De Novo Design, Synthesis, and Biological Evaluation of 3,4

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De Novo Design, Synthesis and Biological Evaluation of 3, 4-Disubstituted Pyrrolidine Sulfonamides as Potent and Selective Glycine Transporter 1 Competitive Inhibitors Ying Wang, Hongyu Zhao, Jason T. Brewer, Huanqiu Li, Yanbin Lao, Willi Amberg, Berthold Behl, Irini Akritopoulou-Zanze, Justin Dietrich, Udo E. W. lange, Frauke Pohlki, Carolin Hoft, Wilfried Hornberger, Stevan W Djuric, Jens Sydor, Mario Mezler, Ana-Lucia Relo, and Anil Vasudevan J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00295 • Publication Date (Web): 03 Jul 2018 Downloaded from http://pubs.acs.org on July 3, 2018

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Journal of Medicinal Chemistry

De Novo Design, Synthesis and Biological Evaluation of 3, 4-Disubstituted Pyrrolidine Sulfonamides as Potent and Selective Glycine Transporter 1 Competitive Inhibitors Ying Wang,*, † Hongyu Zhao,† Jason T. Brewer,† Huanqiu Li,† Yanbin Lao,† Willi Amberg, ‡ Berthold Behl,‡ Irini Akritopoulou-Zanze,† Justin Dietrich,† Udo E. W. Lange, Frauke Pohlki,‡ Carolin Hoft,‡ Wilfried Hornberger,‡ Stevan W. Djuric,† Jens Sydor,‡ Mario Mezler,‡ Ana Lucia Relo‡ and Anil Vasudevan† †

AbbVie Inc., 1 North Waukegan Road, North Chicago, Illinois 60064, United States

‡

AbbVie Deutschland GmbH & Co. KG, Neuroscience Research, Knollstrasse, 67061

Ludwigshafen KEYWORDS. Glycine Transporter 1, GlyT1, competitive inhibitor, target occupancy, pyrrolidine sulfonamide, brain penetration

ABSTRACT. The development of Glycine Transporter 1 (GlyT1) inhibitors may offer putative treatments for schizophrenia and other disorders associated with hypofunction of the glutaminergic N-methyl-D-aspartate (NMDA) receptor. Herein, we describe the synthesis and biological evaluation of a series of 3, 4-disubstituted pyrrolidine sulfonamides as competitive GlyT1 inhibitors that arose from de novo scaffold design. Relationship of chemical structure to drug-drug interaction (DDI) and bioactivation was mechanistically investigated. Murine studies were strategically incorporated into the screening funnel to provide early assessments of in vivo target occupancy (TO) by ex vivo binding studies. Advanced compounds derived from iterative structure-activity relationship (SAR) studies possessed high potency in ex vivo binding studies

ACS Paragon Plus Environment

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and good brain penetration, promising preliminary in vivo efficacy, acceptable preclinical pharmacokinetics and manageable DDI and bioactivation liabilities.

INTRODUCTION GlyT1 inhibitors. Impaired glutamatergic neurotransmission involving NMDA receptors has been linked to the etiology of schizophrenia. Major support for such a link comes from clinical observations that NMDA receptor antagonists induce the whole spectrum of psychotic symptoms1,2,3,4,5 and from several susceptibility genes converging on NMDA receptor signaling. 6,7,8

In terms of the pathophysiological mechanisms involved, NMDA dysfunction is thought to

result in a disturbed local balance between excitation and inhibition associated with disrupted micro-circuitries and large-scale dis-connectivities.9 One approach to reverse the postulated NMDA receptor hypofunction in schizophrenia is the facilitation of NMDA receptor activity via its modulatory glycine-site. Glycine is an obligatory co-agonist for NMDA receptors that is required for their activation by glutamate. The concentration of glycine in the synaptic cleft is regulated by glycine transporters (GlyTs). Two GlyTs have been classified, namely, GlyT1 and GlyT2. GlyT1 is localized at glutamatergic synapses.10 It has been shown to be physically associated with synthaxin 1A and with postsynaptic density protein 95,11 an NMDA receptorassociated protein, thus indicating a localization close to NMDA receptors. GlyT1 inhibitors have repeatedly been shown to be effective in animal models of schizophrenia across different symptom domains.12 As such, several pharmaceutical companies have focused on the development of selective GlyT1 inhibitors. Both non-competitive and competitive inhibitors have been reported in the literature (Figure 1).13,14,15,16,17,18 A number of these GlyT1 inhibitors have been clinically assessed for


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schizophrenia. The most advanced compound, RG1678 (Bitopertin), a non-competitive GlyT1 inhibitor developed by Roche, had positive phase II results with the treatment of negative symptoms of schizophrenia patients.18 However, RG1678 failed to meet its endpoint in two phase III clinical trials that assessed its efficacy in reducing negative symptoms of schizophrenia.19 Based on our preclinical studies that suggested competitive inhibitors might offer potential advantages with respect to their efficacy and safety, at the onset of our GlyT1 program, we decided to focus on the discovery and development of competitive GlyT1 inhibitors.20,21 Figure 1. Representative Literature GlyT1 Inhibitors.13,16,18

It is widely acknowledged that hit generation via a knowledge-based approach is potentially the fastest entry to novel leads for a given target.22,23 With this in mind, and the structurally diverse chemotypes of reported GlyT1 inhibitors in the literature, we initiated knowledge-based de novo design of GlyT1 inhibitors to complement our internal GlyT1 high throughput screening (HTS) campaign.21 Efflux Ratio (ER). In practice, through several iterative design-synthesis-data cycles, compound 4, a novel GlyT1 inhibitor scaffold, comprised of a 3, 4-disubstituted pyrrolidine core, was


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identified.24 Compound 4 (Figure 2) had acceptable GlyT1 in vitro potency (Ki =198 nM) as a starting point but it also had an efflux ratio (ER) value of 8.7 in the Madine Darby canine kidney (MDCK) – multidrug resistance protein 1 (MDR1) assay. The drug-like structure, combined with the perceived synthetic modularity of compound 4 and its analogs, made this compound a particularly attractive hit despite being a potential P-glycoprotein (P-gp) substrate. As with other central nervous system (CNS) targets, sufficient brain availability is paramount to discovering efficacious GlyT1 inhibitors. Achieving good brain penetration can be more challenging than enhancing potency.18,25 P-glycoprotein (P-gp, MDR1) is an important efflux transporter that is expressed in blood-brain barrier (BBB) and limits the drug entry into CNS. The MDCK-MDR1 assay is a valuable in vitro tool for the identification and characterization of P-gp substrates. Recognizing this, we strategically incorporated the MDCK-MDR1 assay early on in the screening funnel. This assay, along with the in vitro GlyT1 potency assay and liver microsomal stability assay was the first tier assays used collectively to triage compounds and perform multi-parametric optimization. Good correlation between the ER values obtained from our MDCK-MDR1 assay and the P-gp substrate liability of the CNS drugs has been reported in the literature.26 Furthermore, our internal validation data demonstrated that compounds with in vitro ER values greater than two from the assay, correlated well with limited in vivo brain penetration, as represented by free brain to plasma drug concentration ratio of less than 0.3 in rodent brain pharmacokinetic (PK) studies (data not shown). The good correlation between in vitro ER values and in vivo brain penetration further supported that P-gp in BBB plays an important role in restricting brain penetration. Initial SAR studies quickly showed that efforts to replace the sulfonamide functionality on the pyrrolidine nitrogen in compound 4 with amides, carbamates, and tertiary amines were futile,


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resulting in analogs with micromolar GlyT1 in vitro potency. Extensive SAR studies of the amides on the 3-amino group (C-3) in compound 4 only yielded analogs with potency within 5fold that of compound 4, all with high ER values. The breakthrough came from functionalizing the 3-amino group with aryl substituents. One of the early analogs, compound 5 (Figure 2), showed good in vitro GlyT1 potency (Ki = 17 nM). Most importantly, it exhibited dramatically lowered ER value compared to that of compound 4 (0.9 vs. 8.7). As expected, the low ER value translated to a good free brain to plasma drug concentration ratio of 0.6 in rat, suggesting that compound 5 is unlikely to be a P-gp substrate. Figure 2. De Novo Design of GlyT1 Inhibitor

Competition study. We have previously reported studies to assess the competitive and noncompetitive mode of inhibition by GlyT1 inhibitors by measuring the glycine-induced current in GlyT1 expressing oocytes from Xenopus laevis.20 In this assay, competitive inhibitors are characterized by a reduced relative inhibition of glycine-induced current in the presence of high glycine concentrations while non-competitive Glyt1 inhibitors show the same degree of inhibition of glycine-induced currents at high and low glycine concentrations (see experimental section). To our delight, compound 6 showed a concentration dependent effect on glycine-


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mediated currents with inhibition of 77% at 10 µM of glycine and 48% at 3 mM glycine, suggesting a competitive binding mode for the inhibitor (Figure 3). Selected compounds from this series were tested in this study throughout the course of the optimization process. All compounds tested were proven to be reversible competitive GlyT1 inhibitors.

Figure 3. Oocyte experiments with compound 6.20

Oocytes expressing GlyT1c were stimulated with 10 µM or 3 mM glycine to induce a current through the transporter. 3 µM compound 6 was added to the glycine solution and the inhibition of the current was calculated (N>4, P = 0.0006)

Ex vivo target occupancy study. Encouraged by the promising ER values and the competitive nature of the initial analogs, compounds with a good balance of in vitro profiles (potency, liver microsomal stability, ER and selectivity) were further profiled in preclinical PK studies. An ex vivo binding assay was developed to estimate ED50 or EC50 values for in vivo target occupancy.


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In this assay, forebrain homogenates from mice intraperitoneally treated with different doses of the test compound, were evaluated for binding of a GlyT1 radioligand ex vivo (experimental section). The ED50 values for the estimated in vivo target occupancy could be derived from the specific radioligand binding vs. the dose. In addition, EC50 values for target occupancy were calculated by plotting target occupancies assessed ex vivo vs the compound concentrations in the homogenates. The values of ED50 determined from these ex vivo binding assays were used as surrogates for in vivo potency. Our preliminary studies showed that the values of ED50 correlated to the ED50’s obtained from the in vivo efficacy models. As the program progressed, we collected a large in vitro and in vivo dataset from different chemical series, including in vitro potency Ki, in vivo rodert brain PK, and ex vivo GlyT1 target occupancy (TO) in forebrain. As described earlier, TO ED50 value was an important parameter to triage the compounds. However, the radioligand binding study to determine ex vivo TO was labor-extensive with low throughput. It would be nice to be able to estimate the TO outcome earlier in the program to triage compounds. Hence, we studied the relationship between brain exposure and TO to see if we would be able to estimate the TO from routine rodent brain PK study, which is much higher throughput. For CNS targets, only free drug in brain is available to occupy the targets, thus free brain drug concentration should directly correlate with target occupancy. From routine brain PK study, total drug concentrations in brain homogenates could be determined. The unbound fraction in brain homogenate could be measured separately. Together, free rodent brain drug concentration could be calculated. In order to normalize a wide range of in vitro Ki values of the compounds tested from different chemical series, we corrected the free brain drug concentrations with in vitro Ki. When plotted, a good correlation was observed between ex vivo forebrain GlyT1 TO and free brain drug concentration corrected by in


