Natural product leads for drug discovery: Isolation, synthesis and biological evaluation of 6-cyano-5-methoxyindolo[2,3-a]carbazole based ligands as antibacterial agents

Article abstract of DOI:10.1016/j.bmc.2009.08.061

Indolo[2,3-a]carbazole based inhibitors were synthesized from readily available indigo via a seven-step linear synthetic sequence with a moderate overall yield. The inhibitors were selectively and readily functionalized at the nitrogen on the indole porti

Full text of DOI:10.1016/j.bmc.2009.08.061

Bioorganic & Medicinal Chemistry 17 (2009) 7126–7130
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Natural product leads for drug discovery: Isolation, synthesis and biological
evaluation of 6-cyano-5-methoxyindolo[2,3-a]carbazole based
ligands as antibacterial agents
Songpo Guo a, , Suresh K. Tipparaju a, , Scott D. Pegan b, Baojie Wan c, Shunyan Mo a,b, Jimmy Orjala a,b
,
Andrew D. Mesecar a,b,c, Scott G. Franzblau c, Alan P. Kozikowski a,
*
a Drug Discovery Program, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 833 S. Wood Street, Chicago, USA
b Center for Pharmaceutical Biotechnology, University of Illinois at Chicago, 900 S. Ashland Avenue, Chicago, USA
c Institute for Tuberculosis, Research College of Pharmacy, University of Illinois at Chicago, 833 S. Wood Street, Chicago, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Indolo[2,3-a]carbazole based inhibitors were synthesized from readily available indigo via a seven-step
linear synthetic sequence with a moderate overall yield. The inhibitors were selectively and readily func-
tionalized at the nitrogen on the indole portion of the carbazole unit. The synthesized analogs displayed
moderate inhibitory activities toward Bacillus anthracis and Mycobacterium tuberculosis, indicating that
indolo[2,3-a]carbazoles could serve as promising leads in the development of new drugs to combat
anthrax and tuberculosis infections.
Received 5 August 2009
Revised 20 August 2009
Accepted 29 August 2009
Available online 4 September 2009
Keywords:
Carbazole
Anthrax
Published by Elsevier Ltd.
Tuberculosis
1. Introduction
(strain EX-5-1) and identified by Moore and co-workers in 1990.5
In their studies, compound 1b showed modest antiviral activity
The heterocyclic indolo[2,3-a]carbazole system is found in sev-
eral biologically active natural products which are available from a
variety of organisms. Examples of such natural products include
the protein kinase inhibitor staurosporine (STS) from the bacte-
rium Streptomyces staurosporeus,1 anti-cancer agents such as the
arcyriaflavins isolated from the slime mold Arcyria denudate,2 and
the topoisomerase I inhibitor rebeccamycin, which originates from
the bacterium Nocardia aerocoligenes (Figs. 1 and 2).3 Due to the
wide spectrum of biological activities exhibited by the naturally
occurring carbazoles, a variety of synthetic analogs based on the
indolo[2,3-a]carbazole scaffold have garnered considerable atten-
tion in the development of medicines for psoriasis, hypertension,
cancer, and HIV-infection.4 During a screening effort for antibacte-
rial agents from cyanobacteria, we identified a strong inhibitory
activity towards Bacillus anthracis (B. anthracis) for the extract of
Nostoc muscorum (UTEX 2301). Bioassay guided fractionation led
to the isolation and identification of 6-cyano-5-methoxyindo-
lo[2,3-a]carbazole (1a, Fig. 2) as the active principle.5 Compound
1a and 6-cyano-5-methoxy-12-methylindolo[2,3-a]carbazole (1b)
were first isolated from the blue-green alga Nostoc sphaericum
against herpes simplex virus type 2; in infected mink lung cells,
the virus titer is reduced 95% at 1 g/mL, and weak, non-selective
g/mL) against KB and LoVo human carcinoma
l
cytotoxicity (MIC 5
cell lines.5
l
As part of an ongoing, multi-faceted program aimed toward
developing novel antiinfective chemotherapeutics against poten-
H
H
N
N
O
O
O
O
R1
R2
N
N
N
N
H
H
H
Cl
Cl
O
OH
Arcyriaflavin A: R1 = R2 = H
HO
Arcyriaflavin B: R1 = 2-OH, R2 = H
Arcyriaflavin C: R1 = 2-OH, R2 = 10-OH
Arcyriaflavin D: R1 = 2-OH, R2 = 9-OH
OH
OMe
Rebeccamycin
* Corresponding author. Tel.: +1 312 996 7577; fax: +1 312 996 7107 (A.P.K.).
