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Neuroactive Steroids. 2. 3α-Hydroxy-3β-methyl-21-(4cyano‑1H‑pyrazol-1′-yl)-19-nor-5β-pregnan-20-one (SAGE-217): A Clinical Next Generation Neuroactive Steroid Positive Allosteric Modulator of the (γ-Aminobutyric Acid)A Receptor Gabriel Martinez Botella,†,∥ Francesco G. Salituro,*,† Boyd L. Harrison,† Richard T. Beresis,‡ Zhu Bai,§ Maria-Jesus Blanco,† Gabriel M. Belfort,†,⊥ Jing Dai,† Carlos M. Loya,†,# Michael A. Ackley,† Alison L. Althaus,† Scott J. Grossman,† Ethan Hoffmann,† James J. Doherty,† and Albert J. Robichaud† †

Sage Therapeutics, Inc. 215 First Street, Cambridge, Massachusetts 02142, United States Shanghai Chempartner, 998 Halei Road, Shanghai 201203, China § WuXi AppTec, 288 Fute Zhong Road, Shanghai 200131, China ‡

S Supporting Information *

ABSTRACT: Certain classes of neuroactive steroids (NASs) are positive allosteric modulators (PAM) of synaptic and extrasynaptic GABAA receptors. Herein, we report new SAR insights in a series of 5β-nor-19-pregnan-20-one analogues bearing substituted pyrazoles and triazoles at C21, culminating in the discovery of 3α-hydroxy-3β-methyl-21-(4-cyano-1H-pyrazol-1′-yl)-19nor-5β-pregnan-20-one (SAGE-217, 3), a potent GABAA receptor modulator at both synaptic and extrasynaptic receptor subtypes, with excellent oral DMPK properties. Compound 3 has completed a phase 1 single ascending dose (SAD) and multiple ascending dose (MAD) clinical trial and is currently being studied in parallel phase 2 clinical trials for the treatment of postpartum depression (PPD), major depressive disorder (MDD), and essential tremor (ET).

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INTRODUCTION Neuroactive steroids (NASs) have been shown to impact central nervous system (CNS) function through multiple mechanisms, included but not limited to, positive allosteric modulation (PAM) of the (γ-aminobutyric acid)A (GABAA) receptor.1−4 The family of GABAA receptors are quite complex, with 19 known subtypes, and are broadly expressed in the brain. These receptors are pentameric ion channels primarily composed of two alpha subunits (α1−α6), two beta subunits (β1−β3) and one additional subunit (γ1−γ3, δ, ε, π, or θ).5 The unique subunit composition determines the biophysical and pharmacological characteristics of the channel as well as influencing its location at synaptic or extrasynaptic sites. As the primary inhibitory neurotransmitter in the nervous system, GABA, acting via the GABAA receptor, can influence a wide range of brain circuits that are central to a variety of behavioral states, such as anxiety levels, seizures, sleep, vigilance, and memory. Not surprisingly, given its critical role in the function of neuronal circuits, GABAA receptors are the target for numerous clinically relevant drugs such as benzodiazepines, barbiturates, and anesthetics. The differentiating ability of NASs to potently modulate both synaptic and extrasynaptic GABAA receptors3−7 may be relevant in the context of a variety of seizure disorders where the synaptic GABAA receptors are down-regulated, or weakened in their activity,8,9 thus effectively removing the target of pharmacological intervention. This is thought to be the primary mechanism of benzodiazepine tolerance. The importance of extrasynaptic receptor modu© 2017 American Chemical Society

lation should not be lost, as this property may potentially confer an advantage for NAS GABA modulators in certain indications compared to compounds belonging to the benzodiazepine class, which only modulate synaptic GABAA receptors.10−12 A parenteral, continuous infusion formulation of the first-generation NAS, allopregnanolone, 3α-hydroxy-5αpregnan-20-one (SAGE-547,13 brexanolone, 1, Figure 1), is currently in phase 3 clinical trials for the treatment of super refractory status epilepticus (SRSE), a life-threatening condition in which the brain is in a state of persistent seizure that fails to respond to standard treatments.13−15 In addition, compound 1 has completed a phase 2 clinical trial in severe postpartum depression (PPD)16 and an exploratory study in essential tremor (ET).17 Simultaneous with the development program in SRSE, compound 1 is being studied in a phase 3 program in moderate and severe postpartum depression.16 There is a considerable body of evidence supporting the potential for GABAA modulation to have benefit in a number of mood disorders.18 Studies using biochemical measures of GABA in plasma and cerebrospinal fluid seemed to indicate decreases in GABA levels in patients with depression.19 Postmortem studies using brain tissue from neocortex and hippocampus also describe changes in the GABA system in individuals with mood disorders. Furthermore, and in regard to postpartum depression in particular, there is a rationale Received: June 9, 2017 Published: July 28, 2017 7810

DOI: 10.1021/acs.jmedchem.7b00846 J. Med. Chem. 2017, 60, 7810−7819

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Figure 1. Structures of 1 (allopregnanolone, SAGE-54713−17), 2 (SGE-51622), and 3 (SAGE-21726).

molecular properties within a series of pyrazole substituents, which eventually led to the preparation of orally bioavailable pyrazole 3, was a significant evolution of the strategy described in our earlier disclosure.21 Compound 3 was prepared following the route depicted in Scheme 1. The key step was the stereoand regio-selective introduction of the 3β-methyl group, via methyl Grignard addition to the ketone 5 using a bulky aluminum based ligand [methyl aluminum bis-(2,6-di-tertbutyl-4-methylphenoxide), MAD] with excellent selectivity and yields. Significantly bulky oxygenophilic organoaluminum reagents such as MAD have been used efficiently for stereoselective activation of carbonyl moieties. Combination of MAD with Grignard reagents generates a new amphiphilic reaction system25 that allows for regio- and stereochemical nucleophilic addition. All compounds in Table 1 were prepared following the same approach as that for the synthesis of 3.26,27 Our profiling cascade to evaluate GABAA receptor activity involved a high throughput assessment of compound binding by first using the [35S]-tert-butylbicyclophosphorothionate ([35S]TBPS) binding assay28 as an indirect measurement of NAS PAM activity. The TBPS assay has been commonly used to identify compounds that bind to the GABAA receptor family, although there is not necessarily a linear relationship between NAS functional potency and inhibition of TBPS binding because these activities result from binding to different sites of the receptor. Compounds that showed significant potency (IC50 < 100 nM) were then progressed into more rigorous synaptic and extrasynaptic GABAA receptor functional assays, using recombinant α1β2γ2 GABAA receptor in LTK cells in a Q-patch formatted assay and in α4β3δ GABAA receptor in CHO cells manual patch assay, respectively. Following on the early results21 that led to analogue 2, we decided to concentrate optimization efforts through a focused SAR expansion of pyrazole analogues, such as 11. Key to this exploration was an optimized synthetic approach, access to rapid molecular profiling, and availability of a variety of substituted pyrazoles as a surrogate scaffold. From this exercise, we then envisioned selecting only the most promising pyrazole substitutions to apply to the triazole scaffold, thus generating a series of matched pairs. SAR trends started to develop quickly around these two related scaffolds. Pyrazole analogue 11 was equally potent at α1β2γ2 and α4β3δ GABAA receptors yet suffered from >5-fold lowering in potency (EC50s ∼1 μM) compared to triazole 2 (Table 1). The addition of a methyl group to the pyrazole led to three regio-isomeric NASs (12−14). The 3-methyl and 5methylpyrazole analogues, 13 and 14 respectively, were less potent in the [35S]TBPS assay and both showed >3-fold decrease in functional activity at the synaptic α1β2γ2 GABAA receptor in comparison to pyrazole 11. However, the 4methylpyrazole analogue 12 achieved a ∼3-fold improvement at the synaptic α1β2γ2 GABAA receptor, and the functional activity against the extrasynaptic α4β3δ GABAA receptor

