2-HETEROARYL-3-OXO-2,3-DIHYDROPYRIDAZINE-4-CARBOXAMIDES FOR THE TREATMENT OF CANCER

Abstract

The present invention covers 2-heteroaryl-3-oxo-2,3-dihydropyridazine-4-carboxamide compounds of general formula (1):

##STR00001##

in which X, R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 are as defined herein, methods of preparing said compounds, intermediate compounds useful for preparing said compounds, pharmaceutical compositions and combinations comprising said compounds and the use of said compounds for manufacturing pharmaceutical compositions for the treatment or prophylaxis of diseases, in particular of cancer or conditions with dysregulated immune responses or other disorders associated with aberrant AHR signaling, as a sole agent or in combination with other active ingredients.

Claims

1. A compound having the structure: ##STR00501## or a hydrate or solvate thereof, or a pharmaceutically acceptable salt of any of the foregoing.

2. The compound of claim 1, which is ##STR00502## or a pharmaceutically acceptable salt thereof.

3. The compound of claim 1, which is ##STR00503## or a pharmaceutically acceptable salt thereof.

4. A composition, comprising 6-(4-chlorophenyl)-N-(1-hydroxypropan-2-yl)-2-(1-methyl-1H-pyrazol-4-yl)-3-oxo-2,3-dihydropyridazine-4-carboxamide, or a pharmaceutically acceptable salt thereof.

5. The composition of claim 4, comprising a racemic mixture of: 6-(4-chlorophenyl)-N-[(2S)-1-hydroxypropan-2-yl]-2-(1-methyl-1H-pyrazol-4-yl)-3-oxo-2,3-dihydropyridazine-4-carboxamide, or a pharmaceutically acceptable salt thereof; and 6-(4-chlorophenyl)-N-[(2R)-1-hydroxypropan-2-yl]-2-(1-methyl-1H-pyrazol-4-yl)-3-oxo-2,3-dihydropyridazine-4-carboxamide, or a pharmaceutically acceptable salt thereof.

6. The composition of claim 4, comprising 6-(4-chlorophenyl)-N-[(2S)-1-hydroxypropan-2-yl]-2-(1-methyl-1H-pyrazol-4-yl)-3-oxo-2,3-dihydropyridazine-4-carboxamide, or a pharmaceutically acceptable salt thereof.

7. A pharmaceutical composition, comprising a compound of claim 2, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.

8. A pharmaceutical composition, comprising a compound of claim 3, or a pharmaceutically acceptable salt thereof.

9. A method of inhibiting AHR in a subject in need thereof, comprising administering to the subject a compound of claim 2, or a pharmaceutically acceptable salt thereof.

10. The method of claim 9, wherein the subject is a human.

11. A method of inhibiting AHR in a subject in need thereof, comprising administering to the subject a compound of claim 3, or a pharmaceutically acceptable salt thereof.

12. The method of claim 11, wherein the subject is a human.

13. A method of treating a disease or disorder associated with aberrant AHR signaling in a subject in need thereof, comprising administering to the subject a compound of claim 2, or a pharmaceutically acceptable salt thereof.

14. The method of claim 13, wherein the subject is a human.

15. A method of treating a disease or disorder associated with aberrant AHR signaling in a subject in need thereof, comprising administering to the subject a compound of claim 3, or a pharmaceutically acceptable salt thereof.

16. The method of claim 15, wherein the subject is a human.

17. The method according to claim 15, wherein the disease is a cancer or a condition with dysregulated immune responses.

18. The method according to claim 15, wherein the disease is a liquid tumor or a solid tumor.

19. The method according to claim 15, wherein the disease is a respiratory tract cancer, head and neck cancer, or a kidney cancer.

20. The method according to claim 19, wherein the disease is small-cell lung carcinoma, non-small-cell lung carcinoma, bronchial adenoma, or pleuropulmonary blastoma.

21. The method according to claim 20, wherein the disease is non-small-cell lung carcinoma.

22. The method according to claim 19, wherein the disease is renal cell carcinoma, urothelial cell carcinoma, juxtaglomerular cell tumor (reninoma), angiomyolipoma, renal oncocytoma, Bellini duct carcinoma, clear-cell sarcoma of the kidney, mesoblastic nephroma, or Wilms' tumor.

