8-azatetracyclines: Synthesis and evaluation of a novel class of tetracycline antibacterial agents

Article abstract of DOI:10.1021/jm1015389

A novel series of fully synthetic 8-azatetracyclines was prepared and evaluated for antibacterial activity. Compounds were identified that overcome both efflux (tet(K)) and ribosomal protection (tet(M)) tetracycline resistance mechanisms and are active against Gram-positive and Gram-negative organisms. Two compounds were identified that exhibit comparable efficacy to marketed tetracyclines in in vivo models of bacterial infection.

Full text of DOI:10.1021/jm1015389

ARTICLE
pubs.acs.org/jmc
8-Azatetracyclines: Synthesis and Evaluation of a Novel Class
of Tetracycline Antibacterial Agents
Roger B. Clark,* Minsheng He, Corey Fyfe, Denene Lofland, William J. O’Brien, Louis Plamondon,
Joyce A. Sutcliffe, and Xiao-Yi Xiao
Tetraphase Pharmaceuticals, Inc., 480 Arsenal Street, Watertown, Massachusetts 02472, United States
S Supporting Information
b
ABSTRACT: A novel series of fully synthetic 8-azatetracyclines
was prepared and evaluated for antibacterial activity. Compounds
were identified that overcome both efflux (tet(K)) and ribosomal
protection (tet(M)) tetracycline resistance mechanisms and are
active against Gram-positive and Gram-negative organisms. Two
compounds were identified that exhibit comparable efficacy to marketed tetracyclines in in vivo models of bacterial infection.
’ INTRODUCTION
In 2005, Myers and co-workers published a fully synthetic
route to the tetracyclines, the key step of which involves the
Michael-Dieckmann condensation of the AB precursor 8 with
structurally diverse D-ring precursors.15,16 This route provides
potential access to a broad range of tetracyclines that are inacces-
sible via traditional semisynthesis.17 One example of this is the
introduction of a nitrogen atom into the D-ring, resulting in
“azatetracyclines.” One 7-azatetracycline and one 9-azatetracycline
were disclosed in Myers original work, resulting in compounds
with only modest antibacterial activity. Herein we demonstrate
this powerful approach using pyridyl D-ring precursors 7 to
generate novel 8-azatetracyclines, 10 (Scheme 1).
The tetracycline class of antibacterial agents has seen wide-
spread clinical use for over 50 years due to its broad spectrum anti-
bacterial activity.1 Tetracyclines inhibit bacterial growth by prevent-
ing protein biosynthesis through binding to the 30S ribosome,
thereby blocking the binding of aminoacyl-tRNA to the acceptor
site.2 This class includes a number of naturally occurring com-
pounds, such as chlorotetracycline (1)3 and tetracycline (2),4 as
well as several semisynthetic analogues, such as doxycycline (3)5
and minocycline (4) (Figure 1).6 In addition to their use as anti-
biotics for serious infections, tetracyclines have also been pre-
scribed for use in acne and rosacea.7 As with many classes of anti-
biotics, widespread use of tetracyclines has led to resistance and
has limited their use in more complicated bacterial infections.8,9
Two major bacterial resistance mechanisms have been identi-
fied for tetracyclines: (1) active transport from bacterial cells via
efflux pumps10 and (2) ribosomal protection.11 The efflux pumps
are comprised of inner membrane tetracycline efflux proteins that
were first identified in Gram-negative organisms (tet(A-E))10a,10b
and were later found in Gram-positive bacteria (tet(K-L)).10c The
ribosomal protection mechanism (tet(M-O)) involves the expres-
sion of proteins that bind to the ribosome and allow protein
synthesis to proceed even in the presence of tetracyclines. The
exact mechanism by which this occurs remains unclear. Since
these early discoveries, the number of efflux and ribosomal protec-
tion mechanisms identified has increased.12
’ RESULTS AND DISCUSSION
Chemistry. To explore 7- and 9-substituted-8-azatetracyclines,
we required intermediates that could be readily modified either prior
to or after construction of the protected compound 9. Thus, the
halopyridines 15 and 16 were prepared according to Schemes 2
and 3. 3,5-Dichloroisonicotinic acid was treated with sodium
benzylate to give compound 12, which was esterified to the
phenyl ester 13 by conversion to the acid chloride, followed by
treatment with phenol and 4-(dimethylamino)pyridine (DMAP).
Suzuki coupling18 using methylboronic acid and PdCl2Cy2
catalyst19 gave the parent D-ring precursor 14 in good yield.
N-Oxide formation with hydrogen peroxide in acetic acid
followed by treatment with either phosphorousoxychloride or
phosphorousoxybromide gave 15a and 15b, respectively. For
15a, the reaction gave a 4.5:1.0 ratio of the desired regioi-
somer, while a 25:1 ratio was observed for 15b. Both desired
regioisomers were isolated in pure form by recrystallization.
The regiochemistry was assigned by conversion to the dimethyla-
mino compound 15c through a palladium-mediated amination.20
1H NMR studies indicated an nOe between the methyl
The presence of the strongly activating 10-hydroxy group
readily enables chemical modification of the tetracycline D-ring
at the 7- and 9-positions. This strategy led to the introduction of
the 7-dimethylamino group in compounds 4-6, all of which
have demonstrated improved activity in bacterial strains bear-
ing efflux pump resistance mechanisms. More recently, the
9-substituted glycylcyclines, such as tigecycline (5),13 and the
aminomethylcyclines, such as amadacycline (6),14 have shown
improved activity for resistant organisms bearing the ribosomal
protection mechanism.
Received: December 1, 2010
Published: February 08, 2011
r
2011 American Chemical Society
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Figure 1. Examples of naturally occurring (1 and 2) and semisynthetic (3-6) tetracyclines.
Scheme 1. Fully Synthetic Approach to the 8-Azatetracyclines
Scheme 2. Synthesis of D-Ring Precursors for 7-Substituted-
8-azatetracyclinesa
groups of the amine and the adjacent ring methyl group. The
methoxy derivative 15d was prepared from 15b by treatment
with sodium methoxide in 2-methylpyrrolidinone (NMP) at
120 °C. This gave the acid directly, which was re-esterified to the
phenyl ester.
The dihalo compounds 16a and 16b were prepared by N-
oxidation of 15a and 15b followed by treatment with POBr3
(Scheme 3). Yields were typically moderate (45-50%), with
reduction of the N-oxide to give the starting pyridines 15 observed
as major side reactions in both cases. Palladium-mediated coupling
of 16b with t-butylcarbamate gave a ∼2.5:1 mixture of regioi-
somers, which were readily separated after conversion to the free
amine 16c upon treatment with HCl. Treatment of 16c with
sodium nitrite in the presence of HF-pyridine gave the fluoro
derivative 16d in good yield.21 Similarly, a 2.4:1.0 ratio of
regioisomers was observed in the palladium-mediated reaction
of 16b with dimethylamine to give 16e and 16f. Interestingly, the
regioselectivity could be reversed by heating 16b in the presence of
dimethylamine without catalyst to give 16f as the major isomer in a
3:1 ratio.
a Reagents and conditions: (i) NaH (2 equiv), BnOH, NMP,
80 °C; (ii) (COCl)2, cat. DMF, CH2Cl2; (iii) PhOH, Et3N,
DMAP, CH2Cl2; (iv) CH3B(OH)2, PdCl2Cy2, K3PO4, to-
luene, water, 100 °C; (v) H2O2, AcOH, 80 °C; (vi) POCl3,
toluene, 100 °C; (vii) POBr3, toluene, 100 °C; (viii) dimethy-
lamine in THF, Pd2dba3, Xantphos, K3PO4, 1,4-dioxane,
100 °C (sealed); (ix) NaOMe, NMP, 100-120 °C.
The 9-amino D-ring precursors 18a and 18b were prepared
according to Scheme 4. Once again, palladium-mediated cou-
plings using t-butylcarbamate proved to be highly effective for
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Scheme 3. Synthesis of D-Ring Precursors for 7,9-Disubstituted-8-azatetracyclinesa
a Reagents and conditions: (i) for 15a, mCPBA, CH2Cl2; for 15b, H2O2, AcOH, 80 °C; (ii) POBr3, toluene, 100 °C; (iii) BocNH2, Pd2dba3, Xantphos,
Cs2CO3, 1,4-dioxane, 80 °C; (iv) HCl, 1,4-dioxane; (v) HF-pyridine, NaNO2, pyridine; (vi) dimethylamine in THF, Pd2dba3, Xantphos, Cs2CO3, 1,4-
dioxane, 60 °C (sealed).
Scheme 4. Synthesis of D-Ring Precursors for 9-Amino-8-azatetracyclinesa
a Reagents and conditions: (i) BocNH2, Pd2dba3, Xantphos, Cs2CO3, 1,4-dioxane, 80 °C; (ii) Boc2O, DMAP, DMF.
Scheme 5. Synthesis of 8-Azatetracyclinesa
a Reagents and conditions: (i) for 14, 15b-d, 16e, and 18a, LDA, TMEDA, -78 °C, THF then 8, -78 °C to -20 °C; (ii) for 16a, 16d, and 18b,
LHMDS, 8, THF, -78 °C to -20 °C; (iii) CH3B(OH)2 or PhB(OH)2, PdCl2dppf2-CH2Cl2, K3PO4, toluene, 1,4-dioxane, water, 100 °C; (iv) BocNH2,
Pd2dba3, Xantphos, K3PO4, 1,4-dioxane, 100 °C; (v) aqueous HF, CH3CN or 1,4-dioxane; (vi) 10% Pd/C, H2, HCl, MeOH, 1,4-dioxane.
introduction of the amino group. Further protection of the mono-
Boc intermediates 17 with Boc2O and DMAP in DMF gave the
di-Boc precursors 18.
Synthesis of the 8-azatetracyclines is outlined in Scheme 5.
Most of the D-ring precursors were lithiated with lithium
diisopropylamide (LDA) at -78 °C, followed by treatment with
the enone 8. Warming to -20 °C yielded the fully protected
tetracycline precursors 19. For D-ring precursors 16a, 16d,
and 18b, a one-pot reaction was performed in which lithium
bis(trimethylsilyl)amide (LHMDS) was added to a -78 °C
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Scheme 6. Synthesis of 9-Glycylamido-8-azatetracyclinesa
a Reagents and conditions: (i) HCl, 1,4-dioxane; (ii) bromoacetylchloride, THF then t-BuNH2, 50 °C; (iii) aqueous HF, CH3CN or 1,4-dioxane; (iv)
10% Pd/C, H2, HCl, MeOH, 1,4-dioxane.
Scheme 7. Synthesis of 7,9-Disubstituted-8-azatetracyclinesa
a Reagents and conditions: (i) R2R3NH2, Pd2dba3, Xantphos, K3PO4 or Cs2CO3, 1,4-dioxane, 100 °C or n-propylamine, CuI, 2-acetylcyclohexanone,
K3PO4, DMF, 100 °C; (ii) LHMDS, THF then Boc2O or CbzCl; (iii) LDA, TMEDA, -78 °C, THF then 8, -78 °C to -20 °C or LHMDS, 8, THF, -
78 °C to -20 °C; (iv) aqueous HF, CH3CN, or 1,4-dioxane; (v) 10% Pd/C, H2, HCl, MeOH, 1,4-dioxane; (vi) R2B(OH)2, (Ph3P)4Pd, K3PO4, toluene,
1,4-dioxane, H2O, 90 °C.
solution of the precursor and the enone 8 followed by
warming to -20 °C. Yields varied from 20 to 84% depending
on substrate, with larger reaction scales generally providing
better results. The LHMDS procedure also typically gave
higher yields but was only possible on the more highly electron
deficient D-ring precursors. The 7-bromo intermediate 19c was
further derivatized at this stage via Suzuki couplings with methyl-
boronic acid and phenylboronic acid to give 19d and 19e,
respectively. Similarly, palladium-mediated amination with 19h
and t-butylcarbamate provided the 7-chloro-9-amino compound
19j. Deprotection of the intermediates 19 with HF followed by
hydrogenation in the presence of palladium on carbon gave the
final 8-azatetracyclines 20a-k.
by excess t-butylamine gave the acylated intermediates, which
were deprotected under standard conditions to give the final
analogues 21a-c.
The bromopyridine intermediates proved to be useful for the
introduction of alkylamines via palladium-mediated aminations
and alkyl groups via Suzuki couplings, ultimately providing the
9-alkylamino-8-azatetracyclines 24 and 9-alkyl-8-azatetracyclines
26 (Scheme 7). Thus, 19h was treated with various amines in
the presence of Pd2dba3 and 9,9-dimethyl-4,5-bis-(diphenyl-
phosphino)xanthenes (Xantphos) in 1,4-dioxane at 100 °C.
Potassium phosphate was found to be the most effective base
for this transformation, with cesium carbonate and sodium t-
butoxide leading to rapid decomposition. Low yields were
generally observed due to decomposition of both the starting
material and products upon heating. Reaction times of 2-4 h
were eventually settled on as a balance between product forma-
tion and decomposition. Copper-catalyzed aminations were also
To compare the 8-azatetracyclines directly to tigecycline, the
9-glycylamido-8-azatetracyclines 21a-cwere prepared (Scheme 6).
The Boc groups of compounds 19j-lwere deprotected with HCl in
1,4-dioxane. Treatment with bromoacetylchloride in THF followed
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Table 1. In Vitro Antibacterial Activity of 7-Substituted-8-azatetracyclines
MIC (μg/mL)
S. pneumoniae
S. aureus
E. coli
K. pneumoniae
compd
R1
wild typea
tet(M)b
tet(K)c
wild typed
tet(M)c
wild typee
tet(A)c
wild typef
20a
20b
H
1
32
32
32
32
16
8
16
32
0.5
1
0.25
1
32
32
>32
4
0.5
2
>32
>32
>32
>32
>32
>32
>32
>32
8
1
Cl
1
4
20d
CH3
1
0.25
1
1
20e
Ph
0.063
0.031
0.125
0.063
1
0.031
0.063
0.063
0.016
0.25
0.5
0.125
0.25
0.25
2
2
20f
(CH3)2N-
CH3O-
F
2
8
0.25
1
20g
4
16
8
20i
8
8
0.5
4
tetracycline
minocycline
>32
16
32
0.25
32
8
0.125
<0.016
0.5
1
a ATCC 13709. b Obtained from Micromyx (Kalamazoo, MI). c Obtained from Marilyn Roberts’ lab at the University of Washington. d ATCC 49619.
e ATCC 25922. f ATCC 13883.
explored, but rapid decomposition of the starting material was
observed. Hindered amines generally gave low yields as did
reactions with the 7-dimethylamino compound 19m. Alter-
natively, the alkylamines could be introduced prior to the
Michael-Dieckmann reaction using either palladium or cop-
per-mediated aminations22 with 16a, 16d, and 16e. Protection
of the resulting aminopyridines upon treatment with LHMDS
followed by Boc2O or CbzCl gave the D-ring precursors 22.
Michael-Dieckmann reaction then gave the protected inter-
mediates 23. Suzuki reactions with 19h or 19m and various
boronic acids were also carried out in moderate yields,
providing the 9-alkyl intermediates 25. Deprotection of the
intermediates under standard conditions gave the final 8-aza-
tetracyclines 24 and 26.
