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DIAZINYL AMINO ACRIDINES AND MEDICAL USES THEREOF

20200405707 ยท 2020-12-31

    Inventors

    Cpc classification

    International classification

    Abstract

    There is provided herein compounds of formula (I) and pharmaceutically-acceptable salts and/or detectably-labelled derivatives thereof, wherein R.sup.1 to R.sup.3, X, Y, n and m have meanings as provided in the description, together with formulations and products comprising the same. There is also provided the use of such compounds, formulations and products in the treatment of cancers characterised by increased MYC activity.

    ##STR00001##

    Claims

    1. A compound of formula I ##STR00029## or a pharmaceutically-acceptable salt and/or detectably-labelled derivative thereof, wherein: R.sup.1 represents C.sub.1-12 alkyl, C.sub.2-12 alkenyl or C.sub.2-12 alkynyl, each optionally substituted by one or more groups independently selected from G.sup.1a, heterocyclyl 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; R.sup.2 and R.sup.3 each independently represent H, R.sup.a1, CN, S(O).sub.pR.sup.b1, S(O).sub.pN(R.sup.c1)R.sup.d1 or S(O).sub.pOR.sup.e1; m represents 1, 2, 3 or 4; n represents 0, 1, 2 or 3; each X independently represents independently represents halo, R.sup.a2, CN, -A.sup.a1-C(Q.sup.a1)R.sup.b2, -A.sup.b1-C(Q.sup.b1)N(R.sup.c2)R.sup.d2, -A.sup.c1-C(Q.sup.c1)OR.sup.e2, -A.sup.d1-S(O).sub.pR.sup.f2, -A.sup.e1-S(O).sub.pN(R.sup.g2)R.sup.h2, -A.sup.f1-S(O).sub.pOR.sup.i2, N.sub.3, N(R.sup.j2)R.sup.k2, N(H)CN, NO.sub.2, ONO.sub.2, OR.sup.l2, SR.sup.m2, B(OR.sup.n2).sub.2, P(O)(N(R.sup.o2)R.sup.p2).sub.2, P(O)(N(R.sup.o2)R.sup.p2)(OR.sup.q2) or P(O)(OR.sup.r2).sub.2; each A.sup.a1 to A.sup.f1 independently represents a single bond, C(O)N(R.sup.s2), N(R.sup.t1) or O; each Q.sup.a1 to Q.sup.c1 independently represents O, S, C(H)NO.sub.2, N(CN), NR.sup.u2, NN(R.sup.v2)R.sup.w2 or N(OR.sup.x2); NS(O).sub.pN(R.sup.v1)R.sup.w2; each Y independently represents halo, R.sup.a3, CN, -A.sup.a2-C(O)R.sup.b3, -A.sup.b2-C(O)N(R.sup.c3)R.sup.d3, -A.sup.c2-C(O)OR.sup.e3, -A.sup.d2-S(O).sub.pR.sup.f3, -A.sup.e2-S(O).sub.pN(R.sup.g3)R.sup.h3, -A.sup.f2-S(O).sub.pOR.sup.i3, N(R.sup.j3)R.sup.k3, OR.sup.l3 or SR.sup.m3; each A.sup.a2 to A.sup.f2 independently represents a single bond, N(R.sup.n3) or O; each G.sup.1a independently represents halo, CN, -A.sup.a3-C(Q.sup.a2)R.sup.b4, -A.sup.b3-C(Q.sup.b2)N(R.sup.c4)R.sup.d4, -A.sup.c3-C(Q.sup.c2)OR.sup.e4, -A.sup.d3-S(O).sub.pR.sup.f4, -A.sup.e3-S(O).sub.pN(R.sup.g4)R.sup.h4, -A.sup.f3-S(O).sub.pOR.sup.i4, N.sub.3, N(R.sup.j4)R.sup.k4, N(H)CN, NO.sub.2, ONO.sub.2, OR.sup.l4, SR.sup.m4B(OR.sup.n4).sub.2, P(O)(N(R.sup.o4)R.sup.p4).sub.2, P(O)(N(R.sup.o4)R.sup.p4)(OR.sup.q4) or P(O)(OR.sup.r4).sub.2 or =Q.sup.d2; each G.sup.1b, G.sup.1c and G.sup.1d independently represents halo, R.sup.a4, CN, -A.sup.a3-C(Q.sup.a2)R.sup.b4, -A.sup.b3-C(Q.sup.b2)N(R.sup.c4)R.sup.d4, -A.sup.c3-C(Q.sup.c2)OR.sup.e4, -A.sup.d3-S(O).sub.pR.sup.f4, -A.sup.e3-S(O).sub.pN(R.sup.g4)R.sup.h4, -A.sup.f3-S(O).sub.pOR.sup.i4, N.sub.3, N(R.sup.j4)R.sup.k4, N(H)CN, NO.sub.2, ONO.sub.2, OR.sup.l4, SR.sup.m4B(OR.sup.n4).sub.2, P(O)(N(R.sup.o4)R.sup.p4).sub.2, P(O)(N(R.sup.o4)R.sup.p4)(OR.sup.q4) or P(O)(OR.sup.r4).sub.2 or =Q.sup.d2; each A.sup.a3 to A.sup.f3 independently represents a single bond, C(O)N(R.sup.s4), N(R.sup.t4) or O; each Q.sup.a2 to Q.sup.d2 independently represents O, S, C(H)NO.sub.2, N(CN), NR.sup.u4, NN(R.sup.v4)R.sup.w4 or N(OR.sup.x4), NS(O).sub.pN(R.sup.v4)R.sup.w4; R.sup.a1, R.sup.b1 and R.sup.e1 represent C.sub.1-12 alkyl, C.sub.2-12 alkenyl or C.sub.2-12 alkynyl, each optionally substituted by one or more groups independently selected from G.sup.2a, or heterocyclyl optionally substituted by one or more groups independently selected from G.sup.2b; R.sup.c1 and R.sup.d1 independently represent H, C.sub.1-12 alkyl, C.sub.2-12 alkenyl or C.sub.2-12 alkynyl, each optionally substituted by one or more groups independently selected from G.sup.2a, or heterocyclyl optionally substituted by one or more groups independently selected from G.sup.2b, or alternatively R.sup.c1 and R.sup.d1 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 halo, C.sub.1-3 alkyl, C.sub.2-3 alkenyl or C.sub.2-3 alkynyl each optionally substituted by one or more halo, and O; each R.sup.a2 and R.sup.f2 independently represents C.sub.1-12 alkyl, C.sub.2-12 alkenyl or C.sub.2-12 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; 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.o2, R.sup.p2, R.sup.q2, R.sup.r2, R.sup.s2, R.sup.t2, R.sup.u2, R.sup.v2, R.sup.w2 and R.sup.x2 independently represents H, C.sub.1-12 alkyl, C.sub.2-12 alkenyl or C.sub.2-12 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, or alternatively any of R.sup.c2 and R.sup.d2, R.sup.g2 and R.sup.h2, R.sup.j2 and R.sup.k2, R.sup.o2 and R.sup.p2 and/or R.sup.v2 and R.sup.w2 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 halo, C.sub.1-3 alkyl, C.sub.2-3 alkenyl or C.sub.2-3 alkynyl each optionally substituted by one or more halo, and O, or alternatively any two R.sup.n2 are linked together to form, along with the boron, and the oxygen atoms to which they are attached, a 5- to 8-membered heterocyclic ring, which ring optionally contains one or more further heteroatoms 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, or alternatively two R.sup.r2 are linked together to form, along with the phosphorus, and the oxygen atoms to which they are attached, a 5- to 8-membered heterocyclic ring, which ring optionally contains one or more further heteroatoms 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.a3 and R.sup.f3 independently represents C.sub.1-6 alkyl optionally substituted by one or more F; 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 and R.sup.n3 independently represents H or C.sub.1-6 alkyl optionally substituted by one or more F, or alternatively any of R.sup.c3 and R.sup.cd, R.sup.fg and R.sup.h3 and/or R.sup.j3 and R.sup.k3, 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 F, C.sub.1-3 alkyl optionally substituted by one or more F, and O; each R.sup.a4 and R.sup.f4 independently represents C.sub.1-8 alkyl, C.sub.2-8 alkenyl or C.sub.2-8 alkynyl each optionally substituted by one or more groups independently selected from G.sup.4a, heterocyclyl 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.4, or heteroaryl optionally substituted by one or more groups independently selected from G.sup.4d; 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, R.sup.p4, R.sup.q4, R.sup.r4, R.sup.s4, R.sup.t4, R.sup.u4, R.sup.v4, R.sup.w4 and R.sup.x4 independently represents H or C.sub.1-8 alkyl, C.sub.2-8 alkenyl or C.sub.2-8 alkynyl each optionally substituted by one or more groups independently selected from G.sup.4a, heterocyclyl 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 alternatively any of R.sup.c4 and R.sup.d4, R.sup.g4 and R.sup.h4, R.sup.j4 and R.sup.k4, R.sup.o4 and R.sup.p4 and/or R.sup.v4 and R.sup.w4 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 halo, C.sub.1-3 alkyl, C.sub.2-3 alkenyl or C.sub.2-3 alkynyl each optionally substituted by one or more halo, and O, or alternatively two R.sup.n4 are linked together to form, along with the boron, and the oxygen atoms to which they are attached, a 5- to 8-membered heterocyclic ring, which ring optionally contains one or more further heteroatoms and which ring optionally is substituted by one or more groups independently selected from halo, C.sub.1-3alkyl optionally substituted by one or more halo, and O, or alternatively two R.sup.r4 are linked together to form, along with the phosphorus, and the oxygen atoms to which they are attached, a 5- to 8-membered heterocyclic ring, which ring optionally contains one or more further heteroatoms 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 G.sup.2a independently represents halo, CN, -A.sup.a4-C(Q.sup.a3)R.sup.b5, -A.sup.b4-C(Q.sup.c3)N(R.sup.c5)R.sup.d5, -A.sup.c4-C(Q.sup.c3)OR.sup.e5, -A.sup.d4-S(O).sub.pR.sup.f5, -A.sup.e4-S(O).sub.pN(R.sup.g5)R.sup.h5, -A.sup.f4-S(O).sub.pOR.sup.i5, N.sub.3, N(R.sup.j5)R.sup.k5, N(H)CN, NO.sub.2, ONO.sub.2, OR.sup.l5, SR.sup.m5B(OR.sup.n5).sub.2, P(O)(N(R.sup.o5)R.sup.p5).sub.2, P(O)(N(R.sup.o5)R.sup.p5)(OR.sup.q5) or P(O)(OR.sup.r5).sub.2 or =Q.sup.d3; each G.sup.2b independently represents halo, R.sup.a5, CN, -A.sup.a4-C(Q.sup.a3)R.sup.b5, -A.sup.b4-C(Q.sup.c3)N(R.sup.c5)R.sup.d5, -A.sup.c4-C(Q.sup.c3)OR.sup.e5, -A.sup.d4-S(O).sub.pR.sup.f5, -A.sup.e4-S(O).sub.pN(R.sup.g5)R.sup.h5, -A.sup.f4-S(O).sub.pOR.sup.i5, N.sub.3, N(R.sup.j5)R.sup.k5, N(H)CN, NO.sub.2, ONO.sub.2, OR.sup.l5, SR.sup.m5B(OR.sup.n5).sub.2, P(O)(N(R.sup.o5)R.sup.p5).sub.2, P(O)(N(R.sup.o5)R.sup.p5)(OR.sup.q5) or