Bioisosteric Replacement of Amide Group with 1,2,3-Triazoles in Acetaminophen Addresses Reactive Oxygen Species-Mediated Hepatotoxic Insult in Wistar Albino Rats

Article abstract of DOI:10.1021/acs.chemrestox.9b00392

Acetaminophen (AP) is a popularly recommended over-the-counter analgesic-antipyretic in clinical use. However, the drug is handicapped by the occurrence of hepatotoxic insult following acute ingestion. Consequently, AP-induced hepatotoxicity is often impl

Full text of DOI:10.1021/acs.chemrestox.9b00392

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Article
Bioisosteric replacement of amide group with 1,2,3-
triazoles in Acetaminophen (AP) addresses ROS
mediated hepatotoxic insult in Wistar albino rats.
Adarsh Sahu, Debashree Das, Preeti Sahu, Shweta Mishra,
Ayyamperumal Sakthivel, Asmita Gajbhiye, and Ramkishore Agrawal
Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.9b00392 • Publication Date (Web): 18 Dec 2019
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Bioisosteric replacement of amide group with 1,2,3-triazoles in acetaminophen (AP)
addresses ROS mediated hepatotoxic insult in Wistar albino rats.
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Adarsh Sahu*1, Debashree Das1, Preeti Sahu2, Shweta Mishra1, Ayyamperumal Sakthivel2, Asmita Gajbhiye1,
Ramkishore Agrawal1,
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1Department of Pharmaceutical Sciences, Dr. Harisingh Gour Vishwavidyalaya, Sagar (MP), India
2*Department of chemistry, Central University of Kerala, Kerala, India
E-mails: adarshniper@gmail.com
ABSTRACT:
Acetaminophen (AP) is a popularly recommended over the counter analgesic-antipyretic in
clinical use. However the drug is handicapped by the occurrence of hepatotoxic insult
following acute ingestion. Consequently AP induced hepatotoxicity is often implicated in
accidental or suicidal overdose. In the current study, we investigated the potential of
bioisosteric replacement of amide in AP with 1,2,3-triazoles in curbing AP induced
hepatotoxicity. The therapeutic utility of synthesized bioisosteres was established by careful
tailoring and optimization of the synthetic methodology along with detailed toxicological
testing of pharmacologically potent APTCs (Acetaminophen triazole conjugates). In lieu with
the same, we herein report a series of novel 17 APTCs synthesized via aromatic substitution
using sodium azide, L-proline and copper iodide followed by click reaction with substituted
alkynes using copper sulphate and sodium ascorbate. Pharmacological evaluation of
synthesized APTCs revealed that out of the series of 17 compounds 5a and 5e were found to
be most efficacious in exerting anti-inflammatory, analgesic and antipyretic activity in animal
model. Further toxicity studies documented that in both acute as well as sub acute toxicology;
AP administration caused significant hepatotoxicity which was found to be a consequence of
ROS mediated oxidative stress. Potent APTCs (5a and 5e) on the other hand revealed no
adverse event both in acute and sub toxicological analysis. Median lethal dose (LD50) and No
observed adverse effect level (NOAEL) values for 5a and 5e were found to be > 1000 mg/kg
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and 2000 mg/kg respectively. Human equivalent dose defining the maximum safe
concentration of a compound in human’s physiology was found to be 27.68 mg/kg for the
most potent APTCs (5a and 5e). Thus it can be concluded that triazole incorporation into AP
nucleus produced conjugates devoid of hepatotoxic manifestations having added advantage of
anti-inflammatory efficacy along with analgesic and antipyretic potency.
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Keywords: AP, NAPQI, 1,2,3-triazole, amide bond, Acute toxicity, Sub-Acute toxicity, HED
1. INTRODUCTION
Acetaminophen (AP) chemically acknowledged as N-acetyl-p-aminophenol and commonly
addressed as paracetamol, is an excellent analgesic-antipyretic agent. The virtues of the drug
can be traced back to inhibition of prostaglandin synthesis in CNS with added advantage of
not causing NSAID (non-steroidal anti-inflammatory agents) associated gastrointestinal side
effects. Consequently AP is the most widely used OTC medication for pain and fever often
recommended over other popular NSAIDs for conditions related to non-inflammatory algesia
and hyperpyrexia1. However, despite an obvious edge, the efficacy of AP is handicapped by
narrow therapeutic window, weak anti-inflammatory activity2 and potential to elicit
hepatotoxicity3. Furthermore as a consequence of its popular status and inconveniently large
therapeutic dose the drug is often implicated in cases of accidental and suicidal overdose.
Also, increasing prescriptions of AP combination products is yet another cause for its chronic
overdose accounting for more than 50% cases of acute liver failure especially in developed
nations like UK and USA. Currently, AP is an emerging cause of liver transplantation4 with
an increasing global burden is observed with more than 10 lakhs hospitalization being
reported annually because of acute hepatotoxic episode following AP administration5-9.
The cause of AP hepatotoxicity can be elucidated on metabolic basis. Whilst, the usual
route of the drug’s biotransformation is via glucouronide and sulphate conjugation, AP also
enters a minor metabolic pathway by cytochrome P450 enzymes involving the generation of
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N-acetyl-p-benzoquinoneimine (NAPQI). At therapeutic dose, NAPQI form adducts with
reduced glutathione (GSH) and gets excreted as mercapturic acid or cysteine conjugate.
However, at supra-therapeutic dose, hepatic GSH stores are depleted, rendering NAPQI free
to form covalent associations with cellular nucleophiles, thereby precipitating manifestations
of acute hepatotoxicity10-11. Previous investigations have implicated amide linkage in AP
molecule for NAPQI generation12. It has also been reported that, NAPQI causes production
of superoxide’s and reactive oxygen species (ROS), which subsequently precipitates lipid
per oxidation and consequent inflammatory cascade. The inflammatory mediators thus
generated derails hepatocellular (ROS) balance, thereby causing oxidative stress and eventual
pathogenesis of AP-induced hepatotoxicity13. Thus, we hypothesized that replacing the amide
bond in the AP nucleus with a therapeutically viable bio-isostere would in turn aid in
addressing the occurrence of toxic manifestations as a result of its untoward
biotransformation.
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The term bio-isosterism is used to define a phenomenon in which molecules with similar
physical and chemical properties exhibit broadly similar biological properties14. In
contemporary practice of drug designing, the concept of bio-isosteric replacement has been
adopted as a tactical approach in order to introduce structural changes in the target moiety
that can be beneficial in improving potency, enhancing selectivity, and altering physical
properties, reducing or redirecting metabolisms, eliminating or modifying toxicophores15. In
the present study, we have employed the concept of bioisosterism with the aim of addressing
ROS mediated acetaminophen toxicity. For the said purpose we utilized 1,2,3-triazole
scaffold for creating AP triazole conjugated pharmacophore with increased efficacy and no
toxicity16-17
.
