigated polymorphisms within the 5-HT2A gene A-1438 is associated with the antidepressant response. 28 Regarding the 5-HT2C receptor, the Cys23ser polymorphism is associated with the changes in function, and is found to influence the vulnerability to affective disorders.29,30 However whether 5-HT2C receptor activation or inactivation is required to produce an antidepressant-like response in preclinical studies remains controversial. Evidence also suggests the involvement of the 5HT2C receptor in anxiety. Recently, the 5-hydroxytryptamine6 (5-HT6) receptor has emerged as a promising molecular target which interacts with several drugs that act on the central nervous system. The 5-HT6 receptor is distributed mainly in the central nervous system, such as the stratum, nucleus accumbens, olfactory tubercle, and cortex, with moderate expression in the hippocampus, amygdala, cerebellum, and thalamus.31 5-HT6 antagonists improve cognition, learning, and memory, and agents such as latrepirdine are being developed as novel treatments for Alzheimer's disease and other forms of dementia. The 5-HT6 receptor has become an attractive target for the development of new medicines to treat different diseases and impairments of the central nervous system. A large number of publications in scientific journals as well as patents covering new inhibitors of the 5-HT6 receptors

32-35

reflects the the

pharmaceutical research community’s interest. Therefore a series of 4-nitroindoles were added to our continuous study to learn more details of 5-HT2A and 5-HT6 ligands. All the compounds were evaluated in vitro against the human recombinant 5-HT2A, 5-HT2C, and 5-HT6 serotonin receptors in a stable CHO cell line. Most of prepared compounds showed good binding affinity to the 5-HT2A receptor with IC50 values ranging from 0.1 – 1.6 µM, but only a few compounds demonstrated binding affinity for 5-HT2C receptors. The most active compound 14, which has a 2, 3-dihydro-1,4-benzodioxinyl sulfonyl amide moiety, had

an IC50 value of 0.1 µM. The methoxypheny sulfonamide containing

compounds 9 and 10 showed good activity with IC50 values of 0.2 µM. The 1- naphtyl, or 2naphtyl sulfonamides (12 or 13) showed good affinity with IC50 values of ca. 0.5 µM, but 5dimethylamino-,1-naphtyl ,or 1-napthyl ethyl sulfonamides showed no affinity.(data not shown) The bulky group-substituted sulfonamides, such as 4-butylphenyl (8) and 2,2,4,6,7-pentamethyl5-dihydrobenzofuryl sulfonamides, showed low binding affinity. (data not shown) The halogen substituted phenyl sulfonamides 5 and 6 showed fairly good affinity with IC50 values of 0.702

and 0.247 µM, respectively.

When the compounds were further evaluated for their selectivity on the 5-HT2A receptor over the 5-HT2C receptor (Table 1), only 2-naphthyl, 4-methylphenyl, and 4-methoxyphenyl sulfonamides (7, 9, and 13) showed affinity for 5-HT2C receptor (IC50 values 0.480, 1.040, and 0.463 µM respectively). Therefore, a different structural requirement is necessary for 5-HT2C antagonism.

Also, the synthesized compounds were evaluated in vitro against the human recombinant 5-HT6 serotonin receptor. The functional efficacy of each compound was evaluated by measuring the 5HT-induced Ca2+ increases using a HeLa cell line expressing the cloned human 5-HT6 receptor (Table 2). Most of prepared compounds significantly inhibited the 5-HT-induced Ca2+ increases with IC50 values ranging from 0.1 -6.3 µM. The three compounds (9; IC50 = 0.74 µM, 10; IC50 = 1.4 µM, and 14: IC50 = 0.1 µΜ) were more active than the other derivatives, indicating that compounds 9, 10, and 14 are potent 5-HT6 receptor antagonists. However, these compounds showed a high affinity for the 5-HT2A receptor, demonstrating little selectivity over the 5-HT2A receptor. Some 5HT6 receptor antagonists, such as MS-254, SAX-187, E-6837, SAM-532, and MS-245, have an indole, or indole like ring as the central aromatic core, and have showed promising biological activity. They are based on its pharmacophore model 36-38, which includes a central aromatic or heterocyclic scaffold (AR), separating a positive ionizable group (PI: usually, piperazine, methyl piperidine or N,N-dimethyl ethyl group) 23,39, from a strong multiple hydrogen bond acceptor group (mHBA: usually sulfonyl or sulfamide group)

