Neuroactive Steroid N-Methyl-D-aspartate Receptor (NMDAR

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Neuroactive Steroid N-Methyl-D-aspartate Receptor (NMDAR) Positive Allosteric Modulators: Synthesis, SAR and Pharmacological Activity Daniel S. La, Francesco G. Salituro, Gabriel Martinez Botella, Andrew Griffin, Zhu Bai, Michael Ackley, Jing Dai, James Doherty, Boyd Harrison, Ethan Hoffman, Tatiana Kazdoba, Michael Lewis, Michael Quirk, and Albert Robichaud J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00591 • Publication Date (Web): 07 Aug 2019 Downloaded from pubs.acs.org on August 7, 2019

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Journal of Medicinal Chemistry

Neuroactive Steroid N-Methyl-D-aspartate Receptor (NMDAR) Positive Allosteric Modulators: Synthesis, SAR and Pharmacological Activity Daniel S. La,*† Francesco G. Salituro, † Gabriel Martinez Botella,† Andrew M. Griffin,† Zhu Bai,‡ Michael A. Ackley,† Jing Dai,† James J. Doherty,† Boyd L. Harrison,† Ethan C. Hoffmann,† Tatiana M. Kazdoba,† Michael C. Lewis,† Michael C. Quirk,† Albert J. Robichaud† † SAGE Therapeutics, 215 First Street, Cambridge, MA 02142. ‡ Wuxi AppTec, 288 Fute Zhong Road, Shanghai, China 200131 ABSTRACT: Neuroactive steroids (NASs) play a pivotal role in maintaining homeostasis is the CNS. We have discovered that one NAS in particular, 24(S)-hydroxycholesterol (24(S)-HC), is a positive allosteric modulator (PAM) of NMDA receptors. Using 24(S)HC as a chemical starting point, we have identified other Me Me OH OH NASs that have good in vitro potency and efficacy. Herein, Me Me 24 Me 17 we describe the structure activity relationship and Me Me Me H Me H Me pharmacokinetic optimization of this series that ultimately led to SGE-301 (42). We demonstrate that SGE-301 H H H H enhances long-term potentiation (LTP) in rat hippocampal HO 3 5 HO 6 Me slices and in a dose-dependent manner, improves cognition 24(S)-HC SGE-301 (42) in a rat social recognition study.

INTRODUCTION The N-methyl-D-aspartate (NMDA) receptors, in addition to the -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors, comprise three subclasses of ionotropic glutamate receptors (iGluRs).1 NMDAR receptors (NMDARs) are known to play a central role in synaptic transmission and plasticity and, over the past 30 years, have been the subject of intense active research, especially since its initial cloning results reported in 1991. 2 Multiple forms of modulation (agonist, co-agonists, selective and non-selective antagonists, channel blockers, positive and negative allosteric modulators) have been identified through rigorous research which is exemplified by a vast library of patents and publications.3 Accordingly, modulation of receptor function presents therapeutic opportunities due to strong links between imbalances in glutamatergic activity and various central nervous system (CNS) diseases. 4 For example, excessive activation of NMDARs has been implicated in a variety of neurological disorders such as traumatic brain injury (TBI), neurodegeneration and depression. Conversely, NMDAR hypofunction has been associated with diseases such as schizophrenia, ADHD and dementia. In contrast to compounds that directly activate or inhibit the receptor, there is a growing interest in the use of allosteric modulators of NMDAR as tractable therapeutics. Too much modulation of NMDAR

activity either up or down has been associated with undesirable outcomes. Finely tuned increases or decreases in receptor activity, whilst maintaining its temporal activation by neuronal activity may result in improved tolerability for therapies targeting the NMDA receptor system. 5, 6 Therefore, compounds that have varying degrees of modulation provide tremendous latitude by which to probe NMDAR pharmacology and ultimately address severe CNS maladies. We describe here the identification of neuroactive steroids (NASs) as positive allosteric modulators (PAMs) of NMDARs.7 Each NMDAR subtype exists as a heterotetramer comprised of two GluN1 and two GluN2(A-D) or GluN3(A,B) subunits. 8 There are four domains on each receptor subtype; the amino terminal domain (ATD), ligand binding domain (LBD), the transmembrane domain (TMD) and the cytoplasmic carboxy terminal domain (CTD). The TMD contains a series of four helices, M1-4, which undergo structural changes in response to conformational movement upon binding of neuro transmitter co-agonists, glutamate and glycine or D-serine, to the LBD thereby coupling ligand association with channel gating. NMDARs are selectively permeable to cations, specifically allowing passage of calcium and sodium ions into the cell (For comprehensive reviews see reference 1). NMDARs are subject to a voltage dependent block by magnesium ions. At resting

