Note: Descriptions are shown in the official language in which they were submitted.
~ro 94t275~ 2 16 ~ G ~ O PCT~S94/~7
Descr;pt;on
STABLE COPPER(I) COMPLEXES
AND METHODS RELATED THERETO
Techn'c~l F;el~
This invention is generally directed to a copper(I)
complex and methods relating to the use thereof and, more
specifically, to copper(I) complexed by a multi-dentate
ligand such that the +1 oxidation state for copper is
favored in the resulting complex.
R~ckgrol~n~ of the Invent;o~
Copper is found in both plants and animals, and a
number of copper-containing proteins, including enzymes,
have been isolated. Copper may exist in a variety of
oxidation states, including the 0, +1, +2 and +3 oxidation
states (i.e., copper(0), copper(I), copper(II) and
copper(III), respectively), with copper(I) and copper(II)
the most common. The relative stabilities of copper(I)
and copper(II) in aqueous solution depend on the nature of
the anions or other ligands present in the solution.
Moreover, only low equilibrium concentrations of copper(I)
in aqueous solutions (i.e., c 10-2M) can exist. This
instability is due, in part, to the tendency of copper(I)
to disproportionate to copper(II) and copper(0). Most
copper(I) compounds readily oxidize to copper(II)
compounds, although further oxidation to copper(III) is
difficult (see, generally, A.F. Cotton and G. Wilkinson,
30 A~v~nce~ Inorg~n;c ~he~i stry, 5th ed., John Wiley & Sons,
New York, pp. 903-922, 1988).
Due to the relatively well-defined aqueous chemistry
of copper(II), a large number of copper(II) salts and
complexes are known. For example, a great deal of
research has been directed to the biological activity of
peptide/copper(II) complexes, and such copper(II)
WOg4/27594 ~ ~636 4 PCT~S94/06~'
complexes have been shown to possess utility for a variety
of therapeutic and cosmetic purposes. In particular, the
naturally occurring glycyl-histidyl-lysine:copper(II)
complex ("GHK-Cu(II)") has been shown to be an effective
agent in the enhancement of wound healing in warm-blooded
animals, as well as generally serving as an anti-
inflammatory agent (see U.S. Patent No. 4,760,051).
Various derivatives of GHK-Cu(II) possess similar activity
(see U.S. Patent Nos. 4,665,054 and 4,877,770). GHK-
Cu(II) and other peptide-copper(II) complexes have also
been shown to be effective for stimulating hair growth
(U.S. Patent Nos. 5,177,061 and 5,120,831), for inducing
biological coverings in wounds (U.S. Patent No.
4,810,693), for preventing ulcers (U.S. Patent Nos.
4,767,753, 5,023,237, 5,145,838), for cosmetic
applications (U.S. Patent No. 5,135,913), and for healing
bone (U.S. Patent No. 5,509,588). Moreover, anti-
oxidative and anti-inflammatory activity of metal(II)-
peptide complexes has been disclosed (U.S. Patent No.
5,118,665), as well as the use of copper(II)-containing
compounds to accelerate wound healing (U.S. Patent No.
5,164,367).
Although great strides have been made in the study of
copper(II) complexes, and particularly peptide/copper(II)
complexes, there is still a need in the art for additional
copper complexes which posses biological activity. The
present invention fulfills this need, and provides further
related advantages.
S77mmr7ry of the Invention
This invention is generally directed to stable
copper(I) complexes and methods relating thereto. More
specifically, the stable copper(I) complexes of the
present invention comprise copper(I) complexed by a multi-
dentate ligand such that the +1 oxidation state for copperis favored.
~o g4,27594 2 1 ~ 3 6 ~ ~ ~ IUS94l06247
The stable copper(I) complexes have utility for
enhancing wound healing -in warm-blooded animals, for
enhancing or restoring the resistance of warm-blooded
animals to oxidative or inflammatory damage associated
5 with reactive oxygen species and/or lipid mediators, for
stimulating the growth of hair in warm-blooded animals,
for modulating lipid metabolism, for modulating signal
transduction in cells by inhibiting protein kinases, and
for inhibiting viral activity, including (but not limited
to) HIV replication in an HIV-infected animal. Methods of
the present invention comprise administering an effective
amount of a stable copper(I) complex to the animal.
Other aspects of this invention will become evident
upon reference to the attached figures and following
detailed description. All references identified in the
detailed description, including the examples, are hereby
incorporated by reference in their entirety
Descr;pt;on of the F'gures
Figure 1 illustrates the activity of a representative
copper(I) complex of this invention (i.e., bathocuproine
disulfonic acid ("BCDS") copper(I)) to accelerate wound
healing.
Figure 2 illustrates the ability of a representative
copper(I) complex of the present invention, BCDS
copper(I), to inhibit viral (i.e., HIV) replication.
Figure 3 illustrates synthesis pathways for
prostaglandins and leukotrienes, as well as certain key
enzymes associated therewith.
Figure 4 illustrates a synthesis pathway for
cholesterol formation, including the intermediates acetyl
CoA and HMG-CoA and the enzymes acetyl CoA synthetase and
HMG-CoA reductase.
Figure 5 illustrates the action of Protein Kinase C
(PKC) and protein tyrosine kinase in signal transduction
(PI = phosphatidyl inositol, IP3 = inositol triphosphate,
wo94n7sg4 ~ PCT~S94/~7
?,1,,636~
PG = phosphatyl glycerol, P-Protein = phosphorylated
protein, CDR PK = calmoduln-regulated protein kinase,
PKA = Protein Kinase A, Protein Kinase = Protein Tyrosine
Kinase (cytoplasmic), and EGF-R Protein Kinase = Epidermal
growth factor receptor protein tyrosine kinase).
Det~'le~ Descr~ption
This invention is generally directed to copper(I)
complexes and methods relating to the use thereof, and
more specifically, to copper(I) complexed by a multi-
dentate ligand to form a stable copper(I) complex. As
used herein, a "stable copper(I) complex" is copper(I)
chelated by at least one multi-dentate ligand such that
the resulting complex favors the +1 oxidation state of
copper. The most common states of copper(I) are
associated with four coordination sites, and are generally
of a tetrahedral configuration. In general, chelating
agents are coordination compounds in which a single ligand
occupies more than one coordination position of a metal
ion. If the ligand occupies two coordination positions,
it is considered a bi-dentate ligand; if more than two
coordination positions are occupied by the ligand, it is
considered a poly-dentate ligand (such as a tri-dentate
ligand or a tetra-dentate ligand). As used herein, a
"multi-dentate ligand" is a bi-, tri- or tetra-dentate
ligand which occupies two, three or four coordination
sites, respectively, of copper (I).
The stable copper(I) complexes of this invention
include all complexes of copper(I) chelated by at least
one multi-dentate ligand which structurally favors the +1
oxidation state of copper. Copper(I) complexes may be
formed by reacting a multi-dentate ligand with a source of
copper(I) (such as CuCl, Cu2O or CuCN) in aqueous solution.
The resulting copper(I) complex may then be observed by
suitable analytical techniques, such as ESR, NMR and/or
W-VIS, to determine the oxidation state of the copper in
~094/27594 PCT~S94/06~7
-21636qo
the complex (see Munakata et al., Copper Coor~;n~t;on
~he~;stry: R;oche~;c~1 ~n~ Inorg~n;c Perspect;ves, Karlin
and Zubieta editors, Adenine Press, Guilderland, N.Y., pp.
473-495, 1983). For example, copper(I) complexes can be
5 identified by their characteristic absence of an ESR
signal, while copper(II) complexes will generally possess
an ESR signal. Furthermore, copper(II) complexes exhibit
broadening of proton NMR signals, and copper(I) complexes
exhibit relatively sharp proton NMR signals. Following
identification of the copper(I) complex, its stability can
be evaluated by determining its susceptibility to
oxidation by, for example, exposing the copper(I) complex
to air. As used herein, a "stable" copper(I) complex has
a half-life of at least 5 minutes, preferably of at least
one hour, and more preferably of 24 hours or more (i.e.,
half of the copper(I) complex remains in the +1 oxidation
state) upon exposure to air, at room temperature (23C) and
atmospheric pressure. In other words, stable copper(I)
complexes of this invention resist oxidation, while non-
stable copper(I) complexes are readily oxidized to yieldcopper(II) complexes upon exposure to air.
As mentioned above, any multi-dentate ligand which
chelates copper(I) to yield a stable copper(I) complex is
suitable in the practice of this invention. However, in a
preferred embodiment, the multi-dentate ligands of this
invention are selected from the following general
structures I through VII:
WO 94/27S94 ~63G PCTJUS94/06247
A B ~ --A a ~ ~ A B
II III IV
A B --A B
V VI VI I
wherein A and B represent heteroatoms which may occupy
coordination sites of copper(I), and are preferably
selected from nitrogen, oxygen, sulfur and phosphorous.
The rings of structures I through VII may be
aromatic, non-aromatic or a mixture of both aromatic and
non-aromatic rings. For example, the following structures
are representative of such combinations:
Ia IIa IIIa
A B
IIIa' IVa
Va VIa VI Ia
~094127594 PCT~S94/06247
_ ~6
7 36
Representative examples of multi-dentate ligands of
this invention having structures I through VII are set
forth in Table 1. Specifically, Table 1 identifies the
structure of the representative multi-dentate ligand,
lists the corresponding chemical name, identifies the
Chemical Abstracts Registration Number ("CA Reg. No."),
and provides a corresponding reference (if available)
describing the synthesis and/or chemistry of the
identified multi-dentate ligand.
T~hle 1
Strl1ctl~re Name CA Reg. Reference
No.
h~ benzo (2,1-b:3,4- 211-53-0 Sturaro et al.,
b) dithiophene ~eterocycl.
~S S ~ Chem. 27:1867,
1990
h-\\ benzo (2,1-b:3,4- 211-47-2 Rene et al.,
r~~~ b) difuran ~l~r. J. Med.
O O l'h.om . - rh i m .
~L_ 13:435,
1978
~ thieno (3,2-g~ 438-31-9 Cagniant and
1, `> - ~ I benzofuran Kirsch, Hebd.
O S S~nces Acad.
Sci. C.
2~2:465, 1976
h~\ 2H-furo(3,2-g) 103671-62-1 Lawrence Jr.,
> _ ~ ; indole Eur. Pat. Appl.
H EP 173~820
1986
2H-benzo (2,1- 112149-08-3 Berlin et al.,
~ ; b:3,4-b') J. ~h~m. SOC.
H H dipyrrole ~h~m. Com~-ln.
(15):1176, 1987
W O 94~7~94 PCTAUS94/06247
?,~636 4~
lH-cyclopenta 42262-29-3
~N N J(2,1-b:3,4-b')
blpyrldlne
/ = \ 1,10- 66-71-7
< - ~ N = > phenanthroline
/ = \ furo ~3,2-h) 234-28-6
/ ~ 1~ quinoline
2,2'-bipyridyl 366-18-7
N N
In structures I through VII above, further ring
substitutions with heteroatoms are permitted. Preferably,
such heteroatoms are selected from nitrogen, oxygen,
sulfur, and phosphorus. For example, the compounds listed
in Table 2 illustrate further representative multi-dentate
ligands of the present invention having additional ring
substitutions. As with Table 1, Table 2 identifies the
structure of the representative multi-dentate ligands,
lists the corresponding chemical name, identifies the CA
Reg. No., and provides a corresponding reference (if
available) describing the synthesis and/or chemistry of
the identified multi-dentate ligand.
~0 94127594 PC~IUS94/06247
- .2l636~a
T~hl e 2.
Strl~ctl~re ~m~ ~P. Reg. Reference
~ furano (3,2-g) 25885-39-6
N ~ n7o~7ole
O O
furano (2,3-e) 66037-80-1 Turin et al.,
benzoxazole Fr. Demande
N O ~ 2,338,041, 1977
~ thieno (3,2-g) 58188-85-5 Iddon et al.,
N ~ ~ ~ b~n7n~7Ole J ~h~m . SOC . .
O S Perki n Tr~n.~ . I
17:1686, 1975
thieno (3,2-g) 72121-58-5
Njl ~ _ ¢ ~Ibenzothiazole
S S
r \\ thieno (2,3-e) 211-36-9
? s benzothiazole
~ benzo (1,2-d:3,4- 211-50-7Dallacker and
O ~ _ 0 d') bis (1,3) Weiner, Ju~t
O dioxide T.i ebigs A~n,
Chem. l~:99
1969
/ - \ benzo (1,2-d:3,4- 211-10-9
N ~ ~ - Nd') diimidazole
-``N N--
/ = \ pyrrolo(2,3-e) 53068-46-5Chetverikov et
N ~ ~ ` benzimidazole al., U.S.S.R.
-``N N ~ 425,906, 1974
~ benzo (2,1-d:3,4- 211-54-1
O ~ ~ d~) bis (1,3)
S S~ oxathiole
WO 94n7s94 PCTAUS94/06247
?~636 4~ lo
~ 2H-imidazo (4,5- 42341-40-2
S ~ ~ Ne) benzothiazole
H
/ - \2H-imidazo (4,5- 211-23-4
N ~ - ~ Ng) benzothiazole
S N ~
~ 1,3-dioxolo (4,5- 77482-58-7 Foerster et
~ ~3 e) benzothiazole al. Ger.
O N ' Offen.
2,903,966, 1980
~ benzo (1,2-d:3,4- 211-37-0
N ~ ~ ~ Sd') bisthiazole
S N
~ \~benzo (2,1-d:3,4- 23147-19-5
1 ~ 3 d ) bisthiazole
- N N ~
benzo (1,2-d:4,3- 10558-80-2Grandolini et
Nd') bisthiazole al., A~n. ~him.
S S 58:91, 1968
~ thiazolo(5,4-e) 211-35-8
N ~\~ /~ Obenzoxazole
`S N-~
thiazolo (5,4-g) 51273-21-3
Sbenzoxazole
O N '
~ thiazolo (4,5-e) 315-47-9
O ~ ~ /~ Sbenzoxazole
"N N-~
\thiazolo (4,5-f)67239-73-0Fridman et al.,
~ ~ benzoxazole Ikr. ~h;~, Zh.
O S 44:399,1978
~ benzo (2,1-d:3,4- 211-19-8
O ~ ~ ~ d') bisoxazole
``N N '
~ benzo (1,2-d:3,4- 211-20-1
N -`~\ ~ Od') bisoxazole
\ O N
2 1 6 3 6 4 0 PCTrUS94/06247
11
benzo (1,2-d:4,3- 54935-19-2Barker et al.,
~Nld') bisoxazole J. ~h~m . Res.
0 0 Sy~op. (9):328,
1986
,N ~ furo (2,3-d) 110665-19-5
jl ~ ~ 9~ thieno (3,2-b)
S pyridine
~ N, lH-imidazo (4,5- 111163-54-3Takada et al.,
N ~ d) thieno (3,2- Eur. Pat. Appl.
\ N S b)-pyridine EP 223,420,
1987
N ~ dithieno (3,2- 40826-38-8Yang et al.,
j b:2',3'-d) Sy~th~is
\ S S pyridine 2:130, 1989;
Heeres et al.,
Syrl. Comm~ln.
2:365, 1972
/N ~ 5H-oxazolo (4,5-211-46-1
~ e) thiazolo (3,2-
" N S c) pyrimidine
N-N dithieno (3,2- 51974-92-6 Nonciaux et
c:2',3'-e) al., Bull. Soc.
S S pyridazine Chim. Fr. 12 Pt
2, 3318, 1973
/N ~ lH-(1,2,4) 387-96-2
N -\ ~ N triazolo (5,1-b)
`'HN N purine
N-N` N-N bis (1,2,4) 55366-22-8Vercek et al.,
triazolo (1, 5- Tetr~he~on
N N d 5~ -c) L~_
pyrazine (51/52):4539,
1974
/ \ benzo (2,1-b:3,4- 231-29-8 Mnn~ts~h
_ // \ b') dipyran 80:743, 1949
"~ O O
WO 94t27~94 ` PCT/US94/06247
2~636 4~
/ = \ benzo (1,2-b:4,3- 231-34-5
// ~ b') bis (1,4)-
- S S oxathiin
/ = \ benzo (1,2-e:3,4-
N ~// N e') dipyrazine
\=N N=/
/ = \ benzo (1,2-d:3,4- 211-10-9
N~N d') diimidazole
--N N~
/ = \ pyrazino (2,3-f) 231-23-2Shim et al.,
N - ~// N ~lnox~l ine Sy~th~is
- N N= 2:116, 1980;
Nasielski-
Hinkins et al.,
J. ~h~m. Soc.
Perkln Tr~n~.
1:1229, 1975
/ = \ bis (1,2,4) 74382-83-5
~ o~ olo (2,3-
- N N~ d:3',2'-c)
pyrazine
/=N (1,2,4)- 56248-95-4Miura et al.,
O ~ ~ N oxadiazolo (3,2- ~h~m. Ph~rm.
``` N N ~ i) purine ~ :464,
1975
/ = \bis (1,2,4) 51519-32-5Polanc et al.,
N ~ ~ N triazolo (1,5- J. Org. ~hem.
"N N/ b:5',1'-f) ~:2143, 1974
pyridazine
/ = \bis (1,2,4) 76044-62-7Brown and
~_ ~ ~triazolo (1,5- Shinozuka,
\N N~ d:1',5'-c) A-~t. J ~h~m,
pyrimidine 33:1147, 1980
~094n7sg4 21 635~o rCT~S94/~247
General structures I through VII identified above may
possess further chemical moieties covalently attached to
the structural backbone, as illustrated below:
R~ 7 R~ ~7
Ib IIb
R2 ~ R
R1 R7
IIIb IVb
à R6
Vb VIb
\4 5
~R 6
2~ R
1 B 7
VIIb
wherein R1 through R8 are the same or different, and are
selected from the following chemical moieties: -H, -OH,
-X, -OX, -COOH, -COOX, -CHO, -CXO, -F, -Cl, -Br, -I, -CN,
W094~7594 PCT~S94/~6~7
2 ~ 6 3 6 ~ 14
-NH2, -NHX, -NX2, -PX2, -S03H, -so3Na~ -S03K, -S03X,
-P03H, -OP03H, -P03X, -OP03X and -NO2 As used herein,
"X" represents and an alkyl moiety or an aryl moiety. An
"alkyl moiety" is a straight chain or branched, cyclic or
noncyclic, saturated or unsaturated, substituted or
unsubstituted carbon chain containing from 1-20 carbon
atoms; and an ~aryl moiety" is a straight chain or
branched, cyclic or noncyclic, saturated or unsaturated,
substituted or unsubstituted carbon chain containing at
least one substituted or unsubstituted aromatic moiety and
containing from 6-20 carbon atoms. Such chemical moieties
may also be covalently attached to the ring fusion atoms.
Representative examples of the chemical moieties of this
invention include, but are not limited to, the moieties
identified in Table 3 below.
T~hle 3
-H -CH3 -CH2Br
-CH20H -CH2Cl -cBr3
-CH2c6H5 -C6H5 - (CH2) 1-12CH3
-Cl -CHO -COOH
-COOMe - CH=NOH -CH2NH2
-CH2C_CH -CH=CH2 -p(c6H5)2
-CH2CH(C02H)2 -CON(CH2COOH)2 -CH2N(cH2cOoH)2
_CH2 CH3 -CH2N(CH2)11CH3
N ~CH20H _N-CH-CH-~H5 CH3
~H CH3 OH
-Ph-SO3Na
Representative examples of the multi-dentate ligands
possessing further chemical moieties covalently attached
to the structural backbone of structures I through VII are
presented in Table 4. In particular, Table 4 identifies
the structure of the representative multi-dentate ligands,
lists the corresponding chemical name, identifies the CA
'~094l27594 21 63 6qO PCT~S94/06~7
Reg. No., and provides a corresponding reference (if
available) describing the -synthesis and/or chemistry of
- the multi-dentate ligand.
T~hle 4
Str1~ctllre ~m~ CA Reg.Reference
CO2H CO2H 2,2'- 6813-38-3
4,4'-
dicarboxylic
acid
CH3 2,2'-bis (4,5- 69286-06-2J. Org~nnm~t.
~ ~\ ~ ~ 3dimethyl Chem. 307:39,
H3C NIN CH3 imidazole) 1986
~ 2,3-bis (2- 25005-96-3(Aldrich:
N/ ~ pyridyl) 28,164-16)
pyrazine
N~"
H3C~ ~ S ~ ~ S CH3 5,5'-dimethyl- 16303-58-5
_I, \\_ 1 2,2'-
bithiophene
6,6'-dimethyl- 4411-80-7Kauffmann et
H3C~ ,N , N ~ " CH3 2,2~-dipyridine al., ~h~m Ber.
, lQ~:3864, 1976
The chemical moieties covalently attached to the
structural backbone may be joined to yield an aromatic or
nonaromatic cyclic chemical moiety. Representative
examples of such cyclic chemical moieties are set forth in
Table 5, which identifies the structure of the
representative multi-dentate ligands, lists the
W094/27594 636 ~Q PCT~S94/06247
~ 16
corresponding chemical name, identifies the CA Reg. No.,
and provides a corresponding reference (if available)
describing the synthesis and/or chemistry of the multi-
dentate ligand.
T~hle 5
.~trllctl~re ~m~ CA Reg. Reference
No.
H3C ~ CH3 6,7-dihydro- 5298-71-5
~ ~ ~ 5,8-dimethyl
/\/ N N ~ ~ dibenzo
(b)(l,lo)
phenanthroline
N H bibenzimidazole 123067-51-6
N ~ ~ N ~ ~ 2,2'- 119-91-5 (Aldrich:
bisquinoline B3,540-7)
The synthesis of representative examples of the
multi-dentate ligands of this invention are disclosed in
Table 6 and Table 7 below. Specifically, in these tables
the structure of the multi-dentate ligands are identified
along with their CA Reg. No. and one or more references
disclosing their synthesis and/or chemistry.
V094/275g4 21636qO PCT~S94/06247
',:
17
T~hle 6
Synthes;s of Repres~nt~t~ve Copper(I) Co~lexes
H~vlng the Strl~ctl]re:
3 ~ R6
R 8
(R2 through R7 = hydrogen, unless indicated)
R1 ~ CA Reg. No. Reference
-CH3 -CH3 484-11-7 O'Reilly et al.,
Aust. J . rh ~m .
1~:145, 1960
-CH2Br -CH2Br 78831-37-5 Weijen et al., J.
Org. ~h~m , 57:7258,
1992; Jukkala
et al., ~elv. ~h i m .
~a~ 75:1621, 1992
Chandler et al., J.
~terocycl. ~hem.
l8:599, 1981
-CH2Br -CH2OH 142470-16-4Weijen et al., J.
Org. ~h~m. 57:7258,
1992
-CBr3 -CBr3 Chandler et al., J.
~eterocycl. rhem.
18:599, 1981
-CH2Cl -CH2Cl Newkome et al., J.
Org. ~h~m . 50:3807,
1985; Newcome et
al., J. Org. ~h~m.
48:5112, 1983
-CC13 -CC13 Chandler et al., J.
terocycl. rh~m,
18:599, 1981;
Ne~ et al., J.
Org. ~h~m. 48:5112,
1983
-CN -CN 57709-63-4Chandler et al., J.
~eterocycl. ~h~m.
18:599, 1981;
Sjoegren et al.,
OrgAnome~Al lics
ll:3954, 1992
wog4n7sg4 ~ : PCT~S94/~ ~7
?~6~6 4 ~-
-CH2C6H5 -CH2C6H5 223-20-1 Sjoegren et al.,
OrgAnnm~tAllics
- 11:3954, 1992
-(CH2)11CH3-(CH2)11CH3 Menger et al., J.
,~m. rh~m. SOC.
11~:4017, 1991
-(CH2)3CH3 -(CH2)3CH3 85575-93-5PSugihara et al., JP
02096578 A2, JDn.
KokA- Tokkyo Kohn
1l3(15):132159v
(R3=R6=H, Ph
-(CH2)3CH3 -(CH2)3CH3 Delton et al., EP
339973 A1, ~l~r. Pat.
112(21):19835p, 1989
(R4=Rs=-CH3)
-Cl -Cl 29176-55-4Sjoegren et al.,
OrgAnometallics
11:3954, 1992;
Delton et al., EP
339973 A1, ~llr. Pat.
112(21):19835p, 1989
-CH2OH -CH2OH 78831-36-4Chandler et al., J.
~terocycl. rh~m.
18:599, 1981; Delton
et al., EP
339973 A1, ~l~r. Pat.
AD~)l .
112(21):19835p,
1989; Newcome et
al., J. Org. ~h~m.
~Q:5112, 1983
-CHO -CHO 57709-62-3Ziessel, TetrAh.o~rnn
Lett. 30:463, 1989;
Toner, EP 288256 A2,
~l~r. Pat. A~Dl.
111(15):130322c;
Bell et al., J.
Incll~.~io~ Phf~nom.
~:149, 1987
- COOH - COOH ~handler et al ., ~,
~eterocycl. Chem.
1~:599, 1981
-COOMe -COOMe Chandler et al., J.
~eterocycl. ~h~m.
1~:599, 1981;
New~..o et al., J.
Org. ~h~m. 48:5112,
1983
"V094/275g4 19 ~-
-CH=NOH -CH=NOH Chandler et al., J.
~eterocycl. ~h~m,
1~:599, 1981
-CH2NH2 -CH2NH2 Chandler et al., J.
~eterocycl rh~
18:599, 1981
-CHO -H 33795-37-8Toner, EP 288256 A2,
~l~r Pat. A~
111(15):130322c
-COOH -H 1891-17-4Toner, EP 288256 A2,
~l]r Pat. A~Dl.
111(15):130322c
-CH2C--CH -CH2C--CH Sj oegren et al.,
Org~nnm~t~llics
11:3954, 1992
-C6H5 -C6H5 Dietrich-Buchecker
et al., Tetr~he~ron
Lett. ~:5291, 1982
-Cl -CH3 Newcome et al., J.
Org. rh~m. 54:1766,
1989
-CH=CH2 -CH=CH2 Newkome et al., J.
Org. rh~m. 50:3807,
1985
-P(C6H5)3 -P(C6H5)3 Zlessel, Tetr;3h r n
CH2CH(C2H)2 -cH2cH(co2H)2 Newc ? et al.,
Innrg. ~hem. 24:811,
1985
-CH2N(CH2)11CH CH2H Weljen et al., J
CH3 ~N~ 1992
I , H
CH2 _CH2 Weijen et al., J.
N ~CH20H N ~CH20H Org. ~'h~m. 57:7258,
~H ~H
-CH OH _CH2 Weijen et al., J.
2 CH20H Org. rh.om. 57:7258,
~N,,~ 1992
H
CH3 N-CH-CH-C6H Org. rh~m. 57 7258,
CH3 OH
-CH2N(CH2 -CH2N(CH2 Mukkala et al.,
COOH)2 COOH)2 ~elv rh-m Acta
Toner, EP 288256 A2,
~r P~t. ~pDl.
111(15):130322c
WOg4/27~94 ~ ~636 ~ PCT~S94/06247
-CON(CH2 -CON(CH2 Toner, EP 288256 A2,
COOH)2 COOH)2 F~l~r. Pat. P~7Dl.
` 11l(15):130322c
-CH3 -CH3 52698-84-7 Blair et al, T~l~nta
(R3=R6= 1:163, 1961
-Ph-SO3Na
T~hle 7
.Synthes;s of Re~resent~t~ve Co~er(I~ Co~plexes
H~v~g the Strl]ctl]re:
R2 ,,~ 7
N N
Rl R8
(R2 through R7 = hydrogen, unless indicated)
Bl ~ CA Reg. No. Refer~nce
-CN -CN 4411-83-0Sjoegren et al.,
Org~n~met~llics
:3954, 1992
-CH2Cl -CH2C1 74065-64-8 Bell et al., J.
Tncll~ n Ph~n~ m.
~:149, 1987
-CHO -CHO Newkome et al., J. Org.
Chem. 50:3807, 1985
-CH=CH2 -CH=CH2 Newkome et al., J. Org.
Chem. 50:3807, 1985
(Rl and (R7 and 119-91-5 (Aldrich: B3,540-7)
R2= benzo R8 =
moiety) benzo
moiety)
In one embodiment of this invention, the multi-
dentate ligands are selected from the following
structures:
-~094/27D4 21 ~1636 PCT~S94/06~7
R~ 7 ~ ~ 7
R/1 N N \R R~1 N N \R
Ic IIc
wherein R1 through R8 are the same or different, and are
selected from hydrogen, an alkyl moiety and an aryl
moiety.
In a preferred embodiment, the multi-dentate ligand
is 6,6'-dimethyl-2,2'-dipyridine having structure Id:
~-N N
CH3 CH3
Id
In a further preferred embodiment, the multi-dentate
ligand is neocuproine (2l9-dimethy~ lo-phenanthroline)
having structure IId, or is bathocuproine disulfonic acid
(~'BCDS") having one of the isomeric structures IIe, IIe'
or IIe'':
, N N
CH3 CH3
IId
wog4n7s94 ~636 PCT~S94/~7
S03Na S03Na S03Na
Na
~H3N CH3 ~H3N CH3
IIe IIe'
NaO3S ~ ~ O3Na
~ N N
CH3 CH3
IIe''
Unless otherwise indicated, BCDS refers to a physical
mixture of the above isomers (i.e., IIe, IIe' and IIe'').
Typically, the ratio of the various isomers (i.e.,
IIe:IIe':IIe'') vary depending upon the commercial source
of BCDS as follows: Aldrich Chemical Co., Inc. (Milwaukee,
Wisconsin) 9.1:38.6:41.2; Spectrum Chemical Manufacturing
Corp. (Gardena, California) 8.5:39.7:45.2; GFS Chemicals
(Columbus, Ohio) 8.4:38.5:45.3; Janssen Pharmaceutica
(subsidiary of Johnson & Johnson) (Beerse, Belgium)
4.6-8.7:36.4-39.4:44.4-55.9.
As discussed above, stable copper(I) complexes of
this invention may be made by contacting a multi-dentate
ligand with a copper(I) source. The multi-dentate ligands
may be obtained from commercial sources, or may be
synthesized by known organic synthesis techniques from
commercially available reagents. Preferably, water
soluble multi-dentate ligands are complexed with the
copper(I) in aqueous solution, employing CuCl, Cu2O or
CuCN as the copper(I) source. The resulting copper(I)
V094/27~94 PCT~S94/06247
21 63 6~o
complex may then be recovered by evaporation of solvent to
yield the copper(I) complex. Alternatively, if the multi-
- dentate ligand is not readily soluble in water, copper(I)
complexes may be formed by the above procedure employing a
suitable non-aqueous (e.g., organic) solvent.
In the practice of this invention, the ratio of the
multi-dentate ligand to copper(I) may be any ratio which
results in a stable copper(I) complex. Preferably, the
ligand to copper ratio is at least 1:1. In a more
preferred embodiment, the ligand to copper ratio ranges
from 1:1 to 3:1 (including 2:1). Such copper(I) complexes
may be made by the procedures identified in the preceding
paragraph by reacting the appropriate molar ratios of the
multi-dentate ligand and the copper(I) ion source.
Although not intending to be limited by the following
theory, it is believed that copper(I) has enhanced
biological activity over copper(II) in certain biological
events. For example, it is believed that copper(I) may be
an important intermediate for copper metabolism, including
copper uptake and/or transfer, as well as cellular
delivery. Thus, the reduction of copper(II) to copper(I)
is bypassed by direct delivery of copper(I). Furthermore,
the stable copper(I) complexes of this invention are
suitable for systemic delivery to warm blooded animals,
and may provide a sustained release of copper to the
animal.
The stable copper(I) complexes of this invention
possess utility as therapeutic substances, including
utility as anti-oxidative and anti-inflammatory agents
generally and, more specifically, as wound healing agents.
The copper(I) complexes of this invention also possess
activity as hair growth agents, lipid modulation agen~s,
signal transduction modulating agents, and anti-viral
agents. For purpose of clarity, the various biological
activities of the stable copper(I) complexes of this
invention are addressed individually below.
W094/27594 ~636 4 PCT~S94/~24-
24
Highly reactive oxygen species such as the superoxide
anion (2~ hydrogen peroxide (H2O2), hydroxyl radical
(HO-), and lipid peroxides (LOOH) are involved in a number
of human diseases. For example, such oxygen species have
been implicated in autoimmune diseases, arthritis, tissue
damage caused by environmental pollutants, cigarette smoke
and drugs, tissue injury during, for example, surgery and
transplantation, as well as a variety of other conditions
(see, e.g., Halliwell, B., Fe~. Amer. Soc. F.~p. ~3;ol.
1:358-364, 1987). Reactive oxygen species are also
generated during the response to injury by phagocytic
cells. One of the early events in the wound healing
response i5 the cleansing and sterilization of the wound
by neutrophils and macrophages. A mechanism for this
sterilization is the generation of the superoxide anion
and hydrogen peroxide, and generally results in an
inflammatory response. Moreover, superoxide anion and
hydrogen peroxide will, in the presence of iron or other
redox active transition metal complexes, generate the
hydroxyl radical. The hydroxyl radical is a potent
oxidant which initiates the free radical oxidation of
fatty acids, as well as the oxidative degradation of other
biomolecules. For example, an important area in which
reactive oxygen species cause tissue damage is in post-
injury damage to the brain and spinal chord, and inreperfusion injury to ischemic tissue following surgery
and transplantation (such as heart surgery and/or
transplantation). A sudden inrush of oxygenated blood and
activated phagocytic cells leads to superoxide anion and
hydrogen peroxide formation. These species do direct
damage to tissue, and also react with iron (as discussed
above) to generated the very reactive hydroxyl radical.
The stable copper(I) complexes of this invention
generally serve as anti-oxidative agents which prevent or
limit the oxidative damage caused by reactive oxygen
species, and further serve as anti-inflammatory agents by
-vog4n7~94 21 ~ 6~ PCT~S94/~7
reducing the inflammatory response associated with such
reactive oxygen species. More specifically, the copper(I)
complexes of the present invention are useful in the
enhancement and/or restoration of the defense of warm-
- 5 blooded animals to oxidative or inflammatory damage caused
by the highly reactive oxygen species, and may be used in
pharmaceutical preparations to inhibit oxidative and
inflammatory processes which lead to tissue damage.
Moreover, the stable copper(I) complexes of this invention
accelerate the wound healing process by "detoxifying~
tissue damage by the highly reactive oxygen species.
In addition to highly reactive oxygen species,
macrophages and neutrophils induce or continue an
inflammatory response through the generation of certain
lipid mediators of inflammation (e.g., leukotrienes and
prostaglandins). The involvement of such mediators in
inflammatory bowel disease (IBD) and related chronic
inflammatory conditions, such as arthritis, is evidenced
by a strong correlation between disease progression and
the levels and presence of leukotrienes and prostaglandins
in the circulation and effected tissue. Prostaglandins
enhance vasodilation and edema formation, while
leukotrienes are potent chemoattractive agents for
leukocytes, especially neutrophils, and stimulate
degranulation and the release of damaging lysosomal
enzymes and superoxide production.
The distribution of the two major pathways leading
either to prostaglandins or to leukotrienes varies
according to cell type. While most cells possess the
cyclooxygenase pathway, the 5-lipoxygenase pathway leading
to the leukotrienes is less widely distributed and is
prominent in inflammatory cells, such as neutrophils,
macrophages, monocytes and mast cells. The general scheme
for lipid mediator synthesis is illustrated in Figure 3.
The stable copper(I) complexes of this invention
inhibit the formation of prostaglandins and/or
WOg4/275g4 2 ~63 6 ~ PCT~S94/~7
26
leukotrienes by inhibiting the enzymes involved in their
formation. Referring to Figure 3, the stable copper(I)
complexes are effective inhibitors of both cyclooxygenase-
1 and cyclooxygenase-2, thereby inhibiting the formation
of prostaglandins. Similarly, the stable copper(I)
complexes are effective inhibitors of 5-lipoxygenase and
leukotriene C4 (LCT4) synthetase, thereby inhibiting the
formation of leukotrienes.
In addition, proteolysis of various cellular targets
by elastase (a neutrophil-released serine protease) at the
site of inflammation has been implicated in a number of
pathologic conditions, including emphysema, rheumatoid
arthritis, and psoriasis. Thus, inhibitors of elastase
may be used to treat, prevent or limit the breakdown of
normal tissue at the site of inflammation, and the stable
copper(I) complexes of this invention are effective
inhibitors of elastase.
The stable copper(I) complexes of this invention may
also be used in the regulation and/or modulation of lipid
metabolism in general. For example, hypercholesterolemia
and hyperlipidemia are common and serious health problems
which are treatable with the stable copper(I) complexes of
this invention.
Hypercholesterolemia has been observed in marginal
and severely copper-deficient rats, as well as other
animals, including humans (Lei, "Plasma Cholesterol
Response in Copper Deficiency," Role of Copper ;n T.ip;d
Met~hol;sm, ed: Lei, CRC Press, pages 1-24, 1990).
Elevation in serum cholesterol level has been linked to
increases in the activity of hepatic 3-hydroxy-3-
methylglutaryl coenzyme A reductase (HMG CoA reductase,
E.C.1.1.1.34) and glutathione levels (Bunce,
"Hypercholesterolemia of Copper Deficiency is Linked to
Glutathione Metabolism and Regulation of HMG CoA
Reductase," Nlltr. Rev. 51: 305-307, 1993; Kim et al.,
"Inhibition of Elevated Hepatic Glutathione Abolishes
~094~7594 PCT~S94/06~7
- ` 21636~o
Copper Deficiency Cholesterolemia," FA~R J. 6: 2467-2471,
1992).
Similar increases in the synthesis and level of other
hepatic lipids (fatty acids, triacylglycerols and
phospholipids) have been observed in copper deficient rats
(al-Othman et al., "Copper Deficiency Increases In Vivo
Hepatic Synthesis of Fatty Acids, Triacylglycerols, and
Phospholipids," Proc. Soc. ~y~. R;ol . Me~. 204(1): 97-103,
1993) and treatment with a copper(II) complex has been
shown to lower the activity of liver enzymes involved in
lipid metabolism, including acetyl CoA synthetase in vivo
(Hall et al., "Hypolipidemic Activity of Tetetrakis-mu-
(trimethylamine-boranecarboxylato)-bis(trimethylamine-
carboxylborane)-dicopper(II) in Rodents and its Effect on
Lipid Metabolism," J. Ph~r~. Sc; . 73(7): 973-977, 1984).
Conversely, it has been reported that treatment by
injection of copper(II) increased serum cholesterol
concentrations in rats, possibly by increasing the
activity of the HMG CoA reductase (Tanaka et al., "Effect
of Cupric Ions on Serum and Liver Cholesterol Metabolism,"
T.;p~ 1016-1019, 1987). Accordingly, it is believed
that copper may be an important factor in the regulation
of lipid levels.
Acetyl CoA synthetase catalyzes the formation of
acetyl CoA from acetate. As illustrated in Figure 4,
acetyl CoA can be further metabolized along many different
pathways leading primarily to the formation of cholesterol
and fatty acids or energy production. Agents which
inhibit this enzyme influence the biosynthesis of various
lipids. HMG-CoA reductase (3-hydroxy-3-methylglutaryl
coenzyme A reductase) is located biochemically later in
the lipid synthesis scheme and converts HMG-CoA to
mevalonic acid, and is the rate limiting reaction in
cholesterol biosynthesis (see Figure 4). Stable copper(I)
compounds of this invention inhibit certain key enzymes
involved in the formation of lipids, and thus serve as
W094l27594 ~ PCT~S94/~247
lipid modulating or regulating agents. (The ability of
stable copper(I) complexes to inhibit enzymes in the
formation of lipids is disclosed in further detail in
Examples 12-13.)
The stable copper(I) complexes of this invention may
also serve as modulating agents of signal transduction in
cells. Most intracellular signaling processes are
regulated by reversible phosphorylation of specific
proteins by kinases. Breakdown of phosphatidylinositol
leads to the formation of diacylglycerol and inositol
triphosphate, the former acting synergistically with
calcium to activate Protein Kinase C (PKC), resulting in
translocation of the enzyme from cytosol to the membrane.
Phosphorylation of proteins by PKC has been implicated as
a pivotal regulatory element in signal transduction,
cellular regulation and tumor promotion. Inhibitors of
PKC, as well as other protein kinases, have the potential
to block proliferative signaling in tumor induction,
atherosclerosis and immune modulation.
Examples of factors which stimulate the G-protein
linked phospholipase C breakdown of phosphatidylinositol
include angiotensin II, bradykinin, endothelin, f-Met-Leu-
Phe, and vasopressin. These protein kinase C enzymes are
also directly activated by tumor promoters such as phorbol
esters. Examples of Receptor linked tyrosine kinases
include Epidermal Growth Factor, Nerve Growth Factor, and
Platelet Derived Growth Factor. Examples of cytoplasmic
tyrosine kinase activators include cytokines such as
Interleukin 2, Interleukin 3, and Interleukin 5. These
factors bind to specific lymphocyte receptors which
activate the cytoplasmic tyrosine kinase.
The action of PKC and protein tyrosine kinase action
is illustrated in Figure 5. The stable copper(I) complexes
of this invention serve as signal transduction modulating
agents by inhibiting one or more enzymes involved in
vog4n7sg4 1 636~ PCT~S94/06~7
intracellular signal transduction, including PKC and
protein tyrosine kinases.
- When administered to an animal to treat the
conditions discussed above, the stable copper(I) complexes
may first be combined with one or more suitable carriers
or diluents to yield a pharmaceutical preparation suitable
for topical, oral or parenteral application. Such
diluents or carriers, however, should not interact with
the stable copper(I) complex to significantly reduce the
effectiveness thereof, or oxidize copper(I~. Effective
administration will preferably deliver a dosage of
approximately 0.01 to 100 mg of the stable copper(I)
complex per kg of body weight.
Methods for encapsulating compositions (such as in a
coating of hard gelatin) for oral administration are well
known in the art (see, e.g., Baker, Richard, Controlled
Rele~se of R;olog;c~l Act;ve Ag~nts, John Wiley and Sons,
1986)(incorporated herein by reference). Suitable
carriers for parenteral application (such as intravenous,
subcutaneous or intramuscular in~ection) include sterile
water, physiological saline, bacteriostatic saline (saline
containing 0.9 mg/ml benzyl alcohol) and phosphate-
buffered saline. The stable copper(I) complexes may be
topically applied in the form of liquids, containing
pharmaceutically acceptable diluents (such as saline and
sterile water) or may be applied as lotions, creams or
gels, containing additional ingredients to impart the
desired texture, consistency, viscosity and appearance.
Such additional ingredients are familiar to those skilled
3 0 in the art and include emulsifying agents such as non-
ionic ethoxylated and nonethoxylated surfactants, fatty
alcohols, fatty acids, organic or inorganic bases,
preserving agents, wax esters, steroid alcohols,
triglyceride esters, phospholipids such as lecithin and
cephalin, polyhydric alcohol esters, fatty alcohol esters,
hydrophilic lanolin derivatives, hydrophilic beeswax
W094~7594 PCT~S94/~247
216~64 ~~
- 30
derivatives, hydrocarbon oils such as palm oil, coconut
oil, mineral oil, cocoa butter waxes, silicon oils, pH
balancers and cellulose derivatives.
Topical administration may by accomplished by
applying an amount of the preparation directly to the
desired area, such as a wound or an inflamed area. The
required dosage will vary according to the particular
condition to be treated, the severity of the condition,
and the duration of the treatment. Preferably, when the
stable copper(I) complex is topically applied in the form
of a lotion, cream or gel, the preparation may contain
about 1% to about 20% of a penetration enhancing agent.
Examples of penetration enhancing agents include
dimetnylsulfoxide (DMSO), urea and eucalyptol. In the
case of a liquid preparations for topical application, the
concentration of penetration enhancing agent (such as
DMSO) may comprise about 30% to about 80~ of the
preparation.
In addition to the activity discussed above, the
stable copper(I) complexes of this invention also possess
utility as hair growth agents. Hair loss is a common
affliction of humans, the most common being "alopecia"
where males lose scalp hair as they get older (also called
"male pattern baldness"). Other hair loss afflications
include alopecia areata (AA), female pattern baldness and
secondary alopecia (e.g., hair loss associated with
chemotherapy and/or radiation treatment). The stable
copper(I) complexes of this invention are particularly
useful in stimulating hair growth associated with any hair
30 loss afflication, including the specific afflications
identified above.
Hair is normally divided into two types, "terminal"
and "vellus" hairs. Terminal hair is coarse, pigmented
hair which arises from follicles which are developed deep
within the dermis. Vellus hairs are typically thin, non-
pigmented hairs which grow from hair follicles which are
V094~7594 ~ 636 PCT~S94/06~7
smaller and located superficially in the dermis. As
alopecia progresses, there is a change from terminal to
vellus type hair. Other changes that contribute to
alopecia are alterations in the growth cycle of hair.
Hair typically progresses through three cycles, anagen
(active hair growth), catagen (transition phase), and
telogen (resting phase during which the hair shaft is shed
prior to new growth). As baldness progresses, there is a
shift in the percentages of hair follicles in each phase,
with the majority shifting from anagen to telogen. The
size of hair follicles is also known to decrease while the
total number remains relatively constant.
As mentioned above, the stable copper(I) complexes of
this invention have utility as stimulating agents for the
growth of hair in warm-blooded animals. In one embodiment
of the present invention, the copper(I) complex may be
administered intradermally in the area to be treated,
along with a suitable vehicle, at a concentration of
approximately 100-500 micrograms of copper(I) complex per
0.1 ml of vehicle. Suitable vehicles in this regard
include saline, sterile water, and the like.
In another embodiment, the stable copper(I) complex
may be topically applied in the form of a liquid, lotion,
cream or gel by applying an effective amount of the
topical preparation directly to the scalp. Any quantity
sufficient to stimulate the rate of hair growth is
effective, and treatment may be repeated as often as the
progress of hair growth indicates. Preferably, suitable
topical hair growth preparations contain from about 0.1~
30 to about 20~ by weight of the stable copper(I) complex
(based on the total weight of the preparation).
Topical hair growth preparations of the present
invention may contain about 0.5~ to about 10~ of an
emulsifying or surface active agent. Non-ionic surface
active agents and ionic surface active agents may be used
for the purposes of the present invention. Examples of
WOg4127~94 ~ PCT~S94/~24-
2~636 ~ ~
suitable non-ionic surface active agents are
nonylphenoxypolyethoxy - ethanol (Nonoxynol-9),
polyoxyethylene oleyl ether (Brij-97), various
polyoxyethylene ethers (Tritons), and block copolymers of
ethylene oxide and propylene oxide of various molecular
weights (Pluronic 68, for example). Acceptable
preparations may also contain about 1~ to about 10~ of
certain ionic surface active agents. These ionic surface
active agents may be used in addition to or in place of,
the non-ionic surface active agents. Examples of ionic
surface active agents are sodium lauryl sulfate and
similar compounds.
In addition to, or in place of, the emulsifying or
surface active agent, topical hair growth preparations of
this invention may contain about 1~ to about 20~ of a
penetration enhancing agent. Examples of penetrating
enhancing agents are DMSO and Urea. In the case of a
liquid preparation to be applied topically, the
concentration of a penetrating enhancing agent, such as
DMSO, may comprise about 30~ to about 80~ of the topical
preparation. The balance of the topical hair growth
preparation may comprise an inert, physiologically
acceptable carrier. Suitable carriers include, but are
not limited to, water, physiological saline,
bacteriostatic saline (saline containing 0.9 mg/ml benzyl
alcohol), petrolatum based creams (e.g., USP hydrophilic
ointments and similar creams, Unibase, Parke-Davis),
various types of pharmaceutically acceptable gels, and
short chain alcohols and glycols (e.g., ethyl alcohol and
propylene glycol).
The following are examples of suitable hair growth
preparations within the context of the present invention:
Prep~ r~ t;on A:
Copper(I) Complex 10.0~ (w/w)
Hydroxy Ethyl Cellulose 3.0~
'094/27594 636~o PCT~S94/~247
33
Propylene Glycol 20.0~
Nonoxynol-9 - 3.0%
Sodium Lauryl Sulfate 2.0
Benzyl Alcohol 2.0
0.2N Phosphate Buffer 60.0
Prep~rat;on R
Copper(I) Complex 10.0~ (w/w)
Nonoxynol-9 3.0%
Ethyl Alcohol 87.0
Prep~r~tion C:
Copper(I) Complex 5.0~ (w/v)
Ethyl Alcohol 47.5
Isopropyl Alcohol 4.0
Propylene Glycol20.0
Laoneth-4 1.0
Water 22.5
Preparat;on D:
Copper(I) Complex 5.0~ (w/v)
Water 95.0
Prepar~t;on ~:
Copper(I) Complex 5.0% (w/v)
Hydroxypropyl Cellulose 2.0
Glycerin 20.0
Nonoxynol-9 3.0
Water 70.0
Prep~r~tlon F:
Copper(I) Complex 1.0~ (w/w)
Nonoxynol-9 5.0
Unibase Cream 94.0
Prep~r~tion G:
Copper(I) Complex 2.0~ (w/w)
Nonoxynol-9 3.0
Propylene Glycol50.0
Ethanol 30.0
Water 15.0
W094/27S94 36~ PCT~S94/06247
~,~6
34
The copper(I) complexes of the present invention also
posses utility as anti-viral agents, and are particularly
effective in the inhibition of the AIDS virus. Human
acquired immunodeficiency syndrome or "AIDS" is a fatal
disease for which there is presently no cure. The disease
is believed to be caused by a virus known as the human
immunodeficiency virus, commonly referred to as "HIV."
The virus is transmitted by HIV-infected individuals
through the exchange of bodily fluids. HIV infection
results most commonly from sexual contact with an infected
partner and the sharing among intravenous drug users of
hypodermic syringes previously used by an infected
individual. A pregnant HIV-infected mother may infect her
unborn child by trans-placental transmission, and HIV-
contaminated blood is a possible source of infection forindividuals subject to blood transfusion.
HIV infection causes a suppression of the immune
system. The immune suppression renders the infected
individual vulnerable to a variety of opportunistic
infections and conditions that are otherwise kept in
balance by a healthy immune system. Fatalities result
from HIV infection due to the inability of AIDS patients
to respond to treatment of the opportunistic infections
and conditions as a consequence of their compromised
immune systems. Because the virus may often remain
dormant, the manifestation of AIDS from HIV infection may
take as long as ten years.
One approach to the treatment of AIDS has targeted
the opportunistic infections or conditions which result
from HIV infection. The treatment of such infections or
conditions, however, is ultimately ineffective and, while
prolonging the life of the infected individual, does not
treat the underlying HIV infection. A second approach to
the treatment of AIDS targets the cause of the disease
itself. Because AIDS results from viral infection, it is
believed that viral inactivation may ultimately provide a
'094/27~94 21 636q~ PCT~S94/06~7
cure. Materials which are capable of viral inactivation
or inhibition are referred to herein as "antiviral
- agents."
To understand the mode of action of antiviral agents
in the treatment of AIDS, an understanding of the process
of HIV infection is necessary. HIV chronically infects
specific immune cells known as T-helper cells, which are
required for normal immune response. The HIV infected T-
helper cells serve as hosts to the virus and facilitate
the reproduction of the virus (the process of viral
reproduction is commonly referred to as "replication").
After HIV infection, the infected host cell eventually
dies, the replicated HIV virus is released, and the
infection spreads to additional cells. This cycle
continues unabated, depleting the population of T-helper
cells and, in time, weakens the immune system to the onset
of AIDS symptoms. Because T-helper cells are continuously
produced by the body, the population of these cells may be
reestablished in the absence of further HIV infection.
Therefore, the progression of HIV infection (and the
subsequent onset of AIDS) may be arrested by the
prevention or inhibition of viral replication, and
antiviral agents capable of inhibiting or preventing the
replication of HIV should be effective in the treatment of
AIDS.
At the genetic level, HIV replication requires the
insertion of viral deoxyribonucleic acid ("DNA") into the
genome of the host cell. The genome of the host cell
consists of the cell's own DNA, and is responsible for the
synthesis of materials essential to the cell's own
function and proliferation. Once the viral DNA is
inserted into the host genome, the host facilitates
replication of HIV. The inserted viral DNA is an
enzymatic product derived from viral ribonucleic acid
("RNA") and the action of an enzyme known as HIV reverse
transcriptase. Inhibition of HIV reverse transcriptase
W094l27594 ~636 PCT~S94/06247
36
precludes the formation of viral DNA required for
insertion into the genome of the host. Viral replication
is prevented by the absence of viral DNA in the host cell
genome. Antiviral agents which inhibit HIV reverse
transcriptase are thus potential therapeutic drugs for
treatment of AIDS.
Accordingly, in yet another embodiment of the present
invention, antiviral agents are disclosed for inhibiting
HIV replication, as well as methods relating to the
administration thereof to an HIV-infected patient. The
antiviral agents of this invention are the stable
copper(I) complexes discloses above, and the methods
include administration of a therapeutically effective
amount of a composition which includes a stable copper(I)
complex in combination with a pharmaceutically acceptable
carrier or diluent. Although not limited by the following
theory, it is believed that the copper(I) complexes of
this invention enhance transport of copper(I) into HIV
infected cells which, in turn, inhibits or inactivates HIV
protease and thus inhibits the replication of HIV. As
used herein, the term "HIV" includes the various strains
of the virus such as HIV-1 and HIV-2.
Administration of the stable copper(I) complexes of
the present invention may be accomplished in any manner
which will result in a systemic dose of a therapeutically
effective amount of the copper(I) complex to an HIV-
infected animal or patient (including human patients).
For example, such administration may be by injection
(intramuscular, intravenous, subcutaneous or intradermal),
oral, nasal, or suppository applications. Typically,
preparations of the present invention include stable
copper(I) complexes in solution for various forms of
injection, or in preparations which are formulated for the
sustained release of the stable copper(I) complexes for
oral, nasal, or suppository dosage application and
generally include one or more inert, physiological
-V094/27594 21 63 6~D PCT~S94/06247
acceptable carriers. As used herein, the term "effective
amount" means an amount of the stable copper(I) complex
which inhibits HIV replication in the patient. Suitable
dosages may range from approximately 0.01 to 100 mg of
stable copper~I) complex per kg body weight.
The stable copper(I) complexes of this invention may
be screened for their ability to inhibit HIV replication
using known techniques. For example, HIV virus
replication may be monitored using the Cytopathic Effect
(CPE) assay disclosed by Bergeron et al. (J. V;rol.
~:5777-5787, 1992). In this assay, the degree of
infection is monitored by the appearance of fused cellular
membranes ("syncitium"). Alternatively, assays directed
to activity of HIV protease may be employed. For example,
the assays and techniques disclosed in the following
references may be employed: Ashorn et al., Proc. Natl.
Aca~. Sc'. U.S.A. 87:7472-7476, 1990; Schramm et al.,
B;ochem. R;op~ys. Res. Comml]n. 179:847-851, 1991; Sham et
al., R;ochem. R;ophys. Res. Comml]n. 175:914-919, 1991; and
Roberts et al., Sc;ence 2~:358-361, 1990. Moreover, the
ability of the stable copper(I) complexes of this
invention to inhibit HIV replication may be determined by
the assay disclosed in Example 5 herein below.
The stable copper(I) complexes of this invention, in
addition to inhibiting HIV replication, may also inhibit
replication of other viruses, including human T-cell
leukemia (HTLV) I and/or II, human herpes virus,
cytomegalo virus (CMV), encephalomyocarditis virus (EMCV),
Epstein Barr virus (EBV), human hepatitis virus, Varicella
3 0 Zoster virus, Rhinovirus, and rubella virus. One skilled
in the art could readily assay the stable copper(I)
complexes of this invention for their inhibitory activity
with regard to these viruses. For example, Example 15
illustrates the inhibitory affect of stable copper(I)
complexes of this invention on both encephalomyocarditis
virus (EMCV) and cytomegalo virus (CMV).
wo 94n7s94 ~636 4 PCT~S94/06~7
38
In addition to the biological activity of the stable
copper(I) complexes of the present invention, the multi-
dentate ligands of this invention also possess biological
activity when administered alone as the "free" multi-
dentate ligand (i.e., without copper(I)). Such biologicalactivity includes the activities identified above,
including anti-viral activity, as well as a preventative
agent against gastric tissue damage. Although not
intending to be limited to the following theory, when the
multi-dentate ligands of this invention are administered
as the free ligand, it is believed that they function, at
least in part, by scavenging copper(I) to yield the stable
copper(I) complex ' n V ' VO .
The following examples are offered by way of
illustration, and not by way of limitation.
F.XZ~MPT .F..C~
The examples which follow illustrate the preparation
and utility of certain exemplary embodiments of the stable
copper(I) complexes of the present invention. To
summarize the examples that follow: Example 1 illustrates
the synthesis of neocuproine copper(I) at a molar ratio of
1:1 and 2:1; Example 2 illustrates the superoxide
dismutase (SOD)-mimetic activity of representative
copper(I) complexes of this invention (employing a
copper(II)-peptide complex as a positive control); Example
3 illustrates the wound healing activity of a
representative copper(I) complex of this invention;
Example 4 illustrates hair growth activity of a
representative copper(I) complex of this invention;
Example 5 illustrates inhibition of HIV replication by a
representative copper(I) complex of this invention;
Example 6 illustrates the activity of a representative
"free" multi-dentate ligand of this invention for both
wound healing and protection against ethanol-induced
gastric mucosal damage; Examples 7 and 8 illustrates the
vog4n7594 6~o PCT~S94/06247
39
inhibition of cyclooxygenase-1 and cyclooxygenase-2,
respectively, by representative stable copper(I)
- complexes; Example 9 illustrates the inhibition of 5-
lipoxygenase by representative stable copper(I) complexes;
Example 10 illustrates the inhibition of leukotriene C4
synthetase by representative stable copper(I) complexes;
Example 11 illustrates the inhibition of elastase by a
representative stable copper(I) complex; Example 12
illustrates the inhibition of acetyl coenzyme A synthetase
by representative stable copper(I) complexes; Example 13
illustrates the inhibition of HMG-CoA reductase by
representative stable copper(I) complexes; Example 14
illustrates the inhibition of HIV-1 activity by various
isomers of a representative stable copper(I) complex;
Example 15 illustrates the anti-viral activity of
representative stable copper(I) complexes and a
representative free multi-dentate ligand; Example 16
illustrates inhibition of HIV-1 and HIV-2 proteases by
representative stable copper(I) complexes; Example 17
illustrates the inhibition of HIV reverse transcriptase by
representative stable copper(I) complexes; and Examples 18
and 19 illustrate the inhibition of Protein Kinase C and
various tyrosine kinases, respectively, by representative
stable copper(I) complexes.
F~xi 3 Tr~ l e
Synthesis of Copper(I)-Neocl~roine
Neocuproine hydrate was used as received from Aldrich
Chemical Company, having the following properties: mpl61-
163C; 1H NMR (500MHz, DMS0-d6) ~ 8.32 (2H, d, J = 8.2),
7.85 (2H, s), 7.60 (2H, d, J = 8.1), 2.79 (6H, s); 13C NMR
(125MHz, DMSO-d6) ~ 158.0, 144.6, 136.1, 126.4, 125.3,
123.1, 24.9.
W094/27594 PCT~S94/0624~
"636~
A. Neocl~ro;ne Copper(I) (1:1)
Cuprous chloride (1.98g, 20.Ommol) was added to a
stirred, vacuum-degassed solution of neocuproine hydrate
(4.53g, 20.Ommol) in acetonitrile (150mL). This solution
was stirred for 2 hours. The resulting suspension was
warmed to boiling and filtered. The filtrate was boiled to
a volume of about lOOmL. This solution was allowed to
cool slowly to give dark red needles: mp280-284C(decomp.,
lit. 310-320C)(Healy et al., J. Chem. Soc. D~lton Tr~n.s.
2531, 1985); 1H NMR (500MHz, DMSO-d6) ~ 8.74 (2H, d, J =
8.2), 8.21 (2H, s), 7.95 (2H, d, J = 8.2), 2.38 (6H, s);
3C NMR (125MHz, DMSO-d6) ~ 157.6, 142.2, 137.4, 127.1,
125.9, 125.6, 25.1; Anal. calcd. for Cl4Hl2ClCuN2: C,
54.73; H, 3.94; N, 9.12; Cl, 11.54. Found: C, 54.67; H,
3.89; N, 9.04; Cl, 11.40.
B. Neocupro~ne Copper(I) (2:1)
A vacuum degassed solution of neocuproine hydrate
(4.53g, 20.Ommol) in absolute ethanol (150mL) was added to
cuprous chloride (99Omg, lO.Ommol) via cannula under an
atmosphere of nitrogen. The resulting bright red solution
was stirred at room temperature for 2 hours. This mixture
was filtered, to remove a small amount of insoluble
matter, and evaporated to give 5.64g (100~) of bright red
solid. Recrystallization from aqueous methanol gave very
fine needles: mp231-233C; W-vis ~max (95~ ethanol) 207nm
(~ = 63,750M~lcm~l), 226nm (~ = 76,250), 272nm ( = 60,000),
454nm (~ = 6,750), 1H NMR (500MHz, DMSO-d6) ~ 8.75 (2H, br
s), 8.22 (2H, s), 7.96 (2H, br s), 2.40 (6H, s); 13C NMR
(125MHz, DMSO-d6) ~ 157.6, 142.2, 137.3, 127.1, 125.8,
125.6, 25.0; Anal. calcd. for C28H24ClCuN4: c, 65.24; H,
4.69; N, 10.87; Cl, 6.88; Cu, 12.33. Found: C, 65.01; H,
4.73; N, 10.75; Cl, 6.84; Cu, 12.70.
YO94/27594 PCT~S94/06~7
2l636~o
F.~ e
.~l~erox;~e D;sml~t~.~e M;~et;c Act;v;ty
of Copper(I) Co~plex
As used herein, compounds which pos$ess activity in a
superoxide dismutase (SOD) assay are termed "SOD
mimetics." In this example, representative copper(I)
complexes of this invention were evaluated for SOD mimetic
activity as measured by the Xanthine Oxidase/NBT method
(see Oberly and Spitz, H~n~hook of Metho~.q for Oxygen
~;c~l Rese~rch, R. Greenwald (ed.), pp. 217-220, 1985;
Auclair and Voisin, H~n~hook of Metho~.~ for Oxygen R~;cal
Rese~rch, R. Greenwald (ed.), pp. 123-132, 1985). The
reactions contained the following: 100 uM Xanthine, 56 uM
NBT (Nitro Blue Tetrazolium), 1 unit of Catalase, 50 mM
Potassium Phosphate Buffer, pH 7.8. The reaction was
initiated by the addition of Xanthine Oxidase in
sufficient quantity to obtain an increase in absorbance at
560 nm of approximately 0.025/min. in a total volume of
1.7 ml. The Xanthine Oxidase was prepared fresh daily and
stored on ice until used. All the components of the
reaction are added except the Xanthine Oxidase and the
spectrophotometer was adjusted to zero at 560 nm. The
reaction was initiated by the addition of the Xanthine
Oxidase. All reagents were obtained from Sigma Chemical
Co .
Measurements of the Absorbance at 560 nm were taken
at 1-2 minute intervals for at least 16 minutes. The
control consisted of reactions containing zero copper(I)
30 complex. The copper(I) complexes tested in this example
were as follows: bathocuproine disulfonate copper(I)
("BCDS:Cu(I)"); neocuproine copper(I) ("NC:Cu(I)~); and
2,2'-biquinoline copper(I) ("BQ:Cu(I)"). As a positive
control, reactions containing a peptide-copper(II) complex
(i.e., glycyl-L-histidyl-L-lysine:copper(II) or "GHK:Cu"),
which is a known SOD mimetic (see U.S. Patent No.
W094~7594 PCT~S94/06~-
636 ~
4,760,051), were also employed. One unit of SOD activity
was taken as that amount -of sample in micromoles which
inhibits the control reaction with the NBT by 50~. The
relative activity is then obtained by comparing the
micromoles of copper(I) complex necessary to product a 50
inhibition of the control reactions. The lower the value,
the more active the compound is as an SOD mimetic. The
results of this experiment are presented in Table 8 below.
T~hle 8
SOD-Mimet;c Act;vity of Copper(I) Co~plexes
Cop~er Activity Rel~tive
~xp. No. Co~o-~n~ Ratio (~1 per Activity to
(lig~nd:Cu) ~M~x. I~h;h. ) Control
1GHK:Cu(II) 2:1 0.055 ---
BCDS:Cu~I) 2:1 0.034 1.6
2GHK:Cu(II) 2:1 0.0503 ---
BCDS:Cu(I) 2:1 0.0278 1.8
BCDS:Cu(I) 2:1 0.0018 28
NC:Cu(I) 2:1 0.0014 36
3GHK:Cu(II) 2:1 0.0479 ---
NC:Cu(I) 1:1 0.0018 27
BQ:Cu(I) 2:1 0.0028 17
Fx~m~l e 3
Wol]n~ He~l;ng Activity
of Copper(I) Co~lexes
The subcutaneous implantation of stainless steel
20 wound chambers in rats provides a model for the healing of
open cavity wounds. Implantation of these chambers
triggers a series of responses which reflect the series of
~rog4/27594 21636~D PCT~S94/06247
43
phases involved in wound healing - fibrin clot formation,
infiltration of white cells, collagen synthesis, and new
blood vessel formation.
This assay involves the implantation of a stainless
steel chamber (1 X 2.5 cm cylindrical 312 SS, 20 mesh,
with Teflon end caps) on the dorsal mid-line of rats.
After one week to allow for encapsulation of the chamber,
the chamber on each rat was injected with a 0.2 ml saline
solution containing 2.7 ~mol of the copper(I) complex
(i.e., BCDS copper(I) 1:1 or 2:1), or with the same volume
of saline (0.2 ml) without the copper(I) complex (i.e.,
control). Injections were made on days 5, 7, 9, 12, 14,
16 and 19. The chambers were then removed on day 21.
The chambers were lyophilized and the interior
contents removed for biochemical analysis. The
biochemical parameters examined included the total dry
weight, protein content, collagen content (i.e.,
hydroxyproline content after acid hydrolysis) and
glycosaminoglycan content or "GAG" (i.e., uronic acid
content after acid hydrolysis).
The protein was determined by the method of Lowry
et al. (J. B;ol. Che~. 193: 265-275, 1951) using Bovine
Serum Albumin (BSA) as a standard. The collagen content
was determined by acid hydrolysis and a colorimetric assay
for hydroxyproline (Bergman et al., Cl;n. ~h;m. Acta
~1:347-349, 1970), an amino acid specific for collagen.
Glycosaminoglycan content was determined by quantitation
of the amount of uronic acid (UA). Aliquots of the
homogenate were dissolved in 0.5M NaOH, precipitated and
washed with ethanol, and uronic acid was determined by a
colorimetric assay using 2-phenylphenol as a reagent
(Vilim V., Rlome~. R;ochem. Act~. 44(11/12s):1717-1720,
1985). Glycosaminoglycan content was expressed as ~g of
uronic acid per chamber.
The results of this experiment are illustrated in
Figure 1. Specifically, BCDS copper(I) at both the 1:1
W094/27594 ~63~ PCT~S94/06247
and 2:1 ratio significantly stimulated the
glycosaminoglycan content- of the injected chamber.
Moreover, BCDS copper(I) at both ratios stimulated the
collagen content of the injected chambers. Collagen and
glycosaminoglycans are two of the critical extracellular
matrix components important for tissue regeneration
associated with wound healing.
F~x;~ e 4
St;m~ tlon of H~;r Growth hy Copper(I) Co~lexes
The following example illustrates the stimulation of
hair growth in warm-blooded animals after intradermal
injection of a copper(I) complex of this invention.
The backs of C3H mice (60 days old, telogen hair
growth phase) were closely clipped on day 1 using an
electric clipper. A sterile saline solution containing
the indicated copper complex was then injected
intradermally (i.e., infiltrated under the skin) at two
locations within the clipped areas of the mice. Injection
at two locations provided two test locations within the
clipped area of each mouse. Each injection (0.1 ml)
contained the indicated amount of the copper(I) complex
(i.e., BCDS copper(I) (1:1) complex at 0.14 ~mol and 1.4
~mol) within a sterile saline solution. A group of saline
injected mice (0.1 ml) served as controls. Following
injection of the copper(I) complex, indications of hair
growth were seen within 10 days. The first visual signs
were a darkening of the skin in a circular region
surrounding the injection site. The size of this region
is generally dose dependent, increasing with an increase
in dose. The 0.1 ml injections used in this experiment
produced a circle of hair growth measuring approximately
0.5 cm2 to 5 cm2 in diameter. Active hair growth occurred
between 14-20 days following injection, with a maximum
effect seen by day 29. Both the number of mice growing
~094/27594 ~ 6~ us94/06247
hair at the injection site and the diameter of the hair
growth region were determined at day 21. A positive
response was expressed as the number of mice exhibiting
hair growth at the injection sites compared to the total
number of mice injected in the study. The results of this
experiment are presented in Table 9 below.
T~hle 9
H~-r Growth Act-v;ty of RCDS Copper(I) Co~plex
Amollnt Injecte~ ~ol) Growth Area (cm~
0.0 (control) o.o
0.14 1.35 (Std. Dev. 0.42)
1.4 3.06 (Std. Dev. 0.47)
F~x~ e 5
~h;h;t;on of HIV Repl;cation
of Copper(I) Co~lex
In this experiment, the inhibitory effect of
bathocuproine disulfonic acid (BCDS) copper(I) (2:1)
complex on phytohemagglutinin (PHA) stimulated peripheral
blood mononuclear cells is demonstrated.
PHA stimulated peripheral blood mononuclear cells
(PBMC) were infected by HIVIIIB in the presence of the
copper(I) complex identified above and cultured in the
presence of the copper(I) complex for two weeks. The
extent of HIV replication was assayed at 1 and 2 weeks by
a p24 antigen capture ELISA assay. More specifically,
PBMC was stimulated with PHA for 24 to 72 hours in basal
medium, containing RPMI-1640, 10~ fetal bo~ine serum, and
50 ~g/mL gentamicin, and then cultured overnight in the
presence of 250 units/ml IL-2. Treated PBMC were pelleted
by centrifugation and resuspended to 0.75 x 106/mL in basal
medium with appropriate dilutions of the copper(I) complex
or with no copper(I) complex added (i.e., control). To
each 0.5 mL aliquot of cells, 0.5 mL of appropriate HIV
W094~7594 PCT~S94/06~-
~,~636 ~ '
46
dilution was added. The virus-cell mixture was incubated
for 2 hours at 37C in a-5~ CO2 humidified atmosphere.
Following the incubation period, the PBMC were washed
twice in phosphate-buffered saline. Cells were
resuspended in 5 mL to 7 x 104 cells/mL in basal medium
with (or without) the copper(I) complex. Each cell
aliquot was dispensed into four replicate wells of a 48
well tissue culture plate. Cells were fed twice a week
with appropriate medium.
At one week and two week culture timepoints the
extent of HIV replication was assayed by a p24 antigen
capture assay kit (Coulter Corp., Hialeah, Florida). PBMC
were treated with buffered detergent to release viral
proteins. The cell extract was absorbed to immunoassay
titer plates and p24 was detected by binding of a
monoclonal anti-p24 antibody coupled to an enzyme.
Following the addition of a chromogenic substrate, the
amount of p24 was quantified spectrophotometrically.
The results of this experiment are presented in
Figure 2. In particular, a 50 uM concentration of the
BCDS copper(I) (2:1) complex completely inhibited HIV
replication at both week 1 and week 2 at the identified
virus dilutions. Furthermore, the 5 uM concentration of
BCDS copper(I) (2:1) complex completely inhibited HIV
replication at week 2 at the 10-6 virus dilution.
~x~mple 6
Activlty of "Free" Mnltl-Dent~te T,l g~nd
This example illustrates the activity of the free
multi-dentate ligands of this invention. As used herein,
the free ligand is not complexed to the copper(I) ion
prior to administration.
YO 94n7594 63~ PCT/US94/06247
47
A. Inh;h;t;on of F.th~nol-In~nce~ G~str;c Mncos~l D~m~ge
Juvenile Sprague-Dawley rats were used in this
- example. After fasting for 24 hours, the rats were
treated by oral gavage with bathocuproine disulfonic acid
-5 (BCDS) as the copper(I)-free ligand at various dosages
(i.e., 0, 7.6 and 37.6 mg/kg body weight). One hour after
BCDS treatment, the animals were challenged with 1 ml of
95~ ethanol by oral gavage to cause erosion of the gastric
mucosa. As shown in Table 10, BCDS pre-treatment led to a
dose-dependent protection against the mucosal damage
observed in the control animals.
T~hle 10
~ffect of BCDS on ~th~nol-In~uce~
G~str;c Ml~cosal Damage
Dosage Mucosal D~m~ge
mg/kg hody we;ght ~ of total ~rea
~n S.~.M.
0.0 45.48 6.94
7.6 32.95 7.49
37.6 23.45 8.18
B. Wonn~ He~l;ng Act;v;ty
The BCDS ligand was also examined in the rat wound
20 chamber model as disclosed above in Example 3. The
results of this experiment are presented in Table 11.
T~hle 11
~ffect of RCDS on wonn~ He~ling
~g/;nject;on ug nron;c ~c;~/mg prote;n
0.0 (control) 28.3 ( Std. Dev. + 8.7)
1.5 57.6 (Std. Dev. + 9.1)
7.5 79.2 (Std. Dev. + 10.8)
W094~7594 ~ ~ 6 3 ~ 4 PCT~S94/~24
48
These results indicate that glycosaminoglycan synthesis is
stimulated by administration of the free BCDS ligand.
~x~mple 7
Inh; h;t;o~ of Cyclooxygen~e-1 hy
Neocl~ro;ne ~n~ RCDS Copper(I) Co~plexes (2:1)
Cyclooxygenase is involved in the formation of
prostaglandins and thromboxanes by the oxidative
metabolism of arachidonic acid (see Figure 3).
In this experiment, cyclooxygenase-1 from ram seminal
vesicles was incubated with arachidonic acid (100 uM) for
2 minutes at 37 C in the presence or absence of
neocuproine copper(I) (2:1) or BCDS copper(I) (2:1) at
increasing concentrations of neocuproine copper(I) or BCDS
copper(I) from 0.3 to 300 ~M. The assay was terminated by
the addition of trichloroacetic acid (TCA), and
cyclooxygenase-1 activity was determined by reading the
absorbance at 530 nm (Evans et al., "Actions of Cann~his
Constituents on Enzymes of Arachidonate Metabolism:Anti-
inflammatory Potential," R;ochem. Ph~r~col. 36: 2035-
2037, 1987; Boopathy and Balasubramanian, "Purification
and Characterization of Sheep Platelet cyclooxygenase,~
B;ochem J. ~: 371-377, 1988).
Neocuproine copper(I) (2:1) was found to inhibit
cyclooxygenase-1 with an IC50 of 23~M (see Table 12).
BCDS copper(I) (2:1) complex produced approximately 44~
inhibition at a concentration of 300 ~M. These results
demonstrate that the stable copper(I) complexes of this
invention are potent inhibitors of prostaglandin synthesis
through inhibition of cyclooxygenase-1.
-v094~7594 PCT~S94/06~7
2~6~6
T~hle 12
T~h; h; t;on of Cyclooxyg~n~se-l hy
St~hle Copper(I) Co~plexes
CO~ol~n~ Conc.Percent Inhl h; t;on
(~M)(Mean + SEM)
BCDS Copper(I) (2:1) 300 43.5 + 1.5
Neocuproine Copper(I) (2:1) 300 77.3 + 1.5
54.5 + 0.5
3.0 15.5 + 2.5
0.3 6.5 + 0.5
F.x~m~l e 8
I~hi h;t;o~ of Cyclooxygen~se-2 hy
Neocl~ro;ne ~n~ RCDS Copper(I) Co~lexes (2:1)
Cyclooxygenase-2, also known as prostaglandin H
synthase-2, catalyzes the oxygenation of un@sterified
precursors to form cyclic endoperoxide derivatives,
including prostaglandin H (see Figure 3).
In this experiment, cyclooxygenase-2 from sheep
placenta, 80 units/tube, was pre-incubated with 1 mM
glutathione (GSH), 1 mM hydroquinone, 2.5 ~M hemoglobin,
and either neocuproine copper(I) (2:1) or BCDS copper(I)
(2:1) at increasing concentrations of neocuproine
copper(I) or BCDS copper(I) from 0.3 to 300 ~M for 1
minute at 25C. The reaction was initiated by the addition
of arachidonic acid (100 ~M), and terminated after 20
minutes at 37 C by the addition of TCA. Following
centrifugal separation of the precipitated protein,
thiobarbiturate was added and cyclooxygenase activity was
determined by absorbance at 530 nm (see Evans et al.,
- sl~ra; Boopathy and Balasubramanian, sl~r~; O'Sullivan et
al., 'ILipopolysaccharide Induces Prostaglandin H Synthese-
2 in Alveolar Macrophages," R; ochem. R; Op~ys . Res. Comml~n.
187:1123-1127, 1992).
wo94n7594 PCT~S94/06247
~ 1636 ~ 50
Neocuproine copper(I) (2:1) was found to inhibit
cyclooxygenase-2 at an estimated IC50 of 25~M (see Table
13), which is similar to the results of Example 7 with
cyclooxygenase-1. BCDS copper(I) (2:1) produced
approximately 34~ inhibition at the screening
concentration of 300 ~M. These results show that stable
copper(I) complexes of this invention are also potent
inhibitors of prostaglandin synthesis through inhibition
of cyclooxygenase-2.
T~hle 13
I~h;h;t;on of Cyclooxyge~se-2 hy
StAhle Copper(I) Com~lexes
Com~ol~n~ Conc. Percent Inh'hit'on
(~M) (Mean + SEM)
BCDS Copper(I) (2:1) 30034.0 + 1.0
Neocuproine Copper(I) (2:1) 30063.8 + 0.5
30 54.0 + 1.0
3.0 7.0 + 1.0
0.3 6.5 + 2.5
~x~m~1e 9
I~h;h;t;on of 5-T.;poxygen~se hy
Neocl~pro;ne ~n~ RCDS Copper(I) Complexes (2:1)
The 5-lipoxygenase is the principal lipoxygenase in
basophils, polymorphonuclear (PMN) leukocytes,
macrophages, mast cells, and any organ undergoing an
inflammatory response. As illustrated in Figure 3, ~he
action of 5-lipoxygenase leads to the formation of 5-HPETE
and 5-HETE, which are precursors to the leuokotriene LTB4
and LTC4.
In this experiment, 5-lipoxygenase assays were run
using a crude enzyme preparation prepared from rat
~094/27594 51 ~1 PCT~S94/06~7
basophilic leukemia cells (RBL-1). Neocuproine copper(I)
(2:1) or BCDS copper(I) (2:-1) at increasing concentrations
from 0.3 to 300 ~M were pre-incubated with the 5-
lipoxygenase for 5 minutes at room temperature, and the
reaction was initiated by addition of arachidonic acid
substrate. After incubation at room temperature for 8
minutes, the reaction was terminated by the addition of
citric acid. The levels of 5-HETE were determined by a
specific 5-HETE RIA (Shimuzu et al., "Enzyme with Dual
Lipoxygenase Activities Catalyzes Leukotriene A4 Synthesis
from Arachidonic Acid," Proc. N~tl. Aca~. Sc;. U.S.A.
81:689-693, 1984; Egan and Gale, "Inhibition of Mammalian
5-Lipoxygenase by Aromatic Disulfides," J. Ri ol . Chem.
260:11554-11559, 1985).
Both BCDS copper(I) (2:1) and neocuproine copper (I)
(2:1) were found to be inhibitors of 5-lipoxygenase with
estimated IC50's of less than 10 ~M (see Table 14). These
results show that stable copper(I) complexes of this
invention are potent inhibitors of neutrophil 5-
lipoxygenase, thus preventing the accumulation of
inflammatory lipid mediators at the sites of inflammation.
T~hle 14
Inh;h;tion of 5-T,;poxygenase h~
St~hle Copper(I) Co~lexes
Co~pol~nd Conc.Percent Inh;hit;on
(~M)(Mean + SEM)
BCDS Copper(I) (2:1) 30 71.3 i 2.5
3.029.0 + 5.0
0.3 5.5 + 3.5
0-03 4.0 + 1.0
Neocuproine Copper(I) (2:1) 30 99.0 + 0.6
3.051.0 t 6.0
0.315.5 + 2.5
0.03 7.0 + 0.0
wo 94n7sg4 ~6 52 PCT~S94/0624~
~x~mple 10
Inh'h;t;on of T.ellkotr;ene C1 Synthet~se by
Neocl~ro;ne ~n~ BCDS Copper(I) Co~lexes (2:1)
Leukotriene C4 (LTC4) Synthetase is involved in the
formation of LTC4 from LTA4, as illustrated in Figure 3,
by the addition of a reduced glutathione at the C6 site.
In this example, LTC4 Synthetase was prepared as a
crude fraction from rat basophilic leukemia cells (RBL-1).
The crude enzyme fraction was incubated with test
compounds, LTA4 methyl ester, albumin (to stabilize the
product), and serine borate (to prevent conversion of LTC4
to LTD4) for 15 minutes at 37 C. The reaction was
terminated by the addition of ice cold methanol, and LTC4
concentration was determined by a specific RIA (Bach et
al., "Inhibition by Sulfasalazine of LTC4 Synthetase and
of Rat Liver Glutathione S-Transferases," R; ochem.
Pharmacol. 34:2695-2704, 1985; Fitzpatrick et al.,
"Albumin Stabilizes Leukotriene A4," J. Rlol . Chem.
257:4680-4683, 1982).
Both BCDS copper(I) (2:1) and neocuproine copper(I)
(2:1) were found to be inhibitors of LTC4 Synthetase with
estimated IC50's of 87 and 285 ~M, respectively (see Table
15). These results show that stable copper(I) complexes
are potent inhibitors of neutrophil LTC4 Synthetase, thus
preventing the accumulation of inflammatory lipid
mediators at the sites of inflammation.
-~o s4n7ss4 636~o PCT/US94/06247
T~hle 15
I~h;h;t;on of T,el~kotr;e~e Cl (TTCl) Sy~thet~se by
St~hle Copper(I) Co~lexes
Co~pol~n~ Conc. Percent Inh;h;t;on
(~M)(Mean + SEM)
BCDS Copper(I) (2:1) 1000 77.8 + 1.9
100 51.0 + 4.0
- 10 26.5 + 1.5
1 11.0 + 2.0
Neocuproine Copper~I) (2:1)1000 71.0 + 1.9
100 32.5 + 0.5
15.0 + 1.0
9.0 + 1.0
Ex~m~le 11
I~h;h;t;on of ~l~stase hy
RCDS Copper(I) (2:1)
Proteolysis of various cellular targets by elastase
has been implicated in a number of pathologic conditions,
including emphysema, rheumatoid arthritis, and psoriasis.
In this experiment, human neutrophil was the source
of the elastase. In particular, human neutrophil elastase
was prepared in crude form from fresh blood following
dextran sedimentation, leukocyte isolation, cell lysis and
homogenization of sub-cellular granules containing the
elastase. BCDS copper(I) (2:1) was incubated with the
enzyme and substrate (methoxysuccinly-alanyl-alanyl-
propyl-valine-4-nitroanalide) for 8 minutes at 25C. The
reaction is terminated by immersing the test tubes in
boiling water for 5 minutes. Spectrophotometric analysis
of the proteolytic product is measured at 410 nm (Baugh
and Travis, "Human Leukocyte Granule Elastase, Rapid
W0941275g4 ~636 PCT~S94/06~7
Isolation and Characterization," R; ochem;stry 1~:836-841,
1976).
BCDS copper(I) ~2:1) was found to inhibit human
neutrophil elastase with an estimated IC50 of 12 ~M (see
Table 16). These results show that stable copper(I)
complexes of this invention are potent inhibitors of
neutrophil elastase, thus preventing or limiting the
breakdown of normal tissue at the sites of inflammation.
T~hle 16
Inhihition of Hl~m~n Neutroph;l ~1~stase hy
St~hle Copper(I) Com~lexes
Com~ol]nd Conc.Percent Inh;bition
(~M) (Mean + SEM)
BCDS Copper(I) (2:1) 30 65.8 + 3.1
3.0 25.0 + 5.0
0.3 18.5 + 0.5
0.03 5.5 + 0.5
Fx~m~le 12
Inh;h;t;on of Acetyl Coenzyme A (CoA) Synthetase hy
Neocuproine ~n~ BCDS Copper(I) (2:1)
In this experiment, the ability of two stable
copper(I) complexes, neocuproine copper(I) (2:1) and BCDS
copper (I) (2:1), to inhibit certain key enzymes involved
in the formation of lipids is demonstrated.
CoA synthetase (yeast) activity was monitored by
utilization of a labeled substrate, sodium [3H]acetate
(Grayson and WestKaemper, "Stable Analogs of Acyl
Adenylaes, Inhibition of Acetyl and Acy~ (acyl-CoA) CoA
Synthetase by Adenosine 5'-alkylphosphates," T~i fe Sci. ~:
437-444, 1988). A reaction buffer including 0.1 M
glycine-NaOH (pH 9.0), ATP, and the substrate was pre-
-~os4n7s~4 6~o
incubated for 5 minutes at 27C, followed by addition of 2
nM coenzyme A for an additional 5 minute incubation at 27
C. The reaction was terminated by addition of HCl, and
the remaining substrate determined by scintillation
counting.
The results of this experiment are presented in Table
17. Both BCDS copper(I) (2:1) and neocuproine copper(I)
(2:1) were found to inhibit acetyl CoA synthetase
activity.
T~hle 17
I~hih;tion of Acetyl CoA Synthet~se hy
St~hle CoDper(I) Co~Dlexes
Co~ol]n~ 1~50 (~M)
BCDS Copper(I) (2:1) 29
Neocuproine Copper(I) (2:1) 47
Reference compounds:
Ethyl-5-AMP 60
Lovastatin >100
Orotic Acid ~100
Both stable copper(I) complexes tested were found to
inhibit acetyl CoA synthetase with estimated ICs0's of 30-
50 ~M. These results indicate that the stable copper(I)
complexes of this invention may serve as lipid modulating
(e.g., lipid lowering) agents.
~x~m~le 13
Inh;h;t;on of H~G-CoA Re~llct~se hy
Neocl~ro;ne ~n~ RCDS CoDDer(I) (2:1)
In this experiment, HMG-CoA reductase was isolated
from rat liver and incubated with [14C]HMG-CoA and either
neocuproine copper(I) (2:1) or BCDS copper(I) (2:1) for 15
W094~75g4 PCT~S94/06~7
2~ 636 40
56
minutes at 37C. The reaction is terminated by addition of
HCl, and [14C] MVA is separated from the intact substrate
by column filtration (Kubo and Strott, "Differential
Activity of 3-hydroxy-3-methylglutaryl Coenzyme A
Reductase in Zones of the Adrenal Cortex," F~n~ocrlnology
120: 214-221, 1987; Heller and Gould, "Solubilization and
Partial Purification of Hepatic 3-hydroxy-3-methylglutaryl
Coenzyme A Reductase," R;ochem. R;ophys. Res. Co~m. 50:
859-865, 1973).
Testing at 30 ~M indicated that both neocuproine
copper(I) (2:1) and BCDS copper(I) (2:1) inhibited the
HMG-CoA reductase enzyme. The results of this experiment
are presented in Table 18.
T~hle 18
Inh; h; t;on of H~G-CoA Re~llct~se h~
St~hle CopDer(I) Com~lexes
Co~pol~n~ IC50
BCDS Copper(I) (2:1) >30 ~M, <50 ~M
Neocuproine Copper(I) (2:1) >30 ~M, ~50 ~M
Reference compound:
Lovastatin >12 nM
Both stable copper(I) complexes tested were found to
inhibit HMG-CoA reductase with ICsO's estimated at greater
than 30 ~M. These results indicate that the stable
copper(I) complexes of this invention may serve as lipid
modulating (e . g., lipid lowering) agents.
vo94n7sg4 2l 63 6~a PCT~S94/~247
F.x~le 14
Tnh;h;t;on of HIV-l Act;v;ty hy
RCDS Copper(I) (~:1) Tsomers
The experiments presented in this example demonstrate
the effect on anti-HIV activity of different isomers of
BCDS copper(I) (2:1). Two experiments utilized p24
antigen capture as a marker for viral replication, while
two further experiments utilized reverse transcriptase
activity to monitor the course of infection. The
infection in all three experiments was performed in
cultures of human peripheral blood mononuclear cells
( PBMC) treated with HIV-l.
The positional isomers of BCDS copper(I) employed in
this experiment are identified above as structures IIe,
IIe~ and IIe'', and are set forth below:
3 SO3Na SO3Na
Na
/ N N \ N N -
CH3 ~H3 ~H3 ~H3
IIe IIe'
NaO3 S ~03Na
~ N N
CH3 ~H3
IIe''
Structure IIe is referred to herein as the para-para
("PP") BCDS isomer since both disulfonic acid/sodium salt
W094~7594 216 3 6 ~ PCT~S94/06~'
58
moieties are located in the para position. Similarly,
structure IIe' and IIe'' are referred to herein as the
meta-para ("MP") and meta-meta ("MM") BCDS isomers,
respectively. In addition, a mixture of the PP, MP and MM
BCDS isomers was also tested (referred to herein simply as
"BCDS"), having a ratio of PP:MP:MM of approximately
5:39:56.
In the first experiment, the anti-HIV activity of
BCDS, MP-BCDS and MM-BCDS copper(I) (2:1) at two
concentrations (i.e., 10 and 25 ~M) was compared. These
concentrations had been previously determined to be
partially and completely effective, respectively, for
inhibition of HIV replication by BCDS copper(I).
The same methodology as described above in Example 5
for evaluating inhibition of HIV replication was employed
in the experiment. The results of this experiment are
present in Table 19.
T~hle 19
20Inh; h; t;on of HIV Repl;c~t;on hy
RCDS MP-RCDS ~n~ MM-BCDS Copper(I) (2:1) as Measure~ by
p24 ~ntigen C~pture (@1:1000 v;ral ~ ltlon)
Week 1
Co~olln~ (SEM) % Inhihition
Control (infected cells)30910.00 3770.00 --
BCDS Copper(I) (lO~M)1959.00317.16 93.66
BCDS Copper(I) (25~M)0.25 0.25 99.99
MP-BCDS Copper(I) (lO~M) 404.50 124.66 98.69
MP-BCDS Copper(I) (25~M) 0.50 0.50 99.99
MM-BCDS Copper(I) (lO~M) 346.50 106.27 98.88
MM-BCDS Copper(I) (25~M) 0.00 0.00 100.00
~~094/~7594 ~163 ~ PCT~594/~6~47
Week 2
CO~ol~n~ p24 (S~M) ~ Inh;h't;on
Control (infected cells)10483.801109.73 --
BCDS Copper (I) (lO~M)3286.00242.36 68.66
BCDS Copper (I) (25~M)`0.00 0.00 100.00
MP-BCDS Copper (I) (lO~M)901.75277.26 91.40
MP-BCDS Copper (I) (25~M)0.00 0.00 100.00
MM-BCDS Copper (I) (lO~M)549.50176.25 94.76
MM-BCDS Copper (I) (25~M)0.00 0.00 100.00
In a second experiment, the activity of BCDS
copper(I) and PP-BCDS copper(I) was compared in the manner
described above. The results of this experiment are set
forth in Table 20. In this experiment the p24
concentrations were lower than in the above experiment.
This is due to a different ELISA technique used in this
experiment. The standard curve for p24 detection
maximizes at 300 pg/ml. Any values over 300 require a
kinetic extrapolation to estimate the p24 concentration.
Such extrapolation gives a substantial underestimation of
the actual p24 concentration. To obtain a more accurate
estimate, a series of dilutions of the sample was made to
arrive at a reading that is in the middle of the standard
curve, and the dilution factor applied to the reading to
give the p24 concentrations. This method (which was used
in the first experiment, see Table 19 above) while more
accurate, yields an overestimate due to the errors of
dilution. Nevertheless, the comparisons from one sample
to the next in each experiment reflect the inhibitory
effects of stable copper(I) complexes tested.
W094~75g4 2 ~ 6 3 6 4 PCT~S94/06247
T~hle ~0
I~h;h;t;o~ of HIV Repl;c~t;o~ hy
BCDS ~n~ PP-RCDS Copper(I) (2~ s Me~sllre~ hy
p~4 ~nt;ge~ t~re (@1:1000 v;r~ t;on)
Week 1
Compolln~ ~2~ (SEM) ~ I~h;h;t'on
Control (infected cells)1649.7529.32 --
BCDS Copper(I) (lO~M)474.25 41.22 71.25
BCDS Copper (I) (25~M)39.50 6.06 97.61
PP-BCDS Copper (I) (lO~M)480.00 49.65 70.90
PP-BCDS Copper (I) (25~M)34.50 4.57 97.91
Week 2
Compolln~ (S~M) ~ Inh;h;t;on
Control (infected cells)2256.5045.93 --
BCDS Copper (I) (lO~M)1785.00 49.03 20.90
BCDS Copper (I) (25~M)22.00 6.38 99.02
PP-BCDS Copper (I) (lO~M)1915.7569.75 15.10
PM-BCDS Copper(I) (25~M)33.25 6.60 98.53
In a third experiment, the anti-HIV activity of BCDS,
PP-BCDS, MP-BCDS and MM-BCDS copper(I) (2:1) was
determined by monitoring the same type of culture (i.e.,
HIV-1, PBMC) by measuring the reverse transcriptase
activity as an infection marker. The PBMC culture
conditions for this experiment are described above in
Example 5. Following 6 days of incubation, the activity
of HIV-1 reverse transcriptase in cellular extracts was
determined as a marker for the replication of the virus in
culture. The measurement of HIV-1 reverse transcriptase
in PBMC cultures may be performed by known techniques
(Chattopadhyay et al., "Purification and Characterization
of Heterodimeric Human Immunodeficiency Virus Type
(HIV-1) Reverse Transcriptase Produced by an In Vitro
Processing of p66 with Recombinant HIV-1 Protease," J.
W094~7594 ~ 636~D PCT~S94/06247
61
Riol. Chem. 267:14227-14232, 1992). The results of this
experiment are presented in Table 21.
TAhle ~1
5Inhi hi t;o~ of HIV Replic~t;on ~y
BCDS. PP-RCDS. MP-~CDS ~n~ MM-RCDS Copper(I) (2:1)
~s Me~llre~ ~y Reverse Tr~nscript~se Act;v;ty
Reverse Tr~n~cript~se Activ;ty
Con~polln~ COI1C . (~M) ~1~ Inhi hi tion
None(control) 0 29283 NA
BCDS Copper(I) 0.001 23963 18.17
0.01 19585 33.12
0.1 17340 40.78
1 17623 39.82
4974 83.01
100 585 98.00
PP-BCDS Copper(I)0.001 26934 8.02
0.01 28097 4.05
0.1 12742 56.49
1 12247 58.18
1846 93.70
100 566 98.07
MP-BCDS Copper(I)0.001 19966 31.82
0.01 15040 48.64
0.1 12369 57.76
1 9880 66.26
1408 95.19
100 540 98.16
MM-BCDS Copper(I)0.001 22679 22.55
0.01 18212 37.81
0.1 18464 36.95
1 2085 92.88
583 98.01
W094~7594 ~636 62 PCTN594/~47
In a fourth experiment, inhibition of HIV-1, HIV-2
and SIV, as compared to AZT, was determined for BCDS
copper(I), PP-BCDS copper(I), MP-BCDS copper(I) and MM-
BCDS copper(I). The experimental conditions described
above where employed utilizing Reverse Transcription assay
to monitor infection. The results of this experiment are
presented in Table 22. It should be noted that the data
presented in Table 22 are report in a different format
from that of Table 21. In particular, the data of Table
22 represent the calculated EC50 values. The EC50 is
determined by non-linear regression from inhibition data
(such as that presented in Table 21), and extrapolated for
the concentration of the test compound required to
accomplish a 50~ inhibition of reverse transcriptase
activity.
T~hle ~
Inh;h;t;on of HIV-1. HIV-2 ~n~ SIV Repllc~t;on hy
BCDS. PP-BCDS. MP-RCDS ~nd MM-RCDS Copper(I) (2:1)
20~s Me~sure~ hy Reverse Tr~n~cr;pt~se Act;v;ty
~C50 (~M)
Co~ol]nd HIV-1 HIV-2 ~1
BCDS Copper(I) 1.7 17.6 4.6
PP-BCDS Copper(I) 0.25 1.2 6.4
MP-BCDS Copper(I) 0.04 12.1 4.3
MM-BCDS Copper(I) 0.13 0.62 6.1
AZT 0.004-0.009 0. 0004 0 . 0066
~x~mple 15
25~nt;-V;r~l Act;v;ty of St~hle Copper(I) Co~lexes
This example illustrates that the stable copper (I)
compounds of this invention, as well as the free ligands,
~094n7594 1 636~o PCT~S94/06247
63
have general anti-viral activity. In this experiment,
BCDS copper(I) and BCDS alone (i.e., the free ligand) were
assayed for the ability to inhibit the murine virus
encephalomyocarditis (EMCV) and the cytomegalo virus
- 5 (CMV).
I~h;h;t;on of FMCV
Cultures of As4g cells (human lung) were infected
with EMCV for 24-48 hours in the presence of either BCDS
copper(I) or BCDS alone. The cells were cultured in DMEM
(10~ FBS) for 3-4 days prior to use. The medium was then
removed, and the cells incubated with sufficient EMCV in
serum free DMEM to kill between 30-90~ of the cells in the
culture. After 2-3 hours of incubation of the cells with
EMCV in their presence (or absence) of the test compounds,
complete medium (DMEM + 10~ FBS) was added and the cells
allowed to incubate for 1-2 days in the presence or
absence of the test compounds at concentrations ranging
from 0.0001-0.0005 M.
The viability of the cultures was then measured by
mitochondrial function test (Mossman, J. Imm-]nol. Meth.
65:55-63,1983). The ability of the test compounds to
protect the cells from the lethality of the EMCV infection
was calculated as a percent protection compared to the
mitochondrial activity of parallel, uninfected cells. The
results of this experiment are presented in Table 23.
T~hle 23
I~h;h;t;on of ~MCV hy
St~hle CopDer(I~ Com~lex ~n~ Free T; g~nd
~ Protect;on
Co~c. (~M) R~nS Copper(I) (2:1) RCDS T.;g~n~
100 30.2 10.7
200 67.2 3.2
400 97.7 22.2
500 153.2 84.2
WOg4~7594 PCT~S94/06247
~,~,636 ~
I~h'h;t;on of CMV
Normal Diploid Human Fibroblasts were isolated and
cultured with Minimal Essential Medium (MEM) containing
Earles balanced salts and supplemented with 10~ Fetal
Bovine Serum (FBS). Cytomegalo virus (CMV) was added to
the cultures in the presence or absence of BCDS and BCDS
copper(I) (2:1). Five cultures were employed in each test
group, with the exception of the uninfected cell groups
which utilized 8 cultures. The uninfected cell groups
were used to ensure that antiviral activity was achieved
in the absence of any direct cytotoxic effect of the test
compounds.
After one week of incubation, cellular viability
(i.e., mitochondrial function) was determined, and the
ability of the test compounds to prevent the cytopathic
effect (CPE) of the virus was calculated as percent
protection by the following formula:
~ Protection = (Vt-Vv)/Vu-VV) x 100
where Vt represents viability of the test culture, Vv
represents the viability of culture with virus alone, and
Vu represents the viability of uninfected cells.
The results of this experiment are presented in Table
24. No cytotoxic effects were observed on the uninfected
compounds treated with the test compounds.
T~hle 24
I~h1hltion of CMV hy
St~hle Copper(I) Com~lex ~n~ Free T.l g~nd
~ Protect;on (SF.~)
Co~c. (~M)RCDS Copper(I) (~:1) RCDS T.;g~n~
13.1 (7.7) 34.2 (8.1)
100 117.3 (13.3) 35.4 (6.2)
250 92.9 (5.2) 23.6 (8.3)
W094~7594 PCT~S94/06~7
~1 636~o
~ le 16
T~h;h;t~on of ~IV-l ~n~ HIV-2 Prote~ses ~y
St~hle Copper(I) Co~1exes
This example illustrates the ability of stable
copper(I) complexes of this invention to inhibit HIV-l and
HIV-2 proteases.
HIV-l Prote~se 1~5I-SPA A~s~y
In this experiment, SPA beads (Scintillation
Proximity Assay) were coupled with a peptide substrate to
assay for HIV-l protease. The substrate was a 12 residue
peptide with the following sequence:
AcN-Tyr-Arg-Ala-Arg-Val-Phe-Phe-Val-Arg-Ala-Ala-Lys-COOH
The peptide was monoiodinated on the terminal tyrosine
residue, biotinylated through the ~-amino group on the
terminal lysine, and linked to the SPA bead via a
streptavidin link.
HIV-l protease cleaves the peptide substrate at the
Phe-Phe bond, releasing the l25I-fragment from the bead.
Once the peptide is cleaved, it can no longer stimulate
the scintillant in the SPA bead and the signal is reduced.
The rate of reduction is proportional to the activity of
the HIV-l protease. Recombinant HIV-l protease, affinity
purified for kinetic and assay studies, was used in this
3 0 expe~iment .
Two types of controls were conducted with this assay,
one without enzyme to test for possible scintillation
quenching by the test compound (i.e., BCDS copper(I)
(2:l)), and another positive control with acetyl
pepstatin. At concentrations l0 times that used in the
WOg4~75g4 ~ PCT~S94/~7
~ ~636~ 66
assay, there was no quench detected in the presence of
BCDS copper(I) (2:1). -
The results of this experiment are presented in Table25. The data presented is the mean + SD of the percent
inhibition relative to a no enzyme control reaction. As
discussed above, the ICsO was estimated from the point at
which the dose inhibition line crossed the 50~ inhibition
line. The estimated ICso with this HIV-1 protease assay
was ll~M.
T~hle 25
I~hih;t;o~ of ~IV-l Prote~e h~y
St~hle Copper(I) Complexes
Com~olln~ Conc.Percent I~h;h;t;on
(~M)(Mean + SEM)
BCDS Copper(I) (2:1) 25 86.7 + 2.1
45.2 + 2.3
17.6 + 2.3
2 12.2 + 1.8
1 8.5 + 4.6
0.5 1.8 + 0.6
0.1 0.0 + 1.9
Reference Compound:
Acetyl Pepstatin 0.5 67.4 i 1.1
0.25 50.1 + 0.4
0.1 28.4 + 7.7
0.05 16.6 + 0.5
0.025 10.2 + 2.6
0.01 2.4 + 3.5
HIV-2 Prote~se 125I-SPA A~s~y
As in the above experiment, SPA beads were coupled
with a peptide substrate to assay for HIV-2 protease. The
substrate was the 12 residue peptide identified above and
~0~4~7594 ~3~D rCT~S94/0~47
monoiodinated on the terminal tyrosine residue,
biotinylated through the ~-amino group on the terminal
lysine, and linked to the SPA bead via a streptavidin
link.
HIV-2 protease cleaves the peptide substrate at the
Phe-Phe bond, releasing the 125I-fragment from the bead.
Once the peptide is cleaved, it can no longer stimulate
the scintillant in the SPA bead and the signal is reduced.
The rate of reduction is proportional to the activity of
the HIV-2 protease. Recombinant HIV-2 protease, affinity
purified for kinetic and assay studies, was used in this
experiment. HIV-2 protease has about 50~ sequence
homology with HIV-l protease, and is similar to simian
immunodeficiency virus (SIV) protease.
Two types of control assays were again run, one
without enzyme and the other using acetyl pepstatin as a
positive control.
The results of this experiment are presented in Table
26. The data presented is the mean + SD of the percent
inhibition relative to a no enzyme control reaction. The
IC50 was estimated from the point at which the dose
inhibition line crossed the 50~ inhibition line. The
estimated IC50 with this HIV-2 protease assay was 10~M.
W094~7594 PCT~S94/06247
?,~.636 ~
- 68
T~hle ~6
Inh; h;tlon of HIV-~ Prote~e hy
.~t~hle Copper(I) Co~plexes
Co~pol~n~ Conc.Percent I~h; h; t;on
(~M) (Mean + SEM)
BCDS Copper(I) (2:1)25 51.9 + 5.5
49.6 + 2.9
32.2 + 2.8
2 14.1 + 1.1
1 5.3 + 0.9
0.5 0.8 + 5.3
0.1 2.5 + 0.6
Reference Compound:
Acetyl Pepstatin 5.0 88.8 + 0.5
2.5 70.6 + 2.3
1.0 45.5 + 1.8
0.5 37.2 + 0.3
0.25 19.9 + 5.6
0.1 4.3 + 12.3
~x~mple 17
I~h;h;t;o~ of HIV Reverse Tr~nscr;ptase ~hy
St~hle Copper(I) Co~p1exes
This example illustrates the ability of a stable
copper(I) complex of this invention, BCDS copper(I) (2:1),
to inhibit HIV reverse transcriptase activity.
As in Example 16 above, SPA (Scintillation Proximity
Assay) beads were used to assay for the reverse
transcriptase activity. The reverse transcriptase (10 uL)
was incubated with the 3H-deoxyribonucleotides (10 uL),
the DNA primer linked to biotin (10 uL), and the RNA
template. After incubation at 37 for 20 minutes, the
reaction was stopped and the labeled product was recovered
V094~7594 ~ PCT~S94/06247
~6~
by addition of the SPA beads coupled to streptavidin which
binds to the biotin linked DNA primer.
The extent of the reaction was determined by
scintillation counting. Increasing concentrations of BCDS
copper(I) (2:1) were added and the extent of the reaction
determined by the method described above.
The results of this experiment are presented in Table
27. The data show the mean + SD of the percent inhibition
relative to a no test compound control reaction. The IC50
is estimated from the point at which the dose inhibition
like crosses the 50~ inhibition line. The estimated IC50
was ll~M.
T~hle 27
Inh;h;t'on of HIV Reverse Tr~n.~cr;ptase hy
St~hle Copper(I) Co~lexes
Co~ol~n~ Conc. Percent Inh;hit;on
(~M) (Mean)
BCDS Copper(I) (2:1) 25 63.2
35.4
26.8
2 27.0
1 9.8
0-5 0.2
0.1 1.8
F.xampl e 18
Inh;h;t;on of Protei n Kln~se C hy
Stahle CoppertI) Co~plexes
- This example illustrates the ability of the
representative stable copper(I) complexes, BCDS copper(I)
(2:1) and neocuproine copper(I) (2:1), to inhibit enzymes
involved in intracellular signal transduction. The
WOg4/27594 PCT~S94/06247
~636 ~o
enzymes tested in this experiment were various protein
kinase C isozymes [and protein tyrosine kinases specific
for growth factors and cytokines].
Prote;n K;n~se C (non-select;ve~ A~s~y
In this experiment, the reaction mixture included 20
mM Tris-HCl, pH 7.4, [32P]-ATP, phosphatidylserine,
partially purified PKC from rat brain, and one of the test
compounds (Hunnun, et al. "Activation of Protein Kinase C
by Triton X-100 Mixed Micelles Containing Diacylglycerol
and Phosphatidylserine," J. R;ol . ~hem. 260:10039-10043,
1985; Jeng, et al., "Purification of Stable Protein Kinase
C from Mouse Brain Cytosol by Specific Ligand Elution
Using Fast Protein Liquid Chromatography," C~ncer. Res.
