Chemoproteomics of an indole-based quinone epoxide identifies druggable vulnerabilities in vancomycin-resistant staphylococcus aureus

Article abstract of DOI:10.1021/acs.jmedchem.9b00774

The alarming global rise in fatalities from multidrug-resistant Staphylococcus aureus (S. aureus) infections has underscored a need to develop new therapies to address this epidemic. Chemoproteomics is valuable in identifying targets for new drugs in different human diseases including bacterial infections. Targeting functional cysteines is particularly attractive, as they serve critical catalytic functions that enable bacterial survival. Here, we report an indole-based quinone epoxide scaffold with a unique boat-like conformation that allows steric control in modulating thiol reactivity. We extensively characterize a lead compound (4a), which potently inhibits clinically derived vancomycin-resistant S. aureus. Leveraging diverse chemoproteomic platforms, we identify and biochemically validate important transcriptional factors as potent targets of 4a. Interestingly, each identified transcriptional factor has a conserved catalytic cysteine residue that confers antibiotic tolerance to these bacteria. Thus, the chemical tools and biological targets that we describe here prospect new therapeutic paradigms in combatting S. aureus infections.

Full text of DOI:10.1021/acs.jmedchem.9b00774

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Article
Chemoproteomics of an Indole-Based Quinone-Epoxide identifies druggable
vulnerabilities in Vancomycin Resistant Staphylococcus aureus (VRSA)
Amogh Kulkarni, Isha Soni, Dhanashree Kelkar, Allimuthu T Dharmaraja,
Rathinam Sankar, Gaurav Beniwal, Abinaya Rajendaran, Sharavari
Tamhankar, Sidharth Chopra, Siddhesh S Kamat, and Harinath Chakrapani
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00774 • Publication Date (Web): 26 Jun 2019
Downloaded from http://pubs.acs.org on June 27, 2019
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Chemoproteomics of an Indole-Based Quinone-Epoxide identifies druggable
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vulnerabilities in Vancomycin Resistant Staphylococcus aureus (VRSA)
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Amogh Kulkarni,a Isha Soni,b Dhanashree S. Kelkar,c Allimuthu T. Dharmaraja,a,d Rathinam K.
Sankar,a,e Gaurav Beniwal,a Abinaya Rajendaran,c Sharvari Tamhankar,c Sidharth Chopra,b,*
Siddhesh S. Kamat,c,* and Harinath Chakrapania,*
aDepartment of Chemistry, Indian Institute of Science Education and Research Pune, Dr. Homi
b
Bhabha Road, Pashan, Pune-411008, Maharashtra, India. Division of Microbiology, CSIR-
Central Drug Research Institute, Sector 10, Janakipuram Extension, Sitapur Road, Lucknow-
226021, Uttar Pradesh, India.
cDepartment of Biology, Indian Institute of Science Education and Research Pune, Dr. Homi
Bhabha Road, Pashan, Pune-411008, Maharashtra, India.
dPresent address: Department of Chemistry, Indian Institute of Technology, Kanpur, India.
ePresent address: Faculty of Pharmaceutical Sciences, University of Iceland, 107 Reykjavik,
Iceland.
Abstract
The alarming global rise in fatalities from multi-drug resistant Staphylococcus aureus (S.
aureus) infections has underscored a need to develop new therapies to address this epidemic.
Chemoproteomics is valuable in identifying targets for new drugs in different human diseases
including bacterial infections. Targeting functional cysteines is particularly attractive, as they
serve critical catalytic functions that enable bacterial survival. Here, we report an indole-
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based quinone epoxide scaffold with a unique boat-like conformation that allows steric
control in modulating thiol reactivity. We extensively characterize a lead compound (4a),
which potently inhibits clinically derived Vancomycin-Resistant S. aureus. Leveraging diverse
chemoproteomic platforms, we identify and biochemically validate important transcriptional
factors as potent targets of 4a. Interestingly, each identified transcriptional factor has a
conserved catalytic cysteine residue that confers antibiotic tolerance to these bacteria. Thus,
the chemical tools and biological targets that we describe here, prospect new therapeutic
paradigms in combatting S. aureus infections.
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Introduction
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Clinical incidences of antimicrobial resistance (AMR) are on the rise globally, and will
likely render the existing pool of antimicrobial drugs ineffective. Resistance developed
against these drugs makes treatment difficult and expensive.1 The bacterium
Staphylococcus aureus (S. aureus) is associated with numerous nosocomial infections
and can hence rapidly spread among susceptible patient populations. The emergence
of resistance against vancomycin (VRSA) has further weakened our defence against
this particular pathogen.2 Since only a few new classes of antibiotics are in the
pipeline,3 identification of novel druggable biological targets is of high priority. The
chemoproteomics technique, activity-based protein profiling (ABPP)4 has recently
emerged as a powerful tool to study and understand the native activity of enzymes in
complex physiological settings, mainly utilizing the unique reactivity of serine,5-6 lysine7
and cysteine8 residues on the proteins. Among these, the cysteine-based ABPP has
received considerable attention since thiols are excellent nucleophiles, and are known
to play crucial physiological roles.9-10 In order to study such cysteines, thiol-selective
probes are being designed to pharmacologically perturb the activity of such proteins
in order to understand their specific biological functions. Such probes not only help
identify new druggable protein sites, but can also eventually emerge as potential drug
candidates. An integral part of this strategy is to identify functional cysteine residues
that are vital to survival, and modification of which, may result in stress and eventual
lethality to bacteria. While a number of probes are reported in literature,11 a significant
portion of the proteome still remains unmined.8 Thus, there is an increasing need to
develop new chemical tools to profile the thiol proteome. As a testament to the
success of ABPP in identifying new bacterial proteins as targets, Sieber and co-workers
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have reported a suite of reactive probes to interrogate the proteome for druggable
targets.12-16 For example, using a Michael acceptor, they studied the thiol proteome of
S. aureus and found a number of virulence factors as druggable targets.13 This and
other pioneering studies12 lay a strong foundation for our approach to overcome VRSA.
Here, we report the design of a new scaffold that is cell-permeable, selective in its
reactivity with thiols and has in-built structural features for modulating thiol-reactivity,
which helps in profiling the S. aureus thiol proteome.
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Although several electrophilic species have been developed as thiol-selective probes,
many of them suffer from diminished cell permeability and indiscriminate reactivity.8
The diversity of pKa of cysteine residues in the proteome also suggests that a single
probe may likely be insufficient. The 2,3-epoxy-1,4-benzoquinone functional group has
been previously reported to react with protein thiols,14, 17 and was used as the primary
thiol-reactive scaffold for this study (Figure 1).18-19 We hypothesized that reactivity of
this scaffold with a thiol can be modulated by systematic introduction of substituents
around the epoxide (Figure 1).
