[3+2]-Annulation of pyridinium ylides with 1-chloro-2-nitrostyrenes unveils a tubulin polymerization inhibitor

Abstract

Indolizines and pyrazolo[1,5-a]pyridines were prepared via 3+2]-cycloaddition of pyridinium ylides to 1-chloro-2-nitrostyrenes. The synthesized molecules were evaluated for antiproliferative activities against a BE(2)-C neuroblastoma cell line with several compounds decreasing viability of cancer cells. Indolizine 9db showed a higher potency than that of all trans-retinoic acid, an approved cancer drug. Mechanistically, it was found to inhibit tubulin polymerization and it is thus proposed that the discovered chemistry can be exploited for the development of novel microtubule-targeting anticancer agents.

Graphical Abstract

Heterocycles prepared via [3+2]-cycloaddition of pyridinium ylides to 1-chloro-2-nitrostyrenes, were evaluated as microtubule-targeting anticancer agents potent against BE(2)-C neuroblastoma cells.

Introduction

It is hard to overstate importance of indolizine and pyrazolo[1,5-a]pyridine scaffolds for modern organic and medicinal chemistry.^1–7 These molecules are isosteric to indoles and display a wide array of biological activities:^8–10 anti-microbial, anti-cancer, anti-inflammatory, anti-oxidant, and anti-histaminic. Probably, most famous medicinal agents possessing indolizine core are Topotecan, Irinotecan, and Camtobell, which are all synthetic analogs of natural alkaloid Camptothecin, isolated from Chinese “tree of life” (Camptotheca acuminata) and demonstrating remarkable anti-cancer^11–14 and anti-HIV^{15, 16} activity. Family of Lamellarine alkaloids isolated from mollusks of Lamellaria genus and other marine species, also demonstrated potent anti-cancer activities.^17–21 In material science and chemical biology indolizine-based molecules are used to create highly sensitive and tunable fluorescent probes, such as Seoul-Fluor.²² Fantofarone is an indolizine-based calcium channel blocker, used in treatment of vasospasms.^23–26 Also, indolizines are commonly utilized as synthetic platforms for preparation of several important classes of natural indolizidine alkaloids.^27–29 Expectedly, colossal efforts by many research groups were placed for elaboration of synthetic approaches to these heterocyclic moieties, and multiple efficient synthetic protocols were successfully developed, documented and reviewed.^{10, 30–33} Herein we wish to report a full account of our synthetic exercises towards indolizine and pyrazolo[1,5-a]pyridine scaffolds employing [3+2] dipolar cycloaddition of pyridinium ylides to 2-chloro-1-nitroalkenes and evaluation of anti-cancer activity of the synthesized molecules.

Results and discussion

Perhaps, one of the most versatile and straightforward synthetic methods allowing for expeditious and facile assembly of both indolizine and pyrazolo[1,5-a]pyridine bicyclic systems involves [3+2]-cycloaddition of pyridinium ylides to alkynes or alkenes. This approach is outlined in Schemes 1 and 2. Initial treatment of quaternary pyridinium salts 1 allows for generation of dipole 2, typically stabilized by the presence of electron-withdrawing substituent Z¹. This species reacts with olefin 3 with Michael acceptor character, possessing at least one electron-withdrawing substituent Z² (Scheme 1). Resulting tetrahydroindolizine adduct 4 could be further aromatized via oxidation to afford indolizine 6. Alternatively, product 5 could be formed after elimination of HZ¹, HZ² entities, if groups both Z¹ and Z² groups are sufficiently nucleofugal. Furthermore, disubstituted olefin 7 could be employed as dipolarophile, in which Z² is an electron-withdrawing group, and Z³ is electron-donating or electron-neutral, but both are good nucleofuges. This reaction would provide trisubstituted tetrahydroindolizine intermediate 8, which can be aromatized after elimination of HZ² and HZ³ moieties to afford product 9 retaining substituent Z¹ (Scheme 1). However, this process can compete with aerobic oxidation affording more substituted products, for example 6 (Scheme 1).

Scheme 1.

General methods for synthetic preparation of indolizines employing [3+2]-cycloaddition of pyridinium ylides.

Scheme 2.

General methods for synthetic preparation of pyrazolo[1,5-a]pyridines employing [3+2]-cycloaddition of pyridinium ylides.

In a quite similar way, zwitterionic pyridin-1-ium-1-ylamide 11 can be generated via deprotonation of 1-aminopyridin-1-ium 10 (Scheme 2). Subsequent dipolar cycloaddition of alkene 7 would provide intermediate species 12, which can be aromatized either via elimination of HZ² and HZ³ moieties to afford 13 or via an alternative pathway, involving a concurrent oxidation and leading to formation of more substituted products, for example 14 (Scheme 2).

Since our research group is greatly instrumental in development of synthetic chemistry of aliphatic nitro-compounds,^34–38 we decided to utilize alkenes substituted with nitro-group (Z² = NO₂) as reactants. This approach is not entirely original; the reported results, however, are quite scattered. The employment of “regular” nitroalkenes is quite well documented in conjunction with oxidizing agents (Cu(OAc)₂, TEMPO, or MnO₂). These reactions are accompanied with elimination of nitrous acid and lead to the formation of indolizine products 5 (Scheme 1) without incorporation of nitro-groups.^39–44 Second group of reactions employ 1-nitro-2-(phenylthio)ethylene (Z³ = PhS) or 1,1-bis(methylthio)-2-nitroethylene, offering a second (and third) nucleofugal moiety for elimination. This typically does not require any oxidants and also results in formation of indolizines 9 that lack nitro-substitution (Scheme 1), although incorporation of alkylthio-moiety was well documented.^45–48 Finally, the use of halogenated nitroalkanes was also reported. Nenaidenko recently demonstrated preparation of fluorinated indolizines and pyrazolo[1,5-a]pyridines employing 1-fluoro-1-nitroalkenes.^{43, 49} Reactions of 1,1-diiodo-2,2-dinitroethylene to assemble 2-iodo-1-nitroindoizines (hence, elimination of one HI and one HONO moieties occurs) were also reported.^{50, 51} However, to the best of our knowledge there was no communications on successful utilization of 2-halo-1-nitroalkenes in such reaction. In the past, this could be well excused by poor synthetic availability of such nitroolefins. However, they have become routinely accessible since after 2016, when Xu reported very concise and convenient method for copper-mediated chloronitration of acetylenes.⁵² This breakthrough prompted us to assess the classical approach towards indolizines and pyrazolo[1,5-a]pyridines employng 1-chloro-2-nitrostyrenes as new affordable feedstock.

