Design of selective PI3Kδ inhibitors using an iterative scaffold-hopping workflow

Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx

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Design of selective PI3Kδ inhibitors using an iterative scaffold-hopping workflow Xavier Fraderaa, , Joey L. Methotb, Abdelghani Achabb, Matthew Christopherb, Michael D. Altmana, Hua Zhoub, Meredeth A. McGowanb, Sam D. Kattarb, Kevin Wilsonb, Yudith Garciab, Martin A. Augustind, Charles A. Lesburga, Sanjiv Shahc, Peter Goldenblattc, Jason D. Katzb ⁎

a

Computational and Structural Chemistry, Merck & Co., Inc., Boston, MA, USA Discovery Chemistry, Merck & Co., Inc., Boston, MA, USA In vitro Pharmacology, Merck & Co., Inc., Boston, MA, USA d Proteros Biostructures GmbH, Martinsried, Germany b c

ARTICLE INFO

ABSTRACT

InChIKeys: UHHXNKYPVVAPEH-UHFFFAOYSA-N KQGDDLRQLMABOG-UHFFFAOYSA-N IDFNWMWUIBGXDM-UHFFFAOYSA-N PXPGVPPGCPSCSN-UHFFFAOYSA-NKeywords: PI3Kδ Scaffold-hopping Structure-based drug design Oxindole

PI3Kδ mediates key immune cell signaling pathways and is a target of interest for multiple indications in immunology and oncology. Here we report a structure-based scaffold-hopping strategy for the design of chemically diverse PI3Kδ inhibitors. Using this strategy, we identified several scaffolds that can be combined to generate new PI3Kδ inhibitors with high potency and isoform selectivity. In particular, an oxindole-based scaffold was found to impart exquisite selectivity when combined with several hinge binding motifs.

PI3Kδ is a lipid kinase involved in multiple signaling pathways related to cell survival and proliferation, cytokine secretion, and immune regulation and activation. There are 4 PI3K isoforms (α, β, γ, and δ), of which the δ isoform is expressed primarily in leukocytes and is of interest as a target for many indications such as oncology, asthma, COPD, or arthritis.1,2 Recently, a selective PI3Kδ inhibitor, Zydelig,3 has been approved for the treatment of hematological malignancies Another PI3Kδ inhibitor, Umbralisib, has been given breakthrough therapy status for the treatment of lymphoma, and is also being evaluated for treatment of hematological malignancies.4,5 Several organizations are currently developing PI3Kδ inhibitors for oncology or inflammatory diseases.4–12 Progress in the design of inhibitors for PI3Kδ and related isoforms has been reviewed recently.11,12 As part of our PI3Kδ lead identification program, we have utilized several approaches to generate new chemical matter. One of these is to take advantage of available structural data to design new compounds by using computational scaffold-hopping techniques. Many PI3Kδ inhibitors are a modular assembly of three components: 1) a “hinge binder” scaffold, making one or more hydrogen bonds to the hinge region of the kinase; 2) an “affinity piece”, filling the back pocket and

⁎

contributing significantly to potency; 3) a “selectivity piece”, exposed to solvent and interacting with Trp 760, a key residue for isoform selectivity.13–15 As an example, Fig. 1 illustrates this concept for a representative compound (1-0). In general, one can hold constant two of the three components, and use scaffold-hopping techniques to replace the third. For example, one could use a combination of a known affinity piece and hinge binder to screen in silico a library of scaffolds for the selectivity piece. When trying to find new hinge binders, it can be advantageous to use only one of the affinity or selectivity pieces. This process can be repeated several times, holding different regions constant, to generate structurally diverse chemical matter that is very different from the initial starting points. Similar strategies have been reported recently.20,21 The inspiration for our scaffold-hopping effort was a pyridine-sulfonamide scaffold exemplified by compound 1-0 (see Fig. 1). The pyridine-sulfonamide motif binds into the affinity pocket and SAR on the scaffold indicated that it is an important factor by itself to the overall binding energy between the ligand and the receptor. As such, we decided to use this affinity piece as the starting point for our optimization efforts. We built a virtual library by combining this affinity piece

Corresponding author. E-mail address: [email protected] (X. Fradera).

https://doi.org/10.1016/j.bmcl.2019.08.004 Received 6 June 2019; Received in revised form 1 August 2019; Accepted 2 August 2019 0960-894X/ © 2019 Elsevier Ltd. All rights reserved.

