COVALENTLY BINDING IMAGING PROBES
The present invention relates to molecular probes of the formula (I) L1-R1-L-A-X (I) as defined herein that allow for the observation of the catalytic activity of a selected caspase, cathepsin, MMP and carboxypeptidase in in vitro assays, in cells or in multicellular organisms, a method for their preparation and the use thereof.
1 . A molecular probe for proteases of the formula (I)
L1R1-L-A-X (I)
wherein
X is an electrophilic warhead; or X is a hydrogen;
A is a group recognizable by a protease;
R1 is a linker;
L is a bond or a group allowing for a facile conjugation of the group R1, and
L1 is a label optionally bound to a solid support.
2 . The molecular probe according to claim 1 , wherein the protease is a cysteine protease from the cathepsin or caspase subfamily, or a metalloprotease from the MMP or carboxypeptidase subfamily.
3 . The molecular probe according to claim 2 , wherein the cysteine protease from the cathepsin or caspase subfamily is selected from cathepsin K, cathepsin S, cathepsin B, caspase-1, caspase-3 or caspase-8, and the metalloprotease from the MMP or carboxypeptidase subfamily is selected from MMP-13 or TAFI.
4 . The molecular probe according to claim 1 , wherein L is a direct bond or a group selected from
—(NRx)-, —O—, —C═N—, —C(═O)—, —C(═O)—NH—, —NH—C(═O)—, —C(═O)H, —CRx=CRy-, —C≡C— and phenyl, wherein Rx and Ry are independently H or (C 1 -C 6 )alkyl.
5 . The molecular probe according to claim 1 , wherein
R1 is a straight or branched chain alkylene group with 1 to 300 carbon atoms, wherein optionally
(a) one or more carbon atoms are replaced by oxygen;
(b) one or more carbon atoms are replaced by nitrogen carrying a hydrogen atom, and the adjacent carbon atoms are substituted by oxo, representing an amide function —NH—CO—;
(c) one or more carbon atoms are replaced by an ester function —O—CO—;
(d) the bond between two adjacent carbon atoms is a double or a triple bond; or
(e) two adjacent carbon atoms are replaced by a disulfide linkage,
or a combination thereof.
6 . The molecular probe according to claim 5 , wherein when
(a) one or more carbon atoms are replaced by oxygen, then every third carbon atom is replaced by oxygen.
7 . The molecular probe according to claim 6 , wherein R1 is a polyethyleneoxy group with 1 to 100 ethyleneoxy units;
8 . The molecular probe according to claim 1 , wherein R1 is alkyl or a straight or branched chain alkylene group with 1 to 50 carbon atoms, wherein one or more carbon atoms are replaced by oxygen.
9 . The molecular probe according to claim 8 , wherein every third carbon atom is replaced by oxygen.
10 . The molecular probe according to claim 9 , wherein R1 is a polyethyleneoxy group with 1 to 20 ethyleneoxy units.
11 . The molecular probe according to claim 1 , wherein L1 is a spectroscopic probe; a chromophore; a magnetic probe; a contrast reagent; a molecule which is one part of a specific binding pair which is capable of specifically binding to a partner; a molecule covalently attached to a solid support, where the support may be a glass slide, a microtiter plate or any polymer known to those proficient in the art; a biomolecule with desirable enzymatic, chemical or physical properties; or a molecule possessing a combination of any of the properties listed above; or a positively charged linear or branched polymer.
12 . The molecular probe according to claim 11 , wherein L1 is a spectroscopic probe which is a fluorophore
13 . The molecular probe according to claim 1 , wherein X is an electrophilic warhead.
14 . The molecular probe according to claim 13 , wherein X is a group selected from
wherein R=alkyl, aryl.
