Synthesis and Biological Evaluation of 8-Aminomethyltetracycline

Article pubs.acs.org/jmc

Synthesis and Biological Evaluation of 8‑Aminomethyltetracycline Derivatives as Novel Antibacterial Agents Roger B. Clark,*,† Minsheng He,† Yonghong Deng,† Cuixiang Sun,† Chi-Li Chen,† Diana K. Hunt,† William J. O’Brien,‡ Corey Fyfe,‡ Trudy H. Grossman,‡ Joyce A. Sutcliffe,‡ Catherine Achorn,‡ Philip C. Hogan,§ Christopher E. Katz,§ John Niu,§ Wu-Yan Zhang,§ Zhijian Zhu,§ Magnus Ronn,§ and Xiao-Yi Xiao† †

Discovery Chemistry, ‡Microbiology, and §Process Chemistry R&D, Tetraphase Pharmaceuticals, Inc., 480 Arsenal Street, Watertown, Massachusetts 02472, United States

ABSTRACT: The C-8 position of the tetracyclines has been largely underexplored because of limitations in traditional semisynthetic techniques. Employing a total synthetic approach allowed for modifications at the C-7 and C-8 positions, enabling the generation of structure−activity relationships for overcoming the two most common tetracycline bacterial-resistance mechanisms: ribosomal protection (tet(M)) and efflux (tet(A)). Ultimately, several compounds were identified with balanced activity against both Gram-positive and Gram-negative bacteria, including pathogens bearing both types of tetracycline-resistance mechanisms. Compounds were screened in a murine systemic infection model to rapidly identify compounds with oral bioavailability, leading to the discovery of several compounds that exhibited efficacy when administered orally in murine pyelonephritis and pneumonia models.

■

INTRODUCTION The ability of bacteria to rapidly adapt and develop resistance to the current arsenal of approved antibacterial agents necessitates the continuous discovery and development of new antibiotics.1−4 Despite significant effort in the pharmaceutical industry, novel classes of antibacterials, in particular those that target novel mechanisms, have generally failed to make it through clinical trials and onto the market.5 Thus, attention continues to be given to known classes of compounds with proven mechanisms of action as well as proven safety and efficacy. One of the most clinically important antibacterial mechanisms involves the blockade of protein synthesis by inhibition of the bacterial ribosome, the target of several major classes of antibacterial agents, including the aminoglycosides, the macrolides, the oxazolidinones, and the tetracyclines.1d,6 Within the tetracycline class, this approach has led to the 7,9disubstituted compounds tigecycline (2, marketed, Pfizer),7 omadacycline (3, phase 3, Paratek Pharmaceuticals),8 and eravacycline (4, phase 3, Tetraphase Pharmaceuticals),9 all of which have shown marked improvements in activity against the two major bacterial resistance mechanisms identified for tetracyclines: (1) active transport from bacterial cells via efflux pumps3d,10 (tet(A−E)10a,b and tet(K−L))10c and (2) ribosomal protection (tet(M−O)).11 Previously reported structure−activity relationships (SAR)12,13 as well as the recently solved tetracycline 30Sribosome cocrystal structure have shown that the hydroxyl and carbonyl groups of the lower and right-hand portions of the © 2013 American Chemical Society

molecule interact directly with the ribosome through an extensive series of hydrogen bonds and are required for tight ribosomal binding.14 In contrast, the left-hand region, encompassing C-7, C-8, and C-9 of the D-ring, is not directly involved in binding to the ribosome and can tolerate substitution without loss in activity. Indeed, C-9 substituents can dramatically increase both intrinsic bacterial potency and activity against tetracycline-resistance mechanisms. The earlier generations of tetracyclines were prepared by semisynthesis, largely limiting their scope to substitution at the C-7 and C-9 positions and functional groups that could be introduced by, or were derived from, electrophilic aromatic substitution reactions because of the presence of the highly electron-rich, ortho-, paradirecting C-10 hydroxyl group.15 The total synthetic methodology developed by Myers and co-workers16−18 and further expanded by Tetraphase has opened up this chemical space significantly, allowing for easier access to a more diverse set of substitutions at C-7 and C-919 as well as for the introduction of heterocyclic20 and polycyclic21 ring systems (Figure 1). The key step involves a Michael−Dieckmann reaction between a lefthand side D-ring precursor 5 and the AB-ring precursor, enone 6 (Figure 1). In addition to influencing in vitro potency and susceptibility to tetracycline-specific resistance mechanisms, this has also allowed for the modulation of ADMET properties, leading to enhanced in vivo activity. Received: August 6, 2013 Published: September 18, 2013 8112

dx.doi.org/10.1021/jm401211t | J. Med. Chem. 2013, 56, 8112−8138

Journal of Medicinal Chemistry

Article

Figure 1. Tetracycline (1) and its semisynthetic (2 and 3) and fully synthetic (4 and 7) derivatives.

Scheme 1. Synthesis of 7-Fluoro-8-aminomethyltetracyclinesa

a Reagents and conditions: (i) Br2, AcOH; (ii) BnBr, K2CO3, acetone; (iii) LDA, THF, −78 °C, then DMF, −78 to −20 °C; (iv) CH3OH, (CH3O)3CH, TsOH-H2O, reflux; (v) (a) LDA, TMEDA, THF, −78 °C and (b) enone 6, −78 to −10 °C; (vi) TFA, CH2Cl2; (vii) R1NH2 or R1R2NH, AcOH, Na(OAc)3BH, DCE; (viii) for R1NH2 in step vii: R2′CHO, AcOH, Na(OAc)3BH, DCE; (ix) HF, 1,4-dioxane; and (x) H2, Pd/C, HCl, CH3OH, 1,4-dioxane.

groups introduced via Suzuki reactions from the bromide precursors. Previous reports by Myers and co-workers and ourselves have described C-8, C-9 polycyclic derivatives prepared by total synthesis.18,21 These analogs demonstrated reasonable antibacterial activity, particularly against Grampositive bacteria, indicating that substitution at the C-8 position is tolerated. Herein, we detail our work on a series of 8aminomethyl substituted tetracycline derivatives, including both

Because of the limited accessibility by semisynthesis, the C-8 position of the tetracyclines has been largely underexplored. Due to the aforementioned reactivity of the C-7 and C-9 positions, the few examples of substitution at the C-8 position generally require substitution at the C-9 position and amino substitution at either C-7 or C-9.22 In a handful of examples, the C-9 amino group has been removed after introduction of a halide at the C-8 position. In all of these examples, reported C8 analogs have been limited to halogen, hydroxyl, and aryl 8113

dx.doi.org/10.1021/jm401211t | J. Med. Chem. 2013, 56, 8112−8138

Journal of Medicinal Chemistry

Article

Scheme 2. Alternative Synthesis of 7-Fluoro-8-aminomethyltetracyclinesa

a

Reagents and conditions: (i) R1NH2 or R1R2NH, AcOH, Na(OAc)3BH, DCE; (ii) R1NH2 or R1R2NH, Ti(O-iPr)4, DCE, then NaBH4, CH3OH; (iii) LDA, TMEDA, THF, −78 °C, then enone 6, −78 to −10 °C; (iv) for R1NH2 in step ii: R2′CHO, AcOH, Na(OAc)3BH, DCE; (v) HF, 1,4dioxane; and (vi) H2, Pd/C, HCl, CH3OH, 1,4-dioxane.

Scheme 3. Synthesis of 7-Chloro-8-aminomethyltetracycline D-Ring Precursora

Reagents and conditions: (i) Br2, AcOH, CH3OH, 0 °C to rt; (ii) NaNO2, HCl, water, 0 °C, then Na2CO3, CuCN, NaCN, water, 0 to 50 °C; (iii) DIBALH, THF, −78 °C to rt; (iv) NaClO2, NaH2PO4, 2-methyl-2-butene, t-BuOH, water; (v) (COCl)2, DMF, CH2Cl2, then PhOH, Et3N, DMAP, CH2Cl2; (vi) NCS, CH3CN, 60 °C; (vii) BBr3, CH2Cl2, −78 to 0 °C; (viii) BnBr, K2CO3, acetone; (ix) i-PrMgCl-LiCl, THF, −78 to 0 °C, then DMF, rt; and (x) CH3OH, (CH3O)3CH, TsOH-H2O, 65 °C. a

−10 °C to provide the fully protected precursor 12.18 Deprotection of the acetal with trifluoroacetic acid (TFA) in CH2Cl2 proceeded with simultaneous removal of one of the benzyl protecting groups. Using crude compound 13, the amino substituent was then introduced by reductive amination with either primary or secondary amines in the presence of AcOH and Na(OAc)3BH to give aminomethyl compounds 14.23 When primary amines were used, the resulting secondary amines could be further functionalized by reductive alkylation with aldehydes to give tertiary amines. Desilylation of 14 with aqueous HF and catalytic hydrogenation in the presence of palladium on carbon gave 7-fluoro-8-aminomethyltetracyclines 15a−v.18 An alternative strategy for the synthesis of 7-fluoro-8aminomethyltetracyclines involved the introduction of the amino group prior to the Michael−Dieckmann reaction (Scheme 2). This method was particularly useful for the introduction of sterically hindered tertiary amines, for preparing a large set of compounds in which R1 remained constant while R2 was varied, and for scaling up larger quantities of a single

in vitro SAR and in vivo efficacy in several murine models of bacterial infection.

■

CHEMISTRY

Among the most common methods for preparing benzylic amines is the reductive alkylation of amines with benzaldehydes. During the course of our work in the 7-fluorotetracyclines, we observed that the D-ring precursor 9 (Scheme 1) was metalated by lithium diisopropylamide (LDA) on the open ring position rather than as expected at the methyl group required for the Michael−Dieckmann reaction. Using this to our advantage for the synthesis of 7-fluoro-8-aminomethyltetracylcines, the anion could be trapped with DMF to provide benzaldehyde 10 in high yield. Compound 9 was readily prepared by bromination of phenol 89a followed by benzylation. Compound 10 was protected under standard conditions to give dimethylacetal 11. The Michael−Dieckmann reaction was then carried out in good yield by treatment of 11 with LDA and N,N,N′,N′-tetramethylethylenediamine (TMEDA) in THF at −78 °C followed by the addition of enone 6 and warming to 8114

dx.doi.org/10.1021/jm401211t | J. Med. Chem. 2013, 56, 8112−8138

Journal of Medicinal Chemistry

Article

Scheme 4. Synthesis of 7-Methoxy-8-aminomethyltetracycline D-Ring Precursora

Reagents and conditions: (i) BBr3, CH2Cl2, −78 to 0 °C; (ii) PhI(OAc)2, CH3OH, 1,4-dioxane, then Zn powder, AcOH; (iii) Boc2O, DMAP, CH2Cl2; (iv) i-PrMgCl-LiCl, THF, 0 °C, then DMF, rt; and (v) CH3OH, (CH3O)3CH, TsOH-H2O, 65 °C.

a

Scheme 5. Synthesis of 7-Trifluoromethyl-8-aminomethyltetracycline D-Ring Precursora

Reagents and conditions: (i) 2,4,6-trivinylcyclotriboroxane-pyridine complex, Pd(Ph3P)4, K2CO3, 1,4-dioxane, water, reflux; (ii) O3, CH2Cl2, −78 °C, then (CH3)2S, −78 °C to rt; (iii) Br2, AcOH; (iv) BBr3, CH2Cl2, −78 °C to rt; (v) BnBr, K2CO3, DMF; (vi) CH3O2CCF2SO2F, CuI, DMF, 100 °C; and (vii) CH3OH, (CH3O)3CH, TsOH-H2O, 65 °C. a

magnesium−halogen exchange with i-PrMgCl-LiCl and subsequent alkylation with DMF.26 The final 7-chloro dimethylacetal D-ring precursor 26 was formed upon refluxing in CH3OH in the presence of TsOH-H2O and trimethylorthoformate. The 7-methoxy D-ring precursor was also prepared from bromo intermediate 22b (Scheme 4). Phenol 27 was first prepared by demethylation of 22b with BBr3 in 90% yield. This was oxidized to the quinone ketal with PhI(OAc)2 and subsequently reduced to methoxyphenol 28 with zinc and acetic acid, albeit in moderate yield.27 Following Boc protection of the phenol, compound 29 was converted to aldehyde 30 by magnesium−halide exchange and DMF quench. Treatment with TsOH-H2O and trimethylorthoformate in CH3OH gave the acetal product along with partial loss of the Boc protecting group. The crude mixture was reprotected using Boc2O and 4dimethylaminopyridine (DMAP) to give 7-methoxy D-ring precursor 31. The 7-trifluoromethyl D-ring precursor was prepared according to Scheme 5. A Suzuki coupling28 of compound 22b with 2,4,6-trivinylcyclotriboroxane-pyridine complex and Pd(Ph3P)4 gave styrene 32, which was then converted to aldehyde 33 by ozonolysis. Bromination, demethylation, and benzylation then gave aldehyde 34. The trifluoromethyl group was introduced by CuI-mediated coupling between 34 and

analogue. Thus, benzaldehyde 10 was treated with primary or secondary amines in the presence of AcOH and Na(OAc)3BH to give benzylic amines 16. For more sterically hindered amines, aldehyde 10 and the amine were treated with Ti(iPrO)4 in 1,2-dichloroethane (DCE) followed by reduction with NaBH4 in CH3OH.24 Standard Michael−Dieckmann reaction conditions gave the protected compounds 17. In the case of secondary amines, a second amino substituent could be introduced by reductive alkylation with aldehydes in the presence of AcOH and Na(OAc)3BH to give tertiary amines. Compounds 17 and 18 were then deprotected as described above to yield 7-fluoro-8-aminomethyltetracyclines 15w−yy. Similar approaches were taken for the 7-chloro, 7-methoxy, and 7-trifluoromethyl compounds, with analogues generally prepared using the dimethylacetal derivatives, analogous to the route outlined in Schemes 1 and 2. Thus, aniline 19 was brominated, and the aniline was then diazotized and converted to cyano compound 21 in good overall yield (Scheme 3).25 The material was then reduced with diisobutylaluminum hydride (DIBALH) and was hydrolyzed to the aldehyde. Oxidation to benzoic acid 22a with NaClO2 and a two-step esterification process with oxalyl chloride followed by phenol gave phenyl ester 22b. Chlorination with N-chlorosuccinimide (NCS), demethylation with BBr3, and benzylation of the resulting phenol gave compound 24. Aldehyde 25 was then prepared by 8115

dx.doi.org/10.1021/jm401211t | J. Med. Chem. 2013, 56, 8112−8138

Journal of Medicinal Chemistry

Article

Scheme 6. Synthesis of 7-Substituted-8-Aminomethyltetracyclinesa

Reagents and conditions: (i) LDA, TMEDA, THF, −78 °C, then enone 6, −78 to −10 °C; (ii) 6 N HCl, THF, or TFA, CH2Cl2, water; (iii) R1NH2 or R1R2NH, AcOH, Na(OAc)3BH, DCE; (iv) for R1NH2 in step iii: R2′CHO, AcOH, Na(OAc)3BH, DCE; (v) HF, 1,4-dioxane; and (vi) H2, Pd/C, HCl, CH3OH, 1,4-dioxane. a

Scheme 7. Synthesis of 7-Dimethylamino-8-aminomethyltetracyclinesa

Reagents and conditions: (i) HNO3, H2SO4; (ii) (COCl)2, CH2Cl2, then PhOH, Et3N, DMAP, CH2Cl2; (iii) BBr3, CH2Cl2, −78 to 0 °C; (iv) BnBr, K2CO3, acetone; (v) Zn, AcOH; (vi) aqueous formaldehyde, AcOH, Na(OAc)3BH, CH3CN; (vii) LDA, TMEDA, THF, −78 °C, then enone 6, −78 to −10 °C; (viii) PhLi, n-BuLi, THF, −78 °C, then DMF; (ix) R1NH2 or R1R2NH, AcOH, Na(OAc)3BH, DCE; (x) for R1NH2 in step ix: R2′CHO, AcOH, Na(OAc)3BH, DCE; (xi) HF, 1,4-dioxane; and (xii) H2, Pd/C, HCl, CH3OH, 1,4-dioxane. a

methyl 2,2-difluoro-2-(fluorosulfonyl)acetate at 100 °C in DMF in high yield.29 Conversion to the dimethylacetal was carried out as described above, yielding 7-trifluoromethyl Dring precursor 35. The 7-chloro-, 7-methoxy-, and 7-trifluoromethyl-8-aminomethyltetracycline derivatives were prepared according to Scheme 6. D-ring precursors 26, 31, and 35 were lithiated with LDA in the presence of TMEDA, and the subsequent

addition of enone 6 gave the fully protected tetracycline intermediates 36. Deprotection of the acetal could be carried out either by treating with aqueous HCl in THF or aqueous TFA in CH2Cl2. These conditions generally left all of the benzyl protecting groups intact, although the Boc group on the phenol of the 7-methoxy compound was removed. Various amines were then introduced via reductive amination with primary or secondary amines and Na(OAc)3BH and AcOH. 8116

dx.doi.org/10.1021/jm401211t | J. Med. Chem. 2013, 56, 8112−8138

Journal of Medicinal Chemistry

Article

Table 1. In Vitro Antibacterial Activity of 7-Fluoro-8-aminomethyltetracyclines with Acyclic Amines

MIC (μg/mL)a

cmpd

R1

R2

15w 15x 15a 15b 15c 15d 15e 15f 15g 15y 15z 15aa 15bb 15cc 15dd 15ee 15ff 15gg 15hh 15ii 15jj 15kk 15ll 15h 15i 15mm 15nn

CH3 CH3 Et Et Et 2-fluoroethyl n-Pr n-Pr n-Pr cyclopropyl cyclopropyl i-Pr i-Pr t-Bu t-Bu t-Bu 2-methylpropyl 2-methylpropyl 2-methylpropyl 2,2-dimethylpropyl 2,2-dimethylpropyl 2,2-dimethylpropyl t-amyl Ph benzyl cyclopentyl cyclopentyl tetracycline omadacycline tigecycline

H CH3 H CH3 Et Et H CH3 Et CH3 Et H CH3 H CH3 Et H CH3 Et H CH3 Et CH3 H CH3 H CH3

b

SA158

c

SA100

SA161

ATCC 13709

MRSA tet(M)

tet(K)

1 0.25 0.25 0.25 0.25 0.5 0.13 0.25 0.5 0.25 0.25 0.25 0.13 0.25 0.13 0.13 0.13 0.016 0.25 0.25 0.063 0.13 0.013 0.13 0.25 0.13 0.031 0.13 0.25 0.13

>32 32 32 32 16 4 32 16 32 2 1 16 16 8 8 4 16 2 1 4 0.13 0.25 4 0.13 0.25 8 8 64 1 0.13

1 0.13 0.25 0.13 0.13 0.25 0.13 0.13 0.25 0.13 0.13 0.13 0.13 0.13 0.063 0.063 0.031 0.031 0.13 0.031 0.063 0.13 0.016 NT 0.13 0.063 0.031 32 0.25 0.13

SP160c

EC107

tet(M)

ATCC 49619

>32 16 32 16 16 4 16 16 16 2 1 32 16 16 16 4 8 2 1 4 0.13 0.25 4 0.13 0.25 16 8 64 0.25 0.063

0.25 0.031 0.063 0.031 0.031 0.031 0.016 0.016 0.063 0.063 0.13 0.063 0.031 0.031 0.016 0.016 0.016 0.016 0.031 0.016 0.13 0.5 0.016 0.5 0.25 0.031 0.016 0.25 0.031 0.016

EF159

c

SP106

EC155c

KP109

KP153c

tet(M)

ATCC 25922

AB110

PA111

tet(A)

ATCC 13883

tet(A)

ATCC 19606

ATCC 27853

4 1 2 1 1 4 1 2 4 4 2 1 1 1 1 0.5 0.5 2 2 0.5 1 4 1 8 2 0.5 1 32 0.063 0.016

0.5 0.25 0.25 0.13 0.25 1 0.25 0.13 0.5 0.5 0.5 0.25 0.13 0.25 0.13 0.25 0.13 0.13 0.5 0.13 1 2 0.13 32 1 0.13 0.13 1 1 0.13

>32 8 8 4 2 4 8 4 8 16 8 8 1 2 0.5 1 2 2 4 2 2 8 0.5 >32 4 2 1 >64 16 1

1 0.5 0.5 0.25 0.5 2 0.25 0.25 1 1 1 0.5 0.25 0.5 0.25 0.5 0.25 0.5 1 0.5 1 4 0.25 >32 2 0.25 0.25 2 2 0.25

32 4 8 4 2 16 8 2 4 8 4 8 1 2 0.5 1 2 2 4 1 2 8 0.5 >32 8 2 2 >64 16 1

8 0.25 4 0.13 0.13 0.063 0.5 0.016 0.016 0.016 0.031 2 0.063 1 0.031 0.016 0.031 0.016 0.031 0.016 0.031 0.5 0.016 2 0.13 0.25 0.016 1 1 0.5

16 4 8 8 16 >32 8 32 >32 32 32 8 16 16 16 32 16 32 >32 32 >32 >32 32 >32 >32 16 32 16 >32 8

Strains were obtained from the American Type Culture Collection (ATCC, Manassas, VA) unless otherwise noted. The first six strains from the left are Gram-positive strains. The last six strains are Gram-negative strains. Strains with tet(A), tet(K), or tet(M) noted underneath are tetracyclineresistant strains. SA, Staphylococcus aureus; EF, Enterococcus faecalis; SP, Streptococcus pneumoniae; EC, Escherichia coli; AB, Acinetobacter baumannii; PA, Pseudomonas aeruginosa; and KP, Klebsiella pneumoniae. bObtained from Micromyx (Kalamazoo, MI). cObtained from Marilyn Roberts’ laboratory at the University of Washington. a

followed by reductive methylation with aqueous formaldehyde and Na(OAc)3BH. Michael−Dieckmann reaction under standard conditions gave protected tetracycline intermediate 46. The aldehyde was then introduced by deprotonation of the enolic proton with PhLi, lithium−bromine exchange with n-BuLi, and quenching with DMF to give 47. Reductive amination conditions as previously described gave protected 8-aminomethyl derivatives 48, which were then deprotected under standard conditions to provide desired 7-dimethylamino-8aminomethyltetracyclines 49a−g.

