Polyhalonitrobutadienes as Versatile Building Blocks for the Biotargeted Synthesis of Substituted N-Heterocyclic Compounds †

Substituted nitrogen heterocycles are structural key units in many important pharmaceuticals. A new synthetic approach towards heterocyclic compounds displaying antibacterial activity against Staphylococcus aureus or cytotoxic activity has been developed. The selective synthesis of a series of 64 new N-heterocycles from the three nitrobutadienes 2-nitroperchloro-1,3-butadiene, 4-bromotetrachloro-2-nitro-1,3-butadiene and (Z)-1,1,4-trichloro-2,4-dinitrobuta-1,3-diene proved feasible. Their reactions with N-, O- and S-nucleophiles provide rapid access to push-pull substituted benzoxazolines, benzimidazolines, imidazolidines, thiazolidinones, pyrazoles, pyrimidines, pyridopyrimidines, benzoquinolines, isothiazoles, dihydroisoxazoles, and thiophenes with unique substitution patterns. Antibacterial activities of 64 synthesized compounds were examined. Additionally, seven compounds (thiazolidinone, nitropyrimidine, indole, pyridopyrimidine, and thiophene derivatives) exhibited a significant cytotoxicity with IC50-values from 1.05 to 20.1 µM. In conclusion, it was demonstrated that polyhalonitrobutadienes have an interesting potential as structural backbones for a variety of highly functionalized, pharmaceutically active heterocycles.


Introduction
Halogenated nitrobutadienes are part of a relatively small group of selectively reactive aliphatic nitro compounds [1].
Representatives with one or two nitro and three to five halogen groups are easily accessible by introduction of an activating and directing nitro group into polyhalo-1,3-butadienes [2]. These can be easily obtained in high purity and multigram scale by radical dimerization of industrial solvents such as trichloroethene and 1,2-dichloroethene with subsequent dehydrohalogenation-halogenation, followed by nitration reactions. 2-Nitroperchlorobutadiene (1) has been synthesized in three steps from trichloroethene [3,4] (Scheme 1). 4-Bromotetrachloro-2-nitrobutadiene (2) could be obtained from trichloroethene in five steps [5].

Imidacloprid Analogues
The imidazolidine Imidacloprid (N-{1-[(6-chloro-3-pyridyl)methyl]-4,5-dihydroimidazol-2-yl}nitramide) has been the most widely used systemic insecticide worldwide. A first synthesis of analogues from nitropolychloroalkenes has been reported [12,13], new types of derivatives are presented here. For instance, 11a and 11b were obtained from nitrodiene 1 and chloropyridines 10a and 10b, respectively [13]. Reaction of imidazolidine 11a with a 2.5-fold excess of N-nucleophiles such as ethyl piperidine-4-carboxylate and 1,2,3,4-tetrahydroisoquinoline in methanol at 35-50 °C leads to compounds 12a and 12b in 60-85% yield, respectively. By using 2-mercaptoethan-1-ol as Snucleophile for the reaction with 11a in the presence of sodium ethanolate, the corresponding sulfane 12c was obtained in 63% yield. Treatment of 11b with a fivefold excess of dimethylamine at rt led to the formation of oxazolidine 12d (70%). By the reaction of bromonitrodiene 2 with an equimolar amount of 4-fluorobenzenethiol in DCM at rt, sulfane 13 was obtained as mixture of two isomers in a total yield of 74%. The subsequent vinylic substitution of the monothio compound 13 by means of 10a gave imidazolidine 14 (44%) as well as ketene dithioacetal 15 (30% yield). Arylthiols are known to be both, good nucleophiles as well as good leaving groups. Compound 14 was previously synthesized in 40% yield directly from nitrodiene 2 and diamine 10a [12]. 1,1-Dithio compound 15 could be obtained in 83% yield from diene 2 and two equivalents of 4-fluorobenzenethiol using sodium methanolate as a base. The reaction of diene 2 with diamine 16 [13] at optimized conditions furnished the imidazolidine 17 as a mixture of two isomers in a total yield of 89% (Scheme 4).

