Analytical Profile Of Diethylstilbestrol

ANALYTICAL PROFILE OF DIETHYLSTILBESTROL

Abdullah A. Al-Badr** M e 1 G. Mekkawi*

**Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University

*Riyadh Health Institute, Ministry of Health, Riyadh.

ANALYTICAL PROFILES OF DRUG SUBSTANCES VOLUME 19

145

Copyright B 1990 by Academic Press,Inc. All rights of reproduction in any form reserved

ABDULLAH A. AL-BADR AND ABDEL G. MEKKAWI

146

CONTENTS 1. Description.

Nomenclature Formulae Molecular Weight Elemental Composition 1.5 Appearance, Color and Odor

1.1 1.2 1.3 1.4

2. Physical Properties. 2 . 1 Solubility 2 . 2 Melting Range 2 . 3 Spectral Properties 2.3.1 2.3.2 2.3.3 2.3.4

Ultraviolet Infrared Nuclear Magnetic Resonance Mass

2 . 4 Crystalography and X-Ray Crystalography 2 . 5 Thermal Analysis 2 . 6 X-Ray Powder Diffraction 3. Synthesis. 4. Physiology, Pharmacology and Uses.

5. Pharmacokinetics.

6. Methods of Analysis. 6.1 6.2 6.3 6.4 6.5 6.6

Identification Titrimetric Methods Spectrophotometric Methods Polarographic Methods Chromatographic Methods Radioirnmunoassay Methods

7. Metabolism

Acknowledgement References.

147

DIETHYLSTILBESTROL

1. DESCRIPTION 1.1

1.1.1

Nomenclature Chemical Name 3,4-Di(p-hydroxyphenyl)hex-3-ene. 3,4-Bis(p-hydroxyphenyl)-3-hexene. 4,4(1,2-Diethyl-1,2-ethenediyl)bis(E)phenol. -4,4*-Dihydroxy-GP-diethylstilbene.

a,&-Diethylstilbenediol.

a,&-Diethyl-(E)-4,4’-Stilbenediol. trans-a,&-diethyl-4,4’-Stilbenediol

1.1.2

(1-4).

Generic Names Diethylstilbestrol; Diethylstilboestrol; DES, Stilbestrol; Stilboestrol; Stilbol (2,3,5).

1.1.3

Properietary Names Antigestil; Bio-des; Biofon; Cyren A; Distilbene; Domestrol; Estrobene; Estrosyn; F o r n a t o l ; Grafestrol; Hi-Bestrol; Microest; Neo-Oestranol 1, Oes trogenine; O e s t r o m e n i n; O e s t r o m e n s y 1 ; Oe st romon; Palest rol ; Percutacrine Oestrogenique Iscovesco; Serral; Sexocretin; Sibol; S t ilbetin; Stilboef ra; Stilboestroform; Stilkap; Synestrin (Tablets); Synthoestrin (1-5).

1.2 Formulae 1.2 1 Empirical 18*20°2

1.22 Structural

ABDULLAH A. AL-BADR AND ABDEL G. MEKKAWI

148

1.23 CAS registry No. [ 56-53-1

1.

1.3 Molecular Weight 268.4 1.4 Elemental Composition C 80.56%, H 7.51%, 0 11.92%. 1.5 Appearance, Color and Odor

White or almost white, crystalline powder and is odorless (2,4). 2. PHYSICAL PROPERTIES 2.1 Solubility Very slightly soluble in water, soluble solutions of alkali hydroxides, soluble in ether, in 5 parts of 95% v/v alcohol and in arachis oil, slightly soluble in chloroform

in aqueous 3 parts of 40 parts of (2,4).

2.2 Melting range 169-175'C

(1).

2.3 Spectral Properties 2.3.1

Ultraviolet Spectrum

The ultraviolet spectrum of diethylstilbestrol in 0.1N sodium hydroxide, maximum at 259 nm ( E l % , 1 cm 764). (2). The ultraviolet spectrum of diethylselbestrol in ethanol is obtained using Cary, 219 spectrophotometer and is shown in Figure [l]. The spectrum shows a major band at 240 nm and a minor band at 280 nm. 2.3.2

Infrared Spectrum

The infrared spectrum of diethylstelbestrol as KBr disc is presented in Figure [2] and is recorded in PerkinElmer spectrophotometer model 580 B. The structural

DIETHYLSTILBESTROL

149

Wavelength ( n m )

300

400

Figure I : Ultrwiokl spectrum of Diethylstilbcstrd in ethanol

MICRONS I

4olm 3000 2500 2000 la00 1600 1400 1000 800 Wavenumber Figure 2 : Infrared spectrum of Diethylstilbestrol, KBr disc.

600

400 200

ABDULLAH A. AL-BADR AND ABDEL G. MEKKAWI

150

assignments have been correlated with band frequencies and are given in the following table :Frequency (cm")

Assignment

1592,1612 2990 3420 1340 830

C=C stretch (aromatic) C-H stretch OH stretch C-H bending C-H bending (aromatic)

Clarke (2) reported the following principal peaks as KBr disc: 1198, 1515 and 1165 cm-'. 2.3.3 Nuclear Magnetic Resonance Spectra 2.3.3.1

Proton Magnetic Resonance Spectrum (PMR)

The PMR spectrum of diethylstilbestrol was recorded on varian T-60 A spectrometer with DMSOd6 as a solvent and TMS (tetramethylsilane) as internal reference. The spectrum is shown in Figure [31 and the signal are assigned as follows:Proton

6.6 - 7.1 0.65 2.05 9.16

Phenyl protons CH3 CH2 OH 2.3.3.2

Mu1t iplicity

Chemical shift (ppm)

Multiplet Triplet Quartet

Carbon-13 NMR Spectra

The Carbon-13 NMR noise-decoupled and of fresonance spectra are presented in Figures [ 4 ] and 151 respectively. The samples were dissolved in DMSO-d6 and the spectra were obtained on Jeol-XL100 NMR spectrometer using tetramethylailane (TMS) a s i n t e r n a l r e f e r e n c e standard. Spectral assignments are listed below: a'

9'

CH2CH3 140

-' H

!(

3

CH 8 2

3'

2'

ioo

300

200

0

c , . ' . . * * 1 * - .

.

~

-

.

"

~

.

"

'

~

-

.

.

.

~

.

.

.

.

~

.

.

.

.

~

.

.

~

.

8.0 7.0 6.0 s.oppM( 6 40 3.0 2.0 LO 0 Figure 3: Proton nuclear magnetic resonance spectrum of Diethylstilbestrol in DMSO-dg using TMS as reference standard.

1

152

ABDULLAH A. AL-BADR AND ABDEL G. MEKKAWI

figure 4 : Proton -decoupled carbon-I3 NMR spectrum

Of

DiethylsteLkstrol i n

DMSO-d6 using TMS as reference standard.

-

Figure 5: Off-Resonance carbon I3 NMR spectrum of Diethylstelbestrol in D M s 0 - d ~using TMS as reference standard.

DIETHYLSTILBESTROL

Carbon No.