Triazoles are an interesting class of heterocyclics with broad spectrum of pharmacological
activities. The molecule achieved legendary fame in exerting anti-microbial efficacy18. The
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clinical versatility of triazoles can be further highlighted by successful studies revealing the
compound’s anti-inflammatory19, anti-neoplastic20, anti-diabetic21, anti-oxidant22, anti-
convulsant23, antiparkinsonism24, immunomodulatory and antimalarial potency25. In the
context of anti-inflammatory (AI) activity, scientific literature reveals selective COX-2
inhibitory potency of compounds bearing 1,2,3-triazole moiety26.
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The predominant imperative of the current study was to evaluate the toxicology and
pharmacology of APTCs especially in context of addressing the expression of ROS mediated
hepatotoxicity as observed with AP therapy. For ensuring the therapeutic success of the
conceptualized bioisostere careful tailoring and optimization of the synthetic methodology
was done for obtaining the candidate pharmacophore27. Herein, we report the synthesis of 17
APTCs via aromatic substitution using sodium azide, L-proline and copper iodide followed
by click reaction with substituted alkynes using copper sulphate and sodium ascorbate.
Comparative potency of these conjugates with the parent AP molecule was evaluated to
ensure therapeutic efficacy of the synthesized APTCs. The onset and expression of toxicity
was assessed for the most potent APTCs with the objective of bench to bedside translation of
the APTC pharmacophore as potent and safe therapeutic moiety devoid of hepatotoxicity or
any other systemic adversity as observed in context of currently used antipyretic, analgesic
and anti-inflammatory agents.
2. MATERIAL AND METHOD
2.1.
Test chemicals and instrumentation
All reagents were purchased from commercial sources (Sigma-Aldrich and Alfa Aesar) and
used without further purification. Proton nuclear magnetic resonance experiments and
carbon-13 nuclear magnetic resonance experiments were recorded on Bruker Advance DX
400 MHz spectrometer. Chemical shifts were reported in parts per million relative to
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tetramethylsilane as internal standard. The H NMR spectra were referenced with respect to
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the residual CHCl3 proton of the solvent CDCl3 at 7.27 ppm, residual MeOH proton of the
solvent MeOD at 4.90 and residual DMSO proton of the solvent DMSO-d6 at 3.33 and 2.51.
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Coupling constants were reported in hertz (Hz). C NMR spectra were fully decoupled and
were referenced to the middle peak of the solvent at 77.00, 48.00 and 72.00 ppm. Splitting
pattern were designated as s, singlet; d, doublet; dd, doublet of doublet; m, multiplet.
Compounds were routinely analyzed for their purity on the silica gel GF-254 visualized under
UV at wavelength 254 nm. UPLC HRMS LOW spectra were measured in the ESI mode at an
ionization potential of 70 eV. Chromatographic purification of synthesized compounds was
performed by column chromatography on silica gel (60-120 mesh) eluting with ethyl acetate
in hexane.
2.2. Synthesis of acetaminophen triazole conjugates (APTCs)
2.2.1. General procedure for the aromatic substitution28: A mixture of 2 mmol of aryl
bromide, 4 mmol of sodium azide, 0.2 mmol of copper iodide, 0.6 mmol of L-proline and 0.6
mmol of NaOH in 4 mL EtOH/H2O (7:3) in a closed flask was heated to 95 ºC under argon.
After the reaction completion 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 MgSO4, and concentrated in vacuo. The residual oil was loaded on a silica gel
column and eluted with petroleum ether/ethyl acetate to afford the product.
2.2.2. Procedure for the Boc-protection29: Suspended 76.9 mmol of 4-aminophenol in a
mixture of 60 ml of dioxane 60 ml of water in a 250 ml round bottom flask containing a
magnetic stir bar. Added 16.4 ml of di-t-butyl dicarbonate all at once and adjust the pH 8-9 by
addition of 4N NaOH. Completion of reaction was checked by TLC in DCM:MeOH (9:1).
Reaction mix was extracted with 40 ml of ether to remove the dioxane (Evaporated the
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reaction mixture with 40 ml of ether to remove the dioxane). Chill the aqueous phase with ice
and acidified with citric acid to pH 3. Extracted the separated oil layer with ethyl acetate (50
ml) and washed the combined extracts with water and dried it over anhydrous sodium
sulphate.
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2.2.3. Procedure of O- and N-alkylation30, 31: To a solution of alcohol or aniline 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 portion wise 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. General procedure for the 1,2,3-triazole synthesis32, 33: The azide (1 mmol) and
alkyne (1.1 equiv) mixture was taken in a stainless steel (SS) jar (capacity,50mL) containing
10 SS balls (10mmo.d.) and sodium ascorbate (0.4 equiv) was added to it followed by the
addition of CuSO4 (0.2 equiv). The mixture was then ground in a planetary ball mill (Retsch
PM-100, Retsch GmbH, Germany) at 300 rpm. After completion of their action the mixture
was dissolved in EtOAc and was purified by column chromatography (EtOAc:Hex) to yield
analytically pure product.
2.2.5. Spectral data of the synthesized series.
2.2.5.1. 4-(4-methyl-1H-1,2,3-triazol-1-yl)phenol (5a): Yield 90%; mp: 89 °C; Brownish
Black solid; δH (400 MHz, MeOD); 8.11 (1H, s, Triazole-H), 7.61-7.57 (2H, m, ArH), 6.96-
6.93 (2H, m, ArH) 2.39 (3H, s, CH3); δC (100 MHz, CDCl3);158.16, 143.43, 129.41, 121.87,
120.50, 115.69, 9.04; IR (KBr) νmax: 3373.62, 2950.54, 2923.43, 2857.14, 1744.86, 1459.64,
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1371.7, 1275.50, 1259.29, 750.07; HRMS: m/z calculated for C9H9N3O2: 176.0746 [M + H]+,
Found 176.0813 [M + H]+.
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2.2.5.2.
4-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)phenol (5b): Yield 93%;
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Brownish solid; mp: 72 °C; δH (400 MHz, MeOD); 8.30 (1H, s, Triazole-H), 7.64 (2H, d, J
2.8 Hz, ArH), 6.98 (2H, d, J 2.8 Hz, ArH) 3.36 (1H, q, J 12 Hz, CH2OH),; δC (100 MHz,
CDCl3); 158.16, 129.32, 122.04, 121.04, 115.74, 54.04, 48.01; IR (KBr) νmax: 3383.10, 3200,
2927, 1602.05, 1524.26, 1485.14, 1425.30, 1404.70, 1371.46, 1339.61, 1310.51, 1272.60,
1254.50, 1230.17, 1173.90, 1112.25, 1069.96, 1050.23, 1001.15, 854.28, 829.80, 770.91,
682.39, 547.67, 525.87, 468.23; HRMS: m/z calculated for C9H9N3O2: 191.0695 [M + 1]+,
214.0592 [M + Na]+ Found 214.0580 [M + Na]+.
