Changing for the Better: Discovery of the Highly Potent and Selective CDK9 Inhibitor VIP152 Suitable for Once Weekly Intravenous Dosing for the Treatment of Cancer

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Changing for the Better: Discovery of the Highly Potent and

Selective CDK9 Inhibitor VIP152 Suitable for Once Weekly

Intravenous Dosing for the Treatment of Cancer

̈

Ulrich Lucking, Dirk Kosemund, Niels B ö hnke, Philip Lienau, Gerhard Siemeister, Karsten Denner,

Rolf Bohlmann, Hans Briem, Ildiko Terebesi, Ulf B ö mer, Martina Sch ä fer, Stuart Ince,

Dominik Mumberg, Arne Scholz, Raquel Izumi, Stuart Hwang,* and Franz von Nussbaum

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ABSTRACT: Selective inhibition of exclusively transcription-regulating positive

transcription elongation factor b/CDK9 is a promising new approach in cancer

therapy. Starting from atuveciclib, the ﬁrst selective CDK9 inhibitor to enter

clinical development, lead optimization eﬀorts aimed at identifying intravenously

(iv) applicable CDK9 inhibitors with an improved therapeutic index led to the

discovery of the highly potent and selective clinical candidate VIP152. The

evaluation of various scaﬀold hops was instrumental in the identiﬁcation of

VIP152, which is characterized by the underexplored benzyl sulfoximine group.

VIP152 exhibited the best preclinical overall proﬁle in vitro and in vivo, including

high eﬃcacy and good tolerability in xenograft models in mice and rats upon once

weekly iv administration. VIP152 has entered clinical trials for the treatment of cancer with promising longterm, durable

monotherapy activity in double-hit diﬀuse large B-cell lymphoma patients.

therapy. However, the discovery of selective CDK9 inhibitors

is challenging due to the high degree of homology among the

kinases of the CDK family. So far, four selective CDK9

inhibitors, atuveciclib (BAY 1143572),¹⁷AZD4573,¹⁸KB-

0742,¹⁹and VIP152 (previously BAY 1251152),²⁰are reported

to have entered clinical trials (Figure 1). We previously

disclosed the preclinical proﬁle of atuveciclib, the ﬁrst selective,

orally available CDK9 inhibitor to enter clinical develop-

ment.¹⁷In vitro, atuveciclib shows low nanomolar CDK9

inhibitory activity, high selectivity against other CDKs, and

antiproliferative activity against various tumor cell lines with

submicromolar IC₅₀values. Moreover, atuveciclib demonstra-

ted single-agent in vivo eﬃcacy at tolerated doses in various

xenograft tumor models upon oral administration. To fully

explore future treatment options using selective CDK9

inhibitors, we initiated a follow-up program to identify novel

selective CDK9 inhibitors suitable for intravenous (iv)

administration in patients. We now outline the discovery of

the highly potent and selective CDK9 inhibitor VIP152, which

has entered phase 1 clinical trials in patients.

INTRODUCTION

■

The cyclin-dependent kinase (CDK) family consists of closely

related serine/threonine kinases with specialized functions

related to the regulation of the cell cycle (CDKs 1, 2, 4, and 6)

or transcription (CDKs 7, 8, 9, 12, 13, and 19).¹⁻³Due to their

critical role in cell division and gene transcription, CDKs have

been considered strong prospective targets for a new

generation of anticancer drugs.^4,5However, while multi-CDK

inhibitors such as alvocidib,⁶dinaciclib,⁷or roniciclib⁸revealed

promising results in xenograft models, they have displayed

limited treatment responses and signiﬁcant adverse events in

clinical trials, with the exception of certain hematological

malignancies, which have been attributed to their CDK9

inhibitory activity.^9,10CDK9 associates with cyclins T1, T2a,

and T2b to form the heterodimer positive transcription

elongation factor b (PTEFb), which activates transcription

via phosphorylation of RNA polymerase II.¹¹Overexpression

and/or hyperactivity of CDK9 is associated with a variety of

human pathological conditions such as cancer, virally induced

infectious diseases, and cardiovascular diseases.^12,13PTEFb-/

CDK9-mediated transcription of short-lived antiapoptotic

survival proteins and oncogenes such as MCL-1 and MYC

plays a critical role in a variety of cancers, especially in several

hematological malignancies.¹⁴⁻¹⁶Selective, transient inhibition

of CDK9 leads to a rapid depletion of short-lived messenger

RNA (mRNA) transcripts of these important survival proteins

and oncogenes, providing a promising rationale for cancer

Received: June 2, 2021

https://doi.org/10.1021/acs.jmedchem.1c01000

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Journal of Medicinal Chemistry

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Table 1. Lead Optimization Eﬀorts at the Hinge Binding

Core and the Corresponding Key In Vitro Properties

Figure 1. Structures of the clinical CDK9 inhibitors atuveciclib,

AZD4573, KB-0742, and VIP152.

RESULTS AND DISCUSSION

■

Atuveciclib as a Starting Point for the Identiﬁcation

of the New Lead Structure BAY-332. Atuveciclib

demonstrated potent and highly selective CDK9 inhibitor in

our biochemical in vitro assay under low adenosine 5′-

triphosphate (ATP) conditions (ATP concentration: 10 μM;

IC₅₀CDK9/CycT1: 13 nM; ratio of IC₅₀values CDK2/

CDK9: 100, see Table 3; and selectivity within the CDK

family: >145).¹⁷However, under high ATP conditions (ATP

concentration: 2 mM), which reﬂect the approximate cellular

ATP concentration in humans, a signiﬁcant drop in CDK9

inhibitory activity, by a factor of ca. 30, was recorded (IC₅₀

CDK9/CycT1: 380 nM). Atuveciclib revealed promising

antiproliferative activity against a variety of human cancer

cell lines, for example, against A2780 (IC₅₀: 380 nM) and

MOLM-13 (IC₅₀: 310 nM) cells, and high aqueous solubility

(S_w, pH 6.5: 479 mg/L). In in vivo pharmacokinetic (PK)

studies in rats, atuveciclib showed low blood clearance (CL_b:

1.1 L/h/kg), a moderate to high volume of distribution (V_ss:

1.0 L/kg), and a short half-life (t_1/2: 0.6 h) (see Table 3).

Moreover, atuveciclib demonstrated eﬃcacy in human

xenograft tumor models [e.g., models of acute myeloid

leukemia (AML) in mice and rats]. The compound is active

upon once daily dosing, as well as upon certain intermittent

dosing schedules (3 on/2 oﬀ). Due to the promising overall

proﬁle in vitro and in vivo, atuveciclib entered clinical trials in

patients with advanced cancer and leukemia (NCT01938638

and NCT02345382). In a phase 1 study to determine the

safety, tolerability, PKs, maximum tolerated dose (MTD), and

preliminary antitumor activity in patients with advanced

cancer, neutropenia became the most prominent adverse

event upon treatment with atuveciclib.²¹

Since inhibition of MCL-1 induces apoptosis in circulating

neutrophils and other white blood cell types,²²permanent on-

target inhibition of MCL-1 in circulating white blood cells by

daily oral administration of atuveciclib is thought to be the

main reason for the observed neutropenia in these patients. To

fully explore future treatment options using selective CDK9

inhibitors, we initiated a follow-up program aimed at

maximizing the therapeutic index. A highly potent and selective

CDK9 inhibitor with a short half-life was envisaged to enable

deﬁned periods of acute but suﬃcient CDK9 inhibition for the

desired antitumor eﬀect without reducing the therapeutic

window by prolonged transcription inhibition.^23,24Moreover,

switching from oral to iv administration was expected to

reduce the variation in plasma levels and exposure often

associated with oral administration of small-molecule kinase

inhibitors²⁵and thus contribute to the desired increase of the

therapeutic index.

In preclinical studies, atuveciclib was rated not feasible for iv

application in patients. In animal models, the compound

revealed only limited eﬃcacy after once weekly iv application.

Moreover, the aqueous solubility of atuveciclib is not suﬃcient

to enable the formulation of the corresponding predicted

therapeutic human dose of the compound for iv administration

in patients. Thus, we set out to identify a new generation of

potent and highly selective CDK9 inhibitors suitable for

intermittent iv treatment of patients with cancer.

According to docking experiments, binding of atuveciclib to

the hinge region of CDK9 is mediated by the triazine core and

the aniline NH via two hydrogen bonds (Figure 2). The

methoxyphenyl substituent at the triazine points toward the

ribose pocket, whereas the benzylic sulfoximine moiety is

directed toward the exit of the ATP binding pocket.

B

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Table 2. Selected Examples of Lead Optimization Eﬀorts at the 2-Position of the 5-Fluoropyrimidine Scaﬀold and the

Corresponding Key In Vitro Properties

a

b

c

Cells were treated with test compounds for 96 h. The solid state of the test compounds was not characterized. Determined by a high-throughput

screening method using 1 mM dimethyl sulfoxide (DMSO) stock solutions.³²n.d.: not determined. Determined by equilibrium shake ﬂask

d

e

solubility assay from powder.

Sulfoximines, long neglected in drug discovery, have

attracted signiﬁcant interest in medicinal chemistry re-

cently.^26,27Sulfoximines are aza analogues of sulfones, but

their properties diﬀer signiﬁcantly: the introduction of the

nitrogen atom creates asymmetry and oﬀers an additional

point for substitution. The nitrogen atom is basic enough to

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Table 3. Properties of the CDK9 Inhibitors Atuveciclib, BAY-332, 25, and VIP152

atuveciclib

BAY-332

25

VIP152

CDK9/CycT1; IC₅₀[nM] low ATP

13

5

4

3

selectivity vs CDK2, ratio of low ATP IC₅₀values

CDK9/CycT1; IC₅₀[nM] high ATP

100

380

54

37

154

11

150

3

selectivity vs CDK2, ratio of high ATP IC₅₀values

53

380

310

479

107

57

85

24

785

126

80

2

43

1033

45

29

25

a

A2780; IC₅₀[nM]

MOLM-13; IC₅₀[nM]

a

b

S_w, pH 6.5 [mg/L]

b

S_w, pH 4 [mg/L]

309

30

699

P_appA−B [nm/s]

35

168

26

166

eﬄux ratio

6.1

0.17

0.15

1.1

1.0

0.6

1.1

>20

0.61

1.9

1.6

1.3

1.6

1.0

0.88

9.3

1.1

0.91

0.82

1.1

0.74

1.0

0.92

>20

c

CL_bratHep [L/h/kg]

CL_bratLM [L/h/kg]

CL_brat in vivo, iv [L/h/kg]

V_ssrat in vivo, iv [L/kg]

t_1/2rat in vivo, iv [h]

blood/plasma ratio (rat)

CYP inhibition [μM]

CYP1A2 induction

n.d.

c

2C8: 9.2, 2C19: 15, all others: >20

no (up to 41 μg/L)

no (up to 370 μg/L)

no (up to 14 μg/L)

a

b

Cells were treated with test compounds for 96 h. Determined by equilibrium shake ﬂask solubility assay from powder. The solid state of the test

c

compounds was not characterized. n.d.: not determined.

series. The sulfone analogue of atuveciclib, triazine 1, was used

as a point of reference (Table 1). Switching from the triazine

core to pyridine 2 improved the CDK9 inhibitory activity in

vitro under low and high ATP conditions (IC₅₀values: 9 and

254 nM) but also reduced the selectivity against CDK2 (ratio

of low ATP IC₅₀values: 30). The selectivity against cell cycle

kinase CDK2 was used as a ﬁrst indicator for the overall

selectivity within the family of CDKs. Introduction of a

ﬂuorine substituent at the 5-position of the pyridine core (3)

further improved the activity against CDK9 (IC₅₀values: 4 and

99 nM), while ﬂuorine at the 3-position (4) reduced the in

vitro activity signiﬁcantly (IC₅₀values: 144 and >10,000 nM).

However, switching from triazine 1 to 5-ﬂuoropyrimidine 5 as

the central core substantially improved the CDK9 inhibitory

activity under both low and high ATP conditions (IC₅₀values:

3 and 7 nM) while slightly improving the selectivity against

CDK2 (ratio of low ATP IC₅₀values: 77). No further

improvement of in vitro potency and selectivity against CDK2

was observed upon the introduction of a chloro (6), cyano (7),

or triﬂuoromethyl (8) substituent at the 5-position.

Figure 2. Docking mode of atuveciclib in complex with CDK9. The

compound was docked into a published X-ray complex of CDK9/

CycT1 (PDB ID: 3MY1). The aminotriazine moiety can form two

hydrogen bonds to the hinge region of the kinase. For the substituted

phenyl ring attached to the triazine, a π-stacking interaction with

Phe103 and a weak hydrogen bond from the para-ﬂuoro substituent

to Lys48 may be postulated.

Based on the results from our optimization eﬀorts at the

hinge binding core in the sulfone series, the initial lead

optimization eﬀorts in the analogous sulfoximine series led to

the identiﬁcation of the new lead compound BAY-332 which is

characterized by a 5-ﬂuoropyrimidine scaﬀold and an addi-

tional ﬂuorine atom at the 3-position of the benzylic

sulfoximine substituent (see Table 2). In comparison to

atuveciclib, BAY-332³⁰demonstrated signiﬁcantly improved in

vitro activity against CDK9 in the biochemical assays,

especially under high ATP conditions [IC₅₀values: 380 nM

(atuveciclib) vs 37 nM (BAY-332), see Tables 2 and 3]. The

new lead compound also revealed improved selectivity against

CDK2 based on the ratio of IC₅₀values from the

corresponding biochemical in vitro assays under high ATP

conditions [ratio: 53 (atuveciclib) vs 107 (BAY-332)]. BAY-

332 also exhibited improved antiproliferative potency in vitro,

for example, against MOLM-13 cells [IC₅₀values: 310 nM

(atuveciclib) vs 85 nM (BAY-332)] and A2780 cells [IC₅₀

values: 380 nM (atuveciclib) vs 57 nM (BAY-332)]. In vitro

allow metal ion coordination or salt formation. The

heteroatoms bound to the sulfur are hydrogen bond acceptors,

with NH sulfoximines having dual hydrogen bond donor/

acceptor functionality. Moreover, sulfoximines have often

exhibited high hydrolytic and metabolic stability, as well as

favorable physicochemical properties.²⁷⁻²⁹In the lead

optimization approach that led to the identiﬁcation of

atuveciclib, we evaluated a broad variety of functional groups

at the molecular position directed to the exit of the binding

pocket, but the sulfoximine group oﬀered the best overall

proﬁle.¹⁷Thus, we decided to keep the sulfoximine moiety at

this position initially and rather started to investigate the

structural modiﬁcations of the hinge binding core to improve

the CDK9 inhibitory potency in vitro while maintaining high

kinase selectivity. Since relative to sulfoximines, the syntheti-

cally less demanding sulfone analogues in general revealed

comparable in vitro activity against CDK9 in the triazine

inhibitor series; this evaluation was undertaken on the sulfone

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PK studies with BAY-332 demonstrated moderate metabolic

stability in rat hepatocytes (ratHep), resulting in a moderate

predicted blood clearance (CL_b) of 1.9 L/h/kg. In contrast to

atuveciclib, we recorded a high permeability coeﬃcient (P_app

A−B) of 168 nm/s (atuveciclib: 35 nm/s) and a low eﬄux

ratio of 0.61 (atuveciclib: 6.1) raising no concerns about a

possible eﬄux transporter recognition of BAY-332. However,

aqueous solubility at pH 6.5 of BAY-332 was signiﬁcantly

reduced relative to atuveciclib [S_w: 479 mg/L (atuveciclib) vs

24 mg/L (BAY-332)], presumably mainly driven by the switch

from the polar triazine to the 5-ﬂuoropyrimidine core. PK

studies with BAY-332 in rats in vivo revealed a moderate blood

clearance (CL_b: 1.3 L/h/kg), a high volume of distribution

(V_ss: 1.6 L/kg), and an intermediate half-life (t_1/2: 1.0 h). In

comparison to BAY-332, its enantiomer (9) revealed very

similar in vitro properties, well within the limits of measure-

ment accuracy.

good tolerability upon treatment with BAY-332, with a body

weight change of less than 10%, but two fatal toxicities upon

Q3D and Q5D treatment in these unoptimized dosing

schedules. Nevertheless, due to the insuﬃcient solubility

properties of BAY-332, formulation of the predicted human

dose for once weekly iv treatment of patients was rated not

feasible.

Lead Optimization in the 5-Fluoropyrimidine Series.

To enable formulation of a new generation of CDK9 inhibitors

for intermittent iv administration in patients, we set out to

further optimize the biochemical and cellular in vitro potency

to reduce the required therapeutic dose. Complementarily, we

also aimed to improve the solubility properties. In the 5-

ﬂuoropyrimidine series, we focused on structural variation of

the substituent at the 2-position, which is directed to the exit of

the ATP binding pocket. The introduction of polar

substituents at this position to increase aqueous solubility

was envisaged to be more feasible than for the aromatic

substituent at the 4-position pointed toward the ribose pocket.

Moreover, in the lead optimization approach to atuveciclib, the

structural variation of the ribose pocket substituent had

revealed a steep structure−activity relationship with respect

to selectivity against CDK2 and in vitro potency.¹⁷Table 2

presents selected examples of our extensive lead optimization

eﬀorts at the 2-position of the pyrimidine scaﬀold.³¹Various

structural alternatives for the ﬂuorine at the phenyl 3-position

were feasible with respect to in vitro potency. Exchange of the

ﬂuorine atom for a methoxy substituent (10), for instance,

increased the CDK9 inhibitory activity and selectivity against

CDK2, though reduced solubility. Introduction of a second

ﬂuorine atom at the 4-position (11) did not impact in vitro

potency signiﬁcantly but increased metabolic instability in rat

hepatocytes. Use of an N-cyano sulfoximine group in

combination with the additional ﬂuorine at the 4-position of

the phenyl substituent (12) improved the biochemical and

cellular activity signiﬁcantly but further reduced the metabolic

stability. Moreover, the CDK9 inhibitor 12 revealed very low

aqueous solubility. Introduction of an additional nitrogen atom

into the aromatic substituent at the 2-position was promising

with respect to the CDK9 inhibitory activity, selectivity against

CDK2, and aqueous solubility, but pyridine 13 revealed low

permeability (P_appA−B: 23 nm/s) and a high eﬄux ratio

(>10) in the bidirectional Caco-2 permeability assay, raising

concerns about transporter recognition. Introduction of a polar

hydroxy group into the alkyl substituent of the N-cyano

In the MOLM-13 human AML model in mice, BAY-332

administered iv either once daily (QD, 15 mg/kg), once every

3 days (Q3D, 35 mg/kg), or once every 5 days (Q5D, 65 mg/

kg) demonstrated potent antitumor eﬃcacy with T/C ratios of

0.26 (p = 0.006), 0.26 (p = 0.007), and 0.34 (p = 0.029),

respectively (see Figure 3 and Table 4). Additionally, there was

Figure 3. Antitumor eﬃcacy of BAY-332 in the MOLM-13 human

AML model in mice. Tumor growth in mice treated iv with either

vehicle or BAY-332 once daily (QD), once every 3 days (Q3D), or

once every 5 days (Q5D). Treatments were started 5 days after tumor

cell inoculation. Statistical signiﬁcances were calculated using the

mean tumor volumes at the time point when the vehicle group was

sacriﬁced. Asterisks indicate statistical signiﬁcance relative to the

vehicle control (*p < 0.05 and **p < 0.01).

Table 4. Antitumor Eﬃcacy of BAY-332 and VIP152 in the MOLM-13 Human AML Model in Nude Mice and Athymic Rats

a

b

animal model

treatment

dose (mg/kg)

schedule

T/C_volume

fatal toxicity

maximum body weight loss (%)

MOLM-13 AML xenografts in mice

vehicle

0

15

35

65

0

QD

1.00

0.26**

0.34*

0/12

2/12

0/12

2.8

4.5

5.8

4.8

0.0

0.1

4.9

BAY-332

vehicle

VIP152

vehicle

Q3D

Q5D

Q3D

Q5D

QW

QD

MOLM-13 AML xenografts in rats

MOLM-13 AML xenografts in mice

1.00

3

0.11***

0.08***

0.15***

1.00

3.75

4.5

0

VIP152

12.5

QW

0.20**

a

Treatment-to-control (T/C) ratios and statistical signiﬁcances were calculated using the mean tumor volumes at the time point when the vehicle

b

group was sacriﬁced. Asterisks indicate the statistical signiﬁcance relative to the vehicle control (*p < 0.05, **p < 0.01, and ***p < 0.001). The

maximum mean body weight loss expressed as a percentage of the starting weight of the animal. Weight loss greater than 20% is considered toxic.

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sulfoximine group (14) led to signiﬁcant improvement of

CDK9 inhibitory activity and selectivity against CDK2 over

BAY-332. On the other hand, hydroxy derivative 14

demonstrated only moderate antiproliferative activity against

A2780 cells in vitro, low aqueous solubility, and low metabolic

stability. Introduction of an NH₂group into the alkyl chain in

combination with an NH sulfoximine group (15) improved

solubility signiﬁcantly (S_w, pH 6.5: 294 mg/L); however,

amine 15 revealed no permeability (P_appA−B: 0 nm/s)

through the Caco-2 cell monolayer. The aza analogue of

sulfoximine BAY-332, sulfondiimine 16, was prepared with the

hope for similar potency and selectivity but further improved

aqueous solubility. Moreover, no chiral separation of

enantiomers is required in the case of achiral sulfondiimine

16. In comparison to BAY-332, matched analogue 16 revealed

reduced in vitro activity against CDK9 (IC₅₀: 104 nM), as well

as reduced antiproliferative activity against A2780 cells (IC₅₀:

184 nM), yet similar selectivity against CDK2 (ratio of high

ATP IC₅₀values: 101) and signiﬁcantly improved aqueous

solubility at pH 6.5 (S_w: 155 mg/L). In the Caco-2 assay,

sulfondiimine 16 demonstrated a reduced permeability

coeﬃcient [P_appA−B: 168 nm/s (BAY-332) vs 50 nm/s

(16)] and an increased eﬄux ratio [0.61 (BAY-332) vs 5.6

(16)]. Moreover, sulfoximine BAY-332 and matched sulfon-

diimine 16 displayed comparable metabolic stability in rat

hepatocytes in vitro. In a subsequent rat PK study in vivo, an

increased volume of distribution (V_ss: 2.2 L/kg) and

signiﬁcantly increased blood clearance (CL_b: 3.3 L/h/kg) as

well as a short half-life (t_1/2: 0.6 h) of analogue 16 relative to

BAY-332 were recorded. Evaluation of non-sulfur-based

functional groups at the exit of the ATP binding pocket did

not lead to the identiﬁcation of a compound with a suitable

overall proﬁle, similar to the ﬁndings in the lead optimization

approach that led to atuveciclib.¹⁷Benzylamine 17, for

instance, demonstrated reduced in vitro activity in the

biochemical and cellular assays compared to BAY-332, as

well as low metabolic stability and no improvement of aqueous

solubility.

Figure 4. Docking modes of the 5-ﬂuoropyrimidine BAY-332 and the

N-(pyridin-2-yl)pyridin-2-amine VIP152 in complex with CDK9. The

compounds were docked into a published X-ray complex of CDK9/

CycT1 (PDB ID: 3MY1). BAY-332: the aminopyrimidine moiety can

form two hydrogen bonds to the hinge region of the kinase. An

additional hydrogen bond is formed by the sulfoximine NH to the

backbone carbonyl group of Glu107. For the substituted phenyl ring

attached to the pyrimidine, a π-stacking interaction with Phe103 and a

weak hydrogen bond from the para-ﬂuoro substituent to Lys48 may

be postulated. VIP152: the central aminopyridine moiety can form

two hydrogen bonds to the hinge region of the kinase. An additional

hydrogen bond is formed by the sulfoximine NH to the backbone

carbonyl group of Glu107. For the substituted phenyl ring attached to

the central pyridine, a π-stacking interaction with Phe103 and a weak

hydrogen bond from the para-ﬂuoro substituent to Lys48 may be

postulated.

nM (25) vs 57 nM (BAY-332)] and MOLM-13 cells [IC₅₀

values: 80 nM (25) vs 85 nM (BAY-332)] was recorded.

Moreover, 4,6-disubstituted pyrimidine 25 displayed reduced

aqueous solubility at pH 6.5 [S_w: 2 mg/L (25) vs 24 mg/L

(BAY-332)], as well as low permeability (P_appA−B: 26 nm/s)

and a high eﬄux ratio (9.3), which raised concern with respect

to a potential transporter recognition of 25. In contrast, in the

corresponding N-(pyridin-2-yl)pyridin-2-amine series, single

enantiomer VIP152 revealed a very promising preclinical

overall proﬁle.

Additional Scaﬀold Hops Leading to the Clinical

Candidate. Since our lead optimization eﬀorts to identify a

compound with a superior preclinical overall proﬁle to lead

compound BAY-332 proved fruitless in the 5-ﬂuoropyrimidine

series, we turned our attention again to the evaluation of

additional scaﬀold modiﬁcations. However, this time we also

incorporated the neighboring aromatic substituent at the NH

position in our design. The idea was to evaluate a shift of the

nitrogen of the 5-ﬂuoropyrimidine scaﬀold, which is not

involved in hydrogen bonding to the hinge region of the ATP

binding pocket, into the adjacent aromatic ring to yield N-

(pyridin-2-yl)pyridin-2-amine 31 (Figure 4 and Scheme 2). In

the triazine series, the same strategy leads to N-(pyridin-2-

yl)pyrimidin-4-amine 24 (Scheme 1).