supporting the potential utility of NAS, such as allopregnanolone, to have beneficial effects on the depressive20 state through modulation of synaptic and extrasynaptic GABAA receptors. This disease, with no approved drugs to date, is estimated to affect between 10 and 20% of women in the United States after childbirth. Compound 1 is a potent GABAA receptor PAM (see Table 1), active at both synaptic and extrasynaptic GABAA receptors, and is ideally suited for parenteral administration due to its high intrinsic clearance (Tables 2 and 3) and low oral bioavailability. For a next-generation NAS, we sought to develop a molecule with the potential for broader therapeutic applicability not limited by parenteral administration. Ideally, an entity with a robust pharmacological GABAA receptors PAM profile similar to compound 1, but with low intrinsic clearance, high oral bioavailability, and potential for once or twice daily dosing, would have significant potential to reach larger populations of patients. To that end, we have undertaken a broad and multivariate approach to identifying NCEs from our platform of NAS compounds with druglike properties more amenable to oral administration. Throughout this effort, we have been advancing several subclasses of compounds and have recently reported that the addition of specific heterocycles at the C-21 position of 3β-methyl, nor-19 allopregnanolone, and pregnanolone scaffolds affords analogues that can favorably attenuate the physical and pharmacokinetic properties of NASs. These next-generation NASs with high potential to be more suitable for oral administration maintain the modulatory efficacy at the targeted GABAA receptors.21 A key compound from this initial study, the 1,2,5-triazole analogue (SGE-51622), 2, displays a similar molecular pharmacological profile as 1 (Table 1 and Figure 1) with improved aqueous solubility and significantly improved pharmacokinetic properties (Tables 2 and 3). Further profiling of 2 in a broad screening panel of over a hundred biological targets revealed less than desirable cross reactivity at a few sites that we judged suboptimal for the preclinical advancement of 2. In this article, we will unveil further insights on the effect of substitution of five-membered heterocyclic analogues such as 2, leading to the discovery of the clinical candidate 3α-hydroxy-3β-methyl-21-(4-cyano-1H-pyrazol-1′yl)-19-nor-5β-pregnan-20-one, compound 3 (Figure 1), which has completed SAD and MAD phase 1 clinical trials23 and is being progressed to phase 2 clinical trials for the treatment of GABAA receptors dysfunction-related movement such as essential tremor (ET) and mood disorders like postpartum depression (PPD) and major depressive disorder (MDD). Other advanced preclinical compounds are also under evaluation for potential utility in several orphan epilepsy indications.

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RESULTS Our synthetic plan to investigate the SAR at the C-21 position (steroid scaffold numbering24) and further improve the 7811

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Table 1. [35S]TBPS and GABAA Receptor Pharmacology of cis A/B Ring Analoguesa

a α1β2γ2 EC50 (nM)/Emax (%) values were obtained using automated patch clamp electrophysiology.29 α4β3δ EC50 (nM)/Emax (%) values were obtained using manual patch clamp electrophysiology.30 R2 for [35S]TBPS was >0.98 for all compounds; R2 for α1β2γ2 was 0.8 for 14, 0.82 for 21, 0.83 for 13, and 0.89 for 11 and 15, all others were >0.91−0.99; R2 for α4β3δ was 0.87 for 24 and 0.89 for 17, all others were >0.94−0.99.

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Table 1. continued [35S]TBPS IC50 values were fitted using two replicates per concentration. α1β2γ2 and α4β3δ EC50 and Emax (%) values were obtained from three replicates (n = 3) from 4-point dose−response experiments; for compound 3, this data represents an aggregate of multiple testing of different lots (n = 6 for α1β2γ2 and n = 4 for α4β3δ). Each compound was tested for its ability to affect GABA mediated currents at a submaximal agonist dose (GABA EC20 = 2 μM). NT means not tested.

Table 2. Log D, Aqueous Solubility (pH = 7.4), and in Vitro Metabolic Stability (Clhep L/h/kg) Properties for Selected Analoguesa compd

Log D pH 7.4

aq soln (μM)

Hu (Clhep) (L/h/kg)

rat (Clhep) (L/h/kg)

mouse (Clhep) (L/h/kg)

1 2 3 11 12 21 24

4.9 4.6 4.6 4.6 5.0 4.6 4.9

3.3 7.3 2.0 8.0 7.0 1.9 7.0

0.5 0.2 0.2 0.3 0.5 0.3 0.3

3.3 2.7 2.2 2.5 3.0 2.5 3.2

4.0 4.0 4.2 4.9 5.1 5.0 4.9

position without achieving any functional activity improvements at both the synaptic α1β2γ2 and the extrasynaptic α4β3δ GABAA receptors. However, introduction of the smaller cyano group, affording analogue 3, resulted in a >4-fold improvement over the unsubstituted pyrazole analogue 11 in the [35S]TBPS binding assay and matched the 4-methylpyrazole analogue 12 functional activity at the synaptic α1β2γ2 GABAA receptor (EC50 = 375 nM). Interestingly, 3 showed >4-fold improvement in the activity at the extrasynaptic α4β3δ GABAA receptor vs 12, thus matching the profile of allopregnanolone 1 (Table 1). This potency improvement at the extrasynaptic α4β3δ GABAA receptor appears to be consistent with our previous observation that polar rings at C-21 may be favored.21 Hydrolysis of the cyano group yielding both the amide (18) and acid (19) resulted in significant loss of activity, suggesting a disfavorable interaction of polar and charged functional groups within the receptor allosteric binding site. Finally, the addition of an electron-donating group to explore the other end of the SAR, exemplified by the 4-methoxypyrazole analogue (20), led to a ∼2-fold loss of functional activity at both the synaptic α1β2γ2 and the extrasynaptic α4β3δ GABAA receptors compared to pyrazole 11. It could be hypothesized that a richer electronic ring resulted in increased basicity of the nitrogen atoms, which is in line with previous observations on the SAR.21 Although there are no crystal structures available of the GABAA receptor binding with a NAS entity, we speculated that the cyano group yielded an optimal combination of steric and electronic effects, resulting in excellent binding and functional activity as in pyrazole 3. For comparison purposes, we prepared the isomeric 3-cyanopyrazole analogue (21) which showed comparable activity at the extrasynaptic α4β3δ GABAA receptor relative to 3. These results might indicate that the effects of the cyano group on pKa, and ring electronics are the main driving force behind the marked activity improvement when compared to the unsubstituted pyrazole analogue 11. Once we felt that pyrazole substitution was optimized with the 4-cyano substituent, we turned our attention to the C-3 substituent of the steroid scaffold and prepared a narrow set of analogues. Hence the 3β-methyl moiety was replaced by ethyl and methoxymethyl groups, 22 and 23 respectively, while maintaining 4-cyanopyrazole ring at C21. Both analogues showed a significant lower activity at the synaptic α1β2γ2 GABAA receptor, thus suggesting the smaller methyl group was the preferred substituent at this position for this series of analogues. Armed with the data from the pyrazoles containing methyl and cyano substitutions, we then prepared the corresponding analogues in the triazole series. The 4-methyl-2H-1,2,3-triazole analogue (24) showed favorable functional potency at the extrasynaptic α4β3δ GABAA receptors in comparison to pyrazole 12, suggesting that the increased polarity achieved by strategically placing an extra nitrogen was beneficial when modulating extrasynaptic receptors, in agreement with the observations reported previously by our group.21 The isomeric 5-methyl-1H-1,2,3-triazole analogue (25) showed a decrease in

a

Log D and aq soln are single-point estimates. Microsomal stability is estimated by calculating rate of elimination from five time points sampled over 60 min.

