Synthesis and comparative bioefficacy of N-(1-phenethyl-4-piperidinyl) propionanilide (fentanyl) and its 1-substituted analogs in Swiss albino mice

Article abstract of DOI:10.1007/s00044-012-0390-6

Fentanyl [N-(1-phenethyl-4-piperidinyl)propionanilide] is a popular narcotic analgesic agent that is clinically used worldwide. However, fentanyl and its several analogs have caused abuse and fatalities in humans due to overdosing and narrow therapeutic index. The present study reports the synthesis and comparative bioefficacy of fentanyl and its four analogs, viz., N-(1-propyl-4-piperidinyl)propionanilide (1), N-(1-(2-phenoxyethyl)-4- piperidinyl)propionanilide (2), N-(1-(3-phenoxypropyl)-4-piperidinyl) propionanilide (3) and N-(1-(2-cyanoethyl)-4-piperidinyl)propionanilide (4), where the phenethyl chain of fentanyl was replaced by different functional groups, viz., alkyl, ethereal, and nitrile moieties. The median lethal dose (LD₅₀) of the compounds was determined by three different routes and all the analogs were found to be safer than fentanyl. Observational assessment on spontaneous activities of the central nervous system, peripheral nervous system, and autonomic nervous system revealed that all the analogs were similar to fentanyl. Further, the neurotoxic effects of all the analogs were reversed by naloxone hydrochloride (opioid antagonist), confirming that their effects were mediated through opioid receptors. Antinociceptive activity was displayed by all the compounds and their median effective dose (ED₅₀) and analgesic potency ratio were more or less similar to fentanyl. The lowest ED₅₀ (23.7) and the highest potency ratio (1.18) was observed in the case of 2. However, the maximum therapeutic index was afforded by 4. The study indicates the promising role of some new opioid analgesics.

Full text of DOI:10.1007/s00044-012-0390-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:3888–3896
DOI 10.1007/s00044-012-0390-6
ORIGINAL RESEARCH
Synthesis and comparative bioefficacy of N-(1-phenethyl-4-
piperidinyl)propionanilide (fentanyl) and its 1-substituted analogs
in Swiss albino mice
•
Pradeep Kumar Gupta Shiv Kumar Yadav Yangchen Doma Bhutia
•
•
•
Poonam Singh Pooja Rao Niranjan Laxman Gujar
•
•
•
Kumaran Ganesan Rahul Bhattacharya
Received: 24 April 2012 / Accepted: 4 December 2012 / Published online: 15 December 2012
Ó Springer Science+Business Media New York 2012
Abstract Fentanyl [N-(1-phenethyl-4-piperidinyl)propi-
onanilide] is a popular narcotic analgesic agent that is
clinically used worldwide. However, fentanyl and its sev-
eral analogs have caused abuse and fatalities in humans due
to overdosing and narrow therapeutic index. The present
study reports the synthesis and comparative bioefficacy of
fentanyl and its four analogs, viz., N-(1-propyl-4-piperidinyl)
propionanilide (1), N-(1-(2-phenoxyethyl)-4-piperidinyl)pro-
pionanilide (2), N-(1-(3-phenoxypropyl)-4-piperidinyl)propi-
onanilide (3) and N-(1-(2-cyanoethyl)-4-piperidinyl)propionanilide
(4), where the phenethyl chain of fentanyl was replaced by
different functional groups, viz., alkyl, ethereal, and nitrile
moieties. The median lethal dose (LD50) of the compounds
was determined by three different routes and all the analogs
were found to be safer than fentanyl. Observational assess-
ment on spontaneous activities of the central nervous sys-
tem, peripheral nervous system, and autonomic nervous
system revealed that all the analogs were similar to fentanyl.
Further, the neurotoxic effects of all the analogs were
reversed by naloxone hydrochloride (opioid antagonist),
confirming that their effects were mediated through opioid
receptors. Antinociceptive activity was displayed by all the
compounds and their median effective dose (ED50) and
analgesic potency ratio were more or less similar to fentanyl.
The lowest ED50 (23.7) and the highest potency ratio (1.18)
was observed in the case of 2. However, the maximum
therapeutic index was afforded by 4. The study indicates the
promising role of some new opioid analgesics.
Keywords Fentanyl Á Opioids Á 1-Substituted analogs Á
Synthesis Á Bioefficacy Á Mice
Introduction
The treatment of mild to moderate pain can typically be
accomplished with non-narcotic analgesics. These drugs
are relatively inexpensive, but are ineffective for the
treatment of severe pain, for which more potent drugs, viz.,
narcotic analgesics are used. Fentanyl is a popular synthetic
narcotic analgesic that is classified as schedule II-con-
trolled drug and is clinically used worldwide. It has rapid
onset and short duration of action, good overall safety
margin, and is several times more potent than morphine
´
´
(Mather, 1983; Van Nimmen et al., 2004; Micovic et al.,
2000). Fentanyl is a synthetic l-opioid receptor agonist,
widely used for surgical analgesia and sedation (Hug and
Murphy, 1981). Most of the effects like analgesia, eupho-
ria, and respiratory depression displayed by fentanyl are
typical of other opioids. Opioid receptors are coupled with
G-protein receptor and function as both positive and neg-
ative regulators, and are principally found in the central
nervous system (CNS) and the gastrointestinal tract. The
receptors mediate both the beneficial effects and the side
effects of opioids. The analgesic (painkiller) effects of
opioids are due to decreased perception of pain and
impaired reaction to pain, as well as increased pain toler-
ance. In addition to analgesia, alterations in mood,
euphoria and dysphoria, and drowsiness commonly occur.
