A metal-free regioselective multicomponent approach for the synthesis of free radical scavenging pyrimido-fused indazoles and their fluorescence studies

Article abstract of DOI:10.3390/molecules21111571

This study deals with a new and efficient metal-free regioselective synthesis of pyrimido-fused indazoles with nitrogen ring junction motifs. We have developed a metal-free domino type reaction between 3-aminoindazole, aryl aldehydes and aceotophenones in the presence of KOH/DMF that leads to pyrimido[1,2-b]indazole analogues. Response Surface Methodology (RSM) coupled with a Box-Behnken design (BBD) were utilized for exploring the effect of base used (A), temperature of reaction (B) and (C), reaction time. This approach can allow access to a variety of pyrimidoindazole fluorophores and related compounds. The compound N,N-dimethyl-4-(2-phenylpyrimido[1,2-b]indazol-4-yl)aniline (4e) displays the maximum fluorescence intensity at 518 nm and shows a fluorescence quantum yield of 0.068. The synthesized pyramido-fused indazoles have been evaluated for their free radical scavenging activity and compound 4f showed good antioxidant activity.

Full text of DOI:10.3390/molecules21111571

molecules
Article
A Metal-Free Regioselective Multicomponent
Approach for the Synthesis of Free Radical
Scavenging Pyrimido-Fused Indazoles and
Their Fluorescence Studies
Jeyakannu Palaniraja 1, Selvaraj Mohana Roopan 1,*, G Mokesh Rayalu 2,
Naif Abdullah Al-Dhabi 3 and Mariadhas Valan Arasu 3
1
Chemistry of Heterocycles & Natural Product Research Laboratory, Department of Chemistry,
School of Advanced Sciences, VIT University, Vellore 632014, Tamilnadu, India; myne.palani@gmail.com
G Mokesh Rayalu, Department of mathematics, School of Advanced Sciences, VIT University,
2
Vellore 632014, Tamilnadu, India; mokesh.g@gmail.com
Department of Botany and Microbiology, Addiriyah Chair for Environmental Studies, College of Science,
3
King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia; naldhabi@ksu.edu.sa (N.A.A.-D.);
mvalanarasu@gmail.com (M.V.A.)
*
Correspondence: selvarajmohanaroopan@gmail.com or mohanaroopan.s@vit.ac.in;
Tel.: +91-98656-10356 or +91-04162-202352; Fax: +91-416-224-5544 or +91-416-224-5766
Academic Editor: Roman Dembinski
Received: 23 September 2016; Accepted: 11 November 2016; Published: 18 November 2016
Abstract: This study deals with a new and efficient metal-free regioselective synthesis of
pyrimido-fused indazoles with nitrogen ring junction motifs. We have developed a metal-free domino
type reaction between 3-aminoindazole, aryl aldehydes and aceotophenones in the presence of
KOH/DMF that leads to pyrimido[1,2-b]indazole analogues. Response Surface Methodology (RSM)
coupled with a Box-Behnken design (BBD) were utilized for exploring the effect of base used (A),
temperature of reaction (B) and (C), reaction time. This approach can allow access to a
variety of pyrimidoindazole fluorophores and related compounds. The compound N,N-dimethyl-
4-(2-phenylpyrimido[1,2-b]indazol-4-yl)aniline (4e) displays the maximum fluorescence intensity
at 518 nm and shows a fluorescence quantum yield of 0.068. The synthesized pyramido-fused
indazoles have been evaluated for their free radical scavenging activity and compound 4f showed
good antioxidant activity.
Keywords: N-fused pyrimidine; Strecker synthesis; A3 coupling; Box-Behnken design (BBD);
fluorescence
1. Introduction
Multi-component reactions (MCRs) play an important role in combinatorial chemistry [
This method has the capability for producing desired small molecule drugs with various degrees
of structural diversity [ ]. When three or more than three substances or reactants are reacted
simultaneously to produce defined target molecules these reactions are said to be MCRs. The products
display features of all the inputs and thus afford greater possibilities for molecular diversity [ ].
