Activation of carbonic anhydrases from human brain by amino alcohol oxime ethers: towards human carbonic anhydrase VII selective activators

Article abstract of DOI:10.1080/14756366.2020.1838501

The synthesis and carbonic anhydrase (CA; EC 4.2.1.1) activating effects of a series of oxime ether-based amino alcohols towards four human (h) CA isoforms expressed in human brain, hCA I, II, IV and VII, are described. Most investigated amino alcohol derivatives induced a consistent activation of the tested CAs, with K_As spanning from a low micromolar to a medium nanomolar range. Specifically, hCA II and VII, putative main CA targets when central nervous system (CNS) diseases are concerned, were most efficiently activated by these oxime ether derivatives. Furthermore, a multitude of selective hCA VII activators were identified. As hCA VII is one of the key isoforms involved in brain metabolism and other brain functions, the identified potent and selective hCA VII activators may be considered of interest for investigations of various therapeutic applications or as lead compounds in search of even more potent and selective CA activators.

Full text of DOI:10.1080/14756366.2020.1838501

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Activation of carbonic anhydrases from human
brain by amino alcohol oxime ethers: towards
human carbonic anhydrase VII selective activators
Alessio Nocentini , Doretta Cuffaro , Lidia Ciccone , Elisabetta Orlandini ,
Susanna Nencetti , Elisa Nuti , Armando Rossello & Claudiu T. Supuran
To cite this article: Alessio Nocentini , Doretta Cuffaro , Lidia Ciccone , Elisabetta Orlandini ,
Susanna Nencetti , Elisa Nuti , Armando Rossello & Claudiu T. Supuran (2021) Activation of
carbonic anhydrases from human brain by amino alcohol oxime ethers: towards human carbonic
anhydrase VII selective activators, Journal of Enzyme Inhibition and Medicinal Chemistry, 36:1,
48-57, DOI: 10.1080/14756366.2020.1838501
To link to this article: https://doi.org/10.1080/14756366.2020.1838501
© 2020 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
Published online: 25 Oct 2020.
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2021, VOL. 36, NO. 1, 48–57
https://doi.org/10.1080/14756366.2020.1838501
RESEARCH PAPER
Activation of carbonic anhydrases from human brain by amino alcohol oxime
ethers: towards human carbonic anhydrase VII selective activators
Alessio Nocentinia , Doretta Cuffarob
Nutib , Armando Rossellob and Claudiu T. Supurana
, Lidia Cicconeb
, Elisabetta Orlandinib, Susanna Nencettib, Elisa
ꢀ
ꢀ
aSection of Pharmaceutical and Nutraceutical Sciences, Department of Neuroscience, Psychology, Drug Research and Child’s Health
b
(Neurofarba), University of Florence, Sesto Fiorentino, Italy; Department of Pharmacy, University of Pisa, Pisa, Italy
ABSTRACT
ARTICLE HISTORY
Received 29 September 2020
Revised 7 October 2020
Accepted 13 October 2020
The synthesis and carbonic anhydrase (CA; EC 4.2.1.1) activating effects of a series of oxime ether-based amino
alcohols towards four human (h) CA isoforms expressed in human brain, hCA I, II, IV and VII, are described.
Most investigated amino alcohol derivatives induced a consistent activation of the tested CAs, with KAs span-
ning from a low micromolar to a medium nanomolar range. Specifically, hCA II and VII, putative main CA tar-
gets when central nervous system (CNS) diseases are concerned, were most efficiently activated by these
oxime ether derivatives. Furthermore, a multitude of selective hCA VII activators were identified. As hCA VII is
one of the key isoforms involved in brain metabolism and other brain functions, the identified potent and
selective hCA VII activators may be considered of interest for investigations of various therapeutic applications
or as lead compounds in search of even more potent and selective CA activators.
KEYWORDS
Metalloenzyme; activation;
selectivity; CNS-
disease; cognition
1. Introduction
substrate, and the activator. CAAs bind apart from the metal-
coordination system, namely at the middle edge of CAs active site
cavity, where they assist the proton shuttling9–11. As a proof of
this phenomenon, it was demonstrated that efficient activators
possess pKa values in the range of 6–8, values similar to the pKa
of the His imidazole moiety7.
It is seemingly odd that CAs, among the most efficient
enzymes in Nature, may be activated for biomedical purposes8.
However, genetic deficiencies of several CA isoforms (e.g. human
CA I, II, IV, VA, XII and XIV) have been reported in the last decades,
associated to diseases such as osteopetrosis, cerebral calcifications,
retinal problems, hyperammonaemia, hyperchlorhydrosis12–15, and
Activators of the metalloenzymes carbonic anhydrases (CAs; EC
4.2.1.1, CAAs) have been lately going through a second youth in
drug discovery processes1. Early evidence of the CA activation effi-
cacy of amines, such as histamine, dating back to the 1940s was
thereafter long debated up, and even considered as an experimen-
tal artefact2,3. In the early 1990s, the combination of highly purified
enzymes and precise techniques, such as the stopped flow assay,
put an end to the long controversy, testifying the undeniable exist-
ence of CAAs4–7. CAAs intervene in the second and rate determin-
ing step of the catalytic mechanism of carbon dioxide reversible
hydration (Equations 1 and 2), that is the regeneration of the cata- the loss of function of these enzymes would in principle be treat-
able with CA selective activators8. In addition, there is evidence
that CAs activation improves memory deficits, cognitive perform-
lytically active, zinc hydroxide species, by a proton transfer reaction
from the Zn2þ – bound water molecule to the external medium
ance and learning16–18, being nine of the fifteen known human
CA isoforms present in brain19,20. Contrariwise, other evidence
supported that CA inhibitors (CAIs) impair memory in human,
according to studies on the CAIs topiramate and acetazolamide
during acute high-altitude exposure21,22. Thus, CAs represent a
crucial family of new targets for improving cognition, but also in
therapeutic areas, such as phobias, obsessive-compulsive disorder,
generalised anxiety, and post-traumatic stress disorders, for which
few effective therapies are available. In fact, in a recent paper, one
of our groups showed that the CAA D-phenylalanine and the CAI
acetazolamide are respectively able to reinforce and impair extinc-
tion memory, that is, a new memory trace that inhibits the expres-
sion of the memory of a traumatic event23. In this golden period
for CAAs, the discovery of new brain isoform selective CAAs (as
(Equation 2)6–8. This process is assisted by active site residues act-
ing as proton shuttle, such as His residues placed in the middle or
at the entrance of the active site cavity of the a-class human CAs4.
