Base Induced Condensation of Malononitrile with Erlenmeyer Azlactones: An Unexpected Synthesis of Multi-Substituted Δ2-Pyrrolines and Their Cytotoxicity

Article abstract of DOI:10.1002/cbdv.202000014

An efficient, metal free approach to synthesize multi-substituted Δ²-pyrroline derivatives by mild base catalyzed cyclocondensation of malononitrile with Erlenmeyer azlactones via 1,2 addition was developed. The modularity of this reaction was used to assemble a range of poly-substituted pyrrolines. Further, synthesized products were screened for cytotoxic properties on different cancer cell lines such as A549 (Human lung adenocarcinoma cells), HeLa (Human cervical adenocarcinoma cells), Jurkat (Human chronic myeloid leukemia cells) and K562 (Human leukemic T cell Lymphoblast cells). Among the synthesized library of compounds, 6f and 6q displayed potent cytotoxic activity.

Full text of DOI:10.1002/cbdv.202000014

Title: Base Induced Condensation of Malononitrile with Erlenmeyer
Azlactones: An Unexpected Synthesis of Multi-substituted Δ2-
Pyrrolines and their Cytotoxicity Activity
Authors: Seegehalli M Anil, Muddenahalli S Sudhanva, Toreshettahally
R Swaroop, Ajjampura C Vinayaka, Narasimhamurthy Rajeev,
Kuppalli R Kiran, Rangappa Shobith, and Maralinganadoddi
Panchegowda sadashiva
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To be cited as: Chem. Biodiversity 10.1002/cbdv.202000014
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10.1002/cbdv.202000014
Chemistry & Biodiversity
FULL PAPER
Base Induced Condensation of Malononitrile with Erlenmeyer
Azlactones: An Unexpected Synthesis of Multi-substituted Δ2-
Pyrrolines and their Cytotoxicity Activity
Seegehalli M Anil,#a Muddenahalli S Sudhanva,#b Toreshettahally R Swaroop,*c Ajjampura C Vinayaka,a
Narasimhamurthy Rajeev,d Kuppalli R Kiran,d Rangappa Shobith*b and Maralinganadoddi P
Sadashiva*d
[a]
Dr. S. M. Anil, Dr. A. C. Vinayaka
Institute of Plant Science, ARO, Volcani Center, Rishon LeZion 7505101, Israel
Dr. M. S. Sudhanva, Dr. R. Shobith
[b]
Adichunchanagiri Institute for Molecular Medicine, Nagamangala,
Karnataka, 571448, India
E-mail: shobithrangappa@gmail.com (R Shobith)
Dr. T. R. Swaroop
[c]
[d]
Department of Studies in Organic Chemistry, University of Mysore,
Manasagangothri, Mysuru, Karnataka 570 006, India.
E-mail: swarooptr@gmail.com (T R Swaroop)
Mr. N. Rajeev, Mr. K. R. Kiran, Dr. M. P. Sadashiva,
Department of Studies in Chemistry, University of Mysore, Manasagangothri,
Mysuru, Karnataka, 570006, India
E-mail: mpsadashiva@gmail.com (M. P. Sadashiva)
#
Contributed equally
Supporting Information for this article is given via a link at the end of the document
Abstract: An efficient, metal free approach to synthesize multi-
substituted Δ2-pyrroline derivatives by mild base catalyzed cyclo-
condensation of malononitrile with Erlenmeyer azlactones via 1,2
addition was developed. The modularity of this reaction was used to
assemble a range of poly-substituted pyrrolines. Further, synthesized
products were screened for cytotoxic properties on different cancer
cell lines such as A549 (Human lung adenocarcinoma cells), HeLa
(Human cervical adenocarcinoma cellls), Jurkat (Human chronic
concerned with their ring opening reactions. Among these,
arylidene azlactone ring opening with amidines to access
tetrasubstituted 1,3-diazinones through microwave-assisted
transannulation reaction catalyzed by CsF has been reported.[11]
In another study, indenones were synthesized by an
intramolecular Friedel-Crafts reaction catalyzed by Keggin
heteropolyacid H3PW12O40 (supported on Al2O3) under microwave
irradiation.[12] Further, ring opening of Erlenmeyer azlactones
gave anti-α,β-diamino acid derivatives, an important building
block for several bioactive molecules.[13] As Erlenmeyer azlactone
is one of the precious intermediates, its synthesis and further
exploration of its versality in organic synthesis is of interest to the
scientific community. Therefore, we present an easy protocol to
access this Erlenmeyer azlactones in acceptable yields. In
addition, we explore a novel method for the synthesis of Δ2-
pyrrolines from Erlenmeyer azlactones.