First, we examined the reaction between 1-phenacylpyridinium bromide (1a) and 2-(1-chloro-2-nitrovinyl)naphthalene (7a) in different solvents (Table 1). Initial tests were performed in ethanol in the presence of triethylamine as a base (1 equiv.). This choice of solvent and base was made since both pyridinium salt 1a and thirethylammonium salts forming in the reaction are well soluble in ethanol, so the process could be carried out in homogenous solution. The reaction, however, proceeded quite sluggishly, and by the time the starting material was fully consumed (1.5 hr at room temperature), indolizine product 9aa was formed in very marginal yield (Table 1, entry 1). It was rationalized, that the stoichiometric amount of base was not sufficient, since both eliminated species HCl and HONO would consume an equivalent of base for neutralization. Indeed, an attempt to increase the concentration of the base had a positive effect (entries 2, 3). However, even in the presence of 3 equiv. of NEt₃, the yield remained quite marginal (entry 3), with the rest of the material 1a converted to pyridine (15) and 2-ethoxy-1-phenylethan-1-one (16) via S_N2-type solvolytic cleavage. Reaction in acetic acid in the presence of sodium acetate afforded similar yields, probably, also due to partial solvolysis of 1a (entry 4). To address this issue, we attempted to employ acetonitrile as a polar aprotic solvent. Three different bases were tested (NEt₃, DBU, and DABCO), but the yields of the target indolizine 9aa remained marginal (entries 5–7). Evidently, base strength is much less important in formation of indolizine 9aa, since requisite deprotonation of extremely acidic C-H bond in 1a already proceeds quite easily. Theoretically, the poor results obtained in the polar aprotic solvent could be explained by an adverse process of formal [3+3] homo-dimerization of intermediate dipole 2, generated in high concentrations. Such course of reaction was previously documented in the literature,⁵³ although in our experiments we failed to detect formation of any dimeric species. These considerations brought us to conclusion, that performing the reaction in less polar aprotic solvents could actually be beneficial. Indeed, poor solubility of 1a in the medium would result in slow generation of zwitterionic dipole 2, which would lower its effective concentration. Therefore, it would preferentially react with excess of well-soluble nitroalkene affording target indolizine product. Indeed, 9aa was obtained in excellent yield when reaction was performed in dichloromethane (entry 8).

Table 1.

Optimization of reaction conditions for reaction towards indilizine 9aa

#	Solvent	Base	Yielda
1	EtOH	NEt3 (1 equiv.)	16%
2	EtOH	NEt3 (2 equiv.)	32%
3	EtOH	NEt3 (3 equiv.)	43%
4	AcOH	AcONa (3 equiv.)	52%
5	MeCN	NEt3 (3 equiv.)	49%
6	MeCN	DBU (3 equiv.)	48%
7	MeCN	DABCO (3 equiv.)	50%
8	СH2Cl2	NEt3 (3 equiv.)	88%b

^aAll the reaction were performed in 0.15 mmol scale upon stirring for 1.5 hr at room temperature. NMR yields are reported unless specified otherwise.

^bThis reaction was performed in 0.50 mmol scale and isolated yield of purified product is provided.

We also opted to test formation of pyrazolo[1,5-a]pyridines in a similar interaction base-assisted between 2-halo-1-nitroalkenes and N-aminopyridinium salts. Since both optimizations described in Tables 1 and 2 were executed side-by-side, they involve identical initial steps. Thus, reaction of N-aminopyridinium iodide (10a) with (1-chloro-2-nitrovinyl)benzene (7b) in ethanol in the presence of 1 equiv. of triethylamine also provided quite marginal yield (Table 2, entry 1), which also improved insignificantly with increased concentration of the base (entries 2–3). Remarkably, in this case reaction was accompanied by elimination of HCl species only, and aromatization proceeded via aerobic oxidation, leading to formation of product 14ab featuring nitro-substituent at C-3. Migration into polar aprotic solvent medium proved much more beneficial for the reaction performance, especially in the presence of stronger bases (entries 4–7). Reaction in acetonitrile in the presence of DBU afforded nearly quantitative yield of 14ab. Interestingly, in less polar solvent, such as CH₂Cl₂ yields were quite lower (entry 8). Most likely, poor solubility of the starting material in this case plays a detrimental role.

Table 2.

Optimization of reaction conditions for reaction towards pyrazolo[1,5-a]pyridine 14ab

#	Solvent	Base	Yielda
1	EtOH	NEt3 (1 equiv.)	27%
2	EtOH	NEt3 (2 equiv.)	40%
3	EtOH	NEt3 (3 equiv.)	45%
4	MeCN	NEt3 (3 equiv.)	53%
5	MeCN	DBU (3 equiv.)	95%b
6	MeCN	DABCO (3 equiv.)	90%
7	MeCN	TMEDA (3 equiv.)	93%
8	СH2Cl2	DBU (3 equiv.)	62%

^aAll the reaction were performed in 0.15 mmol scale upon stirring for 1.5 hr at room temperature. NMR yields are reported unless specified otherwise.

^bThis reaction was performed in 0.50 mmol scale and isolated yield of purified product is provided.

With optimized reaction conditions in hand, we proceeded to the assessment of the scope and limitation of the featured synthetic method. The results of preparation of various indolizines are depicted in Scheme 3. It should be pointed out, that not only 2-nitrostyrenes 7a,b, but also aliphatic nitroalkene 7c was successfully employed in the reaction, to provide a little lower yields. Functional substituents, such as nitro or ester groups were tolerated in either penacyl or pyridinium moieties, affording functionalized indolizines 9db, 9ec, 9fc, 9ga and 9ha (Scheme 3). Quaternary derivative of isoquinoline 1g also reacted smoothly affording the corresponding pyrrolo[2,1-a]isoquinoline 9ga. Interestingly, reactions involving N-acetonylpyridinium bromide 1c were accompanied by partial aerobic oxidation leading to formation of 1-nitroindolizine derivatives 6cb and 6cc along with comparable quantities of “normal” products 9cb and 9cc, respectively (Scheme 3). Evidently, less stericallyhindered intermediates of type 4 are more susceptible towards aerobic oxidation. Formation of the indolizine ring system in the featured transformation was unambiguously confirmed by a single crystal X-ray diffraction of compound 9db (Figure 2).

Scheme 3.

Preparation of indolizines via dipolar [3+2] cycloaddition of pyridinium ylides to 2-chloro-1-nitroalkenes

Figure 2.

ORTEP drawing showing atom numbering scheme and 50% probability ellipsoids for X-ray structure of compound 9db (CCDC #2083857).

The scope of reaction between N-aminopyridinium derivatives and 2-chloro-1-nitroalkenes (7a-c) was also evaluated, and the results are shown in Scheme 4. In addition to N-aminopyridinium salt 10a, the reactions of N-amino derivatievs of isoquinolinium (10b) and quinolinium (10c) were also tested. It should be pointed out that all these reactions proceeded smoothly affording the corresponding pyrazolo[1,5-a]pyridines (14aa, 14ab, 14ac), pyrazolo[5,1-a]isoquinolines (14ba, 14bb, 14bc), or pyrazolo[1,5-a]quinolines (14ca, 14cb, 14cc), respectively (Scheme 4). It should be pointed out, that 2-chloro-1-nitroalkenes provides the best results in the featured transformation. Indeed, reaction of 10a with (1-bromo-2-nitrovinyl)benzene⁵⁴ afforded only 54% yield of product 14ab. At the same time, in reactions involving (1-iodo-2-nitrovinyl) benzene⁵⁵ or (1-methoxy-2-nitrovinyl)benzene⁵⁶ product 14ab was not formed at all, as these nitro-compounds decomposed.

Scheme 4.