EZn2þꢁOHꢁ þ CO2 À EZn2þꢁHCO3ꢁ þ H2O À EZn2þꢁOH2
þ HCOꢁ3
(1)
EZn2þꢁOH2 À EZn2þꢁOHꢁ þ Hþ
(2)
ꢀ
ꢁ
EZn2þ ꢁ OH2 þ A À EZn2þ ꢁ OH2 ꢁ A À EZn2þ ꢁ OHꢁ ꢁ AHþ
½
ꢂ
À EZn2þ ꢁ OHꢁ þ AHþ
(3)
The activators non-competitively activate the CAs through the
formation of a ternary complex consisting of the enzyme, the well as CAIs) is invaluable to elucidate the role of CA isoforms in
CONTACT Armando Rossello
claudiu.supuran@unifi.it
armando.rossello@unipi.it
Department of Pharmacy, University of Pisa, via Bonanno 6, Pisa, 56126, Italy; Claudiu T. Supuran
Section of Pharmaceutical and Nutraceutical Sciences, Department of Neuroscience, Psychology, Drug Research and Child’s Health
(Neurofarba), University of Florence, via Ugo Schiff 6, Sesto Fiorentino, 50019, Italy
ꢀ
These authors contributed equally to this work.
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
49
brain processes. In addition, CAAs are considered relevant in artifi-
cial tissues and in CO2 capture and sequestration processes1,24
(E)-2,3-Dihydro-1H-inden-1-one O-(3-(tert-butylamino)-2-hydroxy-
propyl) oxime maleate 1: Maleate salt. H NMR (400 MHz, CDCl3): d
1
.
Here, we extend the knowledge of CAA chemotypes by 9.51 (s, 1H), 7.94 (s, 1H), 7.61 (d, J ¼ 7.6 Hz, 1H), 7.34–7.31(m, 2H),
describing the synthesis and CA activating effects of a series of 7.26–7.23 (m, 1H), 6.18 (s, 2H), 4.43–4.38 (m, 1H), 4.29 (dd,
J ¼ 4.08 Hz, J ¼ 12 Hz, 1H), 4.19 (dd, J ¼ 6.0 Hz, J ¼ 11.6 Hz, 1H),
oxime ether based amino alcohols towards four hCA isoforms
expressed in human brain.
3.21–3.28 (m, 1H); 3.05–3.02 (m, 3H), 2.90–2.87 (m, 2H), 1.43 (s,
9H). 13 C NMR (100 MHz, DMSO-d6): d 167.7, 163.5, 148.9, 136.6,
135.8, 131.1, 127.5, 126.4, 121.4, 75.7, 66.1, 56.8, 44.4, 28.5,
26.8, 25.45.
2. Material and methods
(E)-Benzaldehyde O-(3-(tert-butylamino)-2-hydroxypropyl) oxime
maleate 2: Maleate salt. 1H NMR (400 MHz, DMSO-d6): d 8.32 (s,
1H), 7.65–7.62 (m, 2H), 7.45–7.44 (m, 3H), 6.02 (s, 2H), 4.14–4.13
(m, 2H), 3.70–3.67 (m, 1H), 3.11–3.08 (m, 1H), 2.88–2.83 (m, 1H),
1.28 (s, 9H). 13 C NMR (100 MHz, DMSO-d6): d 167.2, 149.5, 136.1,
131.7, 130.1, 128.8, 126.9, 75.4, 65.5, 56.4, 43.9, 25.0.
2.1. Chemistry
1H and 13 C NMR spectra were recorded on a Bruker Avance III HD
400 MHz spectrometer. Chemical shifts (d) are reported in parts
per million and coupling constants (J) are reported in hertz (Hz).
13 C NMR spectra were fully decoupled. The following abbrevia-
tions were used to explain multiplicities: singlet (s), doublet (d),
triplet (t), double doublet (dd), broad (br), and multiplet (m).
Chromatographic separations were performed on silica gel col-
umns by flash column chromatography (Kieselgel 40,
0.040ꢁ0.063 mm, Merck). Reactions were followed by thin-layer
chromatography (TLC) on Merck aluminium silica gel (60 F254)
sheets that were visualised under a UV lamp. Evaporation was per-
formed in vacuo (rotating evaporator). Sodium sulphate was
always used as the drying agent. Commercially available chemicals
were purchased from Sigma-Aldrich.