Thiazolidinones
Thiazolidin-4-ones represent a class of compounds that has proven to exhibit distinctive bio-activity, e.g. antifungal, antibacterial, antitubercular, and anticonvulsant properties [14][15][16][17]. Our research in this area is presented through an efficient synthesis of functionalized (Z)-2-allylidene-thiazolidin-4-ones [18]. Nitrodiene 1 reacts with ethyl 2-mercaptoacetate to give the sulfane 18 as single E-isomer [19]. For the subsequent reactions of sulfane 18, we used two aniline derivatives, an activated (ERG) and an desactivated (EWG) one. In both cases, the expected thiazolidinones 19a,b were obtained in good yields (73-76%). Treatment of 19a,b with a 5-to 8-fold excess of hydrazine led to pyrazoles 20a,b. The assumed mechanism for this ring-opening and subsequent ring-closure transformation forming 20a,b has been presented [18]. Heating of thiazolidinones 19a,b with five membered 2-formyl heterocycles in acetic acid in the presence of trimethylamine furnished hetarylmethylidenethiazolidinones 21a,f in good to excellent yields, as single diastereomers. The Z-configuration was assigned according to literature data. The presence of only one signal for the methylidene proton at 7.72-8. 15 ppm in the 1 H nmr spectra of compounds 21a,f suggested the formation of a single isomer, which was assigned 2.1. 6

. Pyrimidines
In the course of the studies concerning polyhalogenated nitrobutadienes, a new ring closure approach to perfunctionalized 5-nitropyrimidines was also developed [33]. Using this protocol starting from 25c-f, four new nitropyrimidines 27c-f were obtained. Even under optimum conditions, yields of the products 27c-f remained moderate, reaching 49-65%. The assumed mechanism for the formation of pyrimidines 27 has been presented in the literature [33]. 5-Nitrosubstituted pyrimidines are interesting precursors for the synthesis of a wide range of polysubstituted pyrimidines and other heterocyclic systems with potential biological activity [34]. Among numerous applications, some examples are noteworthy: cytotoxic activity is documented [35,36] as well as the potential to inactivate the human DNA repair process [37]. The broad variety of medicinal applications is further illustrated, e.g. with the activity against chronic obstructive pulmonary disease [38], applicability against herpes simplex [39], and other viral diseases [40]. Furthermore, one field of application of 5-nitropyrimidines uses their positive modulating effect of the GABAB receptor [41,42]. Pyrimidin-4-yl-1H-indoles are a very rare class of organic compounds; to the best of our Scheme 6. Synthesis of benzazetines 26 and benzotriazoles 25.

