Low melting mixture glycerol:proline as an innovative designer solvent for the synthesis of novel chromeno fused thiazolopyrimidinone derivatives: An excellent correlation with green chemistry metrics

Article abstract of DOI:10.1016/j.molliq.2019.03.058

The expansion of proficient and environmentally benign synthetic protocols has engrossed increasing attention in recent organic syntheses in view of the mounting concern over the environment. As part of this green concept, our group designed novel LTTM by

Full text of DOI:10.1016/j.molliq.2019.03.058

Accepted Manuscript
Low melting mixture glycerol:proline as an innovative designer
solvent for the synthesis of novel chromeno fused
thiazolopyrimidinone derivatives: An excellent correlation with
green chemistry metrics
Priyanka P. Mohire, Dattatray R. Chandam, Reshma B. Patil,
Ajinkya A. Patravale, Jai S. Ghosh, Madhukar B. Deshmukh
PII:
S0167-7322(18)34197-7
DOI:
Reference:
https://doi.org/10.1016/j.molliq.2019.03.058
MOLLIQ 10599
To appear in:
Journal of Molecular Liquids
Received date:
Revised date:
Accepted date:
13 August 2018
6 March 2019
10 March 2019
Please cite this article as: P.P. Mohire, D.R. Chandam, R.B. Patil, et al., Low melting
mixture glycerol:proline as an innovative designer solvent for the synthesis of novel
chromeno fused thiazolopyrimidinone derivatives: An excellent correlation with green
chemistry metrics, Journal of Molecular Liquids, https://doi.org/10.1016/
j.molliq.2019.03.058
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ACCEPTED MANUSCRIPT
Low Melting Mixture Glycerol: Proline as an Innovative Designer Solvent
for the Synthesis of Novel Chromeno Fused Thiazolopyrimidinone
Derivatives: An Excellent Correlation with Green Chemistry Metrics
Priyanka P. Mohirea, Dattatray R. Chandamc, Reshma B. Patila, Ajinkya A. Patravaled, Jai. S.
Ghosha, Madhukar B. Deshmukh*a,b
aChemistry Research Laboratory, Department of Agrochemicals & Pest Management, Shivaji University,
Kolhapur-416004, Maharashtra, India.
bHeterocyclic Chemistry Laboratory, Department of Chemistry, Shivaji University, Kolhapur-416004,
Maharashtra, India.
cDepartment of Chemistry, BhogawatiMahavidyalaya, Kurukali, Kolhapur-416001, Maharashtra, India.
dDepartment of Chemistry, Vivekananda College Kolhapur-416004, Maharashtra, India.
Email: shubhlaxmi111@gmail.com
ghoshjai@gmail.com
Graphical Abstract

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Abstract
The expansion of proficient and environmentally benign synthetic protocols has
engrossed increasing attention in recent organic syntheses in view of the mounting concern
over the environment. As part of this green concept, our group designed novel LTTM by
using Glycerol and proline (1:1) at reflux condition, and effectiveness of novel DES or
LTTM has been studied for the synthesis of 7-(aryl)-10-methyl-6H,7H-chromeno[4,3-
d]thiazolo[3,2-a]pyrimidin-6-one derivatives via Multicomponent reaction. The synthetic
methodology with the use of novel LTTM as green solvent and inexpensive, biodegradable,
plays a dual role (recyclable catalyst and solvent) with its operational simplicity and
maximum greener synthetic efficiency with green chemistry metrics calculations will be
attractive for academic and industrial research. The synthesized derivatives have been
evaluated for mosquito larvicidal bioassay using two mosquito species namely Aedes aegypti
and Culex quinquefasciatus.
Keywords
Low Transition Temperature Mixture; Green Metrics; Multicomponent Reactions;
chromene; Green Chemistry