The cascade/domino/tandem process [ ], involves subsequent transformations of functionalities
produced in the previous step [ ]. Normally, these reactions are easier to carry out by one pot
synthesis than multi-step synthesis [ ]. In addition, the isolation of an intermediates are not required
in MCR conditions and they are perfect candidates for drug discovery and combinatorial synthesis [ ].
1].
2
3
4–6
7
8
9
The most desirable potential drug candidates are libraries of small organic molecules which have
limitations as bioavailable therapeutics. We can built up very large libraries with in short time by
Molecules 2016, 21, 1571; doi:10.3390/molecules21111571
www.mdpi.com/journal/molecules

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using a small set of reactants [10]. Conversely, the significance of MCRs for discovery of novel drugs
has been verified certified by substantial industries with academic aspects [11]. The studies have
aimed to develop efficient MCR protocols for the generation of a series of heterocyclic compounds.
MCRs such as the Strecker synthesis [12], Hantzsch synthesis [13], Biginelli synthesis [14], Mannich
reaction [15], Kabachnik-Fields reaction [16], Bucherer-Bergs reaction [17], Gewald reaction [18],
Willgerodt-Kindler reaction [19], Ugi reaction [20] and A3 coupling [21] are named reactions which
include the carbonyl compounds as one of the reactants. The synthetic arylation via transition
metal-free conditions [22] and synthesis of a series of unreported N-fused heterocycles [23,24] was
reported by our group. Very recently, our research group proposed a metal catalysed method for the
facile preparation of pyrimido-fused indazoles [25]. This is a metal catalysed A3 coupling reaction
between aminoindazoles, alkynes and aldehydes, that further proceeds via 6-endo-dig cyclization
focusing on the functionalization of pyrimido[1,2-b]indazole derivatives. As a continuation in our
synthetic strategy, we report now a method to synthesize highly functionalized pyrimido-fused
indazole derivatives in good to excellent yields via cascade/tandem/domino reactions under
metal-free conditions. It offers simple reaction conditions, easy protocols and cheap starting materials.
2. Results and Discussion
The proposed protocol for the preparation of 2,4-diphenylpyrimido[1,2-b] indazole (4a) via
the proposed metal-free cascade process is illustrated in Scheme 1. Initially, acetophenone 2a and
benzaldehyde 3a were undergo an aldol reaction in the presence of base providing α,β-unsaturated
carbonyl intermediate II. When intermediate II is coupled with an amine with the removal of one
water molecule it gives an imine (intermediate III). Then intermediate III undergoes electrocyclization
followed by auto-oxidation leading to the formation of 4a.
Scheme 1. Plausible mechanism of the synthesis of compound 4a.
Substituted aldehydes and acetophenones were transformed into the desired products without any
difficulty, which clearly indicates that steric hindrance will not affect product formation. Unfortunately,
nitro- and hydroxyl-substituted aromatic aldehydes failed to provide the target coupling products.
We have investigated the gram scale preparation of compound 4d (Scheme 2) from 20 mmol of
3-aminoindazole (1), 20 mmol of acetophenone (5a) and 20 mmol of 4-methoxy-benzaldehyde (3d)
with good yield (89%). The synthesized compound 4d was characterized by melting point, proton and
carbon NMR and HRMS. Along with this data, we have confirmed the structure of 4d by single crystal
XRD data (CCDC 1456539). The ORTEP diagram is illustrated in Figure 1.

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Scheme 2. Compounds 4d gram scale preparation.
Figure 1. The ORTEP representation of compound 4d.
