Note: Descriptions are shown in the official language in which they were submitted.
~ WO9S/11908 21~ 2 3 ~ 3 PCT~S94/12109
NOVEL PHOSPHORUS-CONTAINING SPIN-TRAP COMPOSITIONS
TECHNICAL FIELD OF THE lNV~llON
This invention relates to the field of spin-trap
molecules useful for trapping free radicals in biological
systems and methods for preparation thereof.
WO 95/11908 PCTIUS94/12109 ~
21~23~3
BACKGROUND OF THE lNV~;NllON
Scientists are continually researching for effective
free radical trapping compounds, known as "spin-traps,"
since free radicals are believed to be involved in
disease initiation and mediation in a~imals. Ischemia
and inflammation are two examples of ~iological events in
which free radicals have been implicated. Spin traps are
important for diagnostic and therapeutic purposes. Known
spin-traps include ~-phenyl N-tert-butyl(-)nitrone (PBN),
~-(4-pyridyl-1-oxide)-N-tert-butyl nitrone (POBN), 2-
methyl-2-nitrosopropane (MNP), and 5,5-dimethyl-1-
pyrroline N-oxide (DMPO).
Despite the discovery of several spin-trap
molecules, the need remains for additional compounds
which are of increased stability and which work more
effectively to trap free radicals in biological systems.
Another problem in the art has been that proposed
structures of desired spin-traps, which theoretically may
provide some of the desired properties, are postulated
from time to time, but synthesis has been difficult or
impossible by known methods. It therefore has been
desired that a convenient method of synthesis be
available for a spin-trap agent having some or all of the
above-described properties.
2152363
WO95/11908 PCT~S94/12109
SUMMARY OF THE lNV ~:N'l'lON
A new family of spin trap molecules comprising
dialklphosphoryl nitrones ("DAP-DMPO") substituted in the
~-position (or 2-position) is disclosed.
A new spin trap molecule, 5,5-dimethyl-2-
diethylphosphoryl-l-pyrroline N-oxide ("2-
diethylphosphoryl-DMPO" 2-"DEP-DMPO") has been
synthesized and characterized. The synthetic method for
making the compound is also new and is expected to be
useful for making the family of ~-dialkylphosphoryl
nitrones.
In another embodiment, PBN type phosphoryl
derivatives and a method for making the same are
disclosed.
~ 21 S 2 3 ~ 3 PCT~S94/12109 ~
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 demonstrates the synthetic route for
preparation of 3-diethylphosphonanyl-DMPO.
FIG. 2 demonstrates the synthetic scheme for PBN-
type 2-phosphoryl nitrones.
FIG. 3 demonstrates the synthetiG~gcheme for DMPO-
type 2-phosphoryl nitrones.
WO 95/11908 215 2 3 6 3 PCT/US9~/12109
DETATT~n DESCRIPTION
A new spin trap derivative of 5,5-dimethyl-1-
pyrroline N-oxide ("DMPO") which includes a
dialkylphosphoryl group at the 2-position has been
successfully synthesized, and iB known as
dialkylphosphoryl-DMPO or "DAP-DMPO." The new compound
has the following structure:
H3 ~ / - R
where R = an alkyl group having 1-18 carbons, preferably,
R=-CH2CH3.
The novel spin trap is effective in trapping free
radicals, as shown in Table 1.
Because of its chemical nature, DAP-DMPO can be used
as a spin trap in cell membrane regions to trap free
radicals which are formed in these areas.
Another related utility is believed to be site-
specific defense against reactive free radicals created
in the polar interface and outer aqueous layers of
membranes. Prophylactic treatment of free-radical
disorders is expected.
The utility of the compounds of the present
invention in preventing or treating diseases is believed
to be initiated or mediated by free-radical generation in
the body. Exemplary doses range from 25 to 125 mg/kg of
body weight in rats. The effective range of dosage in
humans and other m~mm~l S iS expected to be between about
25 to about 125 mg/kg, and preferably between about 25 to
WO95/11908 P~T~S94/12109
215 ~63 6
about 35 mg/kg of body weight. Particular dosage may
vary depending on the particular derivative selected.
