Synthesis of N-alkylated pyrazolo[3,4-d]pyrimidine analogs and evaluation of acetylcholinesterase and carbonic anhydrase inhibition properties

Article abstract of DOI:10.1002/ardp.202000330

Fused pyrimidines, especially pyrazolo[3,4-d]pyrimidines, are among the most preferred building blocks for pharmacology studies, as they exhibit a broad spectrum of biological activity. In this study, new derivatives of pyrazolo[3,4-d]pyrimidine were synthesized by alkylation of the N1 nitrogen atom. We synthesized 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine 2 from commercially available aminopyrazolopyrimidine 1 using N-iodosuccinimide as an iodinating agent. The synthesis of compound 2 started with nucleophilic substitution of 3-iodo-1H-pyrazolo[3,4-d]pyrimidin-4-amine with R–X (X: –OMs, –Br, –Cl), affording?N-alkylated pyrazolo[3,4-d]pyrimidine. We performed this synthesis using a weak inorganic base?and the mild temperature was also used for a two-step procedure to generate N-alkylated pyrazolo[3,4-d]pyrimidine derivatives. Also, all compounds were tested for their ability to inhibit acetylcholinesterase (AChE) and the human carbonic anhydrase (hCA) isoforms I and II, with K_i values in the range of 15.41 ± 1.39–63.03 ± 10.68 nM for AChE, 17.68 ± 1.92–66.27 ± 5.43 nM for hCA I, and 8.41 ± 2.03–28.60 ± 7.32 nM for hCA II. Notably, compound 10 was the most selective and potent CA I inhibitor with a significant selectivity ratio of 26.90.

Full text of DOI:10.1002/ardp.202000330

Received: 3 September 2020
DOI: 10.1002/ardp.202000330
Revised: 1 January 2021
Accepted: 4 January 2021
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F U L L P A P E R
Synthesis of N‐alkylated pyrazolo[3,4‐d]pyrimidine analogs
and evaluation of acetylcholinesterase and carbonic
anhydrase inhibition properties
Busra O. Aydin1
| Derya Anil1,2
| Yeliz Demir3
1Department of Chemistry, Faculty of Science,
Ataturk University, Erzurum, Turkey
Abstract
2Department of Chemistry and Chemical
Process Technologies, Technical Sciences
Vocational School, Ataturk University,
Erzurum, Turkey
Fused pyrimidines, especially pyrazolo[3,4‐d]pyrimidines, are among the most preferred
building blocks for pharmacology studies, as they exhibit a broad spectrum of biological
activity. In this study, new derivatives of pyrazolo[3,4‐d]pyrimidine were synthesized
by alkylation of the N1 nitrogen atom. We synthesized 3‐iodo‐1H‐pyrazolo[3,4‐d]
pyrimidin‐4‐amine 2 from commercially available aminopyrazolopyrimidine 1 using
N‐iodosuccinimide as an iodinating agent. The synthesis of compound 2 started with
nucleophilic substitution of 3‐iodo‐1H‐pyrazolo[3,4‐d]pyrimidin‐4‐amine with R–X (X:
–OMs, –Br, –Cl), affording N‐alkylated pyrazolo[3,4‐d]pyrimidine. We performed this
synthesis using a weak inorganic base and the mild temperature was also used for a
two‐step procedure to generate N‐alkylated pyrazolo[3,4‐d]pyrimidine derivatives. Also,
all compounds were tested for their ability to inhibit acetylcholinesterase (AChE) and
the human carbonic anhydrase (hCA) isoforms I and II, with Ki values in the range of
15.41 1.39–63.03 10.68 nM for AChE, 17.68 1.92–66.27 5.43 nM for hCA I, and
8.41 2.03–28.60 7.32 nM for hCA II. Notably, compound 10 was the most selective
and potent CA I inhibitor with a significant selectivity ratio of 26.90.
3Department of Pharmacy Services, Nihat
Delibalta Göle Vocational School, Ardahan
University, Ardahan, Turkey
Correspondence
Derya Anil, Technical Sciences Vocational
School, Ataturk University, Erzurum 25240,
Turkey.
Email: derya.anil@atauni.edu.tr and
daktas61@gmail.com
K E Y W O R D S
acetylcholinesterase, carbonic anhydrase, enzyme inhibition, pyrazolopyrimidine
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1
INTRODUCTION
pyrimidines, pyrazolopyrimidine is a significant scaffold present in
numerous biologically active compounds. As pyrazolopyrimidines
Heterocyclic compounds have a broad range of applications, in
medicinal chemistry, agrochemicals, polymers, and products from
various industries.[1] More specifically, nitrogen‐containing hetero-
cycles are plentiful in nature, existing in many natural products such
as alkaloids, vitamins, antibiotics, and hormones.[2] Approximately
60% of FDA‐approved drugs contain heterocyclic nitrogen in their
structure. The pyrimidine nucleus is a significant class of heterocyclic
compounds for their medicinal properties due to the presence of
pyrimidine base in uracil, cytosine, and thymine, which form the
building blocks of DNA and RNA.[3]
have a structural resemblance with purines, they are considered
biologically active isomeric purine analogs present in DNA and
RNA.[4] Pyrazolo[3,4‐d]pyrimidines are reported to have various
pharmacological properties as antiviral, anticoagulant, antimicrobial,
antitumor, analgesic, and antileukemic agent.[5] Recently, the
pyrazolo[3,4‐d]pyrimidine ring system has been a significant phar-
macophore for anticancer drug discovery.[6–8] Pyrazolo[3,4‐d]pyr-
imidines are potent and selective inhibitors of many kinases, which
have a key role in cancer cell proliferation.[9–11]
The pyrazolopyrimidine nucleus has been the core of synthetic
drug molecules and is still a frequently occurring motif in many
biologically active compounds (Figure 1). For example, allopurinol,
Pyrimidine and its fused analogs have attracted considerable
attention due to various biological activities. Among fused
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Arch Pharm. 2021;354:e2000330.