15 . The molecular probe according to claim 1 , selected from one of the compounds 1.-61.:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
wherein independently of each other the variables in the compounds 1. to 61. are defined as indicated in the definition next to the respective compound; Y is -L-R1-L1; and R1, L and L1 are as described in claim 1 ; R is H or (C 1 -C 6 )-alkyl;
or W═X,
wherein X is defined as a group selected from
and R′ is H; or C 1 -C 6 -alkyl optionally substituted by
OH, —O—C 1 -C 6 -alkyl, —O—(C═O)—O—C 1 -C 6 -alkyl, SH, —S—C 1 -C 6 -alkyl, NH 2 , —NH—C 1 -C 6 -alkyl, —N(C 1 -C 6 -alkyl) 2 , —(C═O)—NH—C 1 -C 6 -alkyl, —COOH, —C(═O)O—C 1 -C 6 -alkyl or —NH—C(C═O)—C 1 -C 6 -alkyl; or aryl.
16 . The molecular probe according to claim 15 wherein the aryl optional substituent group is a phenyl.
17 . The molecular probe according to claim 1 , selected from one of the compounds 62.-114.:
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
82.
83.
84.
85.
86.
87.
88.
89.
90.
91.
92.
93.
94.
95.
96.
97.
98.
99.
100.
101.
102.
103.
104.
105.
106.
107.
108.
109.
110.
111.
112.
113.
114.
wherein independently of each other the variables in the compounds 62. to 114. are defined as indicated in the definition next to the respective compound; Y is -L-R1-L1; and R1, L and L1 are as described in claim 1 ;
or W═X,
wherein X is defined as a group selected from
and R′ is H; or C 1 -C 6 -alkyl optionally substituted by
OH, —O—C 1 -C 6 -alkyl, —O—(C═O)—O—C 1 -C 6 -alkyl, SH, —S—C 1 -C 6 -alkyl, NH 2 , —NH—C 1 -C 6 -alkyl, —N(C 1 -C 6 -alkyl) 2 , —(C═O)—NH—C 1 -C 6 -alkyl, —COOH, —C(═O)O—C 1 -C 6 -alkyl or —NH—C(C═O)—C 1 -C 6 -alkyl; or aryl.
18 . The molecular probe according to claim 17 wherein the aryl optional substituent group is a phenyl.
19 . The molecular probe according to claim 1 , selected from one of the compounds 115.-116.:
115.
wherein R2 is a halogen and R3 is COOH or H.
116.
wherein R2 is COOH or H.
wherein Y is -L-R1-L1; and R1, L and L1 are as described in claim 1 .
20 . The molecular probe according to claim 1 , selected from one of the compounds 117.-142.:
117.
117a.
118.
118a.
119.
wherein
R is (C 1 -C 5 )alkyl, phenyl, (C 5 -C 6 )cycloalkyl;
R2 is alkyl; and
n and m are independently of each other 0-3.
120.
121.
122.
wherein n is 0 or 1;
123.
124.
wherein
n is 1-4;
m is 1 or 2; and
R is methyl or methoxy.
125.
wherein n is 1-4;
126.
wherein n is 1-4;
127.
128.
129.
130.
wherein
U is S or S(O) 2 , and
Ar is aryl or heteroaryl;
131.
wherein
Ar is an aryl or heteroaryl group selected from phenyl,
benzothiophene, isoquinolyl, cinnamyl, naphthyl, which is
optionally once or independently twice substituted by
methoxy, chloro, methyl, CF 3 , and
wherein
means either a single or a double bond
132.
133.
134.
135
136.
137.
138.
139.
wherein
Ar is aryl or heteroaryl;
U is CH 2 , O or NR9 wherein R9 is hydrogen or (C 1 -C 6 )alkyl,
aryl, heteroaryl, heterocyclyl;
R 2a , R 2a′ , R 2b and R 2b′ are each independently hydrogen, hydroxyl,
N(R 6 ) 2 , halogen, (C 1 -C 4 )alkyl, (C 1 -C 4 )alkoxy, and mixtures
thereof wherein R 6 is hydrogen, (C 1 -C 6 )alkyl, cycloalkyl,
(C 6 -C 10 )aryl; or R 2a and R 2b can taken together to form a double
bond;
139a.
Compound 139. in the all-(S) configuration;
140.
wherein
Ar is aryl or heteroaryl;
R 2a , R 2a′ , R 2b and R 2b′ are each independently hydrogen, hydroxyl,
N(R 6 ) 2 , halogen, (C 1 -C 4 )alkyl, (C 1 -C 4 )alkoxy, and mixtures
thereof wherein R 6 is hydrogen, (C 1 -C 6 )alkyl, cycloalkyl,
(C 6 -C 10 )aryl; or R 2a and R 2b can taken together to form a double
bond;
140a.
Compound 140. in the all-(S) configuration;
141.
wherein
Ar is aryl or heteroaryl; and
U is independently selected from: C(R 1 ) 2 ; C(O); NR 2 ; S; S(O);
S(O) 2 ; wherein R 1 and R 2 are independently hydrogen,
[C(R 3 ) 2 ] p (CH═CH) q R 3 , C(═Z)R 3 , C(═Z)[C(R 3 ) 2 ] p (CH═CH) q R 3 ,
C(═Z)N(R 3 ) 2 , C(═Z)NR 3 N(R 3 ) 2 , CN, CF 3 , N(R 3 ) 2 , NR 3 CN,
NR 3 C(═Z)R 3 , NRC(═Z)N(R 3 ) 2 , NHN(R 3 ) 2 , NHOR 3 , NO 2 , OR 3 ,
OCF 3 , F, Cl, Br, I, SO 3 H, OSO 3 H, SO 2 N(R 3 ) 2 , SO 2 R 3 ,
P(O)(OR 3 )R 3 , P(O)(OR 3 ) 2 ; wherein p is 0 to 12; wherein q is 0 to
12; wherein Z is O, S, NR 3 ; wherein R 3 is independently
hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl;
141a.
Compound 141. in the all-(S) configuration;
142.
wherein
Ar is aryl or heteroaryl; and R 4 and R 5 are independently selected
from: C(R 1 ) 2 ; C(O); NR 2 ; S; S(O); S(O) 2 ; wherein R 1 and R 2 are
independently hydrogen, [C(R 3 ) 2 ] p (CH═CH) q R 3 , C(═Z)R 3 ,
C(═Z)[C(R 3 ) 2 ] p (CH═CH) q R 3 , C(═Z)N(R 3 ) 2 ,
C(═Z)NR 3 N(R 3 ) 2 , CN, CF 3 , N(R 3 ) 2 , NR 3 CN, NR 3 C(═Z)R 3 ,
NRC(═Z)N(R 3 ) 2 , NHN(R 3 ) 2 , NHOR 3 , NO 2 , OR 3 , OCF 3 ,
F, Cl, Br, I, SO 3 H, OSO 3 H, SO 2 N(R 3 ) 2 , SO 2 R 3 , P(O)(OR 3 )R 3 ,
P(O)(OR 3 ) 2 ; wherein p is 0 to 12; wherein q is 0 to 12; wherein
Z is O, S, NR 3 ; wherein R 3 is independently hydrogen, alkyl,
cycloalkyl, aryl, heteroaryl, heterocyclyl;
142a.
Compound 142. in the all-(S) configuration;
wherein independently of each other the variables in the compounds 117. to 142a. are defined as indicated in the definition next to the respective compound; Y is -L-R1-L1; and R1, L and L1 are as described in claim 1 ; and
or W═X,
wherein X is a group selected from
and R′ is H or C 1 -C 6 -alkyl.
21 . The molecular probe according to claim 1 , selected from one of the compounds 143.-155.:
143.
144.
145.
wherein R A and R B are independently hydrogen, (C 1 -C 6 )alkyl,
hydroxyl, (C 1 -C 6 )alkoxy or halogen;
146.
147.
148.
