Electrophilically Activated Nitroalkanes in Double Annulation of [1,2,4]Triazolo[4,3-a]quinolines and 1,3,4-Oxadiazole Rings

Nitroalkanes activated with polyphosphoric acid could serve as efficient electrophiles in reactions with amines and hydrazines, enabling various cascade transformations toward heterocyclic systems. This strategy was developed for an innovative synthetic protocol employing simultaneous or sequential annulation of two different heterocyclic cores, affording [1,2,4]triazolo[4,3-a]quinolines with 1,3,4-oxadiazole substituents.


Introduction
There is significant emphasis on the role of [1,2,4]triazolo [4,3-a]quinolines in modern-day drug discovery and medicinal chemistry. This privileged scaffold was utilized in the design of potent and selective aldosterone synthase inhibitors with antihypertensive activity [1], as well as prospective antitumor [2,3], anticonvulsant [4][5][6][7][8][9][10], anti-inflammatory [11,12], and antimicrobial [13,14] agents. It was also found that an introduction of a heterocyclic substituent at C-5 could allow for the preparation of chimeric scaffolds with improved biological activities. This strategy was employed in the development of cytotoxic and antiviral agents [15], as well as analgesic and antiinflammatory drug candidates [16]. Recently, we described the preparation of novel antitumor agents with in vitro differentiation activity against neuroblastoma cancer cell lines [2]. These compounds were assembled via an unusual annulation reaction between electrophilically activated nitroalkanes [17] and 2-hydrazinylquinolines. We also reported on the cyclocondensation of nitroalkanes with acylhydrazides furnishing 1,3,4-oxadiazole rings [18,19]. With continuous SAR studies, we had a task of building a focused library of perspective "chimeric" antitumor drug candidates possessing both [1,2,4]triazolo [4,3-a]quinoline and 1,3,4-oxadiazole rings. An expeditious and concise synthetic method was needed to allow for a highly efficient installation of both heterocyclic cores in a single-pot fashion. In this report, we disclosed the results of these synthetic studies.

Results and Discussion
As we previously reported, a highly electrophilic phosphorylated nitronate species A is generated upon the interactions between nitroalkanes 1 and polyphosphoric acid (PPA) (Scheme 1). These entities readily react with amines B to afford amidinium intermediates C, which can be used for the highly efficient assembly of imidazoles or oxazoles (D) [20,21], as well as imidazolines [22] and other nitrogen-based heterocycles [23,24]. Fur-Furthermore, the reaction with 2-hydrazinylpyridines H enacts formation of th sponding (hydrazineyl)alkaniminium species I, which was found to undergo int ular cyclocondensations to obtain 1,2,4-triazolo [4,3-a]pyridines (J) (Scheme 1) [ larly, a mechanistically related reaction with acylhydrazides E proved useful for tion toward 1,3,4-oxadiazoles G. (Scheme 1) [18,19]. Remarkably, both latter r could be carried out under similar reaction conditions. This prompted us to pu idea of performing these reactions in a one-pot fashion en route to chimeric het structures 3 (Scheme 1). To test the possibility of the simultaneous installation of t heterocyclic moieties, we carried out the reaction of 2-hydrazineylisonicotinohy (2a) in PPA in the presence of excess 1-nitropropane (1a, 4 equiv. used to compen loss due to evaporation). Initial tests were performed in 80% PPA (which corresp H4P2O7 composition). The reaction was carried out at 130 °C for 30 min, when it slowed down, most likely due to the loss of relatively volatile nitroalkane (bp Two more equivalents of 1a was added, and the reaction was stirred for an addi min to afford the desired 2-ethyl-5-(3-ethyl- [1,2,4]triazolo [4,3-a]pyridin-7-yl)-1,3 azole (3aa) with a 71% yield ( Table 1, entry 1). To evaluate the influence of the m we also performed the same reaction in 87% PPA, which corresponds to polymer with lowered acidity, but enhanced anhydride activity. These parameters were m aiming for an