23. The method according to claim 22, wherein the disease is urothelial cell carcinoma.

24. The method according to claim 19, wherein the disease is squamous cell cancer of the head and neck, laryngeal cancer, hypopharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer, salivary gland cancer, or lip and oral cavity cancer.

25. The method according to claim 24, wherein the disease is head and neck squamous cell carcinoma.

Description

DESCRIPTION OF THE FIGURES

[1629] FIG. 1 describes the sequence listing of the light chain of the TPP-3911 antibody (anti-PD-L1-mIgG1Kappa_RG7446chimera|light_chain|pTT5-anti-PD-L1-huVH-muIgG1-CH1-CH3-kappa-chimera)

[1630] FIG. 2 describes the sequence listing of the heavy chain of the TPP-3911 antibody (anti-PD-L1-mIgG1Kappa_RG7446chimera|heavy_chain|pTT5-anti-PD-L1-huVH-muIgG1-CH1-CH3-kappa-chimera

EXPERIMENTAL SECTION—BIOLOGICAL ASSAYS

[1631] Examples were tested in selected biological assays one or more times. When tested more than once, data are reported as either average values or as median values, wherein [1632] the average value, also referred to as the arithmetic mean value, represents the sum of the values obtained divided by the number of times tested, and [1633] the median value represents the middle number of the group of values when ranked in ascending or descending order. If the number of values in the data set is odd, the median is the middle value. If the number of values in the data set is even, the median is the arithmetic mean of the two middle values.

[1634] Examples were synthesized one or more times. When synthesized more than once, data from biological assays represent average values or median values calculated utilizing data sets obtained from testing of one or more synthetic batch.

[1635] The in vitro activity of the compounds of the present invention can be demonstrated in the following assays:

Transactivation Assay in Human Cell Line (In Vitro Assays 1 and 2)

[1636] Transactivation assays were carried out in U87 glioblastoma cells (ATCC) endogenously expressing AHR. In addition the cells were stably transfected with an AHR inducible firefly luciferase reporter gene construct that carried AHR-binding sites (DRE) in its promoter and a renilla reporter gene construct with constitutively active promoter. Kynurenic acid is an endogenous AHR activating ligand and was used to prestimulate test cells prior to testing the antagonistic properties of compounds.

In Vitro Assay 1: Antagonism in Human Cell Line

[1637] Cells in medium (tryptophan free RPMI, 1% FCS, 2 mM Glutamine) supplemented with 150 uM kynurenic acid were grown for 20 hours in absence (negative control) or presence of increasing concentrations of test compounds (typical dilutions: 72 pmol/L, 0.25 nmol/L, 0.89 nmol/L; 3.1 nmol/L, 11 nmol/L, 38 nmol/L, 130 nmol/L, 470 nmol/L, 1.6 μmol/L, 5.7 μmol/L and 20 μmol/L in duplicates). As positive inhibition control cells supplemented with 150 uM kynurenic acid were incubated in presence of 5 uM Staurosporin. Normalization was done by positive and negative controls.

[1638] Firefly luciferase and Renilla activity was determined by the DualGlo Luciferase Assay System (Promega, #2920). Renilla activity was used to assess toxic effects of compounds.

In Vitro Assay 2: Agonism in Human Cell Line

[1639] Cells in medium (tryptophan free RPMI, 1% FCS, 2 mM Glutamine) were grown for 20 hours in absence (negative control) or presence of increasing concentrations of test compounds (typical dilutions: 72 pmol/L, 0.25 nmol/L, 0.89 nmol/L; 3.1 nmol/L, 11 nmol/L, 38 nmol/L, 130 nmol/L, 470 nmol/L, 1.6 μmol/L, 5.7 μmol/L and 20 μmol/L in duplicates). As positive activation control cells were incubated with 300 uM kynurenic acid. Normalization was done by positive and negative controls.

[1640] Firefly luciferase activity was determined by the SteadyGlo Luciferase Assay System (Promega, #2520).

