Fluorocyclines. 2. Optimization of the C-9 side-chain for antibacterial activity and oral efficacy

Article

pubs.acs.org/jmc

Fluorocyclines. 2. Optimization of the C-9 Side-Chain for

Antibacterial Activity and Oral Efficacy

Roger B. Clark,*^,†Diana K. Hunt,^†Minsheng He,^†Catherine Achorn,^‡Chi-Li Chen,^†Yonghong Deng,^†

Corey Fyfe,^‡Trudy H. Grossman,^‡Philip C. Hogan,^§William J. O’Brien,^‡Louis Plamondon,^†

Magnus Ronn,^§Joyce A. Sutcliffe,^‡Zhijian Zhu,^§and Xiao-Yi Xiao^†

̈

‡

§

^†Discovery Chemistry, Microbiology, and Process Chemistry R&D, Tetraphase Pharmaceuticals, 480 Arsenal Street, Watertown,

Massachusetts 02472, United States

S

* Supporting Information

ABSTRACT: Utilizing a fully synthetic route to tetracycline analogues, the C-9 side-chain of the fluorocyclines was optimized

for both antibacterial activity and oral efficacy. Compounds were identified that overcome both efflux (tet(K), tet(A)) and ribo-

somal protection (tet(M)) tetracycline-resistance mechanisms and are active against Gram-positive and Gram-negative organ-

isms. A murine systemic infection model was used as an oral efficacy screen to rapidly identify compounds with oral

bioavailability. Two compounds were identified that exhibit both oral bioavailability in rat and clinically relevant bacterial sus-

ceptibility profiles against major respiratory pathogens. One compound demonstrated oral efficacy in rodent lung infection

models that was comparable to marketed antibacterial agents.

inaccessible via semisynthesis. While 7-fluorosancycline (7a,

⁹R = H) has been prepared by fluorination of sancycline, the

harsh conditions and subsequent low yields render the route

impractical for generating sufficient quantities of material.¹⁶

Furthermore, substitution at other ring positions on 7-fluoro-

sancycline has not been reported. By employing a tandem

Michael−Dieckmann condensation between a D-ring precursor

5 and enone 6, the 7-fluorocyclines (7) can be readily accessed

in sufficient quantities and with appropriate chemical

protection to allow for rapid and efficient analoguing.

A particularly attractive aspect of the tetracyclines in general

is their oral bioavailability (F) in humans.¹⁷In fact, almost all of

the older tetracyclines can be administered orally, with mino-

cycline and doxycycline having nearly 100% oral bioavail-

ability.¹⁸One complication for drug discovery in the tetra-

cycline class, however, is that oral bioavailability in rodents (rats

and mice), the most commonly used and cost-effective species

for infection models and pharmacokinetics, can be significantly

lower than that in humans. For example, tetracycline (2) is

generally reported to have oral bioavailability of 50−70% in

humans,¹⁹whereas we have found the oral bioavailability to be

only about 15% in rats and 6.3% in mice. Omadacycline appears

to have a similar profile as it has very limited oral bioavailability

in rats (0.7% F) and mice (1.5% F) but has been reported to

have 33% oral bioavailability in man in a phase 2 clinical trial.¹⁰

The C-9 glycylamide side chains found in tigecycline and 1 also

INTRODUCTION

■

Widespread use of the current arsenal of approved antibacterial

agents and the resulting development of bacterial resistance

continues to necessitate the discovery and development of new

antibiotics.¹⁻⁴In the preceding paper (DOI: 10.1021/

jm201465w),²⁵we outlined the discovery of TP-434 (1),⁵a

new broad-spectrum tetracycline analogue active against multi-

drug-resistant Gram-positive and Gram-negative pathogens that

is currently undergoing phase 2 clinical trials in patients with

complicated intra-abdominal infections (cIAI) as well as phase

1 trials to explore its potential for use as an oral therapy. This

compound is a member of the broader class of 7-fluoro-6-

demethyl-6-deoxytetracyclines which we have termed “fluo-

rocyclines”. Importantly, many of these compounds have been

shown to overcome the two major bacterial resistance mecha-

nisms identified for tetracyclines: (1) tetracycline-specific efflux

pumps⁶(i.e., tet(A-E))^6a,band tet(K-L)^6c) and (2) ribosomal

protection (i.e., tet(M-O)).⁷

Traditionally, tetracyclines have been prepared by semisyn-

thesis, a technique which is largely limited to electrophilic aro-

matic substitution reactions at the 7- and 9-positions of the

tetracycline D-ring.⁸Recent examples of this include the 9-

substituted glycylcyclines, such as tigecycline (3),⁹and the

aminomethylcyclines, such as omadacycline (4),¹⁰both of

which have shown improved activity for tetracycline-resistant

organisms (Figure 1). More recently, we have extended the

total synthetic methodology developed by Myers and co-

workers¹¹⁻¹³to the preparation of 8-azatetracyclines¹⁴and 8,9-

pentacyclines,¹⁵generating completely novel analogues that are

Received: October 31, 2011

Published: December 9, 2011

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

appear to impart low oral bioavailability in rats and mice, while

data in humans has not been reported. As previously reported,

the oral bioavailability of 1 appears to improve in larger ani-

mals, leading us to believe low bioavailability in rats may not

adequately predict oral bioavailability in humans.²⁰In this

paper, we describe a detailed study of 9-substituted-7-fluoro-

cyclines with two goals: (a) to explore a more diverse set of

chemical space at the C-9 position and (b) to identify com-

pounds with improved oral efficacy in lower animal species

(rodents), enabling a more complete pharmacological and

pharmacokinetic evaluation of compound activity in animal

models.

12 was nitrated, the phenol was protected with benzyl bromide,

and the nitro group was reduced using sodium hydrosulfite to

give compound 13 in 80% overall yield. Protection of the

aniline by alkylation with allylbromide gave the diallylamino

compound 14 in 89% yield. Other aniline protecting groups

were explored but were either incompatible with the Michael−

Dieckmann annulation conditions (i.e., mono- or di-Boc) or

were not orthogonal (i.e., dibenzyl). The Michael−Dieckmann

annulation was carried out by deprotonation of 14 with lithium

diisopropylamide (LDA) at −78 °C, followed by addition

of the enone 6 and subsequent addition of lithium bis-

(trimethylsilyl)amide (LHMDS). Warming to −10 °C gave the

fully protected intermediate 15 in moderate yield. The aniline

intermediate 16 was then obtained in 71% yield upon treatment

of compound 15 with (Ph₃P)₄Pd and N,N-dimethylbarbituric

acid.

Compound 16 was readily derivatized as outlined in Scheme 3.

Acylation upon reaction with acid chlorides or with car-

boxylic acids mediated by HATU provided the protected amide

intermediates 17, which were desilylated with aqueous HF and

hydrogenated to give the final compounds 9v−bb. A palladium-

mediated amination was carried out on compound 16 using

4,6-dichloropyrimidine as the coupling partner, Pd₂dba₃and

9,9-dimethyl-4,5-bis-(diphenylphosphino)xanthenes (Xant-

phos) as catalysts, and K₃PO₄as base.²²Deprotection as above

gave compound 18. The 9-amino group of 16 was also func-

tionalized via reductive aminations with aldehydes, acetic acid,

and Na(OAc)₃BH to give the protected intermediates 19,

which were deprotected as described above to yield the 9-

aminoalkyl compounds 20a−e.

RESULTS AND DISCUSSION

■

Chemistry. As described in the preceding paper,²⁵many of

the 7-F-9-amino derivatives were prepared from compound 8.

Treatment of 8 with either acid chlorides or carboxylic acids

and 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium

hexafluorophosphate (HATU) gave the 9-amido compounds

9 (Scheme 1). For the saturated heterocyclic compounds 9j−o

and 9s−u, the side chains were introduced as the benzyl-

oxycarbonyl (Cbz) protected amines and were deprotected

under hydrogenation conditions to give the secondary amines

9j, 9k, 9s, and 9u. These compounds were further function-

alized by reductive aminations with aldehydes or ketones to

give compounds 9l−o and 9t. The 9-sulfonamides 10a−c were

prepared by reaction of 8 with the corresponding sulfonyl

chlorides, while the 9-urea substituted fluorocycline 11 was

generated from 8 upon treatment with methylisocyanate.

While derivatization of compound 8 as described above was

effective in many cases, we found that reactions involving more

forcing conditions, such as strong base or prolonged heating,

led to significant decomposition. Additionally, it is known that

epimerization of the C-4 dimethylamino group can occur in the

pH range of 2−6.²¹Thus, we have developed chemistry to enable

functionalization of the 9-position prior to deprotection to the

final tetracycline compounds (Scheme 2). Aromatic compound

9-Aminomethyl-7-fluorocyclines were prepared from 7-

fluorocycline 7a (Scheme 4). Thus, compound 7a was treated

with benzyl N-(hydroxymethyl)carbamate in TFA/CH₃SO₃H

to provide the 9-methylamino compound 21. Reductive amina-

tion with pivaldehyde and Na(OAc)₃BH provided the alkylated

compound 22. Acylation of 21 with 2-t-butylaminoacetyl-

chloride gave the amide 23.

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a

Scheme 1. Synthesis of 9-Amido-, 9-Sulfonamido-, and 9-Urea-7-fluorocyclines

a

Reagents and conditions: (i) for 9a−9k, RCOCl, DMF or RCOCl, Na₂CO₃, THF; (ii) for 9p−s and 9u, RCO₂H, HATU, Et₃N, DMF; (iii) H₂,

Pd/C, MeOH; (iv) R₁R₂CO, Et₃N, Na(OAc)₃BH, DMF; (v) RSO₂Cl, DIEA, THF; (vi) CH₃NCO, Et₃N, CH₂Cl₂.

a

Scheme 2. Alternate Synthesis of 9-Amino-7-fluorocyclines

a

Reagents and conditions: (i) HNO₃, n-Bu₄NBr, 1,2-dichloroethane, water; (ii) BnBr, K₂CO₃, KI, acetone, 56 °C; (iii) Na₂S₂O₄, THF, water, 10 °C

to room temp; (iv) allylbromide, K₂CO₃, KI, NMP, 100 °C; (v) (a) LDA, TMEDA, −78 °C, (b) 6, LHMDS, −78 °C to −10 °C; (vi) (Ph₃P)₄Pd,

N,N-dimethylbarbituric acid, CH₂Cl₂, 35 °C.

Biology. The in vitro antibacterial activities of all analogues

were determined for a panel of Gram-positive (e.g., Staph-

ylococcus aureus and Streptococcus pneumoniae) and Gram-

negative (e.g., Escherichia coli and Klebsiella pneumoniae)

bacteria. Initially, a set of 7-fluorocyclines was prepared and

assayed in order to explore the structure−activity relationships

(SAR) of the C-9 glycylamido and aminomethyl side chains

(Table 1). The unsubstituted 9-amino-7-fluorocycline 8 showed

comparable minimum inhibitory concentrations (MICs) to

tetracycline for the tetracycline-susceptible strains but had

significantly improved activity for the strains bearing tet(M)

and tet(K) resistance mechanisms. Addition of the N-t-butyl-

glycylamido side chain (9a) resulted in further improvement in

all of the tetracycline-resistant strains, including tet(A). Activity

was comparable to tigecycline for the Gram-positive strains but

was 2−6-fold less potent for the Gram-negative strains.

Removal of the carbonyl (20a) gave a 4−8-fold loss in activity

across all strains, but activity similar to compound 8 was

retained. Further removal of the secondary amino group to an

alkylamino side-chain (20c−e) resulted in improved activity for

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a

Scheme 3. Alternate Synthesis of 9-Amino-7-fluorocyclines

a

Reagents and conditions: (i) for 9v−z, RCOCl, Et₃N, THF; (ii) for 9aa−bb, RCO₂H, HATU, Et₃N, THF or CH₂Cl₂; (iii) aq HF, 1,4-dioxane or

CH₃CN; (iv) H₂, Pd/C, MeOH; (v) 4,6-dichloropyrimidine, Pd₂dba₃, Xantphos, K₃PO₄, 1,4-dioxane, 80 °C; (vi) R₁CHO, AcOH, Na(OAc)₃BH,

CH₂Cl₂or DCE.

a

Scheme 4. Synthesis of 9-Aminomethyl-7-fluorocyclines

a

Reagents and conditions: (i) HOCH₂NHCO₂Bn, TFA, CH₃SO₃H; (ii) t-BuCHO, AcOH, Na(OAc)₃BH, DMF; (iii) t-BuNHCH₂COCl·HCl, DMF.

the S. aureus strains, but further reduction in potency for both

the S. pneumoniae and Gram-negative strains. Moving to the

smaller and more polar dimethylamino analogue 20b gave

somewhat improved MICs for the Gram-negative strains but

reduced activity for the S. aureus strains. The omadacycline-type

side chain was also prepared and assayed (22), yielding a

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Table 1. In Vitro Antibacterial Activity of 9-Substituted-7-fluorocyclines

a

b

c

d

ATCC 13709. Obtained from Micromyx (Kalamazoo, MI). Obtained from Marilyn Roberts’ lab at the University of Washington. ATCC 49619.

e

f

ATCC 25922. ATCC 13883.

similar overall profile to omadacycline, albeit with a 2−8-fold

decrease in potency. The presence of the benzylic amine did

result in improved Gram-negative and S. pneumoniae activity

relative to similar aniline compounds (20c−e) although not

nearly to the extent as the glycylamido side chain in 9a, 1, or

tigecycline. The combination of the tigecycline- and omadacy-

cline-type side chains (23) led to significant loss in activity

across all strains. Overall from this initial study it appeared that

a more polar side chain (i.e., small amino group, amide, or

secondary amine) was a key factor in obtaining Gram-negative

and S. pneumoniae activity, whereas the presence of a larger

group was the greatest factor in determining S. aureus, and in

particular, tet(M) activity.

