Previously we discovered a tricyclic indoline, N-[2-(6-bromo-4-methylidene-2,3,4,4a,9,9a-hexahydro-1H-carbazol-4a-yl)ethyl]-4-chlorobenzene-1-sulfonamide (1, Of1), from bioinspired synthesis of a highly diverse polycyclic indoline alkaloid library, that selectively resensitizes methicillin-resistant Staphylococcus aureus strains to β-lactam antibiotics. Herein, we report a thorough structure-activity relationship investigation of 1, which identified regions of 1 that tolerate modifications without compromising activity and afforded the discovery of a more potent analogue with reduced mammalian toxicity.
Previously we discovered a tricyclic indoline, N-[2-(6-bromo-4-methylidene-2,3,4,4a,9,9a-hexahydro-1H-carbazol-4a-yl)ethyl]-4-chlorobenzene-1-sulfonamide (1, Of1), from bioinspired synthesis of a highly diverse polycyclic indolinealkaloid library, that selectively resensitizes methicillin-resistant Staphylococcus aureus strains to β-lactam antibiotics. Herein, we report a thorough structure-activity relationship investigation of 1, which identified regions of 1 that tolerate modifications without compromising activity and afforded the discovery of a more potent analogue with reduced mammaliantoxicity.
Antibiotic resistance is an urgent world
health concern that is
aggravated by a lack of novel antibiotic discovery.[1] The vast majority of antibiotic classes on the market today
were discovered between 1940 and 1960, a period of time known as “the
golden era” of antibiotic discovery.[2] Since this time, however, only two new classes of antibiotics have
been brought to the clinic. Microbial antibiotic resistance has prompted
the pharmaceutical industry to develop analogues of known antibiotics;
however, bacteria have been observed to develop resistant phenotypes
quickly, and there are not currently enough analogues in the antibiotic
pipeline to combat imminent resistance emergence.[3] Methicillin-resistant Staphylococcus aureus (MRSA) is particularly concerning in this regard. Prior to the introduction
of penicillin in the 1940s, the mortality of patients who developed
invasive staphylococcusinfections was nearly 80%, but this statistic
was drastically reduced by the introduction of penicillin in the clinic.[4] In just a few years, however, resistance to penicillin,
mediated by β-lactamases, emerged, which led to the introduction
of β-lactam antibiotics such as methicillin, which are resistant
to degradation by β-lactamase. It was not long, however before S. aureus developed methicillin resistance in the
form of alternative penicillin binding proteins (PBPs), such as PBP2a.[5] In addition to methicillin resistance, a number
of MRSA strains have developed resistant phenotypes against multiple
drugs used in the clinic, thus limiting treatment options for bacterial
infections and threatening the onset of a postantimicrobial era.[6] Of the estimated 2 million illnesses and 23 000
deaths last year that are directly associated with antibiotic resistant
bacteria in the United States, MRSA was directly responsible for 80 000
illnesses and 11 000 deaths.[7] Although
vancomycin as well as some antibiotic analogues are still effective
for the treatment of MRSA, strains that are resistant to these last-line-of-defense
treatments have already become a problem of their own.[8]Resistance-modifying agents (RMAs) offer a promising
solution.[9] These target nonessential, resistance-conferring
genes and restore antibiotic sensitivity. A notable advantage of RMAs
is that they are capable of extending the market lifespan of known
antibiotics that have already been optimized for large-scale production
with well-studied toxicity profiles. For example, clavulanic acid
is a serine β-lactamase inhibitor that is commonly used in combination
with amoxicillin, under the brand name Augmentin, among others, to
treat infections resulting from β-lactamase-producing bacteria.[10] Clavulanic acid deactivates β-lactamase
by irreversibly acylating the catalytic serine residue in the active
site of the β-lactamase enzyme that would otherwise function
to breakdown the β-lactam antibiotic. Although the clavulanic
acid/β-lactam combination is effective for serine β-lactamases,
it is ineffective against metallo-β-lactamases and does nothing
to combat β-lactam resistance mediated by PBP2a.[11]Although efforts have been underway to discover
more novel RMAs, thus far, only those that are β-lactamase inhibitors
have been successfully brought to market. β-Lactam antibiotics
are one of the most widely used antibiotics and have inspired numerous
research efforts to overcome bacterial resistance. In addition to
β-lactamase inhibitors, other classes of compounds have been
reported to potentiate the activity of β-lactam antibiotics,
such as compounds interfering bacterial cell wall synthesis and compounds
affecting resistance-sensing pathways.[12−15]Recently, our group discovered
a tricyclic indoline, 1, with RMA activity against MRSA
that selectively potentiates a variety
of β-lactam antibiotics.[16] Interestingly,
in addition to β-lactam antibiotics that are susceptible to
breakdown by β-lactamase, 1 also potentiates those
that are β-lactamase resistant, such as methicillin, oxacillin,
and meropenem. Intrigued by the unique activity of 1,
we sought to conduct a structure–activity relationship (SAR)
study in order to identify regions of the structure of 1 that may be modified to improve its activity and toxicity profile,
as well as to allow synthesis of functional analogues to facilitate
the discovery of the cellular target of 1. Herein, we
report a thorough SAR study of our previously reported tricyclic indolineRMA, 1.
Results and Discussion
Modification of 1
In order to conduct
our SAR investigation of 1 (Figure 1), we synthesized several series of 1 analogues, which
were then evaluated for their ability to resensitize MRSA to a collection
of β-lactam antibiotics. For these activity tests, we chose
to use amoxicillin in combination with clavulanic acid (a.k.a., Augmentin,
one of the top three most prescribed antibiotics), cefazolin (a first-generation
cephalosporin), and meropenem (an ultrabroad-spectrum carbapenem).
Amoxicillin/clavulanic acid and cefazolin resensitizing experiments
were performed using MRSA ATCC BAA-44, for which the MICs of these
two antibiotics were found to be 32/16 and 128 μg/mL, respectively.
Experiments using meropenem were performed using MRSA ATCC 33592,
since this strain has demonstrated greater level of resistance to
meropenem, with an MIC of 16 μg/mL. To assess activity of each
analogue as RMA, we employed a modified broth microdilution assay,
as described previously.[16] Briefly, this
involves incubating MRSA with 1 or each of its analogues
in 2-fold serial dilution in the presence of each individual antibiotic
at its Clinical Laboratory Standards Institutes (CLSI)-defined sensitive
concentrations. For amoxicillin/clavulanic acid, this concentration
was 4/2 μg/mL (8-fold potentiation); for cefazolin, 8 μg/mL
(16-fold potentiation); and for meropenem, 4 μg/mL (4-fold potentiation).[17] Following overnight incubation, plates were
examined for bacterial growth, or lack thereof. Analogues of 1were tested at concentrations ranging from 0.5 to 32 μg/mL.
