New diarylsulfonamide inhibitors of Leishmania infantum amastigotes

Article abstract of DOI:10.1016/j.ijpddr.2021.02.006

New drugs against visceral leishmaniasis with mechanisms of action differing from existing treatments and with adequate cost, stability, and properties are urgently needed. No antitubulin drug is currently in the clinic against Leishmania infantum, the causative agent of visceral leishmaniasis in the Mediterranean area. We have designed and synthesized a focused library of 350 compounds against the Leishmania tubulin based on the structure-activity relationship (SAR) and sequence differences between host and parasite. The compounds synthesized are accessible, stable, and appropriately soluble in water. We assayed the library against Leishmania promastigotes, axenic, and intracellular amastigotes and found 0, 8, and 16 active compounds, respectively, with a high success rate against intracellular amastigotes of over 10%, not including the cytotoxic compounds. Five compounds have a similar or better potency than the clinically used miltefosine. 14 compounds showed a host-dependent mechanism of action that might be advantageous as it may render them less susceptible to the development of drug resistance. The active compounds cluster in five chemical classes that provide structure-activity relationships for further hit improvement and facilitate series development. Molecular docking is consistent with the proposed mechanism of action, supported by the observed structure-activity relationships, and suggests a potential extension to other Leishmania species due to sequence similarities. A new family of diarylsulfonamides designed against the parasite tubulins is active against Leishmania infantum and represents a new class of potential drugs with favorable cost, stability, and aqueous solubility for the treatment of visceral leishmaniasis (VL). These results could be extended to other clinically relevant species of Leishmania spp.

