Starting from an HTS derived hit 1, application of biostructural data facilitated rapid optimization to lead 22, a novel AMPA receptor modulator. This is the first demonstration of how structure based drug design can be exploited in an optimization program for a glutamate receptor

Basten, Stephanie

Campbell, Robert A.

Cumming, Iain A.

Gillen, Kevin J.

Gillespie, Jonathan

Jamieson, Craig

Kazemier, Bert

Kiczun, Michael

Lamont, Yvonne

Lyons, Amanda J.

Maclean, John K.F.

Moir, Elizabeth M.

Morrow, John A.

Papakosta, Marianthi

Rankovic, Zoran

Smith, Lynn

University of Strathclyde Institutional Repository

Jamieson, Craig and Basten, Stephanie and Campbell, Robert A. and Cumming, Iain A. and Gillen, Kevin J. and Gillespie, Jonathan and Kazemier, Bert and Kiczun, Michael and Lamont, Yvonne and Lyons, Amanda J. and Maclean, John K.F. and Moir, Elizabeth M. and Morrow, John A. and Papakosta, Marianthi and Rankovic, Zoran and Smith, Lynn (2010) A novel series of positive modulators of the AMPA receptor : discovery and structure based hit-to-lead studies. Bioorganic and Medicinal Chemistry Letters, 20 (19). pp. 5753-5756. ISSN 0960-894X , http://dx.doi.org/10.1016/j.bmcl.2010.07.138This version is available at http://strathprints.strath.ac.uk/27808/Strathprints is  designed  to  allow  users  to  access  the  research  output  of  the  University  of Strathclyde. Unless otherwise explicitly stated on the manuscript, Copyright © and Moral Rights for the papers on this site are retained by the individual authors and/or other copyright owners. Please check the manuscript for details of any other licences that may have been applied. You may  not  engage  in  further  distribution  of  the  material  for  any  profitmaking  activities  or  any commercial gain. You may freely distribute both the url (http://strathprints.strath.ac.uk/) and the content of this paper for research or private study, educational, or not-for-profit purposes without prior permission or charge. Any correspondence concerning this service should be sent to the Strathprints administrator: strathprints@strath.ac.ukThe Strathprints institutional repository (http://strathprints.strath.ac.uk) is a digital archive of University of Strathclyde research outputs. It has been developed to disseminate open access research outputs, expose data about those outputs, and enable the management and persistent access to Strathclyde's intellectual output.          A Novel Series of Positive Modulators of the AMPA Receptor: Discovery & Structure Based Hit to Lead Studies. Craig Jamiesona*, Stephanie Bastenb, Robert A. Campbella, Iain A. Cumminga, Kevin J. Gillena, Jonathan Gillespiea, Bert Kazemierb, Michael Kiczuna, Yvonne Lamonta, Amanda J. Lyonsa, John K.F. Macleana, Elizabeth M. Moira, John A. Morrowa, Marianthi Papakostaa, Zoran Rankovica and Lynn Smitha. a)Merck Research Laboratories, MSD, Motherwell, Lanarkshire, ML1 5SH, UK. b)Merck Research Laboratories, MSD, PO Box 20, Oss, 5340 BH, Netherlands. This is where the receipt/accepted dates will go; Received Month XX, 2000; Accepted Month XX, 2000 [BMCL RECEIPT] Abstract²  . Starting from an HTS derived hit 1, application of biostructural data facilitated rapid optimization to lead 22, a novel AMPA receptor modulator. This is the first demonstration of how structure based drug design can be exploited in an optimization program for a glutamate receptor.©2010 Elsevier Science Ltd. All rights reserved.                                                  * Corresponding author at current address: Dept of Pure & Applied Chemistry University of Strathclyde, 295 Cathedral St, Glasgow, G1 1XL. Phone: +44  141 548 4830; Fax +44 141 548 4822; email: craig.jamieson@strath.ac.uk The D-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors are ionotropic glutamate receptors which are abundantly expressed in the central nervous system and are believed to facilitate the majority of fast excitatory amino acid neurotransmission. [1] The role of AMPA receptors appears to be critical to mediating synaptic plasticity and long-term potentiation (LTP), the use dependent enhancement in synaptic efficacy which is thought to encode various forms of learning and memory. AMPA receptor modulators have been shown to enhance LTP and are, therefore, under serious consideration as therapeutic agents for a range of neurological disorders LQFOXGLQJ VFKL]RSKUHQLD $O]KHLPHU¶V 'LVHDVH3DUNLQVRQ¶VGLVHDVHDQG$'+'>2,3]   Subunits of the AMPA receptor are encoded by 4 distinct genes (GluA1 to 4), with each subunit comprising 4 domains: N-terminal, extracellular glutamate binding site (Ligand Binding domain, LBD), a transmembrane region and a C-terminal domain.[4]  In the last decade or so, significant advances have been made in studying the three-dimensional structure of the AMPA receptor with structures reported from a construct relating to the LBD (S1S2) [5] and latterly the     full-length receptor.[6]  Our research efforts were aimed at the identification of a novel series of AMPA receptor positive modulators which are of potential utility in the treatment of the kinds of neurological disorders delineated above.  Following a high-throughput screening campaign using a functional assay with GluA1 overexpressed in HEK cells [7], we identified compound 1 (Figure 1) as a hit with confirmed activity from a well characterized solid sample. Compounds were inactive in the absence of glutamate, thus confirming their allosteric nature.   While compound 1 represented a tractable chemotype amenable to optimization and was deemed to have satisfactory potency (pEC50 = 6.7), further in vitro profiling revealed a number of deficiencies that required attention.  Specifically, measured solubility was low (< 1mg/L) and there were attendant issues with microsomal stability (Rat/human intrinsic clearance, CLi > 270 PL/min/mg protein).    From the outset of the program, we had access to biostructural data using the isolated S1S2 LBD GluA2 construct [5] thus enabling an understanding of how our compounds interacted with the receptor.[8] Figure 1 depicts hit compound 1 in complex with the S1S2 LBD GluA2 construct.[9] Although our biological assay used the GluA1 receptor (wild type GluA2 is not permeable to calcium), in our hands x-ray crystallographic study of selected compounds in both GluA1 and GluA2 constructs showed the structures to be identical.[10]  Given that structures could be more expediently obtained with GluA2, we selected this construct for routine examination. Modulators of the AMPA receptor bind remotely to the orthosteric site and essentially stabilize a protein-protein interaction between the receptor subunits of each ligand binding domain.[11] Analysis of our X-ray data revealed that the interactions made with the receptor were primarily hydrophobic in nature, with key contacts made at the distal ends of the molecule (CF3 moiety and tetrahydrobenzothiophene system).  From consideration of the biostructural data, three principal regions of the molecule were targeted (shown in red, green and blue) in order to address the solubility & metabolism issues (Figure 1).    Modification of the tetrahydrobenzothiophene portion (shown in blue, Figure 1B) was expected to reduce lipophilicity and concomitantly improve potency and reduce propensity for hepatic metabolism.  However, it was also anticipated that modulatory activity at the AMPA receptor may be negatively impacted by such changes.         Figure 1. A: Hit compound 1 in complex with S1S2 LBD of GluA2. Receptor surface is depicted, colored by atom type. Several residues have been omitted from this and subsequent figures for clarity. B: Highlighted in green, red and blue are the three regions targeted.  Indeed, stripping back the fused cyclohexyl ring system to furnish e.g. compound 2 (Table 1) resulted in around a 30 fold reduction in potency, albeit with improvement observed in solubility (2 showed kinetic solubility of 10 mg/L). Other truncated analogues (e.g. 3 and 4) showed a similar reduction in potency without any improvement in solubility. Retention of the fused ring system with incorporation of a heteroatom was next explored in order to attenuate lipophilicity.  