Azobenzene-based inhibitors of human carbonic anhydrase II

Article abstract of DOI:10.3762/bjoc.11.127

Aryl sulfonamides are a widely used drug class for the inhibition of carbonic anhydrases. In the context of our program of photochromic pharmacophores we were interested in the exploration of azobenzene-containing sulfonamides to block the catalytic activ

Full text of DOI:10.3762/bjoc.11.127

Azobenzene-based inhibitors of human carbonic anhydrase II
Leander Simon Runtsch1, David Michael Barber1, Peter Mayer1, Michael Groll2,
Dirk Trauner*1 and Johannes Broichhagen*1
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2015, 11, 1129–1135.
1Department of Chemistry, Ludwig-Maximilians-University Munich and
Munich Center for Integrated Protein Science, Butenandtstrasse
5–13, 81377 Munich, Germany and 2Department of Biochemistry,
Technical University Munich and Munich Center for Integrated Protein
Science, Lichtenbergstr. 4, 85748 Garching, Germany
doi:10.3762/bjoc.11.127
Received: 02 May 2015
Accepted: 19 June 2015
Published: 07 July 2015
Email:
Associate Editor: D. Spring
Dirk Trauner* - dirk.trauner@lmu.de; Johannes Broichhagen* -
johannes.broichhagen@cup.lmu.de
© 2015 Runtsch et al; licensee Beilstein-Institut.
License and terms: see end of document.
* Corresponding author
Keywords:
azobenzene chemistry; enzyme inhibitors; human carbonic anhydrase
II; sulfonamide; X-ray crystallography
Abstract
Aryl sulfonamides are a widely used drug class for the inhibition of carbonic anhydrases. In the context of our program of
photochromic pharmacophores we were interested in the exploration of azobenzene-containing sulfonamides to block the catalytic
activity of human carbonic anhydrase II (hCAII). Herein, we report the synthesis and in vitro evaluation of a small library of nine
photochromic sulfonamides towards hCAII. All molecules are azobenzene-4-sulfonamides, which are substituted by different func-
tional groups in the 4´-position and were characterized by X-ray crystallography. We aimed to investigate the influence of electron-
donating or electron-withdrawing substituents on the inhibitory constant Ki. With the aid of an hCAII crystal structure bound to one
of the synthesized azobenzenes, we found that the electronic structure does not strongly affect inhibition. Taken together, all com-
pounds are strong blockers of hCAII with Ki = 25–65 nM that are potentially photochromic and thus combine studies from chem-
ical synthesis, crystallography and enzyme kinetics.
Introduction
Carbonic anhydrase (CA) is an ubiquitously found zinc- which the product appearance can be observed colorimetrically
containing metalloenzyme with many isoforms, which all (Figure 1a, right) [2]. In humans, isoform II (human carbonic
catalyze the conversion of carbon dioxide and water to bicar- anhydrase II; hCAII) is found in many tissues and is respon-
bonate and a proton (Figure 1a, left) [1]. Despite its native sible for maintaining the inner eye pressure among other regula-
purpose of pH and pressure regulation, its intrinsic esterase tory tasks [1]. Consequently, its failure is associated with glau-
activity can be utilized to measure the catalytic activity by coma [1,3]. Treatment of this severe disease, that leads to blind-
hydrolysis of p-nitrophenyl actetate (pNPA) to a phenolate, of ness, is achieved with the application of aryl sulfonamides [3].
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Beilstein J. Org. Chem. 2015, 11, 1129–1135.
Figure 1: Function and inhibition of hCAII. a) hCAII (pdb: 2vva [7]) catalyzes the hydration of carbon dioxide to bicarbonate and a proton (left) as well
as the hydrolysis of pNPA to acetate and a colored phenolate (λmax = 400 nm). b) Aryl sulfonamide-containing pharmacophores of hCAII. c) Aryl
sulfonamide merged to azobenzenes.
Being a transition-state analogue [4], this functional group of sulfanilamide and subsequent reaction with phenol, N,N-
exhibits excellent blocking characteristics of hCAII and culmi- diethylaniline or N-phenylmorpholine led to azobenzenes 1a
nates its power in many modern marketed drugs, such as acet- [6], 1b [9] and 1c, in moderate to low yields (43%, 38% and
azolamide (AAZ) or dorzolamide (Figure 1b) [5]. Furthermore, 25%, respectively) (Scheme 1a). Employing methylene-
sulfonamide-containing azobenzenes exhibit affinity and protected aniline 2 (crystal structure depicted in Scheme 1e)
blocking ability for hCAII (Figure 1c) [6]. With our knowledge according to the procedure from Supuran and co-workers [6],
in azobenzene chemistry and photopharmacology, we aimed to amino azobenzene 1d was isolated after a one-pot reaction over
further understand how electronic substitution patterns on three steps in 25%. By diazotization of 1d and trapping the salt
azobenzenes correlate to changes in enzyme affinity.
