Further SAR on the (phenylsulfonyl)piperazine scaffold as inhibitors of the Aedes aegypti Kir1 (AeKir) channel and larvicides

Abstract

Zika virus (ZIKV), dengue fever (DENV) and chikungunya (CHIKV) are arboviruses that are spread to humans from the bite of an infected adult female Aedes aegypti mosquito. As there are no effective vaccines or therapeutics for these diseases, the primary strategy for controlling the spread of these viruses is to prevent the mosquito from biting humans through the use of insecticides. Unfortunately, the commonly used classes of insecticides have seen a significant increase in resistance, thus complicating control efforts. Inhibiting the renal inward rectifier potassium (Kir) channel of the mosquito vector Aedes aegypti has been shown to be a promising target for the development of novel mosquitocides. We have shown that Kir1 channels play key roles in mosquito diuresis, hemolymph potassium homeostasis, flight, and reproduction. Previous work from our laboratories identified a novel (phenylsulfonyl)piperazine scaffold as potent AeKir channel inhibitors with activity against both adult and larval mosquitoes. Herein, we report further SAR work around this scaffold and have identified additional compounds with improved in vitro potency and mosquito larvae toxicity.

Graphical Abstract

Mosquito killers: A series of 5-membered heterocyclic analogs have been designed, synthesized and biologically evaluated agains the Aedes aegypti Kir channel. The compounds were found to be potent inhibitors of the ion channel at nanomolar concentrations. Additionally, select compounds were shown to be toxic to multiple strains of Aedes aegypti larvae at 48h.

Introduction

Zika virus (ZIKV) is an arbovirus originating in Africa and Asia that was introduced to South America in 2015 and has quickly expanded throughout the Americas, and is responsible for the high number of reported cases of microcephaly and Guillain-Barré syndrome.^[1] In 2016, the World Health Organization declared the emerging Zika situation as a “public health emergency of international concern”. The virus is primarily transmitted by mosquitoes of the genus Aedes, but the urban-dwelling, anthropophilic Aedes aegypti is suspected as the most important species contributing to the transmission of ZIKV. In addition to ZIKV, Ae. aegypti is the primary vector of the arboviruses that cause chikungunya (CHIKV) and dengue fever (DENV) in humans, which are important diseases that are emerging and reemerging around the globe. Hundreds of millions of people are infected with DENV each year causing hundreds of thousands of hospitalizations and tens of thousands of deaths.^{[2, 3]} Furthermore, there exists substantial economic burdens from DENV with estimated costs of $2.1 billion in the Americas ^[4] and nearly $1 billion in Southeast Asia.^[5] CHIKV was originally confined to Africa and Southeast Asia, but within the last decade has spread to Europe and further spread to the Americas, where over 400,000 cases and autochthonous transmission have already been reported.^{[6, 7]}

There are no effective vaccines and therapeutics available for these viruses and thus, the primary strategy for controlling these diseases is to prevent the mosquito vectors from biting humans, typically by the use of insecticides and/or insecticide-treated materials.^[8–10] However, the progression of resistance in mosquitoes to commonly used classes of insecticides that target the nervous system (e.g., pyrethroids) has severely complicated control efforts.^[10] In addition, as these commonly used insecticides are typically broad-spectrum (i.e., not mosquito selective) their use has been implicated in the decline of pollinators in natural and managed ecosystems (e.g., the honeybee Apis mellifera).^[11] Thus, to overcome the challenge of insecticide resistance in mosquitoes, new control agents with novel modes of action and improved specificity for mosquitoes are needed.

We have previously shown that an inwardly rectifying potassium (Kir1) channel is a promising target for the development of mosquitocides with novel modes of action. Kir1 is expressed in the mosquito Malpighian tubules and ovaries and plays a critical role in renal excretory capacity, hemolymph K⁺ homeostasis, flight and reproduction. We have published on a variety of scaffolds showing toxicity to adult female mosquitoes.^[12–15] More recently, we discovered a new (phenylsulfonyl)piperazine scaffold with superior in vitro potency against AeKir in both a Thallium-flux high-throughput assay as well as in patch clamp assays (Figure 1).^[16] Herein we report further efforts on the structure-activity relationships around this scaffold and their effects on toxicity against mosquito larvae.

Figure 1.

Previously reported (phenylsulfonyl)piperazine inhibitors of AeKir.

Results and Discussion

Chemistry

The synthetic scheme for the synthesis of the 4-nitrophenylpiperazine analogs has been previously reported and is shown in Scheme 1.^[16] First, piperazine, 4, is reacted with the sulfonyl chloride, 5, to yield the key intermediate, 6, in quantitative yield. Next, 6 and 4-bromo-2-fluoro-nitrobenzene, 7, were coupled via palladium catalysis (Pd(OAc)₂, (±)-BINAP) to yield the penultimate intermediate, 8, in good yield (68%). The final targets were completed by the addition of the appropriate amine to 8 under basic conditions to yield compounds 9a-u, in varying yields (4–68%). The additional synthetic targets outlined in Table 1 procedures are compiled in the Supplemental Information.^[16]

Scheme 1.

General synthetic scheme for analogs.

Reagents and conditions: (a) CH₂Cl₂, 0°C, quant.; (b) Pd(OAc)₂, (±)-BINAP, K₂CO₃, dioxane, reflux, 16 h; 68%; (c) R-NH₂, Et₃N, DMSO, 60 °C, 16h, 4–68%

Table 1.

SAR of key nitro- and cyanobenzene derivatives.