23,39

, and a

hydrophobic site (HYD). There are also some known essential pharmacophores of 5-HT2A receptors for antagonism, which are two aryl rings and a basic nitrogen. It is necessary for geometric characteristics that the distance between two aromatic rings is 4.6-7.3Å and the distances between each aromatic ring and the basic amine nitrogen are 5.2-8.4Å and 5.7-8.5Å.40 In this novel 4-nitroindole series, methyleneamino-N, N-dimethylformamidine system was employed as a positive ionizable group. The prepared compounds have a methyleneamino-N,N-dimethylformamidine system which provides multiple nitrogens, and was expected to act as a PI anchoring group for receptor binding. However it failed to map with the dimethylamino nitrogen in the subsequent mapping studies.

Since PI was proposed as an essential feature for 5-HT2A antagonistic activity, omission in the present analysis does not provide a high correlation with the activity profile of the compounds. The details of the computational modeling studies are discussed further. The pharmacophore mapping demonstrated that all prepared compounds showed fit values of < 5.0 for both 5-HT2A- and 5-HT6 antagonists-based hypotheses. The fitting of Hypo1 features with the most active compound 14 and the inactive compound 8 showed fit values of 2.84 and 2.45, respectively, for 5-HT2A. Likewise, for 5-HT6, the Hypo 2-directed mapping with 14 and 8 resulted in fit values of 3.83 and 2.25, respectively. In both instances, the key PI element was omitted from mapping, however, it was positioned adjacent to the nitrogen atom of the methyleneamino group of 14 in the first mapped conformation. The 5-HT2A receptor antagonists possess diverse chemotypes, hence, a comprehensive model capable of describing all the binding characteristics has not yet been formulated. However, the minimal specifications for the activity were suggested as two AR centers and a PI anchoring group 22. In a striking contradiction to this, Ladduwahetty et al41 have reported that a basic nitrogen is not an essential requirement for 5HT2A activity. Our mapping analysis showed modest concordance with these propositions. The chemical features in 14 were compatible with both 5-HT2A and 5-HT6 ligand-based pharmacophores. The methyleneamino-N,N-dimethylformamidine was introduced as a strong cationic interaction center, but the modeling data implied that it is not a favoring factor for PI mapping. This could be due to the increased flexibility added to the structure by the aliphatic chain. Nevertheless, further studies are needed to resolve the uncertainties regarding PI’s role and to redefine the prototypical 5-HT2A pharmacophoric elements.

Figure 2. Figure 3. Figure 4.

3. Conclusion A novel series of 4-nitroindoles was synthesized as 5-HT2A receptor antagonists with methyleneamino-N, N-dimethylformamidine group present. The compounds showed selectivity over the 5-HT2C receptor, but displayed little selectivity over the 5-HT6 receptor. Further studies to profile these compounds, including in vivo experiments, are in progress. Finally, the modeling studies using 5-HT2A and 5-HT6 receptor antagonists-based pharmacophores showed that the merging of key structural components with essential pharmacophoric elements matches the activity data.

4. Experimental 4.1 General remarks All melting points of the synthesized compounds were taken in Pyrex capillaries using an electrothermal digital melting point apparatus (Buchi) and were not corrected. 1H NMR spectra were recorded on a 400 MHz Varian FT-NMR using tetramethylsilane as an internal standard. Chemical shifts are expressed in ppm (d), and peaks are listed as singlet (s), doublet (d), triplet (t), quintet (q), multiplet (m), with coupling constants (J) expressed in Hertz. Mass spectra data were obtained on an Agilent 6220 Accerate Mass TOF LC/MS. Most of the reagents were purchased from Aldrich Chemical Company and Merck Company. TLC was run on the silica gel coated plastic sheets (Silica Gel 60, 230–400 mesh, Merck, Germany) and visualized in UV light (254 or 365 nm).