with TMS-azide, we obtained azido azobenzene 1e in 63% yield
through a [3 + 2] and retro-[3 + 2] cycloaddition (Scheme 1b)
according to the procedure of Barral et al [10]. For the Mills
Results and Discussion
Azobenzenes can be synthesized by a variety of known chem- reaction, different nitroso compounds were generated that were
ical transformations [8]. Among them the most widely used is all used without further purification for the following conden-
the diazotization of aniline, followed by trapping of the diazo- sation reactions. For example, sulfanilamide reacted with
nium salt with an electron-rich aromatic compound (such as Oxone® in a biphasic DCM/water mixture to its nitroso coun-
anilines and phenols). Another commonly used method is the terpart, which was condensed with p-toluidine to give methyl
condensation between anilines and aryl nitroso compounds, azobenzene 1f (Scheme 1c) in 45% yield over two steps. Addi-
known as the Mills reaction. According to these transforma- tionally, several in situ generated nitroso compounds bearing a
tions, nine sulfonamide containing azobenzenes 1a–i with nitro and a carboxylic acid ester were reacted with sulfanil-
different moieties in the 4´-position were synthesized. The amide to obtain nitro azobenzene 1g and ethyl ester azobenzene
substitution in the 4´-position will have the biggest impact on 1i, respectively. Commercially available nitrosobenzene gave
the electronic properties of the sulfonamide group due to com- rise to the unsubstituted sulfonamide 1h (Scheme 1d). The yield
munication through the conjugated π-system of the aromatic was poor for nitro azobenzene 1g (9%). Furthermore, the reac-
units and the diazene unit. Commencing with the diazotization tion of nitrosobenzene to obtain 1h proceeded in low yield
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Scheme 1: Synthesis and characterization of azobenzene-containing aryl sulfonamides by different strategies. a) Diazotization and trapping of the
diazonium salt with an electron-rich aromatic compound yields azobenzenes 1a–c. b) Reaction of methylene sulfonate-protected aniline and one-pot
deprotection yields 1d, which can be converted to 1e. c) Mills condensation to obtain 1f. d) Mills condensation to obtain 1g–i. e) Crystal structure of
sulfonate 2.
(38%), while 1i was isolated in quantitative yield (99%). Crys- for electron-withdrawing groups (λmax (1g) = 328 nm;
tals suitable for X-ray diffraction were obtained for 2 λmax (1i) = 342 nm) and more pronounced for electron-donating
(Scheme 1e) and all sulfonamide-containing azobenzenes 1a–i groups (λmax (1f) = 338 nm, λmax (1a) = 358 nm,
(Figure 2). The crystallization conditions can be found in λmax (1d) = 404 nm, λmax (1c) = 414 nm, and
Supporting Information File 1).
λmax (1b) = 460 nm). Interestingly, azide 1e with a Hammett
constant of σ = 0.08 [12], exhibits a bathochromic shift
The substitution patterns on the aromatic core together with (λmax (1e) = 356 nm) close to hydroxy azobenzene 1a although
their electronic characteristics determines the absorption spectra it can be considered neither electron-withdrawing, nor electron-
of the individual azobenzenes [11]. We assessed the π–π*-band donating.
wavelength of maximal absorption (λmax) by RP–LCMS
equipped with a UV–vis diode array detector, when deter- To gain a deeper understanding into the binding mode of the
mining the purity of our library. Therefore, λmax is determined synthesized azobenzene-containing sulfonamides we set out to
in a water/acetonitrile mixture, which mimics aqueous condi- co-crystallize an inhibitor with the wild-type enzyme. Protein
tions that are also used for the biological assays. The results are crystals bound to 1d (Figure 3a, pdb: 5byi) were obtained using
given in Table 1. While the “naked” azobenzene 1h has the a previously reported method [13]. Due to the rigidness of the
absorbance maximum in the bluest part of the spectrum azobenzene the sulfonamide nitrogen and the 4´-position are far
(λmax (1h) = 322 nm), substitution on the azobenzene in the apart (>12 Å). The moiety in this position is solvent exposed
4´-position leads to a bathochromic shift, which is smaller and should therefore not contribute to the binding affinities by
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Figure 2: Crystal structures for compounds 1a–i (co-solvents and/or multiple molecules in the asymmetric cell are omitted for clarity).