Cmpd	Structure	AeKir Thallium Flux IC50 [μM][a]	% Inhibition[b]
1		0.47 ± 0.02	100
9a		1.29 ± 0.10	96
9b		0.713 ± 0.084	110
9c		1.65 ± 0.13	95
9d		0.546 ± 0.045	92
9e		4.51 ± 0.451	85
9f		0.736 ± 0.011	109
9g		0.441 ± 0.019	110
9h		0.496 ± 0.69	97
9i		1.50 ± 10.4	107
9j		0.820 ± 0.091	111
9k		0.177 ± 0.010	98
9l		0.905 ± 0.032	109
9m		0.108 ± 0.010	99
9n		0.147 ± 0.001	100
9o		0.609 ± 0.035	106
9p		0.190 ± 0.109	109
9q		0.359 ± 0.005	109
9r		30.4[c]	99
9s		0.381 ± 0.005	61
9t		0.381 ± 0.005	10
9u		6.12 ± 1.68	29
12a		0.237 ± 0.012	101
12b		0.404 ± 0.026	89
12c		1.56 ± 0.092	104
12d		0.555 ± 0.028	105
15a		17.5 ± 26.4	15
15b		>50	16
15c		11.1 ± 7.9	8.4
15d		>50	18
21a		9.8 ± 11.5	17
8		2.43 ± 0.28	100

^[a]aThallium flux AeKir; IC₅₀ values represent the average (mean ± SEM) of values obtained from at least three individual experiments.

^[b]% inhibition is the inhibition at the highest concentration (30 mM). The data were normalized to baseline thallium flux in cells where the channel was left uninduced by tetracycline in the HEK293 cell line).

^[c]n = 1 test.

Biology and SAR

The SAR efforts started with the previously reported 2-furylmethyl derivative (9a) which lost 3-fold in potency compared to the previous compound, 1, (9a, IC₅₀ = 1.29 mM vs. 1, IC₅₀ = 0.467 mM) (Table 1)^[16]. From this compound, we then turned our attention to a more detailed scan of 5-membered heterocycles in this area of the molecule. For the SAR testing, we utilized a fluorescence-based in vitro thallium (Tl⁺) flux assay previously described, which allows for a high-throughput screening of molecules.^{[12, 16, 17]} Moving from the 2-furyl to the 3-furyl regained some activity (9b, IC₅₀ = 0.713 mM) against AeKir. Addition of a methyl group to the benzylic position led to a further erosion of activity (9c, IC₅₀ = 1.65 mM). Interestingly, addition of a methyl to the 3-position of the furan (9d, IC₅₀ = 0.546 mM) was a productive change to the molecule, bringing the activity back in line with the 3-pyridine derivative 1; however, moving the methyl to the 5-position led to a loss of activity (9e, IC₅₀ = 4.65 mM). Moving to the isoxazole led to an interesting and surprising result as the 5-methylisoxazole (9f, IC₅₀ = 0.736 mM) regained activity, even though the methyl group is in a similar orientation as the 5-methylfuran, 9e. Further improvements in potency were realized with the 5-methyl-1,3,4-oxadiazole moiety, 9g (IC₅₀ = 0.441 mM). Similar activity trends were seen when moving to the thiazole analogs (9h-j) as were seen with the furan analogs. The 2-thiazole (9h, IC₅₀ = 0.496 mM) retained activity; however, when the benzylic position was methylated, a 3-fold loss in potency was seen (9i, IC₅₀ = 1.50 mM). The 2-methyl-5-thiazole regained 2-fold potency (similar to what was seen in the isoxazole analogs) (9j, IC₅₀ = 0.820 mM), potentially giving some information regarding the placement of the heteroatoms in the 5-membered ring system.

The next set of analogs we analyzed were the 2-oxazole and 5-oxazole ring systems (9k-o), which provided our most potent compounds to date. The 2-oxazole analog, 9k (IC₅₀ = 0.177 mM), was 3-fold more potent than the corresponding 2-thiazole, 9h (IC₅₀ = 0.496 mM) and 7-fold more active than the 2-furan, 9a (IC₅₀ = 1.29 mM). Addition of a methyl substituent to the benzylic position, again, led to a loss of potency (9l, IC₅₀ = 0.905 mM). The regioisomeric 5-oxazole compound was the most potent compound from this scaffold that we have identified (9m, IC₅₀ = 0.108 mM). Addition of a methyl group to the oxazole led to potent compounds; however, the 2-methyl-5-oxazole lost considerable potency versus the 5-methyl-2-oxazole (9o, IC₅₀ = 0.609 mM vs. 9n, IC₅₀ = 0.147 mM). The potency was retained moving from the oxazole to the 1,2,4-oxadiazole (9p, IC₅₀ = 0.190 mM) or the 5-methyl-1,2,4-oxadiazole (9q, IC₅₀ = 0.359 mM) albeit with a slight loss of activity for the 5-methyl derivative. Interestingly, the 3-isoxazole analog lost all activity (9r, IC₅₀ = 30.4 mM). Lastly, the 5-methyl-1,3,4-thiadiazole analog retained potency (9s, IC₅₀ = 0.381 mM), which is 2-fold more potent than the similarly substituted 2-methyl-5-thiazole, 9j. Lastly, extending the ring system in this area to a benzofuran or benzoisoxaole led to dramatic results. The 2-benzofuran, 9t, retained activity compared to the 2-benzisoxazole, 9u, which lost significant potency (IC₅₀ = 0.381 mM vs. 6.12 mM, respectively).

Moving from the nitrobenzene core to the benzonitrile core led to a few interesting observations. First, the unsubstituted 2-amino analog, 12a, was the most potent compound in this series (IC₅₀ = 0.237 mM). Further substitution of the aniline with the oxazole moieties led to potent compounds (12b-d, IC₅₀ = 0.404 – 1.56 mM); however, the benzonitrile compounds were less potent than their nitrobenzene counterparts. Additional analogs were synthesize moving the nitro or cyano group from para to the piperazine to ortho was an unproductive change as all compounds were significantly less potent (15a-d, 21a, IC₅₀ > 9.8 mM), which is in contrast to the pyridine analog, 2. Lastly, the simplified 3-fluoro-4-nitro derivative, 8, had reduced activity (IC₅₀ = 2.43 mM).

