Transition metal free hydrolysis/cyclization strategy in a single pot: Synthesis of fused furo N-heterocycles of pharmacological interest

Article abstract of DOI:10.1039/c3ob41069b

A transition metal free tandem two-step strategy has been developed involving hydrolysis of 2-chloro-3-alkynyl quinoxalines/pyrazines followed by in situ cyclization of the corresponding 2-hydroxy-3-alkynyl intermediates in a single pot leading to fused furo N-heterocycles as potential inhibitors of sirtuins. A representative compound showed promising pharmacological properties in vitro and in vivo.

Full text of DOI:10.1039/c3ob41069b

Organic &
Biomolecular Chemistry
View Article Online
View Journal | View Issue
COMMUNICATION
Transition metal free hydrolysis/cyclization strategy
in a single pot: synthesis of fused furo N-heterocycles
of pharmacological interest†
Ali Nakhi,a Md. Shafiqur Rahman,a,b Guru Pavan Kumar Seerapu,a
Rakesh Kumar Banote,a,c Kummari Lalith Kumar,a Pushkar Kulkarni,a,c
Devyani Haldar*a and Manojit Pal*a
Cite this: Org. Biomol. Chem., 2013, 11,
4930
Received 23rd May 2013,
Accepted 17th June 2013
DOI: 10.1039/c3ob41069b
www.rsc.org/obc
A
transition metal free tandem two-step strategy has been
developed involving hydrolysis of 2-chloro-3-alkynyl quinoxa-
lines/pyrazines followed by in situ cyclization of the corres-
ponding 2-hydroxy-3-alkynyl intermediates in
a single pot
leading to fused furo N-heterocycles as potential inhibitors of
sirtuins. A representative compound showed promising pharma-
cological properties in vitro and in vivo.
The development of chemical approaches that focus on the
use of renewable sources and reduction of waste generation is
of prime interest due to the worldwide concern of environ-
mental safety and global warming. Thus the use of aqueous
media in organic synthesis has gained high popularity
as water is naturally abundant, safe, inexpensive and can be
recycled.1
Fig. 1 Design of B/C as novel inhibitors of sirtuins.
Over the years, functionalized fused N-heteroaromatics have
played key roles in the early stage of drug discovery and many
of them have been marketed as successful drugs,2 e.g.
blockbuster antibiotic levofloxacin. Fused furo N-heterocycles,
e.g. furoquinoxalines, on the other hand have not been
explored extensively as potential bioactive agents3a perhaps
due to their limited or cumbersome accessibility. However,
this class of heterocycles is attractive not only for chemical
biology/bioorganic chemistry efforts but also as transient inter-
mediates in organic synthesis.3b This and our interest in novel
heteroaromatics4 prompted us to explore a greener route to
furo[2,3-b]quinoxalines/furo[2,3-b]pyrazine (B, Fig. 1) as poten-
tial inhibitors of sirtuins. Since their over expression has been
Fig. 2 Binding mode of C in yeast Sir2 (PDB ID: 1Q1A).
implicated in several types of cancer, sirtuins (class III NAD-
dependent deacetylases) are considered as promising targets
for cancer therapeutics.5 Inhibition of sirtuins allows re-
expression of silenced tumor suppressor genes leading to the
reduced growth of cancer cells. Our earlier observation on
activities of pyrrolo[2,3-b]quinoxalines6 A against a panel of
cancer cell lines prompted us to design structurally similar B,
supported by the in silico binding studies of a representative
compound C (Fig. 1) in the catalytic pocket of yeast Sir2
(Fig. 2). The study (docking score −10.2) showed the position-
ing of the central ring and the side chain of C near adenine
aDr. Reddy’s Institute of Life Sciences, University of Hyderabad Campus, Gachibowli,
Hyderabad 500 046, India. E-mail: manojitpal@rediffmail.com;
Tel: +91 40 6657 1500
bChemical Synthesis and Process Technologies, Department of Chemistry,
University of Delhi, New Delhi 110 007, India
cZephase Therapeutics (an incubated company at the DRILS), University of
Hyderabad Campus, Gachibowli, Hyderabad 500046, India
†Electronic supplementary information (ESI) available: Experimental pro-
cedures, spectral data for all new compounds, and results of in vitro/in vivo and
docking studies. CCDC 935821. For ESI and crystallographic data in CIF or other
electronic format see DOI: 10.1039/c3ob41069b
4930 | Org. Biomol. Chem., 2013, 11, 4930–4934
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Communication
Table 2 Synthesis of 3-alkynyl-2-chloropyrazines 3o–sa
Scheme 1 Hydrolysis–cyclization strategy leading to furo derivatives 4.
