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Steroid conjugates I. The use of sulfamic acid for the preparation of steroid sulfates
577
S T E R O I D C O N J U G A T E S I.
T H E USE O F S U L F A M I C ACID F O R
THE PREPARATION OF STEROID SULFATES J o s e p h P. J o s e p h , John P. Dusza and S e y m o u r B e r n s t e i n Organic Chemical Research Section, Lederle Laboratories, A D i v i s i o n of A m e r i c a n C y a n a m i d C o m p a n y , P e a r l R i v e r , New Y o r k
Received
10965
April 26, 1966
T h e u t i l i t y of s u l f a m i c a c i d f o r t h e p r e p a r a t i o n of d i v e r s e s t e r o i d s u l f a t e s h a s b e e n i n v e s t i g a t e d . S u l f a t e s of t h e f o l l o w i n g steroids were prepared and characterized: testosterone, cholesterol, pregnenolone, dehydroisoandrosterone, estrone, 2-methoxyestrone, ethynylestradiol and ethynylestradiol-3-methyl ether.
S t e r o i d c o n j u g a t e s , 1 f o r m e r l y r e g a r d e d s o l e l y as d e t o x i c a t i o n end p r o d u c t s , h a v e r e c e n t l y b e e n d e m o n s t r a t e d as i n t e r m e d i a t e s
in
m e t a b o l i c p a t h w a y s . 2 In v i e w o f t h e w i d e n i n g i n t e r e s t in t h i s i m p o r t a n t a r e a of r e s e a r c h ,
o u r e f f o r t s h a v e b e e n d i r e c t e d to t h e
s y n t h e s i s a n d c h a r a c t e r i z a t i o n of c o n j u g a t e s s u c h as s u l f a t e s a n d glucuronides.
More particularly,
in t h i s p a p e r t h e u t i l i t y of s u l f a m i c
a c i d as a r e a g e n t f o r t h e s y n t h e s i s of s u l f a t e s is f u r t h e r e s t a b l i s h e d . Generally,
steroid sulfates have been prepared by the
a p p l i c a t i o n of t h e f o l l o w i n g r e a g e n t s :
chlorosulfonic acid B and sulfur
trioxide-pyridine complex. 4
T h e e m p l o y m e n t of t h e s e r e a g e n t s ,
to t h e i r i n h e r e n t p r o p e r t i e s ,
has required anhydrous reaction con-
d i t i o n s a n d c a r e f u l c o n t r o l of t e m p e r a t u r e
a n d pH.
These experi-
m e n t a l c o n d i t i o n s h a v e b e e n c i r c u m v e n t e d b y t h e u s e of s u l f a m i c
due
578
S T E R O I D S
7:6
a c i d d e s c r i b e d b y S h o r t and O x l e y 5 in t h e p r e p a r a t i o n estrogen sulfates,
of synthetic
and b y H a s b r o u c k 6 and P r i c e 7 in t h e p r e p a r a t i o n
of e s t r o n e s u l f a t e s a l t s .
The a p p a r e n t s i m p l i c i t y of the m e t h o d ,
w h e r e a n h y d r o u s r e a g e n t s a r e not n e c e s s a r y , its u s e to t h e p r e p a r a t i o n
p r o m p t e d us to e x t e n d
of o t h e r s t e r o i d s u l f a t e s .
Most recently
t h e s y n t h e s i s of v i t a m i n DZ s u l f a t e e m p l o y i n g s u l f a m i c a c i d h a s b e e n reported by Japanese workers. 8 T h e u s e of s u l f a m i c a c i d f o r t h e p r e p a r a t i o n s u l f a t e s h a s b e e n k n o w n f o r m a n y y e a r s .9
of a l k y l and a r y l
Primary
alcohols are
c o n v e n i e n t l y c o n v e r t e d to t h e i r s u l f a t e s a l t s at 1 7 0 - 1 8 0 ° w h i l e s e c o n d a r y a l c o h o l s do n o t r e a c t o r g i v e v e r y p o o r y i e l d s u n d e r t h e s e conditions. for example,
Tertiary
a l c o h o l s do n o t y i e l d s u l f a t e e s t e r s ,
g i v e s r i s e to i s o b u t y l e n e .
t-butanol,
In t h e c a s e of p h e n o l s , t h e
reaction conditions favor rearrangement
of t h e p h e n y l s u l f a t e s to
the sulfonic acids. W ith t h e r e c o g n i t i o n t h a t t e r t i a r y effective catalysts for the reaction, for the esterification temperatures
amines
and a m i d e s
11
were
m o s t of the above q u a l i f i c a t i o n s
c o u l d be d i s m i s s e d .
(85-950), decreased
10
The lowered reaction
reaction times (0.5-1.5
hrs.),
and w e a k l y b a s i c n a t u r e of the r e a c t i o n m e d i a u n d o u b t e d l y c o n t r i b u t e d to t h e i s o l a t i o n of s u l f a t e s a l t s p r e v i o u s l y n o t s t a b l e to t h e o l d e r reaction conditions e.g. reaction temperature
t-butyl ammonium
105 ° - 3 h r s . 1 2
s u l f a t e (37% y i e l d ) -
T h e u s e o f p y r i d i n e as
c a t a l y s t an d s o l v e n t f o r t h e r e a c t i o n is i d e a l l y s u i t e d to t he p r e p a r a t i o n of s t e r o i d s u l f a t e s . L i s t e d in T a b l e I a r e s u l f a t e s p r e p a r e d procedures
o u t l i n e d in t h e E x p e r i m e n t a l .
by the g e n e r a l
It s h o u l d be e m p h a s i z e d
June
1966
S T E R O I D S
579
at t h i s p o i n t t h a t t h e s u l f a m i c a c i d - p y r i d i n e s u l f a t i o n t e c h n i q u e represents
an i m p r o v e d a n d / o r s i m p l i f i e d p r e p a r a t i v e p r o c e d u r e
for the compounds outlined h e r e i n .