4.2 Preparation of (Z)-1-((4-nitro-1H-indol-3-yl) methylene)hydrazine (2): A mixture of substituted 4-nitroindole 3-carboxaldehydes (1 mmol) and hydrazine hydrate (1.5 mmol) in 10 mL dry ethanol was heated to reflux under N2 until no starting material could be

detected by TLC. The resulting mixture was allowed to cool at room temperature and poured into water and extracted with EtOAc. The combined organic layers were washed with saturated aqueous NaHCO3 and brine solution and dried over anhydrous Na2SO4. The solvent was removed in vacuo and the crude product was purified by column chromatography using hexane: ethyl acetate (7:3) as an eluent. Yellow solid (70%), mp>250℃, 1H NMR (DMSO-D6, 400 MHz) δ 12.463(s, 1H), 8.662(s, 1H), 8.250(s, 1H), 7.784-7.915(m, 3H), 7.339-7.389(m, 2H), HR-FABMS Calcd for C9H9N4O2 (M++H): 205.0720, Found: 205.0723

4.3.(1E)-N'-((4-nitro-1H-indol-3-yl)methyleneamino)-N,N-dimethylformamidine (3) To a solution of (Z)-1-((4-nitro-1H-indol-3-yl)methylene)hydrazine (1 mmol), DMF DMA (4 mmol) in 5 mL dry methanol was stirred at rt for 30 minutes. After stirring, the solvent was removed in vacuo. The crude product was collected and dried at room temperature for 1 hour. Orange solid (54%), mp>250℃, 1H NMR (DMSO-D6, 400 MHz) δ 12.200(s, 1H), 8.512(s, 1H), 8.032(d, J=2.0Hz, 2H), 7.778-7.870(m, 2H), 7.247-7.316(m, 1H), HR-FABMS Calcd for C12H14N5O2 (M++H):260.1142, Found: 260.1142

4.4.General procedure for the synthesis of (1E)-N'-((Arylsulfonyl-4-nitro-1H-indol-3yl)methyleneamino)-N,N-dimethylformamidine derivatives (4-14) (1E)-N'-((Arylsulfonyl-4-nitro-1H-indol-3-yl)methyleneamino)-N,N-dimethylformamidine

(1

mmol) was dissolved in 5 mL THF/DMF, was and added slowly to 25 mL flask containing a suspension of potassium hydride (1.17 mmol) in 3 mL THF under nitrogen atmosphere, while maintaining the mass temperature below 10 oC. The reaction mixture was then stirred for a period of 1 h at 25 oC. Ar-sulfonyl chloride (1 mmol) was added slowly to the mixture, and well stirred solution while maintaining the mass temperature below 10 oC. The reaction mixture was further stirred overnight. After the reaction was complete, the reaction mixture was poured on to ice water and extracted with ethyl acetate (3 x 20 mL). The combined ethyl acetate extracts were

then washed with water (20 mL), brine (20 mL) and dried over anhydrous sodium sulfate. The volatiles were removed under reduced pressure and the resulting mixture was purified over a silica gel column using hexane: ethyl acetate as an eluent. 4.4.1.

(1E)-N'-((1-phenylsulfonyl-4-nitro-1H-indol-3-yl)methyleneamino)-N,N-

dimethylformamidine (4) Brown oil (15%) : 1H NMR (chloroform-d6,400 MHz) δ 8.50 (s, 1H), 8.29 (d, J = 7.6 Hz, 1H), 8.14 (s, 1H), 8.06 (s, 1H), 7.92-7.90 (m, 2H), 7.84-7.82 (m, 1H), 7.62-7.58 (m, 1H), 7.51-7.47 (m, 2H), 7.41 (t, J = 8.0 Hz, 1H), 3.01 (s, 6H). HR-EI HR-EI Calcd for C18H17N5O4S (M+H)+: 400.1074 found: 400.1068.

4.4.2.