wileyonlinelibrary.com/journal/ardp
© 2021 Deutsche Pharmazeutische Gesellschaft
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FIGURE 1 Clinically approved drugs containing the pyrazolopyrimidine ring
ibrutinib, and parsaclisib have pyrazolopyrimidine core structures
containing anticancer agents currently approved by the Food and
Drug Administration (FDA). Sapanisertib is an experimental kinase
inhibitor, which is currently in clinical trials for several cancer in-
dications.[12] Zaleplon and sildenafil are other FDA‐approved drugs
containing the pyrazolopyrimidine core structure.[13]
docking of six pyrimidine derivatives with EFGR and CA IX, proposing
their role as inhibitors in cancer studies. They tested these compounds in
silico for drug‐likeness and anticancer activity by docking with the pro-
tein via pyrx docking software.
According to the above information, in this study, we worked on
the synthesis of 13 novel N‐alkylated pyrazolo[3,4‐d]pyrimidine de-
rivatives. Also, we studied their in vitro biological effects on AChE
and hCA I and II isoforms.
The carbonic anhydrases (CAs) play a role in the reversible hy-
dration of CO2 into bicarbonate and protons.[14,15] They also play a
key role in numerous pathological and biological processes such as
tumor growth, respiration, secretion of electrolytes in various tissues
and organs, homeostasis of pH, biosynthetic reactions (i.e., ur-
eagenesis and lipogenesis), bone resorption, and calcification.[16–20]
Hence, CA isoforms play a crucial role as therapeutic goals; their
inhibitors have a significant task for pharmacological areas with a
wide range of ailments such as cancer. osteoporosis, glaucoma,
edema, epilepsy, and obesity.[21,22] Inhibitors of cytosolic human
carbonic anhydrase (hCA) isoforms I and II are mainly employed as
antiepileptic drugs, antiedema drugs, antiglaucoma drugs, and
diuretics.[23,24]
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2
RESULTS AND DISCUSSION
Chemistry
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2.1
The synthesis of N‐alkylated pyrazolo[3,4‐d]pyrimidine derivatives is
an important research area in the literature.[35–38] Minor structural
modifications, which are sometimes single‐atom or group changes,
can have a dramatic impact on the activities of the compounds. In
this study, we report the synthesis of N‐alkylated 3‐iodo‐1H‐pyrazolo
[3,4‐d]pyrimidin‐4‐amine derivatives. N‐Alkylation was performed
using alkyl or benzyl derivatives under basic conditions.
Alzheimer's disease (AD) is a neuropathological disorder, which
mostly affects older people.[25] AD may comprise thinking or problem‐
solving, memory loss, and difficulties with language. AD is characterized
by multiple cortical disturbances, such as intellectual function disorders,
inability to perform daily life activity, decline of learning skills, and loss of
memory.[26,27] There are various hypotheses put forward to explain the
AD. Among them, the cholinergic hypothesis (covering butyr-
ylcholinesterase or BuChE enzymes and acetylcholinesterase or AChE) is
the most considerable hypothesis for the treatment of AD.[28] AChE is
primarily responsible for the hydrolysis of acetylcholine into acetate and
choline.[29] Inhibition of the AChE enzyme gives rise to the enhancement
of cognitive abilities such as memory functions, attention span, and
language skills in AD.[30,31] Hence, the development of potent AChE
inhibitors may be an essential approach for AD treatment.
The alkylation of 1H‐pyrazolo[3,4‐d]pyrimidines has typically
been observed to be selective for N1 under various conditions such
as phase‐transfer catalysis in benzene, Cs2CO3 in dimethylforma-
mide (DMF), strong base, Mitsunobu reaction, or weak base under
high‐temperature conditions.[39,40] We developed useful synthetic
methodologies for the synthesis of new N‐alkylated pyrazolo[3,4‐d]
pyrimidine derivatives with the use of inorganic weak base at 70°C.
All synthesized compounds contain iodine atoms in their struc-
ture and they can be easily functionalized by the coupling reaction.