The compounds of the present invention are
preferably administered systemically. The compounds can
be administered at once, or can be di~vided into a number
of smaller doses to be administered àt varying intervals
of times. The compounds may be administered orally or by
other methods including intravenous, subcutaneous,
topical and intraperitoneal administration.
A method of administration of the compounds of the
present invention is oral delivery. The compounds may be
enclosed in capsules, compressed into tablets,
microencapsulated, entrapped in liposomes, in solution or
suspension, alone or in combination with a substrate
immobilizing material such as starch or poorly absorbable
salts. Pharmaceutically compatible binding agents and/or
adjuvant materials can be used as part of a composition.
Tablets or capsules can contain any of the following
ingredients, or compounds of similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or
gelatin; and excipient such as starch or lactose, an
integrating agent such as alginic acid, corn starch; a
lubricant such as magnesium stearate; a glidant such as
colloidal silicon dioxide; and sweetening and flavoring
agents. When a capsule form is used the liquid carrier
such as a fatty oil may be used. Capsules and tablets
can be coated with sugar, shellac and other enteric
agents as is known.
In a preferred embodiment, 2-DEP-DMPO was
synthesized and tested. Characterization of 2-DEP-DMPO
using Electron Paramagnetic Resonance Spectroscopy (EPR)
revealed distinctive spin trapping chemistry which
provides an optimum condition for this type of analysis.
WO95/11908 21 5 2 3 6 3 PCT~S94/12109
All spin adducts give a large phosphorous hyperfine
splitting which varies in magnitude with the kind of free
radical trapped.
As is shown in Table 1 "EPR HFSC' s of 2-DEP-DMPO
ADDUCTS", about 20 free radicals have been tested. Of
this eighteen (18) successfully give EPR spectra.due to
the spin adducts. The solvent can be either benzene
(selected as typical of lipophilic environments) or
water. Only the bulky radicals such as tert-butoxyl or
trichloromethyl are not apparently trapped by 2-DEP-DMPO.
The lifetime characteristic is an important feature
of the spin adduct. As can be seen with a hydrocarbon
adduct like phenyl (Ph ) the lifetime of the spin adduct
is very long even in water (no decay within 16.5 hours).
Even more remarkable is the long lifetime of the acyl
adduct ( COCH3), namely 52.4 hour half-life. Prior to
this invention, no other spin trap was known or available
which allows detection of acyl radicals because the life-
times of the spin adducts are too short (e.g., PBN or
DMPO itself are not suitable). Also the new spin trap
produces long spin adduct life-times of the alkoxyl
radicals (e.g., i-AmylO , 31.5 hour half-life) and
hydroxyl radical ( OH, t~ = 3.0 hours).
WO 95/11908 PCTIUS94/12109 ~
2~3~3
~o ,.., o
r~ ~ r~ rr~ ,
q~11 11 11 11 ~.,
-
.
~,
~1 0 ~1 ~` ~ O d' ~r co OD ~ ~ O
~ . . . . . . . . . . . .
W H a)
r ~ ~ o
o,~ O ~0 O,~ ~0 r ~ ~ ~.o ~ ~a ~ o ~
rr~ U ~ U~C U ~ U
U r~
r,~ ~ r~l _
p,, r~ + ,
W H ~ ; + C
O +,~ ~ o >~ ~ 5
O d~ I I I I ~ ~,C
p r,~ ,1 r + ~ +
U
O O r~
_ O O o ~ ~ r`~
O ~ q O G
O O I I u rJ
P ~ ~
~ WO 95/11908 21 S 2 3 ~ 3 PCTIUS94112109
.C J
m
,~ a ~ U
O
O O
~1 ~1 1` ~ o ~1 In ~ ~1 0 o al o
co o ~ ~ ~r o ~ o~ ~ ~ a~ oo
? L~ o
c ~ m
~ H
qO S 0 5' ~ ~ ~ ~ ~ ~ N ~ 5~ ~ 5~
~,~ c U~ U 5 5 :C 5 ~ 5 5 U :~ U 5
~ :1 Z ~ 5 ~) ~i o N ,~
W H m o o o o 4 1~ +
-- ~ _ 5 X ~ -- + + +,~ G G
~ C o o o o o o o ~I
0 5 ~ ~ 5~ 5.~ 5 ~ 5N ~ ~ O
X ~ a
~, o,~ ~5 ~ c~ c~ ,, ~ 5. 5'
C,~ V C O O C~) C) C.
