Structure-based design, synthesis and biological evaluation of a newer series of pyrazolo[1,5-a]pyrimidine analogues as potential anti-tubercular agents

Article abstract of DOI:10.1016/j.bioorg.2019.02.044

In-depth study of structure-based drug designing can provide vital leads for the development of novel, clinically active molecules. In this present study, twenty six novel pyrazolo[1,5-a]pyrimidine analogues (6a-6z) were designed using molecular docking studies. The designed molecules were synthesized in good yields. Structural elucidation of the synthesized molecules was carried out using IR, MS, ¹H NMR and ¹³C NMR spectroscopy. All the synthesized compounds were evaluated for their in-vitro anti-tubercular activity against H37Rv strain by Alamar Blue assay method. Most of the synthesized compounds displayed potent anti-tubercular activities. Amongst all the tested compounds 6p, 6g, 6n and 6h exhibited promising anti-tubercular activity. Further, these potent compounds were gauged for MDR-TB, XDR-TB and cytotoxic study. None of these compounds exhibited potent cytotoxicity. Stability of protein ligand complex was further evaluated by molecular dynamics simulation for 10 ns. All these results indicate that the synthesized compounds could be potential leads for further development of new potent anti-tubercular agents.

Full text of DOI:10.1016/j.bioorg.2019.02.044

Accepted Manuscript
Structure-based design, synthesis and biological evaluation of a newer series of
pyrazolo[1,5-a]pyrimidine analogues as potential anti-tubercular agents
Palmi Modi, Shivani Patel, Mahesh Chhabria
PII:
S0045-2068(18)31401-9
DOI:
https://doi.org/10.1016/j.bioorg.2019.02.044
YBIOO 2819
Reference:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
1 December 2018
23 January 2019
20 February 2019
Please cite this article as: P. Modi, S. Patel, M. Chhabria, Structure-based design, synthesis and biological evaluation
of a newer series of pyrazolo[1,5-a]pyrimidine analogues as potential anti-tubercular agents, Bioorganic
Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.02.044
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Structure-based design, synthesis and biological evaluation of a newer series of pyrazolo[1,5-
a]pyrimidine analogues as potential anti-tubercular agents.
PalmiModia,b,d, ShivaniPatela,c ,Mahesh Chhabriaa
aDepartment of Pharmaceutical Chemistry, L. M. College of Pharmacy, Navrangpura,
Ahmedabad – 380009 (Gujarat), India
bDepartment of Pharmacy, Dharmsinh Desai University, Nadiad – 387001(Gujarat), India
cDivision of biological and life sciences, Ahmedabad University, Ahmedabad –
380009(Gujarat), India
dDepartment of Pharmaceutical Chemistry, L. J. Institute of Pharmacy, Ahmedabad-382210
(Gujarat), India.
Corresponding author
✉Mahesh T. Chhabria
E-mail address- mahesh.chhabria@rediffmail.com
1

Abstract:
In-depth study of structure-based drug designing can provide vital leads for the development
of novel, clinically active molecules. In this present study, twenty six novel pyrazolo[1,5-
a]pyrimidine analogues (6a-6z) were designed using molecular docking studies. The
designed molecules were synthesized in good yields. Structural elucidation of the synthesized
1
molecules was carried out using IR, MS, H-NMR and 13C-NMR spectroscopy. All the
synthesized compounds were evaluated for their in-vitro anti-tubercular activity against
H37Rv strain by Alamar Blue assay method. Most of the synthesized compounds displayed
potent anti-tubercular activities. Amongst all the tested compounds 6p,6g, 6n and 6h
exhibited promising anti-tubercular activity. Further, these potent compounds were gauged
for MDR-TB, XDR-TB and cytotoxic study. None of these compounds exhibited potent
cytotoxicity. Stability of protein ligand complex was further evaluated by molecular
dynamics simulation for 10ns. All these results indicate that the synthesized compounds
could be potential leads for further development of new potent anti-tubercular agents.
Key words: Mycobacterium tuberculosis; molecular docking; Molecular dynamics;
Pyrazolo[1,5-a]pyrimidine hybrids
2

1. Introduction
Tuberculosis is the life-threatening infectious disease caused by the bacterium,
Mycobacterium tuberculosis [1]. In addition to this MDR-TB and XDR-TB associated with
HIV is one of the continual diseases with its widespread characteristic [2]. Approximately 13
million people need to be treated by the year 2020. The biggest challenge in management of
tuberculosis treatment is the hostility of the mycobacterial infection [3]. To overcome this
increased resistance new class of anti-tubercular agents acting through novel mechanism of
action are one of the urgent requirement to combat M. tuberculosis infections. Mycolic acid is
the central constituent of causative pathogen. Most of the currently available anti-tubercular
drugs act through inhibition of the mycolic acid synthesis, which is the long chain fatty acid
of 60-90 carbons with α-alkyl β-hydroxy groups [4, 5]. InhA (enoyl acyl reductase enzyme)
is the major targeted enzyme involved in the fatty acid biosynthesis pathway of
Mycobacterium tuberculosis [6]. It is one of the emerging targets for the development of
novel anti-tubercular agents [7].
Heterocyclic derivatives have extensive range of biological importance and applications.
Individual pyrazole and pyrimidine nucleus are the nitrogen containing heterocyclic
molecules which constitute the largest portion of medicinal important moieties [8, 9]. They
exhibits a wide spectrum of biological activities including adenosine receptor antagonists
[10], anti-schistosomal, anti-trypanosomal and sedative [11], anxiolytic [12], HMG-CoA
reductase inhibitors [13], KDR kinase inhibitors [14], COX-1, selective COX-2 inhibitors
[15], kinase inhibitors [16], HIV reverse transcriptase inhibitors [17], anti-malarial and
antifungal [18] and many more. The lipophilicity of these chemophores plays an important
role in their physicochemical properties and biological activity.
Inspired by the activities of these potent motifs, computational methods were applied to
incorporate these two individual pharmacophores in a single framework as pyrazolo[1,5-
a]pyrimidine hybrid analogues. These condensed hetrocyclic derivatives are known to act as
potential anti-tubercular agents by inhibiting FAB-I enzyme involved in the fatty acid
synthesis of the mycobacterium cell wall [19]. The current work describes the synthesis of
novel pyrazolo[1,5-a]pyrimidine hybrid analogues with encouraging anti-mycobacterial
activity against M. tuberculosis H37Rv strain.
In the present study, molecular docking technique was applied on enoyl acyl reductase
protein enzyme (PDB: 2H7M) to identify the important interactions and predict potent hit
molecules [20]. We have designed a new class of pyrazolo[1,5-a]pyrimidine hybrids to
improve the anti-tubercular activity especially against the drug resistance M.
3

tuberculosisstain. Also the designed compounds were further filtered through ADMET
properties to check drug likeliness of predicted molecules. All the designed molecules were
synthesized and screened for their anti-tubercular activity using Alamar Blue Assay Method
against H37Rv strain. Potent hit molecules from preliminary screening were further opted for
MDR-TB, XDR-TB and cytotoxicity on mammalian VERO cell line [20]. Based on the
biological activities the most potent compound was subjected to molecular dynamics
simulation to check its stability with respect to the protein structure.
2. Results and Discussion
2.1. Chemistry
The route of synthesis for the final derivatives of 7-hydroxy-5-methyl-N-substituted phenyl-
2-(substituted phenylamino)pyrazolo[1,5-a]pyrimidine-3-carboxamide (6a-6z) has been
summarised in scheme 1. First step involves the synthesis of 2-cyano-N-
(substitutedphenyl)acetamide(2a-2o) derivatives by nucleophilic addition reaction between
ethyl cyanoacetate(1) andsubstituted anilines. Further, in presence of base and carbon
disulphide it undergo for nucleophilic addition reaction on active methylene of 2-cyano-N-
(substitutedphenyl)acetamide(2a-2o) followed by methylation with dimethyl sulphate, yields
S, S acetal(3a-3o). These S, S acetal(3a-3o) derivatives further undergo for nucleophilic
substitution reaction by addition of substituted anilines and cyclic aliphatic aminesfor the
formation of Keten S, N- acetals(4a-4z). Due to presence of electrophilic and nucleophilic
centres these Keten S, N- acetal possesses high reactivity and that can be utilised in
regioselective ring closure strategies with hydrazine hydrate for the formation of 5-amino-N-
substituted phenyl-3-(substituted phenylamino)-1H-pyrazole-4-carboxamide (5a-5z).
Pyrazole derivatives, (5a-5z) on cyclocondensation with ethyl aceto acetate give the targeted
compound
7-hydroxy-5-methyl-N-substituted
phenyl-2-(substituted
phenylamino)pyrazolo[1,5-a]pyrimidine-3-carboxamide (6a-6z). All the synthesised
1
compounds (6a-6z) characterized by IR, MS, H-NMR, 13C-NMR spectroscopy after
purification. Spectrum values of all the synthesized compounds are presented in the
experimental part.
2.2. In-silico molecular docking approach
Molecular docking is a structure based drug design approach which is an essential part of the
drug discovery assigning knowledge of enzyme-inhibitor complex with respect to the binding
affinity and binding mode. Here we have used Glide module of Schrodinger software
(Maestro v10.1, Schrodinger, LLC, NEW YORK, NY) for carrying out docking studies.The
predicted New Chemical Entities (6a-6z) followed by the Lipinski’s rule of five were docked
4

