S64315 | Microrna-2

Discovery of S64315, a Potent and Selective Mcl‑1 Inhibitor

Zoltan Szlavik, Marton Csekei, Attila Paczal, Zoltan B. Szabo, Szabolcs Sipos, Gabor Radics, Agnes Proszenyak, Balazs Balint, James Murray, James Davidson, Ijen Chen, Pawel Dokurno, Allan E Surgenor, Zoe Marie Daniels, Roderick E. Hubbard, Gaeẗane Le Toumelin-Braizat, Audrey Claperon, Gael̈le Lysiak-Auvity, Anne-Marie Girard, Alain Bruno, Maia Chanrion,

ABSTRACT: Myeloid cell leukemia 1 (Mcl-1) has emerged as an attractive target for cancer therapy. It is an antiapoptotic member of the Bcl-2 family of proteins, whose upregulation in human cancers is associated with high tumor grade, poor survival, and resistance to chemotherapy. Here we report the discovery of our clinical candidate S64315, a selective small molecule inhibitor of Mcl-1. Starting from a fragment derived lead compound, we have conducted structure guided optimization that has led to a signiﬁcant (3 log) improvement of target aﬃnity as well as cellular potency. The presence of hindered rotation along a biaryl axis has conferred high selectivity to the compounds against other
members of the Bcl-2 family. During optimization, we have also established predictive PD markers of Mcl-1 inhibition and achieved both eﬃcient in vitro cell killing and tumor regression in Mcl-1 dependent cancer models. The preclinical candidate has drug-like properties that have enabled its development and entry into clinical trials.

⦁ INTRODUCTION
Apoptosis, an evolutionary highly conserved form of
ImageReceived: July 27, 2020
programmed cell death, is an essential process for the elimination of no longer needed and dangerous cells.1 Evasion of apoptosis is recognized as a critical element of the development as well as sustained expansion of tumors and also underlies resistance to diverse anticancer treatments.2 Mcl-1 is a member of the Bcl-2 family, critical regulatory proteins of the mitochondrial apoptotic pathway, and is frequently upregulated in cancer.3 Moreover increased expression of the MCL1 gene through transcriptional or post-transcriptional mechanisms was observed as a down- stream consequence of several key oncogenic pathways.4 Mcl-1 is needed to sustain the growth of diverse tumors, including acute myeloid leukemia (AML),5 MYC-6 or BCR-ABL-driven pre-B/B lymphomas,7 certain breast cancers as well as non- small-cell lung carcinoma (NSCLC) derived cell lines that carry MCL1 gene ampliﬁcations.8 Some compounds that broadly inhibit gene transcription or protein translation exert their cytotoxic eﬀects in tumor cells (at least in part) by therapy.10 Until recently, only compounds showing weak cellular potency on Mcl-1 (high μM range) were available and therefore useful only as in vitro chemical tools.11 Starting in late 2016, a series of potent and selective Mcl-1 inhibitors were disclosed (Figure 1) some of which have also recently entered clinical development.12 The long-standing interest in Mcl-1 as a target and the late emergence of Mcl-1 targeting drug candidates suggest that drugging Mcl-1 is highly desirable but also very challenging. We have recently reported our successful eﬀorts to establish a drug discovery platform for antiapoptotic targets13 and its use in the identiﬁcation of fragment hits for Mcl-1 and their development into a lead compound targeting Mcl-1.14 The present manuscript describes our eﬀorts to optimize this lead into a clinical candidate and details the unexpected events encountered in this process.

■RESULTS AND DISCUSSION
Our starting point (1a) exhibited aﬃnity on Mcl-1 (28 nM
Ki)14 and remarkable selectivity against other Bcl-2 family
downregulating Mcl-1.9
In clinic, the highly promising activity of the Bcl-2-selective inhibitor venetoclax (Venclexta), which led to its approval in relapsed/refractory chronic lymphocytic leukemia (CLL) patients with 17p deletion and in AML in combination, has validated the concept of direct apoptosis activation in cancer

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© XXXX American Chemical Society
Ahttps://dx.doi.org/10.1021/acs.jmedchem.0c01234
J. Med. Chem. XXXX, XXX, XXX−XXX

Structures of some recently disclosed potent and selective Mcl-1 inhibitors with reported in vivo eﬃcacy members with an impact on the viability of Mcl-1 dependent multiple myeloma cell line H92912a at the single digit micromolar level. The primary objective of our lead optimization eﬀort was to further enhance the aﬃnity of our inhibitor. We determined the X-ray structure of 1a bound to Mcl-1 (Figure 2), which suggested two main areas of
Structure of our Mcl-1 lead compound 1a and the X-ray structure of its complex with Mcl-1 (PDB code 6QYO). S2 is highlighted in cyan, S4 and S5 are in pink. Residues of interest are as labeled.exploitation: the more occluded hydrophobic S2 pocket that the 6-ethyl occupies and the more solvent exposed S4−S5 area extending from the benzyl across Thr266. The 6-ethyl moiety occupies the S2 pocket surrounded by hydrophobic residues (Val 249, Val253, Met231, and Met250) from helixes 3 and 4. Our ﬁrst attempts were directed at optimizing the ﬁlling of the S2 pocket. Since the X-ray structure of 1a suggested that we only have limited space, a set of analogues (1b−d) carrying a small apolar substituent were synthesized and tested. While these modiﬁcations had only a minor eﬀect on the target aﬃnity, their inﬂuence on the cellular activity was more marked. The biggest improvement was achieved by the rigid 1- propynyl substituted analogue (1d), which improved activity 10-fold, probably through improved cell penetration. We also synthesized the cyclopropylated analogue 1e that should not ﬁt into the S2 pocket but surprisingly we observed no detrimental eﬀect on the on-target aﬃnity, suggesting that Mcl-1 was able to accommodate bigger substituents in this region through conformational rearrangement.

