Synthesis and biological evaluation of 8-aminomethyltetracycline derivatives as novel antibacterial agents

Article abstract of DOI:10.1021/jm401211t

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.

Full text of DOI:10.1021/jm401211t

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.
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-, para-
directing C-10 hydroxyl group.15 The total synthetic method-
ology 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 left-
hand 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.
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 pharma-
ceutical 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 7,9-
disubstituted 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 30S-
ribosome cocrystal structure have shown that the hydroxyl and
carbonyl groups of the lower and right-hand portions of the
Received: August 6, 2013
Published: September 18, 2013
© 2013 American Chemical Society
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Figure 1. Tetracycline (1) and its semisynthetic (2 and 3) and fully synthetic (4 and 7) derivatives.
a
Scheme 1. Synthesis of 7-Fluoro-8-aminomethyltetracyclines
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.
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 C-
8 analogs have been limited to halogen, hydroxyl, and aryl
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 Gram-
positive bacteria, indicating that substitution at the C-8 position
is tolerated. Herein, we detail our work on a series of 8-
aminomethyl substituted tetracycline derivatives, including both
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a
Scheme 2. Alternative Synthesis of 7-Fluoro-8-aminomethyltetracyclines
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, 1,4-
dioxane; and (vi) H2, Pd/C, HCl, CH3OH, 1,4-dioxane.
a
Scheme 3. Synthesis of 7-Chloro-8-aminomethyltetracycline D-Ring Precursor
a
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.
in vitro SAR and in vivo efficacy in several murine models of
bacterial infection.
−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-8-
aminomethyltetracyclines 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
CHEMISTRY
■
Among the most common methods for preparing benzylic
amines is the reductive alkylation of amines with benzalde-
hydes. During the course of our work in the 7-fluorotetracy-
clines, 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-aminomethyltetracyl-
cines, 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
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a
Scheme 4. Synthesis of 7-Methoxy-8-aminomethyltetracycline D-Ring Precursor
a
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 Precursor
a
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.
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(i-
PrO)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
magnesium−halogen exchange with i-PrMgCl-LiCl and sub-
sequent alkylation with DMF.26 The final 7-chloro dimethyla-
cetal D-ring precursor 26 was formed upon refluxing in
CH3OH in the presence of TsOH-H2O and trimethylortho-
formate.
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 4-
dimethylaminopyridine (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
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a
Scheme 6. Synthesis of 7-Substituted-8-Aminomethyltetracyclines
a
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-aminomethyltetracyclines
a
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.
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 D-
ring precursor 35.
The 7-chloro-, 7-methoxy-, and 7-trifluoromethyl-8-amino-
methyltetracycline 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.
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Table 1. In Vitro Antibacterial Activity of 7-Fluoro-8-aminomethyltetracyclines with Acyclic Amines
a
MIC (μg/mL)
b
c
c
c
c
c
SA100 SA161
SA158 EF159
SP106 SP160
ATCC
EC107 EC155
ATCC
KP109 KP153
AB110 PA111
ATCC ATCC
ATCC MRSA
ATCC
13883
cmpd
R1
R2
13709
tet(M)
tet(K) tet(M) 49619 tet(M) 25922
tet(A)
tet(A)
19606
27853
15w
15x
CH3
CH3
Et
H
1
>32
32
32
32
16
4
1
>32
16
32
16
16
4
0.25
4
0.5
>32
8
1
32
4
8
16
4
CH3
H
0.25
0.25
0.25
0.25
0.5
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
0.031
0.063
0.031
0.031
0.031
0.016
0.016
0.063
0.063
0.13
1
0.25
0.25
0.13
0.25
1
0.5
0.5
0.25
0.5
2
0.25
4
15a
2
8
8
8
15b
Et
CH3
Et
1
4
4
0.13
0.13
0.063
0.5
8
15c
Et
1
2
2
16
>32
8
15d
2-fluoroethyl
n-Pr
Et
4
4
16
8
15e
H
0.13
0.25
0.5
32
16
32
2
16
16
16
2
1
0.25
0.13
0.5
8
0.25
0.25
1
15f
n-Pr
CH3
Et
2
4
2
0.016
0.016
0.016
0.031
2
32
>32
32
32
8
15g
n-Pr
4
8
4
15y
cyclopropyl
cyclopropyl
i-Pr
CH3
Et
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
4
0.5
16
8
1
8
15z
1
1
2
0.5
1
4
15aa
15bb
15cc
15dd
15ee
15ff
15gg
15hh
15ii
15jj
15kk
15ll
H
16
16
8
32
16
16
16
4
0.063
0.031
0.031
0.016
0.016
0.016
0.016
0.031
0.016
0.13
1
0.25
0.13
0.25
0.13
0.25
0.13
0.13
0.5
8
0.5
0.25
0.5
0.25
0.5
0.25
0.5
1
8
i-Pr
CH3
H
1
1
1
0.063
1
16
16
16
32
16
32
>32
32
>32
>32
32
>32
>32
16
32
16
>32
8
t-Bu
1
2
2
t-Bu
CH3
Et
8
1
0.5
1
0.5
1
0.031
0.016
0.031
0.016
0.031
0.016
0.031
0.5
t-Bu
4
0.5
0.5
2
2-methylpropyl
2-methylpropyl
2-methylpropyl
2,2-dimethylpropyl
2,2-dimethylpropyl
2,2-dimethylpropyl
t-amyl
H
16
2
8
2
2
CH3
Et
2
2
2
1
1
2
4
4
H
4
4
0.5
1
0.13
1
2
0.5
1
1
CH3
Et
0.13
0.25
4
0.13
0.25
4
2
2
0.5
4
2
8
4
8
CH3
H
0.016
0.5
1
0.13
32
0.5
>32
4
0.25
>32
2
0.5
>32
8
0.016
2
15h
Ph
0.13
0.25
8
0.13
0.25
16
8
8
15i
benzyl
CH3
H
0.13
0.063
0.031
32
0.25
2
1
0.13
0.25
0.016
1
15mm
15nn
cyclopentyl
cyclopentyl
tetracycline
omadacycline
tigecycline
0.031
0.016
0.25
0.5
1
0.13
0.13
1
2
0.25
0.25
2
2
CH3
8
1
2
64
1
64
0.25
0.063
32
0.063
0.016
>64
16
1
>64
16
1
0.25
0.13
0.031
0.016
1
2
1
0.13
0.13
0.25
0.5
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 tetracycline-
resistant strains. SA, Staphylococcus aureus; EF, Enterococcus faecalis; SP, Streptococcus pneumoniae; EC, Escherichia coli; AB, Acinetobacter baumannii;
b
c
PA, Pseudomonas aeruginosa; and KP, Klebsiella pneumoniae. Obtained from Micromyx (Kalamazoo, MI). Obtained from Marilyn Roberts’
laboratory at the University of Washington.
