- Email: [email protected]
Investigation of orexin-2 selective receptor antagonists: Structural modifications resulting in dual orexin receptor antagonists
Accepted Manuscript Investigation of orexin-2 selective receptor antagonists: structural modifications resulting in dual orexin receptor antagonists Jason W. Skudlarek, Christina N. DiMarco, Kerim Babaoglu, Anthony J. Roecker, Joseph G. Bruno, Mark A. Pausch, Julie A. O'Brien, Tamara D. Cabalu, Joanne Stevens, Joseph Brunner, Pamela L. Tannenbaum, W. Peter Wuelfing, Susan L. Garson, Steven V. Fox, Alan T. Savitz, Charles M. Harrell, Anthony L. Gotter, Christopher J. Winrow, John J. Renger, Scott D. Kuduk, Paul J. Coleman PII: DOI: Reference:
S0960-894X(17)30132-4 http://dx.doi.org/10.1016/j.bmcl.2017.02.012 BMCL 24684
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
13 January 2017 3 February 2017 5 February 2017
Please cite this article as: Skudlarek, J.W., DiMarco, C.N., Babaoglu, K., Roecker, A.J., Bruno, J.G., Pausch, M.A., O'Brien, J.A., Cabalu, T.D., Stevens, J., Brunner, J., Tannenbaum, P.L., Peter Wuelfing, W., Garson, S.L., Fox, S.V., Savitz, A.T., Harrell, C.M., Gotter, A.L., Winrow, C.J., Renger, J.J., Kuduk, S.D., Coleman, P.J., Investigation of orexin-2 selective receptor antagonists: structural modifications resulting in dual orexin receptor antagonists, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.02.012
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Investigation of orexin-2 selective receptor antagonists: structural modifications resulting in dual orexin receptor antagonists Jason W. Skudlareka,*, Christina N. DiMarcoa, Kerim Babaoglub, Anthony J. Roecker a, Joseph G. Brunoc, Mark A. Pauschc, Julie A. O’Brienc, Tamara D. Cabalud, Joanne Stevense, Joseph Brunnerf, Pamela L. Tannenbaume, W. Peter Wuelfingg, Susan L. Garsonf, Steven V. Foxe, Alan T. Savitze, Charles M. Harrellf, Anthony L. Gotterf, Christopher J. Winrowf, John J. Renger f, Scott D. Kuduk#, and Paul J. Colemana a
Department of Medicinal Chemistry, MRL, Merck & Co., Inc., West Point, PA 19486, USA Department of Chemical Capabilities & Screening, MRL, Merck & Co., Inc., West Point, PA 19486, USA c Department of In Vitro Pharmacology, MRL, Merck & Co., Inc., West Point, PA 19486, USA d Department of Drug Metabolism, MRL, Merck & Co., Inc., West Point, PA 19486, USA e Department of In Vivo Pharmacology, MRL, Merck & Co., Inc., West Point, PA 19486, USA f Department of Neuroscience, MRL, Merck & Co., Inc., West Point, PA 19486, USA g Discovery Pharmaceutical Sciences, MRL, Merck & Co., Inc., West Point, PA 19486, USA b
_______________ * Corresponding author. Tel.: + 1-215-652-2748 ; fax: + 1-215-652-3971. E-mail address: [email protected] #
Current affiliation Novira Therapeutics, Inc., a part of Janssen Pharmaceuticals, Doylestown, PA 18902, USA
Abstract In an ongoing effort to explore the use of orexin receptor antagonists for the treatment of insomnia, dual orexin receptor antagonists (DORAs) were structurally modified, resulting in compounds selective for the OX2R subtype and culminating in the discovery of 23, a highly potent, OX2R-selective molecule that exhibited a promising in vivo profile. Further structural modification led to an unexpected restoration of OX1R antagonism. Herein, these changes are discussed and a rationale for selectivity based on computational modeling is proposed.
Since 1999, the pharmaceutical industry has been targeting the design of orexin receptor antagonists for the treatment of insomnia following the identification of orexin neuropeptides and receptors. 1-2 Orexin secreting neurons in the lateral hypothalamus release excitatory neuropeptides (OXA and OX-B) that activate two G-protein-coupled receptors (GPCRs): orexin receptor 1 (OX 1R) and orexin receptor 2 (OX2R).3 Upon activation, these receptors promote wakefulness in a circadian manner, with signals peaking during normal wake periods and falling dormant during sleep.4-5 Merck & Co., Inc., Kenilworth, NJ, USA received FDA and PMDA approval of suvorexant (Belsomra®, Fig. 1) for the treatment of insomnia recently.6 In addition to suvorexant, several additional DORAs and selective orexin receptor antagonists (SORAs) have also been disclosed by Merck,7-10 and several excellent reviews of the orexin field have recently been published which describe additional clinical compounds from SmithKline Beecham (now GlaxoSmithKline), Actelion, and Eisai.9,11-20 In a previously published article, we disclosed the identification of MK-8133 (1, Fig. 1), a potent and selective orexin-2 receptor antagonist (OX2R SORA), for the treatment of insomnia.21 Therein, we briefly described the general SAR trends which led us to a new series of potent OX2R SORAs, beginning with the unexpected discovery that potent DORAs could be transformed into SORAs via specific structural changes.22 In summation, it was found that piperidine ethers could be converted from DORAs into OX2R SORAs (isoquinoline 2 and quinoline 3 respectively, Fig. 1) by moving the heteroatom from the 2- to the 4-position. This observation inspired us to explore substituted pyridines as replacements (4 and 5, Fig. 1).
Figure 1. Figure 1. Evolution of piperidine SORAs. During our OX2R SORA optimization efforts, we discovered additional examples of structural changes which restored OX1R antagonism. While this shifting between SORA and DORA profiles should not be completely unexpected given the highly conserved ligand binding pockets of OX1R and OX2R,23 at the time we were surprised by the subtlety of structural alterations required to effect such variation. Herein, alongside a broader discussion of OX2R SORA SAR, we will focus on a subset of these structures and attempt to rationalize the selectivity profiles of our ligands using computational methods. Compounds were evaluated for binding affinity (expressed as Ki values) for OX2R and OX1R via an in vitro radioligand competition binding assay with membranes from CHO-K1 cells which overexpress the human orexin receptors. Additionally, cell-based functional FLIPR (fluorometric imaging plate reader) assays, in which Ca2+ flux was measured as a functional determinant of orexin receptor antagonist activity, were also conducted for determination of receptor subtype selectivity (expressed as IC50 values).24 Historically, we have used the FLIPR selectivity as the more reliable arbiter for the identification of selective compounds; the binding data is included herein because it is a more appropriate comparator for structure-based hypotheses of selectivity.
Our earliest efforts at optimizing OX2R SORAs began with the identification of a 4-cyano-3methyl-2-pyridyl ether moiety bound to our 2-methyl-piperidine linker (a monocyclic replacement of the isoquinoline ring of 2) and a 2-triazolobenzene amide functionality (6, Table 1). Whereas 2 presented a DORA profile, 6 exhibited excellent selectivity for OX2R in both binding and FLIPR assays (285-fold and 125-fold over OX1R, respectively), achieving a crucial benchmark for the program. Modification of the amide substituent in 6 provided additional OX2R-selective antagonists shown in Table 1.
