One-Pot Synthesis of Densely Substituted Pyrazolo[3,4-b]-4,7

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One-Pot Synthesis of Densely Substituted Pyrazolo[3,4-b]-4,7-dihydropyridines H. Surya Prakash Rao, Lakshmi Narayana Adigopula, and Krishna Ramadas ACS Comb. Sci., Just Accepted Manuscript • DOI: 10.1021/acscombsci.6b00156 • Publication Date (Web): 10 Apr 2017 Downloaded from http://pubs.acs.org on April 13, 2017

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One-Pot Synthesis of Densely Substituted Pyrazolo[3,4-b]-4,7-dihydropyridines H. Surya Prakash Raoǂ,§,* Lakshmi Narayana Adigopulaǂ,§, Krishna Ramadas§ ǂ

Department of Chemistry, Pondicherry University, Puducherry – 605 014, India.

§

Center for Bioinformatics, Pondicherry University, Puducherry – 605 014, India.

E-mail: [email protected], [email protected]; Tel: +91-413-2654411; Fax: +91-4132656230. Graphical Abstract: O

R1

CN R1

SMe NO2 R2 O

NHR3

H2N-NHR4 AcOH (10 mol%) EtOH 55 oC, 2 h

R2

R4 N N R1

OH NO2

NH2 EtOH

NO2

85 oC, 4-12 h

R2 3

O NHR Intermediate if R4 = H; Isolated and characterized; 4 Examples if R4 = Me or Ph reaction stops at this stage Structural diversity possible in four domains Podophyllotoxin mimics

N N R4

N H

NHR3

when R4 = H 20 Examples More than 85% yield Products precipitate out of reaction mixture. Structural diversity possible in three domains Podophyllotoxin mimics

Abstract We have achieved a facile synthesis of a combinatorial library of densely substituted pyrazolo[3,4-b]-4,7-dihydropyridines - the mimics of anti-genital wart drug podophyllotoxin from 5-aminopyrazoles and 4-(methylthio) 4H-chromenes. The C(4) pyrazolyl 4H-chromenes, which also possess structural features of podophyllotoxin, were isolable intermediates in the twostep, one-pot condensation. The condensation took place in a one-pot, multi-component manner when 3-oxo-3-phenylpropanenitriles, hydrazine (precursors for 5-aminopyrazoles) and 4(methylthio)-4H-chromenes were heated in refluxing ethanol. The condensation, however, stops at 4H-chromene stage when methyl hydrazine or phenylhydrazine were employed. Our findings Page 1 of 19 ACS Paragon Plus Environment

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offer an opportunity for synthesis of a combinatorial library of podophyllotoxin mimics, thus paving the way for discovery of lead candidates for cancer treatment. Introduction The majority of the new chemical entities (NCEs) that are in use for medicinal purposes have been synthesized by drawing inspiration from pharmacophore features of bio-active natural products.1 Many NCEs mimic actions of the molecules found in the human biological system. These compounds (agonists or antagonists) compete with natural ligands for binding to target enzymes, resulting in their inhibition or sometime activation.2 In recent years, several NCEs have been developed and approved for treatment of cancer, a major health threat.3 Among plant-based natural products podophyllotoxin (POD) 1 (Figure 1) and its congeners exhibit anti-cancer properties. 4 POD is an anti-proliferative and apoptosis inducing agent. 5 POD, isolated from rhizomes of Phodophyllum peltatum, Phodophyllum emodi and Sinopodophyllum hexandrum, is a non-alkaloid lignin.6 Although, production of POD is sustainable, its isolation in pure form is not cost-effective and its availability is scarce.7 To overcome such problems, efforts have been made to synthesize its mimics. 8 For example, the POD analogues like 4-aza-2,3-didehydropodophyllotoxin 2 (Figure 1) are easy to synthesize by one-pot multi-component condensation reactions from readily available aromatic aldehydes, tetronic acid and aromatic amines.9 Such NCEs exhibited potent apoptosis inducing activity similar to or better than the natural product 1. Moreover, unlike 1, which has four stereogenic centers, NCEs like 2 have only one stereogenic center, therefore have fewer problems in purification. Even the lone stereogenic center at C(4) is prone to racemization as the C(4)H is labile.