153

Chemical Shift (ppm) relative to TMS

1 and 1’ 2,2‘ and 6,6’ 3,3‘ and 5,5’ 4 and 4’ 7 and 7’ 8 and 8‘ 9 and 9’ 2.3.4

155.58 114.78 129.22 137.85 132.45 28.00 13.27

Multiplicity Singlet doublet doublet singlet singlet triplet quartet

Mass Spectrum

The electron impact (EI) mass spectrum at 70 eV recorded on Varian Mat 311 mass spectrometer and the methane-derived chemical ionization (CI) mass spectrum obtained with Finnigan 4000 mass spectrometer are shown in Figures [ 6 ] and [ 7 ] respectively. Scheme ( 1 ) shows the proposed fragmentation pathway. Low resolution mass spectra for diethylstilbestrol, dienestrol, hexestrol, the acetates of them, dimethyl, the bis-(trimethylsilyl) ethers, and some deuterated derivatives of these were studied by Engel -et a1 ( 6 ) . 2.4 X-Ray Chrystallography

Simley and Rossmann ( 7 ) have determined the crystal structure of diethylstilbestrol because of the interest in its biological activity. Crystal of the drug are orthorhombic with a = 19.18, b = 5.32 c = 15.01 A . , 2 = 4 , calculated d = 1.164, experimental d = 1 . 1 6 2 , and space group Pcab. The planes of the two benzene rings were paralleled but not coplanar. This loss of coplanarity resulted from steric effect between the methylene carbon of the ethyl group and the orthohydrogen atoms. The bond length A and angles ( 0 ) are listed below (Table 1) and Figure I81 gives a respective view of the molecule. Busetta and Hospital ( 8 ) have studied the crystalline structure of diethylstilbestrol. The drug was crystallized by sublimation in the form of orthorhombic needles, space group Pbca with a = 18.992 + 0.005, b = 14.931 + 0.005 and c = 5.296 + 0.005 A; Z =-4. The unit

ABDULLAH A. AL-BADR AND ABDEL G . MEKKAWI

154

loo.(

2

i680.

ME50 100 I50 200 250 300 Figure 6 : Electron impact ( E l lmass spectrum of Diethylstilbestrol

1000-

5 0.0-

'4472.

m/e268

m / e 253

m / e 1-19

czH5 -HO-Q-c=C-Q-OH Scheme 1:

m/e268

m/e91

C2H5

l+

m / e 239

m/e210 Proposed fragmentation pathway of diethylstilbestrol.

-r

A

ro r tm V

6

4

E

I

-

-

f I' "II ' s

$0 I V

Ot I

0

N

I

to

G

I

I '

\

aJ E

0 c

fi

E

\

aJ

6

8 r q 4-

156

Scheme 1 :

Proposed fragmentation (continued).

pathway of

diethylstilbestrol

158

ABDULLAH A. AL-BADR AND ABDEL G . MEKKAW

Figun8:Molecular stereochemistry of

-

aa/diethylstilbene 44’-d io I

DIETHYLSTILBESTROL

159

of symmetry i s h a l f a molecule, and t h e molecules a r e bonded t o e a c h o t h e r w i t h hydrogen bonds 3A long and with a d i h e d r a l a n g l e of 88’. Table (1) Bondlength A

and Bond a n g l e s

C( l.)-C(l)-C(8) C( l)-C(8)-C(9) C( 1)-C( 1 )-C(2) C(2)-C( 1)-C(8) C(3)-C(2)-C(7) C(2)-C(3)-C(4) C(3)-C(4)-C(5) C(4)-C(5)-C(6) C( 5)-C( 6)-C( 7) C(2)-C(7)-C(6) C( 4)-C( 5)-0 C(6)-C(5)-0

(0)

124.07 109.32 122.22 113.78 116.05 125.15 116.27 122.65 119.15 121.65 119.57 117.80

The c r y s t a l s t r u c t u r e of d i e t h y l s t i l b e s t r o l was a l s o s t u d i e d i n r e s p e c t t o e s t r o g e n i c a c t i v i t y by Weeks et a 1 (9). The d r u g ( s i n g l e c r y s t a l s ) were grown by s l o w l y c o o l i n g a s o l u t i o n of t h e d r u g i n a 0.01 M s o l u t i o n of p - c h l o r o p h e n o l i n i s o - o c t a n e . The m o l a r r a t i o of p - c h l o r o p h e n o l of t h e drug i n t h i s s o l u t i o n was 2:l. The s y s t e m a t i c absences (OK1 f o r K odd, h01 f o r 1 odd, and hKO f o r h o d d ) i n t h e d i f f r a c t i o n p a t t e r n , were c o n s i s t a n t w i t h t h e o r t h o r h o m b i c s p a c e group Pbca and t h e c r y s t a l d a t a a r e :

2 0.004, b = 14.929 + 0.001 5.291 + 0.001 A ( a t 2OoC, Cu Kal = 1.5045 A) V = 1497.29 A3, Dm = 1.14 g.cm Dc = 1.19 g . ~ m ‘ ~ , u = 6.1 cm-I (by f l o t a t i o n ) , Z = Space group Pbca, D1”no.61. a = 18.954

c

=

The a u t h o r s have p r e s e n t e d a t a b l e showing t h e agreement b e t w e e n t h e o b s e r v e d and c a l c u l a t e d s t r u c t u r e f a c t o r amplitudes. Table 2 shows a l i s t of t h e r e f i n e d atomic c o o r d i n a t e s and t h e thermal parameters. The i n t e r a t o m i c d i s t a n c e s and valency a n g l e s i n v o l v i n g n o n h y d r o g e n a t o m s a r e shown i n F i g u r e [9] (Standard d e v i a t i o n a r e i n t h e range of 0.006-0.01 A and 0.2-0.7 An u n u s u a l l y s h o r t a p p a r e n t d i s t a n c e of respectively). 1.498 A between C(8) and C(9) r e s u l t from t h e l a r g e thermal motion of C(9). C-H d i s t a n c e l i e s i n t h e range

exp [ 2

0.15324(27) 0.21313(25) 0.21507(22) 0.153%(25) 0.09949(23) 0.09571(23)

0.03131(24) 0.04616(27) 0.07880(32) 0.27396(16)

(U h2a* + 2UI2hka*b* +

0.03507(33) 0.08731(33) 0.14632(28) 0.15144(31)

0.09820(31) 0-04043(28) 0.01726(25) 0.11730(29) 0.14524(35) 0.20083(20)

-0.0921 4.0548 -0.1462 4.3118 -0.2722 -0-0669 -0.0233 -0.0418 -0.2873 4.17M

9) 9) 8) 9) 9) 8) 8) (10) (12) ( 6) ( ( ( ( ( ( (

...11

0.0680(30) 0.0576(26) O.W5(22) 0.0563(27) 0.0479(24) 0.0555(24) 0.0601(23) 0.0633(28) 0.0818(36) 0.0508(17)

0.0474(24) 0.0505(24) 0.0378(21) 0.0523(27) 0.0518(25) 0.0357(20) 0.0291(21) 0.0302(21) 0.0482(28) 0.0456(16)

Table 2 ( b ) : Atomic coordinates of t h e hydrogen atoms. 0.1543 0.2524 0.1707 0.0618 0.0849 -0.01 18 0.0630 0.1342 0.0804 0.2729

-0.0025 -0.0827 -0.1876 -0.1044 -0.1348 -0.1470 -0.1087 -0.1313 -0.2026 -0.2250

-0.2554 -0.1638 -0.4613 -0.3899 -0.0820 -0.0545 -0.4219 -0.2757 -0.3422 -0.3303

0.0533(26) 0.0548(26) 0.0587(26) 0.0556(27) 0.0581(26) 0.047’l(23) 0.0536(24) 0.0811(34) 0.0876(41) 0.0700(23)

-0).O020(22) -0.0058(22)

-0.a)69(2l) -0.0087(19) -O.a)82(22) d0093(21) -0.0071(22) -0.0011(21) -0.U32l(l8) -0.0110(19) -0.0040(17) -0.OllX21) 4-0042(20) -0.0140(25) -0&092(26) -0JB88(30) -0.0126(14) -0.0014(16) -0.0056(2l) -0.0001(17)

-0.0081(20) -0.0056(20)

-0.0033(18) -0.0106(20) -0.0102(21) -0.002l( 17) 4.0004(17) 4.00l2(21) -0.0213(27) -0).M)56(16)

Ho OH

Figure9:lnteratomic distances and valency angles. Corrected for thermal motion ( 9 ) .

162

ABDULLAH A. AL-BADR AND ABDEL G. MEKKAWI

of 0.91 t o 1.19 A, with average v a l u e of 1.00 A. 0 -H d i s t a n c e i s 0.92 A.