into the active site of InhA enzyme (PDB ID: 2H7M) retrieved from the protein data bank
(He et al. 2007). The co-crystalline structure 1-cyclohexyl-N-(3,5-dichlorophenyl)-5-
oxopyrrolidine-3-carboxamide derivative possess -8.17 docking score with Ile21, Met103,
Met147, Phe149, Pro156, Tyr158, Lys165, Ile 202 and Leu218 interacting aminoacid
residues.The MIC values and docking score of the NCE’s were dealing with the numbers. So
we have selected some of the most potent inhibitors 6g, 6h, 6n and 6p which possess -8.23, -
7.27 and -8.89 and -9.23 docking scores respectively and discussed them in detail below.
All the NCE’s anchors same co-ordinates in the substrate binding loop Gly 96, Met 147,
Phe149, Pro 156, Tyr 158, Met161, Lys 165, Ala198, Met199, Ile202 and Val203 as
observed for the native ligand. Compound [2-((p-Chlorophenyl)amino)-7-hydroxy-5-methyl-
N-(p-tolyl)pyrazolo[1,5-a]pyrimidine-3- carboxamide(6g)] Figure 1(a) showed π-π
interaction of its phenyl ring with Lys165 and hydrophobic interactions with Ile21, Met103,
Met147, Phe149, Pro156, Tyr158, Ile 202 and Leu218. Pyrrazolo[1,5-a]pyrimidine moiety
was occupied because of the hydrophobic interaction with Ile21, Met147, Phe149, Lys165,
Gly192 and Pro193. The compound [7-Hydroxy-N-(o-methoxyphenyl)-5-methyl-2-(m-
tolylamino)pyrazolo[1,5-a]pyrimidine-3-carboxamide (6n)] Figure 1(b) having hydrogen
bond interactions with Ile194 and shows that the major important hydrophobic interacting
residues are Ile21, Met103, Met147, Phe149, Pro156, Tyr158, Lys 165, Ile 194, Leu218, and
Ile202.
Compounds
2-((3,4-difluorophenyl)amino)-N-(p-fluorophenyl)-7-hydroxy-5-
methylpyrazolo[1,5-a]pyrimidine-3-carboxamide (6p) Figure 1(c) and 2-((3,4-
dichlorophenyl)amino)-7-hydroxy-5-methylpyrazolo[1,5-a]pyrimidine-3-carboxamide (6h)
Figure 1(d) have molecular interactions with Ile194, and hydrophobic interaction with Ile21,
Met147, Phe149, Tyr158, Lys 165, Leu218, Pro156, Met103, Ile202 indicating similar
interaction as the co-crystalline structure which can be novel M. tuberculosis inhibitor. These
findings suggest that the newly designed compounds may be considered as potential scaffolds
for anti-tubercular drug discovery.
2.3. In-silico ADME prediction
Satisfactory ADMET profile with good effectiveness is one of the major benchmark for the
completion of a drug. Obtained potential leads of Pyrazolo[1,5-a]pyrimidine derivatives
based on molecular docking study and anti-tubercular potential (6a-6z) were further opted for
in-silico ADMET prediction to check its druggability using QikProp module in Schrodinger.
The results are shown in Table 1.
2.4. Biological Evaluation
2.4.1. Anti-tubercular studies
5

Anti-tubercular potential was performed using MicroplateAlamar Blue Assay (MABA)
method for the preliminary screening of the newly synthesized targeted compounds against
Mycobacterium tuberculosis H37Rv strain (Cho et al. 2015). Some of the finally synthesized
pyrazolo[1,5-a]pyrimidine derivatives retains reasonable inhibitory activity compared with
the standard drugs Ciprofloxacin, Pyrazinamide and Streptomycin. The results of the in-vitro
anti-tubercular activity of the title compounds (6a–6z) inMIC levels have shown in Table 2.
Amongst screened all the final molecules, 6p with electron withdrawing substitutions on both
side of the aryl ring yields excellent activity against M. tuberculosis H37Rv strain with MIC
value 0.8 µg/mL. This obtained result found to be more potent that the compared all the
standard drugs. Compounds 6g and 6n have shown MIC of 3.12 µg/mL which are
comparable to the standard drugs Ciprofloxacin and Pyrazinamide. One of the compound 6h
showed MIC of 6.25 mg/mL which is comparable to the standard drug streptomycin.
However with electron donating substitutions 6a, 6b, 6w and 6y starts to loss potency with
MIC of 12.5 µg/mL. Remaining other molecules 6e, 6s and 6x possess anti-tubercular
activity with MIC of 25 µg/mL, while 6c, 6d, 6f, 6i-6m, 6o, 6q, 6r, 6t, 6v and 6z have MIC
of 50 µg/mL each. Compound 6u exhibited MIC of 100 µg/mL.In view of good anti-
tubercular activity of compounds 6g, 6h, 6n and 6p were further evaluated for their MDR-
TB, XDR-TB and in vitro cytotoxicity study using Vero cell line.
2.4.2. Structure Activity Relationship (SAR)
The whole structure activity relationship has been summarised in Figure 2. A good anti-TB
activity may be attributed to the presence of electron withdrawing substitutions at R or R’ or
at both the aryl rings attached to pyrrazolo[1,5-a]pyrimidine. Similarly, replacement of
electron donating substituents at one or both of the phenyl rings attenuated the anti-tubercular
activity. Here Heteroaromatic rings like N-ethyl piperazine, Pyrrolidine and Morpholine at R’
position were found to have decrease the potency.
2.4.3. MDR-TB and XDR-TB
On the basis of anti-tubercular activity, Lowenstein-Jensen medium (L.J. medium) method
was used for the compound 6g, 6h, 6n and 6p to check its vulnerability towards multidrug
resistant Tb (MRD-TB) and extensively drug-resistant TB (XDR-TB). The results are
summarized in Table 3.
2.4.4. Cytotoxicity assay
Selected potent molecules 6g, 6h, 6n and 6p based on anti-tubercular activitywere further
evaluated for its cytotoxic activity against a mammalian Vero cell line using 3-(4,5-
dimethylthiazo-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Here isoniazid was
6

used as a positive control(Maria et al. 2017). The result of study summarised in Table 3.
Compounds 6g had 20.99 μg/mL of IC50, whereas compounds 6h and 6n shows weaker effect
on cancer cell line with IC50 value of 29.02 μg/mLand 21.2 μg/mLrespectively. However,
compound 6p has the lowest sensitivity with IC50 of 13.57 μg/mL. The resulted active
molecules found relatively non-toxic against mammalian Vero cell line. So, it could be
inferred that the presence of electron-withdrawing substitutions on the phenyl ring increase
the cytotoxicity potential of the pyrazolo[1,5-a]pyrimidine.
2.5. Molecular dynamic simulations
The protein-ligand complex was further subjected to MD simulation after demonstrating
significant docking interactions. The stability of simulated system was analysed through
RMSD and RMSF values with respect to the unbound protein structure(John et al. 2015;
Schuttelkopf et al. 2004). The temporal RMSD plot of co-crystalline and 6p complex is
shown in Figure 3(a). The curve exhibited minor fluctuation of 2.0 Å, which demonstrates
the stability of the ligand within the binding pocket. Further, the RMSF plot of complex
revealed that most of the residues fluctuate below 2.5 Å and few residues show fluctuation up
to 4.5 Å Figure 3(b) (Martyna et al. 1994). High fluctuations were observed in N- and C
terminal region compared to any other part of protein. Subsequently, the favourable contacts
between the protein residues and ligand atoms were found with Tyr 158, Lys 165, Ile 194,
Met 199 and Ile 202 conserved for MD stimulation which has been displayed in Figure 4.
The H-bonding interactions were formed with Lys 165 and Tyr 196 and Ile 194 through
water molecule which contributed to the binding affinity of the ligand. These essential
residual interactions with 6p supported its inhibitory potency as anti-tubercular agent.
3. Conclusion
In the present work we have designed and synthesized newer series of 7-hydroxy-5-methyl-
N-substitutedphenyl-2-(substitutedphenylamino)pyrazolo[1,5-a]pyrimidine-3-carboxamide
(6a-6z) derivatives in which pyrazole and pyrimidine both the heterocyclic rings were fused.
Activity of synthesized final potent derivatives 6g, 6h, 6n and 6p possesspotent anti-
tuberculosis activities compared to the reference standards drugs. Molecular modelling and
docking studies also suggested that compounds 6g, 6h, 6n and 6p interacted with InhA
enzyme more efficiently. So, it is concluded that compounds with electron withdrawing
substituents increases the potency. Further this powerful supposition opens up the discovery
of newer potential targeted molecules as novel anti-tuberculosis agents.
4. Experimental
4.1. Chemistry
7

