TFAA/H3PO4 mediated unprecedented N-acylation of carbazoles leading to small molecules possessing anti-proliferative activities against cancer cells

Article abstract of DOI:10.1039/c4ob00686k

For the first time TFAA/H₃PO₄ has facilitated the direct and metal-free N-acylation of carbazoles leading to a number of N-acylated derivatives. Several of these compounds were found to be promising when tested for their anti-proliferative properties against oral cancer cell lines. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob00686k

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Biomolecular Chemistry
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TFAA/H3PO4 mediated unprecedented N-acylation
of carbazoles leading to small molecules
possessing anti-proliferative activities against
cancer cells†
Cite this: Org. Biomol. Chem., 2014,
12, 6080
Received 1st April 2014,
Accepted 1st July 2014
Sunder Kumar Kolli,a Bagineni Prasad,b P. Vijaya Babu,b Mohd Ashraf Ashfaq,b
DOI: 10.1039/c4ob00686k
www.rsc.org/obc
Nasreen Z. Ehtesham,b R. Ramesh Raju*a and Manojit Pal*b
For the first time TFAA/H3PO4 has facilitated the direct and metal-
free N-acylation of carbazoles leading to a number of N-acylated
derivatives. Several of these compounds were found to be promis-
ing when tested for their anti-proliferative properties against oral
cancer cell lines.
Fig. 1 Design of bioactive N-acyl carbazole (A) derived from B.
The new and effective yet simple strategies for the rapid access
to small molecules of potential pharmacological interest are of
great demand both in academic and industrial R&D. Motivated
by this fact we have developed a simple strategy for the N-acyla-
tion of carbazoles.
While N-acylation of indoles has been studied well, only a
few reports are available on the N-acylation of carbazoles. The
N-acyl carbazoles are generally prepared by the action of (i)
acyl halides and anhydrides on carbazoles, (ii) acyl halides on
potassium carbazole and (iii) acyl halides on carbazole mag-
nesium halides.22 These methods, however, have several draw-
backs such as moderate yields, longer reaction times, and the
use of volatile and environmentally harmful organic solvents.
Moreover, the use of a common acylating agent, i.e. moisture
sensitive acid chlorides, often causes environmental pollution
and their preparation typically involves the use of thionyl
chloride or oxalyl chloride. Thus, we were in need of an
alternative and practical method for the preparation of com-
pound A. The use of TFAA (trifluoroacetic anhydride)/H3PO4 as
an efficient catalyst system for C-acylation of various aromatic
compounds is reported in the literature.23 However, we have
observed that the reaction of carbazole (1) with various car-
boxylic acids (2) in the presence of TFAA and H3PO4 afforded
the corresponding N-acyl carbazoles (3 or A, path a) instead of
C-acylated derivatives (path b, Scheme 1). Herein we present
our preliminary findings on this mild, single-step and metal-
free method for the synthesis of 3 via an unusual C–N bond
forming reaction. A similar C–N bond formation under acidic
conditions is not common in the literature and to the best of
our knowledge the use of TFAA/H3PO4 for the N-acylation
purpose has not been explored earlier.
In addition to its natural occurrences in alkaloids isolated
from plants, the carbazole framework has been found to be an
integral part of many bioactive compounds and therefore (like
indole) is considered as a privileged scaffold in the area of new
drug discovery. For example, various carbazole derivatives have
shown a wide range of biological activities, e.g. antioxidant,1 anti-
diabetic,1,2
antimitotic,3
antimicrobial,4–6
anti-vascular,7
antitumor,8–10 anticancer,11–15 antipsychotic,16 and anticonvul-
sant17 activities. Carbazole derivatives are also found to be inhibi-
tors of cyclin dependent kinases (CDKs)18 and checkpoint kinase
(Chk 1)19 and promising peroxisome proliferation activated recep-
tors (PPAR α, β and γ).20 In view of the reported antitumor pro-
perties of condensed N-acylindoles21a the N-acyl carbazole
framework attracted our particular attention due to their common
structural features. We anticipated that small molecules based on
N-acyl carbazoles (A, Fig. 1) might show anticancer properties.
Our anticipation was further supported by an earlier report that
the carbazole derivative B (Fig. 1) was found to be cytotoxic when
tested against mouse mammary carcinoma FM3A cells.21b
aDepartment of Chemistry, Acharya Nagarjuna University, Nagarjuna Nagar,
Guntur 522510, A.P., India
bDr Reddy’s Institute of Life Sciences, University of Hyderabad Campus, Gachibowli,
Hyderabad 500 046, India. E-mail: manojitpal@rediffmail.com;
To establish the optimized reaction conditions we used the
reaction of 1 with benzoic acid (2a) as a model reaction that
was performed in the presence of TFAA (2.093 mol) and
varying amounts of 85% H3PO4 at room temperature (Table 1).
Tel: +91 40 6657 1500
†Electronic supplementary information (ESI) available: Experimental pro-
cedures, spectral data for all new compounds, copies of spectra, results of
in vitro and docking study. See DOI: 10.1039/c4ob00686k
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Scheme 1 TFAA/H3PO4-mediated reaction of carbazole (1) with
various carboxylic acids (2).