P(O)(OR.sup.r5).sub.2 or =Q.sup.d3; each A.sup.a4 to A.sup.f4 independently represents a single bond, C(O)N(R.sup.s5), N(R.sup.t5) or O; each Q.sup.a3 to Q.sup.d3 independently represents O, S, C(H)NO.sub.2, N(CN), NR.sup.u5, NN(R.sup.v5)R.sup.w5 or N(OR.sup.x5), NS(O).sub.pN(R.sup.v5)R.sup.w5; each G.sup.3a and G.sup.3b independently represents halo, R.sup.a6, CN, -A.sup.a5-C(Q.sup.a4)R.sup.b6, -A.sup.b5-C(Q.sup.b4)N(R.sup.c6)R.sup.d6, -A.sup.c5-C(Q.sup.c4)OR.sup.e6, -A.sup.d5-S(O).sub.pR.sup.f6, -A.sup.e5-S(O).sub.pN(R.sup.g6)R.sup.h6, -A.sup.f5-S(O).sub.pOR.sup.i6, N.sub.3, N(R.sup.j6)R.sup.k6, N(H)CN, NO.sub.2, ONO.sub.2, OR.sup.l6, SR.sup.m6B(OR.sup.n6).sub.2, P(O)(N(R.sup.o6)R.sup.p6).sub.2, P(O)(N(R.sup.o6)R.sup.p6)(OR.sup.q6) or P(O)(OR.sup.r6).sub.2 or =Q.sup.d4; each A.sup.a5 to A.sup.f5 independently represents a single bond, C(O)N(R.sup.s6), N(R.sup.t6) or O; each Q.sup.a4 to Q.sup.d4 independently represents O, S, C(H)NO.sub.2, N(CN), NR.sup.u6, NN(R.sup.v6)R.sup.w6 or N(OR.sup.x6), NS(O).sub.pN(R.sup.v6)R.sup.w6; each R.sup.a5 and R.sup.f5 independently represents C.sub.1-6 alkyl optionally substituted by one or more F; each R.sup.b5, R.sup.c5, R.sup.d5, R.sup.e5, R.sup.g5, R.sup.h5, R.sup.i5, R.sup.j5, R.sup.k5, R.sup.l5, R.sup.m5, R.sup.n5, R.sup.o5, R.sup.p5, R.sup.q5, R.sup.r5, R.sup.s5, R.sup.t5, R.sup.u5, R.sup.v6, R.sup.x6 and R.sup.y6 independently represents H or C.sub.1-6 alkyl optionally substituted by one or more F, or alternatively any of R.sup.c5 and R.sup.d5, R.sup.g5 and R.sup.h5, R.sup.j5 and R.sup.k5, R.sup.o5 and R.sup.p5 and/or R.sup.v5 and R.sup.w5, 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 F, C.sub.1-3 alkyl optionally substituted by one or more F, and O; each R.sup.a6 and R.sup.f6 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.5a, heterocyclyl optionally substituted by one or more groups independently selected from G.sup.5b, aryl optionally substituted by one or more groups independently selected from G.sup.5c, or heteroaryl optionally substituted by one or more groups independently selected from G.sup.5d; each R.sup.b6, R.sup.c6, R.sup.d6, R.sup.e6, R.sup.g6, R.sup.h6, R.sup.i6, R.sup.j6, R.sup.k6, R.sup.l6, R.sup.m6, R.sup.n6, R.sup.o6, R.sup.p6, R.sup.q6, R.sup.r6, R.sup.s6, R.sup.t6, R.sup.u6, R.sup.v6, R.sup.w6 and R.sup.x6, independently represents H or 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.5a, heterocyclyl optionally substituted by one or more groups independently selected from G.sup.5b, aryl optionally substituted by one or more groups independently selected from G.sup.5c, or heteroaryl optionally substituted by one or more groups independently selected from G.sup.5d, or alternatively any of R.sup.c6 and R.sup.d6, R.sup.g6 and R.sup.h6, R.sup.j6 and R.sup.k6, R.sup.o6 and R.sup.p6 and/or R.sup.v6 and R.sup.w6, 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 F, C.sub.1-3 alkyl optionally substituted by one or more F, and O; each G.sup.4a, G.sup.4b, G.sup.4c and G.sup.4d independently represents halo, R.sup.a7, CN, -A.sup.a6-C(Q.sup.a5)R.sup.b7, -A.sup.b6-C(Q.sup.b5)N(R.sup.c7)R.sup.d7, -A.sup.c6-C(Q.sup.c5)OR.sup.e7, -A.sup.d6-S(O).sub.pR.sup.f7, -A.sup.e6-S(O).sub.pN(R.sup.g7)R.sup.h7, -A.sup.f6-S(O).sub.pOR.sup.i7, N.sub.3, N(R.sup.j7)R.sup.k7, N(H)CN, NO.sub.2, ONO.sub.2, OR.sup.l7, SR.sup.m7B(OR.sup.n7).sub.2, P(O)(N(R.sup.o7)R.sup.p7).sub.2, P(O)(N(R.sup.o7)R.sup.p7)(OR.sup.q7) or P(O)(OR.sup.r7).sub.2 or =Q.sup.d5; each A.sup.a6 to A.sup.f6 independently represents a single bond, C(O)N(R.sup.s7), N(R.sup.t7) or O; each Q.sup.a5 to Q.sup.d5 independently represents O, S, C(H)NO.sub.2, N(CN), NR.sup.u7, NN(R.sup.v7)R.sup.w7 or N(OR.sup.x7), NS(O).sub.pN(R.sup.v7)R.sup.w7; each R.sup.a7 and R.sup.f7 ndependently 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.6a, heterocyclyl optionally substituted by one or more groups independently selected from G.sup.6b, aryl optionally substituted by one or more groups independently selected from G.sup.6c, or heteroaryl optionally substituted by one or more groups independently selected from G.sup.6d; each R.sup.b7, R.sup.c7, R.sup.d7, R.sup.e7, R.sup.g7, R.sup.h7, R.sup.i7, R.sup.j7, R.sup.k7, R.sup.l7, R.sup.m7, R.sup.n7, R.sup.o7, R.sup.p7, R.sup.q7, R.sup.t7, R.sup.s7, R.sup.t7, R.sup.u7, R.sup.v7, R.sup.w7 and R.sup.x7 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.6a, heterocyclyl optionally substituted by one or more groups independently selected from G.sup.6b, aryl optionally substituted by one or more groups independently selected from G.sup.6c, or heteroaryl optionally substituted by one or more groups independently selected from Gd, or alternatively any of R.sup.c7 and R.sup.d7, R.sup.g7 and R.sup.h7, R.sup.j7 and R.sup.k7, R.sup.n7 and R.sup.o7 and/or R.sup.v7 and R.sup.w7, 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 F, C.sub.1-3 alkyl optionally substituted by one or more F, and O; each G.sup.5a, G.sup.5b, G.sup.5c and G.sup.5d independently represents halo, R.sup.a8, CN, -A.sup.a7-C(Q.sup.a6)R.sup.b8, -A.sup.b7-C(Q.sup.b6)N(R.sup.c8)R.sup.d8, -A.sup.c7-C(Q.sup.c6)OR.sup.e8, -A.sup.d7-S(O).sub.pR.sup.f8, -A.sup.e7-S(O).sub.pN(R.sup.g8)R.sup.h8, -A.sup.f7-S(O).sub.pOR.sup.i8, N.sub.3, N(R.sup.j8)R.sup.k8, N(H)CN, NO.sub.2, ONO.sub.2, OR.sup.l8, SR.sup.m8B(OR.sup.n8).sub.2, P(O)(N(R.sup.o8)R.sup.p8).sub.2, P(O)(N(R.sup.o8)R.sup.p8)(OR.sup.q8) or P(O)(OR.sup.r8).sub.2 or =Q.sup.d6; each A.sup.a7 to A.sup.f7 independently represents a single bond, C(O)N(R.sup.s8), N(R.sup.t8) or O; each Q.sup.a6 to Q.sup.d6 independently represents O, S, C(H)NO.sub.2, N(CN), NR.sup.u8, NN(R.sup.v8)R.sup.w8 or N(OR.sup.x8), NS(O).sub.pN(R.sup.v8)R.sup.w8; each G.sup.6a, G.sup.6b, G.sup.6c and G.sup.6d independently represents halo, R.sup.a9, CN, -A.sup.a8-C(Q.sup.a7)R.sup.b9, -A.sup.b8-C(Q.sup.b7)N(R.sup.c9)R.sup.d9, -A.sup.c8-C(Q.sup.c7)OR.sup.e9, -A.sup.d8-S(O).sub.pR.sup.f9, -A.sup.e8-S(O).sub.pN(R.sup.g9)R.sup.h9, -A.sup.f8-S(O).sub.pOR.sup.i9, N.sub.3, N(R.sup.j9)R.sup.k9, N(H)CN, NO.sub.2, ONO.sub.2, OR.sup.l9, SR.sup.m9B(OR.sup.n9).sub.2, P(O)(N(R.sup.o9)R.sup.p9).sub.2, P(O)(N(R.sup.o9)R.sup.p9)(OR.sup.q9) or P(O)(OR.sup.r9).sub.2 or =Q.sup.d7; each A.sup.a8 to A.sup.f8 independently represents a single bond, C(O)N(R.sup.s9), N(R.sup.t9) or O; each Q.sup.a7 to Q.sup.d7 independently represents O, S, C(H)NO.sub.2, N(CN), NR.sup.u9, NN(R.sup.v9)R.sup.w9 or N(OR.sup.x9), NS(O).sub.pN(R.sup.v9)R.sup.w9; each R.sup.a8 and R.sup.f8 independently represents H or C.sub.1-6 alkyl optionally substituted by one or more F; each R.sup.b8, R.sup.c8, R.sup.d8, R.sup.e8, R.sup.g8, R.sup.h8, R.sup.i8, R.sup.j8, R.sup.k8, R.sup.l8, R.sup.m8, R.sup.n8, R.sup.o8, R.sup.p8, R.sup.q8, R.sup.r8, R.sup.s8, R.sup.t8, R.sup.u8, R.sup.v8, R.sup.w8 and R.sup.x8 independently represents H or C.sub.1-6 alkyl optionally substituted by one or more F, or alternatively any of R.sup.c8 and R.sup.d8, R.sup.g8 and R.sup.h8, R.sup.j8 and R.sup.k8, R.sup.n8 and R.sup.o8 and/or R.sup.v8 and R.sup.w8, 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 F, C.sub.1-3 alkyl optionally substituted by one or more F, and O; each R.sup.a9 and R.sup.f9 independently represents H or C.sub.1-6 alkyl optionally substituted by one or more F; each R.sup.b9, R.sup.c9, R.sup.d9, R.sup.e9, R.sup.g9, R.sup.h9, R.sup.i9, R.sup.j9, R.sup.k9, R.sup.l9, R.sup.m9, R.sup.n9, R.sup.o9, R.sup.p9, R.sup.q9, R.sup.r9, R.sup.s9, R.sup.t9, R.sup.u9, R.sup.v9, R.sup.w9 and R.sup.x9 independently represents H or C.sub.1-6 alkyl optionally substituted by one or more F, or alternatively any of R.sup.c9 and R.sup.dg, R.sup.g9 and R.sup.h9, R.sup.j9 and R.sup.k9, R.sup.n9 and R.sup.o9 and/or R.sup.v9 and R.sup.w9, 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 F, C.sub.1-3 alkyl optionally substituted by one or more F, and O; and each p independently represents 1 or 2, with the proviso that the following compounds are excluded: 6-((4-(dimethylamino)phenyl)diazenyl)-2-ethoxyacridin-9-amine; 6,6-(diazene-1,2-diyl)bis(2-ethoxyacridin-9-amine); 6-((1H-indol-2-yl)diazenyl)-2-ethoxyacridin-9-amine; 2-((9-amino-7-ethoxyacridin-3-yl)diazenyl)-4-((diethylamino)methyl)-3,6-dimethylphenol; N.sup.4-(7-chloro-2-methoxy-3-(naphthalen-1-yldiazenyl)acridin-9-yl)-N,N-diethylpentane-1,4-diamine; (E)-N.sup.4-(7-chloro-2-methoxy-3-(naphthalen-1-yldiazenyl)acridin-9-yl)-N,N-diethylpentane-1,4-diamine; 3-((9-amino-6-ethoxyacridin-3-yl)diazenyl)pyridine-2,6-diamine; 3-((9-amino-7-ethoxyacridin-3-yl)diazenyl)pyridine-2,6-diamine; 4-amino-2-((9-amino-7-ethoxyacridin-3-yl)diazenyl)naphthalene-1-sulfonic acid; 4-((9-amino-7-ethoxyacridin-3-yl)diazenyl)-2,5-dimethylphenol 4-((9-amino-7-ethoxyacridin-3-yl)diazenyl)-2,6-dimethylphenol; and 4-amino-N-(5-((9-amino-7-ethoxyacridin-3-yl)diazenyl)thiazol-2-yl)benzenesulfonamide.