Compounds derived from primary amines could be further derivatized via reductive alkylations with aldehydes and Na(OAc)3BH and AcOH. Standard HF and hydrogenation conditions gave the fully deprotected 8-aminomethyltetracyclines 39−42. In the case of the 7-chloro compounds, partial reduction of the chlorine−carbon bond was generally observed to some degree, leading to the isolation of the 7-H compounds 40a and 40c. The 7-dimethylamino-8-aminomethyltetracyclines were prepared by a slightly modified route wherein the aldehyde was introduced post-Michael−Dieckmann (Scheme 7). Nitration of benzoic acid 22a with HNO3 in H2SO4 followed by esterification gave phenyl ester 43. Demethylation with BBr3 and benzylation then gave compound 44 in 47% overall yield for the four steps. 7-Dimethylamino D-ring precursor 45 was then prepared by Zn-mediated reduction of the nitro group

■

BIOLOGY Compounds were initially screened against a panel of Grampositive (Staphylococcus aureus, Enterococcus faecalis, and Streptococcus pneumoniae) and Gram-negative (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomo8117

dx.doi.org/10.1021/jm401211t | J. Med. Chem. 2013, 56, 8112−8138

Journal of Medicinal Chemistry

Article

nas aeruginosa) bacteria that included both tetracyclinesusceptible (SA100, SP106, EC107, KP109, and AB110) and tetracycline-resistant (SA161, SA158, EF159, SP160, EC155, and KP153) strains. The tetracycline-resistant strains were selected to address the most common resistance mechanisms: ribosomal protection (tet(M)) and tetracycline-specific efflux (tet(K) and tet(A)). The P. aeruginosa strain (PA111) did not have any tetracycline-specific resistance genes but was only modestly susceptible to tetracycline (MIC = 16 μg/mL) because of the organism’s intrinsic resistance mechanisms including nonspecific efflux pumps.30 We first explored a series of 7-fluoro-8-aminomethyltetracyclines with different substitutions on the amine (Table 1). Overall, all of the compounds maintained good activity against the tetracycline-susceptible Gram-positive strains, with MIC values generally falling within two dilutions of those for tetracycline. Against the Gram-negative tetracycline-susceptible strains, several SAR trends began to emerge. For E. coli and K. pneumoniae, most of the compounds were either more potent than or equivalent to tetracycline (MIC = 1 and 2 μg/mL, respectively), with the notable exception of phenyl-substituted compound 15h (MIC = 32 and >32 μg/mL, respectively). The amine of this compound is significantly less basic (pKB ∼3.8) than most of the alkyl-substituted compounds (pKB ∼6−8), a property of several of the other compounds [15d (pKB ∼4.5), 15y (pKB ∼4.5), 15z (pKB ∼4.5), and 15i (pKB ∼5.0)] that also had relatively lower activity against these two strains.31 Another factor in the reduced activity against these two strains appears to be the size of the substituents, with larger alkyl groups (i.e., compounds 15jj and 15kk) leading to reduced Gram-negative activity overall. Activity against A. baumannii (AB110) appeared to follow the opposite trend, with larger substituents yielding improved activity. For example, small secondary amines 15w, 15a, 15e, 15aa, and 15cc all had significantly lower activity against A. baumannii when compared to their N-CH3 (15x, 15b, 15f, 15bb, and 15dd) or N-Et (15b, 15c, 15g, and 15ee) derivatives. When the size of the alkyl group increased further (15ff and 15ii), the activity became similar to the tertiary amine derivatives (15gg, 15hh and 15jj, 15kk). Basicity also appeared to have little influence on activity against A. baumannii, with compounds 15c (pKB ∼5.8), 15d (pKB ∼4.5), 15f (pKB ∼5.8), and 15y (pKB ∼4.5) having nearly identical activity. Activity against P. aeruginosa was generally modest to poor, and small alkyl groups were clearly favored. Only the smallest substituents (CH3, Et, n-Pr, and i-Pr) gave compounds that were more active than tetracycline (MIC = 16 μg/mL), and only the dimethyl substituted compound 15x (MIC = 4 μg/mL) had an MIC that was two-dilutions better. Clear SAR trends could also be seen for activity of the 7fluoro-8-aminomethyltetracycline derivatives against bacterial strains with known tetracycline resistance mechanisms. For the S. aureus (SA161) tet(M) and E. faecalis (EF159) tet(M) strains, larger alkyl groups generally led to improved activity. For example, in the series of secondary amines, activity followed the sequence of CH3 < Et, n-Pr, i-Pr < 2-methylpropyl, t-Bu < 2,2dimethylpropyl (compounds 15w, 15a, 15e, 15aa, 15ff, 15cc, and 15ii), with MIC values improving from >32 to 4 μg/mL across the series for both strains. The same general trend was also seen for the S. pneumoniae (SP160) tet(M) strain, with activity improving from 4 to 0.5 μg/mL across this series. Addition of a second substituent on the amine had minimal effect on activity for the smaller alkyl amines (i.e., compounds 15a−g and 15w−ee) against the tet(M) strains, providing at

most a one-dilution improvement with increasing size from H to CH3 to Et. For the larger 2-methylpropyl and 2,2dimethylpropyl groups, however, activity against the S. aureus and E. faecalis tet(M) strains improved significantly in going from the secondary amines (15ff and 15ii) to the N-CH3 (15gg and 15jj) and N-Et (15hh and 15kk) tertiary amine derivatives, with activity improving 16-fold in each case. In contrast, activity against the S. pneumoniae tet(M) strain decreased 4- and 8-fold within the same series. We have previously observed this divergence between S. aureus and S. pneumoniae SAR, with S. pneumoniae behaving more similarly to the Gram-negative strains than the Gram-positive strains.19 A second factor in activity against the tet(M) strains is the position of branching on the alkyl chain. Whereas branching groups on the carbon directly attached to the nitrogen atom (i.e., 15bb and 15dd) had little effect on activity relative to the unbranched analogues (i.e., 15f), branching at the 2-position (i.e., 15gg and 15jj) led to significant improvement in activity against the S. aureus and E. faecalis tet(M) strains (4- and 8-fold for 15gg vs 15dd and 32-fold for 15jj vs 15ll) as compared to the α-branched derivatives. Activity for the S. pneumoniae tet(M) strain was unchanged in either case. Finally, activity against the tet(M) strains was also affected by the basicity of the aminomethyl group. In the case of 2-fluoroethyl compound 15d, a 4-fold improvement in activity was seen against the S. aureus and E. faecalis tet(M) strains relative to the more basic ethyl compound 15c, and an 8-fold improvement was seen for the less basic cyclopropyl compound 15y versus isopropyl derivative 15bb (pKB ∼5.8). In contrast, in each case, the S. pneumoniae activity decreased by 4-fold. Similar trends were seen for the less basic aniline 15h and benzylic amine 15i, with MIC values of 0.13 and 0.25 μg/mL, respectively, for both the S. aureus and E. faecalis tet(M) strains in comparison to the more basic compounds 15 mm (pKB ∼8.4) and 15nn (pKB ∼6.7) with bulky cyclic alkyl substituents (MIC = 8−16 μg/ mL). Here again, the S. pneumoniae tet(M) activity was better in the more basic compounds. For the S. aureus strain with the tet(K) resistance gene (SA158), all of the compounds showed significantly improved activity relative to tetracycline itself (MIC = 32 μg/mL). All of the compounds had MIC values less than or equal to 0.25 μg/mL with the exception of compound 15w, which had an MIC value of 1 μg/mL. The Gram-negative bacterial strains with the tet(A) resistance gene (EC155 and KP153), however, proved to be more challenging to overcome, with SAR diverging from those seen with the tet(M) strains in several key ways. Increasing the size of the alkyl group for the secondary amines (compounds 15w, 15a, 15e, 15aa, 15cc, 15ff, and 15ii) did give improved activity for the tet(A) strains. For the corresponding N-methyl tertiary amines, the same trend was true for the smaller alkyl groups up to t-butyl (compounds 15x, 15b, 15f, 15bb, and 15dd), but the 2-methylpropyl and 2,2-dimethylpropyl compounds (15gg and 15jj) lost activity relative to the t-butyl derivative 15dd. In addition, the N-ethyl compounds (15g, 15ee, 15hh, and 15kk) were all one-to-two dilutions less active than the corresponding N-methyl analogues (15f, 15dd, 15gg, and 15jj). The position of the branching group was also critical for tet(A) activity, but the SAR was the opposite from that seen for the S. aureus and E. faecalis tet(M) strains. Thus, compounds with α-branching (15dd and 15ll) were significantly more potent against the tet(A) strains relative to the unbranched compounds (15b and 15f), giving three dilution improvements in each case. The αbranching compounds were also two-dilutions more potent 8118

dx.doi.org/10.1021/jm401211t | J. Med. Chem. 2013, 56, 8112−8138

Journal of Medicinal Chemistry

Article

Table 2. In Vitro Antibacterial Activity of 7-Fluoro-8-aminomethyltetracyclines with Cyclic Amines

a Strains were obtained from the American Type Culture Collection (ATCC, Manassas, VA) unless otherwise noted. The first six strains from the left are Gram-positive strains. The last six strains are Gram-negative strains. Strains with tet(A), tet(K), or tet(M) noted underneath are tetracyclineresistant strains. SA, Staphylococcus aureus; EF, Enterococcus faecalis; SP, Streptococcus pneumoniae; EC, Escherichia coli; AB, Acinetobacter baumannii; PA, Pseudomonas aeruginosa; and KP, Klebsiella pneumoniae. bObtained from Micromyx (Kalamazoo, MI). cObtained from Marilyn Roberts’ laboratory at the University of Washington.

8119

dx.doi.org/10.1021/jm401211t | J. Med. Chem. 2013, 56, 8112−8138

Journal of Medicinal Chemistry

Article

Table 3. In Vitro Antibacterial Activity of 7-R-8-Aminomethyltetracyclines

Strains were obtained from the American Type Culture Collection (ATCC, Manassas, VA) unless otherwise noted. The first six strains from the left are Gram-positive strains. The last three strains are Gram-negative strains. Strains with tet(A), tet(K), or tet(M) noted underneath are tetracyclineresistant strains. SA, Staphylococcus aureus; EF, Enterococcus faecalis; SP, Streptococcus pneumoniae; EC, Escherichia coli; AB, Acinetobacter baumannii; PA, Pseudomonas aeruginosa; and KP, Klebsiella pneumoniae. bObtained from Micromyx (Kalamazoo, MI). cObtained from Marilyn Roberts’ laboratory at the University of Washington. a

against the tet(A) strains than the corresponding β-branched analogues (15jj). In another divergence from the tet(M) SAR, the less basic compounds (15d, 15y, 15h, and 15i) were less active against the tet(A) strains relative to more basic derivatives. Overall, in the 7-fluoro-8-aminomethyltetracycline series with alkyl groups, compounds with either good tet(M) activity (i.e., compounds 15jj and 15i) or tet(A) activity (i.e., compounds 15dd and 15ll) were identified. The most balanced derivative for both tet(A) and tet(M) activity was compound

15gg, with MIC values of 2 μg/mL against all strains with these resistance mechanisms. A series of 7-fluoro-8-aminomethyltetracyclines with cyclic amines was also prepared (Table 2), and in general the SAR for these compounds paralleled that for the acyclic amines. Once again, all of the compounds showed good activity against the tetracycline-susceptible Gram-positive strains (SA100 and SP106), and all but the compounds with the least basic amines [15n (pKB ∼0.4), 15q (pKB ∼2.7), and 15t (pKB ∼3.9)] 8120

dx.doi.org/10.1021/jm401211t | J. Med. Chem. 2013, 56, 8112−8138

Journal of Medicinal Chemistry

Article

Table 4. In Vitro Antibacterial Activity of 7-R-8-Aminomethyltetracyclines with Balanced Activity

a Strains were obtained from the American Type Culture Collection (ATCC, Manassas, VA) unless otherwise noted. The first six strains from the left are Gram-positive strains. The last three strains are Gram-negative strains. Strains with tet(A), tet(K), or tet(M) noted underneath are tetracyclineresistant strains. SA, Staphylococcus aureus; EF, Enterococcus faecalis; SP, Streptococcus pneumoniae; EC, Escherichia coli; AB, Acinetobacter baumannii; PA, Pseudomonas aeruginosa; and KP, Klebsiella pneumoniae. bObtained from Micromyx (Kalamazoo, MI). cObtained from Marilyn Roberts’ laboratory at the University of Washington.

8121

dx.doi.org/10.1021/jm401211t | J. Med. Chem. 2013, 56, 8112−8138

Journal of Medicinal Chemistry

Article

polar suggests that greater lipophilicity of the side chain also plays a significant role in improving tet(M) activity. We next turned our attention to changes at the 7-position of the tetracycline ring (Table 3), including chloro, methoxy, dimethylamino, trifluoromethyl, and, in some cases, the 7unsubstituted analogues of several of the more promising 8aminomethyl substituents from the 7-fluoro examples above. All of the compounds showed good activity against the tetracycline-susceptible Gram-positive and Gram-negative strains, with the exception of the P. aeruginosa strain (MIC ≥ 8 μg/mL). For the S. aureus and E. faecalis tet(M) strains, the 7trifluoromethyl derivatives tended to have the best activity in a given series, whereas the 7-dimethylamino compounds tended to be the least potent. The other 7-substituted analogues generally fell in between the 7-CF3 and 7-dimethylamino compounds in activity, although the differences were generally only one-to-two dilutions. For the S. pneumoniae tet(M) activity, this trend continued for the smaller 8-aminomethyl groups (pyrrolidino and dimethylamino), with the 7-CF3 compounds (42a and 42b) having the best activity (0.5 μg/ mL). For the larger 8-aminomethyl groups, however, 7methoxy compounds 41c−e had the best activity, whereas the 7-CF3 analogs (42c−e) were one-to-two dilutions less active. The 7-dimethylamino compounds continued to be among the least active compounds in each series. For the tet(A) strains, the 7-Cl substituted compounds (39a−d) had the best activity regardless of the 8-aminomethyl substituents. Once again, the 7-CF3 compounds were among the best compounds for the smaller 8-aminomethyl groups (42a and 42b), but activity relative to the other 7-substitutions decreased with increased 8-aminomethyl substituent size (42c−e). The 7OCH3 and 7-dimethylamino compounds showed the opposite trend, with relatively poor activity for the dimethylamino (41b and 49b) and pyrrolidino (41a and 49a) compounds and improved activity for the larger 8-aminomethyl groups (41c−e and 49c−e). The 7-F compounds were generally similar in activity to the 7-Cl derivatives, especially for the larger 8aminomethyl groups. Overall, this data suggests that tet(M) activity is largely driven by the size and/or lipophilicity of the groups at both the 7- and 8-positions. Activity against strains carrying tet(A) genes, however, appears to require a balance of size and/or lipophilicity between the groups at the 7- and 8positions. With a smaller, more polar 8-aminomethyl group, tet(A) activity will be improved with a more lipophilic group (i.e., CF3) at the 7-position. As the size of the 8-aminomethyl group increases, however, a smaller or more polar group at the 7-position is favored. Compound 49f was also prepared as a direct comparison between the 8-aminomethyltetracyclines and omadacycline, a 9-aminomethyltetracycline. From the data, it is clear that tet(M) activity is significantly better (8−64-fold) for the 9-substituted compound, whereas tet(A) activity is better (4-fold) for the 8-aminomethyl compound. On the basis of the SAR trends observed for the compounds above, a set of compounds was prepared to find analogues with balanced tet(M) and tet(A) activity, a subset of which is shown in Table 4. In the acyclic series, the first approach was to combine one α-branched amino substituent with a second larger amino substituent, as in compounds 15v and 39f. This yielded compounds with more potent S. aureus and E. faecalis tet(M) activity but came with a sacrifice in tet(A) and S. pneumoniae tet(M) activities. A second, more successful approach for the acyclic series was to choose one amino substituent with both α-branching and β-branching in

maintained activity against the tetracycline-susceptible Gramnegative strains (EC107, KP109, and AB110). All compounds also showed good activity against the S. aureus tet(K) strain (SA158), with MIC values ≤0.25 μg/mL. The increasing ring size from azetidine 15j to pyrrolidine 15k gave a small improvement in activity of one-to-two dilutions against the tet(M) strains and a larger two-to-three dilution improvement in tet(A) activity. Further increasing ring size to piperidine 15pp and homopiperidine 15u provided only a modest one dilution further improvement in MIC for both resistance types. It does appear that the larger cyclic amines have improved activity against both the S. pneumoniae tet(M) and Gramnegative tet(A) strains relative to their acyclic counterparts. For example, piperidine compound 15pp was two-to-three dilutions more potent against these strains than the 5-carbon acyclic compound 15g. For the smaller compounds (i.e., 15k vs 15c), no difference was seen. The β-fluoro-substituted compounds 15n−15q also paralleled the activity of the less basic acyclic compounds above, with activity against S. aureus and E. faecalis tet(M) improving substantially and activity against S. pneumoniae tet(M) and tet(A) decreasing with decreasing basicity. A series of methyl substituted pyrrolidines (15l−m, 15oo) and piperidines (15qq−15vv) provided additional insight into tet(M) and tet(A) SAR. In both ring systems, the addition of one methyl group at the 2-position had essentially no effect on tet(M) activity and minimal effect (i.e., one dilution in some cases) on tet(A) activity. Methyl substitution at the 4-position in the piperidine series (15rr), however, gave a one-to-two dilution improvement in activity against S. aureus and E. faecalis tet(M) while having no effect on the S. pneumoniae tet(M) or Gram-negative tet(A) activity. 4-Phenylpiperidine derivative 15r had significantly improved activity against S. aureus and E. faecalis tet(M) (MIC = 0.5 μg/mL) but decreased S. pneumoniae tet(M) and Gram-negative tet(A) activity. The 2,2-dimethyl substituted pyrrolidine (15oo) and piperidine (15tt) generally had one-to-two dilution improvements in activity against all of the tet(M) and tet(A) strains. Moving the dimethyl substitution to the 3-position (15uu) gave three-tofour dilution improvements in activity against S. aureus and E. faecalis tet(M) relative to unsubstituted piperidine 15pp but led to a one-to-two dilution decrease in activity against S. pneumoniae tet(M) and the Gram-negative tet(A) strains. This SAR also parallels that seen with the α- and β-branched acyclic compounds above. 4,4-Dimethylpiperidine derivative 15vv turned out to be a reasonable compromise between the 2and 3-substituted compounds, with improved activity for all three tet(M) strains while maintaining the tet(A) activity of compound 15pp. This compound proved to be the most balanced compound in terms of tet(M) and tet(A) activity within the 7-fluoro-8-aminomethyltetracycline series. 2,6Dimethyl substitution (compound 15ss) also gave improved tet(M) activity while maintaining tet(A) activity, providing nicely balanced broad-spectrum activity. Several heterocyclic amines were also explored (15ww, 15s, and 15t). Although morpholine analogue 15ww (pKB ∼3.7) had similar activity against S. aureus and E. faecalis tet(M) strains when compared to piperidine 15pp (pKB ∼5.7), piperazine derivatives 15s (pKB ∼0.2) and 15t (pKB ∼3.9) were significantly less potent, and the S. pneumoniae tet(M) and overall Gram-negative activities were worse for all three compounds. The amines of all three of these compounds have reduced basicity relative to the piperidines and might have been expected to yield improved tet(M) activity. The fact that these analogues are also more 8122

dx.doi.org/10.1021/jm401211t | J. Med. Chem. 2013, 56, 8112−8138

Journal of Medicinal Chemistry

Article

combination with a second small substituent (H or CH3), as in compounds 15xx, 39g, 41f, 41g, and 49g. Among these compounds, 7-Cl derivative 39g had slightly better tet(M) activity than the 7-methoxy or 7-dimethylamino compounds, whereas 7-methoxy compounds 41f and 41g had the best tet(A) activity. Once again, the 7-dimethylamino analogue proved to have the worst profile among the 8-aminomethyl compounds. Introducing a very large β-branching group, such as the t-butyl of compound 15xx, gave significantly improved S. aureus and E. faecalis tet(M) activity but resulted in a loss in tet(A) and S. pneumoniae tet(M) activities. The final approach we explored was to combine the 7-F, 7-Cl, or 7-methoxy groups with larger cyclic amines (15yy and 39h) or with α-substituted cyclic amines (39i, 39j, and 41h). Here again, the 7-Cl compounds had the best S. aureus and E. faecalis tet(M) activity, with MICs of 1 or 2 μg/mL, whereas 7-methoxy derivative 41h was one-to-two dilutions less potent. For the tet(A) and S. pneumoniae tet(M) activities, however, the 7-methoxy compound was one-to-three dilutions more potent than the 7-Cl compounds. In fact, compound 41h had the best tet(A) activity of any of the 8-aminomethyltetracyclines. Not surprisingly, introduction of the cyclopropyl group of compound 15yy gave very good activity against the S. aureus and E. faecalis tet(M) strains but resulted in some loss in tet(A) and S. pneumoniae tet(M) activities. Overall, these strategies led to the discovery of several compounds with broad spectrum and balanced activity against all of the tetracycline-resistant strains (MICs ≤ 4 μg/ mL) in the panel. Compounds 39h, 39j, and 41f−h were of particular interest given their potent Gram-negative activity. We next turned our attention to evaluation of the 8aminomethyltetracyclines for their therapeutic potential as oral antibacterial agents. As has been previously reported, the oral bioavailability (% F) of tetracyclines in rodents is compound dependent and is not necessarily predictive of that seen in humans.19 We have utilized a screening paradigm to identify compounds with oral potential based on efficacy in a mouse systemic infection model with the idea that compounds with even limited oral bioavailability in mice may have a significant chance of exhibiting good oral bioavailability in humans. Thus, mice were challenged with S. aureus ATCC 13709 and 1 h later were dosed either intravenously (IV) at 3 mg/kg or orally (PO) at 30 mg/kg, and survival was recorded at 48 h post-treatment (Table 5). Three control tetracyclines were dosed and behaved as expected, with all three compounds providing 100% protection in the IV arm. Tetracycline, the only control with significant oral bioavailability in rat (15%), also provided 100% protection in the PO arm. Omadacycline and tigecycline failed to provide significant protection in the PO arm, consistent with the low rodent oral bioavailability of these compounds (0.7 and 1.1%, respectively). After screening a number of the 8aminomethyltetracyclines, three populations of compounds emerged with distinct efficacy/in vitro activity profiles. The first group consisted of compounds that gave 100% protection in the IV arm and good protection (≥83%) in the PO arm. These compounds all fell into the in vitro activity pattern of good tet(A) and S. pneumoniae tet(M) activities with somewhat reduced S. aureus and E. faecalis tet(M) activities (two or more dilutions less potent). These compounds generally have the more polar α-branched or cyclic amine substituents. The second group consisted of compounds that had poor efficacy (32/>32 4/8 0.25/0.5 32/32 0.031/0.031

0.5/1 1/4 0.5/2 1/2 1/2 1/2 0.5/1 1/2 1/2 1/2 8/>64 8/16 0.5/1 16/64 0.031/0.063

K. pneumoniae (ESβL/KPC) (n = 29)

Log10 CFU/g kidney reduction

E. coli EC200 (ESβL)

K. pneumoniae KP453 (ESβL)

EC200 pyelonephritis 2 mg/kg PO

0.25 0.25 0.13 0.25 0.25 0.25 0.5 0.13 0.25 0.25 >32 2 0.13 0.063 0.016

1 2 1 1 1 1 0.5 1 2 1 4 8 0.5 32 0.031

2.78 ND 2.51 1.91 2.82 1.85 2.99 2.31 (1 mg/kg) 2.27 2.3 ND ND ND 3.78 3.10 (20 mg/kg IV)

0.5/1

1/2 8/>32 8/16 1/1 32/>32 0.063/>32

KP453 pyelonephritis 50 mg/kg PO 2.15 ND 2.15 1.27 1.45 1.04 2.93 1.55 1.32 2.16 1.1 ND ND 1.17 1.44 (30 mg/kg IV)

Table 7. Oral Efficacy of 7-R-8-Aminomethyltetracyclines in a Mouse Lung Infection Model MIC50/MIC90 (μg/mL)

infection model isolates (μg/mL)

Log10 CFU/g lung reduction

cmpd

S. aureus (n = 13)

S. pneumoniae (n = 17)

S. aureus SA191 (MRSA)

S. pneumoniae SP160 (tet(M))

SA191 lung 30 mg/kg PO

SP160 lung 30 mg/kg PO

39h 39i 39j 41f 49g tetracycline omadacycline tigecycline levofloxacin linezolid

0.25/2 0.25/0.5 0.13/2 0.25/4 0.5/2 32/>32 0.5/2 0.13/0.5 16/>64 4/4

0.5/1 0.25/0.5 0.031/0.5 0.5/1 32/>32 0.016/0.031 0.016/0.016 1/1 0.5/1

1 0.5 1 1 2 >64 1 0.5 0.25 2

0.5 1 0.25 0.25 0.5 32 2 0.016 1 1

ND ND ND 1.98 1.27 0.09 ND ND 1.97 2.26

0.22 0.19 0.12 ND 4.31 ND ND ND 0.03 1.79

further assayed in a K. pneumoniae panel composed of extended spectrum β-lactamase (ESβL, n = 20) and K. pneumoniae carbapenemase-producing (KPC, n = 9) strains. Both compounds had MIC90 values similar to tigecycline, whereas omadacycline, levofloxacin, and meropenem demonstrated fairly poor activity (MIC90 values ≥16 μg/mL). All of the compounds except 15ll were screened in two mouse pyelonephritis models using extended spectrum β-lactamase (ESβL) strains of E. coli and K. pneumoniae. Compounds were administered orally BID at 2 mg/kg/dose (E. coli model, dosed at 12 and 24 h postinfection) or 50 mg/kg/dose (K. pneumoniae, dosed at 9 and 24 h postinfection), and the bacterial burden in kidney was determined at 36 h postinitiation of treatment. For the E. coli pyelonephritis model, most of the compounds showed good oral efficacy with >2 log10 CFU/g kidney reductions in bacterial load relative to untreated controls. Compounds 15dd, 15oo, 39c, and 41e performed the best with >2.5 log10 CFU/g kidney reductions. Interestingly, compound 49e with the best combination of % F (32%) and MIC (0.25 μg/mL) was the worst performer within the group, whereas compound 41e performed the best with the worst combination of % F (12%) and MIC (0.5 μg/mL). This is likely due to differences in compound distribution to the kidney and urine. Compound 41e performed comparably to

large number of compounds with oral bioavailability in rodents and led us to conclude that the 8-aminomethyltetracycline series has significant potential for oral bioavailability in humans. On the basis of their Gram-negative activity profiles, a number of compounds were assayed against larger panels of E. coli (n = 14) and K. pneumoniae (n = 19) recent clinical isolates for determination of MIC50 and MIC90 values (Table 6). The majority of the isolates in these panels were either nonsusceptible or resistant to tetracycline (MIC50 values of >32 and 8 μg/mL, respectively) and resistant to levofloxacin (MIC50 values of 32 and 16 μg/mL, respectively) but were susceptible to meropenem (MIC90 values of 0.031 and 0.063 μg/mL, respectively). Omadacycline also had limited activity in the panels, with MIC90 values of 8 and 16 μg/mL against E. coli and K. pneumoniae, respectively, whereas tigecycline had good activity, with MIC90 values of 0.5 and 1 μg/mL, respectively. The 8-aminomethyltetracyclines all had good MIC90 values for E. coli, ranging from 0.25 to 1 μg/mL. Four compounds had MIC90 values equal to tigecycline, with compound 39j having an MIC90 that was one dilution better (0.25 μg/mL). For K. pneumoniae, most of the compounds had MIC90 values of ≤2 μg/mL, with only compound 15ll having an MIC90 of 4 μg/ mL. Both compounds 15dd and 41e had MIC90 values of 1 μg/ mL, the same as tigecycline. Compounds 41e and 41f were 8124

dx.doi.org/10.1021/jm401211t | J. Med. Chem. 2013, 56, 8112−8138

Journal of Medicinal Chemistry

Article

g lung reduction). It is noteworthy that compound 41f demonstrated good oral efficacy in both Gram-negative kidney infection models and the Gram-positive MRSA lung infection model, indicating that the compound has potential as a broadspectrum oral therapy for complicated infections.

