Article pubs.acs.org/jmc

Design, Synthesis, and Biological Evaluation of Hexacyclic Tetracyclines as Potent, Broad Spectrum Antibacterial Agents Cuixiang Sun,† Diana K. Hunt,† Chi-Li Chen,† Yonghong Deng,† Minsheng He,† Roger B. Clark,† Corey Fyfe,‡ Trudy H. Grossman,‡ Joyce A. Sutcliffe,‡ and Xiao-Yi Xiao*,† †

Discovery Chemistry, ‡Microbiology, Tetraphase Pharmaceuticals, 480 Arsenal Street, Watertown, Massachusetts 02472, United States S Supporting Information *

ABSTRACT: A series of novel hexacyclic tetracycline analogues (“hexacyclines”) was designed, synthesized, and evaluated for antibacterial activity against a wide range of clinically important bacteria isolates, including multidrug-resistant, Gram-negative pathogens. Valuable structure−activity relationships were identified, and several hexacyclines displayed potent, broad spectrum antibacterial activity, including promising anti-Pseudomonas aeruginosa activity in vitro and in vivo.

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INTRODUCTION New, potent antibacterial agents are in urgent need to combat life threatening infections caused by multidrug-resistant (MDR), Gram-negative and Gram-positive organisms such as carbapenem-resistant Enterobacteriaceae (CRE)1 and methicillin-resistant Staphylococcus aureus (MRSA).2 Tetracyclines are a proven class of broad spectrum antibiotics first discovered in the mid-1940s.3 While revolutionary at the time, seven decades of widespread use has largely rendered legacy tetracyclines ineffective due to progressing tetracycline resistance. 4a Renewed interest in the class, stemming from advances in chemical synthesis, has resulted in marked improvements against tetracycline-specific resistance, leading to the most recently approved tetracycline, tigecycline,5 in 2005, as well as newer generations of tetracycline antibiotics currently in late stage development such as eravacycline.6,7 Two major bacterial resistance mechanisms4b,c for tetracyclines have been identified: (1) tetracycline-specific efflux pumps, including tet(A)−tet(D) and tet(K)−tet(L), which are frequently found in both Gram-positive and Gram-negative pathogens, and (2) ribosomal protection, including tet(M)− tet(O), which are more commonly seen in Gram-positive bacteria. A number of semisynthetic tetracyclines, including doxycycline, minocycline, and tigecycline, have been developed to overcome bacterial resistance. However, the exploitation of the tetracycline scaffold has been impeded by the limited chemical methods available for semisynthetic tetracyclines until the recent development and application of the tetracycline total synthesis platform, which enables the creation and study of previously difficult-to-access, fully synthetic tetracycline analogues.6,8−10 Prior structure−activity relationship (SAR) studies5,6,8a,9 from our laboratory and other laboratories indicated that the tetracycline scaffold can tolerate structural modifications on © XXXX American Chemical Society

positions from C4 to C9, including various substitutions and additional fused ring systems. These chemical modifications often lead to modulations of tetracyclines’ chemical−physical properties, ribosomal binding, as well as the ability to overcome active drug efflux. We have therefore designed a new hexacyclic tetracycline scaffold (1, Figure 1) that consists of a bicyclic EF-

Figure 1. A hexacyclic tetracycline scaffold.

ring appended to the original tetracycline D-ring. A series of hexacyclic tetracycline analogues (“hexacyclines”) with structural variations at a number of positions was synthesized using our fully synthetic tetracycline strategy.6,8b,10 These hexacyclines were evaluated for antibacterial activity against a broad range of pathogens, especially activities against tetracycline resistant, Gram-negative bacteria.

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RESULTS AND DISCUSSION Chemistry. Using our tetracycline total synthesis approach,6,8b,10 a series of 7-fluorohexacyclines with a wide range of substitutions at the 8-position nitrogen (N8) was prepared from a tricyclic DEF-ring precursor 6 and an AB-ring enone 7.10,11 As shown in Scheme 1, aniline 29b was treated Received: February 13, 2015

A

DOI: 10.1021/acs.jmedchem.5b00262 J. Med. Chem. XXXX, XXX, XXX−XXX

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Scheme 1. Synthesis of 7-Fluorohexacycline Analoguesa

a

Reagents: (a) Br2, NaOAc, HOAc; (b) LHMDS, THF, then Boc2O, and then allyl bromide; (c) n-BuLi, THF, then DMF; (d) N-allyl glycine, DMF; (e) LDA, TMEDA, THF, then enone 7; (f) Pd(PPh3)4, N,N′-dimethylbarbituric acid, CH2Cl2; (g) (i) N-substitution (see Experimental Section), (ii) 48% aq HF, THF, (iii) 10% Pd-C, HCl, CH3OH.

and iodine to give compound 13, which was benzylated under standard conditions to afford intermediate 14. Compound 14 was then reacted at 80 °C with BocNH2 in the presence of Pd(OAc)2, Xantphos,14 and cesium carbonate to yield intermediate 15 in 28% yield over three steps. Aryl bromide 15 was treated with PhLi and n-BuLi followed by DMF to afford aldehyde 16 (37% yield), which was allylated with allyl bromide and NaH to give compound 17 in 82% yield. Cycloaddition12 of compound 17 with a range of cyclic and acyclic N-alkylated amino acids under the conditions described above gave the desired DEF-ring precursors 18 in good yields. Michael−Dieckmann annulation10 of 18 with enone 7, followed by desilylation and hydrogenation, gave the desired 7trifluoromethoxy hexacycline analogues 20a−20f in yields similar to those in Scheme 1 after reverse phase HPLC purification (Table 2). Biology. The novel hexacyclines were evaluated for in vitro antibacterial activity in minimal inhibitory concentration (MIC) assays against a panel of tetracycline-susceptible and tetracycline-resistant, Gram-positive and Gram-negative bacterial strains (Tables 1 and 2). All isolates in the panel were multidrug-resistant (MDR, i.e., resistant to ≥3 different classes of antibiotics) except S. aureus SA101, Escherichia coli EC107, and S. aureus SA158, a tet (K) tetracycline-resistant strain and otherwise drug susceptible. MIC data of representative hexacycline analogues are listed in Tables 1 and 2. As mentioned above, most of the final compounds were isolated as two separate cis-diastereomers. Although it is known that the “north-west” portion of tetracyclines is not involved in ribosomal binding, the two diastereomers displayed considerably different MIC values across the entire panel of bacterial strains, as exemplified by the diastereomer pair 10b and 10c in Table 1. For brevity, only the relatively more active diastereomers from each pair are listed in Tables 1 and 2, except for compounds 10a, 10h, and 20a, which were isolated as mixtures of the two diastereomers (∼1:1).

with bromine in the presence of sodium acetate to give the bromide intermediate 3 in 85% yield. Full protection of the aniline group by treatment with lithium bis(trimethylsilyl)amide (LHMDS) and Boc2O, followed by allyl bromide yielded compound 4 (88% yield). Compound 4 was formylated by treatment with n-BuLi at −100 °C in THF followed by DMF addition to give aldehyde 5 in 94% isolated yield. Cycloaddition12 of aldehyde 5 with N-allyl glycine in DMF at 80 °C afforded the desired tricyclic DEF-ring precursor 6 in 71% yield with a cis-configuration between the fused 5- and 6-membered heterocyclic rings (the corresponding trans isomer was not detected). Michael−Dieckmann annulation10 of 6 with the ABring enone 7 by treatment with lithium diisopropylamide (LDA) and N,N,N′,N′-tetramethylethylenediamine (TMEDA) at −78 °C in THF gave the fully protected intermediate 8 in 98% yield as a mixture of two diastereomers. The allyl group on N8 was removed by standard deallylation conditions to afford intermediate 9 (the two diastereomers were separated by preparative HPLC, 67% combined yield). The two diastereomers of secondary amine 9 were then separately alkylated by reductive alkylation with a variety of aldehydes to yield the N8 substituted intermediates, and these were deprotected by aqueous HF treatment followed by hydrogenation as previously described6 to afford the desired hexacycline analogues 10a− 10h in reasonable yields (20−60%) after reverse phase HPLC purification (Table 1). Similarly, several analogues with a methyl substituent at the bridgehead carbon C10a were also prepared using 3-bromo-2methylpropene instead of ally bromide in Scheme 1, step b (11a, 11b, and 11c, Table 1). Subsequently, to explore the effect of other C7 substitutions, a series of 7-trifluoromethoxy hexacycline analogues was prepared according to Scheme 2. In addition, a range of cyclic and acyclic amino acids was also incorporated in the cycloaddition step to explore structural variations on the Fring. Thus, phenol 1213 was iodinated by treatment with NaH B