2.2.5.3. 4-(4-(1-hydroxycyclohexyl)-1H-1,2,3-triazol-1-yl)phenol (5c): Yield 85%; Off-
white solid; mp: 77 °C; δH (400 MHz, MeOD); 8.22 (1H, s, Triazole-H), 7.64-7.60 (2H, m,
ArH), 6.97-6.93 (2H, m, ArH), 2.13-2.06 (2H, m, Tr-C-(CH2)2-(CH2)2CH2), 1.92-1.88 (2H,
m, Tr-C-(CH2)2-(CH2)2CH2), 1.87-1.78 (2H, m, Tr-C-(CH2)2-(CH2)2CH2), 1.65-1.69 (3H, m,
Tr-C-(CH2)2-(CH2)2CH2), 1.58-1.42 (1H, m, Tr-C-(CH2)2-(CH2)2CH2); δC (100 MHz,
CDCl3);158.10, 158.12, 129.43, 121.97, 119.30, 115.70, 69.89, 37.44, 25.18, 21.68; IR
(KBr) νmax: 3158.04, 3100, 1771.83, 1608.39, 1519.58, 1449.75, 1367.51, 1276.97, 1231.67,
1080.14, 1052.69, 1003.13, 963.59, 897.49, 846.64, 805.65, 728.55, 630.75, 543.63, 521.13,
487.84; HRMS: m/z calculated for C9H9N3O2: 260.1321 [M + 1]+, 282.1218 [M + Na]+
Found 282.1211 [M + Na]+.
2.2.5.4. 4-(4-hexyl-1H-1,2,3-triazol-1-yl)phenol (5d): Yield 92%; mp: 56 °C; Off-yellow
solid; δH (400 MHz, CDCl3); 7.95 (1H, s, Triazole-H), 7.61 (2H, d, J 4 Hz, ArH), 7.02 (2H, s,
ArH) 4.69 (1H, d, J 8 Hz, CH2 CH2(CH)3CH3), 2.12 (2H, s, CH2CH2(CH)3CH3), 1.68 (6H, m,
CH2(CH)3CH3), 1.31 (1H, t, J 6 Hz, CH2CH2(CH)3CH3); δC (100 MHz, CDCl3); 157.05,
145.21, 122.55, 121.30, 116.44, 29.70, 26.28, 21.40, 14.22; IR (KBr) νmax: 3383.10, 3200,
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2927, 1602.05, 1524.26, 1485.14, 1425.30, 1404.70, 1371.46, 1339.61, 1310.51, 1272.60,
1254.50, 1230.17, 1173.90, 1112.25, 1069.96, 1050.23, 1001.15, 854.28, 829.80, 770.91,
682.39, 547.67, 525.87, 468.23; HRMS: m/z calculated for C9H9N3O2: 245.1528 [M + 1]+,
282.1211 [M + K]+ Found 282.1211 [M + K]+.
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2.2.5.5. tert-butyl (4-(prop-2-yn-1-yloxy)phenyl)carbamate (4e): Yield 93%; Yellowish
white; mp: 56 °C; δH (400 MHz, CDCl3); 7.31 (2H, d, J 12 Hz, ArH), 6.95-6.93 (1H, m,
ArH), 6.95-6.93 (1H, d, J 8, ArH), 4.68 (2H, s, OCH2CCH), 2.53 (1H, t, J 2.4
Hz,OCH2CCH), 1.53 (9H, s, OC(CH)3); δC (100 MHz, CDCl3);153.55, 153.08, 132.33,
120.38, 120.38, 115.49, 78.64, 75.44, 56.24, 29.71, 28.36; IR (KBr) νmax:3433.87, 2917.58,
2078.68, 1634.24, 1518.61, 1231.15, 1159.42, 1015.70, 832.91; HRMS: m/z calculated for
C14H17NO3: 247.1208 [M + 1]+, 270.1106 [M + Na]+ Found 270.1102 [M + Na]+.
2.2.5.6.
tert-butyl (4-((1-(4-hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)
carbamate (5e): Yield 93%; Yellowish white; colour solid; mp: 56 °C; δH (400 MHz,
DMSO); 9.95 (1H, s, OH), 9.16 (2H, s, N-H, Tr-H), 8.71 (1H, s, ArH), 7.67 (2H, d, J 8 Hz,
ArH), 7.37 (2H, d, J 8 Hz, ArH), 6.97-6.92 (4H, m, ArH), 5.14 (2H, s, TrOCH2); δC (100
MHz, CDCl3);159.85, 154.25, 153.08, 132.33, 120.38, 120.38, 115.49, 78.64, 75.44, 56.24,
29.71, 28.36; IR (KBr) νmax:3433.87, 2917.58, 2078.68, 1634.24, 1518.61, 1231.15, 1159.42,
1015.70, 832.91; HRMS: m/z calculated for C14H17NO3: 382.1641 [M + 1]+, 405.1538 [M +
Na]+ Found 405.1525 [M + Na]+.
2.2.5.7. Ethyl (1-(4-hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)prolinate (5f); Yellow-
Brownish Liquid.
2.2.5.8. 3-(4-methyl-1H-1,2,3-triazol-1-yl) phenol (6a): Yield 90%; solid; mp: 89 °C; δH
(400 MHz, DMSO); 10.01 (1H, s, Ar-OH), 8.47 (1H, s, Triazole-H), 7.38-7.36 (1H, m, ArH),
7.34 (2H, s, ArH), 6.87-6.84 (1H, m, ArH) 2.32 (3H, s,CH3); δC (100 MHz, CDCl3);158.89,
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143.52, 138.25, 131.15, 120.91, 115.74, 110.65, 107.21, 10.98, 0.57; IR (KBr) νmax: 3373.62,
2950.54, 2923.43, 2857.14, 1744.86, 1459.64, 1371.7, 1275.50, 1259.29, 750.07; HRMS:
m/z calculated for C9H9N3O2: 176.0746 [M + H]+, 198.0643 [M + Na]+ Found 176.0775 [M
+ H]+.
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2.2.5.9. 3-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)phenol (6b): Yield 75%; Dark brown
solid, mp: 75-78 °C; δH (400 MHz, DMSO); 10.51 (1H, s, Ar-OH), 8.23 (1H, s, Triazole-H),
7.61 (1H, d, ArH, J 8 Hz), 7.23 (1H, d, ArH, J 12 Hz); 7.11 (1H, d, ArH, J 8 Hz), 6.97 (1H, s,
ArH) 5.45 (1H, s, CH2OH); 4.98 (2H, s, CH2OH); δC (100 MHz, CDCl3);158.80, 144.64,
143.35, 130.23, 122.75, 120.01, 115.17, 108.30, 54.65; HRMS: m/z calculated for C9H9N3O2:
214.1798 [M + H]+, Found 214.1230 [M + H]+.