The general method used for the N‐alkylation of 1H‐pyrazolo[3,4‐d]
pyrimidin‐4‐amine is shown in Scheme 1. First, a commercially available
In the literature, pyrimidine analogs have been studied as AChE and
CAs inhibitors. For instance, Zhi et al.[32] designed and synthesized some
6‐acetyl‐5H‐thiazolo[3,2‐a]pyrimidine derivatives and studied their in
vitro AChE inhibitory activity. Kuday et al. [33] synthesized 11 new pyrido
[2,3‐d]pyrimidine derivatives containing indole rings and assayed their
effects on hCA I and II. They also examined a structure–activity
relationship. Sharma and Shukla[34] analyzed the in silico molecular
SCHEME 1 General synthesis of N‐alkylated pyrazolopyrimidines

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4‐amino‐1H‐pyrazolo[3,4‐d]pyrimidine (1) compound was treated with N‐
iodosuccinimide in DMF at 70°C to afford compound 2.[41] We aimed for
alkylation with derivatives that have a simple structure and different
electronic properties (halogen‐containing benzyl, ethyne, alkyne, or pro-
pyl). The N‐alkylation of compound 3‐iodo‐1H‐pyrazolo[3,4‐d]pyrimidin‐
4‐amine (2) was performed using R–X (X: –Cl, –Br, –OMs) derivatives in
the presence of potassium carbonate in DMF.
obtained using 4‐bromobenzyl bromide in a reaction. Unlike other deri-
vatives, 4‐bromobenzyl was found to be attached to both C3 and N1
positions. This was evidenced by all spectral data (1H nuclear magnetic
resonance [NMR], 13C NMR, high‐resolution mass spectrometry
[HRMS]). Reaction yield rates were excellent, as seen in Table 2.
In the next step of our study, alkylation was performed using 17,
19, 21, 23, 25, 27, 29, and 31. The OH group in the structure of the
reagents was converted to an easily purified mesylate with MsCl and
NEt3. The N‐alkylation reaction previously performed with alkyl
bromine and chlorine reagents was carried out under the same re-
action conditions using reagents containing the –OMs functional
group. Compound 20 was synthesized according to a procedure in
the literature, using alkyl bromide and higher equivalent K2CO3 at
130°C, and it resulted in a 42% yield.[38] We obtained compound
20 using phenethyl methanesulfonate with 66% yield using low
equivalent K2CO3 at 70°C. Also, compound 18 was synthesized ac-
cording to a procedure in the literature, but it yielded different
results.[43] The compound was an intermediate, so results were not
clear enough to be compared. Compounds 29 and 31 contain OH
groups in the aromatic ring. As aromatic OH groups have acidic
properties, an alkylation step was performed in these groups after
being converted to an –OMs derivative. Obtained –OMs inter-
mediates (17, 19, 21, 23, 25, 27, 29, and 31) were used for the N1
First, we started with chloroalkyl derivatives. Compound 2 was
made to react with benzyl chloride in the presence of potassium
carbonate in DMF and heated at 70°C. Benzyl‐alkylated product
4 was obtained with a 44% yield. To obtain epoxy and hydroxy
alkylation products (6 and 8), a reaction was carried out under the
same conditions using epichlorohydrin (5) and 2‐chloroethanol (7),
but desired products could not be generated (Table 1).
The alkylation reaction with alkyl bromide, benzyl bromide, and
cinnamyl bromide reagents yielded compounds 10, 12, 14, and 16, as
shown in Table 2. Treatment of compound 2 with 9, 11, 13, and 15 and
potassium carbonate in dry DMF afforded
a
series of
N‐substituted pyrazolo[3,4‐d]pyrimidine derivatives that include 10, 12,
14, and 16 with good‐to‐excellent yield. Compound 16 was synthesized
using the alkyl halide mentioned in the literature with a 73% yield using
high equivalent K2CO3 at 60°C.[42] We obtained compound 16 with 87%
yield using low equivalent K2CO3 at 70°C. Here, compound 10 was
TABLE 1 N‐Alkylated pyrazolopyrimidines from alkyl chloride
Entry
1
Reagent
N‐Alkylated product
Yield (%)
44
2
No reaction
3
No reaction

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TABLE 2 N‐Alkylated pyrazolopyrimidines from alkyl bromide
Entry
1
Reagent
N‐Alkylated product
Yield (%)
87
2
79
3
70
4
57
alkylation of pyrazolo[3,4‐d]pyrimidine without any purification.
Products 18, 20, 22, 24, 26, 28, 30, and 32 were obtained with
67–79% yield rates, as seen in Table 3.
tested against AChE (associated with AD) and cytosolic isoforms
hCA I and II (associated with glaucoma and epilepsy) by using the
Ellman's and esterase assay methods. The standard inhibitors, ta-
crine (TAC) and acetazolamide (AZA) were employed as a reference
for AChE and hCA isoforms, respectively. According to Tables 4 and
5, it is shown that all N‐alkylated pyrazolo[3,4‐d]pyrimidine deriva-
tives are potent inhibitors against AChE, hCA I, and II. The inhibition
data (IC50 values and Ki values) are listed in the tables.
