Bio-isosteric replacement of amide group with 1,2,3-triazole in phenacetin improves the toxicology and efficacy of phenacetin-triazole conjugates (PhTCs)

Article abstract of DOI:10.1016/j.lfs.2019.05.004

Aim: Inflammatory algesia and pyresia are common pathological consequences of physiological defense. Phenacetin introduced as effective analgesic anti-pyretic agent, was proscribed from therapeutic use because of associated systemic toxicity. The aim of t

Full text of DOI:10.1016/j.lfs.2019.05.004

Accepted Manuscript
Bio-isosteric replacement of amide group with 1,2,3-triazole in
phenacetin improves the toxicology and efficacy of phenacetin-
triazole conjugates (PhTCs)
Adarsh Sahu, Debashree Das, Ram Kishore Agrawal, Asmita
Gajbhiye
PII:
S0024-3205(19)30342-X
https://doi.org/10.1016/j.lfs.2019.05.004
LFS 16448
DOI:
Reference:
To appear in:
Life Sciences
Received date:
Revised date:
Accepted date:
7 April 2019
30 April 2019
1 May 2019
Please cite this article as: A. Sahu, D. Das, R.K. Agrawal, et al., Bio-isosteric replacement
of amide group with 1,2,3-triazole in phenacetin improves the toxicology and efficacy
of phenacetin-triazole conjugates (PhTCs), Life Sciences, https://doi.org/10.1016/
j.lfs.2019.05.004
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ACCEPTED MANUSCRIPT
Bio-isosteric replacement of amide group with 1,2,3-triazole in phenacetin improves the
toxicology and efficacy of phenacetin-triazole conjugates (PhTCs)
*1
1
1
1
Adarsh Sahu , Debashree Das , Ram Kishore Agrawal , Asmita Gajbhiye
1Department of Pharmaceutical Sciences, Dr. Harisingh Gour central university, Sagar (MP), India
E-mails: adarshniper@gmail.com
Graphical Abstract
ABSTRACT
Aim: Inflammatory algesia and pyresia are common pathological consequences of
physiological defense. Phenacetin introduced as effective analgesic anti-pyretic agent, was
proscribed from therapeutic use because of associated systemic toxicity. The aim of the study
was to evaluate the potency of 1,2,3-triazole conjugation in reducing toxicity and increasing
efficacy of the phenacetin nucleus.
Main methods: The amide bond implicated as the cause of phenacetin toxicity was
bioisosterically replaced with 1,2,3-triazoles to yield a series of PhTCs (PhTC1, PhTC2 and
PhTC3). The toxicology of the synthesized conjugates in reference to phenacetin was
evaluated in accordance with OECD test guidelines 420, 425 and 407. For the purpose of

ACCEPTED MANUSCRIPT
evaluating anti-inflammatory potency carrageenan induced paw edema and croton oil induced
ear edema models were evaluated. Anti-nociceptive efficacy was assessed using Eddy’s hot
plate and acetic acid induced writhing experimental models. For anti-pyretic efficacy, the
conjugates were submitted to Brewer’s yeast antipyretic assay.
Key findings: Toxicological examination of PhTCs in comparison to phenacetin revealed
that, phenacetin treatment caused considerable nephrotoxicity and hepatotoxicity in
experimental models PhTCs were devoid of such toxic manifestations. Results of
pharmacological assays showed that the entire series of PhTCs possessed better anti-
inflammatory, anti-nociceptive and anti-pyretic potential than phenacetin. Furthermore it was
revealed that the pharmacological profile of PhTC1 with triazole substitution at para position
of the phenol ring exhibited potency even better than that exhibited by the reference
standards.
Conclusion: Bioisosteric replacement of amide bond by 1,2,3-triazole in the phenacetin
moiety yields conjugates with superior efficacy and diminished toxicity, thus opening neo
avenues in treatment of inflammatory syndromes.
Keywords: 1,2,3-triazole, phenacetin, amide bond, toxicity, anti-inflammatory, anti-pyretic,
analgesic
1
. Introduction
Inflammation is implicated both as a cause and consequence of many pathological events
such as hay fever, arthritis, atherosclerosis to cancer (Paprocka et al., 2015). Algesia and
pyresis are the predominant markers of inflammation consequently anti-inflammatory agents
with inherent analgesic and anti-pyretic efficacy are highly desirable (Dennis and Norris,
2
015). Till date NSAIDs reign as the major class of anti-inflammatory agents. Paracetamol
along with fellow NSAIDs are most commonly used over the counter anti-pyretic and anti-

