Article

pubs.acs.org/jmc

Design, Synthesis, and Biological Evaluation of Hexacyclic

Tetracyclines as Potent, Broad Spectrum Antibacterial Agents
Cuixiang Sun,† Diana K. Hunt,† Chi-Li Chen,† Yonghong Deng,† Minsheng He,† Roger B. Clark,†
Corey Fyfe,‡ Trudy H. Grossman,‡ Joyce A. Sutcliffe,‡ and Xiao-Yi Xiao*,†
†
Discovery Chemistry, ‡Microbiology, Tetraphase Pharmaceuticals, 480 Arsenal Street, Watertown, Massachusetts 02472, United
States
*
S Supporting Information

ABSTRACT: A series of novel hexacyclic tetracycline analogues (“hexacy-

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

■ INTRODUCTION
New, potent antibacterial agents are in urgent need to combat
positions from C4 to C9, including various substitutions and
additional fused ring systems. These chemical modifications
often lead to modulations of tetracyclines’ chemical−physical
life threatening infections caused by multidrug-resistant
properties, ribosomal binding, as well as the ability to overcome
(MDR), Gram-negative and Gram-positive organisms such as
active drug efflux. We have therefore designed a new hexacyclic
carbapenem-resistant Enterobacteriaceae (CRE)1 and methicil-
tetracycline scaffold (1, Figure 1) that consists of a bicyclic EF-
lin-resistant Staphylococcus aureus (MRSA).2 Tetracyclines are a
proven class of broad spectrum antibiotics first discovered in
the mid-1940s.3 While revolutionary at the time, seven decades
of widespread use has largely rendered legacy tetracyclines
ineffective due to progressing tetracycline resistance. 4a
Renewed interest in the class, stemming from advances in
chemical synthesis, has resulted in marked improvements
against tetracycline-specific resistance, leading to the most
recently approved tetracycline, tigecycline,5 in 2005, as well as
newer generations of tetracycline antibiotics currently in late
stage development such as eravacycline.6,7 Figure 1. A hexacyclic tetracycline scaffold.
Two major bacterial resistance mechanisms4b,c for tetracy-
clines have been identified: (1) tetracycline-specific efflux ring appended to the original tetracycline D-ring. A series of
pumps, including tet(A)−tet(D) and tet(K)−tet(L), which are hexacyclic tetracycline analogues (“hexacyclines”) with struc-
frequently found in both Gram-positive and Gram-negative tural variations at a number of positions was synthesized using
pathogens, and (2) ribosomal protection, including tet(M)− our fully synthetic tetracycline strategy.6,8b,10 These hexacy-
tet(O), which are more commonly seen in Gram-positive clines were evaluated for antibacterial activity against a broad
bacteria. A number of semisynthetic tetracyclines, including range of pathogens, especially activities against tetracycline
resistant, Gram-negative bacteria.

doxycycline, minocycline, and tigecycline, have been developed
to overcome bacterial resistance. However, the exploitation of
the tetracycline scaffold has been impeded by the limited
chemical methods available for semisynthetic tetracyclines until
the recent development and application of the tetracycline total
synthesis platform, which enables the creation and study of
previously difficult-to-access, fully synthetic tetracycline ana-
logues.6,8−10
Prior structure−activity relationship (SAR) studies5,6,8a,9
from our laboratory and other laboratories indicated that the
tetracycline scaffold can tolerate structural modifications on
■
RESULTS AND DISCUSSION
Chemistry. Using our tetracycline total synthesis ap-
proach,6,8b,10 a series of 7-fluorohexacyclines with a wide
range of substitutions at the 8-position nitrogen (N8) was
prepared from a tricyclic DEF-ring precursor 6 and an AB-ring
enone 7.10,11 As shown in Scheme 1, aniline 29b was treated
Received: February 13, 2015

© XXXX American Chemical Society A DOI: 10.1021/acs.jmedchem.5b00262

J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
Scheme 1. Synthesis of 7-Fluorohexacycline Analoguesa
a
Reagents: (a) Br2, NaOAc, HOAc; (b) LHMDS, THF, then Boc2O, and then allyl bromide; (c) n-BuLi, THF, then DMF; (d) N-allyl glycine,
DMF; (e) LDA, TMEDA, THF, then enone 7; (f) Pd(PPh3)4, N,N′-dimethylbarbituric acid, CH2Cl2; (g) (i) N-substitution (see Experimental
Section), (ii) 48% aq HF, THF, (iii) 10% Pd-C, HCl, CH3OH.
with bromine in the presence of sodium acetate to give the
bromide intermediate 3 in 85% yield. Full protection of the
aniline group by treatment with lithium bis(trimethylsilyl)-
amide (LHMDS) and Boc2O, followed by allyl bromide yielded
compound 4 (88% yield). Compound 4 was formylated by
treatment with n-BuLi at −100 °C in THF followed by DMF
addition to give aldehyde 5 in 94% isolated yield. Cyclo-
addition12 of aldehyde 5 with N-allyl glycine in DMF at 80 °C
afforded the desired tricyclic DEF-ring precursor 6 in 71% yield
with a cis-configuration between the fused 5- and 6-membered
heterocyclic rings (the corresponding trans isomer was not
detected). Michael−Dieckmann annulation10 of 6 with the AB-
ring enone 7 by treatment with lithium diisopropylamide
(LDA) and N,N,N′,N′-tetramethylethylenediamine (TMEDA)
at −78 °C in THF gave the fully protected intermediate 8 in
98% yield as a mixture of two diastereomers. The allyl group on
N8 was removed by standard deallylation conditions to afford
intermediate 9 (the two diastereomers were separated by
preparative HPLC, 67% combined yield). The two diaster-
eomers of secondary amine 9 were then separately alkylated by
reductive alkylation with a variety of aldehydes to yield the N8
substituted intermediates, and these were deprotected by
aqueous HF treatment followed by hydrogenation as previously
described6 to afford the desired hexacycline analogues 10a−
10h in reasonable yields (20−60%) after reverse phase HPLC
purification (Table 1).
Similarly, several analogues with a methyl substituent at the
bridgehead carbon C10a were also prepared using 3-bromo-2-
methylpropene instead of ally bromide in Scheme 1, step b
(11a, 11b, and 11c, Table 1).
Subsequently, to explore the effect of other C7 substitutions,
a series of 7-trifluoromethoxy hexacycline analogues was
prepared according to Scheme 2. In addition, a range of cyclic
and acyclic amino acids was also incorporated in the
cycloaddition step to explore structural variations on the F-
ring. Thus, phenol 1213 was iodinated by treatment with NaH
B DOI: 10.1021/acs.jmedchem.5b00262
Article
and iodine to give compound 13, which was benzylated under
standard conditions to afford intermediate 14. Compound 14
was then reacted at 80 °C with BocNH2 in the presence of
Pd(OAc)2, Xantphos,14 and cesium carbonate to yield
intermediate 15 in 28% yield over three steps. Aryl bromide
15 was treated with PhLi and n-BuLi followed by DMF to
afford aldehyde 16 (37% yield), which was allylated with allyl
bromide and NaH to give compound 17 in 82% yield.
Cycloaddition12 of compound 17 with a range of cyclic and
acyclic N-alkylated amino acids under the conditions described
above gave the desired DEF-ring precursors 18 in good yields.
Michael−Dieckmann annulation10 of 18 with enone 7, followed
by desilylation and hydrogenation, gave the desired 7-
trifluoromethoxy hexacycline analogues 20a−20f in yields
similar to those in Scheme 1 after reverse phase HPLC
purification (Table 2).
Biology. The novel hexacyclines were evaluated for in vitro
antibacterial activity in minimal inhibitory concentration (MIC)
assays against a panel of tetracycline-susceptible and tetracy-
cline-resistant, Gram-positive and Gram-negative bacterial
strains (Tables 1 and 2). All isolates in the panel were
multidrug-resistant (MDR, i.e., resistant to ≥3 different classes
of antibiotics) except S. aureus SA101, Escherichia coli EC107,
and S. aureus SA158, a tet (K) tetracycline-resistant strain and
otherwise drug susceptible. MIC data of representative
hexacycline analogues are listed in Tables 1 and 2. As
mentioned above, most of the final compounds were isolated
as two separate cis-diastereomers. Although it is known that the
“north-west” portion of tetracyclines is not involved in
ribosomal binding, the two diastereomers displayed consid-
erably different MIC values across the entire panel of bacterial
strains, as exemplified by the diastereomer pair 10b and 10c in
Table 1. For brevity, only the relatively more active
diastereomers from each pair are listed in Tables 1 and 2,
except for compounds 10a, 10h, and 20a, which were isolated
as mixtures of the two diastereomers (∼1:1).
J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry Article

