Synthesis and pharmacological evaluation of some novel thebaine derivatives: N-(tetrazol-1H-5-yl)-6,14-endoethenotetrahydrothebaine incorporating the 1,3,4-oxadiazole or the 1,3,4-thiadiazole moiety

Article abstract of DOI:10.1002/ardp.201200451

In this study, we synthesized some novel N-(tetrazol-1H-5-yl)-6,14- endoethenotetrahydrothebaine 7α-substituted 1,3,4-oxadiazole and 1,3,4-thiadiazole derivatives as potential analgesic agents. The structures of the compounds were established on the basis

Full text of DOI:10.1002/ardp.201200451

Arch. Pharm. Chem. Life Sci. 2013, 346, 455–462
455
Full Paper
Synthesis and Pharmacological Evaluation of Some Novel
Thebaine Derivatives: N-(Tetrazol-1H-5-yl)-6,14-
endoethenotetrahydrothebaine Incorporating the
1,3,4-Oxadiazole or the 1,3,4-Thiadiazole Moiety
1
Serkan Yavuz , Yusuf Unal , Ozgu¨r Pamir , Demet Yılmazer , Omer Kurtipek , Mustafa Kavutc¸u ,
2
1
3
2
4
¨
¨
¨
Mustafa Arslan4, Mustafa Ark5, and Yılmaz Yıldırır1
1 Faculty of Science, Department of Chemistry, Gazi University, Ankara, Turkey
2 Faculty of Medicine, Department of Anesthesiology and Reanimation, Gazi University Bes¸evler, Ankara,
Turkey
3 Dıskapı Yıldırım Bayezit Research and Education Hospital, Department of Pathology, Dıs¸kapı, Ankara,
Turkey
4 Faculty of Medicine, Department of Clinical Biochemistry, Gazi University, Bes¸evler, Ankara, Turkey
5 Faculty of Pharmacy, Department of Pharmacology, Gazi University, Etiler, Ankara, Turkey
In this study, we synthesized some novel N-(tetrazol-1H-5-yl)-6,14-endoethenotetrahydrothebaine
7a-substituted 1,3,4-oxadiazole and 1,3,4-thiadiazole derivatives as potential analgesic agents. The
structures of the compounds were established on the basis of their IR, 1H NMR, 13C NMR, 2D NMR, and
high-resolution mass spectral data. The analgesic activity was evaluated by a rat-hot plate test model
and a rat tail-flick model. Compound 12 showed analgesic activity higher than that of morphine. In
addition to a histopathological and biochemical evaluation, the LD50 dose for the most active
compound 12 was determined.
Keywords: Analgesic activity / Oxadiazole / Tetrazole / Thebaine / Thiadiazole
Received: November 30, 2012; Revised: March 12, 2013; Accepted: March 18, 2013
DOI 10.1002/ardp.201200451
Introduction
lar, the derivatives of 7a-substituted-6,14-endoethenomor-
phinan are very strong analgesic compounds that show
high affinity for each of the m, k, and d opioid receptor [5–
7]. For example, etorphine (19-(R)-propylorvinol), used for
immobilization of large animals for game conservation
and veterinary purposes, has an analgesic potency 1000 times
that of morphine [8].
Morphinan alkaloids and their semi-synthetic derivatives are
used as narcotic analgesics and painkiller with opioid agonist
properties [1, 2]. In some, morphinan derivatives are used as
opioid antagonist for the treatment of narcotic overdosage
and opioid addiction [3]. These properties depend on sub-
stituents in different positions of the morphinan skeleton.
Among these, 6,14-endoethenotetrahydrothebaine deriva-
tives have been extensively studied, as has the relationship
between structure and analgesic activity for many years. This
class of opioids is directly availabile with the Diels–Alder
reaction of thebaine and various dienophiles [4]. In particu-
In the present study, we reported the synthesis of some
N-(tetrazol-1H-5-yl)-6,14-endo-ethenotetrahydrothebaine 7a-
substituted 1,3,4-oxadiazoles and 1,3,4-thiadiazoles deriva-
tives which are potential clinical analgesics. The structures
of synthesized compounds were verified by various physical
and spectroscopic methods. Analgesic activity was evaluated
by a rat hot-plate test model and rat tail-flick model. In
addition, histopathological, biochemical evaluation with
alanine aminotransferase (ALT) and glutathione S-transferase
(GST) enzymes analyses and lethal dose (LD50) determination
for the most active compound 12 were performed.
Correspondence: Dr. Serkan Yavuz, Faculty of Science, Department of
Chemistry, Gazi University, 06500 Ankara, Turkey
E-mail: syavuz@gazi.edu.tr
Fax: þ9 0312 2122279
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Arch. Pharm. Chem. Life Sci. 2013, 346, 455–462
Scheme 1. Synthesis of N-(tetrazol-1H-5-yl)-6,14-endoethenotetrahydrothebaine 7a-substituted 1,3,4-oxadiazoles and 1,3,4-thiadi-
azoles derivatives. Reaction condition: (a) methylacrylate, benzene; (b) 99% hydrazinhydrate, ethoxyethanol; (c) benzoylchloride or
p-tolylchloride, toluene; (d) phenylisocyanate or p-tolylisocyanate, toluene; (e) phenylisothiocyanate or p-tolylisothiocyanate, toluene;
(f) POCl3; (g) H3PO4; (h) CNBr, CHCl3; (i) NaN3, NH4Cl, DMF.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 455–462
Pharmacological Evaluation of Some Novel Thebaine Derivatives
457
Results and discussion
action of POCl3. The reaction of carbohydrazide with phenyl
isocyanates and phenyl isothiocyanate gave semicarbazides
(12 and 13) and thiosemicarbazide derivatives (14 and 15),
respectively. The 1,3,4-oxadiazole and 1,3,4-thiadiazole ring
can be synthesized with subsequent cyclization of the inter-
mediate acylsemicarbazides and acylthiosemicarbazides.
Further, 1,3,4-oxadiazole and 1,3,4-thiadiazole derivatives
10–15 were refluxed with cyanogen bromide in dry CHCl3
to obtain cyanamides 16–21. At last, the subsequent reaction
of 16–21 with NaN3 and NH4Cl in dry DMF (dimethylform-
amide) furnished tetrazole compounds 22–27.
Chemistry
The synthesis of the target compounds was accomplished
according to the reaction sequence illustrated in
Scheme 1. The structures of the products were determined
by analysis of their IR, 1H NMR, 13C APT, 2D NMR (COSY,
NOESY, HMQC, HMBC) and high resolution mass spectra for
the molecular weights.
