7446 J. Med. Chem. 2009, 52, 7446–7457 DOI: 10.1021/jm900729s
Discovery of Azetidinyl Ketolides for the Treatment of Susceptible and Multidrug Resistant Community-Acquired Respiratory Tract Infections
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Thomas V. Magee,* Sharon L. Ripp, Bryan Li, Richard A. Buzon, Lou Chupak,† Thomas J. Dougherty,‡ Steven M. Finegan, Dennis Girard,§ Anne E. Hagen, Michael J. Falcone, Kathleen A. Farley, Karl Granskog, Joel R. Hardink, Michael D. Huband, Barbara J. Kamicker, Takushi Kaneko, Michael J. Knickerbocker, Jennifer L. Liras, Andrea Marra,§ Ivy Medina, Thuy-Trinh Nguyen, Mark C. Noe, R. Scott Obach, John P. O’Donnell, Joseph B. Penzien, Usa Datta Reilly, John R. Schafer, Yue Shen, Gregory G. Stone,‡ Timothy J. Strelevitz, Jianmin Sun, Amelia Tait-Kamradt, Alfin D. N. Vaz, David A. Whipple, Daniel W. Widlicka, Donn G. Wishka, Joanna P. Wolkowski, and Mark E. Flanagan*
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Pfizer Global Research & Development, Pfizer, Inc., Groton, Connecticut 06340. †Current address: Bristol-Meyers Squibb Research and Development, Wallingford, CT 06492. ‡Current address: Astra-Zeneca Research Center, Waltham, MA 02451. §Current address: Rib-X Pharmaceuticals, New Haven, CT 06511. Current address: TB Alliance, New York, NY 10005. Received May 28, 2009
Respiratory tract bacterial strains are becoming increasingly resistant to currently marketed macrolide antibiotics. The current alternative telithromycin (1) from the newer ketolide class of macrolides addresses resistance but is hampered by serious safety concerns, hepatotoxicity in particular. We have discovered a novel series of azetidinyl ketolides that focus on mitigation of hepatotoxicity by minimizing hepatic turnover and time-dependent inactivation of CYP3A isoforms in the liver without compromising the potency and efficacy of 1.
Introduction The macrolide class of antibiotics has been very successful in the treatment of serious bacterial infections.1 These natural product agents, first discovered over 50 years ago,2 target the bacterial ribosome to inhibit protein synthesis. Two of the most successful members of this class are azithromycin and clarithromycin, first put on the market in the early 1990s and used extensively to treat community-acquired respiratory tract infections. The antibacterial spectrum is targeted primarily against Gram-positive bacterial strains including Streptococcus pneumoniae and S. pyogenes, fastidious Gramnegative strains including Haemophilus influenzae and Moraxella catarrhalis, atypicals Mycoplasma pneumoniae, Chlamydia pneumoniae, and Legionella pneumophilia, and Helicobacter pylori.3 However, these older agents are becoming less effective as resistance has emerged over time, creating a medical need for novel macrolides addressing these resistant strains.4 The two primary macrolide resistance mechanisms expressed in streptococci are erythromycin ribosome methylation (erma)5 and active efflux (mef).6 Newer or “third” generation macrolides (ketolides) have the advantage that they are poor inducers of the erm resistance mechanism and are also poor substrates for mef efflux-mediated resistance. Telithromycin (1, Figure 1)7 is a third generation macrolide that covers macrolide sensitive and *To whom correspondence should be addressed. For T.V.M.: phone, (860) 715-0653; fax, (860) 715-4693; e-mail, thomas.v.magee@pfizer. com. For M.E.F.: phone, (860) 441-0205; fax, (860) 715-4693; e-mail,
[email protected]. a Abbreviations: erm, erythromycin ribosome methylation genotype; mef, macrolide efflux genotype; RTI, respiratory tract infection; OM, otitis media; ER, in vitro human hepatic microsomal extraction ratio; TDI, time-dependent inhibition of cytochrome P450; CYP3A, cytochrome P450 isoform 3A (includes 3A4 and 3A5); DDI, drug-drug interactions; AO, aldehyde oxidase.