E-mail address: kozikowa@uic.edu (A.P. Kozikowski).
Figure 1. Indolo[2,3-a]carbazole based natural products: arcyriaflavins A–D and
These authors contributed equally to this work.
rebeccamycin.
0968-0896/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmc.2009.08.061

S. Guo et al. / Bioorg. Med. Chem. 17 (2009) 7126–7130
7127
Figure 2. An overlay of chemical structures of STS and indolo[2,3-a]carbazoles 1a and 1b.
tial bio-warfare agents6 and neglected diseases,7 we synthesized
and screened a set of compounds containing the indolo[2,3-a]car-
bazole system as a primary scaffold against B. anthracis and Myco-
bacterium tuberculosis (M. tuberculosis). Herein, we report the
synthesis and biological activity of some of these new indolo[2,3-
a]carbazoles.
We noted that the chemical scaffold of antiviral indolo[2,3-
a]carbazoles 1a and 1b was similar to the aglycone portion of the
protein kinase inhibitor STS (Fig. 2). It is well known that the sub-
stituents on the indole nitrogens play a significant role in deter-
mining the biological activity of the indolocarbazoles. Varying
indole portions of the carbazole scaffold. However, this method re-
lied heavily on a very substrate-selective Suzuki–Miyaura and
Stille cross-coupling reactions of the two indole units that require
elaborate synthesis themselves.
In our own efforts, we found that the syntheses described ear-
lier were not readily amenable to modifications that enabled an
easy build-up of a library of N-functionalized indolocarbazoles.
For example, our attempts to modify Somei’s synthesis by intro-
ducing a tert-butoxycarbonyl group as R1 at the stage of intermedi-
ate 5a failed (Scheme 1).
Alternatively, we were successful in functionalizing intermedi-
ate 5a with an easily-cleavable benzyl group as R1 and then selec-
tively modifying R2. The synthesis started from 3-acetoxy-2,20-bi-
1H-indole14 (3), readily made from indigo (2). Intermediate 3
was reacted with dichloroacetyl chloride in refluxing anhydrous
ethyl acetate to afford the 3-acetoxy-30-dichloroacetyl-2,20-biind-
olyl intermediate 4.12d,e Subsequent cyclization of 4 using aqueous
ammonia in MeOH/DMF at ambient temperature provided the tet-
rahydroindolo[2,3-a]carbazole 5a.12c Methylation of 5a with di-
methyl sulfate in the presence of K2CO3 as a base resulted in the
12-methyl intermediate 5b in quantitative yield, while benzylation
of 5a afforded 5c under similar conditions in 83% yield. Reductive
cyanation12c of 5a using only 5 equiv of NaCN instead of the
30 equiv reported in the literature12c gave 6a in 34% yield in com-
parison to the reported yield of 60%. However, using the lower
amounts of NaCN, the reductive cyanation of 5b and 5c took place
in higher yields of 60% and 90%, respectively. Subsequent methyl-
ation of intermediates 6a–c with diazomethane provided 1a–c in
excellent yields. The free indole nitrogen as well as the phenolic
hydroxyl group of intermediates 6b and 6c were methylated to
give the corresponding compounds 7a and 7b. Compound 7b was
subsequently treated with AlCl3 in anisole at 110 °C to remove
the N-benzyl group to form 8 in quantitative yield.15 The nitrile
group at the 6-position of the indolo[2,3-a]carbazole 1b was read-
ily hydrolyzed in the presence of hydrogen peroxide to give the
new carboxamide 9.
these substituents with various uncommon sugars via an a- or b-
N-glycosidic linkage was a key element for modulating their cyto-
toxicity, anti-cancer activity, and topoisomerase inhibition.8 For in-
stance, rebeccamycin and its analogs with different sugar moieties
were much more active than the corresponding aglycone.9
2. Chemistry
In view of the foregoing facts, we wished to evaluate the antibi-
otic activities of indolo[2,3-a]carbazoles (1a–c, 6a–c, 7a, 8 and 9)
with varying R1 and R2 substituents.