Because the amide bond appeared to be important for good

broad-spectrum activity, we prepared and assayed a set of amide

isosteres to further probe this space (Table 2). The parent

benzamide compound 9b retained balanced activity across the

tetracycline-sensitive and resistant S. aureus strains but lacked

significant activity for the S. pneumoniae and Gram-negative

strains. The more polar sulfonamide compounds (10a−c) were

able to pick up the S. pneumoniae activity, with the most polar

methyl analogue 10c also having good activity for the two

tetracycline-sensitive Gram-negative strains. Unfortunately, this

compound had poor activity for the two strains with efflux

resistance mechanisms, tet(K) and tet(A). The urea analogue 11

showed similar activity to 10c, although the activity for all of

the resistant strains was further reduced. The pyrimidine ana-

logue 18 had fairly similar Gram-positive activity to the benz-

amide 9b and the sulfonamide 10a, while the Gram-negative

activity was improved for the tetracycline-sensitive Gram-

negative strains. The compound still lacked activity for the

tet(A) strain.

Expanding on this SAR information, a series of substituted

aryl and heteroaryl amides was prepared with the goal of in-

creasing polarity to improve Gram-negative activity (Table 3).

As with the benzamide 9b, all of the compounds except 9i had

good activity against S. aureus, including the tet(M) and tet(K)

strains, while none of the compounds exhibited significant anti-

bacterial activity for the Gram-negative strains tested. The main

differences were seen in the S. pneumoniae activity. Substitution

at the 2-position (9e) or the 3-position (9c, 9d, 9f) was not

beneficial for activity. In contrast to the corresponding 2- and 3-

substituted compounds 9e and 9f, the 4-dimethylamino

analogue 9g had improved activity for both S. pneumoniae

strains. All of the heteroaryl compounds had some activity for S.

pneumoniae, with the more polar analogues (9i, 9v, 9x−z)

having 2−3 dilution more potent activity than the less polar

compounds (9h, 9w). Interestingly, the 2-pyridyl compound 9v

and the isosteric 2-thiazolyl analogue 9z were significantly more

potent against S. aureus than the 3-pyridyl compound 9i.

On the basis of results from the heteroaryl amides, we next

explored a series of saturated heterocyclic amides (Table 4).

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Table 2. In Vitro Antibacterial Activity of 7-Fluorocyclines with Amide Bond Replacements at C9

a

b

c

d

ATCC 13709. Obtained from Micromyx (Kalamazoo, MI). Obtained from Marilyn Roberts’ lab at the University of Washington. ATCC 49619.

e

f

ATCC 25922. ATCC 13883.

Table 3. In Vitro Antibacterial Activity of 9-Heteroarylamido-7-fluorocyclines

a

b

c

d

ATCC 13709. Obtained from Micromyx (Kalamazoo, MI). btained from Marilyn Roberts’ lab at the University of Washington. ATCC 49619.

e

f

ATCC 25922. ATCC 13883.

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For the most part, these compounds had significantly improved

antibacterial activity, especially for the S. pneumoniae and

the Gram-negative strains tested. One exception was the tet(A)

E. coli strain for which only compound 9bb had an MIC of

1 μg/mL or less. Ring size was a key factor in potency,

with antibacterial activity improving as ring size was reduced

(9bb > 9l > 9t). The stereochemistry of the α-position of the

amide side chain was also important, with the (S)-stereochemis-

try providing the best activity in each case. For the 4- and 5-

membered ring compounds, the N-methyl amines (9bb, 9l)

were more potent than the corresponding N-H amines (9aa,

9j), while the opposite was seen for the 6-membered ring

Table 4. In Vitro Antibacterial Activity of 9-Heterocyclicamido-7-fluorocyclines

a

b

c

d

ATCC 13709. Obtained from Micromyx (Kalamazoo, MI). Obtained from Marilyn Roberts’ lab at the University of Washington. ATCC 49619.

e

f

ATCC 25922. ATCC 13883.

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compounds (9s vs 9t). Substitution on the nitrogen was further

explored in the pyrrolidine series. Here, the N-ethyl compound

9n was 1−2 dilutions more potent than 9l, while the N-

isopropyl compound 9o was less active than either compound.

Finally, several fluorine substituted compounds were prepared

(9p−r) in order to explore the effect of changes in the basicity

of the nitrogen atom on activity. For the monofluoro com-

pound 9q, the Gram-positive activity remained similar to the

parent compound 9l, while the Gram-negative activity was

clearly reduced. The corresponding diastereomer 9p was

somewhat less potent at all strains tested. The difluoro

compound 9r was the least potent compound in the series, in

particular for the Gram-negative strains. Once again, the

overall data suggests that a more polar or more basic

compound gives better antibacterial activity, particularly for

Gram-negative bacteria.

We chose to screen and rank compounds for oral bioavail-

ability using a mouse systemic infection model challenged with

S. aureus ATCC 13709. Six mice per group were dosed at either

3 mg/kg intravenously (IV) or 30 mg/kg orally (PO), and

survival was recorded at 48 h post dose. To validate this

screening paradigm, three control tetracyclines were tested in

the efficacy screen and oral bioavailability was assessed in mice

and rats (Table 5). Tetracycline, with 6.3% F in mice and 14.7%

only modest oral efficacy. The two heterocyclic amides (9bb,

9l) performed the best in the efficacy screen, providing nearly

complete protection in both the IV and PO arms. On the basis

of these results, several other pyrrolidinyl amides (9j, 9n, 9q)

were also screened. All three compounds showed reasonable

efficacy following IV dosing. Despite differing only by the

amino substituent (NH or N-Et vs N-Me) or by a fluorine

atom on the ring, none of the compounds afforded greater than

40% survival when dosed orally. To ensure against false

negatives, PD₅₀s were determined for several of the com-

pounds. All of the compounds that showed less than 50% sur-

vival in the screen had PD₅₀s greater than 30 mg/kg.

Compound 9bb, with 83% survival in the oral screen, had a

PD₅₀of 14.3 mg/kg. Compounds 9bb and 9l were selected for

pharmacokinetic (PK) analysis in rats and were found to have

oral bioavailabilities of 4.6% and 5.7%, respectively. While these

values would generally be considered low, they are significantly

better than the glycylcyclines and omadacycline. It is pos-

sible that oral bioavailability for these compounds will improve

in higher mammals as has been observed for tetracycline,

omadacycline, and 1.

On the basis of the efficacy and oral bioavailability data,

compounds 9bb and 9l were advanced for further profiling

against panels of recently isolated respiratory pathogens (Table 6).

Linezolid and levofloxacin were screened as relevant standard

of care therapies for respiratory infections. For methicillin-

resistant S. aureus (MRSA), both compounds had superior

MIC₉₀values as compared to linezolid, although 9l distin-

guished itself with an MIC₉₀value of 0.13 μg/mL. Most

of these strains were resistant to levofloxacin. Against

S. pneumoniae, 9bb and 9l were very potent, with MIC₉₀values

of 0.016 μg/mL, while both marketed agents had MIC₉₀values

of 1 μg/mL. The two tetracycline analogues were both superior

to linezolid against Hemophilis influenzae, with MIC₉₀values of

0.5 and 0.25 μg/mL vs 16 μg/mL for linezolid. Levofloxacin

was more potent, with an MIC₉₀value of 0.031 μg/mL.

Compounds 9l and 9b were also screened against Streptococcus

pyogenes and Moraxella catarrhalis and were both found to have

MIC₉₀values of 0.016 μg/mL against these species, significantly

more potent than linezolid and levofloxacin. Compounds were

tested for activity against Acinetobacter baumannii, an

opportunistic multidrug-resistant Gram-negative pathogen and

cause of nosocomial infections including pneumonia.²³Against

a panel of 25 A. baumannii isolates, both 9b and 9l had MIC₉₀

values of 1 vs 32 μg/mL for levofloxacin. Overall, compound 9l

was more potent against almost all species and appeared to be

able to provide broader coverage across these species than the

two marketed comparator antibiotics.

Table 5. Oral Efficacy Screen Results

a

S. aureus

survival (%)

IV, PO,

PD₅₀

PO

% F % F

MIC

compd

9bb

(μg/mL)

3 mg/kg 30 mg/kg (mg/kg) mouse rat

0.016

0.25

0.13

0.031

0.13

0.063

0.13

0.25

1

100

67

83

0

14.3

4.6

9j

9l

100

40

33

50

0

5.7

9n

9q

83

9v

0

>30

9z

17

10c

100

83

50

33

100

0

>30

>60

11

20d

17

tetracycline

tigecycline

omadacycline

100

6.9

6.3

BLQ

1.5

14.9

1.1

0.063

0.5

33

30.3

0.7

a

ATCC 13709.

F in rats, was used as a positive control and provided 100%

survival after both IV and oral administration in the mouse

efficacy model. As negative controls, tigecycline (below

level of quantitation (BLQ) in mice and 1.1% F in rats) and

omadacycline (1.5% in mice and 0.7% F in rats) both

provided minimal protection after oral administration (0%

and 33% survival, respectively) while affording 100%

survival when dosed IV in the efficacy screen. As additional

validation for the screen, oral PD₅₀s were determined for

tetracycline (6.9 mg/kg) and omadacycline (30.3 mg/kg),

the results of which appear to correlate well with the oral

bioavailability data.

Compounds were selected based on both antibacterial activ-

ity and chemical diversity and screened in the mouse sepsis

model. Neither the heteroaryl amides (9v, 9z) nor the alkyl-

amine (20d) exhibited substantial efficacy in either the IV or

PO arm. Both the sulfonamide (10c) and the urea (11) ana-

logues provided good protection by IV administration but had

Compound 9l was profiled in two neutropenic mouse lung

infection models with tet(M) resistant strains (Figure 2A,B). In

the MRSA lung infection model, 9l (MIC = 0.25 μg/mL)

exhibited similar efficacy to linezolid (MIC = 2 μg/mL) with a

1.99 vs 2.46 log₁₀reduction in colony forming units (CFUs)/g

lung tissue when dosed orally at 50 mg/kg. In the IV arm, 9l

had greater efficacy than linezolid (3.03 vs 1.29 log₁₀reduction

in CFU/g lung tissue). With an even larger advantage in MIC

for the S. pneumoniae lung infection model, 9l (MIC ≤ 0.016

μg/mL) showed greater efficacy than linezolid (MIC = 0.5 μg/mL)

in both the PO and IV arms of the study. Two immuno-

competent lung infection models were also examined. In an

S. pneumoniae mouse lung infection model (Figure 2C),

compounds were dosed orally at 30 mg/kg, with 9l (MIC ≤

0.008 μg/mL) exhibiting a 4.75 log₁₀reduction in CFU/g lung

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Table 6. MIC₅₀and MIC₉₀Values for 9l and 9bb

species

MIC (μg/mL)

9l

9bb

linezolid

levofloxacin

S. aureus (MRSA, n = 32)

MIC₅₀

0.031

0.13

0.031

1

2

4

16

>32

MIC₉₀

MIC range

0.016−1

0.016−4

2−4

0.25−>32

S. pneumoniae (n = 19)

H. influenzae (n = 14)

S. pyogenes (n = 14)

MIC₅₀

0.016

1

MIC₉₀

MIC range

0.016−0.016

0.5−1

MIC₅₀

0.25

0.5

0.063

0.25

8

0.016

0.031

MIC₉₀

16

MIC range

0.031−0.5

0.016−0.5

8−16

0.016−0.031

MIC₅₀

0.016

1

2

0.5

2

MIC₉₀

MIC range

0.016−0.016

1−2

0.25−2

M. catarrhalis (n = 14)

A. baumannii (n = 25)

MIC₅₀

0.016

8

0.031

0.063

MIC₉₀

MIC range

0.016−0.063

2−8

0.016−0.063

MIC₅₀

0.13

1

0.13

1

NT

4

32

MIC₉₀

MIC range

0.016−2

0.13−>32

Figure 2. Efficacy of 9l in rodent respiratory infection models.

tissue vs a 3.56 log₁₀reduction in CFU/g lung tissue for line-

zolid (MIC = 0.5 μg/mL). In an H. influenzae rat lung infection

model, 9l (MIC = 0.063 μg/mL) produced a 1.77 log₁₀

reduction in CFU/g lung tissue when dosed orally at 100

mg/kg. Compound 9l exhibited good efficacy when dosed at

25 mg/kg IV, with a 4.87 log₁₀reduction in CFU/g lung tissue.

The bacterial count was below the level of quantitation (≥6

log₁₀reduction in CFU/g lung tissue) for azithromycin ((MIC

= 1 μg/mL) when dosed orally at 50 mg/kg. In summary,

compound 9l exhibited significant oral activity in all four

respiratory infection models, with efficacy comparable to or

greater than linezolid.

CONCLUSIONS

■

Utilizing a fully synthetic chemistry platform to generate novel

tetracyclines that in many cases are not readily accessed by

traditional semisynthesis, we were able to significantly expand

the chemical space at the C-9 position of the fluorocyclines.

This approach demonstrates the ability to efficiently optimize

compounds for both antibacterial activity, including overcoming

614

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Journal of Medicinal Chemistry

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solution of compound 8 (20 mg, 0.045 mmol) in THF was added

Na₂CO₃(9.5 mg, 0.089 mmol) and 0.1 mL of a benzoyl chloride

solution (54 μL in 1 mL THF, 0.047 mmol). The reaction mixture

was stirred at room temperature for 1 h. HCl in MeOH (1 mL, 4 N)

was added to the mixture at 0 °C. After 2 min, the mixture was

concentrated under reduced pressure and the residue 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 5.5 mg (21%) of the title compound as a

both efflux and Tet(M)-mediated ribosomal protection tetracy-

cline-resistance mechanisms, and pharmacokinetic properties.