The

A c l o s e i n t e r m o l e c u l a r c o n t a c t of 3.03 A occurs between oxygen atoms. The a n g l e OH---0 i s 171°, and t h e H----0 d i s t a n c e i s 2.11 A. The system of h y d r o g e n bonds a n d t h e packing of t h e molecules a r e i l l u s t r a t e d i n f i g u r e [ l o ] which i s a p r o j e c t i o n of one u n i t c e l l down t h e c axis.

Neither t h e benzene r i n g s nor t h e a l k y l groups l i e i n t h e plane of t h e c e n t r a l e t h y l e n i c l i n k a g e . Since t h e atoms comprising t h e benzene r i n g l i e n e a r l y i n a p l a n e a s do t h o s e a t t a c h e d t o t h e c e n t r a l d o u b l e bond, t h e g e o m e t r y of t h e m o l e c u l e i s f i x e d when t h e t o r t i o n a l a n g l e s a b o u t t h e C(6)-C(7) and C(7)-C(8) bonds a r e d e f i n e d . T h e s e a n g l e s a r e l i s t e d i n T a b l e ( 3 ) . The a n g l e formed by t h e l e a s t - s q u a r e p l a n e t h r o u g h t h e e t h y l e n i c l i n k a g e and t h e p l a n e t h r o u g h t h e benzene r i n g i s 62.8'. Since t h e molecule l i e on c r y s t a l l o g r a phic c e n t r e of symmetry, t h e a n g l e s of r o t a t i o n of t h e two r i n g s a r e i d e n t i c a l . The t h r e e dimensional configur a t i o n of t h e molecule i s i l l u s t r a t e d i n F i g u r e [ l l ] . Rotation of t h e r i n g s o u t of t h e c e n t r a l plane g i v e t h e m o l e c u l e a t h i c k n e s s of a b o u t 4.5 A , which i s comparable t o t h e t h i c k n e s s of a s t e r o i d a l e s t r o g e n a t C(18). The r e s u l t s of t h i s X-ray i n v e s t i g a t i o n c o n f i r med t h a t t h e s y n t h e t i c e s t r o g e n , d i e t h y l s t i l b e s t r o l h a s a non p l a n a r conformation. A s shown by t h e d i s t a n c e between t h e p h e n o l i c oxygen atoms, it i s a molecule s l i g h t l y l o n g e r t h a n t h e n e u t r a l e s t r o g e n s , b u t t h e r o t a t i o n of t h e b e n z e n e r i n g s o u t of t h e p l a n e of t h e c e n t r a l d o u b l e bond r e s u l t s i n a molecular dimention similar t o the t h i c k n e s s of a s t e r o i d a l e s t r o g e n .

a

0

U

2

U

7 2 :

7

c

0

U

.s

.-c .u

8 m

.-C

a

-I V

P

Figure1l:Three dimensional conformation of diethylstilbestrol ( 9 1.

DIETHYLSTILBESTROL

165

Table 3 : T o r s i o n a l a n g l e s f o r one asymmetric u n i t of the structure. Angle

C(l)-C(6)-C(7)-C(7’) C(l)-C(6)-C(7)-C(8) C(5)-C(6)-C(7)-C(7’) C(5)-C(6)-C(7)-C(8) C(6)-C(7)-C(8)-C(9) C(9)-C(8)-C( 7)-C( 7‘)

-118.6’ 62.1 63.4 -115.9 56.0 -123.3

The c e n t e r of symmetry c r e a t e s a n g l e s i n t h e o t h e r h a l f of t h e molecule which have t h e o p p o s i t e s i g n .

2.5 Thermal A n a l y s i s The t h e r m a l a n a l y s i s o f d i e t h y l s t i l b e s t r o l was done between 100°C and 250°C a t a h e a t i n g r a t e of 10°/minute ( F i g u r e [12]). P u r i t y of sample was found t o be 99.86%. Heat of f u s i o n of t h e s a m p l e was found t o be 31.9 Km/mole (7.62 Kcal/mole).

2.6 X-Ray Powder D i f f r a c t i o n The X-ray d i f f r a c t i o n p a t t e r n s of d i e t h y l s t i l b e s t r o l was d e t e r m i n e d u s i n g P h i l i p s f u l l a u t o m a t e d X-Ray d i f f r a c t i o n Spectrogoniometer equipped with PW 1730/10 g e n e r a t o r . R a d i a t i o n was p r o v i d e d by a c o p p e r t a r g e t (Cu anode 2000 w, 8 = 1.5480 A) h i g h i n t e n s i t y x-ray The monochromator t u b e o p e r a t e d a t 40 Kv and 35 mA. was a c u r v e d s i n g l e c r y s t a l o n e (PW 1752/00). Divergance s l i t and t h e r e c e i v i n g s l i t were 1 and 0.1’ respectively. The scanning speed of t h e goniometer (PW 1050/81) used was 0.02-28 p e r second. The i n s t r u m e n t i s combined w i t h P h i l i p s P M 8210 p r i n t i n g r e c o r d e r w i t h b o t h a n a l o g u e r e c o r d e r and d i g i t a l p r i n t e r . The goniometer was a l i g n e d u s i n g s i l i c o n sample b e f o r e use. The x-ray p a t t e r n of D i e t h y l s t i l b e s t r o l i s p r e s e n t e d i n F i g u r e [13]. The i n t e r p l a n n e r d i s t a n c e s d(A) and r e l a t i v e intensity 1/10 a r e shown i n t h e f o l l o w i n g table:-

166

ABDULLAH A. AL-BADR AND ABDEL G . MEKKAWI 171.6

0-

L--.

'...

-

-1-

-171.4 -171. 2

'....DO

3

.'?

.c0

,. 0.q

0

E -2-

g-,

P.*

Purity : 99.86 yole '1. Melting pt : 171.5: Depression : 0.07C * 31.9kJlmole LL 4- Delta H Correction : O.OO*/.

-8 -

-

2IV -5 -6-7-

-171.0 "m

-170.8

"U..

-170.6 b...,

b..., 'b

-169.8

Ip

5

I5

7. 6.

5-

4. 32I07065

55

.

-170.2 -170.0

Total Area I Partial Area

0

45

35

25

15

5

28 Figure 13: The X-Ray ditfraction pattern of diet hy Ist i I best rol

-5L

Y

3

n

-170.4 E

b....

Mol.Weight : 268.3g1Mole Cellconst : 1.282 Onset slope : 7.90mw1d

-

*V

$

167

DIETHYLSTILBESTROL

X-Ray Diffraction Patterns of Diethylstilbestrol d(A)

1/10

9.54 8.06 7.51 5.90 4.85 4.54 4.43 4.33 4.22 4.01 3.94 3.74 3.57 3.44 3.39 3.17 3.10 3.05 3.03 2.91 2.85 2.75 2.56 2.52

16.40 14.03 20.14 48.03 100.00 51.49 65.06 18.11 27.88 17.98 27.23 12.93 35.93 39.11 22.00 11.35 10.49 7.97 8.02 20.58 5.27 3.35 8.31 7.46

d(A)

1/10 9.47 4.42 6.59 6.22 5.44 3.28 3.84 6.29 9.91 3.08 3.54 3.04 3.14 2.93 3.14 3.38 3.49 3.80 2.48 2.42 2.66 1.40 1.37

2.50 2.41 2.38 2.32 2.30 2.24 2.20 2.17 2.14 2.10 2.03 2.00 1.96 1.93 1.89 1.88 1.83 1.75 1.68 1.63 1.56 1.39 1.38

3. SYNTHESIS

Several methods have been reported f o r the synthesis of diethylstelbestro1:1)

et a1 ( 1 0 T h e d r u g w a s f i r s t prepare1 by DO( ds a,b) according to the following scheme:OH

ABDULLAH A. AL-BADR AND ABDEL G. MEKKAWI

168

, , C O G

H3C0

H3C0

72H5

c-

a :.;!.f

co

--@

OCH3

C2H5 Mg Br

PBr

dI

OCH3

t

4

-H20 -

C2H5

-@C2H5 A ~= -@ ocu3 * - ethanolic

KOH

1

2)

C H 2 5 Kharasch and Kleiman ( 1 1 ) p r e p a r e d t h e d r u g by t r e a t i n g a n e t h o l e hypobromide w i t h sodamide i n l i q u i d ammonia t o form a c a r b a n i o n which r e a c t w i t h u n r e a c t i n g s t a r t i n g m a t e r i a l and t h e n t h e e l i m i n a t i o n of h y d r o g e n b r o m i d e f o r m s a n i n t e r m e d i a t e w h i c h on d e m e t h y l a t i o n g i v e diethylstilbestrol.