Full text of DOI:10.1016/j.ijpddr.2021.02.006

Journal Pre-proof
New diarylsulfonamide inhibitors of Leishmania infantum amastigotes
Myriam González, Pedro José Alcolea, Raquel Álvarez, Manuel Medarde, Vicente
Larraga, Rafael Peláez
PII:
S2211-3207(21)00009-9
https://doi.org/10.1016/j.ijpddr.2021.02.006
IJPDDR 395
DOI:
Reference:
To appear in: International Journal for Parasitology: Drugs and Drug
Resistance
Received Date: 13 October 2020
Revised Date: 22 February 2021
Accepted Date: 22 February 2021
Please cite this article as: González, M., Alcolea, Pedro.José., Álvarez, R., Medarde, M., Larraga, V.,
Peláez, R., New diarylsulfonamide inhibitors of Leishmania infantum amastigotes, International Journal
for Parasitology: Drugs and Drug Resistance (2021), doi: https://doi.org/10.1016/j.ijpddr.2021.02.006.
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New diarylsulfonamide inhibitors of Leishmania infantum amastigotes
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Myriam González , Pedro José Alcolea , Raquel Álvarez , Manuel Medarde , Vicente Larraga ,
a,b,c*
Rafael Peláez
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Laboratorio de Química Orgánica y Farmacéutica, Departamento de Ciencias Farmacéuticas, Facultad de
Farmacia, Universidad de Salamanca, Salamanca, Spain.
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Instituto de Investigación Biomédica de Salamanca (IBSAL), Facultad de Farmacia, Universidad de
Salamanca, Salamanca, Spain.
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Centro de Investigación de Enfermedades Tropicales de la Universidad de Salamanca (CIETUS), Facultad de
Farmacia, Universidad de Salamanca, Salamanca, Spain.
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Laboratorio de Parasitología Molecular, Departamento de Microbiología Molecular y Biología de las
Infecciones, Centro de Investigaciones Biológicas Margarita Salas, Consejo Superior de Investigaciones
Científicas, Madrid, Spain.
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pelaez@usal.es (RP)
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Abstract
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New drugs against visceral leishmaniasis with mechanisms of action differing from existing treatments and
with adequate cost, stability, and properties are urgently needed. No antitubulin drugs are currently in the
clinic against Leishmania infantum, the causative agent of visceral leishmaniasis in the Mediterranean area.
We have designed and synthesized a focused library of 350 compounds against the Leishmania tubulin
based on the known structure-activity relationship (SAR) and the sequence differences between the hosts
and the parasite and we have ascertained that the synthesized compounds are accessible, stable, and with
acceptable water solubility. We have assayed the library against Leishmania promastigotes, axenic, and
intracellular amastigotes and found 0, 8, and 16 active compounds, respectively, with a high success rate
against intracellular amastigotes of over 10 % after discarding the cytotoxic compounds. Five compounds
were of similar or better potency than the clinically used miltefosine. 14 compounds showed a host-
dependent mechanism of action that might be advantageous as it may render them less susceptible to the
development of drug resistance. The active compounds cluster in five chemical classes that provide
structure-activity relationships for further hit improvement and facilitate the development of the series.
Molecular docking is consistent with the proposed mechanism of action, is supported by the observed
structure-activity relationships, and suggests a potential extension to other Leishmania species due to the
observed sequence similarities. A new family of diarylsulfonamides designed against the parasite tubulins
was shown to be active against Leishmania infantum and represents a new class of potential drugs with
favorable cost, stability, and aqueous solubility for the treatment of visceral leishmaniasis (VL). These
results could be extended to other clinically relevant species of Leishmania spp.
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Keywords
Leishmania; amastigote; sulfonamides; tubulin.
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1. Introduction
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Leishmaniasis is a neglected tropical disease caused by protozoan parasites classified into the genus
Leishmania (Kinetoplastida: Trypanosomatidae). The incidence is 700,000-2,000,000 cases causing 20,000-
30,000 annual deaths (Alvar et al., 2012). The main clinical forms are kala-azar or visceral (VL), cutaneous
(CL), and mucocutaneous leishmaniasis (MCL). VL is fatal without treatment. Anthroponotic VL (AVL) is
caused by Leishmania donovani in Southeastern Asia and Western Africa, whereas zoonotic VL (ZVL) is
caused by Leishmania infantum and distributes in the Mediterranean basin and South America. Dogs are
the main reservoirs of ZVL, whose incidence is limited in humans in developed countries. Nevertheless,
about a decade ago, an important outbreak in humans was registered in Spain (Arce et al., 2013; Jiménez et
al., 2014; Molina et al., 2012).
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The life cycle of Leishmania spp. is digenetic and develops in two stages. The promastigote is a
motile fusiform extracellular stage and the amastigote is a round intracellular stage whose flagellum does
not emerge from the cellular body. Promastigotes undergo a differentiation process known as
metacyclogenesis within the sand fly vector (Diptera: Psychodidae) gut. The vector injects highly infective
forms called metacyclic promastigotes in the mammalian host's dermis during blood feeding. Metacyclic
promastigotes are internalized by phagocytes and differentiate into amastigotes, which multiply within
infected cells, affecting different tissues depending on the causative species. When a sand fly feeds on an
infected host amastigotes transform into procyclic promastigotes within the peritrophic membrane and
begin differentiation into metacyclic forms as they migrate towards the anterior midgut (Alcolea et al.,
2016; Escudero-Martínez et al., 2017).
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The number of drugs available against the parasite is limited and they present toxicity, side effects,
resistance (Jain and Jain, 2018), long-term treatment, and cost limitations [reviewed in (Nagle et al., 2014;
Ponte-Sucre et al., 2017; Rama et al., 2015)]. Their efficacy is variable, depending on the species, the clinical
development these species cause, and the host (Tiuman et al., 2011). Combination therapy of current
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treatments is being explored leading to moderate improvement (Zulfiqar et al., 2017). Hence, new drugs
are required to control this challenging disease (Zhang et al., 2018). The most used drugs during the last 70
years have been pentavalent antimonials, administered by the intramuscular or the intravenous route. The
long-term treatments cause serious side effects, including cardiac arrhythmia and acute pancreatitis
(Monzote, 2009; Nagle et al., 2014). Efficacy has decreased over time by resistance, associated with
multidrug resistance phenotypes (Légaré et al., 2001), mutations in the macrophage aquaporin AQP1 gene,
and IL10-mediated up-regulation of the macrophage multiple resistance protein MDR1 (Marquis et al.,
2005). Amphotericin B is a polienic macrolide antibiotic with powerful antifungal and antileishmanial
activity. This drug also causes important side effects, is expensive, poorly soluble in water, not stable in the
gastric environment, and poorly membrane permeable. Fungizone®, a micellar suspension of sodium
deoxycholate is administered by the intravenous route and the patients must be hospitalized and
monitored (Abu Ammar et al., 2019; Monzote, 2009; Nagle et al., 2014). In the 1990s it replaced
pentamidine as second-line therapy for refractory VL cases in India (Nagle et al., 2014). High-cost lipid
formulations (AmBisome®) allow lower dosages and side effects, and are successfully used in the control of
VL in the Indian subcontinent, but ineffective against other species in other countries (De Rycker et al.,
2018). Resistance has emerged associated with changes in ergosterol biosynthesis and oxidative stress
prevention (Mbongo et al., 1998). Miltefosine was approved as a first-line drug in 2002 to replace
antimonials in several regions (Nagle et al., 2014; Sundar et al., 2002). It initially showed powerful
antileishmanial activity, but a gradual increase in resistances related to transporters (Mondelaers et al.,
2016; Pérez-Victoria et al., 2006, 2003) and relapses has followed (Rijal et al., 2013; Sundar and Murray,
2005). Paromomycin as an ointment works against CL but is not frequently used due to its side effects (e.g.
ototoxicity) (Monzote, 2009; Sundar et al., 2007). Several drug classes, such as the aminopyrazoles, the
nitroimidazoles, the oxaboroles, the proteasome inhibitors, and the kinase inhibitors, are currently in
development against VL, but oral, safe, effective, low cost, and of short course administration new
chemotypes acting on alternative targets are still required (Alves et al., 2018).
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The sulfonamides are synthetically accessible, stable, drug-like compounds that have a long history
of clinical success (Drews, 2000). Their antiparasitic and antitumor effects have been linked to inhibition of
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the microtubule dynamics (Dumontet and Jordan, 2010; Vicente-Blázquez et al., 2019). None of the current
antileishmanial drugs in the clinical practice or clinical trials target tubulin. Diarylsulfonamides bind at the
colchicine site of tubulin, inhibiting microtubule dynamics, and eliciting antimitotic activity
(Vicente-Blázquez et al., 2019). They combine being a privileged scaffold for the generation of
pharmacological activities with synthetic accessibility and adequate pharmacokinetic profiles, arising from a
favorable combination of chemical stability, hydrogen bonding ability, polarity, hydrophilic-lipophilic
balance, adjustable pK values, solubility, and conformational preferences (Alcolea et al., 2010; Laurence et
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al., 2009; Perlovich et al., 2014).
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The microtubules of eukaryotic cells are made up of αβ-tubulin dimers, and most drugs affecting
microtubule dynamics bind to the tubulin dimer, the microtubule lattice, or microtubule-associated
proteins and motors. Tubulins are highly conserved throughout evolution, but certain differences between
the mammal and parasite orthologs suggest sufficient binding selectivity for drug development. These
cytoskeletal supramolecular structures are involved in structural support, cell motility, cell division,
organelle transport, maintenance of cell morphology, and signal transduction (Jordan et al., 1998).
Specifically, Leishmania tubulin is an essential component of the flagellum and the subpellicular
microtubules. These structures are related to parasite survival (Sinclair and de Graffenried, 2019; Sunter
and Gull, 2017). At least seven distinct drug-binding sites have been identified in tubulin and the
microtubules, named after their prototypical drugs: the taxanes, the Vinca minor alkaloids, the maitansin,
the peloruside/laulimalide, the eribulin, the pironetin, and the colchicine binding sites (Vicente-Blázquez et
al., 2019). Different parasitic species show sequence variations compared to their hosts which vary
depending on the sites, thus making them more or less susceptible to specific drug classes and
representatives, thus allowing for specific treatments (Dostál and Libusová, 2014). Leishmania parasites are
not susceptible to colchicine (Luis et al., 2013), the archetypical ligand of the mammalian eponymous
domain. Hence, an opportunity for selective ligand development arises. This has also been empirically
exploited in the treatment of helminth and fungal parasitosis with antimitotic benzimidazoles binding at
the colchicine site, such as triclabendazole or albendazole (Lacey, 1990).
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2. Materials and methods
2.1 Chemical synthesis
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General chemical techniques. Reagents were used as purchased without further purification. Solvents
(THF, DMF, CH Cl , toluene) were dried and freshly distilled before use according to procedures described
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in the literature. TLC was performed on precoated silica gel polyester plates (0.25 mm thickness) with a UV
fluorescence indicator 254 (Polychrom SI F254). Chromatographic separations were performed on silica gel
columns by flash (Kieselgel 40, 0.040-0.063; Merck) chromatography. Melting points were determined on a
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Buchi 510 apparatus and are uncorrected. H NMR and C NMR spectra were recorded in CDCl , CD OD, or
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Acetone-d on a Bruker WP 200-SY spectrometer at 200/50 MHz or a Bruker SY spectrometer at 400/100
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MHz. Chemical shifts (δ) are given in ppm downfield from tetramethylsilane and coupling constants (J
values) are in Hertz. IR spectra were run on a Nicolet Impact 410 Spectrophotometer. Electrospray-
ionisation (ESI) high-resolution mass spectra (HRMS) were obtained on a VG-TS250 apparatus (70 eV). A
Helios-α UV-320 from Thermo-Spectronic was used for UV spectra.
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1,4-dimethoxy-2-nitrobenzene (74). To a solution of 1,4-dimethoxybenzene (2.35 g, 17 mmol) in acetic
acid (30 mL) at 0°C, nitric acid (1.13 mL, 17 mmol) in acetic acid (20 mL) was added dropwise under
nitrogen atmosphere. The reaction mixture was stirred at 0°C for 4 h and then poured onto ice with
NaHCO 5% and extracted with ethyl acetate. The organic layers were washed to neutrality with brine,
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dried over anhydrous Na SO , filtered and concentrated in vacuum to obtain 2.92 g (94%) of 74. M.p.: 71.8-
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72.5 °C (CH Cl /Hexane). IR (KBr): 1528, 874, 763 cm . H NMR (400 MHz, CDCl ): δ 3.82 (3H, s), 3.92 (3H, s),
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7.03 (1H, d, J = 9.6), 7.12 (1H, dd, J = 9.6 and 3.2), 7.4 (1H, d, J = 3.2). C NMR (100 MHz, CDCl ): δ 55.9
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(CH ), 56.9 (CH ), 109.9 (CH), 115.0 (CH), 120.8 (CH), 139.3 (C), 147.3 (C), 152.7 (C). GC-MS (C H NO ): calcd
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183, found 183.
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2,5-dimethoxyaniline (75). 1,4-dimethoxy-2-nitrobenzene (74, 2.92 g, 15.95 mmol) was suspended in
ethyl acetate (100 mL) and was palladium-catalyzed (Pd (C) 10 mg) reduced under H atmosphere for 48 h.
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The reaction mixture was filtered through celite and the solvent evaporated in vacuum to isolate 2.42 g
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(99%) of 75. Crude reaction product was obtained and used without further purification. IR (KBr): 3459,
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1519, 839 cm . H NMR (400 MHz, CDCl ): δ 3.72 (3H, s), 3.79 (3H, s), 6.24 (1H, dd, J = 9.2 and 3.2), 6.33
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(1H, d, J = 3.2), 6.69 (1H, d, J = 9.2). C NMR (100 MHz, CDCl ): δ 54.5 (CH ), 55.1 (CH ), 100.9 (CH), 101.1
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(CH), 110.3 (CH), 136.2 (C), 140.9 (C), 153.3 (C). GC-MS (C H NO ): calcd 153, found 153.
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N-(2,5-dimethoxyphenyl)-4-methoxybenzenesulfonamide (76). To a solution of 75 (2.42 g, 15.84
mmol) in CH Cl (50 mL) and pyridine (2 mL), was slowly added 4-methoxybenzenesulfonyl chloride (3.27 g,
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15.84 mmol). The mixture was stirred at room temperature for 4 h. Then the reaction was treated with HCl
2N and NaHCO 5%, washed with brine, dried over anhydrous Na SO and the solvent evaporated to obtain
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4.9 g (95%) of 76. It was purified by crystallization in CH Cl /Hexane (4.29 g, 84%). M.p.: 114-115 °C
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(CH Cl /Hexane). IR (KBr): 3313, 1578, 830 cm . H NMR (400 MHz, CDCl ): δ 3.62 (3H, s), 3.74 (3H, s), 3.81
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(3H, s), 6.53 (1H, dd, J = 9.2 and 3.2), 6.65 (1H, d, J = 9.2), 6.86 (2H, d, J = 9.2), 7.01 (1H, s), 7.14 (1H, d, J =
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3.2), 7.72 (2H, d, J = 9.2). C NMR (100 MHz, CDCl ): δ 55.5 (CH ), 55.7 (CH ), 56.2 (CH ), 196.8 (CH), 109.5
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(CH), 111.4 (CH), 113.9 (2CH), 126.8 (C), 129.4 (2CH), 130.7 (C), 143.4 (C), 153.8 (C), 163.0 (C). HRMS
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(C H NO S + H ): calcd 324.0900 (M + H ), found 324.0900.
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N-(2,5-dimethoxy-4-nitrophenyl)-4-methoxybenzenesulfonamide (96). To a stirred solution at 0°C
of 76 (2.07 g, 6.42 mmol) in acetic acid (30 mL), nitric acid (0.44 mL, 6.42 mmol) in acetic acid (20 mL) was
slowly added under nitrogen atmosphere. After 4 h at 0°C, the reaction mixture was poured onto ice and
basified with 5% NaHCO solution. Then it was extracted with ethyl acetate. The organic layers were
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washed with brine, dried over anhydrous Na SO , filtered and concentrated in vacuum to obtain 2.14 g
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(90%) of 96. By crystallization in CH Cl /Hexane 1.53 g (65%) of purified product were isolated. M.p.: 161-
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163 °C (CH Cl /Hexane). IR (KBr): 3277, 1522, 1450, 822 cm . H NMR (400 MHz, CDCl ): δ 3.76 (3H, s), 3.79
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(3H, s), 3.88 (3H, s), 6.9 (2H, d, J = 9.2), 7.26 (1H, s), 7.39 (1H, s), 7.56 (1H, s), 7.77 (2H, d, J = 9.2). C NMR
(100 MHz, CDCl ): δ 55.7 (CH ), 56.6 (CH ), 57.0 (CH ), 103.2 (CH), 108.3 (CH), 114.4 (2CH), 129.4 (2CH),
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129.9 (C), 132.8 (C), 133.1 (C), 141.1 (C), 149.2 (C), 163.6(C). HRMS (C H N O S + H ): calcd 369.0751 (M +
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H ), found 369.0753.
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N-(4-amino-2,5-dimethoxyphenyl)-4-methoxybenzenesulfonamide (104). The nitro sulfonamide 96
(1.44 g, 3.91 mmol) in ethyl acetate (100 mL) and Pd (C) (10 mg) was stirred at room temperature under H2
atmosphere for 48 h. By filtration through celite and solvent evaporation, hydrogenated sulfonamide 104
(1.28 g, 97%) was obtained. 1,11 g (84%) of 104 were isolated by crystallization in CH Cl /Hexane. M.p.:
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164-166 °C (CH Cl /Hexane). IR (KBr): 3430, 3291, 1451, 834 cm . H NMR (400 MHz, CDCl ): δ 3.38 (3H, s),
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3.79 (3H, s), 3.82 (3H, s), 6.15 (1H, s), 6.54 (1H, s), 6.82 (2H, d, J = 9.2), 7.04 (1H, s), 7.58 (2H, d, J = 9.2). C
NMR (100 MHz, CDCl ): δ 55.5 (CH ), 55.9 (CH ), 56.2 (CH ), 99.2 (CH), 108.4 (CH), 113.5 (2CH), 115.3 (C),
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129.4 (2CH), 130.7 (C), 134.6 (C), 141.0 (C), 145.8 (C), 162.7 (C). HRMS (C H N O S + H ): calcd 339.1009
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(M + H ), found 339.1012.
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75
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2-chloro-N-(2,5-dimethoxy-4-((4-methoxyphenyl)sulfonamido)phenyl)acetamide (129). To
a
solution of amine 104 (160 mg, 0.47 mmol) in CH Cl (25 mL) 2-chloroacetyl chloride (46.4 µL, 0.57 mmol)
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was added dropwise under nitrogen atmosphere. After 12 h at room temperature the reaction was washed
with water, dried over anhydrous Na SO and the solvent evaporated in vacuum to give 171 mg (87%) of
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129. The crude reaction product was purified by crystallization in methanol (55 mg, 28%). M.p.: 175-177 °C
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(MeOH). IR (KBr): 3372, 3265, 1677, 1598, 829 cm . H NMR (400 MHz, CD OD): δ 3.47 (3H, s), 3.81 (3H, s),
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3.86 (3H, s), 4.25 (2H, s), 6.94 (2H, d, J = 8.8), 7.14 (1H, s), 7.62 (2H, d, J = 8.8), 7.74 (1H, s). C NMR (100
MHz, Acetone-d ): δ 43.2 (CH ), 55.1 (CH ), 55.8 (CH ), 56.0 (CH ), 104.1 (CH), 106.9 (CH), 113.8 (2CH), 124.8
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(C), 129.3 (2CH), 131.4 (C), 138.1 (C), 142.4 (C), 144.9 (C), 163.0 (C), 164.1 (C). HRMS (C H ClN O S + H ):
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calcd 415.0725 (M + H ), found 415.0700.
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85
86
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3-chloro-N-(2,5-dimethoxy-4-((4-methoxyphenyl)sulfonamido)phenyl)propanamide (138). To a
stirred solution at room temperature of 104 (143 mg, 0.42 mmol) in CH Cl (30 mL) 3-chloropropanoyl
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chloride (49.4 µL, 0.51 mmol) was slowly added under nitrogen atmosphere. After 12 h, the reaction
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mixture was crystalized in CH Cl to obtain 88 mg (48%) of 138. M.p.: 193-197 °C (CH Cl ). H NMR (400
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MHz, CD OD): δ 2.89 (2H, t, J = 6.4), 3.47 (3H, s), 3.81 (3H, s), 3.83 (3H, s), 3.83 (2H, t, J = 6.4), 6.95 (2H, d, J
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= 9.2), 7.12 (1H, s), 7.63 (2H, d, J = 9.2), 7.68 (1H, s). C NMR (100 MHz, Acetone-d ): δ 39.6 (CH ), 40.1
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(CH ), 55.1 (CH ), 55.7 (CH ), 55.9 (CH ), 104.4 (CH), 107.1 (CH), 113.7 (2CH), 120.9 (C), 125.7 (C), 129.3
2
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(2CH), 131.7 (C), 142.2 (C), 144.9 (C), 162.9 (C), 167.8 (C). HRMS (C H ClN O S + H ): calcd 429.0882 (M +
18 21 2 6
+
H ), found 429.0879.
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96
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02
4-methoxy-3-nitrobenzonitrile (250). To a solution of 4-methoxy-3-nitrobenzaldehyde (870 mg, 4.80
mmol) in MeOH (30 mL) hydroxylamine hydrochloride (334 mg, 4.80 mmol) and two drops of pyridine were
added. After 24 h at reflux, the solvent was evaporated, the obtained residue was dissolved in CH Cl and
2
2
washed with water, dried over anhydrous Na SO , evaporated under vacuum, and dissolved in 15 mL of
2
4
pyridine. Finally, acetic anhydride (1 mL) was added to the mixture. After 9 h at room temperature, the
reaction was treated with HCl 2N, extracted with CH Cl and the solvent evaporated to obtain 685 mg (80%)
2
2
1
of 250 Crude reaction product was obtained and used without further purification. H NMR (400 MHz,
CDCl ): δ 4.05 (3H, s), 7.20 (1H, d, J = 8.8), 7.83 (1H, dd, J = 8.8 and 2), 8.15 (1H, d, J = 2). GC-MS (C H N O ):
3
8
6
2
3
calcd 178, found 178.
2
2
2
2
2
03
04
05
06
07
3-amino-4-methoxybenzonitrile (258). To a solution of 250 (685 mg, 3.84 mmol) in ethyl acetate (100
mL) Pd (C) (10 mg) was added and the reaction was stirred at room temperature under H atmosphere for
2
48 h. By filtration through Celite© and solvent evaporation, 529 mg (93%) of crude reaction 258 was
1
obtained and used without further purification. H NMR (400 MHz, CDCl ): δ 3.83 (3H, s), 6.71 (1H, d, J = 8),
3
6.84 (1H, d, J = 2), 6.87 (1H, s) 6.98 (1H, dd, J = 8 and 2). GC-MS (C H N O): calcd 148, found 148.
8
8
2
2
08
N-(5-cyano-2-methoxyphenyl)-4-methoxybenzenesulfonamide (276A) and 4-cyano-2-((4-
methoxyphenyl)sulfonamido)phenyl 4-methoxybenzenesulfonate (276B). To a solution of 258 (529
mg, 3.57 mmol) in CH Cl (50 mL) and pyridine (2 mL) 4-methoxybenzenesulfonyl chloride (1.106 g, 5.35
2
2
2
2
2
2
2
2
09
10
11
12
13
14
15
16
2
2
mmol) was slowly added. The mixture was stirred at room temperature for 4 h. Then the reaction was
treated with HCl 2N and NaHCO 5%, washed with brine, dried over anhydrous Na SO and the solvent
3
2
4
evaporated in vacuum to give a residue that was purified by silica gel chromatography using hexane/EtOAc
1
(7:3) to yield the sulfonamides 276A (466 mg, 41%) and 276B (205 mg, 30%). 276A: H NMR (400 MHz,
CDCl ): δ 3.80 (3H, s), 3.83 (3H, s), 6.80 (1H, d, J = 8), 6.91 (2H, d, J = 9.2), 7.09 (1H, s), 7.33 (1H, dd, J = 8 and
3
1
3
2), 7.75 (2H, d, J = 9.2), 7.76 (1H, d, J = 2). C NMR (100 MHz, CDCl ): δ 55.6 (CH ), 56.1 (CH ), 104.5 (C),
3
3
3
9

