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Noscapine
NOSCAPN Mohammed A. Al-Yahya and Mahmoud M . A . Hassan
408
1. Description 1. I Nomenclature 1.2 Formulae 1.3 Molecular Weight 1.4 Elemental Composition 1.5 Appearance, Color, Odor, and Taste 2. Physical Properties 2.1 X-Ray Diffraction 2.2 Solubility 2.3 Dissociation Constant 2.4 Optical Rotation 2.5 Spectral Properties 3. Preparation 3.1 Isolation from Opium 4. Synthesis of Noscapine 4.1 Tissue Culture Method 4.2 Chemical Methods 5 . Biosynthesis of Noscapine 6. Metabolism 7. Methods of Analysis 7.1 Identification Tests 7.2 Microcrystal Tests 7.3 Titrimetric Methods 7.4 Complexometric Methods 7.5 Spectrophotometric Methods 7.6 Chromatographic Methods 8. References
Analytical Profilesof Drug Substances Volume 1 I
408
408
414 414 414 414 414 415 415 415 416 429 429 429 429 429 429 43 3 436 436 436 439 441 441 445 45 6
407
Copyight 0 1982 by The AmdUn phmn.ce~iiulAmciation ISBN 0-12-260811-9
MOHAMMED A. AL-YAHYA AND MAHMOUD M. A. HASSAN
408
1. D e s c r i p t i o n 1.1
Nomenclature
1.1.1 Chemical Names a-
(S)-6,7-Dimethoxy-3-(5, 6, 7 , 8tetrahydro-4-methoxy-6-methyl-l , 3-d i o x o l o [ 4,5-g] i s o q u i n o l i n - 5 - y l ) - l (1) (3 H) -isobenzof uranone.
b-
1-a -2-methyl-8-methoxy-6,7-methylene dioxy-1- ( 6 , 7-dimethoxy-3-phthalidyl) 1, 2 , 3 , 4-tetra-hydroisoquinoline.
-
(1)*
1.1.2
c-
1 (3 H) Isobenzofuranone, 6 , 7 dimethoxy-3-(5, 6, 7 , 8-tetrahydro-4methoxy-6-methyl-1, 3-dioxolz-[4, 5-g] isoquinolin-5-y1)-, [s-(R* s ) I . (2)
d-
(3 S)-6, 7-dimethoxy-3-[(5R)-5, 6, 7 , 8-tetra-hydro-4-methoxy-6-methyl-1,3-dioxolo [ 4 , 5-g] isoquinolin-5-y1] phthalide. (3).
Generic Names d-Gnoscapine; 1-6-Narcot i n e ; I-Narcot i n e ; N a r c o t i n e ; Noscapine.
1.1.3
Trade Names Capval; Coscopin; Coscotabs; Keyt u s s c a p i n e ; Longatin; Lyobex; Fethoxyh y d r a s t i n e ; Narcompren; Narcosine; Marcotussin; N e i t a c l o n ; Nicolane; Nipaxon; Noscapal; Noscapalin; NSC 5366; Opian; Opianine; Terbenol; Tusscapine; Vadebex.
1.2
Formulae
1.2.1
Empirical
409
NOSCAPINE 1.2.2
Structural
OCH3
Nos c a p i n e The a l k a l o i d n o s c a p i n e can be c l e a v e d v e r y r e a d i l y i n t o two m o i e t i e s ; w i t h d i l u t e s u l f u r i c a c i d , c o t a r n i n e and o p i a n i c a c i d a r e g e n e r a t e d . Under a c i d i c r e d u c i n g c o n d i t i o n s , e.g., zinc i n hydrochloric acid o r s u l f u r i c a c i d , h y d r o c o t a r n i n e and meconine a r e formed (Scheme 1 ) . With t h e s t r u c t u r a l e l u c i d a t i o n of c o t a r n i n e , o p i a n i c a c i d , h y d r o c o t a r n i n e and meconine, and g i v e n t h e p r e s e n c e of a l a c t o n e r i n g i n n o s c a p i n e , the structure o f t h i s a l k a l o i d was e s s e n t i a l l y e s t a b l i s h e d ( 4 ) . 1.2.3
CAS No
(12 8- 62 - 1) 1.2.4
Niswesser L i n e N o t a t i o n T C566 DO FO K N EH & & T J H 01 K J T56 B VO D H J HOL 1 0 1 "ALPHA" LV.
1.2.5
S t e r e o c h e m i s t r y and A b s o l u t e C o n f i g u r a t i o n T h e s t e r e o c h e m i s t r y of n o s c a p i n e h a s been s t u d i e d by many w o r k e r s (5-9). The prolonged a c t i o n of h o t m e t h a n o l i c p o t a s s i u m h y d r o x i d e on n a t u r a l (-)- a - n a r c o t i n e r e s u l t s i n t h e f o r m a t i o n of a n e q u i l i b r i u m m i x t u r e of t h e o r i g i n a l b a s e and a new o p t i c a l l y
410
MOHAMMED A. AL-YAHYA AND MAHMOUD M. A. HASSAN
'n'
'2
OCH3
Noscapine
OCH 3
OC!I
Hydrocotarnine
Cotarnine
+
+
CRO
OCH
OCH
Opianic acid
Meco n ine Scheme 1
41 1
NOSCAPINE a c t i v e d i a s t er eoisomer , (-) -6 -narc0 t i n e , which can be w r i t t e n a s shown i n Scheme 2. Lithium aluminum h y d r i d e r e d u c t i o n of t h e a- and 8-noscapines r e a d i l y a f f o r d s anarcotinediol and 6 - n a r c o t i n e d i o l respectively
.
1
2
A c e t y l a t i o n of t h e s e d i o l s g i v e s r i s e t o t h e corresponding d i a c e t a t e s 2 and k , b u t subsequent c a t a l y t i c h y d r o g e n o l y s i s y i e l d s one and t h e same d e x t r o r o t a t o r y benzyli s o q u i n o l i n e 2. The f o r e g o i n g sequence c l e a r l y e s t a b l i s h e s t h a t a- and 8-noscapine must d i f f e r from each o t h e r o n l y i n t h e i r s t e r e o c h e m i s t r y a t C-9. The b e n z y l i s o q u i n o l i n e 5 shows a p o s i t i v e Cotton e f f e c t near 295 m u , s o t h a t i t s C-1 hydrogen must b e a l p h a as i n d i c a t e d . I t f o l l o w s t h a t t h e C-1 hydrogen i n (-)-an a r c o t i n e and i n (-)- 6 - n a r c o t i n e must a l s o be a l p h a (Scheme 2 ) .
A 1 t e r n a t i v e l y , a-narco t ined i o l w a s c y c l i z ed v i a i t s monomesylate d e r i v a t i v e t o t h e Nm e t h o t e t ra h y d r o p r o t o b e r b e r i n e s a l t 5. T h i s material underwent N-demethylation on p y r o l y s i s t o y i e l d t h e p r o t o b e r b e r i n e b a s e 7. Reductive removal of t h e hydroxyl group w a s achieved i n e t h a n o l i c p e r c h l o r i c a c i d o v e r a palladium c a t a l y s t . The t e t r a h y d r o p r o t o b e r b e r i n e 8 t h u s o b t a i n e d showed a s t r o n g n e g a t i v e r o t a t i o n , so t h a t i t s C-14 hydrogen must be a l p h a . The i d e n t i c a l sequence w a s c a r r i e d o u t using p n a r c o t i n e d i o l t o y i e l d t h e tetrah y d r o p r o t o b e r b e r i n e b a s e ?. Hydrogenolytic c l e a v a g e of t h i s s p e c i e s t h e n provided t h e same 1evor o t a t o r y t e t r a hyd r o pro t o b er b er i n e 8. The c o n c l u s i o n i s t h a t t h e C-1 hydrogens i n b o t h a- and 6-noscapine a r e a l p h a (Scheme 3 ) . Turning t o t h e s t e r e o c h e m i s t r y a t C-9 f o r (-)-a and (-)- B-narcotine, m o l e c u l a r models i n d i c a t e d t h a t t h e d i h e d r a l a n g l e between t h e p r o t o n s a t C-13 and C-14 of t h e 13-a-hydroxy
-
412
MOHAMMED A. AL-YAHYA AND MAHMOUD M . A. HASSAN
CH3
OCH3 (-) -6-Narcotine
(-)-a-Narcotine ( n a t u r a l isomer)
1
1 --
L1A1H4
LiA1H4
OCH~
a-Narcotinediol
1.
1 -
B-Narcotinediol
1 --
Ac 0, pyridine
2
-
-
Ac 20 , P Y ~
idine
OAc CH20Ac OCH3 0CH3
Scheme 2
413
NOSCAPINE
H
........OH
pyridine
6 @CH3 d-Narcotinediol
7 -
as o u t l i n e d
9 OCH3 B-Narcotinediol Scheme 3
MOHAMMED A. AL-YAHYA AND MAHMOUD M. A. HASSAN
414
b a s e 7 i s a b o u t 160'. On t h e o t h e r hand, f o r the 13-6 -hydroxy b a s e 2 d e r i v e d from 6 - n a r c o t i n e , t h i s a n g l e is o n l y a b o u t 60'. Following exchange of t h e h y d r o x y l i c p r o t o n s f o r d e u t e r i u m , i t w a s determined t h a t t h e s p l i t t i n g c o n s t a n t .J13,14 w a s 9 Hz f o r and o n l y a b o u t 1 . 5 Hz f o r 9. species The l a r g e c o u p l i n g v a l u e of 9 Hz i s accord w i t h a t r a n s arrangement of t h e C-13, 1 4 hydrogens i n I_, and t h e s m a l l c o u p l i n g c o n s t a n t of 1 . 5 Hz a r g u e s f o r a c i s r e l a t i o n s h i p i n 9, t h u s s e t t l i n g t h e s t e r e o c h e m i s t r y a t C-9 f& a- and 8-noscapine.
1,
1.3
iz
Molecular Weight 413.43
1.4
E l m e n t a l Composition C, 63.91%; H, 5.61%; N , 3.39%; 0 , 27.09%
1.5
Appearance, C o l o r , Odor
and Taste
Noscapine o c c u r s i n t h e form of Orthorhombic b i s p h e r o i d a l p r i s m s , t a b l e t s from d i a c e t o n e o r a s f i n e , almost w h i t e c r y s t a l l i n e powder. T r i b o l u m i n e s c e n t d 1.395. I t i s o d o r l e s s and tasteless. 2.
Physical Properties 2.1.1
X-ray d i f f r a c t i o n Crystallographic d a t a f o r noscopine are s c a r c e . The o n l y r e p o r t e d d a t a is due t o Love11 (10) and Steward and P l a y e r (11). These a r e a s f o l l o w s : Long needle-shaped c r y s t a l s were o b t a i n e d by r e c r y s t a l l i s a t i o n of t h e commercial n o s c a p i n e from e t h a n o l o r methanol. Weissenberg photographs t a k e n w i t h Cu Ka (1.5418 A)' r a d i a t i o n revealed the following systematic absences:
hOO, h
= 2n
+
1
OKO, K = 2n -I-1 001, 1 = 2n + 1
415
NOSCAPINE
d e f i n i n g unambiguously t h e space group P212121. C e l l dimensions were o b t a i n e d from 28 v a l u e s of 32 r e f l e x i o n s from n o s c a p i n e u s i n g two a x e s i n each c a s e , measured w i t h a counter diffractometer. The f o l l o w i n g d a t a were o b t a i n e d : 413.41
M.W. M.p. (OC)
r
C r y s t a l system Space group Cell
Dimensions
(8)
d 3 )
z
O r t hor homb i c
P212121 15.398(12) b 32.686(36) c ( p r i m ) 8.022(8) 4037(11)
8
Qcalc (g. ~ m - ~ )
1.360
Qexp (g ~ m - ~ )
1.38
.
2.1.2
178
Melting P o i n t 174-176OC(3) 176OC s u b l i m e s a t 15OoC-16O0C under 11 mm p r e s s u r e a t 2 mm d i s t a n c e (1)
2.2
Solubility I t i s i n s o l u b l e i n water; s l i g h t l y s o l u b l e i n a l c o h o l ( 9 5 % ) , i n e t h e r and i n carbon t e t r a c h l o r i d e . S o l u b l e i n chloroform, benzene and v e r y s o l u b l e i n a c e t o n e (12).
2.3
D i s s o c i a t i o n Constant I t i s a v e r y weak b a s e , pKa 7.8 ( 1 ) and 4.85 i n 80% m e t h y l c e l l o s o l v e ( 1 3 ) .
2.4
Optical Rotation
+
42'
to
+
(2% w/v i n 0 . 1 M h y d r o c h l o r i c a c i d ) (3) [ u I D - 198O (1% w/v i n c h l o r o f o r m ) ,
[a],
48'
MOHAMMED A. AL-YAHYA AND MAHMOUD M. A. HASSAN
416
[a],
[a], [a],
2.5
- 146 (2% w/v i n t o l u e n e ) , - 147' (1.59% i n b e n z e n e ) ,
+
50 (1% w/v i n h y d r o c h l o r i c a c i d ) (14)
Spectral Properties 2.5.1
U l t r a v i o l e t Spectrum The W spectrum of n o s c a p i n e i n methanol w a s scanned from 200 t o 400 nm u s i n g V a r i a n Carry 119 Spectrophotometer. I t e x h i b i t s a c h a r a c t e r i s t i c UV spectrum ( F i g . 1) w i t h two maxima: Xmax 310.2 290.6
1%
cm
114.9 106.4
(C, 9.42 mg p e r 100 ml) (C, 9.42 mg p e r 1 0 0 ml)
Other UV s p e c t r a l d a t a of n o s c a p i n e have a l s o been r e p o r t e d : Xmax 209 291 309-310 Xmax 291 310 291 3 09 2.5.2
Log
E
4.86 3.60 3.69
) ) )
i n e t h a n o l (1, 15)
(E)
a b o u t 1.1 ) a b o u t 1.4 ) 3981 4898
1 )
i n a l c o h o l 95% (3) i n methanol (12)
I n f r a r e d Spectrum The I R s p e c t r a of n o s c a p i n e a s K B r d i s c and n u j o l m u l l were r e c o r d e d on a P e r k i n E l m e r FT-680B s p e c t r o p h o t o m e t e r and shown in F i g . 2 t?, F i g . 3 respectively. The s t r u c t u r a l a s s i g n e m e n t s have been c o r r e l a t e d w i t h t h e f o l l o w i n g band f r e q u e n c i e s (Table 1)
417
NOSCAPINE
Fig.
1.
W Spectrum of N o s c a p i n e i n M e t h a n o l .
Wavelmgt h F
Fig.
*O
2.
5.0
6.0
70
8.0
s.0
I R Spectrum of Noscapine as K B r d i s c .
f0
12
!4
2500
Fig. 3 .
2000
1
.
I R Spectrum of Noscapine as Nujol Mull.
800
700
420
MOHAMMED A . AL-YAHYA A N D MAHMOUD M. A . HASSAN T a b l e 1. I R C h a r a c t e r i s t i c s of Moscapine Frequency c m
-1
Assignement
3000, 2945, 2880, 2845, 2800
Methylened i o x y and C-H and -CH frequencies. 3 ( y - l a c t o n e ) 3-C=O g r o u p
17 60 1625 1600,1505,1480, 1280-1 22 5
-c=c-
Aromatic Aromatic m e t h o x y - a r y l C-0 stretching vibrations.
790, 815, 8 3 5 , 885
2 a d j a c e n t H atoms, i s o l a t e d H atom C-H o u t of p l a n e d e f o r m a t i o n . Tetra and pen t a s u b s t i t u t e d b e n z e n e s
.
Other c h a r a c t e r i s t i c a b s o r p t i o n bands are: 1460, 1430, 1405, 1 3 9 0 , 1 3 8 0 , 1 3 6 5 , 1 3 3 0 , 1310, 1 2 0 0 , 1120, 1 0 8 5 , 1040, 1 0 1 0 , 980, 930, 900, B O O , 765, 750, 735, 725, 715, and 700 cm-'. O t h e r I R d a t a a r e a l s o r e p o r t e d (16) 2.5.3
N u c l e a r Magnetic Resonance S p e c t r a 2 . 5 . 3 . 1 P r o t o n Spectrum The PMR s p e c t r u m of n o s c a p i n e i n d e u t e r a t e d chloroform w a s r eco r d ed on a V a r i a n XL200, 2 0 0 MHz NMR s p e c t r o meter u s i n g t e t r a m e t h y l s i l a n e a s a r e f e r e n c e s t a n d a r d ( F i g . 4 ) . The following s t r u c t u r a l a s s i g n m e n t h a v e been made ( T a b l e 2 ) . 4
I, 0
7
F i g . 4.
6
5
4
PMR Spectrum of Noscapine and T e t r a m e t h y l s i l a n e
3
2
i n Deuterated Chloroform.
MOHAMMED A . AL-YAHYA AND MAHMOUD M.A. HASSAN
422
Table 2.
