Functionalization of fluorinated benzenesulfonamides and their inhibitory properties toward carbonic anhydrases

DOI: 10.1002/cmdc.201402490

Full Papers

Functionalization of Fluorinated Benzenesulfonamides and

Their Inhibitory Properties toward Carbonic Anhydrases

[a]

˙

Virginija Dudutiene, Asta Zubriene, Alexey Smirnov, David D. Timm,

[a]

˙

ˇ

Joana Smirnoviene, Justina Kazokaite, Vilma Michailoviene, Audrius Zaksauskas,

[b]

[a]

ˇ

Elena Manakova, Saulius Grazulis, and Daumantas Matulis*

Substituted tri- and tetrafluorobenzenesulfonamides were de-

signed, synthesized, and evaluated as high-affinity and iso-

form-selective carbonic anhydrase (CA) inhibitors. Their bind-

ing affinities for recombinant human CA I, II, VA, VI, VII, XII, and

XIII catalytic domains were determined by fluorescent thermal

shift assay, isothermal titration calorimetry, and a stopped-flow

CO₂hydration assay. Variation of the substituents at the 2-, 3-,

and 4-positions yielded compounds with a broad range of

binding affinities and isoform selectivities. Several 2,4-substitut-

ed-3,5,6-trifluorobenzenesulfonamides were effective CA XIII

inhibitors with high selectivity over off-target CA I and CA II.

3,4-Disubstituted-2,5,6-trifluorobenzenesulfonamides bound

CAs with higher affinity than 2,4-disubstituted-3,5,6-trifluoro-

benzenesulfonamides. Many such fluorinated benzenesulfona-

mides were found to be nanomolar inhibitors of CA II, CA VII,

tumor-associated CA IX and CA XII, and CA XIII. X-ray crystal

structures of inhibitors bound in the active sites of several CA

isoforms provide structure–activity relationship information for

inhibitor binding affinities and selectivity.

Introduction

Human carbonic anhydrases (CA) are zinc metalloenzymes that

catalyze the reversible hydration of carbon dioxide. This reac-

tion is very important in the human body, as it regulates

a broad range of physiological functions such as respiration,

CO₂/bicarbonate transport between lungs and metabolizing

tissues, pH and CO₂homeostasis, and electrolyte secretion in

many tissues and organs.^[1,2]There are 12 active CA isoforms,

which differ in cellular localization, distribution in organs and

tissues, expression levels, and kinetic properties.^[3–6]The struc-

tures of these isozymes are highly homologous, and only

subtle differences between isozyme active sites are ob-

served.^[7,8]The increased activity or expression of various CA

isoforms (e.g., CA II, IV, VA, VB, VII, IX, XII, XIII, and XIV) is often

associated with different human diseases.^[1,9,10]In 1954 acet-

azolamide, the first marketed systemic carbonic anhydrase in-

hibitor for the treatment of glaucoma, was reported.^[11]To

date, approximately 25 drugs or drug candidates are used as

diuretics, antiglaucoma drugs, anticonvulsants, anti-altitude

sickness, and anticancer agents. Many drugs in current use

have side effects that are thought to be due to inhibition of

CA isoforms not involved in the target disease.^[12]Therefore,

there is a need to improve the inhibition and selectivity pro-

files of CA inhibitors (CAIs).

The main class of CAIs is aromatic and heterocyclic sulfona-

mides. Benzenesulfonamides are the most investigated and ef-

fective CAIs.^[4]The binding affinity of benzenesulfonamides de-

pends highly on the pK_aof the sulfonamide group. The high-

est-affinity arylsulfonamides should have a pK_anear the pH of

buffered medium (~7.4).^[13,14]Without the electronegative

groups, the pK_aof benzene sulfonamide is ~11. The presence

of electronegative substituents decreases the pK_aof sulfona-

mide, and this correlates with an increase in CA inhibitory

properties. Introduction of halogen atoms, especially fluorine,

as substituents is one possible way toward better CA inhibi-

tors.^[15,16]The unique features of fluorine enable the synthesis

of compounds with extraordinary properties that cannot be at-

tained using any other element. There are numerous studies

that have investigated benzenesulfonamides containing one

fluorine atom in the benzene scaffold as CA inhibitors.^[17–28]

However, the introduction of several fluorine atoms is less

common.^[13,29–32]

˙

[a] Dr. V. Dudutiene, Dr. A. Zubriene, A. Smirnov, D. D. Timm, J. Smirnoviene,

ˇ

J. Kazokaite˙, V. Michailoviene˙, A. Zaksauskas, Prof. D. Matulis

Department of Biothermodynamics and Drug Design

Institute of Biotechnology, Vilnius University

Polyfluoroaromatic compounds such as hexafluoroben-

zene^[33,34]or pentafluoropyridine^[35]readily undergo nucleophil-

ic aromatic substitution reactions. Even complete nucleophilic

replacement of all fluorine atoms is possible. A second nucleo-

philic replacement for hexafluorobenzene always occurs at the

position para to the first substituent.^[33]The same reactivity

profile is common for other C₆H₅X compounds.^[36]However,

there are exceptions for which ortho substitution is exclusive

or predominant.^[36,37]The ortho-directing effects depend on the

ˇ

Graiciuno 8, Vilnius 02241 (Lithuania)

¯

E-mail: matulis@ibt.lt

daumantas.matulis@bti.vu.lt

ˇ

[b] Dr. E. Manakova, Dr. S. Grazulis

Department of Protein–DNA Interactions

Institute of Biotechnology, Vilnius University

ˇ

Graiciuno 8, Vilnius 02241 (Lithuania)

¯

Supporting information for this article is available on the WWW under

http://dx.doi.org/10.1002/cmdc.201402490.

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Full Papers

character of the X group and the reaction conditions that

allow specific interaction with the nucleophilic reagent. Exclu-

sive ortho substitution was observed for substituents X (C₆F₅X)

containing carbon–oxygen, carbon–nitrogen, and sulfur–

oxygen double bonds in reactions with metal-containing re-

agents such as Grignard, organolithium, or halomagnezyla-

mines. The interactions between the X substituent and the

metal-containing reagent cause the ortho substitution. Analo-

gous interactions between substituent X and the incoming nu-

cleophiles can be enhanced in nonpolar solvents. Reactions of

C₆F₅SO₂CH₃, C₆F₅CO₂Et, C₆F₅CO₂H, C₆F₅COCH₃, and

Results and Discussion

Chemistry

A series of 2,4-substituted-3,5,6-trifluorobenzenesulfonamides,

2-substituted-3,5,6-trifluorobenzenesulfonamides, 2-substitut-

ed-3,4,5,6-tetrafluorobenzenesulfonamides,

3,4-substituted-

2,5,6-trifluorobenzenesulfonamides, and 3,4,5-substituted-2,6-

difluorobenzenesulfonamides were designed and synthesized

(Schemes 1 and 2). The synthesis of starting compounds 1–5,

C₆F₅NO₂with MeNH₂and Me₂NH in benzene and ni-

tromethane have been investigated in detail.^[38]

A

substantial shift in the ortho/para ratio was observed

upon changing from a polar to a nonpolar solvent.

The least common site of attack for C₆F₅X is that of

meta substitution. Strong electron-donating substitu-

ents X (O^À, NH₂) deactivate ortho and para positions

more than meta toward nucleophilic substitution,

and replacement of meta-fluorine occurs.^[39–41]

Owing to the high reactivity of polyfluorinated

compounds (C₆F₅X), monosubstitution (C₆F₄XY) or

even further substitutions are possible. An example

of such compounds is pentafluoronitrobenzene. Re-

actions between pentafluoronitrobenzene and a varie-

ty of nucleophiles have been established and have

been found to be regioselective in the majority of

cases in which polar solvents were used, giving para-

substituted products. In addition, high ortho selectivi-

ty was observed for cases in which nonpolar solvents

were used. 4-Substituted-2,3,5,6-tetrafluoronitroben-

zenes are still activated toward nucleophilic attack,

and 2,4-substituted-3,5,6-trifluoronitrobenzenes can

be obtained.^[42]A further sequential nucleophilic sub-

stitution process affords 2,4,6-substituted-3,5-di-

Scheme 1. Structures of compounds 1(a–o), 2(h–k,m–o), 3(h–k,m, n), 4h, and 5h.

fluoronitrobenzenes.^[43]The reactivity profile established for

pentafluoronitrobenzene in which the 4-, 2-, and 6-positions

are sequentially substituted has allowed chemists to synthesize

a diverse range of compounds.^[42–46]

9, 10 was described previously by our research group.^[47]Reac-

tions of compounds 1–5 with various nitrogen-centered nucle-

ophiles were carried out in DMSO in the presence of Et₃N. The

ortho-substituted (according to the sulfonamide group) prod-

ucts 1(a–o), 2(h–k,m–o), 3(h–k,m,n), 4h, and 5h were ob-

tained. The substitution profile of sulfonamides 1–5 is similar

to that of 4-substituted-2,3,5,6-tetrafluoronitrobenzenes.^[42,43]

Spectral elucidation of purified products proved substitution at

the ortho position. In the ¹³C NMR spectra of isolated products

1(a–o) there were triplets for methylene groups (SCH₂CH₂Ph)

at 35.3–35.5 ppm due to the coupling of two identical fluo-

rines to carbon (coupling constants were 3.2–4.0 Hz). These flu-

orines are in the ortho position relative to the SCH₂CH₂Ph

group. The substituents that appeared from incoming N-cen-

tered nucleophiles exhibited similar CÀF couplings. The signals

of carbons (NÀC) appeared as doublets (due to one fluorine

coupling to carbon) at 40–66 ppm with coupling constants of

10–12 Hz (6 Hz for 1e) for compounds 1(a–o). The H,C HETCOR

spectrum of compound 1m confirmed our assignment of

carbon signals in the ¹³C NMR spectra where the triplet at

35.5 ppm belongs to the carbon of group SCH₂CH₂Ph and the

doublet at 62.3 ppm belongs to the carbon of fragment NHCH.

In a previous study,^[47]we assayed a series of 4-substituted-

2,3,5,6-tetrafluorobenzenesulfonamides as inhibitors of CA iso-

zymes I, II, VII, XII, and XIII. All fluorinated benzenesulfonamides

exhibited higher binding potency toward tested CAs (especial-

ly toward CA I) than nonfluorinated compounds. These studies

suggested that a polyfluorinated benzenesulfonamide scaffold

is a good tool for CA inhibitor development. To understand

the contribution of substituents toward isoform selectivity and

affinity, herein we focus on modifications of 4-substituted-

2,3,5,6-tetrafluorobenzenesulfonamides at the ortho and meta

positions.

A series of fluorinated benzenesulfonamides were synthe-

sized, namely 2,4-substituted-3,5,6-trifluorobenzenesulfona-

mides, 2-substituted-3,5,6-trifluorobenzenesulfonamides, 2-sub-

stituted-3,4,5,6-tetrafluorobenzenesulfonamides, 3,4-substitut-

ed-2,5,6-trifluorobenzenesulfonamides, and 3,4,5-substituted-

2,6-difluorobenzenesulfonamides, in search of CA inhibitors

with high affinity and isoform selectivity.

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The same couplings were observed for compounds 2(h–k,m–

o) and 4h in the ¹³C NMR spectra.

Table 1. 7a/8a and 7b/8b product ratios in various solvents.^[a]

The previously described para-substituted compounds 1–5,

9, and 10^[47]were synthesized from pentafluorobenzensulfona-

mide by nucleophilic substitution using DMSO as solvent

(other solvents were not attempted). Investigation of penta-

fluorobenzensulfonamide nucleophilic substitution in other

solvents such as dioxane and benzene showed the possibility

of obtaining the ortho-substituted (relative to the sulfonamide

group) products (Scheme 2). In the case of previously de-

Solvent

7a/8a

7b/8b

dioxane

C₆H₆

DMSO

43:57

84:16

100% 8a

34:66

71:29

100% 8b

[a] Ratios determined by NMR peak integration of crude products.

Reactions of compounds 9 and 10 with various nitrogen-

centered nucleophiles were accomplished, and meta-substitut-

ed (relative to sulfonamide group) products were obtained.

Sulfonamides 9(d,e,g–q) were obtained from compound 9 by

using the appropriate nucleophile in DMSO in the presence of

triethylamine. Compound 9a was synthesized with excess

methylamine (solution in methanol) in methanol. Fluorinated

derivative 9b was obtained by using 2 equivalents of nucleo-

phile in DMSO. Compound 10, bearing a (2-hydroxyethyl)sul-

fonyl “tail”, was sensitive to such bases as Et₃N or K₂CO₃; in-

stead of these bases the equivalent of nitrogen-centered nu-

cleophile was used. Sulfonamides 10(d,h–k,n–p) were obtained

from compound 10 by using 2 equivalents of the appropriate

nucleophile in DMSO. The synthesis of compounds 1i, 2h, and

10h was described previously.^[52]

Scheme 2. Synthetic route for compounds 7(a,b) and 8(a,b). Reagents and

conditions: a) dioxane or C₆H₆or DMSO, Et₃N (see Experimental Section for

reaction temperatures and duration).

The spectral elucidation of purified products led us to sup-

pose that the nucleophilic substitution for compounds bearing

(2-phenylethyl)sulfonyl and (2-hydroxyethyl)sulfonyl “tails” took

place at the meta position. We expected to observe splittings

for a methylene group (SO₂CH₂CH₂Ph and SO₂CH₂CH₂OH) as in

the case of compounds 1(a–o), 2(h–k,m–o), 4h in the ¹³C NMR

spectra. However, such splittings were observed only for com-

pounds 9h (recorded in CDCl₃), 9p, and 9q (both recorded in

[D₆]DMSO). In the ¹³C NMR spectra of isolated products 9h, 9p,

scribed compounds 1(a–o), 2(h–k,m–o), 4h, and 5h, ortho sub-

stitution was achieved after para substitution. The formation

of ortho-substituted products 7(a,b) is due to the ortho-direct-

ing capacity of the sulfonamide group. This feature of the sul-

fonamide group is readily recognizable in nonpolar solvents.

The ortho-directing capacity of nitro^[48,49]and carboxylate

groups^[50]in S_NAr reactions has been known for more than 50

years. The regioselectivity of 2,4-dihaloaromatic compounds

with various functionalities in reactions with piperidine were

investigated:^[51]Wendt and Kunzer concluded that compounds

which display a strong preference for ortho substitution in

nonpolar solvents possess functionalities that can interact with

the incoming nucleophile, biasing it toward ortho attack via

a hydrogen bond or electrostatic interactions. Among other

compounds, 2,4-difluorobenzenesulfonamide was investigated

in reaction with piperidine using dioxane and DMSO as sol-

vents. The significant shift in regioselectivity toward the ortho

product was observed upon changing DMSO to dioxane (ratio

from 32 in DMSO to 96 in dioxane).

and 9q, there were doublets for

a methylene group

(SO₂CH₂CH₂Ph) at 58.7, 58.0, and 58.0 ppm, with respective

coupling constants of 4, 2, and 2 Hz.

The splitting was due to the coupling of one fluorine atom

to carbon. This fluorine is at the ortho position relative to the

SO₂CH₂CH₂Ph group. The H,C HETCOR spectrum of compound

9h confirmed our assignment of carbon signals in the ¹³C NMR

spectrum, in which the doublet at 58.7 ppm belongs to the

methylene carbon of SO₂CH₂CH₂Ph, and the doublet at

56.2 ppm belongs to the carbon of fragment NHCH. The sub-

stituents that appeared from incoming N-centered nucleo-

philes similarly to compounds 1(a–o), 2(h–k,m–o), 3(h–k,m,n),

4h, and 5h exhibit CÀF couplings. The signals of carbons

(NÀC) appeared as doublets (due to one fluorine coupling to

carbon) at 34–66 ppm with coupling constants of 11–13 Hz

(4 Hz for 9b,g,l) for compounds 9(a,b,d,e,g–q) and 10(d,h–k,n–

p).

Furthermore, meta substitution for compounds 9d, 10d,

and 10p was demonstrated by X-ray structures of CA crystals

soaked with these inhibitors. The position of the substituent

was well resolved in the electron density of the compounds

bound in active centers of CAs (see Crystallography section

below). The substitution profile of compounds 9 and 10 differs

from that of compounds 1 and 2. The oxidized groups

The substrate pentafluorobenzensulfonamide S_NA reactions

with the primary amine 4-methoxybenzylamine and the secon-

dary amine piperidine were investigated in such solvents as di-

oxane, C₆H₆, and DMSO (Scheme 2, Table 1). Exclusive para

substitution with both amines was observed using DMSO. Pre-

dominant ortho substitution was observed in C₆H₆, and espe-

cially for primary amine. The ortho-para substitution ratios

were nearly equal in dioxane for the primary amine; however,

predominant para substitution was observed for the secondary

amine. Primary amine-4-methoxybenzylamine showed a greater

ability for ortho substitution in both solvents (dioxane and

C₆H₆) than the secondary amine piperidine.

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SO₂CH₂CH₂Ph and SO₂CH₂CH₂OH exhibit stronger electron-

withdrawing properties than SO₂NH₂, and direct nucleophiles

to the meta position relative to the SO₂NH₂group.

double meta-substituted products, and allowed us to elucidate

the correct structure of the compounds. As an example, we an-

alyzed the ¹³C NMR spectrum of compound 12k. The signal of

benzene ring carbon C4 appeared as a triplet at 115.5 ppm

with a coupling constant of 3 Hz. The splitting was due to the

coupling of two identical fluorines to meta-positioned carbon

C4. The signal of benzene ring carbon C1 appeared as a triplet

as well (127.9 ppm). However, in this case, the coupling con-

stant was larger (16 Hz) because two identical fluorines were in

the ortho positions relative to C1.

In the ¹³C NMR spectrum, there was a double doublet for

carbons C3 and C5 at 135.7 ppm, with respective coupling

constants of 10 and 6 Hz. The splitting was due to coupling of

two fluorines to ortho-, para-positioned carbons. The signal of

benzene ring carbons C2 and C6 appeared as double doublet

as well. However, in this case the first coupling constant was

much larger (247 Hz) due to flu-

The 3-substituted compounds bearing (2-phenylethyl)sulfon-

yl and (2-hydroxyethyl)sulfonyl “tails” are activated toward a nu-

cleophilic attack and thus the 3,5-substituted compounds were

obtained. The second nucleophilic substitution occurred at the

meta position again. Compound 11 h was obtained from com-

pound 9 by using 2 equivalents of cyclooctylamine in DMSO in

the presence of Et₃N. Compound 12k was obtained from com-

pound 10 by using 4 equivalents of 3,4-dimethoxybenzylamine

in DMSO. Compounds 13(c,f) with different substituents at the

meta position were obtained (Scheme 3). These compounds

were synthesized from compound 9i by using 2 equivalents of

the appropriate nucleophile in DMSO. The reactivity profile of

compounds 9 and 10 is similar to that of pentafluoronitroben-

orines coupling to ipso carbons

(directly substituted by fluo-

rines). The substituents that ap-

peared from incoming N-cen-

tered nucleophiles exhibited CÀF

couplings.

Interestingly,

the

signal of carbon (NHCH₂R) ap-

peared as a triplet with a cou-

pling constant of 6 Hz instead of

doublet or double doublet. The

same splitting was observed for

compound 11 h as well. We

assume that the triplet appeared

as a result of double doublet

overlapping. However, the split-

ting into doublets was observed

for carbon signals of compounds

13(c,f) bearing different substitu-

ents at the meta position. H,C

HETCOR spectrum of compound

12k confirmed our assignment

of carbon signals in the ¹³C NMR

Scheme 3. Structures of compounds 9(a,b,d,e,g–q), 10(d,h–k,n–p), 11 h, 12k, 13(c,f).

spectrum, where the triplet at

51.2 ppm belongs to the methyl-

zene, in which the 4-, 2-, and 6-positions were sequentially

substituted. However, two similar electron-withdrawing groups

were present in our case, so the asymmetric substitution was

possible, as expected. Nevertheless, only symmetric substitu-

tion products were obtained.

ene group of NHCH₂R, and the singlet at 58.3 ppm belongs to

the methylene carbon of substituent SO₂CH₂CH₂OH.

Binding studies

Spectral elucidation of the purified products showed

a double meta substitution. There was one signal as singlet in

the ¹⁹F NMR spectra of isolated products 11 h and 12k. This

proved the presence of two identical fluorine atoms. The

¹³C NMR spectra of these compounds also confirmed the

double meta substitution. The signals of benzene ring carbons

were split by fluorine coupling to carbon. The CÀF couplings

involved in the functional groups attached to a benzene ring

were previously discussed. These couplings were most impor-

tant for the determination of substitution direction. The split-

ting of benzene ring carbon signals is very informative in

The 4-substituted-2,3,5,6-tetrafluorobenzenesulfonamides have

been previously described as potent inhibitors of CA I, II, VII,

XII, and XIII, exhibiting particularly strong binding to CA I (sub-

nanomolar affinity) and being selective for CA I.^[47]The repre-

sentative derivatives 1, 2, 9, and 10 bound to CA I with K_d

values in the range of 0.1–0.25 nm and had higher binding af-

finity than tetrafluorobenzenesulfonamide 3 (K_d=2.4 nm) and

pentafluorobenzenesulfonamide 6 (PFBSA; K_d=2.2 nm). Thus,

it appeared that the additional contacts between the para sub-

stituent on the benzenesulfonamide ring and CA have

emerged. Indeed, the crystal structure of 4-phenylethylsulfon-

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yl-2,3,5,6-tetrafluorobenzenesulfonamide 9 in complex with

CA II shows that the van der Waals interactions with the pro-

tein side chains participate in binding, and the sulfonyl group

of the para “tail” also makes hydrogen bonds with the protein

active site residues, which also contribute favorably to the

binding affinity.^[47]

Because CA I is involved in retinal cerebral edema,^[53]we

found such derivatives to be interesting candidates for the de-

velopment of anti-edema drugs. However, for targeting of any

other isozyme involved in certain pathology (for example,

CA XII, which is a potential target in cancer), the inhibition of

widespread cytosolic isozymes CA I and II is undesirable be-

cause it can lead to side effects.

Introduction of substituents at the ortho position (com-

pounds 1(a–o), 2(h–o), 3(h–n), 4h, 5h, 7(a,b)) in most cases

lowered the affinity for all CA isozymes, especially for CA I

(Table 2). The binding of all these compounds to CA I was in

the micromolar or millimolar range (or even lower affinity), sig-

nificantly weaker than their analogues without ortho substitu-

ents (compounds 1–3, 6) which bound to CA I in the nanomo-

lar range. Most of the compounds were also weak inhibitors of

CA II, VA, and VI (K_din the micromolar range). ortho-Substituted

compounds were the most effective and selective inhibitors

toward CA XIII. Compound 1d was the most potent inhibitor

of CA XIII (K_d=40 nm) in the series of 1(a–p), 2i (K_d=11 nm)

was most potent among the compounds of series 2(h–o), and

3k (K_d=140 nm) was the most potent of the series 3(h–n). Fig-

ure 1A shows CA I and CA XIII stabilization by 2k, resulting in

the >100-fold differences in their K_dvalues.

Therefore, we focus on the modification of 4-substituted-

2,3,5,6-tetrafluorobenzenesulfonamides at the ortho and meta

positions in order to show the contribution of substituents to

the selectivity and affinity of in-

hibitors. The first group of com-

pounds were ortho-substituted

compounds at the para position

bearing a long-chain (phenyle-

thylthio or hydroxyethylthio)

substituent (compounds 1(a–p),

2(h–o), 4h, 5h), fluorine (7(a, b))

or nonsubstituted compounds

(3(h–n)) (Schemes 1 and 2,

Table 2). The second group of

compounds included the meta-

substituted ligands bearing (2-

phenylethyl)sulfonyl (9a–q) and

(2-hydroxyethyl)sulfonyl (10(d–

p)) “tails” at the para position

(Scheme 3, Table 3). Double

meta-substituted

compounds

with (2-phenylethyl)sulfonyl

(11 h) and (2-hydroxyethyl)sul-

fonyl (12k, 13c, 13 f) “tails” at

the para positions were also syn-

thesized (Scheme 3, Table 4).

The fluorinated compounds

bearing substitutions at ortho or

meta positions were tested for

binding to seven CA isoforms:

CA I, II, VA, VI, VII, XII, and XIII.

The binding affinity for all CAs

was determined by the fluores-

cent thermal shift assay (FTSA)

Figure 1. Selected compound binding curves as determined by fluorescent thermal shift assay. A) Compound 2k

binding to CA isozymes I and XIII. B) Compounds 7a and 7b binding to CA I. Panels at top show the protein raw

melting curves at several added ligand concentrations. Panels on the bottom show the dependence of the pro-

tein melting temperatures (T_m) on ligand concentrations. Data points are from the experimental values of the raw

melting curves (upper panels) and the curve fits are simulated according to the model.^[55]

and confirmed for some compounds (2h and 10d) by

ITC (Tables 2–4). In addition to the binding measurements

by FTSA and ITC, IC₅₀values for compounds 2h and 10h

for CA II were also determined by the stopped-flow CO₂hydra-

tion assay. There was good agreement between FTSA, ITC,

and inhibition data, with discrepancies between K_dand

IC₅₀values not exceeding twofold. For example, the K_dvalues

for 2h binding to CA II were 1300 and 730 nm as determined

by FTSA and ITC, respectively, and the IC₅₀was equal to

910 nm.

Compounds of the series 1(a–o) with a phenylethylthio

group at the para position were, in most cases, weaker inhibi-

tors of CAs than compounds 2(h–o), bearing the same sub-

stituents at the ortho position, but a different (hydroxyethylth-

io) substituent at the para position. By comparing two com-

pounds bearing SCH₂CH₂Ph (compound 1h) and SCH₂CH₂OH

(compound 2h) groups at the para position with the same do-

decylamino substituent at the ortho position, the latter dis-

played higher binding affinity for CA VII, XII, and XIII. Com-

pounds 2h and 3h bound CA VA, VI, VII, XII, and XIII similarly,

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but the binding with CA I and II differed (for example, the K_d

for binding of CA II with 3h is ~5-fold lower than with 2h).

Similarly, the addition of a para-phenylethylthio group weak-

ened the binding to all isoforms more strongly, as compared

with the SCH₂CH₂OH group at the para position (compare 1h

and 2h vs. 3h). On the other hand, the absence of a para

group (compounds of series 3) causes less selectivity across

various isoforms due to better binding of CA I and CA II (com-

pound 3h, 3i and 3k vs. 2h, 2i and 2k).

Substitution of the cyclohexylamino group in 1 f with bulkier

groups in 1g–i resulted in a significant decrease in binding af-

finities for all CAs. Compound 1i, bearing the largest cyclo-

Table 2. 2,4-Disubstituted-3,5,6-trifluorobenzenesulfonamide dissociation constants for seven human recombinant CA isoform catalytic domains.