myeloid leukemia cells) and K562 (Human leukemic
T cell
Lymphoblast cells). Among the synthesized library of compounds 6f
and 6q displayed potent cytotoxic activity.
Keywords: Erlenmeyer azlactones • Pyrrolines • Condensation
reaction • 1,2 Addition reaction • Cytotoxicity
Pyrrolines and pyrrolidine derivatives are ubiquitous structural
motifs found in an array of natural products,[14, 15] amino acid
derivatives[16, 17] and pharmaceuticals[18, 19] with diverse range of
biological and medicinal properties. Pyrrolines are common
structural frameworks present in porphyrin, which is essential
Introduction
Erlenmeyer azlactone is one of the most useful and important
intermediates in organic synthesis.[1, Erlenmeyer azlactones
21]
components in haemoglobin, chlorophyll and cobalamin.[20,
2]
This framework is also present in thienamycin, one of the most
potent naturally produced antibiotics known so far.[22] Besides,
pyrroline derivatives act as anticancer agents,[23] antibiotics,[24, 25]
hepatitis C inhibitors[26] and antitubercular agents.[27] In organic
synthesis, they have been used as key intermediates in total
synthesis of natural products and to access other classes of
important heterocycles.[28-30] Because of aforementioned natural,
pharmaceutical and synthetic importance, development of an
efficient synthetic method to access pyrrolines is intensively
pursued by the scientific community.[31-33]
Most of the approaches describe the synthesis of Δ3-pyrrolines,
while less attention has been devoted towards synthesis of Δ2-
pyrrolines. The major successful routes, which yielded Δ2-
pyrrolines or 2,3-dihydropyrroles are Michael addition/cyclization
have been used in a wide variety of reactions as precursors for
the generation of biologically active peptides, herbicides,
fungicides, pesticides and agrochemical intermediates.[3] One of
the most important reactions is conversion of glycine into different
amino acids via azlactone intermediate.[4] Some other important
reactions are aza-ene reaction with enamines,[5, 6] lactone ring
oxygen replacement from other hetero atoms like N or S-
containing substrates,[7] incorporation of allyl functionality in a
selective manner to the Erlenmeyer azlactones core structure.[8]
Alkylidene azlactones have been used in asymmetric hetero-
Diels–Alder reactions, undergo reaction with isatins[9] and vinyl
cyclopropanes,[10] which process through [4+2] annulations and
[3+2]-cycloaddition respectively. Although, most reactions of
Erlenmeyer azlactones rely on cycloaddition, few of them are
1
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10.1002/cbdv.202000014
Chemistry & Biodiversity
FULL PAPER
sequence[34, 35] and cyclization strategies in a one-pot sequential
reaction.[36, 37] Other methods include the ring closing metathesis
of enamides,[38] cyclization of sulfonamide anions with
alkynyliodonium triflates,[39] ring expansion of aziridines,[40]
hydroamination of alkynyl sulfonamides,[41] copper-catalyzed
double N-alkenylation,[42] homopropargylamines reaction with
I2/AgOAc,[43]
isomerization
of
3,4-dihydropyrroles,[44]
cycloadditions of nitro-olefins to isocyanoesters[45] and reactions
of 1-cyano or 1-nitro cyclopropyl ketones with aniline.[46] In
addition, a more convergent approach to synthesize Δ2-pyrrolines
is via [4+1][47-49] or [3+2][50, 51] cycloaddition reactions. The latter
has been exploited with 1,3-oxazolium-5-oxides,[52] metallated
azomethine ylides,[53, 54] isocyanoesters,[55, 56] and aziridines.[57, 58]
Although, these methods are quite suitable for obtaining Δ2-
pyrrolines, there are several limitations. For example: use of
costly organo (rosin-derived tertiary amine-thiourea),[35] metal
(gold),[36] organometallics (iron[49] and silver complexes[50]) or
phosphines[47] as catalyst, unstable substrates like sulfur
ylides,[48] alkyl diazo acetate[49] and aziridines,[58] long reaction
51]
time[49,
and sophisticated reaction media like supercritical
carbon dioxide,[57] etc. Thus, the development of a more efficient,
cost-effective and metal free strategy with stable precursors for
the rapid construction of structurally diverse Δ2-pyrrolines is
particularly appealing.
