Discovery, characterization and biological evaluation of a novel (R)-4,4-difluoropiperidine scaffold as dopamine receptor 4 (D4R) antagonists

Bioorganic & Medicinal Chemistry Letters 26 (2016) 5757–5764

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Discovery, characterization and biological evaluation of a novel (R)-4,4-difluoropiperidine scaffold as dopamine receptor 4 (D4R) antagonists Daniel E. Jeffries a, Jonathan O. Witt a, Andrea L. McCollum a, Kayla J. Temple b,d, Miguel A. Hurtado b, Joel M. Harp c, Anna L. Blobaum b,d, Craig W. Lindsley a,b,d, Corey R. Hopkins a,b,d,e,⇑ a

Department of Chemistry, Vanderbilt University, Nashville, TN 37232, United States Department of Pharmacology, Vanderbilt University, Nashville, TN 37232, United States c Department of Biochemistry, Vanderbilt University, Nashville, TN 37232, United States d Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University, Nashville, TN 37232, United States e Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE 58198, United States b

a r t i c l e

i n f o

Article history: Received 23 September 2016 Accepted 14 October 2016 Available online 17 October 2016 Keywords: Dopamine 4 receptor Difluoropiperidine Addiction PD-LIDs Selectivity

a b s t r a c t Herein, we report the synthesis and structure–activity relationship of a novel series of (R)-4,4-difluoropiperidine core scaffold as dopamine receptor 4 (D4) antagonists. A series of compounds from this scaffold are highly potent against the D4 receptor and selective against the other dopamine receptors. In addition, we were able to confirm the active isomer as the (R)-enantiomer via an X-ray crystal structure. Ó 2016 Elsevier Ltd. All rights reserved.

The dopamine receptors are a class of G protein-coupled receptors (GPCRs) that are prominent in the central nervous system (CNS), with dopamine being the endogenous ligand for this class of receptors.1,2 This family of receptors contains at least five subtypes of receptors that are further subgrouped into two families.3,4 The D1-like receptor family are coupled to Gsa which activates adenylyl cyclase resulting in the increase of intracellular cAMP and contains the D1 and D5 receptors. The D2-like receptor family are coupled to Gia which inhibits cAMP and contains the D2, D3, and D4 receptors. Significant work has been dedicated to the dopamine receptor class due to the dopamine hypothesis of schizophrenia—resulting in numerous approved medications as antipsychotics, predominantly non-selective D2/D3 mixed receptor modulators.5 The D4 receptor received considerable attention for schizophrenia; however, a key compound, L-745,870, failed in the clinic due to a lack of efficacy.6–8 Nearly all of the antagonists developed at this time were based on a piperazine scaffold and the compounds possessed varying degrees of selectivity. In addition to the potential as an antipsychotic, the D4 receptor has been implicated as a

⇑ Corresponding author. Tel.: +1 402 559 9729; fax: +1 402 559 5643. E-mail address: [email protected] (C.R. Hopkins). http://dx.doi.org/10.1016/j.bmcl.2016.10.049 0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.

potential target for Parkinson’s disease—especially for L-DOPAinduced dyskinesia’s,9 and for the treatment of substance abuse.10 L-745,870 has been shown to offer a positive result in both rat and non-human primate models of L-DOPA-induced dyskinesia’s, however, the compound produced some locomotor deficits that suggest the compound reduces the overall benefit in the rat model.11,12 Despite the promising results of these initial studies, there has been limited effort over the past several years on the development of new and selective D4 antagonists. Recently, our laboratory has identified two series based on a chiral morpholine scaffold that has produced potent and selective D4 antagonists (Fig. 1).13,14 The initial compound, ML398, 1, was brain penetrant and active in a cocaine-induced hyperlocomotion assay; however, the SAR was limited due to synthetic access to the upper-right hand portion.13 Thus, the right-hand phenethyl moiety was replaced with the alkoxymethyl group which allowed for substantial synthetic modifications, leading to 2, another potent, selective and brain penetrant D4 antagonist.14 Compound 2, was shown to drastically reduce global AIM scores in a 6-OHDA mouse study for the reversal of L-DOPA-induced dyskinesias.15 Next, we turned our attention to replacing the morpholine core moiety with another novel group, a difluoropiperidine scaffold. The synthesis of this new scaffold is shown in Scheme 1. The b-keto

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Figure 1. Structures of previously disclosed chiral morpholine D4 anatgonists, ML398, 1, and alkoxymethyl morpholine, 2.