Pyrimidines
In the course of the studies concerning polyhalogenated nitrobutadienes, a new ring closure approach to perfunctionalized 5-nitropyrimidines was also developed [33]. Using this protocol starting from 25c-f, four new nitropyrimidines 27c-f were obtained. Even under optimum conditions, yields of the products 27c-f remained moderate, reaching 49-65%. The assumed mechanism for the formation of pyrimidines 27 has been presented in the literature [33]. 5-Nitro-substituted pyrimidines are interesting precursors for the synthesis of a wide range of poly-substituted pyrimidines and other heterocyclic systems with potential biological activity [34]. Among numerous applications, some examples are noteworthy: cytotoxic activity is documented [35,36] as well as the potential to inactivate the human DNA repair process [37]. The broad variety of medicinal applications is further illustrated, e.g. with the Molecules 2020, 25, 2863 7 of 41 activity against chronic obstructive pulmonary disease [38], applicability against herpes simplex [39], and other viral diseases [40]. Furthermore, one field of application of 5-nitropyrimidines uses their positive modulating effect of the GABAB receptor [41,42]. Pyrimidin-4-yl-1H-indoles are a very rare class of organic compounds; to the best of our knowledge, only 4 representatives are known till today [43][44][45]. With the aim to synthesize a new pyrimidin-4-yl-1H-indole with potent biological activity, we made an attempt to oxidize the 2,3-dihydroindole 27f. Indeed, by using DDQ as oxidizing agent (ratio 27f: DDQ 1: 2.25, toluene, reflux 5 h), the expected indole 28 was obtained in 66% yield (Scheme 7).
starting from 25c-f, four new nitropyrimidines 27c-f were obtained. Even under optimum conditions, yields of the products 27c-f remained moderate, reaching 49-65%. The assumed mechanism for the formation of pyrimidines 27 has been presented in the literature [33]. 5-Nitrosubstituted pyrimidines are interesting precursors for the synthesis of a wide range of polysubstituted pyrimidines and other heterocyclic systems with potential biological activity [34]. Among numerous applications, some examples are noteworthy: cytotoxic activity is documented [35,36] as well as the potential to inactivate the human DNA repair process [37]. The broad variety of medicinal applications is further illustrated, e.g. with the activity against chronic obstructive pulmonary disease [38], applicability against herpes simplex [39], and other viral diseases [40]. Furthermore, one field of application of 5-nitropyrimidines uses their positive modulating effect of the GABAB receptor [41,42]. Pyrimidin-4-yl-1H-indoles are a very rare class of organic compounds; to the best of our knowledge, only 4 representatives are known till today [43][44][45]. With the aim to synthesize a new pyrimidin-4-yl-1H-indole with potent biological activity, we made an attempt to oxidize the 2,3dihydroindole 27f. Indeed, by using DDQ as oxidizing agent (ratio 27f: DDQ 1: 2.25, toluene, reflux 5 h), the expected indole 28 was obtained in 66% yield (Scheme 7). Scheme 7. Synthesis of pyrimidines 27, 28.

Benzo[h]quinolines
In the course of our studies on polyhalogenated nitrobutadienes, a new ring closure approach to benzo[h]quinolines was also developed [61]. Starting from nitrodiene 1 in three steps, the target benzo[h]quinolines with a unique substitution pattern at the pyridine ring were obtained in good yields. In detail, after mono substitution of one chlorine group in diene 1 sulfanes 34a,b were formed as single isomers each in yields of about 80% according to the literature [62] for the benzyl derivative 34a and literature [9] for the 4-chlorophenyl derivative 34b. In a second step, we synthesized the aminothiobutadienes 35a,b by interaction of sulfane 34a,b with an excess of 1-naphthylamine in methanol at −10 °C to rt. Dienes 35a,b were also formed (76-85% yield) as single E-isomers due to the stable six membered hydrogen bridge between the amino and nitro group. Finally, the ring closure at optimum conditions (twofold excess of triethylamine as a base) proceeded under formation of the expected benzo[h]quinolines 36a,b in good yields (76-85%). The assumed mechanism for the formation of benzo[h]quinolines is depicted in the literature [61]. Benzo[h]quinolines are a precious class of organic compounds and show interesting biological properties [63][64][65][66][67]. Oxidation of quinolines 36a,b with excess of hydrogen peroxide in a mixture of acetic acid and chloroform lead to the formation of sulfoxides 37a,b in 89-91% yield. The sulfinyl group is known to be a good leaving

Benzo[h]quinolines
In the course of our studies on polyhalogenated nitrobutadienes, a new ring closure approach to benzo[h]quinolines was also developed [61]. Starting from nitrodiene 1 in three steps, the target benzo[h]quinolines with a unique substitution pattern at the pyridine ring were obtained in good yields. In detail, after mono substitution of one chlorine group in diene 1 sulfanes 34a,b were formed as single isomers each in yields of about 80% according to the literature [62] for the benzyl derivative 34a and literature [9] for the 4-chlorophenyl derivative 34b. In a second step, we synthesized the aminothiobutadienes 35a,b by interaction of sulfane 34a,b with an excess of 1-naphthylamine in methanol at −10 • C to rt. Dienes 35a,b were also formed (76-85% yield) as single E-isomers due to the stable six membered hydrogen bridge between the amino and nitro group. Finally, the ring closure at optimum conditions (twofold excess of triethylamine as a base) proceeded under formation of the expected benzo[h]quinolines 36a,b in good yields (76-85%). The assumed mechanism for the formation  [63][64][65][66][67]. Oxidation of quinolines 36a,b with excess of hydrogen peroxide in a mixture of acetic acid and chloroform lead to the formation of sulfoxides 37a,b in 89-91% yield. The sulfinyl group is known to be a good leaving group [68][69][70]. Indeed, treatment of sulfoxide 37a with an excess of pyrrolidine in toluene at 100 •

