Sulfonamides incorporating piperazine bioisosteres as potent human carbonic anhydrase I, II, IV and IX inhibitors

Sulfonamides incorporating piperazine bioisosteres as potent human Carbonic Anhydrase I,

II, IV and IX inhibitors

a

b

a

Niccolò Chiaramonte, Silvia Bua, Andrea Angeli, Marta Ferraroni, Ilaria Picchioni, Gianluca

a

a*

Bartolucci, Laura Braconi, Silvia Dei, Elisabetta Teodori, Claudiu T. Supuran, Maria Novella

Romanelli^a^*

a

University of Florence, Department of Neuroscience, Psychology, Drug Research and Child’s

Health, Section of Pharmaceutical and Nutraceutical Sciences, Via Ugo Schiff 6, 50019, Sesto

Fiorentino (Italy)

b

University of Florence, Department of Chemistry, via della Lastruccia, 50019 Sesto Fiorentino

(Italy)

*

Corresponding authors:

Maria Novella Romanelli: phone 0039 055 4573691, e-mail: novella.romanelli@unifi.it;

Claudiu T Supuran: phone 0039 055 4573729, e-mail: claudiu.supuran@unifi.it

Keywords

Carbonic anhydrase, sulfonamides, piperazines, piperazine bioisosteres, carbonic anhydrase

inhibitors, isoform selectivity.

Abstract

Starting

from

the

molecular

simplification

of

(R)

4-(3,4-dibenzylpiperazine-1-

carbonyl)benzenesulfonamide 9a, a compound endowed with selectivity for human Carbonic

Anhydrase (hCA) IV, a series of piperazines and 4-aminopiperidines carrying a 4-

sulfamoylbenzamide moiety as Zn-binding group have been designed and tested on human isoforms

hCA I, II, IV and IX, using a stopped flow CO hydrase assay. The aim of the work was to derive

2

structure-activity relationships useful for designing isoform selective compounds. These structural

modifications changed the selectivity profile of the analogues from hCA IV to hCA I and II, and

improved potency. Several of the new compounds showed subnanomolar activity on hCA II. X-ray

crystallography of ligand-hCAII complexes was used to compare the binding modes of the new

piperazines and the previously synthesized 2-benzyl-piperazine analogues, explaining the inhibition

profiles.

1

. Introduction

Carbonic Anhydrase (CA) is a metallo-enzyme widely expressed in nature. Eight genetically-

different families (α-ι) [1] have been found up until now. In humans, sixteen different isoforms

belonging to the α family have been characterized, differing for cellular and tissue distribution, and

catalytic activity. The reversible hydration of CO is the most important reaction catalysed by these

2

enzymes, affecting several important physiological processes such as pH and electrolyte balance. An

abnormal activity of these enzymes often leads to pathological effects [2]. CA inhibitors (CAI) are

used in the clinic for more than 70 years mainly as diuretics, antiglaucoma and antiepileptic agents,

although their use may be limited by side effects due to poor isoform selectivity [3]. Some examples

of clinically relevant CAI are reported in Figure 1. However, recent findings suggest also other

important uses, such as in renal and central nervous system diseases [4], pain [5], and cancer [6]; for

the latter use, it is worth mentioning compound SLC-0111 which is under development as an

anticancer agent in hypoxic tumors [7]. The wide number of possible therapeutic applications fosters

the research for new potent and selective CAI.

NH

S

CH₃

O

N N

S

N

O

H C

N

H

SO2NH2

3

S

SO NH

2

MeO

O

1

(Acetazolamide)

2

(Brinzolamide)

O

N

NH

SO NH

O

2

SO NH

2

F

2

3

(Zonisamide)

4 (SLC-0111)

Figure 1. Structure of some CAIs used in the clinic or under clinical development.

The catalytic site of α-CA contains a Zn(II) ion, coordinated to three histidine residues and to the

reactive water molecule/hydroxide ion. Inhibitors can block enzymatic activity with different

mechanisms [8]. A primary sulfonamide on an aryl moiety is the metal ion-chelator group most

studied in the field of CAI, since it potently and irreversibly displaces the active water molecule. The

highly conserved amino-acid sequence of the region surrounding the zinc ion does not allow a

selective interaction of the sulfonamide group; therefore, isoform selectivity can be achieved by

adding structural tails that interact with neighbouring less conserved regions [3].

2

R₁

R

N

R

N

R

N

SO NH₂

SO2NH2

NH

2

N

O

N

SO NH

2

HN

O

N

H

N

O

SO NH₂

2

5

6

7

8: R = H (a), F (b)

1

Figure 2. Structure of some piperazine CAIs reported in the literature. R substituents in compounds

-7 are mainly aryl or arylmethyl groups

5

The piperazine ring is widely used as a scaffold in medicinal chemistry, and it has been used also in

the field of CAI [9-11]. The piperazine moiety has been often decorated with aryl or benzyl

substituents on one N atom, while the other N atom has been linked to a benzenesulfonamide moiety

directly or through a spacer of variable length and flexibility; some examples (compounds 5-7) are

shown in Figure 2 [12-15]. The activity of compound 8a and 8b as CAI was reported in the literature

while the present work was in progress [16].

R₁

ZBG

R

N

Ph

R

N

R₁

N

NR R

1

2

(

CH )n

(CH )n

2

N

ZBG

ZBG Ph

ZBG

Bn

ZBG

9

10

11

12

13

R = H, CH benzyl, acyl,

sulphonyl

n = 0-2;

n = 0,1;

R = H, CH

R R = H,

1, 2

3,

R = CH C H X;

1,

3

CH Bn

3,

1

2

6

4

9

a, 10a: R = benzyl;

b: R = CH ; 10b: R = H 3-NH₂_,4-Cl, 4-NO₂_,

X = 3-OMe, 3-NO₂_,

3

4-NH₂

ZBG =

SO NH

2

O

Figure 3. Structure of the lead compounds and of the newly designed piperazines.

The activity of a series of 2- or 3-benzylpiperazines as CAI (general formulas 9 and 10, Figure 3) has

been recently reported by our group [17]. The compounds carried on one nitrogen atom a sulfamoyl

benzamide group as Zn binding group, while the other nitrogen atom was decorated with

alkyl/acyl/sulfonyl moieties. In general, all compounds were able to inhibit hCA I, II, IV and IX with

3

potency ranging from nanomolar to micromolar, hCA IV being the most sensitive and hCA IX the

least sensitive isoform. (R)-9a (Figure 3, R = Bn) resulted particularly interesting for its selectivity

ratios: this compound was found to be 165, 26, and 182 times more potent on hCA IV than on hCA

I, II and IX, respectively (Table 1). Both the absolute configuration and the position of the benzyl

group affected potency and selectivity: in fact, (S)-9a, the S-enantiomer, showed a slight preference

for hCA II over hCA I and hCA IV (3 and 9 times, respectively) while being 30 times less potent on

hCA IX. Shifting the position of the benzyl group on the piperazine ring, from C3 to C2, significantly

reduced potency and selectivity: as an example, the activity of (S)-10a (R = Bn) on CA IX was largely

improved.

With these premises, in order to expand structure-activity relationships of this class of compounds,

the effect of structural changes on the piperazine ring and of different aromatic substituents on activity

and selectivity was investigated, hoping to improve potency on CA IV. To this aim, some structural

analogues of compounds 9 and 10 (compounds 11-13, Figure 3) were prepared, characterized by the

presence of a piperazine or another 5-7-membered ring carrying two endocyclic nitrogen atoms, or

one endocyclic and the other one exocyclic. The activity and selectivity of the new compounds was

assessed on isoforms hCA I, II, IV and IX, and compared to the previously reported derivatives 9 and

1

0.

2

. Results and discussion

.1 Chemistry

Compounds 11a,b were synthesized as reported in Scheme 1. Reaction of commercially available N-

benzylhomopiperazine, or of N-benzylimidazolidine [18] with 4-sulfamoylbenzoyl chloride gave

compounds 11a,b in good yields; compound 8a [16] was prepared in the same way from N-

benzylpiperazine. Reaction of N-Boc-piperazine with the suitable benzyl bromide or chloride,

followed by deprotection with trifluoroacetic acid gave compounds 14a-e [19-23], which were treated

with 4-sulfamoylbenzoyl chloride to obtain compounds 8b [16], 11c-f. The nitro group of 11d and

1

1f were then reduced to NH using different reagents: in fact, the reaction with SnCl was successful

2 2

only for the 3-nitro derivative 11d, easily giving 11g. Catalytic hydrogenation of 11f gave the 4-NH₂

derivative 11h.