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Figure 1. Structures of podophyllotoxin 1, 4-aza-2,3-didehydro-podophyllotoxin

2, 2-

methylamino-3-nitro-4-aryl-4H-chromene 3. In recent years, we have been working on the synthesis and evaluation of anti-cancer properties of 2-methylamino-3-nitro-4-aryl-4H-chromenes like 3 (Figure 1). 10 They possess pharmacophores features of POD 1, which include (i) presence A, B and C rings, (ii) C(3) nitro group in place of lactone, (iii) intramolecularly hydrogen bond stabilized C(2) alkyl amino group in place of the methylene (Figure 1). Besides the similarities, 4-aryl-4H-chromenes like 3, display hydrogen bond donor and acceptor characteristics at C(2) secondary amine. More importantly, they can be assembled in two easy, high-yielding and environmentally compatible steps.11 To continue these efforts, we planned substitution of C(4) SMe in 4H-chromene 4 with 5-aminopyrazole 5 (Scheme 1). The reactions of 4 with 5 can take place at C(5) of the pyrazole to afford 4-pyrazolo-4H-chromenes 6 or on the primary amine to give 4-pyrazolylamino-4Hchromenes 7. The reaction of 4 with 5 conducted in EtOH at 55 oC gave 6 exclusively (Scheme 1). Furthermore, upon heating in refluxing EtOH 6 rearranged to the densely substituted 4,7dihydro-1H-pyrazolo[3,4-b]pyridine 8. We describe herein details of this study for the synthesis of a combinatorial library of 4,7-dihydropyrazolo[3,4-b]pyridines. The 4,7-dihydropyrazolo[3,4b]pyridine ring is a medicinally privileged scaffold.12 It possesses structural features of POD 1 like aromatic rings A, C, dihydro aromatic ring B, electron rich aromatic ring C and C(5) nitro group which is an isostere of the lactone (Figure 2). In addition, 8 has C(6) NHR which has


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hydrogen bond donor acceptor characteristics and C(3) aryl group which can show hydrophobic and stacking interactions. Moreover, functionalized pyrazolo[3,4-b]pyridines like 9 (Scheme 1), which can be derived from 8 by dehydrogenation can mimic anxiolytic drugs like etazolate, cartazolate, and tracazolate.13

Scheme 1. Synthesis of 4-pyrazolo-4H-chromene 6, 4,7-dihydropyrazolo[3,4-b]pyridine 8 and pyrazolo[3,4-b]pyridine 9 from 4H-chromene 4.

Figure 2. Pharmacophore features of 4,7-dihydropyrazolo[3,4-b]pyridines. Results and Discussion Initially, we conducted the condensation of 5-aminopyrazole 5 and 4H-chromene 4 in EtOH at rt and the reaction after 24 h provided the C-alkylated product 6 exclusively in 25% yield; there was no trace of N-aryl product 7 (Scheme 1). At 55 oC the reaction took only 1.5 h to afford 6 in 62% yield. Notably, the reaction to form 6 took place on the more-crowded C(4) Page 4 of 19 ACS Paragon Plus Environment


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carbon of pyrazole 5 instead of less-crowded C(5) primary amino group as confirmed by the NMR spectral data. The characteristic signal of C(4)H of 6 appeared at δ 5.36 ppm in its 1H NMR spectrum recorded in a mixture of DMSO-d6 and CCl4 (1:1). The chemical shift of the signal indicates that C(4) arylation rather than C(4) amination took place. The N-aryl product 7 is anticipated to exhibit a singlet at about 6.7 ppm for C(4)H.14 The HMBC NMR (2D) spectrum of N-phenyl analogue 6 (Scheme 4, vide supra) displayed cross peaks for C(4)H to C(3) of chromene and to C(4) of pyrazole.15 The 1H NMR spectral signals due to the pyrazolyl NH2 and aromatic CH of 6 were broad. Its