In the next round we tested some sterically more demanding aromatic groups. The monocyclic furyl (1f), thienyl (1g), and phenyl (1h) substituents were equally accommodated by the protein resulting in aﬃnities like 1a, while their increased lipophilicity led to a marked improvement of their cellular activity. Further increasing the substituent to the bicyclic benzofurane (1i) resulted in a considerable drop in aﬃnity marking the inability of Mcl-1 to rearrange suﬃciently to accommodate a bicyclic moiety in this region. Further exploring the limits of this pocket, we investigated some monosubstituted aromatics. Introducing a methyl group next to the biaryl link of thiophene (1j) was detrimental, due probably to the fact that this change does not allow the two aromatic rings to adopt a beneﬁcial coplanar conformation. If the substituent was shifted into the 3- or 4-position of the monocycle, then it was well tolerated on both the ﬁve (1k) and six membered (1l) substituent. On the other hand, the replacement of the phenyl ring of 1h by 4- pyridyl (1m) resulted in a decrease of both the aﬃnity and activity in H929 cells, due probably to the polarity of the latter
leading to increased desolvation and decreased cell penetrance.

The next set of experiments explored the structure−activity relationship with analogues carrying a methoxy substituent on the lactic acid part (Table 1, 2a−l). The reference compound 2a exhibited an aﬃnity for Mcl-1 of 20 nM, while its cellular activity was in the low micromolar range. The change of the 6- ethyl substituent to propynyl (2b) led to a similar change as observed for 1a. The aﬃnity was slightly decreased while the cellular activity improved considerably. Next we tested the ﬂuorophenyl analogues 2c−f. Neither the position of the ﬂuorine on the benzene ring nor the number of ﬂuorine substituents had a major eﬀect. All four compounds had similar aﬃnity for Mcl-1 (9−20 nM) and showed good cellular activity varying in a narrow range between 190 nM for 2c and 230 nM for 2e. Changing the ﬂuorine to chlorine (2g) did not lead to signiﬁcant changes. Replacement of the 3-chlorophenyl substituent by 3-pyridyl (2h) was well tolerated for target aﬃnity but was detrimental for the cellular activity. Finally, we introduced a series of furane derivatives into the 6-position (2i−l).

The parent compound 2j showed a signiﬁcant improvement both in aﬃnity (4.0 nM vs 20 nM for 2a) and in cellular activity (110 nM). The halogenated furane derivatives were equally potent. Their aﬃnity for Mcl-1 ranged between 3 and 4 nM and achieved sub-100 nM cellular activities for the ﬁrst time. The crystal structure of 2g bound to Mcl-1 was obtained, and overlaying the bound structures of 1a and 2g (Figure 3) nicely demonstrated the conformational changes of Mcl-1 necessary to accommodate the latter compound. The superimposition revealed the movement of helix 4 as its backbone peels away from the binding site along with some side chain reorientation to give a more capacious pocket, which is necessary to hold a bigger group such aschlorophenyl from 2g.

We also assessed the in vitro ADME properties of selected compounds (Table 1 in Supporting Information). In general, the modiﬁcations in the 6-position had only a minor eﬀect on the predicted absorption (low to moderate) and liver Comparison of the X-ray structures of the Mcl-1 inhibitors 1a (orange, PDB code 6QYO) and 2g (blue, 6YBG) bound to Mcl-1. The backbone and side chain movements of Helix 4 improve the binding of inhibitor 2g.
microsomal stability (high in rodents, intermediate to low in human). Running the experiments with human hepatocytes for selected compounds in the presence of plasma resulted in high stability due probably to the very high plasma protein binding of these molecules (unbound fraction (fu) below 1%). The in vitro properties of 2i (good cellular activity and high metabolic stability) suggested that this compound could also exhibit some activity in vivo. Mice bearing the Mcl-1 dependent human multiple myeloma AMO112a tumors, as sensitive to Mcl-1 inhibition as H929, were treated intravenously with 2i at25 mg/kg and 75 mg/kg, respectively. The tumors were harvested 2 h after treatment, and the amount of cleaved PARP, a well-established apoptosis marker, was measured (Figure 4a). Marked cleaved PARP dose dependent eﬀect was observed after 2i treatment, with 80 and 120 fold increase (at 25 mg/kg and 75 mg/kg, respectively) compared to untreated tumors, showing in vivo apoptosis induction in the AMO-1 tumors after 2i treatment.