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

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

When the pyrrolidine nitrogen (N8) was not substituted, the
hexacycline analogues had only modest antibacterial activity, as
shown by MIC data of compounds 10a, 11a, and 20a.
However, when the nitrogen was substituted with a methyl
group, the potency was dramatically improved by 2- to >32-fold
against most strains in the panel except PM385 (10c vs 10a,
20b vs 20a). Further increases in lipophilicity of the nitrogen
substituent tended to decrease potency, especially against
Gram-negative strains, by varying degrees depending on the
C DOI: 10.1021/acs.jmedchem.5b00262
amount of increase in lipophilicity (10d and 11c). The
presence of additional polar groups, such as hydroxyl, amino,
and amide groups, on the nitrogen substituent drastically
decreased the compounds’ antibacterial activities (10e−10h).
Interestingly, the potency of the difluoroethylaminoethyl
substituted analogue 10g was decreased by a much lesser
degree, especially against Gram-positive pathogens, probably
due to the decreased basicity and/or polarity of the
difluoroethylamino group. Alkyl substitution at the C10a
J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry Article

Scheme 2. Synthesis of 7-Trifluoromethoxyhexacycline Analoguesa

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

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

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

position decreased potency against most strains in the panel, as (20f) or lipophilicity (either by alkyl substitution on the F-
shown by the 10a-methyl substituted analogues 11a, 11b, and ring (20c) or by incorporation of a seventh ring (G-ring, 20d,
11c. 20e)) decreased potency. Interestingly, compared to the 7-
Similar general SAR trends were also observed for the 7- fluoro analogue 10c, the corresponding 7-trifluoromethoxy
trifluoromethoxy series (Table 2). Thus, a methyl group on the analogue 20b was more potent against most Gram-positive
pyrrolidine nitrogen (N8) was the optimum substitution for strains (≥4-fold) as well as some of the Gram-negative strains
antibacterial activity (compound 20b). Additional polarity (Escherichia coli strains EC107 and EC155, and Stenotropho-
D DOI: 10.1021/acs.jmedchem.5b00262
J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
Figure 2. In vivo efficacy of compound 10c in (A), mouse lung infection model challenged with P. aeruginosa PA1145; (B) mouse thigh infection
model challenged with P. aeruginosa PA694.
monas maltophilia SM256) but 2−4-fold less active against
Proteus mirabilis PM385, Pseudomonas aeruginosa PA555, and
Acinetobacter baumannii AB250.
Overall, compounds 10c and 20b displayed high in vitro
potency against a broad range of MDR Gram-positive and
Gram-negative pathogens. Particularly, compound 10c also
demonstrated promising in vitro activity against P. aeruginosa
PA555 with an MIC of 4 μg/mL, which is among the lowest
MIC values in the tetracycline class. Ribosomal inhibition was
confirmed by an in vitro P. aeruginosa coupled transcription/
translation assay,15 in which compound 10c was 10-fold more
potent than tetracycline with an IC50 value of 0.21 μM.
Compound 10c was further tested for in vivo efficacy in two
mouse infection models challenged with P. aeruginosa. In a
mouse lung infection model challenged with P. aeruginosa
PA1145 (Figure 2A), compound 10c (MIC = 4 μg/mL)
demonstrated a 4-log colony-forming unit (CFU) reduction in
bacterial burden when dosed intravenously (IV) at 40 mg/kg,
twice daily. Comparator compounds meropenem (MIC = 4
μg/mL) and amikacin (MIC = 1 μg/mL) showed bacterial
burden reductions of about 2−2.5 log CFU when administered
at the same doses. In the mouse thigh infection model
challenged with P. aeruginosa PA694 (Figure 2B), compound
10c (MIC = 8 μg/mL) showed promising dose-proportional
efficacy when dosed at 1.5, 5, 15, and 40 mg/kg, IV, twice daily
and was equally efficacious at 40 mg/kg as comparator
levofloxacin (MIC = 1 μg/mL) dosed IV at 5 mg/kg twice
daily (1.8 log CFU reduction from T = 0 and 4.5 log CFU
reduction from T = 24 h).
■ CONCLUSIONS
A series of hexacycline analogues based on scaffold 1 was
designed and synthesized with a range of substitutions at C7,
N8, C9, and C10a, and their antibacterial activity was evaluated
against a broad range of Gram-negative and Gram-positive
pathogens, including multidrug-resistant and tetracycline-
resistant bacterial strains. Analogues 10c, a 7-fluorohexacycline
analogue, and 20b, a 7-trifluoromethoxyhexacycline analogue,
demonstrated potent, broad spectrum in vitro antibacterial
activity. Compound 10c also displayed promising anti-P.
aeruginosa potency with MIC values ranging from 4 to 8 μg/
mL in this study. In vivo efficacy studies in thigh and lung
E DOI: 10.1021/acs.jmedchem.5b00262
Article
mouse infection models challenged with P. aeruginosa
demonstrated that compound 10c was equally or more
efficacious than various comparator compounds at comparable
dose levels. These data support the further optimization of the
hexacycline scaffold for the discovery and development of new
tetracycline antibiotics against a broad range of pathogens,
including MDR Gram-negative bacteria.
■
EXPERIMENTAL SECTION
Chemistry. All commercially available reagents and solvents,
including anhydrous solvents, were used without further purification.
All reactions under dry conditions were performed under nitrogen
atmospheres. 1H and 13C NMR (nuclear magnetic resonance) spectra
were recorded at room temperature (25 °C) on a 400 MHz JEOL
ECX-400 spectrometer. CDCl3 (for intermediates) or CD3OD (for
final compounds) were used as the solvents. Chemical shifts (δ) are
expressed in parts per million (ppm). The signals of the deuterated
solvents were used as the internal standards. Thin-layer chromatog-
raphy (TLC) analysis was performed on Merck Silica Gel 60 F254 and
visualized under UV light. Flash chromatography was performed on a
Biotage Isolera One using SNAP cartridges (KP-Sil). Purity of tested
compounds was determined to be ≥95% by reverse phase analytical
HPLC/MS analysis (high-performance liquid chromatography/mass
spectrometry) performed on a Waters Alliance system (column,
SunFire C18, 5 μm, 4.6 mm × 50 mm; solvent A, water with 0.1%
formic acid; solvent B, acetonitrile with 0.1% formic acid; MS detector,
Waters 3100) unless otherwise noted. Reverse phase preparative
HPLC was performed on a Waters Autopurification system with mass-
directed fraction collection (for final compounds: column, Polymerx
RP-1 100A, 10 μm, 150 mm × 21.20 mm; flow rate, 20 mL/min;
solvent A, water with 0.05 N HCl; solvent B, acetonitrile; for
intermediates: column, SunFire Prep C18 OBD, 5 μm, 19 mm × 50
mm; flow rate, 20 mL/min; solvent A, water with 0.1% formic acid;
solvent B, acetonitrile with 0.1% formic acid) unless otherwise noted.
Phenyl 3-Amino-2-(benzyloxy)-4-bromo-5-fluoro-6-methyl-