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vitro Ki (Figure 4). The data was fitted into a sigmoidal model with a R2 value of 0.60 and a Hill coefficient of 0.93. To achieve 50% TO, free brain drug concentration was estimated to be approximately 0.3 fold of in vitro Ki (figure 4, horizontal and vertical lines). With this brain exposure-TO relationship established, we were able to estimate TO ED50 of promising compounds based on brain drug concentration and dose from the in vivo rodent brain PK study. This allowed us to rapidly triage the compounds earlier in the screening funnel without laborintensive ex vivo TO studies. In practice, routine in vivo rodent brain PK study was incorporated as one of the key second tier assays in the optimization process. Figure 4. Brain Exposure -TO Relationship between forebrain GlyT1 occupancy and free forebrain concentration adjusted by Ki


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Herein, we describe structure-activity relationships (SARs) of the novel 3,4-disubstituted pyrrolidine sulfonamides which led to the discovery of several highly potent and selective GlyT1 competitive inhibitors with excellent in vivo brain penetration. RESULTS AND DISCUSSION Chemistry. As shown in Scheme 1, the 3,4-disubstituted pyrrolidine ring was constructed via a 1,3-dipolar cycloaddition reaction between the nitro vinyl substrate 7 and the dipolar reagent 8. Only the trans isomer 9 was observed. This held true for all the analogs in this series. The nitro vinyl starting materials were either commercially available or could be synthesized via Henry reaction between nitromethane and the corresponding aldehydes. Reduction of the nitro group, protection of the 3-amino group with the BOC and debenzylation led to structure 10, which reacted with 1-methyl-1-imidazole-4-sulfonyl chloride to yield compounds with structure 11. BOC deprotection afforded 12, which was the precursor used for further derivatization at the 3amino group. Chiral SFC separation of the racemic 11 or 12 gave enantiomerically pure precursors to be further carried through the synthesis to final compounds. For all chiral compounds studied in the GlyT1 binding assay, the trans enantiomer configuration as shown in structure 13 is preferred. This is the synthetic route to get to the majority of the analogs with different R1 group quickly and effectively. Scheme 1a


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For acetal compounds 60 and 61, compound 14 (Scheme 2) was obtained in a similar manner as depicted in Scheme 1 (R1 = (CH3O)2CH). Condensation with 1,4-butanediol and 1,3-propanediol then gave the precursors to compounds 60 and 61, respectively. Scheme 2a


10

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With precursor 12 in hand, final structures like 15-17, shown in Table 1, could be synthesized by standard procedures as illustrated in Scheme 3. This is how compounds 4-5, 31-48, 51, and 58 61 were synthesized. Scheme 3a O R

N a

R

N N

N

NH2

N S O O

R

HN R1 N S O O HN Ar

b N N

15

N S O O

16

12 c R

N N

HN Alkyl

N S O O

17

a.Reagents

and conditions. (a) EDCI, DMF:Pyridine (1:1), RT, overnight; (b) Pd2(dba)3, XPhos, NaOt-Bu, dioxane, 110 ºC, 2 hr; (c) pH=4 buf fer, aldehyde or ketone, MP-cyanoborohydride (microporous polymer supported cyanoborohydride), RT, overnight Compound 52-54 and compound 57 in Table 3 were synthesized via the routes depicted in Scheme 4. Cycloaddition between structures18 and 19 yielded the pyrrolidine 20, which underwent debenzylation and sulfonylation to afford structure 22. Analogs with general structures as 25, thus, could be accessed from structure 22 by reduction of the ester followed by a Mitsunobu reaction. Ketone analogs 26 can be obtained via Weinreb amide formation from


11


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structure 23 followed by the treatment with Grignard reagents. Acylation of general structure 23 followed by reduction of the amide gave compound 27. These highly divergent synthetic routes allowed us to rapidly generate diverse analogs and to explore SAR, one of the attractive features of this de novo designed GlyT1 chemical series. Scheme 4a F Si

COOMe

N

+

F

O

O

a

b

O O

O

F

N N H

18

19 O

F

20 O

F

O

21

d

c

N

N O S O

N N

22

OR

N O S O

N N

24

23 g,h

F

R e

N O S O

N

H N

O

F

OH

f

i, j

O

F

R

H N

F

R

N N

N O S O

N N

25

N O S O

26

N N

N O S O

27

a.Reagents

and conditions: (a) TFA (Cat.), CH2Cl2, RT, overnight; (b) Pd(OH)2/C, H2, 30 psi, RT, THF, 16 hr; (c) 1-Methyl-1-imidazole-4-sulf onyl chloride, Et3N, CH2Cl2, DMAP(cat), RT, 1hr; (d) LiOH, MeOH/H2O, RT, 5 hr; (e) RNH2,EDCI, DMF/Pyridine, RT; (f ) BH3, dimethylsulf ide, THF, 60oC, 5hr, then RT overnight; (g) LiAlH4, THF, -30oC, 30 mins; (h) Phenol (ROH), DBAD, PS-PPh3, THF, RT, overnight; (i) dimethylhydroxylamine hydrochloride, TBTU, Et3N, DMF, RT; (j) RMgBr, THF, RT


12

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Compound 56 was synthesized via a different route (Scheme 5). Epoxide ring opening of compound 28 followed by BOC deprotection gave compound 30. Sulfonylation followed by nucleophilic substitution of compound 30 gave compound 56. Scheme 5a

SAR studies to improve GlyT1 potency and ER values. Having developed practical synthetic routes to assess a diverse array of analogs, we initiated extensive SAR studies on the pyrrolidine ring. As mentioned previously, 3-amido analogs such as compound 4 (Table 1) generally have high ER values with sub-optimal in vitro GlyT1 potency. However, replacing the amido groups with aryl groups afforded a cohort of potent analogs, many of which had single digit nanomolar GlyT1 potencies, for example, compounds 34-37 and compounds 43-47 in Table 1. Especially of note were the dramatically lowered ER values from amido analogs (ER = 8.5 for compound 4) to the anilino analogs with most of which were within the range of 0.6 to 1.5 (Table 2). This means these aniline analogs were less susceptible to P-gp efflux at the blood-brain barrier. Achieving


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good brain penetration with a safe and efficacious concentration profile remains one of the biggest hurdles in CNS drug discovery and development. Many parameters are in play and it is not always easy to optimize the brain penetration property later in a medicinal chemistry program.27 Thus, the overall desirable ER values of the aniline analogs at the onset of the lead optimization process prompted us to focus intensively on this subseries. Table 1. Structure-Activity Relationship for Compound 4, 5, 31-48

hGlyT1 Ki [uM]

ER

mClint,u (L/hr/kg) (human/r at)

4

0.198

8.7

7/14

31

0.199

0.9

15/57

32

1.51

1.3

55/180

5

0.018

0.9

49/119

33

1.25

0.6

21/140

34

0.008

-

-

Compo und

R1

R2

Free Brain/freePlasma Drug Concentration (Rat)

0.6


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35

0.003

0.9

85/255

2.1

36

0.003

1.1

203/350

1.7

37

0.005

0.8

83/158

38

1.16

10

10/41

39

0.042

1

87/266

40

0.021

0.9

37/194

41

0.118

18.5

2/5

42

0.766

-

-

43

0.002

0.8

52/300

44

0.004

0.7

227/328

2.9

45

0.008

1.3

15/50

0.6

46

0.005

7.5

5/11

0.2

47

0.002

0.7

129/304

2.1


15


0.001

48

1.5

60/88

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0.5

Table 2. Selected Profiling Data on Lead Compound 46-48, 58 and 61

Compou nd Structure

47

46

48

58

61

ED50 [mg/kg,i. p.] Clp (L/h/kg, rat) t1/2 (h, rat) F% (rat) Clp (L/h/kg, Monkey) t1/2 (h, Monkey) F% (Monkey)

1.3

4.9

4.4

0.9

0.9

2.2

0.7

2.8

1.6

1.4

2.0

2.3

0.3

1.2

0.4

25 1.2

71 -

6 -

32 0.9

35 1.2

4.6

-

-

2.4

1.1

17

-

-

1.7

1.0

In general, this series possessed excellent selectivity as evidenced from the CEREP study results where >10 µM affinities were observed against a panel of seventy-five diverse receptors (see supporting information). In light of the potential genotoxicity liability of anilines, analogs were


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screened early on extensively in MiniAmes assay. Results were negative for all the analogs tested (see supporting information). Replacing the sulfonamide group (N-1 in compound 4) with amides, tertiary amines, carbamates and ureas gave analogs with micromolar GlyT1 potency. More than one hundred sulfonamides were quickly synthesized and evaluated via parallel library synthesis. The only replacement that showed comparable in vitro potency and ER profile was the 1-methyl-1,2,3-triazole sulfonamide. For example, compound 51 in Table 3, had a Ki of 0.003 µM and an ER value of 0.5, which were comparable to compound 37 in Table 1 (Ki = 0.005 µM, ER = 0.8). However, the analogs with triazole sulfonamides in general had worse liver microsomal stability than the corresponding analogs with 1-methyl imidazole sulfonamides (compound 51 vs compound 37). This suggested that the 1-methyl imidazole sulfonamide group in compound 4-6 was a critical pharmacophore for the observed profile. Consequently, we chose to retain this moiety for all subsequent analogs designed and synthesized. Many 5-membered and 6-membered heterocycles at position R1 were synthesized and evaluated (structures not shown), most of which had inferior in vitro potency and high ER values compared to most of the compounds in Table 1, which indicated that they are likely P-gp substrates and could have limited brain penetration. Thus, the medicinal chemistry efforts were concentrated on substituted phenyl derivatives at R1 position (Table 1). A fluorine substituent on the phenyl ring at R1 position (Table 1) could be tolerated without negatively impacting the ER value. In terms of potency though, a para-F analog (compound 36, Ki = 0.003 µM) was more potent than ortho and meta-F analogs (compound 39, Ki = 0.042 µM and compound 40, Ki = 0.021 µM ). Compared to the unsubstituted analogs, for example,


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compound 34 ( Ki = 0.008 µM ) vs. compound 35 (Ki = 0.003 µM), para-F analogs were either equally or slightly more potent. Other substituents were extensively investigated and in general, afforded analogs with reduced potency and worse ER values. The best aromatic group at R1 was deemed to be the para-F phenyl group, which showed consistently good potency and low ER values, albeit high unbound intrinsic microsomal clearance (mClint,u). 28 With R2 as a substituted phenyl, the effects of different substituents at different positions were examined. From Table 1, it is evident that meta substitution on the phenyl ring at the R2 position boosted the in vitro potency significantly, while in general ortho and para substitutions were detrimental to the in vitro potency, as exemplified in compound 31-33 and compound 5. Although aromatic groups and long chain alkyl groups were tolerated at the meta position on the phenyl ring at R2 position (data not shown), the best groups were the small meta substituents such as Cl, Methyl, CF3 and OCF3, all of which in general gave single digit nanomolar in vitro potency as well as low ER values (compound 5, 34-36, 43-44). In these cases, a para-F group on the phenyl ring at R2 position could also be introduced into the molecule without losing in vitro potency such as in compound 47 (Ki = 0.002 µM ). In addition, the favorable low ER values translated to high free brain to free plasma drug concentration ratios in rodents (Table 1). For example, compound 47 has an ER value of 0.7 and in rodent studies, the ratio of free brain to free plasma drug concentration was determined to be 2.1. This further confirmed that these compounds most likely were not P-gp substrates, and possessed good brain penetration in vivo. Compounds with nitrogen atoms introduced into the R1 phenyl rings generally had much worse GlyT1 potency, as exemplified by compound 42 ( Ki = 0.766 µM ), compared to compound 34 (Ki = 0.008 µM). When nitrogen atoms were introduced into the R2 phenyl rings, these analogs