After a successful synthesis (see Scheme 1), the enantiomers

of N-(pyridin-2-yl)pyrimidin-4-amine 24 exhibited very similar

in vitro properties, much the same as the results previously

recorded with the corresponding enantiomers in the triazine

and 5-ﬂuoropyrimidine series. The slightly more potent

enantiomer 25 revealed signiﬁcantly improved CDK9 inhib-

itory activity (IC₅₀: 11 nM) and selectivity against CDK2

under high ATP conditions (ratio of IC₅₀values: 785) in

comparison to lead structure BAY-332 (IC₅₀: 37 nM and ratio

of IC₅₀values: 107; Table 3). However, no increased

antiproliferative activity against A2780 cells [IC₅₀values: 126

Preclinical Properties of VIP152. N-(Pyridin-2-yl)-

pyridin-2-amine VIP152 (see Scheme 2 and Table 3) revealed

very potent CDK9 inhibitory activity (IC₅₀: 3 nM) and high

selectivity against the structurally related kinases CDK2 (ratio

of IC₅₀values: 1033) and CDK7 (ratio of IC₅₀values: >5000)

in the Bayer kinase panel under high ATP conditions. The high

kinase selectivity of VIP152 in vitro, even within the CDK

family, was conﬁrmed in the external kinase panels with a

selectivity factor of at least 50 against all tested CDKs (see the

Supporting Information). Outside the CDK family, substantial

inhibitory activity with a selectivity factor of less than 50 was

only recorded against GSK3α and IRAK1 (DiscoverX K_D

values: CDK9, 1.3 nM; GSK3α, 7.4 nM; and IRAK1, 61

nM) (see the Supporting Information). In a recent

comprehensive survey of CDK inhibitor selectivity in live

cells with energy-transfer probes, VIP152 demonstrated the

strongest target aﬃnity and selectivity among the tested CDK9

inhibitors.³³VIP152 exhibited very potent antiproliferative

activity in vitro, especially against A2780 cells (IC₅₀: 45 nM)

and MOLM-13 cells (IC₅₀: 29 nM). In comparison to VIP152,

its enantiomer 32 revealed very similar in vitro properties, well

within the limits of measurement accuracy. However, with

multiple batches of enantiomer 32, there was a trend toward a

slightly reduced activity against CDK9 in the biochemical assay

under high ATP conditions (IC₅₀: 6 nM) and antiproliferative

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a

Scheme 1. Synthesis of N-(Pyridin-2-yl)pyrimidin-4-amines

a

Reagents and conditions: (a) Pd(dppf)Cl₂, K₂CO₃(aq), 1,2-dimethoxyethane (DME), 90 °C, 150 min, and 79%; (b) Xantphos, Pd₂(dba)₃,

Cs₂CO₃, 1,4-dioxane, 100 °C, 22 h, and 60%; (c) CF₃C(O)NH₂, NaOt-Bu, 1,3-dibromo-5,5-dimethylhydantoin, 1,4-dioxane, tetrahydrofuran

(THF), 10 °C, and 63%; (d) KMnO₄, acetone, 50 °C, and crude; (e) K₂CO₃, MeOH, room temperature (rt), and 15% (over two steps); and (f)

chiral preparative HPLC.

a

Scheme 2. Synthesis of VIP152

a

Reagents and conditions: (a) Pd(PPh₃)₄, K₂CO₃(aq), DME, 100 °C, 4 h, and 95%; (b) Xantphos, Pd₂(dba)₃, Cs₂CO₃, 1,4-dioxane, 100 °C, 5 h,

and 94%; (c) CF₃C(O)NH₂, NaOt-Bu, 1,3-dibromo-5,5-dimethylhydantoin, 1,4-dioxane, THF, 10 °C, and crude; (d) KMnO₄, acetone, 50 °C, and

crude; (e) K₂CO₃, MeOH, rt, and 10% (over three steps); and (f) chiral preparative HPLC.

activity against A2780 cells (IC₅₀: 52 nM) and MOLM-13 cells

(IC₅₀: 42 nM). VIP152 demonstrated low aqueous solubility at

pH 6.5 (S_w: 25 mg/L), but in contrast to atuveciclib and BAY-

332, N-(pyridin-2-yl)pyridin-2-amine VIP152 exhibited a

signiﬁcant increase in solubility at pH 4 (S_w: 699 mg/L),

which is still within the physiologically acceptable range for iv

application in humans.³⁴Nevertheless, VIP152 exhibited high

Caco-2 permeability (P_appA−B: 166 nm/s) and no eﬄux

(eﬄux ratio: 1.1) in the Caco-2 assay.

In an in vivo PK study in rats (Table 3), VIP152 showed low

blood clearance (CL_b: 1.1 L/h/kg), a moderate volume of

distribution (V_ss: 0.74 L/kg), and a short to moderate half-life

(t_1/2: 1.0 h). Furthermore, VIP152 exhibited a blood/plasma

ratio of about 1 and did not show signiﬁcant inhibition of

cytochrome P450 activity, with IC₅₀values >20 μM. Induction

of CYP1A2 in vitro was not observed.

(4.5 mg/kg) demonstrated similar potent antitumor eﬃcacies

with T/C ratios of 0.11, 0.08, and 0.15, respectively, and

excellent tolerability (all p < 0.001; Figure 5A and Table 4).

Antitumor activity upon once weekly iv dosing was then

conﬁrmed in a second species using the MOLM-13 xenograft

model in mice. Once weekly (QW) administration of VIP152

at 12.5 mg/kg resulted in a T/C ratio of 0.20 (p = 0.001;

Figure 5B and Table 4) and was well tolerated, as indicated by

no signiﬁcant body weight reduction (body weight change:

−5%) and no fatal toxicities throughout the study.

The MV4-11 xenograft model in rats was used to compare

the activity of daily oral administration of atuveciclib (12 mg/

kg × 15 days) compared with once weekly iv dosing of VIP152

(4.5 mg/kg, Q7D × 3). Treatment with VIP152 leads to

complete remission in all animals with no measurable tumor

burden for an additional 30 days without treatment compared

with regrowth in all of the atuveciclib-treated animals.

The MOLM-13 xenograft model in rats was used to

determine the optimum application frequency regarding the

antitumor activity of VIP152. VIP152 administered iv at its

respective MTD Q3D (3 mg/kg), Q5D (3.75 mg/kg), or QW

Since the once weekly application maybe the best regimen to

allow for recovery of the treatment-induced acute reduction of

circulating neutrophil levels, this application schedule was

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the desired sulfoximine 24, which was separated into the

enantiomers by chiral high-performance liquid chromatog-

raphy (HPLC). The absolute conﬁguration of the single

stereoisomers 25 and 26 at the sulfur was not determined.

The research synthesis of N-(pyridin-2-yl)pyridin-2-amine

31 was accomplished with a similar synthetic strategy. Suzuki

coupling of 2-chloro-5-ﬂuoro-4-iodopyridine (27) and boronic

acid 19 aﬀorded the desired building block 28 in very high

yield. A subsequent palladium-catalyzed cross-coupling reac-

tion of chloropyridine 28 and commercial pyridin-2-amine 21

resulted in 2,2′-dipyridylamine 29 in very high yield, too.

Finally, thioether 29 was converted into the racemic

sulfoximine 31 relying again on the conditions of Gries and

Kru

̈

ger.³⁵Chiral HPLC separation of racemate 31 yielded

VIP152 and its enantiomer 32. The stereochemistry at the

sulfur of the sulfoximine group of VIP152 was determined by

X-ray crystallography (Figure 6).

Figure 6. Small-molecule X-ray structure of VIP152 (accession code

CCDC 2005811).

CONCLUSIONS

■

Transient modulation of CDK9 by highly selective inhibitors

leads to rapid depletion of short-lived mRNA transcripts of

important survival proteins and oncogenes such as MCL-1 and

MYC,³⁶providing a promising rationale for cancer therapy,

especially for hematological malignancies. Atuveciclib (BAY

1143572), the ﬁrst selective, orally available CDK9 inhibitor

that entered clinical development, revealed promising results in

xenograft models. In a phase 1 clinical trial in patients with

advanced cancer, atuveciclib displayed a limited therapeutic

index with oral administration with neutropenia being the most

prominent adverse event putatively mediated, at least in part,

by the on-target inhibition of MCL-1 in circulating white blood

cells leading to apoptosis induction in neutrophils. With the

aim of improving the therapeutic index of anticancer therapy

with CDK9 inhibitors, a follow-up program was initiated to

identify a new generation of highly potent and selective CDK9

inhibitors enabling once weekly iv treatment of patients by a

more pronounce “oncogenic shock”-like²⁴disruption of

oncogene transcription, followed by a time without target

inhibition needed for the recovery of circulating neutrophils.

Our lead optimization eﬀorts started with atuveciclib focused

on potency, selectivity, and aqueous solubility which led to the

identiﬁcation of a new generation of CDK9 inhibitors.

Sulfoximine containing VIP152 clearly exhibited the most

promising preclinical overall proﬁle with respect to potency,

selectivity, physicochemical properties, and in vivo PK, as well

as in vivo potency. VIP152 is eﬃcacious in human xenograft

tumor models of AML in mice and rats upon once weekly iv

administration with good tolerability. The evaluation of various

scaﬀold hops and the employment of the underexplored benzyl

sulfoximine group were crucial for overcoming hurdles in this

Figure 5. Antitumor eﬃcacy of atuveciclib and VIP152 in human

AML models in rats and mice. (A) Tumor growth in rats treated iv

with either vehicle or VIP152 once every 3 days (Q3D), once every 5

days (Q5D), or once weekly (QW). Treatments were started 6 days

after tumor cell inoculation. (B) Tumor growth in mice treated iv

with either vehicle or VIP152 once weekly (QW). Treatments were

started 5 days after tumor cell inoculation. (C) Tumor growth in rats

treated with atuveciclib p.o. daily (15 days) dosing or VIP152 iv once

weekly (3 doses). Statistical signiﬁcances were calculated using the

mean tumor volumes at the time point when the vehicle group was

sacriﬁced. Asterisks indicate statistical signiﬁcance relative to the

vehicle control (**p < 0.01 and ***p < 0.001).

deﬁned as the target regimen for the clinical development of

VIP152.

Due to the very promising overall proﬁle in vitro and in vivo,

sulfoximine VIP152 was selected as the development candidate

and entered phase 1 clinical trials in patients (NCT02635672

and NCT02745743).

Chemistry. The synthesis of racemic N-(pyridin-2-yl)-

pyrimidin-4-amine 24 started with the reaction of 4,6-

dichloropyrimidine (18) and boronic acid 19 under Suzuki

conditions to aﬀord building block 20. A palladium-catalyzed

cross-coupling reaction of chloropyrimidine 20 and commer-

cial pyridin-2-amine 21 yielded 4,6-disubstituted pyrimidine

22. The thioether was ﬁrst converted into the racemic

sulﬁlimine 23 using the conditions of Gries and Kru

Oxidation and cleavage of the triﬂuoroacetyl group aﬀorded

̈

ger.³⁵

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LC−MS (for a detailed description of the LC−MS methods, see the

Supporting Information). Compound names were generated using

ICS software.

project, which highlights the ongoing important role of

medicinal chemistry in small-molecule drug discovery.

The discovery of VIP152, in optimizing atuveciclib,

demonstrates promising translation of our eﬀorts in early

clinical studies.³⁷The improved selectivity on CDK9 starting

from removal of the CDK2 activity, ATP independence, and a

short-lived, iv strategy resulted in a promising 38% disease

control rate in the ﬁrst 31 patients in the dose escalation phase

of VIP152’s ﬁrst-in-human study. Several patients experienced

extended beneﬁt beyond 15 cycles including two patients with

double-hit diﬀuse large B-cell lymphoma (manuscript in

prep).^37,38This early clinical experience demonstrates that

VIP152 is well tolerated with a manageable safety proﬁle for

extended periods and opens the door to test the oncogenic

shock hypothesis with a once weekly dosing schedule in

patients with cancer.

Synthetic Procedures. Compound 1. 1-[(Methylsulfanyl)-

methyl]-3-nitrobenzene. Sodium methanethiolate (13.5 g, 192

mmol) was added in two portions to a stirred solution of 1-

(chloromethyl)-3-nitrobenzene (30.0 g, 175 mmol) in EtOH (360

mL) at −15 °C. The cold bath was removed, and the mixture was

stirred at rt for 3 h. Then, it was diluted with brine and extracted with

EtOAc (2×). The combined organic phases were washed with H₂O,

dried (Na₂SO₄), ﬁltered, and concentrated to aﬀord the desired

product (32.2 g) that was used without further puriﬁcation. ¹H NMR

(400 MHz, CDCl₃): δ 8.18 (m, 1H), 8.11 (m, 1H), 7.66 (m, 1H),

7.50 (m, 1H), 3.75 (s, 2H), 2.01 (s, 3H).

1-[(Methylsulfonyl)methyl]-3-nitrobenzene. m-Chloroperoxyben-

zoic acid (77%; 26.9 g, 120 mmol) was added to a stirred solution of

1-[(methylsulfanyl)methyl]-3-nitrobenzene (10.0 g) in dichloro-

methane (DCM, 1305 mL) at 0 °C. The mixture was stirred at 0

°C for 30 min and then at rt for 2.5 h. Then, it was diluted with H₂O

(300 mL) and NaHCO₃(11.0 g) was added. The mixture was

extracted with DCM (2×). The combined organic phases were

ﬁltered using a Whatman ﬁlter and concentrated. The residue was

puriﬁed by ﬂash chromatography (DCM/EtOH 95:5) and ﬁnally

recrystallized from EtOAc to aﬀord the desired product (6.2 g, 28.9

EXPERIMENTAL SECTION

■

General Methods and Materials. Commercially available

reagents and anhydrous solvents were used as supplied, without

further puriﬁcation. All air- and moisture-sensitive reactions were

carried out in oven-dried (at 120 °C) glassware under an inert

atmosphere of argon. A Biotage Initiator Classic microwave reactor

was used for reactions conducted in a microwave oven. Reactions

were monitored by thin-layer chromatography (TLC) and ultra-

performance liquid chromatography (UPLC) analysis with a Waters

Acquity UPLC MS Single Quad system; column: Acquity UPLC BEH

C18 1.7 μm, 50 × 2.1 mm; basic conditions: eluent A: H₂O + 0.2 vol

% aq NH₃(32%), eluent B: MeCN; gradient: 0−1.6 min 1−99% B,

1.6−2.0 min 99% B; ﬂow: 0.8 mL/min; acidic conditions: eluent A:

H₂O + 0.1 vol % formic acid (99%), eluent B: MeCN; gradient: 0−

1.6 min 1−99% B, 1.6−2.0 min 99% B; ﬂow: 0.8 mL/min;

temperature: 60 °C; and DAD scan: 210−400 nm. Analytical TLC

was carried out on aluminum-backed plates coated with a Merck

Kieselgel 60 F254, with visualization under UV light at 254 nm. Flash

chromatography was carried out using a Biotage Isolera One system

with a 200−400 nm variable detector. Preparative HPLC was carried

out with a Waters AutoPuriﬁcation MS Single Quad system; column:

Waters XBridge C18 5 μm, 100 × 30 mm; basic conditions: eluent A:

H₂O + 0.2 vol % aq NH₃(32%), eluent B: MeCN; gradient: 0−0.5

min 5% B, ﬂow: 25 mL/min; 0.51−5.50 min 10−100% B, ﬂow: 70

mL/min; 5.51−6.5 min 100% B, ﬂow: 70 mL/min; acidic conditions:

eluent A: H₂O + 0.1 vol % formic acid (99%), eluent B: MeCN;

gradient: 0−0.5 min 5% B, ﬂow: 25 mL/min; 0.51−5.50 min 10−

100% B, ﬂow: 70 mL/min; 5.51−6.5 min 100% B, ﬂow: 70 mL/min;

temperature: 25 °C; and DAD scan: 210−400 nm. NMR spectra were

recorded at rt (22 1 °C), unless otherwise noted, on Bruker Avance

1

mmol, and 53% yield over two steps). H NMR (400 MHz, DMSO-

d₆): δ 8.28 (m, 1H), 8.22 (m, 1H), 7.83 (m, 1H), 7.69 (m, 1H), 4.68

(s, 2H), 2.93 (s, 3H).

3-[(Methylsulfonyl)methyl]aniline. TiCl₃solution (ca. 15% in ca.

10% HCl, 162 mL) was added to a stirred solution of 1-

[(methylsulfonyl)methyl]-3-nitrobenzene (5.1 g, 23.8 mmol) in

THF (250 mL) at rt, and the mixture was stirred for 16 h. The pH

was increased to 10 by the addition of 1 N NaOH; then, the reaction

mixture was extracted with EtOAc (2×). The combined organic

phases were washed with brine, ﬁltered using a Whatman ﬁlter, and

concentrated to aﬀord the desired product (4.5 g) that was used

without further puriﬁcation. ¹H NMR (400 MHz, DMSO-d₆): δ 6.97

(m, 1H), 6.51 (m, 3H), 5.13 (br, 2H), 4.23 (s, 2H), 2.83 (s, 3H).

4-Chloro-N-{3-[(methylsulfonyl)methyl]phenyl}-1,3,5-triazin-2-

amine. DIPEA (3.7 mL, 21.3 mmol) was added to a stirred solution

of 2,4-dichloro-1,3,5-triazine (1.60 g, 10.7 mmol) in THF/i-PrOH

(1:1, 20 mL) at −40 °C. Then, a suspension of 3-[(methylsulfonyl)-

methyl]aniline (1.97 g, 10.7 mmol) in THF/i-PrOH (1:1, 10 mL)

was added at this temperature. Under stirring, the temperature of the

reaction mixture was slowly raised over 3 h to 0 °C. The mixture was

concentrated in vacuo to aﬀord the crude product (5.2 g) that was

used without further puriﬁcation.

4-(4-Fluoro-2-methoxyphenyl)-N-{3-[(methylsulfonyl)methyl]-

phenyl}-1,3,5-triazin-2-amine (1). A mixture of crude 4-chloro-N-{3-

[(methylsulfonyl)methyl]phenyl}-1,3,5-triazin-2-amine (1000 mg),

(4-ﬂuoro-2-methoxyphenyl)boronic acid (569 mg, 3.35 mmol;

Aldrich), and tetrakis(triphenylphosphine)palladium(0) [Pd(PPh₃)₄]

(580 mg, 0.50 mmol) in DME (10.3 mL) and 2 M aq K₂CO₃(3.4

mL) was degassed using argon. The mixture was stirred under argon

for 90 min at 100 °C. After cooling, the mixture was diluted with

EtOAc and washed with brine. The organic phase was ﬁltered using a

Whatman ﬁlter and concentrated. The residue was puriﬁed by ﬂash

chromatography (DCM to DCM/EtOH 95:5) to aﬀord 1 (402 mg,

1.03 mmol) as a colorless solid. ¹H NMR (400 MHz, CDCl₃): δ 8.82

(s, 1H), 7.96 (br s, 1H), 7.69−7.87 (m, 2H), 7.38−7.46 (m, 2H),

7.16 (d, J = 7.53 Hz, 1H), 6.75−6.81 (m, 2H), 4.26 (s, 2H), 3.94 (s,

3H), 2.79 (s, 3H). ESI-HRMS m/z: [M + H]⁺calcd for

C₁₈H₁₈FN₄O₃S, 389.1084; found, 389.1080.

1

III HD spectrometers. H NMR spectra were obtained at 300, 400,

500, or 600 MHz and referenced to the residual solvent signal (7.26

ppm for CDCl₃and 2.50 ppm for DMSO-d₆). ¹³C NMR spectra were

obtained at 100 or 150 MHz and also referenced to the residual

solvent signal (39.52 ppm for DMSO-d₆). ¹H NMR data are reported

as follows: chemical shift (δ) in ppm, multiplicity (s = singlet, d =

doublet, t = triplet, q = quartet, quin = quintet, br = broad, and m =

multiplet), coupling constant(s), and integration. High-resolution

mass spectra were recorded on a Xevo G2-XS Tof (Waters)

instrument. Low-resolution mass spectra (electrospray ionization,

ESI) were obtained via HPLC−MS (ESI) using a Waters Acquity

UPLC system equipped with an SQ 3100 Mass Detector; column:

Acquity UPLC BEH C18 1.7 μm, 50 × 2.1 mm; eluent A: H₂O +

0.05% formic acid (99%), eluent B: MeCN + 0.05% formic acid

(99%); gradient: 0−0.5 min 5% B, 0.5−2.5 min 5−100% B, 2.5−4.5

min 100% B; total run time: 5 min; and ﬂow: 0.5 mL/min. Optical

rotations were recorded on a JASCO P-2000 polarimeter. IR spectra

were obtained on a Bruker Vertex 70 FTIR spectrometer (2 cm⁻¹

resolution, 32 scans, and 400−4000 cm⁻¹range) using single-bounce

diamond attenuated total reﬂection (ATR) or KBr techniques. The

purity of all target compounds was at least 95%, as determined by

Compound 2. 2-Chloro-4-(4-ﬂuoro-2-methoxyphenyl)pyridine.

A mixture of 4-bromo-2-chloropyridine (1 g, 5.20 mmol; Aldrich), (4-

ﬂuoro-2-methoxyphenyl)boronic acid (971 mg, 5.72 mmol), and

Pd(PPh₃)₄(600 mg, 0.52 mmol) in toluene (26 mL) and a solution of

K₂CO₃(861 mg, 6.23 mmol) in H₂O (5.2 mL) was degassed using

argon. The mixture was stirred under argon for 3 h at 90 °C. After

cooling, the mixture was diluted with EtOAc and washed with brine.

The organic phase was ﬁltered using a Whatman ﬁlter and

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concentrated. The residue was puriﬁed by column chromatography

(hexane/EtOAc 4:1) to aﬀord the desired product (210 mg, 0.88

mmol, 17% yield). ¹H NMR (400 MHz, DMSO-d₆): δ 8.40−8.44 (m,

1H), 7.60−7.63 (m, 1H), 7.54 (dd, J = 1.52, 5.07 Hz, 1H), 7.50 (dd, J

= 6.84, 8.62 Hz, 1H), 7.11 (dd, J = 2.53, 11.41 Hz, 1H), 6.92 (dt, J =

2.53, 8.36 Hz, 1H), 3.83 (s, 3H). UPLC (ESI+) m/z: [M + H]⁺238.

4-(4-Fluoro-2-methoxyphenyl)-N-{3-[(methylsulfonyl)methyl]-

phenyl}pyridin-2-amine (2). A mixture of 3-[(methylsulfonyl)-

methyl]aniline (123 mg, 0.66 mmol), 2-chloro-4-(4-ﬂuoro-2-

methoxyphenyl)pyridine (105 mg, 0.44 mmol), tris-

(dibenzylideneacetone)dipalladium(0) [Pd₂(dba)₃] (48 mg, 0.05

mmol), 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (BINAP) (102

mg, 0.16 mmol), and sodium tert-butoxide (85 mg, 0.88 mmol) in

DMF (4.1 mL) and THF (8.3 mL) was stirred under argon for 4.5 h

at 110 °C. After cooling, the mixture was diluted with EtOAc and

washed with brine. The organic phase was ﬁltered using a Whatman

ﬁlter and concentrated. The residue was puriﬁed by column

chromatography (hexane/EtOAc) to aﬀord 2 (55 mg, 0.14 mmol,

32% yield). ¹H NMR (400 MHz, DMSO-d₆): δ 9.16 (s, 1H), 8.15 (d,

J = 5.32 Hz, 1H), 7.78 (br d, J = 7.86 Hz, 1H), 7.63 (s, 1H), 7.39 (dd,

J = 6.84, 8.36 Hz, 1H), 7.28 (t, J = 7.86 Hz, 1H), 7.07 (dd, J = 2.28,

11.41 Hz, 1H), 6.97 (s, 1H), 6.86−6.95 (m, 2H), 6.85 (dd, J = 1.27,

5.32 Hz, 1H), 4.44 (s, 2H), 3.83 (s, 3H), 2.93 (s, 3H). ¹³C NMR

ATR, cm⁻¹): 3370 (w), 3330 (w), 3105 (w), 2924 (w), 1624 (m),

1601 (s), 1483 (s), 1447 (m), 1379 (m), 1298 (s), 1213 (m), 1194

(m), 1153 (m), 1026 (m), 953 (m), 878 (w), 837 (w), 798 (m).

UPLC (ESI+) m/z: [M + H]⁺405. ESI-HRMS m/z: [M + H]⁺calcd

for C₂₀H₁₉F₂N₂O₃S, 405.1085; found, 405.1082.

Compound 4. 2-Chloro-3-ﬂuoro-4-(4-ﬂuoro-2-methoxyphenyl)-

pyridine. A mixture of 2-chloro-3-ﬂuoro-4-iodopyridine [1 g, 3.88

mmol; Tokyo Chemical Industry (TCI)], (4-ﬂuoro-2-

methoxyphenyl)boronic acid (660 mg, 3.88 mmol), and Pd(PPh₃)₄

(449 mg, 0.38 mmol) in DME (10 mL) and 2 M aq K₂CO₃(5.8 mL)

was degassed using argon. The mixture was stirred under argon for 3.5

h at 100 °C. After cooling, the mixture was diluted with EtOAc and

washed with brine. The organic phase was ﬁltered using a Whatman

ﬁlter and concentrated. The residue was puriﬁed by column

chromatography (hexane to hexane/EtOAc 1:1) to aﬀord the desired

product (569 mg, 2.23 mmol, 57% yield). ¹H NMR (400 MHz,

DMSO-d₆): δ 8.27 (d, J = 5.05 Hz, 1H), 7.45 (t, J = 5.05 Hz, 1H),

7.37 (dd, J = 6.95, 8.46 Hz, 1H), 7.09 (dd, J = 2.53, 11.37 Hz, 1H),

6.90 (dt, J = 2.53, 8.46 Hz, 1H), 3.77 (s, 3H).

3-Fluoro-4-(4-ﬂuoro-2-methoxyphenyl)-N-{3-[(methylsulfonyl)-

methyl]phenyl}pyridin-2-amine (4).

A mixture of 3-

[(methylsulfonyl)methyl]aniline (135 mg, 0.73 mmol), 2-chloro-3-

ﬂuoro-4-(4-ﬂuoro-2-methoxyphenyl)pyridine (125 mg, 0.49 mmol),

Pd₂(dba)₃(54 mg, 0.06 mmol), BINAP (113 mg, 0.18 mmol), and

sodium tert-butoxide (94 mg, 0.98 mmol) in DMF (4.5 mL) and THF

(9 mL) was stirred under argon for 2 h at 100 °C. After cooling, the

mixture was diluted with EtOAc and washed with brine. The organic

phase was ﬁltered using a Whatman ﬁlter and concentrated. The

residue was puriﬁed by preparative HPLC (basic conditions) to aﬀord

1

(100.67 MHz, DMSO-d₆): δ 163.18 (d, J_CF= 244.99 Hz, 1C_q),

3

157.54 (d, J_CF= 10.60 Hz, 1C_q), 155.83 (s, 1C_q), 147.01 (s, 1CH),

3

145.95 (s, 1C_q), 141.94 (s, 1C_q), 131.17 (d, J_CF= 10.17 Hz, 1CH),

4

129.32 (s, 1C_q), 128.78 (s, 1CH), 123.93 (d, J_CF= 3.39 Hz, 1C_q),

122.99 (s, 1CH), 120.05 (s, 1CH), 117.95 (s, 1CH), 115.35 (s,

2

1CH), 110.91 (s, 1CH), 107.25 (d, J_CF= 21.19 Hz, 1CH), 100.28

1

2

4 (78 mg, 0.19 mmol, 39% yield). H NMR (400 MHz, CDCl₃): δ

(d, J_CF= 25.86 Hz, 1CH), 59.72 (s, 1CH₂), 56.17 (s, 1CH₃), 39.50

(s, 1CH₃). IR (diamond ATR, cm⁻¹): 3597 (br s), 3364 (w), 3290

(w), 3009 (w), 2922 (w), 1597 (s), 1529 (m), 1479 (m), 1443 (m),

1379 (m), 1279 (s), 1192 (m), 1113 (s), 1024 (s), 951 (s), 876 (m),

833 (m), 798 (m). ESI-HRMS m/z: [M + H]⁺calcd for

C₂₀H₂₀FN₂O₃S, 387.1179; found, 387.1175.