In continuation of our efforts to synthesize biologically important
heterocyclic compounds,[59-64] we focused on fusion of oxazole
five- and pyran six-membered heterocycles, which are common
structural motifs in pharmaceutically active compounds[65, 66] and
methods for their synthesis remain an active field in modern
organic chemistry.[67] Very recently, Zamani et. al., successfully
developed a high-throughput methodology for the synthesis of
fused chromeno[3,2-d]oxazoles starting from the same
precursors via Erlenmeyer azlactones.[68] However, we failed to
synthesize the fused pyrano-oxazoles due to unexpected
Erlenmeyer azlactone ring opening reaction in the formation of Δ2-
pyrrolines.
We herein, present an easy way to access Erlenmeyer azlactones
in acceptable yields and their unexpected ring opening reaction
with malononitrile via cyclo-condensation to yield Δ2-pyrroline
derivatives catalyzed by base. Furthermore, it is demonstrated
that two of the synthesized Δ2-pyrroline derivatives, effectively
reduced cell viability in A549, HeLa, Jurkat and K562 cancer cell
lines.
Scheme 1. Unexpected synthesis of 2,3-dihydro pyrroline derivatives.
Reagents and reaction conditions: i. (a) 5% NaOH, 2 hrs; (b) H3O+; ii. Cl-CO-
OEt, NEt3, toluene, 5 °C, 2 hrs; iii. NEt3, ethanol, r.t., 30 min.
A reaction with benzaldehyde (4a), hippuric acid (3) and ethyl
chloroformate was considered as model reaction to survey key
parameters (temperature, solvent and base) to obtain Erlenmeyer
azlactones (5). At 5 °C toluene and triethylamine were compatible
and achieved higher yield compared to other standard organic
solvents and bases (The compatibility of the solvents and bases
are given in Supporting Information (Table S1)). Application of
these conditions to a selected aryl/hetero aryl aldehydes and
hippuric acid provided a variety of Erlenmeyer azlactones 5 in
moderate to good yields (54-62%).
As a model reaction to achieve fused pyrano-oxazoles (X), we
assessed the reaction of 4-benzylideno-5(4H)-oxazolone (5a) and
malononitrile to survey key parameters of the optimal reaction
process. In the solvents compatibility check, dimethyl
formaldehyde (DMF), tetrahydrofuran (THF), acetonitrile and
toluene with triethylamine base were not efficient and gave lower
product yields of 28-63%. However, the solvents viz., methanol
and ethanol were gave higher product yield and found to be
optimal at room temperature with respect to reaction time (30
minutes) and yields (72 and 81%). The same reaction at reflux
temperatures of methanol and ethanol gave a product with 28 and
30% yields, respectively. Indicating that the reaction is more
sensitive to changes in temperature and opting room temperature
as the most favourable condition for this cyclo-condensation
reaction. A survey of different bases showed the highest yield
(81%) of product to be obtained with triethylamine. Thus, ethanol
with triethylamine as a base at room temperature was found to be
the optimal conditions to explore the substrate scope of the
reaction.
