Discovery of a Tetracyclic Quinoxaline Derivative as a Potent and

Article pubs.acs.org/jmc

Discovery of a Tetracyclic Quinoxaline Derivative as a Potent and Orally Active Multifunctional Drug Candidate for the Treatment of Neuropsychiatric and Neurological Disorders Peng Li,*,† Qiang Zhang,† Albert J. Robichaud,‡,⊥ Taekyu Lee,‡,∥ John Tomesch,† Wei Yao,† J. David Beard,† Gretchen L. Snyder,† Hongwen Zhu,†,# Youyi Peng,† Joseph P. Hendrick,† Kimberly E. Vanover,† Robert E. Davis,§ Sharon Mates,† and Lawrence P. Wennogle† †

Intra-Cellular Therapies, Inc., 3960 Broadway, New York, New York 10032, United States Medicinal Chemistry, Bristol-Myers Squibb Research and Development, Princeton, New Jersey 08543, United States § 3D Pharmaceutical Consultants, Inc., 13272 Glencliff Way, San Diego, California 92130, United States ‡

S Supporting Information *

ABSTRACT: We report the synthesis and structure−activity relationships of a class of tetracyclic butyrophenones that exhibit potent binding affinities to serotonin 5-HT2A and dopamine D2 receptors. This work has led to the discovery of 4-((6bR,10aS)-3-methyl-2,3,6b,9,10,10a-hexahydro-1H,7H-pyrido[3′,4′:4,5]pyrrolo[1,2,3-de]quinoxalin-8-yl)-1-(4-fluorophenyl)-butan-1-one 4-methylbenzenesulfonate (ITI-007), which is a potent 5-HT2A antagonist, postsynaptic D2 antagonist, and inhibitor of serotonin transporter. This multifunctional drug candidate is orally bioavailable and exhibits good antipsychotic efficacy in vivo. Currently, this investigational new drug is under clinical development for the treatment of neuropsychiatric and neurological disorders.

■

INTRODUCTION Schizophrenia is a chronic neuropsychiatric illness affecting approximately 1% of the global population.1,2 This disorder is characterized principally by the appearance of hallucinations, delusions, agitation, and conceptual disorganization among other so-called positive symptoms. These positive symptoms can be accompanied by varying degrees of social withdrawal, blunted affect and emotion withdrawal, and asociality (negative symptoms) as well as reduction in working memory, attention, and verbal fluency (cognitive dysfunction).3 The etiology and pathology of schizophrenia remain largely unresolved. Yet a disturbed and hyperactive dopaminergic signal transduction system clearly contributes to some symptoms in this disorder.3,4 Further, the relative potencies of antipsychotic drugs as dopamine receptor antagonists correlate with their clinical potencies. The first-generation antipsychotics, such as chlorpromazine and haloperidol (1), are potent D2 receptor antagonists. Use of these agents caused extrapyramidal symptoms (EPS) including tardive dyskinesias and hyperprolactinemia.5 While effective in controlling some of the positive symptoms, these first-generation antipsychotic drugs are relatively ineffective and may exacerbate negative symptoms as well as cognitive dysfunction. The discovery of the dibenzodiazepine, clozapine (2),6,7 ushered in a new generation of potentially superior antipsychotic drugs. Clozapine was able to block dopamine © 2014 American Chemical Society

mediated behavior in animals and exerted antipsychotic effects in humans without EPS or elevation of prolactin levels at efficacious doses. This profile was sufficiently different from the first-generation antipsychotic drugs such that clozapine was labeled “atypical”, and clozapine became the blueprint for the development of other newer so-called atypical antipsychotic drugs. It was later appreciated that clozapine exerted potent antagonist actions at 5-HT2A receptors, leading to the study of selective 5-HT2A antagonists as stand-alone antipsychotic agents.8 The potent and selective 5-HT2A antagonist, MDL100907 (3), showed activity in several preclinical animal models of antipsychotic activity.9,10 However, it failed, like other selective 5-HT2A antagonists, to exhibit sufficient antipsychotic efficacy in clinical trials, and its development for this indication was discontinued.11 It is now apparent that these second-generation antipsychotics, including clozapine and olanzapine,12 are antagonists at both serotonin 5-HT2A and dopamine D2 receptors and activity at both of these receptors is necessary for the antipsychotic efficacy by this class of drugs. These atypical antipsychotics are efficacious in controlling the positive symptoms of schizophrenia while reducing the liability for EPS and hyperprolactinemia.13−15 However, treatment with these antipsyReceived: December 20, 2013 Published: February 21, 2014 2670

dx.doi.org/10.1021/jm401958n | J. Med. Chem. 2014, 57, 2670−2682

Journal of Medicinal Chemistry

Article

Figure 1. Structures of representative antipsychotics and 5.

Scheme 1a

Reagents and conditions: (a) NaNO2, HOAc, 0−5 °C, 60−80%; (b) Zn, HOAc, 10 °C; (c) ethyl 4-oxopiperidine-1-carboxylate, HOAc, HCl, 100 °C, 40−60% (two steps); (d) NaBH3CN, TFA, rt, 85−95%; (e) R2X, K2CO3 or NaH, 80−95%; (f) borane, THF, reflux, then 6N HCl, reflux, 85− 95%; (g) KOH, nBuOH, reflux, 90−99%; (h) 4-chloro-1-(4-fluorophenyl)butan-1-one, NEt3, dioxane, toluene, reflux, 50−70%; (i) chiral HPLC separation, 30−45%. a

stereochemistry at different parts of the heterocyclic cores as well as the ring systems and linker types of the side chains, we identified a novel scaffold, 2,3,6b,9,10,10a-hexahydro-1H,7Hpyrido[3′,4′:4,5]pyrrolo[1,2,3-de]quinoxaline and found compounds with this quinoxaline core generally exhibited better physicochemical and pharmacological properties and consequently showed better in vivo efficacy than compounds with other polycyclic cores. This work eventually led to the discovery of the quinoxaline-type compound 5, a putative antipsychotic that has recently completed a large multicenter placebo controlled human clinical trial in patients with acutely exacerbated schizophrenia (Figure 1). The synthesis and structure−activity relationships (SAR) of 5 and its tetracyclic analogues are described in this article, along with their unique receptor binding profiles and efficacy in animal behavioral models.

chotics is accompanied by various other side effects such as excessive weight gain, type II diabetes, hyperglycemia, and dyslipidemia,5 which are primarily caused by high affinity binding to other off-target receptors such as serotonin 5-HT2C and histamine H1 receptors.16−18 Therefore, there are still urgent unmet medical needs to develop novel antipsychotics with good in vivo efficacy against a broad array of symptoms that accompany schizophrenia, improved pharmacological properties, and reduced side effect profiles. 1,2,7,8,9,10-Hexahydropyrido[3′,4′:4,5]pyrrolo[1,2,3-de][1,4]benzoxazines19 and 6,7,9,10,11,12-hexahydro-5H-pyrido[3′,4′:4,5]pyrrolo[1,2,3-ef ][1,5]benzothiazepines20 were reported to have antidepressant activity over three decades ago. The corresponding hydrogenated derivatives, (cis)/(trans)octahydropyridopyrrolo-benzoxazines and benzothiazepines, were also documented.21 However, the biochemical mechanism of action of these compounds was not clear at that time. By linking this (cis)-octahydropyridopyrrolo-benzothiazepine core with 2′-aminobutyrophenone side chains, a series of potent 5HT2A and D2 antagonists were identified, as represented by compound 4.22 In an effort to develop more efficacious and safer antipsychotics based upon these findings, we designed and evaluated a number of polycyclic cores linked with various side chains. By systematically varying ring sizes, heteroatoms, and

■

CHEMISTRY The synthesis of these tetracyclic compounds with various cores and side chains is illustrated in Schemes 1−3. The general synthetic route used for the preparation of compounds listed in Table 1 is depicted in Scheme 1. 3,4-Dihydroquinoxalin-2(1H)one 6 was treated with sodium nitrite and acetic acid to give 4nitroso-3,4-dihydro-quinoxalin-2(1H)-one 7, which was re2671



Article

Table 1. SAR of Compounds with Variations of Substituents on A- and D-Ring

Ki (nM)a

ratio

compd

R1

R2

5-HT2A

5-HT2C

D2

2C/2A

5 31 32 33 34 35 36 37

H H H H H H Br MeO

Me H ethyl isopropyl n-butyl benzyl Me Me

0.54 0.94 2.1 7.5 14 4.7 4.0 3.5

173 280 287 498 240 385 582 1090

32 30 66 ND ND 86 ND 46

320 298 137 66 17 82 146 311

b

D2/2Ac 59 32 31 ND ND 18 ND 13

a Ki values are the means of at least two experiments. bRatio of Ki(5-HT2C)/Ki(5-HT2A). cRatio of Ki(D2)/Ki(5-HT2A). ND stands for not determined.

Scheme 2a

a

Reagents and conditions: (a) 4-piperidone monohydrate hydrochloride, HCl, EtOH, reflux, 6 h, 76%; (b) TFA, Et3SiH, rt, 19 h, 83%; (c) ethyl chloroformate, NEt3, THF, rt, 1 h, 84%; (d) benzophenone imine, Pd2(dba)3, NaOBut, BINAP, toluene, 105 °C, 93%; (e) ethyl bromoacetate, Na2CO3, KI, acetone, reflux, 16 h; (f) 2N HCl, THF, rt, 1.5 h, 73% (two steps); (g) MeI, K2CO3, acetone, 109 °C, 6 h, 99%; (h) BH3-THF, THF, reflux, 3 h, 99%; (i) KOH, nBuOH, 120 °C, 3 h, 99%; (j) R3X, NEt3, dioxane, toluene, reflux, 50−70%; (k) chiral HPLC separation, 30−45%.

duced with zinc in acetic acid and 2-propanol to afford 4amino-3,4-dihydroquinoxalin-2(1H)-one. The one-step construction of the tetracyclic core 8 was accomplished by Fisher-indole cyclization of 4-amino-3,4-dihydroquinoxalin2(1H)-one with ethyl 4-oxopiperidine-1-carboxylate. Subsequent cis-reduction of 8 with sodium cyanoborohydride in trifluoroacetic acid afforded the indoline derivative (cis)-9, which was reacted with various alkyl or benzyl halides to give (cis)-10. The (cis)-2,3,6b,9,10,10a-hexahydro-1H,7H-pyrido[3′,4′:4,5]pyrrolo[1,2,3-de]quinoxaline core, (cis)-12, was produced by reduction of (cis)-10 with borane in THF, followed by deprotection with potassium hydroxide in n-butanol. The para-fluoro butyrophenone side chain was introduced by Nalkylation under basic conditions to furnish the racemic (cis)-

13, which was then resolved by chiral chromatograph to afford the (6bR,10aS)-14 (entries 31−37 and 5, Table 1) and (6bS,10aR)-15 (entries 51 and 54, Table 3). The synthetic route illustrated in Scheme 2 was developed to expedite the side chain SAR development at the piperidinyl ring. 2-Bromophenylhydrazine 16 was treated with 4piperidone to produce the tricyclic indole derivative 17, which upon treatment with triethylsilane in trifluoroacetic acid furnished the racemic indoline derivative (cis)-18. Reaction of (cis)-18 with ethyl chloroformate afforded (cis)-19, which was coupled with benzophenone imine, employing Buchwald− Hartwig cross-coupling method,23 to produce intermediate (cis)-20. N-Substitution of (cis)-20 with ethyl bromoacetate and sodium carbonate, followed by acidic hydrolysis of the 2672