Having optimized the substituent in the S2 pocket, we continued with the variation of the phenyllactic acid moiety for compounds bearing the 4-ﬂuorofuryl (3) or the 4-ﬂuorophenyl(4) moiety in position-6 in parallel (Table 2). We switched to a new assay format more suitable to assess the aﬃnity of very potent binders toward Mcl-1, a static quenching assay. We introduced a set of ﬁve diﬀerent substituents onto the ortho- position of the benzene ring, which was suggested to be the preferred vector toward the S4−S5 pockets based on the X-ray structures (Figure 2). The triﬂuoroethyl derivatives (3a, 4a) showed a marked diﬀerence in aﬃnity with 3a being 10 times more potent, and its cellular activity was also markedly better (31 nM vs 114 nM, Table 2). A very similar behavior was observed for the tetrahydrofurylmethyl (3b, 4b) and pyridylmethyl (3c, 4c) molecule pairs. The ﬂuorofuryl analogues (3b, 3c) showed 4- to 5-fold higher aﬃnity and 3−4 times higher cellular activity than the corresponding ﬂuorophenyl derivatives 4b and 4c. Interestingly, replacing the pyridine by pyrazine (3d and 4d) maintained the diﬀerence in the aﬃnities (cf. 0.90 nM vs 5.0 nM), but the cellular activities became equipotent (36 nM vs 42 nM). Finally, replacing the pyrazine by N-methylpyrazole (3e, 4e) broadened the gap in aﬃnity (0.86 nM vs 21 nM). In cellular activity we observed a marked but smaller diﬀerence with 3e registering at 44 nM and
4e at 114 nM. We selected a potent compound from each series, 3e and 4d, with similar ADME properties (Table 1 in Supporting Information) and assessed their activity in the in vivo PD study. Groups of xenografted mice bearing AMO1 tumors were (A) Dose dependent apoptosis induction of 2i in AMO1 xenografted mice evidenced through PARP cleavage induction 2 h after treatment (n = 3). (B) Time and dose dependent apoptosis induction by the Mcl-1 inhibitors 3e and 4d in AMO1 xenografted mice evidenced through PARP cleavage induction (n = 3). (C) Dose dependent antitumor activity of 3e and 4d in AMO1 xenografted mice following iv administration, for 5 consecutive days (n = 8).

1Ki measured in Mcl-1 FP assay.13 2Measured in H929 cell line with 10% FCS, 48 h.treated with 25 mg/kg or 50 mg/kg doses, and the fold changes in the quantity of cleaved PARP were determined at diﬀerent time points after iv bolus treatment. The results are shown in Figure 4b. In each case we observed a robust onset of apoptosis as evidenced by PARP cleavage registered after 6 h. For both compounds we observed a dose eﬀect at the tested doses at time points 6 and 16 h. Interestingly, data suggest that in the PD study 3e might be about twice as potent as 4d, showing similar fold activation at the diﬀerent time points at half the dose. In the case of 3e we see a stronger dose dependence of PARP activation at 16 h than at 6 h. Irrespective of the compound or dose, the level of cleaved PARP after 30 h was close to the baseline in all samples. In order to test whether those PD results were predictive of antitumor activity, we evaluated 4d and 3e eﬃcacy on AMO-1 grafted mice. Mice were treated intravenously (iv) with 25 mg/ kg or 50 mg/kg of the respective compound for 5 consecutive days. In the case of 4d we observed moderate dose dependent tumor growth inhibition (TGImax = 96.2% and 125.6% at 25 mg/kg and 50 mg/kg, respectively) (Figure 4c and Table 4). The antitumor activity of 3e was greater and led to tumor regression on treatment at the tested doses. At 25 mg/kg, the tumor regrew at the end of the treatment period, while at 50 mg/kg the eﬀect was lasting, and the regrowth of the tumors started only 5 weeks later. The marked diﬀerence between antitumor activity of 3e and 4d suggests that evaluation of PARP cleavage 16 h after treatment might be used as a PD marker for compound screening.

To further explore the chemistry in these two subseries, we obtained the Mcl-1 bound X-ray structures of 3e and 4d (Figure 5). Both compounds bind in a similar manner to previous compounds of the series, and the heteroaromatic rings sit on top of the ridge between the so-called S2 and S4 pockets providing suitable vectors to grow into the latter region. The lasting tumor regression on 3e treatment after iv bolus treatment at 12.5 mg/kg dose (n = 3).prompted us to explore ﬁrst the ﬁlling of the shallow S4 pocket through the variation of the substituent on the pyrazole ring. The methyl substituent in 3e as shown in the X-ray does not directly interact with Mcl-1 We expected that the conforma- tional ﬂexibility of the two-atom linker should allow the pyrazole ring to ﬂip and orient its substituent toward S4 to interact with Mcl-1 more productively. A collection of alkyl- substituted analogues was prepared and tested both in the 4- ﬂuorofuryl (5a−e) and 4-ﬂuorophenyl (6a−e) series. Increas- ing the length of the alkyl chain led to considerable improvement in both subseries. Growing from methyl (3e, 4e) through ethyl (5a, 6a) to butyl (5c, 6c) resulted in a signiﬁcant improvement of both aﬃnity and cellular activity. Increasing the branching to isopropyl (5b, 6b) and tert-butyl (5d, 6d) was equally tolerated reaching sub-100 pM aﬃnities of 10 nM or better cellular activity for both 5d and 6d. The triﬂuoroethyl-substituted molecules were also synthesized. In the ﬂuorofuryl subseries with 5e we reached 6 nM activity in the viability assay, while the ﬂuorophenyl analogue 6e was less active at 41 nM. At this stage we also wanted to see if the replacement of the hydroxy acid by an amino acid or the propynyl substitution in the 6-position that was beneﬁcial for less decorated molecules (e.g., 1d and 2b) is tolerated in 5c.