benzoate (3). To a solution of phenyl 3-amino-2-(benzyloxy)-5-
fluoro-6-methylbenzoate (2,9b 15.00 g, 42.69 mmol) and NaOAc
(10.50 g, 128.07 mmol, 3 equiv) in HOAc (100 mL) was added a
solution of Br2 (2.20 mL, 42.69 mmol, 1 equiv) in HOAc (10 mL)
dropwise via a syringe at 17 → 19 °C while cooled in a cold water
bath. After stirring at 20 °C for 20 min, more Br2 (66 μL) in HOAc (1
mL) was added. After stirring for 5 min, the reaction was poured into
ice/water. The resulting mixture was extracted with EtOAc (600 mL).
The organic phase was separated, washed with 10% aqueous Na2S2O3
solution, water, saturated aqueous sodium bicarbonate, and brine. The
organic phase was dried over sodium sulfate, filtered, and concentrated
J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
under reduced pressure. Flash chromatography on silica gel using 5%
→ 6% EtOAc/hexanes yielded the desired product 3 as a thick pale-
yellow oil (15.59 g, 85%). 1H NMR (400 MHz, CDCl3) δ 7.44−7.35
(m, 7 H), 7.28−7.25 (m, 1 H), 7.15−7.13 (m, 2 H), 5.01 (s, 2 H), 4.27
(br s, 2 H), 2.32 (d, J = 2.4 Hz, 3 H). MS (ESI) m/z calcd for
C21H18BrFNO3 [M + H]+ 430.05, 432.05; found 429.94, 431.92.
Phenyl 2-(Benzyloxy)-4-bromo-3-{[(tert-butoxy)carbonyl]-
(prop-2-en-1-yl)amino}-5-fluoro-6-methylbenzoate (4). To a
solution of aniline 3 (908 mg, 2.11 mmol) in anhydrous THF (8
mL) was added a solution of LHMDS in THF (4.43 mL, 1.0 M, 4.43
mmol, 2.1 equiv) dropwise over 7 min while maintaining the internal
temperature below −70 °C (dry ice−acetone bath). The reaction
solution was stirred at −78 °C for 15 min. A solution of Boc2O (484
mg, 2.22 mmol, 1.05 equiv) in THF (1 mL) was added dropwise while
maintaining the internal temperature below −71 °C. The reaction was
stirred at −78 °C for 30 min, and then the dry ice was removed from
the cold bath. The reaction was then warmed up to −50 °C, and allyl
bromide (0.20 mL, 2.32 mmol, 1.1 equiv) was added. The reaction was
warmed up to rt in 20 min and heated at 50 °C for 3 h. More allyl
bromide (0.20 mL, 2.32 mmol, 1.1 equiv) was added. The reaction was
heated at 50 °C for 2 h, cooled to rt, and diluted with EtOAc (40 mL).
The reaction solution was washed with saturated aqueous NH4Cl (2 ×
30 mL) and brine (30 mL), dried over sodium sulfate, filtered, and
concentrated under reduced pressure. Flash chromatography on silica
gel using 2% → 5% EtOAc/hexanes yielded the desired product 4
(1.06 g, 88%, ∼3:1 rotamers). 1H NMR (400 MHz, CDCl3) δ 7.39−
7.34 (m, 7 H), 7.29−7.25 (m, 1 H), 7.04−7.00 (m, 2 H), 6.00−5.90
(m, 1 H), 5.09−5.04 (m, 1 H), 5.03−5.00 (m, 2.25 H), 4.92 (d, J =
10.4 Hz, 0.75 H), 4.50 (dd, J = 6.1, 14.6 Hz, 0.75 H), 4.24 (dd, J = 6.1,
15.3 Hz, 0.25 H), 4.04−3.97 (m, 1 H), 2.42 (d, J = 2.4 Hz, 2.25 H),
2.40 (d, J = 2.4 Hz, 0.75 H), 1.54 (s, 2.25 H), 1.44 (s, 6.75 H). MS
(ESI) m/z calcd for C29H29BrFNO5Na [M + Na]+ 592.11, 594.11;
found 591.99, 593.98.
Phenyl 2-(Benzyloxy)-3-{[(tert-butoxy)carbonyl](prop-2-en-
1-yl)amino}-5-fluoro-4-formyl-6-methylbenzoate (5). To a
solution of bromide 4 (1.06 g, 1.86 mmol) in anhydrous THF (30
mL) was added a solution of n-BuLi in hexanes (1.16 mL, 1.6 M, 1.86
mmol, 1 equiv) dropwise while maintaining the internal temperature
below −100 °C (liquid nitrogen−ethanol bath). After stirring for 3
min, a solution of DMF (0.22 mL, 2.79 mmol, 1.5 equiv) in THF (1
mL) was added dropwise while maintaining the internal temperature
below −100 °C. The reaction solution was then allowed to warm up to
−78 °C (dry ice−acetone bath) and stirred at that temperature for 35
min. Saturated aqueous NH4Cl was added. The resulting mixture was
allowed to warm up to rt and extracted with EtOAc (40 mL). The
organic phase was washed with brine, dried over sodium sulfate,
filtered, and concentrated under reduced pressure. Flash chromatog-
raphy on silica gel using 3% → 12% EtOAc/hexanes yielded the
desired product 5 (0.91 g, 94%, ∼2:1 rotamers). 1H NMR (400 MHz,
CDCl3) δ 10.22 (s, 1 H), 7.38−7.33 (m, 7 H), 7.28−7.24 (m, 1 H),
7.02−6.99 (m, 2 H), 5.93−5.79 (m, 1 H), 5.04−4.96 (m, 3.35 H), 4.89
(d, J = 9.8 Hz, 0.65 H), 4.64 (dd, J = 5.5, 14.6 Hz, 0.65 H), 4.32 (dd, J
= 5.5, 14.6 Hz, 0.35 H), 3.97 (dd, J = 7.9, 14.6 Hz, 0.35 H), 3.90 (dd, J
= 8.5, 14.6 Hz, 0.65 H), 2.40 (d, J = 1.8 Hz, 2 H), 2.37 (d, J = 1.8 Hz, 1
H), 1.51 (s, 3 H), 1.36 (s, 6 H). MS (ESI) m/z calcd for
C30H30FNO6Na [M + Na]+ 542.20; found 542.1.
5-tert-Butyl 7-Phenyl 6-(Benzyloxy)-9-fluoro-8-methyl-1-
(prop-2-en-1-yl)-1H,2H,3H,3aH,4H,5H,9bH-pyrrolo[3,2-c]-
quinoline-5,7-dicarboxylate (6). A solution of aldehyde 5 (570 mg,
1.09 mmol) and N-allylglycine (338 mg, 2.23 mmol, 2 equiv) in DMF
(11 mL) was heated at 90 °C for 17 h. The reaction mixture was
cooled to rt, poured into water (125 mL), and extracted with EtOAc
(100 mL, 2 × 50 mL). The combined organic layers were washed with
water and brine, dried over anhydrous Na2SO4, filtered, and
concentrated under reduced pressure. The resulting oil was purified
via column chromatography (Biotage 100 g column, 4−40% EtOAc in
hexanes gradient), yielding 444 mg (71%) of the desired product 6 as a
clear oil (cis). 1H NMR (400 MHz, CDCl3) δ 7.40−7.21 (m, 8 H),
7.08−7.01 (m, 2 H), 5.79−5.64 (m, 1 H), 5.16−5.08 (m, 1 H), 5.01−
4.92 (m, 2 H), 4.85−4.75 (m, 1 H), 4.30−4.20 (m, 1 H), 3.80−3.77
F DOI: 10.1021/acs.jmedchem.5b00262
Article
(m, 1 H), 3.09−2.99 (m, 1 H), 2.85−2.54 (m, 2 H), 2.34 (s, 3 H),
2.20−2.09 (m, 2 H), 2.06−1.94 (m, 1 H), 2.45−2.22 (m, 10 H). MS
(ESI) m/z calcd for C34H36FN2O5 [M − H]− 571.26; found 571.14.
tert-Butyl (1R,19S,26S,27S)-14,22-Bis(benzyloxy)-19-[(tert-
butyldimethylsilyl)oxy]-26-(dimethylamino)-4-fluoro-18-hy-
droxy-16,20-dioxo-7-(prop-2-en-1-yl)-24-oxa-7,12,23-
triazaheptacyclo[15.11.0.03,15.05,13.06,10.019,27.021,25]octacosa-
3,5(13),14,17,21(25),22-hexaene-12-carboxylate (8). LDA (1.3
equiv) was prepared at −40 °C from n-butyllithium (1.6 M solution in
hexanes, 845 μL, 1.35 mmol) and diisopropylamine (216 μL, 1.50
mmol) in THF (10 mL). The LDA solution was cooled to −78 °C
(dry ice−acetone bath) and TMEDA (217 μL, 1.45 mmol, 1.4 equiv)
was added, followed by dropwise addition of a solution of compound 6
(655 mg, 1.14 mmol, 1.1 equiv) in THF (4.5 mL) with a 1.5 mL rinse.
This resulted in a deep-red solution. After 30 min, the reaction was
cooled to −100 °C (liquid nitrogen−ethanol bath), and a solution of
enone 7 (503 mg, 1.04 mmol) in THF (3.5 mL) was added dropwise,
maintaining the internal temperature below −95 °C. After complete
addition, the reaction was allowed to warm to −70 °C over 40 min,
and a solution of LHMDS (1 M in THF, 1.04 mL, 1.04 mmol, 1
equiv) was added. The reaction was allowed to warm to −20 °C over 1
h and quenched by the addition of NH4Cl (saturated aqueous
solution, 30 mL). The reaction mixture was extracted with EtOAc (2 ×
100 mL). The combined extracts were dried over Na2SO4, filtered, and
concentrated under reduced pressure. The resulting foam was purified
