Design and Synthesis of Triazole-Phthalimide Hybrids with Anti-inflammatory Activity

Article abstract of DOI:10.1248/cpb.c18-00607

Phthalimido-alkyl-1H-1,2,3-triazole derivatives 3a–d and 4a–d were efficiently synthesized using 1,3-di-polar cycloaddition reaction. Anti-inflammatory activity and toxicity studies were performed. The results demonstrated that all the tested compounds reduced carrageenan-induced paw edema and indicated no lethality for toxicity against Artemia salina and acute toxicity in vivo (LD₅₀ up to 1gkg ^?1). Furthermore, the structure of phthalimide linked to phenyl group proved to be more active than the compounds containing benzothiazole moiety. Structural modifications such as removal of the phthalimide group and subsequent acetylation, to exemplify a non-cyclic amide, demonstrate that the phthalimide and triazole moieties are important for design of potent candidates with anti-inflammatory drug proprieties. Docking into the cyclooxy-genase-2 (COX-2) confirms the importance of the phthalimide and triazole groups in the anti-inflammatory activity. The histopathological studies showed that the compounds 3a–d and 4a–d did not cause serious pathological lesions liver or kidneys.

Full text of DOI:10.1248/cpb.c18-00607

96
Chem. Pharm. Bull. 67, 96–105 (2019)
Vol. 67, No. 2
Regular Article
Design and Synthesis of Triazole-Phthalimide Hybrids with Anti-
inflammatory Activity
Shalom P. de O. Assis,a,b,c Moara T. da Silva,a Filipe Torres da Silva,a,c Mirella P. Sant’Anna,c
Carolina M. B. de Albuquerque Tenório,c Caroline F. Brito dos Santos,c
,b
,a
Caíque S. M. da Fonseca,b Gustavo Seabra,d Vera L. M. Lima,* and Ronaldo N. de Oliveira*
a Laboratório de Síntese de Compostos Bioativos, Departamento de Química, Universidade Federal Rural de
Pernambuco; Rua Dom Manoel de Medeiros s/n, Dois Irmãos, CEP 52171–900, Recife-PE, Brazil: b Departamento
de Bioquímica, Universidade Federal de Pernambuco; Av. Prof. Moraes Rego, s/n, CEP 50.670–420. Recife-PE,
c
Brazil: Departamento de Medicina, Universidade Católica de Pernambuco; Av. do Príncipe, 526, CEP 50.050–900,
d
Recife-PE, Brazil: and Departamento de Química Fundamental; Av. Jornalista Anibal Fernandes, s/nº Cidade
Universitária, CEP 50.740–560, Recife-PE, Brazil.
Received August 8, 2018; accepted November 6, 2018
Phthalimido-alkyl-1H-1,2,3-triazole derivatives 3a–d and 4a–d were efficiently synthesized using 1,3-di-
polar cycloaddition reaction. Anti-inflammatory activity and toxicity studies were performed. The results
demonstrated that all the tested compounds reduced carrageenan-induced paw edema and indicated no le-
thality for toxicity against Artemia salina and acute toxicity in vivo (LD50 up to 1gkg ꢀ1). Furthermore, the
structure of phthalimide linked to phenyl group proved to be more active than the compounds containing
benzothiazole moiety. Structural modifications such as removal of the phthalimide group and subsequent
acetylation, to exemplify a non-cyclic amide, demonstrate that the phthalimide and triazole moieties are im-
portant for design of potent candidates with anti-inflammatory drug proprieties. Docking into the cyclooxy-
genase-2 (COX-2) confirms the importance of the phthalimide and triazole groups in the anti-inflammatory
activity. The histopathological studies showed that the compounds 3a–d and 4a–d did not cause serious
pathological lesions liver or kidneys.
Key words phthalimide; triazole; anti-inflammatory activity; toxicity
Phthalimido-alkyl-1H-1,2,3-triazole derivatives were ef-
Since the discovery of click chemistry by Fokin and
ficiently synthesized. All synthesized compounds reduced Sharpless,10) and Tornøe and Meldal,11) reactions involving
carrageenan-induced paw edema and indicated no lethality for copper-catalyzed azide-alkyne cycloaddition (CuAAC) have
toxicity. Phthalimide linked to phenyl group demonstrated to gained popularity as a general strategy for the synthesis of
be more active than those containing benzothiazole moiety. 1,4-disubstituted 1,2,3-triazoles. Recently, several of these
The histopathological studies showed that the compounds did compounds have been prepared employing the click chemis-
not cause serious pathological lesions liver or kidneys.
