Investigation of inhibitory properties of some hydrazone compounds on hCA I, hCA II and AChE enzymes

Article abstract of DOI:10.1016/j.bioorg.2019.02.008

Recently, inhibition of carbonic anhydrase (hCA) and acetylcholinesterase (AChE) have appeared as a promising approach for pharmacological intervention in a variety of disorders such as glaucoma, epilepsy, obesity, cancer, and Alzheimer's disease. Keeping this in mind, N,N′-bis[(1-aryl-3-heteroaryl)propylidene]hydrazine dihydrochlorides, N1-N11, P1, P4-P8, and R1-R6, were synthesized to investigate their inhibitory activity against hCA I, hCA II, and AChE enzymes. All compounds in N, P, and R-series inhibited hCAs (I and II) and AChE more efficiently than the reference compounds acetazolamide (AZA), and tacrine. According to the activity results, the most effective inhibitory compounds were in R-series with the K_i values of 203 ± 55–473 ± 67 nM and 200 ± 34–419 ± 94 nM on hCA I, and hCA II, respectively. N,N′-Bis[1-(4-fluorophenyl)-3-(morpholine-4-yl)propylidene]hydrazine dihydrochlorides, N8, in N-series, N,N′-Bis[1-(4-hydroxyphenyl)-3-(piperidine-1-yl)propylidene]hydrazine dihydrochlorides, P4, in P-series, and N,N′-bis[1-(4-chlorophenyl)-3-(pyrrolidine-1-yl)propylidene]hydrazine dihydrochlorides, R5, in R-series were the most powerful compounds against hCA I with the K_i values of 438 ± 65 nM, 344 ± 64 nM, and 203 ± 55 nM, respectively. Similarly, N8, P4, and R5 efficiently inhibited hCA II isoenzyme with the K_i values of 405 ± 60 nM, 327 ± 80 nM, and 200 ± 34 nM, respectively. On the other hand, P-series compounds had notable inhibitory effect against AChE than the reference compound tacrine and the K_i values were between 66 ± 20 nM and 128 ± 36 nM. N,N′-Bis[1-(4-fluorophenyl)-3-(piperidine-1-yl)propylidene]hydrazine dihydrochlorides, P7, was the most potent compound on AChE with the K_i value of 66 ± 20 nM. The other most promising compounds, N,N′-bis[1-(4-hydroxyphenyl)-3-(morpholine-4-yl)propylidene]hydrazine dihydrochlorides, N4 in N-series and N,N′-bis[1-(4-hydroxyphenyl)-3-(pyrrolidine-1-yl)propylidene]hydrazine dihydrochlorides, R4 in R-series were againts AChE with the K_i values of 119 ± 20 nM, 88 ± 14 nM, respectively.

Full text of DOI:10.1016/j.bioorg.2019.02.008

Accepted Manuscript
Investigation of Inhibitory Properties of Some Hydrazone Compounds on hCA
I, hCA II and AChE Enzymes
Kaan Kucukoglu, Halise Inci Gul, Parham Taslimi, Ilhami Gulcin, Claudiu T.
Supuran
PII:
S0045-2068(18)31417-2
DOI:
https://doi.org/10.1016/j.bioorg.2019.02.008
YBIOO 2783
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 December 2018
28 January 2019
3 February 2019
Please cite this article as: K. Kucukoglu, H. Inci Gul, P. Taslimi, I. Gulcin, C.T. Supuran, Investigation of Inhibitory
Properties of Some Hydrazone Compounds on hCA I, hCA II and AChE Enzymes, Bioorganic Chemistry (2019),
doi: https://doi.org/10.1016/j.bioorg.2019.02.008
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.

Investigation of Inhibitory Properties of Some Hydrazone Compounds on hCA I, hCA II
and AChE Enzymes
Kaan Kucukoglu1*, Halise Inci Gul2, Parham Taslimi3, Ilhami Gulcin3, Claudiu T.
Supuran4
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Selcuk University, Konya-
Turkey
2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Atatürk University,
Erzurum-Turkey
3Department of Chemistry, Faculty of Science, Atatürk University, Erzurum-Turkey.
4Neurofarba Department, Section of Pharmaceutical and Nutriceutical Sciences, Universita
degli Studi di Firenze, Florence-Italy.
Corresponding author: Assoc. Prof. Dr. Kaan Kucukoglu, PhD, Department of
Pharmaceutical Chemistry, Faculty of Pharmacy, Selcuk University, Konya-Turkey; e-mail:
kucukogluk35@hotmail.com
1

Abstract
Recently, inhibition of carbonic anhydrase (hCA) and acetylcholinesterase (AChE) have
appeared as a promising approach for pharmacological intervention in a variety of disorders
such as glaucoma, epilepsy, obesity, cancer, and Alzheimer’s disease. Keeping this in mind,
N,N’-bis[(1-aryl-3-heteroaryl)propylidene]hydrazine dihydrochlorides, N1-N11, P1, P4-P8,
and R1-R6, were synthesized to investigate their inhibitory activity against hCA I, hCA II,
and AChE enzymes. All compounds in N, P, and R-series inhibited hCAs (I and II) and
AChE more efficiently than the reference compounds acetazolamide (AZA), and tacrine.
According to the activity results, the most effective inhibitory compounds were in R-series
with the Ki values of 203±55-473±67 nM and 200±34-419±94 nM on hCA I, and hCA II,
respectively.
N,N’-Bis[1-(4-fluorophenyl)-3-(morpholine-4-yl)propylidene]hydrazine
dihydrochlorides, N8, in N-series, N,N’-Bis[1-(4-hydroxyphenyl)-3-(piperidine-1-
yl)propylidene]hydrazine dihydrochlorides, P4, in P-series, and N,N’-bis[1-(4-chlorophenyl)-
3-(pyrrolidine-1-yl)propylidene]hydrazine dihydrochlorides, R5, in R-series were the most
powerful compounds against hCA I with the Ki values of 438±65 nM, 344±64 nM, and
203±55 nM, respectively. Similarly, N8, P4, and R5 efficiently inhibited hCA II isoenzyme
with the Ki values of 405±60 nM, 327±80 nM, and 200±34 nM, respectively. On the other
hand, P-series compounds had notable inhibitory effect against AChE than the reference
compound tacrine and the Ki values were between 66±20 nM and 128±36 nM. N,N’-Bis[1-(4-
fluorophenyl)-3-(piperidine-1-yl)propylidene]hydrazine dihydrochlorides, P7, was the most
potent compound on AChE with the Ki value of 66±20 nM. The other most promising
compounds,
N,N’-bis[1-(4-hydroxyphenyl)-3-(morpholine-4-yl)propylidene]hydrazine
dihydrochlorides, N4 in N-series and N,N’-bis[1-(4-hydroxyphenyl)-3-(pyrrolidine-1-
yl)propylidene]hydrazine dihydrochlorides, R4 in R-series were againts AChE with the Ki
values of 119±20 nM, 88±14 nM, respectively.
2