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inflammatory agents (Moore et al., 2015). Not only NSAIDs associated side effects severely
limit their use in chronic conditions such as neuropathic algesia, their popular clinical status
makes them liable to be abused leading to fatal consequences such as hepatotoxicity and
perforated gastric ulcers (Tittareli et al., 2017). For that reason there lies a considerable scope
in development of anti-inflammatory agents with anti-nociceptive and anti-pyretic potential.
In the current study we propose the development of such anti-inflammatory agents with
increased efficacy and reduced toxicity.
Phenacetin and Paracetamol were contemporarily introduced in the 1890s. Although
initially phenacetin enjoyed a superior status in alleviating pain, continued occurrence of
nephrotoxicity and hepatotoxicity led to USFDA banning the drug’s use in 1983 (Clissold et
al., 1986). Our study herein attempts to revive the therapeutic status of phenacetin by
addressing its toxicity issue. The cause of phenacetin toxicity can be elucidated on
metabolism basis. The mechanism of the renal and hepatic toxicity of phenacetin is proposed
to occur in two steps, N-hydroxylation at amide bond of phenacetin followed by conjugation
and subsequent decomposition to a reactive intermediate N-hydroxyphenacetin (NHP) and
phenetidine (Mulder et al., 1977; 1978). Furthermore, NHP is an important metabolite of
phenacetin, in terms of its carcinogenicity. It has been suggested to be the metabolite of
phenacetin most likely to induce tumors due to its chemical similarity to the known
carcinogenic N-arylhydroxamic acids (Calder and Williams, 1975). It has also been
suggested as the metabolic product of phenacetin which could best account for the covalent
binding of phenacetin to cell protein (Nery,1971a) and as an intermediate in the formation of
other products of phenacetin metabolism (Nery,1971b; Calder et al.,1974). Thus, we
hypothesized that replacing the amide bond in the phenacetin molecule with a therapeutically
viable bio-isostere of the same would in turn aid in addressing the occurrence of toxic
manifestations as consequence of untoward biotransformation.

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The triazole nucleus is known to be present in numerous molecules of biological
interest. Their therapeutic potency is already established in context of anti-fungal efficacy.
Numerous evidences in scientific literature advocate the molecules efficacy in context of
inflammation and algesia. Their ability to intervene in the selective cox-2 inhibition and
inhibiting the 5-lipo-oxygenase enzyme is said to be the cardinal factor associated with their
anti-inflammatory potential (Kraljevic et al, 2016).
From these considerations we conceptualized that bio-isosteric replacement of amide
in the phenacetin nucleus with 1,2,3-triazoles would invariably yield conjugates with reduced
potential to cause nephrotoxicity and hepatotoxicity. Furthermore, since 1,2,3-triazoles have
documented anti-inflammatory and analgesic potency (Salgin et al, 2007), the synthesized
conjugates would be of increased efficacy thereby enabling formulation with reduced
recommended dose. We herein report a series of novel aminophenol 1,2,3-triazoles
synthesized via aromatic substitution by azide displacement followed by azide-alkyne
cycloaddition then O-alkylation by ethyl iodide and potassium carbonate. The predominant
imperative of the study was to investigate the onset and expression of toxicity, if any in the
neo triazole conjugated phenacetin analogues. Comparative potency of the same with the
parent molecule was also evaluated with the aspiration to justify bench to bedside translation
of the laboratory created molecules as potent and efficacious, anti-inflammatory, analgesic
and anti-pyretic agent.
2
2
. Material and Methods
.1. Animals
For acute toxicological analysis as well as determination of LD50 female while for
sub-acute toxicological as well as pharmacological analysis (in-vivo anti-inflammatory, anti-
analgesic and anti-inflammatory studies) either sex Wistar albino rats were used. All the

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experimental protocols were approved by the Institutional Animal Ethical Committee
(
IAEC). The experimental animals were housed at constant temperature (21±2ºC) and RH
5±5% with 12hrs alternate light and dark cycles and free access to food and water. The
5
protocol used in context of the study was pre-approved by the Institutional Animal Ethical
Committee. (Reg No: 379/CPCSEA/IAEC-2018/030)
2
.2. Chemicals
Phenacetin triazole conjugates (PhTCs) were synthesized as described in section 2.3.
drugs used as reference standards (Paracetamol, Phenacetin and Aspirin) for the study were
purchased from Sigma-Aldrich all other reagents were purchased from commercial sources
(Sigma-Aldrich and Alfa Aesar) and used without further purification.
2
2
.3. Chemistry
.3.1. General Procedure for the aromatic substitution (Ma et al. 2008): A mixture of 2
mmol of aryl bromide, 4 mmol of sodium azide, 0.2 mmol of copper iodide, 1 mmol of
sodium methoxide, 0.6 mmol of L-proline and 0.6 mmol of NaOH in 4 mL EtOH/H O (7:3)
2
in a closed flask was heated to 95 ºC under argon. After the completion of reaction it was
monitored by TLC, the cooled mixture was partitioned between ethyl acetate and water. The
organic layer was separated, and the aqueous layer was extracted with ethyl acetate twice. The
combined organic layers were washed with brine, dried over MgSO , and concentrated in
4
vacuo. The residual oil was loaded on a silica gel column and eluted with petroleum
ether/ethyl acetate to afford the product.
2
.3.2. General Procedure for the 1,2,3-triazole synthesis (Mukherjee et al, 2013): The
azide (1 mmol) and alkyne (1.1 mol equiv) mixture was taken in a RBF and sodium ascorbate
0.4 mol equiv) was added to it followed by the addition of CuSO (0.2 mol equiv) and
stirring it in mixture of methanol and water (1:1). After completion of their action the mixture
(
4

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was dissolved in EtOAc and was purified by column chromatography (EtOAc:Hex) to yield
analytically pure product.
2
.3.3. Procedure of O-alkylation (Johnstone, 1979 et al, Morita, 2005 et al): To a solution
of alcohol in anhydrous DMF (50 ml) maintained over an ice-bath was added NaH (60%
suspension in mineral oil) with stirring at 0 °C. After 15 min alkyl halide was added in
portion at 0 °C. After the completion of reaction 30 mL methanol was added to it for
quenching the excess of NaH. The solvent was evaporated off under reduced pressure. The
crude product was then taken up in dichloromethane and was washed with saturated brine.
The organic layer was then dried over anhydrous sodium sulphate and was concentrated
under reduced pressure.
2
2
.4. Characterization of the synthesized series.
.4.1. 1-(4-ethoxyphenyl)-4-methyl-1H-1,2,3-triazole: Yield 85%; mp: 113-117 °C; δ
H
(
400 MHz, CDCl
3
); 8.11 (1H, s, Triazole-H), 7.65 (2H, d, J 8 Hz) 6.90 (2H, q, J 8 Hz), 4.12
); 1.30 (3H, t, J 4 Hz); δ (100 MHz, CDCl ); 154.76,
44.64, 130.91, 119.58, 116.13, 115.41, 67.24, 16.73, 11.73. HRMS: m/z calculated for
(2H, q, J 8 Hz); 2.44 (3H, s, CH
3
C
3
1
+
+
C
11
H
13
N O: 226.0956 [M + H] , Found 226.0899 [M + 1] .
3

