THIOREDOXIN REDUCTASE INHIBITORS FOR USE IN THE TREATMENT OF CANCER
20210038577 ยท 2021-02-11
Inventors
Cpc classification
A61K39/395
HUMAN NECESSITIES
A61K31/436
HUMAN NECESSITIES
A61K39/3955
HUMAN NECESSITIES
C07K2317/73
CHEMISTRY; METALLURGY
A61K39/3955
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K31/4433
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
A61K31/439
HUMAN NECESSITIES
A61K31/439
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K31/506
HUMAN NECESSITIES
A61K31/44
HUMAN NECESSITIES
A61K31/501
HUMAN NECESSITIES
A61K31/166
HUMAN NECESSITIES
A61K31/4433
HUMAN NECESSITIES
A61K31/44
HUMAN NECESSITIES
C07K2317/24
CHEMISTRY; METALLURGY
A61K9/0019
HUMAN NECESSITIES
A61K31/501
HUMAN NECESSITIES
A61K31/444
HUMAN NECESSITIES
A61K31/436
HUMAN NECESSITIES
A61K39/395
HUMAN NECESSITIES
A61K31/506
HUMAN NECESSITIES
A61K31/166
HUMAN NECESSITIES
International classification
A61K31/444
HUMAN NECESSITIES
A61K31/55
HUMAN NECESSITIES
A61K9/00
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
Abstract
The present invention provides inhibitors of thioredoxin reductase, in particular selenium compromised thioredoxin reductase-derived apoptotic protein (SecTRAP) forming agents, for use in treating an immune cell infiltrated cancer (e.g. a T-cell infiltrated cancer) in a subject, wherein said agents stimulate an anti-cancer immune response. The present invention also provides combinations comprising a SecTRAP forming agent and other therapeutic agents for use in treating cancer.
Claims
1. A selenium compromised thioredoxin reductase-derived apoptotic protein (SecTRAP) forming agent for use in treating an immune cell infiltrated cancer in a subject, wherein said agent stimulates an anti-cancer immune response.
2. The selenium compromised thioredoxin reductase-derived apoptotic protein (SecTRAP) forming agent for use according to claim 1, wherein said immune cell infiltrated cancer is a T-cell infiltrated cancer.
3. The selenium compromised thioredoxin reductase-derived apoptotic protein (SecTRAP) forming agent for use according to claim 1 or claim 2, wherein said anti-cancer immune response is characterized by (i) a reduction in the level of Tregs; and/or (ii) an increase in the level of CD8+ T-cells and/or other cytotoxic immune cells; and/or (iii) a reduction in the ratio of Tregs to CD8+ T-cells and/or other cytotoxic immune cells.
4. The selenium compromised thioredoxin reductase-derived apoptotic protein (SecTRAP) forming agent for use according to any one of claims 1 to 3, wherein said SecTRAP forming agent is is a compound of formula XI ##STR00114## or a pharmaceutically acceptable salt thereof, wherein: L represents S(O).sub.2 or S(O) X represents a heteroaryl group or heterocyclyl, connected to L via a carbon atom, or C.sub.1-12 alkyl, C.sub.2-12 alkenyl, C.sub.2-12 alkynyl, or phenyl, each optionally substituted by one or more groups independently selected from Y; R.sup.1, R.sup.2 and R.sup.3 each independently represent H, halo, R.sup.a1, CN, -A.sup.a1-C(Q.sup.a1)R.sup.b1, -A.sup.b1-C(Q.sup.b1)N(R.sup.c1)R.sup.d1, -A.sup.c1-C(Q.sup.c1)OR.sup.e1, -A.sup.d1-S(O).sub.pR.sup.f1, -A.sup.e1-S(O).sub.pN(R.sup.g1)R.sup.h1, -A.sup.f1-S(O).sub.pOR.sup.i1, N.sub.3, N(R.sup.j1)R.sup.k1, N(H)CN, NO.sub.2, ONO.sub.2, OR.sup.l1 or SR.sup.m1; each A.sup.a1 to A.sup.f1 independently represents a single bond, N(R.sup.p1) or O; each Q.sup.a1 to Q.sup.c1 independently represents O, S, NR.sup.n1 or N(OR.sup.o1); each R.sup.a1 and R.sup.f1 independently represents C.sub.1-6 alkyl, C.sub.2-6 alkenyl or C.sub.2-6 alkynyl each optionally substituted by one or more groups independently selected from G.sup.1a, or heterocyclyl optionally substituted by one or more groups independently selected from G.sup.1b; each R.sup.b1, R.sup.c1, R.sup.d1, R.sup.e1, R.sup.g1, R.sup.h1, R.sup.i1, R.sup.j1, R.sup.k1, R.sup.l1, R.sup.m1, R.sup.n1, R.sup.o1 and R.sup.p1 independently represents H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl or C.sub.2-6 alkynyl each optionally substituted by one or more groups independently selected from G.sup.1a, or heterocyclyl optionally substituted by one or more groups independently selected from G.sup.1b; or any of R.sup.c1 and R.sup.d1, R.sup.g1 and R.sup.h1 and/or R.sup.j1 and R.sup.k1 are linked together to form, together with the nitrogen atom to which they are attached, a 3- to 6-membered ring, which ring optionally contains one further heteroatom and which ring optionally is substituted by one or more groups independently selected from G.sup.1b, C.sub.1-3 alkyl, C.sub.2-3 alkenyl or C.sub.2-3 alkynyl each optionally substituted by one or more G.sup.1a, and O; each G.sup.1a and G.sup.1b independently represents halo, CN, N(R.sup.a2)R.sup.b2, OR.sup.c2, SR.sup.d2 or O; each R.sup.a2, R.sup.b2, R.sup.c2 and R.sup.d2 independently represents H or C.sub.1-6 alkyl, C.sub.2-6 alkenyl or C.sub.2-6 alkynyl each optionally substituted by one or more fluoro; or R.sup.a2 and R.sup.b2 are linked together to form, along with the nitrogen atom to which they are attached, a 3- to 6-membered ring, which ring optionally contains one further heteroatom and which ring optionally is substituted by one or more groups independently selected from fluoro and C.sub.1-3 alkyl, C.sub.2-3 alkenyl or C.sub.2-3 alkynyl each optionally substituted by one or more fluoro; each Y independently represents halo, R.sup.a3, CN, -A.sup.a2-C(Q.sup.a2)R.sup.b3, -A.sup.b2-C(Q.sup.b2)N(R.sup.c3)R.sup.d3, -A.sup.c2-C(Q.sup.c2)OR.sup.e3, -A.sup.d2-S(O).sub.qR.sup.f3, -A.sup.e2-S(O).sub.qN(R.sup.g3)R.sup.h3, -A.sup.f2-S(O).sub.qOR.sup.i3, N.sub.3, N(R.sup.j3)R.sup.k3, N(H)CN, NO.sub.2, ONO.sub.2, OR.sup.l3, SR.sup.m3 or O each Q.sup.a2 to Q.sup.c2 independently represents O, S, NR.sup.n3 or N(OR.sup.o3); each A.sup.a2 to A.sup.f2 independently represents a single bond, N(R.sup.p3) or O; each R.sup.a3 and R.sup.f3 independently represents C.sub.1-6 alkyl, C.sub.2-6 alkenyl or C.sub.2-6 alkynyl each optionally substituted by one or more groups independently selected from G.sup.2a, heterocyclyl optionally substituted by one or more groups independently selected from G.sup.2b, aryl optionally substituted by one or more groups independently selected from G.sup.2c, or heteroaryl optionally substituted by one or more groups independently selected from G.sup.2d; each R.sup.b3, R.sup.c3, R.sup.d3, R.sup.e3, R.sup.g3, R.sup.h3, R.sup.i3, R.sup.j3, R.sup.k3, R.sup.l3, R.sup.m3, R.sup.n3, R.sup.o3 and R.sup.p3 independently represents H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl or C.sub.2-6 alkynyl each optionally substituted by one or more groups independently selected from G.sup.2a, heterocyclyl optionally substituted by one or more groups independently selected from G.sup.2b, aryl optionally substituted by one or more groups independently selected from G.sup.2c, or heteroaryl optionally substituted by one or more groups independently selected from G.sup.2d; or any two R.sup.c3 and R.sup.d3, R.sup.g3 and R.sup.h3 and/or R.sup.j3 and R.sup.k3 are linked together to form, along with the nitrogen atom to which they are attached, a 3- to 6-membered ring, which ring optionally contains one further heteroatom and which ring optionally is substituted by one or more groups independently selected from halogen, C.sub.1-3alkyl optionally substituted by one or more halogens, O, heterocyclyl optionally substituted by one or more groups independently selected from G.sup.2b, aryl optionally substituted by one or more groups independently selected from G.sup.2c, or heteroaryl optionally substituted by one or more groups independently selected from G.sup.2d; each G.sup.2a independently represents halo, CN, N(R.sup.j4)R.sup.k4, OR.sup.l4, SR.sup.m4 or O; each G.sup.2b independently represents halo, R.sup.a4, CN, N(R.sup.j4)R.sup.k4, OR.sup.l4, SR.sup.m4 or O; each G.sup.2c and G.sup.2d independently represents halo, R.sup.a4, CN, -A.sup.a3-C(Q.sup.a4)R.sup.b4, -A.sup.b3-C(Q.sup.b3)N(R.sup.c4)R.sup.d4, -A.sup.c3-C(Q.sup.c3)OR.sup.e4, -A.sup.d3-S(O).sub.qR.sup.f4, -A.sup.e3-S(O).sub.qN(R.sup.g4)R.sup.h4, -A.sup.f3-S(O).sub.qOR.sup.i4, N.sub.3, N(R.sup.j4)R.sup.k4, N(H)CN, NO.sub.2, ONO.sub.2, OR.sup.l4 or SR.sup.m4; each Q.sup.a3 to Q.sup.c3 independently represents O, S, NR.sup.n4 or N(OR.sup.o4); each A.sup.a3 to A.sup.f3 independently represents a single bond, N(R.sup.p4) or O; each R.sup.a4 and R.sup.f4 independently represents C.sub.1-6 alkyl, C.sub.2-6 alkenyl or C.sub.2-6 alkynyl each optionally substituted by one or more groups independently selected from G.sup.3a, heterocyclyl optionally substituted by one or more groups independently selected from G.sup.3b, aryl optionally substituted by one or more groups independently selected from G.sup.3c, or heteroaryl optionally substituted by one or more groups independently selected from G.sup.3d; each R.sup.b4, R.sup.c4, R.sup.d4, R.sup.e4, R.sup.g4, R.sup.h4, R.sup.i4, R.sup.j4, R.sup.k4, R.sup.l4, R.sup.m4, R.sup.n4, R.sup.o4 and R.sup.p4 independently represents H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl or C.sub.2-6 alkynyl each optionally substituted by one or more groups independently selected from G.sup.3a or heterocyclyl optionally substituted by one or more groups independently selected from G.sup.3b, aryl optionally substituted by one or more groups independently selected from G.sup.3c, or heteroaryl optionally substituted by one or more groups independently selected from G.sup.3d; or any of R.sup.c4 and R.sup.d4, R.sup.g4 and R.sup.h4 and/or R.sup.j4 and R.sup.k4 are linked together to form, together with the nitrogen atom to which they are attached, a 3- to 6-membered ring, which ring optionally contains one further heteroatom and which ring optionally is substituted by one or more groups independently selected G.sup.3b; each G.sup.3a and G.sup.3b independently represents halo, R.sup.a5, CN, N(R.sup.b5)R.sup.c5, OR.sup.d5, SR.sup.e5 or O; G.sup.3c and G.sup.3d independently representing halo, R.sup.a5, CN, -A.sup.a4-C(Q.sup.a4)R.sup.b5, -A.sup.b4-C(Q.sup.b4)N(R.sup.c5)R.sup.d5, -A.sup.c4-C(Q.sup.c4)OR.sup.e5, -A.sup.d5-S(O).sub.qR.sup.f5, -A.sup.e4-S(O).sub.qN(R.sup.g5)R.sup.h5, -A.sup.f4-S(O).sub.qOR.sup.i5, N.sub.3, N(R.sup.j5)R.sup.k5, N(H)CN, NO.sub.2, ONO.sub.2, OR.sup.l5 or SR.sup.m5, each Q.sup.a4 to Q.sup.c4 independently represents O, S, NR.sup.n5 or N(OR.sup.o5); each A.sup.a4 to A.sup.f4 independently represents a single bond, N(R.sup.p5) or O; with each R.sup.f5 to R.sup.p5 independently representing H, or C.sub.1-6 alkyl, C.sub.2-6 alkenyl or C.sub.2-6 alkynyl each optionally substituted by one or more groups independently selected from G.sup.4, or with each R.sup.g5 and R.sup.h5, and R.sup.j5 and R.sup.k5 being linked together to form, together with the nitrogen atom to which they are attached, a 3- to 6-membered ring, which ring optionally contains one further heteroatom and which ring optionally is substituted by one or more groups independently selected from G.sup.4; each R.sup.a5 independently represents C.sub.1-6 alkyl, C.sub.2-6 alkenyl or C.sub.2-6 alkynyl each optionally substituted by one or more groups independently selected from G.sup.4; each R.sup.b5, R.sup.c5, R.sup.d5 and R.sup.e5 independently represents H, or C.sub.1-6 alkyl, C.sub.2-6 alkenyl or C.sub.2-6 alkynyl each optionally substituted by one or more groups independently selected from G.sup.4; or each R.sup.b5 and R.sup.c5 are linked together to form, together with the nitrogen atom to which they are attached, a 3- to 6-membered ring, which ring optionally contains one further heteroatom and which ring optionally is substituted by one or more groups independently selected from G.sup.4; each G.sup.4 independently represents halo, R.sup.a6, CN, N(R.sup.b6)R.sup.c6, OR.sup.d6 or O; each R.sup.a6 independently represents C.sub.1-6 alkyl, C.sub.2-6 alkenyl or C.sub.2-6 alkynyl each optionally substituted by one or more fluoro; each R.sup.b6, R.sup.c6 and R.sup.d6 independently represents H, or C.sub.1-6 alkyl, C.sub.2-6 alkenyl or C.sub.2-6 alkynyl each optionally substituted by one or more fluoro; and each p and q independently represents 1 or 2.