Keywords: hydrazone; Mannich base; carbonic anhydrase; acetylcholinesterase; enzyme
inhibition
3

Highlights
 N,
P,
R-series
hydrazone
derivatives,
N,N’-bis[(1-aryl-3-
heteroaryl)propylidene]hydrazine dihydrochlorides, were tested against hCAs and
AChE.
 Ki values were in the range of 203±55 - 738±84 nM against hCA I.
 Ki values were in the range of 200±34 - 710±88 nM against hCA II.
 Ki values were in the range of 66±20 - 308±109 nM against AChE.
 The compounds had potent activity against hCA I, hCA II and AChE.
4

GRAPHICAL ABSTRACT
R5 (Ki 203±55 nM against hCA I)
P7 (Ki 66±20 nM against AChE)
(Ki 200±34 nM against hCA II)
Lead compounds of the study
5

1. Introduction
Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous zinc containing metallo-
enzymes and catalyze the hydration reaction of carbon dioxide into bicarbonate in living
organisms1. There are seven genetically distinct CA families in Bacteria, Archaea, and
Eukarya: α-, β-, γ-, δ-, ζ-, η-, and θ-1-5. CA isoforms present in various tissues in the
cytoplasm, cell membrane, and mitochondria in humans6 are involved in many physiological
and pathological processes such as pH and CO2 homeostasis, respiration, calcification, bone
resorption, electrolyte secretion, biosynthetic reactions (as lipogenesis and gluconeogenesis),
tumorigenicity, etc.7,8 CA inhibitors are used for decades as diuretics9, antiglaucoma
agents1,10, antiepileptics11,12. CA inhibitors have potential as anti-obesity and anti-infective
agents13,14. More recently, it has been shown that not only CA IX and CA XII but also CA I
and CA II isoenzymes have possible roles in tumors as potential targets for cancer therapy15-
17. Because of involving in these vital processes, CA isozymes have been considerable targets
for medicinal chemists16-28
.
Acetylcholinesterase enzyme (AChE, E.C. 3.1.1.7) which is available in all over the
peripheral and central neural systems of humans and animals catalyzes the hydrolysis of the
neurotransmitter acetylcholine (ACh) to choline and acetate29-33. In accordance with
cholinergic hypothesis, imbalances in the cholinergic pathways cause the emerging of
neurodegenerative illnesses such as depression, schizophrenia, and Alzheimer’s disease
(AD)19,34,35. AChE inhibitors have been shown to improve cognitive function and these
inhibitor compounds including donepezil, tacrine, huperzine A, galanthamine, and
rivastigmine have been used as fundamental drugs in AD therapy. Furthermore, in the
treatment of glaucoma and Myasthenia gravis AChE inhibitors are used to modulate
cholinergic function32,33
.
6

Hydrazones are a special group of compounds which are synthesized generally by the
reaction of a stoichiometric amount of substituted hydrazines/hydrazides and carbonyl
compounds such as aldehydes and ketones in suitable solvent under reflux condition36.
Hydrazones, RR-C=N-R’R’’, have two connected nitrogen atoms with different nature. C=N
double bond conjugated with a lone electron pair of the terminal nitrogen atom is available in
hydrazone molecule. The physical and chemical properties of hydrazones are usually
connected to these structural frangments. Nitrogen atoms that are in the hydrazone group have
nucleophilic character, moreover the amino type nitrogen is more reactive. In contrast, the
carbon atom of hydrazone group has electrophilic and nucleophilic character37. Hydrazones
and their derivatives have a great importance in chemistry since they are used as intermediates
for the syntheses of heterocyclic compounds, which are possible ligands for metal complexes
and drug design38. Hydrazones can be easily synthesized, crystallized, and have increased
hydrolytic stability relative to imines. Because of these favourable properties, hydrazones
have been highly studied compounds for a long time. Hydrazones have been reported to have
antibacterial39,40
,
anticonvulsant41,42
,
antitubercular43, antiplatelet44, antitumoral45,46
,
cytotoxic47-51 and antiviral52 activities.
A reactive hydrogen atom, formaldehyde, and secondary amines react together to
synthesize aminomethylated compounds, namely Mannich bases, ordinarily53. Mannich bases
have a great importance in medicinal chemistry and there are some sub Mannich base types
such as carbon Mannich bases and nitrogen Mannich bases54. Various biological activities had
been found in compounds which had Mannich base scaffold as antimicrobial55-57
,
antioxidant58, anti-inflammatory59,60, antifungal61,62, cytotoxic and anticancer23,63-73 and CAs
inhibitory28,74 activities.
In our research laboratory, we designed and synthesized some hydrazone compounds,
N,N’-bis[(1-aryl-3-heteroaryl)propylidene]hydrazine dihydrochlorides, N, P, and R-series by
7

using precursor mono-Mannich bases having 1-aryl-3-heteroaryl-1-propanone structures, and
evaluated their cytotoxic activities, and already published (Table 1)49-51. Here, we investigated
the inhibitory properties of these hydrazone compounds we had presented before against hCA
I, hCA II, and AChE (Table 2).
2. Results and discussion
2.1. Chemistry
The
synthesis
of
N,N’-bis[(1-aryl-3-heteroaryl)propylidene]hydrazine
dihydrochlorides, N1-N11, P1, P4-P8, and R1-R6, was outlined in Scheme 1. First,
corresponding acetophenones were reacted with paraformaldehyde, amine (morpholine HCl;
N-series, piperidine HCl; P-series or pyrrolidine; R-series) and HCl (37%) in ethanol. In the
second step, mono-Mannich bases obtained were stirred with hydrazine hydrate to give final
hydrazone compounds (N, P, and R-series) in ethanolic acetic acid (%3 w/v). Experimental
details, data, and spectral analysis of hydrazones had been presented in our previous studies
(Table 1)49-51
.
2.2. Enzyme inhibition results
In
this
paper,
we
evaluated
the
effects
of
N,N’-bis[(1-aryl-3-
heteroaryl)propylidene]hydrazine dihydrochlorides, N1-N11, P1, P4-P8, and R1-R6
derivatives on hCA I, hCA II, and AchE enzymes.
α-CAs are made up of 16 isoenzymes and expressed widespread in mammals and
humans. These 16 isoenzymes have thioesterase or esterase activity75. In some diseases such
as cancer, activation or aberrant expression of some isoenzymes of α-CAs is observed so
medicinal chemists are interested in the design and development of novel compounds having
CAs inhibition properties76. Methazolamide, acetazolamide, and dorzolamide which inhibited
8