In Vitro Assay 3: AHR-Regulated CYP1A1 Expression in Human Cell Line

[1641] To assess the AHR inhibitory activity of the substances described in this application, the ability thereof to antagonise ligand-induced AHR gene regulation in a dose-dependent manner was quantified. For this purpose, quantitative PCR analysis was used to determine expression of the AHR-regulated gene CYP1A1 in a human monocytic U937 cell line upon stimulation with 200 uM KA in the presence and absence of AHR inhibitor. U937 cells were sown at a concentration of 2×10.sup.5 cells/well in 100 ul of growth medium (RPMI 1640, 20% FCS) in 96-well microtitre plates. CYP1A1 expression was induced with 200 uM KA (positive control) in the presence or absence of the substances for 6 hours. Human U937 cells were typically incubated with eight different concentrations of the substances (1 nM, 3 nM, 10 nM, 30 nM, 100 nM, 300 nM, 1 uM and 3 uM) and analyzed in duplicate on the same microtitre plate. After stimulation, cells were lysed with Nucleic Acid Lysis Solution (#4305895, Applied Biosystems) and RNA was isolated using the 6100 Nucleic Acid Preparation Station (Applied Biosystems) and reverse-transcribed to cDNA using SuperScript VILO cDNA synthesis kit (#11754-250, Invitrogen). Unstimulated cells were used as the negative control. Taqman probes for human CYP1A1 (Hs01054797_g1) and human HPRT (Hs02800695_m1) were used to analyze fold expression of CYP1A1 of HPRT. Quantitation was performed on a Taqman SDS7900HT.

