Synthesis and Antiproliferative Activity of 2,4,6,7-Tetrasubstituted-2H-pyrazolo[4,3-c]pyridines

A library of 2,4,6,7-tetrasubstituted-2H-pyrazolo[4,3-c]pyridines was prepared from easily accessible 1-phenyl-3-(2-phenylethynyl)-1H-pyrazole-4-carbaldehyde via an iodine-mediated electrophilic cyclization of intermediate 4-(azidomethyl)-1-phenyl-3-(phenylethynyl)-1H-pyrazoles to 7-iodo-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridines followed by Suzuki cross-couplings with various boronic acids and alkylation reactions. The compounds were evaluated for their antiproliferative activity against K562, MV4-11, and MCF-7 cancer cell lines. The most potent compounds displayed low micromolar GI50 values. 4-(2,6-Diphenyl-2H-pyrazolo[4,3-c]pyridin-7-yl)phenol proved to be the most active, induced poly(ADP-ribose) polymerase 1 (PARP-1) cleavage, activated the initiator enzyme of apoptotic cascade caspase 9, induced a fragmentation of microtubule-associated protein 1-light chain 3 (LC3), and reduced the expression levels of proliferating cell nuclear antigen (PCNA). The obtained results suggest a complex action of 4-(2,6-diphenyl-2H-pyrazolo[4,3-c]pyridin-7-yl)phenol that combines antiproliferative effects with the induction of cell death. Moreover, investigations of the fluorescence properties of the final compounds revealed 7-(4-methoxyphenyl)-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridine as the most potent pH indicator that enables both fluorescence intensity-based and ratiometric pH sensing.

The obtained alcohols 3-7 were further converted into azides 9-12, respectively. Many methods have been developed for such a transformation, including Mitsunobu-type displacements [60,61], two-step procedures that involve a halogenated [62] or mesylated intermediate [63], the one-pot halogenation-azidation of alcohols [64], reactions with phosphitine intermediates [65], N-methyl-2-pyrolidone hydrosulphate, and trimethylsilylazide (TMSN 3 ) [51]. The latter method was chosen for the synthesis, and the reactions were performed in DCM with a catalytic amount of boron trifluoride diethyl etherate (BF 3 ·Et 2 O). The reactions were carried out at room temperature under an argon atmosphere and with a dry solvent in order to protect both the boron trifluoride and TMSN 3 from moisture (Scheme 1). Conversion was completed in 30 minutes, and the reaction products 8-12 were furnished in 50-93% yields.
The newly synthesized azides 8-12 were further used to form the pyrazolo [4,3c]pyridine core with iodine in the 7-position by adopting electrophilic substitution reaction conditions that were previously used to obtain 1,3,4-trisubstituted isoquinolines from 2-alkynyl benzyl azides [66]. Namely, azides 8-12 were dissolved in DCM and treated with iodine and a proper base (Scheme 1). Five equivalents of K 3 PO 4 were used for the primary azides 8 and 12, while one equivalent of NaHCO 3 was used for the secondary azides 9-11. The reactions were carried out at room temperature in the dark for 12 h, furnishing compounds 13-17 in 70-88% yields. An attempt to make use of a weaker base NaHCO 3 for the reaction with the primary azide 6 led to the formation of the dehalogenated side product 2,6-diphenyl-2H-pyrazolo [4,3-c]pyridine, which resulted in a troublesome purification and a lower yield of the target product.
The obtained 7-iodo-2,6-diphenyl-2H-pyrazolo[4,3-c]pyridines 13-17 were further used in palladium-catalysed Suzuki-Miyaura cross-coupling reaction (Scheme 2) by adopting a previously reported procedure [67]. Namely, aromatic boronic acids were reacted with compounds 13-17 using palladium acetate as a catalyst and Cs 2 CO 3 as a base in an aqueous ethanol solution under an argon atmosphere. To ensure a short reaction time, cross-coupling reactions were carried out under microwave irradiation, thus giving rise to compounds 18-37. Compounds 18 and 20-37 (Scheme 2) were obtained in fair to excel-lent yields (48-96%), but the full cross-coupling conversion of 13 using 2-methoxyphenyl boronic acid could not be achieved, resulting in a lower yield of compound 19. Scheme 3. Synthesis of compounds 40-42. Reagents and conditions: i: NaH, RI, and DMF at 70 • C for 1 h.