hCA are used for the treatment of glaucoma. Furthermore, acetazolamide is the most
widespread hCA inhibitor77. As AChE inhibitors have been used for the symptomatic
treatment of AD, which is characterized by decreased cholinergic transmission, formation of
tangles, and amyloid plaques and neuronal loss, they have a great utilization worldwide78,79
.
However, most of the AChE inhibitors available have intense side effects, novel molecules
with more powerful and decreased non-desirable effects are urgently needed80. In this study,
inhibitory effects of N,N’-bis[(1-aryl-3-heteroaryl)propylidene]hydrazine dihydrochlorides,
N1-N11, P1, P4-P8, and R1-R6 on the activity of hCA I, hCA II, and AChE enzymes were
tested under in vitro conditions. The following results are presented in Table 2:
Abnormal levels of CA I enzyme in the blood is a marker for hemolytic anemia81. All
the compounds, N,N’-bis[(1-aryl-3-heteroaryl)propylidene]hydrazine dihydrochlorides, N1-
N11, P1, P4-P8, and R1-R6, inhibited the slow cytosolic isoform hCA I with Ki values
ranging between 203±55 and 738±84 nM. N-series compounds showed the inhibitory effect
on hCA I with the Ki values of 438±65-738±84 nM, P-series compounds inhibited hCA I with
the Ki values of 344±64-608±53 nM. 4-Fluoro derivative N8 was the most powerful
compound with the Ki values of 438±65 nM in N-series compounds, 4-hydroxy derivative P4
had the best inhibitory effect on hCA I enzyme with the Ki values of 344±64 nM in P-series
compounds. The best inhibitory results on hCA I enzyme was found with R-series compounds
bearing pyrrolidine as a heteroaryl ring with the Ki values of 203±55-473±67 nM. And among
N, P, and R-series compounds the most powerful compound was R5, N,N’-bis[1-(4-
chlorophenyl)-3-(pyrrolidine-1-yl)propylidene]hydrazine dihydrochlorides, with the Ki values
of 203±55 nM. The standard and clinically used drug acetazolamide (AZA) demonstrated a Ki
value of 983±119 nM (Table 2). Thus, the investigated compounds had better inhibitory
properties compared to AZA.
9

Additionally, CA II isozyme is often related to some diseases such as glaucoma,
osteoporosis, and renal tubular acidosis76. All hydrazone compounds tested againts hCA II
showed notable inhibitory effects with the Ki values ranging between 200±34 and 710±88
nM. N, P, R-series compounds inhibited hCA II with the Ki values of 405±60 nM -710±88
nM for N-series, 327±80 nM-483±102 nM for P-series, and 200±34 nM-419±94 nM for R-
series. The most potent compounds among them were N8, P4, and R5 on hCA II isoenzyme.
N8 had the Ki value of 405±60 nM whereas P4 inhibited hCA II with the Ki value of 327±80
nM. The most effective compound was R5 that had the Ki value of 200±34 nM against hCA II
in all hydrazone compounds. The reference compound AZA had the Ki value of 904±127 nM
against hCA II, so all hydrazone compounds tested had better inhibitory profile compared to
AZA (Table 2).
Overall, N, P, and R-series compounds showed excellent inhibitory activity on AChE
with the Ki values of 119±20-290±59 nM for N-series, 66±20-128±36 nM for P-series, and
88±14-308±109 nM for R-series. Unlike the inhibitory results on hCA I and hCA II, P-series
compounds had the most excellent inhibitory effect on AChE. N,N’-Bis[1-(4-
hydroxyphenyl)-3-(morpholine-4-yl)propylidene]hydrazine dihydrochlorides, N4, had the Ki
value of 119±20 nM in N-series compounds whereas R4 which was a 4-hydroxy derivative
showed inhibitory effect with the Ki value of 88±14 in R-series compounds towards AChE.
The
most
potent
compound
was
N,N’-bis[1-(4-fluorophenyl)-3-(piperidine-1-
yl)propylidene]hydrazine dihydrochlorides, namely P7, which had a 66±20 nM of the Ki
value in three hydrazone series tested. Tacrine, used as a standard AChE inhibitor in this
study, inhibited AChE with the Ki value of 358±72 nM. Thus, these results show N, P, R-
series compounds had better inhibitory profile than the reference compound tacrine. In
addition, P-series were more selective than the others (Table 2).
10

3. Conclusion
In
this
study,
some
hydrazones
synthesized,
N,N’-bis[(1-aryl-3-
heteroaryl)propylidene]hydrazine dihydrochlorides, N1-N11, P1, P4-P8, and R1-R6, tested
against hCA I, hCA II, and AChE. All compounds effectively inhibited metabolic enzymes
of carbonic anhydrase and acetylcholinesterase. The most potent compounds having
inhibitory effect on hCA I and hCA II were N8, P4, and R5. They inhibited efficiently hCA I
with the Ki values of 438±65 nM, 344±64 nM, and 203±55 nM, respectively. And the Ki
values of N8, P4, and R5 against hCA II were 405±60 nM, 327±80 nM, and 200±34 nM,
respectively. In the inhibitory activity results against AChE, N4, P7, and R4 were the most
promising compounds with the Ki values of 119±20 nM, 66±20 nM, and 88±14 nM,
respectively. These compounds stand out promising candidates for further studies.
4. Experimental section
4.1. General information
All commercially available reagents were purchased from Merck AG, Fluka AG,
Acros Organics, Riedel-de Haën, J. T. Baker or Sigma-Aldrich Chemie and used without
further purification. Melting points were measured on an Electrothermal 9100 melting point
1
apparatus (IA9100, Electrothermal, Essex, UK). H (400 MHz) and 13C NMR (100 MHz)
spectra were recorded employing a Varian 400 MHz FT spectrometer (Danbury, USA) for N,
1
P, and R-series hydrazone derivatives, while H NMR (60 MHz) spectra were recorded on a
Varian EM-360 spectrometer for Nm, Pm, and Rm compounds (precursor mono-Mannich
bases).
4.2. Synthesis of precursor mono-Mannich bases, 1-aryl-3-(heteroaryl)-1-propanone
hydrochlorides, (N1m-N11m, P1m, P4m-P8m, and R1m-R6m), (Scheme 1)
They were reported in our previous studies49-51
.
11

