Fragment-based lead discovery to identify novel inhibitors that target the ATP binding site of pyruvate dehydrogenase kinases

Bioorg. Med. Chem. 44 (2021) 116283

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Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier.com/locate/bmc

Fragment-based lead discovery to identify novel inhibitors that target the

ATP binding site of pyruvate dehydrogenase kinases

,

Tatsuo Akaki^{a *}, Yuki Bessho^a, Takashi Ito^a, Shingo Fujioka^a, Minoru Ubukata^a, Genki Mori^a,

Kenji Yamanaka^b, Takuya Orita^a, Satoki Doi^a, Tomoko Iwanaga^a, Kazutaka Ikegashira^a,

Yoshiji Hantani^b, Isao Nakanishi^c, Tsuyoshi Adachi^a

^aChemical Research Laboratories, Central Pharmaceutical Research Institute, Japan Tobacco Inc, 1-1, Murasaki-cho, Takatsuki, Osaka 569-1125, Japan

^bBiological/Pharmacological Research Laboratories, Central Pharmaceutical Research Institute, Japan Tobacco Inc, 1-1, Murasaki-cho, Takatsuki, Osaka 569-1125,

Japan

^cComputational Drug Design and Discovery, Department of Pharmaceutical Sciences, Kindai University, 3-4-1, Higashi-osaka, Osaka 577-8502, Japan

A R T I C L E I N F O

A B S T R A C T

Keywords:

A fragment-based lead discovery approach was applied to Pyruvate Dehydrogenase Kinases (PDHKs) to discover

inhibitors against the ATP binding site with novel chemotypes. X-ray fragment screening toward PDHK4 pro-

vided a fragment hit 1 with a characteristic interaction in a deep pocket of the ATP binding site. While known

inhibitors utilize several water molecules in a deep pocket to form water-mediated hydrogen bond interactions,

the fragment hit binds deeper in the pocket with a hydrophobic group. Displacement of a remaining water

molecule in the pocket led to the identiﬁcation of lead compound 7 with a notable improvement in inhibition

potency. This lead compound possessed high ligand efﬁciency (LE) and showed decent selectivity proﬁle. Two

additional lead compounds 10 and 13 with new scaffolds with tricyclic and bicyclic cores were generated by

merging structural information of another fragment hit 2. The characteristic interaction of these novel inhibitors

in a deep pocket provides new structural insights about PDHKs ATP binding site and opens a novel direction for

the development of PDHKs inhibitors.

Fragment-based drug discovery (FBDD)

X-ray fragment hit

Pyruvate Dehydrogenase Kinases (PDHKs)

ATP binding site

1. Introduction

and these known ATP-binders inhibit all PDHK isozymes. ^9–12

Fig. 2 shows the AMP-PNP bound X-ray structure of PDHK4.¹³The

The human pyruvate dehydrogenase complex (PDC) catalyzes the

oxidative decarboxylation of pyruvate with formation of acetyl-CoA.

PDC activity is regulated by pyruvate dehydrogenase kinase (PDHK),

which phosphorylates and inactivates PDC. The reduction of PDC ac-

tivity, which is caused by the induction and activation of PDHK, have

been observed in various diseases such as type 2 diabetes, ischemic heart

disease, peripheral artery disease, pulmonary hypertension, and can-

cers.^1–6There are four isozymes of PDHK (PDHK1, 2, 3, and 4),⁷and it

has been reported that the glucose levels of PDHK2/4 double knockout

mice are lower than single knockout mice bearing only PDHK2 or

PDHK4.⁸It is also known that the expression levels of PDHKs are higher

in patients with the diseases mentioned above.

ATP binding site is located in the N-terminal domain of PDHKs. PDHK is

a member of the GHKL ATPase kinase superfamily, and like other GHKL

family proteins, the adenine binding site includes four conserved water

molecules. WAT1, which bridges Asp293 and the adenine ring, is a

structural water as it forms four hydrogen bonds. Three other conserved

water molecules are involved in a hydrogen bond network in a deeply

buried pocket, mediating interactions with the nearby Leu255, Asn258,

Ile291, and Asp293 residues. Known inhibitors which bind at the

adenine binding site utilize several water molecules to form a water-

mediated interaction in the deep pocket. The resorcinol core, which is

frequently used as a GHKL ATPase inhibitor as in the case of PDHK,

replaces one water molecule (WAT4) with one phenolic hydroxyl group

that is involved in a hydrogen bond network. The loop consisting of

residues 317–330, known as the ‘ATP lid’ trapping the ATP phosphate, is

partially visible in the electron density of the X-ray structure.