improved reaction performance or lowering of the reaction temp Indeed, even at 120 °C the yield of product 3aa was notably higher (entry 2), and it 84% at 130 °C (entry 3). We also tested for the intermediate value of 85% P2O5 co the reaction medium, which was found to be optimal for the featured process. increase in reaction temperature from 110-120 °C and then to 130 °C, the yield o proved from 52-79% and then to 90%, respectively (entries 4-6). A further increas perature to 140 °C, however, was found to be detrimental, as it caused partial d sition of the product (entry 7). Scheme 1. Electrophilic activation of nitroalkanes in synthesis of heterocycles. With optimized reaction conditions in hand, we proceeded with the scope and limitation studies, the results of which are shown in Scheme 2. Nitromethane (1c) reacted smoothly, but the yields were somewhat lower due to partial loss of this reagent through its high volatility. The addition of nitromethane (up to 7 equivalents) to compensate for evaporation allowed for the isolation of polyheterocyclic products 3ac and 3dc in moderate to high yields (Scheme 2). Reactions involving three homologous nitroalkanes with higher boiling points, nitroethane (1g), 1-nitropropane (1a), and 1-nitrooctane (1b), proceeded much more efficiently, generally providing notably higher yields (Scheme 2). Furthermore, we managed to carry out the reaction of ethyl 2-nitroacetate, efficiently generating a bis-annulation product 3ae with two newly introduced ester functionalities (Scheme 2). Moreover, p-tolyl(2-nitroethane) (1h) reacted smoothly, affording compound 3ch in excellent yield (Scheme 2). A putative mechanistic rationale of the featured transformation is depicted in Scheme 3. The reaction begins with two nucleophilic attacks by both hydrazine and hydrazide groups of the substrate at two of the phosphorylated nitronate species A. After subsequent elimination of the two molecules of ortho-phosphoric acid, the resulting species 4 underwent a double-fold 5-endo-trig nucleophilic cyclization employing both N-(1-hydrazineylalkylidene)-O-phosphonohydroxylammonium moieties. Next, tautomeric form 5 would undergo re-protonation to produce heterocyclic intermediate 6. The latter experiences an elimination of two equivalents of hydroxylamine O-phosphate to afford the aromatic final product 3 (Scheme 3). With optimized reaction conditions in hand, we proceeded with the scope and limitation studies, the results of which are shown in Scheme 2. Nitromethane (1c) reacted smoothly, but the yields were somewhat lower due to partial loss of this reagent through its high volatility. The addition of nitromethane (up to 7 equivalents) to compensate for evaporation allowed for the isolation of polyheterocyclic products 3ac and 3dc in moderate to high yields (Scheme 2). Reactions involving three homologous nitroalkanes with higher boiling points, nitroethane (1g), 1-nitropropane (1a), and 1-nitrooctane (1b), proceeded much more efficiently, generally providing notably higher yields (Scheme 2). Furthermore, we managed to carry out the reaction of ethyl 2-nitroacetate, efficiently generating a bis-annulation product 3ae with two newly introduced ester functionalities (Scheme 2). Moreover, p-tolyl(2-nitroethane) (1h) reacted smoothly, affording compound 3ch in excellent yield (Scheme 2). A putative mechanistic rationale of the featured transformation is depicted in Scheme 3. The reaction begins with two nucleophilic attacks by both hydrazine and hydrazide groups of the substrate at two of the phosphorylated nitronate species A. After subsequent elimination of the two molecules of ortho-phosphoric acid, the resulting species 4 underwent a double-fold 5-endo-trig nucleophilic cyclization employing both N-(1-hydrazineylalkylidene)-O-phosphonohydroxylammonium moieties. Next, tautomeric form 5 would undergo re-protonation to produce heterocyclic intermediate 6. The latter experiences an elimination of two equivalents of hydroxylamine O-phosphate to afford the aromatic final product 3 (Scheme 3).