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1H-NMR spectra of 1-(4-ethoxyphenyl)-4-methyl-1H-1,2,3-triazole (PhTC1)
13C-NMR spectra of 1-(4-ethoxyphenyl)-4-methyl-1H-1,2,3-triazole (PhTC1)

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2
.4.2. 1-(3-ethoxyphenyl)-4-methyl-1H-1,2,3-triazole: Yield 85%; mp: 133-189 °C; δ
H
(
400 MHz, CDCl ); 8.28 (1H, s, Triazole-H), 7.61 (1H, s, Triazole-H), 7.31 (1H, q, J 8 Hz),
3
7
.13, (1H, q) 7.00 (1H, q, J 8 Hz), 5.27 (1H, t, 4 Hz), 4.09 (2H, t, J 8 Hz); 1.45 (3H, t, J 4Hz,
+
CH
3
); 1.24 (3H, s); HRMS: m/z calculated for C11
H
13
N
3
O: 226.0956 [M + H] , Found
2
26.0961 [M + 1]+.
1HNMR spectra of 1-(3-ethoxyphenyl)-4-methyl-1H-1,2,3-triazole (PhTC2)
2
.4.3. 1-(2-ethoxyphenyl)-4-methyl-1H-1,2,3-triazole: Yield 85%; mp: 113-117 °C; δ
H
(400 MHz, CDCl
3
); 8.32 (1H, s, Triazole-H), 7.59 (1H, d, J 8 Hz) 7.32 (2H, t, J 8 Hz), 7.11
(
1H, t, J 8 Hz); 7.00 (1H, d, J 8 Hz); 5.28 (1H, t, OCH ); 4.08 (2H, d, J 8 Hz); 1.46 (3H, s,
2
CH
3
); 1.24 (3H, t, J 4 Hz); δ
C
3
(100 MHz, CDCl ); 152.37, 145.76, 130.05, 128.62, 124.68,
1
20.89, 116.47, 113.74, 64.38, 15.45, 11.73. HRMS: m/z calculated for C11
H
13
N
3
O: 226.0956
+
+
[
M + H] , Found 226.0956 [M + 1] .

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1H-NMR of the 1-(2-ethoxyphenyl)-4-methyl-1H-1,2,3-triazole (PhTC3)
13C-NMR of the 1-(2-ethoxyphenyl)-4-methyl-1H-1,2,3-triazole (PhTC3)

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2
2
.5. Toxicological Analysis
.5.1. Experimental design
Toxicological analysis of the synthesized compounds was accomplished by acute
toxicity study, LD50 determination and sub-acute toxicity study performed as per OECD test
guidelines 420, 425 and 407 respectively. For the purpose of dosing the synthesized
conjugates (PhTC1, PhTC2 and PhTC3) as well as Phenacetin were suspended in
physiological saline. The experimental animals were seggregated randomly into control and 4
treatment groups (n=6). The control group received vehicle treatment while the wistar albino
rats of either sex were randomly assigned to control and 4 treatment groups. Control group
animals received vehicle treatment. The treatment groups received Phenacetin, PhTC1,
PhTC2 and PhTC3 respectively orally. The dose for sub-acute toxicity study was determined
after performing LD50 study for Phenacetin as well as PhTCs.
2
.5.2. Fixed dose acute toxicity study
Acute toxicological analysis of the synthesized compounds is reference to Phenacetin
was performed in accordance with OECD test guidelines 420 with slight modification. The
animals were fasted overnight and dosed orally as per the study design. Dosing was done in
stepwise procedure (50, 100, 300, 500 and 1000mg/Kg b.w.). 300mg/kg was used as the
starting dose for the study. Post dosing the animals were observed periodically for 24hrs and
daily thereafter for any signs of morbidity or mortality. From the observation NOAEL (No
Observed Adverse Effect Level) was determined (Table 1) (Table 2) (Dekanski et al.,2018).
2
.5.3. Determination of LD50
To determine the LD50 of the synthesized conjugates OECD test guideline 425 (up
and down procedure) was followed with slight modification. As per the described study

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design animals were segregated into control and treatment groups and fasted overnight prior
to the study. The test compounds were orally administered in a single odered test progression
(175, 550, 1000 and 2000mg/Kg), one at a time at 48 hour interval. Subsequent animals were
administered a lower or higher dose on the basis of appearance of either morbidity or
mortality. Dosing was discontinued if three consequtive animals survived at the upper bound
dose. Following which an estimate of LD50 was calculated using the maximum likelyhood
method (Table 1) (Vakili et al., 2017).
2
.5.4. Repeated dose sub-acute toxicity study
Sub-acute toxicological analysis was performed as per OECD TG 407 with slight
modification. The animals were orally dosed as per study design daily 7days each week for a
period of 28 days. The animals were periodically observed post dosing for any signs of
morbidity or mortality. At the end of the study protocol body weight (Table 3), biochemical
(Table 4), necroscopic and histopathological analysis were performed to asses the extent of
toxic manifestation.
2
2
.6. Pharmacological evaluation
.6.1. Experimental design
The experimental animals were randmoly seggregated into following six experimental
groups (n=6): Control (vehicle treated), Negative control (disease induced untreated),
Positive control (treated with the reference standards) and three test groups treated
individually with PhTC1, PhTC2 and PhTC3.
2
2
.6.2. Anti-inflammatory assay
.6.2.1. Carrageenan induced paw assay

