Note: Descriptions are shown in the official language in which they were submitted.
L3~
Description
Derivatives of All-trans- nd 13-cis P~tinoic ~cid
and thods for PreFaring ~e
Field o~ Lh~ Invention
. . _ . .
Ihe presen-t invention relates to ncvel derivatives of
al7-trans- and 13~cis-retinoic acid.
Backgrou~d o~ the Inv nt on
In 1967 r 13~cis-retino.ic acid was iæ n-tified in rat
tissue ex*rzcts and it was postulated to be a natural
me~bolite of all-trans-retinoic acid.l Ihe 13-cis-retinoic
aci~ has ~een found to be at least as efiective as all-
trars-retinoic acid in promo-ting vitamin A-~ependent growth as
well as in controlling epithelial cell differentia-tion.
Recent ~rk s~ggests that 13-c retinoic acid is not solely
'~ 15 pro~u oed as c~n artifact of isolation but ~hat all trans-
re inoic acid is iscmerized in mammals to scme extent to
13-cis-retLnoic acid.2 The utlity of 13-cis-retinoic acid in
treatiny den~atological conditions, i~s t~Imor inhibitory
prcperties, a~ its reduced toxicity relat_ve to all-trans-
re, ~oic acid are kncwn.
Disclo~ure of the Invention
.
I~e pr.esent invention relates to ~cve1 derivatives of
a1l-trans- and 13-cis-retinoic acid. k~re specifically, this
irventicn re1~t~s to N-hydroxysuccini~idyl and coenzyme A
esters of all-trans- and 13-cis-retinoic acid.
Ihe der-~ative of the present i~Jention can be
represented by the following foDmula:
\
13a~s~3~
wherein ~ and ~ are each selected frGm the f~G~ crnsisting
of:
(a~ hydrcgen,
O ~
(b) - C - O ~ and
(c) ~ ~ C o ~
.
where CQ~ represents cc~nzym~ A, except that both of ~ anc~ R2
cannot be (a), but one of ~ and R2 must be (a).
The compounds of the present invention can be p ~pared :in
S accordance with the following schematic c~iac~ram and
descrip~tic~. ~ ~ - o- H~
co~7 y.~,te A
A/ hy~%y~ de 2 ~ o
~Co~ft ~ c-s - c f~h
\~/ d;~ cLahe~ L-
~d;"~;~o aOe ~ E A
~c~
where 1 = all tran~-retinoic acid
2 = N-hydroxysucclnimldyl all trans-retinoa~
3 = all trans-retinoic acid anh~dride
104 = all trans-retinc~l CoA
~3~5136
~U~ ~ - O~
s ~ /o o .
C~ ~e A
(-5 co~
wr~ e 5 = 13-cis~retinoic acid
6 = N-hydroxysuccinimidyl 13- _ -reti~ate
7 = 13~cis-retir.cyl CoA
De~ailed Description of Processes
C~ icals
All _rans-retinoic acid and the sodium salt of coenzyme A
we~e purchased from Sigma Chemical Co. (St. Louis, ~).
M-~droxysuccinimicle ~nd N,N'-dicyclohexylcarbodii~ide were
f~om the Aldrich Chemical Co. (Milwaukee, ~ Disposable
silica gel Sep-Pak cartridges ~Waters Pssociates, ~lilford, MA)
we-e used for purification of synthetic intermRdiates.
All operations irvolving the use of retinoic acid analogs
e~2 perfor~d under yellow light in a nitrogen a~mosphere.
Gl3ssware silanized ~th aimethyldichlorosilane was used for
~r~ synthesis, handling and storage of retinoic acid CoA
t~oesters~ All reac*ions were done using l and 5 ml
Reacti-Vials (Pierce Chen. Corp., Rockford, IL).
Ch~cmato~raphic ~ e
Analytical and preparatlv~ thin-layer chromatography
(~C) was done usiny pre ate alu~inum silica gel sheets wi-~h
W indica~or and glass pre-coated silica gel plates (20~20 cm,
2 m~ thick) without fluorescence indicator, respectively,
c~,ained fro~ EM Science (Gibbstown, NJ). Plates were
c~eloped using sol~ent sys-t~ms: A; hexane-ethyl acetate l:l
~-~ B; n-butanol-acetic acid-watex 5:2:3.
~L3
sæc =
~ . .