pubs.acs.org/jmc
Published on Web 09/23/2009
resistant strains and is currently the only marketed ketolide. Unfortunately, although a promising compound, 1 has been plagued by safety concerns.8 Cethromycin (2) is the next most advanced ketolide and is currently in late stage development.9 Both structures derive the moniker ketolide from the ketone function at position C3. Both also have bicyclic aryl rings tethered to the macrocylic ring, but in the case of 1 the tether springs from the N11 of the 11,12-carbamate fused to the macrocycle, while for 2 the attachment point is the O6 position. This disclosure focuses on the discovery of a novel series of N11 ketolides addressing some of the safety issues facing 1. Synthetic Chemistry The synthesis of the azetidinyl ketolide template (5) began with the conversion of the clarithromycin starting material (3) to the known acylimidazole intermediate (4),10 as shown in Scheme 1. This was carried out by bis-acetylation of the desosamine and cladinose sugars of 3 followed by dehydration of the 10-hydroxyl and concomitant transformation of the 11-hydroxyl to the acyl imidazole utilizing 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and carbonyl diimidazole (CDI). These reactions proceeded very smoothly in near-quantitative yield, requiring no further purification for the subsequent steps. Incorporation and cyclization of 1-benzhydrylazetidine-3-ylamine into intermediate 4 were unsuccessful under the standard conditions (triethylamine or diisopropylethylamine in acetonitrile with heating) for reacting amine tethers with this system likely because of steric crowding intrinsic to the azetidinylamine and its large benzhydryl protecting group. A stronger organic base (DBU) proved to be very effective for both the displacement of the imidazole and cyclization steps. Subsequent removal of the 400 -acetylcladinose using aqueous r 2009 American Chemical Society
Article
Figure 1. Structures of leading ketolides.
Scheme 1. Synthesis of the Azetidinyl Ketolide Templatea
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the methyl ketone 10, used in the synthesis of diastereomeric isomers 6p and 6q. The isomers were separated by reversephase HPLC and the more active 6q recrystallized from ethyl acetate to unambiguously assign the structure (see Supporting Information). For the synthesis of the 3-hydroxy intermediate 14 a modified Friedlander reaction12 was found to be much more efficient than the Skraup approach. Thus, converting 7 to pivalamide 11 followed by treatment with chlorine gas yielded the 3-chloro intermediate 12, which was dilithiated with tertbutyllithium in ethyl ether and subsequently condensed with 3-dimethylaminopropenal. Upon treatment with strong acid the intermediate obtained from the anion addition collapsed in relatively good overall yield to the [1,8]naphthyridine 13. Selenium dioxide oxidation followed by methoxide displacement of the chloride proceeded smoothly. Methyl ether cleavage was then carried out in 6 N HCl to yield 14. The route used to access the [1,5]naphthyridine coupling partner 22, used in the synthesis of 6s, is given in Scheme 3. Thus, treatment of dibromide 15 with sodium methoxide in DMF gave 16 in good yield, which in turn was converted to 17 using palladium-catalyzed coupling of tert-butyl carbamate. Installation of a methyl group at the 4-position via dilithiation and methyl iodide treatment yielded 18 after acidic removal of the protecting group. The Skraup reaction to form the [1,5]naphthyridine system was generally better than the [1,8] disposition, and this was particularly true with an electron donating group in the 5-position (methoxy in this case). Thus, the conversion of 18 to 19 was affected in modest but workable (45%) yield. Oxidation of 19 with selenium dioxide was not successful, so we then resorted to a longer sequence of bromination followed by displacement with potassium acetate and ester hydrolysis to give the benzylic alcohol 20. Oxidation of 20 with IBX yielded aldehyde 21, which was subsequently treated with strong acid to cleave the methyl ether, giving 22. Results and Discussion
a
Reagents and conditions: (i) Ac2O, TEA, DMAP, DCM; (ii) DBU, CDI, THF, isopropyl ether; (iii) DBU, 1-benzhydrylazetidine-3-ylamine, MeCN, 50 °C; (iv) 2 N HCl, EtOH, 40 °C; (v) DMSO, pyridinium trifluoroacetate, EDC; (vi) MeOH, 50 °C; (vii) Pearlman’s catalyst, H2 (40 psi), MeOH, conc aq HCl (2-3 equiv) in Parr shaker at 35 °C, 2 h, then TEA (3 equiv), 35% yield over five steps; (viii) aldehyde or ketone, HOAc, TEA, 4 A˚ powdered molecular sieves, Na(OAc)3BH, 30-70% yields.