p . ~ p: 5 ~ 5 ~ ~ ~ ~
WO 95/11908 , 21~ 2 3 6 3 PCT/US94/12109 ~
' R '~ X
.~ 3 ~ ~ Q ~ , 1l ~
'~. R ~
~ ~ ~D~ ~ o " ~n ~ o
~i d',i ~ ~ ~r _ h R a) U~ ~ D ~ 5 L'
a) ~ ~ ~ ~ ~ ~ ~, ~ a 3 1 D tJ~ ~D
a L~ L
e,, ,~ ~,O 'O ~ ~ ~ ~ R--
.D O ~ D o,~ ~ O I ~ ~ '
H ~ S _
o R 0 5~ ~ ON ~J~ L ~ L L-- '' -
~ C) ~ m~ LD, O a
U O ~ ~ ~ S~ _ ~D - rt
U~ ~ ~ ~ R ~ D
tD ~ ~D ~ L D
~ D~
L'; a) a) ~ ~; L3 ~ R ID
W H + +~ L F G ~ _, o F,~
~LJ L~ y ~~ I J ~ ~1 C
P~ O :~ tD U J R 1
q~ ~ a) ~ O ~D q~ -
R L U
L -- U~
6 c - IJ :~
~: , ID ~ ~ L 11
C.i ~ I ~D ~D
L Ll I O
p ~ ~ ~ z; a~
WO95/11908 21 5 2 3 ~ 3 PCT~S94112109
General Method of Producinq 2-Phosphoryl Nitrones
for SPin TrapPinq
A new general method for the synthesis of 2-
phosphoryl nitrones useful as spin traps is disclosed.
Since the reaction of the lithium salt of the
dialkylphosphoryl anion is simply an addition to a nitrone
function, it should be readily possible to produce a
variety of new nitrones starting with aldo-nitrones of the
following type:
WO 95/11908 '3 6 3 PCT/US94/12109
PBN- TYPE
Rl_f=~ .R~
(R30)2P=o
The synthetic scheme is given in FIG. 2.
where Rl = phenyl, aryl, alkyl, tert-butyl, H
R2 = phenyl, aryl, alkyl, tert-butyl
R3 = alkyl ( CH2 ) nH where n = (1, 2 . . . .18)
DMPO - TYPE
R4 R5
where R3 i9 alkyl (CH2)nH where n = (1, 2 . . . .18);
where R4 is alkyl (CH2)nH where n = (1, 2 . . . .18); aryl;
( CH2 ) n COOR where n = (0, 1, 2 . . . .18) and R = H ~ CH3 ~
CH3-CH2, or Group IA metal ions; (CH2)n P(0) (OR)2 where n =
(0, 1, 2, . . . .18), R = H , CH3 ~ CH3 - CH2 , or Group IA
metal ions;
and Rs is alkyl (CH2)nH where n = (1, 2 . . . .18);
aryl; ( CH2 ) n COOR where n = (0, 1, 2 . . . .18) and R = H
CH3~ CH3-CH2, or Group IA metals; (CH2)n P(0) (OR)2 where n
= (0, 1, 2, . . . .18), R = H ~ CH3 , CH3-CH2, or Group IA
metal ions; and wherein
WO95/11908 2 1 ~ 2 ~ ~ ~ PCT~S94/12109
R4 can be the same or different from R3 in a given
molecule.
The synthetic scheme is given in FIG. 3.