Results and Discussion
Chemistry: The Δ2-pyrroline derivatives were constructed from
easily available starting materials, beginning from sequential
azlactone formation and base induced 1,2-cyclo condensation
reaction. N-benzoyl glycine/ hippuric acid (3) was prepared by
reacting glycine (2) with benzoyl chloride (1). The prepared
benzoyl glycine was reacted with ethyl chloroformate in the
presence of triethylamine, followed by treatment with various
aldehydes (4), resulted in the formation of functionalized
Erlenmeyer azlactone precursors (5a-s) in 54-62% yields
(Scheme 1). Selected Erlenmeyer azlactones (5) were cyclo-
condensed with malononitrile in mild basic conditions resulted in
the generation of multi-substituted dihydropyrroles (6), instead of
predicted pyrano[3,2-d]oxazoles X (Scheme 1).
The purified product formed was characterized by NMR and
HRMS analysis, indicating pyrano[3,2-d]oxazole i.e. X (R = Ph)
as anticipated. Surprisingly, single crystal X-ray diffraction (XRD)
2
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10.1002/cbdv.202000014
Chemistry & Biodiversity
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studies revealed the genuine structure of the product as 6a ((Z)-
2-amino-1-(benzenecarbonyl)-5-benzylidene-4-oxo-4,5-dihydro-
1H-pyrrole-3-carbonitrile). The HRMS and NMR analysis could
not distinguish between these two structures, since both of them
are identical in their molecular weight and nuclei environments.
Finally, with the help of single crystal X-ray diffraction we have
elucidated the structure of unexpected product as 6a. The ORTEP
and packing diagram of 6a is given in Figure 1. The
crystallographic data is deposited at the CCDC No. 1873630.
Scheme 2. Plausible mechanism for the synthesis of Δ2-pyrrolines.
Confirmation of genuine structure from XRD analysis, prompted
us to identify the mechanism of the reaction to yield Δ2-pyrrolines
(6). Scheme 2 shows a suggested reaction mechanism consistent
with the clean formation of Δ2-pyrrolines. At first, The nucleophile
(7) generated by the abstraction of acidic proton of malononitrile
preferred to attack higher electropositive carbonyl carbon of
azlactone (Path B; 1,2-addition) rather than addition to exocyclic
double bond of the azlactone (Path A; 1,4-addition or Michael
addition). According to our prediction path A, conjugate base of
malononitrile 7, attacks β-carbon of exocyclic double bond of 5 to
form oxazol-5-olate 8. This undergoes intramolecular cyclization
to give pyrano[3,2-d]oxazole X via intermediate 9. Whereas,
experimental Path B, nucleophile 7 attacked carbonyl carbon of
Figure 1. ORTEP and packing diagram of compound (Z)-2-
amino-1-benzoyl-5-benzylidene-4-oxo-4,5-dihydro-1H-pyrrole-3-
carbonitrile (6a), (C19H13N3O2)
The compound 6a crystallized in orthorhombic space group P21
21 21. The lattice parameters are Cell: a = 10.548Å; b = 11.474Å;
c = 12.946Å; α = 90°; β = 90°; γ = 90°. The final residual value,
R1 is 0.09 with π-π stacking interaction running parallel to (010)
direction. Further, XRD data revealed the geometrical isomerism
of the exocyclic double bond as stable Z over E form.
Application of these conditions to
a selected Erlenmeyer
azlactones (5) and malononitrile provided a variety of Δ2-pyrroline
derivatives 6 (Table 1) in good to excellent yields (65−89%). Thus,
various azlactones bearing halogens at different positions were
able to yield corresponding products 6b-h in 65-89% (Table 1).
