Heterocyclyl Tetracyclines. 1. 7-Trifluoromethyl-8-Pyrrolidinyltetracyclines: Potent, Broad Spectrum Antibacterial Agents with Enhanced Activity against Pseudomonas aeruginosa

Article abstract of DOI:10.1021/acs.jmedchem.6b01903

Utilizing a total synthesis approach, the first 8-heterocyclyltetracyclines were designed, synthesized, and evaluated against panels of tetracycline- and multidrug-resistant Gram-positive and Gram-negative pathogens. Several compounds with balanced, highly potent in vitro activity against a broad range of bacterial isolates were identified through structure-activity relationships (SAR) studies. One compound demonstrated the best antibacterial activity against Pseudomonas aeruginosa both in vitro and in vivo for tetracyclines reported to date.

Full text of DOI:10.1021/acs.jmedchem.6b01903

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Article
Heterocyclyl Tetracyclines. 1. 7-Trifluoromethyl-8-
Pyrrolidinyltetracyclines: Potent, Broad Spectrum Antibacterial
Agents with Enhanced Activity against Pseudomonas aeruginosa
Yonghong Deng, Cuixiang Sun, Diana K. Hunt, Corey Fyfe, Chi-
Li Chen, Trudy H. Grossman, Joyce A. Sutcliffe, and Xiao-Yi Charlie Xiao
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01903 • Publication Date (Web): 01 Mar 2017
Downloaded from http://pubs.acs.org on March 1, 2017
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Heterocyclyl Tetracyclines. 1. 7ꢀTrifluoromethylꢀ8ꢀPyrrolidinyltetracyclines:
Potent, Broad Spectrum Antibacterial Agents with Enhanced Activity against
Pseudomonas aeruginosa
*†
†
†
‡
†
Yonghong Deng, Cuixiang Sun, Diana K. Hunt, Corey Fyfe, ChiꢀLi Chen, Trudy H.
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‡
‡
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Grossman, Joyce A. Sutcliffe, XiaoꢀYi Xiao
†
‡
Discovery Chemistry, Microbiology, Tetraphase Pharmaceuticals, 480 Arsenal Way,
Watertown, MA 02472, United States
ABSTRACT
Utilizing a total synthetic approach, the first 8ꢀheterocyclyltetracyclines were designed,
synthesized and evaluated against panels of tetracyclineꢀ and multidrugꢀresistant Gramꢀpositive
and Gramꢀnegative pathogens. Several compounds with balanced, highly potent in vitro activity
against a broad range of bacterial isolates were identified through structureꢀactivity relationships
(
SAR) studies. One compound demonstrated the best antibacterial activity against Pseudomonas
aeruginosa both in vitro and in vivo for tetracyclines reported to date.
INTRODUCTION
1
Since the discovery of chlortetracycline (1) in 1948, the tetracycline class of antibiotics is
generally regarded to be safe and has been used as firstꢀline drugs for the treatment of many
2
,3
bacterial infections caused by both Gramꢀpositive and Gramꢀnegative pathogens. As with all
antibiotic classes, the extensive use of tetracyclines for over 60 years, both in the clinic and in
agriculture, has resulted in increased pathogenic bacterial resistance and threatens the continued
4
,5
effectiveness of many tetracyclines used today. Of the identified mechanisms by which
bacteria become resistant to tetracyclines, tetracyclineꢀspecific efflux pumps [tet(A)ꢀtet(D), and
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6ꢀ8
9ꢀ10
tet(K)ꢀtet(L)], and ribosomal protection proteins [tet(M)ꢀtet(O)],
are by far the two most
common forms encountered in the clinic. Therefore, it is highly desirable to develop new
tetracyclines with improved in vitro potency and in vivo efficacy for use against current and
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emerging
multidrugꢀresistant
(MDR)
pathogens,
including
carbapenemꢀresistant
Enterobacteriaceae, MDR Acinetobacter species, and Pseudomonas aeruginosa.
As the number of naturally occurring new tetracyclines from microbial sources is limiting,
semisynthetic approaches that chemically modify natural antibiotics obtained by fermentation,
have emerged as a dominating discovery effort to seek new scaffolds with improved safety and
potency profiles. However, due to the densely functionalized structure and the chemical
instability of tetracyclines, chemical modifications have generally been rather challenging and
limited largely to the Cꢀ7 and Cꢀ9 positions using electrophilic aromatic substitution reactions
(
e.g., halogenation and nitration) followed by further functional group transformations.
1
1,12
13ꢀ15
Examples of this include minocycline (2)
and tigecycline (3).
While a few total synthetic
routes have been developed successfully, most were lengthy as well as low overall yielding and
therefore were impractical to scale both in terms of the number and quantity of new tetracycline
1
6
17,18
analogues. A more recent total synthetic methodology developed by Myers and coꢀworkers,
and expanded by Tetraphase Pharmaceuticals, has enabled the efficient introduction of a broad
array of substituents at many more positions including Cꢀ4, Cꢀ4a, Cꢀ5, Cꢀ5a, Cꢀ6, Cꢀ7, Cꢀ8, Cꢀ9,
1
9ꢀ21
and Cꢀ12a,
as well as the incorporation of heterocyclic and polycyclic ring systems into the
22ꢀ24
tetracycline core structure,
creating completely novel tetracycline derivatives that were
previously inaccessible (or extremely difficult to access) through semisynthesis. More than 3000
tetracycline analogues have been synthesized by Tetraphase using the total synthesis approach.
Many of them are highly active against both Gramꢀpositive and Gramꢀnegative bacteria and a
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number of them have progressed into various clinical and preꢀclinical stages, with the lead
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development candidate, eravacycline (4), a broad spectrum antibiotic with improved activity
against multidrug resistant pathogens, currently undergoing late stage clinical studies.
Cyclic amines such as pyrrolidines and piperidines are an important class of pharmacophores
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found in a variety of natural products and approved drugs. In an effort to continue the search
for new tetracyclines, a series of 8ꢀheterocycle substituted tetracyclines has been designed and
synthesized in our laboratories. Their antimicrobial activities were evaluated against panels of
Gramꢀpositive and Gramꢀnegative pathogens including those with various tetracycline resistance
mechanisms. Herein, we report the details of these studies.
H C
CH3
H C
3
CH3
H C
CH3
3
3
Cl
CH
N
N
N
HO
3
H
H
H
H
OH
NH2
OH
NH2
O
O
OH
O
HO H O
O
OH
O
HO
O
O
H
Chlortetracycline (1)
Minocycline (2)
H C
3
CH3
H C
CH3
H C
CH3
3
3
N
N
F
N
H
H
H
5 H
6
4
7
OH
NH2
OH
NH2
O
O
H
N
D
C
B
A
H C
N
3
N
H
N
1
0
11
12
1
H C
3
O
H
O
CH3
OH
O
HO H O
O
OH
O
HO H O
O
Tigecycline (3)
Eravacycline (4)
Figure 1. Structures of Natural (1), Semiꢀsynthetic (2 and 3) and Fully Synthetic (4)
Tetracycline.
RESULTS AND DISCUSSION
Chemistry. The initial method leading to the 7ꢀchloroꢀ8ꢀpyrrolidinyl tetracyclines is described
in scheme 1. Compound 6 was readily prepared in quantitative yield by benzylation of phenol
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27
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.
A Suzuki coupling of aryl bromide 6 with NꢀBocꢀ2ꢀpyrroleboronic acid catalyzed by
dichloro[1,1’ꢀbis(diphenylphosphino)ferrocene] palladium(II) dichloromethane adduct gave
compound 7 in 60% yield. Reduction of the pyrrole by catalytic hydrogenation at atmospheric
pressure proceeded with concurrent removal of the benzyl protecting group. The crude product
was reprotected using benzyl bromide and potassium carbonate to give compound 8 in 81% yield
over two steps. Chlorination with Nꢀchlorosuccinimide followed by deprotection of the Boc
group with hydrogen chloride in dioxane yielded the Dꢀring precursor 9. The tandem Michaelꢀ
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Dieckmann annulation was then carried out by treatment of 9 with lithium diisopropylamide in
o
the presence of N,N,N’,N’ꢀtetramethylethylenediamine in THF at ꢀ78 C followed by the addition
1
7, 28
of ABꢀring precursor, enone 10,
generating intermediate 11 in 46% yield. Further
elaboration of the pyrrolidinyl group via reductive alkylation with various aldehydes in the
29
presence of acetic acid and sodium triacetoxyborohydride gave compound 12. Desilylation of
1
2 with aqueous HF followed by catalytic hydrogenation in the presence of palladium on carbon
in ethyl acetate gave the desired 7ꢀchloroꢀ8ꢀ(2ꢀpyrrolidinyl)tetracyclines 13. Partial deꢀ
chlorination was observed when polar solvents such as methanol were used for hydrogenation.
a
Scheme 1. Synthesis of 7-Chloro-8-pyrrolidinyltetracyclines
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a
Reagents and conditions: (a) BnBr, K CO , acetone; (b) NꢀBocꢀ2ꢀpyrroleboronic acid,
2
3
PdCl (dppf)·CH Cl , Na CO , toluene/dioxane/H O; (c) H , Pd/C, CH OH; (d) BnBr, K CO ,
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3
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2
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3
o
acetone; (e) NCS, CH CN; (f) HCl/dioxane; (g) LDA, TMEDA, THF, ꢀ78 C, then enone 10, ꢀ78
3
o
to ꢀ10 C; (h) RCHO, AcOH, Na(OAc) BH, ClCH CH Cl; (i) HF, CH CN; (j) H , Pd/C, EtOAc.