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Table 1. In Vitro Antibacterial Activity of 7-Fluorohexacycline Analogues

a

Single diastereomer unless otherwise noted. bStrains were obtained from the American Type Culture Collection (ATCC, Manassas, VA) unless otherwise noted. The first six strains from the left are Gram-positive strains. The last nine strains are Gram-negative strains. Strains with “tet(A)”, “tet(B)”, “tet(K)”, or “tet(M)” genes noted underneath are tetracycline-resistant strains. SA, Staphylococcus aureus; EFs, Enterococcus faecalis; EFm, Enterococcus faecium; SP, Streptococcus pneumoniae; EC, Escherichia coli; KP, Klebsiella pneumoniae (KP457 contains a blacTX‑M‑15 extended spectrum β-lactamase gene); PM, Proteus mirabilis; PA, Pseudomonas aeruginosa; ECl, Enterobacter cloacae; AB, Acinetobacter baumannii; SM, Stenotrophomonas maltophilia; BC, Burkholderia cenocepacia. cObtained from Micromyx (Kalamazoo, MI). dObtained from Marilyn Roberts’ laboratory at the University of Washington. eObtained from Eurofins-Medinet, Chantilly, VA. fObtained from R. K. Ernst’s Laboratories at University of Maryland. gA mixture of two diastereomers (∼1:1).

When the pyrrolidine nitrogen (N8) was not substituted, the hexacycline analogues had only modest antibacterial activity, as shown by MIC data of compounds 10a, 11a, and 20a. However, when the nitrogen was substituted with a methyl group, the potency was dramatically improved by 2- to >32-fold against most strains in the panel except PM385 (10c vs 10a, 20b vs 20a). Further increases in lipophilicity of the nitrogen substituent tended to decrease potency, especially against Gram-negative strains, by varying degrees depending on the

amount of increase in lipophilicity (10d and 11c). The presence of additional polar groups, such as hydroxyl, amino, and amide groups, on the nitrogen substituent drastically decreased the compounds’ antibacterial activities (10e−10h). Interestingly, the potency of the difluoroethylaminoethyl substituted analogue 10g was decreased by a much lesser degree, especially against Gram-positive pathogens, probably due to the decreased basicity and/or polarity of the difluoroethylamino group. Alkyl substitution at the C10a C

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Scheme 2. Synthesis of 7-Trifluoromethoxyhexacycline Analoguesa

a

Reagents: (a) NaH, toluene, then I2; (b) BnBr, K2CO3, DMF; (c) BocNH2, Pd(OAc)2, Xantphos, Cs2CO3, 1,4-dioxane; (d) PhLi, n-BuLi, THF, then DMF; (e) NaH, DMF, then allyl bromide; (f) N-alkylated amino acid, DMF; (g) LDA, TMEDA, THF, then enone 7; (h) (i) 48% aq HF, THF, (ii) 10% Pd-C, HCl, CH3OH.

Table 2. In Vitro Antibacterial Activity of 7-Trifluoromethoxyhexacycline Analogues

a

Single diastereomers unless otherwise noted. bSee footnotes b-f for Table 1. cA mixture of two diastereomers (∼1:1).

(20f) or lipophilicity (either by alkyl substitution on the Fring (20c) or by incorporation of a seventh ring (G-ring, 20d, 20e)) decreased potency. Interestingly, compared to the 7fluoro analogue 10c, the corresponding 7-trifluoromethoxy analogue 20b was more potent against most Gram-positive strains (≥4-fold) as well as some of the Gram-negative strains (Escherichia coli strains EC107 and EC155, and Stenotropho-

position decreased potency against most strains in the panel, as shown by the 10a-methyl substituted analogues 11a, 11b, and 11c. Similar general SAR trends were also observed for the 7trifluoromethoxy series (Table 2). Thus, a methyl group on the pyrrolidine nitrogen (N8) was the optimum substitution for antibacterial activity (compound 20b). Additional polarity D

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Figure 2. In vivo efficacy of compound 10c in (A), mouse lung infection model challenged with P. aeruginosa PA1145; (B) mouse thigh infection model challenged with P. aeruginosa PA694.

mouse infection models challenged with P. aeruginosa demonstrated that compound 10c was equally or more efficacious than various comparator compounds at comparable dose levels. These data support the further optimization of the hexacycline scaffold for the discovery and development of new tetracycline antibiotics against a broad range of pathogens, including MDR Gram-negative bacteria.

monas maltophilia SM256) but 2−4-fold less active against Proteus mirabilis PM385, Pseudomonas aeruginosa PA555, and Acinetobacter baumannii AB250. Overall, compounds 10c and 20b displayed high in vitro potency against a broad range of MDR Gram-positive and Gram-negative pathogens. Particularly, compound 10c also demonstrated promising in vitro activity against P. aeruginosa PA555 with an MIC of 4 μg/mL, which is among the lowest MIC values in the tetracycline class. Ribosomal inhibition was confirmed by an in vitro P. aeruginosa coupled transcription/ translation assay,15 in which compound 10c was 10-fold more potent than tetracycline with an IC50 value of 0.21 μM. Compound 10c was further tested for in vivo efficacy in two mouse infection models challenged with P. aeruginosa. In a mouse lung infection model challenged with P. aeruginosa PA1145 (Figure 2A), compound 10c (MIC = 4 μg/mL) demonstrated a 4-log colony-forming unit (CFU) reduction in bacterial burden when dosed intravenously (IV) at 40 mg/kg, twice daily. Comparator compounds meropenem (MIC = 4 μg/mL) and amikacin (MIC = 1 μg/mL) showed bacterial burden reductions of about 2−2.5 log CFU when administered at the same doses. In the mouse thigh infection model challenged with P. aeruginosa PA694 (Figure 2B), compound 10c (MIC = 8 μg/mL) showed promising dose-proportional efficacy when dosed at 1.5, 5, 15, and 40 mg/kg, IV, twice daily and was equally efficacious at 40 mg/kg as comparator levofloxacin (MIC = 1 μg/mL) dosed IV at 5 mg/kg twice daily (1.8 log CFU reduction from T = 0 and 4.5 log CFU reduction from T = 24 h).