DIETHYLSTILBESTROL

hH

169

O e Y = h * O H

C2H5

C2H5 3)

Kuwada -e t a1 ( 1 2 ) have prepared the drug according t o the f o l l o w i n g scheme :-

CN I C2H5MgBr C2H5-CH-OH

OH I H3COC6H4MgBr C2H5-CO-CH-C2H5 c

72H5

c -CH--

H3C0G-,

H3C0

I

I

OH

‘2*’5

@Z12H Y C O - C H

2 5

=( @ ’;

30% H,SO4

OH

b

H3CO-C 6H 4-MgBr

b

OH

H3C0

C2H5

@-OC3H

C&-?C=

I

C2H5

C2H5

OCH3

HC 1

.----+

H

OCH3-

R MgBr

+

ABDULLAH A. AL-BADR AND ABDEL G. MEKKAWI

170

4)

Alder et a1 (13) have reported the synthesis of diethylstilbestrol according to the following scheme:-

OH

C2H5

H C 1 gas

E-co

._____)

-H,O

OH

*OAc

Na/AmOH 140°

L

C2H5 HO

-@[2H5

C2H5

H

H C 1 gas -H20

bI -@OH

OH

>

f2H5 HO-@

5)

f=c C2H5 Shishido and Nozaki (14) reported the synthesis of diethylstilbestrol through the pinacol-pinacolone compounds according to the following scheme:

CH

Mg B r

f

DIETHYLSTILBESTROL

171

6)

Yoshida and Akagi (15) have reported the synthesis of the drug by treating p-CH30C6H4CHEtC(OH)EtC6H40CH3-P with POC13 in boiling toluene.

7)

A method for the synthesis of diethylstilbestrol have been reported by Slager (16). It involves the

conversion of 3,4-bis-(p-hydroxyphenyl)- 3,4hexandiol to 3,3-bis(p-hydroxyphenyl)-4-hexanone, treatment with Et4NBr and Et4NOH and electrical reduction to give the drug. OH

OH &OH

-H20*0

F2HS C-COC2H5 I

C2HS

4. PHYSIOLOGY, PHARMACOLOGY AND USES Diethylstilbestrol is a synthetic nonsteroidal estrogenic compound that exhibits the characteristic physiological actions of the hormonal class estrogens, these hormones are largely responsible for the changes that take place at puberty in girls and they go a long way for the tangible and intangible attributes of femininity (17). They affect the body, building and function; the biological role, the physiology, the thinking and psychology of the female. Diethylstilbesterol is indicated clinically for purposes where estrogenic effects are needed. On molecular basis, 1.6 mg. of diethylstilbestrol diphosphate is approximately equivalent to 1 mg. of diethylstilbestrol. The drug is indicated for the following clinical purposes: 1) Cancer treatment

Diethylstilbestrol exerts the usual inhibitory effects of estrogens on androgen dependent carcinomas and also exerts a direct cytotoxic action due to release of free s t i 1 b e s t r ol w i thin the neoplasm ( 1 8 , 1 9 , 2 0) Suc h cancers include prostatic cancer where the level of the

.

ABDULLAH A. AL-BADR AND ABDEL G. MEKKAWI

172

hormone is prostatic tissues was 100 times greater than that in the fat and muscle (20,211. The hormone also suppresses pituitary Lutenizing hormones and hence suppression of androgenic stimuli (20-23). 2) Suppression of Lactation

Diethylstilbestrol is used to suppresses lactation particularly post-partum lactatibn and galactorrhea (24-26) 3) Polycythemia Vera Treatment

Wirth (27) used diethylstilbesterol to improve cases of polycythemia Vera. 4) Symptomatic Relief of Menopause

Diethylstilbesterol has been used with success in symptomatic relief of menopause. It is reported that diethylstilbesterol supplementation may delay the onset of oesteo porisis in post-menopausal women (28,29).

5) Post-hysterectomy Use Diethylstilbesterol like other estrogens is a definite indication for post hysterectomy menopause to prevent vaginal atrophy and hot flashes (29-31). 6) Contraception Diethylstilbestrol use in oral contraceptives is no longer popular but its indication in post coital contraception is still widely used (FDA drug bulletin, 1973). 7) Other Indications

Other indications where diethylstilbestrol is used include: Postpartum breast engorgement, Duchennes muscular dystrophy, after sexual assault, sickle cell disease, dysfunctional uterine bleeding, failure of ovarian development and acne (17,32,33).

173

DIETHYLSTILBESTROL

8 ) Adverse E f f e c t s A d v e r s e s i d e e f f e c t may i n c l u d e h e a d a c h e , n a u s e a , v o m i t t i n g and m i l d d i a r r h o e a . R e c e n t l y i t h a s b e e n p o s t u l a t e d t h a t long d u r a t i o n t h e r a p y may p r e c i p i t a t e some c o m p l i c a t i o n s i n some p a t i e n t s , s u c h a s v o i c e c h a n g e , m a l i g n a n t changes, p e r e p h e r a l venous thrombos i s , pulmonary e m b o l i s m , h y p e r t e n s i o n and s t r o k e ( 1 7 , 3 0 , 32-35). The d r u g i s t e r a t o g e n i c , may induce malformation and malfunction i n o f f s p r i n g s of pregnant women who use it. 5. PHARMACOKINETICS D i e t h y l s t i l b e s t r o l i s r e a d i l y absorbed from t h e g a s t r o i n t e s t i n a l t r a c t . The d r u g i s d i s t r i b u t e d t h r o u g h o u t body t i s s u e s . I t i s bound i n 50-80% t o p r o t e i n s ( 3 4 , 3 5 ) . Borozdina e t . a l . (36) detected r e s i d u a l d i e t h y l s t i l b e s t r o l i n meat o f a n i m a l s f e d t h e drug. The compound was d e t e c t e d i n t h e f a t , m e a t , l i v e r and kidneys. A f t e r a s i n g l e subcutaneous i n j e c t i o n t h e drug was completely e l i m i n a t e d a f t e r 5 d a y s , i n r o o s t e r s ( 3 6 ) , t h e drug i s slowly i n a c t i v a t e d i n t h e l i v e r and e x c r e t e d i n u r i n e and f a e c e s , p r i n c i p a l l y a s g l u c u r o n i d e (17,35-37) and a s t h e s u l p h a t e (37-39). I n p r o s t a t i c cancer p a t i e n t s t h e l e v e l of t h e hormone was 100 times h i g h e r i n p r o s t a t i c t i s s u e s t h a n i n f a t and muscle ( 2 0 , 2 1 ) . It i s n a t u r a l t o a n t i c i p a t e h i g h e r c o n c e n t r a t i o n s of t h e hormone i n i t s n a t u r a l t a r g e t t i s s u e s t h a n t h e r e s t of body t i s s u e s (17,32,33,39). 6. METHODS OF ANALYSIS 6.1 I d e n t i f i c a t i o n T e s t s The f o l l o w i n g t e s t s a r e d e s c r i b e d i n B.P.

