Structure-Based Discovery of Potent BCL-XL Inhibitors through Rescaffolding

Abstract

Evasion of apoptosis is a hallmark of cancer. Deregulation of BCL-XL, a member of the BCL-2 family of proteins, has been linked to the development of various tumor types. This study presents the design and synthesis of BCL-XL inhibitors with novel mono- and bicyclic cores. The new structural features were optimized to combine high binding efficiency with the opening of diverse novel vectors for additional modifications. The lead compounds exhibited picomolar affinities and significant cellular potency in the BCL-XL-dependent MOLT-4 cell line, which also translated into marked tumor growth inhibition in a xenograft study. These findings highlight the potential of BCL-XL inhibitors as therapeutic agents in cancer treatment by targeting the apoptotic intrinsic pathway.

Introduction

Apoptosis is a cell death pathway that plays an important role in development, tissue homeostasis, and removal of damaged cells. Deregulation of apoptosis contributes to different types of human diseases, including cancer. The intrinsic apoptotic cell death pathway is regulated by the BCL-2 family of proteins which consists of three classes of proteins that contain between one and four of the mainly helical BCL-2 homology domains (BH1-BH4). Upon activation, the pro-apoptotic proteins such as BAX and BAK containing BH domains 1–3 form mitochondrial pores, which trigger the release of caspases and cell death. The pro-survival members such as BCL-2, BCL-XL, and MCL-1 contain BH domains 1–4. The third class of proteins such as BIM and PUMA are pro-apoptotic containing just one BH domain (BH3) and play a role in activating BAX and BAK. Cell fate is dependent on the extent of pairing between pro-survival and pro-apoptotic family members in which the BCL-2 homology 3 (BH3) domain of a pro-apoptotic protein binds in a groove on the surface of a pro-survival protein. Overexpression of the pro-survival members of the BCL-2 family is a hallmark of cancer, playing an important role in tumor development, maintenance, and resistance to anticancer therapies. The gene coding for BCL-XL (BCL2L1 for Bcl-2-like protein 1) is frequently amplified in cancer and its expression inversely correlates with sensitivity to several anticancer therapies. Small molecules that mimic the BH3 peptide have been designed to bind to the pro-survival proteins of the BCL-2 family, releasing pro-apoptotic proteins that can then activate and trigger apoptosis.

WEHI-539 (Figure a) was one of the first selective BCL-XL inhibitors, with high affinity and selectivity for BCL-XL, killing cells by antagonizing its pro-survival activity. , The benzothiazole moiety of WEHI-539 binds deep into the mainly hydrophobic P2 pocket (Figure b) of the protein, making a hydrogen-bonding interaction with the backbone NH of L108 (shown in Figure c). The NH group of the hydrazone moiety interacts with the backbone carbonyl of S106 in the P2 pocket and links the benzothiazole and tetralin rings. This arrangement positions a thiazole carboxylic acid that makes polar interactions with the guanidinium side chain of R139 and the side chain of N136. The remainder of the compound, connected through a flexible chain to the thiazole ring, makes nonpolar interactions with BCL-XL. Further development led to A-1155463 and A-1331852 containing the same benzothiazole but linked via an amide moiety to the bicyclic tetrahydroisoquinoline (THIQ) core. All three of these literature compounds (WEHI-539, A-1155463, and A-1331852) bind to the P2–P4 pocket of BCL-XL in a similar fashion. Although the tertiary nitrogen of A-1155463 does not make any direct polar interactions with the protein, the increased solubility gives a potent and selective BCL-XL inhibitor that demonstrated activity in xenograft models of human tumors.

1.

(a) The first selective BCL-XL inhibitors. (b) Detail showing the molecular surface of BCL-XL in the crystal structure (PDB ID 3ZLR) bound to WEHI-539 (in stick representation with white carbons), indicating polar protein–ligand interactions and the P2 and P4 pockets. (c) WEHI-539 (white carbons) overlaid on 1 (WEHI-539 core with green carbons) in crystal structures bound to BCL-XL in a view approximately 90° rotated from that in (a) with key interactions in dashed lines. See the Experimental Section and Figure S1 for details of the BCL-XL construct used for the crystallographic structures reported here.

The central THIQ and tetralin cores of these three inhibitors occupy a defined cleft in the protein but make only weak hydrophobic interactions. Their principal role is to position their substituents for optimal interactions with the P2 and P4 pockets. Both the tetralin and THIQ cores are known to be sensitive to oxidation, and the exploration of the additional vectors that they offer for further derivatization is synthetically challenging. We therefore decided to replace these cores to facilitate the introduction of new vectors while conserving the orientation of the P2 and P4 filling groups to maintain affinity and selectivity. These new cores were then optimized to generate a lead series of very potent and selective BCL-XL inhibitors that induced tumor regression in an animal model and provided new attachment points for further development.

Results and Discussion

The strategy to design core replacements is outlined in Figure . The position and orientation of the benzothiazole and linker of WEHI-539 in the BCL-XL structure are conserved in the published crystal structures of A-1155463 and A-1331852 with different linkers. We synthesized 1 and 2 which represent the benzothiazole-linked cores of WEHI-539 and A-1155463, respectively, and determined their crystal structures bound to BCL-XL. An overlay of these structures with those of A-1155463 and A-1331852 (Figure a) confirmed that this side of the literature inhibitors is firmly anchored in pocket P2 but also suggested designs for replacements for the tetralin and THIQ cores (Figure b). A new cycle incorporating the linker would replace one of the core rings and connect key atoms. While one side of the newly formed ring would be close to the BCL-XL protein, the other side of the ring or the “freed” nitrogen could provide different new vectors for further development. To test our core replacement hypotheses, we first generated some tool compounds with a diaminoaryl-core and compared them to the corresponding cores of the literature compounds (Table ). The first core replacements, such as the tetrahydronaphthalene in 3 resulted in a significant drop of affinity for binding to BCL-XL compared to 1 or 2. However, the introduction of two nitrogen atoms such as in the phthalazine 4, recovered most of the affinity. Partial saturation of the core (5) further increased the affinity, in part probably due to decreased rigidity, bringing it to the same affinity range as the cores of the literature compounds. Elimination of the second ring of the core (6) led to maintained affinity, improved ligand efficiency as well as compound properties. The position of the nitrogens in the core was found to be important since the pyrazine derivatives (7 and 8) had greatly reduced affinity for BCL-XL, as did the naphthalene analog 3. At this point, our inhibitor 6 had similar affinity and ligand efficiency (0.39) for BCL-XL as the WEHI-539 core 1, binding in the same position and orientation in BCL-XL (Figure a), with some small variations in the vectors from the thiazole (Figure b). There is a lone pair interaction of the pyridazine nitrogens with the vacant sulfur d-orbitals of the thiazole and benzothiazole moieties. This preorientates the substituents in the conformation necessary for efficient binding although the nitrogens make no polar interactions with the protein. The significant decrease in binding affinity observed for compounds 7 and 8 can be attributed to the pyrazine ring’s nitrogen atoms, which stabilize the thiazole (for 7) and benzothiazole moieties (for 8) in an inactive conformation.

2.

Core replacement strategy to obtain selective BCL-XL inhibitors with new vectors. (a) Overlay of stick representations of A-1155463 (PDB ID 4QVX, white carbons), A-1331852 (PDB ID 9AQZ, white carbonsnote that the ligand in the crystal structure is the methyl ester), A-1155463 core 2 (PDB ID 9IGB, purple carbons) and WEHI-539 core 1 (PDB ID 9I9E, green carbons) in crystal structures bound to BCL-XL in the same view as in Figure c. (b) Design of the replacement core.

1. BCL-XL Affinities and Ligand Efficiencies of the Various Core Replacements.

^aeKi measured in the BCL-XL FP assay in micromolar.

^bLigand efficiency calculated as ΔG°/heavy atom count based on BCL-XL FP assay.

3.

(a) Comparison of 1 (PDB ID 9I9E, stick with white carbons) and 6 (PDB ID 9IGC, green carbons) bound to the surface of BCL-XL. (b) View approximately 90° from that in (a) of the two inhibitors alone, overlaid on the common benzothiazole moiety.

The next step was to investigate the optimization of the substitution pattern of the aminopyridazine moiety (hereafter Core A) at three positions (Table ). Replacement of the R¹ methyl group with a slightly bigger cyclopropyl moiety (9) was tolerated, while the longer and more polar methoxymethyl group (10) led to a 4-fold reduction in affinity. The same methyl-to-methoxymethyl replacement in the R² position (11) or even the introduction of a much larger N-methylpiperazinepropyl group into the same position (12) was tolerated without any change of affinity, which is in line with the expectation that these substituents point toward the solvent-exposed area. The importance of the methyl group in the R¹ position is highlighted by the fact that its removal led to a 20–30-fold drop of BCL-XL affinity irrespective if R² was also replaced by a hydrogen (13) or not (14). In line with this observation, elimination of the methyl group in the R² position (15) is well tolerated. Interestingly, trimethyl substitution (R,¹ R,² R³ = Me, 16) leads to a significant 200-fold drop in affinity compared to the parent dimethylpyridazine analog (6) probably due to the steric clash of R² and R³ forcing a suboptimal conformation of the P4-filling side chain. When the two methyl groups were in positions R¹ and R³, and R² was hydrogen, compound 17 had much improved 7.5 nM affinity with 0.41 ligand efficiency. Larger substituents in the R³ position on the central amino group, such as a propyl (18) or 3,4-dihydroxybutyl (19) were well tolerated, with 19 displacing waters in a pocket to make additional polar interactions with R146 side chain amidine, D140 main chain (mc) NH, L137 main chain O and D140 side chain carboxylic acid (as shown in Figure S2).

2. BCL-XL Affinities and Ligand Efficiencies of Ligands with Different Substitution Patterns.

^aeKi measured in BCL-XL FP assay in micromolar.

^bLigand efficiency based on the BCL-XL FP assay.

^ccKi measured in BCL-XL Quench assay in micromolar.

^dLigand efficiency based on BCL-XL Quench Assay.