4

X

a

X

R₁

N

a

b, c

(

)n

( )n

n = 0,1

R = Benzyl

N

H

n = 1,

R = Boc

1

N

H

1

O

1

4a: X = 3-OMe

4b: X = 3-NO₂

4c: X = 4-Cl

SO NH

2 2

2

1

8

b: X = 4-F

8

a: n = 1

1a: n = 2

1b: n = 0

1

1c: X = 3-OMe

¹^d^:^X⁼³^-^N^O2

1e: X = 4-Cl

1f: X = 4-NO₂

1g: X = 3-NH₂

1h: X = 4-NH₂

4d: X = 4-F

4e: X = 4-NO₂

1

d

e

Scheme 1. Synthesis of compounds 8a,b and 11a-h. Reagent and conditions: a) 4-sulfamoylbenzoyl

chloride, Et N; b) substituted benzyl halide, Et N; c) TFA; d) SnCl ; e) H /Pd/C.

3

2

Compounds 12a-c (Scheme 2) were obtained by reacting 4-sulfamoylbenzoyl chloride or 2,5-

dioxopyrrolidin-1-yl 4-sulfamoylbenzoate [24] with commercially available 1-benzylpiperidin-4-

amine, 1-benzylpirrolidin-3-amine, or with 1-benzyl-N-methylpiperidin-4-amine, prepared according

to Pryde [25].

O

X

O

R₁

NH

N

SO NH

2

SO NH

2

(

)n

( )n

Ph

N

R = CH n = 1

a) or b)

12a: R = H, n = 1

1

3,

1

R = H, n = 1

12b: R = CH n = 1

1

3,

Ph

R = H, n = 0

1

12c: R = H, n = 0

1

O

a)12a: X = Cl, Et N; b) 12b, 12c: X =

N O

3

O

Scheme 2. Synthesis of compounds 12a-c.

Scheme 3 shows the synthesis of 4-aminopiperidines 13a,b. At first we thought to obtain the desired

compounds starting from commercially available 4-(Boc-amino)piperidine, which was reacted with

2

,5-dioxopyrrolidin-1-yl 4-sulfamoylbenzoate to give 15, then deprotected, obtaining compound 16

as trifluoroacetate salt. However, the reaction of 16 with benzyl bromide gave 13a in poor yield, and

for this reason a new strategy was applied. Commercially available 4-(amino)piperidine-1-Boc was

5

treated with benzaldehyde, reduced with NaBH , then protected as Cbz derivative; removal of Boc

4

gave compound 17 [19], which was transformed into 18 by reaction with 2,5-dioxopyrrolidin-1-yl 4-

sulfamoylbenzoate. Deprotection with HBr/AcOH gave 13a, which was transformed into 13b by

reaction with methyl iodide.

HN

N

Ph

NH

N

2.TFA

NHBoc

N

a

b

c

N

H

O

SO NH

2

SO NH

2

15

16

13a

f

g

Cbz

H C

3

NH₂

N

Ph

N

Ph

N

Ph

d,e,b

a

N

H

Boc

O

1

7

SO NH

2 2

2

18

13b

Scheme 3. Synthesis of compounds 13a,b. Reagents and conditions: a) 2,5-dioxopyrrolidin-1-yl 4-

sulfamoylbenzoate; b) TFA; c) PhCH Br, NaHCO ; d) PhCHO, NaBH : e) ClCOOBn, Et N; f)

2

3

4

3

HBr, AcOH; g) MeI, NaHCO₃.

1

13

All final compounds were characterized through H, C-NMR and HRMS spectra; representative

NMR spectra have been reported in Appendix A. As far as piperazines 11c-11h are concerned, in

1

13

both H and C-NMR spectra the presence of the amide group split the aliphatic protons in four

unresolved signals, centred around 2.30 and 2.50 ppm (CH NBn) and at 3.20 and 3.6 ppm (CH NCO).

2

1

3

In addition, the C-NMR spectra of these compounds showed four peaks for the four piperazine C

atoms, suggesting that the symmetry of the molecule has been disrupted by the presence of the amide

conformers. The presence of amide conformers was evident also in other compounds: for example,

1

3

in the C-NMR spectrum of 12b the peaks for the CH , CH and the benzylic CH groups were

3

2

1

doubled, and the piperidine CH moieties gave each a different signal. In the CDCl H-NMR

2

3

spectrum of 12b, two signals were found for the NMe group (a singlet at 2.75 ppm and another one

enclosed in a multiplet between 2.90 and 3.10 ppm), for the CH proton (two multiplets centred at.

6

5

.13 and 3.29), and for the benzylic CH (two singlets at 3.51 and 3.39 ppm). On the contrary, in the

2

1

H NMR spectrum (CD OD) of the isomer 13b the N-Me and the benzylic methylene groups gave

3

only the expected singlets at 2.22 and 3.63 ppm, respectively, while the piperidine protons gave each

a different signal.

1

Also the H NMR spectrum of 11b clearly showed a mixture of two conformers, as all the aliphatic

and the benzenesulfonamide protons gave two sets of similar peaks at different ppm and with half

1

3

integrals; in addition, the C-NMR spectrum showed two peaks for each C atom.

2

.2 CA inhibition study

The inhibitory activity of the new compounds on several hCA isoforms was assessed using a stopped

flow CO hydrase assay [26]. The ubiquitous cytosolic hCA I and hCA II, the membrane-anchored

2

hCA IV, and the transmembrane, tumor-associated hCA IX were chosen for biological testing.

Results are reported in Table 1; the standard sulfonamide inhibitor acetazolamide (AAZ) and both

enantiomers of the previously synthesized 9a,b and 10a,b were taken as reference compounds.

The activity of the unsubstituted piperazine 8a could help to understand the contribution of the C-

benzyl moiety of compounds 9 and 10; its biological activity, already reported for hCA I, II and IX

[16], was further measured on hCA IV. Indeed, 8a was almost equipotent with (R)-9a on hCA IV

(Table 1) but it lost the selectivity for this one with respect to the other tested isoforms. To check the

importance of ring size the piperazine moiety was expanded to diazepane (11a) or contracted to

imidazolidine (11b). These modifications brought a substantial reduction of potency on hCA IV

(about 12-15 times) and on CA IX (12-23 times). Activity on hCA I and II was not modified by ring

expansion while it was improved by ring contraction, since imidazolidine 11b was, respectively, 8

and 5 times more potent than piperazine 8a. Therefore, these modifications introduced in 11a and

1

1b some selectivity toward hCA I and II.

Then the attention was focused on the N-benzyl moiety. Compounds 11c-h, carrying different

substituents in position 3 and 4 on the aromatic ring, were prepared aiming to investigate how these

modifications could affect potency or selectivity. Compound 8b, whose activity on hCA I, II and IX

had been already reported [16], was tested also on hCA IV. On this isoform only 4-NO and 4-NH₂

2

groups were tolerated, since 11f and 11h were only twice less potent than 8a; all the other substituents

decreased activity (11-53 times). Aromatic substitution did not substantially modify activity on hCA

IX, with the exception of the 3-OMe group (11c), which was 23 times less active than 8a. On hCA I

only the 4-Cl moiety (11e, 8 times less active than 8a) and the 4-NO group (11f, 3 times more active

2

than 8a) modulated potency. On hCA II aromatic substitution slightly modulated activity (3-4 times

7

increase or decrease of Ki), with compounds 11c (3-OMe) and 11f (4-NO ) showing subnanomolar

2

Ki values, similarly to the already described 8b. Therefore, aromatic substitution, irrespective of the

position and electronic characteristics of the group, changed the selectivity profile of 8a, since

compounds 11c-h were more active on CA I and II with respect to IV and IX. Compound 11c,

carrying a 3-OMe group, showed the best selectivity ratios, being 8, 248 and 769 times more active

on hCA II than on hCA I, IV and IX, respectively.