Propan-2-one O-(3-(tert-butylamino)-2-hydroxypropyl) oxime oxal-
ate 3: oxalate salt. 1H NMR (400 MHz, DMSO-d6): d 4.00–3.87 (m,
3H), 2.99 (dd, J ¼ 2.4 Hz, J ¼ 12.4 Hz, 1H), 2.76 (dd, J ¼ 9.1 Hz,
J ¼ 12.4 Hz, 1H), 1.81 (s, 3H), 1.26 (s, 9H). 13 C NMR (100 MHz,
DMSO-d6): d 164.7, 155.0, 74.4, 65.5, 56.0, 44.1, 25.0, 21.2.
(E)-4-Methoxybenzaldehyde O-(3-(tert-butylamino)-2-hydroxypropyl)
1
oxime maleate 4: Maleate salt. H NMR (400 MHz, DMSO-d6): d 8.24 (s,
1H), 7.58 (m, 2H), 7.02 (m, 2H), 6.01 (s, 2H), 4.27–4.07 (m, 6H), 3.33 (s,
3H), 3.15–3.05 (m, 1H), 2.94–2.89 (m, 1H), 1.27 (s, 9H). 13 C NMR
(100 MHz, DMSO-d6): d 165.4, 161.2, 149.4, 128.9, 124.7, 114.8, 75.8,
65.9, 56.5, 55.7, 44.6, 25.4.
(E)-3-Methoxybenzaldehyde O-(3-(tert-butylamino)-2-hydroxypropyl)
oxime oxalate 5: Oxalate salt. H NMR (400 MHz, DMSO-d6): d 8.27 (s,
1
2.1.1. General procedure for the synthesis of amino alcohols 1–13
A solution of the proper oxime (17–27) (10 mmol) in anhydrous
DMF (10 ml) was added portionwise to a solution of MeONa in
MeOH (30 ml), prepared from anhydrous MeOH (30 ml) and Na
(11 mmol). The reaction mixture was stirred at 60 ꢃC for 1 h and
then cooled at rt. Epichlorohydrin (0.86 ml, 11 mmol) dissolved in
anhydrous DMF (10 ml) was added dropwise, and the resulting
mixture was stirred for 1 h at rt, poured into water (100 ml) and
extracted with CHCl3 (2 ꢄ 100mL). The organic phases were com-
bined, washed with water (2 ꢄ 200 ml), dried (Na2SO4) filtered and
evaporated under reduced pressure. The crude was distilled in
vacuo affording an oil corresponding to the proper epoxide
(28–38, yield 75–85%). NMR data of 28–35 were in accordance
1H), 7.35 (t, J¼ 7.6 Hz, 2H), 7.21–7.17 (m, 2H), 7.01 (dd, J¼ 1.6 Hz,
J¼ 8 Hz, 1H), 4.12–4.05 (m, 3H), 3.77 (s, 3H), 3.09–3.06 (m, 1H),
2.86–2.81 (m, 1H), 1.28 (s, 9H). 13 C NMR (100 MHz, DMSO-d6): d 165.4,
159.9, 149.8, 133.6, 130.4, 119.9, 116.5, 112.1, 76.1, 65.9, 56.5, 55.6,
44.6, 25.4.
(E)-2-Methoxybenzaldehyde O-(3-(tert-butylamino)-2-hydroxypropyl)
1
oxime oxalate 6: Oxalate salt. H NMR (400 MHz, DMSO-d6): d 8.42 (s,
1H), 7.65 (dd, J¼ 1.6 Hz, J¼ 8 Hz, 2H), 7.43 (m, 1H), 7.10 (d, J¼ 8Hz,
1H), 6.99 (t, J¼ 7.2 Hz, 1H), 4.11–4.05 (m, 3H), 3.83 (s, 3H), 3.07 (bd,
J¼ 12 Hz, 1H), 2.86–2.83 (m, 1H), 1.28 (s, 9H). 13 C NMR (100 MHz,
DMSO-d6): d 165.2, 157.7, 145.2, 132.2, 126.3, 121.1, 120.0, 112.4, 76.0,
65.6, 56.2, 50.2, 47.3, 19.2.
with those reported in literature25–33
.
(E)-3-Chlorobenzaldehyde O-(3-(tert-butylamino)-2-hydroxypropyl)
1
(E)-4-Chlorobenzaldehyde O-oxiran-2-ylmethyl oxime (36): yield
76%; 1H NMR (400 MHz, CDCl3): d 8.40 (s, 1H), 7.35 (m, 2H), 7.30
(m, 2H), 4.38 (dd, J ¼ 12.3 Hz, J ¼ 3.5 Hz, 1H), 4.13 (dd, J ¼ 12.3 Hz,
J ¼ 3.5 Hz, 1H), 3.28–3.32 (m, 1H), 2.88 (dd, J ¼ 5.2 Hz, J ¼ 4.2 Hz,
1H), 2.70 (dd, J ¼ 5.2 Hz, J ¼ 4.2 Hz, 1H).
oxime oxalate 7: Oxalate salt. H NMR (400 MHz, DMSO-d6): d 8.32
(s, 1H), 7.68 (m, 1H), 7.60–7.59 (m, 1H), 7.50–7.47 (m, 2H),
4.15–4.05 (m, 3H), 3.08–3.05 (bd, J ¼ 12 Hz, 1H), 2.83 (dd,
J ¼ 8.8 Hz, J ¼ 12.4, 1H), 1.27 (s, 9H). 13 C NMR (100 MHz, DMSO-d6):
d 165.4, 148.6, 134.4, 131.3, 130.3, 126.9, 125.9, 76.3, 65.9, 56.5,
44.6, 25.5.