The starting material was thebaine which was treated with
methyl acrylate to give the Diels–Alder adducts 2 [9]. The
reaction predominantly gave rise to 7a-substituted tetra-
hydrothebaine. The key intermediate compound 3 was pro-
duced from the reaction of compound 2 with hydrazine
hydrate in ethoxyethanol. Compound 3 reacted with benzoyl
chloride derivatives in toluene producing diacylhydrazine
compounds 4 and 5. In 7a position, oxadiazole ring substi-
tuted thebaine derivatives 10 and 11 were obtained by the
cyclodehydration of diacylhydrazines 4 and 5 under the
Analgesic activity
The analgesic activities of compounds 10, 12, 13, 14, 15, 25, 26,
and 27 were determined in rat using the hot-plate and tail-
flick method in comparison with morphine as a reference
drug. A comparative study of the analgesic activity of the test
compound relative to the reference drug (Tables 1 and 2) at
different time intervals indicated the following: after 0.5 and
Table 1. Analgesic activities of some compounds (10, 12, 13, 14, 15, 25, 26, and 27) and morphine in the hot-plate test.
Compound
The average reaction time (s) at different time intervals after compound administration
Control
0.5 h
1 h
2 h
3 h
10
12
13
14
15
25
26
27
3.28 ꢀ 1.21
4.28 ꢀ 1.23
3.36 ꢀ 0.84
5.44 ꢀ 3.69
3.74 ꢀ 0.71
2.92 ꢀ 0.54
4.08 ꢀ 1.68
3.12 ꢀ 0.54
4.24 ꢀ 1.39
7.04 ꢀ 2.32ꢁꢁ
9.78 ꢀ 3.79ꢁ,ꢁꢁ
6.88 ꢀ 1.42
5.40 ꢀ 1.66
6.44 ꢀ 2.20
4.44 ꢀ 0.50
5.78 ꢀ 1.58
3.74 ꢀ 0.67
6.28 ꢀ 0.46
2.88 ꢀ 0.87
8.92 ꢀ 3.26ꢁ,ꢁꢁ
4.80 ꢀ 1.94
4.36 ꢀ 0.78
6.68 ꢀ 1.28
4.30 ꢀ 1.35
3.42 ꢀ 1.50
4.06 ꢀ 1.14
5.00 ꢀ 1.82
3.26 ꢀ 0.82
3.64 ꢀ 1.41
5.58 ꢀ 1.40
4.78 ꢀ 1.54
5.28 ꢀ 1.35
4.38 ꢀ 1.10
4.48 ꢀ 1.27
3.18 ꢀ 0.54
4.76 ꢀ 0.70
3.58 ꢀ 0.29
4.34 ꢀ 0.66
4.56 ꢀ 2.10
2.90 ꢀ 0.51
5.32 ꢀ 1.24
4.12 ꢀ 0.73
2.74 ꢀ 0.33
3.18 ꢀ 0.60
4.66 ꢀ 1.08
Morphine
ꢁ
Each value represents the mean ꢀ SD and all showed at least significant difference at p < 0.05 in comparison with the morphine
group and ꢁꢁp < 0.05 compared with the control value.
Table 2. Analgesic activities of some compounds (10, 12, 13, 14, 15, 25, 26, and 27) and morphine in the tail-flick test.
Compound
The average reaction time (s) at different time intervals after compound administration
Control
0.5 h
1 h
2 h
3 h
10
12
13
14
15
25
26
27
5.72 ꢀ 3.85
6.92 ꢀ 3.82
7.18 ꢀ 3.54
7.34 ꢀ 3.98
10.24 ꢀ 4.64
10.26 ꢀ 6.16
7.32 ꢀ 3.61
8.32 ꢀ 4.03
6.56 ꢀ 1.51
15.82 ꢀ 7.14ꢁꢁ
21.40 ꢀ 0.00ꢁ,ꢁꢁ
8.04 ꢀ 4.80
7.20 ꢀ 7.89
20.48 ꢀ 2.06ꢁꢁ
13.66 ꢀ 4.15
18.32 ꢀ 2.96ꢁꢁ
18.00 ꢀ 2.91
14.74 ꢀ 4.59
10.58 ꢀ 6.50
11.84 ꢀ 8.21
15.64 ꢀ 8.13
8.58 ꢀ 1.21
18.62 ꢀ 6.22ꢁꢁ
5.94 ꢀ 2.64
10.06 ꢀ 5.63
16.00 ꢀ 1.32
15.74 ꢀ 2.65
8.58 ꢀ 2.03
10.76 ꢀ 6.40
14.32 ꢀ 6.76
10.36 ꢀ 2.33
15.70 ꢀ 5.32ꢁꢁ
10.02 ꢀ 4.66
9.26 ꢀ 2.00
15.60 ꢀ 3.39
12.76 ꢀ 4.45
11.82 ꢀ 5.37
9.74 ꢀ 5.06
10.42 ꢀ 4.19
14.06 ꢀ 5.63ꢁꢁ
13.28 ꢀ 3.46
14.52 ꢀ 4.48
10.40 ꢀ 3.40
12.44 ꢀ 5.94
15.90 ꢀ 5.94
Morphine
ꢁ
Each value represents the mean ꢀ SD and all showed at least significant difference at p < 0.05 in comparison with the morphine
group and ꢁꢁp < 0.05 compared with the control value.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 455–462
1 h compound 12 was found to be more potent than the
reference drug. Moreover, compounds 15 exhibited promis-
ing analgesic activity in comparison to morphine at the 3 h
interval. Conversely, compounds 13 and 14 were moderately
active, whereas compounds 10, 25, 26, and 27 were poorly
active or practically inactive. Additionally, it indicates that
the oxadiazole group on 7a position of thebaine could affect
the activity. It seems that the compounds with N-methyl
group are better than that with N-tetrazole group in analgesic
activity.
for Grade 0 (minimal or no evidence of injury), Grade 1 (mild
injury, cytoplasmic vacuolization, focal nuclear pycnosis),
Grade 2 (moderate-severe injury, common nuclear pycnosis,
cytoplasmic hyper eosinophily), Grade 3 (severe necrosis,
hemorrhage, the presence of polymorphonuclear leukocytes
(PMNL) infiltration), respectively. These results show that the
drug 4 h after the damage to the liver. Similarly, with the
degree of liver damage paralleled shows the number of PMNL
cells in that region. The control group values, the number of
cells with the compound was similar in look PMNL. This
finding also supports the histopathology.
Acute toxicity (LD50
)
The median lethal dose (LD50) of the most active compound
12 was determined intraperitoneally (i.p.) in rat according to
reported procedures [10]. The animals got injection i.p. of a
certain grade. The acute study of compound 12 shows
that 187 mg/kg is a lethal dose. The LD50 for thebaine is
20 mg/kg if you consider that reduction of acute toxicity
of an acceptable level.