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2.2
Pharmacology/biology
Biological evaluation
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2.2.1
The novel synthesized 13 N‐alkylated pyrazolo[3,4‐d]pyrimidine de-
rivatives (4, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, and 32) were
1. All the novel synthesized N‐alkylated pyrazolo[3,4‐d]pyrimidine
derivatives moderately inhibited cytosolic isoform hCA I, with

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TABLE 3 N‐Alkylated pyrazolopyrimidines from alkyl mesylate
Entry
1
Reagent
N‐Alkylated product
Yield (%)
75
2
66
3
65
4
64
5
67
6
74
7
79
(Continues)

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TABLE 3 (Continued)
Entry
8
Reagent
N‐Alkylated product
Yield (%)
75
IC50 values ranging from 16.12 to 69.30 nM and Ki values ranging
from 17.68 1.92 to 66.27 5.43 nM. Moreover, all compounds
displayed the best inhibition values, compared with AZA, which is
a standard inhibitor, and they were found to be more effective
inhibitors in the range between 7.16 and 26.90 as compared with
AZA (Ki: 475.55 10.62 nM). Compound 10 displayed the best in-
hibition values (Ki: 17.68 1.92 nM) (Figure 2a). Compound 32 dis-
played low inhibition values according to results. The inhibitor
properties of N‐alkylated pyrazolo[3,4‐d]pyrimidines against hCA I
were found to be decreased in the following order: 10 > (Ki:
17.68 1.92 nM) 18 > (Ki: 19.51 3.33 nM) 14 > (Ki: 19.75 3.95 nM)
22 > (Ki: 20.46 4.98 nM) 20 > (Ki: 23.10 2.37 nM) 12 > (Ki:
30.76 0.72 nM) 26 > (Ki: 31.60 3.46 nM) 4 > (Ki: 33.75 4.66 nM)
28 > (Ki: 35.22 6.22 nM) 16 > (Ki: 44.25 5.32 nM) 24 > (Ki:
51.35 7.32 nM) 30 > (Ki: 60.44 3.89 nM) 32 > (Ki: 66.27 5.43 nM)
(Tables 4 and 5 and Figure 3a; the corresponding plots are given in
the Supporting Information). According to results, the addition of an
ethyl group to compound 4 caused an increase in the inhibition effect
against hCA I (compound 20, Ki: 23.10 2.37 nM). The replacement of
the 4‐chlorobenzyl (26, Ki: 31.60 3.46 nM) moieties with methoxy
(28, Ki: 35.22 11.77 nM) and methanesulfonate (30, Ki:
60.44 3.89 nM) groups caused a decrease in the inhibition values.
The reduction in the length of the alkyl chain at N2 from butyl (16) to
propyl (18) led to a decrease in the inhibitory activity. Finally, the
addition of a methoxy group to compound 30 led to a more active
analog (32, Ki: 66.27 5.43 nM).
2. N‐Alkylated pyrazolo[3,4‐d]pyrimidine derivatives effectively inhibited
cytosolic isoform hCA II, with IC50 values ranging from 12.83 to
36.47 nM and Ki values ranging from 8.41 2.03 to 28.60 7.32 nM.
These 13 analogs displayed a better inhibition effect than the stan-
dard inhibitor AZA (Ki: 97.73 1.62 nM). Compound 14 (Ki:
8.41 2.03 nM) showed the best inhibitory profile (Figure 2b),
TABLE 4 IC50 and Ki values of hCA isoforms (I–II) and AChE with N‐alkylated pyrazolo[3,4‐d]pyrimidine compounds
IC50 (nM)
Ki (nM)
Compounds
hCA I
49.50
25.67
33.00
19.80
51.44
34.65
40.76
38.50
53.31
43.41
36.47
66.00
69.30
223.90
–
r2
hCA II
28.88
18.73
30.13
16.12
23.90
31.50
33.00
34.65
36.47
31.50
27.72
23.10
12.83
96.67
–
r2
AChE
36.47
23.10
33.00
40.76
34.65
25.67
28.88
31.50
49.50
69.30
73.65
63.00
20.38
–
r2
hCA I
hCA II
AChE
4
0.9847
0.9845
0.9844
0.9946
0.9822
0.9834
0.9876
0.9852
0.9945
0.9844
0.9849
0.9960
0.9849
0.9988
–
0.9902
0.9851
0.9776
0.9911
0.9790
0.9885
0.9812
0.9738
0.9822
0.9945
0.9893
0.9808
0.9806
0.9999
–
0.9740
0.9869
0.9811
0.9858
0.9934
0.9841
0.9781
0.9967
0.9909
0.9854
0.9796
0.9911
0.9978
–
33.75 4.66
17.68 1.92
30.76 0.72
19.75 3.95
44.25 5.32
19.51 3.33
23.10 2.37
20.46 4.98
51.35 7.32
31.60 3.46
35.22 6.22
60.44 3.89
66.27 5.43
475.55 10.62
–
26.15 3.15
18.24 2.77
26.65 4.64
8.41 2.03
9.90 2.21
17.18 0.66
15.61 0.87
16.44 3,72
15.48 0.40
28.60 7.32
24.45 4.73
10.61 0.87
11.55 1.21
97.73 1.62
–
33.04 5,12
22.30 3.94
31.81 0.79
37.28 3.78
32.56 4.41
18.62 2.70
24.97 5.87
27.75 5.20
49.03 4.21
59.12 9.74
63.03 10.68
55.27 11.29
15.41 1.39
–
10
12
14
16
18
20
22
24
26
28
30
32
AZAa
TACb
430.10
0.9998
159.64 0.87
Abbreviations: AChE, acetylcholinesterase; AZA, acetazolamide; hCA, human carbonic anhydrase; TAC, tacrine.