Compounds with a bicyclic core (hereafter, Core B), formed by connecting the central amine substituent with the proximal position of the pyridazine ring, were also investigated. It was found that the pyrido­[2,3-c]­pyridazine (21) having a [6 + 6] bicyclic core had high affinity (2 nM) with 0.41 ligand efficiency, while in the case of the [5 + 6] (20, 9 nM) and [7 + 6] (22, 115 nM) derivatives lower affinities were observed. Modeling suggested that the piperidine ring might offer a good vector for further functionalization. The 6-(R)-hydroxy substitution of the pyrido­[2,3-c]­pyridazine core (23) was very well tolerated, maintaining BCL-XL affinity. This was confirmed by a more sensitive BCL-XL Quench assay (5.5 and 1.4 nM, respectively), which was used for all high-affinity compounds later. A comparison of the crystal structures of various of the compounds in the two series (Figure ) confirms that the binding mode of the identical left-hand side of the molecules is conserved, but that variations in the central moiety of the core provide opportunities for subtly different vectors for further derivatization from the thiazole carboxylic acid. In addition, one other vector for optimization was demonstrated at the R³ position (Table ) where longer substituents such as the 3,4-dihydroxybutyl chain in 19 can form additional interaction(s) with a polar pocket (see Figure S2c for details).

In conclusion, we have successfully identified and characterized two series of BCL-XL ligands featuring mono- or bicyclic cores. These scaffolds exhibit nanomolar affinity for BCL-XL and demonstrate enhanced ligand efficiency relative to the core structures of the parent molecules 1 and 2. Furthermore, these series provide novel exit vectors suitable for further medicinal chemistry optimization and development. By utilizing our new scaffolds, we have successfully eliminated both the sensitive THIQ core and the amide linkage, thereby effectively addressing their associated medicinal chemistry liabilities.

None of the compounds in Table showed any notable cellular activity in a BCL-XL-sensitive cell line MOLT-4, presumably due to a lack of cell penetration. To gain cellular potency, we therefore introduced different P4-filling side chains that had previously been shown to confer such cellular activity (Table ).

3. BCL-XL Affinities and Cellular Potency of Inhibitors with Different Side Chains.

^acKi measured in BCL-XL Quench assay in picomolar.

^bIC₅₀ was measured in MTT cell viability assay in nanomolar in MOLT-4 cells.

The combination of the phenoxypropargylamine side chain (hereafter side chain a) with the pyridazine (24) and the pyrido­[2,3-c]­pyridazine (25) cores led to single-digit picomolar BCL-XL affinities and nanomolar cell killing in the BCL-XL-dependent MOLT-4 cell line. Substitution of the central nitrogen of the pyridazine core with a 4-hydroxybutyl group (26) was well tolerated, resulting in a slight elevation of the affinity and maintaining cellular activity. Introduction of the more polar and charged 2-(methylamino)­ethoxy moiety into the 6-position of the pyrido­[2,3-c]­pyridazin core (27) had no impact on its affinity, while the cellular activity decreased by an order of magnitude, probably as a consequence of decreased cell penetration due to its increased polarity compared to 25.

The carboxylic acid moiety is carried by a pyridine rather than a thiazole in A-1331852 in the adamantly pyrazole side chain (hereafter side chain b). This was combined with our pyridazine (28) and pyrido­[2,3-c]­pyridazine (29) cores. These modifications gave a significant boost to the BCL-XL affinity and cellular potency, similar to side chain a. Also, as reported by others previously, inhibitors containing side chain b had significantly increased PBS solubility at pH 7.4 compared to their analogs with side chain a (23 and 22 μM for 28 and 29, respectively, compared to <1 μM for inhibitors with side chain a).

Introduction of a solubilizing moiety onto the adamantane ring gave side chain c, which when combined with the pyridazine core (30) resulted in high affinity and outstanding cellular activity. 4-Methylaminobutyl group on the central nitrogen (31) was added as a further polar group, which further elevated the BCL-XL affinity, while the cellular activity was similar to 28. Not surprisingly, we observed elevated solubility (88 μM and 66 μM, respectively) due to the presence of the polar amine moieties. In all cases shown, the incorporation of the P4-filling side chains significantly increased the binding affinity to the picomolar level and improved cellular potency, sometimes below the nanomolar level, similar to A-1155463 and A-1331852 (Table ).

4. BCL-XL Selectivity vs BCL-W, BCL-2, and MCL-1.

compound	BCL-XL	BCL-W	BCL-2	MCL-1	MOLT-4	RS4;11
24	4.40 × 10–12	7.77 × 10–9	4.64 × 10–7	>1 × 10–5	0.46	>1000
29	7.96 × 10–12	1.56 × 10–8	4.26 × 10–7	>1 × 10–6	4.85	>1000
A-1155463	5.30 × 10–12	2.40 × 10–7	nd	nd	6.30	>1000
A-1331852	3.20 × 10–11	2.77 × 10–8	3.94 × 10–7	nd	0.44	>1000
venetoclax	5.06 × 10–8	1.30 × 10–6	9.50 × 10–10	nd	3000	3.64

^acKi [M] measured in BCL-XL Quench assay.

^beKi [M] measured in the FP assay.

^cIC₅₀ (nanomolar) was measured in MTT cell viability assay.

On the basis of the data available at the time, compounds 24 and 29 were selected as leads. Both compounds exhibited exceptional selectivity for BCL-XL over BCL-W, -MCL-1, and -BCL-2 (Table ). These findings are consistent with the potent activity observed in the BCL-XL-dependent MOLT-4 cell line and the corresponding lack of activity in the BCL-2-dependent RS4;11 cell line. This selectivity profile mirrors those of A-1155463 and A-1331852, while remaining distinct from the BCL-2-selective profile of venetoclax. The mode of action of these compounds (24, 29) was further supported by the dose-dependent caspase-3 induction and PARP cleavage in the MOLT-4 cell line in vitro following a 4 h treatment (Figure S4). As anticipated, the effect was completely ablated by the addition of caspase inhibitor Q-VD. These data confirm that these compounds induce cell death in the MOLT4 cell line through apoptosis as expected.

The pharmacokinetics (PK) in mice were measured following low-dose intravenous (IV) and oral (PO) administration (Table ). After IV administration, 24 showed a moderate total plasma clearance, an intermediate terminal half-life, and a 4-fold lower drug exposure, whereas 29 showed a low total plasma clearance and a longer terminal half-life. On oral administration, 24 showed very limited exposure (C _max <0.01 μM) and bioavailability (nc), while 29 showed good exposure and oral bioavailability (F, 53%) with a C _max of 1 μM, suitable for pharmacodynamic (PD) and efficacy studies.

5. SCID Mouse IV and Oral PK Parameters for 24 and 29 .

	SCID mouse IV PK, 1 mpk					SCID mouse PO PK, 3 mpk
compound	C ∞ (μM)	t 1/2 (h)	V ss (L/kg)	CLp (mL/min/kg)	AUC (μM·h)	C max (μM)	t 1/2 (h)	AUC (μM·h)	F (%)
24	4.23	1.5	0.9	27.2	0.97	0.01	nc	nc	nc
29	6.70	3.9	1.1	7.56	3.44	1.07	3.8	5.46	53

^anc= not calculated since compound concentration in plasma samples was under the limit of quantitation (BLQ < 0.004 μM).

To evaluate the pharmacodynamic response, mice bearing MOLT-4 tumor xenografts were administered a single 7.5 mg/kg dose of compound 29 via either IV or oral routes. Tumor samples were harvested and analyzed 6 h postadministration (Table ). Regardless of the administration route, the compound elicited a robust induction of apoptotic markers, with cleaved PARP increasing 24.1–38.6-fold and cleaved caspase 3 increasing 7.2–18.9-fold relative to untreated controls. Furthermore, consistent with the established role in regulating platelet lifespan, ,, both treatment modalities resulted in a significant, transient reduction in platelet counts to 1% of control values, followed by full recovery.

6. Cleaved PARP and Cleaved Caspase 3 Activation in Tumor, and Platelet Loss in MOLT-4-Grafted Female NOD SCID Mice 6 h after Treatment (PO or IV) with 29 at 7.5 mg/kg .

dose (mg/kg) and route	time point (h)	cleaved PARP (fold increase over control)	cleaved caspase 3 (fold increase over control)	platelet count (103/μL)	% remaining platelets (vs controls)
7.5, PO	6	24.1	7.2	7	1
7.5, IV	6	38.6	18.9	8	1

^aCleaved PARP and caspase 3 were measured in tumor sample lysates by using the apoptosis immunoassay from MSD (Meso Scale Discovery). Platelet count was performed in whole blood using the Hematology Analyzer Coulter AcT diff (Beckman Coulter). Mean of 3 mice per group is shown.

Given the robust pharmacodynamic response observed following both IV and PO administration, the therapeutic efficacy of compound 29 was also evaluated. Mice bearing MOLT-4 xenograft tumors were administered varying IV doses of compound 29 (2.5, 5, and 7.5 mg/kg) or A-1331852 (7.5 mg/kg) on a Q3D6 schedule, with tumor volumes monitored at least twice weekly (Figure and S5). Treatment with compound 29 elicited significant tumor regression across all dose levels, which was sustained throughout the dosing period. While tumors in the lower dose cohorts (2.5 and 5 mg/kg) relapsed more rapidly, the 7.5 mg/kg group exhibited a prolonged response. Notably, no clinically significant body weight loss was observed, indicating a favorable safety profile under these conditions (Figure ). While A-1331852 at 7.5 mg/kg achieved tumor regression comparable to the 2.5 mg/kg dose of compound 29, only compound 29 at the 7.5 mg/kg dose induced complete regression (CR, defined as tumor volume <25 mm³ for at least three consecutive measurements; Figure S6). On day 17 post-treatment initiation, compound 29 exhibited tumor growth inhibition (TGI) values of 122%, 124%, and 133% at dosages of 2.5, 5, and 7.5 mg/kg, respectively (Figure , Table ). In comparison, A-1331852 resulted in a TGI of 115% at a 7.5 mg/kg dose, which was significantly lower than the inhibition achieved by compound 29 at all tested dose levels.

4.

(left) Tumor volume (mm³) of MOLT-4-grafted female NOD SCID mice upon treatment with vehicle (HPBCD/HCl) or 29 (2.5, 5, and 7.5 mg/kg) or A-1331852 (7.5 mg/kg), administered IV, Q3D6, n = 7. (right) Body weight (g) of MOLT-4-grafted NOD SCID female mice upon treatment with vehicle (HPBCD/HCl) or 29 (2.5, 5, and 7.5 mg/kg) or A-1331852 (7.5 mg/kg), administered IV, Q3D6, n = 7.

7. MOLT-4 Tumor Growth Inhibition Upon Treatment with Compound 29 (2.5, 5, and 7.5 mg/kg) or A-1331852 (7.5 mg/kg) Administered IV, Q3D6.

compound	dose (mg/kg)	TGI on day 17 (%)
29	2.5	122
29	5	124
29	7.5	133
A-1331852	7.5	115

Chemistry

The preparations of all compounds 1–31 are described in the Supporting Information. To highlight the two main synthetic strategies used for the synthesis of the two main series, the preparations of the lead molecules, 24 and 29, are shown in Schemes and , respectively, and described in the Experimental Section.