Statistical analysis
Statistical Package for the Social Sciences (SPSS, Chicago,
IL, USA) 12.0 program was used for statistical analysis.
Variations in plasma oxidative state parameters, erythrocyte
deformability between study groups were assessed by using
Kruskal–Wallis test. Bonferroni adjusted Mann–Whitney U
test was used after significant Kruskal–Wallis to determine
which group differs from the other. Results were expressed as
mean ꢀ SD. Statistical significance was set at p < 0.05 for all
analyses.
Histopathologic and biochemical analyses
The activities of ALT and GST markers for liver damage were
evaluated in rat liver tissues and serum. The activities in
different groups are presented in Tables 3 and 4. After i.p. Experimental
injection of the agent applied to the microscopic damage to
the liver tissue with the control group (morphine group) were
not use this drug results suggest that constitute damage after
4 h in the liver. The degree of liver damages sequential scale
General
All the reagents for syntheses were commercially available and
used without further purification or purified by standard
methods prior to use. Melting points were determined using
an Electrothermal 9100 apparatus (Thermo Fisher Scientific
Inc. in Great Britain), uncorrected. Reactions were monitored
by TLC, which was carried out on 0.25 mm Silica Gel F254 plates
(Acros) using either UV light, a 5% EtOH solution of H2SO4 with
heat as developing agent or a chamber with silica-gel saturated
with I2. All NMR spectra were recorded on a Bruker 400 (1H:
400 MHz, 13C: 100 MHz) NMR spectrometer (Bruker Corporation,
in Germany), in CDCl3 or (d6) DMSO. Chemical shift values were
reported in ppm relative to that for TMS used as an internal
reference standard, J in Hz. MS were obtained from Waters LCT
Premier XE LTOF (TOF MS) instruments (Waters Corporation,
Milford MA, USA). FTIR spectra were recorded on a Mattson
1000 spectrometer (Mattson Instruments, Baton Rouge, LA,
USA) using KBr pellets. The progress of reactions was monitored
Table 3. Histopathological evaluation of liver damage of scores and
PMNL values of compound 12.
Morphine
group
(n ¼ 5)
The study group
(compound
12, n ¼ 5)
p
Liver damage 0.40 ꢀ 0.54
0.50 ꢀ 0.54
7.17 ꢀ 3.37
–
0.785
PMNL
6.62 ꢀ 2.88
PMNL, polymorphonuclear leukocytes.
Each value represents the mean ꢀ SD.
Table 4. The results of measuring levels of Serum ALT, Liver ALT, serum GST and Liver of. compound 12.
Morphine group (n ¼ 5)
The study group (12) (n ¼ 5)
p
Liver damage
PMNL
ALT serum (IU/L)
ALT liver (IU/mg protein)
GST serum (IU/L)
GST liver (IU/mg protein)
0.40 ꢀ 0.54
6.62 ꢀ 2.88
0.50 ꢀ 0.54
7.17 ꢀ 3.37
–
0.785
0.111
0.662
0.413
0.004
53.15 ꢀ 8.25
522.68 ꢀ 81.27
71.15 ꢀ 23.57
824.90 ꢀ 68.47
64.50 ꢀ 9.11
493.30 ꢀ 103.29
81.26 ꢀ 17.43
574.78 ꢀ 160.49ꢁ
ALT, alanine aminotransferase; GST, glutathione S-transferase.
Each value represents the mean ꢀ SD and all showed at least significant difference at ꢁp < 0.05 in comparison with morphine group.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 455–462
Pharmacological Evaluation of Some Novel Thebaine Derivatives
459
by TLC using Silufol UV-254 plates from Merck (Darmstadt,
Germany). The products were isolated by column chromatog-
raphy on silica gel. The compounds 1–5, 10–13, 16, 17, 22, and 23
were synthesized using a published method. All the physical
and spectral data were in line with the previously reported
results [4, 11–14].
General procedure for the synthesis of compounds 10–15
A stirring mixture of the compounds 4–9 (0.5 mmol) in H3PO4
(6 mL) was heated at 908C for 3 h (TLC control). Then, the mixture
was allowed to cool to room temperature. After stirring for
an additional 30 min, the resulting solution was poured
into ice-cold water and made alkaline to pH 8 with NaOH
solution. The precipitated product was filtered, purified by
CC (chloroform/methanol, 9:1) and recrystallized from an appro-
priate solvent.
General procedure for the synthesis of compounds 4–9
A mixture of 6,14-endoethenotetrahydrothebaine-7a-carbohy-
drazide (3, 1 mmol) and a mol-equivalent amount of the benzoyl
chloride, phenyl isocyanate or phenyl isothiocyanate in toluene
was heated at 708C for 2 h (TLC control). Then, after evaporation
of toluene under reduced pressure, a solid appeared. This crude
product was recrystallized from an appropriate solvent to afford
the desired compound.
2-(6,14-Endoethenotetrahydrothebaine-7a-yl)-5-N-
phenylamin-1,3,4-thiadiazole 14
Recrystallization from ethanol; 82% yield; mp 274–2758C;
IR (n, cmꢂ1): 3204, 3076, 3038, 2971, 1620. 1H NMR (400 MHz,
DMSO-d6, d, ppm): 10.21 (s, 1H, NH), 7.62 (d, J ¼ 7.8 Hz, 2H, H-20),
7.38 (t, J ¼ 7.5 Hz, 2H, H-30), 7.04 (t, J ¼ 7.5 Hz, 1H, H-40), 6.70
(d, J ¼ 8.2 Hz, 1H, H-2), 6.60 (d, J ¼ 8.2 Hz, 1H, H-1), 5.77
(d, J ¼ 8.7 Hz, 1H, H-17), 5.71 (d, J ¼ 8.7 Hz, 1H, H-18), 4.97
(s, 1H, H-5b), 3.93 (dd, J ¼ 9.3 Hz, J ¼ 5.5 Hz, 1H, H-7b), 3.74
(s, 3H, 30-OCH3), 3.77 (s, 3H, 3-OCH3), 3.45 (s, 3H, 6-OCH3), 3.34
(m, 1H, H-8b), 3.28 (d, J ¼ 6.5 Hz, 1H, H-9a), 3.18 (d, J ¼ 18.6 Hz,
1H, H-10b), 2.51 (m, 1H, H-16eq), 2.48 (m, 1H, H-10a), 2.33
(s, 3H, N–CH3), 2.30 (m, 1H, H-16ax), 2.22 (m, 1H, H-15ax), 1.77
(m, 1H, H-15eq), 1.44 (dd, J ¼ 13.1 Hz, J ¼ 5.5 Hz, 1H, H-8a).