Optical Properties
The fluorescence properties of all final compounds 18-42 were first investigated in THF, with the excitation wavelength λ ex being set to 350 nm (Table S1, Supplementary File). The emission maxima λ em of all the compounds were located in the 437-487 nm range, which corresponds to the blue part of the visible light spectrum. A polar 4-hydroxyphenyl substituent at the 7-position bearing compounds 23, 29, and 37, as well as derivatives 31-32, 38, 40-42 (all of which bear 4-alkoxyphenyl substituents at the 7-position and ethyl or isopropyl substituents at the 4-position), possessed the most pronounced fluorescence properties. Namely, the Stokes shifts for these compounds were in 199-205 nm range, and the quantum yield reached approximately 60-85%.
Intracellular pH plays an important role in many biological processes, and its changes from normal to abnormal levels can lead to cellular dysfunction, various diseases, and a decrease in physical performance [68]. pH-sensitive fluorescent indicators enable the precise measurement of intracellular pH, which consequently provides valuable information about ongoing physiological and pathological processes at the cellular and sub-cellular levels [69]. To assess whether the fluorescence properties of the prepared compounds are pH-dependent, they were all analysed in pH 5, 7, and 9 buffers with the excitation wavelength λ ex set to 360 nm (Table S2, Supplementary File). The quantum yield of compounds 18, 24, and 30, all of which bear phenyl substituent at the 7-position, increased at acidic pH without substantial shifts in emission maxima, which were observed to be in the 435-447 nm range. The further analysis of compound 18 in a range of pH 2-11 buffers revealed a gradual decrease in fluorescence intensity with the increase of pH (Figure S1A, Supplementary File). On the other hand, the quantum yield of 2-methoxyphenyl or 4-methoxyphenyl substituent at the 7-position possessing compounds 19, 21, 25, 27, and 31 was higher in basic pH; moreover, in the case of 4-methoxyphenyl substituent at the 7-position bearing compounds 21, 27, and 31, an acidic pH caused the red shift of the emission maxima. For instance, in the case of compound 21, the emission spectrum was found to be composed of two partly overlapping bands ( Figure S1B, Supplementary File). The short-wavelength part is pH-sensitive. It is dominant in the basic environment, and decreasing the pH from 11 to 6 caused a decrease in fluorescence intensity without a shift in emission maxima, which was maintained at 458 nm. After a further decrease of pH, it was the long-wavelength band that became more dominant, which was manifested as a gradual shift of emission maxima to 519 nm. Any other 4-alkoxyphenyl substituent at the 7-position bearing compounds only exhibited a drop of quantum yield at acidic pH without the shift of the emission maxima. A polar 4-hydroxyphenyl substituent at the 7-position bearing compounds 23, 29, and 37, which possessed the highest quantum yields in THF, had the lowest quantum yields of up to 0.3% in an aqueous solution. It is well known that hydroxyphenyl groups are sensitive to photochemical reactions [70]. Typically, their pK a drops from~10 in the ground state to~3 in the excited state, and excited-state proton transfer reactions are common in aqueous solutions. For our molecules, these reactions resulted in fluorescence quenching. Our preliminary observations suggested that most of the compounds, except for the polar derivatives 23, 29, and 37, could be of potential interest as pH indicators. Considering the approximately 5-fold quantum yield increase and 40 nm blue shift of the emission spectrum maximum when moving from pH 5 to 9, the compound 21 seems to be the best pH indicator from the set of examined molecules, enabling both fluorescence-intensity-based and ratiometric pH sensing.
Subsequently, the effects of the most active compound 23, its less active 4-substituted analogues 29 and 37, and derivative 21 were studied on K562 leukemic cells. Asynchronously growing K562 cells were treated with 10 µM concentrations of selected compounds for 24, 48, and 72 h and analysed using immunoblotting and flow cytometry ( Figure 2). Immunoblotting revealed that 48 h treatment with the most potent compound 23 was sufficient for the induction of poly(ADP-ribose) polymerase 1 (PARP-1) cleavage [71] and the activation of initiator enzyme of apoptotic cascade caspase 9 [72]. Interestingly, in addition to the clear pro-apoptotic effects, we also observed the time-dependent fragmentation of microtubule-associated protein 1-light chain 3 (LC3), which has appeared during autophagy [73]. Similar outcomes with lower efficiencies were observed in all tested compounds. In addition to cell-death-related proteins, the expression levels of proliferating cell nuclear antigen (PCNA), which plays a key role in DNA replication [74], were analysed. The results revealed that all studied compounds reduced the levels of PCNA time-dependently, with the most pronounced effect observed for compounds 23 and 29. To independently support this observation, immunoblotting was complemented with the flow cytometric analysis of bromodeoxyuridine (BrdU) incorporation, which allowed us to recognize replicating BrdU-positive cells in the population [75] ( Figure 2B). In control samples the number of proliferating cells came up to 40%, but the 10 µM treatment with tested compounds 21, 23, 29, and 37 reduced the proportion of actively proliferating BrdU-positive cells in up to approximately 10% for the most active compounds 23 and 29. Overall, the obtained results suggest the complex action of the compounds, combining antiproliferative effects with the induction of cell death.

