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Analytical Profile of Azintamide
ANALYTICAL PROFILE OF AZINTAMIDE
Ezzat M. Abdel-Moety and Hamad A . Al-Khamees
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P . O . Box 2457, Riyadh-11451, Saudi Arabia.
ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 18 1
Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved
EZZAT M. ABDEL-MOETY AND HAMAD A. AL-KHAMEES
2
CONTENTS
1.
I NTRODUCT0R.Y
2.
DESCRIPTION 2-1. Name 2-2. Formulae 2-3. The Chemical Abstract Registry (CAS) Number 2-4. Appearance, C,olor, Odor and Taste 2-5. Physical Characteristics 2-6. Crystal Characteristics 2 - 7 . Spectral Characterization
3.
SYNTHESIS
4.
PHARMACOLOGY
5.
THERAPEUTIC CATEGORATION AND USES 5-1. Categoration 5-2. Uses
6.
TOXICOLOGY
7.
STABILITY AND DEGRADATION
8.
PHARMACOKINETICS 8-1. Biotransformation 8-2. Absorption 8-3. Excretion
9.
METHODS OF ANALYSIS 9-1. Qualitative Methods 9-2. Quantitative Methods ACKNOWLEDGEMENT REFERENCES
3
AZINTAMIDE
1.
INTRODUCTORY Azintamide is a true potent choleretic drug, which is totally synthesized in 1 9 5 9 . The drug has the registered trade name OragallinB In spite of the potent choleretic activity, with moderate cholepoietic action, and its wide therapeutic applications in different conditions and countries, no detailed informations about its physical, chemical, clinical, and bioavailability characteristics have been yet collectively summarized in simple presentation. The present Analytical Profile is an effort in this direction.
.
2.
DESCRIPTION 2-1.
Names
2.11. Chemical: thiol-N,N-diethylacetamide.
Other
chemical
names
2-[(6-Chloro-3-pyridazinyl)
are,
N,N-diethyl-2-[6-(3-
chloropyridaziny1)thiolacetamide; N,N-diethyl-2-[6-(3chloropyridaziny1)-mercapto]acetamide; and (3-chloro-6pyridaziny1thio)acetic acid diethylamide ( 1 ) .
2.12. Properioritg: Oragallin, Ora-gallin, and ST 9067. Azintamide has been registered under the trade name Oragallin 8 for Osterreichische Stickstoffwerke, AG, Linz/Donau - Austria.
2-2.
Formula and Molecular Weight
CI
[CioHi4ClN30S (259.77)]
EZZAT M. ABDEL-MOETY AND HAMAD A . AL-KHAMEES
4
2-3.
The Chemical Abstract Registry (CAS) Number: [ 1830-32-61-
2-4.
Appearance. Color, Odor, and Taste
Microcrystalline, white, odorless powder with bitter taste. 2-5.
Physical Characteristics 2-51.
Melting Range
The melting of azintamide was carried out at a heating rate of 1"C.min-1 on a Kofler hot-stage microscope. Table 1: Melting point and range of azintamide* Start temperature ('C)
90
Melting range
Mid-point
("C)
("C)
Literature
t 'C)
95.0-97.0 (2.0)
96.0
98-100 (2)
95.5-97.5 (2.0,
96.5
97-98 ( ~ 3 )
*Sample from Bsterreichishe Stickstoffwerke, AG, Linz/DonauAustria, BN: 23540/524699 - all values ( ' C ) are uncorrected. 2-52. Differentional Thermal Scanning (DSC) The DSC-curve was obtained on a DuPont TAto a data processing unit. Figure 1 shows the DSC-curve of azintamide. The running was between 50-150°C at heating rate of 10"C.min-1. The heat of activation and the purity of the sample was determined using purity program. 9900 Thermal Analyzer attached
2-53.
Solubility
Azintamide is freely soluble in benzene, chloroform, ethyl acetate and acetone, its solubility in water is 5 mg.ml-1 (1).
Sample : AZII~ITAMICJE-8 Size : 4 . 6 0 mg :lethod : DSC 50 TO 150 Comment:
@10 C/M.
DSC
Run Date:
02/11/88
19: 13
97.60 097.59
-1
-z
\ x
-
D
97.58
O
D1
L
r
:: LL
-
-u 97.97
-2-
4J 3
-3-
21
I m
I -4
-
0 1
Purity : Melting P t :
Oepresslan : Oelta H : Corrcctlon : I.101. I l e l g h t :
c e l l const : -5 - O n s e t S l a p c :
97.55
100.01 Mole X 9 7 . 5 'C -O.OOn'C 3 0 . 6 kJ/molB 1 . 0 7 x: 259.8 g / ~ o l e
1.283
-7.90
mN/'C
nn
-6.
,
50
.
0
',
60
Fiq. 1 :
.
, 10' .
70
I
80
a*
2 I-
37.55
37.54
J
T a t a l Ar.ea/Pdrt i d 1 A r e a 20 30 40
.
L
9b id0 Temperature
110
('Cl
[he d i f f e r e n t i a - t Scanning
50
120
140
L L J I - V-cOSC) ~
60
140
37.33 120 PURITY V l . l . 4
of fizintnmide.
EZZAT M. ABDEL-MOETY AND HAMAD A. AL-KHAMEES
6
Optical Rotation
2-54.
Azintamide
species. 2-6.
is
an
optically-inactive
Crystal Characteristics Crystallization
2-61.
acetone (1). 2-62.
Azintamide
is readily crystallizable from
Crystal Forms
Microscopic examination of the microcrystals of azintamide was carried out by using a Leitz Camera Lucida (X = 4 0 ) attached to a Leitz projector. shows the different crystal forms of Figure 2 azintamide.
Fig.2:
Different Crystal Forms of Azintamide
7
AZINTAMIDE
2-63.
);-Ray Powder Diffraction
The X-ray powder diffraction pattern of azintamide was obtained on a Philips’ PW1710 Diffractometer with single crystal monochromator and copper Ka radiations. The patterns were recorded on a Philips’ PM 8210 printing recorder. The values of 2 0 , d-spacing ( A ) , and counts were automatically obtained on a Digital printer. Table 2 summarizes the obtained characteristic lines; while figure 3 shows the obtained X-ray diffractometric curve. Table 2:
7.179 7.978 10.363 12.029 12.557 13.051 15.925 16.639 18.088 18.704 19.727 20.788 21.532 21.983 22.904 23.995 24.664 25.211 25.889 26.875 27.365 27.779 28.868 30.053 30.711 32.075
Characteristic lines of the X-ray diffraction of azintamide powder.
12.3132 11.0815 8.5357 7.3571 7.0488 6.7832 5.5650 5.3278 4.9041 4 7439 4.5003 4.2724 4.1269 4.0433 3.8827 3.7087 3.6094 3.5324 3.4414 3.3173 3.2591 3.2114 3.0927 2.9734 2.9112 2.7904
13.14 52.52 20.10 7.85 6.63 100.00 7.48 4.49 3.06 5.13 45.39 27.82 2.90 7.72 2.80 20.02 7.42 4.78 2.83 11.39 4.99 2.09 10.18 3.07 44.95 30.49
33.064 34.213 35.445 35,870 36.454 36.913 37.390 39.936 40.407 41.317 41.985 42.377 43.129 44.420 45.775 46.789 47.036 48.034 49.226 50.482 52.793 56.196 56.572 58.426 60.288
2.7092 2.6208 2.5324 2.5034 2.4647 2.4350 2.4051 2.2574 2.2322 2.1851 2.1519 2.1329 2.0974 2.0394 1.9821 1.9415 1.9319 1,8941 1.8510 1.8078 1.7340 1.6368 1.6268 1.5795 1.5351
2.52 12.79 2.67 4.91 4.18 2.77 4.28 6.63 2.19 2.87 2.91 15.98 8.66 6.11 2.70 2.63 2.08 3.67 8.22 5.36 2.99 4.13 2.16 5.00 5.06
EZZAT M. ABDEL-MOETY AND HAMAD A. AL-KHAMEES
8
I
,
,
60
55
.
50
45
40 35 30 2 5 20
2 0-Value
15
10
5
I
9
AZINTAMIDE
2-7.
Spectral Characterization Ultraviolet ( U V ) Spectrum
2-71.