13C NMR (100 MHz, DMSO-d6, d, ppm): 22.2 C(10); 32.9 C(8);
35.2 C(15); 39.4 C(7); 43.2 C(14); 43.6 MeN; 45.5 C(16); 46.8
C(13); 51.1 MeO-C(6); 56.5 MeO-C(3); 59.6 C(9); 81.7 C(6); 93.1
C(5); 113.9 C(2); 117.7 C(20); 119.9 C(1); 122.1 C(40); 127.7 C(18);
128.6 C(11); 129.5 C(30); 134.5 C(12); 138.3 C(17); 141.2 C(3); 141.8
C(10); 147.8 C(4); 161.5 (S–C¼N); 164.8 (S–C¼N). HR-MS: 515.2057
([MþH]þ, C29H31N4O3Sþ, calc. 515.2117).
1-[(6,14-Endoethenotetrahydrothebaine-7a-yl)-carbonyl]-
4-phenylthiosemicarbazide 8
Recrystallization from methanol; 78% yield; mp 169–1708C;
IR (n, cmꢂ1): 3493, 3307, 3260, 3061, 3019, 2980, 1738, 1673,
1329. 1H NMR (400 MHz, DMSO-d6, d, ppm): 10.03 (s, 1H, NH), 9.78
(s, 1H, NH), 8.92 (br, 1H, NH), 7.49 (d, J ¼ 7.7 Hz, 2H, H-20), 7,35
(t, J ¼ 7.7 Hz, 2H, H-30), 7.16 (t, J ¼ 6.7 Hz, 1H, H-40), 6.63
(d, J ¼ 8.2 Hz, 1H, H-2), 6.52 (d, J ¼ 8.2 Hz, 1H, H-1), 5.63
(d, J ¼ 8.7 Hz, 1H, H-18); 5,51 (d, J ¼ 8.6 Hz, 1H, H-17), 4.61
(s, 1H, H-5b), 3.70 (s, 3H, 3-OCH3), 3.49 (s, 3H, 6-OCH3), 3.20
(d, J ¼ 5.5 Hz, 1H, H-9a), 3.12 (d, J ¼ 18.6 Hz, 1H, H-10b), 2.92
(m, 1H, H-8b), 2.80 (m, 1H, H-7b), 2.51 (m, 1H, H-16eq), 2.43
(dd, J ¼ 18.6 Hz, J ¼ 6.0 Hz, 1H, H-10a), 2.30 (s, 3H, N–CH3),
2.26 (m, 1H, H-16ax), 1.95 (m, 1H, H-15ax), 1.75 (m, 1H,
H-15eq), 1.38 (dd, J ¼ 11.1 Hz, J ¼ 5.4 Hz, 1H, H-8a). 13C NMR
(100 MHz, DMSO-d6, d, ppm): 21.7 C(10); 30.6 C(8); 32.9 C(15);
40.8 C(7); 42.6 C(14); 43.6 MeN; 45.0 C(16); 46.7 C(13); 52.3
MeO-C(6); 56.4 MeO-C(3); 59.0 C(9); 81.1 C(6); 92.8 C(5);
113.4 C(2); 118.8 C(20); 119.4 C(1); 122.5 C(40); 127.1 C(18);
128.2 C(11); 129.2 C(30); 133.7 C(12); 135.1 C(17); 139.9 C(10);
141.2 C(3); 147.4 C(4); 171.9 (C¼S); 180.0 (C¼O). HR-MS:
533.2222 ([MþH]þ, C29H33N4O4S, calc. 533.2223).
2-(6,14-Endoethenotetrahydrothebaine-7a-yl)-5-N-40-
methoxyphenylamin-1,3,4-thiadiazole 15
Recrystallization from ethanol; 72% yield; mp 194–1968C;
IR (n, cmꢂ1): 3238, 3052, 3056, 2927, 1600. 1H NMR (400 MHz,
DMSO-d6, d, ppm): 9.85 (s, 1H, NH), 7.38 (d, J ¼ 8.3 Hz, 2H, H-20),
6.82 (d, J ¼ 8.3 Hz, 2H, H-30), 6.55 (d, J ¼ 8.1 Hz, 1H, H-2), 6.44
(d, J ¼ 8.1 Hz, 1H, H-1), 5.59 (d, J ¼ 8.3 Hz, 1H, H-17), 5.54
(d, J ¼ 8.3 Hz, 1H, H-18), 4.81 (s, 1H, H-5b), 3.95–3.96 (m, 1H,
H-7b), 3.63 (s, 3H, 40-OCH3), 3.62 (s, 3H, 3-OCH3), 3.29 (s, 3H,
6-OCH3), 3.13–3.15 (m, 2H, H-8b, H-9a), 3.05 (d, J ¼ 18.2 Hz, 1H,
H-10b), 2.35–2.42 (m, 2H, H-16eq, H-10a), 2.21 (s, 3H, N–CH3), 1.27–
2.18 (m, 4H, H-16ax, H-15ax, H-15eq, H-8a). 13C NMR (100 MHz,
DMSO-d6, d, ppm): 22.2 C(10); 32.9 C(8); 35.2 C(15); 38.7 C(7);
43.2 C(14); 43.6 MeN; 45.5 C(16); 46.8 C(13); 51.1 MeO-C(6); 55.7
MeO-C(40); 56.5 MeO-C(3); 59.6 C(9); 80.7 C(6); 91.1 C(5); 113.9 C(2);
114.7 C(30); 119.6 (20); 119.9 C(1); 127.8 C(18); 128.6 C(11);
134.5 C(12); 134.8 C(10); 138.2 C(17); 141.8 C(3); 147.8 C(4);
154.8 C(40), 160.7 (S–C¼N); 165.4 (S–C¼N). HR-MS: 545.2203
([MþH]þ, C30H33N4O4Sþ, calc. 525.2223).
