U.S. patent application number 10/176824 was filed with the patent office on 2002-12-26 for dimeric non-azo naphthalimides and uses for same.
This patent application is currently assigned to MICROBIOMED CORP.. Invention is credited to Judy, Millard M., Lewis, David E., Matthews, J. Lester, Utecht, Ronald E..
Application Number | 20020198385 10/176824 |
Document ID | / |
Family ID | 23740123 |
Filed Date | 2002-12-26 |
United States Patent
Application |
20020198385 |
Kind Code |
A1 |
Lewis, David E. ; et
al. |
December 26, 2002 |
Dimeric non-azo naphthalimides and uses for same
Abstract
A class of predominantly hydrophilic 1,8-naphthalimide dyes. The
dye contains at least two 1,8-naphthalimide ring systems, joined by
a spacer moiety. Each of the 1,8-naphthalimide ring system has a
ring nitrogen atom and bears, at the 4 position, an amino nitrogen
atom, carrying a hydrogen. The remaining unsatisfied valences, if
present, of the ring nitrogen atoms or the amino nitrogen atoms, or
all, are occupied by one or more alkyl substituents. Each of the
1,8-naphthalimide ring system is free of an azo substituent and is
also free of a nucleofuge. Upon activation by an activating agent
in an environment independent of the presence or absence of oxygen,
these compounds generate activated species. The activated species
can cause structural changes in lipid and any associated proteins
and polypeptides, extra- or intra-cellular or transmembrane,
leading to polymerization and crosslinking.
Inventors: |
Lewis, David E.; (Brookings,
SD) ; Utecht, Ronald E.; (Volga, SD) ; Judy,
Millard M.; (Dallas, TX) ; Matthews, J. Lester;
(Dallas, TX) |
Correspondence
Address: |
JACKSON WALKER LLP
2435 NORTH CENTRAL EXPRESSWAY
SUITE 6000
RICHARDSON
TX
75080
US
|
Assignee: |
MICROBIOMED CORP.
|
Family ID: |
23740123 |
Appl. No.: |
10/176824 |
Filed: |
June 21, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10176824 |
Jun 21, 2002 |
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09438306 |
Jun 28, 1999 |
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6410505 |
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09438306 |
Jun 28, 1999 |
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08472139 |
Jun 7, 1995 |
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5917045 |
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Current U.S.
Class: |
546/122 |
Current CPC
Class: |
C07D 221/14
20130101 |
Class at
Publication: |
546/122 |
International
Class: |
C07D 471/02 |
Claims
What is claimed is:
1. A compound comprising: a first 1,8-naphthalimide ring system
having a first ring nitrogen atom and bearing, at a first
4-position, a first amino nitrogen atom carrying a first hydrogen,
wherein the first 1,8-naphthalimide ring system is free of azo
substituent and is free of nucleofuge; a second 1,8-naphthalimide
ring system having a second ring nitrogen atom and bearing, at a
second 4-position, a second amino nitrogen carrying a second
hydrogen, wherein the second 1,8-naphthalimide ring system is free
of azo substituent and is free of nucleofuge; and a spacer moiety
joining the first and the second 1,8-naphthalimide ring systems,
wherein the spacer moiety joins the first and the second ring
nitrogen atoms, the first and the second amino nitrogen atoms, or
the first ring nitrogen atom and the second amino nitrogen atom,
and wherein remaining unsatisfied valences, when present, of the
first and the second ring nitrogen atoms and of the first and the
second amino nitrogen atoms are occupied by one or more alkyl
substituents.
2. A compound having the formula: 1which is a mixture of
stereoisomers, wherein: R and R' are the same or different and are:
a straight-chain or branched chain alkyl.
C.sub.nH.sub.kX.sub.mY.sub.p where X is a Group VIA element
selected from the group O, S and Se. Y is a Group VA element
selected from the group N. P and As (n=2-200, 1.ltoreq.m.ltoreq.n,
1.ltoreq.p.ltoreq.n, 2n<m+p<4n, 1.ltoreq.k.ltoreq.2n+1+p) and
wherein they are free of X--X, X--Y or Y--Y bonds: or monocyclic,
polycyclic, fused-ring. bridged ring or spirocyclic alicyclic alkyl
C.sub.nH.sub.2q+p+1X.sub.mY.sub.p where X is a Group VIA element
selected from the group consisting of O, S and Se, Y is a Group VA
element selected from the group consisting of N, P and As (n=2-200,
q<n, 1.ltoreq.m.ltoreq.n, 1.ltoreq.p.ltoreq.n, 2n<m+p<4n,
1.ltoreq.m.ltoreq.n) with saturated or unsaturated side-chains,
branched or unbranched, and wherein they are free of X--X, X--Y or
Y-Y bonds; or a straight-chain or branched chain alkyl,
C.sub.nH.sub.kX.sub.mY.sub.pAr.su- b.r where X is a Group VIA
element selected from the group consisting of O, S and Se, Y is a
Group VA element selected from the group consisting of N, P and As,
Ar is an aryl group derived from an aromatic compound selected from
the group consisting of benzene, pyridine, furan, pyrrole,
thiophene, indole, imidazole, oxazole, isozazole, thiazole,
benzofuran, benzothiophene. benzimidazole, quinoline. isoquinoline,
naphthalene, anthracene, acridine, phenanthrene and their
alkylated, halogenated, nitrated or sulfonated derivatives
(n=2-200, 1.ltoreq.m.ltoreq.n, 1.ltoreq.p.ltoreq.n,
2n<m+p<4n, 1.ltoreq.r.ltoreq.20, 1.ltoreq.k.ltoreq.2n+1+p-r)
and wherein they are free of X--X, X--Y or Y--Y bonds; or a
straight-chain or branched chain alkyl,
C.sub.nH.sub.kX.sub.mY.sub.pA.sub.a where X is a Group VIA element
selected from the group consisting of O, S and Se, Y is a Group VA
element selected from the group consisting of N, P and As, A is a
substituent selected from the group consisting of F, Cl, Br, I,
SiZ.sub.3, BZ.sub.2 or AlZ.sub.2, Z is selected from the group
consisting of F, Cl, Br, I, OH, OC.sub.sH.sub.2t+1 (n=2-200,
1.ltoreq.m.ltoreq.n, 1.ltoreq.p.ltoreq.n, 2n<m+p<4n,
1.ltoreq.a.ltoreq.2n, 1.ltoreq.s.ltoreq.20, 1.ltoreq.t.ltoreq.s,
1.ltoreq.k.ltoreq.2n+1+p-a) and wherein they are free of X--X, X--Y
or Y--Y bonds; or a straight-chain or branched chain alkyl,
C.sub.nH.sub.kX.sub.mY.sub.pB.sub- .b where X is a Group VIA
element selected from the group consisting of O, S and Se, Y is a
Group VA element selected from the group consisting of N, P and As,
A is a poly(.alpha.-aminoacid) containing up to 300 residues,
(n=2-200, 1.ltoreq.m.ltoreq.n, 1.ltoreq.p.ltoreq.n,
2n<m+p<4n, 1.ltoreq.b.ltoreq.20, 1.ltoreq.s.ltoreq.20,
1.ltoreq.t.ltoreq.s, 1.ltoreq.k.ltoreq.2n+1+p-b) and wherein they
are free of X--X, X--Y or Y--Y bonds; Q is: a straight-chain or
branched chain alkyl, C.sub.nH.sub.kX.sub.mY.sub.p where X is a
Group VIA element selected from the group consisting of O, S and
Se, Y is a Group VA element selected from the group consisting of
N, P and As (n=2-200, 1.ltoreq.m.ltoreq.n, 1.ltoreq.p.ltoreq.n
2n<m+p<4n, 1.ltoreq.k.ltoreq.2n+p) and wherein they are free
of X--X, X--Y or Y--Y bonds; or monocyclic, polycyclic, fused-ring,
bridged ring or spirocyclic alicyclic alkyl
C.sub.nH.sub.2q+pX.sub.mY.sub.p where X is a Group VIA element
selected from the group consisting of O, S and Se. Y is a Group VA
element selected from the group consisting of N, P and As (n=2-200,
q<n, 1.ltoreq.m.ltoreq.n, 1.ltoreq.p.ltoreq.n, 2n<m+p<4n,
1.ltoreq.m.ltoreq.n) with saturated or unsaturated side-chains,
branched or unbranched, and wherein they are free of X--X, X--Y or
Y--Y bonds; or a straight-chain or branched chain alkyl,
C.sub.nH.sub.kX.sub.mY.sub.pAr.- sub.r where X is a Group VIA
element selected from the group consisting of O, S and Se, Y is a
Group VA element selected from the group consisting of N, P and As,
Ar is an aryl group derived from an aromatic compound selected from
the group consisting of benzene, pyridine, furan, pyrrole.
thiophene. indole, imidazole, oxazole, isozazole. thiazole,
benzofuran, benzothiophene, benzimidazole, quinoline, isoquinoline,
naphthalene, anthracene, acridine, phenanthrene and their
alkylated, halogenated, nitrated or sulfonated derivatives
(n=2-200, 1.ltoreq.m.ltoreq.n, 1.ltoreq.p.ltoreq.n,
2n<m+p<4n, 1.ltoreq.r.ltoreq.20, 1.ltoreq.k.ltoreq.2n+p-r)
and wherein they are free of X--X, X--Y or Y--Y bonds; or a
straight-chain or branched chain alkyl,
C.sub.nH.sub.kX.sub.mY.sub.pA.sub.a where X is a Group VIA element
selected from the group consisting of O, S and Se, Y is a Group VA
element selected from the group consisting of N, P and As, A is a
substituent selected from the group consisting of F, Cl, Br, I,
SiZ.sub.3, BZ.sub.2 or AlZ.sub.2, Z is selected from the group
consisting of F, Cl, Br, I, OH, OC.sub.sH.sub.2t=1, (n=2-200,
1.ltoreq.m.ltoreq.n, 1.ltoreq.p.ltoreq.n, 2n<m+p<4n,
1.ltoreq.a.ltoreq.2n, 1.ltoreq.s.ltoreq.20, 1.ltoreq.t.ltoreq.s,
1.ltoreq.k.ltoreq.2n+p-a) and wherein they are free of X--X, X--Y
or Y--Y bonds; or a straight-chain or branched chain alkyl,
C.sub.nH.sub.kX.sub.mY.sub.pB.sub.b where X is a Group VIA element
selected from the group consisting of O, S and Se, Y is a Group VA
element selected from the group consisting of N, P and As, B is a
poly(.alpha.-aminoacid) containing up to 300 residues, (n=2-200,
1.ltoreq.m.ltoreq.n, 1.ltoreq.p.ltoreq.n, 2n<m+p<4n,
1.ltoreq.b.ltoreq.20, 1.ltoreq.s.ltoreq.20, 1.ltoreq.t.ltoreq.s,
1.ltoreq.k.ltoreq.2n+p-b) and wherein they are free of X--X, X--Y
or Y--Y bonds; or pharmaceutically acceptable salts thereof.
3. A compound having the formula 2which is a mixture of
stereoisomers, wherein: R and R' are the same or different and are:
a straight-chain or branched chain alkyl,
C.sub.nH.sub.kX.sub.mY.sub.p where X is a Group VIA element
selected from the group consisting of O, S and Se. Y is a Group VA
element selected from the group consisting of N, P and As (n=2-200,
1.ltoreq.m.ltoreq.n, 1.ltoreq.p.ltoreq.n, 2n<m+p<4n,
1.ltoreq.k.ltoreq.2n+1+p) and wherein they are free of X--X, X--Y
or Y--Y bonds: or monocyclic, polycyclic, fused-ring, bridged ring
or spirocyclic alicyclic alkyl C.sub.nH.sub.2q+1+pX.sub.mY.sub.p
where X is a Group VIA element selected from the group consisting
of O, S and Se. Y is a Group VA element selected from the group
consisting of N, P and As (n=2-200, q<n, 1.ltoreq.m.ltoreq.n,
1.ltoreq.p.ltoreq.n, 2n<m+p<4n, 1.ltoreq.m.ltoreq.n) with
saturated or unsaturated side-chains, branched or unbranched, and
wherein they are free of X--X, X--Y or Y--Y bonds: or a
straight-chain or branched chain alkyl,
C.sub.nH.sub.kX.sub.mY.sub.pAr.- sub.r where X is a Group VIA
element selected from the group consisting of O, S and Se, Y is a
Group VA element selected from the group consisting of N, P and As,
Ar is an aryl group derived from an aromatic compound selected from
the group consisting of benzene, pyridine. furan, pyrrole,
thiophene, indole, imidazole, oxazole, isozazole. thiazole,
benzofuran, benzothiophene, benzimidazole, quinoline. isoquinoline,
naphthalene, anthracene, acridine, phenanthrene and their
alkylated, halogenated, nitrated or sulfonated derivatives (n=2-
200, 1.ltoreq.m.ltoreq.n, 1.ltoreq.p.ltoreq.n, 2n<m+p<4n,
1.ltoreq.r.ltoreq.20, 1.ltoreq.k.ltoreq.2n+1+p-r) and wherein they
are free of X--X, X--Y or Y--Y bonds; or a straight-chain or
branched chain alkyl, C.sub.nH.sub.kX.sub.mY.sub.pA.sub.a where X
is a Group VIA element selected from the group consisting of O, S
and Se, Y is a Group VA element selected from the group consisting
of N, P and As, A is a substituent selected from the group
consisting of F, Cl, Br, I. SiZ.sub.3, BZ.sub.2, or AlZ.sub.2, Z is
selected from the group consisting of F, Cl, Br, I, OH,
OC.sub.sH.sub.2t+1 (n=2-200, 1.ltoreq.m.ltoreq.n,
1.ltoreq.p.ltoreq.n, 2n<m+p<4n, 1.ltoreq.a.ltoreq.2n,
1.ltoreq.s.ltoreq.20, 1 .ltoreq.t.ltoreq.s,
1.ltoreq.k.ltoreq.2n+1+p-a) and wherein they are free of X--X, X--Y
or Y--Y bonds: or a straight-chain or branched chain alkyl,
C.sub.nH.sub.kX.sub.mY.sub.pB.sub.b where X is a Group VIA element
selected from the group consisting of O, S and Se, Y is a Group VA
element selected from the group consisting of N, P and As, B is a
poly(.alpha.-aminoacid) containing up to 300 residues, (n=2- 200,
1.ltoreq.m.ltoreq.n, 1.ltoreq.p.ltoreq.n, 2n<m+p<4n,
1.ltoreq.b.ltoreq.20, 1.ltoreq.k.ltoreq.2n+1+p-b)and wherein they
are free of X--X, X--Y or Y--Y bonds; Q is: a straight-chain or
branched chain alkyl, C.sub.nH.sub.kX.sub.mY.sub.p where X is a
Group VIA element selected from the group consisting of O, S and
Se, Y is a Group VA element selected from the group consisting of
N, P and As (n=2-200, 1.ltoreq.m.ltoreq.n, 1.ltoreq.p.ltoreq.n,
2n<m+p<4n, 1.ltoreq.k.ltoreq.2n+p) and wherein they are free
of X--X, X--Y or Y--Y bonds; or monocyclic, polycyclic, fused-ring,
bridged ring or spirocyclic alicyclic alkyl
C.sub.nH.sub.2q+pX.sub.mY.sub.p where X is a Group VIA element
selected from the group consisting of O, S and Se, Y is a Group VA
element selected from the group consisting of N, P and As (n=2-200,
q<n, 1.ltoreq.m.ltoreq.n, 1.ltoreq.p.ltoreq.n, 2n<m+p<4n,
1.ltoreq.m.ltoreq.n) with saturated or unsaturated side-chains,
branched or unbranched, and wherein they are free of X--X, X--Y or
Y--Y bonds; or a straight-chain or branched chain alkyl,
C.sub.nH.sub.kX.sub.mY.sub.pAr.- sub.r where X is a Group VIA
element selected from the group consisting of O, S and Se, Y is a
Group VA element selected from the group consisting of N, P and As,
Ar is an aryl group derived from an aromatic compound selected from
the group consisting of benzene, pyridine. furan, pyrrole,
thiophene, indole, imidazole, oxazole, isozazole. thiazole,
benzofuran, benzothiophene, benzimidazole, quinoline, isoquinoline,
naphthalene, anthracene, acridine, phenanthrene and their
alkylated. halogenated. nitrated or sulfonated derivatives
(n=2-200, 1.ltoreq.m.ltoreq.n, 1.ltoreq.p.ltoreq.n,
2n<m+p<4n, 1.ltoreq.r.ltoreq.20, 1.ltoreq.k.ltoreq.2n+p-r)
and wherein they are free of X--X, X--Y or Y--Y bonds; or a
straight-chain or branched chain alkyl,
C.sub.nH.sub.kX.sub.mY.sub.pA.sub.a where X is a Group VIA element
selected from the group consisting of O, S and Se, Y is a Group VA
element selected from the group consisting of N, P and As, A is a
substituent selected from the group consisting of F, Cl, Br, I,
SiZ.sub.3, BZ.sub.2 or AlZ.sub.2, Z is selected from the group
consisting of F, Cl, Br, I, OH, OC.sub.sH.sub.2t+1 (n=2-200,
1.ltoreq.m.ltoreq.n, 1.ltoreq.p.ltoreq.n, 2n<m+p<4n,
1.ltoreq.a.ltoreq.2n, 1.ltoreq.s.ltoreq.20, 1.ltoreq.t.ltoreq.s,
1.ltoreq.k.ltoreq.2n+p-a) and wherein they are free of X--X, X--Y
or Y--Y bonds; or a straight-chain or branched chain alkyl,
C.sub.nH.sub.kX.sub.mY.sub.pB.sub.b where X is a Group VIA element
selected from the group consisting of O, S and Se, Y is a Group VA
element selected from the group consisting of N, P and As, B is a
poly(.alpha.-aminoacid) containing up to 300 residues, (n=2-200,
1.ltoreq.m.ltoreq.n, 1.ltoreq.p.ltoreq.n, 2n<m+p<4n,
1.ltoreq.d.ltoreq.20, 1.ltoreq.k.ltoreq.2n+p-b) and wherein they
are free of X--X, X--Y or Y--Y bonds; or pharmaceutically
acceptable salts thereof.
4. A compound having the formula 3which is a mixture of
stereoisomers wherein: R and R' are the same or different and are:
a straight-chain or branched chain alkyl.
C.sub.nH.sub.kX.sub.mY.sub.p where X is a Group VIA element
selected from the group consisting of O, S and Se, Y is a Group VA
element selected from the group consisting of N, P and As (n=2-200,
1.ltoreq.m.ltoreq.n, 1.ltoreq.p.ltoreq.n, 2n<m+p<4n,
1.ltoreq.k.ltoreq.2n+1+p) and wherein they are free of X--X, X--Y
or Y--Y bonds; or monocyclic, polycyclic. fused-ring. bridged ring
or spirocyclic alicyclic alkyl C.sub.nH.sub.2q-1+pX.sub.mY.sub.p
where X is a Group VIA element selected from the group consisting
of O, S and Se, Y is a Group VA element selected from the group
consisting of N, P and As (n=2-200, q<n, 1.ltoreq.m.ltoreq.n,
1.ltoreq.p.ltoreq.n, 2n<m+p<4n, 1.ltoreq.m.ltoreq.n) with
saturated or unsaturated side-chains, branched or unbranched. and
wherein they are free of X--X, X--Y or Y--Y bonds; or a
straight-chain or branched chain saturated alkyl,
C.sub.nH.sub.kX.sub.mY.sub.pAr.sub.r where X is a Group VIA element
selected from the group consisting of O, S and Se, Y is a Group VA
element selected from the group consisting of N, P and As, Ar is an
aryl group derived from an aromatic compound selected from the
group consisting of benzene, pyridine, furan. pyrrole, thiophene,
indole, imidazole. oxazole. isozazole, thiazole, benzofuran,
benzothiophene, benzimidazole. quinoline, isoquinoline,
naphthalene, anthracene, acridine, phenanthrene and their
alkylated. halogenated, nitrated or sulfonated derivatives
(n=2-200, 1.ltoreq.m.ltoreq.n, 1.ltoreq.p.ltoreq.n,
2n<m+p<4n, 1.ltoreq.r.ltoreq.20, 1.ltoreq.k.ltoreq.2n+1+p-r)
and wherein they are free of X--X, X--Y or Y--Y bonds; or a
straight-chain or branched chain saturated alkyl,
C.sub.nH.sub.kX.sub.mY.sub.pA.sub.a where X is a Group VIA element
selected from the group consisting of O, S and Se, Y is a Group VA
element selected from the group consisting of N, P and As, A is a
substituent selected from the group consisting of F, Cl, Br, I,
SiZ.sub.3, BZ.sub.2, or AIZ.sub.2, Z is selected from the group
consisting of F, Cl, Br, I, OH, OC.sub.sH.sub.2t+1 (n=2-200,
1.ltoreq.m.ltoreq.n, 1.ltoreq.p.ltoreq.n, 2n<m+p<4n,
1.ltoreq.a.ltoreq.2n, 1.ltoreq.s.ltoreq.20, 1.ltoreq.t.ltoreq.s,
1.ltoreq.k.ltoreq.2n+1+p-a) and wherein they are free of X--X, X--Y
or Y--Y bonds; a straight-chain or branched chain alkyl,
C.sub.nH.sub.kX.sub.mY.sub.pB.sub.b where X is a Group VIA element
selected from the group consisting of O, S and Se, Y is a Group VA
element selected from the group consisting of N, P and As, B is a
poly(.alpha.-aminoacid) containing up to 300 residues. (n=2-200,
1.ltoreq.m.ltoreq.n, 1.ltoreq.p.ltoreq.n, 2n<m+p<4n,
1.ltoreq.b.ltoreq.20, 1.ltoreq.k.ltoreq.2n+1+p-b)and wherein they
are free of X--X, X--Y or Y--Y bonds; Q is: a straight-chain or
branched chain alkyl, C.sub.nH.sub.kX.sub.mY.sub.p where X is a
Group VIA element selected from the group consisting of O, S and
Se, Y is a Group VA element selected from the group consisting of
N, P and As (n=2-200, 1.ltoreq.m.ltoreq.n, 1.ltoreq.p.ltoreq.n,
2n<m+p<4n, 1.ltoreq.k.ltoreq.2n+p) and wherein they are free
of X--X, X--Y or Y--Y bonds; or monocyclic, polycyclic, fused-ring,
bridged ring or spirocyclic alicyclic alkyl
C.sub.nH.sub.2q+pX.sub.mY.sub.p where X is a Group VIA element
selected from the group consisting of O, S and Se, Y is a Group VA
element selected from the group consisting of N, P and As (n=2-200,
q<n, 1.ltoreq.m.ltoreq.n, 1.ltoreq.p.ltoreq.n, 2n<m+p<4n,
1.ltoreq.m.ltoreq.n) with saturated or unsaturated side-chains,
branched or unbranched, and wherein they are free of X--X, X--Y or
Y--Y bonds; a straight-chain or branched chain alkyl,
C.sub.nH.sub.kX.sub.mY.sub.pAr.su- b.r where X is a Group VIA
element selected from the group consisting of O, S and Se, Y is a
Group VA element selected from the group consisting of N, P and As,
Ar is an aryl group derived from an aromatic compound selected from
the group consisting of benzene, pyridine, furan, pyrrole,
thiophene, indole, imidazole, oxazole, isozazole, thiazole.
benzofuran, benzothiophene, benzimidazole, quinoline, isoquinoline.
naphthalene, anthracene, acridine, phenanthrene and their
alkylated, halogenated, nitrated or sulfonated derivatives
(n=2-200, 1.ltoreq.m.ltoreq.n, 1.ltoreq.p.ltoreq.n,
2n<m+p<4n, 1.ltoreq.r.ltoreq.20,
1.ltoreq.k.ltoreq.2n+p.ltoreq.r) and wherein they are free of X--X,
X--Y or Y--Y bonds; a straight-chain or branched chain alkyl,
C.sub.nH.sub.kX.sub.mY.sub.pA.sub.a where X is a Group VIA element
selected from the group consisting of O, S and Se, Y is a Group VA
element selected from the group consisting of N, P and As, A is a
substituent selected from the group consisting of F, Cl, Br, I,
SiZ.sub.3, BZ.sub.2 or AlZ.sub.2, Z is selected from the group
consisting of F, Cl, Br, I, OH, OC.sub.sH.sub.2t+1 (n=2-200,
1.ltoreq.m.ltoreq.n, 1.ltoreq.p.ltoreq.n, 2n<m+p<4n,
1.ltoreq.a.ltoreq.2n, 1.ltoreq.s.ltoreq.20, 1.ltoreq.t.ltoreq.s,
1.ltoreq.k.ltoreq.2n+p-a) and wherein they are free of X--X, X--Y
or Y--Y bonds; a straight-chain or branched chain alkyl,
C.sub.nH.sub.kX.sub.mY.sub.pB.sub.b where X is a Group VIA element
selected from the group consisting of O, S and Se, Y is a Group VA
element selected from the group consisting of N, P and As, B is a
poly(.alpha.-aminoacid) containing up to 300 residues, (n=2-200,
1.ltoreq.m.ltoreq.n, 1.ltoreq.p.ltoreq.n, 2n<m+p<4n,
1.ltoreq.d.ltoreq.20, 1.ltoreq.k.ltoreq.2n+p-d) and wherein they
are free of X--X, X--Y or Y--Y bonds; or pharmaceutically
acceptable salts thereof.
5. A compound having the formula 4which is a mixture of
stereoisomers. wherein 1.ltoreq.n.ltoreq.20, and R and R' are the
same or different and are: a straight-chain or branched chain
alkyl. C.sub.mH.sub.k (1.ltoreq.m.ltoreq.20 :
1.ltoreq.k.ltoreq.2m+1): or a straight-chain or branched chain
alkyl. C.sub.mH.sub.k+qO.sub.pN.sub.q (1.ltoreq.m.ltoreq.20:
1.ltoreq.k.ltoreq.2m+1; 2.ltoreq.2p.ltoreq.m:
2m.ltoreq.p+q.ltoreq.4m), wherein they are free of O--O, N--N or
N--O bonds: R" is: H, CH.sub.3, C.sub.2H.sub.5, C.sub.3H.sub.7,
C.sub.4H.sub.9, C.sub.5H.sub.11, C.sub.6H.sub.13; and X is: O, NH,
NCH.sub.3, NC.sub.2H.sub.5; or pharmaceutically acceptable salts
thereof.
6. A compound having the formula 5which is a mixture of
stereoisomers. wherein 1.ltoreq.n.ltoreq.20, and R and R' are the
same or different and are: a straight-chain or branched chain
alkyl, C.sub.mH.sub.k (1.ltoreq.m.ltoreq.20:
1.ltoreq.k.ltoreq.2m+1): or a straight-chain or branched chain
alkyl. C.sub.mH.sub.k+qO.sub.pN.sub.q (1.ltoreq.m.ltoreq.20:
1.ltoreq.k.ltoreq.2m+1; 2.ltoreq.2p.ltoreq.m;
2m.ltoreq.p+q.ltoreq.4m), wherein they are free of O--O, N--N or
N--O bonds: R" is: H, CH.sub.3, C.sub.2H.sub.5, C.sub.3H.sub.7,
C.sub.4H.sub.9, C.sub.5H.sub.11, C.sub.6H.sub.13: and X is: O, NH,
NCH.sub.3, NC.sub.2H.sub.5: or pharmaceutically acceptable salts
thereof.
7. A compound having the formula 6which is a mixture of
stereoisomers, wherein 1.ltoreq.n.ltoreq.20, and R and R' are the
same or different and are: a straight-chain or branched chain
alkyl, C.sub.mH.sub.k (1.ltoreq.m.ltoreq.20 ;
1.ltoreq.k.ltoreq.2m+1): or a straight-chain or branched chain
alkyl. C.sub.mH.sub.k+qO.sub.pN.sub.q (1.ltoreq.m.ltoreq.20;
1.ltoreq.k.ltoreq.2m+1; 2.ltoreq.2p.ltoreq.m;
2m.ltoreq.p+q.ltoreq.4m), wherein they are free of O--O , N--N or
N--O bonds: R" is: H, CH.sub.3, C.sub.2H.sub.5, C.sub.3H.sub.7,
C.sub.4H.sub.9, C.sub.5H.sub.11, C.sub.6H.sub.13; and X is: O, NH,
NCH.sub.3, NC.sub.2H.sub.5; or pharmaceutically acceptable salts
thereof.
8. A compound having the formula 7or pharmaceutically acceptable
salts thereof.
9. A compound having the formula 8or pharmaceutically acceptable
salts thereof.
10. A compound having the formula 9or pharmaceutically acceptable
salts thereof.
11. A composition for cross-linking protein, which comprises an
effective amount of a compound of claim 1, or its derivative, in
combination with an acceptable carrier.
12. A method for affixing a compound of claim 1, or its derivative,
to a protein, comprising: admixing an effective amount of the
compound of claim 1 with the protein to give a resultant mixture;
and activating the resultant mixture with a sufficient amount of an
activating agent to affix the compound of claim 1 to the
protein.
13. A method for cross-linking a first protein to a second protein,
the method comprising: admixing the first protein and the second
protein with an effective amount of a compound of claim 1, or its
derivative, to give a resultant mixture; activating the resultant
mixture with a sufficient amount of an activating agent to
cross-link the first protein to the second protein.
14. A method of bonding a first body tissue to a second body
tissue, comprising: coating a surface of each of the first and the
second body tissues with a sufficient amount of a compound of claim
1, or its derivative, to give a first coated body tissue and a
second coated body tissue; placing the first coated body tissue and
the second coated body tissue in close proximity to give a
resultant mixture; activating the resultant mixture with a
sufficient amount of an activating agent to bond the first and the
second body tissues.
15. A method claim 14, wherein the first tissue is meniscal
cartilage, articular cartilage or cornea.
16. A method claim 14, wherein the second tissue is meniscal
cartilage, articular cartilage or cornea.
17. A bonded tissue, comprising at least two tissues, prepared in
accordance with the method of claim 14.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to dimeric non-azo
naphthalimides, or non-azo bis-(benz[de]isoquinoline-1 ,3-dione)
derivatives, their compositions, their preparations, and their
uses. More specifically, the present invention relates to
predominantly hydrophilic dimeric non-azo naphthalimides joined by
a spacer moiety, their compositions, their preparations, and their
uses in tissue welding.
[0002] Maintenance of close contact between opposed biological
tissue surfaces is necessary for access of cellular and molecular
species required for formation of new connective tissue components
and is thus critical to tissue healing. Ideally, methods of
maintaining close contact between healing tissue surfaces should
interfere only minimally with tissue or organ function and only
minimally affect the healing process itself.
[0003] In numerous surgical procedures mechanical closure, e.g.
clamping or suturing with biocompatible materials, suffices for
functionally adequate wound closure. However these mechanical
methods of approximating and holding together wound surfaces can be
contraindicated where they interfere seriously with structure or
function or their presence complicates healing and restoration of
tissue and organ function. For example, sutures cannot be
satisfactorily used to hold together surfaces of small blood
vessels with diameters 1-2 mm or less. Sutures actually impinge
upon the vessel lumen and the fibrous tissue, which always forms in
response to the presence of the suture material, further impinges
upon the lumen, compromising blood flow.
[0004] As another example, anchoring sutures introduce frequent
complications during healing of corneal implants. To perform the
implant, a full-thickness, circular portion of central recipient
cornea is removed using a trephine. A corresponding section of
cornea is cut form the donor eye and inserted in the recipient eye
and sutured in place. Sutures are left in place until sufficient
healing occurs and structural integrity is restored. Sutures can be
removed typically as early as 6 months or up to 2 years later.
[0005] Surgical complication can arise from the sutures themselves.
First, the suture material can initiate an inflammatory response
with resulting pain, irritation, and invitation to invasion by
vision-impairing blood vessels. Second, suturing makes a hole in
the cornea that allows entry into the cornea of infectious agents
(e.g., virus) that are ordinarily kept out by the epithelium.
Third, sutures do not apply perfectly even pressure along the
circumference of the wound (although running sutures are better in
this regard). Consequently, areas of the wound not firmly held
together are susceptible to leakage of aqueous humor and epithelial
downgrowth (into the incision), both serious complications.
Together, the problems of inflammation, irritation, wound gape,
leakage, and epithelial downgrowth pose significant clinical
problems in post surgical management of corneal transplant and are
frequent causes for transplant failure.
[0006] Use of sutures in affixing bone joint cartilage is precluded
because of their mechanical interference with the smooth mutual
sliding of cartilage surfaces important to unimpeded joint
motion.
[0007] As an alternative to suturing, thermally-setting
proteinaceous glues have been used with infra-red emitting laser
beams to affix or weld, for example, small blood vessels,
intestine, bone, and meniscal cartilage. Although mechanical
affixation using this method generally has been satisfactory,
tissue damaged thermally during lasing and the proteinaceous glue
volume promote the formation of an increased volume of connective
tissue which increased healing time and mechanically interfered
with tissue function.
[0008] Different dyes have been used in classical photodynamic
therapy, which superficially resembles the new invention described
herein is a technique by which membrane alterations can be made in
a living cell by irradiation of a compound or dye incorporated into
the membrane of the cell. See, U.S. Pat. Nos. 4,613,322; 4,684,521;
4,649,151; and 4,878,891.
[0009] Non-azo naphthalimide dyes that are predominantly
hydrophobic have been used to cross-link proteins and tissues. See,
Lewis et al., U.S. Pat. No. 5,235,045, the entire content of which
is hereby incorporated by reference. There is, however, a need for
predominantly hydrophilic compounds to better weld or cross-link
proteins and tissues having high proteoglycan or water content,
such as meniscal cartilage, articular cartilage, and cornea. A
means of affixing tissue surfaces in wound closure which results in
requisite mechanical strength during healing, seals against leakage
of fluid contents, minimal deformation or occlusion of tissue and
tissue lumen, and healing without the formation of large volumes of
associated connective tissue is needed.
SUMMARY
[0010] According to the present invention, predominantly
hydrophilic dimeric non-azo naphthalimide dyes are provided. These
dyes are "dimeric" in that they contain at least two
1,8-naphthalimide ring systems, joined by a spacer moiety or group.
Each of the 1,8-naphthalimide ring system is free of an azo
substituent and is also free of a nucleofuge. Further, the use of
these new compounds in welding or cross-linking proteins or tissues
are given.
[0011] More specifically, the present invention relates to a
predominantly hydrophilic dimeric 1,8-naphthalimide compounds,
having at least two 1,8-naphthalimide ring systems each having a
ring nitrogen atom and each bears, at its 4-position, a
heteroatomic electron-releasing group which is being characterized
as having a heteroatom directly linked to the 4-position of the
ring system and having at least one hydrogen directly attached to
the heteroatom, such as amino nitrogen atom. Each of the
1,8-naphthalimide ring systems is free of any azo substituent and
is also free of any nucleofuge. The two 1,8-naphthalimide ring
systems are joined by a spacer moiety or group. The spacer moiety
can join the two ring nitrogen atoms, or the two amino nitrogen
atoms, or the ring nitrogen atom from one 1,8-naphthalimide ring
system and the amino nitrogen atom from the other 1,8-naphthalimide
ring system. The remaining unsatisfied valences, if presence, of
the ring nitrogen atoms or the amino nitrogen atoms, or all, are
occupied by one or more alkyl substituents. These new naphthalimide
dyes are further characterized as giving activated derivatives or
species after being activated by a sufficient amount of activating
agent in an environment independent of the presence or absence or
oxygen.
[0012] These new non-azo 1,8-naphthalimide dyes can be activated in
the simultaneous presence of an activating agent and the target
tissue or protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 shows the structural formula of "Type I" dimeric
non-azo 1,8-naphthalimide dyes, each having a spacer moiety joining
the amino nitrogen atom at the 4-position of the first
1,8-naphthalimide ring system and the amino nitrogen atom at the
4'-position of the second 1,8-naphthalimide ring system;
[0014] FIG. 2 shows the structural formula of "Type II" dimeric
non-azo 1,8-naphthalimide dyes, each having a spacer moiety joining
the first ring nitrogen atom of the first 1,8-naphthalimide ring
system and the second ring nitrogen atom of the second
1,8-naphthalimide ring system;
[0015] FIG. 3 shows the structural formula of "Type III" dimeric
non-azo 1,8-naphthalimide dyes, each having a spacer moiety joining
the amino nitrogen at the 4-position of the first 1,8-naphthalimide
ring system and the ring nitrogen atom of the second
1,8-naphthalimide ring system;
[0016] FIG. 4 shows the plot of shear strength vs. energy density
in human meniscus cartilage (Parallel); and
[0017] FIG. 5 shows the plot of shear strength vs. energy density
in human femoral articular cartilage.
DETAILED DESCRIPTION OF THE INVENTION
[0018] The generic formula for non-azo 1,8-naphthalimide dyes of
the present invention are represented by Types I, II, and III
compounds as shown in FIGS. 1, 2, and 3, respectively. The "Q" is
the spacer moiety.
[0019] Types I, II and III compounds, having mixtures of
stereoisomers. are compounds wherein:
[0020] R and R' are the same or different and are:
[0021] a straight-chain or branched chain alkyl,
C.sub.nH.sub.kX.sub.mY.su- b.p where X is a Group VIA element
selected from the group O, S and Se, Y is a Group VA element
selected from the group N, P and As (n=2-200, 1.ltoreq.m.ltoreq.n,
1.ltoreq.p.ltoreq.n, 2n<m+p<4n, 1.ltoreq.k.ltoreq.2n+1+p) and
wherein they are free of X--X, X--Y or Y--Y bonds; or
[0022] monocyclic, polycyclic, fused-ring, bridged ring or
spirocyclic alicyclic alkyl C.sub.nH.sub.2q+p+1X.sub.mY.sub.p where
X is a Group VIA element selected from the group consisting of O, S
and Se, Y is a Group VA element selected from the group consisting
of N, P and As (n=2-200, q<n, 1.ltoreq.m.ltoreq.n,
1.ltoreq.p.ltoreq.n, 2n<m+p<4n, 1.ltoreq.m.ltoreq.n) with
saturated or unsaturated side-chains, branched or unbranched, and
wherein they are free of X--X, X--Y or Y--Y bonds; or
[0023] a straight-chain or branched chain alkyl,
C.sub.nH.sub.kX.sub.mY.su- b.pAr.sub.r where X is a Group VIA
element selected from the group consisting of O, S and Se, Y is a
Group VA element selected from the group consisting of N, P and As,
Ar is an aryl group derived from an aromatic compound selected from
the group consisting of benzene, pyridine, furan, pyrrole,
thiophene, indole, imidazole, oxazole, isozazole, thiazole,
benzofuran, benzothiophene, benzimidazole, quinoline, isoquinoline,
naphthalene, anthracene, acridine, phenanthrene and their
alkylated, halogenated, nitrated or sulfonated derivatives
(n=2-200, 1.ltoreq.m.ltoreq.n, 1.ltoreq.p.ltoreq.n,
2n<m+p<4n, 1.ltoreq.r.ltoreq.20, 1.ltoreq.k.ltoreq.2n+1+p-r)
and wherein they are free of X--X, X--Y or Y--Y bonds; or
[0024] a straight-chain or branched chain alkyl,
C.sub.nH.sub.kX.sub.mY.su- b.pA.sub.a where X is a Group VIA
element selected from the group consisting of O, S and Se, Y is a
Group VA element selected from the group consisting of N, P and As,
A is a substituent selected from the group consisting of F, Cl, Br,
I, SiZ.sub.3, BZ.sub.2 or AlZ.sub.2, Z is selected from the group
consisting of F, Cl, Br, I, OH, OC.sub.sH.sub.2t+1 (n=2-200,
1.ltoreq.m.ltoreq.n, 1.ltoreq.p.ltoreq.n, 2n <m+p<4n,
1.ltoreq.a.ltoreq.2n, 1.ltoreq.s.ltoreq.20, 1.ltoreq.t.ltoreq.s,
1.ltoreq.k.ltoreq.2n+1+p-a) and wherein they are free of X--X, X--Y
or Y--Y bonds; or
[0025] a straight-chain or branched chain alkyl,
C.sub.nH.sub.kX.sub.mY.su- b.pB.sub.b where X is a Group VIA
element selected from the group consisting of O, S and Se, Y is a
Group VA element selected from the group consisting of N, P and As,
A is a poly(.alpha.-aminoacid) containing up to 300 residues,
(n=2-200, 1.ltoreq.m.ltoreq.n, 1.ltoreq.p.ltoreq.n,
2n<m+p<4n, 1.ltoreq.b.ltoreq.20, 1.ltoreq.s.ltoreq.20,
1.ltoreq.t.ltoreq.s, 1.ltoreq.k.ltoreq.2n+1+p-b) and wherein they
are free of X--X, X--Y or Y--Y bonds;
[0026] Q is:
[0027] a straight-chain or branched chain alkyl,
C.sub.nH.sub.kX.sub.mY.su- b.p where X is a Group VIA element
selected from the group consisting of O, S and Se, Y is a Group VA
element selected from the group consisting of N, P and As (n=2-200,
1.ltoreq.m.ltoreq.n, 1.ltoreq.p.ltoreq.n, 2n<m+p<4n,
1.ltoreq.k.ltoreq.2n+p) and wherein they are free of X--X, X--Y or
Y--Y bonds; or
[0028] monocyclic, polycyclic, fused-ring, bridged ring or
spirocyclic alicyclic alkyl C.sub.nH.sub.2q+pX.sub.mY.sub.p where X
is a Group VIA element selected from the group consisting of O, S
and Se, Y is a Group VA element selected from the group consisting
of N, P and As (n=2-200, q<n, 1.ltoreq.m.ltoreq.n,
1.ltoreq.p.ltoreq.n, 2n<m+p<4n, 1.ltoreq.m.ltoreq.n) with
saturated or unsaturated side-chains, branched or unbranched, and
wherein they are free of X--X, X--Y or Y--Y bonds; or
[0029] a straight-chain or branched chain alkyl,
C.sub.nH.sub.kX.sub.mY.su- b.pAr.sub.r where X is a Group VIA
element selected from the group consisting of O, S and Se, Y is a
Group VA element selected from the group consisting of N, P and As,
Ar is an aryl group derived from an aromatic compound selected from
the group consisting of benzene, pyridinefuran, pyrrole, thiophene,
indole, imidazole, oxazole, isozazole, thiazole, benzofuran,
benzothiophene, benzimidazole, quinoline, isoquinoline,
naphthalene, anthracene, acridine, phenanthrene and their
alkylated, halogenated, nitrated or sulfonated derivatives
(n=2-200, 1.ltoreq.m.ltoreq.n, 1.ltoreq.p.ltoreq.n,
2n<m+p<4n, 1.ltoreq.r.ltoreq.20, 1.ltoreq.k.ltoreq.2n+p-r)
and wherein they are free of X--X, X--Y or Y--Y bonds; or
[0030] a straight-chain or branched chain alkyl,
C.sub.nH.sub.kX.sub.mY.su- b.pA.sub.a where X is a Group VIA
element selected from the group consisting of O, S and Se, Y is a
Group VA element selected from the group consisting of N, P and As,
A is a substituent selected from the group consisting of F, Cl, Br,
I, SiZ.sub.3, BZ.sub.2, or AlZ.sub.2, Z is selected from the group
consisting of F, Cl, Br, I, OH, OC.sub.sH.sub.2t=1 (n=2-200,
1.ltoreq.m.ltoreq.n, 1.ltoreq.p.ltoreq.n, 2n<m+p<4n,
1.ltoreq.a.ltoreq.2n, 1.ltoreq.s.ltoreq.20, 1.ltoreq.t.ltoreq.s,
1.ltoreq.k.ltoreq.2n+p-a) and wherein they are free of X--X, X--Y
or Y--Y bonds; or
[0031] a straight-chain or branched chain alkyl,
C.sub.nH.sub.kX.sub.mY.su- b.pB.sub.b where X is a Group VIA
element selected from the group consisting of O, S and Se, Y is a
Group VA element selected from the group consisting of N, P and As,
B is a poly(.alpha.-aminoacid) containing up to 300 residues,
(n=2-200, 1.ltoreq.m.ltoreq.n, 1.ltoreq.p.ltoreq.n,
2n<m+p<4n, 1.ltoreq.b.ltoreq.20, 1.ltoreq.s.ltoreq.20,
1.ltoreq.t.ltoreq.s, 1.ltoreq.k.ltoreq.2n+p-b) and wherein they are
free of X--X, X--Y or Y--Y bonds;
[0032] or pharmaceutically acceptable salts thereof.
[0033] As used herein, the word "dye" is interchangeable with the
word "compound," as referred to non-azo 1,8-naphthalimides.
[0034] A "non-azo compound" or dye is one that does not possess a
functional grouping having two nitrogen atoms connected by a double
bond.
[0035] A nucleofuge is any group which can be displaced from a
molecule by a nucleophile. Examples of nucleofuge includes
halogens, sulfonate esters, quaternary ammonium salts.
[0036] As used herein, the words "unsatisfied valences" mean less
than tervalent. Thus, any nitrogen atom which is less than
tervalent or tri-coordinate contains unsatisfied valences.
[0037] The "activating agent" as used herein denotes a means or an
agent that is capable of activating, exciting. or sensitizing a
photoactive compound. The activating agent can be radiated energy,
electromagnetic energy, laser, electric current, electrons, thermal
neutrons or chemicals. The electromagnetic spectrum can include
visible light, xenon light, laser light, near infrared and
ultraviolet light. The laser or other radiation energy can be
continuous or pulsed. The pulsed energy used is such that the
energy supplied has a multiple number of short pulses of relatively
high energy, but at the same time, has a much lower average energy
rate. The laser could be a Helium-Cadmium laser, argon ion laser, a
solid state laser, a gas discharge laser, krypton laser, argon ion
pumped dye laser, or hollow cathode metal vapor laser or
semiconductor diode laser, and others. Even sources such as
conventional filament lamp source with appropriate filtering, an
arc lamp source with appropriate filtering, even a pulsed xenon
flash lamp with appropriate filtering could be used, or light
emitting semiconductor such as GaN and ZnSe diodes.
[0038] The term "body tissue" as used herein is to be understood to
include "body fluid," red blood cells, white blood cells,
platelets, cryo precipitate from blood plasma, other plasma
proteins, bone marrow, skin, blood vessel wall, nerve sheath,
meniscal cartilage, fermoral articular cartilage, cornea, ligament,
tendon and other tissues from an animal or a human.
[0039] The term "animal" as used herein is to denote any animal;
this includes human and other domestic and farm animals.
[0040] The term "carrier" as used herein denotes a vehicle, a
solution containing water, buffers, serum, serum proteins,
lipoproteins, artificial bio-membranes, micelles, liposomes,
monoclonal antibodies, carbohydrates, cyclodextrans, organic
solvents or other pharmaceutically acceptable, or compatible,
solutions. The carrier, or vehicle, used is pharmaceutically
compatible in that it is relatively non-toxic to the normal cells
and normal tissues and it does not react with the solute or
therapeutic agent contained therein.
[0041] The phrase "effective amount" as used herein is to denote
the concentration or level of the compound that can attain a
particular end, such as cross-linking, without producing pronounced
toxic symptoms.
[0042] In general, liphophilic 1,8-naphthalimide dyes are readily
incorporated into lipid bilayers, membranes and micelles of natural
or synthetic origin. Due to the sequestration of these lipophilic
dyes into the surface regions of proteins, both cellular and
liposomal membranes, and tissues having a high density of
biomolecular nucleophilic groups, the covalent reactions initiated
by the activated form of these dyes can result in chemical
alteration of amino acid residues, of protein and peptide
conformation and function, and can cross-link the amino acid
residues, peptides, and proteins. Thus, this class of dyes can be
used to link desired molecular and biomolecular species to
peptides, proteins, cells, and biological tissues as well as other
substrates containing nucleophilic groups, and to cross-link
peptides, proteins, tissues, and other substrates containing
nucleophilic groups selectively upon application of an activating
agent, such as electromagnetic radiation with wavelength
corresponding in absorption spectrum of the dye absorption
spectrum. See, U.S. Pat. No. 5,235,045.
[0043] The appropriate electromagnetic radiation absorption
spectrum includes the ultraviolet through visible light to near
infrared and the K-alpha, etc., X-ray absorption energies of the
molecular halogen substituent. Other activating agents include
thermal neutrons which could be used to activate boron-containing
1,8-naphthalimide dyes.
[0044] The partitioning of non-azo 1,8-naphthalimide dyes into
hydrophobic or hydrophilic regions of a tissue, and the capability
of activating covalent chemical reactions with nucleophilic amino
acid residues allows cross-linking of peptides or proteins located
either extra- or intra-cellularly or associated with the bilayer
membrane selectively upon exposure to light. No cross-linking
occurs until the dye is being activated by an activating agent,
such as light. This is in contrast to the use of conventional
dark-acting chemical cross-linking or linking agents such as
formaldehyde, gluteraldehyde, succinimidyl esters, iodoactamides,
or maleimides which act immediately upon contact with the
appropriate protein residues during mixing by physical agitation or
diffusion.
[0045] Thus, with the use of conventional dark-acting agents, it is
difficult to delay initiation of the cross-linking chemical
reaction until a chosen time during appropriate mixing or
sequestration of mixture components. The use of the light
activatable 1,8-naphthalimide dyes allows temporary delay of
cross-linking until desired mixing or sequestration of liposomal
membrane constituents and internalized components has been
accomplished. This capability allows, for example, synthesis of
liposomes containing a completely cross-linked network of
Gramacidin-D peptide units within the lipid bilayer and which
contain a peptide or protein or other molecular species within the
interior such as hemoglobin. The structural and functional
integrity of these molecules has been maintained during the
cross-linking process because the lipophilic cross-linking dye is
physically isolated from the hydrophilic interior and incapable of
chemical reaction with the internalized molecular species.
Additionally, the lack of cross-linking in the dark by
1,8-naphthalimide dyes allows greater facility of mixing of the
constituent in the dark without fear of reaction.
[0046] The newly synthesized class of light-activated protein
cross-linking bifunctional naphthalimide dyes offer the possibility
of an entirely new approach to hydrophilic tissue bonding that
overcomes present difficulties with sutures, thermal welding and
exogenous glues. This class of compounds is typified structurally
by two photochemically active naphthalimide ring systems linked
together by an inert chain, and each ring is also substituted with
an inert ligand chain. These "dimeric" naphthalimides absorb light
in the range of from about 350 nm to about 550 nm. Preferably, they
absorb in the 410-480 nm range of the visible light spectrum with
maximum absorption (.epsilon.about 13,000 l/mole cm) at about 430
nm. Following light absorption, the excited molecule can lose
energy either by fluorescent emission or through photochemical
reaction and covalent bond formation with an available reactive
substrate. Incorporation of polyether chains and charged amino
groups render these dyes polar and hydrophilic.
[0047] The water solubility of the new class of naphthalimide dyes
and their ability to penetrate various biological tissues depends
strongly upon their molecular structure. Tissues such as cornea,
meniscus and articular cartilage tend to exclude hydrophilic forms
of dyes. This probably stems from the highly polar and ionically
charged nature of proteoglycans and other structural molecules
present in high concentrations in these tissues. The hydrophilic
nature of the molecular environment of these tissues would exclude
the close positioning of the hydrophobic dye required for
photochemical linking, necessary for welding two opposing
surfaces.
[0048] The dyes of the present invention are hydrophilic and most
are soluble in water and other polar solvents. They readily
associate with and penetrate the surface of tissues, such as,
corneal stroma, meniscus and articular cartilage upon exposure to
solution.
[0049] Light induced covalent linking of the non-azo
1,8-naphthalimide dyes with a substrate also allows their use in
linking a desired biomolecular or pharmaceutical agent to target
biological cells or to the surface of a biological tissue at a
desired time.
[0050] Use of light activation of these dyes also allows
cross-linking of proteins at different tissue surfaces in order to
bond or weld biological tissues together and avoid unwanted
intermediate reaction. The advantage of light induced tissue
welding over thermally induced welding is less damage to tissue
adjacent to the welded area due to lack of heating of surrounding
tissues by diffusion during the procedure.
CHEMICAL SYNTHESES
[0051] Given below are different general synthetic methods that can
be used for the syntheses of the new non-azo8,1-naphthalimide dyes.
The synthetic methods are Methods A-H given below:
[0052] A. 4-Chloro-N-alkyl-1,8-naphthalimides may be prepared as
adapted from Lewis, et al., U.S. Pat. No. 5,235,045 (Aug. 10,
1993). A typical procedure is as follows:
[0053] To a suspension or solution of freshly-recrystallized
4-chloro-1,8-naphthalic anhydride (1 eq.) in a solvent (acetic acid
or ethanol; 10-15 mL per gram of anhydride) is added the amine
R--NH.sub.2 or R'--NH.sub.2 (2 eq.). The solution is heated under
reflux for 16-18 hours, after which the mixture is poured into
water (approximately 5 mL of water per mL of solution). The solid
which precipitates is collected by vacuum filtration and washed
with water and ethanol. The material is sufficiently pure for
conversion directly to the 4-aminonaphthalimide; it may be further
purified by recrystallization from acetic acid.
[0054] B.
2-{.omega.'-[6"-chloro-(1"H)benz[de]isoquinolin-1",3"(2"H)-dion--
2"-yl]-1'-alkyl
}-6-chloro-(1H)benz[de]isoquinolin-1,3(2H)-dione[N,N'-alky-
lene-bis-(4chloro-1,8-naphthalimide].
[0055] To a solution of freshly-recrystallized
4chloro-1,8-naphthalic anhydride (2 eq.) in acetic acid (10-15 mL
per gram of anhydride) is added the diamine H.sub.2N--Q--NH.sub.2
(1eq.). The solution is stirred at 120.degree. C. for 16-18 hours,
after which the solution is poured into water (approximately 5 mL
water per mL of solution). The precipitated solid is collected by
vacuum filtration and washed with water or ethanol. The material is
sufficiently pure for conversion directly to the
4-aminonaphthalimide; it may be further purified by
recrystallization from acetic acid.
[0056] C. Water-insoluble
2-{.omega.'-[6"-alkylamino-(1"H)benz[de]isoquino-
lin-1",3"(2"H)-dion-2"-yl]-1'-alkyl}-6-alkylamino-(1H)benz[de]isoquinolin--
1,3(2H)-diones.
[0057] The N,N'-alkylene-bis-4-chloro-1,8-naphthalimide (1 eq.) is
heated with an excess of the primary amine, R--NH.sub.2 or
R'--NH.sub.2 (usually 5 mL of the amine per gram of chloroimide) at
a temperature below 130.degree. C. for 16-18 hours. After this time
the solution is poured into water, and the solid precipitated is
collected by vacuum filtration. The crude product is purified by
recrystallization from acetic acid.
[0058] D. Water-soluble
2-{.omega.'-[6"-alkylamino-(1"H)benz[de]isoquinoli-
n-1",3"(2"H)-dion-2"-yl]-1'-alkyl}-6-alkylamino-(1H)benz[de]isoquinolin-1,-
3(2H)-diones.
[0059] The N,N'-alkylene-bis-(4-chloro-1,8-naphthalimide (1 eq.) is
heated with an excess of the primary amine R--NH.sub.2 or
R'--NH.sub.2, (usually 5 mL of the amine per gram of chloroimide)
at a temperature below 130.degree. C. for 16-18 hours. After this
time the solution is poured into ethanol, and the solid
precipitated is collected by vacuum filtration. The crude product
is purified by recrystallization from ethanol.
[0060] E. One-Pot Method for Water-soluble
2-{.omega.'-[6"-alkylamino-(1"H-
)benz[de]isoquinolin-1",3"(2"H)-dion-2"-yl]-1'-alkyl}-6-alkylamino-(1H)ben-
z[de]isoquinolin-1,3(2H)-diones.
[0061] Freshly recrystallized 4-chloro-1,8-naphthalic anhydride (1
eq.) is heated with an excess of the primary amine R--NH.sub.2 or
R'--NH.sub.2, (usually 5 mL of the amine per gram of chloroimide)
at a temperature below 130.degree. C. for 16-18 hours. After this
time the solution is poured into ethanol, and the solid
precipitated is collected by vacuum filtration. The crude product
is purified by recrystallization from ethanol.
[0062] F. One-Pot Method for
2-{.omega.'-[6"-alkylamino-(1"H)benz[de]isoqu-
inolin-1",3"(2"H)-dion-2"-yl]-1"-alkyl}-6-alkyl-(1H)benz[de]isoquinolin-1,-
3(2H)-diones.
[0063] The 4-chloro-N-alkyl-1,8-naphthalimide is heated with an
excess of the primary amine R'--NH.sub.2, (usually 10 mL of the
amine per gram of chloroimide) at a temperature below 130.degree.
C. for 20-25 hours. After this time the solution is poured into
water or ethanol, and the solid precipitated is collected by vacuum
filtration. The crude product is purified by recrystallization from
ethanol.
[0064] G.
N,N'-bis-{[2"-alkyl-(1"H)benz[de]isoquinolin-1",3"(2"H)-dion-6"--
yl]alkyl}alkane-diamides.
[0065] A
2-alkyl-6-(.omega.'-aminoalkyl)amino-(1H)benz[de]isoquinolin-1,3(-
2H)-dione (2 eq.) is dissolved in dichloromethane (.apprxeq.10 mL
per gram of imide) and pyridine (2 eq.) and an alkanedioyl chloride
(1 eq) are added in sequence. The reaction mixture is allowed to
stand at ambient temperature for 24 h, after which time t.l.c.
analysis indicates that all the starting material has been
consumed. The solvent is evaporated under reduced pressure to
affords the crude product as a red-orange oil which is
recrystallized from 1-butanol or acetic acid.
[0066] H.
6-{.omega.'-[6"-alkylamino-(1"H)benz[de]isoquinolin-1",3"(2"H)-d-
ion-2"-yl]-1'-alkyl}amino-2-alkyl-(1H)benz[de]isoquinolin-1,3(2H)-diones.
[0067] The
2-alkyl-6-(.omega.'-aminoalkyl)amino-(1H)benz[de]isoquinolin-1,-
3(2H)-dione (1 eq.) is heated in ethanol or acetic acid
(.apprxeq.10 mL per gram of imide) with freshly recrystallized
4-chloro-1,8-naphthalic anhydride (1 eq.) for 18-24 h. After this
time, the solution is poured into water and the solid precipitated
is collected by vacuum filtration. The crude product is heated
under reflux for 24 h with an excess of 1-butylamine (usually 10 mL
per gram of crude bis-imide). After this time, the reaction mixture
is poured into ethanol, and the crude product is collected by
vacuum filtration. The product is purified by recrystallization
from ethanol or acetic acid.
[0068] Generally, Type I compounds can be prepared by, first, using
Method A, followed by Method F. Alternatively, Type I compounds can
be prepared by, using Method A, followed by Method C, and, lastly,
followed by Method G.
[0069] Type II compounds can generally be synthesized by, first,
using Method B, followed by Method C. Alternatively, Type II
compounds can be synthesized by, first, using Method B, followed by
Method D. Still another alternate method to synthesize Type II
compound is to use the "one-pot" Method E.
[0070] As for Type III compounds, the sequence to use is, first,
Method A, followed by Method H.
[0071] The following compounds have been prepared by these
Methods:
1 MONOMERIC NAPHTHALIMIDES USED AS INTERMEDIATES: R R' METHOD MP
YIELD Cl C.sub.6H.sub.13 A 188-9 65% Cl C.sub.4H.sub.9 A 176-8 53%
Cl CH.sub.2COOH A 222-7 80% Cl CH.sub.2COOC.sub.2H.sub.5 A 133-6
27% Cl (CH.sub.2).sub.2OH A 187-9 89% Cl
(CH.sub.2).sub.2OCOCH.sub.3 A 100-3 88% C.sub.6H.sub.13
C.sub.6H.sub.13 E 59-60 65% (CH.sub.2).sub.2NH.sub.2
C.sub.6H.sub.13 F 59-60 65% (CH.sub.2).sub.6NH.sub.2
C.sub.6H.sub.13 F 152-4 85% CH.sub.2(CH.sub.2 C.sub.4H.sub.9 F 96-9
70% OCH.sub.2).sub.2CH.sub.2NH.sub.2 (CH.sub.2).sub.2OH
C.sub.6H.sub.13 F 123-5 66% CH.sub.2(CH.sub.2 CH.sub.2COOH F 219-22
79% OCH.sub.2).sub.2CH.sub.2NH.sub.2 C.sub.4H.sub.9 CH.sub.2COOH F
192-6 41% C.sub.4H.sub.9 (CH.sub.2).sub.2 F 255-60 83%
OCOCH(Br)CH.sub.3 (CH.sub.2).sub.2OH CH.sub.2COOH F 202-6 31%
C.sub.4H.sub.9 (CH.sub.2).sub.2OH F 160-2 95%
[0072]
2 DIMERIC NAPHTHALIMIDE INTERMEDIATES R = R' Q METHOD MP YIELD Cl
CH.sub.2(CH.sub.2OCH.sub.2).sub.3CH.sub- .2 B 110-2 74% Cl
CH.sub.2(CH.sub.2OCH.sub.2).sub.2CH.sub.2 B 185-7 62%
[0073]
3 REPRESENTATIVE DIMERIC NAPHTHALIMIDES Type R=R' Q METHOD MP YIELD
II C.sub.5H.sub.13 CH.sub.2(CH.sub.2OCH.su- b.2).sub.2CH.sub.2 C
121-3 41% II CH.sub.2(CH.sub.2OCH.sub.2).sub.- 2CH.sub.2NH.sub.2
CH.sub.2(CH.sub.2OCH.sub.2).sub.2CH.sub.2 E 156-60 23% II
CH.sub.2(CH.sub.2OCH.sub.2).sub.3CH.sub.2NH.sub.2
CH.sub.2(CH.sub.2OCH.sub.2).sub.3CH.sub.2 E 96-8 18% I
C.sub.4H.sub.9
[CH.sub.2(CH.sub.2OCH.sub.2).sub.2CH.sub.2NHCO(CH.sub.2).s-
ub.2].sub.2 G 67-69 67% III C.sub.4H.sub.9
CH.sub.2CONH(CH.sub.2CH.- sub.2O)CH.sub.2CH.sub.2 H Crude III
C.sub.6H.sub.13 CH.sub.2CH.sub.2 H Crude
BIOLOGICAL EVALUATION
[0074] 1. Methods
[0075] The methods of photochemical bonding and determination of
tissue bond shear strength are as follows. Briefly, one surface of
each of two tissue samples was painted with a solution of a Type II
compound wherein R and R' is
--[(CH.sub.2).sub.2O].sub.3(CH.sub.2).sub.2NH.sub.2 and wherein Q
is --(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2-
--O--(CH.sub.2).sub.2-- (concentrations 6-16 mM) (see FIG. 2) and
the painted surfaces opposed to give an overlapped region of about
0.3.times.0.3 cm in area and approximately one-third of the length
of the rectangular tissue sample. The overlapped strips were
carefully wrapped in Mylar film to minimize subsequent water
evaporation and clamped between two Pyrex microscope slides with a
pressure of approximately 1.6 kg/cm.sup.2. The samples were then
individually exposed to 457.9 nm light from an Argon ion laser at
250 mW/crn.sup.2 irradiance (1 cm diameter beam) for preselected
exposure durations. Following light exposure, the bonded sample was
axially loaded in tension using discrete weights to determine bond
tearing (ultimate shear) strength. Values of bond shear strength
were calculated as the ratio of applied force (kg) and overlapped
tissue area (cm.sup.2) and plotted as a function of light radiant
exposure (J/cm.sup.2) for each dye concentration (see FIGS. 4 and
5). Each data point is the result obtained for one sample with
error bars computed using error propagation theory from
uncertainties in applied load (.+-.0.0025 kg) and in the measured
dimensions of the overlapped area (.+-.0.01 cm).
[0076] Dye concentrations and exposure duration were systematically
varied to determine their effects on tissue bond shear strength for
(a) porcine cornea (split and reopposed internal surfaces of the
stroma), (b) human meniscal cartilage (adjacent sheets cut parallel
to the tibial surface and reopposed), and (c) human femoral
articular cartilage (adjacent sheets cut approximately parallel to
articular surface and reopposed).
[0077] 2. Tissue Bonding Results
[0078] Human Meniscal Cartilage
[0079] As shown in FIG. 4, the shear strength of the photochemical
weld in meniscus increased with light exposure and asymptotically
approached a limiting value of light exposure for each dye
concentration studied. The limiting shear strength value increased
with increasing concentration of dye. Shear strengths exceeding 1.3
kg/cm.sup.2 were obtained with blue light exposures (457.9 nm) of
1031 J/cm.sup.2 and a dye concentration of 4 mM; greater shear
strengths were achieved with 8 mM and higher dye concentrations.
Optical and electron microscopic examination of the welded region
of meniscus disclosed intimate contact and sealing of the two
bonded sheets.
[0080] Human Fermoral Articular Cartilage
[0081] As shown in FIG. 5, the bond shear strength behavior of
photochemically welded articular cartilage sheets with increasing
light exposure and dye concentration values are similar to that
obtained with human meniscal cartilage bonds. Ultimate shear
strength values, however, were somewhat smaller than values
obtained in meniscus, being in the range of approximately 0.8-1.2
kg/cm.sup.2.
[0082] Porcine Cornea
[0083] Strips of porcine cornea, 5 mm wide, were separated along a
lamellar plane, parallel to the corneal surface. Hydrophilic dye
was applied to the inner (stromal) sides of the tissue and the
dye-coated surfaces opposed as described above. Light exposure of
1,031 J/cm.sup.2 and dye-solution concentration of 6mM resulted in
a bond shear strength of 0.5.+-.0.1 kg/cm.sup.2 in cornea (N=3).
This strength is approximately 130 times larger than the calculated
3.81 g/cm.sup.2 shell stress in the (assumed spherical) cornea due
to the intraocular pressure (22 mm Hg). Thus, photochemical bonding
would yield closure of the corneal transplant wound with sufficient
strength to prevent leakage of aqueous humour.
[0084] 3. Summary Results
[0085] Bond shear strength values obtained for both meniscal and
articular cartilage are significantly greater than values
.about.0.6 kg/cm.sup.2 obtained in the same tissue with
transglutaminase glues (K. Jurgensen, et al., "A New Biological
Glue for Cartilage-Cartilage Interfaces in Orthopaedic Surgery,"
presented at the Fourth International Conference on Tissue
Transglutaminase and Protein Cross-Linking Reaction, Debrecen,
Hungary, Aug. 26-31, 1991) and the value obtained with laser-heated
thermal welds in collagenous arterial wall of .about.0.3
kg/cm.sup.2 (R. D. Jenkins, et al., "Laser Balloon Angioplasty:
Effect of Tissue Temperature on Weld Strength of Human Postmortem
Intima-Media Separations," Lasers Surg Med 8:30-39, 1988). The
entire contents of both of those two references are hereby
incorporated by reference.
[0086] While the present invention has been particularly described
in terms of specific embodiments thereof, it will be understood in
view of the present disclosure that numerous variations upon the
invention are now enabled to those skilled in the art, which
variations yet reside within the scope of the present invention.
Accordingly, the invention is to be broadly construed, and limited
only by the scope and spirit of the claims now appended hereto.
* * * * *