To suppress four isozymes of PDHK, we have started our research

program targeting the isoform conserved ATP binding site. Fig. 1 shows

some examples of known PDHK inhibitors against the ATP binding site,

* Corresponding author.

E-mail address: tatsuo.akaki@jt.com (T. Akaki).

https://doi.org/10.1016/j.bmc.2021.116283

Received 13 April 2021; Received in revised form 13 June 2021; Accepted 17 June 2021

Available online 8 July 2021

T. Akaki et al.

Bioorganic & Medicinal Chemistry 44 (2021) 116283

Fig. 1. PDHK inhibitors actting at ATP site.

Fig. 2. X-ray Structure of AMP-PNP bound on PDHK4. (PDB ID: 2E0A). (a)

Protein surface is coloured with red oxygen, blue nitrogen, yellow sulfur and

gray carbon atoms. AMP-PNP is shown in sticks with light green carbons. Water

molecules are represented as red spheres, and dashed lines indicate hydrogen

bonds. (b) Detailed view around adenine binding site. Protein interiors are

shown in gray mesh. Four water molecules (red spheres) are coordinated in the

binding pocket.

To ﬁnd novel chemotypes of inhibitors that target the ATP binding

site of PDHKs, we applied a fragment-based drug discovery (FBDD)

strategy. FBDD has emerged as an effective methodology to identify new

chemotypes utilizing low-molecular-weight fragments.¹⁴As the proba-

bility of ﬁtting into the binding pocket falls dramatically for a bigger

sized compound, FBDD generally gives a higher hit rate compared to

traditional high-throughput screening. As the afﬁnities of the fragment

hits are weak, optimization is generally carried out utilizing X-ray

structural information so that the afﬁnity is increased effectively. Higher

hit rates followed by the rapid afﬁnity increase with structural infor-

mation makes the fragment-based lead discovery approach a potential

strategy to gain novel chemotypes.

Fig. 3. Selected fragment hits from X-ray screening toward PDHK4. (a) PDHK4

is shown in gray cartoons. The fragment hits are shown in sticks with light

green carbons for 1 and cyan carbons for 2. Location number is indicated in

italic on compound 2. (b) Detailed view around fragment binding site. Protein

interiors are shown in gray mesh. Water molecules are represented as red

spheres, and dashed lines indicate hydrogen bonds. (c) X-ray structure of a

resorcinol ligand bound on PDHK2 (PDB ID: 4V25). VER-246608 is shown in

sticks with yellow carbons.

conserved water molecules in the deep pocket to form water-mediated

hydrogen bond interactions with surrounding residues, fragment 1

binds deeper by displacing the WAT3 and WAT4 conserved waters. The

phenyl ring of fragment 1 displaced these waters without forming

hydrogen-bond interaction with surrounding residues. Fragment 2 also

participated in a hydrogen-bonding network with Asp293 and WAT1,

The cyano group displaces one conserved water molecule (WAT4) to

form a hydrogen-bond interaction with Asn258. The hydrogen at the 2-

position of fragment 2 is in close contact with WAT3 to form a CH-O

interaction, and the 3-position cyano group is considered to

strengthen this interaction through an inductive effect. Inhibition ac-

tivity of these fragment hits was measured by a RapidFire mass assay

which detected the PDH reaction product acetyl-coenzyme A, and these

In this report, we describe our FBDD process leading to the genera-

tion of lead compounds with novel chemotypes at the PDHK ATP

binding site.

2. Results and discussion

2.1. Identiﬁcation of fragment hits with novel chemotypes from X-ray

fragment screening

X-ray fragment screening was applied to discover novel chemotypes

of binders against the ATP binding site of PDHKs. X-ray fragment

screening toward PDHK4 using our in-house fragment library of 638

fragments led to the identiﬁcation of 17 fragment hits, corresponding to

a hit rate of 2.66%. All these fragments were found to bind on the ATP

site. Among the gained fragment hits, compounds 1 and 2 were selected

for further structure-guided medicinal chemistry (Fig. 3). These com-

pounds showed a clear binding pose at the ATP binding site in the X-ray

structure. Fragment 1 participated in a hydrogen-bonding network with

Asp293 and the structural water molecule (WAT1) through an amide of

the lactam core, and showed characteristic interaction in a deep pocket.