via column chromatography (Biotage 100 g column, 4−60% EtOAc in
hexanes), yielding 983 mg (98%, mixture of diastereomers) of the
desired product 8 as a yellow solid. MS (ESI) m/z calcd for
C54H64FN4O9 [M − H]− 959.44; found 959.28.
tert-Butyl (1R,19S,26S,27S)-14,22-Bis(benzyloxy)-19-[(tert-
butyldimethylsilyl)oxy]-26-(dimethylamino)-4-fluoro-18-hy-
droxy-16,20-dioxo-24-oxa-7,12,23-triazaheptacyclo-
[15.11.0.03,15.05,1306,10.019,27.021,25]octacosa-3,5(13),14,17,21-
(25),22-hexaene-12-carboxylate (9a and 9b). A solution of
compound 8 (945 mg, 0.983 mmol), N,N′-dimethylbarbituric acid
(1.53 g, 9.80 mmol, 10 equiv), and tetrakis(triphenylphosphine)-
palladium(0) (77.5 mg, 0.067 mmol, 0.068 equiv) in CH2Cl2 (10 mL)
was degassed by bubbling nitrogen for 5 min. The reaction flask was
fitted with a nitrogen-flushed water-cooled reflux condenser, and the
reaction was heated to 35 °C in an oil bath. After 4.5 h, the reaction
was cooled to rt and saturated aqueous NaHCO3 (20 mL) was added
slowly over 5 min, followed by the addition of pH 7 phosphate buffer
(30 mL). The aqueous layer was extracted with CH2Cl2 (2 × 75 mL),
and the combined organic layers were dried over Na2SO4, filtered, and
concentrated under reduced pressure. The crude material was purified
on a Waters Autopurification system equipped with a Sunfire Prep
C18 OBD column [5 μm, 19 mm × 50 mm; flow rate, 20 mL/min;
solvent A, H2O with 0.1% HCO2H; solvent B, CH3CN with 0.1%
HCO2H; gradient, 30 → 80% B; mass-directed fraction collection] to
provide the two diastereomers as yellow solids (9a, 291 mg; 9b, 284
mg; 9a + 9b, 31 mg; 67% total yield). 9a: 1H NMR (400 MHz,
CDCl3) δ 16.11−15.68 (br m, 1 H), 7.52−7.45 (m, 2 H), 7.41−7.25
(m, 8 H), 5.36 (s, 2 H), 4.90−4.65 (br m, 1 H), 4.58−4.38 (br m, 1
H), 4.28−4.04 (br m, 2 H), 3.94 (d, J = 10.38 Hz, 1 H), 3.25−3.14 (m,
1 H), 3.09−2.88 (m, 3 H), 2.62−2.29 (m, 10 H), 2.19−2.03 (m, 2 H),
1.65−1.43 (br m, 1 H), 1.42−1.35 (m, 10 H), 0.82 (s, 9 H), 0.28 (s, 3
H), 0.12 (s, 3 H); MS (ESI) m/z calcd for C51H62FN4O9Si [M + H]+
921.43, found 921.48. 9b: 1H NMR (400 MHz, CDCl3) δ 16.30−
15.79 (br m, 1 H), 7.52−7.44 (m, 2 H), 7.41−7.24 (m, 8 H), 5.36 (s, 2
H), 5.06−4.51 (br m, 2 H), 4.38−4.06 (br m, 2 H), 3.96 (d, J = 10.38
Hz, 1 H), 3.26−3.14 (m, 1 H), 3.12−2.86 (m, 3 H), 2.60−2.32 (m, 10
H), 2.18−2.02 (m, 2 H), 1.62−1.50 (br m, 1 H), 1.50−1.02 (br m, 10
H), 0.81 (s, 9 H), 0.27 (s, 3 H), 0.12 (s, 3 H); MS (ESI) m/z calcd for
C51H62FN4O9Si [M + H]+ 921.43, found 921.47.
(5S,9S,10S,12R)-9-(Dimethylamino)-15-fluoro-4,5,8,25-tetra-
hydroxy-18-(2-hydroxyethyl)-2,6-dioxo-18,23-diazahexacyclo-
[12.11.0.03,12.05,10.016,24,.017,21]pentacosa-1(25),3,7,14,16(24)-
pentaene-7-carboxamide (10e). To a solution of 9a (12.5 mg,
0.0135 mmol) in 1,2-dichloroethane (300 μL) was added (tert-
butyldimethylsilyloxy)acetaldehyde (90%, 14 μL, 0.068 mmol, 5
equiv), acetic acid (3.9 μL, 0.068 mmol, 5 equiv), and sodium
J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
triacetoxyborohydride (13 mg, 0.61 mmol, 5 equiv). After stirring at rt
for 18 h, the reaction was quenched with saturated aqueous NaHCO3
(10 mL). The reaction mixture was extracted with EtOAc (2 × 20
mL). The combined organic layers were dried over Na2SO4, filtered,
and concentrated under reduced pressure to provide the N8 alkylated
intermediate. MS (ESI) m/z calcd for C59H80FN4O10Si [M + H]+
1079.54; found 1079.23. Final deprotections using previously
described desilylation and hydrogenation procedures6 provided the
desired compound 10e as a yellow solid after reverse phase HPLC
purification (2.8 mg, HCl salt, 27% over three steps). 1H NMR (400
MHz, CD3OD) δ 4.98−4.95 (m, 1 H), 4.07 (s, 1 H), 3.93−3.84 (m, 1
H), 3.80−3.36 (m, 6 H), 3.20−2.64 (m, 11 H), 2.54−2.40 (m, 1 H),
2.30−2.08 (m, 3 H), 1.68−1.55 (m, 1 H). MS (ESI) m/z calcd for
C28H34FN4O8 [M + H]+ 573.24; found 573.12.
(5S,9S,10S,12R)-9-(Dimethylamino)-18-ethyl-15-fluoro-
4,5,8,25-tetrahydroxy-2,6-dioxo-18,23-diazahexacyclo-
[12.11.0.03,12.05,10.016,24.017,21]pentacosa-1(25),3,7,14,16(24)-
pentaene-7-carboxamide (10d). By similar procedures, compound
10d was prepared from 9a using acetaldehyde in the reductive
alkylation step. 1H NMR (400 MHz, CD3OD, HCl salt) δ 4.85 (d, J =
6.4 Hz, 1 H), 4.11 (s, 1 H), 3.72−3.66 (m, 1 H), 3.63−3.58 (m, 1 H),
3.46 (dd, J = 5.0, 12.8 Hz, 1 H), 3.42−3.33 (m, 2 H), 3.19−2.97 (m,
10 H), 2.80 (br s, 1 H), 2.54−2.45 (m, 1 H), 2.25−2.03 (m, 3 H),
1.64−1.54 (m, 1 H), 1.38 (t, J = 6.9 Hz, 3 H). MS (ESI) m/z calcd for
C28H34FN4O7 [M + H]+ 557.24; found 557.15.
(5S,9S,10S,12R)-9-(Dimethylamino)-15-fluoro-4,5,8,25-tetra-
hydroxy-2,6-dioxo-18,23-diazahexacyclo-
[12.11.0.03,12.05,10.016,24.017,21]pentacosa-1(25),3,7,14,16(24)-
pentaene-7-carboxamide (10a). Compound 9a was deprotected
using previously described procedures6 to provide compound 10a. 1H
NMR (400 MHz, CD3OD, HCl salt) δ 4.80 (d, J = 6.4 Hz, 1 H), 4.10
(s, 1 H), 3.49−3.40 (m, 3 H), 3.12−2.96 (m, 9 H), 2.89 (t, J = 11.9
Hz, 1 H), 2.79−2.71 (m, 1 H), 2.49−2.39 (m, 1 H), 2.24−2.16 (m, 2
H), 2.12−2.02 (m, 1 H), 1.65−1.56 (m, 1 H). MS (ESI) m/z calcd for
C26H30FN4O7 [M + H]+ 529.21; found 529.07.
The following analogues (10b, 10c, 10f−10h, and 11a−11c) were
prepared using similar procedures. See details in Supporting
Information.
(5S,9S,10S,12R)-9-(Dimethylamino)-15-fluoro-4,5,8,25-tetra-
hydroxy-18-methyl-2,6-dioxo-18,23-diazahexacyclo-
[12.11.0.03,12.05,10.016,24.017,21]pentacosa-1(25),3,7,14,16(24)-
pentaene-7-carboxamide. Diastereomer A, 10b: 1H NMR (400
MHz, CD3OD, HCl salt) δ 4.77 (d, J = 6.4 Hz, 1 H), 4.12 (s, 1 H),
3.79−3.74 (m, 1 H), 3.45 (dd, J = 5.5, 12.8 Hz, 1 H), 3.37−3.31 (m, 1
H), 3.14−2.97 (m, 13 H), 2.76−2.75 (m, 1 H), 2.58−2.49 (m, 1 H),
2.50−2.18 (m, 2 H), 2.07−2.00 (m, 1 H), 1.65−1.55 (m, 1 H); MS
(ESI) m/z calcd for C27H32FN4O7 [M + H]+ 543.23, found 543.07.
Diastereomer B, 10c: 1H NMR (400 MHz, CD3OD, HCl salt) δ 4.77
(d, J = 6.0 Hz, 1 H), 4.10 (s, 1 H), 3.79−3.73 (m, 1 H), 3.47 (dd, J =
5.5, 12.8 Hz, 1 H), 3.37−3.32 (m, 1 H), 3.12−2.97 (m, 13 H), 2.78 (br
s, 1 H), 2.56−2.51 (m, 1 H), 2.23 (br d, J = 10.1 Hz, 1 H), 2.14 (t, J =
14.2 Hz, 1 H), 2.05−1.98 (m, 1 H), 1.63−1.54 (m, 1 H); 13C NMR
(400 MHz, CD3OD, HCl salt) δ 194.96, 187.82, 174.60, 174.26,
153.09, 150.77, 147.47, 137.16, 115.57, 112.40, 109.68, 105.77, 96.12,
75.47, 70.75, 63.21, 54.89, 43.07, 42.03, 41.81, 40.97, 35.91, 35.27,
35.12, 33.88, 31.12, 27.20; MS (ESI) m/z calcd for C27H32FN4O7 [M
+ H]+ 543.23, found 543.07.
(5S,9S,10S,12R)-9-(Dimethylamino)-18-[2-(dimethylamino)-
ethyl]-15-fluoro-4,5,8,25-tetrahydroxy-2,6-dioxo-18,23-
diazahexacyclo[12.11.0.0 3,12 .0 5,10 .0 16,24 .0 17,21 ]pentacosa-1-
(25),3,7,14,16(24)-pentaene-7-carboxamide (10f). 1H NMR
(400 MHz, CD3OD, HCl salt) δ 5.00−4.94 (m, 1 H), 4.12−3.97
(m, 2 H), 3.90−3.75 (m, 2 H), 3.74−3.58 (m, 2 H), 3.52−3.37 (m, 2
H), 3.27−2.85 (m, 17 H), 2.84−2.78 (m, 1 H), 2.62−2.48 (m, 1 H),
2.26−2.04 (m, 3 H), 1.68−1.54 (m, 1 H). MS (ESI) m/z calcd for
C30H39FN5O7 [M + H]+ 600.29; found 600.09.