(1E)-N'-((1-4-fluorophenylsulfonyl-4-nitro-1H-indol-3-yl)methyleneamino)-N,N-

dimethylformamidine (5) Orange solid (43%), mp 72–75 oC: 1H NMR (chloroform-d6,400 MHz) δ 8.49 (s, 1H), 8.27(d, J = 7.6 Hz, 1H), 8.11 (s, 1H), 8.06 (s, 1H), 7.96-7.92 (m, 2H), 7.84 (d, J = 8.2 Hz, 1H), 7.43 (t, J = 8.2 Hz, 1H), 7.18-7.14 (m, 2H), 3.01(s, 6H). HR-EI HR-EI Calcd for C18H16N5O4SF (M+H)+: 418.098 found: 418.0992. 4.4.3. (1E)-N'-((1-4-Iodophenylsulfonyl-4-nitro-1H-indol-3-yl)methyleneamino)-N,Ndimethylformamidine (6) Yellow solid (22%), mp 62–68 oC: 1H NMR (chloroform-d6,400 MHz) δ 8.49 (s, 1H), 8.25 (d, J = 7.6 Hz,1H), 8.102 (s, 1H),8.06 (s, 1H), 7.86-7.82 (m, 3H), 7.59 (d, J = 8.8 Hz, 2H), 7.42 (t, J = 8.2 Hz, 1H), 3.01(s, 6H). HR-EI HR-EI Calcd for C18H16N5O4SI (M+H)+: 526.004 found: 526.0031. 4.4.4. (1E)-N'-((1-4-methylphenylsulfonyl-4-nitro-1H-indol-3-yl)methyleneamino)-N,Ndimethylformamidine (7) Brown solid (32%), mp 145–146 oC: 1H NMR (chloroform-d6,400 MHz) δ 8.51 (s, 1H), 8.28 (d, J = 7.6 Hz,1H), 8.13 (s, 1H), 8.06 (s, 1H), 7.83-7.77 (m, 3H), 7.40 (t, J = 8.2 Hz, 1H), 7.27-7.25

(m, 2H), 3.01(s, 6H), 2.37 (s, 3H). HR-EI HR-EI Calcd for C19H19N5O4S (M+H)+: 414.1231 found: 414.1226. 4.4.5. (1E)-N'-((1-4-Butylphenylsulfonyl-4-nitro-1H-indol-3-yl)methyleneamino)-N,Ndimethylformamidine (8) Yellow solid (57%), mp 105–107 oC: 1H NMR (chloroform-d6,400 MHz) δ 8.51 (s, 1H), 8.29 (d, J = 7.6 Hz, 1H), 8.13 (s, 1H), 8.06 (s, 1H), 7.83-7.79 (m, 3H), 7.40 (t, J = 8.2 Hz, 1H), 7.27 (d, J = 7.6 Hz, 2H), 3.00 (s, 6H), 2.61 (t, J = 8.0 Hz, 2H), 1.56-1.52 (m, 2H), 1.33-1.24 (m, 2H), 0.88 (t, J = 7.4 Hz, 3H). HR-EI HR-EI Calcd for C22H25N5O4S (M+H)+: 456.1700 found: 456.1702. 4.4.6. (1E)-N'-((1-4-methoxyphenylsulfonyl-4-nitro-1H-indol-3-yl)methyleneamino)-N,Ndimethylformamidine (9) Yellowsolid (64%), mp 135–138 oC: 1H NMR (chloroform-d6,400 MHz) δ 8.51 (s, 1H), 8.27 (d, J = 7.2 Hz, 1H), 8.13 (s, 1H), 8.06 (s, 1H), 7.86-7.81 (m, 3H), 7.40 (t, J = 8.2 Hz, 1H), 6.91 (t, J = 9.2 Hz, 2H), 3.81 (s, 3H), 3.01 (s, 6H). HR-EI HR-EI Calcd for C19H19N5O5S (M+H)+: 430.1180 found: 430.1174. 4.4.7. (1E)-N'-((1-2,5-dimethoxyphenylsulfonyl-4-nitro-1H-indol-3-yl)methyleneamino)N,N-dimethylformamidine (10) Orange solid (71%), mp 215–220 oC: 1H NMR (chloroform-d6,400 MHz) δ 8.56 (s, 1H), 8.19 (s, 1H), 8.09 (d, J = 7.6 Hz, 1H), 8.06 (s, 1H), 7.80 (d, J = 6.8 Hz, 1H), 7.66 (s, 1H), 7.33 (t, J = 8.2 Hz, 1H), 7.09 (dd, J = 3.2 Hz, 9.2 Hz, 1H), 6.82 (d, J = 8.8 Hz, 1H), 3.85 (s, 3H), 3.63 (s, 3H), 3.01 (s, 6H). HR-EI HR-EI Calcd for C20H21N5O6S (M+H)+: 460.1285 found: 460.1286. 4.4.8.