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EXPERIMENTAL SECTION

Chemistry. All commercially available reagents and solvents, including anhydrous solvents, were used without further purification. All reactions under dry conditions were performed under nitrogen atmospheres. 1H and 13C NMR (nuclear magnetic resonance) spectra were recorded at room temperature (25 °C) on a 400 MHz JEOL ECX-400 spectrometer. CDCl3 (for intermediates) or CD3OD (for final compounds) were used as the solvents. Chemical shifts (δ) are expressed in parts per million (ppm). The signals of the deuterated solvents were used as the internal standards. Thin-layer chromatography (TLC) analysis was performed on Merck Silica Gel 60 F254 and visualized under UV light. Flash chromatography was performed on a Biotage Isolera One using SNAP cartridges (KP-Sil). Purity of tested compounds was determined to be ≥95% by reverse phase analytical HPLC/MS analysis (high-performance liquid chromatography/mass spectrometry) performed on a Waters Alliance system (column, SunFire C18, 5 μm, 4.6 mm × 50 mm; solvent A, water with 0.1% formic acid; solvent B, acetonitrile with 0.1% formic acid; MS detector, Waters 3100) unless otherwise noted. Reverse phase preparative HPLC was performed on a Waters Autopurification system with massdirected fraction collection (for final compounds: column, Polymerx RP-1 100A, 10 μm, 150 mm × 21.20 mm; flow rate, 20 mL/min; solvent A, water with 0.05 N HCl; solvent B, acetonitrile; for intermediates: column, SunFire Prep C18 OBD, 5 μm, 19 mm × 50 mm; flow rate, 20 mL/min; solvent A, water with 0.1% formic acid; solvent B, acetonitrile with 0.1% formic acid) unless otherwise noted. Phenyl 3-Amino-2-(benzyloxy)-4-bromo-5-fluoro-6-methylbenzoate (3). To a solution of phenyl 3-amino-2-(benzyloxy)-5fluoro-6-methylbenzoate (2,9b 15.00 g, 42.69 mmol) and NaOAc (10.50 g, 128.07 mmol, 3 equiv) in HOAc (100 mL) was added a solution of Br2 (2.20 mL, 42.69 mmol, 1 equiv) in HOAc (10 mL) dropwise via a syringe at 17 → 19 °C while cooled in a cold water bath. After stirring at 20 °C for 20 min, more Br2 (66 μL) in HOAc (1 mL) was added. After stirring for 5 min, the reaction was poured into ice/water. The resulting mixture was extracted with EtOAc (600 mL). The organic phase was separated, washed with 10% aqueous Na2S2O3 solution, water, saturated aqueous sodium bicarbonate, and brine. The organic phase was dried over sodium sulfate, filtered, and concentrated

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CONCLUSIONS A series of hexacycline analogues based on scaffold 1 was designed and synthesized with a range of substitutions at C7, N8, C9, and C10a, and their antibacterial activity was evaluated against a broad range of Gram-negative and Gram-positive pathogens, including multidrug-resistant and tetracyclineresistant bacterial strains. Analogues 10c, a 7-fluorohexacycline analogue, and 20b, a 7-trifluoromethoxyhexacycline analogue, demonstrated potent, broad spectrum in vitro antibacterial activity. Compound 10c also displayed promising anti-P. aeruginosa potency with MIC values ranging from 4 to 8 μg/ mL in this study. In vivo efficacy studies in thigh and lung E

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under reduced pressure. Flash chromatography on silica gel using 5% → 6% EtOAc/hexanes yielded the desired product 3 as a thick paleyellow oil (15.59 g, 85%). 1H NMR (400 MHz, CDCl3) δ 7.44−7.35 (m, 7 H), 7.28−7.25 (m, 1 H), 7.15−7.13 (m, 2 H), 5.01 (s, 2 H), 4.27 (br s, 2 H), 2.32 (d, J = 2.4 Hz, 3 H). MS (ESI) m/z calcd for C21H18BrFNO3 [M + H]+ 430.05, 432.05; found 429.94, 431.92. Phenyl 2-(Benzyloxy)-4-bromo-3-{[(tert-butoxy)carbonyl](prop-2-en-1-yl)amino}-5-fluoro-6-methylbenzoate (4). To a solution of aniline 3 (908 mg, 2.11 mmol) in anhydrous THF (8 mL) was added a solution of LHMDS in THF (4.43 mL, 1.0 M, 4.43 mmol, 2.1 equiv) dropwise over 7 min while maintaining the internal temperature below −70 °C (dry ice−acetone bath). The reaction solution was stirred at −78 °C for 15 min. A solution of Boc2O (484 mg, 2.22 mmol, 1.05 equiv) in THF (1 mL) was added dropwise while maintaining the internal temperature below −71 °C. The reaction was stirred at −78 °C for 30 min, and then the dry ice was removed from the cold bath. The reaction was then warmed up to −50 °C, and allyl bromide (0.20 mL, 2.32 mmol, 1.1 equiv) was added. The reaction was warmed up to rt in 20 min and heated at 50 °C for 3 h. More allyl bromide (0.20 mL, 2.32 mmol, 1.1 equiv) was added. The reaction was heated at 50 °C for 2 h, cooled to rt, and diluted with EtOAc (40 mL). The reaction solution was washed with saturated aqueous NH4Cl (2 × 30 mL) and brine (30 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. Flash chromatography on silica gel using 2% → 5% EtOAc/hexanes yielded the desired product 4 (1.06 g, 88%, ∼3:1 rotamers). 1H NMR (400 MHz, CDCl3) δ 7.39− 7.34 (m, 7 H), 7.29−7.25 (m, 1 H), 7.04−7.00 (m, 2 H), 6.00−5.90 (m, 1 H), 5.09−5.04 (m, 1 H), 5.03−5.00 (m, 2.25 H), 4.92 (d, J = 10.4 Hz, 0.75 H), 4.50 (dd, J = 6.1, 14.6 Hz, 0.75 H), 4.24 (dd, J = 6.1, 15.3 Hz, 0.25 H), 4.04−3.97 (m, 1 H), 2.42 (d, J = 2.4 Hz, 2.25 H), 2.40 (d, J = 2.4 Hz, 0.75 H), 1.54 (s, 2.25 H), 1.44 (s, 6.75 H). MS (ESI) m/z calcd for C29H29BrFNO5Na [M + Na]+ 592.11, 594.11; found 591.99, 593.98. Phenyl 2-(Benzyloxy)-3-{[(tert-butoxy)carbonyl](prop-2-en1-yl)amino}-5-fluoro-4-formyl-6-methylbenzoate (5). To a solution of bromide 4 (1.06 g, 1.86 mmol) in anhydrous THF (30 mL) was added a solution of n-BuLi in hexanes (1.16 mL, 1.6 M, 1.86 mmol, 1 equiv) dropwise while maintaining the internal temperature below −100 °C (liquid nitrogen−ethanol bath). After stirring for 3 min, a solution of DMF (0.22 mL, 2.79 mmol, 1.5 equiv) in THF (1 mL) was added dropwise while maintaining the internal temperature below −100 °C. The reaction solution was then allowed to warm up to −78 °C (dry ice−acetone bath) and stirred at that temperature for 35 min. Saturated aqueous NH4Cl was added. The resulting mixture was allowed to warm up to rt and extracted with EtOAc (40 mL). The organic phase was washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. Flash chromatography on silica gel using 3% → 12% EtOAc/hexanes yielded the desired product 5 (0.91 g, 94%, ∼2:1 rotamers). 1H NMR (400 MHz, CDCl3) δ 10.22 (s, 1 H), 7.38−7.33 (m, 7 H), 7.28−7.24 (m, 1 H), 7.02−6.99 (m, 2 H), 5.93−5.79 (m, 1 H), 5.04−4.96 (m, 3.35 H), 4.89 (d, J = 9.8 Hz, 0.65 H), 4.64 (dd, J = 5.5, 14.6 Hz, 0.65 H), 4.32 (dd, J = 5.5, 14.6 Hz, 0.35 H), 3.97 (dd, J = 7.9, 14.6 Hz, 0.35 H), 3.90 (dd, J = 8.5, 14.6 Hz, 0.65 H), 2.40 (d, J = 1.8 Hz, 2 H), 2.37 (d, J = 1.8 Hz, 1 H), 1.51 (s, 3 H), 1.36 (s, 6 H). MS (ESI) m/z calcd for C30H30FNO6Na [M + Na]+ 542.20; found 542.1. 5-tert-Butyl 7-Phenyl 6-(Benzyloxy)-9-fluoro-8-methyl-1(prop-2-en-1-yl)-1H,2H,3H,3aH,4H,5H,9bH-pyrrolo[3,2-c]quinoline-5,7-dicarboxylate (6). A solution of aldehyde 5 (570 mg, 1.09 mmol) and N-allylglycine (338 mg, 2.23 mmol, 2 equiv) in DMF (11 mL) was heated at 90 °C for 17 h. The reaction mixture was cooled to rt, poured into water (125 mL), and extracted with EtOAc (100 mL, 2 × 50 mL). The combined organic layers were washed with water and brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The resulting oil was purified via column chromatography (Biotage 100 g column, 4−40% EtOAc in hexanes gradient), yielding 444 mg (71%) of the desired product 6 as a clear oil (cis). 1H NMR (400 MHz, CDCl3) δ 7.40−7.21 (m, 8 H), 7.08−7.01 (m, 2 H), 5.79−5.64 (m, 1 H), 5.16−5.08 (m, 1 H), 5.01− 4.92 (m, 2 H), 4.85−4.75 (m, 1 H), 4.30−4.20 (m, 1 H), 3.80−3.77

