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Heterocyclyl Tetracyclines. 1. 7-Trifluoromethyl-8Pyrrolidinyltetracyclines: Potent, Broad Spectrum Antibacterial Agents with Enhanced Activity against Pseudomonas aeruginosa Yonghong Deng, Cuixiang Sun, Diana K. Hunt, Corey Fyfe, ChiLi Chen, Trudy H. Grossman, Joyce A. Sutcliffe, and Xiao-Yi Charlie Xiao J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01903 • Publication Date (Web): 01 Mar 2017 Downloaded from http://pubs.acs.org on March 1, 2017
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Heterocyclyl Tetracyclines. 1. 7-Trifluoromethyl-8-Pyrrolidinyltetracyclines: Potent, Broad Spectrum Antibacterial Agents with Enhanced Activity against Pseudomonas aeruginosa Yonghong Deng,*† Cuixiang Sun,† Diana K. Hunt,† Corey Fyfe,‡ Chi-Li Chen,† Trudy H. Grossman,‡ Joyce A. Sutcliffe,‡ Xiao-Yi Xiao† † Discovery Chemistry, ‡Microbiology, Tetraphase Pharmaceuticals, 480 Arsenal Way, Watertown, MA 02472, United States ABSTRACT
Utilizing a total synthetic approach, the first 8-heterocyclyltetracyclines were designed, synthesized and evaluated against panels of tetracycline- and multidrug-resistant Gram-positive and Gram-negative pathogens. Several compounds with balanced, highly potent in vitro activity against a broad range of bacterial isolates were identified through structure-activity relationships (SAR) studies. One compound demonstrated the best antibacterial activity against Pseudomonas aeruginosa both in vitro and in vivo for tetracyclines reported to date. INTRODUCTION Since the discovery of chlortetracycline (1) in 1948,1 the tetracycline class of antibiotics is generally regarded to be safe and has been used as first-line drugs for the treatment of many bacterial infections caused by both Gram-positive and Gram-negative pathogens.2,3 As with all antibiotic classes, the extensive use of tetracyclines for over 60 years, both in the clinic and in agriculture, has resulted in increased pathogenic bacterial resistance and threatens the continued effectiveness of many tetracyclines used today.4,5 Of the identified mechanisms by which bacteria become resistant to tetracyclines, tetracycline-specific efflux pumps [tet(A)-tet(D), and 1 ACS Paragon Plus Environment
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tet(K)-tet(L)],6-8 and ribosomal protection proteins [tet(M)-tet(O)],9-10 are by far the two most common forms encountered in the clinic. Therefore, it is highly desirable to develop new tetracyclines with improved in vitro potency and in vivo efficacy for use against current and emerging
multidrug-resistant
(MDR)
pathogens,
including
carbapenem-resistant
Enterobacteriaceae, MDR Acinetobacter species, and Pseudomonas aeruginosa. As the number of naturally occurring new tetracyclines from microbial sources is limiting, semisynthetic approaches that chemically modify natural antibiotics obtained by fermentation, have emerged as a dominating discovery effort to seek new scaffolds with improved safety and potency profiles. However, due to the densely functionalized structure and the chemical instability of tetracyclines, chemical modifications have generally been rather challenging and limited largely to the C-7 and C-9 positions using electrophilic aromatic substitution reactions (e.g., halogenation and nitration) followed by further functional group transformations. Examples of this include minocycline (2)11,12 and tigecycline (3).13-15 While a few total synthetic routes have been developed successfully, most were lengthy as well as low overall yielding and therefore were impractical to scale both in terms of the number and quantity of new tetracycline analogues.16 A more recent total synthetic methodology developed by Myers and co-workers,17,18 and expanded by Tetraphase Pharmaceuticals, has enabled the efficient introduction of a broad array of substituents at many more positions including C-4, C-4a, C-5, C-5a, C-6, C-7, C-8, C-9, and C-12a,19-21 as well as the incorporation of heterocyclic and polycyclic ring systems into the tetracycline core structure,22-24 creating completely novel tetracycline derivatives that were previously inaccessible (or extremely difficult to access) through semisynthesis. More than 3000 tetracycline analogues have been synthesized by Tetraphase using the total synthesis approach. Many of them are highly active against both Gram-positive and Gram-negative bacteria and a
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number of them have progressed into various clinical and pre-clinical stages, with the lead development candidate, eravacycline (4),25 a broad spectrum antibiotic with improved activity against multidrug resistant pathogens, currently undergoing late stage clinical studies. Cyclic amines such as pyrrolidines and piperidines are an important class of pharmacophores found in a variety of natural products and approved drugs.26 In an effort to continue the search for new tetracyclines, a series of 8-heterocycle substituted tetracyclines has been designed and synthesized in our laboratories. Their antimicrobial activities were evaluated against panels of Gram-positive and Gram-negative pathogens including those with various tetracycline resistance mechanisms. Herein, we report the details of these studies. Cl HO CH3 H
H3C CH3 N H OH
H 3C
N
CH3
H3C CH3 N H H OH
NH2 OH
O O HO H O
NH2
O
OH
Chlortetracycline (1)
H3C
H3C H3C
H N CH3
N
CH3
O N H
F 6
7
O
NH2 OH
O
Minocycline (2)
H 3C CH3 N H H OH
O O HO H O
O O HO H O
N
O
Tigecycline (3)
D
N H
10
OH
C 11
H 3C CH3 N H 5 H 4 OH B 12
A
O O HO H O
NH2 1
O
Eravacycline (4)
Figure 1. Structures of Natural (1), Semi-synthetic (2 and 3) and Fully Synthetic (4) Tetracycline.
RESULTS AND DISCUSSION Chemistry. The initial method leading to the 7-chloro-8-pyrrolidinyl tetracyclines is described in scheme 1. Compound 6 was readily prepared in quantitative yield by benzylation of phenol
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5.21 A Suzuki coupling27 of aryl bromide 6 with N-Boc-2-pyrroleboronic acid catalyzed by dichloro[1,1’-bis(diphenylphosphino)ferrocene] palladium(II) dichloromethane adduct gave compound 7 in 60% yield. Reduction of the pyrrole by catalytic hydrogenation at atmospheric pressure proceeded with concurrent removal of the benzyl protecting group. The crude product was reprotected using benzyl bromide and potassium carbonate to give compound 8 in 81% yield over two steps. Chlorination with N-chlorosuccinimide followed by deprotection of the Boc group with hydrogen chloride in dioxane yielded the D-ring precursor 9. The tandem MichaelDieckmann annulation18 was then carried out by treatment of 9 with lithium diisopropylamide in the presence of N,N,N’,N’-tetramethylethylenediamine in THF at -78 oC followed by the addition of AB-ring precursor, enone 10,17,
28
generating intermediate 11 in 46% yield. Further
elaboration of the pyrrolidinyl group via reductive alkylation with various aldehydes in the presence of acetic acid and sodium triacetoxyborohydride29 gave compound 12. Desilylation of 12 with aqueous HF followed by catalytic hydrogenation in the presence of palladium on carbon in ethyl acetate gave the desired 7-chloro-8-(2-pyrrolidinyl)tetracyclines 13. Partial dechlorination was observed when polar solvents such as methanol were used for hydrogenation. Scheme 1. Synthesis of 7-Chloro-8-pyrrolidinyltetracyclinesa
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a
Reagents and conditions: (a) BnBr, K2CO3, acetone; (b) N-Boc-2-pyrroleboronic acid,
PdCl2(dppf)·CH2Cl2, Na2CO3, toluene/dioxane/H2O; (c) H2, Pd/C, CH3OH; (d) BnBr, K2CO3, acetone; (e) NCS, CH3CN; (f) HCl/dioxane; (g) LDA, TMEDA, THF, -78 oC, then enone 10, -78 to -10 oC; (h) RCHO, AcOH, Na(OAc)3BH, ClCH2CH2Cl; (i) HF, CH3CN; (j) H2, Pd/C, EtOAc. Giovannini A. et al. reported that N-Boc-ω-amino ketones could be prepared from N-Boc fiveto eight-membered lactams via organometallic ring-opening reactions.30 Deprotection of the Boc group with trifluoroacetic acid (TFA) provided the corresponding 2-substituted cyclic imines, which could be further reduced to provide cyclic amine moieties. Using this strategy, we then explored the synthesis of 8-piperidinyl tetracyclines, as well as 8-azepanyl tetracyclines. Thus, lithium-halogen exchange of bromide 1421 with n-butyllithium at -100 oC followed by addition of 1-N-Boc-2-piperidone provided N-Boc-δ-amino ketone 15 with various levels of cyclic N5 ACS Paragon Plus Environment
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Boc-enamine 16 (Scheme 2). Treatment of ketone 15 with TFA gave the corresponding amine, which cyclized readily to imine 19 under the reaction conditions (87% yield). Compound 19 was also obtained from N-Boc-enamine 16 via Boc deprotection by treatment with TFA (76% yield). In the synthesis of azepanes using N-Boc-ε-caprolactam, formation of imine 20 was achieved by treatment of the acyclic aminoketone 17 with titanium (IV) isoproxide. The D-ring precursors 21 and 22 were then prepared by reduction of the imines (19 and 20) with sodium borohydride. Analogous 7-chloro-8-(2-piperidinyl)tetracyclines 25 and 7-chloro-8-(2-azepanyl)tetracyclines 26 were then synthesized via Michael-Dieckmann annulation, reductive alkylation, de-silylation, and hydrogenation according to similar procedures used in the preparation of compound 13. Scheme 2. Synthesis of 7-Chloro-8-heterocyclyltetracyclinesa
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a
Reagents and conditions: (a) n-BuLi, THF, then 1-N-Boc-2-piperidone or N-Boc-ε-
caperolactam; (b) TFA, CH2Cl2; (c) Ti(O-iPr)4, THF; (d) NaBH4, CH3OH; (e) LDA, TMEDA, THF, -78 oC, then enone 10, -78 to -10 oC; (f) HCHO, AcOH, Na(OAc)3BH, ClCH2CH2Cl; (g) HF, CH3CN; (h) H2, Pd/C, EtOAc. Several 8-heteroaryl substituted tetracycline analogues were also synthesized for the structureactivity relationships (SAR) study (Scheme 3). Michael-Dieckmann reaction between D-ring precursor 14 and enone 10 under standard conditions gave fully protected intermediate 27, and subsequent Suzuki coupling with heteroaryl boronic acids gave compounds 28 and 29. The two-
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step deprotection sequence as described above (HF and hydrogenation) yielded 7-chloro-8heteroaryltetracyclines 30 and 31. Scheme 3. Synthesis of 7-Chloro-8-heteroaryltetracyclinesa
a
Reagents and conditions: (a) LDA, TMEDA, THF, -78 oC, then enone 10, -78 to -10 oC; (b)
ArB(OH)2, PdCl2(dppf)·CH2Cl2, Na2CO3, toluene/dioxane/H2O; (c) HF, CH3CN; (d) H2, Pd/C, EtOAc. We next turned our attention to explore analogues with different substituents at C-7 position. Since 8-(2-pyrrolidinyl)tetracyclines showed better antimicrobial activities against a panel of selected bacteria in the initial screening, further studies were focused on 8-(2-pyrrolidinyl) analogues. As a typical example, the synthetic approach to the 7-trifluoromethyl-8-(2pyrrolidinyl)tetracyclines is shown in scheme 4. Following similar approaches as outlined in Scheme 2, imines 32 were prepared in a two-step process from aryl bromide 6 and N-Boc-2pyrrolidinone
or
N-Boc-3-methyl-2-pyrrolidinone
or
N-Boc-5-methyl-2-pyrrolidinone.