Compound 5 displayed promising activity at the receptor but lacked any appreciable degree of solubility (< 1 mg/L) or metabolic stability (human and rat Cli > 270 PL/min/mg protein). Insertion of a basic amine (6) or introduction of a pendant alcohol (7) led to a more than ten-fold reduction in potency.  Table 1. Modifications to the tetrahydrobenzothiophene region  NNNHSONH2F3COR2R1  Compds R1 R2 pEC50a 1 -(CH2)4- 6.7 2 H H 5.2 3 Me Me 6.1 4 Et Me 6.2 5 -CH2-CH2-O-CH2- 6.7 6 -CH2-CH2-NH-CH2- 5.3 7 CH2OH H 5.5 aValues are means of two experiments performed in duplicate.   In parallel, the contribution of the tetrahydroindazole system (shown in green, Figure 1B) to AMPA modulatory activity was examined (Table 2). Deletion of the fused cyclohexyl ring (8) resulted in approximately a 30-fold reduction in potency presumably due to loss of a non-specific hydrophobic interaction.  Activity could be partially restored by introduction of a 5-CF3 substituent (9), however again this was accompanied by low solubility and microsomal stability (Solubility < 1 mg/L, Cli (human) = 174 PL/min/mg protein, Cli (rat) = 200 PL/min/mg protein). A fully aromatic system (10) exhibited promising GluA1 activity, however, was again compromised by inadequate solubility (< 1 mg/L) and less than optimal microsomal stability (Cli (human) = 153 L/min/mg protein, Cli (rat) = 91 PL/min/mg protein). As we expected from the biostructure of 1, having a correctly orientated trifluromethyl moiety is essential for activity. Replacement with a methyl (11) or altering the relative position (12) gave rise to inactive compounds.  Table 2 Modifications to the tetrahydroindazole  region NHSONH2OR Compds R pEC50a  8   5.3 NNFFFNNNHSONH2F3CO   9    5.8   10   6.4  11     <4.5   12    <4.5 aValues are means of two experiments performed in duplicate.   The most effective means of meeting our optimization goals emerged from modification of the amide region of 1. From consideration of the biostructural data, a hydrophilic pocket adjacent to the primary carboxamide motif (highlighted in red, Figure 1B) could be accessed by suitably functionalized entities. We, therefore, focused our efforts in making modifications to this region. Intially, simple carboxamide derivatives were prepared (e.g. 13, 14, Table 3) which confirmed that substitution at this position would be tolerated.  Encouraged by these results, we next prepared a number of more functionalized derivatives with pendant solubilising groups. Evaluation of compound 15 in the GluA1 assay showed the compound had modest potency compared to our progenitor hit 1. However, it was sufficiently potent to obtain biostructural data (Figure 2) which revealed that contact with an Asp residue at the side of the hydrophilic pocket could be further optimized. We hypothesized that either modification of the linker length, or removal of one or both of the N,N-dimethyl moieties could be beneficial to potency. In compound 15, the binding mode of the tetrahydroindazole region was essentially identical to that observed with the progenitor compound (1).  Table 3. Modifications to the tetrahydrobenzothiophene region  NNNHSONHF3COR Compds R pEC50a 13 Et 6.5 14 cPropyl 6.2 15 -CH2CH2NMe2 5.2 16 -(CH2)3-NH2 6.4 17 -CH2CH2NHMe 5.7 18 -(CH2)4-NH2 5.9 19 -CH2CH2NH2 5.8 20 -CH2CH2OH 7.3 21 -CH2CH2NHSO2Me 7.0 aValues are means of two experiments performed in duplicate  These considerations led to the design of compound 16 which had comparable potency to the original hit. Other amine derivatives (e.g. 17-19), although more potent than the progenitor 15, were not as active at the receptor compared to compound 16.  The hydrophilic region proved to be relatively tolerant of other functional groups with alcohols typified by 20 and sulfonamide derivatives such as 21 displaying impressive potencies but in common with many other members of the series lacked solubility or microsomal stability.    