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For the purpose of evaluating the anti-inflammatory activity carrageenan induced rat
paw edema assay was employed. The animals were starved overnight with ad libitum access
to water. Prior to carrageenan challenge the animals allocated to different experimental
groups were pre-treated with vehicle, reference standard and the test compounds as per study
design. After 60mins, 0.05mL of 1% carrageenan solution was injected (s.c.) into the plantar
region of the left hind paw. The right paw served as control and the change in paw volume
was measured plethysmographically at 0, 3 and 6 hrs post carrageenan challenge (Table 4,
Kim et al, 2008).
2
.6.2.2. Croton oil induced ear edema
Croton oil solution was prepared by mixing croton oil, ethanol, pyridine and ethyl
ether in the ratio (4:1:2:3). The animals where pretreated as per study design following which
.02mL of the prepared croton oil solution was applied to the right ear while left ear was left
0
untreated. After 4hrs, the animals were euthanized. Both ear were removed and weighed. The
difference between the treated and untreated ear in recorded indicating the degree of
inflammatory edema (Table 5, De Morais et al, 2018).
2
2
.6.3. Anti-nociceptive assay
.6.3.1. Eddy’s Hot Plate
The animals were placed in an Eddy’s Hot Plate Analgesiometer maintained at 55-56
ºC and time to elicit paw withdrawal response was measured. A cut-off time of 15 secs was
ensured to avoid any cardinal tissue damage. Analgesic potential was evaluated on the basis
of latency of response before and 30, 60, 90 and 120 mins post dosing. The treatment
received by the animals allocated to different experimental groups was in accordance with the
study design (Table 6) (Won et al, 2006).

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2
.6.3.2. Acetic acid induced writhing
Injection of irritant into the peritoneal cavity is known to induce pain and
characteristic writhing in rodents. 1ml of 0.5% acetic acid solution was injected i.p. in the
experimental animals following pre-treatment with the test and standard drugs as per study
design (Table 7). The animals were placed individually in glass beaker and number wriths
per 10 min were observed 05 mins post challenge (Ali et al, 2008).
2
.6.4. Anti-pyretic assay
Brewer’s yeast induced pyrexia model was employed to evaluate the anti-pyretic
potential of the synthesized TP analogues. The protocol for the assay is in accordance with
that mentioned by Shukla et al, 2010 with slight modification. A 15% suspension of Brewer
Yeast was prepared in 0.9% saline. Initial rectal temperature was measured following which,
the experimental animals were challenged s.c. with 10mL/Kg of brewer yeast suspension. 18
hrs post challenge, the animals that exhibited 0.3º-0.5º C increase in body temperature were
used for further analysis. The animals received the test and standard drug as per study design
(Table 8). Rectal temperature was measured again after 0, 30, 60, 120 and 180 mins post
dosing.
2
.7. Statistical analysis
All data regarding in-vivo experimental studies were subjected to statistical analysis.
The results are presented as mean ± standard error of the mean (SEM). The difference in
mean values was analyzed by one way ANOVA followed by Dunnett’s test using software
GraphPad Insat 3 software. p value <0.05 was considered statistically significant.
3
3
. Result
.1. Chemistry

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In lieu with the concept of Bio-isosterism, we designed the bio-isosteres of phenacetin
with the imperative to create metabolically stable and less toxic scaffolds. Amide bond
present in the structure of phenacetin is less stable at alkaline environment, leading to rapid
metabolism of the compounds. With the motive to create a molecule denial of toxic
metabolite, we replaced the amide with 1,2,3-triazole resulting in metabolically stable
compounds. The key intermediate in the synthesis of the proposed triazolyl phenacetin analog
PhTCs is the 4-azidophenol 2a, 3-azidophenol 2b and 2-azidophenol 2c. It is likely that
copper-catalyzed cycloaddition between 2a-c and appropriate alkyne 4 will furnish the
desired triazolyl phenacetin analogs PhTCs. The azido phenol 2a-c could also be accessed
starting from bromophenol 1a-c. Thus, azidophenol was synthesized in a mixed solvent
(ethanol and water (7:3)) using 10 mol% Copper iodide, 30 mol% L-proline, sodium azide.
Scheme 1. Synthesis of azido phenol from bromophenol
3
.1.1. Synthesis of Copper catalysed 1,2,3-triazolyl analogs
To identify the optimum conditions for Cu(I) catalyzed cycloaddition, the reaction of
azidophenol 2a-c with trimethyl silyl propyne was carried out in a mixture of methanol: water
1:1). To deprotect the trimethylsilyl from propyne, sodium methoxide was used. Cu(I)-
(
catalysed reaction of azide 2a-c with alkyne 4 resulted in triazoles 3a , 3b, and 3c in excellent
yield, furnishing the desired triazolyl compounds in 80-85% yield (Scheme 2). The 2a-c was
coupled with aromatic and alkynes 4 using click chemistry in the presence of copper sulphate

