Discovery of the S1P2 Antagonist GLPG2938 (1-[2-Ethoxy-6-(trifluoromethyl)-4-pyridyl]-3-[[5-methyl-6-[1-methyl-3-(trifluoromethyl)pyrazol-4-yl]pyridazin-3-yl]methyl]urea), a Preclinical Candidate for the Treatment of Idiopathic Pulmonary Fibrosis

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Discovery of the S1P2 Antagonist GLPG2938 (1-[2-Ethoxy-6-

(

triﬂuoromethyl)-4-pyridyl]-3-[[5-methyl-6-[1-methyl-3-

triﬂuoromethyl)pyrazol-4-yl]pyridazin-3-yl]methyl]urea), a

(

Preclinical Candidate for the Treatment of Idiopathic Pulmonary

Fibrosis

Oscar Mammoliti,* Adeline Palisse,* Caroline Joannesse, Sandy El Bkassiny, Brigitte Allart, Alex Jaunet,

Christel Menet, Beatrice Coornaert, Kathleen Sonck, Inge Duys, Philippe Cl é ment-Lacroix, Line Oste,

Monica Borgonovi, Emanuelle Wakselman, Thierry Christophe, Nicolas Houvenaghel, Mia Jans,

Bertrand Heckmann, Laurent Saniere, and Reginald Brys

̀

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*

sı Supporting Information

ABSTRACT: Mounting evidence from the literature suggests that blocking S1P2 receptor (S1PR2) signaling could be eﬀective for

the treatment of idiopathic pulmonary ﬁbrosis (IPF). However, only a few antagonists have been so far disclosed. A chemical

enablement strategy led to the discovery of a pyridine series with good antagonist activity. A pyridazine series with improved

lipophilic eﬃciency and with no CYP inhibition liability was identiﬁed by scaﬀold hopping. Further optimization led to the discovery

of 40 (GLPG2938), a compound with exquisite potency on a phenotypic IL8 release assay, good pharmacokinetics, and good

activity in a bleomycin-induced model of pulmonary ﬁbrosis.

INTRODUCTION

■

Sphingolipids are a class of membrane lipids. Apart from their

structural role in membrane biology, they can also act as

signaling messengers. Among them, lysophospholipid sphingo-

sine-1-phosphate (S1P, Figure 1) plays a role in regulating a

wide range of biological processes, including cell growth, cell

1

survival, angiogenesis, and lymphocyte traﬃcking. S1P is

generated from sphingosine by phosphorylation of the C1

hydroxyl group. This process is mediated by sphingosine

kinases (SPHK1 and SPHK2). The intracellular level of S1P is

negatively regulated by S1P phosphatases (SGPP1 and

SGPP2) and by sphingosine-1-phosphate lyase (SPL), which

catalyzes the irreversible degradation of S1P to ethanolamine

phosphate and 2-trans hexadecenal. The intracellular S1P is

exported outside the cell by speciﬁc transporters, including

SPNS2 (Sphingolipid Transporter 2). Extracellular S1P then

signals through ﬁve G coupled protein receptors (GPCRs),

S1P1, S1P2, S1P3, S1P4, and S1P5. S1P1, S1P2, and S1P3 are

Figure 1. Structures of sphingosine-1-phosphate (S1P, 1), FTY720

(2), and its active form, FTY720-P (3).

1b

Received: January 24, 2021

Published: May 3, 2021

©

2021 American Chemical Society

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Figure 2. Examples of published S1P2 antagonists (4−8) and allosteric antagonist CYM-5520 (9).

widely expressed, while the expression of S1P4 and S1P5 is

restricted to particular organs (lymphoid, hematopoietic, and

pulmonary tissue for S1P4 and brain and spleen for S1P5).

has been reported that S1P2-deﬁcient mice showed increased

liver regeneration capacity and ameliorated ﬁbrosis in liver

2

11g

injury models.

S1P is increased in the bronchoalveolar

S1P1 activation promotes the lymphocytes egress from

lymph nodes to sites of inﬂammation. In fact, modulation of

the S1P1 receptor has demonstrated to be eﬀective in multiple

models of auto-immune diseases by depleting the circulating

lavage (BAL) ﬂuid and in plasma of idiopathic pulmonary

ﬁbrosis (IPF) patients; it was also shown that S1P promotes

endothelial to mesenchymal transition (EMT), a hallmark of

11h

ﬁbrosis, in TGFβ-stimulated A549 cells by activation of S1P2

3

11h

lymphocytes. Moreover, Fingolimod (FTY720/Gilenya, com-

and S1P3.

Very recently, it has been shown that S1P2-

pound 2, Figure 1), a functional antagonist of S1P1, has been

approved for relapsing multiple sclerosis. FTY720 is a

deﬁcient mice have reduced inﬂammation and ﬁbrosis markers

in the bleomycin (BLM)-induced pulmonary ﬁbrosis model.

4

12

prodrug, which is activated in vivo by phosphorylation. The

active form (FTY720-P, 3) negatively modulates S1P1 by

acting as an internalizing agonist.

Another account reported that genetic deletion of S1P2

resulted in attenuated lung ﬁbrosis in the BLM mouse model.

These eﬀects were recapitulated by prophylactic administration

of an undisclosed S1P2 antagonist. All this evidence suggests

that blocking S1P-mediated S1P2 signaling may be eﬀective

against ﬁbrotic diseases.

13

On the other hand, FTY720 has also been associated to pro-

5

ﬁbrotic events in preclinical toxicological studies, in animal

6

7

models, and in various in vitro settings. In particular, Sobel et

al. showed that FTY720-P caused a robust stimulation of

extracellular matrix (ECM) deposition and induced ﬁbrotic

It has been shown that in approximately half of the cases

−

/−

S1P2

mice develop cell lymphomas after 1.5−2 years of

8

14

gene expression in normal human lung ﬁbroblasts (NHLF).

age. This eﬀect has been linked to the increase in germinal

center (GC) B cells. Successive research, however, showed

that, in humans, also another Gα13 coupled receptor, P2RY8,

for which no orthologues have been identiﬁed in mice,

promotes growth regulation and local conﬁnement of GC B

These eﬀects were attributed to S1P2 and S1P3 activation.

This was in contrast with a previous report that showed that

33

FTY720-P was not able to displace S1 P from membranes

9

derived from S1P2 overexpressing CHO cells. Sobel and

15

colleagues, however, were able to show FTY720-P-mediated

S1P2 receptor activation with impedance assays in diﬀerent

cell types. FTY720-P also induced an increased ECM synthesis

in myoﬁbroblasts; this eﬀect was ascribed only to S1P2

modulation as expression levels of S1P3 were considerably low

in those cells. Sobel et al. also showed that FTY720-P induced

myoﬁbroblast contraction, a phenomenon associated with

cells, suggesting a possible redundant role for S1P2.

Despite the attractiveness of S1P2 as a target, only few

16

documents describe the optimization of S1P2 antagonists

and there are no reports of clinical studies. This is in stark

contrast with the number of S1P1 modulators that have

17

entered clinical development. Some representative S1P2

antagonists are highlighted in Figure 2. Notably, the ﬁrst report

10

18

ﬁbrosis, in a S1P2-dependent manner.

of synthetic S1P2 antagonists

exempliﬁes the S1P2

These reports came to join many other lines of evidence

linking S1P2 activation to ﬁbrotic diseases. Among those, it

antagonist JTE-013 (compound 4, Figure 2), a compound

that has been widely reported in the literature. Other

11

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noteworthy reports are from Novartis, from Ono Pharma-

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19

cases, the 1H-pyrazolo[3,4-b]pyridine was redeployed, with

diﬀerent substituents.

1

6,20

21

27b,28

ceutical,

and, more recently, from Timothy Hla, who

Synthesis and decoration of fused

describes S1P2 antagonists (e.g., compound 8, Figure 2) whose

bicyclic scaﬀolds may entail long and laborious syntheses and

also reduce the breadth of the explorable chemical space. For

this reason, we opted for designs based on chemical

enablement, which is “the eﬃcient bond disconnection that

facilitates the synthesis of libraries that probe multiple

hypotheses”. To this end, the focus was placed on a new

pyridine scaﬀold, which would allow to introduce a broad

diversity of groups. For ease of synthesis, a methyl group was

introduced instead of the recurring bulkier isopropyl group at

the C4 position (Figure 3). This was also aiming at having a

smaller pharmacophore. The newly introduced substituents

may cause the scaﬀold to tilt in the binding pocket. In that

case, the smaller methyl group could be a better ﬁt for avoiding

potential clashes with the protein.

structures are reminiscent of an allosteric agonist, CYM-5520

2

(

compound 9).

Some of the antagonists so far described may have potential

shortcomings. Novartis reported biological activity data only

for a limited set of compounds, including compounds 5 and 6

29

(

Figure 2). Compound 5 has a high TPSA (125.5) and a low

23

clogD at pH 7.4 (−0.63, SimPlus) combined with the

presence of a carboxylic acid and three other carbonyls. The

combination of these attributes could very well result in low

permeability and, therefore, low oral absorption. On the other

hand, compound 6 features highly lipophilic and ﬂexible

groups, which are potentially prone to oxidative metabolism.

Ono Pharmaceutical has also been active in the ﬁeld.

Compound 7 constitutes an improvement from molecules

previously reported, which were suﬀering from low bioavail-

ability. However, despite good bioavailability in mice and dogs,

Two vectors were chosen for exploration, from the C5 and

C6 position of the pyridine core, respectively. A structurally

diverse set of groups was initially employed, with more than 40

compounds synthesized (a selection is presented in Table 1).

To ensure the most eﬀective receptor blockade, antagonists

able to shut down S1P2 signaling across multiple pathways

1

6c

bioavailability in monkeys was low (12%).

Moreover,

compound 7 possesses three benzene rings and the number

of carboaromatic rings has been shown to correlate negatively

24

30

with drug developability. JTE-013 (compound 4) features a

hydrazine moiety, a functional group that has been associated

with hepatotoxicity and drug-induced systemic lupus eryth-

were sought. To this end, potential antagonists were tested in

a calcium ﬂux assay (Gq involved) in CHO cells over-

expressing the human S1P2 receptor, as well as in a GTPγS

2

5

ematosus. JTE-013 has been extensively used as chemical

probe in cellular assays. However, even though it has been

proved to be a bona ﬁde S1P2 antagonist, some reports call

assay (mainly G involved), also based on the human S1P2

i

receptor. Small substituents, such as dimethylamino or CN,

showed modest potency in both assays (compounds 12−15).

Aromatic groups, on the other hand, showed an improvement,

with compound 19 displaying the best combination of

potencies in the GTPγS and calcium ﬂux assays, when tested

in antagonist mode. None of the compounds showed any

activity when tested in agonist mode (Table 1; this was also

the case for all compounds reported in this manuscript; for this

reason, it will not be mentioned further). Six-membered

aromatic rings (such as 21) were also active. However, the

pyrazole motif was considered more attractive, in virtue of the

reduced lipophilicity and size. The ethyl ester 16 showed

submicromolar potency in the calcium ﬂux assay. Ester groups,

however, are potentially hydrolyzed very quickly by esterases,

making them, most of the times, unsuitable for progression.

For this reason, a few amide replacements were explored

26

into question its selectivity and speciﬁcity.

The biological evidence so far accumulated on the role of

S1P2 in ﬁbrosis prompted us to initiate a drug discovery

campaign aiming at identifying S1P2 antagonists to validate

this therapeutic hypothesis. Moreover, the limitations of

existing S1P2 antagonists served also as a stimulus to provide

improved chemical matter.

RESULTS AND DISCUSSION

■

To ﬁnd new starting points, two consecutive HTS campaigns

were conducted; however, they failed to identify progressable

hits. We therefore decided to focus on a pyridine core to

generate new chemical matter (Figure 3). Some S1P2

antagonists are based on fused bicyclic scaﬀolds, which are

reminiscent of the 1H-pyrazolo[3,4-b]pyridine scaﬀold of the

(

compounds 22−24). These derivatives, however, showed a

27

marked drop in potency. Compounds 16, 17, and 19 were able

to compete with radiolabeled S1P in membranes derived from

stable cell lines overexpressing the human S1P2 receptor. This

indicated that these compounds were most probably

orthosteric antagonists. Again, compound 19 was the most

potent and, in virtue of this, it was selected for further

optimization.

JTE-013 (e.g., compounds 10 and 11 , Figure 3 ). In some

Finding a replacement for the hydrazine urea was the next

focus of the optimization. Some examples from the literature

showed that it was possible to replace the hydrazine linker on

27a

fused bicyclic scaﬀolds. It could not be anticipated, however,

if those replacements would have been compatible with the

pyridine scaﬀold. Designs were guided by the idea of keeping a

hydrogen bond donor and a hydrogen bond acceptor at the

same distance from the core as in the hydrazine-based

molecules showed in Table 1. The hydrogen bond donor

was connected to the core by a benzylic CH instead of an NH

2

spacer (Table 2). Compounds were also proﬁled for liver

microsomal stability. Scaled clearance values, corrected for

microsomal binding (fu, mic), were used (Clint, u, see eqs

Figure 3. Chemical enablement to obtain S1P2 antagonists.

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Table 1. Initial Exploration of the Pyridine Core: LHS

Modiﬁcations

proﬁled in a phenotypic assay based on S1P-stimulated IL8

production in human lung ﬁbroblasts (HFL1). Elevated levels

of IL8, a neutrophilic chemoattractant, have been linked to

32

IPF and S1P has been shown to stimulate IL8 secretion by

33

signaling through S1P2. The majority of the compounds in

Table 2 presented IC values in the IL8 assay in line with the

5

0

GTPγS assay. Compounds 32 and 33 showed the highest shift

5x).

To ameliorate clearance and further improve potency, a

(

broader exploration of the C5 position of the pyridine was

undertaken (Figure 4). To this end, a chemical enablement

strategy based on a urea protecting group was implemented

(

Scheme 5). The protecting group allowed us to perform

certain reactions, such as Buchwald and Ullmann couplings, in

a convergent fashion. The 2-ethoxy-6-(triﬂuoromethyl)pyridin-

4

-amine group was selected for the right-hand side. This group

conferred a level of potency similar to the 2-chloro-6-

ethoxypyridin-4-amine group (cfr 34 and 98, Supporting

Information), but it had also the advantage to be unreactive in

cross-coupling conditions, while the 2-chloro-6-ethoxypyridin-

4

-amine could potentially react on the C2 position. Following

this and other less convergent approaches, some of them

described above, more than 60 analogs were prepared to

broadly investigate the C5 position (the full SAR is shown in

Table S4, Supporting Information). Essentially three chemo-

types were explored: CC-linked (hetero)aryl groups, (hetero)-

alkyl/(hetero)aryl secondary amines, and (hetero)alkyl tertiary

amines (green, orange, and blue, respectively, in Figure 4). The

CC-linked (hetero)aryls and the secondary amines showed

high potency in both calcium ﬂux and GTPγS assays, while the

tertiary amines were in general less potent, especially in the

GTPγS assay. Some of the compounds were proﬁled in the IL8

phenotypic assay and in the microsomal stability assay. Only

few compounds showed a good balance between potency and

clearance. The best compound in this aspect for each

chemotype is shown in Table 3.

The CF group in 34 was an improvement in comparison

3

with the methyl group of matched pair 32, especially in the IL8

phenotypic assay (7x improvement, Table 3). To measure the

phenotypic activity in a more physiological setting, compounds

3

2 and 34 were tested in the presence of 2% human serum

a

b

Assay run in antagonist mode. Assay run in agonist mode.

albumin (HSA, equivalent to approximately 40% serum).

Serum could not be used as it contains S1P. The presence of

HSA could also oﬀset potential binding to plastics and medium

for more lipophilic compounds, leading to a better estimate of

the relevant free IC . The free IC values were obtained

estimating the fraction unbound in the medium (fu, medium)

from the human plasma protein binding (hPPB) according to

eqs 1 and 2 in the Experimental Section. For a set of

compounds, the binding to the assay medium containing HSA

was directly measured and it was in agreement with the

binding calculated from PPB values. For 32 and 34, the

absolute IC₅₀values in the presence of HSA were 3039 and

572 nM, while the free IC₅₀values were 28 and 1.4 nM,

respectively. The improvement for 34 was thus greater than

the 1 log unit increase in lipophilicity.

Also, compounds 35 and 36 showed good potency (with 36

being somewhat weaker in the GTPγS assay) and improved

clearance. However, none of them was superior to 34. The

secondary amines, like 35, showed a similar proﬁle to CC-

linked derivatives, with no apparent advantage to compensate

for the extra H bond donor. Therefore, they were not

investigated further. For the tertiary amines, like 36, the data

c

^E^x^p^e^rⁱ^m^eⁿ^t^s^aⁱ^mⁱⁿ^g^a^t^d^e^t^e^r^mⁱⁿⁱⁿ^g^t^h^e^I^C⁵0 values were performed

at least twice, unless otherwise stated. When multiple experiments

were performed, the geometrical mean is provided. n = 1. IC for

each run (n = 5) in Supporting Information, Table S10. NT = not

tested.

d

e

5

0

5

0

50

34

3

−5, Experimental Section). The benzimidazole-based com-

pounds 25 and 26 showed good potency, but they also showed

high in vitro clearance in mouse liver microsomes (MLMs).

Indoles 27 and 28 possessed good potency, but, also in this

case, the in vitro clearance was too high (Clint, u > 34 L/h/kg).