(m, 1 H), 3.09−2.99 (m, 1 H), 2.85−2.54 (m, 2 H), 2.34 (s, 3 H), 2.20−2.09 (m, 2 H), 2.06−1.94 (m, 1 H), 2.45−2.22 (m, 10 H). MS (ESI) m/z calcd for C34H36FN2O5 [M − H]− 571.26; found 571.14. tert-Butyl (1R,19S,26S,27S)-14,22-Bis(benzyloxy)-19-[(tertbutyldimethylsilyl)oxy]-26-(dimethylamino)-4-fluoro-18-hydroxy-16,20-dioxo-7-(prop-2-en-1-yl)-24-oxa-7,12,23triazaheptacyclo[15.11.0.03,15.05,13.06,10.019,27.021,25]octacosa3,5(13),14,17,21(25),22-hexaene-12-carboxylate (8). LDA (1.3 equiv) was prepared at −40 °C from n-butyllithium (1.6 M solution in hexanes, 845 μL, 1.35 mmol) and diisopropylamine (216 μL, 1.50 mmol) in THF (10 mL). The LDA solution was cooled to −78 °C (dry ice−acetone bath) and TMEDA (217 μL, 1.45 mmol, 1.4 equiv) was added, followed by dropwise addition of a solution of compound 6 (655 mg, 1.14 mmol, 1.1 equiv) in THF (4.5 mL) with a 1.5 mL rinse. This resulted in a deep-red solution. After 30 min, the reaction was cooled to −100 °C (liquid nitrogen−ethanol bath), and a solution of enone 7 (503 mg, 1.04 mmol) in THF (3.5 mL) was added dropwise, maintaining the internal temperature below −95 °C. After complete addition, the reaction was allowed to warm to −70 °C over 40 min, and a solution of LHMDS (1 M in THF, 1.04 mL, 1.04 mmol, 1 equiv) was added. The reaction was allowed to warm to −20 °C over 1 h and quenched by the addition of NH4Cl (saturated aqueous solution, 30 mL). The reaction mixture was extracted with EtOAc (2 × 100 mL). The combined extracts were dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting foam was purified via column chromatography (Biotage 100 g column, 4−60% EtOAc in hexanes), yielding 983 mg (98%, mixture of diastereomers) of the desired product 8 as a yellow solid. MS (ESI) m/z calcd for C54H64FN4O9 [M − H]− 959.44; found 959.28. tert-Butyl (1R,19S,26S,27S)-14,22-Bis(benzyloxy)-19-[(tertbutyldimethylsilyl)oxy]-26-(dimethylamino)-4-fluoro-18-hydroxy-16,20-dioxo-24-oxa-7,12,23-triazaheptacyclo[15.11.0.03,15.05,1306,10.019,27.021,25]octacosa-3,5(13),14,17,21(25),22-hexaene-12-carboxylate (9a and 9b). A solution of compound 8 (945 mg, 0.983 mmol), N,N′-dimethylbarbituric acid (1.53 g, 9.80 mmol, 10 equiv), and tetrakis(triphenylphosphine)palladium(0) (77.5 mg, 0.067 mmol, 0.068 equiv) in CH2Cl2 (10 mL) was degassed by bubbling nitrogen for 5 min. The reaction flask was fitted with a nitrogen-flushed water-cooled reflux condenser, and the reaction was heated to 35 °C in an oil bath. After 4.5 h, the reaction was cooled to rt and saturated aqueous NaHCO3 (20 mL) was added slowly over 5 min, followed by the addition of pH 7 phosphate buffer (30 mL). The aqueous layer was extracted with CH2Cl2 (2 × 75 mL), and the combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude material was purified on a Waters Autopurification system equipped with a Sunfire Prep C18 OBD column [5 μm, 19 mm × 50 mm; flow rate, 20 mL/min; solvent A, H2O with 0.1% HCO2H; solvent B, CH3CN with 0.1% HCO2H; gradient, 30 → 80% B; mass-directed fraction collection] to provide the two diastereomers as yellow solids (9a, 291 mg; 9b, 284 mg; 9a + 9b, 31 mg; 67% total yield). 9a: 1H NMR (400 MHz, CDCl3) δ 16.11−15.68 (br m, 1 H), 7.52−7.45 (m, 2 H), 7.41−7.25 (m, 8 H), 5.36 (s, 2 H), 4.90−4.65 (br m, 1 H), 4.58−4.38 (br m, 1 H), 4.28−4.04 (br m, 2 H), 3.94 (d, J = 10.38 Hz, 1 H), 3.25−3.14 (m, 1 H), 3.09−2.88 (m, 3 H), 2.62−2.29 (m, 10 H), 2.19−2.03 (m, 2 H), 1.65−1.43 (br m, 1 H), 1.42−1.35 (m, 10 H), 0.82 (s, 9 H), 0.28 (s, 3 H), 0.12 (s, 3 H); MS (ESI) m/z calcd for C51H62FN4O9Si [M + H]+ 921.43, found 921.48. 9b: 1H NMR (400 MHz, CDCl3) δ 16.30− 15.79 (br m, 1 H), 7.52−7.44 (m, 2 H), 7.41−7.24 (m, 8 H), 5.36 (s, 2 H), 5.06−4.51 (br m, 2 H), 4.38−4.06 (br m, 2 H), 3.96 (d, J = 10.38 Hz, 1 H), 3.26−3.14 (m, 1 H), 3.12−2.86 (m, 3 H), 2.60−2.32 (m, 10 H), 2.18−2.02 (m, 2 H), 1.62−1.50 (br m, 1 H), 1.50−1.02 (br m, 10 H), 0.81 (s, 9 H), 0.27 (s, 3 H), 0.12 (s, 3 H); MS (ESI) m/z calcd for C51H62FN4O9Si [M + H]+ 921.43, found 921.47. (5S,9S,10S,12R)-9-(Dimethylamino)-15-fluoro-4,5,8,25-tetrahydroxy-18-(2-hydroxyethyl)-2,6-dioxo-18,23-diazahexacyclo[12.11.0.03,12.05,10.016,24,.017,21]pentacosa-1(25),3,7,14,16(24)pentaene-7-carboxamide (10e). To a solution of 9a (12.5 mg, 0.0135 mmol) in 1,2-dichloroethane (300 μL) was added (tertbutyldimethylsilyloxy)acetaldehyde (90%, 14 μL, 0.068 mmol, 5 equiv), acetic acid (3.9 μL, 0.068 mmol, 5 equiv), and sodium F