Reduction of the imines with sodium borohydride followed by Boc protection gave compounds
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33. Regioselective iodination with iodine/silver trifluoroacetate31 yielded aryl iodides 34, which were subjected to copper-mediated trifluoromethylation using methyl 2,2-difluoro-2(fluorosulfonyl)acetate/CuI as a trifluoromethyl anion equivalent32 to afford compounds 35 after Boc deprotection. The requisite D-ring precursors 36-38 were then prepared by reductive alkylation with either formaldehyde or benzaldehyde or alkylation with allyl bromide. Michael– Dieckmann reaction under standard conditions gave fully protected tetracycline intermediates 39, 40, and 42. Deprotection of the allyl group in compound 40 with Pd(PPh3)4 and N,Ndimethylbarbituric acid provided compound 41a, which was further converted to 41b via reductive alkylation with formaldehyde. Desilylation with aqueous HF and subsequent hydrogenation in the presence of palladium on carbon in methanol gave the final compounds 4345. Scheme 4. Synthesis of 7-Trifluoromethyl-8-pyrrolidinyltetracyclinesa
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R2 Br
R1
CH3 a, b
R2 R1
CH3
N
CO2Ph
c, d
CH3
N Boc
CO2Ph
OBn
CO2Ph
OBn
OBn
32
6
33 e
R2 R1
R2 CF3 h or i or j
CH3
N R
R1
R2 CF3
N H
CO2Ph
R1
f, g
CH3
I CH3
N Boc
CO2Ph
OBn
CO2Ph
OBn
36: R1 = R2 = H, R = CH3 37: R1 = CH3, R2 = H, R = allyl 38a: R1 = H, R2 = CH3, R = Bn 38b: R1 = H, R2 = CH3, R = CH3
OBn 34
35
k R2 R
CF3
1
N R
a
R
m, n N
OBn O
l
R2
H3 C CH3 N H H O
HO
O OTBS
OBn
39: R1 = R2 = H, R = CH3 40: R1 = CH3, R2 = H, R = allyl 41a: R1 = CH3, R2 = R = H 41b: R1 = CH3, R2 = H, R = CH3 42a: R1 = H, R2 = CH3, R = Bn 42b: R1 = H, R2 = CH3, R = CH3
CF3
1
N R
H
H3 C CH3 N H OH NH2
OH
O O HO H O
O
43a: R = CH3, R1 = R2 = H, 44a: R = R2 = H, R1 = CH3 44b: R = R1 = CH3, R2 = H 45a: R = R1 = H, R2 = CH3 45b: R = R2 = CH3, R1 = H
h
Reagents and conditions: (a) n-BuLi, THF, then N-Boc-2-pyrrolidinone or 3-methyl-N-Boc-2-
pyrrolidinone or 5-methyl-N-Boc-2-pyrrolidinone; (b) TFA, CH2Cl2; (c) Na(OAc)3BH, ClCH2CH2Cl; (d) Boc2O, DMAP, DMF; (e) I2, CF3CO2Ag, CHCl3; (f) CH3O2CCF2SO2F, CuI, DMF; (g) HCl, dioxane; (h) HCHO, AcOH, Na(OAc)3BH, ClCH2CH2Cl; (i) allylbromide, K2CO3, NaI, NMP; (j) PhCHO, AcOH, Na(OAc)3BH, ClCH2CH2Cl; (k) LDA, TMEDA, THF, -
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78 oC, then enone 10, -78 to -10 oC; (l) Pd(PPh3)4, N,N-dimethylbarbituric acid, CH2Cl2; (m) HF, CH3CN; (n) H2, Pd/C, CH3OH. The Michael–Dieckmann reaction between the achiral 4-(2-pyrrolidinyl) substituted D-ring precursors and enone 10 generated two diastereomers, and it was found that one of the diastereomers was more active across the entire panel of selected bacterial strains. We then investigated the asymmetric synthesis of 8-(2-pyrrolidinyl)tetracycline analogues applying the methodology developed by Ellman et al. for the enantioselective preparation of 2-substituted pyrrolidines.33 Thus, enantiomerically pure 4-(2-pyrrolidinyl) substituted D-ring precursor 49 was prepared in three steps (Scheme 5): titanium (IV)-mediated condensation of tertbutanesulfinamide with aldehyde 4621 gave N-(tert)-butanesulfinyl imine 47 in 81% yield; addition of Grignard reagent 2‐[2‐(bromomagnesio)ethyl]‐1,3‐dioxane prepared from 2-(2bromoethyl)-1,3-dioxane to the imine 47 at -78 oC to -48 oC afforded the desired sulfinamide 48 in excellent yield (95%) and diastereoselectivity; deprotection of the acetal with 1:1 TFA:H2O proceeded with simultaneous cleavage of the sulfinamide group, and subsequent in-situ reduction with Na(OAc)3BH gave enantiomerically pure 49 in moderate yield. Standard MichaelDieckmann reaction gave compound 50. Various alkyl groups were then introduced via reductive alkylation with aldehydes or ketones. Aqueous HF treatment, and palladium catalyzed hydrogenation
provided
the
desired
enantiomerically
pure
7-trifluoromethyl-8-(2-
pyrrolidinyl)tetracycline analogues 43b-h. Scheme 5. Asymmetric Synthesis of 7-Trifluoromethyl-8-pyrrolidinyltetracyclinesa
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a
Reagents
and
conditions:
(a)
(S)-tert-butanesulfinamide,
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Ti(OEt)4,
THF;
(b)
2‐[2‐
(bromomagnesio)ethyl]‐1,3‐dioxane, THF; (c) TFA–H2O, then Na(OAc)3BH; (d) LDA, TMEDA, THF, -78 oC, then enone 10, -78 to -10 oC; (e) RCHO or RCOR’, AcOH, Na(OAc)3BH, ClCH2CH2Cl; (f) HF, CH3CN; (g) H2, Pd/C, CH3OH. Similar approaches were taken for the preparation of 7-fluoro, 7-methoxy, 7-dimethylamino, 7-trifluoromethoxy, and 7-hydro-8-pyrrolidinyltetracycline derivatives, and the synthetic schemes are included in the Supporting Information. Biology. The fully synthetic 8-heterocyclyl tetracycline analogues were evaluated for in vitro antibacterial activity in minimal inhibitory concentration (MIC) assays as per Clinical Laboratory Standards Institute methodology34 against panels of tetracycline-resistant and 12 ACS Paragon Plus Environment
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tetracycline-susceptible Gram-negative and Gram-positive bacteria, which also included multidrug-resistant (MDR) organisms (Tables 1 and 2). When one or more additional chiral centers were introduced on the C8 substituent by non-asymmetric synthetic methods, as we also observed in earlier studies,24 the resulting diastereomers showed varying levels of antibacterial activity, despite the belief that the “north-western” region (i.e. the C4 to C8 portion) of the tetracycline molecule is not directly involved in binding with the 30S ribosome based on cocrystal structure studies.35 For brevity, only the relatively more active diastereomer from each diastereomeric set was included in Tables 1 and 2, except for compounds 13, 25 and 78, which were isolated and tested as mixtures of the respective two diastereomers (~ 1:1), and except for compounds 43c and 43d, which were listed as an example of a diasteromeric pair. As shown in Table 1, when the C7 substituent was fixed as chlorine, increasing the alkyl heterocyclic ring size at C8 from 5-member (compound 13) to 6-member and 7-member (compounds 25 and 26) decreased antibacterial activity by 2- to 8-fold against most strains in the panel. Likewise, replacing the alkyl heterocyclic substitution at C8 with an aromatic heterocycle (compounds 30 and 31) further diminished potency, especially against the Gram-negative isolates, most likely due to the decreased basicity of the aromatic nitrogen.21,24
Insert Table 1 Here.
We then fixed the C8 substituent as a 2-pyrrolidine ring and investigated the structure-activity relationships (SAR) of analogues with a range of substituents at C7 (R7), as well as with various substitutions on the pyrrolidine ring at C8. As shown in Table 2, when R7 was trifluoromethyl and the C8 pyrrolidine was not substituted, the diastereomer with a (2S)-configured pyrrolidine
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(compound 43c) was 2- to greater than 64-fold more potent than the corresponding (2R)configured diastereomer (compound 43d) against P. aeruginosa PA555 and all Gram-positive strains tested. When the pyrrolidine nitrogen was substituted by alkyl groups with increasing lipophilicity, varying from methyl, ethyl, isopropyl, propyl, cyclopropylmethyl, to isobutyl group (compounds 43a, 43f, 43e, 43g, 43b, and 43h), the corresponding analogues’ antibacterial potency decreased progressively against most organisms in the panel, especially against Gramnegative strains, with compound 43h losing almost all Gram-negative activity. Interestingly, the increased lipophilicity of the N-substituent on the pyrrolidine ring had much less effect, and sometimes had even a slightly reversed effect, on activity against the tet(M) strains, with potency in some cases increased by 2- to 4-fold (Staphylococcus aureus SA161, Enterococcus faecalis EFs327, and Enterococcus faecium EFm404). When a methyl group was present on one of the ring carbons, the compound (44a) displayed slightly increased activity (2- to 4-fold) against S. aureus SA161, Escherichia coli EC155, Acinetobacter baumannii AB250, Stenotrophomonas maltophilia SM256, and Burkholderia cenocepacia BC240, while having slightly decreased potency (2-fold) against S. aureus SA101, Klebsiella pneumoniae KP457, and P. aeruginosa PA555. Moving the methyl group to another ring carbon and/or to the ring nitrogen (compounds 44b, 45a, and 45b) generally decreased potency against most isolates in the panel. When the substitution on C7 was changed from trifluoromethyl to hydrogen, fluorine, dimethylamino, methoxy, and trifluoromethoxy (compounds 86, 76, 78, 77, and 79), the analogues’ antibacterial activity decreased from 2- to more than 512-fold against most strains tested, with the 7-methoxy and 7-dimethylamino compounds (77 and 78) retaining the least
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activity and the 7-trifluoromethoxy compound (79) retaining the most activity as compared to the 7-trifluoromethyl analogue 43c.
Insert Table 2 Here.
Overall, compounds 43c, 43a, 44a, and 45a were highly potent and displayed a balanced spectrum of activity against a broad range of MDR Gram-negative and Gram-positive pathogens, with no MIC values greater than 8 µg/mL. Furthermore, due to its better potency against P. aeruginosa with MIC values of 2-4 µg/mL, which were among the best in vitro antiPseudomonas activity for tetracyclines reported to date, compound 43c was selected as the first compound from this 8-heterocyclyl tetracycline series to be evaluated for in vivo antibacterial activity in mouse models of infections. As shown in Figure 2, in a mouse lung infection model challenged with P. aeruginosa PA1145, compound 43c (MIC = 2 µg/mL against P. aeruginosa PA1145) demonstrated dose-proportional bacterial burden reductions of 1.7, 2.3, and 3.7 mean log10 colony-forming units (CFUs) from T = 24 h when administered intravenously (IV) at 5, 15, and 40 mg/kg, respectively, twice daily. In the same model, comparator antibiotics amikacin (MIC = 4 µg/mL), tobramycin (MIC = 1 µg/mL), and tigecycline (MIC = 16 µg/mL) showed 3.6, 4.2, and essentially no mean log10 CFU reductions from untreated control group (T = 24 h), respectively, when dosed IV (amikacin and tigecycline) or IN (tobramycin) at 40 mg/kg, twice daily. In a mouse thigh infection model challenged with P. aeruginosa PA694, compound 43c (MIC = 4 µg/mL against P. aeruginosa PA694) also displayed dose-proportional efficacy with mean log10 CFU reductions of 0.4, 0.9, and 1.9 versus the 24 hour untreated control when dosed IV at 5, 15, and 40 mg/kg, respectively, twice daily. The comparator meropenem had better in
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vitro potency (MIC = 0.125 µg/mL), which also translated into greater in vivo efficacy of 2.9, 4.2, and 4.9 mean log10 CFU reductions from the 24 hour untreated control group when administered similarly in the same model.
Insert Figure 2 Here.
CONCLUSIONS A series of fully synthetic 7-R-8-heterocyclyl tetracyclines were designed and prepared using the tetracycline total synthesis platform.17 Antibacterial activities of the new tetracyclines were evaluated against panels of tetracycline- and multidrug-resistant Gram-positive and Gramnegative pathogens. Structure-activity relationships regarding the substitutions at the C7 and C8 positions were investigated. A number of analogues (43c, 43a, 44a, and 45a) displayed balanced, highly potent in vitro activity against a broad range of bacterial isolates. Furthermore, compound 43c,
a
7-trifluoromethyl-8-[(2S)-2-pyrrolidinyl]-6-demethyl-6-deoxytetracycline,
showed
enhanced in vitro anti-P. aeruginosa activity with MIC values of 2-4 µg/mL, which were among the best antibacterial activity against P. aeruginosa for tetracyclines reported to date.24 Subsequent in vivo efficacy studies in mouse lung and thigh models of infections challenged with P. aeruginosa (PA1145 and PA694, respectively) demonstrated that the in vitro antibacterial activity of compound 43c translated into in vivo efficacy against P. aeruginosa. Our data on this and other new tetracycline scaffolds24 suggested that considerable in vitro and in vivo anti-P. aeruginosa activity is attainable by exploring greater chemical diversity as enabled by the fully-synthetic tetracycline platform.17 EXPERIMENTAL SECTION