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membrane potentials, the channel is blocked by magnesium ions, so that even if the channel is opened by agonist binding, no current will flow through the channel. By progressively depolarizing the membrane, for example by repetitive activation of neighboring AMPA receptors, this magnesium block is relieved and allows sodium and calcium to enter the cell. As such, NMDARs can act as coincidence detectors, being activated by summating inputs from divergent pathways. The influx of calcium through the NMDAR can activate several downstream effectors and biochemical changes within the cell. One example of this is during long term plasticity, whereby temporal elevations in calcium influx through NMDAR can result in strengthening or weakening of synapses by increasing or decreasing AMPA receptor surface expression and protein synthesis. Such long-term plastic changes in areas such as the hippocampus have been postulated as a cellular basis for learning and memory. In accordance with this understanding, herein we highlight pharmacological data consistent with NMDAR potentiation as a mechanism by which: 1) long term potentiation (LTP) is increased in in vitro hippocampal slices and 2) social recognition, an in vivo model that is dependent on NMDAR function,9 is improved in aged rats. OMe

O

Me OH

MeO N

O O Cl

O

O OMe

N

Me N H

OMe

CIQ (1)

PDY-106 (2) N

HO O

O

N

Me S

N

F3C

O

GNE-6901 (3)

CF3

N

N

N

Cl

GNE-0723 (4) 21

Me 20 22 Me O

Me N N N

N H

H N

S O O Me

2

17

19 1 Me

H H

HO 3

GNE-9278 (5)

4

5

6

identified by the labs of Traynelis and Liotta,10, 11 selectively potentiates GluN2C and D, which could be a tool to explore potentiation of brain regions specific to these isoforms. The Traynelis and Liotta labs also discovered PDY-106 (2), a PAM that is selective for GluN2C.12, 13 Genentech has also been very active in studying NMDA PAMs and has disclosed GNE-6901 (3) and GNE-0723 (4) which selectively modulate GluN2A containing NMDARs.14, 15 Through X-ray crystallographic characterization, the binding site for these compounds has been revealed as the GluN1-GluN2A LBD interface. An additional modulator, GNE-9278 (5), bears a similar 5,6-bicyclic ring system,16 however, this compound binds to the transmembrane domain region and does not appear to display subunit selectivity. We have previously identified an endogenous oxysterol, 24(S)-hydroxycholesterol (24(S)-HC, 6), as a robust PAM of NMDARs.17 Whereas the exact binding site of 24(S)HC is not fully understood, chimera experiments which exchanged domains between rat NMDAR and GluK2 kainate receptors revealed that binding of 6 requires the TMD. Alternative binding sites for NAS could also exist as another steroid, pregnenolone sulfate (PS), requires both the TMD and LBD of the NMDA receptor for potentiation.18 The authors suggest that while more extensive mechanistic studies are required to elucidate the exact site of binding for the NAS modulators, these regions could contain the site of association. Thus, although 24(S)-HC displays unsurprisingly poor PK (rat Cl > 5.5 L/hr/kg, T1/2 < 0.6 h and %F < 10; See Table 2), the robust potential for compounds in this class to be effective modulators of NMDA, coupled with the data supporting the opportunity to treat indications arising from hypo-glutamatergic activity, we felt this compound serves as an ideal lead for drug discovery. We therefore initiated an SAR campaign to optimize properties and identify compounds with suitable pharmacokinetics and a pharmacological profile to produce a once daily oral therapeutic.

N

S F

Page 2 of 24

OH 24

Me

Me H

7

24(S)-HC (6)

Figure 1. Recently Disclosed NMDAR Positive Allosteric Modulators.