try protocol12,13) providing rapid access to small molecules as
Phthalimides are considered an important synthon for or- privileged medicinal scaffolds.14,15) 1,2,3-Triazole has shown
ganic synthesis and several of their derivatives, after biologi- a broad range of biological activities, such as anti-nocicep-
cal evaluation, have been reported to have interesting phar- tive,16) enzyme inhibitive17,18) and, as recently reported by us,
macological proprieties.1) A molecular diversity has emerged antitumoral19) and anti-inflammatory.20,21)
from the phthalimide scaffold linked to other heterocyclic
In 2012, Shafi et al.16) analyzed the efficacy of 1,2,3-triazole
nucleus; for example, benzothiazoles have shown significant compounds based on studies of structure–activity relation-
cytotoxic activity against human cancer cells lines, such as ships. They focused on the three structural components: i) the
Burkitt’s lymphoma CA46, chronic myelogenous leukaemia nature of the group attached to triazolic ring; ii) the substitu-
K562 and human hepatoma SKHep1.2)
ent (functional groups); iii) the position of the substitutions on
The search for molecular hybrids from bioactive chemical the aromatic ring. They synthesized bis-heterocycle containing
entities to enhance their original biological activities has been 2-mercapto-benzothiazole and triazole, which demonstrated
a goal of the scientific community. Triazole is an attractive potential anti-inflammatory activity.
heterocycle which has the potential of having a wide range of
Pharmacophoric moieties of phthalimide analogues were
applications in the field of medicinal chemistry.3–6)
studied by Lima et al.22) The authors concluded that
1,2,3-Triazole has particular proprieties such as metabolic phthalimide ring plays an important role in the anti-inflam-
stability and slow degradation. This specific triazolic structure matory activity. These analogues were evaluated and revealed
can also be an acceptor and/or donor to hydrogen bond forma- ability to inhibit tumor necrosis factor (TNF-α) levels in the
tion.7) For instance, our research reported an intramolecular bronchoalveolar lavage fluid of mice lungs treated with them.
CH–O hydrogen bond between H5-triazolic and the endocyclic
Phthalimide and 1,2,3-triazole nucleus have been applied
oxygen of N-glucopyranoside.8) In addition, an assisted version as a strategy to design new molecular hybrids. In previ-
of a classical E2 elimination mechanism to furnish conjugated ous studies, we reported on the synthesis23,24) and biological
alkene after triazole protonation was described by de Oliveira activities19,21,25,26) of these structures to valorize them as prom-
et al.9)
ising “drug-like” molecules. One example of phthalimide-
*To whom correspondence should be addressed. e-mail: lima.vera.ufpe@gmail.com; ronaldo.noliveira@ufrpe.br
© 2019 The Pharmaceutical Society of Japan

Vol. 67, No. 2 (2019)
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97
Fig. 1. Triazole and Phthalimide Based Hybrid Molecules
Chart 1. Synthesis of 1,2,3-Triazole Compounds 3a–d and 4a–d
triazole molecule was synthesized and studied for its anti-
Recently, our research group developed an efficient click
protocol for the preparation of N-phthalimidoalkyl 1H-1,2,3-
inflammatory activities.20)
The present work concerns the synthesis of phthalimide- triazoles using N,N-dimethylformamide (DMF) as the solvent,
linked to 1,2,3-triazole using a protocol based on click chem- copper iodide (CuI) and triethylamine (Et3N) under ultrasound
istry. The toxicity and anti-inflammatory activities were tested irradiation for 30min at room temperature.24) Under these
on mice, and docking studies show the mode of action of the conditions, eight compounds 3a–d and 4a–d were synthesized
compounds. Based on the heterocycles, namely phthalimide, in good to excellent yields of 70–96% (Chart 1 and Table 1).
triazole and benzothiazole, this research proposed two strate-
The structures of the compounds 3a–d and 4a–d were
1
gies for molecular hybridization towards new anti-inflammato- characterized by IR, H- and 13C-NMR spectra and elemen-
1
ry compound leads (cf. Fig. 1).
tal analysis. The H-NMR data for alkyl groups were well
characterized, showing the presence of methylene groups
with multiplicity according to the structural sizes chain. The
Results and Discussion
Chemistry We started with the synthesis of 1 and 2a–d. phthalimide protons appeared between δ 7.64 and 7.91ppm as
The propargyl bromide and 1,3-benzothiazole-2-thiol reacted second-order multiplets. The presence of the H5-triazolic in
to afford 1 with yield of 70%. The azide-phthalimides (2a–d) the aromatic region was identified as a singlet in the downfield
were synthesized, according to the literature description, in region as a shielded proton (δ 7.64–8.64ppm) confirming the
60–93% yields.24)
1,3-dipolar cycloaddition reaction.

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Table 1. Chemical Structures, Melting Points, Yields and LogP of the Compounds 3a–d and 4a–d
*Partition coefficient log P values were calculated using Advanced Chemistry Development (ACD/Labs Algorithm Version: v12.01).
Pharmacology
Toxicity against Artemia salina
decreased respiratory rate, in the treated mice.
In Vivo Anti-inflammatory Activity T h e a n t i - i n fl a m m a -
Artemia salina has been used as an indicator of toxicity for tory assays were preceded by the acute toxicity tests in mice.
evaluating chemicals, pesticides and polluting products among Anti-inflammatory tests were performed for compounds 3a–d
other substances. A. salina has been reported as a good way and 4a–d in the groups of mice treated with a dose of 250mg
to determine and evaluate the biological activity (toxicity) of kg−1. All the compounds exhibited anti-inflammatory proper-
a particular chemical compound, or a natural product by using ties when compared with acetylsalicylic acid (ASA) and ibu-
the median lethal concentration (LC50). None of the eight profen in the same dosage (Table 2, Figs. 2 and 3).
compounds synthesized in this study showed lethality against
Artemia salina for concentrations up to 1000ppm.