Article

used during the production of 5 and its analogues on large scales. To delineate the SAR of compounds with various tetracyclic cores, we have developed a versatile synthetic route for producing the cores with different ring sizes, heteroatoms, and stereochemistry, as illustrated in Scheme 3. Some of the synthetic methods have been described in a previous paper.22 In general, the tetracyclic indole derivatives 27a−27f were synthesized by Fisher-indole cyclization of 26a−26e with 4piperidone or azepan-4-one. Cis-reduction of 27a−27f with sodium cyanoborohydride or triethylsilane in trifluoroacetic acid, followed by Boc-protection, provided (cis)-28a−28f, which were separated by chiral chromatography, followed by Boc-deprotection to afford the optically pure tetracyclic cores 29a−29f along with the corresponding enantiomers 30a−30f. Reaction of these key intermediates with 4-chloro-1-(4fluorophenyl)butan-1-one afforded compounds 59−65 in Table 3. Compounds 52 and 53 are a pair of trans-enantiomers, which were synthesized using a method similar to the one described in Scheme 1 except for the trans-indole reduction step, which was accomplished by employing the previously reported method utilizing borane and hydrochloric acid.24 The synthetic route for compounds 55 and 56 is similar to the route illustrated in Scheme 2. Azepan-4-one was used in the Fisherindole cyclization step to construct the core with a sevenmembered C-ring. Compounds 57 and 58 were prepared using the synthetic method explicated in Scheme 2 as well. In step g, methyl iodide and sodium hydride were used to achieve the Nmethylation and α-carbon methylation in one step. Mono- or dimethylation of the α-carbon can be controlled by controlling the equivalency of sodium hydride and reaction time.

diphenylketimine moiety and spontaneous ring closure, furnished the tetracyclic intermediate (cis)-21. Subsequent Nmethylation with methyl iodide and reduction with borane in THF afforded (cis)-22. Removal of the ethoxycarbonyl protective group from (cis)-22 gave the tetracyclic core, which was used in subsequent alkylation reactions to introduce various side chains for SAR studies. The obtained racemic (cis)23 was resolved by chiral chromatography to produce the desired (6bR,10aS)-24 (entries 38−50 (Table 2) and 5 (Table Table 2. SAR of Compounds with Variations of Substituents on the C-Ring

■

RESULTS AND DISCUSSION In Vitro Activities and SAR. The binding affinities of synthesized compounds against the human full-length recombinant serotonin 5-HT2A and 5-HT2C receptors, expressed in HEK-293E cells, were determined by radioligand displacement methods. (±)-1-(2,5-Dimethoxy-4-[ 125 I]iodophenyl)-2-aminopropane (125DOI) was used as a radioligand.25,26 Binding affinities of compounds against the rat recombinant dopamine D2 short receptor, expressed in CHO cells, were determined with [3H]-N-methylspiperone as a radioligand. To delineate the SAR around the investigational new drug 5, three representative sets of compounds were selected as summarized in Tables 1−3. The SAR of compounds with different substituents at position 3 of the D-ring and position 5 of the A-ring is summarized in Table 1. N-Methylated compound 5 and its des-methyl analogue 31 exhibit the most potent binding affinities for 5-HT2A receptor and show greater than 200-fold selectivity against 5-HT2C receptor. As the size of the alkyl substituent increases, the binding activity for 5-HT2A decreases (32−34 vs 5 and 31). N-Benzyl substituted compound 35 binds to 5-HT2A, with a Ki of 4.7 nM, which is 9-fold weaker relative to 5. We also prepared analogues of 35 containing various substituents on the benzyl group, and they did not show any improvement in 5-HT2A binding relative to the parent. In general, binding affinity for 5-HT2A decreases as the size and hydrophobicity of a substituent at the 3-position increases. Conversely, the affinity for 5-HT2C is relatively insensitive to the variation of substituents at the position. The high selectivity against 5-HT2C has been suggested to be crucial to minimize potential metabolic side effects associated with

a Ki values are the means of at least two experiments. bPercent of inhibition at 100 nM. cNo inhibition at 100 nM. ND stands for not determined.

1)) and other enantiomer (6bS,10aR)-25 (entry 51, Table 3). Alternatively, racemic (cis)-18 was resolved to give the desired (4aS,9bR)-18 by forming a diastereomeric salt with (S)mandelic acid with >99% enantiomeric excess. Absolute stereochemistry was determined by single crystal X-ray crystallography using (4aR,9bS)-18 (R)-mandelate. The desired (4aS,9bR)-18 was then converted to (6bR,10aS)-22 using the same synthetic sequence. This route was easily scalable and 2673



Article

Table 3. SAR of Compounds with Variations of the Tetracyclic Cores

Ki (nM)a

chirality

ratio

compd

X

a

b

R4

R5

m

n

5-HT2A

5-HT2C

D2

5-HT2C/5-HT2A

D2/5-HT2A

5 51 52b,d 53b 54 55 56 57 58 59 60 61 62 63 64 65

N-Me N-Me N-Me N-Me NH N-Me N-Me N-Me N-Me CH2 CH2 CH2 CH2 S O O

R S rel-S rel-R S Sc Rc R R R S R R R R Sc

S R rel-S rel-R R S R S S S R S S S S S

H H H H H H H H Me H H H H H H H

H H H H H H H Me Me H H H H H H H

1 1 1 1 1 1 1 1 1 1 1 0 2 1 1 1

1 1 1 1 1 2 2 1 1 1 1 1 1 1 1 2

0.54 4.6 >1000 324 5.5 1.7 17 5.7 5.2 1.0 7.7 2.9 1.1 1.3 0.89 14

173 532 >1000 >1000 702 503 1280 280 862 83 110 507 195 150 520 641

32 95 97 52 ND 148 ND ND ND 27 ND 71 ND 50 41 69

320 116 ND >3 128 296 75 49 166 83 14 175 177 115 584 46

59 21 1000 nM. ND stands for not determined.

Scheme 3a

a

Reagents and conditions: (a) 4-piperidone hydrochloride monohydrate or azepan-4-one, HCl, EtOH, reflux, 50−80%; (b) TFA, NaBH3CN or Et3SiH, 80−95%; (c) Boc2O, aq NaOH, dioxane, rt, 80−90%; (d) chiral HPLC separation, 30−45%; (e) TFA, CH2Cl2, rt, 90−99%.

tetracyclic core unit binds, is the most restrictive among the receptors that were tested. Compound 5 exhibits the highest binding affinities for both 5-HT2A and D2 receptors, with a D2/ 5-HT2A affinity ratio of 59. To explore the SAR of compounds with different side chains at position 8 of the C-ring, analogues 38−50 were synthesized, as listed in Table 2. Comparing compound 5 with 38, the 4′fluoro group on the butyrophenone side chain increases the 5HT2A binding potency by 11-fold and improves selectivity against 5-HT2C by 21-fold. Compound 39, containing a 4pyridyl headpiece on the side chain, maintains similar 5-HT2A

antagonizing the receptor. Compound 5 exhibits 320-fold higher affinity for 5-HT2A receptor relative to 5-HT2C. These results suggest that a small alkyl group, such as a methyl group, is optimal at the position for better 5-HT2A binding potency and selectivity against 5-HT2C. Substitution at position 5 of the A-ring is generally unfavorable for 5-HT2A binding (36 and 37 vs 5). Compounds with potent 5-HT2A binding affinity were also tested in dopamine D2 binding assays, and they show comparable potency regardless of the substituents at positions 3 and 5. The SAR trends described above suggest that the 5HT2A receptor subsite, where the position 3 and 5 region of the 2674



Article

Table 4. Receptor Profiling of Selected Compoundsa serotonin receptors

adrenergic receptors

ratio

dopamine receptors

ratio

compd

5-HT2A

5-HT1A

5-HT2C

2C/2A

SERT

H1

α1A

α1B

D1

D2

D4

D2/5-HT2A

5 31 43 44 59 64 clozapine olanzapine

0.54 0.94 3.3 2.5 1.0 0.89 9.6 2.5

1480 ND ND 364 206 1330 140 2720

173 280 287 300 83 520 13 7.1

320 298 87 120 83 584 1.4 3

61 31−78 ND ND 98 ND 3900c >15000c

>1000 12%b ND ND 9%b ND 1d 2d

73 ND 314 ND ND 262 19 60

31 ND 294 66 ND 104 12e 69e

52 110 ND ND ND ND 290c 52c

32 30 174 192 27 41 190 31

108 ND ND ND 60 320 40 28

59 32 53 77 27 46 20 12

a Ki (nM) values are the means of at least two experiments. bPercent of inhibition at 100 nM. cData from ref 28. dData from ref 29. eData from ref 30. ND stands for not determined.

of these tetracyclic compounds for SERT is similar to that of the 5-HT2A receptor. Compounds with nonaromatic heterocycles did not show any SERT inhibitory activity (40, 49, and 50). Compound 5 exhibits more potent SERT inhibition than 42, which contains the ethylcarbamoyl linker. However, the propoxy linker containing compound 41 demonstrates improved SERT inhibitory activity while showing reduced binding potency to 5-HT2A and D2 receptors relative to 5. The isosteric indazole analogue 45 exhibits comparable SERT and D2 receptor binding potencies to phenone 5 but 33-fold weaker 5-HT2A binding affinity than 5. As a result, 45 binds to the 5HT2A, D2, and SERT with the double-digit nanomolar Ki’s. Intriguingly, we also identified potent and selective SERT inhibitors, 47 and 48, by incorporating 6-chloro-indolin-2-one and 2,8-dimethyl-pyrido[1,2-a]pyrimidin-4-one headpieces using an ethyl linker. Compound 47 inhibits SERT with a Ki of 28 nM without significant binding to 5-HT2A, 5-HT2C, and D2 receptors. These results suggest that binding affinity and selectivity profiles of these tetracyclic compounds among 5HT2A, 5-HT2C, D2, and SERT can be customized by the side chains. To obtain a preferred binding profile, a butyryl linker and an aromatic pharmacophore on the side chain are required, as exemplified by compound 5. Effects on receptor binding profiles by structural modifications of the core unit were explored by varying ring size, heteroatoms, and stereochemistry at different parts of the tetracyclic core, as exemplified in Table 3. Compound 5 has a quinoxaline core with absolute configuration of (6bR,10aS). Its enantiomer 51 is 9-fold weaker in 5-HT2A binding affinity and 3-fold weaker in D2 binding potency. Similarly, the des-methyl analogue 31 with the preferred (6bR,10aS) stereochemistry is more potent than its enantiomer 54. Compounds 52 and 53 are a pair of trans-stereoisomers of 5. Both compounds exhibit significantly weaker 5-HT2A binding affinity relative to 5. Notably, compound 52 selectively binds to D2 receptor with a Ki of 97 nM, while its binding affinity to 5-HT2A, 5-HT2c, and SERT becomes insignificant. Expanding the C-ring of the tetracyclic core into a seven-membered ring decreases 5-HT2A and D2 receptor binding potency by 3−5-fold (55 vs 5). 55, with an equivalent configuration at the B/C ring junction as 5, is also preferred by the 5-HT2A receptor and shows 10-fold tighter binding than its enantiomer 56. Methylations at the position 1 of the D-ring weaken the 5-HT2A binding potency (57 and 58 vs 5). Replacement of the nitrogen at position 3 of the D-ring with a carbon leads to a tetracyclic quinoline scaffold. In comparison with the quinoxaline derivative 5, the corresponding quinoline analogue 59 gives comparable 5-HT2A

potency while showing significantly improved selectivity over both 5-HT2C and D2 receptors relative to analogue 38. Compounds 40, 49, and 50, containing nonaromatic side chain headpieces, exhibit total loss of binding affinities for all tested receptors. This observation suggests that an aromatic ring on the side chain is a critical component to maintain potent binding. According to a homology model of the human D2 receptor in complex with compound 5, the butyrophenone side chain has both π−π and hydrophobic interactions with hydrophobic residues, including Trp386, Phe389, Phe390, and Ile184. This model is shown in Figure S-2 of the Supporting Information. According to this model, these interactions would be significantly weakened if the phenyl ring on the side chain was replaced with a cycloalkyl group, which is consistent with the observed SAR summarized in Table 2. The linker unit between the tetracyclic quinoxaline core and the aromatic headpiece on the side chain also has a significant impact on receptor binding profiles. The butyryl linker maximizes binding potency for 5-HT2A and D2 receptors and selectivity against 5HT2C compared to other linkers (5 vs 41, 42, and 46). Remarkably, compound 5 with the butyryl linker exhibits 1200and 24-fold superior binding for 5-HT2A and D2 receptors over the ethylcarbamoyl linker containing compound 42. Replacing the carbonyl group in the butyryl linker with oxygen or restraining the side chain flexibility by forming a dihydroisoxazole ring leads to weaker 5-HT2A binding (41 and 46 vs 5). Compounds 43, 44, and 45 contain bioisosteres of the phenone pharmacophore. Indeed, compound 43 shows similar potency for 5-HT2A compared to the parent 38, while 44 is 5−6-fold weaker in both 5-HT2A and D2 binding potency than 5. Interestingly, the indazole analogue 45 exhibits 7-fold enhanced binding affinity to D2 receptor but a similar degree of reduced 5-HT2A activity relative to benzoisoxazole 44 to become almost equipotent for both receptors. During the development of 5, we found several of these pyrido[3′,4′:4,5]pyrrolo[1,2,3-de]quinoxalines also potently inhibited serotonin transporter (SERT), a major target for most antidepressant drugs. Depression is a prominent symptom that can be associated with schizophrenia. SERT inhibitory activity in antipsychotic drugs could add beneficial effects in the treatment of schizophrenia. The atypical antipsychotic drug aripiprazole, which has been approved by FDA as an adjunct to treat major depressive disorders, also has some SERT inhibitory activity.27 As shown in Table 2, compound 5 has a Ki of 61 nM for SERT inhibition. The binding affinity against SERT in human platelet membranes was determined using [3H]-Nmethyl-citalopram as a radioligand. The overall side chain SAR 2675