Specifically, two compounds (9bb, 9l) were identified that

showed superior potency versus standard of care therapies

against panels of clinically important respiratory pathogens.

Using a mouse sepsis model to screen for oral efficacy, the oral

bioavailability of compound 9l was initially demonstrated,

enabling a more extensive evaluation of oral efficacy in several

rodent lung infection models. While oral bioavailability in rat

was found to be low, precedents set by other members of the

tetracycline class suggest further examination of compound 9l

as an oral therapy in higher mammals is warranted.

1

yellow solid. H NMR (400 MHz, CD₃OD) δ 8.23 (d, J = 10.8 Hz,

1 H), 7.97 (d, J = 7.6 Hz, 2 H), 7.66−7.54 (m, 3 H), 4.11 (s, 1 H),

3.21−2.90 (m, 9 H), 2.37−2.24 (m, 2 H), 1.72−1.66 (m, 1 H). MS

(ESI) m/z 552.1 (M + H).

The following compounds were prepared by similar methods to 9b:

(4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12a-

tetrahydroxy-1,11-dioxo-9-C-[3-(trifluoromethyl)benzene]-

1,4,4a,5,5a,6,11,12a-octahydrotetracene-2,9-dicarboxamide

EXPERIMENTAL PROCEDURES

■

General. Air and moisture sensitive liquids and reagents were

transferred via syringe or cannula and were introduced into flame-

dried glassware under a positive pressure of dry nitrogen through

rubber septa. All reactions were stirred magnetically. Commercial

reagents were used without further purification. Analytical thin-layer

chromatography was performed on EM Science precoated glass-

backed silica gel 60 Å F-254 250 μm plates. Visualization of the plates

was effected by ultraviolet illumination and/or immersion of the plate

in a basic solution of potassium permanganate in water followed by

heating. Column chromatography was performed on a 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 Auto-

purification 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 tetracycline 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). Unless otherwise described, all final

tetracycline compounds were isolated as mono-, di-, or trihydrochlo-

ride salts following freeze-drying. ¹H NMR spectra were recorded on a

JEOL ECX-400 (400 MHz) spectrometer and are reported in ppm

using residual solvent as the internal standard (CDCl₃at 7.24 ppm,

DMSO-d₆at 2.50 ppm, or CD₃OD at 3.31 ppm). High performance

liquid chromatography−electrospray mass spectra (LC-MS) were

obtained using an Waters Alliance HPLC system equipped with a

binary pump, a diode array detector, a Waters Sunfire C18 (5 μm,

4.6 mm I.D. × 50 mm) column, 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 = 0.5 min, 100% A; (c) time = 3.5 min, 100% B; (d)

time = 5 min, 100% B; (e) time = 6 min, 100% A; (f) time = 7 min,

100% A. All final products were ≥95% purity as assessed by this

method.

1

Hydrochloride (9c). Yellow solid. H NMR (400 MHz, CD₃OD)

δ 8.25 (s, 1 H), 8.21 (d, J = 8.0 Hz, 1 H), 8.14 (d, J = 10.4 Hz, 1 H),

7.92 (d, J = 8.0 Hz, 1 H), 7.76 (t, J = 8.0 Hz, 1 H), 4.08 (s, 1 H),

3.21−2.89 (m, 9 H), 2.35−2.22 (m, 2 H), 1.71−1.61 (m, 1 H). MS

(ESI) m/z 620.1 (M + H).

(4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-3,10,12,12a-

tetrahydroxy-9-C-(3-methoxybenzene)-1,11-dioxo-

1,4,4a,5,5a,6,11,12a-octahydrotetracene-2,9-dicarboxamide

1

Hydrochloride (9d). Yellow solid. H NMR (400 MHz, CD₃OD)

δ 8.10 (d, J = 10.8 Hz, 1 H), 7.41−7.33 (m, 3 H), 7.09−7.07 (m, 1 H),

4.00 (s, 1 H), 3.78 (s, 3 H), 3.12−2.86 (m, 9 H), 2.23−2.13 (m, 2 H),

1.60−1.50 (m, 1 H). MS (ESI) m/z 582.1 (M + H).

( 4 S , 4 a S , 5 a R , 1 2 a S ) - 4 - ( D i m e t h y l a m i n o ) - 9 - C - [ 2 -

(dimethylamino)benzene]-7-fluoro-3,10,12,12a-tetrahydroxy-

1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2,9-dicar-

boxamide Dihydrochloride (9e). Yellow solid. ¹H NMR (400

MHz, CD₃OD) δ 8.19−8.14 (m, 2 H), 8.05 (d, J = 8.4 Hz, 1 H),

7.91−7.89 (m, 1 H), 7.76−7.74 (m, 1 H),.4.12 (s, 1 H), 3.32 (s, 6 H),

3.21−2.96 (m, 9 H), 2.41−1.98 (m, 2 H), 1.72−1.59 (m, 1 H). MS

(ESI) m/z 595.0 (M + H).

( 4 S , 4 a S , 5 a R , 1 2 a S ) - 4 - ( D i m e t h y l a m i n o ) - 9 - C - [ 3 -

(dimethylamino)benzene]-7-fluoro-3,10,12,12a-tetrahydroxy-

1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2,9-dicar-

boxamide Dihydrochloride (9f). Yellow solid. ¹H NMR (400 MHz,

CD₃OD) δ 8.13−8.06 (m, 2 H), 7.98 (d, J = 7.6 Hz, 1 H), 7.77 (d, J =

7.2 Hz, 1 H), 7.67 (t, J = 8.0 Hz, 1 H), 4.01 (s, 1 H), 3.26 (s, 6 H),

3.14−2.83 (m, 9 H), 2.27−2.13 (m, 2 H), 1.64−1.52 (m, 1 H). MS

(ESI) m/z 595.1 (M + H).

( 4 S , 4 a S , 5 a R , 1 2 a S ) - 4 - ( D i m e t h y l a m i n o ) - 9 - C - [ 4 -

(dimethylamino)benzene]-7-fluoro-3,10,12,12a-tetrahydroxy-

1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2,9-dicar-

boxamide Dihydrochloride (9g). Yellow solid. ¹H NMR (400

MHz, CD₃OD) δ 8.08 (d, J = 10.8 Hz, 1 H), 7.98 (d, J = 8.4 Hz, 2 H),

7.49 (d, J = 8.4 Hz, 2 H), 4.02 (s, 1 H), 3.19 (s, 6 H), 3.12−2.88 (m,

9 H), 2.24−2.13 (m, 2 H), 1.60−1.51 (m, 1 H). MS (ESI) m/z 595.1

(M + H).

N-[(5aR,6aS,7S,10aS)-9-Carbamoyl-7-(dimethylamino)-4-flu-

o r o - 1 , 8 , 1 0 a , 1 1 - t e t r a h y d r o x y - 1 0 , 1 2 - d i o x o -

5,5a,6,6a,7,10,10a,12-octahydrotetracen-2-yl]thiophene-2-car-

(4S,4aS,5aR,12aS)-9-[2-(tert-Butylamino)acetamido]-4-(di-

methylamino)-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo-

1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-

drochloride (9a). 2-t-Butylaminoacetylchloride hydrochloride (4.2 mg,

0.022 mmol) was added to a solution of 8 (5 mg, 0.011 mmol) in

DMF (0.1 mL). The reaction was stirred for 30 min. The reaction

mixture was diluted with 0.05 N aqueous HCl (2 mL) and was purified

directly 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 3.9 mg (62%) of the title compound

1

boxamide Hydrochloride (9h). Yellow solid. H NMR (400 MHz,

CD₃OD) δ 8.12 (d, J = 10.8 Hz, 1 H), 7.89 (d, J = 3.2 Hz, 1 H), 7.78

(d, J = 4.8 Hz, 1 H), 7.22 (t, J = 8.8 Hz, 1 H), 4.10 (s, 1 H), 3.20−2.98

(m, 9 H), 2.36−2.20 (m, 2 H), 1.68−1.61 (m, 1 H). MS (ESI) m/z

558.1 (M + H).

N-[(5aR,6aS,7S,10aS)-9-Carbamoyl-7-(dimethylamino)-4-flu-

o r o - 1 , 8 , 1 0 a , 1 1 - t e t r a h y d r o x y - 1 0 , 1 2 - d i o x o -

5,5a,6,6a,7,10,10a,12-octahydrotetracen-2-yl]pyridine-3-car-

boxamide Dihydrochloride (9i). Yellow solid. ¹H NMR (400 MHz,

CD₃OD) δ 9.34 (s, 1 H), 9.04−9.00 (m, 2 H), 8.20−8.15 (m, 2 H),

4.07 (s, 1 H), 3.27−2.94 (m, 9 H), 2.34−2.18 (m, 2 H), 1.68−1.59

(m, 1 H). MS (ESI) m/z 553.1 (M + H).

1

as a yellow solid. H NMR (400 MHz, CD₃OD) δ 8.25 (d, J = 11.0

Hz, 1 H), 4.11 (br s, 1 H), 4.09 (s, 2 H), 3.22−2.86 (m, 3 H), 3.05

(s, 3 H), 2.97 (s, 3 H), 2.33−2.20 (m, 2 H), 1.69−1.57 (m, 1 H), 1.42

(s, 9 H). MS (ESI) m/z 561.39 (M + H).

(4S,4aS,5aR,12aS)-9-C-Benzene-4-(dimethylamino)-7-fluoro-

3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-oc-

tahydrotetracene-2,9-dicarboxamide Hydrochloride (9b). To a

(2S)-N-[(5aR,6aS,7S,10aS)-9-Carbamoyl-7-(dimethylamino)-

4 - f l u o r o - 1 , 8 , 1 0 a , 1 1 - t e t r a h y d r o x y - 1 0 , 1 2 - d i o x o -

5,5a,6,6a,7,10,10a,12-octahydrotetracen-2-yl]pyrrolidine-2-

carboxamide Dihydrochloride (9j). To a solution of compound

615

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Journal of Medicinal Chemistry

Article

8 (17.0 mg, 0.038 mmol) in DMF (200 μL) was added

N-benzyloxycarbonyl-^L-proline acid chloride (1.0 M in toluene,

57 μL). After 50 min, the reaction mixture was diluted to 3 mL

with 0.05 N aqueous HCl and filtered to remove solids. The solution

was purified directly by preparative HPLC (Phenomenex Polymerx

10 μ RP 100A column, 10−20% B gradient). Fractions with the

desired MW were collected and freeze-dried. The material was dis-

solved in 1,4-dioxane:MeOH (1:3, 2.3 mL), and palladium on carbon

(10%, 10 mg) was added. The flask was fitted with a septum and

evacuated and backfilled three times with hydrogen gas. The reaction

mixture was stirred under an atmosphere of hydrogen for 1.7 h, was

filtered through Celite, and was concentrated under reduced pressure.

Half of the resulting residue was purified by preparative HPLC

(Phenomenex Polymerx 10 μ RP 100A column, 0−35% B gradient).

Fractions with the desired MW were collected and freeze-dried to yield

(m, 1 H), 4.09 (s, 1 H), 3.78−3.60 (m, 2 H), 3.42−3.30 (m, 2 H),

3.20−2.95 (m, 8 H), 2.70−2.58 (m, 1 H), 2.38−2.00 (m, 5 H), 1.70−

1.60 (m, 1 H), 1.44−1.36 (m, 6 H). MS (ESI) m/z 587.36 (M + H).

(2S,4R)-N-[(5aR,6aS,7S,10aS)-9-Carbamoyl-7-(dimethylami-

no)-4-fluoro-1,8,10a,11-tetrahydroxy-10,12-dioxo-

5,5a,6,6a,7,10,10a,12-octahydrotetracen-2-yl]-4-fluoro-1-

methylpyrrolidine-2-carboxamide Dihydrochloride (9p). To a

solution of compound 8 (20 mg, 0.045 mmol) in THF was added

Na₂CO₃(9.5 mg, 0.089 mmol), (4R)-4-fluoro-1-methyl-L-proline (9.8

mg, 0.067 mmol), and HATU (34.6 mg, 0.047 mmol). The reaction

mixture was stirred at room temperature for 20 h. HCl in MeOH

(1 mL, 4 N) was added to the mixture at 0 °C. After 2 min, the mixture

was concentrated under reduced pressure and the residue 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 6.0 mg (21%) of the title compound as a

1

1.9 mg (30%) of compound the title compound as a yellow solid. H

1

yellow solid. H NMR (400 MHz, CD₃OD) δ 8.18 (d, J = 10.8 Hz,

NMR (400 MHz, CD₃OD) δ 8.16 (d, J = 11.0 Hz, 1 H), 4.59−4.56

(m, 1 H), 4.10 (s, 1 H), 3.48−3.33 (m, 2 H), 3.18−2.95 (m, 9 H),

2.59−2.50 (m, 1 H), 2.34−2.05 (m, 5 H), 1.70−1.60 (m, 1 H). MS

(ESI) m/z 545.38 (M + H).

1 H), 5.51 (d, J = 51.6 Hz, 1 H), 4.76−4.72 (m, 1 H), 4.22−4.16 (m,

1 H), 4.10 (s, 1 H), 3.74−3.63 (m, 1 H), 3.21−2.97 (m, 14 H), 2.35−

2.21 (m, 2 H), 1.69−1.60 (m, 1 H). MS (ESI) m/z 577.1 (M + H).