(E)-2-Chlorobenzaldehyde O-oxiran-2-ylmethyl oxime (37): yield
82%; 1H NMR (400 MHz, CDCl3): d 8.34 (s, 1H), 7.37 (m, 2H),
7.28–7.24 (m, 2H), 4.35 (dd, J ¼ 12.3 Hz, J ¼ 3.5 Hz, 1H), 4.06 (dd,
J ¼ 12.3 Hz, J ¼ 3.5 Hz, 1H), 3.30–3.34 (m, 1H), 2.90 (dd, J ¼ 5.2 Hz,
J ¼ 4.2 Hz, 1H), 2.76 (dd, J ¼ 5.0 Hz, J ¼ 4.2 Hz, 1H).
(E)-4-Chlorobenzaldehyde O-(3-(tert-butylamino)-2-hydroxypropyl)
1
oxime oxalate 8: Oxalate salt. H NMR (400 MHz, DMSO-d6): d 8.32
(s, 1H), 7.68 (m, 1H), 7.65 (m, 2H), 7.51 (m, 2H), 4.14–4.09 (m, 3H),
3.09–3.05 (bd, J ¼ 12.4 Hz, 1H), 2.83 (m, 1H), 1.27 (s, 9H). 13 C NMR
(100 MHz, DMSO-d6): d 165.4, 148.6, 134.4, 131.2, 130.2, 126.9,
125.9, 76.3, 65.9, 56.5, 44.6, 25.4.
Cyclohexanone O-(2-hydroxy-3-(isopropylamino)propyl) oxime
oxalate 9: Oxalate salt. 1H NMR (400 MHz, DMSO-d6): d 3.90–3.83
(m, 3H), 3.01–2.98 (m, 1H), 2.80–2.77 (m, 1H), 2.67–2.63 (m, 1H),
2.40–2.38 (m, 2H), 2.12 (t, J ¼ 6.8 Hz, 2H), 1.61–1.48 (m, 6H), 1.08
(d, J ¼ 6.4 Hz, 6H). 13 C NMR (100 MHz, DMSO-d6): d 165.35, 160.0,
75.5, 67.0, 49.4, 31.8, 27.0, 25.7, 25.2.
(E)-3-Chlorobenzaldehyde O-oxiran-2-ylmethyl oxime (38): yield
79%; 1H NMR (400 MHz, CDCl3): d 8.51 (s, 1H), 7.34–7.28 (m, 1H),
7.26–7.23 (m, 3H), 4.32 (dd, J ¼ 12.1 Hz, J ¼ 3.7 Hz, 1H), 4.18 (dd,
J ¼ 12.1 Hz, J ¼ 3.7 Hz, 1H), 3.31–3.35 (m, 1H), 2.88 (dd, J ¼ 5.0 Hz,
J ¼ 4.1 Hz, 1H), 2.67 (dd, J ¼ 5.0 Hz, J ¼ 4.1 Hz, 1H).
A stirred solution of epoxide (28–38, 10 mmol) in dry benzene
(6 ml) was treated with an excess of isopropylamine or tert-butyl-
amine (50 mmol). The reaction mixture was stirred for 12 h at
90 ꢃC, and then evaporated. The crude was dissolved in a mixture
of MeOH/EtOH 3:7 (20 ml) and treated with 1.2 equivalent of the
(E)-1,7,7-Trimethylbicyclo[2.2.1]heptan-2-one O-(2-hydroxy-3-(iso-
propylamino)propyl) oxime fumarate 10: Fumarate salt. 1H NMR
proper organic acid (oxalic, malic or fumaric acids) to give the cor- (400 MHz, DMSO-d6): d 6.47 (s, 1H), 4.00–3.91 (m, 2H), 3.87–3.82
responding amino alcohol (1–13) as a white solid salt. (m, 1H), 3.24–3.21 (m, 1H), 2.96–2.93 (m, 1H), 2.78–2.76 (m, 1H),

50
A. NOCENTINI ET AL.
2.50–2.40 (m, 2H), 1.93 (dd, J ¼ 2.4 Hz, J ¼ 17.6 Hz, 1H), 1.86 (t, 2H), 7.27 (m, 1H), 6.90 (d, J ¼ 7.8 Hz, 1H), 6.85 (t, J ¼ 7.6 Hz, 1H),
J ¼ 3.2 Hz, 1H), 1.78–1.74 (m, 1H), 1.71–1.64 (m, 1H), 1.32–1.28 (m, 6.01 (s, 2H), 5.76 (m, 1H), 4.12–4.07 (m, 3H), 3.33–3.30 (m, 1H),
3.09–3.07 (m, 1H), 2.90–2.89 (m, 1H), 1.22 (m, 6H). 13 C NMR
1H), 1.18 (t, J ¼ 6.0 Hz, 6H), 0.91 (s, 3H), 0.87 (s, 3H), 0.71 (s, 3H).
13 C NMR (100 MHz, DMSO-d6): d 169.2, 168.3, 135.5, 75.3, 65.9,
(100 MHz, DMSO-d6): d 167.6, 156.5, 147.4, 132.0, 127.4, 119.9,
117.8, 116.7, 75.9, 65.7, 50.3, 47.1, 19.3, 18.5.
51.8, 49.7, 48.2, 43.5, 33.9, 27.2, 19.6, 19.5, 18.9, 18.7, 11.6.