H o w e v e r , we h a v e n o t e d in o u r
e x p e r i m e n t s t h a t c o m p o u n d s p o s s e s s i n g the d i h y d r o x y a c e t o n e s i d e chain undergo extensive d e c o m p o s i t i o n under these r e a c t i o n conditions.
T h i s t e c h n i q u e is t h u s n o t r e c o m m e n d e d f o r s u c h c o m p o u n d s .
In T a b l e II a r e g r o u p e d t h e a n a l y t i c a l r e s u l t s f o r t h e v a r i o u s s u l f a t e salts.
T h e c o m p o u n d s w i t h o u t e x c e p t i o n a r e h y d r a t e d to a v a r y i n g
degree.
In t h i s r e g a r d c o m p l e t e a n a l y t i c a l d a t a a r e r a r e l y r e p o r t e d
in t h e l i t e r a t u r e w i t h p a r t i a l d e t e r m i n a t i o n s b e i n g t h e g e n e r a l r u l e . Paper and thin layer chromatographic as t h e o n l y s t a n d a r d of p u r i t y .
results are frequently cited
We h a v e e n d e a v o r e d to r e l a t e t h e
e l e m e n t a l a n a l y s e s to t h e w a t e r c o n t e n t as d e t e r m i n e d b y K a r l Fischer analysis. It is of i n t e r e s t h e r e to m e n t i o n t h e n u c l e a r m a g n e t i c r e s o n a n c e s p e c t r a of s t e r o i d a m m o n i u m s u l f a t e s .
As m e a s u r e d in d e u t e r a t e d
d i m e t h y l s u l f o x i d e , t h e c o m p o u n d s in g e n e r a l e x h i b i t e d a b r o a d p e a k c e n t e r e d a t 7 . 1 Z p. p . m . D20.
which underwent rapid proton exchange with
One of t h e s a l t s s t u d i e d , 3 ~ - h y d r o x y p r e g n - 5 - e n - Z 0 - o n e
sulfate
a m m o n i u m s a l t (IIb), e x h i b i t e d t h r e e p e a k s of e q u a l i n t e n s i t y c e n t e r e d at 7 . 1 Z p . p . m . characteristic
a n d s p l i t b y 50 c . p . s .
(N 14 c o u p l i n g ) ,
of a m i n e s a l t s of s t r o n g a c i d s d e m o n s t r a t i n g s l o w
e x c h a n g e of p r o t o n s . 13
A m a s s s p e c t r a l s t u d y of 3 ~ - h y d r o x y a n d r o s t -
5 - e n - 1 7 - o n e s u l f a t e a m m o n i u m s a l t (IV) s h o w e d no m o l e c u l a r ion a n d e x h i b i t e d t h e b a s e p e a k at m / e 270 {M-115).
The temperature
re-
q u i r e d f o r s i g n a l r e s p o n s e l e d to d e c o m p o s i t i o n of t h e m a s s ion.
MOSOz
/
MOSO
2
III
Compound
~ v ~
CH =O
OSO2OM
218-220
b. K
c NH 4 214-215
194-I 95
210-212
a. N a
a. K
c. N H 4 :201-203
I
-24 °a
-21 °a
-19 °a
+24 °
+70 °
+58 °
260
D
240(15,000)
240(i 4, 800)
240(16,300)
, ~max
[ct..725 ~AMeOHm~.
b. I~
C°
+74 °
P.
213-215
M.
a. N a
M
54
57
53
42
35
Z0
40
34
~/o
Yield c Reference
4 b , m o n o h y d r a t e m . p. 210°(239 ° )
4b,hexahydrate m . p. l 77-I 78.5 °
2a, m . p . 198-201 °
15, m. p. 215° ;Ea_]D 74.5° ;)kmax241 mu
Physical Properties of Steroid Sulfate Salts P r e p a r e d with Sulfamic A c i d - P y r i d i n e
Table I
O
O'I OO CD
MOSO
MOSO
2
2
I MOSO 2
Compound
M.P.c.o
K
K
>260
219-220
NH 4 206-207
i
-
+Iii °
+19 °
z.7~s
Table
57
55
90
Yield
222(8800) 66 282(3200)
270(6oo),
215(9800)
/ \max
kieO.~.
I (Cont'd)
l 7,m , p. 2 0 3 - 2 0 6
Reference
O~ CO J--J
m= O
md
%O O% O%
v
VIII
OSO
OM
---C---CH
OS O zOM
K
b.
K
a. N a
M
165-170
130
130
M. P° C. °
_
2 ob
_6 °
m u
270(550)
2_.15(9700)
270(790)
215(8700)
m a x
MeOH
25 d
62 d
29 d
%
Yield c
a. ) D i m e t h y l f o r m a m i d e - M e t h a n o l b. ) Dimethylsulfoxide c. ) Recrystallized d. ) P r o n o u n c e d decomposition w a s noted on storage at r o o m - t e m p e r a t u r e .