(5S,9S,10S,12R)-18-{2-[(2,2-Difluoroethyl)amino]ethyl}-9-(di-
methylamino)-15-fluoro-4,5,8,25-tetrahydroxy-2,6-dioxo-
18,23-diazahexacyclo[12.11.0.03,12.05,10.016,24.017,21]pentacosa-
1(25),3,7,14,16(24)-pentaene-7-carboxamide (10g). 1H NMR
(400 MHz, CD3OD, HCl salt) δ 6.35 (J = 2.4 Hz, J [19F−1H] = 53.7
G DOI: 10.1021/acs.jmedchem.5b00262
Article
Hz, 1 H), 4.98−4.92 (m, 1 H), 4.11−3.92 (m, 2 H), 3.91−3.79 (m, 1
H), 3.80−3.53 (m, 5 H), 3.51−3.39 (m, 2 H), 3.27−2.85 (m, 10 H),
2.83−2.67 (m, 1 H), 2.63−2.49 (m, 1 H), 2.26−2.04 (m, 3 H), 1.68−
1.53 (m, 1 H). MS (ESI) m/z calcd for C30H37F3N5O7 [M + H]+
636.27; found 636.19.
(5S,9S,10S,12R)-9-(Dimethylamino)-15-fluoro-4,5,8,25-tetra-
hydroxy-18-[2-(N-methylacetamido)ethyl]-2,6-dioxo-18,23-
diazahexacyclo[12.11.0.0 3,12 .0 5,10 .0 16,24 .0 17,21 ]pentacosa-1-
(25),3,7,14,16(24)-pentaene-7-carboxamide (10h). 1H NMR
(400 MHz, CD3OD, HCl salt) δ 4.07 (s, 1 H), 3.92−3.69 (m, 3
H), 3.68−3.62 (m, 1 H), 3.59−3.39 (m, 4 H), 3.14−2.91 (m, 13 H),
2.88−2.61 (m, 1 H), 2.56−2.43 (m, 1 H), 2.29−2.02 (m, 6 H), 1.67−
1.55 (m, 1 H). MS (ESI) m/z calcd for C31H39FN5O8 [M + H]+
628.27; found 628.11.
(5S,9S,10S,12R)-9-(Dimethylamino)-15-fluoro-4,5,8,25-tetra-
hydroxy-21-methyl-2,6-dioxo-18,23-diazahexacyclo-
[12.11.0.03,12.05,10.016,24.017,21]pentacosa-1(25),3,7,14,16(24)-
pentaene-7-carboxamide (11a). 1H NMR (400 MHz, CD3OD,
HCl salt) δ 4.31 (s, 1 H), 4.08 (s, 1 H), 3.47−3.40 (m, 2 H), 3.18−
3.11 (m, 2 H), 3.08−2.95 (m, 9 H), 2.23−2.13 (m, 4 H), 1.65−1.55
(m, 1 H), 1.15 (s, 3 H). MS (ESI) m/z calcd for C27H32FN4O7 [M +
H]+ 543.23; found 543.14.
(5S,9S,10S,12R)-9-(Dimethylamino)-15-fluoro-4,5,8,25-tetra-
hydroxy-18,21-dimethyl-2,6-dioxo-18,23-diazahexacyclo-
[12.11.0.03,12.05,10.016,24.017,21]pentacosa-1(25),3,7,14,16(24)-
pentaene-7-carboxamide (11b). 1H NMR (400 MHz, CD3OD,
HCl salt) δ 4.36 (s, 1 H), 4.08 (s, 1 H), 3.82−3.75 (m, 1 H), 3.41−
3.33 (m, 1 H), 3.22−3.11 (m, 3 H), 3.08−2.96 (m, 11 H), 2.34−2.18
(m, 3 H), 2.10−2.04 (m, 1 H), 1.65−1.56 (m, 1 H), 1.09 (s, 3 H). MS
(ESI) m/z calcd for C28H34FN4O7 [M + H]+ 557.24; found 557.13.
(5S,9S,10S,12R)-9-(Dimethylamino)-15-fluoro-4,5,8,25-tetra-
hydroxy-21-methyl-2,6-dioxo-18-propyl-18,23-
diazahexacyclo[12.11.0.0 3,12 .0 5,10 .0 16,24 .0 17,21 ]pentacosa-1-
(25),3,7,14,16(24)-pentaene-7-carboxamide (11c). 1H NMR
(400 MHz, CD3OD, HCl salt) δ 4.40 (s, 1 H), 4.10 (s, 1 H),
3.75−3.68 (m, 1 H), 3.49−3.42 (m, 1 H), 3.38−3.31 (m, 1 H), 3.24−
3.15 (m, 3 H), 3.12−3.07 (m, 1 H), 3.04−2.96 (m, 8 H), 2.30−2.18
(m, 3 H), 2.13−2.07 (m, 1 H), 1.83−1.75 (m, 1 H), 1.72−1.56 (m, 2
H), 1.08 (s, 3 H), 0.98 (t, J = 7.3 Hz, 3 H). MS (ESI) m/z calcd for
C30H38FN4O7 [M + H]+ 585.27; found 585.26.
Phenyl 4-Bromo-2-hydroxy-3-iodo-6-methyl-5-
(trifluoromethoxy)benzoate (13). To phenol 1213 (5.20 mmol,
obtained from treatment of 2.50 g of the corresponding benzyl ether
with TFA/anisole, containing inseparable impurities, ∼75% pure) in
toluene (20 mL) at rt was added NaH (0.83 g, 20.80 mmol, 60% in
mineral oil, 4 equiv) in small portions. The reaction mixture was
stirred at rt for 20 min. Iodine (5.28 g, 20.80 mmol, 4 equiv) was
added. The reaction mixture was stirred at rt overnight, diluted with
EtOAc (200 mL), washed with 1 N aqueous HCl (100 mL × 1), 5%
aqueous Na2S2O3 (100 mL × 2), and brine (100 mL × 1), dried over
sodium sulfate, and concentrated under reduced pressure to yield the
crude product 13 as a pale oil. MS (ESI) m/z calcd for C15H8BrF3IO4
[M − H]− 514.86; found 514.81.
Phenyl 2-(Benzyloxy)-4-bromo-3-iodo-6-methyl-5-
(trifluoromethoxy)benzoate (14). To the above crude phenol 13
(5.20 mmol) in DMF (10 mL) at rt was added potassium carbonate
(1.44 g, 10.44 mmol, 2 equiv) and benzyl bromide (0.74 mL, 6.23
mmol, 1.2 equiv). The reaction mixture was stirred at rt for 3 h, diluted
with EtOAc (200 mL), washed with water (200 mL × 1, 100 mL × 1)
and brine (50 mL × 1), dried over sodium sulfate, and concentrated
under reduced pressure. Flash column chromatography on silica gel
with 0−3% EtOAc/hexane yielded the desired product 14 as a pale oil
(3.48 g, ∼90% pure). 1H NMR (400 MHz, CDCl3) δ 7.55−7.00 (m,
10 H), 5.11 (s, 2 H), 2.44 (s, 3 H). MS (ESI) m/z calcd for
C22H14BrF3IO4 [M − H]− 604.91; found 604.83.
Phenyl 2-(Benzyloxy)-4-bromo-3-{[(tert-butoxy)carbonyl]-
amino}-6-methyl-5-(trifluoromethoxy)benzoate (15). To com-
pound 14 (5.20 mmol, 90% pure) was added cesium carbonate (2.54
g, 7.80 mmol, 1.5 equiv), BocNH2 (0.67 g, 5.70 mmol, 1.1 equiv),
Xantphos (1.20 g, 2.07 mmol, 0.4 equiv), Pd(OAc)2 (224 mg, 1.00
mmol, 0.2 equiv), and anhydrous 1,4-dioxane (10 mL). Nitrogen gas
J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
was bubbled through the mixture for 5 min. The reaction vessel was
sealed and heated at 80 °C for 48 h with vigorous stirring. After
cooling down to rt, water (100 mL) was added. The reaction mixture
was extracted with methylene chloride (100 mL × 1, 50 mL × 2). The
combined extracts were dried over sodium sulfate and concentrated
under reduced pressure. Flash column chromatography on silica gel
with 0−15% EtOAc/hexane yielded the desired product 15 as a white
solid (0.87 g, 28% overall yield). 1H NMR (400 MHz, CDCl3) δ
7.45−7.20 (m, 8 H), 7.03 (d, J = 7.3 Hz, 2 H), 6.07 (br s, 1 H), 5.03
(s, 2 H), 2.46 (s, 3 H), 1.46 (s, 9 H). MS (ESI) m/z calcd for
C27H24BrF3NO6 [M − H]− 594.01; found 594.01.
Phenyl 2-(Benzyloxy)-3-{[(tert-butoxy)carbonyl]amino}-4-
formyl-6-methyl-5-(trifluoromethoxy)benzoate (16). To com-
pound 15 (0.68 g, 1.14 mmol) in anhydrous THF (6 mL) at −78 °C
(dry ice−acetone bath) was added PhLi (0.95 mL, 1.80 M/n-Bu2O,
1.71 mmol, 1.5 equiv) dropwise over 1 min. After stirring at −78 °C
for 10 min, n-BuLi (0.86 mL, 1.60 M/hexanes, 1.38 mmol, 1.2 equiv)
was added dropwise over 2 min. The reaction was stirred at −78 °C
for 5 min. Dry DMF (0.26 mL, 3.36 mmol, 3 equiv) was added
dropwise. The reaction was stirred from −78 to 0 °C over 1 h and
quenched with saturated aqueous sodium bicarbonate (50 mL). The
reaction mixture was extracted with methylene chloride (50 mL × 3).
The combined extracts were dried over sodium sulfate and
concentrated under reduced pressure. Flash column chromatography
on silica gel with 0−15% EtOAc/hexane yielded the desired product
16 as a pale solid (232 mg, 37%). 1H NMR (400 MHz, CDCl3) δ
10.21 (s, 1 H), 7.90 (br s, 1 H), 7.45−7.20 (m, 8 H), 7.05 (d, J = 7.3
Hz, 2 H), 5.00 (s, 2 H), 2.42 (s, 3 H), 1.43 (s, 9 H). MS (ESI) m/z
calcd for C28H25F3NO7 [M − H]− 544.16; found 544.22.
Phenyl 2-(Benzyloxy)-3-{[(tert-butoxy)carbonyl](prop-2-en-
1-yl)amino}-4-formyl-6-methyl-5-(trifluoromethoxy)benzoate
(17). To compound 16 (232 mg, 0.43 mmol) in dry DMF (2 mL) at rt
was added NaH (21 mg, 60% in mineral oil, 0.52 mmol, 1.2 equiv).
After stirring at rt for 30 min, allyl bromide (56 μL, 0.64 mmol, 1.5
equiv) was added. The reaction mixture was stirred at rt for 1 h,
diluted with EtOAc (50 mL), washed with water (50 mL × 2) and
