α-Carbonic anhydrases are strongly activated by spinaceamine derivatives

Article abstract of DOI:10.1016/j.bmc.2019.01.017

A series of 4-substituted-spinaceamine (4,5,6,7-tetrahydro-imidazolo[4,5-c]pyridine) were prepared from histamine and aromatic aldehydes Schiff bases, and investigated as activators of four human (h) carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic hCA I, II and VII, and the membrane-associated hCA IV. All isoforms were effectively activated by the new derivatives, and the nature of the moiety in position 4 of the bicyclic system was the factor influencing activation properties against all isoforms. For hCA I, these compounds showed K_As in the range of 2.52–21.5 μM, the most effective activator being 4-(2-hydroxyphenyl)-spinaceamine. For hCA II the activation constants ranged between 0.60 and 17.2 μM, with 4-(2,3,5,6-tetrafluorophenyl)- spinaceamine the best activator. Affinity for hCA IV was in the range of 0.52–63.8 μM, and the same compound as for hCA II was the most effective activator. The most sensitive isoform for activation was the brain-associated hCA VII, for which K_As in the range of 82 nM–4.26 μM were observed. Effective hCA VII activators were the (2-bromophenyl)-, 2,3,5,6-tetrafluorophenyl- and furyl-substituted spineaceamines (K_As of 82–95 nM). As CA activators may have pharmacologic applications in various fields, this work provides interesting derivatives for further studies.

Full text of DOI:10.1016/j.bmc.2019.01.017

Accepted Manuscript
α-Carbonic anhydrases are strongly activated by spinaceamine derivatives
Suleyman Akocak, Nabih Lolak, Silvia Bua, Alessio Nocentini, Gulcin
Karakoc, Claudiu T. Supuran
PII:
S0968-0896(19)30001-X
https://doi.org/10.1016/j.bmc.2019.01.017
BMC 14709
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 January 2019
18 January 2019
21 January 2019
Please cite this article as: Akocak, S., Lolak, N., Bua, S., Nocentini, A., Karakoc, G., Supuran, C.T., α-Carbonic
anhydrases are strongly activated by spinaceamine derivatives, Bioorganic & Medicinal Chemistry (2019), doi:
https://doi.org/10.1016/j.bmc.2019.01.017
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

α-Carbonic anhydrases are strongly activated by spinaceamine derivatives
Suleyman Akocak a*, Nabih Lolak a, Silvia Bua b, Alessio Nocentini b, Gulcin Karakoc a,
Claudiu T. Supuran b*
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Adiyaman University, 02040
Adiyaman, Turkey
bUniversità degli Studi di Firenze, Dipartimento Neurofarba, Sezione di Scienze Farmaceutiche e
Nutraceutiche, Via U. Schiff 6, 50019 Sesto Fiorentino (Florence), Italy.
Abstract. A series of 4-substituted-spinaceamine (4,5,6,7-tetrahydro-imidazolo[4,5-c]pyridine)
were prepared from histamine and aromatic aldehydes Schiff bases, and investigated as
activators of four human (h) carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic hCA I,
II and VII, and the membrane-associated hCA IV. All isoforms were effectively activated by the
new derivatives, and the nature of the moiety in position 4 of the bicyclic system was the factor
influencing activation properties against all isoforms. For hCA I, these compounds showed KAs
in the range of 2.52 – 21.5 µM, the most effective activator being 4-(2-hydroxyphenyl)-
spinaceamine. For hCA II the activation constants ranged between 0.60 – 17.2 µM, with 4-
(2,3,5,6-tetrafluorophenyl)- spinaceamine the best activator. Affinity for hCA IV was in the
range of 0.52 – 63.8 µM, and the same compound as for hCA II was the most effective activator.
The most sensitive isoform for activation was the brain-associated hCA VII, for which KAs in the
range of 82 nM – 4.26 µM were observed. Effective hCA VII activators were the (2-
bromophenyl)-, 2,3,5,6-tetrafluorophenyl- and furyl-substituted spineaceamines (KAs of 82-95
nM). As CA activators may have pharmacologic applications in various fields, this work
provides interesting derivatives for further studies.
Keywords: carbonic anhydrase; activator; spinaceamine; histamine; proton shuttle.
_____
*Corresponding authors: Tel: +90 (416) 223 3800-2836, Fax: +90 (416) 223 3842, E-mail:
akocaksuleyman@gmail.com, sakocak@adiyaman.edu.tr, (Suleyman Akocak);
Tel: +39-055-4573729, Fax: +39-055-4573385, E-mail: claudiu.supuran@unifi.it (Claudiu T.
Supuran)