13

C NMR spectrum did not exhibit signals due to quaternary

carbons of the pyrazole and phenyl rings.16 This observation indicates the presence of slowly interconverting conformational isomers of 6 in DMSO-d6 and CCl4 (1:1). The conjugates of 4Hchromene and 5-phenyl-1H-pyrazol-3-amine e.g. 6 could exhibit restricted rotation around the CC bond joining 4H-chromene and pyrazole ring, as well as around the C-C bond joining phenyl and pyrazole rings. Moreover 6 can be present as an equilibrium mixture of 6A and 6B which are generated by a thermally allowed suprafacial 1,5-H shift (Figure 3). We believe that broadening of the relevant signals in the 1H NMR spectrum and the absence of quaternary carbons in the 13C NMR spectrum indicate restricted rotation and equilibrium between tautomeric structures. H

H N N

O 6A

N N

C D

NH2

NH2

NO2

NO2

NHMe

O 6B

NHMe

OH NO2

OH NO2 N

A

N H

HN

B

N H 8A

NHMe

N

N H 8B

NHMe

Figure 3. Structures of tautomeric forms of C(4) pyrazolyl 4H-chromene 6 and pyrazolopyridine 8. We noticed that 6 was not stable at room temperature (30 oC). On standing, it slowly rearranged into densely substituted 4,7-dihydropyrazolo[3,4-b]pyridine 8, a new product of Page 5 of 19 ACS Paragon Plus Environment

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higher polarity (TLC, silica gel; hexane EtOAC, 2:8, Rf = 0.1; Scheme 1). Complete conversion of 6 into 8 took place upon heating in refluxing EtOH for 2 h. Structure of 8 was deduced from its spectra (1H,

13

C, DEPT-135 and 2D NMR) and HRMS. The C4(H) of 8 appeared at δ 5.72

ppm as singlet, 0.4 ppm down field shifted from that of corresponding C(4)H of 6. The 13C NMR spectrum exhibited signals at δ 154.8 ppm for C(2) and δ 101.5 ppm for C(3), each of which are up-field shifted by about 5 ppm compared to corresponding carbons in 6. Interestingly, unlike that of 6, both 1H and 13C NMR signals of 8 in DMSO-d6 were sharp, which indicates that there is free rotation in the C-C single-bond between C(4) of dihyropyridine and the phenyl group attached to it. Similar to 6, the pyrazole ring in 8 can, in principle, have two tautomeric structures 8A and 8B (Figure 3), but unlike in 6, the pyrazole ring in 8 stabilizes in structure 8A as confirmed by single crystal X-ray structure analysis (Figure 4). The X-ray crystal structure showed that C(4) aryl group of dihydropyridine occupies pseudo-equatorial position and rings C and D are orthogonal to each other (see structures 8A given in Figure 3).



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Figure 4. X-Ray crystal structure of 2-(6-(methylamino)-5-nitro-3-phenyl-4,7-dihydro-1Hpyrazolo[3,4-b]pyridin-4-yl)phenol 8; thermal ellipsoids are set at 50% probability (CCDC 1502099). Next, we explored the possibility of conducting a multi-component, one-pot, domino condensation reaction involving 4H-chromene 4{1}, 3-oxo-3-phenylpropanenitrile 10{1} (phenacyl nitrile) and hydrazine hydrate 11 to form pyrazolodihydropyridine 8{1,1} (Scheme 2). We reasoned that in the first-step 3-oxo-3-phenylpropanenitrile 10{1} and hydrazine hydrate 11 condense to form 5-aminopyrazole 5, which will then react with 4H-chromene 4{1} in a dominofashion to form 4H-chromene 6{1,1}. Finally, 6{1,1} will rearrange to form the densely substituted pyrazolodihydropyridine 8{1,1}. The surmise proved to be correct, when we realized an 85% yield of 8{1,1} from the one-pot domino condensation of 3-oxo-3-phenylpropanenitrile 10{1}, hydrazine hydrate 11 and 4H-chromene 4{1} in refluxing EtOH (Scheme 2). The domino-condensation did not proceed when the reaction was conducted at a lower temperature (55 oC). To optimize the reaction conditions, we conducted the multi-component reaction at the reflux temperatures of solvents like diethyl ether (no product formation), di-isopropyl ether (24 h, 35%), tert-butyl methyl ether (20 h, 55%), tetrahydrofuran (22 h, 40%), 1,4-dioxane (12 h, 74%), toluene (10 h, 61%), MeCN (10 h, 70%), MeOH (12 h, 60%), EtOH (8 h, 85%) and nPrOH (10 h, 74%), which showed that EtOH was the best solvent. Moreover, the product 8{1,1} precipitated from the reaction mixture almost completely upon cooling to rt.