3
2
2
3
2
Giovannini A. et al. reported that NꢀBocꢀωꢀamino ketones could be prepared from NꢀBoc fiveꢀ
3
0
to eightꢀmembered lactams via organometallic ringꢀopening reactions. Deprotection of the Boc
group with trifluoroacetic acid (TFA) provided the corresponding 2ꢀsubstituted cyclic imines,
which could be further reduced to provide cyclic amine moieties. Using this strategy, we then
explored the synthesis of 8ꢀpiperidinyl tetracyclines, as well as 8ꢀazepanyl tetracyclines. Thus,
2
1
o
lithiumꢀhalogen exchange of bromide 14 with nꢀbutyllithium at ꢀ100 C followed by addition
of 1ꢀNꢀBocꢀ2ꢀpiperidone provided NꢀBocꢀ
δꢀamino ketone 15 with various levels of cyclic Nꢀ
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Bocꢀenamine 16 (Scheme 2). Treatment of ketone 15 with TFA gave the corresponding amine,
which cyclized readily to imine 19 under the reaction conditions (87% yield). Compound 19 was
also obtained from NꢀBocꢀenamine 16 via Boc deprotection by treatment with TFA (76% yield).
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In the synthesis of azepanes using NꢀBocꢀεꢀcaprolactam, formation of imine 20 was achieved by
treatment of the acyclic aminoketone 17 with titanium (IV) isoproxide. The Dꢀring precursors 21
and 22 were then prepared by reduction of the imines (19 and 20) with sodium borohydride.
Analogous 7ꢀchloroꢀ8ꢀ(2ꢀpiperidinyl)tetracyclines 25 and 7ꢀchloroꢀ8ꢀ(2ꢀazepanyl)tetracyclines
26 were then synthesized via MichaelꢀDieckmann annulation, reductive alkylation, deꢀsilylation,
and hydrogenation according to similar procedures used in the preparation of compound 13.
a
Scheme 2. Synthesis of 7-Chloro-8-heterocyclyltetracyclines
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a
Reagents and conditions: (a) nꢀBuLi, THF, then 1ꢀNꢀBocꢀ2ꢀpiperidone or NꢀBocꢀεꢀ
caperolactam; (b) TFA, CH Cl ; (c) Ti(OꢀiPr) , THF; (d) NaBH , CH OH; (e) LDA, TMEDA,
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o
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THF, ꢀ78 C, then enone 10, ꢀ78 to ꢀ10 C; (f) HCHO, AcOH, Na(OAc) BH, ClCH CH Cl; (g)
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2
2
HF, CH CN; (h) H , Pd/C, EtOAc.
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2
Several 8ꢀheteroaryl substituted tetracycline analogues were also synthesized for the structureꢀ
activity relationships (SAR) study (Scheme 3). MichaelꢀDieckmann reaction between Dꢀring
precursor 14 and enone 10 under standard conditions gave fully protected intermediate 27, and
subsequent Suzuki coupling with heteroaryl boronic acids gave compounds 28 and 29. The twoꢀ
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step deprotection sequence as described above (HF and hydrogenation) yielded 7ꢀchloroꢀ8ꢀ
heteroaryltetracyclines 30 and 31.
a
Scheme 3. Synthesis of 7-Chloro-8-heteroaryltetracyclines
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Reagents and conditions: (a) LDA, TMEDA, THF, ꢀ78 C, then enone 10, ꢀ78 to ꢀ10 C; (b)
ArB(OH) , PdCl (dppf)·CH Cl , Na CO , toluene/dioxane/H O; (c) HF, CH CN; (d) H , Pd/C,
2
2
2
2
2
3
2
3
2
EtOAc.
We next turned our attention to explore analogues with different substituents at Cꢀ7 position.
Since 8ꢀ(2ꢀpyrrolidinyl)tetracyclines showed better antimicrobial activities against a panel of
selected bacteria in the initial screening, further studies were focused on 8ꢀ(2ꢀpyrrolidinyl)
analogues. As a typical example, the synthetic approach to the 7ꢀtrifluoromethylꢀ8ꢀ(2ꢀ
pyrrolidinyl)tetracyclines is shown in scheme 4. Following similar approaches as outlined in
Scheme 2, imines 32 were prepared in a twoꢀstep process from aryl bromide 6 and NꢀBocꢀ2ꢀ
pyrrolidinone or NꢀBocꢀ3ꢀmethylꢀ2ꢀpyrrolidinone or NꢀBocꢀ5ꢀmethylꢀ2ꢀpyrrolidinone.
Reduction of the imines with sodium borohydride followed by Boc protection gave compounds
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3
3. Regioselective iodination with iodine/silver trifluoroacetate yielded aryl iodides 34, which
were subjected to copperꢀmediated trifluoromethylation using methyl 2,2ꢀdifluoroꢀ2ꢀ
3
2
(
fluorosulfonyl)acetate/CuI as a trifluoromethyl anion equivalent to afford compounds 35 after
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Boc deprotection. The requisite Dꢀring precursors 36ꢀ38 were then prepared by reductive
alkylation with either formaldehyde or benzaldehyde or alkylation with allyl bromide. Michael–
Dieckmann reaction under standard conditions gave fully protected tetracycline intermediates 39,
4
0, and 42. Deprotection of the allyl group in compound 40 with Pd(PPh ) and N,Nꢀ
3 4
dimethylbarbituric acid provided compound 41a, which was further converted to 41b via
reductive alkylation with formaldehyde. Desilylation with aqueous HF and subsequent
hydrogenation in the presence of palladium on carbon in methanol gave the final compounds 43ꢀ
45.
a
Scheme 4. Synthesis of 7-Trifluoromethyl-8-pyrrolidinyltetracyclines
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R2
R2
R1
R1
Br
CH3
CO Ph
CH3
CH3
N
N
a, b
c, d
Boc
CO Ph
CO Ph
2
2
2
OBn
OBn
OBn
e
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33
6
R2
R2
R2
CF3
CF3
I
R1
R1
R1
f, g
CH3
CO Ph
h or i or j
CH3
CH3
N
N
N
H
Boc
R
CO Ph
CO Ph
2
2
2
OBn
OBn
OBn
1
2
36: R = R = H, R = CH
34
3
35
1
2
37: R = CH , R = H, R = allyl
3
1
2
38a: R = H, R = CH , R = Bn
3
1
2
3
8b: R = H, R = CH , R = CH
3
3
k
R2
H C
3
R2
CH3
H3C
H
CH3
CF3
N
CF3
OH
N
R1
H
H
R1
H
OH
NH2
O
N
m, n
N
N
R
R
O
OBn
OBn O HO
O
OTBS
O
HO H O
O
1
2
1
2
3
4
4
4
9: R = R = H, R = CH
43a: R = CH , R = R = H,
3
3
1
2
2
1
0: R = CH , R = H, R = allyl
44a: R = R = H, R = CH
3
3
l
1
2
1
2
1a: R = CH , R = R = H
44b: R = R = CH , R = H
3
3
h
1
2
1
2
1b: R = CH , R = H, R = CH
45a: R = R = H, R = CH
3
3
3
3
1
2
2
1
42a: R = H, R = CH , R = Bn
45b: R = R = CH , R = H
3
3
1
2
42b: R = H, R = CH , R = CH
3
a
Reagents and conditions: (a) nꢀBuLi, THF, then NꢀBocꢀ2ꢀpyrrolidinone or 3ꢀmethylꢀNꢀBocꢀ2ꢀ
pyrrolidinone or 5ꢀmethylꢀNꢀBocꢀ2ꢀpyrrolidinone; (b) TFA, CH Cl ; (c) Na(OAc) BH,
2
2
3
ClCH CH Cl; (d) Boc O, DMAP, DMF; (e) I , CF CO Ag, CHCl ; (f) CH O CCF SO F, CuI,
2
2
2
2
3
2
3
3
2
2
2
DMF; (g) HCl, dioxane; (h) HCHO, AcOH, Na(OAc) BH, ClCH CH Cl; (i) allylbromide,
3
2
2
K CO , NaI, NMP; (j) PhCHO, AcOH, Na(OAc) BH, ClCH CH Cl; (k) LDA, TMEDA, THF, ꢀ
2
3
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o
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8 C, then enone 10, ꢀ78 to ꢀ10 C; (l) Pd(PPh ) , N,Nꢀdimethylbarbituric acid, CH Cl ; (m) HF,
3 4 2 2
CH CN; (n) H , Pd/C, CH OH.