1. Synthesis of 24 .

^a (i) 2-Chloro-1,3-benzothiazole, XantPhos, Pd₂(dba)₃, Cs₂CO₃, DIPEA, DMF, (ii) SEMCl, DMAP, DIPEA, DCM, 0 °C; (iii) methylthiourea, TEA, EtOH, 0 °C; (iv) N,N-dimethyl- propargylamine, Pd­(PPh₃)₂Cl₂, CuI, DIPA, THF, 60 °C; (v) XantPhos, Pd₂(dba)₃, Cs₂CO₃, DIPEA, 1,4-dioxane, reflux; (vi) NaI, acetone, reflux, (vii) Cs₂CO₃, acetone; (viii) HF × Pyr, MeCN, 60 °C; (ix) LiOH × H₂O, 1,4-dioxane,water, 60 °C.

2. Synthesis of 29 .

^a (i) 3,6-Dichloro-1,2,4,5-tetrazine, THF, 160 °C in a sealed tube; (ii) BCl₃, DCM; (iii) (COCl)₂, TEA, DMSO, DCM, −78 °C; (iv) Pd­(dppf)­Cl₂.CH₂Cl₂, K₂CO₃, THF, reflux; (v) NaCNBH₃, MeOH-AcOH, reflux; (vi) Cs₂CO₃, α,α,α-trifluorotoluene, 160 °C, sealed tube; (vii) 2-aminobenzothiazole, Xantphos, Pd₂(dba)₃, DIPEA, 1,4-dioxane, 160 °C, sealed tube; (viii) LiOHxH₂O, 1,4-dioxane, reflux.

For the synthesis of 24 (Scheme ), a SEM-protected chloromethylpyridazinylbenzothiazol-imine intermediate (33) was prepared from commercially available 3-amino-6-chloro-4-methylpyridazine (32) and 2-chloro-1,3-benzothiazole in two steps by a Buchwald coupling and subsequent SEM protection. This intermediate was coupled with the aminothiazole ester 35 under Buchwald conditions, affording 36. In both Buchwald couplings the use of a two-base (Cs₂CO₃, DIPEA) XantPhos system proved to be the most efficient. After activation of the haloalkyl side chain through halogen exchange, the O-alkylation of the phenoxypropargyl reagent (39), prepared by the Sonogashira coupling of the commercially available N,N-dimethyl-propargyl amine and 2-fluoro-4-iodophenol, led to the desired 24 inhibitor after removal of the protecting groups.

For the synthesis of 29 (Scheme ), the core was built up from the pyridyl side, and the 2-aminobenzothiazole moiety was introduced at the end. O-Benzyl-hex-4-yn-1-ol (40) was reacted with 3,6-dichloro-1,2,4,5-tetrazine in an inverse-electron-demand Diels–Alder reaction. The obtained 41 pyridazine was debenzylated and Swern-oxidized to the corresponding 43 aldehyde. The reductive amination of 43 pyridazinylpropanal with the 3-substituted ethyl 6-aminopyridine-2-carboxylate (46), which was synthesized from the commercially available ethyl 3-bromo-6-aminopyridine-2-carboxylate (44) and boronic acid 45 in a Suzuki coupling, led to key intermediate 47. Intramolecular nucleophilic substitution between the chloropyridazine and aminopyridine moieties of 47 in a sealed tube led to the formation of the bicyclic core (48). In the concluding steps, the 2-aminobenzothiazole moiety was introduced by a Buchwald coupling and hydrolysis of the ester moiety gave the desired 29 inhibitor.

Conclusions

We report the discovery of novel, very potent inhibitors of BCL-XL, which exhibit in vivo efficacy in tumor xenografts by displacing pro-apoptotic BH3-containing proteins. The inhibitors conserve some of the interactions made by previously reported inhibitors, − with the main modification being new central cores that feature a 1,4-arylenediamine motif in a monocyclic or bicyclic layout. This feature ensures the proper distance and orientation of the benzothiazole moiety in the deep-P2 pocket of BCL-XL and the aromatic carboxylic acid moiety on the phenylalanine-bridge. While the affinity for binding to BCL-XL of the initial compounds was low, incorporation of two nitrogens into the aryl ring and extensive optimization of the substituents ensured the optimal orientation of the two main binding moieties, which led to single-digit nanomolar affinities. Incorporation of different P4-filling side chains from the literature into our binders not only enhanced their affinities to the single-digit nanomolar range but also led to high cellular potency in the BCL-XL-dependent MOLT-4 cell line. The lead compounds (24 and 29) display very high selectivity for BCL-XL over other BCL-2 family pro-survival proteins. Furthermore, the in vivo activity of compound 29 was demonstrated following both intravenous and oral administration. Treatment resulted in significant tumor regression and the induction of apoptosis within tumor cells. A pronounced reduction in circulating platelets was also observed; however, platelet counts recovered following treatment discontinuation. In a comparative in vivo study against the established BCL-XL inhibitor A-1331852, compound 29 exhibited superior efficacy with statistical significance. While A-1331852 has previously shown efficacy across various xenograft models, the clinical development of potent and selective BCL-XL inhibitors was never initiated due to the emergence of cardiotoxicity in higher species. Although the aforementioned cardiotoxicity had not been identified at the time of this studyrendering its mitigation outside the current scopethe profound thrombocytopenia observed during treatment ultimately led to the termination of our small-molecule inhibitor program. A potential advantage of the newly developed inhibitors, including 26, 27, and 31, is their feature of offering novel exit vectors. These attachment points make them suitable for further derivatization, which may be strategically exploited for the development of novel conjugates, such as Antibody-Drug Conjugates (ADCs), bivalent degraders (i.e., PROTACs), and other targeted modalities.

Experimental Section

All reagents obtained from commercial sources were used without any further purification. Anhydrous solvents were obtained from commercial sources and used without further drying.

All tested compounds are >95% pure by HPLC analysis.

All experiments were conducted in accordance with the European and French regulations in force in 2016. (Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes, Decree n° 2013–118 from 01 February 2013 relative to the protection of animals used for scientific purposes, modified by decree n°2020–274 from 17 March 2020, and 5 orders of 01 February 2013’ (Code rural et de la pêche maritimearticle R. 214–87 à R. 214–137 Section 6: Utilization d’animaux vivants à des fins scientifiques). The experimental site approval reference is Institut de Recherche Servier Croissy sur Seine, C 78–100–2. The experimental project (Evaluation de l’activité antitumorale d’un composé chez la souris porteuse de tumeur humaine) was endorsed by the Servier Ethics Committee (registered by the French Ministry of Research under No. 31) and authorized by the Ministry of Research.

Protein Production

Proteins were generated from a number of BCL-XL constructs.

For X-ray crystal structure determination, the construct used encoded a mutated loop-deleted chimera of human BCL-2 and BCL-XL: BCL-2 (9–33)–BCL-XL (28–155) [Δ46–85, S28A, M45 V, I114L, Q121G, E124A, Q125T]–BCL-2 (163–206) as introduced in the legend to Figure (the BCL-XL surrogate). A His–TEV-tagged construct of this was generated by gene synthesis (DNA2.0, USA) in pJ express411. For protein expression, the construct was used to transform E. coli BL21 (DE3) pLysS cells, which were subsequently cultured in LB supplemented with 50 μg/mL kanamycin. Protein expression was induced at O.D.600 nm = 0.6 by the addition of 1 mM IPTG (final concentration), and the temperature reduced to 4 °C overnight before harvesting by centrifugation. For protein purification, cell pellets were resuspended in lysis buffer (20 mM Tris-HCl pH8.0, 500 mM NaCl containing EDTA-free protease inhibitor cocktail (Roche) and DNase I (Sigma)) and lysed by mechanical homogenization. The lysate was clarified by centrifugation and applied to a 1 mL HisTrap FF column (Cytiva). Protein was eluted using a linear gradient of lysis buffer supplemented with 500 mM imidazole. Fractions containing the expressed protein were pooled and concentrated. The protein was then applied to a HiLoad 16/600 Superdex pg75 column pre-equilibrated in 20 mM Tris-HCl pH7.8, 10 mM EDTA, 1 mM DTT. The N-terminal His-tag was removed by incubation overnight at RT, with AcTEV protease (ThermoFisher). A repeat of the affinity purification resulted in BCL-XL surrogate protein in the flowthrough fraction. The protein was concentrated before size exclusion chromatography was performed again in 20 mM Tris-HCl pH7.8, 100 mM NaCl, 1% glycerol, 2 mM DTT. The sample was then reapplied to a 1 mL HisTrap FF column with the buffers used previously. The flowthrough was concentrated then loaded onto a HiLoad 16/600 Superdex pg75 column pre-equilibrated in 20 mM Tris-HCl pH7.7, 100 mM NaCl, 1% glycerol, 2 mM DTT. The resulting protein was concentrated to 13.6 mg/mL, aliquoted, and snap-frozen prior to being stored at −80 °C.

For the fluorescence polarization assay, a construct was made by cloning DNA encoding BCL-XL (2–209) into pGex5X-1 between EcoRI and NotI sites. Protein was expressed as above (except using 100 μg/mL ampicillin instead of kanamycin), and cell pellets resuspended in buffer A (20 mM Tris-HCl pH8, 200 mM NaCl, 1 mM DTT, and 2 mM EDTA with EDTA-free protease inhibitor cocktail (Roche) and DNase I (Sigma)) before lysis by chemical homogenization. The lysate was clarified by centrifugation and applied to a 5 mL GSTrap FF column (Cytiva). Protein was eluted in buffer A containing 10 mM glutathione, and the eluate was applied to a HiLoad 16/600 Superdex pg75 column pre-equilibrated in buffer A. Eluted protein was aliquoted and stored at −80 °C.

For the quench assay, a plasmid encoding a N-terminal double 6His-tagged BCL-XL (2–197) [N197C] was generated in pET41a. Protein was expressed as described above, and cell pellets were resuspended in buffer B (20 mM HEPES pH7.4, 500 mM NaCl, 1 mM TCEP with EDTA-free protease inhibitor cocktail (Roche) and DNase I (Sigma)) before lysis by chemical homogenization. The lysate was clarified by centrifugation and applied to a 5 mL HisTrap FF crude column (Cytiva). Protein was eluted in buffer B containing 500 mM imidazole, and the eluate was applied to a HiLoad 16/600 Superdex pg75 column pre-equilibrated in 20 mM HEPES pH7.4, 200 mM NaCl, 1 mM TCEP. Eluted protein was aliquoted and stored at −80 °C.

