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Discovery of GDC-0853, a Potent, Selective, and Non-Covalent Bruton’s Tyrosine Kinase Inhibitor in Early Clinical Development James J. Crawford, Adam R. Johnson, Dinah L. Misner, Lisa D. Belmont, Georgette M. Castanedo, Regina Choy, Melis Coraggio, Liming Dong, Charles Eigenbrot, Rebecca Erickson, Nico Ghilardi, Jonathan Hau, Arna Katewa, Pawan Bir Kohli, Wendy Lee, Joseph W. Lubach, Brent S. McKenzie, Daniel Fred Ortwine, Leah Schutt, Suzanne Tay, Binqing Wei, Karin Reif, Lichuan Liu, Harvey Wong, and Wendy B. Young J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 19 Feb 2018 Downloaded from http://pubs.acs.org on February 20, 2018

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Journal of Medicinal Chemistry

Discovery of GDC-0853, a Potent, Selective, and Non-Covalent Bruton’s Tyrosine Kinase Inhibitor in Early Clinical Development. James J. Crawford*, Adam R. Johnson, Dinah L. Misner, Lisa D. Belmont, Georgette Castanedo, Regina Choy, Melis Coraggio, Liming Dong,$ Charles Eigenbrot, Rebecca Erickson, Nico Ghilardi, Jonathan Hau, Arna Katewa, Pawan Bir Kohli, Wendy Lee, Joseph W. Lubach, Brent S. McKenzie, Daniel F. Ortwine, Leah Schutt, Suzanne Tay, BinQing Wei, Karin Reif, Lichuan Liu, Harvey Wong, and Wendy B. Young* Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA $

formerly of ChemPartner, No. 1 Building, 998 Halei Road, Zhangjiang Hi-Tech Park, Pudong New Area, Shanghai, China 201203

KEYWORDS Kinase inhibitor, Bruton's tyrosine kinase, Btk, Rheumatoid arthritis, Lupus, kinase mutants, C481S, GDC-0853.

ABSTRACT Btk is a non-receptor cytoplasmic tyrosine kinase involved in B-cell and myeloid cell activation, downstream of B-cell and Fcγ receptors, respectively. Pre-clinical studies have indicated that inhibition of Btk activity might offer a potential therapy in autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. Here we disclose the discovery

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and pre-clinical characterization of a potent, selective, and non-covalent Btk inhibitor currently in clinical development. GDC-0853 (29) suppresses B cell- and myeloid cell-mediated components of disease, and demonstrates dose-dependent activity in an in vivo rat model of inflammatory arthritis. It demonstrates highly favorable safety, pharmacokinetic (PK), and pharmacodynamic (PD) profiles in pre-clinical and Phase 2 studies are ongoing in patients with rheumatoid arthritis, lupus and chronic idiopathic urticaria. Based on its potency, selectivity, long target residence time, and non-covalent mode of inhibition, 29 has the potential to be a bestin-class Btk inhibitor for a wide range of immunological indications.

Introduction Bruton’s tyrosine kinase (Btk) is a non-receptor Tec family tyrosine kinase that is broadly expressed in hematopoietic cells, with the exception of T cells.1,2 Btk plays a crucial role in signaling through the B-cell antigen receptor (BCR) and the Fcγ receptor (FcγR) in B cells and myeloid cells, respectively.1,3-5 Spontaneous mutations in the pleckstrin homology domain of the Btk gene found in xid mice,6 and targeted mutations in Btk knockout mice, both result in B-cell development and proliferation defects.2,7 These deficiencies are manifested by reduced numbers of mature circulating B cells, greatly reduced serum titers of certain immunoglobulin (Ig) isotypes—particularly IgM and IgG3—and poor T-independent antibody responses. X-linked agammaglobulinemia (XLA) is a human congenital immunodeficiency condition that occurs in males,2,7 which is associated with low to undetectable btk mRNA levels and Btk protein expression and, as a consequence, low to no Btk kinase activity.8 In contrast to the phenotype observed in xid mice, B-cell development in XLA patients is almost completely blocked at the pro-B-cell to pre-B-cell transition leading to extremely low to undetectable levels of mature B


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cells and, in turn, immunoglobulins.2,7 Left untreated, XLA patients succumb to recurring infections, but can be treated with Ig replacement therapy.9-11 Taken together, these factors indicate that inhibition of Btk represents an attractive potential therapeutic approach for the treatment of immunological disorders in which B cells and myeloid cells induce or sustain an excessive autoimmune response such as rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE).4,5,7,12-14 For over a decade, there have been significant efforts expended towards the goal of identifying Btk inhibitors for clinical application in chronic inflammatory diseases as well as hematologic malignancies.15-23 While the irreversible Btk inhibitor ibrutinib (PCI-32765, Imbruvica®)15,24 has been successful in treating B-cell malignancies and is approved for chronic lymphocytic leukemia (CLL),25,26 relapsed or refractory mantle-cell lymphoma (MCL),27,28 and Waldenström's macroglobulinemia (WM),29 there is currently no approved Btk-targeted therapy for chronic autoimmune indications. A number of Btk inhibitors have shown efficacy in several preclinical models of inflammatory or autoimmune diseases that are driven by pathogenic B cells or myeloid cells,5,14,24,30-37 but thus far none of these has progressed into advanced clinical trials for the treatment of autoimmune diseases. This slow progress in the autoimmune/inflammatory arena may, at least in part, be due to the stringent safety requirements these indications demand. There continues to be significant interest in the pursuit of Btk inhibitors and the leading inhibitors to date are covalent: ibrutinib (1), spebrutinib31 (2) (CC-292, AVL-292), tirabrutinib38 (3) (GS-4059, ONO-4059) and acalabrutinib39 (4) (ACP-196) (Figure 1). All of these compounds react covalently and irreversibly with cysteine-481 in the ATP binding site of Btk. This dependence on irreversible inhibition poses a selectivity risk – since ten other human kinases possess an equivalent cysteine residue in their active site40 – and raises the spectre of unwanted


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off-target inhibition of these kinases. Indeed, the limited kinase selectivity data published so far for these covalent inhibitors align with this expectation, as off targets include some or all of these 10 kinases that contain an analogous cysteine residue.24,31 Furthermore, due to their ATP-like binding mode, these covalent molecules can also bind non-covalently to and inhibit even more kinases, further increasing safety concerns due to selectivity. Finally, the reactive groups on covalent compounds may lead to haptenization of serum proteins, potentially leading in rare cases to serum sickness-like reactions in patients. Together, these off-target effects could theoretically erode the safety profiles of these molecules. Because autoimmune indications such as RA and SLE require exquisitely safe drugs, our strategy was to develop non-covalent inhibitors that interact with Btk in a different manner than existing ATP-mimetic covalent inhibitors. Figure 1. Selected examples of clinical stage Btk inhibitors.


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Results We previously reported the discovery and structure-activity relationships (SAR) of our first clinical-stage inhibitor, GDC-0834, 5,20,21 which suffered from rapid amide bond hydrolysis in humans21, but which we evolved into more advanced compounds including G-278, 6 (Figure 2).22 With a Btk enzyme IC50 of 4 nM and a BCR-induced CD69 IC50 of 35 nM in human whole blood, 6 had a desirable profile. This, in addition to good kinase selectivity and an acceptable pre-clinical pharmacokinetic profile, allowed us to evaluate 6 in rat and dog tolerability studies with the intent of advancing this compound into the clinic. Unfortunately, 6 induced doselimiting toxicities with unacceptably low safety margins in both species. In Sprague-Dawley rats, an unusual pancreatic toxicity was also observed with this as well as a number of other Btk


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inhibitors, and this was the subject of previously reported investigations (vide infra),41 whereas in dogs liver toxicity was observed with 6 (as well as related analogues). The observed hepatotoxicity in dogs was characterized by increases in ALT and AST liver enzymes, as well as other findings in the liver such as perivascular mixed cell infiltrates, Kupffer cell hypertrophy, and hepatocellular degeneration. In addition, increased circulating neutrophils were observed at higher dose levels (Supporting Information). As a result, hepatotoxicity was the primary issue that we sought to eliminate in next generation analogues. Figure 2. Progression of Genentech’s published Btk inhibitors from GDC-0834 to G-278.