1. Introduction
Activation of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) was already
reported in the early ‘40s by several independent groups1-5, with amines (e.g., histamine), amino
acids and some short peptides. However, this field became rather controversial soon thereafter,
since many researchers working on CAs considered that such effective catalysts do not “need to
be activated”6. It has even been suggested that this phenomenon does not exist at all, being an
artefact due to the complexation of adventitious heavy metals (which inhibit the enzyme activity)
present in the enzyme preparations by the activators6-8. This rather long period of controversy
dated till the early ‘90s, when work with highly purified enzymes and very precise techniques,
such as the stopped-flow assay and the X-ray crystallography, undoubtedly demonstrated that the
CA activators (CAAs) exist and that they take part to the catalytic cycle9-11. In 1990, a general
mechanism of action for the CAAs has been proposed based on equation 1 9:
EZn2+-OH2 + A[EZn2+-OH2 - A] [EZn2+HO- - AH+] EZn2+HO- + AH+ (1)
enzyme - activator complexes
According to this model, the activator binds within the enzyme active site with the formation of
enzyme – activator complexes9, in which the activator molecule participates to the rate-
determining step of the catalytic cycle, i.e., proton shuttling between the zinc-coordinated water
molecule and the environment, with the formation of the zinc hydroxide species of the enzyme.
In fact, in many CA isoforms, it has been shown that residue His64 placed in the middle of the
active site cavity is involved in this phenomenon, acting as a proton shuttle during the catalytic
cycle12. Tu et al.12 demonstrated this by means of site-directed mutagenesis and extensive kinetic
measurements, showing that His64 through its imidazole moiety (with a pKa of around 7), is
crucial for proton shuttling and the generation of the nucleophilically active species of the
enzyme, according to equations 2 and 3 which describes the CA catalytic cycle:
-
-
EZn2+OH- + CO2  EZn2+HCO3  EZn2+-OH2 + HCO3 (2)
EZn2+-OH2  EZn2+HO- + H+
(3)

Work from several groups showed that the rate limiting step of the entire catalytic cycle
for the CO2 hydration reaction catalyzed by CAs is the proton transfer step (equation 3) 11-13
.
Confirmation that activators have a similar role to His64, i.e., shuttling of the protons from the
active site to the environment and facilitation of the formation of the nucleophilic enzyme
species, came from the first X-ray crystallographic structure of isoform CA II complexed with
histamine as activator11. Histamine was observed bound at the entrance of the active site cavity,
far away from the metal ion, and participating in a network of hydrogen bonds involving several
water molecules, which, as in the case of His64, favor the release of the proton from the water
molecule coordinated to the zinc, to the reaction medium11. X-ray crystal structures with many
other amine and amino acid CAAs were thereafter reported14-21, which reinforced the above
findings: all activators bind in the same active site region, at the entrance of the cavity, from
where they can enhance the formation of the zinc hydroxide species of the enzyme, by favoring
the proton shuttling between the cavity and the reaction medium13. Furthermore, recently it has
also been shown the CAAs may have pharmacological applications for enhancing cognition, in
the management of CA deficiencies, for therapy memory and for obtaining artificial tissues22,23
.
Thus, there is a strong interest in designing CAAs belonging to various chemical classes, with a
more effective and isoform-selective profile compared to amines and amino acids from the first
generation of such derivatives13, such as histamine, dopamine, serotonin and natural amino acids,
which may have other pharmacological activities connected to the histaminergic, dopaminergic,
serotoninergic activities, in addition to their activating effects in many CA isoforms. Here, we
explored the synthesis and CA activating effects on four human (h) CA isoforms with a series of
spinaceamine derivatives.
2. Results and Discussion
2.1. Chemistry
Spinaceamine (SP), an alkaloid isolated from the skin of amphibians from Australia and
Papua New Guinea, may be considered a cyclization product of histamine24. This bicyclic
derivative incorporates two basic nitrogen atoms (in the imidazolic and six-membered cycle)
which in principle may be protonated and thus participate to proton shuttling when bound to the
CA active site. The rationale for designing CAAs presented in this work is based on our previous

data which showed efficient CA activating effects for derivatized histamine Schiff base
compounds25. Therefore, in this work, a number of structurally diverse spinaceamine substituted
compounds (ring-closure product of histamine Schiff bases)26,27 were synthesized according to
general synthetic route illustrated in Scheme 1. In order to generate chemical diversity, different
substituted aldehydes were chosen, possessing aromatic and heterocyclic moieties, and they were
reacted with histamine leading to the new spinaceamine substituted compounds SP(1-14)
(Scheme 1). All the synthesized compounds SP(1-14) were fully characterized by using several
analytical and spectral data (see experimental part for details).
In the current work, the synthesis of the series of substituted spinaceamines SP(1-14) was
carried out with some modifications of the literature procedures26,27. Briefly, histamine
dihydrochloride was coupled with substituted aromatic/heterocyclic aldehydes, leading after
oxidative ring-closure under heating to the desired bicylic derivatives. The structures of
substituted spinaceamines SP(1-14) were confirmed by using several analytical and spectral data
(FT-IR, 1H-NMR, 13C-NMR, etc.) as described in the experimental part.
Scheme 1. General synthetic route for the synthesis of 4-substituted spinaceamines SP(1-14).

2.2. Carbonic anhydrase activation
Considering the fact that the new heterocyclic derivatives SP(1-14) reported here
incorporate in their molecules functionalities with an appropriate pKa for acting as proton
shuttles in the CA catalytic cycle, we have investigated them as CAAs against the following four
CA isoforms with important physiological functions: the three cytosolic enzymes hCA I, II and
VII29, and the membrane-associated hCA IV30. They are involved in various pathologies, both in
the CNS, kidneys, eyes and other organs in which they are highly abundant29-31
.
Table 1. CA activation data with histamine spinaceamines SP1-SP4 and histamine (HST) as a
standard activator by a stopped-flow CO2 hydrase assay28.
Compound
KA (µM)*
hCA IV
hCA I
hCA II
hCA VII
SP1
8.12
17.6
6.27
6.01
3.45
4.89
2.52
4.30
12.7
4.90
3.76
9.28
9.92
21.5
2.10
16.3
8.49
17.2
7.31
7.44
7.25
1.62
2.80
15.5
1.03
0.71
3.26
0.60
5.12
125
63.8
50.1
7.06
6.37
3.14
5.80
3.59
4.05
4.42
10.9
6.17
2.84
0.52
2.40
4.03
2.69
0.84
1.03
0.73
0.59
4.26
0.11
0.082
0.38
0.12
0.26
0.093
0.11
0.095
37.6
SP2
SP3
SP4
SP5
SP6
SP7
SP8
SP9
SP10
SP11
SP12
SP13
SP14
HST