2
2
2
2
2
2
2
17
18
19
20
21
22
23
110.9 (CH), 114.2 (2CH), 118.6 (C), 122.6 (CH), 127.2 (C), 129.4 (2CH), 129.5 (CH), 130.1 (C), 152.1 (C), 163.4
+
+
(C). HRMS (C H N O S + Na ): calcd 341.0569 (M + Na ), found 341.0566. 276B: IR (KBr): 2233, 1462, 833,
15
14
2
4
-
1 1
749 cm . H NMR (400 MHz, CDCl ): δ 3.83 (3H, s), 3.91 (3H, s), 6.91 (2H, d, J = 9.2), 7.01 (2H, d, J = 8.8),
3
7.03 (1H, d, J = 8), 7.15 (1H, s), 7.25 (1H, dd, J = 8 and 2), 7.73 (2H, d, J = 9.2), 7.75 (2H, d, J = 8.8), 7.78 (1H,
1
3
d, J = 2). C NMR (100 MHz, CDCl ): δ 55.6 (CH ), 55.9 (CH ), 111.5 (C), 114.5 (2CH), 115.0 (2CH), 117.4 (C),
3
3
3
123.7 (CH), 124.4 (CH), 124.8 (C), 128.4 (CH), 129.4 (2CH), 129.8 (C), 130.8 (2CH), 131.5 (C), 142.4 (C), 163.6
+
+
(C), 165.1 (C). HRMS (C H N O S + Na ): calcd 497.0448 (M + Na ), found 497.0409.
21
18
2
7 2
2
2
2
2
2
2
2
24
25
26
27
28
29
30
4-methoxy-3-nitrobenzenesulfonyl chloride (86). To
a
stirred solution at 0°C of 4-
methoxybenzenesulfonyl chloride (4.41 g, 21.36 mmol) in CH Cl (20 mL) and H SO (5 mL) nitric acid (0.95
2
2
2
4
mL, 21.36 mmol) was dropwise added under nitrogen atmosphere. After 4 h, the reaction was poured onto
ice and the mixture was kept at 4°C for 30 min. Then, the precipitate was filtered under vacuum to dryness
1
to obtain 4.97 g (92%) of 86. H NMR (400 MHz, CDCl ): δ 4.11 (3H, s), 7.33 (1H, d, J = 8.8), 8.20 (1H, dd, J =
3
1
3
8.8 and 2.4), 8.48 (1H, d, J = 2.4). C NMR (100 MHz, CDCl ): δ 57.0 (CH ), 114.0 (CH), 124.8 (CH), 127.1 (C),
3
3
132.2 (CH), 135.0 (C), 157.1 (C). GC-MS (C H ClNO S): calcd 251, found 251.
7
6
5
2
2
2
2
2
2
2
2
2
2
31
32
33
34
35
36
37
38
39
40
N-(2,5-dimethoxyphenyl)-4-methoxy-3-nitrobenzenesulfonamide (183). To 650 mg of the amine 75
(4.24 mmol) in CH Cl (50 mL) and pyridine (2 mL), 1.07 g of the sulfonyl chloride 86 was slowly added (4.24
2
2
mmol) and stirred at room temperature for 6 h. The reaction was treated with HCl 2N and NaHCO 5%,
3
washed with brine, dried over anhydrous Na SO and the solvent evaporated to obtain 4.9 g (95%) of 76.
2
4
The residue was crystallized CH Cl /Hexane to afford the purified compound (4.29 g, 84%). M.p.: 121-123 °C
2
2
1
(CH Cl /Hexane). H NMR (400 MHz, CDCl ): δ 3.61 (3H, s), 3.72 (3H, s), 3.96 (3H, s), 6.56 (1H, dd, J = 8.8 and
2
2
3
3.2), 6.65 (1H, d, J = 8.8), 7.07 (1H, d, J = 8.8), 7.09 (1H, d, J = 3.2), 7.16 (1H, s), 7.88 (1H, dd, J = 8.8 and 2.4),
1
3
8.22 (1H, d, J = 2.4). C NMR (100 MHz, CDCl ): δ 55.7 (CH ), 56.0 (CH ), 57.0 (CH ), 108.1 (CH), 110.5 (CH),
3
3
3
3
111.4 (CH), 113.6 (CH), 125.2 (CH), 125.5 (C), 130.9 (C), 133.1 (CH), 138.8 (C), 143.9 (C), 153.8 (C), 155.8 (C).
+
+
HRMS (C H N O S + Na ): calcd 391.0563 (M + Na ), found 391.0570.
15
16
2
7
1
0

2
2
2
2
2
2
2
2
2
2
2
2
41
42
43
44
45
46
47
48
49
50
51
52
N-(4-bromo-2,5-dimethoxyphenyl)-4-methoxy-N-methyl-3-nitrobenzenesulfonamide (204). To a
stirred solution of 183 (86 mg, 0.23 mmol) in CH Cl (25 mL) N-bromosuccinimide (41 mg, 0.23 mmol) was
2
2
added. After 2 h the solvent was evaporated in vacuum and the residue was re-dissolved in CH CN. 25 mg
3
of crushed KOH (0.36 mmol) and 17 µL of methyl iodide (0.27 mmol) were added to the reaction mixture
and it was stirred at room temperature for 24 h. Finally, it was concentrated, re-dissolved in CH Cl , washed
2
2
with brine, dried over anhydrous Na SO , filtered and concentrated in vacuum. The residue was purified by
2
4
1
silica gel chromatography using toluene/EtOAc (8:2) to afford 87 mg (80%) of 204. H NMR (400 MHz,
CDCl ): δ 3.18 (3H, s), 3.43 (3H, s), 3.88 (3H, s) 4.01 (3H, s), 6.96 (1H, s), 6.99 (1H, d, J = 9.2), 7.01 (1H, s),
3
1
3
7.68 (1H, dd, J = 9.2 and 2.4), 7.72 (1H, d, J = 2.4). C NMR (100 MHz, CDCl ): δ 37.7 (CH ), 55.5 (CH ), 56.6
3
3
3
(CH ), 56.9 (CH ), 111.0 (CH), 112.0 (C), 116.1 (CH), 116.6 (CH), 127.2 (CH), 127.9 (C), 130.2 (CH), 131.6 (C),
3
3
+
+
139.0 (C), 149.9 (C), 150.0 (C), 157.6 (C). HRMS (C H BrN O S + H ): calcd 462.9877 (M + H ), found
16
17
2
7
462.9992.
2
2
2
2
2
2
2
2
2
2
53
54
55
56
57
58
59
60
61
62
N-(2,5-dimethoxyphenyl)-4-nitrobenzenesulfonamide (130). To a solution of 75 (1.06 g, 6.89 mmol)
in CH Cl (50 mL) and pyridine (2 mL), was slowly added 4-nitrobenzenesulfonyl chloride (1.53 g, 6.89
2
2
mmol). The mixture was stirred at room temperature for 4 h. Then the reaction was treated with HCl 2N
and NaHCO 5%, washed with brine, dried over anhydrous Na SO and the solvent evaporated to obtain
3
2
4
2.09 g (90%) of the sulfonamide 130. The crude reaction product was purified by crystallization in
1
CH Cl /Hexane (1.274 g, 55%). M.p.: 164-168 °C (CH Cl /Hexane). H NMR (400 MHz, CDCl ): δ 3.57 (3H, s),
2
2
2
2
3
3.74 (3H, s), 6.58 (1H, dd, J = 9.2 and 2.8), 6.65 (1H, d, J = 9.2), 7.15 (1H, d, J = 2.8), 7.93 (2H, d, J = 9.2), 8.22
1
3
(2H, d, J = 9.2). C NMR (100 MHz, CDCl ): δ 55.8 (CH ), 56.0 (CH ), 108.3 (CH), 110.6 (CH), 111.5 (CH), 123.9
3
3
3
+
(2CH), 125.3 (C), 128.5 (2CH), 143.8 (C), 144.7 (C), 150.1 (C), 153.8 (C). HRMS (C H N O S + Na ): calcd
14
14
2
6
+
361.0465 (M + Na ), found 361.0463.
2
2
2
2
63
64
65
66
N-(4-bromo-2,5-dimethoxyphenyl)-4-nitrobenzenesulfonamide (296). To a stirred solution of 130
(1.95 g, 5.76 mmol) in CH Cl (100 mL) N-bromosuccinimide (1.23 g, 6.92 mmol) was added. After 4 h at
2
2
room temperature the reaction was washed with water, dried over anhydrous Na SO and the solvent
2
4
evaporated in vacuum to give 2.35 g (98%) of 296. The crude reaction product was purified by
1
1

1
2
2
2
2
2
67
68
69
70
71
crystallization in methanol (1.26 g, 52%). M.p.: 190-196 °C (MeOH). H NMR (400 MHz, CDCl ): δ 3.57 (3H,
3
13
s), 3.88 (3H, s), 6.94 (1H, s), 6.97 (1H, s), 7.24 (1H, s), 7.90 (2H, d, J = 8.8), 8.27 (2H, d, J = 8.8). C NMR (100
MHz, CDCl ): δ 56.8 (CH ), 57.5 (CH ), 107.6 (CH), 108.7 (C), 116.6 (CH), 124.6 (2CH), 124.8 (C), 129.0 (2CH),
3
3
3
+
144.7 (C), 145.1 (C), 150.8 (C), 151.0 (C). HRMS (C H BrN O S + Na ): calcd 438.9580 and 440.9583 (M +
14
13
2
6
+
Na ), found 438.9570 and 440.9549.
2
2
2
2
2
2
2
2
2
72
73
74
75
76
77
78
79
80
4-amino-N-(4-bromo-2,5-dimethoxyphenyl)benzenesulfonamide (302). To an EtOH/HOAc/H O
2
mixture (2:2:1, 12.5 mL) HCl (c) (1 drop), 296 (2.35 g, 5.63 mmol) and Fe (3.15 g, 56.3 mmol) were added
and the reaction stirred for 2 h at 100°C. After extraction with CH Cl , filtration through celite and
2
2
treatment with NaHCO 5%, the crude reaction mixture was purified by silica gel chromatography using
3
-
1 1
hexane/EtOAc (7:3) to yield 1.02 g (47%) of 302. IR (KBr): 3368, 1498, 822 cm . H NMR (400 MHz, CD OD):
3
13
δ 3.55 (3H, s), 3.77 (3H, s), 6.57 (2H, d, J = 8.8), 7.00 (1H, s), 7.11 (1H, s), 7.39 (2H, d, J = 8.8). C NMR (100
MHz, Acetone-d ): δ 56.2 (CH ), 56.3 (CH ), 105.0 (C), 105.9 (CH), 112.9 (2CH), 116.3 (CH), 125.6 (C), 127.4
6
3
3
+
(C), 129.3 (2CH), 144.5 (C), 150.1 (C), 152.9 (C). HRMS (C H BrN O S + Na ): calcd 408.9828 and 410.9808
14
15
2
4
+
(M + Na ), found 408.9825 and 410.9793.
2
2
2
2
2
2
2
2
2
2
81
82
83
84
85
86
87
88
89
90
N-(4-(N-(4-bromo-2,5-dimethoxyphenyl)sulfamoyl)phenyl)formamide (315). 690 mg of 302 (1.78
mmol) were dissolved in CH Cl (70 mL), pyridine (5 mL) and formic acid (10 mL) and stirred at room
2
2
temperature. After 24 h, the reaction mixture was poured onto ice and treated with HCl 2N and NaHCO3
5%. The organic layers were washed to neutrality with brine, dried over anhydrous Na SO , filtered and
2
4
evaporated to dryness to afford 708 mg (95%) of 315. The crude reaction product was purified by
-
1
crystallization in methanol (307 mg, 41%). M.p.: 202-207 °C (MeOH). IR (KBr): 3337, 1702, 1593, 831 cm .
1
H NMR (400 MHz, CD OD): δ 3.49 (3H, s), 3.81 (3H, s), 7.01 (1H, s), 7.15 (1H, s), 7.67 (4H, bs), 8.30 (1H, s).
3
13
C NMR (100 MHz, DMSO-d ): δ 56.9 (CH ), 57.0 (CH ), 107.1 (C), 109.8 (CH), 117.1 (CH), 119.1 (2CH), 126.2
6
3
3
+
(C), 128.6 (2CH), 134.8 (C), 142.8 (C), 146.9 (C), 149.6 (C), 160.6 (CH). HRMS (C H BrN O S + Na ): calcd
15
14
2
5
+
436.9777 and 438.9757 (M + Na ), found 436.9772 and 438.9750.
1
2