(E)-3-Hydroxybenzaldehyde O-(3-(tert-butylamino)-2-hydroxypropyl)
oxime fumarate 15: Fumarate salt. 1H NMR (400 MHz, DMSO-d6): d
8.17 (s, 1H), 7.21 (t, J¼ 7.6 Hz, 1H), 7.04–7.00 (m, 2H), 6.82–6.80 (m,
2H), 6.40 (s, 1H), 4.07–4.06 (m, 2H), 4.02–3.94 (m, 1H), 2.89–2.86 (m,
1H), 2.71–2.66 (m, 1H), 1.18 (s, 9H). 13 C NMR (100 MHz, DMSO-d6): d
169.8, 158.2, 149.7, 133.5, 130.3, 118.6, 117.7, 113.3, 76.4, 66.6, 54.5,
45.0, 26.5.
Propan-2-one O-(2-hydroxy-3-(isopropylamino)propyl) oxime oxal-
ate 11: Oxalate salt. H NMR (400 MHz, DMSO-d6): d 4.04–3.99 (m,
1
1H), 3.94 (dd, J ¼ 5.36 Hz, J ¼ 11.2 Hz, 1H), 3.86 (dd, J ¼ 5.32 Hz,
J ¼ 11.2 Hz, 1H), 3.34–3.29 (m, 2H), 3.00 (dd, J ¼ 2.64 Hz,
J ¼ 12.56 Hz, 1H), 2.85–2.80 (m, 1H), 1.80 (s, 6H), 1.21 (t, J ¼ 6.04 Hz,
6H). 13 C NMR (100 MHz, DMSO-d6): d 165.4, 155.4, 75.0, 66.0, 50.1,
47.5, 21.7, 19.2, 18.5.
(E)-4-Hydroxybenzaldehyde O-(3-(tert-butylamino)-2-hydroxypropyl)
oxime fumarate 16: H NMR (400 MHz, DMSO-d6): d 8.12 (s, 1H), 7.42
(m, 2H), 6.80 (m, 2H), 6.40 (s, 1H), 4.02–3.97 (m, 3H), 3.04–3.00 (m,
1H), 2.87–2.83 (m, 1H), 2.70–2.65 (m, 1H), 1.10 (d, J¼ 6.4 Hz, 6H).13C
NMR (100 MHz, DMSO-d6): d 169.0, 159.8, 149.3, 129.0, 123.1, 116.1,
76.4, 66.9, 49.4, 48.9, 21.1, 20.7.
(E)-4-Chlorobenzaldehyde O-(2-hydroxy-3-(isopropylamino)propyl)
oxime oxalate 12: Oxalate salt. 1H NMR (400 MHz, DMSO-d6): d
8.31 (s, 1H), 7.65 (m, 2H), 7.51 (m, 2H), 4.13–4.10 (m, 3H),
3.33–3.28 (m, 1H), 3.08–3.04 (m, 1H), 2.91–2.86 (m, 1H), 1.21 (t,
J ¼ 6.4 Hz, 6H). 13 C NMR (100 MHz, DMSO-d6): d 165.5, 148.6,
134.4, 134.0, 131.2, 130.2, 127.7, 125.9, 76.4, 65.6, 50.1, 47.3,
19.24, 18.7.
1
(E)-2-Chlorobenzaldehyde O-(2-hydroxy-3-(isopropylamino)propyl)
oxime oxalate 13: Oxalate salt. 1H NMR (400 MHz, DMSO-d6): d
8.49 (s, 1H), 7.81 (dd, J ¼ 1.6 Hz, J ¼ 7.6 Hz, 1H), 7.55 (dd, J ¼ 1.2 Hz,
J ¼ 8 Hz, 1H), 7.48 (dt, J ¼ 1.6 Hz, J ¼ 7.2 Hz, 1H), 7.41 (m, 1H),
4.16–4.11 (m, 3H), 3.34–3.31 (m, 1H), 3.10–3.07 (m, 1H), 2.93–2.88
(m, 1H), 1.22 (t, J ¼ 6.4 Hz, 6H). 13 C NMR (100 MHz, DMSO-d6): d
165.25, 146.2, 133.2, 132.2, 130.4, 129.6, 128.1, 127.7, 76.5, 65.6,
50.2, 47.2, 19.2, 18.6.
2.2. Carbonic anhydrase activation
A stopped-flow method34 has been used for assaying the CA cata-
lysed CO2 hydration activity with Phenol red as indicator, working
at the absorbance maximum of 557 nm, following the initial rates
of the CA-catalysed CO2 hydration reaction for 10–100 s. For each
activator, at least six traces of the initial 5–10% of the reaction
have been used for determining the initial velocity. The uncata-
lyzed rates were determined in the same manner and subtracted
from the total observed rates. Stock solutions of activator (0.1 mM)
were prepared in distilled-deionised water and dilutions up to
0.1 nM were done thereafter with the assay buffer. The activation
constant (KA), defined similarly with the inhibition constant (KI),
was obtained by considering the classical Michaelis–Menten equa-
tion (Equation 4), which has been fitted by nonlinear least squares
by using PRISM 3:
2.1.2. General procedure for the synthesis of aminoalcohols 14–16
Epichlorohydrin (0.88 ml,11.2 mmol) was added dropwise to a
stirred solution of N-hydroxy-5-norbornene-2,3-dicarboximide (2 g,
11.2 mmol) and Et3N (3.12 ml, 22.4 mmol) in anhydrous DMF (8 ml).
After stirring for 18 h at rt the reaction mixture was poured into
water (50 ml) and extracted with CHCl3 (2ꢄ50mL). The organic
phases were combined, washed with water (2 ꢄ 100 ml), dried
(Na2SO4) filtered and evaporated under reduced pressure. The
crude epoxide 39 was purified by crystallisation with n-hexane.