PMR C h a r a c t e r i s t i c s of Noscapine
Ass ignemen t (Group)
Po s i t i o n
Chemical S h i f t ( 6 )
3 , 4 of i s o q u i n o l i n e
2.32 (m)
N- CH3
2 of i s o q u i n o l i n e
2.53 ( s )
OCH3
8 of i s o q u i n o l i n e
3.84 ( s )
OCH3
&'of p h t h a l i d y l
4.02 (s)
OCH3
5'of
4.08 ( s )
-CH2-CH2
phthalidyl
-CH-
1 of i s o q u i n o l i n e
4.37 (d)
-CH-
9 of p h t h a l i d y l
5.55 (d)
-CH2-
methylened i o x y
5.92 ( s )
-CH-
2'0f
6.05 (d)
-CH-
5 of i s o q u i n o l i n e
6.29 ( s )
-CH-
3'of
6.94 (d)
phthalidyl phthalidyl
s = s i n g l e t , d = doublet, m = multiplet
Other PMR s p e c t r a l d a t a was a l s o r e p o r t e d ( 1 7 an.d 5 5 ) . 2.5.3.2
I3C-NMR
Spectra
I3C-NMR c o m p l e t e l y decoupled and o f f - r e s o n a n c e s p e c t r a are shown i n Fig. 5 and Fig. 6 r e s p e c t i v e l y . Both were r e c o r d e d o v e r 11001.1 HZ r a n g e , i n d e u t e r a t e d c h l o r o f o r m (CDC13) on XL-200,200 MHz NMR s p e c t r o m e t e r . Using 10 mm sample t u b e and tetramethylsilane as reference standard a t 25OC. The c a r b o n chemical s h i f t s a s s i g n e d on t h e b a s i s of t h e a d d i t i v i t y p r i n c i p a l s and o f f - r e s o n a n c e s p l i t t i n g p a t t e r n (Table 3) (18).
9
a Fig. 5.
7
6
5
4
1 3
2
f
l3C-NMR Spectrum of Noscapine i n Deuterated Chloroform.
0
3
1
424
50
1
T
60
70
80
1
90
100 110 120 130 1 4 0 150 160
170
190 200 210 220 230 240 250 260 270 280 290 300
180
1
310
1
1 2
320 330
340 350 360 370 380 390 400 410 420 430 440 4 5 0 Fig. 7.
EI-Mass
Spectrum of Noscapine.
MOHAMMED A . AL-YAHYA AND MAHMOUD M. A. HASSAN
426
’
22
OCH3
Table 3. Carbon No.
c-1 c- 2
Carbon Chemical S h i f t s of Noscapine
Chemical S h i f t PPm 60.84 49.99 28.03 134.03 117.65 140.45 100.73 141.14 152.18 132.09 59.36
c-3
c-4
c-5 C-6
c-7
C-8 c- 9 c-10
c-11 2.5.4
(d) (t) (t)
(s)
(d) (s) (t) (s)
(s) (s) (q)
Carbon No.
Chemical S h i f t Ppm
c-12 C-13 C-14 C-15 C-16 C-17 C-18 c-19 c-20 c-21 c-22
46.29 81.83 120.17 118.19 102.29 147.67 148.37 117.11 168.06 56.78 62.22
(q) (d) (s) (d)
(d) (s) (s) (s) (s) (4)
(q)
Mass Spectrum
The mass spectrum of n o s c a p i n e by e l e c t r o n impact i o n i z a t i o n and recorded on Ribermag R-10-10 mass equibbed w i t h d i r e c t i n l e t probe. ( F i g . 7) shows m o l e c u l a r i o n peak a b a s e peak a t m / e 220.
obtained which w a s spectrometer The spectrum and shows
The mass spectrum of n o s c a p i n e o b t a i n e d by butane chemical i o n i z a t i o n ( F i g . 8) shows a m o l e c u l a r i o n peak PI+ a t m / e 413 w i t h a r e l a t i v e i n t e n s i t y of 2.8% and a b a s e peak a t m / e 220. The most prominent f r a g m e n t s ,
90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 2t
1
290 300 31 0 320 330 340 350 360 370 380 390 4-00410 420 430 440 450 460 470 Fig.
8.
CI-Mass
Spectrum of Noscapine.
428
MOHAMMED A. AL-YAHYA AND MAHMOUD M. A. HASSAN t h e i r r e l a t i v e i n t e n s i t i e s and s t r u c t u r e s a r e l i s t e d i n Table 4 . Other mass s p e c t r a l d a t a f o r p h t h a l i d e i s o q u i n o l i n e s was a l s o r e p o r t e d (19, 2 0 ) . Table 4 .
m/e 413
Mass Fragments of Moscapine Relative Intensity
96
Fragment
2.8
M+
221
220
195
8.0
OH
193 OCH3
429
NOSCAPINE
3.
P r eDara t i o n 3.1
I s o l a t i o n from Opium Noscapine o c c u r s up t o 11%n a t u r a l l y i n opium (Papaver sornniferum L . (Fam. papaveraceae) I t w a s f i r s t d i s c o v e r e d by Derosne i n 1803 ( 2 0 ) , and i s o l a t e d by Robinquet i n 1817 ( 2 1 ) . Noscapine can be s e p a r a t e d from o t h e r opium a l k a l o i d s by t h e procedure o u t l i n e d i n Scheme 4 (90).
.
Another p a t e n t method h a s been a l s o d e s c r i b e d f o r i t s i s o l a t i o n on a n i n d u s t r i a l scale ( 2 2 ) .
4.
S y n t h e s i s of Noscapine
4.1
By T i s s u e C u l t u r e Method Khanna e t a 1 (23) d e s c r i b e d a method f o r t h e s y n t h e s i s of noscapine a l o n g w i t h o t h e r a l k a l o i d s by t i s s u e c u l t u r e of Papaver somniferum Linn.
4.2
By Chemical Methods P e r k i n and Robinson (24) d i s c o v e r e d t h a t h e a t i n g a m i x t u r e of c o t a r n i n e 1 and meconine 1i n e t h a n o l r e s u l t e d i n a s m a l l y i e l d of noscapine 3 . The expected second isomer of noscapine-(because of t h e presence of 2 a s s y m e t r i c c e n t r e s ) w a s n o t found. The s y n t h e t i c noscapine was t h e n r e s o l v e d and t h e n o s c a p i n e o b t a i n e d shown t o b e i d e n t i c a l w i t h t h e n a t u r a l p r o d u c t . (Scheme 5).
A q u i t e e f f i c i e n t s y n t h e s i s of noscapine w a s developed by Hope and Robinson i n 1914 ( 2 5 ) , i n which c o t a r n i n e i s condensed w i t h iodomeconine 2 and t h e adduct w a s reduced w i t h sodium amalgum t o g i v e t h e d e s i r e d p r o d u c t , corresponding t o t h e n a t u r a l s e r i e s (Scheme 6 ) .
L
5.
B i o s y n t h e s i s of Noscapine I t has been p o s t u l a t e d t h a t t h e ph t h a l i d e i soqu i n o 1i n e s a r e formed i n n a t u r e by o x i d a t i v e m o d i f i c a t i o n of t e t r a h y d r o p r o t o b e r b e r i n e s , and p r e v i o u s work w i t h l a b e l e d precursors supports t h i s hypothesis (26).
MOHAMMED A. AL-YAHYA AND MAHMOUD M. A. HASSAN Powdered Op i um
430
+
+
Shake w i t h warm calcium c h l o r i d e s o l u t i o n F i 1t e r Insoluble matter C (discard)
F i ltrate (hydroch l o r i des o f a 1ka l o i ds)
4
Reduce volume (evaporate under reduced p r e s s u r e ) t o syrupy 1 i q u i d
4
Add 10% NaOH s o l u t i o n Precipitates p k (noscapine, papaverine, thebaine)
4
a
l ine solution (morphine, codeine,
E x t rn aa c rt cw e i nt he ) ci h l o r o f o r m
Dissolve i n d i l u t e a l c o h o l
$.
Add a c e t i c a c i d t o make s l i g h t l y
0
acidii
Add 3 volumes o f b o i l i n g water
A Solution
Aqueous extract alkaline (containsolution i n g codeine) (morphine, narceine)
C h 1 o r o f orm
4
rz 1
Precipitate (papave r ine, noscap ine)
(theba ine)
\
4
Further pur i f ic a t i o n
Make a c i d i c
4
Aqueous a c i d i c s o l n . ( s a l t s of morphine and na r c e i ne)
Dissolve i n b o i l i n g 0.33%(aqueous)oxali c a c i d soln.
4
4
Make s l i g h t l y a l k a l i n e w i t h ammonia
A l l o w t o stand
Bring t o b o i l i n g A l l o w t o stand
C rys t a 1 s (papave r ine ac id oxalate)
4
Further purification
Sol u t i o n (noscap ine oxa 1 a t e )
+
Repeat
Precipitate (morphine)
I
Further p u r i f ic a t ion
Solution (narceine)
Make a l k a l i n e w i t h ammonia
Precipitate (noscapine)
+
1 i
+
s o l u t on (discard)
Dissolve i n b o i l i n g alcohol Crystallization Scheme 4:
i
Further pur i f i cat ion
Isolation of
from powdered op i um .
NOSCAPINE
43 1
OCH3
OCH 2 Plecoriine
I
Cotarnine
D
C2H50H
0
0ch3 3
(2)-a-Narco t ine
Scheme 5
432
MOHAMMED A. AL-YAHYA AND MAHMOUD M . A. HASSAN
Cotarnine 1
Iodomeconine
\
\
CH 3
H..
... *..s . 0
2
CH 3
CH 3O
Na/Hg
OCH3
OCIl
3
(+) -a-Narcotine
Scheme 6
NOSCAPINE
433
S e v e r a l f e e d i n g e x p e r i m e n t s ( 6 , 8 , 94 ) have been r u n t o e l u c i d a t e t h e b i o g e n e s i s of n o s c a p i n e i n Papaver somnif erum L . (Papaveraceae) When l a b e l e d (+) t y r o s i n e was f e d t o t h e p l a n t , r a d i o a c t i v e n a r c o t i n e l a b e l e d s p e c i f i c a l l y and e q u a l l y a t C-1 and C-3 was o b t a i n e d . The b e n z y l i s o q u i n o l i n e s y s t e m of n o s c a p i n e i s t h u s d e r i v e d b i o l o g i c a l l y from two Ar-C-C u n i t s which c a n a r i s e from t y r o s i n e .
.
The c a r b o n atoms t h a t a r i s e from t h e S-methyl of m e t h i o n i n e were c l e a r l y p i n p o i n t e d when, a f t e r f e e d i n g radioactive methionine, noscapine labeled a t t h e l a c t o n e c a r b o n y l , t h e m e t h y l e n e d i o x y g r o u p , and t h e N- and 0-methyl c a r b o n atoms w a s o b t a i n e d ( 2 7 , 2 8 ) . Progressing f u r t h e r along the biogenetic locus, t h e b e n z y l i s o q u i n o l i n e (+)- n o r l a u d a n o s o l i n e l a b e l e d C-1 l e d t o n o s c a p i n e a l s o l a b e l e d C-1 ( 2 9 ) . Even more s i g n i f i c a n t l y , when q u a d r u p l y l a b e l e d (+)- and (-) r e t i c u l i n e were f e d s e p a r a t e l y t o P. somniferum, i t w a s found t h a t b o t h enantiomers were i n c o r p o r a t e d i n t o n o s c a p i n e , b u t w i t h t h e (+)-isomer d o i n g so s l i g h t l y more e f f i c i e n t l y . E v i d e n t l y e p i m e r i z a t i o n of t h e wrong b e n z y l i s o q u i n o l i n e p r e c u r s o r must o c c u r , p r o b a b l y by o x i d a t i o n - r e d u c t i o n a t C-1. I n keeping w i t h t h i s c o n c l u s i o n c o n s i d e r a b l e l o s s of t r i t i u m o c c u r e d i n t h e c o u r s e of i n c o r p o r a t i o n of b o t h r e t i c u l i n e s . Another i m p o r t a n t o b s e r v a t i o n i s t h a t t h e l a c t o n e c a r b o n y l of t h e p h t h a l i d e i s o q u i n o l i n e must b e d e r i v e d from t h e N-methyl group of t h e b e n z y l i s o q u i n o l i n e p r e c u r s o r ( 2 7 , 29, 3 0 ) . F i n a l l y , i t h a s been found t h a t t h e f e e d i n g of l a b e l e d ( - ) - s c o u l e r i n e r e s u l t s i n t h e f o r m a t i o n of r a d i o a c t i v e noscapine. Protoberberines are, therefore, t h e precursors f o r t h e phthalideisoquinolines i n plants. S i g n i f i c a n t l y , ( - ) - s c o u l e r i n e , which p o s s e s s e s t h e same a b s o l u t e c o n f i g u r a t i o n a s ( + ) - r e t i c d i n e and ( - ) - a - n a r c o t i n e , w a s more t h a n one hundred t i m e s more e f f i c i e n t t h a n i t s enantiomer a s a p r e c u r s o r f o r ( - ) - a - n a r c o t i n e . The b i o g e n e t i c sequence i n p l a n t s i s , t h e r e f o r e , b e n z y l i s o qu i n o l i n e s +- t e t r a h y d r o p r o t o b e r b e r i n e s + p h t h a l i d e i s o q u i n o l i n e s . The b i o s y n t h e s i s of (-)-cC-narcotine i s shown i n Scheme 7 .
6.
Metabolism The m e t a b o l i s m of n o s c a p i n e was r e p o r t e d
MOHAMMED A. AL-YAHYA AND MAHMOUD M. A. HASSAN
434
&:
Labeled t y r o s i n e
*<:w bCH3
H $H3-S-(CH
1
Labeled noscapine
Papaver
) -C-COOH 2 2 1 somn i f e r u m )
NH2 Labeled methionine
I
N\*
CH 3O
CH 3
- 0
OgH3 L a b e l e d nos cap i n e
Laheled ( a - n o r l a u d a n o s o l i n e Scheme 7
L a b e l e d n‘bscapine
435
NOSCAPINE
Scheme 7 ( c o n t i n u e d )
(4)
"H3
Labeled (+) - r e t i c u l i n e (Labeled (-) - r e t i c u l i n e somewhat less e f f i c i e n t )
R = H o r T Labeled n o s c a p i n e ( a p p r e c i a b l e l o s s of tritium)
CH30 HO
OCH3
Labeled (-) - s c o u l e r i n e
Labeled noscapine (Some t r i t i u m l o s s )
MOHAMMED A . AL-YAHYA AND MAHMOUD M . A . HASSAN
436
(31, 3 2 ) . Oral a d m i n i s t r a t i o n of to male r a b b i t s and e x a m i n a t i o n o f t h e 24 h o u r s u r i n e by p r e p a r a t i v e TLC and methane c h e m i c a l i o n i z a t i o n mass s p e c t r o m e t r y r e v e a l e d t h e p r e s e n c e of two O-monodemet h y l a t e d compounds a s f r e e m e t a b o l i t e s 2 and 5, One 0 - d i d e m e t h y l a t e d d e r i v a t i v e 2 o r 5 and t h e i r c o n j u g a t e d forms. Noscapine g i v e n orally to rats was m e t a b o l i s e d t o di-0-demethyl-noscapine 3 or 4 , cotarnine 5 , h y d r o c o t a r n i n e 1,o x y c o t a r n i n e 2, a n d 0-demethylmeconine 5. T h e s e m e t a b o l i t e s were i s o l a t e d from u r i n e . A l l p o s s i b l e m e t a b o l i t e s of n o s c a p i n e a r e shown i n Scheme 8.
7.
Methods of A n a l y s i s
7.1
I d e n t i f i c a t i o n Tests The f o l l o w i n g i d e n t i f i c a t i o n t e s t s a r e d e s c r i b e d by t h e B r i t i s h Pharmacopoeia ( 1 9 8 0 ) . a)
The l i g h t a b s o r p t i o n , i n t h e r a n g e 230 t o 350 nm, of a 0.005 p e r c e n t w/v s o l u t i o n i n m e t h a n o l e x h i b i t s two maxima, a t 291 nm and 310 nm and a minimum a t 263 nm; r a t i o of t h e a b s o r b a n c e a t t h e maximum a t 310 nm t o t h a t a t t h e maximum a t 291 nm, a b o u t 1 . 2 .
b)
To 1 0 mg add 0 . 5 m l of s u l f u r i c a c i d and mix; a g r e e n i s h - y e l l o w s o l u t i o n i s formed which t u r n s r e d and f i n a l l y v i o l e t o n h e a t i n g .
c)
7-2
S o l u t i o n s i n organic s o l v e n t s , such as
methanol and c h l o r o f o r m , a r e l e v o r o t a t o r y ; aqueous a c i d i c s o l u t i o n s are d e x t r o r o t a t o r y .
M i c r o c r y s t a l Tests a)
According t o t h e method of C l a r k e and W i l l i a m s (33) , i n potassium chromate s o l u t i o n , n o scap i n e f o r m s f e a t h e r y r o s e t t e s o r b u n c h e s of b l a d e s , s e n s i t i v i t y b e i n g 1 i n 1500 ( F i g . 9 ) .
b)
I n sodium c a r b o n a t e s o l u t i o n r o s e t t e s and b u n c h e s of n e e d l e s a r e s e e n a t t h e same s e n s i t i v i t y (Fig. 10).
NOSCAPINE
437
Fig. 9 .
C r y s t a l s of Noscapine with Potassium Chromate S o l u t i o n .
Fig. 10.
C r y s t a l s of Noscapine with Sodium Carbonate S o l u t i o n .
MOHAMMED A . AL-YAHYA AND MAHMOUD M. A. HASSAN
43 8
Scheme 8 .