2
2
2
2
2
2
2
2
2
3
3
91
92
93
94
95
96
97
98
99
00
01
N-(4-bromo-2,5-dimethoxyphenyl)-4-(methylamino)benzenesulfonamide (323). To a solution of
the formamide 315 (680 mg, 1.64 mmol) and NaBH (93 mg, 2.45 mmol) in dry THF (15 mL) at 0°C,
4
trichloroacetic acid (401 mg, 2.45 mmol) in dry THF (10 mL) was added dropwise under nitrogen
atmosphere. The reaction mixture was stirred at 0°C to room temperature for 24 h and then concentrated
and re-dissolved in EtOAc, washed with brine, dried over anhydrous Na SO , filtered and solvent
2
4
evaporated in vacuum. The residue was purified by silica gel chromatography using hexane/EtOAc (8:2) to
-
1 1
yield 246 mg (37%) of 323. IR (KBr): 3420, 3251, 1599, 820 cm . H NMR (400 MHz, CD OD): δ 2.76 (3H, s),
3
1
3
3.56 (3H, s), 3,78 (3H, s), 6.51 (2H, d, J = 8.8), 7.00 (1H, s), 7.13 (1H, s), 7,45 (2H, d, J = 8.8). C NMR (100
MHz, CDCl ): δ 30.0 (CH ), 56.5 (CH ), 56.8 (CH ), 105.3 (CH), 105.5 (C), 111.1 (2CH), 115.8 (CH), 125.0 (C),
3
3
3
3
+
126.6 (C), 129.3 (2CH), 143.5 (C), 150.2 (C), 152.7 (C). HRMS (C H BrN O S + Na ): calcd 422.9985 and
15
16
2
4
+
424.9964 (M + Na ), found 422.9985 and 424.9959.
3
3
3
3
3
3
3
3
3
3
02
03
04
05
06
07
08
09
10
11
N-benzyl-N-(4-bromo-2,5-dimethoxyphenyl)-4-(methylamino)benzenesulfonamide (332). 35 mg
(0.25 mmol) of K CO were added to a stirred solution of 323 (50 mg, 0.12 mmol) in 3 mL of dry DMF. After
2
3
1 h at room temperature 21.7 µL (0.19 mmol) of benzyl chloride were added and stirred for 24 h. The
reaction mixture was concentrated, re-dissolved in CH Cl , washed with brine, dried over anhydrous
2
2
Na SO , filtered and concentrated in vacuum to obtain 59 mg (96%) and crystalized in MeOH (23 mg, 38%).
2
4
1
M.p.: 177-183 °C (MeOH). H NMR (400 MHz, CDCl ): δ 2.79 (3H, s), 3.33 (3H, s), 3.57 (3H, s), 4.64 (2H, s),
3
1
3
6.46 (2H, d, J = 8.8), 6.52 (1H, s), 6.85 (1H, s), 7.14 (5H, m), 7.44 (2H, d, J = 8.8). C NMR (100 MHz, CDCl ): δ
3
30.1 (CH ), 53.3 (CH ), 55.7 (CH ), 56.7 (CH ), 110.8 (2CH), 111.49 (C), 116.5 (CH), 117.5 (CH), 126.5 (C),
3
2
3
3
126.9 (C), 127.4 (CH), 128.2 (2CH), 128.7 (2CH), 129.7 (2CH), 136.7 (C), 149.4 (C), 151.0 (C), 152.4 (C). HRMS
+
+
(C H BrN O S + Na ): calcd 515.0415 (M + Na ), found 515.0434.
22
23
2
4
3
12
3,4,5-trimethoxy-2-((4-methoxyphenyl)sulfonamido)benzoic acid (63A) and 3,4,5-trimethoxy-2-
(3,4,5-trimethoxy-2-((4-methoxyphenyl)sulfonamido)benzamido) benzoic acid (63B) To a stirred
solution of 2-amino-3,4,5-trimethoxybenzoic acid (300 mg, 1.32 mmol) in CH Cl (50 mL) and pyridine (2
3
3
3
3
13
14
15
16
2
2
mL) 4-methoxybenzenesulfonyl chloride (273 g, 1.32 mmol) was slowly added. After 6 h, the reaction
mixture was poured into HCl 2N solution and extracted with CH Cl . The organic layers were washed to
2
2
1
3

3
3
3
3
3
3
3
3
3
3
3
3
3
17
18
19
20
21
22
23
24
25
26
27
28
29
neutrality with saturated NaCl, dried over anhydrous Na SO , filtered and evaporated to dryness. The
2 4
residue was purified by two successive crystallizations in CH Cl /hexane, compounds 63A (40 mg, 8 %) and
2
2
63B (17 mg, 4 %) were isolated. 63A: M.p.: 165-167 °C (CH Cl /Hexane). IR (KBr):3264, 2939, 1668, 1458,
2
2
-
1 1
837 cm . H NMR (400 MHz, CDCl ): δ 3.45 (3H, s), 3.84 (3H, s), 3.88 (3H, s), 3.91 (3H, s), 6.93 (2H, d, J =
3
1
3
8.8), 7.28 (1H, s), 7.77 (2H, d, J = 8.8), 8.7 (1H, s). C NMR (100 MHz, CDCl ): δ 55.6 (CH ), 56.1 (CH ), 60.3
3
3
3
(CH ), 61.1 (CH ), 109.0 (CH), 113.7 (2CH), 116.5 (C), 128.0 (C), 129.4 (2CH), 132.0 (C), 147.7 (C), 148.4 (C),
3
3
+
+
150.5 (C), 162.8 (C), 171.4 (C). HRMS (C H NO S + H ): calcd 398.0901 (M + H ), found 398.0905. 63B: M.p.:
17
19
8
1
170-171 °C (CH Cl /Hexane). H NMR (400 MHz, CDCl ): δ 3.35 (3H, s), 3.75 (3H, s), 3.79 (3H, s), 3.84 (3H, s),
2
2
3
3.88 (3H, s), 3.96 (3H, s), 3.99 (3H, s), 6.85 (2H, d, J = 8.8), 7.20 (1H, s), 7.25 (1H, s), 7.68 (2H, d, J = 8.8), 8.15
1
3
(1H, s), 9,22 (1H, s). C NMR (100 MHz, CDCl ): δ 57.1 (CH ), 59.0 (2CH ), 61.9 (CH ), 62.4 (CH ), 62.6 (CH ),
3
3
3
3
3
3
63.0 (CH ), 108.9 (CH), 110.2 (CH), 115.2 (2CH), 120.9 (C), 124.1 (C), 126.6 (C), 127.4 (C), 131.3 (2CH), 133.2
3
(C), 147.0 (C), 148.3 (C), 149.7 (C), 149.9 (C), 152.9 (C), 153.4 (C), 153.9 (C), 165.3 (C), 167.5 (C). HRMS
+
+
(C H N O S + H ): calcd 607.1599 (M + H ), found 607.1593.
27
30
2
12
3
3
3
3
3
3
3
3
3
3
30
31
32
33
34
35
36
37
38
39
N-(3,5-dimethoxyphenyl)-4-methoxybenzenesulfonamide (259). To
a
solution of 3,5-
dimethoxyaniline (290 mg, 1.89 mmol) in CH Cl (50 mL) and pyridine (2 mL), was slowly added 4-
2
2
methoxybenzenesulfonyl chloride (469 mg, 2.27 mmol). The mixture was stirred at room temperature for 4
h. Then the reaction was treated with HCl 2N and NaHCO 5%, washed with brine, dried over anhydrous
3
Na SO and the solvent evaporated to obtain 596 mg (97%) of the sulfonamide 259. The crude reaction
2
4
product was purified by crystallization in CH Cl /Hexane (438 mg, 71%). M.p.: 115-122 °C (CH Cl /Hexane).
2
2
2
2
-
1 1
IR (KBr): 3234, 1595, 824 cm . H NMR (400 MHz, CDCl ): δ 3.66 (6H, s), 3.77 (3H, s), 6.13 (1H, t, J = 2), 6.17
3
13
(2H, d, J = 2), 6.85 (2H, d, J = 8.8), 7.68 (2H, d, J = 8.8). C NMR (100 MHz, CDCl ): δ 55.3 (2CH ), 55.5 (CH ),
3
3
3
97.0 (CH), 98.9 (2CH), 114.2 (2CH), 129.5 (2CH), 130.4 (C), 138.6 (C), 161.1 (2C), 163.1 (C). HRMS
+
+
(C H NO S + H ): calcd 324.0909 (M + H ), found 324.0900.
15
17
5
3
3
3
40
41
42
N-benzyl-N-(3,5-dimethoxyphenyl)-4-methoxybenzenesulfonamide (270). To a stirred solution of
259 (90 mg, 0.28 mmol) in dry DMF (3 mL) 78 mg (0.56 mmol) of K CO were added. After 1 h at room
2
3
temperature, 48.5 µL (0.42 mmol) of benzyl chloride were added and stirred for 24 h. The reaction mixture
1
4

3
3
3
3
3
3
3
43
44
45
46
47
48
49
was concentrated, re-dissolved in CH Cl , washed with brine, dried over anhydrous Na SO , filtered and
2 2 2 4
concentrated under vacuum to produced 104 mg (90%) of crude reaction product from which 81 mg (70%)
-
1 1
of 270 were purified by crystallization. M.p.: 146-150 °C (MeOH). IR (KBr): 3467, 1458, 806 cm . H NMR
(400 MHz, CDCl ): δ 3.62 (6H, s), 3.87 (3H, s), 4.65 (2H, s), 6.12 (2H, d, J = 2), 6.28 (1H, t, J = 2), 6.94 (2H, d, J
3
1
3
= 8.8), 7.22 (5H, m), 7.63 (2H, d, J = 8.8). C NMR (100 MHz, CDCl ): δ 54.7 (CH ), 55.3 (2CH ), 55.6 (CH ),
3
2
3
3
100.0 (CH), 107.2 (2CH), 113.9 (2CH), 127.5 (CH), 128.3 (2CH), 128.5 (2CH), 129.8 (2CH), 130.3 (C), 136.1 (C),
+
+
140.9 (C), 160.4 (2C), 162.9 (C). HRMS (C H NO S + H ): calcd 414.1370 (M + H ), found 414.1369.
22
23
5
3
3
50
51
N-benzyl-N-(4-bromo-3,5-dimethoxyphenyl)-4-methoxybenzenesulfonamide (326A), N-benzyl-
N-(2-bromo-3,5-dimethoxyphenyl)-4-methoxybenzenesulfonamide (326B) and N-benzyl-N-(2,4-
dibromo-3,5-dimethoxyphenyl)-4-methoxybenzenesulfonamide (326C). To a solution of 270 (195
mg, 0.47 mmol) in CH Cl (40 mL) N-bromosuccinimide (168 mg, 0.94 mmol) was added and stirred for 48 h
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
2
2
at room temperature. After that, the reaction was washed with water, dried over anhydrous Na SO and
2
4
the solvent evaporated in vacuum to produce 183 mg of crude reaction. The residue was flash
chromatographed on silica gel (hexane/EtOAc 8:2) to afford the purified compounds: 326A (116 mg, 50%),
-
1 1
326B (24 mg, 10%) and 326C (6 mg, 2%). 326A: M.p.: 199-203 °C (MeOH). IR (KBr): 3435, 1589, 836 cm . H
NMR (200 MHz, CDCl ): δ 3.65 (6H, s), 3.88 (3H, s), 4.68 (2H, s), 6.12 (2H, s), 6.97 (2H, d, J = 9), 7.22 (5H, bs),
3
1
3
7.65 (2H, d, J = 9). C NMR (100 MHz, CDCl ): δ 54.9 (CH ), 55.7 (CH ), 56.4 (2CH ), 100.5 (C), 105.8 (2CH),
3
2
3
3
114.0 (2CH), 127.8 (CH), 128.4 (2CH), 128.6 (2CH), 129.9 (C), 130.0 (2CH), 135.7 (C), 139.4 (C), 156.7 (2C),
+
+
163.1 (C). HRMS (C H BrNO S + H ): calcd 492.0475 and 494.0454 (M + H ), found 492.0473 and 494.0440.
22
22
5
-
1 1
326B: IR (KBr): 2938, 1593, 831 cm . H NMR (200 MHz, CDCl ): δ 3.57 (3H, s), 3.80 (3H, s), 3.87 (3H, s), 4.60
3
(1H, d, J = 14.4), 4.89 (1H, d, J = 14.4), 6.14 (1H, d, J = 2.8), 6.38 (1H, d, J = 2.8), 6.94 (2H, d, J = 9) 7.20 (5H,
1
3
bs), 7.74 (2H, d, J = 9). C NMR (100 MHz, CDCl ): δ 54.4 (CH ), 55.5 (CH ), 55.6 (CH ), 56.3 (CH ), 100.0 (CH),
3
2
3
3
3
105.9 (C), 109.4 (CH), 113.9 (2CH), 127.7 (CH), 128.2 (2CH), 129.4 (2CH), 130.1 (2CH), 131.8 (C), 135.7 (C),
+
+
138.8 (C), 157.2 (C), 158.9 (C), 163.0 (C). HRMS (C H BrNO S + H ): calcd 492.0475 and 494.0454 (M + H ),
22
22
5
-
1 1
found 492.0467 and 494.0447. 326C: IR (KBr): 2935, 1595, 835 cm . H NMR (200 MHz, CDCl ): δ 3.60 (3H,
3
s), 3.77 (3H, s), 3.87 (3H, s), 4.55 (1H, d, J = 14.4), 4.97 (1H, d, J = 14.4), 6.30 (1H, s), 6.95 (2H, d, J = 9), 7.22
1
5