4.3. Synthesis of hydrazone compounds, N,N'-bis[(1-aryl-3-heteroaryl)propylidene]hydrazine
dihydrochlorides, (N1-N11, P1, P4-P8, R1-R6, Scheme 1)
They were reported in our previous studies49-51
.
4.4. Biochemical studies
4.4.1. hCA I and hCA II isoenzymes purification and inhibition studies
To observe the inhibition effects of N, P, R-series hydrazone compounds (N1-N11,
P1, P4-P8, and R1-R6) on hCA I, and II isoforms, these enzymes were purified from fresh
human erythrocyte using an affinity chromatography by the procedures of Verpoorte et al.82
as in our previous studies18-25,27,83,84 and the inhibitory effects were determined by
spectrophotometric procedure16-28. In this procedure, changes in activity were obtained during
3 min at 22oC. p-Nitrophenylacetate (PNA) compound was used as a substrate, and it was
converted by both isoforms to p-nitrophenolate ions. The quantity of protein was measured
according to the previously described Bradford method85 and bovine serum albumin was used
as the standard. After the purification method of the CA isoforms, samples were subjected to
SDS polyacrylamide gel electrophoresis (SDS-PAGE). The change in activity was
spectrophotometrically obtained at 348 nm. The IC50 values were calculated from activity (%)
against compounds inhibition. Three different concentrations were used to calculate Ki values.
4.4.2. AChE activity determination
The inhibitory efficacy of the N, P, R-series hydrazone compounds (N1-N11, P1, P4-
P8, and R1-R6) on AChE activity was tested following the spectrophotometric process of
Ellman’s test18,19,24,86. Acetylthiocholine iodide (AChI) was used as substrates. For the
mensuration of the AChE activity, 5,5′-dithio-bis(2-nitro-benzoic)acid compound (DTNB,
D8130-1G, Sigma-Aldrich, Steinheim, Germany) was used. Briefly, 50 μl DTNB and 100 μl
of Tris–HCl solution (1 M, pH 8.0), 750 ml of sample solution dissolved in distilled water at
12

disparate concentrations, and 50 μl AChE (5.32 × 10−3 U) solution were incubated and
mixed for 15 min at 30°C. Finally, the reaction was started by adding 50 μl of AChI. The
enzymatic hydrolysis of this substrate that produces a yellow 5-thio-2-nitrobenzoate anion as
the result of the product of thiocholine with DTNB was recorded spectrophotometrically at a
wavelength of 412 nm.24 Tacrine (TAC) was used as a reference compound.
Acknowledgements
This study was supported by the Research Foundation of Atatürk University Erzurum
(Turkey).
Conflict of interest
There is no conflict of interest.
ORCID
Kaan Kucukoglu
http://orcid.org/0000-0001-8977-9775
References
1. Supuran CT. How many carbonic anhydrase inhibition mechanisms exist? J Enzyme
Inhib Med Chem. 2016;31:345-60.
2. Supuran CT, Capasso C. Carbonic anhydrase from Porphyromonas gingivalis as a
drug target. Pathogens. 2017;6:E30.
3. Capasso C, Supuran CT. Bacterial, fungal and protozoan carbonic anhydrases as drug
targets. Expert Opin Ther Targets. 2015;19:1689-704.
4. Del Prete S, De Luca V, De Simone G, Supuran CT, Capasso C. Cloning, expression
and purification of the complete domain of the η-carbonic anydrase from Plasmodium
falciparum. J Enzyme Inhib Med Chem. 2016;31:54-9.
5. Del Prete S, De Luca V, Vullo D, Osman SM, AlOthman Z, Carginale V, Supuran CT,
Capasso C. A new procedure for the cloning, expression and purification of the β-
carbonic anhydrase from the pathogenic yeast Malassezia globosa, an anti-dandruff
drug target. J Enzyme Inhib Med Chem. 2016;31:1156-61.
13

6. Supuran CT, Scozzafava A. Carbonic anhydrases as targets for medicinal chemistry.
Bioorg Med Chem. 2007;15:4336-50.
7. Ceylan M, Kocyigit UM, Usta NC, Gürbüzlü B, Temel Y, Alwasel SH, Gülçin İ.
Synthesis, carbonic anhydrase I and II isoenzymes inhibition properties, and
antibacterial activities of novel tetralone-based 1,4-benzothiazepine derivatives. J
Biochem Mol Toxicol. 2017;31:e21872.
8. Supuran CT. Advances in structure-based drug discovery of carbonic anhydrase
inhibitors. Expert Opin Drug Discov. 2017;12:61-88.
9. Carta F, Supuran CT. Diuretics with carbonic anhydrase inhibitory action: A patent
and literature review (2005-2013). Expert Opin Ther Pat. 2013;23:681-91.
10. Masini E, Carta F, Scozzafava A, Supuran CT. Antiglaucoma carbonic anhydrase
inhibitors: A patent review. Expert Opin Ther Pat. 2013;23:705-16.
11. Masereel B,
Thiry A,
Dogn M,
Supuran CT. Anticonvulsant
sulfonamides/sulfamates/sulfamides with carbonic anhydrase inhibitory activity: drug
design and mechanism of action. Curr Pharm Des. 2008;14:661-71.
12. Thiry A, Dogn M, Masereel B, Supuran CT. Carbonic anhydrase inhibitors as
anticonvulsant agents. Curr Top Med Chem. 2007;7:855-64.
13. Coban TA, Beydemir S, Gülçin I, Ekinci D. Morphine inhibits erythrocyte carbonic
anhydrase in vitro and in vivo. Biol Pharm Bull. 2007;30:2257-61.
14. Coban TA, Beydemir S, Gülçin I, Ekinci D. The effect of ethanol on erythrocyte
carbonic anhydrase isoenzymes activity: an in vitro and in vivo study. J Enzyme Inhib
Med Chem. 2008;23:266-70.
15. Mboge MY, Mahon BP, McKenna R, Frost SC. Carbonic anhydrases: role in pH
control and Cancer. Metabolites. 2018;8:E19.
16. Gul HI, Yamali C, Bulbuller M, Kirmizibayrak PB, Gul M, Angeli A, Bua S, Supuran
CT. Anticancer effects of new dibenzenesulfonamides by inducing apoptosis and
autophagy pathways and their carbonic anhydrase inhibitory effects on hCA I, hCA II,
hCA IX, hCA XII isoenzymes. Bioorg Chem. 2018;78:290-7.
17. Gul HI, Yamali C, Sakagami H, Angeli A, Leitans J, Kazaks A, Tars K, Ozgun DO,
Supuran CT. New anticancer drug candidates sulfonamides as selective hCA IX or
hCA XII inhibitors. Bioorg Chem. 2018;77:411-9.
18. Yamali C, Gul HI, Ece A, Taslimi P, Gulcin I. Synthesis, molecular modeling, and
biological evaluation of 4-[5-aryl-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl]
benzenesulfonamides toward acetylcholinesterase, carbonic anhydrase I and II
enzymes. Chem Biol Drug Des. 2018;91:854-66.
14