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Scheme 2. Domino multi-component one-pot condensation to form pyrazolodihydropyridine 8{1,1}. The proposed mechanism for the domino-multicomponent condensation is given in Scheme 3. Initial dehydrative condensation of 3-oxo-3-phenylpropanenitrile 10 with hydrazine hydrate 11 gives the corresponding hydrazone 12. Further cyclization gives the cyclic product 13, which on tautomerization leads to the stable 5-aminopyrazole 5. The displacement of C(4)SMe group in 4, possibly via benzopyrylium cation gives 6. In refluxing EtOH, the amino group in 6 reacts at C(2) position of the chromene ring in the intra-molecular fashion to generate the strained tetracyclic intermediate 14. Opening of the pyran ring in 14 leads to 8. We have carried out theoretical (MM2) calculations on 6{1,1} and 8{1,1} to understand the driving force for the rearrangement.17 The calculations showed that 6 was more stable than 8 by about 7.1 kcal mol-1. However, the amino-group of the pyrazole was 127 pm from the C(2) of 4H-chromene ring in 6, compared to 282 pm for the phenolic hydroxy to the C(2) of pyrazolodihydropyridine. This observation indicates that proximity of the amino group to C(2) in 6 is crucial to the rearrangement. After the rearrangement the phenolic hydroxy group in 8 is far from C(2). Alternatively, the proposed mechanism could involve the initial substitution of the C(4)SMe in the 4H-chromene 4 with 3-oxo-3-phenylpropanenitrile 10 ahead of pyrazole ring formation. To evaluate this possibility, we reacted 10 with 4 without hydrazine. The substitution reaction did not proceed even after 12 h of refluxing in EtOH. This experiment shows 5-aminopyrazole Page 8 of 19 ACS Paragon Plus Environment


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formation takes place ahead of substitution. Support for the proposed mechanism also came from the observation that 6 could be isolated by conducting the reaction of 4 and 5 at 55 oC (Scheme 1), which in turn could be subjected to rearrangement to 8 by heating in refluxing EtOH.

Scheme 3. Possible mechanism for the formation of pyrazolo[3,4-b]-4,7-dihydropyridine 8{1,1}. To evaluate the generality of the above domino multi-component condensation and to generate a combinatorial library of pyrazolodihydropyridines 8{1-4, 1-4}, we conducted threecomponent condensation reaction involving the C(4)SMe 4H-chromenes 4{1-4}, the 3-oxo-3phenylpropanenitriles 10{1-4} and hydrazine 11 to realize the sixteen pyrazolodihydropyridines 8{1-4, 1−4} (Table 1) in good yield. Electron-withdrawing (Cl, Br) and electron-donating groups (Me) at C(4) position of 3-oxo-3-phenylpropanenitrile or electron-withdrawing (Cl, Br) and electron-donating groups (OMe) in the aryl ring of 4H-chromene did not have a significant influence on the outcome of the reaction (Table 1). Finally, we varied the N-alkyl in 4 from methyl to n-butyl and subjected resulting 4{5} to reaction with 10{1-4} and hydrazine hydrate to realize four more pyrazolodihydropyridines 8{5, 1-4} in good yield (Table 1). The n-butyl


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substitution in 4{5} was meant to increase the lipophilic characteristics of the pharmacophore in 8. The spectral data (IR, 1H NMR, 13C NMR, DEPT-135) and analytical data (HRMS) of 8{1-5, 1-4} supported the assigned structures and they compared well with the parent 8{1,1} for characteristic signals. Table 1. Substrate scope for the preparation of pyrazolo[3,4-b]-4,7-dihydropyridines 8

