CA1261856A - Conjugates of leukotrienes with proteins - Google Patents

Abstract

A B S T R A C T

A compound having the following formula:

or wherein n is 0 to 10, and the lactones form thereof.
These compounds are useful far the preparation of compounds comprising leukotrienes conjugated with proteins such as Bovine Serum Albumin and Hemocyanin from Giant Keyhole Limpets.

Description

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This is a divisional of Canadian Application S.N. 466,742, filed October 31, 1984.

RELATIONSHIP TO THE PRIOR ART
The concept of usiny conjugates of leuko-trienes in a radioimmunoassay was described, by L.
Levine, R.A. Morgan, R.A. Lewis, K.F. Austin, D.A.
Clark, A. Marfat, and E.J. Corey, Proceeding of the National Academy of Sciences, U~S~Ao~ Vol. 78, No. 12 7692 (1981). This method uses direct coupling through an activated acid derivative to the protein. This method is much less effective than the present in-vention.
Bifunctional cross-linking reagents useful in preparation of protein-hapten conjugates have also been prepared, see Kitagawa, J. Biochem. 79, 233-236;
and Kitagawa, Chem. Pharm. Bull. 29(4), 1130-1135;

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describing maleimido-succinimide derivatives. The present invention relates to conjugates of leukotrienes C4, B4, D4 or E4 (preferably C4 and B4) with a protein selected from hemo-cyanine from giant keyhole limpets (KLH), bovineserum albumin (BSA), human serum albumin, tetanus antigen, diphtheriae toxoid, or CRM 197 (a diphtheriae toxoid produced by a mutant of Corynebacterium diphtheriae), through the coupling agents 1,5-difluoro-2,4-dinitrobenzene or 6-N-maleimido-alkanoic acid chloride,-preferably 6-N-maleimido-hexanoic acid chloride, wherein the alkanoic moiety has 2 to 8 carbon atoms. The conjugates are useful in a sensitive and specific immunoassay and are also useful immunotherapeutic agents in the treatment of various allergic and chronic inflammatory diseases of the skin, lung and airways, including asthma, allergic rhinitis, rheumatoid arthritis,~and skin diseases such as psoriasis and eczema.~ The present invention also relates to useful reagents for preparing such conjugates. ~ ~
Leukotriene C4 (LTC4) has the~following structure: ~

~ COOH
S ~ COOH
: J~

: : ~ ~ :
~ ` ~
COOH ~ 4- ; ;
SH

::: :

: :
.

2298P/0832A ~ 3 - 16777IB

Leukoteiene B4 (LTB4) has the following structure:

H` ? \C02H
~ LTB4_ Leukotriene D4 (LTD4) has the following structure:

~1 S ~ N ~ CO H
~ ~H2 2 ~ CO2N

LTD4_ :
Leukotriene E4 (LTE4) has~the following structure:

~ NH2 O2H~

TE

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35~
- 4 - 16777Is The present invention also relates to the following compounds which are useful in preparing the conjugates (especially the conjugates of LTB4):

1) HO ~ CONH(CH2~nNH2 ~-/~/

wherein n is O to lOj preferably O or 2 to lO, more preferabIy O or 3.

2) HO ~ ~ CoNH~cH2)nNH ~ ~

:
wherein n is O ~o lO, preferably O or 2 to lO, more preferably O or 3.

3) ~V 1' : :: : : :

wherein n is 0 to 10, preferably 0 or 2 to 10, more preferably 0 or 3.

In the above three compounds, the compounds where n is 1 are likely to be less stable than the other compounds having the same generic formula.
The preparation of the conjugates of the present invention may be illustrated by reference to conjugates of LTC4 and LTB4.
For LTC4, coupling procedures were selected so that the reactions took place on the free amino group of the glutamyl residue, thus retaining the most important parts of the LTC4 molecule unchanged.
The general conjugation procedure utilized a stepwise method with well characterized intermedlates. The strong W absorption of the triene chromophore in LTC4 ~ = 40,000~at 280 nm) was used as a~probe for determining coupling efficiencies and for monitoring the state of the LTC4 molecules throughout the procedures.
Coupling ratios~in~th~e ranges~of 5 to 15 equivalents~of LTC4 per 100,000 daltons~of prot~in were desired.
Con~ugates of LTC4 Conjugations using 1,5-difluoro-2,4-; dinitrobenzene as~coupl~ing ~
~Th~e reagent, 1,~5~-d~lfluoro-2,~4-dinitrob;enzene (DFDNB) reacts~quite specifically with amino functions~, allowing clean~stepw~ise replacement~of the two ~fluorine atoms (the second fluorine being~

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5~i replaced at a much slower rate). In addition, the strong and characteristic W absorptions of the reagent, and its mono- and diamino substituted derivatives allows one to follow the course of the coupling procedure and to quantitate the final adducts by W spectroscopy.
LTC4 was found to react essentially quantitatively with excess DFDNB in pH 7.2 buffered aqueous methanol within 30 minutes. The intermediates thus formed could be characterized by HPLC analysis, by the appearance of a strong W band at 345 nm characteristic of 1-amino-5-fluoro-2,4-dinitrobenzenes. After removal of methanol from the reaction the excess DFDNB could be removed by ether extraction. The intermediatés could be further purified by HPLC but this was found to not offer any advantage and, in general, the cr~ude reaction mixture was then allowed to react with protein in pH 8.5 bufer for two days in the~dark. Final separation of the conjugates from unreacted LTC4 or reagents was achieved by filtration on Sephadex G-50~ The derived coupled products now showed~W absorptions at 342 and 420 nm characteristic of 1,5-diaminodinitrobenzenes as well as the characteristic absorptions of the triene system at 271, 282, and 291 nm in the case of the LTC4 conjugates. In this manner, S-p-chloro- `
phenacylglutathione when reacted in 10:1 molar ratio with BSA gave a~con~ugat;e wlth~about 6~moles of hapten per mole of BSA.
~ ~ Simila~rly,~LTC4~1n 30 fold molar excess gave a conjugate with BSA with~9-10 moles LTC4 per mole BSA, and LTC4~in ca. 30 fold molar excess :

.

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(calculated per 100,000 daltons protein), gave a conjugate with KLH with 11-12 equivalents LTC4 per 100,000 daltons KLH.