19. Gul HI, Demirtas A, Ucar G, Taslimi P, Gulcin I. Synthesis of Mannich bases by two
different methods and evaluation of their acetylcholine esterase and carbonic
anhydrase inhibitory activities. Lett Drug Des Discov. 2017;14:573-80.
20. Gul HI, Mete E, Taslimi P, Gulcin I, Supuran CT. Synthesis, carbonic anhydrase I and
II inhibition studies of the 1,3,5-trisubstituted-pyrazolines. J Enzyme Inhib Med
Chem. 2017;32:189-92.
21. Mete E, Comez B, Inci Gul H, Gulcin I, Supuran CT. Synthesis and carbonic anydrase
inhibitory activities of new thienyl-substituted pyrazoline benzenesulfonamides. J
Enzyme Inhib Med Chem. 2016;31:1-5.
22. Kucukoglu K, Oral F, Aydin T, Yamali C, Algul O, Sakagami H, Gulcin I, Supuran
CT, Gul HI. Synthesis, cytotoxicity and carbonic anhydrase inhibitory activities of
new pyrazolines. J Enzyme Inhib Med Chem. 2016;31:20-4.
23. Inci Gul H, Yamali C, Tugce Yasa A, Unluer E, Sakagami H, Tanc M, Supuran CT.
Carbonic anhydrase inhibition and cytotoxicity studies of Mannich base derivatives of
thymol. J Enzyme Inhib Med Chem. 2016;31:1375-80.
24. Ozgun DO, Yamali C, Gul HI, Taslimi P, Yanik T, Supuran CT. Inhibitory effects of
isatin Mannich bases on carbonic anhydrases, acetylcholinesterase, and
butyrylcholinesterase. J Enzyme Inhib Med Chem. 2016;31:1498-501.
25. Gul HI, Yazici Z, Tanc M, Supuran CT. Inhibitory effects of benzimidazole
containing new phenolic Mannich bases on human carbonic anhydrase isoforms hCA I
and II. J Enzyme Inhib Med Chem. 2016;31:1540-4.
26. Yamali C, Tugrak M, Gul HI, Tanc M, Supuran CT. The inhibitory effects of phenolic
Mannich bases on carbonic anhydrase I and II isoenzymes. J Enzyme Inhib Med
Chem. 2016;31:1678-81.
27. Gul HI, Kucukoglu K, Yamali C, Bilginer S, Yuca H, Ozturk I, Taslimi P, Gulcin I,
Supuran CT. Synthesis of 4-(2-substituted hydrazinyl)benzenesulfonamides and their
carbonic anhydrase inhibitory effects. J Enzyme Inhib Med Chem. 2016;31:568-73.
28. Bilginer S, Unluer E, Gul HI, Mete E, Isik S, Vullo D, Ozensoy-Guler O, Beyaztas S,
Capasso C, Supuran CT. Carbonic anhydrase inhibitors. Phenols incorporating 2-or 3-
pyridyl-ethenylcarbonyl and tertiary amine moieties strongly inhibit Saccharomyces
cerevisiae β-carbonic anhydrase. J Enzyme Inhib Med Chem. 2014;29:495-9.
29. Öztaşkın N, Çetinkaya Y, Taslimi P, Göksu S, Gülçin İ. Antioxidant and
acetylcholinesterase inhibition properties of novel bromophenol derivatives. Bioorg
Chem. 2015;60:49-57.
30. Sujayev A, Garibov E, Taslimi P, Gulçin İ, Gojayeva S, Farzaliyev V, Alwasel SH,
Supuran CT. Synthesis of some tetrahydropyrimidine-5-carboxylates, determination of
their metal chelating effects and inhibition profiles against acetylcholinesterase,
15