Table 3. In Vivo Parameters for Selected Compounds in Rats (R) and Mice (M), following iv (5 mg/kg) and Oral (20 mg/kg) Administrationa compd

clearance (L/h/kg) (R/M)

oral bioavailability (%) (R/M)

brain:plasma ratio (R/M)

1 2 3 11 12 21 24

4.8/12 2.3/2.6 1.4/1.5 1.7/4.5 3.8/3.6 1.5/2.6 3.0/3.7

2/NT 25/17 32/62 19/5 9.5/6 56/26 6/39

1.8/1.9 1.8/1.0 4.2/1.6 1.1/ND 2.0/ND 1.9/1.4 ND/1

a

Brain:plasma ratios are obtained by single point at 30 min post iv. Clearance, oral bioavailability, and brain:plasma ratios calculated from mean plasma concentrations from two rats. NT equals “not tested”.

remained unchanged when compared to pyrazole 11. The >12fold difference in functional activity at the synaptic α1β2γ2 GABAA receptor between isomeric pyrazole analogues 12, 13, and 14 arose from a point change in the location of a nitrogen atom (Table 1) and a methyl substituent. We hypothesized that the “ortho-like” position of the methyl was sterically disfavored in analogue 14. In the case of 13, the low activity could be the result of a combination of unfavorable sterics and an inability of the unsubstituted nitrogen to engage in a hydrogen bond interaction with the receptor. Because the 4-substituted analogue 12 afforded the most promising result, we then focused on varying the electronic character of the pyrazole ring by changing the nature of the substituent at the 4-position (Table 1). Replacing the methyl moiety by a trifluoromethyl group (15) led to a marked improvement in the [35S]TBPS binding assay (IC50 = 8 nM), but the functional activity at the synaptic α1β2γ2 GABAA receptor was weakened, suggesting the extra lipophilicity or steric hindrance afforded by the trifluoromethyl group had a detrimental effect. On the other hand, a very high Emax (2587%) was observed at the extrasynaptic α4β3δ GABAA receptor, without an improvement in potency. Electron withdrawing substituents such as chlorine (16) and nitro group (17) were incorporated at the 4-pyrazole 7813

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Scheme 1. Synthesis of Compound 3

functional potency at both the synaptic α1β2γ2 and the extrasynaptic α4β3δ GABAA receptors. As in corresponding pyrazole analogue 14, steric interference with the receptor might afford an explanation for the decrease in activity. Finally, the 4-methyl-1H-1,2,3-triazole analogue (26) was less potent at the synaptic α1β2γ2 GABAA receptor than the corresponding isomeric triazole analogue 24 and the matched pair 4methypyrazole analogue 12. This suggests that the nitrogen adjacent to the methyl substituted carbon led to a disfavored interaction with the receptor. Finally, substitution of the triazole ring with a cyano group (27−28) had a marked detrimental effect, especially when compared to the cyanopyrazole analogues 3 and 21. In vitro DMPK properties of key compounds with desired pharmacological profiles were then assessed for their physicochemical properties (e.g., Log D) and metabolic stabilities using human, rat, and mouse microsomes (see Table 2). The thermodynamic aqueous solubility measured at pH = 7.4 was between 2 and 8 μM, and the measured Log D ranged between 4.6 and 5.0. The majority of these novel NAS compounds described herein displayed improved stability in human microsomes compared to 1 except for the 4methypyrazole analogue 12. The methyl substituent at the pyrazole ring in 12 might be subjected to CYP mediated oxidation, leading to a decrease in metabolic stability. However, the corresponding methyl triazole analogue 24 was more stable in human microsomes. We next evaluated the most promising compounds in rat and mouse in vivo PK studies (Table 3). As we have reported earlier, allopregnanolone 1 is rapidly cleared in both rats and mice and displays low oral bioavailability. The addition of a C21 unsubstituted pyrazole (11) or triazole (2) ring yielded analogues with significantly improved rat oral bioavailability (F = 19−25%). However, high clearance and modest bioavailability in mice, more importantly, led to deprioritization of

these analogues. The introduction of methyl substituents within the pyrazole and triazole rings, analogues 12 and 24 respectively, did not increase significantly oral bioavailability relative to the corresponding unsubstituted heterocyclic compounds. However, both 3- and 4-cyanopyrazole analogues, 21 and 3 respectively, displayed suitable PK parameters for the intended oral administration specifically, low to moderate clearance, and oral bioavailability higher than 25% in the preclinical rodent species. Additionally, all compounds were able to maintain a significant blood−brain penetration as demonstrated by the brain to plasma ratios measured from preclinical in vivo experiments (B/P > 1, Table 3). Both cyanopyrazole analogues 3 and 21 achieved comparable in vitro potency and efficacy to compounds 1 and 2 at the GABAA receptors (Table 1). More importantly, the SAR changes that led to 3 and 21 yielded oral exposures consistent with our goals for a potential orally administered development candidate (Figure 2). To that end, pyrazole 3 was selected for further profiling. Compound 3 was further evaluated in dog PK studies as well as CYP inhibition. The results are shown in Table 4. Pyrazole 3 showed low in vivo clearance with 68% oral bioavailability in dog PK studies. In vitro CYP inhibition (IC50) was >30 μM for CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP2B6, indicating low probability of drug−drug interactions. Compound 3 showed >30 μM inhibition in a cardiac panel of eight relevant cardiac ion channels (Table 4). Compound 3 was submitted for broad binding selectivity assessment to an offtarget panel of more than 75 enzymes and receptors. At 10 μM concentrations of 3, only binding at the glycine (57%), sigma receptors (88%), and inhibition of the transient receptor potential vanilloid 1 (TRPV1, 95%) was noted (Table 4). When tested against a panel of 22 nuclear hormone receptors, compound 3 displayed no significant functional activity in either agonist or antagonist mode (Table 4). 7814

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(although still suffering from high clearance).21 We chose to expand the SAR to explore the effects on the potency and DMPK properties of this series of NASs by introducing substituted pyrazole and triazole rings at C-21. Our initial data from Table 1 with methyl substituted pyrazole analogues (12− 14) suggested a focus on the 4-position, among a plethora of potent subclasses, to expand our SAR studies. Except for the carboxylic acid analogue 19, and triazole 25 (IC50 > 3000 nM), all other substituents were well tolerated in the [35S]TBPS binding assay, with the cyano group proving to be the most potent analogue (3, IC50 = 7 nM). The 4-cyano, 3-cyano, and 4-methylpyrazole analogues, 3, 21, and 12, showed comparable activity at the synaptic α1β2γ2 GABAA receptors to analogues 1 and 2 (EC50 = 375, 225, and 291 nM vs 1 and 2, EC50 = 237 and 125 nM, respectively). Throughout our studies, we learned that the receptor appears to be sensitive to subtle changes in steric hindrance and electronics, with either large polar groups (CONH2, 18) or electron-donating groups (OCH3, 20) displaying lower activity. The most potent compounds at the extrasynaptic α4β3δ GABAA receptors were also the cyano analogue 3, 21, and triazole 24 (EC50 = 299, 110, and 192 nM, respectively), comparable to the potency of 1 and 2 (EC50 = 75.5 and 240 nM, respectively). Overall, there seems to be a preference for electron-withdrawing groups and reduced lipophilicity when attempting to achieve compounds with a potent and balanced functional activity against both α1β2γ2 and α4β3δ GABAA receptors. Unexpectedly, the beneficial effect of the cyano group was lost when added to the triazole ring, as 27 and 28 were less active than 2 and 3 analogues. Furthermore, when a methyl group replaced the cyano group on the triazole ring, yielding 24, the activity was recovered with a preference for extrasynaptic α4β3δ GABAA receptors. The two most promising analogues, 3 and 21, were further assessed for their physicochemical and pharmacokinetic properties, showing a similar profile (Table 2 and 3). The pharmacological profile, preclinical rodent PK profile consistent with oral development (Table 3), as well as a more favorable developability assessment (data not shown),32 led to the selection of 3 over 21 for further studies. Additional PK studies of 3 in dog showed low clearance (30

plasma protein binding (% bound) mouse/rat/dog/human

99/98.8/98.4/98.8

cardiac ion channel panel, IC50 (μM) Nav1.5, hERG, KCNQ1/minK, Cav1.2, Kv4.3/KChIP2.2, Kv1.5, Kir2.1, HCN2