Please cite this article as: Xavier Fradera, et al., Bioorganic & Medicinal Chemistry Letters, https://doi.org/10.1016/j.bmcl.2019.08.004

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X. Fradera, et al.

Table 1 “Hinge Screening” with aryl-sulfonamide affinity piece. See19 for details on biochemical assays and selectivity calculations.

Fig. 1. (a) Highlight of the affinity piece, hinge binding scaffold, and selectivity piece for compound 1-0 (GSK 2292767, Ref. 16, and PDB:5AE9).

Fig. 2. Ligand Binding Efficiency (LBE)17 vs. isoform selectivity for compounds from the hinge screening library. LBE was calculated based on PI3Kδ IC50 values, and selectivity was defined as the minimum fold-difference between PI3Kδ and other isoforms.19 Compounds denoted with a red cross are listed in Table 1.

PI3Kδ IC50 (nM) and LBE, (in parentheses)

Selectivity δ/α

δ/β

δ/γ

1-1

43 (0.46)

31

17

26

1-2

33 (0.47)

129

22

14

1-3

23 (0.50)

2

2

3

1-4

7.2 (0.53)

4

6

4

1-5

11 (0.52)

3

4

3

1-6

150 (0.44)

8

3

9

1-7

58 (0.47)

9

4

17

1-8

10 (0.52)

1

3

2

1-9

61 (0.47)

4

2

6

1-10

20 (0.50)

2

4

6

that it would also provide a better dynamic range with respect to potency, as new chemical matter was identified. Based on a model of compound 2-1 bound to PI3Kδ, we realized that position 3 of the imidazopyrimidine scaffold was the optimal vector to interact with the selectivity region. Addition of an aryl-morpholine scaffold in 2-2 led to a great improvement in potency, but limited selectivity, likely due to suboptimal interactions with Trp 760. 2-3 and 2-4, with a similar shape, did not show significant improvements in potency or selectivity. In contrast, homologated morpholine 2-5 had an approximately 10-fold improvement in selectivity versus all non-PI3Kδ isoforms. The methylene spacer between the phenyl and morpholine rings imparted more flexibility to the selectivity piece, which could allow the morpholine ring to stack on top of the Trp 760 side-chain (Fig. 3) and account for this observation. Similarly, compound 2-6, which can position a phenyl ring for interaction with Trp 760, had a potency and selectivity profile comparable to 2-5. Amide (2-7) and sulfone linkers (2-8) had weaker potency and an inferior isoform selectivity profile. Compounds 2-2 to 2-5 confirmed that moderate potency and selectivity could be obtained for the pyrazolopyrimidine scaffold. The next step was to design a selectivity piece optimized for potency and selectivity (at least 100-fold against all non-δ isoforms). To this end, we screened a library of fragments based on structures from the Cambridge Structural Database, using MOE software.24 Each fragment was coupled to the pyrazolopyrimidine core, and only molecules that could interact with the side-chain of Trp 760 were retained. The approximately 7,000

with a collection of approx. 20,000 hinge-binding scaffolds (without consideration of a selectivity piece at this point) and docked all the ligands into the active site of PI3Kδ.18 Based on docking scores and visual inspection, 105 ligands were selected and synthesized, spanning a wide range of potency and selectivity profiles, as depicted in Fig. 2. Table 1 lists 10 of the actives that displayed higher efficiency and/or isoform selectivity. Three criteria were used to choose hinge binder scaffolds: 1) Ligand Binding Efficiency (LBE)17 2) Selectivity over nonPI3Kδ isoforms, and 3) Opportunity for further growth into the selectivity region. Some compounds had > 10-fold selectivity against all isoforms (1-1, 1-2), or LBE > 0.50 (1-3, 1-4, 1-5, 1-8, 1-10). From these, compounds 1-1, 1-2, 1-8, and 1-10 were predicted to have a convenient vector for the introduction of a selectivity piece. The pyrazolopyrimidine 1-8 was chosen for progression, because this was the most efficient scaffold which would allow growth towards the selectivity region. The polar pyridine-sulfonamide affinity motif can be a limiting factor for optimization of PK and physicochemical properties, as it is ionized at pH 7.4. SAR from other molecular series indicated that 4-Me pyrimidine can also be an efficient affinity piece without the liabilities of the sulfonamide.23 Incorporation into the new hinge binder resulted in a stark loss of potency (compound 2-1, Table 2), but previous SAR had informed us that potency and selectivity could be recovered by adding an appropriate selectivity piece. In addition, we hypothesized