In an attempt to better understand the hepatotoxicity that was primarily observed in dogs, a cell viability (cytotoxicity) assay using cryopreserved human hepatocytes was used to identify highrisk compounds (Supporting Information).42,43 Compounds with an IC50 < 50 µM in this assay were classified as having potentially high risk for cytotoxicity while compounds with IC50 >100 µM were considered low risk. Once a sufficient data set was obtained, we set out to develop an in silico model for the prediction of hepatocyte cytotoxicity to be applied prior to compound synthesis and to flag any possible causative factors (Figure S-1, Supporting Information). A


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training data set consisting of 70 Btk inhibitors was used, with their cytotoxicity classified into three toxicity risk bins: high (IC50 < 50 µM), medium (IC50 = 50-100 µM), or low (IC50 > 100 µM). We explored a wide range of commonly used structural and physicochemical descriptors and a variety of modeling algorithms. A decision tree model was chosen based on its performance in 5-fold cross-validation as well as its easy interpretability. It was subsequently tested using the data for 121 molecules outside the training set, including some compounds that originated from other internal discovery projects. The model performed reasonably well on this more structurally diverse set of molecules (see details in Supporting Information), which supported its application on this programme. The model relied on only three calculated properties (pKa of the most basic nitrogen, plasma protein binding (PPB), and logP)44 and assigned a predicted hepatocyte IC50 range of a given molecule into one of the three defined toxicity risk categories. While we had previously hypothesized basicity (i.e., high pKa) to be a major risk factor for cytotoxicity, the decision tree model identified logP and PPB as two additional predictive factors and defined specific thresholds for each of them. Although often related—logP and PPB within this set of compounds were linked enough to give a modest correlation (R2=0.55)—removing either parameter significantly reduced the performance of the model.

Interestingly, inclusion of structure

descriptors did not improve the accuracy of predictions. This suggested that, within this series of Btk inhibitors, physicochemical properties rather than particular substructures were the likely drivers of hepatocyte cytotoxicity (for more details, see the Supporting Information). Given the influence of basicity and logP, moving forward we focused our efforts on avoiding the incorporation of strongly basic groups, and lowering logD (i.e., taking into account the combined impact of pKa and logP), while aiming to maintain or improve whole blood potency and


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pharmacokinetic (PK) properties.

SAR: Increasing Potency, Improving Metabolic Stability, and Minimizing Toxicity One region of this Btk inhibitor scaffold that we sought to optimize was the hydroxymethylsubstituted aryl ring that links the pyridone hinge-binding motif to the selectivity pocket (or ‘H3’) motif. While hydroxymethyl and F substitution had been shown to improve potency,22,45 changes to the ring itself had not.

We sought to lower logD by replacing the aryl ring with a heterocycle such as pyridine. The three isomeric pyridyl analogues were prepared and tested (7-9, Table 1).

In all cases,

incorporation of the nitrogen reduced the calculated and measured logD by at least one unit and resulted in improved metabolic stability in human liver microsomes as compared to the benzene analogue 6. Examples 7 and 8 maintained whole blood potency within two-fold of the benzene analogue 6, however, the isomer with the pyridyl nitrogen ortho to the H3 motif (7) retained the most intrinsic potency with a Btk Ki of 1.8 nM and thus was regarded as the preferred isomer. Table 1. Initial SAR around the core aryl ring.

cmpd

Core

Btk Ki

WB CD69 IC50

HLM CLhep

clogDd / logD


8

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6

7

8

9

(nM)a

(nM)b

(mL/min/kg)c

(pH 7.4)e

0.69±0.44

36.5±10.2 n=7

12.3

2.6 / 3.1

1.80±1.20

54.9±19.4 n=3

8.1

1.6 / 2.0

6.30±2.80

64.4±6.1 n=2

7.9

1.3 / 1.9

6.90±2.20

387.0±101.8 n=2

8.0

1.2 / 1.8

a

Inhibitor Ki values were determined in at least triplicate in an assay that monitors Btk-catalyzed phosphorylation of a synthetic peptide substrate. See Supporting Information for assay details. bCD69 expression on CD19+ B cells in human whole blood was measured after stimulation with anti-IgM (Supporting Information). cHuman liver microsome-predicted hepatic clearance. dCalculated logD (logD was calculated using v2 of the MoKa program, available from Molecular Discovery Ltd. (www.moldiscovery.com)). eLogD measured in house using a high throughput microscale shake flask with liquid chromatography tandem mass spectrometry quantitation.46

Over time, many additional pyridyl compounds were prepared and the same trends toward improved metabolic stability and LLE were apparent as illustrated in matched pair plot analyses (Figure 3).47,48

Figure 3. Matched pair plots for Btk inhibitors containing an Ar-F (l) or pyridyl (r) linker. Lines join matched pairs. (a) HLM stability (mL/min/kg);

(a) HLM (mL/min/kg)

(b)

LLE = pKi – clogD47,48

(b) LLE = pKi - clogD


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LLE

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

CLhep (mL/min/kg)


HO

HO

N

N

As previously noted, much of the potency and selectivity of our chemical scaffold results from the ability of these compounds to stabilize and interact with binding site in Btk that is generated via a conformational rearrangement of the activation loop of the kinase domain.5,22,50 It is instructive to note that, given our induced-fit binding mode, we used our whole blood assay as the main driver of SAR in this program since it could encompass the effects of a combination of therapeutically relevant parameters, including Btk affinity, cell permeability, PPB, and favorable target-binding kinetics. In this respect, potency in whole human blood was determined as previously described14 and monitored via either Btk Y223 phosphorylation or anti-IgM-induced CD69 expression on B cells. The generally lipophilic groups at the tricyclic H3 motif termini present hydrophobic surfaces that interact with Y551 of the activation loop of Btk (among other residues), ultimately occupying a lipophilic specificity/selectivity pocket. We decided to synthesize a set of inhibitors


10

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with a variety of H3 groups in combination with the pyridyl linker to see if we could take advantage of improved LLE, allowing us to incorporate groups that might afford improved whole blood potency. In the case of thiophene-containing examples 10 and 11, the resulting increase in lipophilicity relative to pyrrole analogue 7 led to increased logD and a drop in metabolic stability in human liver microsomes (Table 2). The dimethyl substituted cyclopentyl (11) offered improved whole blood potency relative to the corresponding cyclohexyl analogue (10), so the next compound prepared was the corresponding pyrrole-containing derivative, 12. This molecule displayed improved whole blood potency (IC50 = 15.4 nM), and maintained the metabolic stability and physicochemical property profile of 6 (kinetic solubility = 79.8 µM for 12 vs. 12 µM for 6). Small changes such as unsaturation of the lactam ring or fluoro substitution of the pyrrole ring—albeit not in tandem, as was subsequently reported to be beneficial49—did not afford any appreciable whole blood potency benefits in our series. Lastly, although the tertbutyl-substituted bicyclic thiophene lactam 15 was a potent inhibitor, it had an unfavorable high measured logD (>3) like the other thiophene-containing examples.