1
3
3
3
3
3
69
70
71
72
(5H, m), 7.73 (2H, d, J = 9). C NMR (100 MHz, CDCl ): δ 54.2 (CH ), 55.6 (CH ), 56.5 (CH ), 60.5 (CH ), 108.8
3 2 3 3 3
(C), 112.4 (C), 112.8 (CH), 114.0 (2CH), 128.0 (CH), 128.3 (2CH), 129.4 (2CH), 130.0 (2CH), 131.5 (C), 135.4
+
(C), 137.4 (C), 155.6 (C), 155.7 (C), 163.2 (C). HRMS (C H Br NO S + H): calcd 569.9580 and 571.9559 (M +
22
21
2
5
H), found 569.9577 and 571.9558.
3
3
3
3
3
3
3
3
3
3
73
74
75
76
77
78
79
80
81
82
N-(3,5-dimethoxyphenyl)-4-nitrobenzenesulfonamide (242). To a solution of 3,5-dimethoxyaniline
(1.39 g, 9.98 mmol) in CH Cl (50 mL) and pyridine (2 mL), was slowly added 4-nitrobenzenesulfonyl
2
2
chloride (2.21 g, 9.07 mmol) and stirred at room temperature for 12 h. Then the mixture reaction was
treated with HCl 2N and NaHCO 5%. The organic layers were washed to neutrality with brine, dried over
3
anhydrous Na SO and concentrated under vacuum to yield 2.66 g (87%) of the sulfonamide 242. The crude
2
4
reaction product was purified by crystallization in CH Cl /Hexane (2.43 g, 79%). M.p.: 131-139 °C
2
2
1
(CH Cl /Hexane). H NMR (400 MHz, CDCl ): δ 3.71 (6H, s), 6.22 (1H, t, J = 2.4), 6.25 (2H, d, J = 2.4), 7.98 (2H,
2
2
3
1
3
d, J = 8.8), 8.28 (2H, d, J = 8.8). C NMR (100 MHz, CDCl ): δ 55.4 (2CH ), 97.4 (CH), 99.7 (2CH), 124.3 (2CH),
3
3
+
+
128.6 (2CH), 137.4 (C), 144.4 (C), 150.1 (C), 161.3 (2C). HRMS (C H N O S + H ): calcd 339.0653 (M + H ),
14
14
2
6
found 339.0645.
3
3
3
3
3
3
3
3
83
84
85
86
87
88
89
90
4-amino-N-(3,5-dimethoxyphenyl)benzenesulfonamide (245). To a solution of 242 (2.60 g, 7.69
mmol) in ethyl acetate (150 mL) an MeOH (5 mL), Pd (C) (10 mg) was added and the reaction was stirred at
room temperature under H atmosphere for 24 h. By filtration through celite and solvent evaporation, 2.30
2
g (97%) of 245 was obtained and purified by crystallization in MeOH (1.53 g, 65%). M.p.: 149-155 °C
-
1 1
(MeOH). IR (KBr): 3450, 3370, 1458, 821 cm . H NMR (400 MHz, CD OD): δ 3.67 (6H, s), 6.13 (1H, t, J = 2.4),
3
1
3
6.25 (2H, d, J = 2.4), 6.60 (2H, d, J = 8.8), 7.46 (2H, d, J = 8.8). C NMR (100 MHz, CD OD): δ 54.2 (2CH ),
3
3
95.6 (CH), 98.2 (2CH), 112.8 (2CH), 125.3 (C), 128.8 (2CH), 139.8 (C), 152.8 (C), 161.1 (2C). HRMS
+
+
(C H N O S + H ): calcd 309.0904 (M + H ), found 309.0915.
14
16
2
4
3
3
3
91
92
93
N-(3,5-dimethoxyphenyl)-4-(dimethylamino)benzenesulfonamide (254). To a solution of p-
formaldehyde (534 mg, 17.77 mmol) in MeOH (40 mL), 548 mg of 245 (1.77 mmol) were added and stirred
for 30 min, then NaCNBH (223 mg, 10.62 mmol) was added and the reaction was heated at reflux for 24 h.
3
1
6

3
3
3
3
3
3
94
95
96
97
98
99
The reaction mixture was concentrated, poured onto ice and extracted with EtOAc, dried over Na SO ,
2 4
filtered through celite and the solvent evaporated in vacuum to afford 580 mg (97%) of 254. M.p.: 158-164
-
1 1
°C (MeOH). IR (KBr): 3228, 1498, 812 cm . H NMR (400 MHz, CD OD): δ 2.99 (6H, s), 3.67 (6H, s), 6.12 (1H,
3
1
3
t, J = 2.4), 6.26 (2H, d, J = 2.4), 6.68 (2H, d, J = 9.2), 7.58 (2H, d, J = 9.2). C NMR (100 MHz, CDCl ): δ 39.9
3
(2CH ), 55.3 (2CH ), 96.6 (CH), 98.4 (2CH), 110.8 (2CH), 123.9 (C), 129.1 (2CH), 139.1 (C), 152.9 (C), 161.1
3
3
+
+
(2C). HRMS (C H N O S + H ): calcd 337.1217 (M + H ), found 337.1205.
16
20
2
4
4
4
4
4
4
4
4
4
4
4
00
01
02
03
04
05
06
07
08
09
N-benzyl-N-(3,5-dimethoxyphenyl)-4-(dimethylamino)benzenesulfonamide (275). 77 mg (0.55
mmol) of K CO were added to a stirred solution of 254 (93 mg, 0.27 mmol) in 3 mL of dry DMF. After 1 h at
2
3
room temperature 48.2 µL (0.41 mmol) of benzyl chloride were added and stirred for 24 h. The reaction
mixture was concentrated, re-dissolved in EtOAc, washed with brine, dried over anhydrous Na SO , filtered
2
4
and concentrated in vacuum to obtain 112 mg (95%) and crystalized in MeOH (62 mg, 52%). M.p.: 155-160
-
1 1
°C (MeOH). IR (KBr): 3471, 1455, 811cm . H NMR (400 MHz, CDCl ): δ 3.05 (6H, s), 3.63 (6H, s), 4.63 (2H, s),
3
13
6.18 (2H, d, J = 2.4), 6.26 (1H, t, J = 2.4), 6.64 (2H, d, J = 8.8), 7.20 (5H, m), 7.53 (2H, d, J = 8.8). C NMR (100
MHz, CDCl ): δ 40.1 (2CH ), 54.5 (CH ), 55.3 (2CH ), 99.9 (CH), 107.1 (2CH), 110.6 (2CH), 123.9 (C), 127.4
3
3
2
3
(CH), 128.2 (2CH), 128.5 (2CH), 129.6 (2CH), 136.4 (C), 141.4 (C), 152.8 (C), 160.3 (2C). HRMS (C H N O S +
23
26
2
4
+
+
H ): calcd 427.1686 (M + H ), found 427.1658.
4
4
4
4
4
4
4
4
4
4
10
11
12
13
14
15
16
17
18
19
N-(6-methoxypyridin-3-yl)-4-nitrobenzenesulfonamide (283). To a solution of 6-methoxypyridin-3-
amine (1.82 g, 14.66 mmol) in CH Cl (50 mL) and pyridine (2 mL), was slowly added 4-nitrobenzenesulfonyl
2
2
chloride (3.9 g, 17.59 mmol). The mixture was stirred at room temperature for 4 h. Then the reaction was
treated with HCl 2N and NaHCO 5%, washed with brine, dried over anhydrous Na SO and the solvent
3
2
4
evaporated to obtain 3.10 g (68%) of 273. It was purified by crystallization in MeOH 2.67 g (59%). M.p.: 139-
-
1 1
143 °C (MeOH). IR (KBr): 3208, 1610, 1350, 826 cm . H NMR (400 MHz, CDCl ): δ 3.88 (3H, s), 6.49 (1H, s),
3
6.70 (1H, d, J = 8.8), 7.43 (1H, dd, J = 8.8 and 2.8), 7.72 (1H, d, J = 2.8), 7.88 (2H, d, J = 9.2), 8.31 (2H, d, J =
1
3
9.2). C NMR (100 MHz, CDCl ): δ 53.8 (CH ), 111.5 (CH), 124.4 (2CH), 125.2 (C), 128.6 (2CH), 136.3 (CH),
3
3
+
+
143.2 (CH), 144.3 (C), 150.3 (C), 163.1 (C). HRMS (C H N O S + H ): calcd 310.0492 (M + H ), found
12
11
3
5
310.0487.
1
7

4
4
4
4
4
4
4
4
4
20
21
22
23
24
25
26
27
28
4-amino-N-(6-methoxypyridin-3-yl)benzenesulfonamide (287). 3.00 g of 283 (9.71 mmol) was
suspended in ethyl acetate (120 mL) and was palladium-catalyzed (Pd (C) 10 mg) reduced under H2
atmosphere for 72 h. The reaction mixture was filtered through celite and the solvent evaporated in
vacuum to isolate 2.67 g (98%) of 287. Crude reaction product was purified by crystallization in MeOH (539
-
1 1
mg, 20%). M.p.: 176-180 °C (MeOH). IR (KBr): 3485, 3281, 1619, 1500, 794 cm . H NMR (400 MHz, CD OD):
3
δ 3.82 (3H, s), 6.59 (2H, d, J = 8.4), 6.68 (1H, d, J = 8.8), 7.33 (2H, d, J = 8.4), 7.41 (1H, dd, J = 8.8 and 2.4),
1
3
7.71 (1H, d, J = 2.4). C NMR (100 MHz, CD OD): δ 52.7 (CH ), 110.1 (CH), 112.8 (2CH), 124.6 (C), 128.4 (C),
3
3
+
+
128.8 (2CH), 135.2 (CH), 141.2 (CH), 152.9 (C), 161.9 (C). HRMS (C H N O S + H ): calcd 280.0750 (M + H ),
12
13
3
5
found 280.0745.
4
4
4
4
4
4
4
4
4
4
29
30
31
32
33
34
35
36
37
38
N-(4-(N-(6-methoxypyridin-3-yl)sulfamoyl)phenyl)formamide (309). 970 mg of 287 (3.57 mmol)
were dissolved in CH Cl (100 mL), pyridine (5 mL) and formic acid (15 mL) and stirred at room temperature.
2
2
After 24 h, the reaction mixture was poured onto ice and treated with HCl 2N and NaHCO 5%. The organic
3
layers were washed to neutrality with brine, dried over anhydrous Na SO , filtered and evaporated to
2
4
dryness to afford 555 mg (52%) of 309. The crude reaction product was purified by crystallization in
-
1 1
methanol (368 mg, 34%). M.p.: 189-193 °C (MeOH). IR (KBr): 3349, 3273, 1698, 1592, 823 cm . H NMR
(400 MHz, CD OD): δ 3.82 (3H, s), 6.69 (1H, d, J = 8.8), 7.42 (1H, dd, J = 8.8 and 2.8), 7.62 (2H, d, J = 8.8),
3
1
3
7.70 (2H, d, J = 8.8), 7.71 (1H, d, J = 2.8), 8.31 (1H, s). C NMR (100 MHz, Acetone-d6): δ 52.8 (CH ), 110.6
3
(CH), 118.9 (2CH), 128.0 (C), 128.4 (2CH), 133.9 (C), 134.8 (CH), 141.6 (CH), 142.3 (C), 159.6 (C), 161.9 (CH).
+
+
HRMS (C H N O S + H ): calcd 308.0703 (M + H ), found 308.0700.
13
13
3
4
4
4
4
4
4
4
4
39
40
41
42
43
44
45
N-(6-methoxypyridin-3-yl)-4-(methylamino)benzenesulfonamide (316). To a solution of the
formaldehyde 309 (485 mg, 1.58 mmol) and NaBH (90 mg, 2.37 mmol) in dry THF (15 mL) at 0°C,
4
trichloroacetic acid (387 mg, 2.37 mmol) in dry THF (10 mL) was added dropwise under nitrogen
atmosphere. The reaction mixture was stirred at 0°C to room temperature for 24 h and then concentrated
and re-dissolved in EtOAc, washed with brine, dried over anhydrous Na SO , filtered and solvent
2
4
evaporated in vacuum. The crude reaction product (334 mg, 72%) was purified by crystallization in MeOH
-
1 1
to yield 137 mg (30%) of 316. M.p.: 183-186 °C (MeOH). IR (KBr): 3387, 3277, 1500, 821 cm . H NMR (400
1
8