The UV-scanning of 5 pg-ml-l solutions of azintamide solutions in water, 0.1-N HC1, and 0.1-N NaOH is given in figure 4. The sepctral running was carried out on a DMS 90 Varian double-beam UV/visible spectrophotorneter attached to a Hewlett-Packard 7015 B X-Y chart recorder and using 1-cm quartz cells. Table 3 presents collectively the obtained A ( l % , 1 cm)values, molar absroptivities, and the ratios of absorbances (Aratios) at about 316 nm, 306 nm, and 2 5 8 nm of az intamide solution in 95% ethanol.
-
I\
0.0
~
200
,
,
,
,
,
,
L , ,
250
3 00
350
Wavelength ( n m )
Fig. 4: The UV-scanning of 5 pg.1~1-lSolutions of 4 and 0.1N NaOH '.... Azintamide (-
EZZAT M. ABDEL-MOETY AND HAMAD A . AL-KHAMEES
10
Table 3 :
The ultraviolet absorDtion of azintamide (in ethanol)
*
1 cm)
A(l%,
Wavelength* (nm)
E
316
54.08
1.404 X 103
306
57.43
1.492 X l o 3
258
547.00
1 . 4 2 1 X 104
A-ratios = A 258/316 = 10.114 and A 258/306 = 9.525 2-72.
Infrared (IR) Spectrum
The IR spectrum was carried out on a Perkin-Elmer 1310 IR-Spectrometer, in KBr (ca. 1 % ) . Figure 5 shows the IR spectrum of azintamide, and table 4 collects the IR band assignments of the drug. Table 4:
The infrared band assignments for azintamide
Wave No. (cm-')*
Assignment
2980 ( s ) , 2945 ( m )
CH-stretching in heteroaromatic ring
1630-1636
C
= 0
1580 (m)
N
=
1500-1420 ( m )
CHz-scissoring with CO and S
1410 ( s )
C-N stretching, amide
860 (m)
C-C1 and C-N stretching
~
*
(s)
~~
~
m = medium, s = strong
stretching, amide
N stretching
Fig. 5 :
The Infrared (It?) Spectrum of Azintamlde.
i--l%,KBy).
EZZAT M . ABDEL-MOETY AND HAMAD A . AL-KHAMEES
12
2-73.
Mass Spectrum
The low resolution mass spectrum of azintamide is presented in figure 6 . The running was made on a Varian CH-7 Mass Spectrometer. Table 5 shows the possible mass fragmentations obtained from mass spectrometric measurement of the drug by introducing it directly using shove pole. The following scheme shows the possible mass fragmentation pattern of azintamide
r 42 I I
I
5 -CH2 - co
145.5l
159.5
I
, -558
I
CH2- CH3
iN\ CH2- CH3
1187.5
-
Base-line (100%) - k 7 2 - i
Az intamide
2-74.
Nuclear Magnetic Resonance (NMR) Spectra
The proton as well as the 13carbon nuclear magnetic resonances of azintamide were carried out on the drug solution in CDC13 by using TMS as internal standard on a Varian XL-200 spectrometer.
'"1
-30
8ol
90
70
- 20
10
30 0 N Lo
I: M.2
SPEC i 3221002
AZI NTAMIOE
STEP MASS
= 10 , I /B/.S/C
: 1%
EZZAT M. ABDEL-MOETY AND HAMAD A. AL-KHAMEES
14
Table 5:
Low resolution mass spectrometric assignments of azintamide
Measured mass*
Structural Assignment
262 ( 4 )
C i o H i 6 C 1 N 3 0s
M t 2
c1
Formula
187 ( 1 9 )
C6H3 C 1 N 2 0 S
159 ( 1 5 )
C5 H3 C l N 2 S
113 ( 5 0 )
C4HClNz
100 ( 5 0 ) 85 ( 4 6 )
72 (100)
base-peak
71 (37)
,CH2 CHJCON, CH2' L C2 H5 N' 'c2 H5
[
N:C2H5-
CH2 -CH3
57 (8)
CH3CON
56 ( 2 4 )
CHiCON CH3
42 ( 2 5 )
N
:
* CHCON,
I]
c2 H5
58 ( 1 2 )
44 ( 2 2 )
*
1
C 5 H6 NO
'c2 H5
c4 H7 NO
C4 H6 N CiHsN c2 H5
#
CHCONHz
C2 H3 NO
0
CCONH2
C2 Hz NO
kY2# CH3
CH2
CH3
H 2 N CH y]
C2H6N C2 H4 N
Figures in parenthesis are the percent relative intensity of the peak.
15
AZINTAMIDE
2-741.
Proton Nuclear Magnetic Resonance (lH-NMR) Spectrum
The 200 MHz 1H-NMR spectrum of azintamide is shown in Pigure 7. The spectral peak assignments of the drug are presented in Table 6. Table 6:
The lH-NMR spectral assignments for azintamide
CI
Chemical shift ( 6 , ppm)
Proton assignments (CDCL3)*
7.46
[d, 1 H ( 1 or 2 1 , aromatic]
7.37
[d, 1 H (1 or 2 1 , aromtic]
4.33
[ s , 2H ( 3 ) ,
3.52-3.41
[m, 4H ( 4 & 5 1 , 2CHz.CH.31
1.32-1.25
[t, 3H ( 6 1 , CHzCH31'
1.18-1.11
[ t , 3H 1 7 1 , CHzCH31t
SCHzCOI
* s , d, t, m , are symbols f o r singlet, doublet, triplet, and multiplet, in order; figure in parentheses are the location numbers. + The non-equivalence of the two methyl groups of the aliphatic amide in azintamide is due to restricted steric
' '\
0
rotation about C
N
(5).
Fig. 7:
1 h e 200-MIiz
1 Ii-NMR
Spectui-m of_fizintam~dq.
17
AZINTAMIDE
2-742. 13Carbon Nuclear Magnetic Resonance Spectrum (13C-NMR) The 200 MHz 13C-NMR spectrum of azintamide is shown in Figure 8. The spectral peak assignments of the drug are presented in Table 7. Table 7:
The I3C-NMR spectral assignments for azintamide
Chemical shift ( 6 , ppm)
* +
Carbon assignment (CDCl3) *
12.85
[lC (lo), C.CIH31'
14.28
[lc
34.10
[1C ( 5 1 , SCH2COl
40.76
[1C (7), N.CHz.C]*
42.52
[lC (81, N._CHz.Cl+
(91, C.CH3It
127.39
[ l C (2), CH-aromatic]
128.16
[lC (3), CH-aromatic]
153.60
[lc (l), C-aromatic)]
161.21
[1C
166.01
[lC (6), C.CO.N ]
(4), C-aromatic]
Figure in parentheses are the location numbers. Non-equivalence due to restricted steric rotation about the aliphatic amide (5).
I O I '.E
-
' S Z 151
18
-
m I 0
19
8
"
x 0
I
",I"".,
EZZAT M. ABDEL-MOETY AND HAMAD A. AL-KHAMEES
20
The Distortionless Enhancement by Polarization Transfer (DEPT) at 200 MHz for azintamide was runned on the same sample of the drug in CDC13 against TMS as internal standard; figure 9 shows the typical classification of protonated carbons (CHX) to CH-, CHz- and CH3- carbons. 3.
SYNTHESIS Azintamide has been synthesized in 1959 by Schonbeck ( 6 ) among various pyridazine derivatives having Azintamde (ST different choleretic activities ( 3 ) . 9067) was assigned and patented (German Patents: 1188604, Nov. 1965) to Lentia GmbH, Munich-F.R. Germany (7).
The following scheme illustrate pathway of azintamide:
the
synthetic
CI
+
CICH2CON(CH2CH3)2
-
( i n ethanol) SH
.-c
E
8 CI
\
c
U
C
rc
<
SCH2CO N
F 2 H5 \
C2H5
Az in ta rnide
AZINTAMIDE
21
Azintamide, [3-chloropyridazinyl-6-thiol-acetic acid diethylamide, could be obtained by reacting 7 . 3 parts of 3-chloro-6-methylthiopyridazine (8)dissolved in 20 parts of 10% NaOH with a solution of 7 . 5 parts chloroacetic acid diethylamide in 20 parts alcohol at 60'C for 30 min. Crystallization can occur by cooling to O'C and recrystallization from acetone { 3 ) . 4.