Amides 29 and 30 resulted in total loss of activity. Urea 31, on

the other hand, displayed submicromolar potency in both

GTPγS and calcium ﬂux assays. A quick exploration of the

aromatic right-hand side led to urea 32, which had improved

potency in comparison with 31. Compound 32 showed also a

31

marked improved LipE (obtained from the calcium ﬂux assay

and clogD). The right-hand side pyridine conferred to 31 a

much greater microsomal stability comparing with the phenyl

ring of matched pair 33 (>10x), highlighting the importance of

this group for metabolic stability. The compounds were also

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Table 2. Linker Optimization in the Pyridine Scaﬀold

a

b

Antagonist mode. Experiments aiming at determining the IC₅values were performed at least twice, unless otherwise stated. When multiple

0

c

d

experiments were performed, the geometrical mean is provided. n = 1. Fraction unbound in microsomes (fu, mic) experimentally determined,

unless otherwise stated. Fu, mic calculated. Precipitation at high dose. NT = not tested.

e

f

indicated that it could be more challenging to obtain a good

balance between potency and clearance (Figure 4). For this

reason, their progression was also put on hold.

retained good potency with lower lipophilicity, thus resulting

in an improved LipE (4.4 vs 5.0). CYP inhibition was

mitigated (recombinant 2C19, 2C9, and 3A4, 61%, 58%, and

23% inhibition at 10 μM, respectively, Table S5, Supporting

Information). Clearance was improved, especially in human

liver microsomes. A broader analysis of matched pairs between

the pyridine and the pyridazine scaﬀolds clearly showed the

latter to be an improvement as all matched pairs had better

LipE (Figure 5).

The new lead 34 displayed strong CYP inhibition

(

recombinant 2C19, 2C9, and 3A4, 100%, 83%, and 83%

inhibition at 10 μM, respectively, Table S5 , Supporting

Information). CYP inhibition was a general feature of the

pyridine series. To remove this liability, and to improve

clearance, a scaﬀold hopping strategy privileging more polar

cores was envisaged. A pyridazine-based scaﬀold was selected

and synthesized. The beneﬁts of the CH to N switch have

The exploration of the right-hand side was the next focus of

the optimization. At ﬁrst, pyridine-4-amines with two

substituents (C2 and C6 positions) were evaluated (37−45,

35

already been reviewed elsewhere. Compound 37 (Table 4)

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hand, more polar groups were poorly tolerated (42 and 45) or

lost all activity (43 and 44).

In a second set of compounds (46−51), the C3 position of

the right-hand side pyridine-4-amine was investigated. This

position was selected in reason of the ortho directing

properties of the alkoxy groups, which allowed for a

regioselective bromination at the C3 position for further

derivatization (Scheme S4, Supporting Information). Methyl

substitution in compounds 46 and 47 resulted in similar

potency in the calcium ﬂux and GTPγS assays comparing with

matched pairs 37 and 39. This change resulted, however, in a

marked increase in potency in the IL8 phenotypic assay (about

1

0x for both compounds). When this change was applied to

4

1, an increase in potency of about 10x in all assays was

36

obtained (compound 48). This “magic methyl” eﬀect was

also accompanied by retention of good metabolic stability

(

human) and improved LipE. A chlorine atom had a very

similar eﬀect for compound 50. Replacement with CN resulted

in total loss of activity (compound 51). Other groups were

investigated, but, in general, they resulted to be worse than the

37

methyl group and chlorine atom.

The exploration of the right-hand side resulted in molecules

with very good potency and microsomal stability. Compounds

3

7, 40, and 48 were therefore selected for pharmacokinetic

studies in preclinical species. The potent compound 47 was

excluded because it showed tolerability issues after single dose

administration (both iv and po) in mice. Compound 37

showed low to moderate half-life across species and low

bioavailability in general (Table 5). Therefore, it was not

progressed further, also considering its low solubility.

Compound 40, on the other hand, showed good pharmaco-

kinetics, with long half-life, low clearance, and good

bioavailability in all species, especially in dogs. Plasma protein

binding was quite high; from the PK data, however, a free

concentration covering the IL8 IC₅₀was attainable. Although

solubility was low both in FaSSGF and FaSSIF media, this did

not forbid to obtain good bioavailability with simple

formulations, at least at low doses in mice and dogs.

Compound 48, on the other hand, showed shorter half-life

and higher clearance in all species comparing with 40, while

having a markedly increased solubility in the FaSSGF medium

(>200x). This improvement resulted most probably from the

basicity of the pyridine group (measured pKa: 4.32), which

allowed the compound to have high solubility in the FaSSGF

medium (pH = 1.6). The pKa of 40 could not be measured,

Figure 4. Broad exploration of pyridine’s C5 position by a chemical

enablement strategy. (a) Representation of chemotypes. (b) Potency

in calcium ﬂux assay vs potency in GTPγS assay. (c) Clearance

unbound in mouse liver microsomes vs potency in IL8 assay.

Table 4). In the presence of the ethoxy group, electron-

withdrawing substituents showed good potencies (Cl, CN, and

CF in 37, 39, and 40, respectively). 40 was exquisitely potent

3

in the IL8 assay (free IC : 0.60 nM). Replacement of the

50

electron-withdrawing groups by a methyl group in 41 resulted

in a considerable loss of potency (5−10x) across all assays. The

compounds from this set showed in general good microsomal

stability, especially in humans. Replacement of the ethyl group

in 37 by a CH CHF group was tolerated (38). On the other

2

Table 3. Broad Investigation of Pyridine’s C5 Position: Best Compounds per Chemotype

a

b

d

Cpd

clogD

S1P2 calcium ﬂux IC₅₀(nM) (LipE)

S1P2 GTPγS IC₅₀(nM)

IL8 IC₅₀(nM)

Clint, u mouse/human (L/h/kg)

c

34

35

36

3.6

3.1

3.6

11 (4.4)

12 (4.8)

16 (4.2)

22

23

7.4

23

27/9.0

52/16

30/7.5

c

55

105

a

b

Antagonist mode. Experiments aiming at determining the IC₅values were performed at least twice, unless otherwise stated. When multiple

0

c

d

experiments were performed, the geometrical mean is provided. n = 1. Fraction unbound in microsomes (fu, mic) experimentally determined,

unless otherwise stated. NT = not tested.

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Table 4. SAR of Right-Hand Side Groups in the Pyridazine-

Based Series

Compound 40 conﬁrmed to be active against S1P2 in the

radioligand-based binding assay as well as in an arrestin-based

assay. It was also potent against the murine S1P2 receptor

(

mS1P2, arrestin assay, Table S1, Supporting Information). In

terms of selectivity, 40 did not have any meaningful activity

against any of the other S1P receptors (Table S1, Supporting

Information). When proﬁled against a broad panel of

receptors, transporters, and enzymes (Cerep Diversity Panel,

compound tested at 10 μM), only a few targets were picked up

with inhibition, binding, or activation >50%. Follow up dose

responses on eight targets showed IC values to be only in the

5

0

micromolar range for all these targets (Table S3 , Supporting

Information). Compound 48 was also active against mS1P2

and showed complete selectivity against the other S1P

receptors (Table S2, Supporting Information). No meaningful

inhibition, binding, or activation was picked up in the Cerep

Diversity Panel. Both 40 and 48 showed no meaningful

inhibition against a panel of 154 kinases when tested in a single

dose at 1 μM.

Compound 40 was tested in a cell contraction assay with

human pulmonary ﬁbroblasts (HPFs). Fibroblast contraction

is a phenomenon observed during wound healing as well as in

1

0

many ﬁbrotic disorders. In the cell contraction assay,

myoﬁbroblasts that have been pre-treated with TGF-β for 4

days are suspended in a collagen gel lattice mixture. Stress

build-up continues for another 3 days. Upon release of the gels

from the well, the gel lattice will start to shrink due to the

myoﬁbroblast contraction, which can be further stimulated by

S1P addition, resulting in a reduced surface area. As it could be

seen in Figure 6, compound 40 signiﬁcantly prevented the

S1P-mediated contraction at all tested concentrations.

From the data presented above, compounds 40 and 48

emerged as potent and selective S1P2 antagonists with good

ADMET and PK. This and their conﬁrmed potency against the

murine S1P2 receptor made them suitable candidates for the

bleomycin-induced pulmonary ﬁbrosis in vivo model. The

BLM-induced ﬁbrosis is one of the most accepted models of

38

IPF. Compounds 40 (dosed at 1, 3, and 10 mg/kg BID) and

8 (3, 10, and 30 mg/kg BID) were therefore evaluated in a

4

prophylactic setting, together with the reference compound

pirfenidone (dosed at 50 mg/kg BID), an approved treatment

for IPF. In this model, BLM was dosed intranasally to mice.

Pirfenidone or an S1P2 antagonist was administered twice a

day orally for 14 days. The eﬃcacy of the treatments was

evaluated by assessing the histopathological changes in lung

39

architecture. To this end, the modiﬁcation of the Ashcroft

a

b

40,41

Antagonist mode. Experiments aiming at determining the IC₅₀

score introduced by Matsuse was used.

After 14 days,

values were performed at least twice, unless otherwise stated. When

multiple experiments were performed, the geometrical mean is

provided. n = 1. Fraction unbound in microsomes (fu, mic)

experimentally determined, unless otherwise stated. NT = not tested.

administration of BLM caused widespread epithelial damage,

inﬂammation in the lungs, ﬁbrosis, and lung structural

distortion, as evidenced by the Ashcroft score in the vehicle

groups (orange squares and dots, Figure 7). Pirfenidone and

compounds 40 and 48 displayed a marked protective eﬀect at

all dosed tested, resulting in statistically signiﬁcant reduction of

the Ashcroft score.

A steady-state PK study was performed on the doses of 3

and 10 mg/kg BID for compound 40. The exposures appeared

to be in both cases well above the IC₅₀values obtained from

the IL8 assay (Figure S1, Supporting Information).

As both compounds showed very good activity in the BLM

model, 40 and 48 were both engaged in rat toxicology studies.

After analysis of the toxicological proﬁles and ensuing

therapeutic windows, compound 40 was selected for further

progression.

c

d

indicating lack of protonation even at low pH values.

Solubility, even when limited to low pH values, was deemed

important in order to achieve dose-proportional exposure in

toxicology studies, when usually high doses are administered.

Other compounds were designed and synthesized to

improve solubility, speciﬁcally by replacing the methyl group

at the N1 position of the pyrazole or the methyl at the C5

position of the pyridazine core with more polar groups. None

of these compounds, however, possessed a balanced proﬁle in

terms of potency, metabolic stability, and solubility in order to

be progressed further.

37

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Figure 5. Lipophilicity vs pIC₅₀(calcium ﬂux) for matched pairs between pyridine and pyridazine series.

Table 5. Pharmacokinetics in Preclinical Species and Selected In Vitro Data for 37, 40, and 48

a

b

iv PK preclinical species (dose: 1 mg/kg)

thermodynamic solubility

g

cpd

species

t₁_/₂(h)

Cl (L/h/kg)

Vss (L/kg)

F (po dose)

PPB (% bound)

FaSSGF (μg/mL)

FaSSIF (μg/mL)

c

37

40

48

mouse

rat

dog

0.55

1.1

3.1

2.3

2.7

7.6

0.45

1.0

1.6

0.62

0.25

0.47

0.24

0.091

0.10

0.73

0.37

0.66

0.49

0.38

2.1

0.81

0.35

1.1

0.47

0.54

1.5

20% (5 mg/kg)

10% (5 mg/kg)

30% (5 mg/kg)

64% (5 mg/kg)

29% (5 mg/kg)

99.8

99.9

98.4

99.9

>99.9

99.7

98.6

99.6

97.8

5

36

d

e

d

mouse

2.7

23

15

f

rat

e

dog

mouse

rat

100% (5 mg/kg)

d

29% (5 mg/kg)

505

d

84% (10 mg/kg)

d

dog

39% (1 mg/kg)

a

Male CD1 mice (n = 6), male Sprague Dawley rats (n = 3, unless otherwise stated), and male Beagle dogs (n = 3) were used in these studies.

b

c

Numbers of animals are referring to each study. All values were obtained from plasma. Vehicle was PEG200/water (60/40; v/v). Vehicle was

Tween 80/MC 0.5%. Vehicle was PEG400/MC 0.5%. Vehicle was MC 0.5%. n = 2 for the iv route. 4 h incubation at a 5 μM compound

d

e

f

g

concentration in plasma.

Figure 6. Activity of compound 40 in a cell contraction assay in HPF cells. S1P was used as trigger at 1 μM. Bar height: mean values. Error bars:

95% CI.

Compound 40 displayed no hERG inhibition and limited

in the production of amorphous batches, which resulted in

improved solubility. This allowed the smooth running of dose-

ranging studies in dogs and rats.

CYP inhibition and had no liabilities in terms of chemical or

plasma stability (Tables S5 −S8 , Supporting Information). No

genotoxicity was detected. 40 was therefore nominated as a

preclinical candidate (GLPG2938) for treatment in IPF.

Subsequent CMC work in preclinical development resulted

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Figure 7. Ashcroft score at D14 following intranasal bleomycin challenge. Data are given as group median. *p < 0.05 vs BLM/vehicle control

(

Mann−Whitney test). #p < 0.05 vs BLM/Vehicle (Wilcoxon signed-rank test); cpd 40: n = 15 per group; cpd 48: n = 14 per group. Animals: male

C57BL/6 mice (40: 8 weeks old on arrival; 48: 7 weeks old on arrival. One week acclimation).

a

Scheme 1. Synthesis of Compounds 12−21

a

Reagents and conditions: (a) tert-butyl hydrazinecarboxylate, Cs CO , Pd (dba) , dppf, toluene, 100 °C; (b) 2,6-dichloro-4-isocyanatopyridine,

2

3

2

3

THF, rt; (c) 4 M HCl in 1,4-dioxane, DCM, 40 °C; (d) Ar-B(OR) , 2 M K CO , Pd(dppf)Cl , 1,4-dioxane, 130 °C, μw; (e) Ar-B(OR) , 2 M

Na CO , Pd(dppf)Cl , 1,4-dioxane, 150 °C, μw.

2

3

2

3

2

a

Scheme 2. Synthesis of Compounds 22−24

a

Reagents and conditions: (a) 10a, LiOH, H O/THF; (b) 2,6-dichloro-4-isocyanatopyridine, THF, rt; (c) 4 M HCl in 1,4-dioxane, DCM, 40 °C;

2

1

2

(d) R R NH, HATU, DIPEA, DMF, DCM, rt.

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a

Scheme 3. Synthesis of Compounds 25 and 27−34

a

Reagents and conditions: (a) Zn(CN) , Pd(PPh ) , DMF, 150 °C; (b) Ar-B(OR) , Cs CO , Pd(dppf)Cl , 1,4-dioxane, H O, 100 °C; (c) BH ·

2

3

4

2

3

2

3

SMe , THF, 45 °C; (d) 2,5,6-trichloro-1H-benzo[d]imidazole, MeCN, 170 °C, μw; (e) RCOOH, HATU, DIPEA, DMF, DCM, rt or 45 °C; (f)

RNCO, THF, rt; (g) 2-chloro-6-ethoxypyridin-4-amine, CDT, pyridine, DCM, rt.

2

a

Scheme 4. Synthesis of Compound 26

a

Reagents and conditions: (a) 1,3-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole, Cs CO , Pd(dppf)Cl , 1,4-dioxane, H O,

2

3

2

1

00 °C; (b) EtOH, NaH, rt; (c) Zn, NH Cl, MeOH, rt; (d) CNBr, EtOH, 45 °C; (e) Ti(OiPr) , EtOH, 60 °C.

4

CHEMISTRY

ates 2−3c were obtained after Suzuki coupling. Reduction of

the nitrile group in the presence of BH ·SMe yielded primary

■

3

2

Final compounds 12−21 were synthesized according to

Scheme 1. In summary, Buchwald−Hartwig conditions with

tert-butyl hydrazinecarboxylate were used to obtain inter-

mediates 6−10a, 12a, and 17a from the respective aryl halides.

Intermediates 12a and 17a were coupled with aromatic

boronic acids/esters to obtain intermediates 13−15a and

amine intermediates 4−5c. S Ar reaction with 2,5,6-trichloro-

N

1

H-benzo[d]imidazole yielded compound 25. A series of

HATU-mediated couplings between 4c and the corresponding

carboxylic acids delivered compounds 27−30. Ureas 31 and 33

were obtained after coupling of 4c with the corresponding

isocyanates. Intermediates 4−5c were coupled with 2-chloro-6-

ethoxypyridin-4-amine using 1,1′-carbonyl-di-(1,2,4-triazole)

1

8−19a. Finally, the hydrazine derivatives 6−10a, 13−15a,

and 18−19a underwent a two-step sequence featuring urea

formation by reaction with 2,6-dichloro-4-isocyanatopyridine

followed by Boc deprotection in acidic medium.

(

CDT) to obtain ureas 32 and 34.

The synthetic sequence described in Scheme 4 led to

compound 26. The bromide 1d underwent Suzuki coupling

with 1,3-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-

yl)-1H-pyrazole to aﬀord intermediate 2d. Reductive amina-

tion with the aminobenzimidazole 6d gave the desired product

26. During the optimization of the reductive amination step, it

was observed that the reducing agent was not necessary for the

reduction of the imine intermediate. It was concluded that the

titanium tetraisopropoxide played the roles of both Lewis acid

Compounds 22−24 were synthesized according to Scheme

2

. Intermediate ester 10a was hydrolyzed by LiOH to acid 1b,

which underwent urea formation. Final compounds were

obtained after Boc deprotection and HATU-based amide

coupling with the corresponding amines.