3
2
3
The Michael–Dieckmann reaction between the achiral 4ꢀ(2ꢀpyrrolidinyl) substituted Dꢀring
precursors and enone 10 generated two diastereomers, and it was found that one of the
diastereomers was more active across the entire panel of selected bacterial strains. We then
investigated the asymmetric synthesis of 8ꢀ(2ꢀpyrrolidinyl)tetracycline analogues applying the
methodology developed by Ellman et al. for the enantioselective preparation of 2ꢀsubstituted
10
11
12
13
14
15
16
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44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
3
pyrrolidines. Thus, enantiomerically pure 4ꢀ(2ꢀpyrrolidinyl) substituted Dꢀring precursor 49
was prepared in three steps (Scheme 5): titanium (IV)ꢀmediated condensation of tertꢀ
2
1
butanesulfinamide with aldehyde 46 gave Nꢀ(tert)ꢀbutanesulfinyl imine 47 in 81% yield;
addition of Grignard reagent 2‐[2‐(bromomagnesio)ethyl]‐1,3‐dioxane prepared from 2ꢀ(2ꢀ
o
o
bromoethyl)ꢀ1,3ꢀdioxane to the imine 47 at ꢀ78 C to ꢀ48 C afforded the desired sulfinamide 48
in excellent yield (95%) and diastereoselectivity; deprotection of the acetal with 1:1 TFA:H O
2
proceeded with simultaneous cleavage of the sulfinamide group, and subsequent inꢀsitu reduction
with Na(OAc) BH gave enantiomerically pure 49 in moderate yield. Standard Michaelꢀ
3
Dieckmann reaction gave compound 50. Various alkyl groups were then introduced via reductive
alkylation with aldehydes or ketones. Aqueous HF treatment, and palladium catalyzed
hydrogenation provided the desired enantiomerically pure 7ꢀtrifluoromethylꢀ8ꢀ(2ꢀ
pyrrolidinyl)tetracycline analogues 43b-h.
a
Scheme 5. Asymmetric Synthesis of 7-Trifluoromethyl-8-pyrrolidinyltetracyclines
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a
Reagents and conditions: (a) (S)ꢀtertꢀbutanesulfinamide, Ti(OEt) , THF; (b) 2‐[2‐
4
(bromomagnesio)ethyl]‐1,3‐dioxane, THF; (c) TFA–H O, then Na(OAc) BH; (d) LDA,
2
3
o
o
TMEDA, THF, ꢀ78 C, then enone 10, ꢀ78 to ꢀ10 C; (e) RCHO or RCOR’, AcOH,
Na(OAc) BH, ClCH CH Cl; (f) HF, CH CN; (g) H , Pd/C, CH OH.
3
2
2
3
2
3
Similar approaches were taken for the preparation of 7ꢀfluoro, 7ꢀmethoxy, 7ꢀdimethylamino,
ꢀtrifluoromethoxy, and 7ꢀhydroꢀ8ꢀpyrrolidinyltetracycline derivatives, and the synthetic
7
schemes are included in the Supporting Information.
Biology. The fully synthetic 8ꢀheterocyclyl tetracycline analogues were evaluated for in vitro
antibacterial activity in minimal inhibitory concentration (MIC) assays as per Clinical
3
4
Laboratory Standards Institute methodology against panels of tetracyclineꢀresistant and
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tetracyclineꢀsusceptible Gramꢀnegative and Gramꢀpositive bacteria, which also included
multidrugꢀresistant (MDR) organisms (Tables 1 and 2). When one or more additional chiral
centers were introduced on the C8 substituent by nonꢀasymmetric synthetic methods, as we also
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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36
37
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
24
observed in earlier studies, the resulting diastereomers showed varying levels of antibacterial
activity, despite the belief that the “northꢀwestern” region (i.e. the C4 to C8 portion) of the
tetracycline molecule is not directly involved in binding with the 30S ribosome based on coꢀ
3
5
crystal structure studies. For brevity, only the relatively more active diastereomer from each
diastereomeric set was included in Tables 1 and 2, except for compounds 13, 25 and 78, which
were isolated and tested as mixtures of the respective two diastereomers (~ 1:1), and except for
compounds 43c and 43d, which were listed as an example of a diasteromeric pair.
As shown in Table 1, when the C7 substituent was fixed as chlorine, increasing the alkyl
heterocyclic ring size at C8 from 5ꢀmember (compound 13) to 6ꢀmember and 7ꢀmember
(
compounds 25 and 26) decreased antibacterial activity by 2ꢀ to 8ꢀfold against most strains in the
panel. Likewise, replacing the alkyl heterocyclic substitution at C8 with an aromatic heterocycle
(
compounds 30 and 31) further diminished potency, especially against the Gramꢀnegative
2
1,24
isolates, most likely due to the decreased basicity of the aromatic nitrogen.
Insert Table 1 Here.
We then fixed the C8 substituent as a 2ꢀpyrrolidine ring and investigated the structureꢀactivity
7
relationships (SAR) of analogues with a range of substituents at C7 (R ), as well as with various
7
substitutions on the pyrrolidine ring at C8. As shown in Table 2, when R was trifluoromethyl
and the C8 pyrrolidine was not substituted, the diastereomer with a (2S)ꢀconfigured pyrrolidine
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5
6
(
compound 43c) was 2ꢀ to greater than 64ꢀfold more potent than the corresponding (2R)ꢀ
configured diastereomer (compound 43d) against P. aeruginosa PA555 and all Gramꢀpositive
strains tested. When the pyrrolidine nitrogen was substituted by alkyl groups with increasing
lipophilicity, varying from methyl, ethyl, isopropyl, propyl, cyclopropylmethyl, to isobutyl group
(compounds 43a, 43f, 43e, 43g, 43b, and 43h), the corresponding analogues’ antibacterial
potency decreased progressively against most organisms in the panel, especially against Gramꢀ
negative strains, with compound 43h losing almost all Gramꢀnegative activity. Interestingly, the
increased lipophilicity of the Nꢀsubstituent on the pyrrolidine ring had much less effect, and
sometimes had even a slightly reversed effect, on activity against the tet(M) strains, with potency
in some cases increased by 2ꢀ to 4ꢀfold (Staphylococcus aureus SA161, Enterococcus faecalis
EFs327, and Enterococcus faecium EFm404).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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0
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0
1
2
3
4
5
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7
8
9
0
When a methyl group was present on one of the ring carbons, the compound (44a) displayed
slightly increased activity (2ꢀ to 4ꢀfold) against S. aureus SA161, Escherichia coli EC155,
Acinetobacter baumannii AB250, Stenotrophomonas maltophilia SM256, and Burkholderia
cenocepacia BC240, while having slightly decreased potency (2ꢀfold) against S. aureus SA101,
Klebsiella pneumoniae KP457, and P. aeruginosa PA555. Moving the methyl group to another
ring carbon and/or to the ring nitrogen (compounds 44b, 45a, and 45b) generally decreased
potency against most isolates in the panel.
When the substitution on C7 was changed from trifluoromethyl to hydrogen, fluorine,
dimethylamino, methoxy, and trifluoromethoxy (compounds 86, 76, 78, 77, and 79), the
analogues’ antibacterial activity decreased from 2ꢀ to more than 512ꢀfold against most strains
tested, with the 7ꢀmethoxy and 7ꢀdimethylamino compounds (77 and 78) retaining the least
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activity and the 7ꢀtrifluoromethoxy compound (79) retaining the most activity as compared to the
7ꢀtrifluoromethyl analogue 43c.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
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37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Insert Table 2 Here.