P. K. Gupta Á K. Ganesan
Division of Synthetic Chemistry, Defence Research and
Development Establishment, Jhansi Road, Gwalior 474 002,
Madhya Pradesh, India
S. K. Yadav Á Y. D. Bhutia Á P. Singh Á P. Rao Á
N. L. Gujar Á R. Bhattacharya (&)
Division of Pharmacology and Toxicology, Defence Research
and Development Establishment, Jhansi Road, Gwalior 474 002,
Madhya Pradesh, India
e-mail: rbhattacharya41@rediffmail.com
123

Med Chem Res (2013) 22:3888–3896
3889
It depresses the respiratory centers and cough reflex, and
constricts the pupils (Fine, 2004; McNicol et al., 2008).
Although, fentanyl is very popular, its several analogs have
caused abuse and fatalities in humans, which led to syn-
thesis of some more potent and safer analogs (Higashikawa
and Suzuki, 2008).
antagonist) to ascertain that their effects were mediated
through opioid receptors. Antinociceptive activity and
toxicity of all the compounds were measured by median
effective dose (ED50) and median lethal dose (LD50),
respectively. The study reveals that replacing the phenyl
group of phenethyl tail of fentanyl with different functional
groups results in decreased toxicity of the molecules
without sacrificing their potency. Thereby, enhanced ther-
apeutic index could be achieved.
Over the last four decades, structure–activity relation-
ship and molecular modeling studies of fentanyl analogs
provided considerable insight into the key structural fea-
tures and pharmacophore elements required for high-
affinity binding to the l-receptor (Helsley et al., 1969;
Van Bever et al., 1974). From these studies, it was pro-
posed that a number of structural elements were required
for the analgesic activity of fentanyl class of compounds,
and variations in these pharmacological parameters led to
the synthesis of novel potent analogs of fentanyl
Materials and methods
Synthesis of fentanyl and its analogs
All the chemicals of highest purity, including dimethyl sulf-
oxide (DMSO), naloxone hydrochloride, and formalin were
purchased from Sigma-Aldrich Inc. (St. Louis, USA). Fentanyl
[N-(1-phenethyl-4-piperidinyl)propionanilide], N-(1-propyl-4-
piperidinyl)propionanilide (1), N-(1-(2-phenoxyethyl)-4-pipe-
ridinyl)propionanilide (2), N-(1-(3-phenoxypropyl)-4-piperid-
inyl)propionanilide (3), and N-(1-(2-cyanoethyl)-4-piperidinyl)
propionanilide (4) were synthesized in the Synthetic Chemistry
Division of Defence Research and Development Establishment
(DRDE), Gwalior. The test compounds were prepared as per the
following scheme by the modification of the classical method
reported elsewhere (Janssen, 1965).
ˇ
(Vuckovic et al., 1998). The strong potency of fentanyl in
relation to that of morphine is largely due to its high
lipophilicity that facilitates its easy penetration into the
´
CNS (Ivanovic et al., 1995). The presence of an aromatic
ring attached to the piperidine ring nitrogen was supposed
to be responsible for the favorable lipophilic interaction
of the molecules at the receptor site. Its replacement with
groups with differing polarities may produce clinically
more useful compounds with better pharmacological
profile. In the present study, different fentanyl analogs,
where the phenyl group of the phenethyl chain of fentanyl
was replaced with groups of differing polarities, viz.,
alkyl, ethereal and nitrile moieties, were synthesized and
evaluated for their bioefficacy.
The conversion of commercially available N-benzyl-4-
piperidone (NBP; 5) to 4-anilino-N-benzylpiperidine
(ANBP; 6) was carried out in two steps, first involving the
reaction of 5 with aniline to give corresponding imine, which
was then reduced with sodium borohydride to give 6. In the
present method, this conversion was effected in single step
involving the reaction of 5 and aniline in the presence of zinc
and acetic acid to give the desired product 6. Transformation
of 6 to N-(4-piperidinyl)propionanilide (PP; 8) was carried
out as per the reported method involving acylation with
propionyl chloride to give N-(1-benzyl-4-piperidinyl)pro-
pionanilide (BPP; 7) followed by its debenzylation in pres-
ence of 10 % palladium–charcoal. Compound 8 acted as a
common precursor and once this key intermediate was in
hand, (N-alkylpiperidin-4-yl)propionanilides, all the com-
pounds were prepared by its reaction with different alkyl
At normal environmental temperature, a single intra-
peritoneal (ip) administration of fentanyl to unrestrained
rats has been shown to evoke analgesia and behavioral
effects such as catalepsy, hyperexcitability, loss of righting
reflex, and corneal reflex, accompanied by changes in body
ˇ
´
temperature (Vuckovic et al., 1998; Ivanovic et al., 1995).