Article

ogy in humans depending on the dose administered. 5 rapidly penetrates into the brain and engages targets in human brain in a dose dependent manner as estimated by receptor occupancy studies using positron emission tomography, first saturating the 5-HT2A receptors at low doses and then occupying dopamine and serotonin transporters at higher doses. The details of receptor occupancy studies will be published elsewhere. In the HEK-293E cells expressing human recombinant 5HT2A receptors, compound 5 was found to antagonize serotonin (30 nM) induced increase in calcium fluorescence with an IC50 of approximately 7 nM.33 In the same cell system, but measuring serotonin induced [3H]-inositol phosphate accumulation,34 compound 5 inhibited second messenger formation by right-shifting the serotonin dose−response curve. These assay results indicate compound 5 acts as a 5HT2A antagonist. Compound 5 was found to block europiumlabeled GTP analogue (Eu-GTP) binding to 10 nM 2-(Nphenethyl-N-propyl)amino-5-hydroxytetralin (PPHT)-activated G proteins in CHO cells expressing human recombinant D2-short dopamine receptors.35 100% antagonism was observed at 10 μM of compound 5. At this concentration, in the absence of dopamine, 0% activation of the system was seen with compound 5 alone at up to 1 μM concentration, indicating a pure antagonist nature of compound 5 in this system. Compound 5 was evaluated for inhibition of [3H]-serotonin uptake in whole human platelets cells, an indication of transport inhibition, and showed potent inhibition with a Ki of 72 nM. Overall, therefore, compound 5 displayed strong antagonist properties versus 5-HT2A and D2 receptors as evaluated in these systems, as well as potent inhibition of SERT. By virtue of its unique pharmacological profile and superior selectivity against undesired targets, this putative antipsychotic 5 is being developed for multiple indications and is expected to have good tolerability. In Vivo Studies. The in vivo efficacy of compound 5 and selected analogues was evaluated in the rat quipazine head twitch model. This model measures blockade of head twitches induced by quipazine which is a 5-HT2A agonist.36 As shown in Table 5, compounds 5 and 31 have ED50 values of 0.12 and

binding affinity but lower selectivity against 5-HT2C with no change of D2 binding potency. Its enantiomer 60 is weaker in 5HT2A binding and less selective against 5-HT2C, which is consistent with the SAR trends of the quinoxaline series. Shrinking or expanding the size of the D-ring has little effect on 5-HT2A binding potency while providing a marginal improvement in 5-HT2C selectivity (61 and 62 vs 59). Benzothiazine and benzoxazine derivatives, 63 and 64, are slightly weaker in 5HT2A and D2 binding than the quinoxaline 5. Finally, benzoxazine 65 with a seven-membered C-ring exhibits weaker 5-HT2A binding affinity with reduced 5-HT2C selectivity in comparison with 64, while both compounds have similar D2 binding potency. The SAR of these compounds suggests that the stereochemistry of the tetracyclic core has a profound impact on compound receptor binding profiles, and an absolute configuration of (6bR,10aS) at the junction of the B/C rings is preferred. Among different tetracyclic ring systems illustrated in Table 3, the 2,3,6b,9,10,10a-hexahydro-1H,7H-pyrido[3′,4′:4,5]pyrrolo[1,2,3-de]quinoxaline core was found to provide compounds with potent 5-HT2A and D2 binding affinity as well as good selectivity against 5-HT2C, as exemplified by 5. By systematic optimization of the core and side chain, we have also identified selective SERT inhibitor 47 and compound 52 that selectively binds to D2 receptor. Most importantly, this work led to the discovery of compound 5, which exhibiting an optimal receptor binding profile as an antipsychotic drug candidate. Receptor Profiling. Compound 5 and selected analogues were profiled against a panel of receptors, transporters, ion channels, and enzymes. Targets inhibited by more than 50% at 100 nM of 5 are included in the Table 4. The complete list of targets and inhibition data are provided in the Supporting Information. Histamine H1 receptor binding data are also included in the table in order to compare with the representative atypical antipsychotics, clozapine and olanzapine. 5 and its close analogues have similar receptor binding profiles, with 5 possessing a relatively better overall profile. In comparison with clozapine and olanzapine, 5 and its analogues demonstrate much better selectivity against 5-HT2C and H1 receptors. Binding to these targets are thought to contribute to metabolic side effects and weight gain.16−18 Clozapine actually possesses higher binding affinity for H1 and 5-HT2C receptors relative to the therapeutic targets, 5-HT2A and D2 receptors. Olanzapine binds to H1, 5-HT2C, and 5-HT2A in similar potency, while 5 demonstrates >1800-fold selectivity against H1 relative to 5-HT2A and 320-fold selectivity over 5-HT2C, which suggests 5 is expected to have a more favorable metabolic side effect profile than these antipsychotics. The ratio of affinity for adrenergic α1A and α1B receptors to that of serotonin 5-HT2A receptor for compound 5 (135 and 57, respectively) is far greater than the ratios for clozapine (2.0 and 1.3, respectively) and olanzapine (24 and 28, respectively). This indicates that compound 5 should interact with serotonin 5-HT2A receptor without significantly interacting with adrenergic α1A and α1B receptors, predicting less liability for potential side effects associated with adrenoceptors.31 Notably, the 5-HT2A binding affinity of 5 is 59-fold more potent relative to D2 receptors. The high D2/5-HT2A ratio will result in differential dose-dependent engagement of 5-HT2A and D2 receptors by compound 5 and should enable this putative antipsychotic to be used in the treatment of other disorders including insomnia, agitation, aggression, and depression at low doses.32 In keeping with this concept, we have shown that 5 exhibits differential pharmacol-

Table 5. In Vivo Efficacy of Selected Compounds in the Rat Quipazine Head Twitch Modela compd

ED50 (mg/kg)

compd

ED50 (mg/kg)

5 31 32 36 clozapine

0.12 0.08 0.99 0.66 0.92

39 41 43 64 olanzapine

1.8 1.1 1.0 0.25 0.20

a

Test compound suspended in 0.25% methylcellulose was dosed to animals orally by gavage. Assay protocol was described in the Experimental Section.

0.08 mg/kg, respectively, which are more efficacious than their analogues and the reference compounds, clozapine and olanzapine. The relatively weaker efficacy of analogues is nicely correlated with in vitro 5-HT2A binding potency. Compound 5 was also evaluated in the rat conditioned avoidance response model37,38 and was found to show good efficacy (ED50 = 1.5 mg/kg). Inhibition of conditioned avoidance behavior without affecting escape behavior, as observed with compound 5, predicts antipsychotic efficacy consistent with D2 receptor antagonism. Tested up to a dose of 30 mg/kg, compound 5 2676



■

never produced frank catalepsy as tested in a step-down latency model in rats.39 In contrast, the positive control, haloperidol, produced a full cataleptic response across a similar dose range where haloperidol showed efficacy in the conditioned avoidance response test. Compound 5 showed a wide separation between its antipsychotic ED50 in conditioned avoidance and doses that increased step-down latency. These data predict antipsychotic efficacy with compound 5 and a low liability for extrapyramidal motor side effects. Properties of 5. Compound 5 is being developed as a tosylate salt, displaying a plate-like crystalline material. This salt can be consistently produced as a nonhygroscopic, nonhydrated, and nonsolvated single polymorph and has demonstrated good stability under stressed conditions. Compound 5 has good physicochemical and biophysical properties, as summarized in Table 6. This compound is highly

a

3.38 0.55 6.5; 9.1 0.33 97.4 15.8 1.3

Apical-to-basolateral permeability in MDR1-MDCK cells. ratio (Papp B→A)/(Papp A→B).

