- Email: [email protected]
Discovery of novel potent and selective ligands for 5-HT2A receptor with quinazoline scaffold
Accepted Manuscript Discovery of Novel Potent and Selective Ligands for 5-HT2A Receptor with Quinazoline Scaffold Xinxian Deng, Lin Guo, Lili Xu, Xuechu Zhen, Kunqian Yu, Weili Zhao, Wei Fu PII: DOI: Reference:
S0960-894X(15)00734-9 http://dx.doi.org/10.1016/j.bmcl.2015.07.030 BMCL 22919
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
25 March 2015 15 June 2015 13 July 2015
Please cite this article as: Deng, X., Guo, L., Xu, L., Zhen, X., Yu, K., Zhao, W., Fu, W., Discovery of Novel Potent and Selective Ligands for 5-HT2A Receptor with Quinazoline Scaffold, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.07.030
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Discovery of Novel Potent and Selective Ligands for 5-HT2A Receptor with Quinazoline Scaffold
Xinxian Deng, Lin Guo, Lili Xu, Xuechu Zhen, Kunqian Yu, Weili Zhao, Wei Fu
1
Bioorganic & Medicinal Chemistry Letters j ou r n a l h o m ep a g e : w w w . e ls e vi e r . c o m
Discovery of Novel Potent and Selective Ligands for 5-HT2A Receptor with Quinazoline Scaffold Xinxian Denga§, Lin Guoc§, Lili Xua§, Xuechu Zhenb*, Kunqian Yuc, Weili Zhaoa*, Wei Fua** a
Department of Medicinal Chemistry & Key Laboratory of Smart Drug Delivery, Ministry of Education & PLA, School of Pharmacy, Fudan University, Shanghai 201203, China b Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-disorders & Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, 199 Ren’ai Road, Suzhou, Jiangsu Province, China. 215123 c Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
A RT I C L E I N F O
A BS T RA C T
Article history: Received Revised Accepted Available online
A series of compounds with quinazoline scaffold were designed, synthesized and evaluated as novel potent 5-HT2A receptor ligands. N-(4-chlorophenyl)-2-(piperazin-1-yl)quinazolin-4-amine (5o) has a Ki value of 14.04 ± 0.21 nM , with a selectivity more than 10000 fold over 5-HT1A receptors (D1 and D2-like receptors). The functional assay showed that this compound is an antagonist to 5-HT2A receptor with an IC50 value of 1.66 µM.
Keywords: 5-HT2A receptor antagonist; quinazoline derivatives; Synthesis; Molecular Docking
2009 Elsevier Ltd. All rights reserved .
* Corresponding authors Wei Fu, Prof./Ph.D. Department of Medicinal Chemistry, School of Pharmacy, Fudan University 826 Zhangheng Road, Shanghai 201203, P.R. China; Fax: +86-21-51980010, Phone: +86-21-51980010; E-mail: [email protected]. Xuechu Zhen, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-disorders & Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, 199 Ren’ai Road, Suzhou, Jiangsu Province, China. Email: [email protected] Weili Zhao, Department of Medicinal Chemistry, School of Pharmacy, Fudan University; 826 Zhangheng Road, Shanghai 201203, China; Email: [email protected]. §These authors contributed equally to this work. 2
alleviating “negative symptom” of schizophrenia and reducing extrapyramidal system (EPS). Great efforts in identifying novel chemicals with 5-HT2A antagonistic activity have been made, which are mainly based on tricycle scaffold and long-chain arylpiperazine (LCAP), represented by clozapine (Ki = 5.35 nM) and aripiprazole (Ki = 8.7 nM), respectively (Figure 1). Limited novel scaffold has been reported. Herein, the discovery of a structurally novel class of 5-HT2A inhibitors exemplified by compound 5o which derived from a quinazoline scaffold, was described.
5-HT2A receptor is a G protein-coupled receptor (GPCR) coupling with Gq/G11 protein which generally mediates excitatory neurotransmission1, 2. One of the most fundamental biological functions of 5-HT2A receptor is the regulation of dopamine release in three dopaminergic pathways3-5. 5-HT2A is involved in a wide array of etiology processes including schizophrenia, depression, anxiety, migraine, hallucinations, insomnia and addiction. Nevertheless, 5-HT2A antagonists have joined with D2 dopamine antagonists as the most used atypical antipsychotic agents6. The importance of 5-HT2A antagonism in atypical antipsychotic drug is believed to associate with
Figure 1 Structures of clozapine and aripiprazole.