RSM is a statistical and mathematical method for empirical modelling and we have optimized
a response (output variables) influenced by numerous independent variables (input variables) with
a careful design of experiments. It can analyse the effect of multiple or combined variables in
a minimal number of experiments and to evaluate the influences of experimental factors on the response
by an accurate second order polynomial equation [26–32]. Aldehydes react with phenylacetylene
and aminoindazole to give pyrimido[1,2-b]indazoles by C-2 subsitution in the presence of a metal
(Scheme 1). Contrarily, aldehyde substitution occurs at the C-4 position in the absence of a metal
catalyst. As part of our continuing effort, our research group has disclosed a simple and highly effective
method for the preparation of N-fused pyrimido[1,2-b]indazoles 4(a–s) via a domino process. RSM
coupled with BBD was used for various conditions to fine-tune the working parameters. The selection
level is based on teach variables (Table 1).
Table 1. A selection of levels and variables used for BBD.
Levels
Reaction
Variables
Code
Units
−1
1
0
2
+1
3
Base used (equivalent)
Reaction temperature
Reaction time
A
B
mg
Metal free cascade Reaction
◦
C
110
4
120
8
130
12
C
h

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From the optimized A3 coupling method [25], we have observed that the aldehyde easily
substituted at the C-2 position in the pyrimido-fused indazoles. In order to increase the scope
of the products, we have developed another novel synthetic route to synthesize pyrimido-fused
indazoles via metal-free one pot multi-MCRs. In this case, we found that the aldehydes are certainly
substituted at C-4 position in the core structure. This cascade reaction proceeds under metal-free
conditions with the reaction between aldehydes, ketones and amines. Our research group started
performing reactions between 3-aminoindazole (1), acetophenone (2a) and benzaldehyde (3a) under
neat conditions for 8 h, but unfortunately the expected compound was not identified (Table 2, entries 1
and 2). The conditions of reactions are optimized by varying organic solvent, organic bases and
inorganic bases (Table 1, entries 3–15) but the expected target molecule was obtained between 16%–93%.
After the various combinations of bases and solvents, we have found that the expected product
2,4-diphenylpyrimido[1,2-b]indazole 4a in 56% yield was noted in KOH and EtOH combination at
80 ◦C for 8 h (Table 1, entry 9). In order to increase the yield further, we have tried with KOH base in
various organic solvents like MeOH, DMSO, DMF, 1,4-dioxane, THF and acetonitrile. Finally, we have
attained upto 93% yield in KOH/DMF combination at 120 ◦C for 8 h (Table 1, entry 12).
Table 2. Optimization of cascade reaction to synthesis compound 2,4-diphenylpyrimido[1,2-b]indazole
(4a) via metal free conditions a.
◦
Yield b (%)
Entry
Base
Solvent
Temp ( C)
1
2
3
4
5
6
7
8
9
-
-
-
EtOH
EtOH
EtOH
EtOH
t-BuOH
t-BuOH
EtOH
100
80
80
80
80
100
100
80
NR c
NR c
NR c
Traces
NR c
40
30
50
56
Triethylamine
Na2CO3
Piperidine
NaOtBu
KOtBu
NaOH
KOH
EtOH
80
10
11
12
13
14
15
KOH
KOH
KOH
KOH
KOH
KOH
MeOH
DMSO
DMF
1,4-Dioxane
THF
70
40
45
93
25
16
16
120
120
80
80
80
Acetonitrile
a
Reactions proceed with 1 mmol of
1
,
2a and 3a in 5 mL of solvent. Optimized condition are denoted in bold
b
letters there are none. The reactions were carried out at various temperatures for 8 h. Isolated yield and
c
NR-No reaction. Letters refer to wrong things.
The BBD statistical design is presented in Table 3. We have utilized three variables such as
base (A), temperature (B) and time (C) for the reactions without metal. By using multi-regression and
backward eradication, the best-fitting models were determined.