>30

radioligand binding and functional cellular and enzymatic assays (CEREP SA) at 10 μM (78 targets) >50% baseline

sigma-R glycine-R TRPV1

Functional nuclear hormone receptor assays (22 receptors) (5-fold induction threshold agonist model/50% inhibition threshold antagonist mode) at 0.4, 2, and 10 μM

no hits

In conclusion, pyrazole 3 was selected as the lead entity for further development due to its overall suitable profile, the in vitro PAM activity, as well as its pharmaceutical properties.32

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DISCUSSION AND CONCLUSION In our quest for an orally bioavailable, potent positive allosteric modulator at the synaptic and extrasynaptic GABAA receptors, we have continued to optimize our platform of NASs that belong to the 3α-hydroxy-3β-methyl-19-nor-5β-pregnan-20one series.21 The addition of unsubstituted, small aromatic five-membered rings at C-21 yielded promising compounds with excellent modulatory activity at the GABAA receptors and improved in vivo PK properties over previous molecules 7815

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204.54 (CO). HRMS m/z 399.3017 calcd for C25H39N2O2+ 399.3006. 3α-Hydroxy-3β-methyl-21-(3-methyl-1H-pyrazol-1′-yl)-19nor-5β-pregnan-20-one (13). Yield: 11 mg (14%) as an off-white solid. LC-MS: tR = 0.95 min, m/z = 399 (M + 1). 1H NMR (400 MHz, CDCl3) δ 7.28 (d, J = 2.3 Hz, 1H), 6.09 (d, J = 2.3 Hz, 1H), 4.89−4.75 (m, 2H), 2.56 (t, J = 8.8 Hz, 1H), 2.28 (s, 3H), 2.23−2.11 (m, 1H), 2.09−1.99 (m, 1H), 1.90−1.73 (m, 4H), 1.69−1.57 (m, 2H), 1.52− 1.21 (m, 15H), 1.18−1.16 (m, 1H), 1.17−1.04 (m, 3H), 0.68 (s, 3H). 13 C NMR (100 MHz, CDCl3) δ 13.70, 13.85 (2 × CH3), 23.37, 24.49, 25.57, 25.80, 26.19 (5 × CH2), 26.68 (CH3), 31.49, 34.67 (2 × CH2), 34.84, 37.76 (2 × CH), 39.21 (CH2), 40.42 (CH), 41.28 (CH2), 41.80 (CH), 45.21 (C), 55.99, 60.69 (2 × CH), 61.51 (CH2), 72.17 (C), 106.16, 131.53 (2 × CH), 149.17 (C), 204.49 (CO). HRMS m/z 399.3017 calcd for C25H39N2O2+ 399.3006. 3α-Hydroxy-3β-methyl-21-(5-methyl-1H-pyrazol-1′-yl)-19nor-5β-pregnan-20-one (14). Yield: 9 mg (11%) as an off-white solid. LC-MS: tR = 0.973 min, m/z = (M + 1). 1H NMR (400 MHz, CDCl3): δ 7.50−7.40 (m, 1H), 6.10−6.00 (m, 1H), 4.90−4.75 (m, 2H), 2.65−2.50 (m, 1H), 2.25−1.95 (m, 5H), 1.90−1.55 (m, 7H), 1.50−1.20 (m, 15H), 1.20−1.00 (m, 3H) 0.69 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 11.26, 13.87 (2 × CH3), 23.40, 24.51, 25.58, 25.85, 26.20 (5 × CH2), 26.68 (CH3), 31.49, 34.67 (2 × CH2), 34.84, 37.77 (2 × CH), 39.25 (CH2), 40.41 (CH), 41.29 (CH2), 41.81 (CH), 45.35 (C), 56.05 (CH), 59.65 (CH2), 60.63 (CH), 72.19 (C), 105.89, 139.26 (2 × CH), 139.34 (C), 204.20 (CO). HRMS m/z 399.3017 calcd for C25H39N2O2+ 399.3006. 3α-Hydroxy-3β-methyl-21-(4-trifluoromethyl-1H-pyrazol-1′yl)-19-nor-5β-pregnan-20-one (15). Yield: 69 mg (48%) as an offwhite solid. LC-MS: tR = 1.11 min, m/z = 453 (M + 1). 1H NMR (400 MHz, CDCl3): δ 7.72 (s, 2H), 5.08−4.75 (m, 2H), 2.75−2.50 (m, 1H), 2.30−2.00 (m, 2H), 1.95−1.59 (m, 6H), 1.53−1.30 (m, 16H), 1.25−1.00 (m, 3H), 0.68 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 13.92 (CH3), 23.24, 24.47, 25.55, 25.79, 26.17 (5 × CH2), 26.69 (CH3), 31.46, 34.64 (2 × CH2), 34.80, 37.74 (2 × CH), 39.27 (CH2), 40.38 (CH), 41.24 (CH2), 41.78 (CH), 45.49 (C), 56.05, 61.13 (2 × CH), 61.73 (CH2), 72.17 (C), 114.43 (C, J = 38 Hz), 122.62 (CF3, J = 264 Hz), 130.20−130.50 (m, CH), 137.25−137.50 (m, CH), 202.79 (CO). HRMS m/z 453.2763 calcd for C25H36N3O2+ 453.2723. 3α-Hydroxy-3β-methyl-21-(4-chloro-1H-pyrazol-1′-yl)-19nor-5β-pregnan-20-one (16). Yield: 8 mg (21%) as an off-white solid. LC-MS: tR = 1.05 min, m/z = 419 (M + 1). 1H NMR (400 MHz, CDCl3) δ 7.45 (s, 1H), 7.40 (s, 1H), 4.94−4.77 (m, 2H), 2.57 (t, J = 9.2 Hz, 1H), 2.26−2.12 (m, 1H), 2.07−1.98 (m, 1H), 1.90−1.58 (m, 8H), 1.50−1.23 (m, 13H), 1.19−1.01 (m, 4H), 0.66 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 13.90 (CH3), 23.26, 24.47, 25.55, 25.79, 26.17 (5 × CH2), 26.68 (CH3), 31.46, 34.64 (2 × CH2), 34.80, 37.73 (2 × CH), 39.25 (CH2), 40.38 (CH), 41.24 (CH2), 41.78 (CH), 45.41 (C), 56.02, 60.90 (2 × CH), 62.14 (CH2), 72.15, 110.79 (2 × C), 128.86, 138.39 (2 × CH), 203.41 (CO). HRMS m/z 419.2462 calcd for C24H36ClN2O2+ 419.2460. 3α-Hydroxy-3β-methyl-21-(4-nitro-1H-pyrazol-1′-yl)-19-nor5β-pregnan-20-one (17). Yield: 12 mg (31%) as an off-white solid. LC-MS: tR = 1.01 min, m/z = 430 (M + 1). 1H NMR (400 MHz, CDCl3) δ 8.18 (s, 1H), 8.08 (s, 1H), 5.04−4.85 (m, 2H), 2.62 (t, J = 8.8 Hz, 1H), 2.27−2.16 (m, 1H), 2.08−2.00 (m, 1H), 1.89−1.61 (m, 7H), 1.52−1.24 (m, 15H), 1.18−1.04 (m, 3H), 0.68 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 13.97 (CH3), 23.24, 24.46, 25.56, 25.80, 26.17 (5 × CH2), 26.73 (CH3), 31.44, 34.63 (2 × CH2), 34.79, 37.74 (2 × CH), 39.31 (CH2), 40.37 (CH), 41.23 (CH2), 41.78 (CH), 45.65 (C), 56.09, 61.34 (2 × CH), 62.24 (CH2), 72.17 (C), 130.42, 136.07 (2 × CH), 136.57 (C) 201.80 (CO). HRMS m/z 430.2696 calcd for C24H36N3O4+ 430.2700. 3α-Hydroxy-3β-methyl-21-(4-carbamoyl-1H-pyrazol-1′-yl)19-nor-5β-pregnan-20-one (18). Yield: 91 mg (46%) as an offwhite solid. LC-MS: tR = 0.78 min, m/z = 410 (M − 18). 1H NMR (400 MHz, CDCl3): δ 7.88 (s, 1H), 7.79 (s, 1H), 5.57 (br s, 2H), 5.04−4.83 (m, 2H), 2.65−2.55 (m, 1H), 2.27−2.14 (m, 1H), 2.09− 1.98 (m, 1H), 1.91−1.66 (m, 7H), 1.53−1.01 (m, 20H), 0.68 (s, 3H). 13 C NMR (100 MHz, CDCl3) δ 3.95 (CH3), 23.27, 24.48, 25.57,