From this exploration,

compound 12 offered the best balance of properties and was not cytotoxic in primary human hepatocytes (IC50 >100 µM). However, when dosed in a cassette rat PK study, 12 exhibited high plasma clearance (CLp = 165 mL/min/kg) and a relatively short half-life (0.4 hours), so SAR efforts continued in order to improve the metabolic stability of this scaffold. Table 2. Selectivity pocket / H3 SAR. a


11


cmpd

7

10

11

12

13

14

15

a

H3 group

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Btk Ki (nM)

WB CD69 IC50 (nM)

HLM CLhep (mL/min/kg)

clogD / logD (pH 7.4)

1.80±1.20

54.9±19.4 n=3

8.1

1.6 / 2.0

5.97±0.32

121.1±24.5 n=2

13.0

2.1 / 3.4

0.58±0.01

39.0±20.4 n=3

13.5

2.3 / 3.3

1.80±0.56

15.4±8.2 n=5

7.2

1.8 / 1.9

4.20±0.51

77.8±52.9 n=3

9.2

0.8 / 2.5

2.50±1.20

42.4 n=1

8.1

1.7 / 2.5

1.00±0.22

61.9±11.3 n=3

10.0

2.1 / 3.6

a

See footnotes from Table 1 for experimental details.

We previously reported that a pyridopiperazine moiety, such as 12 (Table 3), that spans a flat, lipophilic channel leading to a partially solvent exposed region of the protein (“H2 pocket”), optionally as the N-oxetanyl derivative, displayed improved kinetic solubility and pharmacokinetic properties.22 With a preference for compounds with a lower pKa, we next set


12

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out to examine the SAR around the H2 region of our inhibitors. Moving the oxetanylpiperazine ortho to the pyridyl nitrogen (example 16) was tolerated with respect to Btk inhibition (Ki maintained relative to 12), but resulted in a significant drop in whole blood potency and metabolic stability (Table 3). Structural modeling suggested that substitution at the other ring positions would not be tolerated, so our subsequent efforts focused on 2,5-disubstituted pyridyl inhibitors. Replacement of the oxetane with an acyl group, either as the piperazine (17) or the bis-azetidine isostere thereof (18), was tolerated, but offered no advantage. Similarly, while the introduction of other substituents that allowed for modulation of the piperazine nitrogen pKa (1922) did not result in a significant loss in potency (with the exception of 21, with a whole blood potency of 116 nM), each of these suffered from decreased metabolic stability in human liver microsomes (CLhep of 12-16 mL/min/kg) relative to 12. A close-in analogue of 12 was prepared in the form of oxetanylazetidine 23. While 23 maintained much of the Btk affinity and whole blood potency relative to 12, it suffered from poor metabolic stability and a higher measured logD value and, therefore, was not advanced.

Table 3. Piperazine replacements.a


13

R2

BTK Ki (nM)

WB CD69 IC50 (nM)

HLM CLhep (mL/mi n/kg)


MDCK AB:ERb

H

1.80±0.56

15.4±8.2 n=5

7.2

1.8 / 1.9

15.7 / 1.8

1.80±0.08

240.9 n=1

17

2.1 / 3.5

-/-

H

1.80±0.44

66.5 n=1

8.8

1.7 / 2.9

11.1 / 1.2

H

3.30±0.55

33.4 n=1

10.0

1.5 / 2.5

-/-

H

1.20±0.28

36.6±22.2 n=2

12.0

1.9 / 3.2

12.5 / 1.0

H

1.00±0.27

32.2 n=1

12.0

H

4.00±1.60

116.1 n=1

16.0

3.4 / 4.4

14.2 / 0.6

H

0.45±0.08

31.6 n=1

14.0

2.3 / 3.0

-/-

H

0.43±0.15

23.5 n=1

16.0

1.6 / 3.0

48.5 / 0.4


cmpd 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

R1

12

16

H

17

18

19

20

21

22

23

2.3 / 3.3

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9 / 1.3

a

See footnotes from Table 1. b Permeability in Madin-Darby Canine Kidney (MDCKI) cells, Apical-to-Basolateral (AB); Efflux Ratio (ER).


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One aspect of the piperazine SAR that remained relatively unexplored was substitution of the piperazine ring itself. Modeling suggested that we might be able to increase van der Waals contacts with the edge of the protein and/or perhaps influence the conformation of the ring itself using small alkyl substituents, e.g., methyl (Figure 4A). We subsequently synthesized and tested a set of such compounds, with a methyl group in the four possible positions on the ring, using available intermediates containing the F-aryl linker motif (Table 4). Figure 4. Solvent exposed surface of Btk around the oxetanylpiperazine motifs: (A) Model of oxetanylpiperazine.22 (B) From the X-ray co-crystal structure of 29 bound to Btk (see Figure 5).

(A)

(B)


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In each case whole blood potency improved without any negative impact on other properties. Most significantly, in the case of (S)-2,4-dimethyl-1-(pyridin-3-yl)piperazine 26 (as in an X-ray co-crystal structure of 29, Figure 4B), we observed a 5-fold improvement in CD69 whole blood potency relative to 24. Since this particular example had a higher logD and pKa than desired (per our in silico model for prediction of human hepatotoxicity), we applied this change to compound 12.

Table 4. SAR for piperazine ring substitution. a

cmpd 24

25

26

Piperazine

BTK Ki (nM)a

WB CD69 IC50 (nM)

HLM CLhep (mL/min/kg)


0.35±0.11

27.6±19.9 n=7

15.0

2.5 / 3.1

0.37±0.13

16.9 n=1

13.0

2.9 / 3.1

0.65±0.28

4.6±3.5 n=4

14.0

2.9 / 3.2


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27

28

a

0.32±0.01

27.5±0.6 n=2

13.0

2.9 / 2.3

0.43±0.23

12.7±4.5 n=4

13.0

2.9 / 3.3

See footnotes from Table 1.

We chose to transfer the (S)-methyl group of 26 onto 12, which afforded a compound with an overall excellent profile. This resulting compound, GDC-0853, 29 (Table 5), had a ~2-fold improvement in whole blood B cell CD69 potency (IC50 = 8.4 nM) relative to 12 (IC50=15.4 nM) and greater than 4-fold relative to 6 (IC50 = 36.5 nM). The corresponding enantiomer of 29 (30) suffered from poor metabolic stability, and had whole blood potency of only 40 nM. Increasing the size of the substituent to an ethyl group (31) resulted in an expected logD increase and eroded stability in human liver microsomes and hepatocytes. Adding an additional methyl to the ring as in 32 gave a similar result, albeit with excellent whole blood potency.

Finally,

incorporation of nitrogen atoms as in hinge motif pyridazinone 33 and H2 pyrazine 34 yielded no further benefit. Compound 29 exhibited the best overall profile of any compound prepared, and as such was further characterized. When tested for cytotoxicity in primary human hepatocytes, 29 had a clean profile with an IC50 > 300 µM, in contrast to 6, which had an IC50 of 39 µM.