The minimum resensitizing concentration (MRC) was defined as the concentration
of 1’s analogues at which no overnight growth
was observed in the presence of a sensitive concentration of antibiotic.
Compounds that displayed similar or improved RMA activity relative
to 1 were further tested for their toxicity against the
growth of human cervical adenocarcinoma HeLa cells by incubating a
range of concentrations of each compound with cells for 24 h and assessing
viability at each concentration using the CellTiter Glo mammalian
viability assay (Promega). The half growth inhibitory concentration
(GI50) of each analogue was determined by fitting the data
using KaleidaGraph (v4.1.1, Synergy Software).
Figure 1
The structure of 1.
The structure of 1.We began our SAR investigation
by testing a number of functional
group modifications to the indoline and sulfonamidenitrogens of 1. Due to the different pKa, different
bases were used in the functionalization steps. As shown in Scheme 1, for compound 1a, selective methylation
was carried out in the presence of potassium carbonate in anhydrous
CH3CN; compounds 1b and 1c were
prepared with corresponding electrophilic reagents under similar condition.
While compound 1d was generated by using sodium hydride
as a base, triethylamine was employed to activate the sulfonamidenitrogen selectively for the preparation of analogues 1e and 1f. We observed that when either or both of these
moieties are modified, the RMA activity of the compound is abolished
(Table1). We thus concluded that both these
nitrogens must not be modified.
Scheme 1
Functionalization of the Indoline
and Sulfonamide Nitrogens
Reagents and conditions:
(a)
R1X, K2CO3, 0 °C, CH3CN; (b) for 1d, NaH 0 °C, then MeI, 0–60
°C, DMF; for 1e,f, R2X,
Et3N, 0 °C, DCM.
Table 1
MRC Values for Indoline and Sulfonamide
Nitrogen Modifications
entry
compd
R1
R2
amox/clava,b
cefazolina,b
meropenema,c
1
1a
Me
H
>32
>32
>32
2
1b
SO2PhpCl
H
>32
>32
>32
3
1c
COPhpCl
H
>32
>32
>32
4
1d
H
Me
>32
>32
>32
5
1e
H
SO2PhpCl
>32
>32
>32
6
1f
H
COPhpCl
>32
>32
>32
All MRC values are in μg/mL.
MRSA ATCC BAA-44.
MRSA ATCC 33592.
Functionalization of the Indoline
and Sulfonamide Nitrogens
Reagents and conditions:
(a)
R1X, K2CO3, 0 °C, CH3CN; (b) for 1d, NaH 0 °C, then MeI, 0–60
°C, DMF; for 1e,f, R2X,
Et3N, 0 °C, DCM.All MRC values are in μg/mL.MRSA ATCC BAA-44.MRSA ATCC 33592.
Modifications of the Aromatic Substitution
of Indoline
We next sought to modify the aromatic portion
of the indoline ring.
To prepare these analogues, we adapted the three-step synthetic route
that we previously used for synthesis of the original library. As
depicted in Scheme 2, cyclic imine 2 was reacted with different aryl hydrazines 3 and 4-chlorobenzenesulfonyl
chloride to afford the alkynyl indole product 4 using
a modified Fischer indole synthesis protocol.[18] Cyclization under our standard gold catalysis conditions (5 mmol
% Ph3PAuNTf2)[19] followed
by a reductive ring-opening reaction afforded a series of 1 analogues (6a–l). In order to test
the necessity of the bromine (R2) at the 5-position of
the indoline, we synthesized analogues replacing it with a methyl
group (6b), a methoxy group (6c), or a hydrogen
(6e). In the case of each of these modifications, RMA
activity was abolished or greatly diminished for all three antibiotics
tested. Moving the bromine to other positions on the indoline (e.g., 6i, 6h, and 6g) also significantly
reduced its RMA activity. We thus concluded that the presence of a
halogen at the R2 position is necessary for RMA activity.
We further evaluated whether the identity of the halogen at the R2 position affects its activity. We found that RMA activity
is optimal when bromine is maintained as the R2 halogen.
However, although replacing the R2 bromine with chlorine
(6a) reduced RMA activity somewhat, this analogue also
showed significantly reduced toxicity against mammalian cells (Table 2). In addition, we also synthesized several 1 analogues with an additional halogen at the 7-position of
indoline. We found that when the R2 halogen is chlorine,
fluorine at R4 significantly reduces RMA activity (6j); however, when the identity of both R2 and
R4 are chlorine (6k), the RMA activity remains
similar to that of 1. Maintaining bromine at the R2 position and adding fluorine at R4 (6l), however, yield slightly improved RMA activity and reduced mammaliantoxicity with respect to 1. On the basis of this series
of modifications on the aromatic indoline ring, we concluded that
in order to maintain RMA activity, the identity of the R2 position must be a halogen (bromine or chorine) and that the R2 position may be functionalized with a second halogen to improve
or maintain activity.
Scheme 2
Synthesis of 6a–l
Reagents and conditions: (a) ClPhSO2Cl, DMAP, 23 °C, 0.5
h, DMF; 2, 0.5 h; MsOH, then 3, 60–120
°C, 20 h; (b) Ph3PAuNTf2, 50 °C, toluene,
1–12 h; (c) AcOH, NaBH3CN, MeOH, 0 °C, 0.5
h. [Ph3PAuNTf2 = [bis(trifluoromethanesulfonyl)imidate](triphenylphosphine)gold(I).]
Table 2
MRC and GI50 Values for
the Substitutions of Indoline Aromatic Ring
entry
compd
R1
R2
R3
R4
amox/clava,b
cefazolina,b
meropenema,c
GI50d
1
1
H
Br
H
H
4
4
4
17.1
2
6a
H
Cl
H
H
8
4
8
35
3
6b
H
Me
H
H
16
16
32
–
4
6c
H
MeO
H
H
>32
>32
>32
–
5
6d
H
F
H
H
16
16
16
–
6
6e
H
H
H
H
16
16
16
–
7
6f
H
H
phenylene
>32
>32
>32
–
8
6g
H
H
H
Br
>32
32
8
–
9
6h
H
H
Br
H
16
16
16
–
10
6i
Br
H
H
H
32
16
16
–
11
6j
H
Cl
H
F
16
16
32
–
12
6k
H
Cl
H
Cl
4
4
4
13.6
13
6l
H
Br
H
F
2
4
4
18.1
MRC values are in μg/mL.
MRSA ATCC BAA-44.
MRSA
ATCC 33592.
HeLa cells;
GI50 values
are in μg/mL.
Synthesis of 6a–l
Reagents and conditions: (a) ClPhSO2Cl, DMAP, 23 °C, 0.5
h, DMF; 2, 0.5 h; MsOH, then 3, 60–120
°C, 20 h; (b) Ph3PAuNTf2, 50 °C, toluene,
1–12 h; (c) AcOH, NaBH3CN, MeOH, 0 °C, 0.5
h. [Ph3PAuNTf2 = [bis(trifluoromethanesulfonyl)imidate](triphenylphosphine)gold(I).]MRC values are in μg/mL.MRSA ATCC BAA-44.MRSA
ATCC 33592.HeLa cells;
GI50 values
are in μg/mL.