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-aminomethyltetracy-
clines 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 pre-
pared 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
followed by reductive methylation with aqueous formaldehyde
and Na(OAc)3BH. Michael−Dieckmann reaction under stand-
ard 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-amino-
methyl derivatives 48, which were then deprotected under
standard conditions to provide desired 7-dimethylamino-8-
aminomethyltetracyclines 49a−g.
BIOLOGY
■
Compounds were initially screened against a panel of Gram-
positive (Staphylococcus aureus, Enterococcus faecalis, and
Streptococcus pneumoniae) and Gram-negative (Escherichia coli,
Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomo-
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nas aeruginosa) bacteria that included both tetracycline-
susceptible (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
most a one-dilution improvement with increasing size from H
to CH3 to Et. For the larger 2-methylpropyl and 2,2-
dimethylpropyl 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 α-
branching compounds were also two-dilutions more potent
We first explored a series of 7-fluoro-8-aminomethyltetracy-
clines 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 7-
fluoro-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 < 2,2-
dimethylpropyl (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
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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 tetracycline-
resistant strains. SA, Staphylococcus aureus; EF, Enterococcus faecalis; SP, Streptococcus pneumoniae; EC, Escherichia coli; AB, Acinetobacter baumannii;
b
c
PA, Pseudomonas aeruginosa; and KP, Klebsiella pneumoniae. Obtained from Micromyx (Kalamazoo, MI). Obtained from Marilyn Roberts’
laboratory at the University of Washington.
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Table 3. In Vitro Antibacterial Activity of 7-R-8-Aminomethyltetracyclines
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 tetracycline-
resistant strains. SA, Staphylococcus aureus; EF, Enterococcus faecalis; SP, Streptococcus pneumoniae; EC, Escherichia coli; AB, Acinetobacter baumannii;
b
c
PA, Pseudomonas aeruginosa; and KP, Klebsiella pneumoniae. Obtained from Micromyx (Kalamazoo, MI). Obtained from Marilyn Roberts’
laboratory at the University of Washington.
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)]
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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 tetracycline-
resistant strains. SA, Staphylococcus aureus; EF, Enterococcus faecalis; SP, Streptococcus pneumoniae; EC, Escherichia coli; AB, Acinetobacter baumannii;
b
c
PA, Pseudomonas aeruginosa; and KP, Klebsiella pneumoniae. Obtained from Micromyx (Kalamazoo, MI). Obtained from Marilyn Roberts’
laboratory at the University of Washington.
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maintained activity against the tetracycline-susceptible Gram-
negative 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 Gram-
negative 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. pneumo-
niae 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-to-
four 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 2-
and 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. 2,6-
Dimethyl 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
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 7-
unsubstituted analogues of several of the more promising 8-
aminomethyl 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 7-
trifluoromethyl 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, 7-
methoxy 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 7-
OCH3 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 8-
aminomethyl 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 8-
positions. 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
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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 com-
pound 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 8-
aminomethyltetracyclines 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 8-
aminomethyltetracyclines, 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
(<50% survival) in both the IV and PO arms (15hh, 15jj, 15i,
15xx, and 15yy). These compounds were characterized by
having more potent S. aureus and E. faecalis tet(M) activity
Table 5. Oral Efficacy Screening Results of 7-R-8-
Aminomethyltetracyclines
survival (%)
S. aureus ATCC 13709
MIC (μg/mL)
IV
PO
%F
compd
15dd
(3 mg/kg) (30 mg/kg) rat
0.13
0.25
0.063
0.013
0.25
0.13
0.031
0.13
0.031
0.25
0.25
0.25
0.25
0.13
0.13
0.25
0.063
0.063
0.13
0.13
0.25
0.13
0.25
0.063
100
33
100
20
9
15hh
15jj
0
0
15ll
100
0
100
0
45
15i
15oo
15rr
100
100
100
100
100
100
50
100
83
15
15
15tt
100
100
100
100
83
39c
21
32
12
49e
41e
42e
15v
50
75
15xx
15yy
39f
33
0
0
0
50
50
39g
67
50
39h
100
100
100
100
100
100
100
100
100
100
100
100
33
39j
41f
49g
tetracycline
omadacycline
tigecycline
15
0.7
1.1
0
(MIC ≤ 0.5 μg/mL) but reduced tet(A) and S. pneumoniae
tet(M) activities (MIC ≥ 1 μg/mL). These compounds
generally have the more lipophilic (15i and 15xx), β-branching
(15hh and 15jj), or less basic (15yy) amino side chains.