TABLE-US-00003 TABLE 3 IC.sub.50 values of examples in in vitro assays 1-3 Assay 1: Assay 2: Assay 3: AHR-luc Hum AHR-luc Hum Hum CYP1A1 Antagonism Agonism Antagonism Example IC.sub.50 [M] IC.sub.50 [M] IC.sub.50 [M] 1 7.38E−9 >2.00E−5 4.30E−9 2 2.57E−9 >2.00E−5 3.03E−9 3 1.05E−7 >2.00E−5 4 9.52E−9 >2.00E−5 5 5.99E−9 >2.00E−5 3.45E−9 6 1.81E−7 >2.00E−5 7 2.05E−8 >2.00E−5 8 1.90E−8 >2.00E−5 1.58E−8 9 1.64E−7 >2.00E−5 10 4.25E−9 >2.00E−5 1.02E−8 11 4.55E−9 >2.00E−5  1.50E−10 12 2.32E−8 >2.00E−5 1.85E−8 13 3.25E−8 >2.00E−5 2.00E−8 14 1.21E−8 >2.00E−5 2.55E−8 15 2.65E−8 >2.00E−5 1.22E−7 16 3.69E−9 >2.00E−5 2.49E−9 17 1.50E−8 >2.00E−5 2.50E−8 18 1.42E−8 >2.00E−5 1.13E−8 19 3.92E−9 >2.00E−5 6.43E−9 20 2.67E−8 >2.00E−5 6.31E−8 21 2.80E−8 >2.00E−5 1.82E−8 22 2.00E−8 >2.00E−5 1.12E−8 23 4.57E−8 >2.00E−5 24 8.75E−8 >2.00E−5 25 6.82E−8 >2.00E−5 9.11E−8 26 8.75E−8 >2.00E−5 27 1.77E−7 >2.00E−5 28 2.93E−7 >2.00E−5 29 7.61E−8 >2.00E−5 30 6.08E−8 >2.00E−5 31 2.44E−7 >2.00E−5 32 3.56E−8 >2.00E−5 2.19E−8 33 2.66E−9 >2.00E−5 34 1.30E−7 >2.00E−5 35 1.50E−9 >2.00E−5 4.10E−8 36 >2.00E−5 37 2.64E−9 >2.00E−5  4.23E−10 38 3.43E−8 >2.00E−5 3.62E−8 39 2.08E−9 >2.00E−5 3.39E−9 40 1.10E−6 >2.00E−5 41 1.65E−6 >2.00E−5 42 1.57E−6 >2.00E−5 43 2.30E−8 >2.00E−5 44 1.16E−8 >2.00E−5 9.43E−9 45 4.15E−8 >2.00E−5 8.35E−8 46 1.48E−9 >2.00E−5 1.36E−9 47 1.52E−9 >2.00E−5 1.66E−9 48 2.49E−9 >2.00E−5 49 6.50E−9 >2.00E−5 7.55E−9 50 8.39E−9 >2.00E−5 6.74E−9 51 9.54E−9 >2.00E−5 52 1.15E−8 >2.00E−5 53 1.16E−8 >2.00E−5 1.11E−8 54 1.43E−8 >2.00E−5 55 1.79E−8 >2.00E−5 56 2.56E−8 >2.00E−5 57 7.48E−9 >2.00E−5 58 1.86E−8 >2.00E−5 1.08E−8 59 2.00E−8 >2.00E−5 60 2.18E−8 >2.00E−5 61 2.84E−8 >2.00E−5 3.89E−8 62 2.95E−8 >2.00E−5 1.91E−8 63 8.10E−8 >2.00E−5 64 8.16E−8 >2.00E−5 65 2.55E−8 >2.00E−5 66 2.59E−7 >2.00E−5 67 4.30E−9 >2.00E−5 68 2.42E−8 >2.00E−5 2.18E−8 69 2.14E−7 >2.00E−5 70 1.59E−7 >2.00E−5 71 3.80E−8 >2.00E−5 6.07E−8 72 1.82E−7 >2.00E−5 4.95E−7 73 2.83E−7 >2.00E−5 3.55E−7 74 1.52E−7 >2.00E−5 75 8.34E−8 >2.00E−5 76 1.19E−8 >2.00E−5 1.38E−8 77 1.12E−8 >2.00E−5 8.70E−9 78 6.68E−9 >2.00E−5 4.81E−9 79 3.95E−8 >2.00E−5 8.04E−8 80 2.71E−8 >2.00E−5 3.26E−8 81 1.06E−7 >2.00E−5 82 3.76E−8 >2.00E−5 3.45E−8 83 4.61E−9 >2.00E−5 84 1.63E−7 >2.00E−5 85 3.60E−8 >2.00E−5 86 5.59E−8 >2.00E−5 87 2.96E−8 >2.00E−5 2.15E−8 88 7.80E−8 >2.00E−5 89 4.80E−9 >2.00E−5 2.64E−9 90 2.17E−8 >2.00E−5 4.02E−8 91 4.81E−8 >2.00E−5 92 1.90E−8 >2.00E−5 2.76E−9 93 2.48E−8 >2.00E−5 4.32E−9 94 8.44E−9 >2.00E−5 1.65E−8 95 6.23E−8 >2.00E−5 96 3.67E−9 >2.00E−5 3.77E−9 97 7.06E−9 >2.00E−5 98 2.51E−7 >2.00E−5 99 1.29E−7 >2.00E−5 100 1.27E−8 >2.00E−5 101 9.54E−9 >2.00E−5 102 1.88E−9 >2.00E−5 1.69E−9 103 1.33E−8 >2.00E−5 2.45E−8 104 2.02E−8 >2.00E−5 1.48E−8 105 2.37E−9 >2.00E−5 1.86E−9 106 >2.00E−5 107 1.23E−8 >2.00E−5 108 >2.00E−5 109 1.63E−9 >2.00E−5 110 3.31E−9 >2.00E−5 111 2.65E−8 >2.00E−5 112 1.15E−9 >2.00E−5  9.05E−10 113 4.83E−8 >2.00E−5 1.05E−7 114 8.55E−8 >2.00E−5 115 2.63E−9 >2.00E−5 1.09E−8 116 1.15E−7 >2.00E−5 117 >2.00E−5 118 4.51E−8 >2.00E−5 5.70E−8 119 3.06E−9 >2.00E−5 5.72E−9 120 6.79E−8 >2.00E−5 121 2.54E−9 >2.00E−5 122 2.65E−8 >2.00E−5 123 5.70E−9 >2.00E−5 124 3.64E−9 >2.00E−5 125 3.82E−9 >2.00E−5 4.40E−9 126 4.60E−9 >2.00E−5 5.15E−9 127 4.17E−9 >2.00E−5 128 5.05E−9 >2.00E−5 129 5.53E−9 >2.00E−5 130 6.54E−9 >2.00E−5 6.42E−9 131 6.23E−9 >2.00E−5 7.29E−9 132 6.88E−9 >2.00E−5 133 7.52E−9 >2.00E−5 134 7.68E−9 >2.00E−5 9.89E−9 135 7.91E−9 >2.00E−5 136 6.04E−8 >2.00E−5 137 4.23E−9 >2.00E−5 138 9.04E−9 >2.00E−5 7.61E−9 139 9.20E−9 >2.00E−5 9.16E−9 140 9.55E−9 >2.00E−5 141 1.48E−7 >2.00E−5 142 8.32E−9 >2.00E−5 143 9.61E−9 >2.00E−5 144 3.14E−8 >2.00E−5 145 6.90E−9 >2.00E−5 146 1.03E−8 >2.00E−5 147 1.22E−8 >2.00E−5 1.11E−8 148 1.53E−8 >2.00E−5 149 1.88E−8 >2.00E−5 1.12E−8 150 1.99E−8 >2.00E−5 151 2.11E−8 >2.00E−5 152 2.41E−8 >2.00E−5 2.13E−8 153 2.42E−8 >2.00E−5 2.17E−8 154 3.68E−8 >2.00E−5 155 9.05E−8 >2.00E−5 156 2.50E−8 >2.00E−5 157 3.93E−8 >2.00E−5 158 2.18E−8 >2.00E−5 2.52E−8 159 5.70E−8 >2.00E−5 9.79E−8 160 7.92E−8 >2.00E−5 161 1.03E−7 >2.00E−5 162 2.73E−8 >2.00E−5 3.73E−8 163 1.34E−7 >2.00E−5 164 1.57E−7 >2.00E−5 165 2.09E−7 >2.00E−5 166 1.80E−7 167 2.39E−7 >2.00E−5 168 2.40E−7 169 3.17E−8 >2.00E−5 170 5.73E−8 >2.00E−5 2.24E−8 171 5.84E−8 >2.00E−5 172 2.95E−8 >2.00E−5 2.34E−8 173 5.49E−8 >2.00E−5 1.40E−7 174 6.15E−8 >2.00E−5 175 6.61E−8 >2.00E−5 1.12E−7 176 6.61E−8 >2.00E−5 3.88E−8 177 4.65E−7 >2.00E−5 178 5.94E−7 >2.00E−5 179 7.36E−7 >2.00E−5 180 7.94E−7 >2.00E−5 181 9.11E−7 >2.00E−5 182 6.11E−6 >2.00E−5 183 1.97E−6 >2.00E−5 184 3.81E−6 >2.00E−5 185 3.71E−8 >2.00E−5 186 1.31E−7 >2.00E−5 187 3.35E−8 >2.00E−5 188 1.25E−8 >2.00E−5 189 3.03E−9 >2.00E−5 190 6.56E−8 >2.00E−5 191 3.35E−9 >2.00E−5 192 3.41E−7 >2.00E−5 4.30E−9 193 3.52E−7 >2.00E−5 3.03E−9 194 3.09E−9 >2.00E−5 7.60E−9 195  3.63E−10 >2.00E−5 196  4.18E−10 >2.00E−5 197  5.02E−10 >2.00E−5 198  5.99E−10 >2.00E−5 199  6.94E−10 >2.00E−5 200  7.34E−10 >2.00E−5 201  8.23E−10 >2.00E−5 202  8.25E−10 >2.00E−5 203  8.52E−10 >2.00E−5 204 1.00E−9 >2.00E−5 1.55E−9 205 1.06E−9 >2.00E−5 206 1.07E−9 >2.00E−5 207 1.21E−9 >2.00E−5 208 1.22E−9 >2.00E−5 209 1.24E−9 >2.00E−5 210 1.46E−9 >2.00E−5 2.02E−9 211 1.62E−9 >2.00E−5 2.67E−9 212 1.69E−9 >2.00E−5 213 1.92E−9 >2.00E−5 214 2.07E−9 >2.00E−5 215 2.21E−9 >2.00E−5 216 2.38E−9 >2.00E−5 217 2.44E−9 >2.00E−5 7.01E−9 218 2.78E−9 >2.00E−5 4.01E−9 219 3.21E−9 >2.00E−5 220 3.36E−9 >2.00E−5 1.96E−9 221 3.63E−9 >2.00E−5 222 4.27E−9 >2.00E−5 223 4.31E−9 >2.00E−5 224 4.37E−9 >2.00E−5 225 5.04E−9 >2.00E−5 226 5.91E−9 >2.00E−5 227 5.99E−9 >2.00E−5 1.16E−8 228 1.33E−8 >2.00E−5 229 7.09E−9 >2.00E−5 1.22E−8 230 7.57E−9 >2.00E−5 1.04E−8 231 7.89E−9 >2.00E−5 9.93E−9 232 8.16E−9 >2.00E−5 7.38E−9 233 8.23E−9  1.72E−5 234 8.42E−9 >2.00E−5 9.60E−9 235 8.87E−9 >2.00E−5 236 9.03E−9 >2.00E−5 237 9.38E−9 >2.00E−5 7.86E−9 238 9.88E−9 >2.00E−5 1.47E−8 239 1.00E−8 >2.00E−5 240 1.06E−8 >2.00E−5 2.27E−8 241 1.09E−8 >2.00E−5 3.63E−8 242 1.11E−8 >2.00E−5 243 1.18E−8 >2.00E−5 244 1.19E−8 >2.00E−5 245 1.28E−8 >2.00E−5 246 1.32E−8 >2.00E−5 247 1.39E−8 >2.00E−5 248 1.41E−8 >2.00E−5 3.13E−8 249 1.53E−8 >2.00E−5 250 1.69E−8 >2.00E−5 1.05E−8 251 1.79E−8 >2.00E−5 252 1.80E−8 >2.00E−5 253 1.94E−8 >2.00E−5 254 2.29E−8 >2.00E−5 255 2.58E−8 >2.00E−5 256 2.60E−8 >2.00E−5 257 2.63E−8 >2.00E−5 258 2.75E−8 >2.00E−5 259 2.81E−8 >2.00E−5 260 2.94E−8 >2.00E−5 1.44E−8 261 3.30E−8 >2.00E−5 262 4.12E−8 >2.00E−5 263 4.19E−8 >2.00E−5 264 4.44E−8 >2.00E−5 8.56E−8 265 4.88E−8 >2.00E−5 266 5.68E−8 >2.00E−5 267 5.69E−8 >2.00E−5 268 5.69E−8 >2.00E−5 269 6.17E−8 >2.00E−5 270 6.45E−8 >2.00E−5 271 7.20E−8 >2.00E−5 272 9.11E−8 >2.00E−5 273 1.01E−7 >2.00E−5 274 1.11E−7 >2.00E−5 275 1.30E−7 >2.00E−5 276 1.31E−7 >2.00E−5 277 1.39E−7 >2.00E−5 278 1.40E−7 >2.00E−5 279 1.68E−7 >2.00E−5 280 1.78E−7 >2.00E−5 281 2.61E−7 >2.00E−5 282 3.04E−7 >2.00E−5 283 4.30E−7 >2.00E−5 284 5.71E−7 >2.00E−5 285 6.02E−7 >2.00E−5 286 6.72E−7 >2.00E−5 287 7.21E−7 >2.00E−5 288 8.20E−7 >2.00E−5 289 8.75E−7 >2.00E−5 290 2.82E−9 >2.00E−5 291 4.38E−9 >2.00E−5 292 4.59E−8 >2.00E−5 4.33E−8 293 8.22E−8 >2.00E−5 294 4.98E−8 >2.00E−5 295 3.19E−8 >2.00E−5 8.99E−8 296 1.18E−7 >2.00E−5 297 1.49E−9 >2.00E−5 298 3.69E−9 >2.00E−5 299 1.33E−8 >2.00E−5 300 >2.00E−5 301 3.10E−8 >2.00E−5 302 8.15E−8 >2.00E−5 303 1.33E−7 >2.00E−5 304 3.49E−7 >2.00E−5 305 1.64E−9 >2.00E−5 306 1.33E−9 >2.00E−5 307 7.39E−8 >2.00E−5 308 2.19E−8 >2.00E−5 3.37E−8 309 1.64E−8 >2.00E−5 4.03E−8 310 1.70E−9 >2.00E−5 1.69E−9 311 1.82E−9 >2.00E−5 1.03E−9 312 1.85E−9 >2.00E−5 313 2.63E−9 >2.00E−5 3.81E−9 314 5.80E−9 >2.00E−5 1.09E−8 315 1.59E−8 >2.00E−5 1.19E−8 316 2.06E−8 >2.00E−5 317 3.03E−8 >2.00E−5 3.73E−8 318 3.20E−8 >2.00E−5 319 3.55E−8 >2.00E−5 2.23E−8 320 4.41E−8 >2.00E−5 321 5.06E−8 >2.00E−5 322 5.67E−8 >2.00E−5 3.11E−8 323 6.04E−8 >2.00E−5 324 1.22E−7 >2.00E−5 325 2.42E−7 >2.00E−5 326  5.04E−10 >2.00E−5 327 2.58E−9 >2.00E−5 328 3.06E−8 >2.00E−5 329 2.94E−8 >2.00E−5 330 9.27E−8 >2.00E−5 331 2.34E−7 >2.00E−5 332 9.45E−8 >2.00E−5 333 7.49E−8 >2.00E−5 334 6.02E−8 >2.00E−5 335 1.13E−8 >2.00E−5 336 4.29E−8 >2.00E−5 337 2.08E−8 >2.00E−5 338 2.41E−8 >2.00E−5 339 2.76E−8 >2.00E−5 340 4.97E−8 >2.00E−5 341 1.02E−7 >2.00E−5 342 1.32E−7 >2.00E−5 343 1.71E−7 >2.00E−5 344 1.74E−7 >2.00E−5 345 5.53E−8 >2.00E−5
In Vitro Assay 4: Rescue of TNFα Production from Human Primary Monocytes