Conjugation using 6-N-maleimidohexanoic acid chloride as c upling agent Since this invention provides a second LTC4 protein conjugate using a~different spacer group, a number of potential coupling methods were examined. A direct coupling using a reagent such as DCC or ECDI (6) was considered but quickly rejected due to the expectation that a heterogeneous mixture of adducts would be formed. Also, preliminary experiments indicated that the efficiency of such a~
coupling would be low. The known agents, toluene diisocyanate and m-maleimidobenzoyl-N-hydroxy-succinimide ester~were not used due to the possibility of immunological~cross reactivity with~ ~
respect to the spacer un~its between the two ~ ;
conjugatesO `~ ~ ~
The coupling agent 6-N-maleimidohexanoic acid ch;loride provide~s rapld,~selective~
functionalizati~on of~the~gl~utamyl~amino~group~of LTC4, as well as high coupling~eficiency.~
The agent chosen w~as 6-N-maleimidohexanoic acid~ch~loride~which was readlly~pre~pared~from~
6-amino:hexanolc~acld.~ 0ther~analogous~rea~g~ents ;~
having~from 2-~8~carbon atoms~in~ the;chain can~be used`, e.g~.~, 2-aminoace~tic ac~ up~to 8-amino octanoic ;
acid.
The 6-N-maleimidohmxanoic~acid~amide~of ~LTC4 wa~s~prepared~by~reac~ti~ng~à~methanolic solution :

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of LTC4 tripotassium salt with the reagent (1.5 equivalents in dry THF) in the presence of excess Et3N~ HPLC analysis showed essentially complete conversion to the amide (eluting before LTC4 on RP-HPLC). A portion of this adduct, isolated from HPLC, had W characteristics essentially unchanged from those of LTC4. For subsequent coupling with thiolated protein (KLH) the crude mixture (in pH 7.2 borate buffer) was used as such.
The thiolated protein used, in this case derived from KLH, was prepared by reaction with S-acetylmercaptosuccinic anhydride. As no report of thiolation of XLH could be found in the literature, trials were done to determine conditions for obtaining KLH with about 20 S-acetyl groups per 100,000 daltons protein [thio] content, after hydrolysis of the acetyl groups, was determined by Elleman's method. The S-acetylmercaptosuccinyl derivatized KLH was highly unstable to oxygen until further reacted with N-ethyl maleimide (NEM).
However, once any free SH groups were thus reacted, the material could be handled and purified by Sephadex~G-50 filtration.
Concentration of the resulting purified protein was accomplished by dialysis agalnst a packing of anhydrous Sephadex~G-200 resin. Just prior to coupling with derivatized LTC4, the thiol groaps were liberated by hydrolysis of the rigorously deoxygenated ~solution at pH 11.5 followed by reduction of the pH to 7.2.
This mixture was then reacted with the deoxygenated solution of the 6-N-maleimidohexanoic , ,: :

ra.~ s6 acid amide of LTC4 in a ratio o~ 80 equivalents LTC4 per 100,000 daltons RLH. After stabilization with NEM and purification by Sephadex~G-50, the protein conjugate showed 7-10 moles LTC4 per 100,000 daltons KLH by W analysis.
The protein solution has proven to be very stable during several months storage frozen at -78C.
More detailed examples follow. It is noted that IR spectra were recorded on a Perkin-Elmer 267 Grating Spectrophotometer. PMR spectra were recorded on a Varian EM-390 spectrometer. UV spectra were recorded on a Cary 210 spectrophotometer. Spectra were recorded in water unless otherwise indicated.
Sephadex~G-50 (medium grade) was obtained from Pharmacia Fine Chemicals.
Bov1ne Serum Albumin was obtained as crystallized and lyophilized grade from Sigma Chemical Co. and Hemocyanin (Keyhole Limpet) was obtained as lyophilized powder~from Calbiochem Behring Co~p. Lèukotriene C4 was synthetic material prepared in our laboratories using known procedures, Rokach et al~, Tet~. Lett., 21, 1485 tlg8o).

Preparation o~LTB4 conjugates is illustrated by the following reaction schemes:

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Scheme 1 HO_~ CO 2 Et H~ O~Ph II

H 2N/V\NH 2 ¢~

H

15 H = ~CON~\ H 2 1 ~ ~N~IIO~

HQ
~\CON~V\NH : ~ F

~/ ; ~( H2N-BSA ~:

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2298P/0832A ~ 16777IB
r H~CON~M~NH~BSA

L v 02N~\N0 This method makes use of the immediate synthetic precursor to LTB4, Ethyl 5(S)benzoyloxy-12(R)-hydroxy-6,14(2)-8,10(E)-eicosatetraenoate (II). We reasoned that reaction of II with a volatile diamine such as l,3-diaminopropane would at the same time remove the benzoate protecting group and convert the ethyl e~ster to the ~-aminopropyl-amide, all under mild weakly basic conditions. The solvents could then be removed under vacuum leaving only a mixture of the product (IIIj and N-~-amino-propylbenzamide. In model studies, using ethyl 5-(4-octylphenyl)-5-benzoyloxypentanoate this reaction was found to~be extremely sluggish,~even in neat 1,3-diaminopropane. ~However, when a oatalytic amount of 2-hydroxypyridine was~added to~the reaction mixture~,~the~diester;was~smoothly converted ~o the desired~aminoamide.~Wh~en~appl~ied~to the~ protec~ed ~;
LTB4 lII) a similar smooth con~version to III was effected~ could~be ~reacted~directly~in~the nex~
step, after~removal~of the~volatile components. The aminoamide~ was~réacted~with;excess~1,5-difluoro-~ 2,4~-dinitr~obenzene ~n~th~e~presen~e of t~rlethylamine 30 ~ to~ prov~ide the ~: ~.dduct~ IV~in hlgh yield. ~ This ~product was~purif~ied~by~reverse~pha~se~HPLC;and was~fully characte~rized~by W and~PMR~spectroscopyO~ Finally,~

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IV reacted smoothly with bovine serum albumin (BSA) (mole ratio-12:1) in a mixture of dimethylformamide and pH 8.5 borate buffer to provide the conjugate V
which was puriPied by chromatography on Sephadex G-50. UV spectral analysis indicated that the triene chromophore was unchanged and allowed the estimation that 5.5-8.3 moles of LTB4 were coupled per mole of BSA. (45-70% coupling efficiency).
The aminoamide III could also be prepared by direct reaction of LTB4 -lactone with 1,3-diaminopropane at room temperature. This provided III in quantitative yield free of side products.