8

Table 1. Inhibitory activity of compounds 11-13, 15 and 18 on human (h) CA isoforms I, II, IV and

IX. The standard sulfonamide inhibitor acetazolamide (AAZ), 9a,b and 10a,b were used as reference

compounds.^a

R₂

R₁

N

Bn

N

(CH₂)_n

R1

O

(CH2)n

A

N

C

B

O

SO NH₂

SO2NH2

SO NH

2

a

K (nM)

I

Cmp

Structure

R₁

R₂

n

1

hCA I

hCA II

hCA IV

hCA IX

R-9a^b

S-9a^b

R-10a^b

S-10a^b

380.2±12

22.1±1.2

60.7±4.1 2.3±0.12 418.5±32

8.3±0.37 73.3±3..7 249.2±15

455.7±24 176±15.4 66.9±2.8 2639±89

88.9±7.4 46.0±2.1 79.3±5.0 27.9±1.9

6.8±0.31^c3.0 ±0.14^c4.2±0.23 33.1±0.98^c

8

a^c

b^c

1a

1b

1c

1d

1e

1f

1g

1h

2a

2b

2c

3a

3b

A

B

C

-

Bn

-

8

4-F-Bn

Bn

-

1 0.69±0.02^c0.50±0.01^c68.4±3.0 45.1 ±2.8^c

1

2

0

1

0

-

7.7±0.40

0.8±0.06

7.2±0.35

6.4±0.27

2.3±0.11 52.9±2.9 407.6±33

0.6±0.02 63.9±5.1 750.7±62

0.9±0.04 224.9±12 761.4±48

9.5±0.44 48.7±3.5 40.4±2.0

1

Bn

1

3-OMe-Bn -

1

3-NO -Bn -

2

1

4-Cl-Bn

-

54.8±2.9 13.3±0.92 89.4±4.7 48.4±2.3

2.4±0.13 0.77±0.05 9.7±0.63 52.3±1.6

1

4-NO -Bn -

2

1

3-NH -Bn -

7.4±0.50

5.8±0.24

0.9±0.07

7.9±0.61

36.2±1.8

39.9±2.0

9.3±0.48

6.5±0.13

56.1±4.0

6.9±0.30 74.9±4.9 36.3±1.7

3.5±0.22 9.1±0.75 40.2±0.9

0.5±0.04 17.5±1.1 19.7±0.86

7.9±0.47 272.0±14 353.7±19

4.4±0.25 25.3±1.9 21.6±1.0

44.5±1.8 92.2±6.7 38.4±3.1

8.9±0.31 37.4±2.6 129.6±6.9

3.3±0.15 42.4±3.1 75.7±3.8

86.4±3.8 632.5±24 421.4±26

2

1

4-NH -Bn -

2

1

H

-

1

Me

H

1

H

Bn

1

Me

H

-

1

5

8

Boc -

1

Cbz

Bn

-

AAZ

250.0±13

12.0±0.8 74.0±3.5 25.0±1.7

a

b

c

Mean from 3 different assays, by a stopped flow technique. From ref [17]. From ref [16]

9

The effect of moving one nitrogen atom of 8a outside the ring was checked by testing 4-

aminopiperidine derivatives 12a-c and 13a,b. The shift of the sulfamoylbenzamide group outside the

six-membered ring gave a compound (12a) which showed subnanomolar potency on CA I and II and

2

-digit nanomolar activity on CA IV and IX. Its N-methyl derivative 12b showed a selectivity profile

similar to 12a but a decreased potency, particularly on hCA IV and IX; this effect may be due to the

removal of a H-bond donor group, or to steric hindrance. Decreasing ring size was not productive,

since 12c was less potent than 12a and showed only a slight preference for CA II (5-8 times) over the

other tested isoforms.

An exocyclic N-benzyl group was detrimental for activity on hCA I, II and IV; in fact, on these

isoforms the activity of 13a was decreased, respectively, 6, 15 and 22 times when compared to 8a,

resulting in a loss of selectivity. By adding a Me group of the NH moiety of 13a (compound 13b)

potency was restored on CA I and II. It seems that the addition of a NMe group, possibly by increasing

lipophilic interactions, improves activity on hCA I, II and IV, but not hCA IX. Synthetic intermediates

1

5 and 18 were also tested: the good activity of 15 suggests that a basic N atom in this part of the

molecule is not required, at least on hCA I and II, but a Cbz group may be too large thus producing

steric hindrance on all the four isoforms. The structure-activity relationships are summarized in

Figure 4.

Figure 4. Summary of the structure-activity relationships found in this study.

2

.3 X-ray crystallography

1

0

To study the differences in binding mode between the new derivatives and the previously synthesized

-benzylpiperazine analogues, compounds 11a, 11b, (R)-9b and (R)-10b were crystallized with hCA

II. Compounds (R)-9b and (R)-10b were chosen among those having higher potency on this isoform

Ki 5.4 nM and 8.1 nM, respectively [17]; see their structures in Figure 3). Crystal parameters and

2

(

reﬁnement data of the four complexes are summarized in Table 2.

Figure 5. Active site region of hCA II/(R)-10b (PDB: 6RG3) (A) and hCA II/(R)-9b (PDB: 6RG4)

(B) adducts. Inhibitors showed as σA-weighted |Fo−Fc| density map at 2.0 σ. Hydrogen bonds, van

der Waals interactions and water bridges are also shown (see text for explanation).

In all the four complexes, the sulfonamide moiety was directly bound to the zinc ion in the active-

site. Two additional hydrogen bond interactions, evidenced in Figures 5 and 6, between the

-

sulfonamide and Thr199 contributed to stabilize the complexes (NH with the OH in the side chain

and the sulfonamide oxygen with the peptide nitrogen). These interactions are typical of this zinc

binding group [27-29]. The binding of (R)-9b and (R)-10b was further stabilized through a water

molecule bridging the benzoate carbonyl group and Gln92; a second water bridge was observed

between the carbonyl moiety of (R)-10b and the NH group of Asn67. On the contrary, compound

2

(R)-9b showed an additional water molecule linking the hydroxyl portion of the side-chain of Thr200

with the NH of the sulfonamide group (Figure 5). The tail portion of (R)-10b showed limited

interaction with hCA II active site and, in particular, only a hydrophobic interaction of the benzyl

group with residues Trp5 was observed (Figure 5A). On the other hand, compound (R)-9b showed

1

different hydrophobic interactions with hCA II arranging the benzyl portion in a different pocket of

the active-site via interactions with Val135, Pro202, and Leu204 (Figure 5B).

Figure 6. Active site region of hCA II/11a (PDB:6RHJ) (A) and hCA II/11b (PDB:6RHK) (B)

adducts. Inhibitors are showed as σA-weighted |Fo−Fc| density map at 2.0 σ. Hydrogen bonds, van

der Waals interactions and water bridges are also shown.

The benzyl portion located on the N atom of diazepane 11a or imidazolidine 11b scaffolds moved to

the opposite side of the cavity with respect to (R)-10b (Figure 3); the higher potency of compounds

1

1a and 11b (Ki 2.3 and 0.6 nM, respectively, Table 1) for hCA II could result from this different

orientation. Both 11a and 11b showed a water bridge linking Thr200 and the sulfonamide NH group

Figure 6A and 6B) as found for compound (R)-9b (Figure 5B). Moreover, the size of the heterocyclic

(

ring could influence the hydrophobic interactions made by the benzyl group. In fact, a bulkier scaffold

such as the diazepane ring pushed the benzyl portion away from the hydrophobic region delimited by

residues Phe131, Val135, Leu198, and Pro202 (Figure 6A). On the contrary, the less bulky

imidazolidine scaffold of 11b allowed its N-benzyl moiety to form stronger hydrophobic interactions

with residues Phe131 and Pro202 (Figure 6B). Unfortunately, the poor electron density for the benzyl

groups of both inhibitors made their orientation not easily inferable.

1

2

Interestingly, comparison of the binding modes of compounds (R)-10b and (R)-9b (Figure 7A)

showed that the presence of a methyl group on the piperazine nitrogen atom of (R)-9b led to a different

ring conformation which shifted the carbonyl moiety of 1.8 Å (Figure 7B) and placed the benzyl

group in the opposite side of the cavity with respect to (R)-9b. This orientation was different also

with respect to the position of the 4-fluorobenzyl moiety in the structure of the complex hCA II/8b

[16]. As previously discussed for 11b and 11a (Figure 4B), these substantial differences prove the

importance of the inhibitor tail in order to modulate its potency.

Figure 7. (A) Structural superposition between (R)-10b (green) and (R)-9b (cyan) bound to the hCA

II active site. (B) Structural superposition between (R)-10b (green), (R)-9b (cyan), 11a (magenta) and

1

1b (purple).