b

EXPERIMENTAL SECTION

Chemistry. Unless otherwise noted, all materials were obtained from commercial suppliers and used without further purification. All reactions involving air- or moisture-sensitive reagents were performed under an argon atmosphere. All microwave assisted reactions were conducted with an Initiator Eight EXP microwave system from Biotage, Charlotte, NC, USA. Silica gel or alumina column chromatography was performed with a CombiFlash Companion purification system from Teledyne ISCO, Lincoln, NE. Filtrations were generally performed with Waterman paper filters. All products, unless otherwise noted, were purified by column chromatography and/or preparative reverse-phase HPLC. A Waters preparative HPLC system equipped with a Delta 600EF pump, 2996 PDA detector, and WFCIII fraction collector was used for compound purification using the following general HPLC method. Column: Gemini, AXIA packed, 10 μm C18 110 Å, 250 mm × 21.2 mm; mobile phase A, 0.1% formic acid in water; mobile phase B, 0.1% formic acid in acetonitrile; gradient was adjusted and optimized based on compound polarity; HPLC run time was 22 min; flow rate was 24 mL/min; detection was at 210−350 nm. Purification of optically pure enantiomers was conducted on semipreparative Chiralpak AD-H or Chiralcel OD chiral columns with ethanol or 2-propanol−hexanes as an eluent. 1H NMR spectra were determined in the cited solvent on a Bruker DRX 300 or Avance III (400 or 500 MHz) spectrometer. 13C NMR spectra were recorded at 75 or 126 MHz. Chemical shifts are reported in delta (δ) units, parts per million (ppm) downfield from tetramethylsilane. Coupling constants are reported in hertz (Hz). Splitting patterns are designated as follows: s, singlet; br, broad singlet; d, doublet; t, triplet; q, quartet; m, multiplet. Purity of all final products was >95% as determined by reverse-phase UPLC/HPLC using method A and/or method B. Method A (UPLC): Waters ACQUITY UPLC system, ACQUITY HSS T3 column, 50 mm × 2.1 mm, 1.8 μm, 25 °C; mobile phase A, 0.1% formic acid in water/acetonitrile (95/5); mobile phase B, 0.1% formic acid in acetonitrile; gradient, 0.0−3.0 min, 5−95% B; 3.0−4.0 min, 95% B; 4.0−5.0 min, 95−5% B; flow rate 0.3 mL/min; detection at 210−400 nm. Method B (HPLC), Waters Alliance HT 2795 system, Ace 5 C18 column, 150 mm × 4.6 mm, 5 μm, 30 °C; mobile phase A, 0.1% HClO4 in water/acetonitrile (95/5); mobile phase B, 0.1% HClO4 in acetonitrile; gradient, 0.0−2.0 min, 0% B; 2.0−22 min, 0−100% B; 22−25 min, 100% B; flow rate 1.5 mL/min; detection at 254 nm and/or 280 nm. Chiral purity was determined by HPLC on a Daicel Chiralpak AD-H 4.6 mm × 250 mm chiral column; mobile phase, ethanol at 0.7 mL/min; UV detection at 250 and 280 nm. Mass spectral (MS) data were determined on a Micromass Quattro micro API or LCT Premier XE mass spectrometer from Waters. High-resolution mass spectral (HRMS) data were determined on the LCT Premier XE mass spectrometer. The chemical yields reported below are not optimized and correspond to specific examples of one preparation. Representative Synthetic Procedures of the Tetracyclic Butyrophenone Derivatives Shown in Table 1 via Scheme 1. 4-((6bR,10aS)-3-Methyl-2,3,6b,9,10,10a-hexahydro-1H,7Hpyrido[3′,4′:4,5]pyrrolo[1,2,3-de]quinoxalin-8-yl)-1-(4-fluorophenyl)-butan-1-one 4-methylbenzenesulfonate (5). Step a: 4Nitroso-3,4-dihydroquinoxalin-2(1H)-one (7; R1 = H). A solution of 3,4-dihydroquinoxalin-2(1H)-one 6 (9.5 g, 64 mmol) in acetic acid (100 mL) and water (50 mL) was cooled to 5 °C, and then a solution of NaNO2 (4.6 g, 67 mmol) in water (20 mL) was added dropwise. The reaction mixture was stirred at 0−5 °C for an hour and then filtered. The filter cake was washed with water and then dried under vacuum to give the title compound (7.6 g, 67% yield). 1H NMR (DMSO-d6, 400 MHz) δ 10.96 (s, 1H), 7.94 (dd, J = 8.2, 1.3 Hz, 1H), 7.40−7.26 (m, 1H), 7.20−7.06 (m, 2H), 4.53 (s, 2H). Steps b and c: 2-Oxo-2,3,9,10-tetrahydro-1H,7H-pyrido[3′,4′:4,5]pyrrolo[1,2,3-de]quinoxaline-8-carboxylic Acid Ethyl Ester (8; R1 = H). A suspension of 7 (4.0 g, 23 mmol) in acetic acid (60 mL) was cooled to 5−10 °C, and then zinc powder (8.0 g, 122 mmol) was added in portions. The reaction mixture was stirred at 10−20 °C until all of the starting material had been consumed. Ethyl 4-oxopiperidine1-carboxylate (3.4 mL, 22.6 mmol) was added to the reaction mixture,

Table 6. Properties of Compound 5 Log D at pH 7.4 Log D at pH 1.7 pKa (free base) solubility in PBS at pH 7.4 (mg/mL) human plasma protein binding (%) permeability, AB (× 10−6 cm/s)a efflux ratio BA/ABb

Article

Efflux

permeable and is not a p-glycoprotein substrate, as determined in the bidirectional permeability assays in the MDR1-trnsfected MDCK cells. Pharmacokinetic properties of 5 in rat, dog, and human subjects have been extensively studied, and good pharmacokinetic characteristic were demonstrated. Compound 5 is safe and well tolerated after wide range of single and multiple once daily doses in normal volunteers and patients with schizophrenia. Also, in phase II studies, low doses of 5, predominantly occupying 5-HT2A receptors, have been shown to increase slow wave sleep and reduce waking after sleep onset while restoring normal sleep architecture in patients with sleep maintenance insomnia. At a higher dose, compound 5 demonstrated antipsychotic efficacy in a phase II clinical trial in patients with acutely exacerbated schizophrenia.

■

CONCLUSION A new series of tetracyclic quinoxaline derivatives has been synthesized and selected candidates have been evaluated as potential new antipsychotic agents. Among this series, compound 5 is a potent serotonin 5-HT2A and dopamine D2 antagonist with high affinity for SERT. This compound has a desirable selectivity profile against other receptors, such as serotonin 5-HT2C, histamine H1, and adrenergic α1A receptors, which are known to be associated with adverse effects of marketed antipsychotics. The high D2/5-HT2A ratio of 5 provides dose-dependent engagement of 5-HT2A and D2 receptors and enables 5 to be used both in treating psychiatric illness such as schizophrenia at high doses and in treating other disorders, such as sleep or mood alterations, at low doses. 5 has suitable physicochemical, biophysical, and pharmacokinetic properties as a CNS-active drug candidate. The human clinical trials of 5 (ITI-007)40 are ongoing, and the results will be reported in due course. 2677



Article

and then the mixture was filtered. The filter cake was rinsed with acetic acid (4 × 50 mL). The filtrate was combined, followed by adding 1.0 M HCl in ether (1 mL). The mixture was heated at 100 °C for an hour. Acetic acid was removed under reduced pressure, and the residue was treated with ethanol (20 mL). After filtration, the filter cake was washed with cold methanol (2 × 5 mL) to give the title compound (2.8 g, 42% yield) as a white solid. 1H NMR (CDCl3, 500 MHz) δ 7.76 (s, 1H). 7.14 (d, J = 8.0 Hz, 1H), 7.02−6.92 (m, 1H), 6.52 (d, J = 7.4 Hz, 1H), 4.87 (s, 2H), 4.79−4.61 (m, 2H), 4.21(q, J = 7.1 Hz, 2H), 3.97−3.79 (m, 2H), 2.89−2.70 (m, 2H), 1.31 (t, J = 7.1 Hz, 3H). MS (ESI) m/z 300.1 [M + H]+. Step d: (cis-)-2-Oxo-2,3,6b,9,10,10a-hexahydro-1H,7H-pyrido[3′,4′:4,5]pyrrolo[1,2,3-de]quinoxaline-8-carboxylic Acid Ethyl Ester (cis-9; R1 = H). To a vigorously stirred solution of 8 (12 g, 40 mmol) in trifluoroacetic acid (125 mL) was added sodium cyanoborohydride (4.0 g, 65 mmol) in small portions at 0−5 °C. After the addition was complete, the reaction mixture was stirred at room temperature for 30 min and then poured slowly into ammonium hydroxide (300 mL) containing ice, followed by the addition of enough 1N NaOH to make the mixture basic. The mixture was extracted with CH2Cl2 twice. The combined organic phase was washed with water, dried over MgSO4, concentrated, and dried under vacuum to give the title compound (10.9 g, 90% yield) as an off-white powder. 1H NMR (DMSO-d6, 500 MHz) δ 10.37 (s, 1H), 6.81 (d, J = 7.3 Hz, 1H), 6.65 (t, J = 7.5 Hz, 1H), 6.59 (d, J = 7.7 Hz, 1H), 4.09−3.98 (m, 2H), 3.97−3.85 (m, 1H), 3.84 (d, J = 14.6 Hz, 1H), 3.65 (dt, J = 13.2, 4.3 Hz, 1H), 3.51− 3.42 (m, 1H), 3.38 (d, J = 14.5 Hz, 1H), 3.31−3.23 (m, 1H), 3.20− 3.02 (m, 1H), 2.95−2.59 (m, 1H), 2.03−1.88 (m, 1H), 1.85−1.72 (m, 1H), 1.18 (t, J = 7.1 Hz, 3H). MS (ESI) m/z 302.2 [M + H]+. Step e: (cis)-3-Methyl-2-oxo-2,3,6b,9,10,10a-hexahydro-1H,7Hpyrido[3′,4′:4,5]pyrrolo[1,2,3-de]quinoxaline-8-carboxylic Acid Ethyl Ester (cis-10; R1 = H, R2 = Me). Sodium hydride (900 mg of 60% dispersion in mineral oil, 22.5 mmol) was washed with hexanes and suspended in anhydrous DMF (5 mL). The suspension was added to a stirred solution of cis-9 (6.02 g, 20 mmol) in anhydrous DMF (50 mL) under nitrogen atmosphere. After gas evolution had subsided, the mixture was cooled in an ice−water bath and treated with methyl iodide (3.55 g, 25 mmol). The mixture was stirred at room temperature for an hour and then concentrated. The residue was treated with water and extracted with CH2Cl2 twice. The combined organic phase was washed with brine, dried over MgSO4, and evaporated to dryness to give the title compound (5.48 g, 87% yield) as a tan solid. 1H NMR (DMSO-d6, 500 MHz,) δ 6.92 (d, J = 7.3 Hz, 1H), 6.86 (d, J = 7.9 Hz, 1H), 6.77 (t, J = 7.7 Hz, 1H), 4.09−3.85 (m, 4H), 3.67 (dt, J = 13.6, 4.2 Hz, 1H), 3.51−3.42 (m, 2H), 3.35−3.26 (m, 1H), 3.22 (s, 3H), 3.17−3.01 (m, 1H), 2.90−2.61 (m, 1H), 2.01− 1.91 (m, 1H), 1.87−1.74 (m, 1H), 1.18 (t, J = 7.1 Hz, 3H). MS (ESI) m/z 316.1 [M + H]+. Step f: (cis)-3-Methyl-2,3,6b,9,10,10a-hexahydro-1H,7H-pyrido[3′,4′:4,5]pyrrolo[1,2,3-de]quinoxaline-8-carboxylic Acid Ethyl Ester (cis-11; R1 = H, R2 = Me). A solution of borane in THF (1.0 M, 33 mL, 33 mmol) was added dropwise to a stirred solution of cis-10 (5.24 g, 16.6 mmol) in anhydrous THF (25 mL) under nitrogen atmosphere. After the addition was complete, the mixture was stirred and heated at reflux for an hour and then cooled and treated with 6N HCl (15 mL). The mixture was then heated under reflux for 30 min, cooled, and evaporated to dryness under reduced pressure. The residue was dissolved in a minimum quantity of water, and the solution was basified with 1N NaOH and then extracted with CH2Cl2 twice. The combined organic phase was washed with water, dried over MgSO4, concentrated, and dried under vacuum to give the title compound (4.65 g, 93% yield) as a viscous oil. 1H NMR (CDCl3, 300 MHz) δ 6.67 (t, J = 8.1 Hz, 1H), 6.61 (d, J = 8.1 Hz, 1H), 6.41 (d, J = 8.1 Hz, 1H), 4.16 (q, J = 7.0 Hz, 2H), 3.87−3.79 (m, 1H), 3.75−3.56 (m, 2H), 3.38−3.26 (m, 2H), 3.26−3.05 (m, 2H), 2.90−2.81 (m, 5H), 1.93−1.78 (m, 2H), 1.78−1.68 (m, 1H), 1.28 (t, J = 7.0 Hz, 3H). MS (ESI) m/z 302.3 [M + H]+. Step g: (cis)-3-Methyl-2,3,6b,7,8,9,10,10a-octahydro-1H-pyrido[3′,4′:4,5]pyrrolo[1,2,3-de]quinoxaline (cis-12; R1 = H, R2 = Me). To a stirred solution of cis-11 (4.52 g, 15.0 mmol) in warm n-butanol (50