    2. The compound of claim 1, wherein R.sup.1 represents: 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.

    3. The compound of claim 1 or claim 2, wherein R.sup.1 represents: phenyl optionally substituted by one or more groups independently selected from G.sup.1c; or pyridinyl optionally substituted by one or more groups independently selected from G.sup.1d.

    4. The compound of any one of claims 1 to 3, wherein each G.sup.1c and G.sup.1d independently represents halo, R.sup.a4, N(R.sup.j4)R.sup.k4 or OR.sup.l4.

    5. The compound of any one of claims 1 to 4, each G.sup.1c and G.sup.1d independently represents: C.sub.1-12 alkyl optionally substituted by one or more groups independently selected from G.sup.4a; N(R.sup.j4)R.sup.k4; or OR.sup.l4.

    6. The compound of any one of claims 1 to 5, wherein the compound of formula I is a compound of formula Ia ##STR00030## or a compound of formula Ia ##STR00031## wherein R.sup.1 to R.sup.3, X, Y, m and n are as claimed in any one of claims 1 to 5.

    7. The compound of any one of claims 1 to 6, wherein n represents 0.

    8. The compound of any one of claims 1 to 7, wherein R.sup.3 represents H.

    9. The compound of any one of claims 1 to 8, wherein R.sup.2 and R.sup.3 each represent H.

    10. The compound of any one of claims 1 to 9, wherein m represents at least 1.

    11. The compound of any one of claims 1 to 10, wherein m represents 1.

    12. The compound of any one of claims 1 to 11, wherein the compound of formula I is a compound of formula Ib ##STR00032## or a compound of formula Ib ##STR00033## wherein R.sup.1 to R.sup.3, X, Y and n are as claimed in any one of claims 1 to 11 and t represents 0, 1, 2 or 3.

    13. The compound of any one of claims 1 to 12, wherein each X independently represents halo (e.g. fluoro) or OR.sup.l2.

    14. The compound of any one of claims 1 to 13, wherein: t represents 0; and X represents OR.sup.l2.

    15. The compound of any one of claims 1 to 14, wherein each R.sup.l2 represents C.sub.1-6 alkyl optionally substituted by one or more groups independently selected from G.sup.3a.

    16. The compound of any one of claims 1 to 15, wherein each R.sup.l2 represents C.sub.2 alkyl optionally substituted by one or more fluoro.

    17. A compound of formula I as claimed in any one of claims 1 to 16, or a pharmaceutically acceptable salt and/or detectably labelled derivative thereof, for use as a pharmaceutical, with the proviso that the following compounds are excluded: 3-((9-amino-6-ethoxyacridin-3-yl)diazenyl)pyridine-2,6-diamine; 3-((9-amino-7-ethoxyacridin-3-yl)diazenyl)pyridine-2,6-diamine; 4-amino-2-((9-amino-7-ethoxyacridin-3-yl)diazenyl)naphthalene-1-sulfonic acid; 4-((9-amino-7-ethoxyacridin-3-yl)diazenyl)-2,6-dimethylphenol; and 4-amino-N-(5-((9-amino-7-ethoxyacridin-3-yl)diazenyl)thiazol-2-yl)benzenesulfonamide.