While known inhibitors like VER-246608 keep at least WAT2 and WAT3

hits did not show measurable inhibition activity at levels up to 1000 μM

on PDHK4. X-ray structures of PDHK2 with these fragment hits were also

obtained, and similar binding poses were observed for each fragment

(PDB codes: see the later “Accession codes” section).

2.2. SBDD optimization of fragment hit 1 into a lead compound

Firstly, a structure-based design approach was applied to fragment

hit 1 to improve PDHKs inhibition activity. Analysis of the X-ray struc-

ture of 1 with PDHK4 revealed that there is a hydrophobic space around

Ile303 (Fig. 4). Compound 3 was purchased to fulﬁll the space with a Me

2

T. Akaki et al.

Bioorganic & Medicinal Chemistry 44 (2021) 116283

bulk water (enthalpy =+3.99 kcal/mol, entropy = +3.12 kcal/mol).

Water molecules at the WAT2 position in MD trajectories are within

hydrogen bonding distance with the Asp293 sidechain, The358 back-

bone, and Ile291 backbone, and as Thr358 and Ile291 are in the β-sheet,

hydrogen bond geometries with these residues are far from ideal. The

unstable enthalpic energy proﬁle on the WAT2 position suggested that

the polar interaction on this hydration site was not enough to overcome

the desolvation penalty of the bound ligand. The thermodynamic proﬁle

of the WaterMap calculation result led to the strategy to hold stable the

conserved water molecule (WAT1), and to displace another unstable

water molecule (WAT2) with a hydrophobic group.

The crystal structure of compound 1 indicated that the 7-position of

the lactam core was the ideal position for attaching substituents to

displace the unstable WAT2 water. Table 1 shows the SAR result of

installing several chemical groups at the R₁position. Compound 4 and 5

were designed to displace the WAT2 water molecule with the alkyl

groups. A methyl aniline compound 7 was designed to displace the

WAT2 water with a methyl substituent, and to form an additional

hydrogen bond interaction with Asp293. A primary aniline compound 6

was also synthesized to form a hydrogen bond interaction with Asp293.

The inhibitory activities toward PDHK2 and PDHK4 were measured by

spectrophotometric assay which detected the residual PDH activity by

measuring the absorbance of NADH at 340 nm. Ethyl compound 4

improved the potency by 20-fold against PDHK2, and cyclopropyl

compound 5 also showed moderate inhibitory activity. Methyl amine

compound 7 further improved the potency which showed IC₅₀values

Fig. 4. (a) Detailed View around Ile303 of X-ray structure of 1 with PDHK4.

Protein surface is coloured with red oxygen, blue nitrogen, yellow sulfur and

gray carbon atoms. Compound 1 is shown in sticks with light green carbons. (b)

PDHK2 and PDHK4 IC₅₀were determined by RapidFireMass Spectroscopy.

Location number is indicated in italic on compound 1. (c) Values in parthness

were percent inhibition at the indicated concentration.

0.59

μM and 12.8 μM against PDHK2 and PDHK4, respectively. As the

activity of the amine compound 6 was very weak (PDHK2 and PDHK4

IC₅₀> 300 μM), this demonstrates the importance of the methyl sub-

stituent of compound 7.

substituent from the 3-position of the lactam core, and exhibited an

Compound 7 was selected as a lead compound and several proﬁles

were measured. PDHK1 and PDHK3 inhibition activities were measured

for 7, and ligand efﬁciency was derived for all PDHK isozymes (Table 2).

Compound 7 inhibited all PDHK isozymes and possessed high ligand

efﬁciency.

increased activity in the RapidFire mass assay (PDHK2 IC₅₀= 50.2 μM,

PDHK4 IC₅₀= 890 μM).

To further improve the inhibition activity, hydration site analysis

using the WaterMap¹⁵calculation was carried out on the compound 1 X-

ray structure (Fig. 5). WaterMap is a molecular dynamics (MD)-based

computational method to detect hydration sites from resultant MD tra-

jectories, and also to calculate the thermodynamic properties (free en-

ergy (ΔG), enthalpy (ΔH) and entropy (-TΔS)) relative to bulk water.