HCl and ethanol yielded the C3 hydroxyl intermediate, which was oxidized to the C3 ketone using DMSO, pyridinium triflouroacetate, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. After removal of the 20 -acetyl group by heating in methanol, the benzhydryl protecting group was removed by hydrogenolysis using Pearlman’s catalyst to give the azetidinyl template 5. From this template target analogues 6a-s were synthesized by reductive amination using standard conditions. Many of the coupling partners for the azetidinyl template 5 were either commercially available or synthesized using well precedented chemistries. The naphthyridine aromatic headpieces, however, had to be constructed from more basic building blocks as illustrated in Schemes 2 and 3. The [1,8]naphthyridines were initially accessed via the Skraup reaction11 on 7, although in poor yields as is typical for such systems. The Skraup product 8 was subsequently converted to 9 (used to make 6m, Figure 2) by way of selenium dioxide benzylic oxidation. Aldehyde 9 was further homologated to
Gram-positive potency criteria for advancement of a compound was MIC e 1 μg/mL against sensitive and resistant streptococcal strains, including the constitutive erm strain of S. pyogenes 1079 (superior to 1, MIC = 32 μg/mL). The fastidious Gram-negative H. influenzae, with its restriction on entry into and efflux out of the bacterium, has always been a challenge for large molecules such as macrolides.13 The H. influenzae 1325 strain (Table 1) was one of the least susceptible in our laboratories, and its modal MIC values were more or less representative of the MIC90 results obtained from larger populations of this pathogen (N g 30 strains). Thus, MIC e 4 μg/mL vs H. influenzae 1325 was the standard for further advancement of a compound, matching the Gramnegative profile of 1 for its least susceptible strain. Because 1 has generated serious safety concerns,8 particularly with respect to its hepatotoxicity profile,14 we focused on lowering the liabilities of our analogues by reducing its elevated hepatic turnover and the high degree of time-dependent inactivation (TDI) of cytochrome P450 liver enzymes (specifically 3A isoforms). In addition to mitigating the hepatic liabilities associated with 1, this strategy would have the added benefit of improving the pharmacokinetics through reduction of metabolic clearance and drug-drug interactions (DDI). Human liver microsomes were used to screen compounds for metabolic clearance.15 The measured human liver microsomal extraction ratio (ER) of 1 was 0.7, consistent with the
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Scheme 2. Synthesis of [1,8]Naphthyridinesa
Reagents and conditions: (i) m-nitrobenzene sodium sulfonate, conc H2SO4, glycerol, 120 °C, 8 h, then basify, 11% yield; (ii) SeO2, dioxane, water, 80 °C, 77% yield; (iii) MeMgBr, toluene, THF, 24% yield; (iv) IBX, EtOAc, 80 °C, 86% yield; (v) pivoyl chloride, TEA, DCM, 82% yield; (vi) Cl2(g), Na2HPO4, water, DCM; 86% yield; (vii) t-BuLi (2.1 equiv), Et2O, -65 to -15 °C, then add 3-dimethylaminopropenal, -15 °C to room temp, treat with concentrated HCl, 52% yield; (viii) NaOMe, MeOH, 67% yield; (ix) 6 N HCl, reflux, 98% yield. a
Scheme 3. Synthesis of [1,5]Naphthyridinesa
a Reagents and conditions: (i) NaOMe, DMF, 50 °C, 90% yield; (ii) BocNH2, Pd2(dba)3-CHCl3, xantphos, Cs2CO3, dioxane, 100 °C for 30 h, 84% yield; (iii) n-BuLi, THF, MeI, MTBE, 100% yield; (iv) 4 N HCl, MeOH, 84% yield; (v) sodium 3-nitrobenzenesulfonate, MeSO3H, FeSO4 3 7H2O, glycerol, 125-130 °C, 45% yield; (vi) NBS, dibenzoyl peroxide, CCl4, 95% yield; (vii) KOAc, DMF, MeOH, K2CO3, 63% yield; (viii) IBX, EtOAc; (ix) 6 N HCl, reflux, 90% yield combining steps viii and ix.