Scheme 1. Reagents and conditions: (a) R-PhCH2CH2I, K2CO3, acetone, reflux, 52– 67%; (b) LiCl, DMF, 150 °C, 42–50%; (c) Deoxo-Fluor, EtOH CH2Cl2, 48–64%; (d) NH4HCO2, Pd/C, MeOH, reflux, 90–96%; (e) ArCH2X, Cs2CO3, THF, rt, 47–74%; (f) ArCHO, MP-CNBH3, CH2Cl2, lW, 110 °C, 19–48%.

ester piperidine, 3, was alkylated with an appropriate phenethyl halide resulting in 4 (K2CO3, acetone, reflux) in moderate yield (52–68%); which was then immediately decarboxylated (LiCl, DMF, 150 °C) yielding the alkylated piperidinone, 5.16 The ketone was treated with Deoxo-FluorÒ17,18 yielding the gemdifluoropiperidine, 6, (48–64%) which was then deprotected under reductive conditions (NH4HCO2, Pd/C, MeOH, reflux) leaving the penultimate compound, 7, in good yields. The final compounds were synthesized either via alkylation of the nitrogen (ArCH2X, Cs2CO3) for 8 or via reductive amination (ArCHO, MP-CNBH3, lW, 100 °C) for 9. The racemic final compounds were then separated by chiralSFC to yield the desired chiral final compounds.19 The SAR evaluation started with the southern N-linked portion of the molecule, keeping the upper right-hand phenethyl portion constant (Table 1). The compounds were originally evaluated at a single concentration (10 lM) for the ability to inhibit D4.4 using a radioligand (spiperone). Those compounds that were able to produce 80% inhibition or greater were then evaluated for an IC50 and Ki (www.EuroFins.com). The 4-chlorophenyl, 8a, a direct comparator to ML398, 1, was active (Ki = 210 nM); however, it was less potent than 1. Expanding the steric bulk to the naphthalene derivative produced an equipotent molecule (8b, Ki = 220 nM). Moving the 3,4-dimethyl derivative produced the most potent compound of the series to date (8c, Ki = 82 nM), as the racemic mixture. Both the 4-methoxy (8d) and 4-thiomethyl (8g) retained activity, 120 nM and 140 nM, respectively, as did the 6-quinoline, 8h (Ki = 380 nM). The 3-fluoro-4-methoxyphenyl derivative, 8j, a direct comparator to 2, was the most potent compound tested in this set of analogs (Ki = 31 nM). A number of compounds were