Assessment of in vivo pharmacological activity was performed by measuring the anticonvulsant effects of 3 in a number of animal models.31 Acute administration of 3 (0.3−10 mg/kg, ip) effectively reduced pentylenetretazol (PTZ)induced seizures in mice (MECplasma = 85 nM; 3 (0.3−3 mg/ kg, ip)) as well as producing a dose-dependent anticonvulsant effect in the mouse 6 Hz electrical stimulation model. In the rat lithium-pilocarpine model of status epilepticus (SE), 3 (0.3−5 mg, iv) abolished both behavioral and electrographic seizure activity, even when administered 60 min after induction of SE. In sharp contrast, diazepam (10 mg/kg, iv) and lamotrigine (3−30 mg/kg, iv) both failed to ablate seizure activity under these model conditions. This latter result illustrates the potential advantage of modulating extrasynaptic GABAA receptors by NASs when compared to compound classes that only modulate synaptic GABAA receptors such as benzodiazepines. Detailed accounts of the in vivo pharmacological effects of 3, with an emphasis on the underlying pharmacology, will be published in a separate manuscript33 highlighting the properties of this compound. On the basis of its overall in vitro and in vivo profile including a full in vivo toxicological package (data not reported here), compound 3, was selected for advancement to phase 1 clinical trials. SAD and MAD phase 1 clinical trials of compound 3 have been completed,23 and subsequent phase 2 clinical trials as an oral treatment for a range of mood and movement disorder indications, such as major depressive disorder (MDD), essential tremor (ET), and postpartum depression (PPD), are currently underway.34

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EXPERIMENTAL SECTION

The synthesis of intermediates has been published elsewhere.21,26,27 Experimental procedures and characterization data for compounds 2 and 11 can be found elsewhere.21 LC/MS, preparative HPLC, and accurate mass methods can be found elsewhere.21 All compounds were isolated using preparative HPLC. 1H NMR spectra (δ) were recorded on a Bruker or Varian 400 MHz or Bruker 500 MHz instrument, and TMS was used as an internal standard. The purity of the final compounds was assessed on the basis of analytical LC-MS, and the results were greater than 95%. All compounds are named according to the steroid atom numbering convention.24 All compounds presented in Table 1 were synthesized according to procedures recently published.21,26,27 3α-Hydroxy-3β-methyl-21-(4-cyano-1H-pyrazol-1′-yl)-19nor-5β-pregnan-20-one (3). Yield: 28 g (49%) as an off-white solid. LC-MS: tR = 1.00 min, m/z = 410 (M + 1). 1H NMR (400 MHz, CDCl3): δ 7.86 (s, 1H), 7.80 (s, 1H), 5.08−4.84 (m, 2H), 2.70−2.55 (m, 1H), 2.25−2.15 (m, 1H), 2.10−2.00 (m, 1H), 1.88−1.59 (m, 7H), 1.53−1.30 (m, 15H), 1.25−1.00 (m, 3H), 0.67 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 13.92 (CH3), 23.20, 24.44, 25.54, 25.78, 26.15 (5 × CH2), 26.69 (CH3), 31.43, 34.61 (2 × CH2), 34.77, 37.71 (2 × CH), 39.26 (CH2), 40.35 (CH), 41.21 (CH2), 41.75 (CH), 45.56 (C), 56.04, 61.24 (2 × CH), 61.78 (CH2), 72.14 (C), 93.25 (C), 113.35 (CN), 136.16, 142.49 (2 × CH), 202.23 (CO). HRMS m/z 410.2803 calcd for C25H36N3O2+ 410.2802. 3α-Hydroxy-3β-methyl-21-(4-methyl-1H-pyrazol-1′-yl)-19nor-5β-pregnan-20-one (12). Yield: 8 mg (21%) as an off-white solid. LC-MS: tR = 0.98 min, m/z = 399 (M + 1). 1H NMR (400 MHz, CDCl3): δ 7.34 (s, 1H), 7.17 (s, 1H), 4.92−4.75 (m, 2H), 2.57 (t, J = 8.8 Hz, 1H), 2.25−2.12 (m, 1H), 2.09 (s, 3H), 2.08−2.00 (m, 1H), 1.90−1.73 (m, 4H), 1.67−1.57 (m, 3H), 1.50−1.20 (m, 15H), 1.16− 1.03 (m, 3H), 0.67 (s, 3H). 13C NMR (100 MHz, CDCl3): δ 9.07 13.88 (2 × CH3), 23.33, 24.50, 25.57, 25.81, 26.19 (5 × CH2), 26.68 (CH3), 31.49, 34.66 (2 × CH2), 34.83, 37.75 (2 × CH), 39.22 (CH2), 40.40 (CH), 41.27 (CH2), 41.80 (CH), 45.30 (C), 56.01, 60.65 (2 × CH), 61.70 (CH2), 72.17 (C), 116.96 (C), 129.52, 140.51 (2 × CH), 7816