Overall, compounds 43c, 43a, 44a, and 45a were highly potent and displayed a balanced
spectrum of activity against a broad range of MDR Gramꢀnegative and Gramꢀpositive pathogens,
with no MIC values greater than 8 µg/mL. Furthermore, due to its better potency against P.
aeruginosa with MIC values of 2ꢀ4 µg/mL, which were among the best in vitro antiꢀ
Pseudomonas activity for tetracyclines reported to date, compound 43c was selected as the first
compound from this 8ꢀheterocyclyl tetracycline series to be evaluated for in vivo antibacterial
activity in mouse models of infections. As shown in Figure 2, in a mouse lung infection model
challenged with P. aeruginosa PA1145, compound 43c (MIC = 2 µg/mL against P. aeruginosa
PA1145) demonstrated doseꢀproportional bacterial burden reductions of 1.7, 2.3, and 3.7 mean
log colonyꢀforming units (CFUs) from T = 24 h when administered intravenously (IV) at 5, 15,
1
0
and 40 mg/kg, respectively, twice daily. In the same model, comparator antibiotics amikacin
MIC = 4 µg/mL), tobramycin (MIC = 1 µg/mL), and tigecycline (MIC = 16 µg/mL) showed
.6, 4.2, and essentially no mean log CFU reductions from untreated control group (T = 24 h),
(
3
1
0
respectively, when dosed IV (amikacin and tigecycline) or IN (tobramycin) at 40 mg/kg, twice
daily. In a mouse thigh infection model challenged with P. aeruginosa PA694, compound 43c
(
MIC = 4 µg/mL against P. aeruginosa PA694) also displayed doseꢀproportional efficacy with
mean log CFU reductions of 0.4, 0.9, and 1.9 versus the 24 hour untreated control when dosed
1
0
IV at 5, 15, and 40 mg/kg, respectively, twice daily. The comparator meropenem had better in
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vitro potency (MIC = 0.125 µg/mL), which also translated into greater in vivo efficacy of 2.9,
4.2, and 4.9 mean log10 CFU reductions from the 24 hour untreated control group when
administered similarly in the same model.
0
1
2
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8
9
0
Insert Figure 2 Here.
CONCLUSIONS
A series of fully synthetic 7ꢀRꢀ8ꢀheterocyclyl tetracyclines were designed and prepared using
17
the tetracycline total synthesis platform. Antibacterial activities of the new tetracyclines were
evaluated against panels of tetracyclineꢀ and multidrugꢀresistant Gramꢀpositive and Gramꢀ
negative pathogens. Structureꢀactivity relationships regarding the substitutions at the C7 and C8
positions were investigated. A number of analogues (43c, 43a, 44a, and 45a) displayed balanced,
highly potent in vitro activity against a broad range of bacterial isolates. Furthermore, compound
43c,
a
7ꢀtrifluoromethylꢀ8ꢀ[(2S)ꢀ2ꢀpyrrolidinyl]ꢀ6ꢀdemethylꢀ6ꢀdeoxytetracycline, showed
enhanced in vitro antiꢀP. aeruginosa activity with MIC values of 2ꢀ4 µg/mL, which were among
2
4
the best antibacterial activity against P. aeruginosa for tetracyclines reported to date.
Subsequent in vivo efficacy studies in mouse lung and thigh models of infections challenged
with P. aeruginosa (PA1145 and PA694, respectively) demonstrated that the in vitro
antibacterial activity of compound 43c translated into in vivo efficacy against P. aeruginosa. Our
24
data on this and other new tetracycline scaffolds suggested that considerable in vitro and in
vivo antiꢀP. aeruginosa activity is attainable by exploring greater chemical diversity as enabled
1
7
by the fullyꢀsynthetic tetracycline platform.
EXPERIMENTAL SECTION
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Chemistry. All reactions involving airꢀsensitive reagents were performed under nitrogen in
flameꢀdried glassware using syringeꢀseptum cap technique. Unless it is specified, all reagents
were used as received without further purifications. Thin layer chromatography (TLC) analysis
was performed on Merck silicaꢀgel 60 F254 and visualized under UV light or immersion of the
plate in a basic solution of potassium permanganate in water followed by heating. Flash
chromatography was performed on a Biotage Isolera One purification system using Biotage
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
SNAP cartridges. Proton ( H) NMR (nuclear magnetic resonance) spectra were recorded on a
400 MHz JEOL ECXꢀ400 spectrometer. Chemical shifts are reported in parts per million (ppm)
using residual solvent as the internal standard (CDCl at 7.24 ppm, DMSOꢀd at 2.50 ppm,
3
6
CD OD at 3.30 ppm). Reverse phase preparative HPLC was performed on a Waters
3
Autopurification system with massꢀdirected fraction collection (For final compounds: column,
Polymerx RPꢀ1 100A, 10 ꢁm, 250 mm × 21.20 mm; flow rate, 20 mL/min; solvent A, water with
0
.05 N HCl; solvent B, acetonitrile. For intermediates: column, SunFire Prep C18 OBD, 5 ꢁm,
9 mm × 50 mm; flow rate, 20 mL/min; solvent A, water with 0.1% formic acid; solvent B,
1
acetonitrile with 0.1% formic acid). Unless otherwise described, all final tetracycline compounds
were isolated as monoꢀ, diꢀ, or trihydrochloride salts following freezeꢀdrying. Analytical high
performance liquid chromatographyꢀelectrospray mass spectra (HPLCꢀMS) were obtained using
a Waters Alliance system (column, SunFire C18, 5 ꢁm, 4.6 mm × 50 mm; flow rate, 1 mL/min;
solvent A, water with 0.1% formic acid; solvent B, acetonitrile with 0.1% formic acid; MS
detector, Waters 3100). Purity of tested compounds was determined to be ≥95% by analytical
HPLCꢀMS analysis.
Phenyl 2-(Benzyloxy)-4-bromo-6-methylbenzoate (6). To a solution of 5 (9.53 mmol, 1
equiv) in acetone (19 mL) was added K CO (2.63 g, 15.00 mmol, 1.5 equiv) and BnBr (1.19
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2
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3
3
3
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
mL, 10.00 mmol, 1.05 equiv). The mixture was stirred at room temperature overnight and
filtered through a Celite pad. The Celite pad was washed with EtOAc. The combined filtrate was
concentrated under reduced pressure. Flash chromatography on silica gel with 0%ꢀ5%
0
1
2
3
4
5
6
7
8
9
0
1
2
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7
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1
EtOAc/hexanes yielded the desired product 6 as a white solid (3.61 g, 96%). H NMR (400
MHz, CDCl3)
19.1 (M + Na).
tert-Butyl
δ
7.20ꢀ7.45 (m, 8H), 7.03ꢀ7.09 (m, 4H), 5.13 (s, 2H), 2.43 (s, 3H). MS (ESI) m/z
4
2-[3-(benzyloxy)-5-methyl-4-(phenoxycarbonyl)phenyl]-1H-pyrrole-1-
carboxylate (7). To a pressure vial was charged with compound 6 (852 mg, 2.14 mmol, 1
equiv), NꢀBocꢀ2ꢀpyrroleboronic acid (543 mg, 2.57 mmol, 1.2 equiv), dichloro[1,1’ꢀ
bis(diphenylphosphino)ferrocene] palladium(II) dichloromethane adduct (88 mg, 0.11 mmol,
0.05 equiv), and sodium carbonate (1.14 g, 10.7 mmol, 5 equiv). The vial was briefly evacuated
and filled with N . Toluene (5 mL), 1,4ꢀdioxane (5 mL), and H O (1 mL) were added. The
2
2
o
reaction mixture was heated with a 90 C oil bath for 2 h, cooled to rt, diluted with EtOAc,
washed sequentially with aqueous phosphate buffer (pH = 7) and brine, dried over Na SO , and
2
4
concentrated. Purification of the residue by Biotage flash chromatography gave compound 7 as a
1
colorless oil (621 mg, 60%). H NMR (400 MHz, CDCl )
δ 7.22ꢀ7.48 (m, 9H), 7.12 (d, J = 7.8
3
Hz, 2H), 6.89 (d, J = 7.8 Hz, 2H), 6.20ꢀ6.26 (m, 2H), 5.15 (s, 2H), 2.48 (s, 3H), 1.41 (s, 9H). MS
(ESI) m/z 484.4 (M + H).
tert-Butyl
2-[3-(benzyloxy)-5-methyl-4-(phenoxycarbonyl)phenyl]pyrrolidine-1-
carboxylate (8). Compound 7 (621 mg, 1.28 mmol, 1 equiv) was dissolved in methanol. PdꢀC
(10 wt%, 186 mg) was added. The reaction flask was briefly evacuated and reꢀfilled with
hydrogen. The reaction mixture was stirred under 1 atm H at rt for 2 h and filtered through a
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Celite pad. The Celite pad was washed with methanol. The filtrate was concentrated to give the
intermediate as white foam.
