Discovery of Potent, Selective, and Orally Bioavailable Small-Molecule Modulators of the Mediator Complex-Associated Kinases CDK8 and CDK19

Article

pubs.acs.org/jmc

Discovery of Potent, Selective, and Orally Bioavailable Small-

Molecule Modulators of the Mediator Complex-Associated Kinases

CDK8 and CDK19

Aurelie Mallinger,*^,†Kai Schiemann,^‡Christian Rink,^†Frank Stieber,^‡Michel Calderini,^‡

́

Simon Crumpler,^†Mark Stubbs,^†Olajumoke Adeniji-Popoola,^†Oliver Poeschke,^‡Michael Busch,^‡

Paul Czodrowski,^‡Djordje Musil,^‡Daniel Schwarz,^‡Maria-Jesus Ortiz-Ruiz,^†Richard Schneider,^‡

Ching Thai,^†Melanie Valenti,^†Alexis de Haven Brandon,^†Rosemary Burke,^†Paul Workman,^†

Trevor Dale,^§Dirk Wienke,^‡Paul A. Clarke,^†Christina Esdar,^‡Florence I. Raynaud,^†Suzanne A. Eccles,^†

Felix Rohdich,^‡and Julian Blagg*^,†

^†Cancer Research UK Cancer Therapeutics Unit at The Institute of Cancer Research, London, SW7 3RP, U.K.

^‡Merck KGaA, Darmstadt, 64293, Germany

^§School of Bioscience, Cardiﬀ University, Cardiﬀ, CF10 3AX, U.K.

S

* Supporting Information

ABSTRACT: The Mediator complex-associated cyclin-de-

pendent kinase CDK8 has been implicated in human disease,

particularly in colorectal cancer where it has been reported as a

putative oncogene. Here we report the discovery of 109

(CCT251921), a potent, selective, and orally bioavailable

inhibitor of CDK8 with equipotent aﬃnity for CDK19. We

describe a structure-based design approach leading to the

discovery of a 3,4,5-trisubstituted-2-aminopyridine series and

present the application of physicochemical property analyses

to successfully reduce in vivo metabolic clearance, minimize

transporter-mediated biliary elimination while maintaining

acceptable aqueous solubility. Compound 109 aﬀords the

optimal compromise of in vitro biochemical, pharmacokinetic, and physicochemical properties and is suitable for progression to

animal models of cancer.

down of CDK8 protein reduces the growth of HT29 and

Colo205 colorectal cancer human tumor xenograft animal

models harboring CDK8 gene ampliﬁcation.¹⁴Notably, CDK8

expression transforms NIH3T3 cells into a malignant

phenotype whereas a kinase-dead mutant does not, thereby

implicating the kinase function of CDK8 in oncogenesis.¹⁵The

function and role of CDK19 are less well explored. CDK19 has

been reported to form Mediator complexes independent of

CDK8; however their context-dependent roles are the subject

of ongoing study.⁷

Previously reported small molecule ligands for CDK8 and its

paralog CDK19 have been described in a recent comprehensive

review.¹⁶In brief, the steroidal natural product cortistatin A (1)

was the ﬁrst-reported high aﬃnity and selective ligand for

CDK8/19 (Chart 1);¹⁷recent disclosures include a patent

describing cortistatin A analogs and a report demonstrating

potent in vitro and in vivo antileukemic activity of cortistatin A

through dual CDK8/19 inhibition.^18,19The marketed kinase

INTRODUCTION

■

The Mediator complex is a multiprotein assembly comprising

at least 30 subunits that functions as a regulator of gene

transcription in multiple contexts including stem cell function,

the immune response, inﬂammation, cell adhesion, the

epithelial to mesenchymal transition and development.¹⁻⁵

The Mediator complex-associated kinase CDK8 and its paralog

CDK19 are cyclin C-dependent enzymes that, along with

MED12 and MED13, form the kinase module of the Mediator

complex.^6,7CDK8 has been reported to regulate basal

transcription by phosphorylation of RNA polymerase II⁸and

to phosphorylate E2F1, thereby activating WNT signaling.⁹

Interestingly, CDK8 gene expression correlates with activation

of β-catenin, a core transcriptional regulator of canonical WNT

signaling, in colon and gastric cancers.^10,11CDK8 gene

expression also correlates with increased mortality in colorectal,

breast, and ovarian cancers;¹²furthermore CDK8 is overex-

pressed and essential for cell proliferation in melanoma.¹³

Consistent with these reports, CDK8 is located in a region of

chromosome 13 known to undergo copy number gain in ∼60%

of colorectal cancers and inducible shRNA-mediated knock-

Received: October 29, 2015

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Chart 1. Small Molecule CDK8/19 Ligands: Cortistatin A (1), Sorafenib (2), Linifanib (3), Ponatinib (4), Senexin B (5), and 6

a

Scheme 1. General Synthetic Routes to 3,4,5-Trisubtituted Pyridines

a

The asterisk (∗) indicates diﬀerent solvent and/or slightly diﬀerent conditions were used for compounds 17, 18, 42, 47, 50, and 52.

inhibitor sorafenib (2) has been cocrystallized with CDK8/

cyclin C,²⁰and subsequently the same group reported a

fragment-based approach to CDK8 ligands building from the

urea moiety associated with the type II binding mode of

sorafenib.²¹Type II kinase inhibitors linifanib (3) and

ponatinib (4) have also been reported to bind both CDK8

and CDK19.²²Recently, a cell-based HTS campaign seeking

inhibitors of p21-activated transcription was reported; this

eﬀort led to the discovery of aminoquinazoline-based CDK8/

19 ligands, exempliﬁed by senexin B (5) (Chart 1).²³Other

series of small molecule CDK8 inhibitors have also been

reported in the patent literature.²⁴⁻²⁶

We have previously reported the discovery of 6

(CCT251545), a potent, orally bioavailable small molecule

inhibitor of WNT signaling from a cell-based pathway screen

(Chart 1).²⁷We identiﬁed protein kinases CDK8 and CDK19

as the primary targets of this trisubstituted pyridine series and

demonstrated a strong correlation between CDK8 and CDK19

binding aﬃnities in this chemical series.²²Here we describe the

medicinal chemistry optimization of 6 to compound 109, a

potent, selective, and orally bioavailable inhibitor of CDK8 with

equipotent aﬃnity for CDK19 that demonstrates potent cell-

based activity together with improved pharmacokinetic and

pharmaceutical properties. We demonstrate inhibition of CDK8

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a

Scheme 2. General Synthetic Routes to 2-Aminopyridine Derivatives

a

The asterisk (∗) indicates that KF was added for the synthesis of compounds 76 and 82.

function concomitant with reduced proliferation in a human

tumor xenograft animal model of colorectal cancer.

2);²⁷it also demonstrates potent activity in LS174T human

colorectal carcinoma cells that harbor a reporter-based readout

measuring constitutive β-catenin mutation-driven WNT path-

CHEMISTRY

way activity (IC₅₀= 23

11 nM).²⁷Compound 6 displays

■

moderate in vivo clearance in both mouse and rat; however,

high predicted human clearance (∼76% liver blood ﬂow)

prevented further progression (Table 1). Furthermore, the

The general synthetic method for the preparation of 3-Cl and

3-F substituted pyridine analogues involved initial SN_Ar

displacement at the 4-position of 3-bromo-4,5-dichloropyridine

(7) or 3-bromo-4-chloro-5-ﬂuoropyridine (57) to give

intermediates 8, 26−36, 58, 59, and 96, which were then

subject to Suzuki cross-coupling to give ﬁnal compounds 17−

25, 40−56, 60−63, and 100−105 (Scheme 1 and Tables 2, 3,

4, and 7). The corresponding CF₃-substituted pyridines (70,

71, and 72, Table 4) were synthesized by two alternative routes.

Compound 71 was prepared by selective copper-mediated

triﬂuoromethylation at the bromo-substituted carbon atom of

8,²⁸followed by Suzuki coupling at the pyridine chloro

substituent. Alternatively, compounds 70 and 72 were prepared

by SN_Ardisplacement at the 4-position of 3,5-diiodo-4-

chloropyridine 64, followed by copper-mediated triﬂuorome-

thylation at one iodo-substituted carbon followed by Suzuki

cross-coupling at the other.

a

Table 1. Pharmacokinetic Proﬁle of Compound 6

species

mouse

Cl (L/h/kg) LBF (%) V_d(L/kg) F (%) t_1/2(h)

1.87

1.54

31

35

1.08

1.53

54

88

0.55

0.97

rat

dog

0.84

33

0.74

126

∼70

0.70

human prediction

∼0.88

∼76

∼0.85

∼0.70

a

Dose: 0.2 mg/kg (iv), 0.5 mg/kg (po).

volumes of distribution (V_d) across all species tested were low

to medium and the aqueous kinetic and thermodynamic

solubilities (94 μM and 0.006 mg/mL, respectively) were

suboptimal. We therefore turned our attention to lowering the

lipophilicity of compound 6 (measured log D = 3.5) with the

aim of decreasing the microsomal and in vivo clearance as well

as improving aqueous solubility. To increase the V_d, we also

attempted the introduction of a weakly basic center.^29,30In the

course of improving physicochemical, pharmaceutical, and

pharmacokinetic properties, we monitored in vitro biochemical

aﬃnity for CDK8 and CDK19 which we found to consistently

predict for cell-based activity in both the reporter-based 7dF3

and LS174T assays;²²we also observed consistent SAR in these

two reporter-based cellular assays for compounds described in

this manuscript (Figure S1 in Supporting Information).

Upon the basis of our work to identify the molecular targets

of 6,²²we knew that extended linear substituents were tolerated

at C-5 of the pyridine ring; therefore, we introduced a variety of

polar groups on the C-5 phenylpyrazole anticipating that these

would enhance metabolic stability and aqueous solubility

(Table 2, entries 2−5). Pleasingly, replacement of the pyrazole

N1-methyl substituent by hydroxyethyl or 2-hydroxy-2-

2-Aminopyridines (88, 92−95, 108, and 111, Tables 5 and

8) were prepared by SN_Ar-mediated displacement of the 4-

chloro substituent in pyridines 73, 74, and 75 followed by

Suzuki cross-coupling (Scheme 2). In the case of 85−87, 89−

91, 109, and 110, we found that protection of the primary

amine with a PMB group improved both the SN_Ardisplacement

and the subsequent palladium-mediated cross-coupling reac-

tion; deprotection was achieved using triﬂuoroacetic acid at

room temperature.

RESULTS AND DISCUSSION

■

Compound 6 is a high aﬃnity ligand for CDK8 and CDK19

(IC₅₀of 7.2

1.4 and 6.0

1.0 nM, respectively) and

demonstrates potent inhibition of WNT-dependent signaling

using our previously described inducible luciferase reporter

assay in human embryonic kidney cells (HEK293) that contains

both estrogen receptor-dishevelled (DVL2) and TCF-lucifer-

ase-IRES-GFP constructs (7dF3 IC₅₀= 5.0 2.0 nM, Table

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Table 2. Introduction of Polarity at the Pyridine C-5 Substituent

a

b

c

d

Free base. TFA salt. Run in diﬀerent conditions (see reporter displacement assay in Experimental Section). In this case the chloro substituent is

at C-5 according to nomenclature 8-(3-(3-amino-1H-indazol-6-yl)-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one.

methylpropyl led to a signiﬁcant improvement in microsomal

stability in all species; furthermore, aqueous solubility also

increased (Table 2, entries 2 and 3) while CDK8/19

biochemical aﬃnity and cell-based 7dF3 potency were

maintained. Introduction of a basic center resulted in a 4-fold

drop in potency in both biochemical and cell-based assays,

suggesting that permeability is not responsible for the observed

decrease (Table 2, entries 4−6). However, we noticed that

compounds such as 17, 18, and 21 (Table 2, entries 2, 3, 6) had

reduced microsomal clearance (Cl_int), which conﬁrmed that the

introduction of polarity could be fruitful. Replacement of

methylpyrazole by fused 6,5-heterocycles was also investigated.

Introduction of 5-substituted-1,3-dihydrobenzo[c]isothiazole

2,2-dioxide (henceforth abbreviated to sultam) improved the

microsomal stability of the molecule across all species tested

(Table 2, entry 7). Furthermore, the thermodynamic aqueous

solubility of 22 was improved by 120-fold; however a 23-fold

drop in cell-based potency was also observed. In order to

improve cell permeability by lowering H-bond donor count, the

N-methyl sultam derivative 23 was prepared (Table 2, entry 8);

while this compound was potent in the 7dF3 cell-based assay,

both 23 and its regioisomer 24 exhibited signiﬁcant metabolic

instability (Table 2, entries 8 and 9). The aminoindazole 25

gave an acceptable in vitro clearance proﬁle across all species

and high aqueous solubility (Table 2, entry 10); however, as for

most of the derivatives in Table 2, we observed low oral

bioavailability and no improvement of the in vivo mouse

clearance compared to 6.

To understand whether the introduction of polarity

inﬂuenced binding mode, we determined the crystal structure

of aminoindazole derivative 25 in complex with the kinase

domain of CDK8 and cyclin C (Figure 1, panel A). As

previously observed with 6, analog 25 occupies the ATP

binding site (Figure 1, panel B).²²All interactions were

conserved including a cation−π interaction of the indazole

phenyl ring with Arg365 due to insertion of the C-terminal

domain of CDK8 into the ATP binding site.²²Notably, binding

to the hinge via the pyridine is conserved despite the

D

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Figure 1. Crystal structure of 25 in CDK8/cyclin C (panel A, PDB code 5FGK). Overlay of 6 (pink) and 25 (gray) in CDK8/cyclin C (panel B).

DMSO is colored cyan.

introduction of a potential alternative 3-aminoindazole hinge-

binding motif; pleasingly, the only diﬀerence we observe

between the binding modes of compounds 6 and 25 is the

presence of a DMSO molecule in a small pocket adjacent to 25

(see Figure 1, panel A).

fold improvement in potency in the 7dF3 assay compared to 23

(Table 3, entry 2) but suﬀered from poor metabolic stability

which was not overcome by exchanging the methyl for a

triﬂuoromethyl group (Table 3, entry 13). Given that the lower

dynamic range of our in vitro biochemical assays for CDK8 and

CDK19 is approximately 3 nM due to the concentration of

enzyme (5 and 6.7 nM, respectively), it is possible that the

exquisite cell-based potency for compound 49 is on-target.

Introduction of a basic center (compound 51, entry 14)

demonstrated that polarity was tolerated at the piperidine C-4

position, but all analogs were metabolically unstable. Un-

surprisingly, spiropyrrolidine 52 (Table 3, entry 15) led to a

less active compound due to removal of the carbonyl group and

loss of interaction with Lys52.

As noted above, a DMSO-ﬁlled pocket was observed in the

X-ray crystal structure of 25 (Figure 1, panel A), and we

postulated that potency could be regained by targeting this

region. Addition of a linker on the pyrrolidine nitrogen (Table

3, entries 16−19) led to a 4-fold improvement in potency;

however, the metabolic stability of such compounds was poor

and, disappointingly, cocrystallization of 54 in CDK8/cyclin C

did not demonstrate electron density consistent with binding of

the pendant methoxyethyl moiety in the DMSO pocket (Figure

3). Despite its high in vivo clearance, the spirocarbamate 42

(Table 3, entry 5) was attractive from its in vitro proﬁle and we

considered spirocarbamate as a potential replacement for the

spirolactam in our further optimization.

Next, we investigated variation of the pyridine C-3 position

(Table 4). We had previously demonstrated that small

lipophilic residues were tolerated at this position consistent

with the protein−ligand crystal structure of 6, 25, and 42 in

CDK8/cyclin C where the pyridine C3 substituent is proximal

to the lipophilic gatekeeper residue Phe97.²⁷3-Fluoro

derivatives (Table 4, entries 1−4) led to a decrease in both

biochemical and cell-based potency consistent with a weaker

lipophilic interaction of ﬂuorine compared to chlorine with

Phe97.³¹Pleasingly, the combination of either spirolactam or

spirocarbamate with the N-methyl sultam and C-3 ﬂuoro group

resulted in acceptable potency and good solubility; however,

both 61 and 63 showed high in vivo clearance in mouse

pharmacokinetics (Table 4, entries 2 and 4). Introduction of a

C-3 triﬂuoromethyl group restored potency for spirolactams 70

and 71 (Table 4, entries 5−6); however, they proved

Although the N-methyl sultam group conferred metabolic

instability (Table 2, entry 8), its beneﬁcial eﬀect on the potency

was very attractive and we were keen to explore replacement of

the spirolactam in combination either with the N-methyl sultam

or with our hitherto preferred 4-(1-methyl-1H-pyrazol-4-

yl)phenyl substituent at C-5. Consistent with our desire to

replace the spirolactam with more polar heterocycles, mass

spectrometry-mediated metabolite identiﬁcation for 6 indicated

that the spirolactam was the main site of oxidative metabolism

(data not shown). Introduction of a carbamate (Table 3, entry

3) resulted in >100-fold decrease in CDK8 aﬃnity, whereas

isomer 41 had a potency proﬁle similar to 6 suggesting that a

hydrogen-bond-mediated interaction from the carbamate NH

of compound 41 with Asp173 is required for potency (Table 3,

entry 4). Compound 42 (Table 3, entry 5), which combines the

optimal carbamate isomer and an N-methyl sultam, gave very

potent CDK8/19 aﬃnity with improved metabolic stability

compared to 6. However, in vivo clearance in mice was higher

than that of compound 6. Addition of two methyl groups to

block metabolically vulnerable sites in the spirocycle did not

lead to improved metabolic stability, and a drop in potency was

also observed (Table 3, entry 6). Crystallization of compound

42 (Table 3, entry 5) in CDK8/cyclin C conﬁrmed the

carbamate NH hydrogen bond interaction with Asp173 and

showed interaction of the carbamate carbonyl group with Lys52

(Figure 2).

Cognizant of blocking oxidative metabolism on the

spirolactam, we prepared additional polar derivatives where

the lactam was replaced by imidazolidin-4-one, imidazolidine-

2,4-dione, 2-methylimidazol-5-one, and piperazine-2,5-dione

(Table 3, entries 7−11). Pleasingly, all derivatives were

signiﬁcantly more stable in microsomal clearance assays;

however, translation to cell-based potency was eroded for the

imidazolidine-2,4-dione, 2-methylimidazole-5-one, and piper-

azine-2,5-dione derivatives (Table 3, entries 8−11) and no

examples demonstrated signiﬁcant improvement in in vivo

mouse clearance (Table 3, entries 7−11). Surprisingly, the 3-

methylpyrazolone derivative 49 (Table 3, entry 12) led to a 13-

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Table 3. Introduction of Polarity at the Pyridine C-4 Substituent

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Table 3. continued

a

b

c

Free base. TFA salt. The chloro substituent is at C-5 according to 5-(5-chloro-4-(1-methyl-1,8-diazaspiro[4.5]decan-8-yl)pyridin-3-yl)-1-methyl-

d

1,3-dihydrobenzo[c]isothiazole 2,2-dioxide. The chloro substituent is at C-5 according to 5-(5-chloro-4-(1-(2-methoxyethyl)-1,8-diazaspiro[4.5]-

decan-8-yl)pyridin-3-yl)-1-methyl-1,3-dihydrobenzo[c]isothiazole 2,2-dioxide. The chloro substituent is at C-5 according to 5-(5-chloro-4-(1-(3-

methoxypropyl)-1,8-diazaspiro[4.5]decan-8-yl)pyridin-3-yl)-1-methyl-1,3-dihydrobenzo[c]isothiazole 2,2-dioxide. The chloro substituent is at C-5

e

f

according to 5-(5-chloro-4-(1-(3-(methylsulfonyl)propyl)-1,8-diazaspiro[4.5]decan-8-yl)pyridin-3-yl)-1-methyl-1,3-dihydrobenzo[c]isothiazole 2,2-

dioxide.