Depending on the reaction phenomenon, we have found the yield range was 41%–93%. Due to
the reaction performed by the above method the correlation between the process variables and the
isolated yield (Y) with a quadratic polynomial model is represented in Equation (1):
Y = 90.80 + 8.25A + 2.88 B + 17.13C − 2.50 AB − 1.00 AC − 1.25 BC − 13.03 A − 9.28 B − 21.27C (1)
where A, B and C represents the variables coded and Y represents isolated yield. Figure 2 depicts the
respectable correlation linear between actual and predicted yields.

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Table 3. BBD matrix response and its design a.
Metal Free Condition
Run
A
B
C
Y (%)
X (%)
2
2
1
1
3
2
3
3
2
3
1
2
2
1
2
2
2
120
120
120
130
130
110
110
120
120
120
110
110
130
120
120
130
120
8
8
4
8
8
4
8
4
8
12
8
12
4
12
8
12
8
93.01
89.00
40.33
62.03
82.09
34.12
80.31
50.41
91.01
71.00
50.21
82.07
41.09
65.21
88.44
84.51
93.01
90.80
90.80
30.12
65.62
77.12
39.00
76.37
48.62
90.80
80.88
54.87
75.75
47.25
66.37
90.80
79.00
90.80
a
Y—Experimental Yield, X—Predicted Yield.
Figure 2. The predicted and actual isolated yield values.
ANOVA results for response surface quadratic models are shown in Table 4. The F-value model is
11.57 and it suggests the model was significant. F value has a chance of 0.002% of being due to noise.
For this model A, C, A2, B2, C2 are significant model terms. The values <0.1 represent that the model
terms are non-significant. The lack of fit F value of 25.69 suggests that lack of fit is significant. The “Pred
R-Squared” of 0.0372 is not as close to the “Adj R-Squared” of 0.8560 as one might usually expect;
i.e., the difference is more than 0.002. All empirical models should be tested by doing confirmation
runs. “Adeq Precision” noise to signal ratio measures and ratio <4 is desirable. The adequate signal
with the ratio of 10.194 implies that we utilized the above model for space design navigation. Effect of
interaction between base (A) and temperature of the reaction (B) time constant of reaction of 8 h (C)
(Figure 3).

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Table 4. Quadratic model by ANOVA for response surface a.
Source
Sum of Squares
DF
Mean Square
F Value
p-Value Prob > F
Model
A
B
C
AB
AC
BC
A
B
6271.98
544.50
66.13
2346.12
25.00
9
1
1
1
1
1
1
1
1
1
7
3
4
696.89
544.50
66.13
2346.12
25.00
11.57
9.04
1.10
38.96
0.42
0.066
0.10
11.86
6.01
31.65
0.0020
0.0197
0.3295
0.0004
0.5399
0.8040
0.7567
0.0108
0.0439
0.0008
S
4.00
6.25
4.00
6.25
714.32
362.21
1905.79
421.55
400.75
20.80
714.32
362.21
1905.79
60.22
133.58
5.20
C
Residual
Lack of Fit
Pure Error
Correlation Total
25.69
0.0045
S
6693.53
16
a
Where S—Significant, DF—Degree of freedom.
Figure 3. Contour plots for parameters combined yield for reaction conditions without metal. (
I) Effect
of A and B; (II) Effect of A and C; (III) Effect of B and C.
Plots of contour base (A) and temperature (B) (Figure 3IB) are circular, which result in lower
a interaction between A and B. Plots of contour base (A) and time (C) (Figure 3IIB), temperature (B)
and time (C) (Figure 3IIIC) are elliptical, which shows a good relation of interaction between A and C,
and between B and C. The optimized condition for predicted and actual isolated yields is revealed
in Table 5.
Table 5. Model validation for metal free reaction.