M
CH3OV
~
Compound
Table I (Cont'd)
Reference
cn
O
CO
IIIc
salt
IV
IIlb
salt
3 ~ - Hydr oxyandr o st- 5 - en- 17 -one Sulfate a m m o n i u m salt
ammonium
potassium
Cholest-5-en-3~-ol Sulfate sodium salt Ilia
IIb
salt
ammonium
Ic
IIa
salt
ammonium
Ib
Ia
3 ~- Hydr oxypr e gn- 5 - en- 20 -one Sulfate potassium salt
salt
potassium
17~- Hydr oxyandr o st- 4- en- 3 -one Sulfate sodium salt
Compound
• 1/2 H 2 0
C I 9 H 3 1 0 5 S N " 3/4 H 2 0
C27H4904SN
C 2 7 H 4 5 0 4 S K . I i/4 H 2 0
C 2 7 H 4 5 0 4 SNa" H 2 0
Czl H 3 5 0 5 S N " H 2 0
C 2 1 H 3 1 0 5 S K . I I/4 H 2 0
C I 9 H 3 1 0 5 S N . 1/4 H 2 0
C19H2705SK" HzO
C I 9 H 2 7 0 5 S N a . 1/2 H 2 0
Form ula
8. 70 9. 14
9.37 9.33
8.64 8.47
7.37 7.67
8.14 8.38
6.41 6. 85
7.07 7.09
H
Calc'd 57.19 F o u n d 57. 79
8. 21 7.93
Calc'd 65.81 I0.03 F o u n d 66. 27 i0. 25
Calc'd 61.49 F o u n d 61. 70
Calc'd 64.00 F o u n d 63. 70
Calc'd 58.44 F o u n d 58.52
Calc'd 55. 25 F o u n d 54.95
Calc'd 58.51 F o u n d 58. 71
Calc'd 53. 74 F o u n d 53. 77
Calc'd 57.12 F o u n d 57.36
C
8.04 7. 84
6.51 6. 87
6.08 6.09
6.33 6.58
7.42 7.84
7.03 6.53
8. 23 8. 28
7.55 7. 72
8. 03 7.96
Sa
Analyse s H20'
3.51 3.39 3.82 3.15
2.84 2.83 3.10 2. 07
7.41 4. 27 7.67 4.36
4.53 3.60 4. 78 2.60
3. 25 4.18 3.32 3.90
8.58 4.95 8.78 4.48
3.59 1.15 3.98 3.05
9.21 4. 25 9.35 4.54
5. 76 2. Z5 5.86 2. 71
M b
%0
Analytical Results of Steroid Sulfate Salts
f.2n GO CO
O
Cn
0% 0%
m (D
Table II
V
Formula
C 2 0 H 2 2 0 8 S 2 K 2 "I 1/4 H 2 0
C 2 0 H z 2 0 8 S 2 N a 2 .I 1 / 2 H 2 0
C 2 1 H 2 5 0 5 S K •3/4 H 2 0
C I 9 H 2 3 0 6 S K . 1/2 H z O
C I 8 H 2 1 0 5 S K . 1/2 H 2 0
a. ) G r a v i m e t r i c s u l f u r d e t e r m i n a t i o n s b . ) Na, K, or N c. ) B a s e d o n r e c r y s t a l l i z e d y i e l d
Vlllb
VIIIa
17a -Ethynylestra- 1,3,5(i 0)triene-3,17~-diol sulfate disodium salt
dipotas sium salt
VII
17a -Ethynyl- 3 -methoxyestraI, 3,5(10)-trien-I 7~-ol sulfate potassium salt
3 - H y d r o x y - 2 - m e t h o x y e s t r a - 1 , 3 , 5 VI (10)-trien- 17-one sulfate potassium salt
3-Hydroxyestra-1,3,5(10)+trien17-one sulfate p o t a s s i u m salt
Compound
T a b l e II ( C o n t ' d )
8.07 7. 73
5.58 5.97
H2OC
4.78 11.15 5.16 11.13
Calc'd 45.54 Found 45.93 Calc'd 43. 26 Found 4 2 . 6 8
8.71 5.10 8.93 4.89
7.71
6.39
4.44 ii.55 13. 62 4. 05 5.10 11.62 14.08 3.78
8.83 3.10
8.39 2.76
7. 25
6.06
Calc'd 57.05 Found 56.78
9.15 2.11 9.03 2.01
9.8612. 26 9.53 1.79
M b
5.66 5.60
7.50 7. 75
Sa
H
Calc'd 53.38 Found 53. 00
Calc'd 54.39 Found 53. 77
C
Analyse s
Oo 4=
June
1966
S T E R O I D S
585 +
÷
o
o
qH40
>
m/e
0
270 (M-115)
P r o m i n e n t p e a k s w e r e o b s e r v e d f o r SO + ( m / e 48) a n d S O 2 + ( m / e 64). When 3 - h y d r o x y - 2 - m e t h o x y e s t r a - 1 , 3 , 5 ( 1 0 ) - t r i e n - 1 7 - o n e s u l f a t e p o t a s s i u m s a l t (VI) w a s s t u d i e d , h e r e a l s o no m o l e c u l a r ion p e a k was observed.
T h e b a s e p e a k w a s m / e 300 ( M - 1 1 9 ) , t h e m o l e c u l a r
ion of 3 - h y d r o x y - 2 - m e t h o x y e s t r a - 1 , 3 , 5 ( 1 0 ) - t r i e n - 1 7 - o n e , i n d i c a t i n g c l e a v a g e of t h e S - O b o n d . since the t e m p e r a t u r e
No p e a k s w e r e o b s e r v e d f o r SO + a n d SO2 +
e m p l o y e d w a s not s u f f i c i e n t to c a u s e d e c o m p o -
s i t i o n of t h e r e s u l t a n t p o t a s s i u m s a l t . E x p e r i m e n t a l 14 General Procedures A.
The P r e p a r a t i o n
of S o d i u m a n d P o t a s s i u m S t e r o i d S u l f a t e s
P u l v e r i z e d s u l f a m i c a c i d ( 0 . 0 6 - 0 . 0 8 m o l s ) w a s a d d e d to p y r i d i n e (25-75 m l . ) a n d the s u s p e n s i o n w a s h e a t e d (85-95 ° ) and agitated vigorously under a nitrogen atmosphere.
The s t e r o i d (0.02
mols) was then i n t r o d u c e d and the r e a c t i o n conditions w e r e continued f o r 1-Z h r s .