Fluorescence Polarization Assay

The assay measures the change in polarization of fluorescence from a labeled PUMA peptide on displacement by a compound from binding to a protein as previously described. For MCL-1 and BCL-2, the protein used was generated as described previously. − For BCL-XL, the GST-tagged construct was used, and for BCL-W, the His-tagged construct.

Fluorescence Quench Assay

The assay measures changes in fluorescence intensity of cyanine 5 (Cy5 from Lumiprobe) labeled protein bound to a PUMA peptide labeled at the C-terminus with TQ5WS (from AAT Bioquest), as previously described for studies on MCL-1¹⁷. The fluorescence of the complex is quenched, and displacement of the peptide by the compound increases the fluorescence intensity. The His-tagged BCL-XL protein was used for the data reported here,

MTT MOLT-4 and RS4-11 Cell Viability Assay

The MTT colorimetric assay is based on the mitochondrial reduction of tetrazolium salt by living cells. The viable cell number is proportional to the production of formazan salts, which can be read spectrophotometrically at 540 nm. MOLT-4 cells were purchased from ATCC and cultivated in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 IU/ml), streptomycin (100 μg/mL), and l-glutamine (2 mM). RS4–11 cells were also purchased from ATCC and cultivated in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 IU/mL), streptomycin (100 μg/mL), and glucose (4.5 g/L). Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. Cells were seeded in 96 microwell plates (150 μL per well) and exposed to the compounds for 48 h (3,16-fold serially diluted; 9 concentrations each, triplicates). At the end of the incubation time, 15 μL of MTT solution (5 mg/mL) were added per well, and the cells were incubated for another 4 h. Then, 100 μL of 10% sodium dodecyl sulfate (SDS)/HCl 10 mM were added per well, and the plate was incubated overnight, before measurement of optical density at 540 nm. IC50s were calculated using standard four-parametric curve fitting. IC50 is defined as the compound concentration at which the MTT signal is reduced to 50% of that measured for the control.

Crystallization and X-ray Structure Determination

Crystallization

In all cases, BCL-XL surrogate protein (6–7 mg/mL, ∼0.4 mM in 20 mM Tris buffer pH 7.8, 0.2 M NaCl, 1 mM DTT, 1% glycerol) was mixed with 5–10-fold excess of compound from 20 mM stock DMSO solution and incubated at 4 °C for 4–6 h to form a complex, which was centrifuged to separate out nondissolved compound. The resulting solution was then used with a selection of conditions from commercial crystallization screens that had been most successful previously in generating crystals of BCL-XL and BCL-2 complexes. Crystallization was performed using the vapor diffusion technique with sitting droplets (0.4–0.6 μL in size), combining equal volumes of the protein/compound complex and well reservoir, and set up by Mosquito (TTP Lab Tech) robot. Transparent crystals (prisms or plates) appeared after a period of 2 days to 4 weeks at 284 K. These were transferred to a cryoprotecting solution prior to flash freezing in liquid nitrogen. Diffraction-quality crystals were obtained under the conditions shown in Table .

8. Crystallization Conditions.

compound	crystallization	cryoprotectant
1	0.1 M KSCN, 0.1 M sodium acetate buffer pH 5.0, 20% Peg3350	0.1 M KSCN, 0.1 M sodium acetate buffer pH 5.0, 30% Peg3350
2	2.5 M sodium formate, 0.1 M sodium acetate buffer pH 4.5	2.5 M sodium formate, 0.1 M sodium acetate buffer pH 4.5, 20% glycerol
6	0.2 M calcium acetate, 0.1 M sodium acetate buffer pH 5.5, 10% PegMME550, 10% Peg3350	0.2 M calcium acetate, 0.1 M sodium acetate buffer pH 5.5, 20% PegMME550, 10% Peg3350
11	3.0 M sodium formate, 0.1 M sodium acetate buffer pH 5.5	3.0 M sodium formate, 0.1 M sodium acetate buffer pH 5.5, 20% glycerol
17	4.0 M sodium formate, 0.1 M sodium acetate buffer pH 5.0	4.0 M sodium formate, 0.1 M sodium acetate buffer pH 5.0, 20% glycerol
19	0.1 M KSCN, 0.1 M sodium acetate buffer pH 5.5, 10% Peg8k, 10% Peg1k	0.1 M KSCN, 0.1 M sodium acetate buffer pH 5.5, 10% Peg8k, 20% Peg1k
20	4.0 M sodium formate, 0.1 M sodium acetate buffer pH 5.5	4.0 M sodium formate, 0.1 M sodium acetate buffer pH 5.5, 20% glycerol
21	0.2 M calcium acetate, 0.1 M sodium acetate buffer pH 5.5, 10% PegMME550, 10% Peg3350	0.2 M calcium acetate, 0.1 M sodium acetate buffer pH 5.5, 20% PegMME550, 10% Peg3350

X-ray Data Collection

Suitable crystals for data collection were flash-frozen in liquid nitrogen after cryoprotection using the crystallization reservoir with added glycerol (for crystals obtained in high-salt conditions) or enriched in low MW Peg/PegMME solutions. Table S2 summarizes the data collection statistics, with data obtained from DLS and Soleil synchrotrons or single-crystal Bruker D8Venture X-ray diffraction equipment (installed at Vernalis premises) consisting of a rotating anode generator TXS coupled with Photon100 CMS image detector. Data were processed and scaled with XDS/Aimless software from synchrotron automatic processing pipelines for all structures, except with 6 where SAINT/SADABS software was used for data processing.

X-ray Structure Determination

All software was from the CCP4 package. The structure of the first compoundsurrogate BCL-XL complex was solved by molecular replacement with MolRep using the published structure of BCL-XL complexed with ABT-737 (PDB ID 2YXJ ) stripped of the compound and solvent. From the initial electron density maps, it became apparent that the compound is present in the binding site of BCL-XL. Initial maps allowed the extension of the refinement resolution range and the correct placement of side chains. At this point, sequence differences between the published structure and our surrogate BCL-XL sequence were clearly visible, and appropriate changes were made in the model. For all subsequent structures, the first refined structure of the BCL-XL surrogate (stripped of the compound) was used as a starting model for solving by molecular replacement. After structure solution, several cycles of refinement were conducted with Refmac5, alternating with manual rebuilding using the program COOT. Compounds were modeled in the surplus peaks in difference Fourier electron density maps. Topology files for the ligands were created by ProDrg. Refinement statistics for each new structure reported in this paper are provided in Table S2.

Pharmacokinetic Study

A pharmacokinetic study was performed at 1 mg/kg IV and 3 mg/kg PO in female SCID mice (3 mice/route of administration). Compounds were formulated in HPBCD/HCl (IV bolus, 5 mL/kg) and in PEG300/EtOH/Phosal (30/10/60) (PO gavage, 10 mL/kg). Blood PK samples (3 mice/time points) were collected, by microsampling, over 24 h on each mouse: 0.05, 0.33, 1.0, 2.0, 6.0, and 24 h after IV dosing, and 0.25, 0.5, 1.0, 2.0, 6.0, and 24 h after PO dosing. Plasma samples were analyzed by LC-MS/MS.

Pharmacodynamics and Tumor Regression Study

The in vivo therapeutic and pharmacodynamic effects of BCL-XL-targeting small molecules were determined in the MOLT-4 T-cell acute lymphoblastic leukemia (T-ALL) model upon intravenous (IV) or oral (PO) administration.

Materials and Other Methods

MOLT-4 cells (ATCC No. CRL-1582) were cultured in RPMI supplemented with 10% FBS. Cells were resuspended in 50% matrigel (BD Biosciences) and 0.1 mL containing 5 × 10⁶ cells was subcutaneously inoculated into the right flank of female NOD SCID mice (Charles River). For efficacy studies, when tumors reached the appropriate volume, mice were randomized (7 animals per group) using EasyStat software. Control vehicle (HPBCD/HCl, 50/50), 29 (2.5, 5, or 7.5 mg/kg) or A-1331852 (7.5 mg/kg) were injected IV (twice weekly for 3 weeksQ3D6). Mice body weight was monitored at least twice a week, and tumor size was measured using electronic calipers. Tumor volume (V _t) was estimated from the measured minimum tumor diameter (d _min) and maximum tumor diameter (d _max) using the formula V _t = d _min ² d _max/2. Individual curves representing tumor volumes of individual mice are shown in Figure S6. At day 17, tumor growth inhibition (TGI) was calculated using the following formula:

$$\mathrm{TGI}=[1-\frac{\mathrm{mean}(\mathrm{\Delta }{V}_{\mathrm{t}}\mathrm{at}\mathrm{day}x\mathrm{in}\mathrm{treated}\mathrm{group})}{\mathrm{mean}(\mathrm{\Delta }{V}_{\mathrm{t}}\mathrm{at}\mathrm{day}x\mathrm{in}\mathrm{control}\mathrm{group})}]\times 100\%$$

where ΔV _t at day x = (V _t at day x) – (V _t at randomization).

Mice were sacrificed at the first measurement for which the tumor volume exceeded 2000 mm³ or at the first signs of animal health deterioration.

For pharmacodynamic studies and platelet analysis, when tumors reached the appropriate volume, mice were randomized (3 animals per group) using EasyStat software. Twenty-nine (7.5 mg/kg) was dosed IV (once per dayQD) in HPBCD/HCl or PO (once per dayQD) in PEG300/EtOH/Phosal (30/10/60).

Tumor samples from 3 mice/group were collected 6 h after dosing and lysed (10 mM HEPES pH 7.4, 142.5 mM KCl, 5 mM MgCl₂, 1 mM EDTA, 1% NP40, protease and phosphatase inhibitor cocktailsCalbiochem). Cleared lysates were prepared for immunodetection of cleaved PARP and Caspase 3 by using the immunoassay MSD (Meso Scale Discovery) apoptosis panel whole-cell lysate kit in 96-well plates, according to manufacturer’s instructions and were analyzed on the QuickPlex SQ 120. The mean of the triplicates was expressed as fold change relative to untreated controls in Table .

For platelet assessment, whole blood samples from 3 mice/group were analyzed on the Hematology Analyzer Coulter Ac•T diff (Beckman Coulter). Results were expressed as platelet count mean of n = 3 mice and percentage of untreated controls in Table .