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1. Introduction
Within the scope of green chemistry, green solvents occupy an imperative position.
Green solvents are accomplished as green medium; because they have been meeting the
criteria of twelve green chemistry principles such as availability, non-toxicity,
biodegradability, reusability, economical, etc [1]. Ionic liquids are highlighted as green
solvents and extensively used in different synthetic methods. To overcome the drawbacks of
the ionic liquid, Deep Eutectic Solvents (DES) have been arising as an eco-friendly solvent as
well as catalyst for several organic transformations over the years [2]. They have been well
known to make progress in MCR with excellent green metrics such as high atom economy
and low E factor [3].
Deep Eutectic Solvent (DES) or Low Transition Temperature Mixtures (LTTMs)
were introduced by A. P. Abott in this century [4] and work has been continued by M. C.
Kroon and coworkers. This new designer solvent LTTM is a mixture of Hydrogen Bond
Donor (HBD) and Acceptor (HBA) which has been proved as bio-solvents [5]. Preparation of
LTTM is very simple, eco-friendly and straightforward and 100% atom economic,
inexpensive and non-toxic materials. In several organic transformations LTTM acts as
catalyst as well as solvent due to physical properties such as low vapor pressure, non-
inflammability, biocompatibility, biodegradability and cost efficiency [6-9]. Other than this,
LTTMs or DESs are accounted as innovative and sustainable solvent and has several
applications [10-13]. Each LTTM has its own properties which make them suitable as green
solvent [14, 15].
Chromene ring widely present in plant products including edible vegetables and fruits
[16]. Also, various chromene-based bioactive natural products have been recognized, and
associated with the capacity to prevent diseases [17]. Even though, it shows wide applications
in laser dyes and fluorescence, pigments and cosmetics [18-20]. Chromene fused derivatives

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are vital class of natural compounds because of its bioactivity in pharmaceutical and
agrochemicals field [21]. Chromene scaffolds with thiazolopyrimidine moiety have also wide
applications in heterocyclic chemistry [22] such as anticancer [23-27] activities, anti-
tuberculotic [28], anti-microbial [29-34], anti-inflammatory [33] and analgesic [35],
insecticidal [36], herbicidal [37, 38], antioxidant [34, 39], acetylcholinesterase inhibitors [40],
antimalarial [41], anti-HIV [42]. Reddy et al. have been reported the synthesis of chromeno
fused thiazolopyrimidine molecules [43]. However, these methods have been reported with
some limitations such as use of expensive reagents and solvents, longer reaction time, and
lower selectivity of catalyst, environmental hazards and catalyst reusability problem. To
overcome these limitations, it is necessary to implement versatile alternatives and new
strategies for the development of sustainable chemical processes in the framework of green
chemistry principles [44].
Taking into consideration, the significance of LTTMs and in search for new eco-
friendly and bio-renewable solvents, thus our group has been taking efforts towards synthesis
of several organic reactions and novel bioactive molecules by using biodegradable ionic
liquid, DES or LTTM [45-51]. LTTM of glycerol and proline have been explored in this
work. By selecting easily available, cost effective starting materials such as glycerol and
proline with proportion (1:1) LTTM is prepared. The physical and thermal properties of novel
LTTM were studied. Similarly the catalytic efficiency and the role LTTM or DES as a
reaction promoting medium were investigated for the synthesis of chromeno[4,3-
d]thiazolo[3,2-a]pyrimidin motifs.
Considering reviews on LTTM or DES, it is applicable for several multicomponent
reactions (MCRs). MCRs represents environmentally and eco-friendly pathway with high
atom economy [52, 53]. In extension of our constant efforts in the development of novel
synthetic methodologies for the synthesis of bioactive scaffolds, we report an expedient

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synthesis of chromeno fused thiazolopyrimidne derivatives (Scheme 1) and performed in-
vitro mosquito larvicidal bioassay of selected motifs using two well known species of
mosquitoes, Aedes aegypti and Culex quinquefasciatus.
R
CHO
OH
O
O
N
S
LTTM
Reflux
N
O
NH2
R
+
H3C
+
O
H3C
S
N
Scheme 1 Glycerol: Proline (1:1) LTTM mediated synthesis of chromeno[4,3-d]thiazolo[3,2-
a]pyrimidin derivatives
2. Experimental
All the chemicals were purchased from Alfa Aesar and Spectrochem (PVT. Ltd,
Mumbai, India), Sigma Aldrich and used without purification. The reaction was monitored by
TLC. The desired structures of the synthesized compounds were confirmed by their relevant
spectral data. The melting points were determined in open glass capillary method and are
uncorrected. The Compounds were confirmed by IR, 1H NMR and 13C NMR. The IR spectra
were recorded on a JASCO FT-IR 4600 spectrum spectrophotometer and the values are
1
expressed as ν max cm-1. The H NMR and 13C NMR, DEPT spectra were recorded on
Bruker Spectrospin Avance II-300 MHz and 75 MHz spectrophotometer relative to TMS as
an internal standard using DMSO-d6 as a solvent. Thermal analysis of newly prepared LTTM
was performed on (SDT Q600 V20.9 Build 20) instrument.
2.1 General synthetic procedure for the preparation of Glycerol: Proline (1:1) LTTM
The LTTM have been prepared by selecting glycerol as a hydrogen bond donor and
proline as a hydrogen bond acceptor. A mixture of glycerol (100 mmol, 9.2 g) and proline
(100 mmol, 11.5 g) in the ratio 1:1was heated at 80˚C with continuous stirring for 30 min
(Scheme 2). The resulting LTTM forms dark yellowish viscous liquid with an excellent atom
economy, was subsequently allowed to cool at room temperature and was used for the