Similarly, Δ2-pyrrolines tethered with electron-donating groups
(methyl, methoxy, ethyl, isopropyl and phenoxy) formed
respective products 6i-q in 57-74% yield. Finally, azlactones
derived from cinnamaldehyde and furfural afforded corresponding
products 6r and 6s in 80 and 71% yield respectively. Almost all
the substituents presented were well tolerated, different
substituents on the aromatic aldehydes showing that the
electronic effects of substituents had little influence on the
reaction. However, Aldehydes with electron-withdrawing groups
had a slightly faster reaction time and higher yield, compared to
aldehydes containing electron donating groups.
azlactone
5
to produce enamide 10, which underwent
via
intramolecular cyclization to generate Δ2-pyrrolines
6
electronic rearrangement through 11 and 12 (Scheme 2). The
final product formed 6 has an exocyclic double bond, which is
susceptible to undergo Michael addition with another molecule of
malononitrile to give a new bicyclic compound. But in our reaction
condition, the reaction stopped at the Δ2-pyrroline step, even with
use of excess of malononitrile and base. This is one of the added
benefits of the herein developed methodology for the synthesis of
Δ2-pyrrolines tethered with an active exocyclic double bond.
3
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10.1002/cbdv.202000014
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Jurkat and K562 with respective IC50 values of 16.91, 18.92, 11.43
and 17.22 µM. Position of chloro group on the phenyl ring of Δ2-
pyrroline (6e, 6f and 6g) has significant effect on cytotoxicity.
Surprisingly, the chloro moiety at meta-position (6f) enhances the
cytotoxic potential, while substitution on ortho-position (6e) results
in decreased activity and substitution on para-position (6g)
substantially diminishes the activity of the compound, reason for
which is uncertain. The in vitro cytotoxic effect of active
compounds 6f and 6q on cancer cell lines are depicted in Fig 2.
Interestingly, both of the active molecules exhibited their cytotoxic
effect in a dose and time dependent manner against the screened
cancer cell lines.
Table 1. Substrate Scope for Synthesis of Δ2-Pyrroline Derivatives.
NC
O
H2N
N
O
6a
Yield = 81%
Biology: in vitro cytotoxicity assay
Figure 2. Cytotoxic effect of Δ2-pyrrolines 6f and 6q on various cancer
cells. Cells were treated with increased concentration of 6f and 6q (12.5 μM, 25
μM, and 50 μM) for 48h and 72h, cells were harvested and subjected for MTT
assay to unveil the cytotoxic potential of compounds, DMSO treated cells served
as vehicle control. Different cancer cells used for screening of 6f and 6q are
Jurkat (a), A549 (b), K562 (c) and HeLa (d). Each experiment was repeated a
minimum of three times and error bars indicate the SEM and P values compared
with mean control groups with mean of 6f and 6q treated group, p < 0.05.
The synthesized novel Δ2-pyrrolines (6a-s) were screened for
their in vitro cytotoxic activity against A549 (Human lung
adenocarcinoma cells), HeLa (Human cervical adenocarcinoma
cells), Jurkat (Human chronic myeloid leukemia cells) and K562
(Human leukemic T cell Lymphoblast cells) through MTT assay.