meropenem/cilastatin (dosed IV), with a 3.10 log10 CFU/g kidney reduction in bacterial load, whereas none of the compounds were as good as levofloxacin (essentially 100% oral bioavailability), with a 3.78 log10 CFU/g kidney reduction in bacterial load. Efficacy in the K. pneumoniae pyelonephritis model turned out to be more challenging, with only four of the nine compounds yielding >2 log10 CFU/g kidney reductions in bacterial load relative to untreated controls. Levofloxacin, with an MIC of 32 μg/mL and tetracycline, with an MIC of 4 μg/ mL failed to show good efficacy, as expected. The meropenem/ cilastatin control, dosed at 30 mg/kg IV, demonstrated only marginal efficacy in this model, with a 1.44 log10 CFU/g kidney reduction in bacterial load. Once again, compound 41e had the best activity (2.93 log10 CFU/g kidney reduction), with compounds 15dd, 15oo, and 41f also having ∼2.15 log10 CFU/g kidney reductions. These data indicate that the 8aminomethyltetracyclines have significant potential as oral treatments for serious Gram-negative infections. Compounds that displayed good tet(M) activity and were active in the oral efficacy screen were further profiled against larger panels of methicillin-resistant S. aureus (MRSA) and S. pneumoniae (Table 7), relevant pathogens in communityacquired bacterial pneumonia (CABP). The majority of isolates in these panels were resistant to tetracycline (MIC50 = 32 μg/ mL), and the majority of MRSA isolates were resistant to levofloxacin (MIC50 = 16 μg/mL). Both omadacycline and tigecycline had good activity in the MRSA (MIC90 = 2 and 0.5 μg/mL, respectively) and S. pneumoniae (MIC90 = 0.031 and 0.016 μg/mL, respectively) panels. Linezolid, prescribed for CABP to cover methicillin-resistant staphylococci in particular, had MIC90 values of 4 and 1 μg/mL for the two pathogens, respectively, whereas levofloxacin had similar activity in the S. pneumoniae panel (MIC90 = 1 μg/mL) but showed poor activity against the MRSA panel (MIC90 >64 μg/mL). All of the 8aminomethyltetracyclines had MIC90 values that were equivalent to or better than linezolid in both panels and were also comparable to omadacycline in the MRSA panel. Compound 39i was the most potent, with an MIC90 of 0.5 μg/mL in the MRSA panel, equivalent to tigecycline. None of the 8aminomethyltetracyclines were as potent as omadacycline or tigecycline in the S. pneumoniae panel, but all were comparable to linezolid and levofloxacin. The compounds were then screened in murine lung infection models using either a tet(M) MRSA strain (SA191) or a tet(M) S. pneumoniae strain (SP160). Compounds were dosed at 30 mg/kg/dose orally BID (2 and 12 h postinfection), and lungs were cultured for bacterial burden at 24 h postinitiation of treatment. The S. pneumoniae lung model proved to be very difficult, with linezolid providing only 1.79 log10 CFU/g lung reduction in bacterial load and levofloxacin failing to demonstrate efficacy. Of the four 8-aminomethyltetracyclines tested, only compound 49g demonstrated significant efficacy, providing a 4.31 log10 CFU/g lung reduction in bacterial load, significantly better than the control antibiotics. In the MRSA lung infection model, both linezolid and levofloxacin demonstrated good efficacy, with 2.26 and 1.97 log10 CFU/g lung reductions in bacterial load, respectively. Tetracycline, with an MIC of >64 μg/mL, failed to demonstrate significant efficacy in the MRSA model. Compound 49g, with a relatively higher MRSA versus S. pneumoniae MIC value (2 vs 0.5 μg/mL), had reduced efficacy in the MRSA model (1.27 log10 CFU/g lung reduction). Compound 41f, however, demonstrated efficacy that was comparable to both levofloxacin and linezolid (1.98 log10 CFU/

■

CONCLUSIONS The first 8-aminomethyltetracyclines were prepared via a total synthesis approach and were assayed for activity against a panel of Gram-positive and Gram-negative bacteria. Initial SAR led to compounds that were active either against tetracycline-resistant Gram-positive isolates expressing the tet(M) gene or against Gram-negative isolates expressing the tet(A) gene. Combining structural elements from these two subsets gave several compounds with balanced activity against both resistance mechanisms. Compounds from the Gram-negative and the broad-spectrum subsets were identified and demonstrated activity in an in vivo IV/oral efficacy screen, whereas members of the Gram-positive subset failed to show efficacy upon either IV or PO administration. Furthermore, compounds from the first two subsets generally showed oral bioavailability in rats that was significantly better than the known 9-substituted tetracyclines and was either comparable to or better than that seen for tetracycline. Four compounds were identified that demonstrated good oral efficacy in both E. coli and K. pneumoniae mouse pyelonephritis infection models, including compounds 41e and 41f. Compound 41f was also found to be orally active in a mouse MRSA lung infection model, with efficacy comparable to linezolid. Thus, compound 41f shows promise as a broad-spectrum oral therapy for complicated bacterial infections. Overall, the 8-aminomethyltetracyclines appear to have significant potential as oral antibacterial agents for use against serious bacterial infections caused by difficult to treat Gram-negative and Gram-positive bacterial pathogens.

■

EXPERIMENTAL PROCEDURES

General Procedures. Air- and moisture-sensitive liquids and reagents were transferred via syringe or cannula and were introduced into flame-dried glassware under a positive pressure of dry nitrogen through rubber septa. All reactions were stirred magnetically. Commercial reagents were used without further purification. Analytical thin-layer chromatography was performed on EM Science precoated glass-backed silica gel 60 Å F-254 250 μm plates. Visualization of the plates was effected by ultraviolet illumination and/or immersion of the plate in a basic solution of potassium permanganate in water followed by heating. Column chromatography was performed on a Biotage Isolera One purification system using Biotage SNAP cartridges. Preparative reversed-phase HPLC chromatography (HPLC) was accomplished using a Waters Autopurification system with massdirected fraction collection. All intermediate compounds were purified with a Waters Sunfire Prep C18 OBD column (5 μm, 19 × 50 mm; flow rate = 20 mL/min) using a mobile phase mixture of H2O (A) and CH3CN (B) containing 0.1% HCO2H. The final tetracycline compounds were purified using a Phenomenex Polymerx 10 μ RP 100A column (10 μm, 30 × 21.2 mm; flow rate = 20 mL/min) using a mobile phase mixture of 0.05 N HCl in H2O (A) and CH3CN (B). Unless otherwise described, all final tetracycline compounds were isolated as mono-, di-, or trihydrochloride salts following freeze-drying. 1 H NMR spectra were recorded on a JEOL ECX-400 (400 MHz) spectrometer and are reported in ppm using residual solvent as the internal standard (CDCl3 at 7.24 ppm, DMSO-d6 at 2.50 ppm, or CD3OD at 3.31 ppm). High-performance liquid chromatography− electrospray mass spectra (LC−MS) were obtained using an Waters Alliance HPLC system equipped with a binary pump, a diode array detector, a Waters Sunfire C18 (5 μm, 4.6 mm i.d. × 50 mm) column, 8125

dx.doi.org/10.1021/jm401211t | J. Med. Chem. 2013, 56, 8112−8138

Journal of Medicinal Chemistry

Article

and a Waters 3100 Series mass spectrometer with electrospray ionization. Spectra were scanned from 100 to 1200 amu. The eluent was a mixture of H2O (A) and CH3CN (B) containing 0.1% HCO2H at a flow rate of 1 mL/min. Purity of the final compounds was assessed by the following gradient: (a) time = 0, 100% A; (b) time = 0.5 min, 100% A; (c) time = 3.5 min, 100% B; (d) time = 5 min, 100% B; (e) time = 6 min, 100% A; and (f) time = 7 min, 100% A. All final products were ≥95% purity as assessed by this method. Phenyl 2-(Benzyloxy)-3-bromo-5-fluoro-6-methylbenzoate (9). Phenyl 3-fluoro-6-hydroxy-2-methylbenzoate (4.92 g, 20.0 mmol) was dissolved in acetic acid (50 mL), and bromine (1.54 mL, 30.0 mmol) was added via syringe at rt. After 2 h, the reaction mixture was diluted with EtOAc, was washed with water (3 × 100 mL) and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give 7.06 g of the crude arylbromide as a lightyellow solid. 1H NMR (400 MHz, CDCl3) δ 11.14 (s, 1H), 7.52 (d, J = 9.2 Hz, 1H), 7.49−7.43 (m, 2H), 7.36−7.30 (m, 1H), 7.21−7.16 (m, 2H), 2.55 (d, J = 2.3 Hz, 3H). Benzyl bromide (0.540 mL, 4.45 mmol) was added dropwise to a 0 °C mixture of the above crude arylbromide (1.06 g, 2.97 mmol) and K2CO3 (0.82 g, 5.94 mmol) in acetone (20 mL). After 2 h, the reaction mixture was heated at 50 °C for 1 h. The reaction mixture was diluted with EtOAc (100 mL), washed with water and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The material was purified by column chromatography (Biotage 10 g column, 2−5% EtOAc in hexanes gradient), yielding 1.03 g (84%, two steps) of the title compound as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.50−7.47 (m, 2H), 7.40−7.33 (m, 6H), 7.25 (t, J = 7.3 Hz, 1H), 7.04 (d, J = 8.6 Hz, 2H), 5.09 (s, 2H), 2.32 (d, J = 1.8 Hz, 3H). Phenyl 2-(Benzyloxy)-3-bromo-5-fluoro-4-formyl-6-methylbenzoate (10). n-BuLi (1.6 M in hexanes, 5.10 mL, 8.16 mmol) was added to diisopropylamine (1.15 mL, 8.16 mmol) in THF (15 mL) at −78 °C. The reaction mixture was warmed to −20 °C, stirred for 15 min, and cooled to −78 °C. A solution of compound 9 (2.26 g, 5.44 mmol) in THF (5 mL) was added dropwise, resulting in an orange-red solution. After 10 min, DMF (1.26 mL, 16.30 mmol) was added dropwise. The reaction mixture was allowed to warm to −20 °C over 1 h and was quenched with NH4Cl (saturated, aqueous solution). The reaction mixture was diluted with EtOAc (100 mL), washed with water and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. The material was purified by column chromatography (Biotage 24 g column, 5−10% EtOAc in hexanes gradient), yielding 2.23 g (92%) of the title compound as a light-yellow solid. 1H NMR (400 MHz, CDCl3) δ 10.37 (s, 1H), 7.51−7.47 (m, 2H), 7.40− 7.33 (m, 5H), 7.27 (t, J = 7.3 Hz, 1H), 7.06−7.02 (m, 2H), 5.12 (s, 2H), 2.37 (d, J = 2.3 Hz, 3H). Phenyl 2-(Benzyloxy)-3-bromo-4-(dimethoxymethyl)-5-fluoro-6-methylbenzoate (11). To a solution of compound 10 (10 g, 22.6 mmol) in CH3OH were added trimethylorthoformate (4.8 g, 45.2 mmol) and p-toluenesulfonic acid monohydrate (0.13 g, 0.68 mmol). The reaction mixture was heated to reflux overnight and was then concentrated under reduced pressure. The residue was diluted with water and extracted with EtOAc. The organic layer was dried over Na2SO4 and evaporated to dryness. The material was purified by column chromatography on silica gel (1−3% EtOAc in petroleum ether gradient) to give 10 g (91%) of the title compound as a lightyellow solid. 1H NMR (400 MHz, CDCl3) δ 7.41−7.45 (m, 2H), 7.25−7.35 (m, 5H), 7.16−7.21 (m, 1H), 6.98 (d, J = 8.0 Hz, 2H), 5.71 (s, 1H), 5.04 (s, 2H), 3.46 (s, 6H), 2.29 (d, J = 2.4 Hz, 3H). (1R,3S,4S,11S)-8,16-Bis(benzyloxy)-17-bromo-11-[(tertbutyldimethylsilyl)oxy]-18-(dimethoxymethyl)-4-(dimethylamino)-19-fluoro-12-hydroxy-6-oxa-7-azapentacyclo[11.8.0.03,11.05,9.015,20]henicosa-5(9),7,12,15,17,19-hexaene10,14-dione (12). n-BuLi (4.8 mL, 2.5 M in hexanes, 12 mmol) was added to a −78 °C solution of diisopropylamine (1.1 g, 10.9 mmol) and TMEDA (5 mL) in THF (5 mL) and was stirred for 1 h. A portion of the LDA/TMEDA solution (0.71 M in THF, 7.9 mL, 5.6 mmol) was added to a −78 °C solution of compound 11 (1.37 g, 2.80 mmol) and compound 6 (0.45 g, 0.93 mmol) in THF (5 mL). After 10 min, the reaction mixture was allowed to warm to −10 °C over 20

min. The reaction mixture was quenched with NH4Cl (saturated, aqueous solution, 50 mL) and extracted with EtOAc (3 × 50 mL). The organic phase was dried over Na2SO4 and evaporated under reduced pressure. The material was purified by column chromatography on silica gel (2−6% EtOAc in petroleum ether gradient) to give 0.60 g (74%) of the title compound. 1H NMR (400 MHz, CD3OD) δ 15.97 (s, 1H), 7.61−7.59 (m, 2H), 7.55−7.53 (m, 2H), 7.42−7.40 (m, 6H), 5.83 (s, 1H), 5.40 (s, 2H), 5.02−4.97 (m, 2H), 3.96 (d, J = 10.8 Hz, 1H), 3.57 (s, 6H), 3.35−3.26 (m, 1H), 3.09−2.95 (m, 1H), 2.67−2.58 (m, 1H), 2.53 (s, 6H), 2.50−2.39 (m, 1H), 2.21−2.10 (m, 1H), 1.58 (s, 9H), 0.31 (s, 3H), 0.16 (s, 3H). MS (ESI) m/z: 877.3 (M + H). (1R,3S,4S,11S)-16-(Benzyloxy)-17-bromo-11-[(tertbutyldimethylsilyl)oxy]-4-(dimethylamino)-19-fluoro-8,12-dihydroxy-10,14-dioxo-6-oxa-7-azapentacyclo[11.8.0.03,11.05,9.015,20]henicosa-5(9),7,12,15,17,19-hexaene-18carbaldehyde (13). Compound 12 (50 mg, 0.057 mmol) was dissolved in dry dichloromethane (1 mL). TFA (0.5 mL) was added. The solution was stirred at 10 °C for 1 h. The reaction mixture was washed with water (10 mL × 3) and concentrated under reduced pressure to give crude 13, which was used for the next step without further purification. MS (ESI) m/z: 741.1 (M + H). General Experimental A. Conversion of 13 to 14 by Reductive Amination. Compound 13 (crude, 0.057 mmol, 1.0 equiv) was dissolved in 1,2-dichloroethane (2 mL). Acetic acid (20 μL) and the amine (0.34 mmol, 6.0 equiv) were added. The mixture was stirred for 1 h. Na(OAc)3BH (0.34 mmol, 6.0 equiv) was added, and the resulting mixture was stirred for 1 h. The mixture was washed with water (10 mL) and concentrated to give crude 14, which was used for the next step without further purification. General Experimental B. Conversion of Secondary Amines (14) to Tertiary Amines (14) by Reductive Alkylation. Secondary amine 14 (crude, 0.057 mmol, 1.0 equiv) was dissolved in 1,2dichloroethane (2 mL). Acetic acid (20 μL) and the aldehyde (0.34 mmol, 6.0 equiv) were added. The mixture was stirred for 1 h. Na(OAc)3BH (0.34 mmol, 6.0 equiv) was added, and the resulting mixture was stirred for 1 h. The mixture was washed with water (10 mL) and concentrated to give crude tertiary amine 14, which was used for the next step without further purification. General Experimental C. Deprotection of 14 to Give 8Aminomethyltetracyclines (15). Compound 14 (crude, 0.057 mmol) was dissolved in THF (5 mL) in a polypropylene tube at rt. Aqueous HF (2 mL, 48−50%) was added. The reaction mixture was stirred at rt for 1 h. The resulting mixture was carefully poured into an aqueous solution of K2HPO4. The pH of the mixture was adjusted to 7 to 8 by adding more aqueous K2HPO4. The mixture was extracted with EtOAc (20 mL), and the EtOAc extract was concentrated under reduced pressure. The material was dissolved in CH3OH (5 mL). HCl (4 M solution in CH3OH, 1 mL) and 10% Pd−C (15 mg) were added. An atmosphere of hydrogen (balloon) was introduced, and the mixture was stirred at rt for 1 h. The mixture was filtered and concentrated. The crude compound was purified by preparative HPLC (Phenomenex Polymerx 10 μ RP 100A column, 0−100% B gradient). Fractions with the desired MW were collected and freeze-dried to yield compounds 15 as HCl salts. (4S,4aS,5aR,12aS)-4-(Dimethylamino)-8-[(ethylamino)methyl]-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15a). Prepared by general experimental methods A (ethylamine) and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.02 (d, J = 5.6 Hz, 1H), 4.25 (s, 2H), 4.11 (s, 1H), 3.22−2.98 (m, 11H), 2.36−2.23 (m, 2H), 1.68−1.58 (m, 1H), 1.33 (t, J = 7.2 Hz, 3H). MS (ESI) m/z: 490.4 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-8-{[ethyl(methyl)amino]methyl}-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15b). Prepared by general experimental methods A (ethylmethylamine) and C, yellow solid. 1H NMR (400 MHz, CD3OD/DCl) δ 7.11 (d, J = 5.48 Hz, 1H), 4.51 (dd, J = 13.3, 6.4 Hz, 1H), 4.31 (dd, J = 13.3, 6.4 Hz, 1H), 4.16 (s, 1H), 3.42−3.10 (m, 4H), 3.10−2.97 (m, 7H), 2.85 (s, 3H), 2.39−2.25 (m, 2H), 1.62 (dd, J = 8126

dx.doi.org/10.1021/jm401211t | J. Med. Chem. 2013, 56, 8112−8138

Journal of Medicinal Chemistry

Article

(4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12at e t ra hy d ro xy - 1, 11 -d i o xo - 8- (p yr ro li d i n -1 - yl m e t hy l )1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15k). Prepared by general experimental methods A (pyrrolidine) and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.06 (d, J = 6.0 Hz, 1H), 4.46 (s, 2H), 4.09 (s, 1H), 3.04 (s, 3H), 2.97 (s, 3H), 3.27−2.97 (m, 3H), 2.37 (m, 1H), 2.28−2.14 (m, 3H), 2.08− 2.00 (m, 2H), 1.72−1.62 (m, 1H). MS (ESI) m/z: 516.4 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12atetrahydroxy-8-{[(2R)-2-methylpyrrolidin-1-yl]methyl}-1,11dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15l). Prepared by general experimental methods A ((R)-2-methylpyrrolidine) and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.01 (d, J = 5.6 Hz, 1H), 4.56 (d, J = 13.2 Hz, 1H), 4.13 (d, J = 13.2 Hz,1H), 4.01 (s, 1H), 3.56−3.36 (m, 2H), 3.16−2.86 (m, 10H), 2.33−2.16 (m, 3H), 2.07−1.91 (m, 2H), 1.73− 1.67 (m, 1H), 1.61−1.52 (m, 1H), 1.42 (d, J = 6.4 Hz, 3H). MS (ESI) m/z: 575.2 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12atetrahydroxy-8-{[(2S)-2-methylpyrrolidin-1-yl]methyl}-1,11dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15m). Prepared by general experimental methods A ((S)-2-methylpyrrolidine) and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.01 (d, J = 5.6 Hz, 1H), 4.56 (d, J = 13.2 Hz, 1H), 4.13 (d, J = 13.2 Hz,1H), 4.01 (s, 1H), 3.56−3.36 (m, 2H), 3.16−2.86 (m, 10H), 2.33−2.16 (m, 3H), 2.07−1.91 (m, 2H), 1.73− 1.67 (m, 1H), 1.61−1.52 (m, 1H), 1.42 (d, J = 6.4 Hz, 3H). MS (ESI) m/z: 575.2 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12atetrahydroxy-1,11-dioxo-8-{[(2S)-2-(trifluoromethyl)pyrrolidin1-yl]methyl}-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15n). Prepared by general experimental methods A ((2S)-2-(trifluoromethyl)pyrrolidine) and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 6.98 (d, J = 5.5 Hz, 1H), 4.25 (d, J = 14.7 Hz, 1H), 4.05 (d, J = 14.7 Hz, 1H), 4.08 (s, 1H), 3.81−3.73 (m, 1H), 3.04 (s, 3H), 2.96 (s, 3H), 3.27−2.97 (m, 4H), 2.76 (m, 1H), 2.35−2.17 (m, 3H), 2.09−1.99 (m, 1H), 1.99−1.87 (m, 2H), 1.69−1.59 (m, 1H). MS (ESI) m/z: 584.3 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-8-{[(3S)-3-fluoropyrrolidin-1-yl]methyl}-3,10,12,12a-tetrahydroxy-1,11dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15o). Prepared by general experimental methods A ((3S)-3-fluoropyrrolidine) and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.11 (d, J = 5.6 Hz, 1H), 5.47 (d, J = 52 Hz, 1H), 4.55 (s, 2H), 4.12 (s, 1H), 3.95−3.47 (m, 4H), 3.24−2.98 (m, 9H), 2.70−2.62 (m, 1H), 2.39−2.25 (m, 3H), 1.69−1.61 (m, 1H). MS (ESI) m/z: 534.1 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-8-{[(3R)-3-fluoropyrrolidin-1-yl]methyl}-3,10,12,12a-tetrahydroxy-1,11dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15p). Prepared by general experimental methods A ((3R)-3-fluoropyrrolidine) and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.10 (d, J = 6 Hz, 1H), 5.47 (d, J = 52.4 Hz, 1H), 4.54 (s, 2H), 4.11 (s, 1H), 3.92−3.39 (m, 4H), 3.26−2.98 (m, 9H), 2.70−2.62 (m, 1H), 2.40−2.23 (m, 3H), 1.71−1.62 (m, 1H). MS (ESI) m/z: 534.1 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-8-[(3,3-difluoropyrrolidin-1-yl)methyl]-4-(dimethylamino)-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15q). Prepared by general experimental methods A (3,3-difluoropyrrolidine) and C, yellow solid. 1 H NMR (400 MHz, CD3OD) δ 7.00 (d, J = 5.6 Hz, 1H), 4.48 (s, 2H), 4.01 (s, 1H), 3.87 (t, J = 11.6 Hz, 2H), 3.87 (t, J = 7.6 Hz, 2H), 3.18−2.88 (m, 9H), 2.18−2.07 (m, 2H), 2.33−2.11 (m, 2H), 1.56− 1.51 (m, 1H). MS (ESI) m/z: 552.2 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12atetrahydroxy-1,11-dioxo-8-[(4-phenylpiperidin-1-yl)methyl]1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15r). Prepared by general experimental methods A (4phenylpiperidine) and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.24−7.11 (m, 5H), 7.07 (d, J = 4.8 Hz, 1H), 4.35 (s, 2H), 4.04 (s, 1H), 3.60−3.57 (m, 3H), 3.16−2.80 (m, 11H), 2.31−2.17 (m, 2H),