2.2.5.10.
3-(4-(1-hydroxycyclohexyl)-1H-1,2,3-triazol-1-yl)phenol (6c): Yield 95%;
Whitish brown solid, mp: 110-115 °C; δH (400 MHz, DMSO); 9.98 (1H, s, Ar-OH), 8.53
(1H, s, Triazole-H), 7.38-7.31 (3H, m), 6.87-6.84 (1H, s, ArH) 4.98 (1H, s, OH); 1.95 (2H, q,
J 8 Hz); 1.76 (2H, d, J 8 Hz); 1.73 (2H, d, J 8 Hz); 1.73 (1H, d, J 8 Hz); 1.48 (1H, d, J 8 Hz);
1.45 (1H, d, J 8 Hz); δC (100 MHz, CDCl3);158.88, 131.16, 115.74, 110.67, 107.22, 68.38,
39.37, 38.11, 25.71, 22.16; IR (KBr); HRMS: m/z calculated for C14H17N3O2: 259.1321 [M +
H]+, 282.1218 [M + Na]+ Found 282.1214 [M + Na]+.
2.2.5.11. 3-(4-tetradecyl-1H-1,2,3-triazol-1-yl)phenol (6d): Yield 90%; mp: 119-123 °C;
δH (400 MHz, DMSO); 9.55 (1H, s, Ar-OH), 8.15 (1H, s, Triazole-H), 7.37 (1H, d, J 8 Hz),
7.23 (1H, d, J 8 Hz); 6.80 (1H, s, ArH); 2.68 (2H, q, CH2(CH2)13CH3); 1.74-1.72 (2H, p, J 4
Hz); 1.73 (1H, d, J 8 Hz); 1.28-1.27 (22H, m); 0.98 (3H, t, J 8 Hz); δC (100 MHz, CDCl3);
149.37, 148.55, 129.34, 122.78, 120.02, 119.45, 119.05, 118.47, 84.80, 68.01, 33.82, 31.93,
29.69, 29.66, 29.54, 29.51, 29.36, 29.27, 29.22, 28.78, 28.51, 25.56, 22.69, 18.40. LC-MS:
m/z calculated for C22H35N3O: 358.27 [M + H]+, Found 358.26 [M + H]+.
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2.2.5.12. 3-(4-((4-aminophenoxy)methyl-1H-1,2,3-triazol-1-yl)phenol (6e): Yield 86%;
Yellowish White; mp: 112-119 °C; δH (400 MHz, DMSO); 9.96 (1H, s, Ar-OH), 8.75 (1H, s,
Triazole-H), 7.67 (2H, d, J 8 Hz), 7.35 (2H, d, J 8 Hz); 6.95 (4H, s, ArH); 1.48 (9H, d, J 8
Hz); δC (100 MHz, CDCl3); 149.37, 148.55, 129.34, 122.78, 120.02, 119.45, 119.05, 118.47,
84.80, 68.01, 33.82, 31.93, 29.69, 29.66, 29.54, 29.51, 29.36, 29.27, 29.22, 28.78, 28.51,
25.56, 22.69, 18.40. HRMS: m/z calculated for C14H17NO3: 382.1641 [M + 1]+, 405.1538 [M
+ Na]+ Found 405.1839 [M + Na]+.
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2.2.5.13. tert-butyl(4-((1-(3-hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)
carbamate (6f): Yield 82%; mp: 130-135 °C; δH (400 MHz, CDCl3); 8.13 (1H, s, Tr-H), 8.03
(2H, s, Ar-H); 7.44 (2H, d, J 8 Hz), 7.32 (2H, d, J 8 Hz); 7.00 (4H, s, ArH); 3.78 (2H, d);
2.50 (2H, d, J 8 Hz); 2.18 (2H, d, J 8 Hz); 1.31 (2H, d, J 8 Hz); 0.91 (3H, t, J 8 Hz) δC (100
MHz, CDCl3); 173.68, 162.67, 151.65, 146.36, 138.35, 130.48, 120.03, 116.46, 108.73,
96.28, 89.56, 59.09, 51.23, 43.10, 39.99, 36.52, 31.43, 26.06, 19.36, 14.93. HRMS: m/z
calculated for C14H17NO3: 382.1641 [M + 1]+, 405.1538 [M + Na]+ Found 405.1525 [M +
Na]+.
2.2.5.14. 2-(4-methyl-1H-1,2,3-triazol-1-yl)phenol (7a): Yield 86%; mp: 137-139 °C; Dark
Brown solid δH (400 MHz, CDCl3); 10.11 (1H, s, OH), 7.86 (1H, s, Triazole-H); 7.41 (1H, d,
J 4 Hz), 7.39 (1H, d, J 4 Hz); 7.00 (1H, d, ArH, J 8 Hz); 2.45 (3H, s); δC (100 MHz,
CDCl3);149.35, 148.55, 129.36, 122.79, 120.05, 119.43, 119.10, 118.52, 14.06. HRMS: m/z
calculated for C9H9N3O: 176.0746 [M + H]+, Found 176.01 [M + H]+.
2.2.5.15. 2-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)phenol (7b): Yield 76%; mp: 120-
122 °C; Blackish color; δH (400 MHz, CDCl3); 10.09 (1H, s, OH), 8.01 (1H, s, Triazole-H);
7.45 (1H, d, J 12 Hz), 7.29 (1H, d, J 4 Hz); 7.18 (1H, d, J 4 Hz) 7.10 (1H, d, ArH, J 8 Hz);
6.99 (1H, d, ArH, J 8 Hz); 5.39 (1H, s, OH); δC (100 MHz, CDCl3);193.51, 178.55, 160.90,
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144.76, 130.33, 127.33, 119.03, 115.17, 107.76, 102.83, 102.00, 55.41. HRMS: m/z
calculated for C9H9N3O2: 214.1798 [M + H]+, Found 214.1230 [M + H]+.
