Our long-standing collaboration with Prof. Stefan Krauss and Dr. Jo Waaler’s Cell Signaling and Drug Discovery research groups (Oslo University Hospital) has been a fruitful one. Symeres has contributed to the publication of three journal articles, three patents, and eight cocrystal structures of novel tankyrase inhibitors to date, and we continue to provide the project team with practical and intellectual expertise, creating novel molecules and high-quality scientific content, with great valorization potential.
A summary of the 2021 J. Med. Chem. publication can be found below. For the previous publication in this series, click here.
In 2016, our customer from Oslo approached us to advance their early lead molecule OD336, [RL1] a TNKS1/2 inhibitor, in order to improve its physicochemical and biological profile (Figure 1). TNKS1/2 inhibitors, which influence key proteins within the WNT/β-catenin and Hippo signaling pathways, show attractive potential for effective cancer treatment and valorization, as they have already been shown to display anticancer efficacy in mouse models against colorectal cancer, osteosarcoma, and melanoma (as monotherapy or in combination therapies). As reported in our first joint J. Med. Chem. publication, we were able to provide our client with 1,2,4-triazole analog 1, which showed an improved cellular efficacy and more favorable early ADME parameters compared to the initial lead compound OD336 (Figure 1). However, triazole 1 displayed sub-optimal pharmacokinetics in mice, justifying further optimization iterations.
Figure 1: Early lead molecule OD336, and our initially optimized lead compound 1.
During the first optimization round, we had already identified East-analogs with annulated aromatic heterocycles such as quinoxalines and naphthyridines, which displayed picomolar potencies in biochemical assays. In the previous publication, these compounds were not advanced due to their poor stability in mouse microsomes. However, our medicinal chemistry experts hypothesized that in this optimization phase, more polar annulated aromatic heterocycle analogs could provide attractive next-generation inhibitors with improved microsomal stability.
Using the optimized synthetic pathway, described in our previous publication, our medicinal chemists synthesized single-point modifications of lead compound 1, displaying various moieties on the West-, South-, and East-side of the triazole core (Figure 2). The structure-activity relationships of these analogs were evaluated in a TNKS2 biochemical assay and a cellular WNT/β-catenin signaling pathway reporter assay in human HEK293 cells, based on luciferase activity. A structurally diverse and potent set of six compounds was subsequently further evaluated in mouse peroral pharmacokinetic studies.
Initial attempts to generate potent, but more metabolically stable annulated aromatic heterocycle analogs were unsuccessful: South-pyridyl analogs of naphthyridine compound 15, such as compound 18b,were all less potent than their South-fluorophenyl predecessor (Figure 2). However, South-methylthiophene analogs 22 and 23 did yield more potent compounds than compound 15. East-quinoxaline analog 24 displayed picomolar cellular activity and increased TNKS2 inhibition compared to naphthyridine analog 15. West-pyridine analogs of 24b (30b, 31a, 31b) all yielded highly potent compounds.
Figure 2: Exemplary compounds in the lead-optimization campaign.
Compounds 18b, 22a, 24, 30b, 31a and 31b were shortlisted, based on their potency and structural diversity, and were further evaluated in mouse peroral pharmacokinetic studies (Table 1). Analogs 18b, 22a, 30b, and 31a were all deemed less attractive than lead compound 1: 18b and 22a both showed relatively lower exposure, while 31a showed similar exposure compared to 1. Compound 30b did display a high AUC, but was dismissed because of its small volume of distribution. Analogs 24 and 31b both showed higher exposure levels than lead compound 1, but ethoxypyridine analog 24 showed superior PK values: Compound 24 had a threefold lower Cmax than 31b, and displayed a more favorable volume of distribution and kinetic solubility profile. Compared to 1, quinoxaline 24 showed a higher exposure, combined with a 40% reduction in Cmax, half the clearance and 30-fold higher cellular efficacy.