Table 2 Summary of Data Collection and Atomic Model Refinement Statistics

HCAII + 9b HCAII + 10b

HCAII + 11a HCAII + 11b

PDB ID

Wavelength (Å)

Space Group

Unit cell

6RG4

0.827

P21

6RG3

0.827

P21

6RHJ

1.000

P21

6RHK

1.000

P21

42.37, 41.30, 42.36, 41.26,

42.37, 41.37, 42.60, 41.63,

(

a, b, c, α, β, γ)

71.93 90.0,

104.4, 90.0

71.92 90.0,

104.3, 90.0

71.76 90.0,

104.4 90.0

72.35 90.0,

104.54, 90.0

(Å,°)

Limiting resolution 41.07-1.25

41.08-1.32

(1.41-1.32)

69.49-1.44

(1.44-1.47)

70.03-1.44

(1.44-1.47)

(Å)

(1.25-1.33)

Unique reflections

66753

188539 (10131) 43292 (2186) 44092 (2184)

(11257)

1

3

Rsym (%)

Rmeas (%)

Redundancy

5.8 (99.2)

6.9 (118.9)

3.3 (3.2)

4.7 (77.4)

5.5 (92.1)

3.3 (3.3)

7.6 (18.5)

9.5 (28.2)

2.7 (2.3)

5.8 (19.4)

7.1 (24.4)

2.9 (2.4)

Completeness

overall (%)

99.1 (99.1)

99.2 (98.7)

99.4 (99.0)

99.3 (98.8)

<

I/σ(I)>

9.39 (0.79)

99.8 (54.1)

12.39 (1.24)

99.9 (64.3)

7.2 (2.2)

10.0 (3.4)

CC (1/2)

98.9 (82.9)

99.6 (93.1)

Refinement statistics

Resolution range

41.07-1.25

41.08-1.32

50.0-1.44

(Å)

Unique reflections, 62531/3239

working/free

53621/2719

41141/2125

41791/2061

Rfactor (%)

Rfree(%)

15.5

20.0

17.7

19.7

17.6

20.0

15.4

17.5

r.m.s.d. bonds(Å)

r.m.s.d. angles (°)

0.015

2.008

0.0127

1.427

0.0126

1.8699

0.0134

1.9184

Ramachandran statistics (%)

Most favored

97.3

2.7

96.9

2.7

96.9

3.1

97.3

2.7

additionally

allowed

outlier regions

0.0

0.4

0.0

Average B

factor (Å²)

All atoms

inhibitors

solvent

18.16

34.36

30.31

17.60

17.68

28.91

13.53

28.17

21.90

13.36

32.19

23.29

3

. Conclusion

A series of benzenesulfonamides carrying a piperazine nucleus or its bioisosteres have been prepared

in order to further investigate the structure-activity relationships of this class of compounds. The

removal of the C-benzyl moiety from 9a to give 8a resulted in a loss of selectivity for the CA IV

isoform, while structural modification on 8a shifted the selectivity toward the cytosolic hCA I and II

isoforms. In particular, the reduction of ring size from 6 to 5 atoms to give 11b, the introduction of a

NO group on the para position of the N-benzyl ring (7f) or the extrusion of the sulfamoylbenzamide

2

moiety out the six-membered ring (8a) gave compounds showing subnanomolar potency on hCA II.

1

4

This information can be useful in the design of selective CAI carrying a piperazine ring; in particular,

the decoration of the piperazine C-atoms with a benzyl group seems tolerated only on hCA IV but

not on the other tested isoforms. Work is underway to check the contribution of other C-substituents

in order to expand the structure-activity relationships in this class of potent sulfonamide CA

inhibitors.

4

. Experimental

.1 General remarks and instrumentation. All melting points were taken on a Büchi apparatus and

are uncorrected. When reactions were performed under anhydrous conditions, the mixtures were

maintained under nitrogen. NMR spectra were recorded on a Brucker Avance 400 spectrometer (400

1

13

MHz for H NMR, 100 MHz for C). Chromatographic separations were performed on a silica gel

column by gravity chromatography (Kieselgel 40, 0.063- 0.200 mm; Merck) or flash chromatography

(Kieselgel 40, 0.040-0.063 mm; Merck). Yields are given after purification, unless differently stated.

The purity of the tested compounds has been assessed by means of LC-DAD analyses, carried out on

an Agilent 1200 system (Agilent, Palo Alto CA, USA). High resolution mass spectrometry (HR-MS)

analysis was performed with a Thermo Finnigan LTQ Orbitrap mass spectrometer equipped with an

electrospray ionization source (ESI). The analysis was carried out in positive ion mode monitoring

+

protonated molecules, [M+H] species, and it was used a proper dwell time acquisition to achieve

6

0,000 units of resolution at Full Width at Half Maximum (FWHM). Elemental composition of

compounds was calculated on the basis of their measured accurate masses, accepting only results with

an attribution error less than 5 ppm and a not integer RDB (double bond/ring equivalents) value, in

order to consider only the protonated species [30]. Compounds were named following IUPAC rules

by means of MarvinSketch 18.1.

4

.1.1 General procedure for the insertion of the Zinc Binding Group. To a solution of the suitable

amine (30-100 mg) in anhydrous CH CN (5-30 mL), stirred at room T, Et N (2 eq) and 4-

3

sulfamoylbenzoyl chloride (1.5 eq) were sequentially added (Method A); alternatively, (Method B),

the amine was treated with 2,5-dioxopyrrolidin-1-yl 4-sulfamoylbenzoate [24]. The mixture was left

stirring at room T for 6-20 hrs. After removal of the solvent under vacuum, the residue was partitioned

between ethyl acetate and sat. aqueous NaHCO₃; drying and removal of the solvent gave a residue

which was purified by flash chromatography, when necessary. In this way the following compounds

were prepared:

1

5

4

-(4-Benzylpiperazine-1-carbonyl)benzene-1-sulfonamide 8a [16] (Method A): from

1

commercially-available N-benzylpiperazine. White solid, m.p. 204-5 °C, 60% yields. [ H-NMR]

(

DMSO) δ: 7.84 (d, J=8.2 Hz, 2H, Ar), 7.54 (d, J=8.2 Hz, 2H, Ar), 7.44 (s, 2H, SO₂NH₂), 7.36-7.20

m, 5H, Ar), 3.61 (bs, 2H), 3.48 (s, 2H, CH₂Ph), 3.40-3.20 (m, 2H+water), 2.41 (bs, 2H), 2.32 (bs,

(

2

H) ppm.

4

-{4-[(4-Fluorophenyl)methyl]piperazine-1-carbonyl}benzene-1-sulfonamide 8b [16] (Method

A): from 14d [22] after purification using DCM/MeOH 90:10 as eluent. White solid, m.p.209 °C (d)

1

(

lit [16] 206-207 °C), 41% yields. [ H-NMR] (DMSO) δ: 7.84 (d, J = 8.4 Hz, 2H, Ar), 7.54 (d, J =

8

.4 Hz, 2H, Ar), 7.44 (s, 2H, SO NH ), 7.32 (dd, J = 8.3 Hz, 5.8 Hz, 2H, Ar), 7.12 (t, J=8.8 Hz, 2H,

2 2

Ar), 3.70-3.55 (m, 2H), 3.47 (s, 2H, CH Ar), 3.28-3.22 (m, 2H), 2.46-2.28 (m, 4H) ppm.

2

4

-(4-Benzyl-1,4-diazepane-1-carbonyl)benzene-1-sulfonamide 11a (Method A): from

1

commercially-available N-benzyl-homopiperazine. White solid, m.p. 175°C (d), 41% yields. [ H-

NMR] (DMSO) δ: 7.94-7.82 (m, 2H, Ar), 7.64-7.51 (m, 2H, Ar), 7.45 (s, 2H, NH ), 7.35-7.23 (m,

2

5

1

H, Ar), 3.72-3.54 (m, 4H), 3.32 (s, 2H+water), 2.78-2.69 (m, 1H), 2.67-2.53 (m, 3H), 1.89-1.78 (m,

1

3

H), 1.72-1.62 (m, 1H) ppm. [ C NMR] (DMSO, mixture of conformers) δ: 169.57 (CO), 169.50

(

CO), 144.88 (C ), 140.72 (C ), 139.48 (C ), 129.03 (CH ), 128.90 (CH ), 128.67 (CH ), 128.64

Ar Ar Ar Ar Ar Ar

(

CH ), 127.53 (CH ), 127.43 (CH ), 127.35 (CH ), 126.29 (CH ), 126.26 (CH ), 61.57 (CH Ph),

Ar Ar Ar Ar Ar Ar 2

6

4

1.37 (CH Ph), 55.53 (CH ), 55.19 (CH ), 54.43 (CH2), 53.84 (CH ), 49.55 (CH ), 48.50 (CH ),

2 2 2 2 2 2

+

5.70 (CH ), 45.00 (CH ), 28.56 (CH ), 26.99 (CH ) ppm. ESI-HRMS (m/z) [M+H] : calculated for

2

C H N O S 374.1533; found 374.1532.