* Mean from 3 different determinations (errors in the range of 5-10 % of the reported values,
data not shown).
The following structure-activity relationship (SAR) can be evidenced from data of Table 1:
(i)
The cytosolic isoform hCA I was moderately activated by all compounds reported
here with KA values in the range of 2.52-21.5 µM. The substituted spinaceamines
were slightly weaker activators compared to the starting, standard compound
histamine (HST, KA = 2.10 µM), which has a moderate activity on hCA I. In
particular, compound SP7 bearing the 2-OH-phenyl substitution showed the best
activation against this isoform with a KA of 2.52 µM. The activation constants of the
other derivatives were close to each other and were ranging between 3.45 and 9.92
µM, except for compounds SP2 (4-Cl), SP9 (2-Me, 5-Br) and SP14 (furyl) which
were less effective activators compared to the other derivatives (KA values of 17.6,
12.7 and 21.5 µM, respectively).
(ii)
All compounds reported here SP(1-14) were more active CAAs against one of most
abundant cytosolic isoform, hCA II, compared to histamine, which is a quite weak
activator of this isoform with a KA of 125 µM. In this series of compounds, the best
hCA II activators were SP7 (2-OH), SP10 (2-OH, 5-Br), SP11 (3,4-diMeO) and
SP13 (2,3,5,6-F) with KAs in the range of 0.60-1.62 µM, being thus several orders of
magnitude better CAAs compared to histamine. Compounds SP1 (4-H), SP3 (4-Me)
and SP9 (2-Me, 5-Br) were the least active CAAs in the series, with KA values of
16.3, 17.2, and 15.5 µM. The structure-activity data demonstrate that the
spinaceamine derivatives SP(1-14) show much better hCA II activation potency than
their counterparts, non-cyclic histamine Schiff base derivatives, which were reported
recently by us and showed ineffective activity against this isoform25.
(iii) The membrane-bound isoform hCA IV was moderately activated by most of the
compounds reported here. SP13 (2,3,5,6-F) showed a good potency, with an
activation constant of 0.52 µM. The activation constants of the remaining compounds
ranged from 2.40 µM to 10.9 µM, except the SP1 (4-H) and SP2 (4-Cl), which were
the weakest activators in the series, with KAs of 63.8 and 50.1 µM, respectively.

(iv)
The other cytosolic isoform investigated here, hCA VII, was efficiently activated by
most of the compounds reported in this work. All derivatives showed much better
activation profile than the lead histamine (KA of 37.6 µM) against hCA VII. Only
three compounds showed KAs > 1 µM, i.e., compounds SP1 (4-H), SP3 (4-Me) and
SP6 (4-CN) with KA values of 2.69, 1.03 and 4.26 µM, respectively. The remaining
derivatives were much more effective CAAs: SP8 (2-Br), SP12 (2,3,4-triMeO) and
SP14 (furyl) showed nanomolar activation potency, with KAs in the range of 82-95
nM. As this isoform is one of the most widely spread in the brain, and probably
involved in crucial metabolic/pH regulation processes, our results seem to be very
promising in the search of more effective CA VII activators than the currently
available such derivatives. The rest of the investigated compounds were also good
activators, with KA values ranging between 0.11 µM and 0.84 µM. Another important
finding in the current work is the good selectivity observed for hCA VII activation
over the other physiologically related isoforms, such as hCA I, II and IV (e.g., SP8
was a nanomolar hCA VII activators whereas it has micromolar activity against the
remaining three isoforms – Table 1).
3. Conclusions
In the present study, a series of 4-substituted-spinaceamine derivatives SP(1-14) were
synthesized from histamine by using aromatic and heterocyclic aldehydes. The obtained
compounds were investigated and assessed as activators of four human (h) carbonic anhydrase
isoforms, the cytosolic hCA I, II and VII, and the membrane-associated hCA IV. All the
compounds from this study showed great activity against the tested isoforms that most of the
compounds were having better affinity than starting compound histamine. Specifically, hCA VII,
was greatly activated by most of the tested compounds which some of them having nanomolar
potency (82-95 nM). Since CAAs are getting more attention in the memory therapy and
cognitive neurodegenerative disorders, and hCA VII is a key isoform involved in brain
metabolism, these potent hCA VII activators reported here may be considered of interest for in
vivo investigations for possible therapeutic applications.