Compd

X

R

K_d[nm] for CA isoforms^[a]

CA I

CA II

CA VA

CA VI

CA VII

0.83

CA XII

110

CA XIII

0.22

1

F

0.20

1.7

500

200

670

200

3300

1a

NHCH(CH₃)₂

3300

130

2900

67

1b

1c

5000

2500

2200

1000

2500

1700

2900

110

10000

1700

20000

>200000

14000

4000

1300

5000

330

40

1d

3300

200

1400

1e

1 f

>200000

4000

250

33000

20000

>200000

25000

100000

170

500

1g

1h

>200000

1400

>200000

10000

1000

1300

4000

500

710

>200000

1i^[52]

>200000

1700

3300

1j

6700

1k

1700

20000

25000

20000

67

1l

5000

3300

5000

>200000

6700

4000

1700

100

670

1m

10000

>200000

1n

2500

>200000

330

1o

1p

14000

>200000

1100

33000

3300

2500

4000

50

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Table 2. (Continued)

Compd

X

R

F

K_d[nm] for CA isoforms^[a]

CA I

CA II

CA VA

CA VI

CA VII

CA XII

220

CA XIII

8.3

2

0.11

6.7

1300

(580)

330

3300

(ND)^[b]

200

4300

(ND)^[b]

46

330

50000

(>10000)

330

140

2h^[52]

(900)

(180)

(370)

2i

2j

20000

1700

>200000

25000

3300

330

11

6600

4000

33000

670

100000

42000

13000

1200

2k

10000

11 000

6700

10000

77

2m

2n

8300

3000

10000

33000

2900

1700

1100

2000

33

14000

10000

25000

17000

120

2o

>200000

33000

>200000

6700

5600

3

F

2.4

29

290

670

11

330

450

20

3h

420

250

4000

330

200

3i

3j

2500

4000

14000

20000

>200000

111000

33000

6700

1400

4300

1000

560

11 000

20000

H

3k

670

5000

13000

6700

5000

140

3m

3n

14000

10000

3500

2200

10000

20000

3600

7700

12000

21000

2900

390

450

10000

4h

25000

2000

25000

20000

1000

290

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Table 2. (Continued)

Compd

X

R

K_d[nm] for CA isoforms^[a]

CA I

CA II

CA VA

CA VI

CA VII

CA XII

CA XIII

770

5h

>200000

10000

>200000

100000

1700

1400

6

F

2.2

27

400

450

710

25

500

40

7a

67

200

4000

560

1000

140

7b

8a

8b

F

100000

29000

10

14000

500

>200000

1800

>200000

330

14000

F

0.14

20

14

2.5

0.03

13

670

1700

100

13

[a] Determined by FTSA at 378C, pH 7.0 (ITC values are given in parentheses); uncertainties in FTSA and ITC measurements do not exceed 1.6-fold K_d.

[b] Not determined.

dodecylamino group, bound only CA XIII isoform with micro-

molar affinity (K_d=1.7 mm).

2,3,4,5-Tetrafluoro-6-[(4-methoxybenzyl)amino]benzenesulfo-

namide 7a was most similar to 3k (hydrogen at the para posi-

tion), and both compounds showed the same affinity for

Compounds 2(h–o) exhibited remarkable selectivity toward

CA XIII against CA I, II, VI, VII, and XII. For example, 2i bound to

CA XIII ~1800-fold stronger than to CA I, 154-fold stronger

than to CA II, 300-fold stronger than to CA VII, and 30-fold

stronger than to CA XII. Comparison of compounds 2 and 2i

showed that fluorine substitution at the ortho position with

a bulky cyclododecylamino group does not affect the binding

affinity for CA XIII (K_d: 8.3 and 11.0 nm, respectively), but weak-

ened the binding to other CA isoforms, especially with CA I (K_d

from 0.1 nm for 2 to 20 mm for 2i).

Compounds 3(h–n) have no substituent at the para position,

but the fluorine atom at the ortho position is substituted by

a bulky group. They bound with considerably higher affinity to

CA I than the same compounds with a substituent at the para

position (1(a–o), 2(h–o)). For example, 3h, with a cyclooctylami-

no group at the ortho position, was found to be a nanomolar

inhibitor of CA I (K_d=420 nm), but the introduction of phenyle-

thylthio (compound 1h) and hydroxyethylthio (compound 2h)

groups at the para position weakened binding to CA I signifi-

cantly (K_dꢀ200 and 50 mm, respectively). Interestingly, the

compounds 3(h–n) bound CA XIII weaker than 2(h–o) (contrary

to CA I), showing that the SCH₂CH₂OH group in 2(h–o) com-

pounds interacts with the protein and enhances binding affini-

ty (Figure 2A).

The nature of the para substituent on the benzenesulfona-

mide ring with a cyclododecylamino moiety at the ortho posi-

tion (compounds 1h, 2h, 4h, 5h) influenced the binding affin-

ity for all CAs. Compound 2h, with a hydroxyethylthio group,

was found to be the most potent inhibitor among the com-

pounds of this group.

Figure 2. FTSA data for the binding of 10o, 2n, and 3n to A) CA XIII and

B) CA I.

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CA XIII (K_d=140 nm), but with 7a being more active for other

CAs than 3k. Interestingly, an inflexible piperidine group at the

ortho position led to a sharp decrease in binding affinity for all

tested CAs, especially CA I (Figure 1B). Compounds 8a and 8b,

with 4-methoxybenzylamine or piperidine tails, bound CA I

with sub-nanomolar affinity (K_d: 0.14 and 0.03 nm, respectively)

showing that an inflexible piperidine group is more favorable

for CA I binding.

Overall, most of the 2,4-disubstituted-3,5,6-trifluorobenzene-

sulfonamides (Table 2) are potent and highly selective CA XIII

inhibitors, being weak inhibitors of cytosolic isozymes CA I and

CA II. As CA XIII is involved in the sperm motility processes,^[4]

Table 3. 3,4-Disubstituted-2,5,6-trifluorobenzensulfonamide dissociation constants for seven human recombinant CA isoform catalytic domains.

Compd

X

R

K_d[nm] for CA isoforms^[a]

CA I

0.25

67

670

CA II

CA VA

CA VI

CA VII

CA XII

CA XIII

9

9a

9b

F

1.3

5.9

1.7

140

500

770

100

45

1.3

8.3

0.22

77

290

50

0.40

1.3

3.6

NHCH₃

NHC(CH₃)₃

9d

9e

9g

9h

56

500

100

330

6.7

17

250

500

110

670

40

5.0

4.0

2.5

3.3

40

33

2.5

6.7

5.0

3.3

1.3

13

500

150

6.7

2500

200

9i

9j

2500

59

670

22

6700

1000

2500

220

25000

50

3.3

8.3

670

170

9k

210

22

200

400

6.7

250

3.3

9l

500

50

20

14000

830

330

180

5.0

17

36

5.6

1.0

9m

13

9n

9o

1000

6.7

3300

1000

330

130

2.0

40

50

2.5

6.7

13

9p

9q

1700

770

33

91

2000

3300

830

290

10

40

250

400

5.6

6.7

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Table 3. (Continued)

Compd

X

R

F

K_d[nm] for CA isoforms^[a]

CA I

0.20

200

(320)

CA II

CA VA

CA VI

CA VII

CA XII

CA XIII

10

17

83

(120)

290

330

67

100

(220)

7.1

130

250

25

29

14

(ND)^[b]

(350)

(16)

(52)

10d

710

(710)

60

(62)

2500

95

9.8

(140)

3.3

(16)

3.6

(39)

10h^[52]

10i

(ND)^[b]

400

200

36

17

2900

1000

200

50

40

5.0

67

0.8

25

10j

1000

10k

83

25

67

910

14

67

4.3

10n

10o

1000

1400

17

67

3300

1000

100

5.0

10

8.3

13

8.3

5.6

NA^[c]

(48)

10p

20000

50

5000

370

33

7.1

[a] Determined by FTSA at 378C, pH 7.0 (ITC values are given in parentheses for several compounds); uncertainties in FTSA and ITC measurements do not

exceed 1.6-fold K_d. [b] Not determined. [c] Not available due to high compound fluorescence.

such trifluorobenzensulfonamides could be good lead com-

pounds for the development of CA XIII inhibitors as contracep-

tive agents. Our newly synthesized compounds 8a and 8b,

similar to the previously described 4-substituted-2,3,5,6-tetra-

fluorobenzenesulfonamides,^[47]are very effective (sub-nanomo-

lar affinity) and highly selective CA I inhibitors.

Interestingly, the K_dvalue for CA XIII did not significantly

depend on the size of the substituent at the meta position; the

K_dvalues for all tested compounds of series 9(a–q) ranged in

the narrow nanomolar affinity range (K_d: 1.0–8.3 nm). The com-

pounds of series 10(d–p) bound CA XIII with K_dvalues of 0.8–

25 nm. In this series of derivatives, compounds 9h and 10h,

which bound CA XIII (K_d: 3.3 and 3.6 nm, respectively), also

strongly inhibited the cancer-associated isoform CA XII with K_d

values of 6.7 and 3.3 nm, respectively. Compound 9h bound the

cytosolic isoform CA I 49-fold weaker, whereas 10h binding to

CA I was more than 215-fold weaker than for CA XII. However,

the most interesting feature of compounds 10(d–p) was that

they were much better inhibitors of CA XII and XIII than nonsub-

stituted compound 10. Compound 10i was the most potent

and selective inhibitor of CA XIII (K_d=0.8 nm), whereas 9b was

most potent and selective for CA VII (K_d=0.22 nm).

The effect of the substitution at the meta position of 4-phe-

nylethylsulfonyl-2,3,5,6-tetrafluorobenzenesulfonamide

9 and

4-hydroxyethylsulfonyl-2,3,5,6-tetrafluorobenzenesulfonamide

10 was also investigated. It was observed for the series of 9(a–

q) and 10(d–p) that even a slight structural modification at the

meta position causes significant variations in binding affinity

for CA I as well as for other isoforms. For example, fluorine

substitution in compound 9 with an NHCH₃group (compound

9a) resulted in a >260-fold lower K_dvalue for CA I, 4.5-fold for

CA II, 3.5-fold for CA VA, 6.4-fold for CA VII, 3.8-fold for CA XII,

and 3.3-fold for CA XIII. The binding affinity for CA VI did not

change upon introduction of the NHCH₃group. In general, the

bulkier substituent R at the meta position caused weaker bind-

ing to CA I.

CA II inhibition (IC₅₀) showed that the position of the sub-

stituent R on the 4-substituted benzenesulfonamide ring

strongly affects enzyme activity (Figure 3). The IC₅₀value for

compounds 2h and 10h were 910 and 29 nm, respectively.

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Table 4. 3,4,5-trisubstituted-2,6-difluorobenzenesulfonamide dissociation constants for seven human recombinant CA isoform catalytic domains.

Compd

X

R

R¹

K_d[nm] for CA isoforms^[a]

CA I

CA II

CA VA

CA VI

CA VII

CA XII

CA XIII

11 h

> 200000

>200000

25000

2500

>200000

12k

5000

17000

3300

10000

250

13c

13 f

33000

1400

33000

10000

5600

>200000

140

500

330

>200000

[a] Determined by FTSA at 378C, pH 7.0; uncertainties of the FTSA measurements do not exceed 1.6-fold K_d.

forms). Compound 12k, bearing a more flexible group at the

meta position than 11 h, showed micromolar binding affinity

for all CAs (K_dfrom 17 mm for CA VI to 2.5 mm for CA VA),

whereas its affinity for CA XIII was notably higher (K_d=

250 nm). Compounds 13c and 13 f bound CA XII and XIII sig-

nificantly better (K_dvalues in the range of 140–500 nm) than

the other tested CAs. Compound 13c could be distinguished

as the most effective and selective CA XII inhibitor in this

group of compounds.

Crystal structures

The binding modes of three inhibitors, 9d, 10d, and 10p, in

the active centers of CA II, XII, and XIII were determined by X-

ray crystallography. The CA II–10p as CA XII complexes had

four, whereas CA XIII usually had two protein chains in the

asymmetric unit, and binding of the ligand was slightly differ-

ent between subunits. For subsequent analysis, the protein

chains that represent different ligand positions or where the

ligand has alternate orientations, as in the CA XII–10d com-

plex, were used. Selected protein chains are shown in bold in

Supporting Information (SI) Table S1. The residues are num-

bered as in the corresponding crystal structures. The structural-

ly coinciding residues in CA II, XII, and XIII ligand binding pock-

ets can be found in SI Table S2.

Figure 3. Inhibition curves of CA II catalytic activity by 2h and 10h as deter-

mined by stopped-flow CO₂hydration assay.

These values are very close to those determined by FTSA (1300

and 59 nm, respectively). CA inhibition assay confirmed that

ortho-substituted compounds are less potent inhibitors of CAs

than the meta-substituted benzenesulfonamides.

Several double meta-substituted compounds bearing the

same (11 h, 12k) or different (13c, 13 f) substituents at these

positions were synthesized and evaluated (Table 4). The double

substitution at the meta position resulted in much weaker-

binding compounds relative to their single meta-substituted

analogues. Steric reasons may play a role in the significantly

decreased activity of these compounds. For example, com-

pound 10h was found to be a nanomolar inhibitor of CAs

(except CA VA), whereas introduction of the second cyclooctyl-

amine group at the meta position resulted in a completely in-

effective compound 11 h (K_dꢀ 200 mm for all tested CA iso-

Compound 10d in CA XII in all subunits is in a similar posi-

tion, except for protein chain A, where it has two alternate po-

sitions that differ with respect to the orientation of the para

substituent (2-hydroxyethyl)sulfonyl group (Figure 4B). One of

the alternative positions allows a hydrogen bond between the

hydroxy group and Pro200 and with Gln89. The most remark-

able feature of the CA XII active site is the absence of Phe131

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Figure 4. Binding of 10d (A–C), 10p (D–F), and 9d (G–I) in the active sites of CA II (A, D, G), CA XII (B, E, H), and CA XIII (C, F, I). Histidine residues that coordi-

nate Zn²⁺ions are transparent, while the Zn²⁺ions themselves are shown as grey semitransparent spheres. Protein residues are shown in yellow for CA II,

green for CA XII, and pink for CA XIII. Alternate conformations of the protein amino acid side chains are rendered transparent. Inhibitors bound CA II are col-

ored orange. In the crystal structures of CA XII, the alternate conformations in chain A (crystal CA XII–10d) are colored cyan and transparent cyan. Positions of

ligands 10p and 9d from both representative protein chains of CA XII are also shown in cyan and transparent cyan for the protein chains A and B, respective-

ly. Ligands bound to CA XIII are colored blue. In panel F, the positions of 10p are shown for both protein chains of CA XIII (blue for chain A and transparent

blue for chain B). Hydrogen bonds are shown as dashed lines.

analogue, which is replaced by an alanine residue (Ala129).

Therefore, bulky substituents at the meta position of the fluo-

robenzene ring are often oriented in CA XII toward the ala-

nine,^[47]as in this case. In CA II and XIII, where Phe131 is pres-

ent, the fluorobenzene rings of 10d are shifted; they are in the

same plane as in CA XII (Figure 4A,C, SI Figure S1). In contrast

to CA XII and XIII, the N-benzylamine group of 10d in CA II is

rotated toward the opposite side of the ligand binding pocket

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(SI Figure S1 and Figure 4A). In CA XIII, this group interacts hy-

drophobically with Pro204 and Phe133, whereas the para sub-

stituent (2-hydroxyethyl)sulfonyl group makes a hydrogen

bond with His66 (Figure 4C) and water-mediated hydrogen

bond with Pro203.

hydroxy group. With respect to the orientation of the fluoro-

benzene ring, 9d shows remarkable orientational flexibility (SI

Table S1).

The position of the fluorobenzene ring of 9d and 10d in

CA II differs (SI Figure S4A). Compound 9d makes hydrogen

bonds with the protein main chain and Thr200. In the crystal

structure with CA XII (Figure 4H), which has four protein

chains per asymmetric unit, in two of them (protein chains B

and D) the fluorobenzene ring of 9d is found in the same ori-

entation as in CA II (“OÀH bond” position in SI Table S1). In

CA XII (chains A and C) the fluorobenzene rings are oriented

along the nitrogen atom of sulfonamide and shifted toward

His66 (Figure 4H), similar to 9d in CA XIII (Figure 4I), which is

bound uniformly in both protein chains. The orientation of the

compound itself when bound in CA II and CA XII (chains B and

D) exhibits stacking interactions between the para-phenyl and

fluorobenzene ring (Figure 4G–H). The para phenyl in this ori-

entation makes van der Waals contacts with the ligand binding

pocket (His66, Ser67, Asn64 in CA XII). The meta substituent in-

teracts with the loop carrying Ala129 in CA XII (Figure 4H),

whereas in CA II it is shifted to avoid a steric clash with Phe131

(Figure 4G). In CA XIII, the first ring of both 10d and 9d is

positioned in the same plane with the nitrogen atom of the

sulfonamide, and 10d is shifted toward His66 (SI Table S1, Fig-

ure S4B). These two crystal structures nicely illustrate two in-

teresting points: First, the presence of valine residue in CA XIII

instead of threonine in CA II and CA XII could be the reason

why none of the compounds was found with its fluorobenzene

ring positioned along sulfonamide oxygen (the orientation

that allows formation of hydrogen bonds between the fluoro-

benzene ring and protein, “OÀH bond” in SI Table S1). Such an

orientation was found in 9d–CA II and in 9d–CA XII crystal

structures (Figure 4G–H). Second, the para-phenyl group of 9d

is located in the same position as the meta-phenyl in 10d, that

is, between Pro204 and Phe133. The meta-phenyl of 9d over-

laps in space with the hydroxy group of 10d and is located be-

tween His66, Ser64, and Asn69. In common, 9d undergoes

more van der Waals contacts with the protein moiety than

10d, which could explain the better binding of this ligand

with CA II (K_d: 6.7 nm for 9d and 83 nm for 10d) and CA XIII

(K_dby FTSA: 2.5 nm for 9d and 14 nm for 10d).

Compounds 10d and 10p carry different meta substituents

but the same group at the para position. In CA XII–10p, the

meta group is found in two orientations (SI Figure S5A–B). In

the protein chain A of CA XII–10p, the meta substituents of

10p are oriented toward Ala129, opposite to 10d bearing N-

benzylamine at the meta position. The bulky group displaces

the fluorobenzene ring of 10p toward Ala129 (“N-shift” posi-

tion, SI Table S1). In the protein chain B of the 10p–CA XII com-

plex, the meta group is oriented similarly to 10d in CA XII (SI

Figure S5B). No significant displacement of the fluorobenzene

ring occurs in this case, as there are no steric conflicts of the

meta substituent with protein residues. Interestingly, this loca-

tion of 10p in CA XII nearly coincides with that in the protein

chain B of the 10p–CA XIII complex. In this case, one of the

phenyl groups of 10p overlaps with the N-benzylamine group

of 10d in CA XIII (SI Figure S5C).

Compound 10p carries the bulkiest meta substituent

[(1R,2S)-2-hydroxy-1,2-diphenylethyl]amino group. The K_d

values of both compounds 10d and 10p in CA II (K_d: 83 nm

for 10d and 50 nm for 10p), XII (25 nm for 10d and 48 nm for

10p) and XIII (14 nm for 10d and 7.1 nm for 10p) were similar

(Table 3). The electron density of this compound is very good

in all crystal structures (SI Figure S2,D–F). In the CA II–10p

crystal structure in all protein subunits, the ligand was found

in the same orientation that coincides with 10p in CA XII pro-

tein chain B, and CA XIII protein chain B (Figure 4D–F and SI

Figure S3B). This orientation is considered to be the main one.

Here, the phenyl of the bulky meta group is fixed by van der

Waals contacts between Phe131 (in CA II, Ala129 in CA XII, and

Phe133 in CA XIII) and Pro202 (in CA II, Pro201 in CA XII, and

Pro204 in CA XIII) at the entrance into the active site of all

three isoforms. In CA II and CA XIII, Phe131 (Phe133 in CA XIII)

is turned away from the active site, making more space for the

meta group. The second phenyl of the meta group is fixed be-

tween the same phenylalanine residue (Ala129 in CA XII) and

hydrophobic side chains of Val121 (Val119 in CA XII and Val123

in CA XIII), Leu198 (Leu197 in CA XII and Leu200 in CA XIII),

and Leu141 (Leu139 in CA XII and Leu143 in CA XIII). The flexi-

ble (2-hydroxyethyl)sulfonyl group at the para position is di-

rected in a similar way in this orientation (it could not be

bound in the opposite orientation due to steric clash with the

wall of the binding pocket in all three isoforms; SI Figure S3B).

Interestingly, in the CA XIII chain A, the meta group is found to

face the solvent, allowing Phe133 to adopt its usual position

(Figure 4F). An alternate position of 10p is found in the

A chain of CA XII (SI Figure S3A). The meta group is directed

toward the opposite wall of the ligand binding pocket and has

van der Waals contacts with His91, Asn64, Ser67, Lys69,

Leu197, Thr199, Pro201, and Trp4. These interactions push the

fluorobenzene ring in the opposite direction, in the same

plane toward Leu139 (SI Figure S3A).

From comparison of these compounds one can come to the

conclusion that the interaction between ligands and CAs is

mostly hydrophobic; the substituents at the meta position

define the position of the first ring. In most cases, the first ring

is positioned in the same plane with the nitrogen atom of sul-

fonamide (these are referred to as “N-main” and “N-shift” in SI

Table S1). In the case of such an orientation of the fluoroben-

zene ring the meta group of 10p pushes Phe131 (Phe133 in

CA XIII, chain B) away from the active center (Figure 4F). The

group at the para position does not influence binding, al-

though it can make occasional hydrogen bonds with the pro-

tein moiety (with Asn64 or Gnl89 in CA XII, Figure 4E) and it in-

teracts mostly with solvent.

To evaluate the influence of the para substituent on the po-

sitioning of the ligand in the active sites of CA isoforms the

binding of 10d was compared with that of 9d (Figure 4G–I, SI

Figure S4), which has a large phenyl group instead of a small

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(Merck) and visualized with UV light. High-resolution mass spectra

(HRMS) were recorded on a Dual-ESI Q-TOF 6520 mass spectrome-

ter (Agilent Technologies). The purity of final compounds was veri-

fied by HPLC to be >95% using an Agilent 1290 Infinity instru-

ment with a Poroshell 120SB-C₁₈(2.1 mm100 mm, 2.7 mm) re-

versed-phase column. Analytes were eluted using a linear gradient

of H₂O/MeOH (20 mm ammonium formate in both phases) from

60:40 to 30:70 over 12 min, then from 30:70 to 20:80 over 1 min,

and then 20:80 over 5 min at a flow rate of 0.2 mLmin^À1. UV detec-

tion was at l 254 nm.

Both the meta- and para-position substituents were found

to influence compound affinities and selectivities toward

a target CA isoform. No general tendencies could be drawn

based on three compound structures with three isoforms, as

every substituent had a particular effect on each CA isoform.

However, ortho substituents significantly diminish affinities

toward most CA isoforms relative to meta substituents.

Conclusions

General procedure for the syntheses of 1(a–o). A mixture of

2,3,5,6-tetrafluoro-4-[(2-phenylethyl)thio]benzenesulfonamide (com-

pound 1) (0.20 g, 0.55 mmol), Et₃N (0.080 mL, 0.57 mmol), DMSO

(1 mL) and appropriate nucleophile (0.57 mmol) was stirred at

608C for 16 h, compounds 1n,o were obtained after 40 h, com-

pound 1e was obtained after stirring at 708C for 26 h. The mixture

was then diluted with H₂O (20 mL) and extracted with EtOAc (3

10 mL). The combined organic phase was dried over MgSO₄and

evaporated under reduced pressure.

The ortho-substituted compounds bearing

a para-phenyl-

ethylthio group (compounds of series 1) bound CA I, II, VA, VI,

VII, XII, and XIII with lower affinity than ligands carrying para-

hydroxyethylthio (series 2) or no para group (series 3). Both

para groups had a moderate effect on the selectivity toward

CA XIII, which is a common feature of all ortho-substituted li-

gands, because the absence of para substitution results in

stronger binding of CA I and II, thus leading to the loss of ob-

served selectivity.

2-(Isopropylamino)-3,5,6-trifluoro-4-[(2-phenylethyl)thio]benze-

nesulfonamide (1a). The product was purified by chromatography

on a column of silica gel with EtOAc (5%)/CHCl₃, R_f=0.51; yield:

The chemical nature of ortho substituents influenced both

affinity and selectivity. The bulkier substituents usually de-

creased binding affinity and could lead to better selectivity

toward CA XIII, and in few cases for other isoforms.

1

0.12 g, 55%; mp: 47–488C; H NMR (300 MHz, CDCl₃): d=1.23 (6H,

dd, ¹J=6.3 Hz, ²J=1.2 Hz, 2CH₃), 2.94 (2H, t, J=7.8 Hz, SCH₂CH₂),

3.28 (2H, t, J=7.8 Hz, SCH₂CH₂), 3.84–3.95 (1H, m, CH), 5.52 (2H,

brs, SO₂NH₂), 7.17–7.44 ppm (5H, m, ArH); ¹³C NMR (75 MHz,

The nature and size of the substituent at the meta position

of the fluorinated benzenesulfonamide ring did not significant-

ly influence binding affinity for CA XIII, but had a strong effect

on the binding to other tested CA isozymes. 3,4-Disubstituted-

2,5,6-trifluorobenzensulfonamides showed moderate-to-weak

inhibition of CA I, but nanomolar binding to CA II, VII, tumor-

associated XII, and CA XIII.

CDCl₃): d=24 (2CH₃), 35.4 (SCH₂CH₂, t,

J

₁₃_C=3.7 Hz), 36.8

¹⁹FÀ

1

(SCH₂CH₂), 48.4 (CH, d, J ₁₃_C=11 Hz), 117.4 (C1, dd, J ₁₃_C=12 Hz,

¹⁹FÀ

²J ₁₃_C=5 Hz), 119.9 (C4, t, J ₁₃_C=21 Hz), 127.0 (Ar), 128.8 (Ar),

¹⁹FÀ

132.6 (C2, d, J ₁₃_C=16 Hz), 139.4 (Ar), 142.2 (C5, ddd, ¹J

=

¹⁹FÀ

¹⁹FÀ¹³C

240 Hz, ²J ₁₃_C=16 Hz, ³J ₁₃_C=5 Hz), 145.0 (C6, ddd, ¹J

247 Hz, ²J ₁₃_C=16 Hz, ³J ₁₃_C=5 Hz), 148.6 ppm (C3, d, J

243 Hz); ¹⁹F NMR (282 MHz, CDCl₃): d=À125.1 (C3-F, d, J=11 Hz),

À143.4 (C5-F, dd, ¹J=27 Hz, ²J=12 Hz), À148.5 ppm (C6-F, d, J=

26 Hz); HRMS for C₁₇H₁₉F₃N₂O₂S₂[M+H]⁺: calcd 405.0913, found

405.0918.

¹⁹FÀ

The crystal structures of 9d, 10d, and 10p in CA II, CA XII,

and CA XIII showed that all ligands were bound in the active

site mostly hydrophobically. The fluorobenzene ring of 9d in

CA II and XII was found in two alternate positions, one of

which allows the formation of hydrogen bonds between fluo-

rine and the protein (residues 199–200 in CA II). In CA XIII,

there is a Val202 instead of Thr200, and therefore this orienta-

tion was not observed in CA XIII.

2-(Benzylamino)-3,5,6-trifluoro-4-[(2-phenylethyl)thio]benzene-

sulfonamide (1b). The product was purified by chromatography

on a column of silica gel with EtOAc/CHCl₃(1:14), R_f=0.45; yield:

1

0.15 g, 60%; mp: 94–958C; H NMR (300 MHz, [D₆]DMSO): d=2.74

(2H, t, J=7.5 Hz, SCH₂CH₂), 3.19 (2H, t, J=7.5 Hz, SCH₂CH₂), 4.51

(2H, dd, ¹J=6.3 Hz, ²J=4.2 Hz, NHCH₂), 6.81 (1H, td, ¹J=6.3 Hz,

²J=1.8 Hz, NH), 7.07–7.43 (10H, m, ArH), 8.20 (2H, s, SO₂NH₂);

Experimental Section

¹³C NMR (75 MHz, [D₆]DMSO): d=35.3 (SCH₂CH₂, t, J ₁₃_C=3.2 Hz),

¹⁹FÀ

36.3 (SCH₂CH₂), 50.5 (NHCH₂, d, J ₁₃_C=12 Hz), 118.0 (C4, t, J

=

Chemistry

¹⁹FÀ

¹⁹FÀ¹³C

19 Hz), 119.2 (C1, dd, ¹J ₁₃_C=12 Hz, ²J ₁₃_C=4.5 Hz), 127.1 (Ar),

¹⁹FÀ

All starting materials and reagents were commercial products and

were used without further purification. Melting points of the com-

pounds were determined in open capillaries on a Thermo Scientific

9100 Series, and are uncorrected. Column chromatography was

performed using silica gel 60 (0.040–0.063 mm, Merck). ¹H and

¹³C NMR spectra were recorded on Varian Unity Inova (300 and

75 MHz, respectively) or Bruker (400 and 100 MHz, respectively)

spectrometers with TMS as an internal standard; chemical shifts

are expressed in parts per million (ppm) in the indicated solvent.