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pentahydrate and L-sodium ascorbate in a Fritsch Ball mill under solvent free conditions at
3
00rpm furnishing the conjugates PhTCs 80-85% yields (Scheme 3).
Scheme 2. Synthesis of alkynes and clicking with substituted azides to prepare 1,2,3-triazolyl
phenol derivatives
For the synthesis of phenacetin bioisosteres PhTCs were reacted with ethyl iodide in
the presence of potassium carbonate in dichloromethane. The obtained yield was 75, 67 and
4
5% respectively, Scheme 3.
Scheme 3. Synthesis of phenacetin analogs
3
3
.2. Toxicological analysis
.2.1. Single dose acute toxicity study

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Single oral administration of the synthetic PhTCs produced no sign of morbidity from
starting to 1000 mg/Kg dose. However, Phenacetin treated groups were found to be
significantly moribund at 1000mg/Kg dose with significant decrease in body weight in
comparison to the animals of the control group (Table 1). The NOAEL determined for
phenacetin as well as PhTCs (PhTC1, PhTC2 and PhTC3) are documented in Table 2.
Table 1: Effect of synthesized compounds on body weight (gm) after single dose acute toxicity study
Days
Control
Phenacetin
PhTC1
PhTC2
PhTC3
0
7
day
day
120.45±9.55
118.63±9.50
124.37±9.23
119.40±11.84**
115.42±08.41**
104.32±10.36**
120.32±12.26ns
121.41±7.05ns
123.40±9.16ns
118.27±5.52ns
120.47±8.43ns
120.59±6.70ns
123.16±13.52ns
126.85±17.32ns
128.77±15.43ns
1
4 day



Data is represented as mean ± SEM (n=6). Results were analyzed using one way ANOVA.
Values are expressed as mean±SEM
*
*
ns
p value <0.05(Significant ); p value >0.05(Non-Significant )
3
.2.2. Determination of LD50
Results of the acute toxicity study by Up and Down procedure (OECD 425) showed
the LD50 value for PhTCs (PhTC1, PhTC2, PhTC3) to be >2000mg/Kg b.w. while that for the
Phenacetin was found to be <1000mg/Kg b.w. (Table 2).
Table 2: Toxicity profile of compounds
Compound
Phenacetin
PhTC1
LD50 (mg/kg)
<1000
NOAEL (mg/kg)
300
3
.2.3.
>2000
2000
2000
2000
PhTC2
>2000
PhTC3
>2000
Repeated dose sub acute toxicity study

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3
.2.3.1. Body weight and biochemistry
Table 3 illustrates the difference in body weight during the course of the study
duration. No significant (p>0.05) changes in body weight in comparison to the control group
were observed in the groups treated with PhTCs while significant (p<0.05) decrease in body
weight was observed in the groups treated with Phenacetin. The drastic decrease in body
weight was found to be consistent with the moribund status of the phenacetin treated animals.
In context of biochemical analysis, Table 4 documents the results of the liver and
kidney function tests performed for the control and treated groups. In comparison to the
control group the liver enzyme panel (ALT, AST and ALP) was significantly (p<0.05)
elevated in phenacetin treated group. While the liver enzymes of the groups treated with
PhTCs were showed insignificant change (p>0.05) in the comparison to control. Significantly
(p<0.05) elevated levels of BUN (Blood Urea Nitrogen), Creatinine and uric acid were
observed in the phenacetin treated group. The kidney function test values for the groups
treated with PhTCs (PhTC1, PhTC2 and PhTC3) were found to be in range with those of the
control groups. From the data obtained it can be stated that considerable degeneration in
kidney and liver functions is brought to notice at 1000mg p.o,. dose of phenacetin, while at
the same dose, the newly synthesized PhTCs are free of such limitations.
Table 3: Effect of synthesized compounds on body weight (gm) parameters after 28 days
repeated dose oral toxicity studies
Days
Control
Phenacetin
PhTC1
PhTC2
PhTC3
0
day
120.45±9.55
120.47±14.84**
118.08±2.26ns
114.57±4.12ns
120.47±14.84ns

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7
1
2
2
day
117.63±9.50
124.37±9.23
119.49±9.45
121.12±8.71
117.85±13.30**
115.77±14.76**
111.83±12.35**
107.57±9.65**
117.70±2.44ns
120.77±15.16ns
121.52±5.65ns
124.45±15.96ns
119.37±8.43ns
123.15±7.79ns
123.46±8.25ns
121.32±5.76ns
127.85±13.30ns
129.77±14.76ns
129.83±12.35ns
129.57±9.65ns
4 day
1 day
8 day



Data is represented as mean ± SEM (n=6). Results were analyzed using one way ANOVA.
Values are expressed as mean ± SEM
*
*
ns
p value <0.05(Significant ); p value >0.05(Non-Significant )
Table 4: Effect of synthesized compounds on liver and kidney function parameters after 28 days
repeated dose oral toxicity studies
Compound
Liver function test
Kidney function test
No.(Code)
ALT
AST
ALP
BUN
Creatinine
1.40 ± 0.12
Uric acid
3.11±0.17
7.51±1.40
3.07±0.24 ns
Control
Phenacetin
PhTC1
45.71 ± 2.01
58.51±2.17**
162.20 ±1.14
173.12±1.19**
206.17± 5.16
247.38± 1.74**
8.70 ± 3.96
15.42±4.39 **
9.02±1.40 ns
6.09 ± 0.29**
1.39 ± 0.29 ns
45.78 ± 1.23 ns 163.10 ± 2.21 ns 206.17 ± 5.16 ns
PhTC2
4
6.16 ± 2.09 ns 165.30 ± 1.25 ns
210.59 ± 4.71 ns
211.45 ± 3.41 ns
9.25±2.07 ns
9.05±1.32 ns
1.49 ±0.53 ns
1.57 ± 0.39 ns
3.21±0.10 ns
3.11±0.17 ns
PhTC3
4
6.56 ± 1.72 ns
168.18 ± 1.90 ns

ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; ALP: Alkaline Phosphatase ; BUN: blood
Urea Nitrogen.