◦
Parameter
Base Equivalent (A)
Reaction Temperature C (B) Reaction Time (min) (C) % Yield
Predicted
Experimental
2.23
2.0
125.3
120.0
9.17
8.0
93.5
93.0

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To further confirm the suitability for this model for identifying a higher yield, three confirmation
runs were performed using the optimum parameters. The isolated yield results of compound 4a
were 93.0%, 92.4% and 93.7%. To validate the efficiency of the metal-free conditions, the choice of the
reactants were explored under the optimized condition and the results are indicated in Table 6. A series
of amines 1(a–d), aryl aldehydes 3(a–k) and acetophenones 2(a–f) were subsequently converted into
the corresponding pyrimido-fused indazoles 4(a–s) in good to moderate yields.
Table 6. Syntheses of N-fused pyrimidine derivatives 4(a–p).
2
3
1
4(a-p)

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The synthesized N-fused analogues
4
(a
–
s) were orange to red powders. The solvatochromism
spectra of compound 4a was studied with fifteen organic solvents (Figure S1). Ethyl acetate shows
higher UV/Vis absorbance and we have recorded UV/Vis absorbances (Figure S2) and fluorescence
emission spectra for all synthesized compounds 4(a–p) in EtOAc at 10−5 M concentration.
Compound 4e displays a maximum fluorescence intensity at 518 nm (Figure 4). The fluorescence
quantum yield [33] (
Equation (2):
ΦF) of the synthesized compounds 4(a–p) have been calculated by using following
Φ = (Φ ×I × OD × η )/(I × OD × η )
(2)
where ΦR = fluorescence quantum yield of reference, IS and IR = integral area of reference and
sample, respectively, ODS and ODR = excited absorbance of sample and reference respectively, and
s
F
R
S
R
R
S
R
η
s
ηR = refractive index of sample solvent and reference solvent respectively. We have used tryptophan
as a standard for calculating the emission quantum yield.
Figure 4. Fluorescence emission spectra the compounds 4(a–p).
The fluorescence quantum yield of the synthesized compounds 4(a–p) ocurrs in the range of 0.010
to 0.068. The compound N,N-dimethyl-4-(2-phenylpyrimido[1,2-b]indazol-4-yl)aniline (4e) shows
a fluorescence quantum yield of 0.068 (Table 7).
Table 7. The optical properties of the synthesized pyrimido[1,2-b]indazolederivatives 4(a–p).
Entry
Λmax (abs, nm)
Λmax (em, nm)
Stokes Shift (nm)
OD
I
Φ
F
Tryptophan
280
355
75
0.384
158,517
0.130
[32]
4a
4b
4c
306
306
308
270 (Sh)
310
300
344 (Sh)
310
306
533
530
553
553
524
518
518
524
534
534
227
224
245
283
214
218
174
214
228
264
0.774
0.957
1.064
0.838
1.505
0.870
0.788
1.505
1.883
1.251
46,182
90,759
31,279
31,279
115,328
165,016
165,016
115,328
73,195
73,195
0.019
0.031
0.010
0.012
0.025
0.062
0.068
0.025
0.013
0.019
4d
4e
4h
4i
270

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Table 7. Cont.
Entry
Λmax (abs, nm)
Λmax (em, nm)
Stokes Shift (nm)
OD
I
Φ
F
4j
4k
4l
4m
4n
300
314
320
308
272
322 (Sh)
270
306
534
532
540
535
530
530
543
499
499
234
218
220
227
258
208
273
193
231
1.140
1.950
2.070
1.794
0.449
0.439
1.761
1.176
0.827
61,312
83,407
65,088
70,535
54,760
54,760
29,458
59,599
59,599
0.017
0.014
0.010
0.013
0.040
0.041
0.005
0.016
0.023
4o
4p
268 (Sh)
Sh = shoulder; abs = absorbance; em = emission; OD = excited absorbance; I = integral area; ΦF = Fluorescence
quantum yield.
We have evaluated the free radical scavenging property of the synthesized pyrimido-fused
indazoles at 0.001 mM and 0.002 mM and its IC50 values were measured. We have calculated the IC50
values and compound 4f showed excellent antioxidant properties as 3.08 and compounds 4g (8.91),
4n (9.24), 4k (8.59) and 4o (5.60) showed significant antioxidant properties compared to ascorbic acid
(4.50; Supplementary Materials Table S1).
