Schiff bases and their amines: Synthesis and discovery of carbonic anhydrase and acetylcholinesterase enzymes inhibitors

Article abstract of DOI:10.1002/ardp.201800146

Three series of symmetrical Schiff bases were synthesized from 1,2-diaminoethane, 1,3-diaminopropane and 1,4-diaminobutane and substituted benzaldehydes, and reduced by sodium borohydride to the corresponding benzylic diamines 4–6. All of the compounds obtained were characterized using elemental analysis, FT-IR, ¹H NMR, and ¹³C NMR spectroscopy. The enzyme inhibitory properties of these compounds were tested and the influence of the alkane chain length and the substituents on the phenyl group on the enzyme inhibition activity were examined. The novel Schiff bases and their amine derivatives (1a–d, 2a–d, 3b–d, 4a–c, 5a–c, 6a, 6c, 6d) were effective inhibitors of the cytosolic carbonic anhydrase I and II isoforms (hCA I and II), and acetylcholinesterase (AChE) with K_i values in the range of 159.43 ± 30.03 to 563.73 ± 115.30 nM for hCA I, 104.88 ± 18.44 to 524.32 ± 95.03 nM for hCA II, and 3.95 ± 0.74 to 30.83 ± 6.81 nM for AChE.

Full text of DOI:10.1002/ardp.201800146

Received: 14 May 2018
Revised: 20 June 2018
Accepted: 29 June 2018
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DOI: 10.1002/ardp.201800146
FULL PAPER
Schiff bases and their amines: Synthesis and discovery of
carbonic anhydrase and acetylcholinesterase enzymes
inhibitors
Beyhan Yiğit1
Murat Yiğit1
Parham Taslimi2
Yetkin Gök3
İlhami Gülçin2
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1 Faculty of Science and Art, Department of
Chemistry, Adıyaman University, Adıyaman,
Turkey
Abstract
Three series of symmetrical Schiff bases were synthesized from 1,2-diaminoethane,
1,3-diaminopropane and 1,4-diaminobutane and substituted benzaldehydes, and
reduced by sodium borohydride to the corresponding benzylic diamines 4–6. All of the
compounds obtained were characterized using elemental analysis, FT-IR, 1H NMR, and
13C NMR spectroscopy. The enzyme inhibitory properties of these compounds were
tested and the influence of the alkane chain length and the substituents on the phenyl
group on the enzyme inhibition activity were examined. The novel Schiff bases and
their amine derivatives (1a–d, 2a–d, 3b–d, 4a–c, 5a–c, 6a, 6c, 6d) were effective
inhibitors of the cytosolic carbonic anhydrase I and II isoforms (hCA I and II), and
acetylcholinesterase (AChE) with Ki values in the range of 159.43 30.03 to
563.73 115.30 nM for hCA I, 104.88 18.44 to 524.32 95.03 nM for hCA II, and
3.95 0.74 to 30.83 6.81 nM for AChE.
2 Faculty of Science, Department of
Chemistry, Atatürk University, Erzurum,
Turkey
3 Faculty of Science and Art, Department of
Chemistry, Inönü University, Malatya, Turkey
Correspondence
Dr. Parham Taslimi, Faculty of Science,
Department of Chemistry, Ataturk University,
25240-Erzurum, Turkey.
Email: parham_taslimi_un@yahoo.com,
parhamsmkh66@gmail.com
K E Y W O R D S
1,2-diaminoethane, 1,3-diaminopropane, acetylcholinesterase, carbonic anhydrase, enzyme
inhibition, Schiff bases
antiviral activities.[16–24] Schiff bases are also used in synthesis of
amines. The reduction of Schiff bases using borohydride reagents or
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1
INTRODUCTION
Schiff bases are known as imines or azomethines compounds
containing carbon–nitrogen double bond.[1] They are prepared by
the condensation between primary amine and active carbonyl
compounds.[2] Schiff bases are an important class which are widely
used and investigated organic compounds due to their unique
properties and numerous applications in many fields including
analytical, biological, and inorganic chemistry.[3–9] Schiff bases form
stable complexes with most of transition metals owing to their strong
coordinative ability. The catalytic and biological properties of Schiff
bases and their metal complexes have been extensively studied.[10–15]
They have been shown to exhibit a broad range of biological activities
such as antibacterial, antifungal, antimalarial, anti-inflammatory, and
transition metal hydrogenation catalysts is the most efficient and
convenient method for the synthesis of substituted amines which are
extremely important pharmacophores in numerous biologically active
compounds.[25]
Human carbonic anhydrase (hCA) enzymes include a class of
sixteen isoenzymes which their action is to catalyze the reversible
hydration of carbon dioxide (CO2), which generates a proton (H+) and
bicarbonate anion (HCO3−).[26–30] This simple biological act is central
to regulating pH mechanism in the extra- and intracellular space in
disparate organs and tissues. CAs are metalloenzymes available in all
three of life's phylogenetic domains (Eukarya, Archaea, and Bacteria)
and different isoenzymes present in most organisms evaluated so
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Arch Pharm Chem Life Sci. 2018;e1800146.
wileyonlinelibrary.com/journal/ardp
© 2018 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.201800146