1-[(6,14-Endoethenotetrahydrothebaine-7a-yl)-carbonyl]-
4-(40-methoxy)-phenylthiosemicarbazide 9
Recrystallization from ethanol; 88% yield; mp 164–1658C;
IR (n, cmꢂ1): 3499, 3311, 3263, 3061, 3030, 2983, 1676, 1632,
1327. 1H NMR (400 MHz, DMSO-d6, d, ppm): 10.00 (s, 1H, NH), 9.68
(s, 1H, NH), 8.84 (br, 1H, NH), 7.31 (d, J ¼ 8.9 Hz, 2H, H-20), 6.91
(d, J ¼ 8.9 Hz, 2H, H-30), 6.63 (d, J ¼ 8.2 Hz, 1H, H-2), 6.52
(d, J ¼ 8.2 Hz, 1H, H-1), 5.62 (d, J ¼ 8.7 Hz, 1H, H-18), 5.51
(d, J ¼ 8.7 Hz, 1H, H-17), 4.59 (s, 1H, H-5b), 3.79 (s, 3H, 40-OCH3),
3.70 (s, 3H, 3-OCH3), 3.47 (s, 3H, 6-OCH3), 3.21 (d, J ¼ 6.1 Hz, 1H,
H-9a), 3.13 (d, J ¼ 18.6 Hz, 1H, H-10b), 2.51–2.89 (m, 3H, H-8b,
H-7b, H-16eq), 2.43 (dd, J ¼ 18.6 Hz, J ¼ 6.1 Hz, 1H, H-10a), 2.30
(s, 3H, N–CH3), 1.76–2.25 (m, 1H, H-16ax, H-15ax, H-15eq), 1.38
(dd, J ¼ 11.8 Hz, J ¼ 5.8 Hz, 1H, H-8a). 13C NMR (100 MHz,
DMSO-d6, d, ppm): 22.3 C(10); 30.2 C(8); 33.4 C(15); 41.4 C(7); 43.1
C(14); 43.5 MeN; 45.5 C(16); 47.3 C(13); 53.1 MeO-C(6); 56.5 MeO-C(3);
56.7 MeO-C(40); 59.5 C(9); 81.6 C(6); 93.6 C(5); 113.9 C(20); 114.0 C(2);
114.5 C(30); 119.9 C(1); 126.9 C(18); 128.8 C(11); 132.1 C(10); 134.2
C(12); 136.1 C(17); 141.7 C(3); 147.9 C(4); 157.9 C(40); 171.2 (C¼S);
181.0 (C¼O). HR-MS: 563.2333 ([MþH]þ, C30H35N4O5Sþ, calc.
563.2328).
General procedure for the synthesis of compounds 16–21
A solution of corresponding compounds 10–15 (0.5 mmol) and
0.1 g (1 mmol) cyanogen bromide in 10 mL dry chloroform were
refluxed to obtain N-CN compounds for 24 h by monitoring with
TLC. The cooled reaction mixture was evaporated, and the res-
idue was purified by column chromatography on silica gel using
ethyl acetate/petrol ether (2:1) as eluent. The residue was recrys-
tallized from an appropriate solvent.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 455–462
2-(6,14-Endoetheno-6,7,8,14-tetrahydrothebaine-N-
cyano-7a-yl)-5-N-phenylamine-1,3,4-oxadiazole 18
2-(6,14-Endoetheno-6,7,8,14-tetrahydrothebaine-N-
cyano-7a-yl)-5-N-(40-methoxyphenylamine)-1,3,4-
thiadiazole 21
Recrystallized from methanol/ethyl acetate, 43% yield; mp 174–
1758C; IR (n, cmꢂ1): 3059, 2972, 2210. 1H NMR (400 MHz, DMSO-
d6, d, ppm): 9.77 (s, 1H, NH); 7.72 (d, J ¼ 7.4 Hz, 2H, H-20), 7.59
(t, J ¼ 7.1 Hz, 2H, H-30), 7.02 (t, J ¼ 7.1 Hz, 1H, H-40); 6.71
(d, J ¼ 8.2 Hz, 1H, H-2); 6.65 (d, J ¼ 8.2 Hz, 1H, H-1); 5.69
(d, J ¼ 8.7 Hz, 1H, H-17); 5.64 (d, J ¼ 8.7 Hz, 1H, H-18); 4.95
(s, 1H, H-5b); 4.15 (d, J ¼ 5.6 Hz, 1H, H-9a); 4.07–4.11 (m, 1H,
H-7b); 3.73 (s, 3H, 3-OCH3); 3.53 (s, 3H, 6-OCH3); 3.41–3.45 (m, 1H,
H-16eq); 3.17–3.31 (m, 2H, H-16ax, H-10a); 3.15 (d, J ¼ 18.6 Hz, 1H,
H-10b); 2.97 (dd, J ¼ 12.2 Hz, J ¼ 9.1 Hz, 1H, H-8b); 2.37–2.42 (m, 1H,
H-15ax); 1.85–1.91 (m, 2H, H-15eq, H-8a). 13C NMR (100 MHz,
DMSO-d6, d, ppm): 30.6 C(15); 31.3 C(8); 31.8 C(10); 33.6 C(7); 40.4
C(14); 41.5 C(16); 46.3 C(13); 51.4 CH3O-C(6); 56.1 CH3O-C(3); 57.2 C(9);
81.5 C(6); 90.6 C(5); 114.4 C(2); 118.9 (N-CN); 120.3 C(1); 122.7 C(40);
123.5 C(10); 126.5 C(12); 128.3 C(17); 128.2 C(20); 129.7 C(30); 132.6
C(11); 135.2 C(18); 142.3 C(3); 148.2 C(4); 164.4 (C¼N); 169.3 (C¼N).
HR-MS: 509.2055 ([MþH]þ, C29H28N5O4þ, calc. 509.2063).
Recrystallized from methanol/ethyl acetate, 53% yield; mp 159–
1608C; IR (n, cmꢂ1): 3049, 2987, 2213. 1H NMR (400 MHz, DMSO-
d6, d, ppm): 9.61 (s, 1H, NH); 7.59 (d, J ¼ 7.7 Hz, 2H, H-20), 6.96
(d, J ¼ 7.7 Hz, 2H, H-30), 6.67 (d, J ¼ 8.2 Hz, 1H, H-2); 6.61
(d, J ¼ 8.2 Hz, 1H, H-1); 5.70 (d, J ¼ 8.8 Hz, 1H, H-17); 5.58
(d, J ¼ 8.8 Hz, 1H, H-18); 4.91 (s, 1H, H-5b); 4.11 (m, 2H, H-7b,
H-9a); 3.79 (s, 3H, 40-OCH3); 3.70 (s, 3H, 3-OCH3); 3.49 (s, 3H,
6-OCH3); 3.13–3.38 (m, 3H, H-16eq, H-16ax, H-10a); 3.11
(d, J ¼ 18.7 Hz, 1H, H-10b); 2.91 (m, 1H, H-8b); 2.33–2.41
(m, 1H, H-15ax); 1.83–1.89 (m, 2H, H-15eq, H-8a). 13C NMR
(100 MHz, DMSO-d6, d, ppm): 30.9 C(15); 31.7 C(8); 32.5 C(10);
33.7 C(7); 40.5 C(14); 41.7 C(16); 46.6 C(13); 51.6 CH3O-C(6); 56.0
CH3O-C(3); 57.6 C(9); 81.7 C(6); 90.3 C(5); 114.3 C(2); 118.5 C(30);
119.1 (N-CN); 120.5 C(1); 126.3 C(12); 128.5 C(17); 129.5 C(20);
129.9 C(10); 132.7 C(11); 135.5 C(18); 142.3 C(3); 148.1 C(4);
161.5 C(40); 167.1 (–C¼N); 169.3 (–C¼N). HR-MS: 556.2015
([MþH]þ, C30H30N5O4Sþ, calc. 556.2019).