1

9

24

3

4

-(3-Benzylimidazolidine-1-carbonyl)benzene-1-sulfonamide 11b (Method A): from N-

benzylimidazolidine [18], after purification using DCM/MeOH 90:10 as eluent. White solid, m.p.

1

75 °C (d); 10% yields. [ H-NMR] (DMSO, mixture of conformers) δ: 7.85-7.79 (m, 2H, Ar), 7.71

(

d, J=8.0 Hz, 1H, Ar), 7.65 (d, J = 8.0 Hz, 1H, Ar), 7.43 (s, 1H, SO NH ), 7.41 (s, 1H, SO NH ),

2 2 2 2

7

3

.34-7.20 (m, 5H, Ar), 4.10 (s, 1H), 3.97 (s, 1H), 3.66 (s, 1H), 3.55 (s, 1H), 3.52 (t, J = 6.4 Hz, 1H),

1

3

.44 (t, J = 6.0 Hz, 1H), 2.86 (t, J = 6.4 Hz, 1H), 2.80 (t, J = 6.4 Hz, 1H) ppm. [ C-NMR] (DMSO,

mixture of conformers) δ: 167.16 (CO), 166.86 (CO), 145.91 (C ), 145.79 (C ), 139.80 (C ),

Ar

1

39.58 (C ), 138.64 (C ), 138.53 (C ), 129.12 (CH ), 128.94 (CH ), 128.81 (CH ), 128.78

Ar Ar Ar Ar Ar Ar

(

CH ), 128.32 (CH ), 128.10 (CH ), 127.67 (CH ), 126.25 (CH ), 126.13 (CH ), 70.27 (CH ),

Ar Ar Ar Ar Ar Ar 2

6

8.52 (CH ), 57.43 (CH ), 56.98 (CH ), 52.87 (CH ), 51.31 (CH ), 47.00 (CH ), 44.34 (CH ) ppm.

2 2 2 2 2 2 2

+

ESI-HRMS (m/z) [M+H] : calculated for C H N O S 346.1220; found 346.1222.

1

7

20

3

4

-{4-[(3-Methoxyphenyl)methyl]piperazine-1-carbonyl}benzene-1-sulfonamide 11c (Method

A): from 14a [19] after purification using DCM/MeOH 90:10 as eluent. White solid, m.p 186°C (d),

1

4

7% yields. [ H-NMR] (DMSO) δ: 7.82 (d, J = 8.2 Hz, 2H, Ar), 7.52 (d, J = 8.2 Hz, 2H, Ar), 7.41 (s,

1

6

2

H, SO₂NH₂), 7.19 (m, 1H, Ar), 6.87-6.70 (m, 3H, Ar), 3.69 (s, 3H, OCH₃), 3.65-3.55 (m, 2H), 3.43

1

3

(

s, 2H, CH -Ar), 3.30-3.20 (m, 2H+water), 2.45-2.26 (m, 4H) ppm. [ C-NMR] (DMSO) δ: 168.18

2

(

CO), 159.74 (C ), 145.22 (C ), 139.88 (C ), 139.58 (C ), 129.73 (CH ), 127.95 (CH ), 126.31

Ar

(

CH ), 121.52 (CH ), 114.81 (CH ), 112.90 (CH ), 62.19 (CH -Ar), 55.41 (OCH₃), 53.13 (CH₂),

Ar

2

+

5

3

4

2.59 (CH₂), 47.55 (CH₂), 42.07 (CH₂) ppm. ESI-HRMS (m/z) [M+H] : calculated for C H N O S

1

9

24

3

4

90.1482 found 390.1480.

-{4-[(3-Nitrophenyl)methyl]piperazine-1-carbonyl}benzene-1-sulfonamide 11d (Method A):

from 14b [20] after purification using DCM/MeOH/NH₃ 85:15:1.5 as eluent. White solid, m.p, 176°C

1

(

d), 73% yields. [ H-NMR] (DMSO) δ: 8.15 (s, 1H, Ar), 8.11 (d, J = 8.0 Hz, 1H, Ar), 7.85 (d, J = 8.4

Hz, 2H, Ar), 7.77 (d, J = 7.6 Hz, 1H, Ar), 7.62 (t, J = 8.0 Hz, 1H, Ar), 7.56 (d, J = 8.4 Hz, 2H, Ar),

.42 (s, 2H, NH ), 3.64 (bs, 4H), 3.30-3.20 (bs, 2H+water), 2.50-2.40 (bs, 2H+DMSO), 2.40-2.30

7

2

1

3

(

m, 2H) ppm. [ C-NMR] (DMSO) δ: 168.19 (CO), 148.37 (C ), 145.26 (C ), 140.95 (C ), 139.55

Ar Ar Ar

(

C ), 135.95 (CH ), 130.24 (CH ), 127.94 (CH ), 126.30 (CH ), 123.63 (CH ), 122.56 (CH ),

Ar Ar Ar Ar Ar Ar Ar

6

0.99 (CH -Ar), 53.04 (CH₂), 52.49 (CH₂), 47.54 (CH₂), 42.01 (CH₂) ppm. ESI-HRMS (m/z)

2

+

[

M+H] : calculated for C H N O S 405.1227 found 405.1230.

1

8

21

4

5

4

-{4-[(4-Chlorophenyl)methyl]piperazine-1-carbonyl}benzene-1-sulfonamide 11e (Method A):

from 14c [21] after purification using DCM/MeOH/NH₃ 90:10:1 as eluent. White solid, m.p. 253°C

1

(

d); 46% yields. [ H NMR] (DMSO) δ: 7.82 (d, J = 8.4 Hz, 2H, Ar), 7.52 (d, J = 8.4 Hz, 2H, Ar),

7

3

1

.40 (s, 2H, SO NH ), 7.34 (d, J = 8.4 Hz, 2H, Ar), 7.28 (d, J = 8.4 Hz, 2H, Ar), 3.65-3.50 (m, 2H),

2 2

1

3

.45 (s, 2H, CH Ar), 3.30-3.20 (m, 2H+water), 2.42-2.25 (m, 4H) ppm. [ C-NMR] (DMSO) δ:

2

68.17 (CO), 145.22 (C ), 139.55 (C ), 137.35 (C ), 132.04 (C ), 131.12 (CH ), 128.67 (CH ),

Ar

27.95 (CH ), 126.30 (CH ), 61.30 (CH -Ar), 53.07 (CH₂), 52.48 (CH₂), 47.55 (CH₂), 42.02 (CH₂)

Ar

2

+

ppm. ESI-HRMS (m/z) [M+H] : calculated for C H ClN O S 394.0987 found 394.0987.

1

8

21

3

4

-{4-[(4-Nitrophenyl)methyl]piperazine-1-carbonyl}benzene-1-sulfonamide 11f (method A):

from 14e [23] after purification using DCM/MeOH/NH₃ 92:8:0.8 as eluent. White solid, m.p. 226 °C

1

(

d); 65% yields. [ H NMR] (DMSO) δ: 8.18 (d, J = 8.8 Hz, 2H, Ar), 7.85 (d, J = 8.4 Hz, 2H, Ar),

7

3

1

.59 (d, J = 8.8 Hz, 2H, Ar), 7.55 (d, J = 8.4 Hz, 2H, Ar), 7.42 (s, 2H, SO NH ), 3.66 (bs, 4H), 3.30-

2 2

1

3

.20 (m, 2H+water), 2.50-2.31 (m, 4H) ppm. [ C-NMR] (DMSO) δ: 168.19 (CO), 147.13 (C ),

Ar

46.75 (C ), 145.24 (C ), 139.51 (C ), 130.25 (CH ), 127.96 (CH ), 126.30 (CH ), 123.87

Ar

(

CH ), 61.21 (CH -Ar), 53.15 (CH₂), 52.59 (CH₂), 47.55 (CH₂), 42.02 (CH₂) ppm. ESI-HRMS (m/z)

Ar

2

+

[

M+H] : calculated for C H N O S 405.1227 found 405.1225.