    18. A compound of formula I as claimed in any one of claims 1 to 16, or a pharmaceutically acceptable salt and/or detectably labelled derivative thereof, for use in the treatment of a cancer characterised by increased MYC activity.

    19. A method of treating a cancer characterised by increased MYC activity comprising administering to a patient in need thereof a therapeutically effective amount of a compound of formula I as claimed in any one of claims 1 to 16, or a pharmaceutically acceptable salt and/or detectably labelled derivative thereof.

    20. The use of a compound of formula I as claimed in any one of claims 1 to 16, or a pharmaceutically acceptable salt and/or detectably labelled derivative thereof, for the manufacture of a medicament for the treatment of a cancer characterised by increased MYC activity.

    21. The compound for use, method or use of any one of claims 18 to 20, wherein the cancer is selected from the list consisting of: Burkitt's lymphoma; ovarian cancer, such as ovarian cancer with BRCA alterations; basel-like breast cancer; esophageal squamous cell carcinoma; colon cancer; endometrial cancer; neuroblastoma; small cell lung carcinoma; medulloblastoma, such as group 3; pancreatic cancer; head and neck cancer; prostate cancer; and hepatocellular carcinomas.

    22. A pharmaceutical composition comprising a compound of formula I as claimed in any one of claims 1 to 16, or a pharmaceutically acceptable salt and/or detectably labelled derivative thereof, and optionally one or more pharmaceutically-acceptable excipient, with the proviso that the following compounds are excluded: 3-((9-amino-6-ethoxyacridin-3-yl)diazenyl)pyridine-2,6-diamine; 3-((9-amino-7-ethoxyacridin-3-yl)diazenyl)pyridine-2,6-diamine; 4-amino-2-((9-amino-7-ethoxyacridin-3-yl)diazenyl)naphthalene-1-sulfonic acid; 4-((9-amino-7-ethoxyacridin-3-yl)diazenyl)-2,6-dimethylphenol; and 4-amino-N-(5-((9-amino-7-ethoxyacridin-3-yl)diazenyl)thiazol-2-yl)benzenesulfonamide.

    23. A pharmaceutical composition as defined in claim 22, but without the proviso, for use in the treatment of as cancer as defined in any one of claims 18 to 21.

    24. A combination product comprising: (I) a compound of formula I as claimed in any one of claims 1 to 16, or a pharmaceutically acceptable salt and/or detectably labelled derivative thereof with the proviso that the following compounds are excluded: 3-((9-amino-6-ethoxyacridin-3-yl)diazenyl)pyridine-2,6-diamine; and 3-((9-amino-7-ethoxyacridin-3-yl)diazenyl)pyridine-2,6-diamine, and (II) one or more other therapeutic agent that is useful in the treatment of cancer, wherein each of components (I) and (II) is formulated in admixture, optionally with one or more a pharmaceutically-acceptable excipient.

    25. A kit-of-parts comprising: (a) a pharmaceutical composition comprising a compound of formula I as claimed in any one of claims 1 to 16, or a pharmaceutically acceptable salt and/or detectably labelled derivative thereof, and optionally one or more pharmaceutically-acceptable excipient, with the proviso that the following compounds are excluded: 3-((9-amino-6-ethoxyacridin-3-yl)diazenyl)pyridine-2,6-diamine; and 3-((9-amino-7-ethoxyacridin-3-yl)diazenyl)pyridine-2,6-diamine, and (b) one or more other therapeutic agent that is useful in the treatment of cancer, optionally in admixture with one or more pharmaceutically-acceptable excipient, which components (a) and (b) are each provided in a form that is suitable for administration in conjunction with the other.

    26. A process for the preparation of a compound of formula I as claimed in any one of claims 1 to 16, or a pharmaceutically acceptable salt and/or detectably labelled derivative thereof, comprising the step of: reacting a compound of formula II ##STR00034## wherein R.sup.2, R.sup.3, X, Y, n and m are as defined in any one of claims 1 to 16 with suitable source of nitrite in the presence of a suitable solvent and a suitable acid, followed by reaction with a compound of formula III
    H.sub.2NR.sup.1(III) wherein R.sup.1 is as defined in any one of claims 1 to 16 in the presence of a suitable solvent and a suitable base.

    Description

    BRIEF DESCRIPTION OF THE FIGURES

    [0306] FIG. 1: A) Microscale thermophoresis (MST) of fluorescently labeled MAX in a MYC:MAX heterodimer formation assay based on recombinant proteins. MST of labeled MAXbHLHZip after titration of Example 1 pre-incubated with 1 M MYCbHLHZip, or with 1 M MAXbHLHZip. Fluorescence intensity of labeled MAXbHLHZip relative DMSO was plotted against Example 1 concentration. B) Surface plasmon resonance (SPR) of MYC:MAX heterodimer formation assay. MAXbHLHZip was immobilized by an amino coupling procedure to a CM5 sensor chip. MYCbHLHZip pre-incubated with or without compound (as indicated) was injected over MAX for 180 seconds, allowed to dissociate for 240 seconds and regenerated. Reference surface (without MAXbHLHZip) subtracted sensorgrams are shown from one representative experiment. C) MYC binding response units (RU) plotted against Example 1 concentration from which an IC50 of Example 1 inhibition of MYC:MAX heterodimer formation is determined, summary of four experiments.

    [0307] FIG. 2: A) MST assay of Example 1 effect on MYC and MAX, respectively. Recombinant MYC bHLHZip and MAX bHLHZip proteins were titrated, respectively, in a fixed concentration (3 M) of Example 1. Changes in fluorescence were measured and normalized to control (buffer). Data are shown as meanstandard deviation of 6-8 biological repeats. B) MST assay of MYC (reverse MST). A fixed concentration of 200 nM labeled MYC was mixed with different concentrations of Example 1 as indicated. Data are shown as meanstandard deviation of 5 biological repeats. C) MST assay of Example 2 effect on MYC. D) MST assay of MYC with a control compound (non-binder).

    [0308] FIG. 3: A) SPR assay to determine affinity of Example 1 to MYC. MYC bHLHZip protein was immobilized by aminocoupling on a CM5 sensor chip. Example 1 was injected at various concentrations in a kinetic experiment. The reference surface was subtracted from the analyte surface to generate a sensorgram. Association and dissocation rates (k.sub.a=9294 M-1 s-1, k.sub.d=0.02293 s-1) were determined using the Langmuir 1:1 model in the Biacore Evaluation program fitting curves with a constant Rmax of 43 RU (theoretical Rmax), thereby suggesting a K.sub.D of 2.5 M with a Chi.sup.2 value of 0.073. The sensorgram displays one representative experiment. Four kinetic experiments were carried out on two different sensor chips and an average K.sub.D of 1.60.5 M was calculated. B) Four MYC equilibrium binding experiments with Example 1 summarized in an equilibrium binding plot. Binding affinities were estimated from the plot as 50% of Rmax suggesting a K.sub.D of approximately 1.5-2 mM with an experimental Rmax of 25-30 RU (theoretical Rmax=23 RU). Equilibrium binding experiments of MYC with Example 2 and control compound (non-binder) were plotted as well.

    [0309] FIG. 4: A) Endogenous MYC:MAX (upper panel) and FRA1:JUN (lower panel) interactions as visualized as fluorescent red dots by isPLA. Nuclei were counterstained with DAPI (blue). Cells were treated with 10 M of indicated compounds for 16 hours and subjected to isPLA using pairs of MYC and MAX and of FRA1 and JUN antibodies, respectively. As negative control, primary antibody pairs but only one oligo-conjugated secondary antibody was used. B) Quantification of MYC:MAX and C) FRA1:JUN isPLA, representing an average number of nuclear dots per cell from three microscopic fields normalized to corresponding values for DMSO-treated cells. D) Coimmunoprecipitation of endogenous MAX with MYC from MDA-MB231 cells treated with 5 M Example 1 or DMSO for 3.5 hours. 1.sup.st-4.sup.th lanes from top; coimmunoprecipitated MAX, immunoprecipitated MYC, total levels of MAX and ACTIN, respectively, as determined by western blot analysis. E) MYC:MAX GLuc assay in cells. HEK293 cells transfected with MYC-GLuc-C and MAX-GLuc-N, and full length CMV-Luc. 24 hours later cells were treated with 10 mM of Example 1, Example 2 and control compound for 16 hours and thereafter analyzed in a dual luciferase assay. The ratio of Gaussia/Firefly luciferase luminescence were calculated and normalized to DMSO-treated cells.

    [0310] FIG. 5: A) Inhibition of MYC transactivation of target genes (A) ODC1, (B) RSG16, and (C) CR2 as determined by RT-qPCR analysis, based on three biological experiments with three technical repeats each. U2OS-MYC-ER cells were treated with or without 100 nM 4-hydroxy-tamoxifen (HOT) for 4 hours, after which DMSO or Example 1 was added for 24 hours before total RNA was extraction. Fold changes in mRNA expression are presented relative to DMSO in non-HOT-treated cells after normalization to GAPDH, used as reference gene.