PHARMACOLOGY The choleretic activity of azintamide has been predicted firstly by comparing with those of the 2-[(6chloro-3-pyridazinyl)thio] acetic acid (ST 9024), synthesized by Schonbeck (6). The substituted amide showed the highest potency (3,6). The choleretic activity of azintamide has been carried out on rats against dehydrocholic acid as reference substance (91, then on human volunteers ( 1 0 - 1 3 ) . Choleresis due to azintamide in man with rapid onset was manifested within a short administration time. Azintamide is now one of the most recommended choleretic references for naturally occuring plant cholagogues ( 1 4 ) and other (15). synthetic choleretically active compounds Azintamide, in identical doses, evokes a more powerful choleresis than either of dehydrocholic acid and 1phenylpropanol ( 9 ) . The elimination of bromosulfophthalene (BSP) is only influenced in the first 30minutes period following intradoudonal administration of 50 mg.Kg-' dose. The same dose of dehydrocholic acid, in every cases, retarded BSP-elimination over all 30 minutes periods. The choleretic activity of the drug can be demonstrated in selected patients by means of Bartelheimer's double ballon tube. An increase in bile flow, depending on the dose, was stated after This demonstrates administration of 1 g azintamide. the drug has true choleretic increasing secretion of the components of the bile and not merely increasing fluid volume, i.e. hydrocholeresis. No increase of serum bilirubin was observed after azintamide and the maximum effect is reached within 20 to 40 minutes after administration.
EZZAT M . ABDEL-MOETY A N D HAMAD A . AL-KHAMEES
22
5.
THERAPEUTIC CATEGEORATION AND U 5-1.
J
Categoration
Azintamide is a potent choleretic drug substance which exerts also a moderate cholepoietic action as well. The drug can be categorized as antihypercholeserolemic substance because it can reduce the serum cholestrol, non-esterified fatty acid and free glycerol; i.e. the drug can be considered as antihyperlipidemic.
Fatty indigestion, cholangitis, cholecystitis, icteric and posticteric cases, liver protection in conditions of cholecystopathy, meteorism and hepatogenic dermatosis, such as psoriasis vulgaris (16). The drug can be used in some cases of Azintamide can be hypercholesterolemia ( 1 7 , 1 8 ) . prescribed for hepatitis after treatment and for Koemheld-syndromes. 5-21.
Contraindications
As all choleretics, azintam avoided in cases of acute hepatitis inclining to biliary colic due to chole choleresis can cause mobilization of possibly resulting in partial or complete the bile duct.
5-22.
de should be and in cases ithiasis, as gall stones, occlusion of
Dosage
150-300 mg.day-1 is the normal adult dose, which can be taken during meal once or divided to three times. 6.
TOXICOLOGY Acute Toxicity (oral by mice): (1.94-2.48) ( 9 , 10).
A-LDBO is 2.34
g.Kg-1
Chronic Toxicity ( 6 months term): C-LDso is 1 . 1 8 g.Kg-l (0.97-1.43) ( 1 9 ) .
23
AZINTAMIDE
7.
STABILITY AND DEGRADATION Lindner et al. (19) demonstrated tht azintamide decomposes due to the hydrolysis of the amide linkage with formation of diethylamine and [3-chloropyridazinyl-6-thio] acetic acid as the principal degradation products.
8.
PHARMACOKINET1CS
8-1. Biotransformation Through amide-linkage break, [3-chloropyridazinyl-6-thiolacetic acid is identified in rats’ urine Only small amount as the main metabolic product ( 1 9 ) . of the unchanged drug could be traced in plasma and urine. The principal metabolites, i.e. [3-chloropyridazinyl-6-thiolacetic acid and diethylamine, are subject for further biotransformation such as soxidation or detoxification as sulfate and or glucuronate conjugates. 8-2.
Absorption
The main part of the intaken azintamide is absorbed and reabsorbed from intestine (19). Sometimes, azintamide may be dispensed with some digesting enzymes, e.g. Wilzym 600, a standardized pancreatic ferments), in such cases the preparation has to be in enteric-coated form to protect the enzyme components from inactivation by stomach acids. The inactivation of these enzymes can also be caused by excess antacids, such as silicates, magnesia, and bicarbonate, or by absorbents like charcoal powder (20).
8-3.
Excretion
Azintamide is excreted directly or indirectly in Traces of unchanged drug can be the bile (19). investigated in feaces, but the main excretory product is the [3-chforopyridazinyl-6-thio] acetic acid, small amounts in free form and the majority in conjugated bindings.
24
9.
EZZAT M. ABDEL-MOETY AND HAMAD A. AL-KHAMEES
METHODS OF ANALYSIS 9-1. Qualitative Methods 9-11. Elemental Composition Element C H
c1 N 0 S
%-Composition 46.24 5.43 13.65 16.17 6.16 12.35
9-12. Identification with Microchemical Tests Table 8 summarizes the chemical reactions with some common reagents used for identification of azintamide. The color reactions can be also useful for identification of the drug after its chromato-graphic, paper (PC) and thin-layer (TLC), separation. Table 8:
Microchemical tests for identification of azintamide
Reagent
*
2% anisaldehyde/conc.H3P04 t CHJCOOH t CzH5OH (3:1:1, v/v/v) ; heat.
* 0.5% p-dimethylaminobenzal-
dehyde (PDAB) HC1 t CzH50H; heat.
* 0.5% PDAB/70% * Vanilin/H+ : heat *5% Silicotangestic acid/HCl
Observation Violet-red color (19)
Green-blue color (9,19)
Yellow-red (21). Blue-violet color to turbidity (19). White precipitate, dissolves on boiling.
25
AZINTAMIDE
9-13. Chromatographic Methods 9-131. Paper Chromatography (PC)
Table 9 shows the PC-separation and identification of azintamide. Table 9: Mobile phase 30% acetic acid
The PaDer chromatography of azintamide
*
hRf
88
Visualization 0.5% PDAB in HCI
t CzH50H; heat at
Reference (9)
70"C/5 min., or under UV-light; green-blue spots appear *hRf is the travelling rate (Rf) X 100.
9-132. Thin-laver Chromatography (TLC)
Azintamide can be chromatographed on different thin-layers eithing by adopting the one- or the two-dimensional techniques. Table 10 summarizes the TLC-separation and characterization of azintamide.
EZZAT M. ABDEL-MOETY AND HAMAD A. AL-KHAMEES
26
Table 10:
The thin-layer ChromatoEraDhv of azintamide
Mobile phase
Layer
hRf
Visualization Reference
~
1.
One-dimensional TLC
WHCh t CzHsOH
Silica gel GFz54
a. UV-light ( 2 5 0 nm) b. Spray with KMn04/0H- to give yellow spots on violet background.
(2)
65-66
a. UV-light ( 2 5 4 nm) b. 2% PDAB in 5% H2S04; gives yellowish green spots after ca. 30 min.
(21)
74
CH3 COOH
a. 2% anisalde hyde in conc, H 3 W 4 t CH3COOH t CzHsOH ( 3 : 1: 1, v/v/v); heat 1 2 0 " C / 2 0 min., to give violet red spots on white to light rose background.
v/v/v 1
b. PDBA/H+ ; heat
(100:5,
v/v)
*CH30H t conc. NHiOH (100: 1.5, v/v) ( 2 2 )
2.
Silica gel GFz54
Two-dimensional TLC Silica gel G
*P1:
CH3COOCzH5 t CH30H t 3-N( NH4 )2C03
DMF (12:2:1:1,
t
v/v/v/v 1 *PZ :
Cch
55
t
CH30H t
(77.5:20:2.5,
57
12O"C/20 min, to
give green blue spots.
c. Vanilin/H+ ; heat, to give blue violet spots.
21
AZINTAMIDE
9-2.
Quantitative Methods 9-21.
Volumetry 9.211.
Titrimetric determination of sulfer and chlorine contents
Azintamide can be assayed via its sulfer content after decomposition in Schoniger combustion flask and titration with 0.02-N Ba(C104)2 as the and thorine as indicator, taking 1 2 . 3 4 theoretical percent S-contents. Determination of the C1-contents, also after combustion, through titration with 0.01-N Hg(C104 12 and diphenylcarbazone as indicator can be adopted for the drug assay. The theoretical percent content of sulfer is 1 3 . 6 5 ( 2 ) . 9-22.