Modifications
of the Side Chain
We next attempted to
conduct SAR investigations on the side chain of 1. For
this portion of our SAR investigation, we maintained the bromine group
at the 5-position of the indoline moiety. We included several analogues
in this portion of our study with fluorine at the 7-position of indoline,
since 6l showed similar activity but lower mammaliancytotoxicity with respect to 1 (Table 2). The general synthetic route (Scheme 3) was similar to Scheme 2. For this series
of 1 analogues, benzyl chloroformate was used as the
active reagent in the indole synthesis step, which was removed successfully
by treating ring-opening products 10a,b with
boron trifluoride–dimethyl sulfide.[20] The resulting freeamine products 11a,b were further functionalized to afford compounds 12a–n and 13a–m. In addition, analogue 12o was prepared by the reduction
of 12n with zinc dust in the presence of ammonium chloride.[21] We first discovered that removing the side chain
(R2) entirely resulted in severely reduced activity in
the presence or absence of the R1 fluorine (11a,b). Likewise, replacing the sulfonamide with an amide
(e.g., 12a–c) resulted in abolished
RMA activity. We discovered the necessity of the phenyl ring by replacing
it with a pyridine ring (12m), which resulted in a loss
of RMA activity. We next attempted to modify the side chain phenyl
ring. When the chlorine at the para position relative to the sulfonamide
was removed from 1, the RMA activity is reduced at least
2-fold (12d). We thus attempted to modify the phenyl
ring on the side chain with a series of functional groups. For these
experiments, we synthesized analogues in which the chlorine was maintained
and an additional chlorine was added to the meta or ortho position,
respectively (12i,j). These disubstituted
products did not result in improved RMA activity; however, in the
presence of the fluorine at the 7-position of indoline (R1), reduced toxicity was observed for the disubstituted analogue 13f. The remainder of our modifications focused on the para
position of the phenyl ring. Replacing the chlorine with a fluorine
(i.e., 12f) abolished RMA activity entirely; however,
this same modification in combination with the R1 as a
fluorine (i.e., 13c) resulted in similar RMA activity
relative to 1. A similar trend was also observed when
the chlorine was replaced with an iodine in the absence (12h) or presence (13e) of the R1 fluorine. Interestingly,
when the chlorine is replaced with a methyl group (12e), RMA activity is lost; however, it is regained when fluorine is
added at the R1 position (13b). In addition,
the mammaliantoxicity of this analogue is reduced in this case. This
trend was also observed when the chlorine was replaced with a cyanide
(12k, 13h). Although substituting a methoxy group for
the chlorine in the presence of the R1 fluorine (13a) results in slightly reduced activity, the mammaliantoxicity
is significantly reduced. Likewise, replacing the chlorine with a
bromine (12g) resulted in slightly reduced RMA activity,
and this analogue demonstrated a significant decrease in mammaliantoxicity (Table 3). Intriguingly, when we added
the R1 fluorine (13d) to this molecule, we
observed a significant (at least 4-fold) increase in RMA activity
as well as decreased toxicity relative to 1.
Scheme 3
Synthesis
of 12a–n and 13a–i
Reagents and conditions: (a)
CbzCl, DMAP, 23 °C, 0.5 h, DMF; 2, 2–12 h;
MsOH, then 3, 60–120 °C; (b) Ph3PAuNTf2, 50 °C, toluene, 1–12 h; (c) AcOH,
NaBH3CN, MeOH, 0 °C, 0.5 h; (d) BF3·Et2O, Me2S, DCM, 23 °C, 1.5 h; (e) R2X, Et3N, DCM, 0 °C, 0.5–2 h.
Table 3
MRC and GI50 Values for 1 Analogues with Modifications on the Side Chain
entry
compd
R1
R2
amox/clava,b
cefazolina,b
meropenema,c
GI50d
1
1
H
SO2PhpCl
4
4
4
17.1
2
11a
H
H
32
32
32
–
3
11b
F
H
>32
32
16
16.2
4
12a
H
TFA
>32
>32
32
–
5
12b
H
COBu
>32
>32
32
–
6
12c
H
COPhpCl
>32
>32
>32
–
7
12d
H
SO2Ph
8
8
16
–
8
12e
H
SO2PhpMe
>32
>32
>32
–
9
12f
H
SO2PhpF
>32
>32
>32
–
10
12g
H
SO2PhpBr
4
4
8
40
11
12h
H
SO2PhpI
4
4
32
33
12
12i
H
SO2Ph3,4Cl
4
2
4
12.8
13
12j
H
SO2Ph2,4Cl
8
>32
4
–
14
12k
H
SO2PhpCN
16
8
16
–
15
12l
H
SO2PhpNHAc
>32
>32
32
–
16
12m
H
SO25Py
32
32
32
–
17
12n
H
SO2PhpNO2
>32
>32
>32
–
18
12o
H
SO2PhpNH2
16
16
16
–
20
13a
F
SO2PhpOMe
8
4
8
49
21
13b
F
SO2PhpMe
4
4
4
22
22
13c
F
SO2PhpF
4
4
4
18.3
23
6l
F
SO2PhpCl
2
4
4
18.1
24
13d
F
SO2PhpBr
1
1
1
22
25
13e
F
SO2PhpI
4
2
4
19.6
26
13f
F
SO2Ph3,4Cl
4
4
4
31
27
13g
F
SO2PhpNHAc
32
32
16
32
28
13h
F
SO2PhpCN
8
4
4
17.0
29
13i
F
SO2PhpCF3
4
4
4
8.7
MRC values are in μg/mL.
MRSA ATCC BAA-44.
MRSA ATCC 33592.
HeLa cells; GI50 values
are in μg/mL.
Synthesis
of 12a–n and 13a–i
Reagents and conditions: (a)
CbzCl, DMAP, 23 °C, 0.5 h, DMF; 2, 2–12 h;
MsOH, then 3, 60–120 °C; (b) Ph3PAuNTf2, 50 °C, toluene, 1–12 h; (c) AcOH,
NaBH3CN, MeOH, 0 °C, 0.5 h; (d) BF3·Et2O, Me2S, DCM, 23 °C, 1.5 h; (e) R2X, Et3N, DCM, 0 °C, 0.5–2 h.MRC values are in μg/mL.MRSA ATCC BAA-44.MRSA ATCC 33592.HeLa cells; GI50 values
are in μg/mL.