butyrylcholinesterase and carbonic anhydrase. J Enzyme Inhib Med Chem
2016;31:1531-9.
31. Turan B, Şendil K, Şengül E, Gültekin MS, Taslimi P, Gulçin İ, Supuran CT. The
synthesis of some β-lactams and investigation of their metal-chelating activity,
carbonic anhydrase and acetylcholinesterase inhibition profiles. J Enzyme Inhib Med
Chem. 2016;31:79-88.
32. Özbey F, Taslimi P, Gülçin İ, Maraş A, Göksu S, Supuran CT. Synthesis of diaryl
ethers with acetylcholinesterase, butyrylcholinesterase and carbonic anhydrase
inhibitory actions. J Enzyme Inhib Med Chem. 2016;31:79-85.
33. Garibov E, Taslimi P, Sujayev A, Bingol Z, Çetinkaya S, Gulçin İ, Beydemir S,
Farzaliyev V, Alwasel SH, Supuran CT. Synthesis of 4,5-disubstituted-2-thioxo-
1,2,3,4-tetrahydropyrimidines and investigation of their acetylcholinesterase,
butyrylcholinesterase, carbonic anhydrase I/II inhibitory and antioxidant activities. J
Enzyme Inhib Med Chem. 2016;31:1-9.
34. Taslimi P, Sujayev A, Mamedova S, Kalın P, Gulçin İ, Sadeghian N, Beydemir S,
Kufrevioglu OI, Alwasel SH, Farzaliyev V, Mamedov S. Synthesis and bioactivity of
several new hetaryl sulfonamides. J Enzyme Inhib Med Chem. 2017;32:137-45.
35. Topal F, Gulcin I, Dastan A, Guney M. Novel eugenol derivatives: Potent
acetylcholinesterase and carbonic anhydrase inhibitors. Int J Biol Macromol.
2017;94:845-51.
36. Solomons TWG, Fryhle CB. Organic Chemistry Asia, 10th ed., John Wiley & Sons,
Medford, NY, USA, 2011.
37. Alhadi AA, Shaker SA, Wagee AY, Ali HM, Abdullah MA. Synthesis, magnetic and
spectroscopic studies of Ni(II), Cu(II), Zn(II) and Cd(II) complexes of a newly Schiff
base derived from 5-bromo-2-hydroxybezylidene)-3,4,5 trihydroxybenzohydrazide.
Bull Chem Soc Ethiop. 2012;26:95-101.
38. Charles D, Turner JH, Redmond C. The endometrial karyotypic profiles of women
after clomiphene citrate therapy. J Obstet Gynaecol Br Commonw. 1973;80:264-70.
39. Kumar P, Narasimhan B, Sharma D, Judge V, Narang R. Hansch analysis of
substituted benzoic acid benzylidene/furan-2-yl-methylene hydrazides as antimicrobial
agents. Eur J Med Chem. 2009;44:1853-63.
40. Sherman AR, Bicyclic 5-6 systems: two heteroatoms 1:1, in: Katritzky AR, Ramsden
CA, Scriven EFV & Taylor JK (Eds.), Comprehensive Heterocyclic Chemistry III,
Vol. 10, Elsevier, Oxford, 2008, pp 263-338.
41. Dimmock JR, Vashishtha SC, Stables JP. Anticonvulsant properties of various
acetylhydrazones, oxamoylhydrazones and semicarbazones derived from aromatic and
unsaturated carbonyl compounds. Eur J Med Chem. 2000;35:241-8.
16

42. Gul HI, Calis U, Vepsalainen J. Synthesis of some mono-Mannich bases and
corresponding azine derivatives and evaluation of their anticonvulsant activity.
Arzneimittelforschung. 2004;54:359-64.
43. Joshi SD, Vagdevi HM, Vaidya VP, Gadaginamath GS. Synthesis of new 4-pyrrol-1-
yl benzoic acid hydrazide analogs and some derived oxadiazole, triazole and pyrrole
ring systems: a novel class of potential antibacterial and antitubercular agents. Eur J
Med Chem. 2008;43:1989-96.
44. Fraga AGM, Rodrigues CR, Miranda ALP, Barreiro EJ, Fraga CAM. Synthesis and
pharmacological evaluation of novel heterocyclic acylhydrazone derivatives, designed
as PAF antagonists. Eur J Pharm Sci. 2000;11:285-90.
45. Pandey J, Pal R, Dwivedi A, Hajela K. Synthesis of some new diaryl and triaryl
hydrazone
derivatives
as
possible
estrogen
receptor
modulators.
Arzneimittelforschung. 2002;52:39-44.
46. Abadi AH, Eissa AA, Hassan GS. Synthesis of novel 1,3,4-trisubstituted pyrazole
derivatives and their evaluation as antitumor and antiangiogenic agents. Chem. Pharm.
Bull. 2003;51:838-44.
47. Gul HI, Das U, Pandit B, Li PK. Evaluation of the cytotoxicity of some mono-
mannich bases and their corresponding azine derivatives against androgen-
independent prostate cancer cells. Arzneimittelforschung. 2006;56:850-4.
48. Kucukoglu K, Gul M, Atalay M, Mete E, Kazaz C, Hanninen O, Gul HI. Synthesis of
some Mannich bases with dimethylamine and their hydrazones and evaluation of their
cytotoxicity against transformed Jurkat cells. Arzneimittelforschung. 2011;61:366-71.
49. Kucukoglu K, Gul HI, Cetin-Atalay R, Baratli Y, Charles AL, Sukuroglu M, Gul M,
Geny B. Synthesis of new N,N’-bis[1-aryl-3-(piperidine-1-yl)propylidene]hydrazine
dihydrochlorides and evaluation of their cytotoxicity against human hepatoma and
breast cancer cells. J Enzyme Inhib Med Chem. 2014;29:420-6.
50. Kucukoglu K, Gul HI, Gul M, Cetin-Atalay R, Baratli Y, Geny B. Cytotoxicity of
hydrazones of morpholine bearing Mannich bases towards Huh7 and T47D cell lines
and their effects on mitochondrial respiration. Lett Drug Des Discov. 2016;13:734-41.
51. Kucukoglu K, Gul M, Gul HI, Cetin-Atalay R, Geny B. Cytotoxicities of novel
hydrazone compounds with pyrrolidine moiety: inhibition of mitochondrial respiration
may be a possible mechanism of action for the cytotoxicity of new hydrazones. Med
Chem Res. 2018;27:2116-24.
52. Abdel-Aal MT, El-Sayed WA, El-Ashry el-SH. Synthesis and antiviral evaluation of
some sugar arylglycinoylhydrazones and their oxadiazoline derivatives. Arch Pharm
(Weinheim). 2006;339:656-63.
17