Efforts directed toward the identification of 5-HT2A selective inhibitors were initiated by pharmacophore-based virtual screening of our in-house library against 5-HT2A receptor, followed by biological assays. A number of potential candidates were found suitable for lead optimization studies, among them compound 5a was particularly attractive7. It possessed medium activity to 5-HT2A receptor (Ki = 593 nM) with extremely high selectivity over 5-HT1A, D1 and D2 dopamine receptors7. Besides, properites of 5a caclulated by Schrӧdinger conformed to Lipinski’s rule and its extension. 5a has a molecular weight below 500 KDa, a topological polar surface area of 80 Å2, a predicted cLogP of 2, 7 rotable bonds, 3 hydrogen bond donors and 7 hydrogen bond acceptors. It is worth noting that 5a has a novel N4-phenylquinazoline-2,4-diamine scaffold (Tabel 1) as 5-HT2A antagonist, and quinazoline is one of the most developed skeletons as kinase inhibitors8, 9. They showed weak antitumor activity and very narrow therapeutic window in several cell lines including H2228, U87MG, LNCap and A549 (see supporting information, Table S3). Such skeleton has rarely been reported to show affinity to 5-HT2A receptor. We thus thought to further optimize it.
NH group in aniline and the hydroxyl group in ethanol moiety of 5a form hydrogen bond interactions with N7.36 and S3.36, respectively. Our predicted binding mode is quite consistent with available site-directed mutagenesis studies which have proved that D3.3210-12, Ser3.3610, 13, 14, and Y7.4314-17 are important to binding affinities of 5-HT2A ligands. It indicates that the piperazinyl-ethanol arm is critical to the activity, as these indispensable interactions determined the binding orientation of the arm down into the hydrophilic binding pocket (ArmBP) enclosed by D3.32, S3.36 and Y7.43, and the tail up to a hydrophobic binding pocket (TailBP) comprised of the extracellular segments of TM-II and VII. Careful examination showed that the tail part of 5a did not form any interaction with surrounding residues (Figure 2), suggesting that 5a could be optimized by appending favorable groups in aniline part (Table 1, 5b-5l). On the other hand, we consider that the piperazinyl-ethanol arm has high flexibility and the hydrogen bond formed by hydroxyl group of arm with S3.36 may not be conservative in all the quinazoline analogues. Therefore, several different fragments were added in the arm region to examine the effect of piperazinyl-ethanol on activity (Table 2, 5m-5v). The synthesis of target compounds was accomplished according to scheme 1. 2-amino-1-methyl benzoate (1), as starting material, was coupled with KCNO in acetic acid at ambient temperature. Cyclization of methyl 2-ureidobenzoate (6) was done at reflux in KOH methanol solution (pH = 10)18. Chlorination of quinazoline-2,4(1H,3H)-dione (2) was achieved by refluxing with POCl3 at the presence of tri-n-propylamine in toluene19. Then the corresponding phenylamine derivatives were attached to the 4-position of 2,4-dichloroquinazoline (3) through nucleophilic addition under basic condition in THF at room temperature20. The aniline analogs were added to the 2,
Compound 5a is composed by a quinazoline core, an arm (piperazinyl-ethanol) and a tail (dimethoxy-aniline). To rationalize the design of the derivatives, the structural model of the complex 5a-5-HT2A was constructed by combining molecular docking and all available experimental data (Figure 2). 5a is lying at the antagonistic crevice comprised of TM-III, TM-VI and TM-VII10. And several important interactions were identified. The protonated nitrogen atom (N1) of 5a is not only involved in the conserved hydrogen bond with D3.32, but also participates in an additional cation-π interaction with W3.28. 3
published protocol22, 23. For the initial screening, the inhibition percentages of the compounds 5a-5v at 10 μM were measured and the values of Ki were given only when the inhibition rate of compound was greater than 90%. The potent compound of 5-HT2A (+)Butaclamol was used as reference compound to test the binding affinity. The results are summarized in Table 1 and 2, which illustrated that most of these compounds exhibited moderately strong inhibitory activities.
4-dichloroquinazoline THF solution in a dropwise way so that less di-substituted side products were produced. The next step was another nucleophilic addition where three different secondary amines were attached to 2-chloro-N-phenylquinazolin-4-amine derivatives (4), respectively21. Finally, intermediates 5 were treated with conc. HCl to get the corresponding hydrochloride salt. The receptor binding assays were conducted according to our previous
Scheme 1
(i) KCNO; AcOH; H2O; (ii) KOH; MeOH; H2O; (iii) POCl3; PhMe; tri-n-propylamine; (iv) corresponding phenylamine derivatives; TEA; THF; (v) corresponding amine; TEA; acetonitrile; (vi) conc. HCl; THF
4
Figure 2 Predicted Binding mode of 5a, 5f, 5g and 5k with 5-HT2A receptor. Ligands were shown in yellow sticks and surrounding residues in green. 5-HT2A was shown in white ribbon. Hydrogen bond interactions were represented in dotted blue lines and cation-π interactions in orange.