(85, Table 5, entry 1) maintained potency with acceptable

metabolic stability and pharmacokinetics, albeit with poor

aqueous solubility. At this stage, we decided to introduce the 2-

amino functionality into selected optimal compounds from

Tables 2, 3, and 4. Similar to 85, all additional 2-aminopyridines

(Table 5, entries 2−11) were signiﬁcantly more metabolically

stable than their corresponding pyridine matched pair. For

example, the 2-aminopyridine 87 was at least 5-fold more stable

in mouse Cl_intexperiments than 25. The presence of the 2-

amino functionality also led to lower mouse in vivo clearance;

for example, 92 (Table 5, entry 8, Cl = 1.28 L/h/kg) compared

to its pyridine matched pair 61 (Table 4, entry 2, Cl = 7.9 L/h/

kg). However, we became concerned that reducing in vitro

mouse metabolic Cl_intto <10 μL/min/mg (Table 5, entry 6)

did not translate to improved mouse in vivo clearance. Thus, in

vitro Cl_intdid not appear to be a suﬃcient predictor of in vivo

clearance for this series. Predicted mouse blood clearance

(parallel tube model) was estimated from the Cl_intdata to take

Figure 2. Overlay of lactam 6 (brown) and carbamate 42 (cyan) in

CDK8/cyclin C, PDB code 5HBE.

into account the polarity of the compounds and their

nonspeciﬁc protein binding in in vitro clearance assays;

however, this method also failed to explain the discrepancy

between in vitro and in vivo data (Figure 4).

metabolically unstable. Carbamate 72, despite being surpris-

ingly less potent than the spirolactam 70, had an acceptable

Cl_intproﬁle and in vivo mouse clearance similar to 6. However,

due to the consistently poor aqueous solubility of these CF₃

derivatives, we did not progress them further.

The protein−ligand crystal structure of 25 in CDK8/cyclin C

demonstrates that the C2-H of the pyridine is proximal to the

backbone carbonyl of Asp98 (distance of 2.3 Å, Figure 1), an

observation consistent with our previously reported crystal

structure of 6 in CDK8/cyclin C.²²We hypothesized that

introduction of a hydrogen bond donor at the 2 position of the

pyridine could form a favorable interaction with the backbone

carbonyl of Asp98. Pleasingly, introduction of a 2-amino group

We hypothesized that the increased in vivo clearance in

comparison with the in vitro predicted clearance may be driven

by active transport. Caco-2 assay data conﬁrmed that most

compounds had a high eﬄux ratio (ER) (Table S1). However,

it was unclear if the lack of correlation between predicted and

observed blood clearance (calculated from measured plasma

clearance data) was due to a P-glycoprotein (P-gp) transporter

liability (Figure 4). To clarify this matter, we conducted

pharmacokinetic evaluation in P-gp knockout (KO) mice for a

set of compounds (Table 6). We observed lower in vivo

clearance in P-gp KO mice for compounds 42, 48, and 90,

while clearance remained unchanged for both 6 and 89 that did

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Figure 3. Crystal structure of 54 in CDK8/cyclin C (panel A, PDB code 5HBH). Overlay of 25 (gray) with 54 (green) in CDK8/cyclin C (panel B).

Table 4. Introduction of Chlorine Replacements at the Pyridine C-3 Position

a

b

c

Free base. TFA salt. The CF₃substituent is at C-5 according to 8-(3-(4-(1-methyl-1H-pyrazol-4-yl)phenyl)-5-(triﬂuoromethyl)pyridin-4-yl)-2,8-

d

diazaspiro[4.5]decan-1-one. The CF₃substituent is at C-5 according to 8-(3-(1-methyl-2,2-dioxido-1,3-dihydrobenzo[c]isothiazol-5-yl)-5-

(triﬂuoromethyl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one. The CF₃substituent is at C-5 according to 8-(3-(4-(1-methyl-1H-pyrazol-4-

e

yl)phenyl)-5-(triﬂuoromethyl)pyridin-4-yl)-1-oxa-3,8-diazaspiro[4.5]decan-2-one.

not exhibit eﬄux in the Caco-2 assay. Furthermore, in P-gp KO

mice we noted a lower percentage of parent compound cleared

unchanged in the feces compared to wild-type mice for all

compounds (Table 6). Taken together, these data are

consistent with the notion that transporter-mediated hepatic

uptake and elimination contribute to the overall clearance of

some compounds in this series. However, we noted that even in

the P-gp KO mice, compounds 48 and 90, both of which have

low Cl_int, demonstrate moderate in vivo clearance and that

compound 48 is still observed in the feces of P-gp KO mice,

H

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Table 5. 2-Aminopyridine Derivatives

a

b

Free base. TFA salt.

suggesting that additional nonhepatic elimination mechanisms

are also involved.

2.5, TPSA < 80−100, HBA ≤ 7 with no basic center. Analysis

of physicochemical properties versus eﬄux ratio in a Madin−

Darby canine kidney epithelial cell line (MDCK), transfected to

express human P-gp (MDR-MDCK), led to broadly similar

trends. Analysis of Caco-2 data in the 2-aminopyridine series

indicated that physicochemical properties in the range HBD ≤

3 and TPSA < 100 or HBD ≤ 3 and log P > 3 are necessary for

low eﬄux in this chemical series, and a combination of HBD ≤

3, TPSA < 100, and log P > 3 is more likely to lead to a low

eﬄux ratio. These observations are consistent with previously

published studies demonstrating that a ﬁne balance of

physicochemical properties is often required to achieve

To understand the contribution of physicochemical param-

eters to eﬄux, we conducted a more detailed analysis of the

Caco-2 data. Cognizant of the potential for variability in Caco-2

assay data,³²we classiﬁed compounds into two large data sets,

pyridines (n = 165) and aminopyridines (n = 39), seeking

trends to direct our medicinal chemistry design (Figure 5).

Analysis of physicochemical property trends in the pyridine

series showed that HBD ≤ 2 is necessary but not suﬃcient to

give ER < 3. The probability of compounds in this series

exhibiting a low eﬄux ratio could be further improved by

limiting physicochemical properties within the range log P >

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combination with the spirolactam at C-4 of the 2-amino-

pyridine scaﬀold. This tactic led to suboptimal human Cl_intand

an eﬄux ratio of 7 (Table 8, entry 1). We then combined the

spirolactam and spirocarbamate with a methylindazole (Table

8, entries 2 and 3). The spirolactam derivative 109 (measured

log D = 2.5) was potent (CDK8 IC₅₀= 4.9 0.6 nM, CDK19

IC₅₀= 2.6 0.4 nM with residence times of 53 and 86 min,

respectively, in the reporter displacement assay), soluble, and

stable with a low eﬄux ratio and acceptable in vivo mouse

pharmacokinetics (Cl = 0.61 L/h/kg, F = 30%, Table 9);

however, the carbamate matched pair 110 was less potent and

subject to increased eﬄux. To further improve the metabolic

stability, we prepared compound 111 bearing an N-

isopropylindazole; however, this compound was less stable in

all species. Crystallization of 109 in CDK8/cyclin C (Figure 6)

demonstrated a binding mode consistent with those previously

observed, and pleasingly, a new hydrogen bond interaction

between the exocyclic nitrogen of the 2-aminopyridine scaﬀold

and the backbone carbonyl of Asp98 was observed (3.0 Å).

In light of its promising proﬁle, compound 109 was further

proﬁled in rat and dog pharmacokinetics (Table 9). Moderate

clearance was observed in both species; furthermore, the

human pharmacokinetic prediction for 109 was signiﬁcantly

better than for compound 6 (Tables 9 and 1, respectively,

∼31% of liver blood ﬂow for 109 compared to ∼76% for 6).

Pleasingly, compound 109 had acceptable aqueous solubility

(Table 8, entry 2) and demonstrated minimal activity when

tested in a panel of 55 receptors, ion channels, and enzymes at

1 μM (Tables S2 and S3) and in a panel of 279 kinases (Table

S4); weak inhibition of CYPs was observed (Table S5).

Consistent with the proﬁle of chemical probe 6, compound 109

demonstrated potent inhibition of reporter-based readouts

measuring basal WNT pathway activity in human cancer cell

lines that have constitutively activated WNT pathway signaling:

LS174T (β-catenin mutant), SW480 and Colo205 (APC

mutant) or PA-1 human teratocarcinoma cells that are WNT

ligand dependent (Table 10).

Compound 109 was then assessed in the in vivo APC-mutant

SW620 human colorectal carcinoma xenograft model, treating

established tumors in female NCr athymic mice. Mice were

treated orally (30 mg/kg q.d.) for 15 days; a 54.2% reduction in

tumor weight was observed at day 15 (Figure 7, panels A and

B). We monitored inhibition of STAT1^SER727phosphorylation,

which we have previously demonstrated to be an in vitro cell-

based and in vivo pharmacodynamic biomarker of CDK8

inhibition.²²Reduced STAT1^SER727phosphorylation was

maintained for more than 6 h after the last dose (Figure S2)

consistent with measured free plasma and tumor exposures that

remained above CDK8 IC₅₀(Figure 7, panel C, Figure S3, and

Table S6). In light of its potent and selective proﬁle coupled

with good oral pharmacokinetics and duration of in vivo target

engagement on oral dosing, compound 109 (CCT251921) has

been selected for progression into further preclinical in vivo

eﬃcacy and safety studies.

Figure 4. In vitro−in vivo correlation of clearance for compounds

from the pyridine and 2-aminopyridine series. X axis is calculated

blood clearance from measured plasma clearance in mouse PK. Y axis

is predicted blood clearance using the parallel tube model.

Table 6. Pharmacokinetic Proﬁle of a Set of Compounds in

Wild-Type and P-gp KO Mice

wild type mice

parent

P-gp KO mice

parent

plasma

Cl

compd

in feces

(%)

plasma

Cl

compd

in feces

(%)

ER

mouse Cl_int

Caco-2 (μL/min/mg) (L/h/kg)

(L/h/kg)

6

2.5

2.0

141

18.5

2.34

2.59

4.89

4.08

4.43

9.1

17

2.30

2.84

4.00

2.85

2.22

<1

24

89

42

90

48

4.7

57

<10

30

18

14.8

17.6

20

67

potency, metabolic stability, and low eﬄux in a single

molecule.^33,34

Cognizant of these analyses, we set out to reduce eﬄux in

both the pyridine and 2-aminopyridine series. In the pyridine

series, compound 25 (Table 7, entry 1) has an attractive

solubility coupled with low Cl_intand acceptable potency;

however, this compound harbors four hydrogen bond donors

with an eﬄux ratio of >1200. Alkylation of the 3-aminoindazole

nitrogen in 25 signiﬁcantly reduced Caco-2 eﬄux but to the

detriment of metabolic stability and potency (Table 7, entries

2−4). Methylation of the hydantoin (Table 7, entry 6) also led

to a lower eﬄux ratio compared to the parent compound 45,

again to the detriment of microsomal stability. The potent,

metabolically stable, and soluble compound 44 (Table 7, entry

7) bears two hydrogen bond donors and exhibits an eﬄux ratio

of 3.7. Methylation of the N-1 nitrogen of the imidazolidinone

ring reduced the eﬄux ratio (Table 7, entry 8); however, poor

microsomal stability was again observed. Replacement of the

methyl group by a variety of substituents including triﬂuoro-

ethyl did not improve the microsomal stability (Table 7, entry

9). As these attempts proved unsuccessful, we turned our

attention to the aminopyridine series.

In the 2-aminopyridine series, the presence of additional

hydrogen bond donor functionality by virtue of the 2-amino

substituent restricted our freedom to modulate physicochemical

properties in the remainder of the molecule. Thus, the choice of

spirolactam isostere was diminished and we hypothesized that

introduction of more lipophilic C5-pyridine substituents may

be necessary to stay within our desired physicochemical

property range and maximize the chances of low eﬄux ratios.

Thus, we introduced an isopropylpyrazole at C-5 in

CONCLUSIONS

■

Literature evidence⁹⁻¹⁶and our own studies²²point to a role

for the Mediator complex-associated kinases CDK8 and

CDK19 in human disease, particularly in colorectal cancer

where CDK8 has been reported as a putative oncogene.

CDK19, a paralogue of CDK8, is relatively unexplored, and our

previous studies demonstrate that selectivity for CDK8 over

CDK19 with a small molecule ligand is likely to be

J

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Figure 5. Physicochemical property analysis results for pyridine and aminopyridine series.

challenging.²²Indeed, in our recently reported discovery of

CCT251545 (6), a potent and selective chemical probe for the

further exploration of CDK8 and CDK19 pharmacology, we

show strong correlation of CDK8 and CDK19 binding

aﬃnity.²²

Here, we have further optimized the small molecule CDK8/

19-selective chemical probe 6 to give compound 109 by

improving oral pharmacokinetics and pharmaceutical properties

in order to facilitate further in vivo evaluation of CDK8/19

pharmacology and progression into preclinical in vivo eﬃcacy

and safety studies. Chemical probe 6, although a high aﬃnity

ligand for CDK8 and CDK19, displays moderate in vivo

clearance in preclinical species resulting in a clearance

prediction to man that may preclude consistent target

engagement for extended periods of time; in addition, aqueous

solubility was suboptimal and we anticipated that this may limit

the maximum absorbable dose. In attempting to reduce

oxidative metabolism by reducing lipophilicity while maintain-

ing key interactions within the CDK8 kinase domain, we

beneﬁted from detailed knowledge of the binding mode of 6

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d

Table 7. Attempts To Reduce Eﬄux in the Pyridine Series

a

b

c

Free base. TFA salt. In this case the chloro substituent is at C-5 according to nomenclature 8-(3-(3-amino-1-methyl-1H-indazol-6-yl)-5-

d

chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one. M: mouse. R: rat. H: human. log P was calculated using Percepta Batch, version 2015 (www.

acdlabs.com). TPSA was calculated in the program MOE (www.chemcomp.com).

and analogs through protein−ligand crystal structures in the

CDK8 kinase domain. We were able to improve in vitro

metabolic stability by introducing a C-2 amino substituent to

the pyridine scaﬀold which reinforced interactions with the

kinase hinge region and reduced compound lipophilicity.

However, we identiﬁed transporter-mediated hepatic uptake

as a component of in vivo clearance. In particular, we noted that

a signiﬁcant number of compounds in both the pyridine and 2-

aminopyridine series exhibited higher measured in vivo

clearance than predicted by experimental in vitro clearance

assessment. Through careful correlation of in vitro Caco-2

eﬄux ratios with physicochemical properties and subsequent

medicinal chemistry design within desirable physicochemical

property ranges, we were able to identify compounds that

demonstrated reduced clearance without increased suscepti-

bility to active hepatic uptake. Compound 109 was identiﬁed as

the best compromise of in vitro biochemical and pharmacoki-

netic properties that demonstrated acceptable in vivo

pharmacokinetics suitable for progression to in vivo animal

models of cancer. Further in vivo evaluation of 109 will be

reported in due course.

EXPERIMENTAL SECTION

■

Chemistry. Commercially available starting materials, reagents, and

dry solvents were used as supplied. Column chromatography was

performed on a Biotage SP1 puriﬁcation system using Thomson or

Biotage Flash silica cartridges or on a Companion puriﬁcation system

using Interchim silica cartridges. Preparative TLC was performed on

Merck plates. Ion exchange chromatography was performed using

acidic Isolute Flash SCX-II columns or basic Isolute Flash NH₂

columns. Preparative HPLC was conducted according the following

methods. For method A, injections of the sample were made onto a

SunFire C18 OBD column (100 Å, 5 μm, 30 mm × 100 mm).

Chromatographic separation at room temperature was carried out

using Agilent Tehnologies, 1260 Inﬁnity, acetonitrile/water gradient

(both modiﬁed with 0.1% formic acid) at a ﬂow rate of 50 mL/min.

For method B, injections of the sample were made onto a

Phenomenex Gemini column (10 μm, 250 mm × 21.2 mm, C18,

Phenomenex, Torrance, CA, USA). Chromatographic separation at

room temperature was carried out using Gilson GX-281 liquid handler

L

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d

Table 8. Reducing Eﬄux in the 2-Aminopyridine Series

a

b

c

d

free base. TFA salt. besylate salt. M: mouse. R: rat. H: human.

a

Table 9. Pharmacokinetic Proﬁle of 109

Table 10. Potency of 6 and 109 versus Reporter-Based

Readouts of WNT Pathway Activity in Human Cancer Cell

Lines

species

mouse

Cl (L/h/kg) LBF (%) V_d(L/kg) F (%) t_1/2(h)

0.61

1.49

10

34

0.63

2.0

30

57

0.78

1.10

0.99

cell line

WNT pathway activation

6 IC₅₀(nM)

109 IC₅₀(nM)

rat

dog

1.07

43

1.4

68

LS174T

SW480

Colo205

PA-1

β-catenin mutant

APC-mutant

23 11

33 13

human prediction

∼0.36

∼31

∼1.4

∼70

∼2.7

190 30

22

15

2

1

a

APC-mutant

35

3

Dose: 0.2 mg/kg (iv), 0.5 mg/kg (po).

WNT ligand-dependent

20 10

64 34

references were used: CDCl₃(δ_C77.2), CD₃OD (δ_C49.0), and

DMSO-d₆(δ_C39.5); unobserved resonances for quaternary carbon

atoms are denoted by “Cq not observed”. LC/MS and HRMS analyses

were performed on an Agilent 1200 series HPLC and diode array

detector coupled to a 6210 time-of-ﬂight mass spectrometer with dual

multimode APCI/ESI source. Analytical separation was carried out

according to the following methods. For method A, analytical

separation was carried out on a Chromolith Speed ROD column

(RP-18e, 50 mm × 4.6 mm) using a ﬂow rate of 2.4 mL/min in a 3.9

min gradient elution with detection at 220 nm. The mobile phase was

a mixture of water containing 0.05% formic acid (solvent A) and

acetonitrile containing 0.04% formic acid (solvent B). Gradient elution

was as follows: 95:5 (A/B) to 0:100 (A/B) over 2.8 min, 0:100 (A/B)

for 0.5 min, and then reversion back to 95:5 (A/B) over 0.1 min,

ﬁnally 95:5 (A/B) for 0.5 min. For method B, analytical separation was

carried out on a Chromolith Performance column (RP-18e, 100 mm ×

3 mm) using a ﬂow rate of 2.0 mL/min in a 4.8 min gradient elution

with detection at 220 nm. The mobile phase was a mixture of water

(solvent A) and acetonitrile (solvent B) both containing 0.1% TFA.

Gradient elution was as follows: 99:1 (A/B) over 0.2 min, then 99:1 to

0:100 (A/B) over 3.6 min, 0:100 (A/B) for 0.4 min, and then

reversion back to 99:1 (A/B) over 0.1 min and ﬁnally 99:1 (A/B) for

0.5 min. For method C, analytical separation was carried out at 30 °C

on a Merck Purospher STAR column (RP-18e, 30 mm × 4 mm) using

a ﬂow rate of 1.5 mL/min in a 4 min gradient elution with detection at

254 nm. The mobile phase was a mixture of MeOH (solvent A) and

water (solvent B), both containing 0.1% formic acid. Gradient elution

was as follows: 1:9 (A/B) to 9:1 (A/B) over 2.5 min, 9:1 (A/B) for 1

min, and then reversion back to 1:9 (A/B) over 0.3 min, ﬁnally 1:9

(A/B) for 0.2 min. For method D, analytical separation was carried out

at 30 °C on a Merck Purospher STAR column (RP-18e, 30 mm × 4

mm) using a ﬂow rate of 1.5 mL/min in a 4 min gradient elution with

Figure 6. X-ray crystal structure of 109 in CDK8/cyclin C, PDB code

5HBJ.

system combined with a Gilson 322 HPLC pump (Gilson, Middleton,

WI, USA) over a 15 min gradient elution from 40:60 to 100:0 MeOH/

water (both modiﬁed with 0.1% formic acid) at a ﬂow rate of 20 mL/

min. ¹H NMR spectra were recorded on a Bruker Avance 500, Bruker

Avance 400, or Avance II 400. Samples were prepared as solutions in a

deuterated solvent and referenced to the appropriate internal

nondeuterated solvent peak. ¹³C NMR spectra were recorded at 126

MHz using an internal deuterium lock. The following internal

M

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ide (14). 2-Chlorobenzylsulfonyl chloride (1.86 g, 8.26 mmol) was

dissolved in acetone (27 mL), and then ammonium hydroxide (18

mL) was added. The reaction mixture was stirred for 2.5 h at rt, and

the solvent was evaporated. The reaction mixture was diluted with

EtOAc, and water was added. The two layers were separated, and the

aqueous layer was extracted with EtOAc. The combined organic layers

were dried over MgSO₄and concentrated under vacuum. The crude

product was puriﬁed by Biotage column chromatography (cyclo-

hexane/acetone 90:10 to 60:40) to aﬀord (2-chlorophenyl)-

1

methanesulfonamide as a white solid (1.6 g, 94% yield). H NMR

(500 MHz, CDCl₃) δ 7.56−7.53 (m, 1H), 7.47−7.44 (m, 1H), 7.36−

7.30 (m, 2H), 4.66 (bs, 2H), 4.57 (s, 2H); LC−MS (method D, ESI,

m/z) t_R= 1.77 min, parent does not ionize.