Biology. The in vitro antibacterial activities of all analogues
were determined for a panel of Gram-positive (e.g., Staphylococcus
aureus and Streptococcus pneumoniae) and Gram-negative (e.g.,
Escherichia coli and Klebsiella pneumoniae) bacteria. The activities
of the 7-substituted-8-azatetracycline as well as several control
tetracyclines for a representative set of these organisms are found
in Table 1. The parent 8-azatetracycline 20a showed comparable
activity to tetracycline, with reasonable minimum inhibitory con-
centrations (MICs) of 0.25-1 μg/mL for the tetracycline-suscep-
tible strains. Lacking a substituent at either C-7 or C-9, 20a
demonstrated poor activity for the strains bearing tet(M), tet(K),
and tet(A). Overall, the 7-position appears to be very tolerant of
substitution, allowing for bulky (20e), electron withdrawing (20b
and 20i), and electron donating (20f and 20g) groups while
maintaining activity against the tetracycline-susceptible strains. As
with minocycline, introduction of the 7-dimethylamino group in
20f led to improved activities for most strains tested. Modest (1-2
dilution) improvements in MICs were observed for the tet(M)
strains, while a more significant improvement was seen for the
strain bearing thetet(K) resistance mechanism. With the exception
of the 7-chloro analogue, all of the 7-substituted-8-aza compounds
exhibited improved MICs for the tet(K) strain, correlating well
with literature reports on 7-substituted tetracyclines. Unfortunately,
all of the 8-azatetracyclines appear to be substrates for the tet(A)
efflux pump, exhibiting no antibacterial activity at the highest
concentration tested (32 μg/mL). In general, substitution at the
7-position appears to be inefficient at overcoming the ribosomal
protection resistance mechanism of the tet(M) strains. The only
compound with two dilution improvements for both tet(M)
strains was compound 20i, the 7-fluoro analogue. This com-
pound was also one of the more potent antibacterial compounds
in this series.
To make a direct comparison to tigecycline, the 9-glycylami-
do-8-azatetracyclines were prepared (Table 2). Whereas tigecy-
cline is a very potent antibacterial agent against all of the organisms
in our panel, including the four strains bearing tetracycline
resistance mechanisms, the corresponding 8-azatetracycline
analogues 21a-c were found to have poor overall activity.
The aniline precursors 20j and 20k, however, showed signifi-
cant improvements in MICs for both tet(M) strains relative to
the corresponding 9-unsubstituted compounds 20b and 20i.
For the 7-Cl analogue 20j, antibacterial activity also improved
for the tet(K) and wild type strains. These compounds were also
assessed for their ability to inhibit protein synthesis in a cell-free
transcription/translation assay (Table 3). In this assay, com-
pounds 20j and 20k were about 2-fold more potent than
tetracycline, in agreement with their relative MICs for E. coli.
Compounds 21a-c, on the other hand, inhibited protein
synthesis with IC50s between 0.42 and 0.90 μg/mL, similar to
tigecycline, despite having only modest antibacterial activity in
the whole-cell assays. These data suggest that the compounds
are active at the target, but that compounds 21a-c are not
capable of getting to the target in the whole-cell assay, possibly
due to poor cell permeability.
On the basis of the results of compounds 20j and 20k, a series
of 9-amino-8azatetracyclines was prepared (Table 4). Compounds
bearing a small, unbranched alkylamino group at the 9-position
(24a-c) proved to be very potent antibacterial agents against
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Table 2. In Vitro Antibacterial Activity of 9-Glycylamido-8-azatetracyclines
a ATCC 13709. b Obtained from Micromyx (Kalamazoo, MI). c Obtained from Marilyn Roberts’ lab at the University of Washington. d ATCC 49619.
e ATCC 25922. f ATCC 13883.
(24n) and 7-dimethylamino-9-n-propylamino (24o) analogues
were prepared as comparisons to 24c. While the 7-fluoro
compound had MICs that tracked fairly closely to the 7-chloro
analogue, the 7-dimethylamino compound exhibited no anti-
bacterial activity for any of the strains at the highest concen-
tration tested (32 μg/mL) with the exception of the tetracycline-
susceptible S. pneumoniae strain. All three of the compounds had
similar, albeit weak, activity in the transcription/translation assay,
indicating the differences in MICs might be due to poor perme-
ability of the 7-dimethylamino analogue. Introduction of a more
polar heteroatom into the side chain (24g-i,k) resulted in
improved antibacterial activity against the S. pneumoniae and
Gram-negative strains but had minimal effect on activity
toward either resistant S. aureus organisms. Interestingly, the
oxygen bearing side chains (24g and 24h) yielded significantly
improved antibacterial activity toward tetracycline-suscepti-
ble Gram-positive strains relative to the all carbon side chains
(24e and 24f), while the nitrogen analogues (24k and 24i)
were less potent against S. aureus and showed more modest
improvements against S. pneumoniae. The combination of a
branched side chain with a distal amino group gave the most
balanced antibacterial compounds in this series (24l, 24m, and
24j). These compounds combine good antibacterial activity
toward both Gram-positive and Gram-negative organisms, with
modest activity against tet(A) E. coli. Both compounds 24l and
24m are less potent than tigecycline but compare favorably to
amadacycline.
Table 3. Transcription/Translation (TnT) Assay Data
compd
E. colia MIC (μg/mL)
TnT IC50 (μg/mL)
20f
0.125
0.25
0.25
4
0.76
1.1
20j
20k
0.93
0.78
0.36
0.71
0.58
0.70
5.3
21a
21b
8
21c
32
24i
8
24l
0.5
4
24c
24n
2
2.8
24o
32
3.4
tetracycline
2
2.0
tigecycline
0.125
0.47
a ATCC 25922 is a tetracycline-susceptible strain.
the two tetracycline-susceptible Gram-positive strains, with MICs
between 0.016 and 0.25 μg/mL. A 4-fold improvement in activity
against the tet(M) S. aureus strain was observed for all of these
compounds relative to 20j, while MICs for the S. pneumoniae
tet(M) strain were largely unchanged. For the tet(K) S. aureus
strain, antibacterial activity improved with increasing size of the
alkyl group. Among the tetracycline-susceptible Gram-negative
strains, however, there was a clear loss in potency as the size of
the alkyl group increased, a trend that continued for the larger
branched alkyl compounds 24d-f. This is not surprising, as it is
known that Gram-negative antibacterial agents tend to be smaller
and more polar.23 These larger side chains also adversely affected
the antibacterial activity for S. pneumoniae while having less effect
on the MICs for the S. aureus strains. The 7-fluoro-9-n-propylamino
The antibacterial activity of the 9-alkyl and phenyl substituted
compounds were generally modest (Table 5). Here again, a
larger substituent at C-9 yielded improved activity for tet(M) and
tet(K) strains but resulted in loss of activity for wild type S.
pneumoniae and Gram-negative strains. Introduction of a 7-Cl or
7-dimethylamino group also gave improved activity for most of
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Table 4. In Vitro Antibacterial Activity of 9-Alkylamino-8-azatetracyclines
a ATCC 13709. b Obtained from Micromyx (Kalamazoo, MI). c Obtained from Marilyn Roberts’ lab at the University of Washington. d ATCC 49619.
e ATCC 25922. f ATCC 13883.
the strains in the panel. When comparing the 7-Cl-9-alkyl
compounds (26b) to the 7-Cl-9-aminoalkyl compounds (24c),
it is clear that the nitrogen confers significant improvements in
antibacterial activity. In the 7-dimethylamino case, however, the
7-dimethylamino-9-alkyl compound 26a is a balanced Gram-
positive antibacterial agent while the 9-aminoalkyl compound
24o has no significant activity.
Compounds 20f and 24l were selected for further profiling.
Both compounds inhibited protein synthesis in the transcrip-
tion/translation assay with IC50s comparable to tigecycline.
Susceptibility testing was performed, and MIC50 and MIC90
values were calculated for recent clinical isolates of S. aureus, S.
pneumoniae, Haemophilus influenzae, and E. coli (Table 6). The
panels contained both tetracycline-susceptible and resistant
strains, including 12methicillin-resistant S. aureus(MRSA) strains.
As expected from the initial MIC data, compound 20f had
very potent antibacterial activity for the tetracycline-suscep-
tible strains but performed poorly against the resistant
organisms. The compound did have MIC90s that were sig-
nificantly lower than tetracycline. Despite having higher
MICs in the primary screen relative to tigecycline, compound
24l performed similarly in the S. aureus, S. pneumoniae, and H.
influenzae panels, exhibiting clinically relevant MIC90s in each
case. These organisms are particularly relevant, as they are
responsible for infections in a large portion of community-
acquired bacterial pneumonia cases.
The pharmacokinetic profiles of the two 8-azatetracyclines
were determined in Sprague-Dawley rats (Table 7). Compound
20f had very similar AUC, clearance, and volume of distribution
profiles to both tetracycline and tigecycline, although the half-life
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Table 5. In Vitro Antibacterial Activity of 9-Alkyl-8-azatetracyclines
MIC (μg/mL)
S. pneumoniae
S. aureus
E. coli
K. pneumoniae
compd
R1
R2
wild typea
tet(M)b
tet(K)c
wild typed
tet(M)c
wild typee
tet(A)c
wild typef
26a
26b
26c
26d
26e
26f
(CH3)2N
n-Pr
n-Pr
CH3
CH3
Ph
1
1
2
4
1
4
2
4
1
4
1
2
4
16
>32
8
>32
>32
>32
>32
>32
>32
16
32
8
Cl
Cl
H
1
8
16
32
1
0.5
1
8
32
2
16
8
8
8
Cl
8
>32
16
>32
32
H
Ph
8
16
2
8
a ATCC 13709. b Obtained from Micromyx (Kalamazoo, MI). c Obtained from Marilyn Roberts’ lab at the University of Washington. d ATCC 49619.
e ATCC 25922. f ATCC 13883.
Table 6. MIC90 Analysis
species
MIC (μg/mL)
20f
24l
tigecycline
0.063
tetracycline
S. aureus (n = 31)
MIC50
e0.016
16
0.125
2
0.125
MIC90
1
>64
MIC range
e0.016-32
0.063-4
e0.016-1
0.063f64
S. pneumoniae (n = 19)
H. influenzae (n = 11)
E. coli (n = 20)
MIC50
2
e0.016
e0.016
32
MIC90
4
0.063
e0.016
>32
MIC range
e0.016-8
e0.016-0.063
e0.016-e0.016
0.125f32
MIC50
0.5
1
0.5
4
MIC90
2
1
0.5
16
MIC range
0.063-4
0.25-2
0.25-1
0.25-16
MIC50
0.25
1
0.25
2
MIC90
>32
4
0.5
>64
MIC range
e0.016-32
0.5-8
0.125-2
0.25f64
was somewhat shorter. Low oral bioavailability (%F) of 12.7%
was observed but was comparable to tetracycline (12.1%). In
humans, tetracycline is generally reported to have oral bioavail-
ability of 50-70%.24 We and others have found the oral
bioavailability of several tetracycline derivatives to be signifi-
cantly lower in rat than reported data in humans. Tigecycline,
administered only intravenously in humans, had very low oral
bioavailability. Compound 24l had a 4-fold higher AUC and
5-fold lower clearance than the other three compounds as well as
a significantly lower volume of distribution. Unfortunately, oral
bioavailability was comparable to tigecycline.
Table 7. Pharmacokinetic Properties of 20f and 24l in Spra-
gue-Dawley Ratsa
AUC
Clobs
Vzobs
compd
20f
(h ng/mL) (mL/h/kg) (mL/kg) t1/2 (hr)
%F
3
1087
4872
802
919
202
4177
1256
3676
6119
3.1
4.3
4.5
4.6
12.7
0.8
24l
tetracycline
1230
12.1
tigecycline
1052
929
1.1
a Compounds were dosed at 1 mg/kg IV and 10 mg/kg PO with sterile
water as vehicle.
The compounds were also investigated in mouse S. aureus and
E. coli septicemia models (Table 8). Both 8-azatetracyclines
performed well in the S. aureus model following IV administra-
tion, with PD50 values comparable to the two marketed control
tetracyclines. 20f, the most potent of the four compounds, also
provided the best protection, with 100% survival at the lowest
dose at which it was administered (0.30 mg/kg). Despite having
an 8-fold higher MIC, compound 24l provided equivalent
protection relative to tigecycline, likely due to its higher AUC
and lower clearance.25 The E. coli model proved to be more
challenging, requiring higher doses for protection. Compound
20f had a PD50 of 4.3 mg/kg compared to 2.1 mg/kg for
tigecycline. Compound 24l was 4-fold less potent than 20f and
was comparable to tetracycline in this model.
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Table 8. IV Efficacy in Mouse Septicemia Modela
S. aureus
E. coli
compd
20f
MIC (μg/mL)
PD50 (mg/kg)
95% C.I.
MIC (μg/mL)
PD50 (mg/kg)
95% CI
0.031
0.5
<0.30b
0.36
0.13
0.5
1
4.3
17
4.1-4.6
4.1-30
7.3-27
1.8-2.4
24l
0.36-0.56
0.34-0.37
0.24-0.47
tetracycline
tigecycline
0.25
0.35
17
0.063
0.35
0.13
2.1
a S. aureus ATCC 13709 (Smith) or E. coli ATCC 25922 was mixed with 5% mucin and inoculated by intraperitoneal injection at 2.1 ꢀ 106 cfu/mouse (S.
aureus) or 2.0 ꢀ 107 cfu/mouse (E. coli). One hour postchallenge, mice received IV treatment (vehicle = sterile water) with the test article at
concentrations ranging from 0.05-10 mg/kg (S. aureus) or 0.30-30 mg/kg (E. coli). The PD50 in mg/kg was calculated as survival after 48 h. b Lowest
dose was 0.30 mg/kg with 100% survival at all doses.
’ CONCLUSIONS
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 a Waters Alliance HPLC system
equippedwith abinary pump, a diodearraydetector, aWaters Sunfire C18
(5 μm, 4.6 mm I.D. ꢀ 50 mm) column, 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 method: (a) time = 0, 100% A;
(b) time = 0.5min, 100%A;(c) time= 3.5min, 100% B;(d) time= 5 min,
100% B (e) time = 6 min, 100% A; (f) time = 7 min, 100% A. All final
products were g95% purity as assessed by this method.
3-(Benzyloxy)-5-chloroisonicotinic Acid (12). Sodium hydride
(60% dispersion in mineral oil, 4.37 g, 109 mmol) was added portionwise
to a solution of 3,5-dichloroisonicotinic acid (10.2 g, 53.3 mmol) in NMP
(100 mL). After gas evolution ceased, benzyl alcohol (5.52 mL, 53.3 mmol)
was added dropwise. After gas evolution ceased, the reaction mixture was
heated to 80 °C. After 1 h, the reaction was complete and was allowed to
cool to room temperature and stand overnight. The reaction mixture was
diluted with water (300 mL) and was washed with Et2O (2 ꢀ 100 mL,
discarded). The aqueous layer was brought to pH ∼2 with conc HCl, causing
a precipitate to form. The mixture was diluted with brine (100 mL) and was
allowed to stand for 30 min. The solid was collected by filtration, was washed
with water (3ꢀ), and was dried in a 45 °C vacuum oven overnight. This gave
9.36 g (67%) of the title compound as a white solid. 1H NMR (400 MHz,
DMSO-d6) δ 14.30-14.10 (bs, 1 H), 8.52 (s, 1H), 8.34 (s, 1 H), 7.50-7.30
(m, 5 H), 5.33 (s, 2 H). MS (ESI) m/z 264.20 (M þ H).