    [0311] FIG. 6: A-B) Neuroblastoma cell lines with (SK-N-DZ, Kelly and IMR-32) or without (SK-N-F1, SK-N-RA and SK-N-AS) MYCN-amplification were exposed to different concentrations of Example 1, Example 2 and control compound, respectively, for 48 hours. Cell growth and viability was estimated by measuring metabolic activity. C) Growth of TGR-1 (wt), HO15.19 (MYC knockout) and HOmyc3 (MYC reconstituted HO15.19) Rat1 fibroblasts, as measured by the WST-1 assay after treatment with Example 1. Data are shown as meanstandard deviation of 3-5 biological experiments, each with 3 technical repeats.

    [0312] FIG. 7: Example 1 inhibits MYC:MAX interaction, induces apoptosis and reduces tumor cell growth and microvascularity in a MYCN-amplified neuroblastoma mouse tumor model in vivo. SK-N-DZ MYCN-amplified neuroblastoma xenograft tumors reaching a volume of 100-200 mm.sup.3 were treated with Example 1 (20 mg/kg body weight) or vehicle injected i.p. daily for 1-2 weeks. A) Apoptosis was determined by TUNEL staining of tumor tissues from mice treated with Example 1 or vehicle, counterstained with DAPI. Quantification of TUNEL staining was normalized to whole tumor areas as determined by DAPI from three Example 1- and three vehicle-treated mice. Student's t-test of Example 1 vs. vehicle; p=0.0083. B) Cell proliferation determined by Ki67 of tumor tissues from mice treated with Example 1 or vehicle, respectively, and counterstained with DAPI. Quantification of Ki67 negative areas normalized to whole tumor areas by DAPI from three Example 1- and three vehicle-treated mice. Student's t-test of Example 1 vs. vehicle; p=0.0380. C) Microvascular density visualized by CD31 staining in the red. C) Quantification of CD31 staining normalized to whole tumor areas from three Example 1- and three vehicle-treated mice. Student's t-test of Example 1 vs. vehicle; p=0.014. D) Detection of MYCN:MAX protein interaction by isPLA performed on tumor tissue from mice treated with Example 1 or vehicle using antibodies against MYCN and MAX. Quantification of MYCN:MAX isPLA signals in tumor tissue from Example 1- and vehicle-treated mice, presented as average number of dots from four randomly chosen microscopic fields from Example 1 treated mice normalized to corresponding values from vehicle-treated mice (F=28.102, P=0.008). E) Apoptosis determined from TUNEL stained tumor section derived from a MDA-MB231 xenograft mouse treated with Example 2 for 2 weeks. Quantification of apoptotic cells per counted field.

    EXAMPLES

    [0313] The present invention will be further described by reference to the following examples, which are not intended to limit the scope of the invention.

    [0314] In the event that there is a discrepancy between nomenclature and any compounds depicted graphically, then it is the latter that presides (unless contradicted by any experimental details that may be given or unless it is clear from the context).

    Experimental Procedures

    [0315] Starting materials and intermediates used in the synthesis of compounds described herein are commercially available or can be prepared by the methods described herein or by methods known in the art.

    [0316] Experiments were generally carried out under inert atmosphere (nitrogen or argon), particularly in cases where oxygen- or moisture-sensitive reagents or intermediates were used.

    [0317] Mass spectrometry data are reported from liquid chromatography-mass spectrometry (LC-MS) using electrospray ionization. Chemical shifts for NMR data are expressed in parts per million (ppm, ) referenced to residual peaks from the deuterated solvent used.

    [0318] For syntheses referencing general procedures, reaction conditions (such as length of reaction or temperature) may vary. In general, reactions were followed by thin layer chromatography or LC-MS, and subjected to work-up when appropriate. Purifications may vary between experiments: in general, solvents and the solvent ratios used for eluents/gradients were chosen to provide an appropriate R.sub.f and/or retention time.

    [0319] For the preparation of Example 7 onwards, the following will apply (although such techniques may also apply to Examples 1 to 6).

    [0320] 1H NMR spectra were recorded using a Bruker DPX400 spectrometer (400 MHz) using deuterated solvents.

    [0321] All evaporations were carried out in vacuum with a rotary evaporator at 10-30 mmHg. Analytical samples were dried under high vacuum (1-5 mmHg) at room temperature. Thin layer chromatography (TLC) was performed on silica gel plates (Merck) with fluorescent indicator. Spots were visualized by UV light (214 and 254 nm).

    [0322] Flash chromatography was performed using silica gel 60 from Merck. All compounds evaluated in biological tests were purified to >95% as determined by HPLC-MS on an Agilent/HP 1200 system 6110 mass spectrometer with electrospray ionization (ESI+). HPLC-MS methods were the following. Method 1: Waters XBridge C18 3.5 m column (3.0 mm50 mm), 3.5 min gradient mobile phase [CH3CN]/[10 mM NH4HCO3/H2O]. Method 2: ACE C18 3.5 m column (3.0 mm50 mm), mobile phase [0.1% TFA/CH3CN]/[0.1% TFA/H2O]. Absorbance was monitored at 30590 and 254 nm. All solvents used were HPLC grade. Preparative HPLC was performed on a Gilson HPLC system. Acidic pH: column ACE 5 C8 (150 mm30 mm), H2O (containing 0.1% TFA), and MeCN were used as mobile phases at a flow rate of 45 mL/min, with a gradient time of 9 min.

    Intermediate Compounds

    Intermediate 1: 5-[(E)-(9-amino-7-ethoxy-acridin-3-yl)azo]-2-hydroxy-benzaldehyde

    [0323] NaNO.sub.2 (28 mg, 0.42 mmol) in H.sub.2O (150 mL) was added to a stirred mixture of 6,9-diamino-2-ethoxyacridine lactate hydrate (150 mg, 0.41 mmol) and H.sub.2O (3 mL) at 0-5 C., immediately followed by the addition of H.sub.2SO.sub.4 (aq, 5%, 3 mL). The mixture was stirred at this temperature for 20 min and poured into a vigorously stirred 0-5 C. solution of 2-hydroxybenzaldehyde (56 mg, 0.46 mmol) and Na.sub.2CO.sub.3 (0.40 g, 3.8 mmol) in H.sub.2O (6 mL). The mixture was stirred at rt for 2 h and then pH was adjusted to ca 1 with HCl (aq, 2M) and then filtered. The dark solid was triturated in methanol (ca 10 mL) over the weekend and filtered and dried in vacuum to give the title compound, yield 120 mg (75%).

    [0324] H.sup.1 NMR (400 MHz, DMSO-d.sub.6): ppm 1.44 (t, J=6.95 Hz, 3H) 4.21 (q, J=7.06 Hz, 2H) 7.21 (d, J=9.00 Hz, 1H) 7.70 (dd, J=9.16, 2.37 Hz, 1H) 7.88 (d, J=9.16 Hz, 1H) 7.94 (dd, J=9.32, 1.42 Hz, 1H) 7.98 (d, J=2.37 Hz, 1H) 8.15 (dd, J=8.92, 2.61 Hz, 1H) 8.22 (d, J=1.58 Hz, 1H) 8.24 (d, J=2.69 Hz, 1H) 8.74 (d, J=9.32 Hz, 1H) 9.77 (br. s., 2H) 10.38 (s, 1H)

    Example Compounds

    Example 1: 3-((9-amino-7-ethoxyacridin-3-yl)diazenyl)pyridine-2,6-diamine sulphate hydrate

    [0325] ##STR00018##

    [0326] NaNO.sub.2 (2.85 g, 41.31 mmol) in H.sub.2O (150 mL) was added to a stirred mixture of 6,9-diamino-2-ethoxyacridine lactate hydrate (15.00 g, 41.52 mmol) and H.sub.2O (300 mL) at 5 C., immediately followed by addition of H.sub.2SO.sub.4 (aq, 5%, 300 mL) at 5 C. The mixture was stirred at 5 C. for 20 min and poured into a vigorously stirred solution of 2,6-diaminopyridine (4.52 g, 4.14 mmol), Na.sub.2CO.sub.3 (40.0 g, 377 mmol) and H.sub.2O (600 mL). The mixture was stirred at rt for 12 h and filtered. The solids were collected, suspended in H.sub.2O (500 mL) and stirred at 60 C. for 6 h. The solids were collected, washed with H.sub.2O and dried in vacuo over P.sub.2O.sub.5 to give the title compound (15.50 g, 31.66 mmol, 76.3%).

    [0327] .sup.1H NMR (400 MHz, DMSO-d.sub.6): 1.38 (t, J=7.0 Hz, 3H); 4.14 (q, J=7.0 Hz, 2H); 6.02 (d, J=8.8 Hz, 1H); 6.94 (br.s, 2H); 7.42 (dd, J=2.2, 9.2 Hz, 1H); 7.68 (d, J=8.8 Hz, 1H); 7.72 (d, J=2.2 Hz, 1H); 7.77 (d, J=9.2 Hz, 1H); 7.82 (dd, J=1.6, 9.2 Hz, 1H); 7.89 (d, J=1.6 Hz, 1H); 8.39 (d, J=9.2 Hz, 1H).

    [0328] Following an analogous protocol, the compound of Example 1 was also prepared as the corresponding HCl salt. In the biological examples provided herein, these salts were used interchangeably.