2-(6,14-Endoetheno-6,7,8,14-tetrahydrothebaine-N-
cyano-7a-yl)-5-N-(40-methoxyphenylamine)-1,3,4-
oxadiazole 19
General procedure for the synthesis of compounds 22–27
To a solution of corresponding compounds 16–21 (0.3 mmol)
in 5 mL DMF were added 0.19 g (3 mmol) NaN3 and 0.15 g
(3 mmol) NH4Cl. The mixture was heated was for 4 h under
reflux, and the reaction completion was checked by TLC. After
refluxing, the reaction mixture was cooled to room temperature
and evaporated under reduced pressure (20 mbar). The residue
product was extracted with ethyl acetate/water (30:10 mL).
The organic layer was dried over MgSO4 and the solvent
evaporated. The crude product was recrystallized from an appro-
priate solvent.
Recrystallized from methanol/ethyl acetate, 49% yield; mp 199–
2008C; IR (n, cmꢂ1): 3087, 2978, 2215. 1H NMR (400 MHz, DMSO-
d6, d, ppm): 9.79 (s, 1H, NH); 7.61 (d, J ¼ 7.2 Hz, 2H, H-20), 7.09
(d, J ¼ 7.2 Hz, 2H, H-30), 6.69 (d, J ¼ 8.2 Hz, 1H, H-2); 6.63
(d, J ¼ 8.2 Hz, 1H, H-1); 5.67 (d, J ¼ 8.8 Hz, 1H, H-17); 5.61
(d, J ¼ 8.8 Hz, 1H, H-18); 4.93 (s, 1H, H-5b); 4.13 (d, J ¼ 5.6 Hz,
1H, H-9a); 4.05–4.09 (m, 1H, H-7b); 3.77 (s, 3H, 40-OCH3); 3.71
(s, 3H, 3-OCH3); 3.52 (s, 3H, 6-OCH3); 3.39–3.42 (m, 1H, H-16eq);
3.13–3.32 (m, 2H, H-16ax, H-10a); 3.14 (d, J ¼ 18.6 Hz, 1H, H-10b);
2.95 (m, 1H, H-8b); 2.35–2.40 (m, 1H, H-15ax); 1.86–1.92 (m, 2H,
H-15eq, H-8a). 13C NMR (100 MHz, DMSO-d6, d, ppm): 30.6 C(15);
31.5 C(8); 32.3 C(10); 33.9 C(7); 40.6 C(14); 41.7 C(16); 46.5 C(13);
51.5 CH3O-C(6); 56.1 CH3O-C(3); 57.5 C(9); 81.5 C(6); 90.5 C(5);
112.2 C(30); 114.4 C(2); 117.7 (N-CN); 120.3 C(1); 122.5 C(20); 126.5
C(12); 128.3 C(17); 129.9 C(10); 132.6 C(11); 135.3 C(18); 142.4 C(3);
148.0 C(4); 158.7 C(40); 166.7 (–C¼N); 169.1 (–C¼N). HR-MS:
540.2231 ([MþH]þ, C30H30N5O5þ, calc. 540.2245).
2-[N-(Tetrazol-1H-5-yl)-6,14-endoetheno-6,7,8,14-
tetrahydrothebaine-7a-yl]-5-N-4-phenylamine-1,3,4-
oxadiazole (24)
Recrystallized from methanol/ethyl acetate, 73% yield; mp 202–
2038C; IR (n, cmꢂ1): 3383–3111, 3071, 2991. 1H NMR (400 MHz,
DMSO-d6, d, ppm): 9.76 (s, 1H, NH); 7.59 (d, J ¼ 7.4 Hz, 2H, H-20),
7.08–7.20 (m, 4H, H-30, H-40); 6.59 (d, J ¼ 8.2 Hz, 1H, H-2); 6.51
(d, J ¼ 8.2 Hz, 1H, H-1); 5.74 (d, 1H, J ¼ 8.8 Hz, H-17); 5.59 (d, 1H,
J ¼ 8.8 Hz, H-18); 4.87 (s, 1H, H-5b); 4.59 (d, J ¼ 6.4 Hz, 1H, H-9a);
3.86–3.90 (m, 1H, H-7b); 3.74–3.77 (m, 1H, H-16eq); 3.71 (s, 3H,
3-OCH3); 3.49 (s, 3H, 6-OCH3); 3.13–3.25 (m, 2H, H-16ax, H-10a);
2.84 (d, J ¼ 18.7 Hz, 1H, H-10b); 2.68–2.70 (m, 1H, H-8b); 2.27–
2.31 (m, 1H, H-15ax,); 1.92–1.95 (m, 1H, H-15eq); 1.77 (m, 1H,
H-8a). 13C NMR (100 MHz, DMSO-d6, d, ppm): 31.1 C(10); 31.6 C(8);
31.7 C(15); 33.8 C(7); 40.5 C(14); 42.2 C(16); 47.3 C(13); 51.2
CH3O-C(6); 55.4 CH3O-C(3); 56.9 C(9); 81.1 C(6); 91.0 C(5); 112.1
(N¼C–N); 114.4 C(2); 115.9 C(20); 120.4 C(40); 123.7 C(1); 123.5
C(11); 125.9 C(18); 133.1 C(30); 135.7 C(12); 135.9 C(17); 138.1 C(10);
141,0 C(3); 149.3 C(4); 161.1 (–C¼N); 163.9 (–C¼N). HR-MS:
553.2319 ([MꢂH]þ, C29H29N8O4þ, calc. 553.2312).