It should be pointed out that reactions involving α-nitrotoluene as a pro-electrophile proceeded sluggishly affording the corresponding bis-phenylsubstituted products (such as 3ad) with disappointingly marginal yields (10% or below). This limitation, however, can be addressed by substituting α-nitrotoluene with α-nitroacetophenones (1d or 1f, 3.0 equiv.). This tactical trick was first presented in our original report on the preparation of benzimidazoles and benzoxazoles in 2015 [20]. An updated version of this mechanistic rationale adapted for the reaction with bifunctional substrate 2a is shown in Scheme 4. It is assumed that the initial acid-assisted double-fold interaction of 2a with α-nitroacetophenone (1d) leads to the formation of bis-1-(2-nitroethylidene)hydrazin-1-ium as an intermediate 7 (Scheme 3). The electron pairs for the heteroatoms of ketone and pyridine form an intramolecular attack to afford two new five-membered rings as an intermediate 8. The latter experiences a double-fold nucleofugal cleavage of nitromethane to produce molecule 3ad (Scheme 4). Compounds 3cd, 3dd, and 3cf were formed in a similar manner with high yields (Scheme 2). It should be pointed out that reactions involving α-nitrotoluene as a pro-ele proceeded sluggishly affording the corresponding bis-phenylsubstituted produ as 3ad) with disappointingly marginal yields (10% or below). This limitation, h can be addressed by substituting α-nitrotoluene with α-nitroacetophenones (1d equiv.). This tactical trick was first presented in our original report on the prepa benzimidazoles and benzoxazoles in 2015 [20]. An updated version of this me rationale adapted for the reaction with bifunctional substrate 2a is shown in Sch is assumed that the initial acid-assisted double-fold interaction of 2a with α-ni phenone (1d) leads to the formation of bis-1-(2-nitroethylidene)hydrazin-1-ium termediate 7 (Scheme 3). The electron pairs for the heteroatoms of ketone and form an intramolecular attack to afford two new five-membered rings as an inte 8. The latter experiences a double-fold nucleofugal cleavage of nitromethane to molecule 3ad (Scheme 4). Compounds 3cd, 3dd, and 3cf were formed in a similar with high yields (Scheme 2).
In both discussed mechanistic rationales (Schemes 3 and 4), the initial nuc attack of the hydrazine groups and subsequent annulations are occurring at two sites. This would most likely take place independently with different kinetic rate the sake of a concise depiction they are shown here to proceed in parallel.  It should be pointed out that reactions involving α-nitrotoluene as a pro-ele proceeded sluggishly affording the corresponding bis-phenylsubstituted produ as 3ad) with disappointingly marginal yields (10% or below). This limitation, can be addressed by substituting α-nitrotoluene with α-nitroacetophenones (1d equiv.). This tactical trick was first presented in our original report on the prepa benzimidazoles and benzoxazoles in 2015 [20]. An updated version of this me rationale adapted for the reaction with bifunctional substrate 2a is shown in Sch is assumed that the initial acid-assisted double-fold interaction of 2a with α-n phenone (1d) leads to the formation of bis-1-(2-nitroethylidene)hydrazin-1-ium termediate 7 (Scheme 3). The electron pairs for the heteroatoms of ketone and form an intramolecular attack to afford two new five-membered rings as an inte 8. The latter experiences a double-fold nucleofugal cleavage of nitromethane to molecule 3ad (Scheme 4). Compounds 3cd, 3dd, and 3cf were formed in a simila with high yields (Scheme 2).
In both discussed mechanistic rationales (Schemes 3 and 4), the initial nuc attack of the hydrazine groups and subsequent annulations are occurring at two sites. This would most likely take place independently with different kinetic rate the sake of a concise depiction they are shown here to proceed in parallel. In both discussed mechanistic rationales (Schemes 3 and 4), the initial nucleophilic attack of the hydrazine groups and subsequent annulations are occurring at two different sites. This would most likely take place independently with different kinetic rates, but for the sake of a concise depiction they are shown here to proceed in parallel.