A novel, fully synthetic series of 8-azatetracycline analo-
gues was prepared and optimized. Through modification of
the 7- and 9-positions, compounds were found that overcome
both tet(K) efflux and tet(M)-mediated ribosomal protection.
The 7-chloro-9-alkylamino-8-azatetracycline 24l exhibits
clinically relevant MIC90 profiles against both Gram-positive
and Gram-negative organisms that include these resistance
mechanisms. This compound also demonstrated in vivo efficacy
in murine models of infection that is equivalent to currently
marketed tetracycline antibacterial agents. The 7-dimethylami-
no-8-azatetracycline 20f was efficacious when administered
intravenously and had oral bioavailability in rat equivalent to
tetracycline.
The current work demonstrates the ability of this chem-
istry platform to generate antibacterial compounds that are
inaccessible through traditional semisynthesis, providing the
medicinal chemist a broader array of tools through which
essential antibacterial properties can be modulated. Impor-
tantly, this includes the ability to overcome ever evolving
resistance mechanisms and to identify structure-activity
relationships to deliver compounds with desired pharmaco-
kinetic properties.
’ EXPERIMENTAL PROCEDURES
General. Air and moisture sensitive liquids and reagents were trans-
ferred 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 FlashMaster Personal system using ISOLUTE Flash Si II silica
gel prepacked cartridges (available from Biotage). Preparative reversed-
phase HPLC chromatography (HPLC) was accomplished using a Waters
Autopurification system with mass-directed fraction collection. All inter-
mediate compounds were purified with a Waters Sunfire Prep C18 OBD
column (5 μm, 19 mm ꢀ 50 mm; flow rate = 20 mL/min) using a mobile
phase mixture of H2O (A) and CH3CN (B) containing 0.1% HCO2H.
The final 8-azatetracycline compounds were purified using a Phenomenex
Polymerx 10 μ RP 100A column (10 μm, 30 mm ꢀ 21.2 mm; flow rate =
20 mL/min) using a mobile phase mixture of 0.05N HCl in H2O (A) and
CH3CN (B). All final 8-azatetracycline compounds were isolated as
mono-, di-, or trihydrochloride salts following freeze-drying. 1H
NMR spectra were recorded on a JEOL ECX-400 (400 MHz) spectro-
meter and are reported in ppm using residual solvent as the internal
Phenyl 3-(Benzyloxy)-5-chloroisonicotinate (13). Oxalyl chlor-
ide (4.9 mL, 56 mmol) was added to a suspension of 12 (3.71 g, 14.1 mmol)
in CH2Cl2 (50 mL) over ∼2 min. DMF was added dropwise in ∼20 μL
portions every 5 min until complete solution was achieved. After stirring for
an additional 30 min, the reaction mixture was concentrated under reduced
pressure. The resulting solid was dissolved in CH2Cl2 (50 mL), and phenol
(2.65 g, 28.1 mmol), DMAP (0.172 g, 1.41 mmol), and Et3N (9.76 mL,
70.4 mmol) were added. After 1 h, the reaction mixture was diluted with
CH2Cl2 (50 mL) and was washed with water (2 ꢀ 100 mL) and NaHCO3
(saturated, aqueous, 100 mL). The organics were dried over Na2SO4,
filtered, and concentrated under reduced pressure. The material was
purified by column chromatography (Biotage 50 g column, 0-25%
EtOAc in hexanes gradient), yielding 4.20 g (88%) of the product as an
off-white solid. Rf = 0.43 in 30% EtOAc/hexanes. 1H NMR (400 MHz,
DMSO-d6) δ 8.70 (s, 1H), 8.48 (s, 1 H), 7.52-7.30 (m, 8 H), 7.15-
7.08 (m, 2 H), 5.44 (s, 2 H). MS (ESI) m/z 340.25 (M þ H).
Phenyl 3-(Benzyloxy)-5-methylisonicotinate (14). Compound
13 (2.01 g, 5.92 mmol), methyl boronic acid (1.06 g, 17.7 mmol),
dichlorobis(tricyclohexylphosphine)palladium(II) (102 mg, 0.296 mmol),
and K3PO4 (3.76 g, 17.7 mmol) were heated to 100 °C in toluene (20 mL)
and water (2 mL). After 16 h, the reaction mixture was allowed to cool to
room temperature and was diluted with EtOAc (20 mL). This was washed
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Journal of Medicinal Chemistry
ARTICLE
with water (20 mL) and brine (20 mL), dried over Na2SO4, filtered, and
concentrated under reduced pressure. The material was purified by column
chromatography (Biotage 50 g column, 0-40% EtOAc in hexanes
gradient), yielding 1.66 g (88%) of the product as a white solid. Rf = 0.18 in
30% EtOAc/hexanes. 1H NMR (400 MHz, DMSO-d6) δ 8.50 (s, 1H), 8.26
(s, 1 H), 7.52-7.30 (m, 8 H), 7.20-7.10 (m, 2 H), 5.37 (s, 2 H), 2.38 (s,
3 H). MS (ESI) m/z 320.27 (M þ H).
388 mg (40%) of the title compound as a white solid. 1H NMR (400 MHz,
CDCl3) δ 7.96 (s, 1H), 7.45-7.20 (m, 8 H), 7.08 (d, J = 8.2 Hz,
2 H), 5.16 (s, 2 H), 2.76 (s, 6 H), 2.37 (s, 3 H). MS (ESI) m/z 363.45
(M þ H).
Phenyl 5-(Benzyloxy)-2-methoxy-3-methylisonicotinate
(15d). Sodium methoxide (38 mg, 0.70 mmol) was added to a solution
of compound 15b (118 mg, 0.351 mmol) in NMP (2 mL), and the reaction
mixture was heated to 100 °C. After heating overnight, additional sodium
methoxide (57 mg, 1.1 mmol) was added and heating was continued at
100 °C. After 1 h, the reaction mixture was heated to 120 °C. After an
additional 1 h, the reaction mixture was cooled to room temperature, water
(20 mL) was added, and the pH was adjusted to 2 with 6 N aqueous HCl.
This was extracted with EtOAc (3 ꢀ 20 mL), and the combined extracts
were dried over Na2SO4, filtered, and concentrated under reduced pressure.
The crude acid intermediate was dissolved in CH2Cl2 (10 mL), and oxalyl
chloride (0.123 mL, 1.40 mmol) was added. After 1 h, the reaction mixture
was concentrated under reduced pressure. The material was dissolved
in CH2Cl2 (10 mL), and phenol (66 mg, 0.70 mmol), DMAP (4 mg,
0.04 mmol), and Et3N (0.245 mL, 1.76 mmol) were added. After 1 h,
the reaction mixture was diluted with EtOAc (50 mL) and was washed
with water (2 ꢀ 30 mL) and brine (30 mL). The organics were dried
over Na2SO4, filtered, and concentrated under reduced pressure. The
material was purified by column chromatography (Biotage 10 g column,
0-12% EtOAc in hexanes gradient), yielding 20.3 mg (17%) of clean
product. An additional 29.1 mg of product contaminated with some
residual phenol was also isolated. Rf = 0.39 in 20% EtOAc/hexanes.
1H NMR (400 MHz, CDCl3) δ 7.75 (s, 1H), 7.45-7.24 (m, 9 H),
7.10 (d, J = 7.3 Hz, 2 H), 5.13 (s, 2 H), 3.91 (s, 3 H), 2.67 (s, 3 H). MS
(ESI) m/z 336.1, 338.1 (M þ H).
Phenyl 5-(Benzyloxy)-2-chloro-3-methylisonicotinate
(15a). Compound 14 (9.66 g, 30.3 mmol) was heated to 80 °C in acetic
acid (50 mL) and hydrogen peroxide (30% aqueous solution, 17 mL). After
6 h, LC/MS indicated that the starting material was consumed and the
N-oxide present. Most of the acetic acid and water were removed under
reduced pressure. The material was then diluted with EtOAc (200 mL), and
the mixture was washed with NaHCO3 (saturated aqueous solution, 3 ꢀ
50 mL). The organics were dried over Na2SO4, filtered, and concentrated
under reduced pressure, yielding 9.37 g (92%) of the crude N-oxide. The
material was dissolved in toluene (60 mL), phosphorousoxychloride (7.69 g,
50.1 mmol) was added, and the reaction mixture was heated to 100 °C.
After 2 h, LC/MS indicated that the reaction was complete. Upon cooling to
room temperature, the reaction mixture was poured into a solution of
K2CO3 (40 g) in water (200 mL). This mixture was extracted with EtOAc
(3 ꢀ 150 mL), and the combined extracts were dried over Na2SO4, filtered,
and concentrated under reduced pressure. 1H NMR indicated a mixture of
regioisomers (4.5:1). The material was redissolved in EtOAc (30 mL) with
heating and was then allowed to cool to room temperature and stand
overnight. A white crystalline solid was obtained (4.99 g, 51%). Another
1.92 g (19%) of the white crystalline solid was obtained from the mother
liquor after standing in a refrigerator overnight. Both were the desired pure
regioisomer 15a by 1H NMR. 1H NMR (400 MHz, CDCl3) δ8.05 (s, 1H),
7.43-7.30 (m, 7 H), 7.29-7.20 (m, 1 H), 7.07 (d, J = 8.2 Hz, 2 H), 5.21 (s,
2 H), 2.46 (s, 3 H). MS (ESI) m/z 398.22, 400.22 (M þ H).
Phenyl 3-(Benzyloxy)-2-bromo-6-chloro-5-methylisonicoti-
nate (16a). Compound 15a (4.99 g, 14.1 mmol) was dissolved in
CH2Cl2 (30 mL), and 3-chloroperbenzoic acid (77%, 6.33 g, 28.2 mmol)
was added in one portion. After 6 h, an additional portion of 3-chloroper-
benzoic acid (3.15 g, 14.1 mmol) was added, and the reaction was stirred at
room temperature overnight. The reaction mixture was diluted with
CH2Cl2 (200 mL) and was washed with Na2CO3 (saturated aqueous
solution, 100 mL) and brine. The organics were dried over Na2SO4, filtered,
and concentrated under reduced pressure. The material was purified by
column chromatography (Biotage 70 g column, 5-50% EtOAc in hexanes
gradient), yielding 3.90 g (75%) of the pure N-oxide as an off-white
solid (Rf = 0.33 in 66% EtOAc/hexanes). The N-oxide was dissolved in
toluene (30 mL), phosphorousoxybromide (6.36 g, 22.1 mmol) was
added, and the reaction mixture was heated to 80 °C. After 1 h, the
reaction mixture was allowed to cool to room temperature and was
poured into a solution of K2CO3 (10 g) in water (50 mL). This mixture
was extracted with EtOAc (3 ꢀ 100 mL), and the combined extracts
were dried over Na2SO4, filtered, and concentrated under reduced pressure.
The material was purified by column chromatography (Biotage 50 g
column, 0-10% EtOAc in hexanes gradient), yielding 2.06 g (45%) of the
desired product 16a as an oil which slowly solidified on standing overnight.
In addition, 1.01 g (20%) of compound 15a was recovered. 1H NMR (400
MHz, CDCl3) δ 8.05 (s, 1H), 7.43-7.30 (m, 7 H), 7.29-7.20 (m, 1 H),
7.07 (d, J = 8.2 Hz, 2 H), 5.21 (s, 2 H), 2.46 (s, 3 H). MS (ESI) m/z 398.22,
400.22 (M þ H).
Phenyl 5-(Benzyloxy)-2-bromo-3-methylisonicotinate
(15b). Compound 14 (25.9 g, 81.1 mmol) was heated to 80 °C in
acetic acid (160 mL) and hydrogen peroxide (30% aqueous solution,
40 mL). After stirring overnight, LC/MS indicated that the starting
material was consumed and the N-oxide present. The reaction mixture was
concentrated under reduced pressure, was diluted with EtOAc, and was
washed with NaHCO3 (saturated aqueous solution, 3ꢀ). The organics
were dried over Na2SO4, filtered, and concentrated under reduced pressure,
yielding 25.4 g (93% crude) of the N-oxide as a tan solid. The material was
suspended in toluene (200 mL) and was heated to 80 °C, at which point
most of the solid had dissolved. Phosphorousoxybromide (25 g, 87 mmol)
was added in one portion. After 30 min, LC/MS indicated the reaction was
complete. Upon cooling to room temperature, the reaction mixture was
poured into a solution of K2CO3 (83 g) in water (300 mL). This was stirred
for 30 min at room temperature. The layers were separated, and the aqueous
layer was extracted with EtOAc. The combined extracts were dried over
MgSO4, filtered through Celite, and concentrated under reduced pressure.
The material was recrystallized from EtOAc/hexanes (1:1), yielding 20.6 g
(64%) of the product as an off-white solid. Single isomer by 1H NMR. 1H
NMR (400 MHz, CDCl3) δ 8.05 (s, 1H), 7.43-7.30 (m, 7 H), 7.29-7.20
(m, 1 H), 7.07 (d, J = 8.2 Hz, 2 H), 5.21 (s, 2 H), 2.46 (s, 3 H). MS (ESI)
m/z 398.22, 400.22 (M þ H).
Phenyl 5-(Benzyloxy)-2-(dimethylamino)-3-methylisoni-
cotinate (15c). Compound 15b (1.06 g, 2.66 mmol), dimethylamine
(2.0 M solution in THF, 4.0 mL, 8.0 mmol), K3PO4 (1.7 g, 8.0 mmol),
tris(dibenzylideneacetone)dipalladium (120 mg, 0.13 mmol), and Xant-
phos (220 mg, 0.40 mmol) were heated to 100 °C in 1,4-dioxane
(10 mL) in a sealed pressure vessel. After heating overnight, the reaction
mixture was cooled to room temperature, was diluted with EtOAc
(100 mL), and was washed with water (3 ꢀ 50 mL) and brine (50 mL).
The organics were dried over Na2SO4, filtered, and concentrated under
reduced pressure. The material was purified by column chromatography
(Biotage 50 g column, 0-15% EtOAc in hexanes gradient), yielding
Phenyl 3-(Benzyloxy)-2,6-dibromo-5-methylisonicotinate
(16b). Compound 15b (8.54 g, 21.4 mmol) was heated to 80 °C in
acetic acid (120 mL) and hydrogen peroxide (30% aqueous solution,
30 mL). After stirring overnight, LC/MS indicated that the starting material
was mostly consumed and the N-oxide present. The reaction mixture was
cooled to room temperature and was diluted with brine (150 mL). The
mixture was cooled in a -20 °C freezer for 30 min. The resulting precipitate
was collected by filtration and was washed with water/brine (1:1, 2ꢀ). The
solid was dissolved in CH2Cl2 and was dried over Na2SO4, filtered, and
concentrated under reduced pressure. This gave 7.83 g (88% crude) of the
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N-oxide as a tan solid. The material was suspended in toluene (200 mL) and
was heated to 100 °C, at which point most of the solid had dissolved.
