Medicinal chemistry Hecker 2015

Download Medicinal chemistry Hecker 2015

Post on 12-Jan-2016




1 download

Embed Size (px)


beta lactamase inhibitor


<ul><li><p>Discovery of a Cyclic Boronic Acid Lactamase Inhibitor (RPX7009)with Utility vs Class A Serine CarbapenemasesScott J. Hecker,*, K. Raja Reddy, Maxim Totrov, Gavin C. Hirst, Olga Lomovskaya,</p><p>David C. Grith, Paula King, Ruslan Tsivkovski, Dongxu Sun, Mojgan Sabet, Ziad Tarazi,</p><p>Matthew C. Clifton, Kateri Atkins, Amy Raymond, Kristy T. Potts, Jan Abendroth, Serge H. Boyer,</p><p>Jerey S. Loutit, Elizabeth E. Morgan, Stephanie Durso, and Michael N. Dudley</p><p>Rempex Pharmaceuticals, Inc., A Subsidiary of The Medicines Company, 3033 Science Park Rd., Suite 200, San Diego, California92121, United StatesMolsoft L.L.C., 11199 Sorrento Valley Road, San Diego, California 92121, United StatesBeryllium, 3 Preston Court, Bedford, Massachusetts 01730, United States</p><p>ABSTRACT: The increasing dissemination of carbapenemases in Gram-negative bacteria hasthreatened the clinical usefulness of the -lactam class of antimicrobials. A program was initiatedto discover a new series of serine -lactamase inhibitors containing a boronic acid pharmacophore,with the goal of nding a potent inhibitor of serine carbapenemase enzymes that are currentlycompromising the utility of the carbapenem class of antibacterials. Potential lead structures werescreened in silico by modeling into the active sites of key serine -lactamases. Promising candidate molecules were synthesizedand evaluated in biochemical and whole-cell assays. Inhibitors were identied with potent inhibition of serine carbapenemases,particularly the Klebsiella pneumoniae carbapenemase (KPC), with no inhibition of mammalian serine proteases. Studies in vitroand in vivo show that RPX7009 (9f) is a broad-spectrum inhibitor, notably restoring the activity of carbapenems against KPC-producing strains. Combined with a carbapenem, 9f is a promising product for the treatment of multidrug resistant Gram-negative bacteria.</p><p> INTRODUCTIONRapidly rising resistance to multiple antimicrobial agents inGram-negative bacteria, commonly related to healthcare-associated infections, is an emerging public health concern inU.S. hospitals. While the cephalosporin class of -lactams was themainstay of treatment in the 1980s, the dissemination ofextended-spectrum -lactamases (ESBLs) over the past 2decades has dramatically weakened the utility of this class andbrought about a corresponding reliance on the carbapenems.1</p><p>Although carbapenems are widely recognized as a safe andeective class of antimicrobials, carbapenem-resistant Enter-obacteriaceae (CRE) due to the Klebsiella pneumoniaecarbapenemase (KPC) and other -lactamases now threatensthe usefulness of all -lactam antibiotics.2 The Centers forDisease Control (CDC) considers CRE to be an urgentantimicrobial resistance threat that now has been detected innearly every U.S. state, with an alarming increase in incidenceover the past 5 years.3 The failure to develop antimicrobial agentsto manage CRE threatens to have a catastrophic impact on thehealthcare system.4</p><p>A proven strategy to overcome resistance to -lactamantibiotics has been to restore their activity by combining themwith an inhibitor of the -lactamase enzymes responsible for theirdegradation. Examples of clinically important -lactamaseinhibitors (Figure 1) include clavulanic acid (combined withamoxicillin), sulbactam (with ampicillin), and tazobactam (withpiperacillin). The KPC -lactamase is poorly inhibited by these-lactamase inhibitors, and thus, they have no usefulness in the</p><p>treatment of infections due to CRE. More recently, thediazabicyclooctane inhibitors avibactam (NXL-104)5 andrelebactam (MK-7655)6 have entered clinical development, incombination with ceftazidime and imipenem, respectively. Bothcompounds display a broad spectrum of -lactamase inhibitionthat includes the KPC enzyme.Boronic acids have long been explored as inhibitors of serine</p><p>proteases.7 Mechanistically, the anity of boronates for serinehydrolases is due to the formation of a covalent adduct betweenthe catalytic serine side chain and the boronate moiety, whicheectively mimics the tetrahedral transition state on the acylationor deacylation reaction path. -Lactamase inhibition by boronicacids such as phenylboronic acid 1 (Figure 2) was rst reportedfrom researchers at Oxford University,8 who noted an earlierobservation9 that borate ions inhibit -lactamase I. Followingpublication of the high-resolution X-ray crystal structure of theclass A RTEM-1 -lactamase of E. coli,10 the group of Jones(Toronto) described a rationally designed inhibitor 2 thatdisplayed an inhibition constant of 110 nM.11,12 Furtherstructure-guided design yielded two highly potent inhibitors 3and 4 with inhibition constants of 6 and 13 nM, respectively.13</p><p>From the late 1990s to the present, several publications from thelaboratory of B. Shoichet detailed structure-based design eortsin this class (e.g., compounds 5 and 6).14,15 More recently,published patent applications1618 describe analogs (e.g., 7)</p><p>Received: January 22, 2015</p><p>Drug Annotation</p><p></p><p> XXXX American Chemical Society A DOI: 10.1021/acs.jmedchem.5b00127J. Med. Chem. XXXX, XXX, XXXXXX</p></li><li><p>related to earlier compounds 3 and 4, as well as heterocyclicvariants such as 819 (see Figure 2). Notably, despite a high level ofinterest by multiple investigators, there is not a single reportrelated to determination of ecacy in an animal infection modelwith any of the compounds of these classes. Believing thatboronate-containing -lactamase inhibitors might nd utility ininhibiting the problematic serine carbapenemases of CRE, we setout to identify a potent inhibitor of the KPC enzyme for use incombination with a carbapenem.