R1
O
O
R2
O
R3
O
O
N
O
R
Fig. 1 Proposed indole-quinone epoxide (Inset: An epoxide that is the electrophilic reactive site in
the probe and the arrows indicate the approach of the nucleophile)
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In order to achieve this, a naphthoquinone epoxide was fused with an indole ring, and
the resulting indole-based quinone epoxides (IND-QEs) were designed with two major
considerations. First, due to its high rates of occurrence in natural products and drugs,
indole has been considered a privileged structure.20 This typically suggests enhanced
permeability and bioactivity. Second, substituting around the epoxide ring provides
opportunities to modulate thiol reactivity and cell-permeability. These, previously
unutilized structural variations, will provide a systematic way to study the bacterial
thiol proteome. Here, we describe the synthesis of this scaffold and generation of
analogues, its structure activity studies in bacteria, their chemoproteomic profiling and
subsequent identification of the biological targets of a lead compound (4a) for this
scaffold in S. aureus.
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Results and Discussion
Keeping the aforementioned synthetic criteria in mind, we decided to synthesize a
small yet focused library of IND-QEs. In order to synthesize the desired compounds, a
series of indole-based quinones (compounds 1b-15b, Q1-Q2) were first synthesized
using the general procedure outlined in Scheme 1.
O
O
R1
R2
R1
a
b
H
N
R
R3
N
R
O
N
R
Scheme 1. General procedure for the synthesis of indole-quinone epoxides: (a) R1CH2PPh3Br (1.15
eq.), n-BuLi (1.0 eq.), THF, -78 °C; (b) Benzoquinone (2.5 eq.), ethanol, RT.
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The first series of compounds had varying substituents on the indole nitrogen (position
R). These adducts were then converted to an epoxide using NaOCl (Table 1). We tested
the ability of these compounds to inhibit S. aureus ATCC 29213, and the minimum
inhibitory concentration (MIC) was determined using a previously described protocol
(Table 1, entries 1-5).21-22
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Table 1. IND-QE analogues with variations in R, their MIC values and reactivity with thiol
O
O
NaOCl
O
N
O
N
O
R
R
Entry
Compound
R
H
Yield (%)
MICa
% Remainingb
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2
3
4
5
1a
2a
3a
4ac
5a
94
94
96
96
82
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2
33
27
Me
Et
33
iPr
Ph
0.125 - 0.5
2
34d
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aMIC in g/mL against S. aureus ATCC 29213. bDetermined by HPLC analysis: the compound was
treated with L-Cysteine (1 eq) in pH 7.4 phosphate buffer for 60 min. cX-ray analysis, see SI, Figure
S1. dFor HPLC trace, see SI, Figure S2.
Based on this data, the N-isopropyl derivative, 4a, was identified as the most potent
inhibitor with a MIC of 0.125-0.5 g/mL (Table 1, entry 4). To assess the nature of
bacterial growth inhibitory activity of 4a, a time-kill method was employed and this
compound was found to have robust bactericidal activity (see SI, Figure S3 A). Next, 4a
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was tested against a panel of clinical drug-resistant S. aureus strains and was found to
be a potent inhibitor of all of these, including VRSA (Table 2, entries 1-8; for the entire
panel see SI, Table S1). Time-kill analysis with VRSA strain HIP 11714 showed that 4a
had potent bactericidal activity comparable with Daptomycin, a drug which is
frequently used to treat VRSA infections (Figure 2A).23
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Fig. 2 A) Time-kill analysis for 4a (10x MIC, MIC=0.5 g/mL) and Daptomycin (Dap, 10x MIC, MIC=0.5
g/mL) against VRSA strain HIP 11714 showing potent bactericidal activity of 4a. For entire plot (with
Vancomycin), see SI, Figure S4), (n=2); B) Time courses of independent reactions of compounds 4a,
9a, 10a and 11a with 10 eq. L-Cysteine. Curve fitting to an exponential decay afforded pseudo first
order rate-constants, see SI, Table S2, (n=2).
Since IND-QE was designed to react with thiols (See SI, Scheme S1), we tested the
propensity for these compounds to react with L-Cysteine. HPLC analysis of reaction
mixtures containing the epoxide and L-Cysteine revealed comparable thiol reactivity
for 4a and its analogues during 60 min (Table 1, entries 1-5). A time course of
consumption for 4a in the presence of L-Cysteine was next assessed, and this reaction
followed pseudo-first order kinetics with a rate constant of 7.54 x 10-2 min-1 (Figure
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2B). To further confirm the formation of a thiol-adduct, compound 4a was reacted with
4-nitro-benzyl thiol, and HPLC analysis showed the formation of a thiolated-adduct.
This result was further confirmed by co-elution with a synthetic standard (See SI,
Figures S5, S6, Scheme S2). Together, these data suggest that 4a reacts with a thiol to
form a stable covalent adduct.
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Table 2. MIC (g/mL) of 4a against various pathogens including VRSA
MIC of
Entry
Bacterium
Strain
MIC of 4a
Vancomycin
>64
1
2
S. aureus
S. aureus
SA-HIP-14300
SA-HIP-11983
AIS100050
SA-HIP-15178
SA-71080
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
64
32-64
32-64
32-64
32-64
>64
3
S. aureus
4
S. aureus
5
S. aureus
6
S. aureus
HIP11714
7
S. aureus
HIP14300
>64
8
S. aureus
1002434
>64
9
E. coli
ATCC 252922
BAA 1705
<0.01a
64a
10
11
12
13
K. pneumoniae
E. faecalis
P. aeruginosa
A. baumanii
64
B3119
64
0.5
ATCC 27853
BAA 1605
64
0.5a
64
8a
aCiprofloxacin was used.
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In order to test the ability of the IND-QE scaffold to modulate reactivity with thiols,
analogues of 4a, our lead compound, with varying substituents at the position R1, were
synthesized. (Table 3, entries 3-4). We surprisingly found that addition of these
substituents resulted in diminished reactivity (example, 9a, R1 = Et, Figure 2B). The
differences in the rate constants were about 6-fold (See SI, Table S2). While this
difference is not small, it is yet a structural tool that provides remote control over
reactivity. Given the difficulty in synthesis, preparation of compounds with increased
substituent size beyond an ethyl substituent at R1 was unsuccessful.
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Intrigued by this remote substituent effect, we decided to further investigate the
structural aspects that may contribute to diminished reactivity. X-ray diffraction
analysis of crystalline 4a was conducted and a boat-like conformation for the quinone
epoxide was observed21 (Figure 3). In light of this structure, it is expected that during
the thiol attack (attack at C-10, Figure 3B), one flagpole of the boat is expected to
become more planar; and this process is likely hindered by neighbouring substituents.
Similarly, when attack happens at the C-1 position, a similar flattening of the boat is
expected. Our data suggests that the neighbouring substituent does play a role.