Table 5. Combination of core aryl ring, H3, piperazine, and late stage SAR.a


17


Cmpd

R1

29, GDC0853

30

31

32

33

34 a

R2

X

Y

Btk Ki (nM)a

WB CD69 IC50 (nM)

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(mL/min/kg)


9.4 / 7.6

2.2 / 1.6

HLM / HH CLhep

MDCK AB:ER

OH

CH

CH

0.91±0.34

8.4±5.6 n=5

OH

CH

CH

1.80±0.30

40.0±15.5 n=4

15.0 / 14.0

2.2 / 2.8

7.8 / 0.8

OH

CH

CH

0.96±0.21

15.9 n=1

12.0 / 16.0

2.7/ 3.6

9.5 / 0.9

OH

CH

CH

1.30±0.65

10.2±1.5 n=1

12.0 / 12.0

2.7 / 3.5

7.2 / 0.9

OH

N

CH

-

16.1±0.7 n=1

11.0 / 13.0

2.3 / 3.7

17.3 /0.9

OH

CH

N

0.8 n=1

51.5 n=1

12.0 / 10.0

1/4 / -

15.1 / 0.9

15.6 / 1.2

See footnotes from Tables 1 & 3.

We obtained a 1.59-Å resolution X-ray co-crystal structure of GDC-0853 (29) bound to Btk (Figure 5). Within the Btk kinase domain, 29 shows the expected hinge binding by the central


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pyridone, with hydrogen bonding between the pyridone carbonyl and pendant aniline NH hydrogen and the protein backbone atoms of Met477. Consistent with other previously solved crystal structures with earlier molecules, Tyr551 in the activation loop has moved 18Å from its position in apo-Btk to pack against the hydrophobic right hand side of the tricyclic ring system, forming part of the H3 site that confers substantial potency and kinase selectivity. Additionally, the gem-dimethyl cyclopentane presents nonplanar structure that complements the shape of the pocket in this area, in a similar vein to tert-butyl analogues. The hydoxymethyl projects into an interior water-filled cavity, forming hydrogen bonds with Lys430 and Asp539. The pyridyl ring occupies part of the active site cleft and packs against Gly480 and Leu408 while its nitrogen atom accepts a non-traditional H-bond from an aromatic hydrogen on the adjacent pyridone ring, contributing to the molecule’s rigidity. The 2-Me substituent on the piperidine ring imparts a restricted rotation relative to the unsubstituted analogue, favoring a more out-of-plane orientation of the piperidine ring. Energetics from a quantum torsion scan of the piperazine-to-pyridyl bond support this observation (Supporting Information). The unsubstituted piperazine is calculated to have relatively free rotation about this bond, while the 2-Me substituent on the piperazine ring imparts a more restricted rotation.

The conformation observed in the structure of 29 lies

essentially at the calculated global minimum. Thus, in addition to being conformationally rigid, it presents a more non-planar motif at each end of its structure, which aids in conferring favorable physiochemical, cellular potency, and ultimately pharmacokinetic properties. Detailed physicochemical properties of 29 are included in the Supplementary Information. The most stable known crystalline form has a very high melting point of 274 °C, which contributes to the relatively low water solubility of 0.003 mg/mL. When protonated in 0.1 N HCl, aqueous


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solubility jumps to 35.9 mg/mL, affording excellent solubility and dissolution in the, typically acidic, mammalian stomach. Figure 5. X-ray co-crystal structure of the 29/Btk kinase domain complex (PDB 5VFI) with key residues labeled. The molecular surface of 29 is in blue, and protein-ligand H-bonds are shown as dashed lines.


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Pre-clinical PK Profile We assessed the in vivo pharmacokinetic properties of 29 (Table 6) in cassette studies in both rats (0.2 mg/kg IV and 1.0 mg/kg PO) and dogs (0.2 mg/kg IV and 0.5 mg/kg PO). In rats, 29 had moderate clearance of 27.4 mL/min/kg and excellent bioavailability (F=65%) relative to the des-methyl analogue 12. The plasma clearance was reduced 6-fold, from 165 to 27.4 mL/min/kg, while the volume of distribution (Vd) and oral bioavailability were relatively unchanged, which resulted in a 5.5-fold improvement in plasma half-life from 0.4 to 2.2 hours. Because efficacy in the collagen-induced arthritis model in rat likely requires chronic inhibition of Btk throughout the study,30 this improvement enabled adequate exposures following oral dosing such that preclinical efficacy could be assessed. Compound 29 also demonstrated favorable PK properties in dogs. The 3.8-hour half-life (Clp 10.9 mL/min/kg, Vd 2.96 L/kg) and high oral bioavailability (85%) also enabled attainment of sufficient exposures in dog toxicology studies. Overall, these enhancements in the in vivo pharmacokinetics provided adequate exposures to enable rat and dog pre-clinical toxicology studies.


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Table 6: Rat pharmacokinetic parameters estimated from single-dose intravenous (0.2 mg/kg) and oral (1 mg/kg) cassette pharmacokinetic study. 29 12 (GDC-0853) CL (mL/min/kg)

165

27.4

Vd (L/kg)

4.97

5.42

t1/2 (h)

0.4

2.2

F (%)

65.8

65.1

Table 7: In Vitro Safety Profile of 29 (GDC-0853). a Assay

Result

Receptor Binding Profile (41 targets)

No binding > 50% at 10 µM

hERG Channel Inhibition (Functional)

IC50 > 30 µM (>1,500-fold window)

hNav1.5 Channel Inhibition (Functional)

0.6% at 1 µM, 1.9% at 10 µM

hCav1.2 Channel Inhibition (Functional)

1.5% at 1 µM, 3.8% at 10 µM

Ames

Negative

MNT (HPBLs)

Negative

Cytotoxicity in Human Hepatocytes

IC50 > 300 µM

CYP 3A4, 1A2, 2C19, 2D6, 2D9 Inhibition

IC50s > 5 µM

a

See Supporting Information for assay methods and details.


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29 was tested for off-target activity against a panel of in vitro safety assays and was found to have a clean off-target profile (Table 7), was negative in the in vitro Ames and MNT assays, indicating a low risk for genotoxicity, and has low potential cardiovascular risk liability as indicated in the in vitro hERG assay (IC50 > 30 µM). Like other structurally related molecules that bind in the H3 region of Btk, 29 is an extremely selective Btk inhibitor. When compared with six other clinical stage Btk inhibitors, each tested at 1 µM against a broad panel of human kinase biochemical assays, 29 was found to be the most Btk-selective molecule and inhibited only 3 of 286 off-target kinases (Figure 6a). Based on the determined IC50 values, the selectivity for Btk was >100-fold against each of these 3 off-targets: Bmx (153-fold), Fgr (168-fold), and Src (131-fold) (Figure 6b and Supporting Information). In contrast, BMS-98614223,51 showed >50% inhibition of 20 of 219 off-target kinases tested and ibrutinib (1) inhibited 31 of 221 off-target kinases by >50% (Figure 6b and Supporting Information). The IC50 values (Figure 6b and Supporting Information) demonstrate that ibrutinib is rather non-selective, being 80-fold higher than the targeted efficacious exposure, i.e., exceeding for 12 hours the IC70 concentration (from the human whole blood CD69 assay). In Sprague-Dawley rats, 29 and other structurally distinct Btk inhibitors have been shown to be associated with islet-centric pancreatic lesions at clinically relevant doses41. After a thorough investigation involving evaluation of strain and species sensitivity differences, Btk knockout (KO) mice, and literature reports of humans with XLA mutations, we concluded that the GDC-0853-related pancreas findings in the Sprague-Dawley strain were the result of a rat-specific, strain-variable, on-target effect of Btk inhibition that is not relevant for humans.41 These conclusions have been supported by a histologic evaluation of the pancreas of untreated Btk KO Sprague-Dawley rats, that demonstrated the presence of identical pancreatic pathology.57 With a favorable safety profile and evidence that the observed pancreatic toxicity was a rat-specific phenomenon, we selected 29 as our lead candidate for clinical development.