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Chemistry. All reactions involving air-sensitive reagents were performed under nitrogen in flame-dried glassware using syringe-septum cap technique. Unless it is specified, all reagents were used as received without further purifications. Thin layer chromatography (TLC) analysis was performed on Merck silica-gel 60 F254 and visualized under UV light or immersion of the plate in a basic solution of potassium permanganate in water followed by heating. Flash chromatography was performed on a Biotage Isolera One purification system using Biotage SNAP cartridges. Proton (1H) NMR (nuclear magnetic resonance) spectra were recorded on a 400 MHz JEOL ECX-400 spectrometer. Chemical shifts are reported in parts per million (ppm) using residual solvent as the internal standard (CDCl3 at 7.24 ppm, DMSO-d6 at 2.50 ppm, CD3OD at 3.30 ppm). Reverse phase preparative HPLC was performed on a Waters Autopurification system with mass-directed fraction collection (For final compounds: column, Polymerx RP-1 100A, 10 µm, 250 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 described, all final tetracycline compounds were isolated as mono-, di-, or trihydrochloride salts following freeze-drying. Analytical high performance liquid chromatography-electrospray mass spectra (HPLC-MS) were obtained using a Waters Alliance system (column, SunFire C18, 5 µm, 4.6 mm × 50 mm; flow rate, 1 mL/min; solvent A, water with 0.1% formic acid; solvent B, acetonitrile with 0.1% formic acid; MS detector, Waters 3100). Purity of tested compounds was determined to be ≥95% by analytical HPLC-MS analysis. Phenyl 2-(Benzyloxy)-4-bromo-6-methylbenzoate (6). To a solution of 5 (9.53 mmol, 1 equiv) in acetone (19 mL) was added K2CO3 (2.63 g, 15.00 mmol, 1.5 equiv) and BnBr (1.19
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mL, 10.00 mmol, 1.05 equiv). The mixture was stirred at room temperature overnight and filtered through a Celite pad. The Celite pad was washed with EtOAc. The combined filtrate was concentrated under reduced pressure. Flash chromatography on silica gel with 0%-5% EtOAc/hexanes yielded the desired product 6 as a white solid (3.61 g, 96%). 1H NMR (400 MHz, CDCl3) δ 7.20-7.45 (m, 8H), 7.03-7.09 (m, 4H), 5.13 (s, 2H), 2.43 (s, 3H). MS (ESI) m/z 419.1 (M + Na). tert-Butyl
2-[3-(benzyloxy)-5-methyl-4-(phenoxycarbonyl)phenyl]-1H-pyrrole-1-
carboxylate (7). To a pressure vial was charged with compound 6 (852 mg, 2.14 mmol, 1 equiv), N-Boc-2-pyrroleboronic acid (543 mg, 2.57 mmol, 1.2 equiv), dichloro[1,1’bis(diphenylphosphino)ferrocene] palladium(II) dichloromethane adduct (88 mg, 0.11 mmol, 0.05 equiv), and sodium carbonate (1.14 g, 10.7 mmol, 5 equiv). The vial was briefly evacuated and filled with N2. Toluene (5 mL), 1,4-dioxane (5 mL), and H2O (1 mL) were added. The reaction mixture was heated with a 90 oC oil bath for 2 h, cooled to rt, diluted with EtOAc, washed sequentially with aqueous phosphate buffer (pH = 7) and brine, dried over Na2SO4, and concentrated. Purification of the residue by Biotage flash chromatography gave compound 7 as a colorless oil (621 mg, 60%). 1H NMR (400 MHz, CDCl3) δ 7.22-7.48 (m, 9H), 7.12 (d, J = 7.8 Hz, 2H), 6.89 (d, J = 7.8 Hz, 2H), 6.20-6.26 (m, 2H), 5.15 (s, 2H), 2.48 (s, 3H), 1.41 (s, 9H). MS (ESI) m/z 484.4 (M + H). tert-Butyl
2-[3-(benzyloxy)-5-methyl-4-(phenoxycarbonyl)phenyl]pyrrolidine-1-
carboxylate (8). Compound 7 (621 mg, 1.28 mmol, 1 equiv) was dissolved in methanol. Pd-C (10 wt%, 186 mg) was added. The reaction flask was briefly evacuated and re-filled with hydrogen. The reaction mixture was stirred under 1 atm H2 at rt for 2 h and filtered through a
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Celite pad. The Celite pad was washed with methanol. The filtrate was concentrated to give the intermediate as white foam. The above intermediate was dissolved in acetone (12 mL), followed by addition of K2CO3 (350 mg, 2.54 mmol, 2 equiv) and BnBr (0.16 mL, 1.33 mmol, 1.04 equiv). After stirring overnight at rt, the reaction mixture was filtered through a Celite pad. The Celite pad was washed with three portions of EtOAc. The combined filtrate was concentrated. Purification of the residue by Biotage flash chromatography gave compound 8 as a colorless oil (504 mg, 81% over two steps). 1H NMR (400 MHz, CDCl3, rotamer) δ 7.22-7.48 (m, 8H), 7.05-7.15 (m, 2H), 6.63-6.70 (m, 2H), 5.13 (s, 2H), 4.90 and 4.76 (br s, 1H), 3.50-3.65 (m, 2H), 2.43 (s, 3H), 2.25-2.28 (m, 1H), 1.72-1.90 (m, 3H), 1.48 (s, 3H), 1.26 (s, 6H). MS (ESI) m/z 488.4 (M + H). Phenyl 6-(Benzyloxy)-3-chloro-2-methyl-4-(pyrrolidin-2-yl)benzoate (9). To a solution of compound 8 (556 mg, 1.14 mmol, 1 equiv) in 5 mL of CH3CN was added NCS (160 mg, 1.20 mmol, 1.05 equiv) in one portion. The reaction mixture was heated with a 60 oC oil bath for 18 h, cooled to rt, and evaporated to dryness. The residue was suspended in 200 mL CH2Cl2, washed sequentially with aqueous NaOH (1 N), H2O and brine, dried over Na2SO4, and concentrated. Purification of the residue by Biotage flash chromatography gave aryl chloride as a white solid (447 mg, 75%). 1H NMR (400 MHz, CDCl3, mixture of rotamers) δ 7.22-7.48 (m, 8H), 7.05-7.15 (m, 2H), 6.63-6.70 (m, 1H), 5.06-5.26 (m, 3H), 3.47-3.58 (m, 2H), 2.46 (s, 3H), 2.25-2.28 (m, 1H), 1.55-1.88 (m, 3H), 1.48 (s, 3H), 1.26 (s, 6H). MS (ESI) m/z 522.4 (M + H). The aryl chloride (447 mg, 0.86 mmol) was suspended in HCl/1,4-dioxane (4.0 M, 9 mL). After stirring at rt for 1 h, the volatiles were evaporated. The residue was suspended in EtOAc, washed with saturated aqueous NaHCO3 and brine, dried over Na2SO4, and concentrated. Purification of the residue by Biotage flash chromatography gave compound 9 as an off-white
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solid (338 mg, 93%). 1H NMR (400 MHz, CDCl3) δ 7.48 (dd, J = 1.8, 7.8 Hz, 2H), 7.34-7.42 (m, 6H), 7.26 (t, J = 7.8 Hz, 1H), 7.14 (d, J = 7.8 Hz, 2H), 5.20 (s, 2H), 4.57 (t, J = 7.4 Hz, 1H), 3.04-3.18 (m, 2H), 2.52 (s, 3H), 2.34-2.45 (m, 1H), 2.06 (br s, 1H), 1.78-1.85 (m, 2H), 1.44-1.54 (m, 1H). MS (ESI) m/z 422.4 (M + H). (1R,3S,4S,11S)‐‐8,16‐‐Bis(benzyloxy)‐‐11‐‐[(tert‐‐butyldimethylsilyl)oxy]‐‐19‐‐chloro‐‐4‐‐ (dimethylamino)‐‐12‐‐hydroxy‐‐18‐‐(pyrrolidin‐‐2‐‐yl)‐‐6‐‐oxa‐‐7‐‐ azapentacyclo[11.8.0.03,11.05,9.015,20]henicosa-5(9),7,12,15(20),16,18‐‐hexaene‐‐10,14‐‐dione (11). A solution of n-BuLi in hexanes (2.5 M, 0.53 mL, 0.63 mmol, 2.3 equiv) was added dropwise to a solution of i-Pr2NH (0.20 mL, 1.38 mmol, 2.4 equiv) in THF (4 mL) at -78 oC under N2 atmosphere. The reaction solution was stirred at -78 oC for 20 min and at 0 oC for 5 min, and was then re-cooled to -78 oC. N,N,N’,N’-Tetramethylethylenediamine (0.22 mL, 1.50 mmol, 2.6 equiv) was added, followed by addition of a solution of 9 (267 mg, 0.63 mmol, 1.1 equiv) in THF (4 mL) via a cannula. The resulting dark-orange mixture was stirred for 1 h at -78 o
C and was cooled to -100 oC. A solution of enone 10 (277 mg, 0.58 mmol, 1 equiv) in THF (4
mL) was added dropwise via a cannula. The mixture was allowed to gradually warm to -78 oC. LHMDS (0.60 mL, 1.0 M/THF, 0.60 mmol, 1.05 equiv) was added, and the reaction was slowly warmed to -5 oC. Saturated aqueous NH4Cl was added. The mixture was extracted three times with EtOAc. The combined EtOAc extracts were washed with brine, dried over Na2SO4, and concentrated. Purification of the residue by Biotage flash chromatography gave compound 11 as a light yellow foam (214 mg, 46%). 1H NMR (400 MHz, CDCl3) δ 7.26-7.50 (m, 11H), 5.35 (s, 2H), 5.25 (d, J = 11.4 Hz, 1H), 5.23 (d, J = 11.4 Hz, 1H), 4.51 (t, J = 6.9 Hz, 1H), 3.97 (d, J = 10.4 Hz, 1H), 3.43 (dd, J = 4.9, 15.9 Hz, 1H), 2.91-3.09 (m, 3H), 2.28-2.56 (m, 10H), 2.12 (d, J
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= 14.6 Hz, 1H), 1.54-1.80 (m, 3H), 1.34-1.48 (m, 2H), 0.83 (s, 9H), 0.26 (s, 3H), 0.14 (s, 3H). MS (ESI) m/z 810.4 (M + H). (1R,3S,4S,11S)‐‐8,16‐‐Bis(benzyloxy)‐‐11‐‐[(tert‐‐butyldimethylsilyl)oxy]‐‐19‐‐chloro‐‐4‐‐ (dimethylamino)‐‐12‐‐hydroxy‐‐18‐‐(1‐‐methylpyrrolidin‐‐2‐‐yl)‐‐6‐‐oxa‐‐7azapentacyclo[11.8.0.03,11.05,9.015,20]henicosa‐‐5(9),7,12,15(20),16,18‐‐hexaene‐‐10,14‐‐dione (12). Compound 11 (30.8 mg, 0.038 mmol, 1 equiv) was dissolved in 1,2-dichloroethane (1 mL). Aqueous formaldehyde (37%, 8.5 µL, 0.11 mmol, 3 equiv) and acetic acid (6.5 µL, 0.11 mmol, 3 equiv) were added. After stirring at rt for 45 min, sodium triacetoxyborohydride (16 mg, 0.076 mmol, 2 equiv) was added. Stirring was continued overnight. The reaction mixture was poured into saturated aqueous NaHCO3. The mixture was extracted three times with CH2Cl2. The combined organic extracts were washed with brine, dried over Na2SO4, and concentrated to give crude 12, which was used directly for the next step without further purification. MS (ESI) m/z 824.7 (M + H). (4S,4aS,5aR,12aS)-7-Chloro-4-(dimethylamino)-3,10,12,12a-tetrahydroxy-8-(1methylpyrrolidin-2-yl)-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide (13). In a polypropylene vial, crude 12 was dissolved in CH3CN (1.25 mL). Aqueous HF (48 %, 0.25 mL) was added. After stirred at rt for 16 h, the reaction mixture was poured into aqueous K2HPO4 (1.75 g in 12.5 mL water). The mixture was extracted three times with CH2Cl2. The combined organic phases were washed with brine, dried over sodium sulfate, and concentrated to dryness. The above residue was dissolved in EtOAc (3 mL) and added with Pd-C (10 wt%, 7.5 mg). The reaction flask was briefly evacuated and re-filled with hydrogen. The reaction mixture was stirred under 1 atm hydrogen until the reaction was complete as monitored by LC-MS analysis.
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The hydrogen source was removed. Methanol (5 mL) and HCl/methanol (0.5 N, 0.5 mL) were added. The mixture was stirred at rt for 30 min and filtered through a small pad of Celite. The filtrate was concentrated to give the crude product, which was purified by preparative reverse phase HPLC. Fractions with the desired MW were collected and freeze-dried to give compound 13 as a yellow solid (14.7 mg, HCl salt, 63.9% over 3 steps). 1H NMR (400 MHz, CD3OD) δ 7.28 (s, 1H), 5.01-5.07 (m, 1H), 4.14 (s, 1H), 3.97-4.04 (m, 1H), 3.38-3.46 (m, 2H), 2.91-3.13 (m, 11H), 2.62-2.68 (m, 1H), 2.10-2.47 (m, 5H), 1.61-1.71 (m, 1H). MS (ESI) m/z 532.4 (M + H). Phenyl methylbenzoate
6-(Benzyloxy)-4-(5-{[(tert-butoxy)carbonyl]amino}pentanoyl)-3-chloro-2(15)
and
tert-butyl
6-[5-(benzyloxy)-2-chloro-3-methyl-4-
(phenoxycarbonyl)phenyl]-1,2,3,4-tetrahydropyridine-1-carboxylate (16). To a solution of 14 (1.01 g, 2.33 mmol, 1 equiv) in THF (6 mL) was added n-BuLi (0.98 mL, 2.5 M/hexane, 2.45 mmol, 1.05 equiv) dropwise under N2 at -100 oC. The resulting orange solution was stirred at 100 oC for 20 min. A solution of 1-(tert-butoxycarbonyl)-2-piperidone (488 mg, 2.45 mmol, 1.05 equiv) in THF (6 mL) was added. The reaction mixture was stirred for another 2 h, maintaining the temperature below -78 oC, and quenched by the addition of aqueous HCl (1 N). The reaction mixture was warmed to rt and extracted three times with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, concentrated to dryness. Purification of the residue by Biotage flash chromatography gave 15 as a white solid (412 mg, 32%). 1H NMR (400 MHz, CDCl3) δ 7.33-7.43 (m, 7H), 7.24 (t, J = 7.8 Hz, 1H), 7.06 (d, J = 7.8 Hz, 2H), 6.83 (s, 1H), 5.14 (s, 2H), 4.57 (br s, 1H), 3.11-3.16 (m, 2H), 2.90 (t, J = 7.3 Hz, 2H), 2.47 (s, 3H), 1.68-1.75 (m, 2H), 1.50-1.57 (m, 2H), 1.43 (s, 9H). MS (ESI) m/z 574.5 (M + Na). And 16 as white foam (637 mg, 51%). 1H NMR (400 MHz, CDCl3) δ 7.33-7.44 (m, 7H), 7.24 (t, J = 7.8 Hz, 1H), 7.09 (d, J
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= 7.8 Hz, 2H), 6.88 (s, 1H), 5.11-5.13 (m, 3H), 3.75 (br s, 2H), 2.46 (s, 3H), 2.26-2.31 (m, 2H), 1.90-1.97 (m, 2H), 1.10 (s, 9H). MS (ESI) m/z 556.5 (M + Na). Phenyl 6-(Benzyloxy)-3-chloro-2-methyl-4-(3,4,5,6-tetrahydropyridin-2-yl)benzoate (19). Compound 15 (360 mg, 0.65 mmol) was dissolved in CH2Cl2 (3 mL), cooled with an ice bath, and treated with a solution of TFA (3 mL) in CH2Cl2 (3 mL). The reaction mixture was stirred at rt for 1 h and concentrated. The residue was re-dissolved in CH2Cl2, washed with saturated aqueous NaHCO3 and brine, dried over Na2SO4, and concentrated. Purification of the residue by Biotage flash chromatography gave compound 19 as a white solid (246 mg, 87%). Compound 19 was also prepared from 16 (310 mg, 0.58 mmol) according to the same procedure (191 mg, 76%). 1H NMR (400 MHz, CDCl3) δ 7.31-7.44 (m, 7H), 7.22 (t, J = 7.8 Hz, 1H), 7.04 (d, J = 7.8 Hz, 2H), 6.82 (s, 1H), 5.13 (s, 2H), 3.72-3.84 (m, 3H), 3.59-3.66 (m, 1H), 2.48 (s, 3H), 1.80-1.88 (m, 2H), 1.71-1.76 (m, 2H). MS (ESI) m/z 434.4 (M + H). Phenyl 6-(Benzyloxy)-3-chloro-2-methyl-4-(piperidin-2-yl)benzoate (21). To the solution of compound 19 (437 mg, 1.01 mmol, 1 equiv) in methanol (2 mL) was added NaBH4 (43 mg, 1.1 mmol, 1.1 equiv) at 0 oC. The reaction mixture was stirred at rt for 30 min. Aqueous HCl (1 N, 2 mL) was added. Stirring was continued for another 5 min. Solvents were removed with RotoVap. The residue was suspended in EtOAc, washed with saturated aqueous NaHCO3 and brine, dried over Na2SO4, and concentrated. Purification of the residue by Biotage flash chromatography gave 21 as a white solid (377 mg, 85.6%). 1H NMR (400 MHz, CDCl3) δ 7.43 (d, J = 7.8 Hz, 2H), 7.32-7.80 (m, 5H), 7.29 (s, 1H), 7.23 (t, J = 7.8 Hz, 1H), 7.05 (d, J = 7.8 Hz, 2H), 5.15 (s, 2H), 4.09 (dd, J = 2.3, 11.0 Hz, 1H), 3.20-3.23 (m, 1H), 2.83-2.90 (m, 1H), 2.46 (s, 3H), 1.86-1.92 (m, 2H), 1.47-1.70 (m, 4H), 1.24-1.34 (m, 1H). MS (ESI) m/z 436.4 (M + H).