A f t e r c o o l i n g , a n h y d r o u s e t h e r (50 m l . ) w a s a d d e d a n d
t h e s l u r r y of t h e c r u d e s u l f a t i o n m i x t u r e ( a m m o n i u m s t e r o i d s u l f a t e a n d p y r i d i n i u m s u l f a m a t e 6) w a s c o l l e c t e d b y f i l t r a t i o n a n d d r i e d . T h e a b o v e d r i e d p r o d u c t w a s p a r t i t i o n e d b e t w e e n a m i x t u r e of s o d i u m o r p o t a s s i u m h y d r o x i d e (12% - 50 m l . ) a n d p y r i d i n e (25 m l . ). The s e p a r a t e d o r g a n i c p h a s e w a s d i l u t e d w i t h e t h e r (Z00 m l . ). After decanting the initial solution, the solid was slurried with
586
S T E R O I D S
7:6
additional ether (3 x I00 ml. ). The crude sulfate, usually a crystalline solid at this time, was dissolved in methanol (50 ml. ), treated with charcoal, filtered and then concentrated.
The addition of
anhydrous ether to the boiling methanol solution gave a crystalline solid which was collected after cooling.
The purity of the salts were
established by elemental analysis, spectral analysis and thin layer chromatography. B.
The Preparation of A m m o n i u m
Excess a m m o n i u m
Salts
hydroxide or a m m o n i a
gas was added to a
solution of the crude sulfation mixture dissolved in a m i n i m u m volume of methanol.
After the inorganic solid was r e m o v e d by
filtration, the product was crystallized as above. chromatography of the a m m o n i u m
In the thin layer
salts, a yellow spot was evident
at the origin after spraying of the plates with phosphomolybdic
acid.
This probably results from an ion exchange process with the appearance of a m m o n i u m
phosphomolybdate
as the yellow spot.
References I. For an extensive s u m m a r y of steroid conjugation see, Bernstein, S., Cantrall, E. W., Dusza, J. P., and Joseph, J. P. , "Steroid Conjugates - A Bibliography, " Chemical Abstracts Service, A m e r i c a n Chemical Society, Washington, D.C., 1966. 2. Inter alios: (a)Calvin, H. I., Vande Wiele, R. L., and Lieberman, S,, Biochemistry, 2, 648 (1963); (b) Calvin, H. I., and Lieberman, S., Biochemistry, 3, 259 (1964); (c) Roberts, IZ. D., Bondi, Z., Calvin, H. I., D~ucker, W. D., and Lieberman, S., J. A m e r . C h e m . Soc., 86, 958 (1964); (d) Biochemistry, 3, 1983 (1964); {e) Corpechot, C., and Baulieu, E-E., Compt. Rend., 258, 3584 (1964); (f)Baulieu, E-E,, and Corpechot, C., Bull. Soc, Chim. Biol. 4__77,443 {1965); (g) Killinger, D. W., and Solomon, S., J. Clin. Endocrinol. and Metab., 25, 290 (1965). 3. Mandel, J. A., and Neuberg, C., Biochem. Z., 71, 186 (1915); Butenandt, A., Z__. physiol. C h e m . , 259, 222 (193"9-).
June
1966
S T E R O I D S
587
. (a) S o b e l , A. E . ,
D r e k t a , I. J . , and N a t e l s o n , S. J . , J. Biol. C h e m . , 115, 381 (1936); ( b ) S o b e l , A. E . , and S p o e r r i _ P ~ , J.~er. C h e m . S o c . , 63, 1259 (1941); and (c) L e v i t z , M . , ~'tero---~, ~ ~'~).--
. Short, W. F. and Oxley, P., Brit. Patent 577,666 ( M a y 27, 1946); C h e m . Abstr., 41, 2084c (1947). . Hasbrouck, R. B., U.S. Patent 2,642,427 (June 16, 1953); C h e m . Abstr., 48, 6474i (1954). . Price, W . H., U.S. Patent 2,917,522 (Dec. 15, 1959); C h e m . Abstr., 54, 6823f (1960). .
Higaki, M . , T a k a h a s h i , M . , Suzuki, T . , V i t a m i n o l . , 11, 261 (1965).
and S a h a s h i ,
Y . , J.
. F o r a review of the early references to sulfamic acid see, Cupery, M . E., Ind. Eng. C h e m . , 30, 627 (1938). I0. M a r x ,
K . , B r o d e r s e n , K . , and Q u a e d v l i e g , M. G e r . P a t e n t 5 7 0 , 9 5 6 ( F e b . 22, 1933); C h e m . A b s t r . 27, 4246 (1933).
Ii. R a m s a y , J. R . ,
and P o t t e r , D. J . , U . S . P a t e n t 2 , 4 5 2 , 9 4 3 (Nov. 2, 1948); C h e m . A b s t r . , 43, 3024 (1949).
12.
Y a m a g u c h i , S . , J. C h e m . Soc. J a p a n ,
13.
Bible, R. H., Jr., Approach," Plenum
80, 171 (1959).
"Interpretation of NMR Spectraan Empirical Press, New York, 1965, p. 61, 67.
14. All m e l t i n g points a r e u n c o r r e c t e d . The a n a l y s e s w e r e c a r r i e d out b y L. M. B r a n c o n e and a s s o c i a t e s . The s p e c t r a l , n . m . r . ( V a r i a n A60), o p t i c a l r o t a t i o n a l and m a s s s p e c t r a l data ( C o n s o l i d a t e d 103C) w e r e s u p p l i e d by W i l l i a m F u l m o r and associates. The thin l a y e r c h r o m a t o g r a m s w e r e c a r r i e d out at r o o m t e m p e r a t u r e on g l a s s p l a t e s c o a t e d with S i l i c a Gel G ( M e r c k , D a r m s t a d t ) p r e p a r e d a c c o r d i n g to Stahl, E . , C h e m . ~ _ . , 82, 323 (1958). The s o l v e n t s y s t e m e m p l o y e d w a s u p p e r p h a s e b e n z e n e , a c e t o n e , w a t e r (2:1:2) 7 0 % : m e t h a n o l 30%. D e t e c t i o n w a s a c c o m p l i s h e d with 10% m e t h a n o l i c p h o s p h o m o l y b d i c a c i d spray. 15.