Preparation of pyrazolo[1,5-a]pyridines via dipolar [3+2] cycloaddition of pyridinium ylides to 2-chloro-1-nitroalkenes

Mechanistic rationale of the featured transformation is depicted in Scheme 5. Evidently, the reaction should begin after deprotonation of acidic C-H or N-H bond of pyridinium salt 1 or 10, respectively. Formed zwitterionic intermediate 2 acts as nucleophile in addition across electron-deficient C=C bond of nitroalkene 7. Subsequent 5-endo-trig attack on iminium moiety takes place in intermediate zwitterionic species 15 to afford chlorinated bicyclic species 16. Then, a base-assisted dehydrochlorination takes place to afford 1-nitro-1,8a-dihydroindolizine 17 (X = C-Z) or 3-nitro-3,3a-dihydropyrazolo[1,5-a]pyridine 17 (X = N), existing in equilibrium with tautomeric forms 18 (X = C-Z) or 18 (X = N), respectively. The following reactivity cold take one or both of the following directions. If oxidation of these species proceeds easily, like in 3-nitro-1,3a-dihydropyrazolo[1,5-a]pyridines 18 (X = N), it takes place to afford aromatic product 14, bearing nitro group. If such oxidation proceeds slowly (X = C-COAr, C-COOR), base assisted elimination of nitrous acid takes place instead, producing indolizines 9. In intermediate cases (X = C-COAlk) both types of products 9 and 6 are being formed, as oxidation and elimination take place concurrently (Scheme 5).

Scheme 5.

Mechanistic rationale

Due to the “privileged structure” status of the synthesized indolizines and pyrazolo[1,5-a]pyridines, we evaluated them for cytotoxic activity against a neuroblastoma cell line BE(2)-C cultured in our laboratory. The compounds were incubated with cancer cells at a single concentration of 25 μM and cell viability was assessed using the MTT assay. The results are shown in Figure 2. Activity shared by structurally similar pyrazolo[1,5-a]pyridines 14ca, 14cb and 14ba is noteworthy and can probably be attributed to their planar nature and interaction with DNA and/or topoisomerases in cancer cells. The most potent activity, however, resides with indolizine 9db. Further testing of this compound (not shown) revealed an IC₅₀ of 10 μM, a potency higher than that of all trans-retinoic acid (ATRA), an anticancer drug used to treat acute promyelocytic leukemia.⁵⁷ A search of the literature revealed a related structure that was identified in a virtual screen of tubulin-targeting compounds,⁵⁸ although it possessed a 3,4,5-trimethoxy motif in the benzoyl substituent (in the blue benzene ring of 9db in Scheme 3), common for the colchicine site binders.⁵⁹ Our own experience with developing microtubule disrupting compounds binding at the colchicine site of b-tubulin suggested that often one of the methoxy groups can be omitted or substituted with bromine,⁶⁰ possibly indicating that we might accidentally discovered a new tubulin-targeting compound while pursuing this synthetic project. To verify this conjecture, the effect of this compound on in vitro tubulin polymerization were assessed.⁶¹ In this experiment, tubulin polymerization was followed by fluorescence enhancement due to the incorporation of a fluorescent reporter, in this case 4’,6-diamidino-2-phenylindole (DAPI), into microtubules as polymerization occurs. The test compound was compared to three different controls, a known microtubule stabilizer (paclitaxel), a known microtubule destabilizer (colchicine) as well as a DMSO solvent control sample. Whereas paclitaxel induced potent enhancement of microtubule formation relative to the effect of the DMSO control, both runs of indolizine 9db displayed potent inhibition of microtubule assembly in a manner similar to the known tubulin polymerization inhibitor colchicine (Figure 3).

Figure 3.

Effects of the synthesized indolizines and pyrazolo[1,5-a]pyridines on viability of BE(2)-C neuroblastoma cells. Cells were treated with the indicated compounds at the concentration of 25 μM for 4 days and cell viability was assessed with the MTT assay. Experiments were repeated 4 times with consistent results. ATRA = all trans-retinoic acid.

A Glide XP docking of 9db into the colchicine binding pocket revealed a possible binding pose in which the ketone carbonyl forms a hydrogen bond with Asn258, and the bromine is involved in a halogen bond with Thr317 (Fig. 4). The indolizine ring occupies a flat hydrophobic region within the pocket, and the phenyl ring protrudes out of the pocket into the small space located between the monomers.

Figure 4.

Effect of 9db (from two experiments) on in vitro tubulin polymerization. Paclitaxel (3 µM) promotes microtubule formation relative to 1% DMSO control. Both runs of indolizine 9db (25 µM) and colchicine (6 µM) completely suppress tubulin polymerization.

Conclusions

Efficient preparative protocol for [3+2]-cycloaddition of pyridinium ylides and 1-chloro-2-nitrostyrenes was developed and a series of heterocyclic compounds, indolizines and pyrazolo[1,5-a]pyridines were prepared via this method. Also, the current study identified several cytotoxic compounds among which indolizine 9db turned out to be most potent rivaling an established anticancer agent ATRA in its potency toward neuroblastoma cells. The reason for why only a single compound was found to have potent cytotoxic activity probably resides in the favorable combination of the scaffold presenting the two aromatic substituents in the correct orientation (at С−2 and С−3 of 9db) together with the necessary substituents – bromine and methoxy – required to make favorable contacts at the colchicine site of b-tubulin as we established in our previous work with colchicine site-interacting compounds.⁶⁰ Given the potential of colchicine site binders as anticancer agents, with combretastatin A4 being evaluated in multiple clinical trials, and the simplicity of synthetic access to such indolizines using our methodology, the discovery of indolizine 9db sets the stage for a new program in the discovery and potential translation of tubulin-targeting anticancer agents.

Experimental part

General.

¹H and ¹³C NMR spectra were recorded on a Bruker Avance-III spectrometer (400 or 100 MHz, respectively) equipped with a BBO probe in CDCl₃ or DMSO-d₆ using TMS as an internal standard. High-resolution mass spectra were registered with a Bruker Maxis spectrometer (electrospray ionization, in MeCN solution, using HCO₂Na−HCO₂H for calibration). Melting points were measured with a Stuart smp30 apparatus. The reaction progress and purity of isolated compounds were controlled by TLC on Silufol UV-254 plates, with hexanes/EtOAc mixtures used as eluents. All other reagents and solvents were purchased from commercial vendors and used as received.

Cell Viability Assay.

Cell viability was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Briefly, neuroblastoma cell line BE(2)-C cells (obtained from American Type Culture Collection) were maintained in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) media supplemented with 10% Fetalgro bovine growth medium (RMBIO, cat# FGR-BBT) and penicillin (100 U/mL)-streptomycin (100 μg/mL) (Corning, Cat# 30–002 CI). 1,500 cells per well were plated into 96-wells plate, and cells were treated with individual compounds at 25 μM in triplicates for four days. After four days, cells were replaced with MTT reagent (at 0.18 mg/mL in DMEM/F12) in each well and incubated with cells for 1 h at 37 °C. Culture media was removed after the plate was spin at 2000 rpm for 5 minutes. DMSO was then used to dissolve the crystal formed in each 96 well. Optical density values at wavelength 570 nm and 630 nm were measured using Epoch Microplate Spectrophotometer (BioTek Instruments), and the difference in the two optical density values was used to analyze the cell survival.

Tubulin Polymerization Assay.

The fluorescence-based Tubulin Polymerization Assay from Cytoskeleton, Inc. was used. Aqueous solutions of the tested agents (9db, paclitaxel and colchicine) were prepared at a 10^X concentration in a 10% DMSO. The tubulin reaction mixture was prepared per manufacturer’s instructions, kept on ice and used within ~ 15 min of preparation. A Biotek Synergy H4 hybrid multi-mode plate reader was pre-heated to 37 °C. Prior to starting the assay, a half-area black 96-well plate was preheated within the plate reader. A volume of 5 μL of each of the tested samples was pipetted into separate wells of the plate and incubated in the warm plate reader for 1 minute. A volume of 45 μL of the tubulin reaction mixture was then mixed into each of the wells, thereby diluting the samples to their final concentration (9db at 25 μM, paclitaxel at 3 μM, colchicine at 6 μM, 1% DMSO control). The fluorescence of the samples (λ_Ex = 360/20 nm, λ_Em = 485/20 nm) was then recorded every minute for 1 hour. Paclitaxel was used as a tubulin polymerization inducing control, colchicine as a tubulin polymerization inhibitor, and 1 % DMSO was used as a carrier control.

