Augmentation of steroidal β-formylenamide with pyrazolo and benzimidazo moieties: A tandem approach to highly fluorescent steroidal heterocycles

Article abstract of DOI:10.1016/j.tetlet.2021.152893

A facile synthesis of pyrazolo[1,5-a]pyrimidine and benzimidazo[1,2-a]pyrimidine annulated steroids is described from the novel reaction of β-formyl enamides with amino pyrazoles, indazoles and benzimidazoles. Several of the products exhibited fluorescence properties with high quantum yields.

Full text of DOI:10.1016/j.tetlet.2021.152893

Tetrahedron Letters 67 (2021) 152893
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Augmentation of steroidal b-formylenamide with pyrazolo
and benzimidazo moieties: A tandem approach to highly
fluorescent steroidal heterocycles
⇑
Geetmani Singh Nongthombam, Romesh Chandra Boruah
Applied Organic Chemistry Group, Chemical Science & Technology Division, CSIR-NEIST, Jorhat 785006, Assam, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A facile synthesis of pyrazolo[1,5-a]pyrimidine and benzimidazo[1,2-a]pyrimidine annulated steroids is
described from the novel reaction of b-formyl enamides with amino pyrazoles, indazoles and benzimida-
zoles. Several of the products exhibited fluorescence properties with high quantum yields.
Ó 2021 Elsevier Ltd. All rights reserved.
Received 21 December 2020
Revised 23 January 2021
Accepted 27 January 2021
Available online 5 February 2021
Keywords:
b-Formylenamide
Pyrazolo
Benzimidazo
Steroid
Fluorescence
Introduction
pyrimidine and pyrazolo[3,4-b]pyridines exhibit underlying activ-
ities for COX-2 selective inhibitor [23], CK-2 kinase inhibitor [24],
Organic molecules that demonstrate strong fluorescence emis-
sions have found its own niche amongst the elites of the scientific
community [1,2]. In recent years, molecular fluorophores have
been deeply introspected over a wide spectrum of research areas
ranging from chemical, biological to material sciences [3,4]. Fluo-
rescence properties of steroids find their applications in different
diagnostic tools such as fluorescent immunoassay [5,6], membrane
structure and function [7–9], steroid protein interaction [10] as
well as steroid inclusion into cyclodextrins [11]. Nevertheless,
the weak intrinsic fluorescence of steroids requires additional
treatment like denaturation by strong acids [12,13] or attachment
of different fluorescent fluorophores to steroids [14]. Consequently,
discovery of steroidal single fluorophore [15] becomes a challeng-
ing task; and efforts are inevitable in this approach.
and HMG-CoA reductase inhibitor activities [25]. It has also been
found to possess profound applications as selective peripheral ben-
zodiazepine receptor ligands [26], antianxiety agents [27], his-
tamine-3 receptor ligands [28], and as potassium channel ligands
[29]. To the best of our knowledge, no literature report is available
for the study of fluorescence potential of such steroidal
heterocycles.
In 2014, our group first reported the synthesis of several A/D-
ring annulated steroidal pyrazolo[1,5-a]pyrimidines from conju-
gated enones and substituted 3-amino-1H-pyrazoles in the pres-
t
ence of KO Bu under thermal condition (Scheme 1) [30,31].
Subsequently, another synthetic protocol involving b-bro-
movinyl aldehyde was developed for the synthesis of pyrazolo
[1,5-a]pyrimidines and pyrazolo[3,4-b]pyridines under microwave
Recently, Pyrazolo[1,5-a]pyrimidines have emerged as attrac-
tive substrates with efficient synthetic methods [16–19] and fluo-
rescence properties [20,21]. The combination of photophysical
properties with biological activities allows the use of these com-
pounds as lipid droplet biomarkers for HeLa cells (cancer cells)
and L929 cells (normal cells) [22], demonstrating the interesting
versatility of this heterocyclic core. Moreover, pyrazolo[1,5-a]
2
irradiation using Pd(OAc) as catalyst (Scheme 1) [32,33]. How-
ever, these studies were aimed specifically in developing new syn-
thetic strategies for these class of steroidal heterocycles without
any focus to explore their fluorescence potentials.
In continuation of our endeavor, herein, we report a novel appli-
cation of steroidal b-formyl enamides [34] in the synthesis of new
class of fluorescent steroids. Several of our synthesized steroidal
pyrazolo[1,5-a]pyrimidines (3a-d, 3f-h, 3j-k) demonstrated strong
fluorescence properties. It was also noted that steroidal benzimi-
dazo[1,2-a]pyrimidine (5a-b) exhibited high fluorescence.
⇑
Corresponding author.
E-mail address: rc_boruah@yahoo.co.in (R.C. Boruah).
https://doi.org/10.1016/j.tetlet.2021.152893
040-4039/Ó 2021 Elsevier Ltd. All rights reserved.
0