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in general possessed lower mClint,u (Table 1, compound 38, 41, and 46). However, these analogs had higher ER values, likely to be Pg-P substrates with variable potencies. It is widely recognized that molecular polarity descriptors such as H-bond acceptor, H-bond donor, and tPSA could be key factors for CNS exposure and especially for determining unbound brain exposure.29 In our case, the data showed that the effect on ER values is also dependent on the position of the nitrogen in the phenyl ring at R2 position, with the ER values ranged from 7.5-18.5 for compound 38, 41, and 46 in Table 1. Analogs with various alkyl groups at R2 were also extensively synthesized and profiled, most of which though high ER (>>2), plausibly due to the higher basicity of these analogs compared to those analogs where R2 is aryl or heteroaryl. From this effort, the best substituents identified were the indanyl groups, which offered the best balance of potencies and ER values as exemplified in compound 48 (Ki = 0.001µM, ER = 1.5) in Table 1 . The lead compounds were separated via chiral resolution into their enantiomers. The enantiomers with (3R,4S) configurations were proven to give the similar/better in vitro potency values while maintaining the ER profile of the racemic compound, for example, compound 35 vs. compound 43 in Table 1. The other enantiomers with the trans (3S, 4R) configurations gave micromolar GlyT1 potency values. Characterization of compound 47 and elucidation of DDI mechanism. From the initial medicinal chemistry campaign, compound 47 (Tables 1 and 2) quickly emerged as a lead. It displayed excellent in vitro potency of 2 nM in the hGlyT1 assay. It also demonstrated an ED50 of 1.3 mg/kg in the ex vivo TO study in mouse. In addition, compound 47 showed excellent brain availability in in vivo brain PK studies with a free brain to plasma drug concentration ratio of 2.1


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in rat. Competition studies confirmed compound 47 was a competitive GlyT1 inhibitor, similar to other compounds studied in this series. The rat PK profile (Clp = 2.2 L/h/kg and F% = 25) was acceptable to move this series forward (Table 2). Compound 47 was then moved forward in a L687,414-induced hyperactivity in vivo model where the NMDA receptor activation was assessed by the antagonism of hyperlocomotion induced by the NMDA receptor glycine site antagonist L687,414.30 L-687,414 ((3R,4R)-3amino-1-hydroxy-4-methylpyrrolidin-2-one)31 is a known selective and brain-penetrant glycine site NMDA receptor antagonist. Preliminary result showed that at 1 mg/kg, compound 47 showed almost complete inhibition of the L-687,414 effect (experimental section). The hyperlocomotion response to compound 47 alone (in the absence of L-687,414) at 10 mg/kg is consistent with hyperlocomotion observed with other GlyT1 inhibitors when tested at very high doses ( ≥30-fold ED50 for the antagonism of the L-687,414 effect). Consequently, compound 47 was advanced into further in vitro assessment of DDI and bioactivation liabilities. The results from these studies suggested compound 47 carried complex DDI liabilities. Compound 47 was metabolized mainly by CYP3A4 (98%) and exhibited moderate time-dependent inhibition (TDI) of CYP3A4 activity. Compound 47 also underwent glutathione (GSH) conjugation in an in vitro bioactivation assay using GSH-enriched liver microsomes. As such, additional studies were carried out to understand the mechanisms for bioactivation and CYP3A4 TDI. Interestingly, a liver microsomal metabolism study (supporting information) suggested oxidative defluorination (F to OH) was the major metabolic pathway (Scheme 6), which was unexpected as blocking the metabolic hotspot on the aromatic rings with fluorine is a commonly exploited medicinal chemistry strategy. However, a literature search revealed the precedence for similar observations such as in the case of Gefitinib.32,33 A plausible


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mechanism for this biotransformation is proposed in Scheme 6. A reactive benzoquinone imine metabolite, was formed by CYP3A4 mediated oxidative defluorination of compound 47. Upon formation, this reactive benzoquinone imine metabolite reacts with GSH to form a GSH conjugate in the bioactivation assay, and reacts with CYP3A4 cysteine to form a covalent protein adduct in CYP3A4 TDI assay, which in turn inhibits the CYP3A4 activity. The structurally similar quinone imine metabolite has been implicated in gefitinib-induced pulmonary and hepatic toxicities.34 The identification of pharmacophore modifications that could mitigate the DDI, bioactivation and potential drug-induced toxicities was deemed critical at this point in the optimization process. The goal was to search for compounds with low DDI and bioactivation liabilities while maintaining the favorable properties of compound 47. Scheme 6. Proposed Mechanism for Observed CYP3A4 TDI and Bioactivation of Compound 47


21


OCF3

F

F

HN (S)

N N

OCF3

F (S)

OCF3

F

OH

HN

(R)

Page 22 of 61

O

N (S)

(R)

(R)

CYP3A4

N S O O

N N

N S O O

N N

N S O O

47 GSH

OCF3

F

OH

HN (S)

N N

(R)

OCF3

F

OH

HN (S)

(R)

SG

S-CYP

N S O O

N N

49

N S O O

50

GSH Conjugation CYP3A4 TDI

Strategies to address DDI and CYP3A4 TDI liabilities. After the discovery of the inherent liability associated with the aniline moiety in the oxidative defluorination metabolic pathway, initial efforts were made to replace the aniline moiety while focusing on maintaining the favorable in vitro potency and ER values. Table 3 illustrates some examples from this endeavor (compound 52-57) where various linkers were synthesized and profiled to replace the aniline moiety. Unfortunately, none of the compounds designed and synthesized possessed an acceptable combination of in vitro potency, ER value, and liver microsomal stability comparable to those of compound 47.


22

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Table 3. Structure-Activity Relationship for Compound 51-57

6

hGlyT 1 Ki [uM]

ER

mClint,u (L/hr/kg) (human/rat)

F

R6

0.003

0.5

173/601

F

R5

0.007

-

-/1198

R5

0.029

1.3

73/261

Compoun d

R

51

52

3

R5,

4

R

O OCF3

53

F

54

F

R5

0.060

1.2

771/1310

55

H

R5

0.093

1.3

16/320

56

F

R5

0.097

9.8

3/12

57

F

R5

0.36

7.2

8/45

We then turned our attention back to the pool of compounds synthesized where R2 groups were varied while the NH group was kept in the molecule (Table 4). To understand the structureactivity relationships for CYP3A4 TDI and bioactivation liabilities, compounds were selected


23


Page 24 of 61

and funneled through various in vitro assays including liver microsomal metabolism, CYP phenotyping, bioactivation, and CYP3A4 TDI. The results were partially summarized in Table 4. The observations were in accordance with our proposed mechanism for the bioactivation and CYP3A4 liabilities in Scheme 6. Structurally, heteroaryls (structure C and D in Table 4), with the para position blocked by nitrogen atom in the ring, and alkyl groups (Structure E in Table 4), were tested negative in bioactivation and CYP3A4 TDI assays. The challenge would be to improve brain penetration with structure C -E in Table 4, as these compounds tended to have higher ER values compared to the analogs where R2 is a substituted phenyl. Table 4. Bioactivation and CYP3A4 TDI Results

R2

A Bioactivation

YES

B YES

D

C NO

NO

CYP3A4 TDI YES YES NO NO X = small and large substituents, alkyl and aryl, EWG and EDG groups

E NO NO

Through several SAR iterations, two lead compounds 46 and 48 were identified with this approach (Table 2) where the R2 group is heteroaryl and alkyl group, respectively. Compound 46 possessed an improved rat PK profile compared to compound 47 (Table 2), with much lower Clp (0.7 L/h/kg) and much better bioavailability (F% = 71). As expected, incorporate of the


24

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pyrimidine ring at R2 position translated into the negative results in both bioactivation and CYP3A4 TDI assays for compound 46. However, progression of this compound was halted due to borderline brain penetration (free brain/free plasma drug concentration = 0.2 ) and suboptimal ED50 from the TO study (ED50 [mg/kg,i.p.] = 4.9) as seen from Table 1 and Table 2. Compound 48, the other lead form this effort, had excellent in vitro potency (Ki = 0.001µM ) , acceptable brain impairment with free brain to free plasma drug concentration of 0.5 , and was also devoid of bioactivation and CYP3A4 TDI liabilities. In rat PK though, this compound had higher clearance and lower bioavailability (F% = 6) than those of compound 47, as well as suboptimal TO (ED50 [mg/kg,i.p.] = 4.4, Table 2) compared to compound 47. Thus, efforts focusing on moieties devoid of bioactivation and DDI liabilities while improving the in vitro potency and ER values were unsuccessful. Concurrently, in light of the excellent in vitro potency and favorable ER values exhibited by the 3-substituted anilino groups (structure A in Table 4), for example, compound 47, a parallel approach would be to keep the anilino moiety while dramatically modifying R1. The rationale was that the metabolic soft spot of the compound of interest might change with varied R1 in such a way that oxidative defluorination would not be the major metabolic pathway. The relationship between ClogP and mClint,u values of 800 analogs on this series were also analyzed via a box plot (Figure 5). After binned the compounds into four categories based on their ClogP values on the x-axis, each bar shows the distribution of mClint,u (y-axis) within the range of ClogP values on the x-axis. The count in the table in Figure 5 shows the number of analogs of which the ClogP fall into a particular binned value. The median mClint,u value for each bar is also given in the table (Figure 5). From this box plot, a clear trend can be seen that lower ClogP values resulted in improved mClint, u, as demonstrated by the corresponding lowered median values of mClint,u. The


25


Page 26 of 61

improved mClint, u would have a positive impact on lowering the human predicted dose, which would mitigate the bioactivation and DDI risks of these compounds. Figure 5. Box plot of binned ClogP values versus Clint,u (rat). ClogP values were binned into four categories: less than 2.0; between 2.0 and 3.0; between 3.0 and 4.0, and between 4.0 and 5.0. Box plots show minimum and maximum values as whiskers; the 25th, 50th, and 75th percentiles as boxes; and mean values as crosses.


26

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To achieve this, we introduced saturated heterocyclic rings at R1. A series of aliphatic R1 substituents were synthesized and studied. Compound 58 (Table 5) quickly rose to the top of our list with excellent brain availability and in vitro potency. Compared to compound 47, as expected, the lower ClogP translated to much improved mClint, u values. In ex vivo TO study, an ED50 of 0.9 mg/kg was obtained for compound 58 (Table 2). Compound 58 also had a comparable rat PK profile (Clp = 1.6 L/h/kg, t1/2 =1.2, F% =32, Table 2) to compound 47. It was a selective and potent competitive GlyT1 inhibitor. When compound 58 was advanced to a monkey PK study, to our surprise, minimal oral bioavailability was observed (F = 1.7%). This was in stark contrast to that of compound 47 (F = 17%). First pass metabolism in gut was suspected for the unexpectedly inferior monkey PK result since compound 58 was mainly metabolized by CYP3A4. Despite this, preliminary modeling suggested an improved predicted human dose for compound 58 compared to compound 47 (data not shown). However, progression of compound 58 was stopped due to the concerns that the bioactivation and DDI risks might still be high. Table 5. Structure-Activity Relationship for Compound 45, 58-61.