Having identified a number of potent AeKir inhibitors, we next screened a selection of these compounds for larvae toxicity at 100 μM and compared their efficacies to previously reported VU854.[16] As with other compounds, the new analogs had limited activity at 24 h (<25%, data not shown); however, many of the new analogs had significant mortality at 48 h (Figure 2). Compounds 8 (100%), 9p (99%) and 12d (98%) showed significantly greater mortality at 48 h compared to VU854 (shown in red, Figure 2).[16] A number of other compounds (9n, 9q, 9s, 12a and 12b) were similarly efficacious as VU854 (shown in grey, Figure 2), whereas others were less efficacious than VU854 (shown in blue, Figure 2). These results are intriguing as compounds that are active in the larval toxicity assay are not the most potent in vitro (e.g., 8) which raises questions to whether a compound such as 8 is more permeable versus other, more potent, compounds (e.g., 9k). In addition, 8 is devoid of the amino substituent which, previously, has been important for activity (e.g., hydrogen substituents were inactive). Further work concentrated on determining the effects of in vitro potency on larval toxicity are underway and will be reported in due course.

Figure 2.

48 h mortality of first instar (Liverpool, LVP) Ae. aegypti after addition of small molecules (100 μM) to the rearing water. Significance was determined by a one-way ANOVA and Dunnett’s multiple comparison’s test: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Red bars indicate significantly greater than 854. Blue bars indicate significantly lower. Values are mean ± SEM based on at least 12 replicates.

Lastly, we evaluated the top three compounds (8, 9p, 12d) in a concentration response curve (CRC) experiment against both the LVP (pyrethroid-susceptible) and the Puerto Rico (PR) strain (pyrethroid-resistant) to assess their larval toxicity (Figure 3). As can be seen in Figure 3A, within 48 h, VU854 is equipotent against both strains with maximal mortality reaching ~50% at 100 µM. Both 8 and 12d, reached 100% mortality within 48 h at 100 mM, with 12d showing no differences between the strains, whereas 8 was ~2-times more potent against the LVP strain. Disappointingly, in the CRC experiments, 9p did not recapitulate the high efficacy found in the screening experiments (Figure 2) even after repeated testing. Its maximal mortality was limited to ~50% within 48 h at 100 µM. However, 9p consistently produced a unique pathology in the larvae (Figure 3E) wherein an accumulation of a contiguous trail of excrement (magenta arrows) that began in the midgut and extended continuously beyond the rectum, indicative of constipation or excretory failure. We have not seen this result with any other compound tested.

Figure 3.

(A–D) 48 h mortality of first instar (LVP or PR) Ae. aegypti after addition of small molecules to the rearing water at various concentrations. (E) Excretory failure (magenta arrows) observed in larvae treated with 9p.

Conclusion.

We have reported on further SAR evaluation of our previously reported (phenylsulfonyl)piperazine scaffold, by expanding on the heterocyclic moieties explored around the phenyl ring. We have discovered a number of compounds that are more potent in the Thallium-flux in vitro assay (<200 nM). SAR results indicate that the oxazole and oxadiazole heterocycles were the optimal group, and cyano for nitro substitution was also tolerated. In addition, 8, 9p and 12d show excellent efficacy against Aedes aegypti larvae in a 48 h first instar toxicity assay and 8 and 12d were potent in a CRC against both the LVP and PR strains of Ae. aegypti, suggesting these compounds could be used to combat resistance seen in pyrethroids. Compound 8 was also unique as it did not contain the heterocyclic substituted amino group that has been seen to be optimal in previous studies. Further optimization and adult lethality studies are on-going and will be reported in due course.

Experimental Section

Chemistry.

All ¹H and ¹³C NMR spectra were recorded on Bruker AV-400 (500 MHz) instrument. Chemical shifts are reported in ppm relative to residual solvent peaks as an internal standard set to δH 7.26 or δC 77.0 (CDCl₃). Data are reported as follows: chemical shift, multiplicity (br = broad, s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), coupling constant (Hz), and integration. Low resolution mass spectra were obtained on an Agilent 1260 LCMS with electrospray ionization, with a gradient of 5–95% MeCN in 0.1% formic acid water over 4 min. Analytical thin layer chromatography was performed on LuxPlate silica gel 60 F254 plates. Visualization was accomplished with UV light, and/or the use of ninhydrin, anisaldehyde and ceric ammonium molybdate solutions followed by charring on a hot-plate. Chromatography on silica gel was performed using Silica Gel 60Å (230–400 mesh) from Sorbent Technologies. Solvents for extraction, washing and chromatography were HPLC grade. All reagents were purchased from Aldrich Chemical Co. (or similar) and were used without purification. All reagents and solvents were commercial grade and purified prior to use when necessary. Microwave synthesis was performed on an Anton Paar Monowave 400 equipped with an autosampler. Final compounds were purified on a Gilson preparative reversed-phase HPLC system comprised of a 322 aqueous pump with solvent-selection valve, 334 organic pump, GX-271 liquid hander, two column switching valves, and a 159 UV detector. UV wavelength for fraction collection was user-defined, with absorbance at 254 nm always monitored. Column: Phenomenex Axia-packed Luna C18, 50 × 21.2 mm, 5 μm. For Acidic Method: Mobile phase: CH₃CN in H₂O (0.1% formic acid). Gradient conditions: 2.0 min equilibration, followed by user-defined gradient (starting organic percentage, ending organic percentage, duration, typically 15 mins), hold at 95% CH₃CN in H₂O (0.1% TFA) for 2 min, 20 mL/min, 23° C.

General Procedure I. 1-((4-fluorophenyl)sulfonyl)piperazine (6).