inactive or weakly active (8n–8v), analogs containing both electron withdrawing and donating groups. The 5-methoxy-3-indole, 8w, a moiety that previously was shown to impart significant activity was moderately potent in this series (Ki = 200 nM). Dopamine receptor selectivity on selected compounds reveals that the compounds were selective for the D4 receptor (8c and 8cc) and another showed slight activity against D2S (8w, 52% inhibition at 10 lM). The next round of SAR concentrated on a two-prong approach by modification of the areas of the molecule (Table 2). The R group modification centered around both electron-donating (OMe) and electron-withdrawing (F) groups. The southern R1 moieties consisted of the best groups that we have identified previously. To this end, the 2-OMe phenyl R group produced active compounds for all of the R1 groups evaluated, with the exception of the 4-fluoro-3indole, 9d, which was not active in the alkoxymethyl morpholine series either.14 The 6-halogen-3-indole (9a,b) and the 6-methoxy-3-indole (9e) were all sub-100 nM in activity. Moving to the 3-OMe phenyl group produced less active compounds across the board (9f–i), although the best compound, 9i, was similar in potency with its 9e counterpart (150 nM vs. 100 nM, respectively). Much like the 3-OMe compounds, the 4-OMe phenyl groups produced less active compounds compared to the 2-OMe phenyl compounds (e.g., 9a vs 9l). However, moving to the electronwithdrawing fluorine groups, the compounds were much more potent. The 4-fluorophenyl group produced compounds that were sub-20 nM (9m–p), with the 6-fluoro-3-indole (9m, Ki = 7.0 nM), the 6-methoxy-3-indole (9o, Ki = 10.3 nM) and the imidazo[1,5-a] pyridine (9p, Ki = 9.7 nM) being the most potent from this series of racemic compounds. The 3-fluorophenyl analogs were less potent than the 4-fluorophenyl counterparts (e.g., 9m vs 9q and 9p vs 9t). Moving to the 2-fluorophenyl moiety proved beneficial as well, producing compounds that were potent in the single digit nanomolar range (9w, Ki = 8.6 nM; 9z, Ki = 5.9 nM). The difluoropiperidine scaffold has been shown to be a productive scaffold change from the morpholine scaffold. Selectivity was performed on select compounds which again showed variable selectivity against the other dopamine receptors, with 9p and 9q being selective and 9v and 9z showing modest activity against D2S and D3. Up to this point, we have only synthesized and evaluated racemic compounds; however, we were able to synthesize the racemic compounds on scale (150 mg) and then separate the final compounds into the isolated enantiomers utilizing chiral SFC protocols. In order to confirm the structure of the active isomer, the intermediate 7 (R = H) was separated into the corresponding (S) and (R) isomers and then the active isomer was converted to the parabromosulfonamide and then submitted for a single crystal X-ray structure (Fig. 2).20 The X-ray confirms the active isomer as the (R)-enantiomer, which corresponds the same isomer as the previously reported morpholine and alkoxymethyl morpholine D4 antagonists.13,14 Our next set of analogs further establishes the (R)-enantiomer as the active isomer since both enantiomers were made and tested based on previously evaluated racemic compounds, as well as new analogs (Table 3). For many of the analogs, the potency resides completely in the (R)-enantiomer (8c, 9bb–9ee); however, in other compounds the (S)-enantiomer is active as a D4 antagonist (9a, 9kk, 9r, and 9v). The combination of the 4-fluorophenyl and 6-methoxy-3-indole ((R)-9o, Ki = 1.9 nM) is the most potent compound to date from this series of analogs. In fact, the 6-methoxy3-indole southern fragment produced a number of potent analogs. Much like what was seen above, the selectivity of the compounds showed modest activity against D3 and variable and D2L, with only (R)-9kk being the only enantiospecific compound that is selective against all receptors.

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D. E. Jeffries et al. / Bioorg. Med. Chem. Lett. 26 (2016) 5757–5764 Table 1 Structure and D4 activity of the N-linked analogs

Cmpd

R

D4.4a (% inh.)

ML398, 1 2

IC50b (nM)

Kib (nM)

130 56

36 14.3

8a

80

760

210

8b

80

790

220

85 300 <20% inhibition at 10 lM against D1, D2L, D2S, D3 and D5

82

8c

8d

87

430

120

8e

79

8f

55

8g

93

510

140

8h

87

1360

380

8i

70

8j

88

110

31

8k

70

8l

66

8m

88

300

84

8n

77

8o

51

8p

6

8q

10

8r

10

8s

49

8t

18

(continued on next page)

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Table 1 (continued) Cmpd

D4.4a (% inh.)