The following compounds were prepared by similar methods to 9p:

(2S,4S)-N-[(5aR,6aS,7S,10aS)-9-Carbamoyl-7-(dimethylami-

no)-4-fluoro-1,8,10a,11-tetrahydroxy-10,12-dioxo-

5,5a,6,6a,7,10,10a,12-octahydrotetracen-2-yl]-4-fluoro-1-

methylpyrrolidine-2-carboxamide Dihydrochloride (9q).

(2R)-N-[(5aR,6aS,7S,10aS)-9-Carbamoyl-7-(dimethylamino)-

4 - f l u o r o - 1 , 8 , 1 0 a , 1 1 - t e t r a h y d r o x y - 1 0 , 1 2 - d i o x o -

5,5a,6,6a,7,10,10a,12-octahydrotetracen-2-yl]pyrrolidine-2-

carboxamide Dihydrochloride (9k). Prepared as described for 9j.

1

Yellow solid. H NMR (400 MHz, CD₃OD) δ 8.17 (d, J = 11.0 Hz,

1

Yellow solid. H NMR (400 MHz, CD₃OD) δ 8.16 (d, J = 10.8 Hz,

1 H), 4.59−4.53 (m, 1 H), 4.09 (s, 1 H), 3.48−3.37 (m, 2 H), 3.18−

2.90 (m, 9 H), 2.59−2.50 (m, 1 H), 2.34−2.05 (m, 5 H), 1.70−1.59

(m, 1 H). MS (ESI) m/z 545.37 (M + H).

1 H), 5.48 (d, J = 51.2 Hz,1 H), 4.60−4.56 (m, 1 H), 4.11 (s, 1 H),

4.05−3.98 (m, 1 H), 3.67−3.54 (m, 1 H), 3.24−2.96 (m, 13 H), 2.55−

2.44 (m, 1 H), 2.34−2.22 (m, 2 H), 1.70−1.66 (m, 1 H). MS (ESI)

m/z 577.1 (M + H).

(2S)-N-[(5aR,6aS,7S,10aS)-9-Carbamoyl-7-(dimethylamino)-

4 - f l u o r o - 1 , 8 , 1 0 a , 1 1 - t e t r a h y d r o x y - 1 0 , 1 2 - d i o x o -

5,5a,6,6a,7,10,10a,12-octahydrotetracen-2-yl]-1-methylpyrro-

lidine-2-carboxamide Dihydrochloride (9l). Yellow solid. The

second half of the intermediate described in 9j (0.012 mmol) was

dissolved in DMF (500 μL), and formaldehyde (37% aqueous solu-

tion, 5.3 μL, 0.072 mmol), triethylamine (5.0 μL, 0.036 mmol), and

sodium triacetoxyborohydride (8.4 mg, 0.039 mmol) were added

sequentially. After 2 h, the reaction mixture was diluted to 1.8 mL with

0.05 N aqueous HCl, and the solution was purified directly by

preparative HPLC (Phenomenex Polymerx 10 μ RP 100A column, 0−

30% B gradient). Fractions with the desired MW were collected and

freeze-dried to provide a mixture of the desired compound and an

overformylated product. The resulting compound mixture was

dissolved in 4 N aqueous HCl solution (1.5 mL) and stirred for

50 h. The resulting solution was freeze-dried to provide 1.0 mg (15%)

of the title compound as a yellow solid. ¹H NMR (400 MHz, CD₃OD)

δ 8.17 (d, J = 10.4 Hz, 1 H), 4.36 (t, J = 8.6 Hz, 1 H), 4.08 (s, 1 H),

3.82−3.73 (m, 1 H), 3.20−2.90 (m, 12 H), 2.73−2.68 (m, 1 H), 2.35−

2.10 (m, 5 H), 1.70−1.60 (m, 1 H). MS (ESI) m/z 559.38 (M + H).

(2R)-N-[(5aR,6aS,7S,10aS)-9-Carbamoyl-7-(dimethylamino)-

4 - f l u o r o - 1 , 8 , 1 0 a , 1 1 - t e t r a h y d r o x y - 1 0 , 1 2 - d i o x o -

5,5a,6,6a,7,10,10a,12-octahydrotetracen-2-yl]-1-methylpyrro-

lidine-2-carboxamide Dihydrochloride (9m). Prepared as

described for 9l. Yellow solid. ¹H NMR (400 MHz, CD₃OD) δ 8.17 (d,

J = 10.4 Hz, 1 H), 4.45−4.34 (m, 1 H), 4.08 (s, 1 H), 3.84−3.74 (m,

1 H), 3.20−2.90 (m, 12 H), 2.79−2.65 (m, 1 H), 2.33−2.05 (m, 5 H),

1.70−1.58 (m, 1 H). MS (ESI) m/z 559.40 (M + H).

(2S)-N-[(5aR,6aS,7S,10aS)-9-Carbamoyl-7-(dimethylamino)-

4 - f l u o r o - 1 , 8 , 1 0 a , 1 1 - t e t r a h y d r o x y - 1 0 , 1 2 - d i o x o -

5,5a,6,6a,7,10,10a,12-octahydrotetracen-2-yl]-4,4-difluoro-1-

methylpyrrolidine-2-carboxamide Dihydrochloride (9r). Yellow

1

solid. H NMR (400 MHz, CD₃OD) δ 8.18 (d, J = 10.8 Hz, 1 H),

4.76−4.71 (m, 1 H), 4.17−4.12 (m, 1 H), 4.09 (s, 1 H), 3.96−3.86

(m, 1 H), 3.67−3.53 (m, 1 H), 3.55−3.53 (m, 1 H), 3.25−2.73 (m, 12

H), 2.33−2.19 (m, 2 H), 1.68−1.59 (m, 1 H). MS (ESI) m/z 595.3

(M + H).

(2S)-N-[(5aR,6aS,7S,10aS)-9-Carbamoyl-7-(dimethylamino)-

4 - f l u o r o - 1 , 8 , 1 0 a , 1 1 - t e t r a h y d r o x y - 1 0 , 1 2 - d i o x o -

5,5a,6,6a,7,10,10a,12-octahydrotetracen-2-yl]piperidine-2-car-

boxamide Dihydrochloride (9s). To

a solution of

(S)-(−)-1-Cbz-piperidinecarboxylic acid (34.2 mg, 0.13 mmol), and

HATU (50.0 mg, 0.13 mmol) in DMF (200 μL) was added triethylamine

(18 μL, 0.13 mmol). After 30 min, aniline 8 (17.5 mg, 0.039 mmol) was

added. After 16 h, the reaction mixture was diluted to 3 mL with 0.05 N

aqueous HCl and the solution was purified directly by preparative

HPLC (Phenomenex Polymerx 10 μ RP 100A column, 15−70% B

gradient). Fractions with the desired MW were collected and freeze-

dried. The material was dissolved in 1,4-dioxane:MeOH (1:3, 1.2 mL),

and palladium on carbon (10%, 4 mg) was added. The flask was fitted

with a septum and was evacuated and backfilled three times with

hydrogen gas. The reaction mixture was stirred under an atmosphere

of hydrogen gas for 1.5 h, was filtered through Celite, and was

concentrated under reduced pressure. The material was purified by

preparative HPLC (Phenomenex Polymerx 10 μ RP 100A column,

0−35% B gradient). Fractions with the desired MW were collected and

freeze-dried to yield 0.75 mg (4%) of the title compound as a yellow

(2S)-N-[(5aR,6aS,7S,10aS)-9-Carbamoyl-7-(dimethylamino)-

4 - f l u o r o - 1 , 8 , 1 0 a , 1 1 - t e t r a h y d r o x y - 1 0 , 1 2 - d i o x o -

5,5a,6,6a,7,10,10a,12-octahydrotetracen-2-yl]-1-ethylpyrroli-

dine-2-carboxamide Dihydrochloride (9n). Prepared as described

1

solid. H NMR (400 MHz, CD₃OD) δ 8.15 (d, J = 11.0 Hz, 1 H),

1

for 9l using acetaldehyde instead of formaldehyde. Yellow solid. H

4.12−4.06 (m, 2 H), 3.48−3.40 (m, 2 H), 3.20−2.90 (m, 9 H), 2.36−

2.18 (m, 3 H), 2.02−1.90 (m, 2 H), 1.82−1.60 (m, 4 H). MS (ESI)

m/z 559.37 (M + H).

NMR (400 MHz, CD₃OD) δ 8.14 (d, J = 11.0 Hz, 1 H), 4.48−4.40

(m, 1 H), 4.10 (s, 1 H), 3.85−3.76 (m, 1 H), 3.50−3.30 (m, 2 H),

3.25−2.90 (m, 10 H), 2.75−2.62 (m, 1 H), 2.36−2.02 (m, 5 H), 1.70−

1.58 (m, 1 H), 1.35 (t, J = 6.9 Hz, 3 H). MS (ESI) m/z 573.33 (M + H).

(2S)-N-[(5aR,6aS,7S,10aS)-9-Carbamoyl-7-(dimethylamino)-

4 - f l u o r o - 1 , 8 , 1 0 a , 1 1 - t e t r a h y d r o x y - 1 0 , 1 2 - d i o x o -

5,5a,6,6a,7,10,10a,12-octahydrotetracen-2-yl]-1-(propan-2-yl)-

pyrrolidine-2-carboxamide Dihydrochloride (9o). Prepared as

described for 9l using acetone instead of formaldehyde. Yellow solid.

¹H NMR (400 MHz, CD₃OD) δ 8.15 (d, J = 10.5 Hz, 1 H), 4.61−4.53

(2S)-N-[(5aR,6aS,7S,10aS)-9-Carbamoyl-7-(dimethylamino)-

4 - f l u o r o - 1 , 8 , 1 0 a , 1 1 - t e t r a h y d r o x y - 1 0 , 1 2 - d i o x o -

5,5a,6,6a,7,10,10a,12-octahydrotetracen-2-yl]-1-methylpiperi-

dine-2-carboxamide Dihydrochloride (9t). To a solution of

compound 9s (8.7 mg, 0.0138 mmol) in DMF (750 μL) were added

sequentially formaldehyde (37% aqueous solution, 6.2 μL, 0.083 mmol),

triethylamine (5.8 μL, 0.041 mmol), and sodium triacetoxyborohy-

dride (11 mg, 0.051 mmol). After 17 h, the reaction mixture was

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concentrated to remove amine and 6 N aqueous HCl (500 μL) was

added. The solution was purified by preparative HPLC (Phenomenex

Polymerx 10 μ RP 100A column, 15−50% B gradient). Fractions with

the desired MW were collected and freeze-dried to yield 2.4 mg (31%)

of the title compound as a yellow solid. ¹H NMR (400 MHz, CD₃OD)

δ 8.16 (d, J = 11.0 Hz, 1 H), 4.08−4.04 (m, 1 H), 3.59−3.53 (m, 1 H),

3.20−3.10 (m, 5 H), 3.06−2.96 (m, 5 H), 2.90 m (s, 3 H), 2.36−2.25

added to a solution of LDA in THF (0.051 M, 40 mL, 2.03 mmol) and

TMEDA (304 μL, 2.03 mmol) at −78 °C. The reaction was stirred at

−78 °C for 15 min. A solution of enone 6 (784 mg, 1.62 mmol) in

THF (6.5 mL) was added dropwise, followed by addition of LHMDS

(1.0 M in THF, 2.03 mL, 2.03 mmol). The reaction mixture was

slowly warmed to −10 °C over 1 h, was quenched with NH₄Cl

(saturated, aqueous solution, 6 mL), and was warmed to 25 °C. The

solution was poured into NH₄Cl (saturated, aqueous solution, 20 mL)

and was extracted with EtOAc (2 × 75 mL). The combined extracts

were dried over Na₂SO₄, filtered, and concentrated. The material was

purified by preparative HPLC (Sunfire Prep C18 column, 88−100% B

gradient). Fractions with the desired MW were collected and freeze-

(m,

2 H), 2. 11−2. 05 (m, 1 H), 2. 02−1. 94 (m,

2 H), 1.90−1.74 (m, 2 H), 1.71−1.58 (m, 2 H). MS (ESI) m/z

573.33 (M + H).

(2R)-N-[(5aR,6aS,7S,10aS)-9-Carbamoyl-7-(dimethylamino)-

4 - f l u o r o - 1 , 8 , 1 0 a , 1 1 - t e t r a h y d r o x y - 1 0 , 1 2 - d i o x o -

5,5a,6,6a,7,10,10a,12-octahydrotetracen-2-yl]piperidine-2-car-

boxamide Hydrochloride (9u). Prepared as described for 9t. Yellow

1

dried to give 552 mg (41%) of the title compound. H NMR (400

MHz, CDCl₃) δ 16.22 (s, 1 H), 7.49−7.47 (m, 4 H), 7.37−7.31 (m,

6 H), 6.80 (d, J = 11.0 Hz, 1 H), 5.76−5.64 (m, 2 H), 5.35 (s, 2 H),

5.17−5.11 (m, 4 H), 4.98 (d, J = 9.2, 1 H), 4.87 (d, J = 9.8 Hz, 1 H),

3.96 (m, J = 10.4 Hz, 1 H), 3.83−3.71 (m, 4 H), 3.14 (dd, J = 14.7,

4.3 Hz, 1 H), 3.0−2.87 (m, 1 H), 2.55−2.35 (m, 9 H), 2.11 (d, J =

14.7 Hz, 1 H), 0.82 (s, 9 H), 0.26 (s, 3 H), 0.13 (s, 3 H). MS (ESI)

m/z 820.55 (M + H).

1

solid. H NMR (400 MHz, CD₃OD) δ 8.16 (d, J = 11.0 Hz, 1 H),

4.13−4.06 (m, 2 H), 3.50−3.43 (m, 2 H), 3.20−2.90 (m, 9 H), 2.38−

2.18 (m, 3 H), 2.04−1.88 (m, 2 H), 1.83−1.60 (m, 4 H). MS (ESI)

m/z 559.38 (M + H).