P o s s i b l e M e t a b o l i t e s of (-)-a-narcotine
J
be@ ‘ 0
0
OCH3
CH3
nos c a p i n e 1
5
0-demezhyla ted meconine
cotarnine
1
hydrocotarnine
CH3 c o t a r n i n e (pseudo b a s e form) OCH3
ox yc o t a r n i n e
0-d i d eme t h y l a t e d m e t a b o l i t e s
439
NOSCAPINE
7.3
T i t r i m e t r i c Methods The o f f i c i a l methods of d e t e r m i n i n g Noscapine a r e d e s c r i b e d by t h e B.P. (3) and U.S.P. ( 3 4 ) . 7.3.1
Non-Aaueous T i t r a t i o n The B.P. method :-
(3) d e s c r i b e s t h e f o l l o w i n g
D i s s o l v e 0.5 g i n 40 m l of anhydrous g l a c i a l a c e t i c acid previously neutralised t o c r y s t a l v i o l e t , warming g e n t l y . T i t r a t e w i t h 0.1 M p e r c h l o r i c a c i d u s i n g 0.25 m l of c r y s t a l v i o l e t s o l u t i o n a s i n d i c a t o r . Each m l of 0.1 M p e r c h l o r i c a c i d i s e q u i v a l e n t t o 0.04134 g of C22H23 NO,. The U.S.P. met hod :-
describes the following
D i s s o l v e about 1 . 5 g of Noscapine, a c c u r a t e l y weighed, i n 25 m l of g l a c i a l a c e t i c a c i d . Add 25 m l of d i o x a n e and 5 d r o p s of c r y s t a l v i o l e t T . S . , and t i t r a t e with 0.1 N perchloric acid i n g l a c i a l a c e t i c a c i d t o t h e end-point change from p u r p l e t o b l u e . Perform a b l a n k d e t e r m i n a t i o n , and make any n e c e s s a r y c o r r e c t i o n . Each m l of 0.1 N perchloric acid is equivalent t o 41.34 mg of C22H23N07. Another method w a s d e s c r i b e d by T u t h i l l e t a l . (35) u s i n g m a l a c h i t e g r e e n a s b e t t e r indicator than c r y s t a l v i o l e t . 7.3.2
Polarographic T i t r a t i o n a)
Using a dropping mercury e l e c t r o d e a s i n d i c a t o r n o s c a p i n e h y d r o c h l o r i d e c a n be t i t r a t e d w i t h Cadmium I o d i d e i n s o l u t i o n of n e u t r a l s a l t s ( 0 . 1 t I KNO N a C l or 3’ Na2S04) ( 3 6 ) .
b)
Dusinsky (37) d e s c r i b e d a method where noscapine c a n be t i t r a t e d i n a l k a l i n e
440
MOHAMMED A. AL-YAHYA AND MAHMOUD M. A. HASSAN s o l u t i o n (1.25 N NaOH) showing depression i n t h e polarographic curve a t 1.5V. Sodium a l i z a r i n s u l p h o n a t e h a s been used f o r t h e d e t e r m i n a t i o n of n o s c a p i n e (38). A p o t e n t i a l of - 0 . 6 5 V was used and t h e t i t r a t i o n medium was 0.3 N K C 1 a d j u s t e d t o a pH of 4 - 6 . SoucKova and S;ka (39) s t a t e d a method using t u n g s t o s i l i c i c acid. This acid w a s found e s p e c i a l l y u s e f u l s i n c e t h e r e a c t i o n i s v e r y s e n s i t i v e and prec i p i t a t i o n i s immediate. The p o l a r o graphy a l s o gave t h e c o m p o s i t i o n of t h e t u n g s t o s i l i c i c acid organic base complex. The poor s e l e c t i v i t y of T u n g s t o s i l i c i c a c i d i s due t o i t s h i g h s e n s i t i v i t y which a l l o w s a c c u r a t e d e t e r m i n a t i o n of 10-20 mg of b a s e . A 0.01 M aqueous s o l u t i o n of t u n g s t o s i l i c i c a c i d i s used w i t h a dropping mercury c a t h o d e and S . C . E . anode a t 0 . 6 5 V . The pH of s o l u t i o n a d j u s t e d w i t h HC1 ( 0 . 1 t o 0 . 6 N ) . P l o t of c u r r e n t vs a c i d used c o n s i s t s of two s t r a i g h t l i n e s and t h e i n t e r s e c t c o n s i d e r e d as t h e e q u i v a l e n c e p o i n t . Another method f o r p o l a r o g r a p h i c t i t r a t i o n was a l s o r e p o r t e d ( 4 0 ) . T h i s method is based on t h e f o r m a t i o n of complex mercury compounds. S o l u t i o n of K2HgI4 c o n t a i n i n g a n e x c e s s of i o d i d e i s t h e most s u i t a b l e f o r t h e determinat i o n . The t i t r a t i o n i s c a r r i e d o u t w i t h a d r o p p i n g mercury e l e c t r o d e a t t h e p o t e n t i a l -0.8 V t o - 0.9 V (VS. t h e S . C . E . ) w i t h 0 . 1 PI KN03 o r 0 . 1 M H SO a s s u p p o r t i n g e l e c t r o l y t e . 2 4
7.3.3
P o t e n t iometr i c T i t r a t i o n Tungsten rod w a s used a s i n d i c a t o r e l e c t r o d e i n t h e p o t e n t iometr i c t i t r a t i o n of n o s c a p i n e i n a 1 : 6 m i x t u r e of a c e t i c a c i d : a c e t i c anhydride ( 4 1 ) .
NOSCAPINE
7.4
441 Complexometric Noscapine is p r e c i p i t a t e d from 0 . 5 N H C 1 w i t h 0.028 M Bi-EDTA and 0.112 M K I forming iodobismuthate complexes and EDTA i s being set f r e e (42). After c e n t r i f u g a t i o n t h e f r e e EDTA i s determined i n a n a l i q u o t of t h e supern a t e n t l i q u i d w i t h 0.01 M ZnSO4 i n pH 9 . 1 b o r a t e b u f f e r and Eriochrome b l a c k T as i n d i c a t o r . T e r t i a r y amines, q u a t e r n a r y ammonium s a l t s o r analogous sulphonium, phosphonium and arsonium compounds i n t e r f e r e i n the determination.
7.5
Spectrophotometric 7.5.1
Colorimetric
a ) Yoichi and Sano (43) d e s c r i b e a method f o r a n a l y s i s of noscapine i n mixed pharmaceutical preparations. The sample is mixed w i t h a s o l u t i o n of chromotropic a c i d 0.2% i n 70% (v/v) H3PO4 a c i d and h e a t e d a t 100°C f o r 30 m i n u t e s and t h e e x t i n c t i o n i s measured a t 570 nm. A c a l i b r a t i o n c u r v e i s r e c t i l i n e a r f o r 30 t o 150 pg of noscapine h y d r o c h l o r i d e per m l . b) Solochrome Green V 150 ( C . I . Mordant Green 15) h a s been used a s aqueous 1 mM s o l u t i o n . The complex formed by noscapine is e x t r a c t e d i n t o chloroform and t h e absorbance i s measured a t 520 nm ( 4 4 ) . c ) Another method f o r t h e q u a n t i t a t i v e s e p a r a t i o n of p a p a v e r i n e from n o s c a p i n e i n m i x t u r e s w a s a l s o r e p o r t e d (45). T h i s method i s based on t h e f o r m a t i o n of a n insoluble papaverine r e i n e c k a t e i n acid s o l u t i o n i n t h e p r e s e n c e of e x c e s s chloroform. Procedure T r i t u r a t e t h e sample (4.5 g) w i t h g l a c i a l a c e t i c a c i d (25 ml) followed by H 2 0 ( 20 ml) and f i l t e r . E x t r a c t a 1 0 m l a l i q u o t w i t h
442
MOHAMMED A . AL-YAHYA AND MAHMOUD M. A. HASSAN
CHC13 (8 X 1 0 ml) and wash e a c h e x t r a c t i n t u r n w i t h H20 (15 m l ) , H20 (15 ml) p l u s NaOH soln. ( 1 : 1) containing a l i t t l e NaHS03 (15 m l ) , H 2 0 ( 1 5 m l ) , 0 . 1 M H2SO4 (15 m l and 1 0 ml) and 0.05% N a H C 0 3 s o l n . (10 m l ) . E v a p o r a t e t h e combined washed e x t r a c t s t o d r y n e s s o n a water b a t h . D i s s o l v e t h e r e s i d u e i n CCl4 (50 ml) , s t r a i n through cotton-wool and p a s s t h r o u g h a column of Ca(OH)2. Wash t h e column w i t h C C l 4 (2 X 1 0 ml) and e x t r a c t t h e combined CCl4 f r a c t i o n s w i t h 0 . 1 N H C 1 ( 2 X 1 0 m l ) . Shake t h e H C 1 s o l n . w i t h CHC13 (10 ml) f o r 10 m i n . , add 2% ammonium r e i n e c k a t e s o l n . ( 1 0 m l ) , shake f o r 3 0 min. and f i l t e r through s i n t e r e d g l a s s . To d e t e r m i n e papaverine d i s s o l v e t h e ppt. i n acetone and measure t h e e x t i n c t i o n a t 525 mu. To d e t e r m i n e n o s c a p i n e , shake t h e CHC13 l a y e r of t h e f i l t r a t e w i t h 0.25% AgN03soln. ( 4 0 m l ) , s e p a r a t e and f u r t h e r e x t r a c t w i t h CHC13 (2 X 1 0 m l ) ; s t r a i n t h e combined CHC13 f r a c t i o n s through cotton-wool, d i l u t e t o 250 m l , and e i t h e r measure t h e e x t i n c t i o n a t 310 mp o r e v a p o r a t e and t i t r a t e w i t h 0.05 N H C l O 4 i n g l a c i a l a c e t i c a c i d . d) Thomas d e s c r i b e d a method f o r d e t e r m i n a t i o n of some d r u g s c o n t a i n i n g a t e r t i a r y - a m i n e group ( 4 6 ) . The d r u g i s h e a t e d w i t h 10% malonic a c i d i n a c e t i c a n h y d r i d e a t 800 f o r 1 5 min. and, a f t e r d i l u t i o n w i t h e t h a n o l , t h e e x t i n c t i o n i s measured a t 333 nm. t h e l i m i t of d e t e c t i o n f o r n o s c a p i n e hydroDosage c h l o r i d e was 1 0 t o 30 ng m l - 1 . forms r e q u i r e p r e l i m i n a r y e x t r a c t i o n of t h e drug.
7 . 5.2
Infra-red
et a1 (47) d e s c r i b e d a n i n f r a - r e d Bakre s p e c t r o s c o p i c method f o r t h e d e t e r m i n a t i o n of t h e o r i g i n of opium as w e l l a s a s i m u l t a n e o u s a s s a y of n o s c a p i n e , t h e b a i n e and papav e r i n e . 4.5 g f i n e l y ground sample w a s t i t u r a t e d f o r 20 min. i n 25 m l water w a s s l o w l y added w i t h c o n t i n u o u s s t i r r i n g and t h e
443
NOSCAPINE
resulting solution was filtered. 10 m l A l i q u o t of f i l t r a t e w a s e x t r a c t e d f o u r t i m e s i n t o 1 0 m l c h l o r o f o r m and e a c h e x t r a c t w a s washed w i t h 1 0 m l water, 25 m l of 0.2% sodium b i s u l p h i t e i n 30% aqueous sodium h y d r o x i d e , 1 0 m l water and a g a i n 1 0 m l water. The combined c h l o r o f o r m s o l u t i o n was f i l t e r e d t h r o u g h c o t t o n wool and e v a p o r a t e d . The r e s i d u e i s d r i e d i n a d e s i c a t o r t h e n mixed w i t h anhydrous c a r b o n t e t r a c h l o r i d e , f i l t e r e d through s i n t e r e d g l a s s and d i l u t e d t o 25 m l . The I R i s examined from 1100 cm-1 t o 1900 c m - l i n a 1 mm sodium c h l o r i d e c e l l , noscapine b e i n g measured a t 1767 cm-l. The a b s o r b a n c e i s compared w i t h a b s o r b a n c e s of s o l u t i o n s of known c o n c e n t r a t i o n s . Other I R methods f o r d e t e r m i n a t i o n of i s o q u i n o l i n e a l k a l o i d s were a l s o r e p o r t e d (48, 4 9 ) . 7.5.3
Ultra-Violet T e t r a p o n a m i x t u r e of t h e h y d r o c h l o r i d e s of Morphine, Noscapine, c o d e i n e and p a p a v e r i n e w a s a n a l y s e d by J e n s e n ( 5 0 ) . Morphine w a s s e p a r a t e d by e x t r a c t i o n w i t h c h l o r o f o r m from s t r o n g a l k a l i n e s o l u t i o n and d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y w i t h NaN02 a t 440 run. The o t h e r a l k a l o i d s were s e p a r a t e d by T L C on K i e s e l g e l CF 254 w i t h e t h a n o l : benzene 1 : 4 a s s o l v e n t . The s p o t s ( l o c a t e d i n U.V. r a d i a t i o n ) were e x t r a c t e d w i t h methanol and determined a t 215 run f o r c o d e i n e , a t 279 nm f o r p a p a v e r i n e and 312 nm f o r n o s c a p i n e .
7.5.4
Atomic A b s o r p t i o n An i n d i r e c t method f o r t h e a n a l y s i s of Noscapine i n d r u g s w a s r e p o r t e d ( 5 1 ) . A complex i s formed between Noscapine and Reinecke s a l t i n t h e p r e s e n c e of t a r t a r i c a c i d a t pH 1 . 7 . This is extracted i n t o c h l o r o f o r m and Noscapine i s d e t e r m i n e d i n d i r e c t l y by measuring chromium c a t i o n by a t o m i c a b s o r p t i o n s p e c t r op ho tome t er y
.
444
MOHAMMED A . AL-YAHYA AND MAHMOUD M. A. HASSAN 7.5.5
Spectrofluorimetdc a ) Noscapine h a s been determined i n m i x t u r e s of opium a l k a l o i d s (52) by measuring t h e f l u o r e s c e n c e a t 375 run ( e x c i t a t i o n a t 315 nm). The sample i s b u f f e r e d a t pH 9 i n 0 . 1 N s u l p h u r i c a c i d and 0 . 1 N sodium hydroxide. Noscapine i s e x t r a c t e d i n chloroform and a p o r t i o n of t h i s e x t r a c t i s t r e a t e d w i t h t r i c h l o r o a c e t i c a c i d i n chloroform t o quench t h e f l u o r e s c e n c e of papaverine. A s t a n d a r d s o l u t i o n of 2-aminopyridine i n 0 . 1 N s u l p h u r i c a c i d is a l s o measured a t 375 nm ( e x c i t a t i o n a t 315 nm). For t h e c a l c u l a t i o n each f l o u r e s c e n c e r e a d i n g on t h e t e s t s o l u t i o n i s c a l c u l a t e d a s a p e r c e n t a g e of t h a t f o r t h e s t a n d a r d and r e f e r r e d t o c a l i b r a t i o n g r a p h s prepared s i m i l a r l y f o r Noscapine. Sub-microgram amounts of Noscapine c a n b e determined without preliminary separation. b) Vedso s t a t e d a method (53) f o r t h e d e t e r m i n a t i o n of Noscapine i n plasma and urine. I t involves e x t r a c t i o n of 1 m l sample a t pH 1 0 i n t o e t h y l e t h e r and r e - e x t r a c t i o n w i t h d i l u t e HC1. The a c i d i s n e u t r a l i s e d and t h e s o l u t i o n was a d j u s t e d t o PH 9.2 w i t h a borax b u f f e r . Fluorescence i s measured through 480 t o 580 mu f i l t e r ( e x c i t a t i o n a t 365 mu) b e f o r e and a f t e r a u t o c l a v i n g a t 120OC f o r 3 0 min. S t a n d a r d s ( 0 t o 2.5 pg cm-3) are a l s o measured i n t h e same way. I t was s t a t e d t h a t c o n c e n t r a t i o n s from 0.05 ug p e r m l can be determined and a l t h o u g h t h e p r e s e n c e of morphine gave an i n c r e a s e i n f l o u r e s c e n c e about h a l f t h a t f o r n o s c a p h e , c o d e i n e , n a r c e i n e and papaver i n e d i d n o t i n t e r f e r e
.
c ) A r e a c t i o n m i x t u r e of 10%malonic a c i d i n
a c e t i c a n h y d r i d e was used i n a method r e p o r t e d by Rao and Tandon ( 5 4 ) . I n t e r f e r e n c e was caused by T e r t i a r y amines, glucose,magnesium a c e t a t e and some i n o r g a n i c s a l t s , b u t n o t by d i e t h y l a m i n e , a n i l i n e , benzoic a c i d , a s p i r i n and s a c c h a r i n .
NOSCAPINE
445
7.5.6
Nuclear Magnetic Resonance A known amount of t - b u t y l a l c o h o l w a s added as a s t a n d a r d t o n o s c a p i n e i n e t h a n o l f r e e c h l o r o f o r m and t h e peaks a t 3 . 8 3 , 4.00 and 4 . 0 5 ppm; c o r r e s p o n d i n g t o t h e n i n e methoxy group p r o t o n s of n o s c a p i n e were i n t e g r a t e d a l o n g w i t h t h e peak a t 1 . 3 ppm c o r r e s p o n d i n g t o t h e n i n e methyl-group p r o t o n s of t - b u t y l a l c o h o l ( F i g . 11). The amount of n o s c a p i n e i s c a l c u l a t e d from t h e i n t e g r a t i o n r a t i o and t h e known amount of s t a n d a r d ( 5 5 ) .
7.5.7
Mass Noscapine w a s i d e n t i f i e d i n opium (92) w i t h o u t any p r i o r s e p a r a t i o n . Samples a r e introduced d i r e c t l y i n t o t h e ion s our c e us ing a s o l i d sampling probe. Reagent g a s e s were i s o b u t a n e and w a t e r , mass s p e c t r a l measurement was a t m / e 220. Arnold (93) d e s c r i b e d a G C /M S method f o r t h e d e t e r m i n a t i o n of n o s c a p i n e i n opium preparations.
7.6.