Electron i~pact mass spectra (EI-~S) were recorded at 70
eV with al~ AEI l'~-9 spectrGme-ter coupled to a DS-50 data
system.
Ultraviolet (W) absorp-tio~ spectra were recorded in
absolute ethanol or dioxane with a Hitachi Ik~del 10~-60
W -visible spectrcphoto~eter~
~nfrared spectra (IR~ t~iere recorded on a Nicolet ~
Fm-lR spec~rGmeter in CC14 solution or using i~ns of oily
substances.
Proton magnetic resonance spectra (~I-NMR) were taken
t~th a Bruker ~ 270 FT spectr~eter in acetone-d6 solutions
containing tetra~ethylsilane (T~LS) as internal standard.
Bec]~N~l m~del 4500 pH meter equipped with Bech~an C~B
p~-electrode was used for pH adjus-tments.
PREPARATIONS
Preparation of N-hydroxy~succininLidyl ester of
all-trans-retinoic acid (2)
Pll-tr -retinoic acid (1) (20 mg, 64 ~M) in 0.8 ml of
dioxane was trea ed with N-hydroxysuccin~nide (7.6 mg, 64 ~M)
in 0.4 ml of dioxare and dicyclohexylcarbodiimide (13.6 mg, 64
~i) in 0.4 ml of dioxane. ~ne resulting solution was
ragnetically stirred at room temF~rature for 5 hours and then
diluted with 10 ml of ethyl ether. The precipitated
dicyclohexylurea was separated by filtration and the filtrate
evaporated to dryness under reduced pressure. me oily
residue was dissolved in warm metnanol (10 ml) and the
solution concen~rated under reduced pressure until a slight
prec~pitate becare apparent. After standing overnight at
-20C, crystals t~--ere filtered off and washed with cold
~ethanol. Chro~tographically pure product (2) showed TLC Rf
0.47 (solvent syst~m A~. W (ethanol) ~max 377 (36.400); IR
1758, 1734 cm 1; ~ (acetone-d6) ~ 1.05 ~s, 6,(CH3)2C), l.S
(m, 2, 2 ~ I2), 1.6 (m, 2j 3-CH2), 1.72 Is, 3, 5-~13), 2.01 (s,
3, 9{~I3), 2.02 ~2, 4{~l2~ ~ 2.37 (s, 3, 13 CF13), 2.97 (s, 4,
~3CP~;3L36
~ 12 2l-Gn~2); ~ m/e (relative intensity~ ~M3~ 397 (65) ~
3~2 (5) 1 299 ~12) r 2~;3 (40) ~ 267 (19) ~ 255 (10) r 239 (17) ~ ~;9
(lC0).
~ all~_rans-retinoie acid anydride (3)
ALl trans-ret7~oie acid (10 m~, 33 yM) in 0.2 ml o*
tetrahydrofuran ~ias treated with dicyclohexylcarbcdiimide (7.2
~, 34 ~M) in 0.2 ~L of acetonitrile. The _olution was
stirred at ro~ te~~ ature for 2 hours and then diLuted with
ethyl ether. P~ee_?itated dic~clohexylurea was re-~oved by
filtration ~rd solven~ evaporated under reduced pr~ssure. rrhe
residue was susper~a in hexane and chrc~natographed on a
Sep~Pak ear~-c'ge. Anhydride 3 was eluted frc~ the cartridse
with the m~xt~re o 1.5% propanol~2 in hexane (20 ml).
Further elu~i~n wit`n the same ~uxt~re afforded the unreaeted
substrate cx~'~L~.~ed wi~h the produet of condensation of
dieyelohe~yLc~Lrboliimide wi-th all-trans-retinoic acid. rrhe
pure anhydr~e 3 (_olvent system A) ~as obtained as an oily
substance (~ro~ hexa~e). l~C, R~ 0.81 (solven-t system A). W
(eth~nol~ ~rax 38~ ~E 76,0C~3 ~maX 385, 68,300 ~ethanol)~;
IR 1701, 1767 cm ~ (acetc~ne-d6) 0 l.Q4 (S, 12, (CH332C),
1.5 (m, 4, 2-CH2), 1.6 ~m, 4, 3-CH2), 1.74 ~6, S, 5-CH3~, 2.1
(m, 10, 4-C~.2, 9-CE3), 2.44 (S, 6, 13-CH3), MS, m/e ~relative
intensity) (~I)* 582 (7), 444 (3.6), 404 (5~4)~ 381 (9.4), 366
(4.61, 2~3 ~7), 177 (7), 159 (26), 145 (17), 44 (lC0)~
Prèparation o' all-trans-retinoic acid CoA ester ~4)
N-hydro~succirimidyl ester of all-trans-retinoic acid
(2) (1.5 mg, 3.7 ~ in 0.4 ml of tetrahydrofuran was added to
a stirred solUtion O~ coenzyme A (2.5 m~, 2.8 ~M) in 0.2 ml of
water. The ~H o' ~e solution was adjusted to 8.0 - 8.5 wi~h
1% NaHC03. Ih2 re~ction mixture was stirred under nitrogen
atm~sphere at 35C _or 20 hours until most of the CoA was
consumed as ~eterm~d by TIC using the solvent system B.