Acute Toxicity in Vivo
The benzoheterocycle-triazole-phthalimides (BTP) 3a–d
showed less anti-inflammatory activity than that observed for
ASA and ibuprofen. These compounds 3a–d were the least
The acute toxicity study using the eight new phthalimide active of all of the tested drugs. These compounds decreased
triazoles-linked benzoheterocycles indicated that the dose the inflammation in paw edema carrageenan-induced by 14.2,
(250mgkg−1) required for anti-inflammatory activity was in 18.0, 30.1, 20.1%, respectively, at 4h post-carrageenan; for
the safe therapeutic range, with a single dose administered inhibition at 24h the results were 16.8, 11.0, 19.4 and 15.8% of
orally to mice having an LD50 >1gkg−1. Thus, this dose limit inhibitions, respectively (cf. Fig. 3-A).
of the compounds did not result in any lethality or observable
On the other hand, when 2-mercapto benzothiazole was
behavioral changes such as writhing, gasping, palpitation, and replaced by phenyl, the results were improved for the com-

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99
Table 2. Anti-inflammatory Activity of Phthalimide Triazoles-Linked Benzoheterocycles Compounds 3a–d and 4a–d in Carrageenan-Induced Edema in
Mice
Anti-inflammatory activity
Paw size (mm)
(% inhibition)
Compound
Time (h)
Time (h)
4
1
2
3
4
24
3.558 0.307** 3.450 0.400*** 3.496 0.360**
3.180 0.167 3.450 0.400 3.492 0.363
48
2
24
3a
3b
3c
3d
4a
4b
4c
4d
4.356 0.395
3.876 0.581
4.186 0.285
3.866 0.217
4.285 0.194
4.263 0.176
4.395 0.351
4.505 0.267
4.146 0.273
3.550 0.421
3.572 0.259*
3.678 0.258
4.065 0.104
3.885 0.225
3.984 0.159
3.422 0.340
4.8
8.4
14.2
18.0
30.1
20.1
34.2
42.5
40.9
16.8
11.0
19.4
15.8
35.6
20.0
13.6
22.4
3.204 0.413*** 2.928 0.400*** 3.372 0.277** 3.472 0.271**
3.524 0.240 3.088 0.522** 3.256 0.186* 3.326 0.203*
14.7
5.1
3.755 0.089*** 2.820 0.107*** 2.760 0.184*** 3.283 0.164***
3.158 0.099*** 2.463 0.457*** 3.408 0.053*** 3.568 0.092**
5.1
8.8
3.380 0.422*** 3.243 0.309*** 2.595 0.291*** 3.798 0.098*
3.938 0.061
23.1
7.0
4.188 0.179 3.223 0.094*** 2.303 0.332*** 3.498 0.188*** 3.745 0.110***
48.9
(117.8)a)
ASA
4.584 0.121
4.360 0.217*** 4.372 0.190*** 2.670 0.402*** 2.590 0.359*** 2.574 0.296***
4.272 0.190*** 3.234 0.095*** 2.596 0.268*** 2.756 0.323*** 2.634 0.286***
5.2
8.3
41.5
45.2
43.5
41.4
Ibuprofen 4.644 0.200
1% CMC 4.674 0.199
4.915 0.096
5.442 0.359*** 5.567 0.336*** 5.592 0.335*** 4.434 0.191
−3.8 −17.6 −18.1
Data were expressed as mean standard deviation. *p<0.05; **p<0.01; ***p<0.001 vs. left hind paw. One-way ANOVA followed by Bonferroni posttest. a) Potency:
was expressed of the tested compound relative to ASA at 4h as best result (4d: 48.9/41.5=117.8).
Fig. 2. In Vivo Anti-inflammatory Activity of Phthalimide-Linked to Triazoles Compounds (3a–d), (4a–d), Ibuprofen and ASA on Carrageenan-
Induced Edema in Mice
(Color figure can be accessed in the online version.)
pounds 4a–d. In general, the activity was enhanced according anti-inflammatory activity. The compounds 3a–d show log
to the size of the aliphatic chain (cf. Fig. 3-B). The inhibition p=3.75–4.67 values, and % of inhibition from 14 to 30%.
of inflammation induced by compound 4a proved to be time- On the other hand, compounds 4a–d show values of log
dependent as even after 24h compound 4a presented inflam- p=2.85–3.76, and % of inhibition between 34 and 48.9% (Ta-
matory inhibition by 35% (Table 2). In contrast, all the other bles 1, 2). These results indicate that the tendency to penetrate
phenyl derivatives 4b–d had a decrease in the inflammation lipid barriers seems no very important data to be considered
process by the end of 24h.
for pharmacokinetic properties and drug design.