8.01 (d, J = 5.05 Hz, 1H), 7.82 (s, 1H), 7.72 (dd, J = 1.64, 7.96 Hz,

1H), 7.38 (t, J = 7.96 Hz, 1H), 7.22−7.28 (m, 1H), 7.06 (d, J = 7.58

Hz, 1H), 6.72−6.80 (m, 4H), 4.27 (s, 2H), 3.83 (s, 3H), 2.80 (s, 3H).

1

¹³C NMR (100.67 MHz, DMSO-d₆): δ 163.58 (d, J_CF= 244.99 Hz,

3

2

1C_q), 157.87 (d, J_CF= 10.60 Hz, 1C_q), 145.09 (d, J_CF= 11.02 Hz,

1

3

1C_q), 143.94 (d, J_CF= 256.43 Hz, 1C_q), 141.71 (d, J_CF= 7.20 Hz,

1CH), 140.92 (s, 1C_q), 131.65 (d, ³J_CF= 10.17 Hz, 1CH), 131.30 (d,

³J_CF= 11.45 Hz, 1C_q), 129.19 (s, 1C_q), 128.55 (s, 1CH), 124.03 (s,

1CH), 121.62 (s, 1CH), 119.28 (s, 1CH), 118.50 (d, ⁴J_CF= 3.39 Hz,

1C_q), 116.77 (s, 1CH), 107.08 (d, ²J_CF= 21.19 Hz, 1CH), 100.10 (d,

²J_CF= 25.86 Hz, 1CH), 59.77 (s, 1CH₂), 56.21 (s, 1CH₃), 39.47 (s,

Compound 3. 2,5-Diﬂuoro-4-(4-ﬂuoro-2-methoxyphenyl)-

pyridine. A mixture of 2,5-diﬂuoro-4-iodopyridine (500 mg, 2.07

mmol; abcr), (4-ﬂuoro-2-methoxyphenyl)boronic acid (388 mg, 2.28

mmol), and Pd(PPh₃)₄(240 mg, 0.21 mmol) in DME (6.2 mL) and 2

M aq K₂CO₃(3.1 mL) was degassed using argon. The mixture was

stirred under argon for 16 h at 100 °C. After cooling, the mixture was

diluted with EtOAc and washed with brine. The organic phase was

ﬁltered using a Whatman ﬁlter and concentrated. The residue was

puriﬁed by column chromatography (hexane to hexane/EtOAc 3:2)

1CH₃). IR (diamond ATR, cm⁻¹): 3379 (m), 3026 (w), 3013 (w),

2920 (w), 1632 (s), 1609 (s), 1599 (s), 1535 (s), 1491 (s), 1437 (s),

1317 (m), 1283 (s), 1236 (m), 1188 (s), 1109 (s), 1030 (s), 984 (m),

951 (s), 893 (m), 835 (s), 762 (m). ESI-HRMS m/z: [M + H]⁺calcd

for C₂₀H₁₉F₂N₂O₃S, 405.1085; found, 405.1085.

1

to aﬀord the desired product (420 mg, 1.76 mmol, 85% yield). H

NMR (300 MHz, CDCl₃): δ 8.07 (t, J = 1.32 Hz, 1H), 7.33 (br d, J =

1.51 Hz, 1H), 6.95 (dd, J = 2.64, 4.52 Hz, 1H), 6.71−6.83 (m, 2H),

3.83 (s, 3H). UPLC (ESI+) m/z: [M + H]⁺240.

Compound 5. 2-Chloro-5-ﬂuoro-4-(4-ﬂuoro-2-methoxyphenyl)-

pyrimidine. A mixture of 2,4-dichloro-5-ﬂuoropyrimidine (200 mg,

1.20 mmol; Aldrich), (4-ﬂuoro-2-methoxyphenyl)boronic acid (224

mg, 1.31 mmol), and Pd(PPh₃)₄(138 mg, 0.12 mmol) in DME (3.6

mL) and 2 M aq K₂CO₃(1.8 mL) was degassed using argon. The

mixture was stirred under argon for 16 h at 90 °C. After cooling, the

mixture was diluted with EtOAc and washed with brine. The organic

phase was ﬁltered using a Whatman ﬁlter and concentrated. The

residue was puriﬁed by column chromatography (hexane/EtOAc 1:1)

5-Fluoro-4-(4-ﬂuoro-2-methoxyphenyl)-N-{3-[(methylsulfonyl)-

methyl]phenyl}pyridin-2-amine (3). To a solution of 2,5-diﬂuoro-4-

(4-ﬂuoro-2-methoxyphenyl)pyridine (150 mg, 0.63 mmol) and 3-

[(methylsulfonyl)methyl]aniline (140 mg, 0.75 mmol) in DMF (5

mL), NaH (60% suspension in mineral oil; 37 mg, 0.94 mmol) was

added. The mixture was stirred for 2 h at 70 °C. After cooling, the

mixture was diluted with EtOAc and washed with brine. The organic

phase was ﬁltered using a Whatman ﬁlter and concentrated. The

residue was puriﬁed by preparative HPLC (basic conditions) to aﬀord

1

to aﬀord the desired product (106 mg, 0.41 mmol, 35% yield). H

NMR (400 MHz, CDCl₃, 27 °C): δ 8.47 (m, 1H), 7.51 (m, 1H), 6.82

(m, 1H), 6.73 (m, 1H), 3.85 (s, 3H).

1

3 (41 mg, 0.10 mmol, 16% yield). H NMR (300 MHz, CDCl₃): δ

8.11 (d, J = 1.51 Hz, 1H), 7.39−7.48 (m, 2H), 7.30−7.37 (m, 1H),

7.01 (d, J = 7.16 Hz, 1H), 6.82 (d, J = 4.90 Hz, 1H), 6.70−6.79 (m,

2H), 6.51 (s, 1H), 4.22 (s, 2H), 3.83 (s, 3H), 2.78 (s, 3H). ¹³C NMR

5-Fluoro-4-(4-ﬂuoro-2-methoxyphenyl)-N-{3-[(methylsulfonyl)-

methyl]phenyl}pyrimidin-2-amine (5).

A mixture of 3-

[(methylsulfonyl)methyl]aniline (108 mg, 0.58 mmol), 2-chloro-5-

ﬂuoro-4-(4-ﬂuoro-2-methoxyphenyl)pyrimidine (150 mg, 0.58

mmol), Pd₂(dba)₃(96 mg, 0.11 mmol), 4,5-bis-

(diphenylphosphino)-9,9-dimethylxanthene (Xantphos) (88 mg,

0.15 mmol), and Cs₂CO₃(800 mg, 2.46 mmol) in 1,4-dioxane (1.0

mL) was degassed using argon. The mixture was stirred under argon

for 150 min at 100 °C. After cooling, the mixture was ﬁltered and the

ﬁlter was rinsed with DCM and EtOAc. The ﬁltrate was concentrated

in vacuo and the residue was puriﬁed by preparative HPLC (acidic

1

(100.67 MHz, DMSO-d₆): δ 163.64 (d, J_CF= 245.41 Hz, 1C_q),

3

4

157.92 (d, J_CF= 10.60 Hz, 1C_q), 152.41 (d, J_CF= 1.69 Hz, 1C_q),

151.36 (d, ¹J_CF= 244.14 Hz, 1C_q), 147.01 (s, 1CH), 145.95 (s, 1C_q),

141.82 (s, 1C_q), 135.00 (d, J_CF= 15.68 Hz, 1C_q), 133.60 (d, J_CF

26.28 Hz, 1CH), 131.64 (d, ³J_CF= 10.17 Hz, 1CH), 129.40 (s, 1C_q),

128.84 (s, 1CH), 123.10 (s, 1CH), 119.95 (s, 1CH), 118.52 (d, ⁴J_CF

2

=

3.39 Hz, 1C_q), 117.59 (s, 1CH), 115.35 (s, 1CH), 112.63 (s, 1CH),

107.12 (d, ²J_CF= 21.62 Hz, 1CH), 100.16 (d, ²J_CF= 26.28 Hz, 1CH),

59.67 (s, 1CH₂), 56.25 (s, 1CH₃), 39.51 (s, 1CH₃). IR (diamond

1

conditions) to aﬀord 5 (49 mg, 0.12 mmol, 21% yield). H NMR

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(400 MHz, DMSO-d₆, 27 °C): δ 9.83 (m, 1H), 8.51 (m, 1H), 7.69

(m, 2H), 7.50 (m, 1H), 7.25 (m, 1H), 7.08 (m, 1H), 6.91 (m, 2H),

4.37 (s, 2H), 3.80 (s, 3H), 2.86 (s, 3H). ¹³C NMR (100.65 MHz,

Whatman ﬁlter and concentrated. The residue was puriﬁed by column

chromatography (DCM to DCM/EtOH 95:5 and hexane to hexane/

EtOAc 1:1) to aﬀord the desired product (618 mg) as an equimolar

mixture with (Z)-3-amino-N-tert-butyl-3-(4-ﬂuoro-2-methoxyphen-

yl)-2-formylprop-2-enamide.

1

3

DMSO-d₆): δ 164.34 (d, J_CF= 247.11 Hz, 1C_q), 158.55 (d, J_CF

=

4

3

10.60 Hz, 1C_q), 156.53 (d, J_CF= 2.54 Hz, 1C_q), 151.52 (d, J_CF

14.41 Hz, 1C_q), 150.23 (d, ¹J_CF= 250.50 Hz, 1C_q), 145.72 (d, ²J_CF

2-Chloro-4-(4-ﬂuoro-2-methoxyphenyl)pyrimidine-5-carboni-

24.58 Hz, 1CH), 140.67 (s, 1C_q), 132.06 (d, ³J_CF= 10.60 Hz, 1CH),

129.31 (s, 1C_q), 128.67 (s, 1CH), 124.15 (s, 1CH), 120.91 (s, 1CH),

119.05 (m, 1C_q), 118.49 (s, 1CH), 107.30 (d, ²J_CF= 21.62 Hz, 1CH),

100.16 (d, ²J_CF= 25.86 Hz, 1CH), 59.76 (s, 1CH₂), 56.34 (s, 1CH₃),

40.46 (s, 1CH₃). ESI-HRMS m/z: [M + H]⁺calcd for

C₁₉H₁₈F₂N₃O₃S, 406.1037; found, 406.1041.

trile.

A mixture of N-tert-butyl-2-chloro-4-(4-ﬂuoro-2-

methoxyphenyl)pyrimidine-5-carboxamide (564 mg) and thionyl

chloride (10.3 mL) was stirred for 18 h at 80 °C. After cooling, the

volatiles were removed under reduced pressure. The residue was

puriﬁed by column chromatography (hexane to hexane/EtOAc 3:1)

1

to aﬀord the desired product (206 mg, 0.78 mmol). H NMR (300

MHz, CDCl₃): δ 8.85 (s, 1H), 7.60 (dd, J = 6.50, 8.57 Hz, 1H), 6.85

(br d, J = 2.07 Hz, 1H), 6.79 (dd, J = 2.17, 10.46 Hz, 1H), 3.93 (s,

3H). UPLC (ESI+) m/z: [M + H]⁺264.

Compound 6. 2,5-Dichloro-4-(4-ﬂuoro-2-methoxyphenyl)-

pyrimidine. A mixture of 2,4,5-trichloropyrimidine (367 mg, 2

mmol; Combi-Blocks, Inc.), (4-ﬂuoro-2-methoxyphenyl)boronic acid

(374 mg, 2.2 mmol), and [1,1′-bis(diphenylphosphino)ferrocene]-

dichloropalladium(II) [Pd(dppf)Cl₂] (1:1 complex with DCM; 163

mg, 0.2 mmol; Aldrich) in DME (6.1 mL) and 2 M aq K₂CO₃(3 mL)

was degassed using argon. The mixture was stirred under argon for 1

h at 90 °C. After cooling, the mixture was diluted with EtOAc and

washed with brine. The organic phase was dried (Na₂SO₄), ﬁltered,

and concentrated. The residue was puriﬁed by column chromatog-

raphy (hexane/EtOAc 1:1) to aﬀord the desired product (280 mg,

1.03 mmol, 52% yield). ¹H NMR (400 MHz, CDCl₃): δ 8.60 (s, 1H),

7.34 (dd, J = 6.57, 8.34 Hz, 1H), 6.80 (dt, J = 2.40, 8.27 Hz, 1H), 6.73

(dd, J = 2.27, 10.86 Hz, 1H), 3.82 (s, 3H). UPLC (ESI+) m/z: [M +

H]⁺273.

4-(4-Fluoro-2-methoxyphenyl)-2-({3-[(methylsulfonyl)methyl]-

phenyl}amino)pyrimidine-5-carbonitrile (7). A mixture of 2-chloro-

4-(4-ﬂuoro-2-methoxyphenyl)pyrimidine-5-carbonitrile (80 mg, 0.303

mmol), 3-[(methylsulfonyl)methyl]aniline (56 mg, 0.303 mmol),

(XPhos) palladium(II) phenethylamine chloride (XPhos Pd G1, CAS

1028206-56-5) (18.8 mg, 0.023 mmol), 2-dicyclohexylphosphino-

2′,4′,6′-triisopropyl-1,1′-biphenyl (XPhos) (10.8 mg, 0.023 mmol),

and potassium phosphate (322 mg, 1.52 mmol) in toluene (3.2 mL)

and N-methyl-2-pyrrolidone (NMP, 0.8 mL) was degassed using

argon. The mixture was stirred under argon for 3 h at 130 °C. After

cooling, the mixture was diluted with EtOAc and washed with brine.

The organic phase was ﬁltered using a Whatman ﬁlter and

concentrated. The residue was puriﬁed by preparative HPLC (acidic

conditions) to aﬀord 7 (7.6 mg, 0.02 mmol, 7% yield). ¹H NMR (300

MHz, CDCl₃): δ 8.65 (s, 1H), 7.81 (s, 1H), 7.63 (br d, J = 3.39 Hz,

2H), 7.50 (dd, J = 6.88, 8.19 Hz, 1H), 7.40 (t, J = 7.91 Hz, 1H), 7.15

(d, J = 7.72 Hz, 1H), 6.74−6.87 (m, 2H), 4.25 (s, 2H), 3.92 (s, 3H),

2.78 (s, 3H). IR (diamond ATR, cm⁻¹): 3325 (w), 3313 (w), 3296

(w), 3086 (w), 3020 (w), 2976 (w), 2931 (w), 2224 (m), 1606 (m),

1568 (s), 1441 (s), 1282 (m), 1155 (w), 1117 (w), 1030 (w), 955

(w), 800 (w). UPLC (ESI+) m/z: [M + H]⁺413.

Compound 8. 4-Chloro-N-{3-[(methylsulfonyl)methyl]phenyl}-5-

(triﬂuoromethyl)pyrimidin-2-amine. To a solution of 2,4-dichloro-5-

(triﬂuoromethyl)pyrimidine (527 mg, 2.43 mmol; Combi-Blocks,

Inc.) in t-BuOH (8.2 mL) and DCE (8.2 mL), 1 M zinc chloride in

diethyl ether (2.43 mL, 2.43 mmol) was added. The resulting mixture

was stirred for 1 h at rt. Then, 3-[(methylsulfonyl)methyl]aniline

(300 mg, 1.62 mmol) was added, followed by a solution of

triethylamine (0.339 mL, 2.43 mmol) in t-BuOH (0.55 mL) and

DCE (0.55 mL). After 5 h, the mixture was diluted with DCM and

washed with aq citric acid solution−brine, aq NaHCO₃solution, and

brine. The organic phase was dried over Na₂SO₄and concentrated to

aﬀord the desired product (662 mg). ¹H NMR (300 MHz, CDCl₃): δ

8.59 (s, 1H), 7.76 (s, 1H), 7.59 (br dd, J = 7.54, 8.85 Hz, 2H), 7.42 (t,

J = 7.91 Hz, 1H), 7.19 (d, J = 7.54 Hz, 1H), 4.28 (s, 2H), 2.84 (s,

3H). UPLC (ESI+) m/z: [M + H]⁺366.

5-Chloro-4-(4-ﬂuoro-2-methoxyphenyl)-N-{3-[(methylsulfonyl)-

methyl]phenyl}pyrimidin-2-amine (6). To a mixture of 2,5-dichloro-

4-(4-ﬂuoro-2-methoxyphenyl)pyrimidine (136 mg, 0.498 mmol) and

3-[(methylsulfonyl)methyl]aniline (92 mg, 0.49 mmol) in n-BuOH, 4

M HCl in 1,4-dioxane (0.125 mL, 0.498 mmol) was added. The

mixture was stirred for 65 h at 120 °C and an additional 72 h at 140

°C. Then, the mixture was evaporated to dryness and the residue was

taken up in EtOAc and washed with aq NaHCO₃solution, followed

by brine. The organic phase was dried over Na₂SO₄and concentrated.

The residue was puriﬁed by column chromatography (hexane/EtOAc

1:1) to aﬀord 6 (70 mg, 0.16 mmol, 33% yield). ¹H NMR (400 MHz,

CDCl₃): δ 8.42 (s, 1H), 7.79 (d, J = 1.76 Hz, 1H), 7.52−7.58 (m,

1H), 7.29−7.38 (m, 2H), 7.24 (s, 1H), 7.07 (d, J = 7.53 Hz, 1H),

6.79 (dt, J = 2.38, 8.22 Hz, 1H), 6.74 (dd, J = 2.38, 10.67 Hz, 1H),

4.23 (s, 2H), 3.84 (s, 3H), 2.74 (s, 3H). ¹³C NMR (100.67 MHz,

1

DMSO-d₆): δ 163.90 (d, J_CF= 245.84 Hz, 1C_q), 162.03 (s, 1C_q),

3

158.18 (s, 1C_q), 157.94 (d, J_CF= 10.60 Hz, 1C_q), 156.85 (s, 1C_q),

3

140.15 (s, 1C_q), 131.22 (d, J_CF= 10.60 Hz, 1CH), 129.38 (s, 1C_q),

4

128.72 (s, 1CH), 124.63 (s, 1CH), 121.86 (d, J_CF= 2.97 Hz, 1C_q),

121.49 (s, 1CH), 119.78 (s, 1C_q), 119.14 (s, 1CH), 106.99 (d, ²J_CF

=

22.04 Hz, 1CH), 100.04 (d, ²J_CF= 26.28 Hz, 1CH), 59.69 (s, 1CH₂),

56.17 (s, 1CH₃), 39.44 (s, 1CH₃). IR (diamond ATR, cm⁻¹): 3337

(s), 3074 (w), 3020 (w), 2924 (w), 1663 (w), 1607 (m), 1593 (m),

1545 (s), 1508 (m), 1445 (s), 1394 (s), 1312 (w), 1281 (s), 1198

(w), 1151 (m), 1024 (w), 953 (s), 880 (m), 849 (m), 822 (m), 789

(m). UPLC (ESI+) m/z: [M + H]⁺422. ESI-HRMS m/z: [M + H]⁺

calcd for C₁₉H₁₈ClFN₃O₃S, 422.0742; found, 422.0741.

4-(4-Fluoro-2-methoxyphenyl)-N-{3-[(methylsulfonyl)methyl]-

phenyl}-5-(triﬂuoromethyl)pyrimidin-2-amine (8). A mixture of 4-

chloro-N-{3-[(methylsulfonyl)methyl]phenyl}-5-(triﬂuoromethyl)-

pyrimidin-2-amine (100 mg, 0.27 mmol), (4-ﬂuoro-2-

methoxyphenyl)boronic acid (51 mg, 0.3 mmol), and bis-

(triphenylphosphine)palladium(II) chloride (8 mg, 0.01 mmol) in

DME (3.6 mL) and 2 M aq K₂CO₃(1.13 mL) was degassed using

argon. The mixture was stirred under argon for 96 h at 105 °C. After

cooling, the mixture was evaporated to dryness, and the residue was

puriﬁed by preparative HPLC (acidic conditions) to aﬀord 8 (14 mg,

Compound 7. N-tert-Butyl-2,4-dichloropyrimidine-5-carboxa-

mide. To 2,4-dichloropyrimidine-5-carbonyl chloride (5 g, 24

mmol; Combi-Blocks, Inc.) in THF (30 mL), a solution of tert-

butylamine (1.82 g, 24.9 mmol) and triethylamine (3.45 mL) in THF

(30 mL) was added slowly at −5 °C. The mixture was stirred for 4.5 h

at this temperature. After removal of the precipitated salts by

ﬁltration, the ﬁltrate was evaporated to dryness. The resulting material

was used without further puriﬁcation.

1

0.03 mmol). H NMR (400 MHz, CDCl₃): δ 8.72 (s, 1H), 7.83 (s,

1H), 7.57 (dd, J = 1.26, 8.08 Hz, 1H), 7.47 (s, 1H), 7.39 (t, J = 7.83

Hz, 1H), 7.21 (dd, J = 6.32, 8.34 Hz, 1H), 7.13 (d, J = 7.58 Hz, 1H),

6.76 (dt, J = 2.27, 8.21 Hz, 1H), 6.71 (dd, J = 2.27, 10.61 Hz, 1H),

4.24 (s, 2H), 3.77 (s, 3H), 2.74 (s, 3H). ¹³C NMR (151 MHz,

DMSO-d₆): δ 163.8, 163.6, 160.7, 157.8, 156.8, 139.3, 130.5, 129.5,

128.8, 125.7, 124.1, 122.4, 122.4, 120.1, 113.8, 106.5, 99.7, 59.6, 56.0,

39.4. ESI-HRMS m/z: [M + H]⁺calcd for C₂₀H₁₈F₄N₃O₃S, 456.1005;

found, 456.1002.

N-tert-Butyl-2-chloro-4-(4-ﬂuoro-2-methoxyphenyl)pyrimidine-

5-carboxamide. A mixture of N-tert-butyl-2,4-dichloropyrimidine-5-

carboxamide (5.73 g, 23.1 mmol), (4-ﬂuoro-2-methoxyphenyl)-

boronic acid (4.32 g, 25.4 mmol), and [Pd(dppf)Cl₂] (1:1 complex

with DCM; 1.88 g, 2.3 mmol) in DME (70 mL) and 2 M aq K₂CO₃

(34.6 mL) was degassed using argon. The mixture was stirred under

argon for 16 h at 90 °C. After cooling, the mixture was diluted with

EtOAc and washed with brine. The organic phase was ﬁltered using a

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Compound 9 and BAY-332. 1-Fluoro-3-[(methylsulfanyl)-

methyl]-5-nitrobenzene. Sodium methanethiolate (5.18 g, 73.8

mmol, 1.4 equiv) was added portionwise at 0 °C to a solution of 1-

(chloromethyl)-3-ﬂuoro-5-nitrobenzene (10.0 g, 52.8 mmol) in EtOH

(110 mL). After stirring for a further 20 min at that temperature, the

mixture was warmed to rt and stirred for a further 18 h. The

suspension was then poured into dilute aq NaCl solution, and the

mixture was extracted with EtOAc. The organic extract was ﬁltered

through a Whatman ﬁlter, and the ﬁltrate was concentrated under

reduced pressure. The residue was taken up in DCM and again

concentrated under reduced pressure. The oily residue was taken up

in EtOAc and washed with dilute aq NaCl solution, ﬁltered through a

Whatman ﬁlter, and concentrated under reduced pressure to aﬀord

the title compound (9.86 g, 49.0 mmol, 95% purity, 93% yield) as a

sulfoximine−CH₂, H_B, AB system), 3.87 (s, 1H, NH), 2.79 (s, 3H,

sulfoximine−CH₃). UPLC (ESI+) m/z: [M + H]⁺233.

(rac)-Ethyl [(3-Fluoro-5-nitrobenzyl)(methyl)oxido-λ⁶-

s u l f a n y l i d e n e ] c a r b a m a t e . ( r a c ) - 1 - F l u o r o - 3 - [ ( S -

methylsulfonimidoyl)methyl]-5-nitrobenzene (9.36 g) from the

previous step was dissolved in pyridine (380 mL) and cooled to 0

°C. Ethyl chloroformate (5.01 mL, 52.4 mmol, ca. 1.3 equiv) was

added dropwise at 0 °C to this mixture, after which the mixture was

warmed to rt. After stirring for 16 h at that temperature, the mixture

was concentrated under reduced pressure. The residue was taken up

in EtOAc and washed with brine and then ﬁltered through a

Whatman ﬁlter. The ﬁltrate was concentrated under reduced pressure

and the crude product was puriﬁed by ﬂash chromatography (silica

gel, EtOAc/hexane) to aﬀord the title compound (7.09 g, 23.3 mmol,

1

71% yield over two steps). H NMR (300 MHz, CDCl₃, 24 °C): δ

brown oil. H NMR (400 MHz, CDCl₃, 27 °C): δ 8.00 (s, 1H),

8.11 (s, 1H), 8.01 (br d, J = 7.91 Hz, 1H), 7.57 (br d, J = 7.72 Hz,

1H), 4.90 (d, J = 13.94 Hz, 1H, sulfoximine−CH₂, H_A, AB system),

4.76 (d, J = 13.94 Hz, 1H, sulfoximine−CH₂, H_B, AB system), 4.08−

4.22 (q, J = 7.16 Hz, 2H, OCH₂CH₃), 3.12 (s, 3H, sulfoximine−

CH₃), 1.22−1.35 (t, J = 7.16 Hz, 3H, OCH₂CH₃). UPLC (ESI+) m/

z: [M + H]⁺305.

7.80−7.85 (m, 1H), 7.42 (dt, J = 8.59, 1.89 Hz, 1H), 3.74 (s, 2H,

SCH₂), 2.03 (s, 3H, SCH₃). ¹³C NMR (151 MHz, DMSO-d₆): δ

163.8, 163.6, 160.7, 157.8, 156.8, 139.3, 130.5, 129.5, 128.8, 125.7,

124.1, 122.4, 122.4, 120.1, 113.8, 106.5, 99.7, 59.6, 56.0, 39.4.