Changing the linker oxygen in 5c to nitrogen (5f) led to a signiﬁcant decrease of on-target aﬃnity (0.42 nM vs 0.014 nM) as well as cellular activity (47 nM vs 4.8 nM). In contrast the replacement of the 4-ﬂuorofuryl moiety by 1-propynyl (7) led to the highest aﬃnity registered so far at 23 pM accompanied by a slightly disappointing 18 nM cellular activity. Following the identiﬁcation of several compounds that showed high cellular potency, a selection of them was assessed in the in vivo PD study in AMO1 xenografted mice with iv bolus treatment at 12.5 mg/kg. The fold activation results in the tumors harvested 16 h after treatment are listed in Table 3. Compounds 5b and 6b gave the weakest response with PARP increases at 32-fold and 58-fold, respectively. For compounds 5d, 6d, 5e, and 6e we observed a very strong apoptosis induction after 16 h with PARP increases between 118- and 309-fold, highlighting 5e in particular. The amino acid analogue 5f was also active in the PD study registering a 125-fold PARP increase. Replacing the aromatic moiety in the 6-position by an acetylene analogue (7) was well tolerated and gave strong apoptosis induction. In general, we can state that all studied compounds led to apoptosis induction and most of them showed a considerable pharmacodynamic eﬀect.
To probe the predictive power of the PD data, two compounds were selected, 5b as the least active and 5e as the most active, and tested in the AMO1 eﬃcacy model. Mice were treated with 12.5 mg/kg of the respective compound on 5 consecutive days using iv bolus administration. The measured eﬃcacy data are listed in Table 4. Gratifyingly, both compounds induced rapid tumor regression evidenced by the respective TGImax values of 173% and 153% observed 2 days after beginning of the treatment. This eﬀect was persistent at day 7 corresponding to the last day of remaining mice in the

X-ray structures of the Mcl-1 inhibitors 3e (PDB code 6YBJ), 4d (6YBK), and 9m (6YBL) bound to Mcl-1.
control group (TGI = 112% and 119% with 5b and 5e, respectively). Diﬀerentiation between the two compounds was observed on the time to relapse, as shown by the time to reach 500 mm3. Indeed, while time to reach 500 mm3 occurred 18 equipotent registering at 34 pM and 48 pM, respectively. Interestingly the cellular activity of 9i was very high showing a 1.7 nM IC50, while 9j and 9k were in the expected activity range at 3.5 nM and 5.7 nM. Finally, we added an oxygen atom days after treatment for 5b, this same size was reached 32 days
to the ortho-tolyl moiety of 9h resulting in the hydrox after 5e treatment showing a superiority of the latte ymethylphenyl (9l) and methoxyphenyl
(9m) substituted compound.

Although 5e showed very robust tumor regression, some of its other characteristics (e.g., light sensitivity) were suboptimal; therefore, we have also explored the further optimization of 4d. As its Mcl-1 bound X-ray structure suggests (Figure 5), the vector for growing into S4 is the position meta to the oxymethyl moiety. We have established (data not shown) that replacing the pyrazine by pyrimidine is beneﬁcial in terms of both aﬃnity and compound behavior. For the ﬁrst set of compounds bearing diverse substituents on the pyrimidine ring, we prepared both the ﬂuorofuryl (8a−d) and
ﬂuorophenyl (9a−d) analogues. The small polar methoxy compounds. Compared to the tolyl analogue, both 9l and 9m showed some improvement in aﬃnity (28 pM and 29 pM vs 55 pM) as well as in cellular activity (3.5 nM and 1.7 nM vs 4.7 nM), 9m being the more potent compound.

Out of this diverse set of very potent Mcl-1 inhibitors we tested 9e−m in our PD assay at 12.5 mg/kg. All of the tested compounds induced apoptosis, and the highest activities were observed for 9i (237-fold) and 9m (285-fold). Although 9i and 9m were the most active both in the cellular and in vivo PD assays, when we compared the ranking of the other compounds in the cellular and PD assays, it was diﬃcult to see any correlation. We progressed 9i and 9m and tested their in vivo substituent (8a, 9a) was tolerated in both series showing aﬃnities in the several hundred picomolar range and 15 and 24 nM cellular activities, respectively (Table 5). Replacing methoxy by morpholine (8b, 9b) had little eﬀect on the aﬃnity but improved the cellular activity of the compounds. The 2-methoxyethyl analogues showed an increase in aﬃnity for 8c registering at 43 pM while 9c remained at 720 pM, which was accompanied by single digit nanomolar cellular potencies (3.9 and 5.7 nM, respectively). The introduction of an aromatic substituent, the 4-pyridyl moiety, was also beneﬁcial. At this point the so far apparent diﬀerence in aﬃnity between the ﬂuorofuryl and ﬂuorophenyl subseries disappeared, 8d and 9d having alike values of 30 pM and 36 pM, respectively, and their cellular activities were also very close (4.0 nM vs 3.7 nM). To assess the predictivity of the in vitro assays, compounds 8b−c and 9b−c were tested in our PD model. They were injected at a dose of 12.5 mg/kg in AMO-1 grafted mice, and the tumors were harvested 16 h after the treatment. The fold increase values of cleaved PARP are described in Table 5. All four compounds showed strong apoptosis induction, slightly less pronounced for 9c as compared to 8b, 9b, and 8c, and it was diﬃcult to correlate the PD response with their aﬃnity or cellular activity. Interestingly, activity of those ﬂuorophenyl derivatives matched the antitumor activity of the ﬂuorofuryl compound 5e.