Analysis of X-ray crystallographic data showed overlapping van der Waals radii24 for
the carbon attached to the nitrogen and the carbonyl adjacent to it. According to our
data (see Table 1), the N-substituent contributes to only a marginal difference in
relative thiol reactivity, which is consistent with the substituent being far from the
reaction centre. The substituent on the C-3 (R1) and the adjacent carbonyl also have
overlapping van der Waals radii. Here, however, the substituent effect is larger, i.e. 6-
fold. Further evidence for this trend was obtained from compounds 6a and 7a, which
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had a methyl substituent at the C-3 position with varying substituents at the indole
nitrogen (Table 3, entries 1-2). Here, we observed that the reactivity with thiol
uniformly decreased in these cases (Table 3, entries 1-2) when compared with their
unsubstituted counterparts (Table 1, entries 1 and 3). Together, these data support a
preference for attack on C-1 when compared with C-10.
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Table 3. IND-QE analogues with variations in R1, their reactivity with thiol and MIC values
R1
O
O
N
O
R
Entry Compound
R
H
R1
Me
Me
Me
Et
% Remaininga
MICb
>16
2
1
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4
6a
7a
8a
9a
86
93
84
88
Et
iPr
iPr
4
64
aDetermined by HPLC analysis: the compound was treated with L-Cysteine (1 eq) in pH 7.4
phosphate buffer for 60 min. bMIC in g/mL against S. aureus ATCC 29213.
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Fig. 3 A) ORTEP diagram for 4a with van der Waals radii overlap (dotted circles), for groups at R and R1
with carbonyl oxygens; B) ORTEP diagram for 4a showing the boat-like conformation of the quinone
epoxide and schematic showing flagpole positions of quinone epoxide during the thiol attack
Lastly, the analogues containing a phenyl ring on the carbons bearing the epoxide (R2
and R3) were synthesized (Table 4, entries 1-2, See SI, Scheme S3 for synthesis of
substituted benzoquinone). As expected, addition of a phenyl group on the carbon
bearing the epoxide had an impact on the rate of thiol-mediated disappearance of the
epoxide. The rate constant for the reaction of 11a was determined as 0.78 x 10-2 min-1
(Figure 2B). The 10-fold rate difference between 4a and 11a translates to roughly 1.4
kcal/mol difference in the activation barrier (See SI, Table S2). However, the analogue
10a, which had a substituent on the 10-position showed a difference in rate of only
about 2-fold (Figure 2B, See SI, Table S2). Together, these data corroborate our model
that the attack on C-1 is more favorable than nucleophilic substitution at C-10.
Recently, Diness and co-workers have reported fluorobenzene-based probes for
cysteine-selective modification.25 Here, they varied the electronics in the aryl ring and
find that the presence of an electron withdrawing group increased the rate of reaction
with cysteines while an electron-donating group reduced reactivity.26 This elegant
design allows for tuneable reaction with thiols. For example, Pan and co-workers have
earlier reported Ibrutinib-based irreversible Bruton’s Tyrosine Kinase (BTK) inhibitors
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and studied a series of analogues by changing the Michael acceptor group on the
pyrazole.27 This study suggests the reactivity of cysteine residue on BTK is critical for
the inhibition and can be tuned by the structural modifications on the Michael
acceptor installed. Further, leveraging advanced chemoproteomics, Cravatt and co-
workers have further employed alkyne-tagged irreversible probes selectively targeting
EGFR (Epidermal Growth Factor Receptor) and BTK. In another study, Adams and co-
workers have utilized 2-chloropropionamide as a steric-driven low reactivity
electrophile that selectively acts as a PDI (Protein Disulfide Isomerase) inhibitor.28
Recently, Ojida and co-workers have reported reversible modification of kinase
cysteines using chlorofluoroacetamides and have explored new warheads for TCI
(Targeted Covalent Inhibition) by inhibiting EGFRs.29 With IND-QE, the possibility of
tuning the thiol-reactivity with varying substituent size offers a unique and
complementary approach to interrogating cysteines with a large range of pKas. The
precise rationale for preferential attack on one carbon of the epoxide ring over the
other remains to be deciphered. However, to the best of our knowledge, this is the
first report where tunability of reactivity with thiol using an epoxide warhead, is
achieved by modulation of sterics around the electrophile.30-32
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We compared the effects of these substitutions on S. aureus growth inhibitory activity.
In general, we observed that lowering reactivity with a thiol reduced inhibitory activity.
For example, a significant lowering of potency with increased substituent size at the
R1-position (Table 3, entries 3-4). We also found that the MICs of 10a and 11a against
S. aureus were in accordance with their thiol-reactivity (Table 4, entries 1-2).
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Table 4. IND-QE analogues synthesized in the study and their MIC values
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N
O
Entry
Compd
10ab
11ad
12a
R2
R3
Ph
H
MICa
0.0625c,e
>64e
0.5
1
2
3
4
5
6
H
Ph
H
4-OMePh
13a
4-OMePh
H
H
4-BrPh
H
>64
14a
1
15af
4-BrPh
>64
aMIC in g/mL against S. aureus ATCC 29213. bFor X-ray structure of 10a, see SI, Figure S7. cTime-kill
analysis with S. aureus ATCC 29213 showed this compound had bactericidal activity (see SI, Figure S3
B). dFor X-ray structure of 11a, see SI, Figure S8, eFor HPLC trace, See SI, Figure S9, fFor X-ray structure
of 15a, see SI, Figure S10.
Similar observations were made with their analogues (Table 4, entries 3-6), which were
synthesized using similar techniques. To expand the structure activity relationship
within this scaffold, and further understand the role of indole in the antibacterial
activity, we synthesized naphthoquinone epoxide (NQE) derivatives with and without
substituent at the carbon bearing the epoxide and tested their inhibitory activity
against S. aureus ATCC 29213 (See SI, Table S3). Not surprisingly, we found that these
compounds were significantly less effective in inhibiting S. aureus compared to their
indole counterparts. Furthermore, the quinones33 containing indoles were also
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relatively poor inhibitors (See SI, Table S4). Previously, Sieber and co-workers have
reported aminoepoxycyclohexenones with higher MICs; one analogue with an indole
tether was found to have good potency against S. aureus.14 Thus, the indole moiety
plays a major role in enhancing the permeability and activity of this quinone epoxide
scaffold.
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Based on the MIC measurements, the most active compound with comparatively
easier synthetic access, 4a, with antibacterial activity comparable to that of
vancomycin, was chosen as the lead compound for subsequent investigations. To
assess the selectivity of this compound, we screened its antibacterial activity against
Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter
baumannii, Pseudomonas aeruginosa and Enterococcus faecalis (ESKAPE pathogens)
(Table 2, also see SI, Table S5). We found that 4a has exquisitely selective inhibitory
activity against S. aureus, with no significant activity against any other pathogen. Taken
together, IND-QE 4a was a selective and potent inhibitor of VRSA, and thus, becomes
an excellent starting point for chemoproteomic profiling to identify its biological
targets.