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synthesis of chromeno fused thiazolo[3,2-a]pyrimidin derivatives without further
purification.
O
O
H
H
N
OH
O
Heated slowly
upto 80oC
HO
OH
O
.
+
O
H
HN
HO
OH
H
Proline
Glycerol
LTTM
Scheme 2 Preparation of Glycerol: Proline (1:1) LTTM
2.2 General synthetic procedure for the synthesis of chromeno[4,3-d]thiazolo[3,2
a]pyrimidin
A mixture of 4-Hydroxycoumarin (1 mmol, 0.162 g) and 4-chlorobenzaldehyde (1
mmol, 0.140 g), with 5ml LTTM was taken in a 50 ml round bottomed flask at room
temperature to form the Knoevenagel product, monitored by TLC and then 2-mino 5-methyl
thiazole (1 mmol, 0.106 g) were added and continued at reflux condition for 20 min. The
progress of the reaction was monitored by TLC using petroleum ether- ethyl acetate (8:2 v/v).
After completion, the reaction mixture was cooled to room temperature. Water was added to
the reaction mixture and the product was collected by simple filtration, washed with hot
ethanol and diethyl ether. Finally, the crude product was recrystallized from ethanol to obtain
the pure product (Scheme 1).
3. Result and Discussion
A new LTTM is formed by glycerol and proline with proportion (1:1). Generally,
glycerol is obtained from plant and animal sources, occurs as triglycerides. The hydrolysis,
saponification or trans-esterification of triglycerides produces glycerol and fatty acids. On the
other side, proline is α amino acid, oftenly used as an asymmetric catalyst in organic
synthesis. To replace volatile organic solvents, LTTMs has been used as a catalyst or solvent
in the advancement of green sustainable synthetic chemistry. Combining the applicability of

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multicomponent reactions with interesting features of LTTM, with a complete study on
physical and thermal properties of new LTTM has been developed.
3.1 Characterization of Glycerol: Proline (LTTM)
3.1.1 TGA analysis of new LTTM-GP (1:1)
TGA is a conventional and most popular technique used to study the thermal stability
and decomposition of the materials. TGA is performed of LTTM G: P (1:1) to evaluate the
thermal stability by means of the degradation temperature. The thermal decomposition
process is affected by the operating parameters and conditions such as the rate of heating,
temperature, pressure, surrounding environment and moisture content and composition of the
heated sample. The LTTM sample was heated from room temperature to 350˚C under
continuous nitrogen flow. The weight loss of LTTM during the long runs could be endorsed
to the amount of moisture content and volatiles present in the sample.
Figure 1 show that TGA and DTG curves of the glycerol and proline mixture. These
curves reveal two main weight loss regions. The first weight loss is about 13 wt % in the 50–
175˚C temperature range may be due to the loss of physically adsorbed and bound water.
This temperature range is sufficient to promote and catalyze organic synthesis. The second
weight loss of about 81 wt % in the temperature range about 175–300˚C, corresponds to the
thermal degradation/ decomposition of glycerol and proline moiety. The decomposition
values (Tdcp) of LTTM G: P (1:1) in range from 58.42˚C to 344.34˚C. We can conclude that,
the LTTM stay intact under experimental conditions as a solvent.

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Fig. 1 TGA analysis of new LTTM-GP (1:1)
3.1.2 DSC analysis of LTTM-GP (1:1)
Plot of heat flow vs. temperature (Fig. 2) range between 25˚C and 350˚C obtained by
DSC. A broad endothermic peak ~116˚C is observed in the DSC curves on heating the
glycerol-proline mixture. This is associated with water content in the mixture and its
interaction with the LTTM and the exothermic events that start to appear at about ~237˚C,
associated with the degradation backbone of LTTM.

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Fig.2 DSC curve of LTTM-GP (1:1)
3.1.3 FT-IR and NMR Spectral analysis of LTTM-GP (1:1)
The possible structure of LTTM-GP (1:1) was investigated with FT-IR, NMR spectral
analysis to explore the type of interaction and atoms involved in interactions in the given
ratio of components. The FT-IR spectra (Fig. 3) of LTTM of glycerol, proline (molar ratio
1:1) were recorded at room temperature. In glycerol, the stretching vibration band of –OH
observed at νmax3303 and C=O at 1650 cm-1. In proline, stretching vibration band of C=O
observed at 1614 cm-1 and C-OH at 3151 and 3357 cm-1. The mixture of LTTM-GP (1:1)
shows stretching band corresponding to C-OH at 3325cm-1 and the another stretching band
observed at 2360 and 2138cm-1which indicates the formation of a hydrogen bonding (O=C-
O-H----N).