GDC-0853: Tackling Btk Mutations in Oncology


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We have previously reported that our inhibitors bind to Btk in an orientation that is essentially orthogonal to that of the reported clinical covalent inhibitors. While our inhibitors do bind the hinge region, they do not encroach or interact with either the gatekeeper T474 or the C481 residue that is responsible for covalent interaction with Btk (Figure 10).50

Figure 10. Superposition of the X-ray structures of 29 (GDC-0853) (cyan) and 1 (ibrutinib)58 (purple) bound in the kinase domain of Btk. The orthogonal binding modes of these inhibitors are apparent: 1 (ibrutinib) occupies the kinase back pocket behind the gatekeeper T474 and also interacts with C481; 29 does neither.

We hypothesized that if drug resistance appeared in patients being treated with ibrutinib, our unique inhibitors could retain activity against certain mutant forms of Btk. Thus, we proactively generated 9 potential Btk mutant proteins and tested the potency of 29 against them. We have previously reported that our Btk inhibitors retain activity against clinically occurring mutant


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forms of Btk, most notably the C481S active site mutation.50 We extended these studies to 29 and assessed the effect of it and 1 (ibrutinib) on Y223 phosphorylation of wild type Btk and of the C481S mutant in HEK293T cells transiently expressing these proteins.50 Whereas no Btk protein was detected in non-transfected cells (Figure 11a, lane 1), transient transfection with WT Btk or Btk C481S-encoding plasmids (lanes 2 and 3, respectively) yielded similar total Btk protein levels and pY223 tyrosine phosphorylation levels as assessed by western blotting with anti-total or anti-pY223 Btk antibodies, In cells that were transfected with either WT Btk or Btk C481S plasmids and treated with vehicle alone, robust levels of pY223 were observed for both WT (lane 1) and C481S (lane 4) mutant Btk. Treatment of WT Btk-transfected cells with 1 µM of either 1 (Figure 11b, lane 2) or 29 (lane 3) ablated Y223 phosphorylation. In contrast, in C481S Btk-transfected cells, treatment with 1 µM 1 failed to block autophosphorylation of the Btk C481S protein on Y223 (Figure 10b, lane 5), whereas 1 µΜ GDC-0853 fully inhibited Y223 autophosphorylation of the Btk-C481S protein (lane 6). Likewise, in biochemical testing 29 maintained its inhibitory activity against four Btk enzyme mutants at either the active site C481 or the gatekeeper threonine residues: C481S, C481R, T474I, and T474M (Table 10). Similarly, six of our other H3-binding Btk inhibitors also showed equivalent inhibitory activity against WT Btk and the C481S and T474M mutants, while being slightly less active against the T474I mutant (Table S-1, Supporting Information).

Figure 11. 29 (GDC-0853) blocked cellular autophosphorylation of WT Btk and the C481S mutant. a) Btk protein levels and Btk Y223 autophosphorylation was assessed by western blotting, with anti-total or anti-pY223 Btk antibodies, respectively, of HEK293T cells transiently transfected with plasmids encoding WT (lane 2) or C481S Btk (lane 3) or non-transfected cells


36

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(lane 1). b) 1 and 29 both blocked cellular autophosphorylation of Y223 in WT Btk. However, only 29 was able to inhibit cellular autophosphorylation of Y223 of the C481S Btk mutant protein. Ibrutinib (1) was unable to block cellular Y223 autophosphorylation of Btk C481S. The effect of 29 or 1 (ibrutinib) on WT Btk and Btk-C481S mutant phosphorylation on Y223 was assessed and quantified as previously described.50

a)

Btk transfected 1

2

3

pY223 Btk

total Btk

b)

1 (Ibrutinib) -

+

-

-

+

-

29 (GDC-0853) -

-

+

-

-

+

Btk

WT 1

2

C481S 3

4

5

6

pY223 Btk

total Btk


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Table 10. Biochemical potency of 29 against WT Btk and the C481S, C481R, T474I, and T474M mutants. Each Ki is the mean ± SD of at least three inhibitor titrations.

Inhibitor

29, GDC-0853

Ki (nM) against Btk enzyme form WT

C481S

C481R

T474I

T474M

0.91 ± 0.34

1.6 ± 0.9

1.3 ± 0.6

12.6 ± 4.8

3.4 ± 0.6

In 2013, Chang and coworkers reported the first cases of NHL or CLL patients relapsing on ibrutinib.59 Genomic sequencing of these lymphomas showed that the BTKC481S mutation was prevalent in these patients. Because 29 has the potential to treat ibrutinib relapsed patients harboring the BTKC481S mutation, we are currently conducting a first-in-human Phase 1 study in patients with relapsed or refractory B cell NHL and CLL that had relapsed on ibrutinib, and in particular those that were identified to be BTKC481S positive. Initial results of this study will be reported shortly.

Synthesis of GDC-0853 (29) GDC-0853 (29) was synthesized in a highly convergent manner as shown in Schemes 1 and 2. Scheme 1 shows the synthesis of the left portion of the molecule, intermediate 41. Wittig olefination of chlorovinyl aldehyde 3560 gave olefin intermediate 36. Treatment with sodium azide and subsequent heating gave rise to pyrrole 37 following denitrogenative cyclization via the nitrene. The tricylic lactam 40 was then accessed via a three-step sequence: N-alkylation with bromoacetonitrile, nitrile reduction, and base-mediated saponification/lactam cyclization. Finally, palladium catalyzed C-N coupling gave the desired H3-linker intermediate 41 via


38

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coupling with bromo-pyridine aldehyde 42 in the presence of Pd2(dba)3 and xantphos in 32% yield.

Scheme 1. Synthesis of 29 part 1. Synthesis of intermediate 42a

a

Reagents and conditions: (a) Ethoxycarbonylmethylene triphenylphosphoran, benzene, 14h,

37%; (b) NaN3, DMSO, 75 °C, 8h, 37%; (c) NaH, DMF, bromoacetonitrile, 14h, 95%; (d) 10% Pd/C, 12% HCl in EtOH/EtOAc, H2 (50 psi), 6h; (e) NaOEt, EtOH, 55 °C, overnight, 61%, 2 steps; (f) 2-bromo-4-chloronicotinaldehyde (42), Pd2(dba)3, Xantphos, Cs2CO3, dioxane, 100 °C, 5h, 63%.

The synthesis of 29 was completed as shown in Scheme 2. The H2 motif was assembled via two C-N coupling reactions, first between bromopyridine 4322 and Boc-protected piperazine 44 to afford intermediate 45. Subsequent nitro reduction (to 46) set up the second coupling, this time with 3,5-dibromo-1-methylpyridin-2(1H)-one to give 47.

Boc deprotection followed by

reductive amination with oxetan-3-one in the presence of ZnCl2 afforded the fully-formed right hand side of the molecule in the form of 49. Borylation of 49 with bis(pinacolato)diboron set up


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the penultimate step of the synthesis, Suzuki coupling with H3-linker aldehyde 41 to give 51 in 31% yield. Finally, aldehyde reduction of 51 with NaBH4 gave GDC-0853, 29. Analogues 7 – 10, 12 – 15, and 30 – 32 were prepared in a similar manner using the appropriate left hand moiety and H2 motif (Supporting Information). The synthetic details of additional intermediates and characterization details are provided in the Experimental and Supporting Information sections. Scheme 2. Synthesis of 29 part 2. a


40

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a

Reagents and conditions: (a) Pd2(dba)3, BINAP, Cs2CO3, dioxane, 100 °C, 15 h, 63%; (b) H2,

Pd/C, MeOH, 16 h, 93%; (c) 3,5-dibromo-1-methylpyridin-2(1H)-one, Pd2(dba)3, Xantphos, Cs2CO3, dioxane, 100 °C, 16 h, 61%; (d) HCl in dioxane, DCM, 4 h, 95%; (e) oxetan-3-one, NaBH3CN, ZnCl2, MeOH, 50 °C, 4 h, 73%; (f) bis(pinacolato)diboron, Pd2(dba)3, Xphos, KOAc, dioxane, 70 °C, 2 h, 90%; (g) Pd(dppf)Cl2, K3PO4, THF, H2O, reflux, 3 h, 31%; (h) NaBH4, MeOH, 1 h, 44%.