Molecular Docking.

Molecular docking was carried out using the Schrödinger suite of modelling tools (release 2021–2). The tubulin receptor complex was obtained from the PDB (3UT5) and for the purposes of preparation, only chains A and B were retained, with the colchicine binding pocket located at the interface between these two monomers. GTP, also located nearby at the interface was deleted as well as any solvent molecules. Protein preparation included the addition of any necessary side chains. The Glide grid was generated using colchicine to identify the region of the binding pocket, and Cys241 was set as having a rotatable polar hydrogen. Docking was carried out using Glide XP (expanded sampling) the highest scoring pose was selected. This was further subjected to a Prime MM-GBSA binding energy calculation with minimization of the ligand and residues within a 10 Å radius.

3-(Ethoxycarbonyl)-1-(2-(2-nitrophenyl)-2-oxoethyl)pyridin-1-ium bromide⁶² (1f):

To the ethyl pyridine-3-carboxylate (1.51 g, 10 mmol) dissolved in acetone(10 ml) was added 2-bromo-1-(2-nitrophenyl)ethan-1-one (2.44 g, 10 mmol). The mixture was heated under reflux for 90–120 min. The resulting salt was filtered off and crystallized from ethanol. Yield 3.9 g (9.9 mmol, 99%), white solid, m.p. 216–218 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 9.64 (s, 1H), 9.27 (d, J = 6.0 Hz, 1H), 9.17 (d, J = 8.2 Hz, 1H), 8.50 (dd, J = 8.0, 6.3 Hz, 1H), 8.27 (d, J = 8.1 Hz, 1H), 8.17 (d, J = 7.6 Hz, 1H), 8.10 – 8.03 (m, 1H), 8.00 – 7.91 (m, 1H), 6.59 (s, 2H), 4.48 (q, J = 7.1 Hz, 2H), 1.39 (t, J = 7.1 Hz, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 192.1, 161.5, 149.1, 147.0, 146.6, 146.2, 134.6, 133.5, 130.6, 129.9, 129.2, 128.6, 124.7, 67.5, 62.8, 14.0; FTIR (KBr, cm⁻¹):3089, 3012, 2988, 2882, 1713, 1640, 1568, 1522, 1465, 1399, 1339, 1305, 1211, 1199, 1134; HRMS (ES TOF) calc`d for C₁₆H₁₅N₂O₅ (M-Br)⁺ 315.0975, found 315.0972 (1.1 ppm).

2-(2-(2-Nitrophenyl)-2-oxoethyl)isoquinolin-2-ium (1g)

To the ethyl isoquinoline (1.29 g, 10 mmol) dissolved in acetone(10 ml) was added 2-bromo-1-(2-nitrophenyl)ethan-1-one (2.44 g, 10 mmol). The mixture was heated under reflux for 90–120 min. The resulting salt was filtered off and crystallized from ethanol. Yield 3.68 g (9.9 mmol, 99%), white solid, m.p. 245–247 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 10.16 (s, 1H), 8.77 (s, 2H), 8.64 (d, J = 8.3 Hz, 1H), 8.44 (d, J = 8.3 Hz, 1H), 8.34 (t, J = 7.6 Hz, 1H), 8.30 (d, J = 8.2 Hz, 1H), 8.19 (d, J = 7.3 Hz, 1H), 8.13 (t, J = 7.6 Hz, 1H), 8.07 (t, J = 7.5 Hz, 1H), 7.95 (t, J = 7.7 Hz, 1H), 6.61 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 193.0, 151.5, 146.0, 137.7, 137.4, 135.9, 134.7, 133.2, 131.6, 131.4, 130.8, 129.0, 127.5, 126.9, 125.9, 124.7, 67.3; FTIR (KBr, cm⁻¹): 3095, 2975, 2823, 1720, 1651, 1573, 1534, 1479, 1363; HRMS (ES TOF) calc`d for C₁₇H₁₃N₂O₃ (M-Br)⁺ 293.0920, found 293.0921 (0.2 ppm).

2-(1-Chloro-2-nitroethenyl)naphthalene⁵² (7a):

To a 100 mL flask were added 2-ethynylnaphthalene (1.52 g, 10 mmol), Cu(NO₃)₂·3H₂O (3.6240 g, 15 mmol), SnCl₂·2H₂O (2.2565 g, 10 mmol) and MeCN (50 mL). The mixture was stirred at 40 °C under N₂ for 4 h. Then the reaction mixture was cooled down to room temperature and filtered. The filtrate was concentrated and purified by column chromatography on silica gel to give the desired product as a yellow solid (1.85 g, 78%), E/Z = 84/16. Then the solid was recrystallized with hexane/ EtOAc and gave the pure E-product. Z-isomer: Eluent for chromatographic purification: Benzene/Hexane, gradient 1:5 – 1:3. Yield 0.28 g (1.2 mmol, 12%), yellow solid, m.p. 60–62 °C, R_f 0.24 (benzene/hexane 1:3); ¹H NMR (400 MHz, CDCl₃) δ 8.23 (d, J = 2.3 Hz, 1H), 7.95 – 7.86 (m, 4H), 7.69 – 7.59 (m, 3H).¹³C NMR (101 MHz, CDCl₃) δ 141.5, 134.9, 134.6, 132.8, 129.3, 129.2(2C), 128.7, 127.9, 127.6, 124.2, 123.4; FTIR (KBr, cm⁻¹):3103, 2833, 1946, 1595, 1510, 1322, 1277, 1183, 1131, 1007, 958, 898, 864, 821, 756; HRMS (ES TOF) calc`d for C<sub>12</sub>H<sub>8</sub>ClNNaO<sub>2</sub> (M+Na)<sup>+</sup> 256.0136, found 256.0144 (−3.2 ppm). E-isomer: Eluent for chromatographic purification: benzene/hexane, gradient 1:5 – 1:3. Yield 1.54 g (6.6 mmol, 66%), bright yellow solid, m.p. 74–75 °C, R<sub>f</sub> 0.34 (benzene/hexane 1:3); <sup>1</sup>H NMR (400 MHz, CDCl<sub>3</sub>) δ 7.98 (d, J = 2.2 Hz, 1H), 7.92 – 7.85 (m, 3H), 7.64 – 7.52 (m, 3H), 7.46 (dd, J = 8.6, 2.0 Hz, 1H);<sup>13</sup>C NMR (101 MHz, CDCl<sub>3</sub>) δ 147.6, 135.9, 134.4, 132.6, 130.8, 129.1, 129.0, 128.5, 128.3, 128.0, 127.2, 124.7; FTIR (KBr, cm<sup>−1</sup>): 3108, 2930, 2833, 1607, 1525, 1330, 115, 985, 903, 855, 816; HRMS (ES TOF) calc`d for C₁₂H₈ClNNaO₂ (M+Na)⁺ 256.0136, found 256.0144 (−3.2 ppm).