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Fig. 3 FT-IR spectrum of LTTM-GP (1:1)
1
The HNMR analysis of glycerol and proline LTTM mixture (molar ratio 1:1) was
recorded. The 1H NMR (Fig. 4) showed a singlet at δ,4.158 ppm due to (R-OH) proton which
confirms hydrogen bonding between two reactants and δ, 4.867 ppm represents (Ar-OH)
proton of proline in the same mixture. The signals that appeared between δ, 3.267 ppm and
3.471 ppm represent the aliphatic protons (R-H).
Fig. 4 NMR Spectrum of LTTM-GP (1:1)

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3.2 Chemistry
In the view of bioactive applications of 7-(aryl)-10-methyl-6H,7H-chromeno[4,3
d]thiazolo[3,2-a]pyrimidin-6-one derivatives, our current focus has been targeted, to
synthesize these novel scaffolds using simple environmental friendly, facile protocol based
on green chemistry principles. We initially investigated the preliminary reaction of 4-hydroxy
coumarin (1mmol, 0.162 g), 4-chlorobenzaldehyde (1mmol, 0.140 g) and 2-amino 5-methyl
thiazole (1mmol, 0.106 g) using different reaction conditions.
The choice of solvent is also an important aspect in every reaction to prove the
greenness of the protocol. To study the solvent efficiency, the reaction was carried out in a
variety of polar and non-polar solvents at different conditions. Water was used for this
reaction, but according to our expectations, the reaction does not proceed further beyond the
Knoevenagel product and desired product was not obtained. When ethanol was used as a
solvent, the desired product was obtained in 42% yield under reflux conditions (Table 1 Entry
3b). Then, we endeavor same reaction in a combination of water/ethanol mixture and other
organic solvents like DCM, methanol, glycerol at reflux condition, but the desired product is
obtained in very poor yield (Table 1 Entry 4-7). As a new designer solvent, LTTM was used
for the same reaction at reflux condition, astonishingly the yield of the product increased up
to 94% in shorter reaction time (Table 1 Entry 8). We screened the reaction using various
number of LTTM, however the yield of the product was poor (Table 1 Entry 9-11). After
testing various solvents and solvent-free conditions, it is observed that a Glycerol: Proline
(1:1) (LTTM) leads to the formation of the product with better yield (Table 1 Entry 8). The
results are summarized in Table 1.

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Table 1 Optimization of solvent for the synthesis of chromeno[4,3-d]thiazolo[3,2-
a]pyrimidin-6-one
Sr.
No.
Temp.
Condition time (mins.)
Reaction
Solvent
Yieldb(%)
(˚C)
1
Solvent free
Water
30
30
80
30
80
80
80
80
80
80
80
80
80
300
180
120
320
180
120
120
300
90
Knoevenagel product
2a.
2b.
3a.
3b.
4.
Knoevenagel product
Water
Knoevenagel product
Ethanol
Knoevenagel product
Ethanol
42
46
40
52
56
94
70
60
42
Water : Ethanol (1:1)
DCM
5.
6.
Methanol
7.
Glycerol
8.
Glycerol: Proline (1:1)
Glycerol: Proline (1:2)
Choline chloride: oxalic acid (1:1)
Choline chloride: oxalic acid (1:2)
30
9.
80
10.
11.
60
75
Highest yield in shortest reaction time shown in bold
a. Reaction condition: 4-hydroxy coumarin (1mmol), 4-chloro benzaldehyde (1mmol) and 2-amino 5-methyl thiazole
(1mmol) heated at 80˚C in 5 ml of respective solvent.
b. Yields refer to pure isolated products.
Further to evaluate the catalytic effect in order to establish the reaction conditions,
then model reaction was carried out in different catalysts (20mol %) such as citric acid,
PTSA, acetic acid, NaOH and proline but unfortunately inferior results were obtained. The
results are summarized in Table 2.