NMR data were in accordance with those reported in literature25.
A stirred solution of epoxide 39 (611 mg, 2.6 mmol) in dry
EtOH (8 ml) was treated with an excess of iPrNH2 (0.43 ml, 5 mmol)
or t-BuNH2 (0.52 ml, 5 mmol). The reaction mixture was stirred at
50 ꢃC for 4 h, and then the solvent was evaporated. The crude was
constituted principally by the amino alcohol 40 (iPr) or 41(tBu)
and it was used in the next reaction step without further
purification.
A solution of the proper amino alcohol 40 or 41 (1.56 mmol) in
7.8 ml of NH3-MeOH 7 N was stirred rt for 2 h. The resulting mix-
ture was filtered and evaporated. The crude was purified by flash
chromatography with EtOAc/MeOH/Et3N (8: 1.5: 0.5) affording
derivative 42 or 43 (60–63% yield). NMR data were in accordance
with those reported in literature25.
A solution of p-, m-, or o- hydroxybenzaldehyde (6.75 mmol) in
EtOH (11.4 ml) and the proper oxyamine 42 or 43 (6.75 mmol) was
refluxed for 12 h. After cooling, the mixture was evaporated. The
crude was dissolved in a mixture of MeOH/EtOH 3:7 (20 ml) and
treated with 1.2 equivalent of the proper organic acid (oxalic,
malic or fumaric acids) affording the amino alcohols (14–16,
70–80% yields) as white solids after crystallisation from
MeOH/Et2O.
ꢄ
ꢂ
ꢃꢅ
v ¼ vmax= 1 þ KM=½Sꢂ 1 þ ½Aꢂf=KA
(4)
where [A]f is the free concentration of activator.
Working at substrate concentrations considerably lower than
KM ([S]ꢅKM), and considering that [A]f can be represented in the
form of the total concentration of the enzyme ([E]t) and activator
([A]t), the obtained competitive steady-state equation for deter-
mining the activation constant is given by Equation (5):
n
ꢂ
ꢃ ꢂ
ꢃ
2
1=2
v
¼ v0KA=fKA þ ð½Aꢂt–0:5 ½Aꢂt þ ½Eꢂt þ KA – ½Aꢂt þ ½Eꢂt þ KA –4½Aꢂt½Eꢂt gg (5)
where v0 represents the initial velocity of the enzyme-catalysed
reaction in the absence of an activator35–37. Enzyme concentra-
tions in the assay system were in the range of 9–12 nM.
3. Results and discussion
3.1. Chemistry
As mimic of the main proton shuttling residue (i.e. histidine), his-
tamine (Figure 1) is the main lead for designing CAAs8. The bind-
ing mode of histamine with hCA II was elucidated by X-ray
crystallography (Figure 2)6. In the CAA/enzyme adduct, a complex
network of H-bonds involve the Zn-bound water molecule, His64
and the imidazole ring of the activator, located far away from the
metal ion (Figure 2). Successive X-ray crystallographic studies
showed that many other amines and amino acids (Figure 1) bind
(E)-2-Hydroxybenzaldehyde
O-(2-hydroxy-3-(isopropylamino)-
propyl) oxime maleate 14: Maleate salt. 1H NMR (400 MHz, DMSO- in this area and share flexible tails decorated with protonable moi-
d6): d 9.99(bs, 1H), 8.43 (s, 1H), 8.29 (bs, 1H), 7.54 (d, J ¼ 7.6 Hz, eties (Figure 2)38–40. No isoform-selective CAAs were detected so

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
51
far among these amines and amino acid derivatives, except for 41. Oxyamines 42 and 43 were obtained by aminolysis with NH3-
MeOH 7 N of 40 or 41.
few exceptions (e.g. histamine shows a 10 nM KAs against hCAs
VA and XIV but is a micromolar activator of the remaining iso-
forms)1. The molecule of histamine has been extensively modified
(Figure 3), including substituents on the imidazole C atoms
(A)41,42, replacing the imidazole ring with other heterocycles, such
as 2,4,6-trisubstituted pyridinium (B), 1,3,4-thiadiazole (C) or a
combination of these two ring systems (D)43,44 and functionalising
the NH2 group, as in carboxamides/ureas/thioureas (E)45, sulpho-
namides (F)46, arylsulfonylureas (G)47, bis-histamine (H)48,49, oligo-
peptides49,50, or imidazole/imidazoline derivatives of the alkaloyd
spinaceamine51,52 and drug clonidine53 (Figure 3) were reported
to possess improved CA activatory profile when potency and iso-
form selectivity are concerned.
Interestingly, pharmacological agents born for other medical
applications (Figure 4) showed also to possess more or less rele-
vant CA-activating effects, among which psychoactive compounds
of the amphetamine and methamphetamine family54, the selective
serotonin reuptake inhibitors fluoxetine, sertraline and citalo-
pram55, the phosphodiesterase IV inhibitor sildenafil56, and the
b-blocker amino alcohol derivative timolol57. Timolol was taken as
lead compound in the present study to design a new series of
uncommon CAAs, which do not bear imidazole like scaffolds, inte-
sively explored in the field. The enzyme kinetic method showed
that timolol noncompetitively activates hCA I and II by forming of
a ternary complex consisting of the enzyme, the substrate, and
timolol. Docking studies were used to point out that timolol also
binds at the entrance of the active site cavity nearby the proton
shuttle residue His6457. Thus, a series of aminoalcohol oxime ether
derivatives previously shown to possess b-blocking or analgesic/
antiarrhythmic activity (compounds 1–16)25–32, was investigated
for the activation of a panel of brain hCAs.