Table 1 Synthesis of 3-alkynyl-2-chloroquinoxalines 3a–na
Entry
Alkyne (2); R =
T/h
Product (3)
Yieldb (%)
1
2
3
4
5
2a
2g
2h
2k
2
2
4
4
3
3o
3p
3q
3r
64
70
70
62
60c
2m; SiMe3
3s
a See footnote “a” of Table 1. b Isolated yield. c Pd(PPh3)2Cl2
(0.0125 mmol) was used in place of Pd/C and PPh3.
Halide
Product
(3)
Yieldb
(%)
Entry
(1); R1 =
Alkyne (2); R =
T/h
Table 3 Optimization of hydrolysis/cyclization of 3aa
1
2
3
4
5
6
7
8
1a; H
1a
1a
1a
1a
1a
1a
1a
1a
1a
1a
1a
1b; Me
2a; Ph
2
2
2
2
3
2
3
4
4
4
4
4
3
2
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
70
62
65
60
66
69
65
60
68
70
68
60
63
67
2b; C6H4p-Me
2c; (CH2)3Me
2d; (CH2)4Me
2e; (CH2)5Me
2f; (CH2)9Me
2g; CMe3
2h; CMe2OH
2i; CH2OH
2j; (CH2)2OH
2k; 1-hydroxycyclohexyl
2l; 2-pyridinyl
2a
Entry
Solvent
T/h
Yieldb (%)
9
10
11
12
13
14
1
2
3
4
5
DMSOc
4
3
1
4
8
45
62
78
53
28
DMSO–H2O (1 : 1)
DMSO–H2O (1 : 4)
DMSO–H2O (1 : 8)
H2O
1b
2f
a All reactions were performed by using 1 (1.256 mmol), terminal
alkyne 2 (1.256 mmol), 10% Pd/C (0.0125 mmol), PPh3 (0.0502 mmol), a Reactions were carried out using 3a (0.7575 mmol), K2CO3
CuI (0.0125 mmol), and Et3N (1.8844 mmol) in EtOH (4 mL). b Isolated (0.3787 mmol) in solvent (3 mL) at 80 °C. b Isolated yield.
yield.
c Commercially available DMSO was used directly without further
drying.
and nicotinamide binding site respectively, along with two crucial for the success of the reaction) compared to other
H-bond interactions of the quinoxaline nitrogens with the aqueous media including 1,4-dioxane–H2O.7b It was observed
backbone –NH of SER 12 and GLY 30 (see ESI†).
that only DMSO or water alone (entries 1 and 5, Table 3) was
The synthesis of furo[2,3-b]quinoxalines/pyrazines is rather less efficient whereas the best yield of 4a was achieved when
uncommon in the literature.3a,7 While a single compound 1 : 4 DMSO–H2O was used (entry 3 vs. 2 and 4, Table 3). More-
i.e. 2-phenylfuro[2,3-b]quinoxaline was prepared by treating over, we observed that the reaction was relatively faster in
2-chloro-3-phenylethynylquinoxaline with KOH in 1,4-dioxane- aqueous DMSO rather than in aqueous 1,4-dioxane (e.g. 1 vs.