41


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Conclusion and Clinical Evaluation in Immunology Indications. In summary, we have discovered a potential best-in-class Btk inhibitor, GDC-0853 (29), that is currently in clinical investigation for several immune disorders. It is highly potent, and is the most selective Btk inhibitor reported to date. Its pre-clinical pharmacokinetic characteristics are favorable, indicating the potential for QD oral dosing. In addition, the supporting efficacy data reported here suggest that it could have utility in treating rheumatoid arthritis and other B-cell or myeloid cell mediated autoimmune diseases. These findings, combined with a very desirable tolerability and safety profile in multiple species, encouraged us to progress 29 into clinical studies in autoimmune diseases. In a single ascending dose (SAD) study (0.5 mg to 600 mg) and multiple ascending dose (MAD) study for 14 days (250 mg BID to 500 mg QD) in healthy volunteers, GDC-0853 was very well tolerated with no severe adverse events, no safety signals, and no dose limiting toxicities. Additionally, 29 was well absorbed and had linear, doseproportional PK.

Target engagement (CD63, CD69, pBTK) was assessed and complete

suppression of PD markers was maintained over 24 hours. With favorable Phase 1 results, 29 entered Phase 2 clinical studies in rheumatoid arthritis, lupus, and chronic urticaria.61-63 Further details and clinical results will be reported in due course.

EXPERIMENTAL SECTION All chemicals were purchased from commercial suppliers and used as received. 1H NMR spectra were recorded on Bruker Avance 400 or 500 spectrometers. Chemical shifts are expressed in δ ppm referenced to an internal standard, tetramethylsilane (δ = 0 ppm). Abbreviations used in describing peak signals are: br = broad signal, s = singlet, d = doublet, dd = doublet of doublets, t = triplet, q = quartet, m = multiplet. All final compounds were purified to have purity higher than 95% by reverse phase high performance liquid chromatography (HPLC), supercritical fluid chromatography (SFC) or normal phase silica gel flash chromatography. The


42

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purity was assessed by reverse phase HPLC with an isocratic gradient of 5-95% acetonitrile in water (with either acid or base modifier) and monitored by diode array ultraviolet detection at 254 nm. Low-resolution mass spectra were recorded on a liquid chromatography-mass spectrometer in electrospray positive (ES+) mode. HRMS experiments were performed on Dionex LC Ultimate3000 coupled with ThermoScientific Q Exactive orbitrap mass spectrometer using ESI as ionization source and a Phenomenex XB-C18, 1.7mm, 50 × 2.1 mm column with a 0.7 mL / min flow rate at 40 °C for LC separation. Solvent A was 0.1% FA in water and solvent B was 0.1% FA in acetonitrile. The gradient consisted of 2 - 98% solvent B over 7 min and hold 98% B for 1.5 min following equilibration for 1.0 min. The LC was monitored by uv absorbance at 220 nm and 254 nm. MS full scan with 10,000 resolution was applied to all experiments.

(S)-2-(3'-(hydroxymethyl)-1-methyl-5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin2-yl)amino)-6-oxo-1,6-dihydro-[3,4'-bipyridin]-2'-yl)-7,7-dimethyl-3,4,7,8-tetrahydro-2Hcyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one (29). Step 1: A 100-mL single-neck round-bottomed flask equipped with a magnetic stirrer and a reflux condenser was charged with 49 (1.0 g, 2.3 mmol), Pin2B2 (1.4 g, 5.7 mmol), Pd2(dba)3 (105 mg, 0.13 mmol), XPhos (93 mg, 0.23 mmol), KOAc (680 mg, 6.9 mmol), and dioxane (50 mL). After three vacuum purge /argon fill cycles, the mixture was heated to 70 ºC and stirred for 2 h. It was then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was washed with 3:1 petroleum ether / EtOAc (80 mL) to obtain the boronate ester 50 as yellow solid (1.0 g, 90% yield) without further purification. [ES-MS] (ESI+): m/z calcd for C25H37BN5O4 [M + H]+, 482; found, 482. Step 2: A 50-mL single-neck round-bottomed flask equipped with a magnetic stirrer and a reflux condenser was charged with 50 (420 mg, 0.44 mmol), 41 (200 mg, 0.88 mmol), Pd(dppf)Cl2 (36 mg, 0.044 mmol), K3PO4 (279 mg, 1.32 mmol), and THF (20 mL). After three vacuum purge /argon fill cycles, the mixture was heated at reflux for 3 h. It was then cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure and the resulting residue was washed with 3:1 petroleum ether / EtOAc (80 mL) to give (S)-2'-(7,7dimethyl-1-oxo-1,3,4,6,7,8-hexahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-2-yl)-1-methyl5-((5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)amino)-6-oxo-1,6-dihydro-[3,4'-


43


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bipyridine]-3'-carbaldehyde 51 (90 mg, 31% yield) as a solid without further purification. [ESMS] (ESI+): m/z calcd for C37H43N8O4 [M + H]+, 663; found, 663. Step 3: A 50-mL single-neck round-bottomed flask equipped with a magnetic stirrer was charged with 51 (90 mg, 0.11 mmol), NaBH4 (13 mg, 0.33 mmol), and MeOH (10 mL). The mixture was stirred at room temperature for 1 h. It was then filtered and the filtrate concentrated under reduced pressure. The residue was purified by reverse-phase prep-HPLC to afford 40 mg of the title compound 29 as a yellow solid. Yield: 44%. 1H NMR (400 MHz, DMSO-d6) δ 8.62 (d, J = 2.4 Hz, 1H), 8.48 (d, J = 5.0 Hz, 1H), 8.43 (s, 1H), 7.83 (d, J = 2.9 Hz, 1H), 7.47 (d, J = 2.4 Hz, 1H), 7.41 – 7.31 (m, 2H), 7.24 (d, J = 9.0 Hz, 1H), 6.56 (s, 1H), 4.96 (t, J = 5.2 Hz, 1H), 4.59 – 4.52 (m, 2H), 4.51 – 4.35 (m, 4H), 4.29 – 4.15 (m, 3H), 3.89 – 3.80 (m, 1H), 3.72 – 3.63 (m, 1H), 3.60 (s, 3H), 3.44 – 3.35 (m, 1H), 3.14 – 3.04 (m, 1H), 2.99 – 2.90 (m, 1H), 2.65 – 2.52 (m, 3H), 2.43 (s, 2H), 2.39 – 2.23 (m, 2H), 2.24 – 2.14 (m, 1H), 1.22 (s, 6H), 0.93 (d, J = 6.3 Hz, 3H).

13

C NMR (101 MHz, DMSO) δ 159.75, 157.20, 155.30, 149.47, 149.19, 148.36, 141.31,

140.13, 137.08, 131.36, 131.21, 129.11, 127.81, 126.27, 126.00, 124.20, 116.83, 116.26, 113.47, 109.29, 74.93, 74.80, 58.87, 57.65, 55.63, 51.23, 49.83, 48.33, 46.02, 46.01, 42.41, 41.19, 39.44, 38.01, 30.64, 30.52, 14.03. HRMS (ESI+): m/z calcd for C37H45N8O4 [M + H]+, 665.3564; found, m/z 665.3550

7,7-dimethyl-3,4,7,8-tetrahydro-2H-cyclopenta[4,5]pyrrolo[1,2-a]pyrazin-1(6H)-one (40). Step 1: A 500-mL single neck round bottomed flask equipped with a magnetic stirrer and nitrogen inlet was charged with 2-chloro-4,4-dimethylcyclopent-1-enecarbaldehyde (38 g, 240 mmol) in benzene (240 mL). To the solution was added ethoxycarbonylmethylene triphenylphosphorane (84 g, 240 mmol, 1 equiv.). The mixture was stirred for 14 h. After that time, the solvent was evaporated and the residue was triturated with hexanes (2L) to extract the product away from the triphenylphosphine by-products. The organic layer was dried over sodium sulfate and concentrated in vacuo. The residue was purified by column chromatography (SiO2: ethyl acetate / hexane) to afford (E)-ethyl 3-(2-chloro-4,4-dimethylcyclopent-1-enyl)acrylate 36 (20 g, 37% yield). Step 2: A 250-mL single neck round bottomed flask equipped with a magnetic stirrer and nitrogen inlet was charged with 36 (17 g, 74 mmol) in DMSO (100 mL). To the solution was


44

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added sodium azide (9.6 g, 150 mmol, 2 equiv.). The mixture was then heated to 75 °C and stirred for 8 h. After cooling to room temperature, H2O (100 mL) and DCM (200 mL) were added and the organic layer was separated. The aqueous layer was extracted with DCM (50 mL). The combined organic layers were washed with brine, dried over sodium sulfate and concentrated in vacuo. The residue was purified by column chromatography (SiO2: ethyl acetate / hexane) to afford ethyl 5,5-dimethyl-1,4,5,6-tetrahydrocyclopenta[b]pyrrole-2-carboxylate 37 (5.7 g, 37% yield). Step 3: A 250-mL single neck round bottomed flask equipped with a magnetic stirrer and nitrogen inlet was charged with 37 (6.2 g, 30 mmol) in DMF (57 mL). To the solution was added NaH (80% dispersion in mineral oil, 1.3 g, 42 mmol, 1.4 equiv.). The resulting mixture was stirred at room temperature for 90 min. After that time, bromoacetonitrile (2.9 mL, 42 mmol, 1.4 equiv.) was added. The mixture was stirred for 14 h. After that time, water (100 mL) and ethyl acetate (200 mL) were added and the organic layer was separated. The aqueous layer was extracted with ethyl acetate (2 x 50 mL). The combined organic layers were washed with brine, dried over sodium sulfate and concentrated in vacuo. The residue was purified by column chromatography (SiO2: ethyl acetate / hexane) to yield ethyl 1-(cyanomethyl)-5,5-dimethyl1,4,5,6-tetrahydrocyclopenta[b]pyrrole-2-carboxylate 38 (7 g, 95% yield). Step 4: A 500-mL Parr reactor bottle was purged with nitrogen and charged with 10% palladium on carbon (50% wet, 2 g dry weight), 38 (4.5 g, 18 mmol), 12% hydrochloric acid (9.2 mL, 37 mmol) in ethyl acetate (80 mL) and ethanol (52 mL). The bottle was attached to a Parr hydrogenator, evacuated, charged with hydrogen gas to a pressure of 50 psi and shaken for 6 h. After this time, the hydrogen was evacuated, and nitrogen was charged into the bottle. CELITE® (10.0 g) was added and the mixture was filtered through a pad of CELITE®. The filter cake was washed with ethanol (2 x 50 mL), and the combined filtrates were concentrated to dryness under reduced

pressure.

The

crude

residue

ethyl

1-(2-aminoethyl)-5,5-dimethyl-1,4,5,6-

tetrahydrocyclo-penta[b]pyrrole-2-carboxylate hydrochloride 39 was carried onto the next step without further purification. Step 5: A 100-mL single-neck round-bottomed flask equipped with a magnetic stirrer and nitrogen inlet was purged with nitrogen and charged with crude 39 (~18 mmol), sodium ethoxide (6.2 g, 92 mmol) and ethanol (120 mL). The mixture was stirred at 55 °C overnight. After that time, the reaction mixture was concentrated under reduced pressure and the residue was


45


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partitioned between ethyl acetate (200 mL) and water (100 mL). The solution was filtered. The solid was washed with ethyl acetate (15 mL) to give 850 mg of desired product 40. The organic layer was then separated, and the aqueous layer extracted with ethyl acetate (2 x 100 mL). The combined organic layers were dried over sodium sulfate and concentrated under reduced pressure to near dryness. The solution was filtered and the additional solid 40 (1.44 g) was washed with ethyl acetate (15 mL). The combined solids were dried under vacuum to yield the title compound 40 as a yellow solid (2.3 g, 61% yield). 1H NMR (500 MHz, CDCl3) δ 6.70 (s, 1H), 5.82 (s, 1H), 3.96 – 3.93 (m, 2H), 3.65 (s, 2H), 2.50 (s, 2H), 2.47 (s, 2H), 1.23 (s, 6H); [ESMS] (ESI+): m/z calcd for C12H17N2O [M + H]+, 205.1; found, 205.

4-chloro-2-(7,7-dimethyl-1-oxo-1,3,4,6,7,8-hexahydro-2H-cyclopenta[4,5]pyrrolo[1,2a]pyrazin-2-yl)nicotinaldehyde (41). A flask equipped with a magnetic stirrer and a reflux condenser was charged with 2bromo-4-chloronicotinaldehyde (25.0 g, 115 mmol), 40 (24.5 g, 120.0 mmol), Pd2(dba)3 (9.2 mg, 10.0 mmol), XantPhos (5.7 g, 10 mmol), Cs2CO3 (74.7 g, 230.0 mmol), and 1,4-dioxane (1 L). After three cycles of vacuum/argon flush, the mixture was heated at 90 °C for 5 h. It was then cooled to room temperature and filtered, then partitioned between ethyl acetate (500 mL) and water (100 mL). The aqueous layer was separated and extracted with ethyl acetate (500 mL × 3). The combined organic layers were washed with brine (500 mL) and dried over sodium sulfate, filtered and concentrated. The residue was purified by silica gel chromatography (DCM/MeOH = 40/1) to give the title compound (25 g, 63% yield). 1H NMR (400 MHz, DMSO-d6) δ 9.84 (s, 1H), 8.58 (d, J = 5.5 Hz, 1H), 7.53 (d, J = 5.4 Hz, 1H), 6.61 (s, 1H), 4.30 – 4.17 (m, 4H), 2.58 (s, 2H), 2.42 (s, 2H), 1.21 (s, 6H); [ES-MS] (ESI+): m/z calcd for C18H18ClN3O2 [M + H]+, 344.1; found, 344.0. (3S)-tert-butyl-4-(6-aminopyridin-3-yl)-3-methylpiperazine-1-carboxylate (46). Step 1: A 3-L single-neck round-bottomed flask equipped with a magnetic stirrer and reflux condenser was charged with 5-bromo-2-nitropyridine (100 g, 0.49 mol), (S)-tert-butyl 3methylpiperazine-1-carboxylate (99.5 g, 0.49 mol), cesium carbonate (332 g, 1 mol), and 1,4dioxane (1.5 L). After bubbling nitrogen through the resulting solution for 30 min, BINAP (24 g, 0.039 mmol) and Pd2(dba)3 (35.6 g, 0.039 mmol) were added, and the reaction mixture was stirred at 100 °C for 15 h. After this time the reaction was cooled to room temperature,