In the present study, observational assessment on the basis
of behavioral parameters representing the activities of
CNS, peripheral nervous system (PNS), and autonomic
nervous system (ANS) was evaluated after administration
of the compounds by intravenous (iv), ip and per oral (po)
routes in mice. The neurotoxic effects of all the compounds
were challenged by naloxone hydrochloride (opioid
Ph
Ph
COEt
Ph
COEt
COEt
O
NHPh
N
N
N
N
EtCOCl
DCE
Fentanyl: R=CH2CH2Ph
1: R=CH2CH2CH3
2: R=CH2CH2OPh
3: R=CH2CH2CH2OPh
4: R=CH2CH2CN
H2
PhNH2
RBr/ DMF
K2CO3
Zn / AcOH
10 % Pd/C
N
N
N
H
N
R
CH2Ph
CH2Ph
CH2Ph
5
6
7
8
Scheme 1 Synthetic route for fentanyl and its analogs
123

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Med Chem Res (2013) 22:3888–3896
N-(4-piperidinyl)propionanilide (8)
bromide in acetonitrile (Scheme 1) and purified by recrys-
tallization with suitable solvent. The synthesized compounds
were characterized by IR, 1H NMR, 13C NMR, GC-MS, and
elemental analysis.
A solution of 7 (0.07 mol) in 125 ml methanol–acetic acid
mixture (3:2) was taken in a 250 ml thick-walled hydro-
genation vessel containing 10 % palladium on charcoal
catalyst (10 % w/w). The hydrogen gas was then purged
into the vessel using Parr apparatus at 50 °C. When no
more amount of hydrogen was consumed, the vessel was
removed and contents were filtered through Celite. The
filtrate was concentrated on rotary evaporator and the res-
idue was treated with 20 % aqueous sodium hydroxide
solution. The aqueous solution was extracted with ethyl
acetate, dried over anhydrous sodium sulfate, and solvent
was removed under vacuum. The crude compound thus
obtained was recrystallized with petroleum–ether
(40–60 °C), yield 14.62 g (90 %), mp 81–83 °C.
4-Anilino-N-benzylpiperidine (6)
To a mixture of 5 (0.10 mol), aniline (0.10 mol) and acti-
vated zinc (0.40 mol) were taken; 90 % aqueous acetic acid
(1.60 mol) was added portion wise and resulting mixture was
allowed to stir at room temperature for 24 h and at 60–70 °C
on water bath for another 12 h. After completion of the
reaction, the content of the flask was diluted with methanol
and filtered. The filtrate was concentrated under vacuum and
then neutralized with 30 % ammonium hydroxide solution
till pH 10. The crude product was collected by filtration and
recrystallized with petroleum ether (60–80 °C) to get pure
product (85 %), mp 82–83 °C.
IR (m, KBr): 3440 (N–H Str), 3250, 3025, 2930,
2848,1605, 1526, 1492, 1371, 1317, 1250, 1085, 975, 862,
750, 690 cm-1. 1H NMR [400 MHz, CDCl3, d(ppm)]: 1.50
(dq, 2H), 2.10 (bd, 2H), 2.30 (bt, 2H), 2.60 (s, 2H), 2.90
(bd, 2H), 3.35 (m, 1H), 3.50 (sbr, 1H), 7.10-7.40 (m, 109
Ar–H). EI-MS (m/z): 267 [M ? 1]?, 266 [M]?, 173, 158,
146, 132, 118, 91, 82, 65.
IR (m, KBr): 3370, 3029, 2942, 2827, 1656, 1590,
1
695 cm-1. H NMR [400 MHz, CDCl3, d (ppm)]: 0.94 (t,
3H), 1.25 (dq, 2H\), 1.55 (sbr, 1H), 1.75 (bd, 2H), 1.90 (q,
2H), 2.10 (bt, 2H), 3.00 (bd, 2H), 4.65–4.75 (m, 1H), 7.03
(d, 29 Ar–H), 7.05 (d, 19 Ar H), 7.35 (m, 29 Ar–H). EI-
MS (m/z): 175, 146, 132, 118, 82, 77, 68, 57, 55.
(N-alkylpiperidin-4-yl)propionanilides
In a 100-ml two-neck round bottom flask, 25 ml acetoni-
trile was taken and anhydrous K2CO3 (0.03 mol), tetrabu-
tylammonium bromide (10 mol%) and 8 (0.01 mol) were
added. To this, alkyl bromide (0.015 mol) was added
slowly with stirring and the reaction mixture was refluxed
with stirring till completion of the reaction. The reaction
mixture was then filtered to remove solid residues. The
filtrate was concentrated under vacuum to get dark colored
solid residue. The residue was purified by flash chroma-
tography or crystallization to give pure compound.