R

8u

0

8v

48 91 720 <50% inhibition @ 10 lM versus D1, D2L, D3 and D5; D2S = 52%

8w

a b

IC50b (nM)

8x

66

8y

54

8z

69

8aa

16

8bb

68

8cc

90 120 <50% inhibition @ 10 lM versus D1, D2L, D2S, D3 and D5

Kib (nM)

200

34

% inhibition at 10 lM. IC50 and Ki values were run in duplicate in a radioligand binding assay using Spiperone at EuroFins (www.EuroFins.com).

Table 2 Structure and D4 activity of the bidirectional SAR evaluation

R1

IC50b (nM)

Kib (nM)

ML398, 1

130

36

2

56

14.3

Cmpd

R

D4.4a (% inh.)

9a

94

170

48

9b

94

230

64

9c

75

230

63

9d

3

9e

76

370

100

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D. E. Jeffries et al. / Bioorg. Med. Chem. Lett. 26 (2016) 5757–5764 Table 2 (continued) Cmpd

R

R1

D4.4a (% inh.)

IC50b (nM)

Kib (nM)

1540

430

9f

66

9g

88

9h

67

9i

89

550

150

9j

93

430

120

9k

88

640

180

9l

91

480

130

9m

94

25

7.0

9n

95

65

17.9

9o

96

37

10.3

97

35

9.7

9p

<50% inhibition @ 10 lM versus D1, D2L, D2S, D3 and D5 92

77

21

9q

<50% inhibition @ 10 lM versus D1, D2L, D2S, D3 and D5

9r

93

45

12.5

9s

94

160

44

9t

78

9u

59

55

15.3

96 9v

<50% inhibition @ 10 lM versus D1, D2L and D5; D2S, 57%, D3, 69%;

9w

95

31

8.6

9x

92

98

27

(continued on next page)

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Table 2 (continued) Cmpd

R1

R

D4.4a (% inh.)

95

a

Kib (nM)

21

5.9

62

9y

b

IC50b (nM)

9z

<50% inhibition @ 10 lM versus D1, D5, and D2L;D2S, 66%: D3, 80%

9aa

92

150

42

% inhibition at 10 lM. IC50 and Ki values were run in duplicate in a radioligand binding assay using Spiperone at EuroFins (www.EuroFins.com).

Figure 2. ORTEP drawing of the active enantiomer of the difluoropiperidine scaffold.

Finally, having identified a number of potent analogs, we further profiled selected compounds in a battery of Tier 1 in vitro DMPK assays (Table 4). The intrinsic clearance (CLINT) was assessed in liver microsomes and all of the compounds proved to be unstable to oxidative metabolism in both species tested (human and rat).21 In addition, utilizing an equilibrium dialysis approach, the protein binding of the compounds was evaluated and all compounds that were assessed were highly protein bound (Fu < 0.015) in both species. Lastly, we assessed the ability of these compounds to cross the blood–brain barrier (BBB) in a rodent IV cassette experiment.22,23 The compounds evaluated are shown in Table 4, and although the compounds show high clearance in rats, many of the compounds were able to cross the BBB with Kp values >1, with the exception of (R)-9dd and (R)-9v. In conclusion, we have disclosed a novel 4,4-difluoropiperidine scaffold as a potent and selective dopamine receptor 4 antagonist. The work builds upon our previous disclosures of the chiral morpholine and chiral alkoxymethyl morpholine scaffolds. Many of the compounds reported are very potent (D4 Ki < 10 nM) with some showing full selectivity against the other dopamine receptors. As we have shown previously, the scaffold exhibits enantiopreference

Table 3 Structure and D4 activity of the separated enantiomers

D4.4 (% inh)

IC50e (nM)

Kie (nM)

(S)-8c (R)-8c

77

>10,000 230

63

(S)-9bb (R)-9bb

64a 79b

23

6.3

(S)-9cc (R)-9cc

58a 59b

64

17.7

(S)-9dd (R)-9dd

55a 66b

59

16.4

(S)-9ee (R)-9ee

25a 53b

88

24

Cmpd

R

R1

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D. E. Jeffries et al. / Bioorg. Med. Chem. Lett. 26 (2016) 5757–5764 Table 3 (continued) Cmpd