DOI: 10.1021/acs.jmedchem.5b00262 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Hz, 1 H), 4.98−4.92 (m, 1 H), 4.11−3.92 (m, 2 H), 3.91−3.79 (m, 1 H), 3.80−3.53 (m, 5 H), 3.51−3.39 (m, 2 H), 3.27−2.85 (m, 10 H), 2.83−2.67 (m, 1 H), 2.63−2.49 (m, 1 H), 2.26−2.04 (m, 3 H), 1.68− 1.53 (m, 1 H). MS (ESI) m/z calcd for C30H37F3N5O7 [M + H]+ 636.27; found 636.19. (5S,9S,10S,12R)-9-(Dimethylamino)-15-fluoro-4,5,8,25-tetrahydroxy-18-[2-(N-methylacetamido)ethyl]-2,6-dioxo-18,23diazahexacyclo[12.11.0.0 3,12 .0 5,10 .0 16,24 .0 17,21 ]pentacosa-1(25),3,7,14,16(24)-pentaene-7-carboxamide (10h). 1H NMR (400 MHz, CD3OD, HCl salt) δ 4.07 (s, 1 H), 3.92−3.69 (m, 3 H), 3.68−3.62 (m, 1 H), 3.59−3.39 (m, 4 H), 3.14−2.91 (m, 13 H), 2.88−2.61 (m, 1 H), 2.56−2.43 (m, 1 H), 2.29−2.02 (m, 6 H), 1.67− 1.55 (m, 1 H). MS (ESI) m/z calcd for C31H39FN5O8 [M + H]+ 628.27; found 628.11. (5S,9S,10S,12R)-9-(Dimethylamino)-15-fluoro-4,5,8,25-tetrahydroxy-21-methyl-2,6-dioxo-18,23-diazahexacyclo[12.11.0.03,12.05,10.016,24.017,21]pentacosa-1(25),3,7,14,16(24)pentaene-7-carboxamide (11a). 1H NMR (400 MHz, CD3OD, HCl salt) δ 4.31 (s, 1 H), 4.08 (s, 1 H), 3.47−3.40 (m, 2 H), 3.18− 3.11 (m, 2 H), 3.08−2.95 (m, 9 H), 2.23−2.13 (m, 4 H), 1.65−1.55 (m, 1 H), 1.15 (s, 3 H). MS (ESI) m/z calcd for C27H32FN4O7 [M + H]+ 543.23; found 543.14. (5S,9S,10S,12R)-9-(Dimethylamino)-15-fluoro-4,5,8,25-tetrahydroxy-18,21-dimethyl-2,6-dioxo-18,23-diazahexacyclo[12.11.0.03,12.05,10.016,24.017,21]pentacosa-1(25),3,7,14,16(24)pentaene-7-carboxamide (11b). 1H NMR (400 MHz, CD3OD, HCl salt) δ 4.36 (s, 1 H), 4.08 (s, 1 H), 3.82−3.75 (m, 1 H), 3.41− 3.33 (m, 1 H), 3.22−3.11 (m, 3 H), 3.08−2.96 (m, 11 H), 2.34−2.18 (m, 3 H), 2.10−2.04 (m, 1 H), 1.65−1.56 (m, 1 H), 1.09 (s, 3 H). MS (ESI) m/z calcd for C28H34FN4O7 [M + H]+ 557.24; found 557.13. (5S,9S,10S,12R)-9-(Dimethylamino)-15-fluoro-4,5,8,25-tetrahydroxy-21-methyl-2,6-dioxo-18-propyl-18,23diazahexacyclo[12.11.0.0 3,12 .0 5,10 .0 16,24 .0 17,21 ]pentacosa-1(25),3,7,14,16(24)-pentaene-7-carboxamide (11c). 1H NMR (400 MHz, CD3OD, HCl salt) δ 4.40 (s, 1 H), 4.10 (s, 1 H), 3.75−3.68 (m, 1 H), 3.49−3.42 (m, 1 H), 3.38−3.31 (m, 1 H), 3.24− 3.15 (m, 3 H), 3.12−3.07 (m, 1 H), 3.04−2.96 (m, 8 H), 2.30−2.18 (m, 3 H), 2.13−2.07 (m, 1 H), 1.83−1.75 (m, 1 H), 1.72−1.56 (m, 2 H), 1.08 (s, 3 H), 0.98 (t, J = 7.3 Hz, 3 H). MS (ESI) m/z calcd for C30H38FN4O7 [M + H]+ 585.27; found 585.26. Phenyl 4-Bromo-2-hydroxy-3-iodo-6-methyl-5(trifluoromethoxy)benzoate (13). To phenol 1213 (5.20 mmol, obtained from treatment of 2.50 g of the corresponding benzyl ether with TFA/anisole, containing inseparable impurities, ∼75% pure) in toluene (20 mL) at rt was added NaH (0.83 g, 20.80 mmol, 60% in mineral oil, 4 equiv) in small portions. The reaction mixture was stirred at rt for 20 min. Iodine (5.28 g, 20.80 mmol, 4 equiv) was added. The reaction mixture was stirred at rt overnight, diluted with EtOAc (200 mL), washed with 1 N aqueous HCl (100 mL × 1), 5% aqueous Na2S2O3 (100 mL × 2), and brine (100 mL × 1), dried over sodium sulfate, and concentrated under reduced pressure to yield the crude product 13 as a pale oil. MS (ESI) m/z calcd for C15H8BrF3IO4 [M − H]− 514.86; found 514.81. Phenyl 2-(Benzyloxy)-4-bromo-3-iodo-6-methyl-5(trifluoromethoxy)benzoate (14). To the above crude phenol 13 (5.20 mmol) in DMF (10 mL) at rt was added potassium carbonate (1.44 g, 10.44 mmol, 2 equiv) and benzyl bromide (0.74 mL, 6.23 mmol, 1.2 equiv). The reaction mixture was stirred at rt for 3 h, diluted with EtOAc (200 mL), washed with water (200 mL × 1, 100 mL × 1) and brine (50 mL × 1), dried over sodium sulfate, and concentrated under reduced pressure. Flash column chromatography on silica gel with 0−3% EtOAc/hexane yielded the desired product 14 as a pale oil (3.48 g, ∼90% pure). 1H NMR (400 MHz, CDCl3) δ 7.55−7.00 (m, 10 H), 5.11 (s, 2 H), 2.44 (s, 3 H). MS (ESI) m/z calcd for C22H14BrF3IO4 [M − H]− 604.91; found 604.83. Phenyl 2-(Benzyloxy)-4-bromo-3-{[(tert-butoxy)carbonyl]amino}-6-methyl-5-(trifluoromethoxy)benzoate (15). To compound 14 (5.20 mmol, 90% pure) was added cesium carbonate (2.54 g, 7.80 mmol, 1.5 equiv), BocNH2 (0.67 g, 5.70 mmol, 1.1 equiv), Xantphos (1.20 g, 2.07 mmol, 0.4 equiv), Pd(OAc)2 (224 mg, 1.00 mmol, 0.2 equiv), and anhydrous 1,4-dioxane (10 mL). Nitrogen gas