Chemistry

General Synthetic Remarks

The reactions were monitored using LCMS and GCMS instruments. Analytical LCMS: Agilent HP1200 LC with Agilent 6140 quadrupole MS, operating in positive or negative ion electrospray ionization mode. The molecular weight scan range was 100 to 1350 m/z. Parallel UV detection was done at 210 and 254 nm. Samples were supplied as a 1 mM solution in MeCN or in THF/water (1:1) with 5 μL loop injection. LCMS analyses were performed on two instruments, one of which was operated with basic and the other with acidic eluents. Basic LCMS: Gemini-NX, 3 μm, C18, 50 mm × 3.00 mm i.d. column at 23 °C, at a flow rate of 1 mL min^–1 using 5 mM aq NH₄HCO₃ solution and MeCN as eluents. Acidic LCMS: ZORBAX Eclipse XDB-C18, 1.8 μm, 50 mm × 4.6 mm i.d. column at 40 °C, at a flow rate of 1 mL min^–1 using water and MeCN as eluents, both containing 0.02 v/v % formic acid.

Combination gas chromatography and low-resolution mass spectrometry were performed on Agilent 6850 gas chromatograph and Agilent 5975C mass spectrometer using 15 m × 0.25 mm column with 0.25 μm HP-5MS coating and helium as the carrier gas. Ion source: EI+, 70 eV, 230 °C. Quadrupole: 150 °C. Interface: 300 °C.

Flash chromatography was performed on ISCO CombiFlash Rf 200i with prepacked silica-gel cartridges (RediSepRf Gold High Performance). Preparative HPLC purifications were performed on a Teledyne CombiFlash EZ Prep chromatography system with a Gemini-NX 5 μm C18, 250 mm × 50 mm i.d. column running at a flow rate of 118 mL min^–1 with UV diode array detection (210–400 nm).

¹H NMR and proton-decoupled ¹³C NMR measurements were performed on a Bruker Avance III 500 MHz spectrometer and Bruker Avance III 400 MHz spectrometer, using DMSO-d ₆ or CDCl₃ as solvents. ¹H and ¹³C NMR data are in the form of delta values, given in parts per million (ppm), using the residual peak of the solvent as an internal standard (DMSO-d ₆), 2.50 ppm (¹H)/39.5 ppm (¹³C); CDCl₃, 7.26 ppm (¹H)/77.0 ppm (¹³C). Splitting patterns are designated as s (singlet), d (doublet), t (triplet), q (quartet), sp (septet), m (multiplet), br s (broad singlet), dd (doublet of doublets), td (triplet of doublets), and qd (quartet of doublets). In some cases, two sets of signals appear in the spectra due to hindered rotation.

HRMS was determined on a Shimadzu IT-TOF, ion source temperature 200 °C, ESI ±, ionization voltage (±)­4.5 kV. Mass resolution min 10000.

2-[[6-(1,3-Benzothiazol-2-ylamino)-5-methyl-pyridazin-3-yl]-methylamino]-5-[3-[4-[3-(dimethylamino)­prop-1-ynyl]-2-fluoro-phenoxy]­propyl]­thiazole-4-carboxylic Acid (24)

Step 1: Ethyl 5-(3-Chloropropyl)-2-(methylamino)­thiazole-4-carboxylate (35)

A suspension of 2.25 g of methylthiourea (25.0 mmol, 1 equiv) in 100 mL of ethanol was cooled to 0 °C, and then 7.46 g of ethyl 3-bromo-6-chloro-2-oxo-hexanoate (27.5 mmol, 1.1 equiv) was added dropwise at this temperature. After 15 min stirring at 0 °C, 7 mL of TEA (5.06 g, 50 mmol, 2 equiv) was added. The resulting mixture was stirred overnight at rt. Full conversion was observed. The volatiles were removed in vacuo, then the resultant residue was portioned between EtOAc and water. The layers were separated then the organic layer was washed with water then followed with brine. The combined organic layers were dried over Na₂SO₄, filtered and the filtrate was concentrated under reduced pressure. Then it was purified via flash column chromatography using heptane and EtOAc as eluents to give 5 g (76%) of ethyl 5-(3-chloropropyl)-2-(methylamino)­thiazole-4-carboxylate. ¹H NMR (400 MHz, DMSO-d ₆) δ ppm 7.55 (q, J = 4.7 Hz, 1H), 4.21 (q, J = 7.1 Hz, 2H), 3.65 (t, J = 6.4 Hz, 2H), 3.09 (m, 2H), 2.78 (d, J = 4.7 Hz, 3H), 1.98 (m, 2H), 1.26 (t, J = 7.1 Hz, 3H); ¹³C NMR (125 MHz, DMSO-d ₆) δ ppm 165.6, 162.5, 137.4, 135.5, 60.5, 45.0, 34.1, 31.2, 24.4, 14.7. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₀H₁₆ClN₂O₂S: 263.0616, found 263.0615.

Step 2: N-(6-Chloro-4-methylpyridazin-3-yl)-1,3-benzothiazol-2-amine

A 2 L oven-dried, one-necked, round-bottom flask was equipped with a PTFE-coated magnetic stirring bar and fitted with a reflux condenser. It was charged with 34.0 g of 6-chloro-4-methyl-pyridazin-3-amine (237 mmol, 1 equiv), 34 mL of 2-chloro-1,3-benzothiazole (44.2 g, 260 mmol, 1.1 equiv), 124 mL of DIPEA (91.8 g, 710 mmol, 3 equiv) and 137 g of Cs₂CO₃ (710 mmol, 3 equiv), then 1 L of DMF were added and the system was flushed with argon. After 5 min stirring under inert atmosphere 2.01 g of Pd₂(dba)₃ (5.9 mmol, 0.025 equiv) and 6.85 g of XantPhos (11.8 mmol, 0.05 equiv) were added. The resulting mixture was then warmed up to 75 °C and stirred at that temperature for 4 h to reach complete conversion. Reaction mixture was left to cool down to rt, then poured into 3 L of water while it was intensively stirred. After 30 min the precipitated product was removed by filtration, and then it was washed with water for 2 times (2 × 2 L). The product was dried overnight on high vacuum. The dried crude product was stirred in 1 L of heptane: Et₂O (3:2) for 30 min then filtered off to give 64.5 g (98%) of N-(6-chloro-4-methyl-pyridazin-3-yl)-1,3-benzothiazol-2-amine as green powder. ¹H NMR (500 MHz, DMSO-d ₆) δ ppm 11.96 (brs, 1H), 7.86 (d, J = 7.7 Hz, 1H), 7.65 (s, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.38 (t, J = 7.6 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 2.37 (s, 3H); ¹³C NMR (125 MHz, DMSO-d ₆) δ ppm 130.3, 129.5, 126.6, 122.8, 122.3, 17.2. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₂H₁₀ClN₄S: 277.0309, found 277.0305.

Step 3: N-(6-Chloro-4-methylpyridazin-3-yl)-3-(2-trimethylsilylethoxymethyl)-1,3-benzothiazol-2-imine (33)

A 2 L oven-dried, one-necked, round-bottomed flask equipped with a PTFE-coated magnetic stirring bar was charged with 64.5 g of N-(6-chloro-4-methyl-pyridazin-3-yl)-1,3-benzothiazol-2-amine (236 mmol, 1 equiv), 123 mL of DIPEA (9.16 g, 708 mmol, 3 equiv), 14.43 g of N,N-dimethylpyridin-4-amine (11.81 mmol, 0.05 equiv) in 1 L of dry DCM were cooled down to 0 °C under N₂. And during intensive mechanical stirring 46.00 mL of 2-(chloromethoxy)­ethyl-trimethyl-silane (43.32 g, 259 mmol, 1.1 equiv) was added to the mixture dropwise over 5 min period of time. It was stirred at 0 °C for 30 min when the reaction reached complete conversion. 24.5 mL of water was added to the reaction mixture then Celite was added to the reaction mixture and the volatiles were removed under reduced pressure. It was purified via flash column chromatography using heptane and EtOAc as eluents to obtain 46.62 g (48%) of N-(6-chloro-4-methyl-pyridazin-3-yl)-3-(2-trimethylsilylethoxymethyl)-1,3-benzothiazol-2-imine. ¹H NMR (500 MHz, DMSO-d ₆) δ ppm 7.85 (dm, J = 7.8 Hz,1H), 7.72 (m, 1H), 7.53 (dm, J = 8.2 Hz, 1H), 7.47 (m, 1H), 7.29 (m, 1H), 5.89 (s, 2H), 3.70 (m, 2H), 2.39 (d, J = 1.0 Hz, 3H), 0.90 (m, 2H), −0.12 (s, 9H); ¹³C NMR (125 MHz, DMSO-d ₆) δ ppm 159.5, 158.5, 150.0, 138.1, 137.4, 129.5, 127.4, 125.5, 123.8, 123.2, 112.4, 73.0, 66.8, 17.7, 17.1, −1.0. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₈H₂₄ClN₄OSSi: 407.1123, found 407.1120.

Step 4: Ethyl 5-(3-Chloropropyl)-2-[methyl-[5-methyl-6-[(Z)-[3-(2-trimethylsilylethoxymethyl)-1,3-benzothiazol-2-ylidene]­amino]­pyridazin-3-yl]­amino]­thiazole-4-carboxylate (36)

The mixture of N-(6-chloro-4-methyl-pyridazin-3-yl)-3-(2-trimethylsilylethoxymethyl)-1,3-benzothiazol-2-imine (4.07 g, 10 mmol, 1 equiv), ethyl 5-(3-chloropropyl)-2-(methylamino)­thiazole-4-carboxylate (3.15 g, 12 mmol, 1.2 equiv), Cs₂CO₃ (3 equiv), Pd₂(dba)₃ (0.1 equiv), XantPhos (0.2 equiv) and DIPEA (3 equiv) in 1,4-dioxane (5 mL/mmol) was kept at 150 °C for 4 h. After reaching an appropriate conversion the volatiles were removed under reduced pressure, the crude intermediate was purified via flash column chromatography to afford ethyl 5-(3-chloropropyl)-2-[methyl-[5-methyl-6-[(Z)-[3-(2-trimethylsilylethoxymethyl)-1,3-benzothiazol-2-ylidene]­amino]­pyridazin-3-yl]­amino]­thiazole-4-carboxylate (2.6 g, 41%). ¹H NMR (500 MHz, DMSO-d ₆) δ ppm 7.84 (d, J = 7.6 Hz, 1H), 7.65 (s, 1H), 7.45 (m, 1H), 7.43 (m, 1H), 7.25 (tm, J = 7.3 Hz, 1H), 5.85 (s, 2H), 4.30 (q, J = 7.1 Hz, 2H), 3.77 (s, 3H), 3.71 (t, J = 7.2 Hz, 4H), 3.22 (t, J = 7.6 Hz, 2H), 2.48 (s, 3H), 2.10 (quin, J = 7.6 Hz, 2H), 1.31 (t, J = 7.1 Hz, 3H), 0.92 (t, J = 7.9 Hz, 2H), −0.11 (s, 9H). ¹³C NMR (125 MHz, DMSO-d ₆) δ ppm 162.6, 157.4, 156.8, 155.1, 151.7, 140.5, 137.6, 137.1, 135.3, 125.6, 123.5, 123.2, 123.1, 117.6, 111.9, 72.9, 66.7, 60.7, 45.3, 35.4, 34.4, 24.3, 18.0, 17.8, 14.7, −1.0. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₈H₃₈ClN₆O₃S₂Si: 633.1899, found 633.1891.