R

R1

D4.4 (% inh) 75

(S)-9m

b

IC50e (nM)

Kie (nM)

320

89

650% inhibition @ 10 lM versus D1, D2L, D2S and D5; D3 = 52% 59d 7.4 2.1

(R)-9m

<50% inhibition @ 10 lM versus D1, D2S and D5; D2L = 53% and D3 = 85% 64c 63d

(S)-9o (R)-9o

620 7.0

170 1.9

<30% inhibition @ 10 lM versus D1, D2S, and D5; D2L = 67% and D3 = 52% 73a 85c

(S)-9kk (R)-9kk

3200 21

890 5.8

<50% inhibition @ 10 lM versus D1, D2S, D3 and D5; D2L = 51% 62b 83c

(S)-9r (R)-9r

530 18.6

150 5.2

<50% inhibition @ 10 lM versus D1, D2S and D5; D2L = 53% and D3 = 85% (S)-9v (R)-9v

59a 84b

5990 190

1,660 53

66b

490

140

<50% inhibition @ 10 lM versus D1, D2S and D5; D2L = 57%; and D3 = 54% 85c 14.1 3.9

(S)-9w (R)-9w

<50% inhibition @ 10 lM versus D1, D2S and D5; D2L = 51% and D3 = 65% a b c d e

% inhibition a 10 lM. % inhibition at 1 lM. % inhibition at 0.1 lM. % inhibition at 10 nM. IC50 and Ki values were run in duplicate in a radioligand binding assay using Spiperone at EuroFins (www.EuroFins.com).

Table 4 In vitro and in vivo DMPK analysis of select compounds Compd

D4 Ki (nM)

Microsomal intrinsic clearance (mL/min/kg)

Plasma unbound fraction (Fu)

hCLINT

rCLINT

Human

Rat

(R)-8c (R)-9bb (R)-9dd (R)-9kk (R)-9m (R)-9o (R)-9r (R)-9v (R)-9w

63 6.3 16.4 17.7 2.1 1.9 5.2 53 3.9

108 111 70.5 99.0

1673 2488 1908 582

298 300 156

957 1064 1301

0.005 0.001 0.001 0.001 0.004 0.018 0.003 0.005 0.005

0.004 0.014 0.001 0.003 0.011 0.009 0.002 0.002 0.050

Plasma (ng/mL)

Brain (ng/g)

Kp

44.4 28.6 25.3 14.9 13.2 9.2 39.0 54.8 30.4

93.7 100 BLQ 19.6 25.5 30.3 43.5 23.5 38.9

2.1 3.5 N/A 1.3 1.9 3.3 1.1 0.4 1.3

Rodent IV Cassette (0.25 mg/kg, 0.25 h)

(R)-8c (R)-9bb (R)-9dd (R)-9kk (R)-9m (R)-9o (R)-9r (R)-9v (R)-9w Kp = total brain:plasma ratio.