A solution of phosphorousoxybromide (10.8 g, 37.8 mmol) in toluene
(10 mL) was added rapidly. After 45 min, the reaction mixture was allowed
to cool to room temperature and was poured into a solution of K2CO3
(40 g) in water (150 mL). After stirring for 30 min at room temperature, the
layers were separated and the aqueous layer was extracted with EtOAc.
The combined extracts were dried over MgSO4, filtered, and concen-
trated under reduced pressure. The material was purified by column
chromatography (Biotage 50 g column, 0-8% EtOAc in hexanes gradient
to isolate the product then 15% EtOAc in hexanes to recover 15b), yielding
5.02 g (49%) of the product as an off-white solid. Compound 15b was also
recovered. 1H NMR (400 MHz, DMSO-d6) δ 7.49-7.32 (m, 8 H), 7.10
(d, J = 7.3 Hz, 2 H), 5.15 (s, 2 H), 2.41 (s, 3 H). MS (ESI) m/z 476.18,
478.16, 480.16 (M þ H).
Phenyl 3-(Benzyloxy)-2-bromo-6-(dimethylamino)-5-methyli-
sonicotinate (16e) and Phenyl 3-(Benzyloxy)-6-bromo-
2-(dimethylamino)-5-methylisonicotinate (16f). Compound
16b (1.51 g, 3.16 mmol), Cs2CO3 (3.1 g, 9.5 mmol), tris(dibenzylide-
neacetone)dipalladium (145 mg, 0.158 mmol), and Xantphos (267 mg,
0.474 mmol) were weighed into a vial equipped with a septum. This was
evacuated and flushed with nitrogen (3ꢀ), and 1,4-dioxane (7 mL) and
dimethylamine (2.0 M solution in THF, 4.75 mL, 9.50 mmol) were
added. The reaction mixture was heated to 60 °C and was stirred for 4 h.
The reaction mixture was allowed to cool to room temperature, was
filtered through Celite, and was concentrated under reduced pressure.
The material was purified by column chromatography (Biotage 50 g
column, 0-10% EtOAc in hexanes gradient), yielding 868 mg (58%) of
the major regioisomer 16e and 354 mg (24%) of the minor regioisomer
16f. Data for 16e: 1H NMR (400 MHz, CDCl3) δ 7.50-7.46 (m, 2 H),
7.41-7.30 (m, 5 H), 7.30-7.24 (m, 1 H), 7.08-7.02 (m, 2 H), 5.08 (s,
2 H), 2.85 (s, 6 H), 2.32 (s, 3 H); MS (ESI) m/z 441.19, 443.18 (M þ H);
Rf = 0.29 in 10% EtOAc/hexanes. The regiochemistry was confirmed by an
NOE between the methyl protons and the N,N-dimethylamino protons
(0.61%). Data for 16f: 1H NMR (400 MHz, CDCl3) δ 7.41-7.31 (m,
7 H), 7.30-7.20 (m, 1 H), 7.01 (d, J = 7.3 Hz, 2 H), 4.94 (s, 2 H), 3.04 (s,
6 H), 2.33 (s, 3 H); MS (ESI) m/z 441.19, 443.18 (M þ H); Rf = 0.40 in
10% EtOAc/hexanes. The regiochemistry was confirmed by NOE between
the benzylic protons of the O-benzyl group and the N,N-dimethylamino
protons (0.44%).
Phenyl 3-(Benzyloxy)-2-bromo-6-[(t-butoxycarbonyl)-
amino]-5-methylisonicotinate. Compound 16b (2.15 g, 4.50 mmol),
t-butylcarbamate (633 mg, 5.40 mmol), Cs2CO3 (2.93 g, 9.00 mmol), tris-
(dibenzylideneacetone)dipalladium (206 mg, 0.225 mmol), and Xantphos
(380 mg, 0.673 mmol) were weighed into a flask. This was evacuated and
backflushed with nitrogen (3ꢀ), and 1,4-dioxane (15 mL) was added. The
reaction mixture was heated to 80 °C. After 3 h, the reaction mixture was
cooled to room temperature, was diluted with EtOAc (100 mL), and was
washed with water (2 ꢀ 50 mL). The organics were dried over Na2SO4,
filtered, and concentrated under reduced pressure. The material was purified
by column chromatography (Biotage 50 g column, 0-12% EtOAc in
hexanes gradient), yielding 1.43 g (62%) of a 4:1 mixture of the two
regioisomeric compounds. 1H NMR (400 MHz, CDCl3) δ7.51-7.46 (m, 2
H), 7.42-7.32 (m, 7 H), 7.30-7.24 (m, 1 H), 7.12 (d, J = 8.2 Hz, 0.5 H),
7.03 (d, J= 8.2 Hz, 2 H), 6.88 (s, 0.25 H), 6.71 (s, 1 H), 5.12 (s, 2 H), 5.02 (s,
0.5 H), 2.43 (s, 0.75 H), 2.34 (s, 3 H), 1.50 (s, 9 H), 1.47 (s, 2.25 H). MS
(ESI) m/z 535.10, 537.10 (M þ Na).
Phenyl 5-Benzyloxy-6-[(t-butoxycarbonyl)amino]-2-fluoro-3-
methylisonicotinate (17a). Compound 16d (538 mg, 1.29 mmol),
t-butylcarbamate (302 mg, 2.58 mmol), Cs2CO3 (840 mg, 2.58 mmol),
tris(dibenzylideneacetone)dipalladium (59 mg, 0.065 mmol), and
Xantphos (109 mg, 0.190 mmol) were weighed into a flask. This was
evacuated and backflushed with nitrogen (3ꢀ), and 1,4-dioxane (3 mL)
was added. The reaction mixture was heated to 80 °C. After 4 h, the
reaction mixture was cooled to room temperature, was diluted with
EtOAc (50 mL), and was washed with water (2 ꢀ 25 mL). The organics
were dried over Na2SO4, filtered, and concentrated under reduced
pressure. The material was purified by column chromatography (Biotage
20 g column, 0-12% EtOAc in hexanes gradient), yielding 451 mg
(77%) of the product. Rf = 0.35 in 20% EtOAc/hexanes. 1H NMR (400
MHz, CDCl3) δ 7.42-7.24 (m, 8 H), 7.04-6.99 (m, 2 H), 4.99 (s, 2 H),
2.38 (s, 3 H), 1.40 (s, 18 H). MS (ESI) m/z 453.14 (M þ H).
Phenyl 2-Amino-5-(benzyloxy)-6-bromo-3-methylisoni-
cotinate (16c). The above regioisomeric mixture (1.43 g, 2.78 mmol)
was stirred in 4 M HCl in 1,4-dioxane (20 mL) and 1,4-dioxane (5 mL)
overnight. The reaction mixture was diluted with EtOAc (100 mL) and
was washed with NaHCO3 (saturated aqueous solution, 2 ꢀ 100 mL).
The organics were dried over Na2SO4, filtered, and concentrated under
reduced pressure. The material was purified by column chromatography
(Biotage 20 g column, 0-30% EtOAc in hexanes gradient), yielding
805 mg (70%) of the major regioisomer 16c and 270 mg (24%) of the
minor regioisomer. Data for 16c: Rf = 0.39 in 40% EtOAc/hexanes. 1H
NMR (400 MHz, CDCl3) δ 7.50-7.42 (m, 2 H), 7.40-7.30 (m, 5 H),
7.30-7.24 (m, 1 H), 7.04 (d, J = 8.2 Hz, 2 H), 5.06 (s, 2 H), 4.56 (s, 2 H),
2.16 (s, 3 H). MS (ESI) m/z 413.11, 415.01 (M þ H). Data for phenyl
2-amino-3-(benzyloxy)-6-bromo-5-methylisonicotinate: Rf = 0.72 in
Phenyl 3-Benzyloxy-2-[bis(t-butoxycarbonyl)amino]-
6-fluoro-5-methylisonicotinate (18a). Compound 17a (451 mg,
1.00 mmol) was treated with di-t-butyldicarbonate (1.09 g, 5.00 mmol)
and DMAP (24 mg, 0.20 mmol) in DMF (15 mL). After 30 min, the
reaction mixture was diluted with EtOAc (100 mL) and was washed with
water (3 ꢀ 50 mL) and brine (50 mL). The organics were dried over
Na2SO4, filtered, and concentrated under reduced pressure. The material
was purified by column chromatography (Biotage 10 g column, 0-8%
EtOAc in hexanes gradient), yielding 481 mg (87%) of the title
1
40% EtOAc/hexanes; H NMR (400 MHz, CDCl3) δ 7.42-7.34 (m,
8 H), 7.13-7.10 (m, 2 H), 5.01 (s, 2 H), 4.64 (s, 2 H), 2.35 (s, 3 H); MS
(ESI) m/z 413.09, 415.09 (M þ H).
1
Phenyl 3-(Benzyloxy)-2-bromo-6-fluoro-5-methylisonico-
tinate (16d). HF-Pyridine solution (∼70% HF, 2 mL) was added to a
0 °C solution of compound 16c (884 mg, 2.14 mmol) in pyridine (1 mL).
Sodium nitrite (177 mg, 2.57 mmol) was added (bubbling observed),
and the reaction mixture was allowed to slowly warm to room temperature.
After 3 days, the reaction mixture was poured into Na2CO3 (saturated
aqueous solution, 30 mL) and was extracted with EtOAc (3 ꢀ 30 mL). The
combined extracts were washed with HCl (1 N aqueous solution, 2 ꢀ 50
mL) and were dried over Na2SO4, filtered, and concentrated under reduced
pressure. The material was purified by column chromatography (Biotage 20
g column, 0-6% EtOAc in hexanes gradient), yielding 687 mg (77%) of the
product as a colorless oil that slowly solidified on standing. Rf = 0.39 in 10%
compound. Rf = 0.16 in 10% EtOAc/hexanes. H NMR (400 MHz,
CDCl3) δ7.42-7.24(m, 8 H), 7.04-6.99 (m, 2 H), 4.99 (s, 2 H), 2.38 (s,
3 H), 1.40 (s, 18 H). MS (ESI) m/z 575.25 (M þ Na).
Phenyl 5-Benzyloxy-6-[(t-butoxycarbonyl)amino]-2-(di-
methylamino)-3-methylisonicotinate (17b). Compound 16e
(467 mg, 1.06 mmol), t-butylcarbamate (372 mg, 3.17 mmol), Cs2CO3
(1.04 g, 3.17 mmol), tris(dibenzylideneacetone)dipalladium (48 mg,
0.053 mmol), and Xantphos (89.3 mg, 0.159 mmol) were weighed
into a vial. This was evacuated and backflushed with nitrogen (3ꢀ),
and 1,4-dioxane (3 mL) was added. The reaction mixture was heated
to 100 °C. After 4 h, the reaction mixture was cooled to room
temperature and was filtered through Celite. The filtrate was con-
centrated under reduced pressure, and the material was purified by
column chromatography (Biotage 20 g column, 0-16% EtOAc in
hexanes gradient), yielding 493 mg (98%) of the product containing
1
EtOAc/hexanes. H NMR (400 MHz, CDCl3) δ 7.50-7.42 (m, 2 H),
7.40-7.30 (m, 5 H), 7.30-7.24 (m, 1 H), 7.08-7.02 (m, 2 H), 5.14 (s, 2
H), 2.33 (s, 3 H). MS (ESI) m/z 416.17, 418.15 (M þ H).
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Journal of Medicinal Chemistry
ARTICLE
∼20% of an unidentified impurity. Rf = 0.45 in 30% EtOAc/hexanes.
1H NMR (400 MHz, CDCl3) δ 7.43-7.32 (m, 8 H), 7.30-7.24 (m,
1 H), 7.15-7.10 (m, 2 H), 6.80 (s, 1 H), 4.94 (s, 2 H), 2.85 (s, 6 H),
2.31 (s, 3 H), 1.48 (s, 9 H). MS (ESI) m/z 478.27 (M þ H).
5.17 (q, J = 11.0 Hz, 2 H), 4.02 (d, J = 11.0 Hz, 1 H), 3.05 (dd, J = 17.2 Hz,
J = 11.6 Hz, 1 H), 2.98-2.86 (m, 1 H), 2.73 (s, 6 H), 2.62-2.38 (m, 9 H),
2.12 (d, J = 13.4 Hz, 1 H), 0.81 (s, 9 H), 0.26 (s, 3 H), 0.13 (s, 3 H). MS
(ESI) m/z 751.79 (M þ H).
Phenyl 3-Benzyloxy-2-[bis(t-butoxycarbonyl)amino]-6-(di-
methylamino)-5-methylisonicotinate (18b). Compound 17b
(490 mg, 1.03 mmol) was treated with di-t-butyldicarbonate (672 mg,
3.08 mmol) and DMAP (12.5 mg, 0.103 mmol) in DMF (5 mL). After
stirring overnight, the reaction mixture was diluted with EtOAc (25 mL)
and was washed with water (3 ꢀ 20 mL) and brine (20 mL). The organics
were dried over Na2SO4, filtered, and concentrated under reduced pressure.
The material was purified by column chromatography (Biotage 10 g
column, 0-10% EtOAc in hexanes gradient), yielding 424 mg (72%)
of the product. Rf = 0.22 in 15% EtOAc/hexanes. 1H NMR (400 MHz,
CDCl3) δ 7.42-7.24 (m, 8 H), 7.06-7.00 (m, 2 H), 4.92 (s, 2 H), 2.79
(s, 6 H), 2.36 (s, 3 H), 1.39 (s, 18 H). MS (ESI) m/z 578.33 (M þ H).
(4aS,11aR,12aS,13S)-13-(Dimethylamino)-4a-[[(1,1-dimethyl-
ethyl)dimethylsilyl]oxy]-11a,12,12a,13-tetrahydro-5-hydro-
xy-3,7-bis(phenylmethoxy)-isoxazolo[50,40:6,7]naphth[2,3-g]-
isoquinoline-4,6(4aH,11H)-dione (19a). LDA was prepared
at -78 °C from n-butyllithium (1.6 M solution in hexane, 0.495 mL,
0.792 mmol) and diisopropylamine (0.112 mL, 0.792 mmol) in THF
(5 mL). TMEDA (0.318 mL, 2.11 mmol) was added, followed by
dropwise addition of a solution of compound 14 (169 mg, 0.529
mmol) in THF (2 mL). This resulted in a deep-red colored solution.
After 5 min, a solution of 8 (128 mg, 0.264 mmol) in THF (2 mL)
was added. After complete addition, the reaction mixture was
allowed to warm to -20 °C over 1 h. The reaction was quenched
by the addition of ammonium chloride (saturated, aqueous solution,
20 mL) and was extracted with EtOAc (2 ꢀ 20 mL). The combined
extracts were driedoverNa2SO4, filtered, andconcentratedunder reduced
pressure. The material was purified by preparative HPLC (Sunfire Prep
C18 column, 80-100% B gradient), yielding 65.5 mg (35%) of the title
compound as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 15.77 (s,
1 H), 8.36 (s, 1 H), 8.16 (s, 1 H), 7.55-7.24 (m, 10 H), 5.40-5.25 (m,
4 H), 3.93 (d, J = 11.0 Hz, 1 H), 3.16-3.04 (m, 1 H), 2.98-2.90 (m, 1 H),
2.76-2.64 (m, 1 H), 2.60-2.40(m, 8 H), 2.12 (d, J = 14 Hz, 1 H), 0.82(s,
9 H), 0.26 (s, 3 H), 0.13 (s, 3 H). MS (ESI) m/z 708.72 (M þ H).
The following compounds were synthesized by methods similar to 19a.
