Fluorescent Isostere (Fluostere) of the Carboxylate: Design of hDHODH Fluorescent Inhibitors as Proof of Concept

Abstract

Fluorescent probes targeting proteins are used to investigate biological processes, requiring strong binding affinity and favorable fluorescence. In this study, we present the first fluostere with optimized fluorescence properties. We started exploring the fluorescence of acidic pyrazolo­[1,5-a]­pyridin-2-ol, and, by the introduction of EWGs, π-conjugation, incorporation of push–pull systems and rigid structures, we optimized emission profiles and QY, providing a first Structure–Fluorescence relationship (SFR) of the system. To provide proof of concept in biological applications, the established SFR was integrated with hDHODHi, an important oncology target, enabling the SAR designing fluorescent hDHODHi 11a and 14, with 11a being the most potent IC₅₀ = 170 nM. These inhibitors were validated in vitro for their antileukemic and antiviral activity. As they are both environmentally sensitive fluorescent probes that can highlight their binding to the target, their fluorescence was found to colocalize in the mitochondria, where hDHODH is located, in cellular experiments.

Introduction

Protein-targeted fluorescent probes have emerged as a promising and fascinating strategy to study and visualize biological processes within living organisms. Numerous applications are associated with this technique, including studying overexpressed proteins in cancer therapy, , investigating the biodistribution of unidentified targets, and early disease evaluation. The effectiveness of this strategy depends on the development of fluorescent probes endowed with a strong binding affinity to specific targets. Often fluorescent probes are developed by linking a fluorescent tag to a known bioactive ligand. ,

Recently, merging the pharmacophoric and fluorescent features in a single small molecular structure yielding bioactive intrinsically fluorescent ligands (BIFL) has emerged as a new intriguing approach. However, the retention of the fluorescence profile during the modulation of the structure responsible for target affinity still presents a significant challenge in this approach. Thus, the final compound must have an absorption-emission profile with larger Stokes shifts to eliminate spectral overlap while reducing self-quenching and enhancing the imaging signal-to-noise ratio. High emission quantum yield and physicochemical properties compatible with the biological target are also required. In this regard, Sotelo et al. recently reported the design of a green-emitting BIFL (compound 55 in ref with strong cannabinoid receptor subtype 2 (CB2R) affinity, high selectivity, and an agonist profile. ⁷ In the compound 55 structure, a fluorescent nitrobenzoxadiazole (NBD) moiety contributes to compound binding by forming π–π interactions with Phe183 and Phe117, introducing favorable pharmacological properties in the CB2R profile.

Harris Friedman introduced the term “bio-isosteres” in the 1950s to describe a subclass of isosteres that can produce a similar biological effect. Since then, bioisosteric replacement has been recognized as an effective strategy for increasing potency, enhancing selectivity, modulating physical properties, improving metabolic stability, removing or modifying toxicophores, and obtaining new intellectual property. In a further development of the BIFL as well as the bioisosteric fields, in this study, we take a ground-breaking step and present a first example of fluorescent isostere of carboxylic acid as a novel principle for designing intrinsically fluorescent ligands. To our knowledge, this is the first case of introducing “fluo-isosteres” (fluosteres) as a subclass of isosteres with an optimized fluorescence profile. Among the chemical groups used in bioisosteric replacements for carboxylic function and other acidic moieties are the acidic hydroxyazoles. The heterocyclic nature of these systems, acidic due to lactim-lactam tautomerism, is beneficial as it provides, by strategically altering the ring heteronucleus and substituents, the ability to fine-tune pK _a, lipophilicity-polarity balance and improve affinity to a target protein. , Within the hydroxyazole portfolio that we and others have developed for bioisosteric modulations in recent years, − the pyrazolo­[1,5-a]­pyridin-2-ol (1, Figure ) is a fluorescent acidic system (pK _a around 5). Unfortunately, this system is unsuitable for in vitro/in vivo applications when unsubstituted, due to its unfavorable excitation/emission wavelengths. In the first part of this study (Figure , Steps 1–4), the pyrazolo­[1,5-a]­pyridin-2-ol was systematically modulated to identify the structural features responsible for the fluorescent characteristics of the molecule. In other words, initially we focused on constructing a Structure Fluorescence Relationship (SFR, Figure , Step 5) on the system, looking for improvements in quantum yield and emission spectral profile.

1.

Starting from the structure of pyrazolo­[1,5-a]­pyridin-2-ol (1), the pathway followed to improve the fluorescence features and the final proof of concept in the design of fluorescent hDHODH inhibitors.

In the second part (Figure , Step 6), after clearing the preliminary SFR of the developed acid fluosteres, we obtained the first proof of concept by applying this new approach to the design of fluorescent bioactive ligands. In this regard, we decided to use our knowledge in the design of human dihydroorotate dehydrogenase (hDHODH) inhibitors.

The central role of the mitochondrial membrane-bound hDHODH in de novo pyrimidine biosynthesis has sparked engagement by the academic and pharmaceutical industries communities. − hDHODH is considered a consolidated target for treating diseases involving cellular proliferation, like autoimmune diseases and cancer (e.g., Acute Myeloid Leukemia (AML)), − as well as viral infections. ,−

Moreover, high levels of hDHODH expression in solid and pediatric tumors also indicate the significance of this enzyme as a prognostic marker, − highlighting the need for in vivo imaging probes to explore inhibitor efficacy further. The design of the first example of fluorescent hDHODH inhibitors took advantage of the fact that the structure of potent phase I/II hDHODH inhibitors (see example in Figure as brequinar, vidofludimus, AG-636, ASLAN-003 , and Rhizen RP7214 , often contains a carboxylic group that plays a crucial role in the interaction with Arg136 within the so-called lipophilic patch of the enzyme. After merging the Structure Fluorescence Relationship (SFR, Figure , Step 5) with the well-known hDHODH Structure Activity Relationship (SAR), we fine-tuned the fluostere molecule to engage hDHODH Arg136 while retaining optimal optical properties. All the newly developed fluorescent compounds were assayed for hDHODH inhibition activity and validated at the cellular level for two main biological effects of hDHODH inhibitors: the induction of apoptosis in an AML model and the antiviral effect against coronavirus (hCoV-OC43). Then, the most promising fluorescent hDHODH inhibitors were further validated through fluorescence microscopy analyses in different types of target cells, which verified their mitochondrial colocalization with the protein target.

2.

Structure of hDHODH inhibitors bearing an acidic moiety. The PDB IDs of the structures of the inhibitors in complex with hDHODH are indicated where available. The structure of Rhizen Pharmaceutical AG’s compound RP7214, which is involved in clinical trials, has not yet been disclosed. ,, Adapted with permission from reference (Copyright © 2022 by American Chemical Society).

Results and Discussion

Design of Acidic Fluorescent Isosteres

Hydroxyazoles are hydroxylated heterocycles characterized by the presence of a hydroxy group in position 2 or 3 with respect to an endocyclic nitrogen atom in a single or fused five-membered heterocycle.

This combination renders these systems slightly acidic, being the negative charge originates from the deprotonation of the hydroxy group delocalized over the oxygen and nitrogen atoms due to lactim-lactam tautomerism. One member of this class, pyrazolo­[1,5-a]­pyridin-2-ol (Figure , compound 1), is characterized by a nitrogen-bridged fused 5- and 6-membered ring in which the nitrogen in position 1 exhibits a pyrrolic property. Like the parent compounds indolizine and aza-indolizine, this heterocycle has an aromatic nature with 10 π-electrons. With an acidic pK _a value (the experimental pK _a measured on compound 2 is 5.39, Figure ), compound 1 can be considered deprotonated at physiological pH and, thus a carboxylic acid isostere.

3.

Summary of modifications applied to modulate the emission profile of the pyrazolo­[1,5-a]­pyridin-2-ol during the acquisition of the Structure Fluorescence Relationship (SFR).

Since the discovery of its fluorescent nature by Rangnekar et al., the chemistry of this system has been poorly investigated. When designing an effective fluorescent probe with this system, it is essential to consider several key features. Specifically, to reduce interference from biological background signals, an optimal fluorescent dye should emit light at wavelengths (λ_em) longer than 425 nm and exhibit a high Stokes shift. This feature helps prevent the reabsorption of emitted photons, thereby minimizing energy loss and ensuring signal integrity is maintained. Additionally, a high luminescence quantum yield (QY > 0.1), indicating the conversion efficiency of absorbed photons into emitted light, is crucial. We started by analyzing these parameters for 1, finding a promising starting point with a good QY (0.21) and a high Stokes shift (7567 cm^–1). However, these features are associated with an excessively short emission wavelength (λ_em 405 nm), making it unsuitable as a cell-based probe. In the following chapters, we describe the steps we took to systematically investigate the structure of compound 1 to obtain a fluorescent ligand with optimized properties while enriching the SFR of the pyrazolo­[1,5-a]­pyridin-2-ol system.

Step 1. Pyrazolo­[1,5-a]­pyridine: Investigating the Role of the Hydroxy Group on the Fluorescence Profile

A series of compounds was designed to investigate the role of the acidic hydroxyl group, located in position 2 of the pyrazolo­[1,5-a]­pyridine system, on the fluorescence properties.

The acidity introduced by this functional group is crucial for its application as an isosteric replacement of acidic moieties. In azoles, the presence and behavior of lactam-lactim tautomerism strongly depend on the nature of the ring substituents. Besides the fact that spectroscopic techniques suggested that, within lactam-lactim tautomerism, the enol form (OH tautomer) is predominant in solution among the two tautomeric species, alkylation of 1 occurs, although in different ratios, in both the O- and N- positions. Starting from 1, we design and synthesized the nonionizable O-Me (1a) and N-Me (pyrazolone, 1b) analogs. The incorporation of an ester moiety to compound 1 resulted in a slight hypsochromic shift in the violet region (2); thus, we considered this replacement in the following modulations, synthesizing also neutral O-alkyl (2a and 2c) and N-alkyl (pyrazolone, 2b and 2d) analogues (Scheme ).

1. Synthetic Methodologies for the Synthesis of the Targets Compounds 1, 1ac, 2, 2a-d, 3, 3ab .

^a (i) K₂CO₃, diethylmalonate, EtOH abs, 50 °C; (ii) K₂CO₃, MeI or benzyl bromide, dry ACN reflux; (iii) t-BuO^– K⁺, dry THF; (iv) Cs₂CO₃, MeI or benzyl bromide, dry DMF; (v) (a) 5 M NaOH, EtOH, 70 °C, (b) 37% w/w HCl, 70 °C, or * 37% w/w HCl, 0 °C for 1b. (vi) NaNO₂, CH₃COOH; (vii) H₂O₂, CH₃COOH; (viii) Cs₂CO₃, MeI, dry DMF; (ix) SnCl₂, 37% w/w HCl, dioxane; (x) TEA, acyl chloride, dry THF; (xi) TEA, methane sulfonyl chloride, dry THF.

Chemistry

For preparing target compounds 1, 1a–b, 2, 2a–d, the synthetic scheme began from a slightly modified procedure adopted in our previous works. In this case, the synthesis started from commercial 1-aminopyridinium iodide (Scheme ), which was reacted with diethyl malonate in the presence of K₂CO₃ and EtOH as a solvent to yield the common intermediate 15. Subsequently, intermediate 15 was either treated with t-BuO^–K⁺ as a base in dry THF, to afford compound 2 through cyclization or refluxed with an alkylating agent (benzyl bromide or methyl iodide) and a mild base condition (K₂CO₃) in acetonitrile as a solvent, to achieve target compounds 2b and 2d. Moving forward in the scheme, compound 2 was protected on the hydroxyl group with a methyl or benzyl group to afford compounds 2a and 2c, respectively. Moreover, the ester moiety of compounds 2, 2a–c was hydrolyzed in basic conditions to obtain the corresponding acids and immediately decarboxylated in strong acid conditions to afford compounds 1 and 1a–c in good yields.

Fluorescence

The examination of the fluorescence spectra reveals that the pyrazolone analogues (N-alkylated; 1b, 2b, and 2d) present the most favorable emission characteristics in the blue section of the spectrum, along with a significant Stokes shift (Table ). However, they also exhibit lower quantum yield (QY) than the parent hydroxylated compounds (1 and 2) and the O-alkylated analogues (1a, 2a, and 2c, see Table 1). Because the alkylation in both 1 and 2 did not result in major modifications in the luminescence, we focused on assessing for subsequent modifications, when possible, on the alkylated series, as these compounds are easier to handle.

1. Spectral Properties of 1, 2, 3, 1a–b and 2a–d in ACN .

Compound	λabs (nm)	λem (nm)	Stokes shift (cm–1)	QY	Lifetime (ns)
1	310	405	7567	0.21	12
1a	305	400	5942	n.d.	n.d.
1b	335	465	6514	n.d.	n.d.
2	313	373	5139	0.42	7
2a	310	380	5942	0.45	9
2b	345	445	6514	0.26	11
2c	310	380	5942	0.43	9
2d	348	448	6414	0.30	12

^an.d. = Not determined.

Step 2. Pyrazolo­[1,5-a]­pyridine: Investigating the Role of the EWGs in Position 3 on the Fluorescence Profile

To keep exploring the role of the EWGs in position 3 of pyrazole­[1,5-a]­pyridine, other substituents beside the ester were investigated. The Hammett substituent constant (σ _p , Table ) was used as a parameter for judging the moieties nitro (3), acetamido (3a) and sulphonamido (3b) in terms of electronic effects (Scheme ).

2. Spectral Properties of Compounds 3a–b in ACN .

Compound	λabs (nm)	λem(nm)	Stokes shift (cm–1)	QY	Lifetime (ns)	σ p
3	361	n.e.	-	-	-	0.78
3a	312	457	10169	<0.01	-	0.00
3b	315	468	10379	<0.01	2	0.03

^an.e. = No emission observed.

Chemistry

The synthesis of compounds 3, 3a and 3b is outlined in Scheme . The synthesis of compound 3 started from 1. After failing the direct nitration, although following the condition described in the literature, compound 1 was first nitrosylated in the 3-position using NaNO₂ in CH₃COOH as a solvent. In the following, 16 was oxidized with H₂O₂, ending with compound 3 in good yield. The hydroxy function of compound 3 was protected with methyl group using the same procedure outlined in Scheme , obtaining both O- (17a) or exocyclic nitrogen (17b see chemistry characterization) isomers as we earlier described. The nitro group of 17a was then reduced using SnCl₂ as a reducing agent in the presence of hydrochloric acid using dioxane as a solvent, giving intermediate 18 in good yield.

Due to the instability of compound 18 to moisture when isolated as a free base, the latter was isolated as hydrochloride salt and immediately used in the following steps. Compounds 3a and 3b were achieved by a coupling reaction using acetyl chloride or methane sulfonyl chloride, respectively, in the presence of TEA in dry THF.

Fluorescence

Although the nitro group is present in some fluorescent dyes, when used as an EWG on 2-hydroxy-pyrazole­[1,5-a]­pyridine it acts as a fluorescence quencher, leading to a complete loss of emission in 3. On the other hand, a significant bathochromic shift of the emission for compounds 3a and 3b compared to compound 2a is observed, but the QY drops to almost zero.

Step# 3. Pyrazolo­[1,5-a]­pyridine: Investigating the Role of π-conjugation in Position 3 on the Fluorescence Profile

It is widely recognized that increasing π-conjugation could improve the emission characteristics. To investigate how the increase of the conjugation impacts the fluorescence of the pyrazolo­[1,5-a]­pyridin-2-ol, a set of compounds based on the O-methylated scaffold 1a was then designed (Scheme ). Compounds 4a and 4b were designed to evaluate the impact of a combination of a strong EWG and an increase in the π-conjugation of the system. In contrast, compound 5a was designed to evaluate only the effect of the π-conjugation on the luminescence. Moreover, starting from the structure of compound 5a, an electron-donating group (EDG) group (−SCH₃) was introduced (5b) to promote electron donation, while three different EWG groups were incorporated to enhance charge delocalization (5c–e).

2. Synthetic Methodologies for the Synthesis of the Targets Compounds 4a–b, 5a–e, 6, 7a–c .

^a (i) POCl₃, dry DMF, 0 °C; (ii) malononitrile or methyl 2-cyanoacetate, ammonium acetate, CH₃COOH; (iii) phosphonium salt 35 a–e, LiHMDS, dry THF, reflux; (iv) NBS, dry CH₂Cl₂, −10 °C; (v) [Pd­(PPh₃)₄], Cs₂CO₃, boronic acid, dioxane/water 8:2 v/v.

Chemistry

Starting from compound 1a, we followed two different synthetic pathways to afford 4a–b, 5a–e, 6, 7a–c dedicated to modulation of position 3 (Scheme ). In pathway A, 1a underwent a Vilsmeier–Haack reaction at the electron rich position 3 to obtain the corresponding aldehyde 19. Subsequently, this latter was involved in a Knoevenagel reaction to achieve compounds 4a and 4b, with a good yield (75–80%). Furthermore, aldehyde 19 was used as the starting material for synthesizing compounds 5a–e afforded by a Wittig reaction. In the synthetic pathway B, 1a was brominated with NBS and PPh₃ in dry dichloromethane affording 20. Subsequently, a Suzuki-Miyaura cross-coupling was carried out on 20, which resulted in good yields (70–85%) of the target compounds 6 and 7a–c.

Fluorescence

Compounds 4a and 4b were found able to emit yellow light already in the solid state. According to a study conducted by Tigreros et al. on the closely related pyrazolo­[1,5-a]­pyrimidine system, the packing of these heterocycles is predominantly driven by van der Waals forces. Additionally, in the absence of bulky substituents in the core structure, they observed the phenomenon known as the aggregation-induced emission effect (AIE). It is reasonable to assume that the luminescence of 4a and 4b may be attributed to the high level of planarity in the system. Moving to the derivatives 5a–e, the fluorescence falls in the visible region of the spectra. Thus, compounds 5c and 5d show cyan emission, 5a and 5b have a bathochromic shift toward green/yellow, and 5e shows red emission (Table ). Based on the significant bathochromic emission shift observed in compound 5a compared to the lead compound 1, we can conclude that the extension of π-conjugation with a single double bond and a phenyl ring could be sufficient to obtain an emission suitable for biological studies. Interestingly, the absorbance shifts significantly when EWGs of different strengths are introduced in the para position of the terminal phenyl ring. This suggests that the pyrazolo­[1,5-a]­pyridine scaffold would exhibit a more favorable fluorescence profile when acting as a donor group, being conjugated to an EWG.

3. Spectral Properties of Compounds 4a–b, 5a–e and 6 and 7a–c in ACN .

Compound	λabs (nm)	λem (nm)	Stokes shift (cm–1)	QY	Lifetime (ns)
4a	393	n.e.	-	-	-
4b	395	n.e.	-	-	-
5a	335	500	9851	<0.01	3.5
5b	350	510	8964	<0.01	3.5
5c	370	470	5750	<0.01	3.5
5d	378	470	5178	<0.01	3.5
5e	426	662	8368	<0.01	<0.1
6	345	465	7480	0.13	4.3
7a	370	422	3330	0.52	2.8
7b	356	450	5868	0.42	5.5
7c	350	460	6832	0.24	5.3

^an.e. = No emission observed.

Unfortunately, a drastic reduction in quantum yield makes these series unsuitable for biological studies. This decline may be associated with an increase in the internal conversion process, leading to energy dissipation through nonradiative pathways when the molecule returns to the ground state. This behavior may particularly relate to molecular flexibility due to the free rotation around the sigma bonds near the olefinic part.

A common strategy to enhance the brightness or QY of a fluorescent dye is limiting the rotation by elevating the rotational barrier or increasing rigidification while inhibiting quenching via nonradiative relaxation or intersystem crossing. In this sense, ideally starting with compound 5a, the substructure was rigidified by substituting the double bond with a naphthalene or quinoline group. In compound 6, the alkene substructure of 5a was rigidified by incorporating the double bond into a naphthalene moiety. Additionally, based on the inherent fluorescent quantum yield of the “azarene”” heterocycles, a quinoline ring was used to replace the naphthalene substructure, leading to the design of three compounds: 7a–c, where the pyrazolo­[1,5-a]­pyridine group was introduced at positions 2, 6, and 7 on the quinoline ring (Scheme ). The complete rigidification of the scaffold in compound 6 resulted in a modestly diminished blue shift in its emission to a wavelength of 465 nm. Nonetheless, it shows a satisfactory Stokes shift and recovery of the quantum yield. This observation suggests that the reduction in QY in the previous compounds could indeed be due to free rotation around the σ bond. Replacing the naphthalene substructure with a quinoline ring in compounds 7a–c resulted in emission wavelengths ranging from 422 to 460 nm. Significantly, all compounds had improved QY values, ranging from 0.24 to 0.52 (Table ).