brine (50 mL × 1), dried over sodium sulfate, and concentrated under
reduced pressure. Flash column chromatography on silica gel with 0−
8% EtOAc/hexane yielded the desired product 17 as a pale oil (206
mg, 82%). 1H NMR (400 MHz, CDCl3) δ 10.16 (s, 1 H), 7.40−6.95
(m, 10 H), 5.95−5.75 (m, 1 H), 5.10−4.85 (m, 4 H), 4.64, 4.28 (dd,
dd, J = 5.5, 12.8 Hz, J = 4.9, 12.2 Hz, 1 H), 4.00, 3.89 (dd, dd, J = 8.1,
10.2 Hz, J = 8.6, 12.8 Hz, 1 H), 2.46, 2.43 (s, s, 3 H), 1.53, 1.50 (s, s, 9
H). MS (ESI) m/z calcd for C31H29F3NO7 [M − H]− 584.19; found
584.24.
5-tert-Butyl 7-Phenyl 6-(Benzyloxy)-1,8-dimethyl-9-(trifluor-
omethoxy)-1H,2H,3H,3aH,4H,5H,9bH-pyrrolo[3,2-c]quinoline-
5,7-dicarboxylate (18b). To compound 17 (206 mg, 0.35 mmol) in
DMF (2 mL) was added N-methyl glycine (47 mg, 0.53 mmol, 1.5
equiv). The reaction mixture was heated at 100 °C for 24 h. After
cooling to rt, the reaction mixture was diluted with EtOAc (50 mL),
washed with aqueous saturated sodium bicarbonate (50 mL × 2) and
brine (50 mL × 1), dried over sodium sulfate, and concentrated under
reduced pressure. Flash column chromatography on silica gel with 0−
15% EtOAc/hexane yielded the desired product 18b as a white foam
(190 mg, 89%). 1H NMR (400 MHz, CDCl3), broad and complex due
to the presence of various rotamers and/or conformers. MS (ESI) m/z
calcd for C33H36F3N2O6 [M + H]+ 613.25; found 613.36.
tert-Butyl (1R,19S,26S,27S)-14,22-Bis(benzyloxy)-19-[(tert-
butyldimethylsilyl)oxy]-26-(dimethylamino)-18-hydroxy-7-
methyl-16,20-dioxo-4-(trifluoromethoxy)-24-oxa-7,12,23-
triazaheptacyclo[15.11.0.03,15.05,13.06,10.019,27.021,25]octacosa-3-
(15),4,13,17,21(25),22-hexaene-12-carboxylate (19b). To diiso-
propylamine (51 μL, 0.36 mmol, 1.2 equiv) in anhydrous THF (1 mL)
at −78 °C (dry ice−acetone bath) was added n-BuLi (0.23 mL, 1.60
M/hexanes, 0.36 mmol, 1.2 equiv) dropwise. The reaction was stirred
at 0 °C for 10 min and cooled to −78 °C. TMEDA (56 μL, 0.36
mmol, 1.2 equiv) was added, followed by the addition of compound
18b (186 mg, 0.30 mmol) in anhydrous THF (2 mL) dropwise over 2
min. The resulting deep-red solution was stirred at −78 °C for 30 min.
LHMDS (0.36 mL, 1.0 M/THF, 0.36 mmol, 1.2 equiv) was added,
H DOI: 10.1021/acs.jmedchem.5b00262
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followed by the addition of enone 7 (147 mg, 0.30 mmol, 1 equiv) in
anhydrous THF (1 mL) dropwise over 1 min. The reaction was stirred
from −78 to 0 °C for 2 h and quenched with saturated aqueous
sodium bicarbonate (20 mL). The reaction mixture was extracted with
methylene chloride (20 mL × 3). The combined extracts were dried
over sodium sulfate and concentrated under reduced pressure. Flash
column chromatography on silica gel with 0−20% EtOAc/hexane
yielded the two diastereomers of the desired product (diastereomer A,
19ba, 100 mg, yellow foam, 33%; diastereomer B, 19bb, 60 mg, yellow
foam, 20%). MS (ESI) m/z calcd for C53H64F3N4O10Si [M + H]+
1001.44, found 1001.47.
(5S,9S,10S,12R)-9-(Dimethylamino)-4,5,8,25-tetrahydroxy-
18-methyl-2,6-dioxo-15-(trifluoromethoxy)-18,23-
diazahexacyclo[12.11.0.0 3,12 .0 5,10 .0 16,24 .0 17,21 ]pentacosa-1-
(25),3,7,14,16(24)-pentaene-7-carboxamide (20b). Compound
19ba (33 mg, 0.033 mmol) was deprotected according to previously
described desilylation and hydrogenation procedures6 to yield the
desired product 20b as an orange solid after reverse phase HPLC
purification (13 mg, HCl salt, 55% over 2 steps). 1H NMR (400 MHz,
CD3OD) δ 4.75 (d, J = 7.4 Hz, 1 H), 4.08 (s, 1 H), 3.75−3.65 (m, 1
H), 3.50−2.90 (m, 20 H), 2.57−2.47 (m, 1 H), 2.31−2.05 (m, 3 H),
1.68−1.57 (m, 1 H). 13C NMR (400 MHz, CD3OD) δ 194.95, 187.83,
174.92, 174.26, 150.52, 138.79, 137.70, 123.74, 122.26, 121.17, 115.99,
111.49, 109.63, 96.11, 75.48, 70.71, 63.64, 55.25, 43.03, 42.70, 41.97,
41.06, 36.35, 35.81, 35.13, 33.75, 30.38, 27.51. MS (ESI) m/z calcd for
C28H32F3N4O8 [M + H]+ 609.22; found 609.30.
The following compounds (20a, 20c−20f) were prepared similarly.
See experimental details in Supporting Information.
(5S,9S,10S,12R)-9-(Dimethylamino)-4,5,8,25-tetrahydroxy-
2,6-dioxo-15-(trifluoromethoxy)-18,23-diazahexacyclo-
[12.11.0.03,12.05,10.01624.017,21]pentacosa-1(25),3,7,14,16(24)-
pentaene-7-carboxamide (20a). 1H NMR (400 MHz, CD3OD,
HCl salt) δ 4.90−4.80 (m, 1 H), 4.09 (s, 1 H), 3.50−2.85 (m, 10 H),
2.82−2.74 (m, 1 H), 2.43−2.15 (m, 7 H), 2.15−2.05 (m, 1 H), 1.62
(dd, J = 10.6 Hz, 1 H). MS (ESI) m/z calcd for C27H30F3N4O8 [M +
H]+ 595.20; found 595.31.
(5S,9S,10S,12R)-9-(Dimethylamino)-4,5,8,25-tetrahydroxy-
18,19-dimethyl-2,6-dioxo-15-(trifluoromethoxy)-18,23-
diazahexacyclo[12.11.0.0 3,12 .0 5,10 .0 16,24 .0 17,21 ]pentacosa-1-
(25),3,7,14,16(24)-pentaene-7-carboxamide (20c). 1H NMR
(400 MHz, CD3OD, HCl salt) δ 4.85−4.84 (m, 1 H), 4.11 (s, 1
H), 3.67−3.63 (m, 1 H), 3.13−2.93 (m, 15 H), 2.76−2.72 (m, 1 H),
2.35−2.22 (m, 2 H), 1.88−1.83 (m, 1 H), 1.71−1.62 (m, 1 H),1.66 (d,
J = 11.2, 3 H). MS (ESI) m/z calcd for C29H34F3N4O8 [M + H]+
623.24; found 623.43.
(20S,24S,25S,27R)-24-(Dimethylamino)-15,19,20,23-tetrahy-
droxy-17,21-dioxo-2-(trifluoromethoxy)-5,13-diazaheptacyclo-
[14.12.0.03,14.04,11.05,9.018,27.020,25]octacosa-1,3(14),15,18,22-
pentaene-22-carboxamide (20d). 1H NMR (400 MHz, CD3OD,
HCl salt) δ 4.80 (d, J = 6.0, 1 H), 4.41−4.38 (m, 1 H), 4.13 (s, 1 H),
3.80−3.77 (m, 1 H), 3.66−3.60 (m, 1 H), 3.52−3.48 (m, 1 H), 3.16−
3.03 (m, 12 H), 2.42−2.21 (m, 6 H), 2.09−2.01 (m, 1 H), 1.96−1.94
(m, 1 H), 1.69−1.60 (m, 1 H). MS (ESI) m/z calcd for C30H34F3N4O8
[M + H]+ 635.24; found 635.17.
(21S,25S,26S,28R)-25-(Dimethylamino)-16,20,21,24-tetrahy-
droxy-18,22-dioxo-2-(trifluoromethoxy)-5,14-diazaheptacyclo-
[15.12.0.03,15.04,12.05,10.019,28.021,26]nonacosa-1,3(15),16,19,23-
pentaene-23-carboxamide (20e). 1H NMR (400 MHz, CD3OD,
HCl salt) δ 5.00 (d, J = 9.2, 1 H), 4.13 (s, 1 H), 3.73−3.70 (m, 1 H),
3.45−3.38 (m, 1 H), 3.29−3.26 (m, 1 H), 3.16−3.03 (m, 11 H), 2.49−
2.22 (m, 5 H), 1.97−1.79 (m, 5 H), 1.69−1.61 (m, 2 H). MS (ESI)
m/z calcd for C31H36F3N4O8 [M + H]+ 649.25; found 649.12.
(21S,25S,26S,28R)-25-(Dimethylamino)-16,20,21,24-tetrahy-
droxy-18,22-dioxo-2-(trifluoromethoxy)-8-oxa-5,14-
diazaheptacyclo[15.12.0.03,15.04,12.05,10.019,28.021,26]nonacosa-
1,3(15),16,19,23-pentaene-23-carboxamide (20f). 1H NMR
(400 MHz, CD3OD, HCl salt) δ 5.11 (d, J = 7.6, 1 H), 4.14 (s, 1
H), 4.07−4.04 (m, 1 H), 3.94−3.88 (m, 3 H), 3.70−2.90 (m, 8 H),
3.20−2.99 (m, 8 H), 2.66−2.59 (m, 1 H), 2.35−2.22 (m, 2 H), 1.68−
1.62 (m, 1 H). MS (ESI) m/z calcd for C30H34F3N4O9 [M + H]+
651.23; found 651.14.
J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
In Vitro Coupled P. aeruginosa Transcription/Translation
Assay. Antitranslational activity (IC50 values) was assessed in a P.
aeruginosa in vitro coupled transcription/translation assay (TnT) with
a firefly luciferase readout (catalogue no. L1020, Promega, Madison,
WI), as described by Fyfe et al.15 Reactions were run at a volume of 20