N-(1-benzyl-4-piperidinyl)propionanilide (7)
To the solution of 6 (0.08 mol) in 150 ml of 1, 2-dichlo-
roethane, propionyl chloride (0.24 mol) was added drop-
wise, and resulting mixture was stirred at ambient
temperature for 2 h. After completion of the reaction, the
reaction mixture was poured slowly to 4 % aqueous
sodium hydroxide solution with continuous stirring. The
resulting alkaline solution was extracted with dichloro-
methane, organic phase was dried over anhydrous sodium
sulfate and concentrated under reduced pressure to get
crude product which was purified as its hydrochloride salt:
colorless crystals, yield 25.78 g (90 %), mp 232–233 °C
(ethyl acetate). The corresponding free base was obtained
by decomposition of its hydrochloride salt with 20 %
sodium hydroxide solution followed by recrystallization
from petroleum ether (60–80 °C), colorless compound,
yield 23.18 g (90 %), mp 72–73 °C.
Fentanyl
Molecular formula: C22H28N2O, colorless crystalline solid,
°
yield: 65 %, mp 82–84 C (Pet-ether 40–60 °C); HCl salt,
°
mp 220–221 C (acetone). IR (m, KBr): 2941, 2822, 1657,
1592, 1493, 1381, 1264, 1122, 1052, 732, 700, 604 cm-1
.
1H NMR [400 MHz, CDCl3, d(ppm)]: 1.02 (t, 3H), 1.42
(dq, 2H), 1.80 (bd, 2H), 1.91 (q, 2H), 2.14 (bt, 2H), 2.52
(m, 2H), 2.72 (m, 2H), 2.99 (bd, 2H), 4.67 (m, 1H), 7.05
(dd, 29 Ar–H), 7.08–7.09 (m, 39 Ar–H), 7.19 (dd, 29 Ar–
H), 7.36–7.39 (m, 39 Ar–H). EI-MS (m/z): 245, 202, 189,
158, 146 (100), 132, 118, 105, 96, 91, 77, 70, 57, 44, 42.
IR (m, KBr): 3430, 2941, 2822, 1659 (C=O), 1495, 1370,
1260 (C–N Str), 1150, 1090, 705 cm-1
.
1H NMR
[100 MHz, CDCl3, d(ppm)]: 0.94 (t, 3H), 1.30–1.40 (m,
2H), 1.70–1.80 (m, 2H), 1.85 (q, 2H), 2.10 (m, 2H), 2.65
(m, 2H), 3.30 (t, 2H), 4.58–4.67 (m, 1H), 7.10–7.30 (m,
109 Ar–H). EI-MS (m/z): 323 [M ? 1]?, 322 [M]?, 265,
173, 158, 146, 132, 118, 91, 82, 77, 65, 57.
N-(1-propyl-4-piperidinyl)propionanilide (1)
Molecular formula: C17H26N2O, colorless crystalline solid,
°
°
yield: 61 %, mp 89–90 C; HCl salt, mp 220–221 C. IR (m,
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Med Chem Res (2013) 22:3888–3896
3891
KBr): 3050, 2953, 2853, 2527, 1652, 1598, 1494, 1452,
1
rice husk as the bedding materials with free access to food
(Ashirwad Brand, Chandigarh, India) and water. Before
experiment, animals were randomized and acclimatized for
7 days at 12-h light–dark cycle. The care and maintenance
of the animals were as per the approved guidelines of the
Committee for the Purpose of Control and Supervision of
Experiments on Animals (CPCSEA), India. Animals were
fasted overnight and allowed access to food and water 2-h
post-exposure. Animal experiments were carried out with
the approval of Institutional Animal Ethical Committee.
1399, 1268, 1149, 762, 708 cm-1. H NMR [400 MHz,
CDCl3, d(ppm)]: 0.84 (t, 3H), 1.01 (t, 3H), 1.37–1.45 (m,
4H), 1.75 (bd, 2H), 1.91 (q, 2H), 2.04 (bt, 2H), 2.22 (t, 2H),
2.90 (bd, 2H), 4.66 (m, 1H), 7.06 (d, 29 Ar–H), 7.34–7.38
(m, 39 Ar–H). EI-MS (m/z): 276 [M ? 2]?, 275
[M ? 1]?, 274 [M]?, 245, 217, 202, 189, 172, 158, 146,
132, 130, 126, 124, 118, 98, 96, 84, 82, 77, 70, 57, 55, 44,
42.
N-(1-(2-phenoxyethyl)-4-piperidinyl)propionanilide (2)
Determination of LD50
Molecular formula: C22H28N2O2, crystalline solid, yield:
78 %, mp 93–94 °C. IR (m, KBr): 3060, 2953, 2845, 2797,
1649, 1598, 1491, 1465, 1450, 1373, 1258, 1249, 1153,
Acute LD50 (iv, ip, and po) of the compounds was deter-
mined by Dixon’s up and down method using 4-6 animals
for each value (Dixon, 1965). All the compounds were
dissolved in DMSO and administered in a volume\10 ml/
kg body weight. To determine the iv LD50, the compounds
were administered through tail vein using a 27-gauge
needle, and for the po LD50, a 16-gauge oral feeding
cannula (HSE-Harvard, Germany) was used. Although the
animals were observed for 14 days, the mortality invari-
ably occurred within the first 24 h. All the dead animals
were autopsied to see any visceral changes.