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Sc}~eme 2 ~\C00 K ~, COOH
H~ CH ~ H' R ~ T . ~ ~3~ H0 Tl*,R. U~ 1 0 VI VII

~1, Et311 : ~

,, ~ :

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Another type of LTB4 conjugate could be pre-pared as illustrated in Scheme 2. Lactone VI reacted cleanly with hydrazine to provide the hydrazide (VII) in quantitative yield. VII was reacted further with 6-N-maleimidohexanoic acid chloride to give the diacyl hydrazide (VIII). This material could be purified by reverse phase HPLC to remove the excess reagent by-products. However, attempts to concentrate the product in order to obtain a PMR spectrum led to partial de-composition apparently due to hydration or methanolysisof the maleimide system. It was found however, that the crude reaction product could be used in the subse-~uent coupling reaction. VIII was reacted with thiol-ated KLH in a ratio of 50 moles of VIII per 100,000 daltons KLH, to provide the desired conjugate IX which was purified by filtration through Sephadex~ G-50. UV
analysis indicated that 12 equivalents of LTB4 were bound per 100,000 daltons of KLH.

Conjugates of LTC4 Using 1,5-Di~f~luoro-2,4-dinltro-benzene as Coupling Agent A. Conjugation of S-p-Chlorophenacylglutathlone and Bovine Serum Albumin (BSAj 1,5-Difluoro-2~,4-dinltrobenzene (120 mg, 0.59 mmol) in methanol (6 ml) was added~to a solution of S-p-chlorophenacylglutathione (88 mg, 0.19 mmol) in 9 mL of phosphate buffer (pH 7.2, 0 lN). Af;te~r stirring 12 hours at room temperature the me-thanol was~removed ln vacuo and~the~resulting aqueous solution was washed ~ith ether ~ Ihe q F~

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was chromatographed on C-18 Silica Gel (eluting with ~ethanol:water (1:1) to provide the pure adduct intermediate (105 mg). W: ~max ~) 260 (24,000), 347 nm (19,000). PMR (D2O): ~ 8.62 (lH, d, J = 7.5 Hz), 7.6 (2H, d, J = 9Hz, A of AB), 7.1 (2H, d, J =
9Hz, B of AB), 6.7 (lH, d, J = 15Hz), 3.9 (2H, s, phenacyl CH2).
The adduct (1.05 mg, 1.63 X 10 6 mol) in water (0.1 mL) was added to a solution of BSA (10 mg, 1.49 X 10 7 mol) in borate buffer (pH 8.5, 0.2 N, 1 mL). After standing in the dark at room temperature for 71 hours the solution was centrifuged and filtered on Sephadex~G-50 (1.5 X 75 cm) eluting with water. Fractions (10.5 mL) eluting after the void volume (55 mL) contained protein and were~analyzed by W. A sample of this solution diluted 5 times had a W spectrum (in H2O) ~max (Absorbance) 342 (0.359), 425 nm (0.133). Assuming 8 my of protein were recovered and assuming for the l,5-diamino-2,4-dinitrobenzene chromophor of ca. 27,000 (3) at 342 nm the W indicated 6 moles of S-~-chlorophenacyl-glutathione were conjugated per mole o~ BSA.

B. Conjug~ation of 1eukotriene~C4 and Bovine Serum Albumin~ ~
_ _ .
Leukotriene C4 (tripotassium salt)~ (2.5 mg) was dissolved in l mL of phosphate~buffer (pH 7.2, 0.1 N). 1,5-Difluoro-2,4-dinitrobenzene (l mgj~ in~
methanol ~0.6 m~) was;added and the mixture wa5 left 3t min. at~room~temper~ature~.~ The methanol waa removed under a~stream~of~ N2~and then in vacuo to : : .

~26~

remove final traces followed by extraction with ether (3 x 2 mL) to remove unreacted reagent. ~he last traces of ether were removed under N2 and in vacuo. To this mixture was added bovine serum albumin (BSA) (10 mg) in borate buffer (0.2M, pH 8.5, 1 mL), and the mixture was left to stand at room temperature in the dark for two days. The reac~ion mixture was filtered on a column of Sephadex G-50 (1.5 x 75 cm) eluting with water and the yellow protein eluting in 18 mL, after the void volume, of ca. 55 mL, was collected. At a~bout the 140 mL dead volume a peak considered to contain unreacted LTC4 eluted. Direct W analysis on the protein fractions (combined) gave a spectrum ~max (A) 271 (sh), 282 (3.57), 291, 342 (1.835) and 420 nm (0.91). Assuming about 9 mg of protein were recovered, and assuming for the 1,5-diamino 2,4-dinitrobenzene of about 27,000 at 340 nm and for~LTC4 at 280 nm of 40,000, calculations based on the 282 nm absorption about 10.0 mole of LTC4 per mole BSA while calculations based on the absorption of 342~nm indicated 9.1 moles LTC4 per mole BSA.

C. Conjugation of Leukotriene C4 and Hemocyanin ~from Giant Keyhole Limpets (KLH)~
~ Leukotrl~ene C4 (tripotassium salt) (~2.1 mg), and~l,5~-di~luoro-2,4-dinitrobenzene (8 mg)~ were reacted as in react~ion A. ~To the r~sultant~adduct was~added~KLN~;(15~mg~ borate buffer (pH a;.5, 0.2M, 0.8~3 mL)~-and~the~m1~xture~;was ~allowed to stand at room temperature 60 hours. At~this time a precipitate of :

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denatured KLH had formed which was removed by centrif-ugation ~6 mg, dry weight). The supernatant was filtered on Sephadex G-50 as before yielding a yellow protein fraction eluting in 17 mL following the void volume which by UV analysis indicated 11-12 equivalents of LTC4 per 100,000 daltons of KLH.