2-(6,14-Endoetheno-6,7,8,14-tetrahydrothebaine-N-
cyano-7a-yl)-5-N-phenylamine-1,3,4-thiadiazole 20
Recrystallized from methanol/ethyl acetate, 39% yield; mp 212–
2138C; IR (n, cmꢂ1): 3084, 2979, 2212. 1H NMR (400 MHz, DMSO-
d6, d, ppm): 9.81 (s, 1H, NH); 7.59–7.67 (m, 4H, H-20, H-30), 6.93
(t, J ¼ 7.3 Hz, 1H, H-40); 6.69 (d, J ¼ 8.2 Hz, 1H, H-2); 6.63
(d, J ¼ 8.2 Hz, 1H, H-1); 5.67 (d, J ¼ 8.8 Hz, 1H, H-17); 5.62
(d, J ¼ 8.8 Hz, 1H, H-18); 4.93 (s, 1H, H-5b); 4.21 (d, J ¼ 5.6 Hz,
1H, H-9a); 4.15 (dd, J7b,8b ¼ 9.3 Hz, J7b,8a ¼ 6.1 Hz, 1H, H-7b);
3.72 (s, 3H, 3-OCH3); 3.53 (s, 3H, 6-OCH3); 3.18–3.47 (m, 3H,
H-16eq, H-16ax, H-10a); 3.13 (d, J ¼ 18.7 Hz, 1H, H-10b); 2.97
(m, 1H, H-8b); 2.35–2.41 (m, 1H, H-15ax); 1.88–1.93 (m, 2H,
H-15eq, H-8a). 13C NMR (100 MHz, DMSO-d6, d, ppm): 30.3
C(15); 31.5 C(8); 31.7 C(10); 33.5 C(7); 40.2 C(14); 41.6 C(16);
46.1 C(13); 51.3 CH3O-C(6); 56.2 CH3O-C(3); 57.3 C(9); 81.6 C(6);
90.5 C(5); 114.2 C(2); 118.7 (N-CN); 120.2 C(1); 120.8 C(10); 126.3
C(40); 126.5 C(12); 127.1 C(20); 128.6 C(17); 132.4 C(11); 134.3 C(30);
135.3 C(18); 142.7 C(3); 148.5 C(4); 164.3 (C¼N); 169.5 (C¼N).
HR-MS: 526.1911 ([MþH]þ, C29H28N5O3Sþ, calc. 526.1913).
2-[N-(Tetrazol-1H-5-yl)-6,14-endoetheno-6,7,8,14-
tetrahydrothebaine-7a-yl]-5-N-(4-methoxyphenylamine)-
1,3,4-oxadiazole (25)
Recrystallized from methanol/ethyl acetate, 69% yield; mp198–
1998C; IR (n, cmꢂ1): 3391–3124, 3064, 2989. 1H NMR (400 MHz,
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Arch. Pharm. Chem. Life Sci. 2013, 346, 455–462
Pharmacological Evaluation of Some Novel Thebaine Derivatives
461
DMSO-d6, d, ppm): 9,91 (s, 1H, NH); 7.81 (d, J ¼ 8.8 Hz, 2H, H-20);
7.01 (d, J ¼ 8.8 Hz, 2H, H-30); 6.68 (d, J ¼ 8.1 Hz, 1H, H-2); 6.52
(d, J ¼ 8.1 Hz, 1H, H-1); 5.77 (d, 1H, J ¼ 8.7 Hz, H-17); 5.61 (d, 1H,
J ¼ 8.7 Hz, H-18); 4.89 (s, 1H, H-5b); 4.61 (d, J ¼ 6.8 Hz, 1H, H-9a);
3.87–3.91 (m, 1H, H-7b); 3.82 (s, 3H, 40-OCH3); 3.74–3.79 (m, 1H,
H-16eq); 3.72 (s, 3H, 3-OCH3); 3.45 (s, 3H, 6-OCH3); 3.11–3.21
(m, 2H, H-16ax, H-10a); 2.82 (d, J ¼ 18.6 Hz, 1H, H-10b); 2.67–
2.70 (m, 1H, H-8b); 2.25–2,29 (m, 1H, H-15ax,); 1.89–1.92 (m, 1H,
H-15eq); 1.71 (dd, J ¼ 12.2 Hz, J ¼ 6.1 Hz, 1H, H-8a). 13C NMR
(100 MHz, DMSO-d6, d, ppm): 31.0 C(10); 31.4 C(8); 31.6 C(15);
33.9 C(7); 40.5 C(14); 42.1 C(16); 47.4 C(13); 51.2 CH3O-C(6); 55.4
CH3O-C(3); 55.8 CH3O-C(40); 56.6 C(9); 81.3 C(6); 90.7 C(5); 112.1
(N¼C–N); 114.6 C(2); 112.7 C(30); 119.4 C(10); 123.6 C(1); 123.7
C(11); 125.8 C(18); 128.1 C(20); 135.7 C(12); 135.8 C(17); 141,1 C(3);
149.0 C(4); 161.6 (–C¼N); 163.5 (–C¼N); 166.3 C(40). HR-MS:
583.2430 ([MꢂH]þ, C30H31N8O5þ, calc. 583.2417).
Pharmacological screening
Materials and methods
Male adult Wistar rats were obtained from the Gazi University
Laboratory Animals Breeding and Experimental Research Center.
The test compounds and reference drug were suspended in 5%
sodium carboxymethylcellulose (NaCMC) in normal saline.
Animals were housed in separate cages, 5 animals each, in
temperature-controlled rooms at 25 ꢀ 18C. Animals were
allowed free access to food and water and maintained at a
12 h light/dark cycle.
Analgesic activity
The analgesic activity of 10, 12, 13, 14, 15, 25, 26, and 27 were
determined in rat using the hot-plate and tail-flick method [15] in
comparison with morphine as a reference drug. Male Wistar rats
(180–250 g) were placed on a hot-plate apparatus for testing and
the surface temperature was maintained at 55 ꢀ 0.58C. The
reaction time in seconds was taken as the time period from
the instant the animal reached the hot plate until the moment
the animal licked its hind paw or jumped out within a Plexiglas
cylinder placed on the hot plate. The reaction time was taken as
the end point and the increase in hot plate latency was taken as a
measure of the analgesic activity. Animals were randomly div-
ided into groups of five rats each and each animal was used once.
Control values of rats were given morphine and test compounds
were measured and recorded before drug injection. Thirty
minutes later the animals were injected intraperitoneally.
Solutions or suspensions of the test compounds in 5% NaCMC
were injected i.p. at a dose of 10.0 mg/kg and the reference drug
(Morphine animals) were similarly treated with 0.9% NaCl
injected i.p. at a dose of 2.5 mg/kg. Analgesic effects were eval-
uated after waiting for 30 min to the drug absorption. Testing
was done at 0.5, 1, 2, and 3 h after the injection. Mean ꢀ SD was
evaluated for each group. The experimental results are listed
in Tables 1 and 2.
2-[N-(Tetrazol-1H-5-yl)-6,14-endoetheno-6,7,8,14-
tetrahydrothebaine-7a-yl]-5-N-phenylamine-1,3,4-
thiadiazole 26
Recrystallized from methanol/ethyl acetate, 73% yield; mp: 231–
.