Compound

R1

hGlyT1 Ki [uM]

ER

Free brain/plas ma drug concentra tion

mClint,u (L/hr/k g) (human /rat)

ClogP


27


Page 28 of 61

45

0.004

0.5

1.3

227/328

4

58

0.003

0.8

1.0

23/41

2.4

59

0.024

-

-

17/36

2

60

0.002

0.9

0.5

19/30

0.6

61

0.002

1.1

0.4

6/8

0

To further improve mClint,u, analogs with lower ClogP, compounds such as 59- 61 (Table 5) were then synthesized and evaluated in an effort to further reduce the predicted human dose to mitigate the potential DDI risk in clinical studies. Compounds 60-61 had an acetal moiety that has not commonly exploited by medicinal chemists. However, acetal and related moieties are present in marketed oral drugs such as in Topiramate. Acetal groups could be labile when subjected to acidic conditions. In our case, the compounds synthesized all showed good chemical stability at pH=7 with no appreciable amount of hydrolysis at room temperature even after several days. Compound 61 was intact for two days at pH=1 before slow hydrolysis of the acetal group was observed. These compounds had much improved mClint,u compared to the para-F phenyl analog 45 (Table 5) while maintaining high hGlyT1 potency with excellent brain penetration. Based on these results, compound 61 was selected to be further profiled. Similar to the case of compound 58, compound 61 had an excellent ED50 value of 0.9 mg/kg with an acceptable rat PK profile (Clp = 1.4 L/h/kg and F% = 35). Despite the low oral bioavailability in


28

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monkey PK (F% = 1), the preliminary predicted human dose was further improved compared to compound 58, indicating the likelihood that these types of analogs could be advanced into clinical studies despite their bioactivation and DDI liabilities. CONCLUSION. A structurally distinct series of 3,4-disubstituted pyrrolidine sulfonamides were identified from de novo scaffold design as competitive hGlyT1 inhibitors. SAR studies showed that in general, this series possessed excellent in vitro potency, selectivity, in vitro ER values and in vivo brain penetration. DDI and bioactivation liabilities were identified and relationship to structural moieties was mechanistically investigated. Rodent brain PK studies were incorporated early on in the screening funnel to provide an early prediction of TO ED50 values based on brain exposure-TO relationship. Reducing the ClogP of the compounds resulted in leads with lower predicted human doses to mitigate the potential bioactivation and DDI liabilities, while still maintaining high potency and excellent brain penetration.


29


Page 30 of 61

EXPERIMENTAL SECTION Chemistry. Unless otherwise noted, all materials were obtained from commercial suppliers and used without further purification. Flash chromatography was performed using a CombiFlash® Rf automated purification system. Preparative HPLC was performed on either an automated preparative-scale purification system equipped with a Waters Sunfire C8 5m column (150 x 30 mm) or on a Phenomenex Luna C8 5m 100Å AXIA column (50mm × 21.2mm). A gradient of acetonitrile (A) and 0.1% trifluoroacetic acid in water (B) was used, at a flow rate of 30 mL/min (0-0.5 min 5% A, 0.5-6.5 min linear gradient 5-100% A, 6.5-8.5 min 100% A, 8.59.0 min linear gradient 100-5% A, 9.0-10 min 5% A). Proton nuclear magnetic resonance spectra (1H NMR, 500 or 400 MHz) were obtained in deuterodimethylsulfoxide (DMSO-d6) with residual solvent as the internal standard unless otherwise noted. Mass spectra (MS) were obtained by ionizing samples via positive electron spray ionization (ESI) or desorption chemical ionization (DCI) with TOF as the mass analyzer. Analytical LC-MS was performed on a Thermo MSQ-Plus mass spectrometer and Agilent 1100/1200 HPLC system running Xcalibur 2.0.7, Open-Access 1.4, and custom login software. The mass spectrometer was operated under positive APCI or ESI ionization conditions dependent on the system used. The HPLC system comprised an Agilent Binary pump, degasser, column compartment, autosampler and diodearray detector, with a Polymer Labs ELS-2100 evaporative light-scattering detector. The column used was a Phenomenex Kinetex C8, 2.6 µm 100Å (2.1mm × 30mm), at a temperature of 65°C. “TFA method”: A gradient of 5-100% acetonitrile (A) and 0.1% trifluoroacetic acid in water (B) was used, at a flow rate of 1.5 mL/min (0-0.05 min 5% A, 0.05-1.2 min 5-100% A, 1.2-1.4 min 100% A, 1.4-1.5 min 100-5% A. 0.25 min post-run delay). “Ammonium acetate method”: A


30

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gradient of 5-100% acetonitrile (A) and 10 mM ammonium acetate in water (B) was used, at a flow rate of 1.5 mL/min (0-0.05 min 5% A, 0.05-1.2 min 5-100% A, 1.2-1.4 min 100% A, 1.41.5 min 100-5% A. 0.25 min post-run delay). “TFA long with integration method”: A gradient of 5-100% acetonitrile (A) and 0.1% trifluoroacetic acid in water (B) was used, at a flow rate of 0.5 mL/min (0-0.1 min 5% A, 0.1-5.2 min 5-100% A, 5.2-5.7 min 100% A, 5.7-6.0 min 100-5% A. 0.25 min post-run delay). Preparative chiral SFC was performed on a THAR/Waters SFC 80 system running under SuperChrom software control. The sample was dissolved in a 8:1:1 mixture of methanol/dichloromethane/dimethylsulfoxide at a concentration of 15 mg/mL. The sample was loaded into the modifier stream in 1 mL (15 mg) injections. The mobile phase was held isocratically at 30% methanol:CO2. The instrument was fitted with a Chiralpak IA column with dimensions 21 mm i.d. x 250 mm length with 5 µm particles. All compounds described and characterized herein are of ≥95% purity as assessed by 1H NMR and LC-MS. 2-chloro-N-(1-((1-methyl-1H-imidazol-4-yl)sulfonyl)-4-phenylpyrrolidin-3-yl)-3(trifluoromethyl)benzamide (4). Step 1. To a solution of trans-tert-butyl 3-amino-4phenylpyrrolidine-1-carboxylate (0.31 g, 1.16 mmol) and 2-chloro-3-(trifluoromethyl)benzoic acid (0.26 g, 1.16 mmol) in CH2Cl2 (4.7 ml) was added triethylamine (0.49 ml, 3.49 mmol) and HATU (0.66 g, 1.74 mmol) and the mixture was stirred at room temperature for 2 hours. The reaction mixture was partitioned with water, the organic fraction was collected, and the aqueous fraction was washed with dichloromethane. The organic fractions were combined, dried over sodium sulfate and concentrated. The crude product was purified by silica gel chromatography eluting with 30% ethyl acetate / hexanes to afford trans-tert-butyl 3-(2-chloro-3(trifluoromethyl)benzamido)-4-phenylpyrrolidine-1-carboxylate. MS (ESI) m/z 469.3 [M+H]+


31


Page 32 of 61

Step 2. To a solution of 2-chloro-N-((trans-4-phenylpyrrolidin-3-yl)-3(trifluoromethyl)benzamide (0.32 g, 0.68 mmol) in 1,4-dioxane (0.68 ml) was added HCl in 1,4dioxane (4M, 1.7 ml, 6.8 mmol) and the mixture was stirred at room temperature for 2 hours. The solvent was evaporated and the solid was triturated twice with dichloromethane and the solvent was evaporated to afford 2-chloro-N-((trans-4-phenylpyrrolidin-3-yl)-3(trifluoromethyl)benzamide as HCl salt. MS (ESI) m/z 369.2 [M+H]+. Step 3. To a solution of 2-chloro-N-((trans-4-phenylpyrrolidin-3-yl)-3(trifluoromethyl)benzamide (0.075 g, 0.19 mmol) in CH2Cl2 (0.74 ml) was added triethylamine (0.077 ml, 0.56 mmol) and 1-methyl-1H-imidazole-4-sulfonyl chloride (0.033 g, 0.19 mmol). The mixture was stirred at room temperature for 1 hour. The solvent was evaporated. The crude product was purified by HPLC to afford compound 4. 1H NMR (500 MHz, Pyridine-d5) δ 7.98 (dd, J = 10.6, 1.4 Hz, 1H), 7.74 (dd, J = 6.6, 1.3 Hz, 1H), 7.54 – 7.64 (m, 1H), 7.48 (ddd, J = 10.1, 7.5, 1.7 Hz, 1H), 7.45 – 7.36 (m, 2H), 7.33 (td, J = 7.4, 2.5 Hz, 2H), 7.27 (q, J = 7.9, 7.4 Hz, 2H), 7.18 (t, J = 7.7 Hz, 1H), 5.45 (dtd, J = 8.4, 6.1, 4.3 Hz, 1H), 5.17 (dd, J = 8.7, 7.4 Hz, 1H), 4.27 - 4.55 (m, 2H), 4.15 – 4.04 (m, 1H), 4.01 – 3.88 (m, 2H), 3.54 (d, J = 20.7 Hz, 3H). MS (ESI) m/z 513.1 [M+H]+. 1-((1-methyl-1H-imidazol-4-yl)sulfonyl)-4-phenyl-N-(m-tolyl)pyrrolidin-3-amine (5). Step 1. To a solution of (E)-(2-nitrovinyl)benzene (4.2 g, 28.4 mmol) and N- (methoxymethyl)-N(trimethylsilylmethyl)benzylamine (8.1g, 34.0 mmol) in 75 mL of CH2Cl2 at 0 °C under nitrogen was added trifluoroacetic acid (388 mg, 3.4 mmol) in one portion. The reaction was allowed to warm to ambient temperature and stirred for 16 hours. The reaction was then partitioned between CH2Cl2 and saturated sodium bicarbonate solution. The organic fraction was collected. The aqueous portion was washed with additional CH2Cl2 and the combined organic fractions were


32

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washed with water, brine and dried over sodium sulfate. The mixture was filtered, concentrated and purified on a silica gel flash column (7:3 hexane:ethyl acetate) to afford trans-1-benzyl-3phenyl-4-nitropyrrolidine. MS (DCI) m/z 283.1 (M+H)+. Step 2. Trans-1-benzyl-3-phenyl-4-nitropyrrolidine (2.3 g, 15.5 mmol) in tetrahydrofuran 100 mL was added to Raney nickel water slurry (Grace 2800, 5.00 g) in a stainless steel reactor. The vessel was pressurized with 30 psi of hydrogen and shaken at room temperature for 16 hours. The mixture was filtered through a nylon membrane concentrated and purified on a silica gel flash column (95:5 CH2Cl2, 2N ammonia in methanol) to afford trans-1-benzyl-4phenylpyrrolidin-3-amine as colorless oil. MS (DCI) m/z 253.1(M+H)+. Step 3. To a solution of 2.1 g (8.9 mmol) of trans-1-benzyl-4-phenylpyrrolidin-3-amine in tetrahydrofuran (20 ml) was added saturated sodium bicarbonate solution (20 ml) followed by ditert-butyl dicarbonate (1.0 M solution in tetrahyrofuran, 10 ml, 10.0 mmol) at room temperature under nitrogen. The reaction was stirred for 1 hour and then partitioned between ethyl acetate and water. The organic fraction was collected. The aqueous portion was washed several additional times with ethyl acetate and the combined organic extracts were washed with brine and dried over sodium sulfate. The mixture was filtered, concentrated and purified on a silica gel flash column (3:2 ethyl acetate : hexane) to afford tert-butyl trans-1-benzyl-4-phenylpyrrolidin3-ylcarbamate. MS (DCI) m/z 353.2 (M+H)+. Step 4. tert-Butyl trans-1-benzyl-4-phenylpyrrolidin-3-ylcarbamate (2.4 g, 6.9 mmol) and 2,2,2-trifluoroethanol in tetrahydrofuran (40 mL) were added to 20% Pd(OH)2/C (50% water, 0.53 g) in a stainless steel reactor. The vessel was pressurized with 30 psi of hydrogen and shaken at 50 °C for 30 minutes. The mixture was filtered through a nylon membrane, and the