Evaluation
of the Synergistic Activity and Hemolytic Activity
of 13d
In order to assess the synergistic nature
of 13d, we first investigated the antibacterial activity
of 13d against both MRSA and methicillin-sensitive S. aureus (MSSA) strains. For this experiment, we
employed a standard microdilution assay using MRSA ATCC BAA-44, MRSA
ATCC 33592, and MSSA ATCC 25923. The assay was performed by following
the procedure outlined by CLSI.[22] The minimum
inhibitory concentrations (MICs) of 13d against all strains
was higher than 64 μg/mL, the highest concentration tested.
Since 13d exhibited no antiproliferative effect on its
own, we decided to confirm its synergistic activity with the three
antibiotics tested in this report by performing the checkerboard (CB)
test for synergy and calculating the fractional inhibitory concentration
index (FICI) for each antibiotic tested in combination with 13d. CB assays and FICIs were set up and calculated as described
previously.[23] Briefly, 13d was diluted across 96-well microplates in MHB (8 to 0.016 μg/mL),
and to each plate either amoxicillin/clavulanic acid (128 to 1 μg/mL),
cefazolin (128 to 1 μg/mL), or meropenem (16 to 0.125 μg/mL)
was diluted down the plate. Thusly, each well of the 96-well plate
contained a unique concentration combination of 13d and
antibiotic. FICI was calculated by the following formula: FICI = FICA
+ FICB, where FICA is the MIC of drug A in combination with B divided
by the MIC of drug A on its own, and FICB is the MIC of drug B in
combination divided by the MIC of drug B on its own. An FICI that
is less than 0.5 indicates a synergistic drug interaction, an FICI
in the range of 0.5–1 denotes an additive drug interaction,
and an FICI greater than 1 indicates an antagonistic interaction.
The FICI values of 13d for amoxicillin/clavulanic acid,
cefazolin, and meropenem were 0.0315, 0.0156, and 0.0315, respectively.
This result confirms the synergistic action of 13d in
combination with all antibiotics tested in this study.We further
evaluated the toxicity of 13d by conducting a standard
hemolytic assay as previously described.[24,25] This assay measures the amount of hemoglobin leakage in compound-treated
human red blood cells (hRBCs) and is used to measure the amount of
membrane damage induced by drug treatment. The hemolytic activity
assay was conducted for multiple concentrations of 13d. It caused less than 2% hemolysis of red blood cells at 64 μg/mL
(64-fold above its MRC), the highest concentration tested. We thus
concluded that compound 13d shows an insignificant level
of toxicity against hRBCs.Collectively, these results indicate
that 13d is a
more potent analogue of 1, with increased RMA activity,
decreased toxicity, and potent synergy with multiple classes of β-lactam
antibiotics. Furthermore, 13d shows no antiproliferative
effect against MRSA or MSSA on its own.
Conclusions
We
have synthesized a number of structural analogues of our tricyclic
indolineRMA, 1, by taking advantage of the previously
developed highly efficient synthetic approach with high functional
group tolerance. These new analogues have been evaluated for their
ability to potentiate three representative β-lactam antibiotics
(amoxicillin/clavulanic acid, cefazolin, and meropenem) in MRSA and
for their toxicity in mammalian cells. We found that neither the aniline
nor the sulfonamidenitrogen can tolerate further modification. While
the sulfonamide group on the side chain is crucial for the RMA activity,
modifications of both aromatic systems can further fine-tune the RMA
activity and the mammaliantoxicity. Notably, we discovered that adding
fluorine to the 7-position of the indoline increases RMA activity
of the compound in multiple instances, including those in which another
modification has reduced or eliminated RMA activity. Furthermore,
we discovered that a number of substitutions may be added to the phenyl
ring on the side chain, which will allow the development of additional 1 analogues for the discovery of its cellular target, a stepping
stone to understanding the β-lactam resistome.[26] In addition, we have discovered a more potent analogue
of 1, compound 13d, with reduced mammaliantoxicity and low hemolytic activity. We were able to confirm the synergistic
activity of 13d by calculating its FICI and showed that
it is highly synergistic in combination with all antibiotics tested.
Further investigation of the mode of action of 1 and
evaluation of its efficacy in vivo are ongoing and will be reported
in due course.
Exprimental Section
Bacterial
Strains
Strains ATCC BAA-44 (MRSA) and ATCC
25923 (MSSA) were gifts from the laboratories of Daniel Feldheim and
Charles McHenry, respectively. Strain ATCC 33592 (MRSA) was purchased
from ATCC (http://www.atcc.org).
MRC screens were performed as described
previously.[16] Briefly, antibiotic MIC values
where S.
aureus is considered susceptible were determined from
the CLSI handbook supplement.[17] Analogues
of 1 were diluted to 5 mg/mL in DMSO. Antibiotic was
prepared at twice the intended final concentration in Mueller–Hinton
broth (MHB). For amoxicillin/clavulanic acid, the initial concentration
was 8/4 μg/mL; for meropenem, 8 μg/mL; and for cefazolin,
16 μg/mL. A 50 μL portion of the antibiotic containing
media was added to each well of 96-well plates, and 100 μL was
added to the top row. A 1.28 μL portion of of 5 mg/mL alkaloid
solution was added to the top row of each plate to afford a concentration
of 64 μg/mL in the top row of each plate, and 2-fold serial
dilutions were performed down the columns. Once the plates were prepared,
a day culture of MRSA was diluted to OD600 0.002, and 50
μL was added to each well. The final concentration of MRSA added
was OD600 0.001, the final concentration of amoxicillin/clavulanic
acid was 4/2 μg/mL, the final concentration of meropenem was
4 μg/mL, the final concentration of cefazolin was 8 μg/mL,
and the highest concentration of 1 analogue tested was
32 μg/mL. Plates were incubated overnight at 37 °C with
shaking. The MRC value was determined as the concentration of 1 analogue in the presence of antibiotic at which there was
no observable overnight growth.
Microdilution Tests for
Minimal Inhibitory Concentration (MIC)
Determination
The minimal inhibitory concentrations (MICs)
of active 1 analogues were determined by the broth microdilution
method detailed in the CLSI handbook.[22] All antimicrobial compounds were purchased from Sigma-Aldrich. The
growth media used for all MIC experiments was MHB purchased from HIMEDIA
through VWR (cat. 95039-356). The inoculum was prepared by diluting
a bacterial day culture (OD600 0.15–0.4) to OD600 0.002. This dilution was further diluted 2-fold when added
to 96-well microplates (USA Scientific CytoOne 96-well TC plate, cat.
CC7682-7596) for a final inoculum concentration of OD600 0.001. All plates were incubated at 37 °C with shaking for
18 h before results were interpreted.