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far.[31,32] CA inhibitors (CAIs) utilized in the clinical agents are
essentially diuretics and factors for glaucoma-relevant intraocular
hypertension, as showed by the drugs in Figure 1.[33–35] CAIs are
clinically used for decades for the treatment of diverse diseases such as
epilepsy, intracranial hypertension, obesity, glaucoma, and as diuretics.
Recently, they started to be utilized for the therapy of hypoxic tumors,
being also evaluated as important drugs for arthritis, cerebral ischemia,
and neuropathic pain.[36,37]
Agglomeration of β-amyloid, the cholinergic system malfunction-
ing and biometal dyshomeostasis are significant hypothetical deter-
minants that give rise to Alzheimer's disease (AD).[38–40] Scientists
have been working on Tau-hypothesis, cholinergic hypothesis, amyloid
cascade hypothesis, etc. Hence, most marketed drugs (Figure 2)[41] are
based on cholinergic hypothesis that tries to decrease acetylcholine
(ACh) levels in pre-synaptic regions.[42,43] Acetylcholinesterase
inhibitors (AChEIs) are used for the symptomatic therapy of mild-
to-moderate AD. These compounds inhibit AChE, an enzyme
answerable for the breakdown of ACh, a neurotransmitter associated
with memory function.[44]
FIGURE 2 Examples of known AChE inhibitors: donepezil as
indanone derivative, ensaculin and AP2238 as coumarin derivatives
diaminoethane, 1,3-diaminopropane, and 1,4-diaminobutane with two
molar equivalents of the substituted benzaldehydes in ethanol at reflux
temperature. The obtained Schiff bases 1–3 were reduced by
treatment with sodium borohydride in methanol at room temperature
to corresponding benzylic diamines 4–6 in 60–83% yields. All the
compounds are soluble in the common polar solvents such as ethanol
and dichloromethane. The Schiff bases and diamines were character-
ized by 1H and 13C NMR spectroscopy, FTIR and elemental analysis,
which support the proposed structures. In the 1H NMR spectra of 1–3,
In this study, three series of symmetrical Schiff bases 1–3 were
prepared using 1,2-diaminoethane, 1,3-diaminopropane and 1,4-
diaminobutane and substituted benzaldehydes. The reaction of the
Schiff bases with sodium borohydride afforded the corresponding
benzylic diamines 4–6. Carbonic anhydrase and acetylcholinesterase
inhibitory properties of novel Schiff bases and their amines derivatives
(1a–d, 2a–d, 3b–d, 4a–c, 5a–c, 6a, 6c, 6d) were investigated.

characteristic imino protons (N CH) of Schiff bases appear at the
range 8.12–8.23 ppm as sharp singlets. The 13C NMR spectra of the
Schiff bases further supported the assigned structures. The 13C NMR
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N CH carbon atoms in 1–3 appear at the range
shift of
160.0–162.1 ppm as singlet signal. In the IR spectra of the Schiff
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2
RESULTS AND DISCUSSION
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bases, the characteristic C N stretching frequencies were observed
between 1491 and 1590 cm−1. The formation of N,N′-dialkylalkanedi-
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2.1 Synthesis of Schiff bases and
N,N′-dialkylalkanediamines
amines 4–6 was confirmed by the presence of N─H proton resonance
As shown in Scheme 1, three series of symmetrical Schiff bases were
easily prepared in good yields (80–97%) via condensation of 1,2-
FIGURE 1 Clinically used CA inhibitors with antiepileptic activity.
AAZ: acetazolamide, ZNS: zonisamide, TPM: topiramate, MZA:
methazolamide
SCHEME 1 Synthesis of Schiff bases and N,N′-dialkylalkanediamines