The advantage of using 2-DEP-DMPO spin traps is that
the ethyl group could be changed to vary in length as a
hydrocarbon group ~i.e., R3 could be longer than 2-
carbons). Therefore penetration within the biomembrane
could be adjustable. With, for example, an 8-carbon alkyl
group (R3 = C8Hl7) the spin trap could be locked into place
to monitor free radical producing events in the immediate
locality of their source. The molecular compatibility
should be good because phospholipid bilayers have very
similar ester structures in their make-up.
The synthetic route for the preparation of DEP-DMPO
is illustrated in FIG. l. This reaction could be adapted
for synthesis of all dialkyl-DMPOs by substituting the
desired alkyl for the ethyl group in (CH3CH2O)2P(O)H.
Example 1: PreParation of 2-DEP-DMPO
lH NMR spectrum was recorded on a Varian XL-300 NMR
spectrometer using tetramethylsilane (TMS) as an initial
standard. EPR spectra were measured on a Bruker ESP-300E
spectrometer. Chemicals are purchased from Aldrich
Chemical Company, Inc. DMPO was prepared in our
laboratory by known methods.
The procedure for the addition reaction of
dimethylphosphoryl anion to DMPO was adapted from R.
Huber, A. Knierzinger, J.P. Obrechy, and A. Vasella,
Helvetica Chimica Acta, 68: 1730-1747 (1985). Under N2,
lithium diisopropylamide (LDA, l0mL, 2.0 M solution in
heptane/tetrahydrofuran/ethylbenzene, 20 mmoL) was added
dropwise to a solution of diethylphosphite (5.0 g, 36.2
mmoL) in dichloromethane (40mL) which had been precooled
WO95/11908 PCT~S94/12109 ~
2~s~3~
14
to -20C. The mixture was stirred for 15 min. at -20C
and then further cooled to -60C. A solution of DMPO (2.0
g, 17.7 mmoL) in dichloromethane (4 m~) was added. The
reaction solution was allowed to warm slowly to -20OC over
a period of 3.5 h. Water (5 mL) ~as added to quench the
reaction. The resulted solutio~ was warmed to room
temperature and then diluted with 100 ml of
dichloromethane. The solution was washed with NaCl-
saturated aqueous solution (2 X 60 mL), dried over Na2SO4,
filtered and evaporated. The residue was distilled to
give the excess diethyl phosphite (<30C/1 Torr) and the
desired hydroxylamine (2.45 g, b.p. 99-109C/1 Torr). The
hydroxylamine (2.45 g, 9.8 mmoL) was dissolved in 95~ EtOH
and mixed with Cu(OAc) 2 monohydrate (0.1 g) and NH40H (29
aqueous solution, 0.3 mL). The solution was stirred with
bubbling with air until a permanent blue remained (ca. 10
min). The solution was evaporated and the residue was
chromatographed on silica gel eluted with ethyl acetate. A
liquid (1.3 g) was obtained. Overall yield: 29~. lH NMR
(CDCl3/TMS) ~ 4.22 (quintet, JH=Jp=7.7 Hz, 4H, 2 OCH2),
2.74 (dt, JH=7.2 Hz, Jp=3.1 Hz,2H, 3-CH2), 2.06 (t, ~=7.2
Hz, 2H, 4-CH2), 1.35 (s, 6H, 2CH3), 1.29 (t, ~=7.2 Hz, 6H,
2CH3) ppm. The data is consistent with the structure of
2-diethylphosphoryl-5,5-dimethyl-1-pyrrollne N-oxide.
Example 2
The phosphorous derivatives of DMPO or PBN spin traps
will be administered to an animal either orally or
intraperitoneally in amounts of about 25-250 mg/kg.
An effective amount of spin traps will be
administered to trap the anticipated concentration of free
radicals generated in the particular disease state of the
patient.
WO95/11908 21 ~ 2 3 6 3 PCT~Sg~/l2l09
ExamPle 3
A method for in-vivo spin trapping is conducted
according to Lai, et al Arch. Biochem. Biophys. 244 :156-
160 (1986) which is hereby incorporated by reference. The
phosphorous-containing spin traps are tested for
effectiveness in treating various diseases.