33


Page 34 of 61

product was purified on a silica gel column (95:5 CH2Cl2 : 2N ammonia in methanol) to afford tert-butyl 4-phenylpyrrolidin-3-ylcarbamate. MS (DCI) m/z 293.1 (M+H)+. Step 5. To a solution of tert-butyl 4-phenylpyrrolidin-3-ylcarbamate (139 mg, 0.53 mmol), and triethylamine (152 mg, 1.5 mmol) in CH2Cl2 (8 ml) was added 1-methyl-1H-imidazole-4sulfonyl chloride (108 mg, 0.6 mmol) in one portion at room temperature. 4-(dimethylamino)pyridine (4 mg, 0.03 mmol) was added and the reaction stirred for two hours at room temperature. The reaction was concentrated and purified on a silica gel flash column (97:3 dichloromethane: 2N ammonia in methanol) to afford tert-butyl (trans-4-phenyl-1-((1-methyl1H-imidazol-4-yl)sulfonyl)pyrrolidin-3-yl)carbamate. MS (DCI) m/z 407.5 (M+H)+ . Step

6.

tert-butyl

(trans-4-phenyl-1-((1-methyl-1H-imidazol-4-yl)sulfonyl)pyrrolidin-3-

yl)carbamate (187 mg, 0.5 mmol) was stirred with 1,4-dioxane (4 ml) and hydrogen chloride in 1,4-dioxane (4M, 4 ml, 16.0 mmol) at room temperature under nitrogen overnight. The reaction was concentrated and concentrated to afford trans-4-phenyl-1-((1-methyl-1H-imidazol-4-yl) sulfonyl)pyrrolidin-3-amine as the hydrochloride salt. MS (DCI) m/z 307.0 (M+H)+. Step 7. Under nitrogen, a pressure vial was charged with trans-4-phenyl-1-((1-methyl-1Himidazol-4-yl)sulfonyl)pyrrolidin-3-amine as a hydrochloric salt (103 mg, 0.30 mmol), 1-bromo3-methylbenzene (55 mg, 0.3 mmol), 2'-(di-tert-butylphosphino)-N,N-dimethylbiphenyl-2-amine (10 mg, 0.03 mmol), tris(dibenzylidene-acetone)dipalladium(0) (13 mg, 0.02 mmol), sodium tert-butoxide (70 mg, 0.7 mmol), and toluene (2 mL). The reaction mixture was stirred at 80 °C for 6 hours. Then, the reaction mixture was concentrated. Purification via HPLC provided 1-((1methyl-1H-imidazol-4-yl)sulfonyl)-4-phenyl-N-(m-tolyl)pyrrolidin-3-amine with 10% overall yield. 1H NMR (300 MHz, DMSO-d6) δ 7.93 (d, J = 1.3 Hz, 1H), 7.89 (d, J = 1.2 Hz, 1H), 7.32


34

Page 35 of 61 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


– 7.23 (m, 2H), 7.23 – 7.14 (m, 3H), 6.91 – 6.83 (m, 1H), 6.35 – 6.27 (m, 1H), 6.26 – 6.19 (m, 3H), 3.97 – 3.75 (m, 3H), 3.73 (s, 3H), 3.34 – 3.17 (m, 2H), 2.93 (dd, J = 6.9, 9.5 Hz, 1H), 2.11 (s, 3H). MS (ESI) m/z = 397 (M+H)+. (ESI) m/z 397 (M+H)+. (3R,4S)-4-(4-fluorophenyl)-1-((1-methyl-1H-imidazol-4-yl)sulfonyl)-N-(3(trifluoromethyl)phenyl)pyrrolidin-3-amine (43). Tert-butyl trans-4-(4-fluorophenyl)-1-(1methyl-1H-imidazol-4-ylsulfonyl)pyrrolidin-3-ylcarbamate was synthesized on 31.4 mmol scale via similar procedure as described in step 1-5 for compound 5 substituting (E)-1-fluoro-4-(2nitrovinyl)benzene for (E)-(2-nitrovinyl)benzene. A Chiral SFC separation of the racemic compound thus obtained provided the tert-butyl (3R,4S)-4-(4-fluorophenyl)-1-(1-methyl-1Himidazol-4-ylsulfonyl)pyrrolidin-3-ylcarbamate (Instrument: SFC200 Column: AD-H, 50 ×250 mm, 5 µm; Column Temperature: 35 °C; Mobile Phase: CO2/methanol/diethylamine =80/20/0.1; Flow rate: 180 g/min; Back Pressure: 100 Bar; Wavelength: 214 nm; Cycle time: 5.1 min; Injection: 2.0 mL Sample solution: 55 g in 500 mL methanol). Retention time: 5.73 mins. Compound 43 was then synthesized on 5.9 mmol scale via similar steps as described in step 6-7 for compound 5 substituting tert-butyl (3R,4S)-4-(4-fluorophenyl)-1-(1-methyl-1H-imidazol-4ylsulfonyl)pyrrolidin-3-ylcarbamate for tert-butyl (trans-4-phenyl-1-((1-methyl-1H-imidazol-4yl)sulfonyl)pyrrolidin-3-yl)carbamate. 1H NMR (300 MHz, DMSO-d6) δ 7.91 – 7.81 (m, 2H), 7.29 – 7.22 (m, 2H), 7.15 – 7.07 (m, 3H), 6.78 – 6.72 (m, 1H), 6.71 – 6.60 (m, 2H), 6.32 (s, 1H), 3.98 – 3.77 (m, 3H), 3.72 (s, 3H), 3.32 – 3.16 (m, 2H), 2.95 (dd, J = 7.1, 9.5 Hz, 1H). MS (ESI) m/z = 469 (M+H)+. ee = 99% (3R,4S)-N-((R)-2,3-dihydro-1H-inden-1-yl)-4-(4-fluorophenyl)-1-((1-methyl-1H-imidazol4-yl)sulfonyl)pyrrolidin-3-amine (48). (3R,4S)-4-(4-fluorophenyl)-1-((1-methyl-1H-imidazol-


35


Page 36 of 61

4-yl)sulfonyl)pyrrolidin-3-amine was synthesized similarly according to step 6 in the synthesis for compound 5 substituting tert-butyl (3R,4S)-4-(4-fluorophenyl)-1-(1-methyl-1H-imidazol-4ylsulfonyl)pyrrolidin-3-ylcarbamate for tert-butyl (trans-4-phenyl-1-((1-methyl-1H-imidazol-4yl)sulfonyl)pyrrolidin-3-yl)carbamate. To this material (300mg, 0.93 mmol) in 5 ml pH=4 buffer solution ( made from 48 g AcOH and 30.5 g NaOAc in 1 L MeOH) was added 2,3-dihydro-1Hinden-1-one (147 mg, 1.1mmol) and MgSO4 (1.1g, 9.3 mmol). MP-cyanoborohydride (1.3g, 2.8 mmol, loading 2.19mmol/g) was added to above mixture and the reaction was stirred overnight. After flash column separation, compound 48 was obtained with 31% yield. 1H NMR (400 MHz, DMSO-d6) δ 7.90 – 7.77 (m, 2H), 7.31 – 7.17 (m, 2H), 7.17 – 6.98 (m, 6H), 3.91 (t, J = 6.9 Hz, 1H), 3.76 – 3.67 (m, 4H), 3.59 (dd, J = 10.1, 6.8 Hz, 1H), 3.28 – 3.12 (m, 2H), 3.12 – 2.95 (m, 2H), 2.82 – 2.69 (m, 1H), 2.63 – 2.50 (m, 1H), 2.17 – 2.00 (m, 1H), 1.56 – 1.38 (m, 1H). The NH proton was not seen in the spectra due to fast water exchange. MS (ESI) m/z = 441 (M+H)+. 4-((trans-3-(4-fluorophenyl)-4-((3-(trifluoromethoxy)phenoxy)methyl)pyrrolidin-1yl)sulfonyl)-1-methyl-1H-imidazole fluorophenyl)acrylate

(10.52

(52). g,

Step

58.4

1.

A

mmol)

solution and

of

(E)-methyl

3-(4-

N-benzyl-1-methoxy-N-

((trimethylsilyl)methyl)methanamine (19.41 g, 81.8 mmol) in 110 mL of CH2Cl2 was cooled to 0°C. Trifluoroacetic acid (0.495 ml, 6.42 mmol) was slowly added under N2. The reaction mixture was stirred at 0°C for 1 hour, then stirred at room temperature for 18 hours. The reaction mixture was partitioned with saturated sodium bicarbonate. The organic fraction was collected, concentrated, and purified by flash-chromatography on silica gel (20-30% ethyl acetate in hexane) to provide methyl 1-benzyl-4-(4-fluorophenyl)pyrrolidine-3-carboxylate. MS (ESI) m/z 314.3 (M+H)+.


36

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Step 2. methyl 1-benzyl-4-(4-fluorophenyl)pyrrolidine-3-carboxylate (18.0g, 57.4 mmol) and tetrahydrofuran (10 ml) were added to 20% Pd(OH)2/C, wet (3.6 g, 25.6 mmol) in a 250 mL stainless steel pressure bottle and stirred for 16 hours under 30 psi of hydrogen at room temperature. The mixture was filtered through a nylon membrane and concentrated to provide trans-methyl 4-(4-fluorophenyl)pyrrolidine-3-carboxylate. MS (ESI) m/z 224.0 (M+H)+. Step 3. To trans-methyl 4-(4-fluorophenyl)pyrrolidine-3-carboxylate (12.7 g, 57.1 mmol) in 15 mL of

dichloromethane

were

added

triethylamine

(12.1

g,

120

mmol)

and

4-

dimethylaminopyridine (0.35 g, 2.85 mmol). The reaction mixture was cooled to 0 °C. 1methyl-1H-imidazole-4-sulfonyl chloride (10.8 g, 59.9 mmol) was added portion wise at 0 °C. The reaction mixture was slowly warmed up to room temperature and stirred for 1 hour. The reaction mixture was partitioned with dichloromethane, and water. The organic fraction was collected, washed with water, concentrated, and purified by flash-chromatography on silica gel (100% ethyl acetate) to afford trans-methyl 4-(4-fluorophenyl)-1-(1-methyl-1H-imidazol-4ylsulfonyl)pyrrolidine-3-carboxylate. MS (ESI) m/z 368.0 (M+H)+. Step 4. To a solution of trans-methyl 4-(4-fluorophenyl)-1-(1-methyl-1H-imidazol-4ylsulfonyl)pyrrolidine-3-carboxylate (620 mg, 1.7 mmol) in 6 mL of tetrahydrofuran at -30 °C was added lithium aluminum hydride (2N in tetrahydrofuran, 0.93 mL, 1.86 mmol),. The reaction mixture stirred at -30°C for 30 min. The reaction mixture was quenched with a saturated sodium bicarbonate solution, and the solution was partitioned with EtOAc. The organic fraction was collected, washed with water dried over sodium sulfate, and concentrated to give {trans-4-(4-fluorophenyl)-1-[(1-methyl-1H-imidazol-4-yl)sulfonyl]pyrrolidin-3yl}methanol. MS (ESI) m/z 340.0 (M+H)+.