To further understand the pharmacology, two IND-QE alkyne probes, P1 and P2 were
synthesized (Figure 4A; for X-ray data of P2, see SI, Figure S11) based on 4a and 11a
scaffolds respectively, that would enable bioorthogonal approaches (click chemistry)
in pursuit of identifying the biological targets of 4a. The relative thiol-reactivity of these
two probes was evaluated using
a
competition experiment against
monobromobimane (mBBr)34 and both probes were found to be comparable in
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reactivity with their respective parent IND-QE derivatives (See SI, Figure S12, Scheme
S4). We confirmed that P1 (MIC, 1 g/mL) and P2 (MIC, >64 g/mL) had inhibitory
potencies against S. aureus comparable to the parent compounds 4a and 11a
respectively, and proceeded with chemoproteomics experiments using these alkyne
compounds. First, S. aureus cell lysates were titrated with increasing concentrations of
P1 and P2. In this experiment, the compound incubation was done for 1 h, following
which Cu-catalyzed click reaction with rhodamine-azide was carried out, and the
proteomes were resolved and visualized by SDS-PAGE analysis using standard gel-
based ABPP protocols.35,25 The gel-based ABPP experiment showed robust dose
dependent labelling of the S. aureus soluble protein lysate following P1 treatment, but
very minimal labelling with P2 in comparison (Figure 4B). Fractionated membrane
lysates also suggested that P1 has robust protein reactivity, in comparison to P2 (See
SI, Figure S13 a). Qualitatively, we found from these in vitro experiments, that P1 has
more reactivity in the soluble lysates as compared to the membrane proteome.
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Next, to determine if the site of labelling was indeed a protein cysteine residue, we
performed a competitive gel-based ABPP experiment, where fractionated S. aureus
cell lysates (soluble and membrane proteomes) were pre-treated with excess
iodoacetamide (IAM) (10 mM) and chased with P1 (10 M). We found that there was
near complete ablation of P1 protein labelling when lysates were pre-treated with
IAM, suggesting that P1 indeed, and as per literature precedence,36-37 reacted with
protein cysteine residues (Figure 4C). Further, to assess if P1 and P2 were active in situ,
S. aureus cultures were treated with P1 and P2 for 1 h (10 M), following which the
cells were lysed, lysate fractionated into membrane and soluble proteomes, and the
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extent of P1 and P2 protein reactivity in each fraction was analysed by gel-based ABPP,
following click reaction with rhodamine-azide. We found consistent result with the
previously described in vitro studies, that P1 was significantly more reactive than P2 in
S. aureus, and the extent of reactivity was even more pronounced in the soluble
proteome, suggesting that the potential biological target(s) might be soluble proteins
(Figure 4D, See SI, Figure S14 for all coomassie gels)
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Fig. 4 A) Structures of IND-QE alkyne probes P1 and P2 synthesized in this study; B) Dose-dependent
proteome profiling with P1 (0.01 - 100 M) and P2 (0.01 - 100 M) in soluble proteomic fraction of
S. aureus ATCC 29213; C) Proteome profiling with 10 M of P1 in lysates of S. aureus ATCC 29213
compared with Iodoacetamide (IAM) pretreated fractions; D) Proteome profiling with 10 M of P1
and P2 in situ for S. aureus 29213.
Given the exquisite selectivity of the lead compound 4a for S. aureus over other
ESKAPE pathogens, we tried to understand the basis of selectivity, and chose to
chemoproteomically profile P1 in E. coli. We found that in vitro P1 was fairly reactive
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against E. coli lysates, but had virtually no reactivity when P1 was used in an in situ
treatment paradigm in E. coli. (See SI, Figure S13 b). This result suggests that this class
of compounds might lack cell permeability against Gram-negative bacteria. Having
obtained significantly more labelling of soluble proteins over membrane proteins in
the in situ experiments, we hypothesized that the target(s) of interest would likely be
present in the soluble proteome. To find the biological targets of 4a in S. aureus, we
performed a competitive LC-MS/MS based ABPP experiment. In this experiment, we
incubated the S. aureus soluble proteome with DMSO or 4a (10 M, 1 h) or 11a (10
M, 1 h) or IAM (10 mM, 1 h), then chased these pre-treated proteomes with P1 (10
M, 1 h) and following P1 treatment performed click reaction using biotin-azide.
Subsequently, the biotinylated proteins were enriched using avidin-agarose beads.
Following enrichment, we performed on bead trypsin digestion using established
protocols.36-38 The tryptic peptides were subjected to LC-MS/MS analysis to identify
the respective proteins identified and enriched in each treatment group (See
Supporting Information, Proteomics dataset). We hypothesized that proteins absent
(or largely competed) in the 4a and IAM treatment groups (ratio relative to DMSO
group  0.1), but present comparable to control in the 11a treatment group (ratio
relative to DMSO group ~ 1), were likely targets of 4a. Using these filtering criteria, of
the 435 proteins identified by LC-MS/MS, we found only a handful of proteins (17
proteins), that were likely targets of 4a (Figure 5A). Interestingly, we found from this
proteomics experiment, that a significant number of these protein targets of 4a were
in fact transcriptional regulators (also known as virulence factors) (Figure 5B) that have
implications in conferring antibiotic resistance to microbes,26 and have to date been
thought as “undruggable”.
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Fig. 5 A) Targets identified for 4a using LC-MS/MS based proteomics experiment; Proteins that
exhibited ratios of < 0.1 for 4a, and IAM (red color), and ~1 for 11a (blue color) were considered targets
of 4a. B) A pie-chart categorizing the identified targets of 4a.
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Fig. 6. Dose-dependent labelling of targets MarR_12840 and MarR_05815 with varying concentrations
of probe P1, as compared with their alanine point variants; Crystal structures of identified targets with
Cys residues at Cys61 and Cys12 respectively (in black spheres).
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A bioinformatics survey of the amino acid sequences of the protein targets identified
for 4a, showed as predicted, that every protein target identified from the LC-MS/MS
proteomics experiment, had at least one conserved cysteine residue across ESKAPE
pathogens, reinforcing our hypothesis that 4a forms a covalent adduct with a protein
cysteine residue. We were particularly interested in the two protein targets from the
Multiple Antibiotic Resistance Regulon (MarR)39-41 family of transcriptional factors,
namely MarR_12840 and MarR_05815, that were identified as putative targets of 4a,
as previous studies have shown that inactivating mutations to either of these genes
results in increased antibiotic vulnerabilities in E. coli.27
Interestingly, both these MarR proteins have only one conserved cysteine residue,
Cys61 and Cys12 for MarR_12840 and MarR_05815 respectively. To validate whether
these two proteins are indeed targets of 4a, we cloned and purified these recombinant
C-terminal 6X-His-tag proteins from E. coli by Ni-NTA affinity chromatography. We also
made alanine point variants of Cys61 (C61A) and Cys12 (C12A) for MarR_12840 and
MarR_05815 respectively. We then treated the wild type (WT) and the variant proteins
with increasing dose of P1 (0 – 100 M, 1 h) following which we performed click-
reaction using rhodamine-azide, and visualized the activity of P1 labelling by SDS-
PAGE. We found for both proteins, MarR_12840 and MarR_05815, the WT variant
displayed a robust dose dependent labelling with P1, while the C61A and C12A variants
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of both MarR_12840 and MarR_05815 respectively showed negligible labelling for P1
(Figure 6). Of note, the three-dimensional structures are available for both
proteins,28,29 and the Cys61 in MarR_12840 and Cys12 in MarR_05815 are functionally
important residues responsible for transcriptional activation42 and sensing cellular
oxidative stress43 respectively (Figure 6).