1

8

21

4

5

N-(1-Benzylpiperidin-4-yl)-4-sulfamoylbenzamide 12a (Method A): from commercially-available

1

-benzylpiperidinyl-4-amine after purification using DCM/MeOH/NH₃ 90:10:1 as eluent. White

1

solid, m.p. 220 °C (d); 19% yields. [ H] NMR (DMSO) δ: 8.40 (d, J = 7.6 Hz, 1H, NH) 7.92 (d, J =

1

7

8

3

.4 Hz, 2H, Ar), 7.83 (d, J = 8.4 Hz, 2H, Ar), 7.44 (s, 2H, SO₂NH₂), 7.36-7.19 (m, 5H, Ph), 3.80-

.68 (m, 1H), 3.49 (s, 2H, CH₂Ph), 2.77 (d, J = 11.3 Hz, 2H), 2.06-1.88 (m, 1H), 1.74 (d, J = 11.6

1

3

Hz, 2H), 1.65-1.45 (m, 2H) ppm. [ C] NMR (DMSO) δ: 165.12 (CO), 146.62 (C ), 138.09 (C ),

Ar

1

29.29 (CH-Ar), 128.26 (CH ), 128.39 (CH ), 127.42 (CH ), 125.98 (CH ), 62.46 (CH₂), 52.58

Ar

+

(

CH₂), 47.52 (CH), 31.76 (CH₂) ppm. ESI-HRMS (m/z) [M+H] : calculated for C H N O S

1

9

24

3

74.1533, found 374.1531.

N-(1-Benzylpiperidin-4-yl)-N-methyl-4-sulfamoylbenzamide 12b (Method B): from 1-benzyl-N-

methylpiperidin-4-amine [25] after purification using DCM/MeOH/NH₃ 90:10:1 as eluent. White

1

solid, m.p. 207 °C (d); 38% yields. [ H-NMR] (CDCl , mixture of conformers) δ: 7.92-7.80 (m, 2H,

3

Ar), 7.50-7.35 (m, 2H, Ar), 7.35-7.15 (m, 5H, Ar), 5.27-5.05 (m, 2H, SO NH ), 4.60-4.43 (m, 0.5 H),

2

3

2

.58-3.43 (bs, 1H), 3.43-3.37 (bs, 1H), 3.37-3.21 (m, 0.5 H), 3.10-2.90 (m, 2.5 H), 2.90-2.80 (m, 1H),

1

3

.75 (s, 1.5 H), 2.23-2.06 (m, 1H), 1.99-1.62 (m, 4H), 1.62-1.48 (m, 1H) ppm. [ C-NMR] (DMSO,

mixture of conformers) δ: 169.72 (CO), 144.92 (C ), 140.75 (C ), 138.98 (C ), 138.71 (C ), 129.95

Ar

(

CH ), 128.61 (CH ), 127.75 (CH ), 127.36 (CH ), 127.12 (CH ), 126.34 (CH ), 62.44 (CH ),

Ar Ar Ar Ar Ar Ar 2

6

2.11 (CH ), 57.01 (CH), 52.90 (CH ), 52.50 (CH ), 51.83 (CH), 32.31 (CH ), 29.57 (CH ), 28.56

2 2 2 3 2

+

(

CH ), 27.64 (CH ) ppm. ESI-HRMS (m/z) [M+H] : calculated for C H N O S 388.1689, found

2

3

20 26

3

88.1692.

N-(1-Benzylpyrrolidin-3-yl)-4-sulfamoylbenzamide 12c (Method B): from commercially-

available 1-benzylpirrolidin-3-amine after purification using DCM/MeOH/NH₃ 90:10:1 as eluent.

1

White solid, m.p. 174-178 °C; 54% yields. [ H-NMR] (MeOD) δ: 7.98-7.86 (m, 4H, Ar), 7.38-7.20

(m, 5H, Ar), 4.60-4.49 (m, 1H), 3.72-3.60 (m, 2H), 2.94- 2.85 (m, 1H), 2.85-2.77 (m, 1H), 2.65-2.50

1

3

(

m, 2H), 2.41-2.27 (m, 1H), 1.90-1.76 (m, 1H) ppm. [ C NMR] (DMSO) δ: 165.56 (CO), 146.61

(

C ), 139.41 (C ), 137.79 (C ), 129.04 (CH ), 128.60 (CH ), 128.46 (CH ), 127.28 (CH ),

Ar

1

25.96 (CH ), 60.22 (CH₂), 59.86 (CH₂Ph), 52.96 (CH₂), 49.42 (CH₂), 31.12 (CH₂) ppm. ESI-

Ar

+

HRMS (m/z) [M+H] : calculated for C H N O S 360.1376, found 360.1377.

1

8

22

3

tert-Butyl N-[1-(4-sulfamoylbenzoyl)piperidin-4-yl]carbamate 15 (Method B): from

commercially-available tert-butyl piperidin-4-ylcarbamate after purification using AcOEt as eluent.

1

White solid, m.p. 229 °C (d), 65% yields. [ H-NMR] (DMSO) δ: 7.83 (d, J = 8.0 Hz, 2H, Ar), 7.49

(

d, J = 8.0 Hz, 2H, Ar), 7.40 (s, 2H, SO NH ), 6.90-6.84 (m, 1H, NH), 4.32-4.21 (m, 1H), 3.52-3.31

2 2

m, 2H), 3.11-2.98 (m, 1H), 2.98-2.83 (m, 1H), 1.82-1.50 (m, 2H), 1.33 (s, 9H, C(CH ) ), 1.28-1.25

3

1

3

m, 2H) ppm. [ C-NMR] (DMSO) δ: 168.17 (CO), 155.29 (CO), 145.19 (C ), 139.93 (C ), 127.63

Ar

CH ), 126.37 (CH ), 78.16 (CMe ), 47.53 (CH), 46.33 (CH₂), 32.66 (CH₂), 31.79 (CH₂), 28.71

Ar

3

+

CH₃) ppm. ESI-HRMS (m/z) [M+H] : calculated for C H N O S 384.1588, found 384.1586.

1

7

26

3

5

1

8

Benzyl N-benzyl-N-[1-(4-sulfamoylbenzoyl)piperidin-4-yl]carbamate 18 (Method B): from 17

[19] after purification using DCM/MeOH/NH₃ 90:10:1 as eluent. White solid, m.p. 206-209 °C, 98%

1

yields. [ H-NMR] (DMSO) δ: 7.83 (d, J = 8.0 Hz, 2H, Ar), 7.60-7.09 (m, 14 H, Ar + SO NH ), 5.06

2

(

bs, 2H, OCH Ph), 4.60-4.40 (m, 3H, NCH Ph + 1H), 4.23-4.05 (m, 1H), 3.30-3.20 (m, 1H+water),

2

1

3

.14-2.98 (m, 1H), 2.82-2.65 (m, 1H), 1.76-1.60 (m, 3H), 1.59-1.47 (m, 1H) ppm. [ C-NMR]

(

DMSO) δ: 168.10 (CO), 145.07 (C ), 140.04 (C ), 138.86 (C ), 137.25 (C ), 130.34 (CH-Ar),

Ar Ar Ar Ar

1

28.73 (CH ), 128.24 (CH ), 127.92 (CH ), 127.60 (CH ), 127.13 (CH ), 126.95 CH ), 126.27

Ar Ar Ar Ar Ar Ar

+

(

CH ), 66.82 (OCH Ph), 54.88 (CH), 46.91 (NCH ), 41.43 (CH ) ppm. ESI-HRMS (m/z) [M+H] :

Ar 2 2 2

calculated for C H N O S 508.1901, found 508.1903.