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2.2.5.16. 2-(4-(1-hydroxycyclohexyl)-1H-1,2,3-triazol-1-yl)phenol (7c): Yield 88%; mp:
72-75 °C; δH (400 MHz, DMSO); 10.49 (1H, s, OH); 8.25 (1H, d, J 8 Hz); 7.59 (1H, s,
Triazole-H), 7.32 (1H, t, J 8 Hz); 7.12 (1H, t, J 8 Hz); 6.99-6.96 (2H, m, ArH), 4.98 (1H, s,
Cyclohexyl-OH); 2.13-2.06 (2H, m, Tr-C-(CH2)2-(CH2)2CH2), 1.92-1.88 (2H, m, Tr-C-
(CH2)2-(CH2)2CH2), 1.87-1.78 (2H, m, Tr-C-(CH2)2-(CH2)2CH2), 1.65-1.69 (3H, m, Tr-C-
(CH2)2-(CH2)2CH2), 1.58-1.42 (1H, m, Tr-C-(CH2)2-(CH2)2CH2); δC (100 MHz,
CDCl3);158.10, 158.12, 129.43, 121.97, 119.30, 115.70, 69.89, 37.44, 25.18, 21.68; IR
(KBr) νmax: 3158.04, 3100, 1771.83, 1608.39, 1519.58, 1449.75, 1367.51, 1276.97, 1231.67,
1080.14, 1052.69, 1003.13, 963.59, 897.49, 846.64, 805.65, 728.55, 630.75, 543.63, 521.13,
487.84; HRMS: m/z calculated forC9H9N3O2: 260.1321 [M + 1]+, 282.1218 [M + Na]+ Found
282.1215 [M + Na]+.
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2.2.5.17.
(4-((1-(2-hydroxyphenyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)carbamate
(7d): Yield 63%; mp: 156 °C; Orangish-White solid; δH (400 MHz, CDCl3); 10.11 (1H, s,
OH), 9.89 (1H, s, NH); 8.09 (1H, s, Triazole-H); 7.40 (2H, d, J 8 Hz, ArH), 7.31 (1H, t, J 8
Htz, ArH), 7.21 (2H, t, J 8 Htz, ArH), 7.00 (2H, q, Ar-H); 5.22 (2H, s, J 8 Hz, TrOCH2); 2.84
(2H, q, J 8 Hz, O-CH2 ); 1.50 (9H, s, OC-(CH)3); δC (100 MHz, CDCl3);149.35, 148.55,
129.36, 122.75, 120.05, 119.43, 119.10, 118.52, 31.55, 29.23, 28.88, 25.58, 22.55, 14.06;
HRMS: m/z calculated forC14H17NO3: 382.1641 [M + 1]+, Found 382.85 [M + H]+.
2.2.5.18. 2-(2-tetradecyl-1H-1,2,3-triazol-1-yl)phenol (7e): Yield 81%; mp: 120-127 °C;
Yellowish semi-solid; δH (400 MHz, CDCl3); 10.11 (1H, s, Ar-OH), 7.86 (1H, s, Triazole-H),
7.40 (1H, d, J 8 Hz), 7.30 (1H, d, J 8 Hz); 7.21 (1H, s, ArH); 7.00 (1H, t, J 8 Hz, ArH); 2.68
(2H, q, CH2(CH2)13CH3); 1.74-1.72 (2H, p, J 4 Hz); 1.28-1.27 (22H, m); 0.98 (3H, t, J 8 Hz);
1.73 (1H, d, J 8 Hz); 1.48 (1H, d, J 8 Hz); 1.45 (1H, d, J 8 Hz); δC (100 MHz, CDCl3);
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149.36, 148.55, 129.34, 122.76, 120.02, 119.45, LC-MS: m/z calculated for C22H35N3O:
357.27 [M + H]+, Found 357.26 [M + 1]+.
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2.3. Experimental Animals: Healthy nullgravida-nulliparous wistar albino rats were used for
acute and of either sex were used for sub-acute toxicological and pharmacological analysis.
The protocols used in context of pharmacological as well as toxicological analysis were pre-
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(Reg
No:379/Go/ReBi/S/01/CPCSEA)(379/CPCSEA/TAEC-2018/030)
2.4. Pharmacological evaluation
2.4.1. Experimental design
The experimental animals were divided into control and experimental groups (n=3).
The animals in the control group received vehicle while those in the reference control and test
groups were treated with AP and the test compounds as described in Table 1.
Table 1: Study design for pharmacological analysis
Group
Treatment allocation
Vehicle
p.o. dose in mg/kg
Control
-
Reference Standard
Test groups (1-6)
Test groups (7-12)
Test groups (13-17)
AP
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6a, 6b, 6c, 6d, 6e, 6f
7a, 7b, 7c, 7d, 7e
2.4.2. Anti-inflammatory assay (AI)
For the purpose of evaluating anti-inflammatory activity Cox-II enzyme inhibition assay and
Carrageenan induced paw edema assays were employed.
2.4.2.1. Cox-II enzyme assay
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Inhibition assays of synthesized APTCs against COX-2 were performed with modification
using enzyme immunoassay according to the manufacturer's instruction. A reaction mixture
consisting of 0.1 M Tris-HCl pH 8.0, 5 mM EDTA, and 2 mM phenol was mixed with COX-
2. Serial dilutions of the synthesized compounds were prepared and incubated with the
reaction mixture. The reactions were initiated by adding arachidonic acid (AA). After the
reactions were terminated by the addition of hydrochloric acid, the spectrophotometrical
intensity was measured. All experiments were carried out in triplicate. All % inhibition
values were expressed as means of triplicate measurement34. The comparative inhibitory
activity of APTCs against COX-2 in comparison to AP is represented in Figure 1.
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2.4.2.2. Carrageenan induced paw assay
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 were allocated to different experimental
groups and treated as per study design. (Table 1) After 60 mins, 0.05 mL 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 challenge35 (Table 4).
2.4.3. Anti-nociceptive assay
Analgesic (Anti-nociceptive activity) of the compounds were evaluated by Eddy’s hot plate
and acetic acid induced writhing assay.
2.4.3.1. Eddy’s Hot Plate
The animals were placed in Eddy’s hot plate analgesiometer maintained at 55-56 ºC and time
to elicit paw licking/ jumping response was measured. A cut-off time of 15 secs was ensured
to avoid any tissue damage. Analgesic potential was evaluated on the basis of latency of
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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
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2.4.3.2. Acetic acid induced writhing effect
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
1). The animals were placed individually in glass beaker and number wriths per 10 min were
observed for 05 mins post challenge38.
2.4.4. Anti-pyretic assay
Brewer’s yeast induced pyrexia model was employed to evaluate the anti-pyretic potential of
the synthesized APTCs39. 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 10 mL/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 were allocated to control and experimental groups and as per study design (Table 1).
Rectal temperature was measured again after 0, 30, 60, 120 and 180 mins post dosing.
2.5. Toxicological Analysis
2.5.1. Experimental design
Compounds with most favorable pharmacological profile (5a and 5e) were further analyzed
in in vivo toxicological assay paradigms. Toxicological analysis of the synthesized
compounds was accomplished by acute toxicity study, LD50 determination and sub-acute
toxicity study in accordance with OECD test guidelines 420, 425 and 407 respectively. For
the purpose of dosing the synthesized conjugates APTCs (5a and 5e) as well as AP were
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suspended in physiological saline. The experimental animals were segregated randomly into
control and experimental groups. Study design followed for acute and sub-acute toxicological
analysis is described in Table 2. Starting dose for LD50 evaluation was determined after
performing acute toxicological analysis.