Step 5: Ethyl 5-(3-Iodopropyl)-2-[methyl-[5-methyl-6-[(Z)-[3-(2-trimethylsilylethoxymethyl)-1,3-benzothiazol-2-ylidene]­amino]­pyridazin-3-yl]­amino]­thiazole-4-carboxylate (37)

A 100 mL one-necked, round-bottomed flask equipped with a PTFE-coated magnetic stirring bar and fitted with a reflux condenser. It was charged with ethyl 5-(3-chloropropyl)-2-[methyl-[5-methyl-6-[(Z)-[3-(2-trimethylsilylethoxymethyl)-1,3-benzothiazol-2-ylidene]­amino]­pyridazin-3-yl]­amino]­thiazole-4-carboxylate (2.6 g, 4.10 mmol, 1 equiv), NaI (1.23 g, 8.2 mmol, 2 equiv) and dry acetone (20 mL). The reaction mixture was warmed up to 60 °C and stirred at that temperature for 3 days, when the reaction reached complete conversion. The reaction mixture was diluted with the addition of water then the precipitated product was collected by filtration, washed with water, and then dried on high vacuum to obtain ethyl 5-(3-iodopropyl)-2-[methyl-[5-methyl-6-[(Z)-[3-(2-trimethylsilylethoxymethyl)-1,3-benzothiazol-2-ylidene]­amino]­pyridazin-3-yl]­amino]­thiazole-4-carboxylate (2.5 g, 84%). ¹H NMR (500 MHz, DMSO-d ₆) δ ppm 7.82 (d, J = 7.7 Hz, 1H), 7.61 (s, 1H), 7.47–7.39 (m, 1H), 7.47–7.39 (m, 1H), 7.23 (t, J = 7.1 Hz, 1H), 5.83 (s, 2H), 4.29 (q, J = 7.1 Hz, 2H), 3.75 (s, 3H), 3.71 (t, J = 8.0 Hz, 2H), 3.33 (t, J = 6.7 Hz, 2H), 3.16 (t, J = 7.6 Hz, 2H), 2.42 (s, 3H), 2.13 (quint., J = 7.2 Hz, 2H), 1.33 (t, J = 7.1 Hz, 3H), 0.91 (t, J = 8.0 Hz, 2H), −0.12 (s, 9H). ¹³C NMR (125 MHz, DMSO-d ₆) δ ppm 162.6, 157.3, 156.7, 155.1, 151.6, 140.2, 137.6, 137.1, 135.2, 127.1, 125.4, 123.4, 123.2, 117.5, 111.9, 72.8, 66.7, 60.7, 35.2, 35.2, 27.6, 17.8, 17.8, 14.8, 7.8, −1.0. HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₈H₃₈I N₆O₃S₂Si: 725.1255, found 725.1248.

Step 6: 4-[3-(Dimethylamino)­prop-1-ynyl]-2-fluorophenol (39)

The mixture of 2-fluoro-4-iodo-phenol (10.0 g, 42.0 mmol, 1 equiv), N,N-dimethylprop-2-yn-1-amine (5.24 g, 63 mmol, 1.5 equiv), Pd­(PPh₃)₂Cl₂ (0.05 equiv), CuI (0.05 equiv) and DIPA (1 mL/mmol) in THF (5 mL/mmol) was kept at 60 °C. After reaching an appropriate conversion the volatiles were removed under reduced pressure, the crude intermediate was purified via flash column chromatography to afford 4-[3-(Dimethylamino)­prop-1-ynyl]-2-fluoro-phenol (7.3 g, 90%). ¹H NMR (500 MHz, DMSO-d ₆) δ ppm 7.20 (dd, J = 11.9, 2.0 Hz, 1H), 7.07 (dm, J = 8.4 Hz, 1H), 6.91 (m, 1H), 3.39 (m, 2H), 2.21 (m, 3H); ¹³C NMR (125 MHz, DMSO-d ₆) δ ppm 150.9, 146.2, 128.9, 119.5, 118.4, 113.6, 84.5, 84.2, 48.2, 44.3. HRMS-ESI (m/z): [M + H]⁺ calcd for C₁₁H₁₃FNO: 194.0976, found 194.0981.

Step 7: Ethyl 5-[3-[4-[3-(Dimethylamino)­prop-1-ynyl]-2-fluoro-phenoxy]­propyl]-2-[methyl-[5-methyl-6-[(Z)-[3-(2-trimethylsilylethoxymethyl)-1,3-benzothiazol-2-ylidene]­amino]­pyridazin-3-yl]­amino]­thiazole-4-carboxylate

The mixture of ethyl 5-(3-iodopropyl)-2-[methyl-[5-methyl-6-[(Z)-[3-(2-trimethylsilylethoxymethyl)-1,3-benzothiazol-2-ylidene]­amino]­pyridazin-3-yl]­amino]­thiazole-4-carboxylate (1 equiv), 4-[3-(dimethylamino)­prop-1-ynyl]-2-fluorophenol (1–2 equiv), and Cs₂CO₃ (2–3 equiv) in acetone (5 mL/mmol) was stirred at rt. After reaching an appropriate conversion, the volatiles were removed under reduced pressure, and the crude intermediate was purified via flash column chromatography or reverse phase flash column chromatography to afford ethyl 5-[3-[4-[3-(dimethylamino)­prop-1-ynyl]-2-fluoro-phenoxy]­propyl]-2-[methyl-[5-methyl-6-[(Z)-[3-(2-trimethylsilylethoxymethyl)-1,3-benzothiazol-2-ylidene]­amino]­pyridazin-3-yl]­amino]­thiazole-4-carboxylate. ¹H NMR (500 MHz, DMSO-d ₆) δ ppm 7.84 (d, J = 7.8 Hz, 1H), 7.67 (s, 1H), 7.49–7.40 (m, 2H), 7.33 (dd, J = 11.9, 1.8 Hz, 1H), 7.25 (m, 1H), 7.22 (m, 1H), 7.16 (t, J = 8.7 Hz, 1H), 5.86 (s, 2H), 4.26 (q, J = 7.0 Hz, 2H), 4.15 (t, J = 6.0 Hz, 2H), 3.77 (s, 3H), 3.72 (t, J = 8.1 Hz, 2H), 3.49 (brs, 2H), 3.27 (t, J = 7.8 Hz, 2H), 2.46 (s, 3H), 2.27 (s, 6H), 2.13 (qn, J = 7.5 Hz, 2H), 1.29 (t, J = 7.0 Hz, 3H), 0.92 (t, J = 7.1 Hz, 2H), −0.11 (s, 9H). ¹³C NMR (125 MHz, DMSO-d ₆) δ ppm 129.0, 127.2, 123.5, 123.2, 119.2, 117.7, 115.5, 111.9, 72.8, 68.5, 66.7, 60.7, 48.2, 44.0, 35.3, 31.1, 23.2, 17.9, 17.8, 14.6, −0.9. HRMS-ESI (m/z): [M + H]⁺ calcd for C₃₉H₄₉FN₇O₄S₂Si: 790.3035, found 790.3023.

Step 8: 2-[[6-(1,3-Benzothiazol-2-ylamino)-5-methylpyridazin-3-yl]-methylamino]-5-[3-[4-[3-(dimethylamino)­prop-1-ynyl]-2-fluorophenoxy]­propyl]­thiazole-4-carboxylic Acid (24)

The mixture of ethyl 5-[3-[4-[3-(dimethylamino)­prop-1-ynyl]-2-fluoro-phenoxy]­propyl]-2-[methyl-[5-methyl-6-[(Z)-[3-(2-trimethylsilylethoxymethyl)-1,3-benzothiazol-2-ylidene]­amino]­pyridazin-3-yl]­amino]­thiazole-4-carboxylate (1 equiv) and HF × Pyr in MeCN (100 equiv, 15 mL/mmol) was stirred at 60 °C. After reaching an appropriate conversion, the volatiles were removed under reduced pressure and the residue was suspended in a 1:1 mixture of 1,4-dioxane–water (30 mL/mmol), treated with 150 equiv of LiOH × H₂O, and stirred at 60 °C. After reaching an appropriate conversion, the volatiles were removed under reduced pressure, and the crude product was purified via flash column chromatography using DCM and MeOH (containing 1.2% NH₃) as eluents to afford 2-[[6-(1,3-benzothiazol-2-ylamino)-5-methyl-pyridazin-3-yl]-methylamino]-5-[3-[4-[3-(dimethylamino)­prop-1-ynyl]-2-fluoro-phenoxy]­propyl]­thiazole-4-carboxylic acid. ¹H NMR (500 MHz, DMSO-d ₆) δ ppm 11.91 (brs, 1H), 7.88 (d, J = 7.8 Hz, 1H), 7.64 (s, 1H), 7.51 (d, J = 7.9 Hz, 1H), 7.36 (t, J = 7.6 Hz, 1H), 7.29 (dd, J = 11.9, 1.9 Hz, 1H), 7.22–7.16 (m, 2H), 7.14 (t, J = 8.8 Hz, 1H), 4.11 (t, J = 6.6 Hz, 2H), 3.68 (s, 3H), 3.38 (s, 2H), 3.30 (t, J = 7.5 Hz, 2H), 2.43 (s, 3H), 2.19 (s, 6H), 2.09 (m, 2H); ¹³C NMR (125 MHz, DMSO-d ₆) δ ppm 129.0, 126.4, 122.2, 122.2, 119.2, 118.7, 116.9, 115.5, 85.0, 84.2, 68.9, 48.1, 44.2, 35.8, 31.2, 22.7, 17.8; HRMS-ESI (m/z): [M + H]⁺ calcd for C₃₁H₃₁FN₇O₃S₂: 632.1908, found 632.1913.