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in the 1H NMR spectra and by the absence of the Schiff base N CH
Additionally, CA II is often associated with several diseases
such as glaucoma, osteoporosis, and renal tubular acidosis.[53]
hCA II was also efficiently inhibited by the novel Schiff bases and
their amines derivatives (1a–d, 2a–d, 3b–d, 4a–c, 5a–c, 6a, 6c,
6d) investigated here. These compounds appeared to strongly
inhibit hCA II with Ki values ranging from 104.88 18.44 to
524.32 95.03 nM. These values are better than those of the
clinically used drugs AZA (Ki of 603.18 78.44 nM). All the novel
derivatives (1a–d, 2a–d, 3b–d, 4a–c, 5a–c, 6a, 6c, 6d) demon-
strated marked inhibition against hCA II, but the compounds of
1,2-bis(4-methylthiobenzylamino)ethane, 4a, and 1,2-bis(4-ben-
zyloxy-3-methoxybenzylamino)ethane, 4c, showed excellent
inhibitions profile against cytosolic hCA II with Ki values of
129.73 18.47 and 104.88 18.44 nM, respectively (Table 1 and
Figure 3).
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1
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proton resonance in the H NMR spectra and N CH carbon
resonance in the 13C NMR spectra. The 1H NMR spectra of 4–6
exhibit a broad singlet at the range 1.36–2.00 ppm for N─H protons.
The benzylic protons exhibited a singlet at the range 3.66–4.74 ppm.
The benzylic carbon resonance of these N,N′-dialkylalkanediamines
appeared at the range 53.4–56.6 ppm as singlet signal in the 13C NMR
spectra. The elemental analysis data of the Schiff bases and
N,N′-dialkylalkanediamines agree closely with the theoretical require-
ments of their structures.
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2.2 Biochemical results
hCA enzymes are significant not only for pH maintenance, also for bone
resorption, signal transduction, renal acidification, calcification, gastric
acid formation, adaptation to cellular stress, gluconeogenesis, metabo-
lism, biosynthesis, and other processes.[45] Presently, there are over 21
clinically utilized CA inhibitor compounds, but all define little to no
isoenzyme specificity. Accordingly, there is a requirement to extend CA
inhibitor compounds that are isoform selective, for example,
the targeted inhibition of CA IX as an anticancer chemotherapy.[46]
The design of CA isoform specific inhibitor compounds is challenging
due to high sequence similarity and the structural homology within the
active sites of the CA isoenzymes.[47] Hence, there are particular
structural discrepancies that have been used for selective inhibitor
compound development. CA IX isoform has a larger active site hole that
shows amino acid discrepancies in comparison to other CA isoen-
zymes.[48] For instance, phenylalanine in CA II and also residue 131 is a
valine in CA IX, which permits bulkier inhibitor compounds to bind CA
IX.[49] Additionally, many of the CA isoforms are valuable aims for many
pharmacological applications such as diuretics, antiglaucoma drugs,
antiobesity, antitumor, anticonvulsant, agents/diagnostic tools.[50] For
example, hCA I inhibitor are used in cerebral edema and retinal. hCA IV,
II, XIV, and XII inhibitors are utilized as antiglaucoma agents, in the
management of edema, as diuretics, antiepileptic drugs, and also for the
therapy of altitude sickness.[51] In this study, we obtained the effects of
novel Schiff bases and their amines derivatives (1a–d, 2a–d, 3b–d, 4a–c,
5a–c, 6a, 6c, 6d) against hCA I, hCA II, andAChE enzymes. We report the
inhibition effects of these derivatives on the activity of hCA I and hCA II
isoenzymes and AChE enzyme under in vitro conditions. The following
results are presented in Table 1.
The inhibitory effects of the newly synthesized Schiff bases and
their amines derivatives (1a–d, 2a–d, 3b–d, 4a–c, 5a–c, 6a, 6c, 6d) on
AChE are shown in Table 1. The AChE inhibition profiles of the
compounds evaluated here were quite interesting. Overall, the novel
Schiff bases and their amines derivatives (1a–d, 2a–d, 3b–d, 4a–c,
5a–c, 6a, 6c, 6d) had excellent inhibitory activity with Ki values ranging
from 3.95 0.74 to 30.83 6.81 nM. Furthermore, tacrine, used as a
standard AChE inhibitor in this study, demonstrated Ki value of
67.25 16.08 nM toward AChE. The compounds of 1,3-bis
(4-methylthiobenzylamino)propane, 5a, 1,3-bis(4-(1-piperidinyl)-
benzylamino)propane, 5b, 1,3-bis(4-benzyloxy-3-methoxybenzylamino)-
propane, 5c, showed excellent inhibitions profile against AChE with Ki
values of 3.95 0.74, 5.77 1.04, and 7.84 2.76 nM, respectively
(Table 1 and Figure 3).
Recently, drug development and research are based on the
cholinergic hypothesis which offers that the elective loss of
cholinergic neuron cells results in a deficiency of ACh in special
places of the brain that mediate memory functions and learning.
Based on this supposition the cholinergic ratiocination will improve
the recognition in AD.[54,55] Nowadays available therapy for
patients suffering from AD involve approved AChEIs such as
donepezil, galantamine, and rivastigmine which avoid the hydroly-
sis of ACh therewith enhancing its concentrations.[56] Hence,
clinical effect and efficiency is limited as accessible AChEIs can
only ameliorate AD symptoms and, therefore, the search for novel
molecules remains an emerging demand for the therapy of AD. On
the other hand, the design of multi-targeted drugs that can
simultaneously act on ACh and other molecular targets involved in
the development of the disease offer an interesting approach to
manage AD.[57]
Abnormal levels of CA I isoenzyme in the blood are used as a
marker for hemolytic anemia.[52] The slow cytosolic isoform hCA I was
inhibited by the investigated novel Schiff bases and their amines
derivatives (1a–d, 2a–d, 3b–d, 4a–c, 5a–c, 6a, 6c, 6d), with Ki values
ranging between 159.43 30.03 and 563.73 115.30 nM. Further-
more, 1,2-bis(4-benzyloxy-3-methoxybenzylamino)ethane, 4c, dem-
onstrated the most powerful hCA I isoenzyme inhibition properties
with a Ki value of 159.43 30.03 nM. The standard and clinically used
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3
CONCLUSION
drug acetazolamide (AZA) demonstrated
a
Ki value of
Three series of symmetrical Schiff bases and amines were
successfully synthesized in good yields and characterized by
elemental analysis and spectroscopic methods. These compounds
were investigated for their hCA I and II isoforms, and AChE inhibition
620.83 149.27 nM (Table 1 and Figure 3). Thus, the investigated
compounds showed better inhibitory profiles compared to AZA, a
clinically used CA inhibitor.