[1642] The ability of the substances to enhance immune cell activity was determined. The substances were tested for their capacity to reverse KA-induced inhibition of TNFα production by LPS-stimulated human monocytes. Human monocytes were purified by negative selection from donor PBMCs using Miltenyi beads and seeded at 2×10.sup.5 cells/well in complete growth medium (RPMI 1640, 10% FCS). Monocytes were incubated with 10 ng/mL LPS (O127:B38, #L4516, Sigma) and 200 uM KA (#3375, Sigma) and substances were added at concentrations of 1 uM, 0.3 uM and 0.1 uM and cultured for 18 hours. LPS alone served as the positive control. TNFα production in the supernatant was measured by Meso Scale Discovery immunoassay and the ability of the substances to rescue TNFα production was calculated as a percentage of LPS stimulation and KA-induced inhibition and normalized to the donor-specific response with the reference AHR antagonist compound GNF-351 (Smith et al., J Pharmacol Exp Ther, 2011, 338(1):318-27). Table 4 shows highest percent TNFα rescue relative to highest percent rescue with GNF-351 (observed predominantly at 0.3 and 0.1 uM) and the concentration at which highest rescue was observed with the test compound.

TABLE-US-00004 TABLE 4 Human monocytes:Efficacy of selected examples in in vitro assay 4 Individual Donors % rescue TNFα Individual Donors Example normalised to ref cmpd Conc of highest rescue 5 67 1 μM 99 1 μM 61 1 μM 7 126 0.1 μM 63 1 μM 49 1 μM 8 99 1 μM 55 1 μM 104 1 μM 109 1 μM 36 0.1 μM 12 74 0.1 μM 122 1 μM 213 0.1 μM 109 0.3 μM 13 24 1 μM 85 1 μM 16 249 1 μM 165 1 μM 17 438 1 μM 296 1 μM 18 83 1 μM 73 1 μM 76 0.3 μM 105 0.3 μM 21 87 0.3 μM 77 1 μM 22 66 1 μM 71 1 μM 23 274 1 μM 81 1 μM 24 84 0.3 μM 59 1 μM 25 46 1 μM 53 1 μM 27 87 1 μM 105 0.3 μM 29 66 1 μM 88 1 μM 36 74 1 μM 65 1 μM 47 68 1 μM 64 1 μM 69 88 0.1 μM 97 0.1 μM 79 76 0.3 μM 63 0.1 μM 88 69 1 μM 35 1 μM 110 85 0.1 μM 42 0.3 μM 113 91 1 μM 51 1 μM 138 49 0.1 μM 84 0.3 μM 147 50 0.1 μM 89 0.3 μM 184 63 0.1 μM 62 1 μM 186 65 0.1 μM 108 0.3 μM