Fig. 2 Comparison of spectral data of 3a and regioisomeric 9H-carba-
zol-3-yl(phenyl)methanone.24
Initially, the reaction was performed using 2.0 equivalents of
2a and 0.1 equivalent of 85% H3PO4 (0.059 mmol) when a
mixture of products i.e. 3a and 4a was isolated after 3 h
(entry 1, Table 1). Based on spectral (NMR, IR, MS) data com-
pound 3a was identified as an N-acylated product (that con-
tains an amide bond) rather than a C-acylated one24 (Fig. 2).
Compound 4a was identified as a diacylated product i.e.
75% (entry 7, Table 1). However, in spite of several attempts we
failed to find conditions where 3a could be isolated as the only
product. We therefore used the conditions of entry 7 of Table 1
for our further studies. It is worth mentioning that the reac-
tion did not proceed in the absence of H3PO4, indicating its
key role in the present reaction. All these reactions were per-
formed at room temperature. While an increase in reaction
temperature enhanced the reaction rate but decreased the
yield of 3a. The reaction was found to proceed in the absence
of any additional organic solvent thereby decreasing the scope
of environmental pollution. The excess of TFAA used perhaps
played the role of a solvent in this reaction. After completion
of the reaction TFAA converted to TFA can be recovered by dis-
tillation over a dehydrating agent. Nevertheless, the present
reaction does not require the use of any inert or anhydrous
atmosphere indicating the scale-up potential of this method.
With the optimum reaction conditions in hand we then
examined the generality and scope of the present C–N bond
forming reaction. Thus a range of carboxylic acids (2) were
reacted with 1 under the conditions of entry 7 of Table 1 and
yields of the corresponding products are shown in Table 2. It
is evident from Table 2 that the acid 2 may contain a mono or
disubstituted benzene ring and the substituent(s) may be
present at the o-, m- or p-position or any two of these three
positions. Aliphatic carboxylic acids e.g. 2p and 2q also partici-
pated well in the present reaction (entries 16 and 17, Table 2).
However, the use of heteroaromatic acids e.g. thiophene-2-car-
boxylic acid was not successful as a mixture of unidentified
products was isolated in this case. The use of a substituted
carbazole e.g. 3-bromo-9H-carbazole 6 was examined and found
to be effective under the conditions employed (Scheme 2).
A plausible reaction mechanism for the present TFAA/
H3PO4-mediated N-acylation of carbazoles is presented in
Scheme 3.23e,25 The reaction seems to proceed via in situ gen-
eration of the acylation precursor acyl trifluoroacetate X from
TFAA and the carboxylic acid 2. As a covalent catalyst the phos-
phoric acid then converts X into the arylacetyl bis(trifluoroace-
tyl)phosphate Y which then participates in N-acylation of
carbazole 1. Since the reaction does not proceed in the
absence of H3PO4 and it is known that Y participates in a
faster acylation reaction than X25 the direct N-acylation of 1 by
X seems to be unlikely. Moreover, acylation by X could lead to
(9H-carbazole-3,9-diyl)bis(phenylmethanone).
We
were
delighted to observe the formation of 3a in this reaction that
was unprecedented in the literature under the conditions
employed. An increase in the equivalent of H3PO4 used
increased the yield of 3a when the reaction was performed for
1 h (entry 2, Table 1). An increase in the reaction time afforded
4a as the major product (entry 3, Table 1). Further increase of
H3PO4 equivalents afforded 3a as the major product when the
reaction was performed for 1 h (entry 4, Table 1). However,
increasing the reaction time (entry 5, Table 1) to 3 h afforded
4a as the major product. We then performed the reaction
using 1.0 equivalent of 2a and 0.1 equivalent of 85% H3PO4 for
1 h when 3a was isolated in 60% yield (entry 6, Table 1). An
increase in reaction time to 2 h increased the yield of 3a to
Table 1 The effect of conditions on the TFAA/H3PO4-mediated reac-
tion of 1 with 2aa
Yieldb (%)
Entry
H3PO4 (equiv.)
2a (equiv.)
Time (h)
3a
4a
1
0.1
0.5
0.5
1
1
0.1
0.1
2
2
2
2
2
1
1
3
1
3
1
3
1
2
42
61
31
65
26
60
75
45
15
65
18
68
8
2c
3
4c
5
6c
7
10
a All the reactions were carried out using carbazole 1 (0.598 mmol),
benzoic acid 2a, TFAA (2.093 mmol) and 85% H3PO4. b Isolated yield.
c The presence of unreacted 1 was observed in this case.
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Table 2 TFAA/H3PO4-mediated synthesis of N-acyl carbazole (3) (path a,
Scheme 1)a
Scheme 4 TFAA/H3PO4-mediated C-acylation of 3a.