Table 1: Maximal absorbance wavelength (λmax), Hammett constants (σ) and inhibitory characteristics (IC50 and Ki) of 1a–i.
4´-substitution pattern
λmax (nm)
Hammett constant σ [12]
IC50 (nM)
Ki (nM)
1a
1b
1c
1d
1e
1f
OH
358
460
414
404
356
338
328
322
342
–
−0.37
−0.83
−0.83a
−0.66
+0.08
−0.17
+0.78
+0.00
+0.45
–
165.6
139.6
309.2
171.4
257.1
363.2
159.4
249.7
167.9
55.5
29.7
25.0
55.4
30.7
46.1
65.1
28.6
44.8
30.1
10.0
NEt2
N-morpholine
NH2
N3
Me
1g
1h
1i
NO2
H
COOEt
–
AAZ
aTo the best of our knowledge the Hammett constant for morpholine has not been previously determined, therefore we used the parameter for
alkylated amines due to its similar electronic nature.
direct interactions (Figure 3a and b). Apart from primary on the functional group in the 4´-position, as they affect the
binding interactions between the sulfonamide to the zinc center electronic properties of the aromatic system, and this would also
and T199, both of which are well-described [1] (Figure 3b and be reflected by the Hammett constant. Interestingly, the azoben-
c), we were looking for secondary interactions resulting from zene does not adopt a completely planar shape but is distorted
the azobenzene (Figure 3c). Indeed, we found that the methyl with dihedral angles of −143.3° and 23.8° at the N=N bond and
group of L198 interacts with the sulfonamide-bearing aromatic at the second aromatic ring to the diazene unit, respectively. It
core with a carbon to centroid distance of 3.5 Å. Furthermore, should be noted that a water molecule is held by the peptide
P202 and F131 contribute to centroid interactions with the backbone in the gorge (2.6 Å to OH of T200 and 2.7 Å to CO of
second aromatic ring with distances of 3.5 (NCH2 to centroid), P202), which might have interactions with the diazene unit
4.0 (NCH2CH2 to centroid) and 4.7 Å (CH to centroid), respect- although the distance for classical hydrogen bonding is rather
ively. These interactions can be weaker or stronger depending long (3.2 Å).
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Beilstein J. Org. Chem. 2015, 11, 1129–1135.
Figure 3: Crystal structure of hCAII bound to 1d (pdb: 5byi). a) The terminal amine of 1d is solvent-exposed, while the azobenzene is sticking in the
cavity. b) Electron-density map of 1d bound to zinc with primary interactions. c) Interactions of 1d in the catalytic site in angstroms (Å). Primary inter-
actions of the zinc-bound sulfonamide can be seen to T199 and L198 to the aromatic core. Secondary interactions can be observed from F131 and
P202 towards the second aromatic ring of the azobenzene.
In order to determine the half-maximal inhibitory concentra- sized and tested previously (with a CO2 hydration assay), and
tions (IC50) and the inhibitory constants (Ki) towards hCAII for the Ki values determined in the previous work are one order of
our library, we used a colorimetric endpoint measurement of the magnitude higher than in our findings (Ki (1a) = 665 nM or
catalyzed pNPA hydrolysis (Figure 1a). Usually, a dansyl 29.7; Ki (1d) = 106 nM or 30.7) towards hCAII [6]. Interest-
competition assay is employed for this purpose [1,14]. ingly, in our studies the most efficient blocker turned out to be
However, as this assay is fluorescence-based and azobenzenes 1b with a Ki = 25.0 nM, which also shows the greatest red-shift
can quench fluorescence [15], this might cause a distortion in in its maximal absorbance wavelength (π–π* band). Another
the obtained data. Furthermore, irradiation with UV light (i.e., electron-donating blocker bearing a methyl group substituent
λ = 280 nm for tryptophan excitation) can result in azobenzene- (1f), however, had the lowest affinity (Ki = 65.1 nM) of the
cis-isomerization, which could lead to different binding charac- library. Compound 1c offers the second-lowest affinity
teristics. Therefore, we aimed at the endpoint absorbance (Ki = 55.4 nM), which seems counter-intuitive, as the only
system described herein. After expression and purification of difference with respect to 1b is the connection of the ethyl
wild-type hCAII we tested the benchmark blocker AAZ chains by an oxygen atom to a morpholine ring. This does not
(Figure 1b) and obtained a Ki = 10.0 nM, which is in accor- only affect the binding properties, but also the π–π* band,
dance with a previously reported inhibition constant which is 46 nm blue-shifted relative to 1b. The inhibitors with
(Ki (AAZ) = 12 nM [16]). Consequently, we were confident that the proton and azide substituents (1h and 1e, respectively) show
our assay could assess the inhibitory characteristics of our very similar affinities towards hCAII with Ki = 44.8 nM and
library in a robust, reliable and reproducible manner.