5. The selenium compromised thioredoxin reductase-derived apoptotic protein (SecTRAP) forming agent for use according to any one of claims 1 to 4, wherein said SecTRAP forming agent is selected from the group consisting of: ##STR00115## ##STR00116##
6. The selenium compromised thioredoxin reductase-derived apoptotic protein (SecTRAP) forming agent for use according to any one of claims 1 to 5, wherein said SecTRAP forming agent is selected from the group consisting of: ##STR00117##
7. A selenium compromised thioredoxin reductase-derived apoptotic protein (SecTRAP) forming agent for use according to any one of claims 1 to 3, wherein said SecTRAP forming agent is a compound of formula II ##STR00118## or a pharmaceutically acceptable salt thereof, wherein: X represents C.sub.1-12 alkyl optionally substituted by one or more groups independently selected from G.sup.1a, heterocycloalkyl optionally substituted by one or more groups independently selected from G.sup.1b, aryl optionally substituted by one or more groups independently selected from G.sup.1c, or heteroaryl optionally substituted by one or more groups independently selected from G.sup.1d; Y represents C.sub.1-12 alkyl optionally substituted by one or more groups independently selected from G.sup.2a; heterocycloalkyl optionally substituted by one or more groups independently selected from G.sup.2b, aryl optionally substituted by one or more groups independently selected from G.sup.2c, or heteroaryl optionally substituted by one or more groups independently selected from G.sup.2d; Z represents O, S, NR.sup.a or N(OR.sup.b); R.sup.1 and R.sup.2 independently represents H or C.sub.1-6 alkyl, the latter group being optionally substituted by one or more groups independently selected from halo and OC.sub.1-6 alkyl optionally substituted by one or more halo; each G.sup.1a, G.sup.1b, G.sup.1c and G.sup.1d independently represents halo, R.sup.a1, CN, -A.sup.a1-C(Q.sup.a1)R.sup.b1, -A.sup.b1-C(Q.sup.b1)N(R.sup.c1)R.sup.d1, -A.sup.c1-C(Q.sup.c1)OR.sup.e1, -A.sup.d1-S(O).sub.nR.sup.f1, -A.sup.e1-S(O).sub.nC(O)R.sup.g1, -A.sup.f1-S(O).sub.nN(R.sup.h1)R.sup.i1, -A.sup.g1-S(O).sub.nOR.sup.j1, N.sub.3, N(R.sup.k1)R.sup.l1, N(H)CN, NO.sub.2, OR.sup.m1, SR.sup.n1 or =Q.sup.d1; each A.sup.a1 to A.sup.g1 independently represents a single bond, N(R.sup.o1), C(Q.sup.e1)N(R.sup.p1) or O; each Q.sup.a1 to Q.sup.e1 independently represents O, S, NR.sup.q1 or N(OR.sup.r1); R.sup.a and R.sup.b each independently represent H or C.sub.1-6 alkyl, the latter group being optionally substituted by one or more groups independently selected from halo and OC.sub.1-6 alkyl optionally substituted by one or more halo; each R.sup.a1 and R.sup.f1 independently represents C.sub.1-6 alkyl optionally substituted by one or more groups independently selected from G.sup.3a, heterocycloalkyl optionally substituted by one or more groups independently selected from G.sup.3b, aryl optionally substituted by one or more groups independently selected from G.sup.3c, or heteroaryl optionally substituted by one or more groups independently selected from G.sup.3d; each R.sup.b1, R.sup.c1, R.sup.d1, R.sup.e1, R.sup.g1, R.sup.h1, R.sup.i1, R.sup.j1, R.sup.k1, R.sup.l1, R.sup.m1, R.sup.n1, R.sup.q1 and R.sup.r1 independently represents H, C.sub.1-6 alkyl optionally substituted by one or more groups independently selected from G.sup.3a, heterocycloalkyl optionally substituted by one or more groups independently selected from G.sup.3b, aryl optionally substituted by one or more groups independently selected from G.sup.3c, or heteroaryl optionally substituted by one or more groups independently selected from G.sup.3d; or any two R.sup.c1 and R.sup.d1, R.sup.h1 and R.sup.i1 and/or R.sup.k1 and R.sup.l1 are linked together to form, along with the nitrogen atom to which they are attached, a 3- to 6-membered ring, which ring optionally contains one further heteroatom and which ring optionally is substituted by one or more groups independently selected from halo, C.sub.1-3 alkyl optionally substituted by one or more halo, and O; each R.sup.o1 and R.sup.p1 independently represents H or C.sub.1-6 alkyl optionally substituted by one or more halo; each G.sup.2a, G.sup.2b, G.sup.2c and G.sup.2d independently represents halo, R.sup.a2, CN, -A.sup.a2-C(Q.sup.a2)R.sup.b2, -A.sup.b2-C(Q.sup.b2)N(R.sup.c2)R.sup.d2, -A.sup.c2-C(Q.sup.c2)OR.sup.e, -A.sup.d2-S(O).sub.pR.sup.f2, -A.sup.e2-S(O).sub.pC(O)R.sup.g2, -A.sup.f2-S(O).sub.pN(R.sup.h2)R.sup.i2, -A.sup.g2-S(O).sub.pOR.sup.j2, N.sub.3, N(R.sup.k2)R.sup.l2, N(H)CN, NO.sub.2, OR.sup.m2, SR.sup.n2 or =Q.sup.d2; each A.sup.a2 to A.sup.g2 independently represents a single bond, N(R.sup.o2), C(Q.sup.e2)N(R.sup.p2) or O; each Q.sup.a2 to Q.sup.e3 independently represents O, S, NR.sup.q2 or N(OR.sup.r2); each R.sup.a2 independently represents heterocycloalkyl optionally substituted by one or more groups independently selected from G.sup.4b, aryl optionally substituted by one or more groups independently selected from G.sup.4c, or heteroaryl optionally substituted by one or more groups independently selected from G.sup.4d; each R.sup.f2 independently represents C.sub.1-6 alkyl optionally substituted by one or more groups independently selected from G.sup.4a, heterocycloalkyl optionally substituted by one or more groups independently selected from G.sup.4b, aryl optionally substituted by one or more groups independently selected from G.sup.4c, or heteroaryl optionally substituted by one or more groups independently selected from G.sup.4d; each R.sup.b2, R.sup.c2, R.sup.d2, R.sup.e2, R.sup.g2, R.sup.h2, R.sup.i2, R.sup.j2, R.sup.k2, R.sup.l2, R.sup.m2, R.sup.n2, R.sup.q2 and R.sup.r2 independently represents H, C.sub.1-6 alkyl optionally substituted by one or more groups independently selected from G.sup.4a, heterocycloalkyl optionally substituted by one or more groups independently selected from G.sup.4b, aryl optionally substituted by one or more groups independently selected from G.sup.4c, or heteroaryl optionally substituted by one or more groups independently selected from G.sup.4d; or any two R.sup.c2 and R.sup.d2, R.sup.h2 and R.sup.i2 and/or R.sup.k2 and R.sup.l2 are linked together to form, along with the nitrogen atom to which they are attached, a 3- to 6-membered ring, which ring optionally contains one further heteroatom and which ring optionally is substituted by one or more groups independently selected from halo, C.sub.1-3 alkyl optionally substituted by one or more halo, and O; each R.sup.o2 and R.sup.p2 independently represents H or C.sub.1-6 alkyl optionally substituted by one or more halo; each G.sup.3a independently represents halo, CN, -A.sup.a3-C(Q.sup.a3)R.sup.b3, -A.sup.b3-C(Q.sup.b3)N(R.sup.c3)R.sup.d3, -A.sup.c3-C(Q.sup.c3)OR.sup.e3, -A.sup.d3-S(O).sub.qR.sup.f3, -A.sup.e3-S(O).sub.qC(O)R.sup.g3, -A.sup.f3-S(O).sub.qN(R.sup.h3)R.sup.i3, -A.sup.g3-S(O).sub.qOR.sup.j3, N.sub.3, N(R.sup.k3)R.sup.i3, N(H)CN, NO.sub.2, OR.sup.m3, SR.sup.n3 or =Q.sup.d3; each G.sup.3b, G.sup.3c and G.sup.3d independently represents halo, R.sup.a3, CN, -A.sup.3-C(Q.sup.a3)R.sup.b3, -A.sup.b3-C(Q.sup.b3)N(R.sup.c3)R.sup.d3, -A.sup.c3-C(Q.sup.c3)OR.sup.e3, -A.sup.d3-S(O).sub.qR.sup.f3, -A.sup.e3-S(O).sub.qC(O)R.sup.g3, -A.sup.f3-S(O).sub.qN(R.sup.h3)R.sup.i3, -A.sup.g3-S(O).sub.qOR.sup.j3, N.sub.3, N(R.sup.k3)R.sup.l3, N(H)CN, NO.sub.2, OR.sup.m3, SR.sup.n3 or =Q.sup.d3; each A.sup.a3 to A.sup.g3 independently represents a single bond, N(R.sup.o3), C(Q.sup.e3)N(R.sup.p3) or O; each Q.sup.a3 to Q.sup.e3 independently represents O, S, NR.sup.q3 or N(OR.sup.r3); each R.sup.a3 and R.sup.f3 independently represents C.sub.1-6 alkyl optionally substituted by one or more groups independently selected from G.sup.5a, or heterocycloalkyl optionally substituted by one or more groups independently selected from G.sup.5b; each R.sup.b3, R.sup.c3, R.sup.d3, R.sup.e3, R.sup.g3, R.sup.h3, R.sup.i3, R.sup.j3, R.sup.k3, R.sup.l3, R.sup.m3, R.sup.n3, R.sup.q3 and R.sup.r3 independently represents H, C.sub.1-6 alkyl optionally substituted by one or more groups independently selected from G.sup.5a, or heterocycloalkyl optionally substituted by one or more groups independently selected from G.sup.5b; or any two R.sup.c3 and R.sup.d3, R.sup.h3 and R.sup.i3 and/or R.sup.k3 and R.sup.l3 are linked together to form, along with the nitrogen atom to which they are attached, a 3- to 6-membered ring, which ring optionally contains one further heteroatom and which ring optionally is substituted by one or more groups independently selected from halo, C.sub.1-3 alkyl optionally substituted by one or more halo, and O; each R.sup.o3 and R.sup.p3 independently represents H or C.sub.1-6 alkyl optionally substituted by one or more halo; each G.sup.4a independently represents halogen, CN, -A.sup.a4-C(Q.sup.a4)R.sup.b4, -A.sup.b4-C(Q.sup.b4)N(R.sup.c4)R.sup.d4, -A.sup.c4-C(Q.sup.c4)OR.sup.e4, -A.sup.d4-S(O).sub.rR.sup.f4, -A.sup.e4-S(O).sub.rC(O)R.sup.g4, -A.sup.f4-S(O).sub.rN(R.sup.h4)R.sup.i4, -A.sup.g4-S(O).sub.rOR.sup.j4, N.sub.3, N(R.sup.k4)R.sup.l4, N(H)CN, NO.sub.2, OR.sup.m4, SR.sup.n4 or =Q.sup.d4; each G.sup.4b, G.sup.4c and G.sup.4d independently represents