37


Step

5.

To

a

solution

of

Page 38 of 61

{trans-4-(4-fluorophenyl)-1-[(1-methyl-1H-imidazol-4-

yl)sulfonyl]pyrrolidin-3-yl}methanol (52 mg, 0.15 mmol) in tetrahydrofuran (1 mL) was added 3-(trifluoromethoxy)phenol (41 mg, 0.23 mmol), PS-triphenylphosphine (105 mg, 0.34 mmol, loading 3.2 mmol/g), and di-tert-butylazodicarboxylate (70.6 mg, 0.3 mmol). The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was filtered and washed with methanol. The filtrate was concentrated and purified by HPLC to provide 4{[trans-3-(4-fluorophenyl)-4-{[3-(trifluoromethoxy)phenoxy]methyl}pyrrolidin-1-yl]sulfonyl}1-methyl-1H-imidazole. 1H NMR (500 MHz, DMSO-d6) δ 7.87 (dd, J = 9.6, 1.3 Hz, 2H), 7.36 (t, J = 8.3 Hz, 1H), 7.32 – 7.21 (m, 2H), 7.18 – 7.07 (m, 2H), 6.94 – 6.85 (m, 1H), 6.82 (dd, J = 8.4, 2.4 Hz, 1H), 6.75 (d, J = 2.4 Hz, 1H), 3.89 – 3.74 (m, 3H), 3.72 (s, 3H), 3.37 – 3.24 (m, 2H), 3.16 (q, J = 9.3 Hz, 2H), 2.66 – 2.52 (m, 1H). MS (ESI) m/z = 500 (M+H)+. (trans-4-(4-fluorophenyl)-1-((1-methyl-1H-imidazol-4-yl)sulfonyl)pyrrolidin-3-yl)(3(trifluoromethoxy)phenyl)methanone (53). Step 1. To trans-methyl 4-(4-fluorophenyl)-1-(1methyl-1H-imidazol-4-ylsulfonyl)pyrrolidine-3-carboxylate (product from step 3 for compound 51 synthesis) (3.67 g, 9.99 mmol) was added 4 mL of methanol. To this solution was added lithium hydroxide (1 M, methanol:water =5:3, 15 mL, 15 mmol). The reaction mixture was stirred at room temperature for 5 hours. The reaction mixture was concentrated, and the residue was then treated with hydrochloric acid (1 M, aq.) until pH=5. The reaction mixture was partitioned with ethyl acetate and the organic fraction was collected. The aqueous fraction was washed with ethyl acetate (3x), and the organic fractions were combined and concentrated to provide trans-4-(4-fluorophenyl)-1-(1-methyl-1H-imidazol-4-ylsulfonyl)pyrrolidine-3-carboxylic acid. MS (ESI) m/z 354.0 (M+H)+.


38

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Step

To

2.

a

solution

of

trans-4-(4-fluorophenyl)-1-(1-methyl-1H-imidazol-4-

ylsulfonyl)pyrrolidine-3-carboxylic acid (610 mg, 1.73 mmol) in dimethylformamide (5 mL) was added triethylamine (0.52 mL, 3.71 mmol), N,O-dimethylhydroxylamine hydrochloride (236 mg. 2.42

mmol),

and

2-(1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethylisouronium

tetrafluoroborate (665 mg, 2.07 mmol). The mixture was stirred at room temperature for 4 hours. The reaction mixture was partitioned between water and ethyl acetate. The organic fraction was collected. The aqueous fraction was washed with ethyl acetate 3 more times. The combined organic fractions were dried over sodium sulfate, concentrated, and purified by flashchromatography on silica gel (5-10% methanol in dichloromethane (with 0.5% volume triethylamine added) to afford trans-4-(4-fluorophenyl)-N-methoxy-N-methyl-1-(1-methyl-1Himidazol-4-ylsulfonyl)pyrrolidine-3-carboxamide. MS (ESI) m/z 397.0 (M+H)+. Step 3.

To

a solution of trans-4-(4-fluorophenyl)-N-methoxy-N-methyl-1-(1-methyl-1H-

imidazol-4-ylsulfonyl)pyrrolidine-3-carboxamide (113 mg, 0.29 mmol) in tetrahydrofuran (0.5 mL) was added (3-(trifluoromethoxy)phenyl)magnesium bromide (0.57 mL, 0.57 mmol, 1.0 M in tetrahydrofuran) slowly at room temperature. The solution stirred for 1 hour. The reaction mixture was partitioned with saturated ammonium chloride (aq.) and the organic fraction was collected. The organic fraction was dried over sodium sulfate, concentrated, and purified by HPLC to afford compound 53. 1H NMR (400 MHz, DMSO-d6) δ 7.91 – 7.82 (m, 2H), 7.79 (dt, J = 6.0, 1.8 Hz, 1H), 7.64 (d, J = 2.3 Hz, 1H), 7.63 – 7.53 (m, 2H), 7.25 – 7.14 (m, 2H), 7.09 – 6.95 (m, 2H), 4.29 (q, J = 8.5 Hz, 1H), 3.86 (dd, J = 10.3, 8.4 Hz, 2H), 3.79 (dd, J = 10.1, 7.9 Hz, 2H), 3.73 (s, 3H), 3.41 – 3.26 (m, 1H). MS (ESI) m/z = 498.1 (M+H)+. N-((trans-4-(4-fluorophenyl)-1-((1-methyl-1H-imidazol-4-yl)sulfonyl)pyrrolidin-3yl)methyl)-3-(trifluoromethoxy)aniline (54). To a solution of compound 57 (128 mg, 0.25


39


Page 40 of 61

mmol) in dry tetrahydrofuran (0.7 mL) under argon, was added borane dimethyl sulfide complex (0.5 mL, 1.0 mmol, 2M in tetrahydrofuran). The reaction mixture was stirred at 60 °C for 5 hours and stirred at room temperature for 18 hours. 0.5 mL of 0.5 N HCl was carefully added. The reaction mixture was refluxed for 2 hours, then treated with sodium hydroxide (1N) to pH = 8-9. The reaction mixture was portioned with ethyl acetate. The organic fraction was collected. The aqueous fraction was washed with CH2Cl2. The organic fractions were combined, dried over sodium sulfate, concentrated, and purified by HPLC to afford N-((trans-4-(4-fluorophenyl)1-((1-methyl-1H-imidazol-4-yl)sulfonyl)pyrrolidin-3-yl)methyl)-3-(trifluoromethoxy)aniline. 1H NMR (500 MHz, DMSO-d6) 1H NMR (500 MHz, DMSO-d6) δ 7.84 (s, 2H), 7.30 – 7.22 (m, 2H), 7.18 – 7.07 (m, 3H), 6.40 (ddt, J = 10.6, 7.4, 1.5 Hz, 2H), 6.23 (dq, J = 2.4, 1.2 Hz, 1H), 6.07 (t, J = 5.7 Hz, 1H), 3.78 – 3.65 (m, 5H), 3.30 (d, J = 9.8 Hz, 1H), 3.13 (dd, J = 10.4, 8.7 Hz, 1H), 3.04 (q, J = 9.1 Hz, 1H), 2.85 (dd, J = 6.6, 4.6 Hz, 2H), 2.35 (q, J = 7.7 Hz, 1H). MS (ESI) m/z = 499 (M+H)+. 4-{[trans-3-benzyl-4-phenylpyrrolidin-1-yl]sulfonyl}-1-methyl-1H-imidazole (55). Step 1. A solution of N-benzyl-1-methoxy-N-((trimethylsilyl)methyl)methanamine (5.9 g, 0.03 mole) in CH2Cl2 (30 mL) was stirred under nitrogen, in an ice/methanol bath. Chalcone (4.16g, 0.02 mole) was added dropwise and the mixture was stirred in the cold bath overnight. The reaction mixture was washed with aqueous sodium bicarbonate, brine, and dried over sodium sulfate. The solvent was evaporated to give (1-benzyl-4-phenylpyrrolidin-3-yl) (phenyl)methanone . MS (ESI) m/z 342 [M+H]+. Step 2. (1-benzyl-4-phenylpyrrolidin-3-yl)(phenyl)methanone from step one was hydrogenated with 10% Pd/C (0.25 g) in methanol (200 mL). The reaction mixture was filtered, dissolved in methanol (100 mL) and passed through Amberlite resin. The filtrate was concentrated, re-


40

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dissolved in toluene and concentrated. This procedure was repeated several times until the residue was dry. MS (ESI) m/z 338 [M+H]+. Step 3. To a solution of material from step 2 (0.77 g, 0.28 mmol) in dichloromethane (1.1 ml) was added triethylamine (0.16 ml, 1.12 mmol) and 1-methyl-1H-imidazole-4-sulfonyl chloride (0.051 g, 0.28 mmol), The mixture was stirred at room temperature for 15 minutes. The solvent was evaporated. The crude material was purified by HPLC to afford 4-{[trans-3-benzyl-4phenylpyrrolidin-1-yl]sulfonyl}-1-methyl-1H-imidazole. 1H NMR (400 MHz, Methanol-d4) δ 7.82 (d, J = 1.4 Hz, 1H), 7.69 (d, J = 1.3 Hz, 1H), 7.31 (tt, J = 7.7, 1.5 Hz, 2H), 7.26 – 7.07 (m, 6H), 7.04 – 6.93 (m, 2H), 3.91 – 3.69 (m, 4H), 3.48 (dd, J = 10.2, 7.2 Hz, 1H), 3.42 – 3.32 (m, 2H), 3.17 – 3.05 (m, 1H), 2.89 (td, J = 10.0, 7.9 Hz, 1H), 2.62 (dd, J = 13.1, 4.0 Hz, 1H), 2.54 – 2.35 (m, 1H). MS (ESI) m/z 382.2 [M+H]+. 4-((trans-4-(4-fluorophenyl)-1-((1-methyl-1H-imidazol-4-yl)sulfonyl)pyrrolidin-3-yl)oxy)6-(trifluoromethyl)pyrimidine (56). Step

1. To a solution of tert-butyl

6-oxa-3-

azabicyclo[3.1.0]hexane-3-carboxylate (0.93 g, 5 mmol) in tetrahydrofuran (5 mL) at -78 ºC was added copper(I) iodide (85 mg, 0.5 mmol) then (4-fluorophenyl)magnesium bromide (7 mL, 1 M, 7 mol). The mixture was warmed to room temperature and, allowed to stir for 3 hours. The reaction mixture was partitioned with ammonium chloride (aq).