Data is represented as mean ± SEM (n=6). Results were analysed using one way ANOVA.


Values are expressed as mean ± SEM,
*
*
ns
p value <0.05(Significant ); p value >0.05(Non-Significant )
3
.2.3.2. Gross necroscopy
Gross necroscopic observation performed at the end of 28days repeated toxicity study
revealed abnormal growth on the liver of the phenacetin treated group (Figure 1a) indicating
occurrence of hepatotoxic manifestations consistent with the increased liver enzyme panel.

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Also, necroscopy of kidney, revealed the presence of necroscopic lesions (Figure 2a) on the
renal capsule as a consequence of sub-acute phenacetin treatment at 1000mg/Kg p.o. dose.
On the other hand necroscopy of vital organs (liver and kidney) of the groups treated
with PhTC1, PhTC2 and PhTC3(Figure 1b, 1c and 1d and Figure 2b, 2c and 2d) respectively
showed no abnormal manifestations in comparison to the organs of exercised from the
control group (Figure 1e and 2e).
Abnormal
growth
Figure 1: Necroscopy of liver a) Phenacetin treated group b) PhTC1 treated group c)
PhTC2 treated group d) PhTC3 treated group d) Control S

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Necrotic leisons
Figure 2: Necroscopy of kidney a) Phenacetin treated group b) PhTC1 treated group c)
PhTC2 treated group d) PhTC3 treated group d) Control
Necroscopic observation of liver revealed abnormal growth in the phenacetin treated
group while necroscopy of the PhTCs treated groups showed no abnormal manifestation.
Also necroscopy of kidney revealed considerable organ degeneration in the phenacetin
treated group while no such abnormal observation was seen in the groups treated with PhTCs
(PhTC1, PhTC2 and PhTC3)
3
.2.3.3. Histopathology
As indicated in the photomicrograph (Figure 3), light microscopic observation of the
hepatic histoarchitecture shows parenchymal inflammation along with necrotic lesions in the
liver histopathology of phenacetin treated group (Figure 3a). Histoarchitecture of the groups
treated with PhTC1 (Figure 3b), PhTC3 (Figure 3c) and PhTC4 (Figure 3d) were found to be
similar to that observed in the hepatic tissue of the control group (Figure 3e).

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Necrotic leisons
Figure 3: Histomorphology of hepatic tissue a) Phenacetin treated group b) PhTC1 treated
group c) PhTC2 treated group d) PhTC3 treated group e) Control
Necrotic leisons
Figure 4: Histomorphology of renal tissue a) Phenacetin treated group b) PhTC1 treated
group c) PhTC2 treated group d) PhTC3 treated group e) Control
Light microscopic evaluation of the renal histoarchitecture revealed normal tubules
and glomeruli of the control group (Figure 4e). Similar histopathological findings were

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observed in groups treated with PhTC1 (Figure 4b), PhTC2 (Figure 4c) and PhTC3 (Figure
4
d). Kidney histoarchitecture of the phenacetin treated group showed histological lesion
consistent with nephrotoxic renal tubular degeneration, lymphocytic infiltration and necrosis
Figure 4a).
(
3
3
.3. Pharmacological Screening
.3.1. Anti-inflammatory assay
Results of anti-inflammatory assay by carrageenan induced paw edema are revealed
in table 5. The difference between the volume of the normal and inflamed paw is expressed
as edema volume. 120 mins after carrageenan challenge, Phenecetin treated group exhibited
edema volume in range with that observed in the negative control. The Aspirin (reference
standard) treated group was able suppress carrageenan induced edema to a significant extent
(p<0.05). Edema volume of the PhTCs (PhTC1, PhTC2 and PhTC3) treated groups were
significantly less (p<0.05), with PhTC1 treatment allowing least induction of edema.
Table 6. illustrates the results of croton oil induced ear edema method. Form the
recorded observations it was apparent that the PhTCs (PhTC1, PhTC2 and PhTC3) treated
groups were able to significantly (p<0.05) suppress edema formation. With the activity of
PhTC1 surpassing that of the reference standard (Aspirin) as well. Phenacetin treated group
on the other hand showed insignificant difference in edema formation in comparison to the
untreated negative control group (p>0.05).