μL in Costar black 96-well assay plates (Costar catalogue no. 3915) for
60 min at 37 °C. The reaction was stopped by placing on ice for 5 min,
followed by addition of 25 μL of luciferase assay substrate (catalogue
no. E1500, Promega, Madison, WI). Plates were read on a LUMIStar
Optima (BMG Labtech, Ortenberg, Germany) with gain set to 3600,
0.2 s read, 0 s between wells. Percent luminescence was plotted against
inhibitor concentration with 50% inhibition versus untreated controls
marked as the IC50 value.
Susceptibility Testing. Compound stocks were prepared and
serially diluted in sterile deionized water. Minimal inhibitory
concentration (MIC) determinations were performed in liquid
medium in 96-well microtiter plates according to the methods
described by the Clinical and Laboratory Standards Institute (CLSI).16
Cation-adjusted Mueller Hinton broth was obtained from BBL
(catalogue no. 212322, Becton Dickinson, Sparks, MD), prepared
fresh and kept at 4 °C prior to testing. Lysed horse blood (catalogue
no. 205, Quad Five, Ryegate, Montana) was used to supplement
medium, as appropriate. All test methods met acceptable standards
based on recommended quality control ranges for all comparator
antibiotics and the appropriate ATCC quality control strains.
Immunocompetent Lung Infection Model. The mouse lung
infection model was performed at Vivisource, Waltham, MA. Female
BALB/c mice weighing 18−20 g were infected with ∼2 × 107 CFU/
mouse of P. aeruginosa PA1145, a cystic fibrosis isolate from Children’s
Hospital, Boston, via intranasal administration of 0.05 mL of cell
suspension under light anesthesia. One group did not receive drug
treatment, and lungs were harvested at 2 h postinfection. At 2 and 12 h
postinfection, mice were treated with compound 10c, meropenem, or
amikacin intravenously. Six mice per group were treated with each
drug concentration. Twenty-four h post initiation of treatment, mice
were euthanized by CO2 inhalation. The lungs of the mice were
aseptically removed, weighed, homogenized, serially diluted, and
plated on MacConkey medium. The plates were incubated overnight
at 37 °C in 5% CO2. CFU per gram of lung was calculated by
enumerating the plated colonies then adjusting for serial dilutions and
the weight of the lung. Individual animal CFU/g lung data was plotted
using GraphPad Prism.
Immunocompetent Thigh Infection Model. The mouse thigh
infection model was performed at University of North Texas Health
Science Center, Fort Worth, TX. Groups of five female specific-
pathogen-free CD-1 mice weighing 22 ± 2 g were used. On day 0,
animals were inoculated intramuscularly (0.1 mL/thigh) with 3−5 ×
106 CFU/mouse of P. aeruginosa PA694, a clinical isolate from
Eurofins-Medinet, into the right thigh. Two groups did not receive
drug treatment, and thighs were harvested at 2 and 24 h postinfection.
Remaining mice were administered intravenously with either vehicle,
compound 10c, or levofloxacin at 2 and 12 h postinfection. The
muscle of the right thigh of each animal was harvested at 24 h
postinfection. Harvested thigh tissues were homogenized in 2 mL of
PBS (pH 7.4) with a Polytron tissue homogenizer. The homogenates
were serially diluted and plated on Brain Heart Infusion agar + 0.5%
charcoal (w/v) for CFU determination per gram of thigh. Individual
animal CFU/gram thigh data was plotted using GraphPad Prism.
■
ASSOCIATED CONTENT
*
S Supporting Information
Detailed information on the synthesis and analytical data of
compounds 10b, 10c, 10f−10h, 11a−11c, 20a, and 20c−20f.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.5b00262.
I DOI: 10.1021/acs.jmedchem.5b00262
■
Article
AUTHOR INFORMATION
Corresponding Author
*Phone: 617-715-3553. Fax: 617-926-3557. E-mail: xyxiao@
tphase.com.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
We thank Drs. Andrew Myers, Eric Gordon, and Joaquim Trias
for valuable discussions during the course of this work. We also
thank Dr. Shu-hui Chen and his colleagues at WuXi Apptec for
external chemistry support.
■
ABBREVIATIONS USED
aq, aqueous; ATCC, American Type Culture Collection; CFU,
colony-forming units; CLSI, Clinical and Laboratory Standards
Institute; CRE, carbapenem-resistant Enterobacteriaceae; DMF,
N,N-dimethylformamide; HPLC, high-performance liquid
chromatography; IC50, half-minimum inhibitory concentration;
IV, intravenous; LDA, lithium diisopropylamide; LHMDS,
lithium hexamethydisilazide; MDR, multidrug-resistant; MIC,
minimum inhibitory concentration; MRSA, methicillin-resistant
Staphylococcus aureus; MS, mass spectrometry; NMR, nuclear
magnetic resonance; PBS, phosphate buffered saline; SAR,
structure−activity relationship; THF, tetrahydrofuran; TLC,
thin-layer chromatography; TMEDA, N,N,N′,N′-tetramethyle-
thylenediamine; TnT, coupled transcription/translation assay
■
REFERENCES
(1) Gupta, N.; Limbago, B. M.; Patel, J. B.; Kallen, A. J. Carbapenem-
resistant Enterobacteriaceae: Epidemiology and Prevention. Clin. Infect.
Dis. 2011, 53, 60−67.
(2) Mulligan, M. E.; Murray-Leisure, K. A.; Ribner, B. S.; Standiford,
H. C.; John, J. F.; Korvick, J. A.; Kauffman, C. A.; Yu, Y. L. Methicillin-
Resistant Staphylococcus aureus: A Consensus Review of the Micro-
biology, Pathogenesis, and Epidemiology with Implications for
Prevention and Management. Am. J. Med. 1993, 94, 313−328.
(3) Conover, L H. Discovery of Drugs from Microbiological Sources.
In Drug Discovery, Science and Development in a Changing Society;
American Chemical Society: Washington, DC, 1971; Vol. 108, pp 33−
80.
(4) (a) Chopra, I.; Roberts, M. Tetracycline Antibiotics: Model of
Action, Applications, Molecular Biology, and Epidemiology of
Bacterial Resistance. Microbiol. Mol. Biol. Rev. 2001, 65, 232−260.
(b) Levy, S. B. Evolution and Spread of Tetracycline Resistance
Determinants. J. Antimicrob. Chemother. 1989, 24, 1−3. (c) Lomov-
skaya, O.; Watkins, W. J. Efflux Pimps: Their Role in Antibacterial
Drug Discovery. Curr. Med. Chem. 2001, 8, 1699−1711.
(5) Sum, P.-E.; Lee, V. J.; Testa, R. T.; Hlavka, J. J.; Ellestad, G. A.;
Bloom, J. D.; Gluzman, Y.; Tally, F. P. Glycylcyclines. 1. A New
Generation of Potent Antibacterial Agents through Modification of 9-
Aminotetracyclines. J. Med. Chem. 1994, 37, 184−188.
(6) Xiao, X.-Y.; Hunt, D. K.; Zhou, J.; Clark, R. B.; Dunwoody, N.;
Fyfe, C.; Grossman, T. H.; O’Brien, W. J.; Plamondon, L.; Ronn, M.;
Sun, C.; Zhang, W.-Y.; Sutcliffe, J. A. Fluorocyclines. 1. 7-Fluoro-9-
pyrrolidinoacetamido-6-demethyl-6-deoxytetracycline: A Potent,
Broad Spectrum Antibacterial Agent. J. Med. Chem. 2012, 55, 597−
605.
(7) Solomkin, J. S.; Ramesh, M. K.; Cesnauskas, G.; Novikovs, N.;
Stefanova, P.; Sutcliffe, J. A.; Walpole, S. M.; Horn, P. T. Phase 2,
Randomized, Double-blind Study of the Efficacy and Safety of Two
Dose Regimens of Eravacycline versus Ertapenem for Adult
Community-Acquired Complicated Intra-abdominal Infections. Anti-
microb. Agents Chemother. 2014, 58, 1847−1854.
J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry Article