mL) was added powdered KOH (10.0 g, 178 mmol). The reaction mixture was heated under reflux for 5 h and then evaporated under reduced pressure. The residue was treated with water and extracted with CH2Cl2 twice. The combined organic phase was washed with water, dried over MgSO4, concentrated, and dried under vacuum to give the title compound (3.27 g, 95% yield) as a viscous oil. 1H NMR (CDC13, 300 MHz) δ 6.65 (t, J = 7.3 Hz, 1H), 6.51 (d, J = 7.3 Hz, 1H), 6.41 (d, J = 7.3 Hz, 1H), 3.64−3.55 (m, 1H), 3.38−3.26 (m, 3H), 3.12−2.95 (m, 2H), 2.95−2.80 (m, 6H), 2.71−2.57 (m, 1H), 1.93−1.74 (m, 3H). MS (ESI) m/z 230.2 [M + H]+. Step h: (cis)-4-(3-Methyl-2,3,6b,9,10,10a-hexahydro-1H,7Hpyrido[3′,4′:4,5]pyrrolo[1,2,3-de]quinoxalin-8-yl)-1-(4-fluorophenyl)-butan-1-one (cis-13; R1 = H, R2 = Me). A suspension of cis-12 (11.7 g, 51 mmol), 4-chloro-4′-fluoro-butyrophenone (15 g, 75 mmol), triethylamine (30 mL, 214 mmol), and KI (12.6 g, 76 mmol) in dioxane (65 mL) and toluene (65 mL) was refluxed for 7 h. After filtration and evaporation of the solvent, CH2Cl2 (200 mL) was added to dissolve the residue. The CH2Cl2 solution was washed with brine (2 × 200 mL), dried over anhydrous Na2SO4, and concentrated to ca. 55 mL and then added dropwise to a 0.5 N HCl ether solution (600 mL). The resulting brown solid was filtered, washed with ether, and then dissolved in water. The aqueous solution was basified with 2 N NaOH and extracted with CH2Cl2 (2 × 100 mL). The combined organic phase was washed with brine (2 × 200 mL) and dried over anhydrous Na2SO4. Evaporation of the solvent and chromatography of the residue through silica gel gave the title compound (11.5 g, 57% yield) as a brown oil. 1H NMR (CDCl3, 400 MHz) δ 8.01−7.98 (m, 2H). 7.14−7.10 (m, 2H), 6.64 (t, J = 7.6 Hz, 1H), 6.50 (d, J = 7.2 Hz, 1H), 6.40 (d, J = 7.6 Hz, 1H), 3.62−3.55 (m, 1H), 3.31−3.24 (m, 2H), 3.21−3.17 (m, 1H), 3.10−3.04 (m, 1H), 2.96−2.90 (m, 2H), 2.88− 2.78 (m, 5H), 2.67−2.61 (m, 1H), 2.44−2.32 (m, 2H), 2.27−2.20 (m, 1H), 2.00−1.77 (m, 5H). MS (ESI) m/z 394.2 [M + H]+. Step i: 4-((6bR,10aS)-3-Methyl-2,3,6b,9,10,10a-hexahydro1H,7H-pyrido[3′,4′:4,5]pyrrolo[1,2,3-de]quinoxalin-8-yl)-1-(4-fluorophenyl)-butan-1-one 4- methylbenzenesulfonate (5). cis-13 (1.0 g, 2.5 mmol) was dissolved in ethanol (5 mL) and resolved by HPLC on a Chiral AD-H column (2.0 cm × 25 cm) eluting with ethanol. The early eluting peak was collected and concentrated to give the free base form of 5 (0.48 g, 48% yield) as a dense oil. The free base was dissolved in 2-propanol (2 mL), and then a solution of 4methylbenzenesulfonic acid (200 mg, 1.16 mmol) in 2-propanol (1.0 mL) was added slowly at 0 °C. The mixture was stirred at room temperature overnight and then filtered. The filter cake was rinsed with 2-propanol and then dried under vacuum to give 5 (367 mg, 53% yield) as a gray solid. 1H NMR (DMSO-d6, 500 MHz) δ 9.10 (br, 1H), 8.10−8.01 (m, 2H), 7.48 (d, J = 8.0 Hz, 2H), 7.42−7.33 (m, 2H), 7.11 (d, J = 7.8 Hz, 2H), 6.65−6.57 (m, 1H), 6.51 (d, J = 7.3 Hz, 1H), 6.42 (d, J = 7.9 Hz, 1H), 3.59 (dd, J = 12.2, 6.5 Hz, 1H), 3.52−3.37 (m, 3H), 3.37−3.28 (m, 2H), 3.25−3.20 (m, 1H), 3.18−2.99 (m, 5H), 2.81 (s, 3H), 2.71 (td, J = 10.2, 3.0 Hz, 1H), 2.63−2.52 (m, 1H), 2.28 (s, 3H), 2.27−2.22 (m, 1H), 2.15−1.93 (m, 3H). 13C NMR (DMSOd6, 126 MHz) δ 197.2, 165.1 (d, JCF = 252 Hz), 145.6, 137.6, 137.3, 135.2, 133.1, 130.9 (d, JCF = 10 Hz), 128.1, 126.7, 125.5, 120.6, 115.7 (d, JCF = 22 Hz), 112.5, 109.3, 62.2, 55.5, 52.5, 49.8, 47.8, 43.7, 38.6, 37.0, 34.9, 21.7, 20.8, 18.0. MS (ESI) m/z 394.2 [M + H]+. HRMS (ESI) m/z calcd for C24H29FN3O [M + H]+, 394.2295; found, 394.2292. UPLC purity, 97.7%; retention time, 2.06 min (method A). Representative Synthetic Procedures of Tetracyclic Derivatives Shown in Table 2 via Scheme 2. (6bR,10aS)-8-[3-(4Fluorophenoxy)propyl]-3-methyl-2,3,6b,7,8,9,10,10a-octahydro-1H-pyrido[3′,4′:4,5]pyrrolo[1,2,3-de]quinoxaline (41). Step a: 6-Bromo-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole Hydrochloride (17). A 1 L, three-neck flask was equipped with an overhead stirrer, a type-J Teflon covered thermocouple, and a reflux condenser with a N2 inlet. The flask was charged with (2-bromophenyl) hydrazine hydrochloride (50.0 g, 224 mmol), 4-piperidone monohydrate hydrochloride (36 g, 234 mmol), ethanol (500 mL), and hydrochloric acid (50 mL). The resulting mixture was refluxed for 6 h and then cooled to room temperature. After filtration, the filter cake was washed with ethanol (3 × 50 mL) and then dried under vacuum to give the title compound (49.2 g, 76% yield) as a cream-colored solid. 1H NMR 2678



Article

(DMSO-d6, 500 MHz) δ 11.3 (s, 1H), 9.21 (s, 2H),7.49 (d, J = 7.8 Hz, 1H), 7.32 (d, J = 7.6 Hz, 1H), 6.97 (t, J = 7.7 Hz, 1H), 4.31 (s, 2H), 3.48 (t, J = 6.2 Hz, 2H), 3.05 (t, J = 6.2 Hz, 2H). MS (ESI) m/z 251.0 [M + H]+. Step b: (cis)-6-Bromo-2,3,4,4a,5,9b-hexahydro-1H-pyrido[4,3-b]indole (cis-18). To a suspension of 17 (49.2 g, 171 mmol) in TFA (630 mL) in a 1 L flask, triethylsilane (172 mL, 1.08 mol) was added. The resulting mixture was stirred at room temperature under nitrogen atmosphere for 19 h. TFA and triethylsilane were removed under vacuum. The remaining oil was treated with hexanes (550 mL), and the mixture was stirred at room temperature for an hour, after which time the hexanes was decanted. An additional 250 mL of hexanes was added, stirred for an hour, and then decanted. The remaining oil was basified with 2 N NaOH (ca. 360 mL) until it reached pH = 10, and then it was extracted with CH2Cl2 (2 × 400 mL, 2 × 200 mL). The organic layers were combined and washed with brine (2 × 300 mL) and then dried over anhydrous Na2SO4. Evaporation of the solvent gave the title compound (36 g, 83% yield). 1H NMR (CDCl3, 500 MHz) δ 7.19 (d, J = 8.0 Hz, 1H), 7.01 (d, J = 7.2 Hz, 1H), 6.62 (dd, J = 8.1, 7.2 Hz, 1H), 3.98−3.96 (m, 2H), 3.22−3.16 (m, 1H), 3.06 (dd, J = 13.0, 5.8 Hz, 1H), 2.98 (ddd, J = 12.6, 9.0, 3.6 Hz, 1H), 2.89 (dd, J = 13.0, 7.7 Hz, 1H), 2.82−2.73 (m, 1H), 2.02−1.78 (m, 2H), 1.73− 1.65 (m, 1H). MS (ESI) m/z 253.0 [M + H]+. Step c: (cis)-Ethyl 6-Bromo-3,4,4a,5-tetrahydro-1H-pyrido[4,3b]indole-2(9bH)-carboxylate (cis-19). A suspension of cis-18 (36 g, 142 mmol) and triethylamine (24 mL, 171 mmol) in THF (300 mL) was cooled in an ice−water bath, and then ethyl chloroformate (13.5 mL, 142 mmol) was added dropwise via syringe pump over 30 min. The ice−water bath was removed, and the reaction mixture was stirred at room temperature for another hour. The reaction mixture was passed through a pad of Celite. Evaporation of solvent gave the title compound (39 g, 84% yield)) as a cream-colored solid. 1H NMR (CDCl3, 300 MHz) δ 7.20 (d, J = 8.1 Hz, 1H). 7.04 (d, J = 7.2 Hz, 1H), 6.60 (t, J = 7.7 Hz, 1H), 4.21−3.95 (m, 4H), 3.95−3.66 (br, 1H), 3.66−3.52 (m, 1H), 3.52−3.22 (m, 3H), 1.99−1.85 (m, 1H), 1.85− 1.73 (m, 1H), 1.35−1.20 (m, 3H). MS (ESI) m/z 325.0 [M + H]+. Step d: (cis)-Ethyl 6-(Diphenylmethyleneamino)-3,4,4a,5-tetrahydro-1H-pyrido[4,3-b]indole-2(9bH)-carboxylate (cis-20). cis-19 (26 g, 80 mmol), benzophenone imine (16 g, 88 mmol), t-BuONa (15.1 g, 157 mmol), and BINAP (1.53 g, 2.5 mmol) were placed in a 1 L threeneck round-bottom flask equipped with a condenser and a Teflon covered thermocouple. Toluene (300 mL) was added, and nitrogen was bubbled into the suspension through a steel needle through a hole bored in the septum. The temperature was gradually raised to 60 °C via a heating mantle. The heating mantle was then removed, and the flask was cooled to ambient temperature with nitrogen bubbling. Pd2(dba)3 (830 mg, 0.80 mmol) was added, and the flask was warmed up to 60 °C while nitrogen was bubbled through it. The reaction mixture was heated at 105 °C overnight under nitrogen atmosphere and then cooled and diluted with t-butyl methyl ether (1.25 L). The resulting suspension was passed through a pad of Celite and evaporated to dryness under vacuum to give the title compound (31.5 g, 93% yield) as a brown foamy solid. 1H NMR (DMSO-d6, 500 MHz) δ 7.68 (d, J = 7.4 Hz, 2H), 7.52 (m, 1H), 7.45 (m, 2H), 7.34− 7.27 (m, 3H), 7.22−7.14 (m, 2H), 6.64 (d, J = 7.3 Hz, 1H), 6.29 (t, J = 7.5 Hz, 1H), 6.11 (d, J = 7.8 Hz, 1H), 5.23 (s, 1H), 4.04−3.97 (m, 2H), 3.82 (m, 1H), 3.63 (m, 1H), 3.42 (m, 1H), 3.23−3.12 (m, 2H), 2.97 (m, 1H), 1.81−1.74 (m, 1H), 1.70−1.64 (m, 1H), 1.15 (t, J = 7.0 Hz, 3H). MS (ESI) m/z 426.2 [M + H]+. Steps e and f: (cis)-2-Oxo-2,3,6b,9,10,10a-hexahydro-1H,7Hpyrido[3′,4′:4,5]pyrrolo[1,2,3-de]quinoxaline-8-carboxylic Acid Ethyl Ester (cis-21). A suspension of compound cis-20 (63 g, 148 mmol), ethyl bromoacetate (22 mL, 198 mmol), Na2CO3 (22.6 g, 213 mmol), and KI (30.9 g, 186 mmol) in acetone (1.5 L) was refluxed for 16 h. Acetone was removed under vacuum, and the residue was diluted with CH2Cl2 (700 mL) and then washed with water (500 mL) and brine (200 mL) and dried over Na2SO4. Evaporation of the solvent gave an oil, which was then dissolved in THF (400 mL). Then 2 N HCl (140 mL) was added in portions at room temperature. After the reaction was completed (about 1.5 h), THF was removed under