D. Conjugation of 2,4(E),6,9(7)-Pentadecatetraen-l-ol with BSA _ __ A solution of DFDNB (2.04 g, 10 mmol) in dioxane (20 mL) was added to L-proline (0.58 g, 5 mmol) in phosphate buffer (pH 7.5, 0.1 N, 5 mL) and the mixture was stirred 2 hours at room temperature. The mixture was reduced to dryness and the residue was chromatographed on silica gel (eluting with chloro-form:methanol (9:1) to yield W-2,4-dinitro-5-fluoro-phenylproline as a foam (1.1 g).
PMR (CDC13): &9.43 (lH, broad, exchanged by D2O, COOH),` 8.55 (lH, d, JH F = 7~.5 Hz, H-3 of phenyl), 6.62 (lH, d, JH F = 15 Hz, H-6 of phenyl), 4,5 (lH, broad t, J = 6Hz, proline methyne), 3.7-3.1 (2H, m), 2.7-1.9 ppm (4H, m).
To a mixture of 2,4(E),6,9(~)-pentadeca-tetraen-l-ol (123 mg, 0.56 mmol) and the proline deriv-ative above (170 mg, 0.5~7~mmol) in methylene chloride, at -10 C, were added successively,~l-cyclohexyl-3-(2-morpholinoethyl) carbodiimide methyl-p-toluene-sulphonate (266 mg, 0.63 mmol) and~pyrrolidlnopyridine (9 mg, 0.06 mmo1). The solution was~stirred under W2 at room temperature for 7 hours. ~The mixture was filtered and the flltrate was washed with water,~5 NaHCO3, brine and drled~over ; ;~

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22~8P/0832A - 18 - 16777IB

Na2SO4. The residue after concentration was chromatographed on silica gel teluting with chloroform:ethano] ~99.25:0.75)] to yield the pure adduct as an oil PMR (CDC13): ~ 8.57 (lH, d, J = 7.5 Hz), 6.55 (lH, d, J = 15 Hz), 6.7-5.2 ~8H, m, olefinic),

4,65 (2H, d, J = 6Hz, -CO~C~2-), 4.47 (1~, t~ J a 6Hz, proline methyne), 3.45 (2H, m), 2.95 (2H, m) 2.7-1.8 (6H, m), 1.5-1.2 (6H, m), 0.88 (3H, t).
W (dioxane: ~ max (~) 275 (48 "00), 347nm (18,450).
Anal. calcd for C26H32N3O6F: C, 62.26; H, 6.43;
N, 8.38; F, 3.79. Found: C, 61.88; H, 6.72; N, 8.48; F, 3.47.
A suspension of the adduct (5 mg, 1 X 10 5 mol) and BSA (10 mg, 1.5 X 10 7 mol) in dioxane (1 mL) and borate buffer (pH 8.5, 0.2M, 3 mL) was slowly stirred at room temperature for 4 days in the dark.
The mixture~was centrifuged:and the suspernatant was filtered on Sephadex~G-50 (1.5 X 7~5 cm),:eluting with water. The protein fraction eluti:ng in 7 ml after the void volume analyzed by W~for approximately 4 mol~s hapte~n per mole~of BSA.~ : ;

~ ~ ~ : EXAMPLE 2~
Conjugates of LTC4 Using 6~-N-Maleimidohexanoic Acid Chloride as CouPling:~Aqent A. Prepara:tion of 6;-N-Maleimidohexanoic ::; :
Acid~Chloride ~ 6-Amin~ohexanoic~acid (2 g, 0.02 mol) and maleic:anhydride ~(2 9,~ 0.~02:mol) were ref~lùxed togethe~r in~xyl~ene~:(20~mL~)~under a Dean-:Stark~wat~r~ :~
separator such that the internal temperature reaohed :

.

63L1551i;

ca. 165C. The mixture was cooled, diluted with chloroform-methanol and washed with lN hydrochloric acid. The organic layers were washed with water, dried, and reduced to dryness to yield a residue (1 g) which after chromatography on silica gel (eluting with 5% methanol-chloroform) provided pure 6-N-maleimidohexanoic acid, m.p. 84-85C.
IR(KBr): 3300-2500 (COOH), 1700 cm 1 (maleimide and COOH). PMR (CDC13): ~ 11.10 (lH, s, exchanged by D2O, COOH), 6.72 (2H, s, maleimide CH), 3.53 (2H, t, J = 7Hz), 2.34 (2H, t, J = 7Hz), 1.6 ppm (6H, m).
Mass spectrum: m/e 211 (M ).
Anal. Calcd for CloH13NO4: C, 56.87; H, 6.20;
N, 6.63. Found: C, 56.87; H, 6.24; N, 6.62.
6-N-Malelmidohexanoic acid (50 mg, 0.23 mmol) and a,~-dichloromethyl methyl ether (150 ~1, 1.5 mmol) were refluxed~together in anhydrous dichloromethane (1 mL) overnight. ;The mixture was reduced to dryness and the resultant highly hygroscopic solid~ (6-N-malelmldohexanolc acld chloride t54 mg) was used,~freshly prepared~, in the coupling reactions.
IR (fil~m):~1795 (COC~lj, 1700~cm l (maleimide).
PMR (CDC13): ~ 6.60 ~(2H~, s, maleimlde CH),~3.53 (2H, t, J = 7Hz), 2.90 (2H, t, J`= 7Hz), 1.6~ppm (6H, m).
~ : :
B. Reaction of~6-N-Ma~leimidohexanoic Acid Chloride with Leukotriene C~

~LTC4~tr;ipotas~sium~salt~5 mg)~ was~
~dlssolve~d~i~n;~anhydrous~methanol~(l;mL) and~
triethylamine (80 ~L) under~nitrogen and the acid . ~ :

~26~

chloride (25 ~L of a solution of 10 mg acid chloride in 100 ~L anhydrous THF) was added. The reaction was stirred at room temperature and was followed by HPLC
(Whatman Partisil M9 10/25 ODS, eluting with MeOH:H2O:HOAc; 70:30:0.01, 4 mL/min). The adduct eluted at 4.8 min. and LTC4 eluted at 6.6 min.
After 10 and 30 min. about 15% of unreacted LTC4 remained. More of the acid chloride solution (5 ~L) was added and after a further 10 min. 5% unreacted 10 LTC4 remained. The reaction mixture was concentrated to 0.2 mL under a stream of N2, diluted with borate buffer (pH 7.2, 0.IM, 0.5 mL) and the residual methanol was removed ln vacuo. This solution had UV spectrum essentially unchanged from LTC4 itself, and was used as such in reaction with thiolated KLH (see following).