3. Synthetic Methodologies for the Synthesis of the Targets Compounds 8ac .

^a (i) (a) 1.0 M Lithium Hexamethyldisilazane (LiHMDS), Dry THF, −78 °C, Nitrogen Atmosphere, 1 h, (b) Hexachloroethane, Dry THF, r.t., Nitrogen Atmosphere; (ii) 40% w/w Dimethylamine in MeOH, Dry DMF 80 °C; (iii) H₂, Pd/C, EtOH; iv) NaNO₂, CH₃COOH.

Step 4. Pyrazolo­[1,5-a]­pyridine: Introducing a Push–Pull System

Push–pull fluorophores are recognized as prime candidates for developing effective fluorescence. , By functionalizing the core of a molecule with appropriate electron-donating and electron-withdrawing groups, a push–pull dye could be created, ideally capable of emitting light at wavelengths longer than 400 nm, which is necessary to prevent overlap with biological autofluorescence. A push–pull system based on a pyrazolo­[1,5-a]­pyridine core falls under a category of π-conjugated molecules featuring an electron donor and an electron acceptor linked by a π-conjugated segment. Starting with compound 2, we examined how the push–pull system at positions 7 (acting as the push) and 3 (serving as the pull) influences the fluorescence characteristics of pyrazolo­[1,5-a]­pyridine. We initially selected the dimethylamine group as the EDG and identified an ester group as the EWG at position 3 (compound 8a, Scheme ). As for compound 2, we further investigated the role of the nitro group as EWG pull system by inserting this group in positions 6 (8b) and 4 (8c). Because compound 7c, characterized by a quinoline scaffold as the EWG, showed optimal λ_em and QY, we also designed 9b, where a dimethylamine group serves as the EDG.

Chemistry

The scheme for the synthesis of compounds 8a–c (Scheme ), starts from compound 2c that was converted by lithium hexamethyldisilazane (LiHMDS) into the lithium salt by selectively deprotonating position 7. This latter was then quenched with hexachloroethane, affording compound 21 in good yield. A nucleophilic aromatic substitution reaction, using dimethylamine 40% w/w in MeOH as a nucleophile, was employed to achieve compound 22. The latter was subsequently hydrogenated to remove the benzyl-protecting group to afford compound 8a. The application of the nitrosation conditions (NaNO₂ in acetic acid) to compound 8a, surprisingly afforded directly the nitro compound as a mixture of 8b and 8c, obtained the spontaneous oxidation of the nitrous to the nitro group. The mixture was then well resolved by preparative-HPLC (for structure elucidation, see SI).

Compound 9a was synthesized from compound 2a (Scheme ), which was chlorinated at position 7 according to the procedure described in Scheme affording compound 23. The ester of compound 23 was then hydrolyzed in basic environments and the resulting acid was decarboxylated under strongly acidic conditions to give 24. In the following, 24 was iodinated in 3 position using NIS in dry DCM to afford 25. A Suzuki-Miyaura cross-coupling reaction with the quinolin-6-ylboronic acid was applied to 25 affording 9a in good yield. Finally, 9b was afforded through a SNAr reaction using dimethylamine (40% w/w) in MeOH.

4. Synthetic Methodologies for the Synthesis of the Targets Compounds 9a and 9b .

^a (i) (a) Nitrogen Atmosphere, 1.0 M LHMDS, Dry THF, −78 °C, 1 h, (b) Nitrogen Atmosphere, Hexachloroethane r.t.; (ii) (a) 5 M NaOH, EtOH, 70 °C, (b) 37% w/w HCl, 70 °C; (iii) NIS, Dry DCM, −10 °C; (iv) [Pd­(PPh₃)₄], Cs₂CO₃, Quinolin-6-ylboronic Acid, Dioxane/Water 8/2 v/v; v) Dimethylamine 40% in MeOH, Dry DMF 80 °C (Seal Tube).

Fluorescence

When comparing 8a (Table ) with 2 (Table ), it is possible to observe a shift to the red region of the spectrum, while an acceptable quantum yield was retained. Moving to 8b and 8c, also in this case, the nitro group quenches the fluorescence. A similar beneficial effect due to the presence of a dimethylamino EDG in position 7 could be seen comparing 9a (Table ) with 7c (Table ) as a shift to the red region is observed, while the QY is acceptable. Compound 9a, an intermediate for synthesizing 9b, was also included to investigate the retro-donating effect of the chlorine in position 7. Introducing a chlorine atom in 9a also shifts the emission into the red region of the spectrum, although at the cost of a significant reduction in QY, which can be understood considering the heavy atom effect.

4. Spectral Properties of Compounds 8a–c and 9a–b in ACN .

Compound	λabs (nm)	λem (nm)	Stokes shift (cm–1)	QY	Lifetime (ns)
8a	338	411	5255	0.06	3.7
8b	402	n.e.	-	-	-
8c	415	n.e.	-	-	-
9a	350	460	6832	<0.01	5.1
9b	362	552	9508	0.10	2.7

^an.e. = No emission observed.

Step 5. Pyrazolo­[1,5-a]­pyridine: Structure Fluorescence Relationship (SFR)

The data gathered from this investigation were utilized to establish what we refer to as the SFR for the pyrazolo­[1,5-a]­pyridine-2-ol core scaffold (Figure ). While still preliminary and source of continuum expansion, this SFR here proposed could already guide future applications in designing fluorescent ligands.

Because O-/N-alkylated or free–OH derivatives show comparable fluorescent properties, optimized pyrazolo­[1,5-a]­pyridin-2-ol derivatives can be used either as isosteres of carboxylic acids or as a probe to be attached to an already existing bioactive ligand. In the second application, considering the two isomers obtained after scaffold alkylation, the N-alkylated series shows a better emission profile than the O-alkylated. However, both are suitable for biological applications when correctly modulated. While incorporating the -NO₂ group in different positions of the backbone is not allowed due to fluorescence quenching, a small EWG in position 3 is tolerated but causes the loss of QY. Nevertheless, when strategically employed within a π-extended system, it is able to induce a red shift in the emission. To achieve a red shift while retaining acceptable QY, it is also advantageous to introduce an EWG characterized by a rigid structure, such as naphthalene or quinoline. Other groups, such as pyridine or heterocycles, would also be reasonable to suggest and will be soon investigated. Moreover, to achieve a red shift while retaining the QY, it is beneficial to introduce an EWG characterized by a rigid structure in that position. Lastly, to enhance electron density and thus the effectiveness of the electron-donating strength of pyrazolo­[1,5-a]­pyridine, an additional EDG can be introduced at position 7 of the core scaffold as a disubstituted amino group. It must be emphasized that this SFR on the pyrazolo­[1,5-a]­pyridin-2-ol system has to be regarded as provisional and will therefore be expanded in the future. However, it contains sufficient evidence to establish proof of concept in developing fluorescent ligands as well as all the synthetic strategies helpful for easily modulating the core scaffold during the development.

Design of Fluorescent hDHODH Inhibitors

Role and Targeting hDHODH

Human dihydroorotate dehydrogenase (hDHODH) is located in the inner mitochondrial membrane and is involved in de novo pyrimidine biosynthesis. This function links hDHODH activity to rapid cell growth in autoimmune diseases, cancer, and viral infections, where the need for pyrimidines cannot be met by the salvage pathway alone. hDHODH consists of two domains: the C-terminal catalytic domain (Met78-Arg396), which contains the active site where dihydroorotate (DHO) is oxidized into orotate, and an N-terminal domain (Met30-Leu68), which anchors the enzyme to the inner mitochondrial membrane. The N-terminal domain forms a hydrophobic tunnel (termed a lipophilic patch by Baumgartner et al. within the membrane and harbors the FMN binding site, where FMN is reduced to FMNH₂ simultaneously with the oxidation of DHO. This tunnel provides access to the second cofactor, ubiquinone (CoQ), to reach FMN and triggers the second step of the catalytic reaction.

Due to the peculiar ping pong mechanism that characterizes hDHODH, the hDHODH inhibitors described in the literature are designed to bind to the ubiquinone binding site, the cofactor that connects hDHODH to the mitochondrial respiratory chain. The effect of blocking the access of CoQ to FMN, disrupt the ping-pong enzyme mechanism and by reflex block the enzyme activity. Baumgartner et al. first described the topography of the lipophilic patch, subdividing it into five subsites, as shown in Figure . Subsite 1 is highly lipophilic and represents the entrance of the lipophilic patch (Met43, Leu42, Leu46, Ala59, Phe62, Phe98, Leu68, Leu359, and Pro364), which harbors Subsite 2, characterized by two polar amino acids, Arg136 and Gln47. The common structural features of hDHODH inhibitors listed in Figure , the best known of which is brequinar, include a bulky lipophilic tail required for interaction with Subsite 1 and an acidic polar head interacting with Arg136 in polar Subsite 2, the latter being considered a key interaction.

4.

Representation of hDHODH CoQ binding site and subsites classification. On the left image is the representation of the pocket surface in which the hDHODH inhibitors are bound. Zoom-in on right shows the amino acids forming Subsite 1 (green), Subsite 2 (yellow), Subsite 3 (cyan), Subsite 4 (pink), Subsite 5 (orange). ORO and FMN are gray. The images were created using PyMOL starting from PDB ID: 6FMD.

Since 2012, our group at the University of Turin (IT) introduced a new generation of hDHODH inhibitors , designed using scaffold-hopping replacement of brequinar’s acidic moiety with various acidic hydroxylated azoles. ,,,, While different azoles successfully play this role (1,2,5-oxadiazole, thiadiazole, triazole, and pyrazole, Figure ), giving inhibitors with IC₅₀ in the nM range, the pyrazolo­[1,5-a]­pyridine is the most effective being also able to engage contacts with Val134/Val143 (Subsite 4) through its pyridine submoiety. MEDS433 is an orally active best-in-class hDHODH inhibitor (IC₅₀ hDHODH 1.2 nM) currently in advanced preclinical studies (intellectual property owned by the UniTo spinoff Drug Discovery and Clinic s.r.l.). Unfortunately, the pyrazolo­[1,5-a]­pyridin-2-ol moiety in MEDS433 has a poor fluorescence, falling outside the above-described SFR requirements.

5.

Structures of hDHODH inhibitors based on hydroxyazole.

Design of Fluorescent hDHODH Inhibitors: Merging SFR and SAR

With hDHODH inhibitor SAR principles in hand, the next step was to apply them at the initial compound optimization steps. From the SFR/SAR comparison shown in Figure , the first two design steps are pretty obvious: 1) the azole OH group must be maintained because it is required in the interactions with Arg136 and Gln47 inside the Subsite 2, and 2) no suitable EDG substituents, although beneficial for SFR, can be placed in position 7 of pyrazole­[1,5-a]­pyridine because it is known from the SAR that this position does not tolerate substitutions. ,,,

6.

Merging between hDHODH SAR and pyrazolo­[1,5-a]­pyridin-2-ol core SFR for the design of fluorescent hDHODH inhibitors.

Since MEDS433 has a central amide that does not appear to be crucial in the interactions within the binding pocket, the design was focused on its replacement with a substructure able to enhance the conjugation of the system toward a biphenylic scaffold tail while retaining the correct group orientation. The olefinic substructure of compound 11a–b, which could be seen as a close relative of 5a, was first employed to mimic the trans conformation of the amide moiety. The biphenyl was perfluorinated in the internal ring to optimize the dihedral angle as well as introducing a push–pull system with the pyrazolo­[1,5-a]­pyridine acting as an EDG and the tetrafluorobiphenylic system acting as a strong EWG group. As shown by docking experiments, compound 11a well superimposes on the crystallographic pose of MEDS433 (Figure A), retaining the key binding interactions with the pocket. In addition, a good overlap between the biphenyl structure can be observed.

8.

Ubiquinone binding site of hDHODH (PDB ID code: 6FMD). Docking poses of target compounds overlapped with the crystallographic pose of MEDS433. The main amino acidic residues contributing to the binding are shown in the stick representation (carbon backbone is in light gray, nitrogen atoms in blue, oxygen in red, and sulfur in yellow). A yellow dish represents polar interactions. In panel A, the docking pose for compound 11a is blue. Panel B shows the docking pose for compound 12 in pink. Panel C shows the docking pose for compound 13 in cyan. Panel D shows the docking pose for compound 14 in green. The image was created using PyMOL.

Furthermore, although H and F can be considered as isosteres, − the difference in van der Waals radius and, consequently, in the volume they occupy could be beneficial for reducing the free rotation of biphenylic substructure, and potentially restore the QY value, totally lost in the compound 5a–e series. The following design was focused on introducing planarity and reducing free rotation, according to what was learned from SFR. Compound 12 was designed based on compound 7a due to the high QY and good overlap with MEDS433 observed in docking analysis (Figure B). In detail, the position of the nitrogen atom of the quinoline ring of compound 12 could mimic the nitrogen atom of the amide moiety. In addition, the 6-phenyl quinoline substructure fits well into the hydrophobic Subsite 1 pocket, substantially overlapping with the biphenyl moiety (Figure B). Considering the favorable emission and QY of compound 7b, this latter was employed as a basis for developing compounds 13 and 14 (Figures and C–D). In molecules 12 and 13, we investigated how different positions of the quinoline nitrogen can influence the overall hDHODH inhibitory activity.

7.

Structures of the designed fluorescent hDHODH inhibitors.

Moreover, by analyzing the crystallographic pose of MEDS433, it is possible to observe that the presence of two fluorine atoms in the ortho position imposes a rotation of approximately 50° in the terminal ring configuration. Compound 14 was, therefore, designed to mimic the molecular geometry observed in the biphenyl ring of MEDS433.

Chemistry

The synthesis of the target compounds as the first example of fluorescent hDHODH inhibitors is described in Schemes and . To synthesize the final compounds 11a and 11b, the procedure starts from the intermediate 1. After the protection of the hydroxy group with p-methoxybenzyl bromide to afford 26, the latter was converted into the corresponding aldehyde 27 via Vilsmeier–Haack formylation. This latter was involved in a Wittig reaction with the corresponding phosphonium salts (for the synthetic procedure, see SI). In both cases, a mixture of Z and E isomers was isolated. The E isomer 28a, identified by J coupling constant (J ≥ 16 Hz), was isolated together with the defluorinated analogue 28b. Both compounds were deprotected using TFA and thioanisole to afford the desired target compounds 11a and 11b in quantitative yield.

5. Synthetic Methodologies for the Synthesis of the target 11a, b .

^a (i) Cs₂CO₃, 4-methoxybenzyl chloride, dry DMF; (ii) POCl₃, dry DMF; (iii) LiHMDS, dry THF, 41a and b reflux; (iv) TFA, thioanisole, 0 °C.

6. Synthetic Methodologies for the Synthesis of the Target Compounds 12–14 .

^a (i) NIS, PPh₃, dry DCM; (ii) Turbo Grignard, 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, dry THF, 0 °C; (iii) and (v) [Pd­(PPh₃)₄], 30b (for iii) or 30a (for v), Cs₂CO₃, dioxane/water, 8:2 v/v; (iv) 48% w/w HBr, reflux; (vi) [Pd­(OAc)₂], K₃PO₄, boronic acid, X-PHOS, dioxane/water 8:2 v/v; (vii) TFA, thioanisole, 0 °C.

To achieve target compounds 12–14, compounds 1a and 1c were iodinated using NIS and PPh₃ in dry dichloromethane, to achieve intermediates 29a–b in a good yield. In the following, compounds 29a–b were allowed to react with 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane and turbo Grignard reagent in dry THF, leading to the formation of intermediates pinacolic boronic ester 30a–b in good yield. Due to high reactivity, these intermediates tend to undergo deborylation over time. So, they were immediately coupled with compound 43 (see Supporting Information) via Suzuki-Miyaura coupling to afford compound 31. Compounds 33 and 34 were obtained by two consecutive Suzuki cross-coupling reactions starting from intermediate 30a. Subsequently, the final compounds 12–14 were obtained by deprotection of the hydroxy group by heating under reflux in 48% w/w aqueous HBr solution for compound 12 and using the TFA/thioanisole system for compounds 13 and 14.

hDHODH Enzyme Activity Inhibition

The features of the hDHODH inhibitors just described prompted the selection of compounds 11a and 12–14 for the evaluation of their inhibitory activity. These compounds were compared with MEDS433 and Teriflunomide the only FDA/EMA-approved hDHODH inhibitor (Table ).

5. Enzyme Inhibition by MEDS433, Teriflunomide, and 11a and 12–14, with Relative IC₅₀ Values Shown.

Compound	hDHODH a IC50 (C.L. 95%) μM
Teriflunomide	0.39 (0.25–0.52)
MEDS433	0.0012 (0.0010–0.0021)
11a	0.17 (0.14–0.21)
12	16 (7–25)
13	33 (23–42)
14	22 (10–53)

As seen in Table , all the designed compounds act as hDHODH inhibitors presenting IC₅₀ values in the low μM range. The most interesting compound in the series is 11a, which reaches the nM range with an activity 2-fold superior to Teriflunomide.

hDHODH Related AML Cellular Activities

In 2016, a breakthrough discovery showed the association of hDHODH activity with myeloid differentiation in acute myeloid leukemia (AML) , cells, opening new possibilities for addressing a severe pathology with a poor prognosis. In AML, hDHODH is required to maintain the undifferentiated state of leukemia blasts, which are immature cells that cannot mature into adult white blood cells. These blasts proliferate in the blood and target organs, while their accumulation in the bone marrow disrupts normal blood cell production, causing the disease. By inhibiting hDHODH and blocking de novo pyrimidine biosynthesis, AML cells are forced into a state known as “pyrimidine starvation,” forcing them to differentiate and then go to apoptosis. We initially investigated the proapoptotic activity of our compounds in THP1 AML cell lines, a system we frequently explored during the MEDS433 optimization. Table shows the biological activity of compounds 11a and 14 compared to the control MEDS433.

6. Analysis of the Biological Activity (Inhibitory Activity on the Enzyme, ,Apoptosisand Viability) of Compounds 11a and 14, Compared to MEDS433 .

Compound	hDHODHb IC50 μM (C.L. 95%)	Apoptosis EC50 THP1 (nM) (C.L. 95 %)	Viability EC50 K562 (nM) (C.L. 95 %)	Viability EC50 A549 (nM) (C.L. 95 %)	MRC-5 CC50 (μM) (C.L. 95 %)
MEDS433	0.0012 (0.001 – 0.0021)	72 (42 – 124)	32	n.d.	104800
11a	0.17 (0.14–0.21)	693 (567–873)	114 (57 – 186)	423 (375 - −476)	7000 (3000 – 10000)
14	22 (10–53)	n.d.	n.d.	n.d.	n.d.

^aThe apoptotic and viability data are expressed as EC₅₀ on indicated cell lines. “n.d.” Indicates that the compound was not tested in that specific assay.

^bCC₅₀, compound concentration producing 50% cytotoxicity, as determined by cell viability assays performed in MRC-5 cells. Reported values are the means (C.L. 95%) of each group.

The antileukemic pro-apoptotic activity requires inhibitors with IC₅₀ in the one-digit nM range for the isolated enzyme and for this reason, the relatively low inhibitory activity on recombinant hDHODH in vitro could explain the absence of pro-apoptosis efficacy of compound 14. Although 11a is not as potent as MEDS433 on isolated enzymes (0.17 μM and 0.0012 μM, respectively), it still shows significant pro-apoptotic activity on the AML cell line, falling within the nM range. As shown in Table , the antileukemic activity of 11a is nearly 10-fold lower than that of our designed best-in-class MEDS433, despite the latter being 140 times more potent on the isolated enzyme. The pro-apoptotic activity of 11a was completely reversed by adding exogenous uridine (100 μM), suggesting that the induction of apoptosis is mainly due to hDHODH inhibition (see Figure S4). To gain a deeper insight into the antitumor potential of the most promising compound 11a, we extended our viability assays to another leukemic cell line and a solid tumor cell line. Compound 11a demonstrated potent antiproliferative activity against K562 chronic myeloid leukemia cells, with an EC₅₀ value of 114 nM, superior to that observed in THP-1 cells. Moreover, it showed activity against the A549 lung adenocarcinoma cell line, albeit with a slightly higher EC₅₀ of 423 nM.