Because the IV efficacy was poor for these compounds, it is
difficult to draw conclusions on whether the compounds have
reasonable oral bioavailability. Interestingly, very small changes
had significant effects on the in vivo screening results. For
example, compounds 15ll and 15jj vary only in the position of
the dimethyl branching group (α- vs β-), and compounds 15tt
and 15yy differ only in having a 2,2-dimethyl versus a 2-
spirocyclopropyl substituent, yet compounds 15ll and 15tt
provided 100% protection, whereas the latter two compounds
failed to provide any protection. Having noted large differences
among these pairs of compounds in HPLC retention times, we
speculate that the significantly less polar compounds, 15jj and
15yy, might have greater protein or whole-blood binding,
leading to reduced efficacy. The final group of compounds
(42e, 15v, 39f, and 39g) exhibited marginal (50−83% survival)
efficacy in both the IV and PO arms of the screen. Not
surprisingly, these compounds had in vitro profiles that were
intermediate between the other two groups, with MICs for the
tet(M) and tet(A) strains generally within one dilution of each
other. Compounds 15v and 42e were more efficacious in the
PO arms than in the IV arms, indicating that the 30 mg/kg PO
doses gave higher compound exposure levels than the 3 mg/kg
IV doses. Several compounds with good PO efficacy were also
screened for oral bioavailability in rat pharmacokinetic (PK)
studies. All of the compounds had oral bioavailability that was
at least comparable to that of tetracycline, with compounds 15ll
and 49e showing marked improvement with 45 and 32% F,
respectively. Overall, the IV/PO efficacy screen identified a
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Table 6. Oral Efficacy of 7-R-8-Aminomethyltetracyclines in Mouse Pyelonephritis Models
infection model isolates
MIC50/MIC90 (μg/mL)
MIC (μg/mL)
Log10 CFU/g kidney reduction
EC200
pyelonephritis
2 mg/kg PO
E. coli
(n = 14)
K. pneumoniae
(n = 19)
K. pneumoniae
(ESβL/KPC) (n = 29)
E. coli EC200
(ESβL)
K. pneumoniae
KP453 (ESβL)
KP453 pyelonephritis
50 mg/kg PO
cmpd
15dd
15ll
15oo
15rr
39c
49e
41e
39f
0.25/0.5
0.25/0.5
0.5/1
0.5/1
1/4
0.25
0.25
0.13
0.25
0.25
0.25
0.5
1
2.78
2.15
2
ND
ND
0.5/2
1/2
1
2.51
2.15
0.25/1
1
1.91
1.27
0.25/1
1/2
1
2.82
1.45
0.5/1
1/2
1
1.85
1.04
0.25/1
0.5/1
1/2
0.5/1
0.5
1
2.99
2.93
0.25/0.5
0.13/0.25
0.25/0.5
>32/>32
4/8
0.13
0.25
0.25
>32
2
2.31 (1 mg/kg)
1.55
39j
1/2
2
2.27
1.32
41f
1/2
1/2
1
2.3
2.16
tetracycline
omadacycline
tigecycline
8/>64
8/16
0.5/1
16/64
0.031/0.063
8/>32
8/16
4
ND
1.1
8
ND
ND
0.25/0.5
32/32
1/1
0.13
0.063
0.016
0.5
32
0.031
ND
ND
levofloxacin
32/>32
0.063/>32
3.78
1.17
meropenem/
cilastatin (1:1)
0.031/0.031
3.10 (20 mg/kg IV)
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
S. aureus
(n = 13)
S. pneumoniae
(n = 17)
S. aureus SA191
S. pneumoniae SP160
SA191 lung
30 mg/kg PO
SP160 lung
30 mg/kg PO
cmpd
(MRSA)
(tet(M))
39h
39i
39j
41f
49g
0.25/2
0.25/0.5
0.13/2
0.25/4
0.5/2
1
0.5
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
0.5/1
0.5
1
0.25/0.5
0.031/0.5
0.5/1
0.25
0.25
0.5
32
1
2
tetracycline
omadacycline
tigecycline
levofloxacin
linezolid
32/>32
0.5/2
32/>32
0.016/0.031
0.016/0.016
1/1
>64
1
2
0.13/0.5
16/>64
4/4
0.5
0.25
2
0.016
1
0.5/1
1
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 non-
susceptible 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
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. Interest-
ingly, 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
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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 8-
aminomethyltetracyclines have significant potential as oral
treatments for serious Gram-negative infections.
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 broad-
spectrum oral therapy for complicated infections.
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.