Although NMDAR antagonists have been more extensively evaluated in clinical trials, there are limited clinical data on NMDAR positive modulators.3 More recently, several novel molecules have been identified in preclinical studies as PAMs, some of which are illustrated in Figure 1. (3-chlorophenyl [3,4dihydro-6,7-dimethoxy-1-[(4-methoxyphenoxy)methyl]2(1H)-isoquinolinyl]methanone (CIQ, 1), a novel PAM

CHEMISTRY Novel synthetic routes were developed to enable efficient exploration of the C3 substituent as well as the steroid side chain attached to C17, in an attempt to optimize drug-like properties. Although the stereochemistry at C3 for NMDA PAM activity resided in a single configuration of the C3 alcohol, activity was maintained with both the (R) and (S) stereochemistry at the C24 alcohol. Therefore, we synthesized and tested our novel steroids as separated diastereomers at C24, generated from two strategies. Initially, diastereomers were made as racemic mixtures and then separated by supercritical fluid chromatography (SFC). A later approach involved a chiral synthesis using a C22 sulfone which was then alkylated with a range of chiral epoxides. The synthesis of the key sulfone intermediate 12 can be found below in Scheme 1, with the key synthetic steps being the stereoselective introduction of the methyl at C3 using the methylaluminum bis(2,6-di-t-butyl-4methylphenoxide (MAD) reagent developed by Yamamoto,19 followed by the stereoselective hydroboration of alkene 9 to yield alcohol 10 with the desired stereochemistry at C20.

Scheme 1. Synthesis of Sulfone 6


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Journal of Medicinal Chemistry Me

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Me Me

O

H H

Me

MePPh3Br Me

t-BuOK, THF 88%

H

HO

H H

Me

DCM H

Me

H

MAD, THF 51% (2 steps)

Me Me Me

HO

9

Me

Me

Me

Me

TsCl

Me

py 90%

H

Me

H H

HO

H

Me

10

OTs

PhSO2Na, KI DMF, 50 C 50%

Me H HO

O S O

H

Ph

H

Me

11

Sulfone 12 was then deprotonated with n-BuLi, followed by alkylation with a variety of chiral epoxides (Scheme 2). The sulfone was then reductively removed using magnesium and

H

Me

8

Me OH

H H

Me H H HO

MeMgBr

O

7

9-BBN

Me H H

HO

Pregnenolone

NaOH aq, H2O2 81%

Me

DMP

Me

12

NiCl2 to give the target 24-HCs as diastereomerically pure materials.

Scheme 2. Chiral synthesis of 24-HCs Me O S O Me Me Me

Me

Me

(S)

Me

H H

HO

O

Me O S O OH Me Me

H

12

n-BuLi, THF

Me

H H

HO

CH3

Me O S O OH Me Me

(S)

Mg, NiCl2

Me

H 3C

MeOH 27% (2 steps)

H

Me

13

A second approach, as exemplified in Scheme 3, targeted the synthesis of compounds 21 and 22 from the readily available ester 15. The C3 methyl group was again installed using MAD technology and further elaborated to aldehyde 19, thus enabling a focused library of analogs to be accessed by addition of a variety of Grignard reagents. Diastereomers were separated by

H H

HO

Me

CH3

H 3C H

14

SFC and absolute stereochemistry of final products was determined by single crystal X-ray diffraction. Note that for the synthesis of dimethylated analog 36, ester 17 was treated with excess methyllithium to give the desired compound while the synthesis of cyclopropane 40, was furnished from ester 17 upon treatment with EtMgBr and Ti(OiPr)4.

Scheme 3. Grignard method for the synthesis of 24-HCs



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

O

Me

Me

DCM H

H

LiAlH4

Me

Me

OH

Me

PCC

Me

Me

DCM 31%

H

18

H

Me

Me

OH

O Br

t-BuLi, THF 47%

H

19

Me

OH

Me

SFC

H H

Me

H

H H

HO

Me

HO

H

Me

H H

HO

Me

H HO

Me

HO

Me

Me H

21

As depicted in Scheme 4, another variation of the previous method involved olefination of C17 ketone 23 followed by methyl propiolate addition to afford acrylamide intermediate 25. Conversion of the ester to the aldehyde at C24 and subsequent trifluormethylation yielded a mixture of diasteromers which were separated by supercritical fluid chromatography (SFC), providing 29 and 30. Each diastereomer was separately converted to 31 and 32, respectively.