A round bottom flask was charged with piperazine, 4, (2.10 g, 24.0 mmol). The flask was cooled to 0 °C and dichloromethane (40.0 mL) and 4-fluorobenzenesulfonyl chloride, 5, (7.78 mg, 4.00 mmol) were added. After stirring at 0 °C for 3h, the reaction was diluted with dichloromethane (50.0 mL) and washed with saturated NaHCO₃ (50.0 mL). The organic layer was isolated, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced vacuum. The crude 1-((4-fluorophenyl)sulfonyl)piperazine, 3, was isolated as a white solid and used without further purification. Yield = 0.98 g (Quantitative yield). LCMS: R_T = 1.645 min., >95% purity at 215 and 254 nm, m/z = 245.0 [M + H]⁺. Matched previously reported literature.

General Procedure II. 1-(3-fluoro-4-nitrophenyl)-4-((4-fluorophenyl)sulfonyl)piperazine (8).

A round bottom flask was charged with 4-bromo-2-fluoronitrobenzene, 7, (1.10 g, 5.00 mmol), 1-((4-fluorophenyl)sulfonyl)piperazine, 6, (1.22 g, 5.00 mmol), palladium(II) acetate (22.0 mg, 0.100 mmol), (±)-2,2’-Bis(diphenylphosphino)-1,1’-binaphthalene (93.0 mg, 0.150 mmol), potassium carbonate (1.39 g, 10.0 mmol), and dioxane (25 mL). The reaction was purged with argon atmosphere and refluxed for 16h. The reaction was cooled to room temperature and filtered through packed celite. The celite was rinsed with ethyl acetate (50.0 mL) and then the filtrate was concentrated under reduced vacuum. The crude product was purified by column chromatography (0–100% ethyl acetate:hexanes) and isolated as a yellow solid. Yield = 0.26 g (68%). ¹H NMR (499 MHz, CDCl₃) δ 8.00 (t, J = 9.1 Hz, 1H), 7.80 (ddd, J = 7.8, 4.9, 2.4 Hz, 2H), 7.29–7.20 (m, 2H), 6.56 (dd, J = 9.4, 2.6 Hz, 1H), 6.50 (dd, J = 14.5, 2.7 Hz, 1H), 3.54–3.47 (m, 4H), 3.21–3.12 (m, 4H). ¹³C NMR (126 MHz, CDCl₃) δ 166.9, 164.8, 159.3, 157.2, 155.3, 155.2, 131.7, 131.7, 130.9, 130.8, 128.6, 117.1, 116.9, 109.3, 109.3, 102.4, 102.2, 47.0, 45.7. LCMS: R_T = 2.746 min., >98% purity at 215 and 254 nm, m/z = 384.0 [M + H]⁺.

5-(4-((4-fluorophenyl)sulfonyl)piperazin-1-yl)-2-nitroanilines (9a-u).

A vial was charged with 1-(3-fluoro-4-nitrophenyl)-4-((4-fluorophenyl)sulfonyl)piperazine, 8, (50.0 mg, 0.130 mmol). The amine of interest (0.260 mmol), triethylamine (55.0 μL, 0.391 mmol), and dimethyl sulfoxide (1.00 mL) were added to the vial. The reaction was heated at 60 °C for 16h. After cooling to room temperature, the reaction was loaded onto silica gel and purified by flash column chromatography (0–20% methanol:dichloromethane).

5-(4-((4-fluorophenyl)sulfonyl)piperazin-1-yl)-N-(furan-2-ylmethyl)-2-nitroaniline (9a)^[16]

The compound was synthesized following Procedure II. Yield = 60 mg (39%). ¹H NMR (499 MHz, CDCl₃) δ 8.64 (s, 1H), 8.08 (d, J = 9.6 Hz, 1H), 7.87–7.76 (m, 2H), 7.41 (d, J = 11.2 Hz, 2H), 7.26 (d, J = 7.7 Hz, 1H), 6.37 (s, 1H), 6.29 (s, 1H), 6.19 (d, J = 9.6 Hz, 1H), 6.01 (s, 1H), 4.48 (d, J = 4.1 Hz, 2H), 3.48 (s, 4H), 3.16 (s, 4H). ¹³C NMR (126 MHz, CDCl₃) δ 155.2, 150.9, 146.9, 142.3, 130.5, 130.4, 129.1, 125.3, 125.0, 116.7, 116.5, 110.6, 107.6, 104.7, 95.1, 46.8, 45.5, 40.3, 29.7. LCMS: R_t = 2.836 min, >98% purity 215 and 254 nm, m/z = 461.1 [M + H]⁺.

5-(4-((4-fluorophenyl)sulfonyl)piperazin-1-yl)-N-(1-(furan-2-yl)ethyl)-2-nitroaniline (9b).

The compound was synthesized following Procedure II and isolated as a yellow solid. Yield = 40 mg (64%). ¹H NMR (499 MHz, CDCl₃) δ 8.53 (d, J = 6.2 Hz, 1H), 8.04 (d, J = 9.7 Hz, 1H), 7.83–7.74 (m, 2H), 7.34 (d, J = 1.2 Hz, 1H), 7.29–7.19 (m, 2H), 6.31 (dd, J = 3.1, 1.9 Hz, 1H), 6.20–6.10 (m, 2H), 5.91 (d, J = 2.4 Hz, 1H), 4.71 (p, J = 6.7 Hz, 1H), 3.48–3.32 (m, 4H), 3.10 (t, J = 5.1 Hz, 4H), 1.66 (d, J = 6.8 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 207.2, 166.8, 164.8, 156.1, 155.4, 146.6, 142.1, 131.8, 131.8, 130.8, 130.7, 129.3, 125.5, 117.0, 116.8, 110.7, 105.8, 105.0, 96.1, 47.1, 47.1, 45.8, 31.3, 21.4. LCMS: R_T = 2.958 min., >95% purity 215 and 254 nm, m/z = 475.1 [M + H]⁺.