The above intermediate was dissolved in acetone (12 mL), followed by addition of K CO
2
3
10
11
12
13
14
15
16
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23
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31
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43
44
45
46
47
48
49
50
51
52
53
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56
57
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59
60
(
350 mg, 2.54 mmol, 2 equiv) and BnBr (0.16 mL, 1.33 mmol, 1.04 equiv). After stirring
overnight at rt, the reaction mixture was filtered through a Celite pad. The Celite pad was washed
with three portions of EtOAc. The combined filtrate was concentrated. Purification of the residue
by Biotage flash chromatography gave compound 8 as a colorless oil (504 mg, 81% over two
1
steps). H NMR (400 MHz, CDCl , rotamer)
δ 7.22ꢀ7.48 (m, 8H), 7.05ꢀ7.15 (m, 2H), 6.63ꢀ6.70
3
(m, 2H), 5.13 (s, 2H), 4.90 and 4.76 (br s, 1H), 3.50ꢀ3.65 (m, 2H), 2.43 (s, 3H), 2.25ꢀ2.28 (m,
1H), 1.72ꢀ1.90 (m, 3H), 1.48 (s, 3H), 1.26 (s, 6H). MS (ESI) m/z 488.4 (M + H).
Phenyl 6-(Benzyloxy)-3-chloro-2-methyl-4-(pyrrolidin-2-yl)benzoate (9). To a solution of
compound 8 (556 mg, 1.14 mmol, 1 equiv) in 5 mL of CH CN was added NCS (160 mg, 1.20
3
o
mmol, 1.05 equiv) in one portion. The reaction mixture was heated with a 60 C oil bath for 18 h,
cooled to rt, and evaporated to dryness. The residue was suspended in 200 mL CH Cl , washed
2
2
sequentially with aqueous NaOH (1 N), H O and brine, dried over Na SO , and concentrated.
2
2
4
Purification of the residue by Biotage flash chromatography gave aryl chloride as a white solid
1
(447 mg, 75%). H NMR (400 MHz, CDCl , mixture of rotamers)
δ
7.22ꢀ7.48 (m, 8H), 7.05ꢀ7.15
3
(m, 2H), 6.63ꢀ6.70 (m, 1H), 5.06ꢀ5.26 (m, 3H), 3.47ꢀ3.58 (m, 2H), 2.46 (s, 3H), 2.25ꢀ2.28 (m,
1
H), 1.55ꢀ1.88 (m, 3H), 1.48 (s, 3H), 1.26 (s, 6H). MS (ESI) m/z 522.4 (M + H).
The aryl chloride (447 mg, 0.86 mmol) was suspended in HCl/1,4ꢀdioxane (4.0 M, 9 mL).
After stirring at rt for 1 h, the volatiles were evaporated. The residue was suspended in EtOAc,
washed with saturated aqueous NaHCO and brine, dried over Na SO , and concentrated.
3
2
4
Purification of the residue by Biotage flash chromatography gave compound 9 as an offꢀwhite
1
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solid (338 mg, 93%). H NMR (400 MHz, CDCl )
δ
7.48 (dd, J = 1.8, 7.8 Hz, 2H), 7.34ꢀ7.42 (m,
6H), 7.26 (t, J = 7.8 Hz, 1H), 7.14 (d, J = 7.8 Hz, 2H), 5.20 (s, 2H), 4.57 (t, J = 7.4 Hz, 1H),
.04ꢀ3.18 (m, 2H), 2.52 (s, 3H), 2.34ꢀ2.45 (m, 1H), 2.06 (br s, 1H), 1.78ꢀ1.85 (m, 2H), 1.44ꢀ1.54
3
3
0
1
2
3
4
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6
7
8
9
0
1
2
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4
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
m, 1H). MS (ESI) m/z 422.4 (M + H).
(1R,3S,4S,11S)‐8,16‐Bis(benzyloxy)‐11‐[(tert‐butyldimethylsilyl)oxy]‐19‐chloro‐4‐
dimethylamino)‐12‐hydroxy‐18‐(pyrrolidin‐2‐yl)‐6‐oxa‐7‐
(
3
,11 5,9 15,20
azapentacyclo[11.8.0.0 .0 .0
]henicosa-5(9),7,12,15(20),16,18‐hexaene‐10,14‐dione
(
11). A solution of nꢀBuLi in hexanes (2.5 M, 0.53 mL, 0.63 mmol, 2.3 equiv) was added
o
dropwise to a solution of iꢀPr NH (0.20 mL, 1.38 mmol, 2.4 equiv) in THF (4 mL) at ꢀ78 C
2
o
o
under N atmosphere. The reaction solution was stirred at ꢀ78 C for 20 min and at 0 C for 5
2
o
min, and was then reꢀcooled to ꢀ78 C. N,N,N’,N’ꢀTetramethylethylenediamine (0.22 mL, 1.50
mmol, 2.6 equiv) was added, followed by addition of a solution of 9 (267 mg, 0.63 mmol, 1.1
equiv) in THF (4 mL) via a cannula. The resulting darkꢀorange mixture was stirred for 1 h at ꢀ78
o
o
C and was cooled to ꢀ100 C. A solution of enone 10 (277 mg, 0.58 mmol, 1 equiv) in THF (4
o
mL) was added dropwise via a cannula. The mixture was allowed to gradually warm to ꢀ78 C.
LHMDS (0.60 mL, 1.0 M/THF, 0.60 mmol, 1.05 equiv) was added, and the reaction was slowly
o
warmed to ꢀ5 C. Saturated aqueous NH Cl was added. The mixture was extracted three times
4
with EtOAc. The combined EtOAc extracts were washed with brine, dried over Na SO , and
2
4
concentrated. Purification of the residue by Biotage flash chromatography gave compound 11 as
1
a light yellow foam (214 mg, 46%). H NMR (400 MHz, CDCl )
δ 7.26ꢀ7.50 (m, 11H), 5.35 (s,
3
2
H), 5.25 (d, J = 11.4 Hz, 1H), 5.23 (d, J = 11.4 Hz, 1H), 4.51 (t, J = 6.9 Hz, 1H), 3.97 (d, J =
0.4 Hz, 1H), 3.43 (dd, J = 4.9, 15.9 Hz, 1H), 2.91ꢀ3.09 (m, 3H), 2.28ꢀ2.56 (m, 10H), 2.12 (d, J
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14.6 Hz, 1H), 1.54ꢀ1.80 (m, 3H), 1.34ꢀ1.48 (m, 2H), 0.83 (s, 9H), 0.26 (s, 3H), 0.14 (s, 3H).
MS (ESI) m/z 810.4 (M + H).
1R,3S,4S,11S)‐8,16‐Bis(benzyloxy)‐11‐[(tert‐butyldimethylsilyl)oxy]‐19‐chloro‐4‐
dimethylamino)‐12‐hydroxy‐18‐(1‐methylpyrrolidin‐2‐yl)‐6‐oxa‐7-
(
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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(
3
,11 5,9 15,20
azapentacyclo[11.8.0.0 .0 .0
]henicosa‐5(9),7,12,15(20),16,18‐hexaene‐10,14‐dione
(
12). Compound 11 (30.8 mg, 0.038 mmol, 1 equiv) was dissolved in 1,2ꢀdichloroethane (1 mL).
Aqueous formaldehyde (37%, 8.5 ꢀL, 0.11 mmol, 3 equiv) and acetic acid (6.5 ꢀL, 0.11 mmol, 3
equiv) were added. After stirring at rt for 45 min, sodium triacetoxyborohydride (16 mg, 0.076
mmol, 2 equiv) was added. Stirring was continued overnight. The reaction mixture was poured
into saturated aqueous NaHCO . The mixture was extracted three times with CH Cl . The
3
2
2
combined organic extracts were washed with brine, dried over Na SO , and concentrated to give
2
4
crude 12, which was used directly for the next step without further purification. MS (ESI) m/z
24.7 (M + H).
4S,4aS,5aR,12aS)-7-Chloro-4-(dimethylamino)-3,10,12,12a-tetrahydroxy-8-(1-
methylpyrrolidin-2-yl)-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide
13). In a polypropylene vial, crude 12 was dissolved in CH CN (1.25 mL). Aqueous HF (48 %,
8
(
(
3
0
.25 mL) was added. After stirred at rt for 16 h, the reaction mixture was poured into aqueous
K HPO (1.75 g in 12.5 mL water). The mixture was extracted three times with CH Cl . The
2
4
2
2
combined organic phases were washed with brine, dried over sodium sulfate, and concentrated to
dryness.