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 community-
acquired 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 8-
aminomethyltetracyclines had MIC90 values that were equiv-
alent 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 8-
aminomethyltetracyclines 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/
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 mass-
directed 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.
1H 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,
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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 light-
yellow 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).
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
1
(74%) of the title compound. H 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-[(tert-
butyldimethylsilyl)oxy]-4-(dimethylamino)-19-fluoro-8,12-di-
h y d r o x y - 1 0 , 1 4 - d i o x o - 6 - o x a - 7 - a z a p e n t a c y c l o -
[11.8.0.03,11.05,9.015,20]henicosa-5(9),7,12,15,17,19-hexaene-18-
carbaldehyde (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 1,2-
dichloroethane (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 8-
Aminomethyltetracyclines (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.
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
1
steps) of the title compound as a colorless oil. H 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-methyl-
benzoate (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 chromatog-
raphy (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-fluo-
ro-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 light-
(4S,4aS,5aR,12aS)-4-(Dimethylamino)-8-[(ethylamino)-
methyl]-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-
drochloride (15a). Prepared by general experimental methods A
1
yellow solid. H 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).
1
(ethylamine) and C, yellow solid. H 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).
(1R,3S,4S,11S)-8,16-Bis(benzyloxy)-17-bromo-11-[(tert-
butyldimethylsilyl)oxy]-18-(dimethoxymethyl)-4-(dimethyla-
mino)-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-hexaene-
10,14-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
(4S,4aS,5aR,12aS)-4-(Dimethylamino)-8-{[ethyl(methyl)-
amino]methyl}-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-
drochloride (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 =
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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)-4-(Dimethylamino)-7-fluoro-3,10,12,12a-
tetrahydroxy-1,11-dioxo-8-(pyrrolidin-1-ylmethyl)-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-
drochloride (15k). Prepared by general experimental methods A
(4S,4aS,5aR,12aS)-8-[(Diethylamino)methyl]-4-(dimethyla-
mino)-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-
drochloride (15c). Prepared by general experimental method A
1
(pyrrolidine) and C, yellow solid. H 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-3,10,12,12a-
tetrahydroxy-8-{[(2R)-2-methylpyrrolidin-1-yl]methyl}-1,11-
dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxa-
mide Dihydrochloride (15l). Prepared by general experimental
1
(diethylamine) and C, yellow solid. H 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,4aS,5aR,12aS)-4-(Dimethylamino)-8-{[ethyl(2-
fluoroethyl)amino]methyl}-7-fluoro-3,10,12,12a-tetrahydroxy-
1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carbox-
amide 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).
1
methods A ((R)-2-methylpyrrolidine) and C, yellow solid. H 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-3,10,12,12a-
tetrahydroxy-8-{[(2S)-2-methylpyrrolidin-1-yl]methyl}-1,11-
dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxa-
mide Dihydrochloride (15m). Prepared by general experimental
(4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12a-
tetrahydroxy-1,11-dioxo-8-[(propylamino)methyl]-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-
drochloride (15e). Prepared by general experimental methods A
1
methods A ((S)-2-methylpyrrolidine) and C, yellow solid. H 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).
1
(propylamine) and C, yellow solid. H 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-3,10,12,12a-
tetrahydroxy-1,11-dioxo-8-{[methyl(propyl)amino]methyl}-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-
drochloride (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-dioxo-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-
drochloride (15g). Prepared by general experimental methods A
(4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12a-
tetrahydroxy-1,11-dioxo-8-{[(2S)-2-(trifluoromethyl)pyrrolidin-
1-yl]methyl}-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-car-
boxamide Dihydrochloride (15n). Prepared by general exper-
imental methods A ((2S)-2-(trifluoromethyl)pyrrolidine) and C,
1
yellow solid. H 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-flu-
oropyrrolidin-1-yl]methyl}-3,10,12,12a-tetrahydroxy-1,11-
dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxa-
mide Dihydrochloride (15o). Prepared by general experimental
1
1
methods A ((3S)-3-fluoropyrrolidine) and C, yellow solid. H NMR
(propylamine), B (acetaldehyde), and C, yellow solid. H NMR (400
(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).
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-8-{[(3R)-3-flu-
oropyrrolidin-1-yl]methyl}-3,10,12,12a-tetrahydroxy-1,11-
dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxa-
mide Dihydrochloride (15p). Prepared by general experimental
(4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12a-
tetrahydroxy-1,11-dioxo-8-[(phenylamino)methyl]-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-
drochloride (15h). Prepared by general experimental methods A
1
1
methods A ((3R)-3-fluoropyrrolidine) and C, yellow solid. H NMR
(aniline) and C, yellow solid. H NMR (400 MHz, CD3OD) δ 7.59−
(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-difluoropyrro-
lidin-1-yl)methyl]-4-(dimethylamino)-7-fluoro-3,10,12,12a-tet-
rahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetra-
cene-2-carboxamide Dihydrochloride (15q). Prepared by general
experimental methods A (3,3-difluoropyrrolidine) and C, yellow solid.
1H 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-3,10,12,12a-
tetrahydroxy-1,11-dioxo-8-[(4-phenylpiperidin-1-yl)methyl]-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-
drochloride (15r). Prepared by general experimental methods A (4-
phenylpiperidine) 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),
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-(di-
methylamino)-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-
drochloride (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-(dimethylami-
no)-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-
drochloride (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).