As inspired by the binding mode of 5a with 5-HT2A, the effect of substituent at o-, m- and p- positions of aniline was expected to form favorable interaction with residues in the Tail BP. As a result, 5f and 5g were obtained. o-OCH3 substituted compound 5g provided a delightful improvement in the binding affinity (Ki = 145.64 nM), while p-OCH3 substituted 5f exhibited a decreased affinity. It is worth noting that the activities of compounds were sensitive to substituent’s properties and position in tail part of compounds. Then what is the intrinsic reason that causes such sensitivity? The comparison of binding modes of 5a, 5f and 5g unmasks this veil (Figure 2). The hydroxyl group and the protonated amino group in three compounds form the hydrogen bonds with the S3.36 and D3.32, respectively. While the o-OCH3 group in 5g forms the hydrogen bond with the Y7.43, p-OCH3 in 5f does not form any interaction with the surrounding residues; in 5a, the p-OCH3 changes the orientation of o-OCH3 and blocks the formation of this hydrogen bond. Instead, a new hydrogen bond is formed by the amino group of 5a and N7.36 (Figure 2). Therefore, 5g with substituent o-OCH3 shows the strongest activity among three compounds. Unfortunately, the analogue with m-OCH3 substituent was not obtained due to the difficulty in getting the crude reagent, while the compound 5k with m-OH substituent showed improved activity than 5a, 5f and 5g, since the m-OH was located in a favorable position and forms
strong hydrogen bond interaction with Y7.43. The predicted binding energies of compounds 5a, 5f, 5g, 5k, 5m and 5n with 5-HT2A receptor correlated well with their experimental binding activities (Figure S1 in supporting information). Compounds 5b-5e were next prepared to evaluate the influence of different halogens at p-position of aniline on binding affinity. The results indicated that all halogenated derivatives displayed improved binding affinity and the brominated compound performed better than other halogenated ones (p-Br > p-Cl > p-F > p-I), demonstrating the important role of halogens in 5-HT2A ligand design. The compound 5i with o-F substituent on aniline was designed and synthesized to further examine the effect of halogen’s position on binding affinity. Similar to the case with -OCH3 substituent at aniline, 5i with o-F substituent showed 2-fold improvement (65 nM) compared to 5b with p-F substituent in aniline (120 nM). Above hints inspire us to design compounds with -Cl, -Br and -I at o- and p-position of aniline in the future. The effect of larger hydrophobic substituent at p-position of aniline on the affinity was also examined by designing compounds 5h (p-isopropyl) and 5l (Benzyl-amine). It turned out that 5h showed 3-fold improved activity while 5l maintained its activity.
Table 1 Binding affinities of compounds 5a-5l for 5-HT2A. (Ki or percentage displacement of radio-ligand at 10 μM)
Ki ± SD (nM) Entry
R 5-HT2A [3H]-Ketanserin
5a* 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l (+)Butaclamol
o-OMe p-OMe p-F p-Cl p-Br p-I p-OMe o-OMe p-isopropyl o-F o-Me, p-Me m-OH Benzy-amine
593.89 ± 34.10 120.13 ± 17.71 109.17 ± 1.05 92.05 ± 13.45 383.02 ± 7.79 1359.45 ± 51.69 145.64 ± 9.91 149.11 ± 19.01 65.78 ± 17.64 267.70 ± 11.25 83.38 ± 3.38 519.60 ± 15.25 2.97 ± 0.60
5
* Lead compound; not synthetic.
Table 2. Binding affinities of compounds 5m-5v for 5-HT2A. (Ki or percentage displacement of radioligand at 10 μM).
Ki ± SD (nM) Entry
R1
R2
5-HT2A[3H]-Ketanserin
5m
o-OMe
45.77 ± 8.67
5n 5o 5p 5q 5r 5s
o-F p-Cl p-I p-isopropyl p-F o-F
69.99 ± 6.30 14.04 ± 0.21 496.24 ± 31.75 42.14 ± 1.43 234.77 ± 5.66 184.33 ± 28.22
5t
p-Cl
45.27%
5u
p-isopropyl
5.91%
5v
p-isopropyl
75.11%
2.97 ± 0.60 (+)Butaclamol Note: Ki value for 5-HT2A were given only if compounds with inhibition ratio higher than 90%; percentage data represents inhibition ratio.