Scheme 4. Synthesis of trifluoro-analogs 38 and 39


H H

HO

Me

20

Me

H H

29%

OMe

H

17

Me

THF 75%

Me

16

Me

O

Me

MAD, THF 53% (2 steps)

O

15

Me

MeMgBr

OMe

H H

HO

O

Me

OMe

H H

Me

DMP

Me

Page 4 of 24

H

Me

22

OH

Page 5 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Me O Me

PPh3EtBr

H H

HO

O

Me

H

t-BuOK, THF 91%

Me

23

Me

Me

Me H

Et2AlCl, DCM 79%

Me

24

Me

OH

Me

DIBAL-H THF

Me

H HO

OMe

H

Me

H

25

Me

O

Me

MnO2

H H

HO

O

OMe

H H

HO

Me Me

Me

H

H

DCM

Me

HO

H

H H

27 Me

TMSCF3

Me

H HO

H

Me

Me

OH

Me CF3

H

Pd/C, H2 (atm) EtOAc 59%

29

Me

CF3

H H

HO

H

Me

31

SFC, 40% Me

Me H HO

Me

CF3

H

Me

OH

Me

Pd/C, H2 (atm) EtOAc 59%

H

OH

Me Me

30

Where C24 diastereomers were isolated using SFC separation, absolute stereochemistry was determined by X-ray diffraction, as shown in Figure 2 for representative examples. The structures confirmed the stereochemistry of the methyl and hydroxyl substituents at C3, as well as enabled us to assign the configuration of the side chain alcohol at C24.


Me

CF3

H H

HO

H

28 Me

OH

Me Me

HO

CF3

H H

Toluene/THF; TBF 42% (3 steps)

Me

26

OH

Me

H

32

Journal of Medicinal Chemistry Figure 2. X-ray structure of select compounds confirming connectivity and stereochemistry

The synthesis of analogs which are secondary alcohols at C3 followed a similar strategy (see experimental section) to the sequence outlined in Scheme 3. The alcohol of intermediate 15 was protected as a TBPS ether, the ester converted to a Weinreb amide, followed by addition of the appropriate Grignard reagent to afford the targeted intermediates. The resulting C24 ketones were then reduced to the C24 alcohol, the silyl group at C3 removed and the final diastereomeric mixtures separated by SFC to afford pure analogs. RESULTS AND DISCUSSION The ability of compounds to positively modulate NMDA receptors was assessed with electrophysiology methods. Currently, the contribution of specific NMDAR subunits in diand tri-heteromer configurations including multiple GluN2 subunits is not fully understood. Therefore, our approach focused on the discovery of pan-selective NMDAR positive modulators. Stable HEK293 cells, expressing either GluN1/GluN2A or GluN1/GluN2B were placed onto 384 well plates and whole cell patch clamp recordings were obtained using the Ionworks Barracuda system (molecular devices, Sunnyvale, CA). Cells were voltage clamped at -70mV and a voltage step to -45mV was applied during agonist (EC20 Lglutamate and 50 µM glycine) application. Two recordings were then performed, first, during pre-application of the test PAM alone (duration of pre-application - 5 min) and second, during the co-application of the test article and agonist to detect positive modulatory effects of the test article. Compound effects were evaluated in an 8-point concentration-response format and assessed by calculating % potentiation of the peak amplitude of the current in response to agonist application (Figure 3).20 200

% Potentiation

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

control

150

3M 24(S)-HC

100 50

250pA 0 1

10

100

1000

10000 100000

5s

Concentration (nM)

Figure 3. Potentiation of GluN1/GluN2A transfected HEK cells as measured by electrophysiology. A) A representative concentration response curve comparing percent potentiation as a function of compound concentration using 24(S)-HC (6). B) Representative current trace in the presence of NMDA and glycine, with and without 3 M 6.