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2.06−1.96 (s, 4H), 1.63−1.52 (m, 1H). MS (ESI) m/z: 606.2 (M +
H).
reduced pressure. The material was purified by column chromatog-
raphy (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)-7-fluoro-3,10,12,12a-
tetrahydroxy-8-[(4-methylpiperazin-1-yl)methyl]-1,11-dioxo-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Tri-
hydrochloride (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-(di-
methylamino)-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-
drochloride (15t). Prepared by general experimental methods A (4-
acetylpiperazine) 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)-4-(Dimethylamino)-8-{[(2, 2-
dimethylpropyl)amino]methyl}-7-fluoro-3,10,12,12a-tetrahy-
droxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-
carboxamide Dihydrochloride (15ii). Prepared from 17ii by
1
general experimental method C, yellow solid. H 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-3,10,12,12a-
tetrahydroxy-8-[(methylamino)methyl]-1,11-dioxo-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-
1
drochloride (15w). Prepared from methylamine, yellow solid. H
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)-8-(Azepan-1-ylmethyl)-4-(dimethylami-
no)-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-
drochloride (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,4aS,5aR,12aS)-4-(Dimethylamino)-8-{[ethyl(1-
methylcyclopentyl)amino]methyl}-7-fluoro-3,10,12,12a-tetra-
hydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-
2-carboxamide Dihydrochloride (15v). Prepared by general
experimental methods A (1-methylcyclopentylamine), B (acetalde-
hyde), 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.
1H NMR (400 MHz, CDCl3) δ 7.52−7.47 (m, 2H), 7.40−7.33 (m,
(4S,4aS,5aR,12aS)-4-(Dimethylamino)-8-[(dimethylamino)-
methyl]-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-
1
drochloride (15x). Prepared from dimethylamine, yellow solid. H
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-1,11-
dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxa-
mide Dihydrochloride (15y). Prepared from cyclopropylamine and
N-methylated according to general experimental method B (form-
aldehyde), 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-1,11-
dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxa-
mide Dihydrochloride (15z). Prepared from cyclopropylamine and
N-ethylated according to general experimental method B (acetalde-
1
hyde), yellow solid. H 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).
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).
(4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12a-
tetrahydroxy-1,11-dioxo-8-{[(propan-2-yl)amino]methyl}-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-
1
(1R,3S,4S,11S)-8,16-Bis(benzyloxy)-17-bromo-11-[(tert-
butyldimethylsilyl)oxy]-4-(dimethylamino)-18-{[(2,2-
dimethylpropyl)amino]methyl}-19-fluoro-12-hydroxy-6-oxa-7-
azapentacyclo[11.8.0.03,11.05,9.015,20]henicosa-5(9),7,12,15-
(20),16,18-hexaene-10,14-dione (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
drochloride (15aa). Prepared from isopropylamine, yellow solid. H
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-3,10,12,12a-
tetrahydroxy-8-{[methyl(propan-2-yl)amino]methyl}-1,11-
dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxa-
mide Dihydrochloride (15bb). Prepared from isopropylamine and
N-methylated according to general experimental method B (form-
aldehyde), 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-(dimethyla-
mino)-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-
1
drochloride (15cc). Prepared from t-butylamine, yellow solid. H
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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-(di-
methylamino)-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-
drochloride (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-(di-
methylamino)-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-
drochloride (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-3,10,12,12a-
tetrahydroxy-8-{[(2-methylpropyl)amino]methyl}-1,11-dioxo-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-
drochloride (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).
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-3,10,12,12a-
tetrahydroxy-8-{[methyl(2-methylbutan-2-yl)amino]methyl}-
1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carbox-
amide Dihydrochloride (15ll). Prepared from t-amylamine and N-
methylated according to general experimental method B (form-
aldehyde), 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-(dime-
thylamino)-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-
drochloride (15 mm). Prepared from cyclopentylamine, yellow solid.
1H 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-1,11-
dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxa-
mide Dihydrochloride (15nn). Prepared from cyclopentylamine
and N-methylated according to general experimental method B
1
(formaldehyde), yellow solid. H 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-dimethylpyrro-
lidin-1-yl)methyl]-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-
dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxa-
mide Dihydrochloride (15oo). Prepared from 2,2-dimethylpyrroli-
dine, 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-3,10,12,12a-
tetrahydroxy-1,11-dioxo-8-(piperidin-1-ylmethyl)-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-
drochloride (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-3,10,12,12a-
tetrahydroxy-8-{[methyl(2-methylpropyl)amino]methyl}-1,11-
dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxa-
mide Dihydrochloride (15gg). Prepared from 2-methylpropylamine
and N-methylated according to general experimental method B
1
(formaldehyde), yellow solid. H 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,4aS,5aR,12aS)-4-(Dimethylamino)-8-{[ethyl(2-
methylpropyl)amino]methyl}-7-fluoro-3,10,12,12a-tetrahy-
droxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-
carboxamide Dihydrochloride (15hh). Prepared from 2-methyl-
propylamine 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)-7-fluoro-3,10,12,12a-
tetrahydroxy-8-[(4-methylpiperidin-1-yl)methyl]-1,11-dioxo-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-
drochloride (15rr). Prepared from 4-methylpiperidine, yellow solid.