2328C; IR (n, cmꢂ1): 3950–3120, 3059, 2976 cmꢂ1 1H NMR
(400 MHz, DMSO-d6, d, ppm): 9,76 (s, 1H, NH); 7.45
(d, J ¼ 7.9 Hz, 2H, H-20), 7.01–7.12 (m, 4H, H-30, H-40); 6.62
(d, J ¼ 8.2 Hz, 1H, H-2); 6.54 (d, J ¼ 8.2 Hz, 1H, H-1); 5.78
(d, 1H, J ¼ 8.7 Hz, H-17); 5.61 (d, 1H, J ¼ 8.7 Hz, H-18); 4.78
(s, 1H, H-5b); 4.57 (d, J ¼ 6.8 Hz, 1H, H-9a); 3.91 (dd,
J ¼ 9.1 Hz, J ¼ 5.8 Hz, 1H, H-7b), 3.74–3.79 (m, 1H, H-16eq);
3.71 (s, 3H, 3-OCH3); 3.50 (s, 3H, 6-OCH3); 3.15–3.21 (m, 2H,
H-16ax, H-10a); 2.91 (d, J ¼ 18.9 Hz, 1H, H-10b); 2.70–2.73
(m, 1H, H-8b); 2.28–2,33 (m, 1H, H-15ax,); 1.91–1.94 (m, 1H,
H-15eq); 1.76 (m, 1H, H-8a). 13C NMR (100 MHz, DMSO-d6,
d, ppm): 31.0 C(10); 31.7 C(8); 31.8 C(15); 33.7 C(7); 40.4 C(14);
42.4 C(16); 47.8 C(13); 51.1 CH3O-C(6); 55.4 CH3O-C(3); 56.7 C(9);
81.3 C(6); 91.1 C(5); 111.3 C(20); 112.14 (N¼C–N); 114.3 C(2); 123.3
C(40); 123.7 C(1); 123.9 C(11); 125.9 C(18); 135.5 C(12); 135.8 C(17);
138.4 C(10); 139.2 C(30); 141,1 C(3); 149.4 C(4); 160.6 (–C¼N); 163.4
(–C¼N). HR-MS: 569.2089 ([MꢂH]þ, C29H29N8O3Sþ, calc. 569.2083).
Determination of acute toxicity (LD50)
The median lethal dose (LD50) of the most active compound 12
was determined in rat. A group of male adult Wistar rats of
five animals (180–250 g) was injected i.p. at a certain grade.
The percentage of mortality was determined 72 h after
injection. Computation of LD50 was processed by an up and
down method [10].
2-[N-(Tetrazol-1H-5-yl)-6,14-endoetheno-6,7,8,14-
tetrahydrothebaine-7a-yl]-5-N-(4-methoxyphenylamine)-
1,3,4-thiadiazole 27
Recrystallized from methanol/ethyl acetate, 77% yield; mp: 155–
1568C; IR (n, cmꢂ1): 3381–3103, 3041, 2983. 1H NMR (400 MHz,
DMSO-d6, d, ppm): 9,81 (s, 1H, NH); 7.77 (d, J ¼ 8.8 Hz, 2H, H-20);
6.99 (d, J ¼ 8.8 Hz, 2H, H-30); 6.67 (d, J ¼ 8.2 Hz, 1H, H-2); 6.52
(d, J ¼ 8.2 Hz, 1H, H-1); 5.74 (d, 1H, J ¼ 8.7 Hz, H-17); 5.63 (d, 1H,
J ¼ 8.7 Hz, H-18); 4.84 (s, 1H, H-5b); 4.60 (d, J ¼ 6.4 Hz, 1H, H-9a);
3.86–3.79 (m, 4H, H-7b, 40-OCH3); 3.71–3.77 (m, 1H, H-16eq); 3.70
(s, 3H, 3-OCH3); 3.43 (s, 3H, 6-OCH3); 3.14–3.22 (m, 2H, H-16ax,
H-10a); 2.67–2.75 (m, 2H, H-10b, H-8b); 2.23–2,30 (m, 1H, H-15ax,);
1.88–1.93 (m, 1H, H-15eq); 1.69 (dd, J ¼ 12.4 Hz, J ¼ 6.2 Hz, 1H,
H-8a). 13C NMR (100 MHz, DMSO-d6, d, ppm): 31.3 C(10); 31.7 C(8);
31.6 C(15); 33.8 C(7); 40.7 C(14); 42.3 C(16); 47.6 C(13); 51.5
CH3O-C(6); 55.1 CH3O-C(3); 55.2 CH3O-C(40); 56.5 C(9); 81.8 C(6);
90.2 C(5); 114.3 (N¼C–N); 116.1 C(2); 116.7 C(30); 122.1 C(10);
123.8 C(1); 124.9 C(11); 126.2 C(18); 129.6 C(20); 135.1 C(12);
135.4 C(17); 141,3 C(3); 147.4 C(4); 161.5 (–C¼N); 164.3 (–C¼N);
166.6 C(40). HR-MS: 599.2198 ([MꢂH]þ, C30H31N8O4Sþ, calc.
599.2189).
Histopathological and biochemical evaluation
Histological assessment was performed at the pathology clinic of
the Ministry of Health, Yildirim Beyazit Training and Research
Hospital. The study was carried out on male adult Wistar rats
weighing 180–250 g. The histopathological studies were carried
out by a reported method [16]. The rats were sacrificed under
light ether anesthesia after 24 h of the last dosage, the livers
were removed and washed with normal saline and stored in
formalin solution. Sections of 5–6 mm in thickness were cut,
stained with hematoxylin and eosin and then studied under a
light microscope. After the treatment blood was obtained from
all groups of rats by puncturing the retro-orbital plexus. Blood
samples were allowed to clot for 45 min at room temperature
and serum was separated by centrifugation at 3000 rpm for
5 min and analyzed for ALT and GST enzymes and protein
analyses. The GST activities were determined by the method of
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S. Yavuz et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 455–462
Habig et al. [16] and ALT analyses were performed on the Hitachi
902 Automatic Analyzer using the adapted reagents from Roche
(Germany). The results were expressed for serum and liver hom-
ogenate (IU/L) and IU/mg protein, respectively.
[6] J. W. Lewis, S. M. Husbands, J. Med. Chem. 2004, 43, 139–141.
[7] J. W. Lewis, S. M. Husbands, Curr. Pharm. Design. 2004, 10 (7),
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[8] S. Yavuz, Y. Yıldırır, Helv. Chim. Acta. 2010, 93, 2406–2418.
[9] J. W. Lewis, K. W. Bentley, A. Cowan, Ann. Rev. Pharrnacol.
1971, 11, 241–270.
The authors gratefully acknowledge financial support from the Scientific
and Technical Research Council of Turkey (TUBITAK, Project No. 107T676).
We also thank the Turkish Grain Board (TMO) for the supply of thebaine.
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