4. Experimental
4.1. Chemistry
All chemicals and anhydrous solvents were purchased from Sigma-Aldrich, Merck, Alfa
Aesar and TCI and used without further purification. Melting points (mp) were determined with
SMP30 melting point apparatus in open capillaries and are uncorrected. FT-IR spectra were
recorded by using Perkin Elmer Spectrum 100 FT-IR spectrometer. Nuclear Magnetic Resonance
(1H-NMR and 13C-NMR) spectra of compounds were recorded using a Bruker Advance III 300
MHz spectrometer in DMSO-d6 and TMS as an internal standard operating at 300 MHz for 1H-
NMR and 75 MHz for 13C-NMR. Thin layer chromatography (TLC) was carried out on Merck
silica gel 60 F254 plates
4.1.1. General procedure for the synthesis of Spinaceamine substituted compounds
SP(1-14)
To solution of 5 mmol of histamine dihydrochloride in 10 ml of water were added
solutions of 15 mmol of sodium hydroxide (NaOH) and 5 mmol of appropriate aldehyde
derivatives in 15 ml of ethanol. The reaction mixture was heated overnight at around 80 0C and
allowed to slow evaporation of the solvent. The completion of the reaction was monitored by
TLC and FT-IR. After that, the mixture was allowed to cool to room temperature and separated
precipitate was filtered off. The obtained final compounds SP(1-14) recrystallized from hot
water and dried under vacuum at 40 0C and fully characterized by FT-IR, 1H-NMR, 13C-NMR,
and melting points.
4-phenyl-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine (SP1): Yield: 66%; Color: white
powder, mp: 195-198 0C; FT-IR (cm-1): 3312, 3024, 2956, 2917, 2804, 1619, 1438, 1364, 972,
813; 1H-NMR (CD3OD, 300 MHz, δ ppm): 7.80 (s, 1H, H-2 Im), 7.62 (m, 3H, Ar-H), 7.35 (m,
2H, Ar-H), 5.12 (s, 1H, -CH-), 3.32 (m, 2H,-CH2CH2-Im), 2.95 (t, 2H, J = 6.2, -CH2CH2-Im):
13C-NMR (CD3OD, 75 MHz, δ ppm): 140.5, 135.3, 134.4, 129.7, 128.2, 127.4, 113.8, 64.1, 39.7,
30.1.

4-(4-chlorophenyl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine (SP2): Yield: 52%; Color:
white powder, mp: 170-173 0C; FT-IR (cm-1): 3334, 3036, 2955, 2906, 2806, 1622, 1441, 1015,
805; 1H-NMR (CD3OD, 300 MHz, δ ppm): 7.72 (s, 1H, H-2 Im), 7.50 (d, 2H, J = 7.5, Ar-H),
7.28 (d, 2H, J = 7.5, Ar-H), 5.15 (s, 1H, -CH-), 3.25 (m, 2H,-CH2CH2-Im), 2.96 (t, 2H, J = 6.0, -
CH2CH2-Im), 2.41 (s, 3H, -CH3): 13C-NMR (CD3OD, 75 MHz, δ ppm): 139.8, 136.7, 134.8,
134.1, 129.1, 127.8, 114.5, 64.5, 40.6, 30.8, 21.5.
4-(p-tolyl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine (SP3): Yield: 45%; Color: white
powder; mp: 180-183 0C; FT-IR (cm-1): 3345, 3012, 2921, 2841, 1607, 1435, 973, 809; 1H-NMR
(CD3OD, 300 MHz, δ ppm): 7.68 (s, 1H, H-2 Im), 7.48 (d, 2H, J = 7.2, Ar-H), 7.22 (d, 2H, J =
7.2, Ar-H), 5.12 (s, 1H, -CH-), 3.18 (m, 2H,-CH2CH2-Im), 2.90 (t, 2H, J = 6.2, -CH2CH2-Im),
2.38 (s, 3H, -CH3): 13C-NMR (CD3OD, 75 MHz, δ ppm): 139.5, 136.1, 134.6, 133.9, 128.2,
127.5, 114.7, 64.2, 40.3, 30.3, 21.7.
4-(4-methoxyphenyl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine (SP4): Yield: 50%;
Color: light yellow; mp: 180-183 0C; FT-IR (cm-1): 3329, 3028, 2955, 2809, 1609, 1442, 1365,
1009, 813; 1H-NMR (CD3OD, 300 MHz, δ ppm): 7.81 (s, 1H, H-2 Im), 7.58 (d, 2H, J = 7.0, Ar-
H), 7.33 (d, 2H, J = 7.0, Ar-H), 5.16 (s, 1H, -CH-), 3.88 (s, 3H, -OCH3), 3.26 (m, 2H,-CH2CH2-
Im), 2.97 (t, 2H, J = 6.0, -CH2CH2-Im): 13C-NMR (CD3OD, 75 MHz, δ ppm): 158.6, 139.5,
136.2, 134.3, 133.6, 129.4, 127.5, 113.7, 64.0, 55.8, 39.4, 29.3.
4-N,N-dimethyl-phenyl-(4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine (SP5): Yield: 62%;
Color: orange; mp: 90-93 0C; FT-IR (cm-1): 3330, 3024, 2914, 2845, 1605, 1443, 1354, 946, 816;
1H-NMR (CD3OD, 300 MHz, δ ppm): 7.85 (s, 1H, H-2 Im), 7.63 (d, 2H, J = 7.7, Ar-H), 7.42 (d,
2H, J = 7.5, Ar-H), 5.22 (s, 1H, -CH-), 3.32 (m, 2H,-CH2CH2-Im), 3.18 (s, 6H, -N(CH3)2), 2.99
(t, 2H, J = 5.5, -CH2CH2-Im): 13C-NMR (CD3OD, 75 MHz, δ ppm): 150.4, 139.1, 136.7, 134.3,
133.2, 129.1, 126.9, 114.5, 64.2, 40.8, 39.8, 29.7.
4-(4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridin-4-yl)benzonitrile (SP6): Yield: 55%; Color:
light yellow; mp: 200-203 0C; FT-IR (cm-1): 3322, 3158, 3020, 2963, 2848, 2231, 1615, 1452,
1388, 971, 841; 1H-NMR (CD3OD, 300 MHz, δ ppm): 7.88 (s, 1H, H-2 Im), 7.53 (d, 2H, J = 6.9,