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TABLE 1 The inhibition values of novel Schiff bases and their amines derivatives (1a–d, 2a–d, 3b–d, 4a–c, 5a–c, 6a, 6c, 6d) against human
carbonic anhydrase isoenzymes I and II (hCA I and II), and AChE enzyme
IC50 (nM)
Ki (nM)
Compounds
hCA I
r2
hCA II
263.44
235.37
203.77
235.82
158.46
179.03
168.38
143.28
348.11
401.33
398.76
148.91
139.09
124.77
238.67
219.49
213.79
494.38
478.36
422.85
532.48
–
r2
AChE
28.36
19.76
30.18
34.03
14.76
20.22
21.38
21.01
19.77
18.67
20.74
40.17
37.43
36.10
9.03
r2
hCA I
hCA II
AChE
1a
301.23
261.28
248.82
281.33
182.83
201.47
208.02
194.18
402.17
438.32
462.10
199.02
171.48
170.27
291.56
267.83
250.02
504.23
538.27
494.37
604.33
–
0.9722
0.9902
0.9788
0.9889
0.9683
0.9847
0.9903
0.9937
0.9625
0.9528
0.9707
0.9829
0.9902
0.9477
0.9921
0.9683
0.9883
0.9773
0.9880
0.9955
0.9743
–
0.9902
0.9721
0.9732
0.9947
0.9476
0.9770
0.9868
0.9772
0.9954
0.9722
0.9890
0.9937
0.9799
0.9550
0.9930
0.9744
0.9810
0.9308
0.9638
0.9901
0.9699
–
0.9911
0.9692
0.9787
0.9880
0.9630
0.9881
0.9689
0.9722
0.9699
0.9907
0.9627
0.9744
0.9307
0.9633
0.9754
0.9840
0.9927
0.9888
0.9728
0.9825
–
324.74 28.33
257.88 70.12
269.03 38.01
302.12 54.44
172.83 50.05
190.02 14.04
234.18 25.55
201.30 60.84
378.94 100.51
398.58 93.03
449.73 98.11
218.04 24.82
203.49 59.44
159.43 30.03
290.73 88.32
301.55 92.05
231.66 58.32
482.48 103.94
563.73 115.30
488.18 77.03
620.83 149.27
–
281.33 98.03
230.41 91.44
227.47 58.52
254.93 83.99
166.82 47.82
173.66 40.11
149.81 27.48
146.17 36.92
327.15 118.0
385.72 91.23
359.14 103.4
129.73 18.47
154.83 59.22
104.88 18.44
258.47 37.94
245.18 60.34
209.47 27.93
451.03 154.2
524.32 95.03
518.23 118.0
603.18 78.44
–
22.47 1.73
15.33 3.54
25.81 7.31
28.18 10.05
9.14 2.01
13.08 0.88
16.83 3.73
15.87 4.88
14.63 2.66
10.11 3.83
11.08 0.96
28.95 5.87
30.83 6.81
29.77 8.93
3.95 0.74
5.77 1.04
7.84 2.76
16.07 6.41
20.37 9.04
23.55 7.74
–
1b
1c
1d
2a
2b
2c
2d
3b
3c
3d
4a
4b
4c
5a
5b
5c
8.04
10.85
24.83
29.49
29.01
–
6a
6c
6d
AZAa
TACb
103.11
0.9921
67.25 16.08
aAcetazolamide (AZA) was used as a standard inhibitor for both carbonic anhydrase I and II isoenzymes.
bTacrine (TAC) was used as a standard inhibitor for AChE enzyme.
properties. These molecules showed nanomolar inhibition against
these enzymes. Inhibition of AChE and hCA I and II isoforms can
have a significant role in discovery and drug design as well as in
toxicology and medicine. Additionally, these molecules are drug
candidates as anticholinergic, antidiabetic, and antiepileptic appli-
cations. Compounds 4c, 4a, and 5a were good inhibitors for hCA I
and II and AChE enzymes, respectively.
abbreviated as follows: s = singlet, bs = broad singlet, d = doublet,
t = triplet, quint = quintet, and m = multiplet signal. FT-IR spectra
were recorded as KBr pellets in the range 400–4000 cm−1 on Perkin
Elmer Spectrum 100. Melting points were measured in open capillary
tubes with an Electrothermal-9200 melting point apparatus.
Elemental analyses were performed by LECO CHNS-932 elemental
analyzer.
The NMR spectra of the investigated compounds are provided as
Supporting Information. Their InChI codes and some biological activity
data are also provided as Supporting Information.
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4
EXPERIMENTAL
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4.1 Chemistry
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4.1.2 General procedure for the preparation of
4.1.1 General
Schiff bases 1–3
All reactions for the preparation of the Schiff bases and amines were
carried out under air. Chemicals were obtained from Sigma–Aldrich
and Fluka. 1H and 13C NMR spectra were recorded with a Varian AS
400 Merkur spectrometer operating at 400 MHz (1H), 100 MHz (13C)
in CDCl3 with tetramethylsilane as an internal reference. Coupling
constants (J values) are given in hertz. NMR multiplicities are
Diaminoalkane (5 mmol) was added dropwise to a solution of the
aromatic aldehyde (10 mmol) in ethanol (30 mL) over a period of 2 min.
The reaction mixture was stirred at reflux for 3 h, then volatiles were
removed in vacuum to dryness. The crude product was crystallized
from dichloromethane or ethanol/diethyl ether.

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FIGURE 3 Determination of Lineweaver–Burk graphs for excellent inhibitors of hCA I and hCA II isoenzymes, and AChE enzyme
(m, 26H, Ar-H), 8.12 (s, 2H, N CH). 13C NMR (CDCl3) δ: 61.6
(NCH2CH2N), 70.9 and 71.1 (OCH2Ar), 112.4, 113.9, 123.1, 127.1,
127.4, 127.8, 127.9, 128.4, 128.5, 130.0, 136.9, 137.1, 149.2, 151.1

1,2-Bis(4-methylthiobenzylideneamino)ethane 1a
Yield: 1.54 g, 94%; m.p.: 110–111°C, IR, υ: 1590 cm−1 (C N). H NMR
1

(CDCl3) δ: 2.49 (s, 6H, SCH3), 3.94 (s, 4H, NCH2CH2N), 7.22 and 7.59 (d,
8H, J = 8 Hz, Ar-H), 8.20 (s, 2H, N CH). 13C NMR (CDCl3) δ: 15.2
(Ar-C), 162.0 (N CH). Anal. calcd. for C44H40N2O4: C, 80.00; H, 6.06;


(SCH3), 61.6 (NCH2CH2N), 125.7, 128.3, 132.8, 141.9 (Ar-C), 162.0
N, 4.24. Found: C, 80.06; H, 6.08; N, 4.22.

(N CH). Anal. calcd. for C18H20N2S2: C, 65.85; H, 6.09; N, 8.54.
Found: C, 65.88; H, 6.11; N, 8.50.
1,3-Bis(4-methylthiobenzylideneamino)propane 2a
Yield: 1.52 g, 89%; m.p.: 65–66°C, IR, υ: 1641 cm−1 (C N). 1H NMR