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Taken together, we report a new cell active inhibitor class against S. aureus, and
leveraging chemoproteomics identify the biological targets for the lead compound 4a.
The lead compound is highly selective in modifying cysteine residues on proteins, and
inhibits extensively drug-resistant VRSA with a low MIC value. The most important
identified targets are transcriptional factors, which are responsible for conferring
virulence and antibiotic resistance in S. aureus. In a complementary approach,
previously, using Michael-acceptor-based ABPP, Sieber and co-workers have identified
cysteine residues of SarA, SarR and MgrA that bind to DNA as targets for inhibiting
virulence that reduces bacterial pathogenicity.13 The MarR family are novel druggable
targets in S. aureus and this is the first study, to the best of our knowledge, which
reports these two putative MarR proteins as important and novel targets. This study
creates novel paradigms and possibilities in combating antibiotic resistance and
provides new insights into possibly new drug targets.
As the golden era of antibiotics is nearing its end, finding new strategies to overcome
antibiotic resistance are in urgent need, as it is predicted that even routine infections
are likely to be untreatable by current antibiotics in the near future. This has
underscored the importance of finding new druggable targets in highly prevalent
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pathogens such as S. aureus and VRSA. Our study reveals the importance of covalently
modifying cysteine residues of transcription factors, which may have a profound
impact on survival of highly drug-resistant S. aureus. Although covalent modification
of cysteine residues10, 44 is frequently associated with idiosyncratic toxicities, it
presents an attractive alternative to currently used drug design strategies.19, 45 In this
study, cytotoxicity assessment of lead compound 4a against mammalian cell line VERO
cells showed a CC50 > 25 gmL, suggesting higher selectivity index of > 200 (the
selectivity index of 10a was also > 200; see SI, Table S6). Additionally, we found no
evidence for hemolysis at elevated concentrations suggesting that this compound did
not adversely affect human erythrocytes (See SI, Figure S15).
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Conclusions
Projecting ahead, we present a small molecular scaffold that lays the foundation for
new approaches to address the problem of AMR. It is likely that structural optimization
of this scaffold will be necessary for further development and in vivo efficacy. It has
been previously demonstrated that modification of cysteines in transcription factors
reduces DNA binding ability13 and hence, screening for MarR inhibitors may hold
promise for identification of new antibacterial candidates. However, the inhibitory
effects reported in this study might likely be due to the pharmacological disruption of
more than one transcriptional factor(s), and the polypharmacological effects of 4a will
need to be assessed by complementary genetic experiments (Figure 5B). While it may
prove challenging to assess the exact extent of polypharmacological effect from a
medicinal chemistry perspective, the advantages of multiple target engagement may
contribute to positive attributes of 4a. It would be extremely challenging for S. aureus
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to simultaneously generate multiple mutations in the different protein targets to
develop resistance to 4a. With IND-QE, the structural features allowing for tunability
of reactivity with cysteines coupled with excellent S. aureus cell permeability is a major
advancement that can be leveraged to profile thiols in a variety of pathophysiologies.
However, the diminished permeability of IND-QE in Gram-negative bacteria appears to
be a significant impediment to profiling in such pathogens. This will be addressed in
future studies by structural modifications that enhance permeability of the present
scaffold.
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Experimental Procedures (Chemistry)
All reactions were performed under nitrogen atmosphere. All the chemicals were
purchased from Aldrich, Acros, TCI, Spectrochem, Rankem, Alfa Aesar and used as
received unless stated otherwise. Dichloromethane (DCM), ethanol, N, N-
dimethylformamide (DMF), toluene and tetrahydrofuran (THF) for reaction were used
as received, and petroleum ether and ethyl acetate (EtOAc) for chromatography were
distilled before use. Column chromatography was performed on Merck silica gel (60–
120 mesh). 1H and 13C spectra were recorded on JEOL or BRUKER 400 MHz (or 100
MHz for 13C) spectrometers using residual solvent signals as an internal standard
(CHCl3 δH, 7.26 ppm, δC 77.2 ppm) or tetramethylsilane (δH = 0.00, δC = 0.0). Chemical
shifts (δ) are reported in ppm and coupling constants (J) in Hz. The following
abbreviations are used: br (broad), m (multiplet), s (singlet), d (doublet), t (triplet), q
(quartet), h (heptet), ddd (doublet of doublet of doublet), dd (doublet of doublet), dt
(doublet of triplet), dq (doublet of quartet), td (triplet of doublet), ABq (AB quartet).
High-resolution mass spectra were obtained from HRMS-ESI-Q-Time of Flight LC/MS.
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FT-IR spectra were recorded using NICOLET 6700 FT-IR spectrometer as KBr disc for
some compounds and using BRUKER ALPHA FT-IR spectrometer for other samples,
reported in cm-1. Melting point was measured using a VEEGO melting point apparatus.
All melting points were measured in open glass capillary and values are uncorrected.
High performance liquid chromatography (HPLC) was performed on an Agilent model
with Zorbax SB C-18 reversed phase column (250 mm × 4.6 mm, 5 μm) and with
Phenomenex®C-18 reversed phase column (250 mm × 4.6 mm, 5 µm). Fluorimetric and
luminometric measurements were performed using a Thermo Scientific Varioscan
microtiter plate reader. All compounds reported were found to be ≥95% pure, as
determined by elemental analysis and/or HPLC analysis. HPLC traces for key
compounds are included in the Supporting Information.
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(1) General procedure for Diels-Alder reaction:46 To a solution of the Wittig adduct in
ethanol (10 mL), benzoquinone or arylated benzoquinone was added and the reaction
mixture was stirred at RT in an open round bottom flask. Upon complete consumption
of the starting material (TLC analysis), ethanol was evaporated from the reaction
mixture under reduced pressure. Silica gel chromatography of the crude using a
mixture of ethyl acetate and hexane (1:5, v/v) as an eluent gave the pure product. This
procedure was followed for the synthesis of compounds 1b - 15b, Q1 - Q2.