aAZA was used as a positive control for carbonic anhydrase enzyme.[27]
bTAC was used as a positive control for acetylcholinesterase enzyme.[27]

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whereas compound 26 (Ki: 28.60 7.32 nM) showed the weakest
inhibition effect against hCA II. The inhibitory properties of
N‐alkylated pyrazolo[3,4‐d]pyrimidine derivatives against hCA II were
found to be decreased in the following order: 14 > (Ki: 8.41 2.03 nM)
16 > (Ki: 9.90 2.21 nM) 30 > (Ki: 10.61 0.87 nM) 32 > (Ki:
11.55 1.21 nM) 24 > (Ki: 15.48 0.40 nM) 20 > (Ki: 15.61 0.87 nM)
22 > (Ki: 16.44 3.72 nM) 18 > (Ki: 17.18 0.66 nM) 10 > (Ki:
18.24 2.77 nM) 28 > (Ki: 24.45 4.73 nM) 4 > (Ki: 26.15 3.15 nM)
12 > (Ki: 26.65 4.64 nM) 26 > (Ki: 28.60 7.32 nM) (Tables 4 and 5
and Figure 3b). As a general trend, comparing compound 18 with 20,
it is observed that the addition of a phenyl group increased the in-
hibition properties (20, Ki: 15.61 0.87 nM). A similar situation is
observed in hCA
I inhibition. The replacement of the 4‐
methanesulfonate (30, Ki: 10.61 0.87 nM) moiety with chlor-
obenzyl (26, Ki: 28.60 7.32 nM), and methoxybenzyl (28, Ki:
24.45 4.73 nM) groups caused a decrease in the inhibition values.
3. All the synthesized novel series of N‐alkylated pyrazolo[3,4‐d]pyr-
imidines showed potent inhibitory activity against the AChE enzyme
with IC50 values ranging from 20.38 to 73.65 nM and Ki values ran-
ging from 15.41 1.39 to 63.03 10.68 nM. Herein, compound 32
displayed the highest inhibition values, with Ki constant of
15.41 1.39 nM (Figure 2c). This value is 10.36 times stronger than
the standard inhibitor (TAC, Ki: 159.64 0.87 nM). When analyzing
the Ki values of the substances and examining the inhibitory effects,
N‐alkylated pyrazolo[3,4‐d]pyrimidines showed a better inhibition
effect on the AChE enzyme. The inhibitor properties of N‐alkylated
pyrazolo[3,4‐d]pyrimidines against AChE were found to be decreased
in the following order: 32 > (Ki: 15.41 1.39 nM) 18 > (Ki:
18.62 2.70 nM) 10 > (Ki: 22.30 3.94 nM) 20 > (Ki: 24.97 5.87 nM)
22 > (Ki: 27.75 5.20 nM) 12 > (Ki: 31.81 0.79 nM) 16 > (Ki:
TABLE 5 Selectivity index values for Ki constants of the
N‐alkylated pyrazolo[3,4‐d]pyrimidine compounds
Compounds
K
(AZA/hCA I)
K (AZA/hCA II) K i (TAC/AChE)
i
i
4
14.09
26.90
15.46
24.09
10.75
24.37
20.59
23.24
9.26
3.74
5.36
3.67
11.62
9.87
5.69
6.26
5.94
6.31
3.42
4.00
9.21
8.46
4.83
7.16
5.02
4.28
4.90
8.57
6.39
5.75
3.26
2.70
2.53
2.89
10.36
FIGURE 2 Lineweaver–Burk graphs for the best inhibitors. (a)
Human carbonic anhydrase (hCA) I, (b) hCA II,
(c) acetylcholinesterase
10
12
14
16
18
20
22
24
26
28
30
32
32.56 4.41 nM) 4 > (Ki: 33.04 5.12 nM) 14 > (Ki: 37.28 3.78 nM)
24 > (Ki: 49.03 4.21 nM) 30 > (Ki: 55.27 11.29 nM) 26 > (Ki:
59.12 9.74 nM) 28 > (Ki: 63.03 10.68 nM) (Tables 4 and 5 and
Figure 3c). As a general trend, comparing compound 30 with com-
pound 32, it is observed that the addition of a methoxy group caused
a
3.59‐fold decrease in the inhibitory properties (30, Ki:
55.27 11.29 nM). Compound 26 exhibited a more effective inhibi-
tion profile than compound 28. According to this result, it can be said
that the 4‐chlorobenzyl group is more effective than the 4‐
methoxybenzyl in inhibiting the AChE activity. A similar result was
observed with hCA I inhibition. The increase in the length of the
group at N2 position from benzyl (4) to phenethyl (20) improved the
inhibition against hCA II (compound 20, Ki: 15.61 0.87 nM).
15.04
13.50
7.87
7.16
Abbreviations: AChE, acetylcholinesterase; AZA, acetazolamide; hCA,
human carbonic anhydrase; TAC, tacrine.

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and structurally characterized by 1H NMR, 13C NMR, and HRMS. These
compounds were monitored in vitro for their inhibitory potential against
AChE and hCA I and II isoenzymes. The biological studies, which were
determined as significant targets in AD treatment, primarily, were per-
formed using Ellman and Verpoorte methods. In vitro studies revealed
that the synthesized compounds based on our design notably inhibited
hCA I, hCA II, and AChE, even more than reference drugs, namely, AZA
and TAC. Compound 10 displayed the best inhibition against hCA I and
compound 14 displayed the best inhibition against hCA II. Moreover,
compound 32 was defined as the most significant and selective AChE
inhibitor in this series. Finally, all derivatives in this series can be con-
sidered outstanding multitarget inhibitors for further AD treatment
investigations.