1980 (4):-

1)

The l i g h t a b s o r p t i o n , i n t h e range 230 t o 350 nm, of a 2-cm l a y e r of a 0.001 p e r c e n t w/v s o l u t i o n i n a b s o l u t e e n t h a n o l e x h i b i t s a maximum o n l y a t 241 nm; absorbance a t 241 nm, about 1.2.

2)

The l i g h t a b s o r p t i o n , i n t h e range 230 t o 450 nm, of t h e i r r a d i a t e d s o l u t i o n prepared a s d i r e c t e d i n t h e a s s a y , e x h i b i t s two maxima, a t 292 nm and 418 nm.

ABDULLAH A. AL-BADR AND ABDEL G . MEKKAWI

174

3)

Dissolve 0.5 mg in 0.2 ml of glacial acetic acid, add 1 ml of orthophosphoric acid and heat on a water-bath for three minutes; a deep yellow color is produced which almost disappears on dilution with 3 ml of glacial acetic acid.

The following identification tests are described in USP

xx

(1) :

1)

Prepare an alcoholic solution containing 10 ug of USP diethylstilbestrol RS and diethylstilbestrol respectively in each ml. Determine the absorbance of each solution in the range 230 to 350 nm, using alcohol as the blank. The spectrum of diethylstilbestrol exhibits a maximum and an additional inflection at the same wavelength as that of the solution of USP diethylstilbestrol RS, concomitantly measured, and the absorpitivity of diethylstilbestrol at the wavelength of maximum absorbance does not differ from that of the Reference Standard by more than 3.0%.

2)

Prepare the standard preparation as follows: "Dissolve in alcohol a suitable quantity of USP diethylstilbestrol RS, accurately weighed and prepare, by stepwise dilution with alcohol, a solution containing about 20 ug per ml of this solution with an equal volume of dibasic potassium phosphate solution (1 in 55). Transfer 4 ml of this standard preparation to a stoppered, 1-cm quartz cell, place about 5 cm from a low-pressure, short wave mercury lamp, rated at from 2 to 20 watts, and irradiate €or about 5 minutes. Measure the absorption spectrum, in the range of 250 to 4 5 0 nm of this yellow solution exhibits inflections only at the same wavelengths as that of the solution obtained after irradiation of the standard preparation.

6.2 Titrimetric Methods 6.21 Volumetric Determination

Stilbestrol dipropionate was determined volumetrically by Gyenes ( 4 0 ) using the bromination method. 72-82 mg in acetic acid (in 20 ml acetic

DIETHYLSTILBESTROL

175

a c i d ) were t r e a t e d w i t h 0.1 N KBr03 (10 m l ) , H2S04 ( l : l , 0.5 ml) and K B r (300 mg i n 1 m l of H 0 ) i n a s t o p p e r e d f l a s k and l e f t i n t h e dark a t 35' + 3' f o r 80 t o 83 min. K I ( 5 0 0 mg i n 20 m l H20)-was added f o l l o w e d by s t a r c h s o l u t i o n and t i t r a t e d a g a i n s t 0.1 N t h i o s u l p h a t e s o l u t i o n . One molecule o f t h e d r u g consumes 4 e q u i v a l e n t s of bromine. However, f o r s t i l b e s t r o l t h e r e a c t i o n t i m e b e f o r e t i t r a t i o n was 25 minutes. The l i m i t of e r r o r f o r s t i l b e s t r o l was 2 0.5%. Elsayed and Obiakara (41) a s s a y e d s t i l b e s t r o l i n t a b l e t s and powders by s l i g h t l y modifying t h i s method and d i s c u s s e d t h e r e a c t i o n k i n e t i c s i n r e s p e c t t o time of a d d i t i o n and bleaching of t h e r e a g e n t . They c o n c l u d e d t h a t t h e method i s s u i t a b l e f o r r o u t i n e work i f o t h e r p h e n o l i c compounds a r e absent. 6.3 Spectropho tome t r y 6.31 C o l o r i m e t r i c Methods B r i t i s h Pharmacopeia 1980(4) d e s c r i b e d t h e followi n g procedure: D i s s o l v e 20 mg i n s u f f i c i e n t a b s o l u t e e t h a n o l t o produce 100 m l and d i l u t e 10 m l t o 100 m l with t h e same s o l v e n t . To 25 m l of t h e r e s u l t i n g s o l u t i o n add 25 m l of a s o l u t i o n prepared by d i s s o l v i n g 1 g of a n h y d r o u s d i p o t a s s i u m hydrogen orthophosphate i n 55 m l of w a t e r , t r a n s f e r a p o r t i o n of t h e m i x t u r e t o a 1-cm c l o s e d q u a r t z c e l l , p l a c e t h e c e l l 10 cm from a 15-watts, shortwave, u l t r a v i o l e t l a m p , and i r r a d i a t e f o r t e n minutes. Measure t h e a b s o r b a n c e of t h e i r r a d i a t e d s o l u t i o n a t t h e maximum a t about 418 nm, and c a l c u l a t e t h e c o n t e n t of C8H2002 f r o m t h e a b s o r b a n c e o b t a i n e d by r e p e a t i n g t h e o p e r a t i o n using d i e t h y l s t i l b e s t r o l EPCRS i n s t e a d of t h e s u b s t a n c e being examined. United S t a t e Pharmacopia XX( 1 ) d e s c r i b e d t h e f o l l o w i n g procedure: Standard p r e p a r a t i o n : Dissolve i n alcohol a s u i t a b l e q u a n t i t y of USP d i e t h y l s t i l b e s t r o l R S , a c c u r a t e l y w e i g h e d , and p r e p a r e , by s t e p w i s e

176

ABDULLAH A. AL-BADR AND ABDEL G. MEKKAWI

d i l u t i o n w i t h a l c o h o l , a s o l u t i o n c o n t a i n i n g about 20 ug p e r m l . Mix 25 m l of t h i s s o l u t i o n w i t h a n e q u a l volume of d i b a s i c p o t a s s i u m p h o s p h a t e s o l u t i o n ( 1 i n 55). Assay p r e p a r a t i o n : Proceed with a s u i t a b l e q u a n t i t y , a c c u r a t e l y w e i g h e d , of d i e t h y l s t i l b e s t r o l a s d i r e c t e d under Standard p r e p a r a t i o n . Procedure: (Caution - P r o t e c t t h e e y e s from d i r e c t r a y s oE u l t r a v i o l e t l i g h t t h r o u g h o u t t h i s procedure). T r a n s f e r 4 m l of t h e S t a n d a r d p r e p a r a t i o n t o a s t o p p e r e d , 1 cm q u a r t z c e l l , p l a c e a b o u t 5 cm from a low-pressure, shortwave mercury lamp, r a t e d a t f r o m 2 t o 20 w a t t s , and i r r a d i a t e f o r about 5 minutes. P l a c e t h e c e l l i n t h e sample compartment of a s u i t a b l e spectrophotom e t e r , and m e a s u r e t h e a b s o r b a n c e a t t h e wavelength of maximum absorbance a t about 418 nm, u s i n g water a s t h e blank. Continue i r r a d i a t i o n f o r s u c c e s s i v e 1 t o 3-minute i n t e r v a l s , m e a s u r i n g a t 418 nm u n t i l t h e maximum absorbance (about 0.7) has been o b t a i n e d . I f n e c e s s a r y , a d j u s t t h e g e o m e t r y of t h e i r r a d i a t i o n a p p a r a t u s so a s t o o b t a i n maximum, r e p r o d u c i b l e absorbance a t 418 nm. S i m i l a r l y , i r r a d i a t e a 4-ml p o r t i o n of t h e a s s a y p r e p a r a t i o n , r e c o r d i n g t h e a b s o r b a n c e a t 418 nm, a t s u c c e s s i v e s h o r t i n t e r v a l s u n t i l maximum absorbance i s o b t a i n e d . C o n c o m i t a n t l y d e t e r m i n e t h e a b s o r b a n c e s of t h e Assay p r e p a r a t i o n and t h e S t a n d a r d p r e p a r a t i o n i n 1-cm c e l l s a t 418 nm, u s i n g w a t e r a s t h e b l a n k , and s u b t r a c t t h e s e v a l u e s from t h o s e f o r t h e r e s p e c t i v e i r r a d i a t e d s o l u t i o n s , t o o b t a i n t h e c o r r e c t e d maximum a b s o r b a n c e s . C a l c u l a t e t h e q u a n t i t y , i n ug o f C18H2002 i n e a c h m l of t h e Assay p r e p a r a t i o n by t h e formula C(Au/As), i n which C i s t h e c o n c e n t r a t i o n , i n ug p e r m l , of USP d i e t h y l s t i l b e s t r o l RS i n t h e s t a n d a r d p r e p a r a t i o n , and Au and A s a r e t h e c o r r e c t e d maximum absorbances of t h e i r r a d i a t e d Assay p r e p a r a t i o n a n d S t a n d a r d preparation, respectively. De Almedia B a l t a z a r and V e i r a de Arbeu compared d i f f e r e n t methods used i n d e t e r m i n a t i o n of d i e t h y l s t i l b e s t r o l , h e x e s t r o l and d i e n e s t r o l and developed t h e i r own method f o r d i e n e s t r o l (42).