3. Materials and Methods
3.1. General Information
All commercially available reagents were used without any further purification and the reactions
were monitored by TLC. 1H- (400 MHz) and 13C-NMR (100 MHz) were obtained using an Avance
400 Mz spectrometer (Bruker, Oestliche Rheinbrueckenstr, Karlsruhe, Germany, Europe) in CDCl3
with TMS as an internal standard. Chemical shift values (δ) are expressed in parts per million
(ppm). Abbreviations are as follows: s, singlet; d, doublet; t, triplet; m, multiplet. Melting points
were measured on an Elchem microprocessor- based DT apparatus (Geninune Sientific Instruments,
Chennai, Tamilnadu, India) using open capillary tubes and are corrected with benzoic acid. Mass
spectra were obtained on a high resolution mass spectrometer (Bruker, Oestliche Rheinbrueckenstr,
Karlsruhe, Germany, Europe). UV-vis spectra were obtained on a UV-2550 instrument (Shimadzu
Corporation, Kyoto, Japan). The fluorescence spectra were obtained on a F-7000 FL spectrophotometer
(HitachiPerkin-Elmer, Bengaluru, Karnataka, India).
3.2. General Procedure for the Synthesis of 2,4-Diphenylpyrimido[1,2-b]indazoles 4(a–s) via Metal
Free Conditions
To a mixture of 1H-indazol-3-amine (1 mmol), aldehyde (1 mmol) and acetophenone (1 mmol)
in 5 mL of dimethylformamide potassium hydroxide (2.2 mmol) was added at room temperature.
The reaction mixture was heated to 125 ◦C for 9 h. The progress of the reaction was monitored by
TLC. After the completion of the reaction, the mixture was poured into crushed ice. The mixture was
extracted by ethyl acetate and water. The organic layer was separated and dried over sodium sulphate
and the solvent evaporated. The crude product was purified by column chromatography to afford the
product as a solid.
3.3. Characterization Data
4-(4-Isopropylphenyl)-2-phenylpyrimido[1,2-b]indazole (4b); Brown solid; Isolated yield 86%; m.p.:
◦
1
170–172 C; H-NMR (CDCl3)
7.96–7.85 (m, 1H), 7.86 (d, J = 8.0 Hz, 1H), 7.74 (s, 1H), 7.63–7.549 (m, 7H), 7.32–7.28 (m, 1H),
3.08–3.01 (m, 1H), 1.35 (d, J = 6.8 Hz, 6H); 13C-NMR (CDCl3)
23.8, 34.3, 108.4, 113.9, 116.5, 120.6,
δ 8.42 (d, J = 8.0 Hz, 1H), 8.28–8.26 (m, 2H), 8.18–8.16 (m, 2H),
δ

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121.2, 126.5, 127.0, 127.2, 128.0, 128.3, 128.5, 129.0, 129.2, 129.5, 130.0, 133.0, 137.4, 145.0, 145.4, 151.6,
152.2, 152.6; HRMS (EI): m/z calcd. for C25H21N4 363.1735 found 363.1735.
4-(4-Methoxyphenyl)-2-phenylpyrimido[1,2-b]indazole (4d); Yellow solid; Isolated yield 90%; m.p.:
188–190 ◦C; 1H-NMR (CDCl3)
δ
8.40 (d, J = 8.0 Hz, 1H), 8.26–8.22 (m, 4H), 7.84 (d, J = 8.8 Hz, 1H),
7.69 (s, 1H), 7.56–7.49 (m, 4H), 7.29–7.18 (m, 1H), 7.14–7.11 (m, 2H), 3.91 (s, 3H); 13C-NMR (CDCl3)
55.5, 107.8, 113.9, 114.2, 116.5, 120.5, 121.2, 123.9, 127.2, 129.0, 129.7, 130.0, 131.2, 137.5, 145.0, 145.1,
δ
151.5, 152.5, 161.7; HRMS (EI): m/z calcd. for C23H17N3O 351.1372 found 351.1370.