Microdilution Checkerboard
Tests for Drug Synergy
Checkerboard
assays were performed as described previously.[23] Antibiotics were diluted down the columns of a 96-well
microplate, while 13d was diluted across the rows. Plates
were prepared that contained concentrations of antibiotics and 13d 2-fold higher than the intended final concntrations and
were prepared in duplicate. All antimicrobial compounds were purchased
from Sigma-Aldrich. The growth media was MHB purchased from HIMEDIA
through VWR (cat. 95039-356). The inoculum was prepared by diluting
a bacterial day culture (OD600 0.15–0.4) to OD600 0.002. This dilution was further diluted 2-fold when added
to 96-well microplates (USA Scientific CytoOne 96-well TC plate, cat.
CC7682-7596) for a final inoculum concentration of OD600 0.001. All plates were incubated at 37 °C with shaking for
18 h before results were interpreted.
Mammalian Cytotoxicity
of 1 Analogues in HeLa Cells
To evaluate the
cytotoxicity of 1 in mammalian cells,
a cell viability assay was carried out using a CellTiter-Glo luminescent
cell viability assay kit (Promega). Human cervixcal adenocarcinoma
HeLa cells were seeded on white, cell-culture-treated, 96-well plates
(Corning 3917) with Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% fetal calf serum (FCS), 1% penicillin/streptomycin,
at the densities of 20 000 cells/well. The medium volume for each
well was 100 μL. Cells were incubated at 37 °C in 5% CO2/95% air for 16 h. The medium was removed from each well and
replaced with 99 μL of warmed, fresh medium. To each well, 1
μL of 1 analogue was added in DMSO to final concentrations
of 0.5–32 μg/mL. Each series was performed in triplicate.
After incubation at 37 °C for another 24 h, the plates were equilibrated
to room temperature for 30 min, and 100 μL of CellTiter-Glo
reagent (Promega) was added to each well and mixed for 2 min on an
orbital shaker. The plate was incubated at room temperature for another
10 min to stabilize the luminescent signal. The luminescence of each
sample was recorded with an Envision Multilabel Plate Reader (PerkinElmer).
Hemolytic Activity Assay
Eight milliliters of freshly
drawn, heparin-stabilized, human blood was centrifuged at 3500 rpm
for 5 min. The supernatant was removed and the human red blood cells
(hRBCs) were washed with 8 mL of D-PBS three times or until the supernatant
was clear. hRBCs were then resuspended in 80 mL of D-PBS. This was
further diluted in D-PBS to a final concentration of 1% of the original
pellet volume. 13d was dissolved in DMSO and to 1 mL
samples of the hRBC solution to final concentrations of 64, 16, and
4 μg/mL. A 1% Triton X-100 sample was used as the positive control;
this was considered to produce 100% hemolysis. DMSO alone was used
as the negative control. The mixtures were vortexed gently and incubated
at 37 °C for 1 h with shaking. The mixtures were then centrifuged
at 3000 rpm for 10 min, and 50 μL of supernatant from each sample
was transferred to a well of a sterile 96-well plate containing 50 μL
of water. The presence of hemoglobin was measured by absorbance at
415 nm, and the percent hemolysis was calculated using the following
equation:Each condition was assayed in triplicate.
The assay was performed within 3 h of the blood draw.
Chemistry
Unless otherwise noted, reagents were obtained
commercially and used without further purification. CH2Cl2 was distilled from CaH2 under a nitrogen
atmosphere. THF was distilled from sodium–benzophenone under
a nitrogen atmosphere. Toluene was distilled from sodium under a nitrogen
atmosphere. Thin-layer chromatography (TLC) analysis of reaction mixtures
was performed on Dynamicadsorbents silica gel F-254 TLC plates. Flash
chromatography was carried out on Zeoprep 60 ECO silica gel. 1H and 13C NMR spectra were recorded with Varian
INOVA (400, 500 MHz) and Bruker Avance-III (300 MHz) spectrometers.
Mass spectral and analytical data were obtained via the PE SCIEX/ABI
API QSTAR Pulsar iHybrid LC/MS/MS (Applied Biosystems) operated by
the Central Analytical Laboratory, University of Colorado at Boulder.
Infrared (IR) spectra were recorded on a Thermo Nicolet Avatar 370
FT-IR spectrometer. Melting point (mp) determinations were performed
by using a Thomas-Hoover capillary melting point apparatus and are
uncorrected. Compound purity (≥95%) was confirmed on the basis
of the integration of the area under the UV absorption curve at λ
= 254 or 210 nm signals using an Agilent 1260 series HPLC system coupled
with a 6120 Quadrupole mass spectrometer (column: ZORBAX Narrow Bore
SB-C18 RRHT, 2.1 × 50 mm, 1.8 μm, PN 827700-902). The system
was eluted at 0.5 mL/min with a gradient of water/acetonitrile with
0.1% formic acid: 0–5 min, 5–95% acetonitrile; 5–7
min, 95% acetonitrile; 7–7.25 min, 95–5% acetonitrile;
7.25–8.5 min, 5% acetonitrile.
General Protocol for the
One-Pot Three-Component Indole Synthesis
The activating agent
4-chlorobenzenesulfonyl chloride (1.2 equiv)
was added to a solution of 4-dimethylaminopyridine (1.2 equiv) in
anhydrous DMF at 0 °C. The reaction was stirred at 23 °C
for 30 min. A solution of the alkynyl imine 2 (1.0 equiv)
in anhydrous DMF was added and the reaction was stirred at the same
temperature for 0.5 h. Then methanesulfonic acid (3.0 equiv) was next
added to the above mixture at 0 °C. The reaction was then stirred
at 23 °C for 2 h. Arylhydrazine 3 (1.5 equiv) was
added and the mixture stirred for an addition 1 h at 23 °C. The
reaction was then heated to 60–120 °C (60 °C for
electron-rich arylhydrazines and 120 °C for electron-poor arylhydrazines
for 20 h). The reaction was cooled down to room temperature. The residue
was then dissolved in ethyl acetate and washed with brine and a saturated
aqueous solution of sodium bicarbonate. The combined organic layers
were dried over anhydrous Na2SO4, filtered,
and concentrated in vacuo to give a crude product, which was purified
by column chromatography on silica gel to give the indole product 4. 4-Chloro-N-{2-[5-chloro-2-(pent-4-yn-1-yl)-1H-indol-3-yl]ethyl}benzene-1-sulfonamide (4a): TLC (hexanes:ethyl acetate, 3:1 v/v) R = 0.20; light yellow oil, 32%; 1H NMR
(500 MHz, CDCl3) δ 7.99 (s, 1H), 7.66 (d, J = 8.6 Hz, 2H), 7.41 (d, J = 8.6 Hz, 2H),
7.24 (d, J = 2.0 Hz, 1H), 7.21 (d, J = 8.5 Hz, 1H), 7.10 (dd, J = 8.6, 2.0 Hz, 1H),
4.40 (t, J = 6.2 Hz, 1H), 3.25 (q, J = 6.5 Hz, 2H), 2.89 (dt, J = 16.4, 7.1 Hz, 4H),
2.23 (td, J = 6.8, 2.7 Hz, 2H), 2.09 (t, J = 2.6 Hz, 1H), 1.85 (p, J = 7.1 Hz, 2H); 13C NMR (75 MHz, CDCl3) δ 139.1, 138.1, 137.4,
133.7, 129.3, 129.0, 128.3, 125.3, 121.8, 117.3, 111.6, 107.1, 83.5,
69.9, 43.0, 28.2, 24.5, 24.5, 17.7; HRMS (ESI) m/z calcd for C21H21Cl2N2O2S [M + H]+ 435.0695, found 435.0694;
IR (thin film) 3377, 3297, 2924, 2854, 2359, 1575, 1475, 1325, 1160,
1094, 828 cm–1.