Results revealed that among the compounds treated 6e, 6f, 6h,
6l, 6o and 6q affected the viability of HeLa cancer cells. Where
as, compounds 6f and 6q exhibited potent cytotoxic effect against
all four cell lines (Fig 2). The compound 6q is found to be more
selective and has maximum cytotoxic effect on A549 cells with
lower IC50 value of 6.23 μM. Yet, it has good cytotoxic effect on
HeLa and Jurkat cancer cell lines with respective IC50 values of
10.43 and 9.45 µM. This selectivity could be attributed due to the
presence of substituted o-phenoxy group on the phenyl ring of Δ2-
pyrroline.The compound 6f substituted with m-chloro has almost
equal activity on all the cancer cell lines selected viz., A549, HeLa,
The rest of the synthesized Δ2-pyrroline derivatives bearing
different substitutions did not reduce viability in the cancer cell
lines tested. Presence of active groups such as furan (6s) or a
conjugated alkene (6r) on C-5 of Δ2-pyrroline is not as effective
as phenoxy substitution. It was observed from structural activity
relationship studies, it appears that inductively electron-
withdrawing chlorine at m-position (6f) or phenoxy substitution on
o-position (6q) on the phenyl ring of Δ2-pyrroline is essential for
4
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10.1002/cbdv.202000014
Chemistry & Biodiversity
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cytotoxic activity. As of interest the hit compounds 6e, 6f, 6h, 6l,
6o and 6q were minimally cytotoxic to normal human embryonic
kidney cells (HeK) at the tested concentrations (25 μM and 50
μM). The IC50 values of all the compounds are given in Supporting
Information (Table S3).
10 mL), washed with H2O (2 x 20 mL), brine (1 x 20 mL), dried
over Na2SO4 and distilled under reduced pressure to give crude
products, which were purified by column chromatography over
silica gel using hexane-EtOAc (5 : 5) as eluent or recrystallization
with chloroform whichever is permissible.
Acknowledgements
Conclusion
MPS thank UGC-SAP DRS III (Grant No. UGC F-540/10/DRS-III
(SAP-I) Dated 14–09-2016) and Sami Labs Limited, Bangalore.
Authors are grateful to Institution of Excellence, University of
Mysore, Mysuru for spectral analysis. N. Rajeev thank UGC, New-
Delhi for providing RGNF Fellowship.
In summary, we have developed a new, facile and practical metal-
free synthetic route to novel multi-substituted and multi-functional
Δ2-pyrrolines in good to excellent yields. This protocol offers a
promising, cost effective and simple approach to construct new
medicinally and biologically important Δ2-pyrrolines. Further, the
synthesized library of compounds were screened for the
cytotoxicity activity. Among the all, compounds 6f & 6q emerged
as promising candidates without effecting human embryonic
kidney (HeK) normal cells. Therefore, hit compounds 6f and 6q
are worth studying further in vivo and is currently pursued in our
laboratory.
Author Contribution Statement
Dr. S. M. Anil, Mr. N. Rajeev and Mr. K. R. Kiran were performed
the experiments, analyzed the data and wrote the paper.
Dr. M. S. Sudhanva and Dr. A. C. Vinayaka performed all
biological
experiments
and
analyzed
results.
Dr. S. M. Anil, Dr. T. R. Swaroop, Dr. R. Shobith and
Dr. M. P. Sadashiva conceived and designed the experiments.
Experimental Section
General Procedure for the Synthesis of Hippuric Acid:
Glycine (18.75g; 0.25 mol) was dissolved in 300 mL of 5% sodium
hydroxide solution. To this, benzoyl chloride (32 mL; 0.275 mol)
in five portions was added with stirring until benzoyl chloride
completely reacted. The solution was transferred to the beaker
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wise to a mixture of hippuric acid (1 g, 5.5811 mmol) and Et3N
(0.78 mL, 5.5811 mmol) in dry toluene (10 ml) at 5-10 °C with
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General Procedure for the Synthesis of Multi-substituted
Dihydropyrroles:
Mixture of malononitrile (0.053 mL, 0.9628 mmol) and triethyl
amine (0.12 mL, 8.8253 mmol) was added drop-wise to the
selected azlactone (0.8023 mmol) in ethanol (6 ml) at room
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7
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A mild base catalyzed cyclo-condensation of malononitrile with Erlenmeyer azlactones via 1,2 addition to yield Δ2-pyrroline derivatives
was developed. Further, synthetic library of compounds were screened for cytotoxic activity against A549, HeLa, Jurkat and K562
cancer cell lines.
8
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