(rac)-1-Fluoro-3-[(methylsulﬁnyl)methyl]-5-nitrobenzene. To a

solution of 1-ﬂuoro-3-[(methylsulfanyl)methyl]-5-nitrobenzene

(10.3 g, 51.3 mmol) in MeCN (120 mL) was added FeCl₃at rt

(241 mg, 1.49 mmol, 3 mol %), and the mixture was stirred for 10

min at that temperature. Then, periodic acid (12.5 g, 54.8 mmol, 1.07

equiv) was added in one portion, and vigorous stirring was continued

for 90 min during which the temperature of the mixture was

maintained below 30 °C. The resulting suspension was poured into a

solution of sodium thiosulfate pentahydrate (71.2 g) in ice−water

(652 mL), and the mixture was stirred for 50 min. Then, NaCl was

added until saturation, and the mixture was extracted THF (2×). The

combined organic extracts were washed with brine, dried over

Na₂SO₄, and concentrated under reduced pressure. The residue was

stripped oﬀ several times with toluene and DCM and was then

puriﬁed by ﬂash chromatography (silica gel, EtOAc/hexane gradient)

to aﬀord the title compound (8.00 g, 36.8 mmol, 92% purity, 72%

(rac)-Ethyl [(3-Amino-5-ﬂuorobenzyl)(methyl)oxido-λ⁶-

sulfanylidene]carbamate. TiCl₃solution (15% in 10% HCl; 158

mL, 186 mmol, 8.0 equiv) was added dropwise at 0 °C to a solution

of (rac)-ethyl [(3-ﬂuoro-5-nitrobenzyl)(methyl)oxido-λ⁶-

sulfanylidene]carbamate (7.09 g, 23.3 mmol) in THF (300 mL).

Then, the mixture was warmed to rt and stirred for 16 h at that

temperature. Then, ice was added to the mixture, which was stirred

vigorously, after which K₂CO₃was added until the evolution of gas

stopped. After that, the mixture was basiﬁed with 2 N NaOH. The

resulting dark mixture was saturated with NaCl and extracted with

EtOAc (3 × 500 mL). The combined organic extracts were dried over

Na₂SO₄and concentrated under reduced pressure to aﬀord the title

compound (6.37 g, quant) as a yellow oil. ¹H NMR (300 MHz,

CDCl₃, 27 °C): δ 6.38−6.55 (m, 3H), 4.52−4.64 (m, 2H,

sulfoximine−CH₂), 4.08−4.22 (q, J = 7.16 Hz, 2H, OCH₂CH₃),

3.91 (br s, 2H, NH₂), 3.00 (s, 3H, sulfoximine−CH₃), 1.31 (t, J =

7.16 Hz, 3H, OCH₂CH₃). UPLC (ESI+) m/z: [M + H]⁺275.

(rac)-Ethyl [(3-Fluoro-5-{[5-ﬂuoro-4-(4-ﬂuoro-2-

methoxyphenyl)pyrimidin-2-yl]amino}benzyl)(methyl)oxido-λ⁶-

sulfanylidene]carbamate. A mixture of (rac)-ethyl [(3-amino-5-

ﬂuorobenzyl)(methyl)oxido-λ⁶-sulfanylidene]carbamate (436 mg,

1.59 mmol, 1.0 equiv), 2-chloro-5-ﬂuoro-4-(4-ﬂuoro-2-

methoxyphenyl)pyrimidine (530 mg, 2.07 mmol, 1.3 equiv), XPhos

Pd G1 (98.6 mg, 0.12 mmol, 7.5 mol %), XPhos (56.8 mg, 0.12

mmol, 7.5 mol %), and ﬁnely ground potassium phosphate (1.69 g,

7.95 mmol, 5.0 equiv) in toluene (8.5 mL) and NMP (1.3 mL) was

degassed and repressured with argon at rt. The mixture was heated in

a sealed vial to 130 °C in a microwave reactor for 3 h. After cooling to

rt, brine was added, and the mixture was extracted with EtOAc (2 ×

100 mL). The combined organic extracts were ﬁltered through a

Whatman ﬁlter and the ﬁltrate was concentrated under reduced

pressure. The crude product was puriﬁed by silica gel ﬂash

chromatography (ﬁrst chromatography: DCM to DCM/EtOH 93:7

gradient; second chromatography: DCM to DCM/EtOH 95:5

gradient; third chromatography: hexane to hexane/EtOAc 1:1

gradient) to aﬀord the title compound (271 mg, 0.55 mmol, 35%

yield) as a beige solid. ¹H NMR (400 MHz, CDCl₃, 27 °C): δ 8.32−

8.35 (m, 1H), 7.69 (br dd, J = 11.24, 1.89 Hz, 1H), 7.47 (t, J = 7.11

Hz, 1H), 7.38 (s, 1H), 7.28−7.34 (m, 1H), 6.72−6.86 (m, 3H), 4.68

(s, 2H, sulfoximine−CH₂), 4.17 (q, J = 7.07 Hz, 2H, OCH₂CH₃),

3.87 (s, 3H, OCH₃), 3.00 (s, 3H, sulfoximine−CH₃), 1.31 (t, J = 7.07

Hz, 3H, OCH₂CH₃). UPLC (ESI+) m/z: [M + H]⁺495.

1

yield). H NMR (300 MHz, DMSO-d₆, 27 °C): δ 8.04−8.09 (m,

2H), 7.61−7.65 (m, 1H), 4.32 (d, J = 12.81 Hz, 1H, sulfoxide−CH₂,

H_A, AB system), 4.08 (d, J = 12.81 Hz, 1H, sulfoxide−CH₂, H_B, AB

system), 2.46 (s, 3H, SCH₃). UPLC (ESI+) m/z: [M + H]⁺218.

(rac)-2,2,2-Triﬂuoro-N-[(3-ﬂuoro-5-nitrobenzyl)(methyl)oxido-λ⁶-

sulfanylidene]acetamide. To a suspension of (rac)-1-ﬂuoro-3-

[(methylsulﬁnyl)methyl]-5-nitrobenzene (8.00 g, 36.8 mmol), 2,2,2-

triﬂuoroacetamide (8.33 g, 73.7 mmol, 2.0 equiv), magnesium oxide

(5.94 g, 147 mmol, 4.0 equiv), and dirhodium tetraacetate (0.76 g, 3.6

mmol, 10 mol %) in DCM (1.0 L) was added iodosobenzene

diacetate (17.8 g, 55.2 mmol, 1.5 equiv) at rt. The mixture was stirred

at rt for 3 days, after which the mixture was ﬁltered and the ﬁltrate was

concentrated under reduced pressure. The resulting dark oil was

puriﬁed by column chromatography (silica gel, EtOAc/hexane

gradient) to aﬀord the title compound as a light yellow oil, which

1

crystallized upon standing (10.8 g, 33.0 mmol, 90% yield). H NMR

(300 MHz, CDCl₃, 27 °C): δ 8.04−8.16 (m, 2H), 7.55 (dt, J = 7.91,

1.88 Hz, 1H), 4.92 (d, J = 13.94 Hz, 1H, sulfoximine−CH₂, H_A, AB

system), 4.75 (d, J = 13.94 Hz, 1H, sulfoximine−CH₂, H_B, AB

system), 3.32 (s, 3H, sulfoximine−CH₃). UPLC (ESI+) m/z: [M +

H]⁺329.

(rac)-1-Fluoro-3-[(S-methylsulfonimidoyl)methyl]-5-nitroben-

zene. K₂CO₃(22.7 g, 164 mmol, 5.0 equiv) was added at rt to a

solution of (rac)-2,2,2-triﬂuoro-N-[(3-ﬂuoro-5-nitrobenzyl)(methyl)-

oxido-λ⁶-sulfanylidene]acetamide (10.8 g, 33.0 mmol) in MeOH (700

mL). After stirring for 1 h at that temperature, the mixture was

concentrated at 30 °C to 100 mL. After the addition of brine, the

mixture was extracted EtOAc. The organic extract was ﬁltered

through a Whatman ﬁlter, and the ﬁltrate was concentrated under

reduced pressure to aﬀord the crude product (9.36 g), which was

5-Fluoro-4-(4-ﬂuoro-2-methoxyphenyl)-N-{3-ﬂuoro-5-[(S-

methylsulfonimidoyl)methyl]phenyl}pyrimidin-2-amine [Enan-

tiomer 1 (9) and Enantiomer 2 (BAY-332)]. To a solution of

(rac)-ethyl [(3-ﬂuoro-5-{[5-ﬂuoro-4-(4-ﬂuoro-2-methoxyphenyl)-

pyrimidin-2-yl]amino}benzyl)(methyl)oxido-λ⁶-sulfanylidene]-

carbamate (269 mg, 0.54 mmol) in EtOH (5.0 mL) was added at rt a

solution of 1.5 M ethanolic sodium ethoxide (1.81 mL, 2.72 mmol,

1

directly used in the next step without further puriﬁcation. H NMR

(400 MHz, DMSO-d₆, 27 °C): δ 8.18 (s, 1H), 8.07 (dt, J = 8.84, 2.27

Hz, 1H), 7.76 (d, J = 9.12 Hz, 1H), 4.60 (d, J = 13.14 Hz, 1H,

sulfoximine−CH₂, H_A, AB system), 4.50 (d, J = 13.14 Hz, 1H,

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5.0 equiv) freshly prepared by reacting sodium (350 mg) with EtOH

(10 mL). The reaction mixture was heated to 60 °C for 4.5 h, after

which the mixture was cooled to rt and diluted with brine. The

mixture was extracted with EtOAc, and the combined organic extracts

were ﬁltered through a Whatman ﬁlter. The ﬁltrate was concentrated

under reduced pressure and the crude product was puriﬁed by chiral

preparative HPLC [Dionex P 580 pump, Gilson 215 liquid handler,

Knauer K-2501 UV detector; column: CHIRALPAK IC 5 μm, 250 ×

20 mm; eluent: hexane/EtOH 80:20 (v/v); ﬂow: 50 mL/min; rt;

solution: 243 mg in 4 mL DCM/MeOH; injection: 8 × 0.5 mL; and

detection: UV 280 nm].

5-Fluoro-4-(4-ﬂuoro-2-methoxyphenyl)-N-{3-methoxy-5-

[(methylsulfanyl)methyl]phenyl}pyrimidin-2-amine. A mixture of 3-

methoxy-5-[(methylsulfanyl)methyl]aniline (436 mg, 1.59 mmol, 1.0

equiv), 2-chloro-5-ﬂuoro-4-(4-ﬂuoro-2-methoxyphenyl)pyrimidine

(840 mg, 3.24 mmol, 1.0 equiv), XPhos Pd G1 (203 mg, 0.25

mmol, 7.5 mol %), XPhos (117 mg, 0.25 mmol, 7.5 mol %), and ﬁnely

ground potassium phosphate (3.48 g, 16.4 mmol, 5.0 equiv) in

toluene (19 mL) and NMP (4 mL) was degassed and repressured

with argon at rt. The mixture was heated in a sealed vial to 130 °C in a

microwave reactor for 3 h. After cooling to rt, brine was added, and

the mixture was extracted with EtOAc. The combined organic extracts

were ﬁltered through a Whatman ﬁlter and the ﬁltrate was

concentrated under reduced pressure. The crude product was puriﬁed

by ﬂash chromatography (silica gel, hexane to hexane/EtOAc 7:3

gradient), recrystallized from hexane/EtOAc, and again puriﬁed by

ﬂash chromatography (silica gel, hexane to hexane/EtOAc 8:2

gradient). The product-containing fractions were concentrated and

the precipitate was collected by ﬁltration to aﬀord the title compound

Enantiomer 1 (9): t_R= 9.15 min. Yield: 85 mg, 0.20 mmol (37%).

UPLC (ESI+) m/z: [M + H]⁺423. [α]_D+16.6

0.21 (c 1.00,

DMSO).

Enantiomer 2 (BAY-322): t_R= 10.36 min. Yield: 91 mg, 0.22 mmol

(40%). ¹H NMR (400 MHz, CDCl₃, 27 °C): δ 8.35 (d, J = 1.76 Hz,

1H, aniline−NH), 7.76 (d, J = 11.47 Hz, 1H), 7.51 (t, J = 7.26 Hz,

1H), 7.30−7.37 (m, 2H), 6.77−6.87 (m, 3H), 4.35 (d, J = 13.05 Hz,

1H, sulfoximine−CH₂, H_A, AB system), 4.23 (d, J = 13.05 Hz, 1H,

sulfoximine−CH₂, H_B, AB system), 3.90 (s, 3H, OCH₃), 2.97 (s, 3H,

sulfoximine−CH₃), 2.73 (br s, 1H, sulfoximine−NH). ¹³C NMR

1

(655 mg, 1.62 mmol, 50% yield) as a colorless solid. H NMR (400

MHz, CDCl₃, 27 °C): δ 8.30 (m, 1H), 7.51 (m, 1H), 7.36 (m, 1H),

7.15 (br, 1H), 7.03 (m, 1H), 6.80 (m, 1H), 6.74 (m, 1H), 6.55 (m,

1H), 3.86 (s, 3H, OCH₃), 3.80 (s, 3H, OCH₃), 3.63 (s, 2H, SCH₂),

2.02 (s, 3H, SCH₃). UPLC (ESI+) m/z: [M + H]⁺404.

1

(100.56 MHz, DMSO-d₆): δ 164.25 (d, J_CF= 246.90 Hz, 1C_q),

162.13 (d, ¹J_CF= 238.47 Hz, 1C_q), 158.73 (d, ³J_CF= 10.73 Hz, 1C_q),

4

2

156.30 (d, J_CF= 2.68 Hz, 1C_q), 151.70 (d, J_CF= 16.10 Hz, 1C_q),

(rac)-[(3-{[5-Fluoro-4-(4-ﬂuoro-2-methoxyphenyl)pyrimidin-2-

yl]amino}-5-methoxybenzyl)(methyl)-λ⁴-sulfanylidene]cyanamide.

To a mixture of 5-ﬂuoro-4-(4-ﬂuoro-2-methoxyphenyl)-N-{3-me-

thoxy-5-[(methylsulfanyl)methyl]phenyl}pyrimidin-2-amine (638

mg, 1.58 mmol) and cyanamide (133 mg, 3.16 mmol, 2.0 equiv) in

DCM (20 mL) was added at 0 °C iodosobenzene diacetate (560 mg,

1.74 mmol, 1.1 equiv). After stirring for 3 h at that temperature, the

mixture was concentrated under reduced pressure and the residue was

puriﬁed by ﬂash chromatography (silica gel, DCM to DCM/EtOH

9:1 gradient) to aﬀord the title compound (410 mg, 0.92 mmol, 58%

150.53 (d, ¹J_CF= 251.50 Hz, 1C_q), 145.69 (d, ²J_CF= 24.92 Hz, 1CH),

3

142.29 (d, J_CF= 12.27 Hz, 1C_q), 133.00 (d, J_CF= 9.97 Hz, 1C_q),

132.15 (d, ³J_CF= 10.35 Hz, 1CH), 118.98−119.10 (m, 1C_q), 116.73−

116.93 (m, 1CH), 110.24 (d, ²J_CF= 22.62 Hz, 1CH), 107.45 (d, ²J_CF

= 21.85 Hz, 1CH), 104.73 (d, ²J_CF= 26.45 Hz, 1CH), 100.34 (d, ²J_CF

= 26.07 Hz, 1CH), 62.39 (s, 1CH₂), 56.46 (s, 1CH₃), 41.27 (s,

1CH₃). IR (KBr, cm⁻¹): 3428 (br s), 3286 (br s), 3102 (w), 2969

(w), 2923 (w), 1612 (s), 1544 (m), 1434 (s), 1316 (m), 1285 (m),

1213 (m), 1154 (m), 1030 (m), 956 (m), 837 (m), 786 (m). UPLC

(ESI+) m/z: [M + H]⁺423. ESI-HRMS m/z: [M + H]⁺calcd for

C₁₉H₁₈F₃N₄O₂S, 423.1103; found, 423.1099. [α]_D−18.8 0.16 (c

1.00, DMSO).

1

yield) as a brownish solid. H NMR (400 MHz, CDCl₃, 27 °C): δ

8.32 (m, 1H), 7.47 (m, 1H), 7.33 (m, 1H), 7.25 (m, 2H), 6.82 (m,

1H), 6.75 (m, 1H), 6.52 (m, 1H), 4.37 (d, J = 12.6 Hz, 1H), 4.13 (d,

J = 12.6 Hz, 1H), 3.86 (s, 3H), 3.81 (s, 3H), 2.71 (s, 3H). UPLC (ESI

+) m/z: [M + H]⁺444.

Compound 10. 1-Methoxy-3-[(Z)-{3-methoxy-5-

[(methylsulfanyl)methyl]phenyl}-NNO-azoxy]-5-[(methylsulfanyl)-

methyl]benzene. Sodium methanethiolate (2.58 g, 36.8 mmol) was

added under stirring in three portions to a solution of 1-

(chloromethyl)-3-methoxy-5-nitrobenzene (5.30 g, 26.3 mmol; FCH

Group) in EtOH (60 mL) at 0 °C. The cold bath was removed, and

the mixture was stirred at rt overnight. Further sodium methanethio-

late (0.92 g, 13.1 mmol) was added, and the mixture was stirred

overnight. Further sodium methanethiolate (1.66 g, 23.6 mmol) was

added, and the mixture was stirred overnight. The mixture was diluted

with saturated aq NaCl solution and extracted with EtOAc (2×). The

combined organic phases were dried (Na₂SO₄), ﬁltered, and

concentrated. The residue was puriﬁed by chromatography (hexane

to hexane/EtOAc 7:3) to aﬀord the product (2.9 g, 7.66 mmol, 29%

yield). ¹H NMR (400 MHz, CDCl₃, 27 °C): δ 7.85 (m, 1H), 7.73 (m,

2H), 7.68 (m, 1H), 7.08 (m, 1H), 6.96 (m, 1H), 3.91 (s, 3H, OCH₃),

3.87 (s, 3H, OCH₃), 3.73 (s, 2H, SCH₂), 3.70 (s, 2H, SCH₂), 2.04 (s,

3H, SCH₃), 2.03 (s, 3H, SCH₃). UPLC (ESI+) m/z: [M + H]⁺379.

3-Methoxy-5-[(methylsulfanyl)methyl]aniline. To a solution of 1-

methoxy-3-[(Z)-{3-methoxy-5-[(methylsulfanyl)methyl]phenyl}-

NNO-azoxy]-5-[(methylsulfanyl)methyl]benzene (2.80 g, 7.40

mmol) in 1,4-dioxane (60 mL) was added iron (5.98 g, 107 mmol,

14 equiv) at rt. After heating to reﬂux, concd HCl (26 mL) was added

dropwise over 3 h to the mixture. Then, the mixture was cooled to rt

and stirring was continued for 14 h. After that, the reaction mixture

was poured onto ice and the resulting mixture was basiﬁed to pH 9 by

the addition of 2 N aq NaOH. The mixture was extracted with EtOAc

and the organic extract was ﬁltered through a Whatman ﬁlter. After

concentration of the ﬁltrate under reduced pressure, the crude

product was puriﬁed by column chromatography (silica gel, hexane/

EtOAc gradient) to aﬀord the title compound (1.54 g, quant) as a

brown oil. ¹H NMR (400 MHz, CDCl₃, 27 °C): δ 6.28 (m, 2H), 6.12

(m, 1H), 3.76 (s, 3H, OCH₃), 3.66 (br, 2H, NH₂), 3.55 (s, 2H,

SCH₂), 2.02 (s, 3H, SCH₃). UPLC (ESI+) m/z: [M + H]⁺184.

(rac)-[(3-{[5-Fluoro-4-(4-ﬂuoro-2-methoxyphenyl)pyrimidin-2-

yl]amino}-5-methoxybenzyl)(methyl)oxido-λ⁶-sulfanylidene]-

cyanamide. To a mixture of (rac)-[(3-{[5-ﬂuoro-4-(4-ﬂuoro-2-

methoxyphenyl)pyrimidin-2-yl]amino}-5-methoxybenzyl)(methyl)-

λ⁴-sulfanylidene]cyanamide (394 mg, 0.89 mmol) in acetone (10 mL)

was added at rt KMnO₄(281 mg, 1.78 mmol, 2.0 equiv), and the

mixture was heated to 50 °C for 1 h. After cooling to rt, the mixture

was concentrated under reduced pressure and puriﬁed by ﬂash

chromatography (silica gel, DCM to DCM/EtOH 95:5 gradient) to

aﬀord the title compound (307 mg, 0.67 mmol, 75% yield) as a light

brown solid. ¹H NMR (400 MHz, DMSO-d₆, 27 °C): δ 9.92 (m, 1H),

8.53 (m, 1H), 7.53 (m, 2H), 7.34 (m, 1H), 7.09 (m, 1H), 6.92 (m,

1H), 6.62 (m, 1H), 4.87 (m, 2H), 3.80 (s, 3H), 3.70 (s, 3H), 3.31 (s,

3H). UPLC (ESI+) m/z: [M + H]⁺460.

(rac)-5-Fluoro-4-(4-ﬂuoro-2-methoxyphenyl)-N-{3-methoxy-5-

[(S-methylsulfonimidoyl)methyl]phenyl}pyrimidin-2-amine (10).

To a solution of (rac)-[(3-{[5-ﬂuoro-4-(4-ﬂuoro-2-methoxyphenyl)-

pyrimidin-2-yl]amino}-5-methoxybenzyl)(methyl)oxido-λ⁶-

sulfanylidene]cyanamide (302 mg, 0.66 mmol) in DCM (30 mL) was

added at 0 °C triﬂuoroacetic anhydride (TFAA, 0.28 mL, 1.97 mmol,

3.0 equiv). The mixture was warmed to rt and stirred for 2 h at that

temperature. Then, the mixture was concentrated under reduced

pressure (at 40 °C), the residue was taken up in MeOH (5 mL), and

K₂CO₃(454 mg, 3.29 mmol, 5.0 equiv) was added at rt. After stirring

for 2 h at rt and 14 h at 4 °C, the mixture was diluted with EtOAc and

THF and washed with brine. The organic phase was ﬁltered through a

Whatman ﬁlter, the ﬁltrate was concentrated under reduced pressure,

and the crude product was puriﬁed by preparative HPLC (basic

1

conditions) to aﬀord 10 (27 mg, 0.06 mmol, 9% yield). H NMR

(400 MHz, CDCl₃, 27 °C): δ 8.30 (d, J = 2.02 Hz, 1H), 7.49 (t, J =

7.18 Hz, 1H), 7.43−7.46 (m, 1H), 7.25−7.29 (m, 2H), 7.19 (s, 1H),

6.72−6.83 (m, 2H), 6.59−6.63 (m, 1H), 4.32 (d, J = 13.14 Hz, 1H,

M

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sulfoximine−CH₂, H_A, AB system), 4.20 (d, J = 12.88 Hz, 1H,

sulfoximine−CH₂, H_B, AB system), 3.86 (s, 3H, OCH₃), 3.81 (s, 3H,

OCH₃), 2.93 (s, 3H, SCH₃). ¹³C NMR (100.67 MHz, DMSO-d₆): δ

ground potassium phosphate (3.37 g, 15.9 mmol, 5.0 equiv) in

toluene (21 mL) and NMP (4 mL) was degassed and repressured

with argon at rt. The mixture was heated in a sealed vial to 130 °C in a

microwave reactor for 3 h. After cooling to rt, brine was added, and

the mixture was extracted with EtOAc. The combined organic extracts

were ﬁltered through a Whatman ﬁlter and the ﬁltrate was

concentrated under reduced pressure. The crude product was puriﬁed

by ﬂash chromatography (silica gel, hexane to hexane/EtOAc

gradient) to aﬀord the title compound (724 mg, 1.77 mmol, 56%

164.36 (d, ¹J_CF= 246.68 Hz, 1C_q), 159.34 (s, 1C_q), 158.54 (d, ³J_CF

=

4

2

10.60 Hz, 1C_q), 156.49 (d, J_CF= 2.54 Hz, 1C_q), 151.35 (d, J_CF

16.95 Hz, 1C_q), 150.19 (d, ¹J_CF= 251.35 Hz, 1C_q), 145.83 (d, ²J_CF

24.58 Hz, 1CH), 141.52 (s, 1C_q), 132.03 (d, ³J_CF= 11.02 Hz, 1CH),

131.79 (s, 1C_q), 119.1 (m, 1C_q), 113.56 (s, 1CH), 109.59 (s, 1CH),

107.30 (d, ²J_CF= 22.04 Hz, 1CH), 104.10 (s, 1CH), 100.17 (d, ²J_CF

=

1

26.28 Hz, 1CH), 62.82 (s, 1CH₂), 56.36 (s, 1CH₃), 55.00 (s, 1CH₃),

41.00 (s, 1CH₃). IR (diamond ATR, cm⁻¹): 3304 (w), 3256 (w),

3103 (w), 2964 (w), 2943 (w), 1601 (m), 1545 (m), 1454 (m), 1429

(s), 1310 (m), 1150 (m), 1026 (s), 957 (m), 851 (m), 829 (m). ESI-

HRMS m/z: [M + H]⁺calcd for C₂₀H₂₁F₂N₄O₃S, 435.1303; found,

435.1300.

yield) as a yellow solid. H NMR (400 MHz, CDCl₃, 27 °C): δ 8.30

(d, J = 2.02 Hz, 1H), 7.71 (ddd, J = 12.63, 6.82, 2.78 Hz, 1H), 7.48

(dd, J = 8.46, 6.69 Hz, 1H), 7.07−7.22 (m, 2H), 6.78−6.84 (m, 1H),

6.76 (d, J = 10.85 Hz, 1H), 3.87 (s, 3H, OCH₃), 3.70 (s, 2H, SCH₂),

2.06 (s, 3H, SCH₃).

(rac)-[(2,3-Diﬂuoro-5-{[5-ﬂuoro-4-(4-ﬂuoro-2-methoxyphenyl)-

pyrimidin-2-yl]amino}benzyl)(methyl)-λ⁴-sulfanylidene]cyanamide.

To a mixture of N-{3,4-diﬂuoro-5-[(methylsulfanyl)methyl]phenyl}-

5-ﬂuoro-4-(4-ﬂuoro-2-methoxyphenyl)pyrimidin-2-amine (715 mg,

1.75 mmol) and cyanamide (147 mg, 3.49 mmol, 2.0 equiv) in

DCM (10 mL) was added at 0 °C iodosobenzene diacetate (619 mg,

1.92 mmol, 1.1 equiv). After stirring for 4 h at that temperature, the

mixture was concentrated under reduced pressure and the residue was

puriﬁed by ﬂash chromatography (silica gel, DCM to DCM/EtOH

8:2 gradient) to aﬀord the title compound (655 mg, 1.46 mmol, 83%

yield) as a pinkish solid. ¹H NMR (300 MHz, CDCl₃): δ 8.33 (d, J =

1.88 Hz, 1H), 7.89 (ddd, J = 12.53, 7.06, 2.64 Hz, 1H), 7.75 (s, 1H),

7.47 (dd, J = 8.29, 6.78 Hz, 1H), 7.28−7.37 (m, 1H), 6.73−6.87 (m,

2H), 4.33 (m, 2H, SCH₂), 3.87 (s, 3H, OCH₃), 2.82 (s, 3H, SCH₃).