Holden, G . , L e v i , I . , and B r o m l e y , R . , J. Am. C h e m . S o c . , 71, 3844 (1949).
16.
Roy,
17.
R o b e r t s , K . , Bondi, L . , Calvin, H . , David, W. and Lieberman, S . , B i o c h e m i s t r y , 3, 1983 (1964).
A.
B.,
J.
Biol. C h e m . ,
62, 41 (1956).
S T E R O I D C O N J U G A T E S I.
T H E USE O F S U L F A M I C ACID F O R
THE PREPARATION OF STEROID SULFATES J o s e p h P. J o s e p h , John P. Dusza and S e y m o u r B e r n s t e i n Organic Chemical Research Section, Lederle Laboratories, A D i v i s i o n of A m e r i c a n C y a n a m i d C o m p a n y , P e a r l R i v e r , New Y o r k
Received
10965
April 26, 1966
T h e u t i l i t y of s u l f a m i c a c i d f o r t h e p r e p a r a t i o n of d i v e r s e s t e r o i d s u l f a t e s h a s b e e n i n v e s t i g a t e d . S u l f a t e s of t h e f o l l o w i n g steroids were prepared and characterized: testosterone, cholesterol, pregnenolone, dehydroisoandrosterone, estrone, 2-methoxyestrone, ethynylestradiol and ethynylestradiol-3-methyl ether.
S t e r o i d c o n j u g a t e s , 1 f o r m e r l y r e g a r d e d s o l e l y as d e t o x i c a t i o n end p r o d u c t s , h a v e r e c e n t l y b e e n d e m o n s t r a t e d as i n t e r m e d i a t e s
in
m e t a b o l i c p a t h w a y s . 2 In v i e w o f t h e w i d e n i n g i n t e r e s t in t h i s i m p o r t a n t a r e a of r e s e a r c h ,
o u r e f f o r t s h a v e b e e n d i r e c t e d to t h e
s y n t h e s i s a n d c h a r a c t e r i z a t i o n of c o n j u g a t e s s u c h as s u l f a t e s a n d glucuronides.
More particularly,
in t h i s p a p e r t h e u t i l i t y of s u l f a m i c
a c i d as a r e a g e n t f o r t h e s y n t h e s i s of s u l f a t e s is f u r t h e r e s t a b l i s h e d . Generally,
steroid sulfates have been prepared by the
a p p l i c a t i o n of t h e f o l l o w i n g r e a g e n t s :
chlorosulfonic acid B and sulfur
trioxide-pyridine complex. 4
T h e e m p l o y m e n t of t h e s e r e a g e n t s ,
to t h e i r i n h e r e n t p r o p e r t i e s ,
has required anhydrous reaction con-
d i t i o n s a n d c a r e f u l c o n t r o l of t e m p e r a t u r e
a n d pH.
These experi-
m e n t a l c o n d i t i o n s h a v e b e e n c i r c u m v e n t e d b y t h e u s e of s u l f a m i c
due
578
S T E R O I D S
7:6
a c i d d e s c r i b e d b y S h o r t and O x l e y 5 in t h e p r e p a r a t i o n estrogen sulfates,
of synthetic
and b y H a s b r o u c k 6 and P r i c e 7 in t h e p r e p a r a t i o n
of e s t r o n e s u l f a t e s a l t s .
The a p p a r e n t s i m p l i c i t y of the m e t h o d ,
w h e r e a n h y d r o u s r e a g e n t s a r e not n e c e s s a r y , its u s e to t h e p r e p a r a t i o n
p r o m p t e d us to e x t e n d
of o t h e r s t e r o i d s u l f a t e s .
Most recently
t h e s y n t h e s i s of v i t a m i n DZ s u l f a t e e m p l o y i n g s u l f a m i c a c i d h a s b e e n reported by Japanese workers. 8 T h e u s e of s u l f a m i c a c i d f o r t h e p r e p a r a t i o n s u l f a t e s h a s b e e n k n o w n f o r m a n y y e a r s .9
of a l k y l and a r y l
Primary
alcohols are
c o n v e n i e n t l y c o n v e r t e d to t h e i r s u l f a t e s a l t s at 1 7 0 - 1 8 0 ° w h i l e s e c o n d a r y a l c o h o l s do n o t r e a c t o r g i v e v e r y p o o r y i e l d s u n d e r t h e s e conditions. for example,
Tertiary
a l c o h o l s do n o t y i e l d s u l f a t e e s t e r s ,
g i v e s r i s e to i s o b u t y l e n e .
t-butanol,
In t h e c a s e of p h e n o l s , t h e
reaction conditions favor rearrangement
of t h e p h e n y l s u l f a t e s to
the sulfonic acids. W ith t h e r e c o g n i t i o n t h a t t e r t i a r y effective catalysts for the reaction, for the esterification temperatures
amines
and a m i d e s
11
were
m o s t of the above q u a l i f i c a t i o n s
c o u l d be d i s m i s s e d .