The EC50 and Emax for 6 were determined to be 150 nM and 210%, respectively, on GluN1/GluN2A and 530 nM and 150%, respectively, on GluN2B transfected cells (Table 2). In addition, activity in multiple species (human, dog and rat) was examined in GluN1/GluN2A expressing oocytes employing a two-electrode voltage clamp (TEVC) method (Table 1). The EC50 and Emax values from the three species were reasonably similar, with EC50 ranging from 300 – 650 nM and Emax from Table 2. SAR of the C17 side chain

Page 6 of 24

73 – 180%. The similar in vitro activity suggests that preclinical models of in vivo efficacy and exaggerated pharmacology would be translatable across species for any individual compound. Table 1. 6 Potentiates NMDA Mediated Current in Human, Dog and Rat GluN2A Expressed in Oocyctes Receptor

EC50 in nM (95% CI)

% Emax (95% CI)

hGluN2A

300 (13-6810)

73 (40-106)

dGluN2A

650 (17-1570)

130 (59-153)

rGluN2A

650 (28-2378)

180 (104-204)

EC50 and Emax values were obtained using two electrode voltage clamp recordings from oocytes. Values were generated from three replicates (n = 3) from 5-point concentration-response curves. 95% CI = 95% confidence interval.

Having demonstrated that 6 is an efficient PAM across the NMDA subtypes, we focused our attention on using this compound as a starting point in a lead optimization campaign aimed at delivering potent, orally available, centrally penetrant PAMs. Our initial point of attack was to deconvolute the SAR of the steroid side chain while keeping the A-D ring system (i.e. -5,6) and substitution (i.e. secondary alcohol at C3) consistent with 6. (Table 2). Inversion of 6 to give the 24(R) analog (compound 33) resulted in improved potency at GluN2A- and GluN2Bcontaining receptors, however, poorer metabolic stability as assessed by incubation with human and rat microsomes. Simplifying the side chain to the primary alcohol analog 34 (Table 2) maintained efficacy at GluN1/GluN2A and GluN1/GluN2B but resulted in a >10-fold loss in potency. The potency at GluN2A and GluN2B could be regained by introducing a methyl group at the C24 position (compound 35) with the added benefit that the microsomal stability was also improved. Removal of the stereocenter at C24, through addition of a second methyl group (compound 36) lead to a loss in GluN2A potency with respect to compound 33 but with similar activity on GluN2A and B to 24(S)-HC. Cyclizing the two methyl groups at C24 to a cyclopropane (compound 40) resulted in a compound with slightly weaker activity relative to the parent (compound 36). Additional SAR exploration in this series examined the effect of varying the C17 side chain length. Utilizing compound 36 as a reference point, we found that removing one carbon in the side chain (compound 37) or two carbons (compound 38) abolished activity at GluN1/GluN2A and GluN1/GluN2B. Similarly, extending the side chain one carbon to give 25hydroxy cholesterol 39 resulted in complete loss of activity, confirming the optimal side chain length to be three carbon atoms (from C20 the carbon bearing the hydroxyl group). Key activity of the C17 side chain analogs with a hydroxyl group at C24 suggested a critical hydrogen bonding or enthalpic interaction that was sensitive to proximity to the steroid core. As a result, a more focused effort on analogs bearing the C24OH group was investigated.


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Journal of Medicinal Chemistry Me Me Me

R

H H

H

HO

Compound

R

GluN2A EC50, nM (95% CI)

GluN2A % Emax (95% CI)

GluN2B EC50, nM (95% CI)

GluN2B % Emax (95% CI)

150 (46440)

210 (190230)

530 (120240)

150 (120190)

10/52

37 210)

(10-

220 (180270)

55 130)

140 (130160)

28/93

3000 (1400-7300)

290 (230420)

>10000

320 (120850)

150 (120190)

140 (54330)

180 (150220)

7/17

180 (51680)

110 (87140)

410 (1601200)

140 (120170)

10/30

HLM/RLM (L/min mg)

OH Me

24(S)-HC (6)

Me OH Me

33

(20-

Me

OH

34 OH

35 Me

OH 36

37

Me Me Me Me

--

13/25

>10000

--

>10000

--

18/63

>10000

--

>10000

--

6/48

>10000

--

>10000

--

23/31

OH

OH 38

Me Me Me Me

39

OH

OH

40

900

280 (220340)


310

280 (240320)

10/35


EC50 and Emax values were obtained using automated patch clamp electrophysiology. Values were generated from four replicates (n = 4) from 8-point concentration-response curves. 95% CI = 95% confidence interval.