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Table 2 “Selectivity Screening” with pyrazolopyrimidine hinge binder. Compound 2-9 (from Virtual Screening) was not synthesized. Enantiomers for chiral compounds 2-11 and 2-12 were separated, and data reported corresponds to the eutomers. PI3Kδ IC50 (nM)

Selectivity δ/α

δ/β

δ/γ

> 10,000 89

– 1.5

– 22

– 5

2-3

343

1.7

17

4

2-4

60

1.7

24

9

2-5

37

18

18

90

2-6

27

4

30

16

2-7

689

1

715

4

2-8

94

1

25

3

2-9

virtual compound - not synthesized

2-10

1

70

1200

250

2-11

34

15

210

36

2-12

1.9

45

320

200

2-1 2-2

H

Fig. 3. Model of compound 2-5 docked into a crystal structure of PI3Kδ. The pyrazolopyrimidine scaffold has one formal hydrogen-bond to the hinge. The selectivity piece is partially exposed to solvent, with the morpholine ring stacking on top of the side-chain of Trp 760. Key contacts between receptor and ligand are highlighted with yellow dashed lines.

Compound 2-11 was based on the same idea but showed a significant loss of potency and selectivity. The cyclopropylmethyl 2-12 is a direct analog of 2-10 but the smaller cyclopropyl resulted in a slightly inferior potency and selectivity profile. Modeling studies suggested that compound 2-10 would bind with the terminal Ph ring edge to face to the Trp 760 side-chain, and this was later confirmed by crystallography (Fig. 4b). We hypothesized that the interaction between the benzyl group of 2-10 and residues Thr 750, Met 752, and Trp 760 was the cause for isoform selectivity, as suggested by the literature.13,14 However, it was intriguing that selectivity against isoforms PI3Kβ and PI3Kγ was much higher than against PI3Kα. This became clearer when the crystal structure of 2-10 bound to PI3Kα (Fig. 4c) was obtained, revealing a “reverse” binding mode, with the oxindole-based scaffold binding into the affinity pocket, and the 4-Me pyrimidine scaffold exposed to solvent. The “reverse” binding mode in PI3Kα is facilitated by the fact that the pyrazolopyrimidine scaffold has only one classical hydrogen bond to the hinge, which can be maintained in both binding modes. In both cases, 2-10 also makes a weak C-H hydrogen bond to the hinge backbone. For PI3Kδ, the acceptor is the backbone carbonyl of residue Glu 826, while for PI3Kα, this role is taken by Val 851 (equivalent to Glu 828 in PI3Kδ). The oxindole-based scaffold, with its superior selectivity profile, was highly attractive as a selectivity piece. Therefore, we decided to use it as a structural anchor for another round of scaffold-hopping. The objective was to find alternatives to the pyrazolopyrimidine hinge binding core, without considering an affinity piece at this point. We enumerated a library of approx. 3,000 compounds that could be synthesized by coupling oxindole with available reagents. These compounds were computationally docked into the active site of PI3Kδ.18 Based on docking scores and chemical diversity, 40 compounds were selected, and approximately 30 of them were synthesized and tested. Compounds 3-2 to 3-7 in Table 3 are the most potent compounds that resulted from this effort, compared to a reference pyrazolopyrimidine (compound 3-1). Quinoxaline 3-6 had the best combination of

molecules generated were then minimized and scored using the GBVI/ WSA scoring function,22 and the 1,000 highest-ranking ones were inspected visually. Several high-ranking virtual ligands, such as isoindolinone 2-9, could be viewed as rigidified analogs of 2-5, with a phenyl ring in a parallel stacking interaction with the Trp 760 sidechain (see Fig. 4a). A readily available reagent allowed quick access to the structurally similar benzyl analog 2-10, which had a significantly improved potency and selectivity profile compared to compound 2-5.