Figure 2. Biostructure of compound 15 in complex with the S1S2 LBD of GluA2.  In the series as a whole, compound 16 displayed the best balance of solubility (38 mg/L) and microsomal stability (human Cli = 17 PL/min/mg protein, rat Cli = 77 PL/min/mg protein). Permeability as measured by CaCo-2 was deemed to be low, however there was no indication of efflux (A-B = 34 nm/s, B-A = 55 nm/s). In vivo pharmacokinetics in Wistar rats (2 mg/kg i.v., 10 mg/kg p.o.) indicated moderate clearance & half life in the i.v. leg (Clp = 21 mL/min/kg, T1/2 = 3.2 h, Vss = 3.8 L/kg), however, as anticipated from the CaCo-2 data, observed oral bioavailability was low (7.7%). Brain to plasma ratio was low (0.05) which was reasoned to be a function of both the number of hydrogen bond donors and rotatable bonds present in the molecule.  X-ray crystallographic data on the complex of 16 with the S1S2 LBD revealed the binding mode was as anticipated based on our observations from compound 15 (Figure 3). The propylamine side chain was shown to project into the hydrophilic pocket as before, but now forming a salt-bridge with the sidechain of Asp 760. The biostructural data also suggested that conformationally constrained analogues of 16 could be accommodated in the hydrophilic region and this led to the design and synthesis of analogues 22-25 (Table 4) in an effort to improve brain penetration and oral bioavailability.   NNFFFFFFNNFFFNNNNNFFF   Figure 3. X-ray structure of 16 in complex with the S1S2 LBD of GluA2  Comparing 22 and 23 shows a preference for the (R) stereochemistry on the pyrrolidine ring system. This was consistent with our expectations based on the conformation of the propylamine moiety required to facilitate the salt-bridge with Asp 760.  The azetidine analogues 24 and 25 show similar potencies at GluA1 compared to 22.  Table 4. Constrained analogues of compound 16.  NNNHSONHF3COR Compds R pEC50a  22   6.6  23     5.8  24    6.5  25     6.2 aValues are means of two experiments performed in duplicate  Further profiling of 22 indicated that the compound had both reasonable solubility (20 mg/L) and microsomal stability (human Cli = 41 PL/min/mg protein, rat Cli = 50 PL/min/mg protein).  Assessment of permeability in the CaCo-2 assay suggested that absorption might still be an issue (A-B = 19 nm/s, B-A = 22 nm/s).  In vivo pharmacokinetics in rat (2 mg/kg i.v., 10 mg/kg p.o.) indicated that 22 had low clearance and a reasonable half-life (Clp = 2.3 mL/min/kg, T1/2 = 3.8 h, Vss = 0.5 L/kg) and significantly improved brain exposure (brain : plasma ratio = 1.03).  However, measured oral bioavailability was still low (3.8%) indicating that conformational constraint alone was not sufficient to enhance this important parameter.   Synthetic approaches to the compounds discussed above are outlined in Schemes 1 to 3. Compounds 1-7 were prepared through a Gewald cyclisation [12] to prepare the requisite tertrahydrobenzothiophene systems (27) followed by HATU mediated coupling to furnish compounds 1 to 7.  OR2R1SNH2OR1R2NH2 SNH2OR1R2NHONNFFF26a27b, c1 - 7  Scheme 1. Reagents and conditions: a) sulfur, cyanoacetamide, diethylamine, EtOH, reflux 23-51%; b) 3-trifluoromethyl-4,5,6,7-tetrahydroindazol-1-yl)-acetic acid, PS-CDI, PW, 120°C, 10-53%; c) TFA/CH2Cl2, rt, 23% (for 6).  Compounds 8 to 12 were synthesized in a two step fashion from the available amino thiophene derivative 28 (Scheme 2).  Acetylation with bromacetyl bromide followed by alkylation of the appropriate azole derivative furnished the requisite target molecules in an expedient fashion.  SONH2NH2SONH2NHOBrSONH2NHOHet28a b8-1229  Scheme 2. Reagents and conditions: a) bromoacetyl bromide, Et3N, CH2Cl2, rt, 94%; b) azole derivative, NaH, DMF, 0° to rt, 14-49%.  