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in activity with the (R)-enantiomer being the more potent conformer. Further, we were able to confirm via X-ray crystal structure that the (R)-isomer is the active species. Although the compounds possessed less than ideal in vitro DMPK properties, many are highly brain penetrant which, when coupled with the potency, could make these potential radioligands. Further profiling and optimization will be reported in due course. Acknowledgements The authors would like to thank the Michael J. Fox Foundation for Parkinson’s Research for research support for CRH (MJFF Grant ID: 10000) and Jarrett Foster, Sichen Chang, and Xiaoyan Zhan for their contributions to the DMPK screening tier. References and notes 1. Jaber, M.; Robinson, S. W.; Missale, C.; Caron, M. G. Neuropharmacology 1996, 35, 1503. 2. Ye, N.; Neumeyer, J. L.; Baldessarini, R. J.; Zhen, X.; Zhang, A. Chem. Rev. 2013, 113, 3. 3. Girault, J.-A.; Greengard, P. Arch. Neurol. 2004, 61, 641. 4. Van Tol, H. H. M.; Wu, C. M.; Guan, H.-C.; Ohara, K.; Bunzow, J. R.; Civelli, O.; Kennedy, J.; Seeman, P.; Niznik, H. B.; Jovanovic, V. Nature 1992, 358, 149. 5. Tost, H.; Alam, T.; Meyer-Lindenberg, A. Neurosci. Biobehav. Rev. 2010, 34, 689. 6. Patel, S.; Freedman, S. B.; Chapman, K. L.; Emms, F.; Fletcher, A. E.; Knowles, M.; Marwood, R.; McAllister, G.; Myers, J.; Patel, S.; Curtis, N.; Kulagowski, J. J.; Leeson, P. D.; Ridgill, M. P.; Graham, M.; Matheson, S.; Rathbone, D.; Watt, A. P.; Bristow, L. J.; Rupniak, N. M. J.; Baskin, E.; Lynch, J. J.; Ragan, C. I. J. Pharmacol. Exp. Ther. 1997, 283, 636. 7. Bristow, L. J.; Kramer, M. S.; Kulagowski, J.; Patel, S.; Ragan, C. I.; Seabrook, G. R. Trends Pharm. Sci. 1997, 18, 186. 8. Bristow, L. J.; Collinson, N.; Cook, G. P.; Curtis, N.; Freedman, S. B.; Kulagowski, J. J.; Leeson, P. D.; Patel, S.; Ragan, C. I.; Ridgill, M.; Saywell, K. L.; Tricklebank, M. D. J. Pharmacol. Exp. Ther. 1997, 283, 1256. 9. Huot, P.; Johnston, T. H.; Koprich, J. B.; Fox, S. H.; Brotchie, J. M. Pharmacol. Rev. 2013, 65, 171. 10. Di Ciano, P.; Grandy, D. K.; Le Foll, B. Adv. Pharmacol. 2014, 69, 301. 11. Huot, P.; Johnston, T. H.; Koprich, J. B.; Espinosa, M. C.; Reyes, M. G.; Fox, S. H.; Brotchie, J. M. Behav. Pharmacol. 2015, 26, 101. 12. Huot, P.; Johnston, T. H.; Koprich, J. B.; Aman, A.; Fox, S. H.; Brotchie, J. M. J. Pharmacol. Exp. Ther. 2012, 342, 576. 13. Berry, C. B.; Bubser, M.; Jones, C. K.; Hayes, J. P.; Wepy, J. A.; Locuson, C. W.; Daniels, J. S.; Lindsley, C. W.; Hopkins, C. R. ACS Med. Chem. Lett. 2014, 5, 1060. 14. Witt, J. O.; McCollum, A. L.; Hurtado, M. A.; Huseman, E. D.; Jeffries, D. E.; Temple, K. J.; Plumley, H. C.; Blobaum, A. L.; Lindsley, C. W.; Hopkins, C. R. Bioorg. Med. Chem. Lett. 2016, 26, 2481. 15. Sebastianutto, I.; Maslava, N.; Hopkins, C. R.; Cenci, M. A. Neurobiol. Dis. 2016, 96, 156. 16. Krapcho, A. P.; Weimaster, J. F.; Eldridge, J. M.; Jahngen, E. G. E., Jr.; Lovey, A. J.; Stephens, W. P. J. J. Org. Chem. 1978, 43, 138. 17. Singh, R. P.; Shreeve, J. M. Synthesis 2002, 17, 2561. 18. Stanton, M. G.; Hubbs, J.; Sloman, D.; Hamblett, C.; Andrade, P.; Angagaw, M.; Bi, G.; Black, R. M.; Crispino, J.; Cruz, J. C.; Fan, E.; Farris, G.; Hughes, B. L.; Kenific, C. M.; Middleton, R. E.; Nikov, G.; Sajonz, P.; Shah, S.; Shomer, N.; Szewczak, A. A.; Tanga, F.; Tudge, M. T.; Shearman, M.; Munoz, B. Bioorg. Med. Chem. Lett. 2010, 20, 755. 19. Representative synthesis of 2-iodoethyl benzene analogs (yields range 65– 77%). To a solution of 2-(4-fluorophenyl)ethan-1-ol (1.12 g, 7.99 mmol) in DCM (45 mL) at 0 °C was added imidazole (0.60 g, 8.8 mmol), triphenylphosphine (2.31 g, 8.80 mmol), and iodine (2.03 g, 8.00 mmol) in rapid succession. The mixture was allowed to stir at 0 °C for 10 min before being gradually warmed to room temperature and let stir an additional 3 h. Once TLC confirmed complete conversion the reaction was quenched with saturated aqueous Na2S2O3 (90 mL) and the resulting solution extracted with DCM (3  30 mL). The resulting organics were dried over MgSO4 and concentrated in vacuo. The resulting white solid was resuspended in 100 mL hexanes and filtered to