4
4
4
4
46
47
48
49
MHz, CD OD): δ 2.76 (3H, s), 3.82 (3H, s), 6.52 (2H, d, J = 8.8), 6.67 (1H, d, J = 8.8), 7.38 (2H, d, J = 8.8), 7.41
3
1
3
(1H, dd, J = 8.8 and 2.8), 7.71 (1H, d, J = 2.8). C NMR (100 MHz, CDCl ): δ 30.0 (CH ), 53.6 (CH ), 111.0 (CH),
3
3
3
111.3 (2CH), 124.8 (C), 127.0 (C), 129.3 (2CH), 135.9 (CH), 142.5 (CH), 152.7 (C), 162.4 (C). HRMS
+
+
(C H N O S + H ): calcd 294.0907 (M + H ), found 294.0907.
13
15
3
3
4
4
4
4
4
4
4
4
4
50
51
52
53
54
55
56
57
58
N-(4-(N-(6-methoxypyridin-3-yl)sulfamoyl)phenyl)-N-methylformamide (320). The compound 316
(88 mg, 0.30 mmol) was stirred in a mixture of CH Cl (50 mL), pyridine (2 mL) and formic acid (5 mL) at
2
2
room temperature for 24 h. The reaction mixture was poured onto ice and treated with HCl 2N and NaHCO3
5%, washed to neutrality with brine, dried over anhydrous Na SO , filtered and solvent evaporated to
2
4
1
produce 33 mg (34%) of 320, which crystallized in methanol (24 mg, 25%). M.p.: 164-172 °C (MeOH). H
NMR (400 MHz, CD OD): δ 3.30 (3H, s), 3.82 (3H, s), 6.69 (1H, d, J = 8.8), 7.44 (2H, d, J = 8.8), 7.46 (1H, dd, J
3
1
3
= 8.8 and 2.8), 7.71 (1H, d, J = 2.8), 7.74 (2H, d, J = 8.8), 8.65 (1H, s). C NMR (100 MHz, CD OD): δ 29.9
3
(CH ), 52.8 (CH ), 110.7 (CH), 120.2 (2CH), 127.9 (C), 128.6 (2CH), 134.8 (CH), 135.6 (C), 141.5 (CH), 146.3
3
3
+
+
(C), 161.4 (C), 161.9 (CH). HRMS (C H N O S + H ): calcd 322.0856 (M + H ), found 322.0855.
14
15
3
4
4
4
4
4
4
4
4
4
4
4
59
60
61
62
63
64
65
66
67
68
4-methoxy-N-(6-methoxypyridin-3-yl)benzenesulfonamide (240). To a stirred solution of 6-
methoxypyridin-3-amine (312 mg, 2.51 mmol) in CH Cl2 (25 mL) and pyridine (1 mL) 4-
2
methoxybenzenesulfonyl chloride (623 mg, 3.02 mmol) was added under nitrogen atmosphere. After 6 h,
the reaction mixture was treated with HCl 2N and NaHCO 5% solutions. The organic layers were washed to
3
neutrality with saturated NaCl, dried over anhydrous Na SO , filtered and evaporated to yield 631 mg (85%)
2
4
of the sulfonamide 240. The residue was purified by flash chromatography on silica gel using hexane/EtOAc
1
(6:4) to afford 464 mg (62%). H NMR (400 MHz, CDCl ): δ 3.80 (3H, s), 3.83 (3H, s), 6.62 (1H, d, J = 8.8), 6.87
3
13
(2H, d, J = 8.8), 7.40 (1H, dd, J = 8.8 and 2.8), 7.63 (2H, d, J = 8.8), 7.75 (1H, d, J = 2.8). C NMR (100 MHz,
CDCl ): δ 54.3 (CH ), 56.2 (CH ), 111.6 (CH) 114.9 (2CH), 127.5 (C), 130.1 (2CH), 130.6 (C), 136.3 (CH), 143.1
3
3
3
+
+
(CH), 162.9 (C), 163.8 (C). HRMS (C H N O S + H ): calcd 295.0747 (M + H ), found 295.0761.
13
14
2
4
4
4
69
70
N-benzyl-4-methoxy-N-(6-methoxypyridin-3-yl)benzenesulfonamide (279). 92 mg (0.31 mmol) of
240 dissolved in dry DMF (3 mL) were stirred for 1 h in the presence of K CO (97 mg, 0.62 mmol). After
2
3
1
9

4
4
4
4
4
4
4
4
71
72
73
74
75
76
77
78
that, benzyl chloride (54.5 µL 0.47 mmol) was added and stirred for 24 h. The reaction mixture was
concentrated, re-dissolved in EtOAc, washed with brine, dried over anhydrous Na SO , filtered and
2
4
concentrated in vacuum to obtain 112 mg (93%) and crystalized in MeOH (77 mg, 64%). M.p.: 144-146 °C
-
1 1
(MeOH). IR (KBr): 3435, 1490, 823 cm . H NMR (400 MHz, CD OD): δ 3.81 (3H, s), 3.89 (3H, s), 4.72 (2H, s),
3
6.61 (1H, d, J = 8.8), 7.10 (2H, d, J = 8.8), 7.22 (5H, bs), 7.23 (1H, dd, J = 8.8 and 2.8), 7.62 (2H, d, J = 8.8),
1
3
7.67 (1H, d, J = 2.8). C NMR (100 MHz, CDCl ): δ 53.7 (CH ), 54.9 (CH ), 55.7 (CH ), 110.8 (CH), 114.2 (2CH),
3
3
2
3
127.8 (CH), 128.5 (2CH), 128.6 (2CH), 129.2 (C), 129.7 (2CH), 130.7 (C), 135.4 (C), 139.5 (CH), 147.2 (CH),
+
+
163.0 (C), 163.1 (C). HRMS (C H N O S + H ): calcd 385.1217 (M + H ), found 385.1210.
20
20
2
4
4
4
4
4
4
4
4
4
4
4
79
80
81
82
83
84
85
86
87
88
N-(3,4-dimethoxyphenyl)-4-methoxybenzenesulfonamide (8). To 2.49 g of 3,4-dimethoxyaniline
(16.26 mmol) in CH Cl (50 mL) and pyridine (4 mL), 3.61 g of 4-methoxybenzenesulfonyl chloride was
2
2
slowly added (16.26 mmol) and stirred at room temperature for 12 h. The reaction was treated with HCl 2N
and NaHCO 5%, washed with brine, dried over anhydrous Na SO and the solvent evaporated to obtain
3
2
4
5.29 g (99%) of 8. The residue was crystallized in CH Cl /Hexane to afford the purified compound (4.04 g,
2
2
-
1 1
75%). M.p.: 101-102 °C (CH Cl /Hexane). IR (KBr): 3224, 1498, 801 cm . H NMR (400 MHz, CDCl ): δ 3.75
2
2
3
(3H, s), 3.79 (3H, s), 3.80 (3H, s), 6.53 (1H, dd, J = 8.8 and 2.8), 6.66 (1H, d, J = 8.8), 6.70 (1H, d, J = 2.8), 6.86
1
3
(2H, d, J = 8.8), 7.66 (2H, d, J = 8.8). C NMR (100 MHz, CDCl ): δ 55.5 (CH ), 55.9 (CH ), 55.9(CH ), 107.7
3
3
3
3
(CH), 111.1 (CH), 114.0 (2CH), 115.4 (CH), 129.4 (2CH), 129.5 (C), 130.3 (C), 147.2 (C), 149.1 (C), 163.0 (C).
+
+
HRMS (C H NO S + H ): calcd 324.0900 (M + H ), found 324.0906.
15
17
5
4
4
4
4
4
4
4
4
89
90
91
92
93
94
95
96
N-(4,5-dimethoxy-2-nitrophenyl)-4-methoxybenzenesulfonamide (11). To a solution of 8 (588 mg,
1.82 mmol) in CH CN (50 mL) tertbutyl nitrite (120 µL, 0.91 mmol) was added and stirred at 45°C. After 1 h,
3
additional 0.91 mmol tertbutyl nitrite was added to the reaction mixture and it was stirred at 45°C for 24 h.
The mixture was poured into ice and basified with 5% NaHCO solution and extracted with EtOAc. The
3
organic layers were washed with brine, dried over Na SO and concentrated under vacuum to yield 11 (638
2
4
mg, 95%). The residue was purified by crystallization in CH Cl /Hexane to afford 527 mg (78%). M.p.: 152-
2
2
-
1 1
154 °C (CH Cl /Hexane). IR (KBr): 3257, 1521, 1499, 804 cm . H NMR (400 MHz, CDCl ): δ 3.83 (3H, s), 3.87
2
2
3
13
(3H, s), 3.98 (3H, s), 6.90 (2H, d, J = 9.2), 7.35 (1H, s), 7.53 (1H, s), 7.23 (2H, d, J = 9.2). C NMR (100 MHz,
2
0

4
4
4
97
98
99
CDCl ): δ 55.7 (CH ), 56.3 (CH ), 56.7 (CH ), 103.0 (CH), 107.2 (CH), 114.4 (2CH), 129.4 (2CH), 129.7 (C),
3 3 3 3
+
+
129.9 (C), 130.3 (C), 145.2 (C), 155.4 (C), 163.6 (C). HRMS (C H N O S + H ): calcd 369.0751 (M + H ), found
15
16
2
7
369.0752.
5
5
5
5
5
5
5
5
5
00
01
02
03
04
05
06
07
08
N-(2-amino-4,5-dimethoxyphenyl)-4-methoxybenzenesulfonamide (12). To a solution of 11 (620
mg, 1.68 mmol) in ethyl acetate (100 mL) Pd (C) (10 mg) was added and the reaction was stirred at room
temperature under H atmosphere for 48 h. By filtration through celite and solvent evaporation, 550 mg
2
(96%) of crude reaction 12 was obtained. 374 mg (65%) of the purified compound were isolated by
-
1 1
crystallization in CH Cl /Hexane. M.p.: 102-112 °C (CH Cl /Hexane). IR (KBr): 3265, 1458, 835 cm . H NMR
2
2
2
2
(400 MHz, CDCl ): δ 3.46 (3H, s), 3.74 (3H, s), 3.79 (3H, s), 5.72 (1H, s), 5.88 (1H, s), 6.23 (1H, s), 6.87 (2H, d,
3
1
3
J = 8.4), 7.61 (2H, d, J = 8.4). C NMR (100 MHz, CDCl ): δ 55.6 (CH ), 55.8 (CH ), 56.2 (CH ), 101.0 (CH),
3
3
3
3
112.4 (C), 112.9 (CH), 114.1 (2CH), 129.8 (2CH), 130.4 (C), 139.0 (C), 141.5 (C), 149.6 (C), 163.1 (C). HRMS
+
+
(C H N O S + H ): calcd 339.1009 (M + H ), found 339.1018.
15
18
2
5
5
5
5
5
5
5
5
5
5
5
09
10
11
12
13
14
15
16
17
18
N-(2-amino-4,5-dimethoxyphenyl)-4-methoxy-N-methylbenzenesulfonamide (120). To a solution
of 12 (167 mg, 0.49 mmol) in CH CN (25 mL) 68 mg of crushed KOH (0.98 mmol) and 62 µL of methyl iodide
3
(0.98 mmol) were added and stirred at room temperature for 24 h. Then, the reaction mixture was
concentrated, re-dissolved in CH Cl , washed with brine, dried over anhydrous Na SO , filtered and
2
2
2
4
concentrated in vacuum to produce 140 mg (80%) of 120. The crude reaction product was purified by
1
crystallization in MeOH (52 mg, 30%). M.p.: 140-141 °C (MeOH). H NMR (400 MHz, CDCl ): δ 3.11 (3H, s),
3
13
3.46 (3H, s), 3.82 (3H, s), 3.87 (3H, s), 5.80 (1H, s), 6.34 (1H, s), 6.97 (2H, d, J = 8.8), 7.65 (2H, d, J = 8.8). C
NMR (100 MHz, CDCl ): δ 40.2 (CH ), 56.9 (CH ), 57.0 (CH ), 57.6 (CH ), 101.9 (CH), 112.1 (CH), 115.2 (2CH),
3
3
3
3
3
+
119.4 (C), 129.8 (C), 131.6 (2CH), 141.5 (C), 142.2 (C), 151.1 (C), 164.4 (C). HRMS (C H N O S + H ): calcd
16
20
2
5
+
353.1166 (M + H ), found 353.1179.
5
5
5
19
20
21
N-(4,5-dimethoxy-2-((4-methoxy-N-methylphenyl)sulfonamido)phenyl)acetamide (124). 90 mg of
120 (0.25 mmol) were dissolved in CH Cl (45 mL) and pyridine (1 mL). 29 µL of anhydride acetic acid (0.30
2
2
mmol) were added to the solution and stirred at room temperature for 24 h. The reaction mixture was
2
1