Compounds 25 and 27−34 were synthesized according to

Scheme 3. Intermediate 1c was obtained from 11a after a

regioselective coupling under Negishi conditions. Intermedi-

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a

Scheme 5. Synthesis of Compound 35

a

Reagents and conditions: (a) NaBH , NiCl ·6H O, Boc O, MeOH, rt; (b) TFA, DCM, 45 °C; (c) 2-ethoxy-6-(triﬂuoromethyl)pyridin-4-amine,

4

2

CDT, pyridine, DCM, 45 °C; then 2e, DIPEA, THF, rt; (d) HCHO (37% H O), 4-methylmorpholine, tBuNH EtOH/H O, 100 °C; (e) 1,3-

dimethyl-1H-pyrazol-4-amine, Pd (dba) , BINAP, NaOtBu, toluene, 70 °C; (f) 1,4-dioxane/6 M NaOH, 100 °C.

2

2,

2

3

a

Scheme 6. Synthesis of Compounds 37−43, 46−48, 50, and 51

a

Reagents and conditions (a) 3.3 M NaOH, reﬂux; (b) Zn(CN) , Pd (dba) , dppf, DMF, 120 °C; (c) POCl , MeCN, reﬂux; (d) 1-methyl-4-

2

3

(

4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-(triﬂuoromethyl)-1H-pyrazole, Pd(PPh ) , DMF, rt; then 2.5 M Na CO , 85 °C; (e) Pd/C, H gas

3 4 2 3 2

(

1 atm), 6 M HCl, MeOH, rt; (f) NaBH , TFA, NiCl .6H O, MeOH, rt; (g) RNH , CDT, pyridine, DCM, 45 °C; then 6f, DIPEA, THF, rt; (h)

4

2

RNH , CDT, pyridine, DCM, 45 °C; then 7f, rt.

2

and reducing agent in a similar way as the aluminum

separation. The strategy adopted here is not widely exploited

triisopropoxide does in the Meerwein−Schmidt−Ponndorf−

as we could ﬁnd only one account for such protecting group

applied to a urea bearing an aryl motif. The urea so protected

4

2

43

Verley reduction. Compound 122 (Supporting Information,

Scheme S31 ) was also obtained with this procedure. Moreover,

a preliminary investigation showed that these conditions were

working also on other substrates. Two examples are reported

in the Supporting Information (compounds 123 and 124,

Scheme S32). 6d was synthesized in three steps. The ethoxy

underwent Buchwald−Hartwig coupling with 1,3-dimethyl-

1H-pyrazol-4-amine. The product was subsequently depro-

tected in strongly basic conditions to yield the ﬁnal compound

35.

The synthesis of compounds 37−43, 46−48, 50, and 51

(Scheme 6) commenced with the hydrolysis of 3,6-dichloro-4-

methylpyridazine followed by a Negishi coupling to obtain

group was installed by S Ar on the bis-chloropyridine 3d with

N

ethanol. After reduction of the nitro group in 4d with zinc, the

bis-aminopyridine 5d was cyclized using cyanogen bromide to

obtain the 2-aminobenzimidazole 6d.

nitrile 3f. Chlorination with POCl provided 4f, which was

3

coupled with 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-

lan-2-yl)-3-(triﬂuoromethyl)-1H-pyrazole under Suzuki con-

ditions to yield intermediate 5f. The nitrile group was then

reduced using hydrogen gas and palladium on charcoal in the

presence of HCl. The acidic environment was necessary to

prevent intermolecular attack of the amino group on the

partially reduced imine. Alternatively, 5f could be reduced

using a mixture of NaBH , TFA, and NiCl ·6H O. The

Scheme 5 describes the synthesis of compound 35. Nitrile

derivative 1c (Scheme 3) was reduced and Boc protected in a

one-pot procedure to favor puriﬁcation. Intermediate 2e,

obtained after Boc deprotection from 1e, was coupled with

ethoxy-6-(triﬂuoromethyl)pyridin-4-amine to form urea 3e. At

this stage, a protecting group based on a 1,3,5-triazinan-2-one

was envisaged. The tert-butyl group helped for the isolation of

the protected urea, ensuring a straightforward chromatographic

4

2

resulting amines 6f (HCl salt) and 7f (free base) were coupled

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to the corresponding aminopyridine in the presence of 1,1′-

carbonyl-di-(1,2,4-triazole) (CDT) and pyridine to aﬀord the

desired products 37−43, 46−48, 50, and 51. When the HCl

salt 6f was used, DIPEA as a base on top of pyridine and THF

as a co-solvent were necessary to obtain good conversions.

Bromo-4-methylpyridin-2-yl)hydrazine-1-carboxylate (12a).

Pd (dba) (334 mg, 0.365 mmol, 0.05 eq) and dppf (606 mg, 1.09

2

3

mmol, 0.15 eq) were added to a degassed solution of 2,5-dibromo-4-

methylpyridine 11a (1.83 g, 7.29 mmol, 1.0 eq), tert-butyl

hydrazinecarboxylate (963 mg, 7.29 mmol, 1.0 eq), and Cs CO

2

3

(2.38 g, 7.29 mmol, 1.0 eq) in dry toluene (36 mL, 0.2 M). The

reaction mixture was stirred at 100 °C for 2.5 h. The reaction was

cooled down to room temperature and ﬁltered over Celite. The

CONCLUSIONS

■

ﬁltrate was washed with saturated aqueous NaHCO solution. The

compound was extracted with DCM, and the organic layer was dried

In this manuscript, we described the discovery of the

preclinical candidate GLPG2938 (compound 40), a novel

S1P2 receptor inhibitor possessing high potency, exquisite

selectivity, and very good pharmacokinetics. The eﬃcacy

obtained in the BLM model at low doses advocated strongly

for its progression in development for the treatment of IPF. A

key role in this discovery was played by adopting chemical

enablement strategies; this allowed us to identify the 5-pyrazol-

3

over MgSO , ﬁltered, and concentrated to dryness. The crude material

4

was puriﬁed by column chromatography (SiO , petroleum ether/

2

EtOAc, 100:0 to 0:100 over 20 CV) to aﬀord tert-butyl 1-(5-bromo-4-

methylpyridin-2-yl)hydrazine-1-carboxylate (1.07 g, 44% yield).

+

LCMS: m/z = 302.0, 304.0 (M + H) .

tert-Butyl N-Amino-N-[5-(dimethylamino)-4-methyl-2-pyridyl]-

carbamate (6a). This compound was prepared from 1a according

4

-yl-pyridine motif as the key element for further optimization.

to the general procedure used for the preparation of 12a. LCMS: m/z

+

=

267.1 (M + H) .

Chemical enablement also allowed a quick and broad

exploration of the space around the pyridine’s C5 position.

Considerations on lipophilicity were also very important as

they led to the pyridazine-based series. This change brought

molecules with better LipE and helped to solve a potential

CYP inhibition issue.

tert-Butyl N-Amino-N-(5-cyano-4-methyl-2-pyridyl)carbamate

7a). This compound was prepared from 2a according to the general

(

procedure used for the preparation of 12a. LCMS: m/z = 193.2 ((M

+

−

56) + H) .

tert-Butyl N-Amino-N-(6-cyano-4-methyl-2-pyridyl)carbamate

(8a). This compound was prepared from 3a according to the general

procedure used for the preparation of 12a. LCMS: m/z = 249.0 (M +

+

EXPERIMENTAL SECTION

H) .

■

tert-Butyl N-Amino-N-[6-(hydroxymethyl)-4-methyl-2-pyridyl]-

carbamate (9a). This compound was prepared from 4a according

General Experimental Methods. All reagents were of

commercial grade and were used as received without further

puriﬁcation, unless otherwise stated. Commercially available anhy-

drous solvents were used for reactions conducted under an inert

atmosphere. Reagent-grade solvents were used in all other cases,

unless otherwise speciﬁed. Column chromatography was performed

on silica gel 60 (35−70 μm). Thin layer chromatography was carried

out using pre-coated silica gel F-254 plates (thickness 0.25 mm).

Puriﬁcation with preparatory HPLC was performed with a Waters

FractionLynx system coupled to a 2996 PDA detector and a Waters

Mass detector QDA. For the basic method, column used: Waters

XBridge prep (C18, 10 μm OBD, 19 x 100 mm); for the acidic

to the general procedure used for the preparation of 12a. LCMS: m/z

+

=

254.0 (M + H) .

Ethyl 6-[Amino(tert-butoxycarbonyl)amino]-4-methylpyridine-2-

carboxylate (10a). This compound was prepared from 5a according

to the general procedure used for the preparation of 12a. LCMS: m/z

+

=

296.0 (M + H) .

tert-Butyl N-Amino-N-(6-chloro-4-methyl-2-pyridyl)carbamate

17a). This compound was prepared from 16a according to the

(

general procedure used for the preparation of 12a. LCMS: m/z =

+

2

58.0 (M + H) .

General Procedure for Preparation of Boc-Protected 2-

method, column used: Waters XSelect CSH (C18, 5 μm OBD, 19 x

Hydrazineylpyridines 13−15a. tert-Butyl N-Amino-N-[5-(1,3-

dimethylpyrazol-4-yl)-4-methyl-2-pyridyl]carbamate (15a). To a

degassed solution of tert-butyl N-amino-N-(5-bromo-4-methyl-2-

pyridyl)carbamate 12a (200 mg, 0.661 mmol, 1.0 eq), 1,3-dimethyl-

4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (221 mg,

0.993 mmol, 1.5 eq), and K CO (2 M in H O, 1.1 mL, 2.3 mmol, 3.5

1

00 mm). Flow rate: 20 mL/min. H NMR spectra were recorded on

a Bruker DPX 400 NMR spectrometer (400 MHz). Chemical shifts

1

(δ) for H NMR spectra are reported in parts per million (ppm)

relative to tetramethylsilane (δ 0.00) or the appropriate residual

solvent peak, i.e., CHCl₃(δ 7.27), as an internal reference.

Multiplicities are given as singlet (s), doublet (d), triplet (t), quartet

2

3

2

eq) in 1,4-dioxane (7 mL, 0.1 M) was added Pd(dppf)Cl (27 mg,

2

(

q), quintuplet (quin), multiplet (m), and broad (br). Ultraviolet and

0.033 mmol, 0.05 eq), and the reaction was stirred at 130 °C under

microwave irradiation until reaction completion. It was then cooled to

room temperature and partitioned between saturated aqueous

electrospray MS spectra were obtained on a Waters platform LC/MS

spectrometer or with Waters Acquity H-Class UPLC coupled to a

Waters mass detector QDA. Purities were determined by LCMS

analysis (UV traces determined with an Acquity PDA detector) using

two methods. Method A: column used: Waters Acquity UPLC CSH

NaHCO and DCM. The compound was extracted using a phase

3

separator, and the organic layer was concentrated to dryness to obtain

compound 15a, which was used without further puriﬁcation. LCMS:

+

C18 1.7 μm, 2.1 mm ID x 50 mm L; MeCN/H O gradients (H O

contains 0.1% formic acid); Method B: column used: Waters Acquity

UPLC BEH C18 1.7 μm, 2.1 mm ID x 50 mmL; MeCN/H O

gradients (H O contains 13.4 mM NH ). All reported ﬁnal

compounds were analyzed with one of these analytical methods and

had purities ≥95%.

High-resolution mass spectrometry (HRMS) analyses were

performed with a Waters Xevo-G2-XS QTof mass spectrometer

coupled to a Waters UPLC H-class system (column: Acquity BEH

C18, 100*2.1 mm, 1.7 μm particle size, elution: formic acid 0.1% in

water/formic acid 0.1% in acetonitrile 95:5 to 5:95 in 5.2 min, ﬂow:

2

m/z = 318.1 (M + H) .

tert-Butyl N-Amino-N-[4-methyl-5-(1-methylpyrazol-4-yl)-2-

pyridyl]carbamate (13a). This compound was prepared from 12a

according to the general procedure used for the preparation of 15a.

2

3

+

LCMS: m/z = 304.1 (M + H) .

tert-Butyl N-Amino-N-[4-methyl-5-(1-methylpyrazol-3-yl)-2-

pyridyl]carbamate (14a). This compound was prepared from 12a

according to the general procedure used for the preparation of 15a.

+

LCMS: m/z = 304.1 (M + H) .

General Procedure for Preparation of Boc-Protected 2-

Hydrazineylpyridines 18−19a. tert-Butyl N-Amino-N-[6-(4-ﬂuo-

rophenyl)-4-methyl-2-pyridyl]carbamate (19a). To a degassed

solution of tert-butyl 1-(6-chloro-4-methylpyridin-2-yl)hydrazine-1-

carboxylate 17a (200 mg, 0.776 mmol, 1.0 eq), (4-ﬂuorophenyl)-

0.8 mL/min) equipped with a PDA detector (UV detection: total

absorbance 210−400 nm), a column heater module, and controlled

by Masslynx Software. Microwave heating was performed with a

Biotage Initiator. The melting points were measured with a Mettler

Toledo, MP50 melting point system.

General Procedure for Preparation of Boc-Protected 2-

Hydrazineylpyridines 6−10a, 12a, and 17a. tert-Butyl 1-(5-

boronic acid (163 mg, 1.16 mmol, 1.5 eq), and Na

1.36 mL, 2.72 mmol, 3.5 eq) in 1,4-dioxane (5 mL, 0.15 M) was

added Pd(dppf)Cl (32 mg, 0.04 mmol, 0.05 eq), and the reaction

CO (2 M in H O,

2

3 2

2

was stirred at 150 °C under microwave irradiation until reaction

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completion. It was then cooled to room temperature and partitioned

1-(2,6-Dichloro-4-pyridyl)-3-[[5-(1,3-dimethylpyrazol-4-yl)-4-

methyl-2-pyridyl]amino]urea (19). This compound was prepared

from 15a according to the general procedure used for the preparation

between saturated aqueous NaHCO and DCM. The compound was

3

extracted using a phase separator, and the organic layer was

concentrated to dryness to obtain compound 19a, which was used

of 13. LCMS: method A, R

t

: 0.95 min; purity: >99%, m/z = 406.1 (M

+

1

+

H) . H NMR (400 MHz, DMSO-d ) δ 9.70 (s, 1H), 8.81 (s, 1H),

without further puriﬁcation. LCMS: m/z = 318.1 (M + H) .

6

8

.32 (s, 1H), 8.02−7.46 (m, 4H), 6.59 (s, 1H), 3.78 (s, 3H), 2.12 (s,

tert-Butyl N-Amino-N-[4-methyl-6-(1-methylpyrazol-4-yl)-2-

pyridyl]carbamate (18a). This compound was prepared from 17a

according to the general procedure used for the preparation of 19a.

3H), 2.03 (s, 3H).

1-(2,6-Dichloro-4-pyridyl)-3-[[4-methyl-6-(1-methylpyrazol-4-yl)-

2-pyridyl]amino]urea (20). This compound was prepared from 18a

according to the general procedure used for the preparation of 13.

+

LCMS: m/z = 304.2 (M + H) .

General Procedure for Preparation of Ureas 12−21. 2-(5-

Cyano-4-methylpyridin-2-yl)-N-(2,6-dichloropyridin-4-yl)-

hydrazine-1-carboxamide (13). 2,6-Dichloro-4-isocyanatopyridine

LCMS: method A, R

H) . H NMR (400 MHz, DMSO-d ) δ 10.11 (s, 1H), 9.12 (s, 1H),

: 1.11 min; purity: >99%, m/z = 392.1 (M +

t

+

1

6

(

0.10 mL, 0.81 mmol, 1 eq) in THF was added to a mixture of N-

8.38 (s, 1H), 8.10 (s, 1H), 7.70 (s, 2H), 7.14 (s, 1H), 6.63 (s, 1H),

3.91 (s, 3H), 2.34 (s, 3H).

1-(2,6-Dichloro-4-pyridyl)-3-[[6-(4-ﬂuorophenyl)-4-methyl-2-

pyridyl]amino]urea (21). This compound was prepared from 19a

according to the general procedure used for the preparation of 13.

amino-N-(5-cyano-4-methyl-2-pyridyl)carbamate 7a (200 mg, 0.81

mmol, 1 eq) in THF (8 mL ﬁnal volume). The reaction was stirred at

room temperature until the disappearance of the starting material (4

h). The mixture was partitioned between DCM and water. The two

phases were separated; the organic layer was dried (ﬁltered through

phase separator) and concentrated. The residue was taken up in DCM

LCMS: method A, R : 1.44 min; purity: >99%, m/z = 406.6 (M +

t

+

1

H) . H NMR (400 MHz, DMSO-d ) δ 9.76 (s, 1H), 8.85 (s, 1H),

6

8

3

.42 (s, 1H), 8.11−8.03 (m, 2H), 7.84 (s, 2H), 7.27 (t, J = 8.9 Hz,

(

8 mL), and 4 M HCl in 1,4-dioxane (2.0 mL, 8.1 mmol, 10 eq) was

H), 6.48 (s, 1H), 2.31 (s, 3H).

added to the mixture. The reaction was stirred at 40 °C until

completion (1.5 h). The reaction was quenched with saturated

6

-[Amino(tert-butoxycarbonyl)amino]-4-methylpyridine-2-car-

boxylic acid (1b). A solution of LiOH (1.8 g, 43 mmol, 2.8 eq) in

NaHCO , and the two layers were separated. The aqueous layer was

further extracted with DCM. The combined organic layers were dried

3

H O (15 mL) was added to a mixture of ethyl 6-[amino(tert-

2

butoxycarbonyl)amino]-4-methylpyridine-2-carboxylate 10a (4.4 g,

(ﬁltered through phase separator) and concentrated. The residue was

15 mmol, 1 eq) in THF (30 mL). The reaction was stirred for 2 h at

puriﬁed by preparatory HPLC (gradient from 30% MeCN to 55%

MeCN in water/0.1% formic acid) to obtain the title product (11 mg,

4

room temperature. The reaction mixture was acidiﬁed and extracted

with DCM. The two phases were separated. The organic layer was

dried (ﬁltered through phase separator) and concentrated to aﬀord

% yield). LCMS: method A, R : 1.16 min; purity: >99%, m/z = 336.9

t

+

1

(M + H) . H NMR (400 MHz, DMSO-d ) δ 9.78 (s, 1H), 9.22 (s,

+

6

the desired product (3 g, 75% yield). LCMS: m/z = 268.1 (M + H) .