UPLC (ESI+) m/z: [M + H]⁺450.

Compounds 11 and 12. (2,3-Diﬂuoro-5-nitrophenyl)methanol.

To a solution of 2,3-diﬂuoro-5-nitrobenzoic acid (9.00 g, 44.3 mmol)

in THF (85 mL) was added dropwise at 0 °C 1 M borane in THF

(177 mL, 177 mmol, 4.0 equiv). After the addition, the mixture was

warmed to rt and stirred for 14 h at that temperature. Then, the

mixture was cooled to 0 °C and MeOH was added carefully until

evolution of gas ceased. The mixture was then diluted with EtOAc,

washed with 1 N aq NaOH and brine, and then dried over Na₂SO₄.

After removal of the solvent under reduced pressure, the crude

product was puriﬁed by ﬂash chromatography (silica gel, hexane/

EtOAc gradient) to aﬀord the title compound as a yellowish oil which

1

crystallized upon standing (8.20 g, 43.4 mmol, 98% yield). H NMR

(400 MHz, CDCl₃, 27 °C): δ 8.26 (m, 1H), 8.03 (m, 1H), 4.89 (br,

2H), 2.13 (br, 1H). UPLC (ESI−) m/z: [M − H]⁻188.

(rac)-[(2,3-Diﬂuoro-5-{[5-ﬂuoro-4-(4-ﬂuoro-2-methoxyphenyl)-

pyrimidin-2-yl]amino}benzyl)(methyl)oxido-λ⁶-sulfanylidene]-

cyanamide (12). To a mixture of (rac)-[(2,3-diﬂuoro-5-{[5-ﬂuoro-4-

(4-ﬂuoro-2-methoxyphenyl)pyrimidin-2-yl]amino}benzyl)(methyl)-

λ⁴-sulfanylidene]cyanamide (645 mg, 1.44 mmol) in acetone was

added at rt KMnO₄(454 mg, 2.87 mmol, 2.0 equiv). The mixture was

warmed to 50 °C for 1 h, after which it was cooled to rt and

concentrated under reduced pressure. The residue was puriﬁed by

ﬂash chromatography (silica gel, hexane/EtOAc gradient) to aﬀord 12

(5-Amino-2,3-diﬂuorophenyl)methanol. TiCl₃solution (ca. 15%

in ca. 10% HCl; 144 mL) was added to a stirred solution of (2,3-

diﬂuoro-5-nitrophenyl)methanol (4.00 g, 21.2 mmol) in THF (250

mL) at 0 °C, after which the mixture was warmed to rt and stirred for

16 h at that temperature. Then, the mixture was neutralized by the

addition of 1 N NaOH and the resulting dark mass was extracted with

EtOAc. The combined organic phases were washed with brine,

ﬁltered using a Whatman ﬁlter, and concentrated under reduced

pressure. The crude product was puriﬁed by ﬂash chromatography

(silica gel, hexane/EtOAc gradient) to aﬀord the title compound

(3.33 g, 20.9 mmol, 99% yield) as a brown solid. ¹H NMR (400 MHz,

CDCl₃, 27 °C): δ 6.47 (m, 1H), 6.42 (m, 1H), 4.69 (br, 2H). UPLC

(ESI+) m/z: [M + H]⁺160.

3-(Chloromethyl)-4,5-diﬂuoroaniline. To a solution of (5-amino-

2,3-diﬂuorophenyl)methanol (4.13 g, 26.0 mmol) in DCM (78 mL)

and NMP (11 mL) was added at rt thionyl chloride (4.73 mL, 64.9

mmol, 2.5 equiv). After stirring for 14 h at that temperature, the

mixture was poured onto a mixture of ice/NaHCO₃/NaCl and stirred

for 2 h. After cessation of the reaction, the mixture was extracted with

EtOAc, and the organic extracts were ﬁltered through a Whatman

ﬁlter and concentrated under reduced pressure to aﬀord the title

compound as a crude product which was directly used in the next

step.

1

(372 mg, 0.80 mmol, 56% yield) as a colorless solid. H NMR (400

MHz, CDCl₃, 27 °C): δ 8.35 (d, J = 2.02 Hz, 1H), 7.94 (ddd, J =

12.44, 7.14, 2.65 Hz, 1H), 7.43−7.52 (m, 2H), 7.33 (s, 1H), 6.83 (t, J

= 8.11 Hz, 1H), 6.76 (d, J = 10.68 Hz, 1H), 4.65−4.73 (m, 2H,

SCH₂), 3.87 (s, 3H, OCH₃), 3.12 (s, 3H, SCH₃). ¹³C NMR (100.67

1

MHz, DMSO-d₆): δ 164.44 (d, J_C₄_F= 247.11 Hz, 1C_q), 158.59 (d,

³J_CF= 11.02 Hz, 1C_q), 155.96 (d, J_CF= 2.54 Hz, 1C_q), 151.68 (d,

³J_CF= 16.11 Hz, 1C_q), 150.48 (d, ¹J_CF= 251.77 Hz, 1C_q), 149.15 (dd,

¹J_CF= 242.44 Hz, ²J_CF= 13.56 Hz, 1C_q), 145.91 (d, ²J_CF= 25.07 Hz,

1

2

1CH), 143.43 (dd, J_C₄_F= 244.99 Hz, J_CF= 13.56 Hz, 1C_q), 137.32

3

(dd, J_CF= 10.17 Hz, J_CF= 2.97 Hz, 1C_q), 132.07 (d, J_CF= 11.02

Hz, 1CH), 118.77 (m, 1C_q), 117.56 (m, 1CH), 115.74 (m, 1C_q),

111.99 (s, 1C_q), 108.30 (d, ²J_CF= 22.04 Hz, 1CH), 107.38 (d, ²J_CF

=

22.04 Hz, 1CH), 100.25 (d, ²J_CF= 26.28 Hz, 1CH), 56.35 (s, 1CH₃),

53.60 (s, 1CH₂), ∼39.5 (s, overlapped by solvent signals). IR

(diamond ATR, cm⁻¹): 3292 (w), 3096 (w), 3001 (w), 2924 (w),

2193 (s), 1612 (m), 1593 (m), 1508 (s), 1423 (s), 1242 (s), 1192

(s), 1024 (s), 982 (m), 957 (m), 895 (m), 829 (m), 781 (m). UPLC

(ESI+) m/z: [M + H]⁺465. ESI-HRMS m/z: [M + H]⁺calcd for

C₂₀H₁₆F₄N₅O₂S, 466.0962; found, 466.0958.

3,4-Diﬂuoro-5-[(methylsulfanyl)methyl]aniline. Sodium methane-

thiolate (3.61 g, 51.6 mmol) was added portionwise to a solution of 3-

(chloromethyl)-4,5-diﬂuoroaniline from the previous step in EtOH

(75 mL) at 0 °C. The mixture was warmed to rt and stirred for 14 h at

that temperature. After that, the mixture was diluted with brine and

extracted with EtOAc. The organic extracts were ﬁltered through a

Whatman ﬁlter and concentrated under reduced pressure. The residue

was puriﬁed by ﬂash chromatography (silica gel, hexane/EtOAc

gradient) to aﬀord the title compound (1.27 g, 6.71 mmol, 26% yield

(rac)-N-{3,4-Diﬂuoro-5-[(S-methylsulfonimidoyl)methyl]phenyl}-

5-ﬂuoro-4-(4-ﬂuoro-2-methoxyphenyl)pyrimidin-2-amine (11). To

a solution of (rac)-[(2,3-diﬂuoro-5-{[5-ﬂuoro-4-(4-ﬂuoro-2-

methoxyphenyl)pyrimidin-2-yl]amino}benzyl)(methyl)oxido-λ⁶-

sulfanylidene]cyanamide (12; 344 mg, 0.74 mmol) in DCM (30 mL)

was added at 0 °C TFAA (0.78 mL, 5.54 mmol, 7.5 equiv). The

mixture was warmed to rt and stirred for 2 h at that temperature.

Then, the mixture was concentrated under reduced pressure (at 40

°C), the residue was taken up in MeOH (5 mL), and K₂CO₃(511

mg, 3.70 mmol, 5.0 equiv) was added at rt. After stirring for 2 h at rt,

the mixture was diluted with EtOAc and THF and washed with brine.

The organic phase was ﬁltered through a Whatman ﬁlter, the ﬁltrate

1

over two steps) as a brownish oil. H NMR (400 MHz, CDCl₃, 27

°C): δ 6.36 (m, 2H), 3.62 (br, 4H, SCH₂, NH₂), 2.08 (s, 3H, SCH₃).

UPLC (ESI+) m/z: [M + H]⁺190.

N-{3,4-Diﬂuoro-5-[(methylsulfanyl)methyl]phenyl}-5-ﬂuoro-4-(4-

ﬂuoro-2-methoxyphenyl)pyrimidin-2-amine. A mixture of 3,4-

diﬂuoro-5-[(methylsulfanyl)methyl]aniline (600 mg, 3.17 mmol, 1.0

equiv), 2-chloro-5-ﬂuoro-4-(4-ﬂuoro-2-methoxyphenyl)pyrimidine

(1.06 g, 4.12 mmol, 1.3 equiv), XPhos Pd G1 (197 mg, 0.24 mmol,

7.5 mol %), XPhos (113 mg, 0.24 mmol, 7.5 mol %), and ﬁnely

N

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was concentrated under reduced pressure, and the crude product was

puriﬁed by ﬂash chromatography (silica gel, DCM to DCM/EtOH

8:2 gradient) to aﬀord 11 (300 mg, 0.68 mmol, 92% yield). ¹H NMR

(400 MHz, CDCl₃, 27 °C): δ 8.30 (m, 1H), 7.86 (m, 1H), 7.47 (m,

1H), 7.26 (m, 2H), 6.82 (m, 1H), 6.76 (m, 1H), 4.42 (d, J = 13.6 Hz,

1H, sulfoximine−CH₂, H_A, AB system), 4.35 (d, J = 13.6 Hz, 1H,

sulfoximine−CH₂, H_B, AB system), 3.87 (s, 3H, OCH₃), 2.96 (s, 3H,

SCH₃), 2.76 (s, 1H, sulfoximine−NH). ¹³C NMR (100.67 MHz,

pyrimidin-2-amine (730 mg, 1.89 mmol) and cyanamide (159 mg,

3.78 mmol) in DCM (22 mL), iodosobenzene diacetate (670 mg,

2.08 mmol) was added. The mixture was stirred at rt for 2.5 h, then

diluted with DCM, and washed with brine and sodium thiosulfate

solution (10%). The organic layer was dried over Na₂SO₄, ﬁltered,

and concentrated to aﬀord a foam. The crude product was puriﬁed by

column chromatography (EtOAc to EtOAc/MeOH 4:1) to aﬀord the

1

desired product (672 mg, 1.62 mmol, 85%). H NMR (400 MHz,

1

3

DMSO-d₆): δ 10.15 (s, 1H), 8.91 (d, J = 2.27 Hz, 1H), 8.60 (d, J =

2.02 Hz, 1H), 8.24 (t, J = 2.15 Hz, 1H), 8.16 (d, J = 2.02 Hz, 1H),

7.58 (dd, J = 6.82, 8.34 Hz, 1H), 7.13 (dd, J = 2.27, 11.37 Hz, 1H),

6.97 (dt, J = 2.53, 8.46 Hz, 1H), 4.26−4.56 (m, 2H), 3.84 (s, 3H),

2.86 (s, 3H). UPLC (ESI+) m/z: [M + H]⁺415.

DMSO-d₆): δ 164.40 (d, J_CF= 247.11 Hz, 1C_q), 158.57 (d, J_CF

=

4

3

11.02 Hz, 1C_q), 156.09 (d, J_CF= 2.97 Hz, 1C_q), 151.61 (d, J_CF

15.26 Hz, 1C_q), 150.4 (d, ¹J_CF= 251.34 Hz, 1C_q), 149.06 (dd, ¹J_CF

241.60 Hz, ²J_CF= 13.56 Hz, 1C_q), 145.92 (d, ²J_CF= 25.01 Hz, 1CH),

143.35 (dd, ¹J_CF= 242.87 Hz, ²J_CF= 13.14 Hz, 1C_q), 136.86 (dd, ³J_CF

= 10.17 Hz, ⁴J_CF= 2.97 Hz, 1C_q), 132.05 (d, ³J_CF= 10.17 Hz, 1CH),

120.02 (d, ²J_CF= 13.56 Hz, 1C_q), 118.13 (m, 1C_q), 117.22 (m, 1CH),

107.34 (d, ²J_CF= 12.04 Hz, 1CH), 107.15 (d, ²J_CF= 22.04 Hz, 1CH),

(rac)-{[(5-{[5-Fluoro-4-(4-ﬂuoro-2-methoxyphenyl)pyrimidin-2-

yl]amino}pyridin-3-yl)methyl](methyl)oxido-λ⁶-sulfanylidene}-

cyanamide. To a mixture of (rac)-{[(5-{[5-ﬂuoro-4-(4-ﬂuoro-2-

methoxyphenyl)pyrimidin-2-yl]amino}pyridin-3-yl)methyl](methyl)-

λ⁴-sulfanylidene}cyanamide (650 mg, 1.56 mmol) in acetone (15.6

mL) was added at rt KMnO₄(496 mg, 3.13 mmol), and the mixture

was heated to 50 °C for 1 h. After cooling to rt, the mixture was

concentrated under reduced pressure and puriﬁed by chromatography

(silica gel, DCM to DCM/MeOH 4:1 gradient) to aﬀord the title

2

3

100.23 (d, J_CF= 26.28 Hz, 1CH), 56.35 (s, 1CH₃), 56.06 (d, J_CF

=

1.27 Hz, 1CH₂), 41.54 (s, 1CH₃). IR (diamond ATR, cm⁻¹): 3300

(br s), 3099 (w), 2982 (w), 2926 (w), 1634 (w), 1610 (m), 1597

(m), 1506 (s), 1431 (s), 1327 (m), 1207 (s), 1151 (m), 1026 (m),

959 (m), 870 (m), 858 (m), 829 (m), 783 (m). ESI-HRMS m/z: [M

+ H]⁺calcd for C₁₉H₁₇F₄N₄O₂S, 441.1009; found, 441.1007.

1

compound (385 mg, 0.89 mmol, 57% yield) as a colorless foam. H

Compound 13. (5-{[5-Fluoro-4-(4-ﬂuoro-2-methoxyphenyl)-

pyrimidin-2-yl]amino}pyridin-3-yl)methanol. A mixture of 2-

chloro-5-ﬂuoro-4-(4-ﬂuoro-2-methoxyphenyl)pyrimidine (2.13 g,

7.89 mmol), (5-aminopyridin-3-yl)methanol (1.29 g, 9.87 mmol;

ABCR), XPhos Pd G1 (653 mg, 0.79 mmol), XPhos (376 mg, 0.79

mmol), and potassium phosphate (8.38 g, 39.47 mmol) in toluene

(210 mL) and NMP (21 mL) was degassed using argon. The mixture

was stirred under argon for 3 h at 130 °C. After cooling, the reaction

mixture was partitioned between H₂O and EtOAc. The organic layer

was washed with saturated NaCl, dried over Na₂SO₄, ﬁltered, and

concentrated to aﬀord an oil. Puriﬁcation by column chromatography

(silica gel, hexane to hexane/EtOAc 1:1, then DCM to DCM/EtOAc

to EtOAc/MeOH 8:2) aﬀorded the desired product (3.12 g, 9 mmol,

NMR (400 MHz, DMSO-d₆): δ 10.17 (s, 1H), 8.94 (d, J = 2.53 Hz,

1H), 8.61 (d, J = 2.02 Hz, 1H), 8.31 (t, J = 2.15 Hz, 1H), 8.20 (d, J =

2.02 Hz, 1H), 7.56 (dd, J = 6.82, 8.59 Hz, 1H), 7.13 (dd, J = 2.40,

11.49 Hz, 1H), 6.96 (dt, J = 2.40, 8.40 Hz, 1H), 5.01−5.10 (m, 2H),

3.84 (s, 3H), 3.41 (s, 3H). UPLC (ESI+) m/z: [M + H]⁺431.

(rac)-5-Fluoro-4-(4-ﬂuoro-2-methoxyphenyl)-N-{5-[(S-

methylsulfonimidoyl)methyl]pyridin-3-yl}pyrimidin-2-amine (13).

To a cooled (0 °C) solution of (rac)-{[(5-{[5-ﬂuoro-4-(4-ﬂuoro-2-

methoxyphenyl)pyrimidin-2-yl]amino}pyridin-3-yl)methyl](methyl)-

oxido-λ⁶-sulfanylidene}cyanamide (112 mg, 0.26 mmol) in DCM

(11.3 mL), TFAA (0.11 mL, 0.78 mmol) was added. Then, the

mixture was stirred at rt for 2 h. The solvent was evaporated and the

residue was dissolved in MeOH (1.8 mL) and K₂CO₃(180 mg, 1.3

mmol) was added. After 30 min, the mixture was diluted with THF

and partitioned between H₂O and EtOAc. The organic layer was

washed with brine, dried over Na₂SO₄, ﬁltered, and concentrated. The

crude material was puriﬁed by chromatography (EtOAc/MeOH 3%

to EtOAc/MeOH 40%) to aﬀord 13 (63 mg, 0.16 mmol, 59% yield).

¹H NMR (400 MHz, DMSO-d₆): δ 10.20 (s, 1H), 8.95 (br s, 1H),

1

91%). H NMR (400 MHz, DMSO-d₆): δ 9.96 (s, 1H), 8.79 (d, J =

2.53 Hz, 1H), 8.60 (d, J = 2.02 Hz, 1H), 8.08−8.14 (m, 2H), 7.55

(dd, J = 6.82, 8.59 Hz, 1H), 7.13 (dd, J = 2.40, 11.49 Hz, 1H), 6.96

(dt, J = 2.27, 8.46 Hz, 1H), 5.27 (t, J = 5.56 Hz, 1H), 4.50 (d, J = 5.56

Hz, 2H), 3.84 (s, 3H). UPLC (ESI+) m/z: [M + H]⁺345.

N-[5-(Chloromethyl)pyridin-3-yl]-5-ﬂuoro-4-(4-ﬂuoro-2-

methoxyphenyl)pyrimidin-2-amine. (5-{[5-Fluoro-4-(4-ﬂuoro-2-

methoxyphenyl)pyrimidin-2-yl]amino}pyridin-3-yl)methanol (3.1 g,

9 mmol) was dissolved in DCM (66 mL) and NMP (6.6 mL). The

mixture was cooled to 0 °C and thionyl chloride (1.64 mL, 22.5

mmol) was slowly added. The cold bath was removed, and the

mixture was stirred at rt for 2 h. Then, the mixture was diluted with

EtOAc, washed with saturated NaHCO₃solution and brine, dried

over Na₂SO₄, ﬁltered, and concentrated to aﬀord an oil (3.85 g) that

was used without further puriﬁcation. UPLC (ESI+) m/z: [M + H]⁺

363.

5-Fluoro-4-(4-ﬂuoro-2-methoxyphenyl)-N-{5-[(methylsulfanyl)-

methyl]pyridin-3-yl}pyrimidin-2-amine. N-[5-(Chloromethyl)-

pyridin-3-yl]-5-ﬂuoro-4-(4-ﬂuoro-2-methoxyphenyl)pyrimidin-2-

amine (693 mg, 1.91 mmol) was dissolved in EtOH (100 mL) and

sodium methanethiolate (268 mg, 3.82 mmol) was added. The

mixture was stirred at rt for 3 h. Then, it was diluted with brine and

extracted with EtOAc (2×). The combined organic phases were

washed with H₂O, dried (Na₂SO₄), ﬁltered, and concentrated to

aﬀord the desired product (740 mg, 1.98 mmol) that was used

without further puriﬁcation. ¹H NMR (400 MHz, DMSO-d₆): δ 9.98

(s, 1H), 8.76 (d, J = 2.53 Hz, 1H), 8.60 (d, J = 2.02 Hz, 1H), 8.16 (t, J

= 2.15 Hz, 1H), 8.07 (d, J = 2.02 Hz, 1H), 7.55 (dd, J = 6.82, 8.59 Hz,

1H), 7.13 (dd, J = 2.40, 11.49 Hz, 1H), 6.96 (dt, J = 2.27, 8.46 Hz,

1H), 3.84 (s, 3H), 3.68 (s, 2H), 1.96 (s, 3H). UPLC (ESI+) m/z: [M

+ H]⁺375.

8.62 (d, J = 1.52 Hz, 1H), 8.21−8.32 (m, 2H), 7.56 (t, J = 7.73 Hz,

1H), 7.14 (dd, J = 2.03, 11.41 Hz, 1H), 6.96 (dt, J = 2.28, 8.36 Hz,

1H), 4.68−4.81 (m, 2H), 3.84 (s, 3H), 3.12 (s, 3H). ¹³C NMR

1

(100.67 MHz, DMSO-d₆): δ 164.41 (d, J_CF= 247.11 Hz, 1C_q),

3

4

158.56 (d, J_CF= 10.60 Hz, 1C_q), 156.31 (d, J_CF= 2.54 Hz, 1C_q),

151.64 (d, ²J_CF= 15.26 Hz, 1C_q), 150.50 (d, ¹J_CF= 251.35 Hz, 1C_q),

145.97 (d, ²J_CF= 25.01 Hz, 1CH), 144.22 (s, 1CH), 140.13 (s, 1CH),

136.85 (s, 1C_q), 132.12 (d, ³J_CF= 10.60 Hz, 1CH), 127.07 (s, 1CH),

126.39 (s, 1C_q), 118.92 (m, 1C_q), 107.36 (d, ²J_CF= 21.62 Hz, 1CH),

100.18 (d, ²J_CF= 25.86 Hz, 1CH), 59.58 (s, 1CH₂), 56.38 (s, 1CH₃),

41.12 (s, 1CH₃). IR (diamond ATR, cm⁻¹): 3302 (w), 3250 (w),

3125 (w), 2972 (w), 2924 (w), 1682 (m), 1599 (m), 1583 (m), 1543

(s), 1427 (s), 1302 (m), 1281 (s), 1198 (s), 1153 (s), 1030 (s), 953

(m), 881 (m), 837 (s), 785 (s). ESI-HRMS m/z: [M + H]⁺calcd for

C₁₈H₁₈F₂N₅O₂S, 406.1194; found, 406.1145.

Compound 14. 3-[(3-Fluoro-5-nitrobenzyl)sulfanyl]propan-1-ol.

Cs₂CO₃(3.1 g, 9.5 mmol) was added to a solution of 1-

(chloromethyl)-3-ﬂuoro-5-nitrobenzene (1.5 g, 7.91 mmol) and 3-

mercaptopropanol (0.81 mL, 11.1 mmol) in DMF (15 mL). The

mixture was stirred at rt for 90 min. Then, it was diluted with H₂O

and extracted with EtOAc (2×). The combined organic phases were

washed with LiCl solution (5%), saturated NaHCO₃solution, and

brine and dried (Na₂SO₄). The ﬁltrate was concentrated to aﬀord the

desired product (2.32 g) that was used without further puriﬁcation.

¹H NMR (300 MHz, CDCl₃): δ 8.02 (s, 1H), 7.82 (td, J = 2.24, 8.15

Hz, 1H), 7.43 (td, J = 2.05, 8.34 Hz, 1H), 3.79 (s, 2H), 3.74 (t, J =

6.03 Hz, 2H), 2.82 (t, J = 7.06 Hz, 1H), 2.57 (t, J = 7.06 Hz, 2H),

1.77−1.90 (m, 2H). UPLC (ESI−) m/z: [M − H]⁻244.

(rac)-{[(5-{[5-Fluoro-4-(4-ﬂuoro-2-methoxyphenyl)pyrimidin-2-

yl]amino}pyridin-3-yl)methyl](methyl)-λ⁴-sulfanylidene}-

cyanamide. To a cooled solution (0 °C) of 5-ﬂuoro-4-(4-ﬂuoro-2-

methoxyphenyl)-N-{5-[(methylsulfanyl)methyl]pyridin-3-yl}-

O

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3-[(3-Amino-5-ﬂuorobenzyl)sulfanyl]propan-1-ol. TiCl₃solution

(ca. 15% in ca. 10% HCl; 19.4 mL) was added to a stirred solution of

3-[(3-ﬂuoro-5-nitrobenzyl)sulfanyl]propan-1-ol (1.2 g, 4.89 mmol) in

THF (50 mL) at rt, and the mixture was stirred for 116 h. The pH

was increased to 10 by the addition of 1 N NaOH, and then the

mixture was extracted with EtOAc (2×). The combined organic

phases were washed with brine, ﬁltered using a Whatman ﬁlter, and

concentrated. The residue was puriﬁed by column chromatography

(DCM to DCM/EtOH 95:5) to aﬀord the desired product (685 mg,

Compound 15. 3-[(3-Fluoro-5-nitrobenzyl)sulfanyl]propan-1-

amine. Cs₂CO₃(8.93 g, 27.4 mmol) was added to a solution of 1-

(chloromethyl)-3-ﬂuoro-5-nitrobenzene (2 g, 10.5 mmol) and 3-

aminopropane-1-thiol hydrochloride (1.87 g, 14.7 mmol) in DMF (30

mL). The mixture was stirred at rt for 5 h. Then, it was diluted with

H₂O and extracted with EtOAc (2×). The combined organic phases

were washed with LiCl solution (5%), saturated NaHCO₃solution,

and brine and dried (Na₂SO₄). The ﬁltrate was concentrated to aﬀord

the desired product (1.93 g, 7.9 mmol, 74% yield) that was used

without further puriﬁcation.

1

3.18 mmol, 65% yield). H NMR (400 MHz, CDCl₃): δ 6.40−6.45

tert-Butyl {3-[(3-Fluoro-5-nitrobenzyl)sulfanyl]propyl}-

carbamate. To a solution of 3-[(3-ﬂuoro-5-nitrobenzyl)sulfanyl]-

propan-1-amine (1.93 g, 7.9 mmol) in t-BuOH, di-tert-butyl

dicarbonate (2.29 g, 10.5 mmol) was added. The reaction mixture

was stirred at rt for 3 h, then diluted with EtOAc, and concentrated.

Puriﬁcation of the remaining material by column chromatography

(hexanes to hexanes/EtOH 2:1) aﬀorded the desired product (1.84 g,

(m, 2H), 6.27 (td, J = 2.02, 10.36 Hz, 1H), 3.73 (t, J = 6.06 Hz, 2H),

3.60 (s, 2H), 2.56 (t, J = 7.07 Hz, 2H), 1.82 (quin, J = 6.51 Hz, 2H).