Chromatographic
7.6.1
Paper Chromatography The paper-chromatographic method f o r t h e d e t e c t i o n of a l k a l o i d s , e . g . , c o d e i n e , v e r a t r i n e , q u i n i n e and n o s c a p i n e i n s e v e r a l f o o d s ( a 100 g sample) i s d e s c r i b e d . For t h e p r e l i m i n a r y e x t r a c t i o n of t h e a l k a l o i d s , add t o t h e sample 300 m l of e t h a n o l a c i d i f i e d t o l i t m u s p a p e r w i t h H C 1 . D i g e s t o n a water b a t h a t 400 f o r 24 h r . , f i l t e r and r e t a i n t h e f i l t r a t e . Add 100 m l of acidified ethanol to the residue, d i g e s t f o r a n o t h e r 1 2 h r . and f i l t e r a g a i n . Combine t h e f i l t r a t e s and remove t h e a l c o h o l by e v a p o r a t i n g on a w a t e r b a t h a t 4 0 0 . Add 30 m l of water and e x t r a c t w i t h d i e t h y l e t h e r ( 5 X 5 0 m l ) . Add NaOH s o l n . t o t h e a q . l a y e r till i t is a l k a l i n e t o l i t m u s and e x t r a c t w i t h d i e t h y l e t h e r ( 5 X 50 ml). Evaporate t h e e t h e r e x t r a c t t o 50 m l , t r a n s f e r i t t o a s e p a r a t i n g - f u n n e l
446
MOHAMMED A. AL-YAHYA AND MAHMOUD M. A. HASSAN and wash w i t h 2N HCl(3 X 20 m l ) . To t h e combined a c i d washings add NaOH till t h e y a r e a l k a l i n e t o l i t m u s and e x t r a c t w i t h d i e t h y l e t h e r ( 3 X 50 m l ) . E v a p o r a t e t h e e t h e r c o m p l e t e l y and d i s s o l v e t h e extract i n 5 m l of e t h a n o l . R e t a i n t h i s a l c o h o l i c s o h . f o r chromatography by t h e d e s c e n d i n g t e c h n i q u e on Whatman No. 1 p a p e r . Apply d r o p s of t h e t e s t and s t a n d a r d s o l n . t o t h e p a p e r and impregnate i t s whole area above t h e s t a r t i n g l i n e w i t h a f r e s h l y p r e p a r e d e t h a n o l i c s o h . of formamide (1 : 1). Dry t h e paper between two f i l t e r p a p e r s , t h e n a t 400 f o r 3 0 min. Spray t h e s t a r t i n g p o i n t s w i t h t h e e t h a n o l i c s o l n . of formamide (1 : 1) and a f t e r 10 min. t r a n s f e r t h e paper t o a g l a s s c o n t a i n e r c o n t a i n i n g CHC13 a t 250. The development i s complete i n 2.5 h r . Dry t h e chromatogram a t 1050 and s p r a y w i t h a s o h . of potassium i o d o p l a t i n a t e , washing o f f t h e excess w i t h water i n o r d e r t o o b s e r v e t h e s p o t s on t h e w h i t e background. The f o l l o w i n g amounts of a l k a l o i d s can b e d e t e c t e d - c o d e i n e 0.01 t o 0.3 mg, q u i n i n e 0.005 t o 0.015 mg, v e r a t r i n e 0.1 t o 0.15 mg and noscapine 0.05 t o 0.075 mg. ( 5 6 ) . T a b l e 5 d e s c r i b e s methods used i n noscapine a n a l y s i s . A n a l y s i s of noscapine i n opium by paper chromatography and s p e c t r o p h o t o m e t r y involved e x t r a c t i o n of noscapine and measurement of t h e e x t i n c t i o n of t h e s p o t a t 290 mu and comparison w i t h s t a n d a r d s ( 5 7 ) . Other method was a l s o d e s c r i b e d ( 5 8 ) . 7.6.2
Thin Layer Chromatography T h i s t e c h n i q u e h a s been used e x t e n s i v e l y f o r a n a l y s i s of Opium and i t s p r e p a r a t i o n s (63 - 7 1 ) . T a b l e 6 - g i v e s a resume of t e c h n i q u e s and T a b l e 7 - shows s p r a y r e a g e n t s and methods used. S t a h l and co-workers have proposed a s t a n d a r d p r o c e d u r e f o r t h e s e p a r a t i o n of opium a l k a l o i d s ( 6 3 ) . Dried opium ( 0 . 1 g)
... I
S O
. . . .
. - . .
. . . . I . . . . ~ " . . ~ . . . . ~ . . " . ~' I" ' I
I
'
400
300
zbo
I
I
.
I
100
I o ni
H3
1
.
1
I
l . . l . . . ' l . . . ' l . . . . i . . - . l
.
1 . .
.
1
. I . .
1 .
. I
I . .
..
I
II
T a b l e 5.
1 S t a t i o n a r y Phase Paper
Paper
2 Technique Two d i m e n s i o n a l
Two d i m e n s i o n a l
Paper Chromatography Used € o r Noscapine.
4
3 Mobile P h a s e
1. Water s a t . b u t a n o l - a c e t i c acid 5:l 2. e t h e r - 0 . 1 M a c e t i c a c i c 5: 2
6
5 Comment Alkaloids i n Tetrapon 11
1. Dioxan-Formic a c i d - w a t e r 90:0.5 : 9 . 5 2. n - B u t a n o l - a c e t i c a c i d 5:l
11
11
Paper
One d i m e n s i o n a l
1. Dioxan-Formic 90:0.5:9.5
Paper S & S 204 3b.
Ascending o r descending
25% (NH4)2 SO4 i n 0 . 5 N H C 1
Whatman No. 1 paper b u f f e r e d a t pH 3.5
D e s c end i n g
Isobutylalcohol - toluene s a t u r a t e d w i t h water 1 : 1.
Paper
One d i m e n s i o n a l
Butyl acetate - acetic acid 47:9:28:16
acid-water
- butanol - water
11
Separation of n o s c a p i n e from papaver i n e enhancec b e c a u s e of u s e of b u t y l acetate.
rferenct
T a b l e 5.
I 1
c
\D
I
2
(contd
... .)
3
Paper
One d i m e n s i o n a l
Upper l a y e r of a m i x t u r e n-butanol-acet i c a c id-water 5:1:4
Wha tman N o . 1 impregnated above s t a r t i n g l i n e with f ormamidee t h a n o l 1:l then d r i e d between f i l t e r papers t h e n a t 40°C f o r 30 min.
D esc end
Chloroform
4
5
Starting points w e r e sprayed w i t h f ormamide i n e t h a n o l 1:l a f t e r drying t h e paper
6
(56)
T a b l e 6.
S t a t i o n a r y Phase K i e s e l g e l HF
254
TLC Techniques Used f o r Noscapine
Technique Normal chamber
Mobile P h a s e
Rf
Tolune-acetone 95% e t h a n o l 25% aq. NH3
20 : 20 : 3 : 1
P
-
S i l i c a g e l G-Na2C03
tI
Chloroform 4 : l
Silica gel G.
II
E t h y l acetate
K i e s e l g e l 60
It
Chloroform Benzene Acetone 3 : 3 : 1
Silicagel G
I1
Benz ene-methanol
Silica gel G
I1
B u t a n o l - A c e t i c acid-H 0 2 3 : l : l
S i l i c a g e l G impreg. 4% Na2C03
11
Chloroform 4 : l
Silica gel
11
Chloroform-isopropyl a l c o h o l 10% aq.NH3 30 : 10 : 1 Benzene-methanol 4 : 1
Ethanol
VI
0
Silica gel G
-
4 : l
-
Ethanol
Table 7.
TLC S p r a y R e a g e n t s and Methods Used € o r D e t e c t i o n of Noscapine
Reagent
Procedure ~
5% 3,5-dichloro-p-benzoquinonechlorimine i n i s o p r o p y l alcohol
~~
A f t e r s p r a y i n g s p r a y w i t h aqueous
NH3 1 : 1 and o b s e r v e i n d a y l i g h t and u l t r a v i o l e t .
4% Hg(N03)2 i n 3% HN03
After spraying t h e p l a t e i s heated 15 min. a t l l O O C and o b s e r v e d i n d a y l i g h t . D e t e c t i o n l i m i t 2 vg
1. 3% H202 s o l u t i o n 2. 5% K4Fe(CN)6 s o l u t i o n
P l a t e i s f i r s t d r i e d 1 0 min. a t 100°C t h e n s p r a y e d w i t h 1. t h e n d r i e d 1 0 min. a t 100°C and s p r a y e d w i t h 2 and d r i e d 1 0 min. a t 100°C. Brown s p o t s i n t e n s i f i e d t o r e d . D e t e c t i o n l i m i t 1 0 pg.
2.6 g C@(N@3)2 d i s s o l v e d i n 2 m l anhydrous a c e t i c a c i d i s added t o 4 . 4 g o f NaN02 d i s s o l v e d i n 1 0 m l H20 t h e n 20 ml a c e t i c a c i d and 50 m l H 0 i s added t o m i x t u r e . 2
After spraying, t h e p l a t e is heated a t 105 OC f o r 10 min. S p o t s a r e s t a b l e f o r several h o u r s . Noscapine a p p e a r s a s b l u e - g r e e n f l o u r e s c e n t s p o t when viewed under u l t r a - v i o l e t l i g h t .
4% c i t r i c a c i d i n
a c e t i c anhydride
P l a t e i s h e a t e d a t 80 O C f o r 1 0 min. and viewed i n d a v l i g h t ( d e t e c t i o n l i m i t 5 pg) and i n u l t r a - v i o l e t r a d i a t i o n ( d e t e c t i o n l i m i t 0.5 Up).
Ref.
452
MOHAMMED A. AL-YAHYA AND MAHMOUD M. A . HASSAN w a s powdered and t r i t u r a t e d w i t h 5 m l of 70% e t h a n o l . The m i x t u r e w a s warmed a t 50 - 600 f o r 30 min. t h e n f i l t e r e d and d i l u t e d t o 1 0 m l w i t h 70% e t h a n o l . T i n c t u r e of opium (1 m l ) was d i l u t e d w i t h 9 m l 35% e t h a n o l . Three p o r t i o n s ( 5 , 1 0 and 20 u l ) of opium s o l u t i o n and similar p o r t i o n s of s t a n d a r d s o l u t i o n were a p p l i e d t o a l a y e r of K i e s e l g e l H F254 and chromatograph was developed t o 1 5 c m w i t h t o l u e n e - a c e t o n e - 95% e t h a n o l 25% a q . NH3 (20 : 20 : 3 : 1 ) . For d e t e c t i o n , t h e p l a t e w a s h e a t e d a t 110% f o r 1 0 min. and t h e s e p a r a t e d zones l o c a t e d i n u l t r a v i o l e t r a d i a t i o n . The s p o t s a r e t h e n sprayed u s i n g a modified D r a g e n d o r f f ' s r e a g e n t and t h e n w i t h 0.05 N t o 0 . 1 N s u l p h u r i c a c i d . A l s o polyamide h a s been used a s l a y e r f o r chromatography ( 7 6 ) . Where poly-E-caprolactam r e s i n (Amilan CM 10075) was used. Development f o r 2 h r . i n cyclohexane-ethyl a c e t a t e p r o p y l a l c o h o l - Me2NH 3 0 : 2.5 : 0.9 : 0 . 1 showed n o s c a p i n e a t Rf 0 . 6 1 and development i n H20-ethanol - Me2NH 88 : 1 2 : 0 . 1 n o s c a p i n e had Rf 0.00.
7.6.3
Gas Liquid Chromatography
D e r i v i t i z a t i o n of samples i n c l u d e d a c e t y l a t i o n using acetic anhydride i n p y r i d i n e (77) and t r e a t i n g sample w i t h t r i m e t h y l s i l y l acetamide and t r i m e t h y l c h l o r o e t h a n e ( 6 8 ) . I n t e r n a l s t a n d a r d s used were h i s t a p y r r o d i n e h y d r o c h l o r i d e , o e s t r a d i o l v a l e r a t e (78) Phenazone (79) and S q u a l i n e ( 7 7 ) . Another method was a l s o r e p o r t e d ( 8 0 ) . Column t y p e s e t c . a r e r e p o r t e d i n T a b l e 8. 7.6.4
HiPh Performance Liquid Chromatography S e p a r a t i o n s of p h a r m a c e u t i c a l s combined i n v a r i o u s f o r m u l a t i o n s by HPLC on S e p h e r o s i l 5 Um i s o c r a t i c a l y h a s been r e p o r t e d ( 8 1 ) . Noscapine i n a n t i - c o l d p r e p a r a t i o n s w a s s e p a r a t e d on a column of H i t a c h i g e l 3011-0(82)
T a b l e 8. Column Type
S t a t i o n a r y Phase
4 f t x 4 m m glass
D i a p o r t S (80-100) mesh
1 . 5 m x 2.3 mm g l a s s
Chromosorb G-HP AW-DMCS (80-100) mesh
GLC ConditionsUsed f o r Noscapine Flow-ra t e
3 0 cm
SE-30
40 cm
HI-EFF 8 BP
Gas-Chrom 0 (100-120 mesh)
3
min
min
-1
-1
-
Supelcoport (80-100 mesh) 4 f t x 3mm od g l a s s
3
Temperature
2% OV 101
N2
30 cm
3
min
-1
24OoC
tg
I e t e ct o r
Flame i o n i z at i o r
15OoC 235OC a t 1 . 2 5 C min-1
11
2 2 5OC-2 7 O°C a t 2 0 min-1 ~
11
1 8 O o C 5 min. then 7 ' ~ min-l t o 25OoC
11
Ref,
454
MOHAMMED A. AL-YAHYA AND MAHMOUD M. A . HASSAN
u s i n g methanol : 28% aqueous NH3 9 9 : l a s e l u t i n g s o l v e n t and d e t e r m i n a t i o n by spectrophotometry a t 230 nm o r 250 nm. Paracetamol, p h e n a c e t i n , d i p y r o n e , a s p i r i n , c a f f e i n e , etenzamide and m e t h y l e p h e d r i n e d i d n o t i n t e r f e r e . Other methods were a l s o d i s c r i b e d (83, 8 4 ) . 7.6.5
Ion-Exchange Chromatography Knox and Jurand r e p o r t e d a method (85) f o r t h e s e p a r a t i o n of n o s c a p i n e on d r y packed column of Zipax SCX o r SAX (37-44 um) b o r a t e b u f f e r s a t pH 9.2-9.8 c o n t a i n i n g 4% a c e t o n i t r i l e and 1%propanol were used a t 500 t o 1500 l b p e r s q . i n . t o e l u t e n o s c a p i n e .
7.6.6
Ligand-Exchange Chromatography P o r a g e l P.T. r e s i n was used t o s e p a r a t e a l k a l o i d s and n o s c a p i n e h a s been a n a l y s e d on t h i s r e s i n . 0.06 M-aqueous NH3 i n 33% e t h a n o l w a s used as e l u a n t and t h e e l u t i o n volume f o r n o s c a p i n e w a s 6.8 times t h e bulk column volume ( 8 6 ) .
7.6.7
P a r t i t i o n Chromatography I n t h e a s s a y of T e t r a p o n by p a r t i t i o n chromatography t h e s t a t i o n a r y phase i s a phosphate b u f f e r ( 8 7 ) . 5 m l of 0 . 2 N NaOH i s added t o 0.4 g of papaveretum i n 20 m l H 2 0 . The m i x t u r e i s e x t r a c t e d twice w i t h a m i x t u r e of 1 0 m l of c h l o r o f o r m i n 3 0 m l e t h e r and t h e n w i t h 1 0 m l of chloroform. The f i l t e r e d e x t r a c t s a r e e v a p o r a t e d t o 0.5 1 . 0 m l and d i l u t e d w i t h 25 m l of e t h e r b e f o r e t r a n s f e r t o t h e p r e p a r e d column, 200 m l water s a t u r a t e d e t h e r is used t o e l u t e n o s c a p i n e . Other a s s a y w a s a l s o r e p o r t e d ( 8 8 ) .
7.6.8
Paper E l e c t r o p h o r e s i s Due t o f o r m a t i o n of m o l e c u l a r complexes between n o s c a p i n e and 7-(2-hydroxyethyl) t h e o p h y l l i n e , t e t r a m e t h y l u r i c a c i d and 7-carboxymethyltheophyline, n o s c a p i n e h a s
455
NOSCAPINE
been separated from other isoquinoline alkaloids by reversed - phase paper chromatography and electrophoresis. Britton - Robinson buffer pH 3.5 to 4 was used as the mobile phase and o-xylene as stationary phase ( 8 9 ) . Another method was also reported (1).
ACKNOWLEDGEMENTS The authors would like to thank the technicians, Robert Hutchison, K.N. Ludhi and the Research assistant Syed Rafatullah o f the College of Pharmacy, King Saud University, Riyadh, Saudi Arabia for their kind technical assistance for the preparation of the manuscript.