(85-950), decreased
10
The lowered reaction
reaction times (0.5-1.5
hrs.),
and w e a k l y b a s i c n a t u r e of the r e a c t i o n m e d i a u n d o u b t e d l y c o n t r i b u t e d to t h e i s o l a t i o n of s u l f a t e s a l t s p r e v i o u s l y n o t s t a b l e to t h e o l d e r reaction conditions e.g. reaction temperature
t-butyl ammonium
105 ° - 3 h r s . 1 2
s u l f a t e (37% y i e l d ) -
T h e u s e o f p y r i d i n e as
c a t a l y s t an d s o l v e n t f o r t h e r e a c t i o n is i d e a l l y s u i t e d to t he p r e p a r a t i o n of s t e r o i d s u l f a t e s . L i s t e d in T a b l e I a r e s u l f a t e s p r e p a r e d procedures
o u t l i n e d in t h e E x p e r i m e n t a l .
by the g e n e r a l
It s h o u l d be e m p h a s i z e d
June
1966
S T E R O I D S
579
at t h i s p o i n t t h a t t h e s u l f a m i c a c i d - p y r i d i n e s u l f a t i o n t e c h n i q u e represents
an i m p r o v e d a n d / o r s i m p l i f i e d p r e p a r a t i v e p r o c e d u r e
for the compounds outlined h e r e i n .
H o w e v e r , we h a v e n o t e d in o u r
e x p e r i m e n t s t h a t c o m p o u n d s p o s s e s s i n g the d i h y d r o x y a c e t o n e s i d e chain undergo extensive d e c o m p o s i t i o n under these r e a c t i o n conditions.
T h i s t e c h n i q u e is t h u s n o t r e c o m m e n d e d f o r s u c h c o m p o u n d s .
In T a b l e II a r e g r o u p e d t h e a n a l y t i c a l r e s u l t s f o r t h e v a r i o u s s u l f a t e salts.
T h e c o m p o u n d s w i t h o u t e x c e p t i o n a r e h y d r a t e d to a v a r y i n g
degree.
In t h i s r e g a r d c o m p l e t e a n a l y t i c a l d a t a a r e r a r e l y r e p o r t e d
in t h e l i t e r a t u r e w i t h p a r t i a l d e t e r m i n a t i o n s b e i n g t h e g e n e r a l r u l e . Paper and thin layer chromatographic as t h e o n l y s t a n d a r d of p u r i t y .
results are frequently cited
We h a v e e n d e a v o r e d to r e l a t e t h e
e l e m e n t a l a n a l y s e s to t h e w a t e r c o n t e n t as d e t e r m i n e d b y K a r l Fischer analysis. It is of i n t e r e s t h e r e to m e n t i o n t h e n u c l e a r m a g n e t i c r e s o n a n c e s p e c t r a of s t e r o i d a m m o n i u m s u l f a t e s .
As m e a s u r e d in d e u t e r a t e d
d i m e t h y l s u l f o x i d e , t h e c o m p o u n d s in g e n e r a l e x h i b i t e d a b r o a d p e a k c e n t e r e d a t 7 . 1 Z p. p . m . D20.
which underwent rapid proton exchange with
One of t h e s a l t s s t u d i e d , 3 ~ - h y d r o x y p r e g n - 5 - e n - Z 0 - o n e
sulfate
a m m o n i u m s a l t (IIb), e x h i b i t e d t h r e e p e a k s of e q u a l i n t e n s i t y c e n t e r e d at 7 . 1 Z p . p . m . characteristic
a n d s p l i t b y 50 c . p . s .
(N 14 c o u p l i n g ) ,
of a m i n e s a l t s of s t r o n g a c i d s d e m o n s t r a t i n g s l o w
e x c h a n g e of p r o t o n s . 13
A m a s s s p e c t r a l s t u d y of 3 ~ - h y d r o x y a n d r o s t -
5 - e n - 1 7 - o n e s u l f a t e a m m o n i u m s a l t (IV) s h o w e d no m o l e c u l a r ion a n d e x h i b i t e d t h e b a s e p e a k at m / e 270 {M-115).
The temperature
re-
q u i r e d f o r s i g n a l r e s p o n s e l e d to d e c o m p o s i t i o n of t h e m a s s ion.
MOSOz
/
MOSO
2
III
Compound
~ v ~
CH =O
OSO2OM
218-220
b. K
c NH 4 214-215
194-I 95
210-212
a. N a
a. K
c. N H 4 :201-203
I
-24 °a
-21 °a
-19 °a
+24 °
+70 °
+58 °
260
D
240(15,000)
240(i 4, 800)
240(16,300)
, ~max
[ct..725 ~AMeOHm~.
b. I~
C°
+74 °
P.
213-215
M.
a. N a
M
54
57
53
42
35
Z0
40
34
~/o
Yield c Reference
4 b , m o n o h y d r a t e m . p. 210°(239 ° )
4b,hexahydrate m . p. l 77-I 78.5 °
2a, m . p . 198-201 °
15, m. p. 215° ;Ea_]D 74.5° ;)kmax241 mu
Physical Properties of Steroid Sulfate Salts P r e p a r e d with Sulfamic A c i d - P y r i d i n e
Table I
O
O'I OO CD
MOSO
MOSO
2
2
I MOSO 2
Compound
M.P.c.o
K
K
>260
219-220
NH 4 206-207
i
-
+Iii °
+19 °
z.7~s
Table
57
55
90
Yield
222(8800) 66 282(3200)
270(6oo),
215(9800)
/ \max
kieO.~.
I (Cont'd)
l 7,m , p. 2 0 3 - 2 0 6
Reference
O~ CO J--J
m= O
md
%O O% O%
v
VIII
OSO
OM
---C---CH
OS O zOM
K
b.
K
a. N a
M
165-170
130
130
M. P° C. °
_
2 ob
_6 °
m u
270(550)
2_.15(9700)
270(790)
215(8700)
m a x
MeOH
25 d
62 d
29 d
%
Yield c
a. ) D i m e t h y l f o r m a m i d e - M e t h a n o l b. ) Dimethylsulfoxide c. ) Recrystallized d. ) P r o n o u n c e d decomposition w a s noted on storage at r o o m - t e m p e r a t u r e .