46:1966-1971, 1986). Following a 10 minute incubation, 25
ul aliquots are removed, spotted on phosphocellulose
paper, washed three times in cold phosphoric acid, dried,
and counted to determine phosphorylated product. The
results of this experiment are presented in Table 28.
T~hle 28
Inh;h;t;on of Prote;n K;n~se C (non-select;ve) by
St~hle Copper(I) Co~plexes
Co~pol~n~ Conc. Percent Inh;~;t;on
(~M) (Mean + SEM)
BCDS Copper(I) (2:1) 300 87.5 + 2.7
9.5 + 4.5
3.0 7.5 i 4.5
0.3 2.5 + 4.5
Neocuproine Copper(I) (2:1) 300 62.0 + 1.9
22.0 i 7.0
3.0 6.0 + 4.0
0.3 -12.0 + 2.0
-vo94n7sg4 ~ PCT~S94/06247
71 636~o
Prote; n Kln~se Ca A.~s~y
Protein Kinase Ca is one of the major protein kinase
C isoforms. Protein kinase C is a family of
serine/threonine protein kinases that mediate the actions
of a wide variety of growth factor, hormone, and
neurotransmitter action.
In this experiment, protein Kinase Ca was purified to
homogeneity from rat brain using a modification of a the
published procedure(3). The purity of the isolated PKCa
was confirmed by SDS/polyacrylamide gel electrophoresis
and isoform-specific antibodies. The enzyme was pre-
incubated with the test compounds, and its activity is
measured by the ability of the enzyme to phosphorylate
histone H1 in the absence and presence of calcium,
phosphatidylserine, diolein and [32P]ATP. Following a 5
minute incubation, the reaction was terminated by the
addition of acetic acid, 50 ul aliquots are removed,
spotted on phosphocellulose paper, washed three times in
water, dried, and counted to determine phosphorylated
product. The data presented in Table 29 show that the
addition of the stable copper(I) complexes inhibit the
activity of Protein Kinase Ca.
T~hle 29
Inhl h; t;on of Prote;n Kin~se Ca hy
St~hle copper(I) Complexes
Co~ot~n~ Conc. Percent Inh;h-t;on
(~M) (Mean + SEM)
BCDS Copper(I) (2:1) 100 88.3 + 0.6
18.0 i 2.0
1.0 0.0 + 3.0
0.1 -4.5 + 6.5
W094l27594 PCT~S94/06~7
?~636~
Neocuproine Copper(I) (2:1)100 87.5 + 1.8
23.6 + 4.5
1.0 -1.5 + 3.5
0.1 -5.0 + 1.0
Prote;n K;n~e C~ A~s~y
Protein Kinase C~ is another major protein kinase C
isoforms. Protein kinase C is a family of
serine/threonine protein kinases that mediate the actions
of a wide variety of growth factor, hormone, and
neurotransmitter action.
In this experiment, Protein Kinase C~ (which includes
~I and ~II forms) was purified to homogeneity from rat
brain using a modification of a published protocol
(Woodgett and Hunter, "Isolation and Characterization of
Two Distinct Forms of Protein Kinase C," J. R; ol . Chem.
262:4836-4848, 1987). The purity of the isolated PKCa was
confirmed by SDS/polyacrylamide gel electrophoresis and
isoform-specific antibodies. The enzyme was pre-incubated
with test compounds, and its activity is measured by the
ability of the enzyme to phosphorylate histone H1 in the
absence and presence of calcium, phosphatidylserine,
diolein and [32P]ATP. Following a 5 minute incubation,
the reaction was terminated by the addition of acetic
acid, 50 ul aliquots are removed, spotted on
phosphocellulose paper, washed three times in water,
dried, and counted to determine phosphorylated product.
The data presented in Table 30 show that the addition
of the stable copper(I) complexes inhibit the activity of
Protein Kinase C~.
W094~7594 636~ PCT~S94/06247
O
73
T~hle 30
Inh; h;tio~ of Prote; n K- n~se C~ h~y
St~hle Copper(I) Co~lexes
Co~pol]n~Conc. Percent Inh; hit;on
(~M) (Mean i SEM)
BCDS Copper(I) (2:1) 100 96.8 + 2.0
20.0 i 2.0
1.0 3.5 i 6.5
0.1 6.5 i 4.5
Neocuproine Copper(I) (2:1) 100 84.5 + 1.9
25.5 i 1.5
1.0 4.0 i 7.0
0.1 3.5 i 6.5
Prote;~ K;n~se Cy A.~say
Protein Kinase Cy is another major protein kinase C
isoform. Protein kinase C is a family of serine/threonine
protein kinases that mediate the actions of a wide variety
of growth factor, hormone, and neurotransmitter action.
In this experiment, Protein Kinase Cy was purified
from insect cells expressing a baculovirus recombinant
rabbit brain protein kinase Cy isoform. The enzyme was
pre-incubated with the test compounds, and its activity
was measured by the ability of the enzyme to phosphorylate
histone H1 in the absence and presence of calcium,
phosphatidylserine, diolein and [32P]ATP. Following a 5
minute incubation, the reaction was terminated by the
addition of acetic acid, 50 ul aliquots were removed,
spotted on phosphocellulose paper, washed three times in
water, dried, and counted to determine phosphorylated
product.
The data presented in Table 31 show that the addition
of the stable copper(I) complexes inhibit the activity of
Protein Kinase Cy.
wo94n7sg4 PCT~S94/~247
~,~636 ~ '
74
T~hle 31
I~h;h;t;on of Prote;n K;n~se C~ hy
St~hle Copper(I) Co~lexes
Co~pol]n~ Conc. (~M)Percent Inh;h;t;on
BCDS Copper(I) (2:1) 100 99
51
1.0 21
0.1 5
Neocuproine Copper(I) (2:1) 100 97
1.0 28
0.1 17
The data in Tables 28-31 was used to determine the
50~ inhibitory dose (ICso) of each enzyme. This data is
presented below in Table 32. These results show that the
stable copper(I) complexes of this invention are potent
inhibitors of Protein Kinase C.
T~hle 32
Inh;hit;on of Prote; n Kin~se C Isofor~ hy
St~hle Copper(I) Co~plexes
Prote;n K;n~se C 1~50 (~M)
Isoform BCDS Neocllproi~e
Copper(I) (2:1) Co~per(I) (2:1)
Non-selective 97 145
C~ 28 25
C~ 25 25
Cy 8.8 15
094n7594636~o PCT~S94/06~7
F.x~mpl e 19
I~h;h;t;on of Prote;n Tyros;ne K;n~ses by
r St~hle Copper(I) Complexes
5This example illustrates the ability of the
representative stable copper(I) complexes, BCDS copper(I)
(2:1) and neocuproine copper(I) (2:1), to inhibit enzymes
involved in intracellular signal transduction. The
enzymes tested in this experiment were protein tyrosine
kinases specific for growth factors and cytokines.
Fp;~er~l Growth Factor (FGF)
Receptor Tyros;ne Kin~se (hum~n reco~hin~nt) A~say
The binding of EGF or TGF-a (Transforming Growth
Factor a) to the EGF receptor results in activation of the
tyrosine kinase portion of the receptor. This kinase
phosphorylates several cytosolic proteins which lead to
induction of intracellular signaling pathways eventually
leading to cell mitogenesis and in some cases cellular
transformation. Inhibition of the EGF tyrosine kinase is
useful for chemotherapy for malignant cells.
In this experiment, a recombinant form of the human
Epidermal Growth Factor Tyrosine Kinase domain was assayed
(Geissler et al., "Thiazolidine-Diones:Biochemical and
Biological Activity of a Novel Class of Tyrosine Protein
Kinase Inhibitors," J. R;ol . ~hem. 165:22255-22261, 1990;
Wedegartner and Gill, "Activation of the Purified Protein
Kinase Domain of the Epidermal Growth Factor Receptor,ll J.
Biol. Chem. 264:11346-11353, 1989; Yaish et al., "Blocking
of EGF-dependent Cell Prcliferation by EGF-Receptor Kinase
Inhibitors," Sc;ence 24_:933-935, 1988).
The kinase assay measures the activity of the 69kD
kinase domain by employing an immobilized synthetic
polypeptide as a substrate. Following a 10 minute
reaction, phosphorylated tyrosine residues were detected
by incubation with a monoclonal anti-phosphotyrosine
W094127594 2 l 6 3 6 4 PCT~S94/0624
antibody. Bound anti-phosphotyrosine antibody was
quantitated by incubation with a biotin-linked anti-mouse
IgG, followed by streptavidin linked ~-galactosidase
enzyme. Fluorescence resulting from conversion of
fluoroscein-di-~-galactoside to fluorescein was measured.
The results of this experiment are presented in Table 33.
T~hle 33
Inh;h;t;on of ~p;~erm~l Growth F~ctor (EGF) Receptor
Tyros;ne K;nAse (h-~m~n reco~mh;n~nt) hy
St~hle Copper(I) Com~lexes
Co~ol~n~ Conc. Percent Inh;h;t;on
(~M) (Mean + SEM)
BCDS Copper(I) (2:1) 10 102.3 + 2.3
1 40.0 + 3.6
0.1 11.7 + 2.7
0.01 0.3 + 2.4
Neocuproine Copper(I) (2:1) 10 96.7 + 1.0
1 43.7 + 5.5
0.1 12.0 + 4.0
0.01 -5.7 + 0.9
~lç~ Tyros;ne K;n~se A~say
The lck tyrosine kinase is a member of the src family
of cytoplasmic tyrosine kinases. It is expressed only in
T-lymphocytes and NK cells. The p561ck Tyrosine Kinase is
a 56 kD protein that is found associated with the
cytoplasmic side of the plasma membrane of these cells.
It is responsible of transmission of the IL-2 signal
leading to T-lymphocyte activation. The binding of IL-2
to specific IL-2 receptors leads to activation of the p56
tyrosine kinase. In addition, the p561ck Tyrosine Kinase
has been found to function in signal transduction for
antigen activated CD4 and CD8 T-cell receptors.
094/27594 216~6~0 PCT~S94/06247
In this experiment, the p56lck Tyrosine Kinase was
purified from bovine thymus. The kinase assay measures
the activity of the 69kD kinase domain by employing an
immobilized synthetic polypeptide as a substrate. The
test compounds were pre-incubated with the enzyme of 15
minutes. Following a 10 minute reaction with 100 uM ATP,
phosphorylated tyrosine residues are detected by
incubation with a monoclonal anti-phosphotyrosine
antibody. Bound anti-phosphotyrosine antibody was
quantitated by incubation with a biotin-linked anti-mouse
IgG, followed by streptavidin linked ~-galactosidase
enzyme. Fluorescence resulting from conversion of
fluoroscein-di-~-galactoside to fluorescein was measured
(Hatekeyama et al., "Interaction of the IL-2 Receptor with
the src-Family Kinase p56lck: Identification of Novel
Intermolecular Association," Science ~2:1523-1528, 1991;
Caron et al., "Structural Requirements for Enhancement of
T-cell Responsiveness by the Lymphocyte Specific Tyrosine
Protein Kinase p56lck," Mol. Cell R; 01 . lZ: 2720-2729,
1992; Cheng et al., "A Synthetic Peptide Derived from
p34cdc2 is a Specific and Efficient Substrate of src-
Family Tyrosine Kinases." J. Ri ol . Chem. 2~1:9248-9256,
1992).
Both the BCDS copper(I) and neocuproine copper(I)
complexes were found to be potent inhibitors of the kinase
activity. The results of this experiment are presented in
Table 34.
T~hle 34
Inhih;t;on of p56l~k Tyrosine Kin~se Activity
hy St~hle Copper(I) Com~lexes
Compolln~ Conc. Percent Inhihition
(~M) (Mean + SEM)
BCDS Copper(I) (2:1) 10 97.5 + 1.7
1 19.5 + 1.5
wog4n7594 PCT~S94/06247
~,~636 4~ -
0.1 -3.5 + 0.5
0.01 2.5 + 7.5
Neocuproine Copper(I) (2:1)10 90.0 + 2.3
1 19.5 + 0.5
0.1 -8.0 + 2.0
0.01 -1.0 + 6.0
~fyn Tyroslne K-n~se A~s~y
The fyn tyrosine kinase is also a member of the src
family of non-receptor linked cytoplasmic tyrosine
kinases. The p59fYn Tyrosine Kinase is responsible for
mediating signal transduction through the T-cell receptor
(TCR). This receptor is responsible for a signal cascade
leading to lymphokine secretion and cell proliferation.
The p59fyn Tyrosine Kinase is also one of several kinases
associated with the B-cell receptor.
In this experiment, the p59fyn Tyrosine Kinase was
purified from bovine thymus. The kinase assay measures
the activity of the 69kD kinase domain by employing an
immobilized synthetic polypeptide as a substrate. The
test compounds are preincubated with the enzyme of 15
minutes. Following a 10 minute reaction with 100 uM ATP,
phosphorylated tyrosine residues are detected by
incubation with a monoclonal anti-phosphotyrosine
antibody. Bound anti-phosphotyrosine antibody is
quantitated by incubation with a biotin-linked anti-mouse
IgG, followed by streptavidin linked ~-galactosidase
enzyme. Fluorescence resulting from conversion of
fluoroscein-di-~-galactoside to fluorescein is measured
(Cooke et al., "Regulation of T-cell Receptor Signaling by
a src Family Protein Tyrosine Kinase p59fYn," Cell
65:281-291, 1991; Grassman et al, "Protein Tyrosine Kinase
p59fyn is Associated with the T-cell Receptor CD3 Complex
in Functional Human Lymphocytes," Fnr. J. Immllnol. ~:283-
286, 1992; Appleby et al., "Defective T-cell Receptor
~094~7594 21 636 PCT~S94/06247
-
79
Signaling in Mice Lacking the Thymic Isoform of p59fYn,'~
Cell 70:751-763, 1992). -Both the BCDS copper(I) and
neocuproine copper(I) complexes were found to be potent
inhibitors of the kinase activity. The results of this
experiment are presented in Table 35.
T~hle 35
Inh;h't;on of p59fYn Tyros;ne K;n~se Act;v;ty hy
St~hle Copper(I) Co~lexes
Co~onn~ Conc. Percent Inh;h't;on
(~M) (Mean + SEM)
BCDS Copper)(I) (2:1) 10 99.0 + 2.7
1 38.0 + 5.0
0.1 20.5 + 0.5
0.01 2.0 + 8.0
Neocuproine Copper(I) (2:1) 10 91.0 + 3.0
1 25.5 + 1.5
0.1 1.0 + 6.0
0.01 2.5 + 4.5
The data in Tables 33-35 were used to determine the
50~ inhibitory dose (ICso) of each protein tyrosine kinase
tested. This data is shown in Table 36. These results
show that the stable copper(I) complexes of this invention
are potent inhibitors of this class of tyrosine kinase.
wog4n7s94 PCT~S94/~
?'~6364~ 80
T~hle 36
Inh;h;t;on of Prote;n Tyros;ne K;nA~es hy
St~hle Copper(I) Co~lexes
Prote;n Tyros;ne K;n~se 1~50 (~M)
BCDS Neocl~ro;ne
Copper(I) (2:1) Copper(I) (2:1)
EGF Receptor Tyrosine Kinase 1.3 1.4
p561ck Tyrosine Kinase 2.4 2.7
p59fyn Tyrosine Kinase 1.5 2.4
From the foregoing, it will be appreciated that,
although specific embodiments of the invention have been
described herein for purposes of illustration, various
modifications may be made without deviating from the
spirit and scope of the invention. Accordingly, the
invention is not to be limited except as by the appended
claims.