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Table 5: Carrageenan induced paw edema
Compound No.
Code)
Edema volume(ml) ± SEM
(
3
0min
60 min
90 min
120 min
Negative control
Phenacetin
Aspirin
0.91±0.066
0.64±0.031ns
0.43±0.524**
0.26±0.030**
1.19± 0.063
1.10±0.054 ns
0.85± 0.040**
0.69±0.041**
0.99 ± 0.092
0.92± 0.067 ns
0.71 ± 0.104**
0.83± 0.324
0.85± 0.227 ns
0.54± 0.304**
PhTC1
0.53 ± 0.081**
0.76 ± 0.090**
0.34± 0.021**
0.62± 0.160**
0
0
.48±0.027**
.52±0.067**
ns
PhTC2
1.19±0.207
ns
0.88 ± 0.090**
0.69± 0.104**
PhTC3
0.59±0.207



Data is represented as mean ± SEM (n=6). Results were analysed using one way ANOVA.
Values are expressed as mean±SEM
*
*
ns
p value <0.05(Significant ); p value >0.05(Non-Significant )
Table 6: Croton oil induced ear edema
Compound No.
Negative control
Edema mass (mg) ± SEM (% inhibition)
36 ± 0.024**
32 ± 0.081**
14± 0.104**
10 ± 0.091**
Phenacetin
Aspirin
PhTC1
1
1
1± 0.060**
3± 0.124**
PhTC2
PhTC3

Data is represented as mean ± SEM (n=6). Results were analysed using one
way ANOVA.


Values are expressed as mean±SEM
**
ns
p value <0.05(Significant ); p value >0.05(Non-Significant )

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3
.3.2. Anti-nociceptive assay
The findings regarding anti-nociceptive activity by Eddy’s hot plate are recorded in
Table 7. From the study it becomes apparently clear that, PhTCs are more potent central
analgesic than Phenacetin. The latency to heat stimuli was observed to increase significantly
(p<0.05) in the PhTCs (PhTC1, PhTC2 and PhTC3) treated groups with PhTC1
demonstrating unsurpassed activity. The latency of heat stimuli was found to change
insignificantly (p>0.05) in both the reference standard (Aspirin) and phenacetin treated
groups.
Acetic acid induced assay paradigm was used to evaluate the potential of the PhTCs
(PhTC1, PhTC2 and PhTC3) to elicit peripheral analgesic response. The readings of the study
are recorded in Table 8. In comparison to the negative control, number of abdominal wriths
in the reference standard as well as the PhTCs treated groups were significantly less (p<0.05).
Insignificant difference was recorded in the group treated with phenacetin (p>0.05).
Table 7: Analgesic activity by Eddy’s hot plate
Compound No.
Negative control
Phenacetin
Aspirin
Latency time to Heat stimuli (secs)
6.50 ± 0.76ns
7.46 ± 2.06 ns
9.46 ± 2.36 ns
PhTC1
13.95 ± 2.15**
PhTC2
PhTC3
13.43 ± 1.28**
11.90 ± 1.17**

Data is represented as mean ± SEM (n=6). Results were analysed using one way
ANOVA.


Values are expressed as mean±SEM
**
ns
p value <0.05(Significant ); p value >0.05(Non-Significant )

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Table 8: Acetic acid induced writhing assay
Compound No.
Negative control
Phenacetin
Aspirin
No. of Writhes ± SEM
37.50 ± 0.76
31.00 ± 0.85ns
20.20 ± 0.72**
18.00 ± 0.57**
22.00 ± 0.20**
25.33 ± 1.13**
PhTC1
PhTC2
PhTC3

Data is represented as mean ± SEM (n=6). Results were
analysed using one way ANOVA.


Values are expressed as mean±SEM
**
ns
p value <0.05(Significant ); p value >0.05(Non-Significant )
3
.3.3. Anti-pyretic assay
Table 9 describes the observations of the anti-pyretic bioassay performed using
Brewer’s Yeast induced pyrexia. In comparison to the negative control group, the rectal
temperature of the phenacetin treated group was found to increase insignificantly (p>0.05).
The effect was found to be superior to that observed in the paracetamol (reference standard)
treated group. Also the PhTCs (PhTC1, PhTC2 and PhTC3) treated groups were able to
suppress Brewer’s yeast induced pyrexia to certain extent, however, only PhTC1 was able to
surpass the activity of phenacetin. The rectal temperature increased significantly (p<0.05) in
the PhTC2 and PhTC3 treated groups.

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Table 9: Brewer’s yeast induced anti-pyretic assay
Group
Rectal temperature (ºC)
0
h
1h
2h
3h
4h
Negative
Control
3
7.4 ± 0.42
40.5 ± 0.18
39.4± 0.21
39.3± 0.14
39.3± 0.32
Phenacetin
Paracetamol
PhTC1
37.4 ± 0.54
37.5 ± 0.61
37.6 ± 0.06
37.4 ± 0.23
37.4 ± 0.32
38.1± 0.17** 37.9± 0.32ns 37.7± 0.42 ns
38.3± 0.14** 38.3±0.32** 38.1± 0.42**
38.0± 0.31** 37.7± 0.72 ns 37.6± 0.23 ns
38.3± 0.41** 38.3± 0.34** 38.3± 0.12**
39.6 ± 0.18ns 38.9± 0.18** 38.6± 0.16**
37.6± 0.19 ns
37.7± 0.19 ns
37.6± 0.53 ns
38.3± 0.17**
38.7± 0.02**
PhTC2
PhTC3



Data is represented as mean ± SEM (n=6). Results were analysed using one way ANOVA.
Values are expressed as mean±SEM
*
*
ns
p value <0.05(Significant ); p value >0.05(Non-Significant )
4
. Discussion
The acetanilide derivatives paracetamol and phenacetin are contemporaries with
effective anti-pyretic, mild analgesic and anti-inflammatory property (Clissold et al, 1986).
Despite having superior efficacy, phenacetin was discarded because of nephrotoxicity
associated with its therapeutic use. In the current study, we showed that bio-isosteric
replacement of amide in the phenacetin nucleus with 1,2,3-triazole yields conjugates with
superior efficacy and reduced toxicity.
The cause of phenacetin toxicity is mostly attributed to its biotransformatory
metabolites NHP and Phenetidine (Mulder et al.,1977; 1978). Considering that, triazole
conjugation replacing the amide bond in the phenacetin molecule would hinder the formation