halo, R.sup.a4, CN, -A.sup.a4-C(Q.sup.a4)R.sup.b4, -A.sup.b4-C(Q.sup.b4)N(R.sup.c4)R.sup.d4, -A.sup.c4-C(Q.sup.c4)OR.sup.e4, -A.sup.d4-S(O).sub.rR.sup.f4, -A.sup.e4-S(O).sub.rC(O)R.sup.g4, -A.sup.f4-S(O).sub.rN(R.sup.h4)R.sup.i4, -A.sup.g4-S(O).sub.rOR.sup.j4, N.sub.3, N(R.sup.k4)R.sup.l4, N(H)CN, NO.sub.2, OR.sup.m4, SR.sup.n4 or =Q.sup.d4; each A.sup.a4 to A.sup.g4 independently represents a single bond, N(R.sup.o4), C(Q.sup.e4)N(R.sup.p4) or O; each Q.sup.a4 to Q.sup.e4 independently represents O, S, NR.sup.q4 or N(OR.sup.r4); each R.sup.a4 and R.sup.f4 independently represents C.sub.1-6 alkyl optionally substituted by one or more groups independently selected from G.sup.6a, heterocycloalkyl optionally substituted by one or more groups independently selected from G.sup.6b, or aryl optionally substituted by one or more groups independently selected from G.sup.6c; each R.sup.b4, R.sup.c4, R.sup.d4, R.sup.e4, R.sup.g4, R.sup.h4, R.sup.i4, R.sup.j4, R.sup.k4, R.sup.l4, R.sup.m4, R.sup.n4, R.sup.q4 and R.sup.r4 independently represents H, C.sub.1-6 alkyl optionally substituted by one or more groups independently selected from G.sup.6a, or heterocycloalkyl optionally substituted by one or more groups independently selected from G.sup.6b; or any two R.sup.c4 and R.sup.d4, R.sup.h4 and R.sup.i4 and/or R.sup.k4 and R.sup.l4 are linked together to form, along with the nitrogen atom to which they are attached, a 3- to 6-membered ring, which ring optionally contains one further heteroatom and which ring optionally is substituted by one or more groups independently selected from halo, C.sub.1-3 alkyl optionally substituted by one or more halo, and O; each R.sup.o4 and R.sup.p4 independently represents H or C.sub.1-6 alkyl optionally substituted by one or more halo; each G.sup.5a and G.sup.6a independently represents halo or OC.sub.1-6 alkyl optionally substituted by one or more halo; each G.sup.5b, G.sup.6b and G.sup.6c represents halo, C.sub.1-6 alkyl optionally substituted by one or more halogens, or OC.sub.1-6 alkyl optionally substituted by one or more halo; each n independently represents 1 or 2; each p independently represents 1 or 2; each q independently represents 1 or 2; and each r independently represents 1 or 2.
8. The selenium compromised thioredoxin reductase-derived apoptotic protein (SecTRAP) forming agent for use according to any one of claims 1 to 3 or claim 7, wherein said SecTRAP forming agent is ##STR00119##
9. The selenium compromised thioredoxin reductase-derived apoptotic protein (SecTRAP) forming agent for use according to any one of claims 1 to 3, wherein said SecTRAP forming agent is a compound of formula III ##STR00120## or a pharmaceutically acceptable salt thereof, wherein: W represents C.sub.1 alkylene optionally substituted by one or more groups independently selected from R.sup.4; X represents O or S; Y represents C.sub.1-6 alkyl optionally substituted by one or more groups independently selected from G.sup.1a, heterocycloalkyl optionally substituted by one or more groups independently selected from G.sup.1b, aryl optionally substituted by one or more groups independently selected from G.sup.1c, or heteroaryl optionally substituted by one or more groups independently selected from G.sup.1d; Z represents O, S or NR.sup.5; R.sup.1 represents H, halo, R.sup.a1, CN, C(Q.sup.a1)R.sup.b1, C(Q.sup.b1)N(R.sup.c1)R.sup.d1, C(Q.sup.c1)OR.sup.e1, S(O).sub.nR.sup.f1, S(O).sub.pN(R.sup.g1)R.sup.h1, S(O).sub.pOR.sup.i1 or NO.sub.2; R.sup.2 represents H, halo, CN or N.sub.3; R.sup.3 represents H, halo or R.sup.j1; R.sup.4 represents halo or C.sub.1-6 alkyl optionally substituted by one or more groups independently selected from G.sup.1e; R.sup.5 represents H, R.sup.k1, OR.sup.l1 or N(R.sup.m1)R.sup.n1; Q.sup.a1 to Q.sup.c1 each independently represents O, S, NR.sup.o1 or N(OR.sup.p1); each R.sup.a1, R.sup.f1, R.sup.j1 and R.sup.k1 independently represents C.sub.1-6 alkyl optionally substituted by one or more groups independently selected from G.sup.2a, or heterocycloalkyl optionally substituted by one or more groups independently selected from G.sup.2b; each R.sup.b1, R.sup.c1, R.sup.d1, R.sup.e1, R.sup.g1, R.sup.h1, R.sup.i1, R.sup.l1, R.sup.m1, R.sup.n1, R.sup.o1 and R.sup.p1 independently represents H, C.sub.1-6 alkyl optionally substituted by one or more groups independently selected from G.sup.2a, or heterocycloalkyl optionally substituted by one or more groups independently selected from G.sup.2b; or any two R.sup.c1 and R.sup.d1, R.sup.g1 and R.sup.h1 and/or R.sup.m1 and R.sup.n1 are linked together to form, along with the nitrogen atom to which they are attached, a 3- to 6-membered ring, which ring optionally contains one further heteroatom and which ring optionally is substituted by one or more groups independently selected from halogen, C.sub.1-3 alkyl optionally substituted by one or more halogens, and O; each G.sup.1a, G.sup.1b, G.sup.1c and G.sup.1d represent halogen, R.sup.a2, CN, -A.sup.a1-C(Q.sup.a2)R.sup.b2, -A.sup.b1-C(Q.sup.b2)N(R.sup.c2)R.sup.d2, -A.sup.c1-C(Q.sup.c2)OR.sup.e2, -A.sup.d1-S(O).sub.qR.sup.f2, -A.sup.e1-S(O).sub.qC(O)R.sup.g2, -A.sup.f1-S(O).sub.qN(R.sup.h2)R.sup.i2, -A.sup.g1-S(O).sub.qOR.sup.j2, N.sub.3, N(R.sup.k2)R.sup.l2, N(H)CN, NO.sub.2, OR.sup.m2, SR.sup.n2 or =Q.sup.d2; A.sup.a1 to A.sup.g1 each independently represents a single bond, N(R.sup.6), C(Q.sup.e2)N(R.sup.7) or O; Q.sup.a2 to Q.sup.e2 each independently represents O, S, NR.sup.o2 or N(OR.sup.p2); each R.sup.6 and R.sup.7 independently represents H or C.sub.1-6 alkyl optionally substituted by one or more F; each R.sup.a2 and R.sup.f2 independently represents C.sub.1-6 alkyl optionally substituted by one or more groups independently selected from G.sup.3a or heterocycloalkyl optionally substituted by one or more groups independently selected from G.sup.3b; each R.sup.b2, R.sup.c2, R.sup.d2, R.sup.e2, R.sup.g2, R.sup.h2, R.sup.i2, R.sup.j2, R.sup.k2, R.sup.12, R.sup.m2, R.sup.n2, R.sup.o2 and R.sup.p2 independently represents H, C.sub.1-6 alkyl optionally substituted by one or more groups independently selected from G.sup.3a or heterocycloalkyl optionally substituted by one or more groups independently selected from G.sup.3b; or any two R.sup.c2 and R.sup.d2, R.sup.h2 and R.sup.i2 and/or R.sup.k2 and R.sup.l2 are linked together to form, along with the nitrogen atom to which they are attached, a 3- to 6-membered ring, which ring optionally contains one further heteroatom and which ring optionally is substituted by one or more groups independently selected from halogen, C.sub.1-3 alkyl optionally substituted by one or more halogens, and O; each G.sup.1e independently represents halo, R.sup.a2, CN, N(R.sup.a3)R.sup.b3, OR.sup.c3 or SR.sup.d3; R.sup.a3, R.sup.b3, R.sup.c3 and R.sup.d3 each independently represents H or C.sub.1-6 alkyl optionally substituted by one or more F; or R.sup.a3 and R.sup.b3 are linked together to form, along with the nitrogen atom to which they are attached, a 3- to 6-membered ring, which ring optionally contains one further heteroatom and which ring optionally is substituted by one or more groups independently selected from fluoro, C.sub.1-3 alkyl optionally substituted by one or more fluoro, and O; each G.sup.2a and G.sup.2b independently represents halo, CN, N(R.sup.a4)R.sup.b4, OR.sup.c4, SR.sup.d4 or O; each R.sup.a4, R.sup.b4, R.sup.c4 and R.sup.d4 independently represents H or C.sub.1-6 alkyl optionally substituted by one or more F; or R.sup.a4 and R.sup.b4 are linked together to form, along with the nitrogen atom to which they are attached, a 3- to 6-membered ring, which ring optionally contains one further heteroatom and which ring optionally is substituted by one or more groups independently selected from fluoro, C.sub.1-3 alkyl optionally substituted by one or more fluoro, and O; each G.sup.3a and G.sup.3b independently represents halo, CN, N(R.sup.a5)R.sup.b5, OR.sup.c5, SR.sup.d5 or O; each R.sup.a5, R.sup.b5, R.sup.c5 and R.sup.d5 independently represents H or C.sub.1-6 alkyl optionally substituted by one or more fluoro; or R.sup.a5 and R.sup.b5 are linked together to form, along with the nitrogen atom to which they are attached, a 3- to 6-membered ring, which ring optionally contains one further heteroatom and which ring optionally is substituted by one or more groups independently selected from fluoro, C.sub.1-3 alkyl optionally substituted by one or more fluoro, and O; each n independently represents 0, 1 or 2, each p independently represents 1 or 2, each q independently represents 1 or 2.
10. The selenium compromised thioredoxin reductase-derived apoptotic protein (SecTRAP) forming agent for use according to any one of claims 1 to 3, wherein said SecTRAP forming agent is Iniparib.
11. The selenium compromised thioredoxin reductase-derived apoptotic protein (SecTRAP) forming agent for use according to any one of claims 1 to 3, wherein said SecTRAP forming agent is Auranofin.
12. The selenium compromised thioredoxin reductase-derived apoptotic protein (SecTRAP) forming agent for use according to any one of claims 1 to 11, wherein said SecTRAP forming agent is administered systemically.
13. The selenium compromised thioredoxin reductase-derived apoptotic protein (SecTRAP) forming agent for use according to any one of claims 1 to 12, wherein the SecTRAP forming agent is administered intravenously, intraperitoneally or intrathecally.
14. The selenium compromised thioredoxin reductase-derived apoptotic protein (SecTRAP) forming agent for use according to any one of claims 1 to 13, wherein said cancer overexpresses thioredoxin reductase (TrxR) or thioredoxin (Trx) or Protein Disulphide Isomerase (PDI), either individually or in any combination of the three.
15. The selenium compromised thioredoxin reductase-derived apoptotic protein (SecTRAP) forming agent for use according to any one of claims 1 to 14, wherein said cancer is breast cancer, brain cancer, advanced cancer or metastatic cancer.
16. The selenium compromised thioredoxin reductase-derived apoptotic protein (SecTRAP) forming agent for use according to any one of claims 1 to 15, wherein subjects with said cancer overexpress thioredoxin reductase (TrxR) or thioredoxin (Trx) or Protein Disulphide Isomerase (PDI), either individually or in any combination of the three in serum or blood.
17. A combination of (i) a selenium compromised thioredoxin reductase-derived apoptotic protein (SecTRAP) forming agent; and (ii) an immunostimulatory agent for use in treating cancer in a subject.
18. The combination for use according to claim 17, wherein said selenium compromised thioredoxin reductase-derived apoptotic protein (SecTRAP) forming agent is as defined in any one of claims 4 to 11.
19. The combination for use according to claim 17 or claim 18, wherein said selenium compromised thioredoxin reductase-derived apoptotic protein (SecTRAP) forming agent is administered as defined in claim 12 or claim 13.
20. The combination for use according to any one of claims 17 to 19, wherein said cancer or said anti-cancer immune response or said subject is as defined in any one of claims 1, 2, 3, 14, 15 or 16.
21. The combination for use according to any one of claims 17 to 20, wherein said immunostimulatory agent is an immune checkpoint inhibitor.
22. The combination for use according to claim 21, wherein said immune checkpoint inhibitor is an anti-PD-L1 antibody or an anti-PD1 antibody.
23. A combination of (i) a selenium compromised thioredoxin reductase-derived apoptotic protein (SecTRAP) forming agent; and (ii) a Thioredoxin antibody for use in treating cancer in a subject.
24. The combination for use according to claim 23, wherein said use has the features as defined in any one of claims 18 to 20.
25. The combination for use according to 22, wherein said anti-PD1 antibody is Pembrolizumab.
26. A combination of (i) a selenium compromised thioredoxin reductase-derived apoptotic protein (SecTRAP) forming agent; and (ii) a targeted therapeutic agent or a cytotoxic therapeutic agent, for use in treating cancer in a subject.
27. The combination for use according to claim 26, wherein said targeted therapeutic agent is Imatinib, Bevacizumab or Everolimus.
28. The combination for use according to claim 26, wherein said cytotoxic therapeutic agent is carboplatin, a taxol or a vinca alkaloid.
29. The selenium compromised thioredoxin reductase-derived apoptotic protein (SecTRAP) forming agent for use according to any one of claims 1-16 or the combination for use according to any one of claims 17-28, wherein the selenium compromised thioredoxin reductase-derived apoptotic protein (SecTRAP) forming agent is ##STR00121##
30. A method of treating an immune cell infiltrated cancer in a subject, said method comprising administering to a subject in need thereof a therapeutically effective amount of a selenium compromised thioredoxin reductase-derived apoptotic protein (SecTRAP) forming agent, wherein said agent stimulates an anti-cancer immune response.
31. A method of treating cancer in a subject, said method comprising administering to a subject in need thereof a combination of a therapeutically effective amount of a selenium compromised thioredoxin reductase-derived apoptotic protein (SecTRAP) forming agent and an immunostimulatory agent.
32. A method of treating cancer in a subject, said method comprising administering to a subject in need thereof a combination of a therapeutically effective amount of a selenium compromised thioredoxin reductase-derived apoptotic protein (SecTRAP) forming agent and a thioredoxin antibody.
33. A method of treating cancer in a subject, said method comprising administering to a subject in need thereof a combination of a therapeutically effective amount of a selenium compromised thioredoxin reductase-derived apoptotic protein (SecTRAP) forming agent and a targeted therapeutic agent or a cytotoxic therapeutic agent.
34. The method of any one of claims 30 to 33, wherein said method has features as defined in any one of claims 1 to 29.
35. Use of a selenium compromised thioredoxin reductase-derived apoptotic protein (SecTRAP) forming agent in the manufacture of a medicament for treating an immune cell infiltrated cancer wherein said agent stimulates an anti-cancer immune response.
36. Use of a selenium compromised thioredoxin reductase-derived apoptotic protein (SecTRAP) forming agent in the manufacture of a medicament for treating cancer wherein said treatment further comprises the administration of an immunostimulatory agent.
37. Use of a selenium compromised thioredoxin reductase-derived apoptotic protein (SecTRAP) forming agent in the manufacture of a medicament for treating cancer wherein said treatment further comprises the administration of a thioredoxin antibody.
38. Use of a selenium compromised thioredoxin reductase-derived apoptotic protein (SecTRAP) forming agent in the manufacture of a medicament for treating cancer wherein said treatment further comprises the administration of a targeted therapeutic agent or a cytotoxic therapeutic agent.
39. The use of any one of claims 35 to 38, wherein said use has features as defined in any one of claims 1 to 29.
Description
[0577] The invention will be further described with reference to the following non-limiting Example with reference to the following drawings in which:
[0578]
[0579]
[0580]
[0581]
[0582]
[0583]
[0584]
[0585]
[0586]
[0587] Athymic nude mice were inoculated orthotopically with 510.sup.6 MDA-MB-231 breast cancer cells into the mammary fat pad and randomized for treatment when tumors reached an average volume of 80-120 mm.sup.3 (N=12 in each group). Mice were treated with 10 mg/kg OT-1096 via i.v. injection or with vehicle i.v., once a day using a 5 day on two day off (5/2) dosing regimen for the duration of the experiment. Xenograft tumor volume was assessed using caliper measurements for 25 days. Data is represented as mean tumor volumeSEM. Statistical significance (p<0.05) was determined using a Two-way repeated measures ANOVA with Sidak's multiple comparison test. Mean tumor volume for mice treated with OT-1096 was statistically significant compared to vehicle at day 25.
[0588]
[0589] Female BALB/c immunocompetent mice were implanted with 110.sup.5 4T1-luc2 murine tumor cells into the mammary fat pad. Upon growth of the tumors between 60-90 mm.sup.3 the animals were selected for imaging. Mice were randomized and enrolled for treatment based on imaging flux values. Mice were treated once daily with 5 mg/kg of either OT-1096 or vehicle control using a 5/2 (five days on, two days off) dosing protocol. Upon days 1, 8, and 15 whole body imaging was performed on the mice to follow tumor cell luminescence. Analysis consists of primary tumor luminescence in mice. Each mouse was normalized to its own baseline luminescence from day 1. Mice that did not have metastasis present at the first day of imaging were included in the study (N=9). Data is represented as meanSEM. Statistical significance (p<0.05) was determined using a Mann-Whitney test. The relative luminescence flux for mice treated with OT-1096 was significant compared to vehicle at day 15.
[0590]
[0591]
[0592]
[0593]
EXAMPLE 1
General Methods for Chemistry
[0594] All reagents were obtained commercially and used as received. All reactions were run under a nitrogen atmosphere. Reactions were monitored by thin layer chromatography (TLC) with detection using the appropriate staining reagent or by ESI-LCMS (positive ion mode with UV detection at 254 nm). All .sup.1H NMRs were recorded on Bruker Advance 400 MHz spectrometer with multinuclear probe in the appropriate solvent. .sup.1H NMR and .sup.13C NMR spectra were recorded on Bruker Avance 400 (.sup.1H NMR: 400 MHz; .sup.13C NMR: 100 MHz) using tetramethylsilane as internal standard for .sup.1H NMR spectra in CDCl.sub.3. Residual solvent peak for DMSO-d.sub.6 (39.43 ppm), or CDCl.sub.3 (77.00 ppm) for .sup.13C NMR spectra. The residual solvent peak of DMSO-d.sub.6 in .sup.1H NMR is 2.5 ppm. Abbreviations used are: s, singlet; d, doublet; t, triplet; m, multiplet; br, broad singlet. Coupling constants are expressed in Hz.
For all reactions, analytical grade solvents were used. All moisture sensitive reactions were carried out in oven-dried glassware (70 C.).
For crude LCMS monitoring, mass spectra were obtained with API 2000 mass spectrophotometer from Applied Biosystems. Samples were infused at 2 ul/min, and spectra were obtained in positive or negative ionization mode.
Precoated aluminum sheets (Merck, 254 nm) were used for TLC. Column chromatography was performed on Swambe silica gel 100-200 mesh.
Materials and Methods
Determination of Sec-Dependent TrxR Activity (DTNB Assay)
[0595] Sec-dependent TrxR activity was assessed using a 5-5-dithiobis-(2-nitrobenzoic acid) (DTNB) assay. Multiple concentrations of compounds were incubated for various time points in reaction buffer, consisting of 50 mM Tris pH 7.5 with 2 mM EDTA and 0.1 mg/mL bovine serum albumin, and containing recombinant rat TrxR and 250 M NADPH. 2.5 mM DTNB was added to each well and TNB.sup. production was followed at OD.sub.412. Activity was determined following the change in TNB.sup. over time and normalized to DMSO only (Vehicle) and TrxR lacking (blank) controls. The amino acid sequence of rat TrxR1 is available in Gen Bank, with accession number AAF32362.1.
Glutathione Reductase (GR) Activity Assay
[0596] GR activity was determined incubating compounds at various concentrations with GR from baker's yeast and 250 M NADPH for 15 minutes, whereupon 10 mM oxidized glutathione (GSSG) was added to each well and NADPH consumption was followed at OD.sub.340. Activity was determined following the consumption of NADPH over time and normalized to DMSO only (Vehicle) and GR lacking (blank) controls.
Determination of Sec-Independent SecTRAP Activity (Juglone Assay)
[0597] SecTRAP forming capabilities of compounds with TrxR was determined using the Juglone Assay. Compounds were incubated in the presence of recombinant TrxR and 250 M NADPH for 15 minutes at concentrations fully inhibiting enzyme activity in the DTNB assay. 100 M juglone was then added to each well and NADPH consumption was followed at OD.sub.340 to determine sustained reductive capacity at the N-terminus of TrxR (SecTRAP Activity). Activity was determined following the consumption of NADPH over time and normalized to DMSO only (Vehicle) and TrxR lacking (blank) controls.
NADPH-Dependent TrxR Activity
[0598] Irreversible inhibition of TrxR was determined by incubating compounds in the presence of recombinant TrxR, with or without 250 M NADPH, for 90 minutes. Aliquots were used in a DTNB assay to determine inhibition of enzyme activity. Compounds were incubated in the presence of TrxR with 250 M NADPH at concentrations used to fully inhibit Sec-dependent TrxR activity, as confirmed using the DTNB assay. Incubation samples were then added to a reaction buffer containing 250 M NADPH and 100 M juglone, whereupon NADPH consumption was followed at OD.sub.340 to determine sustained SecTRAP activity.
Cellular Assays
Cell Culture
[0599] Cell cultures were maintained at 37 C. in 5% CO.sub.2 in medium containing 20 mg/mL penicillin/streptomycin, 2 mM L-glutamine, and 10% fetal bovine serum (FBS). Experiments were performed in triplicate in medium containing 10% FBS and 25 nM sodium selenite. All compounds were diluted in DMSO, 0.01% final concentration.
Cell Culture Media
[0600] MDA-MB-453 (ATCC HTB-131) cells were grown in media with sodium pyruvate. MDA-MB-231 cells (ATCC HTB-26) were grown in DMEM media or L-15 supplemented with Glutamax (1). U87-MG (ATCC HTB-14) and MDA-MB-468 (ATCC HTB-132) cells were cultured in DMEM supplemented with Glutamax (1). NB-4 (DSMZ ACC-207) cells were cultured in RPMI 1640.
CellQuanti-Blue Cell Viability Assay
[0601] Cells were plated at 2000 cells/well into 96-well plates in media containing 10% FBS. The following day, compounds were added to each well and incubated for 72 hrs. CellQuanti-Blue cell viability reagent was added to each well and the plates were subsequently incubated at 37 C. for 3 hours. Viability was determined fluorometrically using an Enspire plate reader (G.E. Healthcare, USA) excitation: 530 nm, emission: 590 nm. Viability was normalized to DMSO controls and blank wells with media.
Alamar-Blue Cell Viability Assay
[0602] MDA-MB-231 cells were plated 2000 cells/well in 96-well black optical plates in the presence of 10% FBS media containing 25 nM selenite. The following day cells were treated with various concentrations of compounds (0.1% DMSO final) and incubated for 72 hrs. After the incubation Alamar Blue reagent was added to each well and incubated for additional 3 hrs. Fluorescence was read ex:530 nm/em:590 nm, and percent of viability was determined using DMSO vehicle and no cell (blank) controls.
MTT Cell Viability Assay
[0603] Breast cancer and glioblastoma cell lines were plated 4000 cells/well in 96-well plates in the presence of 10% FBS media. The following day cells were treated with various concentrations of the example compounds (0.1% DMSO final) and incubated for 72 hrs. After the incubation an MTT assay was performed to access cell viability. Percent of viability was determined using DMSO vehicle and no cell (blank) controls.
Determination of Trx Intracellular and Extracellular Levels
[0604] The human mammary carcinoma cell line MDA-MB-231 was obtained from the American Type Culture Collection (ATCC; Manassas, Va.) and cultured in Leibovitz's L-15 medium (Thermo Fisher Scientific, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Thermo Fisher Scientific, USA), 10 mM HERBS (Sigma), 25 mM sodium bicarbonate (Sigma), 1 Glutamax (Sigma), 100 IU/mL penicillin and 100 g/ml streptomycin (Sigma) in a 5% CO.sub.2 atmosphere at 37 C. Briefly, cells were seeded at 7500 cells/well in 96-well plates. After 24 h, the cells were treated either with different concentrations of test compounds or DMSO (0.5%; vehicle control) respectively and further incubated at 37 C. in a CO.sub.2 incubator. Culture supernatants were collected at different time points, like, immediately after cell plating (24 h), 0 h (at the time of compound addition), and 1 h, 6 h, 12 h, 24 h, 48 h, 72 h and 96 h following compound addition. Subsequently, cells were lysed from corresponding wells with 0.1% Triton X-100 and the lysates were collected. Trx levels were measured in both the culture supernatants and cell lysates by ELISA using TXN (Human) ELISA Kit [Abnova; Cat no: KA0535] following manufacturer instruction. A standard curve was prepared using different concentrations of the Trx standard (provided in the kit) with the help of GraphPad Prism software (version 5.0; La Jolla, Calif., USA). Trx concentrations for the unknown samples were calculated from the standard curve. The data were expressed as the means of three replicates.
In Vivo Assays
Efficacy of OT-1000 Towards Inhibition of MDA-MB-231 Xenograft Tumor Growth in Athymic Nude Mice
[0605] Athymic nude mice were inoculated orthotopically with 510.sup.6 MDA-MB-231 breast cancer cells into the mammary fat pad, and randomized for treatment when tumors reached an average volume of 80-120 mm.sup.3 (N=12 in each group). Mice were treated with 10 mg/kg OT-1000 via i.v. injection or with vehicle i.v., once a day for the first five days, followed by two days of no treatment, and then three times per week for two weeks. Xenograft tumor volume was assessed using caliper measurements for 22 days. Athymic nude mouse model is described by Richmond A, and Su Y., Disease Models & Mechanisms. 2008; 1(2-3):78-82.
Efficacy of OT-1129 and Iniparib Towards Inhibition of MDA-MB-231 Xenograft Tumor Growth in Athymic Nude Mice
[0606] Athymic nude mice were inoculated orthotopically with 510.sup.6 MDA-MB-231 breast cancer cells into the mammary fat pad, and randomized for treatment when tumors reached an average volume of 80-120 mm.sup.3 (N=12 in each group). Mice were treated with 25 mg/kg OT-1129 via i.v. injection, 25 mg/kg Iniparib via i.p. injection, or with vehicle i.v., once a day for the first five days, followed by two days of no treatment, then three times per week for two weeks and four days totaling 12 doses. Xenograft tumor volume was assessed using caliper measurements for 25 days.
MDA-MB-231 Xenograft Tumor Growth in Immunodeficient Athymic Nude Mice Treated with OT-1096 or Vehicle Control.
Athymic nude mice were inoculated orthotopically with 510.sup.6 MDA-MB-231 breast cancer cells into the mammary fat pad and randomized for treatment when tumors reached an average volume of 80-120 mm.sup.3 (N=12 in each group). Mice were treated with 10 mg/kg OT-1096 via i.v. injection or with vehicle i.v., once a day using a 5 day on two day off (5/2) dosing regimen for the duration of the experiment. Xenograft tumor volume was assessed using caliper measurements for 25 days. Data is represented as mean tumor volumeSEM. Statistical significance (p<0.05) was determined using a Two-way repeated measures ANOVA with Sidak's multiple comparison test. Mean tumor volume for mice treated with OT-1096 was statistically significant compared to vehicle at day 25.
Relative Luminescence Flux in Primary 4T1-Luc2 Immunocompetent Tumors Implanted into the Mammary Fat Pad of BALB/C Mice and Treated with OT-1096 or Vehicle Control.
Female BALB/c immunocompetent mice were implanted with 110.sup.5 4T1-luc2 murine tumor cells into the mammary fat pad. Upon growth of the tumors between 60-90 mm.sup.3 the animals were selected for imaging. Mice were randomized and enrolled for treatment based on imaging flux values. Mice were treated once daily with 5 mg/kg of either OT-1096 or vehicle control using a 5/2 (five days on, two days off) dosing protocol. Upon days 1, 8, and 15 whole body imaging was performed on the mice to follow tumor cell luminescence. Analysis consists of primary tumor luminescence in mice. Each mouse was normalized to its own baseline luminescence from day 1. Mice that did not have metastasis present at the first day of imaging were included in the study (N=9). Data is represented as meanSEM. Statistical significance (p<0.05) was determined using a Mann-Whitney test. The relative luminescence flux for mice treated with OT-1096 was significant compared to vehicle at day 15.
Primary Tumor Growth of TM00098 Patient Derived Xenografts-Triple Negative Breast Cancer in Immunocompetent Humanized NSG Mice (Hu-CD34-NSG) Treated with OT-1096 or Vehicle Control.
Female NSG mice were implanted with human CD34+ hematopoietic stem cells from multiple donors and the level of human CD45+ cells were measured in the peripheral blood 12 weeks post engraftment. Mice with >25% human CD45+ cells in the peripheral blood were determined to have a humanized immune system (Hu-CD34-NSG) mice and were enrolled into the study. The Hu-CD34-NSG mice were implanted with TM00098 patient-derived xenografts (PDX) subcutaneously on the right flank. The TM00098 PDX cancer cells originate from a primary tumor of a patient suffering from a grade 3 TNBC invasive ductal carcinoma. When the tumors reached a volume between 60-120 mm.sup.3 mice were treated with either 10 mg/kg OT-1096 three times a week intravenously (donor 5243 n=5, donor 5252 n=7) or with a vehicle three times a week intravenously (donor 5243 n=3, donor 5252 n=2). In case of tail vein swelling when the test substance or vehicle could not be administered intravenously, Intraperitoneal injection was applied. Tumor volume was measured using a digital caliper two times a week for the duration of the study. Animals that reached a body condition score of 2, a body weight loss of 20% or a tumor volume >2000 mm.sup.3 were euthanized before study terminus. Animals with ulcerated tumors were also euthanized before study terminus. Data is represented as mean tumor volumeSEM. Statistical significance (p<0.05) was determined using a Two-way repeated measures ANOVA with Sidak's multiple comparison test. Mean tumor volume for mice treated with OT-1096 was statistically significant compared to vehicle at day 24 and 28 for CD34+ donor 5243 and at day 31 for CD34+ donor 5252.
Synthesis of Compounds
Synthesis of OT-1000 (2-((4-Chlorophenyl)sulfonyl)-6-methoxy-3-nitropyridine)
[0607] ##STR00106##
To a stirred solution of 6-methoxy-2-chloro-3-nitro pyridine(1; 5.0 g, 26.596 mmol) in dimethyl acetamide (75 mL) was added sodium 4-chlorobenzene-sulphinate (2; 7.92 g, 39.894 mmol) and tetra-N-butylammonium chloride (2.22 g, 7.979 mmol) and Cone HCl 0.75 mL at room temperature. The reaction mixture was stirred at 80 C. for 1 hour. Progress of reaction was monitored by LCMS. The whole reaction mixture was poured on crushed ice to get solid. This solid compound was filtered through sintered funnel and thoroughly dried under vacuum to isolate 2-((4-Chlorophenyl)sulfonyl)-6-methoxy-3-nitropyridine as desired product (7.1 g, 81.21%). .sup.1H NMR (400 MHz, CDCl.sub.3) 8.10 (d, J=8.6 Hz, 1H), 8.0 (d, J=7.9 Hz, 2H), 7.56 (d, J=7.9 Hz, 2H), 6.95 (d, J=8.7 Hz, 1H), 3.69 (s, 3H). LCMS [m/z (M+H)+=(Calculated for C.sub.12H.sub.9N.sub.2O.sub.5SCI+H: 329) found: 329], Purity at =220 nm: 98.73%
Synthesis of OT-1011 (2-Benzylsulfonyl-6-methoxy-3-nitropyridine)
[0608] ##STR00107##
Step-1
[0609] To a solution of 6-methoxy-2-chloro-3-nitro pyridine (1; 5.0 g, 26.596 mmol) in dimethylformamide (20 mL) was added potassium carbonate (4.441 g, 32.181 mmol) and benzyl mercaptan (3.595 g, 28.989 mmol) at room temperature. The reaction mixture was stirred for overnight at room temperature. Progress of reaction was monitored by LCMS. The reaction mixture was quenched with ice cold water (30 ml) and was extracted with ethyl acetate (300 mL). The organic layer was washed with water (350 mL) followed by brine (150 mL). The organic layer was dried over anhydrous sodium sulphate and was evaporated under reduced pressure to give the crude product which was purified by column chromatography eluting with 2% ethyl acetate in hexane affording the step-1 compound (3.2 g, 43.55%) as yellow solid.
Step-2
[0610] To a solution of step-1 compound (Step-1; 15.0 g, 54.348 mmol) in dichloromethane (200 mL) was added m-chloro per benzoic acid (32.71 g, 190.17 mmol) at room temperature. The reaction mixture was stirred at room temperature for overnight.
Progress of reaction was monitored by LCMS. The reaction mixture was diluted with dichloromethane (500 mL) and washed with saturated sodium sulphite solution (2100 mL) followed by brine (1200 mL). The organic layer was dried over anhydrous sodium sulphate and was evaporated under reduced pressure to give the crude product which was purified by column chromatography eluting with 10% ethyl acetate in hexane affording the title compound OT-1011 (8.0 g, 92.15%) as off white solid. .sup.1H NMR (400 MHz, CDCl.sub.3) 8.06 (d, J=8.6 Hz, 1H), 7.38 (m, 5H), 6.99 (d, J=8.7 Hz, 1H), 4.82 (s, 2H), 3.96 (s, 3H). LCMS [m/z (M+H)+=(Calculated for C.sub.13H.sub.12N.sub.2O.sub.5S+H: 309) found: 309], Purity at =220 nm: 100%
Synthesis of OT-1012 (6-Methoxy-3-nitro-2-(pyridin-2-ylsulfonyl)pyridine)
[0611] ##STR00108##
Step-1
[0612] To a solution of 6-methoxy-2-chloro-3-nitro pyridine (1; 5.0 g, 26.596 mmol) in dimethylformamide (20 mL) was added potassium carbonate (4.441 g, 32.181 mmol) and 2-mercapto pyridine (3.22 g, 28.989 mmol) at room temperature. The reaction mixture was stirred for overnight at room temperature. Progress of reaction was monitored by LCMS. The reaction mixture was quenched with ice cold water (30 ml) and was extracted with ethyl acetate (300 mL). The organic layer was washed with water (350 mL) followed by brine (150 mL). The organic layer was dried over anhydrous sodium sulphate and was evaporated under reduced pressure to give the crude product which was purified by column chromatography eluting with 5% ethyl acetate in hexane affording the step-1 compound (2.5 g, 35.7%) as yellow solid.
Step-2
[0613] To a solution of step-1 compound (6.36 g, 24.183 mmol) in dichloromethane (150 mL) was added m-chloro per benzoic acid (14.55 g, 84.639 mmol) at room temperature. The reaction mixture was stirred at room temperature for overnight. Progress of reaction was monitored by LCMS. The reaction mixture was diluted with dichloromethane (300 mL) and washed with saturated sodium sulphite solution (250 mL) followed by brine (150 mL). The organic layer was dried over anhydrous sodium sulphate and was evaporated under reduced pressure to give the crude product which was purified by column chromatography eluting with 5% ethyl acetate in hexane affording the title compound OT-1012 (2.5 g, 36%) as off white solid. .sup.1H NMR (400 MHz, CDCl.sub.3) 8.70-8.69 (m, 1H), 8.29-8.26 (m, 2H), 8.05-8.01 (m, 1H), 7.57-7.54 (m, 1H), 6.98 (d, J=8.8 Hz, 1H), 3.66 (s, 3H). LCMS [m/z (M+H)+=(Calculated for C.sub.11H.sub.9N.sub.3O.sub.5S+H: 296) found: 296], Purity at =220 nm: 98.76%
Synthesis of QT-1096 (6-methoxy-3-nitro-2-(octylsulfonyl)pyridine)
[0614] ##STR00109##
Step-1
[0615] To a solution of 6-methoxy-2-chloro-3-nitro pyridine(1; 250 mg, 0.1.33 mmol) in dimethylformamide (5 mL) was added potassium carbonate (220 mg, 1.596 mmol) and octane-1-thiol (213 mg, 1.463 mmol) at room temperature. The reaction mixture was stirred overnight at room temperature. Progress of reaction was monitored by LCMS. The reaction mixture was quenched with ice cold water (10 ml) and was extracted with ethyl acetate (20 mL). The organic layer was washed with water (310 mL) followed by brine (110 mL). The organic layer was dried over anhydrous sodium sulphate and was evaporated under reduced pressure to give the crude product which was purified by column chromatography eluting with 4% ethyl acetate in hexane affording the step-1 compound (150 mg, 37.8%) as yellow solid.
Step-2
[0616] To a solution of step-1 compound (150 mg, 0.798 mmol) in dichloromethane (10 mL) was added m-chloro per benzoic acid (494 mg, 2.872 mmol) at room temperature. The reaction mixture was stirred at room temperature overnight. Progress of reaction was monitored by LCMS. The reaction mixture was diluted with dichloromethane (20 mL) and washed with saturated sodium sulphite solution (220 mL) followed by brine (120 mL). The organic layer was dried over anhydrous sodium sulphate and was evaporated under reduced pressure to give the crude product which was purified by column chromatography eluting with 20% ethyl acetate in hexane affording the title compound OT-1096 (46 mg, 27.69%) as off white solid. .sup.1H-NMR [CDCl3, 8.13 (d, J=9 Hz, 1H), 7.04 (d, J=8 Hz, 1H), 4.07 (s, 3H), 3.56 (t, J=8 Hz, 2H), 1.89-1.86 (m, 2H), 1.46-1.44 (m, 2H), 1.27-1.25 (m, 8H), 0.86 (m, 3H)]. LCMS [m/z (M+H)+=(Calculated for C.sub.14H.sub.22N.sub.2O.sub.5S+H: 331) found: 331], Purity at =220 nm: 100%
Synthesis of OT-1113 (methyl 3-((6-methoxy-3-nitropyridin-2-yl)sulfonyl)propanoate)
[0617] ##STR00110##
Step-1
[0618] To a solution of 6-methoxy-2-chloro-3-nitro pyridine (250 mg, 1.33 mmol) in dimethylformamide (5 mL) was added potassium carbonate (220 mg, 1.59 mmol) and 3-Mercapto-propionic acid methyl ester (175 mg, 1.46 mmol) at room temperature. The reaction mixture was stirred overnight at room temperature. Progress of reaction was monitored by LCMS. The reaction mixture was quenched with ice cold water (10 ml) and was extracted with ethyl acetate (20 mL). The organic layer was washed with water (310 mL) followed by brine (110 mL). The organic layer was dried over anhydrous sodium sulphate and was evaporated under reduced pressure to give the crude product which was purified by column chromatography eluting with 5% ethyl acetate in hexane affording the step-1 compound (148 mg, 43.42%) as yellow solid.
Step-2
[0619] To a solution of step-1 compound (148 mg, 0.54 mmol) in dichloromethane (10 mL) was added m-chloro per benzoic acid (477.65 mg, 2.72 mmol) at room temperature. The reaction mixture was stirred at room temperature overnight. Progress of reaction was monitored by LCMS. The reaction mixture was diluted with dichloromethane (20 mL) and washed with saturated sodium sulfite solution (220 mL) followed by brine (120 mL). The organic layer was dried over anhydrous sodium sulphate and was evaporated under reduced pressure to give the crude product which was purified by column chromatography eluting with 8% ethyl acetate in hexane affording the title compound OT-1113 (104 mg, 62.87%) as off white solid. .sup.1H-NMR [DMSO-d6, 8.49 (d, J=9 Hz, 1H), 7.37 (d, J=9 Hz, 1H), 4.01-3.97 (m, 5H), 3.59 (s, 3H), 2.84 (t, J=7 Hz, 2H)]. LCMS [m/z (M+H)+=(Calculated for C.sub.10H.sub.12N.sub.2O.sub.7S+H: 305) found: 305], Purity at =220 nm: 98.40%
Synthesis of OT-1129 (2-(ethylsulfonyl)-6-methoxy-3-nitropyridine)
[0620] ##STR00111##
Step-1
[0621] To a solution of 6-methoxy-2-chloro-3-nitro pyridine (250 mg, 1.33 mmol) in dimethylformamide (5 mL) was added potassium carbonate (220 mg, 1.59 mmol) and ethane thiol (1.33 mg, 2.66 mmol) at room temperature. The reaction mixture was stirred overnight at room temperature. Progress of reaction was monitored by LCMS. The reaction mixture was quenched with ice cold water (10 ml) and was extracted with ethyl acetate (20 mL). The organic layer was washed with water (310 mL) followed by brine (110 mL). The organic layer was dried over anhydrous sodium sulphate and was evaporated under reduced pressure to give the crude product which was purified by column chromatography eluting with 2% ethyl acetate in hexane affording the step-1 compound (187 mg, 69.73%) as yellow solid
Step-2
[0622] To a solution of step-1 compound 3 (187 mg, 1.16 mmol) in dichloromethane (10 mL) was added m-chloro per benzoic acid (602 mg, 3.5 mmol) at room temperature. The reaction mixture was stirred at room temperature overnight. Progress of reaction was monitored by LCMS. The reaction mixture was diluted with dichloromethane (20 mL) and washed with saturated sodium sulfite solution (220 mL) followed by brine (120 mL). The organic layer was dried over anhydrous sodium sulphate and was evaporated under reduced pressure to give the crude product which was purified by column chromatography eluting with 12% ethyl acetate in hexane affording the title compound OT-1129 (108 mg, 50.18%) as off white solid. .sup.1H-NMR [DMSO-d6, 8.48 (d, J=9 Hz, 1H), 7.36 (d, J=9 Hz, 1H), 4.02 (s, 3H), 3.72-3.67 (m, 2H), 1.26 (t, J=7 Hz, 3H)]. LCMS [m/z (M+H)+=(Calculated for C.sub.8H.sub.10N2O.sub.5S+H: 247) found: 247], Purity at =220 nm: 100%
Synthesis of OT-1131 ((2-(ethylsulfinyl)-6-methoxy-3-nitropyridine)
[0623] ##STR00112##
Step-1
[0624] To a solution of 6-methoxy-2-chloro-3-nitro pyridine(1; 5 g, 26.59 mmol) in dimethylformamide (50 mL) was added potassium carbonate (4.44 g, 31.95 mmol) and ethane thiol (2,1.81 g, 29.25 mmol) at room temperature. The reaction mixture was stirred overnight at room temperature. Progress of reaction was monitored by LCMS. The reaction mixture was quenched with ice cold water (35 ml) where in solid precipitated from the reaction mixture. The solid were filtered and washed with ice cold water (330 mL) and was dried under reduced pressure affording the compound-3 (4.8 g, 84.24%) as yellow solid.
Step-2
[0625] To a solution of compound-3 (4.8 g, 22.42 mmol) in dichloromethane (100 mL) was added m-chloro per benzoic acid (8.84 g, 51.40 mmol) at room temperature. The reaction mixture was stirred at room temperature overnight. Progress of reaction was monitored by LCMS. The reaction mixture was diluted with dichloromethane (20 mL) and washed with saturated sodium sulphite solution (280 mL) followed by brine (180 mL). The organic layer was dried over anhydrous sodium sulphate and was evaporated under reduced pressure to give the crude product which was purified by column chromatography eluting with 80% ethyl acetate in hexane affording the title compound OT-1131 (2.6 g, 60.61%) as yellow solid. .sup.1H NMR (DMSO-d6, 400 MHz) 8.55 (d, J=8.9 Hz, 1H), 7.16 (d, J=8.9 Hz, 1H), 4.08 (s, 3H), 3.26-3.18 (m, 1H), 2.97-2.88 (m, 1H), 1.23 (t, J=7.3 Hz, 3H). LCMS [m/z (M+H)+=(Calculated for C.sub.8H.sub.10N.sub.2O.sub.4S+H: 231) found: 231], Purity at =220 nm: 99.38%
Synthesis of OT-2056 (exo-4,11-Dibenzyl-4,11-diazatricyclo[5.3.1.0.SUP.2,6.]undec-9-ene-3,5,8-trione)
[0626] ##STR00113##
is as described in the published PCT patent application WO 2017027358 A1.
Iniparib may be synthesised, for example, as described in WO 1994026730A2.
Auranofin may be synthesised, for example, as described in U.S. Pat. No. 4,200,738A
Results
SecTRAP Forming Activity of Compounds
[0627] A compound (agent) may be classified as a SecTRAP forming agent if C-terminal activity of TrxR as assessed by a DTNB assay is inhibited but N-terminal activity of TrxR as a assessed by a juglone assay is not significantly inhibited (or not fully inhibited or not abolished).
[0628] The minimal concentration of compound at which 100% inhibition is observed in the DTNB assay was established and then, using that concentration of the given compound, the effect on juglone reduction in the juglone assay was assessed. Higher % values for juglone activity (juglone reduction activity) in this assay are indicative of stronger prooxidant activity.
[0629] The SecTRAP forming activity of various compounds is summarised in Table A below. TrxR IC50 is the concentration at which 50% of Thioredoxin Reductase activity is inhibited (Molar concentration), as assessed in the DTNB assay. GR IC50 Concentration at which 50% of Glutathione Reductase activity is inhibited (Molar concentration), as assessed by the GR activity assay. Juglone Act (% @ 100% DTNB inhibition) means the % activity observed in the juglone assay when the compound is used at the concentration that achieves 100% inhibition of TrxR in the DTNB assay.
TABLE-US-00003 TABLE A Juglone Act Compound (% @ 100% is an DTNB example Compound TrxR IC50 GR IC50 inhibition) of formula # OT-1000 1.45E08 >30E06 84.35 I OT-1099 1.04E08 >30E06 42.86 I OT-1104 1.849E07 >30E06 43.48 I OT-1109 1.829E08 >30E06 49.28 I OT-1098 1.266E08 >30E06 53.62 I OT-1083 1.145E08 >30E06 60.87 I OT-1084 2.576E10 >30E06 69.57 I OT-1094 1.139E08 >30E06 75.36 I OT-2056 8.335E08 >30E06 107.692 II OT-1218 1.848E06 >30E06 58.6931 III OT-1012 2.684E08 >30E06 89.8 IV OT-1118 1.08E08 >30E06 42.86 IV OT-1119 8.549E10 0.000011 44.64 IV OT-1122 3.991E09 >30E06 53.57 IV OT-1108 6.901E09 >30E06 57.97 IV OT-1128 1.595E07 >30E06 65.31 V OT-1087 9.69E08 >30E06 65.99 V OT-1124 1.032E07 >30E06 73.47 V OT-1129 1.241E07 >30E06 74.83 V OT-1114 1.217E07 >30E06 75.51 V OT-1011 1.82E08 >30E06 83.67 V OT-1127 2.041E07 >30E06 84.35 V OT-1113 6.057E08 >30E06 91.16 V OT-1088 7.599E09 >30E06 33.33 V OT-1117 6.78E09 >30E06 35.71 V OT-1101 6.686E08 >30E06 39.29 V OT-1103 1.882E08 >30E06 39.29 V OT-1090 4.35E09 >30E06 46.38 V OT-1089 7.161E09 >30E06 47.83 V OT-1092 2.03E08 >30E06 52.17 V OT-1100 5.051E09 >30E06 58.93 V OT-1081 1.03E09 >30E06 65.22 V OT-1095 7.33E09 >30E06 71.01 V OT-1091 2.532E08 >30E06 71.01 V OT-1096 1.231E08 >30E06 86.96 V OT-1115 1.622E07 >30E06 70.07 VI OT-1116 9.304E08 >30E06 70.07 VI OT-1086 3.906E07 >30E06 79.59 VI OT-1025 3.157E09 >30E06 89.12 VII Auranofin 7.345E09 >30E06 72.11 ATO 3.100E06 >30E06 82.75 Iniparib 1.770E04 >30E06 90.48
[0630] Example SecTRAP data are shown for various compounds tested (Table Aabove). The data shows TrxR IC50 values for each compound, and retained juglone activities (N-terminal dithiol motif activity) of TrxR in the situation where TrxR is 100% inhibited at the C-terminal active site by compound, and where the concentration of the compound was equal to that required to obtain 100% inhibition.
[0631] The compounds are also specific towards TrxR over GR, as shown by the IC50 values, which is a proof of target specificity (Table A). GR is relevant in the sense that GR represents a main off-target candidate for the compounds in question. The IC50 values for TrxR are generally significantly lower than for GR, meaning that much lower amounts of compound are required to inhibit TrxR than GR. GR inhibition could cause damage to normal cells.
[0632] We have discovered that Iniparib is a SecTRAP forming compound. We believe that in clinical trials Iniparib performed well in cancers where TrxR/Trx is overexpressed and where there is intratumoural immune-cell infiltration, e.g. Triple Negative Breast Cancer. Iniparib has shown clinical benefit, as compared to control, in the 2nd and 3rd line setting, i.e. in patients who have received induction treatment with chemotherapy prior to treatment with Iniparib.
[0633] As mentioned above, we have discovered that Iniparib is a SecTRAP forming compound. As also described herein it has been surprisingly found that SecTRAP forming agents have, in addition to a direct cytotoxic effect, a an ability to confer an anti-cancer immune response. The fact that we have found that Iniparib is a SecTRAP forming agent and that SecTRAP forming agents can confer an anti-cancer immune response is consistent with the positive results in clinical trials in infiltrated tumours.
[0634] ATO (arsenic trioxide, As.sub.2O.sub.3) can increase cellular levels of ROS via several targets and cause apoptosis. Here we have discovered that, and provide definite evidence that, ATO also is a TrxR SecTRAP forming compound.
[0635] Auranofin is related to production of reactive oxygen species as well as the intracellular levels of TrxR. Here we have shown that auranofin is a SecTRAP-forming agent.
[0636] Compounds that inhibit the C-terminal active site of TrxR will lower or prevent reduction of the substrate Trx, which the is normal cellular reducing activity of TrxR. This will lead to a buildup of oxidized Trx. Reduced Trx is required to maintain low levels of reactive oxygen species in the cell. The thioredoxin system directly acts as a reactive oxygen species scavenger (Das, K. C. & Das, C. K. (2000) Biochem. Biophys. Res. Commun. 277, 443-447) and also maintains other intracellular pathways also performing this action. For example, reduced Trx is a direct electron donor to peroxiredoxins or thioredoxin peroxidases, which are major hydrogen peroxide-scavenging enzymes that normally keep the level of reactive oxygen species in the cell under control (Fang J, et al. J Biol Chem. 2005 Jul. 1; 280(26):25284-90). If, in addition, the N-terminal dithiol motif is still active after inhibition of the C-terminal active site, toxicity in the form of reactive oxygen species is produced in the cell via the N-terminal active site.
[0637] We compared OT-1000 to Iniparib with regard to inhibition of C-terminal activity and retained N-terminal activity. In one example, OT-1000 and Iniparib were assayed at various concentrations, with a binding time of 4 hours. Both compounds inhibited TrxR C-terminal activity, and in this assay, OT-1000 (IC50=27 M) was 1000-fold more potent than Iniparib (IC50=9.6 nM) (
[0638] Compared head to head within the same experiment, we also show that for Iniparib at 1 mM, N-terminal juglone reduction activity is retained at a similar level to that obtained for OT-1000 at 1 M (
[0639] We determined the cytotoxic, or cell-killing effect of various SecTRAP-forming compounds on the cancer cell lines MDA-MB-231 (a breast cancer cell line), MDA-MB-468 (a breast cancer cell line), NB-4 (a leukaemia cell line), and U-87 MG (a glioblastoma cell line) (Table X and Y). The data in Table X was obtained using the Alamar Blue cell viability assay. The data in Table Y was obtained using the MTT cell viability assay.
TABLE-US-00004 TABLE X Name MDA-MB-231 (IC50, M) Auranofin 1.40E06 Iniparib 5.44E05 OT-1000 3.53E06 OT-1011 3.81E06 OT-1012 3.61E06 OT-1025 2.92E06 OT-1081 3.70E05 OT-1083 4.20E06 OT-1084 3.44E06 OT-1086 1.23E05 OT-1087 5.60E06 OT-1088 1.80E06 OT-1089 7.90E07 OT-1090 1.30E06 OT-1091 9.45E07 OT-1092 5.24E06 OT-1094 4.21E06 OT-1095 7.13E06 OT-1096 4.51E06 OT-1098 3.52E06 OT-1099 1.73E06 OT-1100 1.85E06 OT-1101 2.66E06 OT-1103 5.22E06 OT-1104 4.17E06 OT-1108 3.09E06 OT-1109 3.82E06 OT-1113 8.09E06 OT-1114 1.14E05 OT-1115 5.08E06 OT-1116 4.53E06 OT-1117 4.36E06 OT-1118 4.54E06 OT-1122 1.09E05 OT-1124 1.25E05 OT-1127 5.13E06 OT-1128 3.31E06 OT-1129 2.95E06
TABLE-US-00005 TABLE Y NB-4 MDA-MB-468 U-87 MG MDA-MB-231 Name (IC50, M) (IC50, M) (IC50, M) (IC50, M) Auranofin 3.10E07 1.72E06 1.66E06 Iniparib 5.76E05 >33 >33E06 >33E06 OT-1000 1.20E06 9.21E06 1.27E05 3.14E06 OT-1011 1.21E06 2.89E06 6.11E06 1.80E06 OT-1012 4.34E06 8.20E06 2.97E06 OT-1025 8.30E07 3.86E06 6.76E06 5.26E06 OT-1084 9.40E07 3.44E06 OT-1086 4.00E06 1.06E05 9.97E06 OT-1087 3.88E06 7.52E06 7.73E06 OT-1096 3.22E06 OT-1113 1.17E06 3.90E06 5.23E06 4.90E06 OT-1114 5.47E06 1.63E05 1.84E05 OT-1115 4.34E06 6.11E06 7.53E06 OT-1116 8.94E06 8.89E06 8.94E06 OT-1117 1.20E06 4.36E06 OT-1124 9.77E06 1.95E05 >33E06 OT-1127 4.90E06 6.00E06 7.10E06 OT-1128 4.68E06 6.92E06 5.91E06 OT-1129 1.12E06 3.16E06 5.95E06 5.12E06 > NL > 4.88E06 OT-1132 >10E06 >33E06 >33E06 OT-1133 6.40E07 2.48E06 1.42E06 OT-1134 >33E06 >33E06 >33E06 OT-1135 9.60E07 2.53E06 1.02E06 OT-1244 >33E06 >33E06 >33E06 OT-1245 1.85E06 9.25E06 3.84E06 OT-1246 >0.000033 >0.000033 >0.000033 OT-1247 2.10E06 9.27E06 3.54E06 OT-1248 >0.000033 >0.000033 >0.000033 OT-1249 2.40E06 3.57E06 OT-1250 >0.000033 >0.000033 >0.000033 OT-1251 5.58E06 1.16E05 OT-1252 3.58E06 OT-1253 >0.000033 OT-2056 4.20E07
[0640] It is also shown that MDA-MB-231 and MDA-MB-453 cultured breast cancer cells are increasingly sensitive to OT-1000 exposure overtime. MDA-MB-231 and MDA-MB-453 are model cell lines for triple-negative breast cancer, and are characterized by basal-like properties, which in turn are associated with aggressiveness in the clinical setting. This time-dependent efficacy means that the mechanistic induction of oxidative stress is not due to a promiscuous reactivity of the compounds, but is an active function resulting from SecTRAP formation (
[0641] During the tumor cell killing process with the compounds described herein, we have shown in vitro, using MDA-MB-231 tumor cells, that the intracellular amount of Trx is decreased. This is shown for OT-1000 and OT-1129 as examples. For OT-1000 and OT-1129 treatments (0.1 M, 1 M and 10 M), intracellular Trx appears to decline during treatment of MBA-MD-231 tumor cells. The effects were greater with increasing concentration of compound (
[0642] Total intracellular Trx at end of experiment was lower in OT-1000 and OT-1129-treated cell cultures than for untreated cell cultures. In these experiments, OT-1129 had a more rapid effect than OT-1000.
[0643] The intracellular Trx level was also assessed during treatment with OT-1011, OT-1131, OT-2056, OT-1012, OT-1096 and OT-1013 (
[0644] Auranofin had a similar effect to the OT compounds (0.1 M, 1 M and 10 M) (
[0645] Iniparib also reduced the intracellular Trx levels in comparison to the level seen with untreated cells, e.g at the 96 h time point (
[0646] ATO also reduced the intracellular Trx levels in comparison to the level seen with untreated cells, e.g at the 96 h time point (
[0647] Eventually, the tumor cells die and stop producing Trx. During cell death there will likely be a transient local increase of extracellular Trx during tumor treatment, after which Trx levels will decline to zero. Without wishing to be bound by theory, in the situation where we treat tumors in vivo with the compounds herein, after a certain amount of time tumor cell-derived Trx will be decreased with the consequence that Treg suppressive activity in the tumor will be diminished. With diminished Treg activity, antitumoral T cell activity should increase. During tumor cell destruction, tumor cell-specific material will be processed by the immune system at the priming stage to further boost an adaptive anti-tumoral response. Further, without being bound by theory, it is possible that Trx, released during the cytolytic burst will attract a new Tcell population migrating into the tumor microenvironment with favorable composition, eg having high CD8+/Treg ratio that will trigger an anti-tumoral response.
[0648] We show herein the anti-cancer effect of compounds including OT-1000 in immunoincompetent (or in other words immunodeficient or immunocompromised) mice. In one example, OT-1000 demonstrated a tumor growth inhibition rate (TGI) of 37% in MDA-MB-231 xenograft-bearing athymic nude mice treated intravenously (IV) with 10 mg/kg OT-1000, with administration of compound occurring 11 times over the course of 22 days (
[0649] The data is also visualized in a waterfall plot which presents individual measured tumor sizes at end of experiment, to visualize distribution of tumor size over treatment arms. More tumors of smaller size are observed for OT-1000 treated animals than vehicle-treated animals, indicating anti-tumor efficacy (
[0650] In the same model system, MDA-MB-231 xenograft-bearing immunodeficient or immunocompromised athymic nude mice, OT-1129 achieved a TGI of 25% when given intravenously and Iniparib achieved a TGI of 9% when given intraperitoneally. The figures show plots of individual tumor growth (
[0651] Final tumor volumes after treatment with OT-1129, Iniparib or vehicle (MDA-MB-231 xenografts in immunodeficient or immunocompromised athymic nude mice) are also visualized in a waterfall plot (
[0652] The SecTRAP forming compound OT-1096 has an % TGI of 19% when treating immunodeficient or immunocompromised athymic nude mice implanted with MDA-MB-231 Xenografts. OT-1096 was administered IV at 10 mg/kg using a 5 day on two day off (5/2) dosing regimen. The % TGI equals 1-(median of tumor volume of treated animals/median tumor volume of vehicle control)100. (
Surprisingly, the SecTRAP forming compound OT-1096 displays a pronounced increased efficacy in immunocompetent mice in comparison with immunoincompetent mice (i.e. immunodeficient or immunocompromised). In BALB/c mice possessing an intact immune system and 4T1-luc2 mammary tumors, representing a TNBC murine tumor, OT-1096 achieved a % TGI of 54% when treated with only 5 mg/kg OT-1096 5/2 for 15 days. The % TGI equals 1-(median of tumor volume of treated animals/median tumor volume of vehicle control)100. The tumor volume was measured by lucipherase bioluminescence). (
Furthermore, in Hu-CD34-NSG mice, possessing a humanized immune system and patient derived TNBC tumor xenografts, OT-1096 elicited a % TGI of 45% and 55% in mice bearing human immune cells from two separate donors. This is again a pronounced increased efficacy demonstrated in immunocompetent mice in comparison with immunoincompetent mice (i.e. immunodeficient or immunocompromised). The % TGI equals 1-(median of tumor volume of treated animals/median tumor volume of vehicle control)100. The tumor volume was measured with caliper (
One important conclusion we draw when comparing treatments with OT-1096 performed in immunoincompetent mice and immunocompetent mice, is that the % TGI is severalfold higher in the immunocompetent models (exemplified by BALB/c mice possessing an intact immune system and 4T1-luc2 mammary tumors as well as in Hu-CD34-NSG mice, possessing a humanized immune system and patient derived TNBC tumor xenografts) compared to the immunodeficient or immunocompromised model (MDA-MB-231 xenografts in immunodeficient or immunocompromised athymic nude mice). Further surprisingly, this was achieved with either a lower dose of OT-1096 (a lower dose was used in the study with immunocompetent BALB/c mice with 4T1-luc2 tumors (
EXAMPLE 2
[0653] TM00098 Patient Derived Xenografts-Triple Negative Breast Cancer in Immunocompetent Humanized NSG Mice (Hu-CD34-NSG) Treated with OT-1096 Alone or in Combination with PembrolizumabAnalysis of Treg Levels.
The study with immunocompetent humanized NSG mice (Hu-CD34-NSG) described in Example 1 herein was expanded and the effect on Treg levels of OT-1096 alone or OT-1096 in combination with Pembrolizumab (an anti-PD1 antibody) was assessed.
Materials and Methods
[0654] Female NSG mice were implanted with human CD34+ hematopoietic stem cells from multiple donors and the level of human CD45+ cells were measured in the peripheral blood 12 weeks post engraftment. Mice with >25% human CD45+ cells in the peripheral blood were determined to have a humanized immune system (Hu-CD34-NSG) mice and were enrolled into the study. The Hu-CD34-NSG mice were implanted with TM00098 patient-derived xenografts (PDX) subcutaneously on the right flank. The TM00098 PDX cancer cells originate from a primary tumor of a patient suffering from a grade 3 TNBC invasive ductal carcinoma. When the tumors reached a volume between 60-120 mm.sup.3 mice were treated with either 10 mg/kg OT-1096 three times a week intravenously (donor 5243 n=5, donor 5252 n=7) or with OT-1096's vehicle three times a week intravenously (donor 5243 n=3, donor 5252 n=2) or with 10 mg/kg initial dose, thereafter 5 mg/kg Pembrolizumab two times a week intraperitoneally (donor 5243 n=3, donor 5252 n=3) or with PBS (Pembrolizumab vehicle) two times a week intraperitoneally (donor 5243 n=1, donor 5252 n=3) or with a combination of OT-1096 and Pembrolizumab using their respective treatment schedule (donor 5243 n=3, donor 5252 n=6). In case of tail vein swelling when the test substance or vehicle could not be administered intravenously, Intraperitoneal injection was applied. Animals that reached a body condition score of 2, a body weight loss of 20% or a tumor volume >2000 mm.sup.3 were euthanized before study terminus. Animals with ulcerated tumors were also euthanized before study terminus. Tumor volume was measured using a digital caliper two times a week for the duration of the study. Treatment occurred until sacrifice at day 41. Tumors from the remaining animals were collected and subjected to flow cytometry measurements of infiltrated Treg levels. Tumors were processed into single cell suspensions and resuspended at a concentration of 1010.sup.6 cells/mL. Tumor suspension (50 L) was incubated for 15-20 minutes in the dark at ART (ambient room temperature) with the following antibodies, Human (hu) CD45 FITC clone HI30, BioLegend, huCD4 PECy7 clone SK3, BioLegend, FoxP3 PE clone 259D, BioLegend, huCD25 APC clone M-A251, BioLegend, huCD3 V605 clone OKT3, BioLegend, 7-AAD, BioLegend. Flow cytometric data acquisition was performed using the FACSCantoll flow cytometer. Data was acquired using BD FACSDiva software. Cell populations was determined by electronic gating (P1=total leukocytes) on the basis of forward versus side scatter. The flow cytometer was set to collect 100,000 P1 events. The percentage of Tregs of CD45+ cells were calculated. Tregs were characterised by being viable CD45+, CD4+, FoxP3+, CD25+, CD3+ cells. The 7-AAD reagent was used to exclude non-viable cells in the flow cytometry analysis. Animals that were sacrificed prior to day 38 and animals that received more than 2 IP doses were excluded from analysis. Data is presented as mean % Tregs of CD45+ cellsSEM. Statistical significance (p<0.05) was determined using a Mann-Whitney test.
Results and Discussion
[0655] The levels of tumor infiltrated Tregs at day 41 decreased for OT-1096 treated tumors for both donors (
[0656] The data in this Example thus provides a further demonstration that a new effect of SecTRAP forming compounds exists, working in concert with (or stimulating) the immune system to combat cancer cell growth. As discussed elsewhere herein, Tregs have an immunosuppressive role in the tumour microenvironment and thus can inhibit an anti-cancer immune response. Thus, depleting Treg populations or inhibiting Treg activity in particular within the tumour microenvironment is desirable.
EXAMPLE 3
[0657] Viability of Isolated Immune Cells after Treatment with OT-1096
Materials and Methods
[0658] Viability of neutrophils after treatment with OT-1096 for 24 h was assessed using donated blood from a healthy male volunteer (HBsAg, HIV I&II negative), Age: 27 yrs (Blood collection date: 1 Jun. 2017) where neutrophils were isolated using dextran sedimentation followed by hypotonic lysis and final isolation by Histopaque 1077 (Sigma-Aldrich). Viability was assessed using an MTT assay.
[0659] Viability of PBMC (peripheral blood mononuclear cells) after treatment with OT-1096 for 24 h was assessed using donated blood from two different donors. Donor-1, a healthy male volunteer (HBsAg, HIV I&II negative), Age: 23 yrs (Blood collection date: 1 Jun. 2017). Donor-2, a healthy male volunteer (HBsAg, HIV I&II negative), Age: 40 yrs (Blood collection date: 8 Jun. 2017). The PBMC was isolated using Histopaque 1077 (Sigma-Aldrich). Viability was assessed using an MTT assay.
[0660] Viability of monocytes after treatment with OT-1096 for 24 h was assessed using donated blood from a healthy male volunteer (HBsAg, HIV I&II negative), Age: 38 yrs (Blood collection date: 8 Jun. 2017) where monocytes was isolated by Histopaque 1077 (Sigma-Aldrich) followed by purification with MiniMACS system of Miltenyi Biotec using CD14 microbeads (Cat No. 130-050-201). Viability was assessed using an MTT assay.
[0661] Viability of CD8+ cells after treatment with OT-1096 for 24 h was assessed using donated blood from a healthy male volunteer (HBsAg, HIV I&II negative), Age: 38 yrs (Blood collection date: 21 Jun. 2017) where CD8+ cells were isolated by Histopaque 1077 (Sigma-Aldrich) followed by purification with MiniMACS system of Miltenyi Biotec using CD8+ T Cell Isolation Reagent (Cat No. 130-096-495). Viability was assessed using an MTT assay.
[0662] Viability of CD4+ cells after treatment with OT-1096 for 24 h was assessed using donated blood from a healthy male volunteer (HBsAg, HIV I&II negative) where CD4+ cells were isolated by Histopaque 1077 (Sigma-Aldrich) followed by purification with MiniMACS system of Miltenyi Biotec using CD4+ T Cell Isolation Reagent (Cat No. 130-096-533). Viability was assessed using an MTT assay.
[0663] Viability of Tregs (CD4+CD25+FOXP3) after treatment with OT-1096 for 24 h was assessed using donated blood from healthy human volunteer (HBsAg, HIV I&II negative) where Tregs were isolated by Histopaque 1077 (Sigma-Aldrich) followed by purification with MiniMACS system of Miltenyi Biotec using CD4+CD25+ Regulatory T Cell Isolation Reagent (130-091-301). Viability was assessed using an MTT assay.
Results
[0664] The viability of the various isolated immune cells populations after treatment with OT-1096 for 24 h was assessed (
EXAMPLE 4
[0665] Intratumoral Immune Cell Populations of Engrafted Tumor Cells after OT-1096, Iniparib, or the Combination of OT-1096 and Iniparib
Materials and Methods
[0666] Intratumoral immune cell populations within engrafted 4T1 cells in BALB/c mice. 4T1 cells are a murine mammary carcinoma cell line. BALB/c mice are immunocompetent. 110.sup.6 4T1 tumor cells in 0% Matrigel were implanted orthotopically into the mammary fat pad. Enrollment of the mice into the treatment arms commenced when the tumors reached an average volume between 175-200 mm.sup.3. Upon enrollment mice were treated bid, twice per day, intratumorally with vehicle, 1 mg/kg Iniparib, 1 mg/kg OT-1096, or the combination of OT-1096 and Iniparib for 5 days (N=10 per group). Tumor volumes were measured using a digital caliper on day 1, 3, and 6 of the study. On day 6 mice were euthanized and tumors were resected for FACs analysis. Populations of murine immune cells including CD45+, CD4+, CD8+, and Tregs were analyzed. CD45+ positive cells were analyzed as the percentage of live cells. CD4+, CD8+, and Tregs cells were analyzed as percentage of CD45+ positive cells. Statistically significant differences between no treatment and treatment groups was determined using a Mann-Whitney test (*p<0.05, **p<0.01, *** p<0.001).
Results
[0667] Direct injection of Iniparib, OT-1096, or the combination of Iniparib and OT-1096 significantly increased CD8+ levels relative to no treatment controls (
The data also indicates that treatment with Iniparib alone increases CD8+ levels in tumours as compared to the no treatment or vehicle controls (
It is known in the art that an increase in the ratio of CD8+/Treg in cancer is correlated to better survival probability and thus the finding that treatment with OT-1096, Iniparib or the combination of Iniparib and OT-1096, increases the ratio of CD8+ cells to Treg cells in tumours indicates that such treatments (and treatments with other SecTRAP forming agents) are useful cancer therapies and that such compounds elicit anti-cancer immune activity and so may be particularly useful in the treatment of cancers (e.g. immune cell infiltrated cancers such as T-cell infiltrated cancers).
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