Phenyl 3-Amino-2-(benzyloxy)-5-fluoro-6-methylbenzoate

(13). To a 250 mL round-bottom flask was added phenyl 3-fluoro-

6-hydroxy-2-methylbenzoate (14.47 g, 56.30 mmol), tetrabutylammo-

nium bromide (0.90 g, 2.80 mmol), 1,2-dichloroethane (60 mL), and

water (60 mL). The clear bilayer was cooled in a 20 °C water bath.

Nitric acid (7.2 mL, 70 wt %, 113 mmol) was added. The reaction was

stirred at room temperature overnight. The organic layer was sepa-

rated, washed with water (60 mL × 2) and brine, and dried over

Na₂SO₄. The solvent was removed to give 17.7 g (100%) of the

nitrated intermediate as a brown oil. The material (17.7 g, 56.30 mmol)

was dissolved in acetone (177 mL), and anhydrous potassium

carbonate (15.6 g, 113.00 mmol), potassium iodide (0.47 g, 2.80

mmol), and benzyl bromide (7.03 mL, 59.10 mmol) were added. The

suspension was then heated to 56 °C for 4 h. The solid was removed

by filtration and washed with acetone (30 mL). The filtrate was

concentrated to give a paste which was partitioned between methyl

t-butyl ether (120 mL) and water (80 mL). The organic layer was

washed with water (80 mL) and brine, dried over anhydrous Na₂SO₄,

and concentrated to give 21.1 g (98%) of the benzylated intermediate

as a brown oil. This material (21.1 g, 55.4 mmol) was dissolved in

THF (230 mL) in a 1 L round-bottom flask. The solution was cooled

in a cold water bath to 10 °C. A solution of sodium hydrosulfite (56.7 g,

276.8 mmol) was added. The temperature quickly rose from 10 to

20.4 °C after the addition. The yellow suspension was stirred while the

cold water bath slowly warmed to room temperature overnight to give

an orange cloudy solution. The reaction mixture was diluted with

EtOAc (460 mL). The organic layer was washed with water (150 mL

× 2) and brine, dried over Na₂SO₄, and concentrated under reduced

pressure. The material was purified by silica gel chromatography (9:1

heptane/EtOAc) to yield 15.8 g (80%, 3 steps) of the title compound.

¹H NMR (400 MHz, CDCl₃) δ 7.45−7.32 (m, 7 H), 7.28−7.23 (m,

1 H), 7.16−7.12 (m, 2 H), 7.57 (d, J = 11.0 Hz, 1 H), 5.00 (s, 2 H),

3.99 (br s, 2 H), 2.26 (d, J = 1.8 Hz, 3 H). MS (ESI) m/z 352.31 (M + H).

Phenyl 2-(Benzyloxy)-3-[bis(prop-2-en-1-yl)amino]-5-fluoro-

6-methylbenzoate (14). To an solution of 13 (10.0 g, 28.5 mmol)

in NMP (50 mL) was added allylbromide (7.65 mL, 85.5 mmol) and

potassium carbonate (11.8 g, 85.5 mmol). Potassium iodide (1 g,

6 mmol) was added, and the reaction mixture was heated to 100 °C.

After 16 h, the reaction was cooled, diluted with water (60 mL), and

extracted with EtOAc (75 mL, then 2 × 50 mL). The combined

organic extracts were washed with water (2 × 35 mL) and were dried

(Na₂SO₄), filtered, and concentrated. Purification via flash column

chromatography on silica gel (RediSep, 125 g, 1−6% EtOAc in

hexanes gradient) gave 10.97 g (89%) of the title compound. ¹H NMR

(400 MHz, CDCl₃) δ 7.42−7.30 (m, 7 H), 7.42−7.20 (m, 1 H), 7.00

(d, J = 8.5 Hz, 2 H), 6.72 (d, J = 11.0 HZ, 1 H), 5.77−5.70 (m, 2 H),

5.20−5.12 (m, 6 H), 3.81 (d, J = 6.1 Hz, 4 H), 2.26 (s, 3 H). MS (ESI)

m/z 432.34 (M + H).

(4aS,11aR,12aS,13S)-8-Amino-13-(dimethylamino)-9-

[(dimethylamino)methyl]-4a-[[(1,1-dimethylethyl)-

dimethylsilyl]oxy]-10-fluoro-11a,12,12a,13-tetrahydro-5-hy-

droxy-3,7-bis(phenylmethoxy)-naphthaceno[2,3-d]isoxazole-4,

6(4aH,11H)-dione (16). A solution of 15 (550 mg, 0.671 mmol) in

degassed CH₂Cl₂(5 mL) was added under nitrogen via syringe to a

flame-dried flask containing N,N-dimethylbarbituric acid (324 mg,

2.07 mmol) and tetrakis(triphenylphosphine)palladium(0) (56.9 mg,

0.0492 mmol). The resulting solution was heated to 35 °C for 4 h and

was concentrated under reduced pressure. The material was purified

by preparative HPLC (Sunfire Prep C18 column, 80−100% B

gradient). Fractions with the desired MW were collected and freeze-

1

dried to give 352 mg (71%) of the title compound. H NMR (400

MHz, CDCl₃) δ 16.10 (s, 1 H), 7.51−7.43 (m, 4 H), 7.9−7.29 (m, 6

H), 6.61 (d, J = 9.8 Hz, 1 H), 5.35 (s, 2 H), 4.87 (dd, J = 22.6, 10.4 Hz,

2 H), 3.96 (d, J = 10.4 Hz, 1 H), 3.91 (s, 2 H), 3.12 (dd, J = 15.3, 10.1

Hz, 1 H), 3.04−2.92 (m, 1 H), 2.55−2.31 (m, 9 H), 2.11 (d, J = 14.7

Hz, 1 H), 0.82 (s, 9 H), 0.27 (s, 3 H), 0.12 (s, 3 H). MS (ESI) m/z

740.44 (M + H).

N-[(5aR,6aS,7S,10aS)-9-Carbamoyl-7-(dimethylamino)-4-flu-

o r o - 1 , 8 , 1 0 a , 1 1 - t e t r a h y d r o x y - 1 0 , 1 2 - d i o x o -

5,5a,6,6a,7,10,10a,12-octahydrotetracen-2-yl]pyridine-2-car-

boxamide Dihydrochloride (9v). To a solution of compound 16

(21.1 mg, 0.028 mmol) was added picolinoyl chloride hydrochloride

(15.8 mg, 0.088) and triethylamine (11.7 μL, 0.084 mmol). Upon

completion, the reaction mixture was filtered through Celite and was

concentrated under reduced pressure. The material was dissolved in

1,4-dioxane (1 mL) and HF (50% aqueous solution, 200 μL) was

added. After stirring overnight, the reaction mixture was poured into a

solution of K₂HPO₄(2.6 g) in water (30 mL) and was extracted with

EtOAc (2 × 25 mL). The combined extracts were dried over Na₂SO₄,

filtered, and concentrated under reduced pressure. The material was

dissolved in 1,4-dioxane:MeOH (1:1, 1 mL), and palladium on carbon

(10%, 10 mg) was added. The flask was fitted with a septum and was

evacuated and backfilled three times with hydrogen gas. The reaction

mixture was stirred under an atmosphere of hydrogen gas for 2 h. The

reaction mixture was filtered through Celite and was concentrated

under reduced pressure. The material was purified by preparative

HPLC (Phenomenex Polymerx 10 μ RP 100A column, 10−60% B

gradient). Fractions with the desired MW were collected and freeze-

dried to provide 5.8 mg (37%) of the title compound as a yellow solid.

¹H NMR (400 MHz, CD₃OD) δ 8.73−8.69 (m, 1 H), 8.58−8.52 (m,

1 H), 8.27−8.21 (m, 1 H), 8.08−8.00 (m, 1 H), 7.66−7.60 (m, 1 H),

4.09 (s, 1 H), 3.29−2.92 (m, 9 H), 2.38−2.18 (m, 2 H), 1.72−1.60

(m, 1 H). MS (ESI) m/z 553.27 (M + H).

The following compounds were prepared by similar methods to 9v:

N-[(5aR,6aS,7S,10aS)-9-Carbamoyl-7-(dimethylamino)-4-flu-

o r o - 1 , 8 , 1 0 a , 1 1 - t e t r a h y d r o x y - 1 0 , 1 2 - d i o x o -

5,5a,6,6a,7,10,10a,12-octahydrotetracen-2-yl]-1-methyl-1H-

pyrrole-2-carboxamide Hydrochloride (9w). Yellow solid. ¹H

NMR (400 MHz, CD₃OD) δ 8.20 (d, J = 11.6 Hz, 1 H), 6.98−6.86

(4aS,11aR,12aS,13S)-13-(Dimethylamino)-4a-[[(1,1-

dimethylethyl)dimethylsilyl]oxy]-8-(di-2-propen-1-ylamino)-

10-fluoro-11a,12,12a,13-tetrahydro-5-hydroxy-3,7-bis(phenyl-

methoxy)-naphthaceno[2,3-d]isoxazole-4,6(4aH,11H)-dione

(15). A solution of 14 (875 mg, 2.03 mmol) in THF (6.5 mL) was

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1

(m, 2 H), 6.17−6.10 (m, 1 H), 4.08 (s, 1 H), 3.94 (s, 3 H), 3.19−2.90

(m, 9 H), 2.33−2.18 (m, 2 H), 1.80−1.56 (m, 1 H). MS (ESI) m/z

555.32 (M + H).

N-[(5aR,6aS,7S,10aS)-9-Carbamoyl-7-(dimethylamino)-4-flu-

o r o - 1 , 8 , 1 0 a , 1 1 - t e t r a h y d r o x y - 1 0 , 1 2 - d i o x o -

5,5a,6,6a,7,10,10a,12-octahydrotetracen-2-yl]-5-methyl-1,2-

oxazole-3-carboxamide Hydrochloride (9x). Yellow solid. ¹H

NMR (400 MHz, CD₃OD) δ 8.32 (d, J = 11.0 Hz, 1 H), 6.59 (s, 1 H),

4.09 (s, 1 H), 3.19−2.90 (m, 9 H), 2.52 (s, 3 H), 2.34−2.18 (m, 2 H),

1.71−1.58 (m, 1 H). MS (ESI) m/z 557.26 (M + H).

and freeze-dried to provide 147 mg (43%) of 17bb. H NMR (400

MHz, CDCl₃) δ 16.04 (s, 1 H), 10.10 (s, 1 H), 8.48 (d, J = 11.0 Hz, 1

H), 7.54−7.48 (m, 4 H), 7.40−7.32 (m, 5 H), 5.36 (s, 2 H), 4.99 (d,

J = 9.8 Hz, 1 H), 4.90 (d, J = 9.8 Hz, 1 H), 3.96 (d, J = 10.4 Hz, 1 H),

3.54 (t, J = 7.9 Hz, 1 H), 3.39−3.34 (m, 1 H), 3.25−3.19 (m, 1 H),

3.05−2.92 (m, 2 H), 2.58−2.36 (m, 10 H), 2.23−2.06 (m, 4 H), 0.81

(s, 9 H), 0.28 (s, 3 H), 0.11 (s, 3 H). MS (ESI) m/z 837.37 (M + H).

To a solution of 17bb (147 mg, 0.175 mmol) in 1,4-dioxane (3.5 mL)

was added HF (50% aqueous solution, 750 μL). After 4 h, the reaction

solution was poured into a solution of K₂HPO₄(9 g) in water (90 mL)

and was extracted with EtOAc (2 × 50 mL). The combined extracts

were dried over Na₂SO₄, filtered, and concentrated under reduced

pressure. The material was dissolved in 1,4-dioxane:MeOH (1:1, 4

mL), and palladium on carbon (10%, 43.5 mg) was added. The flask

was fitted with a septum and was evacuated and backfilled three times

with hydrogen gas. The reaction mixture was stirred under an

atmosphere of hydrogen gas for 3.25 h. The reaction mixture was

filtered through Celite and was concentrated under reduced pressure.

The material was purified by preparative HPLC (Phenomenex

Polymerx 10 μ RP 100A column, 10−35% B gradient). Fractions

with the desired MW were combined. The solution was cooled to

0 °C, and the pH was adjusted to 7.4 via dropwise addition of 0.5 M

aqueous NaOH solution (approximately 7.8 mL). The aqueous

solution was extracted with CH₂Cl₂(3 × 60 mL), and the combined

extracts were dried over Na₂SO₄, filtered, and concentrated under

reduced pressure to provide 79.7 mg (0.146 mmol, 73%) of the free

base. This yellow solid was dissolved in MeOH (3 mL), and MeSO₃H

(19 μL, 0.292 mmol) was added. The solution was concentrated under

reduced pressure, dried under vacuum, and freeze-dried from water to

provide 105 mg of the title compound as a yellow salt. ¹H NMR (400

MHz, CD₃OD) δ 8.22 (d, J = 11.0 Hz, 1 H), 5.16 (t, J = 8.6 Hz, 1 H),

4.21−4.12 (m, 1 H), 4.09−4.02 (m, 2 H), 3.17−2.85 (m, 10 H), 2.68

(s, 6 H, mesylate H), 2.64−2.59 (m, 1 H), 2.34−2.15 (m, 2 H), 1.70−

1.58 (m, 1 H). MS (ESI) m/z 545.18 (M + H).