H2O, other “furano-compounds” could not be prepared by 3 h). Thus the conditions in entry 3 (Table 3) were found to be
using this method.7b On the contrary, we have observed that optimum and used for our further studies.
furo[2,3-b]quinoxalines/furo[2,3-b]pyrazines can be prepared
We then examined the substrate scope and generality of
from 2-chloro-3-alkynyl quinoxalines/pyrazines in the presence this method (Table 4). The heteroaryl alkyne 3 containing con-
of K2CO3 in DMSO–H2O. Herein we report our preliminary trasting groups e.g. aryl (3a–b, 3m and 3o), heteroaryl (3l),
results on this hydrolysis–cyclization strategy leading to the alkyl (3c–g, 3p, 3g and 3n), hydroxyalkyl (3h–k, 3q and 3r) and
desired furo derivatives 4 (or B, Scheme 1).
trimethylsilyl (3s) substituents on the triple bond participated
The key starting material 3 required was prepared via well in the reaction. Both quinoxaline (3a–n) and pyrazine
a selective mono alkynylation of 2,3-dichloroquinoxaline/ (3o–s) derivatives showed similar reactivities affording the
pyrazine (1) under Pd–Cu catalysis (Tables 1 and 2).
desired products in acceptable yields. All the nineteen com-
Initially, the possibility of hydrolysis/cyclization of 2-chloro- pounds synthesized by this method were well characterized
3-(phenylethynyl)quinoxaline 3a leading to 4a was examined by spectral (NMR, IR and MS) data and the molecular structure
in the presence of K2CO3. After assessing a range of solvents of a representative compound 4a was further confirmed
an appropriate combination of DMSO–H2O was found to be unambiguously by single crystal X-ray diffraction (Fig. 3).8 The
effective, perhaps due to the better solubility of 3a (that was study indicated that the supramolecular interactions between
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem., 2013, 11, 4930–4934 | 4931

View Article Online
Communication
Organic & Biomolecular Chemistry
Table 4 Synthesis of furo[2,3-b]quinoxalines/pyrazines 4a
Yieldb (%)
Fig. 4 Supramolecular interactions and crystal packing of compound 4a.
Entry
Alkyne (3)
Product (4); R =
T/h
1
2
3
4
5
6
7
8
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
4a; Ph
1
1
6
4
2
6
3
4
3
3
3
2
1
5
1
2
3
3
4
78
73
71
68
71
62
76
66
62
64
68
60
73
68
76
69c
66
62
58
4b; C6H4p-Me
4c; (CH2)3Me
4d; (CH2)4Me
4e; (CH2)5Me
4f; (CH2)9Me
4g; CMe3
4h; CMe2OH
4i; CH2OH
4j; (CH2)2OH
4k; 1-hydroxycyclohexyl
4l; 2-pyridinyl
4m; Ph
4n; (CH2)9Me
4o; Ph
4p; CMe3
4q; CMe2OH
4r; 1-hydroxycyclohexyl
4s; SiMe3
Scheme 2 TFAA/H3PO4 mediated C-3 acylation of 4a.
9
10
11
12
13
14
15
16
17
18
19
3s
a Reactions were carried out using
3 (0.7575 mmol), K2CO3
(0.3787 mmol) in DMSO–H2O (1 : 4) (3 mL) at 80 °C. b Isolated yield.
c KOH was used in place of K2CO3.
Fig. 5 URA3 silencing assay of known inhibitor splitomicin and 4.
Fig. 3 ORTEP diagram of compound 4a (30% probability, hydrogen atoms and
another asymmetric molecule have been omitted for clarity).
nitrogen and hydrogen are within the range of 2.730–2.770 Å,
which resulted two types of interactions i.e. (N1⋯H15 =
2.734 Å, N2⋯H9 = 2.764 Å). These weak van der Waals inter-
actions resulted in a 1D crystal packing network, as shown in
Fig. 4.
It is evident that the present transition metal9 free hydro-
lysis–cyclization of alkyne 3 proceeds via a 2-hydroxy-3-alkynyl
quinoxaline/pyrazine intermediate (Scheme 1).10 To gain
further evidence, 3-(phenylethynyl)quinoxalin-2-ol (3aa) pre-
Fig. 6 Dose dependent URA3 silencing assay of 4e.
pared from 3a was isolated and treated under the conditions reporter silencing assay, see ESI†).11 As the Sir2 protein is
in entry 3 of Table 3, and 4a was isolated in good yield. The inhibited, the URA3 gene would be de-repressed, resulting in
scope of the present strategy was expanded further by TFAA/ the death of the yeast cell in the presence of 5-FOA. The com-
H3PO4 mediated C-3 benzoylation of 4a (Scheme 2).
pounds were also screened in parallel in the absence of 5-FOA
Most of the compounds 4 along with splitomicin5b were to check their cytotoxicity. In general, the furo[2,3-b]quinoxa-
tested at 50 µM for their ability to inhibit the Sir2 protein lines showed better activities than the furo[2,3-b]pyrazines.