The advances in indolocarbazole chemistry were previously
summarized in a comprehensive review focusing on carbazole
and indolocarbazole synthesis.10 A survey of available literature
on the synthesis of indolocarbazoles indicated that these inhibitors
are generally synthesized through strategies involving either direct
formation of the indolocarbazole skeleton by indole ring synthesis,
elaboration of bisindole precursors by the construction of the cen-
tral carbocyclic ring, or through a combination of these two strat-
egies.11 The first synthesis of 1a and 1b was reported by Somei and
co-workers in 199712c and in the following years, they made exten-
sive efforts toward optimizing those protocols.12a,b In addition,
Snieckus and Cai reported an efficient route to indolo[2,3-a]carba-
zole 1b via a metalation-cross-coupling reaction sequence.13 This
protocol provided a useful way to modify the substituents on the

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S. Guo et al. / Bioorg. Med. Chem. 17 (2009) 7126–7130
O
OAc
OAc
H
H
H
N
N
N
a
b
N
N
N
H
H
H
O
O
4
CHCl
2
2
3
c
O
Cl
HO
NC
OH
NC
OMe
g
f
N
N
N
H
N
R1
N
H
N
R1
R1
6a R1 = H
6b R1 = Me
6c R1 = Bn
1a R1 = H
1b R1 = Me
1c R1 = Bn
OMe
5a R1 = H
d
e
5b R1 = Me
5c R1 = Bn
j
h
H2NOC
NC
OMe
N
H
N
Me
N
N
NC
N
OMe
R2 R1
7a R1 = Me, R2 = Me
7b R1 = Bn, R2 = Me
9
i
N
H
Me
8
Scheme 1. Synthesis of indolocarbazoles 1a–c, 6a–c, 7a, 8 and 9. Reagents and conditions: (a) Sn, AcOH, Ac2O, 64–66 °C; (b) Cl2CHCOCl, EtOAc, reflux; (c) aq 1.3% NH3, MeOH,
DMF, rt; (d) Me2SO4, K2CO3, DMF, rt; (e) BnBr, K2CO3, DMF, rt; (f) NaCN, DMF, H2O, 70 °C; (g) CH2N2, rt; (h) MeI, K2CO3, DMF, rt; (i) AlCl3, anisole, 110 °C, 2 h; (j) 35% H2O2, 3 N
NaOH, EtOH, 30 °C, 20 h.
of M. tuberculosis.16 The results are summarized in Table 1. While
3. Biological studies
there was no clear effect on the activity of indolocarbazoles stem-
ming from the presence or absence of an alkyl group R1 (for exam-
The synthesized derivatives were tested for their inhibitory
ple, compare 1a vs 1b against 6a vs 6c or 7a vs 8), compounds with
R3 = H clearly displayed an improvement in their inhibitory activi-
ties (compare 1b vs 6b and 1a vs 6a). Significantly, it was interest-
ing to note that the presence of an alkyl group as R2 was
detrimental to the activity of these indolocarbazoles (compare 1a
vs 8). Replacement of the cyano functionality in 1b with a carbox-
amide (compound 9) did not alter activity for growth inhibition of
M. tuberculosis. Interestingly, the protein kinase inhibitor STS dis-
played modest activity against M. tuberculosis, while it was inactive
against B. anthracis. The newly synthesized analogs 6a–c clearly
had improved activities against both the pathogens compared to
the known indolocarbazole 1b. Benzyl protected compound 6c
was equipotent to 6a in the inhibition of B. anthracis, while only
fourfold less potent than 6a in the inhibition of M. tuberculosis.
