3-ALKENYL INDOLES AS TRYPTOPHAN 2,3-DIOXYGENASE INHIBITORS FOR THE ENHANCEMENT OF CANCER IMMUNOTHERAPY

(1)

Tryptophan catabolism mediated by indoleamine 2,3-dioxygenase 1 (IDO1) is an important mechanism of peripheral immune tolerance contributing to tumoural immune resistance.1_{Many human tumours constitutively express the enzyme.}2_{IDO1 inhibition has accordingly been an active area of research in drug development.}3

Recently, our group has shown that tryptophan 2,3 dioxygenase (TDO), an unrelated hepatic enzyme also catalysing the first step of tryptophan degradation, is as well expressed in many tumours, where it prevents their rejection by means of locally depleting tryptophan.4_{The complementary role of tryptophan catabolites in this process was demonstrated by others.}5

We therefore set out to develop new, improved TDO inhibitors using as the starting point the only, unoptimised series previously known in the literature.6

Tryptophan catabolism mediated by indoleamine 2,3-dioxygenase 1 (IDO1) is an important mechanism of peripheral immune tolerance contributing to tumoural

immune resistance.1_{Many human tumours constitutively express the enzyme.}2_{IDO1 inhibition has accordingly been an active area of research in drug development.}3

Recently, our group has shown that tryptophan 2,3 dioxygenase (TDO), an unrelated hepatic enzyme also catalysing the first step of tryptophan degradation, is as well expressed in many

tumours, where it prevents their rejection by means of locally depleting tryptophan.4_{The complementary role of tryptophan catabolites in this process was demonstrated by others.}5

We therefore set out to develop new, improved TDO inhibitors using as the starting point the only, unoptimised series previously known in the literature.6

NAMEDIC

3-ALKENYL INDOLES AS TRYPTOPHAN 2,3-DIOXYGENASE INHIBITORS

FOR THE ENHANCEMENT OF CANCER IMMUNOTHERAPY

Eduard Dolušić,a_{Luc Pilotte,}b_{Laurence Moineaux,}a_{Pierre Larrieu,}b_{Vincent Stroobant,}b_{Didier Colau,}b_{Lionel Pochet,}a_{Etienne De Plaen,}b_{Catherine Uyttenhove,}b

Benoît Van den Eynde,b_{Johan Wouters,}a_{Bernard Masereel,}a_{Steve Lanners}a _{and Raphaël Frédérick}a

1_{) Namur Medicine & Drug Innovation Center (NAMEDIC), Namur Research Institute for Life Sciences (NARILIS), University of Namur, B-5000 Namur, Belgium} 2_{) Ludwig Institute for Cancer Research, Brussels Branch, and de Duve Institute, Université Catholique de Louvain, B-1200 Brussels, Belgium}

eduard.dolusic@unamur.be

1. Introduction

2. Synthesis and SAR

7

Scheme 1. Synthetic schemes for (2-pyridin-3-yl)vinylarenes

Table 1. TDO inhibitory potency of analogues 3 - 41. IC₅₀ values tested in cells transfected with mouse TDO (mTDO)

Scheme 2. Synthetic schemes for modifications of the side chain

Scheme 3. Synthetic schemes for linker modifications comp. Ar mTDO IC₅₀ / [mM] comp. R mTDO IC₅₀ / [mM] comp. R mTDO IC₅₀ / [mM] comp. R mTDO IC₅₀ / [mM] 306 ₁ ₁₂ 1-Me, 6-F >200 22 5-Cl 20 32 6-Br >200 3 1 13 2-Me >200 23 5-Br 40 33 6-Me >200 4 >200 14 2-Ph >200 24 5-Me >200 34 6-OMe >200 5 >200 15 4-F 10 25 5-OMe >200 35 6-OH >200 6 >200 16 4-Cl >40 26 5-CN >200 36 6-CO₂Me >200 7 18 17 4-Br >200 27 5-NO₂ >200 37 7-F 50 - 100 8 >200 18 4-CN >200 28 5-CO₂Me >200 38 7-Cl >200 9 >200 19 4-NO₂ >200 29 5-CO₂H >200 39 7-Br >20 10 >200 20 4-CO₂Me >200 30 6-F 1 40 7-Me >200 11 >200 21 5-F 5 31 6-Cl 20 41 7-OMe >200

Table 2. TDO inhibitory potency of indole derivatives with different side chains (tested as above)

comp. R R' R'' mTDO IC₅₀ / [mM] comp. R R' R'' mTDO IC₅₀ / [mM] comp. R R' R'' mTDO IC₅₀ / [mM] 3 H H 1 49 F 3-F-Ph H 10 58 F H 2 30 F H 1 50 F 3-Cl-Ph H >200 59 F CO₂Me H 2 42 H H 20 51 F 3-Br-Ph H >200 60 H CO₂H H 18 43 F H 3 52 F 3-OMe-Ph H 10 61 F CO₂H H 3 44 H H 20 53 F 3-CN-Ph H 1 62 F CH₂OH H 80 45 F H 3 54 F 3-NO₂-Ph H 3 64 F Ph CN >80 46 H Me H >80 55-trans H CN H 13 65 F CO₂Et CN >80 47 F Ph H 40 56-trans F CN H 3 66 F CO₂H CN >80 48 F H >200 57 H H 10 67 F CO₂H CO₂H >80