Antiviral Activity of Target Compounds

Efficient virus replication depends on the availability of pyrimidine nucleotides in infected cells. Therefore, compounds targeting the cellular pathways responsible for providing an appropriate supply of pyrimidines, such as the de novo biosynthetic pathway, have the potential to be used as effective host-acting antiviral (HTA) agents. Moreover, inhibitors of enzymes of the pyrimidine biosynthesis pathway, including hDHODH, can overcome the emergence of viral drug resistance. In addition, being independent of virus-specific replication strategies, they may also be effective against various viruses from different families, acting as broad-spectrum antivirals (BSAs). The emergence of new respiratory virus infections in humans with epidemic or pandemic potential in the last two decades has highlighted the urgent need for effective BSAs to be deployed against future respiratory tract virus infections, with novel coronaviruses and influenza viruses being the most likely to have pandemic potential. − To contribute to this antiviral field, in the past few years, we have characterized the potent hDHODH inhibitor MEDS433 as a BSA candidate effective against several human respiratory viruses, such as coronaviruses, including SARS-CoV-2, as well as influenza A and B viruses, and the respiratory syncytial virus. ,,, On the other hand, these findings further validate the antiviral activity of hDHODH inhibitors as an inherent biological property of such molecules. Therefore, it is interesting to investigate the antiviral activity of compounds 11a and 14 compared to MEDS433 against the representative human endemic beta-coronavirus hCoV-OC43. To this end, FFRAs were performed in HCT-8 cells exposed to target compounds before, during, and after infection with hCoV-OC43 (full treatment).

As shown in Figure , all the target compounds exert a concentration-dependent inhibitory effect on coronavirus replication.

9.

Compounds 11a and 14 inhibit the replication of hCoV-OC43. HCT-8 cell monolayers were infected with the hCoV-OC43 (50 PFU/well), and, where indicated, the cells were treated with increasing concentrations of compounds 1 h before, during virus adsorption, and postinfection. Compounds remained in the culture medium throughout the experiment. hCoV-OC43 replication was quantified at 72 h p.i. by FFRA, and numbers of viral foci microscopically counted were converted into viral titers (PFU/mL). The compounds concentrations producing 50% and 90% reductions of viral replication (EC₅₀ and EC₉₀, respectively) were determined by GraphPad Prism. The data are the means ± SDs (error bars) of n = 5 independent experiments performed in triplicate.

As expected, the calculated EC₅₀ value for MEDS433 is in the low-nanomolar range, while that of 11a is in the low-micromolar range (see Table ). Compound 14 showed a higher EC₅₀ value, albeit in the one-digit micromolar range. Notably, the low 50% cytotoxic concentration (CC₅₀) of 11a and 14, as determined in uninfected HCT-8 cells, confirms that their antiviral activity did not stem from a nonspecific cytotoxicity. Indeed, the Selectivity Index (SI) is about 50 for 14 and 150 for 11a, respectively (Table ). Together, these results confirm the retention of antiviral activity in the low-micromolar range for both the fluorescent hDHODH inhibitors 11a and 14, even though compound 11a showed a greater anticoronavirus potency than 14, consistent with its lower IC₅₀ value in the inhibition of hDHODH activity (Table ). While 11a is not toxic to uninfected HCT-8 cells (301 ± 9 μM), it shows low μM toxicity on the MRC-5 cell line (7 μM (3 – 10, C.L. 95%, Table )).

7. Antiviral Activity of Compounds 11a and 14 Against hCoV-OC43 Replication.

Compound	EC50 (μM)	EC90 (μM)	HCT-8 CC50 (μM)	SI
MEDS433	0.0142 ± 0.0004	0.08 ± 0.01	84 ± 5	5915
11a	2.3 ± 0.5	23.3 ± 0.4	301 ± 9	131
14	8.4 ± 0.8	48 ± 4	360 ± 7	43

^aEC₅₀, the Compound Concentration Inhibiting 50% of Virus replication, as Determined Against hCoV-OC43 by FFRAs in HCT-8 Cells. Reported Values are the Means ± SD of Data Derived from Five Experiments Performed in Triplicate.

^bEC₉₀, the Compound Concentration Inhibiting 90% of Virus replication, as Determined Against hCoV-OC43 by FFRAs in HCT-8 Cells. Reported Values are the Means ± SD of Data Derived from Five Experiments Performed in Triplicate.

^cCC₅₀, Compound Concentration Producing 50% Cytotoxicity, as Determined by Cell Viability Assays Performed in HCT-8 Cells. Reported Values are the Means ± SD of Data Derived from Five Experiments Performed in Triplicate.

^dSI, Selectivity Index Determined as the Ratio of CC₅₀ to EC₅₀.

Photophysical Characterization

To prepare the following cell-based investigation for assessing the subcellular localization of the target compound, fluorescence properties were initially investigated in acetonitrile solution at room temperature (Table ). Compounds 13 and 14, derived from the compound 7 series, show absorbance and emission comparable to the lead SFR-optimized compound 7b. Additionally, they display generally acceptable fluorescence features, making them suitable for an initial investigation in a biological system. Compounds 11a and 11b, ideally developed from compound 5, exhibit yellow emission at 535 and 542 nm, respectively. Surprisingly, the QY value is restored in these two molecules, with compound 11a showing the most favorable outcome. In this case, the insertion of a strong EWG, such as a tetrafluorobiphenyl group, together with the substitution of fluorine atoms in the ortho position relative to the olefin group has significant effects on the photophysical properties of compounds 11a–b. In contrast to the compounds of the 5 series, the strong EWG tetrafluorobiphenyl group can stabilize the excited state by lowering its energy, making the fluorescence more competitive against nonradiative decay processes. In addition, the steric hindrance provided by the diortho-fluoro substituents on the olefin group increases the rigidity of the molecular structure. It restricts free rotation of the substituents around the carbon–carbon double bond, a common pathway for nonradiative decay.

8. Spectral Properties of Compounds 11a–b, and 12, 13, and 14 in ACN.

Compound	λabs (nm)	λem (nm)	Stokes shift (cm–1)	QY	Lifetime (ns)
11a	442	542	4174	0.12	<1
11b	450	535	3531	0.08	<1
12	390	430	2385	<0.01	2.3
13	370	460	5288	0.74	3.7
14	390	465	4136	0.64	3.7

By preventing this rotation, the molecule is less likely to dissipate its energy through nonradiative means such as internal conversion or vibrational relaxation.

Due to an emission profile compatible with biological visualization and a good QY value, the fluorescent properties of 11a and 14 were studied in PBS solution to evaluate the fluorescence in a polar system. As outlined in Figure , the emission of both compounds is completely quenched in polar protic solvent following a behavior that can sometimes be observed in so-called environmentally sensitive fluorescent probes. Fluorescent molecules often lose their fluorescence in polar environments for several reasons related to solvent interactions, but they can maintain or even enhance their fluorescence when bound to proteins. This differential behavior can be explained through various photophysical and structural mechanisms such as twisted intramolecular charge transfer (TICT), quenching resulting from aggregation, or intramolecular proton transfer to the excited state (ESIPT). − Nevertheless, this feature can be highly advantageous when the design aims to apply the probe within a biological system. Indeed, in most cases, when the ligand binding site is located in a lipophilic environment, the probe can recover its fluorescence once it reaches the target. This feature makes these fluorescent probes “smart probes” able to highlight the binding of a molecule to its target or act as a sensor to detect the local biological environment.

10.

Absorption and emission spectra of compounds 11a and 14 in ACN and PBS solution. Plain lines represent the absorption profiles, dashed lines correspond to the emission profiles, 14 is not emissive in PBS.

To assess whether the developed compounds are able to restore their fluorescence in a biological context and to evaluate their potential as environmentally sensitive fluorescent probes, we examined the fluorescence response of compound 11a, the most potent inhibitor within the series, in the presence of a target protein. We evaluated the fluorescence of compound 11a in condition of hDHODH enzymatic inhibition assay, confirming the absence of emission even in TRIS buffer (pH 8), and then observing the restoration of fluorescence in the presence of the recombinant protein. In particular, we observed an increase in fluorescence intensity in correlation with the growing amount of protein, as shown in Figure .

11.

Variation of in the fluorescence emission of compound 11a (1 μM) in the presence of different amount of hDHODH.

Because the isolated hDHODH used for the previous measurements, and in inhibition enzymatic assay, is the GST-tagged protein, we decided to confirm that the observed fluorescence enhancement resulted from specific binding of inhibitor 11a to the target protein and not from nonspecific interactions with the GST portion. When in a control experiment an unrelated GST-conjugated-protein was used, no restoration of fluorescence was observed supporting the conclusion that fluorescence is restored solely by the binding of compound 11a to its specific target protein (Figure S5).

Intracellular Localization of Target Compounds 11a and 14

The results presented in the previous sections highlight that compounds 11a and 14 behave as environmentally sensitive ligands endowed with a sufficient affinity for the target and, importantly, exert two different biological activities typical of hDHODH inhibitors (Tables and , and Figure ). Thus, compounds 11a and 14 were subsequently examined in the context of living cells to determine whether they retained their fluorescence profile after entry into target cells. First, both compounds were incubated for 24 h with THP-1 cells at a concentration of 10 μM. After washing with PBS buffer, cells were analyzed under confocal microscopy using an appropriate laser stimulation (λ_ex: 405 nm, λ_em: 415 and 500 nm for compounds 11a and 14, respectively) to excite the molecules. As shown in Figure , both compounds recovered a strong fluorescence, confirming that they exhibited typical features of environment-sensitive fluorescent probes. Moreover, it was possible to observe that both compounds localize in the cytosol of THP-1 cells.

12.

Fluorescence microscopy imaging of THP-1 cells incubated with 10 μM of 11a and 14. Images were collected Leica TCS SP8 confocal system (Leica Microsystems) and acquired with an HCX PL APO 63×/1.4 NA oil-immersion. (A) THP-1 cells incubated with 11a (green); (B) THP-1 cells not treated; (C) THP-1 incubated with 14 (cyan); (D) THP-1 not treated. White scale bar = 100 μm.

Co-localization experiments were performed to examine whether compound 11a may reach the cellular compartment where hDHODH is located. For this, a specific mitochondrial stain marker in the two cell lines in which the biological activities of 11a were observed, namely THP-1 and HCT-8 cells, was used. The compound was therefore incubated with THP-1 cells (Figure ) or HTC-8 cells (Figure ) at 37 °C for 24 h. Then, cells were stained with Red-MitoTracker (a specific fluorescent probe for mitochondrial targeting) for 30 min at 37 °C. In both THP-1 and HCT-8 cells, merging images obtained using an appropriate laser to excite the molecules confirms the ability of compound 11a to localize into mitochondria, where its protein target is present.

13.

Intracellular localization of the fluorescent inhibitor 11a and Red-MitoTracker (mitochondria-specific dye) in THP-1 cells. Images were collected with a Leica TCS SP8 confocal system (Leica Microsystems) and acquired with a HCX PL APO 63×/1.4 NA oil-immersion: (A) THP-1 cells were incubated with DMSO for 24 h as negative control, and then staining with Red-MitoTracker for cellular localization of mitochondria (30 min at 37 °C); (B) THP-1 cells were treated with 10 μM of 11a for 24 h to assess its cellular localization; (C) THP-1 cells were treated compound 11a for 24 h, and then staining with Red-MitoTracker (30 min at 37 °C). Images were collected with excitation of ligand 11a at λ_ex = 405 nm and emission at λ_em = 500 nm, Red-MitoTracker at λ_ex = 581 nm, and emission at λ_em = 605 nm. White scale bar = 20 μm.

14.

Intracellular localization of the fluorescent inhibitor 11a and Red-MitoTracker (mitochondria-specific dye) in HTC-8 cells. (A) HTC-8 cells were treated with DMSO for 24 h as a negative control and then stained with Red-MitoTracker for intracellular localization of mitochondria (30 min at 37 °C); (B) HTC-8 cells were treated with 10 μM of 11a for 24 h; (C) HTC-8 cells were treated with 10 μM of 11a for 24 h, and then stained with Red-MitoTracker (30 min at 37 °C). Images were acquired by a Leica TCS SP5 multiphoton-inverted confocal microscope (Leica Microsystems) equipped with an HCX PL APO 63×/1.4 NA oil-immersion. Images were acquired with excitation for ligand 11a at λex = 405 nm and emission at λem = 500 nm, for Red-MitoTracker at λex = 581 nm and emission at λem = 605 nm. White scale bar = 10 μm.

To confirm the mitochondrial localization of compound 11a, we performed a competition assay involving a well-known hDHODH inhibitor (Bay2402234) with higher affinity for the target (one digit nM) compared to 11a. THP-1 cells were then incubated with compound 11a, either alone or in combination with Bay2402234, followed by Red-MitoTracker staining to label mitochondria and subsequent analysis by fluorescence microscopy (Figure A-C). At the specific wavelength for compound 11a, it colocalized with the mitochondria, while Bay2402234 showed no detectable fluorescence (Figure A-B). Colocalization between hDHODH inhibitors and mitochondria was quantified using Manders coefficient (Figure D). In the presence of Bay2402234, the colocalization signal of 11a with the mitochondria decreased slightly but statistically significantly, as indicated by the Welch’s t test (p = 0.0024). Altogether, the data demonstrate that Bay2402234 competitively displaced compound 11a from the target, confirming both the mitochondria localization and hDHODH binding specificity of compound 11a.

15.

Colocalization analysis of fluorescent inhibitor 11a with mitochondria of THP-1 cells. THP-1 cells were treated with hDHODH inhibitors at 10 μM for 3 h and then staining with Red-MitoTracker for 30 min at 37 °C. Images were collected with a Leica TCS SP8 confocal system (Leica Microsystems) and acquired with an HCX PL APO 63×/1.4 NA oil-immersion. Scale bar = 10 μm. (A) THP-1 cells were treated with 11a; (B) THP-1 cells were treated with Bayer2402234; (C) THP-1 cells were treated with 11a and Bayer2402234; (D) Manders colocalization coefficient of 11a with mitochondria labeled by Red-MitoTracker. Statistical significance was tested with Welch’s t test (n = 3).

Conclusions

This study introduces the first fluorescent isostere of carboxylic acid, establishing “fluo-isosteres” (fluosteres) as a novel subclass of isosteres with optimized fluorescence. The successful design of the first fluorescent hDHODH inhibitors, compounds 11a and 14, provides proof-of-concept for the fluostere technology, demonstrating its ability to create potent inhibitors optimized for fluorescent properties and able to prove cellular-based activities, as antiviral agents in the micromolar range. Interestingly, both 11a and 14 were identified as environmentally sensitive probes, losing their emission in protic solvents but recovering fluorescence in biological systems by interacting with the target in a lipophilic environment. In colocalization experiments with the Red-MitoTracker in THP-1 and HTC-8 cell lines, compound 11a confirmed its ability to reach the mitochondria, where its biological target hDHODH is situated.

Quite interestingly, the possibility to alkylate the OH group of the pyrazolo­[1,5-a]­pyridine without losing the optimal fluorescent profile allows the possibility to use such a moiety also as probe. In the near future, the fluostere technology will be applied to explore additional SFR and enhance its precision as a research tool.

Experimental Section

General Methods

All chemical reagents were obtained from commercial sources (Sigma-Aldrich, Alfa Aesar, FluoroChem and BLD Pharma), and used without further purification. Analytical-grade solvents (acetonitrile [ACN], diisopropyl ether, diethyl ether, dichloromethane [DCM], dimethylformamide [DMF], ethanol 99.8% v/v, ethyl acetate [EtOAc], hexane, methanol [MeOH], petroleum ether bp 40–60 °C [petroleum ether], toluene), were used without further purification. When needed, solvents were dried over 4 Å molecular sieves. Tetrahydrofuran [THF] was distilled from Na and benzophenone under N₂ immediately prior to use. Anhydrous THF, DCM and DMF were obtained from a Glass Contour Solvent System (SG Water USA). Anhydrous MeOH was obtained by distillation over CaCl₂ and storage over activated 3 Å molecular sieves for a minimum of 24 h. Thin layer chromatography (TLC) was conducted on silica gel on 5 × 20 cm plates at a 0.25 mm layer thickness were used and visualized using UV-light (254 nm) or by TLC visualization reagents (KMnO₄, ninhydrin, iodine and 2,4-dinitrophenylhydrazine). Anhydrous Na₂SO₄ was used as a drying agent for the organic phases. Compound purification was either achieved using flash column chromatography on silica gel (Merck Kieselgel 60, 230–400 mesh ASTM), and the eluents indicated in the procedures for each compound or using CombiFlash Rf 200 (Teledyne Isco) with 5–200 mL/min, 200 psi (with an automatic injection valve), and RediSep Rf Silica columns (Teledyne Isco), with the eluents indicated in the procedures for each compound. Compounds synthesized in our laboratory generally varied between 90% and 99% purity. Purity was assessed by analytical HPLC on an UltiMate HPLC system (Thermo Scientific) consisting of an LPG-3400A pump (1 mL/min), a WPS-3000SL autosampler, and a DAD- 3000D diode array detector using a Gemini- NX C18 column (4.6 × 250 mm, 3 μm, 110 Å); gradient elution 0 to 100% B (ACN/H₂O/TFA, 90:10:0.1) in solvent A (H₂O/TFA, 100:0.1) over 15 min. Data were acquired and processed using Chromeleon Software v. 6.80. Analytical purity is ≥95% unless stated otherwise; retention times (tR) are indicated. Preparative HPLC purification was carried out on a Dionex Ultimate 3000 HLPC system consisting of an LPG-3200BX pump (20 mL/min), a Rheodyne 9725i injector, a 10 mL loop, an MWD-3000SD detector (200, 210, 254, and 281 nm), and an AFC-3000SD automated fraction collector using a Gemini-NX C18 column (21.2 ´ 250 mm, 5 μm, 110 Å); gradient elution was 0 to 80% B (ACN/H₂O/TFA, 90:10:0.1) in solvent A (H₂O/TFA, 100:0.1) over 12 min. Data were acquired and processed using Chromeleon Software v. 6.80. Melting points (m.p.), were measured on the capillary apparatus (Büchi 540). Final mp determination was achieved by placing the sample at a temperature that was 10 °C below the mp and applying a heating rate of 1 °C min¹. All compounds were routinely checked by ¹H- and ¹³C NMR spectroscopy and mass spectrometry. MS spectra were recorded on a Waters Micromass ZQ equipped with an ESCi source for electrospray ionization mass spectra or using either a LC-MS system built from an Agilent 1200 series solvent delivery system equipped with an autoinjector coupled to a DAD and an Agilent 6130A series quadrupole electrospray ionization detector, or a Waters acquity UPLC-MS equipped with a dual-wavelength PDA (214 and 254 nm) combined with electrospray ionization. Gradients of H₂O/ACN/HCOOH (95:5:0.1 v/v/v) (solvent A), and ACN/HCOOH (100:0.1 v/v/v) (solvent B) were used. ¹H–¹³C NMR and 2D-NMR spectra were performed on a JEOL JNM-ECZR 600 spectrometer (¹H NMR operating frequency 600 MHz) or on a Bruker Avance II spectrometer equipped with a 5 mm broad band probe (BBFO) (¹H NMR operating frequency 400 MHz) or a Bruker Avance III HD spectrometer equipped with a cryogenically cooled 5 mm dual probe (¹H NMR operating frequency 600 MHz). Chemical shifts are reported relative to TMS (δ = 0) and referenced against solvent residual peaks. The following abbreviations are used for coupling patterns: br = broad, s = singlet, d = doublet, dd = doublet of doublets, t = triplet, q = quartet, m = multiplet. In this work, protons and carbons are labeled (a, b, c, d, e, f, g, h, l, m, n, o, p, q, r, and s). Values marked with an asterisk (*, ** and ***) are interchangeable. To characterize the E isomer of the double bond, we compare the J coupling constants with those reported in other manuscripts. , The HRMS spectra of the final compounds were recorded on a ZenoTOF 7600 System (Sciex, Framingham, MA, U.S.A.) equipped with an ESI ionization source working in positive mode. LC method: direct infusion flow 0.1 mL/min, mobile phase H₂O (1% TFA)/MeOH 50/50. MS method: spray capillary voltage: 5500 V, declustering potential 40 V, collision energy 10 V TOF mass range scan 100–1000 Da. Compounds 5a–d were obtained according to previously described procedures. Biological experiments were performed on compounds with a purity of at least 95%.