It was reassuring to see that despite its lesser activity in the in vitro models, a ﬂuorophenyl analogue could match the in vivo eﬃcacy of the ﬂuorofuryl derivatives, so at this point we decided to focus our eﬀorts on the ﬂuorophenyl subseries. First, we assessed the position of the nitrogen atom in the pyridyl substituent. The 2-pyridyl derivative (9e) showed a slight decrease of aﬃnity (52 pM vs 36 pM for 9d) and a more marked drop of cellular activity (12 nM vs 3.7 nM), while the 3-pyridyl analogue (9f) lay in between with an aﬃnity of 50 pM and activity of 5.8 nM. Replacing 2-pyridyl by the 2-furyl moiety (9g) led to a modest drop in aﬃnity (81 pM vs 52 pM) that was accompanied by an improvement of cellular activity. On the other hand, forcing the aromatic ring out of coplanarity with the o-tolyl substituent (9h) did not break the aﬃnity (55 pM) with activity retained in the cellular assay at 4.7 nM. We also assessed the eﬀect of methyl substitution on the pyridine substituent. Of the 4-methylpyrid-3-yl (9i), 3-methylpyrid-4-yl (9j), and 2-methyl-5-methoxypyrid-4-yl (9k) analogues, 9i showed a marked drop in aﬃnity while 9j and 9k were nearly
eﬃcacy treating tumor bearing mice on 5 consecutive days with a dose of 6.25 mg/kg. Both compounds induced tumor growth inhibition, and 9m showed the stronger eﬃcacy considering both the magnitude and the duration of tumor growth inhibition (189.5% of TGImax and 38 days to reach 500 mm3).

Having identiﬁed 9m as a very potent compound, we prepared some of its analogues including the amino acid 9n, the hydroxy (10a) and amino acids (10b) bearing a propynyl substituent in the 6-position of the thienopyrimidine core instead of ﬂuorophenyl, and the dimethylamino analogue 11. Besides determining their aﬃnity and cellular activity, these compounds were also characterized in the in vivo PD assay. By comparison of the hydroxy acid−amino acid pairs (9m−9n 10a−10b), 9m showed higher aﬃnity for the target and better cellular activity (1.7 nM vs 14 nM) than 9n, while the aﬃnity diﬀerence between 10a and 10b was marginal, and the propynyl compounds 10b showed a remarkable 0.9 nM activity surpassing its hydroxy acid analogue 10a (3.9 nM). Although the aﬃnity of 11 toward Mcl-1 was only 170 pM, its cellular activity was quite good at 4.7 nM. In the in vivo setting 11 showed a remarkable PD response (251-fold PARP cleavage induction) as compared to the other compounds (9n, 10a−b) (59- to 83-fold). As expected, 11 also showed the strongest antitumor activity at 6.25 mg/kg, although not reaching the in vivo eﬃcacy of 9m.

The synthesis strategies furnishing our Mcl-1 inhibitors are depicted in Scheme 1. All of the developed inhibitors could be constructed from four key building blocks: a halogenated thienopyrimidine core, a lactic acid derivative attached to the 4-position of the thienopyrimidine, a decorated ortho- tolylboronic acid or ester coupled to the 5-position of the core, and a smaller substituent in the 6-position connected to the core through a C−C bond. In the early stages of our work the late stage diversiﬁcation of the 6-position was achieved in two ways. The synthesis of the 1 series started from 6-bromo- 4-chloro-5-iodothieno[2,3-d]pyrimidine and followed a nucle- ophilic substitution (4-position), Suzuki coupling (5-position), Suzuki-coupling (6-position) sequence that was concluded by Mitsunobu coupling and ester hydrolysis to establish the correct substitution pattern. The formed diastereoisomers were separated after the ﬁrst Suzuki coupling. A setback of this approach was the formation of 5,6-disubstituted byproducts in the ﬁrst Suzuki coupling. The alternative route explored in the synthesis of the 2 series started from 4-chloro-5-iodothieno- (A) Coimmunoprecipitation assay of HeLa cells expressing Flag-tagged Mcl-1, BCL-2 or BCL-XL as indicated. Cells were treated with diﬀerent concentrations of 9m for 2 h before lysis. Cleared lysates were used for immunoprecipitation using anti-Flag antibody. The cell lysates (input) and immunoprecipitates (IPs) were analyzed by immunoblotting with anti-Bax, Bak, or Flag antibodies as indicated. ∗ indicates nonspeciﬁc bands. (B) Western blot analysis of endogenous Mcl-1 protein levels in HCT-116 cells treated with 9m for 16 h at the indicated concentrations. Actin level is used as loading control.

(A) H929 cells were treated for 6 h with increasing concentration of 9m. Cleaved PARP was measured using MesoScale Discovery Apoptosis panel. Mean data of two independent biological experiments are represented. (B) Apoptosis induction in WT or BAX/BAK KO THP1 cells treated with 9m at the indicated concentration for 2 h. Cells were analyzed by ﬂow cytometry for PI and annexin V-FITC labeling. Mean and individual points from two biological replicates are shown. CT indicates that cells were treated with DMSO only.[2,3-d]pyrimidine. On this compound the Suzuki coupling in the 5-position was selective, and subsequent iodination of the 6-position yielded R9a. Introduction of the homochiral lactic acid R2b by nucleophilic substitution was followed by the separation of the diastereoisomers (1:1 mixture), derivatization of the 6-position in a second Suzuki coupling, and the aforementioned concluding steps.

For the diversiﬁcation of the phenyl lactate moiety in position 4 (3−11 series) we used a diﬀerent strategy. Exploiting the enhanced reactivity of the thienopyrimidine’s 6-position in cross-coupling reactions, we started from the 6- iodo-4-chloro-thieno[2,3-d]pyrimidine bearing a bromine (R1a) or iodine (R1b) in the 6-position. Selective and high yielding Suzuki or Sonogashira coupling in the 6-position was followed by nucleophilic substitution in the 4-position using the THP-protected enantiopure lactate derivative R2a. We could exploit the stereochemical directing eﬀect of the lactate in the following Suzuki coupling in the 5-position to obtain atropoisomer ratios up to 4:1 in our favor, which could be separated by ﬂash column chromatography. Introduction of the solubilizer (mostly 2-(4-methylpiperazin-1-yl)ethanol) in Mitsunobu coupling and removal of the THP protecting group gave key intermediates R5a−c. Diversiﬁcation of the 4-position followed by the hydrolysis of the ester gave the desired products. We carefully assessed that the stereochemical integrity of our compounds remained intact during the applied reaction conditions. The diastereomeric compounds that would arise from epimerization of one of the chirality elements are easy to detect due to their distinct chromatographic and NMR spectroscopic behavior. In the case of our most advanced compounds (i.e., 9m), we also developed a chiral analytical method that was able to distinguish all four stereoisomers and that conﬁrmed the stereochemical integrity of our compounds. On the basis of the in vitro and in vivo data, 9m was identiﬁed as a potential preclinical candidate and was further characterized. We obtained the X-ray structure of 9m in complex with Mcl-1 (Figure 5).