Instrumental Methods 9-221.
Colorimetry and spectrophotometry i-
Colorimetry
On heating azintamide solution with a solution of p-dimethylaminobenzaldehyde in 70% sulfuric acid, a yellow color is developed with an absorption maximum at 450 nm. Abdel-Moety et al. ( 2 1 ) have adopted the color measurement for quantification of the drug in bulk form and in tablets and effervescent granules containing the drug.
ii-
Direct UV-measurement
The measurement of the light absorption of the drug solution in ethanol at 258 nm is recommended by Abdel-Moety et al. ( 2 1 ) for quantitative determination of the drug in raw materials and only in tablets. Recoveries of 100-16 f 1.10 (n = 6 ) , 9 9 . 8 4 f 1 . 0 0 % (n = 6 ) , in case of tablets, and 100.30 f 1.40% (n = 9 ) , for effervescent granules, could be obtained for added azintamide. iiicribed a procedure
Derivative sDectrophotometrY
Abdel-Moety et al. ( 2 3 ) desfirst-derivative (Di) spectrophotometric for quantification of azintamide in
EZZAT M. ABDEL-MOETY AND HAMAD A . AL-KHAMEES
28
admixtures with papaverine hydrochloride, a smooth muscle relaxant, which is commonly dispensed with the choleretic drug together to inhibit its possible spasmomimetic activity in the gastrointestinal tract. i.e. ppm) and Amounts of azintamide (2-20 pg.ml-', papaverine hydrochloride ( 0 . 5 - 6 pg.ml-1) can be accurately quantified. The concentration ranges of both drugs might allow application of the derivative spectrophotometric method to their determination in biological fluids. The Di(dA/dh )-spectrophotometric measurement of azintamide is recommended at 264 nm, while for the other component a worked out simultaneous equation can be applied. The recoveries were 101.02 2 0.91% (n = 5 ) and 100.48 2 1.33% (n = 5 ) for azintamide and papaverine hydrochloride in order. iv-
PMR-sDectrophotometrp
El-Khateeb and Abdel-Moety described the application of proton-magneticresonance spectrophotometry for quantitative determination of azintamide in pure forms and in dosage formulations. The method involves comparing the integral of both the multiplet centered at about 1 . 1 5 ppm and the sharp singlet at 4 . 3 0 ppm of azintamide molecule to that of the sharp singlet signal at about 6 . 3 0 ppm of maleic acid which is chosen as internal standard. (24)
9-222.
SDectrofluorometry
Abdel-Moety et al. ( 2 5 ) discussed the coupling possibility of TLC-separation (on layers of silica gel 60 F 2 5 4 ) with spectrofluorimetry (EX : 229 nm and EM : 304) for quantificatrion of azintamide in biological fluids. Amounts of 0 . 5 - 2 . 5 ng.ml-1 (ppb) of the drug in urine samples of a healthy volunteer could be accurately traced. Recovery mean percent of 97.25 f 1 . 4 9 (n = 5 ) could be obtained. 9-223.
Flow-injection analysis ( F I A ]
A single-manifold FIA-system f o r quantitative determination of azintamide via spectrophotometric detection at 258 nm is investigated The limit of quantificaby Abdel-Moety et al. (26). tion and detection is about 5 pg.ml-1 of azintamide
AZlNTAMIDE
29
dissolved and/or extracted in ethanol could be accurately analyzed. A good percent mean recovery of 99.24 f 0.89 (n = 4) could be obtained at an introduction rate of about 150 sample.hr-1 or even more. The obtained results were comparable with those of the direct UV-measurement at the same Amax. 9-224. Chromatographic Techniques i-
Gas-liquid chromatographs ( GLC )
GLC-separation and quantification of azintamide in pharmaceutical formulations has been described by Abdel-Moety ( 2 7 ) . The GLC-separation of the drug extracts in chloroform was undertaken on 150 cm X 4 mm i.d. column packed with 10% silar on Diatomite C-AW, 100-120 mesh, at 250°C using Nz as carrier gas with flame ionization detection at 300'C, isothermally. The calibration graph was rectilinear for gm ml-1 of azintamide with concentrations 0 . 5 - 3 recovery of 98.77 t 1.11% (n = 4). The results were compared with those of the spectrophotometric method adopted by the author and others (21). A GC-columns, 150 cm X 4 mm i.d., packed with 5-10% silicone oil on Gas-Chrom Q can elegantly resolve azintamide under the previously mentioned GLC-conditions nearly with identical retention time of about 8 minutes as in case of the silar-column, but the reproducibility in case of the first column was quite better. ii-
High-Performanc Liquid Chromatographs (HPLC)
HPLC-method for assay of azintamide in one-component dosage forms can be achieved on a 5-pm C18-Novapack column by using mixture of methanol and water (98:2, v/v) isocratically as a mobile phase at ambient temperatures (28). Another is described for quantification of HPLC-procedure azintamide and papaverine.HC1 in binary mixtures and dosage formulations containing both drug substances ( 2 9 ) . The separation and quantification can be done on a 5-pm 100 RP-18 LiChrosphere column by using a solvent mixture of acetonitrile and water (56:44, v/v) isocratically at ambient temperature.
EZZAT M. ABDEL-MOETY AND HAMAD A. AL-KHAMEES
30
9-23.
BioloRical Assap
Azintamide can be quantified by the measurement of its choleretic activity according to the procedure described by Stormann ( 9 ) . The biological measurement is done against the activity of a well known choleretic agent, such as dehydrocholic acid and 1-phenylpropanol, The measurement of the elimination times of bromosulfophthalene (BSP) following suitable intradoudenal doses of the drug in the first 30minutes. 9-3.
Other Analytical Possibilites
Different spectroscopic and chromatographic behaviours of azintamide and the various analytical possibilities for the drug have been discussed in a recent communication ( 3 0 ) .
ACKNOWLEDGEMENT The authors would like to thank Dr. R.R. Abou-Shaaban for DSC-investigation and Mr. T.A. Butt for typing the manuscript.
31
AZINTAMIDE
REFERENCES 1.
The Merck Index, 10th edn., M. Windholz (Editor), Merck & Co., Inc., Rahway-NJ, 1983, p . 132-No. 915.
2.
Private communication; Org. of Pharm. Control Lab. of Osterreichische Stickstoffwerke, AG, Linz/DonauAustria, Dec. 1971.
3.
E. Kloimstein, R. Schonbeck, Arzneim.-Forsch.; l4, 261 ( 1 9 6 4 ) .
4.
E.M. Abdel-Moety, A.A. Mostafa, and S.A. Ismaiel: Zentralbl. Pharm. Pharmakother. Laboratoriumsdiagn.;
and H.
Stormann:
124, 730 ( 1 9 8 5 ) .
5.
R.M. Silverstein, G.C. Bassler, and T.C. Morrill (Editors), Spectrometric Identification of Organic ComPounds, 4th edn, J . Wiley & Sons, New York, 1981, p . 198.
Chem.; 90,
6.
R. Schonbeck: Reference 3.
7.
E. Kloimstein, R. Schonbeck, and H. Stormann: German Patents; 1188604 (1965 to Lentia GmbH, Munich-F.R. Germany).
8. 9.
Mh.
284 ( 1 9 5 9 ) ;
J . Druey, K.D. Meier, and K. Eichenberger:
m;37,
133 ( 1 9 5 4 ) .
H. Stormann:
through
Helv. Chim.
Arzneim.-Forsch.; l4, 266 ( 1 9 6 4 ) .
Hitzenberger: Arzneim.-Forsch,; l4, 279 ( 1 9 6 4 ) .
10.
G.
11.
W. Neugebauer:
12.
H.L. Dierel:
13.
A. Miyoshi, M. Ito, S. Nakanishi, T. Hajiro, and T. Takeda: Naika-Hokan; l5, 7 7 ( 1 9 6 8 ) .
14.
R. Hansel:
15.
G.
Landarzt; 41, 33 ( 1 9 6 5 ) .
Wien-Med-Wochenschr.;
115, 613
(1965).
Dtsch. ADoth. Ztg.; 125, 1373 ( 1 9 8 5 ) .
Hofrichter, H. Hampel, H.D. Liehn, and E. Arzneim.-Forsch.; 24, 111 ( 1 9 7 4 ) .
