CA2152363A1

CA2152363A1 - Novel phosphorus-containing spin-trap compositions

Info

Publication number: CA2152363A1
Application number: CA002152363A
Authority: CA
Inventors: Edward G. Janzen; Yong-Kang Zhang
Original assignee: Individual
Current assignee: Oklahoma Medical Research Foundation
Priority date: 1993-10-25
Filing date: 1994-10-20
Publication date: 1995-05-04
Also published as: WO1995011908A1; AU8051894A; JPH08505406A; EP0675892A1

Abstract

Novel spin traps comprising phosphorous containing DMPO and PBN derivatives are disclosed. Effective synthesis methods for these spin traps is also disclosed.

Description

~ 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.

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).

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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

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~

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.

Claims

AMENDED CLAIMS
[received by the International Bureau on 18 April 1995 (18.04.95);
original claims 1 and 7 amended; remaining claims unchanged (2 pages)]

1. A chemical composition of the formula:

where R3 is alkyl (CH2)nH where n = (1, 2 . . . .18);
where R4 is H; 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(O) (OR)2 where n = (O, 1, 2, . . . .18), R = H , CH3 , CH3-CH2 , or Group IA metal ions;
and R5 iS H; alkyl ( CH2) nH where n = (1, 2 . . .
.18); aryl; (CHz)n COOR where n = (0, 1, 2 . . . .18) and R = H, CH3, CH3-CH2, or Group IA metals; (CH2)n P(O) (OR)2 where n = (0, 1, 2, . . . .18), R = H , CH3, CH3-CH2 , or Group IA metal ions; wherein R4 can be the same or different from R5 in a given molecule.

2. A chemical composition of the formula:

3. A chemical composition of the formula:

where R1 = phenyl, aryl, alkyl, tert-butyl, H;
R2 = phenyl, aryl, alkyl, tert-butyl; and R3 = alkyl (CH2) nH where n = (1, 2 . . . .18).

4. A method for synthesizing phosphorous-containlng derivatives of PBN, comprising the steps of:
(a) making an addition product of by adding under appropriate conditions for said addition product to form where R1 = phenyl, aryl, alkyl, tert-butyl, or H;
R2 = phenyl, aryl, alkyl, or tert-butyl; and R3 = alkyl (CH2)nH where n = (1, 2 . . . .18);
(b) subjecting said addition product to water to cause a hydroxylamine to form;
(c) oxidizing said hydroxylamine to said PBN
derivative having the following structure:

5. The method of Claim 4 wherein a mild oxidizing agent is provided in step (c) to catalyze formation of said PBN derivative.

6. The method of Claim 5 wherein said mild oxidating agent is Cu(C2H3O2)2.

7. A method for synthesizing phosphorous-containing derivatives of DMPO, comprising the steps of:
(a) providing to where R3 is alkyl (CH2)nH where n = (1, 2 . . . .18);
where R4 is H ; 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(O) (OR)2 where n = (0, 1, 2, . . . .18), R = H, CH3, CH3-CH2 , or Group IA metal ions;
and R5 is H; 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) P(O) (OR)2 where n = (0, 1, 2, . . . .18), R = H, CH3, CH3-CH2, or Group IA metal ions; and wherein R4 can be the same or different from R5 in a given molecule;
to form an addition product;

(b) adding water to said addition product to form a hydroxylamine;
(c) oxidizing said hydroxylamine to

8. The method of Claim 7 wherein a mild oxidizing agent is provided in step (c) to catalyze formation of said PBN derivative.

9. The method of Claim 8 wherein said mild.
oxidating agent is Cu(C2H3O2)2.