N-[(5aR,6aS,7S,10aS)-9-Carbamoyl-7-(dimethylamino)-4-flu-

o r o - 1 , 8 , 1 0 a , 1 1 - t e t r a h y d r o x y - 1 0 , 1 2 - d i o x o -

5,5a,6,6a,7,10,10a,12-octahydrotetracen-2-yl]-1-methyl-1H-

pyrazole-3-carboxamide Hydrochloride (9y). Yellow solid. ¹H

NMR (400 MHz, CD₃OD) δ 8.38 (d, J = 11.0 Hz, 1 H), 7.68 (s, 1 H),

6.82−6.76 (m, 1 H), 4.09 (s, 1 H), 3.99 (s, 3 H), 3.16−2.90 (m, 9 H),

2.31−2.16 (m, 2 H), 1.70−1.56 (m, 1 H). MS (ESI) m/z 556.31 (M + H).

N-[(5aR,6aS,7S,10aS)-9-Carbamoyl-7-(dimethylamino)-4-flu-

o r o - 1 , 8 , 1 0 a , 1 1 - t e t r a h y d r o x y - 1 0 , 1 2 - d i o x o -

5,5a,6,6a,7,10,10a,12-octahydrotetracen-2-yl]-1,3-thiazole-2-

carboxamide Hydrochloride (9z). Yellow solid. ¹H NMR (400

MHz, CD₃OD) δ 8.38 (d, J = 11.0 Hz, 1 H), 8.02 (d, J = 3.0 Hz, 1 H),

7.95 (d, J = 2.4 Hz, 1 H), 4.09 (s, 1 H), 3.20−2.90 (m, 9 H), 2.34−

2.17 (m, 2 H), 1.70−1.56 (m, 1 H). MS (ESI) m/z 559.23 (M + H).

(2S)-N-[(5aR,6aS,7S,10aS)-9-Carbamoyl-7-(dimethylamino)-4-flu-

oro-1,8,10a,11-tetrahydroxy-10,12-dioxo-5,5a,6,6a,7,10,10a,12-oc-

tahydrotetracen-2-yl]azetidine-2-carboxamide Dihydrochloride

(9aa). To a suspension of 1-Fmoc-^L-azetidine-2-carboxylic acid

(135 mg, 0.42 mmol) and HATU (164 mg, 0.43 mmol) in THF

(1.5 mL) was added triethylamine (60 μL, 0.43 mmol). After 30 min,

compound 16 (106 mg, 0.14 mmol) was added. After 18 h, the

reaction mixture was concentrated under reduced pressure. The

material was purified by preparative HPLC (Sunfire Prep C18 column,

80−100% B gradient). Fractions with the desired MW were collected

and freeze-dried to provide 131 mg of the intermediate 17aa as a

yellow powder. The material was dissolved in CH₂Cl₂(2 mL), and

piperidine (500 μL) was added. After 30 min, the reaction solution

was poured into aqueous pH 7 phosphate buffer and was extracted

with EtOAc (3 × 20 mL). The combined extracts were dried over

Na₂SO₄, filtered, and concentrated under reduced pressure.

Purification by column chromatography on silica gel (Silicycle, 5 g,

0−50% EtOAc in hexane gradient) provided 47.6 mg of the

intermediate. Half of the intermediate (24 mg) was dissolved in

acetonitrile (1 mL), and HF (50% aqueous solution, 200 μL) was

added. After 18.5 h, the reaction mixture was poured into a solution of

K₂HPO₄(2.5 g) in water (20 mL) and was extracted with EtOAc (2 ×

25 mL). The combined extracts were dried over Na₂SO₄, filtered, and

concentrated under reduced pressure. Palladium on carbon (10%, 12.5 mg)

was added to a solution of the intermediate in 1,4-dioxane:MeOH

(1:1, 1 mL). The flask was fitted with a septum and was evacuated and

backfilled three times with hydrogen gas. The reaction mixture was

stirred under an atmosphere of hydrogen gas for 4.5 h, was filtered

through Celite, and was concentrated under reduced pressure. The

material was purified by preparative HPLC (Phenomenex Polymerx

10 μ RP 100A column, 15−50% B gradient). Fractions with the

desired MW were collected and freeze-dried to yield 2.0 mg (5%) of

(4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-9-(thio-

phene-2-sulfonamido)-3,10,12,12a-tetrahydroxy-1,11-dioxo-

1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Hy-

drochloride (10b). To a solution of compound 8 (20 mg, 0.045

mmol) in THF was added DIEA (11.5 mg, 0.089 mmol) and 2-thio-

phenesulfonyl chloride (12.2 mg, 0.067 mmol). The reaction mixture

was stirred for 20 h. HCl in MeOH (1 mL, 4 N) was added to the

mixture at 0 °C. After 2 min, the mixture was concentrated under

reduced pressure and the residue 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

2.0 mg (7%) of the title compound as a yellow solid. H NMR (400

MHz, CD₃OD) δ 7.75 (dd, J = 5.2, 1.2 Hz, 1 H), 7.59 (d, J = 2.8 Hz,

1 H), 7.52 (d, J = 10.4 Hz, 1 H), 7.09 (t, J = 4.4 Hz, 1 H), 4.07 (s, 1

H), 3.11−2.92 (m, 9 H), 2.30−2.18 (m, 2 H), 1.68−1.58 (m, 1 H).

MS (ESI) m/z 593.9 (M + H).

The following compounds were prepared by similar methods to

10b:

(4S,4aS,5aR,12aS)-9-(Benzenesulfonamido)-4-(dimethylami-

no)-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo-

1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Hy-

drochloride (10a). Yellow solid. ¹H NMR (400 MHz, CD₃OD)

δ 7.82 (d, J = 7.6 Hz, 2 H), 7.58−7.46 (m, 4 H), 4.07 (s, 1 H), 3.10−

2.92 (m, 9 H), 2.35−2.25 (m, 2 H), 1.65−1.55 (m, 1 H). MS (ESI)

m/z 552.1 (M + H).

(4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-9-(methane-

sulfonamido)-3,10,12,12a-tetrahydroxy-1,11-dioxo-

1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Hy-

drochloride (10c). Yellow solid. ¹H NMR (400 MHz, CD₃OD)

δ 7.44 (d, J = 10.0 Hz, 1 H), 4.10 (s, 1 H), 3.21−2.90 (m, 12 H), 2.34−

2.22 (m, 2 H), 1.67−1.61 (m, 1 H). MS (ESI) m/z 526.1 (M + H).

(4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-9-

[(methylcarbamoyl)amino]-3,10,12,12a-tetrahydroxy-1,11-

dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxa-

mide Hydrochloride (11). To a suspension of compound 8 (0.260 g,

0.50 mmol) in CH₂Cl₂(5 mL) was added triethylamine (0.139 mL,

1.00 mmol). The reaction was stirred until a clear solution was formed.

1

the title compound as a yellow solid. H NMR (400 MHz, CD₃OD)

δ 8.25 (d, J = 11.0 Hz, 1 H), 5.29−5.24 (m, 1 H), 4.20−4.11 (m, 1 H),

4.09 (s, 1 H), 3.19−2.89 (m, 10 H), 2.69−2.56 (m, 1 H), 2.33−2.19

(m, 2 H), 1.68−1.56 (m, 1 H). MS (ESI) m/z 531.30 (M + H).

(2S)-N-[(5aR,6aS,7S,10aS)-9-Carbamoyl-7-(dimethylamino)-

4 - f l u o r o - 1 , 8 , 1 0 a , 1 1 - t e t r a h y d r o x y - 1 0 , 1 2 - d i o x o -

5,5a,6,6a,7,10,10a,12-octahydrotetracen-2-yl]-1-methylazeti-

dine-2-carboxamide Dimesylate (9bb). To a suspension of

compound 16 (302 mg, 0.408 mmol) and N-methyl-^L-azetidine-2-

carboxylic acid (148 mg, 1.28 mmol) in CH₂Cl₂(6 mL) was added O-

(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate

(395 mg, 1.23 mmol) and DIEA (285 μL, 1.64 mmol). After 16.5 h,

the resulting orange solution was concentrated under reduced pressure

and was purified by preparative HPLC (Sunfire Prep C18 column,

50−90% B gradient). Fractions with the desired MW were collected

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Methylisocyanate (89.4 μL, 1.50 mmol) was added dropwise, and the

reaction mixture was stirred for 1 h. Additional methylisocyanate

(45 μL, 0.75 mmol) was added, and the reaction mixture was stirred

overnight. The reaction mixture was concentrated under reduced

pressure, and the residue was purified by preparative HPLC (Pheno-

menex Polymerx 10 μ RP 100A column, 15−65% B gradient).

Fractions with the desired MW were collected and freeze-dried to yield

B gradient). Fractions with the desired MW were collected and freeze-

dried to provide the title compounds.

The following compounds were prepared by the general procedure

described above:

(4S,4aS,5aR,12aS)-9-{[2-(tert-Butylamino)ethyl]amino}-4-(di-

methylamino)-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-dioxo-

1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Tri-

hydrochloride (20a). Prepared from t-BuN(Cbz)CH₂CHO. Yellow

solid. ¹H NMR (400 MHz, CD₃OD) δ 6.72 (d, J = 11.0 Hz, 1 H), 4.07

(s, 1 H), 3.54−3.46 (m, 2 H), 3.26−3.19 (m, 2 H), 3.03 (s, 3 H), 2.95

(s, 3 H), 3.14−2.92 (m, 3 H), 2.23−2.14 (m, 2 H), 1.67−1.55 (m,

1 H), 1.38 (s, 9 H). MS (ESI) m/z 547.51 (M + H).

(4S,4aS,5aR,12aS)-4,9-Bis(dimethylamino)-7-fluoro-

3,10,12,12a-tetrahydroxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-oc-

tahydrotetracene-2-carboxamide Dihydrochloride (20b). Pre-

pared from aqueous formaldehyde. Yellow solid. ¹H NMR (400 MHz,

CD₃OD) δ 7.46−7.42 (m, 1 H), 4.15 (s, 1 H), 3.33 (s, 6 H), 3.04 (s,

3 H), 2.96 (s, 3 H), 3.17 −2.95 (m, 3 H), 2.44−2.34 (m, 1 H), 2.29−

2.22 (m, 1 H), 1.71−1.60 (m, 1 H). MS (ESI) m/z 476.29 (M + H).

(4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-9-(propyla-

m i n o ) - 3 , 1 0 , 1 2 , 1 2 a - t e t r a h y d r o x y - 1 , 1 1 - d i o x o -

1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-

drochloride (20c). Prepared from n-propanal. Yellow solid. ¹H NMR

(400 MHz, CD₃OD) δ 7.39 (d, J = 9.2 Hz, 1 H), 4.10 (s, 1 H), 3.34 (t,

J = 7.8 Hz, 2 H), 3.04 (s, 3 H), 2.96 (s, 3 H), 3.21 −2.95 (m, 3 H),

2.35 (t, J = 13.7 Hz, 1 H), 2.27−2.20 (m, 1 H), 1.82−1.72 (m, 2 H),

1.71−1.60 (m, 1 H), 1.05 (t, J = 7.4 Hz, 3 H). MS (ESI) m/z 490.32

(M + H).

1

80 mg (32%) of the title compound as a yellow solid. H NMR (400

MHz, CD₃OD) δ 8.12 (d, J = 11.4 Hz, 1 H), 4.07 (s, 1 H), 3.04

(s, 3 H), 2.96 (s, 3 H), 3.13−2.93 (m, 3 H), 2.77 (s, 3 H), 2.27−2.15

(m, 2 H), 1.69−1.57 (m, 1 H). MS (ESI) m/z 505.41 (M + H).

(4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-9-[(pyrimi-

din-4-yl)amino]-3,10,12,12a-tetrahydroxy-1,11-dioxo-

1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Tri-

hydrochloride (18). A vessel containing aniline 16 (18.2 mg, 0.024

mmol), Pd₂dba₃(3.0 mg, 0.0033 mmol), Xantphos (3.4 mg, 0.0059

mmol), K₃PO₄(40 mg, 0.188 mmol), and 4,6-dichloropyrimidine

(6.5 mg, 0.044 mmol) was evacuated and backfilled with nitrogen gas

three times. 1,4-Dioxane (500 μL) was added, and the reaction

mixture was heated at 80 °C for 4.5 h. The reaction mixture was

filtered through Celite and concentrated under reduced pressure. The

material was purified by preparative HPLC (Sunfire Prep C18 column,

80−100% B gradient). Fractions with the desired MW were collected

1

and freeze-dried to provide 7.5 mg (37%) of the intermediate. H

NMR (400 MHz, CDCl₃) δ 15.97 (s, 1 H), 8.48 (s, 1 H), 8.33 (d, J =

5.5 Hz, 1 H), 7.52−7.46 (m, 2 H), 7.40−7.28 (m, 8 H), 7.07 (s, 1 H),

6.11 (s, 1 H), 5.34 (s, 2 H), 4.97 (d, J = 11.6 Hz, 1 H0, 4.88 (d,

J = 11.0 Hz, 1 H), 3.95 (d, J = 10.4 Hz, 1 H), 3.28−3.19 (m, 1 H),

3.09−2.98 (m, 1 H), 2.61−2.54 (m, 1 H), 2.54−2.39 (m, 8 H), 2.16

(d, J = 14.6 Hz, 1 H), 0.83 (s, 9 H), 0.28 (s, 3 H), 0.14 (s, 3 H). MS

(ESI) m/z 852.57 (M + H).

(4S,4aS,5aR,12aS)-4-(Dimethylamino)-7-fluoro-9-[(3-

methylbutyl)amino]-3,10,12,12a-tetrahydroxy-1,11-dioxo-

1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-

drochloride (20d). Prepared from isovaleraldehyde. Yellow solid. ¹H

NMR (400 MHz, CD₃OD) δ 7.44 (d, J = 9.2 Hz, 1 H), 4.12 (s, 1 H),

3.42−3.37 (m, 2 H), 3.05 (s, 3 H), 2.97 (s, 3 H), 3.21−2.97 (m, 3 H),

2.39−2.30 (m, 1 H), 2.29−2.22 (m, 1 H), 1.79−1.59 (m, 4 H), 0.98

(d, J = 6.4 Hz, 6 H). MS (ESI) m/z 518.43 (M + H).