(4aS,11aR,12aS,13S)-10-Chloro-13-(dimethylamino)-4a-
[[(1,1-dimethylethyl)dimethylsilyl]oxy]-11a,12,12a,13-tet-
rahydro-5-hydroxy-3,7-bis(phenylmethoxy)-isoxazolo-
[50,40:6,7]naphth[2,3-g]isoquinoline-4,6(4aH,11H)-dione
(19b). Prepared from 15a in 38% yield, yellow solid. 1H NMR(400 MHz,
CDCl3) δ 15.6 (s, 1 H), 8.12 (s, 1 H), 7.56-7.49 (m, 4 H), 7.42-7.32 (m,
6 H), 5.37 (s, 2 H), 5.07 (s, 2 H), 3.91 (d, J = 11.0 Hz, 1 H), 3.13-3.02 (m,
1 H), 2.97 (dd, J = 15.6 4.9 Hz, 1 H), 2.65-2.45 (m, 3 H), 2.50 (s, 6 H),
2.14 (d, J = 15.6 Hz, 1 H), 0.82 (s, 9 H), 0.27 (s, 3 H), 0.13 (s, 3 H). MS
(ESI) m/z 742.56 (M þ H).
(4aS,11aR,12aS,13S)-13-(Dimethylamino)-4a-[[(1,1-di-
methylethyl)dimethylsilyl]oxy]-11a,12,12a,13-tetrahydro-
5-hydroxy-10-methoxy-3,7-bis(phenylmethoxy)-isoxazolo-
[50,40:6,7]naphth[2,3-g]isoquinoline-4,6(4aH,11H)-dione
(19g). Prepared from 15d in 27% yield, yellow solid. 1H NMR (400 MHz,
CDCl3) δ15.70 (s, 1 H), 7.83 (s, 1 H), 7.55-7.24 (m, 10 H), 5.36 (s, 2 H),
5.17 (q, J = 13.4 Hz, 2 H), 3.98 (d, J = 11.0 Hz, 1 H), 3.72 (s, 3 H), 3.18 (d,
J = 16.5 Hz, 1 H), 3.04 -2.96 (m, 1 H), 2.60-2.30 (m, 9 H), 2.15 (d, J =
14 Hz, 1 H), 0.82 (s, 9 H), 0.26 (s, 3 H), 0.13 (s, 3 H). MS (ESI) m/z
738.66 (M þ H).
2-[(4aS,11aR,12aS,13S)-13-(Dimethylamino)-4a-[[(1,1-di-
methylethyl)dimethylsilyl]oxy]-10-fluoro-4,4a,6,11,11a,
12,12a,13-octahydro-5-hydroxy-4,6-dioxo-3,7-bis(phenyl-
methoxy)isoxazolo[50,40:6,7]naphth[2,3-g]isoquinolin-8-
yl]-imidodicarbonic Acid, 1,3-Bis(1,1-dimethylethyl) Ester
(19k). Prepared from 18a in 56% yield, yellow solid. 1H NMR (400 MHz,
CDCl3) δ 15.59 (s, 1 H), 7.56-7.24 (m, 10 H), 5.36 (s, 2 H), 4.92 (q, J =
67.8 Hz, J = 9.16 Hz, 2 H), 3.91 (d, J = 11.0 Hz, 1 H), 3.20-3.02 (m, 2 H),
2.62-2.45 (m, 9 H), 2.19-2.12 (m, 1 H), 1.37, (s, 18 H), 0.82 (s, 9 H),
0.26 (s, 3 H), 0.13 (s, 3 H). MS (ESI) m/z 941.59 (M þ H).
(4aS,11aR,12aS,13S)-8-Bromo-10,13-bis(dimethylamino)-
4a-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-11a,12,12a,13-
tetrahydro-5-hydroxy-3,7-bis(phenylmethoxy)-isoxazolo-
[50,40:6,7]naphth[2,3-g]isoquinoline-4,6(4aH,11H)-dione
(19m). Prepared from 16e in 54% yield, yellow solid. 1H NMR (400 MHz,
CDCl3) δ 15.65 (s, 1 H), 7.52-7.22 (m, 10 H), 5.36 (s, 2 H), 5.17 (q, J =
11.0 Hz, 2 H), 4.01 (d, J = 11.0 Hz, 1 H), 3.05 (dd, J = 17.2 Hz, J = 11.6 Hz,
1 H), 2.98-2.86 (m, 1 H), 2.73 (s, 6 H), 2.62-2.38 (m, 9 H), 2.12 (d, J =
13.4 Hz, 1 H), 0.82 (s, 9 H), 0.26 (s, 3 H), 0.13 (s, 3 H). MS (ESI) m/z
829.49, 831.48 (M þ H).
(4aS,11aR,12aS,13S)-8-Bromo-10-chloro-13-(dimethyl-
amino)-4a-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-11a,12,
12a,13-tetrahydro-5-hydroxy-3,7-bis(phenylmethoxy)-is-
oxazolo[50,40:6,7]naphth[2,3-g]isoquinoline-4,6(4aH,11H)-
dione (19h). Compound 16a (793 mg, 1.84 mmol) and compound 8
(885 mg, 1.84 mmol) were dissolved in THF (16 mL), and the solution was
cooled to -78 °C. LHMDS (1.0 M solution in THF, 5.5 mL, 5.5 mmol)
was added slowly via syringe. After 10 min, the reaction mixture was allowed
to slowly warm to 0 °C over 1 h. A phosphate buffer solution (pH 7.0,
20 mL) was added, followed by the addition of ammonium chloride
(saturated, aqueous solution, 50 mL). The resulting mixture was
extracted with CH2Cl2 (3 ꢀ 50 mL), and the combined extracts were
dried over Na2SO4, filtered, and concentrated under reduced pressure.
The resulting brown solid was washed with cold methanol (3 ꢀ 5 mL)
to afford 1.11 g (74%) of the title compound as a yellow-brown
powder. The organics were concentrated under reduced pressure and
purified by column chromatography (Biotage 20 g column, 5-20%
EtOAc in hexanes gradient), yielding an additional 150 mg (10%) of
(4aS,11aR,12aS,13S)-10-Bromo-13-(dimethylamino)-4a-
[[(1,1-dimethylethyl)dimethylsilyl]oxy]-11a,12,12a,13-tet-
rahydro-5-hydroxy-3,7-bis(phenylmethoxy)-isoxazolo-
[50,40:6,7]naphth[2,3-g]isoquinoline-4,6(4aH,11H)-dione
(19c). Prepared from 15b in 71% yield, yellow solid. 1H NMR (400 MHz,
CDCl3) δ 15.55 (s, 1 H), 8.12 (s, 1 H), 7.55-7.24 (m, 10 H), 5.40-5.22
(m, 4 H), 3.90 (d, J = 11.0 Hz, 1 H), 3.25 (dd, J = 16.5 Hz, J= 4.88 Hz, 1 H),
3.12-3.02 (m, 1 H), 2.62-2.42 (m, 9 H), 2.14 (d, J = 14.0 Hz, 1 H), 0.82
(s, 9H),0.26(s, 3H),0.13(s, 3H).MS(ESI) m/z786.63, 788.63 (M þ H).
(4aS,11aR,12aS,13S)-10,13-Bis(dimethylamino)-4a-[[(1,
1-dimethylethyl)dimethylsilyl]oxy]-11a,12,12a,13-tetra-
hydro-5-hydroxy-3,7-bis(phenylmethoxy)-isoxazolo-
[50,40:6,7]naphth[2,3-g]isoquinoline-4,6(4aH,11H)-dione
(19f). Prepared from 15c in 50% yield, yellow solid. 1H NMR (400 MHz,
CDCl3) δ 15.65 (s, 1 H), 8.01 (s, 1 H), 7.52-7.22 (m, 10 H), 5.36 (s, 2 H),
1
the product. H NMR (400 MHz, CDCl3) δ 15.45 (br, 1 H), 7.54-
7.48 (m, 4 H), 7.40-7.30 (m, 6 H), 5.36 (s, 2 H), 5.03 (abq, J = 10.4
Hz, 2 H), 3.87 (d, J = 11.0 Hz, 1 H), 3.27-3.23 (m, 1 H), 3.10-3.00
(m, 1 H), 2.65-2.57 (m, 1 H), 2.57-2.43 (m, 8 H), 2.16 (d, J = 11.0
Hz, 1 H), 0.81 (s, 9 H), 0.26 (s, 3 H), 0.12 (s, 3 H). MS (ESI) m/z
820.37, 822.37 (M þ H).
The following compounds were prepared by methods similar to 19h.
(4aS,11aR,12aS,13S)-8-Bromo-13-(dimethylamino)-4a-
[[(1,1-dimethylethyl)dimethylsilyl]oxy]-10-fluoro-11a,12,
12a,13-tetrahydro-5-hydroxy-3,7-bis(phenylmethoxy)-is-
oxazolo[50,40:6,7]naphth[2,3-g]isoquinoline-4,6(4aH,11H)-
dione (19i). Prepared from 16d in 45% yield, yellow solid. 1H NMR
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Journal of Medicinal Chemistry
ARTICLE
(400 MHz, CDCl3) δ 15.55 (s, 1 H), 7.56-7.26 (m, 10 H), 5.36 (s,
2 H), 5.02 (s, 2 H), 3.88 (d, J = 10.4 Hz, 1 H), 3.18-3.04 (m, 2 H), 2.62-
2.58 (m, 1 H), 2.58-2.40 (m, 8 H), 2.17 (d, J= 14.6 Hz, 1 H), 0.81 (s, 9 H),
0.25 (s, 3 H), 0.12 (s, 3 H). MS (ESI) m/z 804.34, 806.34 (M þ H).
2-[(4aS,11aR,12aS,13S)-10,13-Bis(dimethylamino)-4a-
[[(1,1-dimethylethyl)dimethylsilyl]oxy]-4,4a,6,11,11a,12,
12a,13-octahydro-5-hydroxy-4,6-dioxo-3,7-bis(phenyl-
methoxy)isoxazolo[50,40:6,7]naphth[2,3-g]isoquinolin-8-
yl]-imidodicarbonic Acid, 1,3-Bis(1,1-dimethylethyl) Ester
(19l). Prepared from 18b in 53% yield, yellow solid. 1H NMR (400 MHz,
CDCl3) δ 15.50 (s, 1 H), 7.52-7.24 (m, 10 H), 5.36 (s, 2 H), 4.85 (q, J =
86.1 Hz, J = 9.76 Hz, 2 H), 4.02 (d, J = 10.4 Hz, 1 H), 3.08-3.00 (m, 1 H),
3.00-2.82 (m, 1 H), 2.77 (s, 6 H), 2.62-2.38 (m, 9 H), 2.20-2.12 (m,
1 H), 1.35, (br s, 18 H), 0.79 (s, 9 H), 0.26 (s, 3 H), 0.12 (s, 3 H). MS (ESI)
m/z 966.59 (M þ H).
to cool to room temperature, was filtered through Celite, and was
concentrated under reduced pressure. The material was purified by
preparative HPLC (Sunfire Prep C18 column, 80-100% B gradient),
yielding 41.7 mg (40%) of the title compound as a yellow solid. 1H NMR
(400 MHz, CDCl3) δ15.65-15.45 (br s, 1 H), 7.56-7.50 (m, 2 H), 7.43-
7.34 (m, 7 H), 7.29-7.20 (m, 1 H), 5.38 (s, 2 H), 4.94 (dd, J = 29.3 Hz, J =
11.0 Hz, 2 H), 3.90 (d, J = 11.0 Hz, 1 H), 3.27 (dd, J = 16.4 Hz, J = 11.6 Hz,
1 H), 3.12-3.03 (m, 1 H), 2.68-2.60 (m, 1 H), 2.60-2.45 (m, 8 H), 2.18
(d, J=14.6Hz, 1H), 1.47(s, 9H), 0.85(s, 9H), 0.29(s, 3H), 0.15(s, 3H).
MS (ESI) m/z 857.67 (M þ H).
6-Demethyl-6-deoxy-8-azatetracycline Dihydrochloride
(20a). Aqueous HF (48%, 0.4 mL) was added to a solution of 19a
(65.5 mg, 0.093 mmol) in CH3CN (0.6 mL) in a plastic vial. After 18 h,
the reaction mixture was poured into a solution of K2HPO4 (4.8 g) in
water (20 mL). The mixture was extracted with EtOAc (3 ꢀ 20 mL).
The combined extracts were dried over Na2SO4, filtered, and con-
centrated under reduced pressure. The material was dissolved in MeOH
(1 mL) and1,4-dioxane(1 mL), andpalladium oncarbon (Degussa, 10wt
%, ∼5 mg) was added. An atmosphere of hydrogen was introduced, and
the reaction mixture was stirred for 2 h. The reaction mixture was filtered
through Celite, and the filtrate was concentrated under reduced pressure.
The material was purified by preparative HPLC (Phenomenex Polymerx
column, 0-100% B gradient). Fractions with the desired MW were
collected and freeze-dried to yield 18.4 mg (41%, 2 steps) of the title
compound as a yellow solid. 1H NMR (400 MHz, CD3OD with 1 drop
DCl) δ 8.55 (s, 1 H), 8.36 (s, 1 H), 4.19 (s, 1 H), 3.26-2.98 (m, 9 H),
2.71 (t, J = 14.2 Hz, 1 H), 2.39-2.32 (m, 1 H), 1.75-1.63 (m, 1 H). MS
(ESI) m/z 416.44 (M þ H).
(4aS,11aR,12aS,13S)-13-(Dimethylamino)-4a-[[(1,1-di-
methylethyl)dimethylsilyl]oxy]-11a,12,12a,13-tetrahydro-
5-hydroxy-10-methyl-3,7-bis(phenylmethoxy)-isoxazolo-
[50,40:6,7]naphth[2,3-g]isoquinoline-4,6(4aH,11H)-dione
(19d). A solution of compound 19c (52 mg, 0.067 mmol), methylboronic
acid (40 mg, 0.67 mmol), dichloro[1,10-bis(diphenylphosphino)ferrocene]
palladium(II) dichloromethane adduct (3 mg, 0.003 mmol), and K3PO4
(142 mg, 0.667 mmol) in toluene (1 mL), 1,4-dioxane (1 mL), and water
(0.2 mL) was heated to 70 °C. After 2 h, the reaction mixture was heated to
100 °C. After an additional 2 h, the reaction mixture was diluted with EtOAc
(20 mL) and was washed with water (15 mL) and brine (15 mL). The
organics were dried over Na2SO4, filtered, and concentrated under reduced
pressure. The material was purified by preparative HPLC (Sunfire Prep
C18 column, 80-100% B gradient), yielding 18.3 mg (38%) of the title
compound as a yellow solid. 1H NMR (400 MHz, CDCl3) δ15.71 (s, 1 H),
8.24 (s, 1 H), 7.55-7.24 (m, 10 H), 5.36 (s, 2 H), 5.30-5.20 (m, 2 H), 3.95
(d, J = 10.4 Hz, 1 H), 3.10-2.92 (m, 2 H), 2.62-2.42 (m, 12 H), 2.12 (d,
J = 14.0 Hz, 1 H), 0.82 (s, 9 H), 0.26 (s, 3 H), 0.14 (s, 3 H). MS (ESI) m/z
722.72 (M þ H).