46

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partitioned between ethyl acetate (1 L) and water (300 mL), and filtered. The aqueous layer was separated and extracted with ethyl acetate (3 × 500 mL). The combined organic layer was washed with brine (500 mL) and dried over sodium sulfate. The drying agent was removed by filtration and the filtrate was concentrated under reduced pressure. The residue was purified on flash column chromatography (SiO2: petroleum ether/ethyl acetate) to give (3S)-tert-butyl 3methyl-4-(6-nitropyridin-3-yl)piperazine-1-carboxylate 45 (100 g, 63% yield). [ES-MS] (ESI+): m/z calcd for C15H23N4O4, [M + H]+, 323; found, 323. Step 2: A 2-L flask was purged with nitrogen and charged with 45 (100 g, 0.31 mol), 10% palladium on carbon (50% wet, 8 g) and ethanol (1 L). It was then evacuated, charged with hydrogen gas, and stirred at room temperature for 16 h. Hydrogen was evacuated and nitrogen was charged into the flask. The catalyst was removed by filtration through a pad of Celite® and the filtrate was concentrated under reduced pressure to give the title compound 46 (85 g, 93% yield). [ES-MS] (ESI+): m/z calcd for C15H25N4O2 [M + H]+, 293; found, 293. (3S)-5-bromo-1-methyl-3-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridine-2ylamino)pyridin-2(1H)-one (49). Step 1: A 2-L single-neck round-bottomed flask equipped with a magnetic stirrer and reflux condenser was charged with 1,4-dioxane (1 L), 46 (60 g, 210 mmol), 3,5-dibromo-1methylpyridin-2(1H)-one (56 g, 210 mmol), and cesium carbonate (139 g, 427 mmol). After bubbling nitrogen through the resulting solution for 30 min, Xantphos (9.7 g, 16.8 mmol) and Pd2(dba)3 (15.1 g, 16.8 mmol) were added, and the reaction mixture was heated at reflux for 15 h. After this time the reaction was cooled to room temperature, partitioned between ethyl acetate (500 mL) and water (100 mL), and filtered. The aqueous layer was separated and extracted with ethyl acetate (3 × 500 mL). The combined organic layer was washed with brine (500 mL) and dried over sodium sulfate. The drying agent was removed by filtration and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (SiO2:

DCM

/

MeOH)

to

afford

(3S)-tert-butyl-4-(6-(5-bromo-1-methyl-2-oxo-1,2-

dihydropyridin-3-ylamino)pyridine-3-yl)-3-methylpiperazine-1-carboxylate 47 (60 g, 61%). [ESMS] (ESI+): m/z calcd for C21H29BrN5O3 [M + H]+, 478; found, 478. Step 2: To a solution of 47 (55 g, 115 mmol) in DCM (500 mL) was added 3.0 M HCl in dioxane (150 mL). The reaction mixture was stirred at room temperature for 4 h and filtered. The filtrate was reduced under reduced pressure. The residue was basified with aqueous 1.0 M NaOH


47


and

extracted

with

methylene

chloride

to

obtain

Page 48 of 61

(3S)-5-bromo-1-methyl-3-(5-(2-

methylpiperazin-1-yl)pyridin-2-ylamino)pyridine-2(1H)-one 48 (42 g, 95% yield). [ES-MS] (ESI+): m/z calcd for C16H21BrN5O [M + H]+, 378; found, 378. Step 3: A mixture of 48 (40 g, 106 mmol), oxetan-3-one (11.4 g, 159 mmol), NaBH3CN (10.0 g, 159 mmol), and zinc chloride (21.3 g, 159 mmol) in methanol (700 mL) was stirred at 50 °C for 4 h. The mixture was added to H2O (50 mL) and extracted with DCM three times (200 mL × 3). The combined organic layers were concentrated under reduced pressure. The residue was purified by column chromatography (SiO2: DCM / MeOH) to give the title compound 49 (35 g, 73% yield). 1H NMR (500 MHz, CDCl3) δ 8.60 (d, J = 2.5 Hz, 1 H), 8.03 (d, J = 3.0 Hz, 1 H), 7.80 (s, 1 H), 7.30 (dd, J = 9.0 Hz, J = 2.5 Hz, 1 H), 6.95 (d, J = 2.5 Hz, 1 H), 6.76 (d, J = 8.5 Hz, 1 H), 4.72-4.62 (m, 4 H), 3.60 (s, 3 H), 3.54-3.48 (m, 2 H), 3.09 (t, J = 5.5 Hz, 2 H), 2.562.43 (m, 3 H), 2.25-2.22 (m, 1 H), 1.00 (d, J = 6.0 Hz, 3 H). [ES-MS] (ESI+): m/z calcd for C19H25BrN5O2 [M + H]+, 434; found, 434.

X-ray crystallography data Authors will release the coordinates and experimental data upon article publication, PDB accession code 5VFI AUTHOR INFORMATION Corresponding Author * email: [email protected] (J. J. Crawford); [email protected] (W. B. Young) Present Addresses D.L.M.: Alios BioPharma Inc, 260 East Grand Ave., South San Francisco, CA, 94080. M.C.: Food and Drug Administration (FDA), 10903 New Hampshire Ave, Silver Springs, MD 20903.


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H.W.: Faculty of Pharmaceutical Sciences, University of British Columbia, 2405 Wesbrook Mall, Vancouver, BC, Canada, V6T 1Z3. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources Thanks to Genentech, a member of the Roche Group for research funds. Notes ACKNOWLEDGMENT We would like to thank the members of our synthetic chemistry team at Shanghai Chempartner, and in particular Yu Nan, Deng Wei, Zhou Fusheng, and Shi (Stone) Xiaoyong. We would also like to thank Proteros Biostructures GmbH, who performed crystallographic analysis of BTK/GDC-0853, Rama Pai and Patricia Chang for in vitro cytotoxicity data in primary human hepatocytes, and Sergei Romanov (Nanosyn, Inc.) for assistance with the residence time assay. Finally, we would like to acknowledge the Genentech analytical and purification teams, Seth Harris for assistance with graphics, and Joachim Rudolph for helpful comments and insights.

ABBREVIATIONS ALT, alanine aminotransferase; AST, aspartate aminotransferase; BID, twice a day; Btk, Bruton’s tyrosine kinase; CLL, Chronic lymphocytic leukemia; DMPK, Drug metabolism and pharmacokinetics; MS, Multiple sclerosis; MCL, Mantle cell lymphoma; NHL, Non-Hodgkin


49


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lymphoma; NOAEL, No Observed Adverse Effect Level; PK, pharmacokinetic; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; Vd, Volume of distribution; WT, wild type.

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61. RA: Safety and Efficacy Study of GDC-0853 Compared With Placebo and Adalimumab in Participants With Rheumatoid Arthritis (RA); ClinicalTrials.gov Identifier: NCT02833350 https://clinicaltrials.gov/ct2/show/NCT02833350

62. Lupus: A Study of the Safety and Efficacy of GDC-0853 in Participants With Moderate to Severe Active Systemic Lupus Erythematosus; ClinicalTrials.gov Identifier: NCT02908100 https://clinicaltrials.gov/ct2/show/NCT02908100

63. CSU: Efficacy and Safety of GDC-0853 in Participants With Refractory Chronic Spontaneous

Urticaria

(CSU);

ClinicalTrials.gov

Identifier:

NCT03137069

https://clinicaltrials.gov/ct2/show/NCT03137069


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