2-Chloro-1-nitrohept-1-ene (7c):

To a 100 mL flask were added hept-1-yne (0.96 g, 10 mmol), Cu(NO₃)₂·3H₂O (3.6240 g, 15 mmol), SnCl₂·2H₂O (2.2565 g, 10 mmol) and MeCN (50 mL). The mixture was stirred at 40 °C under N₂ for 4 h. Then the reaction mixture was cooled down to room temperature and filtered. The filtrate was concentrated and purified by column chromatography on silica gel to give the desired product as a yellow oil (1.2 g, 67%). Eluent for chromatographic purification: EtOAc/hexane 1:30, yellow oil, R_f 0.4 (Hexane); ¹H NMR (400 MHz, CDCl₃) δ 7.30 (s, 1H), 3.04 – 2.99 (m, 2H), 1.74 – 1.67 (m, 2H), 1.39 – 1.34 (m, 4H), 0.93 – 0.89 (m, 3H);¹³C NMR (101 MHz, CDCl₃) δ 155.0, 137.0, 35.5, 31.1, 27.1, 22.4, 14.0; FTIR (KBr, cm⁻¹):3118, 3964, 1710, 1624, 1563, 1520, 1462, 1431, 1342, 1199, 1134, 1043, 1011, 823;

[2-(Naphthalen-1-yl)indolizin-3-yl](phenyl)methanone (9aa):

Eluent for chromatographic purification: EA/Hexane, gradient 1:6 –1:4. Yield 140 mg (0.4 mmol, 81%), yellow-green oil, R_f 0.32 (EA/Hexane 1:4); ¹H NMR (400 MHz, CDCl₃) δ 9.85 (d, J = 7.2 Hz, 1H), 7.68 – 7.56 (m, 4H), 7.48 – 7.42 (m, 3H), 7.39 (dt, J = 6.5, 3.8 Hz, 2H), 7.19 (dd, J = 8.9, 7.0 Hz, 2H), 6.94 – 6.83 (m, 4H), 6.69 (s, 1H); ¹³C NMR (101 MHz, CDCl₃) δ 187.0, 140.3, 139.7, 137.7, 133.5, 132.9, 132.0, 130.6, 129.5 (2C), 129.2, 128.3, 128.2, 127.9, 127.5, 127.4 (2C), 127.1, 125.9, 125.9, 124.3, 120.3, 118.5, 113.7, 104.5; FTIR (KBr, cm⁻¹):3060, 2939, 2862, 1903, 1737, 1592, 1568, 1505, 1448, 1397, 1342, 1228, 1134, 1060, 1011, 958, 901, 852, 790, 746; HRMS (ES TOF) calc`d for C₂₅H₁₇NNaO (M+Na)⁺ 370.1195, found 370.1202 (2.0 ppm).

(2-Pentylindolizin-3-yl)(phenyl)methanone (9ac):

Eluent for chromatographic purification: EA/Hexane, gradient 1:7 – 1:6. Yield 94 mg (0.32 mmol, 64%), yellow-green oil, R_f 0.74 (EA/Hexane 1:6); ¹H NMR (400 MHz, CDCl3) δ 9.97 (dt, J = 7.1, 1.1 Hz, 1H), 7.82 – 7.77 (m, 2H), 7.55 – 7.46 (m, 4H), 7.20 – 7.14 (m, 2H), 6.92 (td, J = 6.9, 1.4 Hz, 1H), 2.74 – 2.69 (m, 2H), 1.64 (t, J = 7.6 Hz, 2H), 1.34 (q, J = 3.6 Hz, 4H), 0.89 (t, J = 3.2 Hz, 3H); ¹³C NMR (101 MHz, CDCl3) δ 184.0, 141.3, 137.8, 130.7, 129.1 (2C), 129.0, 128.3 (2C), 125.7, 123.7, 117.4, 116.9, 113.9, 31.8, 30.4, 29.9, 25.5, 22.7, 14.2.; FTIR (KBr, cm⁻¹):2939, 2853, 2641, 1910, 1793, 1590, 1563, 1467, 1452, 1414, 1368, 1339, 1236, 1156, 1093, 1019, 970, 874, 778, 739; HRMS (ES TOF) calc`d for C₂₀H₂₁NNaO (M+Na)⁺ 314.1515, found 314.1523 (−2.3 ppm).

1-(2-Phenylindolizin-3-yl)ethenone (9cb):

Eluent for chromatographic purification: EA/Hexane, gradient 1:12 –1:10. Yield 56 mg (0.24 mmol, 48%), orange solid, m.p. 66–67 °C, R_f 0.34 (EtOAc/hexane 1:10); ¹H NMR (400 MHz, CDCl₃) δ 9.99 (dd, J = 7.2, 0.9 Hz, 1H), 7.51 (dt, J = 8.8, 1.3 Hz, 1H), 7.46 – 7.39 (m, 5H), 7.17 (ddd, J = 8.8, 6.7, 1.2 Hz, 1H), 6.89 (td, J = 6.9, 1.4 Hz, 1H), 6.48 (s, 1H), 2.05 (s, 3H).; ¹³C NMR (101 MHz, CDCl₃) δ 188.4, 139.8, 137.4, 137.2, 129.82 (2C), 129.1, 128.3 (2C), 127.9, 124.4, 121.2, 118.2, 113.9, 105.2, 30.2; FTIR (KBr, cm⁻¹): 2943, 2875, 1627, 1509, 1404, 1346, 795; HRMS (ES TOF) calc`d for C₁₆H₁₃NNaO (M+Na)⁺ 258.0889, found 258.0885 (1.5 ppm).

1-(1-Nitro-2-phenylindolizin-3-yl)ethanone (6cb):

Eluent for chromatographic purification: EtOAc/hexane, gradient 1:12 –1:10. Yield 62 mg (0.22 mmol, 44%), pale yellow solid, m.p. 170–171 °C, R_f 0.24 (EA/Hexane 1:6); ¹H NMR (400 MHz, CDCl₃) δ 10.10 (dt, J = 7.2, 1.2 Hz, 1H), 8.64 (dt, J = 9.0, 1.2 Hz, 1H), 7.67 (ddd, J = 9.0, 7.0, 1.2 Hz, 1H), 7.51 (m, 3H), 7.42 – 7.36 (m, 2H), 7.21 (td, J = 7.0, 1.5 Hz, 1H), 1.90 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 190.9, 135.2, 134.2, 133.0, 130.8, 129.6, 129.23, 129.0 (2C), 128.8 (2C), 121.6, 119.2, 117.0, 30.9; FTIR (KBr, cm⁻¹): 2952, 2873, 1723, 1620, 1445, 1218, 1185, 1149, 978; HRMS (ES TOF) calc`d for C₁₆H₁₂N₂NaO₃ (M+Na)⁺ 303.0740, found 303.0744 (−1.2ppm).

1-(2-Pentylindolizin-3-yl)ethanone (9cc):

Eluent for chromatographic purification: EtOAc/hexane, gradient 1:40 –1:30. Yield 52 mg (0.23 mmol, 45%), yellow oil, R_f 0.34 (EtOAc/hexane 1:20); ¹H NMR (400 MHz, CDCl₃) δ ¹H NMR (400 MHz, CDCl₃) δ 9.83 (dt, J = 7.1, 1.2 Hz, 1H), 7.48 (dt, J = 8.9, 1.2 Hz, 1H), 7.31 (s, 1H), 7.09 (ddd, J = 8.9, 6.7, 1.2 Hz, 1H), 6.83 (td, J = 6.8, 1.3 Hz, 1H), 2.75 – 2.70 (m, 2H), 2.54 (s, 3H), 1.74 – 1.64 (m, 4H), 1.37 – 1.36 (m, 2H), 0.91 (d, J = 3.4 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 186.1, 136.9, 128.8, 122.9, 122.8, 116.9, 116.8, 113.6, 31.8, 30.4, 27.3, 25.5, 22.7, 14.2; FTIR (KBr, cm⁻¹): 2967, 2945, 1728, 1566, 1423, 1227, 1145, 1109, 897, 788, 757; HRMS (ES TOF) calc`d for C₁₅H₁₉NNaO (M+Na)⁺ 252.1359, found 252.1356 (1.0 ppm).