The following compounds were synthesized by similar methods
to 20a.
7-Chloro-6-demethyl-6-deoxy-8-azatetracycline Hydro-
chloride (20b). Prepared from 19b in 24% yield, 2 steps, yellow solid.
1H NMR (400 MHz, CD3OD) δ 8.08 (d, J = 6.0 Hz, 1 H), 4.09 (s, 1 H),
3.08-2.92 (m, 9 H), 2.30-2.15 (m, 2 H), 1.70 -1.58 (m, 1 H). MS
(ESI) m/z 450.18 (M þ H).
(4aS,11aR,12aS,13S)-13-(Dimethylamino)-4a-[[(1,1-di-
methylethyl)dimethylsilyl]oxy]-11a,12,12a,13-tetrahydro-
5-hydroxy-10-phenyl-3,7-bis(phenylmethoxy)-isoxazolo-
[50,40:6,7]naphth[2,3-g]isoquinoline-4,6(4aH,11H)-dione
(19e). A solution of compound 19c (31 mg, 0.040 mmol), phenylboronic
acid (24.5 mg, 0.201 mmol), dichloro[1,10-bis(diphenylphosphino)-
ferrocene]palladium(II) dichloromethane adduct (1.6 mg, 0.002 mmol),
and sodium carbonate (21.3 mg, 0.201 mmol) in toluene (1 mL), 1,
4-dioxane (1 mL), and water (0.2 mL) was heated to 100 °C via microwave
reactor for 10 min. The reaction mixture was diluted with EtOAc (10 mL)
and was washed with water (5 mL) and brine (5 mL). The organics were
dried over Na2SO4, filtered, and concentrated under reduced pressure. The
material was purified by preparative HPLC (Sunfire Prep C18 column, 80-
100% B gradient), yielding 16.9 mg (54%) of the title compound as a yellow
solid. 1H NMR (400 MHz, CDCl3) δ 15.66 (s, 1 H), 8.48 (s, 1 H), 7.52-
7.24 (m, 15 H), 5.42-5.30 (m, 4 H), 3.97 (d, J = 10.4 Hz, 1 H), 3.00-2.86
(m, 2 H), 2.78-2.62 (m, 1 H), 2.58-2.30 (m, 8 H), 2.00 (d, J = 14.0 Hz,
1 H), 0.80 (s, 9 H), 0.25 (s, 3 H), 0.14 (s, 3 H). MS (ESI) m/z 784.75
(M þ H).
6-Demethyl-6-deoxy-7-methyl-8-azatetracycline Dihy-
drochloride (20d). Prepared from 19d in 34% yield, 2 steps, yellow
solid. 1H NMR (400 MHz, CD3OD with 1 drop DCl) δ 8.35 (s, 1 H),
4.19 (s, 1 H), 3.25-2.95 (m, 9 H), 2.80-2.25 (m, 5 H), 1.80-1.63 (m,
1 H). MS (ESI) m/z 430.46 (M þ H).
6-Demethyl-6-deoxy-7-phenyl-8-azatetracycline Dihydro-
chloride (20e). Prepared from 19e in 24% yield, 2 steps, yellow solid.
1H NMR (400 MHz, CD3OD with 1 drop DCl) δ 8.53 (s, 1 H), 7.62 (s,
5 H), 4.18 (s, 1 H), 3.14-2.82 (m, 9 H), 2.81-2.66 (m, 1 H), 2.24-2.14
(m, 1 H), 1.66-1.52 (m, 1 H). MS (ESI) m/z 492.48 (M þ H).
6-Demethyl-6-deoxy-7-(dimethylamino)-8-azatetracy-
cline Dihydrochloride (20f). Prepared from 19f in 96% yield, 2 steps,
yellow solid. 1H NMR (400 MHz, CD3OD with 1 drop DCl) δ 8.22 (s,
1 H), 4.19 (s, 1 H), 3.38-3.30 (m, 2 H), 3.24 (s, 6 H), 3.12-2.95 (m,
7 H), 2.60 (t, J = 14.2 Hz, 1 H), 2.42-2.34 (m, 1 H), 1.75-1.63 (m, 1 H).
MS (ESI) m/z 459.50 (M þ H).
6-Demethyl-6-deoxy-7-methoxy-8-azatetracycline Dihy-
drochloride (20g). Prepared from 19g in 51% yield, 2 steps, yellow
solid. 1H NMR (400 MHz, CD3OD with 1 drop DCl) δ 7.80 (s, 1 H),
4.15 (s, 1 H), 3.97 (s, 3 H), 3.40-2.98 (m, 9 H), 2.32-2.20 (m, 2 H),
1.72-1.56 (m, 1 H). MS (ESI) m/z 446.39 (M þ H).
N-[(4aS,11aR,12aS,13S)-10-Chloro-13-(dimethylamino)-
4a-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-4,4a,6,11,11a,12,
12a,13-octahydro-5-hydroxy-4,6-dioxo-3,7-bis(phenyl-
methoxy)isoxazolo[50,40:6,7]naphth[2,3-g]isoquinolin-8-yl]-
carbamic Acid, 1,1-Dimethylethyl Ester (19j). Compound 19h
(100 mg, 0.122 mmol), t-butylcarbamate (42.9 mg, 0.366 mmol), K3PO4
(77.7 mg, 0.366 mmol), tris(dibenzylideneacetone)dipalladium (5.6 mg,
0.006 mmol), and Xantphos (10.3 mg, 0.018 mmol) were weighed into a
vial equipped with a septum. The vial was evacuated and flushed with
nitrogen (3ꢀ), and 1,4-dioxane (0.5 mL) was added. The reaction mixture
was heated to 100 °C and stirred for 2 h. The reaction mixture was allowed
6-Demethyl-6-deoxy-7-fluoro-8-azatetracycline Hydro-
chloride (20i). Prepared from 19i in 68% yield, 2 steps, yellow solid.
1H NMR (400 MHz, CD3OD with 1 drop DCl) δ 7.81 (s, 1 H), 4.17 (s,
1 H), 3.26-2.96 (m, 9 H), 2.42-2.25 (m, 2 H), 1.70-1.58 (m, 1 H).
MS (ESI) m/z 434.27 (M þ H).
9-Amino-7-chloro-6-demethyl-6-deoxy-8-azatetracycline
(20j). Prepared from 19j in 68% yield, 2 steps, yellow solid. 1H NMR
(400 MHz, CD3OD with 1 drop DCl) δ 4.19 (s, 1 H), 3.20-2.98 (m,
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Journal of Medicinal Chemistry
ARTICLE
8 H), 2.35-2.22 (m, 2 H), 1.68-1.56 (m, 1 H). MS (ESI) m/z 465.32
(M þ H).
CD3OD with 1 drop DCl) δ 4.30-4.40 (m, 3 H), 3.40-3.25 (m, 8 H),
3.12-3.04 (m, 4 H), 2.99 (s, 3 H), 2.62-2.52 (m, 1 H), 2.41-2.35
(m, 1 H), 1.73-1.60 (m, 1 H), 1.45 (s, 9 H). MS (ESI) m/z 587.43
(M þ H).
9-Amino-6-demethyl-6-deoxy-7-fluoro-8-azatetracycline
Dihydrochloride (20k). Prepared from 19k in 49% yield, 2 steps,
yellow solid. 1H NMR (400 MHz, CD3OD with 1 drop DCl) δ 4.20 (s,
1 H), 3.40-2.90 (m, 9 H), 2.42-2.22 (m, 2 H), 1.70-1.54 (m, 1 H).
MS (ESI) m/z 449.27 (M þ H).
(4aS,11aR,12aS,13S)-10-Chloro-13-(dimethylamino)-4a-
[[(1,1-dimethylethyl)dimethylsilyl]oxy]-8-(ethylamino)-11a,
12,12a,13-tetrahydro-5-hydroxy-3,7-bis(phenylmethoxy)-is-
oxazolo[50,40:6,7]naphth[2,3-g]isoquinoline-4,6(4aH,11H)-
dione. Compound 19h (50 mg, 0.061 mmol), K3PO4 (39 mg, 0.18
mmol), tris(dibenzylideneacetone)dipalladium (2.8 mg, 0.003 mmol),
and Xantphos (5.1 mg, 0.009 mmol) were added to a small vial equipped
with a septum. After the flask was evacuated and flushed with nitrogen
(3ꢀ), 1,4-dioxane (0.5 mL) and ethylamine (2.0 M solution in THF,
0.091 mL, 0.18 mmol) were added. The reaction mixture was heated to
100 °C and stirred for 3 h. The reaction mixture was allowed to cool to
room temperature and was filtered through Celite. The material was purified
by preparative HPLC (Sunfire Prep C18 column, 90-100% B gradient).
Fractions with the desired MW were collected and freeze-dried to yield
11 mg (23%) ofthe title compound asa yellow solid. 1H NMR (400 MHz,
CDCl3) δ 15.6 (br, 1 H), 7.56-7.49 (m, 4 H), 7.42-7.30 (m, 6 H), 5.37
(s, 2 H), 5.03 (s, 2 H), 3.89 (d, J = 11.0Hz, 1 H), 3.24-3.04 (m, 3 H), 2.97
(dd, J = 15.6, 4.9 Hz, 1 H), 2.64-2.43 (m, 3 H), 2.49 (s, 6 H), 2.14 (d, J =
15.6 Hz, 1 H), 1.25 (t, J = 6.8 Hz, 3 H), 0.81 (s, 9 H), 0.25 (s, 3 H), 0.11 (s,
3 H). MS (ESI) m/z 785.57 (M þ H).
N-[(4aS,11aR,12aS,13S)-10-Chloro-13-(dimethylamino)-
4a-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-4,4a,6,11,11a,12,
12a,13-octahydro-5-hydroxy-4,6-dioxo-3,7-bis(phenyl-
methoxy)isoxazolo[50,40:6,7]naphth[2,3-g]isoquinolin-8-
yl]-2-[(1,1-dimethylethyl)amino]-acetamide. Compound 19j
(68.3 mg, 0.080 mmol) was stirred in 4 M HCl in 1,4-dioxane for 2 h.
The reaction mixture was concentrated under reduced pressure. The
material was dissolved in THF (2 mL), and bromoacetylchloride (12.5 mg,
0.080 mmol) was added. After stirring overnight, an additional portion
of bromoacetylchloride (0.020 mL) was added. After 4 h, the reaction
mixture was concentrated under reduced pressure. MS (ESI) m/z
879.42 (M þ H). t-Butylamine (0.063 mL, 0.60 mmol) was added to a
solution of the intermediate (35 mg, 0.040 mmol) in THF (1 mL),
and the reaction mixture was heated to 50 °C. After stirring overnight,
the reaction mixture was concentrated under reduced pressure and
was purified by preparative HPLC (Sunfire Prep C18 column, 50-
100% B gradient). This gave 16.8 mg (49%) of the title compound as a
yellow solid. 1H NMR (400 MHz, CDCl3) δ 8.20 (br s, 1 H), 7.52-
7.46 (m, 2 H), 7.43-7.30 (m, 8 H), 5.36 (s, 2 H), 4.95 (dd, J = 40.6 Hz, J =
11.0 Hz, 2 H), 3.88 (d, J = 11.0 Hz, 1 H), 3.32-3.24 (m, 1 H), 3.12-3.03
(m, 1 H), 2.66-2.59 (m, 1 H), 2.58-2.42 (m, 8 H), 2.18 (d, J = 14.6 Hz,
1 H), 1.30-1.10 (br s, 9 H), 0.82 (s, 9 H), 0.27 (s, 3 H), 0.12 (s, 3 H). MS
(ESI) m/z 870.60 (M þ H).
7-Chloro-6-demethyl-6-deoxy-9-(ethylamino)-8-azate-
tracycline Dihydrochloride (24a). Aqueous HF (48%, 0.3 mL)
was added to a solution of the preceding intermediate (11 mg, 0.014
mmol) in CH3CN (7 mL) in a plastic vial. After 18 h, the reaction
mixture was poured into a solution of K2HPO4 (2 g) in water (10 mL).
The mixture was extracted with EtOAc (3ꢀ). The combined EtOAc
extracts were dried over Na2SO4 and concentrated under reduced
pressure. The material was dissolved in MeOH (2 mL) and 1,4-dioxane
(2 mL), and palladium on carbon (Degussa, 10 wt %, 5.6 mg) was added.
An atmosphere of hydrogen was introduced, and the reaction mixture
was stirred for 1 h. The reaction mixture was filtered through Celite, and
the filtrate was concentrated under reduced pressure. The material 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 3.2 mg (46%, 2 steps) of the title
compound as a yellow solid. 1H NMR (400 MHz, CD3OD) δ 4.08 (s,
1 H), 3.43 (q, J = 7.4 Hz, 2 H), 3.08-2.92 (m, 9 H), 2.30-2.15 (m, 2 H),
1.67-1.55 (m, 1 H), 1.22 (t, J = 7.4 Hz, 3H). MS (ESI) m/z 493.24
(M þ H).
9-[2-(tert-Butylamino)acetamido]-7-chloro-6-demethyl-
6-deoxy-8-azatetracycline Dihydrochloride (21a). Aqueous
HF (48%, 0.4 mL) was added to a solution of the preceding inter-
mediate (16.8 mg, 0.019 mmol) in CH3CN (0.6 mL) in a plastic vial.
After 18 h, the reaction mixture was poured into a solution of K2HPO4
(7.8 g) in water (15 mL). The mixture was extracted with EtOAc
(3ꢀ). Sodium chloride (10 g) was added to the aqueous layer, and this
was extracted with EtOAc (2ꢀ). The combined extracts were dried
over Na2SO4, filtered, and concentrated under reduced pressure.
The material was dissolved in MeOH (1 mL) and 1,4-dioxane (1 mL),
and palladium on carbon (Degussa, 10 wt %, ∼5 mg) was added. An
atmosphere of hydrogen was introduced, and the reaction mixture was
stirred for 1 h. The reaction mixture was filtered through Celite, and
the filtrate was concentrated under reduced pressure. The material
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 1.2 mg (10%, 2 steps) of the
title compound as a yellow solid. 1H NMR (400 MHz, CD3OD with 1
drop DCl) δ 4.30-4.20 (s, 2 H), 4.19 (s, 1 H), 3.25-2.96 (m, 9 H),
2.48-2.28 (m, 2 H), 1.72-1.58 (m, 1 H), 1.43 (s, 9 H). MS (ESI) m/z
578.48 (M þ H).