Ester 4 ~las s~ar2'~ed from the crude reaction mixture by
preparative T~.C us~ng the solvent system B. Product was
extracted fro~ the ~LC adsorbent using a mixture of acetic
~3~3~
acid and ~ter. ~ .ilization o:E the extract afforded pure
es~r 4 as dete~.~ '. by 'FLC using the solvent system B (Rf
1 0.43, cc~par.~g ~r h ~~ 0.40 and 0.41 for ~-methylcroto.nyl and
¦ r~yris'~l esters ~~ C~). W tH20) AmaX ~5~ (~ lO,S00 and
~'~r~Rx 393 (~ 32rCC~
Ester 4 ~,~.~s ~lso obtained starting fr~n anhydride 3
usi~g the sar~e proc~ure and separation me~hod as a~e. In
this prep~ration, 'r~ er, the fonmation of ester 4 cculd not
ke cc~pleted e~en ^~hen coenzym~ A was exposed to a 2-3-fold
molar excess of 3r~ydride 3 for double the time .in above
pr~paration.
Preparation of N-h~r~ysuccinir~id~l ester o 13-cis-retinolc
acid ~6~
The compcun~ 6 ~as prepared by the rnethod esse~tial'ly the
same as for pr_~ahra'_ion of ester 2 starting fram
13~c s-retinoic aci~ 5. TLC of chrGmatographically pure
ca~pound 6 sh~el ?~ 0.53 ~solvent systern A). W (ethanol)
380 (~ 35 r0~3~; IP~ 1760, 1747 c~n , ~ (acetone-d6) ~
1.05 (s, 6, (~3~2C~, 1.5 (m, 2, 2-CH2), 1.65 (m, 2, 3-CH2),
1.76 ~s, 3, 5 ~ }, 2.05 ~m, 2, 4-CH2~, 2.1 (S, 3t 9~CH3),
2-88 ts, 4~ {~12, 2'-OE12); ~, ~/e (relative intensity~ (M)
3~7 (36), 382 (2), 3~3 ~7), 283 (17), 267 (10], 255 (8), 23g
~19), 41 (100).
Preparation o 13-c~s-retinoic acid CoA ester (7)
Ester 7 ~,~s p~ared and purified by the method described
for the prepa atiç~ or ester 4. TIC of chrcmatographically
pure product S~Y~ _ 0.42 in solvent system B.
T~.e compcun~a O this invention find utility in the
treat~ent of ~e~o~ogical conditions such as acne and
ichthyosis, ar~1 Ga ~bstitutes for all-trans-retinoic acid and
13-cis-retinolc ~^d in their various known applications.
I~y ~ay al~o ~e uced as a substrate for bioche~ical
reactions, such ~s, the transfer of retinoic acid to glycerol
derivatives o~ g'~oe rldes or in the B and omega oxidation of
retinoic acid.
~L3~ 31~i
eferences
1. Zile, M., R. J. E~erick, H. F. DeLuca, "Iclentification of
13-cis-Retinoic ~cid in Tissue Extracts in .-Lts Biological
Activity in Ra~s," Biochi~. Bicp~ 5. Acta, Vol. 141r PP-
639-641, 1967.
2. Zile, M., R. C. Inhorn, H. F. DeLuca, "~tabolites o:E
All-trans Reti Qic Acid in Bile: Identificat.ion of Pll
trans and 13~cis-Retinoyl Glucuronides," J. Biol. Chem.,
Vol. 257, pp. 3537-35~3, lg82.
.
.