M
CH3OV
~
Compound
Table I (Cont'd)
Reference
cn
O
CO
IIIc
salt
IV
IIlb
salt
3 ~ - Hydr oxyandr o st- 5 - en- 17 -one Sulfate a m m o n i u m salt
ammonium
potassium
Cholest-5-en-3~-ol Sulfate sodium salt Ilia
IIb
salt
ammonium
Ic
IIa
salt
ammonium
Ib
Ia
3 ~- Hydr oxypr e gn- 5 - en- 20 -one Sulfate potassium salt
salt
potassium
17~- Hydr oxyandr o st- 4- en- 3 -one Sulfate sodium salt
Compound
• 1/2 H 2 0
C I 9 H 3 1 0 5 S N " 3/4 H 2 0
C27H4904SN
C 2 7 H 4 5 0 4 S K . I i/4 H 2 0
C 2 7 H 4 5 0 4 SNa" H 2 0
Czl H 3 5 0 5 S N " H 2 0
C 2 1 H 3 1 0 5 S K . I I/4 H 2 0
C I 9 H 3 1 0 5 S N . 1/4 H 2 0
C19H2705SK" HzO
C I 9 H 2 7 0 5 S N a . 1/2 H 2 0
Form ula
8. 70 9. 14
9.37 9.33
8.64 8.47
7.37 7.67
8.14 8.38
6.41 6. 85
7.07 7.09
H
Calc'd 57.19 F o u n d 57. 79
8. 21 7.93
Calc'd 65.81 I0.03 F o u n d 66. 27 i0. 25
Calc'd 61.49 F o u n d 61. 70
Calc'd 64.00 F o u n d 63. 70
Calc'd 58.44 F o u n d 58.52
Calc'd 55. 25 F o u n d 54.95
Calc'd 58.51 F o u n d 58. 71
Calc'd 53. 74 F o u n d 53. 77
Calc'd 57.12 F o u n d 57.36
C
8.04 7. 84
6.51 6. 87
6.08 6.09
6.33 6.58
7.42 7.84
7.03 6.53
8. 23 8. 28
7.55 7. 72
8. 03 7.96
Sa
Analyse s H20'
3.51 3.39 3.82 3.15
2.84 2.83 3.10 2. 07
7.41 4. 27 7.67 4.36
4.53 3.60 4. 78 2.60
3. 25 4.18 3.32 3.90
8.58 4.95 8.78 4.48
3.59 1.15 3.98 3.05
9.21 4. 25 9.35 4.54
5. 76 2. Z5 5.86 2. 71
M b
%0
Analytical Results of Steroid Sulfate Salts
f.2n GO CO
O
Cn
0% 0%
m (D
Table II
V
Formula
C 2 0 H 2 2 0 8 S 2 K 2 "I 1/4 H 2 0
C 2 0 H z 2 0 8 S 2 N a 2 .I 1 / 2 H 2 0
C 2 1 H 2 5 0 5 S K •3/4 H 2 0
C I 9 H 2 3 0 6 S K . 1/2 H z O
C I 8 H 2 1 0 5 S K . 1/2 H 2 0
a. ) G r a v i m e t r i c s u l f u r d e t e r m i n a t i o n s b . ) Na, K, or N c. ) B a s e d o n r e c r y s t a l l i z e d y i e l d
Vlllb
VIIIa
17a -Ethynylestra- 1,3,5(i 0)triene-3,17~-diol sulfate disodium salt
dipotas sium salt
VII
17a -Ethynyl- 3 -methoxyestraI, 3,5(10)-trien-I 7~-ol sulfate potassium salt
3 - H y d r o x y - 2 - m e t h o x y e s t r a - 1 , 3 , 5 VI (10)-trien- 17-one sulfate potassium salt
3-Hydroxyestra-1,3,5(10)+trien17-one sulfate p o t a s s i u m salt
Compound
T a b l e II ( C o n t ' d )
8.07 7. 73
5.58 5.97
H2OC
4.78 11.15 5.16 11.13
Calc'd 45.54 Found 45.93 Calc'd 43. 26 Found 4 2 . 6 8
8.71 5.10 8.93 4.89
7.71
6.39
4.44 ii.55 13. 62 4. 05 5.10 11.62 14.08 3.78
8.83 3.10
8.39 2.76
7. 25
6.06
Calc'd 57.05 Found 56.78
9.15 2.11 9.03 2.01
9.8612. 26 9.53 1.79
M b
5.66 5.60
7.50 7. 75
Sa
H
Calc'd 53.38 Found 53. 00
Calc'd 54.39 Found 53. 77
C
Analyse s
Oo 4=
June
1966
S T E R O I D S
585 +
÷
o
o
qH40
>
m/e
0
270 (M-115)
P r o m i n e n t p e a k s w e r e o b s e r v e d f o r SO + ( m / e 48) a n d S O 2 + ( m / e 64). When 3 - h y d r o x y - 2 - m e t h o x y e s t r a - 1 , 3 , 5 ( 1 0 ) - t r i e n - 1 7 - o n e s u l f a t e p o t a s s i u m s a l t (VI) w a s s t u d i e d , h e r e a l s o no m o l e c u l a r ion p e a k was observed.
T h e b a s e p e a k w a s m / e 300 ( M - 1 1 9 ) , t h e m o l e c u l a r
ion of 3 - h y d r o x y - 2 - m e t h o x y e s t r a - 1 , 3 , 5 ( 1 0 ) - t r i e n - 1 7 - o n e , i n d i c a t i n g c l e a v a g e of t h e S - O b o n d . since the t e m p e r a t u r e
No p e a k s w e r e o b s e r v e d f o r SO + a n d SO2 +
e m p l o y e d w a s not s u f f i c i e n t to c a u s e d e c o m p o -
s i t i o n of t h e r e s u l t a n t p o t a s s i u m s a l t . E x p e r i m e n t a l 14 General Procedures A.