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Table 3 Hinge screening with oxindole selectivity piece. Compounds 3-2 to 3-7 were tested as racemates. PI3Kδ IC50 (nM)

Selectivity δ/α

δ/β

δ/γ

3-1

32

235

> 390

> 390

3-2 (rac)

530

1

3

5

3-3 (rac)

540

20

15

13

3-4 (rac)

180

10

> 70

> 70

3-5 (rac)

374

3

10

18

3-6 (rac)

24

20

100

50

3-7 (rac)

179

10

70

50

Table 4 Hinge screening with oxindole selectivity piece. PI3Kδ IC50 (nM)

Selectivity δ/α

δ/β

δ/γ

4-1 (S)

12

328

> 1048

353

4-2 (S)

10

219

> 1195

154

Fig. 4. (a) Structure 2-9 from scaffold-hopping workflow, docked in a PI3Kδ crystal structure (b) x-ray structure of 2-10 in complex with PI3Kδ (PDB:6PYR, resolution 2.21 Å) (c) x-ray structure of 2-10 in complex with PI3Kα (PDB:6PYS, resolution 2.19 Å).

potency and selectivity and was selected for further optimization of the selectivity piece. Based on the crystal structure of 2-9 and modeling, we hypothesized that spiro substituents on the oxindole could be used to present an aliphatic group to the side-chain of Trp-760. This effort resulted in the design of analogs 4-1 and 4-2 (see Table 4). These compounds are highly potent PI3Kδ inhibitors with an exquisite selectivity

profile, even though they lack an affinity piece. An x-ray structure confirmed the proposed binding mode for 4-2, with one classical hydrogen bond to the hinge, and van der Waals contacts between the cyclopropyl ring and C-δ2 and C-ε3 of Trp 760 (CeC distances of 3.4 Å 4

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O O S

O

O

HN

N

N N

O

1-0 0.33 nM 375x selectivity

HN N

O O

1: VS

O S N N

1-8 10 nM no selectivity

N NN

2: Prior knowledge

N N

N

Fig. 5. X-ray structure of compound 4-2 bound to PI3Kδ (PDB:6PYU, resolution 2.54 Å).

NN

HN

and 3.6 Å). Additionally, there are two contacts between the cyclopropyl ring and C-γ2 of Thr 750 at a slightly longer distance (CeC distances of 3.8 Å in both cases) (Fig. 5). The overall iterative design process is summarized below, and in Fig. 6, which highlights key compounds and the structural anchor at each step.

O

N N

H N

3: VS + SBDD 2-10 1 nM 79x selectivity

N NN

1. Optimize hinge binder, with anchor of affinity piece (virtual screen) 2. Replace affinity piece (based on prior knowledge) 3. Optimize selectivity piece, with anchor of hinge binder and affinity pieces (virtual screen, and SBDD) 4. Optimize hinge binder, with anchor of selectivity piece (virtual screen) 5. Final optimization of selectivity piece (SBDD)

2-1 > 10 uM

4: VS

O

3-6 24 nM 100x selectivity

N N

There are several reasons that make this approach highly attractive for PI3K kinases: first, there is a wealth of available structural data. Second, many PI3K inhibitors can be broken down into three modular pieces, making them highly amenable to scaffold-hopping approaches. Third, the hinge region of PI3K can accommodate many different scaffolds, some of them capable of binding in more than one orientation, or with only one hydrogen bond to the hinge. Starting from compound 1-0, designed for inhaled delivery, we were able to remove the ionic sulfonamide piece and build up specific interactions to the hinge and to the selectivity pocket. Although compounds 4-1 and 4-2 are less potent than the starting point, they are structurally different and do not depend on an affinity piece for potency. In particular, oxindole-based scaffolds can impart an exquisite selectivity profile in combination with several hinge binding scaffolds, and are an attractive starting point for design of inhibitors for oral delivery. Additional work on this will be reported elsewhere.

N

H N

5: SBDD O

O

N

N

4-1 12 nM 353x selectivity

N Fig. 6. Optimization steps and key compounds in our iterative design strategy. The “anchor” piece at each step of optimization is highlighted in a blue box. PI3Kδ IC50s and minimum selectivity to non-δ isoforms is listed at the side of each compound.

Appendix A. Supplementary data

Acknowledgment

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bmcl.2019.08.004. These data include MOL files and InChiKeys of the most important compounds described in this article.

We thank Tony Siu for a careful review and helpful discussion.

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References

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