Table 1. Amide SAR.a
BIND ratio (OX1R/2R)
OX2R FLIPR (IC50, nM)
OX1R FLIPR (IC50, nM)
FLIPR ratio (OX1R/2R)
114
285x
8
996
125x
3.0
3568
1189x
21
5928
282x
8
1.7
1887
1110x
32
> 10,000
> 313x
9
1.3
1170
900x
13
6123
471x
10
5.1
1662
326x
29
> 10,000
> 345x
11
8.9
5765
648x
65
> 10,000
> 154x
12
2.0
2517
1259x
22
> 10,000
> 455x
OX2R BIND (Ki, nM)
OX1R BIND (Ki, nM)
0.4
7
Compound
6
R
a
13
0.7
519
741x
9
1741
193x
14
3.1
1155
373x
21
4956
236x
15
0.8
757
946x
16
1967
123x
16
0.7
594
849x
6
2102
350x
17
3.4
790
232x
29
3102
107x
18
1.6
572
358x
7.7
4588
596x
19
2.6
1128
434x
26
4673
180x
20
16.6
1611
97x
69
4446
64x
21
0.7
10
14x
38
71
2x
22
0.6
12
20x
38
74
2x
Values represent the average of n ≥ 2 experiments.
Interestingly, the binding and FLIPR selectivities for 6 could be further maximized. For example, incorporation of a 2-methoxypyridine amide (7) increased binding and FLIPR selectivity 4.2-fold and 2.3fold over 6, respectively. Replacement of the triazole with a cyclopropane in a similar analog (8) proved even more selective in FLIPR (313-fold). Subsequent removal of the pyridine nitrogen (9) likewise improved FLIPR selectivity to 471-fold while only slightly reducing (relative to 7 and 8) binding selectivity to 900-fold. Similar selectivity was achieved by replacing the methoxy group with a bromide
(10) or by replacing the triazole with a pyridine (11). Finally, replacement of the triazole with a thiomethyl (12) gave the most selective compound of all, with 1259-fold binding selectivity and >455fold FLIPR selectivity. Simplification of the amide also provided potent OX2R selective molecules. A series of monosubstituted benzamides exhibited excellent selectivity. Substitution with an ortho-cyclopropane (13), trifluoromethyl (14), trifluoromethoxy (15), ethoxy (16), or pyrrolidine (17) functionality gave potent (629 nM OX2R FLIPR IC50) and selective compounds. Incorporation of a 2-tetrazole in the same position provided 18, which showed exceptional 596-fold selectivity over OX1R in the FLIPR assay. The overwhelming trend for these and many other compounds in this series was towards >30-fold selectivity over OX1R in FLIPR. However, most of these compounds exhibited ancillary liabilities such as PXR inhibition (7, 9, and 16), time-dependent CYP inhibition (8, 12, and 17), or low solubility which precluded further development. An interesting SORA to DORA shift was found in the piperidine ether series with incorporation of an acetamide functionality on two related pyridyl amides. While phenyl-substituted pyridyl amide 20 exhibits less potency and selectivity comparable to compounds 6-18 and especially pyridyl regioisomer 19 above, the addition of an acetamide in both 21 and 22 results in exquisite OX 2R and OX1R potency in both the binding and FLIPR assays, with only 2-fold FLIPR selectivity and 14- to 20-fold binding selectivity. These exceptions were perplexing, but with the recent availability of the X-ray structures for both orexin receptors23,25 we are able to model the key interactions that might be driving receptor selectivities. When compounds 21 or 22, which vary only in the location of the pyridine nitrogen, are docked26 into the crystal structures of OX1R and OX2R, the overall pose of the molecules is very similar to the X-ray coordinates of suvorexant (Fig. 2A). The acetamide group fills the region adjacent to S103/T111 (OX1R/OX2R), a key area of the pocket previously identified to be a hot spot for selectivity.23 The carbonyl of the acetamide is able to pick up a hydrogen bond with the OH of Ser 103 in OX 1R (Fig. 2B). In OX2R this residue is a Thr 111 which, due to the presence of the methyl, has its OH too far away to form a proper hydrogen bond (Fig. 2C). The presence of the acetamide is still favorable as the potency shifts 10-fold for OX2R relative to OX1R. However, the combination of filling the selectivity pocket along with the added H-bond in OX1R presumably causes the significantly greater shift in affinity for OX1R.
Figure 2. A) Docking pose of 22 in cyan superimposed with the X-ray structure of suvorexant in OX1R (PDB 4ZJ8) B) Docking pose for 22 in OX1R suggests a potential hydrogen bond between the acetamide and S103. C) When 22 is docked in OX2R the distance from the acetamide to T111 is too far (3.8Å) for a hydrogen bond. As we learned more about the requirements for potency and selectivity, we sought to simplify 6 by removing the 3-methyl group from the pyridine, resulting in 23 (Fig. 3). This analog proved superior to the methylated parent, with excellent selectivity (293-fold in FLIPR) and good PK properties.
Figure 3. Figure 3. Comparison of 23 and MK-8133.
The in vivo potential for 23 to affect physiology and behavior was assessed in rodents implanted with radio-telemetry recording devices to determine vigilance/sleep state via electroencephalogram/electromyogram (EEG/EMG) polysomnography. In rats, oral administration of 23 at 0.5, 1, and 3 mg/kg resulted in dose-dependent attenuation of active wake relative to vehicle for up to 2 hours following treatment (Fig. 4a). Wake decreases were accompanied by increases in both rapid eye movement (REM) and non-REM sleep. In wild type mice, compound 23 dosed at 30 mg/kg was also effective in reducing wakefulness, most prominently during the 1.5 hours following administration (Fig. 4b). These changes were largely accompanied by increases in NREM sleep in mice, with only marginal increases in REM sleep, a species-dependent difference that has also been observed for OX2R SORA MK-1064.18 Importantly, these changes in active wake and NREM sleep were absent in mice lacking OX2R, indicating that the effects of 23 are mediated selectively through this receptor.
Figure 4. Sleep promoting efficacy of 23 in rodents. The mean time spent in active wake, non-REM sleep (NREM), and REM sleep during 30-min intervals is shown following oral administration of 23 (open circles) relative to vehicle (20% vitamin E TPGS, filled circles) (dose time, vertical grey bar). (a)
Rats were treated with 0.5 mg/kg (n = 16) , 1 mg/kg (n = 14) or 3 mg/kg (n = 12) doses of 23 during the mid active phase (ZT18:00 [6 hrs after lights off]) for 3 consecutive days in a balanced cross-over design. (b) Wildtype (n=6) and OX2R knockout mice (n=5) were treated with 30 mg/kg of 23 during the late active phase (ZT18:00 [6 hrs after lights off]) for 5 consecutive days in a balanced cross-over design. Data from each all days of treatment under each condition were averaged over a 24 hour period as described previously.27 Time points at which significant differences exist between vehicle and 23 responses are indicated by grey vertical lines highlighted by tick marks (short, medium, long; p < 0.05, 0.01, 0.001, respectively; linear mixed-effects model for repeated measures). Unfortunately, 23 also had low biorelevant solubility (0.0137 mg/mL simulated gastric fluid; 0.0135 mg/mL FASSIF) which would require non-conventional formulation in safety assessment studies.28 When the 2-triazole was replaced with the 2-pyrimidine to give 1 (MK-8133) we observed slight decreases in selectivity but improved pH 7 solubility (Fig. 3; biorelevant solubility was also improved to 0.68 mg/mL simulated gastric fluid and 0.66 mg/mL FASSIF), a favorable attribute which led to its clinical development, as shown above.21 An additional discovery made during this extended effort was a SORA to DORA shift effected by replacement of the isonicotinonitrile functionality (23, Table 2) with a nicotinate methyl ester (26, Table 3). This single modification suprisingly resulted in a potent DORA with only 26-fold binding and 9-fold FLIPR selectivity. Moreover, this trend of increased OX1R potency while maintaining OX 2R potency held for the corresponding amine- and sulfide-linked analogs (24 vs. 27 and 25 vs. 28, Tables 2 and 3). We speculate that the ester functionality was able to gain a favorable interaction with OX 1R specifically in the ortho position, as the corresponding isonicotinate methyl ester (29, Table 3) maintained OX2R selectivity, albeit to a lesser extent. Table 2. Isonicotinonitrile SAR.a
OX2R OX1R BIND (Ki, BIND (Ki, nM) nM) O 1.6 829 23 NH 0.9 287 24 S 1.7 324 25 a Values represent the average of n ≥ 2 experiments. Compound
X
BIND ratio (OX1R/2R) 518x 319x 191x
OX2R FLIPR (IC50, nM) 13 29 13
OX1R FLIPR (IC50, nM) 3830 1742 1411
FLIPR ratio (OX1R/2R) 295x 60x 109x
Table 3. Isonicotinate methyl ester SAR. a
OX2R OX1R BIND BIND Compound X R1 R2 (Ki, (Ki, nM) nM) O CO2Me H 2.5 66 26 NH CO2Me H 0.4 12 27 S CO2Me H 0.8 6.3 28 O H CO2Me 3.9 311 29 a Values represent the average of n ≥ 2 experiments.