Chemicals and solvents of laboratory grade were purchased from commercially available
suppliers Rankem India Ltd., Loba, Sigma Aldrich and SpecrochemPvt. Ltd and used after
purification. Melting points of synthesized final compounds were determined by open
capillary method using VMP-D (VEEGO) model and are uncorrected. Completion of the
reaction was monitored by iodine vapours, UV radiations and precoated thin layer
chromatography sheets (Merck60 F254, 0.25 mm). The Infrared (IR) spectra for final
compounds were recorded in KBr using FT-IR 8400S Shimadzu Fourier Transform
spectrophotometer. Mass spectra were taken using Perkin-Elmer LC-MS PE Sciex API/65.
1
13
Bruker AVANCE-II 400 MHz spectrophotometer was used to take H NMR spectra and C
NMR. Chemical shifts are expressed as parts per million (ppm), peak patterns were used to
describe as: s (singlet), d (doublet), t (triplet), q (quartet) or m (multiplet). Tetramethylsilane
(TMS) was used as an internal standard and DMSO-d6 as solvent.
4.1.1. Preparation 2-cyano-N-(substituted phenyl)acetamide (2)
To a round bottom flask ethyl cyanoacetate (0.0468 mol) and substituted anilines(0.0468
mol) was added. The reaction was refluxed for 8 hours using 15 mL DMF as a solvent.
Precoated TLC was used to confirm the completion of the reaction. After completion, the
reaction was poured into ice-cold water to obtain crude solid. Further to yield pure crystalline
product obtained crude solid was filtered, dried and recrystallized from ethanol.
4.1.2. Preparation of 2-cyano-N-(substituted phenyl)-3,3bis(methylthio)acrylamide (3)
2-cyano-N-(substituted phenyl)acetamide (0.00523 mol) was added to the solution of
potassium hydroxide (0.01046mol) containing 5 ml of water at cooled temperature of 0-5oC
with continuous stirring. To that carbon disulphide (0.00523mol) was added drop wise
followed by 15 ml of dimethyl formamide (DMF). Further dimethylsulphate (0.01046 mol)
was added drop wise followed by 2 hours of stirring. After completion of stirring pour the
reaction into ice-cold mixture to obtain the solid. The solid was filtered, washed with water
and recrystallized from methanol to obtain colorless crystalline product [21].
4.1.3. Preparation of 2-cyano-3-(methylthio)-N-substituted phenyl-3-(substituted
phenylamino)acrylamide (4)
Differently synthesized 2-cyano-N-(substituted phenyl)-3,3bis(methylthio)acrylamide (0.003
mol) and substituted aniline (0.003 mol) was dissolved in 10 ml isopropyl alcohol (IPA)
followed by reflux for 14-15 hours. Completion of the reaction was monitored by TLC. After
that the reaction mixture was quenched into ice cold water to obtain dried filtered solid.
Further dried solid was recrystallization from IPA to yielded crystalline product [22].
8

4.1.4. Preparation of 5-amino-N-substituted phenyl-3-(substituted phenylamino)-1H-
pyrazole-4-carboxamide (5)
Above
synthesized
2-cyano-3-(methylthio)-N-substituted
phenyl-3-(substituted
phenylamino)acrylamide (0.0104 mol) and hydrazine hydrate (0.0104 mol) was refluxed for
3-4 hours with 20 ml ethanol in a round bottom flask. Completion of the reaction was
monitored by TLC. After completion of the reaction, ethanol was distilled off and obtained
recrystallized solid was filtered and dried [23].
4.1.5. General procedure for the synthesis of 7-hydroxy-5-methyl-N- substituted phenyl-2-
(substituted phenylamino)pyrazolo[1,5-a]pyrimidine-3-carboxamide (6a-6z)
Mixture of ethyl aceto acetate (0.01029 mol) and 5-amino-N-substituted phenyl-3-
(substituted phenylamino)-1H-pyrazole-4-carboxamide (0.001029 mol) was refluxed in an oil
bath for 1-2 hours. The solid product was separated out from the reaction mixture indicates
the completion of the reaction. The obtained was filtered and recrystallized using chloroform
to obtain pure crystalline product.
4.1.5.1.
7-Hydroxy-N-(4-methoxyphenyl)-2-((4-methoxyphenyl)amino)-5-
methylpyrazolo[1,5- a]pyrimidine-3-carboxamide (6a)
White solid; Yield: 80%;M.P. 276-278°C; IR (KBr, cm-1) 3455 (-OH), 3323 (-NH str.), 1666
(-CO-NH str.), 1240 (C-N str.), 1033 (C-O-C);1H NMR (400 MHz, DMSO-d6, δ, ppm): δ
11.89 (s, 1H, OH-Pyrimidine, D2O exchangeable), δ 9.56 (s, 1H, CONH-Ar., D2O
exchangeable), δ 8.68 (s, 1H, NH-Pyrazole, D2O exchangeable), δ 6.88- 7.61 (m, 8H,-CH-
Ar.), δ 5.75 (s, 1H,-CH-Pyrimidine), δ 3.72- 3.76 (s, 6H, -OCH3-Ar.), δ 2.35 (s, 3H, -CH3-
Pyrimidine); 13C NMR (400 MHz, DMSO-d6, δ, ppm): 161.22, 155.57, 154.82, 153.49,
153.21, 147.20, 140.30, 134.42, 131.59, 122.56, 118.60, 114.04, 113.61, 98.14, 87.33, 55.17,
55.15, 18.79; LCMS (M/Z)Calculated for C22H21N5O4: 419.4; Obtained MS: 420.2 (M+1)
4.1.5.2. 2-((4-Chlorophenyl)amino)-7-hydroxy-N-(4-methoxyphenyl)-5-methylpyrazolo[1,5-
a]pyrimidine-3-carboxamide (6b)
White solid; Yield: 86.42%; M.P 317-320 °C; IR (KBr, cm-1) 3352 (-NH str.), 1666 (-CO-NH
str.), 1238 (C-N str.), 1033 (C-O-C), 825, 551 (C-Cl); 1H NMR (400 MHz, DMSO-d6, δ ppm)
δ 11.96 (s, 1H, OH-Pyrimidine, D2O exchangeable), δ 9.64 (s, 1H, CONH-Ar, D2O
exchangeable), δ 8.97 (s, 1H, NH-Pyrazole, D2O exchangeable), δ 6.93- 7.74 (m, 8H,-CH-
Ar.), δ 5.78 (s, 1H,-CH-Pyrimidine), δ 3.75 (s, 3H, -OCH3-Ar.), δ 2.35 (s, 3H, -CH3-
Pyrimidine); 13C NMR (400 MHz, DMSO-d6, δ ppm): 161.22, 155.57, 154.82, 153.49,
153.21, 147.20, 140.30, 134.42, 131.59, 122.56, 118.60, 114.04, 113.61, 98.14, 87.33, 18.79;
9