5
5
5
5
5
5
5
22
23
24
25
26
27
28
poured onto ice and treated with HCl 2N and NaHCO 5%. The organic layers were washed to neutrality
3
with brine, dried over anhydrous Na SO , filtered and evaporated to dryness. The residue was purified by
2
4
1
silica preparative chromatography (hexane/EtOAc 2:8) to afford 124 (56 mg, 55%). H NMR (400 MHz,
CDCl ): δ 2.21 (3H, s), 3.10 (3H, s), 3.44 (3H, s), 3.85 (3H, s), 3.88 (3H, s), 5.75 (1H, s), 6.96 (2H, d, J = 8.8),
3
1
3
7.55 (2H, d, J = 8.8), 7.89 (1H, s), 8.21 (1H, s). C NMR (100 MHz, CDCl ): δ 24.8 (CH ), 39.5 (CH ), 55.6 (CH ),
3
3
3
3
55.8 (CH ), 56.0 (CH ), 105.9 (CH), 108.9 (CH), 114.0 (2CH), 122.5 (C), 127.1 (C), 130.4 (C), 130.8 (2CH), 144.8
3
3
+
+
(C), 148.8 (C), 163.5 (C), 168.6 (C). HRMS (C H N O S + H ): calcd 395.1271 (M + H ), found 395.1280.
18
22
2
6
5
5
5
5
5
5
5
5
5
5
29
30
31
32
33
34
35
36
37
38
N-(3,4-dimethoxyphenyl)-4-nitrobenzenesulfonamide (23). To a solution of 3,4-dimethoxyaniline
(2.82 g, 18.41 mmol) in CH Cl (50 mL) and pyridine (4 mL), 4-nitrobenzenesulfonyl chloride was slowly
2
2
added (4.49 g, 20.25 mmol) and stirred at room temperature. After 4 h, the reaction was treated with HCl
2N and NaHCO 5%, washed with brine to neutrality, dried over anhydrous Na SO and concentrated under
3
2
4
vacuum to yield 5.43 g (87%) of the sulfonamide 23. The residue was crystallized in EtOAc to afford the
-
1 1
purified compound (4.40 g, 70%). M.p.: 181-183 °C (EtOAc). IR (KBr): 3251, 1532, 1450, 803 cm . H NMR
(400 MHz, CDCl ): δ 3.83 (3H, s), 3.84 (3H, s), 6.38 (1H, s), 6.43 (1H, dd, J = 8.4 and 2.4), 6.69 (1H, d, J = 8.4),
3
1
3
6.77 (1H, d, J = 2.4), 7.87 (2H, d, J = 8.8), 8.28 (2H, d, J = 8.8). C NMR (100 MHz, acetone-d6): δ 55.1 (CH ),
3
55.2 (CH ), 107.6 (CH), 111.9 (CH), 114.9 (CH), 124.1 (2CH), 128.6 (2CH), 129.5 (C), 145.2 (C), 147.6 (C),
3
+
+
149.6 (C), 150.2 (C). HRMS (C H N O S + Na ): calcd 361.0465 (M + Na ), found 361.0469.
14
14
2
6
5
5
5
5
5
5
5
5
39
40
41
42
43
44
45
46
N-(4,5-dimethoxy-2-nitrophenyl)-4-nitrobenzenesulfonamide (334). To a solution of 23 (112 mg,
0.33 mmol) in CH CN (50 mL) tertbutyl nitrite (21.9 µL, 0.16 mmol) was added and stirred at 45°C. After 24
3
h, additional 0.16 mmol of tertbutyl nitrite was added to the reaction mixture and it was stirred at 45°C for
another 24 h. Then, the mixture was concentrated, re-dissolved in EtOAc and treated with 5% NaHCO3
solution. The organic layers were washed with brine, dried over Na SO and concentrated under vacuum to
2
4
yield 334 (109 mg, 86%). The residue was purified by crystallization in MeOH to afford 30 mg (24%). M.p.:
1
169-172 °C (MeOH). H NMR (200 MHz, acetone-d6): δ 3.88 (3H, s), 3.98 (3H, s), 7.28 (1H, s), 7.57 (1H, s),
1
3
8.16 (2H, d, J = 9), 8.41 (2H, d, J = 9). C NMR (100 MHz, Acetone-d6): δ 55.7 (CH ), 56.1 (CH ), 105.3 (CH),
3
3
2
2

5
5
47
48
107.7 (CH), 124.6 (2CH), 127.4 (C), 128.9 (2CH), 144.4 (C), 146.5 (C), 150.7 (C), 150.7 (C), 155.3 (C). HRMS
+
+
(C H N O S + Na ): calcd 406.0316 (M + Na ), found 406.0313.
14
13
3
8
5
5
5
5
5
5
5
5
5
49
50
51
52
53
54
55
56
57
4-amino-N-(3,4-dimethoxyphenyl)benzenesulfonamide (26). To a solution of 23 (1.96 g, 5.81 mmol)
in ethyl acetate (150 mL) an MeOH (5 mL), Pd (C) (10 mg) was added and the reaction was stirred at room
temperature under H atmosphere for 48 h. By filtration through celite and solvent evaporation, 1.71 g
2
(95%) of 26 was obtained and purified by crystallization in EtOAc (1.39 g, 78%). M.p.: 186-187 °C (EtOAc). IR
-
1 1
(KBr): 3449, 3221, 1462, 804 cm . H NMR (400 MHz, CDCl ): δ 3.73 (3H, s), 3.76 (3H, s), 4.01 (2H, s), 6.02
3
(1H, s), 6.41 (1H, dd, J = 8.4 and 2.4), 6.52 (2H, d, J = 8.4), 6.61 (1H, d, J = 8.4), 6.62 (1H, d, J = 2.4), 7.40 (2H,
1
3
d, J = 8.4). C NMR (100 MHz, acetone-d6): δ 55.0 (CH ), 55.3 (CH ), 106.9 (CH), 111.9 (CH), 112.8 (2CH),
3
3
+
113.8 (CH), 126.0 (C), 129.1 (2CH), 131.5 (C), 146.7 (C), 149.4 (C), 152.5 (C). HRMS (C H N O S + Na ): calcd
14
16
2
4
+
331.0723 (M + Na ), found 331.0733.
5
5
5
5
5
5
5
5
5
5
5
58
59
60
61
62
63
64
65
66
67
68
N-(3,4-dimethoxyphenyl)-4-(dimethylamino)benzenesulfonamide (29). To a solution of p-
formaldehyde (1.25 g, 41.11 mmol) in MeOH (40 mL) few drops of acetic acid were added to acid pH, then,
1.27 g of 26 (4.11 mmol) were added and stirred for 30 min. Finally, NaCNBH (517 mg, 24.66 mmol) was
3
added and the reaction was heated at reflux for 24 h. The reaction mixture was concentrated, re-dissolved
in EtOAc and treated with HCl 2N and 5% NaHCO solutions. The organic layers were washed with brine to
3
neutrality, dried over Na SO , filtered through celite and the solvent evaporated in vacuum to afford 1.04 g
2
4
-
1 1
(75%) of 29. M.p.: 161-163 °C (EtOAc). IR (KBr): 3467, 3255, 1596, 795 cm . H NMR (400 MHz, CDCl ): δ
3
3.01 (6H, s), 3.79 (3H, s), 3.82 (3H, s), 6.09 (1H, s), 6.48 (1H, dd, J = 8.4 and 2.4), 6.57 (2H, d, J = 9.2), 6.68
1
3
(1H, d, J = 8.4), 6.70 (1H, d, J = 2.4), 7.51 (2H, d, J = 9.2). C NMR (100 MHz, CDCl ): δ 39.9 (2CH ), 55.8
3
3
(CH ), 55.9 (CH ), 107.2 (CH), 110.6 (2CH), 111.1 (CH), 114.9 (CH), 123.7 (C), 129.1 (2CH), 130.2 (C), 146.7
3
3
+
+
(C), 148.9 (C), 152.8 (C). HRMS (C H N O S + H ): calcd 337.1217 (M + H ), found 337.1204.
16
20
2
4
5
5
5
69
70
71
N-(4,5-dimethoxy-2-nitrophenyl)-4-(dimethylamino)benzenesulfonamide (33). To sulfonamide 29
(1.17 g, 3.48 mmol) in CH CN (50 mL) tertbutyl nitrite was added dropwise by two successive addition (460
3
µL, 3.48 mmol) and the reaction stirred for 24 h at 45°C. Then, the mixture was concentrated, re-dissolved
2
3

5
5
5
5
5
5
5
72
73
74
75
76
77
78
in EtOAc and treated with 5% NaHCO solution. The organic layers were washed with brine, dried over
3
Na SO and concentrated under vacuum to produce 1.22 g of crude reaction product from which 949 mg of
2
4
33 (71%) was isolated by crystallization in EtOAc. M.p.: 190-195 °C (EtOAc). IR (KBr): 3255, 1593, 1525, 793
-
1 1
cm . H NMR (400 MHz, CDCl ): δ 3.01 (6H, s), 3.86 (3H, s), 3.97 (3H, s), 6.57 (2H, d, J = 9.2), 7.35 (1H, s),
3
1
3
7.54 (1H, s), 7.63 (2H, d, J = 9.2). C NMR (100 MHz, CDCl ): δ 40.0 (2CH ), 56.2 (CH ), 56.7 (CH ), 102.4
3
3
3
3
(CH), 107.1 (CH), 110.8 (2CH), 122.9 (C), 129.1 (C), 129.2 (2CH), 131.2 (C), 144.7 (C), 153.2 (C), 155.4 (C).
+
+
HRMS (C H N O S + H ): calcd 382.1067 (M + H ), found 382.1067.
16
19
3
6
5
5
5
5
5
5
5
5
79
80
81
82
83
84
85
86
N-(2-amino-4,5-dimethoxyphenyl)-4-(dimethylamino)benzenesulfonamide (35). To nitroderivative
33 (655 mg, 1.72 mmol) in ethyl acetate (100 mL) under H atmosphere, Pd (C) (10 mg) was added and the
2
reaction stirred at room temperature for 48 h. After filtration through celite and solvent evaporation 577
-
1 1
mg of 35 (95%) was isolated. M.p.: 153-157 °C (MeOH). IR (KBr): 3403, 1449, 824 cm . H NMR (400 MHz,
CDCl ): δ 3.02 (6H, s), 3.49 (3H, s), 3.80 (3H, s), 5.77 (1H, s), 6.00 (1H, s), 6.29 (1H, s), 6.61 (2H, d, J = 9.2),
3
1
3
7.54 (2H, d, J = 9.2). C NMR (100 MHz, CDCl ): δ 41.2 (2CH ), 56.8 (CH ), 57.3 (CH ), 102.1 (CH), 110.0 (CH),
3
3
3
3
111.8 (2CH), 114.3 (C), 124.9 (C), 130.5 (2CH), 139.9 (C), 142.5 (C), 150.5 8C), 154.1 (C). HRMS (C H N O S
16
21
3
4
+
+
+ H ): calcd 352.1326 (M + H ), found 352.1320.
5
5
5
5
5
5
5
5
5
5
87
88
89
90
91
92
93
94
95
96
N-(2-amino-4,5-dimethoxyphenyl)-4-(dimethylamino)-N-methylbenzenesulfonamide (118). To a
solution of 35 (155 mg, 0.44 mmol) in CH CN (25 mL) 61 mg of crushed KOH (0.88 mmol) and 55 µL of
3
methyl iodide (0.88 mmol) were added and stirred at room temperature for 24 h. Then, the reaction
mixture was concentrated, re-dissolved in CH Cl , washed with brine, dried over anhydrous Na SO , filtered
2
2
2
4
and concentrated in vacuum to produce 144 mg of crude reaction product from which 105 mg of 118 (65%)
was isolated by flash chromatography (hexane/EtOAc 4:6). M.p.: 156-157 °C (MeOH). IR (KBr): 3437, 1462,
-
1 1
812 cm . H NMR (400 MHz, CDCl ): δ 3.04 (6H, s), 3.08 (3H, s), 3.47 (3H, s), 3.82 (3H, s), 5.87 (1H, s), 6.33
3
1
3
(1H, s), 6.65 (2H, d, J = 9.2), 7.52 (2H, d, J = 9.2). C NMR (100 MHz, CDCl ): δ 38.8 (CH ), 39.9 (2CH ), 55.7
3
3
3
(CH ), 56.3 (CH ), 100.5 (CH), 110.5 (2CH), 111.0 (CH), 118.7 (C), 121.8 (C), 129.9 (2CH), 140.3 (C), 140.8 (C),
3
3
+
+
149.8 (C), 152.9 (C). HRMS (C H N O S + H ): calcd 366.1482 (M + H ), found 366.1471.
17
23
3
4
2
4

5
97
N-(2-((4-(dimethylamino)-N-methylphenyl)sulfonamido)-4,5-dimethoxyphenyl)
formamide
5
5
6
6
6
6
6
6
6
98
99
00
01
02
03
04
05
06
(132). The compound 118 (60 mg, 0.16 mmol) was stirred in a mixture of CH Cl (30 mL), pyridine (1 mL)
2
2
and formic acid (2 mL) at room temperature for 24 h. Then, the reaction mixture was poured onto ice and
treated with HCl 2N and NaHCO 5%, washed to neutrality with brine, dried over anhydrous Na SO , filtered
3
2
4
and solvent evaporated to produce 51 mg of crude reaction product. The residue was purified by silica
1
preparative chromatography (hexane/EtOAc 2:8) to afford 132 (31 mg, 50%). H NMR (400 MHz, CDCl ): δ
3
3.05 (6H, s), 3.09 (3H, s), 3.48 (3H, s), 3.91 (3H, s), 5.85 (1H, s), 6.65 (2H, d, J = 9.2), 7.45 (2H, d, J = 9.2), 8.00
1
3
(1H, s), 8.42 (1H, s). C NMR (100 MHz, CDCl ): δ 39.4 (CH ), 40.1 (2CH ), 55.8 (CH ), 56.0 (CH ), 105.5 (CH),
3
3
3
3
3
109.1 (CH), 110.5 (2CH), 120.2 (C), 123.0 (C), 129.9 (2CH), 130.3 (C), 144.9 (C), 148.6 (C), 153.2 (C), 159.1
+
+
(CH). HRMS (C H N O S + Na ): calcd 416.1251 (M + Na ), found 416.1250.
18
23
3
5
6
6
6
6
6
6
6
6
6
6
6
07
08
09
10
11
12
13
14
15
16
17
4-((5,6-dimethoxy-1H-benzo[d][1,2,3]triazol-1-yl)sulfonyl)-N,N-dimethylaniline (117). To
a
solution of 35 (100 mg, 0.28 mmol) in MeOH (20 mL) and H O (200 µL) at 0°C, tertbutyl nitrite (33.8 µL, 0.28
2
mmol) was added and the reaction stirred. After 1 h, acetic acid (20 µL) was added to the reaction mixture
and stirred for 24 h to room temperature. Then, the mixture was concentrated, re-dissolved in EtOAc and
treated with 5% NaHCO solution. The organic layers were washed with brine to neutrality, dried over
3
Na SO and concentrated under vacuum. The residue was chromatographed on silica preparative
2
4
1
(hexane/EtOAc 1:1) to afford the purified compound 117 (14 mg, 13%). H NMR (400 MHz, CDCl ): δ 3.03
3
13
(6H, s), 3.94 (3H, s), 4.04 (3H, s), 6.62 (2H, d, J = 9.2), 7.34 (1H, s), 7.46 (1H, s), 7.88 (2H, d, J = 9.2). C NMR
(100 MHz, CDCl ): δ 40.0 (2CH ), 56.2 (CH ), 56.6 (CH ), 92.7 (CH), 99.1 (CH), 110.9 (2CH), 120.8 (C), 127.1
3
3
3
3
+
+
(C), 130.0 (2CH), 139.6 (C), 149.2 (C), 152.7 (C), 154.2 (C). HRMS (C H N O S + H ): calcd 363.1122 (M + H ),
16
18
4
4
found 363.1127.
6
6
6
6
18
19
20
21
Methyl 3,5-dinitrobenzoate (80). 3.24 g of 3,5-dinitrobenzoic acid (15.28 mmol) was stirred in a mixture
of MeOH (100 mL) and H SO (1 mL) for 12 h. Then, anhydrous Na CO was added to the reaction mixture
2
4
2
3
and concentrated in vacuum. The residue was re-dissolved in EtOAc, washed with water to neutrality, dried
over Na SO and solvent evaporated. 3.22 g (94%) of the crude reaction product was obtained and used
2
4
2
5