1H NMR (400 MHz, CD3OD) δ 7.10 (d, J = 5.5 Hz, 1H), 4.35 (s,
(4S,4aS, 5aR, 12aS)-4-(Dimethylamino)-8-{[(2, 2-
dimethylpropyl)(methyl)amino]methyl}-7-fluoro-3,10,12,12a-
tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetra-
cene-2-carboxamide Dihydrochloride (15jj). Prepared from 2,2-
dimethylpropylamine and N-methylated according to general exper-
imental 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).
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-dimethylpiper-
idin-1-yl)methyl]-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-
dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxa-
mide Dihydrochloride (15uu). Prepared from 3,3-dimethylpiper-
1
idine, yellow solid. H 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-{[(2, 2-
dimethylpropyl)(ethyl)amino]methyl}-7-fluoro-3,10,12,12a-
tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetra-
cene-2-carboxamide Dihydrochloride (15kk). Prepared from 2,2-
dimethylpropylamine and N-ethylated according to general exper-
(4S,4aS,5aR,12aS)-4-(Dimethylamino)-8-[(4,4-dimethylpiper-
idin-1-yl)methyl]-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-
dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxa-
mide Dihydrochloride (15vv). Prepared from 4,4-dimethylpiper-
1
imental method B (acetaldehyde), yellow solid. H 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,
1
idine, yellow solid. H 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).
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).
(4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12a-
tetrahydroxy-8-(morpholin-4-ylmethyl)-1,11-dioxo-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-
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
1
drochloride (15ww). Prepared from morpholine, yellow solid. H
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).
1
37.2 g (68%) of the title compound as an off-white solid. H NMR
(4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12a-
tetrahydroxy-8-{[methyl(2,4,4-trimethylpentan-2-yl)amino]-
methyl}-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-
2-carboxamide Dihydrochloride (15xx). Prepared from 2,4,4-
trimethylpentan-2-amine and N-methylated according to general
(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 temper-
ature 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
1
experimental method B (formaldehyde), yellow solid. H 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-(di-
methylamino)-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-
drochloride (15yy). Prepared from 4-azaspiro[2.5]octane, yellow
1
solid. H 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)-2-
methylpiperidin-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-3,10,12,12a-
tetrahydroxy-8-{[(2S)-2-methylpiperidin-1-yl]methyl}-1,11-
dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxa-
mide 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).
1
yield 14.5 g (69%) of the title compound as a light-yellow solid. H
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 off-
white solid. 1H NMR (400 MHz, CDCl3) δ 7.41−7.45 (m, 2H),
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-dime-
thylpiperidin-1-yl]methyl}-7-fluoro-3,10,12,12a-tetrahydroxy-
1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carbox-
amide Dihydrochloride (15ss). Prepared from cis-2,6-dimethylpi-
1
peridine, yellow solid. H 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-dimethylpiper-
idin-1-yl)methyl]-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-
dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxa-
mide Dihydrochloride (15tt). Prepared from 2,2-dimethylpiper-
1
idine, yellow solid. H 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),
8130
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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).
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-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
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
1
white solid. This material was used without further purification. H
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
1
(41%) of the title compound. H 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-methoxy-
2-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
1
1
as a white solid. H NMR (400 MHz, CDCl3) δ 7.33−7.43 (m, 7H),
white solid. H NMR (400 MHz, CDCl3) δ 7.45−7.41 (m, 2H), 7.38
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).
(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-(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
Phenyl 6-{[(tert-Butoxy)carbonyl]oxy}-4-formyl-3-methoxy-
2-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).
1
solid. H 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)-2-
methylbenzoate (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−
Phenyl 4-Ethenyl-2-methoxy-6-methylbenzoate (32). Com-
pound 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 1,4-
dioxane/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
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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
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).
( 1 R , 3 S , 4 S , 1 1 S ) - 8 , 1 6 - B i s ( b e n z y l o x y ) - 1 1 - [ ( t e r t -
butyldimethylsilyl)oxy]-19-chloro-18-(dimethoxymethyl)-4-
(dimethylamino)-12-hydroxy-6-oxa-7-azapentacyclo-
[11.8.0.03,11.05,9.015,20]henicosa-5(9),7,12,15(20),16,18-hex-
aene-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
1
solid. H 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 chromatog-
raphy (100:1 to 50:1 to 30:1 petroleum ether/EtOAc gradient) gave
1
13 g (62%) of the title compound as a light-yellow solid. H 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.
1
3.20 g (90%) of the title compound as a light-yellow solid. H 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.
Phenyl 3-Bromo-4-formyl-2-hydroxy-6-methylbenzoate.
BBr3 (1.90 mL, 19.5 mmol) was added to a solution of phenyl 3-
bromo-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.
( 1 R , 3 S , 4 S , 1 1 S ) - 8 , 1 6 - B i s ( b e n z y l o x y ) - 1 1 - [ ( t e r t -
butyldimethylsilyl)oxy]-19-chloro-4-(dimethylamino)-12-hy-
d r o x y - 1 0 , 1 4 - d i o x o - 6 - o x a - 7 - a z a p e n t a c y c l o -
[11.8.0.03,11.05,9.015,20]henicosa-5(9),7,12,15(20),16,18-hex-
aene-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
1
gave 1.05 g (75%) of the title compound as a light-yellow solid. H
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-6-
methylbenzoate (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 light-
yellow 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).