(Ethoxycarbonyl)­(pyridin-1-Ium-1-yl)­amide (15)

A solution of 1-aminopyridinium iodide (3.0 g, 0.013 mol), diethyl malonate (4.16 g, 0.026 mol), and K₂CO₃ (5.39 g, 0.039 mol) in 130 mL of EtOH was stirred at 50 °C overnight. The reaction was then filtered to remove insoluble inorganic salts and the filtrate was concentrated under reduced pressure. The crude mixture was purified by flash chromatography on Al₂O₂ (eluent: first diethyl ether, then DCM) to afford the title compound as a colorless sticky solid. Yield 68%. ¹H NMR (600 MHz, Chloroform-d): δ 1.29 (t, 3H, J = 7.1 Hz, -OCH₂CH ₃), 3.37 (s, 2H), 4.21 (q, 2H, J = 7.1 Hz, -OCH ₂CH₃), 7.63 – 7.68 (m, 1H), 7.92 (tt, 1H, J = 7.8, 1.3 Hz,), 8.67 – 8.70 (m, 1H). ¹³C NMR (151 MHz, Chloroform-d): δ 14.4, 43.6, 60.9, 126.2, 137.5, 143.3, 169.9, 170.7. MS (ES⁺): 209 (M+1).

Ethyl 2-hydroxypyrazolo­[1,5-a]­pyridine-3-carboxylate (2)

Potassium tert-butoxide (4.88 g, 0.043 mol) was added portion-wise to a solution of 15 (6.0 g, 0.029 mol) in dry THF (300 mL) at 0 °C. The resulting dark-orange suspension was stirred at room temperature for some minutes until complete conversion was observed, after which it was concentrated under vacuum. The residue was taken with water (200 mL) and acidified to pH 2 using 6 M HCl and then extracted with EtOAc (6 × 150 mL). The organic layers were collected, dried over Na₂SO₄, and evaporated under reduced pressure to afford a yellowish crude oil that was purified by flash chromatography (eluent: petroleum ether/EtOAc 60/40 v/v), to afford the desired compound as white solid. (mp 150.0–151.3 °C, from MeOH). Yield 70%. ¹H NMR (600 MHz, Chloroform-d): δ 1.44 (t, 3H, J = 7.0 Hz, −CH₂CH ₃), 4.42 (q, 2H, J = 7.0 Hz, −CH ₂CH₃), 6.88 (t, 1H, J = 6.7 Hz, H-b), 7.39 (t, 1H, J = 7.5 Hz, H-c), 7.75 (d, 1H, J = 8.8 Hz, H-d), 8.34 (d, 1H, J = 6.7 Hz, H-a), 9.06 (br s, 1H, −OH). ¹³C NMR (151 MHz, Chloroform-d): δ 14.7 (−OCH₂ CH₃), 60.6 (−OCH₂CH₃), 86.4 (C-f), 113.3 (C-b), 117.4 (C-d), 128.3 (C-c), 129.5 (C-a), 140.4 (C-e), 160.3 (C-h) *, 167.2 (C-g)*. MS (ES⁺): 207 (M+1).

General Procedure for the Synthesis of Target Compounds 2a and 2c

The correspondent alkylating agent (3.20 mmol) was added dropwise to a mixture of 2 (2.91 mmol) and Cs₂CO₃ (8.73 mmol) in dry DMF (15 mL). The reaction mixture was stirred overnight at room temperature, and then water (100 mL) was added. The mixture was extracted with EtOAc (3 × 25 mL), and the combined organic layers were dried over Na₂SO₄ and evaporated under reduced pressure. Crude compounds were purified following the conditions below.

Ethyl 2-methoxypyrazolo­[1,5-a]­pyridine-3-carboxylate (2a)

The mixture showed two spots on TLC (eluent: petroleum ether/EtOAc 80/20 v/v), ascribed to the two methylated pyrazolo­[1,5-a]­pyridine regio-isomers. The desired regio-isomer was achieved after flash chromatography (eluent: petroleum ether/EtOAc 80/20 v/v) as a white solid (mp 128.9–129.4 °C, from trituration with diisopropyl ether). Yield 85%. ¹H NMR (600 MHz, DMSO-d ₆): δ 1.29 (t, 3H, J = 7.1 Hz, −OCH₂CH ₃), 4.01 (s, 3H, −OCH ₃) 4.24 (q, 2H, J = 7.1 Hz, −OCH ₂CH₃), 7.03 (td, 1H, J = 6.9 Hz, 1.4 Hz, H-b), 7.54 (ddd, 1H, J = 8.8, 7.0, 1.1 Hz, H-c), 7.90 (ddd, 1H, J = 8.9, 1.4, 1.0 Hz, H-d), 8.67 (dt, 1H, J = 6.8, 1.1 Hz, H-a). ¹³C NMR (151 MHz, DMSO-d ₆): δ 14.5 (−OCH₂ CH₃), 56.5 (−OCH₃) *, 59.0 (−OCH₂CH₃)*, 86.7 (C-f), 113.3 (C-b), 117.2 (C-d), 128.8 (C-c), 129.6 (C-a), 142.1 (C-e), 161.9 (C-h)*, 165.0 (C-g)*. MS (ES⁺): 221 (M+1).

Ethyl 2-(Benzyloxy) Pyrazolo­[1,5-a]­pyridine-3-carboxylate (2c)

The mixture showed two spots on TLC (eluent: petroleum ether/EtOAc 80/20 v/v), ascribed to the two benzylated pyrazolo­[1,5-a] pyridine regio-isomers. The desired regio-isomer was achieved after flash chromatography (eluent: petroleum ether/EtOAc 80/20 v/v) purification as a pale-yellow solid (mp 100.0 – 100.8 °C, from trituration with diisopropyl ether). Yield 75%. ¹H NMR (600 MHz, DMSO-d ₆): δ 1.30 (t, J = 7.1 Hz, 3H, OCH₂CH ₃), 4.24 (q, J = 7.1 Hz, 2H, −OCH₂CH₃), 5.44 (s, 2H, −OCH ₂Ph), 7.04 (t, J = 6.9 Hz, 1H, H-b), 7.29–7.45 (m, 3H), 7.47–7.59 (m, 3H), 7.91 (d, J = 8.8 Hz, 1H, H-d), 8.67 (d, J = 6.8 Hz, 1H, H-a). ¹³C NMR (151 MHz, DMSO-d ₆): δ 14.4 (−OCH₂ CH₃), 59.0 (-OCH₂CH₃), 70.2 (−OCH₂Ph), 87.0 (C-f), 113.3 (C-b), 117.2 (C-d), 127.4, 127.9, 128.3, 128.9, 129.6, 136.6, 142.0 (C-e), 161.9 (C-h) *, 164.3 (C-g)*. MS (ES⁺): 297 (M+1).

General Procedure for the Synthesis of Target Compounds 2b and 2d

A solution of compound 15 (6.58 mmol), K₂CO₃ (19.74 mmol), and the correspondent alkylating agent (7.89 mmol) in dry ACN (100 mL) were warm at reflux upon complete conversion of starting material was observed by TLC (eluent: DCM/MeOH 9:1 v/v). The reaction was then filtered to remove insoluble inorganic substances and the filtrate was concentrated under reduced pressure. The crude mixture was purified following the conditions above

Ethyl 1-methyl-2-oxo-1,2-dihydropyrazolo­[1,5-a]­pyridine-3-carboxylate (2b)

The mixture was solubilized in MeOH (10 mL), and cooled diethyl ether (100 mL) was added. The pale-yellow precipitate was filtered off, affording the desired product as a pale-yellow solid (mp 217.8–224.2 °C dec, from diethyl ether). Yield 90%. ¹H NMR (600 MHz, Chloroform-d): δ 1.38 (t, 3H, J = 7.1 Hz, −OCH₂CH ₃), 3.65 (s, 3H, −NCH ₃), 4.36 (q, 2H, J = 7.0 Hz, −OCH ₂CH₃), 6.89 (td, 1H, J = 7.0, 1.3 Hz, H-b), 7.45 (ddd, 1H, J = 8.7, 7.1, 1.0 Hz, H-c), 7.89 (d, 1H, J = 6.8 Hz, H-a), 8.10 (d, 1H, J = 8.9 Hz, H-d). ¹³C NMR (151 MHz, Chloroform-d): δ 14.8 (−CH₂ CH₃), 28.1 (−NCH₃), 59.7 (−CH₂CH₃), 85.6 (C-f), 112.3 (C-b), 117.9 (C-d), 122.2, 131.1, 143.5 (C-e), 160.5, 164.3. MS (ES⁺): 221 (M+1).

Ethyl 1-benzyl-2-oxo-1,2-dihydropyrazolo­[1,5-a]­pyridine-3-carboxylate (2d)

Crude mixture was purified by using RediSep Gold Silica Gel disposable flash column, 40 g of silica (eluent: petroleum ether/EtOAc from 50/50 to 10/90 v/v) to afford the tile compound as a white solid (mp 172.3–174.0 °C; from trituration with EtOAc/diisopropyl ether 1/1 v/v). Yield 70%. ¹H NMR (600 MHz, DMSO-d ₆): δ 1.28 (t, J = 7.1 Hz, 3H, −OCH₂CH ₃), 4.22 (q, J = 7.1 Hz, 2H, −OCH ₂CH₃), 5.43 (s, 2H, −NCH ₂Ph), 6.95 (td, J = 7.1, 1.0 Hz, 1H, H-b), 7.18–7.38 (m, 5H), 7.59 (t, J = 8.0 Hz, 1H, H-c), 7.92 (d, J = 8.8 Hz, 1H, H-d), 8.41 (d, J = 6.9 Hz, 1H, H-a). ¹³C NMR (151 MHz, DMSO-d ₆): δ 14.6 (−OCH₂ CH₃), 43.6 (−NCH₂Ph), 58.5 (−OCH₂CH ₃) 84.4 (C-f), 112.5 (C-b), 116.3 (C-d), 125.2 (C-a), 127.1, 128.0, 128.9, 132.5, 134.0, 142.7 (C-e), 160.0 (C-g) *, 163.2 (C-h)*. MS (ES⁺): 297 (M+1).

General Procedure for the Synthesis of Target Compounds 1, 1a–C

Five M NaOH (10.0 equiv) was added to the correspondent pyrazolo­[1,5-a] pyridine analogue solution (2.40 mmol) in EtOH (20 mL). The reaction mixture was stirred at reflux until complete conversion of the starting material was observed by TLC (eluent: petroleum ether/EtOAc 60/40 v/v for 1a and 1c and DCM/MeOH 9:1 v/v for 1 and 1b). Subsequently, EtOH was removed under reduced pressure. The white solid was taken up with water and warmed at reflux (for 1, 1a and 1c) or stirred at 0 °C (for 1b). Then 37% w/w HCl was slowly added until pH 1 was reached. Subsequently, the mixture was extracted with EtOAc (3 × 20 mL for 1, 1a and 1c) and DCM (7 × 20 mL for 1b). The combined organic layers were dried under Na₂SO₄ and evaporated under reduced pressure to afford a crude oil purified following the above-mentioned conditions.

Pyrazolo­[1,5-a]­pyridin-2-ol (1)

Crude material was purified via flash chromatography (eluent: petroleum ether/EtOAc 9/1 v/v) to afford the title compound as a white solid (mp 126.3–126.6 °C, trituration from diisopropyl ether). Yield: 90%. ¹H NMR (600 MHz, DMSO-d ₆): δ 5.73 (s, 1H, H-f), 6.62 (td, 1H, J = 6.8, 1.2 Hz, H-b), 7.04–7.10 (m, 1H, H-c), 7.35 (d, 1H, J = 8.8 Hz, H-d), 8.33 (d, 1H, J = 6.9 Hz, H-a), 10.40 (s, 1H, -OH). ¹³C NMR (151 MHz, DMSO-d ₆): δ 79.9 (C-f), 109.4 (C-b), 115.7 (C-d), 123.7 (C-c)*, 128.1 (C-a)*, 141.0 (C-e), 163.9 (C-g). MS (ES⁺): 135 (M+1).

2-Methoxypyrazolo­[1,5-a]­pyridine (1a)

The crude material was purified by combiflash using RediSep Gold Silica Gel disposable flash column, 24 g (eluent: petroleum ether/EtOAc from 100 to 80/20 v/v) to afford the title compound as a colorless oil. Yield 90%. ¹H NMR (400 MHz, Chloroform-d): δ 4.00 (s, 3H, −OCH ₃), 5.83 (s, 1H, H-f), 6.59 (td, 1H, J = 6.9, 1.3 Hz, H-b), 7.04 (ddd, 1H, J = 8.8, 6.8, 1.0 Hz, H-c), 7.29 (d, 1H, J = 8.9 Hz, H-d), 8.24 (d, 1H, J = 7.0 Hz, H-a). ¹³C NMR (100 MHz, Chloroform-d): δ 56.8 (−OCH₃), 79.6 (C-f), 109.8 (C-b), 116.5 (C-d), 124.1 (C-c) *, 128.5 (C-a)*, 141.9 (C-e), 166.4 (C-g). MS (ES⁺): 149 (M+1).

1-Methylpyrazolo­[1,5-a]­pyridin-2­(1H)-one (1b)

The crude material was purified by combiflash using RediSep Gold Silica Gel disposable flash column (12 g, eluent: DCM/MeOH 95/5 v/v) to afford the title compound as a pale-pink solid. Yield 75%. ¹H NMR (400 MHz, DMSO-d ₆): δ 3.53 (s, 3H, −NCH ₃), 5.13 (s, 1H, H-f), 6.60 (td, 1H, J = 6.7, 1.7 Hz, H-b), 7.16–7.22 (m, 2H, H-c and d) 8.25 (d, 1H, J = 6.9 Hz, H-a). ¹³C NMR (100 MHz, DMSO-d ₆): δ 27.7 (−NCH₃), 77.6 (C-f), 107.5 (C-b), 114.2 (C-d), 124.2 (C-c) *, 128.3 (C-a)*, 142.2 (C-e), 162.2 (C-g). MS (ES⁺): 149 (M+1).

2-Benzyloxy-pyrazolo­[1,5-a]­pyridine (1c)

This compound was synthesized according to the general procedure and the specific procedure outlined below. Benzyl bromide (0.701 g, 4.10 mmol) was added to a mixture of pyrazolo­[1,5-a] pyridin-2-ol (1, 0.500 g, 3.73 mmol) and Cs₂CO₃ (3.64 g, 11.2 mmol) in dry DMF (10 mL). The reaction mixture was stirred overnight at room temperature, and then water (100 mL) was added. The mixture was extracted with EtOAc (4 × 50 mL), then the combined organic layers were dried over Na₂SO₄ and evaporated under reduced pressure to afford a yellow oil. The mixture was purified using flash chromatography (eluent: petroleum ether/EtOAc 2/1 v/v) to afford the title compound as a white solid (mp 140.8–142.8 °C, trituration from diisopropyl ether). Yield 61%. ¹H NMR (600 MHz, Chloroform-d) δ: 5.34 (s, 2H, −OCH ₂Ph), 5.88 (s, 1H, H-f), 6.60 (t, J = 6.8 Hz, 1H, H-b), 7.01–7.07 (m, 1H, H-c), 7.29 (d, 1H, J = 8.9 Hz, H-d), 7.33 (t, 1H, J = 7.3 Hz), 7.39 (t, 2H, J = 7.5 Hz), 7.50 (d, 2H, J = 7.4 Hz), 8.25 (d, 1H, J = 6.9 Hz, H-a).¹³C NMR (151 MHz, Chloroform-d) δ: 71.1 (-OCH₂Ph), 80.4 (C-f), 109.9 (C-b), 116.6 (C-d), 124.1, 127.9, 128.2, 128.5, 128.6, 137.0, 141.8 (C-e), 165.6 (C-g). MS (ES⁺): 225 (M+1).

3-Nitrosopyrazolo­[1,5-a]­pyridin-2-ol (16)

To a stirred solution of 1 (0.200 g, 1.49 mmol) in acetic acid (2.0 mL) cooled at 0 °C, a solution of NaNO₂ (0.123 g, 1.78 mmol) in water (3.0 mL) was added dropwise. The reaction was then stirred at room temperature until a yellow precipitate was observed, then filtrated and the filtrate dried under vacuum. The resulting solid crude was triturated using a mixture of diisopropyl ether and methanol to afford the title compound as a yellow solid. Yield 84%.¹H NMR (600 MHz, DMSO-d ₆): δ 7.66 (td, 1H, J = 6.3, 3.2 Hz, H-b), 7.84 (td, 1H, J = 7.9, 1.1 Hz, H-c), 8.22 (ddd, 1H, J = 7.9, 1.6, 0.7 Hz, H-d), 8.59 (d, 1H, J = 6.1 Hz, H-a). ¹³C NMR (151 MHz, DMSO-d ₆) δ: 123.7, 127.2, 130.4, 131.1, 134.3, 142.9, 169.2. MS (ES⁺): 164 (M+1).

3-Nitropyrazolo­[1,5-a]­pyridin-2-ol (3)

A solution of H₂O₂ (30% w/w in water) was added dropwise to a cooled suspension of compound 16 (0.150 g, 0.915 mmol) in acetic acid (2.0 mL). The reaction mixture was stirred at room temperature upon complete conversion of starting material was observed. The result suspension was filtered, and the solid was washed with water and dried under vacuum, to afford the title compound as a pale orange solid. Yield 84%.¹H NMR (600 MHz, DMSO-d ₆): δ 7.27 (td, 1H, J = 7.1, 1.3 Hz, H-b), 7.79 (ddd, 1H, J = 8.5, 7.3, 0.9 Hz, H-c), 8.09 (d, 1H, J = 8.7 Hz, H-d), 8.73 (dd, 1H, J = 6.7, 1.1 Hz, H-a), 12.62 (s, 1H, -OH).¹³C NMR (151 MHz, DMSO-d ₆): δ 110.1 (C-f), 116.1, 116.6, 130.2, 132.0, 136.9 (C-e), 159.7 (C-g). MS (ES⁺): 180 (M+1).

2-Methoxy-3-nitropyrazolo­[1,5-a]­pyridine (17a) and 1-Methyl-3-nitropyrazolo­[1,5-a]­pyridine-2­(1H)-one (17b)

Methyl iodide (4.4 mmol) was added dropwise to a mixture of 3 (0.400 g, 2.2 mmol) and Cs₂CO₃ (2.15 g, 6.6 mmol) in dry DMF (15.0 mL). The reaction mixture was stirred overnight at room temperature, and then water (100 mL) was added. The reaction mixture was extracted with EtOAc (8 × 25 mL), and the combined organic layers were dried over Na₂SO₄ and evaporated under reduced pressure. The resulting crude mixture showed two spots on TLC (eluent: petroleum DCM/MeOH 90/10 v/v), ascribed to the two pyrazolo­[1,5-a]-pyridine regio-isomers. Both regio-isomers were achieved after flash chromatography (eluent: DCM/MeOH from 95/5 to 80/20 v/v) as a pale-yellow solid (17a) and green solid (17b).

17a) First isomer eluted. Yield 70%. ¹H NMR (400 MHz, DMSO-d ₆): δ 4.12 (s, 3H, −OCH ₃), 7.33 (dt, 1H, J = 7.0, 1.4 Hz, H-b), 7.87 (ddd, 1H, J = 8.7, 7.2, 1.1 Hz, H-c), 8.15 (ddd, 1H, J = 8.8, 1.4, 1.0 Hz, H-d), 8.87 (dt, 1H, J = 6.7, 1.0 Hz, H-a). ¹³C NMR (151 MHz, DMSO-d ₆): δ 57.4 (−OCH₃), 110.1 (C-f), 116.3 (C-b), 116.8 (C-d), 130.8 (C-a), 132.8 (C-c), 137.5 (C-e), 160.1 (C-g). MS (ES⁺): 194 (M+1).

17b) Second isomer eluted. Yield 30%. ¹H NMR (600 MHz, DMSO-d ₆): δ 3.61 (s, 3H, −NCH ₃), 7.41 (td, 1H, J = 7.4, 1.4 Hz, H-b), 7.95 (ddd, 1H, J = 8.6, 7.4, 1.0 Hz, H-c), 8.21 (ddd, 1H, J = 8.7, 1.3, 0.8 Hz, H-d), 8.79 (d, 1H, J = 6.8 Hz, H-a).¹³C NMR (151 MHz, DMSO-d ₆): δ 28.5\(−NCH₃), 108.7 (C-f), 115.9, 116.1, 125.9, 134.9, 136.4 (C-e), 154.4 (C-g). MS (ES⁺): 194 (M+1).