20. 21. 22. 23.

remove the byproduct triphenylphosphine oxide as a white precipitate. Following filtration the crude material was purified using Teledyne ISCO Combi Flash system (40 g column, solid loading on silica, 100% hexanes, 20 min run) to afford 1-fluoro-4-(2-iodoethyl)benzene (1.43 g, 72%) as a clear oil. Representative synthesis of methyl 1-benzyl-4-oxo-3-phenethylpiperidine-3carboxylate analogs (yields range 52–67%). In a round bottom flask containing a magnetic stir bar was added 1-fluoro-4-(2-iodoethyl)benzene (2.00 g, 7.99 mmol), methyl 1-benzyl-4-oxopiperidine-3-carboxylate HCl salt (1.86 g, 6.57 mmol), and potassium carbonate (3.63 g, 26.3 mmol) which were dissolved in acetone (40 mL) and refluxed for 21 h. After cooling to room temperature the crude material was filtered and condensed in vacuo onto silica gel for purification using Teledyne ISCO Combi Flash system (80 g column, solid loading on silica, 0–15% EtOAc in hexanes, 30 min run) to afford methyl 1benzyl-3-(4-fluorophenethyl)-4-oxopiperidine-3-carboxylate (1.3 g, 54%) as a yellow tinted, viscous oil. Representative synthesis of 1-benzyl-3-phenethylpiperidine-4-one analogs (yields range 42–50%). A mixture of methyl 1-benzyl-3-(4-fluorophenethyl)4-oxopiperidine-3-carboxylate (1.88 g, 5.09 mmol) and LiCl (2.16 g, 50.9 mmol) in DMF (51 mL) was refluxed for 4 h. After cooling to room temperature 1 N HCl (1 mL) was added and the mixture allowed to stir for 5 additional min before being poured onto saturated aqueous NaHCO3 (50 mL). The resulting solution was transferred to a separatory funnel and diluted with EtOAc (125 mL) and washed with 5% aq LiCl (3  75 mL). The organic layer was dried over MgSO4 and condensed onto silica gel in vacuo for purification using Teledyne ISCO Combi Flash system (40 g column, solid loading on silica, 0–20% EtOAc in hexanes, 25 min run) to afford 1-benzyl-3-(4-fluorophenethyl) piperidin-4-one (0.778 g, 50%) as a thick, yellow tinted oil. Representative synthesis of 1-benzyl-4,4-difluoro-3-phenethylpiperidine analogs (yields range 48–64%). A reaction vessel was charged with 1-benzyl3-(4-fluorophenethyl)piperidin-4-one (1.70 g, 5.46 mmol), EtOH (0.06 mL), DCM (2.7 mL), and a stir bar before being briefly purged with argon. DeoxoFluor (2.05 g, 9.29 mmol) was then added and the reaction allowed to stir at room temperature for 22 h before being quenched with the dropwise addition of sat. aqueous NaHCO3. The resulting solution was washed with DCM (2  5 mL) and condensed onto silica gel in vacuo for purification using Teledyne ISCO Combi Flash system (24 g column, solid loading, 0–20% EtOAc in hexanes, 25 min run) to afford 1-benzyl-4,4difluoro-3-(4-fluorophenethyl) piperidine (0.924 g, 51%) as a thick, yellow tinted oil. Representative synthesis of 3-phenylpiperidin-4-one analogs (yields range 90– 96%). 