The WaterMap calculation on the compound 1 X-ray structure repro-

duced the hydration sites at two conserved water positions (WAT1 and

WAT2). The detected hydration site at the WAT1 position gave a stable

free energy value (ꢀ 0.37 kcal/mol) relative to the bulk water, and the

hydration site at WAT2 position gave unstable free energy (+7.11 kcal/

mol). The thermodynamic proﬁle at the WAT2 position was calculated

as both enthalpy and entropy values that were unstable relative to the

To evaluate the selectivity proﬁle of lead compound 7, inhibition

assays toward other GHKL family proteins and a kinase panel assay were

conducted (Table 3). BCKDK and HSP90 were selected from GHKL

family proteins, because BCKDK has the highest sequence similarity to

PDHKs, and some PDHKs inhibitors were reported to inhibit HSP90¹⁰

.

The ﬂuorescence polarization assay using labeled geldanamycin was

used for HSP90, the luminescent assay using ADP-Glo™ was used for

BCKDK inhibition assay, and DiscoverX KINOME scan™ was used for the

protein kinase panel assay. Compound 7 showed little inhibition on

HSP90 and BCKDK, and in the DiscoveRx KINOME scan™ assay, showed

more than 50% inhibition for just 2 kinases of MTOR and MAST1 among

59 kinases at 10

μM. These data demonstrate the decent selectivity

proﬁle of lead compound 7.

The X-ray structure of 7 bound to PDHK4 was obtained and

Table 1

Inﬂuence of R₁substituent on PDHK2 and PDHK4 activity.^a

compound

R₁

PDHK2 IC₅₀

(μ

M)

PDHK4 IC₅₀(μM)

3

4

H

279

>300 (8.0%) ^b

11.0

>300 (50%) ^b

5

4.9

43.6

6

7

>300 (47%) ^b

>300 (40%) ^b

0.59

12.8

Fig. 5. WaterMap calculation of comp 1 X-ray structure on PDHK4. (a) Ther-

modynamic proﬁle of WaterMap detected two hydration sites. Free energy

(ΔG), enthalpy (ΔH) and entropy (-TΔS) relative to a bulk water were calcu-

lated through MD calculation. (b) MD trajectories of the water molecule at

WAT2 hydration site.

a

PDHK2 and PDHK4 IC₅₀determined by absorption spectrophotometry

method.

b

Values in parentheses were percent inhibition at the indicated

concentration.

3

T. Akaki et al.

Bioorganic & Medicinal Chemistry 44 (2021) 116283

displace the WAT2 water molecule, and by superposing the WaterMap

MD trajectories at WAT2 position, the geometry is far from ideal to form

a hydrogen bond interaction with the remaining WAT2 molecule. Based

on these results, large activity difference between compound 6 and 7 is

considered to arise from the difference of WAT2 energy proﬁle after the

ligand binding.

Table 2

Inhibition activity and ligand efﬁciency of compound 7 toward all PDHK

isozymes.

Target

IC₅₀

LE

Target

IC₅₀

LE

PDHK1

PDHK2

0.37

0.59

μ

M

0.63

0.61

PDHK3

PDHK4

8.4

μ

M

0.50

0.48

μ

12.8

μ

M

All PDHKs IC₅₀determined by absorption spectrophotometry method.

2.3. Generation of a new scaffold via merging lead compound 7 with

fragment hit 2

Table 3

The X-ray structure of fragment hit 2 was investigated to generate

other lead compounds. The ﬁve-membered ring of compound 2 and lead

compound 7 both interacts with Asp 293 of PDHK4, and by super-

imposing two X-ray structures of PDHK4, these rings overlay well

(Fig. 7). From this overlay result, we undertook the design of a tri-cyclic

core as a potential new scaffold.

Selectivity proﬁle of compound 7.; GHKL family assay and kinase panel assay

result.