The P r e p a r a t i o n
of S o d i u m a n d P o t a s s i u m S t e r o i d S u l f a t e s
P u l v e r i z e d s u l f a m i c a c i d ( 0 . 0 6 - 0 . 0 8 m o l s ) w a s a d d e d to p y r i d i n e (25-75 m l . ) a n d the s u s p e n s i o n w a s h e a t e d (85-95 ° ) and agitated vigorously under a nitrogen atmosphere.
The s t e r o i d (0.02
mols) was then i n t r o d u c e d and the r e a c t i o n conditions w e r e continued f o r 1-Z h r s .
A f t e r c o o l i n g , a n h y d r o u s e t h e r (50 m l . ) w a s a d d e d a n d
t h e s l u r r y of t h e c r u d e s u l f a t i o n m i x t u r e ( a m m o n i u m s t e r o i d s u l f a t e a n d p y r i d i n i u m s u l f a m a t e 6) w a s c o l l e c t e d b y f i l t r a t i o n a n d d r i e d . T h e a b o v e d r i e d p r o d u c t w a s p a r t i t i o n e d b e t w e e n a m i x t u r e of s o d i u m o r p o t a s s i u m h y d r o x i d e (12% - 50 m l . ) a n d p y r i d i n e (25 m l . ). The s e p a r a t e d o r g a n i c p h a s e w a s d i l u t e d w i t h e t h e r (Z00 m l . ). After decanting the initial solution, the solid was slurried with
586
S T E R O I D S
7:6
additional ether (3 x I00 ml. ). The crude sulfate, usually a crystalline solid at this time, was dissolved in methanol (50 ml. ), treated with charcoal, filtered and then concentrated.
The addition of
anhydrous ether to the boiling methanol solution gave a crystalline solid which was collected after cooling.
The purity of the salts were
established by elemental analysis, spectral analysis and thin layer chromatography. B.
The Preparation of A m m o n i u m
Excess a m m o n i u m
Salts
hydroxide or a m m o n i a
gas was added to a
solution of the crude sulfation mixture dissolved in a m i n i m u m volume of methanol.
After the inorganic solid was r e m o v e d by
filtration, the product was crystallized as above. chromatography of the a m m o n i u m
In the thin layer
salts, a yellow spot was evident
at the origin after spraying of the plates with phosphomolybdic
acid.
This probably results from an ion exchange process with the appearance of a m m o n i u m
phosphomolybdate
as the yellow spot.
References I. For an extensive s u m m a r y of steroid conjugation see, Bernstein, S., Cantrall, E. W., Dusza, J. P., and Joseph, J. P. , "Steroid Conjugates - A Bibliography, " Chemical Abstracts Service, A m e r i c a n Chemical Society, Washington, D.C., 1966. 2. Inter alios: (a)Calvin, H. I., Vande Wiele, R. L., and Lieberman, S,, Biochemistry, 2, 648 (1963); (b) Calvin, H. I., and Lieberman, S., Biochemistry, 3, 259 (1964); (c) Roberts, IZ. D., Bondi, Z., Calvin, H. I., D~ucker, W. D., and Lieberman, S., J. A m e r . C h e m . Soc., 86, 958 (1964); (d) Biochemistry, 3, 1983 (1964); {e) Corpechot, C., and Baulieu, E-E., Compt. Rend., 258, 3584 (1964); (f)Baulieu, E-E,, and Corpechot, C., Bull. Soc, Chim. Biol. 4__77,443 {1965); (g) Killinger, D. W., and Solomon, S., J. Clin. Endocrinol. and Metab., 25, 290 (1965). 3. Mandel, J. A., and Neuberg, C., Biochem. Z., 71, 186 (1915); Butenandt, A., Z__. physiol. C h e m . , 259, 222 (193"9-).
June
1966
S T E R O I D S
587
. (a) S o b e l , A. E . ,
D r e k t a , I. J . , and N a t e l s o n , S. J . , J. Biol. C h e m . , 115, 381 (1936); ( b ) S o b e l , A. E . , and S p o e r r i _ P ~ , J.~er. C h e m . S o c . , 63, 1259 (1941); and (c) L e v i t z , M . , ~'tero---~, ~ ~'~).--
. Short, W. F. and Oxley, P., Brit. Patent 577,666 ( M a y 27, 1946); C h e m . Abstr., 41, 2084c (1947). . Hasbrouck, R. B., U.S. Patent 2,642,427 (June 16, 1953); C h e m . Abstr., 48, 6474i (1954). . Price, W . H., U.S. Patent 2,917,522 (Dec. 15, 1959); C h e m . Abstr., 54, 6823f (1960). .
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14. All m e l t i n g points a r e u n c o r r e c t e d . The a n a l y s e s w e r e c a r r i e d out b y L. M. B r a n c o n e and a s s o c i a t e s . The s p e c t r a l , n . m . r . ( V a r i a n A60), o p t i c a l r o t a t i o n a l and m a s s s p e c t r a l data ( C o n s o l i d a t e d 103C) w e r e s u p p l i e d by W i l l i a m F u l m o r and associates. The thin l a y e r c h r o m a t o g r a m s w e r e c a r r i e d out at r o o m t e m p e r a t u r e on g l a s s p l a t e s c o a t e d with S i l i c a Gel G ( M e r c k , D a r m s t a d t ) p r e p a r e d a c c o r d i n g to Stahl, E . , C h e m . ~ _ . , 82, 323 (1958). The s o l v e n t s y s t e m e m p l o y e d w a s u p p e r p h a s e b e n z e n e , a c e t o n e , w a t e r (2:1:2) 7 0 % : m e t h a n o l 30%. D e t e c t i o n w a s a c c o m p l i s h e d with 10% m e t h a n o l i c p h o s p h o m o l y b d i c a c i d spray. 15.
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