5-(4-((4-fluorophenyl)sulfonyl)piperazin-1-yl)-N-((3-methylfuran-2-yl)methyl)-2-nitroaniline (9c).

The compound was synthesized following Procedure II and isolated as a yellow solid. Yield = 5.0 mg (16%). ¹H NMR (499 MHz, CDCl₃) δ 8.52 (s, 1H), 8.05 (d, J = 9.6 Hz, 1H), 7.87–7.76 (m, 2H), 7.26–7.21 (m, 3H), 6.22 (d, J = 1.6 Hz, 1H), 6.16 (dd, J = 9.7, 2.5 Hz, 1H), 6.02 (d, J = 2.5 Hz, 1H), 4.38 (d, J = 5.2 Hz, 2H), 3.53–3.41 (m, 4H), 3.20–3.09 (m, 4H), 2.05 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 155.2, 147.0, 145.8, 141.4, 130.5, 130.4, 129.1, 125.2, 117.4, 116.7, 116.5, 113.4, 104.6, 95.0, 46.8, 45.5, 38.5, 9.9. LCMS: R_T = 2.940 min., >98% @ 215 and 254 nm, m/z = 475.1 [M+H]⁺.

5-(4-((4-fluorophenyl)sulfonyl)piperazin-1-yl)-N-((5-methylfuran-2-yl)methyl)-2-nitroaniline (9d).

The compound was synthesized following Procedure IV and isolated as a yellow solid. Yield = 11 mg (10%). ¹H NMR (499 MHz, CDCl₃) δ 7.78 (ddd, J = 6.9, 5.0, 2.4 Hz, 2H), 7.30–7.21 (m, 3H), 6.70 (s, 1H), 6.63 (d, J = 8.4 Hz, 1H), 6.47 (d, J = 8.0 Hz, 1H), 4.45 (s, 2H), 3.18–3.03 (m, 8H), 2.29 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 166.7, 164.7, 159.7, 150.2, 147.4, 142.5, 133.6, 133.3, 132.1, 132.1, 130.8, 130.7, 122.8, 117.0, 116.8, 110.3, 108.7, 52.0, 46.6, 41.2, 11.3. LCMS: R_T = 2.943 min., >95% purity 215 and 254 nm, m/z = 475.1 [M + H]⁺.

5-(4-((4-fluorophenyl)sulfonyl)piperazin-1-yl)-N-(furan-3-ylmethyl)-2-nitroaniline (9e).

The compound was synthesized following Procedure II and isolated as a yellow solid. Yield = 41 mg (68%). ¹H NMR (499 MHz, CDCl₃) δ 8.52 (s, 1H), 8.06 (d, J = 9.7 Hz, 1H), 7.83–7.75 (m, 2H), 7.42 (dd, J = 5.2, 3.6 Hz, 2H), 7.26–7.19 (m, 2H), 6.40 (s, 1H), 6.16 (dd, J = 9.7, 2.6 Hz, 1H), 5.91 (d, J = 2.5 Hz, 1H), 4.31 (d, J = 5.2 Hz, 2H), 3.49–3.38 (m, 4H), 3.16–3.08 (m, 4H). ¹³C NMR (126 MHz, CDCl₃) δ 155.5, 147.4, 144.2, 140.2, 130.8, 130.8, 129.4, 125.5, 122.1, 117.0, 116.9, 110.1, 104.9, 95.4, 47.1, 45.8, 38.8. LCMS: R_T = 2.856 min., >95% purity at 215 and 254 nm, m/z = 461.1 [M + H]⁺.

5-(4-((4-fluorophenyl)sulfonyl)piperazin-1-yl)-N-((5-methylisoxazol-3-yl)methyl)-2-nitroaniline (9f).

The compound was synthesized following Procedure II and isolated as a yellow solid. Yield = 6.0 mg (10%). LCMS: ¹H NMR (499 MHz, CDCl₃) δ 8.69 (t, J = 5.9 Hz, 1H), 8.08 (d, J = 9.6 Hz, 1H), 7.85–7.77 (m, 2H), 7.23 (d, J = 8.5 Hz, 2H), 6.20 (dd, J = 9.7, 2.5 Hz, 1H), 6.03 (s, 1H), 5.89 (d, J = 2.5 Hz, 1H), 4.56 (d, J = 6.0 Hz, 2H), 3.48–3.41 (m, 4H), 3.16–3.11 (m, 4H), 2.27 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 168.8, 160.2, 155.3, 146.4, 130.5, 130.5, 129.2, 125.5, 116.7, 116.5, 104.9, 103.0, 94.9, 46.7, 45.5, 39.0, 11.5. R_T = 2.740 min., >98% purity at 215 and 254 nm, m/z = 476.1 [M + H]⁺.

5-(4-((4-fluorophenyl)sulfonyl)piperazin-1-yl)-N-((5-methyl-1,3,4-oxadiazol-2-yl)methyl)-2-nitroaniline (9g).

The compound was synthesized following Procedure II and isolated as a yellow solid. Yield = 16 mg (25%). ¹H NMR (499 MHz, CDCl₃) δ 8.69 (t, J = 6.0 Hz, 1H), 8.11–8.01 (m, 1H), 7.86–7.76 (m, 2H), 7.27–7.21 (m, 2H), 6.26–6.16 (m, 2H), 4.67 (d, J = 6.3 Hz, 2H), 3.55–3.47 (m, 4H), 3.18–3.09 (m, 4H), 2.51 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 164.8, 163.1, 155.2, 146.1, 130.6, 130.5, 129.3, 125.4, 116.7, 116.5, 104.9, 95.1, 46.6, 45.5, 37.7, 11.1. LCMS: R_T = 2.559 min., >98% purity at 215 and 254 nm, m/z = 477.1 [M + H]⁺.

5-(4-((4-fluorophenyl)sulfonyl)piperazin-1-yl)-2-nitro-N-(thiazol-2-ylmethyl)aniline (9h).