Family

Target

Inhibition percentage

GHKL

HSP90

3.2% at 30

14% at 500

μ

M ^a

BCKDK

μ

M ^b

Protein Kinase ^c

44 kinases

13 kinases

MTOR

<20% at 10

μ

M

The cyclopropyl substituent of compound 5 was used for a tricyclic

core as a replacement for the methyl amine of compound 7 in view of the

synthetic tractability, and compound 8 showed the same level of PDHK2

activity as the original compound 5 (Table 4). The weak PDHK4 activity

of compound 8 could be attributed to a limited solubility in the assay

buffer. Addition of a methyl substituent on the pyridine 2-position,

which was designed to form CH-O interaction with Gly295 and

Gly297 backbone carbonyl, led to improve activity of compound 9. And

by changing the core structure from pyridine to pyrimidine, pyrimidine

20–50% at 10

μM

58% at 10

60% at 10

μ

M

MAST1

μM

a

Fluorescence Polarization assay using labeled geldanamycin with BPS

Bioscience assay kits.

b

c

Luminescent kinase assay using ADP-Glo™.

Kinase panel assay was conducted with DiscoverX KINOMEscan™ toward 59

kinases. For details, see the supporting information.

conﬁrmed our design hypothesis (Fig. 6). The carbonyl oxygen keeps the

WAT1-mediated hydrogen bond interaction with Asp293 of fragment hit

1, and one methyl group at the alpha position of the carbonyl occupies

the hydrophobic space around Ile303. The aniline NH of compound 7

formed a polar interaction with the Asp 293 sidechain. The methyl

substituent on aniline, which boosted the activity of compound 7 from

compound 6, displaced the WAT2 water molecule in the buried pocket.

For a primary aniline compound 6, one hydrogen atom on the aniline

substituent is considered to form a polar interaction with Asp 293

sidechain like 7. The second aniline hydrogen is short in length to

10 gave a low μM level IC₅₀inhibition activity for both PDHK2 and

PDHK4. The selectivity toward BCKDK of the same GHKL family protein

was evaluated for compound 10, and this compound showed little

inhibition.

To obtain further chemotypes of inhibitors, the structural and SAR

information of the compound 7 and 10 were transferred into a bicyclic-

core scaffold of fragment hit 2 (Fig. 8). The core structure of fragment hit

2 was changed from a pyridine to a 2-methyl pyrimidine, as the com-

pound 10 improved activity from compound 8 by changing the core

structure in this manner. In addition, to occupy the same space of ter-

minal methyl of compound 7, substituents with the chain length of three

atoms were added at the R₁position of the core structure.

Table 5 shows the SAR result of installing several chemical groups at

the R₁, R₂, and R₃positions on the bicyclic core. Addition of an n-Pr

substituent on the R₁position on compound 2 (compound 11) led to a

slight inhibition activity toward PDHK2. To fulﬁll the pocket interior

with the hydrophobic group like compound 7 and 10, the R₂substituent

was changed from cyano group to halogen and methyl, as in compounds

12, 13, and 14. Compound 14 with a methyl substituent at R₂position

showed IC₅₀values of 14.7 μM for PDHK2, and compound 12 and 13

with halogen substituents further improved activity to a level of single

micromolar IC₅₀for PDHK2. Attaching a chloride on the R₃position

slightly improved activity (from 14 to 15). To further improve activity,

we designed R₁substituents to form an additional polar interaction with

the aspartic acid moiety of PDHKs (Asp293 for PDHK4) as in the methyl

amine of compound 7. Methyl amide 16 and 17 were synthesized to

form a hydrogen bond interaction with the aspartic acid moiety, but

these compounds reduced the activity. Propenyl compound 18 was

–

designed to form a CH-O interaction through alkene C H, and this

compound displayed sub 10 M IC₅₀values both for PDHK2 and PDHK4.

μ

Fig. 6. X-ray Structure of compound 7 on PDHK4. (a) PDHK4 is shown in gray

cartoons. The ligand is shown in sticks with pink carbons. Water molecules are

represented as red spheres, and dashed lines indicate hydrogen bonds. (b)

Detailed view around ligand binding site. Protein interiors are shown in gray

mesh. (c) WaterMap MD trajectories of the water molecule at WAT2 hydration

site on Fig. 5b are superimposed. The methyl substituent of compound 7 dis-

placed the WAT2 position.

Fig. 7. Design strategy of the new tricyclic core scaffold. X-ray poses super-

imposition of a fragment hit 2 (cyan) and a lead compound 7 (purple).