Similarly, 1c with a benzyl protected indole moiety displayed a
activity against the
DANR strain of B. anthracis and H37Rv strain
Table 1
Inhibition of B. anthracis and M. tuberculosis by compounds 1a–c, 6a–c, 7a, 8 and 9
R3O
R4
N
R1
N
R2
Compound R1
R2
R3
R4
MICa (B.
MICb (M.
anthracis) (
l
M) tuberculosis)
(lM)
1a
1b
1c
6a
6b
6c
H
H
H
H
H
H
H
Me CN
Me CN
Me CN
6.3
>128
>128
44.1
15.0
56.6
60.6
>128
>128
>128
14.7
—
Me
Bn
H
Me
Bn
>200
>200
1.6
6.3
1.6
moderate MIC (44.1 lM) against M. tuberculosis. Encouraged by
these preliminary results, we are currently screening more N-func-
tionalized indolo[2,3-a]carbazole derivatives to improve the activ-
ity of these promising leads.
H
H
H
CN
CN
CN
7a
Me Me Me CN
>200
>200
8
H
Me Me CN
4. Conclusions
9
Me
H
Me CONH2 NTg
STSc
Ciprod
RMPe
INHf
128
0.1
—
In conclusion, a variety of indolo[2,3-a]carbazoles were synthe-
sized from the commercially available and widely used indigo dye
in seven linear steps. The described synthetic strategy allows selec-
tive functionalization of either of the indole nitrogens and is ame-
0.1
—
0.6
a
b
c
d
e
f
MIC = minimum inhibitory concentration against
MIC against H37Rv M. tuberculosis.
Staurosporine.
Ciprofloxacin hydrochloride.
Rifampin.
Isoniazid.
DANR B. anthracis.
nable to ready generation of
a library of N-functionalized
indolo[2,3-a]carbazole derivatives. Some of the synthesized com-
pounds displayed reasonable activity against B. anthracis (com-
pounds 1a, 6a and 6c) and moderate activity against M.
tuberculosis (compound 6a). Further rounds of screening and
g
Not tested.

S. Guo et al. / Bioorg. Med. Chem. 17 (2009) 7126–7130
7129
optimization of these new indolo[2,3-a]carbazoles are currently in
progress.
pin and isoniazid as positive controls. Compound stock solutions
were prepared in DMSO at a concentration of 12.8 mM, and the fi-
nal test concentrations ranged from 128
dilutions of compounds were prepared in Middlebrook 7H12 med-
ium (7H9 broth containing 0.1% w/v casitone, 5.6 g/mL palmitic
acid, 5 mg/mL bovine serum albumin, 4 mg/mL catalase, filter-ster-
ilized) in a volume of 100 L in 96-well microplates (black view-
plates, manufacturer). M. tuberculosis H37Rv (ATCC#27294)
(100
L inoculum of 1 ꢀ 105 cfu/mL) was added, yielding a final
testing volume of 200 L. The plates were incubated aerobically
at 37 °C. On the 7 day of incubation 12.5 L of 20% Tween 80,
and 20 L of Alamar Blue (Trek Diagnostic, Westlake, Ohio) were
added to the test plate. After incubation at 37 °C for 16–24 h, fluo-
rescence of the wells was measured (ex 530, em 590 nm). The MICs
ware defined as the lowest concentration effecting a reduction in
fluorescence of P90% relative to the mean of replicate bacteria-
only controls.
lM to 0.5 lM. Two fold
5. Experimental
l
5.1. Biology methods
l
5.1.1. Isolation of 1a from Nostoc muscorum
l
Nostoc muscorum was acquired from the Culture Collection of
Algae at the University of Texas at Austin (UTEX 2301). The cyano-
bacterium was grown in a 2.8 L Fernbach flask containing 1 L of
inorganic media (Allen). Cultures were illuminated with fluores-
cent lamps at 1.93 klx with an 18/6 h light/dark cycle at 22 °C.