Ar-H), 7.33 (d, 2H, J = 7.2, Ar-H), 5.20 (s, 1H, -CH-), 3.28 (m, 2H,-CH2CH2-Im), 2.98 (t, 2H, J
= 6.2, -CH2CH2-Im): 13C-NMR (CD3OD, 75 MHz, δ ppm): 141.2, 137.3, 134.9, 134.0, 128.7,
127.6, 118.8, 114.3, 64.5, 40.4, 30.5.
2-(4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridin-4-yl)phenol (SP7): Yield: 48%; Color: light
brown; mp: 195-198 0C; FT-IR (cm-1): 3342, 3320, 3150, 3035, 2962, 28021, 1610, 1458, 1345,
978, 815; 1H-NMR (CD3OD, 300 MHz, δ ppm): 13.45 (br.s, 1H, -OH), 7.80 (s, 1H, H-2 Im),
7.50 (m, 3H, Ar-H), 7.38 (m, 2H, Ar-H), 5.15 (s, 1H, -CH-), 3.29 (m, 2H,-CH2CH2-Im), 2.95 (t,
2H, J = 6.2, -CH2CH2-Im): 13C-NMR (CD3OD, 75 MHz, δ ppm): 159.3, 136.2, 134.5, 133.6,
128.2, 126.9, 118.1, 113.3, 64.2, 39.4, 29.5.
4-(2-bromophenyl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine (SP8): Yield: 55%; Color:
white powder; mp: 120-122 0C; FT-IR (cm-1): 3125, 3028, 2975, 2816, 1633, 1463, 967, 832;
1H-NMR (CD3OD, 300 MHz, δ ppm): 7.92 (s, 1H, H-2 Im), 7.63 (m, 3H, Ar-H), 7.45 (m, 2H,
Ar-H), 5.21 (s, 1H, -CH-), 3.38 (m, 2H,-CH2CH2-Im), 2.99 (t, 2H, J = 6.5, -CH2CH2-Im): 13C-
NMR (CD3OD, 75 MHz, δ ppm): 141.2, 137.3, 134.9, 133.8, 128.1, 126.5, 118.5, 113.9, 64.7,
39.9, 30.1.
4-(5-bromo-2-methylphenyl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine (SP9): Yield:
70%; Color: white powder; mp: 198-200 0C; FT-IR (cm-1): 3345, 3121, 3020, 2942, 2829, 1613,
1479, 971, 832; 1H-NMR (CD3OD, 300 MHz, δ ppm): 7.88 (s, 1H, H-2 Im), 7.63 (d, 1H, Ar-H),
7.48 (s, 1H, Ar-H), 7.12 (s, 1H, Ar-H), 5.11 (s, 1H, -CH-), 3.32 (m, 2H,-CH2CH2-Im), 2.94 (t,
2H, J = 5.5, -CH2CH2-Im), 2.12 (s, 3H, -CH3): 13C-NMR (CD3OD, 75 MHz, δ ppm): 141.8,
137.9, 134.5, 133.5, 128.7, 126.9, 118.3, 115.6, 113.2, 64.2, 39.5, 30.5, 20.3.
4-bromo-2-(4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridin-4-yl)phenol (SP10): Yield: 73%;
Color: light yellow; mp: 136-139 0C; FT-IR (cm-1): 3284, 3032, 2959, 2816, 1609, 1479, 1251,
963, 816; 1H-NMR (CD3OD, 300 MHz, δ ppm): 13.65 (br.s, 1H, -OH), 7.82 (s, 1H, H-2 Im),
7.58 (d, 1H, Ar-H), 7.42 (s, 1H, Ar-H), 7.10 (s, 1H, Ar-H), 5.05 (s, 1H, -CH-), 3.30 (m, 2H,-
CH2CH2-Im), 2.92 (t, 2H, J = 5.8, -CH2CH2-Im): 13C-NMR (CD3OD, 75 MHz, δ ppm): 157.2,
136.3, 133.9, 133.1, 128.5, 126.3, 118.6, 115.3, 113.1, 64.0, 39.2, 29.4.