14.0, 11.0 Hz, 1H), 1.41 (t, J = 7.3 Hz, 3H). MS (ESI) m/z: 504.2 (M + H). (4S,4aS,5aR,12aS)-8-[(Diethylamino)methyl]-4-(dimethylamino)-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15c). Prepared by general experimental method A (diethylamine) and C, yellow solid. 1H NMR (400 MHz, CD3OD/ DCl) δ 7.10 (d, J = 5.96 Hz, 1H), 4.23 (s, 2H), 4.17 (s, 1H), 3.35− 2.96 (m, 13H), 2.40−2.25 (m, 2H), 1.63 (dd, J = 14.0, 11.0 Hz, 1H), 1.39 (t, J = 7.1 Hz, 6H). MS (ESI) m/z: 518.2 (M + H). ( 4S , 4a S ,5 aR ,1 2a S )- 4- ( Di m e t h yl am in o) -8 - {[ e th yl ( 2fluoroethyl)amino]methyl}-7-fluoro-3,10,12,12a-tetrahydroxy1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15d). Prepared by general experimental methods A (2-fluoroethylamine), B (acetaldehyde), and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.09 (d, J = 6.0 Hz, 1H), 4.96 (t, J = 6.0 Hz, 1H), 4.78 (t, J = 6.0 Hz, 1H), 4.51 (s, 2H), 4.11 (s, 1H), 3.72−3.59 (m, 2H), 3.40 (q, J = 6.8 Hz, 2H), 3.24−2.97 (m, 9H), 2.39−2.24 (m, 2H), 1.69−1.60 (m, 1H), 1.41 (t, J = 7.2 Hz, 3H). MS (ESI) m/z: 536.1 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12atetrahydroxy-1,11-dioxo-8-[(propylamino)methyl]1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15e). Prepared by general experimental methods A (propylamine) and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.03 (d, J = 6.0 Hz, 1H), 4.27 (s, 2H), 4.12 (s, 1H), 3.21 (dd, J = 15.1, 4.6 Hz, 1H), 3.05 (s, 3H), 2.97 (s, 3H), 2.94 (d, J = 6.9 Hz, 2H), 3.14−2.98 (m, 2H), 2.21−2.39 (m, 2H), 1.82−1.71 (m, 2H), 1.70− 1.58 (m, 1H), 1.02 (t, J = 7.6 Hz, 3H). MS (ESI) m/z: 504.4 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12atetrahydroxy-1,11-dioxo-8-{[methyl(propyl)amino]methyl}1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15f). Prepared by general experimental methods A (propylamine), B (formaldehyde), and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.05 (d, J = 5.2 Hz, 1H), 4.48 (d, J = 13.2 Hz, 1H), 4.27 (d, J = 13.2 Hz, 1H), 4.09 (d, J = 4.4 Hz, 1H), 3.22−2.92 (m, 11H), 2.84 (s, 3H), 2.38−2.22 (m, 2H), 1.87−1.70 (m, 2H), 1.68− 1.62 (m, 1H), 1.03 (t, J = 7.2 Hz, 3H). MS (ESI) m/z: 518.0 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-8-{[ethyl(propyl)amino]methyl}-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15g). Prepared by general experimental methods A (propylamine), B (acetaldehyde), and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.08 (d, J = 5.6 Hz, 1H), 4.42 (d, J = 4.0 Hz, 2H), 4.12 (s, 1H), 3.29−2.90 (m, 13H), 2.41−2.22 (m, 2H), 1.90−1.75 (m, 2H), 1.71−1.60 (M, 1H); 1.38 (t, J = 7.2 Hz, 3H), 1.05 (t, J = 7.2 Hz, 3H). MS (ESI) m/z: 532.0 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12at e t r ah y d r o x y- 1 , 1 1- d i o x o - 8 - [ (p h e ny l a m i n o )m e t h y l ]1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15h). Prepared by general experimental methods A (aniline) and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.59− 7.34 (m, 5H), 6.94 (d, J = 5.6 Hz, 1H), 4.63 (s, 2H), 4.10 (s, 1H), 3.19−2.97 (m, 9H), 2.34−2.23 (m, 2H), 1.68 (ddd, J = 13.2, 13.2, 13.2 Hz, 1H). MS (ESI) m/z: 538.2 (M + H). (4S,4aS,5aR,12aS)-8-{[Benzyl(methyl)amino]methyl}-4-(dimethylamino)-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15i). Prepared by general experimental methods A (benzylamine), B (formaldehyde), and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.47−7.41 (m, 5H), 6.95 (d, J = 6.0 Hz, 1H), 4.41− 4.36 (m, 2H), 4.34−4.18 (m, 2H), 4.02 (s, 1H), 3.12−2.88 (m, 9H), 2.72 (s, 3H), 2.28−2.12 (m, 2H), 1.60−1.50 (m, 1H). MS (ESI) m/z: 565.2 (M + H). (4S,4aS,5aR,12aS)-8-(Azetidin-1-ylmethyl)-4-(dimethylamino)-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15j). Prepared by general experimental methods A (azetidine) and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 6.97 (d, J = 6.0 Hz, 1H), 4.45 (s, 2H), 4.25−4.17 (m, 4H), 4.09 (s, 1H), 3.21−2.95 (m, 9H), 2.61−2.51 (m, 1H), 2.49−2.38 (s, 1H), 2.32− 2.18 (m, 2H), 1.65−1.53 (m, 1H). MS (ESI) m/z: 502.1 (M + H). 8127

dx.doi.org/10.1021/jm401211t | J. Med. Chem. 2013, 56, 8112−8138

Journal of Medicinal Chemistry

Article

2.06−1.96 (s, 4H), 1.63−1.52 (m, 1H). MS (ESI) m/z: 606.2 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12atetrahydroxy-8-[(4-methylpiperazin-1-yl)methyl]-1,11-dioxo1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Trihydrochloride (15s). Prepared by general experimental methods A (4-methylpiperazine) and C, yellow solid. 1H NMR (400 MHz, CD3OD/DCl) δ 7.22 (d, J = 5.48 Hz, 1H), 4.60 (s, 2H), 4.16 (s, 1H), 3.98−3.60 (br m, 8H), 3.24−2.94 (m, 12H), 2.40−2.24 (m, 2H), 1.64 (dd, J = 14.0, 11.0 Hz, 1H). MS (ESI) m/z: 545.3 (M + H). (4S,4aS,5aR,12aS)-8-[(4-Acetylpiperazin-1-yl)methyl]-4-(dimethylamino)-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15t). Prepared by general experimental methods A (4acetylpiperazine) and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.10 (d, J = 5.5 Hz, 1H), 4.45 (s, 2H), 4.10 (s, 1H), 3.27−2.98 (m, 7H), 3.04 (s, 3H), 2.97 (s, 3H), 2.37 (t, J = 15.1 Hz, 1H), 2.27−2.17 (m, 1H), 2.14 (s, 3H), 1.70−1.60 (m, 1H). MS (ESI) m/z: 573.3 (M + H). (4S,4aS,5aR,12aS)-8-(Azepan-1-ylmethyl)-4-(dimethylamino)-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15u). Prepared by general experimental methods A (homopiperazine) and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.04 (d, J = 5.6 Hz, 1H), 4.35 (s, 2H), 4.03 (s, 1H), 3.45−3.40 (m, 2H), 3.23−2.90 (m, 11H), 2.32−2.16 (m, 2H), 2.07−1.81 (m, 4H), 1.79−1.65 (m, 4H), 1.63−1.53 (m, 1H). MS (ESI) m/z: 544.2 (M + H). ( 4S , 4a S ,5 aR ,1 2a S )- 4- ( Di m e t h yl am in o) -8 - {[ e th yl ( 1methylcyclopentyl)amino]methyl}-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene2-carboxamide Dihydrochloride (15v). Prepared by general experimental methods A (1-methylcyclopentylamine), B (acetaldehyde), and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.14 (d, J = 5.6 Hz, 1H), 4.60−4.51 (m, 1H), 4.52−4.45 (m, 1H), 4.11 (s, 1H), 3.25−2.92 (m, 11H), 2.41−2.20 (m, 2H), 2.08−1.75 (m, 8H), 1.68− 1.59 (m, 1H), 1.50 (s, 3H), 1.14 (t, J = 7.2 Hz, 3H). MS (ESI) m/z: 572.2 (M + H). Phenyl 2-(Benzyloxy)-3-bromo-4-{[(2,2-dimethylpropyl)amino]methyl}-5-fluoro-6-methylbenzoate (16ii). Compound 10 (0.25 g, 0.56 mmol) was dissolved in 1,2-dichloroethane (4 mL). Neopentylamine (98 mg, 0.11 mmol) was added followed by acetic acid (0.064 mL, 0.11 mmol). After 1 h, Na(OAc)3BH (0.36 g, 1.68 mmol) was added. After stirring overnight, the reaction mixture was diluted with dichloromethane, washed with NaHCO3 (saturated, aqueous solution, 3 × 20 mL) and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. Purification by column chromatography (Biotage 10 g column, 10−20% EtOAc in hexanes gradient) gave 0.25 g (86%) of the title compound as a colorless oil. 1 H NMR (400 MHz, CDCl3) δ 7.52−7.47 (m, 2H), 7.40−7.33 (m, 6H), 7.25 (t, J = 6.9 Hz, 1H), 7.04 (d, J = 8.2 Hz, 2H), 5.10 (s, 2H), 4.04 (d, J = 2.3 Hz, 2H), 2.35 (d, J = 1.8 Hz, 3H), 2.30 (s, 2H), 0.89 (s, 9H). (1R,3S,4S,11S)-8,16-Bis(benzyloxy)-17-bromo-11-[(tertbutyldimethylsilyl)oxy]-4-(dimethylamino)-18-{[(2,2dimethylpropyl)amino]methyl}-19-fluoro-12-hydroxy-6-oxa-7azapentacyclo[11.8.0.0 3,11 .0 5,9 .0 15,20]henicosa-5(9),7,12,15(20),16,18-hexaene-10,14-dione (17ii). n-BuLi (1.6 M solution in hexanes, 0.29 mL, 0.46 mmol) was added to a solution of diisopropylamine (0.065 mL, 0.46 mmol) in THF (3 mL) at −78 °C. The resulting solution was warmed to −20 °C and stirred for 15 min. The LDA solution was cooled to −78 °C, and TMEDA (0.069 mL, 0.46 mmol) was added. A solution of compound 16ii (0.10 g, 0.20 mmol) in THF (1 mL) was added dropwise, resulting in a dark-red solution. After 10 min, a solution of compound 6 (74 mg, 0.15 mmol) in THF (1 mL) was added slowly. The reaction mixture was allowed to slowly warm to −20 °C over 1 h. The reaction mixture was quenched by the addition of phosphate buffer solution (pH 7, 10 mL) followed by NH4Cl (saturated, aqueous solution, 20 mL). This was extracted with dichloromethane (3 × 15 mL), and the combined extracts were dried over Na2SO4, filtered, and concentrated under

reduced pressure. The material was purified by column chromatography (Biotage 10 g column, 10−30% EtOAc in hexanes gradient) to yield 90 mg (65%) of the title compound as a yellow solid. MS (ESI) m/z: 902.7, 904.7 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-8-{[(2,2dimethylpropyl)amino]methyl}-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2carboxamide Dihydrochloride (15ii). Prepared from 17ii by general experimental method C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.09 (d, J = 6.0 Hz, 1H), 4.42 (s, 2H), 4.14 (s, 1H), 3.21 (dd, J = 15.5, 4.6 Hz, 1H), 3.05 (s, 3H), 2.97 (s, 3H), 2.92 (s, 2H), 3.17 −2.97 (m, 2H), 2.39−2.22 (m, 2H), 1.70−1.58 (m, 1H), 1.06 (s, 9H). MS (ESI) m/z: 532.5 (M + H). The following compounds were prepared by methods similar to those used for compound 15ii, substituting the appropriate amine: (4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12at e t r ah y d ro x y- 8 - [( m e t h yl a m i n o) m e t h yl ] - 1, 11 - d i o xo 1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15w). Prepared from methylamine, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.01 (d, J = 6.0 Hz, 1H), 4.17 (s, 2H), 4.10 (s, 1H), 3.21 (dd, J = 15.1, 4.6 Hz, 1H), 3.05 (s, 3H), 2.97 (s, 3H), 2.84 (s, 3H), 3.14−2.98 (m, 2H), 2.21−2.39 (m, 2H), 1.70−1.60 (m, 1H). MS (ESI) m/z: 476.3 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-8-[(dimethylamino)methyl]-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15x). Prepared from dimethylamine, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.08 (d, J = 6.0 Hz, 1H), 4.42 (s, 2H), 4.14 (s, 1H), 3.06 (s, 3H), 2.98 (s, 3H), 2.92 (s, 6H), 3.24−2.97 (m, 3H), 2.37−2.25 (m, 2H), 1.70−1.57 (m, 1H). MS (ESI) m/z: 490.4 (M + H). (4S,4aS,5aR,12aS)-8-{[Cyclopropyl(methyl)amino]methyl}-4(dimethylamino)-7-fluoro-3,10,12,12a-tetrahydroxy-1,11dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15y). Prepared from cyclopropylamine and N-methylated according to general experimental method B (formaldehyde), yellow solid. 1H NMR (400 MHz, CD3OD/DCl) δ 7.13 (d, J = 5.5 Hz, 1H), 4.64−4.46 (m, 2H), 4.17 (s, 1H), 3.26−2.94 (m, 12H), 2.39−2.25 (m, 2H), 1.70−1.56 (m, 1H), 1.14−1.04 (m, 1H), 0.98−0.85 (m, 4H). MS (ESI) m/z: 516.2 (M + H). (4S,4aS,5aR,12aS)-8-{[Cyclopropyl(ethyl)amino]methyl}-4(dimethylamino)-7-fluoro-3,10,12,12a-tetrahydroxy-1,11dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15z). Prepared from cyclopropylamine and N-ethylated according to general experimental method B (acetaldehyde), yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.08 (d, J = 5.5 Hz, 1H), 4.53 (d, J = 6.8 Hz, 2H), 4.10 (s, 1H), 3.41 (q, J = 7.3 Hz, 2H), 3.21 (dd, J = 15.1, 4.6 Hz, 1H), 3.17−2.95 (m, 2H), 3.04 (s, 3H), 2.96 (s, 3H), 2.93−2.85 (m, 1H), 2.36 (t, J = 13.8 Hz, 1H), 2.29−2.20 (m, 1H), 1.69−1.59 (m, 1H), 1.46 (t, J = 7.3 Hz, 3H), 1.10−0.84 (m, 3H), 0.79−0.69 (m, 1H). MS (ESI) m/z: 530.4 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12atetrahydroxy-1,11-dioxo-8-{[(propan-2-yl)amino]methyl}1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15aa). Prepared from isopropylamine, yellow solid. 1H NMR (400 MHz, CD3OD/DCl) δ 7.08 (d, J = 5.96 Hz, 1H), 4.27 (s, 2H), 4.16 (s, 1H), 3.51 (hept, J = 6.9 Hz, 1H), 3.28−2.94 (m, 9H), 2.38−2.26 (m, 2H), 1.60 (dd, J = 13.3, 11.0 Hz, 1H), 1.41 (d, J = 6.9 Hz, 6H). MS (ESI) m/z: 504.3 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12atetrahydroxy-8-{[methyl(propan-2-yl)amino]methyl}-1,11dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15bb). Prepared from isopropylamine and N-methylated according to general experimental method B (formaldehyde), yellow solid. 1H NMR (400 MHz, CD3OD/DCl) δ 7.13 (d, J = 5.92 Hz, 1H), 4.60−4.50 (m, 1H), 4.24−4.14 (m, 2H), 3.76−3.66 (m, 1H), 3.26−2.94 (m, 9H), 2.79 (s, 3H), 2.40−2.26 (m, 2H), 1.70− 1.58 (m, 1H), 1.50−1.40 (m, 6H). MS (ESI) m/z: 518.3 (M + H). (4S,4aS,5aR,12aS)-8-[(tert-Butylamino)methyl]-4-(dimethylamino)-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15cc). Prepared from t-butylamine, yellow solid. 1H 8128

dx.doi.org/10.1021/jm401211t | J. Med. Chem. 2013, 56, 8112−8138

Journal of Medicinal Chemistry

Article

NMR (400 MHz, CD3OD/DCl) δ 7.09 (d, J = 5.92 Hz, 1H), 4.22 (s, 2H), 4.16 (s, 1H), 3.28−2.94 (m, 9H), 2.38−2.26 (m, 2H), 1.60 (dd, J = 14.0, 11.0 Hz, 1H), 1.47 (s, 9H). MS (ESI) m/z: 518.3 (M + H). (4S,4aS,5aR,12aS)-8-{[tert-Butyl(methyl)amino]methyl}-4-(dimethylamino)-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15dd). Prepared from t-butylamine and N-methylated according to general experimental method B (formaldehyde), yellow solid. 1H NMR (400 MHz, CD3OD/DCl) δ 7.10 (d, J = 4.1 Hz, 1H), 4.80−4.72 (m, 1H), 4.15 (s, 1H), 4.04−3.96 (m, 1H), 3.26−2.94 (m, 9H), 2.78−2.74 (m, 3H), 2.39−2.25 (m, 2H), 1.72−1.53 (m, 10H). MS (ESI) m/z: 532.3 (M + H). (4S,4aS,5aR,12aS)-8-{[tert-Butyl(ethyl)amino]methyl}-4-(dimethylamino)-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15ee). Prepared from t-butylamine and N-ethylated according to general experimental method B (acetaldehyde), yellow solid. 1H NMR (400 MHz, CD3OD/DCl) δ 7.21 (d, J = 5.0 Hz, 1H), 4.70−4.61 (m, 1H), 4.32−4.26 (m, 1H), 4.19 (s, 1H), 3.56−3.45 (m, 1H), 3.34−2.95 (m, 10H), 2.40−2.26 (m, 2H), 1.72−1.55 (m, 10H), 1.19 (t, J = 7.3 Hz, 3H). MS (ESI) m/z: 546.3 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12atetrahydroxy-8-{[(2-methylpropyl)amino]methyl}-1,11-dioxo1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15ff). Prepared from 2-methylpropylamine, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.08 (d, J = 6.0 Hz, 1H), 4.28 (s, 2H), 4.14 (s, 1H), 3.21 (dd, J = 15.5, 4.6 Hz, 1H), 3.06 (s, 3H), 2.97 (s, 3H), 2.94 (d, J = 6.9 Hz, 2H), 3.17−2.97 (m, 2H), 2.35−2.25 (m, 2H), 2.12−2.02 (m, 1H), 1.70−1.58 (m, 1H), 1.04 (d, J = 6.9 Hz, 6H). MS (ESI) m/z: 518.5 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12atetrahydroxy-8-{[methyl(2-methylpropyl)amino]methyl}-1,11dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15gg). Prepared from 2-methylpropylamine and N-methylated according to general experimental method B (formaldehyde), yellow solid. 1H NMR (400 MHz, CD3OD/DCl) δ 7.11 (d, J = 5.96 Hz, 1H), 4.63−4.52 (m, 1H), 4.33−4.24 (m, 1H), 4.16 (s, 1H), 3.26−2.94 (m, 11H), 2.87 (s, 3H), 2.38−2.26 (m, 3H), 1.70−1.56 (m, 1H), 1.10−1.02 (m, 6H). MS (ESI) m/z: 532.3 (M + H). ( 4S , 4a S ,5 aR ,1 2a S )- 4- ( Di m e t h yl am in o) -8 - {[ e th yl ( 2methylpropyl)amino]methyl}-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2carboxamide Dihydrochloride (15hh). Prepared from 2-methylpropylamine and N-ethylated according to general experimental method B (acetaldehyde), yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.08 (d, J = 6.0 Hz, 1H), 4.50 (d, J = 13.5 Hz, 1H), 4.34 (d, J = 13.5 Hz, 1H), 4.28 (s, 2H), 4.10 (s, 1H), 3.04 (s, 3H), 2.96 (s, 3H), 3.27−2.97 (m, 7H), 2.36 (t, J = 14.7 Hz, 1H), 2.28−2.12 (m, 2H), 1.70−1.60 (m, 1H), 1.39 (t, J = 6.9 Hz, 3H), 1.05 (dd, J = 15.1, 6.4 Hz, 6H). MS (ESI) m/z: 546.4 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-8-{[(2,2dimethylpropyl)(methyl)amino]methyl}-7-fluoro-3,10,12,12atetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15jj). Prepared from 2,2dimethylpropylamine and N-methylated according to general experimental method B (formaldehyde), yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.12 (d, J = 5.8 Hz, 1H), 4.58 (d, J = 13.3 Hz, 1H), 4.36 (d, J = 13.3 Hz, 1H), 4.11 (s, 1H), 3.05 (s, 3H), 2.97 (s, 3H), 2.95 (s, 5H), 3.26−3.01 (m, 3H), 2.37 (t, J = 14.6 Hz, 1H), 2.29−2.22 (m, 1H), 1.71−1.61 (m, 1H), 1.08 (s, 9H). MS (ESI) m/z: 546.4 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-8-{[(2,2dimethylpropyl)(ethyl)amino]methyl}-7-fluoro-3,10,12,12atetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15kk). Prepared from 2,2dimethylpropylamine and N-ethylated according to general experimental method B (acetaldehyde), yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.09 (d, J = 5.5 Hz, 1H), 4.51 (d, J = 13.3 Hz, 1H), 4.39 (d, J = 13.3 Hz, 1H), 4.07 (s, 1H), 3.03 (s, 3H), 2.95 (s, 3H), 3.25− 2.96 (m, 7H), 2.42−2.33 (m, 1H), 2.26−2.18 (m, 1H), 1.70−1.60 (m,