After 6–8 weeks, the biomass of cyanobacteria was harvested by
centrifugation and then freeze-dried. The freeze-dried biomass
from a total of 7 L of culture (2.09 g) was extracted by repeated
maceration with CH2Cl2:MeOH (1:1) to yield 246.8 mg of crude ex-
tract. The crude extract showed inhibitory activity towards B.
anthracis (MIC 7.5
a gradient with increasing amount of MeOH in CH2Cl2 to afford 16
fractions. Fraction 5 (MIC 0.94 g/mL) eluting with CH2Cl2:MeOH
60:1, was subjected to reversed-phase HPLC (Alltima C18, 10 m,
250 ꢀ 10 mm, 4 mL/min) with a solvent gradient of acetonitrile–
H2O (10:90) to acetonitrile–H2O (90:10) over 35 min to afford
one major compound (tR = 29.8 min, 13.3 mg). The structure was
identified by spectroscopic means (1D, 2D, NMR and MS) and the
data were found to be identical to those previously reported for
6-cyano-5-methoxy-indolo[2,3-a]carbazole.
l
l
l
5.2. Chemistry methods
lg/mL) and was fractionated on silica gel using
1H NMR and 13C NMR spectra were recorded on Bruker spec-
trometer with TMS as an internal standard. Standard abbreviation
indicating multiplicity was used as follows: s = singlet, d = doublet,
t = triplet, q = quadruplet, quin = quintuplet, m = multiplet and
br = broad. HRMS experiment was performed on Q–TOF–2TM
(Micromass). The progress of all reactions was monitored by TLC
on precoated silica gel plates (Merck Silica Gel 60 F254). Column
chromatography was performed using Merck silica gel (230–400
mesh). All reactions were performed under inert atmosphere of
nitrogen, unless otherwise mentioned. All spectral data of know
compounds are identical to those reported.5,12
l
l
5.1.2. MIC against the
D
ANR strain of B. anthracis
ANR (plasmid-
The test compound MIC values against the
D
cured Ames Strain) of B. anthracis were determined using an auto-
mated Microplate-based Alamar Blue assay. Assays were carried
out using a TECAN Freedom Evo 200 liquid-handling robotics plat-
form equipped with a TeMO96 multichannel pipette and TECAN
5.2.1. Cis-6-chloro-6a-hydroxy-5-oxo-12-benzyl-5,6,6s,12-
tetrahydroindolo[2,3-a]carbazole 5c from 5a
K2CO3 (2500 mg, 18.1 mmol) and benzyl bromide (5500 mg,
32.2 mmol) were added to a solution of 5a (1000 mg, 3.23 mmol)
in DMF (30 mL), and the mixture was stirred at room temperature
for 2 h. After addition of the H2O under ice water bath, the whole
solution was extracted with EtOAc. The extract was washed by
brine, dried over Na2SO4, and evaporated under reduced pressure
to leave a residue, which was column-chromatographed on SiO2
with EtOAc–hexane (1:2, v/v) to give 5c (1.1 g, 83%). 1H NMR
(DMSO-d6, 400 MHz) d (ppm): 8.20 (d, J = 7.6 Hz, 1H), 7.87 (d,
J = 7.6 Hz, 1H), 7.77 (d, J = 7.6 Hz, 1H), 7.66 (d, J = 8.0 Hz, 1H),
7.54 (t, J = 7.6 Hz, 1H), 7.50–7.35 (m, 5H), 7.35–7.15 (m, 3H), 6.96
(s, 1H), 6.19 (d, J = 15.6 Hz, 1H), 6.00 (d, J = 15.6 Hz, 1H), 5.45 (s,
1H); 13C NMR (DMSO-d6, 100 MHz) d (ppm): 185.1, 169.4, 154.9,
139.2, 138.9, 136.7, 136.5, 130.6, 128.8, 127.8, 127.4, 126.9,
126.3, 125.4, 124.3, 123.8, 122.2, 121.6, 115.5, 112.3, 87.5, 69.8,
47.8; HRMS (ESI) calculated for C25H17ClN2O2 [M+H]+ 413.1051,
found 413.1073.