As depicted in Figure 2, the flexible group ethoxy tail in 5a, 5f and 5g contributed to the binding affinity by forming a hydrogen bond with S3.36. In order to quantitatively examine the contribution of this flexible ethoxy tail on binding affinity, compounds 5m-5q with eliminated flexible ethoxy group were designed and synthesized. Surprisingly, these designed compounds 5m-5q showed largely improved activity (10-100 nM). Especially, 5o displayed potent activity of 14.04 nM, which is ca. 10 times higher than that of 5c with flexible ethoxy tail (109.1 nM). This extraordinary event inspired us to further investigate the binding mode of these new designed compounds with 5-HT2A receptor. 5o (p-Cl, without ethoxy tail) and 5c (p-Cl, with ethoxy tail) were overlaid in the binding site
of 5-HT2A receptor to uncover the essence of the diminished activity in 5c (Figure 3). Specifically, in the case of 5o (without ethoxyl), the piperazine ring forms hydrogen bond and electrostatic interaction with D3.32 and cation-π interaction with Y7.43. The addition of ethoxyl destroys the hydrogen bond with Y7.43 by anchoring S3.36 through a new hydrogen bond. In the meantime, this anchor action makes the quinazoline core revolve toward helix VI, and this rotation adjusts the aniline arm to stretch into the pocket formed by TM-VII and TM-II from the pocket formed by TM-VI and TM-V. It turns out that the amino group in aniline tail in 5o forms hydrogen bond with N6.55 which might account for the higher activity24.
6
Figure 3 The superimposition of 5g and 5m (up and left), 5c and 5o (down and left) in the binding pocket of 5-HT2A (shown as surface) and detailed binding mode of 5m and 5o with 5-HT2A .
The well-known consensus knowledge in A-class GPCR inhibitory design is that a protonated N in ligand is required for the binding activity because it can form the salt bridge interaction with the conserved residue D3.3210 in the binding site. There are few cases that the ligand without protonated N shows activity toward A-class GPCR25. Therefore, 5r-5u were designed to examine whether this new quinazoline scaffold for GPCR can show activity toward 5-HT2A receptor when the protonated N is eliminated. Interestingly, 5r and 5s with morpholine fragment showed medium activity toward 5-HT2A receptor (234.77 nM and 184.33 nM), while 5t and 5u did not display activity. The molecular docking showed that the hydrophilic morpholine could stay in the hydrophilic pocket composed by the TM-III and TM-VII and atom O acted as hydrogen bond acceptor that formed hydrogen bond with D3.32, while the hydrophobic aniline substituent at this position was unfavorable for the binding. Besides, this type of
compounds with quinazoline scaffold showed higher selectivity over 5-HT1A receptor and dopamine D1, D2 and D3 receptors (supporting information, Table S1). Only four compounds 5d, 5h, 5l and 5q exhibited weak activity toward dopamine receptors. [35S]GTPγS binding assays26 were employed to determine the agonistic or antagonistic activities of compounds with Ki values less than 130 nM (5b, 5c, 5d, 5i, 5k, 5o, 5m 5n), in which olanzapine was used as reference compound. The potent antagonist olanzapine of 5-HT2A was also used as reference compound. As shown in Table 3, compound 5i and 5o produced relatively strong antagonistic activity against 5-HT2A, displaying IC50 values of 1.37 µM and 1.66 µM, respectively. The result demonstrated that the compounds with quinazoline scaffold are a novel type of high potent and selective antagonist for 5-HT2A receptor.
Table 3. [35S]GTPγS binding assays of compounds 5b, 5c, 5d, 5i, 5k, 5o, 5m 5n for 5-HT2A receptor. Compounds 5b 5c 5d 5i 5k 5m 5n 5o Olanzapine 5-HT ND: not determined These compounds are antagonists, no EC50 value.
EC50 (µM)
0.13
In summary, a new scaffold of compounds with the core structure of quinazoline was identified as highly potent and selective 5-HT2A ligands by combining a series of computational approaches, synthetic chemistry and binding assays. Compound 5o showed relatively high binding affinity to 5-HT2A receptor (Ki =14.04 nM) and antagonist activity (IC50 = 1.66 µM). Molecular docking disclosed the binding sites of quinazoline analogues for 5-HT2A: ArmBP and TailBP. The underlying mechanism of the increased activity by eliminating flexible ethoxy tail was also explored. To our best knowledge, this is the first time to find out that the quinazoline
IC50 (µM) 21.26 7.43 27.72 1.37 9.12 ND ND 1.66 0.10 -
analogues can be highly potent and selective ligands for 5-HT2A receptor. Investigations of more potent and selective quinazoline derivatives are required for their practical and therapeutic applications in the future. Experimental Section Experimental procedures for medicinal chemistry, pharmacology and molecular modeling as well as characterization of compounds are provided in the Supporting Information. 7
Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 81172919, 81130023, 31373382), National Basic Research Plan (973) of the Ministry of Science and Technology of China (2011CB5C4403), the State Key Laboratory of Drug Research, and National Drug Innovative Program (No.2009ZX09301-011). Support from Priority Academic Program Development of Jiangsu Higher Education Institutes (PAPD) is also appreciated.