(2) General procedure for epoxidation:18 To a solution of the quinone (compounds 1b
- 15b, Q1 - Q2) in THF, sodium hypochlorite (9-12 wt.% in water, excess) was added
and the reaction mixture was stirred at RT in an open round bottom flask. Upon
complete consumption of the starting material (TLC analysis), the reaction mixture was
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diluted with water (10 mL), and extracted with multiple portions of ethyl acetate (5 ×
5 mL). The combined organic phase was washed with brine (10 mL), dried over
anhydrous Na2SO4 (5 g), filtered and the filtrate was evaporated to dryness to get the
crude product. Silica gel chromatography of the crude using a mixture of ethyl acetate
and hexane (1:5, v/v) as an eluent gave the pure product. This procedure was followed
for the synthesis of compounds 1a - 15a, P1 - P2.
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(3) Preparation and characterization of compounds:
1aH-oxireno[2',3':4,5]benzo[1,2-a]carbazole-2,10(9H,10aH)-dione (1a) - Starting
from 1b (70 mg, 0.28 mmol), 1a (70 mg, 94%) was isolated as an orange yellow
crystalline solid: mp 226 – 228 °C; FT-IR (νmax, cm-1): 3397, 2921, 1681, 1587, 1114; 1H
NMR (400 MHz, DMSO- d6): δ 11.97 (s, 1H), 8.58 (dd, J = 7.9, 1.6 Hz, 1H), 8.23 (d, J =
7.9 Hz, 1H), 7.74 (dd, J = 16.6, 8.2 Hz, 2H), 7.50 (t, J = 7.6 Hz, 1H), 7.25 (t, J = 7.5 Hz, 1H),
13
4.17 (dd, J = 10.4, 3.9 Hz, 2H); C NMR (100 MHz, DMSO- d6): δ 192.3, 191.6, 143.1,
137.1, 130.0, 129.1, 128.6, 127.2, 121.8, 121.2, 121.0, 117.3, 114.5, 113.4, 55.8, 55.7;
HRMS (ESI) for C16H9NO3 [M+H]+: Calculated: 264.0655, Found: 264.0660.
11H-Benzo[a]carbazole-1,4-dione (1b) - Starting from 3-vinyl-1H-indole (510 mg, 3.56
mmol), 1b was isolated as dark red-brown solid (560 mg, 64%): mp 251 – 253 °C; FT-IR
1
(νmax, cm-1): 1646, 1566, 1414, 1303, 1216, 1114, 1019; H NMR (400 MHz, CDCl3): δ
10.40 (br, 1H), 8.33 (d, J= 8.1 Hz, 1H), 8.19 (d, J = 7.8 Hz, 1H), 7.92 (d, J = 7.9 Hz, 1H),
13
7.52 – 7.54 (m, 2H), 7.28 – 7.32 (m, 1H), 6.93 (s, 2H); C NMR (100 MHz, CDCl3): δ
187.0, 185.8, 143.4, 139.4, 139.0, 137.0, 130.1, 129.3, 128.6, 126.5, 121.8, 121.3,
121.0, 119.9, 117.1, 113.4; HRMS (ESI) for C16H9NO2 [M+H]+: Calculated: 248.0712,
Found: 248.0711.
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9-Methyl-1aH-oxireno[2',3':4,5]benzo[1,2-a]carbazole-2,10(9H,10aH)-dione (2a) -
Starting from 2b (50 mg, 0.19 mmol), 2a (50 mg, 94%) was isolated as a yellow
crystalline solid: mp 177 – 179 °C; FT-IR (νmax, cm-1): 2924, 1683, 1471, 1288, 1025; 1H
NMR (400 MHz, CDCl3): δ 8.33 (d, J = 7.8 Hz, 1H), 8.10 (d, J = 7.8 Hz, 1H), 7.87 (d, J = 8.2
Hz, 1H), 7.60 (t, J = 7.8 Hz, 1H), 7.49 (d, J = 8.4 Hz, 1H), 7.32 (t, J = 7.5 Hz, 1H), 4.12 (s,
2H), 3.80 (s, 3H); 13C NMR (100 MHz, CDCl3): δ 192.1, 191.3, 144.6, 138.6, 131.0, 130.0,
128.6, 125.3, 121.6, 121.0, 120.9, 118.2, 110.1, 56.3, 54.9, 34.6; HRMS (ESI) for
C17H11NO3 [M+H]+: Calculated: 278.0812, Found: 278.0825.
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11-Methyl-1H-benzo[a]carbazole-1,4(11H)-dione (2b) - Starting from 1-methyl-3-
vinyl-1H-indole (1 g, 6.36 mmol), 2b was isolated as dark red-brown solid (220 mg,
14%): mp 184 – 186 °C; FT-IR (νmax, cm-1): 1652, 1611, 1466, 1290, 1217, 1122, 1081,
1024; 1H NMR (400 MHz, DMSO-d6): δ 8.55 (d, J = 8.0 Hz, 1H), 8.25 (d, J = 7.8 Hz, 1H),
7.84 (d, J = 8.0 Hz, 1H), 7.70 (d, J = 8.3 Hz, 1H), 7.56 – 7.60 (m, 1H), 7.30 (t, J = 7.3 Hz,
1H), 7.10 (d, J = 10.2 Hz, 1H), 7.00 (d, J = 10.3 Hz, 1H), 3.87 (s, 3H); 13C NMR (100 MHz,
DMSO-d6): δ 185.7, 185.6, 145.3, 141.0, 138.8, 137.0, 131.0, 130.7, 128.9, 125.7, 121.6,
121.4, 118.0, 111.4, 35.8; HRMS (ESI) for C17H11NO2 [M+H]+: Calculated: 262.0868,
Found: 262.0865.
9-Ethyl-1aH-oxireno[2',3':4,5]benzo[1,2-a]carbazole-2,10(9H,10aH)-dione (3a)
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Starting from 3b (50 mg, 0.18 mmol), 3a (51 mg, 96%) was isolated as a bright yellow
crystalline solid: mp 145 – 147 °C; FT-IR (νmax, cm-1): 3042, 2964, 1682, 1626, 1432,
1202, 1131, 1026; 1H NMR (400 MHz, CDCl3): δ 8.29-8.32 (m, 1H), 8.07 (d, J = 7.7 Hz,
1H), 7.83 (dd, J = 9.1, 2.5 Hz, 1H), 7.49-7.58 (m, 2H), 7.30 (t, J = 7.4 Hz, 1H), 4.36-4.51
(m, 2H), 4.10-4.14 (m, 2H), 1.28 (t, J = 7.1 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 192.5,
191.5, 143.6, 136.8, 131.3, 130.1, 128.5, 125.3, 122.0, 121.0, 120.9, 118.1, 117.6,
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110.3, 56.1, 54.7, 40.6, 13.1; Elemental analysis for C18H13NO3calcd. C, 74.22; H, 4.50;
N, 4.81. Found C 74.18; H, 4.33; N, 4.74; HRMS (ESI) for C18H13NO2 [M+H]+: Calculated:
292.0968, Found: 292.0983.