(1E)-N'-((1-5-chloro-2-methoxyphenylsulfonyl-4-nitro-1H-indol-3yl)methyleneamino)-N,N-dimethylformamidine (11)

Orange solid (54%), mp 227–230 oC: 1H NMR (chloroform-d6,400 MHz) δ 8.53 (s, 1H), 8.17 (s, 1H), 8.13 (s, 1H), 8.09 (d, J = 7.6 Hz, 1H), 8.06 (s, 1H), 7.82 (d, J = 6.8 Hz, 1H), 7.55 (dd, J = 2.6 Hz, 12Hz, 1H), 7.37 (t, J = 8.0 Hz, 1H), 6.83 (d, J = 9.2 Hz, 1H), 3.69 (s, 3H), 3.01 (s, 6H). HR-EI HR-EI Calcd for C19H18N5O5SCl (M+H)+: 464.0790 found: 464.0792.

4.4.9. (1E)-N'-((1-napthylsulfonyl-4-nitro-1H-indol-3-yl)methyleneamino)-N,Ndimethylformamidine (12) Orange solid (20%), mp 158–160 oC: 1H NMR (chloroform-d6,400 MHz) δ 8.63 (d, J = 8.0 Hz, 1H), 8.52 (s, 1H), 8.32 (s, 1H), 8.28 (d, J = 6.4 Hz,1H), 8.12-8.09 (m, 2H), 8.07 (s, 1H), 7.91 (d, J = 8.0 Hz, 1H), 7.78 (t, J = 7.2 Hz, 1H), 7.69-7.64 (m, 1H), 7.60-7.55 (m, 2H), 7.33 (t, J = 8.2 Hz, 1H), 3.01(s, 6H). HR-EI HR-EI Calcd for C22H19N5O4S (M+H)+: 450.1231 found: 450.1234. 4.4.10. (1E)-N'-((1-(2-naphtyl)sulfonyl-4-nitro-1H-indol-3-yl)methyleneamino)-N,Ndimethylformamidine (13) Orange solid (35%), mp 93–97 oC: 1H NMR (chloroform-d6,400 MHz) δ 8.56 (s, 1H), 8.51 (s, 1H), 8.35 (d, J = 7.6 Hz,1H), 8.22 (s, 1H),8.07 (s, 1H), 7.98 (d, J = 6.4 Hz, 1H), 7.90-7.75 (m, 4H), 7.68-7.60 (m, 2H), 7.40 (t, J = 6.6 Hz, 1H), 3.01(s, 6H). HR-EI HR-EI Calcd for C22H19N5O4S (M+H)+: 450.1231 found: 450.1239. 4.4.11. (1E)-N'-((1-2,3-dihydro-1,4-benzodioxansulfonyl-4-nitro-1H-indol-3yl)methyleneamino)-N,N-dimethylformamidine (14) Orange solid (58%), mp 180–183 oC: 1H NMR (chloroform-d6,400 MHz) δ 8.51 (s, 1H), 8.28 (d, J = 7.6 Hz, 1H), 8.08 (d, J = 15.2 Hz, 2H), 7.82 (d, J = 6.8 Hz, 1H), 7.43-7.39 (m, 3H), 6.89 (d, J = 8.8 Hz, 1H), 4.28-4.22 (m, 4H), 3.00 (s, 6H). HR-EI HR-EI Calcd for C20H19N5O6S (M+H)+: 58.1129 found: 458.1137.

4.5. Radioligand binding assays 20 4.5.1. [3H]Ketanserin binding to serotonin 5-HT2A receptor For serotonin 5-HT2A receptor binding, an aliquot of frozen membrane from avCHO-K1 cell line expressing the human recombinant 5-HT2A receptor and [3H]Ketanserin (1 nM) were used in the presence of mianserin (0.5 AM) as nonspecific. The reaction mixture was incubated for 15 min at 37 oC using 50 mM Tris–HCl (pH 7.4) buffer, and harvested through Whatman GF/C glass