Supplementary Material Description of 1H NMR, 13C NMR, HRMS and melting point of the target compounds. Description of 5-HT1A, 5-HT2A, D1, D2 and D3 binding assays, [35S]-GTPγS assays and antitumor assays. Description of molecular modeling. This information is available via the Internet.
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8
S0960-894X(15)00734-9 http://dx.doi.org/10.1016/j.bmcl.2015.07.030 BMCL 22919
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
25 March 2015 15 June 2015 13 July 2015
Please cite this article as: Deng, X., Guo, L., Xu, L., Zhen, X., Yu, K., Zhao, W., Fu, W., Discovery of Novel Potent and Selective Ligands for 5-HT2A Receptor with Quinazoline Scaffold, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.07.030
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Graphical Abstract
Leave this area blank for abstract info.
Discovery of Novel Potent and Selective Ligands for 5-HT2A Receptor with Quinazoline Scaffold
Xinxian Deng, Lin Guo, Lili Xu, Xuechu Zhen, Kunqian Yu, Weili Zhao, Wei Fu
1
Bioorganic & Medicinal Chemistry Letters j ou r n a l h o m ep a g e : w w w . e ls e vi e r . c o m
Discovery of Novel Potent and Selective Ligands for 5-HT2A Receptor with Quinazoline Scaffold Xinxian Denga§, Lin Guoc§, Lili Xua§, Xuechu Zhenb*, Kunqian Yuc, Weili Zhaoa*, Wei Fua** a
Department of Medicinal Chemistry & Key Laboratory of Smart Drug Delivery, Ministry of Education & PLA, School of Pharmacy, Fudan University, Shanghai 201203, China b Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-disorders & Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, 199 Ren’ai Road, Suzhou, Jiangsu Province, China. 215123 c Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
A RT I C L E I N F O
A BS T RA C T
Article history: Received Revised Accepted Available online
A series of compounds with quinazoline scaffold were designed, synthesized and evaluated as novel potent 5-HT2A receptor ligands. N-(4-chlorophenyl)-2-(piperazin-1-yl)quinazolin-4-amine (5o) has a Ki value of 14.04 ± 0.21 nM , with a selectivity more than 10000 fold over 5-HT1A receptors (D1 and D2-like receptors). The functional assay showed that this compound is an antagonist to 5-HT2A receptor with an IC50 value of 1.66 µM.
Keywords: 5-HT2A receptor antagonist; quinazoline derivatives; Synthesis; Molecular Docking
2009 Elsevier Ltd. All rights reserved .
* Corresponding authors Wei Fu, Prof./Ph.D. Department of Medicinal Chemistry, School of Pharmacy, Fudan University 826 Zhangheng Road, Shanghai 201203, P.R. China; Fax: +86-21-51980010, Phone: +86-21-51980010; E-mail: [email protected]. Xuechu Zhen, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-disorders & Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, 199 Ren’ai Road, Suzhou, Jiangsu Province, China. Email: [email protected] Weili Zhao, Department of Medicinal Chemistry, School of Pharmacy, Fudan University; 826 Zhangheng Road, Shanghai 201203, China; Email: [email protected]. §These authors contributed equally to this work. 2
alleviating “negative symptom” of schizophrenia and reducing extrapyramidal system (EPS). Great efforts in identifying novel chemicals with 5-HT2A antagonistic activity have been made, which are mainly based on tricycle scaffold and long-chain arylpiperazine (LCAP), represented by clozapine (Ki = 5.35 nM) and aripiprazole (Ki = 8.7 nM), respectively (Figure 1). Limited novel scaffold has been reported. Herein, the discovery of a structurally novel class of 5-HT2A inhibitors exemplified by compound 5o which derived from a quinazoline scaffold, was described.
5-HT2A receptor is a G protein-coupled receptor (GPCR) coupling with Gq/G11 protein which generally mediates excitatory neurotransmission1, 2. One of the most fundamental biological functions of 5-HT2A receptor is the regulation of dopamine release in three dopaminergic pathways3-5. 5-HT2A is involved in a wide array of etiology processes including schizophrenia, depression, anxiety, migraine, hallucinations, insomnia and addiction. Nevertheless, 5-HT2A antagonists have joined with D2 dopamine antagonists as the most used atypical antipsychotic agents6. The importance of 5-HT2A antagonism in atypical antipsychotic drug is believed to associate with
Figure 1 Structures of clozapine and aripiprazole.