1

3

H), 8.99 (s, 1H), 8.45 (s, 1H), 7.68 (s, 2H), 6.64 (s, 1H), 2.38 (s,

6

-[tert-Butoxycarbonyl-[(2,6-dichloro-4-pyridyl)-

carbamoylamino]amino]-4-methylpyridine-2-carboxylic acid (2b).

,6-Dichloro-4-isocyanatopyridine (1.4 mL, 11 mmol, 1 eq) in THF

H).

1

-(2,6-Dichloro-4-pyridyl)-3-[[5-(dimethylamino)-4-methyl-2-

2

pyridyl]amino]urea (12). This compound was prepared from 6a

according to the general procedure used for the preparation of 13.

LCMS: method A, R : 0.90 min; purity: >99%, m/z = 355.0 (M +

H) . H NMR (400 MHz, DMSO-d ) δ 9.65 (s, 1H), 8.80 (s, 1H),

8

was added to a mixture of N-amino-N-(5-cyano-4-methyl-2-pyridyl)-

carbamate 1b (2.9 g, 11 mmol, 1 eq) in THF (110 mL ﬁnal volume).

The reaction was stirred at room temperature until the disappearance

of the starting material (2.5 h). The mixture was partitioned between

DCM and water. The two phases were separated; the organic layer

was dried (ﬁltered through phase separator) and concentrated to

t

+

1

6

.03 (s, 1H), 7.84 (s, 2H), 6.50 (s, 1H), 2.60 (s, 6H), 2.21 (s, 3H).

1

-[(6-Cyano-4-methyl-2-pyridyl)amino]-3-(2,6-dichloro-4-

pyridyl)urea (14). This compound was prepared from 8a according to

the general procedure used for the preparation of 13. LCMS: method

B, R : 1.20 min; purity: >99%, m/z = 337.0 (M + H) . H NMR (400

MHz, DMSO-d ) δ 9.74 (s, 1H), 8.95−8.91 (m, 2H), 7.72 (s, 2H),

7

aﬀord the desired product (4.2 g, 84% yield). LCMS: m/z = 456.1 (M

+

H) .

+

1

t

6

-[2-[(2,6-Dichloro-4-pyridyl)carbamoyl]hydrazino]-4-methyl-

pyridine-2-carboxylic acid (3b). A 4 M HCl solution in 1,4-dioxane

23 mL, 92 mmol, 10 eq) was added to a mixture of 6-[tert-

6

.25 (s, 1H), 6.79 (s, 1H), 2.29 (s, 3H).

(

1

-(2,6-Dichloro-4-pyridyl)-3-[[6-(hydroxymethyl)-4-methyl-2-

butoxycarbonyl-[(2,6-dichloro-4-pyridyl)carbamoylamino]amino]-4-

methylpyridine-2-carboxylic acid 2b (4.2 g, 9.2 mmol, 1 eq) in DCM

pyridyl]amino]urea (15). This compound was prepared from 9a

according to the general procedure used for the preparation of 13.

LCMS: method A, R : 0.87 min; purity: >99%, m/z = 342.0 (M +

H) . H NMR (400 MHz, DMSO-d ) δ 9.77 (s, 1H), 8.88 (s, 1H),

7

(

92 mL). The resulting mixture was stirred at 40 °C for 2 h. The

t

mixture was concentrated, and the residue was washed with methyl

+

1

6

tert-butyl ether to aﬀord the desired product (3.0 g, 92% yield).

.76 (s, 2H), 6.78 (s, 1H), 6.44 (s, 1H), 4.42 (s, 2H), 2.27 (s, 3H).

Ethyl 6-[2-[(2,6-Dichloro-4-pyridyl)carbamoyl]hydrazino]-4-

+

LCMS: m/z = 356.0 (M + H) .

General Procedure for Preparation of Amides 22−24. N-

Cyclopropyl-6-[2-[(2,6-dichloro-4-pyridyl)carbamoyl]hydrazino]-4-

methylpyridine-2-carboxamide (23). A solution of HATU (53 mg,

0.14 mmol, 1 eq) in dry DMF (1 mL) was added to a mixture of

cyclopropanamine (8.8 mg, 0.154 mmol, 1.1 eq), 6-[2-[(2,6-dichloro-

4-pyridyl)carbamoyl]hydrazino]-4-methylpyridine-2-carboxylic acid

3b (50 mg, 0.14 mmol, 1 eq), and DIPEA (50 μL, 0.28 mmol, 2

eq) in dry DCM (1.4 mL). The reaction was stirred at room

temperature until full conversion. The reaction mixture was quenched

methylpyridine-2-carboxylate (16). This compound was prepared

from 10a according to the general procedure used for the preparation

of 13. LCMS: method A, R : 1.28 min; purity: >99%, m/z = 384.1 (M

t

+

1

+

H) . H NMR (400 MHz, DMSO-d ) δ 9.84 (s, 1H), 8.94 (s, 1H),

6

8

.70 (s, 1H), 7.66 (s, 2H), 7.33 (s, 1H), 6.73 (s, 1H), 4.30 (q, J = 7.1

Hz, 2H), 2.32 (s, 3H), 1.29 (t, J = 7.1 Hz, 3H).

-(2,6-Dichloro-4-pyridyl)-3-[[4-methyl-5-(1-methylpyrazol-4-yl)-

-pyridyl]amino]urea (17). This compound was prepared from 13a

1

2

according to the general procedure used for the preparation of 13.

LCMS: method A, R : 0.94 min; purity: >99%, m/z = 392.0 (M +

H) . H NMR (400 MHz, DMSO-d ) δ 9.68 (s, 1H), 8.90 (s, 1H),

8

with saturated NaHCO , and the resulting mixture was extracted with

3

t

DCM. The two phases were separated. The organic layer was dried

(ﬁltered through phase separator) and concentrated. The residue was

puriﬁed by preparatory HPLC (gradient from 35% MeCN to 60%

MeCN in water/0.1% formic acid) to aﬀord the desired product (5

+

1

6

.32 (s, 1H), 8.07 (s, 1H), 7.90 (s, 2H), 7.62 (s, 1H), 6.57 (s, 1H),

.87 (s, 3H), 2.29 (s, 3H).

3

1

-(2,6-Dichloro-4-pyridyl)-3-[[4-methyl-5-(1-methylpyrazol-3-yl)-

mg, 10% yield). LCMS: method B, R : 1.21 min; purity: >99%, m/z =

t

+

1

2

-pyridyl]amino]urea (18). This compound was prepared from 14a

3

8

56.0 (M + H) . H NMR (400 MHz, DMSO-d ) δ 9.84 (s, 1H),

6

according to the general procedure used for the preparation of 13.

.86 (s, 1H), 8.52 (s, 1H), 8.04 (d, J = 4.6 Hz, 1H), 7.83−7.54 (m,

LCMS: method A, R : 0.99 min; purity: >99%, m/z = 392.0 (M +

H) . H NMR (400 MHz, DMSO-d ) δ 8.89 (s, 1H), 8.39 (s, 1H),

8

1

2H), 7.26 (s, 1H), 6.67 (s, 1H), 2.87−2.76 (m, 1H), 2.31 (s, 3H),

0.79−0.64 (m, 2H), 0.50−0.42 (m, 2H).

6-[2-[(2,6-Dichloro-4-pyridyl)carbamoyl]hydrazino]-N,N,4-trime-

thylpyridine-2-carboxamide (22). This compound was prepared

t

+

1

6

.23−8.20 (m, 1H), 7.87 (s, 1H), 7.73 (s, 1H), 6.78 (s, 1H), 6.56 (s,

H), 6.50 (s, 1H), 6.46 (s, 1H), 3.87 (s, 3H), 2.39 (s, 3H).

6

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from 3b according to the general procedure used for the preparation

1H), 7.44 (s, 1H), 7.26 (s, 1H), 6.88 (s, 1H), 4.61 (d, J = 5.9 Hz,

2H), 4.36 (s, 2H), 3.80 (s, 3H), 2.18 (s, 3H), 2.06 (s, 3H), 1.35 (t, J =

7.1 Hz, 3H).

of 23. LCMS: method B, R : 1.14 min; purity: 95%, m/z = 383.0 (M +

t

+

1

H) . H NMR (400 MHz, DMSO-d ) δ 8.94 (s, 1H), 8.49 (s, 1H),

6

7

2

.80 (s, 3H), 6.76 (s, 2H), 6.54 (s, 1H), 6.50 (s, 1H), 2.93 (s, 3H),

.88 (s, 3H), 2.27 (s, 3H).

N-Cyclopropyl-6-[2-[(2,6-dichloro-4-pyridyl)carbamoyl]-

5-Bromo-4-methylpyridine-2-carbonitrile (1c). A mixture of 2,5-

dibromo-4-methylpyridine 11a (5 g, 20 mmol, 1 eq), Zn(CN) (2.81

2

g, 23.9 mmol, 1.2 eq) and Pd(PPh ) (2.3 g, 2.0 mmol, 0.1 eq) in

3

4

hydrazino]-N,4-dimethylpyridine-2-carboxamide (24). This com-

pound was prepared from 3b according to the general procedure used

for the preparation of 23. LCMS: method B, R : 1.22 min; purity:

DMF (100 mL) was heated at 150 °C under microwave irradiation

for 5 min. The mixture was ﬁltered through Celite. The ﬁltrate was

diluted with DCM. The resulting mixture was washed (sat NaHCO₃

and brine), dried (ﬁltered through a phase separator) and

concentrated. The residue was puriﬁed by ﬂash column chromatog-

t

+

1

>

99%, m/z = 409.1 (M + H) . H NMR (400 MHz, DMSO-d ) δ

6

9

6

3

.68 (s, 1H), 8.79 (s, 1H), 8.53 (s, 1H), 7.66 (d, J = 119.1 Hz, 2H),

.76 (s, 1H), 6.54 (s, 1H), 2.90 (s, 3H), 2.81−2.75 (m, 1H), 2.27 (s,

H), 0.43−0.12 (m, 4H).

raphy (SiO , 95:5 petroleum ether/EtOAc) to aﬀord the desired

2

+

product (4.4 g, 100% yield). LCMS: m/z = 198.9 (M + H) .

5

-(1,3-Dimethylpyrazol-4-yl)-4-methylpyridine-2-carbaldehyde

General Procedure for Preparation of Nitriles 2−3c. 5-(1,3-

(

2d). Pd(dppf)Cl (204 mg, 0.25 mmol, 0.1 eq) was added to a

2

Dimethylpyrazol-4-yl)-4-methylpyridine-2-carbonitrile (2c). Pd-

degassed mixture of 1,3-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxa-

(

dppf)Cl (415 mg, 0.51 mmol, 0.1 eq) was added to a degassed

2

borolan-2-yl)-1H-pyrazole (670 mg, 3.0 mmol, 1.2 eq), 5-bromo-4-

methylpicolinaldehyde 1d (500 mg, 2.5 mmol, 1 eq), and Cs CO

mixture containing 5-bromo-4-methylpyridine-2-carbonitrile 1c (1.0

g, 5.1 mmol, 1 eq), 1,3-dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-

dioxaborolan-2-yl)-1H-pyrazole (1.35 g, 6.09 mmol, 1.2 eq), and

Cs CO (4.10 g, 12.7 mmol, 2.5 eq) in 4:1 1,4-dioxane/H O (20

2

3

(

2.04 g, 6.25 mmol, 2.5 eq) in 10:1 1,4-dioxane/H O (12 mL). The

2

reaction was stirred at 95 °C for 4 h. The mixture was ﬁltered through

Celite. The ﬁltrate was partitioned between DCM and H O. The two

phases were separated. The aqueous layer was extracted with DCM.

The combined organic layers were dried (ﬁltered through a phase

2

3

2

mL). The reaction mixture was stirred at 100 °C for 2.5 h. The

mixture was cooled to room temperature and quenched with H O.

2

The resulting mixture was extracted with DCM. The organic layer was

dried (ﬁltered through a phase separator) and concentrated to dryness

separator) and concentrated to aﬀord the desired product (363 mg,

+

6

8% yield). LCMS: m/z = 216.1 (M + H) .

to aﬀord the desired product (550 mg, 51% yield). LCMS: m/z =

6

-Chloro-2-ethoxy-3-nitropyridin-4-amine (4d). NaH in 60%

mineral oil (154 mg, 3.84 mmol, 2 eq) was added to a mixture of

,6-dichloro-3-nitropyridin-4-amine 3d (400 mg, 1.92 mmol, 1 eq) in

+

2

13.0 (M + H) .

4

-Methyl-5-[1-methyl-3-(triﬂuoromethyl)pyrazol-4-yl]pyridine-2-

2

carbonitrile (3c). This compound was prepared from 1c according to

EtOH (6.4 mL). The reaction was stirred at room temperature for 1

h. Water was added, and the resulting mixture was extracted with

DCM. The two phases were separated. The organic layer was dried

the general procedure used for the preparation of 2c. LCMS: m/z =

+

2

67.1 (M + H) .

General Procedure for Preparation of Amines 4−5c. [5-(1,3-

(ﬁltered through phase separator) and concentrated to aﬀord the

Dimethylpyrazol-4-yl)-4-methyl-2-pyridyl]methanamine (4c). BH ·

3

desired product, which was used as such in the next step. LCMS: m/z

=

SMe (2.4 mL, 25 mmol, 5 eq) was slowly added to a mixture of 5-

2

+

218.0 (M + H) .

-Chloro-2-ethoxypyridine-3,4-diamine (5d). Zn dust (1.5 g, 23

(1,3-dimethylpyrazol-4-yl)-4-methylpyridine-2-carbonitrile 2c (1.1 g,

5.1 mmol, 1 eq) in THF at room temperature. The reaction mixture

was stirred at 45 °C for 1 h. The mixture was cooled to 0 °C, and

MeOH was carefully added. The resulting mixture was concentrated.

The residue was taken up in DCM, and 2 M HCl was added. The two

phases were separated. The aqueous layer was basiﬁed and extracted

with DCM. The two phases were separated. The organic layer was

dried (ﬁltered through a phase separator) and concentrated to aﬀord

the desired product (400 mg, 36% yield). LCMS: m/z = 217.1 (M +

6

mmol, 12 eq) and NH Cl (1.23 g, 23 mmol, 12 eq) were added to a

mixture of 6-chloro-2-ethoxy-3-nitropyridin-4-amine 4d (1.92 mmol,

4

1

eq) in MeOH (13 mL). The reaction was stirred at room

temperature for 2.5 h. The mixture was ﬁltered through Celite and

concentrated. The residue was taken up in DCM. The resulting

mixture was washed with saturated NaHCO . The two phases were

separated. The organic layer was dried (ﬁltered through a phase

separator) and concentrated to aﬀord the desired product, which was

used as such in the next step. LCMS: m/z = 188.0 (M + H) .

3

+

H) .

+

[4-Methyl-5-[1-methyl-3-(triﬂuoromethyl)pyrazol-4-yl]-2-

pyridyl]methanamine (5c). This compound was prepared from 3c

according to the general procedure used for the preparation of 4c.

6

-Chloro-4-ethoxy-3H-imidazo[4,5-c]pyridin-2-amine (6d).

CNBr (5 M MeCN solution, 1 mL, 5.8 mmol, 3 eq) was added to

a mixture of 6-chloro-4-ethoxy-3H-imidazo[4,5-c]pyridin-2-amine 5d

+

LCMS: m/z = 271.1 (M + H) .

(1.9 mmol, 1 eq) in EtOH (3.8 mL). The mixture was stirred at 45 °C

5,6-Dichloro-N-[[5-(1,3-dimethylpyrazol-4-yl)-4-methyl-2-

pyridyl]methyl]-1H-benzimidazol-2-amine (25). A mixture of [5-

(1,3-dimethylpyrazol-4-yl)-4-methyl-2-pyridyl]methanamine 4c (100

mg, 0.46 mmol, 1.2 eq) and 2,5,6-trichloro-1H-benzo[d]imidazole

(85 mg, 0.39 mmol, 1 eq) in acetonitrile (0.5 mL) was stirred at 170

°C under microwave conditions for 40 min. The mixture was

overnight. The mixture was concentrated. The residue was taken up in

EtOAc. The organic mixture was washed with saturated NaHCO₃,

and the two phases were separated. The aqueous layer was extracted

with EtOAc. The combined organic layers were dried (Na SO ),

2

4

ﬁltered, and concentrated to aﬀord the desired product (50 mg, 12%

yield). LCMS: m/z = 213.0 (M + H) .

+

partitioned between DCM and H O. The two phases were separated.