Ki = 46.1 nM. Sterics are also restrained, but should not affect
binding, as the 4´-position is solvent-exposed (vide supra).
By using the Cheng–Prusoff equation [17] with a Taking all of these findings into account, we conclude that the
Michaelis–Menten constant of Km = 1092.5 µM for pNPA (see sulfonamide–zinc interaction dominates the binding affinity.
Supporting Information File 1, Figure S2), we calculated the
inhibitory constant Ki (Table 1) for each compound from the The electronic differences of our azobenzene library is
IC50 values obtained from sigmoidal fitting of the activity vs. expressed by their absorption spectra (as an indicator for the
concentration curve (see Figure 4a and Supporting Information electron richness of the azobenzene) or in their Hammett
File 1 for details). Azobenzenes 1a and 1d have been synthe- constants (as an indicator for electron-pushing or pulling
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Beilstein J. Org. Chem. 2015, 11, 1129–1135.
Figure 4: Inhibition of hCAII by electronically different azobenzene sulfonamides and AAZ. a) Endpoint measurement for the determination of IC50 for
compounds 1a–i. b) Hammett constants versus maximal absorbance wavelength shows decreasing trend. c) IC50 versus Hammett constants. d) IC50
versus λmax.
effects). When plotted against each other a trend can be and d can be considered mirror images of each other, com-
observed, which is reflected by a more bathochromic shift when pound 1h does not fit into this picture.
the Hammett constant becomes more negative (Figure 4b).
However, when plotting the Hammett constants or the maximal Conclusion
absorbance wavelength versus the IC50 (Figure 4c and d, res- In conclusion, we have synthesized a small library of nine
pectively), no clear correlation can be found. In both cases azobenzene sulfonamides with differing substitution patterns in
morpholine 1c, azide 1e, methyl 1f and unsubstituted azoben- the 4´-position using either azo coupling reactions or the Mills
zene sulfonamide 1h lie in the same region (50 µM), while all reaction. We determined the π–π* band and the crystal struc-
other inhibitors (hydroxy 1a, alkyl amine 1b, amine 1d, nitro 1h tures of all nine compounds together with the protein crystal
and ethyl carboxylate 1i) show higher affinities at around structure of hCAII bound to inhibitor 1d. The latter structure
30 µM. The latter also shows a distribution from UV to blue highlights the interactions of the sulfonamide and the azoben-
maximal absorbance. We therefore speculate that electronics do zene with the protein cavity. The inhibitory action on hCAII
not have a primary effect on the binding properties towards was tested for all compounds using an endpoint measurement of
hCAII, but rather the sole presence of an aryl sulfonamide is catalytic pNPA hydrolysis. The inhibitory constants were in
sufficient. It should be pointed out that “naked” azobenzene 1h close proximity to each other (Ki = 25–65 nM) and a correla-
does not follow the trend when correlating its Hammett constant tion of electron density as characterized by the Hammett
of σ = 0.00 to its λmax = 322 nm. As the plots from Figure 4c constant σ with the binding affinities was not observed. We
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D.M.B is grateful to the European Comission for a Marie
Skłodowska-Curie Intra-European fellowship (PIEF-GA-2103-
627990). J.B. is grateful to the Studienstiftung des deutschen
Volkes for a Ph.D. fellowship. M.G. and D.T. thank the Munich
Centre for Integrated Protein Science (CIPSM) as well as the
Deutsche Forschungsgemeinschaft SFB749 for financial
support. D.T. acknowledges support of the European Research
Council for an Advanced Grant (268795). We are grateful to the
staff of the beamline X06SA at the Paul Scherrer Institute,
Swiss Light Source, Villigen (Switzerland) for assistance during
data collection. Dr. David H. Woodmansee is acknowledged for
helpful discussion and advice together with providing ethyl
4-nitrosobenzoate as well as Eric P. Trautman and Martin Maier
for excellent synthetic assistance.
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License and Terms
Author Contributions
D.T. supervised the research. J.B. and D.T. conceived and
designed the study. J.B. and L.S.R. performed chemical, bio-
logical and small molecule crystallization experiments. J.B.
performed protein purification, crystallization and binding data
analysis. P.M. collected X-ray datasets of 1a–i and solved the
structures. M.G. collected hCAII X-ray dataset and solved the
structure. J.B., D.M.B and D.T. wrote the manuscript with input
from all authors.
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