The compound was synthesized following Procedure II and isolated as a yellow solid. Yield = 3.0 mg (10%). ¹H NMR (499 MHz, CDCl₃) δ 8.92 (t, J = 5.8 Hz, 1H), 8.07 (d, J = 9.6 Hz, 1H), 7.78 (dd, J = 8.8, 5.0 Hz, 2H), 7.74 (d, J = 3.3 Hz, 1H), 7.32 (d, J = 3.3 Hz, 1H), 7.24 (t, J = 8.5 Hz, 3H), 6.17 (dd, J = 9.6, 2.5 Hz, 1H), 6.01 (d, J = 2.4 Hz, 1H), 4.82 (d, J = 6.0 Hz, 2H), 3.44–3.36 (m, 4H), 3.11–3.06 (m, 4H). ¹³C NMR (126 MHz, CDCl₃) δ 155.0, 152.9, 146.5, 142.5, 130.5, 130.4, 129.2, 120.1, 116.7, 116.6, 116.5, 104.8, 95.6, 46.6, 45.4, 45.1. LCMS: R_T = 2.702 min., >98% purity at 215 and 254 nm, m/z = 478.1 [M + H]⁺.

5-(4-((4-fluorophenyl)sulfonyl)piperazin-1-yl)-2-nitro-N-(1-(thiazol-2-yl)ethyl)aniline (9i).

The compound was synthesized following Procedure II and isolated as a yellow oil. Yield = 14 mg (21%). ¹H NMR (499 MHz, CDCl₃) δ 8.68 (d, J = 5.2 Hz, 1H), 8.05 (d, J = 9.6 Hz, 1H), 7.80–7.75 (m, 2H), 7.71 (d, J = 3.2 Hz, 1H), 7.27 (d, J = 3.3 Hz, 1H), 7.23 (t, J = 8.4 Hz, 2H), 6.14 (dd, J = 9.7, 2.5 Hz, 1H), 5.92 (d, J = 2.5 Hz, 1H), 5.00 (p, J = 6.7 Hz, 1H), 3.41–3.27 (m, 4H), 3.11–3.01 (m, 4H), 1.78 (d, J = 6.8 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 175.9, 155.2, 146.2, 142.0, 130.8, 130.8, 129.4, 125.6, 119.9, 117.0, 116.8, 105.1, 96.6, 52.2, 46.9, 45.7, 23.9. LCMS: R_T = 2.801 min., >95% purity at 215 and 254 nm, m/z = 492.1 [M + H]⁺.

5-(4-((4-fluorophenyl)sulfonyl)piperazin-1-yl)-N-((2-methylthiazol-5-yl)methyl)-2-nitroaniline (9j).

The compound was synthesized following Procedure II and isolated as an orange solid. Yield = 38 mg (59%). ¹H NMR (499 MHz, CDCl₃) δ 8.63 (t, J = 5.4 Hz, 1H), 8.05 (d, J = 9.7 Hz, 1H), 7.83–7.75 (m, 2H), 7.52 (s, 1H), 7.26–7.20 (m, 2H), 6.18 (dd, J = 9.7, 2.5 Hz, 1H), 5.92 (d, J = 2.5 Hz, 1H), 4.62 (d, J = 5.5 Hz, 2H), 3.48–3.39 (m, 4H), 3.18–3.11 (m, 4H), 2.68 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 166.9, 166.8, 164.8, 155.5, 146.8, 140.6, 135.3, 131.8, 131.7, 130.8, 130.8, 129.5, 125.6, 117.0, 116.9, 105.2, 95.3, 47.0, 45.8, 40.1, 31.2, 19.8. LCMS: R_T = 2.711 min., >95% purity at 215 and 254 nm, m/z = 492.1 [M + H]⁺.

5-(4-((4-fluorophenyl)sulfonyl)piperazin-1-yl)-2-nitro-N-(oxazol-2-ylmethyl)aniline (9k).

The compound was synthesized following Procedure II and isolated as a yellow solid. Yield = 5.0 mg (17%). ¹H NMR (499 MHz, CDCl₃) δ 8.77 (t, J = 5.5 Hz, 1H), 8.06 (d, J = 9.6 Hz, 1H), 7.84–7.76 (m, 2H), 7.63 (s, 1H), 7.24 (t, J = 8.5 Hz, 2H), 7.08 (s, 1H), 6.18 (dd, J = 9.6, 2.5 Hz, 1H), 6.12 (d, J = 2.4 Hz, 1H), 4.59 (d, J = 6.0 Hz, 2H), 3.58–3.43 (m, 4H), 3.21–3.09 (m, 4H). ¹³C NMR (126 MHz, CDCl₃) δ 160.5, 155.2, 146.6, 139.6, 130.5, 130.4, 129.2, 127.2, 125.4, 116.7, 116.5, 104.7, 95.3, 46.7, 45.5, 40.3. LCMS: R_T = 2.607 min., >98% purity at 215 and 254 nm, m/z = 462.0 [M + H]⁺.

5-(4-((4-fluorophenyl)sulfonyl)piperazin-1-yl)-2-nitro-N-(1-(oxazol-2-yl)ethyl)aniline (9l).

The compound was synthesized following Procedure II and isolated as a yellow solid. Yield = 20 mg (33%). ¹H NMR (499 MHz, CDCl₃) δ 8.57 (d, J = 6.0 Hz, 1H), 8.03 (d, J = 9.6 Hz, 1H), 7.85–7.75 (m, 2H), 7.59 (s, 1H), 7.30–7.20 (m, 2H), 7.05 (s, 1H), 6.15 (dd, J = 9.6, 2.5 Hz, 1H), 6.09 (d, J = 2.4 Hz, 1H), 4.84 (p, J = 6.8 Hz, 1H), 3.49–3.36 (m, 4H), 3.17–3.05 (m, 4H), 1.75 (d, J = 6.9 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 166.8, 164.8, 164.5, 155.4, 146.3, 139.6, 131.9, 131.8, 130.8, 130.8, 129.4, 127.2, 125.5, 117.0, 116.8, 105.0, 96.0, 47.3, 47.0, 45.8, 31.3, 20.2. LCMS: R_T = 2.741 min., >95% purity at 215 and 254 nm, m/z = 476.1 [M + H]⁺.