BIND ratio (OX1R/2R) 26x 30x 8x 80x
OX2R FLIPR (IC50, nM) 35 38 24 22
OX1R FLIPR (IC50, nM) 309 69 48 1809
FLIPR ratio (OX1R/2R) 9x 2x 2x 82x
SORA to DORA shifting was observed to have even greater molecular complexity as further work was done to explore related molecules. In one such case, the corresponding 4-cyano-3-methyl ester analog (30, Fig. 5) showed modest selectivity (112-fold in binding and 39-fold in FLIPR). We speculate that, in the presence of both nitrile and ester functionalities, the methyl ester is still able to achieve positive interactions with OX 1R, but hindered with regards to positive OX2R interactions with the nitrile or perhaps by steric requirements. However, the dimethylated lactone analog (31) maintained strong SORA characteristics, with binding selectivity of 379-fold and FLIPR selectivity of 414-fold, indicating that the size and shape of the 4-substituent are not as vital as previously hypothesized.
Figure 5. Figure 5. Interchange of SORA and DORA profiles.
At this time, we are unable to explain the differences in selectivity for these closely-related analogs using the previously described computational modeling. Every residue within close proximity to the ester moiety is completely conserved between the two receptors. Given that there are no additional favorable or unfavorable interactions, dynamics or solvation effects are likely to be involved in the selectivity profile observed. Ether-linked compounds were synthesized via the route shown in Scheme 1. Following hydrogenation of 32 to give a 4:1 mixture of trans:cis piperidines (33) and protection with a benzyloxycarbonyl group (34), SFC gave separation of the trans (2R, 5R) enantiomer (35a) and the cis (2R, 5S) enantiomer (35b). A switch to a Boc protecting group provided 36, which was appended with the appropriate pyridine, as exemplified with 2-fluoro-5-cyanopyridine (37). Removal of the Boc group provided the requisite piperidine (38) for subsequent amidation reactions which were mediated by EDC or T3P.29,30
Scheme 1. (a) H2, PtO2, MeOH, HCl, 55 ºC. (b) CbzCl, TEA, CH 2Cl2. (c) Chiral SFC. (d) Boc2O, H2, Pd/C (10%), EtOAc. (e) NaH, DMSO, rt. (f) HCl, dioxane, 0 ºC. (g) EDC, HOAT, Et3N, DMF or T3P, DIEA, DMF.
Amine-linked analogs were synthesized according to Scheme 2. Beginning with cis (2R, 5S) enantiomer 35b, mesylation (39) and displacement with sodium azide (40), followed by reduction with trimethylphosphine, provided amine 41. Protection with a Boc group and removal of the Cbz gave 42, which was coupled with the appropriate carboxylic acid to give 43. Removal of the Boc group and substitution reactions with the requisite fluoro-pyridines provided the targeted analogs.31
Scheme 2. (a) MsCl, DMAP, DIEA, CH2Cl2. (b) NaN3, DMF. (c) Me3P, THF. (d) Boc2O, DMAP, CH2Cl2. (e) H2, Pd/C (10%), 1:1 EtOAc:MeOH. (f) 2-(2H-1,2,3-triazol-2-yl)benzoic acid, EDC, HOAT, Et3N, DMF. (g) HCl, EtOAc. (h) Cs2CO3, DMSO.
Sulfur-linked analogs were made in like-fashion by SN2 displacement of cis mesylates (e.g. 39) with the appropriate 2-thiopyridines. 32 Summary Structural modification of known DORAs gave potent and highly selective OX 2R SORAs. This effort led to the discovery of 23, which exhibited a promising in vivo EEG profile. Low biorelevant solubility prevented further development of this compound and additional work focused on analogs with improved physicochemical properties. During this effort, several analogs exhibited unexpected SORA to DORA shifting. As illustrated with the addition of the acetamide group in compounds 21 and 22, the compounds are clearly able to pick up an additional interaction with OX1R, resulting in a shift from a 2-SORA to a DORA. However, the exquisite selectivity gained by the addition of the nitrile group exemplified in compound 23 and the lack of selectivity with an ester in 26 cannot be readily explained with static structures alone. Under the assumption that the molecules adopt the same binding pose, there are no obvious differences between the bindings of these molecules in the two different receptors. Therefore, either an alternative binding pose exists which the docking was unable to find, or more likely there is a large dynamic and/or solvent effect that is causing the large shifts in observed selectivity. Further exploration of this system with additional crystal structures and molecular dynamics may be required to fully elucidate these mechanisms and will be the focus of future work. Acknowledgments The authors wish to thank the West Point NMR and Mass Spectrometry teams for their assistance in characterization of compounds in this manuscript. We also thank Mr. Jeff Fritzen and Dr. Carmela Molinaro for preparation of key intermediates. References and Notes 1
de Lecea, L.; Kilduff, T. S.; Peyron, C.; Gao, X. B.; Foye, P. E.; Danielson, P. E.; Fukuhara, C.; Battenberg, E. L. F.; Gautvik, V. T.; Bartlett, F. S.; Frankel, W. N.; van den Pol, A. N.; Bloom, F. E.; Gautvik, K. M.; Sutcliffe, J. G. PNAS 1998, 95, 322. 2 Sakurai, T.; Amemiya, A.; Ishii, M.; Matsuzaki, I.; Chemelli, R. M.; Tanaka, H.; Williams, S. C.; Richardson, J. A.; Kozlowski, G. P.; Wilson, S.; Arch, J. R. S.; Buckingham, R. E.; Haynes, A. C.; Carr, S. A.; Annan, R. S.; McNulty, D. E.; Liu, W. S.; Terrett, J. A.; Elshourbagy, N. A.; Bergsma, D. J.; Yanagisawa, M. Cell 1998, 92, 573. 3 Gotter, A.L.; Webber, A. L.; Coleman, P. J.; Renger, J. J.; Winrow, C. J. Pharmacol Rev. 2012, 64, 389. 4 Grady, S. P.; Nishino, S.; Czeisler, C. A.; Hepner, D.; Scammell, T. E. Sleep 2006, 29, 295. 5 Taheri, S.; Sunter, D.; Dakin, C.; Moyes, S.; Seal, L.; Gardiner, J.; Rossi, M.; Ghatei, M.; Bloom, S. Neuroscience Letters 2000, 279, 109. 6 http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm409950.htm 7 Mercer, S. P.; Roecker, A. J.; Garson, S. L.; Reiss, D. R.; Harrell, C. M.; Murphy, K. L.; Bruno, J. G.; Bednar, R. A.; Lemaire, W.; Cui, D.; Cabalu, T. D.; Tang, C.; Prueksaritanont, T.; Hartman, G. D.; Young, S. D.; Winrow, C. J.; Renger, J. J.; Coleman, P. J. Bioorg. Med. Chem. Lett. 2013, 23, 6620. 8 Roecker, A. J.; Reger, T.S.; Mattern, M.C.; Mercer, S.P.; Bergman, J.M.; Schreier, J.D.; Cube, R.V.; Cox, C.D.; Li, D.; Lemaire, W.; Bruno, J. G.; Harrell, C.M.; Garson, S.L.; Gotter, A.L.; Fox, S.V.; Stevens, J.; Tannenbaum, P.L.; Prueksaritanont, T.; Cabalu, T.D.; Cui, D.; Stellabot, J.; Hartman, G. D.; Young, S. D.; Winrow, C. J.; Renger, J. J.; Coleman, P. J. Bioorg. Med. Chem. Lett. 2014, 24, 4884. 9 Coleman, P. J.; Schreier, J. D.; Cox, C. D.; Breslin, M. J.; Whitman, D. B.; Bogusky, M. J.; McGaughey, G. B.; Bednar, R. A.; Lemaire, W.; Doran, S. M.; Fox, S. V.; Garson, S. L.; Gotter, A. L.; Harrell, C. M.; Reiss, D. R.; Cabalu, T.D.; Cui, D.; Prueksaritanont, T.; Stevens, J.; Tannenbaum, P.L.; Ball, R.G.; Stellabott, J.; Young, S.D.; Hartman, G. D.; Winrow, C. J.; Renger, J. J. Chem. Med. Chem. 2012, 7, 415.