(2-Chlorophenyl)methanesulfonamide (450 mg, 2.19 mmol),

tris(dibenzylideneacetone)dipalladium (100 mg, 0.109 mmol), 2-di-

tert-butylphosphino-2′,4′,6′-triisopropylbiphenyl (186 mg, 0.438

mmol), and potassium carbonate (605 mg, 4.38 mmol) were loaded

in a microwave vial, and THF (8.8 mL) was added. The reaction

mixture was stirred at 80 °C in an oil bath for 13 h before being

quenched with a sat. aq NH₄Cl solution. The solvent was then

evaporated and the residue was puriﬁed by Biotage column

chromatography (cyclohexane/acetone 95:5 to 60:40) to aﬀord 1,3-

dihydrobenzo[c]isothiazole 2,2-dioxide as a white solid (296 mg, 80%

yield). ¹H NMR (500 MHz, CDCl₃) δ 7.31−7.26 (m, 1H), 7.26−7.23

(m, 1H), 7.07 (td, J = 7.6, 0.9 Hz, 1H), 6.90 (d, J = 8.0 Hz, 1H), 6.48

(bs, 1H), 4.39 (s, 2H); LC−MS (method D, ESI, m/z) t_R= 1.69 min,

parent does not ionize.

To a suspension of 1,3-dihydrobenzo[c]isothiazole 2,2-dioxide (280

mg, 1.66 mmol) and potassium carbonate (229 mg, 1.66 mmol) in

DMF (5 mL) was added iodomethane (414 μL, 6.62 mmol). The

reaction mixture was stirred for 6 h at rt and was then quenched with a

sat. NH₄Cl solution. The reaction mixture was concentrated and

puriﬁed by Biotage column chromatography (cyclohexane/acetone

90:10 to 70:30) to aﬀord 1-methyl-1,3-dihydrobenzo[c]isothiazole 2,2-

1

dioxide as a white solid (270 mg, 89% yield). H NMR (500 MHz,

CDCl₃) δ 7.37−7.32 (m, 1H), 7.27−7.24 (m, 1H), 7.02 (td, J = 7.6,

1.0 Hz, 1H), 6.73 (d, J = 8.0 Hz, 1H), 4.34 (s, 2H), 3.14 (s, 3H); LC−

MS (method D, ESI, m/z) t_R= 2.07 min, parent does not ionize.

1-Methyl-1,3-dihydrobenzo[c]isothiazole 2,2-dioxide (272 mg, 1.49

mmol) was dissolved in DMF (1.5 mL), and N-bromosuccinimide

(264 mg, 1.49 mmol) was added. The reaction mixture was stirred at rt

for 4 h. After addition of water, the reaction mixture was concentrated.

The residue was puriﬁed by Biotage column chromatography

(cyclohexane/acetone 90:10 to 70:30) to aﬀord 5-bromo-1-methyl-

1,3-dihydrobenzo[c]isothiazole 2,2-dioxide as a white solid (330 mg,

85% yield). ¹H NMR (500 MHz, CDCl₃) δ 7.45−7.41 (m, 1H), 7.37−

7.35 (m, 1H), 6.59 (d, J = 8.5 Hz, 1H), 4.30 (s, 2H), 3.09 (s, 3H);

LC−MS (method C, ESI, m/z) t_R= 2.46 min, parent does not ionize.

5-Bromo-1-methyl-1,3-dihydrobenzo[c]isothiazole 2,2-dioxide (267

mg, 1.02 mmol), bis(pinacolato)diboron (388 mg, 1.53 mmol),

potassium acetate (300 mg, 3.06 mmol), and Pd(dppf)Cl₂·CH₂Cl₂(42

mg, 0.051 mmol) were loaded in a microwave vial, and DME (7.4 mL)

was added. The reaction mixture was stirred in an oil bath at 80 °C

overnight. The reaction was concentrated and puriﬁed by Biotage

column chromatography (cyclohexane/acetone 97:3 to 85:15) to

aﬀord 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3-

dihydrobenzo[c]isothiazole 2,2-dioxide 14 as a white solid (290 mg,

92% yield). ¹H NMR (500 MHz, CDCl₃) δ 7.80−7.77 (m, 1H), 7.69−

7.67 (m, 1H), 6.71 (d, J = 8.0 Hz, 1H), 4.32 (s, 2H), 3.15 (s, 3H), 1.33

(s, 12H); LC−MS (method C, ESI, m/z) t_R= 2.82 min, 309/310 (M

+ H)⁺.

Figure 7. Reduction of (A) tumor volume and (B) tumor weight

versus vehicle-treated controls after chronic oral dosing (30 mg/kg

q.d.) of 109 to an APC-mutant SW620 human colorectal carcinoma

xenograft animal model. (C) Free plasma and free tumor exposure

(nM) at 1, 2, 6, and 24 h after the last dose in the same experiment.

detection at 220 nm. The mobile phase was a mixture of MeOH

(solvent A) and water (solvent B), both containing 0.1% formic acid.

Gradient elution was as follows: 1:9 (A/B) to 9:1 (A/B) over 2.5 min,

9:1 (A/B) for 1 min, and then reversion back to 1:9 (A/B) over 0.3

min, ﬁnally 1:9 (A/B) for 0.2 min. For method E, analytical separation

was carried out on XBridge C8 column (50 mm × 4.6 mm, 3.5 μm)

using a ﬂow rate of 2.0 mL/min in a 10 min gradient elution with

detection at 254 nm. The mobile phase was a mixture of acetonitrile

(solvent A) and water (solvent B), both containing 0.1% TFA.

Gradient elution was as follows: 5:95 (A/B) to 100:0 (A/B) over 8

min, 100:0 (A/B) for 0.1 min, and then reversion back to 5:95 (A/B)

over 0.4 min, ﬁnally 5:95 (A/B) for 1.5 min. The following reference

masses were used for HRMS analysis: caﬀeine [M + H]⁺195.087 652;

(hexakis(1H,1H,3H-tetraﬂuoropentoxy)phosphazene [M + H]⁺

922.009 798) and hexakis(2,2-diﬂuoroethoxy)phosphazene [M + H]⁺

622.028 96 or reserpine [M + H]⁺609.280 657. All compounds

submitted for biological testing were determined to be >95% pure by

method A, B, C, D, or E unless stated otherwise.

8-(3-Chloro-5-(1-methyl-2,2-dioxido-1,3-dihydrobenzo[c]-

isothiazol-5-yl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one

(23). 1-Methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3-

dihydrobenzo[c]isothiazole 2,2-dioxide 14 (58 mg, 0.19 mmol), 8-

(3-bromo-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one²⁷

8

(54 mg, 0.16 mmol), and Pd(dppf)Cl₂·CH₂Cl₂(6.4 mg, 7.9 μmol)

were loaded in a microwave vial, and then 0.5 M sodium carbonate in

water (440 μL, 0.220 mmol) and acetonitrile (2.8 mL) were added.

The reaction was heated at 120 °C for 60 min under microwave

Preparation of Compounds in Table 2, Exempliﬁed by

Compounds 23 and 25. 1-Methyl-5-(4,4,5,5-tetramethyl-1,3,2-

dioxaborolan-2-yl)-1,3-dihydrobenzo[c]isothiazole 2,2-Diox-

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irradiation. The solvent was evaporated and the crude material was

puriﬁed by Biotage column chromatography (DCM/EtOH 99:1 to

85:15) to give 8-(3-chloro-5-(1-methyl-2,2-dioxido-1,3-dihydrobenzo-

[c]isothiazol-5-yl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one 23 as a

8.25 (s, 1H), 7.63 (dd, J = 8.2, 0.9 Hz, 1H), 7.29 (s, 1H), 6.97 (dd, J =

8.2, 1.4 Hz, 1H), 5.66 (s, 1H), 3.26 (t, J = 6.8 Hz, 2H), 3.21−3.13 (m,

2H), 2.74 (t, J = 11.7 Hz, 2H), 1.97−1.87 (m, 4H), 1.37−1.29 (m,

2H); ¹³C NMR (126 MHz, CDCl₃) δ 181.4, 153.2, 150.9, 149.9,

148.9, 142.5, 137.1, 134.0, 128.2, 121.6, 119.6, 114.2, 110.3, 47.7, 41.5,

38.7, 32.5, 32.2; LC−MS (method C, ESI, m/z) t_R= 1.58 min, 397/

399 (M + H)⁺; ESI-HRMS calcd for C₂₀H₂₂³⁵ClN₆O (M + H)⁺

397.1538, found 397.1522.

Preparation of Compounds in Table 3, Exempliﬁed by

Compounds 42 and 54. 1-Oxa-3,8-diazaspiro[4.5]decan-2-one

(38). 4-Aminomethyl-1-benzylpiperidin-4-ol (25.0 g, 113 mmol) was

suspended in DCM (400 mL), and a solution of triphosgene (32.8 g,

110 mmol) dissolved in DCM (200 mL) was added dropwise while

maintaining the temperature between 30 and 35 °C. The solution

turned yellow and the mixture was stirred at rt overnight. To the

reaction mixture 1 N NaOH solution (200 mL) was added, and the

organic phase was separated, washed with water, dried over sodium

sulfate, ﬁltered, and evaporated to dryness. The residue was triturated

with diethyl ether, the solid obtained was washed with diethyl ether

and dried in vacuo to give 13.2 g (82% pure, 39% corrected yield) of 8-

benzyl-1-oxa-3,8-diazaspiro[4.5]decan-2-one³⁵as a yellow solid which

was used in the next step without further puriﬁcation. LC−MS

(method B, ESI, m/z) t_R= 1.49 min, 247 (M + H)⁺.

8-Benzyl-1-oxa-3,8-diazaspiro[4.5]decan-2-one (82% pure, 13.2 g,

43.9 mmol) was dissolved in MeOH (60 mL) and THF (60 mL). Pd/

C 5% (54.1% H₂O, 3.00 g) was added, and the reaction mixture was

stirred at rt under hydrogen for 18 h. Since the reaction was

incomplete, additional Pd/C 5% (54.1% H₂O, 6.00 g) was added and

the reaction mixture was stirred at rt for another 18 h. Another portion

of palladium was added and the reaction was stirred for 18 h. The

catalyst was then ﬁltered oﬀ and the ﬁltrate was evaporated to dryness.

The residue was triturated with diethyl ether, ﬁltered, and dried in

vacuum to yield in 5.39 g (78% yield) of 1-oxa-3,8-diazaspiro[4.5]-

decan-2-one 38 as a brown solid which was used directly in the next

step. LC−MS (method B, ESI, m/z) t_R= 0.39 min, 157 (M + H)⁺.

8-(3-Bromo-5-chloropyridin-4-yl)-1-oxa-3,8-diazaspiro[4.5]-

decan-2-one (27). In a microwave vial 3-bromo-4,5-dichloropyr-

idine²⁷7 (2.00 g, 8.80 mmol) was dissolved in NMP (15 mL). 1-Oxa-

3,8-diazaspiro[4.5]decan-2-one 38 (1.65 g, 10.6 mmol) and triethyl-

amine (2.44 mL, 17.6 mmol) were added. The reaction was stirred for

1 h at 220 °C under microwave irradiation. The brown reaction

solution was treated with 300 mL of water. The beige precipitate was

ﬁltered and washed with water and diethyl ether to give 8-(3-bromo-5-

chloropyridin-4-yl)-1-oxa-3,8-diazaspiro[4.5]decan-2-one 27 (2.34 g,

77% yield) as a beige solid which was used directly in the next step.

LC−MS (method B, ESI, m/z) t_R= 2.01 min, 346/348/350 (M +

H)⁺.

1

white solid (57 mg, 81% yield). H NMR (500 MHz, CDCl₃) δ 8.42

(s, 1H), 8.17 (s, 1H), 7.29−7.24 (m, 2H), 6.81 (d, J = 8.1 Hz, 1H),

6.36 (s, 1H), 4.48 (s, 2H), 3.31 (t, J = 6.8 Hz, 2H), 3.18 (s, 3H), 3.16−

3.10 (m, 2H), 2.81 (t, J = 11.2 Hz, 2H), 1.98 (t, J = 6.8 Hz, 2H), 1.91−

1.80 (m, 2H), 1.39−1.33 (m, 2H); ¹³C NMR (126 MHz, CDCl₃) δ

181.6, 153.0, 150.4, 149.9, 141.5, 133.2, 131.0, 130.3, 128.3, 126.6,

118.0, 109.1, 50.8, 47.7, 41.6, 38.8, 32.4, 32.2, 26.6; LC−MS (method

C, ESI, m/z) t_R= 2.00 min, 447/449 (M + H)⁺; ESI-HRMS calcd for

C₂₁H₂₄³⁵ClN₄O₃S (M + H)⁺447.1252, found 447.1247.

(3-Amino-1H-indazol-6-yl)boronic Acid Hydrochloride (16).

In a screw-capped vessel, 6-bromo-1H-indazol-3-amine (95% purity,

500 mg, 2.36 mmol) and 4-(dimethylamino)pyridine (58 mg, 0.47

mmol) were dissolved in THF (10 mL). Di-tert-butyl dicarbonate

(2.52 mL, 11.8 mmol) and triethylamine (3.27 mL, 23.6 mmol) were

added, and the reaction solution was stirred for 3 days at rt. The

reaction mixture was diluted with water (100 mL) and the aqueous

layer extracted with EtOAc. The organic layer was washed with water,

dried, ﬁltered, and evaporated to dryness to give tert-butyl 3-[bis(tert-

butoxycarbonyl)amino]-6-bromoindazole-1-carboxylate (1.42 g, 73%

pure, 86% corrected yield) as a colorless oil, which was used without

further puriﬁcation. LC−MS (method B, ESI, m/z) t_R= 3.91 min,

534/536 (M + Na)⁺.

In a screw-capped vessel, tert-butyl 3-[bis(tert-butoxycarbonyl)-

amino]-6-bromoindazole-1-carboxylate (73% pure, 1.34 g, 1.91 mmol)

was dissolved in THF (16 mL). Bis(pinacolato)diboron (486 mg, 1.91

mmol), potassium acetate (375 mg, 3.83 mmol), and Pd(dppf)Cl₂·

CH₂Cl₂(78 mg, 0.096 mmol) were added, and the red reaction

mixture was stirred for 15 h at 70 °C. Further bis(pinacolato)diboron

(486 mg, 1.91 mmol), potassium acetate (130 mg, 1.33 mmol), and

Pd(dppf)Cl₂·CH₂Cl₂(78 mg, 0.096 mmol) were added, and stirring

was continued at 70 °C for additional 4 h. The black reaction mixture

was treated with EtOAc, ﬁltered, and evaporated to dryness under

reduced pressure. The dark brown residue was puriﬁed by ﬂash

chromatography (heptane/DCM, gradient) to give tert-butyl 3-

[bis(tert-butoxycarbonyl)amino]-6-(4,4,5,5-tetramethyl-1,3,2-dioxabor-

olan-2-yl)indazole-1-carboxylate (1.00 g, 90% pure, 84% corrected

yield) as a yellow solid which was used directly in the next step. LC−

MS (method B, ESI, m/z) t_R= 3.94 min, 559/560 (M + H)⁺.

tert-Butyl 3-[bis(tert-butoxycarbonyl)amino]-6-(4,4,5,5-tetramethyl-

1,3,2-dioxaborolan-2-yl)indazole-1-carboxylate (90% pure, 1.00 g, 1.61

mmol) was treated with HCl in dioxane (25 mL). The pale yellow

solution was stirred at rt for 15 h. The solution was evaporated to

dryness and the residue was treated with diethyl ether to obtain a beige

solid. The mixture was ﬁltered and the residue was washed with diethyl

ether to aﬀord (3-amino-1H-indazol-6-yl)boronic acid hydrochloride

16 (351 mg, 95% pure, 97% yield) as a beige solid which was used

directly in the next step. LC−MS (method B, ESI, m/z) t_R= 1.34 min,

177/178 (M + H)⁺.

8-(3-(3-Amino-1H-indazol-6-yl)-5-chloropyridin-4-yl)-2,8-

diazaspiro[4.5]decan-1-one (25). 8-(3-Bromo-5-chloropyridin-4-

yl)-2,8-diazaspiro[4.5]decan-1-one²⁷8 (92% pure, 180 mg, 0.480

mmol) and (3-amino-1H-indazol-6-yl)boronic acid hydrochloride 16

(95% pure, 162 mg, 0.720 mmol) were dissolved in acetonitrile (8

mL), and 0.5 M sodium carbonate in water (2.8 mL, 1.4 mmol) and

Pd(dppf)Cl₂·CH₂Cl₂(20 mg, 0.020 mmol) were added. The reaction

mixture was stirred for 1 h at 120 °C under microwave irradiation,

diluted with acetonitrile (10 mL), ﬁltered and the ﬁltrate evaporated to

dryness. The residue was puriﬁed by preparative HPLC (method A, 20

min gradient elution from 2:98 to 20:80 acetonitrile/water). The pure

fractions were combined and evaporated down to a volume of 20 mL.

The solution was neutralized with solid NaHCO₃and extracted with

EtOAc (3 × 30 mL). The organic layer was dried over Na₂SO₄and

evaporated to dryness. The residue was suspended in diethyl ether and

ﬁltered under vacuum to aﬀord 8-(3-(3-amino-1H-indazol-6-yl)-5-

chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one 25 (85 mg, 45%

yield) as a colorless solid. ¹H NMR (500 MHz, CDCl₃) δ 8.45 (s, 1H),

8-(3-Chloro-5-(1-methyl-2,2-dioxido-1,3-dihydrobenzo[c]-

isothiazol-5-yl)pyridin-4-yl)-1-oxa-3,8-diazaspiro[4.5]decan-2-

one (42). 8-(3-Bromo-5-chloropyridin-4-yl)-1-oxa-3,8-diazaspiro-

[4.5]decan-2-one 27 (2.54 g, 7.33 mmol) was suspended in

acetonitrile (60 mL). 1-Methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxabor-

olan-2-yl)-1,3-dihydrobenzo[c]isothiazole 2,2-dioxide 14 (2.72 g, 8.79

mmol), 0.5 M sodium carbonate in water (29.3 mL, 14.6 mmol), and

(Pd(dppf)Cl₂·CH₂Cl₂complex (299 mg, 0.366 mmol) were added.

The reaction mixture was stirred at 80 °C for 3 h. After addition of

EtOAc and water, the organic layer was separated and washed with

water, dried over MgSO₄, ﬁltered, and evaporated to dryness. The

crude material was puriﬁed by ﬂash chromatography (Companion,

DCM/MeOH 100:0 to 90:10) to yield in 1.61 g (49% yield) of 8-(3-

chloro-5-(1-methyl-2,2-dioxido-1,3-dihydrobenzo[c]isothiazol-5-yl)-

pyridin-4-yl)-1-oxa-3,8-diazaspiro[4.5]decan-2-one 42 as a colorless

1

solid. H NMR (500 MHz, DMSO-d₆) δ 8.46 (s, 1H), 8.17 (s, 1H),

7.50 (s, 1H), 7.37 (s, 1H), 7.32 (dd, J = 8.2, 1.9 Hz, 1H), 7.08 (d, J =

8.2 Hz, 1H), 4.72 (s, 2H), 3.20 (s, 2H), 3.10 (s, 3H), 2.96−2.82 (m,

4H), 1.77−1.66 (m, 4H); ¹³C NMR (126 MHz, DMSO-d₆) δ 157.7,

152.1, 150.6, 148.8, 141.2, 133.2, 130.2, 130.0, 127.3, 126.0, 118.7,

109.5, 78.7, 50.1, 50.0, 47.1, 35.8, 26.1; LC−MS (method C, ESI, m/z)

t_R= 1.87 min, 449/451 (M + H)⁺; ESI-HRMS calcd for

C₂₀H₂₂³⁵ClN₄O₄S (M + H)⁺449.1045, found 449.1032.