4-(3,4-dimethoxyphenyl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine (SP11): Yield: 43%;
Color: brown; mp: 162-165 0C; FT-IR (cm-1): 3321, 3018, 2939, 2833, 1627, 1450, 1261, 952,
808; 1H-NMR (CD3OD, 300 MHz, δ ppm): 7.70 (s, 1H, H-2 Im), 7.48 (s, 1H, Ar-H), 7.31 (d, 1H,
J = 6.9, Ar-H), 6.95 (d, 1H, J = 6.9, Ar-H), 5.01 (s, 1H, -CH-), 3.88 (s, 3H, -OCH3), 3.79 (s, 3H,
-OCH3), (3.25 (m, 2H,-CH2CH2-Im), 2.89 (t, 2H, J = 5.8, -CH2CH2-Im): 13C-NMR (CD3OD, 75
MHz, δ ppm): 150.3, 149.6, 134.5, 133.7, 128.2, 126.8, 118.2, 115.1, 113.5, 64.1, 55.7, 55.1,
39.7, 29.8.
4-(3,4,5-trimethoxyphenyl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine (SP12): Yield:
65%; Color: white powder; mp: 148-150 0C; FT-IR (cm-1): 3298, 3109, 2947, 2831, 1588, 1419,
1238, 970, 818; 1H-NMR (CD3OD, 300 MHz, δ ppm): 7.72 (s, 1H, H-2 Im), 7.38 (s, 1H, Ar-H),
7.25 (s, 1H, Ar-H), 5.03 (s, 1H, -CH-), 3.90 (s, 6H, -OCH3), 3.82 (s, 3H, -OCH3), (3.22 (m, 2H,-
CH2CH2-Im), 2.85 (t, 2H, J = 5.5, -CH2CH2-Im): 13C-NMR (CD3OD, 75 MHz, δ ppm): 151.6,
138.6, 134.2, 133.5, 128.1, 126.3, 118.7, 115.6, 114.0, 64.3, 55.9, 55.2, 40.3, 30.4.
4-(2,3,5,6-tetrafluorophenyl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine (SP13): Yield:
55%; Color: white powder; mp: 220-222 0C; FT-IR (cm-1): 3320, 3105, 2967, 2848, 1620, 1435,
1243, 971, 820; 1H-NMR (CD3OD, 300 MHz, δ ppm): 7.75 (s, 1H, H-2 Im), 6.95 (s, 1H, Ar-H),
5.01 (s, 1H, -CH-), (3.20 (m, 2H,-CH2CH2-Im), 2.87 (t, 2H, J = 5.5, -CH2CH2-Im): 13C-NMR
(CD3OD, 75 MHz, δ ppm): 149.6, 137.5, 134.1, 133.7, 128.5, 126.2, 118.4, 115.1, 114.3, 64.5,
40.5, 29.7.
4-(furan-2-yl)-4,5,6,7-tetrahydro-3H-imidazo[4,5-c]pyridine (SP14): Yield: 55%; Color: light
brown mp: 95-97 0C; FT-IR (cm-1): 3284, 3085, 2961, 2825, 1613, 1446, 1365, 1009, 813; 1H-
NMR (CD3OD, 300 MHz, δ ppm): 7.89 (s, 1H, H-2 Im), 7.78 (d, 1H, J = 5.5, furyl ), 6.45 (d,
1H, J = 5.5, furyl), 6.28 (m, 1H, furyl), 5.33 (s, 1H, -CH-), 3.35 (m, 2H,-CH2CH2-Im), 2.99 (t,
2H, J = 6.5, -CH2CH2-Im): 13C-NMR (CD3OD, 75 MHz, δ ppm): 151.7, 143.8, 139.2, 134.1,
118.3, 114.5, 106.3, 63.5, 39.1, 28.7.

4.2. CA activation
An Sx.18Mv-R Applied Photophysics (Oxford, UK) stopped-flow instrument has been
used to assay the catalytic activity of various CA isozymes for CO2 hydration reaction28. Phenol
red (at a concentration of 0.2 mM) was used as indicator, working at the absorbance maximum of
557 nm, with 10 mM Hepes (pH 7.5) as buffer, 0.1 M NaClO4 (for maintaining constant ionic
strength), following the CA-catalyzed CO2 hydration reaction for a period of 10 s at 25 ◦C. The
CO2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters
and inhibition constants. For each activator 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 activators (at 0.1
mM) were prepared in distilled-deionized water and dilutions up to 1 nM were made thereafter
with the assay buffer. Enzyme and activator solutions were pre-incubated together for 15 min
prior to assay, in order to allow for the formation of the enzyme–activator complexes. The
activation constant (KA), defined similarly with the inhibition constant KI, can be obtained by
considering the classical Michaelis–Menten equation (equation 4), which has been fitted by non-
linear least squares by using PRISM 3:
v =vmax/{1+(KM/[S])(1+[A]f/KA)}
(4)
where [A]f is the free concentration of activator.
Working at substrate concentrations considerably lower than KM ([S] <<KM), 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 determining the activation
constant is given by equation 5:
v=v0.KA/{KA+([A]t-0.5{([A]t+[E]t+KA)-([A]t+[E]t+KA)2-4[A]t.[E]t)1/2}} (5)
where v0 represents the initial velocity of the enzyme-catalyzed reaction in the absence of
activator32-35
.