Efforts directed toward the identification of 5-HT2A selective inhibitors were initiated by pharmacophore-based virtual screening of our in-house library against 5-HT2A receptor, followed by biological assays. A number of potential candidates were found suitable for lead optimization studies, among them compound 5a was particularly attractive7. It possessed medium activity to 5-HT2A receptor (Ki = 593 nM) with extremely high selectivity over 5-HT1A, D1 and D2 dopamine receptors7. Besides, properites of 5a caclulated by Schrӧdinger conformed to Lipinski’s rule and its extension. 5a has a molecular weight below 500 KDa, a topological polar surface area of 80 Å2, a predicted cLogP of 2, 7 rotable bonds, 3 hydrogen bond donors and 7 hydrogen bond acceptors. It is worth noting that 5a has a novel N4-phenylquinazoline-2,4-diamine scaffold (Tabel 1) as 5-HT2A antagonist, and quinazoline is one of the most developed skeletons as kinase inhibitors8, 9. They showed weak antitumor activity and very narrow therapeutic window in several cell lines including H2228, U87MG, LNCap and A549 (see supporting information, Table S3). Such skeleton has rarely been reported to show affinity to 5-HT2A receptor. We thus thought to further optimize it.
NH group in aniline and the hydroxyl group in ethanol moiety of 5a form hydrogen bond interactions with N7.36 and S3.36, respectively. Our predicted binding mode is quite consistent with available site-directed mutagenesis studies which have proved that D3.3210-12, Ser3.3610, 13, 14, and Y7.4314-17 are important to binding affinities of 5-HT2A ligands. It indicates that the piperazinyl-ethanol arm is critical to the activity, as these indispensable interactions determined the binding orientation of the arm down into the hydrophilic binding pocket (ArmBP) enclosed by D3.32, S3.36 and Y7.43, and the tail up to a hydrophobic binding pocket (TailBP) comprised of the extracellular segments of TM-II and VII. Careful examination showed that the tail part of 5a did not form any interaction with surrounding residues (Figure 2), suggesting that 5a could be optimized by appending favorable groups in aniline part (Table 1, 5b-5l). On the other hand, we consider that the piperazinyl-ethanol arm has high flexibility and the hydrogen bond formed by hydroxyl group of arm with S3.36 may not be conservative in all the quinazoline analogues. Therefore, several different fragments were added in the arm region to examine the effect of piperazinyl-ethanol on activity (Table 2, 5m-5v). The synthesis of target compounds was accomplished according to scheme 1. 2-amino-1-methyl benzoate (1), as starting material, was coupled with KCNO in acetic acid at ambient temperature. Cyclization of methyl 2-ureidobenzoate (6) was done at reflux in KOH methanol solution (pH = 10)18. Chlorination of quinazoline-2,4(1H,3H)-dione (2) was achieved by refluxing with POCl3 at the presence of tri-n-propylamine in toluene19. Then the corresponding phenylamine derivatives were attached to the 4-position of 2,4-dichloroquinazoline (3) through nucleophilic addition under basic condition in THF at room temperature20. The aniline analogs were added to the 2,
Compound 5a is composed by a quinazoline core, an arm (piperazinyl-ethanol) and a tail (dimethoxy-aniline). To rationalize the design of the derivatives, the structural model of the complex 5a-5-HT2A was constructed by combining molecular docking and all available experimental data (Figure 2). 5a is lying at the antagonistic crevice comprised of TM-III, TM-VI and TM-VII10. And several important interactions were identified. The protonated nitrogen atom (N1) of 5a is not only involved in the conserved hydrogen bond with D3.32, but also participates in an additional cation-π interaction with W3.28. 3
published protocol22, 23. For the initial screening, the inhibition percentages of the compounds 5a-5v at 10 μM were measured and the values of Ki were given only when the inhibition rate of compound was greater than 90%. The potent compound of 5-HT2A (+)Butaclamol was used as reference compound to test the binding affinity. The results are summarized in Table 1 and 2, which illustrated that most of these compounds exhibited moderately strong inhibitory activities.
4-dichloroquinazoline THF solution in a dropwise way so that less di-substituted side products were produced. The next step was another nucleophilic addition where three different secondary amines were attached to 2-chloro-N-phenylquinazolin-4-amine derivatives (4), respectively21. Finally, intermediates 5 were treated with conc. HCl to get the corresponding hydrochloride salt. The receptor binding assays were conducted according to our previous
Scheme 1
(i) KCNO; AcOH; H2O; (ii) KOH; MeOH; H2O; (iii) POCl3; PhMe; tri-n-propylamine; (iv) corresponding phenylamine derivatives; TEA; THF; (v) corresponding amine; TEA; acetonitrile; (vi) conc. HCl; THF
4
Figure 2 Predicted Binding mode of 5a, 5f, 5g and 5k with 5-HT2A receptor. Ligands were shown in yellow sticks and surrounding residues in green. 5-HT2A was shown in white ribbon. Hydrogen bond interactions were represented in dotted blue lines and cation-π interactions in orange.