| ID | t1/2 (h) | tmax (h) | Cmax (ng/mL) |  (ng/mL*h) |  (ng/mL*h) |  (h) | Vd (L/kg) | CL (L/h/kg) | Solubility (µM) |
| 1 | 0.67 | 0.25 | 3203 | 2384 | 2388 | 0.69 | 2.03 | 2.09 | >80 |
| 18b | 1.00 | 0.25 | 285 | 319 | 322 | 1.56 | 22.4 | 15.6 | >80 |
| 22a | 1.17 | 0.25 | 779 | 543 | 547 | 1.39 | 15.5 | 9.14 | 50 |
| 24 | 1.50 | 0.5 | 1967 | 4945 | 5038 | 2.39 | 2.15 | 0.99 | 31 |
| 30b | 0.69 | 0.5 | 6512 | 15083 | 15105 | 1.93 | 0.33 | 0.33 | >80 |
| 31a | 0.76 | 0.25 | 2770 | 3401 | 3404 | 1.24 | 1.61 | 1.47 | >80 |
| 31b | 0.59 | 0.25 | 5796 | 5313 | 5316 | 1.47 | 0.80 | 0.94 | 13 |
Table 1: NKS2/HEK293 IC50 values, mouse pharmacokinetic data (5 mg/kg) and kinetic solubility data of selected compounds.
Early ADME properties were characterized for quinoxaline analog 24, of which microsomal stability, mouse plasma stability and kinetic solubility were measured at Symeres’ ADME facilities. Compound 24’s ADME profile was comparable to that of 1 in terms of plasma and microsomal stability, Caco-2 permeability and efflux, and mouse plasma protein binding. Compound 24 did show lower kinetic solubility than 1, but the obtained value was still in an acceptable range (Table 1). Furthermore, quinoxaline analog 24 showed a favorable off-target activity profile, comparable to 1.
Analysis of a TNKS2-24 cocrystal structure, performed by our computational chemists, revealed an analogous binding mode of 24 in the NAD+ cleft of the catalytic domain, compared to lead compound 1 (Figure 3). Besides the observed hydrogen bonds with the Tyr1060 and Asp1045 backbones, and a water molecule, which were also observed for 1, compound 24 displayed an additional interaction, namely a π−π stacking interaction of the quinoxaline moiety with both His1048 and Phe1035, which resulted in an improved binding affinity.
Figure 3: Cocrystal structure of TNKS2 with triazole inhibitor 24 (PDB code 7O6X).
This improved binding affinity, which was already reflected in the observed increased potency in the biochemical assays (see above), also resulted in greater tumor growth inhibition (GI) of COLO 320DM cancer cells: While treatment with compound 1 decreased COLO 320 DM cell viability with GI50 650 nM and GI25 94 nM, the GI values for quinoxaline analog 24 were more than 40 times smaller (GI50 10.1 nM, GI25 2.5 nM). As a control, treatment of APCwild‑type RKO cells, which are less sensitive to tankyrase inhibition, showed less than 15% tumor growth inhibition at micromolar concentrations of 24.
Compound 24 reduced the level of transcriptionally active β-catenin, in both cytoplasmic and nuclear fractions, while stabilizing AXIN1/2 proteins, as observed in immunoblotting assays. Furthermore, real-time qRT-PCR analyses revealed significant inhibition of the WNT/β-catenin signaling pathway by compound 24, as transcript levels of target genes AXIN2, DKK1, NKD1, and APCDD1 were reduced upon administration of 24 in a dose dependent manner.
With quinoxaline 24 showing a favorable ADME/PK profile while blocking proliferation in COLO320DM cancer cells, we provided our partner with an optimized drug candidate, which was used in additional biochemical profiling and animal PK-PD experiments. The results of these studies were reported in a follow-up publication by the Oslo University team, and co-published by members of our Symeres ADME and medicinal chemistry platform.
To access the J. Med. Chem. article, go to https://doi.org/10.1021/acs.jmedchem.1c01264.
To access the mouse tumor model follow-up publication, go to https://doi.org/10.1158/2767-9764.CRC-22-0027.
Want to explore how Symeres can support and accelerate your academic research? Have a look at our Services page to see what we can offer: https://symeres.com/services/.
Do you prefer a more personal conversation? Don’t hesitate to contact our co-author and Director of Medicinal Chemistry, Anita (anita.wegert@symeres.com). At Symeres, we’re always happy to advance your projects and bring them to the next level!