1,2-Bis(4-(1-piperidinyl)benzylideneamino)ethane 1b
(CDCl3) δ: 2.50 (s, 6H, SCH3), 2.10 (quint, 2H, J = 8 Hz,
NCH2CH2CH2N), 3.69 (t, 4H, J = 4 Hz, NCH2CH2CH2N), 7.25 and
Yield: 1.84 g, 92%; m.p.: 216–218°C, IR, υ: 1605 cm−1 (CN). 1H NMR
(CDCl3) δ: 1.58–1.62 (m, 4H, N(CH2CH2)2CH2), 1.65–1.70 (m, 8H,
N(CH2CH2)2CH2), 3.24 (t, 8H, J = 4 Hz, N(CH2CH2)2CH2), 3.87 (s, 4H,
NCH2CH2N), 6.87 and 7.56 (d, 8H, J = 8 Hz, Ar-H), 8.15 (s, 2H, NCH).
13C NMR (CDCl3) δ: 24.3 (N(CH2CH2)2CH2), 25.6 (N(CH2CH2)2CH2),
49.4 (N(CH2CH2)2CH2), 61.9 (NCH2CH2N), 114.9, 126.4, 129.3, 153.3
7.63 (d, 8H, J = 8 Hz, Ar-H), 8.23 (s, 2H, N CH). 13C NMR (CDCl3) δ:

15.2 (SCH3), 32.0 (NCH2CH2CH2N), 59.2 (NCH2CH2CH2N), 125.8,
128.3, 133.0, 141.8 (Ar-C), 160.6 (NCH). Anal. calcd. for C19H22N2S2:
C, 66.67; H, 6.43; N, 8.19. Found: C, 66.71; H, 6.40; N, 8.22.

(Ar-C), 162.1 (N CH). Anal. calcd. for C26H34N4: C, 77.61; H, 8.46; N,
1,3-Bis(4-(1-piperidinyl)benzylideneamino)propane 2b
13.93. Found: C, 77.65; H, 8.48; N, 13.90.
Yield: 1.79 g, 86%; m.p.: 112–113°C, IR, υ: 1605 cm−1 (C N). H NMR
1

(CDCl3) δ: 1.60 (quint, 4H, J = 4 Hz, N(CH2CH2)2CH2), 1.69 (quint, 8H,
J = 4 Hz, N(CH2CH2)2CH2), 3.26 (t, 8H, J = 4 Hz, N(CH2CH2)2CH2), 2.07
(quint, 2H, J = 8 Hz, NCH2CH2CH2N), 3.64 (t, 4H, J = 8 Hz,
NCH2CH2CH2N), 6.90 and 7.59 (d, 8H, J = 8 Hz, Ar-H), 8.15 (s, 2H,
1,2-Bis(4-benzyloxy-3-methoxybenzylideneamino)ethane 1c
1
Yield: 2.39 g, 94%; m.p.: 151–152°C, IR, υ: 1508 cm−1 (C N). H NMR

(CDCl3) δ: 3.91 (s, 10H, NCH2CH2N, OCH3), 5.17 (s, 4H, OCH2Ar),
6.86–7.43 (m, 16H, Ar-H), 8.16 (s, 2H, NCH). 13C NMR (CDCl3) δ: 56.0
(OCH3), 61.6 (NCH2CH2N), 70.8 (OCH2Ar), 109.2, 112.9, 122.9, 127.2,
127.9, 128.6, 129.8, 136.7, 149.9, 150.4 (Ar-C), 162.1 (NCH). Anal.
calcd. for C32H32N2O4: C, 75.59; H, 6.30; N, 5.51. Found: C, 75.64; H,
6.32; N, 5.49.
CH). 13C NMR (CDCl3) δ: 32.3 (NCH2CH2CH2N), 59.2

N
(NCH2CH2CH2N), 24.3 (N(CH2CH2)2CH2), 25.5 (N(CH2CH2)2CH2),
49.5 (N(CH2CH2)2CH2), 115.0, 126.6, 129.2, 153.3 (Ar-C), 160.9
(NCH). Anal. calcd. for C27H36N4: C, 77.88; H, 8.65; N, 13.46. Found: C,
77.92; H, 8.61; N, 13.44.
1,2-Bis(3,4-dibenzyloxybenzylideneamino)ethane 1d
Yield: 3.2 g, 97%; m.p.: 150–151°C, IR, υ: 1511 cm−1 (CN). 1H NMR
1,3-Bis(4-benzyloxy-3-methoxybenzylideneamino)propane 2c
Yield: 2.09 g, 80%; m.p.: 81–82°C, IR, υ: 1509 cm−1 (C N). 1H NMR

(CDCl3) δ: 3.90 (s, 4H, NCH2CH2N), 5.16 (s, 8H, OCH2Ar), 6.88–7.46
(CDCl3) δ: 3.94 (s, 6H, OCH3), 2.09 (quint, 2H, J = 8 Hz,

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NCH2CH2CH2N), 3.68 (t, 4H, J = 4 Hz, NCH2CH2CH2N), 5.19 (s, 4H,
4.1.3 General procedure for the preparation of
OCH2Ar), 6.87–7.44 (m, 16H, Ar-H), 8.18 (s, 2H, N CH). 13C NMR
N,N′-dialkylalkanediamines 4–6

(CDCl3) δ: 56.0 (OCH3), 32.1 (NCH2CH2CH2N), 59.2 (NCH2CH2CH2N),
70.8 (OCH2Ar), 109.1, 112.9, 122.7, 127.2, 127.9, 128.6, 129.9, 136.7,
149.9, 150.37 (Ar-C), 160.7 (NCH). Anal. calcd. for C33H34N2O4: C,
75.86; H, 6.51; N, 5.36. Found: C, 75.90; H, 6.53; N, 5.38.
Sodium borohydride (15 mmol) was added portionwise to a solution of
diimine (10 mmol) in methanol (30 mL) at room temperature over a
period of 30 min. The reaction mixture was stirred for 12 h and then
heated under reflux for 1 h. Upon cooling to room temperature, the
mixture was treated with 1 N HCl, and organic phase was extracted
with dichloromethane (3 × 30 mL). After drying over MgSO4 and
evaporation, the crude product was crystallized from toluene/
n-hexane.
1,3-Bis(3,4-dibenzyloxybenzylideneamino)propane 2d
Yield: 3.17 g, 94%; m.p.: 104–105°C, IR, υ: 1514 cm−1 (CN). 1H NMR
(CDCl3) δ: 2.08 (quint, 2H, J = 8 Hz, NCH2CH2CH2N), 3.66 (t, 4H,
J = 8 Hz, NCH2CH2CH2N), 5.18 and 5.19 (s, 8H, OCH2Ar), 6.92–7.49
(m, 26H, Ar-H), 8.15 (s, 2H, NCH). 13C NMR (CDCl3) δ: 32.1
(NCH2CH2CH2N), 59.1 (NCH2CH2CH2N), 71.0 and 71.1 (OCH2Ar),
112.3, 114.0, 123.0, 127.1, 127.3, 127.8, 128.4, 128.5, 130.1, 136.9,
137.1, 149.2, 151.0 (Ar-C), 160.6 (NCH). Anal. calcd. for
C45H42N2O4: C, 80.12; H, 6.23; N, 4.15. Found: C, 80.17; H, 6.25;
N, 4.13.
1,2-Bis(4-methylthiobenzylamino)ethane 4a
Yield: 1.99 g, 60%; m.p.: 43–44°C, IR, υ: 1491 cm−1 (C N). 1H NMR