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Table 2 Optimization of catalysts for the synthesis of chromeno[4,3 d]thiazolo[3,2-
a]pyrimidin-6-one
Temp.
Conditions
(˚C)
Sr.
No.
Catalyst Load
(mol %)
Time Yieldb
(mins.) (%)
Catalyst
Citric acid
1.
2.
3.
4.
5.
6.
20
20
20
20
20
-
80
180
120
180
120
120
30
44
40
38
40
68
94
PTSA
80
80
80
80
80
Acetic acid
NaOH
Proline
Glycerol: Proline (1:1)
Highest yield in shortest reaction time shown in bold
a. Reaction condition: 4-hydroxy coumarin (1mmol), 4-chloro benzaldehyde (1mmol) and 2-amino 5-methyl thiazole
(1mmol) heated at 80˚C in 5 ml of respective solvent.
b. Yields refer to pure isolated products.
After the study of effect of solvent and catalyst, it is clear from table 2, the desired
product of 7-(aryl)-10-methyl-6H,7H-chromeno[4,3-d]thiazolo[3,2-a]pyrimidin-6-one was
obtained an excellent yield in Glycerol: Proline (1:1)(LTTM) (Table No. 2, Entry 6).The
results shown in table no.1 and 2 indicate that Glycerol: Proline (1:1)(1:1) LTTM can act as
suitable, eco-friendly reaction promoting medium. Remarkably, the workup procedure for
this reaction involves only simple filtration and washing of residue with hot ethanol and
diethyl ether to result in pure products.
The structures of the products were confirmed on the basis of spectroscopic data. The
infrared (IR) spectra of the compound code CA-4 showed the characteristic band at 1666.20,
1
1608.34 cm-1 due to the presence of a carbonyl group. The H NMR showed a singlet at δ,
6.252 due to benzylic (-CH) proton which confirms the cyclization. The signals appeared
13
between δ, 6.95 and 7.82 ppm represent the aromatic protons. In the C NMR spectrum the
signal appeared at δ, 22.50, 36.11, 58.70, 103.57, 115.80, 120.08, 120.71, 122.59, 129.25,

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124.51, 127.82, 128.70, 130.12, 131.28, 141.20, 152.81, 165.78, 168.70, 169.72 ppm indicate
the presence of aromatic carbons. Characteristic carbonyl carbons appeared at δ, 165.78,
168.70 ppm. Mass spectra of the compound show a molecular ion peak at m/z 380 (Mþ)
which confirmed the proposed structure.
Using these optimized reaction conditions, a series of 7-(aryl)-10-methyl-6H,7H-
chromeno[4,3-d]thiazolo[3,2-a]pyrimidin-6-one derivatives were synthesized in moderate to
an excellent yields from different aromatic aldehydes having electron donating as well as
electron-withdrawing groups. These substrate scopes are listed in Table No. 3.
Table 3 Synthesis of chromeno[4,3-d]thiazolo[3,2-a]pyrimidin-6-one derivatives using
LTTM (Entry 1-20)a
Entry No. Code
Aldehyde
Productc
Time Yieldb M.P.
(mins.)
(Structure)
(%)
(˚C)
O
O
H
N
O
1.
CA-1
30
92
260
H3C
S
N
O
Cl
S
O
35
30
92
92
242
243
CA-2
CA-3
H
N
O
2.
3.
H3C
Cl
O
N
Cl
O
N
O
H3C
H
S
N
Cl

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Cl
O
4.
5.
CA-4
30
94
88
243
256
O
H
N
O
H3C
S
Cl
N
Cl
Cl
O
CA-5
40
O
H
N
O
H3C
Cl
Cl
S
N
O2N
O
O
6.
7.
8.
CA-6
CA-7
CA-8
40
45
35
82
82
86
254
254
252
N
O
H
H3C
S
N
NO 2
NO2
O
O
N
O
H
H3C
S
N
NO 2
NO2
O
O
H
N
O
H3C
O2N
S
N
Br
O
O
9.
CA-9
38
92
258
N
O
H
H3C
S
N
Br

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OCH3
O
10.
CA-10
45
84
262
O
H
N
O
H3C
S
N
H3CO
S
N
O
O
H
S
11.
12.
13.
CA-11
CA-12
CA-13
35
35
45
84
80
88
200
228
264
O
H3C
S
N
O
N
O
O
H
O
O
H3C
S
N
OCH3
OCH3
O
O
H
N
O
H3C
S
N
H3CO
OCH 3
O
14.
15.
CA-14
CA-15
45
30
88
92
276
235
O
O
O
N
O
H3C
S
N
CH3
CH
3
O
N
O
H3C
S
N
CHO

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CH3
CH3
CH
3
16.
17.
35
86
94
˃300
O
CA-16
CH
3
N
O
H3C
S
N
F
CHO
F
F
CA-17
35
280
O
F
N
O
H3C
S
N
CHO
CF3
CF
3
O
18.
19.
20.
CA-18
CA-19
CA-20
40
45
45
82
90
92
238
258
246
N
O
H3C
S
N
CHO
O
O
O
O
O
O
H
N
O
H3C
S
N
F
F
O
N
O
H3C
S
N
CHO
CHO
21.
CA-21
--
720
--
--
H C
3