¹⁹F NMR spectra were recorded on Varian Unity Inova (282 MHz) or

Bruker (376 MHz) spectrometers with CFCl₃as an internal standard,

and fluorine chemical shifts are expressed in ppm in the indicated

solvent. Multiplicity is defined as s (singlet), d (doublet), t (triplet),

q (quartet), dd (double doublet), ddd (double double doublet), m

(multiplet), brs (broad singlet), brd (broad doublet) or brt (broad

triplet). TLC was performed with silica gel 60 F₂₅₄aluminum plates

127.9 (Ar), 128.1 (Ar), 129.0 (Ar), 129.1 (Ar), 129.2 (Ar), 132.9 (C2, d,

J

₁₃_C=14 Hz), 139.8 (Ar), 140.1 (Ar), 141.8 (C5, d, J ₁₃_C=234 Hz),

¹⁹FÀ

144.8 (C6, d, J ₁₃_C=261 Hz), 148.1 (C3, d, J ₁₃_C=242 Hz); ¹⁹F NMR

¹⁹FÀ

(282 MHz, [D₆]DMSO): d=À121.2 (C3-F, d, J=9 Hz), À138.1 (C5-F,

dd, ¹J=27 Hz, ²J=12 Hz), À145.4 ppm (C6-F, d, J=27 Hz); HRMS

C₂₁H₁₉F₃N₂O₂S₂[M+H]⁺: calcd 453.0913, found 453.0917.

2-[(2-Phenylethyl)amino]-3,5,6-trifluoro-4-[(2-phenylethyl)thio]-

benzenesulfonamide (1c). The product was purified by chroma-

tography on a column of silica gel with EtOAc (10%)/CHCl₃, R_f=

0.53; yield: 0.24 g, 92%; mp: 90–918C; ¹H NMR (300 MHz, CDCl₃):

d=2.89–3.01 (4H, m, SCH₂CH₂, NHCH₂CH₂), 3.29 (2H, t, J=7.8 Hz,

SCH₂CH₂), 3.62–3.75 (2H, m, NHCH₂), 5.21 (2H, s, SO₂NH₂), 7.17–

7.42 ppm (10H, m, ArH); ¹³C NMR (75 MHz, CDCl₃): d=35.4

(SCH₂CH₂, t, J ₁₃_C=4 Hz), 36.8 (SCH₂CH₂), 37.1 (NHCH₂CH₂), 48.2

¹⁹FÀ

1

2

(NHCH₂, d, J ₁₃_C=11.5 Hz), 116.3 (C1, dd, J ₁₃_C=12 Hz, J

=

¹⁹FÀ

¹⁹FÀ¹³C

ChemMedChem 2015, 10, 662 – 687

www.chemmedchem.org

675

ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers

5 Hz), 120.0 (C4, t, J ₁₃_C=22 Hz), 126.9 (Ar), 127 (Ar), 128.8 (Ar),

2-(Cycloheptylamino)-3,5,6-trifluoro-4-[(2-phenylethyl)thio]ben-

zenesulfonamide (1g). The product was purified by chromatogra-

phy on a column of silica gel with EtOAc (5%)/CHCl₃, R_f=0.63;

yield: 0.12 g, 48%; ¹H NMR (300 MHz, CDCl₃): d=1.37–1.82 (10H,

m, cycloheptane), 1.89–2.06 (2H, m, cycloheptane), 2.94 (2H, t, J=

7.8 Hz, SCH₂CH₂), 3.27 (2H, t, J=7.8 Hz, SCH₂CH₂), 3.67–3.83 (1H, m,

CH of cycloheptane), 5.57 (2H, s, SO₂NH₂), 6.01 (1H, brs, NH), 7.17–

7.38 ppm (5H, m, ArH); ¹³C NMR (75 MHz, CDCl₃): d=24.1 (cyclo-

¹⁹FÀ

128.9 (Ar), 129.2 (Ar), 133.1 (C2, d, J ₁₃_C=14 Hz), 139.0 (Ar), 139.4

¹⁹FÀ

1

2

3

(Ar), 141.9 (C5, ddd, J ₁₃_C=242 Hz, J ₁₃_C=16 Hz, J ₁₃_C=5 Hz),

¹⁹FÀ

145.0 (C6, ddd, ¹J ₁₃_C=253 Hz, ²J ₁₃_C=11 Hz, ³J ₁₃_C=4 Hz),

¹⁹FÀ

148.2 ppm (C3, d, J ₁₃_C=242 Hz); ¹⁹F NMR (282 MHz, CDCl₃): d=

1

2

À126.5 (C3-F, d, J=11 Hz), À143.6 (C5-F, dd, J=27 Hz, J=12 Hz),

À149.1 ppm (C6-F, d, J=28 Hz); HRMS for C₂₂H₂₁F₃N₂O₂S₂[M+H]⁺:

calcd 467.1069, found 467.1077.

heptane), 28.3 (cycloheptane), 35.4 (SCH₂CH₂, t, J ₁₃_C=4 Hz), 36.1

¹⁹FÀ

2-[(1-Phenylethyl)amino]-3,5,6-trifluoro-4-[(2-phenylethyl)thio]-

benzenesulfonamide (1d). The product was purified by chroma-

tography on a column of silica gel with EtOAc (5%)/CHCl₃, R_f=

(cycloheptane), 36.8 (SCH₂CH₂), 57.5 (CH of cycloheptane, d, J

¹⁹FÀ

1

2

₁₃_C=10 Hz), 117.2 (C1, dd, J ₁₃_C=12 Hz, J ₁₃_C=5 Hz), 119.7 (C4,

¹⁹FÀ

t, J ₁₃_C=19 Hz), 127.0 (Ar), 128.8 (Ar), 132.5 (C2, d, J ₁₃_C=15 Hz),

¹⁹FÀ

1

0.75; yield: 0.15 g, 58%; H NMR (300 MHz, CDCl₃): d=1.59 (3H, dd,

1

2

3

139.5 (Ar), 142.0 (C5, ddd, J ₁₃_C=240 Hz, J ₁₃_C=16 Hz, J

=

¹⁹FÀ

¹⁹FÀ¹³C

2

¹J=6.6 Hz, J=1.2 Hz, CH₃), 2.73–2.82 (2H, m, SCH₂CH₂), 3.13 (2H, t,

5 Hz), 145.0 (C6, ddd, ¹J ₁₃_C=250 Hz, ²J ₁₃_C=16 Hz, ³J

¹⁹FÀ

4 Hz), 148.5 ppm (C3, d, J ₁₃_C=243 Hz); ¹⁹F NMR (282 MHz, CDCl₃):

J=8.4 Hz, SCH₂CH₂), 4.85–4.95 (1H, m, CH), 5.33 (2H, s, SO₂NH₂),

7.07–7.38 ppm (10H, m, ArH); ¹³C NMR (75 MHz, CDCl₃): d=24.6

d=À125.1 (C3-F, d, J=11 Hz), À143.4 (C5-F, dd, ¹J=26 Hz, ²J=

12 Hz), À148.9 ppm (C6-F, d, J=25 Hz); HRMS for C₂₁H₂₅F₃N₂O₂S₂

[M+H]⁺: calcd 459.1382, found 459.1388.

(CH₃), 35.5 (SCH₂CH₂, t, J ₁₃_C=3.5 Hz), 36.7 (SCH₂CH₂), 56.4 (NHCH,

¹⁹FÀ

1

2

d, J ₁₃_C=12 Hz), 117.5 (C1, dd, J ₁₃_C=12 Hz, J ₁₃_C=5 Hz), 119.8

¹⁹FÀ

(C4, t, J ₁₃_C=19.5 Hz), 126.3 (Ar), 126.9 (Ar), 127.7 (Ar), 128.8 (Ar),

¹⁹FÀ

128.9 (Ar), 132.3 (C2, d, J ₁₃_C=13 Hz), 139.4 (Ar), 142.4 (C5, ddd,

¹⁹FÀ

2-(Cyclooctylamino)-3,5,6-trifluoro-4-[(2-phenylethyl)thio]benze-

nesulfonamide (1h). The product was purified by chromatography

on a column of silica gel with EtOAc/CHCl₃(1:6), R_f=0.8; yield:

¹J ₁₃_C=240 Hz, ²J ₁₃_C=15 Hz, ³J ₁₃_C=5 Hz), 144.9 (C6, ddd,

¹⁹FÀ

¹J ₁₃_C=248 Hz, ²J ₁₃_C=16 Hz, ³J ₁₃_C=4 Hz), 148.8 ppm (C3, d,

¹⁹FÀ

₁₃_C=244 Hz);¹⁹F NMR (282 MHz, CDCl₃): d=À122.5 (C3-F, d, J=

1

J

¹⁹FÀ

0.14 g, 54%; H NMR (300 MHz, CDCl₃): d=1.43–1.81 (12H, m, cy-

1

2

11 Hz), À143.2 (C5-F, dd, J=27 Hz, J=12 Hz), À147.4 ppm (C6-F,

d, J=26 Hz); HRMS for C₂₂H₂₁F₃N₂O₂S₂[M+H]⁺: calcd 467.1069,

found 467.1069.

clooctane), 1.85–2.01 (2H, m, cyclooctane), 2.94 (2H, t, J=7.8 Hz,

SCH₂CH₂), 3.27 (2H, t, J=7.8 Hz, SCH₂CH₂), 3.75–3.92 (1H, m, CH of

cyclooctane), 5.45 (2H, s, SO₂NH₂), 7.08–7.41 ppm (5H, m, ArH);

¹³C NMR (75 MHz, CDCl₃): d=23.7 (cyclooctane), 25.8 (cyclooctane),

2-Morpholin-4-yl-3,5,6-trifluoro-4-[(2-phenylethyl)thio]benzene-

sulfonamide (1e). The product was purified by chromatography

on a column of silica gel with EtOAc/CHCl₃(1:3), R_f=0.38; yield:

27.5 (cyclooctane), 33.1 (cyclooctane), 35.4 (SCH₂CH₂, t, J

=

¹⁹FÀ¹³C

4 Hz), 36.8 (SCH₂CH₂), 56.3 (CH of cyclooctane, d, J ₁₃_C=10 Hz),

¹⁹FÀ

117.0 (C1, dd, ¹J ₁₃_C=12 Hz, ²J ₁₃_C=6 Hz), 119.8 (C4, t, J

=

¹⁹FÀ

¹⁹FÀ¹³C

1

0.10 g, 42%; mp: 149–1508C; H NMR (300 MHz, CDCl₃): d=2.82–

18 Hz), 127.0 (Ar), 128.8 (Ar), 132.6 (C2, d, J ₁₃_C=15 Hz), 139.5 (Ar),

¹⁹FÀ

3.05 (4H, m, SCH₂CH₂, morpholine), 3.33 (2H, t, J=7.8 Hz,

SCH₂CH₂), 3.48 (2H, t, J=11.4 Hz, morpholine), 3.73 (2H, t, J=

11.4 Hz, morpholine), 4.00 (2H, d, J=11.4 Hz, morpholine), 6.12

(2H, s, SO₂NH₂), 7.15–7.35 ppm (5H, m, ArH); ¹³C NMR (75 MHz,

141.8 (C5, ddd, ¹J ₁₃_C=240 Hz, ²J ₁₃_C=16 Hz, ³J ₁₃_C=5 Hz),

¹⁹FÀ

145.1 (C6, ddd, ¹J ₁₃_C=245 Hz, ²J ₁₃_C=16 Hz, ³J ₁₃_C=4 Hz),

148.5 ppm (C3, d, J ₁₃_C=243 Hz); ¹⁹F NMR (282 MHz, CDCl₃): d=

1

2

À124.9 (C3-F, d, J=11 Hz), À143.4 (C5-F, dd, J=27 Hz, J=12 Hz),

À149.2 ppm (C6-F, d, J=25 Hz); HRMS for C₂₂H₂₇F₃N₂O₂S₂[M+H]⁺:

calcd 473.1539, found 473.1548.

CDCl₃): d=35.3 (SCH₂CH₂, t, J ₁₃_C=4 Hz), 36.9 (SCH₂CH₂), 51.3

¹⁹FÀ

(morpholine, d, J ₁₃_C=6 Hz), 67.7 (morpholine), 120.3 (C4, t, J

¹⁹FÀ

₁₃_C=21 Hz), 127.1 (Ar), 128.8 (Ar), 128.9 (Ar), 129.5 (C2, d, J

=

¹⁹FÀ¹³C

1

2

7 Hz), 131.7 (C1, dd, J ₁₃_C=16 Hz, J ₁₃_C=5 Hz), 139.1 (Ar), 143.9

¹⁹FÀ

2-(Cyclododecylamino)-3,5,6-trifluoro-4-[(2-phenylethyl)thio]ben-

zenesulfonamide (1i). The synthesis of compound 1i was de-

scribed previously.^[52]

1

2

3

(C5, ddd, J ₁₃_C=260 Hz, J ₁₃_C=16 Hz, J ₁₃_C=5 Hz), 149.6 (C6,

¹⁹FÀ

1

2

3

ddd, J ₁₃_C=250 Hz, J ₁₃_C=17 Hz, J ₁₃_C=6 Hz), 157.8 ppm (C3,

¹⁹FÀ

d, J ₁₃_C=251 Hz); ¹⁹F NMR (282 MHz, CDCl₃): d=À118.8 (C3-F, d,

¹⁹FÀ

J=13 Hz), À131.5 (C6-F, d, J=25 Hz), À143.0 ppm (C5-F, d, ¹J=

24 Hz, ²J=13 Hz); HRMS for C₁₈H₁₉F₃N₂O₃S₂[M+H]⁺: calcd

433.0862, found 433.0863.

2-[(2,6-Dimethoxybenzyl)amino]-3,5,6-trifluoro-4-[(2-phenyle-

thyl)thio]benzenesulfonamide (1j). The product was purified by

chromatography on a column of silica gel with EtOAc (5%)/CHCl₃,

R_f=0.53; yield: 0.20 g, 71%; mp: 118–1198C; ¹H NMR (300 MHz,

CDCl₃): d=2.96 (2H, t, J=7.8 Hz, SCH₂CH₂), 3.30 (2H, t, J=7.8 Hz,

SCH₂CH₂), 3.79 (6H, s, 2CH₃), 4.53 (2H, d, J=1.5 Hz, NHCH₂), 5.25

(2H, s, SO₂NH₂), 6.55 (2H, d, J=8.4 Hz, ArH), 7.18–7.40 ppm (6H, m,

2-(Cyclohexylamino)-3,5,6-trifluoro-4-[(2-phenylethyl)thio]benze-

nesulfonamide (1 f). The product was purified by chromatography

on a column of silica gel with EtOAc/CHCl₃(1:9), R_f=0.63; yield:

1

ArH); ¹³C NMR (75 MHz, CDCl₃): d=35.5 (SCH₂CH₂, t, J ₁₃_C=4 Hz),

0.12 g, 50%; mp: 62–638C; H NMR (300 MHz, CDCl₃): d=1.14–1.47

¹⁹FÀ

(5H, m, cyclohexane), 1.57–1.69 (1H, m, cyclohexane), 1.72–1.85

(2H, m, cyclohexane), 1.92–2.05 (2H, m, cyclohexane), 2.93 (2H, t,

J=7.8 Hz, SCH₂CH₂), 3.27 (2H, t, J=7.8 Hz, SCH₂CH₂), 3.6–3.7 (1H,

m, CH of cyclohexane), 5.57 (2H, s, SO₂NH₂), 6.16 (1H, brs, NH),

7.15–7.38 ppm (5H, m, ArH); ¹³C NMR (75 MHz, CDCl₃): d=25.0 (cy-

clohexane), 25.8 (cyclohexane), 34.4 (cyclohexane), 35.4 (SCH₂CH₂, t,

36.8 (SCH₂CH₂), 40.3 (NHCH₂, d, J ₁₃_C=11 Hz), 56.0 (2CH₃), 104.0

¹⁹FÀ

1

(Ar), 115.3 (Ar), 119.1 (C4, t, J ₁₃_C=19 Hz), 119.6 (C1, dd, J

=

¹⁹FÀ

¹⁹FÀ¹³C

11 Hz, ²J ₁₃_C=4 Hz), 127.0 (Ar), 128.8 (Ar), 128.9 (Ar), 129.6 (Ar),

¹⁹FÀ

1

2

133.6 (C2, dd, J ₁₃_C=15 Hz, J ₁₃_C=3.3 Hz), 139.5 (Ar), 143.3 (C5,

¹⁹FÀ

1

2

3

ddd, J ₁₃_C=242 Hz, J ₁₃_C=16 Hz, J ₁₃_C=5 Hz), 144.4 (C6, ddd,

¹⁹FÀ

¹J ₁₃_C=251 Hz, J ₁₃_C=16 Hz, J ₁₃_C=4 Hz), 150.5 (C3, d, J

=

2

3

¹⁹FÀ

¹⁹FÀ¹³C

245 Hz), 158.8 ppm (Ar); ¹⁹F NMR (282 MHz, CDCl₃): d=À122.4 (C3-

F, d, J=12 Hz), À144.2 (C5-F, dd, ¹J=24 Hz, ²J=12 Hz),

À146.3 ppm (C6-F, d, J=25 Hz); HRMS for C₂₃H₂₃F₃N₂O₄S₂[M+H]⁺:

calcd 513.1124, found 513.1122.

J

₁₃_C=4 Hz), 36.8 (SCH₂CH₂), 55.3 (CH of cyclohexane, d, J

=

¹⁹FÀ

¹⁹FÀ¹³C

1

2

11 Hz), 117.2 (C1, dd, J ₁₃_C=12 Hz, J ₁₃_C=5 Hz), 119.6 (C4, t, J

¹⁹FÀ

₁₃_C=22 Hz), 127.0 (Ar), 128.8 (Ar), 132.6 (C2, d, J ₁₃_C=15 Hz), 139.4

¹⁹FÀ

1

2

3

(Ar), 142.0 (C5, ddd, J ₁₃_C=240 Hz, J ₁₃_C=16 Hz, J ₁₃_C=5 Hz),

¹⁹FÀ

145.0 (C6, ddd, ¹J ₁₃_C=248 Hz, ²J ₁₃_C=16 Hz, ³J ₁₃_C=4 Hz),

¹⁹FÀ

148.5 ppm (C3, d, J ₁₃_C=243 Hz); ¹⁹F NMR (282 MHz, CDCl₃): d=

2-[(3,4-Dimethoxybenzyl)amino]-3,5,6-trifluoro-4-[(2-phenyl-

ethyl)thio]benzenesulfonamide (1k). The product was purified by

chromatography on a column of silica gel with EtOAc (5%)/CHCl₃,

R_f=0.43; yield: 0.13 g, 46%; mp: 104–1058C; ¹H NMR (300 MHz,

1

2

À125.1 (C3-F, d, J=10 Hz), À143.5 (C5-F, dd, J=27 Hz, J=12 Hz),

À149 ppm (C6-F, d, J=27 Hz); HRMS for C₂₀H₂₃F₃N₂O₂S₂[M+H]⁺:

calcd 445.1226, found 445.1235.

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Full Papers

CDCl₃): d=2.87 (2H, t, J=7.8 Hz, SCH₂CH₂), 3.22 (2H, t, J=8.1 Hz,

SCH₂CH₂), 3.81 (3H, s, CH₃), 3.88 (3H, s, CH₃), 4.43 (2H, d, J=3.3 Hz,

NHCH₂), 5.41 (2H, s, SO₂NH₂), 6.76 (1H, d, J=8.1 Hz, ArH), 6.83–6.92

(2H, m, ArH), 7.14–7.36 ppm (5H, m, ArH); ¹³C NMR (75 MHz,

ArH); ¹³C NMR (75 MHz, CDCl₃): d=19.0 (tetrahydronapthalene),

29.3 (tetrahydronapthalene), 30.3 (tetrahydronapthalene), 35.5

(SCH₂CH₂, t, J ₁₃_C=4 Hz), 36.9 (SCH₂CH₂), 54.7 (NHCH, d, J

=

¹⁹FÀ

¹⁹FÀ¹³C

11 Hz), 118.2 (C1, dd, ¹J ₁₃_C=12 Hz, ²J ₁₃_C=5 Hz), 120.0 (C4, t,

¹⁹FÀ

CDCl₃): d=35.4 (SCH₂CH₂, t, J ₁₃_C=3.5 Hz), 36.8 (SCH₂CH₂), 51.0

J

₁₃_C=21 Hz), 126.2 (Ar), 127.0 (Ar), 127.7 (Ar), 128.9 (Ar), 129.1

¹⁹FÀ

(NHCH₂, d, J ₁₃_C=12 Hz), 56.0 (CH₃), 56.1 (CH₃), 111.2 (Ar), 117.6

(Ar), 129.7 (Ar), 132.6 (C2, d, J ₁₃_C=15 Hz), 137.57 (Ar), 137.6 (Ar),

¹⁹FÀ

1

2

1

2

3

(C1, dd, J ₁₃_C=12 Hz, J ₁₃_C=5 Hz), 119.8 (C4, t, J ₁₃_C=21 Hz),

139.4 (Ar), 142.7 (C5, ddd, J ₁₃_C=240 Hz, J ₁₃_C=16 Hz, J

=

¹⁹FÀ

¹⁹FÀ¹³C

120.4 (Ar), 127.0 (Ar), 128.8 (Ar), 131.6 (Ar), 132.8 (C2, d, J

=

5 Hz), 145.0 (C6, ddd, ¹J ₁₃_C=249 Hz, ²J ₁₃_C=12 Hz, ³J

¹⁹FÀ¹³C

¹⁹FÀ

15 Hz), 139.4 (Ar), 142.4 (C5, ddd, ¹J ₁₃_C=240 Hz, ²J ₁₃_C=16 Hz,

4 Hz), 149.3 ppm (C3, d, J ₁₃_C=243 Hz); ¹⁹F NMR (282 MHz, CDCl₃):

¹⁹FÀ

³J ₁₃_C=5 Hz), 144.8 (C6, ddd, ¹J ₁₃_C=250 Hz, ²J ₁₃_C=16 Hz,

d=À123.1 (C3-F, d, J=11 Hz), À142.9 (C5-F, dd, ¹J=27 Hz, ²J=

12 Hz), À147.4 ppm (C6-F, d, J=26 Hz); HRMS for C₂₄H₂₃F₃N₂O₂S₂

[M+H]⁺: calcd 493.1226, found 493.1222.

¹⁹FÀ

³J ₁₃_C=4 Hz), 148.6 (Ar), 148.9 (C3, d, J ₁₃_C=243 Hz), 149.3 ppm

¹⁹FÀ

(Ar); ¹⁹F NMR (282 MHz, CDCl₃): d=À123.9 (C3-F, d, J=13 Hz),

À143.5 (C5-F, dd, ¹J=27 Hz, ²J=12 Hz), À147.7 ppm (C6-F, d, J=

26 Hz); HRMS for C₂₃H₂₃F₃N₂O₄S₂[MÀH]^À: calcd 511.0979, found

511.0982.

2-{[(1R,2S)-2-Hydroxy-1,2-diphenylethyl]amino}-3,5,6-trifluoro-4-

[(2-phenylethyl)thio]benzenesulfonamide (1o). The product was

purified by chromatography on a column of silica gel with EtOAc

1

(10%)/CHCl₃, R_f=0.37; yield: 0.11 g, 35%; H NMR (300 MHz, CDCl₃):

2-(2,3-Dihydro-1H-inden-2-ylamino)-3,5,6-trifluoro-4-[(2-phenyl-

ethyl)thio]benzenesulfonamide (1l). The product was purified by

chromatography on a column of silica gel with EtOAc (10%)/CHCl₃,

R_f=0.55; yield: 0.19 g, 73%; mp: 73–748C; ¹H NMR (300 MHz,

CDCl₃): d=2.85–3.05 (4H, m, SCH₂CH₂, CH₂of indane), 3.25–3.39

(4H, m, SCH₂CH₂, CH₂of indane), 4.58–4.68 (1H, m, NHCH), 5.12

(2H, s, SO₂NH₂), 7.19–7.41 ppm (9H, m, ArH); ¹³C NMR (75 MHz,

d=2.73 (2H, t, J=7.5 Hz, SCH₂CH₂), 3.08 (2H, m, SCH₂CH₂), 5.01

(1H, dd, ¹J=5.4 Hz, ²J=1.8 Hz, CH), 5.06 (1H, d, J=4.8 Hz, CH),

5.58 (2H, s, SO₂NH₂), 7.05–7.32 ppm (15H, m, ArH); ¹³C NMR

(75 MHz, CDCl₃): d=35.5 (SCH₂CH₂, t,

J

₁₃_C=3.4 Hz), 36.6

¹⁹FÀ

(SCH₂CH₂), 66.1 (NHCH, d, J ₁₃_C=11 Hz), 77.3 (CHOH, signal over-

¹⁹FÀ

1

2

laps with CDCl₃signal), 117.7 (C1, dd, J ₁₃_C=12 Hz, J ₁₃_C=5 Hz),

¹⁹FÀ

119.8 (C4, t, J ₁₃_C=21 Hz), 126.8 (Ar), 126.9 (Ar), 128.2 (Ar), 128.3

¹⁹FÀ

CDCl₃): d=35.4 (SCH₂CH₂, t, J ₁₃_C=4 Hz), 36.9 (SCH₂CH₂), 41.2 (CH₂

¹⁹FÀ

of indane), 57.8 (NHCH, d, J ₁₃_C=11 Hz), 117.2 (C1, dd, ¹J

=

(Ar), 128.5 (Ar), 128.7 (Ar), 128.8 (Ar), 131.6 (C2, d, J ₁₃_C=15 Hz),

¹⁹FÀ

¹⁹FÀ¹³C

138.1 (Ar), 139.3 (Ar), 140.4 (Ar), 142.5 (C5, ddd, ¹J ₁₃_C=240 Hz,

2

12 Hz, J ₁₃_C=5.5 Hz), 120.1 (C4, t, J ₁₃_C=22 Hz), 125.2 (Ar), 127

¹⁹FÀ

²J ₁₃_C=16 Hz, ³J ₁₃_C=5 Hz), 144.8 (C6, ddd, ¹J ₁₃_C=248 Hz,

¹⁹FÀ

(Ar), 127.2 (Ar), 128.8 (Ar), 132.2 (C2, d, J ₁₃_C=15 Hz), 139.4 (Ar),

¹⁹FÀ

²J ₁₃_C=12 Hz, ³J ₁₃_C=4 Hz), 148.6 ppm (C3, d, J ₁₃_C=243 Hz);

1

2

3

141.1 (Ar), 142.2 (C5, ddd, J ₁₃_C=239 Hz, J ₁₃_C=16 Hz, J

=

¹⁹FÀ

¹⁹FÀ¹³C

¹⁹F NMR (282 MHz, CDCl₃): d=À122.6 (C3-F, d, J=12 Hz), À143.2

(C5-F, dd, ¹J=25 Hz, ²J=12 Hz), À147.1 ppm (C6-F, d, J=26 Hz);

HRMS for C₂₈H₂₅F₃N₂O₃S₂[M+H]⁺: calcd 559.1331, found 559.1331.

5 Hz), 145.0 (C6, ddd, ¹J ₁₃_C=251 Hz, ²J ₁₃_C=12 Hz, ³J

¹⁹FÀ

4 Hz), 148.2 ppm (C3, d, J ₁₃_C=242 Hz); ¹⁹F NMR (282 MHz, CDCl₃):

d=À126 (C3-F, d, J=12 Hz), À143.3 (C5-F, dd, ¹J=27 Hz, ²J=

12 Hz), À148.3 ppm (C6-F, d, J=25 Hz); HRMS for C₂₃H₂₁F₃N₂O₂S₂

[M+H]⁺: calcd 479.1069, found 479.1077.