UPLC (ESI+) m/z: [M + H]⁺216.

3-[(3-Fluoro-5-{[5-ﬂuoro-4-(4-ﬂuoro-2-methoxyphenyl)-

pyrimidin-2-yl]amino}benzyl)sulfanyl]propan-1-ol. A mixture of 2-

chloro-5-ﬂuoro-4-(4-ﬂuoro-2-methoxyphenyl)pyrimidine (989 mg,

3.85 mmol), 3-[(3-amino-5-ﬂuorobenzyl)sulfanyl]propan-1-ol (638

mg, 2.96 mmol), XPhos Pd G1 (184 mg, 0.22 mmol), XPhos (106

mg, 0.22 mmol), and ﬁnely ground potassium phosphate (3.14 g, 14.8

mmol) in toluene (19 mL) and NMP (3.9 mL) was degassed and

repressured with argon at rt. The mixture was heated in a sealed vial

to 130 °C in a microwave reactor for 3 h. After cooling to rt, brine was

added, and the mixture was extracted with EtOAc. The combined

organic extracts were ﬁltered through a Whatman ﬁlter and the ﬁltrate

was concentrated under reduced pressure. The crude product was

puriﬁed by preparative HPLC (acidic conditions) to aﬀord the title

1

5.35 mmol, 67% yield). H NMR (400 MHz, CDCl₃): δ 7.99−8.02

(m, 1H), 7.82 (td, J = 2.24, 8.15 Hz, 1H), 7.43 (td, J = 1.89, 8.59 Hz,

1H), 4.56 (br s, 1H), 3.77 (s, 2H), 3.20 (br t, J = 6.44 Hz, 2H), 2.47

(t, J = 7.20 Hz, 2H), 1.76 (quin, J = 6.95 Hz, 2H), 1.43 (s, 9H).

tert-Butyl {3-[(3-Amino-5-ﬂuorobenzyl)sulfanyl]propyl}-

carbamate. A solution of tert-butyl {3-[(3-ﬂuoro-5-nitrobenzyl)-

sulfanyl]propyl}carbamate (1.33 g, 3.82 mmol) in EtOH (40.5 mL)

was degassed with argon. 10% palladium on charcoal (3.86 mmol)

was added, and the mixture was stirred at rt under an atmosphere of

hydrogen for 18 h. The suspension was ﬁltered using a Whatman ﬁlter

and concentrated. The resulting residue was puriﬁed by column

chromatography (hexanes to hexanes/EtOAc 1:1) to aﬀord the

1

compound (846 mg, 1.94 mmol, 66% yield). H NMR (400 MHz,

CDCl₃): δ 8.32 (d, J = 2.02 Hz, 1H), 7.59 (td, J = 2.21, 11.24 Hz,

1H), 7.49 (dd, J = 6.57, 8.34 Hz, 1H), 7.29 (br s, 1H), 7.16 (s, 1H),

6.82 (dt, J = 2.40, 8.27 Hz, 1H), 6.76 (dd, J = 2.40, 10.74 Hz, 1H),

6.70 (td, J = 1.77, 9.09 Hz, 1H), 3.87 (s, 3H), 3.71 (t, J = 6.06 Hz,

2H), 3.68 (s, 2H), 2.55 (t, J = 7.07 Hz, 2H), 1.77−1.85 (m, 2H).

UPLC (ESI−) m/z: [M − H]⁻434.

1

desired product (216 mg, 0.69 mmol, 18% yield). H NMR (300

MHz, CDCl₃): δ 6.39−6.49 (m, 2H), 6.29 (br d, J = 10.55 Hz, 1H),

4.60 (br s, 1H), 3.89 (br s, 2H), 3.60 (s, 2H), 3.20 (q, J = 6.34 Hz,

2H), 2.47 (t, J = 7.25 Hz, 2H), 1.75 (quin, J = 6.92 Hz, 2H), 1.46 (s,

9H).

(rac)-[(3-Fluoro-5-{[5-ﬂuoro-4-(4-ﬂuoro-2-methoxyphenyl)-

pyrimidin-2-yl]amino}benzyl)(3-hydroxypropyl)-λ⁴-sulfanylidene]-

cyanamide. To a cooled solution (0 °C) of 3-[(3-ﬂuoro-5-{[5-ﬂuoro-

4-(4-ﬂuoro-2-methoxyphenyl)pyrimidin-2-yl]amino}benzyl)sulfanyl]-

propan-1-ol (774 mg, 1.77 mmol) and cyanamide (150 mg, 3.55

mmol) in DCM (15.4 mL), iodosobenzene diacetate (630 mg, 1.95

mmol) was added. The mixture was stirred at rt for 1 h, then diluted

with DCM, and washed with brine and sodium thiosulfate solution

(10%). The organic layer was dried over Na₂SO₄, ﬁltered, and

concentrated to aﬀord a foam. The crude product was puriﬁed by

column chromatography (DCM to DCM/EtOH 88:12) to aﬀord the

tert-Butyl {3-[(3-Fluoro-5-{[5-ﬂuoro-4-(4-ﬂuoro-2-

methoxyphenyl)pyrimidin-2-yl]amino}benzyl)sulfanyl]propyl}-

carbamate. A mixture of 2-chloro-5-ﬂuoro-4-(4-ﬂuoro-2-

methoxyphenyl)pyrimidine (806 mg, 3.14 mmol), tert-butyl {3-[(3-

amino-5-ﬂuorobenzyl)sulfanyl]propyl}carbamate (800 mg, 2.42

mmol), XPhos Pd G1 (150 mg, 0.18 mmol), XPhos (86 mg, 0.18

mmol), and ﬁnely ground potassium phosphate (2.56 g, 12.1 mmol)

in toluene (15.6 mL) and NMP (3.1 mL) was degassed and

repressured with argon at rt. The mixture was heated in a sealed vial

to 130 °C in a microwave reactor for 2.5 h. After cooling to rt, brine

was added, and the mixture was extracted with EtOAc. The combined

organic extracts were ﬁltered through a Whatman ﬁlter and the ﬁltrate

was concentrated under reduced pressure. The crude product was

puriﬁed by column chromatography (hexanes to hexanes/EtOAc 2:1)

1

desired product (623 mg, 1.33 mmol, 74% yield). H NMR (400

MHz, CDCl₃): δ 8.30−8.36 (m, 1H), 7.68−7.75 (m, 1H), 7.43−7.54

(m, 2H), 7.32 (s, 1H), 6.80−6.86 (m, 1H), 6.76 (dd, J = 2.02, 10.86

Hz, 1H), 6.64−6.73 (m, 1H), 4.11−4.36 (m, 2H), 3.87 (s, 3H),

3.74−3.84 (m, 2H), 3.02−3.31 (m, 2H), 2.04−2.13 (m, 2H). UPLC

(ESI+) m/z: [M + H]⁺476.

1

to aﬀord the desired product (1.21 g, 2.26 mmol, 93% yield). H

NMR (400 MHz, CDCl₃): δ 8.34 (d, J = 1.77 Hz, 1H), 7.65 (br d, J =

11.37 Hz, 1H), 7.52 (dd, J = 6.69, 8.46 Hz, 1H), 7.38 (br s, 1H), 7.17

(br s, 1H), 6.81−6.87 (m, 1H), 6.78 (dd, J = 2.27, 10.86 Hz, 1H),

6.71 (br d, J = 8.84 Hz, 1H), 4.60 (br s, 1H), 3.89 (s, 3H), 3.68 (s,

2H), 3.14−3.23 (m, 2H), 2.46 (t, J = 7.20 Hz, 2H), 1.75 (quin, J =

6.95 Hz, 2H), 1.45 (s, 9H).

(rac)-tert-Butyl {3-[S-(3-Fluoro-5-{[5-ﬂuoro-4-(4-ﬂuoro-2-

methoxyphenyl)pyrimidin-2-yl]amino}benzyl)-N-(triﬂuoroacetyl)-

sulﬁnimidoyl]propyl}carbamate. Under an atmosphere of argon, a

solution of 2,2,2-triﬂuoroacetamide (316 mg, 2.79 mmol) in THF (1

mL) was added dropwise to a solution of sodium tert-butoxide (179

mg, 1.86 mmol) in THF (1.5 mL), so that the temperature of the

mixture remained below 10 °C. Subsequently, a freshly prepared

solution of 1,3-dibromo-5,5-dimethylhydantoin (346 mg, 1.21 mmol)

in THF (1.5 mL) was added dropwise to the stirred mixture, so that

the temperature of the mixture remained below 10 °C. Then, the

mixture was stirred for 10 min at 10 °C. Finally, a solution of tert-

butyl {3-[(3-ﬂuoro-5-{[5-ﬂuoro-4-(4-ﬂuoro-2-methoxyphenyl)-

pyrimidin-2-yl]amino}benzyl)sulfanyl]propyl}carbamate (997 mg,

1.86 mmol) in THF (1.5 mL) was added dropwise to the stirred

mixture, so that the temperature of the mixture remained below 10

(rac)-[(3-Fluoro-5-{[5-ﬂuoro-4-(4-ﬂuoro-2-methoxyphenyl)-

pyrimidin-2-yl]amino}benzyl)(3-hydroxypropyl)oxido-λ⁶-

sulfanylidene]cyanamide (14). (rac)-[(3-Fluoro-5-{[5-ﬂuoro-4-(4-

ﬂuoro-2-methoxyphenyl)pyrimidin-2-yl]amino}benzyl)(3-hydroxy-

propyl)-λ⁴-sulfanylidene]cyanamide (534 mg, 1.12 mmol) was

dissolved in acetone (11 mL). KMnO₄(355 mg, 2.25 mmol) was

added, and the mixture was stirred at 50 °C for 2 h. Then, additional

KMnO₄(89 mg, 0.56 mmol) was added and stirring was continued

for 2 h. The mixture was cooled to rt and ﬁltered. The ﬁlter cake was

rinsed with acetone and EtOH. The combined organic fractions were

concentrated. Puriﬁcation of the crude material by column

chromatography (DCM/EtOH 95:5 to 9:1) aﬀorded 14 (11 mg,

2% yield). ¹H NMR (400 MHz, CDCl₃): δ 8.34 (d, J = 1.77 Hz, 1H),

7.67−7.73 (m, 1H), 7.45−7.55 (m, 3H), 6.74−6.87 (m, 3H), 4.57 (s,

2H), 3.87 (s, 3H), 3.73 (t, J = 5.56 Hz, 2H), 3.27−3.37 (m, 2H),

2.04−2.16 (m, 2H). IR (diamond ATR, cm⁻¹): 3271 (w), 3103 (w),

2920 (w), 2192 (s), 1609 (s), 1587 (s), 1506 (m), 1427 (s), 1331

(w), 1281 (m), 1211 (m), 1151 (m), 1049 (m), 1022 (s), 953 (m),

835 (m), 785 (m). UPLC (ESI+) m/z: [M + H]⁺492. ESI-HRMS m/

z: [M + H]⁺calcd for C₂₂H₂₁F₃N₅O₃S, 492.1318; found, 492.1320.

P

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°C. The mixture was stirred for 1 h at 10 °C. The mixture was diluted

with toluene (4.0 mL) under cooling, and a solution of sodium sulﬁte

(235 mg, 1.86 mmol) in H₂O (7.0 mL) was added so that the

temperature of the mixture remained below 15 °C. Aq NaCl solution

was added, and the mixture was extracted with EtOAc (3×). The

combined organic layers were ﬁltered using a Whatman ﬁlter and

concentrated. The residue was puriﬁed by column chromatography

(DCM to DCM/EtOH 94:6) to aﬀord the desired product (670 mg,

°C perchloric acid (70%, 6.5 mL), and the mixture was stirred for 10

min at that temperature. Cold H₂O (30 mL) was then added to this

mixture, and the resulting emulsion was extracted with DCM (3×).

The combined organic extracts were washed with brine and dried over

Na₂SO₄. The resulting solution of O-(mesitylsulfonyl)hydroxylamine

was then added dropwise at 0 °C to a solution of 5-ﬂuoro-4-(4-ﬂuoro-

2-methoxyphenyl)-N-{3-ﬂuoro-5-[(methylsulfanyl)methyl]phenyl}-

pyrimidin-2-amine (2.50 g, 6.39 mmol) in DCM (6.5 mL). The

mixture was allowed to warm to rt overnight and then concentrated

under reduced pressure to 5 mL. The precipitate was ﬁltered oﬀ, and

the residue was rinsed with diethyl ether. The combined ﬁltrate was

concentrated under reduced pressure to aﬀord the title compound

1

1.04 mmol, 55% yield). H NMR (300 MHz, CDCl₃): δ 8.36 (br s,

1H), 7.76 (br d, J = 11.49 Hz, 1H), 7.46−7.59 (m, 2H), 6.74−6.91

(m, 2H), 6.67 (br d, J = 7.54 Hz, 1H), 4.83 (br s, 1H), 4.45 (br d, J =

12.81 Hz, 1H), 4.22 (br d, J = 13.00 Hz, 1H), 3.89 (s, 3H), 3.14−3.30

(m, 2H), 2.89−3.03 (m, 2H), 1.91−2.04 (m, 2H), 1.44 (br s, 9H).

(rac)-tert-Butyl {3-[S-(3-Fluoro-5-{[5-ﬂuoro-4-(4-ﬂuoro-2-

methoxyphenyl)pyrimidin-2-yl]amino}benzyl)sulfonimidoyl]-

propyl}carbamate. To a solution of (rac)-tert-butyl {3-[S-(3-ﬂuoro-

5-{[5-ﬂuoro-4-(4-ﬂuoro-2-methoxyphenyl)pyrimidin-2-yl]amino}-

benzyl)-N-(triﬂuoroacetyl)sulﬁnimidoyl]propyl}carbamate (561 mg,

0.869 mmol) in MeOH (17 mL), aq KOH solution (25%) was added

until pH 10.5 was reached. Oxone (451 mg, 0.739 mmol) was added,

and the mixture was stirred at a pH of 10−11 for 2 h. Then, the

mixture was ﬁltered, neutralized with HCl (pH 6.6), diluted with

brine, and extracted with EtOAc. The organic layer was washed with

aq sodium thiosulfate solution, ﬁltered using a Whatman ﬁlter, and

concentrated to aﬀord the desired compound (433 mg, 0.77 mmol,

1

(3.04 g, 5.00 mmol, 78% yield) as an oﬀ-white solid. H NMR (400

MHz, DMSO-d₆, 27 °C): δ 10.24 (s, 1H), 8.62 (d, J = 2.02 Hz, 1H),

7.85 (dt, J = 12.19, 2.12 Hz, 1H), 7.54 (t, J = 7.32 Hz, 1H), 7.48 (s,

1H), 7.14 (dd, J = 11.37, 2.27 Hz, 1H), 6.97 (td, J = 8.34, 2.27 Hz,

+

1H), 6.84 (d, J = 8.96 Hz, 1H), 6.73 (s, 2H), 5.96 (s, 2H, SNH₂),

4.58 (d, J = 12.88 Hz, 1H, SCH_AH_B, AB system), 4.38 (d, J = 12.88

Hz, 1H, SCH_AH_B, AB system), 3.83 (s, 3H, OCH₃), 3.03 (s, 3H,

SCH₃), 2.53 (s, 6H, mesitylsulfonate-2-CH₃, -6-CH₃), 2.16 (s, 3H,

mesitylsulfonate-4-CH₃). UPLC (ESI+) m/z: [M + H]⁺407 (free

amine).

5-Fluoro-4-(4-ﬂuoro-2-methoxyphenyl)-N-{3-ﬂuoro-5-[(S-

methylsulfonodiimidoyl)methyl]phenyl}pyrimidin-2-amine (16).

To a solution of (rac)-[(3-ﬂuoro-5-{[5-ﬂuoro-4-(4-ﬂuoro-2-

methoxyphenyl)pyrimidin-2-yl]amino}benzyl)(methyl)-λ⁴-

sulfanylidene]ammonium 2,4,6-trimethylbenzenesulfonate (300 mg,

0.49 mmol) in anhyd DMF (1.3 mL) was added at 0 °C Na₂CO₃

(62.9 mg, 0.59 mmol, 1.2 equiv) and NCS (79.3 mg, 0.59 mmol, 1.2

equiv). After stirring for 15 min at 0 °C, hexamethyldisilazane (0.31

mL, 1.48 mmol, 3.0 equiv) was added, and the mixture was warmed to

rt. After stirring for a further 18 h, brine was added to the solution,

and the mixture was extracted with EtOAc and DCM. The combined

organic extracts were ﬁltered through a Whatman ﬁlter and

concentrated under reduced pressure. The crude product was puriﬁed

by preparative HPLC [Agilent Prep 1200 system (2 × Prep Pump,

DLA, MWD, Prep FC); column: Waters XBridge C18 5 μm, 100 ×

30 mm; eluent A: H₂O + 0.2% NH₃, eluent B: MeCN; gradient: 0−10

min 25−55% B, 10−12 min 100% B; ﬂow: 60 mL/min; rt; solution:

243 mg in 3.2 mL DMSO; injection: 2 × 1.6 mL; detection: UV 225

nm; t_R= 5.72−6.42 min] to aﬀord 16 (40.0 mg, 0.09 mmol, 19%

yield) as a colorless solid. ¹H NMR (400 MHz, DMSO-d₆, 27 °C): δ

10.04 (s, 1H, aniline−NH), 8.59 (d, J = 2.02 Hz, 1H), 7.73 (dt, J =

12.00, 2.21 Hz, 1H), 7.54 (dd, J = 8.34, 6.82 Hz, 1H), 7.46 (s, 1H),

7.12 (dd, J = 11.37, 2.27 Hz, 1H), 6.95 (td, J = 8.46, 2.53 Hz, 1H),

6.83 (br d, J = 9.60 Hz, 1H), 4.24 (s, 2H, SCH₂), 3.83 (s, 3H,

OCH₃), 2.80 (s, 3H, SCH₃). ¹³C NMR (100.65 MHz, DMSO-d₆) δ:

164.39 (d, ¹J_CF= 247.11 Hz, 1C_q), 161.83 (d, ¹J_CF= 239.48 Hz, 1C_q),

1

88% yield). H NMR (400 MHz, CDCl₃): δ 8.35 (d, J = 1.77 Hz,

1H), 7.81 (td, J = 2.02, 11.37 Hz, 1H), 7.48−7.54 (m, 2H), 6.81−

6.87 (m, 1H), 6.76−6.81 (m, 2H), 4.85 (br s, 1H), 4.15−4.34 (m,

2H), 3.89 (s, 3H), 3.27 (q, J = 6.40 Hz, 2H), 3.00−3.13 (m, 2H),

2.10 (td, J = 7.04, 14.46 Hz, 2H), 1.45 (s, 9H).

(rac)-N-(3-{[S-(3-Aminopropyl)sulfonimidoyl]methyl}-5-ﬂuoro-

phenyl)-5-ﬂuoro-4-(4-ﬂuoro-2-methoxyphenyl)pyrimidin-2-amine

(15). To a solution of (rac)-tert-butyl {3-[S-(3-ﬂuoro-5-{[5-ﬂuoro-4-

(4-ﬂuoro-2-methoxyphenyl)pyrimidin-2-yl]amino}benzyl)-

sulfonimidoyl]propyl}carbamate (27 mg, 0.048 mmol) in DCM (2

mL), triﬂuoroacetic acid (TFA, 36.7 μL, 0.447 mmol) was added.

After 2 h, additional TFA (36.7 μL, 0.447 mmol) was added, and the

mixture was stirred for 24 h. Then, the mixture was evaporated to

dryness. The residue was puriﬁed by preparative HPLC (acidic

1

conditions) to aﬀord 15 (14.3 mg, 0.03 mmol, 64% yield). H NMR

(600 MHz, DMSO-d₆): δ 10.17 (br s, 1H), 8.61 (s, 1H), 8.45 (br s,

1H), 7.81 (d, J = 12.1 Hz, 1H), 7.56 (t, J = 7.53 Hz, 1H), 7.46 (br s,

1H), 7.14 (d, J = 10.9 Hz, 1H), 6.97 (t, J = 7.53 Hz, 1H), 6.83 (d, J =

8.66 Hz, 1H), 4.26−4.36 (m, 2H), 3.85 (s, 3H), 3.03 (t, J = 7.15 Hz,

2H), 2.82 (br s, 2H), 1.96 (br s, 2H). ESI-HRMS m/z: [M + H]⁺

calcd for C₂₁H₂₃F₃N₅O₂S, 466.1525; found, 466.1518.

Compound 16. 5-Fluoro-4-(4-ﬂuoro-2-methoxyphenyl)-N-{3-ﬂu-

oro-5-[(methylsulfanyl)methyl]phenyl}pyrimidin-2-amine. A mix-

ture of 2-chloro-5-ﬂuoro-4-(4-ﬂuoro-2-methoxyphenyl)pyrimidine

(750 mg, 2.92 mmol), 3-ﬂuoro-5-[(methylsulfanyl)methyl]aniline

(385 mg, 2.25 mmol), XPhos Pd G1 (139 mg, 0.17 mmol), XPhos

(80 mg, 0.17 mmol), and ﬁnely ground potassium phosphate (2.39 g,

11.2 mmol) in toluene (15 mL) and NMP (3 mL) was degassed and

repressured with argon at rt. The mixture was heated in a sealed vial

to 130 °C in a microwave reactor for 3 h. After cooling to rt, brine was

added, and the mixture was extracted with EtOAc. The combined

organic extracts were ﬁltered through a Whatman ﬁlter and the ﬁltrate

was concentrated under reduced pressure. The crude product was

combined with the crude product from another batch of the same size

and puriﬁed by column chromatography (hexanes to hexanes/EtOAc

2:1) to aﬀord the desired product (1.62 g, 4.14 mmol, 70% yield). ¹H

NMR (400 MHz, CDCl₃): δ 8.32 (d, J = 2.02 Hz, 1H), 7.62 (dt, J =

11.37, 2.15 Hz, 1H), 7.49 (dd, J = 8.46, 6.69 Hz, 1H), 7.22−7.29 (m,

2H), 7.12 (s, 1H), 6.67−6.84 (m, 3H), 3.87 (s, 3H), 3.63 (s, 2H),

2.01 (s, 3H).

3

4

158.57 (d, J_CF= 11.02 Hz, 1C_q), 156.19 (d, J_CF= 2.97 Hz, 1C_q),

151.17 (d, ³J_CF= 14.41 Hz, 1C_q), 150.36 (d, ¹J_CF= 251.77 Hz, 1C_q),

145.88 (d, ²J_CF= 25.00 Hz, 1CH), 141.88 (d, ²J_CF= 12.29 Hz, 1C_q),

133.15 (d, ³J_CF= 10.17 Hz, 1C_q), 132.04 (d, ³J_CF= 11.02 Hz, 1CH),

118.88 (m, 1C_q), 116.97 (d, ⁴J_CF= 1.70 Hz, 1CH), 110.41 (d, ²J_CF

22.04 Hz, 1CH), 107.33 (d, ²J_CF= 22.04 Hz, 1CH), 104.47 (d, ²J_CF

=

26.70 Hz, 1CH), 100.22 (d, ²J_CF= 25.85 Hz, 1CH), 64.32 (s, 1CH₂),

56.35 (s, 1CH₃), 44.99 (s, 1CH₃). IR (diamond ATR, cm⁻¹): 3207

(w), 3190 (w), 3108 (w), 3034 (w), 2971 (w), 2728 (w), 1716 (w),

1599 (m), 1543 (s), 1434 (s), 1317 (m), 1284 (s), 1194 (s), 1155

(s), 1031 (s), 957 (m), 837 (m), 785 (s). UPLC (ESI+) m/z: [M +

H]⁺422. ESI-HRMS m/z: [M + H]⁺calcd for C₁₉H₁₉F₃N₅OS,

422.1263; found, 422.1263.

Compound 17. 5-Fluoro-4-(4-ﬂuoro-2-methoxyphenyl)-

pyrimidin-2-amine. A mixture of 2-chloro-5-ﬂuoro-4-(4-ﬂuoro-2-

methoxyphenyl)pyrimidine (2.00 g, 7.79 mg) in 7 N methanolic

ammonia (20 mL) was heated to 130 °C in a microwave reactor for 2

h. After cooling to rt, the mixture was concentrated under reduced

pressure and the residue was puriﬁed by ﬂash chromatography (silica

gel, hexane to hexane/EtOAc 50:50 gradient) to aﬀord the title

compound (1.11 g, 4.68 mmol, 60% yield) as a beige solid. ¹H NMR

(300 MHz, CDCl₃, 27 °C): δ 8.19 (d, J = 1.70 Hz, 1H), 7.43 (dd, J =

(rac)-[(3-Fluoro-5-{[5-ﬂuoro-4-(4-ﬂuoro-2-methoxyphenyl)-

pyrimidin-2-yl]amino}benzyl)(methyl)-λ⁴-sulfanylidene]-

ammonium 2,4,6-Trimethylbenzenesulfonate. To a solution of

ethyl (1E)-N-[(mesitylsulfonyl)oxy]ethanimidoate (1.82 g, 6.37

mmol, 1.0 equiv) in 1,4-dioxane (6.5 mL) was added dropwise at 0

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8.48, 6.78 Hz, 1H), 6.72−6.84 (m, 2H), 5.02 (br s, 2H, NH₂), 3.86 (s,

3H, OCH₃). UPLC (ESI+) m/z: [M + H]⁺238.

the mixture was stirred in a closed pressure tube for 8 h at 100 °C.

After cooling, additional Xantphos (17 mg, 0.03 mmol) and

Pd₂(dba)₃(9 mg, 0.01 mmol) were added under argon, and the

mixture was stirred in a closed pressure tube for 14 h at 100 °C. After

cooling, the mixture was diluted with DCM and ﬁltered. The solids

were washed with DCM, and the combined ﬁltrates were

concentrated. The residue was puriﬁed by chromatography (silica

gel, DCM/EtOH 95:5) to aﬀord 22 (424 mg, 1.19 mmol, 60% yield).