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N-Boc-ε-caprolactam. To a solution of ε-caprolactam (1.01 g, 8.88 mmol, 1 equiv) in THF (10 mL) at -78 oC was added n-BuLi (2.5 M/hexanes, 3.73 mL, 9.32 mmol, 1.05 equiv) dropwise. The reaction was stirred at -78 oC for 1 h. A solution of Boc2O (2.03 g, 9.32 mmol, 1.05 equiv) in THF (8 mL) was added dropwise over 5 min at -78 oC. The reaction was stirred at -78 oC for 2 h and warmed to rt. Saturated aqueous NH4Cl (10 mL) and water (5 mL) were added. The mixture was extracted with EtOAc (3 times). The combined organic extracts were dried over sodium sulfate and concentrated under reduced pressure. Flash chromatography on silica gel using 0%-20% EtOAc-hexanes yielded the desired product as a colorless oil (1.50 g, 79%). 1H NMR (400 MHz, CDCl3) δ 3,72-3.76 (m, 2H), 2.60-2.68 (m, 2H), 1.68-1.80 (m, 6H), 1.24 (s, 9H). MS (ESI) m/z 236.6 (M + Na). Phenyl 4-(Azepan-2-yl)-6-(benzyloxy)-3-chloro-2-methylbenzoate (22). Following similar procedures used in the preparation of 21, compound 14 (1.02 g, 2.35 mmol, 1 equiv) was reacted with n-BuLi and N-Boc-ε-caprolactam (0.53 g, 2.47 mmol, 1.05 equiv) followed by Boc deprotection with TFA to yield the acyclic aminoketone. This aminoketone intermediate was dissolved in THF (10 mL). Ti(O-iPr)4 (2.06 mL, 7.05 mmol, 3 equiv) was added. The reaction mixture was stirred at rt overnight, diluted with EtOAc, and poured into water. The mixture was filtered through a Celite pad. The organic layer was collected, washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. Flash chromatography on silica gel using 0%-30% EtOAc-hexanes yielded the cyclic imine 20 as an off-white solid (291 mg, 28% over 3 steps). MS (ESI) m/z 448.0 (M + H). The imine (20) was treated with NaBH4 to give the desired product 22 as a waxy solid (292 mg, 100%). 1H NMR (400 MHz, CDCl3) δ 7.41-7.46 (m, 2H), 7.32-7.38 (m, 5H), 7.22 (t, J = 7.3 Hz, 1H), 7.21 (s, 1H), 7.06 (d, J = 7.8 Hz, 2H), 5.16
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(ABq, J = 11.4, 15.1 Hz, 2H), 4.27 (dd, J = 3.6, 9.2 Hz, 1H), 3.08-3.16 (m, 1H), 2.88-2.96 (m, 1H), 2.46 (s, 3H), 1.95-2.05 (m, 1H), 1.50-1.88 (m, 8H). MS (ESI) m/z 450.0 (M + H). (4S,4aS,5aR,12aS)-7-Chloro-4-(dimethylamino)-3,10,12,12a-tetrahydroxy-8-(1methylpiperidin-2-yl)-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide (25). Compound 25 was prepared from 21 via Michael-Dieckmann annulation, reductive alkylation, de-silylation, and hydrogenation according to similar procedures used in the preparation of 13 (HCl salt). 1H NMR (400 MHz, CD3OD) δ 7.30 (s, 1H), 4.14 (s, 1H), 3.66-3.69 (m, 1H), 2.93-3.44 (m, 11H), 2.69 (s, 3H), 2.36-2.46 (m, 1H), 2.26-2.29 (m, 1H), 1.93-2.11 (m, 5H), 1.61-1.81 (m, 2H). MS (ESI) m/z 546.4 (M + H). (4S,4aS,5aR,12aS)-7-Chloro-4-(dimethylamino)-3,10,12,12a-tetrahydroxy-8-(1methylazepan-2-yl)-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-Carboxamide (26). Compound 26 was prepared from 22 via Michael-Dieckmann annulation, reductive alkylation, de-silylation, and hydrogenation according to similar procedures used in the preparation of 13 (HCl salt, diastereomer A). 1H NMR (400 MHz, CD3OD) δ 7.23 (s, 1H), 4.12 (s, 1H), 3.65-3.75 (m, 1H), 3.50-3.60 (m, 1H), 3.38-3.45 (m, 1H), 2.95-3.20 (m, 9H), 2.78 (s, 3H), 2.22-2.45 (m, 3H), 1.58-2.28 (m, 8H). MS (ESI) m/z 560.4 (M + H). (1R,3S,4S,11S)‐‐8,16‐‐Bis(benzyloxy)‐‐18‐‐bromo‐‐11‐‐[(tert‐‐butyldimethylsilyl)oxy]‐‐19‐‐ chloro‐‐4-(dimethylamino)‐‐12‐‐hydroxy‐‐6‐‐oxa‐‐7‐‐azapentacyclo[11.8.0.03,11.05,9.015,20]henicosa‐‐ 5(9),7,12,15(20),16,18‐‐hexaene‐‐10,14‐‐dione (27). n-BuLi in hexanes (2.5 M, 0.29 mL, 0.72 mmol, 1.2 equiv) was added dropwise to a solution of i-Pr2NH (0.11 mL, 0.75 mmol, 1.25 equiv) in THF (4 mL) at -78 oC under N2 atmosphere. The reaction mixture was stirred at -78 oC for 20 min, 0 oC for 5 min, and re-cooled to -78 oC. TMEDA (0.12 mL, 0.78 mmol, 1.3 equiv) was added, followed by the addition of a solution of 14 (285 mg, 0.66 mmol, 1.1 equiv) in THF (4
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mL), maintaining the temperature below -70 oC. The resulting dark-red mixture was stirred for 1 h at -78 oC and was then cooled to -100 oC. A solution of enone 10 (289 mg, 0.60 mmol, 1 equiv) in THF (4 mL) was added dropwise via a cannula. The reaction mixture was allowed to gradually warm to -78 oC. LHMDS (1.0 M/THF, 0.60 mL, 0.60 mmol, 1 equiv) was added. The reaction was allowed to warm to -5 oC slowly over 1 h. Saturated aqueous NH4Cl was added. The mixture was extracted three times with CH2Cl2. The combined CH2Cl2 extracts were washed with brine, dried over Na2SO4, and concentrated. Purification of the residue by Biotage flash chromatography gave compound 27 as a light yellow foam (359 mg, 73%). 1H NMR (400 MHz, CDCl3) δ 15.9 (s, 1H), 7.22-7.52 (m, 10H), 6.81 (d, J = 8.6 Hz, 1H), 5.35 (s, 2H), 5.20 (d, J = 11.4 Hz, 1H), 5.15 (d, J = 11.4 Hz, 1H), 3.93 (d, J = 10.4 Hz, 1H), 3.42 (dd, J = 4.9, 15.9 Hz, 1H), 2.96-3.04 (m, 1H), 2.45-2.58 (m, 9H), 2.12 (d, J = 14.6 Hz, 1H), 0.84 (s, 9H), 0.26 (s, 3H), 0.14 (s, 3H). MS (ESI) m/z 819.4 (M + H). (1R,3S,4S,11S)‐‐8,16‐‐Bis(benzyloxy)‐‐11‐‐[(tert‐‐butyldimethylsilyl)oxy]‐‐19‐‐chloro‐‐4‐‐ (dimethylamino)‐‐12‐‐hydroxy‐‐18‐‐(pyridin‐‐3‐‐yl)‐‐6‐‐oxa‐‐7‐‐ azapentacyclo[11.8.0.03,11.05,9.015,20]henicosa‐‐5(9),7,12,15(20),16,18‐‐hexaene‐‐10,14‐‐dione (28). To a pressure vial was charged with compound 27 (34 mg, 0.041 mmol), 3-pyridine boronic acid (26 mg, 0.21 mmol, 5 equiv), dichloro[1,1’-bis(diphenylphosphino)ferrrocene] palladium(II) dichloromethane adduct (1.7 mg, 0.0021 mmol, 0.05 equiv), and sodium carbonate (22 mg, 0.21 mmol, 5 equiv). The vial was briefly evacuated and filled with N2. Toluene (1 mL), 1,4-dioxane (1 mL), and H2O (0.2 mL) were added. The reaction mixture was heated with an 80 o
C oil bath for 1 h, cooled to rt, diluted with EtOAc, washed with aqueous phosphate buffer (pH
= 7) and brine, dried over Na2SO4, and concentrated. Purification of the residue by Biotage flash chromatography gave compound 28 as a yellow solid (20 mg, 60%). 1H NMR (400 MHz,
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Journal of Medicinal Chemistry
CDCl3) δ 16.0 (s, 1H), 8.63 (dd, J = 1.8, 4.9 Hz, 1H), 8.56 (d, J = 1.8 Hz, 1H), 7.69 (ddd, J = 1.8, 1.8, 7.9 Hz, 1H), 7.26-7.52 (m, 11H), 6.88 (s, 1H), 5.36 (s, 2H), 5.24 (d, J = 11.4 Hz, 1H), 5.18 (d, J = 11.4 Hz, 1H), 3.97 (d, J = 10.4 Hz, 1H), 3.48 (dd, J = 4.9, 15.9 Hz, 1H), 3.01-3.10 (m, 1H), 2.45-2.60 (m, 9H), 2.15 (d, J = 14.6 Hz, 1H), 0.84 (s, 9H), 0.29 (s, 3H), 0.15 (s, 3H). MS (ESI) m/z 818.4 (M + H). (1R,3S,4S,11S)‐‐8,16‐‐Bis(benzyloxy)‐‐11‐‐[(tert‐‐butyldimethylsilyl)oxy]‐‐19‐‐chloro‐‐4‐‐ (dimethylamino)‐‐12‐‐hydroxy‐‐18‐‐(pyridin‐‐4‐‐yl)‐‐6‐‐oxa‐‐7‐‐ azapentacyclo[11.8.0.03,11.05,9.015,20]henicosa‐‐5(9),7,12,15(20),16,18‐‐hexaene‐‐10,14‐‐dione (29). Compound 29 was prepared according to similar procedures used for 28 (yellow solid). 1H NMR (400 MHz, CDCl3) δ 16.0 (s, 1H), 8.68 (d, J = 6.1 Hz, 2H), 7.26-7.52 (m, 12H), 6.85 (s, 1H), 5.36 (s, 2H), 5.23 (d, J = 11.4 Hz, 1H), 5.18 (d, J = 11.4 Hz, 1H), 3.97 (d, J = 10.4 Hz, 1H), 3.48 (dd, J = 4.9, 15.9 Hz, 1H), 3.01-3.10 (m, 1H), 2.45-2.60 (m, 9H), 2.16 (d, J = 14.6 Hz, 1H), 0.84 (s, 9H), 0.28 (s, 3H), 0.16 (s, 3H). MS (ESI) m/z 818.5 (M + H). (4S,4aS,5aR,12aS)-7-Chloro-4-(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-8(pyridine-3-yl)-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide
(30).
In
a
polypropylene vial, compound 28 (20 mg, 0.024 mmol) was dissolved in CH3CN (1.25 mL). Aqueous HF (48%, 0.25 mL) was added. After stirred at rt for 16 h, the reaction mixture was poured into aqueous K2HPO4 (1.75 g in 12.5 mL water). The mixture was extracted three times with CH2Cl2. The combined organic phases were washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The above residue was dissolved in EtOAc (3 mL). Pd-C (10 wt%, 36 mg) was added in four portions. The reaction flask was briefly evacuated and re-filled with hydrogen. The reaction mixture was stirred under 1 atm hydrogen at rt until the reaction was complete as monitored by
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LC-MS analysis. The hydrogen source was removed. Methanol (5 mL) and HCl/methanol (0.5 N, 0.5 mL) were added. The mixture was stirred for 30 min and filtered through a small pad of Celite. The filtrate was concentrated to give the crude product, which was purified by preparative reverse phase HPLC to yield compound 30 as a yellow solid (1.5 mg, HCl salt, 10% over two steps). 1H NMR (400 MHz, CD3OD) δ 9.08 (s, 1H), 8.96 (d, J = 5.6 Hz, 1H), 8.78 (d, J = 5.6 Hz, 1H), 8.22 (t, J = 5.6 Hz, 1H), 7.11 (s, 1H), 4.12 (s, 1H), 3.45 (m, 1H), 2.92-3.15 (m, 8H), 2.47 (dd, J = 15.0, 15.0 Hz, 1H), 2.25-2.28 (m, 1H), 1.60-1.70 (m, 1H). MS (ESI) m/z 526.3 (M + H). (4S,4aS,5aR,12aS)-7-Chloro-4-(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-8(pyridine-4-yl)-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide (31). Compound 31 was prepared from 29 according to similar procedures used for 30. 1H NMR (400 MHz, CD3OD)
δ 8.97 (d, J = 6.4 Hz, 2H), 8.22 (d, J = 6.4 Hz, 2H), 7.09 (s, 1H), 4.12 (s, 1H), 3.45 (dd, J = 4.6, 16.0 Hz, 1H), 2.97-3.21 (m, 8H), 2.47 (dd, J = 15.0, 15.0 Hz, 1H), 2.24-2.30 (m, 1H), 1.63-1.73 (m, 1H). MS (ESI) m/z 526.3 (M + H). Phenyl
2-(Benzyloxy)-4-(3,4-dihydro-2H-pyrrol-5-yl)-6-methylbenzoate
(32).