DIETHYLSTILBESTROL

177

I n t h e v i s i b l e r a n g e Goodyear e t a 1 ( 4 3 ) made d i e t h y l s t i l b e s t r o l to develop a yellow colour a f t e r i r r a d i a t i o n of a n a q u e o u s a c e t i c a c i d s o l u t i o n of t h e drug w i t h UV l i g h t , and measured t h e c o l o u r a b s o r p t i o m e t r i c a l l y . They a l s o measured t h e c o n c e n t r a t i o n by a b s o r b a n c y d i f f e r e n c e s of a c i d and a l k a l i n e s o l u t i o n s i n t h e UV range. Cheng and B u r r o u g h s u s e d SbC15 i n e t h y l e n e c h l o r i d e t o develop a c o l o u r w i t h d i e t h y l s t i l b e s t r o l and used a c o l o r i m e t e r w i t h f i l t e r s a t 525 and 430 mu (44). They found t h a t t h i s method is more s e n s i t i v e t h a n t h e method of Goodyear e t a 1 ( 4 3 ) . The y e l l o w c o l o u r p r o d u c e d by n i t r a t e d d e r i v a t i v e s of s t i l b e s t r o l and d i e n e s t r o l w i t h a l k a l i had been used by Tokar and Simonyi (45). The p h o t o m e t r i c d e t e r m i n a t i o n of 126 p h e n o l i c compounds i n w a t e r u s i n g group-specif i c r e a g e n t s i . e . p - n i t r o a n i l i n e , s u l f a n i l i c a c i d , 4-aminoa n t i p y r i n e and 3-methylbenzothiazoline-2-ylhydraz i n e , was c a r r i e d o u t by Koppe e t a1 ( 4 6 ) . I n i n j e c t a b l e p r e p a r a t i o n s of l i p i d s o l v e n t , t h e p h e n o l i c hormone w a s e x t r a c t e d w i t h aqueous 0.1 N NaOH and determined s p e c t r o p h o t o m e t r i c a l l y a t 259 nm, while i n t a b l e t s a f t e r d i s p e r s i o n i n d i l . H C 1 and e x t r a c t e d by 0.1 N NaOH and measured a t t h e same wavelength. The a b s o r b a n c e obeyed LambertBeer's law a t c o n c e n t r a t i o n s ranging between 2.5 12.5 ug/ml. The s t a n d a r d d e v i a t i o n f o r i n j e c t a b l e s o l u t i o n s d e t e r m i n a t i o n s was 2.06% w h i l e f o r t h e t a b l e t s was 2.52% (47). 6.32 F l u o r i m e t r i c Methods Ponder ( 4 8 ) determined d i e t h y l s t i l b e s t r o l i n a n i m a l f e e d s ( 5 u g / k g ) f l o r i m e t r i c a l l y by c o n v e r t i n g i t t o p h e n a n t h r e n e d i o l d e r i v a t i v e . The f e e d m a t e r i a l was e x t r a c t e d w i t h a c e t o n e - i s o p e n t y l alcohol ( 1 : l ) a f t e r i n i t i a l treatment with a c e t i c e t h a n d i o l ( 1 :4) mixture. A f t e r p u r i f i c a t i o n acid w i t h s o l v e n t p a r t i t i o n i n g , t h e drug was c o n v e r t e d by UV i r r a d i a t i o n i n t o t h e phenenthrenedione which was t h e n o x i d i z e d t o t h e d i o l and d e t e r m i n e d as p r e v i o u s l y d e s c r i b e d by Umberger e t a l ( 4 9 ) . Ponder (50) a s s a y e d t h e drug and i t s monoglucuronides i n b e e f meat. Vogt d e t e r m i n e d d i e t h y l s t i l b e s t r o l i n t h e f e c e s and u r i n e of f e e d e r c a l v e s ( 5 1 ) .

-

ABDULLAH A. AL-BADR AND ABDEL G. MEKKAWI

178

K o l i n s k i e t a 1 ( 5 2 ) e x t r a c t e d t h e drug from e n t e r i c - c o a t e d and p l a i n - c o a t e d t a b l e t s , e x p o s e d t o U V i r r a d i a t i o n (pH 6 - 7 ) and t h e r e a c t i o n product t r e a t e d w i t h 2% c a t e c h o l s o l u t i o n i n 2 M H C 1 a t 7OoC and t h e r e s u l t i n g 9,lO diethylphenathrene-3,6-diol was determined p h o t o m e t r i c a l l y a t 4 1 0 nm w i t h e x c i t a t i o n a t 3 3 5 nm. F o r 1 0 d e t e r m i n a t i o n s a t d i f f e r e n t d a y s , f o r 1 mg t a b l e t s , t h e c o e f f i c i e n t of v a r i a t i o n was 0.6% and r e c o v e r i e s w e r e q u a n t i t a t i v e down t o 0 . 1 mg / t a b l e t

.

A f t e r u s i n g ion-exchange column (Amberlite X AD-2) and ( P o l y c o l o r column) followed by L i Chrosorb RP18 column and an on-line r e a c t i o n system, Verbeke and Vanhee (53) f l u o r i m e t r i c a l l y determined s t i l b e s t r o l r e s i d u e s i n u r i n e and animal t i s s u e s . I n t h e on-line r e a c t i o n system o x i d a t i o n was achieved a t 75OC by SO2 i n a l c o h o l i c s o l u t i o n t o form a h i g h l y f l u o r e s c e n t compound, w h i c h , a f t e r e x c i t a t i o n a t 260 nm was measured a t 370 nm and they c o n c l u d e d t h a t t h i s method i s s u p e r i o r t o H. P L. C. methods

.

.

The f o l l o w i n g f l u o r i m e t r i c procedure i s recommended by t h e B.P. ( 4 ) f o r t h e drug t a b l e t s : Procedure Weigh and powder 20 t a b l e t s . To a q u a n t i t y of powder e q u i v a l e n t t o 5 mg of s t i l b e s t r o l add 50 m l of a b s o l u t e e t h a n o l , s h a k e f o r 1 5 m i n u t e s , add s u f f i c i e n t a b s o l u t e e t h a n o l t o produce 100 m l and c e n t r i f u g e . D i l u t e 20 m l of t h e s u p a r n a t a n t l i q u i d t o 50 m l w i t h a b s o l u t e e t h a n o l and t o 25 m l of t h e r e s u l t i n g s o l u t i o n a d d 25 m l o f a s o l u t i o n prepared 1 gram of anhydrous d i p o t a s s i u m hydrogen o r t h o p h o s p h a t e i n 55 m l of w a t e r . T r a n s f e r a p o r t i o n of t h e mixture t o 1-cm c l o s e d q u a r t z c e l l , p l a c e t h e c e l l 1 0 cm from a 1 5 w a t t s h o r t w a v e , u l t r a v i o l e t lamp, and i r r a d i a t e f o r 10 minutes. Measure t h e absorbance of t h e i r r a d i a t e d s o l u t i o n a t t h e maximum a t about 418 nm and c a l c u l a t e t h e c o n t e n t of s t i l b e s t r o l f r o m t h e a b s o r b a n c e o b t a i n e d by r e p e a t i n g t h e o p e r a t i o n w i t h d i e t h y l s t i l b e s t r o l EPCRS.