The following compounds were prepared by similar methods to 24a:
7-Chloro-6-demethyl-6-deoxy-9-(methylamino)-8-azate-
tracycline Dihydrochloride (24b). Prepared from 19h and methy-
lamine (1.0 M solution in THF) in 21% yield for the amination step, 19%
for the 2 deprotection steps, yellow solid. 1H NMR (400 MHz, CD3OD)
δ 4.08 (s, 1 H), 3.41 (s, 3 H), 3.08-2.92 (m, 9 H), 2.30-2.15 (m, 2 H),
1.67-1.55 (m, 1 H). MS (ESI) m/z 479.22 (M þ H).
7-Chloro-6-demethyl-6-deoxy-9-(propylamino)-8-azate-
tracycline Dihydrochloride (24c). Prepared from 19h and n-propyl-
amine in 24% yield for the amination step, 40% for the 2 deprotection steps,
The following compounds were prepared according to methods
similar to 21a:
1
yellow solid. H NMR (400 MHz, CD3OD with 1 drop DCl) δ 4.16
9-[2-(tert-Butylamino)acetamido]-6-demethyl-6-deoxy-
7-fluoro-8-azatetracycline Dihydrochloride (21b). Prepared
from 19k in 68% yield for the acylation/amine displacement and 58%
yield for the deprotections, yellow solid. 1H NMR (400 MHz, CD3OD
with 1 drop DCl) δ 4.24-4.65 (m, 3 H), 3.26-2.97 (m, 9 H), 2.38-
2.28 (m, 2 H), 1.69-1.56 (m, 1 H), 1.44 (s, 9 H). MS (ESI) m/z 562.37
(M þ H).
(s, 1H), 3.47 (t, J = 7.3 Hz, 2 H), 3.15-2.96 (m, 9 H), 2.32-2.20 (m, 2 H),
1.78-1.55 (m, 3 H), 1.01 (t, J = 7.4 Hz, 3 H). MS (ESI) m/z 507.29
(M þ H).
7-Chloro-6-demethyl-6-deoxy-9-(propan-2-ylamino)-8-
azatetracycline Dihydrochloride (24d). Prepared from 19h and
2-propylamine in 11% yield for the amination step, 16% for the 2
deprotection steps, yellow solid. 1H NMR (400 MHz, CD3OD) δ 4.21-
4.13 (m, 1 H), 4.08 (s, 1 H), 3.14-2.92 (m, 9 H), 2.31-2.15 (m, 2 H),
1.67-1.56 (m, 1 H), 1.23 (dd, J = 6.4, 1.8 Hz, 6 H). MS (ESI) m/z
507.24 (M þ H).
9-[2-(tert-Butylamino)acetamido]-6-demethyl-6-deoxy-
7-(dimethylamino)-8-azatetracycline Trihydrochloride
(21c). Prepared from 19l in 43% yield for the acylation/amine displace-
ment and 55% yield for the deprotections, yellow solid. 1H NMR (400 MHz,
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Journal of Medicinal Chemistry
ARTICLE
7-Chloro-6-demethyl-6-deoxy-9-[(2-methylpropyl)amino]-
8-azatetracycline Dihydrochloride (24e). Prepared from 19h
and 2-methylpropan-1-amine in 15% yield for the amination step, 57%
for the 2 deprotection steps, yellow solid. 1H NMR (400 MHz, CD3OD
with 1 drop DCl) δ 4.18 (s, 1H), 3.38 (d, J = 7.3 Hz, 2 H), 3.20-2.96 (m,
9 H), 2.34-2.20 (m, 2 H), 2.10-1.98 (m, 1H), 1.68-1.54 (m, 1 H),
1.00 (d, J = 6.4 Hz, 6 H). MS (ESI) m/z 521.40 (M þ H).
12a,13-octahydro-5-hydroxy-4,6-dioxo-3,7-bis(phenyl-
methoxy)isoxazolo[50,40:6,7]naphth[2,3-g]isoquinolin-8-yl]-
N-[3-(dimethylamino)-2,2-dimethylpropyl]-carbamic Acid,
Phenylmethyl Ester (23a). A solution of compound 22a (54 mg,
0.090 mmol) in THF (0.5 mL) was added dropwise to a -78 °C
solution of LDA (2.0 M solution in THF, 0.112 mL, 0.224 mmol) and
TMEDA (0.081 mL, 0.54 mmol) in THF (2 mL), resulting in an orange-
colored solution. After 10 min, a solution of 8 (43 mg, 0.090 mmol) in
THF (0.5 mL) was added dropwise over ∼30 s. After complete addition,
the reaction mixture was allowed to warm to -10 °C over 1 h. The
reaction was quenched by the addition of ammonium chloride (saturated,
aqueous solution), was diluted with water, and was extracted with EtOAc
(2ꢀ). The combined extracts were dried over Na2SO4, filtered, and
concentrated under reduced pressure. The material was purified by
preparative HPLC (Sunfire Prep C18 column, 20-100% B gradient),
yielding 22.9 mg (26%) of the desired product as a yellow solid. 1H
NMR (400 MHz, CDCl3) δ 15.55 (br s, 1 H), 7.52-7.20 (m, 15 H),
5.36 (s, 2 H), 5.20-5.00 (m, 2 H), 4.80 (s, 2 H), 3.90 (d, J = 11.0 Hz, 1
H), 3.19-3.05 (m, 2 H), 2.62-2.57 (m, 1 H), 2.56-2.12 (m, 19 H),
0.94-0.74 (m, 15 H), 0.26 (s, 3 H), 0.12 (s, 3 H). MS (ESI) m/z
988.59 (M þ H).
7-Chloro-6-demethyl-6-deoxy-9-[(3-methylbutyl)amino]-
8-azatetracycline Dihydrochloride (24f). Prepared from 19h
and 3-methyl-butan-1-amine in 25% yield for the amination step, 66%
for the 2 deprotection steps, yellow solid. 1H NMR (400 MHz, CD3OD)
δ 4.09 (s, 1 H), 3.45-3.40 (m, 2 H), 3.20 (s, 1 H), 3.08-2.93 (m, 9 H),
2.30-2.16 (m, 2 H), 1.72-1.58 (m, 2 H), 1.55-1.48 (m, 2 H), 0.96 (d, J =
6.4 Hz, 6 H). MS (ESI) m/z 535.30 (M þ H).
7-Chloro-6-demethyl-6-deoxy-9-[(2-methoxyethyl)amino]-
8-azatetracycline Dihydrochloride (24g). Prepared from 19h
and 2-methoxyethylamine in 19% yield for the amination step, 44% for
the 2 deprotection steps, yellow solid. 1H NMR (400 MHz, CD3OD with 1
drop DCl) δ 4.13 (s, 1H), 3.72-3.60 (m, 4H), 3.40 (s, 3 H), 3.12-2.95
(m, 9 H), 2.32-2.21 (m, 2 H), 1.69-1.56 (m, 1 H). MS (ESI) m/z 523.32
(M þ H).
7-Chloro-6-demethyl-6-deoxy-9-[[2-(propan-2-yloxy)ethyl]-
amino]-8-azatetracycline Dihydrochloride (24h). Prepared
from 19h and 2-(propan-2-yloxy)ethan-1-amine in 9% yield for the
6-Demethyl-6-deoxy-9-[[3-(dimethylamino)-2,2-dimethyl-
propyl]amino]-7-fluoro-8-azatetracycline Trihydrochloride
(24j). Aqueous HF (48%, 0.4 mL) was added to a solution of 23a
(22.9 mg, 0.0232 mmol) in CH3CN (0.8 mL) in a plastic vial. After
18 h, the reaction mixture was poured into a solution of K2HPO4
(4.8 g) in water. The mixture was extracted with EtOAc, and the
combined extracts were dried over Na2SO4, filtered, and concen-
trated under reduced pressure. The material was dissolved in MeOH
(1 mL), 0.5 M HCl in MeOH (0.2 mL), and 1,4-dioxane (1 mL), and
palladium on carbon (Degussa, 10 wt %, ∼2 mg) was added. An
atmosphere of hydrogen was introduced, and the reaction mixture
was stirred for 3 h. The reaction mixture was filtered through Celite,
and the filtrate was concentrated under reduced pressure. The
material was purified by preparative HPLC (Phenomenex Polymerx
column, 0-100% B gradient). Fractions with the desired MW were
collected and freeze-dried to yield 9.9 mg (67%, 2 steps) of the
desired product as a yellow solid. 1H NMR (400 MHz, CD3OD with 1
drop DCl) δ 4.16 (s, 1 H), 3.43 (s, 2 H), 3.15-2.90 (m, 17 H), 2.30-
2.22 (m, 1 H), 2.22-2.12 (m, 1 H), 1.65-1.54 (m, 1 H), 1.17, (s, 6 H).
MS (ESI) m/z 562.43 (M þ H).
1
amination step, 30% for the 2 deprotection steps, yellow solid. H
NMR (400 MHz, CD3OD with 1 drop DCl) δ 4.19 (s, 1H), 3.82-
3.69 (m, 5H), 3.20-2.98 (m, 9 H), 2.35-2.22 (m, 2 H), 1.68-
1.56 (m, 1 H), 1.18 (d, J = 6.4 Hz, 6H). MS (ESI) m/z 551.41
(M þ H).
7-Chloro-6-demethyl-6-deoxy-9-[[3-(dimethylamino)propyl]-
amino]-8-azatetracycline Trihydrochloride (24i). Prepared
from 19h and (3-aminopropyl)dimethylamine in 9% yield for the
1
amination step, 21% for the 2 deprotection steps, yellow solid. H
NMR (400 MHz, CD3OD) δ 4.09 (s, 1 H), 3.70-3.46 (m, 4 H),
3.10-2.90 (m, 15 H), 2.31-2.17 (m, 2 H), 2.10-2.00 (m, 2 H),
1.68-1.56 (m, 1 H). MS (ESI) m/z 550.34 (M þ H).
Phenyl 3-(Benzyloxy)-2-[[(benzyloxy)carbonyl][3-(dimethyl-
amino)-2,2-dimethylpropyl]amino]-6-fluoro-5-methylpyr-
idine-4-carboxylate (22a). Compound 16d (100 mg, 0.24 mmol),
Cs2CO3 (235 mg, 0.720 mmol), tris(dibenzylideneacetone)dipalladium
(11 mg, 0.012 mmol), and Xantphos (20 mg, 0.036 mmol) were weighed
into a flask. This was evacuated and backflushed with nitrogen (3ꢀ), and
1,4-dioxane (0.5 mL) and N,N-dimethylneopentanediamine (0.057 mL,
0.36 mmol) were added. The reaction mixture was heated to 70 °C. After
1.5 h, the reaction mixture was cooled to room temperature and was
filtered through Celite. The filtrate was concentrated under reduced
pressure and was purified by column chromatography (Biotage 10 g
column, 0-3% MeOH in CH2Cl2 gradient), yielding 76.9 mg (69%) of
the intermediate. Rf = 0.25 in 5% MeOH/CH2Cl2. MS (ESI) m/z 466.28
(M þ H). LHMDS (1.0 M in THF, 0.22 mL, 0.22 mmol) was added
dropwise to a -78 °C solution of the above intermediate (93 mg, 0.20
mmol) in THF (5 mL). After 5 min, benzyl chloroformate (0.084 mL,
0.60 mmol) was added. After 15 min, the reaction mixture was quenched
by the addition of NH4Cl (saturated, aqueous solution, 15 mL) and was
extracted with EtOAc. The organics were dried over Na2SO4, filtered,
and concentrated under reduced pressure. The material was purified by
column chromatography (Biotage 10 g column, 0-7% MeOH in
CH2Cl2 gradient), yielding 111 mg (93%) of the product. Rf = 0.42 in
10% MeOH/CH2Cl2. 1H NMR (400 MHz, CDCl3) δ 7.45-7.20 (m,
13 H), 7.06-6.98 (m, 2 H), 5.16 (s, 2 H), 4.87 (s, 2 H), 3.78 (br s, 2 H),
2.38 (s, 3 H), 2.18 (br s, 6 H), 1.62 (br s, 2 H), 0.90 (br s, 6 H). MS (ESI)
m/z 600.28 (M þ H).
The following compounds were prepared according to methods
similar to 24j:
7-Chloro-6-demethyl-6-deoxy-9-[[2-(dimethylamino)ethyl]-
amino]-8-azatetracycline Trihydrochloride (24k). Prepared
from 16a and N,N-dimethylethylenediamine. Yellow solid. 1H NMR
(400 MHz, CD3OD) δ 4.13 (s, 1 H), 3.84-3.77 (m, 2 H), 3.44-3.38
(m, 2 H), 3.10-2.92 (m, 15 H), 2.32-2.20 (m, 2 H), 1.68-1.55 (m, 1 H).
MS (ESI) m/z 536.25 (M þ H).
7-Chloro-6-demethyl-6-deoxy-9-[[3-(dimethylamino)-2,
2-dimethylpropyl]amino]-8-azatetracycline Trihydrochlor-
ide (24l). Prepared from 16a and N,N-dimethylneopentanediamine.
Yellow solid. 1H NMR (400 MHz, CD3OD) δ 4.10 (s, 1 H), 3.51-3.36
(m, 4 H), 3.10-2.92 (m, 15 H), 2.33-2.18 (m, 2 H), 1.68-1.57 (m, 1 H),
1.174 (s, 3 H), 1.169 (s, 3 H). MS (ESI) m/z 578.35 (M þ H).
7-Chloro-6-demethyl-6-deoxy-9-[[2-methyl-2-(pyrrolidin-
1-ylmethyl)propyl]amino]-8-azatetracycline Trihydrochlor-
ide (24m). Prepared from 16a and 2,2-dimethyl-3-(pyrrolidin-1-yl)-
1
propan-1-amine. Yellow solid. H NMR (400 MHz, CD3OD) δ 4.09
(s, 1 H), 3.88-3.78 (m, 2 H), 3.50-3.39 (m, 2 H), 3.18 -2.92 (m, 9 H),
2.32-2.08 (m, 6 H), 1.67-1.56 (m, 1 H), 1.15 (s, 6 H). MS (ESI) m/z
604.29 (M þ H).
N-[(4aS,11aR,12aS,13S)-13-(Dimethylamino)-4a-[[(1,1-di-
methylethyl)dimethylsilyl]oxy]-10-fluoro-4,4a,6,11,11a,12,
6-Demethyl-6-deoxy-7-fluoro-9-(propylamino)-8-azate-
tracycline Dihydrochloride (24n). Prepared from 16d and
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Journal of Medicinal Chemistry
ARTICLE
n-propylamine. Yellow solid. 1H NMR (400 MHz, CD3OD) δ 4.09
(s, 1 H), 3.33 (t, J = 6.9 Hz, 2 H) 3.42-3.38 (m, 2 H), 3.20 (s, 1 H),
3.07-2.94 (m, 8 H), 2.91 (dd, J = 14.6, 4.6 Hz, 1 H), 2.23-2.12 (m,
2 H), 1.68-1.55 (m, 3 H), 0.96 (t, J = 7.3 Hz, 3 H). MS (ESI) m/z
491.23 (M þ H).