To
a
solution of 6 (3.01 g, 7.58 mmol) in THF (30 mL) was added nBuLi (2.5 M, 3.18 mL, 7.96 mmol, 1.05 equiv) dropwise under N2 at -100 oC. The resulting orange solution was stirred at this temperature for 30 min, then solution of 1-(tert-butoxycarbonyl)-2-pyrrolidinone (1.52 g, 7.96 mmol, 1.05 equiv) in THF (8 mL) was added. The reaction mixture was stirred for another 3 h, maintaining the temperature below -78 oC, and quenched by the addition of aqueous HCl (1 N). The reaction mixture was warmed to rt and extracted three times with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, concentrated to dryness. The residue was dissolved in CH2Cl2 (20 mL), cooled with an ice bath and treated with a solution of TFA (20 mL) in CH2Cl2 (20 mL). The reaction mixture was stirred at rt for 1 h and concentrated. The
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residue was dissolved in CH2Cl2, washed with saturated aqueous NaHCO3, and brine, dried over Na2SO4, and concentrated. Purification of the residue by biotage flash chromatography gave compound 32a (R1 = R2 = H) as a white solid (2.02 g, 69% over two steps). 1H NMR (400 MHz, CDCl3) δ 7.48 (s, 1H), 7.42 – 7.45 (m, 2H), 7.31 – 7.37 (m, 5H), 7.24 (s, 1H), 7.22 (t, J = 7.8 Hz, 1H), 7.07 (d, J = 7.8 Hz, 2H), 5.19 (s, 2H), 4.08 (t, J = 7.3 Hz, 2H), 2.92 (t, J = 7.8 Hz, 2H), 2.47 (s, 3H), 2.03 – 2.10 (m, 2H). MS (ESI) m/z 386.1 (M + H). tert-Butyl
2-[3-(Benzyloxy)-5-methyl-4-(phenoxycarbonyl)phenyl]pyrrolidine-1-
carboxylate (33). To a solution of compound 32a (2.02 g, 5.24 mmol) in 1,2-dichloroethane (26 mL) were added acetic acid (1.3 mL, 23.6 mmol, 4.5 equiv) and sodium triacetoxyborohydride (5.0 g, 23.6 mmol, 4.5 equiv). The reaction mixture was stirred at rt and monitored by LC-MS. Upon completion, the reaction mixture was diluted with CH2Cl2 (200 mL) and poured into saturated aqueous NaHCO3 (200 mL). The layers were separated and the aqueous layer was further extracted two times with CH2Cl2. The combined organic layers were washed with brine, dried over Na2SO4, and concentrated to dryness. The above residue was dissolved in anhydrous DMF (21 mL). Di-tert-butyl dicarbonate (1.37 g, 6.29 mmol, 1.2 equiv) and DMAP (32 mg, 0.26 mmol, 0.05 equiv) were added. The reaction mixture was stirred at rt for 2 h, and then diluted with EtOAc. The solution was washed sequentially with 1 N HCl, H2O three times, brine, dried over Na2SO4, and concentrated. Purification of the residue by Biotage flash chromatography gave compound 33a (R1 = R2 = H) as white foam (2.27 g, 88.9% over two steps). 1H NMR (400 MHz, CDCl3, rotamer) δ 7.41 – 7.43 (m, 2H), 7.29 – 7.37 (m, 5H), 7.20 – 7.23 (m, 1H), 7.08 – 7.10 (m, 2H), 6.63 – 6.65 (m, 2H), 5.10 (s, 2H), 4.70 – 4.90 (m, 1H), 3.50 – 3.65 (m, 2H), 2.41 (s, 3H), 2.20 – 2.32 (m, 1H), 1.60 – 1.80 (m, 3H), 1.45 (br s, 3H), 1.23 (br s, 6H). MS (ESI) m/z 510.3 (M + Na).
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2-[5-(Benzyloxy)-2-iodo-3-methyl-4-(phenoxycarbonyl)phenyl]pyrrolidine-1-
carboxylate (34). To compound 33a (1.30 g, 2.67 mmol, 1 equiv) in chloroform (13 mL) was added silver trifluoroacetate (648 mg, 2.93 mmol, 1.1 equiv) and iodine (744 mg, 2.93 mmol, 1.1 equiv). The reaction mixture was stirred at rt for 2.5 h and filtered through a Celite pad. The Celite pad was washed with methylene chloride. The combined filtrate was washed with saturated aqueous Na2S2O3, saturated aqueous NaHCO3 and brine, dried over sodium sulfate, and concentrated under reduced pressure. Purification of the residue by Biotage flash chromatography using 0%-20% EtOAc/hexanes yielded 34a (R1 = R2 = H) as white solid (1.55 g, 95%). 1H NMR (400 MHz, CDCl3, rotamer) δ 7.20-7.42 (m, 8H), 7.11 (d, J = 7.8 Hz, 2H), 6.60 and 6.55 (s, s, 1H), 5.05-5.17 (m, 3H), 3.40-3.65 (m, 2H), 2.56 and 2.53 (s, s, 3H), 2.252.40 (m, 1H), 1.50-1.85 (m, 3H), 1.24 and 1.47 (s, s, 9H). MS (ESI) m/z 636.5 (M + Na). Phenyl 6-(Benzyloxy)-2-methyl-4-(pyrrolidin-2-yl)-3-(trifluoromethyl)benzoate (35a, R1 = R2 = H). A solution of compound 34a (1.57 g, 2.56 mmol, 1 equiv), CH3O2CCF2SO2F (3.26 mL, 25.6 mmol, 10 equiv), and CuI (2.44 g, 12.8 mmol, 5 equiv) in DMF (10 mL) in a sealed tube was heated at 80 oC for 24 h. More CH3O2CCF2SO2F (1.63 mL, 12.8 mmol, 5 equiv) and CuI (1.22 g, 6.4 mmol, 2.5 equiv) were added. The reaction was heat at 80 oC for another 20 h, cooled to rt, filtered through a Celite pad. The Celite pad was washed with EtOAc. The combined filtrate was washed with water and brine, dried over sodium sulfate, and concentrated. Purification of the residue by Biotage flash chromatography using 0% - 20% EtOAc/hexanes yielded the corresponding trifluoromethyl derivative as a light yellow oil (1.39 g, contaminated with 34a), which was used in the next step without further purification. A mixture of trifluoromethylbenzene obtained above (1.39 g) and 4 M HCl/1,4-dioxane (12 mL) was stirred at rt for 30 min and concentrated under reduced pressure. The residue was
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dissolved in methylene chloride (200 mL), washed with saturated aqueous NaHCO3 and brine, dried over sodium sulfate, and concentrated. Purification of the residue by Biotage flash chromatography gave compound 35a as tilt oil (376 mg, 32% over 2 steps). 1H NMR (400 MHz, CDCl3) δ 7.68 (s, 1H), 7.20-7.45 (m, 8H), 7.08 (d, J = 7.3 Hz, 2H), 5.23 (ABq, J = 11.4, 18.4 Hz, 2H), 4.65-4.71 (m, 1H), 3.02-3.15 (m, 2H), 2.54 (q, J = 3.2 Hz, 3H), 2.20-2.30 (m, 1H), 1.92 (br s, 1H), 1.72-1.82 (m, 2H), 1.44-1.53 (m, 1H). MS (ESI) m/z 456.0 (M + H). Phenyl 6-(Benzyloxy)-2-methyl-4-(5-methylpyrrolidin-2-yl)-3-(trifluoromethyl)benzoate (35b, R1 = CH3, R2 = H). Compound 35b was prepared from 6 and N-Boc-5-methyl-2pyrrolidinone according to similar procedures used for 35a. 1H NMR (400 MHz, CD3OD) δ 7.88 (s, 1H), 7.45 (d, J = 7.8 Hz, 2H), 7.31 – 7.39 (m, 5H), 7.23 (dd, J = 7.32, 7.32 Hz, 1H), 7.09 (d, J = 7.8 Hz, 2H), 5.23 (ABq, J = 11.4, 18.4 Hz, 2H), 4.63 – 4.70 (m, 1H), 3.30 – 3.39 (m, 1H), 2.52 (q, J = 3.2 Hz, 3H), 2.19 – 2.29 (m, 1H), 1.78 – 1.87 (m, 2H), 1.46 – 1.55 (m, 1H), 1.26 – 1.34 (m, 1H), 1.23 (d, J = 5.9 Hz, 3H). MS (ESI) m/z 470.0 (M + H). Phenyl
6-(Benzyloxy)-2-methyl-4-(3-methylpyrrolidin-2-yl)-3-(trifluoromethyl)benzoate
(35c, R1 = H, R2 = CH3). Compound 35c was prepared from 6 and N-Boc-3-methyl-2pyrrolidinone according to similar procedures used for 35a. 1H NMR (400 MHz, CD3OD) δ 7.70 (s, 1H), 7.44 (d, J = 7.8 Hz, 2H), 7.31 – 7.38 (m, 5H), 7.23 (dd, J = 7.32, 7.32 Hz, 1H), 7.08 (d, J = 7.8 Hz, 2H), 5.22 (ABq, J = 11.4, 18.4 Hz, 2H), 4.65 – 4.71 (m, 1H), 3.15 – 3.22 (m, 1H), 2.93 – 2.99 (m, 1H), 2.53 (q, J = 3.2 Hz, 3H), 2.42 – 2.52 (m, 1H), 2.03 – 2.10 (m, 1H), 1.71 (br s, 1H), 1.38 – 1.48 (m, 1H), 0.49 (d, J = 6.9 Hz, 3H). MS (ESI) m/z 470.0 (M + H). Phenyl
6-(Benzyloxy)-2-methyl-4-(1-methylpyrrolidin-2-yl)-3-(trifluoromethyl)benzoate
(36). Compound 35a (376 mg, 0.83 mmol, 1 equiv) was dissolved in DCE (4 mL). Aqueous formaldehyde (37% in water, 0.18 mL, 2.48 mmol, 3 equiv) and acetic acid (0.19 mL, 3.30
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mmol, 4 equiv) were added. The reaction was stirred at rt for 20 min. Na(OAc)3BH (525 mg, 2.48 mmol, 3 equiv) was added. The reaction mixture was stirred at rt overnight, diluted with methylene chloride, and washed with saturated aqueous NaHCO3 (60 mL). The aqueous layer was extracted with methylene chloride (2 times). The combined organic solutions were washed with brine, dried over sodium sulfate, and concentrated. Purification of the residue by Biotage flash chromatography yielded 36 as a colorless oil (318 mg, 82%). 1H NMR (400 MHz, CDCl3)
δ 7.64 (s, 1H), 7.22-7.46 (m, 8H), 7.08 (d, J = 7.3 Hz, 2H), 5.21 (ABq, J = 11.3, 14.0 Hz, 2H), 3.50-3.58 (m, 1H), 3.18-3.24 (m, 1H), 2.53 (q, J = 3.7 Hz, 3H), 2.20-2.37 (m, 2H), 2.10 (s, 3H), 1.84-1.92 (m, 2H), 1.48-1.62 (m, 1H). MS (ESI) m/z 492.2 (M + Na). Phenyl
6-(Benzyloxy)-2-methyl-4-[5-methyl-1-(prop-2-en-1-yl)pyrrolidin-2-yl]-3-
(trifluoromethyl)benzoate (37). To compound 35b (296 mg, 0.63 mmol, 1 equiv) in N-methyl2-pyrrolidinone (3 mL) at rt was added potassium carbonate (174 mg, 1.26 mmol, 2.0 equiv), allyl bromide (82 µL, 0.94 mmol, 1.5 equiv), and sodium iodide (9.4 mg, 0.063 mmol, 0.1 equiv). The reaction mixture was heated at 50 oC for 2 hrs. Upon cooling to rt, EtOAc (50 mL) was added. The mixture was washed sequentially with H2O (10 mL x 2), brine (10 mL). The organic solution was dried over sodium sulfate and concentrated under reduced pressure. Purification of the residue by Biotage flash chromatography yielded the desired product 37 as a colorless oil (205 mg, 63.8%). 1H NMR (400 MHz, CDCl3) δ 7.79 (s, 1H), 7.22-7.50 (m, 8H), 7.16 (d, J = 7.8 Hz, 2H), 5.52 – 5.63 (m, 1H), 5.24 (s, 2H), 5.01 (d, J = 17.0 Hz, 1H), 4.94 (d, J = 10.1 Hz, 1H), 4.15 – 4.25 (m, 1H), 3.04 (d, J = 6.4 Hz, 2H), 2.80 – 2.88 (m, 1H), 2.54 (q, J = 3.7 Hz, 3H), 2.13-2.21 (m, 1H), 1.81-1.90 (m, 1H), 1.36 - 1.50 (m, 2H), 1.19 (d, J = 5.9 Hz, 3H). MS (ESI) m/z 510.0 (M + H).