1-(1-Nitro-2-pentylindolizin-3-yl)ethanone (6cc):

Eluent for chromatographic purification: EtOAc/hexane, gradient 1:30 – 1:20. Yield 53 mg (0.19 mmol, 38%), yellow oil, R_f 0.35 (EtOAc/hexane 1:10); ¹H NMR (400 MHz, CDCl₃) δ 10.03 (dt, J = 7.2, 1.1 Hz, 1H), 8.61 (dt, J = 8.9, 1.3 Hz, 1H), 7.60 (ddd, J = 9.0, 7.0, 1.2 Hz, 1H), 7.13 (td, J = 7.0, 1.5 Hz, 1H), 3.44 – 3.35 (m, 2H), 2.72 (s, 3H), 1.75 (ddd, J = 14.9, 6.8, 4.1 Hz, 2H), 1.54 – 1.48 (m, 2H), 1.45 – 1.40 (m, 2H), 0.95 (t, J = 7.2 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 189.9, 137.3, 135.2, 130.8, 129.6, 121.7, 119.0, 116.7, 32.4, 31.2, 30.5, 26.5, 22.5, 14.2; FTIR (KBr, cm⁻¹): 2985, 2967, 1739, 1546, 1443, 1378, 1353, 1275, 1227, 1148, 1123, 891, 743; HRMS (ES TOF) calc`d for C₁₅H₁₈N₂NaO₃ (M+Na)⁺ 297.1210, found 297.1212 (−0.7 ppm)

(3-Bromo-4-methoxyphenyl)(2-phenylindolizin-3-yl)methanone (9db):

Eluent for chromatographic purification: benzene/hexane, gradient 1:2 – pure benzene. Yield 73 mg (0.395 mmol, 79%), yellow-green solid, m.p.260–261 °C, R_f 0.43 (EA/Hexane 1:4); ¹H NMR (400 MHz, CDCl₃) δ 9.69 (d, J = 7.2 Hz, 1H), 7.61 (d, J = 2.8 Hz, 1H), 7.55 (d, J = 8.8 Hz, 1H), 7.43 (dd, J = 8.4, 2.2 Hz, 1H), 7.23 – 6.94 (m, 6H), 6.88 (t, J = 6.9 Hz, 1H), 6.59 (s, 1H), 6.53 (d, J = 8.4 Hz, 1H), 3.79 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 184.3, 157.8, 139.6, 137.7, 136.0, 135.4, 133.9, 130.6, 130.1 (2C), 128.1, 127.9 (2C), 126.9, 124.3, 119.9, 118.5, 113.1, 110.7, 110.5, 104.2, 56.4; FTIR (KBr, cm⁻¹):3056,3033, 2964, 1723, 1598, 1231, 1113, 1053, 653; HRMS (ES TOF) calc`d for C₂₂H₁₆BrNNaO₂ (M+Na)⁺ 428.0257, found 428.0253 (0.8 ppm).

Ethyl 3-(2-nitrobenzoyl)-2-pentylindolizine-8-carboxylate (9fc):

Eluent for chromatographic purification: EtOAc/hexane, gradient 1:8 – 1:6. Yield 100 mg (0.245 mmol, 49%), brown oil, R_f 0.42 (EA/Hexane 1:6); ¹H NMR (400 MHz, CDCl₃) δ 10.57 (t, J = 1.3 Hz, 1H), 8.18 (dd, J = 8.2, 1.2 Hz, 1H), 7.78 – 7.73 (m, 2H), 7.66 (td, J = 7.8, 1.5 Hz, 1H), 7.60 (dd, J = 7.5, 1.5 Hz, 1H), 7.54 (dd, J = 9.3, 1.0 Hz, 1H), 6.83 (s, 1H), 4.45 (q, J = 7.1 Hz, 2H), 2.67 – 2.62 (m, 2H), 1.59 (d, J = 7.6 Hz, 2H), 1.44 (t, J = 7.1 Hz, 3H), 1.32 – 1.27 (m, 4H), 0.89 – 0.85 (m, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 180.4, 165.4, 147.3, 138.4, 136.6, 133.7, 132.9, 130.4, 129.8, 126.6, 124.7, 123.7, 122.0, 118.9, 118.4, 116.5, 61.6, 31.7, 30.1, 25.4, 22.6, 14.5, 14.2; FTIR (KBr, cm⁻¹):2973, 2935, 2853, 1718, 1614, 1525, 1472, 1378, 1344, 1286, 1224, 1096, 1014, 917, 848, 758; HRMS (ES TOF) calc`d for C₂₃H₂₄N₂NaO₅ (M+Na)⁺ 431.1577, found 431.1579 (−0.4 ppm).

(2-(Naphthalen-2-yl)pyrrolo[2,1-a]isoquinolin-3-yl)(2-nitrophenyl)methanone (9ga):

Eluent for chromatographic purification: EtOAc/hexane/NEt₃, gradient 10:80:1 –10:50:1. Yield 127 mg (0.325 mmol, 65%), orange solid, m.p. 192–193 °C, R_f 0.70 (EtOAc/Hexane 1:2); ¹H NMR (400 MHz, CDCl₃) 9.78 (d, J = 7.6 Hz, 1H), 8.20 (dt, J = 7.1, 3.5 Hz, 1H), 7.78 (dt, J = 7.3, 3.5 Hz, 1H), 7.71 – 7.66 (m, 1H), 7.60 (ddt, J = 12.1, 8.1, 3.7 Hz, 4H), 7.48 – 7.40 (m, 4H), 7.25 – 7.20 (m, 3H), 7.14 – 7.11 (m, 1H), 7.06 (td, J = 7.5, 1.1 Hz, 1H), 6.85 (ddd, J = 8.3, 7.5, 1.4 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃) δ 182.9, 147.1, 139.8, 137.2, 136.1, 132.8, 132.7, 132.5, 132.2, 130.0, 129.6, 129.4, 129.1, 128.6, 128.1, 127.9, 127.6, 127.4, 127.2, 127.1, 126.3, 126.22, 126.19, 124.4, 123.9, 123.8, 121.9, 114.1, 105.4; FTIR (KBr, cm⁻¹): 2935, 2862, 1739, 1715, 1595, 1520, 1404, 1346, 1296, 1224, 1187, 1146, 954, 915, 857, 819, 795; HRMS (ES TOF) calc`d for C₂₉H₁₈N₂NaO₃ (M+Na)⁺ 465.1210, found 465.1206 (0.9 ppm).