triacetoxyborohydride (13 mg, 0.61 mmol, 5 equiv). After stirring at rt for 18 h, the reaction was quenched with saturated aqueous NaHCO3 (10 mL). The reaction mixture was extracted with EtOAc (2 × 20 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure to provide the N8 alkylated intermediate. MS (ESI) m/z calcd for C59H80FN4O10Si [M + H]+ 1079.54; found 1079.23. Final deprotections using previously described desilylation and hydrogenation procedures6 provided the desired compound 10e as a yellow solid after reverse phase HPLC purification (2.8 mg, HCl salt, 27% over three steps). 1H NMR (400 MHz, CD3OD) δ 4.98−4.95 (m, 1 H), 4.07 (s, 1 H), 3.93−3.84 (m, 1 H), 3.80−3.36 (m, 6 H), 3.20−2.64 (m, 11 H), 2.54−2.40 (m, 1 H), 2.30−2.08 (m, 3 H), 1.68−1.55 (m, 1 H). MS (ESI) m/z calcd for C28H34FN4O8 [M + H]+ 573.24; found 573.12. (5S,9S,10S,12R)-9-(Dimethylamino)-18-ethyl-15-fluoro4,5,8,25-tetrahydroxy-2,6-dioxo-18,23-diazahexacyclo[12.11.0.03,12.05,10.016,24.017,21]pentacosa-1(25),3,7,14,16(24)pentaene-7-carboxamide (10d). By similar procedures, compound 10d was prepared from 9a using acetaldehyde in the reductive alkylation step. 1H NMR (400 MHz, CD3OD, HCl salt) δ 4.85 (d, J = 6.4 Hz, 1 H), 4.11 (s, 1 H), 3.72−3.66 (m, 1 H), 3.63−3.58 (m, 1 H), 3.46 (dd, J = 5.0, 12.8 Hz, 1 H), 3.42−3.33 (m, 2 H), 3.19−2.97 (m, 10 H), 2.80 (br s, 1 H), 2.54−2.45 (m, 1 H), 2.25−2.03 (m, 3 H), 1.64−1.54 (m, 1 H), 1.38 (t, J = 6.9 Hz, 3 H). MS (ESI) m/z calcd for C28H34FN4O7 [M + H]+ 557.24; found 557.15. (5S,9S,10S,12R)-9-(Dimethylamino)-15-fluoro-4,5,8,25-tetrahydroxy-2,6-dioxo-18,23-diazahexacyclo[12.11.0.03,12.05,10.016,24.017,21]pentacosa-1(25),3,7,14,16(24)pentaene-7-carboxamide (10a). Compound 9a was deprotected using previously described procedures6 to provide compound 10a. 1H NMR (400 MHz, CD3OD, HCl salt) δ 4.80 (d, J = 6.4 Hz, 1 H), 4.10 (s, 1 H), 3.49−3.40 (m, 3 H), 3.12−2.96 (m, 9 H), 2.89 (t, J = 11.9 Hz, 1 H), 2.79−2.71 (m, 1 H), 2.49−2.39 (m, 1 H), 2.24−2.16 (m, 2 H), 2.12−2.02 (m, 1 H), 1.65−1.56 (m, 1 H). MS (ESI) m/z calcd for C26H30FN4O7 [M + H]+ 529.21; found 529.07. The following analogues (10b, 10c, 10f−10h, and 11a−11c) were prepared using similar procedures. See details in Supporting Information. (5S,9S,10S,12R)-9-(Dimethylamino)-15-fluoro-4,5,8,25-tetrahydroxy-18-methyl-2,6-dioxo-18,23-diazahexacyclo[12.11.0.03,12.05,10.016,24.017,21]pentacosa-1(25),3,7,14,16(24)pentaene-7-carboxamide. Diastereomer A, 10b: 1H NMR (400 MHz, CD3OD, HCl salt) δ 4.77 (d, J = 6.4 Hz, 1 H), 4.12 (s, 1 H), 3.79−3.74 (m, 1 H), 3.45 (dd, J = 5.5, 12.8 Hz, 1 H), 3.37−3.31 (m, 1 H), 3.14−2.97 (m, 13 H), 2.76−2.75 (m, 1 H), 2.58−2.49 (m, 1 H), 2.50−2.18 (m, 2 H), 2.07−2.00 (m, 1 H), 1.65−1.55 (m, 1 H); MS (ESI) m/z calcd for C27H32FN4O7 [M + H]+ 543.23, found 543.07. Diastereomer B, 10c: 1H NMR (400 MHz, CD3OD, HCl salt) δ 4.77 (d, J = 6.0 Hz, 1 H), 4.10 (s, 1 H), 3.79−3.73 (m, 1 H), 3.47 (dd, J = 5.5, 12.8 Hz, 1 H), 3.37−3.32 (m, 1 H), 3.12−2.97 (m, 13 H), 2.78 (br s, 1 H), 2.56−2.51 (m, 1 H), 2.23 (br d, J = 10.1 Hz, 1 H), 2.14 (t, J = 14.2 Hz, 1 H), 2.05−1.98 (m, 1 H), 1.63−1.54 (m, 1 H); 13C NMR (400 MHz, CD3OD, HCl salt) δ 194.96, 187.82, 174.60, 174.26, 153.09, 150.77, 147.47, 137.16, 115.57, 112.40, 109.68, 105.77, 96.12, 75.47, 70.75, 63.21, 54.89, 43.07, 42.03, 41.81, 40.97, 35.91, 35.27, 35.12, 33.88, 31.12, 27.20; MS (ESI) m/z calcd for C27H32FN4O7 [M + H]+ 543.23, found 543.07. (5S,9S,10S,12R)-9-(Dimethylamino)-18-[2-(dimethylamino)ethyl]-15-fluoro-4,5,8,25-tetrahydroxy-2,6-dioxo-18,23diazahexacyclo[12.11.0.0 3,12 .0 5,10 .0 16,24 .0 17,21 ]pentacosa-1(25),3,7,14,16(24)-pentaene-7-carboxamide (10f). 1H NMR (400 MHz, CD3OD, HCl salt) δ 5.00−4.94 (m, 1 H), 4.12−3.97 (m, 2 H), 3.90−3.75 (m, 2 H), 3.74−3.58 (m, 2 H), 3.52−3.37 (m, 2 H), 3.27−2.85 (m, 17 H), 2.84−2.78 (m, 1 H), 2.62−2.48 (m, 1 H), 2.26−2.04 (m, 3 H), 1.68−1.54 (m, 1 H). MS (ESI) m/z calcd for C30H39FN5O7 [M + H]+ 600.29; found 600.09. (5S,9S,10S,12R)-18-{2-[(2,2-Difluoroethyl)amino]ethyl}-9-(dimethylamino)-15-fluoro-4,5,8,25-tetrahydroxy-2,6-dioxo18,23-diazahexacyclo[12.11.0.03,12.05,10.016,24.017,21]pentacosa1(25),3,7,14,16(24)-pentaene-7-carboxamide (10g). 1H NMR (400 MHz, CD3OD, HCl salt) δ 6.35 (J = 2.4 Hz, J [19F−1H] = 53.7 G