    Example 2: 4-((9-amino-7-ethoxyacridin-3-yl)diazenyl)-3,6-dimethylphenol

    [0329] ##STR00019##

    [0330] The title compound was prepared in accordance with Example 1.

    [0331] .sup.1H NMR (400 MHz, DMSO-d.sub.6): 1.40 (t, J=7.0 Hz, 3H); 2.13 (s, 3H); 2.62 (s, 3H); 4.17 (q, J=7.0 Hz, 2H); 6.78 (s, 1H); 7.35 (dd, J=2.7, 9.4 Hz, 1H); 7.56 (s, 1H); 7.65 (d, J=2.6 Hz, 1H); 7.70 (dd, J=1.9, 9.4 Hz, 1H); 7.79 (d, J=9.4 Hz, 1H); 8.18 (d, J=1.9, 1H); 8.40 (d, J=9.4 Hz, 1H).

    Example 3: 4-((9-amino-7-ethoxyacridin-3-yl)diazenyl)-3,5-dimethylphenol hydrate

    [0332] ##STR00020##

    [0333] The title compound was prepared in accordance with Example 1.

    [0334] .sup.1H NMR (400 MHz, DMSO-d.sub.6): 1.40 (t, J=7.0 Hz, 3H); 2.25 (s, 6H); 4.17 (q, J=7.0 Hz, 2H); 7.36 (dd, J=2.4, 9.2 Hz, 1H); 7.60 (s, 2H); 7.66 (d, J=2.4, 1H); 7.70 (dd, J=1.7, 9.4 Hz, 1H); 7.79 (d, J=9.4 Hz, 1H); 8.18 (d, J=1.7, 1H); 8.40 (d, J=9.4 Hz, 1H).

    Example 4: 5-amino-2-((9-amino-7-ethoxyacridin-3-yl)diazenyl)phenol sulphate hydrate and

    Example 5:3-amino-2-((9-amino-7-ethoxyacridin-3-yl)diazenyl)phenol sulphate hydrate

    [0335] ##STR00021##

    [0336] The mixture of the title compounds was prepared in accordance with Example 1.

    [0337] .sup.1H NMR (400 MHz, DMSO-d.sub.6): 1.38 (t, J=7.0 Hz, 3H); 2.45 (s, 6H); 4.14 (q, J=7.0 Hz, 2H); 6.78 (s, 1H); 7.34 (d, J=9.2 Hz, 1H); 7.57 (s, 2H); 7.64 (unresolved d, 1H); 7.67 (d, J=9.6 Hz, 1H); 7.82 (d, J=9.6 Hz, 1H); 8.17 (d, J=1.9 Hz, 1H); 8.40 (d, J=9.2 Hz, 1H).

    Example 6: 4-((9-amino-7-ethoxyacridin-3-yl)diazenyl)-2,6-dimethylphenol

    [0338] ##STR00022##

    [0339] The title compound was prepared in accordance with Example 1.

    [0340] .sup.1H NMR (400 MHz, DMSO-d.sub.6): 1.40 (t, J=7.0 Hz, 3H), 2.25 (s, 6H), 4.17 (q, J=7.0 Hz, 2H), 7.36 (dd, J=2.4, 9.2 Hz, 1H), 7.66 (s, 2H), 7.70 (dd, J=1.7, 9.4 Hz, 1H), 7.79 (d, J=9.4 Hz, 1H), 8.18 (d, J=1.7 Hz, 1H), 8.40 (d, J=9.4 Hz, 1H).

    Example 7: 5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]-N-[[5-[(E)-(9-amino-7-ethoxy-acridin-3-yl)azo]-2-hydroxy-phenyl]methyl]-N-[2-(dimethylamino)ethyl]pentanamide

    [0341] ##STR00023##

    [0342] The title compound was prepared as follows. 4-[(E)-(9-amino-7-ethoxy-acridin-3-yl)azo]-2-[[2-(dimethylamino)ethylamino]methyl]phenol hydrochlorid (13 mg, 0.028 mmol) and (2,5-dioxopyrrolidin-1-yl) 5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoate (10 mg, 0.029 mmol) was treated with triethylamine (10 L, 0.072 mmol) in DMF (1 mL) at rt on. The reaction is clean and complete. The solution was diluted with some water and then purified by acidic preparative HPLC. The compound came rather early and it would have been a good idea to remove some DMF prior to the chromatography. The pure fractions were pooled and evaporated to give 5 mg of a yellow solid. the solid was treated with some MeOH and 2 M HCl, evaporated and dried in vacuum. The HCl salt was dissolved in a small amount of MeOH and diethyl ether was added. The formed solid was filtered off and dried in vacuum. Yield 3 mg.

    Example 8: 4-[(E)-(9-amino-7-ethoxy-acridin-3-yl)azo]-2-[[2-(dimethylamino)ethylamino]methyl]phenol hydrochloride

    [0343] ##STR00024##

    [0344] Sodium cyanoborohydride (25 mg, 0.16 mmol) was added to a stirred solution of 5-[(E)-(9-amino-7-ethoxy-acridin-3-yl)azo]-2-hydroxy-benzaldehyde (25 mg, 0.065 mmol), N,N-dimethylethane-1,2-diamine (15 L, 0.14 mmol) in acetic acid (10%) in methanol (1 mL) and the reaction was left stirring at room temperature for 30 minutes. The reaction mixture was evaporated and re-dissolved in HCl (aq, 0.1M) and the solution was made basic with NaHCO.sub.3 and the resulting solid was filtered of and washed with water. The crude material was purified by acidic reversed phase chromatography. The evaporated material was re-dissolved in methanol and acidified with HCl (aq, 0.1M) and evaporated to give the title compound, yield 8 mg (25%). The free base can be made by treating the hydrochloric salt with NaHCO.sub.3, evaporating and re-dissolving in methanol.

    [0345] .sup.1H NMR (free base) (400 MHz, METHANOL-d.sub.4) ppm 1.42 (t, J=6.95 Hz, 3H) 2.16 (s, 6H) 2.44 (t, J=7.27 Hz, 2H) 2.70 (t, J=7.19 Hz, 2H) 3.57 (d, J=1.90 Hz, 1H) 3.74 (s, 2H) 4.14 (q, J=6.90 Hz, 2H) 6.60 (d, J=8.53 Hz, 1H) 7.30 (dd, J=9.40, 2.61 Hz, 1H) 7.45 (d, J=2.21 Hz, 1H) 7.65 (dd, J=8.77, 2.13 Hz, 1H) 7.70-7.81 (m, 3H) 8.09 (s, 1H) 8.18 (d, J=9.32 Hz, 1H)

    Example 9:4-[(E)-(9-amino-7-ethoxy-acridin-3-yl)azo]-2-[[2-(dimethylamino)ethyl-methyl-amino]methyl]phenol

    [0346] ##STR00025##

    [0347] Sodium triacetoxyborohydride (60 mg, 0.28 mmol) was added to a stirred solution of 5-[(E)-(9-amino-7-ethoxy-acridin-3-yl)azo]-2-hydroxy-benzaldehyde (24 mg 0.06 mmol), N,N,N-trimethylethane-1,2-diamine (15 L, 0.12 mmol) and DIPEA (100 L) in 1,2-dichloroethane (1.5 mL) and left stirring at room temperature overnight. The dark red solid was filtered off, re-dissolved in methanol and purified by acidic reversed phase chromatography to give the title product, yield 4 mg (14%).

    [0348] .sup.1H NMR (400 MHz, METHANOL-d.sub.4) ppm 1.48 (t, J=6.95 Hz, 3H); 2.91 (s, 3H); 2.99 (s, 6H); 3.58-3.77 (m, 4H); 4.19 (q, J=6.95 Hz, 2H); 4.46 (s, 2H); 7.16 (d, J=8.69 Hz, 1H); 7.65 (dd, J=9.24, 2.61 Hz, 1H); 7.75 (d, J=2.37 Hz, 1H); 7.81 (d, J=9.32 Hz, 1H); 7.95 (dd, J=9.24, 1.82 Hz, 1H); 8.05 (dd, J=8.77, 2.45 Hz, 1H); 8.14 (dd, J=14.45, 1.97 Hz, 2H); 8.54 (d, J=9.16 Hz, 1H).

    Example 10:4-[(E)-2-(9-amino-7-ethoxyacridin-3-yl)diazen-1-yl]-2-(hydroxymethyl)phenol

    [0349] ##STR00026##

    [0350] The title compound was prepared in accordance with the techniques described herein.