GENios Pro spectrophotometer. Using the TeMO96, 50 lL of Cat-
ion-Adjusted Mueller-Hinton Broth (CAHB) was added to each well
of a sterile, Falcon MICROTEST 96-well flat bottom tissue culture
plate. A serial dilution of the desired inhibitors was created by add-
ing 46
quid-handling arm (LiHa), to each well of the first column which
also contained 4 L of inhibitor at 10 mM. Upon mixing, 50 L of
lL of the CAHB, using the Freedom Evo 200’s 8 channel li-
l
l
the first wells was transferred to the next column and mixed. This
routine was repeated ten more times to generate the desired gra-
dient. Sample and positive control wells were inoculated using
the TeMo96 with 50
lL of a diluted B. anthracis DANR bacterial cul-
ture, whereas 50 L of CAHB containing no bacteria was added to
l
the negative control wells. Stock bacterial cultures were grown
to mid-log phase (OD600 = 0.4–0.6), and diluted to the desired
OD600 of 0.004 using fresh CAHB. After the final inoculation with
the bacterial culture, the microplates were incubated at 37 °C over-
night (ꢁ14 h) without agitation. After incubation, 5
lL of alamar
5.2.2. 6-Cyano-5-hydroxy-12-benzylindolo[2,3-a]carbazole (6c)
from 5c
blue dye (Serotec) was added to each well using the TeMo96. Plates
were agitated for 15 min at 900 rpm on a TECAN TeShake, and the
absorbance values at wavelengths of 570 nm and 600 nm were
then measured using the TECAN GENios Pro. Individual wells were
quantitatively assessed using the difference between the absor-
bance values at 600 nm and 570 nm. MICs for each compound
and known antibiotic were assigned based on the first well that
exhibited a negative absorbance difference, representative of no
bacterial growth.
NaCN (654 mg, 13.4 mmol) was added to a solution of 5c
(1100 mg, 2.7 mmol) in DMF (40 mL) and H2O (20 mL), and the
solution was stirred at 70 °C for 30 min. After addition of H2O,
the whole solution was extracted by EtOAc. The extract was
washed by brine, dried over Na2SO4, and evaporated under reduced
pressure to leave a residue, which was column-chromatographed
on SiO2 with EtOAc–hexane (1:2, v/v) to give 6c (947 mg, 90%).
1H NMR (DMSO-d6, 400 MHz) d (ppm): 11.74 (s, 1H), 10.83 (s,
1H), 8.48 (d, J = 7.6 Hz, 1H), 8.41 (d, J = 8.0 Hz, 1H), 7.75–7.60 (m,
2H), 7.55–7.38 (m, 2H), 7.38–7.05 (m, 7H), 6.14 (s, 2H); 13C NMR
(DMSO-d6, 100 MHz) d (ppm): 152.5, 140.4, 139.5, 137.9, 130.7,
128.8, 127.5, 126.7, 125.8, 125.2, 122.8, 122.3, 121.1, 120.8,
5.1.3. MIC against H37Rv strain of M. tuberculosis
The compound MICs against replicating M. tuberculosis were as-
sessed by the Microplate Alamar Blue assay16 (MABA) using rifam-

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S. Guo et al. / Bioorg. Med. Chem. 17 (2009) 7126–7130
119.7, 119.6, 119.5, 119.4, 118.5, 112.3, 111.4, 110.3, 82.2, 47.5;
HRMS (ESI) calculated for C26H17N3O [M+H]+ 388.1444, found
388.1461.