10
Kuduk, S. D.; Skudlarek, J. W.; DiMarco, C. N.; Bruno, J. G.; Pausch, M. A.; O’Brien, J. A.; Cabalu, T. D.; Stevens, J.; Brunner, J.; Tannenbaum, P. L.; Gotter, A. L.; Winrow, C. J.; Renger, J. J.; Coleman, P. J. Bioorg. Med. Chem. Lett. 2014, 24, 1784. 11 Kumar, A.; Chanana, P.; Choudhary, S. Pharmacological Reports 2016, 68 (2), 231. 12 Andrews, S. P.; Aves, S. J.; Christopher, J. A.; Nonoo, R. Curr. Top. Med. Chem. 2016, 16 (29), 3438. 13 Coleman, P. J.; Cox, C. D.; Roecker, A. J. Curr. Top. Med. Chem. 2011, 11, 696. 14 Ruoff, C.; Cao, M.; Guilleminault, C. Curr. Pharm. Des. 2011, 17, 1476. 15 Roecker. A. J. and Coleman, P. J. Curr. Top. Med. Chem. 2008, 8, 977. 16 Gotter, A. L.; Roecker, A. J.; Hargreaves, R.; Coleman, P. J.; Winrow, C. J.; Renger, J. J. Prog. Brain Res. 2012, 198, 163. 17 Mieda, M.; Sakurai, T. CNS Drugs 2013, 27, 83. 18 Gotter, A. L.; Forman, M. S.; Harrell, C. M.; Stevens, J.; Svetnik, V.; Yee, K. L.; Li, X.; Roecker, A. J.; Fox, S. V.; Tannenbaum, P. L.; Garson, S. L.; De Lepeleire, I.; Calder, N.; Rosen, L.; Struyk, A.; Coleman, P. J.; Herring, W. J.; Renger, J. J.; Winrow, C. J. Scientific Reports 2016, 6, 27147. 19 Roecker, A. J.; Mercer, S. P.; Schreier, J. D.; Cox, C. D.; Fraley, M. E.; Steen, J.T.; Lemaire, W.; Bruno, J. G.; Harrell, C. M.; Garson, S. L.; Gotter, A. L.; Fox, S. V.; Stevens, J.; Tannenbaum, P. L.; Prueksaritanont, T.; Cabalu, T. D.; Cui, D.; Stellabott, J.; Hartman, G. D.; Young, S. D.; Winrow, C. J.; Renger, J. J.; Coleman, P. J. ChemMedChem 2014, 9, 312. 20 Roecker, A. J.; Cox, C. D.; Coleman, P. J. J. Med. Chem. 2016, 59, 504. 21 Kuduk, S. D., Skudlarek, J. W.; DiMarco, C. N.; Bruno, J. G.; Pausch, M. A.; O’Brien, J. A.; Cabalu, T. D.; Stevens, J.; Brunner, J.; Tannenbaum, P. L.; Garson, S. L.; Savitz, A. T.; Harrell, C. M.; Gotter, A. L.; Winrow, C. J.; Renger, J. J.; Coleman, P. J. Bioorg. Med. Chem. Lett. 2015, 25, 2488. 22 Raheem, I. T; Breslin, M. R.; Bruno, J.; Cabalu, T. D.; Cooke, A.; Cox, C. D.; Cui, D.; Garson, S.; Gotter, A. L.; Fox, S. V.; Harrell, C. M.; Kuduk, S. D.; Lemaire, W.; Prueksaritanont, T.; Renger, J. J.; Stump, C.; Tannenbaum, P. L.; Williams, P. M.; Winrow, C. J.; Coleman, P. J. Bioorg. Med. Chem. Lett. 2015, 25, 444. 23 Yin, J.; Babaoglu, K.; Brautigam, C. A.; Clark, L.; Shao, Z.; Scheuermann, T. H.; Harrell, C. M.; Gotter, A. L.; Roecker, A. J.; Winrow, C. J.; Renger, J. J.; Coleman, P. J.; Rosenbaum, D. M. Nature Structural and Molecular Biology 2016, 23 (4), 293. 24 Bergman, J. M.; Roecker, A. J.; Mercer, S. P.; Bednar, R. A.; Reiss, D. R.; Ransom, R. W.; Harrell, C. M.; Pettibone, D. J.; Lemaire, W.; Murphy, K. L.; Li, C.; Preuksaritanont, T.; Winrow, C. J.; Renger, J. J.; Koblan, K. S.; Hartman, G. D.; Coleman, P. J. Bioorg. Med. Chem. Lett. 2008, 18, 1425. 25 Yin, J.; Mobarec, J. C.; Kolb, P.; Rosenbaum, D. M. Nature 2015, 519 (7542), 247. 26 The X-ray structures of OX1R and OX2R were prepared using the Protein Preparation protocol 33 in Maestro (Schrodinger LLC) which assigns bond orders, adds hydrogens, analyzes H-bond networks, and does a highly constrained (0.3Å heavy atom convergence) minimization with the OPLS_2005 force field. The docking poses for all compounds were generated using Glide XP34 using default program settings. 27 Gotter, A. L., Winrow, C. J., Brunner, J., Garson, S. L., Fox, S. V., Binns, J., Harrell, C. M., Cui, D., Yee, K. L., Stiteler, M., Stevens, J., Savitz, A., Tannenbaum, P. L., Tye, S. J., McDonald, T., Yao, L., Kuduk, S. D., Uslaner, J., Coleman, P. J., Renger, J. J. BMC Neuroscience 2013, 14, 90. 28 Standard gastric fluid (SGF): 0.01 N HCl with 0.9 wt.% NaCl, pH 1.8; Fasted state intestinal fluid (FASSIF): 3 mM sodium taurocholoate, 0.75 mM lecithin, 100 mM NaCl, NaOH, and NaH2PO4•H2O fixed to pH 6.5. Compound (0.1 mg/mL) was stirred in media at 80 rpm then centrifuged at 14k rpm for 15 mins. Supernatant was analyzed relative to standard via reversed-phase chromatography on a fused-core C18 column (4.6x100 mm, 2.7 µm particles), eluting with 0.1 % aqu. H3PO4 and MeCN. 29 Kuduk, S. D.; Skudlarek, J. W. PCT Int. Appl. 2013, WO 2013059222 A1. 30 Kuduk, S. D.; Skudlarek, J. W. PCT Int. Appl. 2014, WO 2014066196 A1. 31 Kuduk, S. D.; Skudlarek, J. W. PCT Int. Appl. 2014, WO 2014085208 A1. 32 Kuduk, S. D.; Skudlarek, J. W. PCT Int. Appl. 2015, WO 2015095108 A1. 33 Sastry, G. M.; Adzhigirey, M.; Day, T.; Annabhimoju, R.; Sherman, W. Journal of Computer-Aided Molecular Design 2013, 27 (3), 221. 34 Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D. T.; Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K.; Shaw, D. E.; Francis, P.; Shenkin, P. S. J. Med. Chem. 2004, 47 (7), 1739.