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4
EXPERIMENTAL
Chemistry
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4.1
|
4.1.1
General information
All reactions were performed under a nitrogen atmosphere. All com-
mercial reagents and chromatography solvents were used as obtained
unless otherwise stated. Analytical thin‐layer chromatography (TLC) was
performed on Merck silica gel 60 F254. Visualization of TLCs was ac-
complished by UV light (254 nm) and staining with an ethanolic PMA
(phosphomolybdic acid) solution. Elemental analysis was performed on a
Leco CHNS‐932. HRMS was performed using Agilent 7800 ICP‐MS. In-
frared (IR) spectra were recorded on a Perkin Elmer Spectrum One FT‐IR
spectrometer. Melting points were measured with Gallenkamp melting
point devices. NMR spectra were recorded using a Bruker 400 MHz
NMR instrument (1H NMR at 400 MHz and 13C NMR at 100 MHz) or a
Varian 400 MHz instrument (1H NMR at 400 MHz and 13C NMR at
100 MHz) in a CDCl3 solution using tetramethylsilane as an internal
standard. 1H NMR data are reported as follows: chemical shift (δ, ppm),
multiplicity (s = singlet, d = dublet, t = triplet, q = quartet, dd = doublet of
doublets, dt = doublet of triplets, m = multiplet or otherwise stated), in-
tegration, coupling constant (J, Hz). 13C NMR data are given in terms of
chemical shift (δ, ppm) (please see the Supporting Information for the
original spectra).
FIGURE 3 Ki values of the compounds for (a) human carbonic
anhydrase (hCA) I, (b) hCA II, (c) acetylcholinesterase (AChE)
The InChI codes of the investigated compounds, together with some
biological activity data, are provided as Supporting Information.
Similarly, the addition of a fluorine atom to compound 4 caused an
increase in the inhibition effect against AChE (compound 22, Ki:
27.75 5.20 nM).
|
4.1.2
General procedure for the preparation of
N‐alkylated pyrazolo[3,4‐d]pyrimidines 4, 10, 12, 14,
and 16
|
3
CONCLUSION
Anhydrous K2CO3 (2 eq) was added to a mixture of 3‐iodo‐1H‐pyrazolo
[3,4‐d]pyrimidin‐4‐amine (1 eq) and alkyl halide (3, 9, 11, 13, 15) (2 eq) in
dry DMF (50 ml). The reaction mixture was stirred overnight at 70°C
under a nitrogen atmosphere. After cooling, it was poured into water
(80 ml). It was extracted with EtOAc (3 × 60 ml). The combined organic
This study reports an efficient protocol for the N‐alkylation of compound
3‐iodo‐1H‐pyrazolo[3,4‐d]pyrimidin‐4‐amine (2). In total, 13 novel
N‐substituted pyrazolo[3,4‐d]pyrimidine derivatives have been prepared

AYDIN ET AL.
9 of 11
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layers were dried over Na2SO4 and concentrated under reduced pressure
to give the crude product. The crude was precipitated (DCM/hexanes,
1:20, 40 ml) and filtered to give the desired product.
reaction mixture was stirred for 22 h at room temperature, and then
it was quenched with sat. NH4Cl (40 ml) solution and extracted with
DCM (2 × 50 ml). The combined organic layers were dried over
Na2SO4 and filtered. The solvent was evaporated to dryness under
reduced pressure to give alkyl methanesulfonate (17, 19, 21, 23, 25,
29, 31) (82–99%). The yellow liquid was used in the next step without
purification.
1‐Benzyl‐3‐iodo‐1H‐pyrazolo[3,4‐d]pyrimidin‐4‐amine (4)
Yellow solid. Yield: 44%, m.p.: 214–216°C. IR (KBr, cm−1): 3589 and
540. 1H NMR (400 MHz, CDCl3) δ 8.39 (s, 1H), 7.39–7.29 (m, 5H), and
5.57 (s, 2H). 13C NMR (100 MHz, CDCl3) δ 157.6, 156.6, 156.5, 154.1,
136.3, 128.9, 104.3, 86.8, and 51.5. Q‐TOF LC/MS: 352.0044 ([M
+H]+, C12H11IN5+; calc: 352.0059).
Anhydrous K2CO3 (2 eq) was added to a mixture of 3‐iodo‐1H‐
pyrazolo[3,4‐d]pyrimidin‐4‐amine (1 eq) and alkyl methanesulfonate
(17, 19, 21, 23, 25, 29, 31) (2 eq) in dry DMF (50 ml). The reaction
mixture was stirred overnight at 70°C under a nitrogen atmosphere.
After cooling, it was poured into water (80 ml). It was extracted with
EtOAc (3 × 60 ml). The combined organic layers were dried over
Na2SO4 and concentrated under reduced pressure to give the crude
product. The crude was precipitated (DCM/hexanes, 1:20, 40 ml) and
filtered to give the desired product.