General procedure for the syntheses of 2(h–k,m–o). A mixture of

2,3,5,6-tetrafluoro-4-[(2-hydroxyethyl)thio]benzenesulfonamide (2)

(0.20 g, 0.65 mmol), Et₃N (0.093 mL, 0.67 mmol), DMSO (1 mL) and

appropriate nucleophile (0.67 mmol) was stirred at 608C for 16 h.

The mixture was then diluted with H₂O (20 mL) and extracted with

EtOAc (310 mL). The combined organic phase was dried over

MgSO₄and evaporated under reduced pressure.

2-[(1S)-2,3-Dihydro-1H-inden-1-ylamino]-3,5,6-trifluoro-4-[(2-phe-

nylethyl)thio]benzenesulfonamide (1m). The product was puri-

fied by chromatography on a column of silica gel with EtOAc

1

(5%)/CHCl₃, R_f=0.53; yield: 0.08 g, 31%; mp: 101–1028C; H NMR

(300 MHz, CDCl₃): d=2.05 (1H, sext, J=6.6 Hz, indane), 2.56 (1H,

sext, J=6.9 Hz, indane), 2.85–2.97 (1H, m, indane, signal overlaps

with signal of SCH₂CH₂), 2.99 (2H, t, J=7.8 Hz, SCH₂CH₂), 3.07–3.19

(1H, m, indane), 3.33 (2H, t, J=7.8 Hz, SCH₂CH₂), 5.18 (3H, brs,

SO₂NH₂, NHCH), 6.34 (1H, brs, NH), 7.18–7.41 ppm (9H, m, ArH);

¹³C NMR (75 MHz, CDCl₃): d=30.3 (indane), 35.0 (indane), 35.5

2-(Cyclooctylamino)-3,5,6-trifluoro-4-[(2-hydroxyethyl)thio]ben-

zenesulfonamide (2h). The synthesis of compound 2h was de-

scribed previously.^[52]

2-(Cyclododecylamino)-3,5,6-trifluoro-4-[(2-hydroxyethyl)thio]-

benzenesulfonamide (2i). The product was purified by chroma-

tography on a column of silica gel with EtOAc/CHCl₃(1:1), R_f=

(SCH₂CH₂, t, J ₁₃_C=3.8 Hz), 36.9 (SCH₂CH₂), 62.3 (NHCH, d, J

=

¹⁹FÀ

¹⁹FÀ¹³C

11 Hz), 117.6 (C1, dd, ¹J ₁₃_C=12 Hz, ²J ₁₃_C=5 Hz), 120.1 (C4, t,

¹⁹FÀ

1

J

₁₃_C=22 Hz), 124.3 (Ar), 125.3 (Ar), 126.9 (Ar), 127.0 (Ar), 128.4

¹⁹FÀ

0.65; yield: 0.17 g, 55%; mp: 113–1148C; H NMR (300 MHz, CDCl₃):

1

2

(Ar), 128.8 (Ar), 132.9 (C2, dd, J ₁₃_C=15 Hz, J ₁₃_C=3 Hz), 139.4

¹⁹FÀ

d=1.28–1.76 (22H, m, cyclododecane), 2.31 (1H, brs, OH), 3.16

(2H, t, J=6.0 Hz, SCH₂CH₂), 3.75 (2H, t, J=6.0 Hz, SCH₂CH₂), 3.78–

3.86 (1H, m, CH of cyclododecane, signal overlaps with signal of

SCH₂CH₂), 5.59 (2H, s, SO₂NH₂), 6.12 ppm (1H, brs, NH); ¹³C NMR

(75 MHz, CDCl₃): d=21.3 (cyclododecane), 23.3 (cyclododecane),

23.4 (cyclododecane), 24.3 (cyclododecane), 24.6 (cyclododecane),

1

2

3

(Ar), 142.5 (C5, ddd, J ₁₃_C=240 Hz, J ₁₃_C=15 Hz, J ₁₃_C=4 Hz),

¹⁹FÀ

143.7 (Ar), 144.3 (Ar), 145.0 (C6, ddd, ¹J ₁₃_C=248 Hz, ²J

=

¹⁹FÀ

¹⁹FÀ¹³C

16 Hz, ³J ₁₃_C=5 Hz), 148.8 ppm (C3, d, J ₁₃_C=242 Hz); ¹⁹F NMR

¹⁹FÀ

(282 MHz, CDCl₃): d=À124.1 (C3-F, d, J=12 Hz), À143.1 (C5-F, dd,

2

¹J=26 Hz, J=12 Hz), À147.9 ppm (C6-F, d, J=25 Hz); H,C HETCOR

(300 MHz, CDCl₃): d=35.5–3.33 (SCH₂CH₂Ph), 62.3–5.18 ppm

(NHCH); HRMS for C₂₃H₂₁F₃N₂O₂S₂[M+H]⁺: calcd 479.1069, found

479.1063.

30.9 (cyclododecane), 37.6 (SCH₂CH₂, t, J ₁₃_C=3 Hz), 53.6 (CH of

¹⁹FÀ

cyclododecane, d, J ₁₃_C=11 Hz), 61.1 (SCH₂CH₂), 117.5 (C1, dd,

¹⁹FÀ

¹J ₁₃_C=12 Hz, ²J ₁₃_C=5 Hz), 118.4 (C4, t, J ₁₃_C=20 Hz), 133.3

¹⁹FÀ

(C2, d, J ₁₃_C=15 Hz), 141.9 (C5, ddd, ¹J ₁₃_C=239 Hz, ²J

=

¹⁹FÀ

¹⁹FÀ¹³C

2-[(1S)-1,2,3,4-Tetrahydronapthalen-1-ylamino)-3,5,6-trifluoro-4-

[(2-phenylethyl)thio]benzenesulfonamide (1n). The product was

purified by chromatography on a column of silica gel with EtOAc

16 Hz, ³J ₁₃_C=5 Hz), 145.1 (C6, ddd, ¹J ₁₃_C=248 Hz, ²J

16 Hz, ³J ₁₃_C=4 Hz), 148.9 ppm (C3, d, J ₁₃_C=243 Hz); ¹⁹F NMR

¹⁹FÀ

(282 MHz, CDCl₃): d=À124.5 (C3-F, d, J=11 Hz), À142.9 (C5-F, dd,

¹J=27 Hz, ²J=12 Hz), À149.4 ppm (C6-F, d, J=24 Hz); HRMS for

C₂₀H₃₁F₃N₂O₃S₂[M+H]⁺: calcd 469.1801, found 469.1804.

1

(5%)/CHCl₃, R_f=0.79; yield: 0.14 g, 52%; mp: 123–1248C; H NMR

(300 MHz, CDCl₃): d=1.78–2.12 (4H, m, tetrahydronapthalene),

2.71–2.93 (2H, m, tetrahydronapthalene), 2.99 (2H, t, J=7.5 Hz,

SCH₂CH₂), 3.33 (2H, t, J=7.5 Hz, SCH₂CH₂), 4.83 (1H, brs, NHCH),

5.10 (2H, s, SO₂NH₂), 6.26 (1H, brs, NH), 7.11–7.40 ppm (9H, m,

2-[(2,6-Dimethoxybenzyl)amino]-3,5,6-trifluoro-4-[(2-hydroxy-

ethyl)thio]benzenesulfonamide (2j). The product was purified by

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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers

chromatography on a column of silica gel with EtOAc/CHCl₃(1:1),

R_f=0.54; yield: 0.10 g, 34%; ¹H NMR (300 MHz, CDCl₃): d=3.15

(2H, t, J=6.0 Hz, SCH₂CH₂), 3.72 (2H, t, J=6.0 Hz, SCH₂CH₂), 3.79

(6H, s, 2CH₃), 4.51 (2H, s, CH₂), 5.44 (2H, brs, SO₂NH₂), 6.55 (2H, d,

J=8.4 Hz, ArH), 7.22 ppm (1H, t, J=8.4 Hz, ArH); ¹³C NMR (75 MHz,

J

₁₃_C=11 Hz), 61.3 (SCH₂CH₂), 118.7 (C4, t, J ₁₃_C=22 Hz, signal

¹⁹FÀ

1

2

overlaps with signal of C1), 118.9 (C1, dd, J ₁₃_C=12 Hz, J

4 Hz, signal overlaps with signal of C4), 126.1 (Ar), 127.7 (Ar), 129.1

=

¹⁹FÀ

¹⁹FÀ¹³C

(Ar), 129.7 (Ar), 132.7 (C2, d, J ₁₃_C=14 Hz), 137.5 (Ar), 137.6 (Ar),

¹⁹FÀ

142.9 (C5, ddd, ¹J ₁₃_C=240 Hz, ²J ₁₃_C=15 Hz, ³J ₁₃_C=5 Hz),

¹⁹FÀ

CDCl₃): d=37.4 (SCH₂CH₂, brt), 40.3 (NHCH₂, d, J ₁₃_C=11 Hz), 56.0

145.1 (C6, ddd, ¹J ₁₃_C=250 Hz, ²J ₁₃_C=12 Hz, ³J ₁₃_C=5 Hz),

¹⁹FÀ

(CH₃), 61.2 (SCH₂CH₂), 104.0 (Ar), 115.0 (Ar), 117.9 (C4, t, J

=

149.7 ppm (C3, d, J ₁₃_C=243 Hz); ¹⁹F NMR (282 MHz, CDCl₃): d=

¹⁹FÀ¹³C

¹⁹FÀ

19 Hz), 120.3 (C1, dd, ¹J ₁₃_C=11 Hz, ²J ₁₃_C=4 Hz), 129.6 (Ar),

À122.7 (C3-F, d, J=11 Hz), À142.5 (C5-F, dd, J=27 Hz, J=12 Hz),

À147.3 ppm (C6-F, d, J=26 Hz); HRMS for C₁₈H₁₉F₃N₂O₃S₂[MÀH]^À:

calcd431.0716, found 431.0719.

1

2

¹⁹FÀ

133.4 (C2, d, J ₁₃_C=16 Hz), 143.7 (C5, dd, J ₁₃_C=240 Hz, ²J

=

1

¹⁹FÀ

¹⁹FÀ¹³C

1

2

15 Hz), 144.4 (C6, dd, J ₁₃_C=248 Hz, J ₁₃_C=16 Hz), 150.9 (C3, d,

¹⁹FÀ

J

₁₃_C=244 Hz), 158.8 ppm (Ar); ¹⁹F NMR (282 MHz, CDCl₃): d=

¹⁹FÀ

1

2

2-{[(1R,2S)-2-Hydroxy-1,2-diphenylethyl]amino}-3,5,6-trifluoro-4-

[(2-hydroxyethyl)thio]benzenesulfonamide (2o). The product was

purified by chromatography on a column of silica gel with EtOAc/

À121.9 (C3-F, d, J=12 Hz), À143.7(C5-F, dd, J=27 Hz, J=12 Hz),

À145.9 ppm (C6-F, d, J=25 Hz); HRMS for C₁₇H₁₉F₃N₂O₅S₂[M+H]⁺:

calcd 453.0760, found 453.0752.

1

CHCl₃(1:1), R_f=0.43; yield: 0.13 g, 39%; H NMR (300 MHz, CDCl₃):

2-[(3,4-Dimethoxybenzyl)amino]-3,5,6-trifluoro-4-[(2-hydroxy-

ethyl)thio]benzenesulfonamide (2k). The product was purified by

chromatography on a column of silica gel with EtOAc/CHCl₃(1:1),

R_f=0.45; yield: 0.27 g, 91%; mp: 73–748C;. ¹H NMR (300 MHz,

CDCl₃): d=3.08 (2H, t, J=6.0 Hz, SCH₂CH₂), 3.63 (2H, t, J=6.0 Hz,

SCH₂CH₂), 3.84 (3H, s, CH₃), 3.86 (3H, s, CH₃), 4.41 (2H, d, J=3.3 Hz,

CH₂), 5.67 (2H, s, SO₂NH₂), 6.75–6.87 ppm (3H, m, ArH); ¹³C NMR

d=2.38 (1H, brs, OH), 2.88 (2H, t, J=6.0 Hz, SCH₂CH₂), 3.09 (1H,

brs, OH), 3.35–3.45 (2H, m, SCH₂CH₂), 4.97 (1H, brt, CH), 5.05 (1H,

d, J=5.1 Hz, CH), 5.97 (2H, brs, SO₂NH₂), 7.04–7.32 ppm (10H, m,

ArH); ¹³C NMR (75 MHz, CDCl₃): d=37.1 (SCH₂CH₂, brt, J

=

¹⁹FÀ¹³C

2 Hz), 60.9 (SCH₂CH₂), 66.1 (NHCH, d, J ₁₃_C=12 Hz), 77.0 (CHOH,

¹⁹FÀ

signal overlaps with CDCl₃signal), 118.4 (C1, dd, ¹J ₁₃_C=11 Hz,

¹⁹FÀ

²J ₁₃_C=5 Hz, signal overlaps with C4 signal), 118.3 (C4, t, J

=

¹⁹FÀ

¹⁹FÀ¹³C

(75 MHz, CDCl₃): d=37.4 (SCH₂CH₂, t, J ₁₃_C=3 Hz), 51.0 (NHCH₂, d,

¹⁹FÀ

20 Hz, signal overlaps with C1 signal), 126.8 (Ar), 128.0 (Ar), 128.2

J

₁₃_C=12 Hz), 56.1 (CH₃), 56.2 (CH₃), 61.1 (SCH₂CH₂), 111.3 (Ar),

¹⁹FÀ

(Ar), 128.3 (Ar), 128.4 (Ar), 128.5 (Ar), 131.7 (C2, d, J ₁₃_C=15 Hz),

¹⁹FÀ

118.3 (C1, dd, ¹J ₁₃_C=11 Hz, ²J ₁₃_C=5 Hz signal overlaps with

138.1 (Ar), 140.4 (Ar), 142.7 (C5, ddd, ¹J ₁₃_C=241 Hz, ²J

=

¹⁹FÀ

¹⁹FÀ¹³C

15 Hz, ³J ₁₃_C=4 Hz), 144.9 (C6, ddd, ¹J ₁₃_C=251 Hz, ²J

signal of C4), 118.5 (C4, t, J ₁₃_C=21 Hz, signal overlaps with signal

of C1), 120.4 (Ar), 131.5 (Ar), 132.9 (C2, d, J ₁₃_C=16 Hz), 142.6 (C5,

¹⁹FÀ

12 Hz, ³J ₁₃_C=4 Hz), 148.8 ppm (C3, d, J ₁₃_C=243 Hz); ¹⁹F NMR

¹⁹FÀ

1

2

3

ddd, J ₁₃_C=242 Hz, J ₁₃_C=15 Hz, J ₁₃_C=4 Hz), 144.8 (C6, ddd,

¹⁹FÀ

(282 MHz, CDCl₃): d=À122.2 (C3-F, d, J=11 Hz), À142.9 (C5-F, dd,

¹J=26 Hz, ²J=12 Hz), À147.3 ppm (C6-F, d, J=26 Hz); HRMS for

C₂₂H₂₁F₃N₂O₄S₂[M+H]⁺: calcd 499.0968, found 499.0967.

¹J ₁₃_C=249 Hz, ²J ₁₃_C=16 Hz, ³J ₁₃_C=5 Hz), 148.6 (Ar), 149.2

¹⁹FÀ

(Ar), 149.3 ppm (C3, d, J ₁₃_C=243 Hz); ¹⁹F NMR (282 MHz, CDCl₃):

¹⁹FÀ

d=À123.6 (C3-F, d, J=12 Hz), À143.1 (C5-F, dd, ¹J=25 Hz, ²J=

General procedure for the syntheses of 3(h–k,m,n). A mixture of

2,3,5,6-tetrafluorobenzenesulfonamide (3) (0.2 g, 0.87 mmol), Et₃N

(0.124 mL, 0.89 mmol), DMSO (1 mL) and appropriate nucleophile

(0.93 mmol) was stirred at 608C for 8 h, compound 3k was ob-

tained after 16 h, compound 3n was obtained after stirring at

708C for 16 h. The mixture was then diluted with H₂O (20 mL) and

extracted with EtOAc (310 mL). The combined organic phase was

dried over MgSO₄and evaporated under reduced pressure.

12 Hz),

À147.6 ppm

(C6-F,

d,

J=26 Hz);

HRMS

for

C₁₇H₁₉F₃N₂O₅S₂[MÀH]^À: calcd 451.0615, found 451.0621.

2-[(1S)-2,3-Dihydro-1H-inden-1-ylamino]-3,5,6-trifluoro-4-[(2-hy-

droxyethyl)thio]benzenesulfonamide (2m). The product was puri-

fied by chromatography on a column of silica gel with EtOAc/

1

CHCl₃(1:1), R_f=0.71; yield: 0.13 g, 48%; H NMR (300 MHz, CDCl₃):

d=2.02 (1H, sext, J=6.9 Hz, indane), 2.54 (1H, sext, J=6.6 Hz,

indane), 2.88 (1H, quint, J=8.1 Hz, indane), 3.04–3.16 (1H, m,

indane), 3.19 (2H, t, J=6.0 Hz, SCH₂CH₂), 3.77 (2H, t, J=6.0 Hz,

SCH₂CH₂), 5.12–5.21 (1H, m, NHCH), 5.35 (2H, s, SO₂NH₂), 7.15–

7.35 ppm (4H, m, ArH); ¹³C NMR (75 MHz, CDCl₃): d=30.3 (indane),

2-(Cyclooctylamino)-3,5,6-trifluorobenzenesulfonamide (3h). The

product was purified by chromatography on a column of silica gel

with EtOAc/CHCl₃(1:20), R_f=0.32; yield: 0.15 g, 52%; mp: 117–

1

118 8C; H NMR (300 MHz, [D₆]DMSO): d=1.37–1.89 (14H, m, cyclo-

35 (indane), 37.6 (SCH₂CH₂, t, J ₁₃_C=2.5 Hz), 61.3 (SCH₂CH₂), 62.2

(NHCH, d, J ₁₃_C=10 Hz), 118.3 (C1, dd, ¹J ₁₃_C=12 Hz, ²J

¹⁹FÀ

octane), 3.72 (1H, brs, CH of cyclooctane), 6.35 (1H, brs, NH), 7.65–

7.82 (1H, m, ArH), 8.11 ppm (2H, s, SO₂NH₂); ¹³C NMR (75 MHz,

[D₆]DMSO): d=23.6 (cyclooctane), 25.7 (cyclooctane), 27.6 (cyclooc-

=

¹⁹FÀ

¹⁹FÀ¹³C

5 Hz), 118.8 (C4, t, J ₁₃_C=21 Hz), 124.2 (Ar), 125.3 (Ar), 126.9 (Ar),

¹⁹FÀ

128.4 (Ar), 133.0 (C2, d, J ₁₃_C=15 Hz), 142.7 (C5, ddd, ¹J

=

¹⁹FÀ

¹⁹FÀ¹³C

242 Hz, ²J ₁₃_C=15 Hz, ³J ₁₃_C=4 Hz), 143.7 (Ar), 144.2 (Ar), 145.1

tane), 32.8 (cyclooctane), 55.7 (CH of cyclooctane, d, J ₁₃_C=10 Hz),

¹⁹FÀ

1

2

1

2

3

110.2 (C4, t, J ₁₃_C=25 Hz), 120.6 (C1, dd, J ₁₃_C=12 Hz, J

=

¹⁹FÀ

¹⁹FÀ¹³C

(C6, ddd, J ₁₃_C=251 Hz, J ₁₃_C=12 Hz, J ₁₃_C=4 Hz), 149.2 ppm

¹⁹FÀ

5 Hz), 132.9 (C2, dd, ¹J ₁₃_C=13 Hz, ²J ₁₃_C=3 Hz), 141.1 (C5, dt,

(C3, d, J ₁₃_C=242 Hz); ¹⁹F NMR (282 MHz, CDCl₃): d=À123.8 (C3-F,

¹⁹FÀ

¹J ₁₃_C=240 Hz, ²J ₁₃_C=14 Hz), 145.0 (C6, ddd, ¹J ₁₃_C=248 Hz,

1

2

¹⁹FÀ

d, J=12 Hz), À142.7 (C5-F, dd, J=25 Hz, J=12 Hz), À147.7 ppm

(C6-F, d, J=25 Hz); HRMS for C₁₇H₁₇F₃N₂O₃S₂[M+H]⁺: calcd

419.0705, found 419.0714.

²J ₁₃_C=14 Hz, ³J ₁₃_C=5 Hz), 148.3 ppm (C3, dd, ¹J ₁₃_C=236 Hz,

²J ₁₃_C=6 Hz); ¹⁹F NMR (282 MHz, [D₆]DMSO): d=À120.6 (C3-F, t,

J=13 Hz), À133.35–À133.56 (C5-F or C6-F, m), À145.2 ppm

(C5-F or C6-F, dd, ¹J=25 Hz, ²J=11 Hz); HRMS for C₁₄H₁₉F₃N₂O₂S

[M+H]⁺: calcd 337.1192, found 337.1195.

2-[(1S)-1,2,3,4-Tetrahydronapthalen-1-ylamino)-3,5,6-trifluoro-4-

[(2-hydroxyethyl)thio]benzenesulfonamide (2n). The product was

purified by chromatography on a column of silica gel with EtOAc/

CHCl₃(1:1), R_f=0.66; yield: 0.10 g, 36%; mp: 94–958C; ¹H NMR

(300 MHz, CDCl₃): d=1.81–2.05 (4H, m, tetrahydronapthalene),

2.37 (1H, brs, OH), 2.68–3.02 (2H, m, tetrahydronapthalene), 3.19

(2H, t, J=6.0 Hz, SCH₂CH₂), 3.78 (2H, t, J=6.0 Hz, SCH₂CH₂), 4.76–

4.86 (1H, m, NHCH), 5.28 (2H, brs, SO₂NH₂), 6.28 (1H, d, J=9.0 Hz,

NH), 7.1–7.3 ppm (4H, m, ArH); ¹³C NMR (75 MHz, CDCl₃): d=18.9

(tetrahydronapthalene), 29.2 (tetrahydronapthalene), 30.3 (tetrahy-

2-(Cyclododecylamino)-3,5,6-trifluorobenzenesulfonamide (3i).

The product was purified by chromatography on a column of silica

gel with EtOAc/CHCl₃(1:20), R_f=0.49; yield: 0.10 g, 29%; mp: 113–

1

114 8C; H NMR (300 MHz, [D₆]DMSO): d=1.18–1.67 (22H, m, cyclo-

dodecane), 3.71 (1H, brs, CH of cyclododecane), 6.22 (1H, d=

7.8 Hz, NH), 7.64–7.78 (1H, m, ArH), 8.09 ppm (2H, s, SO₂NH₂);

¹³C NMR (75 MHz, [D₆]DMSO): d=21.4 (cyclododecane), 23.4 (cyclo-

dodecane), 23.6 (cyclododecane), 24.1 (cyclododecane), 24.4 (cyclo-

dronapthalene), 37.6 (SCH₂CH₂, brt, J ₁₃_C=3 Hz), 54.7 (NHCH, d,

¹⁹FÀ

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678

ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers

dodecane), 31.1 (cyclododecane), 52.8 (CH of cyclododecane, d,

tography on a column of silica gel with EtOAc (10%)/CHCl₃, R_f=

0.64; yield: 0.10 g, 32%; mp: 124–1258C; ¹H NMR (300 MHz,

[D₆]DMSO): 1.60–2.01 (4H, m, tetrahydronapthalene), 2.61–2.92

(2H, m, tetrahydronapthalene), 4.81 (1H, brs, NHCH), 6.53 (1H, d,

J=8.7 Hz, NH), 7.11–7.25 (3H, m, ArH), 7.41 (1H, d, J=7.5 Hz, ArH),

7.73–7.85 (1H, m, ArH), 8.12 ppm (2H, s, SO₂NH₂); ¹³C NMR

(75 MHz, [D₆]DMSO): d=19.3 (tetrahydronapthalene), 29.3 (tetrahy-

dronapthalene), 30.2 (tetrahydronapthalene), 53.8 (CH of tetrahy-

dronapthalene, d, J ₁₃_C=12 Hz), 110.4 (C4, t, J ₁₃_C=25 Hz), 121.0

1

J

₁₃_C=11 Hz), 110.2 (C4, t, J ₁₃_C=25 Hz), 120.6 (C1, dd, J

=

¹⁹FÀ¹³C

¹⁹FÀ

12 Hz, ²J ₁₃_C=5 Hz), 133.4 (C2, dd, ¹J ₁₃_C=12 Hz, ²J ₁₃_C=2 Hz),

¹⁹FÀ

141.1 (C5, dt, ¹J ₁₃_C=238 Hz, ²J ₁₃_C=13 Hz), 145.0 (C6, ddd,

¹⁹FÀ

¹J ₁₃_C=249 Hz, ²J ₁₃_C=14 Hz, ³J ₁₃_C=4 Hz), 148.1 ppm (C3, dd,

¹⁹FÀ

¹J ₁₃_C=245 Hz, J ₁₃_C=9 Hz); ¹⁹F NMR (282 MHz, [D₆]DMSO): d=

2

À125.4 (C3-F, t, J=13 Hz), À138–138.3 (C5-F or C6-F, m),

À150.1 ppm (C5-F or C6-F, dd, ¹J=25 Hz, ²J=11 Hz); HRMS for

C₁₈H₂₇F₃N₂O₂S [M+H]⁺: calcd 393.1818, found 393.1816.

¹⁹FÀ

1

2

(C1, dd, J ₁₃_C=12 Hz, J ₁₃_C=5 Hz), 126.6 (Ar), 127.9 (Ar), 129.6

¹⁹FÀ

1

2

2-[(2,6-Dimethoxybenzyl)amino]-3,5,6-trifluorobenzenesulfona-

mide (3j). The product was purified by chromatography on

a column of silica gel with EtOAc (5%)/CHCl₃, R_f=0.4; yield: 0.16 g,

(Ar), 129.7 (Ar), 132.7 (C2, dd, J ₁₃_C=12 Hz, J ₁₃_C=2 Hz), 137.6

¹⁹FÀ

1

2

(Ar), 137.9 (Ar), 141.5 (C5, dt, J ₁₃_C=239 Hz, J ₁₃_C=13 Hz), 145.0

¹⁹FÀ

(C6, d, J ₁₃_C=247 Hz), 148.4 ppm (C3, d, J ₁₃_C=239 Hz); ¹⁹F NMR

¹⁹FÀ

1

48%; mp: 137–1388C; H NMR (300 MHz, CD₃OD): d=3.79 (6H, s,

(282 MHz, [D₆]DMSO): d=À123.8 (C3-F, t, J=13 Hz), À137.9–

À138.14 (C5-F or C6-F, m), À149.2 ppm (C5-F or C6-F, dd, ¹J=

2CH₃), 4.51 (2H, d, J=1.5 Hz, CH₂), 4.91 (2H, s, SO₂NH₂), 6.61 (2H,

d, J=8.4 Hz, ArH), 7.22 (1H, t, J=8.4 Hz, ArH), 7.29–7.41 ppm (1H,

2

25 Hz, J=10 Hz); HRMS for C₁₆H₁₅F₃N₂O₂S [MÀH]^À: calcd 355.0734,

m, ArH); ¹³C NMR (75 MHz, CD₃OD): d=39.3 (CH₂, d, J ₁₃_C=12 Hz),

¹⁹FÀ

found 355.0733.