The thienopyridimine part of the 9m binds exactly the same as in 1a, 2g, 3e, and 4d. The oxymethyl linker from the benzyl crosses the Thr266 ridge and positions the pyrimidine-methoxyphenyl in S4. While methox- yphenyl sits in a small hydrophobic cavity surrounded by methoxyphenyl group.
The fact that 9m inhibits the interaction of two proteins at a hydrophobic surface predicted a signiﬁcant plasma protein binding that was in line with the measured free fractions of 0.05% and 0.04% in human and mice plasma, respectively. As a consequence, 9m showed good PK properties in mice, in agreement with the very low in vitro hepatocytes clearance measured in the presence of plasma. Administered at 6.25 or
12.5 mg/kg in mice, the observed clearance was 5.3 and 6.0

Antitumor activity of 9m on AMO-1 grafted mice (n = 10). Mice were treated at diﬀerent doses of 9m by iv for 5 consecutive days (treatment QD) or weekly for 4 weeks (treatment Q7D4). Data are represented as the mean ± SEM.mL min−1 kg−1 leading to high systemic exposures and a terminal half-life of around 5 h at both doses. To conﬁrm that 9m acts in cells through selectively displacing proapoptotic BH3 domain containing proteins from Mcl-1, HeLa cells expressing Flag tagged Mcl-1, Bcl-2, and Bcl-xL were treated with diﬀerent doses of 9m. Following immunoprecipitation using anti-Flag antibody, the endogenous Bak and Bax proteins complexed with Mcl-1, Bcl-2, and Bcl-xL were monitored (Figure 6A). As expected on the basis of its low aﬃnity toward Bcl-2 and Bcl-xL (58 μM and 237 μM, respectively, in the FP assay13), 9m displaced Bak and Bax proteins from Mcl-1 but not from Bcl-2 or Bcl-xL complexes. The shift between the cytotoxic potency and the doses apoptosis readout, was monitored. As shown in Figure 7A, 9m induced PARP cleavage in a dose-dependent manner registering as early as 1 nM and with a maximal eﬀect observed from 30 nM. Finally, to prove that the observed apoptosis was Bax/Bak dependent, THP1 cell line expressing Bax and Bak (WT) and also its BAX/BAK KO analogue were treated with diﬀerent doses of 9m for 2 h. Cells analysis by ﬂow cytometry revealed a dose-dependent onset of apoptosis in the Bax/Bak WT cell line, while the cells devoid of Bax and Bak showed no sign of apoptosis (Figure 7B).

We have also analyzed the activity of 9m in a panel of cell lines from diﬀerent types of hematological malignancies (Table 4 in Supporting Information). We have considered the cell lines to be highly sensitive to 9m when the IC50 was below 0.1 μM, moderately sensitive when the IC50 was between 0.1 μM and 1 μM, and insensitive when the IC50 was higher than 1 μM. Interestingly, all the AML and DLBCL cell lines tested and the majority of the multiple myeloma and other lymphoma cell lines were highly sensitive to 9m. From the three ALL cell lines tested, one was highly sensitive, and two were insensitive, and all ﬁve CML cell lines were insensitive. These results are in line with previous data published by our team with the S63845 Mcl-1 inhibitor compound.12a We next assessed the 9m compound in AMO1 xenografted mice by treating the animals at diﬀerent doses (3.125 mg/kg,
6.25 mg/kg, and 12.5 mg/kg) by iv for 5 consecutive days (Figure 8). A dose-dependent antitumor activity was observed with TGImax of 89.1%, 115.8%, and 162.8% at 3.125 mg/kg, 6.25 mg/kg, and 12.5 mg/kg, respectively, with an outstanding complete regression for all treated animals lasting for 35 days observed at 12.5 mg/kg. Promisingly from a clinical perspective, animals treated at 12.5 mg/kg weekly for 4 weeks experienced similar tumor growth inhibition as compared to animals treated daily (TGI at D10 = 111.6% vs 108.4% after daily or weekly treatment, respectively). Taken together, those results highlight the potential of S64315 as an Mcl-1 inhibitor to be used weekly in hematological malignancies in clinic.