We also decided to take advantage of the reactivity of the electrophilically activated ethyl nitroacetate (1e) to design a sequential approach for [1,2,4]triazolo[4,3a]quinolines bearing 1,3,4-oxadiazole substituent at C-3 (shown in Scheme 5). To this end, 2-hydrazinylquinolines 9 were first utilized in the featured annulation to obtain triazoles 10 bearing an ester function at C-3. These compounds were taken without pu-rification and subjected to the hydrazinolysis reaction to provide heterocyclic products 11. The latter crude acylhydrazide moieties were transformed into 1,3,4-oxadiazole rings this time employing 1-nitropropane (1a) as an electrophilic component. Two compounds, 12a and 12b, were successfully synthesized via this approach with moderate yields (43% and 47%, respectively) (Scheme 5). In principle, the same strategy can potentially be employed for stepwise assembly of longer linear oligomeric chains with repeating 1,3,4-oxadiazole units, provided that greater annulation efficiency could be achieved at every step.
ethyl nitroacetate (1e) to design a sequential approach for [1,2,4]triazolo[4,3-a]quinoline bearing 1,3,4-oxadiazole substituent at C-3 (shown in Scheme 5). To this end, 2-hydrazi nylquinolines 9 were first utilized in the featured annulation to obtain triazoles 10 bearing an ester function at C-3. These compounds were taken without purification and subjected to the hydrazinolysis reaction to provide heterocyclic products 11. The latter crude acylhy drazide moieties were transformed into 1,3,4-oxadiazole rings this time employing 1-ni tropropane (1a) as an electrophilic component. Two compounds, 12a and 12b, were suc cessfully synthesized via this approach with moderate yields (43% and 47%, respectively (Scheme 5). In principle, the same strategy can potentially be employed for stepwise as sembly of longer linear oligomeric chains with repeating 1,3,4-oxadiazole units, provided that greater annulation efficiency could be achieved at every step.
Formation of triazole and oxadiazole cycles in both parallel and sequential modes o the featured annulation was unambiguously confirmed by single-crystal X-ray diffraction of compounds 3ba and 12a, respectively ( Figure 1).

Scheme 5.
Stepwise installation of [1,2,4] Formation of triazole and oxadiazole cycles in both parallel and sequential modes of the featured annulation was unambiguously confirmed by single-crystal X-ray diffraction of compounds 3ba and 12a, respectively (Figure 1). We also decided to take advantage of the reactivity of the electrophilically activated ethyl nitroacetate (1e) to design a sequential approach for [1,2,4]triazolo[4,3-a]quinolines bearing 1,3,4-oxadiazole substituent at C-3 (shown in Scheme 5). To this end, 2-hydrazinylquinolines 9 were first utilized in the featured annulation to obtain triazoles 10 bearing an ester function at C-3. These compounds were taken without purification and subjected to the hydrazinolysis reaction to provide heterocyclic products 11. The latter crude acylhydrazide moieties were transformed into 1,3,4-oxadiazole rings this time employing 1-nitropropane (1a) as an electrophilic component. Two compounds, 12a and 12b, were successfully synthesized via this approach with moderate yields (43% and 47%, respectively) (Scheme 5). In principle, the same strategy can potentially be employed for stepwise assembly of longer linear oligomeric chains with repeating 1,3,4-oxadiazole units, provided that greater annulation efficiency could be achieved at every step.
Formation of triazole and oxadiazole cycles in both parallel and sequential modes of the featured annulation was unambiguously confirmed by single-crystal X-ray diffraction of compounds 3ba and 12a, respectively (Figure 1).

Materials and Methods General
NMR spectra, 1 H and 13 C, were measured in solutions of CDCl 3 or DMSO-d 6 on Bruker AVANCE-III HD instrument (at 400.40 or 100.61 MHz, respectively. Bruker, Billerica, MA, USA). Residual solvent signals were used as internal standards, in DMSO-d 6 (2.50 ppm for 1 H, and 40.45 ppm for 13 C nuclei) or in CDCl 3 (7.26 ppm for 1 H, and 77.16 ppm for 13 C nuclei). HRMS spectra were measured on a Bruker maXis impact (electrospray ionization, in MeCN solutions, employing HCO 2 Na-HCO 2 H for calibration). IR spectra were measured on an FT-IR spectrometer Shimadzu IRAffinity-1S equipped with an ATR sampling module. Reaction progress, purity of isolated compounds, and R f values were monitored with TLC on Silufol UV-254 plates. Column chromatography was performed on silica gel (32-63 µm, 60 Å pore size). Melting points were measured with Stuart SMP30 apparatus. Polyphosphoric acid samples were prepared by dissolving precisely measured amounts of P 2 O 5 in 85% ortho-phosphoric acid. Ethyl 2-chloroquinoline-4-carboxylate, ethyl 6-bromo-2-chloroquinoline-4-carboxylate [25], ethyl 2-chloroisonicotinate [26], and ethyl 6-chloronicotinate [27] were synthesized according to the published methods. All other reagents and solvents were purchased from commercial venders and used as received.
