U.S. patent application number 10/306054 was filed with the patent office on 2003-08-07 for benzodiazepine vitronectin receptor antagonist pharmaceuticals.
Invention is credited to Cheesman, Edward H., Sworin, Michael.
Application Number | 20030149262 10/306054 |
Document ID | / |
Family ID | 27557206 |
Filed Date | 2003-08-07 |
United States Patent
Application |
20030149262 |
Kind Code |
A1 |
Cheesman, Edward H. ; et
al. |
August 7, 2003 |
Benzodiazepine vitronectin receptor antagonist pharmaceuticals
Abstract
The present invention describes novel compounds of the formula:
(Q).sub.d--L.sub.n--C.sub.h, useful for the diagnosis and treatment
of cancer, methods of imaging tumors in a patient, and methods of
treating cancer in a patient. The present invention also provides
novel compounds useful for monitoring therapeutic angiogenesis
treatment and destruction of new angiogenic vasculature. The
pharmaceuticals are comprised of a targeting moiety that binds to a
receptor that is upregulated during angiogenesis, an optional
linking group, and a therapeutically effective radioisotope or
diagnostically effective imageable moiety. The imageable moiety is
a gamma ray or positron emitting radioisotope, a magnetic resonance
imaging contrast agent, an X-ray contrast agent, or an ultrasound
contrast agent.
Inventors: |
Cheesman, Edward H.;
(Luneneberg, MA) ; Sworin, Michael; (Tyngsbord,
MA) |
Correspondence
Address: |
Bristol-Myers Squibb Company
P.O. Box 4000
Princeton
NJ
08543-4000
US
|
Family ID: |
27557206 |
Appl. No.: |
10/306054 |
Filed: |
November 26, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10306054 |
Nov 26, 2002 |
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09281050 |
Mar 30, 1999 |
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60080150 |
Mar 31, 1998 |
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60112715 |
Dec 18, 1998 |
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60112829 |
Dec 18, 1998 |
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60112732 |
Dec 18, 1998 |
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60112831 |
Dec 18, 1998 |
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Current U.S.
Class: |
536/46 ; 540/487;
540/504; 540/513 |
Current CPC
Class: |
B01J 2219/00317
20130101; C07K 7/02 20130101; C07K 5/06191 20130101; C07K 9/003
20130101; C07K 5/0207 20130101; C07D 403/14 20130101; A61K 49/223
20130101; A61K 51/0497 20130101; B01J 2219/00707 20130101; C07D
401/12 20130101; C07K 5/1024 20130101; A61K 49/10 20130101; C07K
5/1008 20130101; A61K 49/0002 20130101; B01J 2219/00495 20130101;
C07D 401/14 20130101; C07K 5/0215 20130101; C07D 403/12 20130101;
C07K 5/06139 20130101; C07K 5/0806 20130101; C07K 5/0205 20130101;
C07K 7/64 20130101; C40B 60/14 20130101; A61K 49/04 20130101; C07K
5/0821 20130101; B01J 2219/00659 20130101; A61K 49/085 20130101;
C07K 7/06 20130101 |
Class at
Publication: |
536/46 ; 540/487;
540/504; 540/513 |
International
Class: |
C08B 037/16; C07D
243/12; C07D 243/24 |
Claims
What is claimed is described below:
1. A compound, comprising: a targeting moiety and a chelator,
wherein the targeting moiety is bound to the chelator, is a
benzodiazepine nonpeptide, and binds to a receptor that is
upregulated during angiogenesis and the compound has 0-1 linking
groups between the targeting moiety and chelator.
2. A compound according to claim 1, wherein the targeting moiety
comprises a benzodiazepine and the receptor is selected from the
group: EGFR, FGFR, PDGFR, Flk-1/KDR, Flt-1, Tek, Tie, neuropilin-1,
endoglin, endosialin, Axl, .alpha..sub.v.beta..sub.3,
.alpha..sub.v.beta..sub.5, .alpha..sub.5.beta..sub.1,
.alpha..sub.4.beta..sub.1, .alpha..sub.1.beta..sub.1, and
.alpha..sub.2.beta..sub.2 and the linking group is present between
the targeting moiety and chelator.
3. A compound according to claim 2, wherein the receptor is the
integrin .alpha..sub.v.beta..sub.3 and the compound is of the
formula:(Q).sub.d--L.sub.n--C.sub.hor(Q).sub.d--L.sub.n--(C.sub.h).sub.d'-
wherein, Q is a compound of Formula (I): 105 wherein: one of R or
R.sup.1 is selected from a bond to L.sub.n or (CH.sub.2).sub.1-4 or
an NH bond to L.sub.n and the other of R or R.sup.1 is selected
from C.sub.1-4 alkyl, benzyl or phenethyl; R.sup.2 is selected from
benzimidazole or imidazole; R.sup.3 is selected from H, C.sub.1-4
alkyl or benzyl; R.sup.4 is selected from H, C.sub.1-4 alkyl or
benzyl; d is selected from 1, 2 and 3; L.sub.n is a linking group
having the formula:(CR.sup.6R.sup.7).sub.g--
-(W).sub.h--(CR.sup.6aR.sup.7a).sub.g'--(Z).sub.k--(W).sub.h'--(CR.sup.8R.-
sup.9).sub.g"--(W).sub.h"--(CR.sup.8aR.sup.9a).sub.g'"--(W).sub.h'"--(CR.s-
up.8bR.sup.9b).sub.g"" provided that g+h+g'+k+h'+g"+h"+g'" is other
than 0; W is independently selected at each occurrence from the
group: O, S, NH, NHC(.dbd.O), C(.dbd.O)NH, C(.dbd.O), C(.dbd.O)O,
OC(.dbd.O), NHC(.dbd.S)NH, NHC(.dbd.O)NH, SO.sub.2,
(OCH.sub.2CH.sub.2).sub.s, (CH.sub.2CH.sub.2O).sub.s',
(OCH.sub.2CH.sub.2CH.sub.2).sub.s",
(CH.sub.2CH.sub.2CH.sub.2O).sub.t, and (aa).sub.t'; aa is
independently at each occurrence an amino acid; Z is selected from
the group: aryl substituted with 0-3 R.sup.10, C.sub.3-10
cycloalkyl substituted with 0-3 R.sup.10, and a 5-10 membered
heterocyclic ring system containing 1-4 heteroatoms independently
selected from N, S, and O and substituted with 0-3 R.sup.10;
R.sup.6, R.sup.6a, R.sup.7, R.sup.7a, R.sup.8, R.sup.8a, R.sup.8b,
R.sup.9, R.sup.9a and R.sup.9b are independently selected at each
occurrence from the group: H, .dbd.O, COOH, SO.sub.3H, PO.sub.3H,
C.sub.1-C.sub.5 alkyl substituted with 0-3 R.sup.10, aryl
substituted with 0-3 R.sup.10, benzyl substituted with 0-3
R.sup.10, and C.sub.1-C.sub.5 alkoxy substituted with 0-3 R.sup.10,
NHC(.dbd.O)R.sup.11, C(.dbd.O) NHR.sup.11, NHC(.dbd.O)NHR.sup.11,
NHR.sup.11, R.sup.11, and a bond to C.sub.h; R.sup.10 is
independently selected at each occurrence from the group: a bond to
C.sub.h, COOR.sup.11, OH, NHR.sup.11, C(.dbd.O)NHR.sup.11,
NH(C.dbd.O)R.sup.11, SO.sub.3H, PO.sub.3H, .dbd.O, R.sup.11, aryl
substituted with 0-3 R.sup.11, C.sub.1-5 alkyl substituted with 0-1
R.sup.12, C.sub.1-5 alkoxy substituted with 0-1 R.sup.12, and a
5-10 membered heterocyclic ring system containing 1-4 heteroatoms
independently selected from N, S, and O and substituted with 0-3
R.sup.11; R.sup.11 is independently selected at each occurrence
from the group: H, C.sub.1-C.sub.10 alkyl substituted with 0-1
R.sup.12, aryl substituted with 0-1 R.sup.12, a 5-10 membered
heterocyclic ring system containing 1-4 heteroatoms independently
selected from N, S, and O and substituted with 0-1 R.sup.12,
C.sub.3-10 cycloalkyl substituted with 0-1 R.sup.12, polyalkylene
glycol substituted with 0-1 R.sup.12, carbohydrate substituted with
0-1 R.sup.12, cyclodextrin substituted with 0-1 R.sup.12, amino
acid substituted with 0-1 R.sup.12, polycarboxyalkyl substituted
with 0-1 R.sup.12, polyazaalkyl substituted with 0-1 R.sup.12,
peptide substituted with 0-1 R.sup.12, wherein the peptide is
comprised of 2-10 amino acids, and a bond to C.sub.h; R.sup.12 is a
bond to C.sub.h; k is selected from 0, 1, and 2; h is selected from
0, 1, and 2; h' is selected from 0, 1, 2, 3, 4, and 5; h" is
selected from 0, 1, 2, 3, 4, and 5; h'" is selected from 0, 1, 2,
3, 4, and 5; g is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and
10; g' is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; g" is
selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; g'" is selected
from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; g"" is selected from 0,
1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; s is selected from 0, 1, 2, 3,
4, 5, 6, 7, 8, 9, and 10; s' is selected from 0, 1, 2, 3, 4, 5, 6,
7, 8, 9, and 10; s" is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,
and 10; t is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; t'
is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; C.sub.h is a
metal bonding unit having a formula selected from the group: 106
A.sup.1, A.sup.2, A.sup.3, A.sup.4, A.sup.5, A.sup.6, A.sup.7, and
A.sup.8 are independently selected at each occurrence from the
group: NR.sup.13, NR.sup.13R.sup.14, S, SH, S(Pg), O, OH,
PR.sup.13, PR.sup.13R.sup.14, P(O)R.sup.15R.sup.16, CO.sub.2H and a
bond to L.sub.n; E is a bond, CH, or a spacer group independently
selected at each occurrence from the group: C.sub.1-C.sub.10 alkyl
substituted with 0-3 R.sup.17, aryl substituted with 0-3 R.sup.17,
C.sub.3-10 cycloalkyl substituted with 0-3 R.sup.17,
heterocyclo-C.sub.1-10 alkyl substituted with 0-3 R.sup.17, wherein
the heterocyclo group is a 5-10 membered heterocyclic ring system
containing 1-4 heteroatoms independently selected from N, S, and O,
C.sub.6-10 aryl-C.sub.1-10 alkyl substituted with 0-3 R.sup.17,
C.sub.1-10 alkyl-C.sub.6-10 aryl-substituted with 0-3 R.sup.17, and
a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms
independently selected from N, S, and O and substituted with 0-3
R.sup.17; R.sup.13 and R.sup.14 are each independently selected
from the group: a bond to L.sub.n, hydrogen, C.sub.1-C.sub.10 alkyl
substituted with 0-3 R.sup.17, aryl substituted with 0-3 R.sup.17,
C.sub.1-10 cycloalkyl substituted with 0-3 R.sup.17,
heterocyclo-C.sub.1-10 alkyl substituted with 0-3 R.sup.17, wherein
the heterocyclo group is a 5-10 membered heterocyclic ring system
containing 1-4 heteroatoms independently selected from N, S, and O,
C.sub.6-10 aryl-C.sub.1-10 alkyl substituted with 0-3 R.sup.17,
C.sub.1-10 alkyl-C.sub.6-10 aryl-substituted with 0-3 R.sup.17, a
5-10 membered heterocyclic ring system containing 1-4 heteroatoms
independently selected from N, S, and O and substituted with 0-3
R.sup.17, and an electron, provided that when one of R.sup.13 or
R.sup.14 is an electron, then the other is also an electron;
alternatively, R.sup.13 and R.sup.14 combine to form
.dbd.C(R.sup.20) (R.sup.21); R.sup.15 and R.sup.16 are each
independently selected from the group: a bond to L.sub.n, --OH,
C.sub.1-C.sub.10 alkyl substituted with 0-3 R.sup.17,
C.sub.1-C.sub.10 alkyl substituted with 0-3 R.sup.17, aryl
substituted with 0-3 R.sup.17, C.sub.3-10 cycloalkyl substituted
with 0-3 R.sup.17, heterocyclo-C.sub.1-10 alkyl substituted with
0-3 R.sup.17, wherein the heterocyclo group is a 5-10 membered
heterocyclic ring system containing 1-4 heteroatoms independently
selected from N, S, and O, C.sub.6-10 aryl-C.sub.1-10 alkyl
substituted with 0-3 R.sup.17, C.sub.1-10 alkyl-C.sub.6-10
aryl-substituted with 0-3 R.sup.17, and a 5-10 membered
heterocyclic ring system containing 1-4 heteroatoms independently
selected from N, S, and O and substituted with 0-3 R.sup.17;
R.sup.17 is independently selected at each occurrence from the
group: a bond to L.sub.n, .dbd.O, F, Cl, Br, I, --CF.sub.3, --CN,
--CO.sub.2R.sup.18, --C(.dbd.O)R.sup.18,
--C(.dbd.O)N(R.sup.18).sub.2, --CHO, --CH.sub.2OR.sup.18,
--OC(.dbd.O)R.sup.18, --OC(.dbd.O)OR.sup.18a, --OR.sup.18,
--OC(.dbd.O)N(R.sup.18).sub.2, --NR.sup.19C(.dbd.O)R.sup.18,
--NR.sup.19C(.dbd.O)OR.sup.18a,
--NR.sup.19C(.dbd.O)N(R.sup.18).sub.2,
--NR.sup.19SO.sub.2N(R.sup.18).sub.2, --NR.sup.19SO.sub.2R.sup.18a,
--SO.sub.3H, --SO.sub.2R.sup.18a, --SR.sup.18,
--S(.dbd.O)R.sup.18a, --SO.sub.2N(R.sup.18).sub.2,
--N(R.sup.18).sub.2, --NHC(.dbd.S)NHR.sup.18- , .dbd.NOR.sup.18,
NO.sub.2, --C(.dbd.O)NHOR.sup.18, --C(.dbd.O)NHNR.sup.18R.sup.18a,
--OCH.sub.2CO.sub.2H, 2-(1-morpholino)ethoxy, C.sub.1-C.sub.5
alkyl, C.sub.2-C.sub.4 alkenyl, C.sub.3-C.sub.6 cycloalkyl,
C.sub.3-C.sub.6 cycloalkylmethyl, C.sub.2-C.sub.6 alkoxyalkyl, aryl
substituted with 0-2 R.sup.18, and a 5-10 membered heterocyclic
ring system containing 1-4 heteroatoms independently selected from
N, S, and O; R.sup.18, R.sup.18a, and R.sup.19 are independently
selected at each occurrence from the group: a bond to L.sub.n, H,
C.sub.1-C.sub.6 alkyl, phenyl, benzyl, C.sub.1-C.sub.6 alkoxy,
halide, nitro, cyano, and trifluoromethyl; Pg is a thiol protecting
group; R.sup.20 and R.sup.21 are independently selected from the
group: H, C.sub.1-C.sub.10 alkyl, --CN, --CO.sub.2R.sup.25,
--C(.dbd.O)R.sup.25, --C(.dbd.O)N(R.sup.25).sub.2, C.sub.2-C.sub.10
1-alkene substituted with 0-3 R.sup.23, C.sub.2-C.sub.10 1-alkyne
substituted with 0-3 R.sup.23, aryl substituted with 0-3 R.sup.23,
unsaturated 5-10 membered heterocyclic ring system containing 1-4
heteroatoms independently selected from N, S, and O and substituted
with 0-3 R.sup.23, and unsaturated C.sub.3-10 carbocycle
substituted with 0-3 R.sup.23; alternatively, R.sup.20 and
R.sup.21, taken together with the divalent carbon radical to which
they are attached form: 107 R.sup.22 and R.sup.23 are independently
selected from the group: H, R.sup.24, C.sub.1-C.sub.10 alkyl
substituted with 0-3 R.sup.24, C.sub.2-C.sub.10 alkenyl substituted
with 0-3 R.sup.24, C.sub.2-C.sub.10 alkynyl substituted with 0-3
R.sup.24, aryl substituted with 0-3 R.sup.24, a 5-10 membered
heterocyclic ring system containing 1-4 heteroatoms independently
selected from N, S, and O and substituted with 0-3 R.sup.24, and
C.sub.3-10 carbocycle substituted with 0-3 R.sup.24; alternatively,
R.sup.22, R.sup.23 taken together form a fused aromatic or a 5-10
membered heterocyclic ring system containing 1-4 heteroatoms
independently selected from N, S, and O; a and b indicate the
positions of optional double bonds and n is 0 or 1; R.sup.24 is
independently selected at each occurrence from the group: .dbd.O,
F, Cl, Br, I, --CF.sub.3, --CN, --CO.sub.2R.sup.25,
--C(.dbd.O)R.sup.25, --C(.dbd.O)N(R.sup.25).sub.2,
--N(R.sup.25).sub.3.sup.+, --CH.sub.2OR.sup.25,
--OC(.dbd.O)R.sup.25, --OC(.dbd.O)OR.sup.25a, --OR.sup.25,
--OC(.dbd.O)N(R.sup.25).sub.2, --NR.sup.26C(.dbd.O)R.sup.25,
--NR.sup.26C(.dbd.O)OR.sup.25a,
--NR.sup.26C(.dbd.O)N(R.sup.25).sub.2,
--NR.sup.26SO.sub.2N(R.sup.25).sub.2, --NR.sup.26SO.sub.2R.sup.25a,
--SO.sub.3H, --SO.sub.2R.sup.25a, --SR.sup.25, --S
(.dbd.O)R.sup.25a, --SO.sub.2N(R.sup.25).sub.2,
--N(R.sup.25).sub.2, .dbd.NOR.sup.25, --C(.dbd.O)NHOR.sup.25,
--OCH.sub.2CO.sub.2H, and 2-(1-morpholino)ethoxy; and, R.sup.25,
R.sup.25a, and R.sup.26 are each independently selected at each
occurrence from the group: hydrogen and C.sub.1-C.sub.6 alkyl; and
a pharmaceutically acceptable salt thereof.
4. A compound according to claim 3, wherein: d is selected from 1,
2, 3, 4, and 5; Z is selected from the group: aryl substituted with
0-1 R.sup.10, C.sub.3-10 cycloalkyl substituted with 0-1 R.sup.10,
and a 5-10 membered heterocyclic ring system containing 1-4
heteroatoms independently selected from N, S, and O and substituted
with 0-1 R.sup.10; A.sup.1, A.sup.2, A.sup.3, A.sup.4, A.sup.5,
A.sup.6, A.sup.7, and A.sup.8 are independently selected at each
occurrence from the group: NR.sup.13, NR.sup.13R.sup.14, S, SH,
S(Pg), OH, and a bond to L.sub.n; E is a bond, CH, or a spacer
group independently selected at each occurrence from the group:
C.sub.1-C.sub.10 alkyl substituted with 0-3 R.sup.17, aryl
substituted with 0-3 R.sup.17, C.sub.3-10 cycloalkyl substituted
with 0-3 R.sup.17, and a 5-10 membered heterocyclic ring system
containing 1-4 heteroatoms independently selected from N, S, and O
and substituted with 0-3 R.sup.17; R.sup.13, and R.sup.14 are each
independently selected from the group: a bond to L.sub.n, hydrogen,
C.sub.1-C.sub.10 alkyl substituted with 0-3 R.sup.17, aryl
substituted with 0-3 R.sup.17, a 5-10 membered heterocyclic ring
system containing 1-4 heteroatoms independently selected from N, S,
and O and substituted with 0-3 R.sup.17, and an electron, provided
that when one of R.sup.13 or R.sup.14 is an electron, then the
other is also an electron; alternatively, R.sup.13 and R.sup.14
combine to form .dbd.C(R.sup.20) (R.sup.21); R.sup.17 is
independently selected at each occurrence from the group: a bond to
L.sub.n, .dbd.O, F, Cl, Br, I, --CF.sub.3, --CN,
--CO.sub.2R.sup.18, --C(.dbd.O)R.sup.18,
--C(.dbd.O)N(R.sup.18).sub.2, --CH.sub.2OR.sup.18,
--OC(.dbd.O).sup.18, --OC(.dbd.O)OR.sup.18a, --OR.sup.18,
--OC(.dbd.O)N(R.sup.18).sub.2, --NR.sup.19C(.dbd.O)R.sup.18,
--NR.sup.19C(.dbd.O)OR.sup.18a,
--NR.sup.19C(.dbd.O)N(R.sup.18).sub.2,
--NR.sup.19SO.sub.2N(R.sup.18).sub.2, --NR.sup.19SO.sub.2R.sup.18a,
--SO.sub.3H, --SO.sub.2R.sup.18a, --S(.dbd.O)R.sup.18a,
--SO.sub.2N(R.sup.18).sub.2, --N(R.sup.18).sub.2,
--NHC(.dbd.S)NHR.sup.18- , .dbd.NOR.sup.18,
--C(.dbd.O)NHNR.sup.18R.sup.18a, --OCH.sub.2CO.sub.2H, and
2-(1-morpholino)ethoxy; R.sup.18, R.sup.18a, and R.sup.19 are
independently selected at each occurrence from the group: a bond to
L.sub.n, H, and C.sub.1-C.sub.6 alkyl; R.sup.20 and R.sup.21 are
independently selected from the group: H, C.sub.1-C.sub.5 alkyl,
--CO.sub.2R.sup.25, C.sub.2-C.sub.5 1-alkene substituted with 0-3
R.sup.23, C.sub.2-C.sub.5 1-alkyne substituted with 0-3 R.sup.23,
aryl substituted with 0-3 R.sup.23, and unsaturated 5-10 membered
heterocyclic ring system containing 1-4 heteroatoms independently
selected from N, S, and O and substituted with 0-3 R.sup.23;
alternatively, R.sup.20 and R.sup.21, taken together with the
divalent carbon radical to which they are attached form:
108R.sup.22 and R.sup.23 are independently selected from the group:
H, and R.sup.24; alternatively, R.sup.22, R.sup.23 taken together
form a fused aromatic or a 5-10 membered heterocyclic ring system
containing 1-4 heteroatoms independently selected from N, S, and O;
R.sup.24 is independently selected at each occurrence from the
group: --CO.sub.2R.sup.25, --C(.dbd.O)N(R.sup.25).sub.2,
--CH.sub.2OR.sup.25, --OC(.dbd.O)R.sup.25, --OR.sup.25,
--SO.sub.3H, --N(R.sup.25).sub.2, and --OCH.sub.2CO.sub.2H; and,
R.sup.25 is independently selected at each occurrence from the
group: H and C.sub.1-C.sub.3 alkyl.
5. A compound according to claim 4, wherein: Q is a peptide
selected from the group: 109 K is an L-amino acid independently
selected at each occurrence from the group: arginine, citrulline,
N-methylarginine, lysine, homolysine, 2-aminoethylcysteine,
d-N-2-imidazolinylornithine, d-N-benzylcarbamoylornithine, and
b-2-benzimidazolylacetyl-1,2-diaminopro- pionic acid; L is glycine;
M is L-aspartic acid; M' is D-aspartic acid; R.sup.1 is L-valine,
D-valine or L-lysine optionally substituted on the e amino group
with a bond to L.sub.n; R.sup.2 is L-phenylalanine,
D-phenylalanine, D-1-naphthylalanine, 2-aminothiazole-4-acetic acid
or tyrosine, the tyrosine optionally substituted on the hydroxy
group with a bond to L.sub.n; R.sup.3 is D-valine; R.sup.4 is
D-tyrosine substituted on the hydroxy group with a bond to L.sub.n;
provided that one of R.sup.1 and R.sup.2 in each Q is substituted
with a bond to L.sub.n, and further provided that when R.sup.2 is
2-aminothiazole-4-acetic acid, K is N-methylarginine; provided that
at least one Q is a benzodiazepine; d is 1, 2, or 3; A.sup.1 is
selected from the group: OH, and a bond to L.sub.n; A.sup.2,
A.sup.4, and A.sup.6 are each N; A.sup.3, A.sup.5, and A.sup.8 are
each OH; A.sup.7 is a bond to L.sub.n or NH-bond to L.sub.n; E is a
C.sub.2 alkyl substituted with 0-1 R.sup.17; R.sup.17 is .dbd.O;
alternatively, C.sub.h is 110 A.sup.1 is selected from the group:
OH, and a bond to L.sub.n; A.sup.2, A.sup.3 and A.sup.4 are each N;
A.sup.5, A.sup.6 and A.sup.8 are each OH; A.sup.7 is a bond to
L.sub.n; E is a C.sub.2 alkyl substituted with 0-1 R.sup.17;
R.sup.17 is .dbd.O; alternatively, C.sub.h is 111 A.sup.1 is
NH.sub.2 or N.dbd.C(R.sup.20) (R.sup.21); E is a bond; A.sup.2 is
NHR.sup.13; R.sup.13 is a heterocycle substituted with R.sup.17,
the heterocycle being selected from pyridine and pyrimidine;
R.sup.17 is selected from a bond to L.sub.n, C(.dbd.O)NHR.sup.18
and C(.dbd.O)R.sup.18; R.sup.18 is a bond to L.sub.n; R.sup.24 is
selected from the group: --CO.sub.2R.sup.25, --OR.sup.25,
--SO.sub.3H, and --N(R.sup.25).sub.2; and, R.sup.25 is
independently selected at each occurrence from the group: hydrogen
and methyl.
6. A compound according to claim 3, wherein the compound is
selected from the group:
(S,S,S)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-m-
ethylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trie-
n-3-yl)propyl)carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(-
carboxymethyl) cyclodecyl)acetylamino)butanoyl amino)butanoic acid;
(S)-2-(2,5-diaza-5-(6((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl)-
)carbonylamino)hexyl)-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4--
oxobicyclo [5.4.0]undeca-1(7),8,10-trien-3-yl) acetic acid;
(S)-2-(2,5-diaza-9-(N-(6-((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyri-
dyl))carbonylamino)hexyl)-N-(benzimidazol-2-ylmethyl)carbamoyl)-5-methyl-4-
-oxobicyclo [5.4.0]undeca-1(7),8,10-trien-3-yl)acetic acid;
(S,S)-2-(2-aza-2-((5-(N-(1,3-bis(N-(6-(aminohexyl-4-oxobicyclo[5.4.0]unde-
ca-1(7),8,10-trien-3-yl)acetic
acid)(2-(2,5-diaza-9-(N-(benzimidazol-2-ylm-
ethyl)propyl)carbamoyl)(2-pyridyl))amino)vinyl) benzenesulfonic
acid;
(S,S,S)-4-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)--
N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)
carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl-
) cyclododecyl)acetylamino)butanoylamino) butanoic acid;
(S,S)-3-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmethyl)-N--
benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)
carbamoyl)-3-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)
cyclododecyl)acetylamino)propanoic acid;
(S,S,S,S,S,S,S,S)-4-(N-1,3-bis(N-
-3-carboxy-1-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarb-
amoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)pr-
opyl)carbamoyl)-4,4-dihydroxypentyl)
carbamoyl)propyl)carbamoyl)-4-(5,5-di-
hydroxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)acety-
lamino) butanoic acid;
(S,S,S,S,S,S,S,S,S,S)-2-(4-(N-(1,3-bis(N-(3-(N-(3-(-
3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxyca-
rbonyl)methyl)-4-oxobicyclo
[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carb-
amoyl)-1-(methoxycarbonyl)propyl)carbamoyl)propyl)carbamoyl)propyl)carbamo-
yl)-4-(2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)
cyclodecyl)acetylamino)-4-carboxybutanoylamino)-4-carboxybutanoylamino)bu-
tanoylamino)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methyl
carbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,1-
0-trien-3-yl)propyl)carbamoyl)butanoic acid;
(S)-2-(2,5-diaza-5-(3-(2-(2-(-
3-((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)
propoxy)ethoxy)ethoxy)propyl)-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarb-
amoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic acid;
(S,S,S,S,S)-4-(N-(1,3-bis(N-(3-(2-(2-(3-(3,6-diaza-10-(N-(benzimidazol-2--
ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1-
(7),8,10-trien-3-yl)propoxy)ethoxy)ethoxy)propyl)carbamoyl)
propyl)carbamoyl)-4-(5,5-dihydroxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(ca-
rboxy methyl)cyclododecyl)acetylamino) hexanoylamino)butanoic acid;
(S,S,S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4--
oxo-5-(6-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-2-(4-(N-(-
(R,S,S,S)-2,3,4,5,6-pentahydroxy
hexyl)carbamoyl)-2-(2-(1,4,7,10-tetraaza--
4,7,10-tris(carboxymethyl)cyclodecyl)
acetylamino)butanoylamino)butanoylam-
ino)hexyl)bicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic acid;
(S,S,S,S)-2-(4-(N-(1-(N-(1-(N-(6-(3,6-diaza-10-(N-(benzimidazol-2-ylmethy-
l)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,1-
0-trien-3-yl)hexyl)carbamoyl)-3-(N-cyclo{Lys-Arg(Mtr)-Gly-Asp(OtBu)-D-Phe}-
[gamma-LysNH]carbamoyl)propyl)carbamoyl)-3-carboxypropyl)
carbamoyl)-4-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)
cyclododecyl)acetylamino)butanoic acid; and or a pharmaceutically
acceptable salt form thereof.
7. A kit comprising a compound of claim 3, or a pharmaceutically
acceptable salt form thereof and a pharmaceutically acceptable
carrier.
8. A kit according to claim, wherein the kit further comprises one
or more ancillary ligands and a reducing agent.
9. A kit according to claim 8, wherein the ancillary ligands are
tricine and TPPTS.
10. A kit according to claim 9, wherein the reducing agent is
tin(II).
11. A diagnostic or therapeutic metallopharmaceutical composition,
comprising: a metal, a chelator capable of chelating the metal and
a targeting moiety, wherein the targeting moiety is bound to the
chelator, is a benzodiazepine nonpeptide and binds to a receptor
that is upregulated during angiogenesis and the compound has 0-1
linking groups between the targeting moiety and chelator.
12. A composition according to claim 11, wherein the
metallopharmaceutical is a diagnostic radiopharmaceutical, the
metal is a radioisotope selected from the group: .sup.99mTc,
.sup.95Tc, .sup.111In, .sup.62Cu, .sup.64Cu, .sup.67Ga, and
.sup.68Ga, the targeting moiety comprises a benzodiazepine and the
receptor is selected from the group: EGFR, FGFR, PDGFR, Flk-1/KDR,
Flt-1, Tek, Tie, neuropilin-1, endoglin, endosialin, Axl,
.alpha..sub.v.beta..sub.3, .alpha..sub.v.beta..sub.5,
.alpha..sub.5.beta..sub.1, .alpha..sub.4.beta..sub.1,
.alpha..sub.1.beta..sub.1, and .alpha..sub.2.beta..sub.2 and the
linking group is present between the targeting moiety and
chelator.
13. A composition according to claim 12, wherein the targeting
moiety is a benzodiazepine and the receptor is
.alpha..sub.v.beta..sub.3.
14. A composition according to claim 12, wherein the radioisotope
is .sup.99mTc or .sup.95Tc, the radiopharmaceutical further
comprises a first ancillary ligand and a second ancillary ligand
capable of stabilizing the radiopharmaceutical.
15. A composition according to claim 14, wherein the radioisotope
is .sup.99mTc.
16. A composition according to claim 15, wherein the
radiopharmaceutical is selected from the group: .sup.99mTc
((S)-2-(2,5-diaza-5-(6((6-(diazeni- do)
(3-pyridyl))carbonylamino)hexyl)-9-(N-(benzimidazol-2-ylmethyl)-N-meth-
ylcarbamoyl)-4-oxobicyclo [5.4.0]undeca-1(7),8,10-trien-3-yl)
acetic acid)(tricine)(TPPTS) and 99mTc (
(S)-2-(2,5-diaza-9-(N-(6-((6-(diazenido-
)(3-pyridyl))carbonylamino)hexyl)-N-(benzimidazol-2-ylmethyl)carbamoyl)-5--
methyl-4-oxobicyclo [5.4.0]undeca-1(7),8,10-trien-3-yl)acetic
acid)(tricine)(TPPTS);
17. A composition according to claim 13, wherein the radioisotope
is .sup.111In.
18. A composition according to claim 17, wherein the
radiopharmaceutical is selected from the group: .sup.111In complex
of 6-(N-(3-(3-aza-10-(N-(b-
enzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo
[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-(2-((2-((carboxyme-
thyl) (2-((carboxymethyl)methylamino)ethyl)amino)
ethyl)(2-((carboxymethyl-
)ethylamino)ethyl)amino)-acetylamino)-4-oxooctane-1,8-dicarboxylic
acid; .sup.111In complex of
(S,S,S)-4-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N--
(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,-
10-trien-3-yl)propyl)
carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,-
10-tris(carboxymethyl) cyclododecyl)acetylamino)butanoylamino)
butanoic acid; and .sup.111In complex of
(S,S)-3-(N-(3-(3,6-diaza-5-(carboxymethyl-
)-10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-
-1(7),8,10-trien-3-yl)propyl)
carbamoyl)-3-(2-(1,4,7,10-tetraaza-4,7,10-tr- is(carboxymethyl)
cyclododecyl)acetylamino)propanoic acid.
19. A composition according to claim 11, wherein the
metallopharmaceutical is a therapeutic radiopharmaceutical, the
metal is a radioisotope selected from the group: .sup.186Re,
.sup.188Re, .sup.153Sm, .sup.166Ho, .sup.177Lu, .sup.149Pm,
.sup.90y, .sup.212Bi, .sup.103Pd, .sup.109Pd, .sup.159Gd,
.sup.140La, .sup.198Au, .sup.199Au, 169Yb, .sup.175Yb, .sup.165Dy,
.sup.166Dy, .sup.67Cu, .sup.105Rh, .sup.111Ag, and .sup.192Ir, the
targeting moiety is a benzodiazepine nonpeptide and the receptor is
selected from the group: EGFR, FGFR, PDGFR, Flk-1/KDR, Flt-1, Tek,
Tie, neuropilin-1, endoglin, endosialin, Axl,
.alpha..sub.v.beta..sub.3, .alpha..sub.v.beta..sub.5,
.alpha..sub.5.beta..sub.1, .alpha..sub.4.beta..sub.1,
.alpha..sub.1.beta..sub.1, and .alpha..sub.2.beta..sub.2 and the
linking group is present between the targeting moiety and
chelator.
20. A composition according to claim 19, the targeting moiety is an
benzodiazepine and the receptor is .alpha..sub.v.beta..sub.3.
21. A composition according to claim 20, wherein the radioisotope
is .sup.153Sm.
22. A composition according to claim 20, wherein the radioisotope
is .sup.177Lu.
23. A composition according to claim 20, the radioisotope is
.sup.90Y.
24. A composition according to claim 11, wherein the
metallopharmaceutical is a MRI contrast agent, the metal is a
paramagnetic metal ion selected from the group: Gd(III), Dy(III),
Fe(III), and Mn(II), the targeting moiety is a benzodiazepine
nonpeptide and the receptor is selected from the group: EGFR, FGFR,
PDGFR, Flk-1/KDR, Flt-1, Tek, Tie, neuropilin-1, endoglin,
endosialin, Axl, .alpha..sub.v.beta..sub.3,
.alpha..sub.v.beta..sub.5, .alpha..sub.5.beta..sub.1,
.alpha..sub.4.beta..sub.1, .alpha..sub.1.beta..sub.1, and
.alpha..sub.2.beta..sub.2 and the linking group is present between
the targeting moiety and chelator.
25. A composition according to claim 24, wherein the targeting
moiety is an indazole and the receptor is
.alpha..sub.v.beta..sub.3.
26. A composition according to claim 25, wherein the metal ion is
Gd(III).
27. A composition according to claim 11, wherein the
metallopharmaceutical is a X-ray contrast agent, the metal is
selected from the group: Re, Sm, Ho, Lu, Pm, Y, Bi, Pd, Gd, La, Au,
Au, Yb, Dy, Cu, Rh, Ag, and Ir, the targeting moiety is a
benzodiazepine nonpeptide, the receptor is
.alpha..sub.v.beta..sub.3, and the linking group is present between
the targeting moiety and chelator.
28. A method of treating rheumatoid arthritis in a patient
comprising: administering a therapeutic radiopharmaceutical of
claim 11 capable of localizing in new angiogenic vasculature to a
patient by injection or infusion.
29. A method of treating cancer in a patient comprising:
administering to a patient in need thereof a therapeutic
radiopharmaceutical of claim 11 by injection or infusion.
30. A method of imaging formation of new blood vessels in a patient
comprising: (1) administering a diagnostic radiopharmaceutical, a
MRI contrast agent, or a X-ray contrast agent of of claim 11 to a
patient by injection or infusion; (2) imaging the area of the
patient wherein the desired formation of new blood vessels is
located.
31. A method of imaging cancer in a patient comprising: (1)
administering a diagnostic radiopharmaceutical of claim 11 to a
patient by injection or infusion; (2) imaging the patient using
planar or SPECT gamma scintigraphy, or positron emission
tomography.
32. A method of imaging cancer in a patient comprising: (1)
administering a MRI contrast agent of claim 24; and (2) imaging the
patient using magnetic resonance imaging.
33. A method of imaging cancer in a patient comprising: (1)
administering a X-ray contrast agent of claim 27; and (2) imaging
the patient using X-ray computed tomography.
34. A compound, comprising: a targeting moiety and a surfactant,
wherein the targeting moiety is bound to the surfactant, is a
benzodiazepine nonpeptide, and binds to a receptor that is
upregulated during angiogenesis and the compound has 0-1 linking
groups between the targeting moiety and surfactant.
35. A compound according to claim 34, wherein the targeting moiety
comprises an benzodiazepine and the receptor is selected from the
group: EGFR, FGFR, PDGFR, Flk-l/KDR, Flt-1, Tek, Tie, neuropilin-1,
endoglin, endosialin, Axl, .alpha..sub.v.beta..sub.3,
.alpha..sub.v.beta..sub.5, .alpha..sub.5.beta..sub.1,
.alpha..sub.4.beta..sub.1, .alpha..sub.1.beta..sub.1, and
.alpha..sub.2.beta..sub.2 and the linking group is present between
the targeting moiety and surfactant.
36. A compound according to claim 35, wherein the receptor is the
integrin .alpha..sub.v.beta..sub.3 and the compound is of the
formula:(Q).sub.d--L.sub.n--S.sub.fwherein, Q is a compound of
Formula (I): 112wherein: one of R or R.sup.1 is selected from a
bond to L.sub.n or (CH.sub.2).sub.1-4 or an NH bond to L.sub.n and
the other of R or R.sup.1 is selected from C.sub.1-4 alkyl, benzyl
or phenethyl; R.sup.2 is selected from benzimidazole or imidazole;
R.sup.3 is selected from H, C.sub.1-4 alkyl or benzyl; R.sup.4 is
selected from H, C.sub.1-4 alkyl or benzyl; d is selected from 1,
2, 3, 4, 5, 6, 7, 8, 9, and 10; S.sub.f is a surfactant which is a
lipid or a compound of the formula: 113 A.sup.9 is selected from
the group: OH and OR.sup.27; A.sup.10 is OR.sup.27; R.sup.27 is
C(.dbd.O)C.sub.1-20 alkyl; E.sup.1 is C.sub.1-10 alkylene
substituted with 1-3 R.sup.28; R.sup.28 is independently selected
at each occurrence from the group: R.sup.30,
--PO3H-R.sup.30,.dbd.O, --CO.sub.2R.sup.29, --C(.dbd.O)R.sup.29,
--C(.dbd.O)N(R.sup.29).sub.2, --CH.sub.2OR.sup.29, --OR.sup.29,
--N(R.sup.29).sub.2, C.sub.1-C.sub.5 alkyl, and C.sub.2-C.sub.4
alkenyl; R.sup.29 is independently selected at each occurrence from
the group: R.sup.30, H, C.sub.1-C.sub.6 alkyl, phenyl, benzyl, and
trifluoromethyl; R.sup.30 is a bond to L.sub.n; L.sub.n is a
linking group having the formula:(CR.sup.6R.sup.7).sub.g--(W-
).sub.h--(CR.sup.6aR.sup.7a).sub.g'--(Z).sub.k--(W).sub.h'--(CR.sup.8R.sup-
.9).sub.g"--(W).sub.h"--(CR.sup.8aR.sup.9a).sub.g'" W is
independently selected at each occurrence from the group: O, S, NH,
NHC(.dbd.O), C(.dbd.O)NH, C(.dbd.O), C(.dbd.O)O, OC(.dbd.O),
NHC(.dbd.S)NH, NHC(.dbd.O)NH, SO.sub.2,
(OCH.sub.2CH.sub.2).sub.20-200, (CH.sub.2CH.sub.2O).sub.20-200,
(OCH.sub.2CH.sub.2CH.sub.2).sub.20-200,
(CH.sub.2CH.sub.2CH.sub.2O).sub.20-200, and (aa).sub.t'; aa is
independently at each occurrence an amino acid; Z is selected from
the group: aryl substituted with 0-3 R.sup.10, C.sub.3-10
cycloalkyl substituted with 0-3 R.sup.10, and a 5-10 membered
heterocyclic ring system containing 1-4 heteroatoms independently
selected from N, S, and O and substituted with 0-3 R.sup.10;
R.sup.6, R.sup.6a, R.sup.7, R.sup.7a, R.sup.8, R.sup.8a, R.sup.9
and R.sup.9a are independently selected at each occurrence from the
group: H, .dbd.O, COOH, SO.sub.3H, PO.sub.3H, C.sub.1-C.sub.5 alkyl
substituted with 0-3 R.sup.10, aryl substituted with 0-3 R.sup.10,
benzyl substituted with 0-3 R.sup.10, and C.sub.1-C.sub.5 alkoxy
substituted with 0-3 R.sup.10, NHC(.dbd.O)R.sup.11,
C(.dbd.O)NHR.sup.11, NHC(.dbd.O)NHR.sup.11, NHR.sup.11, R.sup.11,
and a bond to S.sub.f; R.sup.10 is independently selected at each
occurrence from the group: a bond to S.sub.f, COOR.sup.11, OH,
NHR.sup.11, SO.sub.3H, PO.sub.3H, aryl substituted with 0-3
R.sup.11, C.sub.1-5 alkyl substituted with 0-1 R.sup.12, C.sub.1-5
alkoxy substituted with 0-1 R.sup.12, and a 5-10 membered
heterocyclic ring system containing 1-4 heteroatoms independently
selected from N, S, and O and substituted with 0-3 R.sup.11;
R.sup.11 is independently selected at each occurrence from the
group: H, aryl substituted with 0-1 R.sup.12, a 5-10 membered
heterocyclic ring system containing 1-4 heteroatoms independently
selected from N, S, and O and substituted with 0-1 R.sup.12,
C.sub.3-10 cycloalkyl substituted with 0-1 R.sup.12, amino acid
substituted with 0-1 R.sup.12, and a bond to S.sub.f; R.sup.12 is a
bond to S.sub.f; k is selected from 0, 1, and 2; h is selected from
0, 1, and 2; h' is selected from 0, 1, 2, 3, 4, and 5; h" is
selected from 0, 1, 2, 3, 4, and 5; g is selected from 0, 1, 2, 3,
4, 5, 6, 7, 8, 9, and 10; g' is selected from 0, 1, 2, 3, 4, 5, 6,
7, 8, 9, and 10; g" is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,
and 10; g'" is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
t' is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and a
pharmaceutically acceptable salt thereof.
37. A compound according to claim 36, wherein the compound is of
the formula:Q--L.sub.n--S.sub.fwherein, Q is a compound of Formula
(I): 114 wherein: one of R or R.sup.1 is selected from a bond to
L.sub.n or (CH.sub.2).sub.1-4 or an NH bond to L.sub.n and the
other of R or R.sup.1 is selected from C.sub.1-4 alkyl, benzyl or
phenethyl; R.sup.2 is selected from benzimidazole or imidazole;
R.sup.3 is selected from H, C.sub.1-4 alkyl or benzyl; R.sup.4 is
selected from H, C.sub.1-4 alkyl or benzyl; S.sub.f is a surfactant
which is a lipid or a compound of the formula: 115 A.sup.9 is
OR.sup.27; A.sup.10 is OR.sup.27; R.sup.27 is C(.dbd.O)C.sub.1-15
alkyl; E.sup.1 is C.sub.1-4 alkylene substituted with 1-3 R.sup.28;
R.sup.28 is independently selected at each occurrence from the
group: R.sup.30, --PO.sub.3H-R.sup.30, .dbd.O, --CO.sub.2R.sup.29,
--C(.dbd.O)R.sup.29, --CH.sub.2OR.sup.29, --OR.sup.29, and
C.sub.1-C.sub.5 alkyl; R.sup.29 is independently selected at each
occurrence from the group: R.sup.30, H, C.sub.1-C.sub.6 alkyl,
phenyl, and benzyl; R.sup.30 is a bond to L.sub.n; L.sub.n is a
linking group having the
formula:(CR.sup.6R.sup.7).sub.g--(W).sub.h--(CR.sup.6aR.sup.7a-
).sub.g'--(Z).sub.k--(W).sub.h'--(CR.sup.8R.sup.9).sub.g"--(W).sub.h"--(CR-
.sup.8aR.sup.9a).sub.g'" W is independently selected at each
occurrence from the group: O, S, NH, NHC(.dbd.O), C(.dbd.O)NH,
C(.dbd.O), C(.dbd.O)O, OC(.dbd.O), NHC(.dbd.S)NH, NHC(.dbd.O)NH,
SO.sub.2, (OCH.sub.2CH.sub.2).sub.20-200,
(CH.sub.2CH.sub.2O).sub.20-200,
(OCH.sub.2CH.sub.2CH.sub.2).sub.20-200,
(CH.sub.2CH.sub.2CH.sub.2O).sub.2- 0-200, and (aa).sub.t'; aa is
independently at each occurrence an amino acid; Z is selected from
the group: aryl substituted with 0-3 R.sup.10, C.sub.3-10
cycloalkyl substituted with 0-3 R.sup.10, and a 5-10 membered
heterocyclic ring system containing 1-4 heteroatoms independently
selected from N, S, and O and substituted with 0-3 R.sup.10;
R.sup.6, R.sup.6a, R.sup.7, R.sup.7a, R.sup.8, R.sup.8a, R.sup.9
and R.sup.9a are independently selected at each occurrence from the
group: H, .dbd.O, C.sub.1-C.sub.5 alkyl substituted with 0-3
R.sup.10, and C.sub.1-C.sub.5 alkoxy substituted with 0-3 R.sup.10,
and a bond to S.sub.f; R.sup.10 is independently selected at each
occurrence from the group: a bond to S.sub.f, COOR.sup.11, OH,
NHR.sup.11, C.sub.1-5 alkyl substituted with 0-1 R.sup.12, and
C.sub.1-5 alkoxy substituted with 0-1 R.sup.12; R.sup.11 is
independently selected at each occurrence from the group: H, aryl
substituted with 0-1 R.sup.12, C.sub.3-10 cycloalkyl substituted
with 0-1 R.sup.12, amino acid substituted with 0-1 R.sup.12, and a
bond to S.sub.f; R.sup.12 is a bond to S.sub.f; k is selected from
0, 1, and 2; h is selected from 0, 1, and 2; h' is selected from 0,
1, 2, 3, 4, and 5; h" is selected from 0, 1, 2, 3, 4, and 5; g is
selected from 0, 1, 2, 3, 4, and 5; g' is selected from 0, 1, 2, 3,
4, and 5; g" is selected from 0, 1, 2, 3, 4, and 5; g'" is selected
from 0, 1, 2, 3, 4, and 5; s is selected from 0, 1, 2, 3, 4, and 5;
s' is selected from 0, 1, 2, 3, 4, and 5; s" is selected from 0, 1,
2, 3, 4, and 5; t is selected from 0, 1, 2, 3, 4, and 5; t' is
selected from 0, 1, 2, 3, 4, and 5; and a pharmaceutically
acceptable salt thereof.
38. A compound according to claim 37, wherein the compound selected
from the group: Sodium
1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-(-
S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(6-ami-
nohexyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic
acid-dodecoanoate conjugate;
DPPE-PEG.sub.3400-[(S)-2-(2,5-diaza-9-(N-(be-
nzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(6-aminohexyl)-4-oxobicyclo[5.-
4.0]undeca-1(7),8,10-trien-3-yl)acetic acid]-dodecoanoate
conjugate; and
[(S)-2-(2-aza-(2-((5-(N-(1,3-bis-N-(6-(aminohexyl-4-oxobicyclo[5.4.0]unde-
ca-1(7),8,10-trien-3-yl)acetic
acid)(2-(2,5-diaza-9-(N-(benzimidazol-2-ylm- ethyl) carbamoyl)
propyl) carbamoyl]-w-amino-PEG.sub.3400-dodecanoate-DPPE
conjugate.
39. An ultrasound contrast agent composition, comprising: (a) a
compound of claim 35, comprising: a benzodiazepine that binds to
the integrin .alpha..sub.v.beta..sub.3, a surfactant and a linking
group between the benzodiazepine and the surfactant; (b) a
parenterally acceptable carrier; and, (c) an echogenic gas.
40. An ultrasound contrast agent composition of claim 39, further
comprising: 1,2-dipalmitoyl-sn-glycero-3-phosphotidic acid,
1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, and
N-(methoxypolyethylene glycol 5000
carbamoyl)-1,2-dipalmitoyl-sn-glycero--
3-phosphatidylethanolamine.
41. An ultrasound contrast agent composition of claim 40, wherein
the echogenic gas is a C.sub.2-5 perfluorocarbon.
42. A method of imaging cancer in a patient comprising: (1)
administering, by injection or infusion, a ultrasound contrast
agent composition of claim 35 to a patient; and (2) imaging the
patient using sonography.
43. A method of imaging formation of new blood vessels in a patient
comprising: (1) administering, by injection or infusion, a
ultrasound contrast agent composition of of claim 36 to a patient;
(2) imaging the area of the patient wherein the desired formation
of new blood vessels is located.
44. A therapeutic radiopharmaceutical composition, comprising: (a)
a therapeutic radiopharmaceutical of claim 11; and, (b) a
parenterally acceptable carrier.
45. A diagnostic radiopharmaceutical composition, comprising: (a) a
diagnostic radiopharmaceutical, a MRI contrast agent, or a X-ray
contrast agent of claim 11; and, (b) a parenterally acceptable
carrier.
Description
FIELD OF THE INVENTION
[0001] The present invention provides novel pharmaceuticals useful
for the diagnosis and treatment of cancer, methods of imaging
tumors in a patient, and methods of treating cancer in a patient.
The pharmaceuticals are comprised of a targeting moiety that binds
to the vitronectin receptor that is expressed in tumor vasculature,
an optional linking group, and a therapeutically effective
radioisotope or diagnostically effective imageable moiety. The
therapeutically effective radioisotope emits a gamma ray or alpha
particle sufficient to be cytotoxic. The imageable moiety is a
gamma ray or positron emitting radioisotope, a magnetic resonance
imaging contrast agent, an X-ray contrast agent, or an ultrasound
contrast agent.
BACKGROUND OF THE INVENTION
[0002] Cancer is a major public health concern in the United States
and around the world. It is estimated that over 1 million new cases
of invasive cancer will be diagnosed in the United States in 1998.
The most prevalent forms of the disease are solid tumors of the
lung, breast, prostate, colon and rectum. Cancer is typically
diagnosed by a combination of in vitro tests and imaging
procedures. The imaging procedures include X-ray computed
tomography, magnetic resonance imaging, ultrasound imaging and
radionuclide scintigraphy. Frequently, a contrast agent is
administered to the patient to enhance the image obtained by X-ray
CT, MRI and ultrasound, and the administration of a
radiopharmaceutical that localizes in tumors is required for
radionuclide scintigraphy.
[0003] Treatment of cancer typically involves the use of external
beam radiation therapy and chemotherapy, either alone or in
combination, depending on the type and extent of the disease. A
number of chemotherapeutic agents are available, but generally they
all suffer from a lack of specificity for tumors versus normal
tissues, resulting in considerable side-effects. The effectiveness
of these treatment modalities is also limited, as evidenced by the
high mortality rates for a number of cancer types, especially the
more prevalent solid tumor diseases. More effective and specific
treatment means continue to be needed.
[0004] Despite the variety of imaging procedures available for the
diagnosis of cancer, there remains a need for improved methods. In
particular, methods that can better differentiate between cancer
and other pathologic conditions or benign physiologic abnormalities
are needed. One means of achieving this desired improvement would
be to administer to the patient a metallopharmaceutical that
localizes specifically in the tumor by binding to a receptor
expressed only in tumors or expressed to a significantly greater
extent in tumors than in other tissue. The location of the
metallopharmaceutical could then be detected externally either by
its imageable emission in the case of certain radiopharmaceuticals
or by its effect on the relaxation rate of water in the immediate
vicinity in the case of magnetic resonance imaging contrast
agents.
[0005] This tumor specific metallopharmaceutical approach can also
be used for the treatment of cancer when the metallopharmaceutical
is comprised of a particle emitting radioisotope. The radioactive
decay of the isotope at the site of the tumor results in sufficient
ionizing radiation to be toxic to the tumor cells. The specificity
of this approach for tumors minimizes the amount of normal tissue
that is exposed to the cytotoxic agent and thus may provide more
effective treatment with fewer side-effects.
[0006] Previous efforts to achieve these desired improvements in
cancer imaging and treatment have centered on the use of
radionuclide labeled monoclonal antibodies, antibody fragments and
other proteins or polypeptides that bind to tumor cell surface
receptors. The specificity of these radiopharmaceuticals is
frequently very high, but they suffer from several disadvantages.
First, because of their high molecular weight, they are generally
cleared from the blood stream very slowly, resulting in a prolonged
blood background in the images. Also, due to their molecular weight
they do not extravasate readily at the site of the tumor and then
only slowly diffuse through the extravascular space to the tumor
cell surface. This results in a very limited amount of the
radiopharmaceutical reaching the receptors and thus very low signal
intensity in imaging and insufficient cytotoxic effect for
treatment.
[0007] Alternative approaches to cancer imaging and therapy have
involved the use of small molecules, such as peptides, that bind to
tumor cell surface receptors. An In-111 labeled somatostatin
receptor binding peptide, In-111-DTPA-D-Phe.sup.1-octeotide, is in
clinical use in many countries for imaging tumors that express the
somatostatin receptor (Baker, et al. Life Sci., 1991, 49, 1583-91
and Krenning, et al., Eur. J. Nucl. Med., 1993, 20, 716-31). Higher
doses of this radiopharmaceutical have been investigated for
potential treatment of these types of cancer (Krenning, et al.,
Digestion, 1996, 57, 57-61). Several groups are investigating the
use of Tc-99 m labeled ananlogs of
In-111-DTPA-D-Phe.sup.1-octeotide for imaging and Re-186 labeled
analogs for therapy (Flanagan, et al., U.S. Pat. No. 5,556,939,
Lyle, et al., U.S. Pat. No. 5,382,654, and Albert et al., U.S. Pat.
No. 5,650,134).
[0008] Angiogenesis is the process by which new blood vessels are
formed from pre-existing capillaries or post capillary venules; it
plays a key role in the pathological development of many solid
tumor cancers and their metastases. Tumor released cytokines or
angiogenic factors stimulate vascular endothelial cells by
interacting with specific cell surface receptors for the factors.
The endothelial cells then proliferate and invade into the tumor
tissue. The endothelial cells differentiate to form lumens, making
new vessel offshoots of pre-existing vessels. The new blood vessels
then provide nutrients to the tumor permitting further growth and a
route for metastasis.
[0009] Angiogenesis is also influenced by cell adhesion molecules
(Folkman, J., Nature Medicine, 1995, 1, 27-31). The integrin
.alpha..sub.v.beta..sub.3 is a receptor for a wide variety of
extracellular matrix proteins with an exposed tripeptide
Arg-Gly-Asp moiety and mediates cellular adhesion to its ligands:
vitronectin, fibronectin, and fibrinogen, among others. The
integrin .alpha..sub.v.beta..sub.3 is minimally expressed on normal
blood vessels, but, is significantly upregulated on vascular cells
within a variety of human tumors. The role of the
.alpha..sub.v.beta..sub.3 receptors is to mediate the interaction
of the endothelial cells and the extracellular matrix and
facilitate the migration of the cells in the direction of the
angiogenic signal, the tumor cell population.
[0010] Because of the importance of angiogenesis to tumor growth
and metastasis, a number of chemotherapeutic approaches are being
developed to interfere with or prevent this process. One of these
approaches, involves the use of anti-angiogenic proteins such as
angiostatin and endostatin. Angiostatin is a 38 kDa fragment of
plasminogen that has been shown in animal models to be a potent
inhibitor of endothelial cell proliferation. (O'Reilly et. al. ,
Cell, 1994, 79, 315-328) Endostatin is a 20 kDa C-terminal fragment
of collagen XVIII that has also been shown to be a potent
inhibitor. (O'Reilly et. al., Cell, 1997, 88, 277-285) Systemic
therapy with endostatin has been shown to result in strong
anti-tumor activity in animal models. However, human clinical
trials of these two chemotherapeutic agents of biological origin
have been hampered by lack of availability.
[0011] Another approach to anti-angiogenic therapy is to use
targeting moieties that interact with endothelial cell surface
receptors expressed in the angiogenic vasculature to which are
attached chemotherapeutic agents. Burrows and Thorpe (Proc. Nat.
Acad. Sci, USA, 1993, 90, 8996-9000) described the use of an
antibody-immunotoxin conjugate to eradicate tumors in a mouse model
by destroying the tumor vasculature. The antibody was raised
against an endothelial cell class II antigen of the major
histocompatibility complex and was then conjugated with the
cytotoxic agent, deglycosylated ricin A chain. The same group
(Clin. Can. Res., 1995, 1, 1623-1634) investigated the use of
antibodies raised against the endothelial cell surface receptor,
endoglin, conjugated to deglycosylated ricin A chain. Both of these
conjugates exhibited potent anti-tumor activity in mouse models.
However, both still suffer drawbacks to routine human use. As with
most antibodies or other large, foreign proteins, there is
considerable risk of immunologic toxicity which could limit or
preclude administration to humans. Also, while the vasculature
targeting may improve the local concentration of the attached
chemotherapeutic agents, the agents still must be cleaved from the
antibody carrier and be transported or diffuse into the cells to be
cytotoxic.
[0012] Thus, it is desirable to provide anti-angiogenic
pharmaceuticals and tumor or new vasculature imaging agents which
do not suffer from poor diffusion or transportation, possible
immunologic toxicity, limited availability, and/or a lack of
specificity.
[0013] There is also a growing interest in therapeutic angiogenesis
to improve blood flow in regions of the body that have become
ischemic or poorly perfused. Several investigators are using growth
factors administered locally to cause new vasculature to form
either in the limbs or the heart. The growth factors VEGF and bFGF
are the most common for this application. Recent publications
include: Takeshita, S., et. al., J. Clin. Invest., 1994, 93,
662-670; and Schaper, W. and Schaper, J., Collateral
Circulation:Heart, Brain, Kidney, Limbs, Kluwer Academic
Publishers, Boston, 1993. The main applications that are under
investigation in a number of laboratories are for improving cardiac
blood flow and in improving peripheral vessal blood flow in the
limbs. For example, Henry, T. et. al. (J. Amer. College Cardiology,
1998, 31, 65A) describe the use of recombinant human VEGF in
patients for improving myocardial perfusion by therapeutic
angiogenesis. Patients received infusions of rhVEGF and were
monitored by nuclear perfusion imaging 30 and 60 days post
treatment to determine improvement in myocardial perfusion. About
50% of patients showed improvement by nuclear perfusion imaging
whereas {fraction (5/7)} showed new collatoralization by
angiography.
[0014] Thus, it is desirable to discover a method of monitoring
improved cardiac blood flow which is targeted to new collateral
vessels themselves and not, as in nuclear perfusion imaging, a
regional consequence of new collateral vessels.
[0015] Another therapeutic application of the radiopharmaceuticals
of the present invention that emit cytotoxic radiation (Beta and
Alpha particles and Auger electons) is in treating rheumatoid
arthritis (RA). In RA, the ingrowth of a highly vascularized pannus
is caused by the excessive production of angiogenic factors by the
infiltrating macrophages, immune cells, or inflammatory cells.
Therefore, the radiopharmaceuticals of the present inventions can
be used to destroy the new angiogenic vasculature that results and
thus treat the disease.
[0016] It is one object of the present invention to provide
improved anti-angiogenic pharmaceuticals, comprised of a targeting
moiety that binds to the vitronectin receptor that is expressed in
tumor neovasculature, an optional linking group, and a
radioisotope. The vitronectin receptor binding compounds target the
radioisotope to the tumor neovasculature. The beta or
alpha-particle emitting radioisotope emits a cytotoxic amount of
ionizing radiation which results in cell death. The penetrating
ability of radiation obviates the requirement that the cytotoxic
agent diffuse or be transported into the cell to be cytotoxic.
[0017] It is another object of the present invention to provide
tumor imaging agents, comprised of tumor neovasculature vitronectin
receptor binding compounds conjugated to an imageable moiety, such
as a gamma ray or positron emitting radioisotope, a magnetic
resonance imaging contrast agent, an X-ray contrast agent, or an
ultrasound contrast agent.
SUMMARY OF THE INVENTION
[0018] It is one object of the present invention to provide
anti-angiogenic pharmaceuticals, comprised of a targeting moiety
that binds to a receptor that is expressed in tumor neovasculature,
an optional linking group, and a radioactive metal ion that emits
ionizing radiation such as beta particles, alpha particles and
Auger or Coster-Kronig electrons. The receptor binding compounds
target the radioisotope to the tumor neovasculature. The beta or
alpha-particle emitting radioisotope emits a cytotoxic amount of
ionizing radiation which results in cell death. The penetrating
ability of radiation obviates the requirement that the cytotoxic
agent diffuse or be transported into the cell to be cytotoxic.
[0019] It is another object of the present invention to provide
pharmaceuticals to treat rheumatoid arthritis. These
pharmaceuticals comprise a targeting moiety that binds to a
receptor that is upregulated during angiogenesis, an optional
linking group, and a radioisotope that emits cytotoxic radiation
(i.e., beta particles, alpha particles and Auger or Coster-Kronig
electrons). In rheumatoid arthritis, the ingrowth of a highly
vascularized pannus is caused by the excessive production of
angiogenic factors by the infiltrating macrophages, immune cells,
or inflammatory cells. Therefore, the radiopharmaceuticals of the
present invention that emit cytotoxic radiation could be used to
destroy the new angiogenic vasculature that results and thus treat
the disease.
[0020] It is another object of the present invention to provide
tumor imaging agents, comprised of targeting moiety that binds to a
receptor that is upregulated during angiogenesis, an optional
linking group, and an imageable moiety, such as a gamma ray or
positron emitting radioisotope, a magnetic resonance imaging
contrast agent, an X-ray contrast agent, or an ultrasound contrast
agent.
[0021] It is another object of the present invention to provide
imaging agents for monitoring the progress and results of
therapeutic angiogenesis treatment. These agents comprise of
targeting moiety that binds to a receptor that is upregulated
during angiogenesis, an optional linking group, and an imageable
moiety. Imaging agents of the present invention could be
administered intravenously periodically after the administration of
growth factors and imaging would be performed using standard
techniques of the affected areas, heart or limbs, to monitor the
progress and results of the therapeutic angiogenesis treatment
(i.e., image the formation of new blood vessels).
[0022] It is another object of the present invention to provide
compounds useful for preparing the pharmaceuticals of the present
invention. These compounds are comprised of a peptide or
peptidomimetic targeting moiety that binds to a receptor that is
upregulated during angiogenesis, Q, an optional linking group, Ln,
and a metal chelator or bonding moiety, C.sub.h. The compounds may
have one or more protecting groups attached to the metal chelator
or bonding moiety. The protecting groups provide improved stability
to the reagents for long-term storage and are removed either
immediately prior to or concurrent with the synthesis of the
radiopharmaceuticals. Alternatively, the compounds of the present
invention are comprised of a peptide or peptidomimetic targeting
moiety that binds to a receptor that is upregulated during
angiogenesis, Q, an optional linking group, L.sub.n, and a
surfactant, S.sub.f.
[0023] The pharmaceuticals of the present invention may be used for
diagnostic and/or therapeutic purposes. Diagnostic
radiopharmaceuticals of the present invention are pharmaceuticals
comprised of a diagnostically useful radionuclide (i.e., a
radioactive metal ion that has imageable gamma ray or positron
emissions). Therapeutic radiopharmaceuticals of the present
invention are pharmaceuticals comprised of a therapeutically useful
radionuclide, a radioactive metal ion that emits ionizing radiation
such as beta particles, alpha particles and Auger or Coster-Kronig
electrons.
[0024] The pharmaceuticals comprising a gamma ray or positron
emitting radioactive metal ion are useful for imaging tumors by
gamma scintigraphy or positron emission tomography. The
pharmaceuticals comprising a gamma ray or positron emitting
radioactive metal ion are also useful for imaging therapeutic
angiogenesis by gamma scintigraphy or positron emission tomography.
The pharmaceuticals comprising a particle emitting radioactive
metal ion are useful for treating cancer by delivering a cytotoxic
dose of radiation to the tumors. The pharmaceuticals comprising a
particle emitting radioactive metal ion are also useful for
treating rheumatoid arthritis by destroying the formation of
angiogenic vasculature. The pharmaceuticals comprising a
paramagnetic metal ion are useful as magnetic resonance imaging
contrast agents. The pharmaceuticals comprising one or more X-ray
absorbing or "heavy" atoms of atomic number 20 or greater are
useful as X-ray contrast agents. The pharmaceuticals comprising a
microbubble of a biocompatible gas, a liquid carrier, and a
surfactant microsphere, are useful as ultrasound contrast
agents.
DETAILED DESCRIPTION OF THE INVENTION
[0025] [1] Thus, in a first embodiment, the present invention
provides a novel compound, comprising: a targeting moiety and a
chelator, wherein the targeting moiety is bound to the chelator, is
a benzodiazepine nonpeptide, and binds to a receptor that is
upregulated during angiogenesis and the compound has 0-1 linking
groups between the targeting moiety and chelator.
[0026] [2] In a preferred embodiment, the targeting moiety
comprises a benzodiazepine and the receptor is selected from the
group: EGFR, FGFR, PDGFR, Flk-1/KDR, Flt-1, Tek, Tie, neuropilin-1,
endoglin, endosialin, Axl, .alpha..sub.v.beta..sub.3,
.alpha..sub.v.beta..sub.5, .alpha..sub.5.beta..sub.1,
.alpha..sub.4.beta..sub.l, and .alpha..sub.2.beta..sub.2 and the
linking group is present between the targeting moiety and
chelator.
[0027] [3] In a more preferred embodiment, the receptor is the
integrin .alpha..sub.v.beta..sub.3 and the compound is of the
formula:
(Q)d--L.sub.n--C.sub.h
or
(Q)d--L.sub.n--(C.sub.h)d'
[0028] wherein, Q is a compound of Formula (I): 1
[0029] wherein:
[0030] one of R or R.sup.1 is selected from a bond to L.sub.n or
(CH.sub.2).sub.1-4 or an NH bond to L.sub.n and the other of R or
R.sup.1 is selected from C.sub.1-4 alkyl, benzyl or phenethyl;
[0031] R.sup.2 is selected from benzimidazole or imidazole;
[0032] R.sup.3 is selected from H, C.sub.1-4 alkyl or benzyl;
[0033] R.sup.4 is selected from H, C.sub.1-4 alkyl or benzyl;
[0034] d is selected from 1, 2 and 3;
[0035] L.sub.n is a linking group having the formula:
(CR.sup.6R.sup.7).sub.g--(W).sub.h--(CR.sup.6aR.sup.7a).sub.g'--(Z).sub.k--
-(W).sub.h'--(CR.sup.8R.sup.9)
.sub.g"--(W).sub.h"--(CR.sup.8aR.sup.9a(W).-
sub.h'"--(CR.sup.8bR.sup.9a).sub.g""
[0036] provided that g+h+g'+k+h'+g'+h"+g'" is other than 0;
[0037] W is independently selected at each occurrence from the
group: O, S, NH, NHC(.dbd.O), C(.dbd.O)NHC(.dbd.O), C(.dbd.O)O,
OC(.dbd.O), NHC(.dbd.S)NH, NHC(=O)NH, SO.sub.2,
(OCH.sub.2CH.sub.2).sub.1', (CH.sub.2CH.sub.2O).sub.s,
(OCH.sub.2CH.sub.2CH.sub.2).sub.s",
(CH.sub.2CH.sub.2CH.sub.2O).sub.t, and (aa)ti;
[0038] aa is independently at each occurrence an amino acid;
[0039] Z is selected from the group: aryl substituted with 0-3
R.sup.10, C.sub.3-10 cycloalkyl substituted with 0-3 R.sup.10, and
a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms
independently selected from N, S, and O and substituted with 0-3
R.sup.10;
[0040] R.sup.6, R.sup.6a, R.sup.7, R.sup.7a, R.sup.8, R.sup.8a,
R.sup.8b, R9, R.sup.9a and R.sup.9b are independently selected at
each occurrence from the group: H, .dbd.O, COOH, SO.sub.3H,
PO.sub.3H, C.sub.1-C.sub.5 alkyl substituted with 0-3 R.sup.10,
aryl substituted with 0-3 R.sup.10, benzyl substituted with 0-3
R.sup.10, and C.sub.1-C.sub.5 alkoxy substituted with 0-3 R.sup.10,
NHC(.dbd.O)R.sup.11, C(.dbd.O)NHR.sup.11, NHC(.dbd.O)NHR.sup.11,
NHR.sup.11, R.sup.11, and a bond to C.sub.h;
[0041] R.sup.10 is independently selected at each occurrence from
the group: a bond to C.sub.h, COOR.sup.11, OH, NHR.sup.11,
C(.dbd.O)NHR.sup.11, NH(C.dbd.O)R.sup.11, SO.sub.3H, PO.sub.3H,
.dbd.O, R.sup.11, aryl substituted with 0-3 R.sup.11, C.sub.1-5
alkyl substituted with 0-1 R.sup.12, C.sub.1-5 alkoxy substituted
with 0-1 R.sup.12, and a 5-10 membered heterocyclic ring system
containing 1-4 heteroatoms independently selected from N, S, and O
and substituted with 0-3 R.sup.11;
[0042] R.sup.11 is independently selected at each occurrence from
the group: H, C.sub.1-C.sub.10 alkyl substituted with 0-1 R.sup.12,
aryl substituted with 0-1 R.sup.12, a 5-10 membered heterocyclic
ring system containing 1-4 heteroatoms independently selected from
N, S, and O and substituted with 0-1 R.sup.12, C.sub.3-10
cycloalkyl substituted with 0-1 R.sup.12, polyalkylene glycol
substituted with 0-1 R.sup.12, carbohydrate substituted with 0-1
R.sup.12, cyclodextrin substituted with 0-1 R.sup.12, amino acid
substituted with 0-1 R.sup.12, polycarboxyalkyl substituted with
0-1 R.sup.12, polyazaalkyl substituted with 0-1 R.sup.12, peptide
substituted with 0-1 R.sup.12, wherein the peptide is comprised of
2-10 amino acids, and a bond to C.sub.h;
[0043] R.sup.12 is a bond to C.sub.h;
[0044] k is selected from 0, 1, and 2;
[0045] h is selected from 0, 1, and 2;
[0046] h' is selected from 0, 1, 2, 3, 4, and 5;
[0047] h" is selected from 0, 1, 2, 3, 4, and 5;
[0048] h'" is selected from 0, 1, 2, 3, 4, and 5;
[0049] g is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
[0050] g' is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and
10;
[0051] g" is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and
10;
[0052] g'" is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and
10;
[0053] g"" is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and
10;
[0054] s is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
[0055] s' is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and
10;
[0056] s" is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and
10;
[0057] t is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
[0058] t' is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and
10;
[0059] C.sub.h is a metal bonding unit having a formula selected
from the group: 2
[0060] A.sup.1, A.sup.2, A.sup.3, A.sup.4, A.sup.5, A.sup.6,
A.sup.7, and A.sup.8 are independently selected at each occurrence
from the group: NR.sup.13, NR.sup.13R.sup.14, S, SH, S(Pg), O, OH,
PR.sup.13, PR.sup.13R.sup.14, P(O)R.sup.15R.sup.16, CO.sub.2H and a
bond to L.sub.n;
[0061] E is a bond, CH, or a spacer group independently selected at
each occurrence from the group: C.sub.1-C.sub.10 alkyl substituted
with 0-3 R.sup.17, aryl substituted with 0-3 R.sup.17, C.sub.3-10
cycloalkyl substituted with 0-3 R.sup.17,
heterocyclo-C.sub.1-C.sub.10 alkyl substituted with 0-3 R.sup.17,
wherein the heterocyclo group is a 5-10 membered heterocyclic ring
system containing 1-4 heteroatoms independently selected from N, S,
and O, C.sub.6-10 aryl-C.sub.1-.sub.10 alkyl substituted with 0-3
R.sup.17, C.sub.1-10 alkyl-C.sub.6-10 aryl- substituted with 0-3
R.sup.17, and a 5-10 membered heterocyclic ring system containing
1-4 heteroatoms independently selected from N, S, and O and
substituted with 0-3 R.sup.17;
[0062] R.sup.13, and R.sup.14 are each independently selected from
the group: a bond to L.sub.n, hydrogen, C.sub.1-C.sub.10 alkyl
substituted with 0-3 R.sup.17, aryl substituted with 0-3 R.sup.17,
C.sub.1-10 cycloalkyl substituted with 0-3 R.sup.17,
heterocyclo-C.sub.1-10 alkyl substituted with 0-3 R.sup.17, wherein
the heterocyclo group is a 5-10 membered heterocyclic ring system
containing 1-4 heteroatoms independently selected from N, S, and O,
C.sub.6-10 aryl-C.sub.1-10 alkyl substituted with 0-3 R.sup.17,
C.sub.1-10 alkyl-C.sub.6-10 aryl- substituted with 0-3 R.sup.17, a
5-10 membered heterocyclic ring system containing 1-4 heteroatoms
independently selected from N, S, and O and substituted with 0-3
R.sup.17, and an electron, provided that when one of R.sup.13 or
R.sup.14 is an electron, then the other is also an electron;
[0063] alternatively, R.sup.13 and R.sup.14 combine to form
.dbd.C(R.sup.20) (R.sup.21);
[0064] R.sup.15 and R.sup.16 are each independently selected from
the group: a bond to L.sub.n, --OH, C.sub.1-C.sub.10 alkyl
substituted with 0-3 R.sup.17, C.sub.1-C.sub.10 alkyl substituted
with 0-3 R.sup.17, aryl substituted with 0-3 R.sup.17, C.sub.3-10
cycloalkyl substituted with 0-3 R.sup.17, heterocyclo-C.sub.1-10
alkyl substituted with 0-3 R.sup.17, wherein the heterocyclo group
is a 5-10 membered heterocyclic ring system containing 1-4
heteroatoms independently selected from N, S, and O, C.sub.6-10
aryl-C.sub.1-10 alkyl substituted with 0-3 R.sup.17, C.sub.1-10
alkyl-C.sub.6-10 aryl-substituted with 0-3 R.sup.17, and a 5-10
membered heterocyclic ring system containing 1-4 heteroatoms
independently selected from N, S, and O and substituted with 0-3
R.sup.17;
[0065] R.sup.17 is independently selected at each occurrence from
the group: a bond to L.sub.n, .dbd.O, F, Cl, Br, I, --CF.sub.3,
--CN, --CO.sub.2R.sup.18, --C(.dbd.O)R.sup.18,
--C(.dbd.O)N(R.sup.18).sub.2, --CHO, --CH.sub.2OR.sup.18,
--OC(.dbd.O)R.sup.18, --OC(.dbd.O)OR.sup.18a, --OR.sup.18,
--OC(.dbd.O)N(R.sup.18).sub.2, --NR.sup.19C(.dbd.O)R.sup.18,
--NR.sup.19C(.dbd.O)OR.sup.18a,
--NR.sup.19C(.dbd.O)N(R.sup.18).sub.2,
--NR.sup.19SO.sub.2N(R.sup.18).sub.2, --NR.sup.19SO.sub.2R.sup.18a,
--SO.sub.3H, --SO.sub.2R.sup.18a, --SR.sup.18,
--S(.dbd.O)R.sup.18a, --SO.sub.2N(R.sup.18).sub.2,
--N(R.sup.18).sub.2, --NHC(.dbd.S)NHR.sup.18- , .dbd.NOR.sup.18,
NO.sub.2, --C(.dbd.O)NHOR.sup.18, --C(.dbd.O)NHNR.sup.18R.sup.18a,
--OCH.sub.2CO.sub.2H, 2-(1-morpholino)ethoxy, C.sub.1-C.sub.5
alkyl, C.sub.2-C.sub.4 alkenyl, C.sub.3-C.sub.6 cycloalkyl,
C.sub.3-C.sub.6 cycloalkylmethyl, C.sub.2-C.sub.6 alkoxyalkyl, aryl
substituted with 0-2 R.sup.18, and a 5-10 membered heterocyclic
ring system containing 1-4 heteroatoms independently selected from
N, S, and O;
[0066] R.sup.18, R.sup.18a, and R.sup.19 are independently selected
at each occurrence from the group: a bond to L.sub.n, H,
C.sub.1-C.sub.6 alkyl, phenyl, benzyl, C.sub.1-C.sub.6 alkoxy,
halide, nitro, cyano, and trifluoromethyl;
[0067] Pg is a thiol protecting group;
[0068] R.sup.20 and R.sup.21 are independently selected from the
group: H, C.sub.1-C.sub.10 alkyl, --CN, --CO.sub.2R.sup.25,
--C(.dbd.O)R.sup.25, --C(.dbd.O)N(R.sup.25).sub.2,
C.sub.2-C.sub.10-alkene substituted with 0-3 R.sup.23,
C.sub.2-C.sub.10 1-alkyne substituted with 0-3 R.sup.23, aryl
substituted with 0-3 R.sup.23, unsaturated 5-10 membered
heterocyclic ring system containing 1-4 heteroatoms independently
selected from N, S, and O and substituted with 0-3 R.sup.23, and
unsaturated C.sub.3-10 carbocycle substituted with 0-3
R.sup.23;
[0069] alternatively, R.sup.20 and R.sup.21, taken together with
the divalent carbon radical to which they are attached form: 3
[0070] R.sup.22 and R.sup.23 are independently selected from the
group: H, R.sup.24, C.sub.1-C.sub.10 alkyl substituted with 0-3
R.sup.24, C.sub.2-C.sub.10 alkenyl substituted with 0-3 R.sup.24,
C.sub.2-C.sub.10 alkynyl substituted with 0-3 R.sup.24, aryl
substituted with 0-3 R.sup.24, a 5-10 membered heterocyclic ring
system containing 1-4 heteroatoms independently selected from N, S,
and O and substituted with 0-3 R.sup.24, and C.sub.3-10 carbocycle
substituted with 0-3 R.sup.24;
[0071] alternatively, R.sup.22, R.sup.23 taken together form a
fused aromatic or a 5-10 membered heterocyclic ring system
containing 1-4 heteroatoms independently selected from N, S, and
O;
[0072] a and b indicate the positions of optional double bonds and
n is 0 or 1;
[0073] R.sup.24 is independently selected at each occurrence from
the group: .dbd.O, F, Cl, Br, I, --CF.sub.3, --CN,
--CO.sub.2R.sup.25, --C(.dbd.O)R.sup.25,
--C(.dbd.O)N(R.sup.25).sub.2, --N(R.sup.25).sub.3+,
--CH.sub.2OR.sup.25, --OC(.dbd.O)R.sup.25, --OC(.dbd.O)OR.sup.25a,
--OR.sup.25, --OC(.dbd.O)N(R.sup.25).sub.2,
--NR.sup.26C(.dbd.O)R.sup.25, NR.sup.26C(.dbd.O)OR.sup.25a,
--NR.sup.26C(.dbd.O)N(R.sup.25).sub.2,
--NR.sup.26SO.sub.2N(R.sup.25).sub.2, --NR.sup.26SO.sub.2R.sup.25a,
--SO.sub.3H, --SO.sub.2R.sup.25a, --SR.sup.25,
--S(.dbd.O)R.sup.25a, --SO.sub.2N(R.sup.25).sub.2,
--N(R.sup.25).sub.2, .dbd.NOR.sup.25, --C(.dbd.O)NHOR.sup.25,
--OCH.sub.2CO.sub.2H, and 2-(1-morpholino)ethoxy; and,
[0074] R.sup.25, R.sup.25a, and R.sup.26 are each independently
selected at each occurrence from the group: hydrogen and
C.sub.1-C.sub.6 alkyl;
[0075] and a pharmaceutically acceptable salt thereof.
[0076] [4] In an even more preferred embodiment, the present
invention provides a compound wherein:
[0077] d is selected from 1, 2, 3, 4, and 5;
[0078] Z is selected from the group: aryl substituted with 0-1
R.sup.10, C.sub.3-10 cycloalkyl substituted with 0-1 R.sup.10, and
a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms
independently selected from N, S, and O and substituted with 0-1
R.sup.10;
[0079] A.sup.1, A.sup.2, A.sup.3, A.sup.4, A.sup.5, A.sup.6,
A.sup.7, and A.sup.8 are independently selected at each occurrence
from the group: NR.sup.13, NR.sup.13R.sup.14, S, SH, S(Pg), OH, and
a bond to L.sub.n;
[0080] E is a bond, CH, or a spacer group independently selected at
each occurrence from the group: C.sub.1-C.sub.10 alkyl substituted
with 0-3 R.sup.17, aryl substituted with 0-3 R.sup.17, C.sub.3-10
cycloalkyl substituted with 0-3 R.sup.17, and a 5-10 membered
heterocyclic ring system containing 1-4 heteroatoms independently
selected from N, S, and O and substituted with 0-3 R.sup.17;
[0081] R.sup.13, and R.sup.14 are each independently selected from
the group: a bond to L.sub.n, hydrogen, C.sub.1-C.sub.10 alkyl
substituted with 0-3 R.sup.17, aryl substituted with 0-3 R.sup.17,
a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms
independently selected from N, S, and O and substituted with 0-3
R.sup.17, and an electron, provided that when one of R.sup.13 or
R.sup.14 is an electron, then the other is also an electron;
[0082] alternatively, R.sup.13 and R.sup.14 combine to form
.dbd.C(R.sup.20) (R.sup.21);
[0083] R.sup.17 is independently selected at each occurrence from
the group: a bond to L.sub.n, .dbd.O, F, Cl, Br, I, --CF.sub.3,
--CN, --CO.sub.2R.sup.18, --C(.dbd.O)R.sup.18,
--C(.dbd.O)N(R.sup.18).sub.2, --CH.sub.2OR.sup.18,
--OC(.dbd.O)R.sup.18, --OC(.dbd.O)OR.sup.18a, --OR.sup.18,
--OC(.dbd.O)N(R.sup.18).sub.2, --NR.sup.19C(.dbd.O)R.sup.18,
NR.sup.19C(.dbd.O)OR.sup.18a,
--NR.sup.19C(.dbd.O)N(R.sup.18).sub.2,
--NR.sup.19SO.sub.2N(R.sup.18).sub.2, --NR.sup.19SO.sub.2R.sup.18a,
--SO.sub.3H, --SO.sub.2R.sup.18a, --S(.dbd.O)R.sup.18a,
--SO.sub.2N(R.sup.18).sub.2, --N(R.sup.18).sub.2,
--NHC(.dbd.S)NHR.sup.18- , .dbd.NOR.sup.18,
--C(.dbd.O)NHNR.sup.18R.sup.18a, --OCH.sub.2CO.sub.2H, and
2-(1-morpholino)ethoxy;
[0084] R.sup.18, R.sup.18a, and R.sup.19 are independently selected
at each occurrence from the group: a bond to L.sub.n, H, and
C.sub.1-C.sub.6 alkyl;
[0085] R.sup.20 and R.sup.21 are independently selected from the
group: H, C.sub.1-C.sub.5 alkyl, --CO.sub.2R.sup.25,
C.sub.2-C.sub.5 1-alkene substituted with 0-3 R.sup.23,
C.sub.2-C.sub.5 1-alkyne substituted with 0-3 R.sup.23, aryl
substituted with 0-3 R.sup.23, and unsaturated 5-10 membered
heterocyclic ring system containing 1-4 heteroatoms independently
selected from N, S, and O and substituted with 0-3 R.sup.23;
[0086] alternatively, R.sup.20 and R.sup.21, taken together with
the divalent carbon radical to which they are attached form: 4
[0087] R.sup.22 and R.sup.23 are independently selected from the
group: H, and R.sup.24;
[0088] alternatively, R.sup.22, R.sup.23 taken together form a
fused aromatic or a 5-10 membered heterocyclic ring system
containing 1-4 heteroatoms independently selected from N, S, and
O;
[0089] R.sup.24 is independently selected at each occurrence from
the group: --CO.sub.2R.sup.25, --C(.dbd.O)N(R.sup.25).sub.2,
--CH.sub.2OR.sup.25, --OC(.dbd.O)R.sup.25, --OR.sup.25,
--SO.sub.3H, --N(R.sup.25).sub.2, and --OCH.sub.2CO.sub.2H;
and,
[0090] R.sup.25 is independently selected at each occurrence from
the group: H and C.sub.1-C.sub.3 alkyl.
[0091] [5] In a still more preferred embodiment, the present
invention provides a compound, wherein:
[0092] Q is a peptide selected from the group: 5
[0093] K is an L-amino acid independently selected at each
occurrence from the group: arginine, citrulline, N-methylarginine,
lysine, homolysine, 2-aminoethylcysteine,
d-N-2-imidazolinylornithine, d-N-benzylcarbamoylornithine, and
b-2-benzimidazolylacetyl-1,2-diaminopro- pionic acid;
[0094] L is glycine;
[0095] M is L-aspartic acid;
[0096] M' is D-aspartic acid;
[0097] R.sup.1 is L-valine, D-valine or L-lysine optionally
substituted on the e amino group with a bond to L.sub.n;
[0098] R.sup.2 is L-phenylalanine, D-phenylalanine,
D-1-naphthylalanine, 2-aminothiazole-4-acetic acid or tyrosine, the
tyrosine optionally substituted on the hydroxy group with a bond to
L.sub.n;
[0099] R.sup.3 is D-valine;
[0100] R.sup.4 is D-tyrosine substituted on the hydroxy group with
a bond to L.sub.n;
[0101] provided that one of R.sup.1 and R.sup.2 in each Q is
substituted with a bond to L.sub.n, and further provided that when
R.sup.2 is 2-aminothiazole-4-acetic acid, K is
N-methylarginine;
[0102] provided that at least one Q is a benzodiazepine;
[0103] d is 1, 2, or 3;
[0104] A.sup.1 is selected from the group: OH, and a bond to
L.sub.n;
[0105] A.sup.2, A.sup.4, and A.sup.6 are each N;
[0106] A.sup.3, A.sup.5, and A.sup.8 are each OH;
[0107] A.sup.7 is a bond to Ln or NH-bond to L.sub.n;
[0108] E is a C.sub.2 alkyl substituted with 0-1 R.sup.17;
[0109] R.sup.17 is .dbd.O;
[0110] alternatively, C.sub.h is 6
[0111] is selected from the group: OH, and a bond to L.sub.n;
[0112] A.sup.1, A.sup.2, A.sup.3 and A.sup.4 are each N;
[0113] A.sup.5, A.sup.6 and A.sup.8 are each OH;
[0114] A.sup.7 is a bond to L.sub.n;
[0115] E is a C.sub.2 alkyl substituted with 0-1 R.sup.17;
[0116] R.sup.17 is .dbd.O;
[0117] alternatively, C.sub.h is 7
[0118] A.sup.1 is NH.sub.2 or N.dbd.C (R.sup.20) (R.sup.21);
[0119] E is a bond;
[0120] A.sup.2 is NHR.sup.13;
[0121] R.sup.13 is a heterocycle substituted with R.sup.17, the
heterocycle being selected from pyridine and pyrimidine;
[0122] R.sup.17 is selected from a bond to L.sub.n,
C(.dbd.O)NHR.sup.18 and C(.dbd.O) R.sup.18;
[0123] R.sup.18 is a bond to L.sub.n;
[0124] R.sup.24 is selected from the group: --CO.sub.2R.sup.25,
--OR.sup.25, --SO.sub.3H, and --N(R.sup.25).sub.2; and,
[0125] R.sup.25 is independently selected at each occurrence from
the group: hydrogen and methyl.
[0126] [6] In another even more preferred embodiment, the present
invention provides a compound selected from the group:
[0127]
(S,S,S)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methyl-
carbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-y-
l)propyl)carbamoyl)
-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carb- oxymethyl)
cyclodecyl)acetylamino)butanoyl amino)butanoic acid;
[0128]
(S)-2-(2,5-diaza-5-(6((6-((1-aza-2-(2-sulfophenyl)vinyl)amino)(3-py-
ridyl))carbonylamino)hexyl)-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamo-
yl)-4-oxobicyclo [5.4.0]undeca-1(7),8,10-trien-3-yl) acetic
acid;
[0129]
(S)-2-(2,5-diaza-9-(N-(6-((6-((l-aza-2-(2-sulfophenyl)vinyl)amino)(-
3-pyridyl))carbonylamino)hexyl)-N-(benzimidazol-2-ylmethyl)carbamoyl)-5-me-
thyl-4-oxobicyclo [5.4.0]undeca-1(7),8,10-trien-3-yl)acetic
acid;
[0130]
(S,S)-2-(2-aza-2-((5-(N-(1,3-bis(N-(6-(aminohexyl-4-oxobicyclo[5.4.-
0]undeca-l(7),8,10-trien-3-yl)acetic
acid)(2-(2,5-diaza-9-(N-(benzimidazol-
-2-ylmethyl)propyl)carbamoyl)(2-pyridyl))amino)vinyl)
benzenesulfonic acid;
[0131]
(S,S,S)-4-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylme-
thyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)pr-
opyl)
carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxym-
ethyl) cyclododecyl)acetylamino)butanoylamino) butanoic acid;
[0132]
(S,S)-3-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol-2-ylmeth-
yl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)prop-
yl) carbamoyl)-3-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)
cyclododecyl)acetylamino)propanoic acid;
[0133]
(S,S,S,S,S,S,S,S)-4-(N-1,3-bis(N-3-carboxy-l-(N-(3-(3,6-diaza-lo-(N-
-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyc-
lo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4,4-dihydroxypentyl-
)
carbamoyl)propyl)carbamoyl)-4-(5,5-dihydroxy-2-(2-(1,4,7,10-tetraaza-4,7-
,10-tris(carboxymethyl)cyclodecyl)acetylamino) butanoic acid;
[0134]
(S,S,S,S,S,S,S,S,S,S)-2-(4-(N-(1,3-bis(N-(3-(N-(3-(3,6-diaza-10-(N--
(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-
-oxobicyclo
[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-1-(methox-
ycarbonyl)propyl)carbamoyl)propyl)carbamoyl)p
ropyl)carbamoyl)-4-(2-(2-(1,-
4,7,10-tetraaza-4,7,10-tris(carboxymethyl)
cyclodecyl)acetylamino)-4-carbo-
xybutanoylamino)-4-carboxybutanoylamino)butanoylamino)-4-(N-(3-(3,6-diaza--
lo-(N-(benzimidazol-2-ylmethyl)-N-methyl
carbamoyl)-5-((methoxycarbonyl)me- thyl)-4-oxobicyclo[5.4.0]
undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)but- anoic acid;
[0135]
(S)-2-(2,5-diaza-5-(3-(2-(2-(3-((6-((l-aza-2-(2-sulfophenyl)vinyl)a-
mino)(3-pyridyl))carbonylamino)
propoxy)ethoxy)ethoxy)propyl)-9-(N-(benzim-
idazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10--
trien-3-yl)acetic acid;
[0136]
(S,S,S,S,S)-4-(N-(1,3-bis(N-(3-(2-(2-(3-(3,6-diaza-10-(N-(benzimida-
zol-2-ylmethyl)-N-methylcarbamoyl)
-5-(carboxymethyl)-4-oxobicyclo[5.4.0]u-
ndeca-1(7),8,10-trien-3-yl)propoxy)ethoxy)ethoxy)propyl)carbamoyl)
propyl)carbamoyl)-4-(5,5-dihydroxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(ca-
rboxy methyl)cyclododecyl)acetylamino) hexanoylamino)butanoic
acid;
[0137]
(S,S,S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamo-
yl)-4-oxo-5-(6-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-2-(-
4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxy
hexyl)carbamoyl)-2-(2-(1,4,7,10-tet-
raaza-4,7,10-atris(carboxymethyl)cyclodecyl)
acetylamino)butanoylamino)but- anoylamino)hexyl)bicycl
o[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic acid;
[0138]
(S,S,S,S)-2-(4-(N-(l-(N-(l-(N-(6-(3,6-diaza-10-(N-(benzimidazol-2-y-
lmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(-
7),8,10-trien-3-yl)hexyl)carbamoyl)-3-(N-cyclo{Lys-Arg(Mtr)-Gly-Asp(OtBu)--
D-Phe}[gamma-LysNH]carbamoyl)propyl)carbamoyl)-3-carboxypropyl)
carbamoyl)-4-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)
cyclododecyl)acetylamino)butanoic acid; and
[0139] or a pharmaceutically acceptable salt form thereof.
[0140] [7] In a further preferred embodiment, the present invention
provides a kit comprising a compound of the present invention.
[0141] [8] In an even further preferred embodiment, the kit further
comprises one or more ancillary ligands and a reducing agent.
[0142] [9] In a still further preferred embodiment, the ancillary
ligands are tricine and TPPTS.
[0143] [10] In another still further preferred embodiment, the
reducing agent is tin(II).
[0144] [11] In a second embodiment, the present invention provides
a novel diagnostic or therapeutic metallopharmaceutical
composition, comprising: a metal, a chelator capable of chelating
the metal and a targeting moiety, wherein the targeting moiety is
bound to the chelator, is a benzodiazepine nonpeptide and binds to
a receptor that is upregulated during angiogenesis and the compound
has 0-1 linking groups between the targeting moiety and
chelator.
[0145] [12] In another preferred embodiment, the
metallopharmaceutical is a diagnostic radiopharmaceutical, the
metal is a radioisotope selected from the group: .sup.99mTc,
.sup.95Tc, .sup.111In, .sup.62Cu, .sup.64Cu, .sup.67Ga, and
.sup.68Ga, the targeting moiety comprises a benzodiazepine and the
receptor is selected from the group: EGFR, FGFR, PDGFR, Flk-1/KDR,
Flt-1, Tek, Tie, neuropilin-1, endoglin, endosialin, Axl,
.alpha..sub.v.beta..sub.3, .alpha..sub.v.beta..sub.5,
.alpha..sub.5.beta..sub.1, .alpha..sub.4.beta..sub.1,
.alpha..sub.1.beta..sub.1,and .alpha..sub.2.beta..sub.2 and the
linking group is present between the targeting moiety and
chelator.
[0146] [13] In another more preferred embodiment, the targeting
moiety is a benzodiazepine and the receptor is
.alpha..sub.v.beta..sub.3.
[0147] [14] In another even more preferred embodiment, the
radioisotope is .sup.99mTc or .sup.95Tc, the radiopharmaceutical
further comprises a first ancillary ligand and a second ancillary
ligand capable of stabilizing the radiopharmaceutical.
[0148] [15] In another still more preferred embodiment, the
radioisotope is .sup.99mTc.
[0149] [16] In another further preferred embodiment, the
radiopharmaceutical is selected from the group:
[0150] .sup.99mTc(
(S)-2-(2,5-diaza-5-(6((6-(diazenido)(3-pyridyl))carbony-
lamino)hexyl)-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxobicyc-
lo [5.4.0]undeca-1(7),8,10-trien-3-yl) acetic
acid)(tricine)(TPPTS);
[0151] .sup.99mTc( (S)
-2-(2,5-diaza-9-(N-(6-((6-(diazenido)(3-pyridyl))ca-
rbonylamino)hexyl)-N-(benzimidazol-2-ylmethyl)carbamoyl)-5-methyl-4-oxobic-
yclo [5.4.0]undeca-1(7),8,10-trien-3-yl)acetic acid) (tricine)
(TPPTS);
[0152] [17] In another even more preferred embodiment, the
radioisotope is .sup.111In.
[0153] [18] In another still more preferred embodiment, the
radiopharmaceutical is selected from the group:
[0154] .sup.111In complex of
6-(N-(3-(3-aza-10-(N-(benzimidazol-2-ylmethyl-
)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo
[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-(2-((2-(
(carboxymethyl) (2-((carboxymethyl)methylamino)ethyl)amino) ethyl)
(2-((carboxymethyl)ethylamino)ethyl)amino)-acetylamino)-4-oxooctane-1,8-d-
icarboxylic acid;
[0155] .sup.111In complex of
(S,S,S)-4-(N-(3-(3,6-diaza-5-(carboxymethyl)--
10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1-
(7),8,10-trien-3-yl)propyl)
carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaz-
a-4,7,10-tris(carboxymethyl)
cyclododecyl)acetylamino)butanoylamino) butanoic acid; and
[0156] .sup.111In complex of
(S,S)-3-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-
-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7-
),8,10-trien-3-yl)propyl)
carbamoyl)-3-(2-(1,4,7,10-tetraaza-4,7,10-tris(c- arboxymethyl)
cyclododecyl)acetylamino)propanoic acid.
[0157] [19] In another preferred embodiment, the
metallopharmaceutical is a therapeutic radiopharmaceutical, the
metal is a radioisotope selected from the group: .sup.186Re,
.sup.188Re, .sup.153Sm, .sup.166Ho, .sup.177Lu, .sup.149Pm,
.sup.90y, .sup.212Bi, .sup.103Pd, .sup.109Pd, .sup.159Gd,
.sup.140La, .sup.198Au, .sup.199Au, .sup.169Yb, .sup.175Yb,
.sup.165Dy, .sup.166Dy, .sup.67Cu, .sup.105Rh, .sup.111Ag, and
.sup.192Ir, the targeting moiety is a benzodiazepine nonpeptide and
the receptor is selected from the group: EGFR, FGFR, PDGFR,
Flk-1/KDR, Flt-1, Tek, Tie, neuropilin-1, endoglin, endosialin,
Axl, .alpha..sub.v.beta..sub.3, .alpha..sub.v.beta..sub.5,
.alpha..sub.5.beta..sub.1, .alpha..sub.4.beta..sub.1,
.alpha..sub.1.beta..sub.1, and .alpha..sub.2.beta..sub.2 and the
linking group is present between the targeting moiety and
chelator.
[0158] [20] In another more preferred embodiment, the targeting
moiety is an benzodiazepine and the receptor is
.alpha..sub.v.beta..sub.3.
[0159] [21] In another even more preferred embodiment, the
radioisotope is .sup.153Sm.
[0160] [22] In another even more preferred embodiment, the
radioisotope is .sup.177Lu.
[0161] [23] In another even more preferred embodiment, the
radioisotope is .sup.90Y.
[0162] [24] In another preferred embodiment, the
metallopharmaceutical is a MRI contrast agent, the metal is a
paramagnetic metal ion selected from the group: Gd(III), Dy(III),
Fe(III), and Mn(II), the targeting moiety is a benzodiazepine
nonpeptide and the receptor is selected from the group: EGFR, FGFR,
PDGFR, Flk-1/KDR, Flt-1, Tek, Tie, neuropilin-1, endoglin,
endosialin, Axl, .alpha..sub.v.beta..sub.3,
.alpha..sub.v.beta..sub.5, .alpha..sub.5.beta..sub.1,
.alpha..sub.4.beta..sub.1, .alpha..sub.1.beta..sub.1, and
.alpha..sub.2.beta..sub.2 and the linking group is present between
the targeting moiety and chelator.
[0163] [25] In another more preferred embodiment, the targeting
moiety is a benzodiazepine and the receptor is
.alpha..sub.v.beta..sub.3.
[0164] [26] In another even more preferred embodiment, the metal
ion is Gd(III).
[0165] [27] In another preferred embodiment, the
metallopharmaceutical is a X-ray contrast agent, the metal is
selected from the group: Re, Sm, Ho, Lu, Pm, Y, Bi, Pd, Gd, La, Au,
Au, Yb, Dy, Cu, Rh, Ag, and Ir, the targeting moiety is a
benzodiazepine nonpeptide, the receptor is
.alpha..sub.v.beta..sub.3, and the linking group is present between
the targeting moiety and chelator.
[0166] [28] In another even more preferred embodiment, the present
invention provides a novel method of treating rheumatoid arthritis
in a patient comprising: administering a therapeutic
radiopharmaceutical of the present invention capable of localizing
in new angiogenic vasculature to a patient by injection or
infusion.
[0167] [29] In another even more preferred embodiment, the present
invention provides a novel method of treating cancer in a patient
comprising: administering to a patient in need thereof a
therapeutic radiopharmaceutical of the present invention by
injection or infusion.
[0168] [30] In another even more preferred embodiment, the present
invention provides a novel method of imaging formation of new blood
vessels in a patient comprising: (1) administering a diagnostic
radiopharmaceutical, a MRI contrast agent, or a X-ray contrast
agent of the present invention to a patient by injection or
infusion; (2) imaging the area of the patient wherein the desired
formation of new blood vessels is located.
[0169] [31] In another even more preferred embodiment, the present
invention provides a novel method of imaging cancer in a patient
comprising: (1) administering a diagnostic radiopharmaceutical of
the present invention to a patient by injection or infusion; (2)
imaging the patient using planar or SPECT gamma scintigraphy, or
positron emission tomography.
[0170] [32] In another even more preferred embodiment, the present
invention provides a novel method of imaging cancer in a patient
comprising: (1) administering a MRI contrast agent of the present
invention; and (2) imaging the patient using magnetic resonance
imaging.
[0171] [33] In another even more preferred embodiment, the present
invention provides a novel method of imaging cancer in a patient
comprising: (1) administering a X-ray contrast agent of the present
invention; and (2) imaging the patient using X-ray computed
tomography.
[0172] [34] In a third embodiment, the present invention provides a
novel compound capable of being used in an ultrasound contrast
composition, comprising: a targeting moiety and a surfactant,
wherein the targeting moiety is bound to the surfactant, is a
benzodiazepine nonpeptide, and binds to a receptor that is
upregulated during angiogenesis and the compound has 0-1 linking
groups between the targeting moiety and surfactant.
[0173] [35] In a preferred embodiment, the targeting moiety
comprises an benzodiazepine and the receptor is selected from the
group: EGFR, FGFR, PDGFR, Flk-1/KDR, Flt-1, Tek, Tie, neuropilin-1,
endoglin, endosialin, Axl, .alpha..sub.v.beta..sub.3,
.alpha..sub.v.beta..sub.5, .alpha..sub.5.beta..sub.1,
.alpha..sub.4.beta..sub.1, .alpha..sub.1.beta..sub.1, and
.alpha..sub.2.beta..sub.2 and the linking group is present between
the targeting moiety and surfactant.
[0174] [36] In a more preferred embodiment, the receptor is the
integrin .alpha..sub.v.beta..sub.3 and the compound is of the
formula:
(Q)d--Ln--Sf
[0175] wherein, Q is a compound of Formula (I): 8
[0176] wherein:
[0177] one of R or R.sup.1 is selected from a bond to L.sub.n or
(CH.sub.2).sub.1-4 or an NH bond to L.sub.n and the other of R or
R.sup.1 is selected from C.sub.1-4 alkyl, benzyl or phenethyl;
[0178] R.sup.2 is selected from benzimidazole or imidazole;
[0179] R.sup.3 is selected from H, C.sub.1-4 alkyl or benzyl;
[0180] R.sup.4 is selected from H, C.sub.1-4 alkyl or benzyl;
[0181] d is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
[0182] S.sub.f is a surfactant which is a lipid or a compound of
the formula: 9
[0183] A.sup.9 is selected from the group: OH and OR.sup.27;
[0184] A.sup.10 is OR.sup.27;
[0185] R.sup.27 is C(.dbd.O)C.sub.1-20 alkyl;
[0186] E.sup.1 is C.sub.1-10 alkylene substituted with 1-3
R.sup.28;
[0187] R.sup.28 is independently selected at each occurrence from
the group: R.sup.30, --PO.sub.3H--R.sup.30, .dbd.O,
--CO.sub.2R.sup.29, --C(.dbd.O)R.sup.29,
--C(.dbd.O)N(R.sup.29).sub.2, --CH.sub.2OR.sup.29, --OR.sup.29,
--N(R.sup.29).sub.2, C.sub.1-C.sub.5 alkyl, and C.sub.2-C.sub.4
alkenyl;
[0188] R.sup.29 is independently selected at each occurrence from
the group: R.sup.30, H, C.sub.1-C.sub.6 alkyl, phenyl, benzyl, and
trifluoromethyl;
[0189] R.sup.30 is a bond to L.sub.n;
[0190] L.sub.n is a linking group having the formula:
(CR.sup.6R.sup.7).sub.g--(W).sub.h--(CR.sup.6aR.sup.7a).sub.g'--(Z).sub.k--
-(W).sub.h'--(CR.sup.8R.sup.9).sub.g"--(W).sub.h"--(CR.sup.8aR.sup.9a).sub-
.g'"
[0191] W is independently selected at each occurrence from the
group: O, S, NH, NHC(.dbd.O), C(.dbd.O)NH, C(.dbd.O), C(.dbd.O)O,
OC(.dbd.O), NHC(.dbd.S)NH, NHC(.dbd.O)NH, SO.sub.2,
(OCH.sub.2CH.sub.2).sub.20-200, (CH.sub.2CH.sub.2O).sub.20-200,
(OCH.sub.2CH.sub.2CH.sub.2).sub.20-200,
(CH.sub.2CH.sub.2CH.sub.2).sub.20-200, and (aa).sub.t';
[0192] aa is independently at each occurrence an amino acid;
[0193] Z is selected from the group: aryl substituted with 0-3
R.sup.10, C.sub.3-10 cycloalkyl substituted with 0-3 R.sup.10, and
a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms
independently selected from N, S, and O and substituted with 0-3
R.sup.10;
[0194] R.sup.6, R.sup.6a, R.sup.7, R.sup.7a, R.sup.8, R.sup.8a,
R.sup.9 and R.sup.9a are independently selected at each occurrence
from the group: H, .dbd.O, COOH, SO.sub.3H, PO.sub.3H,
C.sub.1-C.sub.5 alkyl substituted with 0-3 R.sup.10, aryl
substituted with 0-3 R.sup.10, benzyl substituted with 0-3
R.sup.10, and C.sub.1-C.sub.5 alkoxy substituted with 0-3 R.sup.10,
NHC(.dbd.O)R.sup.11, C(.dbd.O)NHR.sup.11, NHC(.dbd.O)NHR.sup.11,
NHR.sup.11, R.sup.11, and a bond to S.sub.f;
[0195] R.sup.10 is independently selected at each occurrence from
the group: a bond to S.sub.f, COOR.sup.11, OH, NHR.sup.11,
SO.sub.3H, PO.sub.3H, aryl substituted with 0-3 R.sup.11, C.sub.1-5
alkyl substituted with 0-1 R.sup.12, C.sub.1-5 alkoxy substituted
with 0-1 R.sup.12, and a 5-10 membered heterocyclic ring system
containing 1-4 heteroatoms independently selected from N, S, and O
and substituted with 0-3 R.sup.11;
[0196] R.sup.11 is independently selected at each occurrence from
the group: H, aryl substituted with 0-1 R.sup.12, a 5-10 membered
heterocyclic ring system containing 1-4 heteroatoms independently
selected from N, S, and O and substituted with 0-1 R.sup.12,
C.sub.3-10 cycloalkyl substituted with 0-1 R.sup.12, amino acid
substituted with 0-1 R.sup.12, and a bond to S.sub.f;
[0197] R.sup.12 is a bond to S.sub.f;
[0198] k is selected from 0, 1, and 2;
[0199] h is selected from 0, 1, and 2;
[0200] h' is selected from 0, 1, 2, 3, 4, and 5;
[0201] h" is selected from 0, 1, 2, 3, 4, and 5;
[0202] g is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;
[0203] g' is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and
10;
[0204] g" is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and
10;
[0205] g'" is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and
10;
[0206] t' is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and
10;
[0207] and a pharmaceutically acceptable salt thereof.
[0208] [37] In another even more preferred embodiment, the compound
is of the formula:
Q--L.sub.n--S.sub.f
[0209] wherein, Q is a compound of Formula (I): 10
[0210] wherein:
[0211] one of R or R.sup.1 is selected from a bond to L.sub.n or
(CH.sub.2).sub.1-4 or an NH bond to L.sub.n and the other of R or
R.sup.1 is selected from C.sub.1-4 alkyl, benzyl or phenethyl;
[0212] R.sup.2 is selected from benzimidazole or imidazole;
[0213] R.sup.3 is selected from H. C.sub.1-4 alkyl or benzyl;
[0214] R.sup.4 is selected from H. C.sub.1-4 alkyl or benzyl;
[0215] S.sub.f is a surfactant which is a lipid or a compound of
the formula: 11
[0216] A.sup.9 is OR.sup.27;
[0217] A.sup.10 is OR.sup.27;
[0218] R.sup.27 is C(.dbd.O)C.sub.1-15 alkyl;
[0219] E.sup.1 is C.sub.1-4 alkylene substituted with 1-3
R.sup.28;
[0220] R.sup.28 is independently selected at each occurrence from
the group: R.sup.30, --PO.sub.3H-R.sup.30, .dbd.O,
--CO.sub.2R.sup.29, --C(.dbd.O)R.sup.29, --CH.sub.2OR.sup.29,
--OR.sup.29, and C.sub.1-C.sub.5 alkyl;
[0221] R.sup.29 is independently selected at each occurrence from
the group: R.sup.30, H, C.sub.1-C.sub.6 alkyl, phenyl, and
benzyl;
[0222] R.sup.30 is a bond to L.sub.n;
[0223] L.sub.n is a linking group having the formula:
(CR.sup.6R.sup.7).sub.g--(W).sub.h--(CR.sup.6aR.sup.7a).sub.g'--(Z).sub.k--
-(W).sub.h'--(CR.sup.8R.sup.9).sub.g"--(W).sub.h"--(CR.sup.8aR.sup.9a).sub-
.g'"
[0224] W is independently selected at each occurrence from the
group: O, S, NH, NHC(.dbd.O), C(.dbd.O)NH, C(.dbd.O), C(.dbd.O)O,
OC(.dbd.O), NHC(.dbd.S)NH, NHC(.dbd.O)NH, SO.sub.2,
(OCH.sub.2CH.sub.2).sub.20-200, (CH.sub.2CH.sub.2O).sub.20-200,
(OCH.sub.2CH.sub.2CH.sub.2).sub.20-200,
(CH.sub.2CH.sub.2CH.sub.2).sub.20-200, and (aa).sub.t';
[0225] aa is independently at each occurrence an amino acid;
[0226] z is selected from the group: aryl substituted with 0-3
R.sup.10, C.sub.3-10 cycloalkyl substituted with 0-3 R.sup.10, and
a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms
independently selected from N, S, and O and substituted with 0-3
R.sup.10;
[0227] R.sup.6, R.sup.6a, R.sup.7, R.sup.7a, R.sup.8, R.sup.8a,
R.sup.9 and R.sup.9a are independently selected at each occurrence
from the group: H, .dbd.O, C.sub.1-C.sub.5 alkyl substituted with
0-3 R.sup.10, and C.sub.1-C.sub.5 alkoxy substituted with 0-3
R.sup.10, and a bond to S.sub.f;
[0228] R.sup.10 is independently selected at each occurrence from
the group: a bond to S.sub.f, COOR.sup.11, OH, NHR.sup.11,
C.sub.1-5 alkyl substituted with 0-1 R.sup.12, and C.sub.1-5 alkoxy
substituted with 0-1 R.sup.12;
[0229] R.sup.11 is independently selected at each occurrence from
the group: H, aryl substituted with 0-1 R.sup.12, C.sub.3-10
cycloalkyl substituted with 0-1 R.sup.12, amino acid substituted
with 0-1 R.sup.12, and a bond to S.sub.f;
[0230] R.sup.12 is a bond to S.sub.f;
[0231] k is selected from 0, 1, and 2;
[0232] h is selected from 0, 1, and 2;
[0233] h' is selected from 0, 1, 2, 3, 4, and 5;
[0234] h" is selected from 0, 1, 2, 3, 4, and 5;
[0235] g is selected from 0, 1, 2, 3, 4, and 5;
[0236] g' is selected from 0, 1, 2, 3, 4, and 5;
[0237] g" is selected from 0, 1, 2, 3, 4, and 5;
[0238] g'" is selected from 0, 1, 2, 3, 4, and 5;
[0239] s is selected from 0, 1, 2, 3, 4, and 5;
[0240] s' is selected from 0, 1, 2, 3, 4, and 5;
[0241] s" is selected from 0, 1, 2, 3, 4, and 5;
[0242] t is selected from 0, 1, 2, 3, 4, and 5;
[0243] t' is selected from 0, 1, 2, 3, 4, and 5;
[0244] and a pharmaceutically acceptable salt thereof.
[0245] [38] In another still more preferred embodiment, the present
invention provides a compound selected from the group:
[0246] Sodium
1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-(S)-2--
(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(6-aminohex-
yl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic
acid-dodecoanoate conjugate;
[0247] DPPE-PEG.sub.3400-[(S) -2-(2,
5-diaza-9-(N-(benzimidazol-2-ylmethyl-
)-N-methylcarbamoyl)-5-(6-aminohexyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10--
trien-3-yl)acetic acid]-dodecoanoate conjugate; and
[0248] [(S)
-2-(2-aza-(2-((S-(N-(1,3-bis-N-(6-(aminohexyl-4-oxobicyclo[5.4-
.0]undeca-l(7),8,10-trien-3-yl)acetic
acid)(2-(2,5-diaza-9-(N-(benzimidazo- l-2-ylmethyl) carbamoyl)
propyl) carbamoyl] -w-amino-PEG.sub.3400-dodecano- ate-DPPE
conjugate;
[0249] [39] In another even more preferred embodiment, the present
invention provides a novel ultrasound contrast agent composition,
comprising:
[0250] (a) a compound comprising: a benzodiazepine that binds to
the integrin .alpha..sub.v.beta..sub.3, a surfactant and a linking
group between the benzodiazepine and the surfactant;
[0251] (b) a parenterally acceptable carrier; and,
[0252] (c) an echogenic gas.
[0253] [40] In another still more preferred embodiment, the
ultrasound contrast agent further comprises:
1,2-dipalmitoyl-sn-glycero-3-phosphotid- ic acid,
1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, and
N-(methoxypolyethylene glycol 5000
carbamoyl)-1,2-dipalmitoyl-sn-glycero--
3-phosphatidylethanolamine.
[0254] [41] In another further preferred embodiment, the echogenic
gas is a C.sub.2-5 perfluorocarbon.
[0255] [42] In another even more preferred embodiment, the present
invention provides a method of imaging cancer in a patient
comprising: (1) administering, by injection or infusion, a
ultrasound contrast agent composition of the present invention to a
patient; and (2) imaging the patient using sonography.
[0256] [43] In another even more preferred embodiment, the present
invention provides a novel method of imaging formation of new blood
vessels in a patient comprising: (1) administering, by injection or
infusion, a ultrasound contrast agent composition of the present
invention to a patient; (2) imaging the area of the patient wherein
the desired formation of new blood vessels is located.
[0257] [44] In another even more preferred embodiment, the present
invention provides a novel therapeutic radiopharmaceutical
composition, comprising:
[0258] (a) a therapeutic radiopharmaceutical of the present
invention; and,
[0259] (b) a parenterally acceptable carrier.
[0260] [45] In another even more preferred embodiment, the present
invention provides a novel therapeutic radiopharmaceutical
composition, comprising:
[0261] (a) a diagnostic radiopharmaceutical, a MRI contrast agent,
or a X-ray contrast agent of the present invention; and,
[0262] (b) a parenterally acceptable carrier.
[0263] Another aspect of the present invention are diagnostic kits
for the preparation of radiopharmaceuticals useful as imaging
agents for cancer. Diagnostic kits of the present invention
comprise one or more vials containing the sterile, non-pyrogenic,
formulation comprised of a predetermined amount of a reagent of the
present invention, and optionally other components such as one or
two ancillary ligands, reducing agents, transfer ligands, buffers,
lyophilization aids, stabilization aids, solubilization aids and
bacteriostats. The inclusion of one or more optional components in
the formulation will frequently improve the ease of synthesis of
the radiopharmaceutical by the practicing end user, the ease of
manufacturing the kit, the shelf-life of the kit, or the stability
and shelf-life of the radiopharmaceutical. The inclusion of one or
two ancillary ligands is required for diagnostic kits comprising
reagent comprising a hydrazine or hydrazone bonding moiety. The one
or more vials that contain all or part of the formulation can
independently be in the form of a sterile solution or a lyophilized
solid.
[0264] Another aspect of the present invention contemplates a
method of imaging cancer in a patient involving: (1) synthesizing a
diagnostic radiopharmaceutical of the present invention, using a
reagent of the present invention, capable of localizing in tumors;
(2) administering said radiopharmaceutical to a patient by
injection or infusion; (3) imaging the patient using planar or
SPECT gamma scintigraphy, or positron emission tomography.
[0265] Another aspect of the present invention contemplates a
method of imaging cancer in a patient involving: (1) administering
a paramagnetic metallopharmaceutical of the present invention
capable of localizing in tumors to a patient by injection or
infusion; and (2) imaging the patient using magnetic resonance
imaging.
[0266] Another aspect of the present invention contemplates a
method of imaging cancer in a patient involving: (1) administering
a X-ray contrast agent of the present invention capable of
localizing in tumors to a patient by injection or infusion; and (2)
imaging the patient using X-ray computed tomography.
[0267] Another aspect of the present invention contemplates a
method of imaging cancer in a patient involving: (1) administering
a ultrasound contrast agent of the present invention capable of
localizing in tumors to a patient by injection or infusion; and (2)
imaging the patient using sonography.
[0268] Another aspect of the present invention contemplates a
method of treating cancer in a patient involving: (1) administering
a therapeutic radiopharmaceutical of the present invention capable
of localizing in tumors to a patient by injection or infusion.
DEFINITIONS
[0269] The compounds herein described may have asymmetric centers.
Unless otherwise indicated, all chiral, diastereomeric and racemic
forms are included in the present invention. Many geometric isomers
of olefins, C.dbd.N double bonds, and the like can also be present
in the compounds described herein, and all such stable isomers are
contemplated in the present invention. It will be appreciated that
compounds of the present invention contain asymmetrically
substituted carbon atoms, and may be isolated in optically active
or racemic forms. It is well known in the art how to prepare
optically active forms, such as by resolution of racemic forms or
by synthesis from optically active starting materials. Two distinct
isomers (cis and trans) of the peptide bond are known to occur;
both can also be present in the compounds described herein, and all
such stable isomers are contemplated in the present invention. The
D and L-isomers of a particular amino acid are designated herein
using the conventional 3-letter abbreviation of the amino acid, as
indicated by the following examples: D-Leu, or L-Leu.
[0270] When any variable occurs more than one time in any
substituent or in any formula, its definition on each occurrence is
independent of its definition at every other occurrence. Thus, for
example, if a group is shown to be substituted with 0-2 R.sup.52,
then said group may optionally be substituted with up to two
R.sup.52, and R.sup.52 at each occurrence is selected independently
from the defined list of possible R.sup.52. Also, by way of
example, for the group --N(R.sup.53).sub.2, each of the two
R.sup.53 substituents on N is independently selected from the
defined list of possible R53. Combinations of substituents and/or
variables are permissible only if such combinations result in
stable compounds. When a bond to a substituent is shown to cross
the bond connecting two atoms in a ring, then such substituent may
be bonded to any atom on the ring.
[0271] The term "nonpeptide" means preferably less than three amide
bonds in the backbone core of the targeting moiety or preferably
less than three amino acids or amino acid mimetics in the targeting
moiety.
[0272] The term "metallopharmaceutical" means a pharmaceutical
comprising a metal. The metal is the cause of the imageable signal
in diagnostic applications and the source of the cytotoxic
radiation in radiotherapeutic applications. Radiopharmaceuticals
are metallopharmaceuticals in which the metal is a
radioisotope.
[0273] By "reagent" is meant a compound of this invention capable
of direct transformation into a metallopharmaceutical of this
invention. Reagents may be utilized directly for the preparation of
the metallopharmaceuticals of this invention or may be a component
in a kit of this invention.
[0274] The term "binding agent" means a metallopharmaceutical of
this invention having affinity for and capable of binding to the
vitronectin receptor. The binding agents of this invention have
Ki<1000 nM.
[0275] By "stable compound" or "stable structure" is meant herein a
compound that is sufficiently robust to survive isolation to a
useful degree of purity from a reaction mixture, and formulation
into an efficacious pharmaceutical agent.
[0276] The term "substituted", as used herein, means that one or
more hydrogens on the designated atom or group is replaced with a
selection from the indicated group, provided that the designated
atom's or group's normal valency is not exceeded, and that the
substitution results in a stable compound. When a substituent is
keto (i.e., .dbd.O), then 2 hydrogens on the atom are replaced.
[0277] The term "bond", as used herein, means either a single or
double bond.
[0278] The term "salt", as used herein, is used as defined in the
CRC Handbook of Chemistry and Physics, 65th Edition, CRC Press,
Boca Raton, Florida, 1984, as any substance which yields ions,
other than hydrogen or hydroxyl ions. As used herein,
"pharmaceutically acceptable salts" refer to derivatives of the
disclosed compounds modified by making acid or base salts. Examples
of pharmaceutically acceptable salts include, but are not limited
to, mineral or organic acid salts of basic residues such as amines;
alkali or organic salts of acidic residues such as carboxylic
acids; and the like.
[0279] The phrase "pharmaceutically acceptable" is employed herein
to refer to those compounds, materials, compositions, and/or dosage
forms which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of human beings and
animals without excessive toxicity, irritation, allergic response,
or other problem or complication, commensurate with a reasonable
benefit/risk ratio.
[0280] As used herein, "pharmaceutically acceptable salts" refer to
derivatives of the disclosed compounds wherein the parent compound
is modified by making acid or base salts thereof. Examples of
pharmaceutically acceptable salts include, but are not limited to,
mineral or organic acid salts of basic residues such as amines;
alkali or organic salts of acidic residues such as carboxylic
acids; and the like. The pharmaceutically acceptable salts include
the conventional non-toxic salts or the quaternary ammonium salts
of the parent compound formed, for example, from non-toxic
inorganic or organic acids. For example, such conventional
non-toxic salts include those derived from inorganic acids such as
hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric
and the like; and the salts prepared from organic acids such as
acetic, propionic, succinic, glycolic, stearic, lactic, tartaric,
citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic,
glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic,
fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic,
oxalic, isethionic, and the like.
[0281] The pharmaceutically acceptable salts of the present
invention can be synthesized from the parent compound which
contains a basic or acidic moiety by conventional chemical methods.
Generally, such salts can be prepared by reacting the free acid or
base forms of these compounds with a stoichiometric amount of the
appropriate base or acid in water or in an organic solvent, or in a
mixture of the two; generally, nonaqueous media like ether, ethyl
acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists
of suitable salts are found in Remington's Pharmaceutical Sciences,
17th ed., Mack Publishing Company, Easton, PA, 1985, p. 1418, the
disclosure of which is hereby incorporated by reference.
[0282] As used herein, "alkyl" is intended to include both branched
and straight-chain saturated aliphatic hydrocarbon groups having
the specified number of carbon atoms, examples of which include,
but are not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl,
i-butyl, sec-butyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl,
and decyl; "cycloalkyl" or "carbocycle" is intended to include
saturated and partially unsaturated ring groups, including mono-,
bi- or poly-cyclic ring systems, such as cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and adamantyl;
"bicycloalkyl" or "bicyclic" is intended to include saturated
bicyclic ring groups such as [3.3.0]bicyclooctane,
[4.3.0]bicyclononane, [4.4.0]bicyclodecane (decalin),
[2.2.2]bicyclooctane, and so forth.
[0283] As used herein, the term "alkene" or "alkenyl" is intended
to include hydrocarbon chains having the specified number of carbon
atoms of either a straight or branched configuration and one or
more unsaturated carbon-carbon bonds which may occur in any stable
point along the chain, such as ethenyl, propenyl, and the like.
[0284] As used herein, the term "alkyne" or "alkynyl" is intended
to include hydrocarbon chains having the specified number of carbon
atoms of either a straight or branched configuration and one or
more unsaturated carbon-carbon triple bonds which may occur in any
stable point along the chain, such as propargyl, and the like.
[0285] As used herein, "aryl" or "aromatic residue" is intended to
mean phenyl or naphthyl, which when substituted, the substitution
can be at any position.
[0286] As used herein, the term "heterocycle" or "heterocyclic
system" is intended to mean a stable 5- to 7- membered monocyclic
or bicyclic or 7- to 10-membered bicyclic heterocyclic ring which
is saturated partially unsaturated or unsaturated (aromatic), and
which consists of carbon atoms and from 1 to 4 heteroatoms
independently selected from the group consisting of N, O and S and
including any bicyclic group in which any of the above-defined
heterocyclic rings is fused to a benzene ring. The nitrogen and
sulfur heteroatoms may optionally be oxidized. The heterocyclic
ring may be attached to its pendant group at any heteroatom or
carbon atom which results in a stable structure. The heterocyclic
rings described herein may be substituted on carbon or on a
nitrogen atom if the resulting compound is stable. If specifically
noted, a nitrogen in the heterocycle may optionally be quaternized.
It is preferred that when the total number of S and O atoms in the
heterocycle exceeds 1, then these heteroatoms are not adjacent to
one another. It is preferred that the total number of S and o atoms
in the heterocycle is not more than 1. As used herein, the term
"aromatic heterocyclic system" is intended to mean a stable 5- to
7- membered monocyclic or bicyclic or 7- to 10-membered bicyclic
heterocyclic aromatic ring which consists of carbon atoms and from
1 to 4 heteroatoms independently selected from the group consisting
of N, O and S. It is preferred that the total number of S and O
atoms in the aromatic heterocycle is not more than 1.
[0287] Examples of heterocycles include, but are not limited to,
1H-indazole, 2-pyrrolidonyl, 2H,6H-1,5,2-dithiazinyl, 2H-pyrrolyl,
3H-indolyl, 4-piperidonyl, 4aH-carbazole, 4H-quinolizinyl,
6H-1,2,5-thiadiazinyl, acridinyl, azocinyl, benzimidazolyl,
benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl,
benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl,
benzisothiazolyl, benzimidazalonyl, carbazolyl, 4aH-carbazolyl,
.beta.-carbolinyl, chromanyl, chromenyl, cinnolinyl,
decahydroquinolinyl, 2H,6H-1, 5,2-dithiazinyl,
dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl,
imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl,
indolinyl, indolizinyl, indolyl, isobenzofuranyl, isochromanyl,
isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,
isothiazolyl, isoxazolyl, morpholinyl, naphthyridinyl,
octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,
1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl,
oxazolidinyl., oxazolyl, oxazolidinylperimidinyl, phenanthridinyl,
phenanthrolinyl, phenarsazinyl, phenazinyl, phenothiazinyl,
phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl,
piperidinyl, pteridinyl, piperidonyl, 4-piperidonyl, pteridinyl,
purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl,
pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole,
pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl,
pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl,
quinuclidinyl, carbolinyl, tetrahydrofuranyl,
tetrahydroisoquinolinyl, tetrahydroquinolinyl,
6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl,
1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl,
thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl,
thiophenyl, triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl,
1,2,5-triazolyl, 1,3,4-triazolyl, xanthenyl. Preferred heterocycles
include, but are not limited to, pyridinyl, furanyl, thienyl,
pyrrolyl, pyrazolyl, imidazolyl, indolyl, benzimidazolyl,
1H-indazolyl, oxazolidinyl, benzotriazolyl, benzisoxazolyl,
oxindolyl, benzoxazolinyl, or isatinoyl. Also included are fused
ring and spiro compounds containing, for example, the above
heterocycles.
[0288] As used herein, the term "alkaryl" means an aryl group
bearing an alkyl group of 1-10 carbon atoms; the term "aralkyl"
means an alkyl group of 1-10 carbon atoms bearing an aryl group;
the term "arylalkaryl" means an aryl group bearing an alkyl group
of 1-10 carbon atoms bearing an aryl group; and the term
"heterocycloalkyl" means an alkyl group of 1-10 carbon atoms
bearing a heterocycle.
[0289] A "polyalkylene glycol" is a polyethylene glycol,
polypropylene glycol or polybutylene glycol having a molecular
weight of less than about 5000, terminating in either a hydroxy or
alkyl ether moiety.
[0290] A "carbohydrate" is a polyhydroxy aldehyde, ketone, alcohol
or acid, or derivatives thereof, including polymers thereof having
polymeric linkages of the acetal type.
[0291] A "cyclodextrin" is a cyclic oligosaccharide. Examples of
cyclodextrins include, but are not limited to,
.alpha.-cyclodextrin, hydroxyethyl-.alpha.-cyclodextrin,
hydroxypropyl-.alpha.-cyclodextrin, .beta.-cyclodextrin,
hydroxypropyl-.beta.-cyclodextrin,
carboxymethyl-.beta.-cyclodextrin,
dihydroxypropyl-.beta.-cyclodextrin,
hydroxyethyl-.beta.-cyclodextrin, 2,6
di-O-methyl-.beta.-cyclodextrin, sulfated-.beta.-cyclodextrin,
.gamma.-cyclodextrin, hydroxypropyl-.gamma.-cyclodextrin,
dihydroxypropyl-.gamma.-cyclodextrin,
hydroxyethyl-.gamma.-cyclodextrin, and sulfated
.gamma.-cyclodextrin.
[0292] As used herein, the term "polycarboxyalkyl" means an alkyl
group having between two and about 100 carbon atoms and a plurality
of carboxyl substituents; and the term "polyazaalkyl" means a
linear or branched alkyl group having between two and about 100
carbon atoms, interrupted by or substituted with a plurality of
amine groups.
[0293] A "reducing agent" is a compound that reacts with a
radionuclide, which is typically obtained as a relatively
unreactive, high oxidation state compound, to lower its oxidation
state by transferring electron(s) to the radionuclide, thereby
making it more reactive. Reducing agents useful in the preparation
of radiopharmaceuticals and in diagnostic kits useful for the
preparation of said radiopharmaceuticals include but are not
limited to stannous chloride, stannous fluoride, formamidine
sulfinic acid, ascorbic acid, cysteine, phosphines, and cuprous or
ferrous salts. Other reducing agents are described in Brodack et.
al., PCT Application 94/22496, which is incorporated herein by
reference.
[0294] A "transfer ligand" is a ligand that forms an intermediate
complex with a metal ion that is stable enough to prevent unwanted
side-reactions but labile enough to be converted to a
metallopharmaceutical. The formation of the intermediate complex is
kinetically favored while the formation of the
metallopharmaceutical is thermodynamically favored. Transfer
ligands useful in the preparation of metallopharmaceuticals and in
diagnostic kits useful for the preparation of diagnostic
radiopharmaceuticals include but are not limited to gluconate,
glucoheptonate, mannitol, glucarate,
N,N,N',N'-ethylenediaminetetraacetic acid, pyrophosphate and
methylenediphosphonate. In general, transfer ligands are comprised
of oxygen or nitrogen donor atoms.
[0295] The term "donor atom" refers to the atom directly attached
to a metal by a chemical bond.
[0296] "Ancillary" or "co-ligands" are ligands that are
incorporated into a radiopharmaceutical during its synthesis. They
serve to complete the coordination sphere of the radionuclide
together with the chelator or radionuclide bonding unit of the
reagent. For radiopharmaceuticals comprised of a binary ligand
system, the radionuclide coordination sphere is composed of one or
more chelators or bonding units from one or more reagents and one
or more ancillary or co-ligands, provided that there are a total of
two types of ligands, chelators or bonding units. For example, a
radiopharmaceutical comprised of one chelator or bonding unit from
one reagent and two of the same ancillary or co-ligands and a
radiopharmaceutical comprised of two chelators or bonding units
from one or two reagents and one ancillary or co-ligand are both
considered to be comprised of binary ligand systems. For
radiopharmaceuticals comprised of a ternary ligand system, the
radionuclide coordination sphere is composed of one or more
chelators or bonding units from one or more reagents and one or
more of two different types of ancillary or co-ligands, provided
that there are a total of three types of ligands, chelators or
bonding units. For example, a radiopharmaceutical comprised of one
chelator or bonding unit from one reagent and two different
ancillary or co-ligands is considered to be comprised of a ternary
ligand system.
[0297] Ancillary or co-ligands useful in the preparation of
radiopharmaceuticals and in diagnostic kits useful for the
preparation of said radiopharmaceuticals are comprised of one or
more oxygen, nitrogen, carbon, sulfur, phosphorus, arsenic,
selenium, and tellurium donor atoms. A ligand can be a transfer
ligand in the synthesis of a radiopharmaceutical and also serve as
an ancillary or co-ligand in another radiopharmaceutical. Whether a
ligand is termed a transfer or ancillary or co-ligand depends on
whether the ligand remains in the radionuclide coordination sphere
in the radiopharmaceutical, which is determined by the coordination
chemistry of the radionuclide and the chelator or bonding unit of
the reagent or reagents.
[0298] A "chelator" or "bonding unit" is the moiety or group on a
reagent that binds to a metal ion through the formation of chemical
bonds with one or more donor atoms.
[0299] The term "binding site" means the site in vivo or in vitro
that binds a biologically active molecule.
[0300] A "diagnostic kit" or "kit" comprises a collection of
components, termed the formulation, in one or more vials which are
used by the practicing end user in a clinical or pharmacy setting
to synthesize diagnostic radiopharmaceuticals. The kit provides all
the requisite components to synthesize and use the diagnostic
radiopharmaceutical except those that are commonly available to the
practicing end user, such as water or saline for injection, a
solution of the radionuclide, equipment for heating the kit during
the synthesis of the radiopharmaceutical, if required, equipment
necessary for administering the radiopharmaceutical to the patient
such as syringes and shielding, and imaging equipment.
[0301] Therapeutic radiopharmaceuticals, X-ray contrast agent
pharmaceuticals, ultrasound contrast agent pharmaceuticals and
metallopharmaceuticals for magnetic resonance imaging contrast are
provided to the end user in their final form in a formulation
contained typically in one vial, as either a lyophilized solid or
an aqueous solution. The end user reconstitutes the lyophilized
with water or saline and withdraws the patient dose or just
withdraws the dose from the aqueous solution formulation as
provided.
[0302] A "lyophilization aid" is a component that has favorable
physical properties for lyophilization, such as the glass
transition temperature, and is added to the formulation to improve
the physical properties of the combination of all the components of
the formulation for lyophilization.
[0303] A "stabilization aid" is a component that is added to the
metallopharmaceutical or to the diagnostic kit either to stabilize
the metallopharmaceutical or to prolong the shelf-life of the kit
before it must be used. Stabilization aids can be antioxidants,
reducing agents or radical scavengers and can provide improved
stability by reacting preferentially with species that degrade
other components or the metallopharmaceutical.
[0304] A "solubilization aid" is a component that improves the
solubility of one or more other components in the medium required
for the formulation.
[0305] A "bacteriostat" is a component that inhibits the growth of
bacteria in a formulation either during its storage before use of
after a diagnostic kit is used to synthesize a
radiopharmaceutical.
[0306] The following abbreviations are used herein:
1 Acm acetamidomethyl b-Ala, beta-Ala 3-aminopropionic acid or bAla
ATA 2-aminothiazole-5-aceti- c acid or 2- aminothiazole-5-acetyl
group Boc t-butyloxycarbonyl CBZ, Cbz or Z Carbobenzyloxy Cit
citrulline Dap 2,3-diaminopropionic acid DCC
dicyclohexylcarbodiimide DIEA diisopropylethylamine DMAP
4-dimethylaminopyridine EOE ethoxyethyl HBTU
2-(1H-Benzotriazol-1-yl)-1,1,3,3- tetramethyluronium
hexafluorophosphate boc-hydrazinonicotinyl group or 2- hynic
[[[5-[carbonyl]-2- pyridinyl]hydrazono]methyl]- benzenesulfonic
acid, NMeArg or MeArg a-N-methyl arginine NMeAsp a-N-methyl
aspartic acid NMM N-methylmorpholine OcHex O-cyclohexyl OBzl
O-benzyl oSu O-succinimidyl TBTU 2-(1H-Benzotriazol-1-yl)-1,1,3,3-
tetramethyluronium tetrafluoroborate THF tetrahydrofuranyl THP
tetrahydropyranyl Tos tosyl Tr trityl
[0307] The following conventional three-letter amino acid
abbreviations are used herein; the conventional one-letter amino
acid abbreviations are NOT used herein:
2 Ala = alanine Arg = arginine Asn = asparagine Asp = aspartic acid
Cys = cysteine Gln = glutamine Glu = glutamic acid Gly = glycine
His = histidine Ile = isoleucine Leu = leucine Lys = lysine Met =
methionine Nle = norleucine Orn = ornithine Phe = phenylalanine Phg
= phenylglycine Pro = proline Sar = sarcosine Ser = serine Thr =
threonine Trp = tryptophan Tyr = tyrosine Val = valine
[0308] As used herein, the term "bubbles", as used herein, refers
to vesicles which are generally characterized by the presence of
one or more membranes or walls surrounding an internal void that is
filled with a gas or precursor thereto. Exemplary bubbles include,
for example, liposomes, micelles and the like.
[0309] As used herein, the term "lipid" refers to a synthetic or
naturally-occurring amphipathic compound which comprises a
hydrophilic component and a hydrophobic component. Lipids include,
for example, fatty acids, neutral fats, phosphatides, glycolipids,
aliphatic alchols and waxes, terpenes and steroids.
[0310] As used herein, the term "lipid composition" refers to a
composition which comprises a lipid compound. Exemplary lipid
compositions include suspensions, emulsions and vesicular
compositions.
[0311] As used herein, the term "lipid formulation" refers to a
composition which comprises a lipid compound and a bioactive
agent.
[0312] As used herein, the term "vesicle" refers to a spherical
entity which is characterized by the presence of an internal void.
Preferred vesicles are formulated from lipids, including the
various lipids described herein. In any given vesicle, the lipids
may be in the form of a monolayer or bilayer, and the mono- or
bilayer lipids may be used to form one of more mono- or bilayers.
In the case of more than one mono- or bilayer, the mono- or
bilayers are generally concentric. The lipid vesicles described
herein include such entities commonly referred to as liposomes,
micelles, bubbles, microbubbles, microspheres and the like. Thus,
the lipids may be used to form a unilamellar vesicle (comprised of
one monolayer or bilayer), an oligolamellar vesicle (comprised of
about two or about three monolayers or bilayers) or a multilamellar
vesicle (comprised of more than about three monolayers or
bilayers). The internal void of the vesicles may be filled with a
liquid, including, for example, an aqueous liquid, a gas, a gaseous
precursor, and/or a solid or solute material, including, for
example, a bioactive agent, as desired.
[0313] As used herein, the term "vesicular composition" refers to a
composition which is formulate from lipids and which comprises
vesicles.
[0314] As used herein, the term "vesicle formulation" refers to a
composition which comprises vesicles and a bioactive agent.
[0315] As used herein, the term "lipsomes" refers to a generally
spherical cluster or aggregate of amphipathic compounds, including
lipid compounds, typically in the form of one or more concentric
layers, for example, bilayers. They may also be referred to herein
as lipid vesicles.
[0316] Angiogenesis is the process of formation of new capillary
blood vessels from existing vasculature. It is an important
component of a variety of physiological processes including
ovulation, embryonic development, wound repair, and collateral
vascular generation in the myocardium. It is also central to a
number of pathological conditions such as tumor growth and
metastasis, diabetic retinopathy, and macular degeneration. The
process begins with the activation of existing vascular endothelial
cells in response to a variety of cytokines and growth factors. The
activated endothelial cells secrete enzymes that degrade the
basement membrane of the vessels. The endothelial cells then
proliferate and migrate into the extracellular matrix first forming
tubules and subsequently new blood vessels.
[0317] Under normal conditions, endothelial cell proliferation is a
very slow process, but it increases for a short period of time
during embryogenesis, ovulation and wound healing. This temporary
increase in cell turnover is governed by a combination of a number
of growth stimulatory factors and growth suppressing factors. In
pathological angiogenesis, this normal balance is disrupted
resulting in continued increased endothelial cell proliferation.
Some of the pro-angiogenic factors that have been identified
include basic fibroblast growth factor (bFGF), angiogenin,
TGF-alpha, TGF-beta, and vascular endothelium growth factor (VEGF),
while interferon-alpha, interferon-beta and thrombospondin are
examples of angiogenesis suppressors.
[0318] Angiogenic factors interact with endothelial cell surface
receptors such as the receptor tyrosine kinases EGFR, FGFR, PDGFR,
Flk-1/KDR, Flt-1, Tek, Tie, neuropilin-1, endoglin, endosialin, and
Axl. The receptors Flk-1/KDR, neuropilin-1, and Flt-1 recognize
VEGF and these interactions play key roles in VEGF-induced
angiogenesis. The Tie subfamily of receptor tyrosine kinases are
also expressed prominently during blood vessel formation.
[0319] The proliferation and migration of endothelial cells in the
extracellular matrix is mediated by interaction with a variety of
cell adhesion molecules. Integrins are a diverse family of
heterodimeric cell surface receptors by which endothelial cells
attach to the extracellular matrix, each other and other cells.
Angiogenesis induced by bFGF or TNF-alpha depend on the agency of
the integrin avb3, while angiogenesis induced by VEGF depends on
the integrin avb5 (Cheresh et. al., Science, 1995, 270, 1500-2).
Induction of expression of the integrins a1b1 and a2b1 on the
endothelial cell surface is another important mechanism by which
VEGF promotes angiogenesis (Senger, et. al., Proc. Natl. Acad, Sci
USA, 1997, 94, 13612-7).
[0320] The pharmaceuticals of the present invention are comprised
of a non-peptide targeting moiety for the vitronectin receptor that
is expressed or upregulated in angiogenic tumor vasculature.
[0321] The ultrasound contrast agents of the present invention
comprise a plurality of vitronectin receptor targeting moieties
attached to or incorporated into a microbubble of a biocompatible
gas, a liquid carrier, and a surfactant microsphere, further
comprising an optional linking moiety, L.sub.n, between the
targeting moieties and the microbubble. In this context, the term
liquid carrier means aqueous solution and the term surfactant means
any amphiphilic material which produces a reduction in interfacial
tension in a solution. A list of suitable surfactants for forming
surfactant microspheres is disclosed in EP0727225A2, herein
incorporated by reference. The term surfactant microsphere includes
nanospheres, liposomes, vesicles and the like. The biocompatible
gas can be air, or a fluorocarbon, such as a C.sub.3-C.sub.5
perfluoroalkane, which provides the difference in echogenicity and
thus the contrast in ultrasound imaging. The gas is encapsulated or
contained in the microsphere to which is attached the biodirecting
group, optionally via a linking group. The attachment can be
covalent, ionic or by van der Waals forces. Specific examples of
such contrast agents include lipid encapsulated perfluorocarbons
with a plurality of tumor neovasculature receptor binding peptides,
polypeptides or peptidomimetics.
[0322] X-ray contrast agents of the present invention are comprised
of one or more vitronectin receptor targeting moieties attached to
one or more X-ray absorbing or "heavy" atoms of atomic number 20 or
greater, further comprising an optional linking moiety, L.sub.n,
between the targeting moieties and the X-ray absorbing atoms. The
frequently used heavy atom in X-ray contrast agents is iodine.
Recently, X-ray contrast agents comprised of metal chelates
(Wallace, R., U.S. Pat. No. 5,417,959) and polychelates comprised
of a plurality of metal ions (Love, D., U.S. Pat. No. 5,679,810)
have been disclosed. More recently, multinuclear cluster complexes
have been disclosed as X-ray contrast agents (U.S. Pat. No.
5,804,161, PCT WO91/14460, and PCT WO 92/17215).
[0323] MRI contrast agents of the present invention are comprised
of one or more vitronectin receptor targeting moieties attached to
one or more paramagnetic metal ions, further comprising an optional
linking moiety, L.sub.n, between the targeting moieties and the
paramagnetic metal ions. The paramagnetic metal ions are present in
the form of metal complexes or metal oxide particles. U.S. Pat Nos.
5,412,148, and 5,760,191, describe examples of chelators for
paramagnetic metal ions for use in MRI contrast agents. U.S. Pat.
No. 5,801,228, U.S. Pat. No. 5,567,411, and U.S. Pat. No.
5,281,704, describe examples of polychelants useful for complexing
more than one paramagnetic metal ion for use in MRI contrast
agents. U.S. Pat. No. 5,520,904, describes particulate compositions
comprised of paramagnetic metal ions for use as MRI contrast
agents.
[0324] The pharmaceuticals of the present invention have the
formulae, (Q).sub.d--L.sub.n--(C.sub.h-X),
(Q).sub.d--L.sub.n--(C.sub.h-X.sup.1).su- b.d', (Q).sub.d13
L.sub.n--(X.sup.2).sub.d", and (Q).sub.d--L.sub.n--(X.su- p.3),
wherein Q represents a non-peptide that binds to a receptor
expressed in angiogenic tumor vasculature, d is 1-10, L.sub.n
represents an optional linking group, C.sub.h represents a metal
chelator or bonding moiety, X represents a radioisotope, X.sup.1
represents paramagnetic metal ion, X.sup.2 represents a
paramagnetic metal ion or heavy atom containing insoluble solid
particle, d" is 1-100, and X.sup.3 represents a surfactant
microsphere of an echogenic gas. The interaction of the non-peptide
recognition sequences of the vitronectin receptor binding portion
of the pharmaceuticals with the .alpha.v.beta.3 receptor results in
localization of the pharmaceuticals in angiogenic tumor
vasculature, which express the .alpha.v.beta.3 receptor.
[0325] The pharmaceuticals of the present invention can be
synthesized by several approaches. One approach involves the
synthesis of the targeting non-peptide moiety, Q, and direct
attachment of one or more moieties, Q, to one or more metal
chelators or bonding moieties, C.sub.h, or to a paramagnetic metal
ion or heavy atom containing solid particle, or to an echogenic gas
microbubble. Another approach involves the attachment of one or
more moieties, Q, to the linking group, L.sub.n, which is then
attached to one or more metal chelators or bonding moieties,
C.sub.h, or to a paramagnetic metal ion or heavy atom containing
solid particle, or to an echogenic gas microbubble. Another
approach involves the synthesis of a non-peptide, Q, bearing a
fragment of the linking group, L.sub.n, one or more of which are
then attached to the remainder of the linking group and then to one
or more metal chelators or bonding moieties, C.sub.h, or to a
paramagnetic metal ion or heavy atom containing solid particle, or
to an echogenic gas microbubble.
[0326] The non-peptide vitronectin binding moieties, Q, optionally
bearing a linking group, L.sub.n, or a fragment of the linking
group, can be synthesized using standard synthetic methods known to
those skilled in the art. Preferred methods include but are not
limited to those methods described below.
[0327] The attachment of linking groups, L.sub.n, to the
non-peptides, Q; chelators or bonding units, C.sub.h, to the
non-peptides, , Q, or to the linking groups, L.sub.n; and
non-peptides, bearing a fragment of the linking group to the
remainder of the linking group, in combination forming the moiety,
(Q).sub.d--L.sub.n, and then to the moiety C.sub.h; can all be
performed by standard techniques. These include, but are not
limited to, amidation, esterification, alkylation, and the
formation of ureas or thioureas. Procedures for performing these
attachments can be found in Brinkley, M., Bioconjugate Chemistry
1992, 3(1), which is incorporated herein by reference.
[0328] A number of methods can be used to attach the non-peptides,
Q, to paramagnetic metal ion or heavy atom containing solid
particles, X.sup.2, by one of skill in the art of the surface
modification of solid particles. In general, the targeting moiety Q
or the combination (Q).sub.dL.sub.n is attached to a coupling group
that react with a constituent of the surface of the solid particle.
The coupling groups can be any of a number of silanes which react
with surface hydroxyl groups on the solid particle surface, as
described in co-pending U.S. Ser. No. (DM6896), and can also
include polyphosphonates, polycarboxylates, polyphosphates or
mixtures thereof which couple with the surface of the solid
particles, as described in U.S. Pat. No. 5,520,904.
[0329] A number of reaction schemes can be used to attach the
non-peptides, Q, to the surfactant microsphere, X.sup.3. These are
illustrated in following reaction schemes where S.sub.f represents
a surfactant moiety that forms the surfactant microsphere.
Acylation Reaction:
S.sub.f--C(.dbd.O)--Y+Q--NH.sub.2
or.fwdarw.S.sub.f--C(.dbd.O)--NH--Q
Q--OH or S.sub.f--C(.dbd.O)--O--Q
[0330] Y is a leaving group or active ester
[0331] Disulfide Coupling:
S.sub.f--SH+Q--SH.fwdarw.S.sub.f--S--S--Q
[0332] Sulfonamide Coupling:
S.sub.f--S(.dbd.O).sub.2--Y+.Q--NH.sub.2.fwdarw.S.sub.f--S(.dbd.O).sub.2---
NH--Q
[0333] Reductive Amidation:
S.sub.f--CHO+Q--NH.sub.2.fwdarw.S.sub.f--NH--Q
[0334] In these reaction schemes, the substituents S.sub.f and Q
can be reversed as well.
[0335] The linking group L.sub.n can serve several roles. First it
provides a spacing group between the metal chelator or bonding
moiety, C.sub.h, the paramagnetic metal ion or heavy atom
containing solid particle, X.sup.2, and the surfactant microsphere,
X.sup.3, and the one or more of the non-peptides, Q, so as to
minimize the possibility that the moieties C.sub.h--X,
C.sub.h--X.sup.1, X.sup.2, and X.sup.3, will interfere with the
interaction of the recognition sequences of Q with angiogenic tumor
vasculature receptors. The necessity of incorporating a linking
group in a reagent is dependent on the identity of Q, C.sub.h--X,
C.sub.h--X.sup.1, X.sup.2, and X.sup.3. If C.sub.h--X,
C.sub.h--X.sup.1, X.sup.2, and X.sup.3, cannot be attached to Q
without substantially diminishing its affinity for the receptors,
then a linking group is used. A linking group also provides a means
of independently attaching multiple non-peptides, Q, to one group
that is attached to C.sub.h--X, C.sub.h--X.sup.1, X.sup.2, or
X.sup.3.
[0336] The linking group also provides a means of incorporating a
pharmacokinetic modifier into the pharmaceuticals of the present
invention. The pharmacokinetic modifier serves to direct the
biodistribution of the injected pharmaceutical other than by the
interaction of the targeting moieties, Q, with the vitronectin
receptors expressed in the tumor neovasculature. A wide variety of
functional groups can serve as pharmacokinetic modifiers,
including, but not limited to, carbohydrates, polyalkylene glycols,
peptides or other polyamino acids, and cyclodextrins. The modifiers
can be used to enhance or decrease hydrophilicity and to enhance or
decrease the rate of blood clearance. The modifiers can also be
used to direct the route of elimination of the pharmaceuticals.
Preferred pharmacokinetic modifiers are those that result in
moderate to fast blood clearance and enhanced renal excretion.
[0337] The metal chelator or bonding moiety, C.sub.h, is selected
to form stable complexes with the metal ion chosen for the
particular application. Chelators or bonding moieties for
diagnostic radiopharmaceuticals are selected to form stable
complexes with the radioisotopes that have imageable gamma ray or
positron emissions, such as .sup.99mTc, .sup.95Tc, .sup.111In,
.sup.62Cu, .sup.60Cu, .sup.64Cu, .sup.67Ga, .sup.68Ga,
.sup.86Y.
[0338] Chelators for technetium, copper and gallium isotopes are
selected from diaminedithiols, monoamine-monoamidedithiols,
triamide-monothiols, monoamine-diamide-monothiols, diaminedioximes,
and hydrazines. The chelators are generally tetradentate with donor
atoms selected from nitrogen, oxygen and sulfur. Preferred reagents
are comprised of chelators having amine nitrogen and thiol sulfur
donor atoms and hydrazine bonding units. The thiol sulfur atoms and
the hydrazines may bear a protecting group which can be displaced
either prior to using the reagent to synthesize a
radiopharmaceutical or preferably in situ during the synthesis of
the radiopharmaceutical.
[0339] Exemplary thiol protecting groups include those listed in
Greene and Wuts, "Protective Groups in Organic Synthesis" John
Wiley & Sons, New York (1991), the disclosure of which is
hereby incorporated by reference. Any thiol protecting group known
in the art can be used. Examples of thiol protecting groups
include, but are not limited to, the following: acetamidomethyl,
benzamidomethyl, 1-ethoxyethyl, benzoyl, and triphenylmethyl.
[0340] Exemplary protecting groups for hydrazine bonding units are
hydrazones which can be aldehyde or ketone hydrazones having
substituents selected from hydrogen, alkyl, aryl and heterocycle.
Particularly preferred hydrazones are described in co-pending U.S.
Ser. No. 08/476,296 the disclosure of which is herein incorporated
by reference in its entirety.
[0341] The hydrazine bonding unit when bound to a metal
radionuclide is termed a hydrazido, or diazenido group and serves
as the point of attachment of the radionuclide to the remainder of
the radiopharmaceutical. A diazenido group can be either terminal
(only one atom of the group is bound to the radionuclide) or
chelating. In order to have a chelating diazenido group at least
one other atom of the group must also be bound to the radionuclide.
The atoms bound to the metal are termed donor atoms.
[0342] Chelators for .sup.111In and .sup.86Y are selected from
cyclic and acyclic polyaminocarboxylates such as DTPA, DOTA, DO3A,
2-benzyl-DOTA,
alpha-(2-phenethyl)1,4,7,10-tetraazazcyclododecane-1-acetic-4,7,10-tris(m-
ethylacetic)acid, 2-benzyl-cyclohexyldiethylenetriaminepentaacetic
acid, 2-benzyl-6-methyl-DTPA, and
6,6"-bis[N,N,N",N"-tetra(carboxymethyl)aminom-
ethyl)-4'-(3-amino-4-methoxyphenyl)-2,2':6',2"-terpyridine.
Procedures for synthesizing these chelators that are not
commercially available can be found in Brechbiel, M. and Gansow,
O., J. Chem. Soc. Perkin Trans. 1992, 1, 1175; Brechbiel, M. and
Gansow, O., Bioconjugate Chem. 1991, 2, 187; Deshpande, S., et.
al., J. Nucl. Med. 1990, 31, 473; Kruper, J., U.S. Pat. No.
5,064,956, and Toner, J., U.S. Pat. No. 4,859,777, the disclosures
of which are hereby incorporated by reference in their
entirety.
[0343] The coordination sphere of metal ion includes all the
ligands or groups bound to the metal. For a transition metal
radionuclide to be stable it typically has a coordination number
(number of donor atoms) comprised of an integer greater than or
equal to 4 and less than or equal to 8; that is there are 4 to 8
atoms bound to the metal and it is said to have a complete
coordination sphere. The requisite coordination number for a stable
radionuclide complex is determined by the identity of the
radionuclide, its oxidation state, and the type of donor atoms. If
the chelator or bonding unit does not provide all of the atoms
necessary to stabilize the metal radionuclide by completing its
coordination sphere, the coordination sphere is completed by donor
atoms from other ligands, termed ancillary or co-ligands, which can
also be either terminal or chelating.
[0344] A large number of ligands can serve as ancillary or
co-ligands, the choice of which is determined by a variety of
considerations such as the ease of synthesis of the
radiopharmaceutical, the chemical and physical properties of the
ancillary ligand, the rate of formation, the yield, and the number
of isomeric forms of the resulting radiopharmaceuticals, the
ability to administer said ancillary or co-ligand to a patient
without adverse physiological consequences to said patient, and the
compatibility of the ligand in a lyophilized kit formulation. The
charge and lipophilicity of the ancillary ligand will effect the
charge and lipophilicity of the radiopharmaceuticals. For example,
the use of 4,5-dihydroxy-1,3-benzene disulfonate results in
radiopharmaceuticals with an additional two anionic groups because
the sulfonate groups will be anionic under physiological
conditions. The use of N-alkyl substituted 3,4-hydroxypyridinones
results in radiopharmaceuticals with varying degrees of
lipophilicity depending on the size of the alkyl substituents.
[0345] Preferred technetium radiopharmaceuticals of the present
invention are comprised of a hydrazido or diazenido bonding unit
and an ancillary ligand, A.sub.L1, or a bonding unit and two types
of ancillary A.sub.L1 and A.sub.L2, or a tetradentate chelator
comprised of two nitrogen and two sulfur atoms. Ancillary ligands
A.sub.L1 are comprised of two or more hard donor atoms such as
oxygen and amine nitrogen (sp.sup.3 hybridized). The donor atoms
occupy at least two of the sites in the coordination sphere of the
radionuclide metal; the ancillary ligand A.sub.L1 serves as one of
the three ligands in the ternary ligand system. Examples of
ancillary ligands A.sub.L1 include but are not limited to dioxygen
ligands and functionalized aminocarboxylates. A large number of
such ligands are available from commercial sources.
[0346] Ancillary dioxygen ligands include ligands that coordinate
to the metal ion through at least two oxygen donor atoms. Examples
include but are not limited to: glucoheptonate, gluconate,
2-hydroxyisobutyrate, lactate, tartrate, mannitol, glucarate,
maltol, Kojic acid, 2,2-bis(hydroxymethyl)propionic acid,
4,5-dihydroxy-1,3-benzene disulfonate, or substituted or
unsubstituted 1,2 or 3,4 hydroxypyridinones. (The names for the
ligands in these examples refer to either the protonated or
non-protonated forms of the ligands.) Functionalized
aminocarboxylates include ligands that have a combination of amine
nitrogen and oxygen donor atoms. Examples include but are not
limited to: iminodiacetic acid, 2,3-diaminopropionic acid,
nitrilotriacetic acid, N,N'-ethylenediamine diacetic acid,
N,N,N'-ethylenediamine triacetic acid, hydroxyethylethylenediamine
triacetic acid, and N,N'-ethylenediamine bis-hydroxyphenylglycine.
(The names for the ligands in these examples refer to either the
protonated or non-protonated forms of the ligands.) A series of
functionalized aminocarboxylates are disclosed by Bridger et. al.
in U.S. Pat. No. 5,350,837, herein incorporated by reference, that
result in improved rates of formation of technetium labeled
hydrazino modified proteins. We have determined that certain of
these aminocarboxylates result in improved yields of the
radiopharmaceuticals of the present invention. The preferred
ancillary ligands A.sub.L1 functionalized aminocarboxylates that
are derivatives of glycine; the most preferred is tricine
(tris(hydroxymethyl)methylglycine).
[0347] The most preferred technetium radiopharmaceuticals of the
present invention are comprised of a hydrazido or diazenido bonding
unit and two types of ancillary designated A.sub.L1, and A.sub.L2,
or a diaminedithiol chelator. The second type of ancillary ligands
A.sub.L2 are comprised of one or more soft donor atoms selected
from the group: phosphine phosphorus, arsine arsenic, imine
nitrogen (sp.sup.2 hybridized), sulfur (sp.sup.2 hybridized) and
carbon (sp hybridized); atoms which have p-acid character. Ligands
A.sub.L2 can be monodentate, bidentate or tridentate, the denticity
is defined by the number of donor atoms in the ligand. One of the
two donor atoms in a bidentate ligand and one of the three donor
atoms in a tridentate ligand must be a soft donor atom. We have
disclosed in co-pending U.S. Ser. No. 08/415,908, and U.S. Ser. No.
60/013360 and 08/646,886, the disclosures of which are herein
incorporated by reference in their entirety, that
radiopharmaceuticals comprised of one or more ancillary or
co-ligands A.sub.L2 are more stable compared to
radiopharmaceuticals that are not comprised of one or more
ancillary ligands, A.sub.L2; that is, they have a minimal number of
isomeric forms, the relative ratios of which do not change
significantly with time, and that remain substantially intact upon
dilution.
[0348] The ligands A.sub.L2 that are comprised of phosphine or
arsine donor atoms are trisubstituted phosphines, trisubstituted
arsines, tetrasubstituted diphosphines and tetrasubstituted
diarsines. The ligands A.sub.L2 that are comprised of imine
nitrogen are unsaturated or aromatic nitrogen-containing, 5 or
6-membered heterocycles. The ligands that are comprised of sulfur
(sp.sup.2 hybridized) donor atoms are thiocarbonyls, comprised of
the moiety C.dbd.S. The ligands comprised of carbon (sp hybridized)
donor atoms are isonitriles, comprised of the moiety CNR, where R
is an organic radical. A large number of such ligands are available
from commercial sources. Isonitriles can be synthesized as
described in European Patent 0107734 and in U.S. Pat. No.
4,988,827, herein incorporated by reference.
[0349] Preferred ancillary ligands A.sub.L2 are trisubstituted
phosphines and unsaturated or aromatic 5 or 6 membered
heterocycles. The most preferred ancillary ligands A.sub.L2 are
trisubstituted phosphines and unsaturated 5 membered
heterocycles.
[0350] The ancillary ligands A.sub.L2 may be substituted with
alkyl, aryl, alkoxy, heterocycle, aralkyl, alkaryl and arylalkaryl
groups and may or may not bear functional groups comprised of
heteroatoms such as oxygen, nitrogen, phosphorus or sulfur.
Examples of such functional groups include but are not limited to:
hydroxyl, carboxyl, carboxamide, nitro, ether, ketone, amino,
ammonium, sulfonate, sulfonamide, phosphonate, and phosphonamide.
The functional groups may be chosen to alter the lipophilicity and
water solubility of the ligands which may affect the biological
properties of the radiopharmaceuticals, such as altering the
distribution into non-target tissues, cells or fluids, and the
mechanism and rate of elimination from the body. Chelators or
bonding moieties for therapeutic radiopharmaceuticals are selected
to form stable complexes with the radioisotopes that have alpha
particle, beta particle, Auger or Coster-Kronig electron emissions,
such as .sup.186Re, .sup.188Re, .sup.153Sm, .sup.166Ho, .sup.177Lu,
.sup.149Pm, .sup.90Y, .sup.212Bi, .sup.103Pd, .sup.109Pd,
.sup.159Gd, .sup.140La, .sup.198Au, .sup.199Au, .sup.169Yb,
.sup.175Yb, .sup.165Dy, .sup.166Dy, .sup.67Cu, .sup.105Rh,
.sup.111Ag, and .sup.192Ir. Chelators for rhenium, copper,
palladium, platinum, iridium, rhodium, silver and gold isotopes are
selected from diaminedithiols, monoamine-monoamidedithiols,
triamide-monothiols, monoamine-diamide-monothiols, diaminedioximes,
and hydrazines. Chelators for yttrium, bismuth, and the lanthanide
isotopes are selected from cyclic and acyclic polyaminocarboxylates
such as DTPA, DOTA, DO3A, 2-benzyl-DOTA,
alpha-(2-phenethyl)1,4,7,10-tetraazacyclododecane-1-acetic-
-4,7,10-tris(methylacetic)acid,
2-benzyl-cyclohexyldiethylenetriaminepenta- acetic acid,
2-benzyl-6-methyl-DTPA, and 6,6"-bis[N,N,N",N"-tetra(carboxym-
ethyl)aminomethyl)-4'-(3-amino-4-methoxyphenyl)-2,2':6',2"-terpyridine.
[0351] Chelators for magnetic resonance imaging contrast agents are
selected to form stable complexes with paramagnetic metal ions,
such as Gd(III), Dy(III), Fe(III), and Mn(II), are selected from
cyclic and acyclic polyaminocarboxylates such as DTPA, DOTA, DO3A,
2-benzyl-DOTA,
alpha-(2-phenethyl)1,4,7,10-tetraazacyclododecane-1-acetic-4,7,10-tris(me-
thylacetic)acid, 2-benzyl-cyclohexyldiethylenetriaminepentaacetic
acid, 2-benzyl-6-methyl-DTPA, and
6,6"-bis[N,N,N",N"-tetra(carboxymethyl)aminom-
ethyl)-4'-(3-amino-4-methoxyphenyl)-2,2':6',2"-terpyridine.
[0352] The technetium and rhenium radiopharmaceuticals of the
present invention comprised of a hydrazido or diazenido bonding
unit can be easily prepared by admixing a salt of a radionuclide, a
reagent of the present invention, an ancillary ligand A.sub.L1, an
ancillary ligand A.sub.L2, and a reducing agent, in an aqueous
solution at temperatures from 0 to 100.degree. C. The technetium
and rhenium radiopharmaceuticals of the present invention comprised
of a tetradentate chelator having two nitrogen and two sulfur atoms
can be easily prepared by admixing a salt of a radionuclide, a
reagent of the present invention, and a reducing agent, in an
aqueous solution at temperatures from 0 to 100.degree. C.
[0353] When the bonding unit in the reagent of the present
invention is present as a hydrazone group, then it must first be
converted to a hydrazine, which may or may not be protonated, prior
to complexation with the metal radionuclide. The conversion of the
hydrazone group to the hydrazine can occur either prior to reaction
with the radionuclide, in which case the radionuclide and the
ancillary or co-ligand or ligands are combined not with the reagent
but with a hydrolyzed form of the reagent bearing the chelator or
bonding unit, or in the presence of the radionuclide in which case
the reagent itself is combined with the radionuclide and the
ancillary or co-ligand or ligands. In the latter case, the pH of
the reaction mixture must be neutral or acidic.
[0354] Alternatively, the radiopharmaceuticals of the present
invention comprised of a hydrazido or diazenido bonding unit can be
prepared by first admixing a salt of a radionuclide, an ancillary
ligand A.sub.L1, and a reducing agent in an aqueous solution at
temperatures from 0 to 100.degree. C. to form an intermediate
radionuclide complex with the ancillary ligand A.sub.L1 then adding
a reagent of the present invention and an ancillary ligand A.sub.L2
and reacting further at temperatures from 0 to 100.degree. C.
[0355] Alternatively, the radiopharmaceuticals of the present
invention comprised of a hydrazido or diazenido bonding unit can be
prepared by first admixing a salt of a radionuclide, an ancillary
ligand A.sub.L1, a reagent of the present invention, and a reducing
agent in an aqueous solution at temperatures from 0 to 100.degree.
C. to form an intermediate radionuclide complex, and then adding an
ancillary ligand A.sub.L2 and reacting further at temperatures from
0 to 100.degree. C.
[0356] The technetium and rhenium radionuclides are preferably in
the chemical form of pertechnetate or perrhenate and a
pharmaceutically acceptable cation. The pertechnetate salt form is
preferably sodium pertechnetate such as obtained from commercial
Tc-99m generators. The amount of pertechnetate used to prepare the
radiopharmaceuticals of the present invention can range from 0.1
mCi to 1 Ci, or more preferably from 1 to 200 mCi.
[0357] The amount of the reagent of the present invention used to
prepare the technetium and rhenium radiopharmaceuticals of the
present invention can range from 0.01 .mu.g to 10 mg, or more
preferably from 0.5 .mu.g to 200 .mu.g. The amount used will be
dictated by the amounts of the other reactants and the identity of
the radiopharmaceuticals of the present invention to be
prepared.
[0358] The amounts of the ancillary ligands A.sub.L1 used can range
from 0.1 mg to 1 g, or more preferably from 1 mg to 100 mg. The
exact amount for a particular radiopharmaceutical is a function of
identity of the radiopharmaceuticals of the present invention to be
prepared, the procedure used and the amounts and identities of the
other reactants. Too large an amount of A.sub.L1 will result in the
formation of by-products comprised of technetium labeled A.sub.L1
without a biologically active molecule or by-products comprised of
technetium labeled biologically active molecules with the ancillary
ligand A.sub.L1, but without the ancillary ligand A.sub.L2. Too
small an amount of A.sub.L1 will result in other by-products such
as technetium labeled biologically active molecules with the
ancillary ligand A.sub.L2 but without the ancillary ligand
A.sub.L1, or reduced hydrolyzed technetium, or technetium
colloid.
[0359] The amounts of the ancillary ligands A.sub.L2 used can range
from 0.001 mg to 1 g, or more preferably from 0.01 mg to 10 mg. The
exact amount for a particular radiopharmaceutical is a function of
the identity of the radiopharmaceuticals of the present invention
to be prepared, the procedure used and the amounts and identities
of the other reactants. Too large an amount of A.sub.L2 will result
in the formation of by-products comprised of technetium labeled
A.sub.L2 without a biologically active molecule or by-products
comprised of technetium labeled biologically active molecules with
the ancillary ligand A.sub.L2 but without the ancillary ligand
A.sub.L1. If the reagent bears one or more substituents that are
comprised of a soft donor atom, as defined above, at least a
ten-fold molar excess of the ancillary ligand A.sub.L2 to the
reagent of formula 2 is required to prevent the substituent from
interfering with the coordination of the ancillary ligand A.sub.L2
to the metal radionuclide.
[0360] Suitable reducing agents for the synthesis of the
radiopharmaceuticals of the present invention include stannous
salts, dithionite or bisulfite salts, borohydride salts, and
formamidinesulfinic acid, wherein the salts are of any
pharmaceutically acceptable form. The preferred reducing agent is a
stannous salt. The amount of a reducing agent used can range from
0.001 mg to 10 mg, or more preferably from 0.005 mg to 1 mg.
[0361] The specific structure of a radiopharmaceutical of the
present invention comprised of a hydrazido or diazenido bonding
unit will depend on the identity of the reagent of the present
invention used, the identity of any ancillary ligand A.sub.L1, the
identity of any ancillary ligand A.sub.L2, and the identity of the
radionuclide. Radiopharmaceuticals comprised of a hydrazido or
diazenido bonding unit synthesized using concentrations of reagents
of <100 .mu.g/mL, will be comprised of one hydrazido or
diazenido group. Those synthesized using >1 mg/mL concentrations
will be comprised of two hydrazido or diazenido groups from two
reagent molecules. For most applications, only a limited amount of
the biologically active molecule can be injected and not result in
undesired side-effects, such as chemical toxicity, interference
with a biological process or an altered biodistribution of the
radiopharmaceutical. Therefore, the radiopharmaceuticals which
require higher concentrations of the reagents comprised in part of
the biologically active molecule, will have to be diluted or
purified after synthesis to avoid such side-effects.
[0362] The identities and amounts used of the ancillary ligands
A.sub.L1, and A.sub.L2 will determine the values of the variables y
and z. The values of y and z can independently be an integer from 1
to 2. In combination, the values of y and z will result in a
technetium coordination sphere that is made up of at least five and
no more than seven donor atoms. For monodentate ancillary ligands
A.sub.L2, z can be an integer from 1 to 2; for bidentate or
tridentate ancillary ligands A.sub.L2, z is 1. The preferred
combination for monodentate ligands is y equal to 1 or 2 and z
equal to 1. The preferred combination for bidentate or tridentate
ligands is y equal to 1 and z equal to 1.
[0363] The indium, copper, gallium, silver, palladium, rhodium,
gold, platinum, bismuth, yttrium and lanthanide
radiopharmaceuticals of the present invention can be easily
prepared by admixing a salt of a radionuclide and a reagent of the
present invention, in an aqueous solution at temperatures from 0 to
100.degree. C. These radionuclides are typically obtained as a
dilute aqueous solution in a mineral acid, such as hydrochloric,
nitric or sulfuric acid. The radionuclides are combined with from
one to about one thousand equivalents of the reagents of the
present invention dissolved in aqueous solution. A buffer is
typically used to maintain the pH of the reaction mixture between 3
and 10.
[0364] The gadolinium, dysprosium, iron and manganese
metallopharmaceuticals of the present invention can be easily
prepared by admixing a salt of the paramagnetic metal ion and a
reagent of the present invention, in an aqueous solution at
temperatures from 0 to 100.degree. C. These paramagnetic metal ions
are typically obtained as a dilute aqueous solution in a mineral
acid, such as hydrochloric, nitric or sulfuric acid. The
paramagnetic metal ions are combined with from one to about one
thousand equivalents of the reagents of the present invention
dissolved in aqueous solution. A buffer is typically used to
maintain the pH of the reaction mixture between 3 and 10.
[0365] The total time of preparation will vary depending on the
identity of the metal ion, the identities and amounts of the
reactants and the procedure used for the preparation. The
preparations may be complete, resulting in >80% yield of the
radiopharmaceutical, in 1 minute or may require more time. If
higher purity metallopharmaceuticals are needed or desired, the
products can be purified by any of a number of techniques well
known to those skilled in the art such as liquid chromatography,
solid phase extraction, solvent extraction, dialysis or
ultrafiltration.
[0366] Buffers useful in the preparation of metallopharmaceuticals
and in diagnostic kits useful for the preparation of said
radiopharmaceuticals include but are not limited to phosphate,
citrate, sulfosalicylate, and acetate. A more complete list can be
found in the United States Pharmacopeia.
[0367] Lyophilization aids useful in the preparation of diagnostic
kits useful for the preparation of radiopharmaceuticals include but
are not limited to mannitol, lactose, sorbitol, dextran, Ficoll,
and polyvinylpyrrolidine(PVP).
[0368] Stabilization aids useful in the preparation of
metallopharmaceuticals and in diagnostic kits useful for the
preparation of radiopharmaceuticals include but are not limited to
ascorbic acid, cysteine, monothioglycerol, sodium bisulfite, sodium
metabisulfite, gentisic acid, and inositol.
[0369] Solubilization aids useful in the preparation of
metallopharmaceuticals and in diagnostic kits useful for the
preparation of radiopharmaceuticals include but are not limited to
ethanol, glycerin, polyethylene glycol, propylene glycol,
polyoxyethylene sorbitan monooleate, sorbitan monoloeate,
polysorbates, poly(oxyethylene)poly(oxyp-
ropylene)poly(oxyethylene) block copolymers (Pluronics) and
lecithin. Preferred solubilizing aids are polyethylene glycol, and
Pluronics.
[0370] Bacteriostats useful in the preparation of
metallopharmaceuticals and in diagnostic kits useful for the
preparation of radiopharmaceuticals include but are not limited to
benzyl alcohol, benzalkonium chloride, chlorbutanol, and methyl,
propyl or butyl paraben.
[0371] A component in a diagnostic kit can also serve more than one
function. A reducing agent can also serve as a stabilization aid, a
buffer can also serve as a transfer ligand, a lyophilization aid
can also serve as a transfer, ancillary or co-ligand and so
forth.
[0372] The diagnostic radiopharmaceuticals are administered by
intravenous injection, usually in saline solution, at a dose of 1
to 100 mCi per 70 kg body weight, or preferably at a dose of 5 to
50 mCi. Imaging is performed using known procedures.
[0373] The therapeutic radiopharmaceuticals are administered by
intravenous injection, usually in saline solution, at a dose of 0.1
to 100 mCi per 70 kg body weight, or preferably at a dose of 0.5 to
5 mCi per 70 kg body weight.
[0374] The magnetic resonance imaging contrast agents of the
present invention may be used in a similar manner as other MRI
agents as described in U.S. Pat. No. 5,155,215; U.S. Pat. No.
5,087,440; Margerstadt et al., Magn. Reson. Med., 1986, 3, 808;
Runge et al., Radiology, 1988, 166, 835; and Bousquet et al.,
Radiology, 1988, 166, 693. Generally, sterile aqueous solutions of
the contrast agents are administered to a patient intravenously in
dosages ranging from 0.01 to 1.0 mmoles per kg body weight.
[0375] For use as X-ray contrast agents, the compositions of the
present invention should generally have a heavy atom concentration
of 1 mM to 5 M, preferably 0.1 M to 2 M. Dosages, administered by
intravenous injection, will typically range from 0.5 mmol/kg to 1.5
mmol/kg, preferably 0.8 mmol/kg to 1.2 mmol/kg. Imaging is
performed using known techniques, preferably X-ray computed
tomography.
[0376] The ultrasound contrast agents of the present invention are
administered by intravenous injection in an amount of 10 to 30
.mu.L of the echogenic gas per kg body weight or by infusion at a
rate of approximately 3 .mu.L/kg/min. Imaging is performed using
known techniques of sonography.
[0377] Other features of the invention will become apparent in the
course of the following descriptions of exemplary embodiments which
are given for illustration of the invention and are not intended to
be limiting thereof.
EXAMPLES
[0378] Representative materials and methods that may be used in
preparing the compounds of the invention are described further
below.
[0379] Manual solid phase peptide synthesis was performed in 25 mL
polypropylene filtration tubes purchased from BioRad Inc., or in 60
mL hour-glass reaction vessels purchased from Peptides
International. Oxime resin (substitution level=0.96 mmol/g) was
prepared according to published procedure (DeGrado and Kaiser, J.
Org. Chem. 1980, 45, 1295), or was purchased from Novabiochem
(substitution level=0.62 mmol/g). All chemicals and solvents
(reagent grade) were used as supplied from the vendors cited
without further purification. t-Butyloxycarbonyl (Boc) amino acids
and other starting amino acids may be obtained commercially from
Bachem Inc., Bachem Biosciences Inc. (Philadelphia, Pa.), Advanced
ChemTech (Louisville, Ky.), Peninsula Laboratories (Belmont,
Calif.), or Sigma (St. Louis, Mo.).
2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluroni- um
hexafluorophosphate (HBTU) and TBTU were purchased from Advanced
ChemTech. N-methylmorpholine (NMM), m-cresol, D-2-aminobutyric acid
(Abu), trimethylacetylchloride, diisopropylethylamine (DIEA),
1,2,4-triazole, stannous chloride dihydrate, and
tris(3-sulfonatophenyl)p- hosphine trisodium salt (TPPTS) were
purchased from Aldrich Chemical Company.
Bis(3-sulfonatophenyl)phenylphosphine disodium salt (TPPDS) was
prepared by the published procedure (Kuntz, E., U.S. Pat. No.
4,248,802). (3-Sulfonatophenyl)diphenylphosphine monosodium salt
(TPPMS) was purchased from TCI America, Inc. Tricine was obtained
from Research Organics, Inc. Technetium-99m-pertechnetate
(.sup.99mTcO.sub.4.sup.-) was obtained from a DuPont Pharma
.sup.99Mo/.sup.99mTc Technelite.RTM. generator. In-111-chloride
(Indichlor.RTM.) was obtained from Amersham Medi-Physics, Inc.
Sm-153-chloride and Lutetium-177-chloride were obtained from the
University of Missouri Research Reactor (MURR). Yttrium-90 chloride
was obtained from the Pacific Northwest Research Laboratories.
Dimethylformamide (DMF), ethyl acetate, chloroform (CHCl.sub.3),
methanol (MeOH), pyridine and hydrochloric acid (HCl) were obtained
from Baker. Acetonitrile, dichloromethane (DCM), acetic acid
(HOAc), trifluoroacetic acid (TFA), ethyl ether, triethylamine,
acetone, and magnesium sulfate were commercially obtained. Absolute
ethanol was obtained from Quantum Chemical Corporation.
EXAMPLES
Example 1
Preparation of
(S,S,S)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)--
N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-t-
rien-3-yl)propyl)carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tr-
is(carboxymethyl) cyclodecyl)acetylamino)butanoyl amino)butanoic
acid
[0380] 12
[0381] Step 1A. Synthesis of tert-butyl 3-(((3-((tert-butoxy)
carbonylamino)propyl)methylamino)methyl)-4-fluorobenzoate 13
[0382] Crude tert-butyl-4-fluoro-3(alpha-bromomethyl)benzoate (4.6
g., 16 mmol), prepared as described in (WO 95/18619,
PCT/US95/00248), was dissolved in 100 mL THF, along with
3-tert-butoxycarbonylamino-1-propylam- ine hydrochloride (2.9 g.,
16.6 mmol) and diisopropylethylamine added (4.6 g., 36 mmol). The
solution was stirred overnight, diluted with 1N NaOH, and extracted
with three portions of ether. The combined organics were washed
with water and sat. NaCl, dried over MgSO.sub.4, and concentrated
under vacuum to 5.7 g. of a yellow oil. This was purified by flash
chromatography (CH.sub.2Cl.sub.2/EtOAc) to afford the product as a
clear oil (2.04 g., .about.35%). .sup.1HNMR (600 MHz, DMSO-d6):
?7.99 (dd, J=2, 5.1 Hz, 1H), 7.78 (ddd, J=2.3, 2.8, 3.0 Hz, 1H),
7.22 (dd, J=8.8, 0.7, 1H), 6.73 (b, 1H), 3.68 (s, 2H), 2.94 (m,
2H), 2.15 (b, 1H), 1.51 (s, 9H), 1.49 (m, 2H), 1.33 (s, 9H); MS
(ES): 765.4 [2M+H].sup.+, 383.3 [M+H].sup.+.
[0383] Step 1B. Synthesis of methyl
(S)-3-N-(3-((tert-butoxyl)carbonyl
amino)propyl)-N-((5-((tert-butyl)oxycarbonyl)-2-fluorophenyl)
methyl)carbamoyl)-3-((phenylmethoxy)carbonylamino)propanoate 14
[0384] The product of Step A (2 g, 5.3 mmol) was dissolved in 20 mL
dry DMF, along with N-Cbz-L-aspartic acid .beta.-methyl ester (1.65
g, 5.9 mmol), and 1-hydroxybenzotriazole hydrate (800 mg, 5.9 mmol)
under a nitrogen atmosphere. Dicyclohexylcarbodiimide (1M in
CH.sub.2Cl.sub.2, 5.9 mL, 5.9 mmol) was added via syringe, and the
solution stirred 18 hr. Ether (25 mL) was added and the solids were
filtered and rinsed with ether. The filtrate was concentrated,
redissolved in ether, filtered, and the filtrate washed with sat.
bicarbonate, water, and sat. NaCl. It was dried (Na2SO4), filtered
and concentrated to a yellow oil which was purified by flash
chromatography (4:1 CH2Cl2/EtOAc) to afford the product (3.0 g,
87%) as a clear oil. .sup.1HNMR (600 MHz, DMSO-d6): mixture of
amide rotamers: ?7.82 (m, 2H), 7.71 (m, 1H), 7.3 (m,6H), 6.72 (bd,
1H), 5.02 (dd, J=12.5, 25.7 Hz, 1H), 4.44-4.88 (m, 4H), 3.52 (d,
2H), 3.27 (d, 3H), 3.10-3.45 (m, 4H)2.45 -2.90 (m, 4H), 1.55 (m,
2H), 1.49 (s, 9H), 1.31 (s, 9H); MS-ES: 590.3 [(M-tBu)+H].sup.+,
646.4 [M+H].sup.+, 668.4 [M+Na].sup.+.
[0385] Step 1C: Synthesis of methyl
(S)-3-amino-3-(N-(3-((tert-butoxy)carb-
onylamino)propyl)-N-((5-((tert-butyl)oxycarbonyl)-2-fluorophenyl)methyl)ca-
rbamoyl)propanoate 15
[0386] The product of step B (2.8 g, 4.4 mmol) was dissolved in
MeOH (50 mL) with 10% Pd/C (530 mg) and shaken under a hydrogen
atmosphere (50 psi) in a Parr shaker for 2 hr. The reaction mixture
was filtered through Celite.RTM. and concentrated to a clear oil
(2.14 g, 94%) under vacuum, which was not further purified. MS-ES:
512.4 [M.sup.+H]+, 1023.5 [2M+H].sup.+;
[0387] Step 1D: Synthesis of methyl
(S)-2-(2,5-diaza-9-((tert-butyl)
oxycarbonyl)-5-(3-((tert-butoxy)carbonylamino)propyl)-4-oxobicyclo[5.4.0]-
undeca-1(7),8,10-trien-3-yl)acetate 16
[0388] The crude oil from C (2.14 g, 4.0 mmol) was dissolved in dry
N-methylpyrollidinone (50 mL) along with 2,6-di-tert-butylpyridine
(2.1 mL, 9.2 mmol) under nitrogen. The solution was heated at
125.degree. C. in an oil bath for 43 hours. The solution was
cooled, poured into 100 mL water, and extracted with ethyl acetate.
The organics were concentrated to an oil and purified by flash
chromatography (CH.sub.2Cl.sub.2/EtOAc) to afford 1.0 g (46%) of
the product. MS-ES: 392.3 [(M-tBoc)+H].sup.+436.3
[(M-tBu)+H].sup.+492.4 [M+H].sup.+, 983.6 [2M+H].sup.+;
[0389] Step 1E: Synthesis of (S)-2,5-diaza-5-(3-((tert-butoxy)
carbonylamino)propyl)-3-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]unde-
ca-1(7),8,10-trien-9-carboxylic acid 17
[0390] The ester from D (880 mg, 1.8 mmol) was dissolved in
dichloromethane (12 mL) and trifluoroacetic acid (6 mL) added with
stirring under nitrogen. The reaction was stirred 2 hours,
concentrated under vacuum, and redissolved in 7 mL dichloromethane.
Acetonitrile (7mL) was added, followed by di-tert-butyldicarbonate
(590 mg, 2.7 mmol) and diisopropylethylamine (1.4 mL, 7.6 mmol).
The reaction was stirred overnight under nitrogen. EtOAc (15 mL)
was added and the entire solution was washed with 5% citric acid
and brine, dried (MgSO4), and concentrated to 1.12 g of oil. This
was purified by flash chromatography (CH2Cl2/EtOAc/MeOH) and the
residue dissolved in 0.1% TFA/acetonitrile (50 mL) and lyophilized
to afford the product (680 mg, 69%) as a white powder. .sup.1HNMR
(600 MHz, DMSO-d6): ?12.14 (b, 1H), 7.62 (d, J=1.8 Hz, 1H), 7.53
(dd, J=1.9 Hz, 8.5 Hz, 1H), 6.66 (bt, J=5.4 Hz, 1H), 6.56 (d, J=8.5
Hz, 1H), 6.55 (m, 1H) 5.41 (d, J=16.6 Hz) 1H), 5.15 (dd, J=5 Hz,
8.8 Hz, 1H), 4.02 (d, 16.7 Hz, 1H), 3.60 (s, 3H), 3.38 (m, 2H),
2.84 (m, 2H), 2.82 (dd, J=8.8 Hz, 16.6 Hz, 1H), 2.67 (dd, J=5.3 Hz,
16.6 Hz, 1H), 1.50 (m, 2H), 1.36 (s, 9H); LRMS(ES): 380.3
[(M-tBu)+H].sup.+, 436.3 [M+H].sup.+.
[0391] Step 1F. Synthesis of methyl
(S)-2-(2,5-diaza-9-(N-(benzimidazol-2--
ylmethyl)-N-methylcarbamoyl)-5-(3-((tert-butoxy)carbonylamino)propyl)-4-ox-
obicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetate 18
[0392] The product of step 1E (476 mg, 1.09 mmol) was dissolved in
dry dimethylformamide along with 2-(methylaminomethyl)benzimidazole
dihydrochloride (290 mg, 1.25 mmol, prepared according to F. Ali
et. al., WO 96/00730), hydroxybenzotriazole hydrate (HOBT) (154 mg,
1.14 mmol), ethyl dimethylaminopropylcarbodiimide hydrochloride
(261 mg, 1.36 mmol), and diisopropylethylamine (1.1 mL, 6 mmol).
The solution was stirred for 23 hr under nitrogen and then
concentrated. The residue was partitioned with ethyl acetate/water,
and the aqueous layer extracted with 2 portions of ethyl acetate.
The combined organic layers were washed with water and brine and
concentrated. The residue was purified by flash chromatography on
silica (95:5 ethyl acetate/methanol) and the product fractions
concentrated to afford the product (435 mg, 69%) as a crunchy foam
after drying under vacuum. LRMS(ES): 579.4 [(M+H].sup.+. .sup.1HNMR
(600.1300 MHz, DMSO-d6): ?12.34 (b, 1H), 7.58 (d, J=1.8 Hz, 1H),
7.48 (dd, J=1.9 Hz, 8.5 Hz, 1H), 7.24 (s, 1H), 7.17 (m, 3H), 6.64
(t, 1H), 6.56 (d, 1H), 6.55 (m, 1H) 6.21 (s, 1H), 5.41 (d, J=16.6
Hz, 1H), 5.10 (dd, J=5 Hz, 8.8 Hz, 1H), 4.76 (q, 2H), 3.89 (d, 16.6
Hz, 1H), 3.60 (s, 3H), 3.37 (m, 2H), 3.04 (s, 3H), 2.82 (m, 3H),
2.64 (dd, J=5.3 Hz, 16.6 Hz, 1H), 1.48 (m, 2H), 1.34 (s, 9H).
[0393] Step 1G: Synthesis of
(S,S)-7-((tert-butyl)oxycarbonyl)-2-(2-((tert-
-butyl)oxycarbonyl)ethyl)-3-oxo-5-((phenylmethoxy)carbonyl
amino)carbonyl)heptanoic acid 19
[0394] Gamma-tert-butoxy-Z-glutamic acid succinimide ester (2.0 g,
4.75 mmol) was dissolved in dimethylformamide, and
gamma-tert-butoxyglutamic acid (0.98 g, 4.8 mmol) added, followed
by diisopropylethylamine (1.75mL, 10.1 mmol). The solution was
stirred 18 hr, concentrated, and the residue partitioned into ethyl
acetate/10% citric acid. The aqueous fraction was extracted with
ethyl acetate and the combined organics were washed with water, 10%
potassium hydrogen sulfate, and brine, and then concentrated. The
residual oil was purified by flash chromatography on silica
(CH.sub.2Cl.sub.2/EtOAc/EtOH, 1:1:0.5%) and the product fractions
combined and evaporated to yield the product (1.3 g, 53%) as a
gummy solid. LRMS (ES): 523.4 [M+H].sup.+, 467.4; .sup.1HNMR
(600.1330 MHz, CDCl.sub.3) 7.30 (m, 6H), 5.80 (d, 1H), 5.09 (m,
2H), 4.53 (m, 1H), 4.29 (m, 1H), 2.36 (m, 4H), 1.88-2.16 (m, 4H),
1.42 (s, 9 H), 1.41 (s, 9H).
[0395] Step 1H: Synthesis of tert-butyl
(S,S,S)-4-(N-(3-(3,6-diaza-5-((met-
hoxycarbonyl)methyl)-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4--
oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-((tert-
-butyl)oxycarbonyl)-2-((phenylmethoxycarbonylamino)butanoylamino)butanoate
20
[0396] The product of 1F (40 mg, 70 .mu.mol) was dissolved in
dichloromethane (1 mL) under nitrogen. To this was added
triethylsilane (110 uL, 0.7 mmol) and trifluoroacetic acid (1 mL).
The reaction was stirred 60 min, concentrated, and reconcentrated
with 5 mL toluene. The residue was dissolved in dry
dimethylformamide (1 mL) and the product of step 1G (40 mg, 77
.mu.mol) added, along with HBTU (33.2 mg, 87 .mu.mol) and
diisopropylethylamine (100 .mu.L, 560 .mu.mol). This was stirred
for 18 hr. The reaction was concentrated, and the residue dissolved
in ethyl acetate. The organics were washed with water, 10%
potassium hydrogen sulfate, water, and brine, and then
concentrated. The residual oil was purified by flash chromatography
on silica (EtOAc/2-PrOH, 1%->10%) and the product fractions
combined and evaporated to yield the product (36 mg, 53%) as a
white solid. LRMS (ES): 983.6 [M+H].sup.+, 492.5 [M+2H].sup.+2;
HRMS (ESI): Calculated for C.sub.51H.sub.67N.sub.8O.sub.12
-983.4878, found -983.4860; .sup.1HNMR (600.1300 MHz, CDCl.sub.3)
7.63 (b, 2H), 7.45 (b, 1H) 7.22-7.41 (m, 11H), 6.90 (b, 1H), 6.54
(d, 1H), 5.99 (b, 1H) 5.39 (d, J=16.6 Hz, 1H), 5.12 (m, 3H),
4.78-4.98 (m, 2H), 4.51 (b, 1H), 4.40 (b, 1H),4.25 (b, 1H), 3.87
(d, J=16.6 Hz 1H), 3.76 (s, 3H), 3.66 (b, 1H), 3.45 (b, 1H), 3.19
(s, 3H), 3.17 (m, 1H), 3.03 (m, 2H), 2.69 (dd, 1H), 2.25-2.45 (m,
4H) 2.05-2.16 (m, 2H), 1.96 (m, 2H), 1.71 (m, 2H), 1.46 (s, 9 H),
1.44 (s, 9H)
[0397] Step 1I: Synthesis of tert-butyl
(S,S,S)-4-amino-4-(N-(3-(3,6-diaza-
-5-((methoxycarbonyl)methyl)-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarba-
moyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-(-
(tert-butyl) oxycarbonyl)propyl)carbamoyl)butanoate acetate salt
21
[0398] The product of Step 1H (33 mg, 33 .mu.mol) was hydrogenated
with 10% palladium on carbon (15 mg) in methanol (6 mL) with acetic
acid (0.1 mL) on a Parr shaker at 40 psi for 1.5 hr. The solution
was filtered on Celite, rinsed with methanol and concentrated. The
residue was dissolved in 20 mL 1:1 acetonitrile/water, frozen, and
lyophilized to afford the product as a white powder (21 mg, 75%).
LRMS (ES): 849.5 [M+H].sup.+, 425.5 [M+2H].sup.+2;
[0399] Step J: Synthesis of tert-butyl
(S,S,S)-4-(N-(1-(N-(3-(3,6-diaza-10-
-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxy
carbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)ca-
rbamoyl)-3-((tert-butyl)oxycarbonyl)
carbamoyl)-4-(2-(1,4,7,10-tetraaza-4,-
5,10-tris(((tert-butyl)oxycarbonyl)
methyl)cyclododecyl)acetylamino)butano- ate trifluoroacetate 22
[0400] The product of step 1I (20 mg, 16.8 .mu.mol) was dissolved
in DMF (1 mL) along with DOTA(OtBu)3-OH (prepared according to EP .
. . ) (26 mg, 25 .mu.mol), HBTU (20 mg, 53 .mu.mol),
diisopropylethylamine (29.1 mg, 225 .mu.mol) and HOBT hydrate (2.5
mg, 18 .mu.mol). This was stirred for 18 hr under nitrogen,
concentrated under vacuum, and purified by preparative HPLC (Vydac
C-18, 2.5 cm.times.15 cm, 0.1%TFA/acetonitrile gradient). The
product fractions were pooled and lyophilized to afford 17.5 mg of
product as a white powder. LRMS (ES) 589.5, 617.8, 646.1, 674.5
[(M-ntBu) +2H]+2, 702.8 [M+2H]+2, 1403.9 [M+H].sup.+
[0401] Step 1K: Synthesis of
(S,S,S)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazo-
l-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]unde-
ca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10--tet-
raaza-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino)butanoyl
amino)butanoic acid 23
[0402] The product of I (16 mg, 7.67 .mu.mol (as .cndot.6TFA salt))
was dissolved in THF/MeOH (1:1, 1 mL) and lithium hydroxide added
(26 .mu.L of a 3M solution in water). The reaction was stirred for
2 hr, concentrated, and treated with trifluoroacetic acid (0.8 mL)
and triethylsilane (0.2 mL) under nitrogen. The solution was
stirred for 21 hr, concentrated under vacuum, and purified by
preparative HPLC (Vydac C-18, 21.5 mm.times.15 cm, 0.1%
TFA/acetonitrile gradient). The product fractions were pooled and
lyophilized to afford the product (6.5 mg, 55%) as a white powder.
LRMS (ES): 370.9 [M+3H]+3, 555.6 [M+2H]+2, 1109.5 [M+H]+; HRMS:
Calculated for C.sub.50H.sub.69O.sub.17N.sub.12: 1109.4904, found:
1109.4890.
Example 2
Preparation of
(S)-2-(2,5-diaza-5-(6((6-((1-aza-2-(2-sulfophenyl)vinyl)ami- no)
(3-pyridyl))carbonylamino)hexyl)-9-(N-(benzimidazol-2-ylmethyl)-N-meth-
ylcarbamoyl)-4-oxobicyclo [5.4.0]undeca-1(7),8,10-trien-3-yl)
acetic acid trifluoroacetate salt
[0403] 24
[0404] Step 2A: Synthesis of tert-butyl 3-(((6-((tert-butoxy)
carbonylamino)hexyl)amino)methyl)-4-fluorobenzoate 25
[0405] This was prepared in the same fashion as Example 1A from
tert-butyl-4-fluoro-3(alpha-bromomethyl)benzoate (5.4 g., 18 mmol)
and 6-tert-butoxycarbonylamino-1-hexylamine hydrochloride (5.0 g.,
19.8 mmol), affording 3.1 g (41%) of product as a yellow oil. LRMS:
425.2 [M+H].sup.+; .sup.1HNMR (270 MHz, DMSO-d6): ?7.95 (dd, 1H),
7.87 (dd, 1H), 7.04 (t, 1H), 4.50 (bs, 1H), 3.83 (s, 2H), 3.07 (q,
2H), 2.59 (t, 2H), 1.57 (s, 9H), 1.42 (s, 9H), 1.60-1.20 (m,
8H)
[0406] Step 2B: Synthesis of methyl
(S)-3-N-(6-((tert-butoxyl)carbonyl
amino)hexyl)-N-((5-((tert-butyl)oxycarbonyl)-2-fluorophenyl)
methyl)carbamoyl)-3-((phenylmethoxy)carbonylamino)propanoate 26
[0407] This was prepared as in Example 1B, starting with 3.06 g of
amine, affording 4.4 g(88%) of the product as a viscous oil. LRMS:
688.4 [M+H].sup.+; .sup.1HNMR (270 MHz, DMSO-d6): Mixture of amide
rotamers, ?7.85 (m, 2H), 7.80 (d, 1H), 7.4-7.2 (m, 6H), 6.73 (br t,
1H), 5.10-4.40 (m, 4H), 3.56, 3.53 (2s, 3H), 3.35 (m, 2H),
3.00-2.55 (m, 4H), 1.51 (s, 9H), 1.35 (s, 9H), 1.70-1.10 (m,
8H);
[0408] Step 2C: Synthesis of methyl
(S)-3-amino-3-(N-(6-((tert-butoxy)carb-
onylamino)hexyl)-N-((5-((tert-butyl)oxycarbonyl)-2-fluorophenyl)methyl)car-
bamoyl)propanoate 27
[0409] This step was done in the same fashion as Example 1C,
starting with 2.3 g of CbZ protected compound, affording 1.71 g
(92%) of the amine as a pale yellow oil. LRMS: 554.3 [M+H].sup.+;
.sup.1HNMR (270 MHz, DMSO-d6) mixture of amide rotamers: ?7.90-7.70
(m, 2H), 7.29 (m, 1H), 6.75 (br, 1H), 4.80 (q, 1H), 4.54 (s, 2H),
4.10 (q, 1H), 3.89 (2t, 1H), 3.53 (2s, 3H) 2.87 (m, 2H), 2.55 (m,
2H), 1.90 (bs, 1H), 1.52 (s, 9H), 1.35 (s, 9H), 1.70-1.10 (m,
8H);
[0410] Step 2D: Synthesis of methyl
(S)-2-(2,5-diaza-9-((tert-butyl)
oxycarbonyl)-5-(6-((tert-butoxy)carbonylamino)hexyl)-4-oxobicyclo[5.4.0]u-
ndeca-1(7),8,10-trien-3-yl)acetate 28
[0411] This step was done in the same fashion as Example 1D,
starting with 1.66 9 of amine, affording 706 mg (44%) of the
benzodiazepine as a pale yellow foam. LRMS: 534.3 [M+H].sup.+;
.sup.1HNMR (270 MHz, DMSO-d6) mixture of amide rotamers: ?7.55 (d,
1H), 7.50 (dd, 1H), 6.70 (br t, 1H), 6.55 (br, 1H), 6.54 (d, 1H),
5.40 (d, 1H), 5.14 (m, 1H), 3.99 (d, 1H), 3.59 (s, 3H) 2.78 (m,
2H), 2.65 (q, 2H), 1.49 (s, 9H), 1.35 (s, 9H), 1.30-1.00 (m,
8H);
[0412] Step 2E: Synthesis of (S)-2,5-diaza-5-(6-((tert-butoxy)
carbonylamino)hexyl)-3-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undec-
a-l(7),8,10-trien-9-carboxylic acid 29
[0413] This step was done in the same fashion as Example 1E,
starting with 301 mg of ester, affording the crude product (394 mg)
as a yellow foam, which was used directly in the next step without
purification. LRMS: 478.2 [M+H].sup.+.
[0414] Step 2F: Synthesis of methyl
(S)-2-(2,5-diaza-9-(N-(benzimidazol-2--
ylmethyl)-N-methylcarbamoyl)-5-(6-((tert-butoxy)carbonyl
amino)hexyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetate
30
[0415] The reaction was carried out as in Example 1F, obtaining 306
mg of crude solid, which was further purified by flash
chromatography to afford the desired product (164 mg, 47% from Step
D) as a pale yellow solid. LRMS: 621.3 [M+H].sup.+; .sup.1HNMR (270
MHz, DMSO-d6): ?12.40 (br, 1H), 7.53 (bs, 2H), 7.20 (m, 4H), 6.71
(br, 1H), 6.52 (d, 1H), 6.23 (bd, 1H), 5.40 (d, 1H), 5.10, (m, 1H),
4.76 (s, 2H), 3.85 (bd, 1H), 3.59 (s, 3H), 3.04 (s, 3H), 2.90-2.55
(m, 2H), 1.35 (s, 9H), 1.40-1.20 (m, 8H)
[0416] Step 2G: Synthesis of
(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethy-
l)-N-methylcarbamoyl)-5-(6-((tert-butoxy)carbonylamino)
hexyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic acid
31
[0417] The product of step F (152 mg, 245 .mu.mol) was stirred with
lithium hydroxide (21 mg, 500 .mu.mol) in THF/H2O (3 mL/2 mL) for
22 hr. THF was removed under vacuum, the residue diluted with water
and acidified with solid citric acid. The precipitated solid and
solution was extracted with dichloromethane, washed with brine,
dried (Na.sub.2SO.sub.4), and concentrated to afford the acid
product (120 mg, 81%) as a pale yellow powder, which was not
purified further. LRMS: 607.2 [M+H].sup.+.
[0418] Step 2H: Synthesis of
(S)-2-(2,5-diaza-5-(6((6-((1-aza-2-(2-sulfoph-
enyl)vinyl)amino)(3-pyridyl))carbonylamino)hexyl)-9-(N-(benzimidazol-2-ylm-
ethyl)-N-methylcarbamoyl)-4-oxobicyclo
[5.4.0]undeca-1(7),8,10-trien-3-yl)- acetic acid trifluoroacetate
32
[0419] The product of step G (87 mg, 143 .mu.mol) was dissolved in
CH.sub.2Cl.sub.2 (4 mL) and trifluoroacetic acid (2 mL) added with
stirring under nitrogen. The solution was stirred for one hour,
concentrated under vacuum, and the residue redissolved in dry DMF
(2.5 mL). To this was added sodium
2-[[[5-[[(2,5-dioxo-1-pyrollidinyl)
oxy]carbonyl]-2-pyridinyl]hydrazono]methyl]-benzenesulfonate (75
mg, 170 .mu.mol) and diisopropylethylamine (500 .mu.L, 2.87 mmol)
with stirring under nitrogen. The reaction was stirred overnight,
concentrated, and the residue purified by preparative HPLC (Vydac
C-18, 2.5 cm.times.15 cm, 0.1% TFA/acetonitrile gradient). The
product fractions were combined and lyophilized to afford the
product as a pale yellow powder (47.3 mg, 35%). LRMS (ES): 810.3
[M+H].sup.+. .sup.1HNMR (600.1300 MHz, DMSO-d6): ?12.40 (b, 2H),
9.24 (bs, 1H), 8.59 (bs, 1H), 8.50 (s, 1H), 8.24 (bs, 1H), 8.20
(bs, 1H), 7.80 (d, 3H), 7.53 (m, 2H), 7.41 (m, 2H), 7.20 (m, 3H),
6.57 (d, 1H), 6.32 (bs, 1H), 5.40 (d, 1H, J=16.4 Hz), 5.10 (m, 1H),
4.76 (s, 2H), 3.85 (d, 1H, J=16.4 Hz), 3.55 (m, 2H), 3.21 (m, 2H),
3.04 (s, 3H), 2.79 (dd, 1 H, J=16.5 Hz, 9 Hz), 2.55 (dd, 1H, J=16.5
Hz, 5 Hz), 1.60 (m, 2H), 1.51 (m, 2H), 1.26 (m, 2H), 1.19 (m,
2H)
Example 3
Synthesis of
(S)-2-(2,5-diaza-9-(N-(6-((6-((1-aza-2-(2-sulfophenyl)vinyl)a-
mino)(3-pyridyl))carbonylamino)hexyl)-N-(benzimidazol-2-ylmethyl)carbamoyl-
)-5-methyl-4-oxobicyclo [5.4.0]undeca-1(7),8,10-trien-3-yl)acetic
acid trifluoroacetate
[0420] 33
[0421] Step 3A: Synthesis of N-(6-((benzimidazol-2-ylmethyl)amino)
hexyl)(phenylmethoxy)formamide dihydrochloride 34
[0422] Both
.alpha.-bromomethyl-(N-tert-butoxycarbonyl)benzimidazole (3.42g, 11
mmol, prepared according to WO96/00730) and
N-(mono-benzyloxycarbonyl)-hexanediamine (4.58 g, 16 mmol, prepared
according to Bioconj. Chem., 1997, 8, 611) were dissolved in THF
(100 mL), along with diisopropylethylamine (8 mL, 45.9 mmol) and
water (3 mL). The mixture was stirred for 20 hr, concentrated, and
the residue partitioned between 1N NaOH and dichloromethane. The
aqueous was reextracted and concentrated to afford a yellow
semi-solid product which was dissolved in ether/dichloromethane
(2:1, 300 mL) and treated with 4N HCl in dioxane (40 mL, 160 mmol)
with stirring at room temperature for 18 hr. The resulting solids
were filtered, dissolved in a minimum amount of 10% sodium
carbonate, extracted into dichloromethane and concentrated to an
oil. This was purified by flash chromatography on silica (9:1
EtOAc/EtOH, 0.1% NH.sub.4OH) and the product fractions
concentrated, dissolved in ether, and treated with 4N HCl/dioxane.
The resulting solids were filtered and washed with ether to afford
745 mg of a white powder. LRMS: 381.3 [M+H].sup.+; .sup.1HNMR (270
MHz, DMSO-d6): ?10.04 (b, 2H), 7.78 (m, 2H), 7.44 (m, 2H), 7.34 (m,
6H) 6.76 (b, 2H), 4.99 (s, 2H), 4.60 (s, 2H), 3.10 (m, 2H), 2.99
(m, 2H), 1.67 (m, 2H), 1.41 (m, 2H), 1.29 (m, 4H)
[0423] Step 3B: Synthesis of methyl
(S)-2-(2,5-diaza-9-(N-(benzimidazol-2--
ylmethyl)-N-(6-((phenylmethoxy)carbonylamino)
hexyl)carbamoyl)-5-methyl-4--
oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetate 35
[0424] The product of Step 3A (300 mg, 0.66 mmol), methyl
(S)-(-)-7-carboxy-2,3,4,5-tetrahydro-4-methyl-3-oxo-1H-1,4-benzodiazepine-
-2-acetate (172 mg, 0.55 mmol, prepared according to
PCT/US95/00248, WO 95/18619), HOBT (89 mg, 0.66 mmol), and
diisopropylethylamine (380 .mu.L, 2.18 mmol) were dissolved in dry
DMF (5 mL) in dry glassware under nitrogen. EDC (89 mg, 0.66 mmol)
was added in one portion and the reaction stirred 20 hr. The
solution was concentrated, partitioned between water and ethyl
acetate, and the aqueous layer extracted with two additional
portions of ethyl acetate. The combined organics were washed with
water and brine, and concentrated. The crude oil was purified by
flash chromatography on silica gel (EtOAc, 0.5% EtOH). The product
fractions were combined and concentrated to yield 145 mg (40%) of
product as a light brown solid. LRMS (ES): 655.3 [M+H].sup.+;
.sup.1HNMR (600.1343 MHz, DMSO-d6): ?12.38 (b, 1H), 7.51 (m, 2H),
7.30 (m, 6H), 7.14 (m, 4H), 6.51 (d, 1H), 6.16 (d, 1H), 5.42 (d,
1H, J=16 Hz), 5.08 (m, 1H), 4.96 (s, 2H), 4.73 (s, 2H), 3.88 (d,
1H, J=16 Hz), 3.57 (s, 3H), 3.33 (m, 2H), 2.89 (m, 2H), 2.85 (s,
3H), 2.78 (dd, 1 H, J=16.5 Hz, 9 Hz), 2.61 (dd, J=16.5 Hz, 5 Hz),
1.52 (m, 2H), 1.30 (m, 2H), 1.15 (m, 4H) .sup.13C NMR (600.1343
MHz, DMSO-d6): 170.9, 169.1, 165.6, 156.0, 151.3, 147.4, 137.3,
129.3, 128.3, 127.8, 127.7, 127.3, 123.0, 118.1, 114.9, 65.0, 59.7,
51.6, 51.3, 50.1, 50.0, 37.4, 35.0, 29.5, 29.2, 26.6, 20.7,
14.1
[0425] Step 3C: Synthesis of methyl
(S)-2-(9-(N-(6-aminohexyl)-N-(benzimid-
azol-2-ylmethyl)carbamoyl)-2,5-diaza-5-methyl-4-oxobicyclo[5.4.0]undeca-1(-
7),8,10-trien-3-yl)acetate 36
[0426] The product of 3B (140 mg, 214 .mu.mol) was dissolved in
methanol (6 mL) with 10% palladium on carbon (30 mg). The slurry
was hydrogenated at one atmosphere pressure for 5.5 hr, filtered
through Celite.RTM. and concentrated to yield the product (100 mg,
90%) as a clear oil which was not further purified, but taken
directly into the next step. LRMS (ES) 521.4 [M+H].sup.+, 275.3,
261.3, 245.2, 231.3.
[0427] Step 3D: Synthesis of
(S)-2-(9-(N-(6-aminohexyl)-N-(benzimidazol-2--
ylmethyl)carbamoyl)-2,5-diaza-5-methyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-
-trien-3-yl)acetic acid 37
[0428] The product of Step 3C (100 mg, 192 .mu.mol) was dissolved
in methanol/tetrahydrofuran (2:1, 1 mL) and lithium hydroxide
hydrate (23 mg, 550 .mu.mol) dissolved in 0.5 mL water was added.
The reaction was stirred for 4 hr, neutralized with 10% potassium
hydrogen sulfate solution, and concentrated. The solids were
dissolved in methanol, filtered, and the filtrate concentrated to
an oil, which was dissolved in water/acetonitrile and lyophilized
to afford 93 mg (96%) of the product as a white solid. LRMS (ES):
507.3 [M+H].sup.+, 459.4, 254.4 [M+2H].sup.+2; .sup.1HNMR (600.1300
MHz, DMSO-d6): ?12.35 (b, 1H), 10.49 (b, 3H), 7.59 (m, 2H), 7.53
(m, 2H), 7.16 (bs, 4H), 6.53 (d, 1H, J=7.4 Hz), 6.18 (s, 1H), 5.44
(d, 1H, J=16.4 Hz), 5.08 (m, 1H), 4.76 (s, 2H), 3.80 (bd, 1H, J=12
Hz), 3.38 (m, 2H), 2.88 (s, 3H), 2.78 (dd, 1 H, J=16.7 Hz, 9 Hz),
2.71 (m, 2H), 2.61 (dd, 1H, J=16.7 Hz, 5 Hz), 1.55 (m, 2H), 1.47
(m, 2H), 1.18 (m, 2H), 1.03 (m, 2H)
[0429] Step 3E: Synthesis of
2-(2,5-diaza-9-(N-(6-((6-((1-aza-2-(2-sulfoph-
enyl)vinyl)amino)(3-pyridyl))carbonylamino)hexyl)-N-(benzimidazol-2-ylmeth-
yl)carbamoyl)-5-methyl-4-oxobicyclo
[5.4.0]undeca-1(7),8,10-trien-3-yl)ace- tic acid trifluoroacetate
38
[0430] The product of Step D (80 mg, 160 .mu.mol) was dissolved in
dry dimethylformamide, along with sodium
2-[[[5-[[(2,5-dioxo-1-pyrolidinyl)
oxy]carbonyl]-2-pyridinyl]hydrazono]methyl]-benzenesulfonate (88
mg, 250 .mu.mol) and diisopropylethylamine (280 .mu.L, 1.6 mmol)
with stirring under nitrogen. The reaction was stirred overnight,
concentrated, and the residue purified by preparative HPLC (Vydac
C-18, 21.5 mm.times.25 cm, 0.1% TFA/acetonitrile gradient). The
product fractions were combined and lyophilized to afford the
product as a white solid (24 mg, 18%). LRMS (ES): 810.3
[M+H].sup.+, 4764.3, 399.3; HRMS (ESI): Calculated for
C.sub.40H.sub.44N.sub.9O.sub.8S (M+H) -810.3033, found
-810.3052.
[0431] .sup.1HNMR (600.1300 MHz, DMSO-d6): ?12.40 (b, 2H), 9.24
(bs, 1H), 8.59 (bs, 1H), 8.50 (s, 1H), 8.24 (bs, 1H), 8.20 (bs,
1H), 7.80 (d, 3H), 7.53 (m, 2H), 7.41 (m, 2H), 7.20 (m, 3H), 6.57
(d, 1H), 6.32 (bs, 1H), 5.47 (d, 1H, J=16.4 Hz), 5.08 (m, 1H), 4.98
(s, 2H), 3.83 (d, 1H, J=16.4 Hz), 3.50 (m, 2H), 3.21 (m, 2H), 2.89
(s, 3H), 2.75 (dd, 1 H, J=16.7 Hz, 9 Hz), 2.53 (dd, 1H, J=16.7 Hz,
5 Hz), 1.65 (m, 2H), 1.48 (m, 2H), 1.26 (m, 2H), 1.19 (m, 2H)
Example 4
Preparation of
(S,S)-2-(2-aza-2-((5-(N-(1,3-bis(N-(6-(aminohexyl-4-oxobicy-
clo[5.4.0]undeca-l(7),8,10-trien-3-yl)acetic
acid)(2-(2,5-diaza-9-(N-(benz-
imidazol-2-ylmethyl)propyl)carbamoyl)(2-pyridyl))amino)vinyl)
benzenesulfonic acid
[0432] 39
[0433] Step 4A. Synthesis of
(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethy-
l)-N-methylcarbamoyl)-5-(6-aminohexyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-
-trien-3-yl)acetic acid 40
[0434] The product of Step 2E (350 mg, 564 .mu.mol) was dissolved
in methanol/tetrahydrofuran (2:1, 8 mL) with stirring. Lithium
hydroxide hydrate (95 mg, 2.25 mmol) was dissolved in water (5 mL)
and added to this solution. It was stirred for two hours,
neutralized with 10% potassium hydrogen sulfate and concentrated to
a gummy solid. This was added to a solution of trifluoroacetic acid
in dichloromethane (4 mL/6 mL) and stirred for two hours. The
solids were filtered off, and the filtrate concentrated to afford
an oil, which was redissolved in water/acetonitrile and lyophilized
to a white powder which was not further purified. LRMS (ES): 507.4
[M+H].sup.+, 254.4 [M+2H].sup.+2.
[0435] Step 4B. Synthesis of
(S,S)-2-(2,5-diaza-(9-(N-benzimidazol-2-ylmet-
hyl))-5-(6-(4-(N-(6-(3,6-diaza-5-(carboxymethyl)-4-oxobicyclo[5.4.0]undeca-
-1(7),8,10-trien-3-yl)hexyl)carbamoyl)-2-((tert-butoxy)carbonylamino)butan-
oylamino)hexyl)-4-oxobicyclo[5.4.0]undeca-1(11),7(8),9-trien-3-yl)acetic
acid 41
[0436] The product of 4A (31 mg, 36.5 .mu.mol) was dissolved in dry
dimethylformamide (1.5 mL), along with diisopropylethylamine (51
.mu.L, 300 .mu.mol). To this was added
bis-(N-hydroxysuccinimide)-N-(tert-butoxy- carbonyl)-glutamate (7.7
mg, 17.5 .mu.mol) with stirring. The solution was allowed to stir
for three hours, when it was concentrated and purified by
preparative HPLC (Vydac C-18, 21.5 mm.times.25 cm, 0.1%
TFA/acetonitrile gradient). The product fractions were combined and
lyophilized to afford the product as a white solid (12 mg, 33%).
LRMS (ES): 1224.7 [M+H].sup.+, 613.1 [M+2H].sup.+2, 409.3
[M+3H].sup.+3. HRMS (ESI): Calculated for
C.sub.64H.sub.82N.sub.13O.sub.12 -1224.6206, found -1224.619.
[0437] Step 4C. Synthesis of
(S,S)-2-(2-aza-2-((5-(N-(1,3-bis(N-(6-(aminoh-
exyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic
acid)(2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)propyl)carbamoyl)(2-pyri-
dyl))amino)vinyl) benzenesulfonic acid 42
[0438] The product of 4B (10 mg 5.5 .mu.mol of .cndot.4TFA salt)
was dissolved in dichloromethane:triflouroacetic acid (1.5 mL/0.5
mL) under nitrogen. It was stirred 20 minutes and concentrated to
an oil, which was resuspended in toluene and reconcentrated to
remove residual TFA. The residue was treated as in step 3E to
afford 2.5 mg (31%) of the product as a white lyophilized solid.
LRMS (ES): 1428.2 [M+H].sup.+, 714.5 [M+2H].sup.+2, 477.3
[M+3H].sup.+3. HRMS (ESI): Calculated for
C.sub.72H.sub.83N.sub.16O.sub.14S -1427.5995, found -1427.601.
Example 5
Preparation of
(S,S,S)-4-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazo-
l-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-
-3-yl)propyl)
carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(-
carboxymethyl) cyclododecyl)acetylamino)butanoylamino) butanoic
acid
[0439] 43
[0440] Step 5A. Synthesis of benzyl((1-(triphenylmethyl)
imidazol-2-yl)methyl) amine 44
[0441] N-tritylimidazole-2-carboxaldehyde (338 mg, 1 mmol, prepared
according to K. L. Kirk; J. Org. Chem., 1978, 43, 4381) was
dissolved in dry toluene (7 mL) and anhydrous magnesium sulfate
(602 mg, 5 mmol) added with stirring under nitrogen. Benzylamine
(131 .mu.L, 1.2 mmol) was added and the solution stirred for 3.5
hr. The solids were filtered under nitrogen and the reaction
concentrated. The residue is redissolved in 1,2-dichloroethane (25
mL) and cooled to 0.degree. C. Sodium triacetoxyborohydride (1.06
g, 5 mmol) was added slowly. The solution was allowed to warm to
room temperature over 2.5 hours. The reaction mixture was added to
water/ethyl acetate and the layers separated. The aqueous layer was
extracted with two portions of ethyl acetate and the combined
organic layers washed with sat. bicarbonate, water, and brine. The
solution was concentrated to an oil and purified by flash
chromatography on silica gel (99:1 EtOAc/EtOH with 0.1%
triethylamine).to afford 330 mg (77%) of product as an oil which
solidified on standing. LRMS (ES): 430.4 [M+H].sup.+, 243.2;
.sup.1HNMR (600.1328 MHz, DMSO-d6): ?7.37 (m, 11H), 7.04 (m, 9 H),
6.92 (d, 1H), 6.64 (d, 1H), 3.34 (s, 2H), 2.77 (2H).
[0442] Step 5B. Synthesis of methyl
(S)-2-(2,5-diaza-5-(3-((tert-butoxy)ca-
rbonylamino)propyl)-4-oxo-9-(N-benzyl-N-((2-(triphenylmethyl)imidazol-2-yl-
)methyl)carbamoyl)bicyclo
[5.4.0]undeca-1(7),8,10-trien-3-yl)acetate 45
[0443] The product of step 5E (150 mg, 0.345 mmol) was treated in
the same manner as step 1F, affording the product (250 mg, 85%) as
a thick oil. LRMS (ES): 847.5 [M+H].sup.+, 430.5, 243.2; .sup.1HNMR
(600.1330 MHz, CDCl.sub.3) This sample gave broad peaks with little
fine splitting, even when refiltered, and was qualitatively similar
to 1E for the benzodiazepine nucleus. Step 5C. Synthesis of methyl
(S)-2-(5-(3-aminopropyl)-2,5-diaza-9-(N-(imidazol-2-ylmethyl)-N-benzylcar-
bamoyl)-4-oxobicyclo [5.4.0]undeca-1(7),8,10-trien-3-yl)acetate
46
[0444] The product of step 5B (220 mg, 0.26 mmol) was added to neat
trifluoroacetic acid (4 mL) containing triethylsilane (1 mL) under
nitrogen and stirred for 1.5 hr. The solution was concentrated and
residual acid removed by reconcentration with toluene. This product
was not purified, but was used directly in the following step. LRMS
(ES): 505.4 [M+H].sup.+, 253.4.
[0445] Step 5D. Synthesis of tert-butyl
(S,S,S)-4-(N-(3-(3,6-diaza-10-(N-(-
imidazol-2-ylmethyl)-N-benzylcarbamoyl)-5-((methoxycarbonyl)
methyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)
carbamoyl)-4-(4-((tert-butyl)oxycarbonyl)-2-((phenylmethoxy)
carbonylamino)butanoylamino)butanoate 47
[0446] A portion of the product of step 5C (65 mg, 130 .mu.mol) was
reacted with step 1G as in Step 1H to afford the product (64 mg,
49% from 5B) as an oil. LRMS (ES): 1009.7 [M+H].sup.+, 505.6
[M+2H].sup.+2,; HRMS (ESI): Calculated for
C.sub.53H.sub.69N.sub.8O.sub.12 -1009.5035, found -1009.502;
.sup.1HNMR (600.1330 MHz, CDCl.sub.3) 7.47 (b, 1H), 7.22-7.41 (m,
14H), 6.99 (s, 2H), 6.93 (b, 1H), 6.44 (d, 1H), 5.98 (b, 1H) 5.32
(d, 1H), 5.13 (d, 1H), 5. 05 (m, 2H) 4. 68 (m, 3H), 4.48 (b, 1H),
4.36 (b, 1H),4.24 (b, 1H), 3.71 (s, 3H), 3.68 (m, 1H), 3.60 (b,
1H), 3.38 (b, 1H), 3.11 (b, 1H), 2.97 dd, 1H), 2.94 (m, 1H), 2.65
(dd, 1H), 2.25-2.45 (m, 4H) 1.88-2.16 (m, 4H), 1.65 (m, 2H), 1.45
(s, 9 H), 1.41 (s, 9H).
[0447] Step 5E: Synthesis of tert-butyl
(S,S,S)-4-amino-4-(N-(1-(N-(3-(3,6-
-diaza-1-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-5-((methoxycarbonyl)m-
ethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3--
((tert-butyl)oxycarbonyl) propyl)carbamoyl)butanoate 48
[0448] The product of 5D (58 mg, 57 .mu.mol) was hydrogenated
according to the procedure of step 1I, to yield the product (44 mg,
88%) as a white solid, which was not further purified but was
lyophilized in 0.1% aqueous trifluoroacetic acid/acetonitrile (1:1)
and used as the trifluoroacetate salt in the next step. LRMS (ES):
875.6 [M+H].sup.+, 438.5 [M+2H].sup.+2,;
[0449] Step 5F: Synthesis of tert-butyl
(S,S,S)-4-(N-(1-(N-(3-(3,6-diaza-1-
0-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-5-((methoxycarbonyl)methyl)--
4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-((tert--
butyl)oxycarbonyl)
propyl)carbamoyl)-4-(2-(1,4,7,10-tetraaza-4,7,10-tris((-
(tert-butyl)oxycarbonyl)methyl)cyclododecyl)acetylamino)butanoate
49
[0450] The product of 5E (24.4 mg, 20 .mu.mol) was reacted with
DOTA tri-tert-butyl ester as in step 1J, to afford the product
(19.6 mg, 55%) as a trifluoroacetate salt after lyophilization.
[0451] LRMS (ES): 1430.0 [M+H].sup.+, 715.7 [M+2H].sup.+2, 477.8
[M+3H].sup.+3; HRMS(ESI): Calculated for
C.sub.73H.sub.113N.sub.12O.sub.1- 7 -1429.8347, found
-1429.838;
[0452] Step 5G: Synthesis of
(S,S,S)-4-(N-(3-(3,6-diaza-5-(carboxymethyl)--
10-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1-
(7),8,10-trien-3-yl)propyl)
carbamoyl)-4-(4-carboxy-2-(2-(1,4,7,10-tetraaz-
a-4,7,10-tris(carboxymethyl)cyclododecyl)acetylamino)butanoylamino)
butanoic acid 50
[0453] The product of 5F (13 mg, 7.4 .mu.mol) was deprotected and
purified as in step 1K, to afford the product (6.5 mg, 55%) as a
trifluoroacetate salt after lyophilization.
[0454] LRMS (ES): 1135.6 [M+H].sup.+, 568.5 [M+2H].sup.+2, 379.6
[M+3H].sup.+3; HRMS(ESI): Calculated for
C.sub.52H.sub.71N.sub.12O.sub.17 -1135.5060, found -1135.503;
Example 6
Preparation of
(S,S)-3-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-(N-(imidazol--
2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-
-yl)propyl)
carbamoyl)-3-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)
cyclododecyl)acetylamino)propanoic acid
[0455] 51
[0456] Step 6A: Synthesis of tert-butyl
(S,S)-3-(N-(3-(3,6-diaza-10-(N-(im-
idazol-2-ylmethyl)-N-benzylcarbamoyl)-5-((methoxycarbonyl)
methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)
carbamoyl)-3-((phenylmethoxy)carbonylamino)propanoate 52
[0457] The product of step 5D (65 mg, 130 .mu.mol) was reacted with
N-(carbobenzyloxy)-.beta.-(tert-butyl)-.alpha.-(N-hydroxysuccinimidyl)
aspartate (66 mg, 156 .mu.mol) and diisopropylethylamine (181
.mu.L, 1.04 mmol) in dimethylformamide (1.5 mL) with stirring at
room temperature under nitrogen for 20 hr. The reaction was
concentrated, and the residue dissolved in ethyl acetate. The
organics were washed with water, 10% potassium hydrogen sulfate,
water, and brine, and then concentrated. The residual oil was
purified by flash chromatography on silica (EtOAc/MeOH, 1%->10%)
and the product fractions combined and evaporated to yield the
product (76 mg, 73%) as an oil. LRMS (ES): 810.5 [M+H].sup.+,
378.0; HRMS (ESI): Calculated for C.sub.43H.sub.52N.sub.7O.sub.9
-810.3826, found -810.3819; .sup.1HNMR (600.1323 MHz, CDCl.sub.3)
7.25-7.38 (m, 12H), 7.18 (m, 2H), 7.07 (b, 1H), 6.99 (s, 2H), 6.39
(d, 1H), 6.18 (b, 1H) 5.30 (d, J=16.2 Hz, 1H), 5.09 (m, 2H), 5.04
(m, 1H) 4.67 (m, 4H), 4.50 (b, 1H), 4.36 (b, 1H), 3.69 (s, 3H),
3.62 (d, J=18.6 Hz, 1H), 3.45 (b, 1H), 3.14 (m, 1H), 2.94 (dd, 1H),
2.86 (m, 2H), 2.62 (m, 2H), 1.60 (m, 2H), 1.39 (s, 9 H)
[0458] Step 6B: Synthesis of tert-butyl
(S,S)-3-amino-3-(N-(3-(3,6-diaza-1-
0-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-5-((methoxycarbonyl)
methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)
carbamoyl)propanoate 53
[0459] The product of 6A (70 mg, 86 .mu.mol) was hydrogenated
according to the procedure of step 1I, to yield the product (55 mg,
95%) as a white solid, which was not further purified but was
lyophilized in 0.1% aqueous trifluoroacetic acid/acetonitrile (1:1)
and used as the trifluoroacetate salt in the next step. LRMS (ES):
676.5 [M+H].sup.+, 339.0 [M+2H].sup.+2, 310.9.
[0460] Step 6C: Synthesis of tert-butyl
(S,S)-3-(N-(3-(3,6-diaza-10-(N-(im-
idazol-2-ylmethyl)-N-benzylcarbamoyl)-5-((methoxycarbonyl)
methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)
carbamoyl)-3-(2-(1,4,7,10-tetraaza-4,7,10-tris(((tert-butyl)
oxycarbonyl)methyl)cyclododecyl)acetylamino)propanoate 54
[0461] The product of 6B (22.4 mg, 22 .mu.mol) was reacted with
DOTA tri-tert-butyl ester and purified as in step 1J, to afford the
product (16.6 mg, 44%) as a trifluoroacetate salt after
lyophilization. LRMS (ES): 1230.9 [M+H].sup.+, 616.2 [M+2H].sup.+2,
411.3 [M+3H].sup.+3; HRMS(ESI): Calculated for
C.sub.63H.sub.96N.sub.11O.sub.14 -1230.7138, found -1230.715;
[0462] Step 6D: Synthesis of
(S,S)-3-(N-(3-(3,6-diaza-5-(carboxymethyl)-10-
-(N-(imidazol-2-ylmethyl)-N-benzylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(7-
),8,10-trien-3-yl)propyl)
carbamoyl)-3-(2-(1,4,7,10-tetraaza-4,7,10-tris(c- arboxymethyl)
cyclododecyl)acetylamino)propanoic acid 55
[0463] The product of 5F (14 mg, 8.3 .mu.mol) was deprotected and
purified as in step 1K, to afford the product (4.6 mg, 47%) as a
trifluoroacetate salt after lyophilization. LRMS (ES): 992.6
[M+H].sup.+, 497.0 [M+2H].sup.+2, 331.8 [M+3H].sup.+3; HRMS(ESI):
Calculated for C.sub.46H.sub.62N.sub.11O.sub.14 -992.4478, found
-992.4457;
Example 7
Synthesis of
(S,S,S,S,S,S,S,S)-4-(N-1,3-bis(N-3-carboxy-1-(N-(3-(3,6-diaza-
-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-ox-
obicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-4,4-dihydroxy-
pentyl)
carbamoyl)propyl)carbamoyl)-4-(5,5-dihydroxy-2-(2-(1,4,7,10-tetraa-
za-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino) butanoic
acid
[0464] 56
[0465] Step 7A: Synthesis of tert-butyl
(S,S,S,S,S,S)4-(N-(1-(N-(3-(3,6-di-
aza-10-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxy
carbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)ca-
rbamoyl)-4-(4-(N-(1-(N-(1-(N-(3-(3,6-diaza-10-10-(N-(benzimidazol-2-ylmeth-
yl)-N-methylcarbamoyl)-5-((methoxy
carbonyl)methyl)-4-oxobicyclo[5.4.0]und-
eca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-3-((tert-butyl)
oxycarbonyl)propyl) carbamoyl)-2-((phenylmethoxy)carbonylamino)
butanoylamino) butanoate 57
[0466] The product of step 1I (65 mg, 54.6 .mu.mol) is dissolved in
DMF (1 mL) along with HBTU (25 mg, 65 .mu.mol),
N-carbobenzyloxy-L-glutamic acid (7.3 mg, 26 .mu.mol), HOBT (7 mg,
52 .mu.mol), and diisopropylethylamine (40 .mu.L, 225 .mu.mol)
under nitrogen. After stirring for 2 hrs, the reaction is
concentrated and purified by preparative HPLC (0.1%
TFA/acetonitrile gradient, Zorbax C8, 21.5 mm.times.25 cm). The
product may be obtained as the trifluoroacetate salt after
lyophilization.
[0467] Step 7B: Synthesis of tert-butyl
(S,S,S,S,S,S)-4-(2-amino-4-(N-(1-(-
N-(3-(3,6-diaza-10-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((-
methoxycarbonyl)methyl)-4-oxobicyclo
[5.4.0]undeca-1(7),8,10-trien-3-yl)pr-
opyl)carbamoyl)-3-((tert-butyl)oxycarbonyl)propyl)carbamoyl)-3-((tert-buty-
l)oxycarbonyl)
propyl)carbamoyl)butanoylamino)-4-(N-(1-(N-(3-(3,6-diaza-10-
-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxy
carbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)ca-
rbamoyl)-3-((tert-butyl)oxycarbonyl)propyl) carbamoyl)butanoate
58
[0468] The product of step 7A is hydrogenated and isolated as in
step 1I. This material is not further purified, but used directly
in the following step.
[0469] Step 7C: Synthesis of tert-butyl
(S,S,S,S,S,S,S,S)-4-(N-(1,3-bis(N--
(3-((tert-butyl)oxycarbonyl)-1-(N-3-((tert-butyl)
oxycarbonyl)-1-(N-(3-(3,-
6-diaza-10-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxy
carbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)
carbamoyl)propyl)carbamoyl)propyl)
carbamoyl)propyl)carbamoyl)-4-(4-((ter-
t-butyl)oxycarbonyl)-2-((phenylmethoxy)carbonylamino)
butanoylamino)butanoate 59
[0470] The product of step 7B is reacted as in step 5D to afford
the product, which is purified by preparative HPLC.
[0471] Step 7D: Synthesis of tert-butyl
(S,S,S,S,S,S,S,S)-4-amino-4-(N-(-(-
N-(1,3-bis(N-(3-((tert-butyl)oxycarbonyl)-1-(N-3-((tert-butyl)oxycarbonyl)-
-1-(N-(3-(3,6-diaza-10-10-(N-(benzimidazol-2-ylmethyl)-N-methyl
carbamoyl)-5-((methoxy
carbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,-
10-trien-3-yl)propyl)carbamoyl) propyl) carbamoyl) propyl)
carbamoyl) propyl) carbamoyl)-3-((tert-butyl) oxycarbonyl) propyl)
carbamoyl) butanoate 60
[0472] The product of step 7C is hydrogenated as in step 1I to
afford the amine, which is not further purified but used directly
in the next step.
[0473] Step 7E: Synthesis of tert-butyl
(S,S,S,S,S,S,S,S)-4-(N-(1-(N-(1,3--
bis(N-(3-((tert-butyl)oxycarbonyl)-1-(N-3-((tert-butyl)
oxycarbonyl)-1-(N-(3-(3,6-diaza-10-10-(N-(benzimidazol-2-ylmethyl)-N-meth-
ylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,-
10-trien-3-yl))propyl)carbamoyl)
propyl)carbamoyl)propyl)carbamoyl)propyl)-
carbamoyl)-3-((tert-butyl)oxycarbonyl)propyl)carbamoyl)-4-(2-(1,4,7,10-tet-
raaza-4,7,10-tris(((tert-butyl)oxycarbonyl)methyl)cyclododecyl)
acetylamino)butanoate 61
[0474] The product of step 7D is reacted with DOTA(OtBu)3-OH as in
step 1J to afford the product as a solid after preparative HPLC
purification and lyophilization. Alternatively, the product of 7B
is reacted with the product of 7I in the presence of HBTU, HOBT,
and diisopropylethylamine in dry dimethylformamide for 2 hours,
after which the reaction is concentrated and the residue purified
by preparative HPLC to afford the product as a solid after
lyophilization.
[0475] Step 7F: Synthesis of
(S,S,S,S,S,S,S,S)-4-(N-1,3-bis(N-3-carboxy-1--
(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(car-
boxymethyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoy-
l)-4,4-dihydroxypentyl)
carbamoyl)propyl)carbamoyl)-4-(5,5-dihydroxy-2-(2--
(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)acetylamino)
butanoic acid 62
[0476] The product of step 7D is deprotected as in step 1K to
afford the product as a solid after preparative HPLC purification
and lyophilization.
[0477] Step 7G: Synthesis of tert-butyl
(S,S)-3,3-dimethyl-3-silabutyl
2-(4-((tert-butyl)oxycarbonyl)-2-((phenylmethoxy)carbonylamino)
butanoylamino)pentane-1,5-dioate 63
[0478] The product of step 1G (1.25 g, 2.4 mmol) was reacted with
2-trimethylsilylethanol (296 mg, 2.5 mmol) in the presence of ethyl
[3-(N,N-dimethylaminopropyl]-carbodiimide hydrochloride (480 mg,
2.5 mmol) and dimethylaminopyridine (250 mg, 1.2 mmol) in
dimethylformamide (10 mL) at 0.degree. C. The reaction was allowed
to warm slowly to room temperature and stirred overnight. It was
concentrated and the residue partitioned between ethyl acetate and
water. The aqueous layer was extracted twice with ethyl acetate,
and the combined organics washed with water, 10% potassium hydrogen
sulfate, and brine, and concentrated. The residue was purified by
flash chromatography (ethyl acetate/hexane) to afford the product
as an oil (1.1 g, 73%). LRMS (ES): 623.5 [M+H].sup.+.
[0479] Step 7H: Synthesis of tert-butyl
(S,S)-3,3-dimethyl-3-silabutyl
2-(2-amino-4-((tert-butyl)oxycarbonyl)butanoylamino)pentane-1,5-dioate
64
[0480] The product of step 7G (1.09 g) was dissolved in 2-propanol
(75 mL) with 10% palladium on carbon (300 mg) and hydrogenated on a
Parr shaker at 45 psi for one hour. The reaction mixture was
filtered on a bed of Celite, washed with 2-propanol, and
concentrated to yield the product (803 mg, 94%) as a clear oil.
LRMS (ES): 489.5 [M+H].sup.+, 977.7 [2M+H].sup.+. .sup.1HNMR
(600.1343 MHz, CDCl.sub.3): 7.78 (m, 1H), 4.53 (m, 1H), 4.22 (m,
2H), 3.53 (m, 1H), 1.80-2.41 (m, 10H), 1.43 (s, 18H), 1.01 (m, 2H),
0.02 (s, 9H).
[0481] Step 7I: Synthesis of tert-butyl
(S,S)-3,3-dimethyl-3-silabutyl
2-(4-((tert-butyl)oxycarbonyl)-2-(2-bromoacetylamino)
butanoylamino)pentane-1,5-dioate 65
[0482] The product of step 7H (397 mg, 0.813 mmol) was dissolved in
dry tetrahydrofuran (5 mL) with diisopropylethylamine (180 .mu.L,
1.05 mmol) and cooled to -10.degree. C. under nitrogen. Bromoacetyl
bromide (85 .mu.L, 0.98 mmol), dissolved in 10 mL tetrahydrofuran,
was added dropwise to the cold solution, keeping T=-5.degree. C.
The reaction was stirred in the cold for 1.5 hr, and 25 .mu.L
methanol added. The solids were filtered and rinsed and the
combined filtrate concentrated to a brown oil, which was purified
by flash chromatography (dichloromethane/ethyl acetate) to afford
the product (388 mg, 78%) as a light tan oil. LRMS (ES):
609.3/611.3 [M+H].sup.+, 631.3/633.3 [M+Na].sup.+, 185.3, 144.2.
.sup.1HNMR (600.1343 MHz, CDCl.sub.3): 7.32 (m, 1H), 7.09 (m, 1H),
4.50 (m, 2H), 4.21 (m, 2H), 3.87 (m, 2H), 2.31 (m, 2H), 2.13 (m,
2H), 1.99 (m, 2H), 1.97 (m, 2H), 1.45 (s, 9H), 1.43 (s, 9H), 1.01
(m, 2H), 0.04 (s, 9H).
[0483] Step 7I: Synthesis of
(S,S)-4-((tert-butyl)oxycarbonyl)-2-(4-((tert-
-butyl)oxycarbonyl)-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(((tert-butyl)oxyca-
rbonyl)methyl)cyclododecyl)acetylamino) butanoylamino)butanoic acid
66
[0484] The product of step 7H (214 mg, 0.416 mmol) was dissolved in
dimethylformamide (3 mL) and added to a solution of triethylamine
(250 .mu.L) and DO3A tri-tert-butyl ester in dimethylformamide
(3mL). The reaction was stirred for 4 days at room temperature,
concentrated, and the residue dissolved in ethyl acetate. This was
washed with water and brine, dried, and concentrated to an oil
which was not further purified but reacted directly with
tetra-butylammonium fluoride (1.0M in tetrahydrofuran, 1.25 mL) in
tetrahydrofuran (2.5 mL). After stirring for 2 hours, the reaction
was treated with ether (50 mL) and water (50 mL) and the layers
separated. The aqueous layer was extracted with three portions of
ethyl acetate, and the combined organic layers concentrated to an
oil. This was purified by preparative HPLC (0.1% trifluoroacetic
acid/acetonitrile, Zorbax C-8, 21.5 mm.times.25 cm) and the product
fractions lyophilized to afford 127 mg (32% for two steps) of the
product as a white solid. LRMS (ES): 943.3 [M+H].sup.+, 887.2,
831.2, 775.5, 719.3, 663.2 (loss of 1-5 tert-butyl) 444.3, 416.2,
388.3, 360.1, 332.1 [M-(1-5 tert butyl) +2H].sup.+2. .sup.1HNMR
(600.1343 MHz, CDCl.sub.3): 9.05 (b, 1H), 8.2 (b, 4H) 7.36 (b, 1H),
4.34 (m, 2H), 2.77-4.23 (very broad humps, 24H), 2.31 (m, 4H), 2.13
(m, 2H), 1.93 (m, 2H), 1.47 (d, 18H), 1.43 (m, 27H).
Example 8
Synthesis of
(S,S,S,S,S,S,S,S,S,S)-2-(4-(N-(1,3-bis(N-(3-(N-(3-(3,6-diaza--
10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)met-
hyl)-4-oxobicyclo
[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-1-(-
methoxycarbonyl)propyl)carbamoyl)propyl)carbamoyl)propyl)carbamoyl)-4-(2-(-
2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)
cyclodecyl)acetylamino)-4--
carboxybutanoylamino)-4-carboxybutanoylamino)butanoylamino)-4-(N-(3-(3,6-d-
iaza-10-(N-(benzimidazol-2-ylmethyl)-N-methyl
carbamoyl)-5-((methoxycarbon-
yl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl-
)butanoic acid
[0485] 67
[0486] Step 8: Synthesis of ditert-butyl (S,S)-2-(4-((tert-butyl)
oxycarbonyl)-2-((phenylmethoxy)carbonylamino)butanoylamino)
pentane-1,5-dioate 68
[0487] Gamma-tert-butyl-N-carbobenzyloxyglutamic acid
N-hydroxy-succinimide ester is dissolved in DMF with
diisopropylethylamine. Bis(tert-butyl)glutamate hydrochloride is
added and the reaction stirred for one hour. The reaction is
concentrated, water added, and the mixture extracted with ethyl
acetate. The combined organic layers are washed with water, 10%
potassium hydrogen sulfate, and brine, and then concentrated. The
product is purified by flash chromatography. Step 8B: Synthesis of
tert-butyl methyl
(S,S,S,S,S)-2-(4-(N-(1,3-bis(N-(3-((tert-butyl)oxycarbonyl)-1-(methoxycar-
bonyl) propyl)carbamoyl)propyl)carbamoyl)-4-((phenylmehtoxycarbonyl
amino)butanoylamino)pentane-1,5-dioate 69
[0488] The product of 8a is dissolved in one volume of
dichloromethane and treated with excess triethylsilane and one
volume of trifluoroacetic acid. The reaction is stirred under
nitrogen for three hours and then concentrated to an oil. The
triacid residue is dissolved in dimethylformamide and treated with
excess gamma-tert-butyl-alpha-methyl glutamate, HBTU, HOBT, and
diisopropylethylamine with stirring under nitrogen for 4-5 hours.
The reaction is concentrated, partitioned into water/ethyl acetate
and extracted with more ethyl acetate. The combined organics are
washed with water and brine and concentrated to an oil, which is
purified by flash chromatography using dichloromethane/ethyl
acetate/methanol.
[0489] Step 8C: Synthesis of methyl
(S,S,S,S,S,S,S,S)-4-(N-(3-(3,6-diaza-1-
0-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)meth-
yl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)carbamoyl)-2-(4--
(N-(1,3-bis(N-(3-(N-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcar-
bamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-tr-
ien-3-yl)propyl)carbamoyl)-1-(methoxycarbonyl)propyl)
carbamoyl)propyl)carbamoyl)-4-((phenylmethoxy)carbonyl
amino)butanoylamino)butanoate 70
[0490] The product of 8b is dissolved in one volume of
dichloromethane and treated with excess triethylsilane and one
volume of trifluoroacetic acid. The reaction is stirred under
nitrogen for three hours and then concentrated to an oil.
[0491] A threefold excess of the product of step 1F is treated in
the same fashion with trifluoroacetic acid and triethylsilane and
concentrated to an oil. The two residues are dissolved in
dimethylformamide, combined, and treated with HBTU, HOBT, and
diisopropylethylamine with stirring under nitrogen, following
disappearance of starting material by HPLC. When complete, the
reaction is concentrated, partitioned into water/ethyl acetate and
extracted with more ethyl acetate. The combined organics are washed
with water and brine and concentrated to an oil, which is purified
by preparative HPLC using a 0.1% trifluoroacetic acid/acetonitrile
gradient to afford the product as a powder after lyophilization.
Step 8D: Synthesis of methyl
(S,S,S,S,S,S,S,S)-2-(4-amino-4-(N-(1,3-bis(N-(3-(N-(3-
-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxy-
carbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propyl)car-
bamoyl)-1-(methoxycarbonyl)
propyl)carbamoyl)propyl)carbamoyl)butanoylamin-
o)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-
-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)-
propyl)carbamoyl)butanoate 71
[0492] The product of step 8C is dissolved in methanol with 10%
palladium on carbon and 2 equivalents of acetic acid in a Parr
bottle. The mixture is hydrogenated at 55 psi in a Parr shaker,
following by HPLC until all the starting material has been reacted.
The reaction is filtered through Celite, concentrated, and the
residual oil lyophilized from water/acetonitrile to yield the
product as a powder, to be used directly in the next step.
[0493] Step 8E: Conjugation of 8D with 7I 72
[0494] The product of step 8D is reacted with the product of step
7I as described in the alternate synthesis of 7E to afford the
product as a solid after preparative HPLC purification and
lyophilization.
[0495] Step 8F: Synthesis of
(S,S,S,S,S,S,S,S,S,S)-2-(4-(N-(1,3-bis(N-(3-(-
N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((met-
hoxycarbonyl)methyl)-4-oxobicyclo
[5.4.0]undeca-1(7),8,10-trien-3-yl)propy-
l)carbamoyl)-1-(methoxycarbonyl)propyl)carbamoyl)propyl)carbamoyl)propyl)c-
arbamoyl)-4-(2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)
cyclodecyl)acetylamino)-4-carboxybutanoylamino)-4-carboxybutanoylamino)bu-
tanoylamino)-4-(N-(3-(3,6-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methyl
carbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,1-
0-trien-3-yl)propyl)carbamoyl)butanoic acid 73
[0496] The product of step 8E is dissolved in 2:1
methanol/tetrahydrofuran and excess lithium hydroxide (3M solution)
added. The solution is stirred, following by HPLC, until all the
methyl esters have been hydrolyzed. The reaction is quenched with
solid citric acid, concentrated, and redissolved in one volume of
dichloromethane. The solids are filtered and the filtrate treated
with excess triethylsilane and one volume of trifluoroacetic acid.
The solution is stirred under nitrogen, following by HPLC, until
all of the tert-butyl esters have been hydrolyzed. The reaction
mixture is concentrated and directly purified by preparative HPLC
using 0.1% formic acid/acetonitrile gradient on a Zorbax C-8 column
to afford the product after lyophilization.
Example 9
Preparation of
(S)-2-(2,5-diaza-5-(3-(2-(2-(3-((6-((1-aza-2-(2-sulfophenyl-
)vinyl)amino)(3-pyridyl))carbonylamino)
propoxy)ethoxy)ethoxy)propyl)-9-(N-
-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxobicyclo[5.4.0]undeca-1(-
7),8,10-trien-3-yl)acetic acid
[0497] 74
[0498] Step 9A: Synthesis of N-(3-(2-(2-(3-aminopropoxy)ethoxy)
ethoxy)propyl)(tert-butoxy)formamide 75
[0499] A solution of at least three equivalents of
4,7,10-trioxa-1,13-trid- ecanediamine in tetrahydrofuran is cooled
to 0.degree. C., and a solution of one equivalent of di-tert-butyl
dicarbonate in acetonitrile is added dropwise with stirring. The
solution is stirred under nitrogen overnight and then concentrated.
The residue is dissolved in ether and washed with five portions of
saturated sodium chloride. The organic layer is dried over
magnesium sulfate, filtered and concentrated to an oil, which is
purified by flash chromatography to afford the monoamine.
[0500] Step 9B: Synthesis of tert-butyl
3-(((3-(2-(2-(3-((tert-butoxy)carb-
onylamino)propoxy)ethoxy)ethoxy)propyl)amino)methyl)-4-fluorobenzoate
76
[0501] The product of step 9A is treated with crude
tert-butyl-4-fluoro-3(alpha-bromomethyl)benzoate, as described in
step 1A, to afford the product after flash chromatography.