2

6

-Chloro-N-[[5-(1,3-dimethylpyrazol-4-yl)-4-methyl-2-pyridyl]-

methyl]-4-ethoxy-3H-imidazo[4,5-c]pyridin-2-amine (26). Ti-

OiPr) (0.21 mL, 0.705 mmol, 3 eq) was added to a mixture of 6-

The organic layer was dried (ﬁltered through phase separator) and

concentrated. The residue was puriﬁed by preparatory HPLC

(

(gradient from 30% MeCN to 55% MeCN in water/0.5% NH ) to

4

3

chloro-4-ethoxy-3H-imidazo[4,5-c]pyridin-2-amine 6d (50 mg, 0.24

mmol, 1 eq) and 5-(1,3-dimethylpyrazol-4-yl)-4-methylpyridine-2-

carbaldehyde 2d (101 mg, 0.47 mmol, 2 eq) in EtOH (0.5 mL). The

reaction was stirred at 60 °C overnight. The mixture was diluted with

aﬀord the desired product (7.5 mg, 5% yield). LCMS: method A, R :

0.95 min; purity: >99%, m/z = 401.1 (M + H) . H NMR (400 MHz,

t

+

1

DMSO-d ) δ 11.09 (s, 1H), 8.26 (s, 1H), 7.74 (s, 1H), 7.55 (t, J = 6.0

6

Hz, 1H), 7.32−7.28 (m, 3H), 4.60 (d, J = 5.9 Hz, 2H), 3.80 (s, 3H),

2.18 (s, 3H), 2.06 (s, 3H).

MeOH. Na SO ·10H O was added, and the mixture was stirred for 30

2

4

2

min. The mixture was ﬁltered, and the ﬁltrate was concentrated. The

residue was taken up in EtOAc and washed with 1 M NaOH. The two

phases were separated. The organic layer was dried (Na SO ) and

General Procedure for Preparation of Amides 27, 29, and

30. 4,6-Dichloro-N-[[5-(1,3-dimethylpyrazol-4-yl)-4-methyl-2-

pyridyl]methyl]-1H-indole-2-carboxamide (27). A solution of

HATU (131 mg, 0.35 mmol, 1 eq) in dry DMF (1 mL) was added

to a mixture of [5-(1,3-dimethylpyrazol-4-yl)-4-methyl-2-pyridyl]-

methanamine 4c (75 mg, 0.35 mmol, 1 eq), 4,6-dichloro-1H-indole-2-

carboxylic acid (88 mg, 0.38 mmol, 1.1 eq), and DIPEA (120 μL, 0.69

mmol, 2 eq) in dry DCM (3.5 mL). The reaction was stirred at room

2

4

concentrated. The residue was puriﬁed by preparatory HPLC

gradient from 20% MeCN to 45% MeCN in water/0.1% formic

acid) to aﬀord the desired product (3.0 mg, 3% yield). LCMS:

(

+

1

method B, R : 1.16 min; purity: >99%, m/z = 412.2 (M + H) . H

t

NMR (400 MHz, DMSO-d ) δ 11.56 (s, 1H), 8.26 (s, 1H), 7.73 (s,

6

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Article

temperature until full conversion. The reaction mixture was

General Procedure for Preparation of Ureas 32 and 34. 1-(2-

partitioned between DCM and saturated NH Cl. The two phases

Chloro-6-ethoxy-4-pyridyl)-3-[[4-methyl-5-[1-methyl-3-

4

(

triﬂuoromethyl)pyrazol-4-yl]-2-pyridyl]methyl]urea (34). A mixture

were separated. The organic layer was dried (ﬁltered through phase

separator) and concentrated. The residue was puriﬁed by preparatory

HPLC (gradient from 35% MeCN to 60% MeCN in water/0.1%

formic acid) to aﬀord the desired product (2.0 mg, 1% yield). LCMS:

of 2-chloro-6-ethoxypyridin-4-amine (25 mg, 0.15 mmol, 0.5 eq),

pyridine (35 μL, 0.44 mmol, 1.5 eq), and 1,1′-carbonyl-di-(1,2,4-

triazole) (36 mg, 0.22 mmol, 0.75 eq) in DCM (1.5 mL) was stirred

at room temperature for 0.5 h. [4-Methyl-5-[1-methyl-3-

+

1

method B, R : 1.19 min; purity: >99%, m/z = 428.1 (M + H) . H

t

(

0

triﬂuoromethyl)pyrazol-4-yl]-2-pyridyl]methanamine 5c (80 mg,

.30 mmol, 1 eq) was added, and the mixture was stirred at room

temperature for 2 h. The mixture was diluted (DCM), washed

saturated NH Cl and saturated NaHCO ), dried (ﬁltered through

NMR (400 MHz, DMSO-d ) δ 12.18 (s, 1H), 9.34 (t, J = 6.0 Hz,

6

1

H), 8.26 (s, 1H), 7.74 (s, 1H), 7.46−7.41 (m, 1H), 7.38 (d, J = 0.9

Hz, 1H), 7.29−7.24 (m, 2H), 4.59 (d, J = 6.0 Hz, 2H), 3.81 (s, 3H),

.20 (s, 3H), 2.07 (s, 3H).

,6-Dichloro-N-[[5-(1,3-dimethylpyrazol-4-yl)-4-methyl-2-

(

4

3

2

phase separator), and concentrated. The residue was puriﬁed by

preparatory HPLC (gradient from 40% MeCN to 65% MeCN in

water/0.1% formic acid) to aﬀord the desired product (14 mg, 20%

2

pyridyl]methyl]pyridine-4-carboxamide (29). This compound was

prepared from 4c according to the general procedure used for the

yield). LCMS: method B, R : 1.36 min; purity: >99%, m/z = 469.2 (M

preparation of 27. LCMS: method A, R : 1.19 min; purity: >99%, m/z

t

+

1

+

1

+ H) . H NMR (400 MHz, DMSO-d ) δ 9.49 (s, 1H), 8.25 (s, 1H),

=

1

=

390.1 (M + H) . H NMR (400 MHz, Chloroform-d) δ 8.35 (s,

H), 7.87 (s, 1H), 7.71 (s, 2H), 7.31 (s, 1H), 7.22 (s, 1H), 4.73 (d, J

4.5 Hz, 2H), 3.94 (s, 3H), 2.29 (s, 3H), 2.19 (s, 3H).

-(3,4-Dichlorophenyl)-N-[[5-(1,3-dimethylpyrazol-4-yl)-4-meth-

6

8

1

2

.07 (s, 1H), 7.29 (s, 1H), 7.18−7.10 (m, 2H), 6.78 (d, J = 1.5 Hz,

H), 4.41 (d, J = 5.5 Hz, 2H), 4.21 (q, J = 7.0 Hz, 2H), 3.99 (s, 3H),

1

3

.16 (s, 3H), 1.28 (t, J = 7.0 Hz, 3H). C NMR (101 MHz, DMSO-

2

^d6⁾^δ¹⁶⁴^.²^,¹⁵⁸^.³^,¹⁵⁴^.⁸^,¹⁵²^.⁰^,¹⁴⁹^.⁸^,¹⁴⁸^.¹^,¹⁴⁷^.⁰^,¹³⁸^.³⁽^d^,^J⁼³⁵^.²

Hz), 134.1, 125.5, 122.5, 126.8−118.0 (m), 116.0, 105.8, 95.7, 62.3,

4.6, 39.9, 19.8, 14.9.

1-(2-Chloro-6-ethoxy-4-pyridyl)-3-[[5-(1,3-dimethylpyrazol-4-yl)-

-methyl-2-pyridyl]methyl]urea (32). This compound was prepared

from 4c according to the general procedure used for the preparation

yl-2-pyridyl]methyl]acetamide (30). This compound was prepared

from 4c according to the general procedure used for the preparation

4

of 27. LCMS: method A, R : 1.23 min; purity: >99%, m/z = 403.1 (M

t

+

1

+

7

(

2

H) . H NMR (400 MHz, Chloroform-d) δ 8.27 (s, 1H), 7.48−

.39 (m, 2H), 7.28 (s, 1H), 7.19 (dd, J = 8.2, 2.1 Hz, 1H), 7.15−7.10

m, 1H), 6.86 (s, 1H), 4.54 (d, J = 5.0 Hz, 2H), 3.93 (s, 3H), 3.61 (s,

H), 2.24 (d, J = 0.6 Hz, 3H), 2.17 (s, 3H).

,6-Dichloro-N-[[5-(1,3-dimethylpyrazol-4-yl)-4-methyl-2-

4

of 34. LCMS: method B, R : 1.23 min; purity: >99%, m/z = 415.2 (M

t

+

1

+

7

H) . H NMR (400 MHz, DMSO-d ) δ 9.46 (s, 1H), 8.26 (s, 1H),

6

4

.75 (s, 1H), 7.25 (s, 1H), 7.13 (s, 2H), 6.78 (s, 1H), 4.39 (d, J = 5.4

pyridyl]methyl]-1H-pyrrolo[3,2-c]pyridine-2-carboxamide (28). A

solution of HATU (131 mg, 0.35 mmol, 1 eq) in dry DMF (1 mL)

was added to a mixture of [5-(1,3-dimethylpyrazol-4-yl)-4-methyl-2-

pyridyl]methanamine 4c (75 mg, 0.35 mmol, 1 eq), 4,6-dichloro-1H-

pyrrolo[3,2-c]pyridine-2-carboxylic acid (88 mg, 0.38 mmol, 1.1 eq),

and DIPEA (120 μL, 0.69 mmol, 2 eq) in dry DCM (3.5 mL). The

reaction was stirred at 45 °C until full conversion. The reaction

Hz, 2H), 4.22 (q, J = 7.0 Hz, 2H), 3.81 (s, 3H), 2.21 (s, 3H), 2.07 (s,

1

3

1

H), 1.28 (t, J = 7.0 Hz, 3H). C NMR (101 MHz, DMSO-d ) δ

6

64.2, 156.6, 154.8, 152.0, 149.7, 148.1, 146.2, 145.6, 131.4, 128.0,

22.8, 115.4, 105.8, 95.7, 62.3, 44.6, 38.8, 20.1, 14.9, 12.6.

tert-Butyl ((5-Bromo-4-methylpyridin-2-yl)methyl)carbamate

(

1e). NaBH (13.5 g, 357 mmol, 7 eq) was added in portions to a

4

mixture of 5-bromo-4-methylpyridine-2-carbonitrile 1c (10 g, 51

mmol, 1 eq), NiCl ·6H O (2.4 g, 10 mmol, 0.2 eq), and Boc O (33 g,

mixture was partitioned between DCM and saturated NH Cl. The

4

2

two phases were separated. The organic layer was dried (ﬁltered

through phase separator) and concentrated. The residue was puriﬁed

by preparatory HPLC (gradient from 35% MeCN to 60% MeCN in

6

.4 mmol, 5 eq) in MeOH (170 mL) at 0 °C. The reaction mixture

was stirred at room temperature for 1 h. A further 50 mg of NaBH₄

was added, and the reaction was stirred for 1 h. The reaction mixture

was ﬁltered through Celite and concentrated. The residue was taken

water/0.5% NH ) to aﬀord the desired product (15 mg, 10% yield).

3

LCMS: method B, R : 1.15 min; purity: >99%, m/z = 429.2 (M +

t

up in DCM and saturated NaHCO . The two layers were ﬁltered and

+

1

3

H) . H NMR (400 MHz, DMSO-d ) δ 12.63 (d, J = 2.1 Hz, 1H),

6

separated. The organic layer was dried (ﬁltered through phase

separator) and concentrated to aﬀord the desired product (7.5 g, 49%

9

1

.45 (t, J = 6.0 Hz, 1H), 8.26 (s, 1H), 7.74 (s, 1H), 7.47 (dd, J = 2.1,

.0 Hz, 1H), 7.43 (d, J = 0.9 Hz, 1H), 7.28 (s, 1H), 4.60 (d, J = 5.9

+

yield). LCMS: m/z = 300.9 (M + H) .

Hz, 2H), 3.81 (s, 3H), 2.20 (s, 3H), 2.06 (s, 3H).

(5-Bromo-4-methylpyridin-2-yl)methanamine TFA Salt (2e). A

General Procedure for Preparation of Ureas 31 and 33. 1-

mixture of tert-butyl ((5-bromo-4-methylpyridin-2-yl)methyl)-

carbamate 1e (7.5 g, 25 mmol, 1 eq) and TFA (14 mL) in DCM

50 mL) was stirred at 45 °C for 1.5 h. The mixture was concentrated.

(

2,6-Dichloro-4-pyridyl)-3-[[5-(1,3-dimethylpyrazol-4-yl)-4-methyl-

2

-pyridyl]methyl]urea (31). 2,6-Dichloro-4-isocyanatopyridine (131

(

mg, 0.69 mmol, 1 eq) in THF was added to a mixture of [5-(1,3-

dimethylpyrazol-4-yl)-4-methyl-2-pyridyl]methanamine 4c (150 mg,

Toluene (100 mL) was added, and the resulting mixture was

concentrated (this operation was done twice) to yield the desired

0.69 mmol, 1 eq) in THF (7 mL ﬁnal volume). The reaction was

+

product (4.6 g, 58% yield). LCMS: m/z = 200.9 (M + H) .

stirred at room temperature until the disappearance of the starting

material (1.25 h). The mixture was partitioned between DCM and

1

-((5-Bromo-4-methylpyridin-2-yl)methyl)-3-(2-ethoxy-6-

triﬂuoromethyl)pyridin-4-yl)urea (3e). A mixture of 1,1′-carbonyl-

di-(1,2,4-triazole) (CDT, 4.14 g, 25.3 mmol, 1.7 eq), 2-ethoxy-6-

triﬂuoromethyl)pyridin-4-amine 4g (Supporting Information) (5.21

(

H O. The two phases were separated; the organic layer was dried

2

(ﬁltered through phase separator) and concentrated. The residue was

(

puriﬁed by preparatory HPLC (gradient from 20% MeCN to 45%

MeCN in water/0.1% formic acid) to aﬀord the desired product (2

g, 25.3 mmol, 1.7 eq), and pyridine (5.6 mL, 69 mmol, 4.6 eq) in

DCM (115 mL) was stirred at 45 °C for 30 min, and then the content

was added to a ﬂask containing (5-bromo-4-methylpyridin-2-yl)-

methanamine TFA salt (4.6 g, 14.6 mmol, 1 eq) and DIPEA (16 mL,

92 mmol, 6.3 eq) in THF (15 mL). The reaction was stirred at room

mg, 1% yield). LCMS: method B, R : 1.19 min; purity: >99%, m/z =

t

+

1

4

05.1 (M + H) . H NMR (400 MHz, DMSO-d ) δ 10.05 (s, 1H),

6

8

.25 (s, 1H), 7.73 (s, 1H), 7.58−7.48 (m, 3H), 7.25 (s, 1H), 4.40 (d,

J = 5.5 Hz, 2H), 3.81 (s, 3H), 2.21 (s, 3H), 2.06 (s, 3H).

temperature for 2 h. The reaction was quenched with H O. The

2

1

-(3,5-Dichlorophenyl)-3-[[5-(1,3-dimethylpyrazol-4-yl)-4-meth-

resulting mixture was extracted with DCM (3x). The combined

organic phase was washed (1 M HCl), dried (ﬁltered through a phase

separator), and concentrated to aﬀord the desired product (3.2 g, 51%

yl-2-pyridyl]methyl]urea (33). This compound was prepared from 4c

according to the general procedure used for the preparation of 31.

+

LCMS: method B, R : 1.31 min; purity: >99%, m/z = 404.1 (M +

t

yield). LCMS: m/z = 435.1 (M + H) .

+

H) . HRMS: calcd mass for C H Cl N O (M + H) 404.1039;

1

9

20

2

5

1-((5-Bromo-4-methylpyridin-2-yl)methyl)-5-(tert-butyl)-3-(2-

ethoxy-6-(triﬂuoromethyl)pyridin-4-yl)-1,3,5-triazinan-2-one (4e).

A mixture of 1-((5-bromo-4-methylpyridin-2-yl)methyl)-3-(2-

ethoxy-6-(triﬂuoromethyl)pyridin-4-yl)urea 3e (3.2 g, 7.4 mmol, 1

1

found 404.1042; diﬀerence 0.6 ppm. H NMR (400 MHz,

Chloroform-d) δ 8.43 (s, 1H), 8.26 (s, 1H), 7.31−7.24 (m, 4H),

.92 (s, 1H), 6.79 (s, 1H), 4.54 (s, 2H), 3.91 (s, 3H), 2.26 (s, 3H),

.14 (s, 3H).

6

2

eq), formaldehyde (37% H O, 12 mL, 148 mmol, 20 eq), 4-

2

6

051

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methylmorpholine (16.3 mL, 148 mmol, 20 eq), and t-BuNH (15.7

(12.4 g, 45 mmol, 0.7 eq) in DMF (275 mL). The resulting mixture

was stirred at room temperature for 1 h. A 2.5 M aqueous Na CO

3

2

mL, 148 mmol, 20 eq) in 1:1 EtOH/H O (74 mL) was stirred at 100

2

°

C for 1 h. The mixture was diluted with EtOAc and H O. The two

solution (64 mL) was added, and the resulting mixture was stirred at

85 °C until completion. The cooled mixture was ﬁltered on Celite and

washed with EtOAc. The organic extract was washed three times with

brine, dried (Na SO ), and concentrated. Puriﬁcation by silica

2

phases were separated, and the aqueous layer was further extracted

with EtOAc. The combined organic layers were dried (Na SO ) and

2

4

concentrated. The residue was puriﬁed by ﬂash column chromatog-

2

4

raphy (SiO , 90:10 to 30:70 petroleum ether/EtOAc) to aﬀord the

chromatography (column: 120 g silica 25 μm; EtOAc/DCM; 0:100

to 10:90) aﬀorded the desired compound (6.8 g, 40% yield). LCMS:

m/z = 268.0 (M + H) .

2

desired product as a yellow oil (2.5 g, 64% yield). LCMS: m/z = 530.1

+

(

M + H) .

1

-[[5-[(1,3-Dimethylpyrazol-4-yl)amino]-4-methyl-2-pyridyl]-

[5-Methyl-6-[1-methyl-3-(triﬂuoromethyl)pyrazol-4-yl]pyridazin-

3-yl]methanamine HCl (6f). Pd (10% on charcoal, 4.25 g, 4 mmol,

0.15 eq) was added to a degassed mixture of 5-methyl-6-(1-methyl-3-

triﬂuoromethyl-1H-pyrazol-4-yl)-pyridazine-3-carbonitrile 5f (7.3 g,

27.3 mmol, 1 eq) and aq. 6 M HCl (10 mL, 60 mmol, 2.2 eq) in

methanol (180 mL). The mixture was stirred at room temperature

under hydrogen (1 atm) until completion of the reaction. The

resulting mixture was ﬁltered on Celite and washed with methanol

and acetonitrile, and the ﬁltrate was concentrated under reduced

methyl]-3-[2-ethoxy-6-(triﬂuoromethyl)-4-pyridyl]urea (35). A mix-

ture of 1-((5-bromo-4-methylpyridin-2-yl)methyl)-5-(tert-butyl)-3-

(

2-ethoxy-6-(triﬂuoromethyl)pyridin-4-yl)-1,3,5-triazinan-2-one 4e

55 mg, 0.10 mmol, 1 eq), 1,3-dimethyl-1H-pyrazol-4-amine (33

mg, 0.3 mmol, 3 eq), Pd (dba) (9 mg, 0.01 mmol, 0.1 eq), BINAP

2

3

(

12 mg, 0.02 mmol, 0.2 eq), and NaOtBu (14 mg, 0.15 mmol, 1.5 eq)

in dry toluene (0.3 mL) was degassed with N . The mixture was

2

stirred at 70 °C overnight. The mixture was diluted with EtOAc and

washed with H O. The two phases were separated, and the aqueous

pressure. The residue was triturated with Et O to aﬀord the

2

layer was extracted with EtOAc. The combined organic layers were

dried (Na SO ) and concentrated. The residue was taken up in 1:1

hydrochloride salt of the desired compound (8.0 g, 95% yield).

+

2

4

LCMS: m/z = 272.1 (M + H) .

1

,4-dioxane/6 M NaOH (2 mL). The resulting mixture was stirred at

00 °C overnight. The mixture was partitioned between EtOAc and

[5-Methyl-6-[1-methyl-3-(triﬂuoromethyl)pyrazol-4-yl]pyridazin-

3-yl]methanamine (7f). NaBH (462 mg, 12.2 mmol, 5 eq) was

4

H O. The two phases were separated. The organic phase was dried

added portionwise to a mixture of 5-methyl-6-(1-methyl-3-triﬂuor-

omethyl-1H-pyrazol-4-yl)-pyridazine-3-carbonitrile 5f (2.44 mmol, 1

eq), TFA (0.94 mL, 12.2 mmol, 5 eq) and NiCl ·6H O (233 mg, 0.98

2

(

Na SO ) and concentrated. The residue was puriﬁed by preparatory

2 4

HPLC (gradient from 20% MeCN to 45% MeCN in water/0.1%

2

formic acid) to aﬀord the desired product (2.2 mg, 5% yield). LCMS:

mmol, 0.4 eq) in MeOH (10 mL) at 0 °C. After 3 h, a further 230 mg

+

1

method B, R : 1.30 min; purity: >99%, m/z = 464.2 (M + H) . H

of NaBH (6.1 mmol, 2.5 eq) was added. The resulting mixture was

t

4

NMR (400 MHz, DMSO-d ) δ 9.64 (s, 1H), 7.54 (d, J = 4.3 Hz, 2H),

stirred at room temperature for 3 h. The mixture was ﬁltered over

Celite and concentrated under reduced pressure. Puriﬁcation by SCX

resin exchange aﬀorded the desired product as a free base. The

product was used as such in the next step. LCMS: m/z = 272.1 (M +

6

7

6

3

.50 (d, J = 1.6 Hz, 1H), 7.11 (t, J = 5.6 Hz, 1H), 7.04−6.98 (m, 2H),

.49 (s, 1H), 4.32−4.20 (m, 4H), 3.75 (s, 3H), 2.20 (s, 3H), 1.95 (s,

H), 1.30 (t, J = 7.0 Hz, 3H).

+

6

-Chloro-4-methyl-pyridazin-3-ol (2f). 3,6-Dichloro-4-methylpyr-

H) .

idazine 1f (2.97 g, 18.2 mmol, 1 eq) in aqueous 3.3 M NaOH (30

mL) was stirred at reﬂux temperature for 1 h. Once cooled at room

temperature, aqueous 50% acetic acid (10 mL) was added to pH ≈ 6

and the resulting solid ﬁltered and washed with water. Puriﬁcation by

silica chromatography (EtOAc/petroleum ether; 25:75 to 100:0)

General Procedure for Preparation of Ureas 37, 42, 43, and

48. 1-(2-Chloro-6-ethoxypyridin-4-yl)-3-[5-methyl-6-(1-methyl-3-

triﬂuoromethyl-1H-pyrazol-4-yl)-pyridazin-3-ylmethyl]urea (37).

1,1′-Carbonyl-di-(1,2,4-triazole) (726 mg, 4.42 mmol, 1.5 eq) was

added to a mixture of 2-chloro-6-ethoxypyridin-4-ylamine (356 mg,

2.07 mmol, 0.7 eq) in pyridine (0.71 mL, 8.85 mmol, 3 eq) and DCM

(15 mL). The reaction mixture was stirred at 45 °C for 30 min. Once

cooled to room temperature, [5-methyl-6-[1-methyl-3-

(triﬂuoromethyl)pyrazol-4-yl]pyridazin-3-yl]methanamine 7f (800

mg, 2.95 mmol, 1.0 eq) was added and the resulting mixture was

stirred at room temperature for 1 h. The mixture was diluted with

aﬀorded the desired compound (1 g, 38% yield). LCMS: m/z = 144.9,

+

1

46.9 (M + H) .

6

-Hydroxy-5-methylpyridazine-3-carbonitrile (3f). A mixture of

6

-chloro-4-methyl-pyridazin-3-ol 2f (21.7 g, 150 mmol, 1 eq),

Zn(CN) (22.9 g, 195 mmol, 1.3 eq), Pd (dba) (6.87 g, 7.5 mmol,

2

3

0

.05 eq), and 1,1′-bis(diphenylphosphino)ferrocene (6.64 g, 12

mmol, 0.08 eq) in DMF (150 mL) was degassed under nitrogen and

DCM and washed with saturated aqueous NH Cl and saturated

4

heated at 120 °C for 1 h. The resulting mixture was diluted with

aqueous NaHCO . The organic phase was ﬁltered through a phase

3

DCM and saturated NaHCO . The aqueous layer was separated and

separator and concentrated under reduced pressure. The residue was

puriﬁed by silica chromatography (MeOH/DCM; 0:100 to 4:96)

followed by triturated in a mixture of methyl tert-butyl ether/

petroleum ether to aﬀord the desired compound (650 mg, 65% yield).

3

extracted with DCM and DCM/iPrOH (4:1), dried (Na SO ),

2

4

ﬁltered, and concentrated. The residue was triturated with a mixture

of petroleum ether/EtOAc (3:1), ﬁltered, washed with petroleum

ether, and dried to aﬀord the desired compound (17.4 g, 86% yield).

LCMS: method B, R : 1.30 min; purity: >99%, m/z = 470.2 (M +

t

+

LCMS: m/z = 135.9 (M + H) .

H) . HRMS: calcd mass for C H ClF N O (M + H) 470.1314;

found 470.1321; diﬀerence 1.6 ppm. H NMR (400 MHz, DMSO-d )

19 20 3 7 2

1

6

-Chloro-5-methylpyridazine-3-carbonitrile (4f). POCl (31 mL,

3

6

3

33 mmol, 3 eq) was added to 6-hydroxy-5-methylpyridazine-3-

δ 9.47 (s, 1H), 8.29 (s, 1H), 7.56 (d, J = 1.0 Hz, 1H), 7.28 (t, J = 5.6

Hz, 1H), 7.13 (d, J = 1.5 Hz, 1H), 6.78 (d, J = 1.5 Hz, 1H), 4.61 (d, J

= 5.6 Hz, 2H), 4.22 (q, J = 7.0 Hz, 2H), 4.02 (s, 3H), 2.26 (d, J = 0.9

carbonitrile 3f (15 g, 111 mmol, 1 eq) in acetonitrile (200 mL), and

the resulting mixture was heated at reﬂux temperature until

completion of the reaction (4 h). The cooled mixture was

concentrated under reduced pressure, and the residue was diluted

in acetonitrile (200 mL). This solution was poured into a stirred

mixture of ice-cold DCM and aqueous NaHCO . Solid NaHCO was

1

3

Hz, 3H), 1.28 (t, J = 7.0 Hz, 3H). C NMR (101 MHz, DMSO-d ) δ

6

164.2, 159.4, 154.9, 153.5, 151.9, 148.2, 138.7 (q, J = 36.1 Hz), 137.8,

134.3, 126.6, 126.3−117.3 (m), 116.7, 105.9, 95.8, 62.4, 43.3, 40.2,

19.2, 14.9. mp = 205 °C.

3

added in portions to attain a pH of approximately 7. Both layers were

separated, and the aqueous layer was extracted with DCM. The

combined organic layer was dried (Na SO ), ﬁltered, and

General Procedure for Preparation of Ureas 38−41, 46, 47,

and 50−51. 1-(2-Ethoxy-6-triﬂuoromethylpyridin-4-yl)-3-[5-meth-

yl-6-(1-methyl-3-triﬂuoromethyl-1H-pyrazol-4-yl)-pyridazin-3-

ylmethyl]urea (40). A mixture of 1,1′-carbonyl-di-(1,2,4-triazole)

(1.14 g, 6.97 mmol, 1.2 eq) was added to a mixture of 2-ethoxy-6-

(triﬂuoromethyl)pyridin-4-amine 4g (Supporting Information ) (1.44

g, 6.97 mmol, 1.2 eq) in dry pyridine (1.41 mL, 17.4 mmol, 3.0 eq)

and dry DCM (29 mL). The resulting mixture was stirred at 45 °C for

30 min. The abovementioned mixture was added to a suspension of

[5-methyl-6-[1-methyl-3-(triﬂuoromethyl)pyrazol-4-yl]pyridazin-3-

2

4

concentrated to aﬀord the desired compound (16.5 g, 97% yield).

+

LCMS: m/z = 154.0 (M + H) .

5

-Methyl-6-(1-methyl-3-(triﬂuoromethyl)-1H-pyrazol-4-yl)-

pyridazine-3-carbonitrile (5f). Pd(PPh ) (7.44 g, 6.64 mmol, 0.1

3

4

eq) was added to a degassed suspension of 6-chloro-5-methylpyr-

idazine-3-carbonitrile 4f (64 mmol, 1.0 eq) and 1-methyl-4-(4,4,5,5-

tetramethyl-[1,3,2]dioxaborolan-2-yl)-3-triﬂuoromethyl-1H-pyrazole

6

052

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yl]methanamine hydrochloride salt 6f (2.0 g, 5.81 mmol, 1.0 eq) and

diisopropylethylamine (4.0 mL, 4.0 eq) in dry THF (8.0 mL). The

resulting mixture was stirred at room temperature for 30 min. The

reaction was performed four times with the same amounts. After

completion, the reactions were combined. The resulting mixture was

diluted with DCM and water. The organic phase was washed with 1

M HCl solution, ﬁltered through a phase separator, and concentrated

under reduced pressure. The crudes were gathered and puriﬁed by

procedure used for the preparation of 37. LCMS: method B, R :

t

1.31 min; purity: >99%, m/z = 547.3 (M + H) . H NMR (400 MHz,

+

1

DMSO-d ) δ 9.75 (s, 1H), 8.31 (s, 1H), 7.57 (s, 1H), 7.51 (s, 1H),

6

7.43 (t, J = 5.8 Hz, 1H), 7.07 (s, 1H), 4.62 (d, J = 5.5 Hz, 2H), 4.33

(t, J = 5.8 Hz, 2H), 4.02 (s, 3H), 2.59 (t, J = 5.8 Hz, 2H), 2.27 (s,

3H), 2.19 (s, 6H).

1-(6-Chloro-2-ethoxy-3-methyl-4-pyridyl)-3-[[5-methyl-6-[1-

methyl-3-(triﬂuoromethyl)pyrazol-4-yl]pyridazin-3-yl]methyl]urea

(

46 ). This compound was prepared from 6f and 3j (Supporting

ﬂash chromatography on silica gel (eluting with Et O/Acetone 95/5

2

Information ) according to the general procedure used for the

preparation of 40. LCMS: method B, R : 1.38 min; purity: >99%,

m/z = 484.2 (M + H) . HRMS: calcd mass for C H ClF N O (M

to 90/10) to give, after trituration in MTBE/MeCN, the desired

compound (11.0 g, 94% yield). LCMS: method B, R : 1.36 min;

t

+

purity: >99%, m/z = 504.3 (M + H) . HRMS: calcd mass for

C H F N O (M + H) 504.1577; found 504.1586; diﬀerence 1.7

ppm. H NMR (400 MHz, DMSO-d ) δ 9.68 (s, 1H), 8.29 (s, 1H),

20 22

3

7

2

+

1

+

H) 484.1470; found 484.1474; diﬀerence 0.8 ppm. H NMR (400

20

6

7

2

1

MHz, DMSO-d ) δ 8.52 (s, 1H), 8.32 (s, 1H), 7.88 (d, J = 12.8 Hz,

6

2

4

H), 7.61 (s, 1H), 4.64 (d, J = 5.3 Hz, 2H), 4.25 (q, J = 7.0 Hz, 2H),

.03 (s, 3H), 2.28 (s, 3H), 2.03 (s, 3H), 1.31 (t, J = 7.1 Hz, 3H).

7

.57 (s, 1H), 7.52 (d, J = 1.6 Hz, 1H), 7.37 (t, J = 5.5 Hz, 1H), 7.05

(

d, J = 1.6 Hz, 1H), 4.62 (d, J = 5.5 Hz, 2H), 4.29 (q, J = 7.0 Hz, 2H),

.02 (s, 3H), 2.26 (d, J = 0.8 Hz, 3H), 1.31 (t, J = 7.0 Hz, 3H). C

1

3

1

-(6-Cyano-2-ethoxy-3-methyl-4-pyridyl)-3-[[5-methyl-6-[1-

methyl-3-(triﬂuoromethyl)pyrazol-4-yl]pyridazin-3-yl]methyl]urea

47 ). This compound was prepared from 6f and 3m (Supporting

4

NMR (101 MHz, DMSO-d ) δ 165.0, 159.4, 155.0, 153.5, 151.0,

6

(

1

9

44.8 (q, J = 33.5 Hz), 138.7 (q, J = 36.1 Hz), 137.8, 134.3, 126.6,

26.0−117.7 (m), 126.1−117.6 (m), 116.7, 104.5 (d, J = 3.8 Hz),

9.8, 62.3, 43.3, 39.8, 19.2, 14.8. mp = 205 °C.

Information ) according to the general procedure used for the

preparation of 40. LCMS: method B, R : 1.32 min; purity: >99%,

t

+

1

m/z = 475.1 (M + H) . H NMR (400 MHz, DMSO-d ) δ 8.68 (s,

6

1

-[2-Chloro-6-(2,2-diﬂuoroethoxy)-4-pyridyl]-3-[[5-methyl-6-[1-

methyl-3-(triﬂuoromethyl)pyrazol-4-yl]pyridazin-3-yl]methyl]urea

38 ). This compound was prepared from 6f and 2h (Supporting

1

4

2

H), 8.35 (s, 1H), 8.30 (s, 1H), 7.87 (t, J = 5.3 Hz, 1H), 7.61 (s, 1H),

.65 (d, J = 5.3 Hz, 2H), 4.31 (q, J = 7.0 Hz, 2H), 4.02 (s, 3H), 2.30−

.25 (m, 3H), 2.11 (s, 3H), 1.33 (t, J = 7.0 Hz, 3H).

(

Information) according to the general procedure used for the

1

-(2-Ethoxy-3,6-dimethyl-4-pyridyl)-3-[[5-methyl-6-[1-methyl-3-

preparation of 40. LCMS: method B, R : 1.32 min; purity: >99%,

t

+

(triﬂuoromethyl)pyrazol-4-yl]pyridazin-3-yl]methyl]urea (48 ). This

compound was prepared from 7f and 2o (Supporting Information)

according to the general procedure used for the preparation of 37.

m/z = 506.3 (M + H) . HRMS: calcd mass for C H ClF N O (M

H) 506.1125; found 506.1134; diﬀerence 1.7 ppm. H NMR (400

1

9

18

5

7

2

+

1

+

MHz, DMSO-d ) δ 9.57 (s, 1H), 8.29 (s, 1H), 7.57 (d, J = 0.9 Hz,

6

LCMS: method B, R : 1.29 min; purity: >99%, m/z = 464.2 (M +

1

H), 7.35 (t, J = 5.6 Hz, 1H), 7.21 (d, J = 1.5 Hz, 1H), 6.91 (d, J = 1.5

Hz, 1H), 6.35 (tt, J = 54.5, 3.4 Hz, 1H), 4.62 (d, J = 5.6 Hz, 2H), 4.50

td, J = 15.1, 3.4 Hz, 2H), 4.02 (s, 3H), 2.26 (d, J = 0.9 Hz, 3H). ¹³

NMR (101 MHz, DMSO-d ) δ 162.9, 159.3, 154.9, 153.5, 152.3,

t

+

H) . HRMS: calcd mass for C H F N O (M + H) 464.2016;

found 464.2027; diﬀerence 2.3 ppm. H NMR (400 MHz, DMSO-d )

21 25

3

7

2

1

(

C

6

δ 8.30 (d, J = 1.1 Hz, 1H), 8.21 (s, 1H), 7.67 (t, J = 5.5 Hz, 1H),

6

7

2

.63−7.56 (m, 2H), 4.62 (d, J = 5.4 Hz, 2H), 4.26 (q, J = 7.0 Hz,

1

47.9, 138.7 (q, J = 36.1 Hz), 137.8, 134.3, 126.6, 121.9 (q, J = 268.9

Hz), 116.7, 114.4 (t, J = 238.9 Hz), 106.9, 95.9, 64.4 (t, J = 26.6 Hz),

3.3, 40.1, 19.2. mp = 221−222 °C.

-(2-Cyano-6-ethoxy-4-pyridyl)-3-[[5-methyl-6-[1-methyl-3-

triﬂuoromethyl)pyrazol-4-yl]pyridazin-3-yl]methyl]urea (39 ). This

H), 4.02 (s, 3H), 2.30−2.24 (m, 6H), 2.00 (s, 3H), 1.29 (t, J = 7.0

1

3

Hz, 3H). C NMR (101 MHz, DMSO-d ) δ 161.4, 159.5, 155.2,

4

6

1

53.4, 152.2, 147.4, 138.7 (q, J = 36.0 Hz), 137.8, 134.3, 126.8, 121.9

1

(

(q, J = 269.2 Hz), 116.7, 106.9, 102.1, 61.3, 43.3, 39.9, 24.6, 19.2,

15.2, 9.6. mp = 195−196 °C.

compound was prepared from 6f and 2i (Supporting Information )

according to the general procedure used for the preparation of 40.

1-(3-Chloro-2-ethoxy-6-methyl-4-pyridyl)-3-[[5-methyl-6-[1-

methyl-3-(triﬂuoromethyl)pyrazol-4-yl]pyridazin-3-yl]methyl]urea

(50 ). This compound was prepared from 6f and 1n (Supporting

Information) according to the general procedure used for the

LCMS: method B, R : 1.24 min; purity: >99%, m/z = 461.1 (M +

t

+

1

H) . H NMR (400 MHz, DMSO-d ) δ 9.62 (s, 1H), 8.29 (s, 1H),

6

7

1

2

.57 (t, J = 1.5 Hz, 2H), 7.40 (t, J = 5.6 Hz, 1H), 7.13 (d, J = 1.8 Hz,

H), 4.62 (d, J = 5.6 Hz, 2H), 4.27 (q, J = 7.0 Hz, 2H), 4.02 (s, 3H),

.26 (d, J = 0.8 Hz, 3H), 1.30 (t, J = 7.0 Hz, 3H).

preparation of 40. LCMS: method A, R : 1.38 min; purity: >99%,

t

+

m/z = 484.1 (M + H) . HRMS: calcd mass for C H ClF N O (M

H) 484.1470; found 484.1479; diﬀerence 1.8 ppm. H NMR (400

2

0

22

3

7

2

+

1

+

1

-(2-Ethoxy-6-methyl-4-pyridyl)-3-[[5-methyl-6-[1-methyl-3-

MHz, DMSO-d ) δ 8.62 (s, 1H), 8.30 (d, J = 1.1 Hz, 1H), 8.15 (t, J =

(triﬂuoromethyl)pyrazol-4-yl]pyridazin-3-yl]methyl]urea (41 ). This

6

5

.5 Hz, 1H), 7.81 (s, 1H), 7.59 (d, J = 0.9 Hz, 1H), 4.63 (d, J = 5.5

compound was prepared from 6f and 2l (Supporting Information )

according to the general procedure used for the preparation of 40.

Hz, 2H), 4.34 (q, J = 7.1 Hz, 2H), 4.02 (s, 3H), 2.30 (d, J = 0.6 Hz,

3

H), 2.27 (d, J = 0.9 Hz, 3H), 1.32 (t, J = 7.0 Hz, 3H).

1-(3-Cyano-2-ethoxy-6-methyl-4-pyridyl)-3-[[5-methyl-6-[1-

LCMS: method B, R : 1.16 min; purity: >99%, m/z = 450.1 (M +

t

+

1

H) . H NMR (400 MHz, DMSO-d ) δ 9.14 (s, 1H), 8.31 (s, 1H),

6

methyl-3-(triﬂuoromethyl)pyrazol-4-yl]pyridazin-3-yl]methyl]urea

7

.56 (s, 1H), 7.19−7.12 (m, 1H), 6.74 (d, J = 4.3 Hz, 2H), 4.60 (d, J

(51 ). This compound was prepared from 6f and 1q (Supporting

=

5.5 Hz, 2H), 4.22 (q, J = 7.0 Hz, 2H), 4.02 (s, 3H), 2.27 (d, J = 3.2

1

3

Information) according to the general procedure used for the

Hz, 6H), 1.27 (t, J = 7.1 Hz, 3H). C NMR (101 MHz, DMSO-d ) δ

6

preparation of 40. LCMS: method B, R : 1.22 min; purity: >99%,

t

1

2

64.2, 159.6, 156.1, 155.2, 153.4, 150.0, 138.7 (d, J = 36.1 Hz), 137.8,

34.3, 126.6, 126.1−117.1 (m), 116.7, 105.9, 94.4, 61.1, 43.2, 39.9,

4.6, 19.2, 15.1.

+

1

m/z = 475.2 (M + H) . H NMR (400 MHz, DMSO-d ) δ 11.16 (s,

6

1

2

3

H), 9.04 (s, 1H), 8.26 (s, 1H), 7.47 (s, 1H), 6.53 (s, 1H), 5.43 (s,

H), 4.46 (q, J = 7.1 Hz, 2H), 4.01 (s, 3H), 2.35 (s, 3H), 2.21 (s,

H), 1.38 (t, J = 7.0 Hz, 3H).

1

-[2-Chloro-6-(2-methoxyethoxy)-4-pyridyl]-3-[[5-methyl-6-[1-

methyl-3-(triﬂuoromethyl)pyrazol-4-yl]pyridazin-3-yl]methyl]urea

42 ). This compound was prepared from 7f and 3h (Supporting

Calcium Flux Assay. The compounds were assessed for they

(

potential agonist activity and, in the same round, for their potential

antagonist eﬀect.

Agonist Assay. CHO cells stably overexpressing human GPCR

sphingosine 1-phosphate receptor 2 were seeded in 384-well sterile

microplates (50 μL; 7500 cells/well) and were incubated overnight at

Information) according to the general procedure used for the

preparation of 37. LCMS: method B, R : 1.21 min; purity: >99%,

t

+

1

m/z = 500.0 (M + H) . H NMR (400 MHz, DMSO-d ) δ 9.49 (s,

6

1

7

2

4

H), 8.29 (s, 1H), 7.56 (d, J = 1.0 Hz, 1H), 7.31 (t, J = 5.6 Hz, 1H),

.15 (d, J = 1.5 Hz, 1H), 6.81 (d, J = 1.6 Hz, 1H), 4.61 (d, J = 5.5 Hz,

H), 4.33−4.26 (m, 2H), 4.02 (s, 3H), 3.65−3.58 (m, 2H), 3.29 (s,

H), 2.26 (d, J = 0.8 Hz, 3H).

3

7 °C under 5% CO . Cells were subsequently washed twice with 25

2

μL/well of starvation medium (F-12 Ham’s medium containing 0.1%

BSA (FAF)) then starved for 1 h at 37 °C, 5% CO and loaded with

1

-[2-[2-(Dimethylamino)ethoxy]-6-(triﬂuoromethyl)-4-pyridyl]-

2

3

-[[5-methyl-6-[1-methyl-3-(triﬂuoromethyl)pyrazol-4-yl]-

25 μL of Fluo 8 dye diluted in HBSS buﬀer plus 20 mM HEPES

following the recommendations of the manufacturer (Tebu-bio)

complemented with 5 mM probenecid. The cells were incubated for 1

pyridazin-3-yl]methyl]urea (43 ). This compound was prepared from

7f and 2k (Supporting Information ) according to the general

6

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h at 37 °C under 5% CO . Dilution series of compounds were diluted

overnight incubation at 37 °C, 5% CO . The medium was replaced

2

in HBSS buﬀer plus 20 mM HEPES plus 0.1% BSA (FAF). Ten

microliters of the diluted compound was added to the cells using the

FDSS/μCELL, and intracellular calcium changes were immediately

measured by reading the ﬂuorescence for 3 min. The ratio of maximal

signal over background before compound injection was used to

determine compound response.

with 64 μL (384 well) or 160 μL (96 well) starvation medium

containing 1% heat-inactivated FBS, 1% P/S with or without 1.95%

HSA (corresponding to approximately 40% serum) for 16−24 h at 37

°C, 5% CO . Eight microliters of a 10-point serial dilution, 30 μM

2

highest ﬁnal concentration, 1/3 dilution was added to the cells. Plates

were incubated for 1 h at 37 °C, 5% CO followed by the addition of

2

Antagonist Assay. After readout of the agonist activity, the plates

were incubated 15 min at 37 °C. Then, the plates were transferred to

the FDSS/μCELL reader and cells were stimulated with 10 μL of

EC₈₀concentration of sphingosine 1-phosphate. Intracellular calcium

changes were immediately measured by reading the ﬂuorescence for 3

min. The ratio of maximal signal over background before compound

injection was used to determine the compound response. For EC /

20 μL (96 format)/8 μL (384 format) S1P at EC₈₀concentration (1

μM, 96 format, or 0.5 μM, 384 format, ﬁnal concentration for plates

without HSA; 5 μM, 96 format, or 2 μM, 384 format, ﬁnal

concentration for plates with HSA) to the sample and vehicle wells.

No trigger was added to the wells to be used as a positive control.

Plates were incubated for 16−24 h at 37 °C, 5% CO . ELISA plates

2

were coated with 40 μL of 1 μg/mL IL8 capture antibody and

incubated overnight at 4 °C. ELISA plates were blocked using 80 μL

of sucrose blocking buﬀer for 4 h at room temperature. Forty

microliters of the supernatant was harvested on the ELISA plates

followed by overnight incubation at 4 °C. Thirty-ﬁve microliters of 50

ng/mL detection antibody was added to the ELISA plates, which were

incubated for 2 h at room temperature. Subsequently, 35 μL (500 ng/

mL) Strep-HRP was added followed by 45−60 min incubation in the

dark. Lastly, 50 μL of luminol substrate was added and

chemiluminescence was read on the Envision after 5 min room

temperature incubation in the dark.

5

0

IC₅₀determination, a 10-point dilution series of compounds starting

from 23.3 μM and 20 μM highest ﬁnal concentration for agonist and

antagonist, respectively, 1/3 dilution was performed. The obtained

ratios for agonist and antagonist readout were normalized versus

vehicle and EC₁₀₀of S1P as controls for agonist mode and versus

vehicle and EC₈₀of S1P for antagonist mode. From these normalized

data, EC₅₀and IC₅₀of the compounds were derived.

3

5

GTPγS Assay. The [ S] GTPγS assay measures the level of G

protein activation following agonist occupation of a GPCR by

3

5

determining the binding of the non-hydrolyzable analog [ S] GTPγS

to Gα subunits. Compounds (serial dilutions, 96-well LIA plate) and

S1P at EC₈₀concentration were incubated with a mixture consisting

of membranes derived from stable cell lines overexpressing

Free IC50 values were calculated from the following equation:

free IC₅₀= IC₅₀× fu, medium

(1)

3

5

recombinant human S1P2, [ S]GTPγS, and SpA beads (Perkin

Elmer, RPNQ0001). Reactions (50 μL cpd + 30 μL S1P2 membrane

preincubated with PVT-WGA SPA beads +20 μL S1P at EC ) were

where fu, medium was determined from the following equation:

1

8

0

fu, medium = ₍

1

incubated at room temperature for 4 h followed by centrifugation (20

min, 2000 rpm). Plates were read on a TopCount reader (Molecular

Devices, ³⁵S-GTPγS, 1 min readout time/well) immediately after

centrifugation.

protein%)₍

− 1 + 1

)

fu

(2)

where fu is the fraction unbound from the hPPB assay and protein% is

the percentage of serum used in the assay.

Radioligand-Based Binding Assay. The following assay can be

used for determination of S1PR2 binding. The binding assay measures

the potential to compete with radioactively labeled S1P for binding to

the receptor. The assay was performed in a 96-well plate where the

following reagents are added (ﬁnal concentrations: 1% DMSO, 7.5

nM radiolabeled S1P, 20 μM to 1 nM 1/3 serial dilutions of test

compounds). First, 50 μL of compound solution (serial dilution; 4%

DMSO/buﬀer) is added into the assay plate followed by addition of

S1P-Induced Collagen Gel Lattice Contraction. Normal

human lung ﬁbroblasts (Promocell) were treated with 1 ng/mL

TGF-β (Gibco, PHG92I4) for 4 days in FGM-2 culture medium

(Lonza, CC3132). 96 h after seeding the cells in medium containing

TGF-β, and the cells were harvested and embedded in collagen

lattices. Collagen lattices were prepared by mixing 8 parts PureCol

(acidic collagen I solution at 3 mg/mL, Advanced Biomatrix, 5005-

100 mL) with 1 part 10X medium or DPBS and 1 part HEPES 0.2 M

at pH 8.0 (to neutralize the pH of the collagen solution and the

stiﬀening of the gel), and cells were added in a 1 part/volume of the

collagen gel at a cell density of 200,000 cells per gel. Per well of a 24

well plate (Greiner Bio-one, 662160), 550 μL of collagen gel with

cells was used, consisting of 400 μL of PureCol, 50 μL of 10X PBS,

and 50 μL of HEPES 0.2 M at pH 8.0 together with 50 μL of cell

100 μL of a mixture consisting of membrane and scintillation

proximity assay (SpA) beads [mixture consists of 20 μg/well

membranes derived from stable cell lines overexpressing S1PR2, 0.5

mg/well polyvinyltoluene-wheat germ-agglutinin (PVT-WGA) beads

(

Perkin Elmer, RPNQ0001)]. All components were diluted in assay

buﬀer containing 20 mM Tris pH 7.5; 10 mM MgCl ; 100 mM NaCl;

2

0

.4% BSA FAF; 1 mM Na VO ) and incubated for 15 min until

suspension. Plates were incubated for 1.5−2 h at 37 °C 5% CO . After

3

4

2

addition to the assay plate. Subsequently, 50 μL of radioactively

2 h of gel formation, 1 mL of FGM without serum supplemented with

labeled S1P (7.5 nM ﬁnal concentration) was added to the wells

1 ng/mL TGF-β was added to the collagen gel. The plates were

3

(

Sphingosine, D-erythro-[3- H] 1-phosphate; ARC; ART0778). After

incubated for 72 h at 37 °C 5% CO for stress build-up. Seventy-two

2

incubation for 2 h at room temperature, plates were centrifuged at

000 rpm for 20 min. Plates were read on a TopCount reader (Perkin

hours after collagen gel formation, the gels were treated with

compounds diluted in FGM-2 serum free medium. Two hours after

compound incubation, 1 μM S1P (Avanti Polar lipids, 860,492) was

added and the treated gels were released from the sides of the wells

2

Elmer) immediately after centrifugation (readout time, 1 min/well).

IL8 Assay (in the Absence and Presence of 2% HSA). The

assay was performed in two formats, 96 and 384 wells. The values

from the two formats were in good agreement. For the compounds

tested in both assays, IC₅₀values are reported as geometrical means.

For compounds tested only in one of the two assays, IC₅₀values are

reported without distinction. Two conditions were applied: with and

without 2% human serum albumin (HSA, equivalent to approximately

after which the plates were incubated at 37 °C 5% CO for another

2

hour. Images were taken at the end of the assay and analyzed with

ImageJ software.

Liver Microsomal Stability Assay. A 10 mM stock solution of

compound in DMSO was diluted three-fold in DMSO. This pre-

diluted compound solution was then diluted to 2 μΜ in a 100 mM

phosphate buﬀer (pH 7.4) and pre-warmed at 37 °C. This compound

dilution was mixed 1/2 with a microsomal/cofactor mix at 37 °C

under shaking at 300 rpm. Final reaction conditions are as follows:

100 pL incubation volume, 1 μΜ test compound (n = 2), 0.2%

DMSO, 0.5 mg/mL microsomes (Xeno-Tech, Kansas City, KS, USA),

0.6 U/mL glucose-6-phosphate-dehydrogenase (G6PDH, Roche,

10127671001), 3.3 mM mgCb (Sigma, M2670), 3.3 mM glucose-6-

phosphate (Sigma, G-7879), and 1.3 mM NADP+ (Sigma, N-0505).