Entry 1 2 3 4 5

Product 8{1,1} 8{1,2} 8{1,3} 8{1,4} 8{2,1}

Yield (%) 85 82 86 76 81

Entry 11 12 13 14 15

Product 8{3,3} 8{3,4} 8{4,1} 8{4,2} 8{4,3}

Yield (%) 68 82 78 65 70 Page 10 of 19

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6 7 8 9 10

80 75 71 72 79

8{2,2} 8{2,3} 8{2,4} 8{3,1} 8{3,2}

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16 17 18 19 20

82 72 82 63 63

8{4,4} 8{5,1} 8{5,2} 8{5,3} 8{5,4}

To clarify the scope of the rearrangement chemistry, we conducted the domino, one-pot, multi-component condensation of 3-oxo-3-phenylpropanenitrile 10{1} and C(4)SMe 4Hchromene 4{1} with methyl hydrazine in refluxing EtOH (Scheme 4). This reaction provided 4H-chromene

15

exclusively.

The

product

did

not

rearrange

to

corresponding

pyrazolodihydropyridine 16. Unlike 6{1,1}, the N-methylpyrazolyl derivative 15 is soluble in CDCl3 and its 1H and

13

C NMR spectra displayed sharp signals indicating free rotation in C-C

bonds linking aryl and heteroaryl rings and arresting of tautomeric structures. The reaction of 10{1} and 4{1} with phenyl hydrazine provided 4H-chromene 17 exclusively (Scheme 4).18 The 4H-chromene

17

did

not

undergo

further

rearrangement

to

corresponding

pyrazolodihydropyridine 18 even after 24 h refluxing in EtOH. i. PhHNNH2 EtOH reflux 6 h, 70%

N N

CN NH2 NO2

O 17 X

O

NHMe ii.

i. MeHNNH3HSO4 Et3N (1 equiv) EtOH reflux, 2 h, 75%

SMe

Me N N NH2

SMe NO2

O NHMe 4{1}

NO2

10{1} ii.

NO2

O NHMe 4{1}

EtOH reflux, 24 h

O 15

NHMe

EtOH reflux, 24 h X

OH NO2

OH NO2

N

N N

N H

N Me

NHMe

N H

NHMe

16 18


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Scheme 4. The domino one-pot multi-component condensation of 3-oxo-3-phenylpropanenitrile 10{1} with C(4)SMe 4H-chromene 4{1} and methyl hydrazine / phenyl hydrazine to provide 4H-chromenes 15 and 17. To demonstrate a synthesis of pyrazolopyridines by our method, as a proof-of-principle, we subjected pyrazolodihydropyridine 8{1,1}

to dehydrogenation with 2,3-dichloro-5,6-

dicyanobenzoquinone (DDQ) to realize densely substituted pyrazolopyridine 9 in good yield (Scheme 1). Conclusion In conclusion, we have described a facile synthesis of densely substituted pyrazolodihydropyridines 8{1-5, 1-4} by a domino, one-pot, multi-component condensation of C(4)SMe 4H-chromenes 4{1-5}, 3-oxo-3-phenylpropanenitriles 10{1-4} and hydrazine. The C(4) 5-aminopyrazolyl 4H-chromenes 6 were isolable intermediates in the reaction. When methyl hydrazine or phenyl hydrazine were used, the condensation stopped at 4H-chromene stage. Our method allows access to a combinatorial library of products that have pharmacophore features of podophyllotoxin. We plan to get each of the pyrazolodihydropyridines and 4Hchromenes evaluated for anti-cancer properties and report the results in due course. Experimental Procedure General: Oven dried glass-ware was used to carry out all the reactions. Progression of reactions was monitored by thin layer chromatography (TLC) plates of 7.5cm × 2.5 cm dimension prepared in-house by using aqueous slurry of silica gel-GF 254 (LOBA Chemie) having 13% CaSO4 as binder. Drying the plates in an oven at 80 ̊C overnight readied them for use. TLC spots were visualized in iodine vapor and in UV light. Mixture of ethyl acetate and hexanes were used as eluents. Methanol and ethanol were distilled according to standard procedures given in