Table 3. TDO inhibitory potency of indole

derivatives with different linkers (tested as above)

comp. X R mTDO IC₅₀ / [mM] comp. X R mTDO IC₅₀ / [mM] 3 1 73 CN >80 68 80 74 CN 44 69 6 57 10 55-trans CN 13 75 >80 55-cis CN >80 76 >80 71 CN >80 77 >80 72 CN >80 78 >80

Table 5. Oral bioavailability of 30 and 58 in mice (administr. 160 mg/kg/day)8 30 58 = LM 10 Figure 1. View of 58 docked

inside the TDO binding clef7

Table 4. Exp. solubility and stability for 30, 58 and 617

Figure 2. Reversal of tumoural immune resistance by systemic inhibition of TDO8

3. Docking,

physicochemistry and in

vivo properties

1) Munn, D. H. and Mellor, A. L., J. Clin. Invest. 2007, 117, 1147-1154; Katz, J. B., et al,

Immunol. Rev. 2008, 222, 206-221; Prendergast, G. C., Oncogene 2008, 27, 3889-3900.

2) Uyttenhove, C., et al, Nat. Med. 2003, 9, 1269-1274.

3) Macchiarulo, A., et al, Amino Acids 2009, 37, 219-229; Liu, X., et al, Blood 2010, 115, 3520-3530; Röhrig, U. F., et al, J. Med. Chem. 2010, 53, 1172-1189; Dolušić, E., et al,

Bioorg. Med. Chem. 2011, 19, 1550-1561.

4) Van den Eynde, B., et al, WO2010008427, 2010; 5) Opitz, C. A., et al, Nature 2011, 478, 197-203;

6) Madge, D. G., et al, Bioorg. Med. Chem. Lett. 1996, 6, 857-860.

7) Dolušić, E., et al, J. Med. Chem. 2011, 54, 5320-5334; Moineaux, L., et al, Eur. J. Med.

Chem. 2012, 54, 95-102;

8) Pilotte, L., et al, Proc. Natl. Acad. Sci. USA 2012, 109, 2497 – 2502. This work was supported in part by FNRS-Télévie (7.4.543.07).

4. References

R' O HO N H 42-45, 47-56 R R' CO₂CH₃ 59 N H F CO₂CH₃ N H F CO₂H 61 N H 57-58 R NH N N N N H H R R = F or H N H F N B S O O O O N H CH₃ 46 N H F 62 OH N H F R' R'' 64-67 O N F N 63 piperidine Et₃N, solvent,  25 - 94% 1. AlCl₃/NaN₃ THF, , 2 h 2. 55-trans or 56-trans , 18 h, 26 - 64%; Ph 1. Pd(PPh₃)_4, aq Na₂CO₃ toluene, mW  (120°C), 30' Br 2. KOH, H₂O/MeOH , 18 h 54% Pd(OAc)₂ Cu(OAc)₂ DMF/DMSO 9/1 70°C, 18 h, 79%; KOH H₂O/EtOH , 1 h, 82% DIBAL, toluene -78°C, 30', 37% 1. POCl₃/DMF 2. aq. NaOH 3. pyrrolidine, toluene , 2.5 h, 98% RCH₂R', pyridine, rt, 1 - 20 h, 3 - 96% or RCH₂R', Et₃N EtOH, rt, 19 h, 81% N H R 3, 55-cis, 57 N H R 68,73, 77 N Br S O O N Si N H 69 N N N H N 70 N H SCN 74 N H X CN N H X HN N N N 71, X = -72, X = CH₂ 74, X = (CH₂)₂S H₂ (1 atm) 3% Pd/C EtOH/THF, rt 1 to 3 days 36 - 92% Ph 1. Pd(OAc)_2, NaOAc PPh_3, (n-Hex)₄NCl DMF, mW 100°C, 15 min 2. sat. aq NaHCO₃ MeOH, 60°C, 72 h 58% 1. LDA, THF/hexanes -78 to 0°C, 1 h N H Br THF/hexanes 0°C to rt, 20 h 75% 2. N H N KSCN, (n-Bu)₄NBr THF, , 24 h ~quant. 1. AlCl₃/NaN_3, THF, , 2 h 2. 71 or 72, , 21 h, 66 - 67% or 74, NaN₃/ZnBr₂ i-PrOH/H₂O , 6 h, 70% 75, X = -76, X = CH₂ 78, X = (CH₂)₂S 28, Ar = N Ar O H O HO ._HCl Ar N 3, 5-10, 12-41 N H O O * N H HO O * 29, Ar = N N H Br N H N 4 N O H O H N H P(Ph)₃ _N 11 N H Br Br 4' 11' 2 Br Ar H 1 ₂ 1. POCl₃/DMF 2. aq. NaOH piperidine Et₃N, solvent,  8 - 96% 2M aq NaOH MeOH/THF 60°C, 3 h, 65% TMSCHN_2, i-PrOH, Ph₃P, 60°C, 24 h, 30% Cu++ N N Cl -Cl -Pd(OAc)_2, Ph₃P, (i-Pr)₂NH, NMP, 140°C, 18 h, 17% ~quant. PPh_3, CH₃CN , 7 h ~quant. NaH, THF, rt, 30' 69% of ~1:1 trans/cis mix.