N-(2-methoxypyrazolo­[1,5-a]­pyridine-3-yl)­acetamide (3a)

Acetyl chloride (0.108 g, 1.38 mmol), triethylamine (0.379 g, 3.75 mmol), and dry DMF (7.0 μL) were solubilized under N₂ atmosphere in 10 mL of dry THF. The solution was cooled down at 0 °C and then, 18 (0.250 g, 1.25 mmol) solubilized in 5.0 mL of dry THF was slowly added to the mixture, keeping the temperature below 0 °C. The reaction mixture was stirred at room temperature upon complete conversion of starting material was observed by TLC (eluent: petroleum ether/EtOAc 6:4 v/v). The mixture was then quenched in NH₄Cl saturated solution (100 mL) and extracted with EtOAc (3 × 25 mL). The combined organic layers were collected, dried over Na₂SO₄ and concentrated under reduced pressure. The crude material was purified by flash chromatography (eluent: petroleum ether/acetone from 100 to 70/30 v/v) to afford the title compound as a white solid. Yield 75%. ¹H NMR (600 MHz, DMSO-d ₆): δ 2.02 (s, 3H, −CONHCH ₃), 3.94 (s, 3H, −OCH ₃), 6.69 (t, 1H, J = 6.5 Hz, H-b), 7.09–7.15 (m, 1H, H-c), 7.20 (d, 1H, J = 8.9, H-d), 8.42 (d, 1H, J = 6.8 Hz, H-a). 9.21 (s, 1H, – CONHCH₃). ¹³C NMR (151 MHz, DMSO-d ₆): δ 22.5 (−CONHCH₃), 56.1 (−OCH₃), 92.9 (C-f), 109.8 (C-b), 115.5 (C-d), 123.9, 128.7, 136.6 (C-e), 159.2, 169.0 (C-g). MS (ES⁺): 206 (M+1).

N-(2-methoxypyrazolo­[1,5-a]­pyridin-3-yl)­methanesulfonamide (3b)

Methane sulfonyl chloride (0.233 g, 2.04 mmol), triethylamine (0.561 g, 5.55 mmol) and dry DMF (7.0 μL) were solubilized under N₂ atmosphere in 10 mL of dry THF. The solution was cooled down and then, 18 (0.370 g, 1.85 mmol) solubilized in 5 mL of dry THF was slowly added to the mixture, keeping the temperature below 0 °C. The reaction was stirred at room temperature upon complete conversion of starting material was observed by TLC (eluent: petroleum ether/EtOAc 6:4 v/v). Then, the mixture was quenched in NH₄Cl saturated solution (100 mL) and extracted with EtOAc (3 × 25 mL). The combined organic layers were dried over Na₂SO₄ and concentrated under reduced pressure. The crude was purified by flash chromatography (eluent: petroleum ether/acetone from 100 to 50/50 v/v) to afford the title compound as a white solid. Yield 78%.¹H NMR (600 MHz, DMSO-d ₆): δ 2.94 (s, 3H, −SO₂CH ₃), 3.98 (s, 3H, −OCH ₃), 6.77 (td, 1H, J = 6.8, 1.4 Hz, H-b), 7.25 (ddd, 1H, J = 8.9, 6.8, 1.1 Hz, H-c), 7.37–7.40 (m, 1H, H-d), 8.48 (dt, 1H, J = 6.9, 1.0 Hz, H-a), 8.91 (s, 1H, −SO₂NH).¹³C NMR (151 MHz, DMSO-d ₆): δ 40.1 (−SO₂ CH₃), 56.4 (-OCH₃), 90.9 (C-f), 110.7, 114.7, 125.3, 128.9, 139.0 (C-e), 160.8 (C-g). MS (ES⁺): 242 (M+1).

2-Methoxypyrazolo­[1,5-a]­pyridine-3-carbaldehyde (19)

A solution of POCl₃ (2.61 g, 17.04 mmol) in dry DMF (20 mL) was stirred for 30 min at rt under a nitrogen atmosphere. After cooling the mixture to 0 °C, a solution of 1a (1.0 g, 5.7 mmol) in dry DMF (10 mL) was added dropwise. The reaction mixture was stirred until complete conversion of the starting material was observed by TLC (eluent: petroleum ether/EtOAc 8:2 v/v). The reaction mixture was quenched with 1 M NaOH (300 mL), and the precipitate obtained was filtered off. The obtained solid was triturated with diisopropyl ether to afford a white fluffy solid (mp 123.1–124.9 °C, from trituration with diisopropyl ether). Yield 94%. ¹H NMR (600 MHz, Chloroform-d): δ 4.11 (s, 3H, −OCH ₃), 6.93 (td, 1H, J = 6.9, 1.4 Hz, H-b), 7.45 (ddd, 1H, J = 8.5, 7.1, 1.0 Hz, H-c), 8.11 (d, 1H, J = 8.7 Hz, H-d), 8.31 (dt, 1H, J = 6.8 Hz, H-a), 9.90 (s, 1H, −COH).¹³C NMR (151 MHz, Chloroform-d): δ 56.8 (−OCH₃), 98.8 (C-f), 114.1 (C-b), 118.3 (C-d), 129.2 (C-c)*, 129.8 (C-a)*, 141.5 (C-e), 167.8 (C-g), 182.2 (C-h). MS (ES⁺): 177 (M+1).

2-((2-Methoxypyrazolo­[1,5-a]­pyridin-3-yl)­methylene) (4a)

Malonitrile (0.082 g, 1.2 mmol) and ammonium acetate (0.174 g, 2.26 mmol) were dissolved in 5.0 mL of acetic acid. The solution was stirred at r.t. for 30 min and then a solution of 19 (0.200 mg, 1.13 mmol) in acetic acid (2.0 mL) was slowly added. The reaction mixture was heated at reflux upon complete conversion of the starting material was observed. The precipitate formed during the reaction was filtered and washed several times with water to afford the title compound as a pale-yellow solid (mp 206.2 – 208.3 °C, from water). Yield 80%.¹H NMR (600 MHz, Chloroform-d): δ 4.13 (s, 3H, −OCH ₃), 7.05 (td, 1H, J = 7.0, 1.2 Hz, H-b), 7.56 (ddd, 1H, J = 8.6, 7.2, 1.1 Hz, H-c), 7.67 (s, 1H, −CHC­(CN)₂), 8.18 (d, 1H, J = 8.9 Hz, H-d), 8.37 (dt, 1H, J = 6.8, 1.1 Hz, H-a). ¹³C NMR (151 MHz, Chloroform-d): δ 57.3 (-OCH₃), 70.5, 94.4 (C-f), 115.1 (C-b), 116.0, 116.6, 119.1 (C-d), 130.5 (C-a), 130.6 (C-c), 140.7 (C-e), 147.2, 166.7 (C-g). MS (ES⁺): 225 (M+1).

Methyl (e)-2-cyano-3-(2-methoxypyrazolo­[1,5-a]­pyridin-3-yl)­acrylate (4b)

Methyl 2-cyanoacetate (0.092 g, 0.93 mmol) and ammonium acetate (0.131 g, 1.7 mmol) were dissolved in 5.0 mL of acetic acid. The solution was stirred at r.t. for 30 min and then a solution of 2-methoxypyrazolo­[1,5-a] pyridine-3-carbaldehyde (0.150 g, 0.85 mmol) in acetic acid (2.0 mL) was slowly added. The mixture was stirred at reflux upon complete conversion of starting material was observed. The reaction mixture was cooled to room temperature and the observed precipitate was filtered off, washed several times with water, and dried in desiccator to afford the title compound as a pale-yellow solid (mp 209.4 – 210.0 °C, from water). Yield 75%. ¹H NMR (600 MHz, Chloroform-d): δ 3.89 (s, 3H, −COOCH ₃)*, 4.12 (s, 3H, −OCH ₃)*, 6.97 (t, 1H, J = 6.9 Hz, H-b), 7.49 (t, 1H, J = 8.0 Hz, H-c), 8.29–8.33 (m, 2H,) 8.34 (d, 1H, J = 6.7, H-a).¹³C NMR (151 MHz, Chloroform-d): δ 52.9 (−COOCH₃)**, 57.1 (−OCH₃)**, 92.1 (C-f), 93.5, 114.3 (C-b), 118.5, 119.5 (C-d), 129.5 (C-c), 130.2 (C-a), 140.8 (C-e), 144.1 (C-h), 165.3 (C-g)***, 167.1 (−COOCH₃) ***. MS (ES⁺): 258 (M+1).

General Procedure for the Synthesis of Compounds 5a–5e

To a stirred suspension of appropriate benzyl bromide (1.0 mmol) in dry toluene, triphenylphosphine (1.0 mmol) was added under nitrogen atmosphere. The reaction mixture was stirred at 40 °C for 48 h until complete conversion of the starting material was observed. Then, the reaction mixture was cooled to room temperature and the precipitate was filtered under nitrogen atmosphere to afford the appropriate phosphonium salt as a white solid (see supporting info for detailed synthesis and characterization).

General Wittig Reaction Procedure for Synthesis of Compounds 5a–e

A suspension of the correspondent phosphonium salt (35 a–e 1.14 mmol) in dry THF (7.0 mL) was cooled at −10 °C with ice/salt bath under nitrogen atmosphere. Then, 1.0 eq. of 1 M solution of LiHMDS in dry THF (1.14 mmol) was added dropwise. The reaction mixture was stirred for 30 min at −10 °C and then, a solution of 2-methoxypyrazolo­[1,5-a]­pyridine-3-carbaldehyde (1.14 mmol), in dry THF (1.0 mL) was slowly added. The reaction mixture was stirred at reflux until completely conversion of the starting material was observed. The mixture was quenched in a saturated solution of NH₄Cl (100 mL) and extracted with EtOAc (3 × 50 mL). The combined organic layers were dried under Na₂SO₄ and evaporated under reduced pressure to afford a dark oil. The crude product was purified by flash chromatography (see below the conditions).

(E)-2-methoxy-3-styrylpyrazolo­[1,5-a]­pyridine (5a)

The crude material was purified by flash chromatography (eluent: petroleum ether/EtOAc 95/5 v/v) to afford the title compound as a pale-yellow solid (mp 117.5–118.3 °C, from trituration with diisopropyl ether). Yield 45%.¹H NMR (600 MHz, Chloroform-d): δ 4.15 (s, 3H, −OCH ₃), 6.63 (td, 1H, J = 6.8, 1.2 Hz, H-b), 7.06–7.16 (m, 3H, H-h, H-i, H-c) 7.20 (t, 1H, J = 7.3 Hz), 7.34 (d, 2H, J = 7.7 Hz), 7.50 (d, 2H, J = 7.3 Hz), 7.53 (d, 1H, J = 8.9 Hz, H-d), 8.22 (d, 1H, J = 6.9 Hz, H-a). ¹³C NMR (151 MHz, Chloroform-d): δ 56.6 (−OCH₃), 94.0 (C-f), 110.2 (C-b), 115.9, 117.0 (C-d), 124.8, 125.0, 125.7, 126.5, 128.7, 128.8, 138.9, 139.1, 163.9 (C-g). MS (ES⁺): 251 (M+1).

(E)-2-methoxy-3-(4-(methylthio)­styryl)­pyrazolo­[1,5-a]­pyridine (5b)

The crude material was purified by flash chromatography (eluent: petroleum ether/EtOAc 90/10 v/v) to affords a mixture of cis–trans isomer. The pure trans isomer was obtained after purification using preparative HPLC (Method: 20 min, H₂O 100% MeOH from 0 to 100%) to afford the title compound as a pale-yellow solid (mp 134.8–136.1 °C, from water). Yield 55%. ¹H NMR (600 MHz, Chloroform-d): δ 2.50 (s, 3H, −SCH ₃), 4.14 (s, 3H, −OCH ₃), 6.63 (1H, t, J = 6.8 Hz, H-b), 7.00–7.16 (m, 3H), 7.23 (d, 2H, J = 8.2 Hz), 7.41 (d, 2H, J = 8.2 Hz), 7.52 (d, 1H, J = 8.9 Hz, H-d), 8.22 (d, 1H, J = 6.9 Hz, H-a). ¹³C NMR (151 MHz, Chloroform-d): δ 16.4 (−SCH₃), 56.7 (−OCH₃), 94.0 (C-f), 110.2 (C-b), 115.9 (C-d), 116.6, 124.4, 124.8, 126.2, 127.3, 128.8, 136.16, 136.17, 139.0 (C-e), 163.9 (C-g). MS (ES⁺): 297 (M+1).

(E)-2-methoxy-3-(4-(methylsulfonyl)­styryl)­pyrazolo­[1,5-a]­pyridine (5c)

The crude material was purified by flash chromatography (eluent: petroleum ether/EtOAc 95/5 v/v) to afford the title compound as a white solid (mp 195.1–196 °C, from trituration with diisopropyl ether). Yield 56% ¹H NMR (600 MHz, Chloroform-d): δ 3.06 (s, 3H, −SO₂CH ₃), 4.16 (s, 3H, -OCH ₃), 6.69 (t, 1H, J = 6.3 Hz, H-b), 7.08 (d, J = 16.3 Hz, 1H) *, 7.21 (t, 1H, J = 7.8 Hz, H-c), 7.29 (d, 1H, J = 16.3 Hz) *, 7.55 (d, 1H, J = 8.8 Hz, H-d), 7.62 (d, 2H, J = 8.2 Hz) 7.86 (d, 2H, J = 8.2 Hz,), 8.24 (d, 1H, J = 6.8 Hz, H-a). ¹³C NMR (151 MHz, Chloroform-d): δ 44.8 (−SO₂ CH₃), 56.7 (-OCH₃), 93.7 (C-f), 110.8 (C-b), 115.7 (C-d), 121.0, 122.3, 125.7, 126.0, 127.9, 129.0, 137.2, 139.5, 144.7, 164.3 (C-g). MS (ES⁺): 329 (M+1).

(E)-4-(2-(2-methoxypyrazolo­[1,5-a]­pyridin-3-yl)­vinyl)­benzonitrile (5d)

The crude material was purified by flash chromatography (eluent: petroleum ether/EtOAc 93/7 v/v) to afford the title compound as a yellow solid (mp 175.7–177 °C, from trituration with diisopropyl ether). Yield 45%. ¹H NMR (600 MHz, Chloroform-d): δ 4.15 (s, 3H, −OCH ₃), 6.68 (t, 1H, J = 6.8 Hz, H-b), 7.03 (d, 1H, J = 16.3 Hz), 7.16–7.21 (m, 1H, H-c), 7.24 (d, 1H, J = 16.3 Hz), 7.50–7.54 (m, 3H), 7.55–7.59 (m, 2H), 8.23 (d, 1H, J = 6.9 Hz, H-a). ¹³C NMR (151 MHz, Chloroform-d): δ 56.7 (−OCH₃), 93.7 (C-f), 109.0 (−CN), 110.8 (C-b), 115.6 (C-d), 119.6, 120.8, 122.5, 125.6, 125.9, 129.0, 132.5, 139.5, 143.7, 164.3 (C-g). MS (ES⁺): 276 (M+1).

(E)-2-methoxy-3-(4-nitrostyryl) Pyrazolo­[1,5-a]­pyridine (5e)

The crude material was purified by flash chromatography (eluent: petroleum ether/EtOAc 95/5 v/v) to afford the title compound as red solid (mp 211.5–213.6 °C, from trituration with diisopropyl ether). Yield: 27%. ¹H NMR (600 MHz, Chloroform-d): δ 4.17 (s, 3H, −OCH ₃), 6.71 (t, 1H, J = 6.6 Hz, H-b), 7.10 (d, 1H, J = 16.3 Hz), 7.19–7.25 (m, 1H, H-c), 7.32 (d, 1H, J = 16.3 Hz), 7.53–7.60 (m, 3H), 8.18 (d, 1H, J = 8.6 Hz), 8.25 (d, 1H, J = 6.7 Hz, H-a). ¹³C NMR (151 MHz, Chloroform-d): δ 56.8 (-OCH₃), 93.9 (C-f), 111.0 (C-b), 115.7 (C-d), 121.9, 122.1, 124.3, 125.7, 125.9, 129.2, 139.6, 145.8, 145.9, 164.4 (C-g). MS (ES⁺): 296 (M+1).

3-Bromo-2-methoxypyrazolo­[1,5-a]­pyridine (20)

A solution of 2-methoxypyrazolo­[1,5-a] pyridine (0.340 g, 2.29 mmol) in dry DCM (10 mL) was cooled at 0 °C by an ice bath. Then a solution of NBS (0.448 g, 2.52 mmol) in dry DCM (2.0 mL) was slowly dropped into the reaction mixture that was stirred until complete conversion of starting material as observed by TLC (eluent: petroleum ether/EtOAc 9:1 v/v). The solvent was removed then under reduced pressure and the reaction crude was purified by flash chromatography (eluent: petroleum ether/EtOAc 90/10 v/v) to afford the title compound as a white solid (mp 55.1–55.6 °C, from trituration with diisopropyl ether). Yield 95%. ¹H NMR (600 MHz, Chloroform-d): δ 4.09 (s, 3H, −OCH ₃), 6.63 (td, 1H, J = 6.9, 1.3 Hz, H-b), 7.14 (ddd, 1H, J = 8.9, 6.7, 1.0 Hz, H-c), 7.27–7.30 (m, 1H, H-d), 8.20 (dt, 1H, J = 7.0, 0.9 Hz, H-a). ¹³C NMR (151 MHz, Chloroform-d): δ 57.0 (−OCH₃), 67.4 (C-f), 110.3 (C-b), 115.2 (C-d), 125.1 (C-c) *, 128.9 (C-a)*, 139.7 (C-e), 162.2 (C-g). MS (ES⁺): 227, 229 (M+1).

General Procedure for the Synthesis of Compounds 6, 7a–c

[Pd­(PPh₃)₄] (4.4 mg, 0.0038 mmol) was added to a solution of 3-bromo-2-methoxypyrazolo­[1,5-a]­pyridine (0.086 mg, 0.38 mmol) and Cs₂CO₃ (0.371 g, 1.14 mmol,) in dioxane/water (8:2 v/v, 10 mL) solution. After stirring the resulting mixture under nitrogen atmosphere for 1 h, the corresponding boronic acid (0.760 mmol) was added. The reaction mixture was stirred at reflux until complete conversion of starting material as observed by TLC (eluent: petroleum ether/EtOAc 8:2 v/v). Then, the reaction mixture was cooled to room temperature and was concentrated under reduced pressure to afford a crude material. The latter was taken-up with water (50 mL), and the aqueous solution was extracted with EtOAc (3 × 10 mL). The combined organic layers were dried over Na₂SO₄ and concentrated under reduced pressure. The crude material was purified by flash chromatography (see below the conditions).

2-Methoxy-3-(naphthalen-2-yl)­pyrazolo­[1,5-a]­pyridine (6)

The mixture was purified by flash chromatography (eluent: petroleum ether/EtOAc from 90/10 to 80/20 v/v) to afford the title compound as a white solid (mp 126.8 – 127.5 °C, from trituration with diisopropyl ether). Yield 76%. ¹H NMR (600 MHz, Chloroform-d): δ 4.17 (s, 3H, −OCH ₃), 6.66 (td, 1H, J = 6.8, 1.3 Hz, H-b), 7.16 (ddd, 1H, J = 8.9, 6.8, 1.1 Hz, H-c), 7.42–7.51 (m, 2H), 7.71 (dt, 1H, J = 9.0, 1.0 Hz, H-d), 7.82–7.88 (m, 3H), 7.90 (d, 1H, J = 8.5 Hz), 8.08 (d, 1H, J = 0.6 Hz), 8.31 (dt, 1H, J = 6.9, 1.0 Hz, H-a). ¹³C NMR (151 MHz, Chloroform-d): δ 56.7 (−OCH₃), 95.6 (C-f), 110.2 (C-b), 116.0 (C-d), 125.0, 125.4, 125.6, 126.2, 126.5, 127.78, 127.82, 128.2, 128.9, 129.9, 131.8, 134.0, 139.2 (C-e), 163.1 (C-g). MS (ES⁺): 275 (M+1).

2-(2-Methoxypyrazolo­[1,5-a]­pyridin-3-yl)­quinoline (7a)

The mixture was purified by Combiflash using RediSep Gold Silica Gel disposable flash column, 40 g of silica (eluent: petroleum ether/EtOAc from 95/5 v/v to 60/40 v/v) to afford the title compound as a pale-yellow solid (mp 129.2–130.3 °C, from trituration with diisopropyl ether). Yield 78%. ¹H NMR (600 MHz, Chloroform-d): δ 4.22 (s, 3H, −OCH ₃), 6.79 (t, J = 6.7 Hz, 1H, H-b), 7.34 (t, J = 7.8 Hz, 1H), 7.43 (t, J = 7.4 Hz, 1H, H-c), 7.66 (t, J = 7.6 Hz, 1H), 7.75 (d, J = 8.0 Hz, 1H), 8.08 (t, J = 8.1 Hz, 2H), 8.16 (d, J = 8.7 Hz, 1H, H-d), 8.31 (d, J = 6.8 Hz, 1H, H-a), 8.89 (d, J = 8.9 Hz, 1H). ¹³C NMR (151 MHz, Chloroform-d): δ 56.8 (−OCH₃), 95.5 (C-f), 111.7 (C-b), 119.9 (C-d), 120.3, 124.9, 126.0, 126.4, 127.6, 128.5, 128.7, 129.2, 135.7, 141.1, 148.4, 153.7, 164.4 (C-g). MS (ES⁺): 276 (M+1).