Ludwig:
EZZAT M. ABDEL-MOETY AND HAMAD A . AL-KHAMEES
32
16.
D.
17.
P. St.-Janiak:
18.
J.N. Pereira and G.T. Holland: Disorders of Lipid Metabolism: Annual Reports in Medicinal Chemistry, R.V. Heinzelman (Editor), Vol. 10, Academic Press, New York,
Konstantinov
(1975).
and L.
Stanoeva: Med-Arh; 29, 117
Drugs Future; 3, 191 ( 1 9 7 8 ) .
1975, p. 182-219.
19.
I . Lindner, H. Stormann, and W. Wendtland: Forsch.; 4, 271 (1964).
Arzneim.-
20.
Private communication; Chemie Linz, AG' Office in Riyadh - Saudi Arabia, Feb. 1989.
Scientific
21.
E.M.
22.
I . Sunshine:
23
E.M. Abdel-Moety, M.E. Mohamed, M.A. Abounassif, and M. Al-Awady: Sci. Pharm.; in press ( 1 9 8 9 ) .
24.
S.Z. El-Khateeb and E.M. 208, 1115 ( 1 9 8 7 ) .
25.
E.M. Abdel-Moety, S.Z. El-Khateeb, and M.A. Abounassif: linder publication.
26.
F.M. Abdel-Moety, A.A. Moustafa, A.K.S. Ahmad, and A.E. El-Gendy: Sci. Pharm.; 55, 259 (1987).
27.
E.M. Abdel-Moety: J. Chromatogr.; 324, 475 ( 1 9 8 5 ) .
28.
S.Z. El-Khateeb and E.M. Abdel-Moety: press (1989).
29.
M.A. Abounassif, E.M. Abdel-Moety, and E.A. Gad-Kariem: Under publication.
30.
E.M.
Abdel-Moety, F.H. Wahdan, N.A. Sharaby, and S.A. Ismaiel: Acta Pharm. Jugosl.; 34, 223 ( 1 9 8 4 ) .
Am. J. Clin. Pathol.; 40, 576 (1963).
Abdel-Moety:
Anal.
Lett.;
Sci. Pharm.;
Abdel-Moety: Zentralbl. Pharm. Laboratoriums-diagn.; 127, 137 ( 1 9 8 8 ) .
Pharmakother.
Literature Survey till Feb. 1989
Ezzat M. Abdel-Moety and Hamad A . Al-Khamees
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P . O . Box 2457, Riyadh-11451, Saudi Arabia.
ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 18 1
Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved
EZZAT M. ABDEL-MOETY AND HAMAD A. AL-KHAMEES
2
CONTENTS
1.
I NTRODUCT0R.Y
2.
DESCRIPTION 2-1. Name 2-2. Formulae 2-3. The Chemical Abstract Registry (CAS) Number 2-4. Appearance, C,olor, Odor and Taste 2-5. Physical Characteristics 2-6. Crystal Characteristics 2 - 7 . Spectral Characterization
3.
SYNTHESIS
4.
PHARMACOLOGY
5.
THERAPEUTIC CATEGORATION AND USES 5-1. Categoration 5-2. Uses
6.
TOXICOLOGY
7.
STABILITY AND DEGRADATION
8.
PHARMACOKINETICS 8-1. Biotransformation 8-2. Absorption 8-3. Excretion
9.
METHODS OF ANALYSIS 9-1. Qualitative Methods 9-2. Quantitative Methods ACKNOWLEDGEMENT REFERENCES
3
AZINTAMIDE
1.
INTRODUCTORY Azintamide is a true potent choleretic drug, which is totally synthesized in 1 9 5 9 . The drug has the registered trade name OragallinB In spite of the potent choleretic activity, with moderate cholepoietic action, and its wide therapeutic applications in different conditions and countries, no detailed informations about its physical, chemical, clinical, and bioavailability characteristics have been yet collectively summarized in simple presentation. The present Analytical Profile is an effort in this direction.
.
2.
DESCRIPTION 2-1.
Names
2.11. Chemical: thiol-N,N-diethylacetamide.
Other
chemical
names
2-[(6-Chloro-3-pyridazinyl)
are,
N,N-diethyl-2-[6-(3-
chloropyridaziny1)thiolacetamide; N,N-diethyl-2-[6-(3chloropyridaziny1)-mercapto]acetamide; and (3-chloro-6pyridaziny1thio)acetic acid diethylamide ( 1 ) .
2.12. Properioritg: Oragallin, Ora-gallin, and ST 9067. Azintamide has been registered under the trade name Oragallin 8 for Osterreichische Stickstoffwerke, AG, Linz/Donau - Austria.
2-2.
Formula and Molecular Weight
CI
[CioHi4ClN30S (259.77)]
EZZAT M. ABDEL-MOETY AND HAMAD A . AL-KHAMEES
4
2-3.
The Chemical Abstract Registry (CAS) Number: [ 1830-32-61-
2-4.
Appearance. Color, Odor, and Taste
Microcrystalline, white, odorless powder with bitter taste. 2-5.
Physical Characteristics 2-51.
Melting Range
The melting of azintamide was carried out at a heating rate of 1"C.min-1 on a Kofler hot-stage microscope. Table 1: Melting point and range of azintamide* Start temperature ('C)
90
Melting range
Mid-point
("C)
("C)
Literature
t 'C)
95.0-97.0 (2.0)
96.0
98-100 (2)
95.5-97.5 (2.0,
96.5
97-98 ( ~ 3 )
*Sample from Bsterreichishe Stickstoffwerke, AG, Linz/DonauAustria, BN: 23540/524699 - all values ( ' C ) are uncorrected. 2-52. Differentional Thermal Scanning (DSC) The DSC-curve was obtained on a DuPont TAto a data processing unit. Figure 1 shows the DSC-curve of azintamide. The running was between 50-150°C at heating rate of 10"C.min-1. The heat of activation and the purity of the sample was determined using purity program. 9900 Thermal Analyzer attached
2-53.
Solubility
Azintamide is freely soluble in benzene, chloroform, ethyl acetate and acetone, its solubility in water is 5 mg.ml-1 (1).
Sample : AZII~ITAMICJE-8 Size : 4 . 6 0 mg :lethod : DSC 50 TO 150 Comment:
@10 C/M.
DSC
Run Date:
02/11/88
19: 13
97.60 097.59
-1
-z
\ x
-
D
97.58
O
D1
L
r
:: LL
-
-u 97.97
-2-
4J 3
-3-
21
I m
I -4
-
0 1
Purity : Melting P t :
Oepresslan : Oelta H : Corrcctlon : I.101. I l e l g h t :
c e l l const : -5 - O n s e t S l a p c :
97.55
100.01 Mole X 9 7 . 5 'C -O.OOn'C 3 0 . 6 kJ/molB 1 . 0 7 x: 259.8 g / ~ o l e
1.283
-7.90
mN/'C
nn
-6.
,
50
.
0
',
60
Fiq. 1 :
.
, 10' .
70
I
80
a*
2 I-
37.55
37.54
J
T a t a l Ar.ea/Pdrt i d 1 A r e a 20 30 40
.
L
9b id0 Temperature
110
('Cl
[he d i f f e r e n t i a - t Scanning
50
120
140
L L J I - V-cOSC) ~
60
140
37.33 120 PURITY V l . l . 4
of fizintnmide.
EZZAT M. ABDEL-MOETY AND HAMAD A. AL-KHAMEES
6
Optical Rotation
2-54.
Azintamide
species. 2-6.
is
an
optically-inactive
Crystal Characteristics Crystallization
2-61.
acetone (1). 2-62.
Azintamide
is readily crystallizable from
Crystal Forms
Microscopic examination of the microcrystals of azintamide was carried out by using a Leitz Camera Lucida (X = 4 0 ) attached to a Leitz projector. shows the different crystal forms of Figure 2 azintamide.
Fig.2:
Different Crystal Forms of Azintamide
7
AZINTAMIDE
2-63.