To a solution of the intermediate (7.5 mg, 0.0088 mmol) in 1,4-

dioxane (1.4 mL) was added HF (50% aqueous solution, 200 μL).

After 15.5 h, the reaction solution was poured into a solution of

K₂HPO₄(2.4 g) in water (20 mL) and was extracted with EtOAc (2 ×

20 mL). The combined extracts were dried over Na₂SO₄, filtered,

and concentrated under reduced pressure. The material was dissolved

in 1,4-dioxane:MeOH (1:1, 1 mL), and palladium on carbon (10%,

10 mg) was added. The flask was fitted with a septum and was

evacuated and backfilled three times with hydrogen gas. The reaction

mixture was stirred under an atmosphere of hydrogen gas for 2.5 h.

The reaction mixture was filtered through Celite and was concentrated

under reduced pressure. The material was purified by preparative

HPLC (Phenomenex Polymerx 10 μ RP 100A column, 10−50% B

gradient). Fractions with the desired MW were collected and freeze-

dried to provide 2.2 mg (48%) of the title compound as a yellow solid.

¹H NMR (400 MHz, CD₃OD) δ 8.83 (s, 1 H), 8.37−8.25 (m, 1 H),

8.18−8.05 (m, 1 H), 7.30−7.20 (m, 1 H), 4.10 (s, 1 H), 3.20−

2.90 (m, 9 H), 2.40−2.29 (m, 1 H), 2.27−2.19 (m, 1 H), 1.72−1.58

(m, 1 H). MS (ESI) m/z 526.31 (M + H).

General Procedure for Reductive Amination Products 20a−e.

Compound 16 (1.0 equiv) was dissolved in 1,2-dichloroethane

(∼0.1 M). HOAc (5 equiv) and the aldehyde (1.5 equiv) were added.

The mixture was stirred for 1 h. Na(OAc)₃BH (3.0 equiv) was added,

and the resulting mixture was stirred for an additional hour. The

mixture was washed with H₂O (10 mL) and concentrated to give the

crude intermediates 19a−e. The crude material was dissolved in 1,4-

dioxane (1.4 mL), and HF (50% aqueous solution, 200 μL) was added.

After 15.5 h, the reaction solution was poured into a solution of

K₂HPO₄(2.4 g) in water (20 mL) and was extracted with EtOAc (2 ×

20 mL). The combined extracts were dried over Na₂SO₄, filtered, and

concentrated under reduced pressure. The material was dissolved in

1,4-dioxane:MeOH (1:1, 1 mL), and palladium on carbon (10%, 10

mg) was added. The flask was fitted with a septum and was evacuated

and backfilled three times with hydrogen gas. The reaction mixture

was stirred under an atmosphere of hydrogen gas for 2.5 h. The

reaction mixture was filtered through Celite and was concentrated

under reduced pressure. The material was purified by preparative

HPLC (Phenomenex Polymerx 10 μ RP 100A column, 10−100%

( 4 S , 4 a S , 5aR , 12aS )-4- (Dimethylamino)-9-[(2, 2-

dimethylpropyl)amino]-7-fluoro-3,10,12,12a-tetrahydroxy-1,11-

dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide

Dihydrochloride (20e). Prepared from 2,2-dimethyl-1-propanal.

1

Yellow solid. H NMR (400 MHz, CD₃OD) δ 6.83 (d, J = 10.5 Hz,

1 H), 4.06 (s, 1 H), 3.34 (t, J = 7.8 Hz, 2 H), 3.03 (s, 3 H), 2.95 (s,

3 H), 3.11−2.93 (m, 5 H), 2.27−2.14 (m, 2 H), 1.67−1.57 (m, 1 H),

1.04 (s, 9 H). MS (ESI) m/z 518.48 (M + H).

(4S,4aS,5aR,12aS)-9-(Aminomethyl)-4-(dimethylamino)-7-

f l u o r o - 3 , 1 0 , 1 2 , 1 2 a - t e t r a h y d r o x y - 1 , 1 1 - d i o x o -

1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide Dihy-

drochloride (21). Benzyl N-(hydroxymethyl)carbamate (92 mg, 0.51

mmol) was added to a TFA/CH₃SO₃H (1 mL/1 mL) solution of

compound 7a (110 mg, 0.25 mmol) and was stirred for 30 min. The

material was purified by preparative HPLC (Phenomenex Polymerx 10

μ RP 100A column, 0−30% B gradient). Fractions with the desired

MW were collected and freeze-dried to yield 23 mg (17%) of the title

compound. ¹H NMR (400 MHz, CD₃OD) δ 7.47 (d, J = 9.2 Hz, 1 H),

4.16 (s, 2 H), 4.13 (s, 1 H), 3.21−2.94 (m, 3 H), 3.06 (s, 3 H), 2.97 (s, 3),

2.37−2.22 (m, 2 H), 1.70−1.58 (m, 1 H). MS (ESI) m/z 462.26

(M + H).

(4S, 4aS, 5aR, 12aS)-4-(Dimethylamino)-9-{[(2, 2-

dimethylpropyl)amino]methyl}-7-fluoro-3,10,12,12a-tetrahy-

droxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-

carboxamide Dihydrochloride (22). Triethylamine (2 μL, 0.014

mmol) was added to a mixture of 21 (3 mg, 0.0065 mmol) and

pivaldehyde (0.8 μL, 0.0072 mmol) in DMF (0.1 mL). The reaction

was stirred for 15 min, and NaBH(OAc)₃(3 mg, 0.013 mmol) and

HOAc (2 μL) were added. The reaction was stirred for 1 h. 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 mg (26%) of the title

compound as a yellow solid. ¹H NMR (400 MHz, CD₃OD) δ 7.52 (d,

J = 9.1 Hz, 1 H), 4.30 (s, 2 H), 4.09 (s, 1 H), 3.23−2.93 (m, 5 H), 3.04

(s, 3 H), 2.95 (s, 3 H), 2.40−2.19 (m, 2 H), 1.71−1.60 (m, 1 H), 1.05

(s, 9 H). MS (ESI) m/z 532.27 (M + H).

619

dx.doi.org/10.1021/jm201467r | J. Med. Chem. 2012, 55, 606−622

Journal of Medicinal Chemistry

Article

(4S,4aS,5aR,12aS)-9-{[2-(tert-Butylamino)acetamido]-

methyl}-4-(dimethylamino)-7-fluoro-3,10,12,12a-tetrahy-

droxy-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-

carboxamide Dihydrochloride (23). 2-t-Butylaminoacetylchloride

hydrochloride (5.8 mg, 0.031 mmol) was added to a solution of 21

(12 mg, 0.026 mmol) in DMF (0.2 mL). The reaction mixture was

stirred for 30 min and was diluted with 0.05 N HCl (2 mL). The

solution 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 3.0 mg (18%) of

days −4 and −1, respectively. Mice were infected with S. pneumoniae

SPN160 via intranasal administration of 0.05 mL under light anes-

thesia. At 2 and 12 h post infection, mice were treated with compound

9l or linezolid at either 30 mg/kg/dose via oral gavage or 10 mg/kg/

dose intravenously. Six mice were treated with each drug concen-

tration. Twenty-four hours post initiation of treatment, mice were

euthanized by CO₂inhalation. The lungs of the mice were aseptically

removed, weighed, homogenized, serially diluted, and plated on TSA-

II medium. The plates were incubated overnight at 37 °C in 5% CO₂.

CFU per gram of lung tissue was calculated by enumerating the plated

colonies then adjusting for serial dilutions and the weight of the lung.

S. pneumoniae Immunocompetent Lung Model. Female CD-

1 mice weighing 18−20 g were infected with S. pneumoniae 1629 (VL-

172) via intranasal administration of 0.05 mL under light anesthesia.

At 5, 24, and 36 h post infection, mice were treated with compound 9l

or linezolid at 30 mg/kg/dose via oral gavage. Six mice were treated

with each drug concentration. Forty-eight hours post initiation of

treatment, mice were euthanized by CO₂inhalation. The lungs of the

mice were aseptically removed, weighed, homogenized, serially diluted,

and plated on TSA-II medium. The plates were incubated overnight at

37 °C in 5% CO₂. CFU per gram of lung tissue was calculated by

enumerating the plated colonies then adjusting for serial dilutions and

the weight of the lung.

H. influenzae Immunocompetent Lung Model. Male Sprague−

Dawley rats weighing 175−200 g were infected with H. influenzae 551

via intratracheal administration of 0.05 mL under light anesthesia. At 5,

24, and 48 h post infection, rats were treated with compound 9l at

either 100 mg/kg/dose orally or 25 mg/kg/dose intravenously, or with

azithromycin at 50 mg/kg/dose orally. Six rats were treated with each

drug concentration. Seventy-two hours post initiation of treatment,

rats were euthanized by CO₂inhalation. The lungs of the rats were

aseptically removed, weighed, homogenized, serially diluted, and

plated on TSA-II medium. The plates were incubated overnight at

37 °C in 5% CO₂. CFU per gram of lung tissue was calculated by

enumerating the plated colonies then adjusting for serial dilutions and

the weight of the lung.

1

the title compound as a yellow solid. H NMR (400 MHz, CD₃OD)

δ 7.34 (d, J = 9.6 Hz, 1 H), 4.46 (s, 2 H), 4.08 (s, 1 H), 3.81 (s, 2 H),

3.18−2.92 (m, 3 H), 3.03 (s, 3 H), 2.96 (s, 3 H), 2.32−2.18 (m, 2 H),

1.69−1.60 (m, 1 H), 1.38 (s, 9 H). MS (ESI) m/z 575.30 (M + H).

Susceptibility Testing. Compound stocks were prepared and

serially diluted in sterile deionized water. Tetracycline-susceptible iso-

lates 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 iso-

lates S. aureus SA158 (tet(K)), S. pneumoniae SP160 (tet(M)),

E. faecalis EF159 (tet(M)), E. coli EC155 (tet(A)), and K. pneumoniae

KP153 (tet(A)) were obtained from Marilyn Roberts’ lab at the

University of Washington. S. aureus SA161 (tet(M)) was obtained

from Micromyx (Kalamazoo, MI). Minimal inhibitory concentration

(MIC) determinations were performed in liquid medium in 96-well

microtiter plates according to the methods described by the Clinical

and Laboratory Standards Institute (CLSI).²⁴Cation-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 Microbio-

logicals, Wilsonville, OR) was used to supplement medium, as appro-

priate. All test methods met acceptable standards based on re-

commended quality control ranges for all comparator antibiotics and

the appropriate ATCC quality control strains.

ASSOCIATED CONTENT

* Supporting Information

■

Animal Efficacy Models. All animal efficacy models were

performed at Vivisource, Waltham, MA.

S

MIC data for the complete panel of bacteria for all compounds

and MIC data for the lung infection model strains. This

material is available free of charge via the Internet at http://

pubs.acs.org.

Mouse Systemic Infection Model. S. aureus ATCC 13709 was

grown to log phase in a liquid culture. Bacteria were diluted in 5%

mucin to a concentration to achieve 90% mortality within 48 h after

infection. The final inoculum concentration of bacteria was 1.98 × 10⁶

CFU/mouse. CD-1 female mice (18−20 g) were infected with 0.5 mL

of bacterial suspension via intraperitoneal injection, 6 per dose con-

centration. For the screening model, mice were treated with the test

article as either a single 30 mg/kg dose via oral gavage or a 3 mg/kg

intravenous dose one hour post infection. Infection control mice were

dosed with vehicle (sterile water). After 48 h, survival was recorded.

For PD₅₀determinations, mice received oral treatment with the test

article at concentrations ranging from 0.30 to 30 mg/kg one hour post

infection. The PD₅₀in mg/kg was calculated as survival after 48 h.

MRSA Neutropenic Lung Model. Female Balb/C mice weighing

18−20 g were rendered neutropenic through two consecutive IP

cyclophosphamide injections of 150 and 100 mg/kg on days −4 and

−1, respectively. Mice were infected with S. aureus VL-137 (MRSA)

via intranasal inoculation under light anesthesia. At 2 and 12 h post

infection, mice were treated with compound 9l or linezolid either

orally at 50 mg/kg/dose or intravenously at 10 mg/kg/dose. Six mice

were treated with each drug concentration. Twenty-four hours post

treatment, mice were euthanized by CO₂inhalation. The lungs were

aseptically removed, weighed, homogenized, serially diluted, and

plated on TSA media. The plates were incubated overnight at 37 °C in

5% CO₂. CFU per gram of lung tissue was calculated by enumerating

the plated colonies then adjusting for serial dilutions and the weight of

the lung.

AUTHOR INFORMATION

Corresponding Author

*Phone: (617) 715-3558. Fax: (617) 926-3557. E-mail: rclark@

tphase.com.

■

ACKNOWLEDGMENTS

■

We thank Professor Andrew Myers, Dr. Eric Gordon,

Dr. Joaquim Trias, and Dr. Robert Zahler for valuable

discussions over the course of this study.