P h e n y l 2 - ( B e n z y l o x y ) - 4 - f o r m y l - 6 - m e t h y l - 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).
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)-3,10,12,12a-
tetrahydroxy-1,11-dioxo-8-(pyrrolidin-1-ylmethyl)-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-
drochloride (39a). Prepared from 37a by general experimental
1
methods A (pyrrolidine) and C, yellow solid. H 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)-3,10,12,12a-
tetrahydroxy-1,11-dioxo-8-[(dimethylamino)methyl]-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-
drochloride (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).
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−
(4S,4aS,5aR,12aS)-8-{[t-Butyl(methyl)amino]methyl}-7-
chloro-4-(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-
dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxa-
mide 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-dime-
thylpyrrolidin-1-yl)methyl]-3,10,12,12a-tetrahydroxy-1,11-
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1
dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxa-
mide Dihydrochloride (39d). Prepared from 37a by general
experimental methods A (2,2-dimethylaminopyrrolidine) and C,
method C in the synthesis of 39c, yellow solid. H 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).
1
yellow solid. H 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).
(1R,3S,4S,11S)-8-(Benzyloxy)-11-[(tert-butyldimethylsilyl)-
oxy]-18-(dimethoxymethyl)-4-(dimethylamino)-12-hydroxy-
19-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-16-
yl 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-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-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)-7-Chloro-8-{[(cyclopropylmethyl)(propan-
2-yl)amino]methyl}-4-(dimethylamino)-3,10,12,12a-tetrahy-
droxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-
carboxamide Dihydrochloride (39f). Prepared from 37a by
general experimental methods A (2-propylamine), B (cyclopropylcar-
1
boxaldehyde), and C, yellow solid. H 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)-3,10,12,12a-
tetrahydroxy-8-({[(2R)-3-methylbutan-2-yl]amino}methyl)-
1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carbox-
amide 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-(dime-
thylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-
drochloride (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-tetrahydroxy-
1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carbox-
amide 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)-4-(Dimethylamino)-3,10,12,12a-tetrahy-
droxy-7-methoxy-1,11-dioxo-8-(pyrrolidin-1-ylmethyl)-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-
drochloride (41a). Prepared from 37b by general experimental
1
methods A (pyrrolidine) and C, yellow solid. H 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-dioxo-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-
drochloride (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-(di-
methylamino)-3,10,12,12a-tetrahydroxy-7-methoxy-1,11-
dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxa-
mide 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-dimethylpyrro-
lidin-1-yl)methyl]-3,10,12,12a-tetrahydroxy-7-methoxy-1,11-
dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxa-
mide Dihydrochloride (41d). Prepared from 37b by general
experimental methods A (2,2-dimethylaminopyrrolidine) and C,
(4S,4aS,5aR,12aS)-7-Chloro-4-(dimethylamino)-8-{[(2S,5S)-
2,5-dimethylpyrrolidin-1-yl]methyl}-3,10,12,12a-tetrahydroxy-
1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carbox-
amide 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-tetrahy-
d r o x y - 1 , 1 1 - d i o x o - 8 - ( p y r r o l i d i n - 1 - y l m e t h y l ) -
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-
drochloride (40a). Isolated as a side product of general experimental
1
method C in the synthesis of 39a, yellow solid. H 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-(di-
methylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-
drochloride (40c). Isolated as a side product of general experimental
1
yellow solid. H NMR (400 MHz, CD3OD) δ 7.02 (s, 1H), 4.54 (d,
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).
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-tetrahy-
droxy-1,11-dioxo-8-(pyrrolidin-1-ylmethyl)-7-(trifluorometh-
yl)-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide
Dihydrochloride (42a). Prepared from 37c by general experimental
(4S,4aS,5aR,12aS)-4-(Dimethylamino)-3,10,12,12a-tetrahy-
droxy-7-methoxy-1,11-dioxo-8-(piperidin-1-ylmethyl)-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-
drochloride (41e). Prepared from 37b by general experimental
1
methods A (pyrrolidine) and C, yellow solid. H 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-(trifluormeth-
yl)-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-(di-
methylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-7-(tri-
fluoromethyl)-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-car-
boxamide 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).
1
methods A (piperidine) and C, yellow solid. H 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-tetrahydroxy-
7-methoxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetra-
cene-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-dimethylbu-
tan-2-yl)(methyl)amino]methyl}-3,10,12,12a-tetrahydroxy-7-
methoxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-
2-carboxamide Dihydrochloride (41g). Prepared from 37b by
general experimental methods A (2,3-dimethylbutan-2-amine), B
(4S,4aS,5aR,12aS)-4-(Dimethylamino)-8-[(2,2-dimethylpyrro-
lidin-1-yl)methyl]-3,10,12,12a-tetrahydroxy-1,11-dioxo-7-(tri-
fluoromethyl)-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-car-
boxamide Dihydrochloride (42d). Prepared from 37c by general
experimental methods A (2,2-dimethylaminopyrrolidine) and C,
1
(formaldehyde), and C, yellow solid. H 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).
1
yellow solid. H 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)-8-{[(2R,6S)-2,6-dime-
thylpiperidin-1-yl]methyl}-3,10,12,12a-tetrahydroxy-7-me-
thoxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-
carboxamide 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).