5.2.6. 5-Methoxy-12-methylindolo[2,3-a]carbazole-6-
carboxylic acid amide (9)
A mixture of the nitrile 1b (44 mg, 0.14 mmol), 35% hydrogen
peroxide (0.1 mL) and 3 N aqueous NaOH (0.03 mL) in ethanol
(2 mL) was stirred at 30 °C for 20 h. The resulting mixture was
acidified with 1 N sulphuric acid, the precipitate was filtered, dried,
which was column-chromatographed on SiO2 with MeOH–CH2Cl2
(1:19, v/v) to afford the compound 9 (33 mg, 70%). 1H NMR (ace-
tone-d6, 400 MHz) d (ppm): 11.10 (br s, 1H), 8.62 (d, J = 8.0 Hz,
1H), 8.35 (d, J = 7.8 Hz, 1H), 7.71 (t, J = 8.2 Hz, 2H), 7.57 (t,
J = 7.2 Hz, 1H), 7.51 (t, J = 7.4 Hz, 1H), 7.37 (q, J = 7.8 Hz, 2H), 4.45
(s, 3H), 4.32 (s, 3H); 13C NMR (acetone-d6, 100 MHz) d (ppm):
170.1, 154.6, 140.3, 140.2, 125.7, 125.3, 121.7, 121.4, 121.2,
120.3, 119.4, 116.9, 112.7, 111.5, 109.0, 86.5, 61.2, 41.7; HRMS
(ESI) calculated for C21H17N3O2 [M+H]+ 344.1395, found 344.1399.
5.2.3. 6-Cyano-5-methoxy-11-methyl-12-methylindolo[2,3-
a]carbazole (7a) from 6b
K2CO3 (38 mg, 0.28 mmol) and MeI (197 mg, 1.4 mmol) were
added to a solution of 6b (43 mg, 0.14 mmol) in DMF (2 mL), and
the mixture was stirred at rt for 2 h. After addition of the H2O un-
der ice water bath, the whole solution was extracted with EtOAc.
The extract was washed by brine, dried over Na2SO4, and evapo-
rated under reduced pressure to leave a residue, which was col-
umn-chromatographed on SiO2 with EtOAc–hexane (1:2, v/v) to
give 7a (41 m g, 90%). 1H NMR (CDCl3, 400 MHz) d (ppm): 8.66
(d, J = 8.0 Hz, 1H), 8.31 (d, J = 7.6 Hz, 1H), 7.70–7.45 (m, 4H),
7.45–7.20 (m, 2H), 4.30 (s, 3H), 4.20 (s, 3H), 4.12 (s, 3H); 13C
NMR (CDCl3, 100 MHz) d (ppm): 154.8, 143.9, 142.3, 132.4, 126.3,
125.7, 125.3, 122.5, 122.0, 121.9, 121.0, 120.8, 120.2, 117.3,
115.2, 110.0, 109.5, 87.5, 61.7, 36.5, 36.0; HRMS (ESI) calculated
for C22H17N3O [M+H]+ 340.1444, found 340.1457.
Acknowledgments
This work was supported in part by DOD (W81XWH-07-1-
0445) and by NIH Grant (1R01GM075856).
5.2.4. 6-Cyano-5-methoxy-11-methyl-12-benzylindolo[2,3-
a]carbazole (7b) from 6c
References and notes
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Y.; Masuma, R. J. Antibiot. 1977, 30, 275.
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1980, 19, 459.
3. Bush, J. A.; Long, B. H.; Catino, J. J.; Bradner, W. T.; Tomita, K. J. Antibiot. 1987,
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1990, 43, 1236.
6. (a) Tipparaju, S. K.; Mulhearn, D. C.; Klein, G. M.; Chen, Y.; Tapadar, S.; Bishop,
M. H.; Yang, S.; Chen, J.; Ghassemi, M.; Santarsiero, B. D.; Cook, J. L.; Johlfs, M.;
Mesecar, A. D.; Johnson, M. E.; Kozikowski, A. P. Chem. Med. Chem. 2008, 3,
1250; (b) Tipparaju, S. K.; Joyasawal, S.; Forrester, S.; Mulhearn, D. C.; Pegan, S.;
Johnson, M. E.; Mesecar, A. D.; Kozikowski, A. P. Bioorg. Med. Chem. Lett. 2008,
18, 3565.
Synthesized by method above by using 6c as a starting material.