Graphical abstract
S0960-894X(17)30132-4 http://dx.doi.org/10.1016/j.bmcl.2017.02.012 BMCL 24684
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
13 January 2017 3 February 2017 5 February 2017
Please cite this article as: Skudlarek, J.W., DiMarco, C.N., Babaoglu, K., Roecker, A.J., Bruno, J.G., Pausch, M.A., O'Brien, J.A., Cabalu, T.D., Stevens, J., Brunner, J., Tannenbaum, P.L., Peter Wuelfing, W., Garson, S.L., Fox, S.V., Savitz, A.T., Harrell, C.M., Gotter, A.L., Winrow, C.J., Renger, J.J., Kuduk, S.D., Coleman, P.J., Investigation of orexin-2 selective receptor antagonists: structural modifications resulting in dual orexin receptor antagonists, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.02.012
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Investigation of orexin-2 selective receptor antagonists: structural modifications resulting in dual orexin receptor antagonists Jason W. Skudlareka,*, Christina N. DiMarcoa, Kerim Babaoglub, Anthony J. Roecker a, Joseph G. Brunoc, Mark A. Pauschc, Julie A. O’Brienc, Tamara D. Cabalud, Joanne Stevense, Joseph Brunnerf, Pamela L. Tannenbaume, W. Peter Wuelfingg, Susan L. Garsonf, Steven V. Foxe, Alan T. Savitze, Charles M. Harrellf, Anthony L. Gotterf, Christopher J. Winrowf, John J. Renger f, Scott D. Kuduk#, and Paul J. Colemana a
Department of Medicinal Chemistry, MRL, Merck & Co., Inc., West Point, PA 19486, USA Department of Chemical Capabilities & Screening, MRL, Merck & Co., Inc., West Point, PA 19486, USA c Department of In Vitro Pharmacology, MRL, Merck & Co., Inc., West Point, PA 19486, USA d Department of Drug Metabolism, MRL, Merck & Co., Inc., West Point, PA 19486, USA e Department of In Vivo Pharmacology, MRL, Merck & Co., Inc., West Point, PA 19486, USA f Department of Neuroscience, MRL, Merck & Co., Inc., West Point, PA 19486, USA g Discovery Pharmaceutical Sciences, MRL, Merck & Co., Inc., West Point, PA 19486, USA b
_______________ * Corresponding author. Tel.: + 1-215-652-2748 ; fax: + 1-215-652-3971. E-mail address: [email protected] #
Current affiliation Novira Therapeutics, Inc., a part of Janssen Pharmaceuticals, Doylestown, PA 18902, USA
Abstract In an ongoing effort to explore the use of orexin receptor antagonists for the treatment of insomnia, dual orexin receptor antagonists (DORAs) were structurally modified, resulting in compounds selective for the OX2R subtype and culminating in the discovery of 23, a highly potent, OX2R-selective molecule that exhibited a promising in vivo profile. Further structural modification led to an unexpected restoration of OX1R antagonism. Herein, these changes are discussed and a rationale for selectivity based on computational modeling is proposed.
Since 1999, the pharmaceutical industry has been targeting the design of orexin receptor antagonists for the treatment of insomnia following the identification of orexin neuropeptides and receptors. 1-2 Orexin secreting neurons in the lateral hypothalamus release excitatory neuropeptides (OXA and OX-B) that activate two G-protein-coupled receptors (GPCRs): orexin receptor 1 (OX 1R) and orexin receptor 2 (OX2R).3 Upon activation, these receptors promote wakefulness in a circadian manner, with signals peaking during normal wake periods and falling dormant during sleep.4-5 Merck & Co., Inc., Kenilworth, NJ, USA received FDA and PMDA approval of suvorexant (Belsomra®, Fig. 1) for the treatment of insomnia recently.6 In addition to suvorexant, several additional DORAs and selective orexin receptor antagonists (SORAs) have also been disclosed by Merck,7-10 and several excellent reviews of the orexin field have recently been published which describe additional clinical compounds from SmithKline Beecham (now GlaxoSmithKline), Actelion, and Eisai.9,11-20 In a previously published article, we disclosed the identification of MK-8133 (1, Fig. 1), a potent and selective orexin-2 receptor antagonist (OX2R SORA), for the treatment of insomnia.21 Therein, we briefly described the general SAR trends which led us to a new series of potent OX2R SORAs, beginning with the unexpected discovery that potent DORAs could be transformed into SORAs via specific structural changes.22 In summation, it was found that piperidine ethers could be converted from DORAs into OX2R SORAs (isoquinoline 2 and quinoline 3 respectively, Fig. 1) by moving the heteroatom from the 2- to the 4-position. This observation inspired us to explore substituted pyridines as replacements (4 and 5, Fig. 1).
Figure 1. Figure 1. Evolution of piperidine SORAs. During our OX2R SORA optimization efforts, we discovered additional examples of structural changes which restored OX1R antagonism. While this shifting between SORA and DORA profiles should not be completely unexpected given the highly conserved ligand binding pockets of OX1R and OX2R,23 at the time we were surprised by the subtlety of structural alterations required to effect such variation. Herein, alongside a broader discussion of OX2R SORA SAR, we will focus on a subset of these structures and attempt to rationalize the selectivity profiles of our ligands using computational methods. Compounds were evaluated for binding affinity (expressed as Ki values) for OX2R and OX1R via an in vitro radioligand competition binding assay with membranes from CHO-K1 cells which overexpress the human orexin receptors. Additionally, cell-based functional FLIPR (fluorometric imaging plate reader) assays, in which Ca2+ flux was measured as a functional determinant of orexin receptor antagonist activity, were also conducted for determination of receptor subtype selectivity (expressed as IC50 values).24 Historically, we have used the FLIPR selectivity as the more reliable arbiter for the identification of selective compounds; the binding data is included herein because it is a more appropriate comparator for structure-based hypotheses of selectivity.
Our earliest efforts at optimizing OX2R SORAs began with the identification of a 4-cyano-3methyl-2-pyridyl ether moiety bound to our 2-methyl-piperidine linker (a monocyclic replacement of the isoquinoline ring of 2) and a 2-triazolobenzene amide functionality (6, Table 1). Whereas 2 presented a DORA profile, 6 exhibited excellent selectivity for OX2R in both binding and FLIPR assays (285-fold and 125-fold over OX1R, respectively), achieving a crucial benchmark for the program. Modification of the amide substituent in 6 provided additional OX2R-selective antagonists shown in Table 1.
Table 1. Amide SAR.a
BIND ratio (OX1R/2R)
OX2R FLIPR (IC50, nM)
OX1R FLIPR (IC50, nM)
FLIPR ratio (OX1R/2R)
114
285x
8
996
125x
3.0
3568
1189x
21
5928
282x
8
1.7
1887
1110x
32
> 10,000
> 313x
9
1.3
1170
900x
13
6123
471x
10
5.1
1662
326x
29
> 10,000
> 345x
11
8.9
5765
648x
65
> 10,000
> 154x
12
2.0
2517
1259x
22
> 10,000
> 455x
OX2R BIND (Ki, nM)
OX1R BIND (Ki, nM)
0.4
7
Compound
6
R
a
13
0.7
519
741x
9
1741
193x
14
3.1
1155
373x
21
4956
236x
15
0.8
757
946x
16
1967
123x
16
0.7
594
849x
6
2102
350x
17
3.4
790
232x
29
3102
107x
18
1.6
572
358x
7.7
4588
596x
19
2.6
1128
434x
26
4673
180x
20
16.6
1611
97x
69
4446
64x
21
0.7
10
14x
38
71
2x
22
0.6
12
20x
38
74
2x
Values represent the average of n ≥ 2 experiments.