55.0 (CH₃), 103.6 (Ar), 108.7 (C4, t, J ₁₃_C=25 Hz), 115.1 (Ar), 121.4

¹⁹FÀ

(C1, dd, ¹J ₁₃_C=12 Hz, ²J ₁₃_C=4 Hz), 129.2 (Ar), 134.3 (C2, dd,

¹⁹FÀ

2-(Cyclooctylamino)-3,5,6-trifluoro-4-(propylthio)benzenesulfo-

namide (4h). A mixture of 2,3,5,6-tetrafluoro-4-(propylthio)benze-

nesulfonamide (4) (0.20 g, 0.66 mmol), Et₃N (0.095 mL, 0.68 mmol),

DMSO (1 mL) and cyclooctylamine (0.10 mL, 0.72 mmol) was stirred

at 608C for 12 h. The mixture was then diluted with H₂O (20 mL)

and extracted with EtOAc (310 mL). The combined organic phase

was dried over MgSO₄and evaporated under reduced pressure.

The product was purified by chromatography on a column of silica

gel with EtOAc (5%)/CHCl₃, R_f=0.64; yield: 0.16 g, 59%; ¹H NMR

(300 MHz, CDCl₃): d=1.03 (3H, t, J=7.2 Hz, CH₃), 1.43–1.75 (14H,

m, CH₂CH₃, cyclooctane), 1.78–1.95 (2H, m, cyclooctane), 2.98 (2H,

t, J=7.2 Hz, SCH₂), 3.72–3.85 (1H, m, CH of cyclooctane), 5.62 ppm

(3H, brs, NH, SO₂NH₂); ¹³C NMR (75 MHz, CDCl₃): d=13.3 (CH₃),

23.5 (cyclooctane), 23.7 (cyclooctane), 25.8 (cyclooctane), 27.5 (cy-

¹J ₁₃_C=13 Hz, ²J ₁₃_C=3 Hz), 142.2 (C5, d, J ₁₃_C=241 Hz), 144.6

¹⁹FÀ

(C6, d, J ₁₃_C=248 Hz), 149.9 (C3, d, J ₁₃_C=249 Hz), 158.8 ppm

¹⁹FÀ

(Ar); ¹⁹F NMR (282 MHz, CD₃OD): d=À124.1 (C3-F, t, J=13 Hz),

1

À141.5À141.7 (C5-F or C6-F, m), À150.3 ppm (C5-F or C6-F, dd, J=

25 Hz, J=10 Hz); HRMS for C₁₅H₁₅F₃N₂O₄S [MÀH]^À: calcd 375.0632,

2

found 375.0634.

2-[(3,4-Dimethoxybenzyl)amino]-3,5,6-trifluorobenzenesulfona-

mide (3k). The product was purified by chromatography on

a column of silica gel with EtOAc (10%)/CHCl₃, R_f=0.29. Recrystalli-

zation was accomplished from EtOH/H₂O (2:1) after chromatogra-

phy; yield: 0.10 g, 33%; mp: 115–1168C; ¹H NMR (300 MHz,

[D₆]DMSO): d=3.73 (3H, s, CH₃), 3.75 (3H, s, CH₃), 4.37–4.45 (2H,

m, CH₂), 6.67–6.76 (1H, m, NH), 6.82–6.98 (3H, m, ArH), 7.65–7.78

(1H, m, ArH), 8.14 ppm (2H, s, SO₂NH₂); ¹³C NMR (75 MHz,

clooctane), 33.0 (CH₂), 36.3 (SCH₂, t, J ₁₃_C=3.6 Hz), 56.5 (CH of cy-

¹⁹FÀ

1

2

clooctane, d, J ₁₃_C=11 Hz), 117.3 (C1, dd, J ₁₃_C=12 Hz, J

=

¹⁹FÀ

¹⁹FÀ¹³C

[D₆]DMSO): d=50.3 (CH₂, d, J ₁₃_C=12 Hz), 56.0 (CH₃), 56.1 (CH₃),

¹⁹FÀ

6 Hz), 120.0 (C4, t, J ₁₃_C=21 Hz), 132.4 (C2, d, J ₁₃_C=13 Hz),

¹⁹FÀ

110.9 (C4, t, J ₁₃_C=24 Hz), 111.9 (Ar), 112.2 (Ar), 120.3 (Ar), 120.4

¹⁹FÀ

142.2 (C5, ddd, ¹J ₁₃_C=239 Hz, ²J ₁₃_C=16 Hz, ³J ₁₃_C=5 Hz),

¹⁹FÀ

(C1, signal overlaps with Ar signal), 132.3 (Ar), 133.5 (C2, d, J

13 Hz), 141.2 (C5 dt, J ₁₃_C=238 Hz, J ₁₃_C=12 Hz), 144.8 (C6, d,

=

¹⁹FÀ¹³C

145.0 (C6, ddd, ¹J ₁₃_C=249 Hz, ²J ₁₃_C=17 Hz, ³J ₁₃_C=4 Hz),

¹⁹FÀ

1

2

¹⁹FÀ

148.9 ppm (C3, d, J ₁₃_C=243 Hz); ¹⁹F NMR (282 MHz, CDCl₃): d=

J

₁₃_C=252 Hz), 148.2 (C3, d J ₁₃_C=240 Hz), 148.6 (Ar), 149.2 ppm

¹⁹FÀ

1

2

À124.8 (C3-F, d, J=11 Hz), À143.5 (C5-F, dd, J=27 Hz, J=12 Hz),

À149.0 ppm (C6-F, d, J=26 Hz); HRMS for C₁₇H₂₅F₃N₂O₂S₂[M+H]⁺:

calcd 411.1382, found 411.1388.

(Ar); ¹⁹F NMR (282 MHz, [D₆]DMSO): d=À124.9 (C3-F, t, J=13 Hz),

1

À138.6–138.8 (C5-F or C6-F, m), À149.6 ppm (C5-F or C6-F, dd, J=

25 Hz, J=10 Hz); HRMS for C₁₅H₁₅F₃N₂O₄S [MÀH]^À: calcd 375.0632,

2

found 375.0631.

2-(Cyclooctylamino)-3,5,6-trifluoro-4-{[2-(4-hydroxyphenyl)ethyl]-

amino}benzenesulfonamide (5h). A mixture of 2,3,5,6-tetrafluoro-

2-[(1S)-2,3-Dihydro-1H-inden-1-ylamino]-3,5,6-trifluorobenzene-

sulfonamide (3m). The product was purified by chromatography

on a column of silica gel with EtOAc/CHCl₃(1:10), R_f=0.38; yield:

0.09 g, 30%; mp: 103–1048C; ¹H NMR (300 MHz, [D₆]DMSO): d=

1.79–1.95 (1H, m, indane), 2.38–2.49 (1H, m, indane), 2.82 (1H,

pent, J=7.8 Hz, indane), 2.91–3.06 (1H, m, indane), 5.09–5.21 (1H,

4-{[2-(4-hydroxyphenyl)ethyl]amino}benzenesulfonamide

(5)

(0.20 g, 0.55 mmol), Et₃N (0.085 mL, 0.61 mmol), DMSO (1 mL) and

cyclooctylamine (0.085 mL, 0.61 mmol) was stirred at 708C for 28 h.

The mixture was then diluted with H₂O (20 mL) and extracted with

EtOAc (310 mL). The combined organic phase was dried over

MgSO₄and evaporated under reduced pressure. The product was

purified by chromatography on a column of silica gel with EtOAc/

1

2

m, NHCH), 6.59 (1H, dd, J=8.4 Hz, J=1.8 Hz, NH), 7.19–7.38 (4H,

m, ArH), 7.75–7.88 (1H, m ArH), 8.13 ppm (2H, s, SO₂NH₂); ¹³C NMR

(75 MHz, [D₆]DMSO): d=30.2 (indane), 35.2 (indane), 61.6 (CH of

1

CHCl₃(1:3), R_f=0.60; yield: 0.13 g, 50%; H NMR (300 MHz, CDCl₃):

d=1.38–1.94 (14H, m, cyclooctane), 2.82 (2H, t, J=6.6 Hz,

NHCH₂CH₂), 3.61–3.75 (3H, m, CH of cyclooctane, NHCH₂CH₂), 5.59

(2H, s, SO₂NH₂), 6.04 (2H, brs, 2NH), 6.79 (2H, d, J=8.4 Hz, ArH),

7.04 ppm (2H, d, J=8.4 Hz, ArH); ¹³C NMR (75 MHz, CDCl₃): d=23.8

(cyclooctane), 25.8 (cyclooctane), 27.5 (cyclooctane), 32.7 (cyclooc-

tane), 36.6 (NHCH₂CH₂), 46.9 (NHCH₂CH₂), 56.4 (CH of cyclooctane,

indane, d, J ₁₃_C=11 Hz), 110.5 (C4, t, J ₁₃_C=25 Hz), 120.5 (C1, dd,

¹⁹FÀ

¹J ₁₃_C=12 Hz, ²J ₁₃_C=5 Hz), 124.7 (Ar), 125.5 (Ar), 127.3 (Ar),

¹⁹FÀ

1

2

128.6 (Ar), 133.2 (C2, dd, J ₁₃_C=13 Hz, J ₁₃_C=3 Hz), 141.4 (C5,

¹⁹FÀ

1

2

dt, J ₁₃_C=238 Hz, J ₁₃_C=14 Hz), 143.6 (Ar), 144.7 (Ar), 144.9 (C6,

¹⁹FÀ

1

2

3

ddd, J ₁₃_C=233 Hz, J ₁₃_C=14 Hz, J ₁₃_C=4 Hz), 148.2 ppm (C3,

¹⁹FÀ

d, J ₁₃_C=226 Hz); ¹⁹F NMR (282 MHz, [D₆]DMSO): d=À120.0 (C3-F,

¹⁹FÀ

1

2

d, J ₁₃_C=10 Hz), 106.3 (C1, dd, J ₁₃_C=12 Hz, J ₁₃_C=5 Hz), 115.7

¹⁹FÀ

t, J=13 Hz), À133.35–133.55 (C5-F or C6-F, m), À144.7 ppm (C5-F

or C6-F, dd, J=25 Hz, J=11 Hz); HRMS for C₁₅H₁₃F₃N₂O₂S [MÀH]^À:

1

2

(C4, signal overlaps with Ar signal), 115.8 (Ar), 130.2 (Ar), 130.3 (Ar),

132.0 (C2, d, J ₁₃_C=13 Hz, signal overlaps with C5 signal), 133.8

¹⁹FÀ

calcd 341.0577, found 341.0580.

1

2

3

(C6, ddd, J ₁₃_C=239 Hz, J ₁₃_C=18 Hz, J ₁₃_C=6 Hz), 138.5 (C3,

¹⁹FÀ

2-[(1S)-1,2,3,4-Tetrahydronapthalen-1-ylamino)-3,5,6-trifluoro-

benzenesulfonamide (3n). The product was purified by chroma-

d, J ₁₃_C=233 Hz), 146.5(C6, ddd, ¹J ₁₃_C=243 Hz, ²J ₁₃_C=14 Hz,

¹⁹FÀ

³J ₁₃_C=3 Hz), 154.8 ppm (Ar); ¹⁹F NMR (282 MHz, CDCl₃): d=

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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers

À144.8 (C5-F, dd, ¹J=23 Hz, ²J=9 Hz), À154.1 (C3-F, s),

À171.4 ppm (C6-F, d, J=23 Hz); HRMS for C₂₂H₂₈F₃N₃O₃S [M+H]⁺:

calcd 472.1876, found 472.1877.

[D₆]DMSO): d=23.7 (CH₂), 26.1 (CH₂), 52.2 (CH₂, J ₁₃_C=3 Hz), 127.2

¹⁹FÀ

(C1), 135.2 (C6, dd, ¹J=11 Hz, ²J=4 Hz), 138.9 (C3 or C4, dt, ¹J=

254 Hz, ²J=14 Hz), 142.8 (C3 or C4, dt, ¹J=257 Hz, ²J=16 Hz),

1

2

144.1 (C2 or C5, dd, J=252 Hz, J=12 Hz), 148.0 ppm (C2 or C5,

dd, ¹J=250 Hz, ²J=10 Hz); ¹⁹F NMR (376 MHz, [D₆]DMSO): d=

À137.4 (d, J=26 Hz), À144.0, À151.0, À157.9 ppm; HRMS for

C₁₁H₁₂F₄N₂O₂S [M+H]⁺: calcd 313.0628, found 313.0631.

2,3,4,5-Tetrafluoro-6-[(4-methoxybenzyl)amino]benzenesulfona-

mide (7a) and 2,3,5,6-tetrafluoro-4-[(4-methoxybenzyl)amino]-

benzenesulfonamide (8a). Method A: A mixture of 2,3,4,5,6-penta-

fluorbenzenesulfonamide (6) (0.20 g, 0.81 mmol), Et₃N (0.11 mL,

0.81 mmol), C₆H₆(3 mL) and 4-methoxybenzylamine (0.11 mL,

0.81 mmol) was stirred at 808C for 7 h. C₆H₆was evaporated under

reduced pressure and the resultant precipitate was washed with

H₂O. A mixture of 7a and 8a was obtained. The compounds were

separated by chromatography on a column of silica gel with

EtOAc/CHCl₃(1:5); yield: 0.14 g, 47% (7a); 0.04 g, 12% (8a).

2,3,5,6-Tetrafluoro-4-piperidin-1-ylbenzenesulfonamide

(8b).

R_f=0.48 (EtOAc/CHCl₃, 1:5); mp: 174–1758C; ¹H NMR (400 MHz,

[D₆]DMSO): d=1.59 (6H, s, 3CH₂), 3.26 (4H, s, 2CH₂), 8.14 ppm (2H,

s, SO₂NH₂); ¹³C NMR (100 MHz, [D₆]DMSO): d=23.9 (CH₂), 26.4

(CH₂), 51.8 (CH₂, t, J ₁₃_C=3 Hz), 114.3 (C1, t, J=15 Hz), 134.1 (C4, t,

¹⁹FÀ

1

2

J=11 Hz), 141.2 (C3, C5, dd, J=245 Hz, J=14 Hz), 144.0 ppm (C2,

1

2

C6, dd, J=252 Hz, J=14 Hz); ¹⁹F NMR (376 MHz, [D₆]DMSO): d=

À141.1–À141.3 (2F, m), À150.9–À151.1 ppm (2F, m); HRMS for

C₁₁H₁₂F₄N₂O₂S [M+H]⁺: calcd 313.0628, found 313.0630.

2,3,4,5-Tetrafluoro-6-[(4-methoxybenzyl)amino]benzenesulfona-

mide (7a). R_f=0.62 (EtOAc/CHCl₃, 1:5); mp: 97–988C; ¹H NMR

(400 MHz, [D₆]DMSO): d=2.87 (3H, s, CH₃), 3.59 (2H, dd, ¹J=

6.4 Hz, ²J=4.4 Hz, CH₂), 6.02 (1H, brs, NH signal overlaps with

signal of Ar), 6.04 (2H, d, J=8.8 Hz, Ar), 6.41 (2H, d, J=8.8 Hz, Ar),

7.34 ppm (2H, s, SO₂NH₂); ¹³C NMR (100 MHz, [D₆]DMSO): d=49.4

2,3,5,6-Tetrafluoro-4-piperidin-1-ylbenzenesulfonamide

(8b).

Method B: A mixture of 6 (0.20 g, 0.81 mmol), Et₃N (0.11 mL,

0.81 mmol), DMSO (1 mL) and piperidine hydrochloride (0.10 g,

0.81 mmol) was stirred at ambient temperature for 24 h. The mix-

ture was then diluted with H₂O (20 mL). The resultant precipitate

was filtered, washed with H₂O. Recrystallization was accomplished

from EtOH/H₂O (2:1); yield: 0.22 g, 88%; mp: 174–1758C.

(CH₂, J ₁₃_C=11.8 Hz), 55.5 (CH₃), 114.4 (Ar), 114.5 (C1), 129.2 (Ar),

¹⁹FÀ

131.3 (Ar), 131.4 (Ar), 131.8 (C3 or C4, dt, ¹J=243 Hz, ²J=16 Hz),

1

2

1

133.5 (C6, dd, J=9 Hz, J=3 Hz), 137.9 (C2 or C5, dd, J=243 Hz,

²J=13 Hz), 143.5 (C3 or C4, d, J=249 Hz), 145.6 (C2 or C5, dd, J=

247 Hz, ²J=9 Hz), 159.0 ppm (Ar); ¹⁹F NMR (376 MHz, [D₆]DMSO):

1

2

3-(Methylamino)-2,5,6-trifluoro-4-[(2-phenylethyl)sulfonyl]benze-

nesulfonamide (9a). A mixture of 2,3,5,6-tetrafluoro-4-[(2-phenyle-

thyl)sulfonyl]benzenesulfonamide (9) (0.20 g, 0.50 mmol), MeOH

(10 mL) and methylamine (2m in MeOH) (0.75 mL, 1,50 mmol) was

held at reflux for 7 h. MeOH was evaporated under reduced pres-

sure and the resultant precipitate was filtered, washed with H₂O.

Recrystallization was accomplished from EtOH/H₂O (2:1); yield:

0.12 g, 57%; mp: 152–1538C; ¹H NMR (300 MHz, [D₆]DMSO): d=

3.01 (3H, dd, ¹J=7.5 Hz, ²J=5.1 Hz, CH₃), 3.08 (2H, t, J=7.5 Hz,

SO₂CH₂CH₂), 3.89 (2H, t, J=7.5 Hz, SO₂CH₂CH₂), 6.62 (1H, brs, NH),

7.13–7.33 (5H, m, ArH), 8.31 ppm (2H, s, SO₂NH₂); ¹³C NMR

d=À136.2 (C2-F or C5-F, dt, J=26 Hz, J=7.5 Hz), À152.0 (C3-F or

1

2

1

C4-F, td, J=23 Hz, J=7.5 Hz), À154.3 (C2-F or C5-F, dt, J=23 Hz,

²J=7.5 Hz), À172.6 ppm (C3-F or C4-F, td, ¹J=23 Hz, ²J=4 Hz);

HRMS for C₁₄H₁₂F₄N₂O₃S [M+H]⁺: calcd 365.0578, found 365.0581.

2,3,5,6-Tetrafluoro-4-[(4-methoxybenzyl)amino]benzenesulfona-

mide (8a). R_f=0.45 (EtOAc/CHCl₃, 1:5); mp: 161–1628C; ¹H NMR

(400 MHz, [D₆]DMSO): d=3.72 (3H, s, OCH₃), 4.47 (2H, d, J=4.0 Hz,

CH₂), 6.89 (2H, d, J=9.2 Hz, Ar), 7.23 (2H, d, J=9.2 Hz, Ar), 7.31

(1H, brt, NH), 7.91 ppm (2H, s, SO₂NH₂); ¹³C NMR (100 MHz,

[D₆]DMSO): d=47.4 (CH₂, t, J ₁₃_C=4 Hz), 55.4 (OCH₃), 108.8 (C1, t,

¹⁹FÀ

J=16 Hz), 114.3 (Ar), 128.4 (Ar), 131.4 (C4, t, J=11 Hz), 132.0 (Ar),

(75 MHz, [D₆]DMSO): d=28.6 (SO₂CH₂CH₂), 34.1 (CH₃, d, J

=

¹⁹FÀ¹³C

13 Hz), 57.5 (SO₂CH₂CH₂), 114.4 (C4, dd, ¹J ₁₃_C=12 Hz, ²J

136.3 (C3, C5, d, J ₁₃_C=243 Hz), 144.0 (C2, C6, d, J ₁₃_C=251 Hz),

¹⁹FÀ

158.7 ppm (Ar); ¹⁹F NMR (376 MHz, [D₆]DMSO): d=À142.1 (2F, d,

J=17 Hz), À159.6 ppm (2F, d, J=17 Hz); HRMS for C₁₄H₁₂F₄N₂O₃S

[M+H]⁺: calcd 365.0578, found 365.0581.

5 Hz), 127.5 (Ar), 128.0 (C1, t, J ₁₃_C=18 Hz), 128.9 (Ar), 129.1 (Ar),

¹⁹FÀ

137.4 (C3, d, J ₁₃_C=12 Hz), 137.6 (Ar), 136.7 (C6, d, J

244 Hz), 144.4 (C2, d, J ₁₃_C=251 Hz), 146.1 ppm (C5, d, J

240 Hz); ¹⁹F NMR (282 MHz, [D₆]DMSO): d=À127.5 (C2-F, s), À135.9

(C6-F, dd, ¹J=25 Hz, ²J=12 Hz), À152.6 ppm (C5-F, dd, ¹J=26 Hz,

²J=7 Hz); HRMS C₁₅H₁₅F₃N₂O₄S₂[M+H]⁺: calcd 409.0498, found

409.0505.

=

¹⁹FÀ

¹⁹FÀ¹³C

¹⁹FÀ

2,3,5,6-Tetrafluoro-4-[(4-methoxybenzyl)amino]benzenesulfona-

mide (8a). Method B: A mixture of 6 (0.20 g, 0.81 mmol), Et₃N

(0.11 mL, 0.81 mmol), DMSO (1 mL) and 4-methoxybenzylamine

(0.11 mL, 0.81 mmol) was stirred at ambient temperature for 24 h.

The mixture was then diluted with H₂O (20 mL). The resultant pre-

cipitate was filtered, washed with H₂O. Recrystallization was ac-

complished from EtOH/H₂O (2:1); yield: 0.25 g, 86%; mp: 161–

1628C.

3-(tert-Butylamino)-2,5,6-trifluoro-4-[(2-phenylethyl)sulfonyl]ben-

zenesulfonamide (9b).

A mixture of compound 9 (0.20 g,

0.50 mmol), DMSO (1 mL) and tert-butylamine (0.11 mL,

0.105 mmol) was stirred at ambient temperature for four days. The

mixture was then diluted with H₂O (20 mL) and the resultant pre-

cipitate was filtered, washed with H₂O. The product was purified

by chromatography on a column of silica gel with EtOAc/CHCl₃

(1:4), R_f=0.62; yield: 0.04 g, 18%; mp: 127–1288C; ¹H NMR

(300 MHz, CDCl₃): d=1.38 (9H, d, J=2.1 Hz, 3CH₃), 3.14 (2H, t, J=

7.8 Hz, SO₂CH₂CH₂), 3.65 (2H, t, J=7.8 Hz, SO₂CH₂CH₂), 5.74 (2H, s,

SO₂NH₂), 6.63 (1H, s, NH), 7.12–7.43 ppm (5H, m, ArH); ¹³C NMR

(75 MHz, CDCl₃): d=28.8 (SO₂CH₂CH₂), 30.9 (CH₃, d, J=7 Hz), 55.7

2,3,4,5-Tetrafluoro-6-piperidin-1-ylbenzenesulfonamide (7b) and

2,3,5,6-tetrafluoro-4-piperidin-1-ylbenzenesulfonamide

(8b).

Method A: A mixture of 6 (0.20 g, 0.81 mmol), Et₃N (0.11 mL,

0.81 mmol), C₆H₆(3 mL) and piperidine hydrochloride (0.10 g,

0.81 mmol) was stirred at 808C for 12 h. C₆H₆was evaporated

under reduced pressure and the resultant precipitate was washed

with H₂O. A mixture of 7b and 8b was obtained. The compounds

were separated by chromatography on a column of silica gel with

EtOAc/CHCl₃(1:5); yield: 0.09 g, 37% (7b); 0.04 g, 17% (8b).

(SO₂CH₂CH₂), 58.8 (NHC, d, J ₁₃_C=4 Hz), 118.5 (C4, dd, ¹J

=

¹⁹FÀ

¹⁹FÀ¹³C

2

12 Hz, J ₁₃_C=6 Hz), 126.3 (C1, t, J ₁₃_C=16 Hz), 127.6 (Ar), 128.5

¹⁹FÀ

1

2

2,3,4,5-Tetrafluoro-6-piperidin-1-ylbenzenesulfonamide

(7b).

(Ar), 129.2 (Ar), 135.8 (C3, dd, J ₁₃_C=18 Hz, J ₁₃_C=3 Hz), 136.4

(Ar), 138.3 (C6, ddd, J ₁₃_C=252 Hz, J ₁₃_C=18 Hz, J ₁₃_C=5 Hz),

145.8 (C2, d, J ₁₃_C=254 Hz), 146.1 ppm (C5, ddd, J ₁₃_C=253 Hz,

²J ₁₃_C=16 Hz, ³J ₁₃_C=4 Hz); ¹⁹F NMR (282 MHz, CDCl₃): d=

¹⁹FÀ

R_f=0.62 (EtOAc/CHCl₃, 1:5); mp: 175–1768C; ¹H NMR (400 MHz,

[D₆]DMSO): d=1.63 (4H, brs, 2CH₂), 1.72 (2H, brs, CH₂), 3.02 (4H,

brs, 2CH₂), 7.59 ppm (2H, s, SO₂NH₂); ¹³C NMR (100 MHz,

1

2

3

¹⁹FÀ ¹⁹FÀ ¹⁹FÀ

1

¹⁹FÀ

ChemMedChem 2015, 10, 662 – 687

www.chemmedchem.org

680

ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers

À122.2 (C2-F, s), À137.6 (C6-F, dd, ¹J=25 Hz, ²J=12 Hz),

À152.9 ppm (C5-F, dd, ¹J=26 Hz, ²J=6 Hz); HRMS for

C₁₈H₂₁F₃N₂O₄S₂[M+H]⁺: calcd 451.0968, found 451.0969.

tography on a column of silica gel with EtOAc/CHCl₃(1:9), R_f=

0.50; yield: 0.22 g, 88%; mp: 90–928C; ¹H NMR (300 MHz, CDCl₃):

d=1.43–1.99 (14H, m, cyclooctane), 3.14 (2H, t, J=7.8 Hz,

SO₂CH₂CH₂), 3.64 (2H, t, J=7.8 Hz, SO₂CH₂CH₂), 3.85–3.95 (1H, m,

cyclooctane), 5.68 (2H, s, SO₂NH₂), 6.91 (1H, d, J=8.7 Hz, NH),

7.14–7.35 ppm (5H, m, ArH); ¹³C NMR (75 MHz, CDCl₃): d=23.5 (cy-

clooctane), 25.7 (cyclooctane), 27.5 (cyclooctane), 28.7 (SO₂CH₂CH₂),

General procedure for the syntheses of 9d,e,g–k,m,n,o. A mix-

ture of 9 (0.20 g, 0.50 mmol), Et₃N (0.071 mL, 0.51 mmol), DMSO

(1 mL) and appropriate nucleophile (0.51 mmol) was stirred at am-

bient temperature. The reaction was monitored by TLC. The reac-

tion proceeded completion after 4–24 h. The mixture was then di-

luted with H₂O (20 mL) and extracted with EtOAc (310 mL). The

combined organic phase was dried over MgSO₄and evaporated

under reduced pressure.

33.2 (cyclooctane), 56.2 (CH of cyclooctane, d, J ₁₃_C=11 Hz), 58.7

¹⁹FÀ

1

2

(SO₂CH₂CH₂, d, J ₁₃_C=4 Hz), 114.7 (C4, dd, J ₁₃_C=12 Hz, J

=

¹⁹FÀ

¹⁹FÀ¹³C

7 Hz), 126.4 (C1, t, J ₁₃_C=16 Hz), 127.6 (Ar), 128.5 (Ar), 129.1 (Ar),

¹⁹FÀ

136.0 (C3, d, J ₁₃_C=13 Hz), 136.5 (Ar), 136.7 (C6, d, J

=

¹⁹FÀ

¹⁹FÀ¹³C

1

251 Hz), 145.8 (C2, d, J ₁₃_C=252 Hz), 146.2 ppm (C5, dd, J

¹⁹FÀ

252 Hz, J ₁₃_C=16 Hz); ¹⁹F NMR (282 MHz, CDCl₃): d=À131.0 (C2-

2

¹⁹FÀ

3-(Benzylamino)-2,5,6-trifluoro-4-[(2-phenylethyl)sulfonyl]benze-

nesulfonamide (9d). Recrystallization was accomplished from

EtOH; yield: 0.21 g, 57%; mp: 59–618C; ¹H NMR (300 MHz,

[D₆]DMSO): d=3.00 (2H, t, J=7.5 Hz, SO₂CH₂CH₂), 3.82 (2H, t, J=

7.5 Hz, SO₂CH₂CH₂), 4.54 (2H, dd, J=6.0 Hz, J=4.2 Hz, NHCH₂), 7.01

(1H, t, J=6.0 Hz, NH), 7.14–7.45 (10H, m, ArH), 8.34 ppm (2H, brs,

1

2

F, s), À138.2 (C6-F, dd, J=25 Hz, J=12 Hz), À156.9 ppm (C5-F, dd,

¹J=26 Hz, ²J=7 Hz); H,C HETCOR (300 MHz, CDCl₃): d=56.2–3.89

(NHCH) 58.7–3.64 ppm (SO₂CH₂CH₂Ph); HRMS for C₂₂H₂₇F₃N₂O₄S₂

[M+H]⁺: calcd 505.1437, found 505.1439.