O

DOI: 10.1021/acs.jmedchem.5b01685

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

8-(3-Bromo-5-chloropyridin-4-yl)-1-(2-methoxyethyl)-1,8-

diazaspiro[4.5]decane (36). To a suspension of 8-boc-1,8-diaza-

spiro[4.5]decane oxalate (202 mg, 0.839 mmol) in acetonitrile (16.8

mL) were added potassium carbonate (348 mg, 2.52 mmol),

potassium iodide (139 mg, 0.839 mmol), and 2-bromoethyl methyl

ether (95 μL, 1.0 mmol). The reaction was heated at 80 °C overnight

and ﬁltered. The ﬁltrate was concentrated and the crude was puriﬁed

by Biotage column chromatography (eluting with 1−8% MeOH/aq

NH₃(10:1) in DCM) to give tert-butyl 1-(2-methoxyethyl)-1,8-

diazaspiro[4.5]decane-8-carboxylate (160 mg, 64% yield) as a colorless

oil. ¹H NMR (500 MHz, CD₃OD) δ 4.11 (br d, J = 14.2 Hz, 2H), 3.53

(t, J = 5.8 Hz, 2H), 3.36 (s, 3H), 3.05−2.67 (m, 6H), 1.89 (s, 4H),

1.67−1.57 (m, 2H), 1.47 (s, 9H), 1.44−1.37 (m, 2H); LC−MS

(method C, ESI, m/z) t_R= 1.75 min, 299 (M + H)⁺.

¹⁹F NMR (500 MHz, CDCl₃) δ −135; LC−MS (method C, ESI, m/z)

t_R= 2.46 min, 328/330 (M + H)⁺.

8-(3-Fluoro-5-(1-methyl-2,2-dioxido-1,3-dihydrobenzo[c]-

isothiazol-5-yl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one

(61). 8-(3-Bromo-5-ﬂuoropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-

one 58 (40 mg, 0.12 mmol), 1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-

dioxaborolan-2-yl)-1,3-dihydrobenzo[c]isothiazole 2,2-dioxide 14 (49

mg, 0.16 mmol), and Pd(dppf)Cl₂·CH₂Cl₂(4.98 mg, 6.09 μmol) were

loaded in a microwave vial, and acetonitrile (2.1 mL) and 0.5 M

sodium carbonate in water (341 μL, 0.171 mmol) were added. The

reaction was stirred at 120 °C for 60 min under microwave irradiation.

After evaporation of the solvents, the crude material was puriﬁed by

Biotage column chromatography (DCM/EtOH 98:2 to 95:5) and by

SCX-2 column chromatography (loading with DCM/MeOH, elution

with 1 N NH₃in MeOH) to give 8-(3-ﬂuoro-5-(1-methyl-2,2-dioxido-

1,3-dihydrobenzo[c]isothiazol-5-yl)pyridin-4-yl)-2,8-diazaspiro[4.5]-

3-Bromo-4,5-dichloropyridine²⁷7 (70 mg, 0.31 mmol) and tert-

butyl 1-(2-methoxyethyl)-1,8-diazaspiro[4.5]decane-8-carboxylate

(120 mg, 0.402 mmol) were introduced in a microwave vial, and

then 1-methoxy-2-propanol (773 μL) and triethylamine (130 μL,

0.928 mmol) were added. The reaction mixture was stirred for 2 h at

220 °C under microwave irradiation. The solvent was evaporated and

the crude material was puriﬁed by Biotage column chromatography

(eluting with 2−5% MeOH/aq NH₃(10:1) in DCM) to give 8-(3-

bromo-5-chloropyridin-4-yl)-1-(2-methoxyethyl)-1,8-diazaspiro[4.5]-

decane 36 (86 mg, 72% yield) as a colorless oil. ¹H NMR (500 MHz,

CD₃OD) δ 8.49 (s, 1H), 8.36 (s, 1H), 3.53 (t, J = 6.0 Hz, 2H), 3.45

(td, J = 12.6, 2.3 Hz, 2H), 3.37 (s, 3H), 3.34−3.28 (m, 2H), 2.91 (t, J

= 6.6 Hz, 2H), 2.76 (t, J = 6.0 Hz, 2H), 2.00−1.85 (m, 6H), 1.46−1.40

(m, 2H); LC−MS (method C, ESI, m/z) t_R= 1.72 min, 388/390/392

(M + H)⁺.

1

decan-1-one 61 (33 mg, 63% yield) as a cream solid. H NMR (500

MHz, CDCl₃) δ 8.26 (d, J = 4.2 Hz, 1H), 8.09 (s, 1H), 7.47 (dd, J =

8.2, 1.7 Hz, 1H), 7.44 (d, J = 1.7 Hz, 1H), 6.83 (d, J = 8.2 Hz, 1H),

5.78 (s, 1H), 4.46 (s, 2H), 3.32 (t, J = 6.9 Hz, 2H), 3.26−3.19 (m,

2H), 3.18 (s, 3H), 3.03−2.94 (m, 2H), 2.05 (t, J = 6.8 Hz, 2H), 1.81

(ddd, J = 13.4, 11.3, 4.2 Hz, 2H), 1.36−1.30 (m, 2H); ¹⁹F NMR (500

MHz, CDCl₃) δ −139; ¹³C NMR (126 MHz, CDCl₃) δ 181.2, 154.6

(d, J = 253.3 Hz), 147.5 (d, J = 3.9 Hz), 143.8, 141.6, 138.1 (d, J =

25.6 Hz), 131.3, 130.3, 130.1, 126.1, 118.1, 109.4, 50.9, 47.5 (d, J = 5.0

Hz), 41.5, 38.7, 32.3, 31.8, 26.6; LC−MS (method C, ESI, m/z) t_R=

1.69 min, 431 (M + H)⁺; ESI-HRMS calcd for C₂₁H₂₄FN₄O₃S (M +

H)⁺431.1548, found 431.1590.

8-(3-Bromo-5-ﬂuoropyridin-4-yl)-1-oxa-3,8-diazaspiro[4.5]-

decan-2-one (59). 8-Benzyl-1-oxa-3,8-diaza-spiro[4.5]decan-2-one

(7.40 g, 98% pure) was dissolved in MeOH (34 mL), THF (17

mL), and acetic acid (8 mL). Pd/C 5% (54.1% H₂O, 4.00 g) was

added, and the reaction mixture was stirred at rt under hydrogen for

16 h. The catalyst was then ﬁltered oﬀ and the ﬁltrate was evaporated

to dryness. The residue was triturated with diethyl ether/diethyl ether,

ﬁltered and dried in vacuum to yield 6.16 g (97% yield) of 1-oxa-3,8-

diazaspiro[4.5]decan-2-one acetate 38 as a white solid which was used

directly in the next step. LC−MS (method B, ESI, m/z) t_R= 0.39 min,

157 (M + H)⁺.

5-(5-Chloro-4-(1-(2-methoxyethyl)-1,8-diazaspiro[4.5]-

decan-8-yl)pyridin-3-yl)-1-methyl-1,3-dihydrobenzo[c]-

isothiazole 2,2-Dioxide (54). 8-(3-Bromo-5-chloropyridin-4-yl)-1-

(2-methoxyethyl)-1,8-diazaspiro[4.5]decane 36 (27 mg, 0.069 mmol),

1-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3-dihydro-

benzo[c]isothiazole 2,2-dioxide 14 (21 mg, 0.069 mmol), and

Pd(dppf)Cl₂·CH₂Cl₂(2.8 mg, 3.5 μmol) were loaded in a microwave

vial, and then acetonitrile (1.2 mL) and 0.5 M sodium carbonate in

water (194 μL, 0.0970 mmol) were added. The reaction was stirred at

120 °C for 60 min. The solvents were evaporated and the crude

material was puriﬁed by Biotage column chromatography (eluting with

1−5% MeOH/aq NH₃(10:1) in DCM) and further puriﬁed by SCX-2

column chromatography (loading with DCM:MeOH, elution with 1 N

NH₃in MeOH) to aﬀord 5-(5-chloro-4-(1-(2-methoxyethyl)-1,8-

diazaspiro[4.5]decan-8-yl)pyridin-3-yl)-1-methyl-1,3-dihydrobenzo[c]-

3-Bromo-4-chloro-5-ﬂuoropyridine 57 (656 mg, 3.12 mmol) and 1-

oxa-3,8-diazaspiro[4.5]decan-2-one acetate 38 (1.34 g, 6.23 mmol)

were introduced in a microwave vial, and 1-methoxy-2-propanol (7.8

mL) and triethylamine (1.34 mL, 9.35 mmol) were added, and the

reaction mixture was stirred at 220 °C for 1 h under microwave

irradiation. The solvent was evaporated and the crude material was

puriﬁed by Biotage column chromatography (DCM/EtOH 98:2 to

95:5) to aﬀord 8-(3-bromo-5-ﬂuoropyridin-4-yl)-1-oxa-3,8-diaza-

1

isothiazole 2,2-dioxide 54 (14 mg, 41% yield) as a colorless oil. H

NMR (500 MHz, acetone-d₆) δ 8.38 (s, 1H), 8.17 (s, 1H), 7.45 (s,

1H), 7.40−7.37 (m, 1H), 7.05 (d, J = 8.2 Hz, 1H), 4.59 (s, 2H), 3.37

(t, J = 6.5 Hz, 2H), 3.27 (s, 3H), 3.16 (s, 3H), 3.10−3.05 (m, 2H),

2.88−2.73 (m, 4H), 2.60 (t, J = 6.5 Hz, 2H), 1.73−1.59 (m, 6H),

1.16−1.09 (m, 2H); ¹³C NMR (126 MHz, CD₃OD) δ 153.8, 149.6,

148.6, 141.8, 134.2, 130.6, 130.3, 128.0, 126.2, 118.5, 109.0, 71.5, 57.6,

50.8, 49.1, 47.2, 32.3, 31.0, 25.1, 20.0 (Cq and CH₂of the sultam not

observed); LC−MS (method C, ESI, m/z) t_R= 1.65 min, 491/493 (M

+ H)⁺; ESI-HRMS calcd for C₂₄H₃₂³⁵ClN₄O₃S (M + H)⁺491.1878,

found 491.1878.

1

spiro[4.5]decan-2-one 59 (380 mg, 37% yield) as a cream solid. H

NMR (500 MHz, CDCl₃) δ 8.42 (s, 1H), 8.25 (d, J = 3.4 Hz, 1H),

6.20 (s, 1H), 3.58−3.50 (m, 2H), 3.42 (s, 2H), 3.31−3.24 (m, 2H),

2.12−2.06 (m, 2H), 1.99−1.92 (m, 2H); ¹⁹F NMR (500 MHz,

CDCl₃) δ −135; LC−MS (method C, ESI, m/z) t_R= 2.32 min, 330/

332 (M + H)⁺.

8-(3-Fluoro-5-(1-methyl-2,2-dioxido-1,3-dihydrobenzo[c]-

isothiazol-5-yl)pyridin-4-yl)-1-oxa-3,8-diazaspiro[4.5]decan-2-

one (63). 8-(3-Bromo-5-ﬂuoropyridin-4-yl)-1-oxa-3,8-diazaspiro[4.5]-

decan-2-one 59 (50 mg, 0.15 mmol), 1-methyl-5-(4,4,5,5-tetramethyl-

1,3,2-dioxaborolan-2-yl)-1,3-dihydrobenzo[c]isothiazole 2,2-dioxide 14

(52 mg, 0.17 mmol), and Pd(dppf)Cl₂·CH₂Cl₂(6.2 mg, 7.6 μmol)

were loaded in a microwave vial, and acetonitrile (2.6 mL) and 0.5 M

sodium carbonate in water (424 μL, 0.212 mmol) were added. The

reaction mixture was stirred at 120 °C for 60 min. After evaporation of

the solvent, the crude material was puriﬁed by Biotage column

chromatography (DCM/EtOH 98:2 to 95:5) and by SCX-2 column

chromatography (loading with DCM/MeOH, elution with 1 N NH₃

in MeOH) to aﬀord 8-(3-ﬂuoro-5-(1-methyl-2,2-dioxido-1,3-dihydro-

benzo[c]isothiazol-5-yl)pyridin-4-yl)-1-oxa-3,8-diazaspiro[4.5]decan-

Preparation of Compounds in Table 4, Exempliﬁed by

Compounds 61, 63, and 72. 8-(3-Bromo-5-ﬂuoropyridin-4-yl)-

2,8-diazaspiro[4.5]decan-1-one (58). 3-Bromo-4-chloro-5-ﬂuoro-

pyridine 57 (300 mg, 1.43 mmol) and tert-butyl 1-oxo-2,8-diaza-

spiro[4.5]decane-8-carboxylate (471 mg, 1.85 mmol) were introduced

in a microwave vial, and 1-methoxy-2-propanol (3.5 mL) and

triethylamine (601 μL, 4.28 mmol) were added. The reaction mixture

was stirred for 1 h at 220 °C under microwave irradiation. The solvent

was evaporated and the crude material was puriﬁed by Biotage column

chromatography (DCM/EtOH 98:2 to 92:8) to aﬀord 8-(3-bromo-5-

ﬂuoropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one 58 (140 mg, 30%

1

yield) as a white solid. H NMR (500 MHz, CDCl₃) δ 8.39 (s, 1H),

2-one 63 (44 mg, 67% yield) as white solid. H NMR (500 MHz,

8.22 (d, J = 3.6 Hz, 1H), 6.86 (s, 1H), 3.51−3.43 (m, 2H), 3.40−3.35

(m, 2H), 3.24−3.16 (m, 2H), 2.16−2.07 (m, 4H), 1.57−1.50 (m, 2H);

CDCl₃) δ 8.26 (d, J = 4.1 Hz, 1H), 8.08 (s, 1H), 7.39 (dd, J = 8.2, 1.8

Hz, 1H), 7.30 (s, 1H), 6.81 (d, J = 8.2 Hz, 1H), 5.78 (s, 1H), 4.40 (s,

P

DOI: 10.1021/acs.jmedchem.5b01685

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

2H), 3.32 (s, 2H), 3.28−3.16 (m, 5H), 3.06−3.03 (m, 2H), 1.92−1.81

(m, 2H), 1.72−1.64 (m, 2H); ¹⁹F NMR (500 MHz, CDCl₃) δ −139;

¹³C NMR (126 MHz, CDCl₃) δ 158.5, 154.4 (d, J = 253.0 Hz), 147.9

(d, J = 4.0 Hz), 143.1 (d, J = 7.4 Hz), 141.5, 138.0 (d, J = 25.5 Hz),

131.0, 130.3, 130.2, 125.6, 117.9, 109.3, 79.9, 51.3, 50.7, 47.0 (d, J =

4.6 Hz), 36.3, 26.5; LC−MS (method C, ESI, m/z) t_R= 1.70 min, 433

(M + H)⁺; ESI-HRMS calcd for C₂₀H₂₂FN₄O₄S (M + H)⁺431.1340,

found 433.1339.

added a solution of 8-(3-iodo-5-(triﬂuoromethyl)pyridin-4-yl)-1-oxa-

3,8-diazaspiro[4.5]decan-2-one 68 (71% pure, 78 mg, 0.13 mmol) in

acetonitrile (4 mL) and 0.5 M sodium carbonate solution (780 μL,

0.390 mmol). The reaction mixture was stirred at 120 °C for 1 h under

microwave irradiation and diluted with acetonitrile (5 mL), ﬁltered

and the ﬁltrate concentrated. The crude product was puriﬁed by

preparative HPLC (method A, 20 min gradient elution from 2:98 to

25:75 acetonitrile/water to give 8-(3-(4-(1-methyl-1H-pyrazol-4-yl)-

phenyl)-5-(triﬂuoromethyl)pyridin-4-yl)-1-oxa-3,8-diazaspiro[4.5]-

decan-2-one 2,2,2-triﬂuoroacetate 72 (18 mg, 24% yield) as a colorless

4-Chloro-3,5-diiodopyridine (64). In a 3 L three-necked round-

bottom ﬂask, 1,4-dihydropyridin-4-one (95% pure, 50 g, 0.50 mol) and

N-iodosuccinimide (97% pure, 232 g, 1.00 mmol) were suspended in

acetonitrile (1 L). The reaction mixture was heated under reﬂux for 3

h and then cooled with an ice bath, ﬁltered and the ﬁltrate washed with

acetonitrile (150 mL). The light yellow solid was dried at 60 °C under

reduced pressure for 15 h to obtain 3,5-diiodopyridin-4-ol³⁶(165 g,

95% yield) as a light yellow solid which was used directly in the next

step. LC−MS (method B, ESI, m/z) t_R= 1.34 min, 347 (M + H)⁺.

In a 3 L three-necked round-bottom ﬂask, 3,5-diiodopyridin-4-ol

(150 g, 432 mmol) was suspended in DMF (1 L). This mixture was

warmed to 70 °C, and phosphoryl chloride (39.7 mL, 432 mmol) was

slowly added in a dropwise manner (caution: slightly exothermic

reaction). After completion of the addition of phosphoryl chloride, the

reaction mixture was then heated to 95 °C for 30 min. The dark brown

mixture was cooled to rt and slowly poured into 6 L of ice−water. A

beige precipitate formed. NaHCO₃was added slowly until no more

gas formation was observed. The solid was ﬁltered and washed with

water (2 L). The residue was suspended in acetonitrile (800 mL) and

ﬁltered again. The residue was washed with acetonitrile (100 mL) and

dried at 60 °C under reduced pressure for 15 h to give 4-chloro-3,5-

diiodopyridine³⁷64 as a yellow solid, which was used without further

puriﬁcation (142 g, 95% pure, 85% corrected yield). LC−MS (method

B, ESI, m/z) t_R= 3.06 min, 366/368 (M + H)⁺.

1

solid. H NMR (500 MHz, DMSO-d₆) δ 8.82 (s, 1H), 8.53 (s, 1H),

8.24 (d, J = 0.8 Hz, 1H), 7.96 (d, J = 0.8 Hz, 1H), 7.71 (d, J = 8.2 Hz,

2H), 7.45 (s, 1H), 7.40 (d, J = 8.2 Hz, 2H), 3.88 (s, 3H), 3.13 (s, 2H),

2.96−2.84 (m, 4H), 1.69−1.56 (m, 4H); ¹⁹F NMR (500 MHz,

DMSO-d₆, TFA salt) δ −74.5; ¹³C NMR (126 MHz, DMSO-d₆, free

base) δ 157.7, 155.9, 155.3, 147.7 (d, J = 7.0 Hz), 136.5, 136.2, 134.1,

132.5, 130.0, 128.2, 124.9, 124.0 (q, J = 273 Hz), 121.3, 78.5, 50.0,

48.2, 38.8, 35.5; LC−MS (method C, ESI, m/z) t_R= 2.64 min, 458 (M

+ H)⁺; ESI-HRMS calcd for C₂₃H₂₃F₃N₅O₂(M + H)⁺458.1798,

found 458.1785.

Preparation of Compounds in Table 5, exempliﬁed by

Compounds 86, 92, and 95. 5-Bromo-3,4-dichloropyridin-2-

amine (73). N-Bromosuccinimide (10.9 g, 61.3 mmol) was added to a

solution of 4-chloro-2-aminopyridine (7.50 g, 58.3 mmol) in dry

acetonitrile (130 mL) at rt under a nitrogen atmosphere. The yellow

solution was stirred for 3 h before the mixture was concentrated in

vacuo. The resulting crude product was puriﬁed by Biotage column

chromatography (cyclohexane/EtOAc 5:1 to 1:1) to give 5-bromo-4-

chloropyridin-2-amine³⁸as a yellow solid (10.0 g, 83% yield). ¹H

NMR (500 MHz, CDCl₃) δ 8.16 (s, 1H), 6.62 (s, 1H), 4.57 (s, 2H);

LC−MS (method C, ESI, m/z) t_R= 2.04 min, 207/209/211 (M +

H)⁺.