relatively high clearance observed in human.16 CYP3A TDI was assessed using human liver microsomes with midazolam as a probe substrate for CYP3A. Compounds were evaluated for time- and concentration-dependent inhibition of midazolam metabolism, and KI and kinact values were determined. These parameters were then used to predict clinical DDI.17 TDI measurements of 1 yielded KI = 2.4 μM and kinact = 0.04 min-1, resulting in a predicted 6-fold increase in area under the curve (AUC) for a concomitantly administered CYP3A substrate, consistent with clinical observations.18 In order to improve on the druglike properties of our analogues relative to 1, the advancement criteria included in vitro microsomal ER e 0.3 and TDI values resulting in predicted AUC increase of e3-fold for concomitantly administered CYP3A substrates. The cardiotoxicity liabilities (QTc) of 1 are not intrinsic to the compound but appear to be a function of its TDI and the effect this has on CYP3A object drugs.18 Thus, the QTc potential of new analogues was monitored in a hERG patch-clamp assay with the goal of not exceeding that of 1 (IC50 g 40 μM). Finally, the most promising compounds were evaluated in preclinical infection models using clinically relevant respiratory strains: S. pneumoniae 1095 (erm) in a murine respiratory tract infection (RTI) model and H. influenzae 1325 (least susceptible of all strains) in a gerbil
otitis media (OM) model to ensure that they demonstrated equivalent or superior oral efficacy to that of 1. With a clear strategy in mind we then set out by cyclizing the tether connecting the critical aryl-alkyl group to the macrocylic ring system.19 Additionally, a nitrogen was placed into the conformationally constrained tether to introduce polarity and to provide a convenient point of attachment for rapid SAR exploration and, significantly, modulation of the physicochemical properties of the final analogues. After considerable empirical evaluation of tether systems, the azetidine ring proved to be a rich vein for the program and is the focus of the present disclosure.20 The unsubstituted azetidinyl template 5 had excellent activity against the susceptible strain of S. pneumoniae (1016), moderate activity against the mef strain (1175), but very poor erm potency (S. pneumoniae 1095 and S. pyogenes 1079; Table 1), which is not unexpected given the well-established need for an aryl ring to capture erm strains from a macrolide template. Appendage of a simple benzyl substituent (6a) gave a dramatic improvement in the erm S. pneumoniae profile, which was not improved upon with the corresponding pyridylmethyl analogues (6b,c). Introduction of a bromine ortho to the methylene bridge of 6c, however, resulted in 6d, which began to show good streptococcal potency, including against erm S. pyogenes 1079.
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Figure 2. Aryl-alkyl attachments to intermediate 5 (Scheme 1) representing key test compounds 6a-s.
Encouragingly, this level of erm potency was clearly differentiated from that of 1. The metabolism of 6d, however, was high (ER > 0.8), and this compound was not pursued any further. Extension into bicyclic aromatics quickly pointed to the advantages of fused systems, which cover the rest of the analogues (6e-s) in Table 1. Similar to the o-bromine effect on the monocyclic ring of 6d, halogens (not shown) and in particular a methoxy substituent (identified by synthesis of a library array) on the bicyclic-fused rings ortho to the methylene bridge demonstrated a distinct potency advantage (6e vs 6f). Quinolines 6g and 6h showed a relative insensitivity to the placement of the nitrogen, although the attachment point to the macrolide made a significant difference, with the R ring junction being consistently preferred over β (6h vs 6i). To reduce the lipophilicity of the target compounds, more polar heterocycles