The above residue was dissolved in EtOAc (3 mL) and added with PdꢀC (10 wt%, 7.5 mg).
The reaction flask was briefly evacuated and reꢀfilled with hydrogen. The reaction mixture was
stirred under 1 atm hydrogen until the reaction was complete as monitored by LCꢀMS analysis.
2
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The hydrogen source was removed. Methanol (5 mL) and HCl/methanol (0.5 N, 0.5 mL) were
added. The mixture was stirred at rt for 30 min and filtered through a small pad of Celite. The
filtrate was concentrated to give the crude product, which was purified by preparative reverse
0
1
2
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8
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0
phase HPLC. Fractions with the desired MW were collected and freezeꢀdried to give compound
1
13 as a yellow solid (14.7 mg, HCl salt, 63.9% over 3 steps). H NMR (400 MHz, CD OD)
δ
3
7
.28 (s, 1H), 5.01ꢀ5.07 (m, 1H), 4.14 (s, 1H), 3.97ꢀ4.04 (m, 1H), 3.38ꢀ3.46 (m, 2H), 2.91ꢀ3.13
m, 11H), 2.62ꢀ2.68 (m, 1H), 2.10ꢀ2.47 (m, 5H), 1.61ꢀ1.71 (m, 1H). MS (ESI) m/z 532.4 (M +
H).
Phenyl
methylbenzoate
(
6-(Benzyloxy)-4-(5-{[(tert-butoxy)carbonyl]amino}pentanoyl)-3-chloro-2-
(15) and tert-butyl 6-[5-(benzyloxy)-2-chloro-3-methyl-4-
phenoxycarbonyl)phenyl]-1,2,3,4-tetrahydropyridine-1-carboxylate (16). To a solution of
(
14 (1.01 g, 2.33 mmol, 1 equiv) in THF (6 mL) was added nꢀBuLi (0.98 mL, 2.5 M/hexane, 2.45
o
mmol, 1.05 equiv) dropwise under N at ꢀ100 C. The resulting orange solution was stirred at ꢀ
2
o
1
00 C for 20 min. A solution of 1ꢀ(tertꢀbutoxycarbonyl)ꢀ2ꢀpiperidone (488 mg, 2.45 mmol, 1.05
equiv) in THF (6 mL) was added. The reaction mixture was stirred for another 2 h, maintaining
o
the temperature below ꢀ78 C, and quenched by the addition of aqueous HCl (1 N). The reaction
mixture was warmed to rt and extracted three times with EtOAc. The combined organic layers
were washed with brine, dried over Na SO , concentrated to dryness. Purification of the residue
2
4
1
by Biotage flash chromatography gave 15 as a white solid (412 mg, 32%). H NMR (400 MHz,
CDCl3) 7.33ꢀ7.43 (m, 7H), 7.24 (t, J = 7.8 Hz, 1H), 7.06 (d, J = 7.8 Hz, 2H), 6.83 (s, 1H), 5.14
(s, 2H), 4.57 (br s, 1H), 3.11ꢀ3.16 (m, 2H), 2.90 (t, J = 7.3 Hz, 2H), 2.47 (s, 3H), 1.68ꢀ1.75 (m,
δ
2H), 1.50ꢀ1.57 (m, 2H), 1.43 (s, 9H). MS (ESI) m/z 574.5 (M + Na). And 16 as white foam (637
1
mg, 51%). H NMR (400 MHz, CDCl )
δ 7.33ꢀ7.44 (m, 7H), 7.24 (t, J = 7.8 Hz, 1H), 7.09 (d, J
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7.8 Hz, 2H), 6.88 (s, 1H), 5.11ꢀ5.13 (m, 3H), 3.75 (br s, 2H), 2.46 (s, 3H), 2.26ꢀ2.31 (m, 2H),
1.90ꢀ1.97 (m, 2H), 1.10 (s, 9H). MS (ESI) m/z 556.5 (M + Na).
Phenyl 6-(Benzyloxy)-3-chloro-2-methyl-4-(3,4,5,6-tetrahydropyridin-2-yl)benzoate (19).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
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60
Compound 15 (360 mg, 0.65 mmol) was dissolved in CH Cl (3 mL), cooled with an ice bath,
2
2
and treated with a solution of TFA (3 mL) in CH Cl (3 mL). The reaction mixture was stirred at
2
2
rt for 1 h and concentrated. The residue was reꢀdissolved in CH Cl , washed with saturated
2
2
aqueous NaHCO and brine, dried over Na SO , and concentrated. Purification of the residue by
3
2
4
Biotage flash chromatography gave compound 19 as a white solid (246 mg, 87%). Compound 19
was also prepared from 16 (310 mg, 0.58 mmol) according to the same procedure (191 mg,
1
7
6%). H NMR (400 MHz, CDCl )
δ
7.31ꢀ7.44 (m, 7H), 7.22 (t, J = 7.8 Hz, 1H), 7.04 (d, J = 7.8
3
Hz, 2H), 6.82 (s, 1H), 5.13 (s, 2H), 3.72ꢀ3.84 (m, 3H), 3.59ꢀ3.66 (m, 1H), 2.48 (s, 3H), 1.80ꢀ1.88
(m, 2H), 1.71ꢀ1.76 (m, 2H). MS (ESI) m/z 434.4 (M + H).
Phenyl 6-(Benzyloxy)-3-chloro-2-methyl-4-(piperidin-2-yl)benzoate (21). To the solution
of compound 19 (437 mg, 1.01 mmol, 1 equiv) in methanol (2 mL) was added NaBH (43 mg,
4
o
1
.1 mmol, 1.1 equiv) at 0 C. The reaction mixture was stirred at rt for 30 min. Aqueous HCl (1
N, 2 mL) was added. Stirring was continued for another 5 min. Solvents were removed with
RotoVap. The residue was suspended in EtOAc, washed with saturated aqueous NaHCO and
3
brine, dried over Na SO , and concentrated. Purification of the residue by Biotage flash
2
4
1
chromatography gave 21 as a white solid (377 mg, 85.6%). H NMR (400 MHz, CDCl )
δ
7.43
d, J = 7.8 Hz, 2H), 7.32ꢀ7.80 (m, 5H), 7.29 (s, 1H), 7.23 (t, J = 7.8 Hz, 1H), 7.05 (d, J = 7.8 Hz,
2H), 5.15 (s, 2H), 4.09 (dd, J = 2.3, 11.0 Hz, 1H), 3.20ꢀ3.23 (m, 1H), 2.83ꢀ2.90 (m, 1H), 2.46 (s,
H), 1.86ꢀ1.92 (m, 2H), 1.47ꢀ1.70 (m, 4H), 1.24ꢀ1.34 (m, 1H). MS (ESI) m/z 436.4 (M + H).
3
(
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N-Boc-
ε
-caprolactam. To a solution of εꢀcaprolactam (1.01 g, 8.88 mmol, 1 equiv) in THF
o
(10 mL) at ꢀ78 C was added nꢀBuLi (2.5 M/hexanes, 3.73 mL, 9.32 mmol, 1.05 equiv)
o
dropwise. The reaction was stirred at ꢀ78 C for 1 h. A solution of Boc O (2.03 g, 9.32 mmol,
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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8
9
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8
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
o
1
.05 equiv) in THF (8 mL) was added dropwise over 5 min at ꢀ78 C. The reaction was stirred at
o
ꢀ78 C for 2 h and warmed to rt. Saturated aqueous NH Cl (10 mL) and water (5 mL) were
4
added. The mixture was extracted with EtOAc (3 times). The combined organic extracts were
dried over sodium sulfate and concentrated under reduced pressure. Flash chromatography on
silica gel using 0%ꢀ20% EtOAcꢀhexanes yielded the desired product as a colorless oil (1.50 g,
1
7
9%). H NMR (400 MHz, CDCl )
δ
3,72ꢀ3.76 (m, 2H), 2.60ꢀ2.68 (m, 2H), 1.68ꢀ1.80 (m, 6H),
3
1.24 (s, 9H). MS (ESI) m/z 236.6 (M + Na).