General Protocol for Gold(I)-Catalyzed
Tandem Cyclization
To a suspension of Ph3PAuNTf2 (as the 2:1
toluene adduct) (0.05 equiv) in anhydrous toluene was added a solution
of indole 4 (1.0 equiv) in anhydrous toluene. The suspension
was heated to 50 °C until TLC showed that there was no starting
material left (1–12 h) under argon atmosphere. The reaction
mixture was then filtered through a short pad of silica gel. The filtrate
was concentrated in vacuo and the residue was purified by column chromatography
on silica gel to afford tetracyclic indoline product 5. 4-Chloro-16-(4-chlorobenzenesulfonyl)-13-methylidene-8,16-diazatetracyclo[7.4.3.01,9.02,7]hexadeca-2,4,6-triene (5a):
TLC (hexanes:ethyl acetate, 3:1 v/v) R = 0.60; light yellow oil, 82%; 1H NMR
(500 MHz, CDCl3) δ 7.61 (d, J =
8.8 Hz, 2H), 7.38 (d, J = 8.8 Hz, 2H), 7.05–7.03
(m, 2H), 6.51 (d, J = 8.1 Hz, 1H), 5.19 (brs, 1H),
4.92 (s, 1H), 4.77 (s, 1H), 3.48 (td, J = 8.7, 1.7
Hz, 1H), 3.03–2.98 (m, 1H), 2.72 (dt, J =
14.2, 3.8 Hz, 1H), 2.37 (ddd, J = 12.7, 10.5, 8.4
Hz, 1H), 2.24–2.12 (m, 3H), 1.93–1.86 (m, 1H), 1.75
(dt, J = 13.7, 4.5 Hz, 1H), 1.63–1.55 (m,
1H); 13C NMR (75 MHz, CDCl3) δ 147.2,
146.4, 138.9, 138.4, 133.0, 129.2, 128.4, 128.3, 124.6, 123.8, 112.8,
111.0, 93.5, 61.1, 47.4, 33.8, 32.0, 30.3, 22.8; HRMS (ESI) m/z calcd for C21H21Cl2N2O2S [M + H]+ 435.0695,
found 435.0691; IR (thin film) 3054, 2986, 2926, 2850, 1478, 1423,
1335, 1265, 1156, 1090, 1001, 740 cm–1.
General Protocol
for the Ring-Opening Reduction
To
a solution of the tetracyclic indoline 5 (1.0 equiv)
in anhydrous methanol was added acetic acid (2.0 equiv) and sodium
cyanoborohydride (4.0 equiv) at 0 °C. The resulting mixture was
stirred at 23 °C for 0.5 h. The solvent was removed in vacuo
to give a residue, which was dissolved in ethyl acetate, and the organic
layers were washed with a saturated aqueous solution of sodium bicarbonate.
The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to give a crude
product, which was purified by column chromatography on silica gel
to afford the product 6. 4-Chloro-N-[2-(6-chloro-4-methylidene-2,3,4,4a,9,9a-hexahydro-1H-carbazol-4a-yl)ethyl]benzene-1-sulfonamide (6a): TLC (hexanes:ethyl acetate, 3:1 v/v) R = 0.20; light yellow oil, 92%; 1H NMR
(500 MHz, CDCl3) δ 7.77 (d, J =
8.6 Hz, 2H), 7.49 (d, J = 8.6 Hz, 2H), 7.01 (dd, J = 8.3, 2.2 Hz, 1H), 6.87 (s, 1H), 6.58 (d, J = 8.3 Hz, 1H), 5.13 (d, J = 5.9 Hz, 1H), 4.94 (s,
1H), 4.67 (s, 1H), 3.63 (t, J = 4.9 Hz, 1H), 3.00–2.89
(m, 1H), 2.20–2.11 (m, 2H), 2.07–2.01 (m, 1H), 1.98–1.93
(m, 1H), 1.81–1.76 (m, 1H), 1.70–1.57 (m, 2H), 1.52–1.46
(m, 1H); 13C NMR (75 MHz, CDCl3) δ 148.6,
148.2, 139.2, 138.4, 135.1, 129.4, 128.5, 127.8, 124.2, 123.8, 112.1,
111.4, 77.5, 77.0, 76.6, 65.4, 52.3, 40.2, 36.1, 32.4, 28.6, 21.7;
HRMS (ESI) m/z calcd for C21H23Cl2N2O2S [M + H]+ 437.0852, found 437.0857; IR (thin film) 2264, 3285, 2934,
2858, 1637, 1604, 1587, 1477, 1428, 1328, 1160, 1093, 1014,905, 827,
617 cm–1.
General Protocol for the Removal of a Cbz
Group
To
a solution of substrate 10 (1.0 equiv) in dry dichloromethane
was added boron trifluoridediethyl etherate (5.0 equiv) and dimethyl
sulfide (10.0 equiv) dropwise at 23 °C. The resulting mixture
was stirred at this temperature for 2 h. After that, TLC showed that
there was no starting material left; the mixture was poured into water
and 10% aqueous ammonium hydroxide and extracted with chloroform three
times. The combined extracts were washed with water and then brine,
dried over anhydrous Na2SO4, filtered, and concentrated
in vacuo to give a crude product, which was purified by column chromatography
on silica gel to afford the product 11. 2-(6-Bromo-4-methylidene-2,3,4,4a,9,9a-hexahydro-1H-carbazol-4a-yl)ethan-1-amine (11a): TLC (chloroform:methanol,
10:1 v/v): R = 0.10;
yellow oil, 72%; 1H NMR (500 MHz, CDCl3) δ
7.20 (d, J = 2.0 Hz, 1H), 7.15 (dd, J = 8.3, 2.1 Hz, 1H), 6.55 (d, J = 8.2 Hz, 1H), 4.88
(s, 1H), 4.60 (d, J = 1.5 Hz, 1H), 3.72 (d, J = 3.2 Hz, 1H), 3.64 (s, 1H), 2.86–2.80 (m, 1H),
2.73–2.67 (m, 1H), 2.25–2.16 (m, 3H), 2.01–1.98
(m, 1H), 1.94–1.88 (m, 1H), 1.85–1.79 (m, 1H), 1.77–1.67
(m, 2H), 1.63–1.58 (m, 1H); 13C NMR (75 MHz, CDCl3) δ 149.8, 149.0, 137.1, 130.2, 127.4, 111.6, 111.6,
110.2, 65.6, 52.5, 39.3, 39.0, 32.3, 29.7, 27.6, 22.0; HRMS (ESI) m/z calcd for C15H20BrN2 [M + H]+ 307.0804, found 307.0811; IR
(thin film) 3367, 2960, 2924, 2853, 1729, 1661, 1600, 1464, 1261,
1092, 870, 802 cm–1.