[0502] Step 9C: Synthesis of methyl
(S)-3-(N-(3-(2-(2-(3-((tert-butoxy)car- bonyl
amino)propoxy)ethoxy)ethoxy)propyl)-N-((5-((tert-butyl)oxy
carbonyl)-2-fluorophenyl)methyl)carbamoyl)-3-((phenylmethoxy)
carbonylamino)propanoate 77
[0503] The product of step 9B is treated with Z-aspartic
acid-.beta.-methyl ester as described in step 1B, to afford the
product after flash chromatography.
[0504] Step 9D: Synthesis of methyl
(S)-3-amino-3-(N-(3-(2-(2-(3-((tert-bu-
toxy)carbonylamino)propoxy)ethoxy)ethoxy)propyl)-N-((5-((tert-butyl)oxycar-
bonyl)-2-fluorophenyl)methyl)carbamoyl)propanoate 78
[0505] The product of step 9C is treated as in step 1C, and used
directly in the following step.
[0506] Step 9E: Synthesis of methyl
(S)-2-(2,5-diaza-9-((tert-butyl)
oxycarbonyl-5-(3-(2-(2-(3-((tert-butoxy)carbonylamino)
propoxy)ethoxy)ethoxy)propyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-
-yl)acetate 79
[0507] The product of step 9D is treated as in step 1D, to afford
the product after flash chromatography.
[0508] Step 9F: Synthesis of
(S)-2,5-diaza-5-(3-(2-(2-(3-((tert-butoxy)car-
bonylamino)propoxy)ethoxy)ethoxy)propyl)-3-((methoxycarbonyl)methyl)-4-oxo-
bicyclo[5.4.0]undeca-1(7),8,10-trien-9-carboxylic acid 80
[0509] The product of step 9E is treated as in step 1E, to afford
the product after flash chromatography.
[0510] Step 9G: Synthesis of methyl
(S)-2-(2,5-diaza-9-(N-(benzimidazol-2--
ylmethyl)-N-methylcarbamoyl)-5-(3-(2-(2-(3-((tert-butoxy)
carbonylamino)propoxy)ethoxy)ethoxy)propyl)-4-oxobicyclo
[5.4.0]undeca-1(7),8,10-trien-3-yl)acetate 81
[0511] The product of step 9F is treated as in step 1F, to afford
the product after flash chromatography.
[0512] Step 9H: Synthesis of
(S)-2-(2,5-diaza-5-(3-(2-(2-(3-((6-((1-aza-2--
(2-sulfophenyl)vinyl)amino)(3-pyridyl))carbonylamino)
propoxy)ethoxy)ethoxy)propyl)-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarb-
amoyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic acid
82
[0513] The product of step 9G is treated as in step 2G, and the
isolated residue then directly treated as in step 2H to afford the
product after preparative HPLC and lyophilization.
Example 10
Preparation of
(S,S,S,S,S)-4-(N-(1,3-bis(N-(3-(2-(2-(3-(3,6-diaza-10-(N-(b-
enzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[-
5.4.0]undeca-1(7),8,10-trien-3-yl)propoxy)-ethoxy)ethoxy)propyl)carbamoyl)
propyl)carbamoyl)-4-(5,5-dihydroxy-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(ca-
rboxy methyl)cyclododecyl)acetylamino) hexanoylamino)butanoic
acid
[0514] 83
[0515] Step 10A: Synthesis of methyl
(S)-2-(5-(3-(2-(2-(3-aminopropoxy)eth-
oxy)ethoxy)propyl)-2,5-diaza-9-(N-(benzimidazol
-2-ylmethyl)-N-methylcarba-
moyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetate 84
[0516] The product of step 9G is treated with trifluoroacetic acid
and triethylsilane in dichloromethane for 30 minutes and the
reaction then concentrated to an oil. Toluene is added and the
solution reconcentrated to an oil, which is used directly in the
next step.
[0517] Step 10B: Synthesis of
(S,S,S,S,S)-4-(N-(1,3-bis(N-(3-(2-(2-(3-(3,6-
-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl-
)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)propoxy)ethoxy)ethoxy)pro-
pyl)carbamoyl)propyl)carbamoyl)-4-(5,5-dihydroxy-2-(2-(1,4,7,10-tetraaza-4-
,7,10-tris(carboxy
methyl)cyclododecyl)acetylamino)hexanoylamino)butanoic acid 85
[0518] The product of step 10A is treated in several steps as
defined in example 7, steps 7A-7F, substituting step 10A product
for step 1I product as a starting material in step 7A. The product
is obtained as a solid after preparative HPLC purification and
lyophilization.
Example 11
Synthesis of
(S,S,S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylc-
arbamoyl)-4-oxo-5-(6-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoy-
l)-2-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)-2-(2-(1,4,7,1-
0-tetraaza-4,7,10-tris(carboxymethyl)cyclodecyl)
acetylamino)butanoylamino-
)butanoylamino)hexyl)bicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic
acid
[0519] 86
[0520] Step 11A: Synthesis of tert-butyl methyl
(S,S)-2-(4-((tert-butyl)ox-
ycarbonyl)-2-((phenylmethoxy)carbonylamino)
butanoylamino)pentane-1,5-dioa- te 87
[0521] This process is carried out as in step 1G, except starting
with alpha-methyl-gamma-tert-butylglutamate.
[0522] Step 11B: Synthesis of methyl
(S,S)-4-(N-((R,S,S,S)-2,3,4,5,6-penta- hydroxy
hexyl)carbamoyl)-2-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)ca-
rbamoyl)-2-((phenylmethoxy)carbonylamino) butanoylamino)butanoate
88
[0523] The product of step 11A is dissolved in dichloromethane,
followed by addition of trifluoroacetic acid (to form a 35%
solution). This is stirred under nitrogen until the starting
material and monoacid have disappeared by HPLC, and then the
solution is concentrated. The residue is dissolved in
dimethylformamide along with 2.5 equivalents of
1-amino-1-deoxysorbitol, 2.5 equivalents of HBTU, 2 equivalents of
hydroxybenzotriazole hydrate, and 3 equivalents
diisopropylethylamine. The solution is stirred for two hours,
concentrated, and the residue purified by preparative HPLC.
[0524] Step 11C: Synthesis of
(S,S)-4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxy- hexyl)
carbamoyl)-2-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl-
)-2-((phenylmethoxy)carbonylamino) butanoylamino)butanoic acid
89
[0525] The product of step 11B is dissolved in
tetrahydrofuran/methanol (1:1) and treated with excess 3N aqueous
lithium hydroxide. The reaction is followed by HPLC for
disappearance of starting material. The reaction is concentrated,
diluted with additional water, and purified by passage down an
acidic ion exchange column. The product fractions are lyophilized
to afford the product as a solid.
[0526] Step 11D: Synthesis of methyl
(S,S,S)-2-(2,5-diaza-9-(N-(benzimidaz-
ol-2-ylmethyl)-N-methylcarbamoyl)-4-oxo-5-(6-(4-(N-((R,S,S,S)-2,3,4,5,6-pe-
ntahydroxyhexyl)carbamoyl)-2-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxy
hexyl)carbamoyl)-2-(phenylmethoxy)carbonylamino)butanoylamino)butanoylami-
no) hexyl)bicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetate 90
[0527] The product of step 2G is dissolved in dichloromethane and
stirred with trifluoroacetic acid and triethylsilane for 15
minutes. The solution is concentrated, and the residue dissolved in
dimethylformamide with the product of step 11C, HBTU,
hydroxybenzotriazole hydrate, and diisopropylethylamine. The
reaction is stirred, following by HPLC for disappearance of
starting materials. When complete, the solution is concentrated and
the residue purified by preparative HPLC. The product solutions are
lyophilized to afford the product.
[0528] Step 11E: Synthesis of methyl
(S,S,S)-2-(5-(6-(2-(2-amino-4(-(N-((R-
,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoyl)
butanoylamino)-4-(N-((R,S,S- ,S)-2,3,4,5,6-pentahydroxyhexyl)
carbamoyl)butanoylamino)hexyl)-2,5-diaza--
9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxo-bicyclo[5.4.0]unde-
ca-1(7),8,10-trien-3-yl)acetate 91
[0529] The product of step11D is treated as in step 11, to afford
the amine after concentration.
[0530] Step11F: Synthesis of
(S,S,S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylm-
ethyl)-N-methylcarbamoyl)-4-oxo-5-(6-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydro-
xyhexyl)carbamoyl)-2-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxyhexyl)carbamoy-
l)-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(((tert-butyl)oxycarbonyl)methyl)cyc-
lododecyl)
acetylamino)butanoylamino)butanoylamino)hexyl)bicyclo[5.4.0]und-
eca-1(7),8,10-trien-3-yl)acetic acid 92
[0531] The product of step11E is reacted as in step 1J to afford
the product after preparative HPLC purification.
[0532] Step 11G: Synthesis of
(S,S,S)-2-(2,5-diaza-9-(N-(benzimidazol-2-yl-
methyl)-N-methylcarbamoyl)-4-oxo-5-(6-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydr-
oxyhexyl)carbamoyl)-2-(4-(N-((R,S,S,S)-2,3,4,5,6-pentahydroxy
hexyl)carbamoyl)-2-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)cyclod-
ecyl)acetylamino)butanoylamino)
butanoylamino)hexyl)bicyclo[5.4.0]undeca-1-
(7),8,10-trien-3-yl)acetic acid 93
[0533] The product of step 11F is treated as in step 1K, to afford
the product after preparative HPLC purification.
Example 12
Synthesis of
(S,S,S,S)-2-(4-(N-(1-(N-(1-(N-(6-(3,6-diaza-10-(N-(benzimidaz-
ol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxobicyclo[5.4.0]und-
eca-1(7),8,10-trien-3-yl)hexyl)carbamoyl)-3-(N-cyclo{Lys-Arg(Mtr)-Gly-Asp(-
OtBu)-D-Phe}[gamma-LysNH]carbamoyl)propyl)carbamoyl)-3-carboxypropyl)
carbamoyl)-4-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)
cyclododecyl)acetylamino)butanoic acid
[0534] 94
[0535] Step 12A: Synthesis of
H-Asp(OtBu)-D-Phe-Lys(Cbz)-Arg(Mtr)-Gly-OH This peptide is prepared
using an Advanced Chemtech Model 90 synthesizer using standard Fmoc
protocols. The starting resin is
4-[4-hydroxymethyl)-3-methoxy-phenoxy]butanoyl benzhydrylamine
resin preloaded with Fmoc-glycine (Fmoc-Gly-HMPB-BHA). Synthesis of
the protected linear peptide is achieved through sequential
coupling (for 3 hrs) of the amino acids
N-alpha-Fmoc-N.sup.9-4-methoxy-2,3,6-trimethylben-
zenesulfonyl-1-arginine,
N-alpha-Fmoc-N-epsilon-benzyloxycarbonyl-L-lysine- ,
Fmoc-phenylalanine, and Fmoc-gamma-tert-butyl aspartic acid, using
HBTU and HOBT as coupling agents. The couplings are carried out
with five equivalents of amino acid, HBTU, HOBT, and
diisopropylethylamine in dimethylformamide. Fmoc deprotections are
accomplished with 20% piperidine in DMF for 30 minutes. The
protected linear peptide is cleaved from the resin with 1%
trifluoroacetic acid in dichloromethane and the peptide solution
collected in 10% pyridine in methanol. The crude peptide is
obtained by concentrating the solvents in vacuo and triturating
with diethyl ether. The peptide is purified by preparative HPLC and
the product fractions are lyophilized.
[0536] Step 12B: Synthesis of
cyclo{Lys(Cbz)-Arg(Mtr)-Gly-Asp(OtBu)-D-Phe} 95
[0537] HBTU (0.7 mmol) and hydroxybenzotriazole (0.5 mmol) are
dissolved in dimethylformamide (10 mL). The solution is warmed to
60.degree. C. under nitrogen and a solution of the product of step
12 A (0.4 g) and diisopropylethylamine (1.5 mmol) in
dimethylformamide (10 mL) added slowly. The solution is stirred at
this temperature for 4 hours under nitrogen. The solution is
concentrated and the residue triturated with ethyl acetate. The
resulting solids are washed with ethyl acetate and dried under
vacuum to afford the product, which is used directly in the next
step.
[0538] Step 12C: Synthesis of
cyclo{Lys-Arg(Mtr)-Gly-Asp(OtBu)-D-Phe} 96
[0539] The product of step 12 B is dissolved in 2-propanol and 10%
palladium on carbon added with stirring. Hydrogen gas is gently
bubbled into the reaction mixture until all of the starting
material is consumed by HPLC analysis. The reaction mixture is
filtered through a bed of Celite and the filtrate concentrated. The
residue is not further purified but used directly in the following
step.
[0540] Step 12D: Synthesis of tert-butyl
(S,S)-4-(N-(6-(3,6-diaza-10-(N-(b-
enzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-o-
xobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)hexyl)carbamoyl)-4-(((phenylme-
thoxy) carbonylamino)butanoate 97
[0541] The product of step 2F is dissolved in dichloromethane and
trifluoroacetic acid added (30% solution). The reaction is stirred
30 minutes and concentrated. The residue is dissolved in
dimethylformamide and
N-carbobenzyloxy-gamma-tert-butyl-alpha-N-hydroxysuccinimidylglutamat-
e added, along with excess diisopropylethylamine. The reaction is
stirred for four hours and concentrated. The residue is purified by
preparative HPLC and the fractions lyophilized to afford the
product as a solid.
[0542] Step 12E: Synthesis of
(S,S)-4-(N-(6-(3,6-diaza-10-(N-(benzimidazol-
-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5-
.4.0]undeca-1(7),8,10-trien-3-yl)hexyl)carbamoyl)-4-(((phenylmethoxy)carbo-
nylamino) butanoyl-cyclo{Lys-Arg(Mtr)-Gly-Asp(OtBu)-D-Phe}
conjugate 98
[0543] The product of step 12D is dissolved in one volume of
dichloromethane, followed by one volume of trifluoroacetic acid na
5 equivalents of triethylsilane. The solution is stirred for four
hours and concentrated. The residue is dissolved in
dimethylformamide containing the product of step 12C, HBTU, and
hydroxybenzotriazole hydrate. Diisopropylethylamine is added to
this mixture with stirring under nitrogen, following by HPLC for
disappearance of the starting materials. When complete, the
reaction is concentrated and the residue purified by preparative
HPLC. The product fractions are combined and lyophilized.
[0544] Step 12F: Synthesis of
(S,S)-4-(N-(6-(3,6-diaza-10-(N-(benzimidazol-
-2-ylmethyl)-N-methylcarbamoyl)-5-((methoxycarbonyl)methyl)-4-oxobicyclo[5-
.4.0]undeca-1(7),8,10-trien-3-yl)hexyl)carbamoyl)-4-amino)butanoyl)-cyclo{-
Lys-Arg(Mtr)-Gly-Asp(OtBu)-D-Phe} conjugate 99
[0545] The product of step 12E is treated as in step 8D. The
product is not further purified, but used directly in the next
step.
[0546] Step 12G: Synthesis of tert-butyl
(S,S,S,S)-4-(N-(1-N-(1-(N-(6-(3,6-
-diaza-10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-4-oxobicyclo[5.4-
.0]undeca-1(7),8,10-trien-3-yl)hexyl)carbamoyl)-3-(N-cyclo{Lys-Arg(Mtr)-Gl-
y-Asp(OtBu)-D-Phe}carbamoyl)propyl)
carbamoyl-3-((tert-butyl)oxycarbonyl)p-
ropyl)carbamoyl)-4-(2-(1,4,7,10-tetraaza-4,7,10-tris(((tert-butyl)oxycarbo-
nyl) methyl)cyclcododecyl)acetylamino)butanoate 100
[0547] The product of step 12F is treated as in step 8E to afford
the product after preparative HPLC purification.
[0548] Step 12H: Synthesis of
(S,S,S,S)-2-(4-(N-(1-(N-(1-(N-(6-(3,6-diaza--
10-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(carboxymethyl)-4-oxo-
bicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)hexyl)carbamoyl)-3-(N-cyclo{Lys--
Arg(Mtr)-Gly-Asp(OtBu)-D-Phe}[gamma-LysNH]carbamoyl)propyl)carbamoyl)-3-ca-
rboxypropyl)carbamoyl)-4-(2-(1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)c-
yclcododecyl) acetylamino)butanoic acid 101
[0549] The product of step 12G is dissolved in tetrahydrofuran and
excess lithium hydroxide added as a 3N solution in water. The
solution is stirred under nitrogen, following by HPLC for
disappearance of starting material. When this is complete, the
reaction is acidified with 10% potassium hydrogen sulfate and
concentrated. The residue is dissolved in neat trifluoroacetic acid
containing thioanisole and stirred at room temperature under
nitrogen, following the multiple deprotections by HPLC, until
complete. The reaction is concentrated and the crude residue
purified by preparative HPLC.
Example 13
Preparation of sodium
1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamin-
e-(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(6--
aminohexyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic
acid-dodecoanoate conjugate
[0550] 102
[0551] Step 13A: Synthesis of sodium
1,2-dipalmitoyl-sn-glycero-3-phosphat-
idylethanolamine-(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl)-N-methylc-
arbamoyl)-5-(6-aminohexyl)-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)-
acetic acid-dodecoanoate conjugate
[0552] 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine monosodium
salt (DPPE) (1.25 g, 0.5 mmol) is dissolved under nitrogen in
chloroform (15 mL) along with disuccinimidyl dodecanoate (0.212 g,
0.5 mmol and the product of step 4A (367 mg, 0.5 mmol). They are
stirred for 5 minutes, when sodium carbonate (0.5 mmol) and sodium
sulfate (0.5 mmol) is added. The reaction is stirred 18 hrs,
filtered, and concentrated. The residue is purified to obtain the
title compound.
[0553] Step 13B: Preparation of contrast agent composition
[0554] The product of step 13A is admixed with three other lipids,
1,2-dipalmitoyl-sn-glycero-3-phosphotidic acid,
1,2-dipalmitoyl-sn-glycer- o-3-phosphatidyl choline, and
N-(methoxypolyethylene glycol
5000)carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine in
relative amounts of 1 wt %:6 wt %:54 wt %:41 wt %. An aqueous
solution of this lipid admixture (1 mg/mL), sodium chloride (7
mg/mL), glycerin (0.1 mg/mL), and propylene glycol (0.1 mL/mL) at
pH 6-7 is then prepared in a 2 cc glass vial. The air in the vial
is evacuated and replaced with perfluoropropane and the vial is
sealed. The ultrasound contrast agent composition is completed by
agitating the sealed vial in a dental amalgamator for 30-45 seconds
to form a milky white solution.
Example 14
Preparation of
DPPE-PEG.sub.3400-[(S)-2-(2,5-diaza-9-(N-(benzimidazol-2-yl-
methyl)-N-methylcarbamoyl)-5-(6-aminohexyl)-4-oxobicyclo[5.4.0]undeca-1(7)-
,8,10-trien-3-yl)acetic acid]-dodecoanoate conjugate
[0555] 103
[0556] Step 14A: Synthesis of
.omega.-amino-PEG.sub.3400-[(S)-2-(2,5-diaza-
-9-(N-(benzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(6-aminohexyl)-4-oxob-
icyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic acid]
[0557] A solution of N-Boc-.omega.-amino-PEG.sub.3400-succinimidyl
ester (1 mmol) and the product of step 4A (1 mmol) in DMF (15 mL)
is treated with diisopropylethylamine (3 mmol) and stirred under
nitrogen for 18 hr. The solution is concentrated and the residue
dissolved in dichloromethane (8 mL) to which trifluoroacetic acid
(6 mL) is added. The solution is stirred for 30 minutes, and then
concentrated under vacuum. The product is isolated by trituration
with diethyl ether.
[0558] Step 14B: Synthesis of
DPPE-PEG.sub.3400-[(S)-2-(2,5-diaza-9-(N-(be-
nzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(6-aminohexyl)-4-oxobicyclo[5.-
4.0]undeca-1(7),8,10-trien-3-yl)acetic acid]-dodecoanoate
conjugate
[0559] A solution of disuccinimidyl dodecanoate (0.5 mmol), DPPE
(0.5 mmol), and the product of step 14A (0.5 mmol) are added to 10
mL chloroform with stirring under nitrogen. Sodium carbonate (1
mmol) and sodium sulfate (1 mmol) are added and the solution is
stirred at room temperature for 18 hrs. The reaction is filtered,
the solvent concentrated, and the residue purified to obtain the
title compound.
[0560] Step 14C: Preparation of contrast agent composition The
product of step 14B is admixed with three other lipids,
1,2-dipalmitoyl-sn-glycero-3- -phosphotidic acid,
1,2-dipalmitoyl-sn-glycero-3-phosphatidyl choline, and
N-(methoxypolyethylene glycol
5000)carbamoyl)-1,2-dipalmitoyl-sn-glycero-- 3-phosphoethanolamine
in relative amounts of 1 wt %:6 wt %:54 wt %:41 wt %. An aqueous
solution of this lipid admixture (1 mg/mL), sodium chloride (7
mg/mL), glycerin (0.1 mg/mL), and propylene glycol (0.1 mL/mL) at
pH 6-7 is then prepared in a 2 cc glass vial. The air in the vial
is evacuated and replaced with perfluoropropane and the vial is
sealed. The ultrasound contrast agent composition is completed by
agitating the sealed vial in a dental amalgamator for 30-45 seconds
to form a milky white solution.
Example 15
Preparation of
[(S)-2-(2-aza-(2-((5-(N-(1,3-bis-N-(6-(aminohexyl-4-oxobicy-
clo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic
acid)(2-(2,5-diaza-9-(N-(benz- imidazol-2-ylmethyl) carbamoyl)
propyl) carbamoyl]-.omega.-amino-PEG.sub.3- 400-dodecanoate-DPPE
conjugate
[0561] 104
[0562] Step 15A: Synthesis of
[(S)-2-(2-aza-(2-((5-(N-(1,3-bis-N-(6-(amino-
hexyl-4-oxobicyclo[5.4.0]undeca-1(7),8,10-trien-3-yl)acetic acid)
(2-(2,5-diaza-9-(N-(benzimidazol-2-ylmethyl) carbamoyl)propyl)
carbamoyl]-.omega.-amino-PEG.sub.3400
[0563] The product of step 4B (1 mmol) was deprotected as described
in step 4C and added to a solution of
N-Boc-.omega.-amino-PEG.sub.3400-succi- nimidyl ester (1 mmol) in
DMF (15 mL). Diisopropylethylamine (3 mmol) is added and the
solution stirred under nitrogen for 18 hr. The solution is
concentrated and the residue dissolved in dichloromethane (8 mL) to
which trifluoroacetic acid (6 mL) is added. The solution is stirred
for 30 minutes, and then concentrated under vacuum. The product is
isolated by trituration with diethyl ether.
[0564] Step 15B: Synthesis of
DPPE-PEG.sub.3400-[(S)-2-(2,5-diaza-9-(N-(be-
nzimidazol-2-ylmethyl)-N-methylcarbamoyl)-5-(6-aminohexyl)-4-oxobicyclo[5.-
4.0]undeca-1(7),8,10-trien-3-yl)acetic acid]-dodecoanoate
conjugate
[0565] A solution of disuccinimidyl dodecanoate (0.5 mmol), DPPE
(0.5 mmol), and the product of step 15A (0.5 mmol) are added to 10
mL chloroform with stirring under nitrogen. Sodium carbonate (1
mmol) and sodium sulfate (1 mmol) are added and the solution is
stirred at room temperature for 18 hrs. The reaction is filtered,
the solvent concentrated, and the residue purified to obtain the
title compound.
[0566] Step 15C: Preparation of contrast agent composition
[0567] The product of step 15B is admixed with three other lipids,
1,2-dipalmitoyl-sn-glycero-3-phosphotidic acid,
1,2-dipalmitoyl-sn-glycer- o-3-phosphatidyl choline, and
N-(methoxypolyethylene glycol
5000)carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine in
relative amounts of 1 wt %:6 wt %:54 wt %:41 wt %. An aqueous
solution of this lipid admixture (1 mg/mL), sodium chloride (7
mg/mL), glycerin (0.1 mg/mL), and propylene glycol (0.1 mL/mL) at
pH 6-7 is then prepared in a 2 cc glass vial. The air in the vial
is evacuated and replaced with perfluoropropane and the vial is
sealed. The ultrasound contrast agent composition is completed by
agitating the sealed vial in a dental amalgamator for 30-45 seconds
to form a milky white solution.
Utility
[0568] The pharmaceuticals of the present invention are useful for
imaging angiogenic tumor vasculature in a patient or for treating
cancer in a patient. The radiopharmaceuticals of the present
invention comprised of a gamma emitting isotope are useful for
imaging of pathological processes involving angiogenic
neovasculature, including cancer, diabetic retinopathy, macular
degeneration, restenosis of blood vessels after angioplasty, and
wound healing. Diagnostic utilities also include imaging of
unstable coronary syndromes (e.g., unstable coronary plaque). The
radiopharmaceuticals of the present invention comprised of a beta,
alpha or Auger electron emitting isotope are useful for treatment
of pathological processes involving angiogenic neovasculature, by
delivering a cytotoxic dose of radiation to the locus of the
angiogenic neovasculature. The treatment of cancer is affected by
the systemic administration of the radiopharmaceuticals resulting
in a cytotoxic radiation dose to tumors.
[0569] The compounds of the present invention comprised of one or
more paramagnetic metal ions selected from gadolinium, dysprosium,
iron, and manganese, are useful as contrast agents for magnetic
resonance imaging (MRI) of pathological processes involving
angiogenic neovasculature.
[0570] The compounds of the present invention comprised of one or
more heavy atoms with atmic number of 20 or greater are useful as
X-ray contrast agents for X-ray imaging of pathological processes
involving angiogenic neovasculature.
[0571] The compounds of the present invention comprised of an
echogenic gas containing surfactant microsphere are useful as
ultrasound contrast agents for sonography of pathological processes
involving angiogenic neovasculature.
[0572] Representative compounds of the present invention were
tested in the following in vitro assays and in vivo models and were
found to be active.
Immobilized Human Placental avb3 Receptor Assay
[0573] The assay conditions were developed and validated using
[I-125]vitronectin. Assay validation included Scatchard format
analysis (n=3) where receptor number (Bmax) and Kd (affinity) were
determined. Assay format is such that compounds are preliminarily
screened at 10 and 100 nM final concentrations prior to IC50
determination. Three standards (vitronectin, anti-avB3 antibody,
LM609, and anti-avB5, P1F6) and five reference peptides have been
evaluated for IC50 determination. Briefly, the method involves
immobilizing previously isolated receptors in 96 well plates and
incubating overnight. The receptors were isolated from normal,
fresh, non-infectious (HIV, hepatitis B and C, syphilis, and HTLV
free) human placenta. The tissue was lysed and tissue debris
removed via centrifugation. The lysate was filtered. The receptors
were isolated by affinity chromatography using the immobilized avb3
antibody. The plates are then washed 3.times. with wash buffer.
Blocking buffer is added and plates incubated for 120 minutes at
room temperature. During this time compounds to be tested and
[I-125]vitronectin are premixed in a reservoir plate. Blocking
buffer is removed and compound mixture pipetted. Competition is
carried out for 60 minutes at room temperature. Unbound material is
then removed and wells are separated and counted via gamma
scintillation.
Oncomouse.RTM. Imaging
[0574] The study involves the use of the c-Neu Oncomouse.RTM. and
FVB mice simultaneously as controls. The mice are anesthetized with
sodium pentobarbital and injected with approximately 0.5 mCi of
radiopharmaceutical. Prior to injection, the tumor locations on
each Oncomouse.RTM. are recorded and tumor size measured using
calipers. The animals are positioned on the camera head so as to
image the anterior or posterior of the animals. 5 Minute dynamic
images are acquired serially over 2 hours using a 256.times.256
matrix and a zoom of 2.times.. Upon completion of the study, the
images are evaluated by circumscribing the tumor as the target
region of interest (ROI) and a background site in the neck area
below the carotid salivary glands.
[0575] This model can also be used to assess the effectiveness of
the radiopharmaceuticals of the present invention comprised of a
beta, alpha or Auger electron emitting isotope. The
radiopharmaceuticals are administered in appropriate amounts and
the uptake in the tumors can be quantified either non-invasively by
imaging for those isotopes with a coincident imageable gamma
emission, or by excision of the tumors and counting the amount of
radioactivity present by standard techniques. The therapeutic
effect of the radiopharmaceuticals can be assessed by monitoring
the rate of growth of the tumors in control mice versus those in
the mice administered the radiopharmaceuticals of the present
invention.
[0576] This model can also be used to assess the compounds of the
present invention comprised of paramagnetic metals as MRI contrast
agents. After administration of the appropriate amount of the
paramagnetic compounds, the whole animal can be placed in a
commercially available magnetic resonance imager to image the
tumors. The effectiveness of the contrast agents can be readily
seen by comparison to the images obtain from animals that are not
administered a contrast agent.
[0577] This model can also be used to assess the compounds of the
present invention comprised of heavy atoms as X-ray contrast
agents. After administration of the appropriate amount of the X-ray
absorbing compounds, the whole animal can be placed in a
commercially available X-ray imager to image the tumors. The
effectiveness of the contrast agents can be readily seen by
comparison to the images obtain from animals that are not
administered a contrast agent.
[0578] This model can also be used to assess the compounds of the
present invention comprised of an echogenic gas containing
surfactant microsphere as ultrasound contrast agents. After
administration of the appropriate amount of the echogenic
compounds, the tumors in the animal can be imaging using an
ultrasound probe held proximate to the tumors. The effectiveness of
the contrast agents can be readily seen by comparison to the images
obtain from animals that are not administered a contrast agent.
Rabbit Matrigel Model
[0579] This model was adapted from a matrigel model intended for
the study of angiogenesis in mice. Matrigel (Becton &
Dickinson, USA) is a basement membrane rich in laminin, collagen
IV, entactin, HSPG and other growth factors. When combined with
growth factors such as bFGF [500 ng/ml] or VEGF [2 .mu.g/ml] and
injected subcutaneously into the mid-abdominal region of the mice,
it solidifies into a gel and stimulates angiogenesis at the site of
injection within 4-8 days. In the rabbit model, New Zealand White
rabbits (2.5-3.0 kg) are injected with 2.0 ml of matrigel, plus 1
.mu.g bFGF and 4 .mu.g VEGF. The radiopharmaceutical is then
injected 7 days later and the images obtained.
[0580] This model can also be used to assess the effectiveness of
the radiopharmaceuticals of the present invention comprised of a
beta, alpha or Auger electron emitting isotope. The
radiopharmaceuticals are administered in appropriate amounts and
the uptake at the angiogenic sites can be quantified either
non-invasively by imaging for those isotopes with a coincident
imageable gamma emission, or by excision of the angiogenic sites
and counting the amount of radioactivity present by standard
techniques. The therapeutic effect of the radiopharmaceuticals can
be assessed by monitoring the rate of growth of the angiogenic
sites in control rabbits versus those in the rabbits administered
the radiopharmaceuticals of the present invention.
[0581] This model can also be used to assess the compounds of the
present invention comprised of paramagnetic metals as MRI contrast
agents. After administration of the appropriate amount of the
paramagnetic compounds, the whole animal can be placed in a
commercially available magnetic resonance imager to image the
angiogenic sites. The effectiveness of the contrast agents can be
readily seen by comparison to the images obtain from animals that
are not administered a contrast agent.
[0582] This model can also be used to assess the compounds of the
present invention comprised of heavy atoms as X-ray contrast
agents. After administration of the appropriate amount of the X-ray
absorbing compounds, the whole animal can be placed in a
commercially available X-ray imager to image the angiogenic sites.
The effectiveness of the contrast agents can be readily seen by
comparison to the images obtain from animals that are not
administered a contrast agent.
[0583] This model can also be used to assess the compounds of the
present invention comprised of an echogenic gas containing
surfactant microsphere as ultrasound contrast agents. After
administration of the appropriate amount of the echogenic
compounds, the angiogenic sites in the animal can be imaging using
an ultrasound probe held proximate to the tumors. The effectiveness
of the contrast agents can be readily seen by comparison to the
images obtain from animals that are not administered a contrast
agent.
Canine Spontaneous Tumor Model
[0584] Adult dogs with spontaneous mammary tumors were sedated with
xylazine (20 mg/kg)/atropine (1 ml/kg). Upon sedation the animals
were intubated using ketamine (5 mg/kg)/diazepam (0.25 mg/kg) for
full anethesia. Chemical restraint was continued with ketamine (3
mg/kg)/xylazine (6 mg/kg) titrating as necessary. If required the
animals were ventilated with room air via an endotrachael tube (12
strokes/min, 25 ml/kg) during the study. Peripheral veins were
catheterized using 20G I.V. catheters, one to serve as an infusion
port for compound while the other for exfusion of blood samples.
Heart rate and EKG were monitored using a cardiotachometer
(Biotech, Grass Quincy, Mass.) triggered from a lead II
electrocardiogram generated by limb leads. Blood samples are
generally taken at .about.10 minutes (control), end of infusion, (1
minute), 15 min, 30 min, 60 min, 90 min, and 120 min for whole
blood cell number and counting. Radiopharmaceutical dose was 300
.mu.Ci/kg adminitered as an i.v. bolus with saline flush.
Parameters were monitored continuously on a polygraph recorder
(Model 7E Grass) at a paper speed of 10 mm/min or 10 mm/sec.
[0585] Imaging of the laterals were for 2 hours with a
256.times.256 matrix, no zoom, 5 minute dynamic images. A known
source is placed in the image field (20-90 .mu.pCi) to evaluate
region of interest (ROI) uptake. Images were also acquired 24 hours
post injection to determine retention of the compound in the tumor.
The uptake is determined by taking the fraction of the total counts
in an inscribed area for ROI/source and multiplying the known
.mu.Ci. The result is .mu.Ci for the ROI.
[0586] This model can also be used to assess the effectiveness of
the radiopharmaceuticals of the present invention comprised of a
beta, alpha or Auger electron emitting isotope. The
radiopharmaceuticals are administered in appropriate amounts and
the uptake in the tumors can be quantified either non-invasively by
imaging for those isotopes with a coincident imageable gamma
emission, or by excision of the tumors and counting the amount of
radioactivity present by standard techniques. The therapeutic
effect of the radiopharmaceuticals can be assessed by monitoring
the size of the tumors over time.
[0587] This model can also be used to assess the compounds of the
present invention comprised of paramagnetic metals as MRI contrast
agents. After administration of the appropriate amount of the
paramagnetic compounds, the whole animal can be placed in a
commercially available magnetic resonance imager to image the
tumors. The effectiveness of the contrast agents can be readily
seen by comparison to the images obtain from animals that are not
administered a contrast agent.
[0588] This model can also be used to assess the compounds of the
present invention comprised of heavy atoms as X-ray contrast
agents. After administration of the appropriate amount of the X-ray
absorbing compounds, the whole animal can be placed in a
commercially available X-ray imager to image the tumors. The
effectiveness of the contrast agents can be readily seen by
comparison to the images obtain from animals that are not
administered a contrast agent.
[0589] This model can also be used to assess the compounds of the
present invention comprised of an echogenic gas containing
surfactant microsphere as ultrasound contrast agents. After
administration of the appropriate amount of the echogenic
compounds, the tumors in the animal can be imaging using an
ultrasound probe held proximate to the tumors. The effectiveness of
the contrast agents can be readily seen by comparison to the images
obtain from animals that are not administered a contrast agent.
[0590] Obviously, numerous modifications and variations of the
present invention are possible in light of the above teachings. It
is therefore to be understood that within the scope of the appended
claims, the invention may be practiced otherwise that as
specifically described herein.
* * * * *