5-(4-((4-fluorophenyl)sulfonyl)piperazin-1-yl)-2-nitro-N-(oxazol-5-ylmethyl)aniline (9m).

The compound was synthesized following Procedure II and isolated as a yellow solid. Yield = 8.0 mg (28%). ¹H NMR (499 MHz, CDCl₃) δ 8.62 (t, J = 5.4 Hz, 1H), 8.08 (d, J = 9.6 Hz, 1H), 7.86 (s, 1H), 7.83–7.76 (m, 2H), 7.24 (d, J = 8.5 Hz, 2H), 7.02 (s, 1H), 6.20 (dd, J = 9.7, 2.5 Hz, 1H), 5.93 (d, J = 2.4 Hz, 1H), 4.55 (d, J = 5.7 Hz, 2H), 3.51–3.42 (m, 4H), 3.21–3.10 (m, 4H). ¹³C NMR (126 MHz, CDCl₃) δ 166.5, 164.5, 155.3, 146.5, 131.4, 130.5, 130.5, 129.2, 125.5, 116.7, 116.6, 104.9, 94.8, 46.7, 45.5, 30.9. LCMS: R_T = 2.607 min., >98% purity at 215 and 254 nm, m/z = 462.0 [M + H]⁺.

5-(4-((4-fluorophenyl)sulfonyl)piperazin-1-yl)-N-((5-methyloxazol-2-yl)methyl)-2-nitroaniline (9n).

The compound was synthesized following Procedure II and isolated as a yellow solid. Yield = 18 mg (57%). ¹H NMR (499 MHz, CDCl₃) δ 8.73 (s, 1H), 8.05 (d, J = 10.2 Hz, 1H), 7.80 (dd, J = 8.5, 5.0 Hz, 2H), 7.24 (dd, J = 14.9, 6.5 Hz, 2H), 6.67 (s, 1H), 6.17 (d, J = 7.0 Hz, 2H), 4.51 (d, J = 5.0 Hz, 2H), 3.53–3.43 (m, 4H), 3.21–3.08 (m, 4H), 2.29 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 166.5, 164.4, 155.1, 146.7, 131.6, 130.5, 130.4, 129.1, 125.3, 116.7, 116.5, 104.6, 95.4, 46.7, 45.5, 40.4, 10.9. LCMS: R_T = 2.722 min., >98% purity at 215 and 254 nm, m/z = 476.1 [M + H]⁺.

5-(4-((4-fluorophenyl)sulfonyl)piperazin-1-yl)-N-((2-methyloxazol-5-yl)methyl)-2-nitroaniline (9o).

The compound was synthesized following Procedure II and isolated as a yellow solid. Yield = 12 mg (10%). ¹H NMR (499 MHz, DMSO) δ 8.60 (t, J = 5.4 Hz, 1H), 7.97–7.83 (m, 4H), 7.54 (t, J = 8.7 Hz, 2H), 6.39 (dd, J = 9.7, 1.3 Hz, 1H), 6.18 (d, J = 1.8 Hz, 1H), 4.42 (d, J = 5.4 Hz, 2H), 3.64–3.54 (m, 4H), 3.08–2.99 (m, 4H), 2.42 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 166.7, 164.7, 162.2, 155.6, 147.7, 137.7, 137.0, 132.1, 132.1, 131.6, 131.5, 129.1, 124.3, 117.8, 117.6, 105.6, 95.2, 46.7, 46.3, 38.9, 14.4. LCMS: R_T = 2.686 min., >95% purity at 215 and 254 nm, m/z = 476.1 [M + H]⁺.

N-((1,2,4-oxadiazol-3-yl)methyl)-5-(4-((4-fluorophenyl)sulfonyl)piperazin-1-yl)-2-nitroaniline (9p).

The compound was synthesized following Procedure II and isolated as an orange solid. Yield = 8.0 mg (13%). ¹H NMR (499 MHz, CDCl₃) δ 8.81 (t, J = 5.9 Hz, 1H), 8.74 (s, 1H), 8.08 (d, J = 9.6 Hz, 1H), 7.88–7.79 (m, 2H), 7.26 (d, J = 8.5 Hz, 2H), 6.21 (dd, J = 9.7, 2.5 Hz, 1H), 6.11 (d, J = 2.4 Hz, 1H), 4.68 (d, J = 6.1 Hz, 2H), 3.50 (dd, J = 15.7, 10.5 Hz, 4H), 3.17 (dd, J = 9.8, 4.7 Hz, 4H). ¹³C NMR (126 MHz, CDCl₃) δ 165.8, 130.9, 130.8, 129.6, 117.1, 117.0, 116.9, 116.9, 105.9, 105.2, 95.5, 95.2, 47.1, 47.1, 45.8, 45.8, 38.6, 29.6. LCMS: R_T = 2.651 min., >95% purity at 215 and 254 nm, m/z = 463.1 [M + H]⁺.

5-(4-((4-fluorophenyl)sulfonyl)piperazin-1-yl)-N-((5-methyl-1,2,4-oxadiazol-3-yl)methyl)-2-nitroaniline (9q).