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11-Ethyl-1H-benzo[a]carbazole-1,4(11H)-dione (3b) - Starting from N-ethyl-3-
vinylindole (1 g, 5.83 mmol), 3b was isolated as dark red-brown solid (285 mg, 18%):
mp 138 – 140 °C; FT-IR (νmax, cm-1): 1740, 1643, 1512, 1443, 1375, 1221, 1038; 1H NMR
(400 MHz, CDCl3): δ 8.32 (d, J = 7.9 Hz, 1H), 8.07 (d, J = 7.7 Hz, 1H), 8.00 (d, J = 8.0 Hz,
1H), 7.51-7.58 (m, 2H), 7.28-7.32 (m, 1H), 6.92 (q, J = 14.0 Hz, 2H), 4.63 (q, J = 10.6 Hz,
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2H), 1.40 (t, J = 7.0 Hz, 3H); C NMR (100 MHz, CDCl3): δ 185.6, 183.4, 144.3, 140.2,
136.9, 131.4, 128.4, 125.1, 122.2, 121.0, 120.9, 118.6, 118.2, 110.8, 42.6, 14.4; HRMS
(ESI) for C18H13NO2 [M+H]+: Calculated: 276.1025, Found: 276.1033.
9-Isopropyl-1aH-oxireno[2',3':4,5]benzo[1,2-a]carbazole-2,10(9H,10aH)-dione (4a) -
Starting from 4b (70 mg, 0.242 mmol), 4a (74 mg, 96%) was isolated as a bright yellow
crystalline solid: mp 204 – 206 °C; FT-IR (νmax, cm-1): 2968, 1683, 1622, 1569, 1416,
1373, 1200, 1116, 1026; 1H NMR (400 MHz, CDCl3): δ 8.24 (d, J = 8.0 Hz, 1H), 8.06 (d, J
= 7.8 Hz, 1H), 7.83 (d, J = 7.8 Hz, 1H), 7.72 (d, J = 8.5 Hz, 1H), 7.50 (td, J = 8.2, 1.0 Hz,
1H), 7.26 (dd, J = 14.1, 7.6 Hz, 1H), 4.64-4.74 (m, 1H), 4.10 (s, 2H), 1.96 (d, J = 6.8 Hz,
3H), 1.39 (d, J = 6.9 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 193.3, 191.1, 142.1, 138.6,
131.0, 130.1, 127.8, 125.0, 123.4, 121.2, 120.7, 118.4, 117.8, 113.9, 56.5, 54.6, 51.3,
20.7, 20.5; HRMS (ESI) for C19H15NO3 [M+H]+: Calculated: 306.1125, Found: 306.1126.
11-Isopropyl-1H-benzo[a]carbazole-1, 4-(11H)-dione (4b) - Starting from N-isopropyl-
3-vinylindole (765 mg, 4.13 mmol), 4b was isolated as dark red-brown solid (280 mg,
23%): mp 138 – 140 °C; FT-IR (νmax, cm-1): 1740, 1643, 1512, 1443, 1375, 1038;1H NMR
(400 MHz, CDCl3): δ 8.32 (d, J = 7.8 Hz, 1H), 8.05 (d, J = 7.7 Hz, 1H), 7.96 (d, J = 7.8 Hz,
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1H), 7.71 (d, J = 8.4 Hz, 1H), 7.44-7.48 (m, 1H), 7.25 (t, J = 7.4 Hz, 1H), 6.92 (d, 2H), 4.85-
4.96 (m, 1H), 1.71 (d, J = 6.9 Hz, 6H); 13C NMR (100 MHz, CDCl3): δ 186.3, 185.8, 142.9,
140.0, 139.7, 136.8, 136.6, 131.5, 131.4, 127.6, 124.9, 123.7, 121.2, 120.8, 118.8,
118.4, 114.4, 52.3, 21.2; HRMS (ESI) for C19H15NO2 [M+H]+: Calculated: 290.1181,
Found: 290.1184.
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9-Phenyl-1aH-oxireno[2',3':4,5]benzo[1,2-a]carbazole-2,10(9H,10aH)-dione (5a) -
Starting from 5b (37 mg, 0.11 mmol), 5a (32 mg, 82%) was isolated as a bright yellow
crystalline solid: mp 187 – 189 °C; FT-IR (νmax): 2935, 1687, 1457, 1283, 1206, 1110; 1H
NMR (400 MHz, CDCl3): δ 8.35 (d, J = 7.9 Hz, 1H), 8.12 (d, J = 7.8 Hz, 1H), 7.90 (d, J = 7.9
Hz, 1H), 7.15 (s, 2H), 7.42-7.49 (m, 2H), 7.31-7.36 (m, 2H), 6.95 (s, 1H), 3.99 (d, J = 4.2
Hz, 1H), 3.76 (d, J = 4.2 Hz, 1H); 13C NMR (100 MHz, CDCl3): δ 191.2, 190.4, 144.8, 139.5,
138.2, 131.3, 129.9, 128.6, 128.1, 127.4, 125.2, 125.1, 121.9, 121.7, 121.0, 118.9,
117.6, 111.2, 55.9, 54.2; HRMS (ESI) for C22H13NO2 [M+H]+: Calculated: 340.0968,
Found: 340.0974.
11-Phenyl-1H-benzo[a]carbazole-1,4(11H)-dione (5b). Starting from N-phenyl-3-
vinylindole (160 mg, 0.73 mmol), 5b was isolated as dark red-brown solid (50 mg, 21%):
mp 218 – 220 °C; FT-IR (νmax): 1655, 1611, 1463, 1412, 1289; 1H NMR (400 MHz, CDCl3):
δ 8.42 (d, J = 7.7 Hz, 1H), 8.14 (d, J = 7.8 Hz, 1H), 8.10 (d, J = 7.6 Hz, 1H), 7.54 (t, J = 7.5
Hz, 2H), 7.45 (t, J = 7.5 Hz, 2H), 7.26 - 7.36 (m, 4H), 6.84 (d, J = 10.0 Hz, 1H), 6.67 (d, J =
13
10.0 Hz, 1H); C NMR (100 MHz, CDCl3): δ 185.6, 184.1, 145.7, 141.1, 139.6, 138.3,
137.1, 136.7, 132.0, 131.2, 129.3, 128.6, 127.7, 126.5, 125.2, 122.0, 121.8, 120.8,
119.4, 117.9, 111.9; HRMS (ESI) for C22H13NO2 [M+H]+: Calculated: 324.1025, Found:
324.1016.