The condensation between derivative 42 or 43 and o-, m- or p-
hydroxybenzaldehyde afforded the desired amino alcohols 14–16,
purified by crystallisation as organic salts.
All the final compounds have been achieved as organic salt
oximes presenting E configuration. The configuration of the C¼N
bound for compounds 1, 2, 4–8, 12 and 13 was assigned by ana-
logy considering the E configuration of starting oximes 17, 18,
20–22, 25–27. As previously reported, the configuration of E-
oximes 17, 18, 20–22, 25–27 was stable under the following
reaction conditions.
The E configuration of amino alcohols 14–16 was conferred by
comparison between the H signals of the iminic protons of com-
pounds 14–16 with the same signals of final compounds 1, 2,
4–8, 12 and 13. The chemical shift of iminic proton signal resulted
around d 8.05–8.50 ppm for all derivatives as usually reported for
oximes with E configuration.
1
3.2. Carbonic anhydrase activation
Amino alcohols 1–16 were here assayed for their activating prop-
erties of 4 catalytically active and physiologically relevant hCA iso-
forms expressed in human brain, that are: the cytosolic hCA I, II,
and VII, and the membrane associated hCA IV58. In the CNS con-
text, hCA I is expressed in the motor neurons in human spinal
cord59. The physiologically dominant isoform hCA II is located
both in the choroid plexus, and in oligodendrocytes, myelinated
tracts, astrocytes and myelin sheaths in the vertebrates brain60.
hCA IV is located on the luminal surface of cerebral capillaries,
associated with the blood–brain barrier, and expressed in layers III
and VI in the cortex, thalamus and hippocampus60,61. CA VII is
expressed in the cortex, hippocampus and thalamus62,63. CA VII
might be considered a brain-associated CA as it is predominantly
expressed in the brain, and absent in most other tissues. CA VII is
Compounds 1–6 and 9–16 were prepared as previously
described (Schemes 1 and 2)25–32. Here, an updated synthetic pro-
cedure is reported. Moreover, new 1H NMR and 13 C NMR spectra
of final compounds are provided in the experimental section. A
fully detailed description is provided for the synthesis of new
compounds 7 and 8, following the same synthetic procedure.
Amino alcohols 1–13 were synthesised as reported in Scheme
126–32. Oximes 17–27 were treated with epichlorohydrin in strong
basic conditions affording epoxides 28–38. The amino alcohols
1–13 were obtained by reaction of oxime ethers 28–38 with an
excess of the proper amine, iPrNH2 or tBuNH2. The final com-
pounds were purified by crystallisation as organic salts.
also considered
pH regulation.
a key molecule in age-dependent neuronal
The CA activation data of these 4 isoforms with amino alcohols
1–16 and histamine as standard CAA are shown in Table 1. The
following structure–activity relationship (SAR) can be worked out:
i. A special mention should be done for phenol derivatives
14–16 which, uniquely among amino alcohols 1–16, did not
produce any activation of the tested hCAs. In fact, it is worth
stressing that CAI properties, rather than as CAAs, are com-
monly ascribed to the phenolic chemotype. Phenols have
been thoroughly reported to anchor to the zinc-bound
nucleophile (water molecule or hydroxide ion), that is, by
one of the four CA inhibition mechanisms known to date.
Surprisingly, neither a significant inhibition was detected by
treating CA I, II, IV and VII with phenols 14–16. One could
speculate that the CAI efficacy of 14–16 is counterpoised by
their CAA action, hindering its detection by the Stopped
Flow assay.
The phenolic derivatives 14–16 were prepared as described in
Scheme 2. N-hydroxy-5-norbornene-2,3-dicarboximide was reacted
with epichlorohydrin to give the epoxide 39. The treatment of 39
with an excess of iPrNH2 or tBuNH2 afforded amino alcohol 40 or
ii. The cytosolic isoform hCA I was moderately activated by all
remaining amino alcohols, that are 1–13, with KA values in
the range of 0.92–12.1 mM. The tert-butylamino derivatives
1–8 induce a slightly greater activation of CA I than isopro-
pylamines 9–13. In the 1–8 subset, it can be noted that the
o- or p-substitution of the aromatic ring bearing the oxime
ether group lower the KA values 4, 6, 8 up to fivefold with
respect to 2 (KAs of 1.37, 1.26 and 0.92 vs 4.25 mM). In con-
trast, m-substitutions are deleterious for the binding to the
Figure 1. Natural amino acids and amines investigated for the activation of cata-
lytically active hCA isoforms.

52
A. NOCENTINI ET AL.
Figure 2. Superimposition of CAA – hCA II complexes as determined by X-ray crystallography. (A) Surface view: the hydrophobic half of the active site is coloured red;
His64 is coloured green and the hydrophilic half in blue. (B) Ribbon active site view. The activators are histamine, in green (PDB 1AVN); L-His, in magenta (PDB 2ABE);
L-Phe, in gold (PDB 2FMG); adrenaline, in cyan (PDB 2HKK).
Figure 3. Histamine-based carbonic anhydrase activators.
target as in 5 and 7 (KAs of 7.10 and 6.02 mM). The cyclisation
of the oxime ether group to the aromatic ring as in 1 led to
in the range 0.079–7.19 mM), except for compounds 4 and 13
(KAs of 6.35 vs 5.90 mM). Surprisingly, the m-anisole derivative
5 reported a 100-fold enhancement of activation efficacy of
hCA II when compared to hCA I (KAs of 0.079 vs 7.10 mM).