);-Ray Powder Diffraction
The X-ray powder diffraction pattern of azintamide was obtained on a Philips’ PW1710 Diffractometer with single crystal monochromator and copper Ka radiations. The patterns were recorded on a Philips’ PM 8210 printing recorder. The values of 2 0 , d-spacing ( A ) , and counts were automatically obtained on a Digital printer. Table 2 summarizes the obtained characteristic lines; while figure 3 shows the obtained X-ray diffractometric curve. Table 2:
7.179 7.978 10.363 12.029 12.557 13.051 15.925 16.639 18.088 18.704 19.727 20.788 21.532 21.983 22.904 23.995 24.664 25.211 25.889 26.875 27.365 27.779 28.868 30.053 30.711 32.075
Characteristic lines of the X-ray diffraction of azintamide powder.
12.3132 11.0815 8.5357 7.3571 7.0488 6.7832 5.5650 5.3278 4.9041 4 7439 4.5003 4.2724 4.1269 4.0433 3.8827 3.7087 3.6094 3.5324 3.4414 3.3173 3.2591 3.2114 3.0927 2.9734 2.9112 2.7904
13.14 52.52 20.10 7.85 6.63 100.00 7.48 4.49 3.06 5.13 45.39 27.82 2.90 7.72 2.80 20.02 7.42 4.78 2.83 11.39 4.99 2.09 10.18 3.07 44.95 30.49
33.064 34.213 35.445 35,870 36.454 36.913 37.390 39.936 40.407 41.317 41.985 42.377 43.129 44.420 45.775 46.789 47.036 48.034 49.226 50.482 52.793 56.196 56.572 58.426 60.288
2.7092 2.6208 2.5324 2.5034 2.4647 2.4350 2.4051 2.2574 2.2322 2.1851 2.1519 2.1329 2.0974 2.0394 1.9821 1.9415 1.9319 1,8941 1.8510 1.8078 1.7340 1.6368 1.6268 1.5795 1.5351
2.52 12.79 2.67 4.91 4.18 2.77 4.28 6.63 2.19 2.87 2.91 15.98 8.66 6.11 2.70 2.63 2.08 3.67 8.22 5.36 2.99 4.13 2.16 5.00 5.06
EZZAT M. ABDEL-MOETY AND HAMAD A. AL-KHAMEES
8
I
,
,
60
55
.
50
45
40 35 30 2 5 20
2 0-Value
15
10
5
I
9
AZINTAMIDE
2-7.
Spectral Characterization Ultraviolet ( U V ) Spectrum
2-71.
The UV-scanning of 5 pg-ml-l solutions of azintamide solutions in water, 0.1-N HC1, and 0.1-N NaOH is given in figure 4. The sepctral running was carried out on a DMS 90 Varian double-beam UV/visible spectrophotorneter attached to a Hewlett-Packard 7015 B X-Y chart recorder and using 1-cm quartz cells. Table 3 presents collectively the obtained A ( l % , 1 cm)values, molar absroptivities, and the ratios of absorbances (Aratios) at about 316 nm, 306 nm, and 2 5 8 nm of az intamide solution in 95% ethanol.
-
I\
0.0
~
200
,
,
,
,
,
,
L , ,
250
3 00
350
Wavelength ( n m )
Fig. 4: The UV-scanning of 5 pg.1~1-lSolutions of 4 and 0.1N NaOH '.... Azintamide (-
EZZAT M. ABDEL-MOETY AND HAMAD A . AL-KHAMEES
10
Table 3 :
The ultraviolet absorDtion of azintamide (in ethanol)
*
1 cm)
A(l%,
Wavelength* (nm)
E
316
54.08
1.404 X 103
306
57.43
1.492 X l o 3
258
547.00
1 . 4 2 1 X 104
A-ratios = A 258/316 = 10.114 and A 258/306 = 9.525 2-72.
Infrared (IR) Spectrum
The IR spectrum was carried out on a Perkin-Elmer 1310 IR-Spectrometer, in KBr (ca. 1 % ) . Figure 5 shows the IR spectrum of azintamide, and table 4 collects the IR band assignments of the drug. Table 4:
The infrared band assignments for azintamide
Wave No. (cm-')*
Assignment
2980 ( s ) , 2945 ( m )
CH-stretching in heteroaromatic ring
1630-1636
C
= 0
1580 (m)
N
=
1500-1420 ( m )
CHz-scissoring with CO and S
1410 ( s )
C-N stretching, amide
860 (m)
C-C1 and C-N stretching
~
*
(s)
~~
~
m = medium, s = strong
stretching, amide
N stretching
Fig. 5 :
The Infrared (It?) Spectrum of Azintamlde.
i--l%,KBy).
EZZAT M . ABDEL-MOETY AND HAMAD A . AL-KHAMEES
12
2-73.
Mass Spectrum
The low resolution mass spectrum of azintamide is presented in figure 6 . The running was made on a Varian CH-7 Mass Spectrometer. Table 5 shows the possible mass fragmentations obtained from mass spectrometric measurement of the drug by introducing it directly using shove pole. The following scheme shows the possible mass fragmentation pattern of azintamide
r 42 I I
I
5 -CH2 - co
145.5l
159.5
I
, -558
I
CH2- CH3
iN\ CH2- CH3
1187.5
-
Base-line (100%) - k 7 2 - i
Az intamide
2-74.
Nuclear Magnetic Resonance (NMR) Spectra
The proton as well as the 13carbon nuclear magnetic resonances of azintamide were carried out on the drug solution in CDC13 by using TMS as internal standard on a Varian XL-200 spectrometer.
'"1
-30
8ol
90
70
- 20
10
30 0 N Lo
I: M.2
SPEC i 3221002
AZI NTAMIOE
STEP MASS
= 10 , I /B/.S/C
: 1%
EZZAT M. ABDEL-MOETY AND HAMAD A. AL-KHAMEES
14
Table 5:
Low resolution mass spectrometric assignments of azintamide
Measured mass*
Structural Assignment
262 ( 4 )
C i o H i 6 C 1 N 3 0s
M t 2
c1
Formula
187 ( 1 9 )
C6H3 C 1 N 2 0 S
159 ( 1 5 )
C5 H3 C l N 2 S
113 ( 5 0 )
C4HClNz
100 ( 5 0 ) 85 ( 4 6 )
72 (100)
base-peak
71 (37)
,CH2 CHJCON, CH2' L C2 H5 N' 'c2 H5
[
N:C2H5-
CH2 -CH3
57 (8)
CH3CON
56 ( 2 4 )
CHiCON CH3
42 ( 2 5 )
N
:
* CHCON,
I]
c2 H5
58 ( 1 2 )
44 ( 2 2 )
*
1
C 5 H6 NO
'c2 H5
c4 H7 NO
C4 H6 N CiHsN c2 H5
#
CHCONHz
C2 H3 NO
0
CCONH2
C2 Hz NO
kY2# CH3
CH2
CH3
H 2 N CH y]
C2H6N C2 H4 N
Figures in parenthesis are the percent relative intensity of the peak.
15
AZINTAMIDE
2-741.
Proton Nuclear Magnetic Resonance (lH-NMR) Spectrum
The 200 MHz 1H-NMR spectrum of azintamide is shown in Pigure 7. The spectral peak assignments of the drug are presented in Table 6. Table 6:
The lH-NMR spectral assignments for azintamide
CI
Chemical shift ( 6 , ppm)
Proton assignments (CDCL3)*
7.46
[d, 1 H ( 1 or 2 1 , aromatic]
7.37
[d, 1 H (1 or 2 1 , aromtic]
4.33
[ s , 2H ( 3 ) ,
3.52-3.41
[m, 4H ( 4 & 5 1 , 2CHz.CH.31
1.32-1.25
[t, 3H ( 6 1 , CHzCH31'
1.18-1.11
[ t , 3H 1 7 1 , CHzCH31t
SCHzCOI
* s , d, t, m , are symbols f o r singlet, doublet, triplet, and multiplet, in order; figure in parentheses are the location numbers. + The non-equivalence of the two methyl groups of the aliphatic amide in azintamide is due to restricted steric
' '\
0
rotation about C
N
(5).
Fig. 7:
1 h e 200-MIiz
1 Ii-NMR
Spectui-m of_fizintam~dq.