DOI: 10.1021/acs.jmedchem.5b00262 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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was bubbled through the mixture for 5 min. The reaction vessel was sealed and heated at 80 °C for 48 h with vigorous stirring. After cooling down to rt, water (100 mL) was added. The reaction mixture was extracted with methylene chloride (100 mL × 1, 50 mL × 2). The combined extracts were dried over sodium sulfate and concentrated under reduced pressure. Flash column chromatography on silica gel with 0−15% EtOAc/hexane yielded the desired product 15 as a white solid (0.87 g, 28% overall yield). 1H NMR (400 MHz, CDCl3) δ 7.45−7.20 (m, 8 H), 7.03 (d, J = 7.3 Hz, 2 H), 6.07 (br s, 1 H), 5.03 (s, 2 H), 2.46 (s, 3 H), 1.46 (s, 9 H). MS (ESI) m/z calcd for C27H24BrF3NO6 [M − H]− 594.01; found 594.01. Phenyl 2-(Benzyloxy)-3-{[(tert-butoxy)carbonyl]amino}-4formyl-6-methyl-5-(trifluoromethoxy)benzoate (16). To compound 15 (0.68 g, 1.14 mmol) in anhydrous THF (6 mL) at −78 °C (dry ice−acetone bath) was added PhLi (0.95 mL, 1.80 M/n-Bu2O, 1.71 mmol, 1.5 equiv) dropwise over 1 min. After stirring at −78 °C for 10 min, n-BuLi (0.86 mL, 1.60 M/hexanes, 1.38 mmol, 1.2 equiv) was added dropwise over 2 min. The reaction was stirred at −78 °C for 5 min. Dry DMF (0.26 mL, 3.36 mmol, 3 equiv) was added dropwise. The reaction was stirred from −78 to 0 °C over 1 h and quenched with saturated aqueous sodium bicarbonate (50 mL). The reaction mixture was extracted with methylene chloride (50 mL × 3). The combined extracts were dried over sodium sulfate and concentrated under reduced pressure. Flash column chromatography on silica gel with 0−15% EtOAc/hexane yielded the desired product 16 as a pale solid (232 mg, 37%). 1H NMR (400 MHz, CDCl3) δ 10.21 (s, 1 H), 7.90 (br s, 1 H), 7.45−7.20 (m, 8 H), 7.05 (d, J = 7.3 Hz, 2 H), 5.00 (s, 2 H), 2.42 (s, 3 H), 1.43 (s, 9 H). MS (ESI) m/z calcd for C28H25F3NO7 [M − H]− 544.16; found 544.22. Phenyl 2-(Benzyloxy)-3-{[(tert-butoxy)carbonyl](prop-2-en1-yl)amino}-4-formyl-6-methyl-5-(trifluoromethoxy)benzoate (17). To compound 16 (232 mg, 0.43 mmol) in dry DMF (2 mL) at rt was added NaH (21 mg, 60% in mineral oil, 0.52 mmol, 1.2 equiv). After stirring at rt for 30 min, allyl bromide (56 μL, 0.64 mmol, 1.5 equiv) was added. The reaction mixture was stirred at rt for 1 h, diluted with EtOAc (50 mL), washed with water (50 mL × 2) and brine (50 mL × 1), dried over sodium sulfate, and concentrated under reduced pressure. Flash column chromatography on silica gel with 0− 8% EtOAc/hexane yielded the desired product 17 as a pale oil (206 mg, 82%). 1H NMR (400 MHz, CDCl3) δ 10.16 (s, 1 H), 7.40−6.95 (m, 10 H), 5.95−5.75 (m, 1 H), 5.10−4.85 (m, 4 H), 4.64, 4.28 (dd, dd, J = 5.5, 12.8 Hz, J = 4.9, 12.2 Hz, 1 H), 4.00, 3.89 (dd, dd, J = 8.1, 10.2 Hz, J = 8.6, 12.8 Hz, 1 H), 2.46, 2.43 (s, s, 3 H), 1.53, 1.50 (s, s, 9 H). MS (ESI) m/z calcd for C31H29F3NO7 [M − H]− 584.19; found 584.24. 5-tert-Butyl 7-Phenyl 6-(Benzyloxy)-1,8-dimethyl-9-(trifluoromethoxy)-1H,2H,3H,3aH,4H,5H,9bH-pyrrolo[3,2-c]quinoline5,7-dicarboxylate (18b). To compound 17 (206 mg, 0.35 mmol) in DMF (2 mL) was added N-methyl glycine (47 mg, 0.53 mmol, 1.5 equiv). The reaction mixture was heated at 100 °C for 24 h. After cooling to rt, the reaction mixture was diluted with EtOAc (50 mL), washed with aqueous saturated sodium bicarbonate (50 mL × 2) and brine (50 mL × 1), dried over sodium sulfate, and concentrated under reduced pressure. Flash column chromatography on silica gel with 0− 15% EtOAc/hexane yielded the desired product 18b as a white foam (190 mg, 89%). 1H NMR (400 MHz, CDCl3), broad and complex due to the presence of various rotamers and/or conformers. MS (ESI) m/z calcd for C33H36F3N2O6 [M + H]+ 613.25; found 613.36. tert-Butyl (1R,19S,26S,27S)-14,22-Bis(benzyloxy)-19-[(tertbutyldimethylsilyl)oxy]-26-(dimethylamino)-18-hydroxy-7methyl-16,20-dioxo-4-(trifluoromethoxy)-24-oxa-7,12,23triazaheptacyclo[15.11.0.03,15.05,13.06,10.019,27.021,25]octacosa-3(15),4,13,17,21(25),22-hexaene-12-carboxylate (19b). To diisopropylamine (51 μL, 0.36 mmol, 1.2 equiv) in anhydrous THF (1 mL) at −78 °C (dry ice−acetone bath) was added n-BuLi (0.23 mL, 1.60 M/hexanes, 0.36 mmol, 1.2 equiv) dropwise. The reaction was stirred at 0 °C for 10 min and cooled to −78 °C. TMEDA (56 μL, 0.36 mmol, 1.2 equiv) was added, followed by the addition of compound 18b (186 mg, 0.30 mmol) in anhydrous THF (2 mL) dropwise over 2 min. The resulting deep-red solution was stirred at −78 °C for 30 min. LHMDS (0.36 mL, 1.0 M/THF, 0.36 mmol, 1.2 equiv) was added,

followed by the addition of enone 7 (147 mg, 0.30 mmol, 1 equiv) in anhydrous THF (1 mL) dropwise over 1 min. The reaction was stirred from −78 to 0 °C for 2 h and quenched with saturated aqueous sodium bicarbonate (20 mL). The reaction mixture was extracted with methylene chloride (20 mL × 3). The combined extracts were dried over sodium sulfate and concentrated under reduced pressure. Flash column chromatography on silica gel with 0−20% EtOAc/hexane yielded the two diastereomers of the desired product (diastereomer A, 19ba, 100 mg, yellow foam, 33%; diastereomer B, 19bb, 60 mg, yellow foam, 20%). MS (ESI) m/z calcd for C53H64F3N4O10Si [M + H]+ 1001.44, found 1001.47. (5S,9S,10S,12R)-9-(Dimethylamino)-4,5,8,25-tetrahydroxy18-methyl-2,6-dioxo-15-(trifluoromethoxy)-18,23diazahexacyclo[12.11.0.0 3,12 .0 5,10 .0 16,24 .0 17,21 ]pentacosa-1(25),3,7,14,16(24)-pentaene-7-carboxamide (20b). Compound 19ba (33 mg, 0.033 mmol) was deprotected according to previously described desilylation and hydrogenation procedures6 to yield the desired product 20b as an orange solid after reverse phase HPLC purification (13 mg, HCl salt, 55% over 2 steps). 1H NMR (400 MHz, CD3OD) δ 4.75 (d, J = 7.4 Hz, 1 H), 4.08 (s, 1 H), 3.75−3.65 (m, 1 H), 3.50−2.90 (m, 20 H), 2.57−2.47 (m, 1 H), 2.31−2.05 (m, 3 H), 1.68−1.57 (m, 1 H). 13C NMR (400 MHz, CD3OD) δ 194.95, 187.83, 174.92, 174.26, 150.52, 138.79, 137.70, 123.74, 122.26, 121.17, 115.99, 111.49, 109.63, 96.11, 75.48, 70.71, 63.64, 55.25, 43.03, 42.70, 41.97, 41.06, 36.35, 35.81, 35.13, 33.75, 30.38, 27.51. MS (ESI) m/z calcd for C28H32F3N4O8 [M + H]+ 609.22; found 609.30. The following compounds (20a, 20c−20f) were prepared similarly. See experimental details in Supporting Information. (5S,9S,10S,12R)-9-(Dimethylamino)-4,5,8,25-tetrahydroxy2,6-dioxo-15-(trifluoromethoxy)-18,23-diazahexacyclo[12.11.0.03,12.05,10.01624.017,21]pentacosa-1(25),3,7,14,16(24)pentaene-7-carboxamide (20a). 1H NMR (400 MHz, CD3OD, HCl salt) δ 4.90−4.80 (m, 1 H), 4.09 (s, 1 H), 3.50−2.85 (m, 10 H), 2.82−2.74 (m, 1 H), 2.43−2.15 (m, 7 H), 2.15−2.05 (m, 1 H), 1.62 (dd, J = 10.6 Hz, 1 H). MS (ESI) m/z calcd for C27H30F3N4O8 [M + H]+ 595.20; found 595.31. (5S,9S,10S,12R)-9-(Dimethylamino)-4,5,8,25-tetrahydroxy18,19-dimethyl-2,6-dioxo-15-(trifluoromethoxy)-18,23diazahexacyclo[12.11.0.0 3,12 .0 5,10 .0 16,24 .0 17,21 ]pentacosa-1(25),3,7,14,16(24)-pentaene-7-carboxamide (20c). 1H NMR (400 MHz, CD3OD, HCl salt) δ 4.85−4.84 (m, 1 H), 4.11 (s, 1 H), 3.67−3.63 (m, 1 H), 3.13−2.93 (m, 15 H), 2.76−2.72 (m, 1 H), 2.35−2.22 (m, 2 H), 1.88−1.83 (m, 1 H), 1.71−1.62 (m, 1 H),1.66 (d, J = 11.2, 3 H). MS (ESI) m/z calcd for C29H34F3N4O8 [M + H]+ 623.24; found 623.43. (20S,24S,25S,27R)-24-(Dimethylamino)-15,19,20,23-tetrahydroxy-17,21-dioxo-2-(trifluoromethoxy)-5,13-diazaheptacyclo[14.12.0.03,14.04,11.05,9.018,27.020,25]octacosa-1,3(14),15,18,22pentaene-22-carboxamide (20d). 1H NMR (400 MHz, CD3OD, HCl salt) δ 4.80 (d, J = 6.0, 1 H), 4.41−4.38 (m, 1 H), 4.13 (s, 1 H), 3.80−3.77 (m, 1 H), 3.66−3.60 (m, 1 H), 3.52−3.48 (m, 1 H), 3.16− 3.03 (m, 12 H), 2.42−2.21 (m, 6 H), 2.09−2.01 (m, 1 H), 1.96−1.94 (m, 1 H), 1.69−1.60 (m, 1 H). MS (ESI) m/z calcd for C30H34F3N4O8 [M + H]+ 635.24; found 635.17. (21S,25S,26S,28R)-25-(Dimethylamino)-16,20,21,24-tetrahydroxy-18,22-dioxo-2-(trifluoromethoxy)-5,14-diazaheptacyclo[15.12.0.03,15.04,12.05,10.019,28.021,26]nonacosa-1,3(15),16,19,23pentaene-23-carboxamide (20e). 1H NMR (400 MHz, CD3OD, HCl salt) δ 5.00 (d, J = 9.2, 1 H), 4.13 (s, 1 H), 3.73−3.70 (m, 1 H), 3.45−3.38 (m, 1 H), 3.29−3.26 (m, 1 H), 3.16−3.03 (m, 11 H), 2.49− 2.22 (m, 5 H), 1.97−1.79 (m, 5 H), 1.69−1.61 (m, 2 H). MS (ESI) m/z calcd for C31H36F3N4O8 [M + H]+ 649.25; found 649.12. (21S,25S,26S,28R)-25-(Dimethylamino)-16,20,21,24-tetrahydroxy-18,22-dioxo-2-(trifluoromethoxy)-8-oxa-5,14diazaheptacyclo[15.12.0.03,15.04,12.05,10.019,28.021,26]nonacosa1,3(15),16,19,23-pentaene-23-carboxamide (20f). 1H NMR (400 MHz, CD3OD, HCl salt) δ 5.11 (d, J = 7.6, 1 H), 4.14 (s, 1 H), 4.07−4.04 (m, 1 H), 3.94−3.88 (m, 3 H), 3.70−2.90 (m, 8 H), 3.20−2.99 (m, 8 H), 2.66−2.59 (m, 1 H), 2.35−2.22 (m, 2 H), 1.68− 1.62 (m, 1 H). MS (ESI) m/z calcd for C30H34F3N4O9 [M + H]+ 651.23; found 651.14. H