1H), 1.43 (t, J = 7.3 Hz, 3H), 1.06 (s, 9H). MS (ESI) m/z: 560.5 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12atetrahydroxy-8-{[methyl(2-methylbutan-2-yl)amino]methyl}1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15ll). Prepared from t-amylamine and Nmethylated according to general experimental method B (formaldehyde), yellow solid. 1H NMR (400 MHz, CD3OD/DCl) δ 7.10 (d, J = 5.96 Hz, 1H), 4.80−4.71 (m, 1H), 4.18 (s, 1H), 4.06−3.98 (m, 1H), 3.26−2.95 (m, 9H), 2.76 (s, 3H), 2.37−2.25 (m, 2H), 2.01−1.89 (m, 2H), 1.69−1.54 (m, 1H), 1.51 (s, 3H), 1.48 (s, 3H), 1.07 (t, J = 6.4 Hz, 3H). MS (ESI) m/z: 546.3 (M + H). (4S,4aS,5aR,12aS)-8-[(Cyclopentylamino)methyl]-4-(dimethylamino)-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15 mm). Prepared from cyclopentylamine, yellow solid. 1 H NMR (400 MHz, CD3OD/DCl) δ 7.07 (d, J = 5.96 Hz, 1H), 4.26 (s, 2H), 4.16 (s, 1H), 3.66 (quint, J = 6.9 Hz, 1H), 3.34−2.94 (m, 11H), 2.36−2.24 (m, 2H), 2.23−2.12 (m, 2H), 1.90−1.54 (m, 5H). MS (ESI) m/z: 530.2 (M + H). (4S,4aS,5aR,12aS)-8-{[Cyclopentyl(methyl)amino]methyl}-4(dimethylamino)-7-fluoro-3,10,12,12a-tetrahydroxy-1,11dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15nn). Prepared from cyclopentylamine and N-methylated according to general experimental method B (formaldehyde), yellow solid. 1H NMR (400 MHz, CD3OD/DCl) δ 7.12 (d, J = 5.5 Hz, 1H), 4.62−4.53 (m, 1H), 4.28−4.20 (m, 1H), 4.17 (s, 1H), 3.84−3.74 (m, 1H), 3.26−2.94 (m, 9H), 2.79 (s, 3H), 2.39− 2.25 (m, 3H), 2.23−2.13 (m, 1H), 1.97−1.81 (m, 4H), 1.80−1.55 (m, 3H). MS (ESI) m/z: 544.3 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-8-[(2,2-dimethylpyrrolidin-1-yl)methyl]-7-fluoro-3,10,12,12a-tetrahydroxy-1,11dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15oo). Prepared from 2,2-dimethylpyrrolidine, yellow solid. 1H NMR (400 MHz, CD3OD/DCl) δ 7.12 (d, J = 5.96 Hz, 1H), 4.56 (d, J = 3.2 Hz, 1H), 4.17 (s, 1H), 4.10 (d, J = 3.2 Hz, 1H), 3.54−3.45 (m, 2H), 3.26−2.96 (m, 9H), 2.39−2.26 (m, 2H), 2.23−1.95 (m, 4H), 1.70−1.56 (m, 4H), 1.44 (s, 3H). MS (ESI) m/z: 544.2 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12atetrahydroxy-1,11-dioxo-8-(piperidin-1-ylmethyl)1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15pp). Prepared from piperidine, yellow solid. 1H NMR (400 MHz, CD3OD/DCl) δ 7.15 (d, J = 5.96 Hz, 1H), 4.37 (s, 2H), 4.17 (s, 1H), 3.56−3.48 (m, 2H), 3.40−2.94 (m, 11H), 2.38− 2.26 (m, 2H), 1.99−1.78 (m, 5H), 1.70−1.48 (m, 2H). MS (ESI) m/z: 530.3 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12atetrahydroxy-8-[(4-methylpiperidin-1-yl)methyl]-1,11-dioxo1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15rr). Prepared from 4-methylpiperidine, yellow solid. 1 H NMR (400 MHz, CD3OD) δ 7.10 (d, J = 5.5 Hz, 1H), 4.35 (s, 2H), 4.12 (s, 1H), 3.51 (m, 2H), 3.24−2.98 (m, 5H), 3.05 (s, 3H), 2.97 (s, 3H), 2.34 (t, J = 15.1 Hz, 1H), 2.29−2.21 (m, 1H), 1.96−1.85 (m, 2H), 1.79−1.56 (m, 2H), 1.54−1.40 (m, 2H), 0.99 (d, J = 6.4 Hz, 3H). MS (ESI) m/z: 544.3 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-8-[(3,3-dimethylpiperidin-1-yl)methyl]-7-fluoro-3,10,12,12a-tetrahydroxy-1,11dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15uu). Prepared from 3,3-dimethylpiperidine, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.10 (d, J = 5.5 Hz, 1H), 4.37 (s, 2H), 4.10 (s, 1H), 3.50 (m, 1H), 3.27−2.93 (m, 5H), 3.05 (s, 3H), 2.97 (s, 3H), 2.85 (m, 1H), 2.36 (t, J = 15.1 Hz, 1H), 2.29−2.21 (m, 1H), 1.99−1.81 (m, 2H), 1.72−1.53 (m, 2H), 1.49− 1.38 (m, 1H), 1.12 (s, 3H), 1.02 (s, 3H). MS (ESI) m/z: 558.5 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-8-[(4,4-dimethylpiperidin-1-yl)methyl]-7-fluoro-3,10,12,12a-tetrahydroxy-1,11dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15vv). Prepared from 4,4-dimethylpiperidine, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.07 (d, J = 5.5 Hz, 1H), 4.39 (s, 2H), 4.09 (s, 1H), 3.38 (m, 2H), 3.24−2.98 (m, 5H), 8129

dx.doi.org/10.1021/jm401211t | J. Med. Chem. 2013, 56, 8112−8138

Journal of Medicinal Chemistry

Article

3.04 (s, 3H), 2.96 (s, 3H), 2.35 (t, J = 15.1 Hz, 1H), 2.27−2.19 (m, 1H), 1.77−1.58 (m, 5H), 1.09 (s, 3H), 1.03 (s, 3H). MS (ESI) m/z: 558.3 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12atetrahydroxy-8-(morp holin-4-ylmet hyl)-1,11-dioxo1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15ww). Prepared from morpholine, yellow solid. 1H NMR (400 MHz, CD3OD/DCl) δ 7.17 (d, J = 5.96 Hz, 1H), 4.45 (s, 2H), 4.16 (s, 1H), 4.08−3.98 (m, 2H), 3.92−3.80 (m, 2H), 3.52−3.42 (m, 2H), 3.38−2.94 (m, 11H), 2.38−2.25 (m, 2H), 1.70−1.55 (m, 1H). MS (ESI) m/z: 532.3 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12atetrahydroxy-8-{[methyl(2,4,4-trimethylpentan-2-yl)amino]methyl}-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene2-carboxamide Dihydrochloride (15xx). Prepared from 2,4,4trimethylpentan-2-amine and N-methylated according to general experimental method B (formaldehyde), yellow solid. 1H NMR (400 MHz, CD3OD/DCl) δ 7.10 (d, J = 5.5 Hz, 1H), 4.82−4.74 (m, 1H), 4.17 (s, 1H), 4.05−3.97 (m, 1H), 3.28−2.95 (m, 9H), 2.76 (s, 3H), 2.38−2.26 (m, 2H), 2.01−1.80 (m, 2H), 1.76−1.56 (m, 7H), 1.12 (s, 9H). MS (ESI) m/z: 588.3 (M + H). (4S,4aS,5aR,12aS)-8-{4-Azaspiro[2.5]octan-4-ylmethyl}-4-(dimethylamino)-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15yy). Prepared from 4-azaspiro[2.5]octane, yellow solid. 1H NMR (400 MHz, CD3OD/DCl) δ 7.15 (br s, 1H), 4.76− 4.58 (m, 2H), 4.17 (s, 1H), 3.40−2.92 (m, 11H), 2.60−2.48 (m, 1H), 2.38−2.15 (m, 3H), 1.99−1.82 (m, 2H), 1.82−1.55 (m, 2H), 1.45− 1.36 (m, 1H), 1.22−1.14 (m, 1H), 1.10−0.99 (m, 1H), 0.99−0.80 (m, 2H). MS (ESI) m/z: 556.2 (M + H). Phenyl 2-(Benzyloxy)-3-bromo-5-fluoro-6-methyl-4-{[(2S)-2methylpiperidin-1-yl]methyl}benzoate (16qq). Compound 10 (89 mg, 0.20 mmol) and (S)-2-methylpiperidine (0.060 mL, 0.50 mmol) were dissolved in 1,2-dichloroethane (2 mL). Titanium(IV) isopropoxide (0.18 mL, 0.60 mmol) was added. After stirring overnight, CH3OH (1 mL) and sodium borohydride (40 mg, 1.1 mmol, in 4 equal portions) were added until the intermediate was completely converted to the product as indicated by LC−MS. The reaction mixture was diluted with dichloromethane, washed with NaHCO3 (saturated, aqueous solution), water (2 × 20 mL), and brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. Purification by column chromatography (Biotage 10 g column, 10− 50% EtOAc in hexanes gradient) gave 86 mg (82%) of the title compound as a colorless oil. (4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12atetrahydroxy-8-{[(2S)-2-methylpiperidin-1-yl]methyl}-1,11dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15qq). Prepared from 16qq following the procedures described for compound 15ii above, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.07 (d, J = 6.0 Hz, 1H), 4.76 (d, J = 13.7 Hz, 1H), 4.15 (d, J = 13.7 Hz, 1H), 4.10 (s, 1H), 3.40−3.32 (m, 2H), 3.05 (s, 3H), 2.97 (s, 3H), 3.27−2.96 (m, 4H), 2.42−2.32 (m, 1H), 2.27− 2.21 (m, 1H), 1.92−1.80 (m, 2H), 1.73−1.58 (m, 3H), 1.56 (d, J = 6.4 Hz, 3H). MS (ESI) m/z: 544.3 (M + H). The following compounds were prepared by methods similar to those used for compound 15qq, substituting the appropriate amine: (4S,4aS,5aR,12aS)-4-(Dimethylamino)-8-{[(2R,6S)-2,6-dimethylpiperidin-1-yl]methyl}-7-fluoro-3,10,12,12a-tetrahydroxy1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15ss). Prepared from cis-2,6-dimethylpiperidine, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.06 (dd, J = 8.2, 6.0 Hz, 1H), 4.40 (s, 2H), 4.09 (s, 1H), 3.68−3.57 (m, 2H), 3.24− 2.98 (m, 3H), 3.04 (s, 3H), 2.96 (s, 3H), 2.40−2.32 (m, 1H), 2.29− 2.21 (m, 1H), 2.05−1.58 (m, 7H), 1.54 (d, J = 6.0 Hz, 3H), 1.35 (d, J = 6.0 Hz, 3H). MS (ESI) m/z: 558.3 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-8-[(2,2-dimethylpiperidin-1-yl)methyl]-7-fluoro-3,10,12,12a-tetrahydroxy-1,11dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (15tt). Prepared from 2,2-dimethylpiperidine, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.04 (d, J = 5.5 Hz, 1H), 4.75 (dd, J = 13.7, 7.3 Hz, 1H), 4.08 (s, 1H), 3.92 (dd, J = 13.7, 7.3 Hz, 1H), 3.32 (m, 2H), 3.24−2.98 (m, 3H), 3.03 (s, 3H),

2.96 (s, 3H), 2.37 (t, J = 15.1 Hz, 1H), 2.27−2.20 (m, 1H), 1.91−1.82 (m, 2H), 1.80−1.63 (m, 5H), 1.63 (s, 3H), 1.50 (s, 3H). MS (ESI) m/ z: 558.3 (M + H). 4-Bromo-2-methoxy-6-methylaniline hydrobromide (20). To an ice-cooled solution of 2-methoxy-6-methylaniline (25.12 g, 183.1 mmol) in CH3OH (79 mL) and acetic acid (25 mL) was added a solution of bromine (9.41 mL, 183.1 mmol) in acetic acid (79 mL) dropwise via an addition funnel. The reaction mixture was allowed to warm to rt and was stirred for 2 h. EtOAc (150 mL) was added, and the solid was collected by filtration and washed with EtOAc, yielding 37.2 g (68%) of the title compound as an off-white solid. 1H NMR (400 MHz, CDCl3) δ 6.82 (d, J = 1.4 Hz, 1H), 6.77 (d, J = 1.4 Hz, 1H), 3.81 (s, 3H), 2.12 (s, 3H). MS (ESI) m/z: 216.1 (M + H). 4-Bromo-2-methoxy-6-methylbenzonitrile (21). Compound 20 (20.00 g, 92.70 mmol) was suspended in concentrated HCl (22 mL) and crushed ice (76 g) and was cooled in an ice bath. A solution of NaNO2 (6.52 g, 94.6 mmol) in water (22 mL) was added dropwise. The resulting mixture was stirred at 0 °C for 30 min and was neutralized with aqueous Na2CO3. A suspension of CuCN (10.40 g, 115.9 mmol) in water (44 mL) was mixed with a solution of NaCN (14.40 g, 294.8 mmol) in water (22 mL) and was cooled in an ice bath. The initial diazonium salt mixture was then added to the CuCN and NaCN mixture with vigorous stirring while maintaining the temperature at 0 °C. Toluene (180 mL) was also added in portions during the addition. The reaction mixture was stirred at 0 °C for 1 h, rt for 2 h, and 50 °C for 1 h. After cooling to rt, the layers were separated. The aqueous layer was extracted with toluene. The combined organic layers were washed with brine, dried over MgSO4, and concentrated. The residue was passed through a silica gel plug, eluting with toluene, to yield 14.5 g (69%) of the title compound as a light-yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.04 (d, J = 1.4 Hz, 1H), 6.93 (d, J = 1.4 Hz, 1H), 3.89 (s, 3H), 2.46 (s, 3H). Phenyl 4-Bromo-2-methoxy-6-methylbenzoate (22b). To a solution of 21 (11.34 g, 50.20 mmol) in THF (100 mL) was added diisobutylaluminum hydride (1.5 M in toluene, 40.1 mL, 60.2 mmol) slowly at −78 °C. The reaction mixture was allowed to warm to rt gradually and was stirred overnight. After cooling to 0 °C, the reaction was carefully quenched with 1 N aqueous HCl. The resulting mixture was stirred at rt for 1 h and was extracted three times with EtOAc. The combined extracts were washed with water, saturated aqueous NaHCO3, and brine, dried over MgSO4, and concentrated. The crude material was suspended in t-BuOH (200 mL), and a solution of NaClO2 (11.34 g, 100.3 mmol) and NaH2PO4 (34.6 g, 251 mmol) in water (100 mL) was added via an addition funnel. After complete addition, 2-methyl-2-butene (42.4 mL, 400 mmol) was added. The resulting homogeneous solution was stirred at rt for 30 min and was concentrated under reduced pressure. The residue was suspended in water (150 mL). The solution was acidified to pH ∼1 with 1 N HCl and was extracted three times with t-butylmethyl ether. The combined extracts were extracted three times with 1 N NaOH. The combined aqueous extracts were acidified with 6 N HCl and were extracted three times with EtOAc. The combined EtOAc extracts were washed with brine, dried over MgSO4, and concentrated to provide 8.64 g (70%) of the benzoic acid intermediate 22a as an off-white solid. To a solution of 22a (8.64 g, 35.2 mmol) in dichloromethane (70 mL) was added oxalyl chloride (3.76 mL, 42.3 mmol) followed by a couple of drops of DMF (caution: gas evolution). The mixture was stirred at rt for 30 min and was evaporated under reduced pressure. The residue was further dried under high vacuum to afford the crude benzoyl chloride. The material was dissolved in dichloromethane (70 mL). Triethylamine (12.3 mL, 88.1 mmol), phenol (3.98 g, 42.3 mmol), and DMAP (0.43 g, 3.5 mmol) were added. The mixture was stirred at rt for 1 h. The solvent was evaporated. The residue was suspended in EtOAc, and the precipitate was filtered off. The organic solution was then washed with 1 N HCl (three times), water, saturated aqueous NaHCO3, and brine, dried over Na2SO4, filtered, and concentrated. Purification by flash chromatography (1−5% EtOAc in hexanes gradient) gave 10.05 g (89%) of the title compound as an offwhite solid. 1H NMR (400 MHz, CDCl3) δ 7.41−7.45 (m, 2H), 8130

dx.doi.org/10.1021/jm401211t | J. Med. Chem. 2013, 56, 8112−8138

Journal of Medicinal Chemistry

Article

7.22−7.27 (m, 3H), 7.04 (d, J = 0.9 Hz, 1H), 6.97 (d, J = 0.9 Hz, 1H), 3.87 (s, 3H), 2.42 (s, 3H). MS (ESI) m/z: 319.0 (M-H). Phenyl 4-Bromo-3-chloro-6-methoxy-2-methylbenzoate (23). To a solution of 22 (2.52 g, 7.87 mmol) in acetonitrile (16 mL) was added N-chlorosuccinimide (1.10 g, 8.27 mmol). The resulting mixture was heated at 60 °C for 45 h. The solvent was evaporated, and the residue was dissolved in diethyl ether (400 mL), washed with 1 N NaOH, water, and brine, dried over Na2SO4, and concentrated to provide 2.76 g (99% crude) of the title compound as a white solid. This material was used without further purification. 1H NMR (400 MHz, CDCl3) δ 7.44 (dd, J = 7.8, 7.8 Hz, 2H), 7.22−7.28 (m, 3H), 7.13 (s, 1H), 3.87 (s, 3H), 2.51 (s, 3H). MS (ESI) m/z: 353.0 (M-H). Phenyl 6-(Benzyloxy)-4-bromo-3-chloro-2-methylbenzoate (24). Compound 23 (2.76 g, 7.76 mmol) was dissolved in dichloromethane (78 mL) and cooled to −78 °C. A solution of boron tribromide (1.0 M in dichloromethane, 7.76 mL, 7.76 mmol) was added dropwise. The resulting yellow solution was stirred at −78 °C for 15 min and at 0 °C for 30 min. Saturated aqueous NaHCO3 was added. The mixture was stirred at rt for 10 min and was extracted with EtOAc (3×). The combined extracts were washed with brine, dried over Na2SO4, and concentrated. The material was dissolved in acetone (40 mL), and K2CO3 (2.14 g, 15.5 mmol) and benzylbromide (0.97 mL, 8.15 mmol) were added. After stirring overnight, the solution was filtered through a bed of Celite. The Celite was washed with EtOAc three times. The combined filtrate was concentrated under reduced pressure. Purification by flash chromatography (1−5% EtOAc in hexanes gradient) gave 2.97 g (89%) of the title compound as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.33−7.43 (m, 7H), 7.19−7.26 (m, 2H), 7.05 (d, J = 7.8 Hz, 2H), 5.11 (s, 2H), 2.51 (s, 3H). MS (ESI) m/z: 429.0 (M-H). Phenyl 6-(Benzyloxy)-3-chloro-4-formyl-2-methylbenzoate (25). To a solution of compound 24 (1.98 g, 4.59 mmol) in THF (23 mL) was added i-PrMgCl·LiCl (1.2 M solution in THF, 7.65 mL, 9.18 mmol) dropwise at −78 °C. After 10 min, the temperature was raised to 0 °C, and the reaction was stirred for 1 h. DMF (1.80 mL, 22.9 mmol) was added, and the reaction mixture was warmed to rt. After 30 min, the reaction was quenched with saturated aqueous NH4Cl. The layers were separated, and the aqueous layer was further extracted with EtOAc (2×). The combined extracts were washed with brine, dried over Na2SO4, filtered, and concentrated under reduced pressure. Purification by flash chromatography (2−10% EtOAc in hexanes gradient) gave 1.45 g (83%) of the title compound as a white solid. 1H NMR (400 MHz, CDCl3) δ 10.51 (s, 1H), 7.33−7.44 (m, 8H), 7.25−7.27 (m, 1H), 7.05 (d, J = 7.8 Hz, 2H), 5.19 (s, 2H), 2.51 (s, 3H). MS (ESI) m/z: 379.1 (M-H). Phenyl 6-(Benzyloxy)-3-chloro-4-(dimethoxymethyl)-2methylbenzoate (26). To a solution of 25 (1.66 g, 4.37 mmol) in CH3OH (22 mL) was added trimethylorthoformate (2.40 mL, 21.9 mmol) and TsOH (83 mg, 0.44 mmol). The reaction mixture was heated at 65 °C for 4 h and was concentrated under reduced pressure. The material was dissolved in EtOAc (200 mL), washed with saturated aqueous NaHCO3 and brine, dried over Na2SO4, and concentrated. Purification by flash chromatography (2−10% EtOAc in hexanes gradient) gave 1.75 g (94%) of the title compound as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.43 (d, J = 7.8 Hz, 2H), 7.34−7.37 (m, 5H), 7.20−7.25 (m, 2H), 7.07 (d, J = 7.8 Hz, 2H), 5.62 (s, 1H), 5.19 (s, 2H), 3.36 (s, 6H), 2.48 (s, 3H). MS (ESI) m/z: 425.1 (M-H). Phenyl 4-Bromo-2-hydroxy-6-methylbenzoate (27). BBr3 (1.0 M solution in CH2Cl2, 28.0 mL, 28.0 mmol) was added to a solution of 22 (8.98 g, 28.0 mmol) in DCM (100 mL) at −78 °C. The reaction mixture was stirred at −78 °C for 20 min and at 0 °C for 15 min. Saturated NaHCO3 solution (120 mL) was added slowly. The mixture was stirred at rt for 20 min and was concentrated under reduced pressure to remove CH2Cl2. The remaining aqueous mixture was extracted with EtOAc (250 mL). The extracts were dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by recrystallization from EtOAc/Hexanes to give 6.76 g (79%) of the title compound as a white solid. The mother liquor was concentrated and purified by column chromatography (2−

10% EtOAc/hexanes gradient) to afford an additional 973 mg (11%) of the title compound. 1H NMR (400 MHz, CDCl3) δ 11.13 (s, 1H), 7.47−7.43 (m, 2H), 7.33−7.29 (m, 1H), 7.19−7.16 (m, 2H), 7.08 (d, J = 1.8 Hz, 1H), 6.96 (d, J = 1.8 Hz, 1H), 2.66 (s, 3H). MS (ESI) m/z: 305.1, 307.1 (M−H). Phenyl 4-Bromo-6-hydroxy-3-methoxy-2-methylbenzoate (28). A solution of PhI(OAc)2 (3.77 g, 11.7 mmol) in CH3OH (20 mL) was added slowly to a solution of 27 (1.71 g, 5.58 mmol) in a mixture of CH3OH (30 mL) and 1,4-dioxane (10 mL) at 0 °C. The reaction mixture was stirred at rt for 17 h. Acetic acid (6 mL) was added followed by Zn dust (1.09 g, 16.7 mmol) (slightly exothermic). The reaction mixture was stirred at rt for 20 min and was filtered through a pad of Celite (EtOAc wash). The filtrate was concentrated, and the residue was partitioned between EtOAc (120 mL) and saturated NaHCO3/brine solution. The organic layer was separated, dried over MgSO4, filtered, and concentrated. Purification by column chromatography (0−4% EtOAc/hexanes gradient) gave 763 mg (41%) of the title compound. 1H NMR (400 MHz, CDCl3) δ 10.70 (s, 1H), 7.47−7.43 (m, 2H), 7.33−7.30 (m, 1H), 7.20−7.17 (m, 2H), 7.16 (s, 1H), 3.75 (s, 3H), 2.67 (s, 3H). MS (ESI) m/z: 335.1, 337.14 (M − H). Phenyl 4-Bromo-6-{[(tert-butoxy)carbonyl]oxy}-3-methoxy2-methylbenzoate (29). Di-tert-butyl dicarbonate (543 mg, 2.49 mmol) and N,N-dimethylaminopyridine (28 mg, 0.23 mmol) were added to a solution of 28 (763 mg, 2.26 mmol) in CH2Cl2 (20 mL). After 20 min, the reaction mixture was concentrated under reduced pressure and was purified by column chromatography (0−5% EtOAc/ hexanes gradient) to afford 783 mg (79%) of the title compound as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.45−7.41 (m, 2H), 7.38 (s, 1H), 7.30−7.26 (m, 1H), 7.24−7.22 (m, 2H), 3.81 (s, 3H), 2.47 (s, 3H), 1.43 (s, 9H). MS (ESI) m/z: 435.1, 437.1 (M − H). Phenyl 6-{[(tert-Butoxy)carbonyl]oxy}-4-formyl-3-methoxy2-methylbenzoate (30). A solution of i-PrMgCl·LiCl (1.2 M in THF, 0.547 mL, 0.657 mmol) was added dropwise to a 0 °C solution of 29 (144 mg, 0.328 mmol) in THF (3.3 mL). After 1 h, DMF (0.127 mL, 1.64 mmol) was added, and the reaction mixture was stirred at 0 °C for 10 min and at rt for 20 min. The reaction mixture was quenched with NH4Cl (saturated, aqueous solution) and brine and was extracted with EtOAc (50 mL). The extracts were dried over MgSO4, filtered, and concentrated. The crude title compound was used directly in the next step. 1H NMR (400 MHz, CDCl3) δ 10.38 (s, 1H), 7.61 (s, 1H), 7.46−7.42 (m, 2H), 7.32−7.28 (m, 1H), 7.26−7.24 (m, 2H), 3.91 (s, 3H), 2.46 (s, 3H), 1.45 (s, 9H). MS (ESI) m/z: 385.2 (M − H). Phenyl 6-{[(tert-Butoxy)carbonyl]oxy}-4-(dimethoxymethyl)3-methoxy-2-methylbenzoate (31). Trimethyl orthoformate (0.180 mL, 1.64 mmol) and p-toluenesulfonic acid (3.1 mg, 0.016 mmol) were added to a solution of 30 in CH3OH (1 mL). The reaction mixture was heated at reflux for 1 h, cooled to rt, and concentrated under reduced pressure. The material was dissolved in EtOAc (40 mL) and was washed with saturated NaHCO3/brine solution (1:1, 20 mL). The organic phase was separated, dried over MgSO4, filtered, and concentrated. The crude 1H NMR indicated partial removal of the Boc group. The material was dissolved in CH2Cl2 (1 mL), and di-tert-butyl dicarbonate (26.8 mg, 0.123 mmol) and N,N-dimethylaminopyridine (1 mg, 0.08 mmol) were added. After 40 min, the reaction mixture was concentrated under reduced pressure. Purification by column chromatography (2−10% EtOAc/ hexanes grade) gave 124 mg (87%, 3 steps) of the title compound as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 7.45−7.41 (m, 2H), 7.33 (s, 1H), 7.30−7.24 (m, 3H), 5.65 (s, 1H), 3.79 (s, 3H), 3.36 (s, 6H), 2.43 (s, 3H), 1.43 (s, 9H). MS (ESI) m/z: 431.3 (M−H). Phenyl 4-Ethenyl-2-methoxy-6-methylbenzoate (32). Compound 22 (20 g, 62.5 mmol), 2,4,6-trivinylcyclotriboroxane-pyridine complex (7.8 g, 31 mmol), Pd(PPh3)4 (2.2 g, 1.9 mmol), and K2CO3 (17.3 g, 125 mmol) were added to a reaction vessel containing 1,4dioxane/water (3:1, v/v). The reaction mixture was purged and backfilled with nitrogen (6×), and the mixture was heated to reflux. After 19 h, the mixture was concentrated, and the material was partitioned between EtOAc and water. The organic layer was dried 8131