2

7

30

3

5

4

.1.2 General procedure to obtain compounds 14a-e. N-Boc-piperazine and Et N (1.2 eq) were

3

dissolved in anhydrous CH Cl , and the suitable benzyl bromide (1 eq) was added dropwise at room

2

T. After completion of the reaction (TLC), the mixture was partitioned between CH Cl and water.

2

Drying and removal of the solvent gave an oily residue used in the following step without further

purification: it was dissolved in CH Cl (5 mL) and trifluoroacetic acid (10 eq) was added. After 6

2

hrs stirring at room T, the solvent was removed and the residue was partitioned between ethyl acetate

and sat. aqueous Na CO₃; drying and removal of the solvent gave a residue which was purified by

2

flash chromatography if necessary.

1

-[(3-Methoxy)methyl]piperazine 14a [19]: oil, 98% yields. [ H-NMR] (CDCl ) δ: 7.08 (t, J = 8.0

3

Hz, 1H, Ar), 6.93-6.85 (m, 2H, Ar), 6.66 (d, J = 8.0 Hz, 1H, Ar), 3.66 (s, 3H, OCH ), 3.33 (s, 2H,

3

CH Ar), 2.87-2.70 (m, 4H), 2.45-2.20 (m, 4H), 1.90 (bs, 1H, NH) ppm.

2

1

-[(3-Nitrophenyl)methyl]piperazine 14b [20]: purified using DCM/MeOH/NH₃ 90:10:1 as eluent.

1

Oil, 96% yields. [ H-NMR] (DMSO) δ: 8.09 (s, 1H, Ar), 8.07 (d, J = 8.0 Hz, 1H, Ar), 7.71 (d, J =

7

4

1

.6 Hz, 1H, Ar), 7.57 (t, J = 8.0 Hz, 1H, Ar), 3.51 (s, 2H, CH Ar), 2.78-2.53 (m, 4H), 2.40-2.17 (m,

2

H) ppm.

1

-[(4-Chlorophenyl)methyl]piperazine 14c [21]: oil, 75% yields. [ H-NMR] (DMSO) δ: 7.31 (d, J

=

8.4 Hz, 2H, Ar), 7.25 (d, J = 8.4 Hz, 2H, Ar), 3.35 (s, 2H, CH Ar), 2.66-2.57 (m, 4H), 2.25-2.15

2

(m, 4H) ppm.

1

-[(4-Fluorophenyl)methyl]piperazine 14d [22]: oil, 70% yields. [ H-NMR] (CDCl ) δ: 7.41-7.25

3

(

m, 2H, Ar), 6.99 (t, J = 8.8 Hz, 2H, Ar), 3.49 (s, 2H, CH Ar), 3.00-2.88 (m, 4H), 2.50-2.83 (m, 4H),

2

1

8

4

.08 (bs, 1H, NH) ppm.

1

-[(4-Nitrophenyl)methyl]piperazine 14e [23]: oil, 92% yields. [ H-NMR] (DMSO) δ: 8.16 (d, J =

.8 Hz, 2H), 7.56 (d, J = 8.8 Hz, 2H), 3.30-3.20 (m, 2H, ArCH ), 2.73-2.60 (m, 4H), 2.33-2.20 (m,

2

H) ppm.

1

9

4

.1.3

4-{4-[(3-Aminophenyl)methyl]piperazine-1-carbonyl}benzene-1-sulfonamide

11g

Compound 11d (0.075 g, 0.186 mmol) was dissolved in abs EtOH (25 mL); the solution was heated

at 75°C, then SnCl₂·2H₂O (0.29 g, 1.29 mmol) was added. The mixture was stirred at 80°C for 3 hrs.

After cooling, the mixture was concentrated and treated with a saturated solution of Na₂CO₃,

producing a white precipitate which was filtered off through celite. The solution was partitioned

between H₂O and CH Cl . Drying (Na₂SO₄) and removal of the solvent gave a residue which was

2

purified by flash chromatography using DCM/MeOH/NH₃ 85:15:1.5 as eluent, obtaining the title

1

product (59% yields). White solid, m.p. 204 °C (d). [ H-NMR] (MeOD) δ: 7.94 (d, J = 8.4 Hz, 2H,

Ar), 7.53 (d, J = 8.4 Hz, 2H, Ar), 7.01 (t, J = 7.6 Hz, 1H, Ar), 6.68 (s, 1H, Ar), 6.67-6.57 (m, 2H, Ar),

3

.80-3.70 (m, 2H), 3.42 (s, 2H, CH Ar), 3.40-3.34 (m, 2H), 2.60-2.46 (m, 2H), 2.45-2.34 (m, 2H)

2

1

3

ppm. [ C-NMR] (DMSO) δ: 168.15 (CO), 149.03 (C ), 145.22 (C ), 139.61 (C ), 138.73 (C ),

Ar

1

29.08 (CH ), 127.95 (CH ), 126.30 (CH ), 116.96 (CH ), 114.85 (CH ), 113.27 (CH ), 62.81

Ar Ar Ar Ar Ar Ar

+

(

CH -Ar), 53.26 (CH₂), 52.66 (CH₂), 47.60 (CH₂), 42.08 (CH₂) ppm. ESI-HRMS (m/z) [M+H] :

2

calculated for C H N O S 375.1485, found 375.1486.

1

8

23

4

3

4

.1.4

4-{4-[(4-Aminophenyl)methyl]piperazine-1-carbonyl}benzene-1-sulfonamide

11h

Compound 11f (0.03 g, 0.0743 mmol) was dissolved in AcOEt (20 mL) and hydrogenated in a Parr

apparatus at 30 psi for 4 hrs, using Pd/C (10 mg) as catalyst. Filtration and removal of the solvent

under vacuum gave a residue which was purified by flash chromatography using DCM/MeOH/NH₃

1

8

5:15:1.5 as eluent, obtaining the title product in 72% yields. White solid, m.p. 180°C (d). [ H-NMR]

(MeOD) δ: 7.96 (d, J = 7.5 Hz, 2H, Ar), 7.55 (d, J = 7.4 Hz, 2H, Ar), 7.05 (d, J = 7.4 Hz, 2H, Ar),

6

2

1

.67 (d, J = 7.4 Hz, 2H, Ar), 3.77 (bs, 2H, CH₂), 3.45 (s, 2H, CH₂Ar), 3.39 (bs, 2H, CH₂), 2.55 (bs,

1

3

H, CH₂), 2.41 (bs, 2H, CH₂) ppm. [ C-NMR] (DMSO) δ: 168.14 (CO), 148.18 (C ), 145.19 (C ),

Ar

39.59 (C ), 130.36 (CH ), 127.94 (CH ), 126.29 (CH ), 114.09 (CH ), 62.15 (CH -Ar), 52.88

Ar

2

+

(

CH₂), 52.39 (CH₂), 47.52 (CH₂), 41.99 (CH₂) ppm. ESI-HRMS (m/z) [M+H] : calculated for

C H N O S 375.1485, found 375.1488.

1

8

23

4

3

4

.1.5 4-[4-(Benzylamino)piperidine-1-carbonyl]benzene-1-sulfonamide 13a Compound 18

(

0.030 g, 0.0592 mmol) was suspended in glacial acetic acid (5 mL) and, at 0 °C, treated with HBr

33% solution in acetic acid, 0.32 mL). The mixture was left stirring at room T for 24 hrs, cooled in

(

ice bath, made alkaline (pH 9) with ammonium hydroxide, and extracted with AcOEt. Drying

Na₂SO₄) and removal of the solvent under vacuum gave a residue which was purified by means of

flash chromatography (DCM/MeOH/NH₃ 90:10:1), obtaining the title compound with 91% yields.

(

2

0

1

M.p.190°C (d). [ H NMR] (CDCl ) δ: 7.80 (d, J = 8.0 Hz, 2H, Ar), 7.35 (d, J = 8.0 Hz, 2H, Ar), 7.30-

3

7

1

.16 (m, 5H, Ar), 4.5-4.37 (m, 1H), 3.77 (s, 2H, CH Ph), 3.60-3.42 (m, 1H), 3.05-2.87 (m, 2H), 2.04-

2

1

3

.85 (m, 1H), 1.82-1.70 (m, 1H), 1.50-1.37 (m, 1H), 1.36-1.20 (m, 1H) ppm. [ C] NMR (DMSO) δ:

68.08 (CO), 145.06 (C ), 141.48 (C ), 140.13 (C ), 128.55 (CH ), 128.38 (CH ), 127.62 (CH ),

Ar

26.96 (CH ), 126.32 (CH ), 53.41 (CH), 50.18 (CH Ph), 46.03 (CH ), 32.58 (CH ), 31.87 (CH )

Ar

2

+

ppm. ESI-HRMS (m/z) [M+H] : calculated for C H N O S 374.1533, found 374.1531.