456
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408
1. Description 1. I Nomenclature 1.2 Formulae 1.3 Molecular Weight 1.4 Elemental Composition 1.5 Appearance, Color, Odor, and Taste 2. Physical Properties 2.1 X-Ray Diffraction 2.2 Solubility 2.3 Dissociation Constant 2.4 Optical Rotation 2.5 Spectral Properties 3. Preparation 3.1 Isolation from Opium 4. Synthesis of Noscapine 4.1 Tissue Culture Method 4.2 Chemical Methods 5 . Biosynthesis of Noscapine 6. Metabolism 7. Methods of Analysis 7.1 Identification Tests 7.2 Microcrystal Tests 7.3 Titrimetric Methods 7.4 Complexometric Methods 7.5 Spectrophotometric Methods 7.6 Chromatographic Methods 8. References
Analytical Profilesof Drug Substances Volume 1 I
408
408
414 414 414 414 414 415 415 415 416 429 429 429 429 429 429 43 3 436 436 436 439 441 441 445 45 6
407
Copyight 0 1982 by The AmdUn phmn.ce~iiulAmciation ISBN 0-12-260811-9
MOHAMMED A. AL-YAHYA AND MAHMOUD M. A. HASSAN
408
1. D e s c r i p t i o n 1.1
Nomenclature
1.1.1 Chemical Names a-
(S)-6,7-Dimethoxy-3-(5, 6, 7 , 8tetrahydro-4-methoxy-6-methyl-l , 3-d i o x o l o [ 4,5-g] i s o q u i n o l i n - 5 - y l ) - l (1) (3 H) -isobenzof uranone.
b-
1-a -2-methyl-8-methoxy-6,7-methylene dioxy-1- ( 6 , 7-dimethoxy-3-phthalidyl) 1, 2 , 3 , 4-tetra-hydroisoquinoline.
-
(1)*
1.1.2
c-
1 (3 H) Isobenzofuranone, 6 , 7 dimethoxy-3-(5, 6, 7 , 8-tetrahydro-4methoxy-6-methyl-1, 3-dioxolz-[4, 5-g] isoquinolin-5-y1)-, [s-(R* s ) I . (2)
d-
(3 S)-6, 7-dimethoxy-3-[(5R)-5, 6, 7 , 8-tetra-hydro-4-methoxy-6-methyl-1,3-dioxolo [ 4 , 5-g] isoquinolin-5-y1] phthalide. (3).
Generic Names d-Gnoscapine; 1-6-Narcot i n e ; I-Narcot i n e ; N a r c o t i n e ; Noscapine.
1.1.3
Trade Names Capval; Coscopin; Coscotabs; Keyt u s s c a p i n e ; Longatin; Lyobex; Fethoxyh y d r a s t i n e ; Narcompren; Narcosine; Marcotussin; N e i t a c l o n ; Nicolane; Nipaxon; Noscapal; Noscapalin; NSC 5366; Opian; Opianine; Terbenol; Tusscapine; Vadebex.
1.2
Formulae
1.2.1
Empirical
409
NOSCAPINE 1.2.2
Structural
OCH3
Nos c a p i n e The a l k a l o i d n o s c a p i n e can be c l e a v e d v e r y r e a d i l y i n t o two m o i e t i e s ; w i t h d i l u t e s u l f u r i c a c i d , c o t a r n i n e and o p i a n i c a c i d a r e g e n e r a t e d . Under a c i d i c r e d u c i n g c o n d i t i o n s , e.g., zinc i n hydrochloric acid o r s u l f u r i c a c i d , h y d r o c o t a r n i n e and meconine a r e formed (Scheme 1 ) . With t h e s t r u c t u r a l e l u c i d a t i o n of c o t a r n i n e , o p i a n i c a c i d , h y d r o c o t a r n i n e and meconine, and g i v e n t h e p r e s e n c e of a l a c t o n e r i n g i n n o s c a p i n e , the structure o f t h i s a l k a l o i d was e s s e n t i a l l y e s t a b l i s h e d ( 4 ) . 1.2.3
CAS No
(12 8- 62 - 1) 1.2.4
Niswesser L i n e N o t a t i o n T C566 DO FO K N EH & & T J H 01 K J T56 B VO D H J HOL 1 0 1 "ALPHA" LV.
1.2.5
S t e r e o c h e m i s t r y and A b s o l u t e C o n f i g u r a t i o n T h e s t e r e o c h e m i s t r y of n o s c a p i n e h a s been s t u d i e d by many w o r k e r s (5-9). The prolonged a c t i o n of h o t m e t h a n o l i c p o t a s s i u m h y d r o x i d e on n a t u r a l (-)- a - n a r c o t i n e r e s u l t s i n t h e f o r m a t i o n of a n e q u i l i b r i u m m i x t u r e of t h e o r i g i n a l b a s e and a new o p t i c a l l y
410
MOHAMMED A. AL-YAHYA AND MAHMOUD M. A. HASSAN
'n'
'2
OCH3
Noscapine
OCH 3
OC!I
Hydrocotarnine
Cotarnine
+
+
CRO
OCH
OCH
Opianic acid
Meco n ine Scheme 1
41 1
NOSCAPINE a c t i v e d i a s t er eoisomer , (-) -6 -narc0 t i n e , which can be w r i t t e n a s shown i n Scheme 2. Lithium aluminum h y d r i d e r e d u c t i o n of t h e a- and 8-noscapines r e a d i l y a f f o r d s anarcotinediol and 6 - n a r c o t i n e d i o l respectively
.
1
2
A c e t y l a t i o n of t h e s e d i o l s g i v e s r i s e t o t h e corresponding d i a c e t a t e s 2 and k , b u t subsequent c a t a l y t i c h y d r o g e n o l y s i s y i e l d s one and t h e same d e x t r o r o t a t o r y benzyli s o q u i n o l i n e 2. The f o r e g o i n g sequence c l e a r l y e s t a b l i s h e s t h a t a- and 8-noscapine must d i f f e r from each o t h e r o n l y i n t h e i r s t e r e o c h e m i s t r y a t C-9. The b e n z y l i s o q u i n o l i n e 5 shows a p o s i t i v e Cotton e f f e c t near 295 m u , s o t h a t i t s C-1 hydrogen must b e a l p h a as i n d i c a t e d . I t f o l l o w s t h a t t h e C-1 hydrogen i n (-)-an a r c o t i n e and i n (-)- 6 - n a r c o t i n e must a l s o be a l p h a (Scheme 2 ) .
A 1 t e r n a t i v e l y , a-narco t ined i o l w a s c y c l i z ed v i a i t s monomesylate d e r i v a t i v e t o t h e Nm e t h o t e t ra h y d r o p r o t o b e r b e r i n e s a l t 5. T h i s material underwent N-demethylation on p y r o l y s i s t o y i e l d t h e p r o t o b e r b e r i n e b a s e 7. Reductive removal of t h e hydroxyl group w a s achieved i n e t h a n o l i c p e r c h l o r i c a c i d o v e r a palladium c a t a l y s t . The t e t r a h y d r o p r o t o b e r b e r i n e 8 t h u s o b t a i n e d showed a s t r o n g n e g a t i v e r o t a t i o n , so t h a t i t s C-14 hydrogen must be a l p h a . The i d e n t i c a l sequence w a s c a r r i e d o u t using p n a r c o t i n e d i o l t o y i e l d t h e tetrah y d r o p r o t o b e r b e r i n e b a s e ?. Hydrogenolytic c l e a v a g e of t h i s s p e c i e s t h e n provided t h e same 1evor o t a t o r y t e t r a hyd r o pro t o b er b er i n e 8. The c o n c l u s i o n i s t h a t t h e C-1 hydrogens i n b o t h a- and 6-noscapine a r e a l p h a (Scheme 3 ) . Turning t o t h e s t e r e o c h e m i s t r y a t C-9 f o r (-)-a and (-)- B-narcotine, m o l e c u l a r models i n d i c a t e d t h a t t h e d i h e d r a l a n g l e between t h e p r o t o n s a t C-13 and C-14 of t h e 13-a-hydroxy
-
412
MOHAMMED A. AL-YAHYA AND MAHMOUD M . A. HASSAN
CH3
OCH3 (-) -6-Narcotine
(-)-a-Narcotine ( n a t u r a l isomer)
1
1 --
L1A1H4
LiA1H4
OCH~
a-Narcotinediol
1.
1 -
B-Narcotinediol
1 --
Ac 0, pyridine
2
-
-
Ac 20 , P Y ~
idine
OAc CH20Ac OCH3 0CH3
Scheme 2
413
NOSCAPINE
H
........OH
pyridine
6 @CH3 d-Narcotinediol
7 -
as o u t l i n e d
9 OCH3 B-Narcotinediol Scheme 3
MOHAMMED A. AL-YAHYA AND MAHMOUD M. A. HASSAN
414
b a s e 7 i s a b o u t 160'. On t h e o t h e r hand, f o r the 13-6 -hydroxy b a s e 2 d e r i v e d from 6 - n a r c o t i n e , t h i s a n g l e is o n l y a b o u t 60'. Following exchange of t h e h y d r o x y l i c p r o t o n s f o r d e u t e r i u m , i t w a s determined t h a t t h e s p l i t t i n g c o n s t a n t .J13,14 w a s 9 Hz f o r and o n l y a b o u t 1 . 5 Hz f o r 9. species The l a r g e c o u p l i n g v a l u e of 9 Hz i s accord w i t h a t r a n s arrangement of t h e C-13, 1 4 hydrogens i n I_, and t h e s m a l l c o u p l i n g c o n s t a n t of 1 . 5 Hz a r g u e s f o r a c i s r e l a t i o n s h i p i n 9, t h u s s e t t l i n g t h e s t e r e o c h e m i s t r y a t C-9 f& a- and 8-noscapine.
1,
1.3
iz
Molecular Weight 413.43
1.4
E l m e n t a l Composition C, 63.91%; H, 5.61%; N , 3.39%; 0 , 27.09%
1.5
Appearance, C o l o r , Odor
and Taste
Noscapine o c c u r s i n t h e form of Orthorhombic b i s p h e r o i d a l p r i s m s , t a b l e t s from d i a c e t o n e o r a s f i n e , almost w h i t e c r y s t a l l i n e powder. T r i b o l u m i n e s c e n t d 1.395. I t i s o d o r l e s s and tasteless. 2.
Physical Properties 2.1.1
X-ray d i f f r a c t i o n Crystallographic d a t a f o r noscopine are s c a r c e . The o n l y r e p o r t e d d a t a is due t o Love11 (10) and Steward and P l a y e r (11). These a r e a s f o l l o w s : Long needle-shaped c r y s t a l s were o b t a i n e d by r e c r y s t a l l i s a t i o n of t h e commercial n o s c a p i n e from e t h a n o l o r methanol. Weissenberg photographs t a k e n w i t h Cu Ka (1.5418 A)' r a d i a t i o n revealed the following systematic absences:
hOO, h
= 2n
+
1
OKO, K = 2n -I-1 001, 1 = 2n + 1
415
NOSCAPINE
d e f i n i n g unambiguously t h e space group P212121. C e l l dimensions were o b t a i n e d from 28 v a l u e s of 32 r e f l e x i o n s from n o s c a p i n e u s i n g two a x e s i n each c a s e , measured w i t h a counter diffractometer. The f o l l o w i n g d a t a were o b t a i n e d : 413.41
M.W. M.p. (OC)
r
C r y s t a l system Space group Cell
Dimensions
(8)
d 3 )
z
O r t hor homb i c
P212121 15.398(12) b 32.686(36) c ( p r i m ) 8.022(8) 4037(11)
8
Qcalc (g. ~ m - ~ )
1.360
Qexp (g ~ m - ~ )
1.38
.
2.1.2
178
Melting P o i n t 174-176OC(3) 176OC s u b l i m e s a t 15OoC-16O0C under 11 mm p r e s s u r e a t 2 mm d i s t a n c e (1)
2.2
Solubility I t i s i n s o l u b l e i n water; s l i g h t l y s o l u b l e i n a l c o h o l ( 9 5 % ) , i n e t h e r and i n carbon t e t r a c h l o r i d e . S o l u b l e i n chloroform, benzene and v e r y s o l u b l e i n a c e t o n e (12).
2.3
D i s s o c i a t i o n Constant I t i s a v e r y weak b a s e , pKa 7.8 ( 1 ) and 4.85 i n 80% m e t h y l c e l l o s o l v e ( 1 3 ) .
2.4
Optical Rotation
+
42'
to
+
(2% w/v i n 0 . 1 M h y d r o c h l o r i c a c i d ) (3) [ u I D - 198O (1% w/v i n c h l o r o f o r m ) ,
[a],
48'
MOHAMMED A. AL-YAHYA AND MAHMOUD M. A. HASSAN
416
[a],
[a], [a],
2.5
- 146 (2% w/v i n t o l u e n e ) , - 147' (1.59% i n b e n z e n e ) ,
+
50 (1% w/v i n h y d r o c h l o r i c a c i d ) (14)
Spectral Properties 2.5.1
U l t r a v i o l e t Spectrum The W spectrum of n o s c a p i n e i n methanol w a s scanned from 200 t o 400 nm u s i n g V a r i a n Carry 119 Spectrophotometer. I t e x h i b i t s a c h a r a c t e r i s t i c UV spectrum ( F i g . 1) w i t h two maxima: Xmax 310.2 290.6
1%
cm
114.9 106.4
(C, 9.42 mg p e r 100 ml) (C, 9.42 mg p e r 1 0 0 ml)
Other UV s p e c t r a l d a t a of n o s c a p i n e have a l s o been r e p o r t e d : Xmax 209 291 309-310 Xmax 291 310 291 3 09 2.5.2
Log
E
4.86 3.60 3.69
) ) )
i n e t h a n o l (1, 15)
(E)
a b o u t 1.1 ) a b o u t 1.4 ) 3981 4898
1 )
i n a l c o h o l 95% (3) i n methanol (12)
I n f r a r e d Spectrum The I R s p e c t r a of n o s c a p i n e a s K B r d i s c and n u j o l m u l l were r e c o r d e d on a P e r k i n E l m e r FT-680B s p e c t r o p h o t o m e t e r and shown in F i g . 2 t?, F i g . 3 respectively. The s t r u c t u r a l a s s i g n e m e n t s have been c o r r e l a t e d w i t h t h e f o l l o w i n g band f r e q u e n c i e s (Table 1)
417
NOSCAPINE
Fig.
1.
W Spectrum of N o s c a p i n e i n M e t h a n o l .
Wavelmgt h F
Fig.
*O
2.
5.0
6.0
70
8.0
s.0
I R Spectrum of Noscapine as K B r d i s c .
f0
12
!4
2500
Fig. 3 .
2000
1
.
I R Spectrum of Noscapine as Nujol Mull.
800
700
420
MOHAMMED A . AL-YAHYA A N D MAHMOUD M. A . HASSAN T a b l e 1. I R C h a r a c t e r i s t i c s of Moscapine Frequency c m
-1
Assignement
3000, 2945, 2880, 2845, 2800
Methylened i o x y and C-H and -CH frequencies. 3 ( y - l a c t o n e ) 3-C=O g r o u p
17 60 1625 1600,1505,1480, 1280-1 22 5
-c=c-
Aromatic Aromatic m e t h o x y - a r y l C-0 stretching vibrations.
790, 815, 8 3 5 , 885
2 a d j a c e n t H atoms, i s o l a t e d H atom C-H o u t of p l a n e d e f o r m a t i o n . Tetra and pen t a s u b s t i t u t e d b e n z e n e s
.
Other c h a r a c t e r i s t i c a b s o r p t i o n bands are: 1460, 1430, 1405, 1 3 9 0 , 1 3 8 0 , 1 3 6 5 , 1 3 3 0 , 1310, 1 2 0 0 , 1120, 1 0 8 5 , 1040, 1 0 1 0 , 980, 930, 900, B O O , 765, 750, 735, 725, 715, and 700 cm-'. O t h e r I R d a t a a r e a l s o r e p o r t e d (16) 2.5.3
N u c l e a r Magnetic Resonance S p e c t r a 2 . 5 . 3 . 1 P r o t o n Spectrum The PMR s p e c t r u m of n o s c a p i n e i n d e u t e r a t e d chloroform w a s r eco r d ed on a V a r i a n XL200, 2 0 0 MHz NMR s p e c t r o meter u s i n g t e t r a m e t h y l s i l a n e a s a r e f e r e n c e s t a n d a r d ( F i g . 4 ) . The following s t r u c t u r a l a s s i g n m e n t h a v e been made ( T a b l e 2 ) . 4
I, 0
7
F i g . 4.
6
5
4
PMR Spectrum of Noscapine and T e t r a m e t h y l s i l a n e
3
2
i n Deuterated Chloroform.
MOHAMMED A . AL-YAHYA AND MAHMOUD M.A. HASSAN
422
Table 2.
PMR C h a r a c t e r i s t i c s of Noscapine
Ass ignemen t (Group)
Po s i t i o n
Chemical S h i f t ( 6 )
3 , 4 of i s o q u i n o l i n e
2.32 (m)
N- CH3
2 of i s o q u i n o l i n e
2.53 ( s )
OCH3
8 of i s o q u i n o l i n e
3.84 ( s )
OCH3
&'of p h t h a l i d y l
4.02 (s)
OCH3
5'of
4.08 ( s )
-CH2-CH2
phthalidyl
-CH-
1 of i s o q u i n o l i n e
4.37 (d)
-CH-
9 of p h t h a l i d y l
5.55 (d)
-CH2-
methylened i o x y
5.92 ( s )
-CH-
2'0f
6.05 (d)
-CH-
5 of i s o q u i n o l i n e
6.29 ( s )
-CH-
3'of
6.94 (d)
phthalidyl phthalidyl
s = s i n g l e t , d = doublet, m = multiplet
Other PMR s p e c t r a l d a t a was a l s o r e p o r t e d ( 1 7 an.d 5 5 ) . 2.5.3.2
I3C-NMR
Spectra
I3C-NMR c o m p l e t e l y decoupled and o f f - r e s o n a n c e s p e c t r a are shown i n Fig. 5 and Fig. 6 r e s p e c t i v e l y . Both were r e c o r d e d o v e r 11001.1 HZ r a n g e , i n d e u t e r a t e d c h l o r o f o r m (CDC13) on XL-200,200 MHz NMR s p e c t r o m e t e r . Using 10 mm sample t u b e and tetramethylsilane as reference standard a t 25OC. The c a r b o n chemical s h i f t s a s s i g n e d on t h e b a s i s of t h e a d d i t i v i t y p r i n c i p a l s and o f f - r e s o n a n c e s p l i t t i n g p a t t e r n (Table 3) (18).