3-(Cyclododecylamino)-2,5,6-trifluoro-4-[(2-phenylethyl)sulfonyl]-

benzenesulfonamide (9i). The product was purified by chroma-

tography on a column of silica gel with EtOAc/CHCl₃(1:10), R_f=

0.37; yield: 0.13 g, 46%; mp: 130–1318C; ¹H NMR (300 MHz,

[D₆]DMSO): d=1.21–1.74 (22H, m, cyclododecane), 3.07 (2H, t, J=

7.5 Hz, SO₂CH₂CH₂), 3.78 (1H, brs, CH of cyclododecane), 3.88 (2H,

t, J=7.5 Hz, SO₂CH₂CH₂), 6.55 (1H, d, J=8.1 Hz, NH), 7.14–7.37 (5H,

m, ArH), 8.36 ppm (2H, s, SO₂NH₂); ¹³C NMR (75 MHz, [D₆]DMSO):

d=21.2 (cyclododecane), 23.3 (cyclododecane), 23.4 (cyclodode-

cane), 24.5 (cyclododecane), 24.7 (cyclododecane), 28.5

(SO₂CH₂CH₂), 30.8 (cyclododecane), 53.5 (CH of cyclododecane, d,

SO₂NH₂); ¹³C NMR (75 MHz, [D₆]DMSO): d=28.5 (SO₂CH₂CH₂), 50.5

1

(NHCH₂, d, J ₁₃_C=13 Hz), 57.7 (SO₂CH₂CH₂), 115.6 (C4, dd, J

=

¹⁹FÀ

¹⁹FÀ¹³C

13 Hz, ²J ₁₃_C=5 Hz), 127.5 (Ar), 128.2 (Ar), 128.4 (Ar), 129 (Ar),

¹⁹FÀ

129.1 (Ar), 129.3 (Ar), 136.0 (C3, d, J ₁₃_C=14 Hz), 137.5 (Ar), 139.7

¹⁹FÀ

(Ar), 139.4 (C6, d, J ₁₃_C=244 Hz), 144.9 (C2, d, J ₁₃_C=253 Hz),

¹⁹FÀ

146.0 ppm (C5, d, J¹⁹₁₃_C=253 Hz); ¹⁹F NMR (282 MHz, [D₆]DMSO):

d=À124.7 (C2-F, s)^F,^ÀÀ134.9 (C6-F, dd, ¹J=25 Hz, ²J=12 Hz),

À150.4 ppm (C5-F, dd, ¹J=26 Hz, ²J=7 Hz); HRMS for

C₂₁H₁₉F₃N₂O₄S₂[M+H]⁺: calcd 485.0811, found 485.0814.

3-[(2-Phenylethyl)amino]-2,5,6-trifluoro-4-[(2-phenylethyl)sulfo-

nyl]benzenesulfonamide (9e). The product was purified by chro-

matography on a column of silica gel with EtOAc/CHCl₃(1:6), R_f=

0.48; yield: 0.18 g, 72%; mp: 141–1428C; ¹H NMR (300 MHz,

[D₆]DMSO): d=2.88 (2H, t, J=7.2 Hz, NHCH₂CH₂), 2.98 (2H, t, J=

7.5 Hz, SO₂CH₂CH₂), 3.60 (2H, brt, NHCH₂CH₂), 3.77 (2H, t, J=

7.2 Hz, SO₂CH₂CH₂), 6.68 (1H, brs, NH), 7.05–7.42 (10H, m, ArH),

8.33 ppm (2H, s, SO₂NH₂); ¹³C NMR (75 MHz, [D₆]DMSO): d=28.6

1

2

J

₁₃_C=12 Hz), 58.0 (SO₂CH₂CH₂), 115.4 (C4, dd, J ₁₃_C=13 Hz, J

¹⁹FÀ

₁₃_C=4 Hz), 127.5 (Ar), 128.2 (C1, t, J ₁₃_C=16 Hz), 129.0 (Ar), 135.8

¹⁹FÀ

(C3, d, J ₁₃_C=16 Hz), 137.6 (Ar), 137.5 (C6, dd, ¹J ₁₃_C=246 Hz,

¹⁹FÀ

²J ₁₃_C=17 Hz), 144.7 (C2, d, J ₁₃_C=250 Hz), 146.2 ppm (C5, dd,

¹⁹FÀ

¹J ₁₃_C=249 Hz, J ₁₃_C=17 Hz); ¹⁹F NMR (282 MHz, [D₆]DMSO): d=

2

¹⁹FÀ

À125.4 (C2-F, s), À134.5 (C6-F, dd, ¹J=27 Hz, ²J=12 Hz),

À151.0 ppm (C5-F, dd, ¹J=27 Hz, ²J=6 Hz); HRMS C₂₆H₃₅F₃N₂O₄S₂

[M+H]⁺: calcd 561.2063, found 561.2071.

(SO₂CH₂CH₂), 36.8 (NHCH₂CH₂), 48.3 (NHCH₂CH₂, d, J ₁₃_C=13 Hz),

¹⁹FÀ

57.5 (SO₂CH₂CH₂), 114.6 (C4, d, J ₁₃_C=12 Hz), 127.1 (Ar), 127.5 (Ar),

¹⁹FÀ

3-[(2,6-Dimethoxybenzyl)amino]-2,5,6-trifluoro-4-[(2-phenyl-

ethyl)sulfonyl]benzenesulfonamide (9j). The product was purified

by chromatography on a column of silica gel with EtOAc/CHCl₃

(1:5), R_f=0.47; yield: 0.13 g, 48%; mp: 133–1378C; ¹H NMR

(300 MHz, [D₆]DMSO): d=2.76 (2H, t, J=8.1 Hz, SO₂CH₂CH₂), 3.47

(2H, t, J=8.1 Hz, SO₂CH₂CH₂), 3.74 (6H, s, 2CH₃), 4.52 (2H, d, J=

5.4 Hz, NHCH₂), 6.62 (2H, d, J=8.4 Hz, ArH), 6.69 (1H, brt, NH),

7.05–7.32 (6H, m, ArH), 8.40 ppm (2H, s, SO₂NH₂); ¹³C NMR

128.1 (C1, t, J ₁₃_C=16 Hz), 128.9 (Ar), 129.0 (Ar), 129.2 (Ar), 129.5

¹⁹FÀ

(Ar), 136.1 (C3, d, J ₁₃_C=13 Hz), 137.5 (Ar), 139.3 (Ar), 137.0 (C6, d,

¹⁹FÀ

J

₁₃_C=244 Hz), 144.3 (C2, d, J ₁₃_C=250 Hz), 145.7 ppm (C5, d,

¹⁹FÀ

₁₃_C=233 Hz); ¹⁹F NMR (282 MHz, [D₆]DMSO): d=À127.0 (C2-F, s),

1

2

1

À135.3 (C6-F, dd, J=27 Hz, J=12 Hz), À152.0 ppm (C5-F, dd, J=

26 Hz, J=7 Hz); HRMS for C₂₂H₂₁F₃N₂O₄S₂[M+H]⁺: calcd 499.0968,

2

found 499.0971.

(75 MHz, [D₆]DMSO): d=28.2 (SO₂CH₂CH₂), 39.4 (NHCH₂, d, J

=

¹⁹FÀ¹³C

3-Morpholin-4-yl-2,5,6-trifluoro-4-[(2-phenylethyl)sulfonyl]ben-

zenesulfonamide (9g). Recrystallization was accomplished from

EtOH; yield: 0.11 g, 46%; mp: 198–1998C; ¹H NMR (300 MHz,

[D₆]DMSO): d=2.90 (2H, d, J=10.8 Hz, morpholine), 3.12 (2H, t,

J=7.8 Hz, SO₂CH₂CH₂), 3.21 (2H, t, J=11.1 Hz, morpholine), 3.57

(2H, t, J=11.1 Hz, morpholine), 3.79 (2H, d, J=10.8 Hz, morpho-

line), 4.06 (2H, t, J=7.8 Hz, SO₂CH₂CH₂), 7.15–7.37 (5H, m, ArH),

8.48 ppm (2H, s, SO₂NH₂); ¹³C NMR (75 MHz, [D₆]DMSO): d=33.3

13 Hz, signal overlaps with signal of DMSO), 56.4 (CH₃), 57.6

(SO₂CH₂CH₂), 104.7 (Ar), 114.7 (Ar), 116.5 (C4, dd, ¹J ₁₃_C=13 Hz,

¹⁹FÀ

²J ₁₃_C=5 Hz), 127.4 (Ar), 127.9 (C1, t, J ₁₃_C=16 Hz), 129.0 (Ar),

¹⁹FÀ

129.1 (Ar), 130.4 (Ar), 136.9 (C3, d, J ₁₃_C=13 Hz), 137.5 (Ar), 138.1

¹⁹FÀ

1

(C6, d J ₁₃_C=251 Hz), 145.5 (C5, d, J ₁₃_C=253 Hz), 146.1 (C2, d,

¹⁹FÀ

J

₁₃_C=254 Hz), 158.7 ppm (Ar); ¹⁹F NMR (282 MHz, [D₆]DMSO): d=

¹⁹FÀ

1

2

1

À121.8 (C2-F, dd, J=11 Hz, J=5 Hz), À135.5 (C6-F, dd, J=27 Hz,

²J=12 Hz), À149.6 ppm (C5-F, dd, ¹J=27 Hz, ²J=5 Hz); HRMS

C₂₃H₂₃F₃N₂O₆S₂[MÀH]^À: calcd 543.0877, found 543.0881.

(SO₂CH₂CH₂), 56.6 (morpholine, d, J ₁₃_C=4 Hz), 62.3 (SO₂CH₂CH₂),

¹⁹FÀ

71.8 (morpholine), 132.2 (Ar), 133.8 (Ar), 134.1 (Ar), 136.1 (C1, t, J

¹⁹FÀ

₁₃_C=6 Hz), 139.7 (C4, dd, ¹J ₁₃_C=16 Hz, ²J ₁₃_C=5 Hz), 142.9 (Ar),

¹⁹FÀ

3-[(3,4-Dimethoxybenzyl)amino]-2,5,6-trifluoro-4-[(2-phenyl-

ethyl)sulfonyl]benzenesulfonamide (9k). Recrystallization was ac-

complished from EtOH; yield: 0.18 g, 67%; mp: 167–1688C;

¹H NMR (300 MHz, [D₆]DMSO): d=2.95 (2H, t, J=7.8 Hz,

SO₂CH₂CH₂), 3.69 (3H, s, CH₃), 3.73 (3H, s, CH₃), 3.82 (2H, t, J=

7.8 Hz, SO₂CH₂CH₂), 4.45 (2H, t, J=4.7 Hz, NHCH₂), 6.78–6.99 (4H,

m, ArH, NH), 7.10–7.32 (5H, m, ArH), 8.38 ppm (2H, s, SO₂NH₂);

¹³C NMR (75 MHz, [D₆]DMSO): d=28.5 (SO₂CH₂CH₂), 50.3 (NHCH₂, d,

1

2

150.7 (C5, C6, dd, J ₁₃_C=261 Hz, J ₁₃_C=17 Hz), 159.3 ppm (C2,

¹⁹FÀ

d, J ₁₃_C=259 Hz); ¹⁹F NMR (282 MHz, [D₆]DMSO): d=À119.1 (C2-F,

¹⁹FÀ

1

d, J=14 Hz), À132.4 (C5-F, d, J=25 Hz), À136.7 ppm (C6-F, dd, J=

25 Hz, ²J=14 Hz); HRMS for C₁₈H₁₉F₃N₂O₅S₂[M+H]⁺: calcd

465.0760, found 465.0765.

3-(Cyclooctylamino)-2,5,6-trifluoro-4-[(2-phenylethyl)sulfonyl]-

benzenesulfonamide (9h). The product was purified by chroma-

ChemMedChem 2015, 10, 662 – 687

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681

ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers

J

₁₃_C=12.5 Hz), 56.0 (CH₃), 56.1 (CH₃), 57.7 (SO₂CH₂CH₂), 112.3 (Ar),

laps with signal of Ar), 129.0 (Ar), 129.1 (Ar), 129.5 (Ar), 129.9 (Ar),

¹⁹FÀ

112.4 (Ar), 115.8 (C4, dd, ¹J ₁₃_C=13 Hz, ²J ₁₃_C=5 Hz), 120.8 (Ar),

135.3 (C3, d, J ₁₃_C=11 Hz), 137.4 (Ar), 137.5 (Ar), 137.6 (Ar), 138.0

¹⁹FÀ

127.5 (Ar), 128.1 (C1, t, J ₁₃_C=16 Hz), 128.9 (Ar), 129.0 (Ar), 131.9

(C6, d, J ₁₃_C=238 Hz), 145.1 (C2, d, J ₁₃_C=254 Hz), 146.1 ppm

1

9

1

9

¹⁹FÀ

F

À

F

À

1

(Ar), 136.0 (C3, d, J ₁₃_C=14 Hz), 137.5 (Ar), 137.7 (C6, dd, J

=

(C5, d, J ₁₃_C=254 Hz); ¹⁹F NMR (282 MHz, [D₆]DMSO): d=À123.5

¹⁹FÀ

¹⁹FÀ¹³C

¹⁹FÀ

2

1

2

249 Hz, J ₁₃_C=18 Hz), 145.1 (C2, d, J ₁₃_C=256 Hz), 146.0 (C5, d,

(C2-F, s), À134.3 (C6-F, dd, J=27 Hz, J=12 Hz), À149.9 ppm (C5-F,

dd, ¹J=27 Hz, ²J=5 Hz); HRMS for C₂₄H₂₃F₃N₂O₄S₂[MÀH]^À: calcd

523.0979, found 523.0983.

¹⁹FÀ

J

₁₃_C=250 Hz), 148.9 (Ar), 149.4 ppm (Ar); ¹⁹F NMR (282 MHz,

¹⁹FÀ

[D₆]DMSO): d=À123.7 (C2-F, s), À134.8 (C6-F, dd, ¹J=27 Hz, ²J=

12 Hz), À150.4 ppm (C5-F, dd, ¹J=27 Hz, ²J=6 Hz); HRMS for

C₂₃H₂₃F₃N₂O₆S₂[MÀH]^À: calcd 543.0877, found 543.0875.

General procedure for the syntheses of 9l,p,q. A mixture of 9

(0.20 g, 0.50 mmol), Et₃N (0.071 mL, 0.51 mmol), DMSO (1 mL) and

appropriate nucleophile (0.52 mmol) was stirred at ambient tem-

perature for three days, compound 9l was obtained after stirring

for five days. The mixture was then diluted with H₂O (20 mL).

3-(2,3-Dihydro-1H-inden-2-ylamino)-2,5,6-trifluoro-4-[(2-phenyl-

ethyl)sulfonyl]benzenesulfonamide (9m). The product was puri-

fied by chromatography on a column of silica gel with EtOAc/

CHCl₃(1:4), R_f=0.60. Recrystallization was accomplished from EtOH

1

3-(1-Adamantylamino)-2,5,6-trifluoro-4-[(2-phenylethyl)sulfonyl]-

benzenesulfonamide (9l). The resultant precipitate was filtered,

washed with H₂O. The product was purified by chromatography

on a column of silica gel with EtOAc/CHCl₃(1:4), R_f=0.75; yield:

0.02 g, 8%; mp: 155–1568C; ¹H NMR (300 MHz, CDCl₃): d=1.69

(6H, brs, adamantane), 1.91 (6H, brs, adamantane), 2.15 (3H, brs,

adamantane), 3.16 (2H, m, SO₂CH₂CH₂), 3.67 (2H, m, SO₂CH₂CH₂),

5.53 (2H, s, SO₂NH₂), 6.41 (1H, s, NH), 7.11–7.40 ppm (5H, m, ArH);

¹³C NMR (75 MHz, CDCl₃): d=28.7 (SO₂CH₂CH₂), 30.2 (adamantane),

36.2 (adamantane), 43.4 (adamantane), 43.5 (adamantane), 56.6

after chromatography; yield: 0.12 g, 45%; mp 155–1568C; H NMR

(300 MHz, [D₆]DMSO): d=2.77–2.95 (4H, m, SO₂CH₂CH₂and CH₂of

indane), 3.26 (1H, d, J=6.3 Hz, indane), 3.31 (1H, d, J=6.3 Hz,

indane), 3.67 (2H, t, J=7.8 Hz, SO₂CH₂CH₂), 4.45–4.55 (1H, m, CH of

indane), 6.87 (1H, d, J=6.6 Hz, NH), 7.07–7.30 (10H, m, ArH),

8.38 ppm (2H, s, SO₂NH₂); ¹³C NMR (75 MHz, [D₆]DMSO): d=28.4

(SO₂CH₂CH₂), 41.1 (CH₂of indane, signal overlaps with signal of

[D₆]DMSO), 57.6 (SO₂CH₂CH₂), 57.7 (CH of indane, d, J ₁₃_C=11 Hz),

¹⁹FÀ

1

2

115.3 (C4, dd, J ₁₃_C=13 Hz, J ₁₃_C=5 Hz), 125.4 (Ar), 127.4 (Ar),

¹⁹FÀ

127.5 (Ar), 128.2 (C1, t, J ₁₃_C=16 Hz), 129.0 (Ar), 129.1 (Ar), 135.4

¹⁹FÀ

(C3, d, J ₁₃_C=14 Hz), 137.5 (Ar), 137.6 (C6, dd, ¹J ₁₃_C=247 Hz,

¹⁹FÀ

(SO₂CH₂CH₂), 58.9 (adamantane, d, J ₁₃_C=4 Hz), 119.6 (C4, dd,

¹⁹FÀ

¹J ₁₃_C=12 Hz, ²J ₁₃_C=6 Hz), 126.0 (C1, t, J ₁₃_C=16 Hz), 127.6

²J ₁₃_C=17 Hz), 141.1 (Ar), 144.4 (C2, d, J ₁₃_C=252 Hz), 146.2 ppm

¹⁹FÀ

(Ar), 128.5 (Ar), 129.2 (Ar), 135.3 (C3, dd, ¹J ₁₃_C=18 Hz, ²J

=

(C5, dd, ¹J ₁₃_C=250 Hz, ²J ₁₃_C=16 Hz); ¹⁹F NMR (282 MHz,

¹⁹FÀ¹³C

¹⁹FÀ

[D₆]DMSO): d=À126.1 (C2-F, s), À134.7 (C6-F, dd, ¹J=27 Hz, ²J=

12 Hz), À150.8 ppm (C5-F, dd, ¹J=27 Hz, ²J=7 Hz); HRMS for

C₂₃H₂₁F₃N₂O₄S₂[M+H]⁺: calcd 511.0968, found 511.0972.

¹⁹FÀ

3 Hz), 137.0 (Ar), 139.0 (C6, d, J ₁₃_C=252 Hz), 146.0 (C5, d, J

254 Hz), 146.4 ppm (C2, d, J ₁₃_C=253 Hz); ¹⁹F NMR (282 MHz,

CDCl₃): d=À120.3 (C2-F, s), À137.6 (C6-F, dd, J=25 Hz, J=12 Hz),

À152.0 ppm (C5-F, dd, ¹J=26 Hz, ²J=6 Hz); HRMS for

C₂₄H₂₇F₃N₂O₄S₂[M+H]⁺: calcd 529.1437, found 529.1440.

¹⁹FÀ

1

2

3-[(1S)-2,3-Dihydro-1H-inden-1-ylamino]-2,5,6-trifluoro-4-[(2-phe-

nylethyl)sulfonyl]benzenesulfonamide (9n). The product was pu-

rified by chromatography on a column of silica gel with EtOAc

(5%)/CHCl₃, R_f=0.38; yield: 0.17 g, 66%; mp: 908C; ¹H NMR

(300 MHz, [D₆]DMSO): d=2.01 (1H, sext, J=6.0 Hz, indane), 2.49

(1H, sext, indane, signal overlaps with signal of [D₆]DMSO), 2.78–

3.23 (4H, m, CH₂of indane, SO₂CH₂CH₂), 3.67–3.89 (2H, m,

SO₂CH₂CH₂), 5.18 (1H, brs, indane), 6.89 (1H, d, J=7.8 Hz, NH),

7.13–7.44 (9H, m, ArH), 8.43 ppm (2H, s, SO₂NH₂); ¹³C NMR

(75 MHz, [D₆]DMSO): d=28.3 (SO₂CH₂CH₂), 30.2 (indane), 35.3

3-{[(1R,2S)-2-Hydroxy-1,2-diphenylethyl]amino}-2,5,6-trifluoro-4-

[(2-phenylethyl)sulfonyl]benzenesulfonamide (9p). The mixture

was then extracted with EtOAc (310 mL). The combined organic

phase was dried over MgSO₄and evaporated under reduced pres-

sure. Recrystallization was accomplished from EtOH/H₂O=2:1;

1

yield: 0.12 g, 40%; mp: 175–1768C; H NMR (300 MHz, [D₆]DMSO):

d=3.06 (2H, t, J=7.5 Hz, SO₂CH₂CH₂), 3.75–3.95 (2H, m,

SO₂CH₂CH₂), 4.89–4.98 (1H, m, CH), 5.10 (1H, d, J=4.5 Hz, CH), 6.01

(1H, brs, OH), 7.05–7.31 (15H, m, ArH), 7.87 (1H, d, J=9.0 Hz, NH),

8.30 ppm (2H, s, SO₂NH₂); ¹³C NMR (75 MHz, [D₆]DMSO): d=28.5

(indane), 57.8 (SO₂CH₂CH₂), 61.7 (CH of indane, d, J ₁₃_C=11.4 Hz),

¹⁹FÀ

1

2

115.6 (C4, dd, J ₁₃_C=14 Hz, J ₁₃_C=5 Hz), 124.6 (Ar), 125.7 (Ar),

¹⁹FÀ

(SO₂CH₂CH₂), 58.0 (SO₂CH₂CH₂, d, J ₁₃_C=2 Hz), 65.4 (CH, d, J

=

¹⁹FÀ

¹⁹FÀ¹³C

127.4 (Ar), 127.5 (Ar), 128.2 (C1, t, J ₁₃_C=16 Hz), 128.9 (Ar), 129.0

¹⁹FÀ

12.8 Hz), 75.6 (CH), 115.3 (C4, dd, ¹J ₁₃_C=13 Hz, ²J ₁₃_C=6 Hz),

¹⁹FÀ

(Ar), 129.1 (Ar), 135.7 (C3, d, J ₁₃_C=15 Hz), 137.5 (Ar), 137.8 (C6, d,

¹⁹FÀ

127.2 (Ar), 127.4 (Ar), 127.8 (Ar), 127.9 (Ar), 128.2 (Ar), 128.3 (Ar),

J

₁₃_C=246 Hz), 143.7 (Ar), 144.2 (Ar), 144.8 (C2, d, J ₁₃_C=252 Hz),

¹⁹FÀ

146.1 ppm (C5, d, J¹⁹₁₃_C=251 Hz); ¹⁹F NMR (282 MHz, [D₆]DMSO):

d=À124.2 (C2-F, s)^F,^ÀÀ134.5 (C6-F, dd, ¹J=27 Hz, ²J=12 Hz),

À150.2 ppm (C5-F, dd, ¹J=27 Hz, ²J=6 Hz); HRMS for

C₂₃H₂₁F₃N₂O₄S₂[M+H]⁺: calcd 511.0968, found 511.0964.

128.9 (Ar), 129.0 (Ar), 129.1 (Ar), 135.2 (C3, d, J ₁₃_C=12 Hz), 137.2

¹⁹FÀ

(C6, d, J ₁₃_C=250 Hz), 137.5 (Ar), 139.7 (Ar), 142.9 (Ar), 144.5 (C2,

¹⁹FÀ

d, J ₁₃_C=254 Hz), 146.0 ppm (C5, d, J ₁₃_C=249 Hz); ¹⁹F NMR

¹⁹FÀ

(282 MHz, [D₆]DMSO): d=À123.3 (C2-F, s), À134.7 (C6-F, dd, ¹J=

25 Hz, ²J=12 Hz), À150.8 ppm (C5-F, dd, ¹J=26 Hz, ²J=7 Hz);

HRMS for C₂₈H₂₅F₃N₂O₅S₂[M+H]⁺: calcd 591.1230, found 591.1220.

3-[(1S)-1,2,3,4-Tetrahydronapthalen-1-ylamino)-2,5,6-trifluoro-4-

[(2-phenylethyl)sulfonyl]benzenesulfonamide (9o). The product

was purified by chromatography on a column of silica gel with

EtOAc/CHCl₃(1:10), R_f=0.37; yield: 0.13 g, 50%; mp: 116–1198C;

¹H NMR (300 MHz, [D₆]DMSO): d=1.71–2.09 (4H, m, tetrahydronap-

thalene), 2.61–2.89 (2H, m, tetrahydronapthalene), 2.94 (2H, t, J=

7.8 Hz, SO₂CH₂CH₂), 3.74 (2H, t, J=7.8 Hz, SO₂CH₂CH₂), 4.78–4.92

(1H, m, CH of tetrahydronapthalene), 6.82 (1H, d, J=9.0 Hz, NH),

7.11–7.43 (9H, m, ArH), 8.41 ppm (2H, s, SO₂NH₂); ¹³C NMR

(75 MHz, [D₆]DMSO): d=19.1 (tetrahydronapthalene), 28.3

(SO₂CH₂CH₂), 29.1 (tetrahydronapthalene), 30.5 (tetrahydronaptha-

3-{[(1S,2R)-2-Hydroxy-1,2-diphenylethyl]amino}-2,5,6-trifluoro-4-

[(2-phenylethyl)sulfonyl]benzenesulfonamide (9q). The resultant

precipitate was filtered, washed with H₂O. Recrystallization was ac-

complished from EtOH/H₂O=2:1; yield: 0.12 g, 40%; mp: 176–

1778C; ¹H NMR (300 MHz, [D₆]DMSO): d=3.06 (2H, t, J=7.5 Hz,

SO₂CH₂CH₂), 3.75–3.95 (2H, m, SO₂CH₂CH₂), 4.90–5.01 (1H, m, CH),

5.10 (1H, t, J=4.2 Hz, CH), 6.01 (1H, d, J=4.2 Hz, OH), 7.02–7.31

(15H, m, ArH), 7.87 (1H, d, J=8.4 Hz, NH), 8.25 ppm (2H, s,

SO₂NH₂); ¹³C NMR (75 MHz, [D₆]DMSO): d=28.5 (SO₂CH₂CH₂), 58.0

lene), 54.1 (CH of tetrahydronapthalene, d, J ₁₃_C=12 Hz), 58.0

(SO₂CH₂CH₂, d, J ₁₃_C=2 Hz), 65.4 (CH, d, J ₁₃_C=12.8 Hz), 75.6

¹⁹FÀ

1

2

(SO₂CH₂CH₂), 116.1 (C4, dd, ¹J ₁₃_C=13 Hz, ²J ₁₃_C=5 Hz), 126.8

(CH), 115.3 (C4, dd, J ₁₃_C=13 Hz, J ₁₃_C=6 Hz), 127.2 (Ar), 127.4

¹⁹FÀ

(Ar), 127.5 (Ar), 128.2 (Ar), 128.2 (C1, t, J ₁₃_C=18 Hz, signal over-

(Ar), 127.8 (Ar), 127.9 (Ar), 128.2 (Ar), 128.3 (Ar), 128.9 (Ar), 129.0

¹⁹FÀ

ChemMedChem 2015, 10, 662 – 687

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ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers

(Ar), 129.1 (Ar), 135.2 (C3, d, J ₁₃_C=12 Hz), 137.2 (C6, d, J

=

118.2 (C4, dd, ¹J ₁₃_C=12 Hz, ²J ₁₃_C=5 Hz), 127.7 (C1, t, J

=

¹⁹FÀ¹³C

¹⁹FÀ

¹⁹FÀ¹³C

¹⁹FÀ

250 Hz), 137.5 (Ar), 139.7 (Ar), 142.9 (Ar), 144.5 (C2, d, J

16 Hz), 130.3 (Ar), 136.8 (C3, d, J ₁₃_C=11 Hz), 137.9 (C6, d,

¹⁹FÀ

254 Hz), 146.0 ppm (C5, d, J ₁₃_C=249 Hz); ¹⁹F NMR (282 MHz,

¹J ₁₃_C=228 Hz), 144.8 (C5, d, J ₁₃_C=242 Hz), 146.0 (C2, d, J

=

¹⁹FÀ¹³C

¹⁹FÀ

[D₆]DMSO): d=À123.3 (C2-F, s), À134.7 (C6-F, dd, ¹J=25 Hz, ²J=

12 Hz), À150.8 ppm (C5-F, dd, ¹J=26 Hz, ²J=7 Hz); HRMS for

C₂₈H₂₅F₃N₂O₅S₂[M+H]⁺: calcd 591.1230, found 591.1221.