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Table 2: Study design for toxicological analysis
Group allocation
Treatment
Dose (mg/kg)
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2.5.2. Fixed dose acute toxicity study
Acute toxicological analysis of the synthesized compounds in reference to AP 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 (Table-2). Dosing was done in
stepwise procedure (50, 100, 300, 500 and 1000 mg/Kg b.w.) using 300 mg/kg 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 mortality40. Biochemical and histopathological
analysis was performed on completion of the study for determining the extent of toxicity
induced in context of acute oral administation of potent APTCs (5a and 5e) in comparision to
AP.
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2.5.3. Determination of median lethal dose (LD50)
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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 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 2000 mg/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. With reference to acute toxicological analysis, the starting dose of 550 mg/Kg was
used. Following which an estimate of LD50 was calculated using the maximum likelyhood
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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), and histopathological analysis were performed to asses the extent of toxic
manifestation. From the observation thus obtained NOAEL (No observed adverse effect
level) and corresponding HED (Human equivalent dose) values for potent APTCs were
determined43. For further mechanistic evaluation of toxicological consequence, hepatocellular
ROS status was determined in experimental animals post sub acute toxicology.
2.5.4.1. Measurement of hepatocellular ROS status
Hepatocellular ROS content was defined by measuring hepatic lipid hydroperoxide content
and level of lipid peroxidation in hepatic tissue isolates from control and treated group
animals post 28-days repeated dose sub-acute toxicological analysis. Liver tissues (50 mg)
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were rinsed, homogenized in 50 mM potassium phosphate buffer (pH 6.0) at a ratio of 50 mg
tissue to 1 ml buffer, and then centrifuged at 10,000 × g for 10 min at 4°C. The supernatant
was discarded, and the pellet was homogenized in 300 µL potassium phosphate buffer
containing 0.5% hexadecyltrimethylammonium bromide (HTAB). The suspension was
sonicated for 20 s on ice and then centrifuged at 10,000 × g for 30 min at 4°C. 50 µL of the
supernatant was mixed with 950 µL of FOX reagent (1 M sorbitol, 25 mM iron(II) sulfate
(FeSO4), 250 mM sulfuric acid (H2SO4), and 1 mM xylenol orange), vortexed, and incubated
in the dark for 30 min at room temperature. The absorbance was read at 560 nm. Lipid
peroxidation level was assessed using the thiobarbituric acid reactive substances (TBARS)
assay as previously described. 200 µL of working solution containing 20% trichloroacetic
acid and 0.5 % thiobarbituric acid in 2.5N hydrochloric acid (HCl) was added to 100 µL liver
homogenate solutions. After 15-min incubation in a boiling water bath, the mixture was
centrifuged at 2,000 × g for 10 min. The concentration of TBARS in the supernatants was
measured spectrophotometrically at 532 nm44.
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2.6. 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. RESULT
3.1. Chemistry
In lieu with the concept of bio-isosterism, we have designed bio-isosteres of AP and its
derivatives with an imperative to create metabolically stable and less toxic scaffolds45. The
amide bond in AP responsible for its toxicity and stability issues is replaced with 1,2,3-
triazole. The key intermediate in the synthesis of the triazolyl AP (TP) analog 1 is the 4-
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azido-phenol 2a, 3-azidophenol 2b and 2-azidophenol 2c. It is likely that copper-catalyzed
cycloaddition between 2 and appropriate alkynes will furnish the desired APTC analogs 1.
The azido phenol 2a-c was accessed 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. Various alkynes of aromatic and aliphatic cycloamines were
used for synthesizing a series of APTC analogues. Some alkynes were purchased and
remaining alkynes were synthesized by treating aliphatic amines and benzyl alcohol with
propargyl bromide in DMF using sodium hydride at 0-5°C.
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R1
R4
R2
R3
R5
R6
10 mol%, CuI, 30 % L-proline
NaN3, 30% NaOH, EtOH, H2O
9 h, 85%
OH
OH
1a, R1= Br, R2= H, R3= H
1b, R1= H, R2= Br, R3= H
1c, R1= H, R2= H, R3= Br
2a, R4= N3, R5= H, R6= H
2b, R4= H, R5= N3, R6= H
2c, R4= H, R5= H, R6= N3
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 various alkynes was carried out in a ball mill at 300rpm. Cu(I)-
catalyzed reaction of azide 2a-c with alkyne 4a-h resulted in triazoles 5a-f, 6a-f and 7a-e in
excellent yield, furnishing the desired triazolyl compounds in 80-85% yield (Scheme 3). We
selected a subset of some terminal alkynes which were closely related to AP and its
metabolite. The 2a-c was coupled with aromatic and aliphatic alkynes using click chemistry
in the presence of copper sulphate pentahydrate and L-sodium ascorbate in a Fritsch Ball mill
under solvent free conditions at 300rpm furnishing the desired APTC analogs. (Scheme 3)
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OH
Boc
1. Boc anhydride
Dioxane:H2O
O
NH
2. CHCCH2Br, NaH,
DMF. 0-5 °C
4c
OH
N
CuSO4, NaAsc
Ball mill, Solvent free
R4
NH2
4a
R5
R6
H3C CH3
400 rpm, 3h
Si CH3
+
4d
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OH
N
2a, R4= N3, R5= H, R6= H
2b, R4= H, R5= N3, R6= H
2c, R4= H, R5= H, R6= N3
R
N
4e-g
R
5a-f
6a-f
7a-e
1. H2SO4, HCl,
NH4OAc
H
N
COOH
APTCS
N
2. CHCCH2Br
NaH, DMF
COOC2H5
4b
4h
Scheme 2. Synthesis of alkynes and clicking with substituted azides to furnish 1,2,3-triazolyl
phenol derivatives
OH
OH
OH
OH
H
OH
N
H3C
N
N
N
H
C
C14H29
N
N
N
N
HO
N
N
N
N
5d
5c
5b
5a
OH
OH
N
HOOC
N
H2N
N
N
N
N
O
N
5f
5e
OH
OH
OH
OH
H
OH
N
N
N
H
C
C6H5
N
H3C
N
N
N
HO
N
N
N
N
N
6c
6d
6b
6a
OH
OH
N
H3C
N
HOOC
N
N
OH
N
H2N
N
N
N
N
O
N
6e
7a
6f
H
OH
N
N
N
N
H
C
C6H5
H2N
N
OH
O
N
N
OH
HO
N
OH
N
OH
N
N
N
7d
7e
7c
7b
Scheme 3. Structure of the synthesized acetaminophen-triazole conjugates (APTC)
3.2. Pharmacological Evaluation
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3.2.1. Anti-inflammatory Assay: For establishing the anti-inflammatory potency of the
synthesized series of APTCs (5a-f, 6a-f and 7a-e), COX-II enzyme inhibition assay and
carrageenan induced paw edema model were used. Cyclo-oxygenase-II or COX-II enzyme is
implicated in mediating pathological pathway of inflammation. Thus activity of test
compound on COX-II enzyme is used as a confirmatory marker of anti-inflammatory efficacy
of the test compounds. For further justification of efficacy, in vivo anti-inflammatory potency
of APTCs was evaluated in carrageenan induced paw edema bioassay in Wistar Albino rats.