C. Reaction of KLH with S-Acetylmercapt succinic Anhvdride KLH (60 mg) was dissolved in borate buffer (0.2M, pH 8, 1.5 mL) and centrifuged to remove denatured protein. The resultant solut~ion analyzed for 24.6 mg/mL by UV;[E278(mg/mL) = 1.36]. The solution was deoxygenated (by three purges alternating high vacuum and pure N2 flush) then treated under N2 with S-acetylmercaptosuccinic anhydride (45 mg~added in~5 mg portions over~one hour). The pH was maintained at 8 by addition of lN
~NaOH (total 400 ~L). Af~ter standing one ~our more, N-ethylmaleimide~(20 mg in 0.1 mL MeOH) w s added to bind any free thiol groups~and stabilize the;solution to air.~ After stand;ing l.5 hours more the solution : :

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~: :

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was centrifuged and applied to a column Sephadex G-50 tl.5 x 75 cm) eluting with 0.1N saline buffered with 0.01N pH 6.2 phospha~e buffer. Two fractions (7 mL) eluting after the void volume contained the bulk of the protein (2.4 mg/mL). An aliquot analyzed for thiol content, after hydrolysis at pH 11.5 for one hour, indicated 18 thiol groups per 100,000 daltons protein.

D. Coupling of 6-N-Maleimidohexanoic Acid Amide of LTC4 and Thiolated KLH
A solution of S-acetylmercaptosuccinate derivative of KLH (from reaction C) (10.8 mg, in 4.5 mL 0.1N salinej buffered to pH 6.2 with 0.01N
phosphate) was rigorously deoxygenated and then the pH was raised to 11.5 with lN NaOH (150 ~L) under N2 and the mixture was left at room temperature for one hour. The pH was then reduced to 7.2 by addition o~ deoxygenated lN HCl (150 ~L) and the~solution of the 6-N-maleimldohexanoic acid amide derivat~lve of LTC4 from reaction B~was added. After standing 2 hours at room temperature, N-ethylmaleimide (1 mg in 10 ~L methanol) was added and the mixture~was left one hour more at room temperature. This so~lution was appli~d to a Sephadex~G-50 column~(l.5~x 7~,5~cm) elu~ing with 0.lN saline~buffered~to pH 6 with 0.01N
phosphate. The protein fraction eluted with 85% in ll;mL after~he vold~volume. Unreacted r~agents eluted at the dead volume (150 mL). The protein i 30 ~solution~was ad~usted to pH 7.2 with lN NaOH ~:~fqr ::
storage. ~

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~26~35~

Analysis of the protein solution by W
indicated 7-10 equivalents of LTC4 were coupled per 100,000 daltons protein.
The conjugates of LTC4 with the proteins BSA and KLH have been used to raise antibodies with rabbits, at a dose of 200 ~g/rabbit, approximately weighing 1 kg; the antibodies specifically recognize Leukotrienes C4, D4, and E4. A detailed description of the antibody production, specificity, and the use of these conjugates in an immunoassay for the leukotrienes follows.
In addition to LTC4 and the specific proteins used, it will be appreciated that other leukotrienes, such as LTD4 and LTE4 can be conjugated with other antigenic proteins such as tetanus antigen, human se~um albumin (HSA), as well as diphtheriae toxide, tetanus antigen,~ and CRM 197 (from coryne bacterium diphtheriae) and other similar antigenic materials. ``

Immunization Usin~ LTC~g~lg~g~
The following ~iS~ thè~immunization reglme used employing KLH-maleimi;do-LTC4~as the immunogen.
Three 4 month old~New~Zealand White rabbits each received sub-cataneoas~injectlons at~mult1ple sites of 200 ~g KLH-LTC4 in complete Freunds~ ~
adjuvant~fôllowed~in~thre~e weeks by sub-cutaneous injections~at mult~iple~sites;with~lOO ~g~LKH-LTC4 3 ~ in~lncomplete~Freunds~adjuvant; . The~rabbits were bled lO~dàys aft~er~the second injection and every~
three~ weeks~thereafter. When~a~significant decline :

8~i~

in the level of antibody was observed, the animals were boosted with 200 ~g KLH-LTC4 in incomplete Freunds adjuvant and the animals bled again on the same schedule.
The antigen BSA-DNP-LTC4 was employed in a solid-phase-immuno-radioassay (SPIRA) in order to be used for the dectectîon of leukotrienes.
Polyvinyl chloride - 96 well microtiter plates (Dynatech Laboratories) were coated with antigen (BSA-DNP-LTC4) by incubating 100 ~1 aliquotes of the antigen at 0.1 mg protein/ml in PBS
for 18 h at 4C. The wells were washed three times with 200 ~1 PBS and then unreacted sites in the wells were blocked by incubating a 200 ~1 aliqout of 10%
horse serum in PBS in the wells for 2 h at 22C. The wells were then washed three times with 200 ~1 of PBS-1.5 H.S.(1.5% horse serum in PBS). One hundred (100)~1 of a reaction m`ixture containing a diiution of the immune or pre-immune rabbit serum was added to the wells and the pl~ates 1ncubated for~4 h~;at 2~2C.
The 100 ~I reaction employed for the ti~ration of rabbit serum consisted of~50 ~1 of dilutions~of the sera in PBS-1.5~H.S. and~50~ 1 bf~P~S-l.5~H.S.~;For competition~analysis~this~reaction~mixture consisted of 50 ~1 of a dilution of~immune serum in PBS-1.5 H.S. which contained a limiting amount~of leukotriene~
specific~antibody and;50 ~1~ of PB5~ .5~. H.S.~
containing various~c;oncent;rations of~leukotrienes or chemically~relat~ed~compounds.~ This 100 ~1 reaction mixture~was prei;ncubated l~h at 22C be~ore it wa.s added~to~the~well of;the~microtlter plate.~

:

: ~

The wells of the microtiter plate were then washed three times with 200 ~1 PBS-1.5 H.S. and then 100 ~1 of 125I-labeled rabbit anti-mouse [F (ab)2 fragments of rabbit anti-mouse IgG (H +
L)] in PBS containing 10% horse serum was added to the wells and the plates incubated 4 h at 22C.
Approximately 2 X 104 cpm of the iodinated reagent was added to each well. After the incubation period, the wells were washed five times with 200 ~1 PBS-1.5 H.S. and once with 200 ~1 PBS. The wells were then cut from the plate and the radioactivity in each well was determined in a gamma counter.
The advantage of this assay is that, although the rabbits were immunized with KLH-maleimido-LTC4, therefore antibodies are present in these animals against KLH, against the maleimido linker and against the hapten-LTC4, however, antibodies directed against the KLH and the maleimido linker do not cross react or bind to the BSA or DNP linker of the~material coated onto the the surface of~ the wells. Therefore the only antibodies that bind to the;material coated on the;wells~
~LTC4-DNP-BSA) are~directed against the LTC4.
These rabbit LTC4~antibodies bind~to;the LTC4 portion of the conjugate and~they~in turn are detected by adding a second species of antibody ~ `~
(125I-labeled goa~t ant~i rabbit ant~ibodiesj.~ Thqre~
a`ntibodies are radio-labeled with iodine and wilI
bind~to the rabbit~antibodies wh~ich in turn are bound 3~. to LTC4.~ The net~result is~ the~more antibodies directed against~LTC4,~the~;more~radloactivity associated~wlth the wel~
~::

~ ~ , .

. ' .

In order to determine if free LTC4 is in a biological sample, an aliquot is added to the plastic well. Some of this free LTC4 will bind to the rabbit anti LTC4 displacing if from the antigen coating which is bound to the surface. This results in a decrease in the number of counts (125I) bound to the surface of the well and by comparing this decrease to the decrease in a standard curve where known amounts of free LTC4 are added, the amount of LTC4 in the sample can be deter-mined.
The other compounds described in Examples land 2 can be similarly used in an assay system, as reagents.
The antisera produced in rabbits by immuni-zation with these conjugates can also be used in con-junction with radio-labelled leukotrienes C4, D4, or E4 as the basis of a radioimmunoassay for Leukotrienes C4, D4, and E4.
These conjugates are useful as chemical immunotherapeutic agents in the treatment various allergic and chronic inflammatory diseases of the~skin, lung, and airways,~ including asthma, allergic rhinitis, rheumatoid arthritis, and skin diseases such as pso-riasis and eczema.
~ LTC4 ANTIBODY ASSAY
In a standard Guinea Pig Ileum assay, 4 t~issues were set up ln 10~ml~baths~ of Kreb'~s~buffer with atropine and pyrilamine both~at 10 6M.
Standard~contractions were observed;using l0 ~l of 2.7 x l0 6M LTC4~solution in a 10 ml~bath, for a final eoneen~ra=l~r of~2.'~x l~0 9M LTC4~

:

:

~ ~ ,q ~.
s~

The standard response tension was 1.1-2. 0 grams.
20 ~1 of stock LTC4 solution was mixed with a varying amount (10 ~1, 40 ~1, 100 ~1 and 400 ~1) of anti-LTC4 serum (rabbit).
(1 ml of serum contained 7.9 x 10 9 M of specific anti-LTC4 antibody).
The serum was incubated on ice (in the dark) for 1/2 hour before use.
Control samples were run using similar~
amounts of normal rabbit serum.
The mixed samples (15 ~1, 30 ~1, 60 ~1 and 210 ~1 respecitvely) were added to baths and the response recorded.

~ RESULTS

Volume o~ Antlbody_Serum ~ % of Control Response 205;~ 100.0 20 ~ 72.7 50 ~ 92.3 200~ yl ` ~ ; 64.7 Volume`of Normal Serum 2Q ~ 85.7 ~ 50~ 102.~6 30 ~ 200~ ?' ~ :114.~3 , .
.

85~

From the above results, clearly the anti-LTC4 serum diminished the effect of LTC4 in each sample. Thus the conjugates can be used to raise antibodies to LTC4, LTD4, LTE4, in humans (in a manner similar to that employed herein in rabbits). The resulting circulating levels of antibodies would serve to diminish plasma levels of LTC4, and LTD4 and LTE4 released during an asthmatic anaphylactic re~ponse and thus serve to alleviate the symptoms. Since the antibodies would be present during long periods of time, this would represent a long term asthma therapy.

EXAMPLE 4_- Conju~ates of LTB4 Materials and Cond_tions PMR spectra were recorded on a Varian EM-390 or Bruker WM-4Q0 spectrometer. W spectra were recorded on a Cary 210 spectrophotometer. Optical rotations were measured using a Perkin Elmer Model 241 Polarimeter. Sephadex G-S0~tmedium grade) was obtained from Pharmacia Fine Chemicals.
Bovine serum albumin was obtained as ;
crystallized~and lyophilized~grade from Sigma Chemical Co. and Hemocyanin (Keyhole Limpet)~ was obtained as lyophilized powder from Calbiochem Behring Corp. ;

1. Conjugatlon of LTB4 with Bovine Serum Albumin A. 5(S),12(R)-dihydroxy-6,14(Z)-8,10(Ej-eicosatetra-eonic acid S;-1actone (LTB4 -lactone) (VI): ~ ~
~ 5(S),12(R)-dihydroxy-6,14(Z)-8,10(E)-eico satetraenoa~te (12 mg) was stirred under nitrogen in ::

: :~: :: :