General Protocol for the
Modification of Free Amine
To a solution of substrate 11 (1.0 equiv) in dry dichloromethane
was added triethyl amine (3.0 equiv) and the corresponding sulfonyl
chloride or acetyl chloride (1.2 equiv) dropwise at 0 °C. The
resulting mixture was stirred at this temperature for 15 min. The
reaction was quenched by aqueous sodium bicarbonate and extracted
with dichloromethane three times. The combined extracts were washed
with water and then brine, dried over anhydrous Na2SO4, filtered, and concentrated in vacuo to give a crude product,
which was purified by column chromatography on silica gel to afford
the product 12. N-[2-(6-Bromo-4-methylidene-2,3,4,4a,9,9a-hexahydro-1H-carbazol-4a-yl)ethyl]-2,2,2-trifluoroacetamide (12a): TLC (hexanes:ethyl acetate, 3:1 v/v) R = 0.40; yellow oil, 82%; 1H NMR (500 MHz, CDCl3) δ 7.20 (dd, J = 8.2, 2.0 Hz, 1H), 7.14 (d, J = 2.0 Hz, 1H), 6.72
(brs, 1H), 6.60 (d, J = 8.2 Hz, 1H), 5.03 (s, 1H),
4.83 (s, 1H), 3.69 (t, J = 5.2 Hz, 1H), 3.46–3.34
(m, 2H), 2.27–2.21 (m, 1H), 2.19–2.14 (m, 1H), 2.13–2.00
(m, 2H), 1.90–1.84 (m, 1H), 1.72–1.60 (m, 2H), 1.53–1.47
(m, 1H); 13C NMR (75 MHz, CDCl3) δ 156.8,
148.3, 135.5, 130.9, 127.0, 112.4, 112.1, 65.4, 52.3, 36.9, 35.4,
32.5, 29.0, 21.6; HRMS (ESI) m/z calcd for C17H19BrF3N2O [M + H]+ 403.0627, found 403.0622; IR (thin film) 3322,
2957, 2928, 2861, 1663, 1599, 1530, 1463, 1378, 1008, 823 cm–1.
General Protocol for the Modification of 1’s
Indoline Amine
To a solution of substrate 1 (1.0
equiv) in dry acetonitrile was added potassium carbonate (3.0 equiv)
and the corresponding iodomethane or sulfonyl chloride or acetyl chloride
(1.2 equiv) dropwise at 0 °C. The resulting mixture was stirred
at this temperature for 15 min. The reaction was quenched by aqueous
sodium bicarbonate and extracted with dichloromethane three times.
The combined extracts were washed with water and then brine, dried
over anhydrous Na2SO4, filtered, and concentrated
in vacuo to give a crude product, which was purified by column chromatography
on silica gel to afford the products 1a–c. N-{2-[6-Bromo-9-(4-chlorobenzenesulfonyl)-4-methylidene-2,3,4,4a,9,9a-hexahydro-1H-carbazol-4a-yl]ethyl}-4-chlorobenzene-1-sulfonamide (1b): TLC (hexanes:ethyl acetate, 2:1 v/v) R = 0.60; light yellow oil, 72%; 1H NMR (500 MHz, CDCl3) δ 7.82 (d, J = 8.6 Hz, 2H), 7.74 (d, J = 8.6 Hz, 2H),
7.57–7.45 (m, 5H), 7.38 (dd, J = 8.6, 2.1
Hz, 1H), 6.99 (d, J = 2.1 Hz, 1H), 5.13 (d, J = 2.0 Hz, 1H), 4.86 (d, J = 2.0 Hz, 1H),
4.11 (dd, J = 7.9, 5.7 Hz, 1H), 3.98 (d, J = 5.1 Hz, 1H), 2.83–2.77 (m, 1H), 2.74–2.64
(m, 1H), 2.29–2.24 (m, 1H), 2.05–1.98 (m, 1H), 1.96–1.89
(m, 1H), 1.69–1.61 (m, 2H), 1.21–1.15 (m, 1H); 13C NMR (75 MHz, CDCl3) δ 144.8, 140.1, 139.4,
139.2, 138.4, 138.2, 137.2, 131.9, 129.8, 129.5, 128.4, 128.3, 127.0,
117.0, 116.8, 113.7, 68.3, 52.1, 39.0, 38.3, 31.6, 29.7, 19.9; HRMS
(ESI) m/z calcd for C27H25BrCl2KN2O4S2 [M + K]+ 692.9448, found 692.9451; IR (thin film) 2292,
2920, 2850, 1638, 1475, 1360, 1338, 1166, 1093, 826, 756 cm–1.
Protocol for the Synthesis of 1d
To a
solution of sodium hydride (0.7 mg, 0.02 mmol) in dry N,N-dimethylformamide was added 1 (6.0
mg, 0.012 mmol) dropwise at 0 °C. After 15 min, iodomethane (3.9
μL, 0.06 mmol) was added dropwise at 0 °C. The resulting
mixture was stirred at this temperature for 15 min and then warmed
up to 60 °C for 30 min. The reaction was quenched by aqueous
ammonium chloride and extracted with ethyl acetate three times. The
combined extracts were washed with water and then brine, dried over
anhydrous Na2SO4, filtered, and concentrated
in vacuo to give a crude product, which was purified by column chromatography
on silica gel to afford the product 1d. N-[2-(6-Bromo-4-methylidene-2,3,4,4a,9,9a-hexahydro-1H-carbazol-4a-yl)ethyl]-4-chloro-N-methylbenzene-1-sulfonamide
(1d): TLC (hexanes:ethyl acetate, 2:1 v/v) R = 0.45; light yellow oil, 5.5 mg, 89%; 1H NMR (500 MHz, CDCl3) δ 7.70 (d, J = 8.5 Hz, 2H), 7.50 (d, J = 8.5 Hz, 2H),
7.16 (dd, J = 8.2, 2.0 Hz, 1H), 7.09 (s, 1H), 6.55
(d, J = 8.2 Hz, 1H), 4.95 (s, 1H), 4.66 (s, 1H),
3.70 (t, J = 4.5 Hz, 1H), 3.18 (ddd, J = 13.6, 11.2, 5.4 Hz, 1H), 2.97 (ddd, J = 13.6,
11.0, 5.1 Hz, 1H), 2.78 (s, 3H), 2.14–2.00 (m, 3H), 1.87–1.79
(m, 1H), 1.73–1.66 (m, 2H); 13C NMR (75 MHz, CDCl3) δ 149.4, 139.1, 135.9, 130.6, 129.5, 129.4, 129.1,
128.7, 126.7, 111.8, 110.0, 109.6, 61.7, 51.4, 47.4, 37.9, 35.2, 33.1,
32.8, 21.8; HRMS (ESI) m/z calcd
for C22H24BrClKN2O2S [M
+ K]+ 533.0062, found 533.0068; IR (thin film) 3371, 2925,
2853, 2359, 1636, 1474, 1344, 1260, 1160, 1104, 1014, 804 cm–1.