253 Hz), 158.7 ppm (Ar); ¹⁹F NMR (282 MHz, [D₆]DMSO): d=À122.1

1

2

(C2-F, s), À135.9 (C6-F, dd, J=26 Hz, J=13 Hz), À150.1 ppm (C5-F,

dd, ¹J=27 Hz, ²J=6 Hz); HRMS for C₁₇H₁₉F₃N₂O₇S₂[MÀH]^À: calcd

483.0513, found 483.0517.

General procedure for the syntheses of 10 (d,h-k,n–p). A mixture

of

2,3,5,6-tetrafluoro-4-[(2-hydroxyethyl)sulfonyl]benzenesulfona-

3-[(3,4-Dimethoxybenzyl)amino]-2,5,6-trifluoro-4-[(2-hydroxye-

thyl)sulfonyl]benzenesulfonamide (10k). The product was puri-

fied by chromatography on a column of silica gel with EtOAc/

CHCl₃(2:1), R_f=0.38. Recrystallization was accomplished from EtOH

mide (10) (0.20 g, 0.59 mmol), DMSO (1 mL) and appropriate nucle-

ophile (1.20 mmol) was stirred at ambient temperature for 24 h.

The mixture was then diluted with H₂O (20 mL) and extracted with

EtOAc (310 mL). The combined organic phase was dried over

MgSO₄and evaporated under reduced pressure.

1

after chromatography; yield: 0.10 g, 29%; mp: 164–1658C; H NMR

(300 MHz, [D₆]DMSO): d=3.65 (2H, t, J=5.7 Hz, SO₂CH₂CH₂), 3.74

(3H, s, CH₃), 3.76 (3H, s, CH₃), 3.82 (2H, brt, SO₂CH₂CH₂), 4.43 (2H,

brs, NHCH₂), 6.78–7.03 (3H, m, ArH), 6.99 (1H, s, NH), 8.38 ppm

(2H, s, SO₂NH₂); ¹³C NMR (75 MHz, [D₆]DMSO): d=50.6 (NHCH₂, d,

3-(Benzylamino)-2,5,6-trifluoro-4-[(2-hydroxyethyl)sulfonyl]ben-

zenesulfonamide (10d). The product was purified by chromatog-

raphy on a column of silica gel with EtOAc/CHCl₃(1:2), R_f=0.19;

yield: 0.11 g, 35%; mp 127–1288C; ¹H NMR (300 MHz, [D₆]DMSO):

d=3.65 (2H, t, J=5.4 Hz, SO₂CH₂CH₂), 3.83 (2H, k, J=5.4 Hz,

SO₂CH₂CH₂), 4.45–4.55 (2H, m, NHCH₂), 5.03 (1H, t, J=5.1 Hz, OH),

6.96 (1H, brt, NH), 7.28–7.52 (5H, m, ArH), 8.36 ppm (2H, s,

SO₂NH₂); ¹³C NMR (75 MHz, [D₆]DMSO): d=50.7 (NHCH₂, d, J

13 Hz), 55.8 (SO₂CH₂CH₂), 60.1 (SO₂CH₂CH₂), 117.6 (C4, dd, J

13 Hz, J ₁₃_C=5 Hz), 127.9 (C1 signal overlaps with signal of Ar),

128.1 (Ar), 128.3 (Ar), 129.3 (Ar), 136.1 (C3, d, J ₁₃_C=13 Hz), 137.8

J

₁₃_C=12 Hz), 55.8 (CH₃), 56.0 (CH₃), 56.1 (SO₂CH₂CH₂), 60.1

¹⁹FÀ

(SO₂CH₂CH₂), 112.2 (Ar), 112.3 (Ar), 117.7 (C4, dd, ¹J ₁₃_C=13 Hz,

¹⁹FÀ

²J ₁₃_C=5 Hz), 120.7 (Ar), 127.8 (C1, t, J ₁₃_C=16 Hz), 131.8 (Ar),

¹⁹FÀ

136.0 (C3, d, J ₁₃_C=16 Hz), 137.8 (C6, d, J ₁₃_C=252 Hz), 145.1

¹⁹FÀ

(C2, d, J ₁₃_C=253 Hz), 146.1 (C5, dd, ¹J ₁₃_C=253 Hz, ²J

=

¹⁹FÀ

¹⁹FÀ¹³C

=

¹⁹FÀ¹³C

16 Hz), 148.9 (Ar), 149.5 ppm (Ar); ¹⁹F NMR (282 MHz, [D₆]DMSO):

d=À124.4 (C2-F, s), À135.2 (C6-F, dd, ¹J=27 Hz, ²J=12 Hz),

À150.7 ppm (C5-F, dd, ¹J=27 Hz, ²J=6 Hz); HRMS for

C₁₇H₁₉F₃N₂O₇S₂[MÀH]^À: calcd 483.0513, found 483.0515.

1

2

¹⁹FÀ

(C6, d, J ₁₃_C=246 Hz), 139.6 (Ar), 144.9 (C2, d, J ₁₃_C=252 Hz),

¹⁹FÀ

146.2 ppm (C5, d, J ₁₃_C=252 Hz); ¹⁹F NMR (282 MHz, [D₆]DMSO):

¹⁹FÀ

3-[(1S)-2,3-Dihydro-1H-inden-1-ylamino]-2,5,6-trifluoro-4-[(2-hy-

droxyethyl)sulfonyl]benzenesulfonamide (10n). The product was

purified by chromatography on a column of silica gel with EtOAc/

d=À125.1 (C2-F, s), À135.3 (C6-F, dd, ¹J=25 Hz, ²J=13 Hz),

À150.7 ppm (C5-F, dd, ¹J=26 Hz, ²J=7 Hz); HRMS for

C₁₅H₁₅F₃N₂O₅S₂[M+H]⁺: calcd 425.0447, found 425.0439.

1

CHCl₃(1:1), R_f=0.38; yield: 0.16 g, 60%; mp: 131–1328C; H NMR

(300 MHz, [D₆]DMSO): d=1.95 (1H, sext, J=6.6 Hz, indane), 2.51

(1H, sext, indane, signal overlaps with signal of [D₆]DMSO), 2.78–

3.08 (2H, m, indane), 3.55–3.68 (2H, m, SO₂CH₂CH₂), 3.71–3.85 (2H,

m, SO₂CH₂CH₂), 5.02 (1H, t, J=5.4 Hz, OH), 5.12–5.25 (1H, m, CH of

indane), 6.88 (1H, d, J=7.8 Hz, NH), 7.21–7.42 (4H, m, ArH),

8.43 ppm (2H, s, SO₂NH₂); ¹³C NMR (75 MHz, [D₆]DMSO): d=30.2

(indane), 35.2 (indane), 55.8 (SO₂CH₂CH₂), 60.2 (SO₂CH₂CH₂), 61.9

(CH of indane, d, J ₁₃_C=12 Hz), 117.6 (C4, dd, ¹J ₁₃_C=13 Hz,

3-(Cyclooctylamino)-2,5,6-trifluoro-4-[(2-hydroxyethyl)sulfonyl]-

benzenesulfonamide (10h). The synthesis of compound 10h was

described previously.^[52]

3-(Cyclododecylamino)-2,5,6-trifluoro-4-[(2-hydroxyethyl)sulfo-

nyl]benzenesulfonamide (10i). The product was purified by chro-

matography on a column of silica gel with EtOAc/CHCl₃(1:1), R_f=

0.50; yield: 0.26 g, 88%; mp: 143–1448C; ¹H NMR (300 MHz,

[D₆]DMSO): d=1.23–1.74 (22H, m, cyclododecane), 3.68 (2H, t, J=

5.4 Hz, SO₂CH₂CH₂), 3.78 (1H, brs, CH of cyclododecane, signal

overlaps with signal of SO₂CH₂CH₂), 3.83 (2H, t, J=5.4 Hz,

SO₂CH₂CH₂), 5.01 (1H, t, J=5.4 Hz, OH), 6.55 (1H, d, J=8.7 Hz, NH),

8.36 ppm (2H, s, SO₂NH₂); ¹³C NMR (75 MHz, [D₆]DMSO): d=21.3

(cyclododecane), 23.4 (cyclododecane), 23.5 (cyclododecane), 24.4

(cyclododecane), 24.6 (cyclododecane), 30.8 (cyclododecane), 53.4

¹⁹FÀ

²J ₁₃_C=5 Hz), 124.7 (Ar), 125.6 (Ar), 127.4 (Ar), 128 (C1, t, J

=

¹⁹FÀ

¹⁹FÀ¹³C

16 Hz), 128.9 (Ar), 135.6 (C3, d, J ₁₃_C=12 Hz), 137.8 (C6, d, J

¹⁹FÀ

253 Hz), 144.4 (Ar), 144.5 (Ar), 144.8 (C2, d,

J

₁₃_C=251 Hz),

¹⁹FÀ

146.3 ppm (C5, d, J¹⁹₁₃_C=258 Hz); ¹⁹F NMR (282 MHz, [D₆]DMSO):

d=À124.6 (C2-F, s)^F,^ÀÀ134.8 (C6-F, dd, ¹J=27 Hz, ²J=12 Hz),

À150.7 ppm (C5-F, dd, ¹J=27 Hz, ²J=6 Hz); HRMS for

C₁₇H₁₇F₃N₂O₅S₂[MÀH]^À: calcd 449.0458, found 449.0461.

(CH of cyclododecane, d, J ₁₃_C=12 Hz), 55.8 (SO₂CH₂CH₂), 60.3

¹⁹FÀ

3-[(1S)-1,2,3,4-Tetrahydronapthalen-1-ylamino)-2,5,6-trifluoro-4-

[(2-hydroxyethyl)sulfonyl]benzenesulfonamide (10o). The prod-

uct was purified by chromatography on a column of silica gel with

EtOAc/CHCl₃(1:1), R_f=0.41; yield: 0.14 g, 51%; mp: 103–1058C;

¹H NMR (300 MHz, [D₆]DMSO): d=1.68–2.11 (4H, m, tetrahydronap-

thalene), 2.63–2.92 (2H, m, tetrahydronapthalene), 3.61 (2H, t, J=

5.4 Hz, SO₂CH₂CH₂), 3.76 (2H, brt, SO₂CH₂CH₂), 4.77–4.90 (1H, m,

CH of tetrahydronapthalene), 5.01 (1H, brs, OH), 6.82 (1H, d, J=

9.0 Hz, NH), 7.11–7.28 (3H, m, ArH), 7.39 (1H, d, J=6.9 Hz, ArH),

8.42 ppm (2H, s, SO₂NH₂); ¹³C NMR (75 MHz, [D₆]DMSO): d=19.3

(tetrahydronapthalene), 29.2 (tetrahydronapthalene), 30.6 (tetrahy-

1

2

(SO₂CH₂CH₂), 117.4 (C4, dd, J ₁₃_C=13 Hz, J ₁₃_C=6 Hz), 127.9 (C1,

¹⁹FÀ

t, J ₁₃_C=16 Hz), 135.7 (C3, d, J ₁₃_C=13 Hz), 137.4 (C6, dd,

¹⁹FÀ

¹J ₁₃_C=246 Hz, ²J ₁₃_C=19 Hz), 144.7 (C2, d,

J

₁₃_C=253 Hz),

¹⁹FÀ

146.3 ppm (C5, d, J¹⁹₁₃_C=247 Hz); ¹⁹F NMR (282 MHz, [D₆]DMSO):

d=À125.4 (C2-F, s)^F,^ÀÀ134.8 (C6-F, dd, ¹J=27 Hz, ²J=12 Hz),

À151.4 ppm (C5-F, dd, ¹J=27 Hz, ²J=6 Hz); HRMS for

C₂₀H₃₁F₃N₂O₅S₂[M+H]⁺: calcd 501.1699, found 501.1701.

3-[(2,6-Dimethoxybenzyl)amino]-2,5,6-trifluoro-4-[(2-hydroxy-

ethyl)sulfonyl]benzenesulfonamide (10j). The product was puri-

fied by chromatography on a column of silica gel with EtOAc

1

(60%)/CHCl₃, R_f=0.45; yield: 0.15 g, 52%; mp: 164–1658C; H NMR

dronapthalene), 54.3 (CH of tetrahydronapthalene, d, J

=

¹⁹FÀ¹³C

1

(300 MHz, [D₆]DMSO): d=3.35 (2H, t, J=5.4 Hz, SO₂CH₂CH₂), 3.63

(2H, t, J=5.4 Hz, SO₂CH₂CH₂), 3.75 (6H, s, 2CH₃), 4.48 (2H, d, J=

5.4 Hz, NHCH₂), 4.93 (1H, brs, OH), 6.58 (1H, brt, NH), 6.66 (2H, d,

J=8.4 Hz, ArH), 7.26 (1H, t, J=8.4 Hz, ArH), 8.42 ppm (2H, s,

SO₂NH₂); ¹³C NMR (75 MHz, [D₆]DMSO): d=39.5 (NHCH₂), 55.4

(SO₂CH₂CH₂), 56.3 (2CH₃), 59.9 (SO₂CH₂CH₂), 104.7 (Ar), 114.6 (Ar),

12 Hz), 55.7 (SO₂CH₂CH₂), 60.3 (SO₂CH₂CH₂), 117.9 (C4, dd, J

2

13 Hz, J ₁₃_C=5 Hz), 126.8 (Ar), 128.0 (C1, t, J ₁₃_C=16 Hz), 128.2

¹⁹FÀ

(Ar), 129.5 (Ar), 129.8 (Ar), 135.1 (C3, d, J ₁₃_C=15 Hz), 137.48 (Ar),

¹⁹FÀ

137.56 (Ar), 137.9 (C6, d, J ₁₃_C=245 Hz), 145.1 (C2, d, J

=

¹⁹FÀ

¹⁹FÀ¹³C

255 Hz), 146.2 ppm (C5, d, J ₁₃_C=254 Hz); ¹⁹F NMR (282 MHz,

[D₆]DMSO): d=À123.9 (C2-F, s), À134.5 (C6-F, dd, ¹J=27 Hz, ²J=

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Full Papers

12 Hz), À150.5 ppm (C5-F, dd, ¹J=27 Hz, ²J=6 Hz); HRMS for

C₁₈H₁₉F₃N₂O₅S₂[M+H]⁺: calcd 465.076, found 465.0760.

General procedure for the syntheses of 13c and 13 f. A mixture

of 10i (0.20 g, 0.40 mmol), DMSO (1 mL) and appropriate nucleo-

phile (0.82 mmol) was stirred, compound 13c was obtained after

stirring at 508C for 14 h, compound 13 f was obtained after stirring

at 608C for 22 h. The mixture was then diluted with H₂O (20 mL)

and extracted with EtOAc (310 mL). The combined organic phase

was dried over MgSO₄and evaporated under reduced pressure.

3-{[(1R,2S)-2-Hydroxy-1,2-diphenylethyl]amino}-2,5,6-trifluoro-4-

[(2-hydroxyethyl)sulfonyl]benzenesulfonamide (10p). The prod-

uct was purified by chromatography on a column of silica gel with

1

EtOAc/CHCl₃(2:1), R_f=0.53; yield: 0.12 g, 39%; H NMR (300 MHz,

CD₃OD): d=3.62 (2H, t, J=5.4 Hz, SO₂CH₂CH₂), 4.04 (2H, t, J=

5.4 Hz, SO₂CH₂CH₂), 4.88 (SO₂NH₂, NH, OH signals overlap with

3-(Butylamino)-5-(cyclododecylamino)-2,6-difluoro-4-[(2-

hydroxyethyl)sulfonyl]benzenesulfonamide (13c). The product

was purified by chromatography on a column of silica gel with

1

2

signal of H₂O), 5.07 (1H, dd, J=5.1 Hz, J=2.1 Hz, CH), 5.14 (1H, d,

J=4.8 Hz, CH), 7.11–7.35 ppm (10H, m, ArH); ¹³C NMR (75 MHz,

CD₃OD): d=55.5 (SO₂CH₂CH₂), 59.8 (SO₂CH₂CH₂), 65.7 (CH, d,

1

EtOAc/CHCl₃(1:2), R_f=0.66; yield: 0.09 g, 42%; H NMR (400 MHz,

CDCl₃): d=0.93 (3H, t, J=7.2 Hz, CH₃), 1.22–1.51 (22H, m, cyclodo-

decane), 1.54–1.71 (4H, m, NHCH₂CH₂CH₂CH₃), 2.56 (1H, brs, OH),

3.31–3.41 (2H, m, NHCH₂), 3.48 (3H, t, J=5.2 Hz, SO₂CH₂CH₂), 3.84

(1H, brs, NHCH), 4.06 (3H, t, J=5.2 Hz, SO₂CH₂CH₂), 5.58 (2H, brs,

SO₂NH₂), 6.12 (1H, brs, NH), 6.35 ppm (1H, brs, NH); ¹³C NMR

(100 MHz, CDCl₃): d=13.7 (CH₃), 20.0 (CH₃CH₂), 20.9 (cyclodode-

cane), 23.0 (cyclododecane), 23.1 (cyclododecane), 24.2

(SO₂CH₂CH₂), 24.4 (cyclododecane), 30.6 (cyclododecane), 32.8

J

₁₃_C=12.5 Hz), 76.3 (CH), 116.8 (C4, dd, ¹J ₁₃_C=13 Hz, ²J

=

¹⁹FÀ¹³C

¹⁹FÀ

5.4 Hz), 126.8 (Ar), 127.3 (Ar), 127.4 (Ar), 127.7 (Ar), 127.8 (Ar), 128.5

(Ar), 135.0 (C3, d, J ₁₃_C=14 Hz), 137.7 (C6, d, J ₁₃_C=250 Hz),

¹⁹FÀ

139.1 (Ar), 141.7 (Ar), 144.8 (C2, d, J ₁₃_C=257 Hz), 146.1 ppm (C5,

¹⁹FÀ

d, J ₁₃_C=247 Hz); ¹⁹F NMR (282 MHz, CD₃OD): d=À123.9 (C2-F, s),

¹⁹FÀ

1

2

1

À136.4 (C6-F, dd, J=25 Hz, J=12 Hz), À152.3 ppm (C5-F, dd, J=

24 Hz, J=6 Hz); HRMS for C₂₂H₂₁F₃N₂O₆S₂[M+H]⁺: calcd 531.0866,

2

found 531.0865.

(NHCH₂CH₂, d, J ₁₃_C=3.2 Hz), 47.1 (NHCH₂, d, J ₁₃_C=12.7 Hz), 53.5

¹⁹FÀ

(CH of cyclododecane, d, J ₁₃_C=12.5 Hz), 56.2 (SO₂CH₂CH₂), 57.0

¹⁹FÀ

(SO₂CH₂CH₂), 111.2 (C4, t, J ₁₃_C=5 Hz), 126.3 (C1, t, J ₁₃_C=16 Hz),

3,5-Bis(cyclooctylamino)-2,6-difluoro-4-[(2-phenylethyl)sulfonyl]-

benzenesulfonamide (11h). A mixture of 9 (0.20 g, 0.50 mmol),

Et₃N (0.142 mL, 1.02 mmol), DMSO (1 mL) cyclooctylamine

(0.142 mL, 1.02 mmol) was stirred at 608C for 32 h. The mixture

was then diluted with H₂O (20 mL) and extracted with EtOAc (3

10 mL). The combined organic phase was dried over MgSO₄and

evaporated under reduced pressure. The product was purified by

chromatography on a column of silica gel with EtOAc (10%)/CHCl₃,

R_f=0.72; yield: 0.15 g, 48%; ¹H NMR (300 MHz, CDCl₃): d=1.43–

1.98 (28H, m, cyclooctane), 3.05–3.15 (2H, m, SO₂CH₂CH₂), 3.51–

3.62 (2H, m, SO₂CH₂CH₂), 3.88 (2H, brs, 2CH of cyclooctane), 5.58

(2H, s, SO₂NH₂), 6.43 (2H, brs, 2NH), 7.14–7.38 ppm (5H, m, ArH);

¹³C NMR (75 MHz, CDCl₃): d=23.8 (cyclooctane), 25.8 (cyclooctane),

27.4 (cyclooctane), 28.5 (SO₂CH₂CH₂), 33.5 (cyclooctane), 55.9

(SO₂CH₂CH₂), 56.2 (CH of cyclooctane, t, J=6 Hz), 111.1 (C4, t,

¹⁹FÀ

135.5 (C3, dd, ¹J ₁₃_C=12.5 Hz, ²J ₁₃_C=3 Hz), 136.0 (C5, dd,

¹⁹FÀ

¹J ₁₃_C=12.7 Hz, ²J ₁₃_C=3 Hz), 139.3 ppm (C2, C6 dd, ¹J

=

¹⁹FÀ

¹⁹FÀ¹³C

243 Hz, ²J ₁₃_C=4.3 Hz); ¹⁹F NMR (376 MHz, CDCl₃): d=À156.44

¹⁹FÀ

(C2-F or C6-F, d, J=11 Hz), À158.1 ppm (C2-F or C6-F, d, J=

10.5 Hz); HRMS for C₂₄H₄₁F₂N₃O₅S₂[M+H]⁺: calcd 554.2528, found

554.2530.

3-(Cyclododecylamino)-2,6-difluoro-4-[(2-hydroxyethyl)sulfonyl]-

5-{[2-(4-hydroxyphenyl)ethyl]amino}benzenesulfonamide (13 f).

The product was purified by chromatography on a column of silica

gel with EtOAc/CHCl₃(1:2), R_f=0.25; yield: 0.05 g, 18%; mp: 158–

1

1598C; H NMR (400 MHz, [D₆]DMSO): d=1.22–1.73 (22H, m, cyclo-

dodecane), 2.75 (2H, brt, CH₂Ph), 3.44 (4H, br t, NHCH₂, SO₂CH₂),

3.73 (3H, brs, NHCH, SO₂CH₂CH₂), 5.02 (1H, t, J=5.4 Hz, OH), 5.94

(1H, brs, NH), 6.28 (1H, brs, NH), 6.70 (2H, d, J=7.6 Hz, ArH), 7.06

(2H, d, J=8.4 Hz, ArH), 8.08 (2H, s, SO₂NH₂), 9.23 ppm (1H, s, OH);

¹³C NMR (100 MHz, [D₆]DMSO): d=21.2 (cyclododecane), 23.2 (cy-

clododecane), 23.3 (cyclododecane), 24.1 (cyclododecane), 24.3 (cy-

clododecane), 30.6 (cyclododecane), 35.9 (CH₂Ph), 49.2 (NHCH₂,

J

₁₃_C=5 Hz), 126.3 (C1, t, J ₁₃_C=16 Hz), 127.4 (Ar), 128.6 (Ar),

¹⁹FÀ

1

2

129.2 (Ar), 135.3 (C3, dd, J ₁₃_C=10 Hz, J ₁₃_C=6 Hz), 137.3 (Ar),

139.4 ppm (C2, dd, ¹J ₁₃_C=244 Hz, ²J ₁₃_C=4.5 Hz); ¹⁹F NMR

¹⁹FÀ

(282 MHz, CDCl₃): d=À144.1 ppm (2F, s); HRMS for C₃₀H₄₃F₂N₃O₄S₂

[M+H]⁺: calcd 612.2736, found 612.2729.

J

₁₃_C=11.4 Hz), 53.0 (NHCH, J ₁₃_C=11.9 Hz), 55.2 (SO₂CH₂), 57.7

¹⁹FÀ ¹⁹FÀ

(SO₂CH₂CH₂), 113.9 (C4), 115.7 (Ar), 127.7 (C1, t, J ₁₃_C=16 Hz),

¹⁹FÀ

129.3 (Ar), 130.1 (Ar), 135.3 (C3, dd, ¹J ₁₃_C=12.2 Hz, ²J

=

3,5-Bis[(3,4-dimethoxybenzyl)amino]-2,6-difluoro-4-[(2-hydroxy-

¹⁹FÀ

¹⁹FÀ¹³C

1

2

ethyl)sulfonyl]benzenesulfonamide (12k).

A mixture of 10

3 Hz),135.5 (C5, dd, J ₁₃_C=12.9 Hz, J ₁₃_C=3 Hz), 140.3 (C2, C6 d,

¹⁹FÀ

₁₃_C=243.4 Hz), 156.3 ppm (Ar); ¹⁹F NMR (376 MHz, [D₆]DMSO):

(0.20 g, 0.59 mmol), DMSO (1 mL) and 3,4-dimethoxybenzylamine

(0.359 mL, 2.38 mmol) was stirred at ambient temperature for five

days. The mixture was then diluted with H₂O (20 mL), the resultant

precipitate was filtered, washed with H₂O. Recrystallization was ac-

J

¹⁹FÀ

d=À135.3 (C2-F or C6-F, s), À136.9 ppm (C2-F or C6-F, s); HRMS

for C₂₈H₄₁F₂N₃O₆S₂[M+H]⁺: calcd 586.2757, found 586.2761.

1

complished from EtOH; yield: 0.20 g, 53%; mp: 99–1028C; H NMR

(300 MHz, [D₆]DMSO): d=3.41 (SO₂CH₂CH₂, signal overlaps with

signal of H₂O), 3.65 (2H, k, J=5.4 Hz, SO₂CH₂CH₂), 3.75 (12H, s,

4CH₃), 4.35 (4H, d, J=5.7 Hz, 2NHCH₂), 5.04 (1H, t, J=5.4 Hz, OH),

6.38 (2H, t, J=5.7 Hz, 2NH), 6.85–7.05 (6H, m, ArH), 8.15 ppm (2H,

s, SO₂NH₂); ¹³C NMR (75 MHz, [D₆]DMSO): d=51.2 (NHCH₂, t,

Protein preparation

The expression and purification of CA I,^[54]II,^[55]VII,^[56]XII,^[57]and

XIII^[56]has been described previously. A DNA oligomer encoding

full-length CA VA was amplified by PCR from the CA VA gene pur-

chased from the Deutsches Ressourcenzentrum für Genomfor-

schung (RZPD; Berlin, Germany), using forward primer with NdeI

recognition site: 5’-GGG CATATG TGT TCT CAG CGT TCC TG-3’ and

reverse primer with XhoI recognition site: 5’-CTA ATG CTCGAG GGA

CCT TGT GCC CTC-3’ (recognition sites in italics). The PCR product

was cloned into the bacterial expression vector pET21a (Novagen)

via NdeI and XhoI restriction sites fusing a 6His tag into the

C terminus of the protein.