N,N-Dimethyl-4-(2-phenylpyrimido[1,2-b]indazol-4-yl)aniline (4e); Brown solid; Isolated yield 85%; m.p.:
180–182 ◦C; 1H-NMR (CDCl3)
δ
8.44 (d, J = 8.0 Hz, 1H), 8.33–8.30 (m, 4H), 7.99 (d, J = 8.4 Hz, 1H),
7.75 (s, 1H), 7.65–7.51 (m, 4H), 7.32–7.28 (m, 1H), 6.92 (d, J = 8.0 Hz, 2H), 3.13 (s, 6H); 13C-NMR (CDCl3)
40.1, 106.8, 111.6, 113.8, 116.4, 118.4, 120.1, 121.3, 127.2, 128.9, 129.5, 129.8, 130.8, 137.8, 145.2, 145.7,
δ
151.5, 152.1, 152.5; HRMS (EI): m/z calcd. for C24H20N4 364.1688 found 364.1688.
4-(Naphthalen-1-yl)-2-phenylpyrimido[1,2-b]indazole (4j); Brown solid; Isolated yield 85%; m.p.:
248–250 ◦C; 1H-NMR (CDCl3)
δ
8.47 (d, J = 8.0 Hz, 1H), 8.30 (d, J = 7.6 Hz, 2H), 8.12 (d, J = 8.0 Hz,
1H), 8.00 (d, J = 8.0 Hz, 1H), 7.86 (d, J = 6.8 Hz, 1H), 7.79 (s, 1H), 7.76–7.65 (m, 2H), 7.57–7.31 (m,
8H); 13C-NMR (CDCl3)
110.8, 114.0, 116.8, 120.8, 121.1, 125.2, 125.4, 126.6, 127.1, 127.2, 128.2, 128.7,
δ
129.1, 129.7, 129.8, 130.2, 130.7, 131.1, 133.7, 137.2, 144.7, 145.1, 151.7, 152.1; HRMS (EI): m/z calcd. for
C26H17N3 371.1422 found 371.1422.
4-(Furan-2-yl)-2-phenylpyrimido[1,2-b]indazole (4k); Brown solid; Isolated yield 78%; m.p.: 172–174 ◦C;
1H-NMR (CDCl3)
δ
8.49–8.48 (m, 1H), 8.36 (d, J = 8.4 Hz, 1H), 8.25 (d, J = 8.0 Hz, 2H), 8.16 (s, 1H), 7.84
(d, J = 8.8 Hz, 1H), 7.70 (s, 1H), 7.83–7.79 (m, 4H), 7.25 (t, J = 8.0 Hz, 1H), 6.73-6.72 (m, 1H); 13C-NMR
(CDCl3) 103.4, 113.2, 113.8, 116.4, 119.7, 120.7, 121.3, 127.2, 129.0, 130.0, 134.4, 137.6, 144.7, 145.0, 145.4,
151.8; HRMS (EI): m/z calcd. for C20H13N3O 311.1059 found 311.1058.
δ
◦
2-Phenyl-4-(thiophen-2-yl)pyrimido[1,2-b]indazole (4l); Brown solid; Isolated yield 86%; m.p.: 190–192 C;
1H-NMR (CDCl3)
δ 8.59–8.58 (m, 1H), 8.42 (d, J = 7.6 Hz, 1H), 8.28 (d, J = 8.0 Hz, 2H), 8.09 (s, 1H), 7.94
(d, J = 8.8 Hz, 1H), 7.79 (d, J = 5.2 Hz, 1H), 7.68–7.52 (m, 4H), 7.36–7.30 (m, 2H); 13C-NMR (CDCl3)
δ 104.8, 113.9, 116.5, 120.8, 121.4, 127.2, 127.7, 129.0, 130.0, 130.1, 131.3, 132.1, 132.2, 137.5, 138.5, 145.2,
151.4, 152.0; HRMS (EI): m/z calcd. for C20H13N3S 327.0830 found 327.0830.