N-Chlorosuccinimide (6.11 g, 45.8 mmol) was added portionwise

to a solution of 5-bromo-4-chloropyridin-2-amine (10.0 g, 48.2 mmol)

in dry acetonitrile (180 mL) at rt under a nitrogen atmosphere. The

reaction mixture was heated to 95 °C for 3 h and then cooled to rt.

The resultant solid was ﬁltered oﬀ and washed with acetonitrile to give

the desired compound as a light brown solid. The ﬁltrate was

concentrated to 50 mL, and the resulting precipitate was ﬁltered and

the resultant solid collected. The two batches of solid material were

combined and recrystallized from hot acetonitrile to give two batches

of 5-bromo-3,4-dichloropyridin-2-amine 73 (P1, 6.8 g; P2, 2.0 g; 75%

yield) which were used in the next step without further puriﬁcation. ¹H

NMR (500 MHz, CDCl₃) δ 8.10 (s, 1H), 5.06 (s, 2H); LC−MS

(method C, ESI, m/z) t_R= 2.93 min, 241/243/245 (M + H)⁺.

5-Bromo-3,4-dichloro-N,N-bis(4-methoxybenzyl)pyridin-2-

amine (81). A solution of 5-bromo-3,4-dichloropyridin-2-amine 73

(2.37 g, 4.91 mmol) in dry DMF (50 mL) at 0 °C was treated

portionwise with sodium hydride (60% in mineral oil) (0.775 g, 18.6

mmol) and the mixture stirred at 0 °C for 15 min before a solution of

p-methoxybenzyl chloride (2.10 mL, 15.5 mmol) in dry DMF (1 mL)

was added. The mixture was stirred at rt for 2 h before sat. NH₄Cl

solution and EtOAc were added. The aqueous layer was extracted

three times with EtOAc. The combined organic layers were dried over

MgSO₄, ﬁltered, and concentrated in vacuo. The resulting brown

residue was puriﬁed by Biotage column chromatography (cyclo-

hexane/DCM 100:0 to 75:25) to give 5-bromo-3,4-dichloro-N,N-

bis(4-methoxybenzyl)pyridin-2-amine 81 (2.4 g, 80% yield) as a

colorless oil. ¹H NMR (500 MHz, CDCl₃) δ 8.26 (s, 1H), 7.20 (d, J =

8.6 Hz, 4H), 6.85 (d, J = 8.6 Hz, 4H), 4.42 (s, 4H), 3.81 (s, 6H); LC−

MS (method C, ESI, m/z) t_R= 3.51 min, 361/363/365 (M + H-

PMB)⁺.

8-(3,5-Diiodopyridin-4-yl)-1-oxa-3,8-diazaspiro[4.5]decan-2-

one (66). In a 50 mL round-bottom ﬂask, 4-chloro-3,5-diiodopyridine

64 (500 mg, 1.37 mmol) and 1-oxa-3,8-diazaspiro[4.5]decan-2-one

acetate 38 (448 mg, 2.05 mmol) were suspended in NMP (10 mL)

and triethylamine (570 μL, 4.11 mmol). The reaction mixture was

stirred at 240 °C for 3 h and then cooled and poured into water (150

mL). The resulting precipitate was ﬁltered and washed with water (20

mL). The solid was dissolved in DCM (150 mL), the solution was

dried over Na₂SO₄, ﬁltered, and concentrated to dryness. The crude

residue was puriﬁed by ﬂash column chromatography (Companion,

DCM/MeOH 100:0 to 90:10) to give 8-(3,5-diiodopyridin-4-yl)-1-

oxa-3,8-diazaspiro[4.5]decan-2-one (196 mg, 30% yield) 66 as a light

brown solid which was used directly in the next step. LC−MS

(method B, ESI, m/z) t_R= 2.26 min, 486 (M + H)⁺.

8-(3-Iodo-5-(triﬂuoromethyl)pyridin-4-yl)-1-oxa-3,8-diaza-

spiro[4.5]decan-2-one (68). Into a 10 mL Schlenck, silver(I)

ﬂuoride (57 mg, 0.45 mmol) was loaded and DMF (3 mL) and

(triﬂuoromethyl)trimethylsilane (81 μL, 0.54 mmol) were added at rt,

and the resulting brown suspension was stirred for 15 min at rt. Then

ﬁnely powdered copper (43 mg, 0.67 mmol) was added and the

resulting dark red suspension was stirred for 1.5h at rt. The reaction

mixture turned green, and a silver precipitate formed on the vessel

wall. 8-(3,5-Diiodopyridin-4-yl)-1-oxa-3,8-diazaspiro[4.5]decan-2-one

66 (196 mg, 0.403 mmol) was added, and the suspension was stirred

at 90 °C for 3 h. The green suspension was diluted with DMF (10

mL) and ﬁltered over Celite. The ﬁltrate was evaporated to dryness.

The crude residue was dissolved in DCM (10 mL) and treated with

diethyl ether (35 mL). The resulting light brown precipitate was

ﬁltered and washed with diethyl ether (10 mL), The ﬁltrate was

evaporated to dryness to yield in 8-(3-iodo-5-triﬂuoromethylpyridin-4-

yl)-1-oxa-3,8-diaza-spiro[4.5]decan-2-one 68 as a yellow oil (78 mg,

71% pure, 32% corrected yield) which was used directly in the next

step. LC−MS (method B, ESI, m/z) t_R= 2.45 min, 428 (M + H)⁺.

8 - ( 3 - ( 4 - ( 1 - M e t h y l - 1 H - p y r a z o l - 4 - y l ) p h e n y l ) - 5 -

(triﬂuoromethyl)pyridin-4-yl)-1-oxa-3,8-diazaspiro[4.5]decan-

2-one 2,2,2-Triﬂuoroacetate (72). To 1-methyl-4-(4-(4,4,5,5-

tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-1H-pyrazole²⁷37 (126

mg, 0.390 mmol) and Pd(dppf)Cl₂·CH₂Cl₂(5 mg, 0.01 mmol) was

8-(2-(Bis(4-Methoxybenzyl)amino)-5-bromo-3-chloropyri-

din-4-yl)-2,8-diazaspiro[4.5]decan-1-one (82). 5-Bromo-3,4-di-

chloro-N,N-bis(4-methoxybenzyl)pyridin-2-amine 81 (5.00 g, 10.4

mmol), 8-boc-2,8-diaza-spiro-[4.5]decan-1-one (2.64 g, 10.4 mmol),

and potassium ﬂuoride (1.21 g, 20.7 mmol) were loaded in a

microwave vial. Triethylamine (4.00 mL, 31.1 mmol) and NMP (25

mL) were added, and the light brown solution was degassed with

argon. The reaction mixture was stirred at 220 °C for 1 h under

Q

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Journal of Medicinal Chemistry

Article

microwave irradiation. After the addition of water, the aqueous layer

was extracted twice with EtOAc and the organic layer was washed with

water, dried over MgSO₄, and concentrated under vacuum. The

resulting brown oil was puriﬁed by Biotage column chromatography

(DCM/EtOH, 97:3 to 90:10) to give 8-(2-(bis(4-methoxybenzyl)-

amino)-5-bromo-3-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one

chloro-3-ﬂuoropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one 77 (105

mg, 64% yield) as a cream solid. H NMR (500 MHz, DMSO-d₆) δ

1

7.67 (s, 1H), 7.59 (s, 1H), 6.17 (s, 2H), 3.35−3.27 (m, 2H), 3.19 (t, J

= 6.8 Hz, 2H), 3.12−3.05 (m, 2H), 2.01 (t, J = 6.8 Hz, 2H), 1.79 (td, J

= 12.4, 4.2 Hz, 2H), 1.44−1.38 (m, 2H); ¹⁹F NMR (500 MHz,

DMSO-d₆) δ −149; LC−MS (method C, ESI, m/z) t_R= 1.73 min,

299/301 (M + H)⁺.

1

82 (3.0 g, 54% yield) as a light brown solid. H NMR (500 MHz,

8-(2-Amino-3-ﬂuoro-5-(1-methyl-2,2-dioxido-1,3-dihydro-

benzo[c]isothiazol-5-yl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-

1-one (92). 8-(2-Amino-5-chloro-3-ﬂuoropyridin-4-yl)-2,8-diazaspiro-

[4.5]decan-1-one 7 (20 mg, 0.067 mmol), 1-methyl-5-(4,4,5,5-

tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3-dihydrobenzo[c]isothiazole

2,2-dioxide 14 (27 mg, 0.087 mmol), and PdCl₂(Pcy₃)₂(2.5 mg, 3.4

μmol) were loaded in a microwave vial. Acetonitrile (1.1 mL) and 0.5

M sodium carbonate in water (187 μL, 0.0940 mmol) were added, and

the reaction mixture was stirred at 150 °C for 30 min under microwave

irradiation. After evaporation of the solvent, the crude material was

puriﬁed by Biotage column chromatography (DCM/EtOH 98:2 to

90:10) to aﬀord 8-(2-amino-3-ﬂuoro-5-(1-methyl-2,2-dioxido-1,3-

dihydrobenzo[c]isothiazol-5-yl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-

CDCl₃) δ 8.15 (br s, 1H), 7.19 (d, J = 8.7 Hz, 4H), 6.84 (d, J = 8.7 Hz,

4H), 5.62 (s, 1H), 4.38 (s, 4H), 3.80 (s, 6H), 3.42−3.32 (m, 6H),

2.25−2.15 (m, 4H), 1.68−1.46 (m, 2H); LC−MS (method C, ESI, m/

z) t_R= 3.68 min, 599/601 (M + H)⁺.

8-(2-Amino-3-chloro-5-(4-(1-(2-hydroxy-2-methylpropyl)-

1H-pyrazol-4-yl)phenyl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-

1-one (86). 8-(2-(Bis(4-Methoxybenzyl)amino)-5-bromo-3-chloro-

pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one 82 (203 mg, 0.339

mmol) and 2-methyl-1-(4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-

2-yl)phenyl)-1H-pyrazol-1-yl)propan-2-ol 9 (73% pure, 190 mg, 0.407

mmol) were dissolved in acetonitrile (5 mL). 0.5 M aqueous sodium

carbonate (1.40 mL, 0.678 mmol) and Pd(dppf)Cl₂·CH₂Cl₂(25 mg,

0.034 mmol) were added. The reaction mixture was stirred at 120 °C

for 1 h under microwave irradiation, and the solvent was evaporated.

The crude residue was puriﬁed by ﬂash chromatography (n-heptane/

EtOAc, gradient and then with DCM/MeOH, gradient) to give 8-(2-

(bis(4-methoxybenzyl)amino)-3-chloro-5-(4-(1-(2-hydroxy-2-methyl-

propyl)-1H-pyrazol-4-yl)phenyl)pyridin-4-yl)-2,8-diazaspiro[4.5]-

decan-1-one (81 mg, 60% pure, 19% corrected yield) as a light-brown

solid which was used directly in the next step. LC−MS (method A,

ESI, m/z) t_R= 2.59 min, 735/737 (M + H)⁺.

1

1-one 92 (17 mg, 57% yield) as a cream solid. H NMR (500 MHz,

DMSO-d₆) δ 7.54 (s, 1H), 7.48 (s, 1H), 7.45 (d, J = 1.8 Hz, 1H), 7.41

(dd, J = 8.3, 1.8 Hz, 1H), 6.97 (d, J = 8.3 Hz, 1H), 6.02 (s, 2H), 4.67

(s, 2H), 3.12 (t, J = 6.8 Hz, 2H), 3.08−3.01 (m, 2H), 3.06 (s, 3H),

2.89 (t, J = 12.2 Hz, 2H), 1.91 (t, J = 6.8 Hz, 2H), 1.61−1.52 (m, 2H),

1.21−1.15 (m, 2H); ¹⁹F NMR (500 MHz, DMSO-d₆) δ −153; ¹³C

NMR (126 MHz, DMSO-d₆) δ 180.5, 150.4 (d, J = 12.8 Hz), 143.9 (d,

J = 5.4 Hz), 142.7 (d, J = 4.3 Hz), 141.1 (d, J = 247 Hz), 140.6, 131.3,

129.5, 126.4, 121.8, 118.7, 109.8, 50.6, 47.5 (d, J = 4.6 Hz), 41.6, 38.3,

32.1, 31.1, 26.6; LC−MS (method C, ESI, m/z) t_R= 1.73 min, 446 (M

+ H)⁺; ESI-HRMS calcd for C₂₁H₂₅FN₅O₃S (M + H)⁺446.1657,

found 446.1656.

8-(2-(Bis(4-methoxybenzyl)amino)-3-chloro-5-(4-(1-(2-hydroxy-2-

methylpropyl)-1H-pyrazol-4-yl)phenyl)pyridin-4-yl)-2,8-diazaspiro-

[4.5]decan-1-one (60% pure, 81 mg, 0.066 mmol) was dissolved in

TFA (1 mL) and stirred at rt overnight. The crude mixture was

concentrated, and the residue was treated with 1 N NaOH. The

mixture was extracted with EtOAc, and the organic layer was dried

over Na₂SO₄, ﬁltered, and concentrated. The crude product was

dissolved in acetonitrile, and the resultant solid precipitate was ﬁltered

and dried in vacuo to aﬀord 8-(2-amino-3-chloro-5-(4-(1-(2-hydroxy-

2-methylpropyl)-1H-pyrazol-4-yl)phenyl)pyridin-4-yl)-2,8-diazaspiro-

5-Bromo-4-chloro-3-(triﬂuoromethyl)pyridin-2-amine (75).

3-(Triﬂuoromethyl)pyridin-2-amine (35.0 g, 0.210 mol) in DMF

(350 mL) was cooled to 0−5 °C under a nitrogen atmosphere. N-

Bromosuccinimide (38.8 g, 0.210 mol) was added portionwise, and the

mixture was stirred at rt for 1 h. The reaction mixture was

concentrated, and the light brown viscous liquid residue was triturated

with ice-cold water (200 mL). The precipitate was ﬁltered and dried to

aﬀord 5-bromo-3-(triﬂuoromethyl)pyridin-2-amine³⁹(45.0 g, 86%

yield) as an oﬀ-white solid that was used directly in the next step.

To a stirred solution of 5-bromo-3-(triﬂuoromethyl)pyridin-2-

amine (25.0 g, 0.100 mol) in THF (250 mL) was added sodium

hydride (60% in mineral oil, 7.35 g, 0.180 mol) portionwise at 0−5 °C

under a nitrogen atmosphere; the reaction mixture was stirred for 1 h

at 0−5 °C. A solution of di-tert-butyl dicarbonate (25.8 mL, 0.110

mol) in THF (50 mL) was added dropwise at 0−5 °C, and stirring was

continued for 3 h. Since the reaction was incomplete, sodium hydride

(60% in mineral oil, 4.08 g, 0.100 mol) was added portionwise at 0−5

°C and stirring continued for 1 h. A solution of di-tert-butyl

dicarbonate (12 mL, 0.050 mol) in THF (20 mL) was added

dropwise, and the reaction mixture was stirred for an additional 2 h at

0−5 °C. The mixture was poured onto ice cold water (250 mL) and

extracted with ethyl acetate (3 × 150 mL). The combined organic

layers were washed with brine (100 mL), dried over Na₂SO₄, and

concentrated under reduced pressure to aﬀord a brown viscous solid.

The residue was puriﬁed by column chromatography (petroleum

ether/EtOAc 100:0 to 94:6) to give tert-butyl (5-bromo-3-

(triﬂuoromethyl)pyridin-2-yl)carbamate (30 g, 74% yield) as an oﬀ-

white solid that was taken to the next step without further puriﬁcation.

To a stirred solution of diisoproylamine (13 mL, 0.090 mol) in

THF (220 mL) cooled to −70 °C under nitrogen atmosphere was

added n-butyllithium (1.6 M solution in hexane, 54 mL, 0.090 mol)

dropwise over a period 0.5 h, and the mixture was stirred for additional

30 min at the same temperature. The mixture was slowly warmed to

−10 °C and stirred for 20 min. After cooling to −70 °C a solution of

(tert-butyl (5-bromo-3-(triﬂuoromethyl)pyridin-2-yl)carbamate (22 g,

0.040 mol) in THF (100 mL) was added dropwise over a period 20

min at −10 °C. The mixture was stirred for 30 min at −70 °C, then a

solution of 1,1,1,2,2,2-hexachloroethane (21 g, 0.090 mol) in THF

1

[4.5]decan-1-one 86 (31 mg, 94% yield) as a beige solid. H NMR

(500 MHz, DMSO-d₆) δ 8.12 (s, 1H), 7.91 (s, 1H), 7.64 (s, 1H), 7.62

(d, J = 8.2 Hz, 2H), 7.51 (s, 1H), 7.23 (d, J = 8.2 Hz, 2H), 6.13 (s,

2H), 4.75 (s, 1H), 4.04 (s, 2H), 3.10 (t, J = 6.8 Hz, 2H), 3.01−2.93

(m, 2H), 2.74−2.61 (m, 2H), 1.82 (t, J = 6.8 Hz, 2H), 1.76−1.65 (m,

2H), 1.25−1.18 (m, 2H), 1.10 (s, 6H); ¹³C NMR (126 MHz, DMSO-

d₆) δ 180.0, 156.3, 153.3, 147.7, 135.8, 135.7, 131.1, 129.6, 128.1,

124.7, 123.6, 121.1, 108.6, 69.3, 62.2, 47.4, 41.4, 37.8, 31.8, 30.4, 27.3;

LC−MS (method C, ESI, m/z) t_R= 1.99 min, 495/497 (M + H)⁺;

ESI-HRMS calcd for C₂₆H₃₂³⁵ClN₆O₂(M + H)⁺495.2270, found

495.2256.

4,5-Dichloro-3-ﬂuoropyridin-2-amine (74). To a solution of

LDA (2 M, 7.97 mL, 15.9 mmol) in THF (31 mL) at −78 °C was

added 5-chloro-3-ﬂuoropyridin-2-amine (934 mg, 6.37 mmol) in

solution in THF (9 mL). After 50 min at −78 °C, hexachloroethane

(1.44 mL, 12.8 mmol) in THF (9 mL) was added. The reaction

mixture was stirred for 40 min and then quenched with NH₄Cl and

extracted with DCM. The crude material was puriﬁed by Biotage

column chromatography (DCM 100%) to aﬀord 4,5-dichloro-3-

1

ﬂuoropyridin-2-amine 74 (950 mg, 82% yield) as a cream solid. H

NMR (500 MHz, CDCl₃) δ 7.92 (d, J = 1.0 Hz, 1H), 4.74 (s, 2H); ¹⁹

F

NMR (500 MHz, CDCl₃) δ −137; LC−MS (method C, ESI, m/z) t_R

= 2.66 min, 181/183/185 (M + H)⁺.

8-(2-Amino-5-chloro-3-ﬂuoropyridin-4-yl)-2,8-diazaspiro-

[4.5]decan-1-one (77). 4,5-Dichloro-3-ﬂuoropyridin-2-amine 74

(100 mg, 0.553 mmol) and tert-butyl 1-oxo-2,8-diazaspiro[4.5]-

decane-8-carboxylate (211 mg, 0.829 mmol) were introduced in a

microwave vial, and NMP (1.4 mL) and triethylamine (233 μL, 1.66

mmol) were added. The reaction mixture was stirred for 2 × 1 h at 220

°C under microwave irradiation. The reaction mixture was

concentrated and the crude material was puriﬁed by Biotage column

chromatography (DCM/EtOH 98:2 to 94:6) to aﬀord 8-(2-amino-5-

R

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Journal of Medicinal Chemistry

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(100 mL) was added dropwise over a period of 30 min and the

reaction mixture stirred for a 1 h at the same temperature. The mixture

was slowly warmed to rt and stirred for additional 20 min, poured onto

ice-cold water (200 mL), and extracted with EtOAc (3 × 200 mL).

The combined organic layers were washed with brine (100 mL), dried

over Na₂SO₄, and concentrated to give the crude product as a brown

viscous solid. The crude product was puriﬁed by column

chromatography (petroleum ether/EtOAc 95:5 to 90:10) to aﬀord

tert-butyl (5-bromo-4-chloro-3-(triﬂuoromethyl)pyridin-2-yl)-

carbamate (9.5 g, 62% yield) as an oﬀ-white solid which was used

directly in the next step.

tert-Butyl (5-bromo-4-chloro-3-(triﬂuoromethyl)pyridin-2-yl)-

carbamate (9.5 g, 0.020 mol) in 1,4-dioxane (48 mL) was cooled to

0−5 °C. HCl in 1,4-dioxane (4 mol/L, 95 mL) was added dropwise

over 20 min. The reaction mixture was stirred at rt for 4 h and then

evaporated to give an oﬀ-white solid. The residue was taken up in

aqueous 10% NaHCO₃. The resulting precipitate was ﬁltered oﬀ and

dried in vacuo. The crude product was puriﬁed by column

chromatography (petroleum ether/EtOAc 100:0 to 90:10) to aﬀord

5-bromo-4-chloro-3-(triﬂuoromethyl)pyridin-2-amine 75 (4.85 g, 70%

yield) as an oﬀ-white solid. ¹H NMR (400 MHz, DMSO-d₆) δ 8.41 (s,

1H), 6.95 (s, 2H); LC−MS (method E, ESI, m/z) t_R= 4.38 min, 275/

277/279 (M + H)⁺.

8-(2-Amino-5-bromo-3-(triﬂuoromethyl)pyridin-4-yl)-1-oxa-

3,8-diazaspiro[4.5]decan-2-one (80). 5-Bromo-4-chloro-3-

(triﬂuoromethyl)pyridin-2-amine 75 (300 mg, 1.09 mmol) and 1-

oxa-3,8-diazaspiro[4.5]decan-2-one 38 (340 mg, 2.18 mmol) were

dissolved in NMP (3 mL), and triethylamine (0.453 mL, 3.27 mmol)

was added. The reaction mixture was stirred for 2 h at 220 °C under

microwave irradiation. The resultant black reaction mixture was

poured into 60 mL of water. The brown precipitate was ﬁltered,

washed with water and diethyl ether, and then dried to give 8-(2-

amino-5-bromo-3-(triﬂuoromethyl)pyridin-4-yl)-1-oxa-3,8-diaza-

spiro[4.5]decan-2-one 80 (280 mg, 89% pure, 58% corrected yield) as

a pale brown solid which was used directly in the next step. LC−MS

(method B, ESI, m/z) t_R= 1.83 min, 395/397 (M + H)⁺.

8-(2-Amino-5-(4-(1-methyl-1H-pyrazol-4-yl)phenyl)-3-

(triﬂuoromethyl)pyridin-4-yl)-1-oxa-3,8-diazaspiro[4.5]decan-

2-one 2,2,2-Triﬂuoroacetate (95). 8-(2-Amino-5-bromo-3-

(triﬂuoromethyl)pyridin-4-yl)-1-oxa-3,8-diazaspiro[4.5]decan-2-one

80 (89% pure, 140 mg, 0.315 mmol) was suspended in acetonitrile (10

mL). 1-Methyl-4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-

phenyl)-1H-pyrazole 37 (81% pure, 166 mg, 0.473 mmol), 0.5 M

aqueous sodium carbonate (1.26 mL, 0.631 mmol), and Pd(dppf)Cl₂·

CH₂Cl₂(13 mg, 0.016 mmol) were added. The reaction mixture was

stirred at 120 °C for 1 h under microwave irradiation. After addition of

EtOAc, the mixture was ﬁltered and the ﬁltrate was concentrated. The

brown oily residue was puriﬁed by preparative HPLC (method A,

gradient elution from 2:98 to 30:70 acetonitrile/water in 20 min) to

aﬀord 8-(2-amino-5-(4-(1-methyl-1H-pyrazol-4-yl)phenyl)-3-

(triﬂuoromethyl)pyridin-4-yl)-1-oxa-3,8-diazaspiro[4.5]decan-2-one

2,2,2-triﬂuoroacetate 95 (46 mg, 25% yield) as a white ﬂuﬀy solid. ¹H

NMR (500 MHz, DMSO-d₆) δ 8.20 (d, J = 0.8 Hz, 1H), 7.92 (d, J =

0.8 Hz, 1H), 7.76 (s, 1H), 7.66 (d, J = 8.2 Hz, 2H), 7.48 (s, 1H), 7.37

(d, J = 8.2 Hz, 2H), 3.87 (s, 3H), 3.13 (s, 2H), 3.12−3.01 (m, 4H),

1.61−1.45 (m, 4H); ¹³C NMR (126 MHz, DMSO-d₆) δ 160.8, 157.6,

154.2, 144.0, 136.2, 133.1, 132.3, 130.1, 128.1, 124.9, 124.0, 121.3,

78.1, 49.8, 48.1, 38.7, 34.5 (1 Cq not observed); LC−MS (method C,

ESI, m/z) t_R= 2.05 min, 473 (M + H)⁺; ESI-HRMS calcd for

C₂₃H₂₄F₃N₆O₂(M + H)⁺473.1907, found 473.1893.

HPLC (method A, gradient elution from 0:100 to 30:70 over 3 min

and to 60:40 over 17 min acetonitrile/water) to give ﬁrst the desired

phenyl (6-bromo-1H-indazol-3-yl)carbamate⁴⁰(54 mg, 3% yield) as a

white solid, which was used directly in the next step, and second

undesired phenyl 3-amino-6-bromo-1H-indazole-1-carboxylate (346

mg) as a white solid. LC−MS (method B, ESI, m/z) t_R= 2.62 min,

332/334 (M + H)⁺.

Phenyl (6-bromo-1H-indazol-3-yl)carbamate (54 mg, 0.16 mmol)

was dissolved in THF (3 mL), and lithium aluminum hydride solution

1.0 M in THF (325 μL, 0.330 mmol) was added dropwise. The

solution turned from colorless to yellow. The mixture was stirred at rt

for 2 h and then diluted with water (2 mL) and THF (8 mL). The

suspension was ﬁltered over Celite and the mother liquor was

evaporated to dryness to give 6-bromo-N-methyl-1H-indazol-3-

amine⁴⁰(41 mg, 89% pure, 99% corrected yield) as a pale brown

solid which was used directly in the next step. LC−MS (method B,

ESI, m/z) t_R= 1.95 min, 226/228 (M + H)⁺.

6-Bromo-N-methyl-1H-indazol-3-amine (89% pure, 41 mg, 0.16

mmol), bis(pinacolato)diboron (82 mg, 0.32 mmol), potassium

acetate (48 mg, 0.48 mmol), and Pd(dppf)Cl₂·CH₂Cl₂(13 mg,

0.020 mmol) were loaded in a microwave vial, and acetonitrile was

added (4 mL). The reaction mixture was stirred for 1 h at 120 °C

under microwave irradiation and then diluted with acetonitrile (10

mL), ﬁltered and the ﬁltrate evaporated to dryness. The crude residue

97 (230 mg, 13% pure, 100% corrected yield) was used in the next

step without puriﬁcation. LC−MS (method B, ESI, m/z) t_R= 1.79

min, 191/192 (M + H)⁺.

8-(3-Chloro-5-(3-(methylamino)-1H-indazol-6-yl)pyridin-4-

yl)-2,8-diazaspiro[4.5]decan-1-one (100). 8-(3-Bromo-5-chloro-

pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one²⁷8 (70.3 mg, 0.20

mmol), (3-(methylamino)-1H-indazol-6-yl)boronic acid 97 (13%

pure, 230 mg, 0.160 mmol) were loaded in a microwave vial, and

acetonitrile (4 mL), 0.5 M aqueous sodium carbonate (1.23 mL, 0.610

mmol) and Pd(dppf)Cl₂·CH₂Cl₂(8 mg, 0.01 mmol) were added. The

reaction mixture was stirred for 1 h at 120 °C under microwave

irradiation. After dilution with acetonitrile (5 mL), the mixture was

ﬁltered and the ﬁltrate was concentrated. The crude product was

puriﬁed by preparative HPLC (method A, 20 min gradient elution

from 2:98 to 20:80 acetonitrile/water) to give 8-(3-chloro-5-(3-

(methylamino)-1H-indazol-6-yl)pyridin-4-yl)-2,8-diazaspiro[4.5]-

1

decan-1-one 100 (13 mg, 12% yield) as a white solid. H NMR (500

MHz, CD₃OD) δ 8.56 (s, 1H), 8.28 (s, 1H), 7.89 (dd, J = 8.4, 0.8 Hz,

1H), 7.41−7.37 (m, 1H), 7.07 (dd, J = 8.4, 1.4 Hz, 1H), 3.41−3.35

(m, 2H), 3.27 (t, J = 6.9 Hz, 2H), 3.06 (s, 3H), 2.97−2.89 (m, 2H),

2.00 (t, J = 6.9 Hz, 2H), 1.92−1.84 (m, 2H), 1.41 (d, J = 13.3 Hz,

2H); LC−MS (method C, ESI, m/z) t_R= 1.74 min, 411/413 (M +

H)⁺; ESI-HRMS calcd for C₂₁H₂₄³⁵ClN₆O (M + H)⁺411.1695, found

411.1680.

tert-Butyl N-tert-Butoxycarbonyl-N-[1-methyl-6-(4,4,5,5-tet-

ramethyl-1,3,2-dioxaborolan-2-yl)indazol-3-yl]carbamate (98).

6-Bromo-1-methyl-1H-indazol-3-amine (500 mg, 2.21 mmol) and

N,N-dimethylpyridin-4-amine (54 mg, 0.44 mmol) were dissolved in

DCM (15 mL). Triethylamine (1.74 mL, 13.3 mmol) and di-tert-butyl

dicarbonate (1.42 mL, 6.64 mmol) were were added, and the reaction

solution was stirred overnight at rt. After addition of water and DCM,

the layers were separated and the aqueous layer was extracted with

DCM. The organic layer was washed with water, dried, ﬁltered, and

evaporated to dryness. The brown solid was puriﬁed by ﬂash-

chromatography (Companion, n-heptane/EtOAc gradient) to give

tert-butyl N-(6-bromo-1-methyl-indazol-3-yl)-N-tert-butoxycarbonyl-

carbamate (150 mg, 16% yield) as a colorless solid which was used

directly in the next step. LC−MS (method B, ESI, m/z) t_R= 3.29 min,

448/450 (M + Na)⁺.

Preparation of Compounds in Table 7, Exempliﬁed by

Compounds 100, 101, and 102. 3-(Methylamino)-1H-indazol-

6-yl)boronic Acid (97). 6-Bromo-1H-indazol-3-amine (95% pure,

1.00 g, 4.48 mmol) was dissolved in pyridine (20 mL) and the mixture

cooled to 0 °C; phenyl chloroformate (0.620 mL, 4.93 mmol) was

added dropwise. and the reaction mixture was stirred at 0 °C for 4 h.

After addition of DCM (50 mL) and water (50 mL), the layers were

separated and the organic layer was washed with brine, dried over

Na₂SO₄, and evaporated to dryness. The residue was puriﬁed by ﬂash

chromatography (n-heptane/EtOAc, gradient) and by preparative

tert-Butyl N-(6-bromo-1-methylindazol-3-yl)-N-tert-butoxycarbonyl-

carbamate (150 mg, 0.352 mmol) was dissolved in THF (5 mL).

Bis(pinacolato)diboron (179 mg, 0.704 mmol), potassium acetate

(104 mg, 1.06 mmol), and Pd(dppf)Cl₂·CH₂Cl₂(29 mg, 0.035 mmol)

were added, and the red reaction mixture was stirred overnight at 70

°C. The resultant black reaction mixture was treated with EtOAc,

ﬁltered and the ﬁltrate evaporated under vacuum. The dark brown

S

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residue was puriﬁed by ﬂash chromatography (Companion, heptane/

DCM gradient) to give a mixture of [3-[bis(tert-butoxycarbonyl)-

amino]-1-methyl-indazol-6-yl]boronic acid and tert-butyl N-tert-

butoxycarbonyl-N-[1-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-

lan-2-yl)indazol-3-yl]carbamate (2:3 ratio, 182 mg, quant) as a pale

yellow solid 98 which was used directly in the next step. LC−MS

(method B, ESI, m/z) t_R= 2.59 min, 413/414 (M + Na)⁺and t_R=

3.49 min, 495/496 (M + Na)⁺.

8-(3-(3-Amino-1-methyl-1H-indazol-6-yl)-5-chloropyridin-4-

yl)-2,8-diazaspiro[4.5]decan-1-one 2,2,2-Triﬂuoroacetate

(101). 8-(3-Bromo-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-

one²⁷8 (84.4 mg, 0.25 mmol) was suspended in acetonitrile (2

mL). A mixture of tert-butyl N-tert-butoxycarbonyl-N-[1-methyl-6-

(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indazol-3-yl]carbamate

and [3-[bis(tert-butoxycarbonyl)amino]-1-methyl-indazol-6-yl]boronic

acid 98 (3:2 ratio, 174 mg, ∼0.368 mmol), 0.5 M aqueous sodium

carbonate (982 uL, 0.491 mmol), and Pd(dppf)Cl₂·CH₂Cl₂(18 mg,

0.025 mmol) were added. The reaction mixture was stirred for 1 h at

120 °C. After addition of EtOAc, the mixture was ﬁltered and the

ﬁltrate was concentrated. The brown residue was puriﬁed by ﬂash

chromatography (Companion, DCM/MeOH gradient) to give tert-

butyl (6-(5-chloro-4-(1-oxo-2,8-diazaspiro[4.5]decan-8-yl)pyridin-3-

yl)-1-methyl-1H-indazol-3-yl)carbamate (47 mg, 71% pure, 27%

corrected yield) as a colorless solid which was used directly in the

next step. LC−MS (method B, ESI, m/z) t_R= 2.19 min, 511/513 (M

+ H)⁺.

reaction mixture was heated at 80 °C overnight and concentrated. The

crude material was puriﬁed by Biotage column chromatography

(cylohexane/EtOAc 95:5 to 70:30) to give N,N,1-trimethyl-6-(4,4,5,5-

tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazol-3-amine 99 (100 mg,

1

84% yield). H NMR (500 MHz, CDCl₃) δ 7.75 (dd, J = 8.1, 0.9 Hz,

1H), 7.72 (s, 1H), 7.39 (d, J = 8.1 Hz, 1H), 3.91 (s, 3H), 3.09 (s, 6H),

1.38 (s, 12H); LC−MS (method C, ESI, m/z) t_R= 3.19 min, 301/302

(M + H)⁺.

8-(3-Chloro-5-(3-(dimethylamino)-1-methyl-1H-indazol-6-

yl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one (102). 8-(3-

Bromo-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one²⁷8 (13

mg, 0.036 mmol), N,N,1-trimethyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxa-

borolan-2-yl)-1H-indazol-3-amine 99 (12 mg, 0.040 mmol), and

Pd(dppf)Cl₂·CH₂Cl₂(1.5 mg, 1.8 μmol) were loaded in a microwave

vial, and acetonitrile (624 μL) and 0.5 M sodium carbonate in water

(101 μL, 0.0510 mmol) were added. The reaction was stirred at 120

°C for 60 min under microwave irradiation. The solvent was

evaporated, and the crude material was puriﬁed by Biotage column

chromatography (DCM/EtOH 98:2 to 91:9) and then by preparative

HPLC (method B). Fractions were concentrated under vacuum and

the product was solubilized in DCM and ﬁltered on a basic Isolute

ﬂash NH₂column to aﬀord 8-(3-chloro-5-(3-(dimethylamino)-1-

methyl-1H-indazol-6-yl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one

1

102 (8 mg, 50% yield). H NMR (500 MHz, CDCl₃) δ 8.46 (s, 1H),

8.27 (s, 1H), 7.82 (dd, J = 8.4, 0.8 Hz, 1H), 7.13 (dd, J = 1.4, 0.8 Hz,

1H), 6.88 (dd, J = 8.4, 1.4 Hz, 1H), 5.68 (s, 1H), 3.92 (s, 3H), 3.28−

3.23 (m, 2H), 3.21−3.15 (m, 2H), 3.13 (s, 6H), 2.71 (t, J = 11.8 Hz,

2H), 2.00−1.90 (m, 4H), 1.36−1.29 (m, 2H); LC−MS (method C,

ESI, m/z) t_R= 2.40 min, 439/441 (M + H)⁺; ESI-HRMS calcd for

C₂₃H₂₈³⁵ClN₆O (M + H)⁺439.2008, found 439.2004.

tert-Butyl (6-(5-chloro-4-(1-oxo-2,8-diazaspiro[4.5]decan-8-yl)-

pyridin-3-yl)-1-methyl-1H-indazol-3-yl)carbamate (71% pure, 47 mg,

0.066 mmol) was dissolved in 5 N hydrogen chloride in dioxane (5

mL) and MeOH (1 mL). The pale brown solution was stirred at rt for

4 h, and the solvents were evaporated. The residue was puriﬁed by

preparative HPLC (method A, 20 min gradient elution from 2:98 to

25:75 acetonitrile/water) to give 8-(3-(3-amino-1-methyl-1H-indazol-

6-yl)-5-chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one 2,2,2-tri-

Preparation of Compounds in Table 8, Exempliﬁed by

Compound 109. 8-(2-Amino-3-chloro-5-(1-methyl-1H-indazol-

5-yl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one (109). Into a

500 mL screw cap jar, 8-(2-(bis(4-methoxybenzyl)amino)-5-bromo-3-

chloropyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one 82 (82% pure, 13.4

g, 18.4 mmol), 1-methylindazole-5-boronic acid 39 (97% pure, 4.99 g,

27.5 mmol), and Pd(dppf)Cl₂·CH₂Cl₂(749 mg, 0.920 mmol) were

weighed in and suspended in acetonitrile (300 mL) and sodium

carbonate solution (0.5 M, 73.4 mL, 36.7 mmol). The reaction mixture

was stirred at 80 °C overnight. The mixture was diluted with

acetonitrile (200 mL), ﬁltered, and evaporated to dryness. The crude

residue was puriﬁed by ﬂash chromatography (DCM/MeOH,

gradient). The isolated product was suspended in acetonitrile (100

mL) and treated with diethyl ether (200 mL). The resultant beige

precipitate was ﬁltered, washed with diethyl ether (100 mL), and dried

under vacuum. The mother liquor was evaporated to 50 mL volume

and treated with diethyl ether (100 mL). The resulting precipitate was

ﬁltered, washed with diethyl ether (40 mL), and dried under vacuum.

The two batches of compound were combined to yield in 8-(2-(bis(4-

methoxybenzyl)amino)-3-chloro-5-(1-methyl-1H-indazol-5-yl)pyridin-

4-yl)-2,8-diazaspiro[4.5]decan-1-one (7.05 g, 59% yield) as a colorless

solid which was used directly in the next step. LC−MS (method B,

ESI, m/z) t_R= 2.78 min, 651/653 (M + H)⁺.

8-(2-(Bis(4-methoxybenzyl)amino)-3-chloro-5-(1-methyl-1H-inda-

zol-5-yl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one (7.05 g, 10.8

mmol) was dissolved in triﬂuoroacetic acid (70 mL) and the reaction

mixture stirred at rt for 4 h. The solution was evaporated to dryness.

The crude residue was suspended in DCM and treated with diethyl

ether (350 mL). The white precipitate was ﬁltered and washed with

diethyl ether (100 mL) and dried under vacuum overnight. The

residue was dissolved in DMSO (50 mL) and poured into sodium

carbonate solution (0.25 M, 500 mL). The resulting light yellow

precipitate was ﬁltered, washed with water (150 mL), and dried at 70

°C under vacuum overnight to give 8-(2-amino-3-chloro-5-(1-methyl-

1H-indazol-5-yl)pyridin-4-yl)-2,8-diazaspiro[4.5]decan-1-one 109

(3.34 g, 75% yield) as a light yellow solid. ¹H NMR (500 MHz,

DMSO-d₆) δ 8.05 (d, J = 1.0 Hz, 1H), 7.69−7.64 (m, 2H), 7.63−7.60

(m, 1H), 7.49 (s, 1H), 7.27 (dd, J = 8.6, 1.6 Hz, 1H), 6.10 (s, 2H),

4.07 (s, 3H), 3.07 (t, J = 6.7 Hz, 2H), 2.98−2.92 (m, 2H), 2.67−2.55

(m, 2H), 1.74 (t, J = 6.7 Hz, 2H), 1.71−1.61 (m, 2H), 1.16 (d, J = 12.6

1

ﬂuoroacetate 101 (15 mg, 43% yield) as a yellow solid. H NMR

(500 MHz, CD₃OD) δ 8.66 (s, 1H), 8.35 (s, 1H), 7.88 (d, J = 8.4 Hz,

1H), 7.47 (s, 1H), 7.04 (d, J = 8.4 Hz, 1H), 3.89 (s, 3H), 3.55−3.47

(m, 2H), 3.27 (t, J = 6.9 Hz, 2H), 3.02−2.95 (m, 2H), 2.01 (t, J = 6.9

Hz, 2H), 1.95−1.87 (m, 2H), 1.50−1.42 (m, 2H); ¹³C NMR (126

MHz, CD₃OD) δ 183.1, 159.7, 148.3, 144.4, 143.3, 143.1, 136.6, 134.7,

127.8, 122.5, 121.2, 115.5, 110.6, 49.8, 43.1, 39.7, 35.1, 33.4, 32.6;

LC−MS (method C, ESI, m/z) t_R= 1.86 min, 411/413 (M + H)⁺;

ESI-HRMS calcd for C₂₁H₂₄³⁵ClN₆O (M + H)⁺411.1695, found

411.1684.

N,N,1-Trimethyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-

2-yl)-1H-indazol-3-amine (99). 4-Bromo-2-ﬂuorobenzonitrile (1.0

g, 5.0 mmol) was dissolved in ethanol (11 mL), and methylhydrazine

(0.756 mL, 14.4 mmol) was added. The reaction mixture was stirred

under microwave irradiation at 120 °C for 50 min. The reaction was

quenched with water, and the product was extracted with EtOAc three

times. The combined organic layers were dried over MgSO₄and

concentrated to give 6-bromo-1-methyl-1H-indazol-3-amine⁴¹(940

mg, 83% yield) as a brown solid. ¹H NMR (500 MHz, CDCl₃) δ 7.47

(dd, J = 8.7, 0.7 Hz, 1H), 7.38 (dd, J = 1.5, 0.7 Hz, 1H), 7.13 (dd, J =

8.7, 1.5 Hz, 1H), 3.80 (s, 3H); LC−MS (method C, ESI, m/z) t_R=

2.50 min, 226/228 (M + H)⁺.

To 6-bromo-1-methyl-1H-indazol-3-amine (736 mg, 3.26 mmol) in

formic acid (3.7 mL, 98 mmol) was added formaldehyde 37% in water

(897 μL, 32.6 mmol). The reaction mixture was heated at 100 °C for 2

h and concentrated. The crude material was puriﬁed by Biotage

column chromatography (cyclohexane/EtOAc 95:5 to 82:18) to aﬀord

6-bromo-N,N,1-trimethyl-1H-indazol-3-amine (85 mg, 10% yield) as a

brown solid. ¹H NMR (500 MHz, CDCl₃) δ 7.60 (dd, J = 8.7, 0.6 Hz,

1H), 7.37 (dd, J = 1.5, 0.6 Hz, 1H), 7.06 (dd, J = 8.7, 1.5 Hz, 1H), 3.82

(s, 3H), 3.07 (s, 6H); LC−MS (method C, ESI, m/z) t_R= 3.11 min,

254/256 (M + H)⁺.

6-Bromo-N,N,1-trimethyl-1H-indazol-3-amine (100 mg, 0.394

mmol), bis(pinacolato)diboron (150 mg, 0.590 mmol), potassium

acetate (116 mg, 1.18 mmol), and Pd(dppf)Cl₂·CH₂Cl₂(16 mg, 0.020

mmol) were loaded in a ﬂask, and DME (2.8 mL) was added. The

T

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Journal of Medicinal Chemistry

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Hz, 2H); ¹³C NMR (126 MHz, DMSO-d₆) δ 180.0, 156.2, 153.5,

148.2, 138.7, 132.4, 130.6, 128.4, 124.2, 123.7, 120.3, 109.2, 108.8,

47.3, 41.4, 37.8, 35.5, 31.8, 30.3; LC−MS (method C, ESI, m/z) t_R=

1.79 min, 411/413 (M + H)⁺. ESI-HRMS calcd for C₂₁H₂₄³⁵ClN₆O

(M + H)⁺411.1695, found 411.1681.

Mouse, Rat, and Human Cl_intDetermination. Microsomes

(ﬁnal concentration 0.5 mg/mL), 50 mM phosphate buﬀer pH 7.4,

and compound (ﬁnal concentration 1 μM) were added to the assay

plate and allowed to preincubate for 5 min at 37 °C. The reaction was

initiated by the addition of NADPH (ﬁnal concentration 1.5 mM), and

the plate was shaken at 800 rpm at 37 °C. After 0, 5, 10, 20, and 30

min aliquots were taken and the reaction was stopped using cold

acetonitrile. The samples were centrifuged at 4000 rpm for 30 min at 4

°C and analyzed by LC−MS/MS. Four test compounds were pooled

for analysis. The in vitro intrinsic clearance was calculated from the

rate of compound disappearance.

DMSO/PEG 200/water, respectively. Consecutive blood samples

were taken by puncture of the vena cephalica from n = 2 animals per

route of administration (crossover design) predose and after 0.1 (only

(iv), 0.25 (only po), 0.5, 1, 2, 4, 6, 24, and 48 h and were further

processed to obtain plasma. The Institute of Cancer Research does not

use non-rodent species in research and, where this is deemed essential,

require ethical approval for use by organizations with whom we

collaborate. Pharmacokinetic analysis of compounds 6 and 109 in

dogs, necessary for prediction of human pharmacokinetics, was

approved by the ICR Ethics Committee. Studies were sponsored

and conducted in full compliance with national regulations at an

Association for Assessment and Accreditation of Laboratory Animal

Care (AAALAC) accredited site of Merck Biopharma.

Bioanalytics. The concentrations of compound in plasma and

feces were quantiﬁed using an UPLC method with tandem mass

spectrometric detection (LC−MS/MS). The LC−MS system

consisted of a Waters Acquity UPLC coupled to an AB Sciex mass

spectrometer API 5500 Q-trap. The UPLC separation was carried out

on a reversed phase column (Acquity UPLC BEC C8, 1.7 μM, 2.1 mm

× 50 mm) using a mobile phase gradient with 0.1% formic acid and

acetonitrile as eluents. The detection of the drug was performed using

multiple reaction monitoring in the positive ionization mode. Plasma

samples were spiked with internal standard, and the analyte was

extracted from the matrix using tert-butyl methyl ether (tBME). The

organic phase was evaporated to dryness under a stream of nitrogen.

The residue was dissolved in acetonitrile/0.1% formic acid for LC−

MS/MS analysis. Feces samples were homogenized with 4 times their

volume ethanol/water (4:1). Aliquots of the aqueous ethanolic extracts

were further diluted with acetonitrile/0.1% formic acid, spiked with

internal standard, and directly injected into the system.

Prediction of Human Clearance. Allometric methods used for

clearance prediction to human were simple allometry (3-species),

allometry with (a) correction by fu, (b) with correction by fu and MLP

(maximum life span), (c) with correction by fu and BrW (brain

weight); in addition, in vitro to in vivo (iviv) scaling from liver

microsomes and hepatocytes was used for clearance prediction (for

compounds 6 and 109, iviv from hepatocytes of 0.53 and 0.215 L/h/

kg may be included in the mean calculation). For compound 6, the

clearance prediction ranged from 1.45 L/h/kg (simple allometry) to

0.485 L/h/kg (BrW). Allometry with correction for fu and MLP was

chosen because of the highest R²value (0.9992) in the linear

regression plot; the mean of all methods gives a clearance prediction of

0.88 L/h/kg. For volume allometry, methods included 3-species

allometry with correction for fu and human−dog proportionality.

Values ranged between 0.7 and 1.0 L/kg; the selected method of

choice was allometry with correction by fu (V_ss= 0.85 L/kg), which

represents roughly the mean of the values. For compound 109 the

corresponding values were as follows: clearance prediction; MLP =

0.511 L/h/kg, BrW = 0.343 L/h/kg. Allometry with correction for fu

and scaling from liver microsomes and hepatocytes was included; the

mean of all methods gave a clearance prediction of 0.36 L/h/kg. For

volume allometry, methods included 3 species allometry with

correction for and human−dog proportionality. Values ranged

between 1.3 and 1.5 L/kg; the mean of all methods gave a volume

prediction of 1.4 L/kg.

In Vitro Cell-Based Reporter Assays. 7dF3 Luciferase

Reporter Assay.²⁷7dF3 cells were seeded at 5000 per well in 50

μL of Dulbecco’s modiﬁed Eagle medium (DMEM) with 4 mM

supplemental ^L-glutamine in 384-well white tissue culture plates

(Corning 3570, USA). After incubating overnight at 37 °C/5% CO₂,

compounds ranging in ﬁnal concentration from 90 μM to 0.3 nM were

added in duplicate to the wells. After 2 h of further incubation as

above, β-oestradiol was added to a ﬁnal concentration of 10 μM. The

cells were incubated for 24 h at 37 °C/5% CO₂, and then 25 μL of

luciferase reagent (SteadyGlo, Promega,USA) was added and mixed.

After leaving the plate for 60 min at room temperature, luminescence

was read on a plate luminescence reader (TopCount, PerkinElmer,

USA). Percentage inhibition for each compound concentration was

calculated using total counts (no compound) and low counts (no β-

oestradiol) as highs and lows, respectively. IC₅₀was subsequently

Caco-2 P_appDetermination. Caco-2 cells were maintained in

DMEM in an atmosphere of 8.5% CO₂. For transport experiments

0.125 × 10⁶cells/well of were seeded on polycarbonate ﬁlter inserts

and allowed to grow and diﬀerentiate for 14 1 days before the cell

monolayers were used for experiments. Drug transport experiments

were carried out using a cocktail approach in a four-dimensional

setting. Apparent permeability coeﬃcients were determined for A → B

and B → A directions with and without the presence of cyclosporine A

as a transporter inhibitor. Up to ﬁve test items and reference

compounds were dissolved in Hank’s balanced salt solution at pH 7.4

to yield a ﬁnal concentration of 1 μM. The assays were performed in

HBSS containing 25 mM HEPES (pH 7.4) in an atmosphere of 5%

CO₂at 37 °C. Prior to the study, the monolayers were washed in

prewarmed HBSS. At the start of the experiments prewarmed HBSS

containing the test items was added to the donor side of the

monolayer and HBSS without test items was added to the receiver

side. The plates were shaken at 150 rpm at 37 °C during the

experiment. After 2 h the Transwell insert containing the monolayer

was carefully removed and placed in a new plate and aliquots of both

the receiver and donor sides were taken and diluted with an equal

volume of acetonitrile containing the internal standard. The mixture

was centrifuged and supernatant analyzed by LC−MS/MS. The

apparent permeability coeﬃcients (P_app) were calculated using the

formula: P_app= [V_rec/(A × C_0,donor)](dC_rec/dt) × 10⁶with dC_rev/dt

being the change in concentration in the receiver compartment with

time, V_recthe volume of the sample in the receiver compartment,

C_0,donorthe concentration in the donor compartment at time 0, and A

the area of the compartment with the cells.

In Vivo Mouse PK In-Life Phase. Female NMRI mice (n = 5)

received either a single intravenous (bolus) injection or single oral

administration (by gavage) of the compound in a cocktail preparation.

Doses of 0.2 mg/kg (per compound, intravenous administration) and

0.5 mg/kg (per compound, perorally) were given as solutions in

DMSO/PEG 200/water. Consecutive blood samples were taken

retroorbitally under isoﬂuorane inhalation from n = 3 animals per

route of administration after 0.1 (iv only), 0.25 (po only), 0.5, 1, 2, 4,

and 6 h and were further processed to obtain plasma. In addition, feces

of 2 mice per route of administration were collected over the time

interval from 0 to 24 h (pooled for analysis, no blood sampling).

In Vivo Rat PK In-Life Phase. Male Wistar rats (n = 6) received

either a single intravenous (bolus) injection or a single oral

administration (by gavage) of the compound in a cocktail preparation.

Doses of 0.2 mg/kg (pre compound, intravenous administration) and

0.5 mg/kg (per compound, perorally) were given as solutions in

DMSO/PEG 200/water. Consecutive blood samples were taken

sublingually under isoﬂuorane inhalation from n = 3 animals per route

of administration after 0.1 (iv only), 0.25 (po only), 0.5, 1, 2, 4, and 6

h and were further processed to obtain plasma. In addition, feces and

urine of 3 rats per route of administration were collected over the time

interval from 0 to 24 h (pooled for analysis, no blood sampling).

In Vivo Dog PK In-Life Phase. Female Beagle dogs received

either a single intravenous (bolus) injection or an oral administration

(by gavage) of the compound in a cocktail preparation. Doses of 0.2

and 0.5 mg/kg were given intravenously and per orally as a solution in

U

DOI: 10.1021/acs.jmedchem.5b01685

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

a

Table 11. Antibodies Used in Western Blotting

antibody name

rabbit anti-Stat1

source

catalogue no.

dilution

type

primary

Santa Cruz

CST

sc-346

8826

1:1000

1:2000

1:10000

1:2500

rabbit anti phospho-Stat1^Ser727

mouse anti GAPDH

Abcam

CST

Ab-8245

7076

loading control

secondary

α-mouse IgG HRP-linked

anti-rabbit IgG HRP-linked

CST

7074

secondary

a

Membranes were cut and incubated at the same time for phospho-Stat^SER727(84, 91 kDa), total Stat1 (84, 91 kDa), and GAPDH (37 kDa).

determined using a curve-ﬁtting software package in Excel (Excelﬁt,

IDBS, U.K.). Data are reported as average values of at least two

determinations with standard deviations unless otherwise noted.

In Vitro Biochemical Assays. CDK8 Lanthascreen Binding

Assay. The assay format was a FRET based Lanthascreen binding

competition assay. A dye-labeled ATP competitive tracer served as a

FRET acceptor upon binding to CDK8. This CDK8 was His-tagged.

The FRET donor was a strepavidin-Eu-chelate bound to a biotinylated

anti-His antibody. When the tracer is competed by an inhibitor, the

respective FRET signal is aﬀected. The CDK8 used for this assay was a

protein coexpressed with CycC. The assay procedure for assays in the

384-well plate format (Greiner PS, white, medium binding no.

784075) was the following: 2 μL of CDK8/biotin-anti-His Ab/SA-Eu

mix in assay buﬀer was pipetted into the wells of the microplates. Then

1 μL compounds in 20 mM Hepes buﬀer/5% DMSO was added. The

plates were shaken for 30 s and incubated for 20 min at rt. Then 2 μL

of Alexa647 tracer in assay buﬀer was added. The plates were shaken

for 30 s and incubated for 60 min at rt in the dark. Finally the plates

were read out on a PerkinElmer Envision 2104 (mode LANCE/TRF,

excitation 340 nm, emissions at 615 and 615 nm). For evaluation the

emission ratios (615 nm/655 nm) were calculated. These were

normalized to data from control wells containing vehicle only or 6 at

30 uM. The assay buﬀer was 50 mM Hepes, pH 7.5 (Merck no.

1.10110), 10 mM MgCl₂(Merck no. 1.05833), 1 mM EGTA (Merck

no. 1.08435), 0.01% Brij-35 (Pierce no. 28316). The ﬁnal

concentrations of the reaction components in 5 μL total assay volume

were 1% DMSO (Merck no. 1.02950), 5 nM CDK8 (CDK8/CycC

Invitrogen no. PV4402), 2 nM biotin-a-His Ab (Invitrogen no.

PV6089), 2 nM SA-europium (Invitrogen no. PV5899), 10 nM

Alexa647 tracer (Invitrogen no. PV5592). Data are reported as average

values of at least two determinations with standard deviations unless

otherwise noted.

Reporter Displacement Assay for CDK8 and CDK19.

Interaction of compounds with human CDK8(aa1-464)/cyclin C-

(aa1-283) and human CDK19(aa1-502)/cyclin C(aa1-283) was

analyzed using the reporter displacement assay provided by Proteros

Biostructures GmbH.⁴²In brief, the reporter displacement assay is

based on reporter probes that are designed to bind to the site of

interest of the target protein. The proximity between reporter and

protein results in the emission of an optical signal. Compounds that

bind to the same site as the reporter probe displace the probe, causing

signal diminution. Reporter displacement is measured over time after

addition of compounds at various concentrations. For IC₅₀

determination, percent probe displacement values are calculated for

the last time point at which the system has reached equilibrium. For

each compound concentration, percent probe displacement values are

calculated and plotted against the compound concentration. IC₅₀-like

values (corresponding to 50% probe displacement) are calculated

using standard ﬁtting algorithms. Data are reported as average values

of at least two determinations with standard deviations unless

otherwise noted.

group) under isoﬂuorothane anesthesia. Tumors were established for 7

days when dosing commenced (day 0) and continued for 16 days.

Compound 109 was prepared 24 h prior to administration to mice.

The compound was weighed into a glass 30 mL universal tube, and

Methocel solution (0.5% Methocel, 0.25% Tween 20 in sterile

phosphate buﬀered saline) was added to provide a ﬁnal concentration

of 3 mg/mL 109. The preparation was agitated at room temperature

by magnetic stirrer to bring it into a ﬁne suspension before use.

Animals were dosed orally by gavage every 24 h at 0.1 mL per 10 g

body weight. Tumors were measured three times weekly by Vernier

calipers and body weights recorded. At the end of the study, animals

were culled at intervals: 3 control and 3 treated at 1, 2, 6, and 24 h

after the ﬁnal dose. Heparinized blood was collected by cardiac

puncture, spun, and plasma snap frozen for analysis of compound

exposure. Tumors were excised, weighed and samples snap frozen for

compound quantiﬁcation and PD analyses. The 30 mg/kg q.d.

schedule was well tolerated with no signiﬁcant body weight loss.

Tumor growth was signiﬁcantly inhibited (T/C = 45.8; P = 0.0241

ﬁnal tumor weights).

Pharmacokinetic Analysis. Plasma and tumor homogenates were

measured by LC−MS/MS on a Water TQS following a separation on

a Waters Acquity BEH C18 column (50 mm × 2.1 mm; 1.7 μm) with

conditions of 0.1% formic acid (mobile phase A) and methanol

(mobile phase B). The column was equilibrated at initial condition of

95% A and 5% B, linear gradient over 3 min to 100% B, held over 1

min, followed by linear gradient back to 5% B over 0.1 min, at 0.6 mL/

min ﬂow rate. Detection was achieved in positive electrospray

ionization mode by multiple reaction monitoring, 412.17 > 376.40

at 33 eV for compound 109, and 299.19 > 91.24 at 33 eV, 299.19 >

177.29 at 26 eV for the internal standard olomoucine. Samples were

quantiﬁed by external standard calibration (8-point calibration ranging

from 2 nM to 50 000 nM). Quality controls were included at 25, 250,

2500, and 7500 nM in duplicate at the beginning and the end of the

analytical run.

Tumor Xenograft Processing and Western Blotting. Tumors

were excised, immediately snap-frozen in liquid nitrogen, and stored at

−80 °C until use. Samples were transferred to MK28 reinforced

homogenizing tubes with metal beads (Stretton Scientiﬁc), and CDK

lysis buﬀer (50 mM Tris-HCl, pH7.4, 1 mM EDTA, 1% Triton X-100

(v/v), 150 mM NaCl, 1 mM activated sodium orthovanandate, 1 mM

PMSF, protease cocktail (Sigma P8340 1:100), and phosphatase

inhibitors (Sigma P5726 and P0044 1:50 dilution) were added

immediately. The samples were ground using a Precellys 24 at 6000

rpm, 2 × 20 s (Stretton Scientiﬁc) and lysates frozen at −80 °C until

use. For analysis, samples were debanked and sonicated for 3 min, on

ice for 10 min. Tubes were spun at 14 000 rpm at 4 °C for 10 min, and

supernatants were collected, aliquoted, and frozen at −80 °C until use.

Tumor lysates were diluted 1:5 in lysis buﬀer and the concentrations

determined using a Direct Detect spectrometer (Merck Millipore).

Samples were aliquoted, then boiled in electrophoresis sample

buﬀer and loaded on SDS-4-12% PAGE gels. After transfer to PVDF

membranes, blots were blocked at room temperature for 1 h in

blocking buﬀer (5% dry milk in TNT: 1 M Tris-HCL, pH 8, 5 M

NaCl, 0.1% Tween 20) and then incubated at 4 °C overnight with the

appropriate antibody (Table 11). After washing with TNT, blots were

incubated with HRP-conjugated secondary antibodies in blocking

buﬀer at room temperature for 1 h, and bands were visualized using

the enhanced chemoluminescence (ECL prime) method.

Human Tumor Xenograft Eﬃcacy Study. All animal procedures

were conducted in accordance with NCRI guidelines⁴³and U.K.

Home Oﬃce regulations. Female athymic nude mice Crl:Nu(NCr)-

Foxn1^nusupplied by Charles River Laboratories were acclimatized for 1

week prior to use. The human colorectal adenocarcinoma cell line,

SW620 (ATCC, no. CCL-227), was expanded in culture, and 5 million

cells were injected sc into the right ﬂanks of animals (n = 12 per

V

DOI: 10.1021/acs.jmedchem.5b01685

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.5b01685.

ABBREVIATIONS USED

■

S

CDK, cyclin-dependent kinase; Cl, clearance; Cl_int, intrinsic

clearance; ER, eﬄux ratio; F, bioavailability; HBA, hydrogen

bond acceptor; HBD, hydrogen bond donor; kin, kinetic; LBF,

liver blood ﬂow; t_1/2, half-life; PMB, p-methoxybenzyl; SN_Ar,

nucleophilic aromatic substitution; therm, thermodynamic;

TPSA, topological polar surface area; V_d, volume of distribution

Caco-2 data for compounds 6, 17−25, 41, 42, 44−49,

51, 54, 61, 63, 72, 85−95 (Table S1), CEREP proﬁle of

109 (Tables S2 and S3), kinase proﬁle of 109 (Table

S4), CYP450 inhibition for 109 (Table S5), plasma and

tumor concentration (Table S6), correlation of 7dF3 and

LS174T cell-based potency (Figure S1), analyses of

phospho-STAT1^SER727(Figure S2), total plasma and

tumor concentration (Figure S3), thermodynamic and

kinetic solubility protocols, preparation of compounds

9−13, 15, 17−22, 24, 26, 28−35, 40, 41, 43−53, 55, 56,

60, 62, 65, 67, 69−71, 76, 78, 79, 83−85, 87−91, 93,

94, 96, 103−108, 110, 111, ¹H NMR spectra for all ﬁnal

compounds, and small molecule X-ray crystallographic

data analysis (including F_o− F_comit maps at 2.5σ cutoﬀ)

for compounds 25, 42, 54, and 109 (PDF)

REFERENCES

■

(1) Allen, B. L.; Taatjes, D. J. The Mediator complex: a central

integrator of transcription. Nat. Rev. Mol. Cell Biol. 2015, 16, 155−166.

(2) Carlsten, J. O.; Zhu, X.; Gustafsson, C. M. The multitalented

Mediator complex. Trends Biochem. Sci. 2013, 38, 531−537.

(3) Kim, S.; Xu, X.; Hecht, A.; Boyer, T. G. Mediator is a transducer

of Wnt/beta-catenin signaling. J. Biol. Chem. 2006, 281, 14066−14075.

(4) Schiano, C.; Casamassimi, A.; Vietri, M. T.; Rienzo, M.; Napoli,

C. The roles of mediator complex in cardiovascular diseases. Biochim.

Biophys. Acta, Gene Regul. Mech. 2014, 1839, 444−451.

(5) Schiano, C.; Casamassimi, A.; Rienzo, M.; de Nigris, F.;

Sommese, L.; Napoli, C. Involvement of Mediator complex in

malignancy. Biochim. Biophys. Acta, Rev. Cancer 2014, 1845, 66−83.

(6) Galbraith, M. D.; Donner, A. J.; Espinosa, J. M. CDK8: a positive

regulator of transcription. Transcription 2010, 1, 4−12.

(7) Tsutsui, T.; Fukasawa, R.; Tanaka, A.; Hirose, Y.; Ohkuma, Y.

Identification of target genes for the CDK subunits of the Mediator

complex. Genes Cells 2011, 16, 1208−1218.

(8) Rickert, P.; Seghezzi, W.; Shanahan, F.; Cho, H.; Lees, E. Cyclin

C/CDK8 is a novel CTD kinase associated with RNA polymerase II.

Oncogene 1996, 12, 2631−2640.

Molecular formula strings (CSV)

Accession Codes

Atomic coordinates and structure factors for the crystal

structures of CDK8/cyclin C with compounds 25, 42, 54,

and 109 can be accessed using PDB codes 5FGK, 5HBE,

5HBH, and 5HBJ, respectively.

(9) Morris, E. J.; Ji, J. Y.; Yang, F.; Di Stefano, L.; Herr, A.; Moon, N.

S.; Kwon, E. J.; Haigis, K. M.; Naar, A. M.; Dyson, N. J. E2F1 represses

beta-catenin transcription and is antagonized by both pRB and CDK8.

Nature 2008, 455, 552−556.

(10) Firestein, R.; Shima, K.; Nosho, K.; Irahara, N.; Baba, Y.;

Bojarski, E.; Giovannucci, E. L.; Hahn, W. C.; Fuchs, C. S.; Ogino, S.

CDK8 expression in 470 colorectal cancers in relation to beta-catenin

activation, other molecular alterations and patient survival. Int. J.

Cancer 2010, 126, 2863−2873.

(11) Kim, M. Y.; Han, S. I.; Lim, S. C. Roles of cyclin-dependent

kinase 8 and beta-catenin in the oncogenesis and progression of gastric

adenocarcinoma. Int. J. Oncol. 2011, 38, 1375−1383.

AUTHOR INFORMATION

Corresponding Authors

*A.M.: e-mail, aurelie.mallinger@icr.ac.uk; phone, +44 (0) 20

8722 4267.

*J.B.: e-mail, julian.blagg@icr.ac.uk; phone, +44 (0) 20 8722

4051.

■

Author Contributions

The manuscript was written with contributions of all authors.

All authors have given approval to the ﬁnal version of the

manuscript.

(12) Porter, D. C.; Liang, J.; Kaza, V.; Chumanevich, A. A.; Altillia, S.;

Farmaki, E.; Chen, M.; Schools, G. P.; Chatzistamou, I.; Pena, M. M.;

Friedhoﬀ, L. T.; Wentland, M. P.; Broude, E.; Kiaris, H.; Roninson, I.

B. Targeting the seed and the soil of cancers with selective small-

molecule inhibitors of CDK8/19: Chemopotentiating, chemopreven-

tive, anti-invasive and anti-metastatic activities. Presented at the AACR

Annual Meeting 2014, San Diego, CA, 2014.

(13) Kapoor, A.; Goldberg, M. S.; Cumberland, L. K.; Ratnakumar,

K.; Segura, M. F.; Emanuel, P. O.; Menendez, S.; Vardabasso, C.;

Leroy, G.; Vidal, C. I.; Polsky, D.; Osman, I.; Garcia, B. A.; Hernando,

E.; Bernstein, E. The histone variant macroH2A suppresses melanoma

progression through regulation of CDK8. Nature 2010, 468, 1105−

1109.

(14) Adler, A. S.; McCleland, M. L.; Truong, T.; Lau, S.; Modrusan,

Z.; Soukup, T. M.; Roose-Girma, M.; Blackwood, E. M.; Firestein, R.

CDK8 maintains tumor dedifferentiation and embryonic stem cell

pluripotency. Cancer Res. 2012, 72, 2129−2139.

(15) Firestein, R.; Bass, A. J.; Kim, S. Y.; Dunn, I. F.; Silver, S. J.;

Guney, I.; Freed, E.; Ligon, A. H.; Vena, N.; Ogino, S.; Chheda, M. G.;

Tamayo, P.; Finn, S.; Shrestha, Y.; Boehm, J. S.; Jain, S.; Bojarski, E.;

Mermel, C.; Barretina, J.; Chan, J. A.; Baselga, J.; Tabernero, J.; Root,

D. E.; Fuchs, C. S.; Loda, M.; Shivdasani, R. A.; Meyerson, M.; Hahn,

W. C. CDK8 is a colorectal cancer oncogene that regulates beta-

catenin activity. Nature 2008, 455, 547−551.

(16) Rzymski, T.; Mikula, M.; Wiklik, K.; Brzozka, K. CDK8 kinase-

An emerging target in targeted cancer therapy. Biochim. Biophys. Acta,

Proteins Proteomics 2015, 1854, 1617−1629.

Notes

The authors declare the following competing ﬁnancial

interest(s): A.M., C.R., S.C., M.S., O.A-P., M.-J.O.-R., C.T.,

M.V., A.d.H.B., R.B., P.W., T.D., P.A.C., F.I.R., S.A.E., and J.B.

are current or former employees of The Institute of Cancer

Research, which has a commercial interest in the development

of WNT pathway inhibitors. K.S., F.S., M.C., O.P., M.B., P.C.,

D.M., D.S., R.S., D.W., C.E. and F.R. are current employees of

Merck.

ACKNOWLEDGMENTS

■

This work was supported by Cancer Research UK [Grant

C309/A11566]. We acknowledge NHS funding to the NIHR

Biomedical Research Centre at The Institute of Cancer

Research and The Royal Marsden. We thank Dr. Amin

Mirza, Meirion Richards, and Dr. Maggie Liu for their

assistance with NMR, mass spectrometry and HPLC, Gary

Nugent and Dr. Christian Herhaus for chemoinformatics

support, and Kate Trotman for administrative support. We

thank the team of Proteros Biostructures GmbH, Bunsenstrasse

7a, D-82152 Martinsried, Germany, for the reporter displace-

ment assay and in particular Dr. Elisabeth V. Schneider and Dr.

Alfred Lammens for the X-ray crystal structures with CDK8/

cyclin C.

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DOI: 10.1021/acs.jmedchem.5b01685

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Journal of Medicinal Chemistry

Article

(17) Cee, V. J.; Chen, D. Y.; Lee, M. R.; Nicolaou, K. C. Cortistatin A

is a high-affinity ligand of protein kinases ROCK, CDK8, and CDK11.

Angew. Chem., Int. Ed. 2009, 48, 8952−8957.

(18) Shair, M. D. Cortistatin analogues and syntheses and uses

thereof. WO2015100420 A1, 2015.

(35) Somanathan, R.; Rivero, I. A.; Nunez, G. I.; Hellberg, L. H.

Convenient synthesis of 1-oxa-3,8-diazaspiro-4,5-decan-2-ones. Synth.

Commun. 1994, 24, 1483−1487.

(36) Yamada, H.; Hayashi, T.; Usuki, T. Bull. Chem. Soc. Jpn. 2015,

88, 673−683.

(37) Kay, A. J.; Woolhouse, A. D.; Gainsford, G. J.; Haskell, T. G.;

Wyss, C. P.; Giffin, S. M.; McKinnie, I. T.; Barnes, T. H. Simple

zwitterionic merocyanines as potential NLO chromophores. J. Mater.

Chem. 2001, 11, 2271−2281.

(38) Bavetsias, V.; Large, J. M.; Sun, C.; Bouloc, N.; Kosmopoulou,

M.; Matteucci, M.; Wilsher, N. E.; Martins, V.; Reynisson, J.; Atrash,

B.; Faisal, A.; Urban, F.; Valenti, M.; de Haven Brandon, A.; Box, G.;

Raynaud, F. I.; Workman, P.; Eccles, S. A.; Bayliss, R.; Blagg, J.;

Linardopoulos, S.; McDonald, E. Imidazo[4,5-b]pyridine derivatives as

inhibitors of Aurora kinases: lead optimization studies toward the

identification of an orally bioavailable preclinical development

candidate. J. Med. Chem. 2010, 53, 5213−5228.

(39) Li, C.; Ai, J.; Zhang, D.; Peng, X.; Chen, X.; Gao, Z.; Su, Y.; Zhu,

W.; Ji, Y.; Chen, X.; Geng, M.; Liu, H. Design, Synthesis, and

Biological Evaluation of Novel Imidazo[1,2-a]pyridine Derivatives as

Potent c-Met Inhibitors. ACS Med. Chem. Lett. 2015, 6, 507−512.

(40) Deng, X.; Zhou, W.; Weisberg, E.; Wang, J.; Zhang, J.; Sasaki,

T.; Nelson, E.; Griffin, J. D.; Janne, P. A.; Gray, N. S. An amino-

indazole scaffold with spectrum selective kinase inhibition of FLT3,

PDGFRalpha and kit. Bioorg. Med. Chem. Lett. 2012, 22, 4579−4584.

(41) Wheeler, R. C.; Baxter, E.; Campbell, I. B.; Macdonald, S. J. F. A

General, One-Step Synthesis of Substituted Indazoles using a Flow

Reactor. Org. Process Res. Dev. 2011, 15, 565−569.

(42) Neumann, L.; von Konig, K.; Ullmann, D. HTS reporter

displacement assay for fragment screening and fragment evolution

toward leads with optimized binding kinetics, binding selectivity, and

thermodynamic signature. Methods Enzymol. 2011, 493, 299−320.

(43) Workman, P.; Aboagye, E. O.; Balkwill, F.; Balmain, A.; Bruder,

G.; Chaplin, D. J.; Double, J. A.; Everitt, J.; Farningham, D. A.;

Glennie, M. J.; Kelland, L. R.; Robinson, V.; Stratford, I. J.; Tozer, G.

M.; Watson, S.; Wedge, S. R.; Eccles, S. A. Guidelines for the welfare

and use of animals in cancer research. Br. J. Cancer 2010, 102, 1555−

1577.

(19) Pelish, H. E.; Liau, B. B.; Nitulescu, II; Tangpeerachaikul, A.;

Poss, Z. C.; Da Silva, D. H.; Caruso, B. T.; Arefolov, A.; Fadeyi, O.;

Christie, A. L.; Du, K.; Banka, D.; Schneider, E. V.; Jestel, A.; Zou, G.;

Si, C.; Ebmeier, C. C.; Bronson, R. T.; Krivtsov, A. V.; Myers, A. G.;

Kohl, N. E.; Kung, A. L.; Armstrong, S. A.; Lemieux, M. E.; Taatjes, D.

J.; Shair, M. D. Mediator kinase inhibition further activates super-

enhancer-associated genes in AML. Nature 2015, 526, 273−276.

(20) Schneider, E. V.; Bottcher, J.; Blaesse, M.; Neumann, L.; Huber,

R.; Maskos, K. The structure of CDK8/CycC implicates specificity in

the CDK/cyclin family and reveals interaction with a deep pocket

binder. J. Mol. Biol. 2011, 412, 251−266.

(21) Schneider, E. V.; Bottcher, J.; Huber, R.; Maskos, K.; Neumann,

L. Structure-kinetic relationship study of CDK8/CycC specific

compounds. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 8081−8086.

(22) Dale, T.; Clarke, P. A.; Esdar, C.; Waalboer, D.; Adeniji-

Popoola, O.; Ortiz-Ruiz, M.; Mallinger, A.; Samant, R. S.; Czodrowski,

P.; Musil, D.; Schwarz, D.; Schneider, K.; Stubbs, M.; Ewan, K.; Fraser,

E.; TePoele, R.; Court, W.; Box, G.; Valenti, M.; de Haven Brandon,

A.; Gowan, S.; Rohdich, F.; Raynaud, F.; Schneider, R.; Poeschke, O.;

Blaukat, A.; Workman, P.; Schiemann, K.; Eccles, S. A.; Wienke, D.;

Blagg, J. A selective chemical probe for exploring the role of CDK8

and CDK19 in human disease. Nat. Chem. Biol. 2015, 11, 973−980.

(23) Roninson, I. B.; Porter, D. C.; Wentland, M. P. CDK8/CDK19

selective inhibitors and their use in anti-metastatic and chemo-

preventative methods for cancer. WO2013116786 A1, 2013.

(24) Hu, H.; Jiang, M.; Jin, T.; Niu, R.; Wang, J.; Yang, S.; Yuan, T.;

Zhou, C.; Wang, M.;, Zhou, Z. Novel phenyl-pyridine/pyrazine amides

for the treatment of cancer. WO2014029726 A1, 2014.

(25) Rzymski, T.; Zarebski, A.; Dreas, A.; Osowska, K.; Kucwaj, K.;

Fogt, J.; Cholody, M.; Galezowski, M.; Czardybon, W.; Horvath, R.;

Wiklik, K.; Milik, M.; Brzozka, K. Substituted tricyclic benzimidazoles

́

as kinase inhibitors. WO2014072435 A1, 2014.

(26) Romero, D. L.; Chaudhary, D.; Robinson, S.; Masse, C. E.;

Morin, M. J. CDK8 inhibitors and uses thereof. WO2014194245 A3,

2014.

(27) Mallinger, A.; Crumpler, S.; Pichowicz, M.; Waalboer, D.;

Stubbs, M.; Adeniji-Popoola, O.; Wood, B.; Smith, E.; Thai, C.;

Henley, A. T.; Georgi, K.; Court, W.; Hobbs, S.; Box, G.; Ortiz-Ruiz,

M. J.; Valenti, M.; De Haven Brandon, A.; TePoele, R.; Leuthner, B.;

Workman, P.; Aherne, W.; Poeschke, O.; Dale, T.; Wienke, D.; Esdar,

C.; Rohdich, F.; Raynaud, F.; Clarke, P. A.; Eccles, S. A.; Stieber, F.;

Schiemann, K.; Blagg, J. Discovery of Potent, Orally Bioavailable,

Small-Molecule Inhibitors of WNT Signaling from a Cell-Based

Pathway Screen. J. Med. Chem. 2015, 58, 1717−1735.

(28) Kremlev, M. M.; Mushta, A. I.; Tyrra, W.; Yagupolskii, Y. L.;

Naumann, D.; Moller, A. Me₃SiCF₃/AgF/CuA new reagents

combination for selective trifluoromethylation of various organic

halides by trifluoromethylcopper, CuCF₃. J. Fluorine Chem. 2012, 133,

67−71.

(29) Gleeson, M. P. Generation of a set of simple, interpretable

ADMET rules of thumb. J. Med. Chem. 2008, 51, 817−834.

(30) Charifson, P. S.; Walters, W. P. Acidic and basic drugs in

medicinal chemistry: a perspective. J. Med. Chem. 2014, 57, 9701−

9717.

(31) Bissantz, C.; Kuhn, B.; Stahl, M. A medicinal chemist’s guide to

molecular interactions. J. Med. Chem. 2010, 53, 5061−5084.

(32) Volpe, D. A. Variability in Caco-2 and MDCK cell-based

intestinal permeability assays. J. Pharm. Sci. 2008, 97, 712−725.

(33) Hann, M. M.; Keseru, G. M. Finding the sweet spot: the role of

nature and nurture in medicinal chemistry. Nat. Rev. Drug Discovery

2012, 11, 355−365.

(34) Johnson, T. W.; Dress, K. R.; Edwards, M. Using the Golden

Triangle to optimize clearance and oral absorption. Bioorg. Med. Chem.

Lett. 2009, 19, 5560−5564.

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DOI: 10.1021/acs.jmedchem.5b01685

J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Discovery of Potent, Selective, and Orally Bioavailable Small-Molecule Modulators of the Mediator Complex-Associated Kinases CDK8 and CDK19

DOI: 10.1021/acs.jmedchem.5b01685

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Article abstract of DOI:10.1021/acs.jmedchem.5b01685

Full text of DOI:10.1021/acs.jmedchem.5b01685

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