1,3‐Bis(4‐bromobenzyl)‐1H‐pyrazolo[3,4‐d]pyrimidin‐4‐amine (10)
White solid. Yield: 57%, m.p.: 190–192°C. IR (KBr, cm−1): 3412 and
655. 1H NMR (400 MHz, dimethyl sulfoxide [DMSO]) δ 8.27 (s, 1H),
7.51–7.48 (m, 4H), 7.30 (d, J = 8.2 Hz, 2H), 7.17 (d, J = 8.2 Hz, 2H),
5.46 (s, 2H), and 4.77 (d, J = 5.9 Hz, 2H). 13C NMR (100 MHz, DMSO)
δ 156.9, 156.7, 153.9, 139.4, 136.8, 132.3, 131.9, 130.5, 129.98,
121.6, 104.0, 89.7, 50.1, and 43.6. Q‐TOF LC/MS: 471. 9770, ([M
+H]+; C19H16Br2N5+; calculated: 471.9772).
3‐Iodo‐1‐propyl‐1H‐pyrazolo[3,4‐d]pyrimidin‐4‐amine (18)
White solid. Yield: 75%, m.p.: 207–209°C. IR (KBr, cm−1): 3428, 1482,
and 541. 1H NMR (400 MHz, DMSO) δ 8.17 (s, 1H), 4.20 (t, J = 7.2 Hz,
2H), 1.77 (q, 7.2 Hz, 2H), and 0.78 (t, J = 7.2 Hz, 3H). 13C NMR
(100 MHz, DMSO) δ 158.4, 156.7, 154.1, 103.7, 89.3, 48.8, 23.2, and
11.6. Elemental analysis for C8H10IN5: Calculated: C, 31.70; H, 3.33;
N, 23.11; found: C, 31.03; H, 3.16; N, 23.21.
(E)‐3‐Iodo‐1‐styryl‐1H‐pyrazolo[3,4‐d]pyrimidin‐4‐amine (12)
White solid. Yield: 70%, m.p.: 182–184°C. IR (KBr, cm−1): 3450, 1651,
and 610. 1H NMR (400 MHz, DMSO) δ 8.21 (s, 1H), 7.41 (d, J = 7.2 Hz,
1H), 7.32–7.16 (m, 3H), 6.55 (d, J = 15.9 Hz, 1H), 6.40 (dt, J = 15.9,
6.1 Hz, 2H), and 5.04 (d, J = 6.1 Hz, 2H). 13C NMR (100 MHz, DMSO)
δ 158.4, 156.8, 154.0, 136.5, 133.4, 129.3, 128.6, 127.2, 124.9, 103.8,
and 90.0. Elemental analysis for C14H12IN5: Calculated: C, 44.58; H,
3.21; N, 18.57; found: C, 44.47; H, 3.49; N, 18.26.
3‐Iodo‐1‐phenethyl‐1H‐pyrazolo[3,4‐d]pyrimidin‐4‐amine (20)
Yellow solid. Yield: 66%, m.p.: 190–192°C. IR (KBr, cm−1): 3438,
3368, 1492, and 539. 1H NMR (400 MHz, CDCl3) δ 8.29 (s, 1H),
7.32–7.15 (m, 5H), 6.29 (bs, 2H), 4.60 (t, 2H), and 3.21 (t, 2H). 13C
NMR (100 MHz, CDCl3) δ 157.5, 156.0, 153.8, 137.6, 128.8, 126.7,
103.9, 86.0, 48.8, and 36.0. Elemental analysis for C13H12IN5: Cal-
culated: C, 42.76; H, 3.31; N, 19.18; found: C, 43.13; H, 3.36;
N, 19.28.
Methyl 2‐[(4‐amino‐3‐iodo‐1H‐pyrazolo[3,4‐d]pyrimidin‐1‐yl)
methyl]‐3‐nitrobenzoate (14)
Yellow solid. Yield: 70%, m.p.: 254–256°C. IR (KBr, cm−1): 3445,
1727, 1555, 1286, and 641. 1H NMR (400 MHz, DMSO) δ 8.18 (s,
1H), 8.11 (d, J = 8.0 Hz, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.72 (t, J = 8.0 Hz,
1H), 5.89 (s, 2H), and 3.78 (s, 3H). 13C NMR (100 MHz, DMSO) δ
167.0, 158.3, 156.8, 154.2, 151.8, 135.1, 130.72, 128.6, 105.8, 103.5,
91.2, 87.9, 53.6, and 43.1. Q‐TOF LC/MS: 454.9947, ([M+H]+;
1‐(3‐Fluorobenzyl)‐3‐iodo‐1H‐pyrazolo[3,4‐d]pyrimidin‐4‐amine (22)
White solid. Yield: 65%, m.p.: 208–210°C. IR (KBr, cm−1): 3454, 1101,
and 602. 1H NMR (400 MHz, DMSO) δ 8.22 (s, 1H), 7.34 (d, J = 8.3 Hz,
1H), 7.24–7.05 (m, 3H), and 5.51 (s, 2H). 13C NMR (100 MHz, DMSO)
δ 161.8, 159.3, 158.4, 157.0, 154.3, 125.3, 124.3, 124.1, 116.0, 103.7,
90.6, and 44.5. Elemental analysis for C12H9FIN5: Calculated: C,
39.05; H, 2.46; N, 18.97; found: C, 39.11; H, 2.61; N, 18.74.