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of the toxic metabolites. Herein, we synthesized a series of Phenacetin triazole conjugates
(PhTCs), having 1,2,3-triazole at the para (PhT1), meta (PhT2) and ortho (PhT-3) positions
of the phenacetin nucleus. In order to establish the toxicological profile of PhTCs, OECD
guidelines were followed. From the data obtained it was made ample clear that, while
phenacetin treatment causes evident toxicity in the experimental animals, treatment with
PhTCs caused no significant toxicological manifestations. The plausible cause for this
difference in toxicity profile can be attributed to bioisosteric replacement of amide by 1,2,3-
triazoles because of which formation of toxic biotransformatory product as formed in mono
phenacetin treatment is inhibited. LD50 values of PhTCs clearly advocate the non-toxic nature
of the conjugates.
For the purpose of evaluating the anti-inflammatory profile, the synthesized PhTCs
were submitted to carrageenan induced paw edema and croton oil induced ear edema
methods. Carrageenan induced paw edema is a highly predictive model of anti-inflammatory
drug activity and doses in this model can be effectively correlated with those in a patient.
Inflammatory response against carrageenan challenge is biphasic, wherein histamine
mediates the response in the first phase followed by prostaglandin mediation in the second
phase (Posadas et al 2004). In second paradigm anti-inflammatory response of the
synthesized conjugates were evaluated with the aid of phlogistic agent croton oil. Topical
application of croton oil causes edema by promoting TNFα and Prostaglandin E2 at the site
of action (Shin et al, 2010). In our study, all PhTCs (PhTC1, PhTC2 and PhTC3) were able to
suppress edema formation in both assay paradigms with PhTC1 exhibiting potency even
better than that of the reference compound (Aspirin). Anti-inflammatory activity of
Phenacetin on the other hand was of very low potency. Independent studies on the
pharmacology of triazoles report anti-inflammatory action of the moiety. These findings
further support the results obtained in our study. Thus it can be stated that the increased anti-

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inflammatory potency of PhTCs in comparison to phenacetin is a consequence of 1,2,3-
triazole incorporation in the phenacetin nucleus.
The anti-nociceptive potency of the conjugates was evaluated by Eddy’s hot plate and
Acetic acid induced writhing experimental models. Eddy’s hot plate is used to assess the
analgesic propensity of the synthesized compounds via central mechanism (Anjareyulu and
chopra et al, 2006). Results of our study indicate that pretreatment with PhTC1 exhibited
significant (p<0.005) increase in latency response time to heat stimuli. Also since the
behavioral components analyzed to determine the latency period are supraspinally integrated
response, the participation of central nociception as drug mechanism can be suggested.
Further since the tenacity of PhTCs to exert anti-nociceptive property was superior to both
phenacetin as well as its reference standard, it can de advocate that triazole to certain extent
contribute to the analgesic behavior of the synthesized conjugates.
Acetic acid induced writhing is acknowledged as a visceral pain model used for
evaluating the propensity of compounds against peripheral nociception. Acetic acid induces
pain by local production of inflammatory mediators (Reichert et al 2001). From our study it
was evident that pretreatment with PhTCs significantly decreased writhing in animal models.
The effect can be attributed to inhibition of inflammatory mediators produced in response to
acetic acid challenge. Furthermore, these results confirm those obtain in context of anti-
inflammatory bioassays. Thereby strengthening the hypothesis of triazole conjunction with
increased efficacy.
Fever is mostly the first physiological defense against pathological offence such as
infection and inflammation (Biomquist and Engbiom, 2018). The anti-pyretic efficacy of
PhTCs was verified by Brewer’s yeast assay. From our study it was evident that potency of
PhTC1 to ameliorate febrile response in experimental animals was superior than produced by

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both phenacetin and the reference standard aspirin. Brewer’s yeast causes pyrexia by
inducing prostaglandin synthesis that eventually irritates the hypothalamus to cause fever
(Abbas et al, 2010). Since PhTCs were able to suppress brewer’s yeast induced fever in the
experimental animals, it can be deduced that the anti-pyretic potency of the compounds is
exerted as a consequence of prostaglandin synthesis inhibitor.
5
. Conclusion
In conclusion, we investigated the toxicological and pharmacological properties of a
series of 1,2,3-triazole conjugated phenacetin molecule (PhTCs). Our findings demonstrate
the superior anti-inflammatory, anti-nociceptive and anti-pyretic potential of para substituted
conjugate (PhTC1) in comparison to the popular NSAIDs (Paracetamol and Aspirin).
Furthermore, it was established that triazole conjugation to phenacetin drastically diminishes
the toxicological impact of the parent nucleus in physiological system, thereby warranting
safe use even in chronic conditions. Thus, these new agents can be a new way to alleviate
suffering in inflammatory syndrome especially when it is a major challenge to treat safely
with clinically established drugs.
Conflict of interest
Authors declare that they have no conflict of interest.
Acknowledgement
The author Adarsh Sahu acknowledges UGC-BSR, New Delhi for providing the Doctoral
fellowship. Debashree Das also acknowledge UGC for providing the Doctoral fellowship.
Authors also acknowledge Dr. KPR Kartha, Department of Medicinal Chemistry, NIPER,
Mohali for allowing AS to work in his laboratory.
References: