Note: Descriptions are shown in the official language in which they were submitted.
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- FIELD OF THE INVE~TION:
The present invention relates to cyclosporin derivatives and
derivative conjugates formed therefrom, as well as to methods useful in
the production of such derivatives and conjugates.
BACXGROUND OF THE INVENTION:
Cyclosporins are produced as secondary metabolites of various fungi
strain~, s~ch as Cylindrocarpon lucidum Booth; and, ~richoderma
polysporum (~ink ex Pers. ) Rifai - aka Tolypocladium in~latum Gams - aka
Beauvaria nivea. Some totally synthetic pathways are also known.
A background on the discovery and early developments relating to
cyclosporins is elaborated in Borel, J., (1983) Cyclosporin: HistoricaL
Perspectives, Transplant Proc. 15, Suppl. 1, 2230 - 2241.
The cyclosporins are biologically active monocyclic peptides and
are variously useful as antibiotics and immunosuppressants (particularly
in human organ transplant applications). The compounds are also
undergoing testing in the treatment of juvenile diabetes and other auto-
immune diseases. See also Stiller, C., and Keown, P., (1984)
CyclosPorin Therapy in Perspective, Progress in Transplantation (Morris
and Tilney, Eds.) pp 11 - 45, Churchill Livingston Publishers, London.
Cyclosporin A is a white amorphous powder (when in amorphous form),
~H,~ Cl~!
C~ f`H-C~
U--
r~--n~- ~....o~~
Cll--CH~--1`1 C~--CII
1."
Cyclosporin A
having a melting point range of 148 to 151 degrees C, an empirical
formula of C62H~l~N"O~, and an elementary analysis as follows: C 61.8%;
H 9.4%; N 13.0%; and, O 15.7%. Cyclosporin A is characterized in
greater detail in US patent 4,215,199, and in even greater detail still
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in the following articles: A. Ruegger, M. Kuhn, H. Lichti, H.R. Loosli,
R. Hugenin, C. Quiquerez, and A. von Wartburg, HELV. CHIM. ACTA., 1976,
Vol. 59; and, TJ Petcher, HP. Weber and A. Ruegger, HELV. CHIM. ACTA.,
1976 Vol. 59; and, M. Dreyfuss, E. Harri, H. Hofmann, H. Kobel, W.
Pasche and H. Tscherter, EUROPEAN J. OF APPL. MICROBIOLOGY, 1976.
Cyclosporin B has also been extensively characterized. It is also
a white amorphous powder (when in the amorphous form), but has a melting
point range of 127 degrees to 130 degrees C. This polypeptide has an
~1 .~ ;,................................. .
J ~CII~
cv~ ~ c~.
~ H C-l--rll~--~H
, , x--cll,
C~
IN--1~""" L ~H~
tH~ ~ N Cll!
' ~n~ fH~ c_
~n~ L t. ~ c-~.
CH! ~ .... OI~C
~CII--CI-~--Cil~ CO--iHl
Cycloæporin B
elementary analysis as follows: C 61.7%; H 9.1%; N 13.1%; and, O 16.5%.
Its empirical formula is C61Hl09N1112.
In general, however, cyclosporins are cyclic, eleven-amino-acid,
peptides containing several unique amino acids. They are highly
methylated, non-polar and highly hydrophobic compounds.
Only a relatively few synthetic schemes for modification of these
cyclosporins are known. See for example: Wenger, R., Traber, R.P.,
Robel, H., and Hofmann, H., (1985) Cyclosporin Derivatives and Their Use
- French Patent 561,651. This approach involved in vivo amino acid
substitution.
Also see: Wenger, R., (1986) Synthesis of Cyclosporin and Analogs:
Structural and Conformational Requirements for Immuno-suppressive
Activity - Prog. Allergy 38, 46 - 64; Wenger, R., (1983) Synthesis of
Cyclosporin and Analoaues: Structure and Activity Relationships of New
Cyclosporin Derivatives - Transplant. Proc. 15, Suppl.1, 2230 - 2241;
and, Wenger, R., (1982) Chemistry of Cyclosporin, Cyclosporin A -
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.. ...
Proceedings of the International Conference (D. White, Ed.) pp l9 - 34,
Elsevier Biomedical Press, Amsterdam, ~etherlands. The approaches set
out in these publications involve the total synthesis of cyclosporin,
starting from tartaric acid.
Also refer to European patent 0283 801, entitled Fluorescence
Polarization Assay for Cyclosporin A and Metabolites and Related
Immunogens and Antibodies, 1988. In this approach, a low-yield
oxidation of cyclosporin is used to produce a derivative which contains
a reactive aldehyde moiety.
The cyclosporins that are of interest here, are essentially
undecapeptides which contain only two chemically reactive sites that can
be modified without collateral destruction of the amide bonds within the
cyclic peptide. Both of these functional groups, (the hydroxyl group
and the double bond) are located on the 2-N-methyl-(R)-((E)-2-butenyl)-
4-methyl-L-Threonine residue. In cyclosporins A and B, the chemical
reactivity of both of these groups is diminished by steric factors
arising from the three-dimensional structure of these cyclosporins in
aqueous solutions. Nevertheless, a few successful attempts have been
made in effecting various modifications at these sites. The essentials
are set out in Traber, R., Loosli, H., Hoffman, H., Kuhn, M., and von
Wartburg, A., (1982) Isolatlon and Structure Determination of New
Cyclosporins E F G H, and I, Helv. Chim. Acta 65 Fasc 5, 1655 - 1677
In Swiss Patentschrift 637,123, for example, the 2-N-methyl-(R)-
((E)-2-butenyl)-4-methyl-L-Threonine residue is modified to produce a
a~ ~
dS-~''r~ ~ ' ~ L
Cyclosporin F
cyclosporin derivative, called Cyclosporin F, substantially as shown.
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Another exemplary approach to modifying the 2-N-methyl-(R)-((E)-2-
butenyl)-4-methyl-L-Threonine residue is described in German
Offenlegungshchrift 28 19 094, in which a derivative family known as
various Cyclosporin D compounds are illustrated.
3~c~l CHl C~
h~ ~112
~cF~ ~cn3 Cl.11 ~C11~ /D~,C~
--C~ cn--~C--I--C~ C11--
Cyclosporin D Dihydro-cyclosporin Iso-cyclosporin D
D
Yet another approach to modifying the 2-N-methyl-(R)-((E)-2-butenyl)-4-
methyl-L-Threonine residue is described in German
Offenlegungshchrift 29 41 080, in which a derivative family known as
various Cyclosporin G compounds are illustrated. These "G" cyclosporins
are the complementary forms,( ie cyclosporin; isocyclosporin; and,
dihydrocyclosporin) of the above illustrated "D" cyclosporin family.
The modification that yields the "iso'~ and "dihydro" forms from the root
cyclosporin molecule are essentially the same in the case of both
families. The close similarity of the root cyclosporin and the
respective complementary modifications will be readily apparent from the
following illustration of the cyclosporin "G" family:
C~ ~h e~3 ~H C~1~C~
C~ ,C~c"
~ C~ ~C~ 111 ~C~ / l",C~C I~C~
_c_~. C~ C~ c l~ C~---
Cyclosporin G Dihydrocyclosporin G Isocyclosporin G
This limited number of successful modifications has been confined
to structurally similar root compounds, and follows very similar and
limited forms of modification. Moreover, the few successes have not met
the needs of the art. Accordingly,
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.`
there continues to be an unsatisfied need for additional and alternate
derivatives, to afford a wider variety of reactive functional groups,
especially in the production of cyclosporin conjugants. -~
In earlier work aimed at producing alternate derivatives, it was
decided that based on the previous, and above referenced work of Traber,
R., Loosli, H., Hoffman, H., Kuhn, M., and von Wartburg, A., (1982)
Isolation and Structure Determination of New Cyclosporins E, F, G, H,
and I, Helv. Chim. Acta 65 Fasc 5, 1655 - 1677, an attempt would be made
to react the hydroxyl group of the 2-N-methyl-(R)-((E)-2-butenyl)-4-
methyl-L-Threonine residue with small carboxylic acid chlorides
containing terminal primary bromide groups ( such as 4-bromobutyric acid
chloride, or 3-bromopropionic acid chloride), with a view to
incoxporating a new fundamental reactive group on the cyclosporin,
through esterificatisn. Notwithstanding the success that had been
enjoyed in the previous work referenced above, (in the production of the
other cyclosporin derivatives through reacting the hydroxyl group with
acetic anhydride to produce o-acetyl-cyclosporin derivatives~, the
carboxylic acid chloride reactions were not successful. This
conventional approach therefore failed to meet the extant need for new
derivative6.
SUMMARY OF THE INVENTION:
In accordance with the present invention, however, there have now
been produced, cyclosporin derivatives and derivative conjugates useful,
inter alia, in fluorescence and immunological studies. The invention
also extends to methods for the production of cyclosporin derivatives
and derivative conjugates.
Accordingly, in one broad aspect of the present invention, there is
provided a acid derivative of cyclosporin, and a method for the
production of same. More -particularly, a cyclosporin derivative is
provided in which the double bond in the 2-N-methyl-(R)-((E)-2-butenyl)-
4-methyl-L-Threonine residue of the cyclosporin molecule is selectively
oxidatively cleaved to produce a carboxylic acid derivative of
cyclosporin. This acid derivative of cyclosporin is a useful
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intermediate in the production of cyclosporin conjugates. Note however,
that with the formation of the acid derivative, there is also formed a
corresponding lactone derivative of the cyclosporin. The relative
concentrations of the two derivative species greatly favours the
lactone, at equiLibrium. However, there is a possibility that the
carboxylic acid derivative may participate in subsequent secondary
reactions.
In accordance with yet another aspect of the present invention,
there is provided an alternative derivation scheme in which cyclosporin
is reacted in known manner to produce an initial O-acetyl cyclosporin
intermediate. This derivative is then selectively oxidized in the same
manner used to produce the above mentioned lactone derivative. Since
the O-acetyl cyclosporin intermediate is incapable of forming a lactone
structure under such conditions, there is formed instead, an O-acetyl
acid derivative of cyclosporin, (hereinafter referred to as O-acetyl
cyclosporic acid). This derivative is not susceptible to the carboxylic
acid equilibrium issues that arise in connection with the last above
mentioned, non-acetylated cyclosporin acid derivative, and hence, is in
general a preferred intermediate for use in the synthesis of cyclosporin
conjugates both from the yield and side products formation points of
view.
The present invention relates generally to the production of
nucleophile-conjugates of cyclosporins, and in particular amino-
conjugates thereof. More specifically, there is provided a process for
the production of amino-(O-acetyl-cyclosporic acid) conjugates, and
exemplary amino-conjugate products thereof, including conjugates and
conjugate complexes based on polyamino nucleophilic r~agents.
In particular, the present invention relates to a method for
producing cyclic cyclosporin-derivatives comprising oxidizing a
cyclosporinhavinga2-N-methyl-(R)-((E)-2-butenyl)-4-methyl-L-threonine
side chain, with or without an O-acetyl group. The method is carried
out under cyclic peptide structure preserving conditions and results in
the production of corresponding derivatives of the group consisting of
O-acetyl cyclosporic acid; and, cyclosporin acid, respectively.
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Preferably the oxidation step is carried out using a
periodate/permanganese oxidizer system, and in particular as an aqueous
solution containing Co~2 ions (from K2CO3); IO4 ions (from NaIO4); and,
MnO4 ions(from KMnO4). That solution is admixed with an organic solvent
solution of the selected cyclosporin, under periodate/permanganese,
cyclosporin-side-chain-oxidizing conditions. A preferred organic
solvent for the selected cyclosporin is tert-butyl alcohol solution.
In accordance with yet another aspect of the present invention,
there is provided a method for producing cyclosporin-derivative
conjugates comprising the steps of reacting O-acetyl cyclosporic acid
with a reactive nucleophile conjugant, in the presence of water and a
reagent operable to reactively modify the terminal carboxylic acid group
of the O-acetyl cyclosporic acid. That modification results in the
formation of a reactive acyl group in aqueous solutions, which then
reacts with the nucleophile, under cyclosporin cyclic peptide structure
preserving conditions, to thereby produce a cyclosporin-nucleophile
conjugate linked through the resulting acyl group on the of the O-acetyl
cyclosporic acid molecule. An exemplary reagent for this purpose is 1-
ethyl-3-[3-(dimethylamino)propyl]carbodiimide.
Preferred nucleophiles are primary amines, such as poly-(L-lysine).
Another preferred nucleophile is 5-(aminoacetamido)fluorescein.
In an especially preferred method according to the present
invention, the nucleophilic conjugant is a poly-nucleophilic conjugant,
such as the aforementioned poly(L-lysine). Such polynucleophiles (ie.
a molecule having a plurality of sterically available nucleophilic
reactive sites), make it possible to utilize a fluorescent, O-acetyl
cyclosporic acid-competitive-binding, carboxylic acid reagent, which is
co-reacted with the poly-nucleophilic conjugant and acyl-modified O-
acetyl cyclosporic acid to produce a mixed poly-conjugant compound. The
cyclosporin and fluorescent reagent residues are therein linked to the
poly-nucleophilic conjugant through respective acyl residues
corresponding to the carboxylic acid groups. An exemplary fluorescent
carboxylic acid reagent for these purposes is 2-~N-methyl-N-(7-
nitrobenz-2-oxa-1,3-diazo-~-yl)amino~ethanoic acid.
2086267
DETAILED DESCRIPTION OF THE INVENTION:
In accordance with particularly preferred aspects of the present
invention, there is provided a novel cyclosporin derivative comprising
cyclosporin lactone having a general formula as follows:
O O
cS r
wherein a acid residue depends from the site marked with the "X" on a
preferred cyclosporin root "Cs", of the general formula:
N~--N;~
NH
0
_4_o ~I~N~
NUh (S=~
0
:,,
Note that for the purposes of this disclosure and the claims, the
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abbreviation "Cs" is used to represent a generic form of an intact
cyclosporin ring structure, which includes the expanded structure that
is illustrated above. In those particular cases where the
abbreviation is styled as l'CsA" it is so used to illustrate the use of
cyclosporin A, in particular.
The above depicted cyclosporin acid was prepared by way of a
modification of the periodate/permanganese oxidation reaction that is
sometimes employed in the cleavage of unsaturated fatty acid
molecules. In a typical preparation, 490 ~L of 0.15 M aqueous K2CO3
(73.5 ~moles) and 490 ~L of 0.20 M aqueous NaIO4
(98.0 ~moles) were added to a solution of 14.7 mg of cyclosporin A
(that was obtained from Sandoz) dispersed as a 12.2 ~molar solution in
1.48 mL of tert-butyl alcohol. Deionized, distilled water was added
dropwise until all NaIO4 had dissolved, then 98.0 ~L of 0.025 M
aqueous KMnO4 (2.45 ~molar) was added. The solution was stirred at
room temperature under a gaseous nitrogen atmosphere for 14 hours, and
then an additional 98 ~L of 0.025 M aqueous KMnO4was added to the
reaction mixture.
Stirring at room temperature under gaseous nitrogen was continued
for an additional six hours to bring the total reaction time to twenty
hours, by which time the solution had taken on a brownish pink
coloration. The reaction pathway is illustrated in schematic
structural form by the following:
OH ,
c.
~
C~AJ~OH _ Cs~
2 3
The reaction was stopped through the addition of 0.147 mL of
freshly prepared 40% (w/v) aqueous Na2S2O5 solution (309 ~moles), 0.250
mL of 1.0 M H2SO4(250 ~molar), and 2.5 mL of deionized, distilled
water. The mixture was stirred for ten minutes and then the aqueous
2086267
solution was extracted using three successive aliquots of 40 mL
diethyl ether.
The solvent was removed from the ether extracts under reduced
pressure, and then the dry product was dissolved in 2 mL of me~hanol
and loaded on 20 grams of Sephadex LH-20 gel filtration media (from
Pharmacia) which had been earlier swollen in methanol, in known
manner.
Separation ensued, and the resulting fractions were analyzed by
TLC (thin layer chromatography). The fractions containing the first
compound to elute from the gel filtration column were collected.
Removal of solvent under reduced pressure furnished 12.2 mg (84%) of a
white solid product, later identified as cyclosporin A acid.
The spectral properties of the product are summarized as follows:
I. 1H NMR (CDCl~L:
NH groups - sigma 8.42, 7.92, 7.50, 7.46, (all d,
each lH); and,
NCH3 groups - 3.46, 3.39, 3.17, 3.05, 2.66, 2.64,
(all s, each 3H);
II. 13C NMR (CDCl~L:
CH2 carbons - delta 49.92, 40.57, 39.05, 35.20,
36.51, 35.90, 35.20, 25.14;
CHOR - 82.77;
CH=CH carbons - 126.72, 130.14;
III. IR.: Carbonyl st~etch (acid), 1787 cm~~;
IV. MS.: m/e expected 1188, m/e found 1188, (M+).
In accordance with another aspect of the present invention, there
is provided a cyclosporin derivative amino-conjugate (for ease of
reference herein designated as compound 8), having the formula:
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1, `
~,,au
a, ~N ~,~IH
wherein an aminoacetamido-fluoroscein residue is linked through a O-
acetyl residue that depends from the site marked with the "Xi' on a
cyclosporin root "Cæ" of the general formula illustrated above.
The preparation of the last above illustrated compound commenced with
the preparation of O-acetyl-cyclosporin, generally in accordance with
the method set out in Traber et al, (1982), Isolation and Structure
Determination of New Cyclosporins E, F, G, H and I, Helv. Chim. Acta
65, Fasc S, 1655 - 1677. A spectrographic analysis, set out below,
confirmed the molecular identity of the prepared compound as O-acetyl-
cyclosporin:
I. 1H NMR (CDCl~L:
NH groups - delta 8.48, 7.97, 7.43, 7.38, (all d,
each 2H); and,
NCH3 groups - 3.38, 3.18, 3.16, 3.13, 2.60, 2.58,
(all s, each 3H);
OC(O)CH3 - 1.93, (s, 3H~
II. 13C NMR (CDCl~L:
CH2 carbons - delta 49.82, 40.79, 37.95, 36.79,
35.61, 33.48, 24.74;
HOC(O)CH3 - 168.21;
CH=CH carbons - 129.21, 126.35;
III. IR.: Carbonyl stretch ( O-acetyl ester), 1746 cm1, (in
2~86267
addition to a strong amide stretch at 1628 cm');
IV. MS.: m/e (FAB) expected 1245, m/e found 1245; (M+).
O-acetyl-cyclosporin was then subjected to generally the same
treatment that had been earlier-used to produce the cyclosporin acid,
as described hereinabove. The two steps of the process to this point
are illustrated in the following structural schematic: ~.
OH
C~
of~ Q,
C~A~ CsA~OI
4 6
In this way, the 0-acetyl cyclosporin derivative, (compound 4), was
further modified to produce O-acetyl cyclosporic acid, (compound 5).
The product yield was virtually 100%. The spectral analysis of the O-
acetyl cyclosporic acid was as follows:
I. ~ :
NH groups - delta 8.38, 8.02, 7.72, 7.45, (all d,
each lH ); and,
NCH3 groups - 3.49, 3.24, 3.09, 2.69, 2.67, (all
preceding s, each 3H ), 3. 25 (s, 6H );
OC(O)CH3 -- 2.01, (s, 3H)
II . 13C NMR ( CDCl~
CH2 carbons - delta 40.50, 38.79, 34.40, 24.92,
(due to multiple conformational species, not all
expected methylene 13C signals could be assigned);
HCOC(O)CH3 - 72.31;
12
2086267
HCOC(O)CH3 - 167.34;
III. IR.: Carbonyl stretch (O-acetyl ester), 1745 cm~;
IV. MS.: m/e expected 1249, m/e found 1249, (M+).
The next step in the process of producing compound 8, involved a
modification of the procedures normally employed in the reaction of
carboxylic acid groups on proteins. In a typical example of the
present process, 33.1 mg (173 ~moles) of 1-ethyl-3-~3-
(dimethylamino)propyl]carbo-diimide, (herein sometimes identified as
compound 6), was mixed with 2.5 mg (5.24 ~moles) of 5-
(aminoacetamido)fluorescein, (herein sometimes identified as compound
7), in solution, in 12 mL of deionized distilled water containing two ~:
drops of 10~ NaOH solution. ~:
o~
.::
oJ~ ~ 7
CU~ L
~ ,N~_~N C.N-U
o~O~ :'
~,C~
C~ Nl ~
A solution of 10 mg (8 ~moles) of O-acetyl cyclosporic acid (compound
5) in 200 ~L of tert-butyl alcohol was added to the aqueous solution
and the solution was transferred to an autotitrator set on pH-stat,
where the pH of the solution was adjusted to 5.50 by titration with
2~8l~267
1.0 M HCl. After 5 hours the reaction was stopped by the addition of
1 ml of pH 4.75 acetate buffer and the solvent was removed by freeze-
drying. The reaction pathway is summarized in the adjacent structural
formulas
The methanol-soluble dried products were dissolved in 2 mL of
methanol and loaded on 20 g of Sephadex LH-20 gel filtration media
(Pharmacia) which had been previously swollen in methanol. The
fastest running coloured compound was collected and the methanol was
removed under reduced pressure. CHCl3 (25 ml) was added to the flask
containing the dried products, and then the solution of the coloured
product in CHCl3 was washed with 3 x 20 mL of pH 7.40 phosphate buffer
to remove side products. Removal of the CHCl3 under vacuum furnished
12.0 mg (87~) of orange solid, which was identified as 5-
~aminoacetamido)fluorescein-O-acetyl cyclosporic acid amide, for which
spectral analysis using 1H NMR (CDCl3), revealed:
NCH3 groups, (for the principal conformer in
solution) - delta 3.48, 3.11, 3.06, 2.74, 2.71,
(all s, each 3H), 3.25 (s, 6H);
Aromatic Region Multiplets - 8.62, 8.34, 7.77,
7.66, 6.78.