dx.doi.org/10.1021/jm401211t | J. Med. Chem. 2013, 56, 8112−8138

Journal of Medicinal Chemistry

Article

7.30 (m, 9H), 7.08−6.90 (m, 2H), 5.60 (s, 1H), 5.20 (s, 2H), 3.73 (s, 6H), 2.56−2.51 (m, 3H). MS (ESI) m/z: 461.1 (M + H). (1R,3S,4S,11S)-8,16-Bis(benzyloxy)-11-[(tertbutyldimethylsilyl)oxy]-19-chloro-18-(dimethoxymethyl)-4(dimethylamino)-12-hydroxy-6-oxa-7-azapentacyclo[11.8.0.0 3,11 .0 5,9 .015,20 ]henicosa-5(9),7,12,15(20),16,18-hexaene-10,14-dione (36a, R3 = Cl). n-BuLi (2.2 M solution in hexanes, 2.37 mL, 5.23 mmol) was added dropwise to a solution of diisopropylamine (0.77 mL, 5.45 mmol) in THF (27 mL) at −78 °C. The resulting solution was stirred at −78 °C for 20 min and at 0 °C for 5 min and was then recooled to −78 °C. TMEDA (0.85 mL, 5.67 mmol) was added followed by the dropwise addition of a solution of 26 (2.05 g, 4.80 mmol) in THF (30 mL). After complete addition, the resulting dark-red mixture was stirred for 1 h at −78 °C and was then cooled to −100 °C. A solution of enone 6 (2.10 g, 4.36 mmol) in THF (30 mL) was added dropwise. The mixture was slowly allowed to warm to −78 °C. LHMDS (1.0 M in THF, 4.36 mL, 4.36 mmol) was added, and the reaction was slowly allowed to warm to −5 °C. The reaction was quenched with NH4Cl (saturated, aqueous solution) and was extracted with EtOAc (3×). The combined extracts were washed with brine, dried over Na2SO4, and concentrated. Purification by column chromatography (5−25% EtOAc in hexanes gradient) gave 3.20 g (90%) of the title compound as a light-yellow solid. 1H NMR (400 MHz, CDCl3) δ 0.16 (s, 3H), 7.22−7.52 (m, 11H), 5.55 (s, 1H), 5.38 (s, 2H), 5.29 (d, J = 11.4 Hz, 1H), 5.24 (d, J = 11.4 Hz, 1H), 3.97 (d, J = 10.4 Hz, 1H), 3.46 (dd, J = 4.9, 15.9 Hz, 1H), 3.38 (s, 3H), 3.29 (s, 3H), 2.96−3.04 (m, 1H), 2.45−2.58 (m, 9H), 2.15 (d, J = 14.6 Hz, 1H), 0.84 (s, 9H), 0.28 (s, 3H). MS (ESI) m/z: 815.3. (1R,3S,4S,11S)-8,16-Bis(benzyloxy)-11-[(tertbutyldimethylsilyl)oxy]-19-chloro-4-(dimethylamino)-12-hydroxy-10,14-dioxo-6-oxa-7-azapentacyclo[11.8.0.0 3,11 .0 5,9 .015,20 ]henicosa-5(9),7,12,15(20),16,18-hexaene-18-carbaldehyde (37a, R3 = Cl). To a solution of compound 36a (1.48 g, 1.82 mmol) in THF (15 mL) was added 3 N HCl (3 mL) at 0 °C. The resulting mixture was stirred at rt for 4 h, diluted with EtOAc, washed with saturated aqueous NaHCO3 and brine, dried over Na2SO4, and concentrated. Purification by column chromatography gave 1.05 g (75%) of the title compound as a light-yellow solid. 1H NMR (400 MHz, CDCl3) δ 15.8 (s, 1H), 10.5 (s, 1H), 7.28−7.50 (m, 11H), 5.36 (s, 2H), 5.28 (d, J = 11.4 Hz, 1H), 5.23 (d, J = 11.4 Hz, 1H), 3.94 (d, J = 10.4 Hz, 1H), 3.48 (dd, J = 4.9, 15.9 Hz, 1H), 2.96− 3.06 (m, 1H), 2.44−2.60 (m, 9H), 2.16 (d, J = 14.6 Hz, 1H), 0.82 (s, 9H), 0.26 (s, 3H), 0.14 (s, 3H). MS (ESI) m/z: 769.3 (M + H). (4S,4aS,5aR,12aS)-7-Chloro-4-(dimethylamino)-3,10,12,12at e t ra hy d ro xy - 1, 11 -d i o xo - 8- (p yr ro li d i n -1 - yl m e t hy l )1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (39a). Prepared from 37a by general experimental methods A (pyrrolidine) and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.20 (s, 1H), 4.58 (s, 2H), 4.11 (s, 1H), 3.69−3.56 (m, 2H), 3.48−3.36 (m, 2H), 3.04 (s, 3H), 2.96 (s, 3H), 3.16−2.93 (comp, 3H), 2.46−2.37 (m, 1H), 2.29−1.98 (comp, 5H), 1.71−1.59 (m, 1H). MS (ESI) m/z: 532.1 (M + H). (4S,4aS,5aR,12aS)-7-Chloro-4-(dimethylamino)-3,10,12,12atetrahydroxy-1,11-dioxo-8-[(dimethylamino)methyl]1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (39b). Prepared from 37a by general experimental methods A (dimethylamine) and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.21 (s, 1H), 4.54−4.47 (m, 2H), 4.12 (s, 1H), 3.39 (dd, J = 16.0 and 4.4 Hz, 1H), 3.15−2.85 (m, 14H), 2.44−2.37 (m, 1H), 2.28−2.22 (m, 1H), 1.70−1.59 (m, 1H). MS (ESI) m/z: 506.1 (M + H). (4S,4aS,5aR,12aS)-8-{[t-Butyl(methyl)amino]methyl}-7chloro-4-(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (39c). Prepared from 37a by general experimental methods A (t-butylamine), B (formaldehyde), and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.20 (s, 1H), 4.80−4.87 (m, 1H), 4.12−4.20 (m, 1H), 4.12 (s, 1H), 3.38−3.40 (m, 1H), 2.96− 3.11 (m, 8H), 2.75 (s, 3H), 2.38−2.40 (m, 1H), 2.24−2.28 (m, 1H), 1.60−1.70 (m, 1H), 1.57 (s, 9H). MS (ESI) m/z: 548.2 (M + H). (4S,4aS,5aR,12aS)-7-Chloro-4-(dimethylamino)-8-[(2,2-dimethylpyrrolidin-1-yl)methyl]-3,10,12,12a-tetrahydroxy-1,11-

over Na2SO4 and was evaporated to dryness. Purification by column chromatography (200:1 to 100:1 to 50:1 petroleum ether/EtOAc gradient) gave 14.8 g (88%) of the title compound as a light-yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.38−7.34 (m, 2H), 7.27−7.16 (m, 3H), 6.83−6.76 (m, 2H), 6.65−6.60 (m, 1H), 5.72 (d, J = 17.6 Hz, 1H), 5.25 (d, J = 11.2 Hz, 1H), 3.83 (s, 3H), 2.38 (s, 3H). MS (ESI) m/z: 269.1 (M + H). Phenyl 4-Formyl-2-methoxy-6-methylbenzoate (33). An ozone-enriched stream of oxygen was bubbled through a −78 °C solution of compound 32 (21 g, 78 mmol) in CH2Cl2 until it turned light blue. The solution was purged with argon at −78 °C for 10 min to remove excess O3. Dimethylsulfide (50 mL) was added, and the reaction mixture was allowed to warm slowly to 25 °C. After 5 h, the reaction mixture was concentrated. Purification by column chromatography (100:1 to 50:1 to 30:1 petroleum ether/EtOAc gradient) gave 13 g (62%) of the title compound as a light-yellow solid. 1H NMR (400 MHz, CDCl3) δ 9.97 (s, 1H), 7.46−7.41 (m, 2H), 7.36−7.22 (m, 5H), 3.92 (s, 3H), 2.51 (s, 3H). MS (ESI) m/z: 271.1 (M + H). Phenyl 3-Bromo-4-formyl-2-methoxy-6-methylbenzoate. Compound 33 (1.8 g, 6.6 mmol) was dissolved in acetic acid. Bromine (1.6 mL, 27 mmol) was added dropwise, and the reaction mixture was stirred for 1 h at rt. The reaction mixture was concentrated, and the material was partitioned between EtOAc and NaHCO3 (saturated, aqueous solution). The organic layer was washed with brine and water, dried over Na2SO4, and concentrated to afford 1.9 g of the crude title compound as a light-yellow solid. The material was used without further purification. Phenyl 3-Bromo-4-formyl-2-hydroxy-6-methylbenzoate. BBr3 (1.90 mL, 19.5 mmol) was added to a solution of phenyl 3bromo-4-formyl-2-methoxy-6-methylbenzoate (3.5 g, 13 mmol) in CH2Cl2 (30 mL) at −78 °C. The reaction mixture was allowed to slowly warm to 25 °C. After 1.5 h, the reaction was quenched with saturated, aqueous NaHCO3 and was extracted with EtOAc. The combined EtOAc extracts were dried over Na2SO4 and were concentrated to yield 3.3 g of the crude title compound. The material was used without further purification. Phenyl 2-(Benzyloxy)-3-bromo-4-formyl-6-methylbenzoate (34). K2CO3 (3.6 g, 26 mmol) and benzylbromide (4.2 g, 26 mmol) were added to a solution of phenyl 3-bromo-4-formyl-2-hydroxy-6methylbenzoate (3.3 g, 13 mmol) in DMF (15 mL). After 2 h, the reaction mixture was filtered (EtOAc wash). The filtrate was washed with water (3×), dried over Na2SO4, and concentrated. Purification by column chromatography (100:1 to 50:1 petroleum ether/EtOAc gradient) gave 3.5 g (62%, 3 steps) of the title compound as a lightyellow solid. 1H NMR (400 MHz, CDCl3) δ 10.43 (s, 1H), 7.46−7.30 (m, 9H), 7.08−7.05 (m, 2H), 5.17 (s, 2H), 2.52 (s, 3H). MS (ESI) m/ z: 425.1 (M + H). Phenyl 2-(Benzyloxy)-4-formyl-6-methyl-3(trifluoromethyl)benzoate. Methyl 2,2-difluoro-2-(fluorosulfonyl)acetate (11 g, 59 mmol) and CuI (4.5 g, 24 mmol) were added to a solution of 34 (5 g, 12 mmol) in anhydrous DMF. The reaction mixture was heated at 100 °C. After 20 h, the mixture was filtered (EtOAc wash). The filtrate was washed with water (3×), dried over Na2SO4, and concentrated to give 7 g of the crude title compound as a brown oil. The material was used without further purification. 1H NMR (400 MHz, CDCl3) δ 10.35−10.32 (m, 1H), 7.40−7.28 (m, 9H), 7.02−6.83 (m, 2H), 5.17 (s, 2H), 2.55−2.51 (m, 3H). MS (ESI) m/z: 415.1 (M + H). Phenyl 2-(Benzyloxy)-4-(dimethoxymethyl)-6-methyl-3(trifluoromethyl)benzoate (35). Trimethylorthoformate (3.6 g, 35 mmol) and p-toluenesulfonic acid (0.23 g, 1.2 mmol) were added to a solution of phenyl 2-(benzyloxy)-4-formyl-6-methyl-3(trifluoromethyl)benzoate (7.0 g crude, 12 mmol) in CH3OH. The reaction mixture was heated to reflux. After 18 h, the reaction mixture was concentrated, and the material was partitioned between EtOAc and water. The organic layer was dried over Na2SO4 and was concentrated. Purification by column chromatography (100:1 to 50:1 petroleum ether/EtOAc gradient) gave 4.9 g (90%, 2 steps) of the title compound as a light-yellow oil. 1H NMR (400 MHz, CDCl3) δ 7.45− 8132

dx.doi.org/10.1021/jm401211t | J. Med. Chem. 2013, 56, 8112−8138

Journal of Medicinal Chemistry

Article

method C in the synthesis of 39c, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.01 (s, 1H), 6.93 (s, 1H), 4.65 (d, J = 12.8 Hz, 1H), 4.08 (s, 1H), 3.90 (d, J = 12.8 Hz, 1H), 2.96−3.11 (m, 9H), 2.70 (s, 3H), 2.58−2.68 (m, 1H), 2.19−2.22 (m, 1H), 1.60−1.70 (m, 1H), 1.53 (s, 9H). MS (ESI) m/z: 514.3 (M + H). (1R,3S,4S,11S)-8-(Benzyloxy)-11-[(tert-butyldimethylsilyl)oxy]-18-(dimethoxymethyl)-4-(dimethylamino)-12-hydroxy19-methoxy-10,14-dioxo-6-oxa-7-azapentacyclo[11.8.0.03,11.05,9.015,20]henicosa-5(9),7,12,15,17,19-hexaen-16yl tert-Butyl Carbonate (36b, R3 = OCH3). Prepared from 31 (124 mg, 0.287 mmol) as described for compound 36a above. Purification by column chromatography (5−30% EtOAc in hexanes gradient) gave 202 mg (95%) of the title compound as a light-yellow solid. 1H NMR (400 MHz, CDCl3) δ 15.67 (s, 1H), 7.49−7.46 (m, 2H), 7.38−7.29 (m, 3H), 5.59 (s, 1H), 5.35, 5.32 (ABq, J = 12.2 Hz, 2H), 3.97 (d, J = 10.4 Hz, 1H), 3.73 (s, 3H), 3.37 (s, 3H), 3.31 (s, 3H), 3.28 (dd, J = 4.9, 15.9 Hz, 1H), 3.02−2.95 (m, 1H), 2.55−2.42 (m, 9H), 2.13 (d, J = 12.0 Hz, 1H), 1.51 (s, 9H), 0.82 (s, 9H), 0.25 (s, 3H), 0.11 (s, 3H). MS (ESI) m/z: 821.5 (M + H). (1R,3S,4S,11S)-8-(Benzyloxy)-11-[(tert-butyldimethylsilyl)oxy]-4-(dimethylamino)-18-formyl-12-hydroxy-19-methoxy10,14-dioxo-6-oxa-7-azapentacyclo[11.8.0.0 3,11 .0 5,9 .0 15,20 ]henicosa-5(9),7,12,15,17,19-hexaen-16-yl tert-Butyl Carbonate (37b, R3 = OCH3). Compound 36b (202 mg, 0.246 mmol) was dissolved in a premixed solution of 6 N HCl (0.34 mL) and THF (3.66 mL). After 40 min, the reaction mixture was diluted with EtOAc (40 mL) and was washed with NaHCO3 (saturated, aqueous solution, 10 mL) and brine (10 mL). The organics were dried over Na2SO4, filtered, and concentrated to give 204 mg (100% crude) of the title compound as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 15.55 (s, 1H), 10.33 (s, 1H), 7.50−7.47 (m, 3H), 7.38−7.31 (m, 3H), 5.36, 5.32 (ABq, J = 12.2 Hz, 2H), 3.94 (d, J = 11.0 Hz, 1H), 3.86 (s, 3H), 3.33 (dd, J = 4.9, 15.9 Hz, 1H), 3.07−2.99 (m, 1H), 2.59−2.44 (m, 9H), 2.16 (d, J = 14.6 Hz, 1H), 1.51 (s, 9H), 0.82 (s, 9H), 0.25 (s, 3H), 0.11 (s, 3H). MS (ESI) m/z: 775.4 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-3,10,12,12a-tetrahydroxy-7-methoxy-1,11-dioxo-8-(pyrrolidin-1-ylmethyl)1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (41a). Prepared from 37b by general experimental methods A (pyrrolidine) and C, yellow solid. 1H NMR (400 MHz, CD3OD, hydrochloride) δ 7.02 (s, 1H), 4.50 (d, J = 12.8 Hz, 1H), 4.32 (d, J = 13.3 Hz, 1H), 4.12 (s, 1H), 3.74 (s, 3H), 3.55−3.50 (m, 2H), 3.25−3.20 (m, 3H), 3.05−2.97 (m, 8H), 2.39 (t, J = 14.6 Hz, 1H), 2.28−2.24 (m, 1H), 2.17−2.03 (m, 4H), 1.70−1.60 (m, 1H). MS (ESI) m/z: 528.3 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-8-[(dimethylamino)methyl]-3,10,12,12a-tetrahydroxy-7-methoxy-1,11-dioxo1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (41b). Prepared from 37b by general experimental methods A (dimethylamine) and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 6.99 (s, 1H), 4.45 (d, J = 12.8 Hz, 1H), 4.25 (d, J = 13.2 Hz, 1H), 4.11 (s, 1H), 3.75 (s, 3H), 3.25−2.98 (m, 9H), 2.90 (s, 3H), 2.85 (s, 3H), 2.40 (dd, J = 14.8, 14.4 Hz, 1H), 2.27−2.24 (m, 1H), 1.66 (ddd, J = 13.2, 13.2, 13.2 Hz, 1H). MS (ESI) m/z: 502.0 (M + H). (4S,4aS,5aR,12aS)-8-{[t-Butyl(methyl)amino]methyl}-4-(dimethylamino)-3,10,12,12a-tetrahydroxy-7-methoxy-1,11dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (41c). Prepared from 37b by general experimental methods A (t-butylamine), B (formaldehyde), and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.01, 6.99 (s, 1H), 4.92 (d, J = 12.8 Hz, 0.6H), 4.60 (d, J = 13.3 Hz, 0.4H), 4.13, 4.12 (s, 1H), 3.96 (d, J = 12.8 Hz, 0.4H), 3.80 (d, J = 12.8 Hz, 0.6H), 3.77, 3.73 (s, 3H), 3.24 (dd, J = 4.6, 15.6 Hz, 1H), 3.27−3.20 (m, 1H), 3.06−2.98 (m, 8H), 2.69 (s, 3H), 2.47−2.40 (m, 1H), 2.28−2.26 (m, 1H), 1.71− 1.64 (m, 1H), 1.55 (s, 9H). MS (ESI) m/z: 544.3 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-8-[(2,2-dimethylpyrrolidin-1-yl)methyl]-3,10,12,12a-tetrahydroxy-7-methoxy-1,11dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (41d). Prepared from 37b by general experimental methods A (2,2-dimethylaminopyrrolidine) and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.02 (s, 1H), 4.54 (d,

dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (39d). Prepared from 37a by general experimental methods A (2,2-dimethylaminopyrrolidine) and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.21 (s, 1H), 4.72 (t, J = 13.2 Hz, 1H), 4.10−4.15 (m, 2H), 3.38−3.55 (m, 2H), 2.95−3.10 (m, 8H), 2.41 (dd, J = 15.0, 15.0 Hz, 1H), 1.96−2.28 (m, 5H), 1.96− 2.28 (m, 5H), 1.66 (s, 3H), 1.63−1.66 (m, 1H), 1.45 (s, 3H). MS (ESI) m/z: 560.3 (M + H). (4S,4aS,5aR,12aS)-7-Chloro-8-{[(cyclopropylmethyl)(propan2-yl)amino]methyl}-4-(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2carboxamide Dihydrochloride (39f). Prepared from 37a by general experimental methods A (2-propylamine), B (cyclopropylcarboxaldehyde), and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.23 (s, 1H), 4.46−4.63 (m, 2H), 4.14 (s, 1H), 3.95−4.02 (m, 1H), 3.40 (dd, J = 4.6, 16.0 Hz, 1H), 2.96−3.21 (m, 10H), 2.41 (dd, J = 15.0, 15.0 Hz, 1H), 2.26−2.29 (m, 1H), 1.60−1.70 (m, 1H), 1.50 (d, J = 6.4 Hz, 3H), 1.41 (d, J = 6.4 Hz, 3H), 1.06−1.17 (m, 1H), 0.72− 0.78 (m, 2H), 0.42−0.44 (m, 2H). MS (ESI) m/z: 574.2 (M + H). (4S,4aS,5aR,12aS)-7-Chloro-4-(dimethylamino)-3,10,12,12atetrahydroxy-8-({[(2R)-3-methylbutan-2-yl]amino}methyl)1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (39g). Prepared from 37a by general experimental methods A ((2R)-3-methylbutan-2-amine) and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.18 (s, 1H), 4.40 (s, 2H), 4.13 (s, 1H), 3.41 (dd, J = 4.6, 16.0 Hz, 1H), 3.32−3.36 (m, 1H), 2.94− 3.11 (m, 8H), 2.40 (dd, J = 15.0, 15.0 Hz, 1H), 2.20−2.28 (m, 2H), 1.60−1.70 (m, 1H), 1.34 (d, J = 6.8 Hz, 3H), 1.06 (d, J = 6.8 Hz, 3H), 1.01 (d, J = 6.8 Hz, 3H). MS (ESI) m/z: 548.2 (M + H). (4S,4aS,5aR,12aS)-8-(Azepan-1-ylmethyl)-7-chloro-4-(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (39h). Prepared from 37a by general experimental methods A (homopiperidine) and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.24 (s, 1H), 4.50 (d, J = 13.7 Hz, 1H), 4.46 (d, J = 13.7 Hz, 1H), 4.12 (s, 1H), 3.57−3.36 (comp, 3H), 3.05 (s, 3H), 2.96 (s, 3H), 3.20−2.94 (comp, 4H), 2.47−2.37 (m, 1H), 2.30−2.21 (m, 1H), 1.99−1.47 (comp, 7H). MS (ESI) m/z: 546.1 (M + H). (4S,4aS,5aR,12aS)-7-Chloro-4-(dimethylamino)-8-{[(2S,6S)2,6-dimethylpiperidin-1-yl]methyl}-3,10,12,12a-tetrahydroxy1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (39i). Prepared from 37a by general experimental methods A ((2S,6S)-2,6-dimethylpiperidine) and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.25 (s, 1H), 4.68 (d, J = 14.4 Hz, 1H), 4.51 (d, J = 14.4 Hz, 1H), 4.11 (s, 1H), 3.88−3.66 (m, 2H), 3.45−3.37 (m, 1H), 3.04 (s, 3H), 2.96 (s, 3H), 3.15−2.94 (comp, 2H), 2.49−2.38 (m, 1H), 2.29−2.21 (m, 1H), 2.19−2.08 (m, 1H), 1.95−1.59 (comp, 6H), 1.48 (d, J = 6.7 Hz, 3H), 1.44 (d, J = 6.7 Hz, 3H). MS (ESI) m/z: 574.3 (M + H). (4S,4aS,5aR,12aS)-7-Chloro-4-(dimethylamino)-8-{[(2S,5S)2,5-dimethylpyrrolidin-1-yl]methyl}-3,10,12,12a-tetrahydroxy1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (39j). Prepared from 37a by general experimental methods A ((2S,5S)-2,5-dimethylpyrrolidine) and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.28 (s, 1H), 4.68−4.48 (m, 2H), 4.13 (s, 1H), 4.12−4.02 (m, 1H), 3.90−3.80 (m, 1H), 3.49− 3.38 (m, 1H), 3.05 (s, 3H), 2.97 (s, 3H), 3.17−2.95 (comp, 2H), 2.52−2.23 (comp, 4H), 1.98−1.60 (comp, 3H), 1.48−1.34 (m, 6H). MS (ESI) m/z: 560.3 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-8-(pyrrolidin-1-ylmethyl)1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (40a). Isolated as a side product of general experimental method C in the synthesis of 39a, yellow solid. 1H NMR (400 MHz, CD3OD) δ 6.99 (s, 1H), 6.94 (s, 1H), 4.33 (s, 2H), 4.09 (s, 1H), 3.57−3.47 (m, 2H), 3.03 (s, 3H), 2.96 (s, 3H), 3.23−2.87 (comp, 5H), 2.64−2.55 (m, 1H), 2.24−1.94 (comp, 5H), 1.66−1.54 (comp, 1H). MS (ESI) m/z: 498.2 (M + H). (4S,4aS,5aR,12aS)-8-{[t-Butyl(methyl)amino]methyl}-4-(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (40c). Isolated as a side product of general experimental 8133