Acknowledgments
This work was partially supported by The Scientific and Technological Research Council
of Turkey (TUBITAK), Research Fund Project No: 215Z484. G.K. acknowledges the TUBITAK
for undergrad research grant (TUBITAK 2209-A undergrad research grant program)
References and notes
1. Leiner, M. Das Ferment Kohlensäureanhydrase im Tierkörper. Naturwiss. 1940; 28: 316-317
2. Leiner M, Leiner G. Die Aktivatoren der Kohlensäureanhydrase. Naturwiss. 1941; 29: 195-
197.
3. Leiner M, Leiner G. Die Messmethoden zur Untersuchung der katalytischen Wirksamkeit der
Kohlensäureanhydrase. Biochem. Z. 1941; 311: 119-145.
4. Main RE, Locke A. Activation of carbonic anhydrase by histamine. J. Biol. Chem. 1941; 140:
LXXXI.
5. Kiese M. Die Aktivierung der Kohlensäureanhydrase. Naturwiss. 1941; 29: 116-117.
6. Clark AM, Perrin DD. A re-investigation of the question of activators of carbonic anhydrase.
Biochem. J. 1951; 48: 495-503.
7. Ho C, Sturtevant JM. Activation of bovine carbonic anhydrase by ethylenediamine tetraacetic
acid. Biochem. Biophys. Res. Commun. 1960; 3: 20-23.
8. Silverman DN, Backman L, Tu C. Role of hemoglobin in proton transfer to the active site of
carbonic anhydrase. J Biol Chem. 1979; 254: 2588-2591.
9. Supuran CT. Carbonic anhydrase activators. Part 4. A general mechanism of action for
activators of isozymes I, II and III. Rev. Roum. Chim. 1992; 37: 411-421.
10. Supuran CT, Balaban, AT. Carbonic anhydrase activators. Part 8. pKa - Activation
relationship in a series of amino acid derivatives activators of isozyme II. Rev. Roum. Chim.
1994; 39: 107-113.
11. Briganti F, Mangani S, Orioli P, Scozzafava A, Vernaglione G, Supuran CT. Carbonic
anhydrase activators: X-ray crystallographic and spectroscopic investigations for the interaction
of isozymes I and II with histamine. Biochemistry. 1997; 36: 10384-92.
12. Tu CK, Silverman DN, Forsman C, Jonsson BH, Lindskog S. Role of histidine 64 in the
catalytic mechanism of human carbonic anhydrase II studied with a site-specific mutant.
Biochemistry. 1989; 28: 7913-7918.
13. Supuran CT. Carbonic anhydrase activators. Future Med Chem. 2018; 10: 561-573.
14. Temperini C, Scozzafava A, Supuran CT. Carbonic anhydrase activation and the drug design.
Curr Pharm Des. 2008; 14: 708-715.

15. Temperini C, Scozzafava A, Vullo D, Supuran CT. Carbonic anhydrase activators.
Activation of isozymes I, II, IV, VA, VII, and XIV with l- and d-histidine and crystallographic
analysis of their adducts with isoform II: engineering proton-transfer processes within the active
site of an enzyme. Chemistry. 2006; 12: 7057-7066.
16. Temperini C, Scozzafava A, Vullo D, Supuran CT. Carbonic anhydrase activators.
Activation of isoforms I, II, IV, VA, VII, and XIV with L- and D-phenylalanine and
crystallographic analysis of their adducts with isozyme II: stereospecific recognition within the
active site of an enzyme and its consequences for the drug design. J Med Chem. 2006; 49: 3019-
3027.
17. Temperini C, Innocenti A, Scozzafava A, Supuran CT. Carbonic anhydrase activators:
kinetic and X-ray crystallographic study for the interaction of D- and L-tryptophan with the
mammalian isoforms I-XIV. Bioorg Med Chem. 2008; 16: 8373-8378.
18. Temperini C, Innocenti A, Scozzafava A, Mastrolorenzo A, Supuran CT. Carbonic anhydrase
activators: L-Adrenaline plugs the active site entrance of isozyme II, activating better isoforms I,
IV, VA, VII, and XIV. Bioorg Med Chem Lett. 2007; 17: 628-635.
19. Temperini C, Scozzafava A, Puccetti L, Supuran CT. Carbonic anhydrase activators: X-ray
crystal structure of the adduct of human isozyme II with L-histidine as a platform for the design
of stronger activators. Bioorg Med Chem Lett. 2005; 15: 5136-5141.
20. Temperini C, Scozzafava A, Supuran CT. Carbonic anhydrase activators: the first X-ray
crystallographic study of an adduct of isoform I. Bioorg Med Chem Lett. 2006; 16: 5152-5156.
21. Bhatt A, Mondal UK, Supuran CT, Ilies MA, McKenna R. Crystal Structure of Carbonic
Anhydrase II in Complex with an Activating Ligand: Implications in Neuronal Function. Mol
Neurobiol. 2018; 55: 7431-7437.
22. Canto de Souza L, Provensi G, Vullo D, Carta F, Scozzafava A, Costa A, Schmidt SD,
Passani MB, Supuran CT, Blandina P. Carbonic anhydrase activation enhances object
recognition memory in mice through phosphorylation of the extracellular signal-regulated kinase
in the cortex and the hippocampus. Neuropharmacology. 2017; 118: 148-156.
23. a) Wang X, Schröder HC, Schlossmacher U, Neufurth M, Feng Q, Diehl-Seifert B, Müller
WE. Modulation of the initial mineralization process of SaOS-2 cells by carbonic anhydrase
activators and polyphosphate. Calcif Tissue Int. 2014; 94: 495-509; b) Sanku RKK, John JS, Ilies
MA, Walker EA. Potential learning and memory disruptors and enhancers in a simple, 1-day
operant task in mice. Behavioural Pharmacol. 2018; 29: 482-492.
24. Roseghini M, Endean R, Temperilli A. New and uncommon indole- and imidazole-
alkylamines in skins of amphibians from Australia and Papua New Guinea. Z Naturforsch C.
1976; 31: 118-120.
25. Akocak S, Lolak N, Vullo D, Durgun M, Supuran CT. Synthesis and biological evaluation of
histamine Schiff bases as carbonic anhydrase I, II, IV, VII, and IX activators. J Enzyme Inhib
Med Chem. 2017; 32: 1305-1312.