J

₁₃_C=6 Hz), 55.5 (SO₂CH₂CH₂), 56.1 (CH₃), 56.2 (CH₃), 58.3

¹⁹FÀ

(SO₂CH₂CH₂), 112.4 (Ar), 116.0 (C4, t, J ₁₃_C=3 Hz), 120.8 (Ar), 127.9

¹⁹FÀ

(C1, t, J ₁₃_C=16 Hz), 132.3 (Ar), 135.7 (C3, dd, ¹J ₁₃_C=10 Hz,

¹⁹FÀ

1

2

²J ₁₃_C=6 Hz), 141.7 (C2, dd, J ₁₃_C=247 Hz, J ₁₃_C=4 Hz), 148.8

¹⁹FÀ

(Ar), 149.4 ppm (Ar); ¹⁹F NMR (282 MHz, [D₆]DMSO): d=

À133.47 ppm (2F, s); H,C HETCOR (300 MHz, [D₆]DMSO): d=51.2–

4.35 (NHCH₂), 56.2–3.75 (CH₃), 55.5–3.65 (SO₂CH₂CH₂), 58.3–

3.41 ppm (SO₂CH₂CH₂); HRMS for C₂₆H₃₁F₂N₃O₉S₂[M+H]⁺: calcd

632.1543, found 632.1548.

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Expression of recombinant CA VA was done in Escherichia coli

BL21(DE3) Codon Plus-RIL strain (Stratagene). Transformed cells

were transferred to LB medium containing 100 mgmL^À1ampicillin,

34 mgmL^À1chloramphenicol and grown at 378C and 220 rpm for

16 h. Then the saturated culture was diluted (1:50) in fresh LB

medium containing 100 mgmL^À1ampicillin, 34 mgmL^À1chloram-

phenicol, 0.05 mm ZnSO₄and grown to OD₆₀₀ꢁ0.8. The expression

of recombinant CA VA was induced with 1 mm isopropyl-b-d-thio-

galactoside (IPTG) and 0.5 mm ZnSO₄. The culture was grown for

4 h at 308C and 250 rpm. The cells were harvested by centrifuga-

tion at 4000 g for 20 min at 48C.

integrated data for each ITC run is provided in the Supporting In-

formation in the ITC Origin Final Figure format.

CO₂hydration assay: The catalytic activity of the recombinant

human CA II catalyzing carbon dioxide hydration was measured

using an Applied Photophysics SX.18MV-R stopped-flow spectrom-

eter as previously described.^[47,58]The reaction of CO₂hydration

leads to a decrease in pH of the medium due to the release of bi-

carbonate and protons. The acidification velocities were measured

by recording the drop in absorbance of bromothymol blue indica-

tor (40 mm, 615 nm). The sample consisted of 20 nm CA II, 0.0–

0.6 mm 10h or 0–10 mm 2h inhibitor (in <0.1% DMSO) and 10 mm

HEPES buffer (pH 7.4) containing 10 mm NaCl. Saturated CO₂solu-

tion was prepared by bubbling CO₂gas in Milli-Q water at 258C for

1 h. The experiments were repeated twice.

The pellet was suspended in lysis buffer (25 mm Tris, 1% Triton X-

100, 0.1m NaCl, 2 mm 2-mercaptoethanol, 0.1m imidazole (pH 7.5)

and 1 mm PMSF) containing protease inhibitor cocktail (Roche Ap-

plied Science, Indianapolis, IN, USA). The cells were incubated at

48C for 60 min and then disrupted by sonication. The supernatant,

containing soluble proteins, was obtained after centrifugation at

20000 g for 25 min. Soluble protein was purified using a Sephar-

ose-IDA-Ni⁺²affinity column (GE Healthcare Bio-Sciences AB, Up-

psala, Sweden). Eluted protein was dialyzed against storage buffer

(25 mm Tris, 0.1m NaCl, 1 mm DTT, 10% glycerol pH 7.5) and

stored at À808C.

The purity of CA VA preparations was analyzed by sodium dodecyl

sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein con-

centrations were determined by UV/Vis spectrophotometry using

an extinction coefficient e₂₈₀=71515m^À1cm^À1and confirmed by

the standard Bradford method.

Crystallization and structure determination

CAs in 20 mm Na-HEPES pH 7.5 and 50 mm NaCl were concentrat-

ed by ultrafiltration to a concentration of 20–60 mgmL^À1. Crystalli-

zation in sitting drop was started by mixing equal volumes (0.5–

2 mL) of protein solution with reservoir buffers. Crystallization buf-

fers are presented in SI Table S3. Complexes of ligands with CA iso-

forms were prepared by soaking of CA crystal with 0.5 mm solution

of ligand prepared by mixing of 50 mm stock solution of ligand in

DMSO with 50 mL of corresponding reservoir solution. Soaked crys-

tals were measured after several days.

Crystal diffraction data were collected at EMBL beam lines on DESY

synchrotron on the PETRA III and DORIS storage ring (Hamburg,

Germany) and on X-ray diffractometer MicroMax 007-HF (Rigaku,

Japan) at the Institute of Biotechnology (Vilnius, Lithuania), as

listed in Table 5. Data collection statistics and refinement details

are presented in Table 5.

Determination of compound binding to CAs

Fluorescent thermal shift assay: Fluorescent thermal shift assay

(also termed ThermoFluor and differential scanning fluorimetry) ex-

periments were performed in a Corbett Rotor-Gene 6000 (Qiagen

Rotor-Gene Q) instrument using the blue channel (excitation

l 365Æ20, detection l 460Æ15 nm). Protein samples containing

various concentrations of inhibitor were heated from 25 to 998C

while recording extrinsic fluorescence and determining the protein

melting temperatures at each inhibitor concentration. Data analysis

was performed as previously described.^[54]Samples contained

10 mm protein, 0–200 mm ligand, 50 mm solvatochromic dye 8-anili-

no-1-naphthalene sulfonate (ANS) and 50 mm sodium phosphate

buffer containing 100 mm NaCl at pH 7.0, with a final DMSO con-

Datasets for all CAs with 10p and CA XIII with 9d were processed

using XDS,^[59]datasets of CA XII and CA II with 9d and 10d as well

CA XIII–10d were processed by MOSFLM.^[60]All structures were

solved by molecular replacement using MOLREP.^[61]Initial phases

for protein structures were obtained using the following PDB en-

tries: 3HLJ for CA II, 1JD0 for CA XII, and 2NNO for CA XIII. A single

protein chain stripped of all ligands was used as initial model in all

molecular replacement procedures. Inhibitor 3D models were cre-

ated using AVOGADRO,^[62]and molecule geometry description was

generated using LIBREFMAC.^[63]Protein models were refined and

manually remodeled using REFMAC^[64]and COOT.^[65]All graphic rep-

resentations were made with MOLSCRIPT,^[66]BOBSCRIPT,^[67]and

RASTER3D.^[68]Coordinates and structure factors were submitted to

the RCSB Protein Data Bank (PDB) with access codes listed in

Table 5.

centration of 2%. The applied heating rate was 18Cmin^À1

.

Isothermal titration calorimetry: ITC experiments were performed

using VP-ITC instruments (Microcal Inc., Northampton, USA) with

5–10 mm protein solution in the cell and 50–100 mm of the ligand

solution in the syringe. Syringe volume was 250 mL, cell volume

was 1.4315 mL. A typical experiment consisted of 25 injections (ini-

tial injection of 3 mL followed by 24 injections of 10 mL) within 3-

or 4-min intervals, a stirring rate of 260 rpm, and a reference

power of 4 mcals^À1. The pre-titration delay was set to 200–600 s.

Experiments were carried out at 378C in a 50 mm sodium phos-

phate or Tris chloride buffer containing 100 mm NaCl at pH 7.0,

with a final DMSO concentration of 1–2%. Protein stock solutions

were dialyzed against buffers that were used to prepare ligand sol-

utions. ITC data were analyzed using MicroCal Origin software. The

first point from the 3 mL injection in the integrated data graph was

deleted. The binding constants, enthalpies, and entropies of bind-

ing were estimated after fitting the data with the single binding

site model. The Wiseman c parameter for all ITC binding measure-

ments was in the range of 7–900. Experiments were repeated at

least twice (except for CA VI due to the lack of protein). Raw and

Acknowledgements

This research was funded by a grant from the Research Council

of Lithuania (No. MIP-067/2011). The authors acknowledge the

COST projects TD0905 and CM0804. X-ray diffraction data were

collected at EMBL/DESY, Hamburg (Germany), P13 and P14

beamline at the PETRA III storage ring and X11 at the DORIS stor-

age ring. Access to the measurement facilities was funded by the

FP7-REGPOT-2009-1 program (project 245721 MoBiLi). A.S. ac-

knowledges support by project “Promotion of Student Scientific

ˇ

Activities” (VP1-3.1-SMM-01V-02-003) from the Research Council

ChemMedChem 2015, 10, 662 – 687

www.chemmedchem.org

685

ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers

Table 5. X-ray crystallographic data collection and refinement statistics.^[a]

CA isoform–ligand: XII–10d

PDB ID: 4QJW

XII–9d

4QJO

XII–10p

4QJ0

II–10d

4QTL

II–9d

4QJM

II–10p

4QIY

XIII–10d

4QJX

XIII–9d

4QJP

XIII–10p

4QIZ

Data collection statistics

Space group

Unit-cell

parameters []

P1

P12₁1

P1

P4₃2₁2

P2₁2₁2₁

a=46.5

b=66.8

c=80.5

a=81.6

b=84.2

g=86.7

a=46.5

b=66.6

c=80.3

a=820

b=84.3

g=86.5

a=77.5

b=74.2

c=91.4

a=42.0

b=40.9

c=71.7

a=42.2

b=41.1

c=720

a=73.6

b=41.2

c=84.0

a=56.8

b=56.8

c=159.6

a=56.0

b=57.5

c=159.5

a=56.6

b=159.6

c=57.1

a=g=90.0 a=g=90.0 a=g=90.0 a=g=90.0 a=b=g=90.0 a=b=g=90.0 a=b=g=90.0

b=108.9 b=103.9 b=104.1 b=109.3

Resolution []

Wavelength []

Radiation source

41.66–1.55 23.59–1.80 86.50–1.55 16.16–1.80 20.62–1.75 79.32–1.30 56.82–1.95

159.52–1.620

0.83

EMBL/DESY

P13

57.10–1.55

0.83

EMBL/DESY

P13

0.82

1.54

0.83

EMBL/DESY Rigaku

MM007-HF P14

1.54

0.83

0.82

EMBL/DESY Rigaku

Rigaku

EMBL/DESY EMBL/DESY

X11

MM007-HF MM007-HF P13

X11

No. unique

reflections

R_mergeoverall

(outer shell)

I/s overall

133601

81977

141122

22117

23685

206964

19979

65652

75960

0.058

(0.319)

41.6

0.050

(0.153)

15.3

0.069

(0.371)

13.1

0.094

(0.380)

19.9

0.057

(0.177)

12.6

0.032

(0.286)

13.0

0.095

(0.537)

20.9

0.038

(0.141)

26.3

0.037

(0.405)

27.2

(outer shell)

Multiplicity overall 1.9

(1.7)

(4.0)

2.0

(4.7)

5.2

(5.6)

5.4

(5.1)

3.6

(3.4)

3.8

(5.8)

14

(8.1)

6.5

(4.4)

6.6

(outer shell)

Completeness [%]

overall (outer shell) (92.2)

(1.5)

96.8

(2.0)

93.3

(89.8)

(5.2)

99.4

(99.7)

(5.2)

99.7

(99.5)

(3.6)

97.4

(95.4)

(3.8)

90.0

(89.6)

(14.2)

100.0

(100.0)

(5.1)

98.5

(90.0)

(6.4)

99.9

(100.0)

Refinement statistics

R_work

R_free

RMSD bond

lengths []

RMSD bond

angles [8]

0.185

0.222

0.021

0.161

0.206

0.019

0.18

0.225

0.023

0.161

0.203

0.018

0.161

0.212

0.019

0.151

0.193

0.023

0.19

0.237

0.018

0.168

0.205

0.025

0.175

0.209

0.025

2.174

1.968

2.237

2.011

2.127

2.286

1.89

2.441

2.332

Average B factors [²]

all

main chain

side chain

inhibitors

waters

15.8

12.6

15.8

26.5

25.2

7.8

19.0

16.2

19.3

43.3

27.6

10.6

34.7

19.0

16.2

19.5

22.4

27.3

10.2

33.8

17.3

14.6

17.4

35.7

27.8

7.4

17.0

14.0

16.8

23.3

27.1

8.7

16.6

13.4

17.2

19.2

26.7

8.0

20.6

18.1

20.9

28.4

28.0

12.9

40.9

21.0

17.9

21.5

34.8

29.6

11.8

39.0

19.5

16.6

20.1

25.7

28.6

10.9

31.4

zinc

other molecules

28.9

35.7

33.5

36.0

Number of atoms

all

protein

inhibitor

water

zinc

9883

8510

135

1206

4

9590

8628

128

818

4

9838

8593

175

1042

4

2376

2126

27

211

1

2453

2111

32

286

1

9716

8499

140

1013

4

2410

2118

27

253

1

4818

4233

64

483

2

4865

4297

70

484

2

other molecules

28

12

24

11

23

60

11

36

12

Ramachandran statistics [%]

most favored

regions

additionally

allowed regions

outliers

97.8

2.2

0

97.1

2.9

0

97.1

2.9

0

96.8

3.2

0

96.6

3.4

0

97.0

3.0

0

97.3

2.7

0

96.2

3.8

1

96.8

3.2

0

[a] All datasets were collected at 100 K; test set size was 10%.

of Lithuania. This project was funded by the Republic of Lithua-

nia and the European Social Fund under the 2007–2013 Human

Resources Development Operational Programme’s priority 3. The

authors thank local contacts at the EMBL beamlines: Dr. Gleb

Bourenkov and Dr. Michele Cianci for assistance in beamline op-

eration. The authors thank Prof. Claudiu Supuran and Daniella

Vullo for instructions on performing the stopped-flow CA inhibi-

ˇ

˙

tion assay. The authors thank Zita Liutkeviciute and Vytautas

¯

Smirnovas (Vilnius University) for help with mass spectrometry

analysis of the synthesized compounds and recombinant pro-

teins.

ChemMedChem 2015, 10, 662 – 687

www.chemmedchem.org

686

ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Papers

[36] G. M. Brooke, J. Fluorine Chem. 1997 , 86 , 1 –7 6.

Keywords: carbonic anhydrases · fluorescent thermal shift

assay · fluorinated benzenesulfonamides · isothermal titration

calorimetry · thermofluor · X-ray crystallography

[37] T. N. Gerasimova, N. A. Orlova, J. Fluorine Chem. 1985 , 28 , 36 1 –3 80.

[38] L. S. Kobrina, G. G. Furin, G. G. Jakobson, Zh. Org. Khim. 1970, 6, 512–

520.

[39] J. G. Allen, J. Burdon, J. C. Tatlow, J. Chem. Soc. 1965 , 632 9 –6 336.

[40] J. Burdon, W. B. Hollyhead, J. C. Tatlow, J. Chem. Soc. 1965 , 515 2 –5 156.

[41] D. G. Holland, G. J. Moore, C. Tamborski, J. Org. Chem. 1964 , 29 , 156 2 –

1565.

[42] J. Zienkiewicz, A. Fryszkowska, K. Zienkiewicz, F. Guo, P. Kaszynski, A. Ja-

nuszko, D. Jones, J. Org. Chem. 2007 , 72 , 351 0 –3 520.

[43] R. J. Holland, I. R. Hardcastle, A. G. Dick, B. P. Nutley, A. Hayes, M.

Jarman, Tetrahedron Lett. 2002 , 43 , 71 9 –7 21.

[44] A. M. Barber, I. R. Hardcastle, M. G. Rowlands, B. P. Nutley, J. H. Marriott,

M. Jarman, Bioorg. Med. Chem. Lett. 1999 , 9 , 62 3 –6 26.

[45] J. H. Marriott, G. W. Aherne, A. Hardcastle, M. Jarman, Nucleosides Nucle-

otides Nucleic Acids 2001 , 20 , 169 1 –1 704.

[1] C. T. Supuran, Nat. Rev. Drug Discovery 2008 , 7 , 16 8 –1 81.

[2] C. T. Supuran, Curr. Pharm. Des. 2008, 14, 603–614.

[3] M. Aggarwal, B. Kondeti, R. McKenna, Bioorg. Med. Chem. 2013 , 21 ,

152 6 –1 533.

[4] V. Alterio, A. D. Fiore, K. D’Ambrosio, C. T. Supuran, G. D. Simone, Chem.

Rev. 2012 , 112 , 442 1 –4 468.

[5] K. M. Gilmour, Comp. Biochem. Physiol. Part A 2010 , 157 , 19 3 –1 97.

[6] C. T. Supuran, Curr. Top. Med. Chem. 2007, 7, 825–833.

[7] M. Aggarwal, C. D. Boone, B. Kondeti, R. McKenna, J. Enzyme Inhib. Med.

Chem. 2013 , 28 , 26 7 –2 77.

[8] K. K. Sethi, D. Vullo, S. M. Verma, M. TanÅ, F. Carta, C. T. Supuran, Bioorg.

Med. Chem. 2013 , 21 , 597 3 –5 982.

[9] M. Aggarwal, R. McKenna, Expert Opin. Ther. Pat. 2012 , 22 , 90 3 –9 15.

[10] R. G. Gieling, K. J. Williams, Bioorg. Med. Chem. 2013 , 21 , 147 0 –1 476.

[11] G. M. Breinin, H. Gortz, AMA Arch. Ophthalmol. 1954 , 52 , 33 3 –3 48.

[12] C. T. Supuran, Future Med. Chem. 2011 , 3 , 116 5 –1 180.

[13] V. M. Krishnamurthy, B. R. Bohall, C.-Y. Kim, D. T. Moustakas, D. W. Chris-

tianson, G. M. Whitesides, Chem. Asian J. 2007 , 2 , 9 4 –1 05.

[14] V. M. Krishnamurthy, G. K. Kaufman, A. R. Urbach, I. Gitlin, K. L. Gudiksen,

D. B. Weibel, G. M. Whitesides, Chem. Rev. 2008 , 108 , 94 6 –1 051.

[15] C. T. Supuran, A. Scozzafava, J. Conway, Carbonic Anhydrase: Its Inhibi-

tors and Activators, CRC Press, Boca Raton, 2004, p. 363.

[16] C. T. Supuran, J. Y. Winum, Drug Design of Zinc-Enzyme Inhibitors: Func-

tional, Structural, and Disease Applications, Wiley, Hoboken, 2009,

p. 912.

[46] J. H. Marriott, A. M. Moreno Barber, I. R. Hardcastle, M. G. Rowlands,

R. M. Grimshaw, S. Neidle, M. Jarman, J. Chem. Soc. Perkin Trans. 1 2000 ,

426 5 –4 278.

˙

[47] V. Dudutiene, A. Zubriene, A. Smirnov, J. Gylyte, D. Timm, E. Manakova,

ˇ

S. Grazulis, D. Matulis, Bioorg. Med. Chem. 2013, 21, 2093–2106.

[48] J. F. Bunnett, R. J. Morath, J. Am. Chem. Soc. 1955 , 77 , 505 1 –5 055.

[49] M. F. Hawthorne, J. Am. Chem. Soc. 1954 , 76 , 635 8 –6 360.

[50] J. F. Bunnett, R. J. Morath, T. Okamoto, J. Am. Chem. Soc. 1955 , 77 ,

505 5 –5 057.

[51] M. D. Wendt, A. R. Kunzer, Tetrahedron Lett. 2010 , 51 , 64 1 –6 44.

˙

[52] V. Dudutiene, J. Matuliene, A. Smirnov, D. D. Timm, A. Zubriene, L. Bara-

nauskiene˙, V. Morkunaite˙, J. Smirnoviene˙, V. Michailoviene˙, V. Juozapai-

¯

˙

ˇ

˙

ˇ

˙

tiene, A. Mickeviciute, J. Kazokaite, S. Baksyte, A. Kasiliauskaite, J.

¯

˙

ˇ

˙

Jachno, J. Revuckiene, M. Kisonaite, V. Pilipuityte, E. Ivanauskaite, G. Mili-

ˇ

˙

ˇ

naviciu¯te, V. Smirnovas, V. Petrikaite, V. Kairys, V. Petrauskas, P. Norvaisas,

[17] S. Biswas, M. Aggarwal, Ö. Güzel, A. Scozzafava, R. McKenna, C. T. Supur-

an, Bioorg. Med. Chem. 2011 , 19 , 373 2 –3 738.

ˇ

˙

ˇ

˙

ˇ

D. Linge, P. Gibieza, E. Capkauskaite, A. Zaksauskas, E. Kazlauskas, E.

Manakova, S. Grazˇulis, J. E. Ladbury, D. Matulis, J. Med. Chem. 2014, 57,

9435–9446.

[18] A. Cecchi, L. Ciani, J.-Y. Winum, J.-L. Montero, A. Scozzafava, S. Ristori,

C. T. Supuran, Bioorg. Med. Chem. Lett. 2008 , 18 , 347 5 –3 480.

[19] A. Cecchi, A. Hulikova, J. Pastorek, S. Pastorekova, A. Scozzafava, J. Y.

Winum, J. L. Montero, C. T. Supuran, J. Med. Chem. 2005 , 48 , 483 4 –

4841.

[53] B.-B. Gao, A. Clermont, S. Rook, S. J. Fonda, V. J. Srinivasan, M. Wojtkow-

ski, J. G. Fujimoto, R. L. Avery, P. G. Arrigg, S.-E. Bursell, L. P. Aiello, E. P.

Feener, Nat. Med. 2007 , 13 , 18 1 –1 88.

˙

[54] L. Baranauskiene, M. Hilvo, J. Matuliene, D. Golovenko, E. Manakova, V.

[20] L. B. Dugad, J. T. Gerig, Biochemistry 1988 , 27 , 431 0 –4 316.

[21] M. A. Ilies, B. Masereel, S. Rolin, A. Scozzafava, G. Campeanu, V. Cimpea-

nu, C. T. Supuran, Bioorg. Med. Chem. 2004 , 12 , 271 7 –2 726.

[22] M. A. Ilies, D. Vullo, J. Pastorek, A. Scozzafava, M. Ilies, M. T. Caproiu, S.

Pastorekova, C. T. Supuran, J. Med. Chem. 2003 , 46 , 218 7 –2 196.

[23] A. D. Scott, C. Phillips, A. Alex, M. Flocco, A. Bent, A. Randall, R. O’Brien,

L. Damian, L. H. Jones, ChemMedChem 2009 , 4 , 198 5 –1 989.

[24] A. Scozzafava, C. T. Supuran, Bioorg. Med. Chem. Lett. 2002 , 12 , 155 1 –

1557.

Dudutiene˙, V. Michailoviene˙, J. Torresan, J. Jachno, S. Parkkila, A. Mares-

ˇ

ca, C. T. Supuran, S. Grazulis, D. Matulis, J. Enzyme Inhib. Med. Chem.

2010, 25, 863–870.

ˇ

˙

[55] P. Cimmperman, L. Baranauskiene˙, S. Jachimoviciute, J. Jachno, J. Torre-

¯

˙

san, V. Michailoviene, J. Matuliene, J. Sereikaite, V. Bumelis, D. Matulis,

Biophys. J. 2008 , 95 , 322 2 –3 231.

ˇ

˙

[56] J. Su¯dzius, L. Baranauskiene, D. Golovenko, J. Matuliene, V. Michailo-

˙

ˇ

viene, J. Torresan, J. Jachno, R. Sukackaite, E. Manakova, S. Grazulis, S.

ˇ

Tumkevicius, D. Matulis, Bioorg. Med. Chem. 2010, 18, 7413–7421.

[25] K. L. Shepard, S. L. Graham, R. J. Hudcosky, S. R. Michelson, T. H. Scholz,

H. Schwam, A. M. Smith, J. M. Sondey, K. M. Strohmaier, R. L. Smith, M. F.

Sugrue, J. Med. Chem. 1991 , 34 , 309 8 –3 105.

˙

[57] V. Jogaite, A. Zubriene, V. Michailoviene, J. Gylyte, V. Morku¯naite, D. Ma-

tulis, Bioorg. Med. Chem. 2013, 21, 1431–1436.

[58] R. G. Khalifah, J. Biol. Chem. 1971, 246, 2561–2573.

[59] W. Kabsch, Acta Crystallogr. Sect. D 2010 , 66 , 12 5 –1 32.

[60] A. G. W. Leslie, Acta Crystallogr. Sect. D 2006 , 62 , 4 8 –5 7.

[61] A. Vagin, A. Teplyakov, J. Appl. Crystallogr. 1997 , 30 , 102 2 –1 025.

[62] Avogadro: an open-source molecular builder and visualization tool, ver-

sion1.0.0, 2009, http://avogadro.openmolecules.net (accessed February

20, 2015).

[63] A. A. Vagin, R. A. Steiner, A. A. Lebedev, L. Potterton, S. McNicholas, F.

Long, G. N. Murshudov, Acta Crystallogr. Sect. D 2004 , 60 , 218 4 –2 195.

[64] G. N. Murshudov, A. A. Vagin, E. J. Dodson, Acta Crystallogr. Sect. D

1997 , 53 , 24 0 –2 55.

[26] W. Vernier, W. Chong, D. Rewolinski, S. Greasley, T. Pauly, M. Shaw, D.

Dinh, R. A. Ferre, S. Nukui, M. Ornelas, E. Reyner, Bioorg. Med. Chem.

2010 , 18 , 330 7 –3 319.

[27] J. Y. Winum, A. Thiry, K. E. Cheikh, J. M. Dogne, J. L. Montero, D. Vullo, A.

Scozzafava, B. Masereel, C. T. Supuran, Bioorg. Med. Chem. Lett. 2007 , 17 ,

268 5 –2 691.

[28] J.-Y. Winum, J.-M. DognØ, A. Casini, X. de Leval, J.-L. Montero, A. Scozza-

fava, D. Vullo, A. Innocenti, C. T. Supuran, J. Med. Chem. 2005 , 48 , 212 1 –

2125.

[29] L. B. Dugad, C. R. Cooley, J. T. Gerig, Biochemistry 1989 , 28 , 395 5 –3 960.

[30] J. Olander, S. Bosen, E. T. Kaiser, J. Am. Chem. Soc. 1973 , 95 , 161 6 –1 621.

[31] D. L. Veenstra, J. T. Gerig, Magn. Reson. Chem. 1998 , 36 , S16 9 –S 178.

[32] W. K. Mung Chong, S. Nukui, L. Li, E. Y. Rui, W. F. Vernier, J. Z. Zhou, J.

Zhu, A. M. Haidle, M. A. Ornelas, M. Teng, (Pfizer Products Inc., Groton,

USA), Int. PCT Pat. No. WO 2008017932, 2008.

[65] P. Emsley, K. Cowtan, Acta Crystallogr. Sect. D 2004 , 60 , 212 6 –2 132.

[66] P. J. Kraulis, J. Appl. Crystallogr. 1991 , 24 , 94 6 –9 50.

[67] R. M. Esnouf, Acta Crystallogr. Sect. D 1999 , 55 , 93 8 –9 40.

[68] E. A. Merritt, D. J. Bacon, Methods Enzymol. 1997 , 277 , 50 5 –5 24.

[33] P. Kirsch, Modern Fluoroorganic Chemistry: Synthesis Reactivity, Applica-

tions, Wiley-VCH, Weinheim 2004, p. 308.

[34] S. Niembro, A. Vallribera, M. Moreno-Manas, New J. Chem. 2008 , 32 , 9 4 –

98.

[35] I. Ojima, Fluorine in Medicinal Chemistry and Chemical Biology, Wiley-

Blackwell, 2009.

Received: November 14, 2014

Revised: February 3, 2015

Published online on March 10, 2015

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Article Doi

Functionalization of fluorinated benzenesulfonamides and their inhibitory properties toward carbonic anhydrases

DOI: 10.1002/cmdc.201402490

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