General Protocol for the Modification of 1’s
Side Chain Amine
To a solution of substrate 1 (1.0 equiv) in dry dichloromethane was added triethylamine (3.0
equiv) and the corresponding sulfonyl chloride or acetyl chloride
(1.2 equiv) dropwise at 0 °C. The resulting mixture was stirred
at this temperature for 15 min. The reaction was quenched by aqueous
sodium bicarbonate and extracted with dichloromethane three times.
The combined extracts were washed with water and then brine, dried
over anhydrous Na2SO4, filtered, and concentrated
in vacuo to give a crude product, which was purified by column chromatography
on silica gel to afford the product 1e or 1f. 2-(6-Bromo-4-methylidene-2,3,4,4a,9,9a-hexahydro-1H-carbazol-4a-yl)-N-(4-chlorobenzenesulfonyl)-S-(4-chlorophenyl)ethane-1-sulfonamido (1e):
TLC (hexanes:ethyl acetate, 2:1 v/v) R = 0.75; colorless oil, 52%; 1H NMR (500
MHz, CDCl3) δ 7.94 (d, J = 8.7 Hz,
4H), 7.55 (d, J = 8.7 Hz, 4H), 7.25–7.12 (m,
2H), 6.57 (d, J = 8.2 Hz, 1H), 4.97 (s, 1H), 4.71
(s, 1H), 3.83 (ddd, J = 15.4, 12.5, 5.3 Hz, 1H),
3.68 (t, J = 4.4 Hz, 1H), 3.51 (ddd, J = 15.2, 12.3, 4.6 Hz, 1H), 2.33–2.12 (m, 4H), 1.89–1.79
(m, 1H), 1.75–1.67 (m, 3H); 13C NMR (75 MHz, CDCl3) δ 148.9, 148.1, 141.0, 138.0, 135.0, 130.8, 129.8,
127.7, 124.6, 112.3, 111.9, 110.2, 65.1, 52.1, 39.4, 35.5, 32.3, 20.0;
HRMS (ESI) m/z calcd for C27H26BrCl2N2O4S2 [M + H]+ 654.9889, found 654.9871; IR (thin film) 2922,
2851, 1736, 1655, 1476, 1377, 1167, 1080, 826, 756, 620 cm–1.
Protocol for the Reduction of 12o
To a
solution of substrate 12n (4.0 mg, 0.008 mmol) in methanol
was added a solution of ammonium chloride (8.7 mg, 0.162 mmol) in
water and zinc dust (5.3 mg, 0.08 mmol) at room temperature. The resulting
mixture was stirred for 6 h and then filtered. The filtrate was removed
under reduce pressure and the residue was diluted with 2 N sodium
hydroxide and extracted with ethyl acetate three times. The combined
extracts were washed with water and then brine, dried over anhydrous
Na2SO4, filtered, and concentrated in vacuo
to give a crude product, which was purified by column chromatography
on silica gel to afford the product 12o. 2-Amino-N-[2-(6-bromo-4-methylidene-2,3,4,4a,9,9a-hexahydro-1H-carbazol-4a-yl)ethyl]benzene-1-sulfonamide (12o): TLC (hexanes:ethyl acetate, 1:1 v/v) R = 0.30; colorless oil, 2.9 mg, 79%; 1H NMR (500 MHz, CDCl3) δ 7.70 (d, J = 7.9 Hz, 1H), 7.35 (t, J = 7.7 Hz, 1H), 7.19–7.12
(m, 1H), 7.01 (s, 1H), 6.84 (t, J = 7.6 Hz, 1H),
6.79 (d, J = 8.2 Hz, 1H), 6.52 (d, J = 8.3 Hz, 1H), 4.97 (d, J = 6.8 Hz, 1H), 4.89 (s,
1H), 4.58 (s, 1H), 3.60 (s, 1H), 2.98–2.88 (m, 2H), 2.13 (q, J = 4.7 Hz, 2H), 1.98 (ddd, J = 15.2, 9.2,
6.2 Hz, 1H), 1.89 (ddd, J = 14.5, 9.0, 5.9 Hz, 1H),
1.77–1.73 (m, 1H), 1.69–1.62 (m, 2H), 1.52–1.47
(m, 1H); 13C NMR (75 MHz, CDCl3) δ 148.7,
147.4, 138.0, 136.0, 135.8, 134.3, 133.1, 130.6, 129.5, 126.7, 121.7,
117.5, 112.0, 111.7, 110.5, 65.2, 52.2, 40.2, 35.4, 32.2, 28.4, 21.6;
HRMS (ESI) m/z calcd for C21H25BrN3O2S [M + H]+ 462.0845,
found 462.0835; IR (thin film) 3370, 2924, 2853, 1712, 1599, 1481,
1454, 1318, 1260, 901, 809, 754 cm–1.
Authors: Hao Wang; Charles J Gill; Sang H Lee; Paul Mann; Paul Zuck; Timothy C Meredith; Nicholas Murgolo; Xinwei She; Susan Kales; Lianzhu Liang; Jenny Liu; Jin Wu; John Santa Maria; Jing Su; Jianping Pan; Judy Hailey; Debra Mcguinness; Christopher M Tan; Amy Flattery; Suzanne Walker; Todd Black; Terry Roemer Journal: Chem Biol Date: 2013-02-21
Authors: Jessica D Podoll; Yongxiang Liu; Le Chang; Shane Walls; Wei Wang; Xiang Wang Journal: Proc Natl Acad Sci U S A Date: 2013-09-09 Impact factor: 11.205
Authors: Chengde Wu; E Radford Decker; Natalie Blok; Huong Bui; Tony J You; Junmei Wang; Andree R Bourgoyne; Vippra Knowles; Kurt L Berens; George W Holland; Tommy A Brock; Richard A F Dixon Journal: J Med Chem Date: 2004-04-08 Impact factor: 7.446
Authors: Patrick M Barbour; Wei Wang; Le Chang; Kasey L Pickard; Rana Rais; Barbara S Slusher; Xiang Wang Journal: Adv Synth Catal Date: 2016-03-03 Impact factor: 5.837
Figure 1
Scheme 1
Scheme 2
Scheme 3