1
1052, 1038, 757, 706 cm-1. H NMR [400 MHz, CDCl3,
d(ppm)]: 1.011 (t, 3H), 1.42 (dq, 2H), 1.76 (bd, 2H), 1.92
(q, 2H), 2.25 (bt, 2H), 2.74 (t, 2H), 3.00 (bd, 2H), 4.02 (t,
2H), 4.68 (m, 1H), 6.84 (d, 29 Ar–H), 6.91 (t, 19 Ar–H),
7.07 (dd, 29 Ar–H), 7.22–7.26 (dd, 29 Ar–H), 7.35-7.40
(m, 39 Ar–H). EI-MS (m/z): 295, 258, 245 (100), 202, 189,
176, 158, 146, 132, 118, 108, 96, 93, 77, 65, 57.
N-(1-(3-phenoxypropyl)-4-piperidinyl)propionanilide (3)
Molecular formula: C23H30N2O2, crystalline solid, yield:
°
Observational assessment
74 %, mp 63–64 C. HCl salt: Crystalline solid, mp
232–233 °C. IR (m, KBr): 3065, 2940, 2857, 2813, 1663,
1596, 1492, 1469, 1450, 1377, 1265, 1249, 1171, 1153,
1133, 1091, 1058, 1036, 950, 789, 766, 707 cm-1. 1H NMR
[400 MHz, CDCl3, d(ppm)]: 1.01 (t, 3H), 1.38 (dq, 2H),
1.79 (bd, 2H), 1.87–1.95 (m, 4H), 2.10 (bt, Ar–H),
7.22–7.41 (m, 59 Ar–H). EI-MS (m/z): 367 [M ? 1]?, 366
[M]?, 337, 309, 245, 216, 190, 189, 175, 158, 146, 132,
118, 107, 98, 96, 82, 77, 70, 57, 44, 42.
The procedure for this test was carried out as per the
modified method of Irwin (Irwin, 1964, 1968). The test is
usually carried out at the start of pharmacological screen-
ing of any compound to find out the psychotropic activity
of the compounds. In addition, toxicity of the compounds
can also be highlighted by this assessment. Mice were
Table 1 Different behavioral effects observed for fentanyl and its
analogs
N-(1-(2-cyanoethyl)-4-piperidinyl)propionanilide (4)
Systems
affected
Signs and symptoms
Molecular formula: C17H23N3O, crystalline solid, yield:
°
CNS
Spontaneous motor activity, restlessness, grooming
behavior, squatting, staggering, ataxic gait, lying
flat on the belly, lying flat on the side, lying flat on
the back, sleeping, narcosis, bizarre behavior,
timidity, Straub’s phenomenon, writhing, tremors,
twitches, opisthotonus, clonic convulsions, tonic
convulsions, rolling and jumping and convulsions
62 %), mp 95–96 C. IR (m, KBr): 3063, 2932, 2634, 2248,
1645, 1595, 1494, 1459, 1390, 1381, 1269, 1102, 1046,
952, 714 cm-1. 1H NMR [400 MHz, CDCl3, d(ppm)]: 1.00
(t, 3H), 1.38 (dq, 2H), 1.80 (bd, 2H), 1.91 (q, 2H), 2.21 (bt,
2H), 2.42 (t, 2H), 2.63 (t, 2H), 2.89 (bd, 2H), 4.65 (m, 1H),
7.05 (dd, 29 Ar–H), 7.37–7.41 (m, 39 Ar–H). EI-MS (m/
z): 245, 228, 202, 189, 146, 137, 135, 118, 96 (100), 82, 77,
70, 57.
PNS
After
manipulations
Auditory stimulus response, escape after touch,
righting reflex, paresis of hind limbs, paresis of
forepaws and catalepsy in induced positions
Animals
Reflexes
ANS
Pinna reflex, corneal reflex and pain following
stimulation
Eyelids (closure or exophthalmus), salivation,
lacrimation, cyanosis, piloerection, defecation and
urination
The Swiss albino male mice (20–30 g body weight) were
obtained from the Animal Facility of DRDE, Gwalior. The
animals were housed in polypropylene cages on dust-free
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Med Chem Res (2013) 22:3888–3896
Table 2 LD50 of fentanyl and its analogs by different routes in mice
Compounds
LD50 (mg/kg)
Intravenous
Intraperitoneal
Oral
Fentanyl
6.9 (5.2–9.4)
17.5 (12.9–23.7)
143.3 (105.9–193.8)
112.2 (82.9–151.8)
90.4 (66.8–122.3)
285.8 (211.2–386.7)
27.8 (20.5–37.6)
1
2
3
4
55.4 (40.9–75.0)
22.1 (16.3–29.9)
18.0 (13.3–24.4)
69.8 (51.6–94.5)
359.8 (265.9-483.9)
453.0 (334.8–612.9)
285.8 (211.2–386.7)
717.9 (530.6–971.4)
Fentanyl and its four analogs, viz., 1–4 were administered through different routes and acute (24 h) LD50 was determined by Dixon’s up and
down method. Values in parentheses are fiducial limits at 95 % confidence interval
divided into six groups of 27 animals each as follows:
(i) vehicle control (DMSO), (ii) Fentanyl, (iii) 1, (iv) 2,
(v) 3, and (vi) 4. Three animals from each group received
0.25, 0.50, 0.75 LD50 each of the compounds by iv, ip, and
po routes. Immediately after administration, the animals
were closely observed for 2 h by a blind observer for
various behavioral effects on CNS, PNS, and ANS as
enumerated below (Table 1).