Thiophenes
In the course of studying nitroperchlorobutadiene 1 as a versatile building block for the directed synthesis of a range of persubstituted heterocycles, we also developed a three-step synthesis to persubstituted 3-amino-4-nitrothiophenes [81]. Incorporating both, an enamine and a thioketene unit, these thiophenes are very electron-rich heterocycles with a unique substitution pattern. Starting from 1, the piperazine derivative 52 was obtained in 90% yield via the dithiolane 51. The push-pull substituted thiophene 53 was efficiently accessible in 85% yield upon treatment of dithiolane 52 with sodium hydroxide using DMSO as solvent. The regioselective ipso-formylation of the 2-chlorothiophene 53 under Vilsmeier-Haack conditions led to the carbaldehyde 54 (64% yield), according to [82].

Thiophenes
In the course of studying nitroperchlorobutadiene 1 as a versatile building block for the directed synthesis of a range of persubstituted heterocycles, we also developed a three-step synthesis to persubstituted 3-amino-4-nitrothiophenes [81]. Incorporating both, an enamine and a thioketene unit, these thiophenes are very electron-rich heterocycles with a unique substitution pattern. Starting from 1, the piperazine derivative 52 was obtained in 90% yield via the dithiolane 51. The push-pull substituted thiophene 53 was efficiently accessible in 85% yield upon treatment of dithiolane 52 with sodium hydroxide using DMSO as solvent. The regioselective ipso-formylation of the 2-chloro-thiophene 53 under Vilsmeier-Haack conditions led to the carbaldehyde 54 (64% yield), according to [82].

Biological Activity of the Polyhalonitrobutadiene Derivatives
Evaluation of the biological activity of the chosen polyhalonitrobutadiene derivatives showed that most of them did not display antibacterial or cytotoxic effects, i.e., residual growth or viability after incubation for 1 and 3 days, respectively, were higher than 50%. Tables with all primary screening data are shown in Supplementary Figures S204-S205. None of the derivatives showed an antibacterial activity against the uropathogenic Escherichis coli strain UPEC 796, whereas some had antibacterial activity against Staphylococcus aureus. The cytotoxic activity of nine compounds could be proven, as these compounds had IC 50 -values < 50 µM in the viability assay. Among those compounds was the "conjugate" 23b of the pyrazole 22 and the thiazolidinone 19b. Whereas the compound series 21 was more or less completely inactive, introduction of the pyrazole group proved successful. In particular, the introduction of a CF 3 substituent resulted in a compound with significant cytotoxicity (IC 50 = 6.2 ± 1.8 µM). Similarly, among the pyrimidines 27, 28, the most potent derivatives were those with the aromatic residues at the nitropyrimidine-core, namely 27c and 28 with IC 50 -values of 1.5 ± 0.4 µM and 1.05 ± 0.2 µM, respectively. The non-aromatic nature of the ring next to the pyrimidine core in 27f prevented the cytotoxic activity. Following the synthesis route of the pyridopyrimidines 32 and 33 revealed that the precursor with the leaving group benzotriazole was the only cytotoxic compound (IC 50 = 6.0 and 5.7 ± 1.0 µM), and that cytotoxicity was lost, when the benzotriazole group was replaced. In addition, the benzo[h]quinolines 36, 38 lost the cytotoxic activity, which was still observed for the intermediate naphthalene-aminothiobutadiene 35. All tested derivatives of both the groups of isothiazoles and dihydroisoxazoles were inactive, whereas among the thiophenes, the derivatives with a cyclic dione residue 58 and 60 represented cytotoxic compounds (IC 50 = 3.1 ± 0.4 µM and 20.1 ± 3.9 µM). Obviously, the morpholino-nitrothiophene structure was not sufficient for biological activity as compound 59 was completely inactive.