fiber filter presoaked in 0.05% Brij. 4.5.2. [3H]Mesulergine binding to serotonin 5-HT2C receptor Frozen membranes from a stable CHO-K1 cell line expressing the human recombinant 5-HT2C receptor were used. For the binding assay, [3H]Mesulergine (1 nM), receptor membrane, and test compounds were added into 50 mM Tris–HCl (pH 7.7) buffer containing 0.1% ascorbic acid and 10 AM pargyline. Nonspecific binding was determined using 0.5 AM mianserin. The incubations were performed for 30 min at 37 oC, and these were terminated by rapid filtration through Whatman GF/C glass fiber filter presoaked in 1% BSA. 4.6. cAMP accumulation assay for 5-HT6 receptors 21 All synthesized compounds were evaluated in vitro against the human recombinant serotonin receptors. They were evaluated for their functional profile by determining adenylate cyclase activity. The HEK293 cell line stably expressed the 5-HT6 receptor (HEK293/6R) and was used for 5-HT6 receptor activities. The known 5-HT6 antagonist, SB258585, was used as reference, with an IC50 of 19.9 ±5.5 nM in our experiment. The results were estimated as the percentage inhibition compared with the untreated controls. To analyze cAMP levels, cAMP dynamic 2 HTRF kits (Cisbio, France) which provide a homogeneous high-throughput assay were used. Cells incubated at 37℃ in 5% CO2 and 95% air atmosphere were suspended in PBS containing 2 mM IBMX (3-isobutyl-1-methylxanthine) and stimulated by 5-HT for 30 min with or without pretreatment with prepared compounds for 10 min. After 30 min, cAMP labeled with the dye d2 and anti-cAMP antibodies labeled with cryptate were added into the cell plates. The plates were incubated at room temperature for 1 hour. The fluorescence intensity of accumulated cAMP level was measured at 314 nm excitation, and 668 and 620 nm emission using Flexstation3 microplate reader (Molecular Devices, Downingtown, PA). The results were estimated as the IC50 inhibition compared with the untreated controls. 4.7. Pharmacophore Generation and Mapping

Two sets of ligands were utilized for the modeling studies. Potential, structurally diverse six 5HT2A receptor antagonists

41

(Figure 2A) constituted the first group. Six 5-HT6 receptor lead

compounds (Figure 2B) which entered the advanced stages of clinical trials formed the second set and were retrieved from the Prous Integrity Drugs & Biologics Database. Using HipHop algorithm (CatHypo/Discovery Studio (DS) 4.0/Accelrys (San Diego, USA)), a common featurebased pharmacophore was derived from each 5-HT class. Maximum number of conformers (Best/255) were generated for each ligand with an energy threshold of 20.0 kcal/mol. The ‘Minimum Interfeature Distance’ was set to 0.5 and 2.0 for 5-HT2A and 5-HT6 datasets respectively. The mapping of best output hypothesis with compounds was carried out by the ‘Ligand Pharmacophore Mapping’ module in DS using the ‘Best/Flexible’ option and ‘MaxOmitFeat’ of 1.

Acknowledgment This work was supported by a Grant (2010-0004-883) from Korea Research Foundation and Korea Institute of Science and Technology (2E25240). .

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Table legands

Table 1. Percent inhibition and IC50 values of the 4-nitrolindole derivatives against 5-HT2A and 5-HT2c receptors. 20 Table 2. Percent inhibition and IC50 values of the 4-nitrolindole derivatives against the 5-HT6 receptor. 21

Figure legands Figure 1. Selected compounds which showed good inhibitory activity on 5-HT2A and 5-HT6 13-17

Figure 2: Chemical structures of the ligands. A) Selected 5HT2A antagonists. B) Potential 5HT6 antagonists. Figure 3. Pharmacophore mapping with Hypo1. A) Hypo1- distance constraints. PI- bright red, AR- orange, HYD-cyan. Mapping of Hypo1 with B) 14 and C) 8. The omitted PI feature is shown as dimmed (maroon). Figure 4. Superimposition of Hypo2 with compounds. A) Hypo2- distance constraints. PI- bright red, AR- orange, HYD- cyan, HBA- green. Hypo2 superimposed with B) 14 and C) 8.

Scheme legand Scheme1. Synthesis of 4-nitroindole derivatives.