A preliminary assessment of the safety pharmacology proﬁle (hERG and oﬀ target activity assays) was performed with 9m. The ﬁrst results showed no signiﬁcant alerts: the hERG inhibition indicated IC50 > 3 μM (maximal solubility of 9m i required to visualize Bax and Bak displacement from Bcl-2 family members is well documented12a,15,16 and could be explained by two reasons. First an experimental bias coming from the fact that once the cell lysates are prepared, part of the compound can dissociate from Mcl-1 allowing Bax and Bak to reassociate, which would result in an apparent loss of potency in this Co-Ip setting. Second, it is not known how much Bax and/or Bak should be freed from Mcl-1 in order to induce cytotoxicity, and this amount might be very little and not easily detectable in a Western blot assay. Selective Mcl-1 inhibitors were also reported to stabilize Mcl-1 protein in a dose- dependent manner.11a,12a,b When the HCT116 cell line (not sensitive to Mcl-1 inhibition12a) was treated with diﬀerent doses of 9m, we observed a signiﬁcant dose-dependent increase of the endogenous Mcl-1 protein. As reported earlier, this assay was very sensitive registering activity at doses as low as 0.3 nM. Having established Mcl-1 target hitting, we next assessed whether cell killing observed following 9m treatment is the consequence of apoptosis induction. To this end, H929 multiple myeloma cell line was treated for 6 h with diﬀerent doses of 9m, and PARP cleavage, a commonly accepted this assay) and the selectivity against all oﬀ targets was over
■ 400-fold. The potential drug−drug interaction in human was assessed with a high throughput assay using human CYP450 transfected cells: 9m was found mainly metabolized by CYP2C8 and exhibited an inhibitory potential toward CYP3A4 (IC50 = 1.8 μM, Table 3 in Supporting Information).

CONCLUSION
The systematic optimization of our lead Mcl-1 inhibitor that possessed nanomolar aﬃnity and micromolar cellular activity led to the identiﬁcation of the preclinical candidate S 64315/ MIK665 (9m). The structure guided optimization revealed the plasticity of Mcl-1, enabling it to accommodate substituents that were not expected to be tolerated on the basis of prior X- ray structures. The ﬁlling of the S2 pocket resulted in a signiﬁcant increase of activity. A signiﬁcant part of our eﬀorts was directed at growing our molecule into the S4−S5 region of Mcl-1. This part of the BH3 groove consists of more shallow pockets so the structural guidance was complemented by systematic modiﬁcations of our inhibitors. Our activities led to the identiﬁcation of a series of selective and highly potent Mcl-inhibitors that showed strong apoptosis induction in in vivo models, which was also translated into eﬃcient tumor growth inhibition. Of the compounds synthesized and tested, 9m stood out and was selected as a preclinical candidate. Its mode of action was conﬁrmed, and its other properties were also favorable for progressing it into preclinical development. This compound, also known as S64315/MIK665, is currently in clinical development against diﬀerent hematological malig- nancies.

⦁ EXPERIMENTAL SECTION
MTT Cell Viability Assay of H929 Cell Line. H929 cells
(purchased from ATCC) were cultured in RPMI 1640 medium supplemented with 10% heat inactivated FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10 mM Hepes, pH = 7.4 at 37 °C, in 5% CO2/95% air. Cells were grown at 37 °C in a humidiﬁed atmosphere with 5% CO2. Cell viability was measured using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay. Cells were seeded in 96-well microplates at a density to maintain control (untreated) cells in the exponential phase of growth during the entire experiment. Cells were incubated with compounds for 48 h followed by incubation with 1 mg/mL MTT for 4 h at 37 °C. Lysis buﬀer (20% SDS) was added, and absorbance was measured at 540 nm 18 h later. All experiments were repeated at least 2 times in triplicate. The percentage of viable cells was calculated and averaged for each well: % growth = (OD treated cells/OD control cells) × 100, and the IC50, concentration reducing by 50% the optical density, was calculated by a linear regression performed on the linear zone of the dose−response curve.

MCL-1 Stabilization. HCT116 cells (purchased from ATCC) were seeded at 0.25× 106 cells in 35 mm dishes in RPMI 1640 medium supplemented with 10% decomplemented fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10 mM Hepes, pH = 7.4. Cells were grown at 37 °C in 5% CO2/95% air. 48 h later, cells were treated with S64315 at the indicated doses for 16 h and harvested in lysis buﬀer (10 mM Hepes, pH7.4, 142.5 mM KCl, 5 mM MgCl2, 1 mM EDTA, 1% NP40, and phosphatase and protease inhibitors). Lysates (20 μg) were analyzed by immunoblot using the following antibodies: anti- Mcl-1 (Santa Cruz sc-819) and anti-actin (Millipore MAB1501R).
Coimmunoprecipitation. HeLa cells (purchased from ATCC) were plated at 2.5 × 105 cells in 60 mm dishes 24 h before transfection in DMEM medium (containing 10% FCS, 1 mM Hepes, 100 U/mL penicillin, 100 μg/mL streptomycin) in 5% CO2 incubator. HeLa cells were transiently transfected, using Eﬀecten reagent (Qiagen), with Flag-tagged Mcl-1, Bcl-xL or Bcl-2 expression vectors. 24 h later, cells were treated with S64315 during 2 h and harvested in lysis buﬀer (10 mM Hepes, pH 7.5, 150 mM KCl, 5 mM MgCl2, 1 mM EDTA, 0.4% TritonX100, protease inhibitors cocktail (Calbiochem 539134) and phosphatase inhibitors cocktail (Calbio- chem 524625)). The cleared lysates were then subjected to immunoprecipitation with anti-Flag M2 agarose beads (Sigma). The immunoprecipitates and inputs were analyzed by immunoblot using the following antibodies: anti-Bax (Santa Cruz sc-493), anti-Bak (BD 556996) and anti-Flag M2 (Sigma).

Apoptosis Induction. H929 cells (purchased from ATCC) were seeded at 2 × 106 cells in 35 mm dishes in RPMI 1640 medium supplemented with 10% decomplemented fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10 mM Hepes, pH = 7.4. Cells were grown at 37 °C in 5% CO2/ 95% air. 24 h after, cells were treated with S64315 for 6 h and harvested in lysis buﬀer (10 mM Hepes, pH 7.4, 142.5 mM KCl, 5 mM MgCl2, 1 mM EDTA, 1% NP40, phosphatases and proteases inhibitors).