Biological Activity of the Polyhalonitrobutadiene Derivatives
Evaluation of the biological activity of the chosen polyhalonitrobutadiene derivatives showed that most of them did not display antibacterial or cytotoxic effects, i.e., residual growth or viability after incubation for 1 and 3 days, respectively, were higher than 50%. Tables with all primary screening data are shown in Supplementary Figures S204-S205. None of the derivatives showed an antibacterial activity against the uropathogenic Escherichis coli strain UPEC 796, whereas some had antibacterial activity against Staphylococcus aureus. The cytotoxic activity of nine compounds could be proven, as these compounds had IC50-values < 50 µM in the viability assay. Among those compounds was the "conjugate" 23b of the pyrazole 22 and the thiazolidinone 19b. Whereas the compound series 21 was more or less completely inactive, introduction of the pyrazole group proved successful. In particular, the introduction of a CF3 substituent resulted in a compound with significant cytotoxicity (IC50 = 6.2 ± 1.8 µM). Similarly, among the pyrimidines 27, 28, the most potent derivatives were those with the aromatic residues at the nitropyrimidine-core, namely 27c and 28 with IC50-values of 1.5 ± Scheme 13. Synthesis of thiophenes 53-60 and dithiolanes 51, 52.

General Information
General Remarks: Solvents and reagents were used as received from commercial sources without further purification. TLC was performed with Merck aluminum-backed TLC plates with silica gel 60, F254. Flash column chromatography was performed with Macherey-Nagel silica gel 60 M (0.040-0.063 mm) with appropriate mixtures of petroleum ether (PE, boiling range 60-70 • C) and ethyl acetate as eluents. Melting points (m.p.) were determined in capillary tubes with a Büchi B-520 instrument and were not corrected. FTIR spectra were recorded with a Bruker "Alpha-T" spectro-meter with solid compounds measured as KBr pellets. ATR-IR spectra were measured on the same instrument with a Bruker "Alpha Platinum ATR" single reflection diamond ATR module. 1 H NMR and 13 C NMR spectra at 600 and 150 MHz, respectively, were recorded with an "Avance III" 600 MHz FT-NMR spectrometer (Bruker, Rheinstetten, Germany). 1 H NMR and 13 C NMR spectra at 400 and 100 MHz, respectively, were recorded with an "Avance" 400 MHz FT-NMR spectrometer (also Bruker). 1 H NMR and 13 C NMR spectra at 200 and 50 MHz, respectively, were recorded with an DPX 200 spectrometer (also Bruker). 14 N and 15 N NMR spectra were measured at their appropriate resonance frequency on the aforementioned spectrometers; 15 N measurements were taken as gs-1 H, 15 N-HSQC or -HMBC experiments with inverse detection. 1 H and 13 C NMR spectra were referenced to the residual solvent peak: CDCl 3 , δ = 7.26 ( 1 H) and 77.0 ppm ( 13 C); DMSO-d 6 , δ = 2.50 ( 1 H), and 39.7 ppm ( 13 C). Mass spectra were obtained with a Hewlett-Packard MS 5989B spectrometer, usually in direct mode with electron impact (70 eV). For chlorinated and brominated compounds, all peak values of molecular ions and fragments refer to the isotope 35 Cl and 79 Br. High resolution mass spectra were recorded with a Waters mass spectrometer "VG Autospec" (EI), with a WATERS mass spectrometer "Q-Tof Premier" coupled with a Waters "Acquity UPLC" (ESI), or with a Micromass mass spectrometer "LCT" coupled with a Waters "Alliance 2965 HPLC" (ESI) at the Institute of Organic Chemistry, Leibniz University of Hannover and at the Georg-August University of Göttingen. (3)