:

methanol (1.5 mL) and water (0.4 mL) with postassium carbonate (22 mg) for 2.5 days at ambient temperature. Most of the methanol was removed under a stream of nitrogen (to leave about 0.4 mL volume) and the mixture was diluted with O.lN pH 6.2 phosphate buffer (2.5 mL). The mixture was extracted with ether (5 x 2 mL) and the combined ether extracts were dried (Na2SO4) and reduced to dryness. UV
analysis of the resulting oil indicated that 8 mg of LTB4 free acid was thus obtained. The oil was dissolved in anhydrous ether (5 mL) and treated with dicyclohexylcarbodiimide (DCC) (20 mg) at 0 under nitrogen for 24 hours. TLC analysis (ethyl acetate:
hexane 2:3) indicated about 50~ conversion of LTB4 to the ~-lactone (Rf LTB4 = 0, Rf LTB4 lactone = 0.6). More DCC (30 mg) was added and after 2 days at 0 TLC indicated essentially complete conversion to the ~-lactone. The mixture was concentrated to 1 mL under N2, filtered, reduced to dryness, taken up in ethyl acetate:hexane (2:3) (1 mL) and chromatographed on silica gel column (10 g) eluting with the same solvent ot provide the ~-lactone contaminated with a small amount of dicyclohexylurea. This material was further purified by HPLC (Wa~ers 10~, ~-porasil; ethylacetate:hexane;
(1:2); 4 mL~min) to provide~the pure LTb4r-lactone ~VI) eluting at 5.7 min (603 mg, 77~ yield~from II).
The lactone crystallized as fine needles from ether:hexàne~, mp 50.0~_50.5 ~]RT~= ~231.0 (C=0.3,~CHC13) W: ~max (e) (MeOH) 260 (37,20Q), 270 (5Q,Q00), 280 nm (39~400). S PMR (400 MHz) (CDC13):
0.87 (3H, t), 1~.2-1.4 (6H, m), 1.65 (2H, m), 1.93 : :~: :
:
:: : :~ :
:

.

5~i (2H, m), 2.03 (2H, q, CH2, C-16), 2.32 ~2H, m, CH, C-13), 2.48 (lH, dt, J=17.5, 7 Hz, one of CH2, C-2), 2.62 (lH, dt, J=18, 5 Hz, one of CH2, C-2), 4.22 (lH, m, methine, C-12), 5.23 (lH, dt, J=10~5, 2Hz, methine, C-5), 5.35 (lH, dd), 5.45 (lH, t), 5.58 (lH, dd), 5.81 (lH, dd), 6.15 (lH, t), 6.29 (2H, m), 6.41 (lH, dd).

B. N-(3-aminopropyl)-5-(S),12(R)-dihydroxy-6,14(Z)-8,10(Ej-eicosatetraenoic acid amide (III):
Method 1. LTB4 ~-lactone (VI) 1.75 mg) was dissolued in redistilled 1,3-diaminopropane (0.5 mL) and the mixture was left at room temperature for 18 hours. The excess diaminopropane was removed under high vacuum to give the amide III, quantitative yield, la~RT = -2 (C=0.17, CHC13).
W: ~max (~)~(MeOH) 259.5 (29,800) 269.5 (46,500), 280 (36,500). o PMR (400 MHz): ~ 2.03 (2H, q,~CH2 C-16), 2.21 (2H, t, -CH2-CONH-), 2~.31~(2H,-m, CH2, C-13) r~ 2.76 (2H,~t, -CH2-NH2)~ 3.33 (2H~,~
q, -CONH-CH2-),~4~20 (lH, q,~ methine, C-12)~, 4.58 (lH, q, methine, C-5j,~5.3-5.43 (2H, m),~5.5S (lH, dd)`, 5.78 (~lH, dd)~, 6.05~(1H, ~t~,~6.18-6.31 ~(2H, m),

6.36 (lH, broad NH, amide), 6.47 (lH,-~dd).

~ Method 2. Ethyl 5(5)~-be~n20yloxy-12(R)-hydroxy-6,1g~(Z)-8~,10(E)-eicosatetraenoate (2.5~mg) and~2-hydroxypyrid~ine~(1.5~ mg)~were dissolved in 1,~3-diaminopropane (0.~5 mL)~and~the~mixture~was left ~ ~
30 àt room;~temperature,~under~nitrogen, for 3~days. The ~;
` excess~diaminopropane was~removed~under hlgh~vacuum ~ ~

: `

: ~ :
:: :: :
:

: :

85il~

at room temperature to provide crude III which was used as such in the next reaction tw: ~ max 227, 260, 270, 280, 298 mm).

C. N-(3-[2,4-dinitro-5-fluorophenyl]aminopropyl)-5(S), 12(R~-dihydroxy-6,14 (Z)-8,10(E)-eicosatetraenoic acid amide (IV)-, _ . . .......... . _.. _ __ .. .
The crude amino amide (III) from Step B, Method 2 (2 mg) in anhydrous methanol (400 ~) and triethylamine (8 ~1) was treated with 1,5-difluoro-2,4-dinitrobenzene (4 mg) in methanol 200 ~1) at room temperature for 15 minutes at which time reverse phase TLC (RPTLC) (acetonitrile:water, 85:15:) indicated complete~reaction of III (Rf=0.1) and the appearance of a new yellow product (Rf=0.7). The mixture was chromatographed on RPHPLC (Waters, 10 ~
~bondapak, C-18, acetonitrile:water, 70:30, l mL/min) to provide the product IV~(1.8 mg) [a]RDT -18~.9 (C=0.37, MeOH).
W: ~ max (MeOH) 260, 270~ 280, 335, 380 (sh). ~PMR
(400 MHz) (acetone-d6:~ r 3.34 (2H,~q,~-CONH~-CH2 3.61 (2H, m, -CH~2-)~, 3.61 ~(2H, m, -CH2-NH-Ar), ~
3.84 (2H, m, 2-OH), 4.14 (lH, m, methine,~C-12) 4.58 (lH, m,~methine, C 5)~, 5.42~(3H,; m), 5.78 ~(~lH, dd, J=14, 6Hz, H-ll), 6.00 (lH,~t, J=llHz,~H-7j, 6.21 (lH, dd, J=14, llHz, H-10), 6.30~(1H, dd~,~J=14, llHz, H-9), 6.57 (lH, dd,;J-`14, llHz, H-8)~,~7.15~(1H,~d,~
JHF=15Hz), 7.27 tlH, broad,~ NH~ amide), 9.00 (lH, d, JH, F=8Hz),~9~.~15 (1H, broad,~NH, amine).

, .
~: .