9
a Fig. 5.
7
6
5
4
1 3
2
f
l3C-NMR Spectrum of Noscapine i n Deuterated Chloroform.
0
3
1
424
50
1
T
60
70
80
1
90
100 110 120 130 1 4 0 150 160
170
190 200 210 220 230 240 250 260 270 280 290 300
180
1
310
1
1 2
320 330
340 350 360 370 380 390 400 410 420 430 440 4 5 0 Fig. 7.
EI-Mass
Spectrum of Noscapine.
MOHAMMED A . AL-YAHYA AND MAHMOUD M. A. HASSAN
426
’
22
OCH3
Table 3. Carbon No.
c-1 c- 2
Carbon Chemical S h i f t s of Noscapine
Chemical S h i f t PPm 60.84 49.99 28.03 134.03 117.65 140.45 100.73 141.14 152.18 132.09 59.36
c-3
c-4
c-5 C-6
c-7
C-8 c- 9 c-10
c-11 2.5.4
(d) (t) (t)
(s)
(d) (s) (t) (s)
(s) (s) (q)
Carbon No.
Chemical S h i f t Ppm
c-12 C-13 C-14 C-15 C-16 C-17 C-18 c-19 c-20 c-21 c-22
46.29 81.83 120.17 118.19 102.29 147.67 148.37 117.11 168.06 56.78 62.22
(q) (d) (s) (d)
(d) (s) (s) (s) (s) (4)
(q)
Mass Spectrum
The mass spectrum of n o s c a p i n e by e l e c t r o n impact i o n i z a t i o n and recorded on Ribermag R-10-10 mass equibbed w i t h d i r e c t i n l e t probe. ( F i g . 7) shows m o l e c u l a r i o n peak a b a s e peak a t m / e 220.
obtained which w a s spectrometer The spectrum and shows
The mass spectrum of n o s c a p i n e o b t a i n e d by butane chemical i o n i z a t i o n ( F i g . 8) shows a m o l e c u l a r i o n peak PI+ a t m / e 413 w i t h a r e l a t i v e i n t e n s i t y of 2.8% and a b a s e peak a t m / e 220. The most prominent f r a g m e n t s ,
90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 2t
1
290 300 31 0 320 330 340 350 360 370 380 390 4-00410 420 430 440 450 460 470 Fig.
8.
CI-Mass
Spectrum of Noscapine.
428
MOHAMMED A. AL-YAHYA AND MAHMOUD M. A. HASSAN t h e i r r e l a t i v e i n t e n s i t i e s and s t r u c t u r e s a r e l i s t e d i n Table 4 . Other mass s p e c t r a l d a t a f o r p h t h a l i d e i s o q u i n o l i n e s was a l s o r e p o r t e d (19, 2 0 ) . Table 4 .
m/e 413
Mass Fragments of Moscapine Relative Intensity
96
Fragment
2.8
M+
221
220
195
8.0
OH
193 OCH3
429
NOSCAPINE
3.
P r eDara t i o n 3.1
I s o l a t i o n from Opium Noscapine o c c u r s up t o 11%n a t u r a l l y i n opium (Papaver sornniferum L . (Fam. papaveraceae) I t w a s f i r s t d i s c o v e r e d by Derosne i n 1803 ( 2 0 ) , and i s o l a t e d by Robinquet i n 1817 ( 2 1 ) . Noscapine can be s e p a r a t e d from o t h e r opium a l k a l o i d s by t h e procedure o u t l i n e d i n Scheme 4 (90).
.
Another p a t e n t method h a s been a l s o d e s c r i b e d f o r i t s i s o l a t i o n on a n i n d u s t r i a l scale ( 2 2 ) .
4.
S y n t h e s i s of Noscapine
4.1
By T i s s u e C u l t u r e Method Khanna e t a 1 (23) d e s c r i b e d a method f o r t h e s y n t h e s i s of noscapine a l o n g w i t h o t h e r a l k a l o i d s by t i s s u e c u l t u r e of Papaver somniferum Linn.
4.2
By Chemical Methods P e r k i n and Robinson (24) d i s c o v e r e d t h a t h e a t i n g a m i x t u r e of c o t a r n i n e 1 and meconine 1i n e t h a n o l r e s u l t e d i n a s m a l l y i e l d of noscapine 3 . The expected second isomer of noscapine-(because of t h e presence of 2 a s s y m e t r i c c e n t r e s ) w a s n o t found. The s y n t h e t i c noscapine was t h e n r e s o l v e d and t h e n o s c a p i n e o b t a i n e d shown t o b e i d e n t i c a l w i t h t h e n a t u r a l p r o d u c t . (Scheme 5).
A q u i t e e f f i c i e n t s y n t h e s i s of noscapine w a s developed by Hope and Robinson i n 1914 ( 2 5 ) , i n which c o t a r n i n e i s condensed w i t h iodomeconine 2 and t h e adduct w a s reduced w i t h sodium amalgum t o g i v e t h e d e s i r e d p r o d u c t , corresponding t o t h e n a t u r a l s e r i e s (Scheme 6 ) .
L
5.
B i o s y n t h e s i s of Noscapine I t has been p o s t u l a t e d t h a t t h e ph t h a l i d e i soqu i n o 1i n e s a r e formed i n n a t u r e by o x i d a t i v e m o d i f i c a t i o n of t e t r a h y d r o p r o t o b e r b e r i n e s , and p r e v i o u s work w i t h l a b e l e d precursors supports t h i s hypothesis (26).
MOHAMMED A. AL-YAHYA AND MAHMOUD M. A. HASSAN Powdered Op i um
430
+
+
Shake w i t h warm calcium c h l o r i d e s o l u t i o n F i 1t e r Insoluble matter C (discard)
F i ltrate (hydroch l o r i des o f a 1ka l o i ds)
4
Reduce volume (evaporate under reduced p r e s s u r e ) t o syrupy 1 i q u i d
4
Add 10% NaOH s o l u t i o n Precipitates p k (noscapine, papaverine, thebaine)
4
a
l ine solution (morphine, codeine,
E x t rn aa c rt cw e i nt he ) ci h l o r o f o r m
Dissolve i n d i l u t e a l c o h o l
$.
Add a c e t i c a c i d t o make s l i g h t l y
0
acidii
Add 3 volumes o f b o i l i n g water
A Solution
Aqueous extract alkaline (containsolution i n g codeine) (morphine, narceine)
C h 1 o r o f orm
4
rz 1
Precipitate (papave r ine, noscap ine)
(theba ine)
\
4
Further pur i f ic a t i o n
Make a c i d i c
4
Aqueous a c i d i c s o l n . ( s a l t s of morphine and na r c e i ne)
Dissolve i n b o i l i n g 0.33%(aqueous)oxali c a c i d soln.
4
4
Make s l i g h t l y a l k a l i n e w i t h ammonia
A l l o w t o stand
Bring t o b o i l i n g A l l o w t o stand
C rys t a 1 s (papave r ine ac id oxalate)
4
Further purification
Sol u t i o n (noscap ine oxa 1 a t e )
+
Repeat
Precipitate (morphine)
I
Further p u r i f ic a t ion
Solution (narceine)
Make a l k a l i n e w i t h ammonia
Precipitate (noscapine)
+
1 i
+
s o l u t on (discard)
Dissolve i n b o i l i n g alcohol Crystallization Scheme 4:
i
Further pur i f i cat ion
Isolation of
from powdered op i um .
NOSCAPINE
43 1
OCH3
OCH 2 Plecoriine
I
Cotarnine
D
C2H50H
0
0ch3 3
(2)-a-Narco t ine
Scheme 5
432
MOHAMMED A. AL-YAHYA AND MAHMOUD M . A. HASSAN
Cotarnine 1
Iodomeconine
\
\
CH 3
H..
... *..s . 0
2
CH 3
CH 3O
Na/Hg
OCH3
OCIl
3
(+) -a-Narcotine
Scheme 6
NOSCAPINE
433
S e v e r a l f e e d i n g e x p e r i m e n t s ( 6 , 8 , 94 ) have been r u n t o e l u c i d a t e t h e b i o g e n e s i s of n o s c a p i n e i n Papaver somnif erum L . (Papaveraceae) When l a b e l e d (+) t y r o s i n e was f e d t o t h e p l a n t , r a d i o a c t i v e n a r c o t i n e l a b e l e d s p e c i f i c a l l y and e q u a l l y a t C-1 and C-3 was o b t a i n e d . The b e n z y l i s o q u i n o l i n e s y s t e m of n o s c a p i n e i s t h u s d e r i v e d b i o l o g i c a l l y from two Ar-C-C u n i t s which c a n a r i s e from t y r o s i n e .
.
The c a r b o n atoms t h a t a r i s e from t h e S-methyl of m e t h i o n i n e were c l e a r l y p i n p o i n t e d when, a f t e r f e e d i n g radioactive methionine, noscapine labeled a t t h e l a c t o n e c a r b o n y l , t h e m e t h y l e n e d i o x y g r o u p , and t h e N- and 0-methyl c a r b o n atoms w a s o b t a i n e d ( 2 7 , 2 8 ) . Progressing f u r t h e r along the biogenetic locus, t h e b e n z y l i s o q u i n o l i n e (+)- n o r l a u d a n o s o l i n e l a b e l e d C-1 l e d t o n o s c a p i n e a l s o l a b e l e d C-1 ( 2 9 ) . Even more s i g n i f i c a n t l y , when q u a d r u p l y l a b e l e d (+)- and (-) r e t i c u l i n e were f e d s e p a r a t e l y t o P. somniferum, i t w a s found t h a t b o t h enantiomers were i n c o r p o r a t e d i n t o n o s c a p i n e , b u t w i t h t h e (+)-isomer d o i n g so s l i g h t l y more e f f i c i e n t l y . E v i d e n t l y e p i m e r i z a t i o n of t h e wrong b e n z y l i s o q u i n o l i n e p r e c u r s o r must o c c u r , p r o b a b l y by o x i d a t i o n - r e d u c t i o n a t C-1. I n keeping w i t h t h i s c o n c l u s i o n c o n s i d e r a b l e l o s s of t r i t i u m o c c u r e d i n t h e c o u r s e of i n c o r p o r a t i o n of b o t h r e t i c u l i n e s . Another i m p o r t a n t o b s e r v a t i o n i s t h a t t h e l a c t o n e c a r b o n y l of t h e p h t h a l i d e i s o q u i n o l i n e must b e d e r i v e d from t h e N-methyl group of t h e b e n z y l i s o q u i n o l i n e p r e c u r s o r ( 2 7 , 29, 3 0 ) . F i n a l l y , i t h a s been found t h a t t h e f e e d i n g of l a b e l e d ( - ) - s c o u l e r i n e r e s u l t s i n t h e f o r m a t i o n of r a d i o a c t i v e noscapine. Protoberberines are, therefore, t h e precursors f o r t h e phthalideisoquinolines i n plants. S i g n i f i c a n t l y , ( - ) - s c o u l e r i n e , which p o s s e s s e s t h e same a b s o l u t e c o n f i g u r a t i o n a s ( + ) - r e t i c d i n e and ( - ) - a - n a r c o t i n e , w a s more t h a n one hundred t i m e s more e f f i c i e n t t h a n i t s enantiomer a s a p r e c u r s o r f o r ( - ) - a - n a r c o t i n e . The b i o g e n e t i c sequence i n p l a n t s i s , t h e r e f o r e , b e n z y l i s o qu i n o l i n e s +- t e t r a h y d r o p r o t o b e r b e r i n e s + p h t h a l i d e i s o q u i n o l i n e s . The b i o s y n t h e s i s of (-)-cC-narcotine i s shown i n Scheme 7 .
6.
Metabolism The m e t a b o l i s m of n o s c a p i n e was r e p o r t e d
MOHAMMED A. AL-YAHYA AND MAHMOUD M. A. HASSAN
434
&:
Labeled t y r o s i n e
*<:w bCH3
H $H3-S-(CH
1
Labeled noscapine
Papaver
) -C-COOH 2 2 1 somn i f e r u m )
NH2 Labeled methionine
I
N\*
CH 3O
CH 3
- 0
OgH3 L a b e l e d nos cap i n e
Laheled ( a - n o r l a u d a n o s o l i n e Scheme 7
L a b e l e d n‘bscapine
435
NOSCAPINE
Scheme 7 ( c o n t i n u e d )
(4)
"H3
Labeled (+) - r e t i c u l i n e (Labeled (-) - r e t i c u l i n e somewhat less e f f i c i e n t )
R = H o r T Labeled n o s c a p i n e ( a p p r e c i a b l e l o s s of tritium)
CH30 HO
OCH3
Labeled (-) - s c o u l e r i n e
Labeled noscapine (Some t r i t i u m l o s s )
MOHAMMED A . AL-YAHYA AND MAHMOUD M . A . HASSAN
436
(31, 3 2 ) . Oral a d m i n i s t r a t i o n of to male r a b b i t s and e x a m i n a t i o n o f t h e 24 h o u r s u r i n e by p r e p a r a t i v e TLC and methane c h e m i c a l i o n i z a t i o n mass s p e c t r o m e t r y r e v e a l e d t h e p r e s e n c e of two O-monodemet h y l a t e d compounds a s f r e e m e t a b o l i t e s 2 and 5, One 0 - d i d e m e t h y l a t e d d e r i v a t i v e 2 o r 5 and t h e i r c o n j u g a t e d forms. Noscapine g i v e n orally to rats was m e t a b o l i s e d t o di-0-demethyl-noscapine 3 or 4 , cotarnine 5 , h y d r o c o t a r n i n e 1,o x y c o t a r n i n e 2, a n d 0-demethylmeconine 5. T h e s e m e t a b o l i t e s were i s o l a t e d from u r i n e . A l l p o s s i b l e m e t a b o l i t e s of n o s c a p i n e a r e shown i n Scheme 8.
7.
Methods of A n a l y s i s
7.1
I d e n t i f i c a t i o n Tests The f o l l o w i n g i d e n t i f i c a t i o n t e s t s a r e d e s c r i b e d by t h e B r i t i s h Pharmacopoeia ( 1 9 8 0 ) . a)
The l i g h t a b s o r p t i o n , i n t h e r a n g e 230 t o 350 nm, of a 0.005 p e r c e n t w/v s o l u t i o n i n m e t h a n o l e x h i b i t s two maxima, a t 291 nm and 310 nm and a minimum a t 263 nm; r a t i o of t h e a b s o r b a n c e a t t h e maximum a t 310 nm t o t h a t a t t h e maximum a t 291 nm, a b o u t 1 . 2 .
b)
To 1 0 mg add 0 . 5 m l of s u l f u r i c a c i d and mix; a g r e e n i s h - y e l l o w s o l u t i o n i s formed which t u r n s r e d and f i n a l l y v i o l e t o n h e a t i n g .
c)
7-2
S o l u t i o n s i n organic s o l v e n t s , such as
methanol and c h l o r o f o r m , a r e l e v o r o t a t o r y ; aqueous a c i d i c s o l u t i o n s are d e x t r o r o t a t o r y .
M i c r o c r y s t a l Tests a)
According t o t h e method of C l a r k e and W i l l i a m s (33) , i n potassium chromate s o l u t i o n , n o scap i n e f o r m s f e a t h e r y r o s e t t e s o r b u n c h e s of b l a d e s , s e n s i t i v i t y b e i n g 1 i n 1500 ( F i g . 9 ) .
b)
I n sodium c a r b o n a t e s o l u t i o n r o s e t t e s and b u n c h e s of n e e d l e s a r e s e e n a t t h e same s e n s i t i v i t y (Fig. 10).
NOSCAPINE
437
Fig. 9 .
C r y s t a l s of Noscapine with Potassium Chromate S o l u t i o n .
Fig. 10.
C r y s t a l s of Noscapine with Sodium Carbonate S o l u t i o n .
MOHAMMED A . AL-YAHYA AND MAHMOUD M. A. HASSAN
43 8
Scheme 8 .