Most remaining tert-butylamino derivatives showed submi-
cromolar KA values, whereas all compounds of the subset
the second-best CA
I activation after 8 (KAs of 0.94
vs 0.92 mM).
iii. The physiologically most relevant isoform hCA II was acti-
vated by most tested amino alcohols more than hCA I (KAs

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
53
Figure 4. Other pharmacological agents showing hCAs activating properties.
Scheme 1. Preparation of Amino alcohols 1–13. Reagents and conditions: (i) epichlorohydrin, MeONa/MeOH, dry DMF, 60 ꢃC, 1 h; (ii) iPrNH2 or tBuNH2, Benzene, 90ꢃC, 12 h.
Scheme 2. Preparation of Amino alcohols 14–16. Reagents and conditions: (i) epichlorohydrin, Et3N, dry DMF, rt, 18 h; (ii) iPrNH2 or tBuNH2, Benzene, 50ꢃC, 4 h; (iii)
NH3 MeOH 7 N, rt, 2 h; (iv) o-, m-, p-hydroxybenzaldehyde, EtOH, 90 ꢃC, 12 h.

54
A. NOCENTINI ET AL.
Table 1. Activation data of human CA isoforms I, II, IV and VII with amino alcohols 1–16 and histamine as reference CAA by a stopped flow CO2 hydrase assay34
.
KA (mM)a
Compound
R1
R2
Ref
6
hCA I
0.94
hCA II
0.76
hCA IV
1.42
hCA VII
0.89
1
tBu
2
tBu
1,6
4.25
0.98
6.13
0.95
3
4
tBu
tBu
6,7
1
7.54
1.37
7.19
6.35
5.94
4.20
1.03
2.40
5
6
tBu
tBu
1
1
7.10
1.26
0.079
0.31
6.01
5.29
0.42
0.13
7
8
tBu
tBu
-
-
6.02
0.92
2.48
0.47
1.01
7.13
0.74
0.24
9
iPr
iPr
2,6
3,6
12.1
8.15
2.50
1.94
7.73
1.08
0.082
0.091
10
11
12
iPr
iPr
6,7
4
8.14
9.20
3.27
4.02
12.9
3.24
8.86
1.02
13
14
15
iPr
iPr
tBu
5
1
1
4.57
n.d
5.90
3.01
n.d
0.51
n.d
n.d
n.d
n.d
n.d
n.d
16
iPr
1
n.d
2.1
n.d
n.d
n.d
Histamine
–
125
25.3
37.5
aFrom three different assays (errors within 10% of the reported values).
9–13 activate hCA II in a low micromolar range (KAs in the
range 1.94–5.90 mM). All CAAs reported here were more
active towards hCA II than histamine, which is a quite weak
activator of this isoform with a KA of 125 mM.
significant differences exist between tert-butyl and isopropyl
derivatives as for the activation of hCA IV.
v. The other cytosolic isoform investigated here, hCA VII, was
rather potently activated, that is chiefly in a submicromolar
range, by most of the compounds reported in this work (KAs
in the range 0.082–8.86 mM). All derivatives showed much
better activation profile than the reference histamine (KA of
37.6 mM) towards hCA VII. Contrariwise to the other hCAs
tested, the most efficient CAAs are the isopropylamino deriv-
atives 9 and 10, ethers of secondary oxime with an aliphatic
iv. No submicromolar KA values were measured for amino alco-
hols 1–13 as hCA IV activators. Indeed, all KAs are in a rather
flat low micromolar range (KAs in the range 1.01–12.9 mM),
making such a membrane associated isozyme the less acti-
vated by the assayed derivatives. Notably, the reference CAA
histamine even less activates hCA IV with a KA of 25.3 mM. No

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
55
pendant showing medium nanomolar KAs values of 82 and
91 nM, respectively. Only the oxime ethers of p-anisaldehyde
and acetone in the isopropylamino and tert-butylamino ser-
ies, respectively, that are compounds 4 and 11, show KAs
steadily in a low micromolar range. As this isoform is one of
the most widely spread in the brain, and probably involved
in crucial metabolic/pH regulation processes, these results
are of interest in the search of more effective CA VII activa-
tors than the currently available such derivatives.
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In the present study, we described the synthesis and CA activating
effects of a series of oxime ether-based amino alcohols towards
four hCA isoforms expressed in human brain, that are CA I, II, IV
and VII. Except for the phenolic compounds 14–16, all amino alco-
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CAs. In this second youth period for CAAs, innovative pharmaco-
logical studies made these lately neglected agents to draw atten-
tion in the memory therapy and cognitive neurodegenerative
disorders, as well as in therapeutic areas, such as phobias, obses-
sive-compulsive disorder, generalised anxiety, and post-traumatic
stress disorders1,20. As hCA VII is a key isoform involved in brain
metabolism, the here identified potent and selective hCA VII acti-
vators may be considered of interest for investigations of possible
therapeutic applications or as lead compounds in search of more
potent and selective CAAs. This work might bring new lights on
the intricate relationship between CA activation and
brain physiology.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
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ORCID
Alessio Nocentini
Doretta Cuffaro
Lidia Ciccone
http://orcid.org/0000-0003-3342-702X
http://orcid.org/0000-0002-8842-542X
http://orcid.org/0000-0002-2762-1929
Elisa Nuti
http://orcid.org/0000-0003-2669-5376
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tic efficacy, spatial learning, and memory through carbonic
Armando Rossello
Claudiu T. Supuran
http://orcid.org/0000-0002-6795-8091
http://orcid.org/0000-0003-4262-0323

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