the therapeutic index was calculated as the ratio of LD50 of
the compounds and their corresponding ED50.
Measurement of analgesic activity
Analgesic activity of all the compounds was measured at
their ED50, following the method discussed elsewhere
(Hunskar and Hole, 1987; Vogel, 2002; Ellis et al., 2008).
Table 3 Observational assessments after intravenous administration
of fentanyl and its analogs in mice
Determination of opioid antagonist activity
In order to ascertain that the effects of all the compounds
were mediated through opioid receptors, naloxone hydro-
chloride (opioid antagonist) was dissolved in normal saline
and subcutaneously (sc) injected (5.0 mg/kg) in mice
10 min before administration of fentanyl and its analogs
(0.50 LD50; ip) (Leavitt, 2009; Matosiuk et al., 2002).
Three animals were used for each compound. Immediately
after administration, the animals were closely observed for
its neurotoxic effects as mentioned in Table 1 and com-
pared with the effects observed in animals receiving the
compounds (0.50 LD50; ip) without naloxone.
Compounds Dose
CNS
PNS
ANS
After
manipulations
Reflexes
Control
–
0
0
0
0
Fentanyl
Low
??
??
??
?
Medium ??? ???
???
???
??
?
High
Low
???? ????
?? ??
?
1
2
3
4
?
Medium ??? ??
???
???
??
?
High
Low
??? ???
??? ??
??
?
Medium ??? ???
???
???
??
?
Determination of ED50, potency ratio, and therapeutic
index
High
Low
???? ???
?
??
??
??
?
Medium ??
??
?
Analgesic ED50 of fentanyl and its analogs was determined
by formalin-induced hind paw licking method (Hunskar
and Hole, 1987). Each compound was assessed using four
different doses, and four animals for each dose. All the
compounds were dissolved in DMSO and administered ip
30 min before administration of formalin (2.5 %, 20 ll)
injected subplantarly in one hind paw. The duration of paw
licking as index of nociception was monitored at 0–5 min
(early phase) and 15–30 min (late phase) intervals. The
ED50 was calculated by the method of Litchfield and
Wilcoxon (1949). The potency ratio was determined as the
ratio of ED50 of fentanyl and the ED50 of each analog and
High
Low
??? ???
???
??
??
?
??
??
??
Medium ??
High
??
??
??
??? ???
???
Mice were intravenously administered 0.25 (Low), 0.50 (Medium),
and 0.75 (High) LD50 of fentanyl and its four analogs, viz., 1–4 The
control animals received DMSO. Immediately after treatment, the
animals were closely observed for 2 h by a blind observer for its
effects on CNS, PNS (effects after manipulation and effects on
reflexes), and ANS activities. The scorings were given as 0 (no
observational change), ? (little activity), ?? (moderate flexibility),
??? (strong response), and ???? (exaggerated response). Each
treatment included three animals
123

Med Chem Res (2013) 22:3888–3896
3893
This method includes useful models, particularly for the
screening of novel compounds, since it encompasses
inflammatory, neurogenic, and central mechanisms of
nociception. Mice were divided into six groups of six
animals each and received various treatments by ip route as
follows: (i) vehicle control (DMSO), (ii) Fentanyl
(28.0 lg/kg), (iii) 1 (26.7 lg/kg), (iv) 2 (23.7 lg/kg), (v) 3
(31.8 lg/kg), and (vi) 4 (33.3 lg/kg). Thirty minutes after
the administration of different compounds, formalin
(2.5 %, 20 ll) was injected subplantarly in one hind paw.
The duration of paw licking as an index of nociception was
monitored at 0–5 min (early phase) and 15–30 min (late
phase) intervals.