</p><p> DESIGNOf the various leads explored by others, we were particularlyinterested in the inhibitors reported by Ness et al.13 for their highpotency in inhibiting some class A -lactamases. These authorsfound that installation of a hydroxyl group on the aromatic ring ofcompound 4a caused a considerable increase in anity to theTEM-1 enzyme. They considered the possibility of formation ofcyclized variant 4b; however, they concluded from examinationof the X-ray crystal structure of this compound bound to TEM-1that it existed in the acyclic form (4a, Figure 3). We wereintrigued by the possibility of using cyclic boronate esterformation to constrain potential lead molecules into thepreferred conformation for enzyme complexation. We alsoanticipated that cyclic boronates may have better selectivitytoward -lactamases versus other serine hydrolases that havelinear substrates. The latter enzymes are likely to have moresterically restricted active sites that can accommodate a linear butnot a cyclic transition state mimetic inhibitor. Several structures</p><p>were proposed and were docked in silico with -lactamaseenzymes from classes A, C, and D. Modeling of the precovalent(Michaelis) complex as well as covalent adduct was performed.High anity of the precovalent complex is expected to facilitaterapid complex formation, while favorable inhibitor/enzymeinteractions within the covalent adduct are important tomaintainthe complex, since the boronate/serine side chain reaction isreversible. On the basis of the docking results, the highest rankingof the proposed inhibitor types was structure 9, from which thefused benzo ring of 4b has been excised.Encouragingly, structure 9 exhibited similar favorable</p><p>precovalent (free docking) and covalently bound poses inrepresentative class A and C enzyme active sites. Comparison toan available X-ray structure of the Michaelis substrate complex inthe active site of AmpC (S64G mutant, Protein Data Bank entry1KVL)20 showed that the putative inhibitor could capture keysubstrateenzyme interactions (Figure 4). The carboxylatemoiety is extensively coordinated within the subpocket thatcoordinates the carboxylate of the substrate -lactam. The amidecarbonyl forms hydrogen bonds to two hydrogen bond donorgroups. The free hydroxyl of the boronic acid moiety enters theoxyanion hole, and the lipophilic portion of the ring engages inhydrophobic interactions with two leucine side chains, againmatching the corresponding interactions of the substrate. Whileno similar experimental structure was available for a class Aenzyme/substrate Michaelis complex at the time, our modelingsuggested that the inhibitor could also mimic the substrateinteractions closely. In contrast to the boron-containing core,</p><p>Figure 1. Currently marketed and promising new -lactamase inhibitors.</p><p>Figure 2. Previously reported boronic acid -lactamase inhibitors.</p><p>Figure 3. Design of cyclic boronate 9.</p><p>Journal of Medicinal Chemistry Drug Annotation</p><p>DOI: 10.1021/acs.jmedchem.5b00127J. Med. Chem. XXXX, XXX, XXXXXX</p><p>B</p></li><li><p>the region of the binding site associated with the N-acylsubstituent was variable across the dierent serine -lactamases;it appeared that this region would benet from a hydrophobicsubstituent but more specic insights could not be gleaned fromthe initial model.</p><p> SYNTHESISThe synthesis of cyclic boronates 9ay was accomplished in sixsteps in an overall yield of about 30% (Scheme 1). Enantiopure-hydroxy ester 10, prepared by lipase-mediated kineticresolution of the corresponding racemate,21 was protected asits silyl ether 11 with TBSCl and imidazole. Regioselectivehydroboration with catalysis by [Ir(COD)Cl]2 gave pinacolboronate 12 which was converted to the more stable pinanediolboronate 13. Stereoselective chloromethylation followingMattesons protocol at 100 C aorded the (S)-chlorohomologue 14 as an 85:15 mixture of diastereomers. Stereo-specic displacement of the chloro group with hexamethyldisi-lazide followed by in situ acylation gave acylamidoboronate 15.</p><p>Acidic removal of all protecting groups aorded the cyclicboronic ester analogs 9ar.