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179

6.4 Polarography

Direct c u r r e n t and a l t e r n a t i n g c u r r e n t p o l a r o g r a p h i c responses of some p h a r m a c e u t i c a l s , i n c l u d i n g e s t o g e n i c compounds, i n a n a p r o t i c o r g a n i c s o l v e n t s y s t e m w e r e i n v e s t i g a t e d by Woodson and Smith (54). The a p p l i c a t i o n of polarography i n s t i l b e s t r o l a n a l y s i s was demonstrat e d by Erb e t a 1 ( 5 5 ) . P u l s e and d i f f e r e n t i a l p u l s e polarography a f t e r i r r a d i a t i o n of t h e drug by U.V. was c a r r i e d o u t by Kubes ( 5 6 ) t o e n a b l e d e t e r m i n a t i o n of t h e compound a s t h e d i k e t o n e , and i t s peak a t -0.8 V (vs the s.c.e) was measured and t h e r e s p o n s e was r e c t i l i n e a r l y r e l a t e d t o c o n c e n t r a t i o n between 0.75 t o 24 u M - s t i l b e s t r o l . 6.5 Chromatographic Methods 6.51 Paper (PLC) and Thin-Layer Chromatography (TLC): The e a r l i e s t of t h e c h r o m a t o g r a p h i c t e c h n i q u e s used i n e s t o g e n i c pharmaceutical p r e p a r a t i o n s was Paper was impregnated paper chromatography (PLC). i n 5% s i l i c o n e i n cyclohexane, d r i e d a t l l O ° C and s p o t s of 5-50 ug of t h e o e s t r o g e n i c compounds were a p p l i e d and descending chromato-graphy was c a r r i e d o u t u s i n g t h e lower l a y e r of t h e mixture: o b t a i n e d by mixing 15 m l of w a t e r , 85 m l of methanol, 30 m l of benzene and 70 m l of hexane a s t h e moving phase ( 5 7 ) . S i l i c a g e l G p l a t e s and i n t e g r a t i n g d e n s i t o m e t r y w e r e c o m b i n e d a f t e r column chromatography i n t h e technique used by J o n e s et a 1 ( 5 8 ) . The s o l v e n t s y s t e m used f o r developing t h e chromatograms was : e t h y l a c e t a t e - l i g h t petroleum - anhydrous a c e t i c acid - ethanol (144:27:20:9). T h y m o p l p h t h a l e i n i n d i c a t o r was s p o t t e d a t RF 1 before s p r a y i n g t h e p l a t e w i t h Folin-Ciocalteu d i l u t e d ( 1 : 4 ) r e a g e n t followed by 0.5 N e t h a n o l i c KOH, u n t i l t h e i n d i c a t o r s p o t was p e r m a n e n t l y b l u e . S e p a r a t i o n of s y n t h e t i c e s t r o g e n s from n a t u r a l e s t r o g e n s was c a r r i e d o u t on s i l i c a g e l H u s i n g chloroform a c e t i c a c i d (85: 1 5 ) ( 5 9 ) . O t h e r s o l v e n t s y s t e m s on T.L.C. were t r i e d by Di b m i z i o and Muscarella (60). Quantitat i o n on s i l i c a g e l G u s i n g two-dimensional chromatography and exposing t h e chromatograms t o 254 nm i r r a d i a t i o n f o l l o w i n g by H2SO4 ( 5 0 % ) and

-

ABDULLAH A. AL-BADR AND ABDEL G. MEKKAWI

180

h e a t i n g a t 95OC was c a r r i e d o u t by Karkosha ( 6 1 ) . High p e r f o r m a n c e t h i n l a y e r c h r o m a t o g r a p h y (H.P.T.L. C. ) u s i n g two-dimensional chromatography u s i n g hexane methylene d i c h l o r i d e ethyl a c e t a t e ( 1 : 2 : 2 ) and c h l o r o f o r m benzene m e t h a n o l ( 3 6 :4: 1) was u s e d , f o r b i o l o g i c a l e x t r a c t s ( f r o m m e a t , l i v e r and u r i n e ) a f t e r p u r i f i c a t i o n by g e l permeation chromatography, by Smets and Verschaeren (62).

-

-

-

Clarke i n h i s monograph ( 2 ) d e s c r i b e d t h e followi n g PLC condition. Sample P r e p a r a t i o n I f t h e s a m p l e i s u r i n e , i t is r e f l u x e d w i t h h y d r o c h l o r i d e a c i d , e x t r a c t e d w i t h e t h e r and t h e e t h e r e a l e x t r a c t is e v a p o r a t e d t o d r y n e s s . R e d i s s o l v e i n e t h a n o l s o t h a t one m l of t h e e t h a n o l i c s o l u t i o n i s e q u i v a l e n t t o 50 m l o f u r i n e . 20 u l of t h i s s o l u t i o n should be a p p l i e d t o chromatogram. Paper Layer: Whatman No.1, a cylinder.

s t a p l e d i n t h e form of

S o l v e n t System: S t r o n g ammonia s o l u t i o n : i s o p r o p r a n o l : water (1:8:1). Equilibration: Development: Location: Rf v a l u e s :

None Ascending

Pauly's

r e a g e n t (yellow).

S t i l b e s t r o l 0.85.

6.52 Gas-Liquid Chromatography (GLC) McGregor e t a 1 (63) a s s a y e d d i e t h y l s t i l b e s t r o l , e x t r a c t e d from u r i n e , u s i n g G.L.C. on a ' c o l u m n of 13.5% polyoxyethylene g l y c o l a d i p a t e on Chromosorb W a t 200'. T h e h o r m o n e was c o n v e r t e d t o t h e d i m e t h y l e t h e r . M o r e t t i e t a 1 ( 6 4 ) derivitized d i e t h y l s t i l b e s t r o l , d i e n o e s t r o l and h e x o e s t r o l a s t h e i r a c e t a t e s and used 3% QF-1 l i q u i d p h a s e a t

--

DIETHYLSTILBESTROL

181

170 OC. The dipropionate of diethylstilbestrol can be determined by direct GLC on 3% JXR on Gas Chrom P at 200°C using stainless steel column. The sample liquid phase was used by Rutherford (65) to determine the cis and trans forms of the estrogenic compound in feeding-stuff pre-mix. The use of a 5% X E - 6 0 o n G a s C h r o m 2 eliminates interference of polyoxyethylene glycol 200 that occurred on 3 % JXR column. The electron-capture G.L.C. of the fluoropropionyl diethylstilbestrol or trifluoroacetic anhydride was successful in determination of concentration as low as 1-2 ppb in animal chew or urine (66,67). The use of columns of OV-17 was described by Kohrman and MacGee (68) and Van de Vaart et a 1 (69) in determinations carried out in biological and cosmetic materials. Clarke in his monograph (2) described the following G.L.C. conditions: Column : 2.5% SE-30 on 80-100 mesh Chromosorb W AWHMDS, 5 ft x 4 mm (i.d.), glass column. Column Temperature : 225OC. Carrier Gas : Nitrogen. Gas Flow Rate : 50 mllminute. Detector : Flame ionization detector (F.I.D.). Hydrogen Flow Rate : 50 mllminute. Air Flow Rate : 300 mllminute. Retention time 0.78 relative to codeine. At a column temperature of 225OC, the relative retention time to codeine (R.Rt) is 0.78. 6.53 High Resolution Liquid Chromatography (H.P.L.C.) and GLC/HPLC HPLC was used by several authors in determination of stilbestrol in various formulations and biological materials. The table summarises some of the HPLC (72-79) conditions of techniques used

Table 4

: Higfrperfomance Liquid chrcooatograply of Diethylstilbestrol.