LCMS (M/Z)Calculated for C21H18ClN5O3: 423.9; Obtained MS (M/Z): 424.2 (M+1), 426.1
(M+2)
4.1.5.3.
2-((2,3-Dimethylphenyl)amino)-7-hydroxy-N-(4-methoxyphenyl)-5-
methylpyrazolo[1,5-a]pyrimidine-3-carboxamide (6c)
White solid; Yield 68.22%;M. P. 280-284 °C; IR (KBr, cm-1) 3363 (-NH str.), 1656 (-CO-NH
str.), 1247 (C-N str.), 1039 (C-O-C); 1H NMR (400 MHZ, DMSO-d6, δ ppm) δ 11.98 (s, 1H,
OH-Pyrimidine, D2O exchangeable), δ 9.54 (s, 1H, CONH-Ar., D2O exchangeable), δ 9.06
(s, 1H, NH-Pyrazole, D2O exchangeable), δ 6.77- 8.21 (m, 8H,-CH-Ar.), δ 5.79 (s, 1H,-CH-
Pyrimidine), δ 3.75 (s, 3H, -OCH3-Ar.), δ 2.17-2.50 (m, 6H, -CH3-Ar.); δ 2.17-2.50 (s, 3H, -
CH3-Pyrimidine); 13C NMR (400 MHz, DMSO-d6, δ ppm) : 161.79, 155.70, 153.71, 138.99,
136.17, 131.38, 125.68, 122.86, 122.67, 122.56, 115.24, 113.72, 55.20, 20.47, 18.81, 12.91;
LCMS (M/Z) Calculatedfor C23H23N5O3: 417.5; Obtained MS (M/Z):418.3 (M+1) 419.4
(M+2)
4.1.5.4.
7-Hydroxy-N-(4-methoxyphenyl)-5-methyl-2-(phenylamino)pyrazolo[1,5-
a]pyrimidine-3-carboxamide (6d)
White solid; Yield 84.24%;M. P. 320-322 °C; IR (KBr, cm-1) 3384 (-NH str.), 1616 (-CO-NH
str.), 1230 (C-N str.), 1035 (C-O-C); 1H NMR (400 MHZ, DMSO-d6, δ ppm) δ 11.94 (s, 1H,
OH-Pyrimidine, D2O exchangeable), δ 9.60 (s, 1H, CONH-Ar., D2O exchangeable), δ 8.88
(s, 1H, NH-Pyrazole, D2O exchangeable), δ 6.89- 7.67 (m, 9H,-CH-Ar.),δ 5.77 (s, 1H,-CH-
13
Pyrimidine), δ 3.42 (s, 3H, -OCH3-Ar.), δ 2.50 (m, 3H, -CH3-Pyrimidine); C NMR (400
MHz, DMSO-d6, δ ppm) : 161.17, 155.62, 154.86, 152.88, 149.93, 140.93, 140.23, 131.52,
128.79, 122.60, 120.42, 117.05, 113.61, 98.14, 87.69, 55.18, 18.84; LCMS (M/Z)Calculated
for C21H19N5O3: 389.1; Obtained MS (M/Z): 390.4 (M+1)
4.1.5.5.
7-Hydroxy-N-(4-methoxyphenyl)-5-methyl-2-morpholinopyrazolo[1,5-
a]pyrimidine-3-carboxamide (6e)
White solid; Yield 48.12%; M.P.291-295 °C; IR (KBr, cm-1) 3359 (-NH str.),1683 (-CO-NH
str.), 1242 (C-N str.), 1033 (C-O-C);1H NMR (400 MHZ, DMSO-d6, δ ppm) : δ 11.94 (s, 1H,
OH-Pyrimidine, D2O exchangeable), δ 9.60 (s, 1H, CONH-Ar., D2O exchangeable), δ 6.90-
7.67 (m, 12H,-CH-Ar., D2O exchangeable), δ 5.87 (s, 1H,-CH-Pyrimidine), δ 3.42 (s, 3H, -
OCH3-Ar), δ 2.27 (m, 3H, -CH3-Pyrimidine); 13C NMR (400 MHz, DMSO-d6, δ ppm)
:159.88, 120.80, 113.83, 90.21, 65.85, 55.17, 16.51; LCMS (M/Z)Calculated for C19H21N5O4:
383.4; Obtained MS (M/Z): 384.2 (M+1)
4.1.5.6.
7-Hydroxy-5-methyl-2-(phenylamino)-N-(p-tolyl)pyrazolo[1,5-a]pyrimidine-3-
carboxamide (6f)
10

White solid; Yield 72.89%;M. P. 283-286 °C; IR (KBr, cm-1) 3377 (-NH str.), 1656 (-CO-NH
str.), 1232 (C-N str.);1H NMR (400 MHZ, DMSO-d6, δ ppm) : δ 11.96 (s, 1H, OH-
Pyrimidine, D2O exchangeable), δ 9.65 (s, 1H, CONH-Ar., D2O exchangeable), δ 8.85 (s, 1H,
NH-Pyrazole, D2O exchangeable), δ 6.89- 7.67 (m, 9H,-CH-Ar.), δ 5.77 (s, 1H,-CH-
Pyrimidine), δ 2.29 -2.35 (s, 3H, -CH3-Ar), δ 2.29 -2.35 (s, 3H, -CH3-Pyrimidine); 13C NMR
(400 MHz, DMSO-d6, δ ppm) :161.29, 154.85, 152.82, 149.98, 140.96, 140.30, 136.07,
132.54, 128.86, 128.79, 120.86, 120.42, 117.06, 98.15, 87.66, 20.49, 18.85; LCMS
(M/Z)Calculated for C21H19N5O2: 373.4; Obtained MS (M/Z): 374.2 (M+1)
4.1.5.7.
2-((4-Chlorophenyl)amino)-7-hydroxy-5-methyl-N-(p-tolyl)pyrazolo[1,5-
a]pyrimidine-3-carboxamide (6g)
White solid; Yield 68.33 %; M. P. 262-264 °C; IR (KBr, cm-1) 3342 (-NH str.), 1668 (-CO-
NH str.), 1234 (C-N str.), 811, 663 (C-Cl); 1H NMR (400 MHZ, DMSO-d6, δ ppm) : δ 11.97
(s, 1H, OH-Pyrimidine, D2O exchangeable), δ 9.67 (s, 1H, CONH-Ar., D2O exchangeable), δ
8.95 (s, 1H, NH-Pyrazole, D2O exchangeable), δ 7.15- 7.73 (m, 8H,-CH-Ar.), δ 5.77 (s, 1H,-
CH-Pyrimidine), δ 2.23-2.47 (s, 3H, -CH3-Ar), δ 2.23-2.47 (s, 3H, -CH3-Pyrimidine); 13C
NMR (400 MHz, DMSO-d6, δ ppm) :161.29, 154.85, 152.82, 149.98, 140.96, 140.30, 136.07,
132.54, 128.86, 128.79, 120.86, 120.42, 117.06, 98.15, 87.66, 20.49; LCMS (M/Z)Calculated
for C21H19ClN5O2: 407.1; Obtained MS (M/Z): 408.5 (M+1)
4.1.5.8.
2-((3,4-Dichlorophenyl)amino)-7-hydroxy-5-methylpyrazolo[1,5-a]pyrimidine-3-
carboxamide (6h)
Yellow solid; Yield 54.43%; M. P. 308-310 °C;IR (KBr, cm-1) 3325, 3238 (-N-H str.), 1658
(-CO-N-H str.)1247 (C-N str.), 850, 698 (C-Cl); 1H NMR (400 MHZ, DMSO-d6, δ ppm) : δ
11.60 (s, 1H, OH-Pyrimidine, D2O exchangeable), δ 9.74 (s, 1H, NH-Pyrazole, D2O
exchangeable), δ 9.68-9.69 (s, 2H, CONH2-Ar., D2O exchangeable), δ 7.31- 8.15 (m, 3H,-
CH-Ar.), δ 5.80 (s, 1H,-CH-Pyrimidine), δ 2.50 (s, 3H, -CH3-Pyrimidine); 13C NMR (400
MHz, DMSO-d6, δ ppm) : 165.55, 164.45, 154.65, 152.83, 149.09, 140.85, 140.44, 131.12,
131.04, 130.52, 130.45, 121.51, 118.04, 117.40, 117.26, 98.49, 86.44, 86.29, 18.82, 16.61;
LCMS (M/Z)Calculated for C14H11Cl2N5O2: 351.1; Obtained MS (M/Z): 352.2 (M+1), 354.3
(M+2)
4.1.5.9.
2-((2,4-Dimethylphenyl)amino)-N-(4-ethoxyphenyl)-7-hydroxy-5-
methylpyrazolo[1,5-a]pyrimidine-3-carboxamide (6i)
White solid; Yield 78.12%;M. P. 271-273 °C; IR (KBr, cm-1) 3334 (-NH str.)1668 (-CO-NH
str.), 1238 (C-N str.), 1049 (C-O-C); 1H NMR (400 MHZ, DMSO-d6, δ ppm) : δ 11.96 (s, 1H,
OH-Pyrimidine, D2O exchangeable), δ 9.52 (s, 1H, CONH-Ar., D2O exchangeable), δ 8.96
11