(2-Nitrophenyl)(2-pentylindolizin-3-yl)methanone (9ec):

Eluent for chromatographic purification: EtOAc/hexane, gradient 1:8 – 1:6. Yield 71 mg (0.21 mmol, 42%), brown oil, R_f 0.45 (EtOAc/hexane 1:6); ¹H NMR (400 MHz, CDCl₃) δ 10.03 (d, J = 7.0 Hz, 1H), 8.21 (dd, J = 8.2, 1.2 Hz, 1H), 7.73 (td, J = 7.5, 1.2 Hz, 1H), 7.65 – 7.61 (m, 1H), 7.51 (dd, J = 7.5, 1.5 Hz, 1H), 7.47 (dt, J = 8.7, 1.3 Hz, 1H), 7.21 (ddd, J = 8.7, 6.8, 1.2 Hz, 1H), 6.91 (td, J = 7.0, 1.4 Hz, 1H), 6.37 (s, 1H), 1.96 (s, 2H), 1.43 – 1.37 (m, 2H), 1.11 m, 2H), 1.02 – 0.94 (m, 2H), 0.78 (t, J = 7.3 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 180.7, 146.4, 141.2, 139.3, 138.3, 134.1, 129.9, 129.5, 129.2, 125.6, 124.9, 120.1, 117.8, 113.9, 104.5, 31.8, 30.0, 28.1, 22.4, 14.1; FTIR (KBr, cm⁻¹): 2964, 2939, 2848, 1734, 1708, 1599, 1527, 1421, 1346, 1231, 1139, 1108, 1021, 970, 898, 857, 787, 749; HRMS (ES TOF) calc`d for C₂₀H₂₀N₂NaO₃ (M+Na)⁺ 359.1366, found 359.1373 (−1.9 ppm).

Ethyl 2-(naphthalen-2-yl)indolizine-3-carboxylate (9ha):

Eluent for chromatographic purification: Acetone/Hexane, 1:10; Yield 180 mg (0.57 mmol, 57%), yellow oil, R_f 0.69 (Acetone/Hexane 1:10); ¹H NMR (400 MHz, CDCl₃) δ 9.57 (d, J = 7.2 Hz, 1H), 7.94 (s, 1H), 7.86 (dd, J = 13.0, 6.4 Hz, 3H), 7.63 (d, J = 8.4 Hz, 1H), 7.53 – 7.40 (m, 3H), 7.06 (t, J = 7.8 Hz, 1H), 6.83 (t, J = 7.1 Hz, 1H), 6.59 (s, 1H), 4.17 (q, J = 7.1 Hz, 2H), 1.00 (t, J = 7.1 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 162.1, 138.1, 137.0, 134.5, 133.1 (2C), 132.7, 129.0, 128.5, 128.2, 128.0, 127.8, 126.7, 126.1, 125.9, 122.3, 118.6, 112.9, 104.2, 59.8, 14.1; FTIR (KBr, cm⁻¹): 2931, 2853, 1717, 1650, 1436, 1329, 1209, 756 ; HRMS (ES TOF) calc`d for C₂₁H₁₇NNaO₂ (M+Na)⁺ 338.1151, found 338.1143 (2.4 ppm).

2-(Naphthalen-2-yl)-3-nitropyrazolo[1,5-a]pyridine (14aa):

Eluent for chromatographic purification: EtOAc/hexane, gradient 1:4 – 1:2. Yield 106 mg (0.37 mmol, 73%), yellow solid, m.p. 77–78 °C (ethanol), R_f 0.29 (EtOAc/ hexane 1:2); ¹H NMR (400 MHz, CDCl₃) δ 8.58 (d, J = 6.8 Hz, 1H), 8.44 (d, J = 7.8 Hz, 1H), 8.34 (s, 1H), 7.98 – 7.85 (m, 4H), 7.70 – 7.63 (m, 1H), 7.55 (p, J = 6.7 Hz, 2H), 7.15 (t, J = 6.9 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃) δ 152.4, 138.4, 134.0, 133.0, 131.0, 130.0, 129.6, 128.8, 127.9 (2C), 127.6, 127.3, 127.0, 126.5, 119.4, 116.1; FTIR (KBr, cm⁻¹): 3094, ,2780, 1636, 1527, 1505, 1489, 1433, 1397, 1380, 1334, 1308, 1250, 1199, 1137, 1060; HRMS (ES TOF) calc`d for C₁₇H₁₁N₃NaO₂ (M+Na)⁺ 312.0743, found 312.0740 (1.2 ppm).

3-Nitro-2-phenylpyrazolo[1,5-a]pyridine (14ab):

Eluent for chromatographic purification: EtOAc/hexane, gradient 1:3 – 1:1. Yield 113 mg (0.48 mmol, 95%), light brown solid, m.p. 195–196 °C (ethanol), R_f 0.51 (EtOAc/hexane 1:2); ¹H NMR (400 MHz, CDCl₃) δ 8.57 (d, J = 6.8 Hz, 1H), 8.44 (d, J = 8.9 Hz, 1H), 7.86 – 7.76 (m, 2H), 7.74 – 7.63 (m, 1H), 7.59 – 7.40 (m, 3H), 7.17 (td, J = 6.9, 1.5 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃) δ 152.4, 138.4, 131.0, 130.2, 130.1, 130.0 (2C), 129.6, 128.3 (2C), 119.42, 116.1; FTIR (KBr, cm⁻¹): 3079, 3026, 2886, 1949, 1831, 1636, 1505, 1481, 1455, 1395, 1310, 1255, 1211, 1181, 1120, 1062; HRMS (ES TOF) calc`d for C₁₃H₉N₃NaO₂ (M+Na)⁺ 262.0587, found 262.0584 (1.1 ppm).

3-Nitro-2-pentylpyrazolo[1,5-a]pyridine (14ac):

Eluent for chromatographic purification: EtOAc/hexane, gradient 1:10 – 1:7. Yield 99 mg (0.43 mmol, 85%), yellow oil, m.p. 77–78 °C (Ethanol), R_f 0.55 (EtOAc/hexane 1:6); ¹H NMR (400 MHz, CDCl₃) δ 8.79 (d, J = 8.4 Hz, 1H), 8.42 – 8.36 (m, 2H), 8.04 – 7.92 (m, 6H), 7.86 (ddd, J = 8.5, 7.0, 1.4 Hz, 1H), 7.70 – 7.62 (m, 1H), 7.60 – 7.53 (m, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 150.8, 136.6, 134.0, 133.7, 133.1, 131.7, 131.5, 130.0, 128.91, 128.86, 128.1, 127.93, 127.89, 127.21, 127.17, 127.0, 126.5, 124.3, 116.5, 116.2; FTIR (KBr, cm^-1):2925, 2848, 1734, 1532, 1493, 1354, 1236, 1161, 1074, 1026, 913, 836, 804; HRMS (ES TOF) calc`d for C₁₂H₁₅N₃NaO₂ (M+Na)⁺ 256.1056, found 256.1047 (3.8 ppm).

2-(Naphthalen-2-yl)-1-nitropyrazolo[5,1-a]isoquinoline (14ba):

Eluent for chromatographic purification: EtOAc/ hexane, gradient 1:6 –1:4. Yield 116 mg (0.34 mmol, 66%), pale yellow solid, m.p. 195–197 °C, R_f 0.36 (EtOAc/hexane 1:4); ¹H NMR (400 MHz, CDCl₃) δ 9.10 – 8.98 (m, 1H), 8.35 (d, J = 7.2 Hz, 1H), 8.24 (s, 1H), 7.98 – 7.86 (m, 4H), 7.81 – 7.71 (m, 3H), 7.60 – 7.51 (m, 2H), 7.36 (d, J = 7.2 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃) δ 151.0, 139.5, 133.9, 133.2, 131.3, 130.7, 129.0, 128.8, 128.8, 128.3, 128.0, 127.9(2C), 127.2, 126.6, 126.5, 126.4, 126.1, 122.6, 116.3; FTIR (KBr, cm⁻¹): 3079, 1990, 1908, 1636, 1534, 1496, 1356, 1293, 1226, 1146, 898, 821, 804; HRMS (ES TOF) calc`d for C₂₁H₁₃N₃NaO₂ (M+Na)⁺ 362.0900, found 362.0898 (0.5 ppm).