¹H NMR (400 MHz, CDCl₃, 27 °C): δ 8.86 (m, 1H), 8.26 (m, 1H),

(3-Fluoro-5-{[5-ﬂuoro-4-(4-ﬂuoro-2-methoxyphenyl)pyrimidin-2-

yl]amino}phenyl)methanol. To a mixture of 5-ﬂuoro-4-(4-ﬂuoro-2-

methoxyphenyl)pyrimidin-2-amine (130 mg, 0.55 mmol, 1.0 equiv),

(3-bromo-5-ﬂuorophenyl)methanol (112 mg, 0.55 mmol, 1.0 equiv),

and 1,4-dioxane (1.3 mL) in a sealable tube was added at rt Cs₂CO₃

(214 mg, 0.66 mmol, 1.2 equiv), and the tube was purged several

times with argon. Then, Pd₂(dba)₃(25.1 mg, 0.027 mmol, 5 mol %)

and Xantphos (31.7 mg, 0.055 mmol, 10 mol %) were added, and the

tube was purged with argon again. After that, the tube was sealed and

heated to 100 °C for 20 h and then cooled to rt upon which further

Pd₂(dba)₃(25.1 mg, 0.027 mmol, 5 mol %) and Xantphos (31.7 mg,

0.055 mmol, 10 mol %) were added. After heating to 100 °C for a

further 22 h, the mixture was cooled to rt and diluted with EtOAc,

washed with brine, and ﬁltered through a Whatman ﬁlter. The ﬁltrate

was concentrated under reduced pressure and puriﬁed by ﬂash

chromatography (silica gel, hexane to hexane/EtOAc 50:50) and

preparative HPLC [Waters AutoPuriﬁcation system (Pump 254,

Sample Manager 2767, CFO, DAD 2996, ELSD 2424, SQD 3100);

column: Waters XBridge C18 5 μm, 100 × 30 mm; eluent A: H₂O +

0.2% NH₃, eluent B: MeCN; gradient: 0−8 min 10−100% B; ﬂow: 50

mL/min; rt; solution: 149 mg in 3.0 mL DMSO; injection: 3 × 1 mL;

detection: DAD scan 210−400 nm; t_R= 5.84−6.00 min] to aﬀord the

8.04 (m, 1H), 7.94 (m, 1H), 7.82 (s, 1H), 7.76 (br, 1H), 6.96 (m,

1H), 6.77 (m, 2H), 3.92 (s, 3H), 3.67 (s, 2H), 2.06 (s, 3H). UPLC

(ESI+) m/z: [M + H]⁺357.

(rac)-2,2,2-Triﬂuoro-N-{[(2-{[6-(4-ﬂuoro-2-methoxyphenyl)-

pyrimidin-4-yl]amino}pyridin-4-yl)methyl](methyl)-λ⁴-

sulfanylidene}acetamide (23). Under argon, a solution of 2,2,2-

triﬂuoroacetamide (200 mg, 1.77 mmol) in THF (1.0 mL) was added

dropwise to a solution of sodium tert-butoxide (113 mg, 1.18 mmol)

in THF (0.8 mL), so that the temperature of the mixture remained

below 10 °C. Subsequently, a freshly prepared solution of 1,3-

dibromo-5,5-dimethylhydantoin (253 mg, 0.88 mmol) in THF (1.5

mL) was added dropwise to the stirred mixture, so that the

temperature of the mixture remained below 10 °C. Then, the mixture

was stirred for 10 min at 10 °C. Finally, a solution of 6-(4-ﬂuoro-2-

methoxyphenyl)-N-{4-[(methylsulfanyl)methyl]pyridin-2-yl}-

pyrimidin-4-amine (22; 420 mg, 1.18 mmol) in 1,4-dioxane (10.0

mL) was added dropwise to the stirred mixture, so that the

temperature of the mixture remained below 10 °C. The mixture

was stirred for 75 min at 10 °C. The mixture was diluted with toluene

(2.0 mL) under cooling, and a solution of sodium sulﬁte (149 mg,

1.18 mmol) in H₂O (2.0 mL) was added so that the temperature of

the mixture remained below 15 °C. Aq NaCl solution was added, and

the mixture was extracted with EtOAc (3×). The combined organic

layers were ﬁltered using a Whatman ﬁlter and concentrated. The

residue was puriﬁed by column chromatography (DCM/EtOH 94:6)

1

title compound (26.0 mg, 0.07 mmol, 13% yield). H NMR (300

MHz, CDCl₃): δ 8.35 (d, J = 1.88 Hz, 1H), 7.63−7.72 (m, 1H), 7.52

(dd, J = 8.48, 6.78 Hz, 1H), 7.26 (br s, 1H), 7.20 (s, 1H), 6.74−6.88

(m, 3H), 4.71 (d, J = 6.03 Hz, 2H, CH₂OH), 3.90 (s, 3H, OCH₃).

UPLC (ESI+) m/z: [M + H]⁺362.

N-[3-(Aminomethyl)-5-ﬂuorophenyl]-5-ﬂuoro-4-(4-ﬂuoro-2-

methoxyphenyl)pyrimidin-2-amine (17). To a solution of (3-ﬂuoro-

5-{[5-ﬂuoro-4-(4-ﬂuoro-2-methoxyphenyl)pyrimidin-2-yl]amino}-

phenyl)methanol (20 mg, 0.055 mmol) in DMF (0.6 mL) was added

dropwise at 0 °C thionyl chloride (10 μL, 0.138 mmol, 2.5 equiv).

The mixture was then allowed to warm to rt over 2 h, after which it

was diluted with EtOAc (100 mL) and slowly basiﬁed in an ice bath

by the addition of aq NaHCO₃/NaCl solution until pH 8. The

mixture was then extracted with EtOAc/THF, the combined extracts

were ﬁltered through a Whatman ﬁlter, and the ﬁltrate was

concentrated under reduced pressure. The crude benzyl chloride

was dissolved in 7 N methanolic ammonia (7.3 mL) and heated in a

sealed tube to 80 °C for 14 h. After cooling to rt, the mixture was

concentrated under reduced pressure, and the crude product was

puriﬁed by preparative HPLC (basic conditions) to aﬀord 17 (9.8 mg,

1

to aﬀord 23 (348 mg, 0.74 mmol, 63% yield). H NMR (400 MHz,

CDCl₃, 27 °C): δ 8.90 (s, 1H, pyrimidine-2-H), 8.38 (d, J = 5.02 Hz,

1H), 8.26 (s, 1H, NH), 8.11 (dd, J = 8.78, 7.03 Hz, 1H), 7.72 (s, 1H),

7.64 (s, 1H), 6.95 (dd, J = 5.02, 1.25 Hz, 1H), 6.84 (t, J = 8.10 Hz,

1H), 6.78 (d, J = 10.84 Hz, 1H), 4.52 (d, J = 13.05 Hz, 1H,

sulﬁlimine−CH₂, H_A, AB system), 4.31 (d, J = 13.05 Hz, 1H,

sulﬁlimine−CH₂, H_B, AB system), 3.95 (s, 3H, OCH₃), 2.74 (s, 3H,

sulﬁlimine−CH₃). UPLC (ESI+) m/z: [M + H]⁺468.

( r a c ) - 6 - ( 4 - F l u o r o - 2 - m e t h o x y p h e n y l ) - N - { 4 - [ ( S -

methylsulfonimidoyl)methyl]pyridin-2-yl}pyrimidin-4-amine (24).

KMnO₄(230 mg, 1.46 mmol) was added to a stirred solution of

(rac)-2,2,2-triﬂuoro-N-{[(2-{[6-(4-ﬂuoro-2-methoxyphenyl)-

pyrimidin-4-yl]amino}pyridin-4-yl)methyl](methyl)-λ⁴-

sulfanylidene}acetamide (23; 340 mg, 0.73 mmol) in acetone (3.4

mL) at rt. The mixture was stirred at 50 °C for 5 h, before additional

KMnO₄(115 mg, 0.73 mmol) was added and stirring was continued

at 50 °C. After 90 min, additional KMnO₄(115 mg, 0.73 mmol) was

added and stirring was continued at 50 °C. After 90 min, a solution of

KMnO₄(230 mg, 1.46 mmol) in acetone (10 mL) was added and

stirring was continued at 50 °C. After 7 h, a solution of KMnO₄(115

mg, 0.73 mmol) in acetone (10 mL) was added and stirring was

continued at 50 °C for 4 h. After cooling, the mixture was ﬁltered, and

the residue was washed with acetone. The combined ﬁltrates were

concentrated. MeOH (100 mL) was added, and the mixture was

ﬁltered. The ﬁltrate was concentrated to aﬀord the crude

triﬂuoroacetamide (180 mg), which was used in the next step.

K₂CO₃(29 mg, 0.21 mmol) was added to a solution of crude 2,2,2-

triﬂuoro-N-{[(2-{[6-(4-ﬂuoro-2-methoxyphenyl)pyrimidin-4-yl]-

amino}pyridin-4-yl)methyl](methyl)oxido-λ⁶-sulfanylidene}-

acetamide (50 mg) in MeOH (9 mL) at rt. The mixture was stirred

for 50 min before H₂O (100 mL) was added. Then, the mixture was

extracted with DCM/i-PrOH (4:1) and EtOAc. The combined

organic layers were ﬁltered using a Whatman ﬁlter and concentrated.

The residue was puriﬁed by preparative HPLC (basic conditions) to

aﬀord 24 (11 mg, 0.03 mmol, 15% yield over two steps).

1

0.027 mmol, 50% yield) as a colorless solid. H NMR (500 MHz,

CDCl₃, 27 °C): δ 8.32 (d, J = 1.59 Hz, 1H), 7.61 (br d, J = 11.13 Hz,

1H), 7.49 (t, J = 7.49 Hz, 1H), 7.19−7.24 (m, 1H), 7.12 (s, 1H), 6.81

(t, J = 8.05 Hz, 1H), 6.76 (d, J = 10.97 Hz, 1H), 6.69 (br d, J = 9.22

Hz, 1H), 3.87 (s, 3H, OCH₃), 3.85 (s, 2H, CH₂NH₂). ESI-HRMS m/

z: [M + H]⁺calcd for C₁₈H₁₆F₃N₄O, 361.1277; found, 361.1276.

Compound 24, Enantiomers 25 and 26 (Scheme 1). 4-Chloro-6-

(4-ﬂuoro-2-methoxyphenyl)pyrimidine (20). Under an atmosphere

of argon, a mixture of 4,6-dichloropyrimidine (18; 5.00 g, 33.56

mmol), (4-ﬂuoro-2-methoxyphenyl)boronic acid (19; 6.27 g, 36.92

mmol), and [Pd(dppf)Cl₂] (1:1 complex with DCM; 2.74 g, 3.36

mmol) in 2 M aq K₂CO₃(50 mL) and DME (101 mL) was stirred for

150 min at 90 °C. After cooling, the mixture was diluted with EtOAc

and washed with dilute aq NaCl solution. The organic layer was

ﬁltered using a Whatman ﬁlter and concentrated. The residue was

puriﬁed by chromatography (hexane/EtOAc 5 to 40%) to aﬀord 20

1

(6.35 g, 26.61 mmol, 79% yield). H NMR (400 MHz, CDCl₃, 27

°C): δ 9.00 (m, 1H), 8.13 (m, 1H), 8.02 (m, 1H), 6.82 (m, 1H), 6.75

(m, 1H), 3.94 (s, 3H). UPLC (ESI+) m/z: [M + H]⁺239.

6-(4-Fluoro-2-methoxyphenyl)-N-{4-[(methylsulfanyl)methyl]-

pyridin-2-yl}pyrimidin-4-amine (22). A mixture of 4-chloro-6-(4-

ﬂuoro-2-methoxyphenyl)pyrimidine (20; 475 mg, 1.99 mmol), 4-

[(methylsulfanyl)methyl]pyridin-2-amine (21; 614 mg, 3.98 mmol;

UkrOrgSynthesis Ltd.), Xantphos (51 mg, 0.09 mmol), and Cs₂CO₃

(972 mg, 2.99 mmol) in 1,4-dioxane (7.1 mL) was degassed using

argon. Pd₂(dba)₃(27 mg, 0.03 mmol) was added under argon, and

R

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6-(4-Fluoro-2-methoxyphenyl)-N-{4-[(S-methylsulfonimidoyl)-

methyl]pyridin-2-yl}pyrimidin-4-amine [Enantiomer 1 (25) and

Enantiomer 2 (26)]. (rac)-6-(4-Fluoro-2-methoxyphenyl)-N-{4-[(S-

methylsulfonimidoyl)methyl]pyridin-2-yl}pyrimidin-4-amine (24; 33

mg) was separated into the enantiomers by chiral preparative HPLC

[Sepiatec Prep SFC 100 system; column: CHIRALPAK IA 5 μm, 250

× 20 mm; eluent: CO₂/MeOH (+0.4% diethylamine) 6:4; ﬂow: 80

mL/min; pressure (outlet): 150 bar; temperature: 40 °C; solution: 33

mg in 2 mL DMF; detection: UV 254 nm].

Enantiomer 1 (25): t_R= 2.90−3.50 min. Yield: 12 mg, 0.03 mmol.

Enantiomer 2 (26): t_R= 3.65−4.50 min. Yield: 13 mg, 0.03 mmol.

¹H NMR (300 MHz, DMSO-d₆): δ 10.27 (s, 1H, aniline−NH), 8.71

(d, J = 1.13 Hz, 1H, pyrimidine-2-H), 8.30−8.32 (m, 1H), 8.28 (d, J

= 4.89 Hz, 1H), 8.00 (dd, J = 8.76, 7.25 Hz, 1H), 7.83 (s, 1H), 7.01−

7.10 (m, 2H), 6.88 (td, J = 8.43, 2.54 Hz, 1H), 4.39 (s, 2H,

sulfoximine−CH₂), 3.89 (s, 3H, OCH₃), 2.86 (s, 3H, sulfoximine−

CH₃). UPLC-MS (basic): t_R= 1.14 min; MS (ESI+) m/z: [M + H]⁺

388.2. ESI-HRMS m/z: [M + H]⁺calcd for C₁₈H₁₉FN₅O₂S,

388.1244; found, 388.1241.

Whatman ﬁlter and concentrated to aﬀord crude 30, which was used

without further puriﬁcation. UPLC (ESI+) m/z: [M + H]⁺485.

(rac)-5-Fluoro-4-(4-ﬂuoro-2-methoxyphenyl)-N-{4-[(S-

methylsulfonimidoyl)methyl]pyridin-2-yl}pyridin-2-amine (31).

Acetone (6.0 mL) and KMnO₄(814 mg, 5.15 mmol) were added

to the residue of the previous step (crude 30), and the mixture was

stirred at 50 °C for 90 min. Additional KMnO₄(223 mg, 1.42 mmol)

was added and stirring was continued at 50 °C for 4 h. Finally,

additional KMnO₄(305 mg, 1.93 mmol) was added and stirring was

continued at 50 °C for 150 min. After cooling, the mixture was

ﬁltered, the residue was washed with acetone, and the combined

ﬁltrates were concentrated. The residue was dissolved in MeOH (60

mL), K₂CO₃(182 mg, 1.32 mmol) was added, and the reaction

mixture was stirred for 20 min at rt. The mixture was diluted with aq

NaCl solution and extracted with DCM (3×). The combined organic

phases were ﬁltered using a Whatman ﬁlter and concentrated. The

residue was puriﬁed by preparative HPLC (basic conditions) to aﬀord

1

31 (50 mg, 0.12 mmol, 10% yield over three steps). H NMR (400

MHz, CDCl₃, 27 °C): δ 8.22 (d, J = 5.31 Hz, 1H), 8.13 (d, J = 1.52

Hz, 1H), 7.80 (s, 1H, aniline−NH), 7.46 (d, J = 5.05 Hz, 1H), 7.25−

7.30 (m, 2H), 6.92 (d, J = 5.05 Hz, 1H), 6.71−6.82 (m, 2H), 4.36 (d,

J = 12.63 Hz, 1H, sulfoximine−CH₂, H_A, AB system), 4.25 (d, J =

12.88 Hz, 1H, sulfoximine−CH₂, H_B, AB system), 3.83 (s, 3H,

OCH₃), 3.00 (s, 3H, sulfoximine−CH₃). ¹³C NMR (100.67 MHz,

Compound 31, Enantiomers 32 and VIP152 (Scheme 2). 2-

Chloro-5-ﬂuoro-4-(4-ﬂuoro-2-methoxyphenyl)pyridine (28). A mix-

ture of 2-chloro-5-ﬂuoro-4-iodopyridine (27; 1.00 g, 3.88 mmol,

APAC Pharmaceutical, LLC), (4-ﬂuoro-2-methoxyphenyl)boronic

acid (19; 660 mg, 3.88 mmol), and Pd(PPh₃)₄(449 mg, 0.38

mmol) in DME (10.0 mL) and 2 M aq K₂CO₃(5.8 mL) was degassed

using argon. The mixture was stirred under an argon atmosphere for 4

h at 100 °C. After cooling, the mixture was diluted with EtOAc and

THF and washed with brine. The organic phase was ﬁltered using a

Whatman ﬁlter and concentrated. The residue was puriﬁed by column

chromatography (hexane to hexane/EtOAc 50%) to aﬀord 28 (947

1

3

DMSO-d₆): δ 163.69 (d, J_CF= 245.41 Hz, 1C_q), 157.92 (d, J_CF

=

1

10.17 Hz, 1C_q), 154.4 (s, 1C_q), 151.88 (d, J_CF= 254.84 Hz, 1C_q),

4

150.82 (d, J_CF= 2.12 Hz, 1C_q), 147.31 (s, 1CH), 140.63 (s, 1C_q),

134.74 (d, ²J_CF= 15.26 Hz, 1C_q), 134.20 (d, ²J_CF= 26.70 Hz, 1CH),

3

4

131.59 Hz (d, J_CF= 10.17 Hz, 1CH), 118.82 (d, J_CF= 2.97 Hz,

1C_q), 118.04 (s, 1CH), 113.42 (d, ²J_CF= 43.23 Hz, 1CH), 113.18 (s,

1CH), 107.15 (d, ³J_CF= 22.04 Hz, 1CH), 100.16 (d, ²J_CF= 25.86 Hz,

1CH), 67.18 (s, 1CH₂), 56.25 (s, 1CH₃), 41.45 (s, 1CH₃). IR

(diamond ATR, cm⁻¹): 3371 (w), 3337 (m), 3289 (br s), 3020 (m),

3005 (m), 2918 (m), 2905 (m), 1622 (m), 1607 (s), 1535 (m), 1481

(m), 1416 (m), 1398 (s), 1283 (m), 1248 (m), 1196 (s), 1030 (s),

953 (s), 829 (s). UPLC (ESI+) m/z: [M + H]⁺405.

1

mg, 3.70 mmol, 95% yield). H NMR (400 MHz, CDCl₃, 27 °C): δ

8.27 (m, 1H), 7.33 (m, 1H), 7.24 (m, 1H), 6.75 (m, 2H), 3.83 (s,

3H). UPLC (ESI+) m/z: [M + H]⁺256.

5-Fluoro-4-(4-ﬂuoro-2-methoxyphenyl)-N-{4-[(methylsulfanyl)-

methyl]pyridin-2-yl}pyridin-2-amine (29). A mixture of 2-chloro-5-

ﬂuoro-4-(4-ﬂuoro-2-methoxyphenyl)pyridine (28; 400 mg, 1.57

mmol), 4-[(methylsulfanyl)methyl]pyridin-2-amine (21; 483 mg,

3.13 mmol), Xantphos (40 mg, 0.07 mmol), and Cs₂CO₃(765 mg,

2.35 mmol) in 1,4-dioxane (6.0 mL) was degassed using argon.

Pd₂(dba)₃(21 mg, 0.02 mmol) was added under argon, and the

mixture was stirred in a closed pressure tube for 5 h at 100 °C. After

cooling, the mixture was diluted with aq NaCl solution and extracted

with DCM (3×). The combined organic phases were ﬁltered using a

Whatman ﬁlter and concentrated. The residue was puriﬁed by

chromatography (hexane to hexane/EtOAc 30%) to aﬀord 29 (556

(R)-5-Fluoro-4-(4-ﬂuoro-2-methoxyphenyl)-N-{4-[(S-

methylsulfonimidoyl)methyl]pyridin-2-yl}pyridin-2-amine (32) and

(S)-5-Fluoro-4-(4-ﬂuoro-2-methoxyphenyl)-N-{4-[(S-

methylsulfonimidoyl)methyl]pyridin-2-yl}pyridin-2-amine (VIP152).

Separation of the two enantiomers of (rac)-31 (3.468 g) via chiral

preparative HPLC yielded 32 and VIP152 [Sepiatec Prep SFC 100

system; column: CHIRALPAK IC 5 μm, 250 × 30 mm; eluent: CO₂/

i-PrOH (+0.4% diethylamine) 7:3; ﬂow: 100 mL/min; pressure

(outlet): 150 bar; temperature: 40 °C; solution: 3.468 g in 55 mL

DCM/MeOH 2:1; detection: UV 254 nm].

1

mg, 1.48 mmol, 94% yield). H NMR (300 MHz, CDCl₃): δ 8.12−

(R)-Enantiomer (32): t_R= 7.0−8.1 min. Yield: 1.31 g, 3.24 mmol.

[α]_D−10.5 0.21 (c 1.00, DMSO).

8.19 (m, 2H), 7.61 (d, J = 5.09 Hz, 1H), 7.36−7.46 (m, 2H), 7.24−

7.31 (m, 1H), 6.71−6.84 (m, 3H), 3.82 (s, 3H, OCH₃), 3.61 (s, 2H,

SCH₂), 2.03 (s, 3H, SCH₃). UPLC (ESI+) m/z: [M + H]⁺374.

(rac)-2,2,2-Triﬂuoro-N-{[(2-{[5-ﬂuoro-4-(4-ﬂuoro-2-

methoxyphenyl)pyridin-2-yl]amino}pyridin-4-yl)methyl](methyl)-

λ⁴-sulfanylidene}acetamide (30). Under argon, a solution of 2,2,2-

triﬂuoroacetamide (195 mg, 1.73 mmol) in 1,4-dioxane (0.5 mL) was

added dropwise to a solution of sodium tert-butoxide (111 mg, 1.15

mmol) in 1,4-dioxane (0.6 mL), so that the temperature of the

mixture remained below 10 °C. Subsequently, a freshly prepared

solution of 1,3-dibromo-5,5-dimethylhydantoin (247 mg, 0.86 mmol)

in 1,4-dioxane (0.6 mL)/THF (1.0 mL) was added dropwise to the

stirred mixture, so that the temperature of the mixture remained

below 10 °C. Then, the mixture was stirred for 10 min at 10 °C.

Finally, a solution of 5-ﬂuoro-4-(4-ﬂuoro-2-methoxyphenyl)-N-{4-

[(methylsulfanyl)methyl]pyridin-2-yl}pyridin-2-amine (29; 430 mg,

1.15 mmol) in 1,4-dioxane (1.0 mL) was added dropwise to the

stirred mixture, so that the temperature of the mixture remained

below 10 °C. The mixture was stirred for 60 min at 10 °C. The

mixture was diluted with toluene (2.0 mL) under cooling and a

solution of sodium sulﬁte (145 mg, 1.15 mmol) in H₂O (2.0 mL) was

added so that the temperature of the mixture remained below 15 °C.

Aq NaCl solution was added, and the mixture was extracted with

EtOAc (3×). The combined organic phases were ﬁltered using a

(S)-Enantiomer (VIP152): t_R= 8.5−10.5 min. Yield: 1.32 mg, 3.26

mmol. ESI-HRMS m/z: [M + H]⁺calcd for C₁₉H₁₉F₂N₄O₂S,

405.1198; found, 405.1196. [α]_D+11.4 0.13 (c 1.00, DMSO).

Pharmacology. Kinase Assays. The inhibitory activities of the

compounds against CDK2/CycE and CDK9/CycT1 were measured

in TR-FRET-based kinase activity inhibition assays using the

biotinylated peptide biotin-Ttds-YISPLKSPYKISEG (C-terminus in

amide form, JPT Peptide Technologies) as the substrate. Recombi-

nant fusion proteins of GST and human CDK2 and of GST and

human CycE, expressed in insect cells (Sf9) and puriﬁed by

glutathione Sepharose aﬃnity chromatography, were purchased

from ProQinase GmbH (product no. 0050-0055-1). Recombinant

full-length His-tagged human CDK9 and CycT1, expressed in insect

cells and puriﬁed by Ni−NTA aﬃnity chromatography, were

purchased from Life Technologies (cat. no. PV4131).

For the assays, 50 nL of a 100-fold concentrated solution of the test

compound in DMSO was pipetted into a black, low-volume, 1536-

well microtiter plate (Greiner Bio-One), 2 μL of enzyme solution in

aqueous assay buﬀer [50 mM Tris HCl pH 8.0, 10 mM MgCl₂, 1.0

mM dithiothreitol, 0.1 mM sodium orthovanadate, 0.01% (v/v)

Nonidet P-40 (Sigma)] was added, and the mixture was incubated for

15 min at 22 °C to allow prebinding of the test compound to the

S

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enzyme before the start of the kinase reaction. Then, the kinase

reaction was started by the addition of 3 μL of a solution of ATP

[ﬁnal concn: 10 μM (=low) or 2 mM (=high)] and substrate (ﬁnal

concn: 0.75 μM) in assay buﬀer, and the resulting mixture was

incubated for a reaction time of 25 min at 22 °C. The concentrations

of the enzymes were adjusted depending on the activity of the

enzyme, lot, and ATP concentration and were chosen appropriate to

have the assays in the linear range. Typical concentrations were in the

range of 10−100 ng/mL for CDK2 and 0.5−1 μg/mL for CDK9. The

reaction was stopped by the addition of 5 μL of a solution of TR-

FRET detection reagents [0.2 μM streptavidin-XL665 (Cisbio

Bioassays), 1 nM anti-RB (pSer807/pSer811)-antibody (BD

Pharmingen, # 558389), and 1.2 nM LANCE Eu-W1024 labeled

anti-mouse IgG antibody (PerkinElmer, product no. AD0077)] in an

aqueous ethylenediaminetetraacetic acid (EDTA) solution [100 mM

EDTA, 0.2% (w/v) bovine serum albumin in 100 mM N-(2-

hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES), pH 7.5].

The resulting mixture was incubated 1 h at 22 °C to allow the

formation of complex between the phosphorylated biotinylated

peptide and the detection reagents. Subsequently, the amount of

phosphorylated substrate was evaluated by measurement of the

resonance energy transfer from the europium chelate to the

streptavidin-XL. Therefore, the ﬂuorescence emissions at 620 and

665 nm after excitation at 350 nm were measured with a PHERAstar

FS reader (BMG Labtech). The ratio of the emissions at 665 and 620

nm was taken as the measure for the amount of phosphorylated

substrate.

monolayers with a TEER of at least 400 Ω·cm²were used. Test

compounds were predissolved in DMSO and added either to the

apical or basolateral compartment at a ﬁnal concentration of 2 μM.

Evaluation was done in triplicate. Before and after incubation for 2 h

at 37 °C, the samples were taken from both compartments and

analyzed, after precipitation with MeOH, by LC−MS/MS. The

apparent permeability coeﬃcient (P_app) was calculated both for the

apical to basolateral (A → B) and the basolateral to apical (B → A)

direction using the following equation: P_app= (V_r/P₀)(1/S)(P₂/t),

where V_ris the volume of medium in the receiver chamber, P₀is the

measured peak area of the test compound in the donor chamber at t =

0, S is the surface area of the monolayer, P₂is the measured peak area

of the test compound in the acceptor chamber after incubation for 2

h, and t is the incubation time. The eﬄux ratio basolateral (B) to

apical (A) was calculated by dividing P_appB−A by P_appA−B.