DOI: 10.1021/acs.jmedchem.5b00262 J. Med. Chem. XXXX, XXX, XXX−XXX

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In Vitro Coupled P. aeruginosa Transcription/Translation Assay. Antitranslational activity (IC50 values) was assessed in a P. aeruginosa in vitro coupled transcription/translation assay (TnT) with a firefly luciferase readout (catalogue no. L1020, Promega, Madison, WI), as described by Fyfe et al.15 Reactions were run at a volume of 20 μL in Costar black 96-well assay plates (Costar catalogue no. 3915) for 60 min at 37 °C. The reaction was stopped by placing on ice for 5 min, followed by addition of 25 μL of luciferase assay substrate (catalogue no. E1500, Promega, Madison, WI). Plates were read on a LUMIStar Optima (BMG Labtech, Ortenberg, Germany) with gain set to 3600, 0.2 s read, 0 s between wells. Percent luminescence was plotted against inhibitor concentration with 50% inhibition versus untreated controls marked as the IC50 value. Susceptibility Testing. Compound stocks were prepared and serially diluted in sterile deionized water. Minimal inhibitory concentration (MIC) determinations were performed in liquid medium in 96-well microtiter plates according to the methods described by the Clinical and Laboratory Standards Institute (CLSI).16 Cation-adjusted Mueller Hinton broth was obtained from BBL (catalogue no. 212322, Becton Dickinson, Sparks, MD), prepared fresh and kept at 4 °C prior to testing. Lysed horse blood (catalogue no. 205, Quad Five, Ryegate, Montana) was used to supplement medium, as appropriate. All test methods met acceptable standards based on recommended quality control ranges for all comparator antibiotics and the appropriate ATCC quality control strains. Immunocompetent Lung Infection Model. The mouse lung infection model was performed at Vivisource, Waltham, MA. Female BALB/c mice weighing 18−20 g were infected with ∼2 × 107 CFU/ mouse of P. aeruginosa PA1145, a cystic fibrosis isolate from Children’s Hospital, Boston, via intranasal administration of 0.05 mL of cell suspension under light anesthesia. One group did not receive drug treatment, and lungs were harvested at 2 h postinfection. At 2 and 12 h postinfection, mice were treated with compound 10c, meropenem, or amikacin intravenously. Six mice per group were treated with each drug concentration. Twenty-four h post initiation of treatment, mice were euthanized by CO2 inhalation. The lungs of the mice were aseptically removed, weighed, homogenized, serially diluted, and plated on MacConkey medium. The plates were incubated overnight at 37 °C in 5% CO2. CFU per gram of lung was calculated by enumerating the plated colonies then adjusting for serial dilutions and the weight of the lung. Individual animal CFU/g lung data was plotted using GraphPad Prism. Immunocompetent Thigh Infection Model. The mouse thigh infection model was performed at University of North Texas Health Science Center, Fort Worth, TX. Groups of five female specificpathogen-free CD-1 mice weighing 22 ± 2 g were used. On day 0, animals were inoculated intramuscularly (0.1 mL/thigh) with 3−5 × 106 CFU/mouse of P. aeruginosa PA694, a clinical isolate from Eurofins-Medinet, into the right thigh. Two groups did not receive drug treatment, and thighs were harvested at 2 and 24 h postinfection. Remaining mice were administered intravenously with either vehicle, compound 10c, or levofloxacin at 2 and 12 h postinfection. The muscle of the right thigh of each animal was harvested at 24 h postinfection. Harvested thigh tissues were homogenized in 2 mL of PBS (pH 7.4) with a Polytron tissue homogenizer. The homogenates were serially diluted and plated on Brain Heart Infusion agar + 0.5% charcoal (w/v) for CFU determination per gram of thigh. Individual animal CFU/gram thigh data was plotted using GraphPad Prism.

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Article

AUTHOR INFORMATION

Corresponding Author

*Phone: 617-715-3553. Fax: 617-926-3557. E-mail: xyxiao@ tphase.com. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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ABBREVIATIONS USED

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REFERENCES

We thank Drs. Andrew Myers, Eric Gordon, and Joaquim Trias for valuable discussions during the course of this work. We also thank Dr. Shu-hui Chen and his colleagues at WuXi Apptec for external chemistry support.

aq, aqueous; ATCC, American Type Culture Collection; CFU, colony-forming units; CLSI, Clinical and Laboratory Standards Institute; CRE, carbapenem-resistant Enterobacteriaceae; DMF, N,N-dimethylformamide; HPLC, high-performance liquid chromatography; IC50, half-minimum inhibitory concentration; IV, intravenous; LDA, lithium diisopropylamide; LHMDS, lithium hexamethydisilazide; MDR, multidrug-resistant; MIC, minimum inhibitory concentration; MRSA, methicillin-resistant Staphylococcus aureus; MS, mass spectrometry; NMR, nuclear magnetic resonance; PBS, phosphate buffered saline; SAR, structure−activity relationship; THF, tetrahydrofuran; TLC, thin-layer chromatography; TMEDA, N,N,N′,N′-tetramethylethylenediamine; TnT, coupled transcription/translation assay

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ASSOCIATED CONTENT

S Supporting Information *

Detailed information on the synthesis and analytical data of compounds 10b, 10c, 10f−10h, 11a−11c, 20a, and 20c−20f. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b00262. I

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DOI: 10.1021/acs.jmedchem.5b00262 J. Med. Chem. XXXX, XXX, XXX−XXX