14
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The present invention also extends to novel cyclosporin poly-
amino conjugate complexes. In one exemplary case, there is provided a
cyclosporin derivative poly-amino conjugate complex (compound llb)
having the formula:
U--N--CU--C --~--CI H--C--N--CH-8- -N--CH-C~--N--ICH-C ~H
CH2 Cl H2 Ctl2 CU~ CH2
CH2 ~H;~ CHa CU~ ~He
Ct~ C~ CUa Cl~ CHt
NH2 4 A, Nlt2 r R2 S
with (q+r~S)ave= 125, and wherein
o
R, = ~ = Rl or H
and wherein the complex is linked through an O-acetyl residue that is
atttached to the site marked with the "X" on a cyclosporin root "Cs"
of the general formula alluded to earlier, hereinabove.
Yet another exemplary cyclosporin derivative poly-amino conjugate
complex (compound llc), according to the present invention, has a
structural formula:
U -N--C~1 C LN--CH--C N--CH-C -N--CH-g --N--CH-C C
, ~ CH H ~tH~ H C, ~ H CH2
C~ C~ 1~ CUa CHa CH2
NH2 4 RNH N~ r NH NHZ S
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with ~q+r+s) ave= 125, and wherein
No~
Q ~ ~
R, ~ R2 = c ~,~N~
and wherein the complex is linked through a O~acetyl residue that is
attached to the site marked with the "X" on a cyclosporin root "Cs" of
the earlier described general formula.
N--CH~
CH~
, N~, ~ ~ ~27 ~
HfH~ II~$H--8~-- -Cl~N~ fl~--~H-qat
~1e q lC~Hp L C~ L ~ ~5 ,,
16
2086267
11a
Iq~r~s~
g ~ l~a,11b. 11c
o $N
11~, Rl = C~N~ R~ or H
oJ~
11b, Fl1 = ,c,~o~ ~e ~ R1 or H
NO~
a ~ ~ ~
11Q R~ _ ,C,--~C A 1~/2 - C~_N~
The last two above mentioned cyclosporin conjugates were prepared as
co-products in the process illustrated above, and described in greater
detail hereinbelow.
In a typical reaction, 623 mg of poly(L-lysine) [0.235 ~moles of
poly~L-lysine), 29.9 ~moles of lysine residues] was combined with 1.5
mg of 2-~N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazo-4yo)amino]ethanoic
acid ~compound 10~ (5.95 ~moles) and 80 mg of EDC (compound 6) (417
~moles) in 12 ml of deionized distilled water which had been rendered
basic through the addition of 2 drops of 10% NaOH solution. A
solution of 10 mg of O-acetyl-cyclosporic acid (compound 5) (8.01
~moles)` in 1 ml of tert-butyl alcohol was added and the solution was
stirred for five hours while the pH was maintained at 5.50. Reaction
products were purified on a column of 20g of Bio-gel P-2 gel
filtration media (Bio-Rad) which had been previously swollen in pH 7.4
phosphate buffer solutionO The desired reaction products eluted in
17
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the first band from the column, along with unreacted poly-(L-lysine).
Unreacted O-acetyl cyclosporic acid and low molecular weight products
from side reactions of compound 10 were collected in later fractions.
1H ~M~ (D20) spectral analysis of products of the first eluted band
revealed poly(L-lysine) peaks at delta 4.05, 2.74. 1.50, and 1.22;
and, cyclosporin related peaks at 2.92, 2.62, 2.60, 1.90, and 0.73.