reduced pressure and the residue was treated with 1 N HCl (200 mL). After filtration, the resulting brown solid was dissolved in CH2Cl2 (250 mL), washed with brine (150 mL), and dried over Na2SO4. Evaporation of the solvent and flash chromatography of the residue on alumina eluting with a gradient of hexanes and ethyl acetate to CH2Cl2 and methanol, gave the title compound (32.5 g, 73% yield). 1 H NMR (CDCl3, 300 MHz) δ 9.46 (s, 1H), 6.85 (d, J = 7.0 Hz, 1H), 6.78−6.67 (m, 2H), 4.23−3.81 (m, 5H), 3.48−3.27 (m, 3H), 3.26− 3.08 (m, 1H), 2.76 (br, 1H), 1.94 (br, 1H), 1.35−1.21 (m, 3H). 13C NMR (CDCl3, 75 MHz) δ 167.6, 155.6, 138.0, 129.3, 123.3, 120.9, 119.2, 113.4, 66.5, 61.6, 52.1, 45.7, 41.4, 39.7, 24.5, 14.9. MS (ESI) m/ z 302.3 [M + H]+. Steps g and h: (cis)-3-Methyl-2,3,6b,9,10,10a-hexahydro-1H,7Hpyrido[3′,4′:4,5]pyrrolo[1,2,3-de]quinoxaline-8-carboxylic Acid Ethyl Ester (cis- 22). cis-21 (17.3 g, 57.5 mmol), K2CO3 (15.8 g, 114 mmol), and methyl iodide (66 mL, 1.06 mol) were placed in a 2 L pressure bottle, and then acetone (500 mL) was added. The bottle was heated in an oil bath (109 °C) for 5.5 h and then cooled to room temperature. After solvents were removed under reduced pressure, the residue was treated with water (200 mL) and then extracted with CH2Cl2 (3 × 200 mL). The combined organic phase was washed with brine (2 × 200 mL), dried over anhydrous Na2SO4, and then evaporated to dryness to provide (cis)-3-methyl-2-oxo-2,3,6b,9,10,10ahexahydro-1H,7H-pyrido[3′,4′:4,5]pyrrolo[1,2,3-de]quinoxaline-8-carboxylic acid ethyl ester (18 g, 99% crude yield), which was taken to the next step without further purification. To a suspension of the above intermediate (18 g, 57 mmol) in THF (50 mL), BH3·THF (1.0 M in THF, 143 mL, 143 mmol) was added slowly at room temperature over 15 min. After the completion of the addition, the reaction mixture was refluxed for 3 h. The reaction mixture was then cooled with an ice−water bath and 6 N HCl (150 mL) was added dropwise. After the THF was removed under reduced pressure, 2 N NaOH was added to adjust the pH to 9. The mixture was extracted with CH2Cl2 (500 mL). The CH2Cl2 layer was washed with brine and water successively, dried over anhydrous Na2SO4, and then evaporated to dryness to give the title compound (17 g, 99% yield) as a dense oil. 1H NMR (CDCl3, 300 MHz) δ 6.66 (t, J = 7.7 Hz, 1H). 6.54 (d, J = 7.2 Hz, 1H), 6.41(d, J = 7.8 Hz, 1H), 4.24−3.78 (m, 4H), 3.64−3.54 (m, 1H), 3.38−3.22 (m, 3H), 3.22−3.02 (m, 2H), 2.92−2.74 (m, 5H), 1.95−1.79 (m, 2H), 1.28 (t, J = 7.0 Hz, 3H). 13C NMR (CDCl3, 75 MHz) δ 155.6, 138.2, 135.1, 128.8, 120.6, 113.3, 109.3, 65.0, 61.4, 50.7, 45.8, 44.4, 41.4, 39.9, 37.7, 24.7, 14.9. MS (ESI) m/z 302.2 [M + H]+. Steps i and j: (cis)-8-[3-(4-Fluorophenoxy)propyl]-3-methyl2,3,6b,7,8,9,10,10a-octahydro-1H-pyrido[3′,4′:4,5]pyrrolo[1,2,3-de]quinoxaline (cis-23, R3 = 3-(4-fluorophenoxy)propyl). cis-22 (17 g, 56 mmol), KOH (12.7 g, 226 mmol), and n-butanol (90 mL) were placed in a 300 mL pressure bottle and heated in an oil bath at 120 °C for 3 h. After n-butanol was removed under vacuum, the residue was treated with water (300 mL) and then extracted with CH2Cl2 (3 × 100 mL). The combined organic phase was washed with brine (2 × 200 mL), dried over anhydrous Na2SO4, and then evaporated to dryness to give (cis)-3-methyl-2,3,6b,7,8,9,10,10a-octahydro-1H-pyrido[3′,4′:4,5]pyrrolo[1,2,3-de]quinoxaline (11.7 g, 91% yield) as a dense oil, which was taken to the next step without further purification. A suspension of the above intermediate (1.2 g, 5.2 mmol), 1-(3chloropropoxy)-4-fluorobenzene (1.4 g, 7.5 mmol), triethylamine (3.0 mL, 21 mmol), and KI (1.3 g, 7.8 mmol) in dioxane (6 mL) and toluene (6 mL) was refluxed for 7 h. The reaction mixture was cooled and then filtered. The filtrate was evaporated, and the residue was dissolved in CH2Cl2 (20 mL), washed with brine twice, dried over Na2SO4, and then filtered. The filtrate was concentrated to one-third of the volume and then added dropwise to a 0.5 N HCl ether solution. The formed solid was collected, washed with ether, and then dissolved in water. The aqueous solution was basified with 2 N NaOH and extracted with CH2Cl2 three times. The combined organic phase was washed with brine twice, dried over Na2SO4, filtered, and then concentrated. The residue was purified by silica-gel flash chromatography to give the title compound (1.1 g, 55% yield) as a dense oil. MS (ESI) m/z 382.2 [M + H]+. 2679



Article

Step k: (6bR,10aS)-8-[3-(4-Fluorophenoxy)propyl]-3-methyl2,3,6b,7,8,9,10,10a-octahydro-1H-pyrido[3′,4′:4,5]pyrrolo[1,2,3-de]quinoxaline (41). cis-23 (100 mg, 0.26 mmol) was dissolved in ethanol and resolved by HPLC using a Chiral AD-H column (2.0 cm × 25 cm) eluting with ethanol containing 0.1% diethylamine. The corresponding peak was collected, concentrated, and dried under vacuum to give the title compound (35 mg, 35% yield) as a pale-yellow oil. 1H NMR (CDCl3, 400 MHz) δ 7.00−6.90 (m, 2H), 6.87−6.78 (m, 2H), 6.65 (t, J = 7.6 Hz, 1H), 6.51 (d, J = 7.3 Hz, 1H), 6.41 (dd, J = 8.0, 1.0 Hz, 1H), 3.97 (t, J = 6.4 Hz, 2H), 3.66−3.54 (m, 1H), 3.37−3.09 (m, 4H), 2.87 (s, 3H), 2.94−2.77 (m, 2H), 2.75−2.60 (m, 1H), 2.58−2.39 (m, 2H), 2.36−2.19 (m, 1H), 2.06−1.83 (m, 5H). 13C NMR (DMSO-d6, 126 MHz) δ 156.4 (d, JCF = 236 Hz), 155.0, 137.7, 134.8, 129.6, 119.7, 115.7 (d, JCF = 23 Hz), 115.6 (d, JCF = 8 Hz), 112.3, 108.6, 66.4, 64.1, 56.1, 54.5, 50.0, 48.8, 43.8, 41.1, 37.1, 26.4, 24.5. MS (ESI) m/z 382.2 [M + H]+. HRMS (ESI) m/z calcd for C23H29FN3O [M + H]+, 382.2295; found, 382.2294. UPLC purity, 98.4%; retention time, 2.08 min (method A). Representative Synthetic Procedures of Tetracyclic Derivatives Shown in Table 3 via Scheme 3. 4-((8aS,12aR)4,5,6,7,9,10,12,12a-Octahydroazepino[3,2,1-hi]pyrido[4,3-b]indol-11(8aH)-yl)-1-(4-fluorophenyl)-1-butanone (62, X = CH2, R4 = R5 = H, m = 2, n = 1, a = R, b = S). Step a: 4,5,6,7,9,10,11,12Octahydroazepino[3,2,1-hi]pyrido[4,3-b]indole (27c). Sodium azide (1.95 g, 30 mmol) was added in small portions to a solution of 3,4dihydro-(2H)-naphthalenone (2.92 g, 20 mmol) in CH3SO3H (50 mL) at 0 °C. The mixture was stirred at 0 °C for 15 min, then 1 h at room temperature and then poured into ice (400 mL). The mixture was basified until pH >8 with 1 N NaOH at 0 °C and extracted with ether (3 × 100 mL). The combined organic phase was dried over MgSO4, concentrated under vacuum, and then purified by silica gel flash column chromatography eluting with 50% ethyl acetate in hexanes to give 1,3,4,5-tetrahydro-2H-1-benzazepin-2-one (2.71 g, 84% yield) as a white solid. 1H NMR (CDCl3, 300 MHz) δ 8.10 (br, 1H), 7.22 (d, J = 7.0 Hz, 2H), 7.13 (td, J = 7.6, 1.5 Hz, 1H),), 6.99 (d, J = 8.1 Hz, 1H), 2.80 (t, J = 7.6 Hz, 2H), 2.36 (t, J = 7.1 Hz, 2H), 2.32−2.18 (m, 2H). A solution of 1,3,4,5-tetrahydro-2H-1-benzazepin-2-one (2.71 g, 16.8 mmol) in THF (40 mL) was added dropwise to a suspension of lithium aluminum hydride (1.27 g, 33.4 mmol) in ether (150 mL) at room temperature. The mixture was refluxed for 16 h. Saturated potassium sodium tartrate salt solution (15 mL) was added to the mixture which was cooled with an ice−water bath. The mixture was stirred for 2 h and the two layers were separated. The aqueous layer was extracted with ether (2 × 25 mL). The combined organic phase was dried over anhydrous Na2SO4, concentrated in vacuum, and purified by flash column chromatography eluting with 30% ethyl acetate in hexanes to give 2,3,4,5-tetrahydro-1H-1-benzazepine (2.40 g, 97% yield) as a yellow liquid. 1H NMR (CDCl3, 300 MHz) δ 7.11 (d, J = 7.4 Hz, 1H). 7.04 (td, J = 7.5, 1.5 Hz, 1H), 6.82 (td, J = 7.3, 1.1 Hz, 1H), 6.74 (dd, J = 7.7, 1.1 Hz, 1H), 3.78 (br, 1H), 3.10−3.00 (m, 2H), 2.82− 2.72 (m, 2H), 1.86−1.72 (m, 2H), 1.70−1.58 (m, 2H). A solution of NaNO2 (1.35 g, 19.6 mmol) in water (4.0 mL) was added dropwise to a solution of 2,3,4,5-tetrahydro-1H-1-benzazepine (2.40 g, 16.3 mmol) in acetic acid (10 mL) at 0−10 °C. The mixture was stirred at 5 °C for 10 min, then at room temperature for 1 h, and then extracted with CH2Cl2 (3 × 20 mL). The combined organic phase was dried over MgSO4, concentrated under vacuum, and then purified by silica gel flash column chromatography to give 1-nitroso2,3,4,5-tetrahydro-1H-1-benzazepine (2.60 g, 91% yield) as a brown liquid. 1H NMR (CDCl3, 300 MHz) δ 7.48−7.40 (m, 1H), 7.40−7.32 (m, 2H), 7.32−7.25 (m, 1H), 3.92 (br, 2H), 2.82−2.70 (m, 2H), 1.85−1.70 (m, 4H). A solution of 1-nitroso-2,3,4,5-tetrahydro-1H-1-benzazepine (2.60 g, 14.7 mmol) in THF (40 mL) was added dropwise under nitrogen atmosphere to a suspension of lithium aluminum hydride (0.56 g, 14.7 mmol) in THF (10 mL) cooled with an ice-bath such that the temperature did not rise above 15 °C. The mixture was stirred at room temperature for an hour, quenched with saturated potassium sodium tartrate salt solution (15 mL), and extracted with ether (3 × 20 mL).