(s, 1H, NH-Pyrazole, D2O exchangeable), δ 6.92- 8.23 (m, 7H,-CH-Ar.) ,δ 5.78 (s, 1H,-CH-
Pyrimidine), δ 2.35-3.58 (s, 5H, -OC2H5-Ar.), δ 2.23 (s, 6H, -CH3-Ar.), δ 1.31-1.34 (s, 3H, -
CH3-Pyrimidine); 13C NMR (400 MHz, DMSO-d6, δ ppm) : 161.78, 154.94, 154.89, 153.69,
149.73, 139.83, 136.61, 131.25, 130.64, 129.02, 126.89, 123.96, 122.87, 116.83, 114.21,
98.25, 87.02, 63.10, 20.27, 18.84, 17.43, 14.64; LCMS (M/Z)Calculated for C24H25N5O3:
431.2; Obtained MS (M/Z): 432.4 (M+1)
4.1.5.10.
N-(4-Chlorophenyl)-2-((2,4-dimethylphenyl)amino)-7-hydroxy-5-
methylpyrazolo[1,5-a]pyrimidine-3-carboxamide (6j)
White solid; Yield 65.13%; M. P. 298-300 °C;IR (KBr, cm-1) 3352 (-NH str.), 1662 (-CO-NH
str.), 1230 (C-N str.), 819, 578 (C-Cl); 1H NMR (400 MHZ, DMSO-d6, δ ppm) : δ 12.02 (s,
1H, OH-Pyrimidine, D2O exchangeable), δ 9.82 (s, 1H, CONH-Ar., D2O exchangeable), δ
8.79 (s, 1H, NH-Pyrazole, D2O exchangeable), δ 7.00- 8.22 (m, 7H,-CH-Ar.), δ 5.80 (s, 1H,-
13
CH-Pyrimidine), δ 2.24 (s, 3H, -CH3-Pyrimidine), δ 2.23 (s, 6H, -CH3-Ar.); C NMR (400
MHz, DMSO-d6, δ ppm) : 162.04, 154.85, 153.59, 149.82, 140.05, 137.59, 136.48, 130.65,
128.17, 128.41, 127.14,126.89, 124.04, 122.41, 116.93, 98.40, 87.09, 20.28, 18.85, 17.40;
LCMS (M/Z)Calculated for C22H20ClN5O2: 421.1; Obtained MS (M/Z): 422.2 (M+1) 424.6
(M+2)
4.1.5.11.
N-(2,4-Dimethylphenyl)-7-hydroxy-2-((4-methoxyphenyl)amino)-5-
methylpyrazolo[1,5-a]pyrimidine-3-carboxamide (6k)
White solid; Yield 85.43%; M. P. 288-290°C;IR (KBr, cm-1) 3344 (-NH str.), 1660 (-CO-NH
str.), 1244 (C-N str.), 1039 (C-O-C); 1H NMR (400 MHZ, DMSO-d6, δ ppm) : δ 11.72 (s, 1H,
OH-Pyrimidine, D2O exchangeable), δ 9.09 (s, 1H, CONH-Ar., D2O exchangeable), δ 9.93
(s, 1H, NH-Pyrazole, D2O exchangeable), δ 6.88- 7.54 (m, 7H,-CH-Ar.), δ 6.87 (s, 1H,-CH-
Pyrimidine), δ 3.64 (s, 3H, -OCH3-Ar.), δ 2.36 (s, 3H, -CH3-Pyrimidine), δ 2.19-2.28 (s, 6H, -
CH3-Ar.); 13C NMR (400 MHz, DMSO-d6, δ ppm) : 161.55, 154.79, 153.54, 134.58, 133.53,
130.85, 126.52, 125.03, 118.53, 114.10, 98.40, 87.18, 55.14, 20.49, 18.88, 17.89; LCMS
(M/Z)Calculated for C23H23N5O3: 417.1;Obtained(M/Z): 418.4 (M+1), 419.2 (M+2)
4.1.5.12.
2-((3,4-Dichlorophenyl)amino)-N-(2,3-dimethylphenyl)-7-hydroxy-5-
methylpyrazolo[1,5-a]pyrimidine-3-carboxamide (6l)
Yellow solid; Yield 89.47%; M. P. 282-285°C;IR (KBr, cm-1) 3350 (-NH str.),1668 (-CO-NH
str.), 1278 (C-N str.), 821, 661 (C-Cl); 1H NMR (400 MHZ, DMSO-d6, δ ppm) : δ 11.82 (s,
1H, OH-Pyrimidine, D2O exchangeable), δ 9.41 (s, 1H, CONH-Ar., D2O exchangeable), δ
9.28 (s, 1H, NH-Pyrazole, D2O exchangeable), δ 7.07- 8.94 (m, 6H,-CH-Ar.), δ 5.83 (s, 1H,-
13
CH-Pyrimidine), δ 2.33 (s, 3H, -CH3-Pyrimidine), δ 2.29-2.20 (s, 6H, -CH3-Ar.); C NMR
12

(400 MHz, DMSO-d6, δ ppm) : 161.46, 152.43, 141.12, 136.99, 135.79, 131.04, 130.49,
127.26, 125.27, 121.47, 118.06, 117.39, 87.55, 20.19, 14.35; LCMS (M/Z)Calculated for
C22H19Cl2N5O2: 455.0; Obtained MS (M/Z): 456.4 (M+1), 458.5 (M+2)
4.1.5.13.
2-((2,4-Dimethylphenyl)amino)-N-(4-fluorophenyl)-7-hydroxy-5-
methylpyrazolo[1,5-a]pyrimidine-3-carboxamide (6m)
Pale yellow solid; Yield 68.16 %; M. P.275-278 °C;IR (KBr, cm-1) 3361 (-NH str.),1658 (-
1
CO-NH str.), 1207 (C-N str.), 833, 1159 (C-F); H NMR (400 MHZ, DMSO-d6, δ ppm) : δ
11.99 (s, 1H, OH-Pyrimidine, D2O exchangeable), δ 9.91 (s, 1H, CONH-Ar., D2O
exchangeable), δ 9.74 (s, 1H, NH-Pyrazole, D2O exchangeable), δ 5.80- 9.24 (m, 7H,-CH-
Ar.), δ 4.10 (s, 1H,-CH-Pyrimidine), δ 2.50 (s, 3H,-CH3-Ar.), δ 2.36 (s, 3H, -CH3-
Pyrimidine), δ 2.32-2.23 (s, 6H, -CH3-Ar.); 13C NMR (400 MHz, DMSO-d6, δ ppm) : 162.04,
154.85, 153.59, 149.82, 140.05, 137.59, 136.48, 130.65, 128.17, 128.41, 127.14,126.89,
124.04, 122.41, 116.93, 98.40, 87.09, 20.28, 18.85, 17.40; LCMS (M/Z)Calculated for
C22H20FN5O2: 405.4; Obtained MS (M/Z): 406.4 (M+1);
4.1.5.14.
7-Hydroxy-N-(2-methoxyphenyl)-5-methyl-2-(m-tolylamino)pyrazolo[1,5-
a]pyrimidine-3-carboxamide (6n)
White solid; Yield 82.45%; M. P. 240-243°C;IR (KBr, cm-1) 3346 (-NH str.), 1674 (-CO-NH
str.), 1247 (C-N str.), 1027 (C-O-C); 1H NMR (400 MHZ, DMSO-d6, δ ppm) : δ 11.79 (s, 1H,
OH-Pyrimidine, D2O exchangeable), δ 9.31 (s, 1H, CONH-Ar., D2O exchangeable), δ 8.82
(s, 1H, NH-Pyrazole, D2O exchangeable), δ 6.70- 8.16 (m, 8H,-CH-Ar.), δ 5.83 (s, 1H,-CH-
Pyrimidine), δ 3.84 (s, 3H,-CH3-Ar.), δ 2.40 (s, 3H, -CH3-Ar.), δ 2.26 (s, 3H, -CH3-
Pyrimidine); 13C NMR (400 MHz, DMSO-d6, δ ppm) : 161.79, 155.70, 153.71, 138.99,
136.17, 131.38, 125.68, 122.86, 122.67, 122.56, 115.24, 113.72, 55.20, 20.47, 12.91; LCMS
(M/Z)Calculated for C22H21N5O3: 403.4; Obtained MS (M/Z): 404.3 (M+1)
4.1.5.15.
2-((4-Chlorophenyl)amino)-7-hydroxy-5-methylpyrazolo[1,5-a]pyrimidine-3-
carboxamide (6o)
Brown solid; Yield 43.67%; M. P. 320-326°C;IR (KBr, cm-1) 3475, 3172 (-N-H str.), 1685 (-
1
CO-N-H str.)1290 (C-N str.), 806, 667 (C-Cl); H NMR (400 MHZ, DMSO-d6, δ ppm) : δ
11.57 (s, 1H, OH-Pyrimidine, D2O exchangeable), δ 9.65 (s, 1H, NH-Pyrazole, D2O
exchangeable), δ 7.68-7.71 (s, 2H, CONH2-Ar., D2O exchangeable), δ 7.33- 7.67 (m, 4H,-
CH-Ar.), δ 5.78 (s, 1H,-CH-Pyrimidine), δ 2.34 (s, 3H, -CH3-Pyrimidine); 13C NMR (400
MHz, DMSO-d6, δ ppm) : 165.55, 164.45, 154.65, 152.83, 149.09, 140.85, 140.44, 131.12,
131.04, 130.52, 130.45, 121.51, 118.04, 117.40, 117.26, 98.49, 86.44, 86.29, 18.82, 16.61;
13