85~

D. Coupling of compound IV with Bovine Serum Albumin (BSA) A solution of compound IV (from step C) (1.5 mg) in dimethylformamide (0.5 mL) was added to a solution of BSA (15 mg) in 0.2N pH 8.5 borate buffer (0.75 mL) and the mixture was allowed to stand in the dark under nitrogen and at room temperature for 4 days. The mixture was centrifuged and the clear supernatant was applied to a column of Sephadex~G-50 (1.5 x 75 cm) eluting with water. The yellow protein fraction eluted cleanly in 20 mL, after the void volume of about 55 mL. At about the 140 mL dead volume a peak containing unreacted IV and byproducts eluted. W analysis of the protein frac~ions gave a spectrum max 266, (sH), 273, 283, 336, 420 nn.
Assuming 100~ recovery of BSA from the column, calculations based on the peak at 273 nm, corrqcting for contributions due to BSA and to the dinitrobenzene chromophore, indicated that 5~.5 moles of L?B4 were coupled per mole of BSA. The absorption at 336 nm (assuming~ for~the ~
1,5-diamin~-2,4-dinitrobenzene~chromophor o~ about 27,000) indicated that~8.3 moles of LT84 were coupled per mole BSA.
2. Con~ugation of LTB4 with Hemocyanin from~
Kevhole Limpets;(KLH) ;~
LT84 ~-la~tone (~I) (4 mg) was disso~lved in a mixture of THF (l mL) and 99~ hydrazine~hydrate ~0c5 mL) and the mixtui~ was~stirred vigorously under nitrogen~at room~-temperature~for 0.5 hours.~The mixture was~estra~cted with ether ~3 x 2 m1)~ and the :: : : : ~ : : ~ : :

:: : : :~ :: : :

359C~

combined organic layers were dried (Na2SO4) and evaporated to dryness under a stream of nitrogen and then ln vacuo to provide the hydrazide VII ~4.2 mg).
~]D =8.9 (C=0.28, MeOH). UV ~max (~)=260 (37,000) 269.5 (50,000), 280 (39,000). PMR (400 MHz) acetone-d6): ~ 2.1 (2H, t), 2.27 (2H, m), 3.82 (lH, m, NH2), 3.99 (lH, broad NH2), 4.14 (lH, m, methine, C-12), 4.56 (lH, m, methine, C-5), 5.3-5.5 (3H, m), 5.77 (lH, dd, J=14, 6Hz, H-ll), 6.00 (lH, t, J=llHz), 6.22 (lH, dd, J=14, llHz, H-10), 6.31 ~lH, dd, J=14, llHz, H-9), 6.56 (lH, dd, J=14, 11 Hz, H-8), 8.22 (lH, broad, -CO-NH-).

B. Reaction of LTB4 hydrazide (VII) with 6-N-Maleimidohexanoic acid chloride-LTB4 hydrazine (VII) 2.5 mg, 7 x 10 moles), in anhydrous methanol (l mL) and triethylamine (20 ~LI was treated with a solution of 6-N-maleimidohexanoic a~id~chloride (8j (3.3 mg, 1.4 x 10 5 moles) in~ anhydrous~ THF~(10~0 ~L) under nitrogen at room~temper~ature. TLC analysis (chloroform~methanol~, 8~5~:15)~ indicated~complete conversion~to~a less~polar product,~ The mixture~was reduced to~dryness,~and the~residue was taken up in deoxygénated methanol~(l.2~mL)~and used as such in the next reac~tion. Th~e prodùct;could~be purified if ;
desired~by~reverse~phase HPLC ~ (Waters~10~, ~-Bondapak~
C-18; methanol:water; 75:25,~2mL/min), to give the ~
pure addu~ct VIII~elu~ting at~4.~5 min. W ~max ~(MeOH) ~ 260~(36,300),~270 ~(50,~000)~, 280.5 nm~(39,400).
On~concent~rat;ion~to~obtain PMR;spectra some decompositlon~was~;noted;by~TLC. NoweVer~the spectrum :
::
: :

: ~ ' :
:

(400 MHz) (acetone d6) contained a weak signal at 6.82 ppm indicating that the malemide unit was present although partially reacted.

C. Coupling of Compound VIII with Thiolated KLH:
S-Acetylmercaptosuccinylated KLH was prepared as previously described (8~. The derivatized protein (KLHSAc) (10 mg) in 0.1 N Saline buffered with 0.01 N pH 6.2 phosphate buffer (PBS) (5 mL) was rigorously deoxygenated; then the pH was raised to llo S by addition of 0. lN NaOH. After standing 1 hour nitrogen at room temperature, the pH
was reduced to 7.~2 by addition of 0.1N HCl. The adduct VIII in methanol (1.2 mL) from reaction B
above, was added~and the mixture was stirred sIowly under nitrogen for 18 hours. N-ethylmalemide (5 mg) in methanol (0.1 mL) was added and the mixture was stirred 1 hour more~ The methanol was~removed under a stream of~nitrogen during l hour, the~mixtu~re was~
centrifuged and the sapernatant was fil~ered on ~
Sephadex G-50 eluting with pH 6.2 PBS. The~protein eluted with 95% in 19 ml after the void~volume and gave a~ W spectrum: ~max ~264 (sH),~273.5,~283.5 nm.
Assuming 9 mg of protein was recovered from the 25 - column and correcting the absorption at 273.5 nm for contributions due to coupled per 100,000 daltons KLH.

`

::

::

:i : : :

Claims (5)

WHAT IS CLAIMED IS:

1. A compound of the formula:

or wherein n is 0 to 10, and the lactones form thereof.

2. A compound according to claim 1, wherein n is 0 or 2 to 10.

3. A compound according to claim 2, wherein n is 3.

4. A compound according to Claim 1, said compound selected from:

5(S),12(R)-dihydroxy-6,14(Z)-8,10(E)-eicosatetraeonic.
acid ?-lactone;
5(S),12(R)-5,12-dihydroxy-6,14(Z),8,10,(E)eicosapena-eonic acid hydrazide;
5(S),12(R)-N'-(.alpha.-oxo-zeta-(2',5'-di-oxo-2',5'-dihydro-pyrrolo)hexane)-5,12-dihydroxy-6,14(Z),8,10(E)-eicosa-tetraenoic acid hydrazide;
N-(3-aminopropyl)-5-(S),12(R)-dihydroxy-6,14(Z)-8,10 (E)-eicosatetraenoic acid amide; and N-(3-[2,4-dinitro-5-fluorophenyl]aminopropyl)-5(S),12-(R)-dihydroxy-6,14(Z)-8,10(E)-eicosatetraenoic acid amide.