Results and discussion
Toxicity of the compounds
Table 2 refers to the LD50 of fentanyl and its analogs by
different routes of administration in mice. On the basis of
iv and ip LD50, toxicity of the compounds were in the
following order: Fentanyl [ 3 [ 2 [ 1 [ 4, while the
same for po route was: Fentanyl [ 3 [ 1 [ 2 [ 4. The
LD50 of fentanyl was 6.9, 17.5, and 27.8 mg/kg for iv, ip,
and po routes, respectively, while the same for 4 was 69.8,
285.8, and 717.9 mg/kg body weight, respectively. By all
the routes of administration, fentanyl was found to be the
most toxic and 4 the least toxic. As per the material safety
data sheet, the LD50 of fentanyl citrate in mice is 2.9, 7.6,
and 368 mg/kg for iv, ip, and po routes, respectively
(MSDS, 2009). Elsewhere, the LD50 values are reported to
be 11.2 and 62 mg/kg for iv and sc routes, respectively
(Gardocki and Yelnosky, 1964) and for po route it is
62 mg/kg (Higashikawa and Suzuki, 2008). This kind of
large variation in LD50 values can be attributed to different
Statistics
Results of analgesic activity were expressed as mean SE
(n = 6). The statistical analysis was performed by Stu-
dent’s t test by means of SigmaStat software (SSP Inc.,
USA). Statistical significance was drawn at p \ 0.01.
Table 4 Observational assessments after intraperitoneal administra-
tion of fentanyl and its analogs in mice
Table 5 Observational assessments after oral administration of fen-
tanyl and its analogs in mice
Compounds Dose
CNS
PNS
ANS
Compounds Dose
CNS
PNS
ANS
After
manipulations
Reflexes
After
manipulations
Reflexes
Control
–
0
0
0
0
Control
–
0
0
0
0
Fentanyl
Low
??
???
?
?
Fentanyl
Low
??
??
??
??
???
?
?
Medium ??? ????
??
???
?
?
Medium ??? ???
?
High
Low
???? ????
?? ??
?
High
Low
???? ???
?
1
2
3
4
?
1
2
3
4
??
??
??
?
Medium ??? ??
??
???
??
???
???
?
??
??
?
Medium ??
??
??
?
?
High
Low
??? ???
?? ??
High
Low
??? ???
?
?
?
?
Medium ??? ??
?
Medium ??? ??
??
???
?
?
High
Low
???? ???
?? ??
?
High
Low
???? ???
?
?
??
??
??
?
Medium ??? ??
??
??
??
??
??
??
??
?
Medium ??
??
???
?
?
High
Low
??? ???
?? ??
High
Low
??? ???
?
??
?
?
Medium ??? ??
High ??? ??
?
Medium ??
High
??
?
??
??
?
??? ???
??
Mice were intraperitoneally administered 0.25 (Low), 0.50 (Medium),
and 0.75 (High) LD50 of fentanyl and its four analogs, viz., 1–4 The
control animals received DMSO. Immediately after treatment, the
animals were closely observed for 2 h by a blind observer for its
effects on CNS, PNS (effects after manipulation and effects on
reflexes), and ANS activities. The scorings were given as 0 (no
observational change), ? (little activity), ?? (moderate flexibility),
??? (strong response), and ???? (exaggerated response). Each
treatment included three animals
Mice were orally administered 0.25 (Low), 0.50 (Medium), and 0.75
(High) LD50 of fentanyl and its four analogs, viz., 1–4. The control
animals received DMSO. Immediately after treatment, the animals
were closely observed for 2 h by a blind observer for its effects on
CNS, PNS (effects after manipulation and effects on reflexes), and
ANS activities. The scorings were given as 0 (no observational
change), ? (little activity), ?? (moderate flexibility), ??? (strong
response), and ???? (exaggerated response). Each treatment
included three animals
123

3894
Med Chem Res (2013) 22:3888–3896
Therapeutic index
Table 6 ED50, potency ratio and therapeutic index of fentanyl and its analogs in mice
Compounds
ED50 (lg/kg)
Potency ratio
Fentanyl
28.0 (14.9–52.4)
26.7 (17.9–39.9)
23.7 (16.3–34.4)
31.8 (18.5–54.7)
33.3 (24.1–45.9)
1.0
625.0 (452.3–865.8)
5367.0 (4857.1–5916.2)
4734.2 (4412.8–5085.9)
2842.8 (2235.8–3610.8)
8582.6 (8424.8–8763.5)
1
2
3
4
1.05 (0.83–1.31)
1.18 (0.91–1.52)
0.88 (0.81–0.96)
0.84 (0.62–1.14)
Analgesic ED50 of fentanyl and its four analogs, viz., 1–4 was determined by formalin-induced hind paw licking method. The potency ratio was
determined as the ratio of ED50 of fentanyl and ED50 of each analog. The therapeutic index was calculated as the ratio of LD50 of the compounds
and their ED50. Values in parentheses are fiducial limits at 95 % confidence interval
animal species and salts of fentanyl used. Mice succumbing
to lethal doses of the compounds administered through
different routes were autopsied immediately after death.
Profuse intestinal hemorrhage was evident in most of the
corpses, which perhaps led to hypovolemic shock and
resultant death. This was very similar to the observations
made previously with methyl-substituted and para-substi-
tuted fentanyl (Higashikawa and Suzuki, 2008).
interneurons (McGinty and Friedman, 1988). Also, in our
study, there were reduced ANS symptoms like defecation
and micturation, typical of opioid analgesics. In the present
study, we evaluated the compounds through parenteral and
po routes of administration, which are the preferred routes
of opioids for pain management (Gardocki and Yelnosky,
ˇ
1964; Vuckovic et al., 2011; Hallenbeck, 2003). In the
present study, to ascertain the efficacy of the compounds,
we evaluated them by three different routes, viz., iv, ip, and
po. Also, most of the motor coordination and behavioral
tests for initial screening remained same as reported earlier
(Gardocki and Yelnosky, 1964).