3-{1-[(Adamantan-1-yl)­methyl]-5-methyl-1H-pyrazol-4-yl}-6-{3-[(1,3-benzothiazol-2-yl)­amino]-4-methyl-5H,6H,7H,8H-pyrido­[2,3-c]­pyridazin-8-yl}­pyridine-2-carboxylic Acid (29)

Step 1: [(Pent-4-yn-1-yloxy)­methyl]­benzene

To an oven-dried flask was added 4-pentyn-1-ol (11.1 mL, 119 mmol, 1 equiv) in THF (100 mL) and the solution was cooled to 0 °C. Sodium hydride (60% dispersion; 7.13 g, 178 mmol, 1.5 equiv) was added portionwise and the mixture was allowed to stir for 30 min at 0 °C before the dropwise addition of benzyl bromide (15.6 mL, 131 mmol, 1.1 equiv). The mixture was allowed to warm to ambient temperature and was stirred for 16 h, then cooled to 0 °C, quenched with saturated aqueous ammonium chloride (30 mL) and diluted with water (30 mL). The mixture was extracted with ethyl acetate (2 × 150 mL), and the combined organic extracts were washed successively with dilute aqueous ammonium hydroxide ammonium hydroxide (150 mL) and brine (100 mL), dried (magnesium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 330 g RediSep silica cartridge) eluting with a gradient of 0–10% ethyl acetate in iso-heptane afforded [(pent-4-yn-1-yloxy)­methyl]­benzeneas a yellow liquid (19.5 g, 112 mmol, 94%). ¹H NMR (400 MHz, Chloroform-d) δ 7.37–7.32 (m, 4H), 7.31–7.27 (m, 1H), 4.52 (s, 2H), 3.58 (t, J = 6.1 Hz, 2H), 2.32 (td, J = 7.1, 2.6 Hz, 2H), 1.95 (t, J = 2.7 Hz, 1H), 1.83 (tt, J = 7.1, 6.2 Hz, 2H). LC/MS-ESI (m/z): [M + H]⁺ calcd for C₁₂H₁₄O: 175, found: 175.

Step 2: [(Hex-4-yn-1-yloxy)­methyl]­benzene (40)

To an oven-dried flask was added [(pent-4-yn-1-yloxy)­methyl]­benzene (19.5 g, 112 mmol, 1 equiv) and tetrahydrofuran (200 mL) and the solution was cooled to −78 °C. n-Butyllithium (66.9 mL, 135 mmol, 1.2 equiv) was added dropwise over 30 min and the reaction was stirred for 1 h then iodomethane (10.5 mL, 168 mmol, 1.5 equiv) was added dropwise and the mixture was allowed to warm to 0 °C over 1 h. The reaction was quenched by the addition of saturated aqueous ammonium chloride (40 mL), diluted with water (40 mL), extracted with ethyl acetate (3 × 100 mL), and the combined organic extracts were successively washed with 2 M aqueous sodium thiosulfate (200 mL) and brine (200 mL), dried (magnesium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 330 g RediSep silica cartridge) eluting with a gradient of 0–10% ethyl acetate in iso-heptane afforded [(hex-4-yn-1-yloxy)­methyl]­benzeneas a yellow liquid (19.2 g, 0.1 mol, 91%). ¹H NMR (400 MHz, DMSO-d ₆) δ 7.41–7.23 (m, 5H), 4.46 (s, 2H), 3.48 (t, J = 6.3 Hz, 2H), 2.23–2.14 (m, 2H), 1.72 (s, 3H), 1.70–1.65 (m, 2H). LC/MS-ESI (m/z): [M + H]⁺ calcd for C₁₃H₁₆O: 189, found: 189.

Step 3: 4-[3-(Benzyloxy)­propyl]-3,6-dichloro-5-methylpyridazine (41)

A solution of 3,6-dichloro-1,2,4,5-tetrazine (5 g, 33.1 mmol, 1 equiv) and [(hex-4-yn-1-yloxy)­methyl]­benzene (7.48 g, 39.8 mmol, 1.2 equiv) in tetrahydrofuran (30 mL) was heated at 160 °C for 19 h in a sealed flask. The reaction was allowed to cool to ambient temperature then concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 220 g RediSep silica cartridge) eluting with a gradient of 0–30% ethyl acetate in iso-heptane afforded 4-[3-(benzyloxy)­propyl]-3,6-dichloro-5-methylpyridazineas an orange oil (7.32 g, 23.5 mmol, 71%). ¹H NMR (400 MHz, DMSO-d ₆) δ 7.45–7.18 (m, 5H), 4.48 (s, 2H), 3.53 (t, J = 5.9 Hz, 2H), 2.96–2.83 (m, 2H), 2.42 (s, 3H), 1.88–1.69 (m, 2H). LC/MS-ESI (m/z): [M + H]⁺ calcd for C₁₅H₁₆Cl₂N₂O: 311, found: 311.

Step 4: 3-(3,6-Dichloro-5-methylpyridazin-4-yl)­propan-1-ol (42)

To a cooled solution of 4-[3-(benzyloxy)­propyl]-3,6-dichloro-5-methylpyridazine­(7.32 g, 23.5 mmol, 1 equiv) in dichloromethane (100 mL) was added boron trichloride solution (1 M in dichloromethane; 58.8 mL, 58.8 mmol, 2.5 equiv) dropwise and the mixture was allowed to stir at ambient temperature for 1 h. The reaction was quenched by the addition of methanol and concentrated in vacuo. The residue was partitioned between dichloromethane (100 mL) and saturated aqueous sodium bicarbonate (150 mL), and the organic phase was washed with brine (150 mL), dried (magnesium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 80 g RediSep silica cartridge) eluting with a gradient of 0–80% ethyl acetate in iso-heptane afforded 3-(3,6-dichloro-5-methylpyridazin-4-yl)­propan-1-olas a yellow oil (4.19 g, 19 mmol, 81%). ¹H NMR (400 MHz, DMSO-d ₆) δ 4.67 (t, J = 5.1 Hz, 1H), 3.49 (td, J = 6.0, 5.1 Hz, 2H), 2.91–2.80 (m, 2H), 2.43 (s, 3H), 1.72–1.59 (m, 2H). LC/MS-ESI (m/z): [M + H]⁺ calcd for C₈H₁₀Cl₂N₂O: 221, found: 221.

Step 5: 3-(3,6-Dichloro-5-methylpyridazin-4-yl)­propanal (43)

To an oven-dried flask was added dimethyl sulfoxide (3.08 mL, 43.4 mmol, 2.4 equiv) and dichloromethane (100 mL) and the solution was cooled to −78 °C. Oxalyl chloride (2 M in dichloromethane; 13.6 mL, 27.1 mmol, 1.5 equiv) was added dropwise and the reaction was allowed to stir for 1 h. A solution of 3-(3,6-dichloro-5-methylpyridazin-4-yl)­propan-1-ol (4 g, 18.1 mmol, 1 equiv) in dichloromethane (20 mL) was then added dropwise and the mixture was allowed to stir for 1 h. Triethylamine (15.1 mL, 109 mmol, 6 equiv) was added and the reaction was allowed to warm to 0 °C over 1 h. The reaction was quenched with water (50 mL), then partitioned between saturated sodium bicarbonate (50 mL) and dichloromethane (200 mL), the aqueous phase was extracted with dichloromethane (200 mL), and the combined organic extracts were washed with brine (100 mL), dried (magnesium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 24 g RediSep silica cartridge) eluting with a gradient of 0–50% ethyl acetate in iso-heptane afforded 3-(3,6-dichloro-5-methylpyridazin-4-yl)­propanal as an off-white solid (2.27 g, 10.4 mmol, 57%). ¹H NMR (400 MHz, DMSO-d ₆) δ 9.71 (s, 1H), 3.03 (dd, J = 8.7, 7.0 Hz, 2H), 2.86–2.69 (m, 2H), 2.44 (s, 3H). LC/MS-ESI (m/z): [M + H]⁺ calcd for C₈H₈Cl₂N₂O: 219, found 219.

Step 6: Ethyl 3-{1-[(adamantan-1-yl)­methyl]-5-methyl-1H-pyrazol-4-yl}-6-aminopyridine-2-carboxylate (46)

A biphasic solution of ethyl 6-amino-3-bromopicolinate (5.14 g, 21 mmol), 1-(1-adamantylmethyl)-5-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)­pyrazole (7.47 g, 21 mmol, 1 equiv) and potassium carbonate (8.7 g, 62.9 mmol, 3 equiv) in tetrahydrofuran (100 mL) and water (20 mL) was mixed vigorously while sparging with nitrogen (10 min). Pd­(dppf)­Cl₂.CH₂Cl₂ (2.57 g, 3.15 mmol, 0.15 equiv) was added and the mixture was kept at reflux for 16 h. The reaction was allowed to cool to ambient temperature and was filtered through Celite. The filtrate was diluted with ethyl acetate (200 mL), washed with water (100 mL), and the organic phase was washed with brine, dried (magnesium sulfate), concentrated in vacuo, and purified by flash column chromatography to give ethyl 3-{1-[(adamantan-1-yl)­methyl]-5-methyl-1H-pyrazol-4-yl}-6-aminopyridine-2-carboxylate­(5.28 g, 64%). ¹H NMR (400 MHz, DMSO-d ₆) δ 7.32 (d, 1H), 7.21 (s, 1H), 6.58 (d, 1H), 6.24 (s, 2H), 4.05 (q, 2H), 3.72 (s, 2H), 2.10 (s, 3H), 1.98–1.88 (m, 3H), 1.71–1.47 (m, 12H), 1.10 (t, J = 7.1 Hz, 3H). LC/MS-ESI (m/z): [M + H]⁺ calcd for C₂₃H₃₀N₄O₂: 395, found: 395.

Step 7: Ethyl 3-{1-[(Adamantan-1-yl)­methyl]-5-methyl-1H-pyrazol-4-yl}-6-{[3-(3,6-dichloro-5-methylpyridazin-4-yl)­propyl]­amino}­pyridine-2-carboxylate (47)

To a solution of 3-(3,6-Dichloro-5-methylpyridazin-4-yl)­propanal (1.92 g, 8.76 mmol, 1 equiv) and ethyl 3-{1-[(adamantan-1-yl)­methyl]-5-methyl-1H-pyrazol-4-yl}-6-aminopyridine-2-carboxylate (3.8 g, 9.64 mmol, 1.1 equiv) in methanol (40 mL) was added acetic acid (15 mL) and sodium cyanoborohydride (2.75 g, 43.8 mmol, 5 equiv) portionwise and the mixture was heated at reflux for 1 h. The reaction was allowed to cool to ambient temperature then poured onto 1N aqueous sodium hydroxide (50 mL) and extracted with ethyl acetate (3 × 50 mL). The organic phase was successively washed with saturated aqueous sodium bicarbonate (100 mL) and brine (100 mL), dried (magnesium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 40 g RediSep silica cartridge) eluting with a gradient of 0–70% ethyl acetate in iso-heptane afforded ethyl 3-{1-[(adamantan-1-yl)­methyl]-5-methyl-1H-pyrazol-4-yl}-6-{[3-(3,6-dichloro-5-methylpyridazin-4-yl)­propyl]­amino}­pyridine-2-carboxylate as a white solid (3.99 g, 6.68 mmol, 76%). ¹H NMR (400 MHz, DMSO-d ₆) δ 7.34 (d, J = 8.6 Hz, 1H), 7.22 (s, 1H), 6.95 (t, J = 5.7 Hz, 1H), 6.63 (d, J = 8.6 Hz, 1H), 4.06 (q, J = 7.1 Hz, 2H), 3.72 (s, 2H), 3.41–3.31 (m, 2H), 2.92–2.83 (m, 2H), 2.42 (s, 3H), 2.10 (s, 3H), 1.98–1.89 (m, 3H), 1.86–1.74 (m, 2H), 1.71–1.48 (m, 12H), 1.08 (t, J = 7.1 Hz, 3H). LC/MS-ESI (m/z): [M + H]⁺ calcd for C₃₁H₃₈Cl₂N₆O₂: 597, found: 597.