Geetmani Singh Nongthombam and Romesh Chandra Boruah
Tetrahedron Letters 67 (2021) 152893
Scheme 1. Synthesis of steroidal pyrazolo[1,5-a]pyrimidines and pyrazolo[3,4-b]pyridines.
Result and discussion
elimination of acetamido group led to intermediate B. Finally, loss
of proton from intermediate B afforded 3a. The isolation of aceta-
b-Formyl enamide was found to be an excellent precursor for
the preparation of pyrazolo(1,5-a)pyrimidines (3a-k) and benzim-
idazo(1,2-a)pyrimidines (5a-c). Initially, the reaction condition of
b-formyl enamide 1a, (1.0 mmol) with equimolar amount of 5-
aminopyrazole 2a (1.2 mmol) was optimized varying solvent, cat-
alyst and reaction temperature.
mide as
mechanism.
a byproduct rendered support to our proposed
In order to corroborate the proposed mechanistic pathway of
the reaction, b-formyl enamide (1a) was reacted with benzamidine
(6a-b) and a similar cyclization pattern was observed to yield
pyrimidine fused product (7a-c) (Scheme 4).
The reaction of 1a with 2a in solvent-less condition did not pro-
ceed under thermal condition; and use of neat DMF as solvent at
elevated temperatures afforded a poor yield of 3a. The best yield
of 3a (87%) was achieved from reaction of 1a and 2a when the reac-
tion was carried in DMF using 5 mol% of CuI at 110 0C. Neither
change of solvent nor catalyst showed any improvement of yields
under thermal conditions (Table 1). Reactions carried at higher
temperature (above 110 °C) did not improve the yield of the
product.
Further, it was also observed that b-formyl enamide (1a) facili-
tated the formation of conjugated enone (9a) with concurrent loss
of acetamido group (Scheme 4). The H NMR of compound 9a
1
exhibited chemical shift for –NH proton at d 10.75. This downfield
shift can be attributed due to hydrogen bonding of –NH with C@O
group; which favours Z isomer. The mechanistic pathway of this
reaction probably proceeds in congruence to the formation of pyra-
zolo[1,5-a]pyrimidines (Scheme 3); however, due to the nucle-
ophilic attack of water molecule the unstable pyrimido[1,6-a]
pyridinium salt intermediate (C) opens up to give intermediate
(D) followed by tautomerization to afford conjugated enone adduct
(9a) (Scheme 5).
Using optimized condition, steroidal as well as non-steroidal
analogs of pyrazolo[1,5-a]pyrimidines (3b-k) were synthesized
(Scheme 2). The scope of the optimized protocol was further
explored with other amino substituted heterocycles. It was found
that 2-aminobenzamidazoles reacted with b-formyl enamide to
afford benzimidazo[1,2-a]pyrimidines (5a-c) in high yields
Several of the synthesized compounds were found to exhibit
fluorescence activity which was ascertained from the strong fluo-
rescent glow exuded by the compounds on exposure to long UV
(
2
Scheme 2). The single-crystal X-ray crystallography data (CCDC
008212) confirmed the proposed structure of the synthesized
radiation (k = 365 nm). All the steroidal as well as non-steroidal
À3
derivatives were dissolved in chloroform (2.0Â10
M) and
compound 5a.
screened for fluorescence activity by illumination under long UV.
It was found that the following compounds 3a-k and 5a-b demon-
strated change in coloration from colorless to fluorescent white or
light yellow on UV365 illumination (Fig. 1). Amongst the synthe-
sized compounds, pyrazolo[1,5-a]pyrimidine 3k and benzimidazo
[1,2-a]pyrimidine 5b demonstrated the highest fluorescence activ-
ities, accounting for fluorescence quantum yield of 88.5% and 90.2%
A probable mechanism for the ring formation reaction was pro-
posed involving nucleophilic displacement of 17-acetamido group
of 1a during the cyclization process (Scheme 3). In the preliminary
step, the amino group of the pyrazole (2a) condenses with the
aldehyde group of the b-formyl enamide (1a) resulting Schiff base
intermediate A [35]. The CuI mediated ring cyclization followed by
2