3.4. Experimental Design and Mathematical Model
An experimental design was established for the series of parameters used for the synthesis of
2,4-diphenylpyrimido [1,2-b] indazoles by two reaction methods such as metal mediated and metal-free
conditions. The model was built by Response Surface Methodology (RSM) with the Design-Expert
Version 9.0.5.1 software (Stat-Ease, Inc., Minneapolis, MN, USA). Levels of selection for each variable
were based on the results of the preliminary studies. The three components for each reaction method,
such as the catalyst loading (A1), reaction temperature (B1) and response time (C1) were utilized
for metal mediated reaction. For metal free reaction, we have used base equivalent (
A2), reaction
temperature ( 2) and reaction time ( 2) as three factors. The actual isolated yields Y1 and Y2 were
B
C
chosen to be the target or response parameter as dependent variables. The values X1 and X2 are
the predicted isolated yields. Seventeen sets of experiments were performed for each both reaction
methods according to Box-Behnken experimental design (BBD). The variables were tested at the three
levels by associating negative sign ( 1) for lower level, Zero (0) indicating the core value and plus
−
signs (+1) for higher stages (Table 1). The experimental design matrix and their effects are presented in
Table S1. The quadratic polynomial equation recommended by RSM was used to predict the optimal

Molecules 2016, 21, 1571
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value and examine the interaction between the response of experimental design (actual yield) and the
variables (process parameters). The general form of quadratic polynomial was as follows:
2
2
Y= β0 + β1X1 + β2X2 + β3X3 + β11X1 + β22X2 + β33X32 + β12X1X2 + β13X1X2+ β23X2X3 (3)
where β0 is constant coefficient of the models. The regression coefficients (β1
, β2 and β3), (β11, β22 and
β33) and (β12 β13 and β23) respectively represent linear, quadratic and interaction effects of the model
,
estimated by multiple regression analysis.
3.5. Antioxidant Activity
The free radical scavenging activity of the pyrimido-fused indazoles was determined according
to the method using our earlier reports [34].
4. Conclusions
A metal-free MCR has been developed to synthesize a series of pyrimido-fused indazoles
via cascade/tandem transformation. This KOH/DMF mediated three component cascade type
transformation offers good to moderate yields. It offers good functional group tolerance. The RSM
coupled with BBD results displayed the significance of the quadratic model and offered fine-tuned
conditions for the preparation of pyrimido[1,2-b]indazole derivatives. The synthesized analogues
showed better fluorescence properties. The synthesized pyrimido-fused indazoles were evaluated as
good free radical scavengers.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/
11/1571/s1.
Acknowledgments: The corresponding author acknowledges the support provided by research grant DST-SERB
(No. SB/FT/CS-126/2012) fund. Naif Abdullah Al-Dhabi and Valan Arasu extend their sincere thanks to
the Deanship of Scientific Research, King Saud University for its funding to the Prolific Research Group
(PRG-1437-28). Also we express our sincere thanks to VIT-SIF for giving permission to record the NMR, UV/Vis
and fluorescence spectra.
Author Contributions: The molecules have been synthesized by Jeyakannu Palaniraja. Also manuscript has been
written by him. The work plan has been suggested by Selvaraj Mohana Roopan. G Mokesh Rayalu helped for
RSM optimization. Mariadhas Valan Arasu grown the single crystal. Antioxidant activity has been carried out by
Naif Abdullah Al-Dhabi. Scientific manuscript has been verified and approved by Selvaraj Mohana Roopan.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
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(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).