7-(2-Methoxypyrazolo­[1,5-a]­pyridin-3-yl)­quinoline (7b)

The mixture was purified by flash chromatography (eluent: petroleum ether/EtOAc from 90/10 v/v to 70/30 v/v) to afford the title compound as a pale-yellow solid (mp 148.1–151.1 °C, from trituration with diisopropyl ether). Yield 70%. ¹H NMR (600 MHz, Chloroform-d): δ 4.17 (s, 3H, −OCH ₃), 6.70 (td, 1H, J = 6.8, 1.2 Hz, H-b), 7.19 (ddd, 1H, J = 8.9, 6.8, 1.0 Hz, H-c), 7.35 (dd, 1H, J = 8.2, 4.2 Hz), 7.82 (d, 1H, J = 9.0 Hz, H-d), 7.85 (d, 1H, J = 8.5 Hz), 7.98 (dd, 1H, J = 8.5, 1.6 Hz), 8.14 (d, 1H, J = 7.4 Hz), 8.31 (d, 1H, J = 6.9 Hz, H-a), 8.37 (s, 1H), 8.90 (dd, 1H, J = 4.1, 1.5 Hz). ¹³C NMR (151 MHz, Chloroform-d): δ 56.7 (−OCH₃), 94.9 (C-f), 110.6 (C-b), 116.1 (C-d), 120.5, 125.5, 126.1, 126.5, 127.0, 128.0, 129.0, 134.1, 135.8, 139.3, 148.9, 150.7, 163. (C-g). MS (ES⁺): 276 (M+1).

6-(2-Methoxypyrazolo­[1,5-a]­pyridin-3-yl)­quinoline (7c)

The mixture was purified by Combiflash using RediSep Gold Silica Gel disposable flash column, 40 g of silica (eluent: petroleum ether/EtOAc from 95/5 v/v to 70/30 v/v) to afford the title compound as a pale-yellow solid (mp 147.5–148.5 °C, from trituration with diisopropyl ether). Yield 85%. ¹H NMR (600 MHz, Chloroform-d): δ 4.16 (s, 3H, -OCH ₃), 6.68 (td, 1H, J = 6.9, 1.2 Hz, H-b), 7.18 (ddd, 1H, J = 8.9, 6.8, 1.0 Hz, H-c), 7.38 (dd, 1H, J = 8.2, 4.2 Hz, H-c), 7.71 (d, 1H, J = 9.0 Hz, H-d), 8.04 (d, 1H, J = 1.8 Hz,), 8.08 (dd, 1H, J = 8.7, 1.9 Hz,), 8.11–8.18 (m, 2H), 8.30 (d, 1H, J = 6.9 Hz, H-a), 8.86 (d, 1H, J = 3.1 Hz). ¹³C NMR (151 MHz, Chloroform-d): δ 56.7 (−OCH₃), 94.9 (C-f), 110.4 (C-b), 115.8, 121.4, 125.0, 125.4, 128.9, 129.0, 129.7, 129.9, 130.8, 135.8, 139.2, 146.8, 149.8, 163.2 (C-g). MS (ES⁺): 276 (M+1).

Ethyl 2-(benzyloxy)-7-chloropyrazolo­[1,5-a]­pyridine-3-carboxylate (21)

LiHMDS (1.0 M THF solution: 1.48 mL, 1.48 mmol) was added dropwise to a solution of 2c (0.400 g, 1.35 mmol) in dry THF (10 mL), previously cooled to a −78 °C with dry ice and acetone. The mixture was stirred at −78 °C for 1 h, and then a solution of hexachloroethane (0.383 g, 1.62 mmol) in dry THF was added. The mixture was left slowly to back at room temperature and, subsequently, the reaction was quenched with saturated solution of NH₄Cl (100 mL). The water phase was extracted with EtOAc (3 × 25 mL). The combined organic layers were dried over Na₂SO₄, filtered, and evaporated to dryness under vacuum. The crude product was purified by flash chromatography (eluent: petroleum ether/ethyl acetate 80:20 v/v) to afford the title compound as a white solid. Yield 81%. ¹H NMR (600 MHz, DMSO-d ₆): δ 1.31 (t, 3H, J = 7.1 Hz, −OCH₂CH ₃), 4.26 (q, 2H, J = 7.1 Hz, -OCH ₂CH₃), 5.48 (s, 2H, −CH ₂Ph), 7.30–7.37 (m, 2H), 7.38–7.44 (m, 2H), 7.50–7.60 (m, 3H), 7.92 (dd, J = 8.8, 0.9 Hz, 1H, H-d). ¹³C NMR (151 MHz, DMSO-d ₆) δ 14.3 (−OCH₂ CH₃), 59.3 (−OCH₂CH₃), 70.4 (−CH₂Ph), 88.8 (C-f), 113.5 (C-b), 115.8 (C-d), 127.6, 127.9 (C-c), 128.3, 129.2, 129.5 (C-a), 136.4, 143.7 (C-e), 161.6 (C-h), 163.9 (C-g). MS (ES⁺): 331 (M+1).

Ethyl 2-(Benzyloxy)-7-(dimethylamino)­pyrazolo­[1,5-a]­pyridine-3-carboxylate (22)

To a stirred solution of compound 21 (0.460, 1.39 mmol) in DMF (5.0 mL), a 30% aqueous solution of dimethylamine (0.35 mL) was added. The reaction mixture was stirred until complete conversion of starting material was observed by TLC. Then, the mixture was diluted with water (50 mL) and extracted with EtOAc (3 × 10 mL). The organic layers were collected, dried over anhydrous MgSO₄, and filtered and concentrated under reduced pressure. The crude obtained was crystallized from methanol to afford the title compound as a white solid. Yield 75%. ¹H NMR (400 MHz, DMSO-d ₆): δ 1.30 (t, 3H, J = 7.1 Hz, −OCH₂CH ₃), 3.02 (s, 6H, −N­(CH ₃)₂), 4.24 (q, 2H, J = 7.1 Hz, −OCH ₂CH₃), 5.49 (s, 2H, −OCH ₂Ph), 6.43 (dd, 1H, J = 7.5, 1.6 Hz, H-b), 7.33 (t, 1H, J = 7.3 Hz), 7.37–7.55 (m, 6H).¹³C NMR (101 MHz, DMSO-d ₆): δ 14.4 (−OCH₂ CH₃), 40.8 (−N­(CH₃)₂), 58.8 (−OCH₂CH₃), 70.0 (−OCH₂Ph), 86.3 (C-f), 99.1 (C-b), 108.3 (C-d), 127.3, 127.7, 128.3, 130.0, 136.9, 144.1 (C-e), 147.4 (C-a), 162.1 (C-h), 163.1 (C-g). MS (ES⁺): 340 (M+1).

Ethyl 7-(Dimethylamino)-2-hydroxypyrazolo­[1,5-a]­pyridine-3-carboxylate (8a)

Palladium on carbon (Pd/C, 8% w/w), was added to a solution of compound 22 (0.105 g, 0.40 mmol), in absolute EtOH (5.0 mL). The resulting mixture was vigorously stirred under a hydrogen atmosphere for 6 h. The suspension was filtered through a cake of Celite, that was washed with methanol. The overall filtrate was concentrated under reduced pressure to afford the crude compound. The latter was triturated with diisopropyl ether to afford the title compounds as a pale-yellow solid. Yield 93%.¹H NMR (400 MHz, DMSO-d ₆): δ 1.29 (t, 3H, J = 7.1 Hz, −OCH₂CH ₃), 3.00 (s, 6H, −N­(CH ₃)₂), 4.24 (q, 2H, J = 7.1 Hz, −OCH ₂CH₃), 6.41 (dd, 1H, J = 7.4, 1.5 Hz, H-b), 7.39–7.48 (m, 2H, H-c and H-d), 10.94 (s, 1H, −OH).¹³C NMR (101 MHz, DMSO-d ₆): δ 14.5 (−OCH₂ CH₃), 40.9 (−N­(CH₃)₂), 58.8 (−OCH₂CH₃), 85.8 (C-f), 99.3 (C-b), 108.3 (C-d), 129.3 (C-c), 143.5 (C-e), 147.2 (C-a), 163.0 (C-g), 163.5 (C-h). MS (ES⁺): 250 (M+1).

Ethyl 7-(Dimethylamino)-2-hydroxy-4-nitropyrazolo­[1,5-a]­pyridine-3-carboxylate (8b) and Ethyl 7-(Dimethylamino)-2-hydroxy-6-nitropyrazolo­[1,5-a]­pyridine-3-carboxylate (8c)

To a stirred solution of 8a (0.050 g, 0.2 mmol) in acetic acid (2.0 mL) at 0 °C, a solution of NaNO₂ (0.016 g, 0.24 mmol) in water (1.0 mL) was added dropwise. The reaction was then stirred at room temperature until complete conversion was observed by LC-MS. Then, the solvent was removed under reduced pressure. The crude was diluted with water (3.0 mL) and extracted with dichloromethane (3 × 2 mL). The organic layers were collected and concentrated under reduced pressure to obtain a dark brown oil. The residue was dissolved in water/acetonitrile (5 mL, 1:1 v/v) and purified by preparative HPLC (Method: 20 min, H₂O 100% MeOH from 0 to 100%). Both fractions were freeze-dried providing two solids, which were triturated using diisopropyl ether to afford compound 8b as a green solid and compound 8c as a red solid.

8b) Yield 14%. ¹H NMR (600 MHz, DMSO-d ₆): δ 1.30 (t, 3H, J = 6.8 Hz, −CH₂CH ₃), 3.06 (s, 6H, −N­(CH ₃)₂), 4.26 (q, 2H, J = 6.8 Hz, −OCH ₂CH₃), 7.49 (d, 1H, J = 9.2 Hz, H-b), 7.98 (d, 1H, J = 9.5 Hz, H-c), 11.66 (s, 1H, −OH).¹³C NMR (101 MHz, DMSO-d ₆): δ 14.4 (−OCH₂ CH₃), 41.7 (−N­(CH₃)₂), 59.4 (−OCH₂CH₃), 89.7 (C-f), 107.9 (C-b), 125.9 (C-d), 128.0 (C-c), 143.8 (C-e), 145.0 (C-a), 162.1, 164.5 (C-g). MS (ES⁺): 295 (M+1).

8c) Yield 8%. ¹H NMR (600 MHz, DMSO-d ₆): δ 1.30 (t, 3H, J = 7.1 Hz, −OCH₂CH ₃), 3.32 (s, 6H, −N­(CH ₃)₂), 4.16 (q, 2H, J = 7.1 Hz, −OCH ₂CH₃), 6.34 (d, 1H, J = 9.2 Hz, H-c), 8.17 (d, 1H, J = 9.2 Hz, H-d), 11.62 (s, 1H, −OH). ¹³C NMR (101 MHz, DMSO-d ₆): δ 14.2 (−OCH₂ CH₃), 42.0 (−N­(CH₃)₂), 59.9 (−OCH₂CH₃), 91.1 (C-f), 95.5, 127.3, 128.9, 135.9, 149.9, 162.5, 162.6. MS (ES⁺): 295 (M+1).

Ethyl 7-Chloro-2-methoxypyrazolo­[1,5-a]­pyridine-3-carboxylate (23)

LiHMDS (1.0 M THF solution: 2.17 mL, 2.17 mmol) was added dropwise to a solution of 2a (0.400 g, 1.82 mmol) in dry THF (10 mL), previously cooled to a −78 °C. The mixture was stirred at −78 °C for 1 h, and then a solution of hexachloroethane (0.572 g, 2.17 mmol) in dry THF was added. The mixture was left slowly to relieve at room temperature and, subsequently, the reaction was quenched with an aqueous saturated solution of NH₄Cl (50 mL). The water phase was extracted with EtOAc (3 × 10 mL). The combined organic phases were dried over Na₂SO₄, filtered, and evaporated to dryness under vacuum. The crude product was purified by flash chromatography (eluent: petroleum ether/ethyl acetate 8:2 v/v) to afford the title compound as a white solid. Yield 81%. ¹H NMR (600 MHz, Chloroform-d): δ 1.41 (t, 3H, J = 7.1 Hz, −CH₂CH ₃), 4.20 (s, 3H, −OCH ₃), 4.39 (q, 2H, J = 7.1 Hz, −CH ₂CH₃), 6.96 (dd, 1H, J = 7.5, 1.3 Hz, H-b), 7.30 (dd, 1H, J = 8.9, 7.5 Hz, H-c), 7.96 (dd, 1H, J = 8.9, 1.3 Hz, H-d). ¹³C NMR (151 MHz, Chloroform-d): δ 14.7 (−CH₂ CH₃), 57.3 (−OCH₃), 60.1 (−CH₂CH₃), 89.8 (C-f), 112.7, 116.5, 127.9, 130.7, 144.6 (C-e), 163.1, 165.7 (C-g). MS (ES⁺): 255 (M+1).

7-Chloro-2-methoxypyrazolo­[1,5-a]­pyridine (24)

Five M NaOH (10.0 equiv) was added to compound 23 (1.00 mmol) in EtOH (20 mL). The reaction mixture was stirred at reflux until complete conversion of the starting material was observed by TLC (eluent: petroleum ether/EtOAc 8:2 v/v). Then, EtOH was removed under reduced pressure. The white solid was taken up with water and warmed at reflux. Then 37% w/w HCl was slowly added until pH 1 was reached. Subsequently, the mixture was extracted with EtOAc (3 × 20 mL), dried under Na₂SO₄, and evaporated under reduced pressure to afford crude oil. The latter was purified by flash chromatography (eluent: petroleum ether/ethyl acetate 90:10 v/v) to afford the title compound as a white solid. Yield 51%. ¹H NMR (400 MHz, Chloroform-d): δ 4.05 (s, 3H, −OCH ₃), 5.97 (s, 1H, H-f), 6.74 (dd, 1H, J = 7.3, 1.2 Hz, H-b), 7.01 (dd, 1H, J = 8.8, 7.3 Hz, H-c), 7.27 (dd, 1H, J = 8.8, 1.2 Hz, H-d). ¹³C NMR (400 MHz, Chloroform-d): δ 57.0 (−OCH₃), 81.7 (C-f), 109.9 (C-b), 114.7 (C-d), 124.2 (C-c), 129.7 (C-a), 143.5 (C-e), 166.4 (C-g). MS (ES⁺): 183 (M+1).

7-Chloro-3-iodo-2-methoxypyrazolo­[1,5-a]­pyridine (25)

A solution of compound 24 (0.113 g, 0.618 mmol) in dry DCM (5.0 mL) was cooled at 0 °C by an ice bath. Successively, NIS (0.121 mg, 0.680 mmol) was slowly dropped into the reaction mixture that was then stirred until complete conversion of starting material as observed by TLC (eluent: petroleum ether/EtOAc 9:1 v/v). Then, the solvent was removed under reduced pressure and the crude residue was purified by flash chromatography (eluent: petroleum ether/EtOAc 95/5 v/v) to afford the title compound as a white solid. Yield 90%. ¹H NMR (600 MHz, Chloroform-d): δ 3.98 (s, 3H, −OCH ₃), 6.62 (dd, 1H, J = 7.3, 1.2 Hz, H-b), 6.94 (dd, 1H, J = 8.9, 7.3 Hz, H-c), 7.06 (dd, 1H, J = 8.9, 1.2 Hz, H-d). ¹³C NMR (151 MHz, Chloroform-d): δ 34.4 (C-f), 57.2 (−OCH₃), 110.6 (C-b), 114.7 (C-d), 125.5 (C-c), 130.3 (C-a), 143.8 (C-e), 165.4 (C-g). MS (ES⁺): 309 (M+1).

6-(7-Chloro-2-methoxypyrazolo­[1,5-a]­pyridin-3-yl)­quinoline (9a)

[Pd­(PPh₃)₄] (1.9 mg, 0.0016 mmol) was added to a solution of 7-chloro-3-iodo-2-methoxypyrazolo­[1,5-a]­pyridine (0.050 g, 0.162 mmol) and Cs₂CO₃ (0.154 mg, 0.486 mmol) in dioxane/water (8:2 v/v, 10 mL) solution. After stirring the resulting mixture under nitrogen atmosphere for 1 h, quinolin-6-ylboronic acid (28 mg, 0.16 mmol) was added and the reaction mixture was stirred at reflux, until the complete conversion of starting material was observed by TLC (eluent: petroleum ether/EtOAc 9:1 v/v). Then, the reaction was cooled to room temperature and was concentrated under reduced pressure to afford a crude material. The latter was taken-up with water (30 mL), and the aqueous solution was extracted with EtOAc (3 × 5 mL). The combined organic layers were dried over Na₂SO₄ and concentrated under reduced pressure. The crude material was purified by flash chromatography (eluent: petroleum ether/EtOAc from 90/10 to 70/30 v/v) to afford the title compound as a pale-yellow solid. Yield 80%. ¹H NMR (600 MHz, Chloroform-d): δ 4.23 (s, 3H, −OCH ₃), 6.84 (d, 1H, J = 7.2, H-b), 7.15 (t, 1H, J = 8.0 Hz, H-c), 7.42 (dd, 1H, J = 8.1, 4.1 Hz), 7.67 (d, 1H, J = 8.8 Hz), 7.97 – 8.12 (m, 2H), 8.20 (t, 2H, J = 7.6 Hz), 8.68 – 9.06 (m, 1H). ¹³C NMR (151 MHz, Chloroform-d): δ 57.0 (−OCH ₃), 96.7 (C-f), 110.6 (C-b), 114.0 (C-d), 121.5, 125.4, 125.5, 128.9, 129.4, 130.3, 130.5, 130.6, 136.4, 140.8, 146.5 (C-e), 149.6, 163.2 (C-g). MS (ES⁺): 310 (M+1).

2-Methoxy-N,N-dimethyl-3-(quinolin-6-yl)­pyrazolo­[1,5-a]­pyridin-7-amine (9b)

To a stirred solution of compound 9a (0.020 g, 0.064 mmol), in DMF (1.0 mL), a 30% aqueous solution of dimethylamine (1.0 mL) was added. The reaction was stirred until complete conversion of starting material was observed by TLC (eluent: petroleum ether/EtOAc 5:5 v/v). Then, the mixture was concentrated under reduced pressure. The crude obtained was purified by flash chromatography (eluent: petroleum ether/EtOAc from 50/50 to 20/80 v/v) to afford the title compound as a pale orange solid. Yield 75%. ¹H NMR (600 MHz, Chloroform-d): δ 3.13 (s, 6H, −N­(CH ₃)₂), 4.23 (s, 3H, −OCH ₃), 6.15 (d, 1H, J = 7.4, 1H, H-b), 7.20 (ddd, 1H, J = 9.0, 7.4, 1.6 Hz, H-c), 7.39 (d, 1H, J = 8.8 Hz, H-d), 7.41–7.46 (m, 1H), 8.08 (s, 1H), 8.15 (dt, 1H, J = 8.8, 2.0 Hz), 8.18–8.26 (m, 2H), 8.88 (s, 1H). ¹³C NMR (151 MHz, Chloroform-d): δ 41.4 (−N­(CH₃)₂), 56.7 (−OCH₃), 94.2 (C-f), 96.8 (C-b), 108.0 (C-d), 121.3, 124.9, 126.8, 128.5, 129.1, 130.8, 131.8, 136.9, 141.3, 145.6, 148.1, 148.6, 162.6 (C-g). MS (ES⁺): 319 (M+1).