Observational assessment
Observational assessment on spontaneous CNS, PNS, and
ANS activities of the animals following administration of
various compounds by iv, ip, and po routes are summarized
in Tables 3, 4, and 5, respectively. The activities produced
by the compounds administered through iv and ip routes
were more intense as compared to po route. Also, the CNS
and PNS activities were more noticeable as compared to
ANS. In general, no appreciable dose–response effects
were observed, but effects in medium and high dose were
certainly more severe as compared to low dose. Effects on
CNS, PNS, and ANS activities were more in fentanyl and 2
as compared to others. The results indicate that all the
analogs exhibited effects almost similar to the parent
compound, fentanyl. The effects were much prominent
when the compounds were given by iv route, in which the
dose was also significantly reduced as compared to po and
ip routes. Morphine and other opioid analgesics exert their
effect through l-opioid receptors. In spite of its in vivo
efficacy, their long term use is limited because of its side
effects such as tolerance, physical dependence, respiratory
depression, and diverse gastrointestinal effects (Per-
likowska et al., 2011; Qi et al., 2011). They cause profound
analgesia, alter gastrointestinal, neuroendocrine, cardio-
vascular, and respiratory function. In the present study,
fentanyl, which is also a l-opioid receptor agonist along
with its analogs showed effects like catalepsy, rigidity,
circling, and stereotypical behavior, characteristic of other
opioid analgesics. Convulsions, characteristic of morphine
intoxication were also observed, which could be due to
inhibition of release of gamma-aminobutyric acid by the
Test for opioid antagonist activity
Short acting naloxone and long acting naltrexone are the
two opioid antagonists that have been most extensively
100
First Phase
Second Phase
80
60
*
*
40
*
*
*
20
0
Control Fentanyl
1
2
3
4
Fig. 1 Analgesic activity of fentanyl and its four analogs, viz., 1–4
was measured by formalin-induced hind paw licking method. Various
groups were as follows: (1) vehicle control (DMSO), (2) Fentanyl
(28.0 lg/kg), (3) 1 (26.7 lg/kg), (4) 2 (23.7 lg/kg), (5) 3 (31.8 lg/
kg), and (6) 4 (33.3 lg/kg). 30 min after intraperitoneal administra-
tion of different compounds, formalin (2.5 %, 20 ll) was injected
subplantarly, in one hind paw. The duration of paw licking as index of
nociception was monitored at 0–5 min (early phase) and 15–30 min
(late phase) intervals. Values are mean SE (n = 6). *p \ 0.01
(Student’s t test)
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Med Chem Res (2013) 22:3888–3896
3895
used to treat opioid addiction (Leavitt, 2009). In the present
study, naloxone quickly reversed the observed neurotoxic
effects of all the compounds, confirming that their effects
excitatory amino acids (Correa and Calixto, 1993; Damas
and Liegeois, 1999). This late phase is inhibited by NSA-
IDs and opioid analgesics. In our study, fentanyl and
analogs were more effective in the later phase rather than
in the first phase, testifying the fact that these compounds
could be inhibitors of the pain mediators during the late
phase.
´ ´
were mediated through opioid receptor (Micovic et al.,
2000; Leavitt, 2009; Matosiuk et al., 2002).
ED50, potency ratio and therapeutic index
of the compounds
Table 6 refers to the analgesic ED50, potency ratio, and
therapeutic index of fentanyl and its analogs determined by
formalin-induced hind paw licking method (Hunskar and
Hole, 1987). The ED50 values and analgesic potency ratio
of all the analogs were more or less similar to fentanyl, but
the lowest ED50 (23.7) and the highest potency ratio (1.18)
was observed in case of 2. The therapeutic index of the
compounds was in the following order: 4 [ 1 [ 2 [
3 [ fentanyl. The highest therapeutic index was exhibited
by 4 (8582.6) as compared to the lowest in fentanyl
(625.0). This indicates that all the analog were 5-14-folds
safer than fentanyl. Previous studies have shown that
4-methylfentanyl was four times more potent than fentanyl
Conclusions
Efficacy of many opioid analgesics is impaired due to
narrow therapeutic window. The present study addresses
the synthesis and bioefficacy studies of 1-substituted fen-
tanyl analogs. The results indicate that by replacing the
phenyl group of phenethyl tail of fentanyl with alkyl,
ethereal, and nitrile moieties, higher therapeutic index could
be achieved as compared to fentanyl. The new molecules
tested could have therapeutic implications in future.
Acknowledgments The authors thank Prof. (Dr.) M.P. Kaushik,
Director, DRDE, Gwalior, for his support and encouragement.
´
´
(Micovic et al., 2000), while introduction of 3-carbome-
thoxy group in the piperidine ring of fentanyl reduced the
potency, but did not affect the tolerability and safety
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