General
All chemicals and solvents were purchased from commercial suppliers and used without further purification unless otherwise specified. The 1 H, 13 C, and 15 N NMR spectra were recorded in CDCl 3 or DMSO-d 6 solutions at 25 • C on either a Bruker Avance III 700 (700 MHz for 1 H, 176 MHz for 13 C, and 71 MHz for 15 N) spectrometer equipped with a 5 mm TCI 1 H-13 C/ 15 N/D z-gradient cryoprobe or a Jeol ECA-500 (500 MHz for 1 H and 126 MHz for 13 C) spectrometer equipped with a 5 mm Royal probe. The chemical shifts, expressed in ppm, were relative to tetramethylsilane (TMS). The 15 N NMR spectra were referenced to neat, external nitromethane (coaxial capillary). The 19 F NMR spectra (376 MHz) were obtained on a Bruker Avance III 400 instrument using C 6 F 6 as an internal standard. FT-IR spectra were collected using the ATR method on a Bruker Vertex 70v spectrometer with an integrated Platinum ATR accessory or on a Bruker Tensor 27 spectrometer in KBr pellets. The melting points of crystalline compounds were determined in open capillary tubes with a Buchi M 565 apparatus (temperature gradient: 2 • C/min) and are uncorrected. Mass spectra were recorded on Q-TOF MICRO spectrometer (Waters), analyses were performed in the positive (ESI + ) mode, and molecular ions were recorded in [M + H] + forms. High-resolution mass spectrometry (HRMS) spectra were obtained in the ESI mode on a Bruker MicrOTOF-Q III spectrometer. All reactions were performed in oven-dried flasks under an argon atmosphere with magnetic stirring. Reaction progress was monitored by TLC analysis on Macherey-Nagel™ ALUGRAM ® Xtra SIL G/UV254 plates. TLC plates were visualized with UV light (wavelengths of 254 and 365 nm) or iodine vapour. Compounds were purified by flash chromatography in a glass column (stationary phase of silica gel, high-purity grade of 9385, pore size of 60 Å, particle size of 230-400 mesh, supplied by Sigma-Aldrich). 1 H, 13

Chemistry
was dissolved in MeOH (12 mL), and the solution was cooled to 0 • C. Subsequently, NaBH 4 (156 mg, 4.12 mmol) was added under an argon atmosphere, and the mixture was stirred for 30 min. Upon completion (monitored by TLC), the reaction mixture was diluted with a saturated aqueous NH 4 Cl solution (20 mL) and extracted with EtOAc (3 × 30 mL). The combined organic layers were dried over anhydrous Na 2 SO 4 , filtered, and evaporated under reduced pressure. The residue was purified by column chromatography (EtOAc/Hex, 1:3 v/v).

General Procedure (A) for the Synthesis of Alcohols 4-7
1-Phenyl-3-(2-phenylethynyl)-1H-pyrazole-4-carbaldehyde 2 (1 equivalent) was dissolved in dry THF under an argon atmosphere. Subsequently, an appropriate Grignard reagent (1.2 equivalents) was added, and the mixture was stirred at room temperature for 10 min. Upon completion (monitored by TLC), the reaction mixture was diluted with water (20 mL) and extracted with EtOAc (3 × 20 mL). The combined organic layers were dried over anhydrous Na 2 SO 4 , filtered, and evaporated under reduced pressure. The residue was purified by column chromatography.

Optical Properties
The UV-vis spectra of 10 −4 mol solutions of the compounds in THF were recorded on a Shimadzu 2600 UV/vis spectrometer. The fluorescence spectra were recorded on an FL920 fluorescence spectrometer from Edinburgh Instruments. The PL quantum yields (Φ f ) were measured from dilute THF solutions by an absolute method using the Edinburgh Instruments integrating sphere excited with a Xe lamp. The optical densities of the sample solutions were ensured to be below 0.1 to avoid reabsorption effects. All optical measurements were performed at room temperature under ambient conditions.

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Stock solutions (4 mM) of the compounds were prepared in DMSO and further diluted in a Britton-Robinson buffer to a final concentration of 2 µM for spectroscopic analyses. Absorption spectra at pH 5, 7, and 9 for all compounds and in the 2-11 pH range with 0.5 step for selected compounds were measured using a Specord 250 Plus spectrophotometer in appropriate Britton-Robinson buffers. The spectra were measured in the 240-450 nm interval with a step of 1 nm, a 1 nm bandpass, and an integration time of 0.5 s. The samples were placed into a quartz cuvette with an optical path of 1 cm. The baseline was measured for the cuvette containing the solvent only.

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The steady-state excitation and emission spectra of 2 µM solutions of all the compounds at pH 5, 7, and 9 and in the 2-11 pH range with a 0.5 step for selected compounds were recorded on a Fluorolog-3 fluorimeter in the quartz cuvette with the 1 cm optical path (both in excitation and emission). Bandpasses in both the excitation and emission monochromator were set to 2 nm, and the spectra were scanned with the 1 nm step and an integration time 0.2 s per data point at 22 • C. Emission spectra were recorded in a 370-700 nm range with excitation at 360 nm.

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The quantum yield was estimated via integration of the fluorescence intensity over a range of 370-700 nm, and a 2.5 µM quinine sulphate solution in 0.05 M H 2 SO 4 was used as a standard (Φ f = 60%) [76].

Data Availability Statement:
The data that support the findings of this study are available upon request.