DOI: 10.1021/acs.jmedchem.7b00846 J. Med. Chem. 2017, 60, 7810−7819

Journal of Medicinal Chemistry

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25.81, 26.19 (5 × CH2), 26.69 (CH3), 31.47, 34.65 (2 × CH2), 34.81, 37.74 (2 × CH), 39.29 (CH2), 40.38 (CH), 41.25 (CH2), 41.79 (CH), 45.50 (C), 56.06, 61.05 (2 × CH), 61.87 (CH2), 72.17 (C), 118.45 (C), 133.13, 139.13 (2 × CH), 164.20 (CONH2), 203.00 (CO). HRMS m/z 428.2913 calcd for C25H38N3O3+ 428.2908. 3α-Hydroxy-3β-methyl-21-(4-carboxy-1H-pyrazol-1′-yl)-19nor-5β-pregnan-20-one (19). Yield: 127 mg (42%) as an off-white solid. LC-MS: tR = 0.85 min, m/z = 429 (M + 1). 1H NMR (400 MHz, CD3OD) δ 8.08 (s, 1H), 7.87 (s, 1H), 5.16−5.04 (m, 2H), 2.74 (t, J = 8.8 Hz, 1H), 2.23−2.09 (m, 2H), 1.95−1.63 (m, 7H), 1.59−1.31 (m, 10H), 1.30−1.07 (m, 8H), 0.69 (s, 3H). 13C NMR (100 MHz, CD3OD) δ 13.96 (CH3), 24.05, 25.33 (2 × CH2), 26.25 (CH3), 26.28, 26.89, 27.25, 32.60, 35.07 (5 × CH2), 35.96, 39.01 (2 × CH), 39.93 (CH2), 41.85 (CH2 + CH), 43.10 (CH), 46.19 (C), 57.08, 61.79 (2 × CH), 62.53 (CH2), 72.47, 116.89 (2 × C), 136.60, 142.27 (2 × CH), 166.47 (COOH), 205.22 (CO). HRMS m/z 429.2733 calcd for C25H37N2O4+ 429.2748. 3α-Hydroxy-3β-methyl-21-(4-methoxy-1H-pyrazol-1′-yl)-19nor-5β-pregnan-20-one (20). Yield: 50 mg (24%) as an off-white solid. LC-MS: tR = 0.95 min, m/z = 415 (M + 1). 1H NMR (400 MHz, CDCl3): δ 7.27 (m, 1H), 7.07 (s, 1H), 4.86−4.71 (m, 2H), 3.75 (s, 3H), 2.55 (s, 1H), 2.23−2.13 (m, 1H), 2.07−1.95 (m, 1H), 1.87−1.78 (m, 3H), 1.73−1.66 (m, 5H), 1.49−1.37 (m, 6H), 1.34−1.22 (m, 8H), 1.14−1.08 (m, 3H), 0.67 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 13.89 (CH3) 23.34, 24.49, 25.57, 25.82, 26.20 (5 × CH2), 26.67 (CH3), 31.49, 34.66 (2 × CH2), 34.83, 37.76 (2 × CH), 39.23 (CH2), 40.41 (CH), 41.27 (CH2), 41.81 (CH), 45.34 (C), 56.03 (CH3), 58.98, 60.68 (2 × CH), 62.40 (CH2), 72.19 (C), 115.43, 127.69 (2 × CH), 147.44 (C), 204.44 (CO). HRMS m/z 415.2962 calcd for C25H39N2O3+ 415.2955. 3α-Hydroxy-3β-methyl-21-(3-cyano-1H-pyrazol-1′-yl)-19nor-5β-pregnan-20-one (21). Yield: 81 mg (53%) as an off-white solid. LC-MS: tR = 1.00 min, m/z = 392 (M + 1). 1H NMR (400 MHz, CDCl3) δ 7.48 (d, J = 2.5 Hz, 1H), 6.73 (d, J = 2.5 Hz, 1H), 5.07−4.88 (m, 2H), 2.60 (t, J = 8.8 Hz, 1H), 2.25−2.14 (m, 1H), 2.09−2.00 (m, 1H), 1.89−1.64 (m, 7H), 1.53−1.23 (m, 15H), 1.18−1.03 (m, 3H), 0.67 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 13.92 (CH3), 23.21, 24.45, 25.54, 25.79, 26.16 (5 × CH2), 26.68 (CH3), 31.44, 34.63 (2 × CH2), 34.78, 37.72 (2 × CH), 39.24 (CH2), 40.36 (CH), 41.22 (CH2), 41.77 (CH), 45.56 (C), 56.04, 61.21 (2 × CH), 62.17 (CH2), 72.15 (C), 112.07 (CH), 114.02 (CN), 125.28 (C), 132.35 (CH), 202.39 (CO). HRMS m/z 410.2795 calcd for C25H36N3O2+ 410.2802. 3β-Ethyl-3α-hydroxy-21-(4-cyano-1H-pyrazol-1′-yl)-19-nor5β-pregnan-20-one (22). Yield: 10 mg (27%) as an off-white solid. LC-MS: tR = 1.04 min, m/z = 446 (M + 23). 1H NMR (400 MHz, CDCl3) δ 7.86 (s, 1H), 7.81 (s, 1H), 5.06−4.84 (m, 2H), 2.61 (t, J = 8.8 Hz, 1H), 2.26−2.14 (m, 1H), 2.10−1.99 (m, 1H), 1.84−1.70 (m, 6H), 1.65−1.60 (m, 2H), 1.54−1.23 (m, 13H), 1.19−1.03 (m, 3H), 0.89 (s, 3H), 0.67 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 7.19, 13.94 (2 × CH3), 23.23, 24.47, 25.02, 25.83, 26.16, 29.79, 31.54, 32.02 (8 × CH2), 34.23, 37.87 (2 × CH2), 38.81, 39.30 (2 × CH2), 40.24, 41.78 (2 × CH), 45.59 (C), 56.07, 61.27 (2 × CH), 61.80 (CH2), 73.46 (C), 93.30 (C), 113.37 (CN), 136.15, 142.51 (2 × CH), 202.23 (CO). HRMS m/z 424.2941 calcd for C26H38N3O2+ 424.2959. 3α-Hydroxy-3β-methoxymethyl-21-(4-cyano-1H-pyrazol-1′yl)-19-nor-5β-pregnan-20-one (23). Yield: 30 mg (24%) as an offwhite solid. LC-MS: tR = 0.95 min, m/z = 440 (M + 1). 1H NMR (400 MHz, CDCl3) δ 7.85 (s, 1H), 7.81 (s, 1H), 5.08−4.84 (m, 2H), 3.45− 3.34 (m, 5H), 2.68−2.57 (m, 2H), 2.26−2.14 (m, 1H), 2.10−1.98 (m, 1H), 1.88−1.59 (m, 8H), 1.53−1.35 (m, 7H), 1.33−1.02 (m, 6H), 0.66 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 13.94 (CH3), 23.22, 24.45, 25.04, 25.75, 25.97, 29.25, 31.33 (7 × CH2), 34.20 (CH), 35.85 (CH2), 37.67 (CH), 39.25 (CH2), 40.22, 41.75 (2 × CH), 45.57 (C), 56.03 (CH), 59.52 (CH3), 61.24 (CH), 61.79 (CH2), 72.69 (C), 76.66 (CH2), 93.27 (C), 113.37 (CN), 136.16, 142.50 (2 × CH), 202.23 (CO). HRMS m/z 440.2897 calcd for C26H38N3O3+ 440.2908. 3α-Hydroxy-3β-methyl-21-(4-methyl-2H-1,2,3-triazol-2′-yl)19-nor-5β-pregnan-20-one (24). Yield: 269 mg (27%) as an offwhite solid. LC-MS: tR = 0.99 min, m/z = 400 (M + 1). 1H NMR (400