1-Nitro-2-phenylpyrazolo[5,1-a]isoquinoline (14bb):

Eluent for chromatographic purification: EA/Hexane, gradient 1:7 –1:6. Yield 128 mg (0.44 mmol, 88%), brown solid, m.p. 120–123 °C, R_f 0.42 (EtOAc/hexane 1:6); ¹H NMR (400 MHz, CDCl₃) δ 8.99 (dd, J = 7.7, 1.9 Hz, 1H), 8.30 (d, J = 7.2 Hz, 1H), 7.87 – 7.85 (m, 1H), 7.77 – 7.70 (m, 4H), 7.52 – 7.48 (m, 3H), 7.33 (d, J = 7.3 Hz, 1H).; ¹³C NMR (101 MHz, CDCl3) δ 150.9, 133.7, 131.2, 130.6, 130.5, 129.7, 129.2(2C), 128.7, 128.6(2C), 127.8, 126.3, 126.0, 122.5, 116.2; FTIR (KBr, cm⁻¹): 2924, 2853, 1739, 1640, 1530, 1493, 1369, 1353, 1226, 1159, 1076, 1029, 917, 837, 806; HRMS (ES TOF) calc`d for C₁₇H₁₁N₃NaO₂ (M+Na)⁺ 312.0743, found 312.0754 (−3.5 ppm).

1-Nitro-2-pentylpyrazolo[5,1-a]isoquinoline (14bc):

Eluent for chromatographic purification: EtOAc/hexane, gradient 1:10 –1:8. Yield 89 mg (0.315 mmol, 61%), yellow-green solid, m.p. 61–62°C, R_f 0.55 (EtOAc/hexane 1:6); ¹H NMR (400 MHz, CDCl₃) δ 9.28 (dd, J = 6.9, 2.6 Hz, 1H), 8.24 (d, J = 7.2 Hz, 1H), 7.83 (dq, J = 7.4, 3.7, 3.1 Hz, 1H), 7.79 – 7.67 (m, 2H), 7.30 (d, J = 7.2 Hz, 1H), 3.17 – 3.09 (m, 2H), 1.83 (p, J = 7.6 Hz, 2H), 1.46 – 1.36 (m, 4H), 0.93 (t, J = 7.0 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 154.7, 134.1, 131.6, 130.6, 128.5, 127.7, 127.4, 126.0, 122.6, 116.0, 31.9, 28.7, 27.9, 22.6, 14.2; FTIR (KBr, cm⁻¹):2968, 2925, 2862, 1734, 1558, 1537, 1452, 1371, 1349, 1211, 782, 746; HRMS (ES TOF) calc`d for C₁₆H₁₇N₃NaO₂ (M+Na)⁺ 306.1213, found 306.1201 (3.8 ppm).

2-(Naphthalen-2-yl)-3-nitropyrazolo[1,5-a]quinoline (14ca):

Eluent for chromatographic purification: EtOAc/ hexane, gradient 1:10 – 1:5. Yield 134 mg (0.395 mmol, 79%), brown solid, m.p. 252–253 °C (ethanol), R_f 0.25 (EtOAc/hexane 1:5); ¹H NMR (400 MHz, CDCl₃) δ 8.78 (d, J = 8.5 Hz, 1H), 8.46 – 8.32 (m, 2H), 8.02 – 7.91 (m, 6H), 7.88 – 7.83 (m, 1H), 7.68 – 7.63 (m, 1H), 7.59 – 7.53 (m, 2H)..; ¹³C NMR (101 MHz, CDCl₃) δ 100.9, 85.9, 83.3, 83.0, 82.4, 81.0 (2С), 80.8, 79.3, 78.2, 78.1, 77.19, 77.15, 76.5, 76.4, 76.2, 75.8, 73.6, 65.8, 65.5; FTIR (KBr, cm⁻¹):2930, 2867, 1934, 1751, 1616, 1551, 1503, 1472, 1424, 1356, 1214, 1178, 1144, 958, 874, 819, 754; HRMS (ES TOF) calc`d for C₂₁H₁₃N₃NaO₂ (M+Na)⁺ 362.0900, found 362.0892 (2.1 ppm).

3-Nitro-2-phenylpyrazolo[1,5-a]quinolone (14cb):

Eluent for chromatographic purification: EtOAc/hexane, gradient 1:8 – 1:6. Yield 113 mg (0.39 mmol, 78%), yellow oil, m.p. 224–225 °C (Ethanol), R_f 0.55 (EtOAc/hexane 1:6); ¹H NMR (400 MHz, CDCl₃) δ 8.73 (d, J = 8.5 Hz, 1H), 8.33 (d, J = 9.4 Hz, 1H), 7.98 – 7.91 (m, 2H), 7.90 – 7.80 (m, 3H), 7.66 – 7.61 (m, 1H), 7.56 – 7.52 (m, 3H).; ¹³C NMR (101 MHz, CDCl₃) δ 150.8, 136.5, 133.7, 131.6, 131.4, 130.6, 130.1 (2C), 129.9, 128.9, 128.4 (2C), 126.9, 124.2, 116.4, 116.2.; FTIR (KBr, cm⁻¹): 3036, 2886, 2795, 1946, 1824, 1718, 1616, 1554, 1520, 1493, 1397, 1344, 1221, 1199, 1137, 1108, 1072, 1021, 966, 889, 836, 814; HRMS (ES TOF) calc`d for C₁₇H₁₁N₃NaO₂ (M+Na)⁺ 312.0743, found 312.0735 (2.7 ppm).

3-Nitro-2-pentylpyrazolo[1,5-a]quinoline (14cc):

Eluent for chromatographic purification: EtOAc/hexane, gradient 1:8 – 1:6. Yield 96 mg (0.34 mmol, 67%), yellow oil, m.p. 64–65 °C (ethanol), R_f 0.64 (EtOAc/hexane 1:6); ¹H NMR (400 MHz, CDCl₃) δ 8.66 (dq, J = 8.4, 0.8 Hz, 1H), 8.27 (d, J = 9.3 Hz, 1H), 7.95 – 7.88 (m, 2H), 7.82 (ddd, J = 8.5, 7.2, 1.4 Hz, 1H), 7.63 – 7.58 (m, 1H), 3.25 – 3.20 (m, 2H), 1.91 – 1.83 (m, 2H), 1.50 – 1.40 (m, 4H), 0.94 (t, J = 7.2 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 153.4, 136.1, 133.7, 131.4, 131.3, 128.8, 126.5, 124.1, 116.4, 116.0, 31.9, 28.5, 27.8, 22.6, 14.2; FTIR (KBr, cm⁻¹): 2973, 2920, 2857, 1996, 1737, 1609, 1558, 1498, 1457, 1421, 1346, 1308, 1226, 1168, 1137, 821; HRMS (ES TOF) calc`d for C₁₆H₁₇N₃NaO₂ (M+Na)⁺ 306.1213, found 306.1218 (−1.6 ppm).

Figure 1.

Medicinally relevant indolizines.

Figure 5.

Glide XP docking of compound 9db using a tubulin complex (PDB ID 3UTP) revealed a possible binding mode within the colchicine binding pocket, located at the monomer interface between chains A and B.