LCMS (M/Z)Calculated for C14H12ClN5O2: 317.7; Obtained MS (M/Z): 318.4 (M+1) 320.3
(M+2)
4.1.5.16.
2-((3,4-Difluorophenyl)amino)-N-(4-fluorophenyl)-7-hydroxy-5-
methylpyrazolo[1,5-a]pyrimidine-3-carboxamide (6p)
Yellow solid; Yield 68.19%; M. P. 290-294°C;IR (KBr, cm-1) 3363 (-NH str.), 1652 (-CO-
NH str.), 1271 (C-N str.), 833, 1159 (C-F); 1H NMR (400 MHZ, DMSO-d6, δ ppm) : δ 11.98
(s, 1H, OH-Pyrimidine, D2O exchangeable), δ 9.91 (s, 1H, CONH-Ar., D2O exchangeable), δ
8.88 (s, 1H, NH-Pyrazole, D2O exchangeable), δ 6.48- 8.02 (m, 7H,-CH-Ar.), δ 5.79 (s, 1H,-
13
CH-Pyrimidine), δ 2.35 (s, 3H, -CH3-Pyrimidine); C NMR (400 MHz, DMSO-d6, δ ppm) :
160.99, 159.44, 157.05, 154.75, 152.24, 150.37, 147.97, 144.61, 142.36, 142.24, 140.45,
138.24, 138.14, 135.10, 122.56, 122.49, 117.35, 117.17, 115.17, 114.95, 113.38, 106.07,
105.85, 98.16, 87.90, 18.85, 16.66; LCMS (M/Z)Calculated for C20H14F3N5O2: 413.3;
Obtained MS (M/Z): 414.4 (M+1)
4.1.5.17.
N-(4-Ethoxyphenyl)-7-hydroxy-5-methyl-2-(phenylamino)pyrazolo[1,5-
a]pyrimidine-3-carboxamide (6q)
White solid; Yield 72.55%; M. P. 280-284°C;IR (KBr, cm-1) 3377 (-NH str.), 1652 (-CO-NH
str.), 1294 (C-N str.),1047 (C-O-C); 1H NMR (400 MHZ, DMSO-d6, δ ppm) : δ 11.92 (s, 1H,
OH-Pyrimidine, D2O exchangeable), δ 9.58 (s, 1H, CONH-Ar., D2O exchangeable), δ 8.89
(s, 1H, NH-Pyrazole, D2O exchangeable), δ 6.89- 7.67 (m, 9H,-CH-Ar.), δ 5.77 (s, 1H,-CH-
Pyrimidine), δ 2.35-3.46 (s, 5H, -OC2H5-Ar.), δ 1.32 (s, 3H, -CH3-Pyrimidine); 13C NMR
(400 MHz, DMSO-d6, δ ppm) : 161.15, 154.84, 152,84, 140.94, 131.47, 128.80, 122.54,
120.40, 117.03, 114.17, 98.14, 87.71, 63.09, 18.84, 14.65; LCMS (M/Z)Calculated for
C22H21N5O3: 403.4; Obtained MS (M/Z): 404.3 (M+1)
4.1.5.18.
N-(2,3-Dichlorophenyl)-2-(4-ethylpiperazin-1-yl)-7-hydroxy-5-
methylpyrazolo[1,5-a]pyrimidine-3-carboxamide (6r)
White solid; Yield 39.20%; M. P. 268-269°C;IR (KBr, cm-1) 3323 (-NH str.), 1663 (-CO-NH
1
str.), 1240 (C-N str.), 1033 (-C-O-C), 829 (P-substituted), 775 (-C-Cl str.); H NMR (400
MHZ, DMSO-d6, δ ppm) : δ 11.96 (s, 1H, OH-Pyrimidine, D2O exchangeable),δ 9.41 (s, 1H,
CONH-Ar., D2O exchangeable), δ 7.07- 8.09 (m, 3H,-CH-Ar.), δ 5.83 (s, 1H,-CH-
Pyrimidine), δ 2.57-3.34 (s, 5H, -OC2H5-Ar.), δ 1.88 (s, 3H, -CH3-Pyrimidine), δ 1.34 (s, 2H,
-CH2-N-ethyl piperazine), δ 1.34 (s, 4H, -CH3-N-ethyl piperazine), δ 1.32 (s, 3H, -CH3-Ar);
13C NMR (400 MHz, DMSO-d6, δ ppm) : 159.88, 154.84, 152.94, 131.47, 128.80, 20.80,
113.83, 90.21, 65.85, 55.17, 16.51; LCMS (M/Z)Calculated for C20H22ClN5O2: 449.3;
Obtained MS (M/Z): 449.4 (M), 451.4 (M+2), 453.1 (M+4)
14