Phenyl 4-(Azepan-2-yl)-6-(benzyloxy)-3-chloro-2-methylbenzoate (22). Following similar
procedures used in the preparation of 21, compound 14 (1.02 g, 2.35 mmol, 1 equiv) was reacted
with nꢀBuLi and NꢀBocꢀεꢀcaprolactam (0.53 g, 2.47 mmol, 1.05 equiv) followed by Boc
deprotection with TFA to yield the acyclic aminoketone. This aminoketone intermediate was
dissolved in THF (10 mL). Ti(OꢀiPr) (2.06 mL, 7.05 mmol, 3 equiv) was added. The reaction
4
mixture was stirred at rt overnight, diluted with EtOAc, and poured into water. The mixture was
filtered through a Celite pad. The organic layer was collected, washed with brine, dried over
sodium sulfate, and concentrated under reduced pressure. Flash chromatography on silica gel
using 0%ꢀ30% EtOAcꢀhexanes yielded the cyclic imine 20 as an offꢀwhite solid (291 mg, 28%
over 3 steps). MS (ESI) m/z 448.0 (M + H). The imine (20) was treated with NaBH to give the
4
1
desired product 22 as a waxy solid (292 mg, 100%). H NMR (400 MHz, CDCl )
δ 7.41ꢀ7.46
3
(
m, 2H), 7.32ꢀ7.38 (m, 5H), 7.22 (t, J = 7.3 Hz, 1H), 7.21 (s, 1H), 7.06 (d, J = 7.8 Hz, 2H), 5.16
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(
ABq, J = 11.4, 15.1 Hz, 2H), 4.27 (dd, J = 3.6, 9.2 Hz, 1H), 3.08ꢀ3.16 (m, 1H), 2.88ꢀ2.96 (m,
1
H), 2.46 (s, 3H), 1.95ꢀ2.05 (m, 1H), 1.50ꢀ1.88 (m, 8H). MS (ESI) m/z 450.0 (M + H).
(4S,4aS,5aR,12aS)-7-Chloro-4-(dimethylamino)-3,10,12,12a-tetrahydroxy-8-(1-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
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39
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60
methylpiperidin-2-yl)-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide
(25). Compound 25 was prepared from 21 via MichaelꢀDieckmann annulation, reductive
alkylation, deꢀsilylation, and hydrogenation according to similar procedures used in the
1
preparation of 13 (HCl salt). H NMR (400 MHz, CD OD)
δ
7.30 (s, 1H), 4.14 (s, 1H), 3.66ꢀ3.69
3
(
m, 1H), 2.93ꢀ3.44 (m, 11H), 2.69 (s, 3H), 2.36ꢀ2.46 (m, 1H), 2.26ꢀ2.29 (m, 1H), 1.93ꢀ2.11 (m,
H), 1.61ꢀ1.81 (m, 2H). MS (ESI) m/z 546.4 (M + H).
(4S,4aS,5aR,12aS)-7-Chloro-4-(dimethylamino)-3,10,12,12a-tetrahydroxy-8-(1-
methylazepan-2-yl)-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-Carboxamide
26). Compound 26 was prepared from 22 via MichaelꢀDieckmann annulation, reductive
5
(
alkylation, deꢀsilylation, and hydrogenation according to similar procedures used in the
1
preparation of 13 (HCl salt, diastereomer A). H NMR (400 MHz, CD OD)
δ 7.23 (s, 1H), 4.12
3
(s, 1H), 3.65ꢀ3.75 (m, 1H), 3.50ꢀ3.60 (m, 1H), 3.38ꢀ3.45 (m, 1H), 2.95ꢀ3.20 (m, 9H), 2.78 (s,
H), 2.22ꢀ2.45 (m, 3H), 1.58ꢀ2.28 (m, 8H). MS (ESI) m/z 560.4 (M + H).
1R,3S,4S,11S)‐8,16‐Bis(benzyloxy)‐18‐bromo‐11‐[(tert‐butyldimethylsilyl)oxy]‐19‐
3
(
3
,11 5,9 15,20
chloro‐4-(dimethylamino)‐12‐hydroxy‐6‐oxa‐7‐azapentacyclo[11.8.0.0 .0 .0
(9),7,12,15(20),16,18‐hexaene‐10,14‐dione (27). nꢀBuLi in hexanes (2.5 M, 0.29 mL, 0.72
mmol, 1.2 equiv) was added dropwise to a solution of iꢀPr NH (0.11 mL, 0.75 mmol, 1.25 equiv)
]henicosa‐
5
2
o
o
in THF (4 mL) at ꢀ78 C under N atmosphere. The reaction mixture was stirred at ꢀ78 C for 20
2
o
o
min, 0 C for 5 min, and reꢀcooled to ꢀ78 C. TMEDA (0.12 mL, 0.78 mmol, 1.3 equiv) was
added, followed by the addition of a solution of 14 (285 mg, 0.66 mmol, 1.1 equiv) in THF (4
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o
mL), maintaining the temperature below ꢀ70 C. The resulting darkꢀred mixture was stirred for 1
o
o
h at ꢀ78 C and was then cooled to ꢀ100 C. A solution of enone 10 (289 mg, 0.60 mmol, 1 equiv)
in THF (4 mL) was added dropwise via a cannula. The reaction mixture was allowed to
0
1
2
3
4
5
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8
9
0
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8
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7
8
9
0
o
gradually warm to ꢀ78 C. LHMDS (1.0 M/THF, 0.60 mL, 0.60 mmol, 1 equiv) was added. The
o
reaction was allowed to warm to ꢀ5 C slowly over 1 h. Saturated aqueous NH Cl was added.
4
The mixture was extracted three times with CH Cl . The combined CH Cl extracts were washed
2
2
2
2
with brine, dried over Na SO , and concentrated. Purification of the residue by Biotage flash
2
4
1
chromatography gave compound 27 as a light yellow foam (359 mg, 73%). H NMR (400 MHz,
CDCl3) 15.9 (s, 1H), 7.22ꢀ7.52 (m, 10H), 6.81 (d, J = 8.6 Hz, 1H), 5.35 (s, 2H), 5.20 (d, J =
11.4 Hz, 1H), 5.15 (d, J = 11.4 Hz, 1H), 3.93 (d, J = 10.4 Hz, 1H), 3.42 (dd, J = 4.9, 15.9 Hz,
δ
1
H), 2.96ꢀ3.04 (m, 1H), 2.45ꢀ2.58 (m, 9H), 2.12 (d, J = 14.6 Hz, 1H), 0.84 (s, 9H), 0.26 (s, 3H),
.14 (s, 3H). MS (ESI) m/z 819.4 (M + H).
0
(1R,3S,4S,11S)‐8,16‐Bis(benzyloxy)‐11‐[(tert‐butyldimethylsilyl)oxy]‐19‐chloro‐4‐
(
dimethylamino)‐12‐hydroxy‐18‐(pyridin‐3‐yl)‐6‐oxa‐7‐
3
,11 5,9 15,20
azapentacyclo[11.8.0.0 .0 .0
]henicosa‐5(9),7,12,15(20),16,18‐hexaene‐10,14‐dione
(
28). To a pressure vial was charged with compound 27 (34 mg, 0.041 mmol), 3ꢀpyridine
boronic acid (26 mg, 0.21 mmol, 5 equiv), dichloro[1,1’ꢀbis(diphenylphosphino)ferrrocene]
palladium(II) dichloromethane adduct (1.7 mg, 0.0021 mmol, 0.05 equiv), and sodium carbonate
(
22 mg, 0.21 mmol, 5 equiv). The vial was briefly evacuated and filled with N . Toluene (1 mL),
2
1
,4ꢀdioxane (1 mL), and H O (0.2 mL) were added. The reaction mixture was heated with an 80
2
o
C oil bath for 1 h, cooled to rt, diluted with EtOAc, washed with aqueous phosphate buffer (pH
=
7) and brine, dried over Na SO , and concentrated. Purification of the residue by Biotage flash
2
4
1
chromatography gave compound 28 as a yellow solid (20 mg, 60%). H NMR (400 MHz,
2
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CDCl3)
δ
16.0 (s, 1H), 8.63 (dd, J = 1.8, 4.9 Hz, 1H), 8.56 (d, J = 1.8 Hz, 1H), 7.69 (ddd, J = 1.8,
1.8, 7.9 Hz, 1H), 7.26ꢀ7.52 (m, 11H), 6.88 (s, 1H), 5.36 (s, 2H), 5.24 (d, J = 11.4 Hz, 1H), 5.18
d, J = 11.4 Hz, 1H), 3.97 (d, J = 10.4 Hz, 1H), 3.48 (dd, J = 4.9, 15.9 Hz, 1H), 3.01ꢀ3.10 (m,
H), 2.45ꢀ2.60 (m, 9H), 2.15 (d, J = 14.6 Hz, 1H), 0.84 (s, 9H), 0.29 (s, 3H), 0.15 (s, 3H). MS
(ESI) m/z 818.4 (M + H).