149.
wherein
m is 0 or 1; and
R 4 , R 5 and R 6 are independently selected from the group consisting
of:
1) H,
2) halogen,
3) (C 1 -C 4 )alkoxy optionally substituted with 1-3 halogen atoms,
4) NO 2 ,
5) OH,
6) benzyloxy, the benzyl portion of which is optionally substituted
with 1-2 members selected from the group consisting of: halogen,
CN, (C 1 -C 4 )alkyl and (C 1 -C 4 )alkoxy, said alkyl and alkoxy being
optionally substituted with 1-3 halogen groups,
7) NH(C 1 -C 4 )acyl,
8) (C 1 -C 4 )acyl,
9) O—(C 1 -C 4 )alkyl-CO 2 H, optionally esterified with a (C 1 -C 6 )alkyl
or a (C 5 -C 7 )cycloalkyl group,
10) CH═CH—CO 2 H,
11) CO 2 H,
12) (C 1 -C 5 )alkyl-CO 2 H,
13) C(O)NH 2 , optionally substituted on the nitrogen atom by 1-2
(C 1 -C 4 )alkyl groups;
14) (C 1 -C 5 )alkyl-C(O)NH 2 , optionally substituted on the nitrogen
atom by 1-2 (C 1 -C 4 )alkyl groups;
15) S(O) 0-2 —(C 1 -C 4 )alkyl;
16) (C 1 -C 2 )alkyl-S(O) 0-2 —(C 1 -C 4 )alkyl;
17) S(O) 0-2 —(C 1 -C 6 )alkyl or S(O) 0-2 -phenyl, said alkyl and phenyl
portions thereof being optionally substituted with 1-3 members
selected from the group consisting of: halogen, CN, (C 1 -C 4 )alkyl
and (C 1 -C 4 )alkoxy, said alkyl and alkoxy being optionally
substituted by 1-3 halogen groups,
18) benzoyl optionally substituted by 1-2 members selected
from the group consisting of: halogen, CN, (C 1 -C 4 )alkyl and
(C 1 -C 4 )alkoxy, said alkyl and alkoxy groups being optionally
substituted by 1-3 halogen groups,
19) phenyl or naphthyl, optionally substituted with 1-2 members
selected from the group consisting of: halogen, CN, (C 1 -C 4 )alkyl
and (C 1 -C 4 )alkoxy, said alkyl and alkoxy being optionally
substituted with 1-3 halogen groups,
20) CN,
21) (C 1 -C 4 )alkylene-HET2, wherein HET2 represents a 5-7
membered aromatic or non-aromatic ring containing 1-4
heteroatoms selected from O, S, NH and N(C 1 -C 4 ) and optionally
containing 1-2 oxo groups, and optionally substituted with 1-3
(C 1 -C 4 )alkyl, OH, halogen or (C 1 -C 4 )acyl groups;
22) O—(C 1 -C 4 )alkyl-HET3, wherein HET3 is a 5 or 6 membered
aromatic or non-aromatic ring containing from 1 to 3 heteroatoms
selected from O, S and N, and optionally substituted with one or
two groups selected from halogen and (C 1 -C 4 )alkyl, and optionally
containing 1-2 oxo groups, and
23) HET4, wherein HET4 is a 5 or 6 membered aromatic or non-
aromatic ring, and the benzofused analogs thereof, containing
from 1 to 4 heteroatoms selected from O, S and N, and is
optionally substituted by one or two groups selected from halogen,
(C 1 -C 4 )alkyl and (C 1 -C 4 )acyl; and wherein halogen includes F, Cl,
Br and I;
149a.
Compound 149. in the all-(S) configuration;
150.
wherein R 4 is selected from the group consisting of:
1) H,
2) halogen,
3) (C 1 -C 4 )alkoxy optionally substituted with 1-3 halogen atoms,
4) NO 2 ,
5) OH,
6) benzyloxy, the benzyl portion of which is optionally substituted
with 1-2 members selected from the group consisting of: halogen,
CN, (C 1 -C 4 )alkyl and (C 1 -C 4 )alkoxy, said alkyl and alkoxy being
optionally substituted with 1-3 halogen groups,
7) NH(C 1 -C 4 )acyl,
8) (C 1 -C 4 )acyl,
9) O—(C 1 -C 4 )alky1-CO 2 H, optionally esterified with a
(C 1 -C 6 )alkyl or a (C 5 -C 7 )cycloalkyl group,
10) CH═CH—CO 2 H,
11) CO 2 H,
12) (C 1 -C 5 )alkyl-CO 2 H,
13) C(O)NH 2 , optionally substituted on the nitrogen atom by 1-2
(C 1 -C 4 )alkyl groups;
14) (C 1 -C 5 )alkyl-C(O)NH 2 , optionally substituted on the nitrogen
atom by 1-2 (C 1 -C 4 )alkyl groups;
15) S(O) 0-2 —(C 1 -C 4 )alkyl;
16) (C 1 -C 2 )alkyl-S(O) 0-2 —(C 1 -C 4 )alkyl;
17) S(O) 0-2 —(C 1 -C 6 )alkyl or S(O) 0-2 -phenyl, said alkyl and phenyl
portions thereof being optionally substituted with 1-3 members
selected from the group consisting of: halogen, CN, (C 1 -C 4 )alkyl
and (C 1 -C 4 )alkoxy, said alkyl and alkoxy being optionally
substituted by 1-3 halogen groups,
18) benzoyl optionally substituted by 1-2 members selected from
the group consisting of: halogen, CN, (C 1 -C 4 )alkyl and
(C 1 -C 4 )alkoxy, said alkyl and alkoxy groups being optionally
substituted by 1-3 halogen groups,
19) phenyl or naphthyl, optionally substituted with 1-2 members
selected from the group consisting of: halogen, CN, (C 1 -C 4 )alkyl
and (C 1 -C 4 )alkoxy, said alkyl and alkoxy being optionally
substituted with 1-3 halogen groups,
20) CN,
21) (C 1 -C 4 )alkylene-HET2, wherein HET2 represents a 5-7
membered aromatic or non-aromatic ring containing 1-4
heteroatoms selected from O, S, NH and N(C 1 -C 4 ) and optionally
containing 1-2 oxo groups,and optionally substituted with 1-3
(C 1 -C 4 )alkyl, OH, halogen or (C 1 -C 4 )acyl groups;
22) O—(C 1 -C 4 )alkyl-HET3, wherein HET3 is a 5 or 6 membered
aromatic or non-aromatic ring containing from 1 to 3 heteroatoms
selected from O, S and N, and optionally substituted with one or
two groups selected from halogen and (C 1 -C 4 )alkyl, and optionally
containing 1-2 oxo groups, and
23) HET4, wherein HET4 is a 5 or 6 membered aromatic or non-
aromatic ring, and the benzofused analogs thereof, containing from
1 to 4 heteroatoms selected from O, S and N, and is optionally
substituted by one or two groups selected from halogen,
(C 1 -C 4 )alkyl and (C 1 -C 4 )acyl; and wherein halogen includes F, Cl,
Br and I;
150a.
Compound 150. in the all-(S) configuration;
151.
152.
153.
154.
155.
wherein independently of each other the variables in the compounds 143. to 155. are defined as indicated in the definition next to the respective compound; Y is -L-R1-L1; and R1, L and L1 are as described in claim 1 ; and
or W═X,
wherein X is a group selected from
and R′ is H or C 1 -C 6 -alkyl.
22 . The molecular probe according to claim 1 , selected from one of the compounds 156.-157.:
156.
157.
wherein independently of each other the variables in the compounds 156. to 157. are defined as indicated in the definition next to the respective compound; Y is -L-R1-L1; and R1, L and L1 are as described in claim 1 ; and
or W═X,
wherein X is a group selected from
and R′ is H or C 1 -C 6 -alkyl.
23 . The molecular probe according to claim 1 , selected from a compound of the formula 158.:
158.
wherein Y is -L-R1-L1; and R1, L and L1 are as described in claim 1 and X is a hydrogen.
24 . The molecular probe according to claim 1 , selected from one of the compounds 159.-167.:
159.
160.
wherein Ar is aryl;
161.