Interestingly, the binding and FLIPR selectivities for 6 could be further maximized. For example, incorporation of a 2-methoxypyridine amide (7) increased binding and FLIPR selectivity 4.2-fold and 2.3fold over 6, respectively. Replacement of the triazole with a cyclopropane in a similar analog (8) proved even more selective in FLIPR (313-fold). Subsequent removal of the pyridine nitrogen (9) likewise improved FLIPR selectivity to 471-fold while only slightly reducing (relative to 7 and 8) binding selectivity to 900-fold. Similar selectivity was achieved by replacing the methoxy group with a bromide
(10) or by replacing the triazole with a pyridine (11). Finally, replacement of the triazole with a thiomethyl (12) gave the most selective compound of all, with 1259-fold binding selectivity and >455fold FLIPR selectivity. Simplification of the amide also provided potent OX2R selective molecules. A series of monosubstituted benzamides exhibited excellent selectivity. Substitution with an ortho-cyclopropane (13), trifluoromethyl (14), trifluoromethoxy (15), ethoxy (16), or pyrrolidine (17) functionality gave potent (629 nM OX2R FLIPR IC50) and selective compounds. Incorporation of a 2-tetrazole in the same position provided 18, which showed exceptional 596-fold selectivity over OX1R in the FLIPR assay. The overwhelming trend for these and many other compounds in this series was towards >30-fold selectivity over OX1R in FLIPR. However, most of these compounds exhibited ancillary liabilities such as PXR inhibition (7, 9, and 16), time-dependent CYP inhibition (8, 12, and 17), or low solubility which precluded further development. An interesting SORA to DORA shift was found in the piperidine ether series with incorporation of an acetamide functionality on two related pyridyl amides. While phenyl-substituted pyridyl amide 20 exhibits less potency and selectivity comparable to compounds 6-18 and especially pyridyl regioisomer 19 above, the addition of an acetamide in both 21 and 22 results in exquisite OX 2R and OX1R potency in both the binding and FLIPR assays, with only 2-fold FLIPR selectivity and 14- to 20-fold binding selectivity. These exceptions were perplexing, but with the recent availability of the X-ray structures for both orexin receptors23,25 we are able to model the key interactions that might be driving receptor selectivities. When compounds 21 or 22, which vary only in the location of the pyridine nitrogen, are docked26 into the crystal structures of OX1R and OX2R, the overall pose of the molecules is very similar to the X-ray coordinates of suvorexant (Fig. 2A). The acetamide group fills the region adjacent to S103/T111 (OX1R/OX2R), a key area of the pocket previously identified to be a hot spot for selectivity.23 The carbonyl of the acetamide is able to pick up a hydrogen bond with the OH of Ser 103 in OX 1R (Fig. 2B). In OX2R this residue is a Thr 111 which, due to the presence of the methyl, has its OH too far away to form a proper hydrogen bond (Fig. 2C). The presence of the acetamide is still favorable as the potency shifts 10-fold for OX2R relative to OX1R. However, the combination of filling the selectivity pocket along with the added H-bond in OX1R presumably causes the significantly greater shift in affinity for OX1R.
Figure 2. A) Docking pose of 22 in cyan superimposed with the X-ray structure of suvorexant in OX1R (PDB 4ZJ8) B) Docking pose for 22 in OX1R suggests a potential hydrogen bond between the acetamide and S103. C) When 22 is docked in OX2R the distance from the acetamide to T111 is too far (3.8Å) for a hydrogen bond. As we learned more about the requirements for potency and selectivity, we sought to simplify 6 by removing the 3-methyl group from the pyridine, resulting in 23 (Fig. 3). This analog proved superior to the methylated parent, with excellent selectivity (293-fold in FLIPR) and good PK properties.
Figure 3. Figure 3. Comparison of 23 and MK-8133.
The in vivo potential for 23 to affect physiology and behavior was assessed in rodents implanted with radio-telemetry recording devices to determine vigilance/sleep state via electroencephalogram/electromyogram (EEG/EMG) polysomnography. In rats, oral administration of 23 at 0.5, 1, and 3 mg/kg resulted in dose-dependent attenuation of active wake relative to vehicle for up to 2 hours following treatment (Fig. 4a). Wake decreases were accompanied by increases in both rapid eye movement (REM) and non-REM sleep. In wild type mice, compound 23 dosed at 30 mg/kg was also effective in reducing wakefulness, most prominently during the 1.5 hours following administration (Fig. 4b). These changes were largely accompanied by increases in NREM sleep in mice, with only marginal increases in REM sleep, a species-dependent difference that has also been observed for OX2R SORA MK-1064.18 Importantly, these changes in active wake and NREM sleep were absent in mice lacking OX2R, indicating that the effects of 23 are mediated selectively through this receptor.
Figure 4. Sleep promoting efficacy of 23 in rodents. The mean time spent in active wake, non-REM sleep (NREM), and REM sleep during 30-min intervals is shown following oral administration of 23 (open circles) relative to vehicle (20% vitamin E TPGS, filled circles) (dose time, vertical grey bar). (a)
Rats were treated with 0.5 mg/kg (n = 16) , 1 mg/kg (n = 14) or 3 mg/kg (n = 12) doses of 23 during the mid active phase (ZT18:00 [6 hrs after lights off]) for 3 consecutive days in a balanced cross-over design. (b) Wildtype (n=6) and OX2R knockout mice (n=5) were treated with 30 mg/kg of 23 during the late active phase (ZT18:00 [6 hrs after lights off]) for 5 consecutive days in a balanced cross-over design. Data from each all days of treatment under each condition were averaged over a 24 hour period as described previously.27 Time points at which significant differences exist between vehicle and 23 responses are indicated by grey vertical lines highlighted by tick marks (short, medium, long; p < 0.05, 0.01, 0.001, respectively; linear mixed-effects model for repeated measures). Unfortunately, 23 also had low biorelevant solubility (0.0137 mg/mL simulated gastric fluid; 0.0135 mg/mL FASSIF) which would require non-conventional formulation in safety assessment studies.28 When the 2-triazole was replaced with the 2-pyrimidine to give 1 (MK-8133) we observed slight decreases in selectivity but improved pH 7 solubility (Fig. 3; biorelevant solubility was also improved to 0.68 mg/mL simulated gastric fluid and 0.66 mg/mL FASSIF), a favorable attribute which led to its clinical development, as shown above.21 An additional discovery made during this extended effort was a SORA to DORA shift effected by replacement of the isonicotinonitrile functionality (23, Table 2) with a nicotinate methyl ester (26, Table 3). This single modification suprisingly resulted in a potent DORA with only 26-fold binding and 9-fold FLIPR selectivity. Moreover, this trend of increased OX1R potency while maintaining OX 2R potency held for the corresponding amine- and sulfide-linked analogs (24 vs. 27 and 25 vs. 28, Tables 2 and 3). We speculate that the ester functionality was able to gain a favorable interaction with OX 1R specifically in the ortho position, as the corresponding isonicotinate methyl ester (29, Table 3) maintained OX2R selectivity, albeit to a lesser extent. Table 2. Isonicotinonitrile SAR.a
OX2R OX1R BIND (Ki, BIND (Ki, nM) nM) O 1.6 829 23 NH 0.9 287 24 S 1.7 324 25 a Values represent the average of n ≥ 2 experiments. Compound
X
BIND ratio (OX1R/2R) 518x 319x 191x
OX2R FLIPR (IC50, nM) 13 29 13
OX1R FLIPR (IC50, nM) 3830 1742 1411
FLIPR ratio (OX1R/2R) 295x 60x 109x
Table 3. Isonicotinate methyl ester SAR. a
OX2R OX1R BIND BIND Compound X R1 R2 (Ki, (Ki, nM) nM) O CO2Me H 2.5 66 26 NH CO2Me H 0.4 12 27 S CO2Me H 0.8 6.3 28 O H CO2Me 3.9 311 29 a Values represent the average of n ≥ 2 experiments.