Entry
Carboxylic acid 2; R
Product 3
Yieldb (%)
H3PO4 (Scheme 4). Further, the selective deprotection of the
N-benzoyl group of 4b performed under the reported condi-
tions26b to afford 5a24,26c indicated the potential of this meth-
odology for the generation of a compound library based on
C-acylated carbazoles (Scheme 5). Indeed, the TFAA/H3PO4-
mediated C-acylation also proceeded well when N-propargyl
carbazole (7) was used as a substrate (Scheme 6).
1
2
3
4
5
6
7
8
2a; Ph
2b; 4-ClC6H4
2c; 2-IC6H4
2d; 2-Cl-5-BrC6H3
2e; 2-BrC6H4
2f; 3-ClC6H4
2g; 2-ClC6H4
2h; 2-MeC6H4
2i; 4-MeC6H4
2j; 4-IC6H4
2k; 2,6-(di-Cl)C6H3
2l; 3,4-(di-Cl)C6H3
2m; 2,4-(di-Cl)C6H3
2n; 2,5-(di-Cl)C6H3
2o; 3,5-(di-Cl)C6H3
2p; Ph2CH
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
75
55
60
64
60
75
60
60
68
70
60
62
70
64
60
61
63
All the synthesized N-acyl carbazoles were tested against
cancer cell lines derived from tongue tissue e.g. CAL 27, breast
cancer cell line e.g. MDA-MB231 and non-cancer cells e.g. HEK
293 T [Human Embryonic Kidney 293 cells] in vitro at 10 μM
9
10
11
12
13
14
15
16
17
using the sulphorhodamine B (SRB) assay.27 Gemcitabine,28
a
well known anticancer agent, was used as a reference com-
2q; n-Pr
a All the reactions were carried out using carbazole 1 (0.598 mmol),
benzoic acid 2a (0.598 mmol), TFAA (2.093 mmol) and 85% H3PO4
(0.059 mmol). b Isolated yield.
Scheme 5 Deprotection of 4a/4b leading to the (9H-carbazol-3-yl)-
(phenyl/p-tolyl)methanone (5a/5b).
Scheme 2 TFAA/H3PO4-mediated reaction of 6 with 2a.
Scheme 6 TFAA/H3PO4-mediated reaction of 7 with 2a.
Table 3 Anti-proliferative activities of N-acyl carbazoles (3) against
Scheme 3 The proposed reaction mechanism for the TFAA/H3PO4-
cancer and normal cells
mediated N-acylation of carbazoles.
%inhibitiona
Compounds
CAL 27
MDA-MB231
HEK 293T
the trifluoroacetylated product of 1 (in addition to 3) that was
not detected in the reaction mixture. While the actual reason
for the preferential N-acylation of 1 is not clear at this stage,
the higher reactivity of the carbazole nitrogen over the ring
carbon towards Y could be the reason for this observation. In
fact the C-acylation of carbazoles was found to proceed
smoothly after N-acylation separately. Thus, the N-benzoyl
carbazole (3a) afforded the C-acylated product 4a26a or 4b
when treated with aromatic acids in the presence of TFAA and
3a
3b
3d
3g
3h
3i
>100
100.84
>100
>100
>100
103.27
93.18
>100
55.54
72.98
78.28
80.42
76.63
74.31
68.10
78.24
51.81
57.74
58.73
65.22
65.47
62.48
46.70
41.61
3l
3n
a Average of at least three determinations.
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pound in this assay. Several of these compounds e.g. 3a, 3b,
3d, 3g, 3h, 3i, 3l, and 3n showed promising growth inhibition
especially of CAL 27 cells (>90% inhibition) after 72 h of treat-
ment (Table 3).30a However, none of them showed notable
effects on normal HEK 293 T cells (highest inhibition being
≤65%) when tested at 10 μM. Similarly, based on their effects
on HEK 293 T cells, compounds 3b, 3d, 3g, 3l and 3n were
found to be active against MDA-MB231 cells (Table 3). Notably,
gemcitabine29 showed ∼100% growth inhibition of CAL 27
cells at 10 μM. In a dose response study compounds 3a, 3d, 3g,
3h and 3n showed consistent growth inhibition when tested
against CAL 27 cells (Fig. 3).30b The IC50 value of 3d and 3n
was found to be 0.028 0.002 and 0.45 0.03 µM, respectively.
Thus these compounds were identified as the best active mole-
cules in the present series. Nevertheless, since the death rate
for oral cancer is higher than that of other cancers globally31
the present class of compounds is of further interest.
In conclusion, TFAA/H3PO4 has been identified for the first
time as an efficient coupling agent for the direct N-acylation of
carbazoles. This metal-free methodology does not require the
use of expensive reagents or catalysts and inert or anhydrous
atmosphere and it works well at room temperature. A number
of N-acylated derivatives were prepared using this methodology
and tested for their anti-proliferative properties against oral
and breast cancer cell lines. Several of them were found to be
promising, indicating that the present series of N-acyl carba-
zoles represent a useful template for the discovery and deve-
lopment of potential anticancer agents.
BP thanks CSIR, for a research fellowship.
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