4

0% serum).

Seven days before seeding, HFL-1 cells were thawed in growth/

seeding medium (F12K medium complemented with 10% heat-

inactivated FBS and 1% Pen/strep) and incubated at 37 °C, 5% CO₂.

Three days before seeding, the cells were split once. Forty (6000

cells/well) or 100 μL (16,000 cells/well) HFL-1 cells were seeded in

growth/seeding medium in 384-well or 96-well plates, respectively.

Plates were incubated at room temperature for 1 h followed by

6

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After 30 min of incubation at 300 rpm and 37 °C, the reaction was

stopped with 600 μL of STOP solution (Acetonitrile with Diclofenac

as internal standard). For the zero-time point, 600 μL of STOP

solution were added to the compound dilution before the microsome

mix was added. The samples of both time points were centrifuged and

ﬁltered, and the supernatant analyzed by LC-MS/MS. The samples

were analyzed on LC/MS-MS with a ﬂow rate of 0.6 mL/min. The

oral route, the compound was formulated in Tween 80/MC 0.5% or

PEG400/MC 0.5%. Before the oral dosing, the animals were deprived

of food for at least 12 h before compound administration and 3 h after

administration. All animals had free access to tap water. Blood

samples were collected under light anesthesia and placed into tubes

containing Li-heparin as an anticoagulant. Blood samples were

collected up to 24 h after dosing (composite design: n = 6 mice

per route, 2 groups of 3 mice sampled at alternate time points). After

centrifugation, the resulting plasma samples were assayed by LC-MS/

MS with a non-good-laboratory-practice (non-GLP)-validated

method. PK parameters were calculated by noncompartmental

analysis using WinNonlin software (Certara, Princeton, NJ, USA).

mobile phase A was 0.1% formic acid in H O, and the mobile phase B

2

was 0.1% formic acid in 90% MeCN and 10% H O. The sample was

2

run under positive or negative ion spray on Pursuit C18, 5 pm (2.0 x

2

0 mm) column, from Agilent. The instrument responses (peak

areas/IS peak area) were referenced to the zero time-point samples

considered as 100%) in order to determine the percentage of

̈

(

PK Rat. These studies were performed with naive male Sprague

compound remaining. Verapamil (1 μΜ) and warfarin (1 μΜ) were

used as reference compounds, as unstable and stable compounds,

respectively. If the microsomal stability values for these controls are

not in the range determined by the historical data, the assay is not

validated. The data on microsomal stability are expressed as a

percentage of the total amount of compound remaining after 30 min

incubation. The solubility of the compound in the ﬁnal test

concentration in 100 mM buﬀer pH 7.4 was checked using a

microscope to indicate whether precipitation occurred. For the

calculation of the half-life, a ﬁrst-order kinetic was assumed. Intrinsic

clearance (Clint) and scaled intrinsic clearance (scaled Clint) values

were obtained with the equations below:

Dawley rats (Janvier France, 6−8 weeks old). Rats were dosed iv via a

bolus in the tail vein with a dose level of 1 mg/kg or orally as a single

oesophageal gavage with a dose level of 5 mg/kg (10 mg/kg for

compound 48). For the iv route, the compound was formulated in

PEG200/water (60/40; v/v). For the oral route, the compound was

formulated in PEG400/MC 0.5%. Before the oral dosing, the animals

were deprived of food for at least 12 h before compound

administration and 3 h after administration. All animals had free

access to tap water. Blood samples were collected under light

anesthesia and placed into tubes containing Li-heparin as an

anticoagulant. Blood samples were collected up to 24 h after dosing

(n = 3 rats per route, n = 2 for the iv PK study of compound 40).

After centrifugation, the resulting plasma samples were assayed by

LC-MS/MS with a non-good-laboratory-practice (non-GLP)-vali-

dated method. PK parameters were calculated by noncompartmental

analysis using WinNonlin software (Certara, Princeton, NJ, USA).

Intrinsic clearance equation:

ln 2

/2^[^mⁱⁿ^]

incubation volume[μL]

Clint[μL mg⁻min ] = _t

1

−1

×

mg of protein

1

̈

(3)

PK Dog. These studies were performed with non-naive male

Beagle dogs (compound 37: age 13.8 months old, origin: Marshall

US, North Rose, NY 14516, USA; compound 40: age 15.8 months

old, origin: Harlan; compound 48: age 17.7 months old, origin:

Harlan). Dogs (n = 3) were dosed iv via a 10 min infusion via a

catheter with a dose level of 1 mg/kg. After a washout of 3 days, they

were dosed orally as a single gavage with a dose level of 5 mg/kg or 1

mg/kg in PEG400/MC 0.5% or MC 0.5%. Before administration by

the po route, animals were fasted for a period of at least 12 h before

treatment, and food was given just after the 3 h blood sampling. All

animals had free access to tap water. Blood samples were taken

without an anesthetic from a jugular or cephalic vein into tubes

containing lithium heparin as an anticoagulant. Blood samples were

taken up to 24 h after the start of infusion. After centrifugation, the

resulting plasma samples were assayed by LC-MS/MS with a non-

good-laboratory-practice (non-GLP)-validated method. PK parame-

ters were calculated by noncompartmental analysis using WinNonlin

software (Certara, Princeton, NJ, USA).

Scaled intrinsic clearance equation:

−

1

−1

scaled Clint[L h kg ]

microsomal protein (mg)

1

−1

=

Clint[μL mg⁻min ] ×

g of liver

liver weight(g)

body weight(kg)

60

1000000

×

(4)

To take into account nonspeciﬁc binding, scaled Clint values were

corrected with the fraction unbound in microsomes (fu, mic),

according to the equation below:

44

Scaled intrinsic clearance unbound (clearance unbound) equation:

−

1

−1

scaled Clint[L h kg ]

−

1

−1

Clint, u[L h kg ] =

fu, mic

(5)

Fu, mic values were experimentally determined. In case it was not

possible, the values were calculated using a global Random Forest

regression model.

Fu, mic Determination. Equilibrium dialysis is a technique used

to measure microsomal binding. Brieﬂy, the assay was performed in a

Prophylactic Bleomycin-Induced Pulmonary Fibrosis 14-

day Mice Model. The aim of these studies is to test the eﬃcacy of

test compounds in a 14-day model of bleomycin-induced pulmonary

ﬁbrosis in mice. The studies were performed at Fidelta Ltd., Zagreb,

Croatia.

45

9

6-well Teﬂon dialysis unit (Dialysis Device, model HTD96b), where

each well consists of two chambers separated by a dialysis membrane

membrane strips, MW cutoﬀ 12−14 kDa, HTDialysis). Inactivated

Animals. These studies were carried out on C57BL/6 N male

mice, supplied by Charles River, Italy, which are acclimatized for 7

days after arrival in an environment maintained at 22 °C, at 55%

relative humidity, with 15−20 air changes per hour under light cycles

of 12 h. Mice were 8 weeks old on arrival for the study involving

compound 40 and 7 weeks old on arrival for the study with

compound 48. Mice pelleted food and water are provided ad libitum.

At least 1 day prior to the start of experiment, all animals were

allocated randomly into groups of 15 (study with compound 40) or

14 (study with compound 48). All animal related research is

conducted in accordance with the 2010/63/EU and National

legislation regulating the use of laboratory animals in scientiﬁc

research and for other purposes (Oﬃcial Gazette 55/13).

(

liver microsomes (pooled human liver microsomes, Xenotech, protein

concentration of 0.5 mg/mL) spiked with a compound (1 μM ﬁnal

concentration, 0.5% DMSO) were added to one chamber and buﬀer

solution (50 mM PBS Buﬀer) to the other side of the well. Each

compound was analyzed in duplicate, for 4 h at 37 °C. At the end of

incubation, both chambers were sampled and analyzed by LC-MS/

MS. The unbound microsomal fraction (fu, mic) was calculated as the

concentration in buﬀer divided by the total concentration in the

microsomal side. Positive controls included in this assay are

terfenadine and verapamil.

̈

PK Mouse. These studies were performed with naive male CD1

Materials. The solvent for the test solutions was prepared by

adding 0.5 g of hydroxyethyl cellulose (Natrosol) into 500 mL of aqua

distillate (0.1%) under continuous stirring without heating for 5 h on

a magnetic stirrer. Anesthetic solution was prepared by adding 1 mL

of Narketan (Narketan 10, Vetoquinol, Bern, Switzerland,

mice (Janvier France, 4−5 weeks old). Mice were dosed iv via a bolus

in the tail vein with a dose level of 1 mg/kg or orally as a single

oesophageal gavage with a dose level of 5 mg/kg. For the iv route, the

compound was formulated in PEG 200/water (60/40; v/v). For the

6

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J. Med. Chem. 2021, 64, 6037−6058

Journal of Medicinal Chemistry

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0

3605877535982) and 0.5 mL of Rompun (Rompun, 2%: Bayer,

Activity of 40 against S1P receptors; activity of 48

against S1P receptors; DR of 40 against receptors and

enzymes from a Cerep broad selectivity panel; steady

state PK and IC coverage of compound 40 (IL8 assay)

Leverkusen, Germany) into 9 mL of saline. The resulting solution was

administered at 10 mL/kg. To prepare a solution for intranasal

challenge (i.n.) challenge, 0.8 mg/mL stock solutions of bleomycin

5

0

(

4

bleomycin sulfate, Enzo Life Sciences, Inc., USA; CAS no. 9041-93-

; cat. no. BML-AP302-0010) were thawed and diluted in 330 μL of

in BLM-induced pulmonary ﬁbrosis model; synthesis of

-aminopyridines intermediates for the synthesis of ﬁnal

4

saline. Prior to i.n. administration, mice were anesthetized i.p. with the

anesthetic solution described above. Fresh pirfenidone formulation

was prepared daily in 0.1% Natrosol formulations to a ﬁnal

concentration of 5 mg/mL. Before dosing, animals were weighed

and the pirfenidone amount administered was adjusted accordingly to

individual weights corresponding to 10 mL/kg body weight, twice

daily p.o., with 7.5 h interval between two administrations. Finally,

test compound solutions were prepared by dissolving a suitable

amount of the test compound in PEG 400 (20% of the ﬁnal volume)

then MC 0.5% (80% of the ﬁnal volume) to reach ﬁnal concentrations

of 1, 0.3, and 0.1 mg/mL, thus yielding compounds for doses of 10, 3,

and 1 mg/kg (compound 40) or in an exact volume of MC 0.5% to

reach a ﬁnal concentration of 0.3, 1, and 3 mg/mL for doses of 3, 10,

and 30 mg/kg (compound 48). Prior to dosing, animals were

weighed, and the amount administered was adjusted accordingly to

individual weights. The application volume of the test doses

corresponds to 10 mL/kg body weight, and the test compounds

were administered p.o. twice daily, with 7.5 h interval between two

administrations.

Study. Animals were examined clinically twice daily. Animals were

weighed daily starting from day 0. On day 14, 2 h post-dosing with

pirfenidone or the test compound, mice were sacriﬁced by anesthetic

overdose. The lungs were excised and weighed individually. For all

groups, the whole superior right lung lobe was placed into a Precellys

tube containing silica beads and immediately snap frozen in liquid

nitrogen and subjected to gene expression analysis. All remaining

lungs were placed into marked bottles containing 10% buﬀered

formalin for further histopathological evaluation. For the study

involving compound 40, two satellite groups were used (n = 6 per

groups) to assess the ssPK for the 3 and 10 mg/kg doses, respectively.

These animals were treated with bleomycin and the test compound

exactly as the other animals in the study. They were sacriﬁced at day

compounds 38−51; synthesis of ﬁnal compounds 44,

45, and 49; synthesis of ﬁnal compounds 36, 52−57, 97,

98, and 101; detailed SAR of investigation of the C5

position of pyridine series; synthesis of ﬁnal compounds

6

5, 72, and 120−124; recombinant CYP inhibition of

compounds 34, 37, and 40; CYP HLM inhibition of

compound 40; chemical stability at diﬀerent pH values

and plasma stability of compound 40; hERG inhibition

of compound 40; LCMS analyses of compounds 34, 40,

and 48 and HRMS spectra of 40 and 48; SAR of

compounds 52−57 (from Figure 5 of the main text);

and IC50 values in S1P2 calcium ﬂux assay (all runs) for

compounds 19 and 20 (PDF )

Molecular formula strings (CSV )

AUTHOR INFORMATION

Corresponding Authors

■

Oscar Mammoliti − Galapagos NV, 2800 Mechelen,

Belgium; orcid.org/0000-0002-1521-9254 ; Phone: +32

5 34 29 00; Email: oscar.mammoliti@glpg.com

Adeline Palisse − Galapagos NV, 2800 Mechelen, Belgium ;

Phone: +32 15 34 29 00; Email: adeline.palisse@glpg.com

1

Authors

Caroline Joannesse − Galapagos NV, 2800 Mechelen,

Belgium

Sandy El Bkassiny − Galapagos NV, 2800 Mechelen, Belgium

Brigitte Allart − Galapagos NV, 2800 Mechelen, Belgium

Alex Jaunet − Galapagos NV, 2800 Mechelen, Belgium

Christel Menet − Galapagos NV, 2800 Mechelen, Belgium

Beatrice Coornaert − Galapagos NV, 2800 Mechelen,

Belgium

7. A minimal target volume of 50 μL of blood was collected into Li-

heparin anticoagulant tubes for each time point. The tubes were kept

on ice until separation. Within maximum 30 min after collection,

blood samples were centrifuged at 2000g for 10 min at 4 °C and the

resulting plasma samples were aliquoted into polypropylene tubes (1 x

Kathleen Sonck − Galapagos NV, 2800 Mechelen, Belgium

Inge Duys − Galapagos NV, 2800 Mechelen, Belgium

Philippe Clément-Lacroix − Galapagos SASU, 93230

Romainville, France

Line Oste − Galapagos NV, 2800 Mechelen, Belgium

Monica Borgonovi − Galapagos SASU, 93230 Romainville,

France

Emanuelle Wakselman − Galapagos SASU, 93230

Romainville, France

Thierry Christophe − Galapagos NV, 2800 Mechelen,

Belgium

Nicolas Houvenaghel − Galapagos NV, 2800 Mechelen,

Belgium

Mia Jans − Galapagos NV, 2800 Mechelen, Belgium

Bertrand Heckmann − Galapagos SASU, 93230 Romainville,

France

̀

Laurent Saniere − Galapagos SASU, 93230 Romainville,

France

Reginald Brys − Galapagos NV, 2800 Mechelen, Belgium

2

5 μL). Samples were stored frozen at −20 °C until analysis. Blood

samples were collected from the tail vein. Lung tissue was collected at

sacriﬁce, after blood sampling for each animal, weighed, placed into

tubes, and stored frozen at −80 °C until analysis.

Sample Analysis, Data Processing, and Statistical Evaluation.

For histopathological evaluation, whole lungs (except sampled

superior right lung) were embedded in paraﬃn and stained with

Mallory’s trichrome. Pulmonary histological changes were assessed

40,41

using Matsuse modiﬁcation of Ashcroft score.

Statistical analysis

and graphical presentation were performed using GraphPad Prism

software (version 5.04). The Mann−Whitney test was employed.

Diﬀerences between groups will be considered statistically signiﬁcant

when p < 0.05. For the ssPK study, after centrifugation, the resulting

plasma samples were assayed by LC-MS/MS with a non-good-

laboratory-practice (non-GLP)-validated method. Lung samples were

homogenized and then processed as the plasma samples. PK

parameters of compound 40 were calculated by noncompartmental

analysis using WinNonlin software (Certara, Princeton, NJ, USA).

All animal experiments performed in the manuscript were

conducted in compliance with institutional guidelines.

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00138

ASSOCIATED CONTENT

■

*

sı Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00138.

Notes

The authors declare no competing ﬁnancial interest.

6

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J. Med. Chem. 2021, 64, 6037−6058

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(

10) Sobel, K.; Monnier, L.; Menyhart, K.; Bolinger, M.; Studer, R.;

ACKNOWLEDGMENTS

■

Nayler, O.; Gatfield, J. FTY720 Phosphate activates sphingosine-1-

phosphate receptor 2 and selectively couples to G α 12/13/rho/rock to

induce myofibroblast contraction. Mol. Pharmacol. 2015, 87, 916−

We are grateful to Stella Adriaens and Tiny Deschrijver for the

work performed in the HPLC puriﬁcation of the ﬁnal

compounds. We thank Marie-Pierre Delage for performing

the HRMS spectra and Pieter Stouten for the description of

the model used for the fu, mic calculations.

9

27.

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M.; de Wries, W.; Julien, B.; Tran-Van-Nhieu, J.; Manin, S.; Poelstra,

K.; Chun, J.; Carpentier, S. The sphingosine 1-phosphate receptor

S1P2 triggers hepatic wound healing. FASEB J. 2007, 21, 2005−2013.

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https://doi.org/10.1021/acs.jmedchem.1c00138

J. Med. Chem. 2021, 64, 6037−6058

Article Doi

Discovery of the S1P2 Antagonist GLPG2938 (1-[2-Ethoxy-6-(trifluoromethyl)-4-pyridyl]-3-[[5-methyl-6-[1-methyl-3-(trifluoromethyl)pyrazol-4-yl]pyridazin-3-yl]methyl]urea), a Preclinical Candidate for the Treatment of Idiopathic Pulmonary Fibrosis

DOI: 10.1021/acs.jmedchem.1c00138

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