1-benzyl-4,4difluoro-3-(4-fluorophenethyl)piperidine (75 mg, 0.23 mmol) was dissolved in MeOH (4.5 mL) and to this solution was added ammonium formate (70.9 mg, 1.13 mmol) and 10% palladium on carbon (75 mg). The resulting solution was heated to 55 °C and allowed to stir for 5 min at which time TLC confirmed complete conversion. The reaction was cooled to room temperature and the crude material was filtered over celite with EtOAc (10 mL) then washed with sat. aqueous NaHCO3 (10 mL). The resulting organics were passed through a phase separator and concentrated in vacuo to afford 3-(4-fluorophenethyl)piperidin-4-one (52.9 mg, 94%) as a thick clear oil. (R)-6-Chloro-3-((4,4-difluoro-3-(4-fluorophenethyl)piperidin-1-yl)methyl)1H-indole (9kk-(R)). Analytical separation was carried out on a Waters (Thar) Investigator SFC: Chiral Technologies CHIRALPAK IF (5 lm, 4.6  250 mm) column, 5–50% MeOH (0.1% DEA) in CO2 over 7 min at 3.5 mL/min, 40 °C, backpressure maintained at 100 bar. Preparative separation was carried out on a PIC Solution Prep 100 SFC: Chiral Technologies CHIRALPAK IF (5 lm, 20  250 mm) column, 10% MeOH (0.1% DEA) in CO2 at 80 mL/min, 40 °C, backpressure maintained at 100 bar. 1H NMR: (600 MHz, CDCl3) d 8.04 (br s, 1H), 7.65 (d, J = 8.5 Hz, 1H), 7.38 (d, J = 5.4 Hz, 1H), 7.09 (m, 2H), 6.94 (m, 2H), 6.89 (m, 2H), 3.68 (q, J = 13.3 Hz, 2H), 2.76 (br s, 2H), 2.49 (m, 2H), 2.37 (br s, 1H), 2.11 (br s 1H), 2.03 (m, 1H), 1.95 (m, 4H). 13C NMR: (150 MHz, CDCl3) d 161.5 (JCF = 242 Hz), 137.2 (JCF = 3.0), 136.7, 129.6 (JCF = 7.7 Hz), 128.2, 126.2, 123.8, 123.4 (JCF = 241 Hz), 120.7, 120.4, 115.0 (JCF = 21 Hz), 113.3, 111.0, 54.5, 52.9, 50.0 (JCF = 9.6 Hz), 42.3 (JCF = 21 Hz), 33.7, 32.6, 27.2. LCMS: RT: 0.977 min., m/z = 407.1 [M+H]+, >99% @ 215 and 254 nm. Specific O.R.: = 14.2 (c = 1 g/dL, MeOH). The structure data were deposited with Cambridge Crystallographic Data Centre Deposition Number: CCDC 1413406. Orbach, R. S. Drug Metab. Disp. 1999, 27, 1350. Smith, N. F.; Raynaud, F. I.; Workman, P. Mol. Cancer Ther. 2007, 6, 428. Bridges, T. M.; Morrison, R. D.; Byers, F. W.; Luo, S.; Daniels, J. S. Pharmacol. Res. Perspect. 2014, 2, e00077.