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Phenyl
4-(1-Benzyl-3-methylpyrrolidin-2-yl)-6-(benzyloxy)-2-methyl-3-
(trifluoromethyl)benzoate (38a). To a solution of compound 35c (38.3 mg, 0.082 mmol, 1 equiv) in DCE (1.0 mL) was added benzaldehyde (24.7 µL, 0.24 mmol, 3 equiv) and acetic acid (18.7 µL, 0.33 mmol, 4 equiv). The reaction was stirred at rt for 45 min. Na(OAc)3BH (51.9 mg, 0.24 mmol, 3 equiv) was added. The reaction mixture was stirred at rt overnight, diluted with methylene chloride, and washed with saturated aqueous NaHCO3 (10 mL). The aqueous layer was extracted with methylene chloride (2 times). The combined organic solutions were washed with brine, dried over sodium sulfate, and concentrated. Purification of the residue by Biotage flash chromatography yielded 38a as a colorless oil (42.4 mg, 92.9%). 1H NMR (400 MHz, CDCl3) δ 7.80 (s, 1H), 7.23-7.46 (m, 13H), 7.11 (d, J = 8.2 Hz, 2H), 5.22 (s, 2H), 4.03 (d, J = 13.3 Hz, 1H), 3.73 (d, J = 13.3 Hz, 1H), 3.07-3.17 (m, 2H), 2.50-2.63 (m, 4H), 2.20-2.28 (m, 1H), 1.99-2.08 (m, 1H), 1.32-1.42 (m, 1H), 0.52 (d, J = 7.3 Hz, 3H). MS (ESI) m/z 560.2 (M + H). Phenyl
6-(Benzyloxy)-4-(1,3-dimethylpyrrolidin-2-yl)-2-methyl-3-
(trifluoromethyl)benzoate (38b). Compound 38b was prepared from 35c and formaldehyde according to similar procedures used for 38a. 1H NMR (400 MHz, CD3OD) δ 7.49 (s, 1H), 7.44 (d, J = 7.8 Hz, 2H), 7.33-7.38 (m, 5H), 7.23 (t, J = 7.3 Hz, 1H), 7.07 (d, J = 7.8 Hz, 2H), 5.20 (ABq, J = 11.3, 14.0 Hz, 2H), 3.72 (s, 3H), 3.31 (br d, J = 7.8 Hz, 1H), 3.22 (ddd, J = 2.3, 8.7, 8.7 Hz, 1H), 2.55 (q, J = 3.7 Hz, 3H), 2.38-2.45 (m, 1H), 2.14-2.22 (m, 1H), 2.10 (s, 3H), 1.921.99 (m, 1H), 1.40-1.48 (m, 1H), 0.93 (d, J = 6.9 Hz, 3H). MS (ESI) m/z 484.2 (M + H). (1R,3S,4S,11S)‐‐8,16‐‐Bis(benzyloxy)‐‐11‐‐[(tert‐‐butyldimethylsilyl)oxy]‐‐4‐‐(dimethylamino)‐‐ 12‐‐hydroxy‐‐18‐‐(1‐‐methylpyrrolidin‐‐2‐‐yl)‐‐19‐‐(trifluoromethyl)‐‐6‐‐oxa‐‐7azapentacyclo[11.8.0.03,11.05,9.015,20]henicosa‐‐5(9),7,12,15(20),16,18‐‐hexaene‐‐10,14‐‐dione 33 ACS Paragon Plus Environment
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(39). To freshly prepared LDA (1.14 mmol, in 10 mL THF, 1.2 equiv) at -78 oC was added TMEDA (185 µL, 1.23 mmol, 1.3 equiv). After stirring for 5 min, a solution of compound 36 (445 mg, 0.95 mmol, 1 equiv) in THF (5 mL) was added dropwise. The resulting red solution was stirred at -78 oC for 30 min. Enone 10 (456 mg, 0.95 mmol, in 5 ml THF, 1 equiv) was added. The reaction was gradually warmed to -50 oC. LHMDS (1.00 mL, 1.0 M/THF, 1.00 mmol, 1.05 equiv) was added. The reaction was allowed to warm to -5 oC. Saturated aqueous NH4Cl was added. The mixture was extracted with EtOAc (3 times). The combined organic extracts were washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. Purification of the residue by Biotage flash chromatography yielded the desired product 39 as a light yellow solid (mixture of two diastereomers, 685 mg, 84%). MS (ESI) m/z 857.9 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-3,10,12,12a-tetrahydroxy-8-(1-methylpyrrolidin-2yl)-1,11-dioxo-7-(trifluoromethyl)-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide (43a). In a plastic vial, compound 39 (55 mg, 0.064 mmol, 1 equiv) was dissolved in CH3CN (1 mL). Aqueous HF (48%, 0.25 mL) was added. After stirred at rt for 6 h, the reaction mixture was poured into aqueous solution of K2HPO4 (1.75 g in 12.5 mL H2O). The resulting mixture was extracted three times with EtOAc. The combined organic phases were washed with brine, dried over sodium sulfate, concentrated to give crude intermediate, which was used directly in the next step without further purification. The above crude was dissolved in 0.5 N HCl in MeOH (256 µL, 2 equiv). The excess volatiles were evaporated. The pre-formed HCl salt was re-dissolved in MeOH (5.0 mL) and to the resulting solution was added palladium on carbon (10 wt%, 20 mg). The reaction flask was briefly evacuated and re-filled with hydrogen. The reaction mixture was stirred at rt and
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monitored by LC-MS. After SM was consumed, the mixture was filtered through a small pad of Celite. The filtrate was concentrated to give crude, which was purified by preparative HPLC to give 43a as a yellow solid (single diastereomer). 1H NMR (400 MHz, CD3OD) δ 7.42 (s, 1H), 4.72-4.78 (m, 1H), 4.14 (s, 1H), 3.86-3.95 (m, 1H), 3.20-3.44 (m, 2H), 2.95-3.10 (m, 8H), 2.76 (s, 3H), 2.60-2.75 (m, 2H), 2.20-2.45 (m, 4H), 1.58-1.72 (m, 1H). MS (ESI) m/z 566.4 (M + H). (4S,4aS,5aR,12aS)-8-[1-(Cyclopropylmethyl)pyrrolidin-2-yl]-4-(dimethylamino)3,10,12,12a-tetrahydroxy-1,11-dioxo-7-(trifluoromethyl)-1,4,4a,5,5a,6,11,12aoctahydrotetracene-2-carboxamide (43b). Compound 50 (30 mg, 0.036 mmol, 1 equiv) was dissolved in 1,2-dichloroethane (1 mL). Cyclopropanecarboxaldehyde (8.0 µL, 0.11 mmol, 3 equiv) and acetic acid (8.1 µL, 0.14 mmol, 4 equiv) were added. After stirring at rt for 30 min, sodium triacetoxyborohydride (22.6 mg, 0.11 mmol, 3 equiv) was added. Stirring was continued for an overnight. The reaction mixture was poured into saturated aqueous NaHCO3. The mixture was extracted three times with CH2Cl2. The combined organic extracts were washed with brine, dried over Na2SO4, and concentrated to give crude intermediate, which was used directly for the next step without further purification. In a polypropylene vial, crude intermediate obtained above was dissolved in CH3CN (1.0 mL). Aqueous HF (48%, 0.25 mL) was added. After stirred at rt for 16 h, the reaction mixture was poured into aqueous K2HPO4 (1.75 g in 12.5 mL water). The mixture was extracted three times with CH2Cl2. The combined organic phases were washed with brine, dried over sodium sulfate, and concentrated to dryness. The above crude material was dissolved in 0.5 N HCl in methanol (200 µL). The excess volatiles were evaporated. The pre-formed HCl salt was re-dissolved in methanol (2.0 mL) and to the resulting solution was added palladium on carbon (10 % wt, 12 mg). The reaction flask
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was briefly evacuated and re-filled with hydrogen. The reaction mixture was stirred at rt and monitored by LC-MS. After SM was consumed, the mixture was filtered through a small pad of Celite. The filtrate was concentrated to give crude, which was purified by preparative HPLC on a Waters Autopurification system to give compound 43b as a yellow solid (8.5 mg, HCl salt, 39% over 3 steps). 1H NMR (400 MHz, CD3OD) δ 7.47 (s, 1H), 4.78-4.85 (m, 1H), 4.13 (s, 1H), 4.024.08 (m, 1H), 3.41-3.48 (m, 1H), 3.23-3.28 (m, 1H), 2.95-3.05 (m, 10H), 2.62-2.74 (m, 2H), 2.17-2.37 (m, 4H), 1.60-1.69 (m, 1H), 0.91-0.97 (m, 1H), 0.58-0.64 (m, 2H), 0.32-0.35 (m, 1H), 0.21-0.25 (m, 1H). MS (ESI) m/z 606.6 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-8(pyrrolidin-2-yl)-7-(trifluoromethyl)-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2carboxamide(43c). Compound 43c was prepared from 50 by de-silylation and hydrogenation according to similar deprotection procedures used for 43b. 1H NMR (400 MHz, CD3OD) δ 7.22 (s, 1H), 4.90-4.99 (m, 1H), 4.12 (s, 1H), 3.57-3.61 (m, 1H), 3.43-3.52 (m, 1H), 3.20-3.28 (m, 1H), 2.95-3.04 (m, 8H), 2.56-2.68 (m, 2H), 2.12-2.33 (m, 4H), 1.60-1.69 (m, 1H). MS (ESI) m/z 552.5 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-8(pyrrolidin-2-yl)-7-(trifluoromethyl)-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2carboxamide (43d). Compounds 43d was prepared from 46 and (R)-tert-butanesulfinamide according to the same procedures used for 43c. 1H NMR (400 MHz, CD3OD) δ 7.26 (s, 1H), 5.05 (t, J = 7.8 Hz, 1H), 4.13 (s, 1H), 3.45-3.59 (m, 2H), 3.20-3.28 (m, 1H), 2.95-3.04 (m, 8H), 2.62 (t, J = 15.1 Hz, 1H), 2.46-2.52 (m, 1H), 2.14-2.33 (m, 4H), 1.59-1.69 (m, 1H). MS (ESI) m/z 552.5 (M + H).
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(4S,4aS,5aR,12aS)-4-(Dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-8-[1(propan-2-yl)pyrrolidin-2-yl]-7-(trifluoromethyl)-1,4,4a,5,5a,6,11,12a-octahydrotetracene2-carboxamide (43e). Compound 43e was prepared from 50 according to similar procedures used in the preparation of 43b. 1H NMR (400 MHz, CD3OD) δ 7.52 (s, 1H), 4.82-4.89 (m, 1H), 4.14 (s, 1H), 3.75-3.80 (m, 1H), 3.43-3.50 (m, 2H), 3.20-3.28 (m, 1H), 2.96-3.05 (m, 8H), 2.632.75 (m, 2H), 2.17-2.34 (m, 4H), 1.60-1.69 (m, 1H), 1.26 (d, J = 6.9 Hz, 3H), 1.23 (d, J = 6.9 Hz, 3H). MS (ESI) m/z 594.5 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-8-(1-ethylpyrrolidin-2-yl)-3,10,12,12atetrahydroxy-1,11-dioxo-7-(trifluoromethyl)-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2carboxamide (43f). Compound 43f was prepared from 50 according to similar procedures used in the preparation of 43b. 1H NMR (400 MHz, CD3OD) δ 7.42 (s, 1H), 4.75-4.80 (m, 1H), 4.13 (s, 1H), 3.91-3.95 (m, 1H), 3.33-3.40 (m, 1H), 3.20-3.28 (m, 1H), 2.96-3.12 (m, 10H), 2.63-2.72 (m, 2H), 2.21-2.34 (m, 4H), 1.60-1.69 (m, 1H), 1.24 (t J = 7.3 Hz, 3H). MS (ESI) m/z 580.5 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-8-(1propylpyrrolidin-2-yl)-7-(trifluoromethyl)-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2carboxamide (43g). Compound 43g was prepared from 50 according to similar procedures used in the preparation of 43b. 1H NMR (400 MHz, CD3OD) δ 7.46 (s, 1H), 4.78-4.80 (m, 1H), 4.14 (s, 1H), 3.93-3.99 (m, 1H), 3.33-3.42 (m, 1H), 3.20-3.28 (m, 1H), 2.94-3.08 (m, 10H), 2.63-2.72 (m, 2H), 2.18-2.33 (m, 4H), 1.58-1.70 (m, 3H), 0.89 (t J = 7.3 Hz, 3H). MS (ESI) m/z 594.6 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-3,10,12,12a-tetrahydroxy-8-[1-(2methylpropyl)pyrrolidin-2-yl]-1,11-dioxo-7-(trifluoromethyl)-1,4,4a,5,5a,6,11,12a-
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octahydrotetracene-2-carboxamide (43h). Compound 43h was prepared from 50 according to similar procedures used in the preparation of 43b. 1H NMR (400 MHz, CD3OD) δ 7.50 (s, 1H), 4.80-4.85 (m, 1H), 4.14 (s, 1H), 3.99-4.05 (m, 1H), 3.33-3.42 (m, 1H), 3.20-3.28 (m, 1H), 2.963.04 (m, 9H), 2.63-2.79 (m, 3H), 2.18-2.33 (m, 4H), 1.89-1.97 (m, 1H), 1.60-1.70 (m, 1H), 0.96 (d, J = 6.9 Hz, 3H), 0.88 (d, J = 6.9 Hz, 3H). MS (ESI) m/z 608.6 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-3,10,12,12a-tetrahydroxy-8-(5-methylpyrrolidin-2yl)-1,11-dioxo-7-(trifluoromethyl)-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide (44a). Compound 44a was prepared from 37 via Michael-Dieckmann annulation, allyl deprotection, de-silylation, and hydrogenation according to similar procedures used in the preparation of 43a (HCl salt, single diastereomer). 1H NMR (400 MHz, CD3OD) δ 7.34 (s, 1H), 5.00 (t, J = 7.8 Hz, 1H), 4.13 (s, 1H), 3.86-3.91 (m, 1H), 3.20-3.28 (m, 1H), 2.95-3.04 (m, 8H), 2.57-2.66 (m, 2H), 2.35-2.46 (m, 1H), 2.17-2.27 (m, 2H), 1.92-2.00 (m, 1H), 1.59-1.68 (m, 1H), 1.53 (d, J = 6.4 Hz, 3H). MS (ESI) m/z 566.2 (M + H). (4S,4aS,5aR,12aS)-4-(dimethylamino)-8-(1,5-dimethylpyrrolidin-2-yl)-3,10,12,12atetrahydroxy-1,11-dioxo-7-(trifluoromethyl)-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2carboxamide (44b). Compound 44b was prepared from 37 via Michael-Dieckmann annulation, allyl deprotection, reductive alkylation, de-silylation, and hydrogenation according to similar procedures used in the preparation of 43a (HCl salt, single diastereomer). 1H NMR (400 MHz, CD3OD) δ 7.43 (s, 1H), 4.80 (t, J = 7.8 Hz, 1H), 4.12 (s, 1H), 3.68-3.74 (m, 1H), 2.96-3.06 (m, 9H), 2.63-2.71 (m, 5H), 2.47-2.55 (m, 1H), 2.21-2.26 (m, 2H), 2.02-2.11 (m, 1H), 1.60-1.69 (m, 1H), 1.55 (d, J = 6.4 Hz, 3H). MS (ESI) m/z 580.2 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-3,10,12,12a-tetrahydroxy-8-(3-methylpyrrolidin-2yl)-1,11-dioxo-7-(trifluoromethyl)-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide
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(45a). Compound 45a was prepared from 38a via Michael-Dieckmann annulation, de-silylation, and hydrogenation according to similar procedures used in the preparation of 43a (HCl salt, single diastereomer). 1H NMR (400 MHz, CD3OD) δ 6.97 (s, 1H), 5.25 (d, J = 7.3 Hz, 1H), 4.13 (s, 1H), 3.62-3.68 (m, 1H), 3.41-3.48 (m, 1H), 3.24-3.28 (m, 1H), 2.85-3.09 (m, 9H), 2.62 (t, J = 15.1 Hz, 1H), 2.44-2.54 (m, 1H), 2.20-2.27 (m, 1H), 1.84-1.93 (m, 1H), 1.60-1.69 (m, 1H), 0.71 (d, J = 6.8 Hz, 3H). MS (ESI) m/z 566.2 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-8-(1,3-dimethylpyrrolidin-2-yl)-3,10,12,12atetrahydroxy-1,11-dioxo-7-(trifluoromethyl)-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2carboxamide (45b). Compound 45b was prepared from 38b via Michael-Dieckmann annulation, de-silylation, and hydrogenation according to similar procedures used in the preparation of 43a (HCl salt, single diastereomer).