P o s s i b l e M e t a b o l i t e s of (-)-a-narcotine
J
be@ ‘ 0
0
OCH3
CH3
nos c a p i n e 1
5
0-demezhyla ted meconine
cotarnine
1
hydrocotarnine
CH3 c o t a r n i n e (pseudo b a s e form) OCH3
ox yc o t a r n i n e
0-d i d eme t h y l a t e d m e t a b o l i t e s
439
NOSCAPINE
7.3
T i t r i m e t r i c Methods The o f f i c i a l methods of d e t e r m i n i n g Noscapine a r e d e s c r i b e d by t h e B.P. (3) and U.S.P. ( 3 4 ) . 7.3.1
Non-Aaueous T i t r a t i o n The B.P. method :-
(3) d e s c r i b e s t h e f o l l o w i n g
D i s s o l v e 0.5 g i n 40 m l of anhydrous g l a c i a l a c e t i c acid previously neutralised t o c r y s t a l v i o l e t , warming g e n t l y . T i t r a t e w i t h 0.1 M p e r c h l o r i c a c i d u s i n g 0.25 m l of c r y s t a l v i o l e t s o l u t i o n a s i n d i c a t o r . Each m l of 0.1 M p e r c h l o r i c a c i d i s e q u i v a l e n t t o 0.04134 g of C22H23 NO,. The U.S.P. met hod :-
describes the following
D i s s o l v e about 1 . 5 g of Noscapine, a c c u r a t e l y weighed, i n 25 m l of g l a c i a l a c e t i c a c i d . Add 25 m l of d i o x a n e and 5 d r o p s of c r y s t a l v i o l e t T . S . , and t i t r a t e with 0.1 N perchloric acid i n g l a c i a l a c e t i c a c i d t o t h e end-point change from p u r p l e t o b l u e . Perform a b l a n k d e t e r m i n a t i o n , and make any n e c e s s a r y c o r r e c t i o n . Each m l of 0.1 N perchloric acid is equivalent t o 41.34 mg of C22H23N07. Another method w a s d e s c r i b e d by T u t h i l l e t a l . (35) u s i n g m a l a c h i t e g r e e n a s b e t t e r indicator than c r y s t a l v i o l e t . 7.3.2
Polarographic T i t r a t i o n a)
Using a dropping mercury e l e c t r o d e a s i n d i c a t o r n o s c a p i n e h y d r o c h l o r i d e c a n be t i t r a t e d w i t h Cadmium I o d i d e i n s o l u t i o n of n e u t r a l s a l t s ( 0 . 1 t I KNO N a C l or 3’ Na2S04) ( 3 6 ) .
b)
Dusinsky (37) d e s c r i b e d a method where noscapine c a n be t i t r a t e d i n a l k a l i n e
440
MOHAMMED A. AL-YAHYA AND MAHMOUD M. A. HASSAN s o l u t i o n (1.25 N NaOH) showing depression i n t h e polarographic curve a t 1.5V. Sodium a l i z a r i n s u l p h o n a t e h a s been used f o r t h e d e t e r m i n a t i o n of n o s c a p i n e (38). A p o t e n t i a l of - 0 . 6 5 V was used and t h e t i t r a t i o n medium was 0.3 N K C 1 a d j u s t e d t o a pH of 4 - 6 . SoucKova and S;ka (39) s t a t e d a method using t u n g s t o s i l i c i c acid. This acid w a s found e s p e c i a l l y u s e f u l s i n c e t h e r e a c t i o n i s v e r y s e n s i t i v e and prec i p i t a t i o n i s immediate. The p o l a r o graphy a l s o gave t h e c o m p o s i t i o n of t h e t u n g s t o s i l i c i c acid organic base complex. The poor s e l e c t i v i t y of T u n g s t o s i l i c i c a c i d i s due t o i t s h i g h s e n s i t i v i t y which a l l o w s a c c u r a t e d e t e r m i n a t i o n of 10-20 mg of b a s e . A 0.01 M aqueous s o l u t i o n of t u n g s t o s i l i c i c a c i d i s used w i t h a dropping mercury c a t h o d e and S . C . E . anode a t 0 . 6 5 V . The pH of s o l u t i o n a d j u s t e d w i t h HC1 ( 0 . 1 t o 0 . 6 N ) . P l o t of c u r r e n t vs a c i d used c o n s i s t s of two s t r a i g h t l i n e s and t h e i n t e r s e c t c o n s i d e r e d as t h e e q u i v a l e n c e p o i n t . Another method f o r p o l a r o g r a p h i c t i t r a t i o n was a l s o r e p o r t e d ( 4 0 ) . T h i s method is based on t h e f o r m a t i o n of complex mercury compounds. S o l u t i o n of K2HgI4 c o n t a i n i n g a n e x c e s s of i o d i d e i s t h e most s u i t a b l e f o r t h e determinat i o n . The t i t r a t i o n i s c a r r i e d o u t w i t h a d r o p p i n g mercury e l e c t r o d e a t t h e p o t e n t i a l -0.8 V t o - 0.9 V (VS. t h e S . C . E . ) w i t h 0 . 1 PI KN03 o r 0 . 1 M H SO a s s u p p o r t i n g e l e c t r o l y t e . 2 4
7.3.3
P o t e n t iometr i c T i t r a t i o n Tungsten rod w a s used a s i n d i c a t o r e l e c t r o d e i n t h e p o t e n t iometr i c t i t r a t i o n of n o s c a p i n e i n a 1 : 6 m i x t u r e of a c e t i c a c i d : a c e t i c anhydride ( 4 1 ) .
NOSCAPINE
7.4
441 Complexometric Noscapine is p r e c i p i t a t e d from 0 . 5 N H C 1 w i t h 0.028 M Bi-EDTA and 0.112 M K I forming iodobismuthate complexes and EDTA i s being set f r e e (42). After c e n t r i f u g a t i o n t h e f r e e EDTA i s determined i n a n a l i q u o t of t h e supern a t e n t l i q u i d w i t h 0.01 M ZnSO4 i n pH 9 . 1 b o r a t e b u f f e r and Eriochrome b l a c k T as i n d i c a t o r . T e r t i a r y amines, q u a t e r n a r y ammonium s a l t s o r analogous sulphonium, phosphonium and arsonium compounds i n t e r f e r e i n the determination.
7.5
Spectrophotometric 7.5.1
Colorimetric
a ) Yoichi and Sano (43) d e s c r i b e a method f o r a n a l y s i s of noscapine i n mixed pharmaceutical preparations. The sample is mixed w i t h a s o l u t i o n of chromotropic a c i d 0.2% i n 70% (v/v) H3PO4 a c i d and h e a t e d a t 100°C f o r 30 m i n u t e s and t h e e x t i n c t i o n i s measured a t 570 nm. A c a l i b r a t i o n c u r v e i s r e c t i l i n e a r f o r 30 t o 150 pg of noscapine h y d r o c h l o r i d e per m l . b) Solochrome Green V 150 ( C . I . Mordant Green 15) h a s been used a s aqueous 1 mM s o l u t i o n . The complex formed by noscapine is e x t r a c t e d i n t o chloroform and t h e absorbance i s measured a t 520 nm ( 4 4 ) . c ) Another method f o r t h e q u a n t i t a t i v e s e p a r a t i o n of p a p a v e r i n e from n o s c a p i n e i n m i x t u r e s w a s a l s o r e p o r t e d (45). T h i s method i s based on t h e f o r m a t i o n of a n insoluble papaverine r e i n e c k a t e i n acid s o l u t i o n i n t h e p r e s e n c e of e x c e s s chloroform. Procedure T r i t u r a t e t h e sample (4.5 g) w i t h g l a c i a l a c e t i c a c i d (25 ml) followed by H 2 0 ( 20 ml) and f i l t e r . E x t r a c t a 1 0 m l a l i q u o t w i t h
442
MOHAMMED A . AL-YAHYA AND MAHMOUD M. A. HASSAN
CHC13 (8 X 1 0 ml) and wash e a c h e x t r a c t i n t u r n w i t h H20 (15 m l ) , H20 (15 ml) p l u s NaOH soln. ( 1 : 1) containing a l i t t l e NaHS03 (15 m l ) , H 2 0 ( 1 5 m l ) , 0 . 1 M H2SO4 (15 m l and 1 0 ml) and 0.05% N a H C 0 3 s o l n . (10 m l ) . E v a p o r a t e t h e combined washed e x t r a c t s t o d r y n e s s o n a water b a t h . D i s s o l v e t h e r e s i d u e i n CCl4 (50 ml) , s t r a i n through cotton-wool and p a s s t h r o u g h a column of Ca(OH)2. Wash t h e column w i t h C C l 4 (2 X 1 0 ml) and e x t r a c t t h e combined CCl4 f r a c t i o n s w i t h 0 . 1 N H C 1 ( 2 X 1 0 m l ) . Shake t h e H C 1 s o l n . w i t h CHC13 (10 ml) f o r 10 m i n . , add 2% ammonium r e i n e c k a t e s o l n . ( 1 0 m l ) , shake f o r 3 0 min. and f i l t e r through s i n t e r e d g l a s s . To d e t e r m i n e papaverine d i s s o l v e t h e ppt. i n acetone and measure t h e e x t i n c t i o n a t 525 mu. To d e t e r m i n e n o s c a p i n e , shake t h e CHC13 l a y e r of t h e f i l t r a t e w i t h 0.25% AgN03soln. ( 4 0 m l ) , s e p a r a t e and f u r t h e r e x t r a c t w i t h CHC13 (2 X 1 0 m l ) ; s t r a i n t h e combined CHC13 f r a c t i o n s through cotton-wool, d i l u t e t o 250 m l , and e i t h e r measure t h e e x t i n c t i o n a t 310 mp o r e v a p o r a t e and t i t r a t e w i t h 0.05 N H C l O 4 i n g l a c i a l a c e t i c a c i d . d) Thomas d e s c r i b e d a method f o r d e t e r m i n a t i o n of some d r u g s c o n t a i n i n g a t e r t i a r y - a m i n e group ( 4 6 ) . The d r u g i s h e a t e d w i t h 10% malonic a c i d i n a c e t i c a n h y d r i d e a t 800 f o r 1 5 min. and, a f t e r d i l u t i o n w i t h e t h a n o l , t h e e x t i n c t i o n i s measured a t 333 nm. t h e l i m i t of d e t e c t i o n f o r n o s c a p i n e hydroDosage c h l o r i d e was 1 0 t o 30 ng m l - 1 . forms r e q u i r e p r e l i m i n a r y e x t r a c t i o n of t h e drug.
7 . 5.2
Infra-red
et a1 (47) d e s c r i b e d a n i n f r a - r e d Bakre s p e c t r o s c o p i c method f o r t h e d e t e r m i n a t i o n of t h e o r i g i n of opium as w e l l a s a s i m u l t a n e o u s a s s a y of n o s c a p i n e , t h e b a i n e and papav e r i n e . 4.5 g f i n e l y ground sample w a s t i t u r a t e d f o r 20 min. i n 25 m l water w a s s l o w l y added w i t h c o n t i n u o u s s t i r r i n g and t h e
443
NOSCAPINE
resulting solution was filtered. 10 m l A l i q u o t of f i l t r a t e w a s e x t r a c t e d f o u r t i m e s i n t o 1 0 m l c h l o r o f o r m and e a c h e x t r a c t w a s washed w i t h 1 0 m l water, 25 m l of 0.2% sodium b i s u l p h i t e i n 30% aqueous sodium h y d r o x i d e , 1 0 m l water and a g a i n 1 0 m l water. The combined c h l o r o f o r m s o l u t i o n was f i l t e r e d t h r o u g h c o t t o n wool and e v a p o r a t e d . The r e s i d u e i s d r i e d i n a d e s i c a t o r t h e n mixed w i t h anhydrous c a r b o n t e t r a c h l o r i d e , f i l t e r e d through s i n t e r e d g l a s s and d i l u t e d t o 25 m l . The I R i s examined from 1100 cm-1 t o 1900 c m - l i n a 1 mm sodium c h l o r i d e c e l l , noscapine b e i n g measured a t 1767 cm-l. The a b s o r b a n c e i s compared w i t h a b s o r b a n c e s of s o l u t i o n s of known c o n c e n t r a t i o n s . Other I R methods f o r d e t e r m i n a t i o n of i s o q u i n o l i n e a l k a l o i d s were a l s o r e p o r t e d (48, 4 9 ) . 7.5.3
Ultra-Violet T e t r a p o n a m i x t u r e of t h e h y d r o c h l o r i d e s of Morphine, Noscapine, c o d e i n e and p a p a v e r i n e w a s a n a l y s e d by J e n s e n ( 5 0 ) . Morphine w a s s e p a r a t e d by e x t r a c t i o n w i t h c h l o r o f o r m from s t r o n g a l k a l i n e s o l u t i o n and d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y w i t h NaN02 a t 440 run. The o t h e r a l k a l o i d s were s e p a r a t e d by T L C on K i e s e l g e l CF 254 w i t h e t h a n o l : benzene 1 : 4 a s s o l v e n t . The s p o t s ( l o c a t e d i n U.V. r a d i a t i o n ) were e x t r a c t e d w i t h methanol and determined a t 215 run f o r c o d e i n e , a t 279 nm f o r p a p a v e r i n e and 312 nm f o r n o s c a p i n e .
7.5.4
Atomic A b s o r p t i o n An i n d i r e c t method f o r t h e a n a l y s i s of Noscapine i n d r u g s w a s r e p o r t e d ( 5 1 ) . A complex i s formed between Noscapine and Reinecke s a l t i n t h e p r e s e n c e of t a r t a r i c a c i d a t pH 1 . 7 . This is extracted i n t o c h l o r o f o r m and Noscapine i s d e t e r m i n e d i n d i r e c t l y by measuring chromium c a t i o n by a t o m i c a b s o r p t i o n s p e c t r op ho tome t er y
.
444
MOHAMMED A . AL-YAHYA AND MAHMOUD M. A. HASSAN 7.5.5
Spectrofluorimetdc a ) Noscapine h a s been determined i n m i x t u r e s of opium a l k a l o i d s (52) by measuring t h e f l u o r e s c e n c e a t 375 run ( e x c i t a t i o n a t 315 nm). The sample i s b u f f e r e d a t pH 9 i n 0 . 1 N s u l p h u r i c a c i d and 0 . 1 N sodium hydroxide. Noscapine i s e x t r a c t e d i n chloroform and a p o r t i o n of t h i s e x t r a c t i s t r e a t e d w i t h t r i c h l o r o a c e t i c a c i d i n chloroform t o quench t h e f l u o r e s c e n c e of papaverine. A s t a n d a r d s o l u t i o n of 2-aminopyridine i n 0 . 1 N s u l p h u r i c a c i d is a l s o measured a t 375 nm ( e x c i t a t i o n a t 315 nm). For t h e c a l c u l a t i o n each f l o u r e s c e n c e r e a d i n g on t h e t e s t s o l u t i o n i s c a l c u l a t e d a s a p e r c e n t a g e of t h a t f o r t h e s t a n d a r d and r e f e r r e d t o c a l i b r a t i o n g r a p h s prepared s i m i l a r l y f o r Noscapine. Sub-microgram amounts of Noscapine c a n b e determined without preliminary separation. b) Vedso s t a t e d a method (53) f o r t h e d e t e r m i n a t i o n of Noscapine i n plasma and urine. I t involves e x t r a c t i o n of 1 m l sample a t pH 1 0 i n t o e t h y l e t h e r and r e - e x t r a c t i o n w i t h d i l u t e HC1. The a c i d i s n e u t r a l i s e d and t h e s o l u t i o n was a d j u s t e d t o PH 9.2 w i t h a borax b u f f e r . Fluorescence i s measured through 480 t o 580 mu f i l t e r ( e x c i t a t i o n a t 365 mu) b e f o r e and a f t e r a u t o c l a v i n g a t 120OC f o r 3 0 min. S t a n d a r d s ( 0 t o 2.5 pg cm-3) are a l s o measured i n t h e same way. I t was s t a t e d t h a t c o n c e n t r a t i o n s from 0.05 ug p e r m l can be determined and a l t h o u g h t h e p r e s e n c e of morphine gave an i n c r e a s e i n f l o u r e s c e n c e about h a l f t h a t f o r n o s c a p h e , c o d e i n e , n a r c e i n e and papaver i n e d i d n o t i n t e r f e r e
.
c ) A r e a c t i o n m i x t u r e of 10%malonic a c i d i n
a c e t i c a n h y d r i d e was used i n a method r e p o r t e d by Rao and Tandon ( 5 4 ) . I n t e r f e r e n c e was caused by T e r t i a r y amines, glucose,magnesium a c e t a t e and some i n o r g a n i c s a l t s , b u t n o t by d i e t h y l a m i n e , a n i l i n e , benzoic a c i d , a s p i r i n and s a c c h a r i n .
NOSCAPINE
445
7.5.6
Nuclear Magnetic Resonance A known amount of t - b u t y l a l c o h o l w a s added as a s t a n d a r d t o n o s c a p i n e i n e t h a n o l f r e e c h l o r o f o r m and t h e peaks a t 3 . 8 3 , 4.00 and 4 . 0 5 ppm; c o r r e s p o n d i n g t o t h e n i n e methoxy group p r o t o n s of n o s c a p i n e were i n t e g r a t e d a l o n g w i t h t h e peak a t 1 . 3 ppm c o r r e s p o n d i n g t o t h e n i n e methyl-group p r o t o n s of t - b u t y l a l c o h o l ( F i g . 11). The amount of n o s c a p i n e i s c a l c u l a t e d from t h e i n t e g r a t i o n r a t i o and t h e known amount of s t a n d a r d ( 5 5 ) .
7.5.7
Mass Noscapine w a s i d e n t i f i e d i n opium (92) w i t h o u t any p r i o r s e p a r a t i o n . Samples a r e introduced d i r e c t l y i n t o t h e ion s our c e us ing a s o l i d sampling probe. Reagent g a s e s were i s o b u t a n e and w a t e r , mass s p e c t r a l measurement was a t m / e 220. Arnold (93) d e s c r i b e d a G C /M S method f o r t h e d e t e r m i n a t i o n of n o s c a p i n e i n opium preparations.
7.6.