BIND ratio (OX1R/2R) 26x 30x 8x 80x
OX2R FLIPR (IC50, nM) 35 38 24 22
OX1R FLIPR (IC50, nM) 309 69 48 1809
FLIPR ratio (OX1R/2R) 9x 2x 2x 82x
SORA to DORA shifting was observed to have even greater molecular complexity as further work was done to explore related molecules. In one such case, the corresponding 4-cyano-3-methyl ester analog (30, Fig. 5) showed modest selectivity (112-fold in binding and 39-fold in FLIPR). We speculate that, in the presence of both nitrile and ester functionalities, the methyl ester is still able to achieve positive interactions with OX 1R, but hindered with regards to positive OX2R interactions with the nitrile or perhaps by steric requirements. However, the dimethylated lactone analog (31) maintained strong SORA characteristics, with binding selectivity of 379-fold and FLIPR selectivity of 414-fold, indicating that the size and shape of the 4-substituent are not as vital as previously hypothesized.
Figure 5. Figure 5. Interchange of SORA and DORA profiles.
At this time, we are unable to explain the differences in selectivity for these closely-related analogs using the previously described computational modeling. Every residue within close proximity to the ester moiety is completely conserved between the two receptors. Given that there are no additional favorable or unfavorable interactions, dynamics or solvation effects are likely to be involved in the selectivity profile observed. Ether-linked compounds were synthesized via the route shown in Scheme 1. Following hydrogenation of 32 to give a 4:1 mixture of trans:cis piperidines (33) and protection with a benzyloxycarbonyl group (34), SFC gave separation of the trans (2R, 5R) enantiomer (35a) and the cis (2R, 5S) enantiomer (35b). A switch to a Boc protecting group provided 36, which was appended with the appropriate pyridine, as exemplified with 2-fluoro-5-cyanopyridine (37). Removal of the Boc group provided the requisite piperidine (38) for subsequent amidation reactions which were mediated by EDC or T3P.29,30
Scheme 1. (a) H2, PtO2, MeOH, HCl, 55 ºC. (b) CbzCl, TEA, CH 2Cl2. (c) Chiral SFC. (d) Boc2O, H2, Pd/C (10%), EtOAc. (e) NaH, DMSO, rt. (f) HCl, dioxane, 0 ºC. (g) EDC, HOAT, Et3N, DMF or T3P, DIEA, DMF.
Amine-linked analogs were synthesized according to Scheme 2. Beginning with cis (2R, 5S) enantiomer 35b, mesylation (39) and displacement with sodium azide (40), followed by reduction with trimethylphosphine, provided amine 41. Protection with a Boc group and removal of the Cbz gave 42, which was coupled with the appropriate carboxylic acid to give 43. Removal of the Boc group and substitution reactions with the requisite fluoro-pyridines provided the targeted analogs.31
Scheme 2. (a) MsCl, DMAP, DIEA, CH2Cl2. (b) NaN3, DMF. (c) Me3P, THF. (d) Boc2O, DMAP, CH2Cl2. (e) H2, Pd/C (10%), 1:1 EtOAc:MeOH. (f) 2-(2H-1,2,3-triazol-2-yl)benzoic acid, EDC, HOAT, Et3N, DMF. (g) HCl, EtOAc. (h) Cs2CO3, DMSO.
Sulfur-linked analogs were made in like-fashion by SN2 displacement of cis mesylates (e.g. 39) with the appropriate 2-thiopyridines. 32 Summary Structural modification of known DORAs gave potent and highly selective OX 2R SORAs. This effort led to the discovery of 23, which exhibited a promising in vivo EEG profile. Low biorelevant solubility prevented further development of this compound and additional work focused on analogs with improved physicochemical properties. During this effort, several analogs exhibited unexpected SORA to DORA shifting. As illustrated with the addition of the acetamide group in compounds 21 and 22, the compounds are clearly able to pick up an additional interaction with OX1R, resulting in a shift from a 2-SORA to a DORA. However, the exquisite selectivity gained by the addition of the nitrile group exemplified in compound 23 and the lack of selectivity with an ester in 26 cannot be readily explained with static structures alone. Under the assumption that the molecules adopt the same binding pose, there are no obvious differences between the bindings of these molecules in the two different receptors. Therefore, either an alternative binding pose exists which the docking was unable to find, or more likely there is a large dynamic and/or solvent effect that is causing the large shifts in observed selectivity. Further exploration of this system with additional crystal structures and molecular dynamics may be required to fully elucidate these mechanisms and will be the focus of future work. Acknowledgments The authors wish to thank the West Point NMR and Mass Spectrometry teams for their assistance in characterization of compounds in this manuscript. We also thank Mr. Jeff Fritzen and Dr. Carmela Molinaro for preparation of key intermediates. References and Notes 1
de Lecea, L.; Kilduff, T. S.; Peyron, C.; Gao, X. B.; Foye, P. E.; Danielson, P. E.; Fukuhara, C.; Battenberg, E. L. F.; Gautvik, V. T.; Bartlett, F. S.; Frankel, W. N.; van den Pol, A. N.; Bloom, F. E.; Gautvik, K. M.; Sutcliffe, J. G. PNAS 1998, 95, 322. 2 Sakurai, T.; Amemiya, A.; Ishii, M.; Matsuzaki, I.; Chemelli, R. M.; Tanaka, H.; Williams, S. C.; Richardson, J. A.; Kozlowski, G. P.; Wilson, S.; Arch, J. R. S.; Buckingham, R. E.; Haynes, A. C.; Carr, S. A.; Annan, R. S.; McNulty, D. E.; Liu, W. S.; Terrett, J. A.; Elshourbagy, N. A.; Bergsma, D. J.; Yanagisawa, M. Cell 1998, 92, 573. 3 Gotter, A.L.; Webber, A. L.; Coleman, P. J.; Renger, J. J.; Winrow, C. J. Pharmacol Rev. 2012, 64, 389. 4 Grady, S. P.; Nishino, S.; Czeisler, C. A.; Hepner, D.; Scammell, T. E. Sleep 2006, 29, 295. 5 Taheri, S.; Sunter, D.; Dakin, C.; Moyes, S.; Seal, L.; Gardiner, J.; Rossi, M.; Ghatei, M.; Bloom, S. Neuroscience Letters 2000, 279, 109. 6 http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm409950.htm 7 Mercer, S. P.; Roecker, A. J.; Garson, S. L.; Reiss, D. R.; Harrell, C. M.; Murphy, K. L.; Bruno, J. G.; Bednar, R. A.; Lemaire, W.; Cui, D.; Cabalu, T. D.; Tang, C.; Prueksaritanont, T.; Hartman, G. D.; Young, S. D.; Winrow, C. J.; Renger, J. J.; Coleman, P. J. Bioorg. Med. Chem. Lett. 2013, 23, 6620. 8 Roecker, A. J.; Reger, T.S.; Mattern, M.C.; Mercer, S.P.; Bergman, J.M.; Schreier, J.D.; Cube, R.V.; Cox, C.D.; Li, D.; Lemaire, W.; Bruno, J. G.; Harrell, C.M.; Garson, S.L.; Gotter, A.L.; Fox, S.V.; Stevens, J.; Tannenbaum, P.L.; Prueksaritanont, T.; Cabalu, T.D.; Cui, D.; Stellabot, J.; Hartman, G. D.; Young, S. D.; Winrow, C. J.; Renger, J. J.; Coleman, P. J. Bioorg. Med. Chem. Lett. 2014, 24, 4884. 9 Coleman, P. J.; Schreier, J. D.; Cox, C. D.; Breslin, M. J.; Whitman, D. B.; Bogusky, M. J.; McGaughey, G. B.; Bednar, R. A.; Lemaire, W.; Doran, S. M.; Fox, S. V.; Garson, S. L.; Gotter, A. L.; Harrell, C. M.; Reiss, D. R.; Cabalu, T.D.; Cui, D.; Prueksaritanont, T.; Stevens, J.; Tannenbaum, P.L.; Ball, R.G.; Stellabott, J.; Young, S.D.; Hartman, G. D.; Winrow, C. J.; Renger, J. J. Chem. Med. Chem. 2012, 7, 415.