4.1.5.19.
N-(3,4-Dichlorophenyl)-7-hydroxy-5-methyl-2-(pyrrolidin-1-yl)pyrazolo[1,5-
a]pyrimidine-3-carboxamide (6s)
White solid; Yield 68.22%; M. P. 273-275°C;IR (KBr, cm-1) 3350 (-NH str.), 1662 (-CO-NH
1
str.), 1298 (-C-N str.), 892 (Di-substituted), 840 (P-substituted), 750 (-C-Cl str.); H NMR
(400 MHZ, DMSO-d6, δ ppm) : δ 11.82 (s, 1H, OH-Pyrimidine, D2O exchangeable), δ 9.41
(s, 1H, CONH-Ar., D2O exchangeable), δ 7.07- 7.57 (m, 3H,-CH-Ar.), δ 5.83 (s, 1H,-CH-
Pyrimidine), δ 3.50-1.88 (s, 5H, -OC2H5-Ar.), δ 1.88 (S, 4H, CH2 pyrrolidine ring), δ 1.34 (S,
4H, CH2 pyrrolidine ring), δ 1.34 (s, 3H, -CH3-Pyrimidine); 13C NMR (400 MHz, DMSO-d6,
δ ppm) : 159.88,155.57, 154.82, 153.49, 153.21, 147.20, 140.30, 120.80, 113.83, 90.21,
65.85, 55.17, 16.51; LCMS (M/Z)Calculated for C18H17Cl2N5O2: 406.2; Obtained MS (M/Z):
406.4 (M), 408.2 (M+2), 410.1 (M+4)
4.1.5.20.
7-Hydroxy-5-methyl-N-phenyl-2-(phenylamino)pyrazolo[1,5-a]pyrimidine-3-
carboxamide (6t)
White solid; Yield 84.24%; M. P. 280-284°C;IR (KBr, cm-1) 3348 (-NH str.), 1662 (-CO-NH
str.),1298 (-C-N str.), 840 (P-substituted); 1H NMR (400 MHZ, DMSO-d6, δ ppm) : δ 11.99
(s, 1H, OH-Pyrimidine, D2O exchangeable), δ 9.77 (s, 1H, CONH-Ar., D2O exchangeable), δ
8.80 (s, 1H, NH-Pyrazole, D2O exchangeable), δ 6.89- 7.69 (m, 10H,-CH-Ar.), δ 5.78 (s, 1H,-
CH-Pyrimidine), δ 3.37 (s, 3H, -CH3-Pyrimidine); 13C NMR (400 MHZ, DMSO-d6, δ ppm) :
161.47, 154.83, 152.99, 149.87, 140.42, 138.69, 138.48, 129.18, 128.46, 123.52, 120.81,
117.15, 98.21, 87.69, 20.30; LCMS (M/Z)Calculated for C20H17N5O2: 359.3; Obtained MS
(M/Z): 360.3 (M+1)
4.1.5.21.
7-Hydroxy-5-methyl-N-phenyl-2-(p-tolylamino)pyrazolo[1,5-a]pyrimidine-3-
carboxamide (6u)
White solid; Yield 49.12%; M. P. 274-276°C;IR (KBr, cm-1) 3365 (-NH str.), 1660 (-CO-NH
str.), 1228 (-C-N str.), 817 (P-substituted); 1H NMR (400 MHZ, DMSO-d6, δ ppm) : δ 11.99
(s, 1H, OH-Pyrimidine, D2O exchangeable), δ 9.77 (s, 1H, CONH-Ar., D2O exchangeable), δ
8.80 (s, 1H, NH-Pyrazole, D2O exchangeable), δ 6.89- 7.69 (m, 10H,-CH-Ar.), δ 5.78 (s, 1H,-
13
CH-Pyrimidine), δ 2.35 (s, 3H, -CH3-Ar), δ 2.08 (s, 3H, -CH3-Pyrimidine); C NMR (400
MHZ, DMSO-d6, δ ppm) : 161.47, 154.83, 152.99, 149.87, 140.42, 138.69, 138.48, 129.18,
128.46, 123.52, 120.81, 117.15, 98.21, 87.69, 20.30, 18.83; LCMS (M/Z)Calculated for
C21H19N5O2: 373.4; Obtained MS (M/Z): 374.3 (M+1)
4.1.5.22.
2-((4-Chlorophenyl)amino)-7-hydroxy-5-methyl-N-phenylpyrazolo[1,5-
a]pyrimidine-3-carboxamide (6v)
15

Pale yellow solid; Yield 51.12%; M. P. 266-268°C;IR (KBr, cm-1) 3365 (-NH str.),1660 (-
1
CO-NH str.), 1294 (-C-N str.), 821 (P-substituted), 754 (-C-Cl str.); H NMR (400 MHZ,
DMSO-d6, δ ppm) : δ 11.99 (s, 1H, OH-Pyrimidine, D2O exchangeable), δ 9.77 (s, 1H,
CONH-Ar., D2O exchangeable), δ 8.80 (s, 1H, NH-Pyrazole, D2O exchangeable), δ 6.89-
7.69 (m, 10H,-CH-Ar.), δ 5.78 (s, 1H,-CH-Pyrimidine), δ 2.50 (s, 3H, -CH3-Pyrimidine); 13C
NMR (400 MHZ, DMSO-d6, δ ppm) : 161.22, 154.81, 152.42, 150.09, 140.48, 139.94,
138.71, 128.90, 128.72, 128.51, 128.47, 123.81, 123.54, 120.75, 118.97, 118.66, 98.16,
88.05, 18.85, 16.71; LCMS (M/Z)Calculated for C20H16ClN5O2: 393.8; Obtained MS (M/Z):
394.3 (M+1), 396.3 (M+2)
4.1.5.23. N-(3-Chloro-4-fluorophenyl)-7-hydroxy-5-methyl-2-(p-tolylamino)pyrazolo[1,5-
a]pyrimidine-3-carboxamide (6w)
White solid; Yield 59.29%; M. P. 265-269°C;IR (KBr, cm-1) 3346 (-NH str.),1649 (-CO-NH
str.), 1207 (-C-N str.), 1130 (-C-F str.), 867 (D-substituted), 821 (P-substituted), 766 (-C-Cl
1
str.); H NMR (400 MHZ, DMSO-d6, δ ppm) : δ 11.90 (s, 1H, OH-Pyrimidine, D2O
exchangeable), δ 9.92 (s, 1H, CONH-Ar., D2O exchangeable), δ 8.53 (s, 1H, NH-Pyrazole,
D2O exchangeable),δ 6.87- 7.96 (m, 7H,-CH-Ar.), δ 5.76 (s, 1H,-CH-Pyrimidine), δ 3.72 (s,
13
3H, -CH3-Ar), δ 2.34 (s, 3H, -CH3-Pyrimidine); C NMR (400 MHZ, DMSO-d6, δ ppm) :
161.50, 154.89, 154.43, 152.96, 152.01, 149.93, 140.63, 138.35, 136.03, 136.00, 129.24,
129.08, 122.00, 120.91, 118.95, 118.77, 117.28, 116.69, 98.29, 87.34, 20.28, 18.94, 13.94;
LCMS (M/Z)Calculated for C21H17ClFN5O2: 425.8; Obtained MS (M/Z): 426.3 (M+1)
4.1.5.24.
N-(3-Chloro-4-fluorophenyl)-7-hydroxy-2-((4-methoxyphenyl)amino)-5-
methylpyrazolo[1,5-a]pyrimidine-3-carboxamide (6x)
Yellow solid; Yield 68.41%; M. P. 260-263°C;IR (KBr, cm-1) 3390 (-NH str.), 1641 (-CO-
NH str.), 1259 (-C-N str.), 1163 (-C-F str.), 1036 (-C-O-C), 877 (D-substituted), 819 (P-
1
substituted), 738 (-C-Cl str.); H NMR (400 MHZ, DMSO-d6, δ ppm) : δ 11.90 (s, 1H, OH-
Pyrimidine, D2O exchangeable), δ 9.92 (s, 1H, CONH-Ar., D2O exchangeable), δ 8.53 (s, 1H,
NH-Pyrazole, D2O exchangeable), δ 6.87- 7.96 (m, 7H,-CH-Ar.),δ 5.76 (s, 1H,-CH-
Pyrimidine), δ 3.58 (s, 3H, -OCH3-Ar), δ 2.34 (s, 3H, -CH3-Pyrimidine); 13C NMR (400
MHZ, DMSO-d6, δ ppm) : 161.50, 154.89, 154.43, 152.96, 152.01, 149.93, 140.63, 138.35,
136.03, 136.00, 129.24, 129.08, 122.00, 120.91, 118.95, 118.77, 117.28, 116.69, 98.29,
87.34, 55.20, 20.28, 18.94; LCMS (M/Z)Calculated for C21H17ClFN5O3: 441.8; Obtained MS
(M/Z): 442.3 (M+1), 443.2 (M+2), 444.4 (M+3)
4.1.5.25. N-(3-Chlorophenyl)-2-((4-fluorophenyl)amino)-7-hydroxy-5-methylpyrazolo[1,5-
a]pyrimidine-3-carboxamide (6y)
16