The anti-inflammatory profile of compound 4d (48.9% in-
In order to investigate the influence of phthalimide scaffold
hibition at 4h and 22.4% inhibition at 24h post-carrageenan) in the inflammatory process, compound 4d was hydrolyzed
was better than standard ASA and ibuprofen when compar- by treatment with a mixture of methanol/hydrazine to furnish
ing at 4h, i.e., ASA 41.5% and ibuprofen 45.2% of inhibition. amino-triazole 5 in 87% yield (Chart 2). Afterwards, acetyla-
Concerning compound 4d, the best result, that 2-mercaptoben- tion of amino-triazole 5 lead to triazole-acetamide 6 in yield
zothiazole was replaced by phthalimide core using our molec- of 61%; thus the structural effect from cyclic imide was also
ular hybridization strategy (see Fig. 1), the results indicate that investigated. On the NMR spectra of N-acetylamino-triazole
anti-inflammatory activity was improved from 46.6%/3h16) to 6 was observed doubling signals due to presence of N-acetyl
48.9%/4h (this work), more active even after 4h.
rotamers (see Experimental data).
The compounds 4b and 4c presented satisfactory anti-
The experiment revealed, after 4h, non-significant variation
inflammatory activities with values of 42.5 and 40.9%, respec- in anti-inflammatory activity of the compound 5 compared
tively; these results were highest for the 4h and comparable with 4d (Table 3). These results also indicate the importance
with ASA and ibuprofen.
In general, the structural correlation of the synthesized Triazole-acetamide 6 was less active (31%) than 4d or 5.
compounds 3a–d and 4a–d considering their aliphatic chain
Histopathological Study Liver and kidneys of the mice
size, relative to individual series, reveals difference in the were evaluated externally and did not show macroscopic dif-
of the amino-triazole moiety in the anti-inflammatory process.

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ferences between the control group (1% carboxymethylcel- with little clinical significance. The foci of necrosis and even-
lulose (CMC)) and the groups treated with compounds 3a–d tual hepatic steatosis, in addition to the single renal impair-
and 4a–d. The livers, sliced with a scalpel, did not show any ment with chronic granulomatous inflammatory process, in all
internal macroscopically significant differences.
that has been demonstrated, are not directly related to the use
The control and mice kidneys tested for compounds 3a–d of 3a–d and 4a–d substances.
and 4a–d showed a mild, sometimes cortical, chronic inflam-
Molecular Docking The anti-inflammatory effects are
matory infiltrate underlying the pelvic, focal and non-specific associated to inhibition of cyclooxygenase-2 (COX-2). The
epithelial lining in the samples (Fig. 4).
binding of the compounds to COX-2 is illustrated in Fig. 6,
In liver microscopy, all the organs studied showed vascular which shows molecule 4d in the active pocket of COX-2. Most
congestion and non-specific chronic inflammatory infiltrates, inhibitors of COX-2 are carboxylic acids and bind in this same
focal and portal. The livers in the control group and the ani- site by establishing H-bonds to Ser530 and Tyr385 through
mals tested for compounds 3a, 3b, and 4a–d exhibited mini- the carboxylate. In all phthalimides, the phthalimide moiety
mal foci of hepatocyte necrosis. Some associated polymorpho- establishes a H-Bond to Ser530 (2.3Å). In 4a, the triazole
nuclear leukocytes could also be observed, with no signs of makes a second weaker bond to Tyr385. With larger linker
specificity (Fig. 5).
sizes this interaction is lost, and in 4d the triazole H-bonds
In the present study, there were minimal foci of hepatocyte to Tyr355 (1.9Å). In all cases, the phenyl makes only hydro-
necrosis in the mice that were tested for 3a–d and 4a–d. The phobic interactions with the surrounding residues. The FlexX
same finding was seen in the mouse that received 1% CMC. score (binding energy) obtained for the best pose in 4d was
Due to the small amount of this alteration found in the evalu- −16.00kJmol−1, which is comparable to the score for ASA
ated organs, and because it was also present in the control (−16.94kJmol−1) and significantly stronger than Ibuprofen
animal, hepatocyte necrosis seems to be a nonspecific finding (−11.68kJmol−1). Substitution of the phenyl ring by mercap-
tobenzothiazole does not improve the interactions, as the size
of the new substituent increases the steric clashes to the sur-
rounding residues, resulting in a −12.26kJmol−1 FlexX score
for 3d, which makes the same H-bonds as 4d. Removing the
phthalimide group, however, results in loss of the important
interactions. Compounds 5 and 6 do not seem enter the bind-
ing site adequately and the FlexX score for the best pose of
compound 5, for example, is only −12.73kJmol−1 (Fig. 6).
Compound 5 is making weak H-bonds to Glu524 and Phe470
but is far away from the catalytic residues Tyr385 and Ser530.
Those are only weak bonds such that in other poses they are
substituted by Asp347 and Glu346. In some poses, the tri-
azole can make H-bonds to Gln350 or Ser530. In agreement
with recent studies on different compounds,30,31) our results
demonstrate the important role of the phthalimide group in
the biological effect, and indicate possible routes for future
improvement.