Chromatographic
7.6.1
Paper Chromatography The paper-chromatographic method f o r t h e d e t e c t i o n of a l k a l o i d s , e . g . , c o d e i n e , v e r a t r i n e , q u i n i n e and n o s c a p i n e i n s e v e r a l f o o d s ( a 100 g sample) i s d e s c r i b e d . For t h e p r e l i m i n a r y e x t r a c t i o n of t h e a l k a l o i d s , add t o t h e sample 300 m l of e t h a n o l a c i d i f i e d t o l i t m u s p a p e r w i t h H C 1 . D i g e s t o n a water b a t h a t 400 f o r 24 h r . , f i l t e r and r e t a i n t h e f i l t r a t e . Add 100 m l of acidified ethanol to the residue, d i g e s t f o r a n o t h e r 1 2 h r . and f i l t e r a g a i n . Combine t h e f i l t r a t e s and remove t h e a l c o h o l by e v a p o r a t i n g on a w a t e r b a t h a t 4 0 0 . Add 30 m l of water and e x t r a c t w i t h d i e t h y l e t h e r ( 5 X 5 0 m l ) . Add NaOH s o l n . t o t h e a q . l a y e r till i t is a l k a l i n e t o l i t m u s and e x t r a c t w i t h d i e t h y l e t h e r ( 5 X 50 ml). Evaporate t h e e t h e r e x t r a c t t o 50 m l , t r a n s f e r i t t o a s e p a r a t i n g - f u n n e l
446
MOHAMMED A. AL-YAHYA AND MAHMOUD M. A. HASSAN and wash w i t h 2N HCl(3 X 20 m l ) . To t h e combined a c i d washings add NaOH till t h e y a r e a l k a l i n e t o l i t m u s and e x t r a c t w i t h d i e t h y l e t h e r ( 3 X 50 m l ) . E v a p o r a t e t h e e t h e r c o m p l e t e l y and d i s s o l v e t h e extract i n 5 m l of e t h a n o l . R e t a i n t h i s a l c o h o l i c s o h . f o r chromatography by t h e d e s c e n d i n g t e c h n i q u e on Whatman No. 1 p a p e r . Apply d r o p s of t h e t e s t and s t a n d a r d s o l n . t o t h e p a p e r and impregnate i t s whole area above t h e s t a r t i n g l i n e w i t h a f r e s h l y p r e p a r e d e t h a n o l i c s o h . of formamide (1 : 1). Dry t h e paper between two f i l t e r p a p e r s , t h e n a t 400 f o r 3 0 min. Spray t h e s t a r t i n g p o i n t s w i t h t h e e t h a n o l i c s o l n . of formamide (1 : 1) and a f t e r 10 min. t r a n s f e r t h e paper t o a g l a s s c o n t a i n e r c o n t a i n i n g CHC13 a t 250. The development i s complete i n 2.5 h r . Dry t h e chromatogram a t 1050 and s p r a y w i t h a s o h . of potassium i o d o p l a t i n a t e , washing o f f t h e excess w i t h water i n o r d e r t o o b s e r v e t h e s p o t s on t h e w h i t e background. The f o l l o w i n g amounts of a l k a l o i d s can b e d e t e c t e d - c o d e i n e 0.01 t o 0.3 mg, q u i n i n e 0.005 t o 0.015 mg, v e r a t r i n e 0.1 t o 0.15 mg and noscapine 0.05 t o 0.075 mg. ( 5 6 ) . T a b l e 5 d e s c r i b e s methods used i n noscapine a n a l y s i s . A n a l y s i s of noscapine i n opium by paper chromatography and s p e c t r o p h o t o m e t r y involved e x t r a c t i o n of noscapine and measurement of t h e e x t i n c t i o n of t h e s p o t a t 290 mu and comparison w i t h s t a n d a r d s ( 5 7 ) . Other method was a l s o d e s c r i b e d ( 5 8 ) . 7.6.2
Thin Layer Chromatography T h i s t e c h n i q u e h a s been used e x t e n s i v e l y f o r a n a l y s i s of Opium and i t s p r e p a r a t i o n s (63 - 7 1 ) . T a b l e 6 - g i v e s a resume of t e c h n i q u e s and T a b l e 7 - shows s p r a y r e a g e n t s and methods used. S t a h l and co-workers have proposed a s t a n d a r d p r o c e d u r e f o r t h e s e p a r a t i o n of opium a l k a l o i d s ( 6 3 ) . Dried opium ( 0 . 1 g)
... I
S O
. . . .
. - . .
. . . . I . . . . ~ " . . ~ . . . . ~ . . " . ~' I" ' I
I
'
400
300
zbo
I
I
.
I
100
I o ni
H3
1
.
1
I
l . . l . . . ' l . . . ' l . . . . i . . - . l
.
1 . .
.
1
. I . .
1 .
. I
I . .
..
I
II
T a b l e 5.
1 S t a t i o n a r y Phase Paper
Paper
2 Technique Two d i m e n s i o n a l
Two d i m e n s i o n a l
Paper Chromatography Used € o r Noscapine.
4
3 Mobile P h a s e
1. Water s a t . b u t a n o l - a c e t i c acid 5:l 2. e t h e r - 0 . 1 M a c e t i c a c i c 5: 2
6
5 Comment Alkaloids i n Tetrapon 11
1. Dioxan-Formic a c i d - w a t e r 90:0.5 : 9 . 5 2. n - B u t a n o l - a c e t i c a c i d 5:l
11
11
Paper
One d i m e n s i o n a l
1. Dioxan-Formic 90:0.5:9.5
Paper S & S 204 3b.
Ascending o r descending
25% (NH4)2 SO4 i n 0 . 5 N H C 1
Whatman No. 1 paper b u f f e r e d a t pH 3.5
D e s c end i n g
Isobutylalcohol - toluene s a t u r a t e d w i t h water 1 : 1.
Paper
One d i m e n s i o n a l
Butyl acetate - acetic acid 47:9:28:16
acid-water
- butanol - water
11
Separation of n o s c a p i n e from papaver i n e enhancec b e c a u s e of u s e of b u t y l acetate.
rferenct
T a b l e 5.
I 1
c
\D
I
2
(contd
... .)
3
Paper
One d i m e n s i o n a l
Upper l a y e r of a m i x t u r e n-butanol-acet i c a c id-water 5:1:4
Wha tman N o . 1 impregnated above s t a r t i n g l i n e with f ormamidee t h a n o l 1:l then d r i e d between f i l t e r papers t h e n a t 40°C f o r 30 min.
D esc end
Chloroform
4
5
Starting points w e r e sprayed w i t h f ormamide i n e t h a n o l 1:l a f t e r drying t h e paper
6
(56)
T a b l e 6.
S t a t i o n a r y Phase K i e s e l g e l HF
254
TLC Techniques Used f o r Noscapine
Technique Normal chamber
Mobile P h a s e
Rf
Tolune-acetone 95% e t h a n o l 25% aq. NH3
20 : 20 : 3 : 1
P
-
S i l i c a g e l G-Na2C03
tI
Chloroform 4 : l
Silica gel G.
II
E t h y l acetate
K i e s e l g e l 60
It
Chloroform Benzene Acetone 3 : 3 : 1
Silicagel G
I1
Benz ene-methanol
Silica gel G
I1
B u t a n o l - A c e t i c acid-H 0 2 3 : l : l
S i l i c a g e l G impreg. 4% Na2C03
11
Chloroform 4 : l
Silica gel
11
Chloroform-isopropyl a l c o h o l 10% aq.NH3 30 : 10 : 1 Benzene-methanol 4 : 1
Ethanol
VI
0
Silica gel G
-
4 : l
-
Ethanol
Table 7.
TLC S p r a y R e a g e n t s and Methods Used € o r D e t e c t i o n of Noscapine
Reagent
Procedure ~
5% 3,5-dichloro-p-benzoquinonechlorimine i n i s o p r o p y l alcohol
~~
A f t e r s p r a y i n g s p r a y w i t h aqueous
NH3 1 : 1 and o b s e r v e i n d a y l i g h t and u l t r a v i o l e t .
4% Hg(N03)2 i n 3% HN03
After spraying t h e p l a t e i s heated 15 min. a t l l O O C and o b s e r v e d i n d a y l i g h t . D e t e c t i o n l i m i t 2 vg
1. 3% H202 s o l u t i o n 2. 5% K4Fe(CN)6 s o l u t i o n
P l a t e i s f i r s t d r i e d 1 0 min. a t 100°C t h e n s p r a y e d w i t h 1. t h e n d r i e d 1 0 min. a t 100°C and s p r a y e d w i t h 2 and d r i e d 1 0 min. a t 100°C. Brown s p o t s i n t e n s i f i e d t o r e d . D e t e c t i o n l i m i t 1 0 pg.
2.6 g C@(N@3)2 d i s s o l v e d i n 2 m l anhydrous a c e t i c a c i d i s added t o 4 . 4 g o f NaN02 d i s s o l v e d i n 1 0 m l H20 t h e n 20 ml a c e t i c a c i d and 50 m l H 0 i s added t o m i x t u r e . 2
After spraying, t h e p l a t e is heated a t 105 OC f o r 10 min. S p o t s a r e s t a b l e f o r several h o u r s . Noscapine a p p e a r s a s b l u e - g r e e n f l o u r e s c e n t s p o t when viewed under u l t r a - v i o l e t l i g h t .
4% c i t r i c a c i d i n
a c e t i c anhydride
P l a t e i s h e a t e d a t 80 O C f o r 1 0 min. and viewed i n d a v l i g h t ( d e t e c t i o n l i m i t 5 pg) and i n u l t r a - v i o l e t r a d i a t i o n ( d e t e c t i o n l i m i t 0.5 Up).
Ref.
452
MOHAMMED A. AL-YAHYA AND MAHMOUD M. A . HASSAN w a s powdered and t r i t u r a t e d w i t h 5 m l of 70% e t h a n o l . The m i x t u r e w a s warmed a t 50 - 600 f o r 30 min. t h e n f i l t e r e d and d i l u t e d t o 1 0 m l w i t h 70% e t h a n o l . T i n c t u r e of opium (1 m l ) was d i l u t e d w i t h 9 m l 35% e t h a n o l . Three p o r t i o n s ( 5 , 1 0 and 20 u l ) of opium s o l u t i o n and similar p o r t i o n s of s t a n d a r d s o l u t i o n were a p p l i e d t o a l a y e r of K i e s e l g e l H F254 and chromatograph was developed t o 1 5 c m w i t h t o l u e n e - a c e t o n e - 95% e t h a n o l 25% a q . NH3 (20 : 20 : 3 : 1 ) . For d e t e c t i o n , t h e p l a t e w a s h e a t e d a t 110% f o r 1 0 min. and t h e s e p a r a t e d zones l o c a t e d i n u l t r a v i o l e t r a d i a t i o n . The s p o t s a r e t h e n sprayed u s i n g a modified D r a g e n d o r f f ' s r e a g e n t and t h e n w i t h 0.05 N t o 0 . 1 N s u l p h u r i c a c i d . A l s o polyamide h a s been used a s l a y e r f o r chromatography ( 7 6 ) . Where poly-E-caprolactam r e s i n (Amilan CM 10075) was used. Development f o r 2 h r . i n cyclohexane-ethyl a c e t a t e p r o p y l a l c o h o l - Me2NH 3 0 : 2.5 : 0.9 : 0 . 1 showed n o s c a p i n e a t Rf 0 . 6 1 and development i n H20-ethanol - Me2NH 88 : 1 2 : 0 . 1 n o s c a p i n e had Rf 0.00.
7.6.3
Gas Liquid Chromatography
D e r i v i t i z a t i o n of samples i n c l u d e d a c e t y l a t i o n using acetic anhydride i n p y r i d i n e (77) and t r e a t i n g sample w i t h t r i m e t h y l s i l y l acetamide and t r i m e t h y l c h l o r o e t h a n e ( 6 8 ) . I n t e r n a l s t a n d a r d s used were h i s t a p y r r o d i n e h y d r o c h l o r i d e , o e s t r a d i o l v a l e r a t e (78) Phenazone (79) and S q u a l i n e ( 7 7 ) . Another method was a l s o r e p o r t e d ( 8 0 ) . Column t y p e s e t c . a r e r e p o r t e d i n T a b l e 8. 7.6.4
HiPh Performance Liquid Chromatography S e p a r a t i o n s of p h a r m a c e u t i c a l s combined i n v a r i o u s f o r m u l a t i o n s by HPLC on S e p h e r o s i l 5 Um i s o c r a t i c a l y h a s been r e p o r t e d ( 8 1 ) . Noscapine i n a n t i - c o l d p r e p a r a t i o n s w a s s e p a r a t e d on a column of H i t a c h i g e l 3011-0(82)
T a b l e 8. Column Type
S t a t i o n a r y Phase
4 f t x 4 m m glass
D i a p o r t S (80-100) mesh
1 . 5 m x 2.3 mm g l a s s
Chromosorb G-HP AW-DMCS (80-100) mesh
GLC ConditionsUsed f o r Noscapine Flow-ra t e
3 0 cm
SE-30
40 cm
HI-EFF 8 BP
Gas-Chrom 0 (100-120 mesh)
3
min
min
-1
-1
-
Supelcoport (80-100 mesh) 4 f t x 3mm od g l a s s
3
Temperature
2% OV 101
N2
30 cm
3
min
-1
24OoC
tg
I e t e ct o r
Flame i o n i z at i o r
15OoC 235OC a t 1 . 2 5 C min-1
11
2 2 5OC-2 7 O°C a t 2 0 min-1 ~
11
1 8 O o C 5 min. then 7 ' ~ min-l t o 25OoC
11
Ref,
454
MOHAMMED A. AL-YAHYA AND MAHMOUD M. A . HASSAN
u s i n g methanol : 28% aqueous NH3 9 9 : l a s e l u t i n g s o l v e n t and d e t e r m i n a t i o n by spectrophotometry a t 230 nm o r 250 nm. Paracetamol, p h e n a c e t i n , d i p y r o n e , a s p i r i n , c a f f e i n e , etenzamide and m e t h y l e p h e d r i n e d i d n o t i n t e r f e r e . Other methods were a l s o d i s c r i b e d (83, 8 4 ) . 7.6.5
Ion-Exchange Chromatography Knox and Jurand r e p o r t e d a method (85) f o r t h e s e p a r a t i o n of n o s c a p i n e on d r y packed column of Zipax SCX o r SAX (37-44 um) b o r a t e b u f f e r s a t pH 9.2-9.8 c o n t a i n i n g 4% a c e t o n i t r i l e and 1%propanol were used a t 500 t o 1500 l b p e r s q . i n . t o e l u t e n o s c a p i n e .
7.6.6
Ligand-Exchange Chromatography P o r a g e l P.T. r e s i n was used t o s e p a r a t e a l k a l o i d s and n o s c a p i n e h a s been a n a l y s e d on t h i s r e s i n . 0.06 M-aqueous NH3 i n 33% e t h a n o l w a s used as e l u a n t and t h e e l u t i o n volume f o r n o s c a p i n e w a s 6.8 times t h e bulk column volume ( 8 6 ) .
7.6.7
P a r t i t i o n Chromatography I n t h e a s s a y of T e t r a p o n by p a r t i t i o n chromatography t h e s t a t i o n a r y phase i s a phosphate b u f f e r ( 8 7 ) . 5 m l of 0 . 2 N NaOH i s added t o 0.4 g of papaveretum i n 20 m l H 2 0 . The m i x t u r e i s e x t r a c t e d twice w i t h a m i x t u r e of 1 0 m l of c h l o r o f o r m i n 3 0 m l e t h e r and t h e n w i t h 1 0 m l of chloroform. The f i l t e r e d e x t r a c t s a r e e v a p o r a t e d t o 0.5 1 . 0 m l and d i l u t e d w i t h 25 m l of e t h e r b e f o r e t r a n s f e r t o t h e p r e p a r e d column, 200 m l water s a t u r a t e d e t h e r is used t o e l u t e n o s c a p i n e . Other a s s a y w a s a l s o r e p o r t e d ( 8 8 ) .
7.6.8
Paper E l e c t r o p h o r e s i s Due t o f o r m a t i o n of m o l e c u l a r complexes between n o s c a p i n e and 7-(2-hydroxyethyl) t h e o p h y l l i n e , t e t r a m e t h y l u r i c a c i d and 7-carboxymethyltheophyline, n o s c a p i n e h a s
455
NOSCAPINE
been separated from other isoquinoline alkaloids by reversed - phase paper chromatography and electrophoresis. Britton - Robinson buffer pH 3.5 to 4 was used as the mobile phase and o-xylene as stationary phase ( 8 9 ) . Another method was also reported (1).
ACKNOWLEDGEMENTS The authors would like to thank the technicians, Robert Hutchison, K.N. Ludhi and the Research assistant Syed Rafatullah o f the College of Pharmacy, King Saud University, Riyadh, Saudi Arabia for their kind technical assistance for the preparation of the manuscript.
456
8.
MOHAMMED A . AL-YAHYA AND MAHMOUD M. A. HASSAN References
1.
M. Windholz, "The Merck Index", 9th e d . , Merck and Co. I n c . , Rahway, U.S.A. , p.872, (1976).
2.
Chemical A b s t r a c t s , I n t r o d u c t i o n t o Volume 66 Subj ect Index, (1967).
3.
" B r i t i s h Pharmacopoeia'', Vol. 1,Her M a j e s t y ' s s t a t i o n a r y o f f i c e , London, p.311, ( 1 9 8 0 ) .
4.
M. Shamma, "The I s o q u i o n o l i n e A l k a l o i d s " P r e s s London, p.360, (1972).
5.
A.R. B a t t e r s b y and H. S p e n c e r , J . Chem. SOC., 1087, (1965).
6.
M. O h t a , H. T a n i , S . Mavozumi, a n d S . Kodaira,Chem. Pharm. B u l l . 2, 1080, ( 1 9 6 4 ) .
7.
M. O h t a , H. T a n i , and S. Movozumi, Chem. pharm. B u l l 12, 1072, (1964).
8.
A.R. B a t t e r s b y , and H. S p e n c e r s , T e t . L e t t . 1, 11, (1964).
9.
S. S a f e , and R.Y.
10.
F.M.
11.
E.G.
12.
J . G . Grasselli, and W.M. R i t c h e y , " A t l a s of S p e c t r a l Data a n d P h y s i c a l C o n s t a n t s for Organic CompoundsI1 2nd ed. Vol.111 CRC P r e s s I r c . , p.670, ( 1 9 7 5 ) .
13.
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