ABBREVIATIONS USED

■

ATCC, American Type Culture Collection; BLQ , below limit

of quantitation; Boc, t-butoxycarbonyl; Cbz, benzyloxycarbon-

yl; CFU, colony forming units; dba, dibenzylideneacetone;

DCE, 1,2-dichloroethane; DIEA, diisopropylethylamine; DMF,

N,N-dimethylformamide; F, oral bioavailability; HATU, 2-(1H-

7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-

phosphate; IV, intravenous; LDA, lithium diisopropylamide;

LHMDS, lithium bis(trimethylsilyl)amide; MIC, minimum inhibitory

concentration; MIC₅₀, minimum inhibitory concentration

required to inhibit growth of 50% of organisms; MIC₉₀,

minimum inhibitory concentration required to inhibit growth

S. pneumoniae Neutropenic Lung Model. Female Balb/c mice

weighing 18−20 g were rendered neutropenic through two con-

secutive IP cyclophosphamide injections of 150 and 100 mg/kg on

620

dx.doi.org/10.1021/jm201467r | J. Med. Chem. 2012, 55, 606−622

Journal of Medicinal Chemistry

Article

Acids Res. 1988, 16, 1218. (c) Connell, S. R.; Tracz, D. M.; Nierhaus,

K. H.; Taylor, D. E. Ribosomal protection proteins and their

mechanism of tetracycline resistance. Antimicrob. Agents Chemother.

2003, 47, 3675−3681.

of 90% of organisms; MRSA, methicillin-resistant Staph-

ylococcus aureus; MW, molecular weight; NMP, 2-methylpyrro-

lidinone; NT, not tested; PD₅₀, dose at which 50% protection

(survival) is observed; PK, pharmacokinetic; PO, oral; SAR,

structure activity relationships; TMEDA, N,N,N′,N′-tetrame-

thylethylenediamine; TFA, trifluoroacetic acid; Xantphos, 9,9-

dimethyl-4,5-bis-(diphenylphosphino)xanthenes

(8) (a) Spencer, J. L.; Hlavka, J. J.; Petisi, J.; Krazinski, H. M.; Boothe,

J. H. 6-Deoxytetracyclines. V. 7,9-Disubstituted Products. J. Med.

Chem. 1963, No. 6, 405−407. (b) Stephens, C. R.; Beereboom, J. J.;

Rennhard, H. H.; Gordon, P. N.; Murai, K.; Blackwood, R. K.;

Wittenau, M. S. 6-Deoxytetracyclines. IV. Preparation, C-6 Stereo-

chemistry, and Reactions. J. Am. Chem. Soc. 1963, 85, 2643−2652.

(c) Martell, M. J.; Boothe, J. H. The 6-Deoxytetracyclines. VII.

Alkylated Aminotetracyclines Possessing Unique Antibacterial Activity.

J. Med. Chem. 1967, 10, 44−46. (d) Church, R. F. R.; Schaub, R. E.;

Weiss, M. J. Synthesis of 7-Dimethylamino-6-demethyl-6-deoxytetra-

cycline (Minocycline) via 9-Nitro-6-demethyl-6-deoxytetracycline. J.

Org. Chem. 1971, 36, 723−725.

(9) (a) Sum, P.-E.; Lee, V. J.; Testa, R. T.; Hlavka, J. J.; Ellestad, G.

A.; Bloom, J. D.; Gluzman, Y.; Tally, F. P. Glycylcyclines. 1. A New

Generation of Potent Antibacterial Agents through Modification of 9-

Aminotetracyclines. J. Med. Chem. 1994, 37, 184−188. (b) Jones, C.

H.; Petersen, P. Tigecycline: a review of preclinical and clinical studies

of the first-in-class glycylcycline antibiotic. Drugs Today 2005, 41,

637−659. (c) French, G. L. A Review of Tigecycline. J. Chemother.

2008, 20, 3−11.

(10) Wang, Y.; Castaner, R.; Bolos, J.; Estivill, C. Amadacycline:

tetracycline antibiotic. Drugs Future 2009, 34, 11−15 Whileoriginally

called amadacycline, the compound is now named omadacycline.

(11) Charest, M. G.; Lerner, C. D.; Brubaker, J. D.; Siegel, D. R.;

Myers, A. G. A Convergent Enantioselective Route to Structurally

Diverse 6-Deoxytetracycline Antibiotics. Science 2005, 308, 395−398.

(12) Brubaker, J. D.; Myers, A. G. A Practical, Enantioselective

Synthetic Route to Key Precursor to the Tetracycline Antibiotics. Org.

Lett. 2007, 9, 3523−3525.

(13) Sun, C.; Wang, Q.; Brubaker, J. D.; Wright, P. M.; Lerner, C. D.;

Noson, K.; Charest, M.; Siegel, D. R.; Wang, Y.-M.; Myers, A. G. A

Robust Platform for the Synthesis of New Tetracycline Antibiotics.

J. Am. Chem. Soc. 2008, 130, 17913−17927.

REFERENCES

■

(1) For a general reviews of antibacterial agents and bacterial

resistance, see: (a) Wilson, D. N. The A−Z of bacterial translation

inhibitors. Crit. Rev. Biochem. Mol. Biol. 2009, 44, 393−433. (b) Chu,

D. T. W.; Plattner, J. J.; Katz, L. New Directions in Antibacterial

Research. J. Med. Chem. 1996, 39, 3853−3874. (c) Neu, H. C. The

Crisis in Antibiotic Resistance. Science 1992, 257, 1064−1078.

(2) For reviews on the tetracyclines, see: (a) Hlavka, J. J.; Ellestad, G.

A.; Chopra, I. Tetracyclines. In Kirk-Othmer Encyclopedia of Chemical

Technology, 4th ed.; John Wiley & Sons, Inc.: New York, 1992; Vol. 3,

pp 331−346. (b) Chopra, I.; Hawkey, R. M.; Hinton, M. Tetracyclines,

molecular and clinical aspects. J. Antimicrob. Chemother. 1992, 29,

245−277. (c) Shlaes, D. M. An Update on Tetracyclines. Curr. Opin.

Invest. Drugs 2006, 7, 167−171. (d) Pereira-Maia, E. C.; Silva, P. P.;

Batista de Almeida, W.; Ferreira dos Santos, H.; Marcial, B. L.;

Ruggiero, R.; Guerra, W. Tetracyclines and Glycylcyclines: an

Overview. Quim. Nova 2010, 33, 700−706.

(3) For reviews of tetracycline resistance mechanisms, see:

(a) Chopra, I; Roberts, M. Tetracycline Antibiotics: Mode of Action,

Applications, Molecular Biology, and Epidemiology of Bacterial

Resistance. Microbiol. Mol. Biol. Rev. 2001, 65, 232−260. (b) Speer,

B. S.; Shoemaker, N. B.; Salyers, A. A. Bacterial resistance to

tetracycline: mechanism, transfer, and clinical significance. Clin.

Microbiol. Rev. 1992, 5, 387−399. (c) Levy, S. B. Evolution and

Spread of Tetracycline Resistance Determinants. J. Antimicrob.

Chemother. 1989, 24, 1−3.

(4) For reviews of the mode of action of the tetracyclines, see:

(a) Chopra, I. Mode of action of the tetracyclines and the nature of

bacterial resistance to them. In The Tetracyclines, Handbook of

Experimental Pharmacology; Hlavka, J. J., Boothe, J. H., Eds.;

Springer-Verlag: Berlin, 1985; Vol. 78, pp 317−392. (b) Brodersen,

D. E.; Clemons, W. M. Jr.; Carter, A. P.; Morgan-Warren, R. J.;

Wimberly, B. T.; Ramakrishnan, V. The structural basis for the action

of the antibiotics tetracycline, pactamycin, and hygromycin B on the

30S ribosomal subunit. Cell 2000, 103, 1143−1154. (c) Pioletti, M.;

Schlunzen, F.; Harms, J.; Zarivach, R.; Gluhmann, M.; Avila, H.;

Bashan, A.; Bartels, H.; Auerbach, T.; Jacobi, C.; Hartsch, T.; Yonath,

A.; Franceschi, F. Crystal structures of complexes of the small

ribosomal subunit with tetracycline, edeine and IF3. EMBO J. 2001,

20, 1829−1839.

(5) Hunt, D. K.; Xiao, X.-Y; Clark, R. B.; O’Brien, W. J.; Fyfe, C.;

Grossman, T. H.; Sutcliffe, J. A.; Plamondon, L. TP-434 is a Novel

Broad-Spectrum Fluorocycline. Presented at the 50th Interscience

Conference on Antimicrobial Agents and Chemotherapy Conference,

Boston, MA, September 12−15, 2010, poster F1−2157.

(6) (a) Levy, S. B.; McMurry, L. Detection of an inductive membrane

protein associated with R-factor mediated tetracycline resistance.

Biochem. Biophys. Res. Commun. 1974, 56, 1080−1088. (b) McMurry,

L.; Petrucci, R. E. Jr.; Levy, S. B. Active efflux of tetracycline encoded

by four genetically different tetracycline resistant determinants in

Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 3974−3977.

(c) McMurry, L.; Park, B. H.; Burdett, V.; Levy, S. B. Energy-

Dependent Efflux Mediated by Class L (tet L) Tetracycline Resistance

Determinant from Streptococci. Antimicrob. Agents Chemother. 1987,

31, 1648−1651.

(14) Clark, R. B.; He, M.; Fyfe, C.; Lofland, D.; O’Brien, W. J.;

Plamondon, L.; Sutcliffe, J. A.; Xiao, X.-Y. 8-Azatetracyclines: Synthesis

and Evaluation of a Novel Class of Tetracycline Antibacterial Agents.

J. Med. Chem. 2011, 54, 1511−1528.

(15) Sun, C.; Hunt, D. K.; Clark, R. B.; Lofland, D.; O’Brien, W. J.;

Plamondon, L.; Xiao, X.-Y. Synthesis and Antibacterial Activity of

Pentacyclines: A Novel Class of Tetracycline Analogs. J. Med. Chem.

2011, 54, 3704−3731.

(16) Hlavka, J. J.; Krazinski, H.; Boothe, J. H. The 6-Deoxytetra-

cyclines. IV. A Photochemical Displacement of a Diazonium Group.

J. Org. Chem. 1962, 27, 3674−3675.

(17) For a review, see: Agwuh, K. N.; MacGowan, A.

Pharmacokinetics and Pharmacodynamics of the Tetracyclines

Including Glycylcyclines. J. Antimicrob. Chemother. 2006, 58, 256−265.

(18) Saivin, S.; Houin, G. Clinical Pharmacokinetics of Doxycycline

and Minocycline. Clin. Pharmacokinet. 1988, 15, 355−366.

(19) Cook, H. J.; Mundo, C. R.; Fonseca, L.; Gasque, L.; Moreno-

Esparza, R. Influence of the diet on bioavailability of tetracycline.

Biopharm. Drug Dispos. 1993, 14, 549−553.

(20) Ronn, M.; Dunwoody, N.; Sutcliffe, J. Pharmacokinetics of

TP-434 in mouse, rat, dog, monkey, and chimpanzee. Presented at

the 50th Interscience Conference on Antimicrobial Agents and

Chemotherapy Conference, Boston, MA, September 12−15, 2010,

paper F1−2163.

(21) McCormick, J. R. D.; Fox, S. M.; Smith, L. L.; Bitler, B. A.;

Reichenthal, J.; Origoni, V. E.; Muller, W. H.; Winterbottom, R.;

Doerschuk, A. P. Studies of the Reversible Epimerization Occurring in

the Tetracycline Family. The Preparation, Properties and Proof of

Structure of some 4-Epitetracyclines. J. Am. Chem. Soc. 1957, 79,

2849−2858.

(22) For recent reviews, see: (a) Schlummer, B.; Scholz, U.

Palladium-Catalyzed C−N and C−O CouplingA Practical Guide

(7) (a) Burdett, V. Streptococcal tetracycline resistance mediated at

the level of protein synthesis. J. Bacteriol. 1986, 165, 564−569.

(b) Sanchez-Pescador, R.; Brown, J. T.; Roberts, M.; Ureda, M. S.

Homology of TetM with translational elongation factors: implications

for potential modes of tetM conferred tetracycline resistance. Nucleic

621

dx.doi.org/10.1021/jm201467r | J. Med. Chem. 2012, 55, 606−622

Journal of Medicinal Chemistry

Article

from an Industrial Vantage Point. Adv. Synth. Catal. 2004, 346, 1599−

1626. (b) Yang, B. H.; Buchwald, S. L. Palladium-catalyzed amina-

tion of aryl halides and sulfonates. J. Organomet. Chem. 1999, 576,

125−146.

(23) (a) Sengstock, D. M.; Thyagarajan, R.; Apalara, J.; Mira, A.;

Chopra, T.; Kaye, K. S. Multidrug-resistant Acinetobacter baumannii: an

Emerging Pathogen Among Older Adults in Community Hospitals

and Nursing Homes. Clin. Infect. Dis. 2010, 50, 1611−1616.

(b) Dijkshoorn, L.; Nemeo, A.; Seifert, H. An Increasing Threat in

Hospitals: Multidrug-Resistant Acinetobacter baumannii. Nature Rev.

Microbiol. 2007, 5, 939−951.

(24) Clinical and Laboratory Standards Institute. Methods for Dilution

Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically,

8th ed.; Approved Standard M07-A8, Vol.29, no. 2; Clinical and

Laboratory Standards Institute: Wayne, PA, 2009.

(25) Xiao, X.-Y.; Hunt, D. K.; Zhou, J.-Y.; Clark, R. B.; Dunwoody,

N.; Fyfe, C.; Grossman, T. H.; O'Brien, W. J.; Plamondon, L.; Ronn,

̈

M.; Sun, C.; Zhang, W.-Y.; Sutcliffe, J. A. Fluorocyclines. 1. 7-Fluoro-9-

pyrrolidinoacetamido-6-demethyl-6-deoxytetracycline: A Potent, Broad

Spectrum Antibacterial Agent. J. Med. Chem. 10.1021/jm201465w.

622

dx.doi.org/10.1021/jm201467r | J. Med. Chem. 2012, 55, 606−622

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