2-Hydrazinylpyridine-4-carbohydrazide (2a). Ethyl 2-chloroisonicotinate (740 mg, 4.00 mmol), hydrazine hydrate (88% solution in water, 2.3 mL, 40.0 mmol), and ethanol (0.7 mL) were combined in a 30 mL G30 vial and covered with a septum. The vial was placed in a Monowave 300 microwave reactor, and the mixture was heated to 160 • C over the course of 5 min (power did not exceed 135 watts), after which this temperature was maintained for 1.5 h (controlled by IR sensor, MW power within 10 watts, 10-15 bar pressure). The resulting mixture was poured into water (40 mL) and filtrated. The resulting precipitate was washed with water several times (2 × 30 mL). It was recrystallized from ethanol to afford 2a as a pale-yellow solid, m.p. 171-172 • C (ethanol); yield 601 mg
Method B with α-nitroacetophenone. A 10 mL Erlenmeyer flask equipped with a magnetic stirrer and a reflux condenser was charged with 2-hydrazinilisonicotinohydrazide (1.00 equiv.) 2, polyphosphoric acid (85% P 2 O 5 , 2 g), and α-nitroacetophenone 1d (3.00 equiv.). The flask was placed in an oil bath and heated to 130 • C while being stirred. The mixture was heated for 1.5-2 h; when TLC analysis showed the reaction was completed, the reaction mixture was cooled. Water was added (5 mL), neutralized with 25% aqueous ammonia solution (4 mL) to pH = 8-9 and extracted with EtOAc (4 × 5 mL). The combined organic phases were concentrated and the crude product was purified by preparative column chromatography eluting with acetone and hexane.
Method C with ethyl nitroacetate. In a 10 mL Erlenmeyer flask equipped with a magnetic stirrer and a reflux condenser, 2-hydrazinilisonicotinohydrazide (1.00 equiv.) 2, PPA 85% (1 g), H 3 PO 3 (1 g), and ethyl nitroacetate 1e (3.0 equiv.) were loaded. The flask was placed in an oil bath and heated to 130 • C while being stirred for 1 h. Then, another 2 equiv. of nitroacetic ether 1e was added and heated for another hour; when TLC analysis showed the reaction was completed, the reaction mixture was cooled. Water was added (5 mL), before neutralizing with 25% aqueous ammonia solution (4 mL) to pH = 8-9 and extracting with EtOAc (4 × 5 mL). The combined organic phases were concentrated, and the crude product was purified by preparative column chromatography eluting with acetone and hexane.

Conclusions
In conclusion, an efficient protocol for simultaneous parallel cyclization of [1,2,4] triazolo[4,3-a]quinoline and 1,3,4-oxadiazole heterocyclic rings was developed. The featured transformation involved an unprecedented double-fold nucleophilic attack by two different hydrazine moieties of 2-hydrazineylpyridinecarbohydrazide substrates on nitroalkanes electrophilically activated in a polyphosphoric acid medium. The method provides an expeditious and direct access to [1,2,4]triazolo[4,3-a]quinolines bearing a 1,3,4oxadiazole substituent, which might be of interest for medicinal chemistry. Furthermore, this research paves the road for development of other acid-mediated cascade transformations for preparation of complex heterocyclic compounds. Synthesis of a focused library for biological studies is currently underway in our laboratories.
Supplementary Materials: Supporting Information data include NMR and HRMS spectral charts and X-ray crystallography data.