26. Smolyar NN, Abramyants MG, Yutilov YM. Dehydrogenation of 4-Phenyl-Substituted
Spinaceamine and Spinacine. Russ. J. Org. Chem. 2006; 42: 541-544.
27. Smolyar NN, Abramyants MG, Zavyazkina TI, Matveeva DI, Borodkin YS, Voloskii IA.
Synthesis and dehydrogenation of Spinaceamine and Spinacine 4-hetaryl derivatives. Russ. J.
Org. Chem. 2009; 45: 1219-1223.
28. Khalifah RG. The carbon dioxide hydration activity of carbonic anhydrase. I. Stop-flow
kinetic studies on the native human isoenzymes B and C. J Biol Chem. 1971; 246: 2561-2573.
29. Supuran CT. Carbonic anhydrase inhibitors and their potential in a range of therapeutic areas.
Expert Opin Ther Pat. 2018; 28: 709-712.
30. a) Supuran CT. Applications of carbonic anhydrases inhibitors in renal and central nervous
system diseases. Expert Opin Ther Pat. 2018; 28: 713-721; b) Lolak N, Akocak S, Bua S, Koca
M, Supuran CT. Design and synthesis of novel 1,3-diaryltriazene-substituted sulfonamides as
potent and selective carbonic anhydrase II inhibitors. Bioorg. Chem. 2018; 77: 542-547; c) Lolak
N, Akocak S, Bua S, Supuran CT. Design, synthesis and biological evaluation of novel ureido
benzenesulfonamides incorporating 1,3,5-triazine moieties as potent carbonic anhydrase IX
inhibitors. Bioorg. Chem. 2019; 82: 117-122.
31. a) Supuran CT. Advances in structure-based drug discovery of carbonic anhydrase inhibitors.
Expert Opin Drug Discov. 2017; 12: 61-88; b) Akocak S, Ilies MA. Next-generation primary
sulfonamide carbonic anhydrase inhibitors. In: Supuran CT, Capasso C (Eds) Targeting Carbonic
Anhydrases, Future Science, London, 2014; 35-51; c) Akocak S, Alam MR, Shabana AM, Sanku
RKK, Vullo D, Thompson H, Swenson ER, Supuran CT, Ilies MA. PEGylated bis-sulfonamide
carbonic anhydrase inhibitors can efficiently control the growth of several carbonic anhydrase
IX-expressing carcinomas. J. Med. Chem. 2016; 59: 5077-5088.
32. Angeli A, Kuuslahti M, Parkkila S, Supuran CT. Activation studies with amines and amino
acids of the α-carbonic anhydrase from the pathogenic protozoan Trypanosoma cruzi. Bioorg
Med Chem. 2018; 26: 4187-4190.
33. a) Angeli A, Del Prete S, Alasmary FAS, Alqahtani LS, AlOthman Z, Donald WA, Capasso
C, Supuran CT. The first activation studies of the η-carbonic anhydrase from the malaria parasite
Plasmodium falciparum with amines and amino acids. Bioorg Chem. 2018; 80: 94-98; b)
Draghici B, Vullo D, Akocak S, Walker EA, Supuran CT, Ilies MA. Ethylene bis-imidazoles are
highly potent and selective activators for isozymes VA and VII of carbonic anhydrase, with a
potential nootropic effect. Chem. Commun. 2014; 50: 5980-5983; c) Akocak S, Lolak L,
Nocentini A, Karakoc G, Tufan A, Supuran CT. Synthesis and biological evaluation of novel
aromatic and heterocyclic bis-sulfonamide Schiff bases as carbonic anhydrase I, II, VII and IX
inhibitors. Bioorg. Med. Chem. 2017; 25(12): 3093-3097.
34. Stefanucci A, Angeli A, Dimmito MP, Luisi G, Del Prete S, Capasso C, Donald WA, Mollica
A, Supuran CT. Activation of β- and γ-carbonic anhydrases from pathogenic bacteria with
tripeptides. J Enzyme Inhib Med Chem. 2018; 33: 945-950.
35. a) Angeli A, Alasmary FAS, Del Prete S, Osman SM, AlOthman Z, Donald WA, Capasso C,
Supuran CT. The first activation study of a δ-carbonic anhydrase: TweCAδ from the diatom
Thalassiosira weissflogii is effectively activated by amines and amino acids. J Enzyme Inhib Med
Chem. 2018; 33: 680-685; b) Akocak S, Lolak N, Bua S, Supuran CT. Discovery of novel 1,3-
diaryltriazene sulfonamides as carbonic anhydrase I, II, VII and IX inhibitors. J Enzyme Inhib
Med Chem. 2018; 33: 1575-1580; c) Akocak S, Lolak L, Bua S, Turel I, Supuran CT. Synthesis
and biological evaluation of novel N,N’-diaryl cyanoguanidines acting as potent and selective
carbonic anhydrase II inhibitors. Bioorg. Chem. 2018; 77: 245-251.

Graphical Abstract

Highlights

In the current study, a series of 4-substituted-spinaceamine derivatives SP(1-14) were
synthesized.


The spinaceamine derivatives were investigated as hCA I, II, IV and VII activators.
Some of the compounds showed nanomolar potency against hCA VII, which is a brain involved
isoform.

Very good selectivity observed for hCA VII activation over the other physiologically related
isoforms, such as hCA I, II and IV.