As inspired by the binding mode of 5a with 5-HT2A, the effect of substituent at o-, m- and p- positions of aniline was expected to form favorable interaction with residues in the Tail BP. As a result, 5f and 5g were obtained. o-OCH3 substituted compound 5g provided a delightful improvement in the binding affinity (Ki = 145.64 nM), while p-OCH3 substituted 5f exhibited a decreased affinity. It is worth noting that the activities of compounds were sensitive to substituent’s properties and position in tail part of compounds. Then what is the intrinsic reason that causes such sensitivity? The comparison of binding modes of 5a, 5f and 5g unmasks this veil (Figure 2). The hydroxyl group and the protonated amino group in three compounds form the hydrogen bonds with the S3.36 and D3.32, respectively. While the o-OCH3 group in 5g forms the hydrogen bond with the Y7.43, p-OCH3 in 5f does not form any interaction with the surrounding residues; in 5a, the p-OCH3 changes the orientation of o-OCH3 and blocks the formation of this hydrogen bond. Instead, a new hydrogen bond is formed by the amino group of 5a and N7.36 (Figure 2). Therefore, 5g with substituent o-OCH3 shows the strongest activity among three compounds. Unfortunately, the analogue with m-OCH3 substituent was not obtained due to the difficulty in getting the crude reagent, while the compound 5k with m-OH substituent showed improved activity than 5a, 5f and 5g, since the m-OH was located in a favorable position and forms
strong hydrogen bond interaction with Y7.43. The predicted binding energies of compounds 5a, 5f, 5g, 5k, 5m and 5n with 5-HT2A receptor correlated well with their experimental binding activities (Figure S1 in supporting information). Compounds 5b-5e were next prepared to evaluate the influence of different halogens at p-position of aniline on binding affinity. The results indicated that all halogenated derivatives displayed improved binding affinity and the brominated compound performed better than other halogenated ones (p-Br > p-Cl > p-F > p-I), demonstrating the important role of halogens in 5-HT2A ligand design. The compound 5i with o-F substituent on aniline was designed and synthesized to further examine the effect of halogen’s position on binding affinity. Similar to the case with -OCH3 substituent at aniline, 5i with o-F substituent showed 2-fold improvement (65 nM) compared to 5b with p-F substituent in aniline (120 nM). Above hints inspire us to design compounds with -Cl, -Br and -I at o- and p-position of aniline in the future. The effect of larger hydrophobic substituent at p-position of aniline on the affinity was also examined by designing compounds 5h (p-isopropyl) and 5l (Benzyl-amine). It turned out that 5h showed 3-fold improved activity while 5l maintained its activity.
Table 1 Binding affinities of compounds 5a-5l for 5-HT2A. (Ki or percentage displacement of radio-ligand at 10 μM)
Ki ± SD (nM) Entry
R 5-HT2A [3H]-Ketanserin
5a* 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l (+)Butaclamol
o-OMe p-OMe p-F p-Cl p-Br p-I p-OMe o-OMe p-isopropyl o-F o-Me, p-Me m-OH Benzy-amine
593.89 ± 34.10 120.13 ± 17.71 109.17 ± 1.05 92.05 ± 13.45 383.02 ± 7.79 1359.45 ± 51.69 145.64 ± 9.91 149.11 ± 19.01 65.78 ± 17.64 267.70 ± 11.25 83.38 ± 3.38 519.60 ± 15.25 2.97 ± 0.60
5
* Lead compound; not synthetic.
Table 2. Binding affinities of compounds 5m-5v for 5-HT2A. (Ki or percentage displacement of radioligand at 10 μM).
Ki ± SD (nM) Entry
R1
R2
5-HT2A[3H]-Ketanserin
5m
o-OMe
45.77 ± 8.67
5n 5o 5p 5q 5r 5s
o-F p-Cl p-I p-isopropyl p-F o-F
69.99 ± 6.30 14.04 ± 0.21 496.24 ± 31.75 42.14 ± 1.43 234.77 ± 5.66 184.33 ± 28.22
5t
p-Cl
45.27%
5u
p-isopropyl
5.91%
5v
p-isopropyl
75.11%
2.97 ± 0.60 (+)Butaclamol Note: Ki value for 5-HT2A were given only if compounds with inhibition ratio higher than 90%; percentage data represents inhibition ratio.