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3-Methyl-1aH-oxireno[2',3':4,5]benzo[1,2-a]carbazole-2,10(9H,10aH)-dione (6a) -
Starting from 6b (50 mg, 0.19 mmol), 6a (45 mg, 85%) was isolated as an orange red
crystalline solid: mp 224 -226 °C; FT-IR (νmax, cm-1): 2252, 1740, 1643, 1512, 1443, 1375,
1038; 1H NMR (400 MHz, CDCl3): δ 10.16 (s, 1H), 8.15 (s, 1H), 8.05 (d, J = 7.8 Hz, 1H),
7.47 – 7.52 (m, 2H), 7.29 (td, J = 5.8 Hz, 2.2 Hz, 1H), 4.03 (q, J = 9.5 Hz, 2H), 2.74 (s, 3H);
13C NMR (100 MHz, CDCl3): δ 193.4, 193.2, 142.1, 137.4, 131.5, 130.2, 129.2, 128.6,
121.4, 121.0, 111.8, 55.7, 55.5, 22.0; HRMS (ESI) for C17H11NO3 [M+H]+: Calculated:
278.0817, Found: 278.0813.
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11-Ethyl-5-methyl-2,3-dihydro-1H-benzo[a]carbazole-1,4(11H)-dione (6b) - Starting
from 3-(1-propenyl) -indole (1 g, 6.36 mmol), 6b was isolated as dark red-brown solid
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(235 mg, 14%): mp 224 – 226 °C; FT-IR (νmax, cm-1): 1739, 1646, 1367, 1224; H NMR
(400 MHz, CDCl3): δ 10.46 (s, 1H), 8.05 (s, 1H), 8.01 (d, J = 8.5 Hz, 1H), 7.48 (dd, J = 1.2,
6.2 Hz, 2H), 7.23 – 7.27 (m, 1H), 6.82 (d, J = 1.9 Hz, 2H), 2.80 (s, 3H); 13C NMR (100 MHz,
CDCl3): δ 188.1, 187.1, 142.5, 140.5, 137.5, 136.9, 132.6, 129.6, 128.5, 126.3, 121.5,
121.2, 120.8, 115.2, 111.7, 23.5; HRMS (ESI) for C17H11NO2 [M+H]+: Calculated:
262.0868, Found: 262.0863.
9-Ethyl-3-methyl-1aH-oxireno[2',3':4,5]benzo[1,2-a]carbazole-2,10(9H,10aH)-dione
(7a) - Starting from 7b (45 mg, 0.15 mmol), 7a (37 mg, 71%) was isolated as a yellow
solid: mp 159 – 161 °C; FT-IR (νmax, cm-1): 1680, 1427, 1258, 1199, 1124; 1H NMR (400
MHz, CDCl3): δ 8.17 (s, 1H), 8.08 (d, J = 7.8 Hz, 1H), 7.57 (t, J = 7.6 Hz, 1H), 7.50 (d, J =
8.3 Hz, 1H), 7.29 (t, J = 7.4 Hz, 1H), 4.40 (q, J = 14.2 Hz, 2H), 4.17 (d, J = 4.6 Hz, 1H), 4.13
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(d, J = 4.5 Hz, 1H), 2.73 (s, 3H), 1.24 (t, J = 7.1 Hz, 3H); C NMR (100 MHz, CDCl3): δ
193.7, 193.4, 143.9, 135.5, 130.5, 130.3, 128.4, 127.8, 121.7, 121.0, 120.6, 118.4,
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110.1, 56.3, 54.6, 40.1, 21.9, 12.8; HRMS (ESI) for C19H15NO3 [M+H]+: Calculated:
306.1125, Found: 306.1133.
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11-Ethyl-5-methyl-1H-benzo[a]carbazole-1,4(11H)-dione (7b) - Starting from N-ethyl-
3-(1-propenyl) -indole (350 mg, 1.88 mmol), 7b was isolated as dark red-brown solid
(70 mg, 13%): mp 132 – 134 °C; FT-IR (νmax, cm-1): 1740, 1645, 1512, 1443, 1375, 1221,
1038; 1H NMR (400 MHz, CDCl3): δ 8.13 (d, J = 3.3 Hz, 1H), 8.05 (d, J = 7.7 Hz, 1H), 7.50-
7.57 (m, 2H), 7.26-7.30 (m, 1H), 6.91 (dd, J = 10.1,1.3 Hz, 1H), 6.84 (dd, J = 10.1 ,1.3,
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1H), 4.51 (dq, J = 7.1, 1.7 Hz, 2H), 2.83 (s, 3H), 1.29 (t, J = 7.1 Hz, 3H); C NMR (100
MHz, CDCl3): δ 187.7, 186.6, 145.1, 138.7, 138.0, 132.7, 131.1, 129.0, 128.4, 121.0,
120.8, 110.8, 100.0, 42.1, 23.8, 13.7; HRMS (ESI) for C19H15NO2 [M+H]+: Calculated:
290.1181, Found: 290.1176.
9-Isopropyl-3-methyl-1aH-oxireno[2',3':4,5]benzo[1,2-a]carbazole-2,10(9H,10aH)-
dione (8a) - Starting from 8b (crude, 55 mg, 0.18 mmol), 8a (48 mg, 84%) was isolated
as a yellow solid: mp 177 – 179 °C; FT-IR (νmax, cm-1): 1680, 1264, 1202, 1192, 1031; 1H
NMR (400 MHz, CDCl3): δ 8.10 (s, 1H), 8.06 (d, J = 7.8 Hz, 1H), 7.69 (d, J = 8.5 Hz, 1H),
7.49 (dt, J = 6.8, 0.9 Hz, 1H), 7.27 (d, J = 7.4 Hz, 1H), 4.55 – 4.66 (m, 1H), 4.12 (ABq, J =
9.5 Hz, 2H), 2.72 (s, 3H), 1.92 (d, J = 6.8 Hz, 3H), 1.38 (d, J = 7.0 Hz, 3H); 13C NMR (100
MHz, CDCl3): δ 194.9, 193.0, 142.5, 137.6, 130.9, 129.9, 128.1, 127.7, 123.1, 121.2,
120.4, 118.6, 113.8, 56.6, 54.5, 51.1, 22.1, 20.7, 20.3; Elemental analysis for
C20H17NO3calcd. C, 75.22; H, 5.37; N, 4.39. Found C, 75.55; H, 5.16; N, 4.03; HRMS (ESI)
for C20H17NO3 [M+H]+: Calculated: 320.1281, Found: 320.1286.
3-ethyl-9-isopropyl-1aH-oxireno[2',3':4,5]benzo[1,2-a]carbazole-2,10(9H,10aH)-
dione (9a) - Starting from 9b (250 mg, 0.79 mmol), 9a (200 mg, 76%) was isolated as a
yellow solid. FT-IR (νmax, cm-1): 1741, 1725, 1706, 1679, 1647, 1626, 1547, 1532, 1516,
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