The combined organic phase was dried over Na2SO4, concentrated under vacuum, and further purified by flash column chromatography using 25% ethyl acetate in hexanes to give 2,3,4,5-tetrahydro-1H-lbenzazepinamine 26c (1.63 g, 68% yield) as a light-yellow solid. 1H NMR (CDCl3, 300 MHz) δ 7.28 (dd, J = 1.4, 8.0 Hz, 1H), 7.21 (td, J = 8.0, 1.4 Hz, 1H), 7.10 (dd, J = 1.1, 7.4 Hz, 1H), 6.91 (td, J = 7.3, 1.5 Hz, 1H), 3.78 (br, 2H), 3.22−3.18 (m, 2H), 2.82−2.70 (m, 2H), 1.92−1.78 (m, 2H), 1.72−1.50 (m, 2H). A mixture of 4-piperidone hydrochloride monohydrate (1.54 g, 10 mmol) and 26c (1.62 g, 10 mmol) in 2-propanol (50 mL) was refluxed for 2 h and then cooled to room temperature. Concentrated HCl (0.82 mL, 10 mmol) was added, and the resulting mixture was refluxed for 3 h before being cooled to room temperature. The solid was filtered, rinsed with cold 2-propanol (2 × 20 mL), and dried under vacuum to give the title compound (1.88 g, 71% yield) as a hydrochloride salt. A small amount (20 mg) of the hydrochloride salt was converted into free base form by basifying with 1N NaOH, followed by extraction with CHCl3, washing with brine, and drying under vacuum to give the title compound as a white foamy solid. 1H NMR (CDCl3, 300 MHz) δ 7.25 (dd, J = 1.0, 7.4 Hz, 1H), 6.98 (t, J = 7.4 Hz, 1H), 6.92 (d, J = 6.3 Hz, 1H), 4.05 (t, J = 1.6 Hz, 2H), 4.02−3.92 (m, 2H), 3.25 (t, J = 5.6 Hz, 2H), 3.20−3.05 (m, 2H), 2.72 (t, J = 5.7 Hz, 2H), 2.20−2.00 (m, 4H), 1.83 (m, 1H). MS (ESI) m/z 227.2 [M + H]+. Step b and c: (cis)-4,5,6,7,9,10,12,12a-Octahydro-pyrido[3′,4′:4,5]pyrrolo[3,2,1-jk][1]benzazepine-11(8aH)-carboxylic Acid, 1,1-Dimethylethyl Ester (cis-28c). Sodium cyanoborohydride (0.94 g, 15 mmol) was added in small portions to a solution of 27c hydrochloride salt (1.32 g, 5.0 mmol) in TFA (15 mL) at 0 °C. After stirring at room temperature for 2 h, the mixture was carefully treated with 6 N HCl (10 mL) and refluxed for 1 h. The mixture was basified with 50% NaOH and extracted with CH2Cl2 (3 × 20 mL). The combined organic phase was dried over MgSO4 and concentrated under vacuum to give (cis)-4,5,6,7,8a,9,10,11,12,12a-decahydro-pyrido[3′,4′:4,5]pyrrolo[3,2,1-jk][1]benzazepine (1.0 g, 88% yield) as a yellow oil. MS (ESI) m/z 229.2 [M + H]+. First, 1N NaOH (10 mL) was added to a solution of (cis)4,5,6,7,8a,9,10,11,12,12a-decahydropyrido[3′,4′:4,5]pyrrolo[3,2,1-jk][1]benzazepine (1.0 g, 4.38 mmol) and di-tert-butyl dicarbonate (1.05 g, 4.8 mmol) in 1,4-dioxane (20 mL), and the mixture was then stirred for 2 h at room temperature. After the solvent was removed under reduced pressure, the residue was treated with ethyl acetate (30 mL). The solution was washed with brine (30 mL), dried over MgSO4, concentrated under vacuum, and then purified by flash column chromatography using 20% ethyl acetate in hexanes to give the title compound (1.2 g, 83% yield) as a white solid. 1H NMR (CDCl3, 500 MHz) δ 6.95 (d, J = 7.2 Hz, 1H), 6.90 (d, J = 7.5 Hz, 1H), 6.68 (t, J = 7.4 Hz, 1H), 3.65−3.55 (m, 1H), 3.48−3.29 (m, 3H), 3.29−3.20 (m, 1H), 3.12−2.71 (m, 2H), 2.71−2.50 (m, 2H), 2.08−1.83 (m, 4H), 1.83−1.62 (m, 1H), 1.62−1.07 (m, 11H). MS (ESI) m/z 329.2 [M + H]+. Steps d and e: (8aS,12aR)-4,5,6,7,8a,9,10,11,12,12a-Decahydropyrido[3′,4′:4,5]pyrrolo[3,2,1-jk][1]benzazepine (29c). (8aS,12aR)4,5,6,7,9,10,12,12a-Octahydro-pyrido[3′,4′:4,5]pyrrolo[3,2,1-jk][1]benzazepine-11(8aH)-carboxylic acid, 1,1-dimethylethyl ester (0.24 g, 0.73 mmol), which was obtained by chiral separation of cis-28c on a Chiracel OD column eluting with 2% 2-propanol in hexane, was stirred in 20% TFA in CH2Cl2 (10 mL) at room temperature for 2 h before the solution was basified with saturated ammonium hydroxide until pH > 10. The layers were separated, and the aqueous layer was extracted with CH2Cl2 (3 × 20 mL). The combined organic phase was washed with brine (20 mL), dried over MgSO4, concentrated, and dried under vacuum to give the title compound (0.16 g, 94% yield) as a white foamy solid. 1H NMR (CDCl3, 500 MHz) δ 6.96−6.87 (m, 2H), 6.69 (t, J = 7.4 Hz, 1H), 4.57−3.58 (br, 1H) 3.35−3.19 (m, 3H), 3.10 (dd, J = 12.7, 6.3 Hz, 1H), 3.03−2.78 (m, 3H), 2.71−2.59 (m, 1H), 2.58−2.44 (m, 2H), 2.10−1.67 (m, 5H), 1.65−1.40 (m, 1H). MS (ESI) m/z 229.2 [M + H]+. 4-((8aS,12aR)-4,5,6,7,9,10,12,12a-Octahydroazepino[3,2,1-hi]pyrido[4,3-b]indol-11(8aH)-yl)-1-(4-fluorophenyl)-1-butanone (62). To a solution of 29c (0.23 g, 1.0 mmol) in 1,4-dioxane (7.0 mL) were 2680



Article #

added 4-chloro-4′-fluorobutyrophenone (0.4 g, 2.0 mmol), KI (83 mg, 0.5 mmol), and K2CO3 (0.28 g, 2.0 mmol). The reaction mixture was heated at 100 °C for 48 h, cooled to 20 °C, and then diluted with CHCl3. The solution was filtered to remove excess K2CO3, and the filtrate was concentrated under vacuum. The residue was purified by silica gel flash column chromatography eluting with a gradient of 0− 2% methanol in ethyl acetate to give the title compound (0.26 g, 66% yield) as a dense oil. 1H NMR (CDCl3, 500 MHz) δ 8.04−7.95 (m, 2H), 7.16−7.08 (m, 2H), 6.95−6.85 (m, 2H), 6.67 (t, J = 7.4 Hz, 1H), 3.27−3.20 (m, 1H), 3.20−3.11 (m, 2H), 2.97 (t, J = 7.2 Hz, 2H), 2.94−2.84 (m, 1H), 2.75 (dd, J = 11.4, 5.1 Hz, 1H), 2.70−2.58 (m, 2H), 2.54−2.43 (m, 1H), 2.43−2.30 (m, 2H), 2.30−2.19 (m, 1H), 2.05−1.89 (m, 5H), 1.89−1.68 (m, 3H), 1.61−1.48 (m, 1H). 13C NMR (DMSO-d6, 126 MHz,) δ 198.4, 164.8 (d, JCF = 251 Hz), 152.4, 133.8 (d, JCF = 3 Hz), 133.4, 130.8 (d, JCF = 9 Hz), 129.0, 126.3, 120.9, 118.9, 115.6 (d, JCF = 22 Hz), 63.7, 57.1, 56.9, 51.4, 48.8, 40.2, 35.7, 34.7, 29.6, 26.9, 25.1, 21.6. MS (ESI) m/z 393.2 [M + H]+. HRMS (ESI) m/z calcd for C25H30FN2O [M + H]+, 393.2342; found, 393.2332. UPLC purity, 99.0%; retention time, 2.29 min (method A). Rat Quipazine Head Twitch. Different groups of male rats (N = 6 rats/dose/test substance) were fasted overnight and then placed in individual cells of a clear Lucite box. Varying concentrations of a test substance were administered by oral gavage to each rat 30 min prior to behavioral testing and 25 min prior to intraperitoneal injection of a standard dose (2.5 mg/kg) of quipazine maleate. Behavioral observations were begun 5 min after quipazine treatment. The number of stereotyped head twitch movements was counted for each rat by a trained observer over a 15 min period after quipazine treatment. The 50% effective dose (ED50 value) for the inhibition of quipazine-induced head twitch was calculated for each test substance. At least two independent dose−response tests were conducted for each test substance, and the average ED50 values were reported. Protocol for the Rat Conditioned Avoidance Response (CAR) Animal Model.37,38 Adult, male Sprague−Dawley rats were trained to consistently escape an electric foot shock (0.75 mA pulsed current, 250 ms on, 750 ms off) delivered to the grid floor of the test chamber. Animals were allowed to escape the shock delivery by climbing onto a pole suspended from the ceiling of the test chamber. Different groups of rats (N = 5−6 rats/dose/test substance) were administered different concentrations of the test substance in a 0.5% methylcellulose vehicle solution or vehicle alone (10 mL/kg volume) by oral gavage. Compound 5 was administered to individual rats at doses ranging from 0.1 to 10 mg/kg. Haloperidol, an antipsychotic medication, was administered to rats at a dose of 2 mg/kg as a positive control. The ability of 5 or haloperidol to inhibit the CAR response was evaluated in rats at 2 h after PO drug administration. The 50% effective dose (ED50) for inhibition of CAR by compound 5 at 2 h postdosing was determined to be 1.5 mg/kg.

■

(H.Z.) Tianjin Hospital, 406 Jiefang Nanlu, Hexi District, Tianjin 300211, P. R. China. Notes

The authors declare the following competing financial interest(s): Peng Li, Qiang Zhang, Wei Yao, J. David Beard, Gretchen L. Snyder, Youyi Peng, Joseph P. Hendrick, Kimberly E. Vanover, Sharon Mates and Lawrence P. Wennogle are fulltime employees of Intra-Cellular Therapies, Inc. (ITI). John Tomesch is a retired employee of ITI. Hongwen Zhu is a former employee of ITI. Albert J. Robichaud and Taekyu Lee are former employees of Bristol-Myers Squibb. Robert E. Davis is a paid consultant to ITI.

■

ACKNOWLEDGMENTS The authors wish to thank all team members from IntraCellular Therapies, Inc., Bristol-Myers Squibb Company and DuPont Pharmaceutical Company for their contributions to this project. We thank Dr. Richard Lerner at the Scripps Research Institute for his thoughtful comments on the manuscript.