2-((4-Methoxybenzyl)­oxy)­pyrazolo­[1,5-a]­pyridine (26)

4-Methoxybenzyl chloride (0.840 g, 5.36 mmol) was added to a mixture of pyrazolo­[1,5-a]­pyridin-2-ol (0.600 g, 4.47 mmol) and Cs₂CO₃ (4.370 g, 13.41 mmol) in dry DMF (15 mL). The reaction mixture was stirred overnight at room temperature, and then water (100 mL) was added. The mixture was extracted with EtOAc (4 × 70 mL), and the combined organic layers were dried over Na₂SO₄ and evaporated under reduced pressure to afford a crude yellow oil. The crude compound was purified by flash chromatography (eluent: petroleum ether/EtOAc 2/1 v/v) to afford the title compound as a pale pink solid (mp 110.2–111.3 °C, trituration from diisopropyl ether). Yield 61%. ¹H NMR (600 MHz, Chloroform-d): δ 3.81 (s, 3H, −OCH ₃), 5.26 (s, 2H, −OCH ₂Ar), 5.85 (s, 1H, H-f), 6.59 (td, 1H, J = 6.9, 0.9 Hz, H-b), 6.92 (d, 2H, J = 8.7 Hz), 7.02–7.07 (m, 1H, H-c), 7.29 (d, 1H, J = 8.9 Hz, H-d), 7.43 (d, 2H, J = 8.7 Hz), 8.24 (dd, 1H, J = 6.9, 0.9 Hz, H-a). ¹³C NMR (151 MHz, Chloroform-d): δ 55.4 (−OCH₃), 70.9 (−OCH₂Ar), 80.4 (C-f), 109.8 (C-b), 114.0, 116.5 (C-d), 124.0, 128.5, 129.1, 129.8, 141.8 (C-e), 159.6, 165.6 (C-g). MS (ES⁺): 255 (M+1).

2-((4-Methoxybenzyl)­oxy)­pyrazolo­[1,5-a]­pyridine-3-carbaldehyde (27)

A solution of POCl₃ (1.08 g, 7.07 mmol) in dry DMF (20.0 mL), was stirred for 30 min at room temperature under a nitrogen atmosphere. After cooling the mixture to 0 °C, a solution of 2-((4-methoxybenzyl)­oxy)­pyrazolo­[1,5-a]­pyridine (0.600 g, 2.35 mmol) in dry DMF (5.0 mL) was added dropwise. The reaction mixture was stirred until complete disappearance of the starting material was observed by TLC. The reaction mixture was quenched with 1 M NaOH (200 mL) and the observed precipitate was filtered off. The solid was triturated with diisopropyl ether to afford a white fluffy solid (mp 149.0 – 151.0 °C, trituration from diisopropyl ether). Yield 90%. ¹H NMR (600 MHz, Chloroform-d): δ 3.82 (s, 3H, -OCH ₃), 5.40 (s, 2H, -OCH ₂Ar), 6.90–6.97 (m, 3H), 7.43–7.48 (m, 3H), 8.14 (d, 1H, J = 8.7 Hz, H-d), 8.33 (d, 1H, J = 6.8 Hz, H-a), 9.92 (s, 1H, −CHO). ¹³C NMR (151 MHz, Chloroform-d): δ 55.4 (−OCH₃), 71.2 (−OCH₂Ar), 99.0 (C-f), 114.1, 114.1, 118.5, 128.2, 129.1, 129.8, 130.2, 141.4 (C-e), 159.9, 167.2 (C-g), 182.4 (-CHO). MS (ES⁺): 283 (M+1).

General Wittig Reaction Procedure for Synthesis of Compounds 28 a–B

A suspension of the correspondent phosphonium salt (1.14 mmol) in dry THF (7.0 mL), was cooled at −10 °C under a nitrogen atmosphere, then, a 1 M solution of LiHMDS in THF (1.14 mmol, 1.14 mL) was added dropwise. The reaction mixture was stirred for 30 min at −10 °C and then, a solution of 2-((4-methoxybenzyl)­oxy)­pyrazolo­[1,5-a]­pyridine-3-carbaldehyde (1.14 mmol), in dry THF (1.0 mL) was slowly added. The reaction was stirred at reflux until complete conversion of the starting material was observed by TLC. The mixture was quenched in a saturated solution of NH₄Cl (100 mL) and extracted with EtOAc (3 × 50 mL). The combined organic layers were dried over Na₂SO₄ and evaporated under reduced pressure to afford a dark oil. The crude product was purified by flash chromatography (see below the conditions).

(E)-2-((4-Methoxybenzyl)­oxy)-3-(2-(2,3,5,6-tetrafluoro-[1,1’-biphenyl]-4-yl)­vinyl)­pyrazolo­[1,5-a]­pyridine (28a)

The crude mixture was purified by flash chromatography (eluent: petroleum ether/acetone from 100 to 95/5 v/v) to afford a mixture Z/E isomer (10:90) as yellow solid. The mixture Z/E was solubilized in CHCl₃ and cooled hexane was added. The yellow precipitate was filtered off, affording the desired E product as yellow solid. Yield 55%. ¹H NMR (600 MHz, Chloroform-d): δ 3.84 (s, 3H, −OCH ₃), 5.46 (s, 2H, −OCH ₂Ar), 6.70 (td, 1H, J = 6.9, 1.3 Hz, H-b), 6.96 (d, 2H, J = 8.7 Hz), 7.14–7.24 (m, 2H), 7.39–7.47 (m, 1H), 7.47–7.56 (m, 7H), 7.59 (d, J = 16.7 Hz, 1H), 8.25 (d, 1H, J = 6.9 Hz, H-a). ¹³C NMR (151 MHz, Chloroform-d): δ 55.5 (−OCH₃), 70.9 (−OCH₂Ar), 94.5 (C-f), 110.0, 111.0, 114.1, 115.6, 116.7 (t, J = 17.2 Hz), 117.6 (t, J = 13.5 Hz), 125.57, 125.63, 125.7, 128.1, 128.6, 128.9, 129.0, 129.4, 130.4, 139.6 (C-e), 144.1 (dd, J = 241.3, 16.1 Hz), 144.5 (dd, J = 246.7, 12.7 Hz), 159.6, 163.6 (C-g). ¹⁹F NMR (565 MHz, Chloroform-d): δ −146.18 (dd, J = 21.6, 10.5 Hz), −144.81 (dd, J = 21.6, 10.4 Hz). MS (ES⁺): 527 (M+Na).

(E)-2-((4-Methoxybenzyl)­oxy)-3-(2-(2,3,6-trifluoro-[1,1’-biphenyl]-4-yl)­vinyl)­pyrazolo­[1,5-a]­pyridine (28b)

The crude mixture was purified by flash chromatography (eluent: petroleum ether/acetone from 100 to 95/5 v/v) to afford a crude compound still slightly impure of the Z isomer that was removed by trituration with diisopropyl ether to afford the title compound as a yellow solid. Yield 22%. ¹H NMR (600 MHz, Chloroform-d): δ 3.84 (s, 3H, −OCH ₃), 5.45 (s, 2H, −OCH ₂Ar), 6.70 (t, 1H, J = 6.8 Hz, C-b), 6.97 (d, 2H, J = 8.5 Hz), 7.13–7.16 (m, 1H), 7.18–7.29 (m, 3H), 7.41 (t, 1H, J = 6.9 Hz), 7.43–7.56 (m, 6H), 7.56 (d, 1H, J = 8.8 Hz, H-d), 8.26 (d, 1H, J = 6.8 Hz, H-a). ¹³C NMR (151 MHz, Chloroform-d): δ 55.5 (−OCH₃), 71.0 (−OCH₂Ar), 94.0 (C-f), 106.5 (d, J = 26.0 Hz), 110.9, 114.1, 114.6 (d, J = 2.2 Hz), 115.8, 116.9 (dd, J = 21.1, 15.2 Hz), 121.4 (d, J = 4.3 Hz), 125.7, 127.9 (dd, J = 12.8, 8.0 Hz), 128.50, 128.52, 128.9, 129.0, 129.7, 130.4, 139.4, 144.9 (d, J = 259.9 Hz), 146.6 (d, J = 246.6 Hz), 155.2 (d, J = 242.7 Hz), 159.7, 163.6. ¹⁹F NMR (565 MHz, Chloroform-d): δ −148.56 (t, J = 17.3 Hz), −139.32 (d, J = 19.8 Hz), −121.18 (t, J = 13.0 Hz). MS (ES⁺): 487 (M+1).

General Procedure Followed for Synthesis of Target Compounds 29a and 29b

A solution of O-protected hydroxy pyrazolo­[1,5-a]­pyridine (4.70 mmol) in dry DCM (10 mL) was cooled at 0 °C by an ice bath. Sub sequentially, NIS (5.17 mmol) was slowly dropped into the reaction. The reaction mixture was stirred until complete conversion of starting material was observed by TLC (eluent: petroleum ether/EtOAc 9:1 v/v). The solvent was removed under reduced pressure and the crude mixture was purified by flash chromatography (see below the conditions).

2-Benzyloxy-3-iodopyrazolo­[1,5-a]­pyridine (29a)

The mixture was purified by flash chromatography (eluent: petroleum ether/EtOAc 95/5 v/v) to afford a crude compound. The latter was triturated with diisopropyl ether, to afford the title compound as a white solid. Yield 93%.¹H NMR (600 MHz, Chloroform-d): δ 5.45 (s, 2H, −CH ₂OAr), 6.64 (td, 1H, J = 6.9, 1.4 Hz, H-b), 7.14 (ddd, 1H, J = 8.9, 6.8, 1.1 Hz, H-c), 7.22–7.27 (m, 1H,), 7.33 (t, 1H, J = 7.4 Hz), 7.40 (t, 2H, J = 7.5 Hz), 7.52 (d, 2H, J = 7.0 Hz), 8.21 (dt, 1H, J = 6.9, 1.0 Hz, H-a). ¹³C NMR (151 MHz, Chloroform-d): δ 32.7 (C-f), 71.2 (−CH₂OAr), 110.7 (C-b), 116.4 (C-d), 125.5, 127.8, 128.1, 128.6, 129.2, 136.9, 142.5 (C-e), 164.6 (C-g). MS (ES⁺): 351 (M+1).

3-Iodo-2-methoxypyrazolo­[1,5-a]­pyridine (29b)

The mixture was purified by flash chromatography (eluent: petroleum ether/EtOAc 90/10 v/v) to afford a crude compound. The latter was triturated with diisopropyl ether, to afford the title compound as a white solid (mp 50.3–51.9 °C, from trituration with diisopropyl ether). Yield 90%. ¹H NMR (400 MHz, Chloroform-d): δ 4.12 (s, 3H, −CH ₃), 6.66 (td, 1H, J = 6.9, 1.4 Hz, H-b), 7.17 (ddd, 1H, J = 8.9, 6.8, 1.0 Hz, H-c), 7.27 (dt, 1H, J = 8.9, 1.1 Hz, H-d), 8.24 (d, 1H, J = 6.9 Hz, H-d). ¹³C NMR (400 MHz, Chloroform-d): δ 32.0 (C-f), 57.0 (−CH₃), 110.6 (C-b), 116.4 (C-d), 125.5 (C-c), 129.2 (C-a), 142.5 (C-e), 165.3 (-OCH₃). MS (ES⁺): 275 (M+1).

General Procedure for the Synthesis of Compounds 30a and 30b

A solution of 3-Iodo-pyrazolo­[1,5-a]­pyridine (1.09 mmol) and 2-isopropoxy-4,4–5,5-tetramethyl-1,3,2-dioxaborolane (0.33 mL, 1.6 mmol) in dry THF (5 mL) was cooled down at −10 °C with an ice salt bath and stirred under nitrogen atmosphere. Isopropyl magnesium chloride, lithium chloride complex solution 1.3 M in THF (2.18 mmol) was added dropwise to the stirred solution over a period of 10 min. Upon completion, the reaction was quenched in a saturated solution of NH₄Cl, and the water phase was extracted with EtOAc (3 × 25 mL). The combined organic layers were dried over Na₂SO₄ and concentrated under reduced pressure. The crude mixture was purified by flash chromatography (see below the conditions).

2-Benzyloxy-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)­pyrazolo­[1,5-a]­pyridine (30a)

The mixture was purified by Combiflash using RediSep Gold Silica Gel disposable flash column, 24 g of silica (eluent: Heptane/EtOAc from 100 to 85/15 v/v) to affords the title compound as a white solid. This latter was immediately used in the next step. MS (ES⁺): 351 (M+1).

2-Methoxy-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)­pyrazolo­[1,5-a]­pyridine (30b)

The mixture was purified by Combiflash using RediSep Gold Silica Gel disposable flash column, 24 g of silica (eluent: Heptane/EtOAc from 100 to 80/20 v/v) to affords the title compound as a white solid. This latter was immediately used in the next step. MS (ES⁺): 275 (M+1).

2-(2-Methoxypyrazolo­[1,5-a]­pyridin-3-yl)-6-phenylquinoline (31)

[Pd­(PPh₃)₄] (5.4 mg, 0.0047 mmol) was added to a solution of 2-chloro-6-phenylquinoline (0.112 g, 0.47 mmol) and Cs₂CO₃ (0.459 g, 1.41 mmol,) in dioxane/water (8:2 v/v, 10 mL) solution. After stirring the resulting mixture under nitrogen atmosphere for 1 h, 2-methoxy-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)­pyrazolo­[1,5-a]­pyridine (0.130 g, 0.47 mmol) was added. The reaction mixture was refluxed for 2 h, then was cooled to room temperature and concentrated under reduced pressure. The crude material was taken-up with water (100 mL) and the mixture was extracted with EtOAc (3 × 25 mL). The combined organic layers were dried over Na₂SO₄ and concentrated under reduced pressure. The crude mixture was purified by Combiflash using RediSep Gold Silica Gel disposable flash column 24 g of silica (eluent:Heptane/EtOAc from 100 to 80/20 v/v)) to afford the title compound as a white solid (mp 182.6–183.3 °C, from trituration with diisopropyl ether). Yield 82%. ¹H NMR (600 MHz, Chloroform-d): δ 4.26 (s, 3H, −OCH ₃), 6.82 (t, 1H, J = 6.8 Hz, H-b), 7.35–7.44 (m, 2H), 7.52 (t, 2H, J = 7.6 Hz), 7.77 (d, 2H, J = 7.5 Hz), 7.95–7.97 (m, 2H), 8.15–8.16 (m, 2H), 8.21 (d, 1H, J = 8.7 Hz, H-d), 8.34 (d, 1H, J = 6.7 Hz, H-a), 8.94 (d, 1H, J = 8.9 Hz). ¹³C NMR (151 MHz, Chloroform-d): δ 56.8 (−OCH₃), 95.6 (C-f), 111.8 (C-b), 120.0, 120.6 (C-d), 125.3, 126.1 (C-c), 126.4, 127.42, 127.47, 128.49 (C-a), 128.8, 129.0, 129.2, 135.9, 137.6 (C-e), 140.9, 141.1, 147.9, 153.7, 164.4 (C-g). MS (ES⁺): 352 (M+1).

7-(2-Benzyloxy-pyrazolo­[1,5-a]­pyridin-3-yl)-3-chloroquinoline (32)

[Pd­(PPh₃)₄] (4.6 mg, 0.004 mmol) was added to a solution of 7-bromo-3-chloroquinoline (0.104 g, 0.43 mmol) and Cs₂CO₃ (0.420 g, 1.29 mmol,) in dioxane/water (8:2 v/v 10 mL) solution. After stirring the resulting mixture under a nitrogen atmosphere for 1 h, 2-benzyloxy-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)­pyrazolo­[1,5-a]­pyridine (0.150 g, 0.43 mmol) was added. The reaction mixture was warm at reflux for 2 h, then was cooled to room temperature and concentrated under reduced pressure. The crude material was taken-up with water (50 mL) and the mixture was extracted with EtOAc (3 × 10 mL). The combined organic layers were dried over Na₂SO₄ and concentrated under reduced pressure. The crude was purified by Combiflash using RediSep Gold Silica Gel disposable flash column, 13 g (eluent: petroleum ether/EtOAc from 100 to 80/20 v/v) to afford the title compound as a white solid (mp 160.9–161.5 °C, from trituration with diisopropyl ether). Yield 58%.¹H NMR (600 MHz, Chloroform-d): δ 5.55 (s, 2H, −CH ₂OAr), 6.72 (td, 1H, J = 6.8, 1.3 Hz, H-b), 7.21 (ddd, 1H, J = 9.0, 6.8, 1.1 Hz, H-c), 7.30–7.35 (m, 1H), 7.39 (t, 2H, J = 7.5 Hz), 7.52–7.55 (m, 2H), 7.76 (d, 1H, J = 8.5 Hz), 7.82 (dt, 1H, J = 9.0, 1.1 Hz, H-d), 8.06 (dd, 1H, J = 8.5, 1.7 Hz), 8.09 (d, 1H, J = 2.2 Hz), 8.32 (dt, 1H, J = 6.9, 1.1 Hz, H-a), 8.33–8.35 (m, 1H), 8.80 (d, 1H, J = 2.4 Hz). ¹³C NMR (151 MHz, Chloroform-d): δ 71.1 (−CH₂OAr), 94.9 (C-f), 110.8 (C-b), 116.1 (C-d), 125.7, 125.8, 126.5, 127.2, 127.6, 127.9, 128.1, 128.3, 128.7, 129.1, 133.8, 134.4, 137.1, 139.3, 146.9, 149.9, 162.6 (C-g). MS (ES⁺): 386 (M+1).

General Procedure for the Synthesis of Compounds 33 and 34

[Pd­(OAc)₂] (4.6 mg, 0.021 mmol) was added to a solution of 7-(2-benzyloxy-pyrazolo­[1,5-a]­pyridin-3-yl)-3-chloroquinoline (0.080 g, 0.21 mmol), X-PHOS (11 mg, 0.042 mmol) and K₃PO₄ (0.133 g, 0.63 mmol) in dioxane/water (8:2 v/v 5 mL) solution. After stirring the resulting mixture under a nitrogen atmosphere for 1 h, the correspondent boronic acid (0.42 mmol) was added and the reaction mixture was stirred at reflux until complete conversion of starting material was observed by TLC. Then, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The crude material was taken up with water (50 mL) and the aqueous layer was extracted with EtOAc (3 × 20 mL). Then the combined organic layers were dried over Na₂SO₄ and concentrated under reduced pressure. The crude material was purified by flash chromatography (see below the conditions).

7-(2-Benzyloxy-pyrazolo­[1,5-a]­pyridin-3-yl)-3-phenylquinoline (33)

The crude was purified by Combiflash using RediSep Gold Silica Gel disposable flash column, 13 g (eluent: petroleum ether/EtOAc from 100 to 80/20 v/v) to afford the title compound as a white solid. Yield 96%. ¹H NMR (600 MHz, Chloroform-d): δ 5.56 (s, 2H, CH ₂OAr), 6.72 (td, 1H, J = 6.9, 1.3 Hz, H-b), 7.21 (ddd, 1H, J = 9.0, 6.8, 1.1 Hz, H-c), 7.31–7.35 (m, 1H), 7.40 (t, 2H, J = 7.5), 7.42–7.45 (m, 1H), 7.52–7.56 (m, 4H), 7.74 (dd, 2H, J = 8.3, 1.2 Hz), 7.87 (dd, 1H, J = 9.0, 1.0 Hz), 7.90 (d, 1H, J = 8.8 Hz), 8.07 (dd, 1H, J = 8.5, 1.7 Hz), 8.28 (d, 1H, J = 2.1 Hz), 8.33 (dd, 1H, J = 6.9, 1.0 Hz, H-a), 8.39–8.40 (m, 1H), 9.17 (d, 1H, J = 2.3 Hz). ¹³C NMR (151 MHz, Chloroform-d): δ 71.1 (CH₂OAr), 95.2 (C-f), 110.7 (C-b), 116.3 (C-d), 125.5, 125.8, 126.2, 127.5, 127.7, 127.8, 128.07, 128.08, 128.2, 128.6, 129.0, 129.3, 133.0, 133.2, 134.1, 137.2, 138.2, 139.3 (C-e), 148.0, 150.2, 162.6 (C-g). MS (ES⁺): 428 (M+1).

7-(2-Benzyloxy-pyrazolo­[1,5-a]­pyridin-3-yl)-3-(2,6-difluorophenyl)­quinoline (34)

The crude mixture was purified by Combiflash using RediSep Gold Silica Gel disposable flash column, 12 g of silica (eluent: petroleum ether/EtOAc from 100 to 85/15 v/v) to affords the title compound as a pale-yellow solid. Yield 90%. ¹H NMR (600 MHz, Chloroform-d): δ 5.56 (s, 2H, −CH ₂OAr), 6.71 (td, 1H, J = 6.8, 1.3 Hz, H-b), 7.02–7.11 (m, 2H), 7.21 (ddd, 1H, J = 9.0, 6.8, 1.1 Hz, H-c), 7.30–7.37 (m, 2H), 7.40 (t, 2H, J = 7.5 Hz), 7.55 (d, 2H, J = 6.9 Hz), 7.84–7.89 (m, 2H), 8.08 (dd, 1H, J = 8.5, 1.7 Hz), 8.25 (s, 1H), 8.32 (dt, 1H, J = 6.9, 1.0 Hz, H-a), 8.40–8.42 (m, 1H), 8.98 (d, 1H, J = 1.5, 1.0 Hz). ¹³C NMR (151 MHz, Chloroform-d): δ 71.1 (−CH₂OAr), 95.2 (C-f), 110.8 (C-b), 112.03 (d, J = 21.1 Hz), 112.06 (d, J = 21.0 Hz), 115.5 (t, J = 18.8 Hz), 116.2, 121.9, 125.62, 125.69, 125.7, 127.6, 127.8, 128.1, 128.3, 128.6, 129.0, 129.8 (t, J = 10.2 Hz), 134.8, 137.1, 137.2, 139.3 (C-e), 148.0, 151.5, 160.4 (d, J = 249.3 Hz), 160.5 (d, J = 250.0 Hz), 162.6 (C-g).¹⁹F NMR (565 MHz, Chloroform-d) δ: −114.19. MS (ES⁺): 464 (M+1).