1
H NMR (400 MHz, CD3OD) δ 7.38 (s, 1H), 4.49 (d, J = 7.3
Hz, 1H), 4.10 (s, 1H), 3.82-3.88 (m, 1H), 3.34-3.43 (m, 2H), 2.91-3.10 (m, 12H), 2.55-2.64 (m, 2H), 2.20-2.27 (m, 1H), 1.78-1.88 (m, 1H), 1.59-1.69 (m, 1H), 1.12 (d, J = 6.8 Hz, 3H). MS (ESI) m/z 580.2 (M + H). Phenyl 6-(Benzyloxy)-2-methyl-4-[(1E)-{[(S)-2-methylpropane-2-sulfinyl]imino}methyl]3-(trifluoromethyl)benzoate (47). To a solution of 46 (4.02 g, 9.70 mmol) in THF (39 mL) was added a solution of Ti(OEt)4 (technical grade, ~20% Ti; 20.1 mL, 19.4 mmol, 2.0 equiv) under N2 atmosphere, followed by (S)-tert-butanesulfinamide (1.76 g, 14.6 mmol, 1.5 equiv). The resulting yellow solution was stirred at rt and conversion was followed by LC-MS. Upon completion, the reaction mixture was poured into 80 mL brine while rapidly stirring, and stirring was continued for another 30 min. The resulting suspension was filtered through a plug of Celite, and the filter cake was washed with EtOAc. The filtrate was transferred to a separation funnel where the organic layer was washed with brine, dried over sodium sulfate, and concentrated
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under reduced pressure. Purification of the residue by Biotage flash chromatography gave compound 47 as an off-white foam (4.07 g, 81%). 1H NMR (400 MHz, CDCl3) δ 8.96 (br s, 1H), 7.23-7.45 (m, 9H), 7.08 (d, J = 7.3 Hz, 2H), 5.25 (s, 2H), 2.58 (q, J = 3.2 Hz, 3H), 1.24 (s, 9H). MS (ESI) m/z 518.5 (M + H). Phenyl-6-(benzyloxy)-4-[3-(1,3-dioxan-2-yl)-1-{[(S)-2-methylpropane-2sulfinyl]amino}propyl]-2-methyl-3-(trifluoromethyl)benzoate (48). A flame dried flask was charged with magnesium turnings (10.94 g, 450 mmol, 3 equiv) and catalytic amounts of I2 (761.4 mg, 3 mmol, 0.02 equiv), which was heated with heat gun under N2 for 2 min. Once they were cooled to rt, THF (150 mL) was added. A small portion solution of 2-(2-bromoethyl)-1,3dioxane (20.3 mL, 150 mmol, 1 equiv) in THF (50 mL) was added. After the reaction commenced, the rest of 2-(2-bromoethyl)-1,3-dioxane solution was added via cannula. The reaction mixture was periodically cooled in a rt water bath to prevent refluxing. After addition of the 2-(2-bromoethyl)-1,3-dioxane solution completed, the reaction mixture was stirred for 2 h. The solution was then transferred to a sure-sealed bottle to remove the remaining Mg and stored in fridge for future use. To a solution of compound 47 (2.32 g, 4.49 mmol) in THF (18 mL) was added the Grignard solution (11.2 mL) prepared above at -78 oC in 10 min. After the mixture was stirred at this temperature for 1.5 h, the cold bath was removed. When the inner temperature reached -48 oC, saturated aqueous NH4Cl (30 mL) was added. The layers were separated. The aqueous layer was extracted with EtOAc (two times). The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated under reduced pressure to yield the crude product as a white solid, which was suspended in 25 mL heptane. The mixture was stirred at rt for 1.5 h, the solid was collected by filtration and washed with small portion of heptane. Further dried under
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Journal of Medicinal Chemistry
high vacuum provided compound 48 as a white solid (2.70 g, 95%). 1H NMR (400 MHz, CDCl3)
δ 7.41 (d, J = 7.3 Hz, 2H), 7.31-7.37 (m, 5H), 7.22 (t, J = 7.3 Hz, 1H), 7.15 (s, 1H), 7.05 (d, J = 7.3 Hz, 2H), 5.20 (s, 2H), 4.88 (dd, J = 7.8, 11.2 Hz, 1H), 4.47 (t, J = 4.6 Hz, 1H), 4.04-4.09 (m, 2H), 3.71-3.75 (m, 3H), 2.52 (q, J = 3.2 Hz, 3H), 1.98-2.09 (m, 1H), 1.81-1.90 (m, 2H), 1.621.71 (m, 1H), 1.47-1.57 (m, 1H), 1.30 (d, J = 11.9 Hz, 1H), 1.17 (s, 9H). MS (ESI) m/z 634.6 (M + H). Phenyl
6-(Benzyloxy)-2-methyl-4-(pyrrolidin-2-yl)-3-(trifluoromethyl)benzoate
(49).
Compound 48 (2.70 g, 4.26 mmol) was added to the mixture of TFA – H2O (21 mL – 21 mL) cooled in an ice bath. The resulting mixture was then stirred at 6 oC and conversion was followed by LC-MS. Upon completion, the reaction mixture was cooled to -20 oC, and NaBH(OAc)3 was added. Temperature was then allowed to warm to rt. After the mixture was stirred at rt for 1 h, it was re-cooled to 0 oC. The pH of the solution was adjusted to ~8 with 45% aqueous KOH. The aqueous solution was extracted with MTBE (three times). The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. Purification of the residue by Biotage flash chromatography gave compound 49 as a light yellow oil (1.29 g, 66%). 1H NMR (400 MHz, CDCl3) δ 7.67 (s, 1H), 7.22-7.46 (m, 8H), 7.08 (d, J = 7.3 Hz, 2H), 5.22 (ABq, J = 11.4, 18.4 Hz, 2H), 4.64-4.69 (m, 1H), 3.02-3.16 (m, 2H), 2.53 (q, J = 3.2 Hz, 3H), 2.21-2.30 (m, 1H), 1.85 (br s, 1H), 1.73-1.80 (m, 2H), 1.44-1.52 (m, 1H). MS (ESI) m/z 456.5 (M + H). (1R,3S,4S,11S)‐‐8,16‐‐Bis(benzyloxy)‐‐11‐‐[(tert‐‐butyldimethylsilyl)oxy]‐‐4‐‐(dimethylamino)‐‐ 12‐‐hydroxy‐‐18‐‐(pyrrolidin‐‐2‐‐yl)‐‐19‐‐(trifluoromethyl)‐‐6‐‐oxa‐‐7‐‐ azapentacyclo[11.8.0.03,11.05,9.015,20]henicosa-5(9),7,12,15,17,19‐‐hexaene‐‐10,14‐‐dione (50). A solution of n-BuLi in hexanes (2.5 M, 0.21 mL, 0.53 mmol, 2.4 equiv) was added dropwise to a
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solution of i-Pr2NH (78 µL, 0.55 mmol, 2.5 equiv) in THF (2 mL) at -78 oC under N2 atmosphere. The reaction solution was stirred at -78 oC for 20 min and 0 oC for 5 min, and recooled to -78oC. TMEDA (86 µL, 0.57 mmol, 2.6 equiv) was added, followed by the addition of a solution of 49 (110 mg, 0.24 mmol, 1.1 equiv) in THF (2 mL) via a cannula. The resulting dark-orange mixture was stirred for 35 min at -78 oC and cooled to -100 oC. A solution of enone 10 (106 mg, 0.22 mmol, 1.0 equiv) in THF (2 mL) was added dropwise via a cannula. The resulting mixture was warmed to -78 oC. LHMDS (1.0 M/THF, 220 µL, 0.22 mmol, 1.0 equiv) was added. The reaction mixture was allowed to gradually warm to -5 oC. Saturated aqueous NH4Cl was added. The mixture was extracted three times with EtOAc. The combined EtOAc extracts were washed with brine, dried over Na2SO4, and concentrated. Purification of the residue by Biotage flash chromatography gave compound 50 as a light yellow foam (61 mg, 33%). 1H NMR (400 MHz, CDCl3) δ 7.66 (s, 1H), 7.27-7.50 (m, 10H), 5.35 (s, 2H), 5.29 (s, 1H), 4.54 (t, J = 6.9 Hz, 1H), 3.97 (d, J = 10.4 Hz, 1H), 3.21 (dd, J = 4.9, 15.9 Hz, 1H), 2.99-3.13 (m, 2H), 2.67-2.88 (m, 2H), 2.17-2.56 (m, 8H), 2.12 (d, J = 14.6 Hz, 1H), 1.54-1.80 (m, 3H), 1.341.48 (m, 2H), 0.83 (s, 9H), 0.26 (s, 3H), 0.14 (s, 3H). MS (ESI) m/z 844.8 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo-8(pyrrolidin-2-yl)-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide (76). 1H NMR (400 MHz, CD3OD) δ 6.98 (d, J = 6.1 Hz, 1H), 4.87-4.79 (m, 1H), 4.09 (s, 1H), 3.49-3.42 (m, 2H), 3.24-2.94 (m, 9H), 2.55-2.12 (m, 6H), 1.70-1.58 (m, 1H). MS (ESI) m/z 502.23 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-3,10,12,12a-tetrahydroxy-7-methoxy-1,11-dioxo-8(pyrrolidin-2-yl)-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide (77). 1H NMR (400 MHz, CD3OD) δ 6.95 (s, 1H), 4.89-4.82 (m, 1H), 4.10 (s, 1H), 3.74 (s, 3H), 3.49-3.43 (m, 1H),
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3.38-3.32 (m, 1H), 3.24 (dd, J = 4.1, 15.1 Hz, 1H), 3.04-2.97 (m, 8H), 2.52-2.47 (m, 1H), 2.41 (t, J = 14.4 Hz, 1H), 2.29-2.16 (m, 4H), 1.71-1.61 (m, 1H). MS (ESI) m/z 514.29 (M + H). (4S,4aS,5aR,12aS)-4,7-Bis(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-8(pyrrolidin-2-yl)-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide (78). 1H NMR (400 MHz, CD3OD) δ 6.91-6.97 (m, 1H), 4.86 (m, 1H), 4.11 (s, 1H), 2.75-3.72 (m, 17H), 2.00-2.56 (m, 6H), 1.72-1.61 (m, 1H). MS (ESI) m/z 527.40 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-8(pyrrolidin-2-yl)-7-(trifluoromethoxy)-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2carboxamide (79). 1H NMR (400 MHz, CD3OD) δ 7.20 (s, 1H), 4.95-4.85 (m, 1H), 4.11 (s, 1H), 3.54-3.45 (m, 2H), 3.20 (dd, J = 5.2, 15.5 Hz, 1H), 3.10-2.95 (m, 2H), 3.05 (s, 3H), 2.96 (s, 3H), 2.55-2.15 (m, 6H), 1.65 (dd, J = 13.4, 24.5 Hz, 1H). MS (ESI) m/z 568.1 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-8(pyrrolidin-2-yl)-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-Carboxamide (86).