dx.doi.org/10.1021/jm401211t | J. Med. Chem. 2013, 56, 8112−8138

Journal of Medicinal Chemistry

Article

J = 12.4 Hz, 0.6H), 4.43 (d, J = 12.8 Hz, 0.4H), 4.11, 4.10 (s, 1H), 4.02 (d, J = 12.8 Hz, 0.4H), 3.88 (d, J = 12.4 Hz, 0.6H), 3.78 (s, 1.2H), 3.74 (s, 1.8H), 3.44−3.39 (m, 2H), 3.26−3.18 (m, 1H), 3.05−2.97 (m, 8H), 2.46−2.35 (m, 1H), 2.29−2.24 (m, 1H), 2.12−2.07 (m, 2H), 2.02−1.97 (m, 2H), 1.71−1.58 (m, 4H), 1.44, 1.43 (s, 3H). MS (ESI) m/z: 556.4 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-3,10,12,12a-tetrahydroxy-7-methoxy-1,11-dioxo-8-(piperidin-1-ylmethyl)1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (41e). Prepared from 37b by general experimental methods A (piperidine) and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.05 (s, 1H), 4.37, 4.24 (ABq, J = 13.3 Hz, 2H), 4.12 (s, 1H), 3.72 (s, 3H), 3.45 (br t, J = 13.7 Hz, 2H), 3.22 (dd, J = 4.1, 15.1 Hz, 1H), 3.07−2.97 (m, 10H), 2.39 (t, J = 14.6 Hz, 1H), 2.27−2.24 (m, 1H), 1.93−1.90 (m, 2H), 1.82−1.75 (m, 3H), 1.70−1.61 (m, 1H), 1.56−1.50 (m, 1H). MS (ESI) m/z: 542.4 (M + H). (4S,4aS,5aR,12aS)-8-{[(2-Cyclopropylpropan-2-yl)(methyl)amino]methyl}-4-(dimethylamino)-3,10,12,12a-tetrahydroxy7-methoxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (41f). Prepared from 37b by general experimental methods A (2-cyclopropylpropan-2-amine), B (formaldehyde), and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.03 (s, 1H), 4.92−4.85 (m, 0.6H), 4.76 (d, J = 12.8 Hz, 0.4H), 4.13, 4.12 (s, 1H), 4.04 (d, J = 12.8 Hz, 0.4H), 3.88 (d, J = 12.8 Hz, 0.6H), 3.77 (s, 1.2H), 3.74 (s, 1.8H), 3.28−3.20 (m, 1H), 3.06−2.98 (m, 8H), 2.76 (s, 3H), 2.48−2.33 (m, 1H), 2.29−2.26 (m, 1H), 1.72−1.62 (m, 1H), 1.42 (s, 3H), 1.37 (s, 3H), 1.32−1.29 (m, 1H), 0.83−0.74 (m, 2H), 0.69−0.66 (m, 2H). MS (ESI) m/z: 570.4 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-8-{[(2,3-dimethylbutan-2-yl)(methyl)amino]methyl}-3,10,12,12a-tetrahydroxy-7methoxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene2-carboxamide Dihydrochloride (41g). Prepared from 37b by general experimental methods A (2,3-dimethylbutan-2-amine), B (formaldehyde), and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.01 (s, 1H), 4.72 (d, J = 12.8 Hz, 0.7H), 4.60 (d, J = 12.8 Hz, 0.3H), 4.12, 4.11 (s, 1H), 3.96 (d, J = 12.8 Hz, 0.3H), 3.81 (d, J = 12.8 Hz, 0.7H), 3.77, 3.73 (s, 3H), 3.26−3.19 (m, 1H), 3.05−2.98 (m, 8H), 2.68 (s, 3H), 2.48−2.32 (m, 1H), 2.28−2.24 (m, 1H), 1.94 (q, J = 7.3 Hz, 2H), 1.72−1.62 (m, 1H), 1.50, 1.48 (s, 6H), 1.11−1.07 (m, 3H). MS (ESI) m/z: 558.4 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-8-{[(2R,6S)-2,6-dimethylpiperidin-1-yl]methyl}-3,10,12,12a-tetrahydroxy-7-methoxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2carboxamide Dihydrochloride (41h). Prepared from 37b by general experimental methods A (cis-2,6-dimethylpiperidine) and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.01, 6.99 (s, 1H), 4.66, 4.58 (ABq, J = 15.1 Hz, 0.67H), 4.43, 4.31 (ABq, J = 15.1 Hz, 1.33H), 4.12 (s, 1H), 3.74, 3.70 (s, 3H), 3.59−3.55 (m, 1.33H), 3.46−3.40 (m, 0.67H), 3.24−3.20 (m, 1H), 3.04−2.97 (m, 8H), 2.39 (t, J = 14.2 Hz, 1H), 2.27−2.24 (m, 1H), 1.98−1.76 (m, 4H), 1.70−1.58 (m, 3H), 1.50 (d, J = 6.0 Hz, 1H), 1.46 (d, J = 6.0 Hz, 1H), 1.32 (d, J = 6.4 Hz, 2H), 1.24 (d, J = 6.4 Hz, 2H). MS (ESI) m/z: 570.5 (M + H). (1R,3S,4S,11S)-8,16-Bis(benzyloxy)-11-[(tertbutyldimethylsilyl)oxy]-18-(dimethoxymethyl)-4-(dimethylamino)-12-hydroxy-19-(trifluoromethyl)-6-oxa-7azapentacyclo[11.8.0.03,11.05,9.015,20]henicosa-5(9),7,12,15,17,19-hexaene-10,14-dione (36c, R3 = CF3). Prepared from 35 (460 mg, 1.25 mmol) as described for compound 36a above. Purification by column chromatography (0−5% to 10−20% EtOAc in hexanes step gradient) gave 380 mg (89%) of the title compound as a light-yellow solid. 1H NMR (400 MHz, CDCl3) δ 15.65 (s, 1H), 7.46−7.22 (m, 11H), 5.49 (s, 1H), 5.30 (s, 2H), 5.25 (s, 2H), 3.91 (d, J = 10.8 Hz, 1H), 3.29 (s, 3H), 3.20 (s, 3H), 2.85−2.62 (m, 2H), 2.53−2.35 (m, 9H), 2.10−2.04 (m, 1H), 0.88 (s, 9H), 0.20 (s, 3H), 0.09 (s, 3H). MS (ESI) m/z: 849.1 (M + H). (1R,3S,4S,11S)-8,16-Bis(benzyloxy)-11-[(tertbutyldimethylsilyl)oxy]-4-(dimethylamino)-12-hydroxy-10,14dioxo-19-(trifluoromethyl)-6-oxa-7-azapentacyclo[11.8.0.03,11.05,9.015,20]henicosa-5(9),7,12,15,17,19-hexaene-18carbaldehyde (37c, R3 = CF3). Compound 36c (0.30 g, 0.35 mmol) was dissolved in CH2Cl2 (3 mL), and TFA (3 mL) was added dropwise. After 1 h, the reaction mixture was concentrated. The

material was taken up in water and was extracted with EtOAc. The combined extracts were dried over Na2SO4 and were concentrated to yield the crude title compound as a yellow solid. This was used without further purification. (4S,4aS,5aR,12aS)-4-(Dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-8-(pyrrolidin-1-ylmethyl)-7-(trifluoromethyl)-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (42a). Prepared from 37c by general experimental methods A (pyrrolidine) and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.26 (s, 1H), 4.71 (d, J = 14.0 Hz, 1H), 4.49 (d, J = 14.0 Hz, 1H), 4.14 (s, 1H), 3.74−3.59 (m, 2H), 3.30−2.96 (m, 11H), 2.69−2.57 (m, 1H), 2.26−2.01 (m, 5H), 1.69−1.59 (m, 1H). MS (ESI) m/z: 566.1(M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-8-[(dimethylamino)methyl]-3,10,12,12a-tetrahydroxy-1,11-dioxo-7-(trifluormethyl)-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (42b). Prepared from 37c by general experimental methods A (dimethylamine) and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.23 (s, 1H), 4.61 (d, J = 14.0 Hz, 1H), 4.45 (d, J = 13.6 Hz, 1H), 4.14 (s, 1H), 3.18−2.93 (m, 15H), 2.69−2.58 (m, 1H), 2.27−2.23 (m, 1H), 1.67−1.58 (m, 1H). MS (ESI) m/z: 540.1 (M + H). (4S,4aS,5aR,12aS)-8-{[t-Butyl(methyl)amino]methyl}-4-(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-7-(trifluoromethyl)-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (42c). Prepared from 37c by general experimental methods A (t-butylamine), B (formaldehyde), and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.25, 7.24 (s, 1 H total), 4.83−4.82 (m, 1H), 4.22−4.10 (m, 1H), 4.12 (s, 1H), 3.20−2.90 (m, 9H), 2.70 (d, J = 10.8 Hz, 3H), 2.65−2.57 (m, 1H), 2.24−2.21 (m, 1H), 1.67−1.61 (m, 1H), 1.53 (s, 9H). MS (ESI) m/z: 582.1 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-8-[(2,2-dimethylpyrrolidin-1-yl)methyl]-3,10,12,12a-tetrahydroxy-1,11-dioxo-7-(trifluoromethyl)-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (42d). Prepared from 37c by general experimental methods A (2,2-dimethylaminopyrrolidine) and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.27 (s, 1H), 4.82− 4.76 (m, 1H), 4.14 (s, 1H), 4.05−4.02 (m, 1H), 3.60−3.47 (m, 2H), 3.11−2.95 (m, 9H), 2.60−2.55 (m, 1H), 2.22−1.95 (m, 5H), 1.71− 1.62 (m, 1H), 1.63 (s, 3H), 1.42 (s, 3H). MS (ESI) m/z: 594.0 (M + H). (4S,4aS,5aR,12aS)-4-(Dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-8-(piperidin-1-ylmethyl)-7-(trifluoromethyl)1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihydrochloride (42e). Prepared from 37c by general experimental methods A (piperidine) and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.35 (s, 1H), 4.58−4.55 (m, 1H), 4.45−4.42 (m, 1H), 4.18 (s, 1H), 3.59−3.49 (m, 2H), 3.28−2.97 (m, 11H), 2.63−2.55 (m, 1H), 2.29−2.26 (m, 1H), 1.94−1.82 (m, 5H), 1.66−1.58 (m, 2H). MS (ESI) m/z: 580.0 (M + H). 4-Bromo-6-methoxy-2-methyl-3-nitrobenzoic acid. A solution of nitric acid (68−70%, 0.56 mL, 8.6 mmol) in concentrated sulfuric acid (2 mL) was added dropwise to a 0 °C solution of 22 (2.00 g, 8.16 mmol) in concentrated sulfuric acid (20 mL). After 10 min, the reaction mixture was poured onto ice (∼200 mL) and was extracted with EtOAc (150 mL). The extracts were washed with brine (2 × 50 mL), dried over MgSO4, filtered, and concentrated to give the crude title compound as an orange solid. The material was used without further purification. 1H NMR (400 MHz, CDCl3) δ 11.5 (br s, 1H), 7.06 (s, 1H), 3.90 (s, 3H), 2.32 (s, 3H). MS (ESI) m/z: 288.0, 290.0 (M − H). Phenyl 4-Bromo-6-methoxy-2-methyl-3-nitrobenzoate (43). The crude 4-bromo-6-methoxy-2-methyl-3-nitrobenzoic acid (8.6 mmol) was dissolved in dichloromethane (16 mL). Oxalyl chloride (0.85 mL, 9.8 mmol) was added followed by a few drops of DMF. After 30 min, the reaction mixture was concentrated and was further dried under high vacuum. The material was redissolved in dichloromethane (16 mL). Phenol (0.92 g, 9.8 mmol), triethylamine (2.84 mL, 20.4 mmol), and DMAP (100 mg, 0.82 mmol) were added. After 1 h, the reaction mixture was concentrated under reduced pressure. The 8134

dx.doi.org/10.1021/jm401211t | J. Med. Chem. 2013, 56, 8112−8138

Journal of Medicinal Chemistry

Article

(75 mL, then 25 mL). The combined extracts were dried over Na2SO4, filtered, and concentrated. Purification by a preparative reverse-phase HPLC on a Waters Autopurification system using a Sunfire Prep C18 OBD column [5 μm, 19 × 50 mm; flow rate, 20 mL/min; solvent A: H2O with 0.1% HCO2H; solvent B: CH3CN with 0.1% HCO2H; injection volume: 4.0 mL (CH3CN); gradient: 80 → 100% B in A over 10 min; mass-directed fraction collection] and freeze-drying of the desired fractions gave 1.71 g (75%) of the title compound as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 16.00 (br s, 1H), 7.50−7.46 (m, 4H), 7.39−7.27 (m, 6H), 7.10 (s, 1H), 5.36 (s, 2H), 5.18, 5.12 (ABq, J = 12.8 Hz, 2H), 4.10 (d, J = 10.4 Hz, 1H), 3.37 (dd, J = 4.3, 15.9 Hz, 1H), 2.88−2.74 (m, 7H), 2.55−2.40 (m, 9H), 2.12 (d, J = 14.0 Hz, 1H), 0.84 (s, 9H), 0.28 (s, 3H), 0.14 (s, 3H). MS (ESI) m/z: 828.3, 830.3 (M + H). (1R,3S,4S,11S)-8,16-Bis(benzyloxy)-11-[(tertbutyldimethylsilyl)oxy]-4,19-bis(dimethylamino)-12-hydroxy10,14-dioxo-6-oxa-7-azapentacyclo[11.8.0.0 3,11 .0 5,9 .0 15,20 ]henicosa-5(9),7,12,15,17,19-hexaene-18-carboxaldehyde (47). Phenyllithium (1.8 M in di-n-butyl ether, 0.10 mL, 0.18 mmol) was added dropwise to a solution of 46 (0.10 g, 0.12 mmol) in THF (3 mL) at −78 °C, resulting in an orange solution. After 5 min, n-BuLi (2.2 M in hexanes, 0.068 mL, 0.15 mmol) was added dropwise followed 2 min later by the addition of N,N-dimethylformamide (0.047 mL, 0.61 mmol). The resulting dark-red reaction mixture was stirred at −78 °C for 65 min. The reaction mixture was allowed to warm to 23 °C, diluted with NH4Cl (saturated, aqueous solution, 20 mL), and extracted with EtOAc (2 × 15 mL). The combined extracts were dried over Na2SO4, filtered, and concentrated. This gave the title compound as an orange oil, which was used directly in the next reactions. 1H NMR (400 MHz, CDCl3) δ 15.85 (s, 1H), 10.32 (s, 1H), 7.50−7.46 (m, 4H), 7.39−7.26 (m, 7H), 5.36 (s, 2H), 5.25, 5.18 (ABq, J = 12.8 Hz, 2H), 4.02 (d, J = 11.0 Hz, 1H), 3.13 (dd, J = 4.9, 15.9 Hz, 1H), 2.98−2.90 (m, 7H), 2.58−2.46 (m, 9H), 2.15 (d, J = 14.0 Hz, 1H), 0.83 (s, 9H), 0.27 (s, 3H), 0.15 (s, 3H). MS (ESI) m/z: 778.3 (M + H). (4S,4aS,5aR,12aS)-4,7-Bis(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-8-(pyrrolidin-1-ylmethyl)1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Trihydrochloride (49a). Prepared from 47 by general experimental methods A (pyrrolidine) and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.04 (s, 1H), 4.62−4.59 (m, 1H), 4.36−4.31 (m, 1H), 4.14 (s, 1H), 3.68−3.63 (m, 2H), 3.23−3.17 (m, 2H), 3.14−2.99 (m, 9H), 2.89−2.83 (m, 6H), 2.54−2.44 (m, 1H), 2.37−2.28 (m, 1H), 2.23− 2.18 (m, 2H), 2.13−2.07 (m, 2H), 1.71−1.62 (m, 1H). MS (ESI) m/z: 541.3 (M + H). (4S,4aS,5aR,12aS)-4,7-Bis(dimethylamino)-8[(dimethylamino)methyl]-3,10,12,12a-tetrahydroxy-1,11dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Trihydrochloride (49b). Prepared from 47 by general experimental methods A (dimethylamine) and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 6.96 (s, 1H), 4.55 (d, J = 13.3 Hz, 1H), 4.23 (d, J = 13.3 Hz, 1H), 4.12 (s, 1H), 3.09−2.82 (m, 21H), 2.47 (t, J = 15.1 Hz, 1H), 2.28−2.25 (m, 1H), 1.70−1.60 (m, 1H). MS (ESI) m/z: 515.3 (M + H). (4S,4aS,5aR,12aS)-8-{[t-Butyl(methyl)amino]methyl}-4,7-bis(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Trihydrochloride (49c). Prepared from 47 by general experimental methods A (t-butylamine), B (formaldehyde), and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 6.99 (s, 0.4H), 6.69 (s, 0.6H), 4.81 (d, J = 13.3 Hz, 0.6H), 4.54 (d, J = 12.8 Hz, 0.4H), 4.24 (d, J = 12.8 Hz, 0.4H), 4.12 (s, 1H), 3.96 (d, J = 13.3 Hz, 0.6H), 3.05−2.72 (m, 18H), 2.54−2.47 (m, 1H), 2.30−2.26 (m, 1H), 1.71−1.62 (m, 1H), 1.55 (s, 9H). MS (ESI) m/z: 557.3 (M + H). (4S,4aS,5aR,12aS)-4,7-Bis(dimethylamino)-8-[(2,2-dimethylpyrrolidin-1-yl)methyl]-3,10,12,12a-tetrahydroxy-1,11-dioxo1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Trihydrochloride (49d). Prepared from 47 by general experimental methods A (2,2-dimethylaminopyrrolidine) and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 6.99 (s, 0.4H), 6.97 (s, 0.6H), 4.68 (d, J = 12.8 Hz, 0.6H), 4.43 (d, J = 12.8 Hz, 0.4H), 4.19 (d, J = 12.8 Hz,

material was dissolved in EtOAc (150 mL) and was washed with 1 N aqueous HCl (50 mL), brine (50 mL), 1 N aqueous NaOH (50 mL), and brine (50 mL). The organics were dried over MgSO4, filtered, and concentrated to afford the crude title compound as a light-yellow solid. The material was used without further purification. 1H NMR (400 MHz, CDCl3) δ 7.45−7.41 (m, 2H), 7.30−7.26 (m, 1H), 7.21−7.16 (m, 2H), 7.09 (s, 1H), 3.94 (s, 3H), 2.38 (s, 3H). MS (ESI) m/z: 364.1, 366.1 (M − H). Phenyl 6-(Benzyloxy)-4-bromo-2-methyl-3-nitrobenzoate (44). BBr3 (1.0 M in CH2Cl2, 8.16 mL, 8.16 mmol) was added slowly to a solution of crude 43 (8.6 mmol) in dichloromethane (32 mL) at −78 °C. The reaction was stirred at −78 °C for 15 min and was then allowed to warm to 0 °C over 50 min. After 10 min at 0 °C, the reaction mixture was poured into saturated aqueous NaHCO3 solution (50 mL) and was stirred at rt for 10 min. The dichloromethane was evaporated under reduced pressure, and the solution was extracted with EtOAc (100 mL, then 30 mL). The combined extracts were dried over MgSO4, filtered, and concentrated to give 2.20 g of the crude phenol intermediate. 1H NMR (400 MHz, CDCl3) δ 11.2 (br s, 1H), 7.48−7.44 (m, 2H), 7.36−7.32 (m, 1H), 7.25 (s, 1H), 7.18−7.16 (m, 2H), 2.63 (s, 3H). MS (ESI) m/z: 350.0, 352.0 (M − H). Benzylbromide (0.78 mL, 6.6 mmol) and K2CO3 (1.73 g, 12.5 mmol) were added to a solution of the above intermediate (2.20 g, 6.25 mmol) in acetone (12 mL). After stirring overnight, the reaction mixture was filtered, and the solids were washed with EtOAc (30 mL). The filtrate was concentrated, and the material was purified by column chromatography (2−20% EtOAc in hexanes gradient) to afford 1.68 g (47%, 4 steps) of the title compound as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.40−7.32 (m, 8H), 7.15 (s, 1H), 7.03−7.01 (m, 2H), 5.18 (s, 2H), 2.39 (s, 3H). MS (ESI) m/z: 440.1, 442.1 (M − H). Phenyl 6-(Benzyloxy)-4-bromo-3-(dimethylamino)-2-methylbenzoate (45). Zinc dust (2.33 g, 35.7 mmol) was added portionwise to a solution of 44 (1.58 g, 3.57 mmol) in a mixture of THF (5 mL) and acetic acid (1 mL) (caution: exothermic!). After 3.5 h, the reaction mixture was diluted with EtOAc and was filtered through a pad of Celite. The Celite was washed with EtOAc. The combined filtrate was washed with saturated aqueous NaHCO3 (60 mL), and the aqueous layer was extracted with EtOAc (40 mL). The combined extracts were dried over Na2SO4, filtered, and concentrated to give the crude aniline intermediate. Formaldehyde (37% aqueous solution, 1.59 mL, 21.4 mmol), acetic acid (0.62 mL, 10.7 mmol), and Na(OAc)3BH (2.27 g, 10.7 mmol) were added to a solution of the intermediate in acetonitrile (30 mL). After 2 h, additional Na(OAc)3BH (0.38 g, 1.8 mmol) was added. After stirring overnight, saturated, aqueous NaHCO3 (70 mL) was added slowly (bubbling). The resulting mixture was stirred for 5 min and was extracted with EtOAc (100 mL, then 50 mL). The combined extracts were dried over Na2SO4, filtered, and concentrated. Purification by column chromatography (2−5% EtOAc in hexanes gradient) gave 1.43 g (91%, 2 steps) of the title compound as a white solid. 1H NMR (400 MHz, CDCl3) δ 7.44−7.35 (m, 7H), 7.26−7.22 (m, 1H), 7.09−7.07 (m, 3H), 5.10 (s, 2H), 2.83 (s, 6H), 2.42 (s, 3H). MS (ESI) m/z: 440.1, 442.1 (M + H). (1R,3S,4S,11S)-8,16-Bis(benzyloxy)-18-bromo-11-[(tertbutyldimethylsilyl)oxy]-4,19-bis(dimethylamino)-12-hydroxy6-oxa-7-azapentacyclo[11.8.0.0 3,11 .0 5,9 .0 15,20 ]henicosa-5(9),7,12,15,17,19-hexaene-10,14-dione (46). n-BuLi (2.17 M in hexanes, 1.46 mL, 3.16 mmol) was added dropwise to a solution of diisopropylamine (0.45 mL, 3.2 mmol) in THF (6 mL) at −78 °C. The reaction mixture was warmed to −20 °C and was then recooled to −78 °C. TMEDA (0.47 mL, 3.2 mmol) was added. After 15 min, a solution of 45 (1.27 g, 2.88 mmol) in THF (3 mL) was added. The resulting deep-red solution was stirred at −78 °C for 55 min and was then cooled to −100 °C. A solution of 6 (1.33 g, 2.75 mmol) in THF (3 mL) was added to the reaction mixture, and the mixture was allowed to warm to −70 °C over 30 min. LHMDS (1.0 M in THF, 3.02 mL, 3.02 mmol) was added, and the reaction mixture was allowed to warm to −5 °C slowly over 1 h and 20 min. The reaction was quenched with a mixture of saturated aqueous NH4Cl and pH 7 phosphate buffer (100 mL, 1:1, v/v) and was extracted with EtOAc 8135

dx.doi.org/10.1021/jm401211t | J. Med. Chem. 2013, 56, 8112−8138

Journal of Medicinal Chemistry

Article

MRSA Neutropenic Lung Model. Female BALB/c mice weighing 18−20 g were rendered neutropenic through two consecutive intraperitoneal (IP) cyclophosphamide injections of 150 and 100 mg/kg on days −4 and −1, respectively. Mice were infected with S. aureus SA191 (MRSA) via intranasal administration of 0.05 mL of inoculum (∼5.0 × 107 CFU per mouse) under light anesthesia. At 2 and 12 h postinfection, mice were treated with test compound or linezolid at 30 mg/kg/dose by oral gavage. Six mice were treated with each dosage. Twenty-four hours post first dose, mice were euthanized by CO2 inhalation. The lungs were aseptically removed, weighed, homogenized, serially diluted, and plated on TSA media. The plates were incubated overnight at 37 °C in 5% CO2. CFU per gram of lung tissue was calculated by enumerating the plated colonies and then adjusting for serial dilutions and the weight of the lung. S. pneumoniae Neutropenic Lung Model. Female BALB/c mice weighing 18−20 g were rendered neutropenic through two consecutive IP cyclophosphamide injections of 150 and 100 mg/kg on days −4 and −1, respectively. Mice were infected with S. pneumoniae SP160 via intranasal administration of 0.05 mL of inoculum (∼1.0 × 107 CFU per mouse) under light anesthesia. At 2 and 12 h postinfection, mice were treated with test compound or linezolid at 30 mg/kg/dose via oral gavage. Six mice were treated with each dosage. Twenty-four hours postinitiation of treatment, mice were euthanized by CO2 inhalation. The lungs of the mice were aseptically removed, weighed, homogenized, serially diluted, and plated on TSA-II medium. The plates were incubated overnight at 37 °C in 5% CO2. CFU per gram of lung tissue was calculated by enumerating the plated colonies and then adjusting for serial dilutions and the weight of the lung. E. coli Kidney Infection Model. Female BALB/c mice weighing 18−20 g were infected with E. coli EC200 via intravenous injection of 0.2 mL of inoculum (∼1.0 × 108 CFU per mouse). At 12 and 24 h postinfection, mice were treated with the test article at 2 mg/kg/dose by oral gavage. Six mice were treated with each dosage. Thirty-six hours post initiation of treatment, mice were euthanized by CO2 inhalation. The kidneys of the mice were aseptically removed, weighed, homogenized, serially diluted, and plated on TSA medium. The plates were incubated overnight at 37 °C in 5% CO2. CFU per gram of kidney was calculated by enumerating the plated colonies and then adjusting for serial dilutions and the weight of the kidney. K. pneumoniae Kidney Infection Model. Female BALB/c mice weighing 18−20 g were infected with K. pneumoniae KP453 via intravenous injection of 0.2 mL of inoculum (7.5 × 105 CFU per mouse) mixed with 0.2% carageenen (Sigma-Aldrich). Mice were treated at 9 and 24 h postinfection with the test compounds at 50 mg/ kg/dose by oral gavage. Six mice were treated with each dosage. Thirty-six hours post initiation of treatment, mice were euthanized by CO2 inhalation. The kidneys of the mice were aseptically removed, weighed, homogenized, serially diluted, and plated on TSA medium. The plates were incubated overnight at 37 °C in 5% CO2. CFU per gram of kidney was calculated by enumerating the plated colonies and then adjusting for serial dilutions and the weight of the kidney.