Conclusion
The phthalimide 1,2,3-triazole compounds 3a–d and 4a–d
were synthesized in good to excellent yields (70–96%). All
the compounds decreased the carrageenan-induced edema in
mice, when compared with ASA and ibuprofen commercial
Table 3. Anti-inflammatory Activity (% Inhibition) of 4d, 5 and 6
Compound
2h
4h (Potency)a)
24h
22.4
4d
5
7.0
16.0
3.8
48.9 (108.0)
47.9 (106.0)
31.0 (68.6)
26.5
37.0 (81.9)a)
Fig. 3. Effect of the Triazole Derivatives on the Development of Edema
Induced by 1% Carrageenan in Mice
6
a) Potency: was expressed of the tested compound relative to Ibuprofen at 4h.
(A) Compounds 3a–d; (B) Compounds 4a–d.
Chart 2. Synthesis of Derivatives 5 and 6

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101
Fig. 4. Renal Histopathological Findings in the Mice Tested for Substances 3a–d, 4a–d and Control Negative: 3a (A), 3b (B), 3c (C), 3d (D), 4b (F),
4d (H) and Control Negative −1% CMC (I)
Renal tissue without inflammatory changes in the animal tested for 4a (E). Area of sclerosis and necrosis in the cortical-medullary transition in mouse kidney tested for
compound 4c (G). (H&E ×40 to ×400).
1
drugs. The best activities were for the compounds containing dure described in the literature.27) The H- and 13C-NMR are
the phenyl group. The results demonstrated that the com- in accordance with previously reported data.27)
pounds 4a and 4d have adequate anti-inflammatory activities
Synthesis of 1,4-Disubstituted 1,2,3-Triazoles (3a–d) and
at 24h (35.6%) and 4h (48.9%) post-carrageenan, respec- (4a–d) The 1,2,3-triazoles 3a–d and 4a–d were prepared ac-
tively. Docking into the COX-2 confirms the importance of cording to the procedure described in the literature.24)
the phthalimide and triazole groups in the anti-inflammatory
4-(Benzothiazol-2-ylsulfanyl)-methyl-1-(N-phthalimi-
activity. The histopathological studies showed that the com- domethyl)-1H-1,2,3-triazole (3a)
pounds 3a–d and 4a–d did not cause serious pathological le-
Brown solid; mp: 136–139°C; Rf 0.4 (Hexane–EtOAc,
sions liver or kidneys. These new compounds may represent a 4:6); IR (KBr) νmax 3127, 1780, 1716, 1467, 1428, 1348,
1
novel class of potent anti-inflammatory agents.
997, 712cm−1; H-NMR (400MHz, CDCl3): δ=8.07 (s, 1H,
Htriaz), 8.01 (d, 1H, J=8.0Hz, Harom), 7.91–7.88 (m, 2H, Phth),
7.78–7.75 (m, 3H, 2HPhth and Harom), 7.47 (t, 1H, J=7.6Hz,
Experimental
Chemistry Melting points were determined in an open Harom), 7.35 (t, 1H, J=7.6Hz, Harom), 6.15 (s, 2H, NCH2),
capillary tube and performed on a PFM II BioSan apparatus. 4.74 (s, 2H, SCH2). 13C-NMR (100MHz, CDCl3): δ=166.3
Elemental analyses were carried out in an EA1110 CHNS-O (2C =O), 165.5 (C=N in benzoimidazole), 152.9 (C–N in ben-
analyzer. The infrared spectra were recorded on an IFS66 zoimidazole), 144.4 (C in triazole), 135.3 (NCH=in triazole),
1
Bruker spectrophotometer using KBr discs. H- and 13C-NMR 134.7 (2C in phthalimide), 131.2 (2C in phthalimide), 126.0
were obtained with a Varian Unity Plus-300 and 400MHz (C-S), 124.3 (C-Ar), 124.1 (C-Ar), 124.0 (2C in phthalimide),
spectrometer using CDCl3 or DMSO-d6 as the solvent. Puri- 121.5 (C-Ar), 121.0 (C-Ar), 49.6 (CH2-triazole), 27.5 (CH2-S).
fication was performed by column chromatography on Merck Anal. Calcd C19H13N5O2S2: C, 56.01; H, 3.22. Found: C, 55.70;
silica gel 60 (70–230 mesh), using a system hexane–EtOAc H, 3.61.
(1:1), and the purity of fractions were monitored by TLC
analysis on a GF254 plate.
4-(Benzothiazol-2-ylsulfanyl)-methyl-1-[2-(phthalimido-2-
yl)ethyl]-1H-1,2,3-triazole (3b)
Synthesis of Terminal Alkyne (1) The acetylene 1 was
Brown solid; mp: 140–142°C; Rf 0.4 (Hexane–EtOAc,
prepared according to the procedure described in the litera- 1:1); IR (KBr) νmax 3140, 2959, 1781, 1721, 1460, 1430,
1
ture, as well as the H- and 13C-NMR data were in agreement 1400, 1313, 1012, 942, 760, 720cm−1; 1H-NMR (400MHz,
with the literature.8)
CDCl3): δ=7.87 (d, 1H, J=8.4Hz, Harom), 7.75–7.72 (m, 4H,
Synthesis of N-(Azidoalkyl)phthalimide (2a–d) The 2HPhth, Harom and Htriaz), 7.66–7.64 (m, 2H, Phth), 7.42 (t, 1H,
azide compounds 2a–d were prepared according to the proce- J=7.2Hz, Harom), 7.31 (t, 1H, J=7.2Hz, Harom), 4.67 (s, 2H,

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Chem. Pharm. Bull.