Amide derivatives 13 to 25 were accessed as depicted in Scheme 3. Starting from cyclohexanone, Gewald cyclisation furnished the aminothiophene ester 30. Amide coupling provided intermediate 31 which was deprotected by acidolysis. Subsequent HATU mediated amide formation provided amides 13 to 15 and 20/21. In the case of 16 to 19 and 22 to 25, a Boc protected diamine derivative is employed in the coupling step with the desired amine unmasked in a final TFA deprotection step. SOONH2SOONHONNF FFOSONHONNF FFNHRa bd, e303113-25 Scheme 3. Reagents and conditions: a) sulfur, tert-butylcyanoacetate, diethylamine, EtOH, reflux, 95%; b) (3-trifluoromethyl-4,5,6,7-tetrahydroindazol-1-yl)-acetic acid, HATU, DIEA, CH2Cl2/DMF, rt, 75%; c) TFA/CH2Cl2, rt, quant.; d) amine/Boc-diamine, HATU, DIEA, DMF, rt 10-70%; e) TFA/CH2Cl2 (for 16 to 19 and 22 to 25), 15-40%.  In summary, we have demonstrated how hit compound 1 was rapidly optimized to improve physicochemical NHNHNHNH parameters and metabolic stability resulting in compounds such as 16 and 22. A central component of our expedient optimization trajectory has been the application of structure-based drug design and is the first example of its use in a glutamate receptor system. Compounds such as 22 are useful tools in further elucidating the role of AMPA receptor modulators in debiOLWDWLQJQHXURORJLFDOGLVRUGHUV VXFKDV$O]KHLPHU¶V'LVHDVH DQG 3DUNLQVRQ¶V  ,Q WKH IROORZLQJ SDSHU ZHdescribe how 22 can be further optimized to an orally bioavailable AMPA receptor modulator with promising in vivo activity.  References and Notes                                                  1. Kew, J. N. C. and Kemp, J. A. Psychopharmacology 2005, 179, 4. 2 . Marenco, S. and Weinberger, D. R. CNS Drugs 2006, 20, 173. 3 . Zarate, J. and Manji, H. K. Exp. Neurology 2008, 211, 7. 4. Mayer, M. L. and Armstrong, N. Annu. Rev Physiol 2004, 66, 161. 5. Armstrong, N.; Sun ,Y.; Chen, G. Q.; Gouaux, E. Nature 1998, 395, 913.  6. Sobolevsky, A. I.; Rosconi, M. P.; Gouaux, E. Nature  2009, 462, 745. 7.  HEK.GluR1(i) cells were maintained in DMEM supplemented with 10% fetaclone II, 1% non-essential amino acidV DQG  ȝJ P/ K\JURP\FLQ DW R&CO2. Twenty-four h prior to the assay, the cells were harvested with trypsin and seeded onto Costar 96 well clear bottomed black plates at a density of 3.5x104 per ZHOO &HOOV ZHUH ORDGHG ZLWK  ȝ0 IOXR-AM in DMEM media in the absence of hygromycin and incubated at 37oC/5% CO2 for one h. After  dye loading, the cells were ZDVKHGRQFHZLWKȝORIORZFDOFLXPVROXWLRQP0hepes, pH 7.4, 160 mM NaCl, 4.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose) containing 0.625 mM of SUREHQHFLGWRUHPRYHWKHG\H7KHQȝORIORZFDOFLXPsolution was added to each well. The Flexstation added 50 ȝORIJOXWDPDWH- test compound in high calcium solution (10 mM Hepes, pH 7.4, 160 mM NaCl, 4.5 mM KCl, 20 mM CaCl2, 1 mM MgCl2 and 10 mM glucose) to each well and the ensuing response was monitored on FLEXstation. 8.  Crystals were grown as described in ref 5. Co-crystals were prepared by soaking test compound (100mM) with crystals for 24h prior to isolation and data collection.  Coordinates and structure factors have been deposited in the Protein Data Bank for complexes of compounds 1 (3O28), 15 (3O29) and 16 (3O2A). 9. The Pymol Molecular Graphics System (DeLano Scientific, Palo Alto, CA, 2002) was used to generate Figures 1-3. 10 . Maclean, J. K. F. and Kazemier, B. manuscript in preparation. 11.  (a) Sun, Y.; Olson, R.; Horning, M.; Armstrong, N.; Mayer, M.; Gouaux, E.. Nature 2002, 417, 245; (b) Ptak,                                                                                 C. P.; Ahmed, A. H.; Oswald, R. E. Biochemistry. 2009, 48, 8594. (c) Sobolevsky, A. I.; Rosconi, M. P.; Gouaux, E. Nature  2009, 462, 745. 12. Gewald, K.; Schinke, E.; Boettcher, H. Chem. Ber.  1966,  99,  94. 

A novel series of positive modulators of the AMPA receptor : discovery and structure based hit-to-lead studies

Basten, S.

Name not available

http://dx.doi.org/10.1016/j.bmcl.2010.07.138

A novel series of positive modulators of the AMPA receptor : discovery and structure based hit-to-lead studies

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