10
Kuduk, S. D.; Skudlarek, J. W.; DiMarco, C. N.; Bruno, J. G.; Pausch, M. A.; O’Brien, J. A.; Cabalu, T. D.; Stevens, J.; Brunner, J.; Tannenbaum, P. L.; Gotter, A. L.; Winrow, C. J.; Renger, J. J.; Coleman, P. J. Bioorg. Med. Chem. Lett. 2014, 24, 1784. 11 Kumar, A.; Chanana, P.; Choudhary, S. Pharmacological Reports 2016, 68 (2), 231. 12 Andrews, S. P.; Aves, S. J.; Christopher, J. A.; Nonoo, R. Curr. Top. Med. Chem. 2016, 16 (29), 3438. 13 Coleman, P. J.; Cox, C. D.; Roecker, A. J. Curr. Top. Med. Chem. 2011, 11, 696. 14 Ruoff, C.; Cao, M.; Guilleminault, C. Curr. Pharm. Des. 2011, 17, 1476. 15 Roecker. A. J. and Coleman, P. J. Curr. Top. Med. Chem. 2008, 8, 977. 16 Gotter, A. L.; Roecker, A. J.; Hargreaves, R.; Coleman, P. J.; Winrow, C. J.; Renger, J. J. Prog. Brain Res. 2012, 198, 163. 17 Mieda, M.; Sakurai, T. CNS Drugs 2013, 27, 83. 18 Gotter, A. L.; Forman, M. S.; Harrell, C. M.; Stevens, J.; Svetnik, V.; Yee, K. L.; Li, X.; Roecker, A. J.; Fox, S. V.; Tannenbaum, P. L.; Garson, S. L.; De Lepeleire, I.; Calder, N.; Rosen, L.; Struyk, A.; Coleman, P. J.; Herring, W. J.; Renger, J. J.; Winrow, C. J. Scientific Reports 2016, 6, 27147. 19 Roecker, A. J.; Mercer, S. P.; Schreier, J. D.; Cox, C. D.; Fraley, M. E.; Steen, J.T.; Lemaire, W.; Bruno, J. G.; Harrell, C. M.; Garson, S. L.; Gotter, A. L.; Fox, S. V.; Stevens, J.; Tannenbaum, P. L.; Prueksaritanont, T.; Cabalu, T. D.; Cui, D.; Stellabott, J.; Hartman, G. D.; Young, S. D.; Winrow, C. J.; Renger, J. J.; Coleman, P. J. ChemMedChem 2014, 9, 312. 20 Roecker, A. J.; Cox, C. D.; Coleman, P. J. J. Med. Chem. 2016, 59, 504. 21 Kuduk, S. D., Skudlarek, J. W.; DiMarco, C. N.; Bruno, J. G.; Pausch, M. A.; O’Brien, J. A.; Cabalu, T. D.; Stevens, J.; Brunner, J.; Tannenbaum, P. L.; Garson, S. L.; Savitz, A. T.; Harrell, C. M.; Gotter, A. L.; Winrow, C. J.; Renger, J. J.; Coleman, P. J. Bioorg. Med. Chem. Lett. 2015, 25, 2488. 22 Raheem, I. T; Breslin, M. R.; Bruno, J.; Cabalu, T. D.; Cooke, A.; Cox, C. D.; Cui, D.; Garson, S.; Gotter, A. L.; Fox, S. V.; Harrell, C. M.; Kuduk, S. D.; Lemaire, W.; Prueksaritanont, T.; Renger, J. J.; Stump, C.; Tannenbaum, P. L.; Williams, P. M.; Winrow, C. J.; Coleman, P. J. Bioorg. Med. Chem. Lett. 2015, 25, 444. 23 Yin, J.; Babaoglu, K.; Brautigam, C. A.; Clark, L.; Shao, Z.; Scheuermann, T. H.; Harrell, C. M.; Gotter, A. L.; Roecker, A. J.; Winrow, C. J.; Renger, J. J.; Coleman, P. J.; Rosenbaum, D. M. Nature Structural and Molecular Biology 2016, 23 (4), 293. 24 Bergman, J. M.; Roecker, A. J.; Mercer, S. P.; Bednar, R. A.; Reiss, D. R.; Ransom, R. W.; Harrell, C. M.; Pettibone, D. J.; Lemaire, W.; Murphy, K. L.; Li, C.; Preuksaritanont, T.; Winrow, C. J.; Renger, J. J.; Koblan, K. S.; Hartman, G. D.; Coleman, P. J. Bioorg. Med. Chem. Lett. 2008, 18, 1425. 25 Yin, J.; Mobarec, J. C.; Kolb, P.; Rosenbaum, D. M. Nature 2015, 519 (7542), 247. 26 The X-ray structures of OX1R and OX2R were prepared using the Protein Preparation protocol 33 in Maestro (Schrodinger LLC) which assigns bond orders, adds hydrogens, analyzes H-bond networks, and does a highly constrained (0.3Å heavy atom convergence) minimization with the OPLS_2005 force field. The docking poses for all compounds were generated using Glide XP34 using default program settings. 27 Gotter, A. L., Winrow, C. J., Brunner, J., Garson, S. L., Fox, S. V., Binns, J., Harrell, C. M., Cui, D., Yee, K. L., Stiteler, M., Stevens, J., Savitz, A., Tannenbaum, P. L., Tye, S. J., McDonald, T., Yao, L., Kuduk, S. D., Uslaner, J., Coleman, P. J., Renger, J. J. BMC Neuroscience 2013, 14, 90. 28 Standard gastric fluid (SGF): 0.01 N HCl with 0.9 wt.% NaCl, pH 1.8; Fasted state intestinal fluid (FASSIF): 3 mM sodium taurocholoate, 0.75 mM lecithin, 100 mM NaCl, NaOH, and NaH2PO4•H2O fixed to pH 6.5. Compound (0.1 mg/mL) was stirred in media at 80 rpm then centrifuged at 14k rpm for 15 mins. Supernatant was analyzed relative to standard via reversed-phase chromatography on a fused-core C18 column (4.6x100 mm, 2.7 µm particles), eluting with 0.1 % aqu. H3PO4 and MeCN. 29 Kuduk, S. D.; Skudlarek, J. W. PCT Int. Appl. 2013, WO 2013059222 A1. 30 Kuduk, S. D.; Skudlarek, J. W. PCT Int. Appl. 2014, WO 2014066196 A1. 31 Kuduk, S. D.; Skudlarek, J. W. PCT Int. Appl. 2014, WO 2014085208 A1. 32 Kuduk, S. D.; Skudlarek, J. W. PCT Int. Appl. 2015, WO 2015095108 A1. 33 Sastry, G. M.; Adzhigirey, M.; Day, T.; Annabhimoju, R.; Sherman, W. Journal of Computer-Aided Molecular Design 2013, 27 (3), 221. 34 Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D. T.; Repasky, M. P.; Knoll, E. H.; Shelley, M.; Perry, J. K.; Shaw, D. E.; Francis, P.; Shenkin, P. S. J. Med. Chem. 2004, 47 (7), 1739.
Graphical abstract