The compound was synthesized following Procedure II and isolated as a yellow solid. Yield = 9.0 mg (15%). ¹H NMR (499 MHz, CDCl₃) δ 8.77 (t, J = 5.8 Hz, 1H), 8.08 (d, J = 9.6 Hz, 1H), 7.91–7.78 (m, 2H), 7.29–7.21 (m, 4H), 6.20 (dd, J = 9.6, 2.5 Hz, 1H), 6.12 (d, J = 2.5 Hz, 1H), 4.57 (d, J = 6.0 Hz, 2H), 3.56–3.45 (m, 4H), 3.20–3.14 (m, 4H), 2.61 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 177.8, 167.6, 164.8, 155.5, 146.8, 131.9, 130.9, 130.8, 129.5, 125.9, 117.0, 116.9, 105.1, 95.5, 47.1, 45.8, 38.8, 31.3, 12.8. LCMS: R_T = 2.693 min., >95% purity at 215 and 254 nm, m/z = 477.1 [M + H]⁺.

5-(4-((4-fluorophenyl)sulfonyl)piperazin-1-yl)-N-(isoxazol-3-ylmethyl)-2-nitroaniline (9r).

The compound was synthesized following Procedure II and isolated as a yellow solid. Yield = 2.0 mg (4%). ¹H NMR (499 MHz, CDCl₃) δ 8.73 (s, 1H), 8.37 (d, J = 1.4 Hz, 1H), 8.05 (d, J = 9.6 Hz, 1H), 7.80 (dd, J = 8.8, 5.0 Hz, 2H), 7.23 (d, J = 8.5 Hz, 2H), 6.35 (d, J = 1.6 Hz, 1H), 6.17 (dd, J = 9.7, 2.5 Hz, 1H), 6.05 (d, J = 2.5 Hz, 1H), 4.59 (d, J = 6.0 Hz, 2H), 3.48–3.40 (m, 4H), 3.16–3.08 (m, 4H). ¹³C NMR (126 MHz, CDCl₃) δ 159.4, 155.1, 146.7, 131.5, 130.5, 130.5, 129.2, 116.7, 116.5, 104.7, 104.6, 103.6, 95.2, 46.6, 45.4, 38.6. LCMS: R_T = 2.709 min., >98% purity at 215 and 254 nm, m/z = 462.1 [M + H]⁺.

5-(4-((4-fluorophenyl)sulfonyl)piperazin-1-yl)-N-((5-methyl-1,3,4-thiadiazol-2-yl)methyl)-2-nitroaniline (9s).

The compound was synthesized following Procedure II and isolated as a yellow solid. Yield = 10 mg (15%). ¹H NMR (499 MHz, CDCl₃) δ 8.82 (t, J = 5.9 Hz, 1H), 8.06 (d, J = 9.6 Hz, 1H), 7.86–7.76 (m, 2H), 7.23 (d, J = 8.5 Hz, 2H), 6.19 (dd, J = 9.6, 2.5 Hz, 1H), 6.14 (d, J = 2.4 Hz, 1H), 4.88 (d, J = 6.2 Hz, 2H), 3.48–3.40 (m, 4H), 3.18–3.08 (m, 4H), 2.75 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 169.6, 167.1, 155.5, 146.5, 130.9, 130.8, 129.6, 125.7, 117.0, 116.8, 105.2, 95.7, 46.9, 45.8, 42.6, 16.2. LCMS: R_T = 2.606 min., >95% purity at 215 and 254 nm, m/z = 493.0 [M + H]⁺.

N-(benzofuran-2-ylmethyl)-5-(4-((4-fluorophenyl)sulfonyl)piperazin-1-yl)-2-nitroaniline (9t).

The compound was synthesized following Procedure II and isolated as a yellow solid. Yield = 34 mg (51%). ¹H NMR (499 MHz, CDCl₃) δ 8.76 (t, J = 5.6 Hz, 1H), 8.07 (d, J = 9.6 Hz, 1H), 7.77 – 7.70 (m, 2H), 7.53 (d, J = 7.6 Hz, 1H), 7.45 (d, J = 8.2 Hz, 1H), 7.35–7.27 (m, 1H), 7.25–7.17 (m, 3H), 6.63 (s, 1H), 6.17 (dd, J = 9.7, 2.5 Hz, 1H), 6.01 (d, J = 2.5 Hz, 1H), 4.63 (d, J = 5.8 Hz, 2H), 3.48–3.40 (m, 4H), 3.10–3.03 (m, 4H). ¹³C NMR (126 MHz, CDCl₃) δ 155.1, 154.9, 153.8, 146.9, 131.4, 130.5, 130.4, 129.2, 128.1, 125.4, 124.4, 123.1, 121.0, 116.7, 116.5, 111.1, 104.7, 104.3, 95.1, 46.7, 45.4, 40.7. LCMS: R_T = 3.304 min., >95% purity at 215 and 254 nm, m/z = 511.1 [M + H]⁺.

N-(benzo[d]oxazol-2-ylmethyl)-5-(4-((4-fluorophenyl)sulfonyl)piperazin-1-yl)-2-nitroaniline (9u).

The compound was synthesized following Procedure II and isolated as a dark yellow solid. Yield = 20 mg (4.0%). ¹H NMR (499 MHz, CDCl₃) δ 8.00 (d, J = 9.1 Hz, 1H), 7.80 (dd, J = 8.7, 5.0 Hz, 2H), 7.72 (dd, J = 6.1, 2.9 Hz, 1H), 7.57–7.50 (m, 1H), 7.37 (p, J = 5.8 Hz, 2H), 7.23 (d, J = 8.5 Hz, 2H), 6.37 (dd, J = 9.1, 1.7 Hz, 1H), 6.29 (s, 1H), 4.68 (s, 2H), 3.15 (m, 8H). ¹³C NMR (126 MHz, CDCl₃) δ 166.8, 164.7, 162.6, 152.4, 151.3, 149.7, 140.8, 133.7, 132.0, 130.8, 130.8, 130.5, 126.0, 125.2, 120.4, 117.0, 116.8, 111.1, 107.0, 103.4, 51.9, 46.5, 41.5. LCMS: R_T = 2.830 min., >95% purity at 215 and 254 nm, m/z = 512.1 [M + H]⁺.