[yeast sirtuin family NAD-dependent histone deacetylase Among the furo[2,3-b]quinoxalines, compounds having
a
(HDAC)] by estimating the inhibition of growth of a yeast linear alkyl side chain at C-2, e.g. 4c, 4d and 4e (or C of Fig. 1
strain containing URA3 gene at a telomeric locus, in the pres- and 2), showed significant inhibition (55, 56 and 65%,
ence of 5-fluoroorotic acid (5-FOA) (the yeast cell based respectively) in the presence of 5-FOA (Fig. 5) and no
4932 | Org. Biomol. Chem., 2013, 11, 4930–4934
This journal is © The Royal Society of Chemistry 2013

View Article Online
Organic & Biomolecular Chemistry
Communication
Fig. 7 Dose dependent mammalian SIRT1 assay of 4e.
Fig. 9 Teratogenicity assay: mean ( S.D.) lesion score of all parameters
different treatment groups. (*p < 0.05, **p < 0.01 and ***p < 0.001). Statistical
significance was analyzed as control group vs. all groups.
Table 5 Results of zebrafish embryo toxicity studies of 4c, 4d and 4e
4c
4d
4e
Statistically significant toxic concentration (µM)
No observed adverse effect level (NOAEL) (µM)
Minimal toxic concentration (MTC) (µM)
50
<1
1
30, 50
1
10
>50
30
50
compared to splitomicin’s 4.2 µM (Fig. 6). The compound 4e
also showed an IC50 ∼ 23.5 µM (Fig. 7) against mammalian
SIRT1, comparable to splitomicin’s 60 µM,5b and no adverse
effects until 30 µM when tested for toxicity in zebrafish em-
bryos12 (Fig. 8) at a range 1.0–50 µM [with no observed adverse
effect level (NOAEL) ∼ 30 µM and minimal toxic concentration
(MTC) ∼ 50 µM]. The results of this teratogenicity assay is
summarized in Fig. 9 and Table 5. Notably, 4c and 4d showed
low to severe abnormalities in zebrafish embryos (Fig. 8 and
9). In a preliminary MTT assay 4e inhibited cell growth sig-
nificantly (>50% inhibition @ ∼ 30 µM) when tested against
human hepatocellular liver carcinoma (HepG2) and cervical
cancer (HeLa) cells.
In conclusion, furo[2,3-b]quinoxalines/pyrazines are syn-
thesized as a novel and unique class of fused furo N-hetero-
cycles via a tandem hydrolysis–cyclization strategy in a single
pot. One of the furo[2,3-b]quinoxalines showed encouraging
pharmacological properties in vitro/in vivo and is of further
interest.
Fig. 8 (a) Control embryo showing normal body; embryo treated with
(b) phenobarbital (positive control) showing severe abnormalities, body bent;
compound 4c and 4e showing slight/no abnormalities at 30 µM and moderate
The authors thank DBT (Grant BT/PR13997/Med/30/310/
2010) and Prof. J. Iqbal for support. AN thanks CSIR for a
abnormalities at 50 µM; 4d showing moderate and severe abnormalities at research fellowship.
30 and 50 µM respectively.
Notes and references
significant toxic effect in the absence of 5-FOA. A longer side
chain or a hydroxyalkyl or aryl group at C-2 was less effective.
In a dose response study 4e showed an IC50 ∼ 15.02 µM in
1 (a) C.-J. Li and L. Chen, Chem. Soc. Rev., 2006, 35, 68;
(b) C. J. Li, Chem. Rev., 2005, 105, 3095.
This journal is © The Royal Society of Chemistry 2013
Org. Biomol. Chem., 2013, 11, 4930–4934 | 4933

View Article Online
Communication
Organic & Biomolecular Chemistry
2 D. Lednicer, Strategies for Organic Drug Synthesis and
Design, John Wiley & Sons, Inc., Hoboken, NJ, USA,
2nd edn, 2009.