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Fig. 5. Hepatic Histopathological Findings in the Mice Tested for Substances 3a–d, 4a–d and Control Negative
Minimum foci of hepatocyte necrosis in animals tested for substances 3a (A), 3b (B), 4a (E), 4b (F), 4c (G), 4d (H) and control group (I). Non-specific, discrete chronic
inflammatory infiltrate in mice tested for 3c (C) and 3d (D). In F image, there are also foci of macro and microvesicular steatosis (mouse tested for compound 4b). (H&E ×400).
SCH2), 4.64 (t, 2H, J 5.6Hz, NCH2), 4.12 (t, 2H, J 5.6Hz,
NCH2). 13C-NMR (100MHz, CDCl3): δ=167.5 (2C=O),
4-Phenyl-1-(N-phthalimidomethyl)-1H-1,2,3-triazole (4a)
Colorless solid; mp: 200–203°C; Rf 0.5 (EtOAc–Hexane,
166.3 (C=N in benzoimidazole), 152.4 (C–N in benzoimid- 8:2); IR (KBr) νmax 3130, 3046, 2940, 1780, 1721, 1543, 1401,
1
azole), 144.2 (C in triazole), 135.2 (NCH=in triazole), 134.2 1358, 1211, 1041, 761, 717cm−1. The H- and 13C-NMR are in
(2C in phthalimide), 131.5 (2C in phthalimide), 126.2 (C-S), accordance with previously reported data.28)
124.5 (C-Ar), 123.4 (2C in phthalimide), 123.3 (C-Ar), 121.3
(C-Ar), 121.1 (C-Ar), 48.0 (CH2-triazole), 37.6 (CH2), 27.7 (4b)
(CH2-S). Anal. Calcd C20H15N5O2S2: C, 56.99; H, 3.59. Found:
4-Phenyl-1-[2-(phthalimido-2-yl)ethyl]-1H-1,2,3-triazole
Colorless solid; mp: 187–189°C; Rf 0.4 (EtOAc–Hexane,
C, 57.35; H, 3.96.
4-(Benzothiazol-2-ylsulfanyl)-methyl-1-[3-(phthalimido-3- 1231, 1078, 765, 720cm−1; 1H-NMR (400MHz, DMSO-d6):
yl)propyl]-1H-1,2,3-triazole (3c) δ=8.64 (s, 1H, Htriaz), 7.87–7.82 (m, 4H, Phth), 7.77 (d, 2H,
1:1); IR (KBr) νmax 3126, 2952, 1774, 1716, 1464, 1432, 1396,
Brown solid; mp: 124–127°C; Rf 0.5 (Hexane–EtOAc, 1:1); J=8.0Hz, Harom), 7.43 (t, 2H, J=7.6Hz, Harom), 7.32 (t, 1H,
1H-NMR (400MHz, CDCl3): δ=7.93 (d, 1H, Harom), 7.84–7.82 J=7.6Hz, Harom), 4.69 (t, 2H, J=6.0Hz, NCH2), 4.06 (t, 2H,
(m, 2H, 2HPhth), 7.76–7.72 (m, 4H, 2HPhth, Htriaz and Harom), 7.41 J=5.6Hz, NCH2). The 13C-NMR is in accordance with previ-
(t, 1H, J=7.2Hz, Harom), 7.29 (t, 1H, J=7.6Hz, Harom), 4.69 (s, ously reported data.29)
2H, SCH2), 4.36 (t, J=6.8Hz, 2H, NCH2), 3.70 (t, J=6.4Hz,
4-Phenyl-1-[3-(phthalimido-3-yl)propyl]-1H-1,2,3-triazole
1
2H, NCH2), 2.30 (qt, 2H, J=6.8Hz, CH2). The IR, H- and (4c)
13C-NMR are in accordance with previously reported data.27)
Colorless solid; mp: 144–146°C; Rf 0.4 (EtOAc–Hexane,
4-(Benzothiazol-2-ylsulfanyl)-methyl-1-[4-(phthalimido-4- 1:1); IR (KBr) νmax 3090, 1767, 1713, 1399, 1338, 1027, 767,
1
yl)butyl]-1H-1,2,3-triazole (3d)
717 cm−1; H-NMR (400MHz, CDCl3): δ=8.00 (s, 1H, Htriaz),
Brown solid; mp: 109–113°C; Rf 0.4 (Hexane–EtOAc, 1:1); 7.86–7.81 (m, 3H, 2HPhth and Harom), 7.75–7.71 (m, 2H, Phth),
1H-NMR (400MHz, CDCl3): δ=7.90 (d, 1H, J=8.0Hz, 7.42 (d, 2H, J=8Hz, Harom), 7.42 (t, 2H, J=8Hz, Harom), 7.33
Harom), 7.81–7.78 (m, 2H, Phth), 7.36 (d, 1H, J=7.6Hz, Harom), (t, 1H, J=8Hz, Harom), 4.46 (t, 2H, J=8Hz, NCH2), 3.79 (t,
7.71–7.68 (m, 2H, Phth), 7.64 (s, 1H, Htriaz), 7.41 (dd, 1H, 2H, J=6.0Hz, NCH2), 2.39 (quintet, 2H, J=6.0Hz, CH2).