The organic fraction was

collected, dried over MgSO4, filtered, and concentrated to provide trans-tert-butyl 3-(4fluorophenyl)-4-hydroxypyrrolidine-1-carboxylate. The crude material was stirred with 1,4dioxane (4 ml) and hydrogen chloride in 1,4-dioxane (4M, 4 ml, 16.0 mmol) at room temperature under nitrogen overnight. The reaction was concentrated and concentrated to afford trans-4-(4fluorophenyl) pyrrolidin-3-ol as the hydrochloride salt. The crude material in dichloromethane (5 mL) was then added triethylamine (2 g, 20 mmol) , 1-methyl-1H-imidazole-4-sulfonyl chloride


41


Page 42 of 61

(900 mg, 5.0 mmol) and 5 mol% dimethylaminopyridine. The mixture was allowed to stir for one hour. It was then concentrated and partitioned between ethyl acetate and NaOH (1 M). The organic fraction was collected, concentrated, and purified by flash chromatography to afford trans-4-(4-fluorophenyl)-1-((1-methyl-1H-imidazol-4-yl)sulfonyl)pyrrolidin-3-ol. Step

2.

To

a

solution

of

trans-4-(4-fluorophenyl)-1-((1-methyl-1H-imidazol-4-

yl)sulfonyl)pyrrolidin-3-ol (110 mg, 0.34 mmol) in dimethylsulfoxide (0.3 mL) was added potassium

2-methylpropan-2-olate

(50

mg,

0.45

mmol)

and

4-bromo-6-

(trifluoromethyl)pyrimidine (70 mg, 0.43 mmol). The mixture was stirred at 100 ºC for 16 hours, and then partitioned between ethyl acetate and water. Purification by HPLC provided the title compound. 1H NMR (400 MHz, DMSO-d6) δ 8.94 (s, 1H), 7.85 (d, J = 1.3 Hz, 1H), 7.80 (d, J = 1.2 Hz, 1H), 7.36 – 7.27 (m, 3H), 7.20 – 7.07 (m, 2H), 5.46 – 5.30 (m, 1H), 3.88 – 3.76 (m, 2H), 3.68 (s, 3H), 3.64 (q, J = 6.5, 6.0 Hz, 1H), 3.44 (ddd, J = 23.6, 11.1, 4.5 Hz, 2H). MS (ESI) m/z = 472(M+H)+. Trans-4-(4-fluorophenyl)-1-((1-methyl-1H-imidazol-4-yl)sulfonyl)-N-(3(trifluoromethoxy)phenyl)pyrrolidine-3-carboxamide (57). To trans-4-(4-fluorophenyl)-1-(1methyl-1H-imidazol-4-ylsulfonyl)pyrrolidine-3-carboxylic acid (product from step 1 for the synthesis of compound 53, 150 mg, 0.42 mmol) in DMF/pyridine (1:1. 15 mL) was added 1-(3dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride ( 85 mg, 0.45 mmol) and 3(trifluoromethoxy)aniline (80 mg, 0.45 mmol).

The reaction mixture was stirred at room

temperature for 18 hours. The reaction mixture was concentrated and purified by HPLC to provide compound 57. 1H NMR (400 MHz, DMSO-d6) δ 10.24 (s, 1H), 7.88 (s, 2H), 7.63 (dq, J = 2.3, 1.1 Hz, 1H), 7.42 – 7.29 (m, 2H), 7.30 – 7.18 (m, 2H), 7.15 – 7.06 (m, 2H), 7.03 – 6.91


42

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(m, 1H), 3.80 (ddd, J = 10.0, 8.1, 6.1 Hz, 2H), 3.72 (s, 3H), 3.52 (td, J = 10.1, 8.0 Hz, 1H), 3.36 – 3.25 (m, 1H), 3.13 (td, J = 9.7, 8.0 Hz, 1H), 2.04 (s, 1H). MS (ESI) m/z = 513.1 (M+H)+. (3R,4S)-1-((1-methyl-1H-imidazol-4-yl)sulfonyl)-4-((R)-tetrahydro-2H-pyran-2-yl)-N-(3(trifluoromethoxy)phenyl)pyrrolidin-3-amine (58). Step 1. To a solution of oxalyl dichloride (131 g, 1.03 mol) in CH2Cl2 (700 mL) was added dimethyl sulfoxide (87 g, 1.12 mol) slowly at 78°C, and keep the reaction for 10 min, and then added (tetrahydro-2H-pyran-2-yl)methanol (100 g, 0.86 mol) dropwise and kept the reaction at -78°C for another 10 min when the addition was completed. Then at -78 °C was added triethylamine (226 g, 2.24 mol) dropwise and the reaction was allowed to come to room temperature for 30 min. Three additional vials were set up as described above. All three reaction mixtures were combined and the mixture was diluted with dichloromethane and filtered through a pad of celite and the filtrate was concentrated with no heat and tetrahydro-2H-pyran-2-carbaldehyde thus obtained was used in next step without further purification. Step 2. Crude material form step 1 (350 g, 3.1 mol) was dissolved in tetrahydrofuran (1000 mL) under N2 atmosphere, and nitromethane (374 g, 6.1 mol) was added in one portion. To the stirred solution was added 1,1,3,3-tetramethylguanidine (17.66 g, 0.15 mol), a mild exothermo reaction ensued, the reaction was stirred for 2.5 hrs at 20°C, then concentrated and purified by silica gel to the intermediate. The intermediate was dissolved in tetrahydrofuran (500 mL) under N2 atmosphere at chilled ice bath, and 2,2,2-trifluoroacetic anhydride (644 g, 3.07 mol) was added rapidly and kept the reaction for 15 min and then triethylamine (621 g, 6.13 mol) was added dropwise and kept the reaction for another 15 min, then the mixture was extracted by ethyl acetate and water, the organic layers were combined and concentrated, the residue obtained was purified by silica gel to give (E)-2-(2-nitrovinyl)tetrahydro-2H-pyran.


43


Page 44 of 61

Chiral Separation and synthesis of compound 58. Tert-butyl (1-((1-methyl-1H-imidazol-4yl)sulfonyl)-4-(tetrahydro-2H-pyran-2-yl)pyrrolidin-3-yl)carbamate was synthesized on 1.6 mmol scale from (E)-2-(2-nitrovinyl)tetrahydro-2H-pyran following the similar procedures as described in step 1-5 for compound 5. The compound thus obtained (22 g, 53.1 mmol) was then dissolved in methanol (500 ml) and kept the concentration at 44 mg/ml, and then chirally separated using the following conditions:{Column: Chiralpak AD-H, 3 cm ID x 25 cm, Mobile Phase SFC CO2, Back pressure: 100 Bar, Pressure drop: 81 Bar, Modifier: methanol 12%. Flow Rate: 100 gm/min, Detector: UV 224 nm, Sample concentration: 40 mg/mL in Methanol, Sample load: 2 mL (80 mg)}. (3R,4S)-1-((1-methyl-1H-imidazol-4-yl)sulfonyl)-4-((R)-tetrahydro-2Hpyran-2-yl)pyrrolidin-3-amine was detected by LCMS at retention time = 2.80 min, 100% purity, m/z = 415.2(M+H)+. Compounds 58 was then synthesized from (3R,4S)-1-((1-methyl-1Himidazol-4-yl)sulfonyl)-4-((R)-tetrahydro-2H-pyran-2-yl)pyrrolidin-3-amine using the similar procedure as described in step 6-7 for the synthesis of compound 5. 1H NMR (400 MHz, DMSOd6) δ 7.88 – 7.78 (m, 2H), 7.11 (t, J = 8.1 Hz, 1H), 6.51 (ddd, J = 8.3, 2.2, 0.8 Hz, 1H), 6.46 (td, J = 2.2, 1.1 Hz, 1H), 6.44 – 6.39 (m, 1H), 6.21 (d, J = 7.0 Hz, 1H), 3.88 – 3.75 (m, 2H), 3.69 (s, 3H), 3.49 (dd, J = 10.3, 6.1 Hz, 1H), 3.40 (dd, J = 10.7, 7.8 Hz, 1H), 3.18 (td, J = 11.1, 3.3 Hz, 1H), 3.12 – 2.97 (m, 2H), 2.81 (ddd, J = 11.1, 7.3, 1.8 Hz, 1H), 2.13 – 2.03 (m, 1H), 1.69 (d, J = 12.1 Hz, 1H), 1.45 – 1.17 (m, 4H), 1.14 – 0.96 (m, 1H). MS (ESI) m/z = 475 (M+H)+. ee = 100%. (3R,4S)-4-(1,3-dioxan-2-yl)-1-((1-methyl-1H-imidazol-4-yl)sulfonyl)-N-(3(trifluoromethoxy)phenyl)pyrrolidin-3-amine (61). Step 1. To 2,2-dimethoxyacetaldehyde (4 mL 60%) in nitromethane (10 mL) was added potassium carbonate (338 mg). The mixture was allowed to stir for 2h, ethyl acetate was added, and water was drained. The crude intermediate


44

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was dissolved in CH2Cl2 (30 mL), triethyl amine (6.1 g, 60 mmol) and trifluoroacetic anhydride (6.3 g, 30 mmol) were added at -20 ºC. The mixture was allowed to stir at -20 ºC for 10 min at room temperature for 1h. CH2Cl2 was removed, ethyl acetate was added and the organic extract was washed with water to afford (E)-3,3-dimethoxy-1-nitroprop-1-ene. Compound 14 was synthesized on 15 mmol scale following the similar procedures as described in step 1-5 for compound 5 substituting (E)-3,3-dimethoxy-1-nitroprop-1-ene for (E)-(2nitrovinyl)benzene followed on 15 mmol scale. Chiral separation of compound 14 gave tert-butyl ((3R,4S)-4-(dimethoxymethyl)-1-((1-methyl-1H-imidazol-4-yl)sulfonyl)pyrrolidin-3yl)carbamate. To this material (2g, 4.9 mmol) in toluene (10 mL) was added propane-1,3-diol (1.5 g, 20 mmol) and toluenesulfonic acid (20 mg, 0.1 mmol). The mixture was heated at 80 ºC for 3 hr. Volatiles were removed to afford tert-butyl (3R,4R)-4-(1,3-dioxan-2-yl)-1-(1-methyl1H-imidazol-4-ylsulfonyl)pyrrolidin-3-ylcarbamate. Compound 61 was then synthesized on 1.6 mmol scale following similar procedures as described in Step 6-7 for compound 5 substituting (3R,4R)-4-(1,3-dioxan-2-yl)-1-(1-methyl-1H-imidazol-4-ylsulfonyl)pyrrolidin-3-ylcarbamate for tert-butyl (trans-4-phenyl-1-((1-methyl-1H-imidazol-4-yl)sulfonyl)pyrrolidin-3-yl)carbamate. 1H NMR (400 MHz, DMSO-d6) δ 7.83 (d, J = 1.3 Hz, 1H), 7.80 (d, J = 1.3 Hz, 1H), 7.12 (ddd, J = 8.4, 7.2, 1.2 Hz, 1H), 6.50 (dt, J = 8.3, 1.2 Hz, 1H), 6.43 (dt, J = 7.1, 1.2 Hz, 2H), 6.23 (d, J = 7.0 Hz, 1H), 4.30 (d, J = 5.2 Hz, 1H), 3.96 – 3.87 (m, 2H), 3.82 – 3.73 (m, 1H), 3.69 (s, 3H), 3.64 – 3.48 (m, 3H), 3.36 (dd, J = 10.5, 8.2 Hz, 1H), 3.22 (dd, J = 10.5, 5.9 Hz, 1H), 3.00 (dd, J = 10.1, 4.5 Hz, 1H), 2.20 (dq, J = 8.2, 5.4 Hz, 1H), 1.86 – 1.69 (m, 1H), 1.34 – 1.20 (m, 1H).MS (ESI) m/z = 477(M+H)+. ee = 99%. Stability of compound 61. A pH=1 aqueous solution (2 mL) of compound 61 (1 mg) was heated at 37°C and the reaction was monitored by LC/MS. No additional peak was observed


45


Page 46 of 61

after 2 days and about 15% of the material was hydrolyzed to the corresponding aldehyde after 5 days based on LC/MS. Same procedure was done at pH=7.4 and no hydrolyzed aldehyde was observed after 5 days.