R2a: Ethyl (2R)-2-Hydroxy-3-(2-tetrahydropyran-2-yloxyphenyl)- propanoate. Step A: [2-(Bromomethyl)phenyl]acetate. 60.07 g 2- methylphenyl acetate (400 mmol), 106.8 g NBS (600 mmol), and 500 mL cyclohexane were placed in a 1 L ﬂask. Then 3.284 g AIBN (20 mmol) was added under vigorous stirring over 30 min. The mixture was stirred at 80 °C until no further conversion was observed, then it was cooled to rt. The precipitate was ﬁltered oﬀ and washed with cyclohexane. The ﬁltrate was concentrated under reduced pressure, and the overweight crude product (100 g) was used in step B without further puriﬁcation. 1H NMR (500 MHz, DMSO-d6) δ ppm: 7.42 (dd, J = 7.8, 1.7 Hz, 1H), 7.35 (td, J = 7.8, 1.7 Hz, 1H), 7.22 (td, J = 7.5, 1.2 Hz, 1H), 7.14 (dd, J = 7.5, 1.2 Hz, 1H), 4.43 (s, 2H), 2.37 (s, 3H).
■ The X-ray structures mentioned in this paper have been deposited in the PDB with the following codes: 2g, 6YBG; 3e, 6YBJ; 4d, 6YBK; 9m, 6YBL. Authors will release the atomic coordinates and experimental data upon article publication

AUTHOR INFORMATION

Corresponding Author
Andras Kotschy − Servier Research Institute of Medicinal Chemistry, H-1031 Budapest, Hungary; orcid.org/0000- 0002-7675-3864; Phone: +36 (1) 881-2000; Email: [email protected]; Fax: +36 (1) 881 2011

Authors
Zoltan Szlavik − Servier Research Institute of Medicinal Chemistry, H-1031 Budapest, Hungary
Marton Csekei − Servier Research Institute of Medicinal Chemistry, H-1031 Budapest, Hungary
Attila Paczal − Servier Research Institute of Medicinal Chemistry, H-1031 Budapest, Hungary
Zoltan B. Szabo − Servier Research Institute of Medicinal Chemistry, H-1031 Budapest, Hungary
Szabolcs Sipos − Servier Research Institute of Medicinal Chemistry, H-1031 Budapest, Hungary
Gabor Radics − Servier Research Institute of Medicinal Chemistry, H-1031 Budapest, Hungary
Agnes Proszenyak − Servier Research Institute of Medicinal Chemistry, H-1031 Budapest, Hungary
Balazs Balint − Servier Research Institute of Medicinal Chemistry, H-1031 Budapest, Hungary
ImageJames Murray − Vernalis (R&D) Ltd., Cambridge CB21 6GB, U.K.; orcid.org/0000-0003-1007-8218
James Davidson − Vernalis (R&D) Ltd., Cambridge CB21 6GB, U.K.
ImageIjen Chen − Vernalis (R&D) Ltd., Cambridge CB21 6GB, U.K.; orcid.org/0000-0001-8865-3193
ImagePawel Dokurno − Vernalis (R&D) Ltd., Cambridge CB21 6GB, U.K.; orcid.org/0000-0002-7332-8889
ImageAllan E Surgenor − Vernalis (R&D) Ltd., Cambridge CB21 6GB, U.K.; orcid.org/0000-0002-9869-1430
Zoe Marie Daniels − Vernalis (R&D) Ltd., Cambridge CB21 6GB, U.K.
Roderick E. Hubbard − Vernalis (R&D) Ltd., Cambridge CB21 6GB, U.K.
Gaeẗane Le Toumelin-Braizat − Institut de Recherche Servier, 78290 Croissy-sur-Seine, France
Audrey Claperon − Institut de Recherche Servier, 78290 Croissy-sur-Seine, France
Gael̈le Lysiak-Auvity − Institut de Recherche Servier, 78290 Croissy-sur-Seine, France
Anne-Marie Girard − Institut de Recherche Servier, 78290 Croissy-sur-Seine, France
Alain Bruno − Institut de Recherche Servier, 78290 Croissy-sur- Seine, France
Maia Chanrion − Institut de Recherche Servier, 78290 Croissy- sur-Seine, France
Fred́eŕic Colland − Institut de Recherche Servier, 78290 Croissy-sur-Seine, France
Ana-Leticia Maragno − Institut de Recherche Servier, 78290 Croissy-sur-Seine, France
Didier Demarles − Technologie Servier, 45000 Orleans, France
Olivier Geneste − Institut de Recherche Servier, 78290 Croissy- sur-Seine, France
Complete contact information is available at: https://pubs.acs.org/10.1021/acs.jmedchem.0c01234

Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the ﬁnal version of the manuscript.

Notes
The authors declare no competing ﬁnancial interest.

⦁ ACKNOWLEDGMENTS
The authors thank co-workers at the Analytical Division of the
Servier Research Institute of Medicinal Chemistry for providing the detailed chemical analysis of the compounds.

⦁ ABBREVIATIONS USED
Mcl-1, myeloid cell leukemia 1; MCL1, Mcl-1 gene; Bcl-2, B-
cell lymphoma 2; Bcl-xL, B-cell lymphoma extra-large; BH3,

https://dx.doi.org/10.1021/acs.jmedchem.0c01234

■ Bcl-2 homology domain3; Bim, Bcl-2-like protein 11; FBS, fetal bovine serum; FP, ﬂuorescence polarization
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