1
6
6
22
23
without further purification. H NMR (400 MHz, CDCl ): δ 4.06 (3H, s), 9.18 (2H, d, J = 2.4), 9.24 (1H, t, J =
3
2.4). GC-MS (C H N O ): calcd 226, found 226.
8
6
2
6
6
6
6
6
6
24
25
26
27
28
Methyl 3,5-diaminobenzoate (81). The compound 80 (3.23 g, 14.27 mmol) was suspended in ethyl
acetate (100 mL) and was palladium-catalyzed (Pd (C) 10 mg) reduced under H atmosphere for 72 h. The
2
reaction mixture was filtered through Celite© and the solvent evaporated in vacuum to isolate 2.18 g (92%)
1
of 81. Crude reaction product was obtained and used without further purification. H NMR (400 MHz,
CDCl ): δ 3.85 (3H, s), 6.18 (1H, t, J = 2), 6.77 (2H, d, J = 2). GC-MS (C H N O ): calcd 166, found 166.
3
8
10
2
2
6
29
Methyl 3-amino-5-((4-methoxyphenyl)sulfonamido)benzoate (84A) and methyl 3,5-bis((4-
6
6
6
6
6
6
6
6
6
6
6
6
6
6
30
31
32
33
34
35
36
37
38
39
40
41
42
43
methoxyphenyl)sulfonamido)benzoate (84B). To a solution of 81 (1.52 g, 9.14 mmol) in CH Cl (50 mL)
2
2
and pyridine (1 mL), was dropwise added 4-methoxybenzenesulfonyl chloride (1.89 g, 9.14 mmol) dissolved
in CH Cl (20 mL). The mixture was stirred at room temperature for 4 h. Then the reaction was treated with
2
2
HCl 0.5N washed with brine, dried over anhydrous Na SO and concentrated in vacuum. The residue was
2
4
purified by flash chromatography on silica gel with toluene/EtOAc (7:3) to yield compounds 84A (375 mg,
12%) and 84B (957 mg, 41%). 84A: M.p.: 165-166 °C (CH Cl /Hexane). IR (KBr): 3468, 3377, 1697, 1497,
2
2
-
1 1
1176, 803 cm . H NMR (400 MHz, CDCl ): δ 3.80 (3H, s), 3.84 (3H, s), 6.82 (1H, t, J = 2), 6.87 (2H, d, J = 8.8),
3
1
3
7.01 (1H, t, J = 2), 7.09 (1H, t, J = 2), 7.71 (2H, d, J = 8.8). C NMR (100 MHz, CD OD): δ 51.1 (CH ), 54.7
3
3
(CH ), 110.2 (CH), 110.8 (CH), 111.5 (CH), 113.8 (2CH), 128.9 (2CH), 130.9 (C), 131.2 (C), 138.8 (C), 148.9 (C),
3
+
+
163.1 (C), 167.0 (C). HRMS (C H N O S + H ): calcd 337.0853 (M + H ), found 337.0855. 84B: M.p.: 174-178
15
16
2
5
-
1 1
°C (CH Cl /Hexane). IR (KBr): 3270, 1724, 1498, 1150, 802 cm . H NMR (400 MHz, CD OD): δ 3.81 (9H, s),
2
2
3
1
3
6.94 (4H, d, J = 8.8), 7.31 (2H, d, J = 2), 7.35 (1H, t, J = 2), 7.63 (4H, d, J = 8.8). C NMR (100 MHz, CD OD): δ
3
51.5 (CH ), 54.8 (2CH ), 113.8 (4CH), 115.1 (CH), 116.0 (2CH), 129.0 (4CH), 130.6 (2C), 131.5 (C), 139.1 (2C),
3
3
+
+
163.2 (2C), 165.9 (C). HRMS (C H N O S + Na ): calcd 529.0710 (M + Na ), found 529.0749.
22
22
2
8 2
6
6
6
44
45
46
Methyl 3,5-bis((4-methoxy-N-methylphenyl)sulfonamido)benzoate (147). To a solution of 84B (132
mg, 0.39 mmol) in acetone (20 mL) K CO (542 mg, 3.92 mmol) and (CH ) SO (281 µL, 2.94 mmol) were
2
3
3 2
4
added, heated at reflux and stirred overnight. Then, the reaction mixture was filtered, poured onto ice and
2
6

6
6
6
6
6
6
47
48
49
50
51
52
extracted with CH Cl . The organic layers were dried over anhydrous Na SO , filtered and evaporated to
2 2 2 4
1
dryness. By crystallization in MeOH compound 147 (73 mg, 51%) was isolated. M.p.: 139-142 °C (MeOH). H
NMR (400 MHz, CDCl ): δ 3.10 (6H, s), 3.84 (6H, s), 3.86 (3H, s), 6.91 (4H, d, J = 8.8), 7.21 (1H, t, J = 2), 7.45
3
1
3
(4H, d, J = 8.8), 7.66 (2H, d, J = 2). C NMR (100 MHz, CDCl ): δ 38.1 (2CH ), 52.9 (CH ), 56.1 (2CH ), 114.7
3
3
3
3
(4CH), 126.0 (2CH), 127.9 (2C), 129.4 (CH), 130.3 (4CH), 131.7 (C), 142.9 (2C), 163.7 (2C), 165.9 (C). HRMS
+
+
(C H N O S + Na ): calcd 557.1023 (M + Na ), found 557.1024.
24
26
2
8 2
6
6
6
6
6
6
6
53
54
55
56
57
58
59
Methyl 3-amino-5-methoxybenzoate (78). 957 mg of 3-amino-5-methoxybenzoic acid (5.73 mmol) was
stirred in a mixture of MeOH (50 mL) and H SO (1 mL) for 24 h. Then, anhydrous Na CO was added to the
2
4
2
3
reaction mixture, filtered and concentrated in vacuum. The residue was re-dissolved in EtOAc, filtered again
and evaporated to dryness. 799 mg (77%) of crude reaction product was obtained and used without further
1
purification. H NMR (400 MHz, CD OD): δ 3.74 (3H, s), 3.83 (3H, s), 6.48 (1H, t, J = 2), 6.84 (1H, t, J = 2), 6.95
3
1
3
(1H, t, J = 2). C NMR (100 MHz, CDCl ): δ 52.1 (CH ), 55.3 (CH ), 104.3 (CH), 105.7 (CH), 109.2 (CH), 132.0
3
3
3
(C), 147.6 (C), 160.6 (C), 167.1 (C). GC-MS (C H NO ): calcd 181, found 181.
9
11
3
6
6
6
6
6
6
6
6
6
6
60
61
62
63
64
65
66
67
68
69
Methyl 3-methoxy-5-((4-methoxyphenyl)sulfonamido)benzoate (79). To a solution of 78 (620 mg,
3.42 mmol) in CH Cl (50 mL) and pyridine (2 mL), was slowly added 4-methoxybenzenesulfonyl chloride
2
2
(707 mg, 3.42 mmol). The mixture was stirred at room temperature for 4 h. Then the reaction was treated
with HCl 2N and NaHCO 5%, washed with brine, dried over anhydrous Na SO and the solvent evaporated
3
2
4
to obtain 1.15 g (95%) of the sulfonamide 79. The crude reaction product was purified by crystallization in
-
1 1
CH Cl (529 mg, 44%). M.p.: 176-177 °C (CH Cl ). IR (KBr): 3258, 1700, 1497, 1152, 802 cm . H NMR (400
2
2
2
2
MHz, CD OD): δ 3.76 (3H, s), 3.81 (3H, s), 3.85 (3H, s), 6.91 (1H, t, J = 2.4), 6.99 (2H, d, J = 8.8), 7.20 (1H, t, J
3
1
3
= 2.4), 7.32 (1H, t, J = 2.4), 7.71 (2H, d, J = 8.8). C NMR (100 MHz, acetone-d6): δ 51.6 (CH ), 54.9 (CH ),
3
3
55.1 (CH ), 109.6 (CH),110.2 (CH), 113.1 (CH), 114.2 (2CH), 129.2 (2CH), 131.2 (C), 132.1 (C), 139.6 (C), 160.3
3
+
+
(C), 163.2 (C), 165.6 (C). HRMS (C H NO S + H ): calcd 352.0849 (M + H ), found 352.0842.
16
17
6
6
6
70
71
3-methoxy-5-((4-methoxy-N-methylphenyl)sulfonamido)benzoic acid (90). To a solution of 79 (100
mg, 0.28 mmol) in CH CN (40 mL) 38 mg of crushed KOH (0.56 mmol) and 36 µL of methyl iodide (0.56
3
2
7

6
6
6
6
6
6
6
6
72
73
74
75
76
77
78
79
mmol) were added and stirred at room temperature for 24 h. Then, the reaction mixture was concentrated,
re-dissolved in CH Cl , treated with HCl 2N, washed with brine to neutrality, dried over anhydrous Na SO ,
2
2
2
4
filtered and concentrated in vacuum. The residue was purified by silica preparative chromatography with
CH Cl (98:2) yielding compound 90 (69 mg, 66%). M.p.: 186-187 °C (CH Cl ). IR (KBr): 3000, 1689, 1502, 806
2
2
2
2
-
1
1
cm . H NMR (400 MHz, CD OD): δ 3.16 (3H, s), 3.79 (3H, s), 3.86 (3H, s), 6.94 (1H, t, J = 2.4), 7.03 (2H, d, J =
3
1
3
8.8), 7.26 (1H, t, J = 2.4), 7.46 (1H, t, J = 2.4), 7.47 (2H, d, J = 8.8). C NMR (100 MHz, CDCl ): δ 37.8 (CH ),
3
3
55.6 (CH ), 55.7 (CH ), 113.8 (CH), 114.0 (2CH), 118.7 (CH), 119.3 (CH), 127.7 (C), 129.9 (2CH), 130.9 (C),
3
3
+
+
143.1 (C), 159.7 (C), 163.2 (C), 170.6 (C). HRMS (C H NO S + H ): calcd 352.0849 (M + H ), found 352.0848.
16
17
6
6
80
2.2 Determination of aqueous solubility
6
6
6
6
6
6
6
81
82
83
84
85
86
87
The aqueous solubility of the sulfonamides was determined in a Helios Alfa Spectrophotometer using an
approach based on the saturation shake-flask method. 1-2 mg of each tested compound was suspended in
300 µL pH 7.0 buffer and stirred for 72 h at room temperature. The resulting mixture was filtrated over a 45
µm filter to discard the insoluble residues. Then, a scan between 270 and 400 nm was performed and the
three maximum wavelengths of absorbance of each compound were selected. Calibration lines were
performed at these wavelengths and the concentration in the supernatant was measured by UV
absorbance.
6
88
2.3 Cells and culture conditions
6
6
6
6
89
90
91
92
The Leishmania strains used in this study were PER/ES/2013/ATE1FL6 and MCAN/ES/MON1/Z001 of
Leishmania infantum. The macrophage human cell line used, originally obtained from a patient with
histiocytic leukemia was U937 (ATCC® CRL1593.2) and human tumor cell lines were HT-29, HeLa, and MCF7,
obtained from a patient with colon, cervical, and breast cancer respectively.
6
6
6
93
94
95
Leishmania promastigotes were cultured at 27°C in RPMI 1640 supplemented with L-glutamine (Lonza-
Cambrex, Karlskoga, Sweden), 10 % heat-inactivated fetal bovine serum (HIFBS) (Lonza-Cambrex), and 100
μg/mL streptomycin-100 IU/mL penicillin (Lonza-Cambrex) in 25 mL culture flasks. Logarithmic and late
2
8