As depicted in Figure 2, the flexible group ethoxy tail in 5a, 5f and 5g contributed to the binding affinity by forming a hydrogen bond with S3.36. In order to quantitatively examine the contribution of this flexible ethoxy tail on binding affinity, compounds 5m-5q with eliminated flexible ethoxy group were designed and synthesized. Surprisingly, these designed compounds 5m-5q showed largely improved activity (10-100 nM). Especially, 5o displayed potent activity of 14.04 nM, which is ca. 10 times higher than that of 5c with flexible ethoxy tail (109.1 nM). This extraordinary event inspired us to further investigate the binding mode of these new designed compounds with 5-HT2A receptor. 5o (p-Cl, without ethoxy tail) and 5c (p-Cl, with ethoxy tail) were overlaid in the binding site
of 5-HT2A receptor to uncover the essence of the diminished activity in 5c (Figure 3). Specifically, in the case of 5o (without ethoxyl), the piperazine ring forms hydrogen bond and electrostatic interaction with D3.32 and cation-π interaction with Y7.43. The addition of ethoxyl destroys the hydrogen bond with Y7.43 by anchoring S3.36 through a new hydrogen bond. In the meantime, this anchor action makes the quinazoline core revolve toward helix VI, and this rotation adjusts the aniline arm to stretch into the pocket formed by TM-VII and TM-II from the pocket formed by TM-VI and TM-V. It turns out that the amino group in aniline tail in 5o forms hydrogen bond with N6.55 which might account for the higher activity24.
6
Figure 3 The superimposition of 5g and 5m (up and left), 5c and 5o (down and left) in the binding pocket of 5-HT2A (shown as surface) and detailed binding mode of 5m and 5o with 5-HT2A .
The well-known consensus knowledge in A-class GPCR inhibitory design is that a protonated N in ligand is required for the binding activity because it can form the salt bridge interaction with the conserved residue D3.3210 in the binding site. There are few cases that the ligand without protonated N shows activity toward A-class GPCR25. Therefore, 5r-5u were designed to examine whether this new quinazoline scaffold for GPCR can show activity toward 5-HT2A receptor when the protonated N is eliminated. Interestingly, 5r and 5s with morpholine fragment showed medium activity toward 5-HT2A receptor (234.77 nM and 184.33 nM), while 5t and 5u did not display activity. The molecular docking showed that the hydrophilic morpholine could stay in the hydrophilic pocket composed by the TM-III and TM-VII and atom O acted as hydrogen bond acceptor that formed hydrogen bond with D3.32, while the hydrophobic aniline substituent at this position was unfavorable for the binding. Besides, this type of
compounds with quinazoline scaffold showed higher selectivity over 5-HT1A receptor and dopamine D1, D2 and D3 receptors (supporting information, Table S1). Only four compounds 5d, 5h, 5l and 5q exhibited weak activity toward dopamine receptors. [35S]GTPγS binding assays26 were employed to determine the agonistic or antagonistic activities of compounds with Ki values less than 130 nM (5b, 5c, 5d, 5i, 5k, 5o, 5m 5n), in which olanzapine was used as reference compound. The potent antagonist olanzapine of 5-HT2A was also used as reference compound. As shown in Table 3, compound 5i and 5o produced relatively strong antagonistic activity against 5-HT2A, displaying IC50 values of 1.37 µM and 1.66 µM, respectively. The result demonstrated that the compounds with quinazoline scaffold are a novel type of high potent and selective antagonist for 5-HT2A receptor.
Table 3. [35S]GTPγS binding assays of compounds 5b, 5c, 5d, 5i, 5k, 5o, 5m 5n for 5-HT2A receptor. Compounds 5b 5c 5d 5i 5k 5m 5n 5o Olanzapine 5-HT ND: not determined These compounds are antagonists, no EC50 value.
EC50 (µM)
0.13
In summary, a new scaffold of compounds with the core structure of quinazoline was identified as highly potent and selective 5-HT2A ligands by combining a series of computational approaches, synthetic chemistry and binding assays. Compound 5o showed relatively high binding affinity to 5-HT2A receptor (Ki =14.04 nM) and antagonist activity (IC50 = 1.66 µM). Molecular docking disclosed the binding sites of quinazoline analogues for 5-HT2A: ArmBP and TailBP. The underlying mechanism of the increased activity by eliminating flexible ethoxy tail was also explored. To our best knowledge, this is the first time to find out that the quinazoline
IC50 (µM) 21.26 7.43 27.72 1.37 9.12 ND ND 1.66 0.10 -
analogues can be highly potent and selective ligands for 5-HT2A receptor. Investigations of more potent and selective quinazoline derivatives are required for their practical and therapeutic applications in the future. Experimental Section Experimental procedures for medicinal chemistry, pharmacology and molecular modeling as well as characterization of compounds are provided in the Supporting Information. 7
Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 81172919, 81130023, 31373382), National Basic Research Plan (973) of the Ministry of Science and Technology of China (2011CB5C4403), the State Key Laboratory of Drug Research, and National Drug Innovative Program (No.2009ZX09301-011). Support from Priority Academic Program Development of Jiangsu Higher Education Institutes (PAPD) is also appreciated.
Supplementary Material Description of 1H NMR, 13C NMR, HRMS and melting point of the target compounds. Description of 5-HT1A, 5-HT2A, D1, D2 and D3 binding assays, [35S]-GTPγS assays and antitumor assays. Description of molecular modeling. This information is available via the Internet.
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