Results of both anti-inflammatory bioassays are revealed in Figure 1. Form the recorded
observations it was apparent that in comparison to AP all members of the synthesized series
exhibited superior anti-inflammatory potency with compound 5a and 5e manifesting
maximum anti-inflammatory efficacy. Since AP is a documented weak anti-inflammatory
agent the efficacy of synthesized APTCs can be attributed to the presence of 1,2,3-triazole in
the structural skeleton of the synthesized moieties.
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*
*
Enzyme inhibition
Paw edema
90
80
70
60
50
40
30
20
10
0
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
5a 5b 5c 5d 5e 5f 6a 6b 6c 6d 6e 6f 7a 7b 7c 7d 7e AP
Treatment
Figure 1. AI assay performed using Cox-II enzyme inhibition and carrageenan induced paw edema
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* p<0.05 (level of significance is in comparison to AP treated group)
5
6
3.2.2. Anti-pyretic Assay: Figure 2. describes the observations of the anti-pyretic bioassay
7
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9
performed using Brewer’s Yeast induced pyrexia. In comparison to AP treatment percentage
inhibition of pyrexia with APTCs treatment was found to vary insignificantly (p>0.05).
However antipyretic efficacy was found to be superior than that observed for AP in groups
treated with 5a and 5e.
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*
90
80
70
60
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40
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10
0
*
5a
5b
5c
5d
5e
5f
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
AP
Treatment
Figure 2. Anti-pyretic bioassay performed using Brewer’s Yeast induced pyrexia
* p<0.05 (level of significance is in comparison to AP treated group)
3.2.3. Analgesic Assay
Analgesic activity of the synthesized series of APTCs (5a-f, 6a-f and 7a-e) was analyzed
using acetic acid induced writhing model and Eddy’s hot plate assay paradigm. Acetic acid
induced writhing in rodents is of help in assessing a compounds potential to elicit peripheral
analgesic response. While Eddy’s hot plate analyses central analgesia of the compounds
under study. The comparative potency of APTCs in both bioassay paradigms is illustrated in
Figure 3. In comparison to the AP treatment, compounds (5a and 5e) exhibited superior
potency in both bioassays paradigms. In context of acetic acid induced writhing assay
numbers of wriths in groups treated with APTCs (5a and 5e) were found to be significantly
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less (p < 0.05) than the vehicle treated control group evidencing the activation of peripheral
mechanism of analgesic action. The latency of heat stimulus in Eddy’s hot plate was also
found to be significantly (p < 0.05 ) more for (5a and 5e) in comparison to the vehicle treated
control. From the findings it can be clearly advocated that of the series of 17 APTCs
compounds 5a and 5e are of superior efficacy than AP in quenching algesia. Furthermore, as
opposed to only analgesic potency in AP, APTCs demonstrate both central as well as
peripheral analgesic potency.
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80
*
Central analgesia
*
Peripheral analgesia
70
*
60
*
50
40
30
20
10
0
5a 5b 5c 5d 5e 5f 6a 6b 6c 6d 6c 6d 6e 6f 7a 7b 7c 7d 7e AP
Treatment
Figure 3 Analgesic bioassay performed using acetic acid induced assay and Eddy’s hot plate
* p<0.05 (level of significance is in comparison to AP treated group)
3.3. Toxicological Evaluation
3.3.1. Single dose acute toxicity Study: Acute toxicology of potent APTCs was performed
in accordance with OECD TG 420. The study was sought with the aim of diagnosing the
presence of toxicity if any after acute administration of compounds 5a and 5e. Table 3
summarizes the findings of acute toxicological study. From the performed study it was
observed that at 500mg/kg single oral dose of AP, experimental animal’s significant
morbidity. However at 1000 mg/kg dose mortality followed the morbidity in AP treated
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group. Further biochemical analysis revealed that the liver enzyme panel (ALT, AST and
ALP) in comparison to the vehicle treated control group was found to be elevated in AP
treated groups (Table 4). Although the rise was found to be insignificant at 500 mg/kg but at
1000 mg/kg dose AP treatment significant (p < 0.05) rise in liver function tests were
observed. In context of kidney function test no significant change was observed for AP
treatment at either doses. Treatment with APTCs (5a and 5e) did not manifest any signs of
morbidity nor mortality after acute oral administration at both doses (Table 3). In comparison
to vehicle treated control no significant variation in hepatic and renal biochemistry was
observed post 5a and 5e acute treatment (Table 4).
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Table 3: Effect of the synthesized compounds on experimental animals after single dose oral acute
toxicity study
Group
Dose (mg/kg)
Sign of morbidity Number of dead animals
Cumulative percent
-
-
-
-
Control
AP-1
500
1000
500
-
0
6
0
0
-
0
AP-2
Present
10.00
5a-1
5a-2
-
-
-
-
0
0
0
0
1000
500
5e-1
1000
-
Table 4: Effect of synthesized compounds on liver and kidney function test after single dose oral acute
toxicity study
Group
Liver function test
AST
Kidney function test
Creatinine
ALT
ALP
BUN
Uric acid
Contro
l
52.69 ± 1.79
160.90 ± 2.36
205.17±3.96
8.50 ± 2.09
1.27±0.76
3.06 ± 1.26
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AP-1
AP-2
5a-1
5a-2
5e-1
5e-2
69.18 ± 2.45
76.70 ± 3.97*
53.70 ± 1.97
52.60 ± 2.09
53.70 ± 1.78
53.90 ± 1.09
167.90 ± 2.36
228.35±2.48
241.90±4.39*
209.35±2.24
210.35±2.48
209.35±1.98
210.35±2.85
9.50±1.98
1.30±0.72
1.78±0.30
1.25±0.81
1.29±0.30
2.08±0.33
2.10±0.50
3.08 ± 1.95
3.27 ± 2.01
3.11 ± 0.61
3.09± 1.76
3.07 ±1.25
3.10±1.35
175.35 ± 3.35*
160.35 ± 4.29
160.90 ± 2.36
160.90 ± 2.36
160.90 ± 2.36
12.56±1.91
8.62±2.64
8.70±2.04
8.52±0.69
8.50±1.21
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Data is represented as mean ± SEM (n=6). Results were analysed using one way ANOVA.