MHz, CDCl3) δ 7.42 (s, 1H), 5.20−5.08 (m, 2H), 2.57 (t, J = 8.8 Hz, 1H), 2.33 (s, 3H), 2.24−2.14 (m, 1H), 2.10−2.03 (m, 1H), 1.90−1.59 (m, 7H), 1.51−1.20 (m, 15H), 1.17−1.03 (m, 3H), 0.70 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 10.83, 13.82 (2 × CH3), 23.32, 24.49, 25.57, 25.79, 26.20 (5 × CH2), 26.69 (CH3), 31.49, 34.66 (2 × CH2), 34.83, 37.76 (2 × CH), 39.14 (CH2), 40.40 (CH), 41.28 (CH2), 41.80 (CH), 45.33 (C), 56.02, 60.72 (2 × CH), 63.79 (CH2), 72.19 (C), 134.45 (CH), 144.88 (C), 203.18 (CO). HRMS m/z 400.2954 calcd for C24H38N3O2+ 400.2959. 3α-Hydroxy-3β-methyl-21-(5-methyl-1H-1,2,3-triazol-1′-yl)19-nor-5β-pregnan-20-one (25). Yield: 250 mg (12%) as an offwhite solid. LC-MS: tR = 0.90 min, m/z = 400 (M + 1). 1H NMR (400 MHz, CDCl3) δ 7.48 (s, 1H), 5.14−5.01 (m, 2H), 2.65 (t, J = 8.8 Hz, 1H), 2.26−2.13 (m, 4H), 2.13−2.02 (m, 1H), 1.90−1.59 (m, 8H), 1.54−1.26 (m, 14H), 1.18−1.03 (m, 3H), 0.69 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 8.55, 13.88 (2 × CH3), 23.30, 24.46, 25.55, 25.81, 26.16 (5 × CH2), 26.67 (CH3), 31.45, 34.63 (2 × CH2), 34.78, 37.73 (2 × CH), 39.27 (CH2), 40.37 (CH), 41.24 (CH2), 41.78 (CH), 45.55 (C), 56.06 (CH), 57.41 (CH2), 61.04 (CH), 72.13 (C), 133.18 (CH), 133.90 (C), 201.93 (CO). HRMS m/z 400.2960 calcd for C24H38N3O2+ 400.2959. 3α-Hydroxy-3β-methyl-21-(4-methyl-1H-1,2,3-triazol-1′-yl)19-nor-5β-pregnan-20-one (26). Yield: 426 mg (21%) as an offwhite solid. LC-MS: tR = 0.89 min, m/z = 400 (M + 1). 1H NMR (400 MHz, CDCl3) δ 7.34 (s, 1H), 5.22−5.01 (m, 2H), 2.62 (t, J = 8.8 Hz, 1H), 2.37 (s, 3H), 2.25−2.10 (m, 1H), 2.10−2.02 (m, 1H), 1.91−1.59 (m, 8H), 1.51−1.24 (m, 14H), 1.18−1.02 (m, 3H), 0.66 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 11.00, 13.90 (2 × CH3), 23.24, 24.45, 25.54, 25.78, 26.16 (5 × CH2), 26.67 (CH3), 31.45, 34.63 (2 × CH2), 34.79, 37.72 (2 × CH), 39.22 (CH2), 40.37 (CH), 41.23 (CH2), 41.78 (CH), 45.52 (C), 56.03 (CH), 59.07 (CH2), 61.24 (CH), 72.14 (C), 122.83 (CH), 143.70 (C), 202.22 (CO). HRMS m/z 400.2962 calcd for C24H38N3O2+ 400.2959. 3α-Hydroxy-3β-methyl-21-(4-cyano-2H-1,2,3-triazol-2′-yl)19-nor-5β- pregnan-20-one (27). Yield: 35 mg (44%) as an offwhite solid. LC-MS: tR = 1.31 min, m/z = 393 (M − 18). 1H NMR (400 MHz, CDCl3) δ 8.02 (s, 1H), 5.35−5.24 (m, 2H), 2.63 (t, J = 8.8 Hz, 1H), 2.27−2.00 (m, 3H), 1.97−1.86 (m, 3H), 1.84−1.75 (m, 5H), 1.71−1.62 (m, 4H), 1.60−1.06 (m, 12H), 0.71 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 13.88, 22.56 (2 × CH3), 23.24, 24.41, 24.89, 25.80, 26.00, 30.57, 31.13 (7 × CH2), 34.29 (CH), 36.92 (CH2), 37.87 (CH), 39.03 (CH2), 39.97, 41.60 (2 × CH), 45.59 (C), 55.84, 61.06 (2 × CH), 64.69 (CH2), 91.37 (C), 111.32 (CN), 122.41 (C), 139.29 (CH), 200.88 (CO). HRMS m/z 411.2390 calcd for C24H35N4O2+ 411.2755. 3α-Hydroxy-3β-methyl-21-(4-cyano-1H-1,2,3-triazol-1′-yl)19-nor-5β-pregnan-20-one (28). Yield: 6.5 mg (99%) as an offwhite solid. LC-MS: tR = 1.26 min, m/z = 393 (M − 18). 1H NMR (400 MHz, CDCl3): δ 8.13 (s, 1H), 5.37−5.13 (m, 2H), 2.72−2.65 (m, 1H), 2.28−2.03 (m, 3H), 1.97−1.85 (m, 3H), 1.84−1.73 (m, 5H), 1.70−1.64 (m, 4H), 1.60−1.23 (m, 9H), 1.20−1.07 (m, 3H), 0.67 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 13.98 (CH3), 22.56 (CH), 23.24, 24.38, 24.87, 25.76, 25.98, 30.54, 31.10 (7 × CH2), 34.26 (CH3), 36.88 (CH2), 37.85 (CH), 39.09 (CH2), 39.93, 41.58 (2 × CH), 45.72 (C), 55.84 (CH), 59.19 (CH2), 61.47 (CH), 91.36, 111.44, 121.54 (3 × C), 131.27 (CH), 200.42 (CO). HRMS m/z 411.2760 calcd for C24H35N4O2+ 411.2755.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00846. General experimental details, protocols for biological assays, details of animal care, and pharmacokinetic experiments (PDF) Molecular formula strings (CSV) 7817