C
14H12IN6O4+; calculated: 454.9964).
1‐Butyl‐3‐iodo‐1H‐pyrazolo[3,4‐d]pyrimidin‐4‐amine (16)
White solid. Yield: 87%, m.p.: 168–170°C. IR (KBr, cm−1): 3444, 1488,
and 610. 1H NMR (400 MHz, DMSO) δ 8.17 (s, 1H), 4.24 (t, J = 7.4 Hz,
2H), 1.82–1.60 (m, 2H), 1.30–1.07 (m, 2H), and 0.84 (t, J = 7.4 Hz, 3H).
13C NMR (100 MHz, DMSO) δ 158.3, 156.6, 154.0, 103.6, 89.2,
46.9, 31.8, 19.9, and 14.1. Elemental analysis for C9H12IN5: Calculated:
C, 34.32; H, 3.90; N, 22.03,; found: C, 34.09; H, 3.81; N, 22.08.
3‐Iodo‐1‐(prop‐2‐yn‐1‐yl)‐1H‐pyrazolo[3,4‐d]pyrimidin‐4‐amine (24)
Yellow solid. Yield: 64%, m.p.: 220–222°C. IR (KBr, cm−1): 3439,
3042, and 597. 1H NMR (400 MHz, DMSO) δ 8.21 (s, 1H), 5.10 (d,
J = 2.3 Hz, 2H), and 3.39 (t, J = 2.3 Hz, 1H). 13C NMR (100 MHz,
DMSO) δ 158.4, 157.1, 153.8, 103.8, 91.4, 78.9, 76.4, and 36.7. Q‐
TOF LC/MS: 299.9746 C8H7IN5+; ([M+H]+ calculated: 299.9746.)
|
4.1.3
General procedure for the preparation of
N‐alkylated pyrazolo[3,4‐d]pyrimidines 18, 20, 22, 24,
26, 28, 30, 32
1‐(4‐Chlorobenzyl)‐3‐iodo‐1H‐pyrazolo[3,4‐d]pyrimidin‐4‐
amine (26)
White solid. Yield: 67%, m.p.: 249–251°C. IR (KBr, cm−1): 3445, and
585. 1H NMR (400 MHz, DMSO) δ 8.22 (s, 1H), 7.37 (d, J = 8.4 Hz,
2H), 7.23 (d, J = 8.4 Hz, 2H), and 5.46 (s, 2H). 13C NMR (100 MHz,
NEt3 (2 eq) and MsCl (2 eq) were added dropwise to a solution of
R‐OH (1 eq) in dry DCM (60 ml) under a nitrogen atmosphere. The

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AYDIN ET AL.
|
|
AChE activity assay
DMSO) δ 158.4, 157.1, 154.2, 136.6, 133.0, 130.3, 129.4, 103.8, 90.6,
and 49.9. Elemental analysis for C12H9ClIN5: Calculated: C, 37.38; H,
2.35; N, 18.16; found: C, 37.08; H, 2.69; N, 18.21.
4.2.2
Acetylcholinesterase from Electrophorus electricus (C2888, Type V‐
S) was used in this study. In vitro effects on the AChE activity of the
target N‐alkylated pyrazolo[3,4‐d]pyrimidine derivatives (4, 10, 12,
14, 16, 18, 20, 22, 24, 26, 28, 30, 32) were evaluated by the method
of Ellman et al.[55] TAC was used as the reference drug. All the
measurements were repeated three times.
3‐Iodo‐1‐(4‐methoxybenzyl)−1H‐pyrazolo[3,4‐d]pyrimidin‐4‐
amine (28)
White solid. Yield: 74%, m.p.: 247–249°C. IR (KBr, cm−1): 3448, 1246,
and 538. 1H NMR (400 MHz, CDCl3) δ 8.39 (s, 1H), 7.36 (d, J = 8.8 Hz,
2H), 6.86 (d, J = 8.8 Hz, 2H), 5.50 (s, 2H), and 3.79 (s, 3H). 13C NMR
(100 MHz, DMSO) δ 159.5, 158.4, 157.0, 156.8, 130.0, 129.8, 114.7,
103.8, 90.1, 55.7, and 50.2. Quadrupole time‐of‐flight liquid
chromatography–mass spectrometry (Q‐TOF LC/MS): 382.0138
ORCID
Busra O. Aydin
https://orcid.org/0000-0002-0045-5254
https://orcid.org/0000-0003-2584-275X
https://orcid.org/0000-0003-3216-1098
Derya Anil
C
13H13IN5O+ ([M+H]+ calculated: 382.0164).
Yeliz Demir
4‐[(4‐Amino‐3‐iodo‐1H‐pyrazolo[3,4‐d]pyrimidin‐1‐yl)methyl]phenyl
methanesulfonate (30)
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SUPPORTING INFORMATION
Additional Supporting Information may be found online in the sup-
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How to cite this article: Aydin BO, Anil D, Demir Y. Synthesis
of N‐alkylated pyrazolo[3,4‐d]pyrimidine analogs and
evaluation of acetylcholinesterase and carbonic anhydrase
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