1
H NMR
(400 MHz, CD3OD) δ 6.94 (s, 1H), 6.91 (s, 1H), 4.64-4.55 (m, 1H), 4.12 (s, 1H), 3.55-3.40 (m, 2H), 3.16-2.83 (m, 9H), 2.57 (t, J = 14.0 Hz, 1H), 2.55-2.45 (m, 1H), 2.32-2.08 (m, 4H), 1.661.52 (m, 1H). MS (ESI) m/z 484.14 (M + H). 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).34 Cation-adjusted Mueller Hinton broth was obtained from BBL (cat. no. 212322, Becton Dickinson, Sparks, MD), prepared fresh and kept at 4 °C prior to testing. Defibrinated horse blood (cat. no. A0432, PML Microbiologicals, Wilsonville, OR) was used to supplement medium, as appropriate. All test methods met acceptable standards
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based on recommended quality control ranges for all comparator antibiotics and the appropriate ATCC quality control strains. Animal Efficacy Models. All animal efficacy models were performed at University of North Texas Health Science Center, Fort Worth, TX. Immunocompetent Lung Infection Model. Female BALB/c mice weighing 18 to 20 grams were infected with ~ 2 x 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 post-infection mice were treated with compound 43c, tigecycline or amikacin intravenously, or tobramycin intranasally. Mice (n = 6 per group) were treated with each drug concentration. Twenty-four hours 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/gram lung data was plotted using GraphPad Prism. Mean and standard deviations were calculated per dose group and statistical significance of dose group vs. T = 0 h or T = 24 h controls was determined by nonparametric Mann-Whitney analysis using GraphPad Prism. Immunocompetent Thigh Infection Model. Groups of 5 female specific-pathogen-free CD-1 mice weighing 22 ± 2 g were used. On day 0, animals were inoculated intramuscularly (0.1 mL/thigh) with 3-5 x 106 CFU/mouse of P. aeruginosa PA694, a recent UTI isolate (Eurofins Medinet), into the right thigh. Two groups did not receive drug treatment and thighs were harvested at 2 h post-infection. Remaining mice were administered intravenously with either
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vehicle, compound 43c, or meropenem at 2 and 12 h post-infection. The muscle of the right thigh of each animal was harvested at 24 h post-infection. 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. Mean and standard deviations were calculated per dose group and statistical significance of dose group vs. T = 0 h or T = 24 h controls was determined by non-parametric Mann-Whitney analysis using GraphPad Prism. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Additional schemes illustrating synthesis of compounds 76, 77, 78, 79, and 86 (PDF) SMILES molecular formula strings (CSV) AUTHOR INFORMATION Corresponding Author Email:
[email protected]; Phone: (617) 715-3582; Fax: (617) 926-3557. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS We thank Professor Andrew Myers, Dr. Eric Gordon, and Dr. 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. ABBREVIATIONS USED
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Boc, t-butoxycarbonyl; CFU, colony forming units; DCE, 1,2-dichloroethane; DMAP, 4dimethylaminopyridine; DMF, N,N-dimethylformamide; Dppf, (diphenylphosphino)ferrocene; IV, intravenous; LDA, lithium diisopropylamide; LiHMDS, lithium bis(trimethylsilyl)amide; MDR, multidrug-resistant; MIC, minimum inhibitory concentration; MTBE, methyl tert-butyl ether; MW, molecular weight; n-BuLi, n-butyllithium; NCS, N-chlorosuccinimide; NMP, 1Methyl-2-pyrrolidinone; NT, not tested; SAR, structure activity relationships; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TMEDA, N,N,N′,N′-tetramethylethylenediamine. REFERENCES (1) (a) Duggar, B. M. Aureomycin: A Product of the Continuing Search for New Antibiotics. Ann. N. Y. Acad. Sci. 1948, 51, 177-181; (b) Duggar, B. M. Aureomycin and Preparation of Same, US2482055, Sept 13, 1949. (2) Conover, L H. Discovery of Drugs from Microbiological Sources. Drug Discovery, Science and Development in a Changing Society: American Chemical Society: Washington, DC, 1971; Vol. 108. pp 33−80. (3) Hlavka, J. J.; Ellestad, G. A.; Chopra, I. Tetracyclines. In Kirk−Othmer Encyclopedia of Chemical Technology, 4th ed.; John Wiley & Sons, Inc.: New York, 1992; Vol. 3, pp 331−346. (4) Hawkey, P. M. The Growing Burden of Antimicrobial Resistance. J. Antimicrob. Chemother. 2008, 62 (Suppl.1), i1−i9. (5) Grossman T. H. Tetracycline Antibiotics and Resistance. Cold Spring Harb. Perspect. Med. 2016, 6(4), a25387. (6) Levy, S. B.; McMurry, L. Detection of an Inductive Membrane Protein Associated with RFactor Mediated Tetracycline Resistance. Biochem. Biophys. Res. Commun. 1974, 56, 1080−1088. 46 ACS Paragon Plus Environment
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(7) Levy, S. B. Evolution and Spread of Tetracycline Resistance Determinants. J. Antimicrob. Chemother. 1989, 24, 1 – 3. (8) Lomovskaya, O.; Watkins, W. J. Efflux Pumps: Their Role in Antibacterial Drug Discovery. Curr. Med. Chem. 2001, 8, 1699−1711. (9) Connell, S. R.; Tracz, D. M.; Nierhaus, K. H.; Taylor, D. E. Ribosomal Protection Proteins and Their Mechanism of Tetracycline Resistance. Antimicrob. Agents Chemother. 2003, 47, 3675−3681. (10) Yonath, A. Antibiotics Targeting Ribosomes: Resistance, Selectivity, Synergism, and Cellular Regulation. Annu. Rev. Biochem. 2005, 74, 649−679. (11) Martell, M. J.; Boothe, J. H. The 6-Deoxytetracyclines. VII. Alkylated Aminotetracyclines Possessing Unique Antibacterial Activity. J. Med. Chem. 1967, 10, 44−46. (12) Church, R. F. R.; Schaub, R. E.; Weiss, M. J. Synthesis of 7-Dimethylamino-6-demethyl-6deoxytetracycline (Minocycline) via 9-Nitro-6-demethyl-6-deoxytetracycline. J. Org. Chem. 1971, 36, 723−725. (13) Sum, P.-E.; Lee, V. J.; Testa, R. T.; Hlavka, J. J.; Ellestad, G. A.; Bloom, J. D.; Gluzman, Y.; Tally, F. P. Glycylcyclines. 1. A New Generation of Potent Antibacterial Agents through Modification of 9-Aminotetracyclines. J. Med. Chem. 1994, 37, 184−188. (14) Jones, C. H.; Petersen, P. Tigecycline: A Review of Preclinical and Clinical Studies of the First-in-Class Glycylcycline Antibiotic. Drugs Today 2005, 41, 637−659. (15) Olson, M. W.; Ruzin, A.; Feyfant, E.; Rush, T. S. III; O’Connell, J.; Bradford, P. A. Functional, Biophysical, and Structural Basis for Antibacterial Activity of Tigecycline. Antimicrob. Agents Chemother. 2006, 50, 2156−2166.
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Z.;
Ronn,
M.;
Xiao,
X.-Y.
Synthesis
and
Biological
Evaluation
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8-
Aminomethyltetracycline Derivatives as Novel Antibacterial Agents. J. Med. Chem. 2013, 56, 8112−8138.
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(22) Clark, R. B.; He, M.; Fyfe, C.; Lofland, D.; O’Brien, W. J.; Plamondon, L.; Xiao, X.-Y. 8Azatetracyclines: Synthesis and Evaluation of a Novel Class of Tetracycline Antibacterial Agents. J. Med. Chem. 2011, 54, 1511−1528. (23) Sun, C.; Hunt, D. K.; Clark, R. B.; Lofland, D.; O’Brien, W. J.; Plamondon, L.; Xiao, X.-Y. Synthesis and Antibacterial Activity of Pentacyclines: A Novel Class of Tetracycline Analogs. J. Med. Chem. 2011, 54, 3704−3731. (24) Sun, C.; Hunt, D. K.; Chen, C.-L.; Deng, Y.; He, M.; Clark, R. B.; Fyfe, C.; Grossman, T. H.; Sutcliffe, J. A.; Xiao, X.-Y. Design, Synthesis, and Biological Evaluation of Hexacyclic Tetracyclines as Potent, Broad Spectrum Antibacterial Agents. J. Med. Chem. 2015, 58, 4703−4712. (25) Zhanel, G. G.; Cheung, D.; Adam, H.; Zelenitsky, S.; Golden, A.; Schweizer, F.; Gorityala, B.; Lagace-Wiens, P. R. S.; Walkty, A.; Gin, A. S.; Hoban, D. J.; Karlowsky, J. A. Review of Eravacycline, a Novel Fluorocycline Antibacterial Agent. Drugs 2016, 76(5), 567 – 588. (26) Gordell, G. A. ed; The Alkaloids: Chemistry and Biology, Academic Press, San Diego, 2000, vol 54. (27) Miyaura, N.; Suzuki, A. Palladium-Catalyzed Cross-Coupling Reactions of Organoboron Compounds. Chem. Rev. 1995, 95(7), 2457–2483. (28) Brubaker, J. D.; Myers, A. G. A Practical, Enantioselective Synthetic Route to a Key Precursor to the Tetracycline Antibiotics. Org. Lett. 2007, 9, 3523−3525. (29) Abdel-Magid, A. F.; Carson, K. G.; Harris, B. D.; Maryanoff, C. A.; Shah, R. D. Reductive Amination of Aldehydes and Ketones with Sodium Triacetoxyborohydride. Studies on Direct and Indirect Reductive Amination Procedures. J. Org. Chem. 1996, 61, 3849-3862.
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(30) Giovannini, A.; Savoia, D.; Umani-Ronchi, A. Organometallic Ring-Opening Reactions of N-Acyl and N-Alkoxycarbonyl Lactams. Synthesis of Cyclic Imines. J. Org. Chem. 1989, 54, 228 – 234. (31) Janssen, D. E.; Wilson, C. V. Organic Syntheses; J. Wiley & Sons: New York, 1963; Collect. Vol. IV, p 547. (32)
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Trifluoromethylating Agent. J. Chem. Soc., Chem. Commun. 1989, 705–706. (33) Brinner, K. M.; Ellman, J. A. A Rapid and General Method for the Asymmetric Synthesis of 2-Substituted Pyrrolidines Using tert-Butanesulfinamide. Org. Biomol. Chem. 2005, 3, 2109 – 2113. (34) Clinical and Laboratory Standards Institute (CLSI). Methods for Dilution of Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically; Approved Standard - 9th Edition. CLSI document M07-A9. Clinical Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania; 2012, Vol. 32, No. 2. (35) Brodersen, D. E.; Clemons, W. M.; Carter, A. P.; Morgan-Warren, R. J.; Wimberly, B. T.; Ramakrishan, V. The Structure Basis for the Action of the Antibiotics Tetracycline, Pactamycin, and Hygromycin B on the 30S Ribosomal Subunit. Cell 2000, 103, 1143 – 1154.
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Table 1. In Vitro Antibacterial Activity of 7-Chloro-8-heterocyclyltetracyclines Cl R8
H3 C CH3 N H H OH NH2
OH
O O HO H O
O
MIC (µg/mL)b Compd.a
SA101
SA161c
SA158d
EF159d
SP160d
SP193
EC107
EC155d
KP153d
KP194
PM112
29213
tet(M)
tet(K)
tet(M)
tet(M)
8668
25922
tet(A)
tet(A)
700603
35659
13f
0.125
1
≤0.0156
1
0.5
≤0.0156
≤0.0156
1
0.5
8
25f
0.25
4
0.0313
2
0.25
≤0.0156
0.125
1
1
0.25
2
0.0313
2
0.5
≤0.0156
0.0625
2
31
0.125
0.5
0.125
0.5
4
0.25
2
30
0.125
1
0.125
1
8
0.25
TET
0.25
>32
32
>32
32
0.125
26
R8
* N CH3
PA169
AB250e
SM256e
BC240e
0.5
16
0.5
0.125
8
16
1
32
2
1
8
1
16
4
32
2
0.5
8
>32
>32
NT
16
>32
2
2
8
2
>32
>32
NT
16
>32
>32
2
8
1
>32
>32
16
32
32
>32
16
32
a
Single diastereomer unless otherwise noted. bStrains obtained from the American Type Culture Collection (ATCC, Manassas, VA) unless otherwise noted. The first six strains from the left are Gram-
positive bacteria. The last nine strains are Gram-negative bacteria. Strains with tet(A), tet(K), or tet(M) noted underneath are tetracycline-resistant strains. SA, Staphylococcus aureus; EF, Enterococcus faecalis; SP, Streptococcus pneumoniae; EC, Escherichia coli; KP, Klebsiella pneumoniae; PM, Proteus mirabilis; PA, Pseudomonas aeruginosa; 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 R. K. Ernst’s Laboratories at University of Maryland. fA mixture of two diastereomers (~ 1:1). TET=tetracycline.
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Table 2. In Vitro Antibacterial activity of 7-R-8-(2-pyrrolidinyl)tetracyclines R7 R8
H3 C CH3 N H H OH NH2
OH
O O HO H O
O MIC (µg/mL)b
Compd.a
R8
R7
SA101
SA161c
SA158d
EFs327e
EFm404e
SP160d
EC107
EC155d
KP457e
29213
tet(M)
tet(K)
tet(M)
tet(M)
tet(M)
25922
tet(A)
CTX-M-15
PM385e
PA555
EC1603e
BAA-47
tet(A)
AB250f
SM256f
BC240f
43c
CF3
0.0625
4
≤0.0156
1
0.25
≤0.0156
≤0.0156
2
0.125
0.5
4
8
2
1
8
43d
CF3
0.125
16
0.0312
16
4
1
≤0.0156
2
0.125
0.5
8
8
2
1
8
43a
CF3
0.125
2
≤0.0156
NT
NT
≤0.0156
0.0312
0.5
2
NT
8
NT
0.25
0.125
4
43f
CF3
0.0312
1
≤0.0156
0.5
0.125
≤0.0156
≤0.0156
0.5
4
4
32
4
1
1
8
43g
CF3
0.25
1
0.125
1
1
≤0.0156
1
2
16
8
>32
32
4
4
16
43h
CF3
2
2
1
1
1
0.5
4
>32
>32
>32
>32
>32
>32
>32
>32
43b
CF3
0.5
1
0.125
1
1
0.0312
1
2
16
8
>32
32
4
4
16
43e
CF3
0.125
2
0.0625
0.5
0.25
≤0.0156
0.125
1
8
4
32
16
2
4
16
44a
CF3
0.125
1
≤0.0156
1
0.25
≤0.0156
≤0.0156
0.5
0.25
0.5
8
8
0.5
0.25
4
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44b
CF3
0.5
1
0.125
2
1
0.0312
0.5
2
8
4
>32
16
2
2
8
45a
CF3
0.125
8
≤0.0156
1
0.5
≤0.0156
≤0.0156
2
1
2
8
2
1
0.5
4
45b
CF3
0.5
4
0.0312
4
2
≤0.0156
0.0625
2
4
8
32
2
8
1
16
86
H
4
>32
0.5
>32
16
8
0.25
4
0.5
0.25
16
4
>32
32
>32
76
F
0.5
>32
0.125
32
4
1
0.125
16
0.5
0.25
8
32
32
8
>32
78g
N(CH3)2
2
32
1
NT
NT
2
2
32
NT
NT
NT
NT
>32
32
>32
77
OCH3
8
>32
2
32
16
4
2
>32
2
4
32
>32
>32
>32
>32
79
OCF3
0.125
2
≤0.0156
1
0.125
≤0.0156
≤0.0156
8
0.25
1
8
16
2
2
8
TET
0.25
>32
32
>32
32
32
1
>32
8
>32
>32
>32
>32
32
32
TGC
0.0625
0.125
0.125
0.125
0.0312
0.0156
0.125
1
1
4
16
4
8
2
16
Single diastereomer unless otherwise noted. bStrains obtained from the American Type Culture Collection (ATCC, Manassas, VA) unless otherwise noted. The first six strains from the left are Gram-
a
positive bacteria. The last nine strains are Gram-negative bacteria. Strains with tet(A), tet(K), or tet(M) 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). TET=tetracycline; TGC=tigecycline.
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Figure 2. In Vivo Efficacy of Compoune 43c in: (A) PA1145 Mouse Lung Infection Model; (B) PA694 Mouse Thigh Infection Model. T=0, mean log10 CFU in lung or thigh prior to first compound administration; T=24 hr, mean log10 CFU in lung or thigh 24 hours post-bacterial challenge.
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Table of Contents Graphic
Pseudomonas aeruginosa PA1145 MIC (µg/mL)
Log10CFU Reduction @ 40 mg/kg
2
3.7
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