Values are expressed as mean ± SEM *p < 0.05 (Significant).
Figure 4 describes the light microscopic as well as CSLM images of hepatic tissue obtained
from excised liver of Wistar albino rats post 14 days acute-toxicological study. In Figure 4a
histopathology of liver post AP treatment is documented while Figure 4c and 4d describes the
hepatic histopathology post acute administration of APTCs at 500mg/Kg and 1000mg/Kg. As
described in Figure 4 histopathology of hepatic tissue revealed slight occurrence of
hepatocellular necrosis at 500mg/kg dose however at 1000mg/kg AP treatment distinct
necrotic lesions were found in the liver tissue. In comparison to the vehicle treated control
group (Figure 4e) no considerable change was observed in the hepatic tissue of APTCs
treated groups.
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3.3.2. Determination of LD50
Results of the acute toxicity study by up and down procedure (OECD 425) showed the LD50
value for APTCs to be > 1000 mg/kg b.w. while that for the AP was found to be < 1000
mg/kg b.w. (Table 5).
Table 5 : Toxicity profile of compounds
Compound
LD50 (mg/kg)
> 1000
NOAEL (mg/kg)
HED (mg/kg)
27.68
5a
5e
2000
300
AP
< 1000
4.152
3.3.2. Repeated dose sub acute toxicity study
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Repeated dose sub acute toxicology was performed in accordance with OECD TG 407. The
study was intended to document the cause and consequence of AP and APTC repeated
administration in experimental animals especially in context of hepatic pathology. The results
of the study are expressed in table 5 in terms of NOAEL and HED. NOAEL states the
maximum dose at which no sign of therapeutic misadventure is documented in experimental
animals which are wistar albino rats in the current study. Human equivalent dose (HED)
quotes the dose corresponding to NOAEL in humans. HED is the maximum dose at which no
toxicity is bound to manifest in human physiology. HED is the maximum concentration of a
compound that is expected to not produce any adverse symptoms in human physiology. HED
is purely a toxicological index which should not be confused with therapeutic dose of a
compound.
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3.3.2.1. Body weight and biochemistry.
Table 6 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 APTCs (5a and 5e) while significant (p<0.05) decrease in
body weight was observed in the groups treated with AP. This drastic decrease in body
weight was found to be consistent with the moribund status of the AP treated animals. In
context of biochemical analysis, Table 7 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
AP treated group. While the liver enzymes of the groups treated with APTCs 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 AP
treated group. The kidney function test values for the groups treated with APTCs (5a, 5e)
were found to be in range with those of the control groups. From the data obtained it can be
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stated that considerable degeneration in kidney and liver functions is brought to notice at
1000mg p.o,. dose of AP, while at the same dose, the newly synthesized APTCs are free of
such limitations.
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Table 6: Effect of synthesized compounds on body weight (gm) parameters after 28 days repeated dose oral
toxicity studies
Days
0 day
7 day
14 day
120.08±2.26
117.70±2.44*
115.34±1.49*
121.92±5.65
124.41±5.25
125.25±10.78
125.35±5.09
21 day
121.57±4.12
116.37±8.43*
112.97±8.43*
123.46±8.25
125.03±7.05
126.05±2.60
125.03±4.55
28 day
120.47±14.84
112.85±13.30*
107.35±4.98*
130.83±12.35
128.58±6.75
129.21±7.25
129.08±8.50
Control
AP-1
AP-2
5a-1
120.45±9.55
122.63±7.90*
121.75±3.57*
119.49±9.45
122.78±5.95
120.81±5.11
124.29±7.59
120.47±14.84
119.85±13.30*
117.25±9.30*
121.83±12.35
124.53±7.79
122.07±9.00
124.94±9.35
5a-2
5e-1
5e-2



Data is represented as mean ± SEM (n=6). Results were analyzed using one way ANOVA.
Values are expressed as mean ± SEM
p value <0.05(Significant **
)
Table 7: Effect of synthesized compounds on liver and kidney function test after 28 days repeated dose
oral toxicity studies
Group
Liver function test
AST
Kidney function test
Creatinine
ALT
ALP
BUN
Uric acid
3.11 ± 1.28
5.04 ± 1.93*
9.17 ± 2.31*
Control
AP-1
52.69 ± 1.38
160.45 ± 5.01
207.67±5.46
8.50 ± 2.09
17.50±1.98*
20.56±1.91*
1.26±0.39
3.07±1.12*
6.18±1.80*
56.78 ± 2.45* 170.90± 4.56* 240.95±4.98*
76.70 ± 3.97* 195.35± 5.95* 291.27±8.54*
AP-2
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5a-1
5a-2
5e-1
5e-2
52.93 ± 3.42
52.98 ± 4.55
52.45 ± 1.54
53.12 ± 1.23
162.65 ± 8.19
208.85±7.84
208.35±3.93
208.75±4.47
212.35±7.80
8.62±2.64
1.34±0.39
1.41±0.67
1.38±0.47
1.48±0.29
3.13 ± 0.89
3.16 ± 1.30
3.23 ±1.56
3.41±1.78
160.90 ± 3.76
163.39 ± 4.89
161.30 ± 3.39
8.70±2.04
8.52±0.69
9.00±1.61
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Data is represented as mean ± SEM (n=6). Results were analysed using one way ANOVA.
Values are expressed as mean ± SEM *p < 0.05 (Significant)
3.3.2.2. Hepatocellular ROS status: To elucidate the mechanism of AP induced
hepatotoxicity and the absence of it with APTCs treatment we examined the hepatocellular
redox status in animals treated with AP and APTCs respectively. As illustrated in Figure 5
level of hepatic hydroperoxide was found to be significantly more in AP treated group
whereas groups treated with APTCs showed insignificant change in level of hydroperoxides
in comparison to the vehicle treated control group.
Also level of lipid peroxidation an indicative of cellular oxidative damage was found to be
significantly more in groups treated with AP. Whereas, as seen in context of hydroperoxide
content lipid peroxidation in liver homogenate of groups treated with APTCs changed
insignificantly in comparison to the vehicle treated control group (Figure 6). Thus from the
obtained observations it can be concluded that hepatotoxicity in response to AP treatment is a
consequence of elevated level of hepatocellular ROS. APTCs, on the other hand are devoid
on ROS mediated hepatotoxic manifestations.
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*
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5e-1
5e-2
Figure 5. Effect of synthesized compounds (5a and 5e) and AP on the TBARS assay
* p<0.05 (level of significance is in comparison to AP treated group)
*
*
5e-1
5e-2
Figure 6. Effect of synthesized compounds and AP on the hydrogen peroxide level
* p<0.05 (level of significance is in comparison to AP treated group)
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