17
AZINTAMIDE
2-742. 13Carbon Nuclear Magnetic Resonance Spectrum (13C-NMR) The 200 MHz 13C-NMR spectrum of azintamide is shown in Figure 8. The spectral peak assignments of the drug are presented in Table 7. Table 7:
The I3C-NMR spectral assignments for azintamide
Chemical shift ( 6 , ppm)
* +
Carbon assignment (CDCl3) *
12.85
[lC (lo), C.CIH31'
14.28
[lc
34.10
[1C ( 5 1 , SCH2COl
40.76
[1C (7), N.CHz.C]*
42.52
[lC (81, N._CHz.Cl+
(91, C.CH3It
127.39
[ l C (2), CH-aromatic]
128.16
[lC (3), CH-aromatic]
153.60
[lc (l), C-aromatic)]
161.21
[1C
166.01
[lC (6), C.CO.N ]
(4), C-aromatic]
Figure in parentheses are the location numbers. Non-equivalence due to restricted steric rotation about the aliphatic amide (5).
I O I '.E
-
' S Z 151
18
-
m I 0
19
8
"
x 0
I
",I"".,
EZZAT M. ABDEL-MOETY AND HAMAD A. AL-KHAMEES
20
The Distortionless Enhancement by Polarization Transfer (DEPT) at 200 MHz for azintamide was runned on the same sample of the drug in CDC13 against TMS as internal standard; figure 9 shows the typical classification of protonated carbons (CHX) to CH-, CHz- and CH3- carbons. 3.
SYNTHESIS Azintamide has been synthesized in 1959 by Schonbeck ( 6 ) among various pyridazine derivatives having Azintamde (ST different choleretic activities ( 3 ) . 9067) was assigned and patented (German Patents: 1188604, Nov. 1965) to Lentia GmbH, Munich-F.R. Germany (7).
The following scheme illustrate pathway of azintamide:
the
synthetic
CI
+
CICH2CON(CH2CH3)2
-
( i n ethanol) SH
.-c
E
8 CI
\
c
U
C
rc
<
SCH2CO N
F 2 H5 \
C2H5
Az in ta rnide
AZINTAMIDE
21
Azintamide, [3-chloropyridazinyl-6-thiol-acetic acid diethylamide, could be obtained by reacting 7 . 3 parts of 3-chloro-6-methylthiopyridazine (8)dissolved in 20 parts of 10% NaOH with a solution of 7 . 5 parts chloroacetic acid diethylamide in 20 parts alcohol at 60'C for 30 min. Crystallization can occur by cooling to O'C and recrystallization from acetone { 3 ) . 4.
PHARMACOLOGY The choleretic activity of azintamide has been predicted firstly by comparing with those of the 2-[(6chloro-3-pyridazinyl)thio] acetic acid (ST 9024), synthesized by Schonbeck (6). The substituted amide showed the highest potency (3,6). The choleretic activity of azintamide has been carried out on rats against dehydrocholic acid as reference substance (91, then on human volunteers ( 1 0 - 1 3 ) . Choleresis due to azintamide in man with rapid onset was manifested within a short administration time. Azintamide is now one of the most recommended choleretic references for naturally occuring plant cholagogues ( 1 4 ) and other (15). synthetic choleretically active compounds Azintamide, in identical doses, evokes a more powerful choleresis than either of dehydrocholic acid and 1phenylpropanol ( 9 ) . The elimination of bromosulfophthalene (BSP) is only influenced in the first 30minutes period following intradoudonal administration of 50 mg.Kg-' dose. The same dose of dehydrocholic acid, in every cases, retarded BSP-elimination over all 30 minutes periods. The choleretic activity of the drug can be demonstrated in selected patients by means of Bartelheimer's double ballon tube. An increase in bile flow, depending on the dose, was stated after This demonstrates administration of 1 g azintamide. the drug has true choleretic increasing secretion of the components of the bile and not merely increasing fluid volume, i.e. hydrocholeresis. No increase of serum bilirubin was observed after azintamide and the maximum effect is reached within 20 to 40 minutes after administration.
EZZAT M . ABDEL-MOETY A N D HAMAD A . AL-KHAMEES
22
5.
THERAPEUTIC CATEGEORATION AND U 5-1.
J
Categoration
Azintamide is a potent choleretic drug substance which exerts also a moderate cholepoietic action as well. The drug can be categorized as antihypercholeserolemic substance because it can reduce the serum cholestrol, non-esterified fatty acid and free glycerol; i.e. the drug can be considered as antihyperlipidemic.
Fatty indigestion, cholangitis, cholecystitis, icteric and posticteric cases, liver protection in conditions of cholecystopathy, meteorism and hepatogenic dermatosis, such as psoriasis vulgaris (16). The drug can be used in some cases of Azintamide can be hypercholesterolemia ( 1 7 , 1 8 ) . prescribed for hepatitis after treatment and for Koemheld-syndromes. 5-21.
Contraindications
As all choleretics, azintam avoided in cases of acute hepatitis inclining to biliary colic due to chole choleresis can cause mobilization of possibly resulting in partial or complete the bile duct.
5-22.
de should be and in cases ithiasis, as gall stones, occlusion of
Dosage
150-300 mg.day-1 is the normal adult dose, which can be taken during meal once or divided to three times. 6.
TOXICOLOGY Acute Toxicity (oral by mice): (1.94-2.48) ( 9 , 10).
A-LDBO is 2.34
g.Kg-1
Chronic Toxicity ( 6 months term): C-LDso is 1 . 1 8 g.Kg-l (0.97-1.43) ( 1 9 ) .
23
AZINTAMIDE
7.
STABILITY AND DEGRADATION Lindner et al. (19) demonstrated tht azintamide decomposes due to the hydrolysis of the amide linkage with formation of diethylamine and [3-chloropyridazinyl-6-thio] acetic acid as the principal degradation products.
8.
PHARMACOKINET1CS
8-1. Biotransformation Through amide-linkage break, [3-chloropyridazinyl-6-thiolacetic acid is identified in rats’ urine Only small amount as the main metabolic product ( 1 9 ) . of the unchanged drug could be traced in plasma and urine. The principal metabolites, i.e. [3-chloropyridazinyl-6-thiolacetic acid and diethylamine, are subject for further biotransformation such as soxidation or detoxification as sulfate and or glucuronate conjugates. 8-2.
Absorption
The main part of the intaken azintamide is absorbed and reabsorbed from intestine (19). Sometimes, azintamide may be dispensed with some digesting enzymes, e.g. Wilzym 600, a standardized pancreatic ferments), in such cases the preparation has to be in enteric-coated form to protect the enzyme components from inactivation by stomach acids. The inactivation of these enzymes can also be caused by excess antacids, such as silicates, magnesia, and bicarbonate, or by absorbents like charcoal powder (20).
8-3.
Excretion
Azintamide is excreted directly or indirectly in Traces of unchanged drug can be the bile (19). investigated in feaces, but the main excretory product is the [3-chforopyridazinyl-6-thio] acetic acid, small amounts in free form and the majority in conjugated bindings.
24
9.
EZZAT M. ABDEL-MOETY AND HAMAD A. AL-KHAMEES
METHODS OF ANALYSIS 9-1. Qualitative Methods 9-11. Elemental Composition Element C H
c1 N 0 S
%-Composition 46.24 5.43 13.65 16.17 6.16 12.35
9-12. Identification with Microchemical Tests Table 8 summarizes the chemical reactions with some common reagents used for identification of azintamide. The color reactions can be also useful for identification of the drug after its chromato-graphic, paper (PC) and thin-layer (TLC), separation. Table 8:
Microchemical tests for identification of azintamide
Reagent
*
2% anisaldehyde/conc.H3P04 t CHJCOOH t CzH5OH (3:1:1, v/v/v) ; heat.
* 0.5% p-dimethylaminobenzal-
dehyde (PDAB) HC1 t CzH50H; heat.
* 0.5% PDAB/70% * Vanilin/H+ : heat *5% Silicotangestic acid/HCl
Observation Violet-red color (19)
Green-blue color (9,19)
Yellow-red (21). Blue-violet color to turbidity (19). White precipitate, dissolves on boiling.
25
AZINTAMIDE
9-13. Chromatographic Methods 9-131. Paper Chromatography (PC)
Table 9 shows the PC-separation and identification of azintamide. Table 9: Mobile phase 30% acetic acid
The PaDer chromatography of azintamide
*
hRf
88
Visualization 0.5% PDAB in HCI
t CzH50H; heat at
Reference (9)
70"C/5 min., or under UV-light; green-blue spots appear *hRf is the travelling rate (Rf) X 100.