53. Dimmock JR, Kumar P. Anticancer and cytotoxic properties of Mannich bases. Curr
Med Chem. 1997;4:1-22.
54. Roman G. Mannich bases in medicinal chemistry and drug design. Eur J Med Chem.
2015;89:743-816.
55. Gul HI, Sahin F, Gul M, Ozturk S, Yerdelen KO. Evaluation of antimicrobial
activities of several mannich bases and their derivatives. Arch Pharm (Weinheim).
2005;338:335-8.
56. Gul M, Atalay M, Gul HI, Nakao C, Lappalainen J, Hänninen O. The effects of some
Mannich bases on heat shock proteins HSC70 and GRP75, and thioredoxin and
glutaredoxin levels in Jurkat cells. Toxicol In Vitro. 2005;19:573-80.
57. Gul M, Gul HI, Das U, Hanninen O. Biological evaluation and structure-activity
relationships of bis-(3-aryl-3-oxo-propyl)-methylamine hydrochlorides and 4-aryl-3-
arylcarbonyl-1-methyl-4-piperidinol hydrochlorides as potential cytotoxic agents and
their alkylating ability towards cellular glutathione in human leukemic T cells.
Arzneimittelforschung. 2005;55:332-7.
58. Shen A-Y, Huang M-H, Liao L-F, Wang T-S. Thymol analogues with antioxidant and
L-type calcium current inhibitory activity. Drug Develop Res. 2005;64:195-202.
59. Gul HI, Suleyman H, Gul M. Evaluation of the antiinflammatory activity of N,N’-
bis(3-dimethylamino-1-phenylpropylidene) hydrazine dihydrochloride. Pharm Biol.
2009;47:968-72.
60. Şahin YN, Demircan B, Süleyman H, Aksoy H, Gul HI. The effects of 3-benzoyl-1-
methyl-4-phenyl-4-piperidinolhydrochloride (C1), indomethacin, nimesulide and
rofecoxib on cyclooxygenase activities in carrageenan-induced paw edema model.
Turk J Med Sci. 2010;40:723-8.
61. Mete E, Gul HI, Bilginer S, Algul O, Topaloglu ME, Gulluce M, Kazaz C. Synthesis
and antifungal evaluation of 1-aryl-2-dimethyl-aminomethyl-2-propen-1-one
hydrochlorides. Molecules. 2011;16:4660-71.
62. Mete E, Ozelgul C, Kazaz C, Yurdakul D, Sahin F, Inci Gul H. Synthesis and
antifungal activity of 1-aryl-3-phenethylamino-1-propanone hydrochlorides and 3-
aroyl-4-aryl-1-phenethyl-4-piperidinols. Arch Pharm (Weinheim). 2010;343:291-300.
63. Bilginer S, Gul HI, Mete E, Das U, Sakagami H, Umemura N, Dimmock JR. 1-(3-
Aminomethyl-4-hydroxyphenyl)-3-pyridinyl-2-propen-1-ones: a novel group of
tumour-selective cytotoxins. J Enzyme Inhib Med Chem. 2013;28:974-80.
64. Tugrak M, Yamali C, Sakagami H, Gul HI. Synthesis of mono Mannich bases of 2-(4-
hydroxybenzylidene)-2,3-dihydroinden-1-one and evaluation of their cytotoxicities. J
Enzyme Inhib Med Chem. 2016;31:818-23.
18

65. Tugrak M, Gul HI, Sakagami H. Synthesis and cytotoxicities of 2-[4-hydroxy-(3,5-
bis-aminomethyl)-benzylidene]-indan-1-ones. Lett Drug Des Discov. 2015;12:806-12.
66. Yerdelen KO, Gul HI, Sakagami H, Umemura N. Synthesis and biological evaluation
of 1,5-bis(4-hydroxy-3-methoxyphenyl)penta-1,4-dien-3-one and its aminomethyl
derivatives. J Enzyme Inhib Med Chem. 2015;30:383-8.
67. Yerdelen KO, Gul HI, Sakagami H, Umemura N, Sukuroglu M. Synthesis and
cytotoxic activities of a curcumin analogue and its bis-Mannich derivatives. Lett Drug
Des Discov. 2015;12:643-9.
68. Gul HI, Tugrak M, Sakagami H. Synthesis of some acrylophenones with N-
methylpiperazine and evaluation of their cytotoxicities. J Enzyme Inhib Med Chem.
2016;31:147-51.
69. Unluer E, Gul HI, Demirtas A, Sakagami H, Umemura N, Tanc M, Kazaz C, Supuran
CT. Synthesis and bioactivity studies of 1-aryl-3-(2-hydroxyethylthio)-1-propanones.
J Enzyme Inhib Med Chem. 2016;31:105-9.
70. Yamali C, Gul HI, Sakagami H, Supuran CT. Synthesis and bioactivities of halogen
bearing phenolic chalcones and their corresponding bis Mannich bases. J Enzyme
Inhib Med Chem. 2016;31:125-31.
71. Kucukoglu K, Mete E, Cetin-Atalay R, Gul HI. Synthesis of 3-aroyl-4-aryl-1-
isopropylamino-4-piperidinols and evaluation of the cytotoxicities of the compounds
against human hepatoma and breast cancer cell lines. J Enzyme Inhib Med Chem.
2015;30:564-8.
72. Tugrak M, Gul HI, Sakagami H, Mete E. Synthesis and anticancer properties of mono
Mannich bases containing vanillin moiety. Med Chem Res. 2017;26:1528-34.
73. Yamali C, Ozgun DO, Gul HI, Sakagami H, Kazaz C, Okuidara N. Synthesis and
structure
elucidation
of
1-(2,5/3,5-difluorophenyl)-3-(2,3/2,4/2,5/3,4-
dimethoxyphenyl)-2-propen-1-ones as anticancer agents. Med Chem Res.
2017;26:2015-23.
74. Büyükkidan N, Büyükkidan B, Bülbül M, Özer S, Gonca Yalçin H. Synthesis and
characterization of phenolic Mannich bases and effects of these compounds on human
carbonic anhydrase isozymes I and II. J Enzyme Inhib Med Chem. 2013;28:337-42.
75. Taslimi P, Gülçin İ, Öztaşkın N, Çetinkaya Y, Göksu S, Alwasel SH, Supuran CT.
The effects of some bromophenols on human carbonic anhydrase isoenzymes. J
Enzyme Inhib Med Chem. 2016;31:603-7.
76. Supuran CT. Carbonic anhydrases and metabolism. Metabolites. 2018;25:1-5.
19