</p><p> EVALUATION OF ANALOGSPotentiation of Carbapenems against the KPC-2</p><p>Carbapenemase. Initial evaluation of early analogs of 9 quicklyestablished that in addition to submicromolar activity againstmost serine -lactamases (vide infra), members of this structureclass displayed exceptional activity in inhibition of KPC-typecarbapenemase enzymes. It was quickly recognized thatinhibition of just this one class of -lactamase would address akey resistance mechanism that threatens the utility of thecarbapenem class. From the outset, we envisioned that ourultimate product would be a combination with eithermeropenem (because of its excellent safety record andregistration status in most of the world) or biapenem (beingthe least euxed of the carbapenems in Pseudomonas aeruginosaand having the best stability to the metallo -lactamase NDM-1).Medicinal chemistry eorts were focused on understanding</p><p>the structureactivity relationships (SARs) of the N-acylsubstituent with respect to potentiation of a carbapenem againstKPC-producing strains. Results of evaluating the ability ofselected inhibitor analogs 9 to potentiate biapenem against aKPC-producing strain of Klebsiella pneumoniae (KP1004) areshown in Table 1. Potency is expressed as MPC1, dened as theminimum concentration of the -lactamase inhibitor required toreduce the biapenem MIC from 32 g/mL (no inhibitor) to 1g/mL; in addition to KPC-2, KP1004 also produces the -lactamase enzymes SHV-11 and TEM-1. The simple N-acetylanalog 9a aorded potent activity, with 0.3 g/mL beingsucient to reduce the biapenem MIC from 32 to 1 g/mL. Inmost cases, further substitution had only a modest impact onpotency. Whereas phenylacetyl analog 9c was only 2-foldimproved relative to 9a (MPC1 = 0.15 g/mL), the 2-thienylacetyl analog 9f (reminiscent of the marketed cephalosporinscephalothin and cefoxitin) was 16-fold more potent than 9a(MPC1 = 0.02 g/mL). Replacement of the thiophene of 9f byother heterocycles such as aminothiazole (9g) or pyridyl (9ik)resulted in lesser activity, although potency was restored byincorporation of an amino group in aminopyridyl analog 9l</p><p>Figure 4. Model of 9 bound at the active site of class C -lactamase(magenta). X-ray structure of cephalothin (core portion, green) boundto AmpC (enzyme is superimposed) is shown in green. Blue ballsrepresent hydrogen bonds.</p><p>Scheme 1. Synthesis of Cyclic Boronate Analogs 9ar</p><p>Journal of Medicinal Chemistry Drug Annotation</p><p>DOI: 10.1021/acs.jmedchem.5b00127J. Med. Chem. XXXX, XXX, XXXXXX</p><p>C</p></li><li><p>(MPC1 = 0.04 g/mL). Various hydroxyl substitutions (9mo)and basic groups (9pr) were tolerated but oered noadvantage. None of these compounds displayed any antibacterialactivity when tested in the absence of a -lactam. At theconclusion of this SAR study, compound 9f (RPX7009)22 wasselected for further evaluation as a potential developmentcandidate because of its high potency as well as precedent for thethienylacetyl side chain in the marketed cephalosporinscephalothin and cefoxitin.</p><p> FURTHER EVALUATION OF COMPOUND 9f INVITRO</p><p>Inhibition of Nitrocen Degradation. The spectrum of -lactamase inhibition of 9f was initially evaluated by measuringinhibition of nitrocen degradation by puried -lactamaseenzymes.23 This compound features a broad spectrum ofinhibition of -lactamases, with particularly potent activityagainst KPC, CTX-M, SHV, and CMY enzymes (Table 2).Notably, 9f is considerably more potent than the -lactamaseinhibitors clavulanic acid and tazobactam against the class Acarbapenemase KPC-2 as well as the class C enzymes P99 andCMY-2.Whole-Cell Activity in Combination with Cefepime.</p><p>The spectrum of -lactamase inhibition was evaluated in whole</p><p>cells in combination with cefepime (Table 3).24 Restoration ofcefepime activity against various clinical strains expressing serineenzymes from classes A, C, and D is observed. In general, weobserved a reasonably good correlation between the biochemicalenzyme inhibition results and cellular activity. For example, 9fhas the lowest potency against the TEM-10 enzyme; likewise, thelowest extent of potentiation is observed against strainsexpressing TEM -lactamases. Still, in addition to intrinsicpotency, other factors most certainly aect the observed extent ofpotentiation, including rate of uptake and active eux of theinhibitor as well as the enzyme expression level.Whole-Cell Activity in Combination with Carbape-</p><p>nems. The potent inhibition of KPC enzymes by our series of -lactamase inhibitors along with the intrinsic stability ofcarbapenems to ESBLs and ampC invited combination of ourinhibitors with these antimicrobials. Table 4 illustrates the abilityof compound 9f to potentiate various carbapenem antibioticsagainst clinical isolates of Enterobacteriaceae expressing class Acarbapenemases, including strains producing other class A -lactamases associated with resistance to cephalosporins. It shouldbe noted that these carbapenems are stable to the -lactamasesrepresented in the strains in Table 3 and thus are highly active ontheir own against these strains. However, when KPC is present,carbapenem MICs are also aected by mutations in the genescoding for major porins such as Omp35 and OmpK36, and thehighest MICs are associated with strains that have double porinmutations (for example, strains KP1084 and KP1087). Even inthes...</p></li></ul>


View more >