9-132. Thin-laver Chromatography (TLC)
Azintamide can be chromatographed on different thin-layers eithing by adopting the one- or the two-dimensional techniques. Table 10 summarizes the TLC-separation and characterization of azintamide.
EZZAT M. ABDEL-MOETY AND HAMAD A. AL-KHAMEES
26
Table 10:
The thin-layer ChromatoEraDhv of azintamide
Mobile phase
Layer
hRf
Visualization Reference
~
1.
One-dimensional TLC
WHCh t CzHsOH
Silica gel GFz54
a. UV-light ( 2 5 0 nm) b. Spray with KMn04/0H- to give yellow spots on violet background.
(2)
65-66
a. UV-light ( 2 5 4 nm) b. 2% PDAB in 5% H2S04; gives yellowish green spots after ca. 30 min.
(21)
74
CH3 COOH
a. 2% anisalde hyde in conc, H 3 W 4 t CH3COOH t CzHsOH ( 3 : 1: 1, v/v/v); heat 1 2 0 " C / 2 0 min., to give violet red spots on white to light rose background.
v/v/v 1
b. PDBA/H+ ; heat
(100:5,
v/v)
*CH30H t conc. NHiOH (100: 1.5, v/v) ( 2 2 )
2.
Silica gel GFz54
Two-dimensional TLC Silica gel G
*P1:
CH3COOCzH5 t CH30H t 3-N( NH4 )2C03
DMF (12:2:1:1,
t
v/v/v/v 1 *PZ :
Cch
55
t
CH30H t
(77.5:20:2.5,
57
12O"C/20 min, to
give green blue spots.
c. Vanilin/H+ ; heat, to give blue violet spots.
21
AZINTAMIDE
9-2.
Quantitative Methods 9-21.
Volumetry 9.211.
Titrimetric determination of sulfer and chlorine contents
Azintamide can be assayed via its sulfer content after decomposition in Schoniger combustion flask and titration with 0.02-N Ba(C104)2 as the and thorine as indicator, taking 1 2 . 3 4 theoretical percent S-contents. Determination of the C1-contents, also after combustion, through titration with 0.01-N Hg(C104 12 and diphenylcarbazone as indicator can be adopted for the drug assay. The theoretical percent content of sulfer is 1 3 . 6 5 ( 2 ) . 9-22.
Instrumental Methods 9-221.
Colorimetry and spectrophotometry i-
Colorimetry
On heating azintamide solution with a solution of p-dimethylaminobenzaldehyde in 70% sulfuric acid, a yellow color is developed with an absorption maximum at 450 nm. Abdel-Moety et al. ( 2 1 ) have adopted the color measurement for quantification of the drug in bulk form and in tablets and effervescent granules containing the drug.
ii-
Direct UV-measurement
The measurement of the light absorption of the drug solution in ethanol at 258 nm is recommended by Abdel-Moety et al. ( 2 1 ) for quantitative determination of the drug in raw materials and only in tablets. Recoveries of 100-16 f 1.10 (n = 6 ) , 9 9 . 8 4 f 1 . 0 0 % (n = 6 ) , in case of tablets, and 100.30 f 1.40% (n = 9 ) , for effervescent granules, could be obtained for added azintamide. iiicribed a procedure
Derivative sDectrophotometrY
Abdel-Moety et al. ( 2 3 ) desfirst-derivative (Di) spectrophotometric for quantification of azintamide in
EZZAT M. ABDEL-MOETY AND HAMAD A . AL-KHAMEES
28
admixtures with papaverine hydrochloride, a smooth muscle relaxant, which is commonly dispensed with the choleretic drug together to inhibit its possible spasmomimetic activity in the gastrointestinal tract. i.e. ppm) and Amounts of azintamide (2-20 pg.ml-', papaverine hydrochloride ( 0 . 5 - 6 pg.ml-1) can be accurately quantified. The concentration ranges of both drugs might allow application of the derivative spectrophotometric method to their determination in biological fluids. The Di(dA/dh )-spectrophotometric measurement of azintamide is recommended at 264 nm, while for the other component a worked out simultaneous equation can be applied. The recoveries were 101.02 2 0.91% (n = 5 ) and 100.48 2 1.33% (n = 5 ) for azintamide and papaverine hydrochloride in order. iv-
PMR-sDectrophotometrp
El-Khateeb and Abdel-Moety described the application of proton-magneticresonance spectrophotometry for quantitative determination of azintamide in pure forms and in dosage formulations. The method involves comparing the integral of both the multiplet centered at about 1 . 1 5 ppm and the sharp singlet at 4 . 3 0 ppm of azintamide molecule to that of the sharp singlet signal at about 6 . 3 0 ppm of maleic acid which is chosen as internal standard. (24)
9-222.
SDectrofluorometry
Abdel-Moety et al. ( 2 5 ) discussed the coupling possibility of TLC-separation (on layers of silica gel 60 F 2 5 4 ) with spectrofluorimetry (EX : 229 nm and EM : 304) for quantificatrion of azintamide in biological fluids. Amounts of 0 . 5 - 2 . 5 ng.ml-1 (ppb) of the drug in urine samples of a healthy volunteer could be accurately traced. Recovery mean percent of 97.25 f 1 . 4 9 (n = 5 ) could be obtained. 9-223.
Flow-injection analysis ( F I A ]
A single-manifold FIA-system f o r quantitative determination of azintamide via spectrophotometric detection at 258 nm is investigated The limit of quantificaby Abdel-Moety et al. (26). tion and detection is about 5 pg.ml-1 of azintamide
AZlNTAMIDE
29
dissolved and/or extracted in ethanol could be accurately analyzed. A good percent mean recovery of 99.24 f 0.89 (n = 4) could be obtained at an introduction rate of about 150 sample.hr-1 or even more. The obtained results were comparable with those of the direct UV-measurement at the same Amax. 9-224. Chromatographic Techniques i-
Gas-liquid chromatographs ( GLC )
GLC-separation and quantification of azintamide in pharmaceutical formulations has been described by Abdel-Moety ( 2 7 ) . The GLC-separation of the drug extracts in chloroform was undertaken on 150 cm X 4 mm i.d. column packed with 10% silar on Diatomite C-AW, 100-120 mesh, at 250°C using Nz as carrier gas with flame ionization detection at 300'C, isothermally. The calibration graph was rectilinear for gm ml-1 of azintamide with concentrations 0 . 5 - 3 recovery of 98.77 t 1.11% (n = 4). The results were compared with those of the spectrophotometric method adopted by the author and others (21). A GC-columns, 150 cm X 4 mm i.d., packed with 5-10% silicone oil on Gas-Chrom Q can elegantly resolve azintamide under the previously mentioned GLC-conditions nearly with identical retention time of about 8 minutes as in case of the silar-column, but the reproducibility in case of the first column was quite better. ii-
High-Performanc Liquid Chromatographs (HPLC)
HPLC-method for assay of azintamide in one-component dosage forms can be achieved on a 5-pm C18-Novapack column by using mixture of methanol and water (98:2, v/v) isocratically as a mobile phase at ambient temperatures (28). Another is described for quantification of HPLC-procedure azintamide and papaverine.HC1 in binary mixtures and dosage formulations containing both drug substances ( 2 9 ) . The separation and quantification can be done on a 5-pm 100 RP-18 LiChrosphere column by using a solvent mixture of acetonitrile and water (56:44, v/v) isocratically at ambient temperature.
EZZAT M. ABDEL-MOETY AND HAMAD A. AL-KHAMEES
30
9-23.
BioloRical Assap
Azintamide can be quantified by the measurement of its choleretic activity according to the procedure described by Stormann ( 9 ) . The biological measurement is done against the activity of a well known choleretic agent, such as dehydrocholic acid and 1-phenylpropanol, The measurement of the elimination times of bromosulfophthalene (BSP) following suitable intradoudenal doses of the drug in the first 30minutes. 9-3.
Other Analytical Possibilites
Different spectroscopic and chromatographic behaviours of azintamide and the various analytical possibilities for the drug have been discussed in a recent communication ( 3 0 ) .
ACKNOWLEDGEMENT The authors would like to thank Dr. R.R. Abou-Shaaban for DSC-investigation and Mr. T.A. Butt for typing the manuscript.
31
AZINTAMIDE
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2.
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32
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23
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