(8) (a) Sun, C.; Hunt, D. K.; Clark, R. B.; Lofland, D.; O’Brien, W. J.;
Plamondon, L.; Xiao, X.-Y. Synthesis and Antibacterial Activity of
Pentacyclines: A Novel Class of Tetracycline Analogs. J. Med. Chem.
2011, 54, 3704−3731. (b) Sun, C.; Wang, Q.; Brubaker, J. D.; Wright,
P. M.; Lerner, C. D.; Noson, K.; Charest, M.; Siegel, D. R.; Wang, Y.-
M.; Myers, A. G. A Robust Platform for the Synthesis of New
Tetracycline Antibiotics. J. Am. Chem. Soc. 2008, 130, 17913−17927.
(9) (a) Clark, R. B.; He, M.; Fyfe, C.; Lofland, D.; O’Brien, W. J.;
Plamondon, L.; Xiao, X.-Y. 8-Azatetracyclines: Synthesis and
Evaluation of a Novel Class of Tetracycline Antibacterial Agents. J.
Med. Chem. 2011, 54, 1511−1528. (b) Clark, R. B.; Hunt, D. K.; Me,
H.; Achorn, C.; Chen, C.-L.; Deng, D.; Fyfe, C.; Grossman, T. H.;
Hogan, P. C.; O’Brien, W. J.; Plamondon, L.; Ronn, M.; Sutcliffe, J. A.;
Zhu, Z.; Xiao, X.-Y. Fluorocyclines. 2. Optimization of the C-9 Side-
Chain for Antibacterial Activity and Oral Efficacy. J. Med. Chem. 2012,
55, 606−622. (c) Clark, R. B.; He, M.; Deng, D.; Sun, C.; Chen, C.-L.;
Hunt, D. K.; O’Brien, W. J.; Fyfe, C.; Grossman, T. H.; Sutcliffe, J. A.;
Achorn, C.; Hogan, P. C.; Katz, C. E.; Niu, J.; Zhang, W.-Y.; Zhu, Z.;
Ronn, M.; Xiao, X.-Y. Synthesis and Biological Evaluation of 8-
Aminomethyltetracycline Derivatives as Novel Antibacterial Agents. J.
Med. Chem. 2013, 56, 8112−8138.
(10) Charest, M. G.; Lerner, C. D.; Brubaker, J. D.; Siegel, D. R.;
Myers, A. G. A Convergent Enantioselective Route to Structurally
Diverse 6-Deoxytetracycline Antibiotics. Science 2005, 308, 395−398.
(11) Brubaker, J. D.; Myers, A. G. A Practical, Enantioselective
Synthetic Route to a Key Precursor to the Tetracycline Antibiotics.
Org. Lett. 2007, 9, 3523−3525.
(12) He, Y.; Mahmud, H.; Moningka, R.; Lovely, C. J.; Dias, H. V. R.
Cyclization Reactions of N-Acryloyl-2-aminobenzaldehyde Deriva-
tives: Formal Total Synthesis of Martinellic Acid. Tetrahedron 2006,
62, 8755−8769.
(13) Chen, C.-L.; Clark, R. B.; Deng, Y.; Plamondon, L.; Sun, C.;
Xiao, X.-Y. Tetracycline Analogs and Their Therapeutic Use against
Infections. PCT Int. Appl. WO 201202712, 2102.
(14) Muci, A. R.; Buchwald, S. L. Practical Palladium Catalysts for
C−N and C−O Bond Formation. Top. Curr. Chem. 2002, 219, 131−
209.
(15) Fyfe, C.; Sutcliffe, J. A.; Grossman, T. H. Development and
Characterization of a Pseudomonas aeruginosa in Vitro Coupled
Transcription−Translation Assay System for Evaluation of Translation
Inhibitors. J. Microbiol. Methods 2012, 90, 256−261.
(16) Methods for Dilution Antimicrobial Susceptibility for Bacteria That
Grow Aerobically: Approved Standard, 9th ed.; CLSI document M07-
A9E; Clinical and Laboratory Standards Institute (CLSI): 940 West
Valley Road, Suite 1400, Wayne, Pennsylvania, 2012; Vol. 32, no. 2.

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