0.1 M CH3aXNH in MeDH - %O ?3:2) # 7.1, I elution at 1 d/mtn.

Electrockmica1 detection limit cyclic voltamretry at + 0.7V

w the

Gradient, lO-lCQ% MeOHorRP-8and 20-100% Me(xf for P 1 8 , 1 ml/m€n.

Zorbax SIL

Et(XHhane (1:39), 1 d/rllin.

RF2 RP-18 Zork

Gradient elution:A: ~3CN/H20(1:9) (;N

s.e.c.

w (254 m),resolved cis

75%Me(xf

Li chrosorb RP-8 or RP-18

Lintit of detection

Detection

Mobile #me

and trans isorners.

_.

Ref. (72)

20 Pg

0.2-2 j.lg/all

(73)

(74)

W 240 nm. Voltamtry with vitreous - carbon and I - (reference) ~ electrodes, Potential sweep rate is 5 mv/sec.

A

0.01 pg.

0.3-0.5

(75)

ng

(76)

.......

Table contd

B: ~ 3 ~ / H 2 0 ( 9 : 1 ) (25% B a t 0 time t o 45%B a t 5 min). Eluent contains 1 ag of L i C 1 and 1 tlg of LiC104. Ijchrosorb

RP-6

Mec€I : $0 (35: 13) (25:23)

Vitreous carbon or plat& counter electrode and a silvefAgC1 reference electrode. 0.4 ml plasm (78) or urine, 1 g tissue.

RP-8

50% acetonitrile (#I 3.5) 1.5

ml/min.

In-line photochemical reaction, fluorimetrically a t 390 rm (excitation a t 280 m).

lw-ppb levels (79) in U r i n e @.==and

sera extracts.

ABDULLAH A. AL-BADR AND ABDEL G. MEKKAWI

184

by v a r i o u s a u t h o r s ( 6 6 , 7 0 - 8 2 ) w h i l e combined GLC/HPLC was t r i e d by o t h e r s (66,691. 6.54 Combined Gas Chromatography Spectrometry (GC/MS)

-

Mass

D e t e r m i n a t i o n s of a n a b o l i c s t e r o i d s and d r u g s , i n c l u d i n g s t i l b e s t r o l i n meat and i t s products by combined gas chromatography mass s p e c t r o m e t r y (GC/MS) were done by S t a n and Abraham (83) a n d Duerbeck and Bueker ( 8 4 ) , i n plasma by Gaskell et a 1 ( 8 5 ) , i n u r i n e by Derks e t a 1 ( 8 6 ) , T u i n s t r a e t a 1 ( 8 7 ) D i e d e r i k e t a 1 ( 8 8 ) and i n baby f o o d s a m p l e s by G a l l i e t a 1 ( 8 9 ) . E.I. mass s p e c t r o m e t r y was u s e d by m o s t o f t h e a u t h o r s w h i l e D i e d e r i k et & ( 8 8 ) u s e d n e g a t i v e 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 u s i n g CHh OR CH4 : N20 ( 4 : l ) as chemical i o n i s a t i o n reagents. However, GC-MS t e c h n i q u e i s t h e most s e n s i t i v e t e c h n i q u e , o t h e r t h a n R.I.A. methods f o r e v a l u a t i o n of such drugs i n b i o l o g i c a l s , food and p h a r m a c e u t i c a l s and t h e s e n s i t i v i t y of t h e technique i s 0.1 1 ppb.

-

-

-

6.55 Assay by NMR Al-Badr and Ibrahim (90) developed an NMR s p e c t r o m e t r i c method f o r t h e q u a n t i t a t i o n of d i e t h y l s t i l b e s t r o l i n t a b l e t s a n d a m p o u l e s . The m e t h o d i n v o l v e d c o m p a r i n g t h e i n t e g r a l of t h e t r i p l e t system of s t i l b e s t r o l spectrum ( p o s i t i o n e d a t 0.73 O) t o t h a t of t h e s i n g l e t ( p o s i t i o n e d a t 6.25 a ) of m a l e i c a c i d , a s a n i n t e r n a l s t a n d a r d . The p r o c e d u r e f o r t h e q u a n t i t a t i o n of t h e a u t h e n t i c drug, t h e t a b l e t s and ampoules was described. 6.6 Radio Immuno Assay (R.1.A.) R.I.A. methods have p r o v e d t o be t h e most s e n s i t i v e methods of a n a l y s i s today. The t e c h n i q u e i s now a r o u t i n e i n most h o s p i t a l s and a n a l y t i c a l l a b o r a t o r i e s . S u c c e s s f u l R.I.A. of d i e t h y l s t i l b e s t r o l and a l l i e d hormones i n b i o l o g i c a l m a t e r i a l s and e s p e c i a l l y i n none t h i c a l u s e ( i n meat p r o d u c t s ) was shown by s e v e r a l a u t h o r s and t h e s e n s i t i v i t y of t h e technique s u r p a s s e s t h a t of GC/MS method. The s e n s i t i v i t y is i n t h e range - l o w 7 ( 9 1 - 9 6 ) . By t h i s t e c h n i q u e i t was

DIETHYLSTILBESTROL

185

determined i n blood of f e e d l o t c a t t l e ( 9 1 ) , i n animal t i s s u e s ( 9 2 , 9 4 , 9 6 ) and i n f e c e s ( 9 5 ) . A r n s t a d t ( 9 2 ) u s e d t h e enzyme immunoassay method by c o m p e t e t i v e b i n d i n g s t i l b e s t r o l and p e r o x i d a s e d l a b e l l e d s t i l b e s t r o l t o a n a n t i b o d y bonded t o DEAE-cellulose. The method is s u i t a b l e f o r a range a s low a s 0.2 pmol (0.05 ng) of s t i l b e s t r o l . G r i d l e y e t a 1 ( 9 3 ) u s e d a n immunogen a n d a s e c o n d a n t i b o d y of g o a t a n t i - r a b b i t - 0 - g l o b u l i n assay f o r s t i l b e s t r o l and i t s m e t a b o l i t e s i n b o v e i n e l i v e r . T r i t i u m l a b e l l e d s t i l b e s t r o l was used and measurement was c a r r i e d by s c i n t i l l a t i o n counting. 7

METABOLISM D i e t h y l s t i l b e s t r o l can be conjugated i n t h e body w i t h H2S04 t o a s m a l l and v a r i a b l e e x t e n t i n r a t s , c a t s and d o g s b u t n o t i n r a b b i t s (97). Metabolism t o monogluaxmiride by s l i c e s o r r a t l i v e r was r e p o r t e d ( 9 8 , 9 9 ) and a l s o i n t h e e v e r t e d s a c s of t h e r a t i n t e s t i n e (99,100,101 ). Formations of d i h y d r o x y - d i e t h y l s t i l b e s t r o l and t h e methoxy d e r i v a t i v e s i n v a r i o u s s p e c i e s were observed (99,102,103,104). The hormones and i t s m e t a b o l i t e s a r e e x c r e t e d v i a t h e kidney, l i v e r b i l e and f e c e s and f r e e s t i l b e s t r o l p r e s e n c e was o b s e r v e d i n f e c e s examined. McLachlan ( 1 0 5 ) s t u d i e d p r e n a t a l exposure t o d i e t h y l stilbestrol i n mice and observed e q u i v a l e n t amounts i n mother and f e t a l plasma. The c o n c e n t r a t i o n of t h e d r u g i n f e t a l g e n i t a l t r a c t was t h r e e - f o l d h i g h e r t h a n t h a t of t h e plasma.

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