77. Scozzafava A, Passaponti M, Supuran CT, Gülçin İ. Carbonic anhydrase inhibitors:
guaiacol and catechol derivatives effectively inhibit certain human carbonic anhydrase
isoenzymes (hCA I, II, IX and XII). J Enzyme Inhib Med Chem. 2015;30:586-91.
78. Akıncıoğlu A, Topal M, Gülçin İ, Göksu S. Novel sulphamides and sulphonamides
incorporating the tetralin scaffold as carbonic anhydrase and acetylcholine esterase
inhibitors. Arch Pharm (Weinheim). 2014;347:68-76.
79. Ofek K, Soreq H. Cholinergic involvement and manipulation approaches in multiple
system disorders. Chem Biol Interact. 2013;203:113-9.
80. Tayeb HO, Yang HD, Price BH, Tarazi FI. Pharmacotherapies for Alzheimer’s
disease: beyond cholinesterase inhibitors. Pharmacol Ther. 2012;134:8-25.
81. Kucuk M, Gulcin I. Purification and characterization of carbonic anhydrase enzyme
from Black Sea trout (Salmo trutta Labrax Coruhensis) kidney and inhibition effects
of some metal ions on the enzyme activity. Environ Toxicol Pharmacol. 2016;44:134-
9.
82. Verpoorte JA, Mehta S, Edsall JT. Esterase activities of human carbonic anhydrases B
and C. J Biol Chem. 1967;242:4221-9.
83. Tugrak M, Inci Gul H, Sakagami H, Gulcin I, Supuran CT. New azafluorenones with
cytotoxic and carbonic anhydrase inhibitory properties: 2-Aryl-4-(4-hydroxyphenyl)-
5H-indeno[1,2-b]pyridin-5-ones. Bioorg Chem. 2018;81:433-9.
84. Gul HI, Tugrak M, Sakagami H, Taslimi P, Gulcin I, Supuran CT. Synthesis and
bioactivity studies on new 4-(3-(4-substitutedphenyl)-3a,4-dihydro-3H-indeno[1,2-
c]pyrazol-2-yl) benzenesulfonamides. J Enzyme Inhib Med Chem. 2016;31:1619-24.
85. Bradford MM. A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal Biochem.
1976;72:248-54.
86. Ellman GL, Courtney KD, Andres V Jr, Feather-Stone RM. A new and rapid
colorimetric determination of acetylcholinesterase activity. 1961;7:88-95.
20

Scheme
1.
Synthesis
of
Hydrazone
Compounds,
N,N’-bis[(1-aryl-3-
heteroaryl)propylidene]hydrazine Dihydrochlorides (N1-N11; P1, P4-P8; and R1-R6).
Reagents and conditions: (a) Paraformaldehyde, piperidine HCl/morpholine HCl/Pyrrolidine,
HCl (37%) and EtOH, 1-9 h reflux for N1m-N11m; P1m, P4m-P8m; R1m-R6m; (b)
Ethanolic acetic acid (3% w/v), hydrazine hydrate stirring for 17-26 h exception R1 for N1-
N11; P1, P4-P8; R2-R6 and 3 h reflux for R1.
21

Table
1.
Synthesized
Hydrazone
Compounds,
N,N’-bis[(1-aryl-3-
heteroaryl)propylidene]hydrazine Dihydrochlorides (N1-N11; P1, P4-P8; and R1-R6).
Compound
Yield (%)
Substitution on Phenyl
Ring
N1
N2
N3
N4
N5
N6
N7
N8
N9
N10
N11
P1
-
2450
2650
6750
5650
6050
1650
950
4-CH3
4-OCH3
4-OH
4-Cl
2-OH
3-OCH3
4-F
5250
5650
8650
7450
5749
6449
4849
8849
1249
1449
3551
651
4-Br
3-OH
2-OCH3
-
P4
4-OH
4-Cl
P5
P6
3-OCH3
4-F
P7
P8
4-Br
R1
R2
R3
R4
R5
R6
-
4-CH3
4-OCH3
4-OH
4-Cl
3451
5051
2451
751
3-OCH3
22

Table 2. Enzyme inhibition results of hydrazone compounds, N1-N11; P1, P4-P8; R1-R6, against hCA I, hCA II and AChE enzymes.
Compound
IC50 (nM)
hCA II
Ki (nM)
hCA II
703±67
683±128
678±105
500±59
602±195
553±94
710±88
405±60
471±54
529±102
666±118
573±92
327±80
421±73
hCA I
704.28
694.18
728.40
548.18
601.73
572.06
737.03
483.08
508.36
551.04
700.88
583.77
359.63
424.63
r2
r2
AChE
308.84
348.03
331.83
173.18
238.37
208.74
385.01
273.98
207.38
198.97
228.16
197.73
116.30
100.43
r2
hCA I
730±100
738±84
709±110
559±78
628±93
601±104
704±203
438±65
501±90
592±148
684±111
608±53
344±64
483±102
AChE
206±39
200±50
248±58
119±20
186±42
149±29
290±59
209±83
146±48
130±35
154±46
105±21
84±17
N1
N2
N3
N4
N5
N6
N7
N8
N9
N10
N11
P1
09814
0.9911
0.9803
0.9716
0.9598
0.9901
0.9881
0.9937
0.9638
0.9810
0.9717
0.9716
0.9812
0.9764
684.73
652.04
692.84
507.83
573.84
538.91
693.84
429.05
483.27
503.98
649.83
522.64
308.94
383.64
0.9598
0.9865
0.9911
0.9793
0.9582
0.9704
0.9793
0.9488
0.9937
0.9858
0.9695
0.9905
0.9728
0.9935
0.9817
0.9811
0.9845
0.9490
0.9709
0.9638
0.9918
0.9726
0.9917
0.9820
0.9672
0.9518
0.9704
0.9822
P4
P5
68±17
23

P6
P7
522.54
403.42
384.51
403.72
484.72
405.17
253.17
234.92
374.92
997.304
-
0.9699
0.9866
0.9682
0.9716
0.9816
0.9529
0.9910
0.9582
0.9717
0.9889
-
461.53
374.15
330.62
371.53
409.64
400.63
218.26
233.83
357.12
915.50
-
0.9816
0.9782
0.9923
0.9822
0.9633
0.9812
0.9726
0.9294
0.9728
0.9719
-
174.62
92.53
0.9712
0.9890
0.9609
0.9712
0.9973
0.9891
0.9498
0.9683
0.9723
-
501±93
439±60
403±111
458±83
473±67
411±134
243±43
203±55
411±99
983±119
-
483±102
388±73
369±71
401±83
419±94
364±49
216±58
200±34
384±107
904±127
-
128±36
66±20
100±32
243±48
308±109
251±79
88±14
100±16
227±98
-
P8
126.93
304.82
369.26
312.55
105.82
113.84
288.02
-
R1
R2
R3
R4
R5
R6
AZA
Tacrine
443.312
0.9948
358±72
24