1R,3S,4S,11S)‐8,16‐Bis(benzyloxy)‐11‐[(tert‐butyldimethylsilyl)oxy]‐19‐chloro‐4‐
(dimethylamino)‐12‐hydroxy‐18‐(pyridin‐4‐yl)‐6‐oxa‐7‐
(
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
(
3
,11 5,9 15,20
azapentacyclo[11.8.0.0 .0 .0
]henicosa‐5(9),7,12,15(20),16,18‐hexaene‐10,14‐dione
1
(29). Compound 29 was prepared according to similar procedures used for 28 (yellow solid). H
NMR (400 MHz, CDCl3)
δ 16.0 (s, 1H), 8.68 (d, J = 6.1 Hz, 2H), 7.26ꢀ7.52 (m, 12H), 6.85 (s,
1
H), 5.36 (s, 2H), 5.23 (d, J = 11.4 Hz, 1H), 5.18 (d, J = 11.4 Hz, 1H), 3.97 (d, J = 10.4 Hz, 1H),
3.48 (dd, J = 4.9, 15.9 Hz, 1H), 3.01ꢀ3.10 (m, 1H), 2.45ꢀ2.60 (m, 9H), 2.16 (d, J = 14.6 Hz, 1H),
.84 (s, 9H), 0.28 (s, 3H), 0.16 (s, 3H). MS (ESI) m/z 818.5 (M + H).
4S,4aS,5aR,12aS)-7-Chloro-4-(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-8-
(pyridine-3-yl)-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide (30). In
0
(
a
polypropylene vial, compound 28 (20 mg, 0.024 mmol) was dissolved in CH CN (1.25 mL).
3
Aqueous HF (48%, 0.25 mL) was added. After stirred at rt for 16 h, the reaction mixture was
poured into aqueous K HPO (1.75 g in 12.5 mL water). The mixture was extracted three times
2
4
with CH Cl . The combined organic phases were washed with brine, dried over sodium sulfate,
2
2
and concentrated under reduced pressure.
The above residue was dissolved in EtOAc (3 mL). PdꢀC (10 wt%, 36 mg) was added in four
portions. The reaction flask was briefly evacuated and reꢀfilled with hydrogen. The reaction
mixture was stirred under 1 atm hydrogen at rt until the reaction was complete as monitored by
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LCꢀMS analysis. The hydrogen source was removed. Methanol (5 mL) and HCl/methanol (0.5
N, 0.5 mL) were added. The mixture was stirred for 30 min and filtered through a small pad of
Celite. The filtrate was concentrated to give the crude product, which was purified by preparative
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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8
9
0
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2
3
4
5
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7
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9
0
1
2
3
4
5
6
7
8
9
0
reverse phase HPLC to yield compound 30 as a yellow solid (1.5 mg, HCl salt, 10% over two
1
steps). H NMR (400 MHz, CD OD)
δ
9.08 (s, 1H), 8.96 (d, J = 5.6 Hz, 1H), 8.78 (d, J = 5.6 Hz,
3
1
H), 8.22 (t, J = 5.6 Hz, 1H), 7.11 (s, 1H), 4.12 (s, 1H), 3.45 (m, 1H), 2.92ꢀ3.15 (m, 8H), 2.47
dd, J = 15.0, 15.0 Hz, 1H), 2.25ꢀ2.28 (m, 1H), 1.60ꢀ1.70 (m, 1H). MS (ESI) m/z 526.3 (M + H).
4S,4aS,5aR,12aS)-7-Chloro-4-(dimethylamino)-3,10,12,12a-tetrahydroxy-1,11-dioxo-8-
(
(
(pyridine-4-yl)-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide (31). Compound 31
1
was prepared from 29 according to similar procedures used for 30. H NMR (400 MHz, CD OD)
3
δ
8.97 (d, J = 6.4 Hz, 2H), 8.22 (d, J = 6.4 Hz, 2H), 7.09 (s, 1H), 4.12 (s, 1H), 3.45 (dd, J = 4.6,
6.0 Hz, 1H), 2.97ꢀ3.21 (m, 8H), 2.47 (dd, J = 15.0, 15.0 Hz, 1H), 2.24ꢀ2.30 (m, 1H), 1.63ꢀ1.73
1
(m, 1H). MS (ESI) m/z 526.3 (M + H).
Phenyl 2-(Benzyloxy)-4-(3,4-dihydro-2H-pyrrol-5-yl)-6-methylbenzoate (32). To a
solution of 6 (3.01 g, 7.58 mmol) in THF (30 mL) was added nBuLi (2.5 M, 3.18 mL, 7.96
o
mmol, 1.05 equiv) dropwise under N at ꢀ100 C. The resulting orange solution was stirred at this
2
temperature for 30 min, then solution of 1ꢀ(tertꢀbutoxycarbonyl)ꢀ2ꢀpyrrolidinone (1.52 g, 7.96
mmol, 1.05 equiv) in THF (8 mL) was added. The reaction mixture was stirred for another 3 h,
o
maintaining the temperature below ꢀ78 C, and quenched by the addition of aqueous HCl (1 N).
The reaction mixture was warmed to rt and extracted three times with EtOAc. The combined
organic layers were washed with brine, dried over Na SO , concentrated to dryness. The residue
2
4
was dissolved in CH Cl (20 mL), cooled with an ice bath and treated with a solution of TFA (20
2
2
mL) in CH Cl (20 mL). The reaction mixture was stirred at rt for 1 h and concentrated. The
2
2
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residue was dissolved in CH Cl , washed with saturated aqueous NaHCO , and brine, dried over
2
2
3
Na SO , and concentrated. Purification of the residue by biotage flash chromatography gave
2
4
1
2
1
compound 32a (R = R = H) as a white solid (2.02 g, 69% over two steps). H NMR (400 MHz,
CDCl3) 7.48 (s, 1H), 7.42 – 7.45 (m, 2H), 7.31 – 7.37 (m, 5H), 7.24 (s, 1H), 7.22 (t, J = 7.8 Hz,
1H), 7.07 (d, J = 7.8 Hz, 2H), 5.19 (s, 2H), 4.08 (t, J = 7.3 Hz, 2H), 2.92 (t, J = 7.8 Hz, 2H), 2.47
s, 3H), 2.03 – 2.10 (m, 2H). MS (ESI) m/z 386.1 (M + H).
tert-Butyl 2-[3-(Benzyloxy)-5-methyl-4-(phenoxycarbonyl)phenyl]pyrrolidine-1-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
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55
56
57
58
59
60
δ
(
carboxylate (33). To a solution of compound 32a (2.02 g, 5.24 mmol) in 1,2ꢀdichloroethane (26
mL) were added acetic acid (1.3 mL, 23.6 mmol, 4.5 equiv) and sodium triacetoxyborohydride
(5.0 g, 23.6 mmol, 4.5 equiv). The reaction mixture was stirred at rt and monitored by LCꢀMS.
Upon completion, the reaction mixture was diluted with CH Cl (200 mL) and poured into
2
2
saturated aqueous NaHCO (200 mL). The layers were separated and the aqueous layer was
3
further extracted two times with CH Cl . The combined organic layers were washed with brine,
2
2
dried over Na SO , and concentrated to dryness.
2
4
The above residue was dissolved in anhydrous DMF (21 mL). Diꢀtertꢀbutyl dicarbonate (1.37
g, 6.29 mmol, 1.2 equiv) and DMAP (32 mg, 0.26 mmol, 0.05 equiv) were added. The reaction
mixture was stirred at rt for 2 h, and then diluted with EtOAc. The solution was washed
sequentially with 1 N HCl, H O three times, brine, dried over Na SO , and concentrated.
2
2
4
1
2
Purification of the residue by Biotage flash chromatography gave compound 33a (R = R = H)
1
as white foam (2.27 g, 88.9% over two steps). H NMR (400 MHz, CDCl , rotamer)
δ 7.41 –
3
7.43 (m, 2H), 7.29 – 7.37 (m, 5H), 7.20 – 7.23 (m, 1H), 7.08 – 7.10 (m, 2H), 6.63 – 6.65 (m,
2H), 5.10 (s, 2H), 4.70 – 4.90 (m, 1H), 3.50 – 3.65 (m, 2H), 2.41 (s, 3H), 2.20 – 2.32 (m, 1H),
1.60 – 1.80 (m, 3H), 1.45 (br s, 3H), 1.23 (br s, 6H). MS (ESI) m/z 510.3 (M + Na).
2
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