    Example 11: 5-[(3aS,4S,6aR)-2-oxo-hexahydro-1H-thieno[3,4-d]imidazol-4-yl]-N-{2-[({5-[(E)-2-(9-amino-7-ethoxyacridin-3-yl)diazen-1-yl]-2-hydroxyphenyl}methyl)(methyl) amino]ethyl}-N-methylpentanamide

    [0351] ##STR00027##

    [0352] The title compound was prepared in accordance with the following general technique. Sodium triacetoxyborohydride (4 mg, 0.42 mmol) was added to a stirred solution of 5-[(E)-(9-amino-7-ethoxy-acridin-3-yl)azo]-2-hydroxy-benzaldehyde (0.005 mmol), N,N,N-trimethylethane-1,2-diamine (0.01 mmol) and DIPEA (100 L) in 1,2-dichloroethane (0.5 mL) and left stirring at room temperature for 1 h. Hplc showed that no sm is left and that the major product is the intended. Another portion of aldehyde, amine and hydride was added and the mixture diluted with more solvent (1 mL) and left stirring over the weekend. Where inefficient amination occurred two small lots were treated with a) more Sodium triacetoxyborohydride and b) diluted with MeOH and treated with NaCNBH3. Reaction (a) led to more reduction of the aldehyde whereas (b) gave more of the intended product. The main reaction was diluted with MeOH (2 mL) and treated with 340 mg of NaCNBH3 during 2 days. Purification was performed using silica gel chromatography (DCM/MeOH 4:1+0.1 Et3N).

    Example 12: 4-[(E)-2-(9-amino-7-ethoxyacridin-3-yl)diazen-1-yl]-2-((dimethylamino) methyl)phenol

    [0353] ##STR00028##

    [0354] The title compound was prepared in accordance with the techniques described herein.

    Biological Assays

    [0355] The biological activity of example compounds as described herein above was assessed using the following biological assays.

    Biological Example 1: Inhibition of the MYC:MAX Interaction In Vitro as Analyzed by MST and SPR

    [0356] To analyze the effect of compounds on the MYC:MAX heterodimer formation, two biophysical assays were used; microscale thermophoresis (MST) and surface plasmon resonance (SPR).

    Assay 1A. Microscale Thermophoresis (MST)

    [0357] MST measures the movement of fluorescent labelled molecules in a microscopic temperature gradient. Changes in charge, size or of the hydration shell of the fluorescent molecule affects its migration in the temperature gradient, which can be detected by the fluorescence of the molecule (See Seidel et al., Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions, METHODS (2013)). Molecular interactions (such as protein-inhibitor interactions) affect the direction and speed of migration allowing quantification of the dissociation constant. MST was carried out on a Monolith NT.115 with blue/green filters according to manufacturer's protocol (NanoTemper). MYCbHLHZip and compounds were diluted and titrated in PBS supplemented with 0.05% Tween-20. Titration of protein was carried out in 16 PCR tubes into which a fixed concentration of fluorescent labelled protein was added. The mixture was applied to capillaries (standard treated, NanoTemper) and placed in the sample chamber of the Monolith NT.115. Capillaries were scanned to measure initial fluorescence of compound. MST was induced and fluorescence of the compound was measured during 40 seconds, indicative of thermophoresis. Double measurements were carried out (MST power of 20% and 40%, or 40% and 60%) for each sample. Relative fluorescence of labeled molecule normalized to control (only buffer) was plotted against titrated molecule concentration. In the MST MYC:MAX interaction assay 1 M fluorescently labelled MAXbHLHZip was combined with a mixture of 1 M MYCbHLHZip and different concentrations of example compound. Titration of Example 1 resulted in a thermophoresis shift of labelled MAX with a K.sub.d of 4.3+/2.9 M, indicating that the compound affected the MYC:MAX conformation, while having minor effects on labelled MAX when pre-mixed with 1 M MAX instead of MYC (FIG. 1A), suggesting that Example 1 discriminates well between MYC:MAX and MAX:MAX interactions.

    Assay 1B. Surface Plasmon Resonance (SPR)

    [0358] The SPR method is a highly sensitive assay to determine the affinity between protein and ligand and to measure the kinetics of the interaction (see Handbook of Surface Plasmon Resonance 2nd edition, Richard B M Schasfoort (Editor), RSC 2017, ca. 500 p., hardcover, ISBN 978-1-78262-730-2). In the SPR assay, the target protein is immobilized on a dextran coated chip, and the ligand is injected over the immobilized protein. The Biacore T200 (GE Healthcare) measures adsorption of molecules on the chip by measuring the refractive index. When a ligand binds to the target protein, the refractive index, presented as response units (RU), is altered proportional to the change in mass on the chip, thereby allowing association and dissociation rates between the ligand and the target to be calculated. The SPR experiments were performed at 25 C. using a Biacore T200 (GE Healthcare) instrument. In the SPR MYC:MAX interaction assay MAXbHLHZip was covalently attached onto a CM5 sensor chip by amino coupling procedure resulting in immobilization levels of approximately 200-500 RU. 100 nM MYCbHLHZip pre-mixed with compound was injected over the MAXbHLHZip-surface and thereafter allowed to dissociate. Example 1 inhibited the MYC:MAX heterodimer formation with an IC50 of 3.8+/1.2 M (FIG. 1B-C). A summary of the results is provided in Table 1.

    TABLE-US-00001 TABLE 1 Summary results for Biological Example 1 Assay 1A. Assay 1B. MYC:MAX MST assay MYC:MAX SPR assay Ex. Kd (M) IC50 (M) 1 4.3 +/ 2.9 3.8 +/ 1.2

    Biological Example 2: Binding and Affinity to MYC Analyzed by MST and SPR

    [0359] To analyze the direct binding of compounds to MYC, the effect on MYC in solution was analyzed by microscale thermophoresis (MST) and the direct binding and affinity to MYC was analyzed by surface plasmon resonance (SPR).

    Assay 2A. MYC Binding MST Assay

    [0360] The MYC MST assay was performed as described above (Biological example 1Assay 1A), except that the fluorescent molecule was either protein or Example 1. The example compound was kept at a fix concentration of 3 M in PBS supplemented with 0.05% Tween-20, while MYCbHLHZip or MAX was titrated. The migration of example compound changed at MYC protein concentrations above 1 M, but was unaffected with MAX up to highest concentration tested (15 M) (FIG. 2A). In a reverse experiment, Example 1 was titrated in a fixed protein concentration in PBS supplemented with 0.05% Tween-20 and 1% DMSO as described above after labelling primary amines of the protein using the Protein labelling kit GREEN-NHS (NanoTemper) according to manufacturer's protocol. MYC protein concentration was estimated to be approximately 200 nM, not counting for protein loss during the labelling procedure. Migration of MYC was affected at compound concentrations above 200-400 nM (FIG. 2B). MST for Example 2 was carried out as in (A). Migration of Example 2 changed dramatically at 2 M (and higher) of MYC (FIG. 2C). No effects on MYC thermophoresis was seen with a non-binding control compound (referred to as 6:1).

    Assay 2B. MYC SPR Assay

    [0361] To analyze the direct binding of compounds to MYC, the direct binding of compounds to MYC was detected by surface plasmon resonance (SPR) as described above (Biological example 1Assay 1B). An amino coupling procedure was used to immobilize MYCbHLHZip protein on a CM5 sensor chip (GE Healthcare) resulting in immobilization levels of approximately 800 respectively 1000 RU. The compounds were injected at different concentrations, one at a time, over the surface for 50-80 seconds with a flow rate of 30 l/min. Afterwards the compounds were allowed to dissociate for 240 seconds in running buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na.sub.2HPO4, 1.8 mM KH.sub.2PO4, 0.005% Tween-20, 1% DMSO, pH 7.4). To remove all remaining analyte the surface was regenerated with 2M Urea for 30 seconds and thereafter 1M NaCl, 2.7 mM KCl, 10 mM Na.sub.2HPO4, 1.8 mM KH.sub.2PO4, 1% DMSO, pH 7.4 for 30 seconds. Sensorgrams were generated by subtraction of the reference (blank immobilized) surface. Binding responses were related to respective compounds' Rmax. Rmax is the maximum response of a compound when it binds with a 1:1 stochiometry to the protein; Rmax=(MW analyte/MW ligand)immobilized ligand level on the chip (RU) x stoichiometry (1:1). Kinetic experiments were carried out where recombinant MYC bHLHZip was immobilized on the chip and different concentrations of Example 1 were injected as above. Association and dissociation rates of the compound were obtained from a 1:1 Langmuir model after subtraction of reference cell values, suggesting a K.sub.D value of 1.60.5 M (FIG. 3A). From equilibrium binding experiments, a K.sub.D value of approximately 1.5-2 M was determined by an equilibrium binding plot (FIG. 3B). Example 2-6 were analyzed for their MYC binding properties by equilibrium binding experiments as described for example 1. A summary of the results is provided in Table 2.

    TABLE-US-00002 TABLE 2 Summary of results from Biological Example 2 Assay 2A. Assay 2B. MYC:MAX MST assay. MYC SPR Assay. Effect on Binding relative to Rmax Ex. thermophoresis (M) (Response Units) 1 1 1.6 2 2 1.2 3 NT 1.5 4-5 NT 0.6 6 NT 0.8 NT = not tested

    Biological Example 3: MYC:MAX Interactions in Cells as Shown by ColP, isPLA and GLuc Assays

    [0362] To analyze the MYC:MAX interaction in cells, three assays were used, in situ proximity ligation assay (isPLA), coimmunoprecipitation (CoIP), and Gaussia luciferase complementation assay (GLuc).