0.4H), 4.10 (s, 1H), 3.93 (d, J = 12.8 Hz, 0.6H), 3.08−2.80 (m, 17H), 2.48 (t, J = 16.0 Hz, 1H), 2.28−2.24 (m, 1H), 2.16−2.14 (m, 2H), 2.04−1.99 (m, 2H), 1.71−1.61 (m, 1H), 1.61 (s, 3H), 1.44 (s, 3H). MS (ESI) m/z: 569.3 (M + H). (4S,4aS,5aR,12aS)-4,7-Bis(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-8-(piperidin-1-ylmethyl)1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide trihydrochloride (49e). Prepared from 47 by general experimental methods A (piperidine) and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.06 (s, 1H), 4.54−4.47 (m, 1H), 4.29−4.26 (m, 1H), 4.14 (s, 1H), 3.51−3.44 (m, 2H), 3.10−2.98 (m, 11H), 2.89−2.83 (m, 6H), 2.51−2.43 (m, 1H), 2.31−2.27 (m, 1H), 1.97−1.92 (m, 2H), 1.88− 1.81 (m, 3H), 1.69−1.52 (m, 2H). MS (ESI) m/z: 555.2 (M + H). (4S,4aS,5aR,12aS)-4,7-Bis(dimethylamino)-8-{[(2,2dimethylpropyl)amino]methyl}-3,10,12,12a-tetrahydroxy1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Trihydrochloride (49f). Prepared from 47 by general experimental methods A ((2,2-dimethylpropyl)amine) and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 6.93 (s, 1H), 4.42 (d, J = 13.7 Hz, 1H), 4.17 (d, J = 13.7 Hz, 1H), 4.10 (s, 1H), 3.04−2.83 (m, 17H), 2.47 (t, J = 14.6 Hz, 1H), 2.28−2.24 (m, 1H), 1.70−1.60 (m, 1H), 1.08 (s, 9H). MS (ESI) m/z: 557.3 (M + H). (4S,4aS,5aR,12aS)-4,7-Bis(dimethylamino)-8-({[(2R)-3,3-dimethylbutan-2-yl]amino}methyl)-3,10,12,12a-tetrahydroxy1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Trihydrochloride (49g). Prepared from 47 by general experimental methods A (((2R)-3,3-dimethylbutan-2-yl)amine) and C, yellow solid. 1H NMR (400 MHz, CD3OD) δ 6.98 (s, 1H), 4.45− 4.42 (m, 1H), 4.31−4.27 (m, 1H), 4.12 (s, 1H), 3.12−2.82 (m, 16H), 2.49−2.42 (m, 1H), 2.31−2.28 (m, 1H), 1.68−1.62 (m, 1H), 1.40 (d, J = 7.2 Hz, 3H), 1.08 (s, 9H). MS (ESI) m/z: 571.3 (M + H). Susceptibility Testing. Compound stocks were prepared and serially diluted in sterile deionized water. Tetracycline-susceptible isolates SA100 (S. aureus ATCC 13709, Smith), SA101 (S. aureus ATCC 29213), SP106 (S. pneumoniae ATCC 49619), EF103 (E. faecalis ATCC 29212), EC107 (E. coli ATCC 25922), KP109 (K. pneumoniae ATCC 13883), AB110 (Acinetobacter baumannii ATCC 19606), EC108 (Enterobacter cloacae ATCC 13047), and PA111 (Pseudomonas aeruginosa ATCC 27853) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Tetracycline-resistant isolates S. aureus SA158 (tet(K)), S. pneumoniae SP160 (tet(M)), E. faecalis EF159 (tet(M)), E. coli EC155 (tet(A)), and K. pneumoniae KP153 (tet(A)) were obtained from Marilyn Roberts’ lab at the University of Washington. S. aureus SA161 (tet(M)) was obtained from Micromyx (Kalamazoo, MI). 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).32 Cation-adjusted Mueller Hinton broth was obtained from BBL (catalog no. 212322, Becton Dickinson, Sparks, MD), prepared fresh, and kept at 4 °C prior to testing. Defibrinated horse blood (catalog no. A0432, PML Microbiologicals, Wilsonville, OR) was used to supplement medium as appropriate. All test methods met acceptable standards based on recommended quality control ranges for all comparator antibiotics and the appropriate ATCC quality control strains. Animal Efficacy Models. All animal efficacy models were performed at Vivisource, Waltham, MA. Mouse Systemic Infection Model. S. aureus ATCC 13709 was grown to log phase in a liquid culture. Bacteria were diluted in 5% mucin to a concentration to achieve 90% mortality within 48 h after infection. The final inoculum concentration of bacteria was 1.98 × 106 CFU/mouse. CD-1 female mice (18−20 g) were infected with 0.5 mL of bacterial suspension via intraperitoneal injection, n = 6 per dose concentration. For the screening model, mice were treated with the test article as either a single 30 mg/kg dose via oral gavage or a 3 mg/ kg intravenous dose 1 h postinfection. Infection control mice were dosed with vehicle (sterile water). After 48 h, survival was recorded. For PD50 determinations, mice received oral treatment with the test article at concentrations ranging from 0.30−30 mg/kg 1 h postinfection. The PD50 in mg/kg was calculated as survival after 48 h.

■

AUTHOR INFORMATION

Corresponding Author

*Email: [email protected]; Phone: (617) 715-3558; Fax: (617) 926-3557. Author Contributions

The manuscript was written through contributions of all authors, the relative significance of which may not be exactly reflected in the order of the authors. Notes

The authors declare the following competing financial interest(s): All authors are current or former employees of Tetraphase Pharmaceuticals, Inc. 8136

dx.doi.org/10.1021/jm401211t | J. Med. Chem. 2013, 56, 8112−8138

Journal of Medicinal Chemistry

■

Article

Structural basis for TetM-mediated tetracycline resistance. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 16900−16905. (4) For reviews of the mode of action of the tetracyclines, see: (a) Chopra, I. Mode of action of the tetracyclines and the nature of bacterial resistance to them. In The Tetracyclines, Handbook of Experimental Pharmacology; Hlavka, J. J., Boothe, J. H., Eds.; Springer-Verlag: Berlin, 1985; Vol. 78, pp 317−392. (b) Brodersen, D. E.; Clemons, W. M., Jr.; Carter, A. P.; Morgan-Warren, R. J.; Wimberly, B. T.; Ramakrishnan, V. The structural basis for the action of the antibiotics tetracycline, pactamycin, and hygromycin B on the 30S ribosomal subunit. Cell 2000, 103, 1143−1154. (c) Pioletti, M.; Schlunzen, F.; Harms, J.; Zarivach, R.; Gluhmann, M.; Avila, H.; Bashan, A.; Bartels, H.; Auerbach, T.; Jacobi, C.; Hartsch, T.; Yonath, A.; Franceschi, F. Crystal structures of complexes of the small ribosomal subunit with tetracycline, edeine and IF3. EMBO J. 2001, 20, 1829−1839. (d) Wilson, D. N. The A-Z of bacterial translation inhibitors. Crit. Rev. Biochem. Mol. Biol. 2009, 44, 393−433. (e) Jenner, L.; Starosta, A. L.; Terry, D. S.; Mikolajka, A.; Filonava, L.; Yusupov, M.; Blanchard, S. C.; Wilson, D. N.; Yusupova, G. Structural basis for potent inhibitory activity of the antibiotic tigecycline during protein synthesis. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 3812−3816. (5) For leading references, see: (a) Payne, D. J.; Gwynn, M. N.; Holmes, D. J.; Pompliano, D. L. Drugs for bad bugs: Confronting the challenges of antibacterial discovery. Nat. Rev. Drug Discovery 2007, 6, 29−40. (b) Projan, S. J. Whither antibacterial drug discovery? Drug Discovery Today 2008, 13, 279−280. (c) Talbot, G. H. What is the Pipeline for Gram-negative pathogens? Expert Rev. Anti Infect. Ther. 2008, 6, 39−49. (d) Joens, D. The antibacterial lead discovery challenge. Nat. Rev. Drug Discovery 2010, 9, 751−752. (e) Silver, L. L. Challenges of antibacterial discovery. Clin. Microbiol. Rev. 2011, 24, 71−109. (f) Butler, M. S.; Cooper, M. A. Antibiotics in the clinical pipeline in 2011. J. Antibiot. 2011, 64, 413−425. (g) Karras, G.; Giannakaki, V.; Kotsis, V.; Miyakis, S. Novel antimicrobial agents against multi-drug-resistant Gram-negative bacteria: An overview. Recent Pat. Anti-Infect. Drug Discovery 2012, 7, 175−181. (h) Giannakaki, V.; Miyakis, S. Novel antimicrobial agents against multi-drugresistant Gram-positive bacteria: An overview. Recent Pat. Anti-Infect. Drug Discovery 2012, 7, 182−188. (6) For recent reviews, see: (a) Franceschi, F.; Duffy, E. M. Structurebased drug design meets the ribosome. Biochem. Pharmacol. 2006, 71, 1016−1025. (b) Wilson, D. N.; Stelzl, U.; Nierhaus, K. H. Protein synthesis inhibitors. In Handbook of Proteins; Cox, M. M., Phillips, G. N., Eds.; Wiley-Interscience: Hoboken, NJ, 2007; Vol. 1, pp 258−270. (c) Shaw, K. J.; Barbachyn, M. R. The oxazolidinones: Past, present, and future. Ann. N.Y. Acad. Sci. 2011, 1241, 48−70. (d) Sutcliffe, J. A. Antibiotics in development targeting protein synthesis. Ann. N.Y. Acad. Sci. 2011, 1241, 122−152. (7) (a) 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 9aminotetracyclines. J. Med. Chem. 1994, 37, 184−188. (b) 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. (c) 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. (d) French, G. L. A review of tigecycline. J. Chemother. 2008, 20, 3−11. (e) Jenner, L.; Starosta, A. L.; Terry, D. S.; Mikolajka, A.; Filonava, L.; Yusupov, M.; Blanchard, S. C.; Wilson, D. N.; Yusupova, G. Structural basis for potent inhibitory activity of the antibiotic tigecycline during protein synthesis. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, S3812-1−S3812-9. (8) Wang, Y.; Castaner, R.; Bolos, J.; Estivill, C. Amadacycline: Tetracycline antibiotic. Drugs Future 2009, 34, 11−15 Although originally called amadacycline, the compound is now named omadacycline. (9) (a) Xiao, X.-Y.; Hunt, D. K.; Zhou, J.; Clark, R. B.; Dunwoody, N.; Fyfe, C.; Grossman, T. H.; O’Brien, W. J.; Plamondon, L.; Ronn, M.; Sun, C.; Zhang, W.-Y.; Sutcliffe, J. A. Fluorocyclines. 1. 7-Fluoro-9-

ACKNOWLEDGMENTS We thank Professor Andrew Myers, Dr. Eric Gordon, Dr. Joaquim Trias, and Dr. Robert Zahler for valuable discussions over the course of this study. We also thank Dr. Shu-Hui Chen and his colleagues at WuXi Apptec for medicinal chemistry support.

■

ABBREVIATIONS USED ADMET, absorption, distribution, metabolism, excretion, and toxicology; ATCC, American Type Culture Collection; BID, twice per day; Boc, t-butoxycarbonyl; CABP, communityacquired bacterial pneumonia; CFU, colony forming units; dba, dibenzylideneacetone; DCE, 1,2-dichloroethane; DIBALH, diisobutylaluminum hydride; DMAP, 4-dimethylaminopyridine; DMF, N,N-dimethylformamide; ESβL, extended spectrum β-lactamase; % F, percent oral bioavailability; IP, intraperitoneal; IV, intravenous; KPC, Klebsiella pneumoniae carbapenemase; LDA, lithium diisopropylamide; MIC, minimum inhibitory concentration; MIC50, minimum inhibitory concentration required to inhibit growth of 50% of a panel of organisms; MIC90, minimum inhibitory concentration required to inhibit growth of 90% of a panel of organisms; MRSA, methicillin-resistant Staphylococcus aureus; MW, molecular weight; NCS, N-chlorosuccinamide; NT, not tested; PG, protecting group; PK, pharmacokinetic; PO, oral; SAR, structure−activity relationships; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TMEDA, N,N,N′,N′-tetramethylethylenediamine

■

REFERENCES

(1) For a general reviews of antibacterial agents and bacterial resistance, see: (a) Neu, H. C. The crisis in antibiotic resistance. Science 1992, 257, 1064−1078. (b) Chu, D. T. W.; Plattner, J. J.; Katz, L. New directions in antibacterial research. J. Med. Chem. 1996, 39, 3853−3874. (c) D’Costa, V. M.; McGrann, K. M.; Hughes, D. W.; Wright, G. D. Sampling the antibiotic resistome. Science 2006, 311, 374−377. (d) Hawkey, P. M. The growing burden of antimicrobial resistance. J. Antimicrob. Chemother. 2008, 62, i1−i9. (e) Wilson, D. N. The A-Z of bacterial translation inhibitors. Crit. Rev. Biochem. Mol. Biol. 2009, 44, 393−433. (f) Davies, J.; Davies, D. Origins and evolution of antibiotic resistance. Microbiol. Mol. Biol. Rev. 2010, 74, 417−433. (2) For reviews on the tetracyclines, see: (a) 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. (b) Chopra, I.; Hawkey, R. M.; Hinton, M. Tetracyclines, molecular and clinical aspects. J. Antimicrob. Chemother. 1992, 29, 245−277. (c) Shlaes, D. M. An update on tetracyclines. Curr. Opin. Invest. Drugs 2006, 7, 167−171. (d) Pereira-Maia, E. C.; Silva, P. P.; Batista de Almeida, W.; Ferreira dos Santos, H.; Marcial, B. L.; Ruggiero, R.; Guerra, W. Tetracyclines and glycylcyclines: An overview. Quim. Nova 2010, 33, 700−706. (3) For reviews of tetracycline resistance mechanisms, see: (a) Levy, S. B. Evolution and spread of tetracycline resistance determinants. J. Antimicrob. Chemother. 1989, 24, 1−3. (b) Speer, B. S.; Shoemaker, N. B.; Salyers, A. A. Bacterial resistance to tetracycline: Mechanism, transfer, and clinical significance. Clin. Microbiol. Rev. 1992, 5, 387− 399. (c) Chopra, I.; Roberts, M. Tetracycline antibiotics: Mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol. Mol. Biol. Rev. 2001, 65, 232−260. (d) Roberts, M. C. Update on acquired tetracycline resistance genes. FEMS Microbiol. Lett. 2005, 245, 195−203. (e) Thaker, M.; Spanogiannopoulos, P.; Wright, G. D. The tetracycline resistome. Cell. Mol. Life Sci. 2010, 67, 419−431. (f) Dönhöfer, A.; Franckenberg, S.; Wickles, S.; Berninghausen, O.; Beckmann, R.; Wilson, D. N. 8137

dx.doi.org/10.1021/jm401211t | J. Med. Chem. 2013, 56, 8112−8138

Journal of Medicinal Chemistry

Article

(21) 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. (22) (a) Huang, Z.; Zhang, H.; Zhou, G. Preparation of guanidylalkylacyl substituted tetracycline derivatives. Faming Zhuanli Shenqing, CN 101684083, 2010. (b) Koza, D. J. The synthesis of 8substituted tetracycline derivatives, the first 8-position carbon-carbon bond formation. Tetrahedron Lett. 2000, 41, 5017−5020. (c) Nelson, M. L.; Koza, D. Preparation of 7,8 and 9-substituted tetracycline derivatives. PCT Int. Application WO 2002004404, 2002. (d) Nelson, M. Preparation of 8-substituted tetracycline derivatives for pharmaceutical use as antibacterial agents. PCT Int. Application WO 2002012170, 2002. (e) Sum, P. E.; Lee, V. J.; Tally, F. P. Synthesis of novel tetracycline derivatives with substitution at the C-8 position. Tetrahedron Lett. 1994, 35, 1835−1836. (f) Sum, P. E.; Lee, V. J.; Hlavka, J. J.; Testa, R. T. Preparation of 7-(substituted)-8(substituted)-9-(substitutedglycyl)amido-6-demethyl-6-deoxytetracyclines. European Patent Application EP 582789, 1994. (g) Sum, P. E.; Lee, V. J.; Hlavka, J. J.; Testa, R. T. Preparation of 7-(substituted)-8(substituted)-9-(substituted amino)-6-demethyl-6-deoxytetracyclines as antibiotic agents. European Patent Application EP 582810, 1994. (23) Abdel-Magid, A. F.; Maryanoff, C. A.; Carson, K. G. Reductive amination of aldehydes and ketones by using sodium triacetoxyborohydride. Tetrahedron Lett. 1990, 31, 5595−5598. (24) Bhattacharyya, S. Titanium(IV) isopropoxide and sodium borohydride: A reagent of choice for reductive amination. Tetrahedron Lett. 1994, 35, 2401−2404. (25) Clarke, H. T.; Read, R. R. o-Tolunitrile and p-tolunitrile. Org. Synth. 1925, 4, 69. (26) Krasovskiy, A.; Knochel, P. A LiCl-mediated Br/Mg exchange reaction for the preparation of functionalized aryl- and heteroarylmagnesium compounds from organic bromides. Angew. Chem., Int. Ed. 2004, 43, 3333−3336. (27) Moriarty, R. M.; Prakash, O. Oxidation of phenolic compounds with organohypervalent iodine reagents. In Organic Reactions; John Wiley & Sons, Inc.: New York, 2001; Vol. 57, pp 327−415. (28) Miyaura, N.; Suzuki, A. Palladium-catalyzed cross-coupling reactions of organoboron compounds. Chem. Rev. 1995, 95, 2457− 2483. (29) Chen, Q.; Wu, S. Methyl (fluorosulfonyl)difluoroacetate; a new trifluoromethylating agent. J. Chem. Soc., Chem. Comm. 1989, 11, 705− 706. (30) Poole, K. Efflux pumps as antimicrobial resistance mechanisms. Ann. Med. 2007, 39, 162−176. (31) pKB values were calculated using ChemAxon’s structure-based calculation software available at http://www.chemaxon.com,. In the case of examples with cyclopropylamine, the ChemAxon software appears to underestimate the reduction in pKB relative to alkyl amines, and the calculated values were lowered by 1 log units based on literature values reported for cyclopropylamine (pKB = 9.10 vs 10.06 calculated) versus isopropylamine (pKB = 10.63 vs 10.43 calculated). See Perrin, C. L.; Fabian, M. A.; Rivero, I. A. Basicities of cycloalkylamines: Baeyer strain theory revisited. Tetrahedron 1999, 55, 5773−5780. (32) Clinical and Laboratory Standards Institute. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically, 8th ed.; Clinical and Laboratory Standards Institute, Wayne, PA, 2009; Vol. 29, No. 2, Approved standard M07-A8.

pyrrolidinoacetamido-6-demethyl-6-deoxytetracycline: A potent, broad spectrum antibacterial agent. J. Med. Chem. 2012, 55, 597−605. (b) Ronn, M.; Zhu, Z.; Hogan, P. C.; Zhang, W.-Y.; Niu, J.; Katz, C. E.; Dunwoody, N.; Gilicky, O.; Deng, Y.; Hunt, D. K.; He, M.; Chen, C.-L.; Sun, C.; Clark, R. B.; Xiao, X.-Y. Process R&D of eravacycline: The first fully synthetic fluorocycline in clinical development. Org. Process Res. Dev. 2013, 17, 838−845. (10) (a) Levy, S. B.; McMurry, L. Detection of an inductive membrane protein associated with R-factor mediated tetracycline resistance. Biochem. Biophys. Res. Commun. 1974, 56, 1080−1088. (b) McMurry, L.; Petrucci, R. E., Jr.; Levy, S. B. Active efflux of tetracycline encoded by four genetically different tetracycline resistant determinants in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 3974−3977. (c) McMurry, L.; Park, B. H.; Burdett, V.; Levy, S. B. Energy-dependent efflux mediated by class L (tet L) tetracycline resistance determinant from Streptococci. Antimicrob. Agents Chemother. 1987, 31, 1648−1651. (11) (a) Burdett, V. Streptococcal tetracycline resistance mediated at the level of protein synthesis. J. Bacteriol. 1986, 165, 564−569. (b) Sanchez-Pescador, R.; Brown, J. T.; Roberts, M.; Ureda, M. S. Homology of TetM with translational elongation factors: Implications for potential modes of tetM conferred tetracycline resistance. Nucleic Acids Res. 1988, 16, 1216−1217. (c) 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. (12) Mitscher, L. A. The Chemistry of the Tetracycline Antibiotics; Marcel Dekker: New York, 1978. (13) Nelson, M. L.; Ismail, M. Y.; McIntyre, L.; Bhatia, B.; Viski, P.; Hawkins, P.; Rennie, G.; Andorsky, D.; Messersmith, D.; Stapleton, K.; Dumornay, J.; Sheahan, P.; Verma, A. K.; Warchol, T.; Levy, S. B. Versatile and facile synthesis of diverse semisynthetic tetracycline derivatives via Pd-catalyzed reactions. J. Org. Chem. 2003, 68, 5838− 5851. (14) Bodersen, D. E.; Clemons, W. M.; Carter, A. P.; MorganWarren, 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. (15) (a) Spencer, J. L.; Hlavka, J. J.; Petisi, J.; Krazinski, H. M.; Boothe, J. H. 6-Deoxytetracyclines. V. 7,9-Disubstituted products. J. Med. Chem. 1963, 6, 405−407. (b) Stephens, C. R.; Beereboom, J. J.; Rennhard, H. H.; Gordon, P. N.; Murai, K.; Blackwood, R. K.; Wittenau, M. S. 6-Deoxytetracyclines. IV. Preparation, C-6 stereochemistry, and reactions. J. Am. Chem. Soc. 1963, 85, 2643−2652. (c) Martell, M. J.; Boothe, J. H. The 6-Deoxytetracyclines. VII. Alkylated aminotetracyclines possessing unique antibacterial activity. J. Med. Chem. 1967, 10, 44−46. (d) Church, R. F. R.; Schaub, R. E.; Weiss, M. J. Synthesis of 7-dimethylamino-6-demethyl-6-deoxytetracycline (minocycline) via 9-nitro-6-demethyl-6-deoxytetracycline. J. Org. Chem. 1971, 36, 723−725. (16) Charest, M. G.; Lerner, C. D.; Brubaker, J. D.; Siegel, D. R.; Myers, A. G. A convergent enantioselective route to structurally diverse 6-deoxytetracycline antibiotics. Science 2005, 308, 395−398. (17) Brubaker, J. D.; Myers, A. G. A practical, enantioselective synthetic route to key precursor to the tetracycline antibiotics. Org. Lett. 2007, 9, 3523−3525. (18) Sun, C.; Wang, Q.; Brubaker, J. D.; Wright, P. M.; Lerner, C. D.; Noson, K.; Charest, M.; Siegel, D. R.; Wang, Y.-M.; Myers, A. G. A robust platform for the synthesis of new tetracycline antibiotics. J. Am. Chem. Soc. 2008, 130, 17913−17927. (19) Clark, R. B.; Hunt, D. K.; He, M.; Achorn, C.; Chen, C.-L.; Deng, Y.; Fyfe, C.; Grossman, T. H.; Hogan, P. C.; O’Brien, W. J.; Plamondon, L.; Ronn, M.; Sutcliffe, J. A.; Zhu, Z.; Xiao, X.-Y. Fluorocyclines. 2. Optimization of the C-9 side-chain for antibacterial activity and oral efficacy. J. Med. Chem. 2012, 55, 606−622. (20) Clark, R. B.; He, M.; Fyfe, C.; Lofland, D.; O’Brien, W. J.; Plamondon, L.; Sutcliffe, J. A.; Xiao, X.-Y. 8-Azatetracyclines: Synthesis and evaluation of a novel class of tetracycline antibacterial agents. J. Med. Chem. 2011, 54, 1511−1528. 8138

dx.doi.org/10.1021/jm401211t | J. Med. Chem. 2013, 56, 8112−8138