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H C
CHO
22.
CA-22
--
720
--
--
3
CH
3
a. Reaction condition: 4-hydroxy coumarin (1mmol), Aromatic aldehyde (1mmol) and 2-amino 5-methyl thiazole
(1mmol) heated at 80˚C in 5 ml of Glycerol: Proline (1:1) LTTM.
b. Yields refer to pure isolated products.
c. Products prepared for first time.
It is noteworthy to reveals that this methodology worked well for hetero-aromatic
aldehydes (Table 3). A plausible mechanism for the synthesis of 7-(aryl)-10-methyl-6H,7H-
chromeno[4,3-d]thiazolo[3,2-a]pyrimidin-6-one derivatives (10) is depicted in (Scheme 3).
The reaction may proceed through the formation of the Knoevenagel condensation of the
aromatic aldehyde (2) and 4-hydroxycoumarin (3) in glycerol: proline as a catalyst and
solvent generates an intermediate. The 2-amino 5-methyl thiazole (4) then undergoes
subsequent aza Michael addition reaction followed by intra-molecular cyclization with
carbonyl group of 4-hydroxycoumarin and amino group of 2-amino 5-methyl thiazole to give
Michael adduct undergoes dehydration to afford the desired product (10). The catalyst may
be increases the electrophilicity of the carbonyl groups in the reaction.

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H
O
OH
O
.
O
H
H
O
O
HN
OH
O
O
OH
O
.
H
O
+
O
H
HN
H
Ar
O
H
O
[3]
[1]
[2]
H
Ar
H
O
O
OH
O
H
.
O
O
O
H
S
HN
HN
-H O
2
O
H
N
O
Ar
[5]
O
H
H
O
O
H
O
O
S
OH
O
[9]
NH
O
N
O
N
O
.
O
H
HN
HO
O
O
Ar
H
H
O
O
O
S
H
O
NH
2
O
[10]
Ar
N
+
H
Ar
O
O
[6]
O
O
N
S
O
OH
H
N
2
Ar
Ar
[4]
O
H
O
O
-H O
2
[8]
[7]
Scheme 3 Mechanistic pathway of the 7-(aryl)-10-methyl-6H,7H-chromeno[4,3-
d]thiazolo[3,2-a]pyrimidin-6-one
4. Reusability of LTTM
The reusability of the catalyst is exceptionally important aspect of green chemistry. In
the present study, reusability of the LTTM was evaluated by using the reaction of 4-
Hydroxycoumarin (1 mmol), 4-Chlorobenzaldehyde (1 mmol), and 2-Amino 5-methyl
thiazole (1 mmol) in the Glycerol: Proline (1:1) LTTM under optimized conditions. After
completion of the reaction, as indicated by TLC, 10 ml of water was added to the reaction
mixture and the crude reaction product was separated by simple filtration. The LTTM was
recovered by evaporating the water at 80˚C under vacuum. The recycled LTTM was
successively reused for the next reaction and recycled another time (Fig. 5).

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Fig.5 Reusability of LTTM
5. Green Metric Calculations
Compliant with principles of green chemistry, green metrics such as Mass Intensity
(MI), Reaction Mass Efficiency (RME), Carbon Efficiency (CE), and Atom Economy (AE)
and E Factor have been considered as a gauge of greenness and environmental sustainability
in diminishing the amount of theoretical waste [54].
In ideal condition MI ≈ 1%, RME ≈ 100%, %CE ≈ 100 and %AE ≈ 100 and E-factor
≈ 0 is expected. To reveal the greenness of the present protocol, we have explored the results
in terms of reported green metric parameters [55].
The high yield of the product shows that the significant RME values and moderate
yield generate moderate RME values. In addition to this, all carbon atoms in the reactants are
present in the product shows excellent values of % CE. MI values and E factor of the reaction
are very close to the ideal values. The percent atom economy of each scaffold indicates
maximum conversion of starting materials into product and minimum waste exclusion. Mass
of catalyst is excluded as it is recyclable. (Fig. 6)

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Fig. 6 Green metrics calculations (The exact values are mentioned in the supporting
information). E- factor, MI mass intensity, % RME percentage reaction mass efficiency,
% CE percentage carbon efficiency, % AE percentage atom economy.
6. Mosquito Larvicidal Bioassay of synthesized chromeno[4,3 d]thiazolo[3,2-
a]pyrimidin derivatives
Mosquito is serious household pest in the society and mosquito borne diseases such
as, malaria, dengue fever, filariasis, yellow fever causing millions of death every year [56]. In
recent years, various numbers of heterocyclic scaffolds exhibited significant insecticidal
activity for the control of insect pests and vectors. Considering all the aspects, we performed
mosquito larvicidal bioassay of synthesized chromeno[4,3-d]thiazolo[3,2-a]pyrimidin
derivatives.
Insects rearing
Laboratory colony of mosquito species Aedes aegypti and Culex quinquefasciatus
were reared and maintained in plastic container containing water and the experiments were
carried out at 270±300 C and 50-90% relative humidity. Larvae were fed a diet of rabbit feed.