Biology/DMPK. Unless stated otherwise the chemicals used for the test systems (reagents, salts, buffers, antibiotics, cell media, etc) were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Thermo Fisher Scientific (Schwerte, Germany or Waltham, Mass, USA). For the animal experiments the experimental procedures were approved by AbbVie’s Animal Welfare Office and were performed in accordance with the German national guidelines as well as recommendations and policies of the U.S. National Institutes of Health “Principles of Laboratory Animal Care” (1996 edition). Animal housing and experiments were conducted in the facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC).

[3H]N-methyl-SSR504734 Radioligand Binding Assays. 20 Radioligand binding to human GlyT1c transporter expressing membranes was measured in duplicate in a total volume of 200 µL in 96-well plates. To 100 µL of membrane suspension in assay buffer (120 mM NaCl, 2 mM KCl, 10 mM HEPES, 1 mM MgCl2, and 1 mM CaCl2, pH 7.5), 80 µL of [3H]N-methylSSR50473435 was added in assay buffer, yielding a final membrane protein concentration of 50 µg/mL. In competition experiments, 10 µL of buffer or unlabeled compound solution were added. The final DMSO concentration was 1% in all cases. Nonspecific binding was determined in the presence of 10 µM SSR504734. After incubation at RT for 1 h, the incubation mixture was harvested (Tomtec Mach III U Harvester) through 96-well GF/B filter plates (Perkin- Elmer Life


46

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and Analytical Sciences, Waltham, MA), presoaked for 1 h with 40 µL per well of 0.1% polyethylenimine. After washing twice with ice-cold buffer (50 mM Tris-HCI, pH 7.4), plates were dried and 35 µL of scintillator (BetaplateScint; PerkinElmer Life and Analytical Sciences) were added per well. The radioactivity was determined by liquid scintillation spectrometry in a MicroBeta (PerkinElmer Life and Analytical Sciences) plate counter. Data Analysis. For binding of [3H]N-methyl-SSR504734 to cell membranes, the calculation of the IC50 values from the displacement binding was performed by iterative nonlinear regression analysis adapted from the Ligand program36 Radioligand displacement curves in the presence of tested compounds were fitted using a one-site model. Apparent Ki values were calculated from the IC50 values and the Kd of 2.5 nM obtained from saturation binding experiments using the Cheng-Prusoff equation.37

P-gp-Efflux. Madin-Darby canine kidney (MDCK II) epithelial cells, transfected with the MDR1 gene were engineered to have lower expression of canine P-gp as described.38 The cells encoding human P-gp are grown on transwell inserts and directional differences in cell monolayer permeability of various test compounds are monitored during a 1 hr experiment. Specifically, Millicell 96-well insert plates (Merck Chemicals AG, Darmstadt, Germany) were seeded with MDCK-MDR1 cells on Day 1 at a density of 300,000 cells/cm2. Media was changed on Day 2 and P-gp interaction assay was performed on Day 4 or Day 5. Compounds were diluted to a concentration of 1 mM in DMSO, and the final assay concentration was 1 µM (added 1 µL of stock to 999 µL of Hank’s Balanced Salt Solution (HBSS) + glucose) (final concentration of 0.1% DMSO). Cell monolayers were dosed on the apical side (A-to-B direction) or basolateral side (B-to-A direction) at a concentration of 1 µM in HBSS + glucose. Incubations were


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performed in duplicate at 37 °C for one hour. At the end of the incubation, 50 µL aliquots were taken from the receiver chambers and 10 µL aliquots were taken from the donor chambers. Prior to the incubation, aliquots of the donor solution were also taken to determine concentration at time equal zero. All donor solutions (both t = 0 min and t = 60 min) are diluted with HBSS + glucose and LC/MS analysis was performed on all samples. Apparent permeability (Papp) values are calculated from area-under- the-curve quantitation from LC-MS/MS (AB Sciex API 4000, 5000 or 6500; AB Sciex Germany GmbH, Darmstadt, Germany) employing Waters Acquity HPLC systems (Waters GmbH, Eschborn Germany). analyses. An internal standard buspirone was added to all wells.The calculation was Papp=(∆Q/∆t)*(1/(A*Co)) where ∆Q is amount of drug solute transported (µmol), ∆t is incubation time (sec), A is filter surface area in cm2, and Co is mean of starting-end drug concentration in the donor well (µM). Drug velocity is expressed as apparent permeability, Papp, with the unit 1 x 10-6 cm/sec. Efflux Ratio (ER) = Papp (B-to-A)/Papp (A-to-B). Functional expression in Xenopus oocytes (competition study). Expression and functional evaluation of GlyT1 was performed as described previously.20 Female Xenopus laevis (Nasco, Fort Atkinson, WI), were anesthetized in solution with 0.2% Tricaine (Sigma, St. Louis, MO) and 2 g/l sodium hydrogen carbonate (Sigma), ovary lobes were removed, and oocytes were released from the follicle tissue with collagenase (type I, 2 mg/mL for 2 h; Roche Applied Science, Mannheim, Germany). Stage V and VI oocytes were selected by hand under a binocular microscope injected 20 nL of cDNA into the nucleus, or 50 nL of prepared cRNA solution into the cytoplasm with GlyT1c expressed in the plasmid pGemHeJuel, and incubated at 18°C in Barth Medium with gentamycin for 3-5 days. The membrane current in whole oocytes was measured through two-electrode voltage clamp (TEVC) employing two microelectrodes filled


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with 1.5 M potassium acetate and 0.1 M potassium chloride with a voltage-clamp amplified (TEC 03X, npi electronics, Tamm, Germany). Alternatively, a robotic measurement system was used (Roboocyte, multichannel systems, Reutlingen, Germany). The measurement chamber was continuously perfused with frog Ringer solution. Compounds were dissolved in this buffer, and the final DMSO concentration was 1% in all cases. To test the surmountability of GlyT1 inhibition, oocytes were voltage clamped at -60 mV, stimulated with glycine (10 µM or 3 mM) for 1 min, and the compounds added to the respective glycine solution for 3 min. At the end of this time period, the relative current, compared to non-inhibited at the same time point was determined. Data were repeated in independent experiments at least 4 times (4-7). Data analysis was performed by the CellWorks Software, the software from the Roboocyte robotic system and subsequent evaluation with GraphPad Prism (GraphPad Software, La Jolla, CA, USA). Results were compared with t-test. Materials and methods for animal behavioral studies. For ex vivo GlyT1 binding assay male, NMRI mice were obtained from Janvier (Le Genest-St-Isle, France) and were housed eight per cage (Macrolon, type III, unless specified otherwise). Animals had a body weight of approximately 26-28 g upon arrival. For L687,414-induced hyperlocomotion experiments, male C57Bl/6 mice were obtained from Janvier (Le Genest-Saint-Isle, France; five weeks old upon arrival) and were housed eight per cage (Macrolon, type III). The animals were maintained under standard conditions (12 hours day/night cycle, light switched on at 0600 hours, room temperature 21±1°C, 55±15% humidity) with free access to food and tap water. Before being used for the experiments, the animals were allowed to recover from transportation for at least one week. All experiments were conducted during the light period of the light/dark cycle.


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All experiments were conducted in facilities with full AAALAC accreditation in accordance with the recommendations and policies of the U.S. National Institutes of Health "Principles of laboratory animal care" (1996 edition), all applicable European and German laws and were approved by AbbVie’s Animal Welfare Officer. Ex vivo GlyT1 Binding Assay. Animals were subjected to single housing one day before the experiment and transferred from the animal facility into the experimental room where they were housed in ventilated containers (Scantainer Type D, Scanbur Ltd., Denmark) until the next day. On the day of the experiment, the mice had a body weight of 28-34 g. Before the experiment, the animals were (pseudo-) randomly assigned to the different treatment groups consisting of five mice per group. The tested compounds or the vehicle was applied intraperitoneally (i.p.). For the determination of non-specific binding, five animals were treated with 100 mg/kg of SSR504734. One hour after drug application, the mice were anaesthetized with isoflurane, blood samples were taken by cardiac puncture. Immediately after blood sampling and still under anesthesia, animals were sacrificed by cervical dislocation. Forebrains were dissected, rapidly frozen in liquid nitrogen and stored at -80°C until investigated in the binding assay. For determination of the ex vivo GlyT1 binding, the dissected forebrains were homogenized in five volumes Trisbuffered saline (50 mM Tris-Cl, pH 7.4; 150 mM NaCl) with the UltraTurrax (9600 UPM, 15 seconds). 100 µL of the homogenate was mixed with 10 µL [3H]N-methyl-SSR504734 to yield a final concentration of 5 nM (0.04 µCi/mL). Three aliquots of 20 µL were transferred to macrowell vials, subjected to filtration with a scatron harvester 60 minutes after addition of the radioligand, and membrane bound radioactivity was determined by liquid scintillation counting. Forebrains from animals treated with 100 mg/kg SSR504734 were used for the determination of non-specific binding. Specific binding was determined by subtracting the mean binding of the


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vehicle- or SSR504734-treated animals from the total binding of each individual sample (triplicates for each animal). Means (± S.E.M.) of the specific binding for the different treatment groups were calculated from the means of individual animals. The forebrain concentration of the compound tested associated with half maximal target occupancy in vivo (EC50

brain,

mean ±

S.E.M.) was estimated by non-linear curve fitting of the specific binding of the means of the individual animals vs. the concentration of the compound in the forebrain homogenate. This estimation is based on the assumption of the formation of a new binding equilibrium during the ex vivo incubation. The apparent ED50 (mean ± S.E.M) was determined by non-linear curve fitting of the inhibition of specific radioligand binding (means of the individual animals) vs. the dose. From this value an ED50 for in vivo TO was estimated by dividing the apparent ED50 by 6.6. This estimation is based on the 6.6-fold dilution of tested compounds by the ex vivo processing of the brain tissue and the assumption of a new binding equilibrium in the binding assay. All nonlinear curve fittings were performed with SigmaPlot 8.0 using a three parametric logistic equation. L687,414-Induced Hyperactivity Model. Non-habituated mice were injected i.p. with 50 mg/kg of L687,414 or its vehicle 15 min prior to the two-hour test. Compound 47 (1, 3 and 10 mg/kg) or its vehicle was given i.p. prior to the test. Each treatment group consisted of eight animals. The data were acquired by Cage Rack Photobeam system (San Diego Instruments, San Diego, CA). The analyzed data were total movements (fine movements + ambulations) during the first 30 min of the test.


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Compound 47 was administered intraperitoneally. Data are shown as mean ± SEM for n=8-10 mice per group. Non-habituated mice were injected i.p. (intraperitoneal injection) with 50 mg/kg of L687,414 or its vehicle 15 min prior to the measurement of total movements (fine movements + ambulations) for two hours. ANOVA revealed a significant main effect of treatment. * P

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