(0.6 mL) in a plastic vial. After 18 h, the reaction mixture was poured into a
solution of K2HPO4 (4.8 g) in water (15 mL). The mixture was extracted
with EtOAc (3ꢀ). The combined EtOAc extracts were dried over Na2SO4,
filtered, and concentrated under reduced pressure. The material was
dissolved in MeOH (1 mL) and 1,4-dioxane (1 mL), and palladium on
carbon (Degussa, 10 wt %, ∼2 mg) was added. An atmosphere of hydrogen
was introduced, and the reaction mixture was stirred for 4 h. The reaction
mixture was filtered through Celite, and the filtrate was concentrated under
reduced pressure. The material was purified by preparative HPLC
(Phenomenex Polymerx, 0-100% B gradient). Fractions with the desired
MW were collected and freeze-dried to yield 2.8 mg (70%, 2 steps) of the
desired product as a yellow solid. 1H NMR (400 MHz, CD3OD with 1 drop
DCl) δ 4.19 (s, 1 H), 3.44 (t, J = 7.6 Hz, 2 H), 3.24-3.12 (m, 7 H), 3.12-
2.97 (m, 8 H), 2.39-2.26 (m, 2 H), 1.73-1.56 (m, 3 H), 0.98 (t, J= 8.4Hz,
3 H); MS (ESI) m/z 516.42 (M þ H).
Phenyl 5-Benzyloxy-2-(dimethylamino)-3-methyl-6-(propyl-
amino)isonicotinate. Copper(I) iodide (4.7 mg, 0.025 mmol) and
K3PO4 (209 mg, 0.984 mmol) were added to a vial equipped with a
septum. After the vial was evacuated and flushed with nitrogen (3ꢀ), a
solution of compound 16e (217 mg, 0.492 mmol) in DMF (2 mL),
1-propylamine (0.162 mL, 1.97 mmol), and 2-acetylcyclohexanone
(0.013 mL, 0.098 mmol) were added. The reaction mixture was heated
to 100 °C for 3 h. The reaction mixture was allowed to cool to room
temperature, was diluted with EtOAc (50 mL), and was washed with
water (3 ꢀ 40 mL). The organics were dried over Na2SO4, filtered, and
concentrated under reduced pressure. The material was purified by
column chromatography (Biotage 10 g column, 0-12% EtOAc in
hexanes gradient), yielding 74.6 mg (36%) of the title compound as a
thick oil. Rf = 0.32 in 10% EtOAc/hexanes. 1H NMR (400 MHz, CDCl3)
δ 7.50-7.46 (m, 2 H), 7.41-7.30 (m, 5 H), 7.30-7.24 (m, 1 H), 7.08-
7.02 (m, 2 H), 5.08 (s, 2 H), 2.85 (s, 6 H), 2.32 (s, 3 H). MS (ESI) m/z
420.34 (M þ H).
(4aS,11aR,12aS,13S)-10,13-Bis(dimethylamino)-4a-[[(1,1-di-
methylethyl)dimethylsilyl]oxy]-11a,12,12a,13-tetrahydro-
5-hydroxy-3,7-bis(phenylmethoxy)-8-(2-propen-1-yl)-is-
oxazolo[50,40:6,7]naphth[2,3-g]isoquinoline-4,6(4aH,11H)-dione
(25a). Compound 19m (105 mg, 0.126 mmol), tetrakis(triphenyl-
phosphine)palladium(0) (15 mg, 0.013 mmol), and K3PO4 (80 mg,
0.38 mmol) were weighed into an 8 mL vial. This was sealed with a
septum and was evacuated and backflushed with nitrogen (3ꢀ).
Toluene (1 mL), 1,4-dioxane (1 mL), and water (0.5 mL) were added,
followed by allylboronic acid pinacol ester (63.7 mg, 0.379 mmol).
The reaction mixture was heated to 90 °C. After 2.5 h, the reaction mixture
was cooled to room temperature and was concentrated under reduced
pressure. The material was purified by preparative HPLC (Sunfire Prep C18
column, 90-100% B gradient), yielding 28.6 mg (29%) of the title
compound as a yellow solid. MS (ESI) m/z 791.64 (M þ H).
Phenyl 5-Benzyloxy-6-[(t-butoxycarbonyl)(propyl)amino]-
2-(dimethylamino)-3-methylisonicotinate (22b). LHMDS
(1.0 M solution in THF, 0.167 mL, 0.167 mmol) was added to a
solution of phenyl 5-benzyloxy-2-(dimethylamino)-3-methyl-6-(propyl-
amino)isonicotinate (63.5 mg, 0.152 mmol) in THF (2 mL), resulting
in a bright-yellow solution. A solution of di-t-butyldicarbonate (99.5 mg,
0.456 mmol) in THF (1 mL) was added. After 10 min, the reaction was
quenched by the addition of ammonium chloride (saturated, aqueous
solution) and was extracted with EtOAc (2ꢀ). The organics were
dried over Na2SO4, filtered, and concentrated under reduced pres-
sure. The material was purified by column chromatography (Biotage
10 g column, 0-12% EtOAc in hexanes gradient), yielding 13.6 mg
6-Demethyl-6-deoxy-7-(dimethylamino)-9-propyl-8-aza-
tetracycline Dihydrochloride (26a). Aqueous HF (48%, 0.4 mL)
was added to a solution of 25a (28.6 mg, 0.036 mmol) in 1,4-dioxane
(1 mL) in a plastic vial. After 18 h, the reaction mixture was poured into a
solution of K2HPO4 (4.8 g) in water. The mixture was extracted with
EtOAc, and the combined extracts were dried over Na2SO4, filtered, and
concentrated under reduced pressure. The material was dissolved in
MeOH (1 mL), 0.5 M HCl in MeOH (0.5 mL), and 1,4-dioxane (1 mL),
and palladium on carbon (Degussa, 10 wt %, ∼5 mg) was added. An
atmosphere of hydrogen was introduced, and the reaction mixture was
stirred for 1 h. The reaction mixture was filtered through Celite, and the
filtrate was concentrated under reduced pressure. The material was
purified by preparative HPLC (Phenomenex Polymerx column, 0-70%
B gradient). Fractions with the desired MW were collected and freeze-
dried to yield 14.3 mg (69%) of the title compound as a yellow solid. 1H
NMR (400 MHz, CD3OD with 1 drop DCl) δ 4.20 (s, 1 H), 3.38-3.15
(m, 7 H), 3.14-2.94 (m, 8 H), 2.91-2.82 (m, 2 H), 2.56 (t, J = 13.8 Hz,
1 H), 2.40-2.32 (m, 1 H), 1.86-1.76 (m, 2 H), 1.74-1.62 (m, 1 H),
1.01 (t, J = 7.34 Hz, 3 H). MS (ESI) m/z 501.35 (M þ H).
1
(17%) of the product. Rf = 0.22 in 10% EtOAc/hexanes. H NMR
(400 MHz, CDCl3) δ 7.40-7.28 (m, 7 H), 7.28-7.22 (m, 1 H),
7.05-7.00 (m, 2 H), 4.90 (s, 2 H), 2.79 (s, 6 H), 2.34 (s, 3 H), 1.76-
1.66 (m, 2 H), 1.56-1.50 (m, 2 H), 0.91 (t, J = 7.56 Hz, 3 H). MS
(ESI) m/z 520.38 (M þ H).
N-[(4aS,11aR,12aS,13S)-10,13-Bis(dimethylamino)-4a-
[[(1,1-dimethylethyl)dimethylsilyl]oxy]-4,4a,6,11,11a,12,
12a,13-octahydro-5-hydroxy-4,6-dioxo-3,7-bis(phenyl-
methoxy)isoxazolo[50,40:6,7]naphth[2,3-g]isoquinolin-8-yl]-
N-propyl-carbamic Acid, 1,1-Dimethylethyl Ester (23b). A
solution of 22b (24 mg, 0.047 mmol) in THF (0.5 mL) was added dropwise
to a -78 °C solution of LDA (0.5 M solution in THF, 0.102 mL, 0.051
mmol) and TMEDA (0.041 mL, 0.27 mmol) in THF (2 mL), resulting
in a yellowish-orange colored solution. After 10 min, a solution of 8
(16.4 mg, 0.034 mmol) in THF (0.5 mL) was added dropwise. After
complete addition, the reaction mixture was allowed to warm to -20 °C
over ∼45 min. The reaction was quenched by the addition of ammo-
nium chloride (saturated, aqueous solution) and was extracted with
EtOAc (2ꢀ). The combined extracts were dried over Na2SO4, filtered,
and concentrated under reduced pressure. The material was purified by
preparative HPLC (Sunfire Prep C18 column, 90-100% B gradient),
yielding 6.1 mg (20%) of the desired product as a yellow solid. 1H NMR
(400 MHz, CDCl3) δ 15.56 (s, 1 H), 7.52-7.24 (m, 10 H), 5.36 (s,
2 H), 4.99-4.64 (m, 2 H), 4.02 (d, J = 11.0 Hz, 1 H), 3.10-2.81 (m, 2 H),
2.78 (s, 6 H), 2.62-2.40 (m, 10 H), 2.20-2.14 (m, 1 H), 1.80-1.45 (m,
2 H), 1.44-1.15 (m, 11 H), 1.00-0.70 (m, 12 H), 0.25 (s, 3 H), 0.12 (s,
3 H). MS (ESI) m/z 908.65 (M þ H).
The following compounds were prepared according to methods
similar to 26a:
7-Chloro-6-demethyl-6-deoxy-9-propyl-8-azatetracycline
Hydrochloride (26b). Prepared from 19h and allylboronic acid
1
pinacol ester, yellow solid. H NMR (400 MHz, CD3OD) δ 4.10 (s,
1 H), 3.22-2.92 (m, 9 H), 2.81-2.74 (m, 2 H), 2.49-2.38 (m, 1 H),
2.26-2.19 (m, 1 H), 1.77 -1.60 (m, 3 H), 0.97 (t, J = 7.3 Hz, 3 H). MS
(ESI) m/z 492.27 (M þ H).
7-Chloro-6-demethyl-6-deoxy-9-methyl-8-azatetracycline
Hydrochloride (26c). Prepared from 19h and methylboronic acid,
1
yellow solid. H NMR (400 MHz, CD3OD) δ 4.09 (s, 1 H), 3.22-
2.92 (m, 9 H), 2.85 (s, 3 H), 2.49-2.38 (m, 1 H), 2.26-2.19 (m, 1 H),
1.77 -1.60 (m, 1H). MS (ESI) m/z 464.30 (M þ H).
6-Demethyl-6-deoxy-7-(dimethylamino)-9-(propylamino)-
8-azatetracycline Dihydrochloride (24o). Aqueous HF (48%,
0.4 mL) was added to a solution of 20o (6.1 mg, 0.007 mmol) in CH3CN
6-Demethyl-6-deoxy-9-methyl-8-azatetracycline Dihydro-
chloride (26d). Prepared from 19h as an over-reduced side product
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Journal of Medicinal Chemistry
ARTICLE
in the synthesis of 26c. 1H NMR (400 MHz, CD3OD) δ 7.78 (s, 1 H),
4.12 (s, 1 H), 3.22-2.91 (m, 9 H), 2.78 (s, 3 H), 2.49-2.21 (m, 1 H),
1.77-1.62 (m, 1H). MS (ESI) m/z 430.46 (M þ H).
’ AUTHOR INFORMATION
Corresponding Author
*Phone: (617) 715-3558. Fax: (617) 926-3557. E-mail: rclark@
tphase.com.
7-Chloro-6-demethyl-6-deoxy-9-phenyl-8-azatetracycline
Hydrochloride (26e). Prepared from 19h and phenylboronic
acid. 1H NMR (400 MHz, CD3OD) δ 8.04-7.98 (m, 2 H),
7.47-7.39 (m, 3 H), 4.08 (s, 1 H), 3.14-2.92 (m, 9 H), 2.57-
2.47 (m, 1 H), 2.28-2.07 (m, 1 H), 1.73-1.65 (m, 1H). MS (ESI)
m/z 526.26 (M þ H).
’ ACKNOWLEDGMENT
We thank members of the Tetraphase Process Chemistry
Group for preparing the enone 8 as well as Professor Andrew
Myers and Bob Zahler for valuable discussions over the course
of this study.
6-Demethyl-6-deoxy-9-phenyl-8-azatetracycline Dihydro-
chloride (26f). Prepared from 19h as an over-reduced side product in
the synthesis of 26e. 1H NMR (400 MHz, CD3OD) δ 8.04-7.98 (m,
2 H), 7.47-7.39 (m, 3 H), 4.08 (s, 1 H), 3.14-2.92 (m, 9 H), 2.57-2.47
(m, 1 H), 2.28-2.07 (m, 1 H), 1.73-1.65 (m, 1H). MS (ESI) m/z
526.26 (M þ H).
’ ABBREVIATIONS USED
Susceptibility Testing. Compound stocks were prepared 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)), 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).26 Cation-adjusted
Mueller Hinton broth was obtained from BBL (cat. no. 212322,
Becton Dickinson, Sparks, MD), prepared fresh and kept at 4 °C
prior to testing. Defibrinated horse blood (cat. no. A0432, PML
Microbiologicals, Wilsonville, OR) was used to supplement med-
ium, 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.
In vitro Transcription/Translation Assay. Compound stocks
prepared and diluted in sterile deionized water were assayed for
inhibition of coupled in vitro transcription/translation using an E. coli
S30 extract system with a firefly luciferase readout from Promega (cat.
no. L1020, Madison, WI). Briefly, compounds were diluted into water
and added to reaction mix aliquoted to black-walled 96-well microtiter
plates (cat. no. 3650, Costar, Corning, NY). An appropriate three-point
titration was used for each compound, and reactions were run in
duplicate. The final total reaction volume was 20 μL. Plates were
incubated at 37 °C for one hour and then placed on ice for 5 min to
arrest transcription/translation. Luciferase substrate (25 μL, Promega
cat. no. E1500) was added to each well and luminescence was detected
on a BMG LabTech LUMIstar-OPTIMA instrument. Positive assay
control values, from reactions without inhibitor, were averaged per plate
to determine percent inhibition of luciferase production. Results were
plotted using Microsoft Excel and 50% inhibition values (IC50) were
determined.
ATCC, American Type Culture Collection; AUC, area under the
curve; Boc, t-butoxycarbonyl; Cbz, benzyloxycarbonyl; Cl, clear-
ance; Cy, cyclohexyl; dba, dibenzylideneacetone; DMAP, 4-(dimethyl-
amino)pyridine; dppf, (diphenylphosphino)ferrocene; %F, percent
oral bioavailability; IV, intravenous; LDA, lithium diisopropyla-
mide; LHMDS, lithium bis(trimethylsilyl)amide; mCPBA,
3-chloroperbenzoic acid; MIC, minimum inhibitory concentra-
tion; MIC50, minimum inhibitory concentration required to
inhibit growth of 50% of organisms; MIC90, minimum inhibitory
concentration required to inhibit growth of 90% of organisms;
MRSA, methicillin-resistant Staphylococcus aureus;NMP, 2-methyl-
pyrrolidinone; nOe, nuclear Overhouser effect; PD50, dose
at which 50% protection is observed; PO, oral; TMEDA, N,
N,N0,N0-tetramethylethylenediamine; Vz, volume of distribu-
tion; Xantphos, 9,9-dimethyl-4,5-bis-(diphenylphosphino)-
xanthenes
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