In Vitro Metabolic Stability in Liver Microsomes. The in vitro

metabolic stability of test compounds was determined by incubation

at 1 μM in a suspension of liver microsomes in 100 mM phosphate

buﬀer pH 7.4 (NaH₂PO₄·H₂O + Na₂HPO₄·2H₂O) and at a protein

concentration of 0.5 mg/mL at 37 °C. The microsomes were

activated by adding a cofactor mix containing 8 mM glucose-6-

phosphate, 4 mM MgCl₂, 0.5 mM nicotinamide adenine dinucleotide

phosphate, and 1 IU/mL glucose-6-phosphate dehydrogenase in

phosphate buﬀer pH 7.4. The metabolic assay was started shortly

afterward by adding the test compound to the incubation at a ﬁnal

volume of 1 mL. The organic solvent in the incubations was limited to

≤0.01% DMSO and ≤1% MeCN. During incubation, the microsomal

suspensions were continuously shaken at 580 rpm and aliquots were

taken at 2, 8, 16, 30, 45, and 60 min, to which an equal volume of cold

MeOH was immediately added. The samples were frozen at −20 °C

overnight and after thawing subsequently centrifuged for 15 min at

3000 rpm. The supernatant was analyzed with an Agilent 1200 HPLC

system with LC−MS/MS detection. The half-life of a test compound

was determined from the concentration−time plot. From the half-life,

the intrinsic clearances and the hepatic in vivo blood clearance (CL)

and maximal oral bioavailability (F_max) were calculated using the

“well-stirred” liver model³⁵together with the additional parameter

liver blood ﬂow, speciﬁc liver weight, and microsomal protein content.

The following parameter values were used for rats: liver blood ﬂow:

4.2 L/h/kg; speciﬁc liver weight: 32 g/kg body weight; and

microsomal protein content: 40 mg/g.

In Vitro Metabolic Stability in Hepatocytes. Hepatocytes from

Han/Wistar rats were isolated via a two-step perfusion method. After

perfusion, the liver was carefully removed from the rat: the liver

capsule was opened and the hepatocytes were gently shaken out into a

Petri dish with ice-cold Williams’ medium E (WME). The resulting

cell suspension was ﬁltered through sterile gauze into 50 mL Falcon

tubes and centrifuged at 50g for 3 min at rt. The cell pellet was

resuspended in WME (30 mL) and centrifuged twice through a

Percoll gradient at 100g. The hepatocytes were washed again with

WME and resuspended in the medium containing 5% FCS. Cell

viability was determined by trypan blue exclusion. For the metabolic

stability assay, liver cells were distributed in WME containing 5% FCS

to glass vials at a density of 1.0 × 10⁶vital cells/mL. The test

compound was added to a ﬁnal concentration of 1 μM. During

incubation at 37 °C, the hepatocyte suspensions were continuously

shaken at 580 rpm, and aliquots were taken at 2, 8, 16, 30, 45, and 90

min, to which an equal volume of cold MeCN was immediately

added. The samples were frozen at −20 °C overnight and after

thawing subsequently centrifuged for 15 min at 3000 rpm. The

supernatant was analyzed with an Agilent 1200 HPLC system with

LC−MS/MS detection. The half-life of a test compound was

determined from the concentration−time plot. From the half-life,

the intrinsic clearances and the hepatic in vivo blood clearance (CL)

and maximal oral bioavailability (F_max) were calculated using the

“well-stirred” liver model⁴⁰together with the additional parameters

liver blood ﬂow, speciﬁc liver weight, and amount of liver cells in vivo

and in vitro. The same parameters for liver blood ﬂow and speciﬁc

liver weight as given above were used; liver cells in vivo: 1.1 × 10⁸

cells/g liver, liver cells in vitro: 1.0 × 10⁶/mL.

The compounds were tested on the same microtiter plate at 11

diﬀerent concentrations in the range of 20 μM to 0.07 nM (dilution

series prepared separately, before the assay, on the level of the 100-

fold concentrated solutions in DMSO by serial dilutions) in duplicate

values for each concentration. IC₅₀values were calculated using

Genedata Screener software.

Merck Millipore CDK Assays. Assays were performed according to

the Merck Millipore KinaseProﬁler standard protocols with an ATP

concentration of 10 μM.

Proliferation Assays. A2780 human ovarian tumor cells (#

93112519) were obtained from the European Collection of

Authenticated Cell Cultures (ECACC) of Public Health England

(Salisbury, UK) and MOLM-13 human AML cells (ACC 554) were

obtained from the Deutsche Sammlung von Mikroorganismen und

Zellkulturen GmbH (DSMZ, German Collection of Microorganisms

and Cell Cultures, Braunschweig). Authentication of cell lines was

conducted at the DSMZ via polymerase chain reaction (PCR)-based

DNA proﬁling of polymorphic short tandem repeats. Cells were

propagated under the suggested growth conditions in a humidiﬁed 37

°C incubator. Proliferation assays were conducted in 96-well plates at

densities of 2500 (A2780) and 5000 (MOLM-13) cells per well in the

growth medium containing 10% fetal calf serum (FCS). Cells were

treated in quadruplicate with serial dilutions of test compounds for 96

h. Relative cell numbers were quantiﬁed by crystal violet staining

(A2780)³⁹or CellTiter-Glo luminescent cell viability assay (Prom-

ega) (MOLM-13). IC₅₀values (inhibitory concentration at 50% of

maximal eﬀect) were determined by means of a four-parameter ﬁt on

measurement data, which were normalized to vehicle (DMSO)-

treated cells (=100%) and measurement readings taken immediately

before compound exposure (=0%).

PK Studies. Caco-2 Permeability Assay. Caco-2 cells were seeded

at a density of 2.5 × 10⁵cells/well on 24-well insert plates, 0.4 μm

pore size, 0.3 cm²(CoStar), and grown for 13−15 days in DMEM

supplemented with 10% FCS, 1% GlutaMAX (100×, Gibco), 100 U/

mL penicillin, 100 μg/mL streptomycin (Gibco), and 1% nonessential

amino acids (100×). Cells were maintained at 37 °C in a humidiﬁed

5% CO₂atmosphere. The medium was changed every 2−3 days.

The bidirectional transport assay for the evaluation of Caco-2

permeability was carried out in 24-well insert plates using a robotic

system (Tecan). Before the assay was run, the culture medium was

replaced by the transport medium (FCS-free HEPES carbonate

transport buﬀer pH 7.2). For the assessment of monolayer integrity,

the transepithelial electrical resistance (TEER) was measured. Only

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Plasma Protein Binding. HT equilibrium dialysis was used to

determine the plasma protein binding, as outlined by Banker et al.⁴¹

In brief, a semipermeable membrane separated the plasma from the

buﬀer compartment. The test compound was added to the plasma

side at a concentration of 3 μM and incubated for 7 h at 37 °C and

5% CO₂with 99% humidity and moderate shaking. Then, 10 μL of

the plasma side was transferred to a deep-well plate containing 90 μL

of buﬀer and 90 μL of the buﬀer side was added to 10 μL of blank

plasma. All samples were precipitated with 400 μL ice-cold MeOH

and frozen overnight at −20 °C. After thawing and mixing, the

samples were centrifuged for 10 min at 3000 rpm. The supernatant

was transferred to a 96-well plate, and LC−MS/MS measurements

were undertaken. From the quotient of buﬀer and plasma

concentration, the unbound fraction (f_u) was calculated.

CYP Inhibition Assay. The inhibitory potency of test compounds

toward cytochrome P450-dependent metabolic pathways was

determined in human liver microsomes (pool) by applying individual

CYP isoform selective standard probes (CYP1A2, phenacetin;

CYP2A6, coumarin; CYP2B6, bupropion; CYP2C8, amodiaquine;

CYP2C9, diclofenac; CYP2C19, (S)-mephenytoin; CYP2D6, dextro-

methorphan; CYP2E1, chlorzoxazone; and CYP3A4, midazolam,

testosterone). Reference inhibitors were included as positive controls.

Incubation conditions (protein and substrate concentration and

incubation time) were optimized with regard to linearity of metabolite

formation. Assays were processed in 96-well plates at 37 °C using a

Freedom EVO Workstation (Tecan, Crailsheim, Germany). The

reactions were stopped by the addition of MeCN (100 μL) containing

the respective stable labeled internal standard. Precipitated proteins

were removed by centrifugation of the well plate (1800 rcf, 10 min, 10

°C). Metabolite formation was quantiﬁed by LC−MS/MS analysis

(QTRAP 6500 system, Sciex, Canada), followed by inhibition

evaluation and IC₅₀calculation.

concentrations in plasma and blood. The PK parameters calculated

from the concentration−time proﬁles after ig administration are as

follows: C_max, maximal plasma concentration (in mg/L); C_max,norm

,

C

_maxdivided by the administered dose (in kg/L); and T_max, time point

at which C_maxwas observed (in h). The parameters calculated from

both iv and ig concentration−time proﬁles are as follows: AUC_norm

,

area under the concentration−time curve from t = 0 to inﬁnity

(extrapolated) divided by the administered dose (in kg·h/L);

AUC_{(0−t last)norm}, area under the concentration−time curve from t =

0 to the last time point for which plasma concentrations could be

measured divided by the administered dose (in kg·h/L); t_1/2, apparent

half-life (in h); and F, oral bioavailability, AUC_normafter ig

administration divided by AUC_normafter iv administration (in %).

Physicochemical Assays. Aqueous Solubility of Compound−

DMSO Solutions. Aqueous solubility at pH 6.5 was determined by an

orientating HTS method.³²Test compounds were applied as 1 mM

DMSO solutions. After addition of buﬀer pH 6.5, the solutions were

shaken for 24 h at rt. The undissolved material was removed by

ﬁltration. The compound dissolved in the ﬁltrate was quantiﬁed by

HPLC−MS/MS.

Equilibrium Shake Flask Solubility Assay. The thermodynamic

solubility of compounds in water was determined by an equilibrium

shake ﬂask method.⁴²A saturated solution of the test compound was

prepared, and the solution was mixed for 24 h to ensure that

equilibrium was reached. The solution was centrifuged to remove the

insoluble fraction, and the concentration of the compound in solution

was determined using a standard calibration curve. To prepare the test

sample, solid compound (2 mg) was weighed in a 4 mL glass vial.

Phosphate buﬀer pH 6.5 (1 mL) was added, and the suspension was

stirred for 24 h at rt. Then, the solution was centrifuged. To prepare

the sample for the standard calibration, solid sample (2 mg) was

dissolved in MeCN (30 mL). After sonication, the solution was

diluted with H₂O to 50 mL. Sample and standard were quantiﬁed by

HPLC with UV detection. For each test sample, two injection

volumes (5 and 50 μL) in triplicate were made. Three injection

volumes (5, 10, and 20 μL) were made for the standard. HPLC

conditions: column: XTerra MS C18 2.5 μm, 4.6 × 30 mm; injection

volume: sample: 3 × 5 μL and 3 × 50 μL, standard: 5, 10, and 20 μL;

ﬂow: 1.5 mL/min; acidic gradient: eluent A: H₂O + 0.01% TFA,

eluent B: MeCN + 0.01% TFA; 0 min 5% B, 0−3 min 65% B linear

gradient, 3−5 min 65% B isocratic, 5−6 min 5% B isocratic; and UV

detection: wavelength near the absorption maximum (between 200

and 400 nm). The areas of sample and standard injections, as well as

the calculation of the solubility values (in mg/L), were determined

using Waters Empower 2 FR software.

CYP Induction Assay. To evaluate the CYP induction potential in

vitro, cultured human hepatocytes from three separate livers were

treated once daily for three consecutive days with vehicle control, one

of eight concentrations of test compound, or known human CYP

inducers (e.g., omeprazole, phenobarbital, and rifampin). After

treatment, the cells were incubated in situ with the appropriate

marker substrates for the analysis of CYP3A4, CYP2B6, and CYP1A2

activity by LC−MS/MS. Following the in situ incubation, the same

hepatocytes from the same treatment groups were harvested for RNA

isolation and analyzed by qRT-PCR to assess the eﬀect of test

compound on CYP1A2, CYP2B6, and CYP3A4 mRNA expression

levels.

In Vivo PK Studies. All animal experiments were conducted in

accordance with the German Animal Welfare Law and were approved

by local authorities. For in vivo PK experiments, test compounds were

administered to male Wistar rats intravenously at doses of 0.3−0.5

mg/kg and intragastrically at doses of 0.6−1 mg/kg formulated as

solutions using solubilizers such as poly(ethylene glycol) 400 (PEG

400) in well-tolerated amounts. For PK after iv administration, the

test compounds were given as iv bolus and blood samples were taken

at 2 min, 8 min, 15 min, 30 min, 45 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 24

h after dosing. For PK after intragastral (ig) administration, the test

compounds were given ig to fasted rats and blood samples were taken

at 5 min, 15 min, 30 min, 45 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h after

dosing. Blood was collected into lithium heparin tubes (Monovette,

Sarstedt) and centrifuged for 15 min at 3000 rpm. An aliquot of 100

μL from the supernatant (plasma) was taken and precipitated by the

addition of cold MeCN (400 μL). The samples were frozen at −20

°C overnight and subsequently thawed and centrifuged at 3000 rpm, 4

°C for 20 min. Aliquots of the supernatants were analyzed using an

Agilent 1200 HPLC system with LC−MS/MS detection. PK

parameters were based on the plasma concentration and time data

and calculated (e.g., using the linear-log trapezoidal rule for AUC

estimation) using an Excel-based program. The PK parameters

derived from the concentration−time proﬁles after iv administration

are as follows: CL_plasma, total plasma clearance of test compound (in

L/h/kg); CL_blood, total blood clearance of test compound, and

CL_plasma× C_p/C_b(in L/h/kg) with C_p/C_bbeing the ratio of

log D Measurement. log D values at pH 7.5 were recorded using

an indirect method for determining hydrophobicity constants by

reversed-phase HPLC.⁴³A homologous series of n-alkan-2-ones (C₃−

C₁₆, 0.02 M in MeCN) was used for calibration. Test compounds

were applied as 0.67 mM DMSO stock solutions in MeCN/H₂O

(1:1). The lipophilicity of compounds was then assessed by

comparison to the calibration curve.

In Vivo Pharmacology Studies. MOLM-13 Human AML

Models in Mice and Rats. All animal experiments were conducted

in accordance with the German Animal Welfare Law and were

approved by local authorities. MOLM-13 human AML cells were

obtained from the DSMZ. Cell lines were authenticated at the DSMZ

via short-tandem repeat DNA ﬁngerprinting before use in experiments

to rule out potential cross-contamination among cell lines. All cell

lines were tested as being free from mycoplasma contamination using

MycoAlert (Lonza) at least every four passages and before a cell line

substock was generated.

For the AML mouse model, 2 × 10⁶MOLM-13 human AML cells

were suspended in 100% Matrigel basement membrane matrix (BD

Biosciences) and inoculated subcutaneously to female NMRI nu/nu

mice (18−21 g, 5−6 weeks, Taconic M&B). For the AML models in

rats, 2 × 10⁶MOLM-13 or MV4-11 human AML cells were

suspended in 100% Matrigel and inoculated subcutaneously to female

athymic nude rats (120−160 g, 5−6 weeks, Harlan). Animals were

stratiﬁed into treatment and control groups (n = 12/group) based on

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primary tumor size. For the MOLM-13 models, treatments were

started 5−6 days after tumor cell inoculation when the average tumor

sizes were 39−41 and 73 mm³for mice and rats, respectively. For the

MV4-11 models, treatments were started on day 13 when the tumor

area was approximately 40 mm².

ASSOCIATED CONTENT

■

sı

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c01000.

BAY-332 was administered once daily (QD, 15 mg/kg), once every

3 days (Q3D, 35 mg/kg), or once every 5 days (Q5D, 65 mg/kg).

VIP152 was administered once weekly (QW, 12.5 mg/kg) for the

MOLM-13 model in mice and Q3D (3 mg/kg), Q5D (3.75 mg/kg),

or QW (4.5 mg/kg) for the MOLM-13 model in rats. PEG 400/

EtOH/H₂O (30:10:60) or PEG 400/H₂O (80:20) was used as the

vehicle control. Unless otherwise indicated, all treatments were

administered intravenously and were continued until the end of the

experiment. Along with body weights, tumors were measured at least

twice weekly by caliper, and the volume was calculated using the

formula (length × width²)/2. Treatment-to-control (T/C) ratios were

calculated by dividing the mean tumor volume of the treatment group

by the mean tumor volume of the vehicle group at the time point

when the vehicle group was sacriﬁced.

Molecular formula strings (CSV)

Kinase selectivity panel data for VIP152 and LC−MS

chromatograms for determination of purity (PDF)

AUTHOR INFORMATION

■

Corresponding Author

Stuart Hwang − Vincerx Pharma, Inc., Palo Alto, California

94306, United States; Email: stuart.hwang@vincerx.com

Authors

Ulrich Lucking − Pharmaceuticals, Research and

̈

Development, Bayer Pharma AG, Berlin 13353, Germany;

orcid.org/0000-0003-2466-5800

All statistical analyses were performed using the statistical

programming language R.⁴⁴Validity of the model assumptions was

checked for each ﬁtted statistical model.

Dirk Kosemund − Pharmaceuticals, Research and

Development, Bayer Pharma AG, Berlin 13353, Germany

Niels Böhnke − Pharmaceuticals, Research and Development,

Bayer Pharma AG, Berlin 13353, Germany

Philip Lienau − Pharmaceuticals, Research and Development,

Bayer Pharma AG, Berlin 13353, Germany

Gerhard Siemeister − Pharmaceuticals, Research and

Development, Bayer Pharma AG, Berlin 13353, Germany

Karsten Denner − Pharmaceuticals, Research and

Development, Bayer Pharma AG, Berlin 13353, Germany

Rolf Bohlmann − Pharmaceuticals, Research and

Development, Bayer Pharma AG, Berlin 13353, Germany

Hans Briem − Pharmaceuticals, Research and Development,

Bayer Pharma AG, Berlin 13353, Germany

Ildiko Terebesi − Pharmaceuticals, Research and

Development, Bayer Pharma AG, Berlin 13353, Germany

Ulf Bömer − Pharmaceuticals, Research and Development,

Bayer Pharma AG, Berlin 13353, Germany

Analyses for the MOLM-13 model in mice were performed by

ﬁtting a linear mixed eﬀects model on the log-transformed outcome.

The model included both a ﬁxed and random intercepts and slopes

for each group and subject. The data for the MOLM-13 model in rats

were analyzed by group comparisons at the start of the study and on

the last day of the vehicle group. Comparisons were performed based

on a linear model estimated using weighted least squares that included

a separate variance term for each group. Group comparisons were

corrected for false positive rate using Dunnett’s method. In all cases, p

< 0.05 was considered as being statistically signiﬁcant.

Docking. Docking calculations were performed using Glide⁴⁵in

the standard precision mode and default settings. The X-ray complex

of CDK9/CycT1 with a benzimidazole inhibitor (PDB ID: 3MY1)

was used.⁴⁶Prior to the docking procedure, all water molecules and

ligands in the protein−ligand complex were removed. Ligands were

pre-processed using LigPrep.⁴⁷Docking poses were selected based on

Glide scoring.

X-ray Structure Determination of VIP152. Single crystals of

VIP152 were obtained by slow evaporation of an acetone/H₂O

solution of the compound at rt. A single crystal with dimensions 0.14

× 0.08 × 0.02 mm³was mounted on a CryoLoop using inert oil. Data

collection was carried out using a Bruker diﬀractometer equipped with

an APEX II CCD area detector, an IμS microsource with Cu Kα

radiation, mirrors as a monochromator, and a Kryoﬂex low-

temperature device (T = 110 K). The program APEX II v.2011.8-0

was used for data collection and reduction.⁴⁸Absorption correction

and scaling were performed using SADABS.⁴⁹The crystal structure

solution was achieved using direct methods as implemented in

SHELXTL version 6.14⁵⁰and visualized using the XP program.⁵⁰

Missing atoms were subsequently located from diﬀerence Fourier

synthesis and added to the atom list. Least-squares reﬁnement on F²

using all measured intensities was carried out using the program

SHELXTL version 6.14.⁵⁰All non-hydrogen atoms were reﬁned

including anisotropic displacement parameters. Final data collection

and structure reﬁnement parameters: λ = 1.54178 Å, T = 110 K, space

group = P2(1), a = 16.6442(11) Å, b = 8.5659(6) Å, c = 26.1351(16)

Å, β = 99.053(5)°, Z = 8, reﬂections collected = 59,133, independent

reﬂections = 12,958 (R_int= 0.0468), completeness = 97.8%, data-to-

parameter ratio = 12.7, R₁(I > 2σ) = 0.0542, wR₂(all data) = 0.1490,

GOF = 1.032, ﬂack parameter = 0.053(9), and largest diﬀerence peak

and hole = 0.897/−0.278 e Å³. CCDC 2005811 (VIP152) contains

the supplementary crystallographic data for this paper. The data can

be obtained free of charge from The Cambridge Crystallographic

Data Centre via www.ccdc.cam.ac.uk/getstructures.

Martina Schäfer − Pharmaceuticals, Research and

Development, Bayer Pharma AG, Berlin 13353, Germany

Stuart Ince − Pharmaceuticals, Research and Development,

Bayer Pharma AG, Berlin 13353, Germany

Dominik Mumberg − Pharmaceuticals, Research and

Development, Bayer Pharma AG, Berlin 13353, Germany

Arne Scholz − Pharmaceuticals, Research and Development,

Bayer Pharma AG, Berlin 13353, Germany

Raquel Izumi − Vincerx Pharma, Inc., Palo Alto, California

94306, United States; orcid.org/0000-0002-0895-2597

Franz von Nussbaum − Pharmaceuticals, Research and

Development, Bayer Pharma AG, Berlin 13353, Germany

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c01000

Author Contributions

The manuscript was written with contributions from all

authors. All authors have given approval to the ﬁnal version of

the manuscript.

Notes

The authors declare the following competing ﬁnancial

interest(s): U.L., D.K., N.B., A.S., P.L., G.S., K.D., R.B., H.B.,

I.T., U.B., M.S., S.I., D.M., and F.v.N. are or have been

employees and stockholders of Bayer AG.R.I. and S.H. are

employees and stockholders of Vincerx Pharma, Inc.

V

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Journal of Medicinal Chemistry

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Article

(12) Krystof, V.; Baumli, S.; Fu

dependent kinase 9 (CDK9) as a drug target. Curr. Pharm. Des. 2012,

18, 2883−2890.

̈

rst, R. Perspective of cyclin-

ACKNOWLEDGMENTS

■

We thank K. Sauvageot-Witzku, R. Golde, A. Glowczewski, C.

Pakebusch, N. Gallus, M. Brands, K. Ziegelbauer, K. Prelle, and

R. Droschinski for technical support; O. Schenk for HPLC

(13) Anshabo, A. T.; Milne, R.; Wang, S.; Albrecht, H. CDK9: A

comprehensive review of its biology, and its role as a potential target

for anti-cancer agents. Front. Oncol. 2021, 11, 678559.

(14) Galbraith, M. D.; Bender, H.; Espinosa, J. M. Therapeutic

targeting of transcriptional cyclin-dependent kinases. Transcription

2019, 10, 118−136.

separations; S. Grundemann, G. Depke, M. Menke, and D.

̈

Tshitenge for analytical support; and U. Ganzer for the

measurement of physicochemical properties. M. Bergmann and

K. Greenﬁeld are acknowledged for valuable technical support

with the manuscript. Aurexel Life Sciences Ltd. (www.aurexel.

com) is acknowledged for editorial support funded by Bayer

AG.

(15) Eyvazi, S.; Hejazi, M. S.; Kahroba, H.; Abasi, M.; Zamiri, R. E.;

Tarhriz, V. CDK9 as an appealing target for therapeutic interventions.

Curr. Drug Targets 2019, 20, 453−464.

(16) Espinosa, J. M. Transcriptional CDKs in the spotlight.

Transcription 2019, 10, 45−46.

ABBREVIATIONS

■

(17) Lucking, U.; Scholz, A.; Lienau, P.; Siemeister, G.; Kosemund,

̈

ATR, ataxia telangiectasia and Rad3-related protein; CL,

clearance; CL_b, blood clearance; ECACC, European Collection

of Authenticated Cell Cultures; FCS, fetal calf serum; MCL-1,

D.; Bohlmann, R.; Briem, H.; Terebesi, I.; Meyer, K.; Prelle, K.;

Denner, K.; Bömer, U.; Schäfer, M.; Eis, K.; Valencia, R.; Ince, S.; von

Nussbaum, F.; Mumberg, D.; Ziegelbauer, K.; Klebl, B.; Choidas, A.;

induced myeloid leukemia cell diﬀerentiation protein; P_app

,

Nussbaumer, P.; Baumann, M.; Schultz-Fademrecht, C.; Ruhter, G.;

̈

Eickhoff, J.; Brands, M. Identification of atuveciclib (BAY 1143572),

the first highly selective, clinical PTEFb/CDK9 inhibitor for the

treatment of cancer. ChemMedChem 2017, 12, 1776−1793.

(18) Cidado, J.; Boiko, S.; Proia, T.; Ferguson, D.; Criscione, S. W.;

San Martin, M.; Pop-Damkov, P.; Su, N.; Roamio Franklin, V. N.;

Chilamakuri, C. S. R.; D’Santos, C. S.; Shao, W.; Saeh, J. C.; Koch, R.;

Weinstock, D. M.; Zinda, M.; Fawell, S. E.; Drew, L. AZD4573 is a

highly selective CDK9 inhibitor that suppresses MCL-1 and induces

apoptosis in hematologic cancer cells. Clin. Cancer Res. 2020, 26,

922−934.

apparent permeability coeﬃcient; RNA Pol II, RNA polymer-

ase II; PTEFb, positive transcription elongation factor b; Q3D,

every 3 days; Q5D, every 5 days; Q7D, every 7 days; ratHep,

rate hepatocytes; S_w, aqueous solubility; TCI, Tokyo Chemical

Industry; T/C, treatment-to-control; UPLC, ultra-performance

liquid chromatography; V_ss, steady-state volume of distribu-

tion; WME, Williams’ media E

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J. Med. Chem. XXXX, XXX, XXX−XXX

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Changing for the Better: Discovery of the Highly Potent and Selective CDK9 Inhibitor VIP152 Suitable for Once Weekly Intravenous Dosing for the Treatment of Cancer

DOI: 10.1021/acs.jmedchem.1c01000

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