    Assay 3A: The isPLA Assay

    [0363] Identification of endogenous protein-protein interactions in fixed cells and has been described (Soderberg, O. et al., Nat Methods, 3, 995-1000 (2006)). MDA-MB231 cells were grown on collagen-coated chamber slides (Falcon), treated with 10 M Example 1 for 24 hours, thereafter washed twice with PBS and fixed in ice cold methanol for 15 min at room temperature. Slides were washed in PBS with 0.05% Tween 20 and incubated in blocking buffer after which isPLA was performed using the Duolink in situ PLA kit (Sigma-Aldrich) according to the manufacturer's protocol. DNA was stained with DAPI. Incubation with primary antibodies directed to MYC and MAX, or FRA-1 and JUN, respectively, were performed at +4 C. overnight. Endogenous MYC:MAX or FRA-1:JUN protein interactions were visualized by fluorescence microscopy as fluorescent dots mainly localized in the cell nucleus. Images were taken using an Axiovert 200M inverted microscope (Zeiss) and fluorescent dots were quantified using semi-automated analysis in ImageJ (http://imagej.net) and averaged to number of dots per cell. Example 1 significantly decreased isPLA signals to 7% of DMSO control, while not affecting the interaction between the bZip transcription factors FRA1 and JUN (FIG. 4A-C).

    Assay 3B: ColP

    [0364] Coimmunoprecipitation of endogenous MYC:MAX proteins were essentially carried out as in Bahram, F. et al. Interferon-gamma-induced p27KIP1 binds to and targets MYC for proteasome-mediated degradation. Oncotarget 7, 2837-2854, doi:10.18632/oncotarget.6693 (2016). MDA-MB231 cells were treated with 5 M Example 1 for 3.5 hours post-harvesting. Endogenous MAX was coimmunoprecipitated with MYC.

    Example 1 Reduced the MYC:MAX Protein Interaction by More than Half of the DMSO Treated Cells (FIG. 4D)

    Assay 3C: GLuc Assay

    [0365] The protein fragment complementation assay using the Gaussia luciferase (GLuc) has been described (Remy, I. & Michnick, S. W. A highly sensitive protein-protein interaction assay based on Gaussia luciferase. Nat Methods 3, 977-979, doi:10.1038/nmeth979 (2006)). 0.2 g of each GLuc-construct (MAX-GLuc-N+MYC-GLuc-C) together with 0.05 g pCMV-Luc (Firefly luciferase) were used for transfection of HEK293 or COS-7 cells. 24 hours later cells were treated with compound or DMSO. After another 17 hours the cells were harvested and lyzed in passive lysis buffer (Promega) supplemented with complete protease inhibitor (Roche). After 60 min incubation at room temperature 20 M D-luciferin was added (substrate of Firefly luciferase) and luminescence was measured in a Lumat LB9501 (Berthold) or OmegaFluostar (BMG Labtech) luminometer. Directly after, the Gaussia luciferase substrate Coelenterazine (Promega) was added to a final concentration of 20 M and the luciferase activity was measured. The ratio between Gaussia and Firefly luciferase values were calculated and normalized to DMSO-treated control cells. Example 1 inhibited exogenous MYC:MAX protein interactions with an IC50 of 10 M. Example 2 inhibited 65% of the MYC:MAX interactions at 10 M (FIG. 4E).

    TABLE-US-00003 TABLE 3 Summary of results from Biological Example 3 MYC:MAX interaction assays in cells Assay 3A: Assay 3B: Assay 3C: isPLA assay CoIP GLuc assay (inhibition at (inhibition at (inhibition at Ex. 10 M) 10 M) 10 M) 1 95% >70% 50% 2 NT NT 65%

    Biological Example 4: MYC Target Gene Expression

    Assay 4: Gene Expression

    [0366] To investigate the effect of the compounds on MYC-driven transcription, we utilized U2OS cells containing a MYC-estrogen receptor (MYC-ER) fusion protein, the activity of which is regulated by the ligand 4-hydroxytamoxifen (HOT). RT-qPCR was performed essentially as described in Bahram, F. et al. Interferon-gamma-induced p27KIP1 binds to and targets MYC for proteasome-mediated degradation. Oncotarget 7, 2837-2854, doi:10.18632/oncotarget.6693 (2016). Activation of MYC (vehicle control+HOT) for 24 hours resulted in increased expression of three previously described direct MYC target genes, OC1, RSG16 and CR2. Treatment with Example 1 significantly reduced HOT-induced expression of all three genes (FIG. 5A-C) indicating that Example 1 inhibits MYC-driven transcription, which is expected of a potent MYC:MAX inhibitor.

    TABLE-US-00004 TABLE 4 Summary of results from Biological Example 4 Assay 4. MYC:MAX inhibition of gene expression at 10 M. Ex. ODC1 gene RSG16 gene CR2 gene 1 100% 68% 100%

    Biological Example 5: MYC-Dependent Cell Growth Inhibition as Measured by Metabolic Activity Assay (WST-1)

    [0367] MYC-dependent cell growth inhibition was analyzed in neuroblastoma cell lines and immortal Rat1 fibroblasts (Tgr) cell lines with different MYC statuses.

    Assay 5A: Neuroblastoma Cell Growth

    [0368] Neuroblastoma cell lines with (SK-N-DZ, Kelly and IMR-32) or without (SK-N-F1, SK-N-RA and SK-N-AS) MYCN-amplification were exposed to different concentrations of example compounds for 48 hours. Cell growth and viability was estimated by measuring metabolic activity by incubating cells in triplicates with WST-1 (Roche) in medium at 37 C. and 5% CO.sub.2 for 2 hours after which absorbance was measured with an Omega Fluostar (BMG Labtech) in a 96 well plate format. Example 1 reduced growth of the MYCN-amplified cell lines significantly stronger than of the MYCN-non-amplified cell lines with an average growth inhibition of 50% (GI50) value of 2.5-6 and >20 M, respectively (FIG. 6A), thereby showing selectivity for MYC-driven tumors cells. Examples 2-6 and 8-9 also discriminated between cells dependent on MYCN status (FIG. 6B). Examples 10-12 have growth inhibition activities in MYCN-driven tumor cells.

    Assay 5B: Tgr Cell Growth

    [0369] Tgr cells were treated at 37 C. and 5% CO.sub.2 at 48 hours with Example and Example 2. H015.19 is a MYC null rat cell line derived from Tgr1 (parental cells), while H0Myc3 cells were generated from the MYC null cells by reconstitution of a MYC gene(Mateyak, M. K., Obaya, A. J., Adachi, S. & Sedivy, J. M. Phenotypes of c-Myc-deficient rat fibroblasts isolated by targeted homologous recombination. Cell Growth Differ 8, 1039-1048 (1997)). Example 1 strongly inhibited growth of wt and reconstituted cells, but did not significantly affect growth of the MYC null cells, thus showing a clear difference in response between MYC expressing and MYC-deficient cells (FIG. 60). Example 2 also discriminated between cells dependent on MYC status (FIG. 60).

    TABLE-US-00005 TABLE 6 Summary of results from Biological Example 5 (first run) MYC-dependent cell growth inhibition Immortal Rat1 fibroblast (Tgr) Neuroblastoma cells cells MYCN- non-MYCN- Tgr and MYC null amplified cells amplified cells H0Myc3 cells (H015.19) cells Ex. (GI50 M) (GI50 M) (GI50 M) (GI50 M) 1 2.5-6 >25 4 >>10 2 4 12.5 NT NT

    TABLE-US-00006 TABLE 7 Summary of results from Biological Example 5 (second run) MYC-dependent cell growth inhibition Neuroblastoma cells MYCN-amplified cells non-MYCN-amplified cells Ex. (GI50 M) (GI50 M) 1 2.5 >25 2 2.5 10 3 2 >25 4 and 5 20 >25 6 1 >25 7 8 4 10 9 6 11 10 15 NT 11 12 25 NT

    Biological Example 6: Bioactivity in MYC-Dependent Mouse Tumor Models

    [0370] To validate the efficacy of Example 1 in tumor tissue in vivo, we utilized a mouse xenograft tumor model based on human MYCN-amplified SK-N-DZ neuroblastoma cells. Tumor cells (510.sup.6) were injected into the flank of athymic nude mice and allowed to form tumors after which Example 1 or vehicle were administered by daily intraperitoneal injection at a dose of 20 mg/kg body weight for 1-2 weeks. TUNEL-staining of tumor sections revealed a dramatic increase in apoptotic tumor areas (FIG. 7A) and a significant increase in non-proliferative areas as determined by Ki67 staining (FIG. 7B) in tumors from compound-treated mice compared with vehicle-treated mice. CD31 staining of endothelial cells revealed a significantly reduced microvascular density (MVD) in compound-treated mice compared with vehicle-treated mice (FIG. 7C). isPLA analysis showed a significant reduction in MYCN:MAX interactions in tumors from compound-treated compared to vehicle-treated mice (FIG. 7D), indicating that Example 1 reaches and is active against its target in vivo.

    TABLE-US-00007 TABLE 7 Summary of results from Biological Example 6 Assay 7. In vivo mouse tumor model Molecular characteristics TUNEL Ki67 Increase in Increase in CD31 MYCN:MAX isPLA apoptotic Ki67-negative Decrease in Decrease in tumor area tumor area microvascular MYCN:MAX isPLA relative to relative to density (% of signal (% of Ex. vehicle (%) vehicle (%) vehicle) vehicle) 1 1420 550 44 66