Yellow solid; Yield 74.44%; M. P. 254-256°C;IR (KBr, cm-1) 3388 (-NH str.), 1643 (-CO-
1
NH str.), 1215 (-C-N str.), 1161 (-C-F str.), 827 (P-substituted), 775 (-C-Cl str.); H NMR
(400 MHZ, DMSO-d6, δ ppm) : δ 11.96 (s, 1H, OH-Pyrimidine, D2O exchangeable), δ 9.95
(s, 1H, CONH-Ar., D2O exchangeable), δ 8.72 (s, 1H, NH-Pyrazole, D2O exchangeable),δ
7.11- 7.86 (m, 8H,-CH-Ar.), δ 5.78 (s, 1H,-CH-Pyrimidine), δ 2.35 (s, 3H, -CH3-Pyrimidine);
13C NMR (400 MHZ, DMSO-d6, δ ppm) : 161.38, 157.71, 155.36, 154.74, 152.61, 150.01,
140.67, 140.37, 137.43, 132.76, 130.15, 123.02, 119.93, 118.86, 118.75, 118.68, 115.27,
115.05, 98.27, 87.73, 18.8; LCMS (M/Z)Calculated for C20H15ClFN5O2: 411.8; Obtained MS
(M/Z): 412.3 (M+1), 414.4 (M+2);
4.1.5.26.
N-(3-Chlorophenyl)-2-((2,3-dimethylphenyl)amino)-7-hydroxy-5-
methylpyrazolo[1,5-a]pyrimidine-3-carboxamide (6z)
White solid; Yield 76.86 %; M. P. 264-266 °C; IR (KBr, cm-1) 3348 (-NH str.), 1656 (-CO-
NH str.), 1234 (-C-N str.), 827 (P-substituted), 773 (-C-Cl str.); 1H NMR (400 MHZ, DMSO-
d6, δ ppm) : δ 12.03 (s, 1H, OH-Pyrimidine, D2O exchangeable), δ 9.88 (s, 1H, CONH-Ar.,
D2O exchangeable), δ 8.85 (s, 1H, NH-Pyrazole, D2O exchangeable), δ 7.76- 8.16 (m, 7H,-
CH-Ar.), δ 5.77 (s, 1H,-CH-Pyrimidine), δ 2.35 (s, 3H, -CH3-Ar), δ 2.26 (s, 3H, -CH3-Ar), δ
13
2.18 (s, 3H, -CH3-Pyrimidine); C NMR (400 MHZ, DMSO-d6, δ ppm) : 162.20, 154.96,
153.71, 140.18, 138.86, 136.17, 132.80, 130.22, 125.61, 123.06, 122.91, 122.70, 120.08,
119.05, 115.47, 98.33, 87.14, 20.45, 18.92, 12.92; LCMS (M/Z)Calculated for
C22H20ClN5O2: 421.8; Obtained MS (M/Z): 422.4 (M+1), 424.4 (M+2)
4.2. Molecular docking study
4.2.1. Ligand Preparation, Protein preparation and grid generation
Molecular docking, a part of computer aided-drug design approach which having crucial role
in the construction of potential lead molecules via interactions with key amino acid residues
of identified targeted protein structure. Newly designed NCE’s surviving to the Lipinski’s
rule of five (6a-6z) were minimized and docked using “Glide Extra precision” (XP) protocol
to identify vital amino acid residues (Maestro v10.1, Schrodinger, LLC, NEW YORK, NY).
For the preparation of protein structure of enoyl-ACP reducates (InhA) inhibitor with co-
crystalline structure (PDB IDs: 2H7M) was selected and extracted from protein data bank
(PDB http://www.rcsb.org/pdb). Further for the refinement of the protein “Protein
Preparation Wizard” in Maestro (Maestro v.10.1) was applied followed by addition of the
hydrogen atoms and removal of the water molecules beyond 5Ǻ with OPLS 2005 force field.
Further “Glide’s Receptor Grid Generation” module was used to generate the receptor grid at
17

the active site of co-crystalline ligand with the centred dimension cubic box of 10 Ǻ × 10 Ǻ ×
10 Ǻ [24].
4.3. In-silico ADME prediction
Acceptable ADMET profile is one of the imperative measures for the accomplishment of
drug. Pyrazole derivatives obtained through molecular docking study further opted for in-
silico ADMET prediction using QikProp module in Schrodinger to check its druggability[20].
This module predicts physical descriptors like molecular weight, partition coefficient, cell
permeability, partition coefficient to brain/blood, blockage of HERG K+ channels, number of
metabolic reactions and percentage human oral absorption and mutagenicity compared to the
properties of a particular known standard drugs. Small molecules that comply with Lipinski’s
rule of five were further opted for the synthesis.
4.4. Biological Evaluation
4.4.1. In-vitro anti-tubercular activity
Micro plate Alamar Blue Assay (MABA) is a non-toxic method which is most commonly
uses thermally stable reagents. It includes Resazurin, oxidized form of Alamar blue on
inoculation of tubercle bacilli reduces to resorufin and gives fluorescent pink colour. The
newly synthesized final analogues of 7-hydroxy-5-methyl-N-substitutedphenyl-2-(substituted
phenylamino)pyrazolo[1,5-a]pyrimidine-3-carboxamide(6a-6z) were screened for its in-vivo
anti-mycobacterial activity against H37Rv strain using Micro plate Alamar Blue Assay
method [25]. Ciprofloxacin, Pyrazinamide and Streptomycin were used as reference standard
drugs. To minimize evaporation of medium in the test wells during incubation 200µl sterile
deionized water was added in 96 wells plate. To that 100 µl of the Middle brook 7H9 broth
and serial dilutions of compounds with 100 to 0.2 µg/ml concentration were added to the
plate. These plates were covered with Para film and incubated at 37ºC for five days. To this
25µl of freshly prepared 1:1 mixture of Alamar Blue reagent and 10% tween 80 was added to
the plate and incubated for 24 hrs. A blue colour in the well was interpreted as no bacterial
growth, and pink colour was recorded as growth and the MIC was defined as lowest drug
concentration which prevented the colour change from blue to pink.
4.4.2. MDR-TB and XDR-TB study
Additionally MDR-TB and XDR-TB study was performed using Lowenstein-Jensen medium
(L. J. medium) on H37Rv strain with potent heat molecules based on in- vitro Micro plate
Alamar Blue Assay method [26]. This method used to check the susceptibility of the
compounds with the strain of multidrug-resistant TB and extensively drug-resistant TB. 4 mL
of sterilized malachite green solution was added to the solution of aseptically broken eggs. To
18

that mineral salt solution was added which consist of magnesium sulphate (0.4 g),
magnesium citrate (1.6 g), potassium phosphate (4.0 g), asparagine (6.0 g), glycerol (20 mL)
and make up the volume up to 1000 mL with distilled water and mixed well to form uniform
medium. Further synthesized targeted compounds dissolved in 10 mL DMSO and from that
0.8 mL of each concentration was transferred into different McCartney bottles. To this 7.2
mL of L. J. medium was added and mixed well and bottles were incubated at 75˚C - 80˚C for
3 days for solidification. Isoniazid was considered as a reference standard for the comparison
of anti-tubercular activity.
4.4.3. MTT Cytotoxicity activity
Compounds active to anti-tubercular study (with MIC < 12.5μg/mL) werefurther evaluated
against mammalian Vero cell line for cytotoxicity study [27]. Vero Cell line was obtained
from National Centre for Cell Sciences (NCCS), Pune. Cell viability was assessed after
exposure to 72 h on the basis of cellular conversion of formazan product from MTT using the
Promega Cell Titer 96 non-radioactive cell proliferation assay. The obtained results are
important for the development of new chemical entities for the treatment of TB.
4.5. Molecular Dynamics simulation
The molecular dynamics study was performed with Berendsen thermostat and barostat
methods for 10 ns for the best dock protein-ligand complex using DESMOND (Schrodinger
Inc., USA) module [28]. The molecular dynamics simulation system study desired protein-
ligand complex was saturated and partial charges were determined. Energy minimization was
performed using OPLS_2005 force field [29]. The solvated system inserted to TIP3P
orthorhombic box of 10 Å × 10 Å × 10 Å [30]. Each system was then neutralised by the
system-built option in the protocol by adding 0.15 M NaCl in buffer. The precision of the
chemical structures was confirmed by Protein Preparation, Ligand preparation, and Epik tools
provided in Desmond. The minimized explicit salvation complex of ligand receptor complex
was simulated for 10 ns using NPT ensemble (temperature of 300 K and pressure of 1.01325
bars) [31]. Steepest Descent and Broyden-Fletcher-Goldfarb-Shanno algorithms were
applied to achieve the relaxation of the system. Dynamics simulation process was maintained
300 K temperature and 1 atm pressure with Nose-Hoover thermostat algorithm and Martyna
Tobias-Klein algorithm respectively. Long-range and short-range coulombic interaction was
controlled using smooth particle mesh ewald method with 9.0 Å endpoint values. MD
simulations were performed for 10 ns, and trajectory information was obtained with the rest
of 2.0 ns [32]. Plots of RMSD, RMSF and hydrogen bonds were generated along with its
dynamic simulation.
19

Acknowledgments
Authors are thankful to Maratha Mandal’s Dental College, Belgaum and NCL Pune for
providing biological screening of these titles compounds. Authors are also thankful to
Schrodinger Inc. for providing the help for the molecular dynamic simulation study.
Conflict of Interest
The authors declare no conflict of interest about this article.
Supporting Information
1
All IR-Spectroscopy, MS, H NMR and 13CMR spectra for final derivatives has been
provided in the “supplementary content” section.
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Scheme 1 synthetic approach for the derivatives of 7-hydroxy-5-methyl-N- substituted
phenyl-2-(substituted phenylamino)pyrazolo[1,5-a]pyrimidine-3-carboxamide (6a-6z)