N N N

N O

S

N

N

O 5-HT2A IC50 = 0.7 nM

OH S N O

5-HT6

IC50 =2.04 ¥ìM

N S N N

5-HT6

N O S O

IC50 = 3.9 ¥ìM

N

O H N S O 5-HT6

N N S IC50 = 0.36 ¥ìM

Figure 1. Selected compounds which showed good inhibitory activity on 5-HT2A and 5-HT6 13-17

A

N O

O

F O

N

N

N

O N H

HO F

1

F O O S N

3

2

H N

N

O N

O S N O

F N

N

F

CN

F N

F F 5

4

6

B H N O

O S

O F N H

O

N

O S

N O

O

N

O

N

N S O O

HN

F 2

1

NH

O O S

N

3 Cl Cl H N

O S O NH

S

O O

Cl

HN S O O

S

O

O N

N N H 4

5

6

Figure 2: Chemical structures of theligands. A) Selected 5HT2A antagonists. B) Potential 5HT6 antagonists.

Figure 3. Pharmacophore mapping with Hypo1. A) Hypo1- distance constraints. PI- bright red, AR- orange, HYD-cyan. Mapping of Hypo1 with A) 14 and B) 8. The omitted PI feature is shown as dimmed (maroon).

Figure 4. Superimposition of Hypo2 with compounds. A) Hypo2- distance constraints. PIbright red, AR- orange, HYD- cyan, HBA- green. Hypo2 superimposed with A) 14 and B) 8.

Scheme1. Method for the synthesis of 4-nitro-indol derivatives.

Table 1. Percent inhibition and IC50 values of the 4-nitrolindole derivatives against 5-HT2a

and 5-HT2c receptors. 5-HT2a %-Inhibition (10µM)

5-HT2c IC50 (µM)

%-Inhibition (10µM)

IC50 (µM)

4

83.9

0.329

48.4

5

94.2

0.702

55.5

6

85.2

0.247

66.2

7

88.3

0.253

82.3

0.480

8

nd

-

nd

-

9

90.7

0.205

67.8

1.040

10

84.4

0.479

48.1

11

75.9

1.593

44.9

12

94.6

0.555

64.2

13

88.0

0.433

80.1

14

96.0

0.105

45.4

0.463

Table 2. Percent inhibition and IC50 values of the 4-nitrolindole derivatives against the 5-HT6 receptor N

NO2

N N

N O O S R

R

% Inhibition (10 µM)

IC 50 (µM) 1.83±0.14

4

phenyl

78.1 ± 8.7

5

4-fluorophenyl

63.9 ± 3.6

4.97±0.27

6

4-iodophenyl

62.4 ± 2.4

6.34±0.18

7

4-methylphenyl

75.2 ± 1.6

2.12±0.18

8

4-butylphenyl

11.3 ± 7.5

-

9

4-methoxyphenyl

78.6 ± 2.8

0.74±0.12

10

2,5-dimethoxyphenyl

78.2 ± 3.4

1.40±0.12

11

3-chloro-6-methoxyphenyl

87.7 ± 4.6

2.44±0.18

12

1-naphthyl

73.9 ± 3.2

2.96±0.77

13

2-naphthyl

68.0 ± 2.2

2.53±0.51

14

2,3-dihydro-1,4-benzodioxinyl

101.0 ± 3.2

0.17±0.02

Graphical Abstract

Synthesis and biological evaluation of 4-nitroindole derivatives as 5-HT2A receptor antagonists Faisal Hayat a, Ambily Nath Indu Viswanath b,d , Ae Nim Pae b,d, Hyewhon Rhim b, Woo-Kyu Park c, Hea-Young Park Choo a,* a

College of Pharmacy & Division of Life & Pharmaceutical Sciences, Ewha Womans University, Seoul 120-750, Republic of Korea b Center for Neuroscience, Korea Institute of Science & Technology, PO Box 131, Cheongryang, Seoul 130-650, Republic of Korea c

Pharmaceutical Screening Research Team, Korea Research Institute of Chemical Technology, Daejeon 305-343 d

Department of Biological Chemistry, School of Science, Korea University of Science and Technology, 52 Eoeun Dong, Yuseong-Gu, 305-333 Daejeon, Republic of Korea