J=7.6 and 7.6Hz, Harom), 7.28 (dd, 1H, J=7.6, 7.6Hz, Harom), 13C-NMR (100MHz, CDCl3): δ=168.4 (2C=O), 147.7 (C in
4.68 (s, 2H, SCH2), 4.34 (t, 2H, J=7.2Hz, NCH2), 3.67 (t, 2H, triazole), 134.2 (2C in phthalimide), 131.8 (NCH=in triazole),
J=6.8Hz, NCH2), 1.90 (qt, 2H, J=7.6Hz, CH2), 1.67 (qt, 2H, 130.5 (C-Ar), 128.8 (2C in phthalimide), 128.1 (2C-Ar), 125.7
1
J=7.2Hz, CH2). The IR, H- and 13C-NMR are in accordance (2C in phthalimide), 123.4 (2C-Ar), 120.3 (C-Ar), 47.8 (CH2-
with previously reported data.27)
triazole), 35.0 (CH2-Phth), 29.4 (CH2). Anal. Calcd C19H16N4O2

Vol. 67, No. 2 (2019)
Chem. Pharm. Bull.
103
Fig. 6. Molecules 4d (Upper) and 5 (Lower) Docked into the Active Site of COX-2
(Color figure can be accessed in the online version.)
(1×H2O): C, 65.13; H, 5.18. Found: C, 65.07; H, 5.26.
mixed. After 12h of reaction, the mixture was co-evaporated
4-Phenyl-1-[4-(phthalimido-4-yl)butyl]-1H-1,2,3-triazole with toluene. The residue was purified by column chromatog-
(4d)
raphy. Yield=61% (ratio rotamers=69:31); yellow solid; mp
1
Yellow solid; mp: 115–118°C; Rf 0.5 (EtOAc–Hexane, 1:1); 167–170°C; Rf 0.6 (MeOH–EtOAc, 1:9). H-NMR (300MHz,
IR (KBr) νmax 3126, 2952, 1766, 1708, 1399, 1364, 1082, 764, CDCl3): δ=7.88 (brs, 1H, Htriaz), 7.83–7.78 (m, 2H, Harom),
1
721 cm−1; H-NMR (400MHz, CDCl3): δ=7.84–7.82 (m, 5H, 7.43 (dd, 2H, J=7.7 and 7.0Hz, Harom), 7.34 (t, 1H, J=7.0 and
2HPhth, 2Harom and Htriaz), 7.72–7.69 (m, 2H, Phth), 7.41 (t, 2H, 7.0Hz, Harom), 6.07 (brs, 1H, NHAc), 4.47 (t, 2H, J=6.4Hz,
J=6.8Hz, Harom), 7.32 (t, 1H, J=7.2Hz, Harom), 4.47 (t, 2H, CH2-triazole), 3.30 (app quartet, 2H, J=6.4Hz, NCH2), 2.15
J=6.8Hz, NCH2), 3.75 (t, 2H, J=6.8Hz, NCH2), 2.00 (qt, (quintet, 2H, J=6.4Hz, CH2), 1.96 (brs, 3H, CH3), 1.87
2H, J=7.6Hz, CH2), 1.76 (qt, 2H, J=7.2Hz, CH2). 13C-NMR (brs, 2H, CH2). Rotamer (visible signals): δ=6.39 (brs, 1H,
(100MHz, CDCl3): δ=167.9 (2C=O), 146.2 (C in triazole), NHAc), 4.54 (t, 2H, J=5.3Hz, CH2-triazole), 3.81 (app quar-
134.3 (2C in phthalimide), 131.6 (NCH=in triazole), 130.8 tet, 2H, J=5.9Hz, NCH2), 1.99 (brs, 3H, CH3). 13C-NMR
(C-Ar), 128.8 (2C in phthalimide), 127.7 (2C-Ar), 125.0 (2C (75.4MHz, CDCl3): δ=170.7 (C=O), 147.9 (C in triazole),
in phthalimide), 122.9 (2C-Ar), 121.2 (C-Ar), 48.9 (CH2- 130.4 (NCH=in triazole), 128.9 (2C-Ar), 128.2 (C-Ar), 125.6
triazole), 36.7 (CH2-Phth), 27.2 (CH2), 25.0 (CH2). Anal. Calcd (2C-Ar), 120.1 (C-Ar), 47.9, 36.5, 30.1, 23.2 (4CH2). Rotamer
C20H18N4O2 (1/2 H2O): C, 67.59; H, 5.39. Found: C, 67.37; H, (visible signals): δ=170.8, 130.2, 128.3, 120.5, 49.6, 39.4, 23.1.