162.
wherein R is alkyl, cycloalkyl; R 2 is halogen; Ar is aryl; and each n
is independently 0 or 1;
163.
wherein R is alkyl, cycloalkyl; and each n is independently 0 or 1;
164.
wherein R is alkyl, cycloalkyl; and n is 0 or 1;
165.
wherein R is alkyl, cycloalkyl; and n is 0 or 1
166.
167.
wherein Y is -L-R1-L1; and R1, L and L1 are as described in claim 1 , and X is a hydrogen atom.
25 . The molecular probe according to any one of claims 17 to 24 , wherein
L is a direct bond or a group selected from
—(NRx)-, —O—, —C═N—, —C(═O)—, —C(═O)—NH—, —NH—C(═O)—, —C(═O)H, —CRx=CRy-, —C≡C— and phenyl, wherein Rx and Ry are independently H or (C 1 -C 6 )alkyl;
R1 is a straight or branched chain alkylene group with 1 to 300 carbon atoms, wherein optionally
(a) one or more carbon atoms are replaced by oxygen;
(b) one or more carbon atoms are replaced by nitrogen carrying a hydrogen atom, and the adjacent carbon atoms are substituted by oxo, representing an amide function —NH—CO—;
(c) one or more carbon atoms are replaced by an ester function —O—CO—;
(d) the bond between two adjacent carbon atoms is a double or a triple bond; or
(e) two adjacent carbon atoms are replaced by a disulfide linkage.
or a combination thereof;
L1 is biotin or methotrexate or a fluorophore selected from the group consisting of a dimethylaminocoumarin derivative, Dansyl, 5/6-carboxyfluorescein and tetramethylrhodamine, BODIPY-493/503, BODIPY-FL, BODIPY-TMR, BODIPY-TMR-X, BODIPY-TR-X, BODIPY630/550-X, BODIPY-650/665-X, Alexa 350, Alexa 488, Alexa 532, Alexa 546, Alexa 555, Alexa 635, Alexa 647, Cyanine 3 (Cy 3), Cyanine 3B (Cy 3B), Cyanine 5 (Cy 5), Cyanine 5.5 (Cy 5.5), Cyanine 7 (Cy 7), Cyanine 7.5 (Cy 7.5), ATTO 488, ATTO 532, ATTO 600, ATTO 655, DY-505, DY-547, DY-632, DY-647; and
X is a group selected from
wherein R is alkyl or aryl.
26 . The molecular probe according to claim 25 , wherein
L is a group selected from
—(NRx)-, —O—, —C═N—, —C(═O)—, —C(═O)—NH—, —NH—C(═O)—, —C(═O)H, —CRx=CRy-, —C≡C— and phenyl, wherein Rx and Ry are independently H or (C 1 -C 6 )alkyl, or a direct bond;
R1 is alkyl or a straight or branched chain alkylene group with 1 to 50 carbon atoms, wherein one or more carbon atoms are replaced by oxygen;
L1 is a fluorophore selected from the group consisting of a dimethylaminocoumarin derivative, Dansyl, 5/6-carboxyfluorescein and tetramethylrhodamine, BODIPY-493/503, BODIPY-FL, BODIPY-TMR, BODIPY-TMR-X, BODIPY-TR-X, BODIPY630/550-X, BODIPY-650/665-X, Alexa 350, Alexa 488, Alexa 532, Alexa 546, Alexa 555, Alexa 635, Alexa 647, Cyanine 3 (Cy 3), Cyanine 3B (Cy 3B), Cyanine 5 (Cy 5), Cyanine 5.5 (Cy 5.5), Cyanine 7 (Cy 7), Cyanine 7.5 (Cy 7.5), ATTO 488, ATTO 532, ATTO 600, ATTO 655, DY-505, DY-547, DY-632, DY-647; and
X is a nitrile group or a group selected from
27 . A method for molecular imaging in vitro, in cell-culture experiments, ex-vivo experiments or in a living organism, the method comprising the use of a probe of the formula (I) according to claim 1 .