(CDCl3) δ: 1.66 (bs, 2H, N-H), 2.47 (s, 6H, SCH3), 2.73 (s, 4H,
NCH2CH2N), 3.72 (s, 4H, NCH2Ar), 7.22 (s, 8H, Ar-H). 13C NMR (CDCl3)
δ: 16.2 (SCH3), 48.7 (NCH2CH2N), 53.4 (NCH2Ar), 126.9, 128.7, 136.7,
137.5 (Ar-C). Anal. calcd. for C18H24N2S2: C, 65.06; H, 7.23; N, 8.43.
Found: C, 65.11; H, 7.21; N, 8.47.
1,4-Bis(4-methylthiobenzylideneamino)butane 3a
This compound was not characterized.
1,2-Bis(4-(1-piperidinyl)benzylamino)ethane 4b
Yield: 2.51 g, 62%; m.p.: 87–89°C, IR, υ: 1510 cm−1 (CN). 1H NMR
(CDCl3) δ: 1.53–1.59 (m, 4H, N(CH2CH2)2CH2), 1.67–1.73 (m, 10H,
N(CH2CH2)2CH2, N-H), 3.12 (t, 8H, J = 4 Hz, N(CH2CH2)2CH2), 2.73 (s,
4H, NCH2CH2N), 3.67 (s, 4H, NCH2Ar), 6.88 and 7.17 (d, 8H, J = 8 Hz,
Ar-H). 13C NMR (CDCl3) δ: 24.3 (N(CH2CH2)2CH2), 25.9 (N-
(CH2CH2)2CH2), 50.3 (N(CH2CH2)2CH2), 48.7 (NCH2CH2N), 53.4
(NCH2Ar), 116.5, 128.9, 131.1, 151.3 (Ar-C). Anal. calcd. for
1,4-Bis(4-(1-piperidinyl)benzylideneamino)butane 3b
Yield: 1.76 g, 82%; m.p.: 166–168°C, IR, υ: 1602 cm−1 (C N). H NMR
1

(CDCl3) δ: 1.73 (quint, 4H, J = 4 Hz, NCH2CH2CH2CH2N), 3.59 (t,
4H, J = 4 Hz, NCH2CH2CH2CH2N), 1.61 (quint, 4H, J = 4 Hz,
N(CH2CH2)2CH2), 1.68 (quint, 8H, J = 4 Hz, N(CH2CH2)2CH2), 3.25
(t, 8H, J = 4 Hz, N(CH2CH2)2CH2), 6.89 and 7.58 (d, 8H, J = 8 Hz, Ar-H),
8.14 (s, 2H, N CH). 13C NMR (CDCl3) δ: 28.9 (NCH2CH2CH2CH2N),

C26H38N4: C, 76.84; H, 9.36; N, 13.79. Found: C, 76.87; H, 9.38; N,
61.5 (NCH2CH2CH2CH2N), 24.3 (N(CH2CH2)2CH2), 25.5
13.76.
(N(CH2CH2)2CH2), 49.5 (N(CH2CH2)2CH2), 115.0, 126.6, 129.2,
153.3 (Ar-C), 160.6 (NCH). Anal. calcd. for C28H38N4: C, 78.14; H,
8.84; N, 13.02. Found: C, 78.19; H, 8.82; N, 13.10.
1,2-Bis(4-benzyloxy-3-methoxybenzylamino)ethane 4c
Yield: 3.28 g, 64%; m.p.: 158–160°C, IR, υ: 1514 cm−1 (CN). 1H NMR
(CDCl3) δ: 2.00 (bs, 2H, N-H), 3.93 (s, 6H, OCH3), 3.67 (s, 4H,
NCH2CH2N), 4.74 (s, 4H, NCH2Ar), 5.13 (s, 4H, OCH2Ar), 6.77–7.42 (m,
16H, Ar-H). 13C NMR (CDCl3) δ: 52.2 (OCH3), 47.3 (NCH2CH2N), 56.6
(NCH2Ar), 71.0 (OCH2Ar), 112.7, 113.9, 121.4, 125.4, 127.3, 127.9,
128.5, 128.6, 136.8, 148.8, 150.4(Ar-C). Anal. calcd. forC32H36N2O4: C,
75.00; H, 7.03; N, 5.47. Found: C, 75.04; H, 7.01; N, 5.49.
1,4-Bis(4-benzyloxy-3-methoxybenzylideneamino)butane 3c
Yield: 2.41 g, 90%; m.p.: 124–126°C, IR, υ: 1513 cm−1 (CN). 1H NMR
(CDCl3) δ: 3.94 (s, 6H, OCH3), 1.72–1.79 (m, 4H, NCH2CH2CH2CH2N),
3.61–3.64 (m, 4H, NCH2CH2CH2CH2N), 5.19 (s, 4H, OCH2Ar),
6.86–7.44 (m, 16H, Ar-H), 8.16 (s, 2H, N CH). 13C NMR (CDCl3) δ:

56.0 (OCH3), 28.8 (NCH2CH2CH2CH2N), 61.4 (NCH2CH2CH2CH2N),
70.8 (OCH2Ar), 109.1, 112.9, 122.7, 127.2, 127.9, 128.5, 129.6, 136.7,
149.9, 150.3 (Ar-C), 160.4 (NCH). Anal. calcd. for C34H36N2O4: C,
76.12; H, 6.72; N, 5.22. Found: C, 76.16; H, 6.70; N, 5.25.
1,3-Bis(4-methylthiobenzylamino)propane 5a
Yield: 2.14 g, 62%; m.p.: 92–94°C, IR, υ: 1511 cm−1 (C N). 1H NMR

(CDCl3) δ: 2.47 (s, 6H, SCH3), 1.67–1.74 (m, 4H, NCH2CH2CH2N, N-H),
2.67–2.70 (m, 4H, NCH2CH2CH2N), 3.73 (s, 4H, NCH2Ar), 7.21 (s, 8H,
Ar-H). 13C NMR (CDCl3) δ: 16.1 (SCH3), 29.9 (NCH2CH2CH2N), 47.9
(NCH2CH2CH2N), 53.4 (NCH2Ar), 126.8, 128.6, 136.7, 137.3 (Ar-C).
Anal. calcd. for C19H26N2S2: C, 65.90; H, 7.51; N, 8.09. Found: C,
65.95; H, 7.53; N, 8.12.
1,4-Bis(3,4-dibenzyloxybenzylideneamino)butane 3d
Yield: 2.96 g, 86%; m.p.: 132–134°C, IR, υ: 1507 cm−1 (C N). H NMR
1

(CDCl3) δ: 1.73–1.76 (m, 4H, NCH2CH2CH2CH2N), 3.60–3.63 (m, 4H,
NCH2CH2CH2CH2N), 5.18 and 5.19 (s, 8H, OCH2Ar), 6.91–7.49 (m,
CH). 13C NMR (CDCl3) δ: 28.8

N
26H, Ar-H), 8.14 (s, 2H,
(NCH2CH2CH2CH2N), 61.4 (NCH2CH2CH2CH2N), 71.0 and 71.1
(OCH2Ar), 112.4, 114.0, 123.0, 127.1, 127.4, 127.8, 128.4, 128.5,
130.1, 136.9, 137.0, 149.2, 151.0 (Ar-C), 160.3 (NCH). Anal. calcd. for
1,3-Bis(4-(1-piperidinyl)benzylamino)propane 5b
Yield: 2.90 g, 69%; m.p.: 60–61°C, IR, υ: 1509 cm−1 (C N). 1H NMR

(CDCl3) δ: 1.53–1.59 (m, 6H, N(CH2CH2)2CH2, N-H), 1.68–1.73
(m, 10H, N(CH2CH2)2CH2, NCH2CH2CH2N), 3.12 (t, 8H, J = 4 Hz,
C
46H44N2O4: C, 80.23; H, 6.40; N, 4.07. Found: C, 80.28; H, 6.43; N,
4.03.