were subsequently incorporated. The benzimidazole 6k was one such example; however, it lacked the requisite in vitro potency. The [1,5]naphthyridine analogue 6l appeared more promising except that both the ER (0.7) and TDI (5-fold) measurements indicated no differentiation from 1 (Table 2). The [1,8]naphthyridine analogue 6m also met the potency criteria and showed a distinct advantage over 6l in the TDI assay (2-fold) but still had a high ER (0.6). Combining the o-methoxy substituent of 6f with quinoline 6g and [1,8]naphthyridine 6m gave analogues 6n and 6o, respectively. While both 6n and 6o had extremely good Gram-positive potencies, their H. influenzae MICs were slightly elevated; furthermore, both had high ERs (6n=0.6; 6o=0.8). Considering the potential for metabolism at the benzylic methylene
bridge connecting the aryl ring to the azetidinyl nitrogen, methyl substitution was used at this position to block metabolism, yielding the diastereomers 6p and 6q. Not unexpectedly, the diastereomers displayed differential activities in the MIC panel, with 6q having a clear potency advantage. This strategy also resulted in lowering the ER of 6q relative to the parent 6m considerably (0.3 vs 0.6); additionally 6q maintained a reasonable TDI value (3-fold) as well as a hERG IC50 (43 μM) comparable to that of 1. Consequently 6q was profiled in the preclinical in vivo RTI and OM models. The oral efficacy results of 6q compared to 1 and negative control 3 are given in Table 3 and were sufficiently encouraging (in combination with its predicted human clearance and safety properties) to nominate 6q for clinical development. Single dose studies in humans, however, were disappointing, with plasma exposures upon oral dosing (300-1000 mg) achieving only approximately 20% of the predicted AUC. Subsequent investigation revealed extensive nonmicrosomally mediated metabolism of 6q catalyzed by aldehyde oxidase (AO),21 which halted development of the compound. Because AO is a cytosolic rather than microsomal enzyme, an in vitro assay with human liver cytosol was retroactively used to analyze the structure-metabolism relationships, in both the presence and absence of an AO inhibitor (raloxifene22), revealing a clear pattern of vulnerability of the [1,8]naphthyridine system to AO metabolism (Table 2). Thus, although the 3-hydroxy[1,8]naphthyridinyl 6r had very promising in vitro potency, it too was revealed to be a substrate for AO and was not progressed further as a consequence. Encouragingly, the 3-hydroxy substituent imparted very favorable ER
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Table 1. Minimum Inhibitory Concentrations (MICs, μg/mL) of Telithromycin (1), Clarithromycin (3), the Azetidinyl Ketolide Template (5), and the Azetidinyl Ketolide Analogues with Varying Aryl-Alkyl Substituents (6a-s) against Susceptible and Resistant Respiratory Pathogens compd
S. pneumoniae 1016 (susceptible)
S. pneumoniae 1095 (ermB)
S. pneumoniae 1175 (mefA)
S. pyogenes 1079 (ermB)
H. influenzae 1325
1 3 5 6a 6b 6c 6d 6e 6f 6g 6h 6i 6j 6k 6l 6m 6n 6o 6p 6q 6r 6s
e0.06 e0.06 e0.06 e0.06 e0.06 e0.06 e0.06 e0.06 e0.06 e0.06 e0.06 e0.06 e0.06 e0.06 e0.06 e0.06 e0.06 e0.06 e0.06 e0.06 e0.06 e0.06
e0.06 >64 64 e0.06 e0.06 0.12 e0.06 0.12 0.12 e0.06 e0.06 e0.06 e0.06 0.125 e0.06 e0.06 e0.06 e0.06 e0.06 e0.06 e0.06 e0.06
e0.06 16 2 0.12 2 0.5 e0.06 e0.06 e0.06 0.25 e0.06 0.25 e0.06 0.5 e0.06 0.12 e0.06 e0.06 0.25 0.25 e0.06 e0.06
32 >64 >64 >64 >64 >64 1 8 1 4 4 16 2 8 1 1 e0.06 e0.06 16 1 0.25 0.5
4 16 16 16 8 4 8 16 16 4 8 16 8 8 8 4 8 8 16 4 4 4
Table 2. Summary of Screening Data Used for Compound Selectiona
compd
hepatic ER
AO metabolism
CYP3A KI (μM)
CYP3A kinact (min-1)
1 6l 6m 6q 6r 6s
0.7 0.7 0.6 0.3 100 35.6d 53.6e
a Mean ( standard deviation (N > 2 studies) or average (N e 2 studies). b N = 5 studies. c N = 3 studies. d N = 1 study. e N = 2 studies.
(