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Vogel’s Text Book of Organic Chemistry.19 VEEGO VMP-DS melting point apparatus was used to determine. Melting points were determined in open end capillaries and are uncorrected. IR spectra were recorded using Nicolet-6700 spectrometer was used as solid solution in KBr. 1H, 13

C and DEPT-135 NMR spectra were recorded on Bruker Avance 400 spectrometer (400 MHz)

using a mixture of DMSO–d6 and CCl4 taken in 1:1 ratio. DEPT-135 spectral data was used to ascertain the number of hydrogen atoms present on each carbon atom. Chemical shift values (δ) are expressed in parts per million units and is measured relative to SiMe4 (d=0.00) as internal standard. Coupling constant (J) are measured in Hz. Multiplicities are expressed as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet) or broad (br). UV spectra were recorded using Shimadzu UV-2450 double-beam spectrometer. Starting products like 2-hydroxybenzaldehydes, acetophenones were purchased from Sigma-Aldrich and were used without further purification and Hydrazine hydrate 80%. N-methyl-4-(methylsulfanyl)-3-nitro-4H-chromene-2-amines and 3aryl-1H-pyrazol-5-amine to literature procedure. Representative procedure for the synthesis of C(4) 3-phenyl-1H-pyrazol-5-amino 2methylamino-3-nitro-4H-chromens. Synthesis of 4-(2-(methylamino)-3-nitro-4H-chromen4-yl)-3-phenyl-1H-pyrazol-5-amine 6{1,1}:

The solution of 5-aminopyrazole 5 (85 mg, 0.50 mmol) and 4H-chromene 4{1} (126 mg, 0.50 mmol) in dry EtOH (5 mL) was heated to 55 oC for 1.5 h by which time the reaction was complete (TLC, 50% EtOAc in hexanes; Rf = 0.4). Evaporation of EtOH at reduced pressure and purification of the crude product by column chromatography 4H-chromenes 6{1,1} resulted as


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colorless solid. (112 mg, yield = 62%). Mp 186-188 °C. IR(υmax) (KBr) 3428, 3291, 2992, 1642, 1365, 1069, 749.cm-1; 1H NMR (400 MHz, DMSO-d6) δ 11.52 (s, 1H), 10.22 (d, J = 4.5 Hz, 1H), 7.31 (d, J = 6.1 Hz, 3H), 7.19 – 7.03 (m, 5H), 6.94 (d, J = 8.0 Hz, 1H), 5.36 (s, 1H), 4.32 (s, 2H), 2.99 (d, J = 4.6 Hz, 3H).

13

C NMR (100 MHz, DMSO-d6) δ 158.3 (C), 146.5 (C), 129.1 (CH),

128.3 (CH), 127.6 (CH), 127.4 (CH), 127.3 (CH), 125.0 (CH), 124.8 (C), 115.2 (CH), 106.4 (C), 30.2 (CH), 27.5 (CH3) ppm. HRMS (ESI) Calcd. for C19H17N5O3 363.1331 amu [M+H] found 364.1402 amu. Representative procedure for the synthesis of C(4)-aryl 4,7-dihydro-1H-pyrazolo[3,4b]pyridines.

Synthesis

of

2-(6-(methylamino)-5-nitro-3-phenyl-4,7-dihydro-1H-

pyrazolo[3,4-b]pyridin-4-yl)phenol 8{1,1}:

To the solution of 3-oxo-3-phenylpropanenitrile 10{1} 20 (87 mg, 0.60 mmol), 4Hchromene 6{1} (126 mg, 0.50 mmol) and aq. hydrazine hydrate (80%) 11 (0.1 gm, 1.00 mmol) in EtOH (14 mL) catalytic amount of acetic acid (10 mol%, 12 mg) in EtOH (1 mL) was added. Resulting solution was heated to reflux for 8 h until the reaction was complete (TLC, 90% EtOAc in hexanes; Rf = 0.2). The product that precipitated from reaction mixture on cooling to rt was separated by filtration with the help of ethanol (3 mL) to get pyrazol-1,4-dihydropyridine 8{1,1 }, as colorless solid (156 mg, yield = 85%). Mp 196-197 °C. IR (υmax) (KBr) 1636, 1486, 1332, 1065, 761cm-1; 1H NMR (400 MHz, DMSO-d6) δ 12.66 (s, 1H), 11.23 (d, J = 4.9 Hz, 1H), 10.56 (s, 1H), 9.15 (s, 1H), 7.56 (d, J = 8.1 Hz, 2H), 7.36 (t, J = 8.0 Hz, 2H), 7.29 (d, J = 7.3 Hz, 1H), 7.06 (s, 1H), 6.85 (t, J = 8.1 Hz, 1H), 6.57 ( t, J = 8.0 Hz, 2H), 5.71 (s, 1H), 3.11 (d, J = 5.1 Page 14 of 19 ACS Paragon Plus Environment


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Hz, 3H);

13

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C NMR (100 MHz, DMSO-d6) δ 154.7 (C), 153.7 (C), 146.7 (C), 137.4 C), 131.0

(CH), 129.6 (C), 129.1 (CH), 128.6 (CH), 128.1 (CH), 127.2 (CH), 126.3 (CH), 118.1 (CH), 115.9 (CH), 109.5 (C), 101.8 (C), 35.8 (CH), 28.8 (CH3) ppm. HRMS (ESI) Calcd. for C19H17N5O3for [M+H] 364.1410 amu, [M+H] found 364.1408 amu Synthesis of 2-(6-(methylamino)-5-nitro-3-phenyl-1H-pyrazolo[3,4-b]pyridin-4-yl)phenol 9:

To the solution of pyrazol-1,4-dihydropyridine 8{1,1} (100 mg 0.275 mmol) and 2,3dichloro-5,6-dicyano-1,4-benzoquinone (0.074 g, 0.57 mmol) in 1,4-dioxane (3 mL) was stirred at 100 oC for 4 h by which time the dehydrogenation was complete (TLC, 50% EtOAc in hexanes; Rf = 0.6). The reaction mixture after cooling was charged on a silica gel column and eluted with 10% ethyl acetate in hexanes to yield pyrazolopyridine 9 yellow color solid after evaporation of solvent from pooled fractions (73 mg, yield = 73%). Mp 163-165 °C. IR (υmax) (KBr) 1636, 1549, 1197, 1065, 883, 747 cm-1; 1H NMR (400 MHz, DMSO-d6) δ 13.38 (s, 1H), 9.53 (s, 1H), 7.19 (d, J = 3.9 Hz, 1H), 7.09 (d, J = 6.5 Hz, 1H), 7.04 – 6.93 (m, 5H), 6.76 (d, J = 7.2 Hz, 1H), 6.64 (d, J = 8.1 Hz, 1H), 6.49 (t, J = 7.3 Hz, 1H), 2.97 (d, J = 3.7 Hz, 3H).

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C

NMR (100 MHz, DMSO-d6) δ 154.2 (C), 151.7 (C), 150.9 (C), 150.5 (C), 138.7 (C), 132.9 (C), 130.8 (CH), 129.8 (CH), 129.3 (CH), 128.1 (CH), 127.0 (CH), 126.9 (CH), 120.1 (C), 118.4 (CH), 115.0 (CH), 113.6 (C), 103.9 (C), 28.6 (CH3) ppm. HRMS (ESI) Calcd. for C19H17N5O3 363.1331 amu, [M+Me] found 378.1572 amu. Supporting Information Page 15 of 19 ACS Paragon Plus Environment

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The Supporting Information that includes experimental details, analytical data, spectroscopic data, copies of 1H,

13

C and DEPT-135 NMR spectra of all the new compounds (PDF) and

crystallographic data for 8{1,1} (CIF file) is available free of charge in the ACS publication website. Acknowledgement H.S.P.R thanks UGC-MRP for financial support and UGC-SAP, DST-FIST for support to Department of Chemistry. L.N.A thanks to CSIR, India for fellowship. We thank CIF, Pondicherry University for spectra.

Reference

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One-Pot Synthesis of Densely Substituted Pyrazolo[3,4-b]-4,7

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