Yield = 95%. 1H NMR (CDCl3, 400 MHz) d (ppm): 8.70 (d, J = 8.0 Hz,
1H), 8.38 (d, J = 7.2 Hz, 1H), 7.60–7.10 (m, 11H), 5.75 (s, 2H), 4.35
(s, 3H), 3.80 (s, 3H); 13C NMR (CDCl3, 100 MHz) d (ppm): 154.8,
144.0, 142.3, 136.5, 132.5, 128.9, 128.2, 127.3, 126.4, 125.8,
125.4, 122.7, 122.4, 122.3, 122.1, 121.5, 120.8, 120.2, 177.2,
115.8, 110.8, 110.0, 88.0, 61.8, 51.8, 36.2; HRMS (ESI) calculated
for C28H21N3O [M+H]+ 416.1757, found 416.1768.
5.2.5. 6-Cyano-5-methoxy-12-methylindolo[2,3-a]carbazole (8)
from 7b
To a stirred suspension of AlCl3 (143 mg, 1.1 mmol) in anisole
(5 mL) in an ice bath was added a solution of 7b (80 mg, 0.2 mmol)
in anisole (5 mL). The mixture was stirred at 110 °C for 2 h. The
reaction mixture was poured into water (20 mL) and extracted
with EtOAc. The extracts were washed with 5% NaHCO3 (10 mL),
water (10 mL), and brine (10 mL) and dried over Na2SO4. The sol-
vent was evaporated, and the residue column-chromatographed
on SiO2 with EtOAc–hexane (1:2, v/v) to give 8 (62 mg, 100%). 1H
7. Tipparaju, S. K.; Joyasawal, S.; Pieroni, M.; Kaiser, M.; Brun, R.; Kozikowski, A. P.
J. Med. Chem. 2008, 51, 7344.
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Pharmacol. 1999, 55, 377.
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Wei, L.; Du, Y.; Sun, D.; Wang, P. G. J. Med. Chem. 2005, 48, 2600.
10. Knolker, H. J.; Reddy, K. R. Chem. Rev. 2002, 102, 4303.
11. Janosik, T.; Wahlstrom, N.; Bergman, J. Tetrahedron 2008, 64, 9159.
12. (a) Somei, M.; Yamada, F.; Suzuki, Y.; Ohmoto, S.; Hayashi, H. Heterocycles
2004, 64, 483; (b) Hayashi, H.; Suzuki, Y.; Somei, M. Heterocycles 1999, 51,
1233; (c) Hayashi, H.; Ohmoto, S.; Somei, M. Heterocycles 1997, 45, 1647; (d)
Somei, M.; Hayashi, H.; Ohmoto, S. Heterocycles 1997, 44, 169; (e) Somei, M.;
Hayashi, H.; Izumi, T.; Ohmoto, S. Heterocycles 1995, 41, 2161.
13. Cai, X.; Snieckus, V. Org. Lett. 2004, 6, 2293.
14. (a) Madelung, W.; Siegert, P. Ber. Dtsch. Chem. Ges. 1924, 57, 222; (b) Bergman,
J.; Eklund, N. Chemica Scripta 1982, 19, 193; (c) McGovern, P. E.; Michel, R. H.
Acc. Chem. Res. 1990, 23, 152.
15. Wada, Y.; Nagasaki, H.; Tokuda, M.; Orito, K. J. Org. Chem. 2007, 72, 2008.
16. Collins, L.; Franzblau, S. G. Antimicrob. Agents Chemother. 1997, 41, 1004.
NMR (DMSO-d6, 400 MHz)
d (ppm): 12.16 (s, 1H), 8.46 (d,
J = 7.6 Hz, 1H), 8.24 (d, J = 8.0 Hz, 1H), 7.90–7.70 (m, 2H), 7.63–
7.45 (m, 2H), 7.45–7.25 (m, 2H), 4.36 (s, 3H), 4.22 (s, 3H); 13C
NMR (DMSO-d6, 100 MHz) d (ppm): 154.4, 140.8, 139.8, 129.2,
126.1, 125.8, 123.9, 121.7, 121.0, 120.9, 120.8, 119.7, 119.6,
117.8, 117.7, 113.8, 112.1, 110.2, 86.1, 62.2, 31.9; HRMS (ESI) cal-
culated for C21H15N3O [M+H]+ 326.1288, found 326.1302.