■

ABBREVIATIONS USED BINAP, 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl; CHO cells, Chinese hamster ovary cells; CNS, central nervous system; EPS, extrapyramidal symptoms; Eu-GTP, europiumlabeled GTP analogue; 5-HT, serotonin or 5-hydroxytryptamine; MDCK, Madin−Darby canine kidney; MDR1, multidrug resistance protein 1; Pd2(dba)3, tris(dibenzylideneacetone)-dipalladium(0); PPHT, 2-(N-phenethyl-N-propyl)amino-5-hydroxytetralin; SERT, serotonin transporter; SAR, structure−activity relationships; TFA, trifluoroacetic acid

■

(1) Wickelgren, I. A new route to treating schizophrenia? Science 1998, 281, 1264−1265. (2) Marino, M. J.; Knutsen, L. J.; Williams, M. Emerging opportunities for antipsychotic drug discovery in the postgenomic era. J. Med. Chem. 2008, 51, 1077−1107. (3) Remington, G.; Agid, O.; Foussias, G. Schizophrenia as a disorder of too little dopamine: implications for symptoms and treatment. Expert Rev. Neurother. 2011, 11, 589−607. (4) Kim, D. H.; Maneen, M. J.; Stahl, S. M. Building a better antipsychotic: receptor targets for the treatment of multiple symptom dimensions of schizophrenia. Neurotherapeutics 2009, 6, 78−85. (5) Muench, J.; Hamer, A. M. Adverse effects of antipsychotic medications. Am. Fam. Physician 2010, 81, 617−622. (6) Meltzer, H. Y. Clozapine: balancing safety with superior antipsychotic efficacy. Clin. Schizophrenia Relat. Psychoses 2012, 6, 134−144. (7) Fakra, E.; Azorin, J. M. Clozapine for the treatment of schizophrenia. Expert Opin. Pharmacother. 2012, 13, 1923−1935. (8) Schmidt, C. J.; Sorensen, S. M.; Kehne, J. H.; Carr, A. A.; Palfreyman, M. G. The role of 5-HT2A receptors in antipsychotic activity. Life Sci. 1995, 56, 2209−2222. (9) Sorensen, S. M.; Kehne, J. H.; Fadayel, G. M.; Humphreys, T. M.; Sullivan, C. K.; Taylor, V. L.; Schmidt, C. J. Characterization of the 5HT2 receptor antagonist MDL 100907 as a putative atypical antipsychotic: behavioral, electrophysiological and neurochemical studies. J. Pharmacol. Exp. Ther. 1993, 266, 684−691. (10) Kehne, J. H.; Baron, B. M.; Carr, A. A.; Chaney, S. F.; Elands, J.; Feldman, D. J.; Frrank, R. A.; van Giersbergen, P. L. M.; Mccloskey, T. C.; Johnson, M. P.; Mccarty, D. R.; Poirot, M.; Senyah, Y.; Siegel, B. W.; Widmaier, C. Preclinical characterization of the potential of the putative atypical antipsychotic MDL 100907 as a potent 5-HT2A

ASSOCIATED CONTENT

S Supporting Information *

Synthesis and analytical data of intermediates and final compounds not listed in the Experimental Section, receptor profiling results, in vitro binding assays, and homology modeling and docking studies. This material is available free of charge via the Internet at http://pubs.acs.org.

■

REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Phone: 212−923−3344. Fax: 212−923−3388. E-mail: pli@ intracellulartherapies.com. Present Addresses ∥

(T.L.) Vanderbilt University, 2201 West End Avenue, Nashville, Tennessee 37235, United States. ⊥ (A.J.R.) Sage Therapeutics, 215 First Street, Cambridge, Massachusetts 02142, United States. 2681



Article

antagonist with a favorable CNS safety profile. J. Pharmacol. Exp. Ther. 1996, 277, 968−981. (11) De Paulis, T. M-100907 (Aventis). Curr. Opin. Invest. Drugs 2001, 2, 123−132. (12) Buckley, P. F. Olanzapine: a critical review of recent literature. Expert Opin. Pharmacother. 2005, 6, 2077−2089. (13) Worrel, J. A.; Marken, P. A.; Beckman, S. E.; Ruehter, V. L. Atypical antipsychotic agents: a critical review. Am. J. Health Syst. Pharm. 2000, 57, 238−255. (14) Meltzer, H. Y.; Matsubara, S.; Lee, J. C. The ratios of serotonin2 and dopamine2 affinities differentiate atypical antipsychotic drugs. Psychopharmacol. Bull. 1989, 25, 390−392. (15) Oakley, N. R.; Hayes, A. G.; Sheehan, M. J. Effect of typical and atypical neuroleptics on the behavioural consequences of activation by muscimol of mesolimbic and nigro-striatal dopaminergic pathways in the rat. Psychopharmocology 1991, 105, 204−208. (16) Reynolds, G. P.; Kirk, S. L. Metabolic side effects of antipsychotic drug treatmentpharmacological mechanisms. Pharmacol. Ther. 2010, 125, 169−179. (17) Deng, C.; Weston-Green, K.; Huang, X. F. The role of histaminergic H1 and H3 receptors in food intake: a mechanism for atypical antipsychotic-induced weight gain? Prog. Neuro-Psychopharmacol. Biol. Psychiatry 2010, 34, 1−4. (18) Kirk, S. L.; Glazebrook, J.; Grayson, B.; Neill, J. C.; Reynolds, G. P. Olanzapine-induced weight gain in the rat: role of 5-HT2C and histamine H1 receptors. Psychopharmacology 2009, 207, 119−125. (19) Rajagopalan, P. Pyridopyrrolobenzoxazine. (Endo Laboratories, Inc.). U.S. Patent 4,013,652, March 22, 1977. (20) Rajagopalan, P. Pyridopyrrolobenzheterocycles for combating depression. (Endo Laboratories, Inc.). U.S. Patent 3,914,421, October 21, 1975. (21) Rajagopalan, P. cis- and trans-Octahydropyridopyrrolobenzheterocycles. (E. I. Du Pont De Nemours and Co.). U.S. Patent 4,219,550, August 26, 1980. (22) Lee, T.; Robichaud, A. J.; Boyle, K. E.; Lu, Y.; Robertson, D. W.; Miller, K. J.; Fitzgerald, L. W.; McElroy, J. F.; Largent, B. L. Novel, highly potent, selective 5-HT2A/D2 receptor antagonists as potential atypical antipsychotics. Bioorg. Med. Chem. Lett. 2003, 13, 767−770. (23) Wolfe, J. P.; Ahman, J.; Sadighi, J. P.; Singer, R. A.; Buchwald, S. L. An ammonia equivalent for the palladium-catalyzed amination of aryl halides and triflates. Tetrahedron. Lett. 1997, 38, 6367−6370. (24) Welch, W. M.; Harbert, C. A.; Weissman, A.; Koe, B. K. Neuroleptics from the 4a, 9b-cis- and 4a, 9b-trans-2,3,4,4a,5,9bhexahydro-1H-pyrido[4,3-b]indole series. 2. J. Med. Chem. 1986, 29, 2093−2099. (25) Fitzgerald, L. W.; Conklin, D. S.; Krause, C. M.; Marshall, A. P.; Patterson, J. P.; Tran, D. P.; Iyer, G.; Kostich, W. A.; Largent, B. L.; Hartig, P. R. High-affinity agonist binding correlates with efficacy (intrinsic activity) at the human serotonin 5-HT2A and 5-HT2C receptors: evidence favoring the ternary complex and two-state models of agonist action. J. Neurochem. 1999, 72, 2127−2134. (26) Nelson, D. L.; Lucaites, V. L.; Wainscott, D. B.; Glennon, R. A. Comparisons of hallucinogenic phenylisopropylamine binding affinities at cloned human 5-HT2A, 5-HT2B and 5-HT2C receptors. NaunynSchmiedeberg’s Arch. Pharmacol. 1999, 359, 1−6. (27) Seo, H. J.; Park, E. J.; Kim, M. J.; Kang, S. Y.; Lee, S. H.; Kim, H. J.; Lee, K. N.; Jung, M. E.; Lee, M.; Kim, M. S.; Son, E. J.; Park, W. K.; Kim, J.; Lee, J. Design and synthesis of novel arylpiperazine derivatives containing the imidazole core targeting 5-HT2A receptor and 5-HT transporter. J. Med. Chem. 2011, 54, 6305−6318. (28) Ablordeppey, S. Y.; Altundas, R.; Bricker, B.; Zhu, X. Y.; Eyunni, S. E. V. K.; Jackson, T.; Khan, A.; Rothb, B. L. Identification of a butyrophenone analog as a potential atypical antipsychotic agent: 4-[4(4-chlorophenyl)-1,4-diazepan-1-yl]-1-(4-fluorophenyl)butan-1-one. Bioorg. Med. Chem. 2008, 16, 7291−7301. (29) Kroeze, W. K.; Hufeisen, S. J.; Popadak, B. A.; Renock, S. M.; Steinberg, S.; Ernsberger, P.; Jayathilake, K.; Meltzer, H. Y.; Roth, B. L. H1-histamine receptor affinity predicts short-term weight gain for

typical and atypical antipsychotic drugs. Neuropsychopharmacology 2003, 28, 519−526. (30) Cahir, M.; King, D. J. Antipsychotics lack α1A/B adrenoceptor subtype selectivity in the rat. Eur. Neuropsychopharmacol. 2005, 15, 231−234. (31) Nourian, Z.; Mow, T.; Muftic, D.; Burek, S.; Pedersen, M. L.; Matz, J.; Mulvany, M. J. Orthostatic hypotensive effect of antipsychotic drugs in Wistar rats by in vivo and in vitro studies of alpha1adrenoceptor function. Psychopharmacology 2008, 199, 15−27. (32) Ancoli-Israel, S.; Vanover, K. E.; Weiner, D. M.; Davis, R. E.; van Kammen, D. P. Pimavanserin tartrate, a 5-HT2A receptor inverse agonist, increases slow wave sleep as measured by polysomnography in healthy adult volunteers. Sleep Med. 2011, 12, 134−141. (33) Porter, R. H. P.; Benwell, K. R.; Lam, H.; Malcolm, C. S.; Allen, N. H.; Revell, D. F.; Adams, D. R.; Sheardown, M. J. Functional characterization of agonists at recombinant human 5-HT2A, 5-HT2B and 5-HT2C receptors in CHO-K1 cells. Br. J. Pharmacol. 1999, 128, 13−20. (34) Conn, P. J.; Sanders-Bush, E. Selective 5-HT-2 antagonists inhibit serotonin stimulated phosphatidylinositol metabolism in cerebral cortex. Neuropharmacology 1984, 23, 993−996. (35) Koval, A.; Kopein, D.; Purvanov, V.; Katanaev, V. L. Europiumlabeled GTP as a general nonradioactive substitute for [35S]GTPγS in high-throughput G protein studies. Anal. Biochem. 2010, 397, 202− 207. (36) Malick, J. B.; Doren, E.; Barnett, A. Quipazine-induced headtwitch in mice. Pharmacol., Biochem. Behav. 1977, 6, 325−329. (37) Wadenberg, M. L. G.; Hicks, P. B. The conditioned avoidance response test re-evaluated: is it a sensitive test for the detection of potentially atypical antipsychotics? Neurosci. Biobehav. Rev. 1999, 23, 851−862. (38) Arnt, J. Pharmacological specificity of conditioned avoidance response inhibition in rats: inhibition by neuroleptics and correlation to dopamine receptor blockade. Acta Pharmacol. Toxicol. 1982, 51, 321−329. (39) Snyder, G. L.; Vanover, K. E.; Zhu, H.; Miller, D. B.; O’Callaghan, J. P.; Tomesch, J.;, Li, P.; Zhang, Q.; Krishnan, V.; Hendrick, J. P.; Nestler, E. J.; Davis, R. E.; Wennogle, L. P.; Mates, S. unpublished results. (40) Snyder, G. L.; Vanover, K. E. Intracellular signaling and approaches to the treatment of schizophrenia and associated cognitive impairment. Curr. Pharm. Des. December 15, 2013, DOI: 10.2174/ 1381612819666131216115417.

2682


Discovery of a Tetracyclic Quinoxaline Derivative as a Potent and

Recommend Documents