DOI: 10.1021/acs.jmedchem.7b00846 J. Med. Chem. 2017, 60, 7810−7819

Journal of Medicinal Chemistry

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(12) Wafford, K. A.; Macaulay, A. J.; Fradley, R.; O’Meara, G. F.; Reynolds, D. S.; Rosahl, T. W. Differentiating the role of gammaaminobutyric acid type A (GABAA) receptor subtypes. Biochem. Soc. Trans. 2004, 32, 553−556. (13) Vaitkevicius, H.; Ng, M. C.; Moura, L.; Rosenthal, E. S.; Westover, M. B.; Rosand, J.; Rogawski, M. A.; Reddy, K.; Cole, A. J. Successful allopregnanolone treatment of new onset refractory status epilepticus (NORSE): First in man experience. In The 4th LondonInnsbruck Colloquium on Status Epilepticus and Acute Seizures; Salzburg, Austria, April 4−6, 2013; 2013; p 124 (abstract P29). (14) Bialer, M.; Johannessen, S. I.; Levy, R. H.; Perucca, E.; Tomson, T.; White, H. S. Progress report on new antiepileptic drugs: a summary of the Twelfth Eilat Conference (EILAT XII). Epilepsy Res. 2015, 111, 85−141 [Allopregnanolone (SAGE-547) injection, p. 91].. (15) A Study With SAGE-547 for Super-Refractory Status Epilepticus; ClinicalTrials.gov; U.S. National Institutes of Health: Bethesda, MD, 2015; https://clinicaltrials.gov/ct2/show/record/ NCT02477618?term=sage+therapeutics&rank=6 (Accessed April 10, 2017). (16) Kanes, S. J.; Colquhoun, H.; Doherty, J.; Raines, S.; Hoffmann, E.; Rubinow, D. R.; Meltzer-Brody, S. Open-label, proof-of-concept study of brexanolone in the treatment of severe postpartum depression. Hum. Psychopharmacol. 2017, 32, e2576. (17) Ellenbogen, A.; Raines, S.; Kanes, S. Exploratory trial results for SAGE-547 in essential tremor. Neurology 2016, 86 (16), P4.297. (18) Brambilla, P; Perez, J; Barale, F; Schettini, G; Soares, J. C. GABAergic dysfunction in mood disorders. Mol. Psychiatry 2003, 8, 721−737. (19) Luscher, B.; Shen, Q.; Sahir, N. The GABAergic deficit hypothesis of major depressive disorder. Mol. Psychiatry 2011, 16, 383−406. (20) Zorumski, C. F.; Paul, S. M.; Izumi, Y.; Covey, D. F.; Mennerick, S. Neurosteroids, stress and depression: potential therapeutic opportunities. Neurosci. Biobehav. Rev. 2013, 37, 109−22. (21) Martinez Botella, G.; Salituro, F. G.; Harrison, B. L.; Beresis, R. T.; Bai, Z.; Shen, K.; Belfort, G. M.; Loya, C. M.; Ackley, M. A.; Grossman, S. J.; Hoffmann, E.; Jia, S.; Wang, J.; Doherty, J. J.; Robichaud, A. J. Neuroactive steroids. 1. Positive allosteric modulators of the (γ-Aminobutyric Acid) A receptor: structure−activity relationships of heterocyclic substitution at C-21. J. Med. Chem. 2015, 58, 3500−3511. (22) Hammond, R. S.; Althaus, A. L.; Ackley, M. A.; Maciag, C.; Martinez Botella, G.; Salituro, F. G.; Robichaud, A. J.; Doherty, J. J. Anticonvulsant profile of the neuroactive steroid, SGE-516, in animal models. Epilepsy Res. 2017, 134, 16−25. (23) Bialer, M.; Johannessen, S. I.; Levy, R. H.; Perucca, E.; Tomson, T.; White, H. S. Progress report on new antiepileptic drugs: a summary of the Thirteenth Eilat Conference on New Antiepileptic Drugs and Devices (EILAT XIII). Epilepsia 2017, 58, 181−221. (24) Lambert, J. J.; Belelli, D.; Hill-Venning, C.; Peters, J. A. Neurosteroids and GABAA receptor function. Trends Pharmacol. Sci. 1995, 16, 295−303. (25) Maruoka, K.; Itoh, T.; Yamamoto, H. Methylaluminum bis(2,6di-tert-butyl-4-alkylphenoxide). A new reagent for obtaining unusual equatorial and anti-Cram selectivity in carbonyl alkylation. J. Am. Chem. Soc. 1985, 107, 4573−4576. (26) Martinez Botella, G.; Harrison, B. L.; Robichaud, A. J.; Salituro, F. G.; Beresis, R. T. 19-Nor-C3,3-disubstituted C21-N-Pyrazolyl Steroids as GABA Receptor Modulators and their Preparation. WO2014169833, 2014. (27) Martinez Botella, G.; Harrison, B. L.; Robichaud, A. J.; Salituro, F. G.; Beresis, R. T. Neuroactive Steroids in Treatment of CNS-related Diseases and their Preparation. WO2015180679, 2015. (28) Gee, K. W. Phenylquinolines PK 8165 and PK 9084 allosterically modulate [35S] t-butylbicyclophosphorothionate binding to a chloride ionophore in rat brain via a novel Ro5 4864 binding site. J. Pharmacol. Exp. Ther. 1987, 240, 747−753. (29) Automated patch clamp method details: see Supporting Information for detailed protocols and ref 21.

AUTHOR INFORMATION

Corresponding Author

*Phone: + 617-299-8396. E-mail: [email protected]. ORCID

Francesco G. Salituro: 0000-0003-1172-7064 Maria-Jesus Blanco: 0000-0003-4333-365X Present Addresses ∥

For G.M.B.: Praxis Precision Medicines For G.M.B.: Takeda Pharmaceuticals, Inc. # For C.M.L.: Collegium Pharmaceutical, Inc. ⊥

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Shanghai Chempartner and WuXi for chemistry and DMPK support. We thank Robert Jansen and the team at OpAns for conducting in vitro ADME assays.

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ABBREVIATIONS USED NAS, neuroactive steroid; GABAA, (γ-aminobutyric acid)A; [35S]TBPS, tert-butylbicyclophosphorothionate; PAM, positive allosteric modulator; SAD, single ascending dose; MAD, multiple ascending dose

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REFERENCES

(1) Martinez Botella, G.; Ackley, M. A.; Salituro, F. G.; Doherty, J. J. Natural and synthetic neuroactive steroid modulators of GABAA and NMDA receptors. Annu. Rep. Med. Chem. 2014, 49, 27−42. (2) Majewska, M. D.; Harrison, N. L.; Schwartz, R. D.; Barker, J. L.; Paul, S. M. Steroid hormone metabolites are barbiturate-like modulators of the GABA receptor. Science 1986, 232, 1004−1007. (3) Hosie, A. M.; Wilkins, M. E.; da Silva, H. M. a.; Smart, T. G. Endogenous neurosteroids regulate GABAA receptors through two discrete transmembrane sites. Nature 2006, 444, 486−489. (4) Hosie, A. M.; Wilkins, M. E.; Smart, T. G. Neurosteroid binding sites in GABA(A) receptors. Pharmacol. Ther. 2007, 116, 7−19. (5) Rudolph, U.; Möhler, H. GABA-based therapeutic approaches: GABAA receptor subtype functions. Curr. Opin. Pharmacol. 2006, 6, 18−23. (6) Belelli, D.; Harrison, N. L.; Maguire, J.; Macdonald, R. L.; Walker, M. C.; Cope, D. W. Extrasynaptic GABAA receptors: Form, pharmacology, and function. J. Neurosci. 2009, 29, 12757−12763. (7) Abramian, A. M.; Comenencia-Ortiz, E.; Modgil, A.; Vien, T. N.; Nakamura, Y.; Moore, Y. E.; Maguire, J. L.; Terunuma, M.; Davies, P. A.; Moss, S. J. Neurosteroids promote phosphorylation and membrane insertion of extrasynaptic GABAA receptors. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 7132−7137. (8) Rogawski, M.; Loya, C. M.; Reddy, K.; Zolkowska, D.; Lossin, C. Neuroactive steroids for the treatment of status epilepticus. Epilepsia 2013, 54, 93−98. (9) Goodkin, H. P.; Joshi, S.; Mtchedlishvili, Z.; Brar, J.; Kapur, J. Subunit-specific trafficking on GABAA receptors during status epilepticus. J. Neurosci. 2008, 28, 2527−2538. (10) Amin, J.; Brooks-Kayal, A.; Weiss, D. S. Two tyrosine residues on the alpha subunit are crucial for benzodiazepine binding and allosteric modulation of gamma-aminobutyric acid A receptors. Mol. Pharmacol. 1997, 51, 833−841. (11) Wieland, H.; Luddens, H.; Seeburg, P. A single histidine in GABAA receptors is essential for benzodiazepine agonist binding. J. Biol. Chem. 1992, 267, 1426−1429. 7818

DOI: 10.1021/acs.jmedchem.7b00846 J. Med. Chem. 2017, 60, 7810−7819

Journal of Medicinal Chemistry

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(30) Manual patch clamp method details: see Supporting Information for detailed protocols and ref 21. (31) Bialer, M.; Johannessen, S. I.; Levy, R. H.; Perucca, E.; Tomson, T.; White, H. S. Progress report on new antiepileptic drugs: a summary of the Twelfth Eilat Conference (EILAT XII). Epilepsy Res. 2015, 111, 85−141 (SAGE-217, p. 128).. (32) Huang, L. F.; Tong, W. Q. Impact of solid state properties on developability assessment of drug candidates. Adv. Drug Delivery Rev. 2004, 56, 321−334. (33) Althaus, A. L.; Belfort, G. M.; Hammond, R. S.; Ackley, M. A.; Quirk, M. C.; Martinez Botella, G.; Salituro, F. G.; Robichaud, A. J.; Doherty, J. J. Unpublished results. (34) SAGE217. ClinicalTrials.gov; U.S. National Institutes of Health: Bethesda, MD, 2017; https://clinicaltrials.gov/ct2/results?term= SAGE217&Search=Search&view=record (Accessed April 10, 2017).

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DOI: 10.1021/acs.jmedchem.7b00846 J. Med. Chem. 2017, 60, 7810−7819