Step 8: Ethyl 3-{1-[(Adamantan-1-yl)­methyl]-5-methyl-1H-pyrazol-4-yl}-6-{3-chloro-4-methyl-5H,6H,7H,8H-pyrido­[2,3-c]­pyridazin-8-yl}­pyridine-2-carboxylate (48)

To ethyl 3-{1-[(adamantan-1-yl)­methyl]-5-methyl-1H-pyrazol-4-yl}-6-{[3-(3,6-dichloro-5-methylpyridazin-4-yl)­propyl]­amino}­pyridine-2-carboxylate (3.99 g, 6.68 mmol, 1 equiv) in α,α,α-trifluorotoluene (150 mL) was added cesium carbonate (4.35 g, 13.4 mmol, 2 equiv) and the mixture was heated in a sealed tube at 160 °C for 3 days. The reaction was allowed to cool to ambient temperature and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 120 g RediSep silica cartridge) eluting with a gradient of 0–70% ethyl acetate in iso-heptane afforded ethyl 3-{1-[(adamantan-1-yl)­methyl]-5-methyl-1H-pyrazol-4-yl}-6-{3-chloro-4-methyl-5H,6H,7H,8H-pyrido­[2,3-c]­pyridazin-8-yl}­pyridine-2-carboxylate as a white solid (0.95 g, 1.69 mmol, 25%). ¹H NMR (400 MHz, DMSO-d ₆) δ 7.85 (d, J = 8.6 Hz, 1H), 7.71 (d, J = 8.6 Hz, 1H), 7.35 (s, 1H), 4.15 (q, J = 7.1 Hz, 2H), 4.01–3.92 (m, 2H), 3.77 (s, 2H), 2.88 (t, J = 6.6 Hz, 2H), 2.31 (s, 3H), 2.19 (s, 3H), 2.04–1.88 (m, 5H), 1.73–1.50 (m, 12H), 1.13 (t, J = 7.1 Hz, 3H). LC/MS-ESI (m/z): [M + H]⁺ calcd for C₃₁H₃₇ClN₆O₂: 561, found: 561.

Step 9: Ethyl 3-{1-[(Adamantan-1-yl)­methyl]-5-methyl-1H-pyrazol-4-yl}-6-{3-[(1,3-benzothiazol-2-yl)­amino]-4-methyl-5H,6H,7H,8H-pyrido­[2,3-c]­pyridazin-8-yl}­pyridine-2-carboxylate

To ethyl 3-{1-[(adamantan-1-yl)­methyl]-5-methyl-1H-pyrazol-4-yl}-6-{3-chloro-4-methyl-5H,6H,7H,8H-pyrido­[2,3-c]­pyridazin-8-yl}­pyridine-2-carboxylate (946 mg, 1.69 mmol, 1 equiv), 2-aminobenzothiazole, (380 mg, 2.53 mmol, 1.5 equiv) and N,N-diisopropylethylamine (0.88 mL, 5.06 mmol, 3 equiv) in 1,4-dioxane (30 mL) was added Xantphos (97.6 mg, 0.17 mmol, 0.1 equiv) and tris­(dibenzylideneacetone)­dipalladium(0) (77.2 mg, 0.08 mmol, 0.05 equiv) and the mixture was heated in a sealed flask at 160 °C for 60 h. The reaction was allowed to cool to ambient temperature and was diluted with ethyl acetate (20 mL), washed with brine (25 mL), dried (magnesium sulfate) and concentrated in vacuo. Purification by automated flash column chromatography (CombiFlash Rf, 80 g RediSep silica cartridge) eluting with a gradient of 0–75% ethyl acetate in iso-heptane afforded ethyl 3-{1-[(adamantan-1-yl)­methyl]-5-methyl-1H-pyrazol-4-yl}-6-{3-[(1,3-benzothiazol-2-yl)­amino]-4-methyl-5H,6H,7H,8H-pyrido­[2,3-c]­pyridazin-8-yl}­pyridine-2-carboxylate as an orange solid (606 mg, 0.9 mmol, 53%). ¹H NMR (400 MHz, DMSO-d ₆) δ 7.95 (d, J = 9.6 Hz, 1H), 7.83 (br s, 1H), 7.69 (d, 1H), 7.57 (br s, 1H), 7.40–7.31 (m, 2H), 7.18 (t, J = 7.5 Hz, 1H), 4.15 (q, J = 7.1 Hz, 2H), 4.05–3.97 (m, 2H), 3.77 (s, 2H), 2.86 (t, J = 6.5 Hz, 2H), 2.33 (s, 3H), 2.20 (s, 3H), 2.04–1.89 (m, 5H), 1.72–1.50 (m, 12H), 1.13 (t, 3H). LC/MS-ESI (m/z): [M + H]⁺ calcd for C₃₈H₄₂N₈O₂S: 675, found: 675.

Step 10: 3-{1-[(Adamantan-1-yl)­methyl]-5-methyl-1H-pyrazol-4-yl}-6-{3-[(1,3-benzothiazol-2-yl)­amino]-4-methyl-5H,6H,7H,8H-pyrido­[2,3-c]­pyridazin-8-yl}­pyridine-2-carboxylic Acid (29)

To ethyl 3-{1-[(adamantan-1-yl)­methyl]-5-methyl-1H-pyrazol-4-yl}-6-{3-[(1,3-benzothiazol-2-yl)­amino]-4-methyl-5H,6H,7H,8H-pyrido­[2,3-c]­pyridazin-8-yl}­pyridine-2-carboxylate­(600 mg, 0.89 mmol, 1 equiv) in 1,4-dioxane (10 mL) was added lithium hydroxide monohydrate (74.6 mg, 1.78 mmol, 2 equiv) and the mixture was heated at reflux for 1 h. The reaction was allowed to cool to ambient temperature and concentrated in vacuo. The residue was taken up in water, acidified to pH 4 and the solids were collected by filtration, washed with water, and dried. Purification by automated flash column chromatography (CombiFlash Rf, 12 g RediSep silica cartridge) eluting with a gradient of 0–10% methanol in dichloromethane afforded 3-{1-[(adamantan-1-yl)­methyl]-5-methyl-1H-pyrazol-4-yl}-6-{3-[(1,3-benzothiazol-2-yl)­amino]-4-methyl-5H,6H,7H,8H-pyrido­[2,3-c]­pyridazin-8-yl}­pyridine-2-carboxylic acid as a yellow solid (93 mg, 0.14 mmol, 16%). ¹H NMR (500 MHz, DMSO-d ₆): δ ppm 7.85–7.75 (m, 2H), 7.59 (d, J = 8.6 Hz, 1H), 7.49 (br., 1H), 7.47 (s, 1H), 7.35 (t, J = 7.4 Hz, 1H), 7.16 (t, J = 7.55 Hz, 1H), 4.00 (t, J = 5.5 Hz, 2H), 3.74 (s, 2H), 2.85 (t, J = 6.3 Hz, 2H), 2.31 (s, 3H), 2.22 (s, 3H), 1.97 (m, 2H), 1.95 (br., 3H), 1.66/1.58 (d+d, J = 41.2, 12.3 Hz, 6H), 1.56 (br., 6H); ¹³C NMR (125 MHz, DMSO-d ₆) δ ppm 139.1, 137.8, 126.4, 122.3, 122.1, 117.2, 60.3, 45.4, 40.7, 36.8, 28.2, 24.4, 21.7, 12.6, 11.2; HRMS-ESI (m/z) [M + H]⁺ calcd for C₃₆H₃₉N₈O₂S: 647.2917, found 647.2913.

Glossary

Abbreviations

Bcl-2

B-cell lymphoma 2

BCL-XL

B-cell lymphoma extralarge

BH3

Bcl-2 homology domain3

Bim

Bcl-2 like protein 11

DEA

diethylamine

DIAD

di-isopropyl azodicarboxylate

DIPA

diisopropylamine

DIPEA

N,N-diisopropylethylamine

DMAP

4-Dimethylaminopyridine

DtBAD

ditert-butyl azodicarboxylate

FBS

fetal bovine serum

FP

fluorescence polarization

HFIP

1,1,1,3,3,3-hexafluoro-2-propanol

ITC

isothermal titration calorimetry

MCL-1

myeloid cell leukemia 1

PBS

phosphate-buffered saline

Pd₂(dba)₃

tris­(dibenzylideneacetone)­dipalladium­(0)

SCID

Severe combined immunodeficient

SEMCl

2-(trimethylsilyl)­ethoxymethyl chloride

TBAF

tetrabutylammoniumfluorid

TEA

triethylamine

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.6c00865.

• Data tables, structural determination details, synthetic protocols, LC chromatograms, and NMR spectra of key compounds (PDF)

• Formula strings and data (CSV)

#.

Mol-Lub Kft, H-2931 Almásfüzitő, Fő út 21, Hungary

∇.

Boehringer Ingelheim RCV GmbH & Co KG, Dr.-Boehringer-Gasse 5-11, 1121 Vienna, Austria

○.

R&D Medical Care, Pierre Fabre Laboratories, 3 Avenue Hubert Curien, 31100 Toulouse, France

◆.

Institut Recherche Pierre Fabre, 3 Avenue Hubert Curien, 31100 Toulouse, France

¶.

YSBL, University of York, Heslington, York YO10 5DD, UK

▲.

Teledyne UK Ltd., 106 Waterhouse Lane, Chelmsford CM1 2QU, UK

A.H., M.D.F.R., F.C., and O.G. contributed to and approved the final version of the manuscript.

The financial support of Ministry of Culture and Innovation of Hungary to M.P.T. through the National Research, Development and Innovation Fund, financed under the KDP-2023 funding scheme (Project C2266727) is gratefully acknowledged.

The authors declare no competing financial interest.