General Procedure for the Synthesis of Compounds 11a, 11b, 13 and 14

TFA (3.0 mL) was added to a 0 °C cooled suspension of the corresponding starting material (0.37 mmol) in thioanisole (436 μL, 3.70 mmol). The reaction mixture was stirred at room temperature, until a complete conversion of starting material was observed by TLC. The mixture was concentrated, and the resulting solid was purified following a different procedure (see details above).

(E)-3-(2-(2,3,5,6-Tetrafluoro-[1,1’-biphenyl]-4-yl)­vinyl)­pyrazolo­[1,5-a]­pyridin-2-ol (11a)

The result sticky solid was triturated with EtOH to afford the title compound as a yellow solid. Yield: quantitative. ¹H NMR (600 MHz, DMSO-d ₆): 6.85 (td, 1H, J = 6.9, 1.3 Hz, H-b), 7.03 (d, 1H, J = 16.6 Hz), 7.34 (ddd, 1H, J = 8.7, 6.9, 1.0 Hz, H-c), 7.47 7.55 (m, 1H), 7.55 (d, 2H, J = 5.7 Hz), 7.59 (d, 1H, J = 16.6 Hz), 7.74 (dt, 1H, J = 8.8, 1.2 Hz, H-d), 8.48 (dt, 1H, J = 6.9, 1.1 Hz, H-a), 11.71 (br, 1H, −OH). ¹³C NMR (151 MHz, DMSO-d ₆) δ 92.7, 107.2, 111.4, 115.0, 115.9 (t, J = 17.3 Hz), 117.3 (t, J = 13.7 Hz), 126.1, 126.9 (t, J = 8.68 Hz), 127.05, 128.7, 128.9, 129.2, 130.1, 138.8, 142.7, 144.3, 162.9 (C-g).¹⁹F NMR (565 MHz, DMSO-d ₆): δ −145.79 (d, J = 13 Hz), −145.08 (d, J = 12.5 Hz). MS (ES⁺): 385 (M + 1). ESI-HRMS (m/z): [M + H]⁺calcd. for C₂₁H₁₃F₄N₂O 385.0959; obsd. 385.0959.

E)-3-(2-(2,3,6-Trifluoro-[1,1’-biphenyl]-4-yl)­vinyl)­pyrazolo­[1,5-a]­pyridin-2-ol (11b)

The resulting oil crude was purified by flash chromatography (eluent: DCM/MeOH from 100 to 90/10 v/v) to afford a title compound as a yellow solid. Yield: 95%. ¹H NMR (600 MHz, DMF-d ₇): δ 6.86 (t, J = 6.7 Hz, 1H, H-b), 7.32–7.38 (m, 2H), 7.48–7.53 (m, 1H), 7.55–7.60 (m, 4H), 7.75–7.86 (m, 2H), 7.97 (d, J = 8.8 Hz, 1H, H-d), 8.48 (d, J = 6.7 Hz, 1H, H-a). ¹³C NMR (151 MHz, DMF-d ₇): δ 93.4 (C-f), 106.7 (d, J = 26.6 Hz), 111.4 (C-b), 112.5, 115.7 (C-d), 116.5 (dd, J = 21.7, 15.7 Hz), 123.6 (d, J = 2.06 Hz), 125.6, 125.8 (C-c), 128.5, 128.9, 128.96, 128.99, 129.1, 129.3, 130.6, 140.0, 155.3 (d, J = 255.51 Hz), 155.5 (d, J = 252.52 Hz), 163.6 (C-g). ¹⁹F NMR (565 MHz, DMF-d ₇): δ −149.88 (t, J = 17.5 Hz), −139.83 (d, J = 21.6 Hz), −120.76 (t, J = 12.5 Hz). MS (ES⁺): 367 (M + 1). ESI-HRMS (m/z): [M + H]⁺calcd for C₂₁H₁₄F₃N₂O 367.1053; obsd. 367.1053.

3-(3-Phenylquinolin-7-yl)­pyrazolo­[1,5-a]­pyridin-2-ol (13)

The crude material was purified by Combiflash using RediSep Gold Silica Gel disposable flash column, 4 g of silica (eluent: DCM/MeOH from 100 to 85/15 v/v) to achieve a crude compound. This latter was solubilized in MeOH and precipitated with water to afford the title compound as an orange solid (mp 281.1–283 °C, dec from water). Yield 75%.¹H NMR (600 MHz, DMSO-d ₆): δ 6.80 (t, 1H, J = 6.4 Hz, H-b), 7.29 (t, 1H, J = 7.8 Hz), 7.41 (t, 1H, J = 7.0 Hz), 7.52 (t, 2H, J = 7.3 Hz) 7.79–7.89 (m, 3H), 8.06 (m, 2H), 8.27 (s, 1H), 8.49 (d, 1H, J = 6.4 Hz, H-a), 8.56 (s, 1H), 9.19 (s, 1H), 11.39 (br, 1H, −OH). ¹³C NMR (600 MHz, DMSO-d ₆): δ 93.1 (C-f), 110.9 (C-b), 115.3 (C-d), 124.4, 125.4, 125.8, 126.7, 127.0, 128.0, 128.4, 129.0, 129.2, 131.8, 132.4, 134.2, 137.3, 138.1, 147.5, 149.6, 161.8 (C-g). MS (ES⁺): 338 (M+1). ESI-HRMS (m/z): [M + H]⁺calcd for C₂₂H₁₆N₃O 338.1288; obsd. 338.1287.

3-(3-(2,6-Difluorophenyl)­quinolin-7-yl)­pyrazolo­[1,5-a]­pyridin-2-ol (14)

The crude compound was purified using a Dowex 50W-X8 (200–400 mesh, capacity 1.7 mequiv/mL wet bed volume) ion-exchange resin, affording the title compound as an orange solid. The resin activation was performed according to the following method. The resin was washed with water (three volumes of resin), 10% w/w HCl (up to acidic pH), water (up to neutral pH), 10% w/w aqueous ammonia (up to basic pH), water (up to neutral pH), and then 10% w/w HCl until acidic pH was reached. The resin was then washed with water until the neutrality of the eluate, and then a solution of the TFA salt, dissolved in slightly acidic water to help solubility, was loaded on the top of the column. The column was eluted with water until neutral pH and then with 10% w/w aqueous ammonia solution to recover the desired compound in a neutral form (mp 295.0–296.6 °C, from water). Yield 20%. ¹H NMR (600 MHz, DMSO-d ₆): δ 6.85 (td, 1H, J = 6.8, 1.3 Hz, H-b), 7.27–7.39 (m, 3H), 7.53–7.61 (m, 1H), 7.89 (d, 1H, J = 8.09 Hz, H-d), 8.04–8.15 (m, 2H), 8.32 (s, 1H), 8.49 (s, 1H), 8.54 (d, 1H, J = 6.9 Hz, H-a), 8.92 (s, 1H), 11.44 (br, 1H, -OH). ¹³C NMR (151 MHz, DMSO-d ₆): δ 92.9 (C-f), 111.0, 112.27 (d, J = 21.0 Hz). 112.30 (d, J = 21.2 Hz), 114.7 (t, J = 18.9 Hz), 115.3, 121.0, 124.3, 124.8, 126.0, 126.7, 128.4, 129.0, 130.8, 135.1, 136.9, 138.1, 147.5, 151.1, 159.61 (d, J = 246.3 Hz), 159.65 (d, J = 246.4 Hz), 161.9 (C-g). ¹⁹F NMR (565 MHz, DMSO-d ₆): −114.79. MS (ES⁺): 374 (M+1). ESI-HRMS (m/z): [M + H]⁺calcd for C₂₂H₁₄F₂N₃O 374.1099; obsd. 374.1100.

3-(6-Phenylquinolin-2-yl)­pyrazolo­[1,5-a]­pyridin-2-ol (12)

2-(2-Methoxypyrazolo­[1,5-a]­pyridin-3-yl)-quinoline (0.01 mmol) was suspended in 1 mL of 48% w/w HBr aqueous solution. The suspension was warm at reflux until complete conversion of starting material was observed by TLC. The resulting solid was filtered off and washed several times with water. The crude material was crystallized with EtOH to afford a yellow solid (mp 230–231.6 °C, from EtOH). Yield 86%. ¹H NMR (600 MHz, DMSO): δ 7.10 (t, 1H, J = 6.8 Hz, H-b), 7.44 (t, 1H, J = 7.3 Hz, H-c), 7.54 (t, 2H, J = 7.6 Hz), 7.61 (t, 1H, J = 7.9 Hz), 7.84 (d, 2H, J = 7.9 Hz), 8.19 (m, 2H), 8.24 (d, 1H, J = 8.9 Hz), 8.37 (s, 1H), 8.40 (d, 1H, J = 8.8 Hz) 8.64–8.70 (m, 2H). ¹³C NMR (151 MHz, DMSO): δ 90.7 (C-f), 113.7 (C-b), 117.3, 120.2, 124.0, 125.5, 126.9, 128.1, 128.6, 129.2, 129.7, 130.8, 137.8, 138.7 (C-e), 139.3, 140.7, 150.5, 164.1 (C-g). MS (ES⁺): 338 (M+1). ESI-HRMS (m/z): [M + H]⁺ calcd for C₂₂H₁₆N₃O 338.1288; obsd. 338.1288.

Molecular Modeling Docking

The structure of hDHODH complexed with MEDS433 (PDB ID: 6FMD) was used to rationally design the new compounds and to perform in-silico simulations. Before docking studies, the protein was prepared by adding hydrogens (all the His residues were considered as protonated on their e₂ nitrogen according to the crystallographic data) and keeping the FMN and ORO cofactors. According to the experimental data, the compounds were simulated in the deprotonated form, bearing the negative charge on the oxygen atom, as this is the most populated tautomer at physiological pH. Compounds were submitted to docking with Glide. Poses were scored with the Chemscore function and ranked accordingly. In order to, validate the docking procedure, the self-docking of the ligand cocrystallized within hDHODH (PDB: 6FMD) was performed, obtaining a pose similar to the crystallographic one.

hDHODH Inhibition Assay

Following the procedure described in Sainas et al., 2018, Inhibitory activity was assessed by monitoring the reduction of 2,6-dichloroindophenol (DCIP), which is associated with the oxidation of dihydroorotate catalyzed by the hDHODH enzyme GST-tagged. The enzyme (around 60 nM) was preincubated for 5 min at 37 °C in Tris-buffer solution (pH 8.0) with coenzyme Q10 (100 μM), tested compounds at different concentrations (final DMSO concentration 0.1% v/v), and DCIP (50 μM). The reaction was initiated by addition of DHO (500 μM) and the reduction was monitored at λ = 650 nm. The initial rate was measured in the first 5 min (ε = 10400 M^–1cm^–1) and the IC₅₀ value was calculated starting from Vi/V0 (i = inhibitor; 0 = control) values using GraphPad Prism 7 software. Values are means ± SE of three independent experiments.

Cell-Based Assays Cell Lines

The AML human cell lines THP1 (acute monocytic leukemia), K562 (chronic myeloid leukemia), A549 (lung adenocarcinoma) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin (GIBCO, Invitrogen, Milan, Italy) were purchased. MRC-5 cell line from ATCC were cultured in EMEM medium (LonzaBioWhittaker) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin (GIBCO, Invitrogen, Milan). hDHODH inhibitors were solubilized in DMSO (Sigma-Aldrich, Milan, Italy) and final dilutions of the drugs were made in culture medium.

Annexin Assay

For the determination of EC₅₀, 1 × 10⁴ THP1 cells were plated in 96-well round-bottom plates and treated with increasing doses of hDHODH inhibitors from 0.001 μM to 10 μM. After 3 days of culture, the apoptotic assay was performed using the Annexin V-FITC Kit (Miltenyi Biotec, Italy), according to the manufacturer’s instructions. The apoptotic cells were acquired on FacsVerse and analyzed using Kaluza software version 2.1 (Beckman Coulter, Fullerton, CA).

Cell Viability Assay

For the determination of EC₅₀, 1 × 10⁴ K562 cells or 3 × 10³ A549 were plated in 96-well plates and treated with increasing doses of hDHODH inhibitors from 0.001 μM to 10 μM. After 3 days of culture, the percentage of live cells was determined using the CellTiter-Glo Luminescent assay (Promega, Milan, Italy), following the manufacturer’s instructions.

Cells and Viruses

The human ileocecal adenocarcinoma cell line HCT-8 (ATCC CCL-244) was purchased from the American Type Culture Collection (ATCC, USA) and cultured in RPMI (Euroclone, Pero (MI), Italy), supplemented with 10% (v/v) fetal bovine serum (FBS), 2 mM glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin, and 100 μg/mL streptomycin sulfate (Euroclone). Human coronavirus OC43 (hCoV-OC43, ATCC VR-1558) was purchased from ATCC, and propagated and titrated as previously described in HCT-8 cells. The AML human cell lines THP1 (acute monocytic leukemia), was cultured in complete RPMI 1640 (Invitrogen Life Technologies, Gaithersburg, MD), supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin (GIBCO, Invitrogen, Milan, Italy). hDHODH inhibitors were solubilized in DMSO (Sigma-Aldrich, Milan, Italy) and final dilutions of the drugs were made in culture medium.

Antiviral Assay

The antiviral activity of 11a and 14 against hCoV-OC43 was evaluated by means of the focus-forming reduction assay (FFRA) procedure as described by Sibille et al. Briefly, HCT-8 cells were seeded on 96-well plates (30,000 cells/well) and, after 24 h, treated with different concentrations of the 11a or 14, or with the vehicle (DMSO, as negative control) or with MEDS433 (as positive control) 1 h prior to and during infection with hCoV-OC43 (50 PFU/well). After virus adsorption (2 h at 33 °C), the infected cell cultures were incubated in medium containing the corresponding compounds plus 1% (w/v) methylcellulose (Merck) and 1% (v/v) FBS. At 72 h post infection (p.i.), cell monolayers were fixed and subjected to indirect immunoperoxidase staining (IPA) with a mAb against the hCoV-OC43 nucleoprotein (N) (Millipore, clone 542-D7; Burlington, MA, USA) (diluted 1:100). Viral foci were microscopically counted, and the mean plaque counts for each drug concentration was converted in viral titer (PFU/mL). GraphPad Prism software version 8.0 was used to determine the concentration of compounds producing 50 and 90% reductions in viral titers (EC₅₀ and EC₉₀).

Cytotoxicity Assay

HCT-8 cells (20,000 cells/well) or MRC-5 (6000 cells/well) cells were seeded in 96-well plates and, after 24 h, exposed to increasing concentrations of MEDS433, 11a, 14, or vehicle (DMSO) as a control. After 72 h of incubation, the number of viable cells was determined using the CellTiter-Glo Luminescent assay (Promega, Milan, Italy). The cytotoxicity of the tested compounds was expressed as cytotoxic concentration (CC₅₀).

Fluorescence Analysis and Live Cell Mitochondrial Imaging

For fluorescence analysis, 10 μM of target compounds were incubated with THP-1 for 24 h. After washing the cells with a PBS buffer solution, they were analyzed under confocal microscopy imaging technique using the appropriate laser to excite the molecule. For the colocalization experiments, HCT-8 cells (55,000 cells/well) were plated on μ-Slide 8 well high glass bottom (IBIDI, Gräfelfing, Germany) and treated for 24 h with 10 μM of 11a at 37 °C. Then, cells were stained with MitoTracker Red CMXRos (ThermoFischer, Carlsbad, CA, USA) (1:4000) for 30′ at 37 °C and after washing with PBS buffer solution, cells were visualized with a Leica TCS SP5 multiphoton-inverted confocal microscope equipped with LAS AF matrix software. Live cells were imaged using an HCX PL APO 63×/1.4 NA oil immersion objective. Images were collected with excitation of ligand 11a at λ_ex = 405 nm and emission at λ_em = 500 nm, Red-MitoTracker at λ_ex = 581 nm and emission at λ_em = 605 nm.

Co-Localization Analysis

THP-1 cells were incubated for 3 h with hDHODHi 11a and Bay240223410 at 10 μM, either alone or in combination. After incubation, the cells were washed and stained with Red-MitoTracker CMXRos (Thermo Fisher, Carlsbad, CA, USA) at a concentration of 100 nM for 30 min at 37 °C. Cells were imaged with a Leica TCS SP8 confocal system (Leica Microsystems) equipped with 4 excitation lasers (405 Diode, Argon, DPSS561, HeNe633). Images were acquired with a HCX PL APO 63×/1.4 NA oil-immersion objective with a resolution of 81 nm × 81 nm and were processed and analyzed with ImageJ software (Rasband, W.S., U.S. National Institutes of Health, Bethesda, MA). In particular, colocalization was analyzed with JACoP plugin and Manders’ coefficient was calculated

Statistical Analysis

Statistical analyses were performed on Prism software, version 8.0 (GraphPad Software, San Diego, CA). All data are reported as means ± SD. A Student’s t test was used as a significance test of different groups. A Wilcoxon test was performed for data not normally distributed. For colocalization analysis, a Welch’s t test was applied. For the determination of EC₅₀, a nonlinear regression model was applied. Moreover, a p-value < 0.05 was considered significant.

Photophysical Characterization

UV–Vis absorption spectra were recorded on a Cary60 spectrometer. Photoemission spectra, luminescence lifetimes, and quantum yields were acquired with a HORIBA Jobin Yvon IBH Fluorolog-TCSPC spectrofluorometer equipped with a Quanta-φ integrating sphere. Luminescence lifetimes were determined by time-correlated single-photon counting; excitation was achieved with nanosecond pulses of light generated by NanoLED pulsed diodes (operating wavelength 295, 370, or 450 nm). Emission-decay data were collected in 2048 channels to 10000 counts in the peak channel. IRF was measures using colloidal silica suspension in H₂O to scatter light. Data were analyzed with the software DAS6 (TCSPC decay-analysis software) using a nonlinear least-squares method to fit of the decay to a sum of exponentials. The value of χ², residuals and the autocorrelation function (Durbin-Watson parameter) were used to determine the quality of the fit. For measurements in the presence of hDHODH, 11a was incubated, at 1 μM concentration in TRIS Buffer 0.1 mM (pH 8) with 0.1% DMSO, alone or with different concentrations of recombinant protein ranging from 31.5 to 504 nM concentration. After 5 min incubation UV–Vis absorption spectra were recorded on a Cary60 spectrophotometer and photoemission spectra were acquired with a HORIBA Jobin Yvon IBH Fluorolog-TCSPC spectrofluorometer with excitation λ of 366 nm and maximum of emission at 500 nm; a.u. data were analyzed with GraphPad Prism using One Site – Specific Binding equation.

Glossary

Abbreviations

AML

acute myeloid leukemia

AIE

aggregation-induced emission effect

BIFL

bioactive intrinsically fluorescent ligands

CB2R

cannabinoid receptor subtype 2

CFSE

carboxyfluorescein diacetate succinimidyl ester

DCIP

dichloroindophenol

DHO

dihydroorotate

EDG

electron-donating group

ETC

electron transport chain

EWGs

electron-withdrawing groups

FMN

flavin mononucleotide

hDHODH

human dihydroorotate dehydrogenase

LiHMDS

lithium hexamethyldisilazane

NBD

nitrobenzoxadiazole

ORO

orotate

QY

quantum yield

SFR

Structure Fluorescence Relationship

SAR

Structure Activity Relationship

TFA

trifluoroacetic acid.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.5c00348.

• Synthesis of intermediates, ¹H NMR, ¹³C NMR, and UHPLC (PDF)

• The docking poses (PDB)

• Molecular formula strings and biological data (CSV)

◆.

S.S. and E.M. contributed equally to this work.

The authors declare no competing financial interest.