In Vivo Assay: Efficacy of Compositions Comprising an Example Compound and a PD-1/-L1 Axis Antagonist

[1643] Animals are ordered from Charles River Sulzfeld, Germany and assigned to the study at the age of 7 weeks. Animal husbandry, feeding and health conditions are according to animal welfare guidelines. B16F10-OVA cells are B16F10 mouse melanoma cells that were virally transduced to express ovalbumin. B16F10-OVA cells were cultivated with RPMI 1640 with 10% FCS+2.5 μg/ml blasticidin and splitted at least 3 times before inoculation. The antibiotic blasticidin is removed 1 passage before inoculation. Female C57/BL6N mice were inoculated with 100000 B16F10OVA tumor cells in 50% medium/50% matrigel subcutaneously in the flank. After 5 days the animals were randomized and therapeutic treatment started. The AhR antagonist was dissolved in Ethanol/Solutol/Water (10/40/50) and given at 30 mg/kg, QD, p.o. The anti-PD-L1 antibody (TPP-3911) was dosed at 10 mg/kg, q3d, i.p. The isotype control mIgG1 was given 10 mg/kg q3d (TPP-3267), i.p.

[1644] The anti-PDL1 antibody is a chimera of the variable domain of atezolizumab with murine IgG1 CH1, 2 and 3 domains. TPP-3911. The isotype antibody is a mouse IgG1 (clone MOPC-21, BioXCell BE0083).

[1645] Tumor size was measured using calipers determining length (a) and width (b). Tumor volume was calculated according to:

[00001]$v=\frac{a\times b^2}{2}$

[1646] Based on tumor volume efficacy was calculated dividing tumor volume of the respective treatment group by tumor volume of the control group (T/C).

TABLE-US-00005 Control: Isotype + aPDL1 + Example 17 + Example 17 + vehicle vehicle isotype aPDL1 T/C 1.00 0.96 0.92 0.71

In Vivo Assay: Efficacy of Compositions Comprising an Example Compound and a CTLA4 Axis Antagonist

[1647] Animals are ordered from Charles River Sulzfeld, Germany and assigned to the study at the age of 8 weeks. Animal husbandry, feeding and health conditions are according to animal welfare guidelines. B16F10-OVA cells are B16F10 mouse melanoma cells that were virally transduced to express ovalbumin. B16F10-OVA cells were cultivated with RPMI 1640 with 10% FCS+2.5 μg/ml blasticidin and splitted at least 3 times before inoculation. The antibiotic blasticidin is removed 1 passage before inoculation. Female C57/BL6J mice were inoculated with 10000 B16F10-OVA tumor cells in 50% medium/50% matrigel subcutaneously in the flank. After 7 days the animals were randomized and therapeutic treatment started on day 8. The AhR antagonists were dissolved in Ethanol/Solutol/Water (10/40/50) and given at 30 mg/kg, QD, p.o. The anti-CTLA4 antibody was dosed at 1 mg/kg, q3d, i.p. The anti-CTLA4 antibody is mouse-specific with syrian hamster IgG1 isotype (Clone: 9H10 (anti mouse CTLA4), Fa. BioXCell BE0131). The isotype antibody is a syrian hamster IgG1 (TPP-9833).

[1648] Tumor size was measured using calipers determining length (a) and width (b). Tumor volume was calculated according to:

[00002]$v=\frac{a\times b^2}{2}$

[1649] Based on tumor volume efficacy was calculated dividing tumor volume of the respective treatment group by tumor volume of the control group (T/C).

TABLE-US-00006 Control: aCTLA4(1 Example Example Example 17 + Example 131 + Isotype + mg/kg) + 17 + 131 + aCTLA4(1 aCTLA4(1 vehicle vehicle isotype isotype mg/kg) mg/kg) T/C 1.00 0.81 0.90 1.05 0.64 0.59

[1650] The clones MOPC-21 and 9H10 (anti mouse CTLA4) can, for example, be purchased via the company Bio X Cell, 10 Technology Dr., Suit 2B, West Lebanon, N.H. 03784-1671 USA (Catalog #:BE0083 (invivoMab quality) #BP0083 (in vivo Plus quality), respectively Catalog #: BE0131).