As mentioned previously, one of the aims of medicinal chemistry program was to reduce the in vivo clearance of our compounds in line with developing an orally bioavailable analog. We hypothesized that one clearance mechanism of our early compounds was likely phase 2 conjugation of the alcohol at the C3 position, however previous work had shown the alcohol at C3 to be necessary for activity in the steroid class, not allowing for the removal or replacement of this functional group. We reasoned that by adding an additional substituent at the C3 position we may be able to sterically shield the metabolism of the C3 alcohol, thus improving the metabolic stability while maintaining the activity as an NMDA modulator. The initial set of C3-methyl compounds are shown in Table 3 and all compounds demonstrated acceptable activity at GluN1/GluN2A and GluN1/GluN2B. A matched pair analysis, comparing compounds with and without a methyl at C3, demonstrated that this functionality did not result in a negative

effect on potency or efficacy and, in several cases, enhanced the activity of the C3 analogs. For instance, compound 14 was found to be about 4 times more potent at GluN1/GluN2A compared to 24(S)-HC and greater than ten times more potent at GluN1/GluN2B. Compound 43 showed a greater than 10fold improvement at GluN1/GluN2A when compared to compound 40. The effect on metabolic stability, as assessed in vitro, was less clear with compound 43 being more stable than compound 40 in human and rat microsomes, whilst compound 42 (SGE-301) was found to be less metabolically stable than compound 36. Furthermore, compound 41 was found to be significantly more stable than compound 14 whilst compound 14 showed improved stability in rat liver microsomes compared to 6. Given that in vitro microsomal clearance is not always predictive of in vivo clearance, i.v. pharmacokinetic parameters in rodents were determined for one matched pair. Compound 14 was found to have an improved clearance (3.1 L/hr/kg. See Table 6) compared to the non C3-methylated analog 24(S)-HC (5.9 L/hr/kg), encouraging us to explore further the SAR in the C3-methyl series. Gratifyingly, compound 42 had an even lower clearance of 1.1 L/hr/kg and a modest bioavailability of 28% in rodents (Table 6).

Table 3. C3 Methylation: Side Chain SAR Me Me Me

HO

R

H H

Page 8 of 24

H

Me


Page 9 of 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Compound

GluN2A EC50 (nM) (95% CI)

R


GluN2B EC50 (nM) (95% CI)



OH Me

14

33 (10-95)

200 230)

(170-

13 (6-25)

230 240)

(210-

230 270)

(190-

10000

--

>10000

--

>10000

--

>10000

--

H

OH Me Me Me

46

OMe Me Me

H H

H

Me Me Me Me

47

OH Me Me

H H

HO

550 2000)


Me

Me

HO


H

Me

Me

GluN2A EC50 (nM) (95% CI) OH Me Me

H

Page 10 of 24

H

Me

EC50 and Emax values were obtained using automated patch clamp electrophysiology. Values were generated from four replicates (n = 4) from 8-point concentration-response curves. 95% CI = 95% confidence interval.


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Having established some basic SAR of the C17 side chain and that introduction of a methyl group at C3 maintained activity with a desired improvement in metabolic stability, we sought to further explore the effect of varying the substituent at the C24 position (Table 5). Compounds 48 and 49, which have a methyl group at C24, showed poor activity at GluN1/GluN2A and GluN1/GluN2B which is in contrast to compound 35 without a methyl group at C3. Furthermore, if a second lipophilic substituent is added at C24 (compound 42), activity is regained. For the secondary alcohol subseries at C24, activity

was regained by increasing the lipophilicity of the substituent at C24 with ethyl, cyclopropyl, cyclobutyl and trifluoromethyl (50-55) all having EC50 and Emax values in the desired range. No clear trend was observed regarding the effect of the alcohol stereochemistry at C24 on activity and overall both diastereomers had acceptable pharmacological activity at GluN1/GluN2A and GluN1/GluN2B. All compounds were found to be very stable in human liver microsomes with stability in rat liver microsomes generally lower, varying from highly stable to moderately stable.

Table 5. SAR at C24. Me Me Me

H H

HO

R

H

Me



Compound

GluN2A EC50 (nM) (95% CI)

R


Page 12 of 24




OH

48

>10000

--

>10000

--

11/67

>10000

--

3000

50

Neuroactive Steroid N-Methyl-D-aspartate Receptor (NMDAR

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