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Larvicidal bioassay
During preliminary screening in the laboratory trial, the larvae of Aedes aegypti and
Culex quinquefasciatus were collected from the insect rearing cage. During the study 5
different concentrations was prepared at the range of 25 μg/ml, 50μg/ml, 100μg/ml,
125μg/ml, 250μg/ml and 500μg/ml. The larvicidal bioassay was performed by the procedure
of WHO guidelines. Groups of 10, some 2nd and 3rd instars larvae of Aedes aegypti and Culex
quinquefasciatus were released in different plastic cups contain 50 ml water and compounds
with different concentrations were added from stock solution. Three replicates were
maintained for the individual compound along with the simultaneous control and untreated
groups. A control group was set up with DMSO-distilled water. During this experiment no
food was offered to larvae. Mortalities of treated larvae were determined after an exposure
period of 24 hrs. The larvae were considered dead, if they were unable to move or respond
when stimulated by probing with a needle. The experimental means in which 100% mortality
of larvae occurs, were selected for dose-response bioassay.
Dosage-response bioassay
Initially, all these synthesized compounds investigated at low to high concentrations.
The control was taken separately. The experiment replicated thrice. The results are expressed
in terms of percent mortality.
Based on the preliminary screening results, preliminary active compounds were
subjected to dose-response bioassay for larvicidal activity against the larvae of Aedes aegypti
and Culex quinquefasciatus. Again, the numbers of dead larvae were counted after 24 hrs of
exposure, and the percentage of mortality was reported from the standard of three replicates.

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Fig.7 Larvicidal assay against 3rd instars larvae of Aedes aegypti
Fig.8 Larvicidal assay against 3rd instars larvae of Culex quinquefasciatus
Statistical Analysis
The average larval mortality data were subjected calculating LC50, LC99, and other
statistics as fiducial limits and Regression equation (table 4 and 5) were calculated using the
SPSS software [57].
The results from the study showed that the chromeno fused scaffolds exhibited
larvicidal activity. No mortality was observed in control. All the compounds possesses

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moderate to excellent mosquito larvicidal activity. Compound CA-17 with LC50 value 68.41,
74.99 ppm and LC99 value 370.363, 170.77 ppm showed the highest larvicidal activity against
3rd and 4th instar larvae of both species Aedes aegypti, Culex quinquefasciatus respectively.
Compound code CA-2 and CA-20 compounds also exhibited larvicidal activity against Aedes
aegypti with LC50 value 141.64 ppm, 98.17 ppm and LC99 value 503.28 ppm, 465.38 ppm
respectively. Whereas CA-2 and CA-20 compounds showed larvicidal activity against Culex
quinquefasciatus with LC50 value 79.78 ppm, 107.42 ppm and LC99 value 391.89 ppm,
371.63 ppm respectively.
Table 4 Statistical data of mosquito larvicidal bioassay of selected derivatives
Statistical data of Aedes aegypti
Entry Compd. LC50
Fiducial Limit
LC99
Fiducial Limit
Regression equation
No.
1.
2.
3.
4.
5.
6.
7.
8.
9.
Code
CA-2
CA-4
CA-5
CA-9
141.64 123.88 – 181.00 503.28 440.61 – 593.84 Y= 0.006 + 0.911 log x
181.73 104.01 – 290.05 641.94 454.74 – 1285.67 Y= 0.005 + 0.191 log x
207.99 124.62 – 356.35 612.27 425.90 – 1367.50 Y= 0.006 + 1.193 log x
375.69 240.09 – 1017.74 898.82 584.67– 7511.33 Y= 0.004 + 1.671 log x
CA-12 271.68 165.60 – 523.26 976.71 647.94 – 2573.86 Y= 0.003 + 0.896 log x
CA-17 68.41 50.18 – 83.95 370.36 318.57 – 450.66 Y= 0.008 + 0.527 log x
CA-19 274.09 184.75 – 453.742 931.05 518.03 – 1506.17 Y= 0.005 + 1.395 log x
CA-20
98.17
--
79.17 – 116.11
465.38 401.56 – 561.73 Y= 0.006 + 0.622 log x
-- -- --
Control
--
LC50 =Lethal concentration 50 at which 50% of target population died.
LC99 = Lethal concentration 99 at which 99% of target population died.