6 B. Prasad, K. S. Kumar, P. V. Babu, K. Anusha,
D. Rambabu, A. Kandale, G. R. Vanaja, A. M. Kalle and
M. Pal, Tetrahedron Lett., 2012, 53, 6059.
3 (a) For anti-inflammatory evaluation of furo[2,3-b]quin-
oxalines, see: M. M. Ismail, Y. A. Ammar, M. K. Ibrahim,
H. S. El-Zahaby and S. S. Mahmoud, Arzneimittelfors-
chung, 2005, 55, 738; (b) P. Roy and B. K. Ghorai,
Beilstein J. Org. Chem., 2010, 6, DOI: 10.3762/bjoc.6.52,
No. 52.
7 (a) C. Iijima and E. Hayashi, Yakugaku Zasshi, 1977, 97,
712; (b) D. E. Ames, J. C. Mitchell and C. C. Takundwa,
J. Chem. Res. (M), 1985, 1683; (c) V. P. Mehta, S. G. Modha,
D. S. Ermolat’ev, K. V. Hecke, L. V. Meervelt and E. V. der
Eycken, Aust. J. Chem., 2009, 62, 27.
8 CCDC No. 935821.
4 For our earlier efforts, see: (a) K. S. Kumar,
P. M. Kumar, K. A. Kumar, M. Sreenivasulu, A. A. Jafar,
D. Rambabu, G. R. Krishna, C. M. Reddy, R. Kapavarapu,
K. S. Kumar, K. K. Priya, K. V. L. Parsa and M. Pal,
9 For a Pd-catalyzed phenol formation/cyclization of 2-chloro-
aryl alkynes leading to benzofurans, see: K. W. Anderson,
T. Ikawa, R. E. Tundel and S. L. Buchwald, J. Am. Chem.
Soc., 2006, 128, 10694.
Chem. Commun., 2011, 47, 5010; (b) K. S. Kumar, P. M. 10 (a) For a Pd-nanoparticle mediated cyclization of 2-hydroxyaryl
Kumar, M. A. Reddy, M. Ferozuddin, M. Sreenivasulu,
A. A. Jafar, G. R. Krishna, C. M. Reddy, D. Rambabu,
K. S. Kumar, S. Pal and M. Pal, Chem. Commun., 2011,
47, 10263; (c) P. M. Kumar, K. S. Kumar, P. K.
Mohakhud, K. Mukkanti, R. Kapavarapu, K. V. L. Parsa
alkynes leading to benzofurans, see: C. A. Witham, W. Huang,
C.-K. Tsung, J. N. Kuhn, G. A. Somorjai and F. D. Toste, Nat.
Chem., 2010, 2, 36; (b) For the cleavage of O–Si bond of o-tri-
isopropylsiloxyphenylacetylenes leading to benzofurans, see:
Y. Ito, T. Aoyama and T. Shioiri, Synlett, 1997, 1163.
and M. Pal, Chem. Commun., 2012, 48, 431; (d) R. Adepu, 11 C. M. Grozinger, E. D. Chao, H. E. Blackwell, D. Moazed
R. Sunke, C. L. T. Meda, D. Rambabu, G. R. Krishna, and S. L. Schreiber, J. Biol. Chem., 2001, 276, 38837.
C. M. Reddy, G. S. Deora, K. V. L. Parsa and M. Pal, 12 (a) M. Westerfield, The Zebrafish Book. A Guide for the
Chem. Commun., 2013, 49, 190.
Laboratory Use of Zebrafish (Danio rerio), University of
Oregon Press, Eugene, 4th edn, 2000; (b) J. M. Panzica-
Kelly, C. X. Zhang, T. L. Danberry, A. Flood, J. W. DeLan,
K. C. Brannen and K. A. Augustine-Rauch, Birth Defects
Res., Part B, 2010, 89, 382.
5 (a) S. Michan and D. Sinclair, Biochem. J., 2007, 404, 1;
(b) A. Bedalov, T. Gatbonton, W. P. Irvine, D. E. Gottschling
and J. A. Simon, Proc. Natl. Acad. Sci. U. S. A., 2001, 98,
15113.
4934 | Org. Biomol. Chem., 2013, 11, 4930–4934
This journal is © The Royal Society of Chemistry 2013