Geetmani Singh Nongthombam and Romesh Chandra Boruah
Tetrahedron Letters 67 (2021) 152893
Table 1
a
Optimization of reaction condition.
Entry
Solvent
Catalyst
Tb (oC)
Yieldc (%)
1
2
3
4
5
6
7
8
9
–
–
–
–
–
100
rt
80
0
0
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMF
DMSO
CHCl
DMF
DMF
DMF
DMF
10
40
42
85
87
45
40
0
110
120
110
110
110
110
120
61
110
110
110
110
–
CuI (10 mol %)
CuI (5 mol %)
Cu(OAc) (10 mol %)
2
CuCl (5 mol %)
CuI (5 mol %)
CuI (5 mol %)
CuBr (5 mol %)
ZnCl
FeCl
10
11
12
13
14
15
3
0
39
35
28
21
2
(5 mol %)
(5 mol %)
3
L-proline
16
17
18
19
20
21
22
THF
CuI (5 mol %)
CuI (5 mol %)
CuI (5 mol %)
CuI (5 mol %)
CuI (5 mol %)
CuI (5 mol %)
CuI (5 mol %)
66
40
56
80
65
78
100
0
CH
Acetone
CH CN
2
Cl
2
trace
0
0
0
0
0
3
Methanol
Ethanol
H O
2
a
Reaction condition: 1a (1 mmol), 2a (1.2 mmol) in solvent (3 mL) for 5 h. bOil bath temperature. cIsolated yields of 3a.
respectively with respect to quinine hemi-sulfate as the reference
90.2%). Because of inherent lipid properties of steroids, the fluores-
cent steroids could provide great scope as a valuable tool in cancer
research and other biological studies.
À5
at 2.0Â10 M concentration (Fig. 2). Compounds 3a-d, 3g-h, 3j-k
and 5a-b showed fluorescence quantum yields ranging 23.9–87.6%.
However, products 3f and 5c exhibited weaker fluorescence and
negligible quantum yields (4.1 and 5.7%).
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared
to influence the work reported in this paper.
Conclusion
In summary, herein, we report a novel application of b-formyl
enamide towards synthesis of fluorescent pyrazolo[1,5-a]pyrim-
idine and benzimidazo[1,2-a]pyrimidine annulated steroidal and
non-steroidal compounds. We demonstrate an interesting phe-
nomenon of b-formyl enamides in nucleophilic reactions facilitat-
ing loss of 17-acetamido group, in contrast to earlier findings,
wherein, acetamido group usually took part in cyclization process
Acknowledgements
RCB thanks CSIR, New Delhi for award of Emeritus Scientist,
CSIR (ES1516Y2144). GSN thanks CSIR, New Delhi for award of
RA and financial support under Emeritus Scientist scheme, CSIR.
The authors are thankful to the Director, CSIR-NEIST, Jorhat for
his keen interests.
[
36]. Furthermore, our studies revealed a novel synthesis of fluo-
rescent steroidal aza heterocycles in high quantum yields (upto
3

Geetmani Singh Nongthombam and Romesh Chandra Boruah
Tetrahedron Letters 67 (2021) 152893
Scheme 2. Copper Iodide catalyzed synthesis of pyrazolo[1,5-a]pyrimidines (3a-k) and benzimidazo[1,2-a]pyrimidine (5a-c).
Scheme 3. Proposed mechanism for the synthesis of pyrazolo[1,5-a]pyrimidines.
4

Geetmani Singh Nongthombam and Romesh Chandra Boruah
Tetrahedron Letters 67 (2021) 152893
Scheme 4. Copper Iodide catalyzed synthesis of pyrimidines (7a-c) and conjugated keto product (9a) from b-formyl enamide.
Scheme 5. Proposed mechanism for the synthesis of conjugated keto system from b-formyl enamide.
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