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N(CH2CH2)2CH2), 2.67 (t, 4H, J = 4 Hz, NCH2CH2CH2N), 3.68 (s, 4H,
NCH2Ar), 6.88 and 7.17 (d, 8H, J = 8 Hz, Ar-H). 13C NMR (CDCl3) δ: 29.9
(NCH2CH2CH2N), 47.8 (NCH2CH2CH2N), 24.3 (N(CH2CH2)2CH2), 25.9
(N(CH2CH2)2CH2), 50.8 (N(CH2CH2)2CH2), 53.5 (NCH2Ar), 116.5,
128.8, 131.1, 151.3 (Ar-C). Anal. calcd. for C27H40N4: C, 77.14; H,
9.52; N, 13.33. Found: C, 77.17; H, 9.55; N, 13.36.
Sepharose-4B-L-tyrosine-sulphanilamide affinity chromatoghraphy. The
protein quantity in the column effluents were evaluated spectrophotomet-
rically at 280 nm as previously described.[61,62] For purity of both
isoenzymes sodium dodecyl sulfate–polyacrylamide gel electrophoresis
(SDS–PAGE) was applied.[63] CA isoenzymes activities were obtained in
conforming with the procedure of Verpoorte et al.[64] as explained
previously.[65] The enhance in absorbance of the reaction medium was
spectrophotometrically obtained at 348 nm. We utilized the esterase
activity procedure for ascertaining the inhibition agents by the Lineweaver–
Burk procedure. [66] CA activity (%) versus inhibitory concentration and 1/V
versus 1/[S] graphs were drawn. The quantity of protein was determined at
595 nm according to Bradford's procedure.[67] Bovine serum albumin was
used as standard protein as given previously in details.[68] For the
designation of the inhibition efficacy of each novel Schiff bases and their
amines derivatives (1a–d, 2a–d, 3b–d, 4a–c, 5a–c, 6a, 6c, 6d), on both hCA
isoenzymes, an activity (%)-[Schiff bases and their amines derivatives] graph
was drawn. The IC50 values were obtained from activity (%) versus
compounds plots. For the calculation of Ki values, three different novel
Schiff bases and their amines derivatives concentrations were used.[69]
1,3-Bis(4-benzyloxy-3-methoxybenzylamino)propane 5c
Yield: 3.78 g, 72%; m.p.: 66–68°C, IR, υ: 1512 cm−1 (CN). 1H NMR
(CDCl3) δ: 1.69–1.79 (m, 4H, NCH2CH2CH2N, N-H), 2.70 (t, 4H,
J = 4 Hz, NCH2CH2CH2N), 3.86 (s, 6H, OCH3), 3.70 (s, 4H, NCH2Ar),
5.13 (s, 4H, OCH2Ar), 6.73–7.44 (m, 16H, Ar-H). 13C NMR (CDCl3) δ:
56.0 (OCH3), 29.9 (NCH2CH2CH2N), 48.0 (NCH2CH2CH2N), 53.8
(NCH2Ar), 71.1 (OCH2Ar), 111.9, 114.0, 120.1, 127.2, 127.7, 128.5,
133.5, 137.3, 147.1, 149.7 (Ar-C). Anal. calcd. for C33H38N2O4: C,
75.29; H, 7.22; N, 5.32. Found: C, 75.33; H, 7.20; N, 5.34.
1,4-Bis(4-methylthiobenzylamino)butane 6a
Yield: 2.56 g, 71%; m.p.: 71–73°C, IR, υ: 1509 cm−1 (CN). 1H NMR
(CDCl3) δ: 1.36 (bs, 2H, N-H), 2.47 (s, 6H, SCH3), 1.52–1.55 (m, 4H,
NCH2CH2CH2CH2N), 2.60 (t, 4H, J = 4 Hz, NCH2CH2CH2CH2N), 3.73 (s,
4H, NCH2Ar), 7.22 (s, 8H, Ar-H). 13C NMR (CDCl3) δ: 16.1 (SCH3), 27.8
(NCH2CH2CH2CH2N), 49.2 (NCH2CH2CH2CH2N), 53.5 (NCH2Ar),
126.9, 128.6, 136.6, 137.5 (Ar-C). Anal. calcd. for C20H28N2S2: C,
66.67; H, 7.78; N, 7.78. Found: C, 66.71; H, 7.76; N, 7.81.
|
4.2.2 AChE activity determination
The inhibitory effects of novel Schiff bases and their amines derivatives
(1a–d, 2a–d, 3b–d, 4a–c, 5a–c, 6a, 6c, 6d) on AChE activitiy were
measured conforming to the spectrophotometric procedure of Ellman
et al.[70] Acetylthiocholine iodide (AChI) was utilized as substrate for the
enzymatic reaction. 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB) was
utilized for the measurement of the AChE activitiy.[71] Briefly, 100 µL of
Tris/HCl buffer (1 M, pH 8.0), 780 µL of sample solution dissolved in
deionized water at different concentrations, and20 µL of AChE solution
were mixed and incubated for 5 min at 25°C. Then, 50 µL of DTNB
(0.5 mM) was added.[72] Then reaction was initiated by the addition of
50 µL of AChI. The hydrolysis of these substrates was monitored
spectrophotometrically by formation of the yellow 5-thio-2-nitro-
benzoate anion as the result of the reaction of DTNB with thiocholine,
released by enzymatic hydrolysis of AChI, with an absorption maximum
at a wavelength of 412 nm.[73–75] For the calculation of Ki values, three
different novel Schiff bases and their amines derivatives concentrations
were used. Finally, the Lineweaver–Burk curves were drawn.[66]
1,4-Bis(4-benzyloxy-3-methoxybenzylamino)butane 6c
Yield: 4.37 g, 81%; m.p.: 97–98°C, IR, υ: 1510 cm−1 (C N). 1H NMR

(CDCl3) δ: 3.88 (s, 6H, OCH3), 1.53–1.56 (m, 6H, NCH2CH2CH2CH2N,
N-H), 2.63 (t, 4H, J = 4 Hz, NCH2CH2CH2CH2N), 3.70 (s, 4H, NCH2Ar),
5.13 (s, 4H, OCH2Ar), 6.74–7.44 (m, 16H, Ar-H). 13C NMR (CDCl3) δ:
56.0 (OCH3), 27.9 (NCH2CH2CH2CH2N), 49.3 (NCH2CH2CH2CH2N),
53.8 (NCH2Ar), 71.1 (OCH2Ar), 111.9, 114.0, 120.1, 127.2, 127.7,
128.5, 133.8, 137.3, 147.1, 149.7 (Ar-C). Anal. calcd. for C34H40N2O4:
C, 75.56; H, 7.41; N, 5.19. Found: C, 75.60; H, 7.44; N, 5.16.
1,4-Bis(3,4-dibenzyloxybenzylamino)butane 6d
Yield: 5.74 g, 83%; m.p.: 69–70°C, IR, υ: 1519 cm−1 (C N). 1H NMR

(CDCl3) δ: 1.47–1.54 (m, 6H, NCH2CH2CH2CH2N, N-H), 2.57 (t, 4H,
J = 4 Hz, NCH2CH2CH2CH2N), 3.66 (s, 4H, NCH2Ar), 5.13 and 5.15 (s,
8H, OCH2Ar), 6.78–7.45 (m, 26H, Ar-H). 13C NMR (CDCl3) δ: 27.8
(NCH2CH2CH2CH2N), 49.2 (NCH2CH2CH2CH2N), 53.6 (NCH2Ar),
71.2 and 71.4 (OCH2Ar), 115.1, 121.0, 127.3, 127.7, 128.4, 134.0,
137.3, 137.4, 147.9, 149.0 (Ar-C). Anal. calcd. for C46H48N2O4: C,
79.77; H, 6.94; N, 4.05. Found: C, 79.73; H, 6.90; N, 4.09.
CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.
ORCID
Parham Taslimi
http://orcid.org/0000-0002-3171-0633
http://orcid.org/0000-0001-5993-1668
İlhami Gülçin
|
4.2 Biochemical studies
|
4.2.1 Carbonic anhydrase I and II isoenzymes
purification and inhibition studies
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SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section at the end of the article.
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How to cite this article: Yiğit B, Yiğit M, Taslimi P, Gök Y,
Gülçin İ. Schiff bases and their amines: Synthesis and
discovery of carbonic anhydrase and acetylcholinesterase
enzymes inhibitors. Arch Pharm Chem Life Sci. 2018;1–9.
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