( 1 R , 3 S , 4 S , 1 1 S ) - 8 , 1 6 - B i s ( b e n z y l o x y ) - 1 1 - [ ( t e r t -
butyldimethylsilyl)oxy]-18-(dimethoxymethyl)-4-(dimethyla-
mino)-12-hydroxy-19-(tri fluoromethyl)-6-oxa-7-
azapentacyclo[11.8.0.03 , 1 1 .05 , 9 .01 5 , 2 0 ]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).
(4S,4aS,5aR,12aS)-4-(Dimethylamino)-3,10,12,12a-tetrahy-
droxy-1,11-dioxo-8-(piperidin-1-ylmethyl)-7-(trifluoromethyl)-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-
drochloride (42e). Prepared from 37c by general experimental
1
methods A (piperidine) and C, yellow solid. H 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 solu-
tion 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
1
further purification. H 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 dichloro-
methane (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
( 1 R , 3 S , 4 S , 1 1 S ) - 8 , 1 6 - B i s ( b e n z y l o x y ) - 1 1 - [ ( t e r t -
butyldimethylsilyl)oxy]-4-(dimethylamino)-12-hydroxy-10,14-
dioxo-19-(trifluoromethyl)-6-oxa-7-azapentacyclo-
[11.8.0.03,11.05,9.015,20]henicosa-5(9),7,12,15,17,19-hexaene-18-
carbaldehyde (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
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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.
(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).
1
The material was used without further purification. H 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
( 1 R , 3 S , 4 S , 1 1 S ) - 8 , 1 6 - B i s ( b e n z y l o x y ) - 1 1 - [ ( t e r t -
butyldimethylsilyl)oxy]-4,19-bis(dimethylamino)-12-hydroxy-
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-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
1
to give 2.20 g of the crude phenol intermediate. H 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
1
(47%, 4 steps) of the title compound as a white solid. H NMR (400
1
reactions. H NMR (400 MHz, CDCl3) δ 15.85 (s, 1H), 10.32 (s,
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-meth-
ylbenzoate (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).
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-tetra-
h y d r o x y - 1 , 1 1 - d i o x o - 8 - ( p y r r o l i d i n - 1 - y l m e t h y l ) -
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Tri-
hydrochloride (49a). Prepared from 47 by general experimental
1
methods A (pyrrolidine) and C, yellow solid. H 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).
( 4 S , 4 a S , 5 a R , 1 2 a S ) - 4 , 7 - B i s ( d i m e t h y l a m i n o ) - 8 -
[(dimethylamino)methyl]-3,10,12,12a-tetrahydroxy-1,11-
dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxa-
mide 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).
(1R,3S,4S,11S)-8,16-Bis(benzyloxy)-18-bromo-11-[(tert-
butyldimethylsilyl)oxy]-4,19-bis(dimethylamino)-12-hydroxy-
6-oxa-7-azapentacyclo[11.8.0.03,11.05,9.015,20]henicosa-5-
(9),7,12,15,17,19-hexaene-10,14-dione (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
(4S,4aS,5aR,12aS)-8-{[t-Butyl(methyl)amino]methyl}-4,7-bis-
(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Tri-
hydrochloride (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-dimethyl-
pyrrolidin-1-yl)methyl]-3,10,12,12a-tetrahydroxy-1,11-dioxo-
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Tri-
hydrochloride (49d). Prepared from 47 by general experimental
1
methods A (2,2-dimethylaminopyrrolidine) and C, yellow solid. H
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,
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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).
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.
(4S,4aS,5aR,12aS)-4,7-Bis(dimethylamino)-3,10,12,12a-tetra-
h y d r o x y - 1 , 1 1 - d i o x o - 8 - ( p i p e r i d i n - 1 - y l m e t h y l ) -
1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide trihy-
drochloride (49e). Prepared from 47 by general experimental
1
methods A (piperidine) and C, yellow solid. H 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-{[(2,2-
dimethylpropyl)amino]methyl}-3,10,12,12a-tetrahydroxy-
1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carbox-
amide 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-di-
methylbutan-2-yl]amino}methyl)-3,10,12,12a-tetrahydroxy-
1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carbox-
amide 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.
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.
AUTHOR INFORMATION
Animal Efficacy Models. All animal efficacy models were
performed at Vivisource, Waltham, MA.
■
Corresponding Author
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 post-
infection. The PD50 in mg/kg was calculated as survival after 48 h.
*Email: rclark@tphase.com; 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.
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Article
Structural basis for TetM-mediated tetracycline resistance. Proc. Natl.
Acad. Sci. U.S.A. 2012, 109, 16900−16905.
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.
(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.;
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ABBREVIATIONS USED
■
ADMET, absorption, distribution, metabolism, excretion, and
toxicology; ATCC, American Type Culture Collection; BID,
twice per day; Boc, t-butoxycarbonyl; CABP, community-
acquired bacterial pneumonia; CFU, colony forming units; dba,
dibenzylideneacetone; DCE, 1,2-dichloroethane; DIBALH,
diisobutylaluminum hydride; DMAP, 4-dimethylaminopyri-
dine; DMF, N,N-dimethylformamide; ESβL, extended spec-
trum β-lactamase; % F, percent oral bioavailability; IP,
intraperitoneal; IV, intravenous; KPC, Klebsiella pneumoniae
carbapenemase; LDA, lithium diisopropylamide; MIC, mini-
mum 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′-tetramethylethyle-
nediamine
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