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Table 5 Statistical data of mosquito larvicidal bioassay of selected derivatives
Statistical data of Culex quinquefasciatus
Entry Compd. LC50
Fiducial Limit
LC99
Fiducial Limit
Regression equation
No.
Code
1.
CA-2
79.78
25.30 – 120.78
391.89 230.58 – 648.49 Y= 0.010 + 0.773 log x
2.
3.
4.
5.
6.
7.
CA-5
CA-9
400.41 298.76 – 681.28 893.09 636.76 – 1871.23 Y= 0.005 + 1.891 log x
294.40 152.85 – 1019.19 856.55 532.74 – 4963.81 Y= 0.004 + 1.218 log x
CA-15 320.57 271.33 – 583.77 876.83 602.24 – 2042.08 Y= 0.004 + 1.341 log x
CA-17
CA-20 107.42
Control --
74.99
68.97 – 81.01
11.18 – 245.87
--
170.77 156.37 – 190.28 Y= 0.024 + 1.822 log x
371.63 298.71 – 2203.36 Y= 0.009 + 0.946 log x
--
--
--
LC50 =Lethal concentration 50 at which 50% of target population died.
LC99 = Lethal concentration 99 at which 99% of target population died.
7. Conclusion
In summary, Glycerol: proline (1:1) LTTM was successively reported and also used
for one pot multi-component synthesis of chromeno[4,3-d]thiazolo[3,2-a]pyrimidin
derivatives as a biocompatible solvent/ catalyst. The LTTM plays a dual role such as
solvent/catalyst with better reusability. This protocol has major advantages such as
operational simplicity, green, efficient protocol in designer solvents, easy work-up procedure,
and good to excellent yields, column chromatography-free technique and an excellent
correlation with green metrics calculations. Hence, by providing a greener alternative route, a
highly efficient protocol was developed which can easily replace existing methods. The
present methodology will be attractive, convenient, and green alternative path for simple
catalytic organic transformations to develop bioactive molecules with structural diversity and
molecular complexity. Also, chromeno[4,3-d]thiazolo[3,2-a]pyrimidin derivatives were
found to have mosquito larvicidal bioassay in laboratory condition with moderate to excellent
results in terms of LC50 and LC99 and percentage mortality after 24hrs.

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8. Spectroscopic data of the synthesized compound
7-(4-chlorophenyl)-10-methyl-6H,7H-chromeno[4,3-d]thiazolo[3,2-a]pyrimidin-
6-one (Table 3 Entry 4, compd. code CA-4)
White powder; yield 94%; mp 243˚C; IR (ν max): 2915.84, 1666.2, 1608.34, 1518.67,
1
1484.92, 1397.17, 1179.26, 1035.59 cm-1; H NMR (300 MHz, DMSO-d6): δ, 2.223
(s, 3H, CH3), 6.252 (s, 1H, -CH), 6.954 (s, 1H, Ar-H), 7.129–7.209 (t, 4H, Ar-H),
7.431–7.454 (d, 1H, Ar-H, J=6.9 Hz), 7.798–7.823 (d, 2H, Ar-H, J=7.5 Hz) ppm; 13C
NMR (75 MHz, DMSO-d6): δ, 22.50, 36.11, 58.70, 103.57, 115.80, 120.08, 120.71,
122.59, 129.25, 124.51, 127.82, 128.70, 130.12, 131.28, 141.20, 152.81, 165.78,
168.70, 169.72 ppm; DEPT90: 75 MHz, DMSO-d6): δ, 36.085, 115.796, 122.240,
123.238, 124.581, 127.863, 128.555, 131.188 ppm; mass (m/z): 380.84742 (M+).
Anal. calcd. For C20H13ClN2O2S: C, 63.07; H, 3.44; N, 7.36%; Found: C, 63.02; H,
3.40; N, 7.32%.
(Remaining all spectral data of synthesized compounds mentioned in supporting information)
Acknowledgement
Authors thank the Department of Science and Technology New Delhi, Govt. of India
for the award of INSPIRE fellowship JRF (IF150988) for financial support. We gratefully
acknowledged to Department of Chemistry, Shivaji University, Kolhapur, for providing
NMR spectral analysis. Also, we would like to thank Ross Life Science, Pune for providing
laboratory facilities.
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