1

9

24

3

4

1

.1.6 4-{4-[Benzyl(methyl)amino]piperidine-1-carbonyl}benzene-1-sulfonamide 13b Compound

3a (0.030 g, 0.08 mmol), dissolved in CH₃CN, was treated with Et N (0.01 mL, 0.08 mmol) and

3

CH₃I (0.01 mL, 0.16 mmol). After stirring at room T for 3 days the solvent was removed under

vacuum and the residue purified by means of flash chromatography (DCM/MeOH 91:9). A white

1

solid was obtained in 46% yields, m.p. 164 °C (d). [ H NMR] (MeOD) δ: 7.98 (d, J = 8.0 Hz, 2H,

Ar), 7.57 (d, J = 8.0 Hz, 2H, Ar), 7.33-7.20 (m, 5H, Ar), 4.75-4.66 (m, 1H), 3.75-3.64 (m, 1H), 3.63

(

s, 2H, CH Ph), 3.20-3.06 (m, 1H), 2.92-2.71 (m, 2H), 2.22 (s, 3H, NCH₃), 2.10-1.99 (m, 1H), 1,90-

2

1

3

1

.80 (m, 1H), 1.75-1.50 (m, 2H) ppm. [ C] NMR (MeOD) δ: 169.40 (CO), 144.94 (C ), 139.24

Ar

(

C ), 137.33 (C ), 129.24 (CH ), 128-13 (CH ), 127.30 (CH ), 127.09 (CH ), 126.18 (CH ),

Ar Ar Ar Ar Ar Ar Ar

6

0.44 (CH), 57.44 (CH Ph), 41.34 (CH ), 36.36 (CH₃), 27.28 (CH ), 26.99 (CH ) ppm. ESI-HRMS

2

+

(

m/z) [M+H] : calculated for C H N O S 388.1689, found 388.1685.

20 26 3 3

4

.1.7 4-(4-aminopiperidine-1-carbonyl)benzene-1-sulfonamide trifluoroacetate salt 16

Compound 15 (125 mg, 0.33 mmol) was dissolved in trifluoroacetic acid (3 mL) and left strring at

room T for 20 hrs. Removal of the solvent under vacuum gave a residue which was crushed with

1

Et₂O. Low melting solid, 82% yields. [ H-NMR] (DMSO) δ: 7.94 (s, 2H, SO NH ), 7.84 (d, J=6.8

2

Hz, 2H, Ar), 7.50 (d, J=6.8 Hz, 2H, Ar), 4.42 (bs, 1H), 3.51-3.40 (m, 2H), 3.20-3.00 (m, 1H), 2.95-

2

.80 (m, 1H), 2.08-1.87 (m, 1H), 1.86-1.77 (m, 1H), 1.60-1.30 (m, 2H) ppm.

4

.1.8 Benzyl N-benzyl-N-(piperidin-4-yl)carbamate 17 This compound was prepared following the

1

procedure reported by Lee [19] for a fluorinated analogue. Oil, 94% yields. [ H NMR] (CDCl ) δ:

3

7

.42-7.04 (m, 10H, Ar), 5.25-5.03 (m, 2H, OCH ), 4.46 (s, 2H, NCH ), 4.22-4.04 (m, 2H), 3.10-2.98

2 2

(m, 2H), 2.70-2.49 (m, 2H), 1.99-1.86 (m, 2H), 1.70-1.50 (m, 4H) ppm.

4

.2 CA inhibition studies. An Sx.18Mv-R Applied Photophysics (Oxford, UK) stopped-flow

instrument has been used to assay the catalytic activity of various CA isozymes for CO hydration

2

reaction as previously described [17, 26]. Phenol red (at a concentration of 0.2 mM) was used as

2

1

indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as buffer and

0 mM Na SO (for maintaining constant the ionic strength), following the initial rates of the CA-

2

4

catalyzed CO hydration reaction for a period of 10−100 s. The CO concentrations ranged from 1.7

2

to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor,

at least six traces of the initial 5−10% of the reaction have been used for determining the initial

velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total

observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled−deionized water, and

dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions

were preincubated together for 6 hours at room temperature prior to assay in order to allow for the

formation of the E−I complex. The inhibition constants were obtained by nonlinear least-squares

methods using PRISM 3 and the Cheng−Prusoff equation, as reported earlier [17], and represent the

mean from at least three different determinations. All CA isofoms were recombinant ones obtained

in-house as reported earlier and their concentrations were in the range of 5.7 – 12 nM [31, 32].

Inhibition data are reported as mean (± standard error) of three independent experiments. Significance

of differences among the groups was assessed by one-way ANOVA followed by Newman-Keuls post

hoc test for multiple comparisons. Calculations were made with Prism 5 statistical software

(GraphPad Software, Inc., San Diego, CA, USA). The results were considered to be statistically

significant when p<0.05.

4

.3 Crystallization and X-ray data collection

Crystals were obtained through the hanging drop vapor diffusion method using 24 well Linbro plate.

2

1

µL of 0.8 mM solution of hCA II in Tris-HCl pH=8.0 were mixed with a solution of 1.5, 1.6 and

.7 M sodium citrate, 50 mM Tris pH 8.0 and were equilibrated against 500 µL of the same solution

at 296 K. Crystals of the protein grew in a few days. hCA II crystals were soaked in 5mM inhibitor

solution for 2 days. The crystals were flash-frozen at 100K using a solution obtained by adding 25%

(v/v) glycerol to the mother liquor solution as cryoprotectant. Data on crystals of the complexes of

hCAII with (R)-9b and (R)-10b were collected using synchrotron radiation at the ID-29 beamline at

ESRF (Grenoble, France) with a wavelength of 0.827 Å and a Pilatus3 6M Dectris CCD detector.

Data on crystals of the complexes of hCAII with 11a and 11b were collected using synchrotron

radiation at the XRD2 beamline at Elettra (Trieste, Italy) with a wavelength of 1.000 Å and a Pilatus3

6

M Dectris CCD detector. Data were integrated and scaled using the program XDS [33].

4

.4 Structure determination

2

The crystal structure of hCA II (PDB accession code: 4FIK) without solvent molecules and other

heteroatoms was used to obtain initial phases of the structures using Refmac5 [34]; 5% of the unique

reﬂections were selected randomly and excluded from the reﬁnement data set for the purpose of Rfree

calculations. The initial |Fo - Fc| difference electron density maps unambiguously showed the

inhibitor molecules. Atomic models for inhibitors were calculated and energy minimized using the

program JLigand 1.0.40 [35]. Reﬁnements proceeded using normal protocols of positional,

anisotropic atomic displacement parameters alternating with manual building of the models using

COOT [36]. Solvent molecules were introduced automatically using the program ARP [37]. The

quality of the ﬁnal models was assessed with COOT and RAMPAGE [38]. Atomic coordinates were

deposited in the Protein Data Bank (PDB accession code: 6RG3, 6RG4, 6RHJ, 6RHK). Graphical

representations were generated with Chimera [39].

5

. Conflict of interest disclosure

The authors declare that they have no conflict of interest.

Acknowledgements

This work was supported by grants from the University of Florence (Fondo Ricerca Ateneo

RICATEN17). The X-ray diffraction experiments were performed on beamline ID-29 at ESRF

(Grenoble, France) and beamline XRD2 at Elettra (Trieste, Italy). We are grateful to local contacts at

the ESRF and Elettra for providing assistance in using the beamlines.

Appendix A. Supplementary material.

1

13

H and C-NMR spectra of 11b, 11e, 12b and 13b.

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6

Highlights



A series of piperazines and 4-aminopiperidines have been prepared as simplified analogues

of a 2-benzylpiperazine lead compound.



The new compounds have been tested on physiologically relevant hCA isoforms.

Several compounds are potent inhibitors of hCA I and II.

Removal of the 2-benzyl group decreased activity and selectivity toward hCA IV.

2

7

Article Doi

Sulfonamides incorporating piperazine bioisosteres as potent human carbonic anhydrase I, II, IV and IX inhibitors

DOI: 10.1016/j.bioorg.2019.103130

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Article abstract of DOI:10.1016/j.bioorg.2019.103130

Full text of DOI:10.1016/j.bioorg.2019.103130

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