5.57.
Anal. Calcd C14H18N4O: C, 65.09; H, 7.02. Found: C, 65.01; H,
Synthesis of 4-(4-Phenyl-1H-1,2,3-triazol-1-yl)butan- 7.31.
1-amine (5) The compound 5 was prepared from 4d accord-
Pharmacology
Drugs
ASA, ibuprofen (Laboratory Teuto Brazilian Ltda., Brazil),
1
ing to the procedure described in the literature.32) The H- and
13C-NMR are in accordance with previously reported data.32)
Synthesis of N-[4-(4-Phenyl-1H-1,2,3-triazol-1-yl)butyl]- CMC and Carrageenan (Sigma, St. Louis, U.S.A.) were used
acetamide (6) 300mg (1.38mmol) of 4-(4-phenyl-1H-1,2,3- in the biological assay.
triazol-1-yl)butan-1-amine 5, 1.5mL of acetic anhydride,
Animals
3.0mL of pyridine and catalytic amount of DMAP were
Three month-old Swiss white mice, 25–30g body weight,

104
Chem. Pharm. Bull.
Vol. 67, No. 2 (2019)
were maintained with water and food (Labina–Agribands in neutral formalin (10%) buffer for 48h then processed in
Brazil Ltd.) ad libitum. Groups of 6 animals were separated automatic processors, embedded in paraffin wax to obtain
for each experiment. All the experimental procedures reported paraffin blocks. Sections of 5µm thicknesses were prepared
here were performed in accordance with the Animal Care and and stained with haematoxylin and eosin (H&E). The slices
Use Committee of the Federal University of Pernambuco and were examined and photographed under a light microscope
guidelines for Care and Use of Laboratory Animals (Of. n° (Olympus® CX 41 and 21 microscopes) at a magnification
098/2002).
Toxicity against Artemia salina
power of ×40, ×100 and ×400.37) Images of the microscopic
findings were obtained through a digital camera (Samsung®
The toxicity test on brine shrimp has been established smartphone).
as a safe, practical, and economic method for the synthetic
Statistical Analysis Results are expressed as the
compounds bioactivity determination.33) The brine shrimp mean standard error of the mean (S.E.M.), and different
lethality bioassay was performed following the reported groups were compared using one way ANOVA followed by
procedure. Initially, the brine was collected from sea water Bonfferone posttests for multiple comparisons.
(approximately 2L) from an uncontaminated site, Tamandaré
Molecular Docking The structure of the Human COX-2
Beach in the state of Pernambuco, Brazil. The growth medium co-crystallized with meclofenamic acid at 2.41Å resolution
was prepared with a simple filtration of the water collected was obtained from the Protein Data Bank (PDBID:5ikq).38)
and sodium carbonate was used to adjust the pH to between The dockings were performed with the FlexX algorithm39)
8 and 9. After 24h, the Artemia salina were counted (using a implemented into BioSolveIT LeadIT v.2.3.2.40) The ligand
Pasteur pipette and watch glass) and placed in tapered tubes. was protonated as in aqueous solution, and we allowed 400
In each tube were placed ten Artemia salina. In addition to maximum solutions per interaction and fragmentation. Only
this, a stock solution was prepared for each compound tested chain A was considered, and the binding site was defined to
3a–d and 4a–d. The stock solutions were prepared with 50mg include all residues with atoms within 12Å of any atom of the
of each compound, in 5mL of 1% CMC (w/v). The stock had crystallographic ligand.
their concentrations measured in parts per million (ppm);
each stock solution was used for testing with the following
Acknowledgments The authors are grateful to National
concentrations: 100ppm (50µL), 250ppm (125µL), 500ppm Council for Scientific and Technological Development-CNPq
(250 µL), 750ppm (375µL) and 1000ppm (500µL). After each (Grant 448082/2014-4), Fundação de Amparo à Ciência e
aliquot was pipetted, the volume was topped up with 5mL of Tecnologia do Estado de Pernambuco-FACEPE (Grant Nos.
sea water. The control group was made up of only 2mL of 1% APQ-0459-1.06/15 and APQ-0741.106/14) and National Coun-
carboxymethylcellulose and the volume topped up with sea cil for the Improvement of Higher Education-CAPES for fi-
water to a total volume of 5mL. The reading was taken after nancial support. We also thanks to BioSolveIT for waiving the
24h with the numbers of dead and live larvae being recorded. licensing fee for LeadIT software. We are indebted to Analyti-
Acute Toxicity in Vivo
cal Centers DQF-UFPE and CENAPESQ-UFRPE.”
Graded doses (50–1000mgkg−1) were administered orally
to groups of 6 mice.34) On the first day, the animals were
Conflict of Interest The authors declare no conflict of
observed every 10min for 4h followed by twice a day ob- interest.
servation at 24, 48 and 72h after administration. Changes
in spontaneous motor activity, reflex, gait, and respiration,
appearance of writhing and piloerection plus mortality were
recorded.
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