4

T. Akaki et al.

Bioorganic & Medicinal Chemistry 44 (2021) 116283

Table 4

Inhibition activity of tricyclic cores on PDHK2 and PDHK4.^a

compound

X

R₁

PDHK2 IC₅₀

M)

PDHK4 IC₅₀

M)

BCKDK IC₅₀

(

μ

(

μ

(μ

M) ^b

8

C

N

H

3.5

>300 (20%) ^c

9

CH₃

2.0

12.0

5.9

10

0.71

>300 (3.0%) ^c

a

PDHK2 and PDHK4 IC₅₀determined by absorption spectrophotometry

Fig. 9. X-ray Structure of compound 13 on PDHK2. (a) PDHK2 is shown in gray

cartoons. The ligand is shown in sticks with purple carbons. (b) Detailed view

around ligand binding site. Protein interiors are shown in gray mesh. Water

molecules are represented as red spheres, and dashed lines indicate

hydrogen bonds.

method.

b

c

Luminescent kinase assay using ADP-Glo™.

Values in parentheses were percent inhibition at the indicated concentration.

Table 6

Inhibition activity and ligand efﬁciency of key compounds.

a

compound

Number of Heavy

Atoms

PDHK2 IC₅₀

PDHK4 IC₅₀

and LE

^aand LE

12 Atoms

192

μ

M ^b(LE =

345

μ

M ^b(LE =

0.42)

0.39)

1

5

10 Atoms

15 Atoms

958

μ

M ^b(LE =

>2000

μ

M ^b

0.41)

Fig. 8. Schematic representation of transferring compound 7 and 10 infor-

mation on fragment hit 2.

4.9

μM (LE =

43.6

μ

M (LE =

0.48)

0.40)

Table 5

Effect of R₁, R₂and R₃substituents on bicyclic pyrimidoindole

7

14 Atoms

17 Atoms

0.59

μ

M (LE =

12.8 μ

0.61)

0.48)

core.

comp

R₁

R₂

R₃

PDHK2 IC₅₀

(

μ

M) ^a

PDHK4 IC₅₀(μ

M) ^a

10

0.71

μ

5.9

μ

0.49)

0.42)

11

12

13

14

15

16

n-Pr

CN

Cl

H

Cl

>30.0 (34%) ^b

>30.0 (8.5%) ^b

1.8

11.0

Br

2.2

12.4

CH₃

14.7

9.9

>30.0 (17%) ^b

>30.0 (45%) ^b

>100 (34%) ^b

13

14 Atoms

2.2

μM (LE =

12.4 μM (LE =

42.8

0.55)

0.48)

17

18

Br

Cl

>30.0 (21%) ^b

>30.0 (3.1%) ^b

a

1.5

8.0

PDHK2 and PDHK4 IC₅₀determined by absorption spectrophotometry

method.

b

PDHK2 and PDHK4 IC₅₀determined by RapidFireMass Spectroscopy

a

PDHK2 and PDHK4 IC₅₀determined by absorption spectrophotometry

method.

b

Values in parentheses were percent inhibition at the indicated

concentration.

corresponds to LE values of 0.42 and 0.39 respectively. A fragment hit 1

binds deeper from a resorcinol core in the pocket, and displayed a same

level of LE value with the resorcinol toward PDHK2. LE values of com-

pound 5 and 7 were improved from fragment hit 1 by displacing a

conserved WAT2 water molecule interior. Compound 7 with a methyl

amine substituent gave higher ligand efﬁciency compared to compound

5 with a cyclopropyl substituent, as the methyl amine forms an addi-

tional polar interaction with the aspartic acid moiety of PDHKs (Asp293

for PDHK4). Starting from X-ray fragment hit 1 with 10 heavy atoms,

addition of 4 heavy atoms increased the PDHK2 activity more than 1000

times with 7. For tricyclic series, weaker cyclopropyl substituent was

used in view of the synthetic tractability, and compound 10 gave

improved LE value from corresponding compound 5. Compound 13 of

bicyclic series, which has n-Pr alkyl substituent like cyclopropyl, also

improved LE value from compound 5 to reach the same level of LE with

Fig. 9 shows the X-ray co-crystal structure of compound 13 with

PDHK2. The core keeps the hydrogen bond interaction with Asp290,

which corresponds to Asp293 of PDHK4, and WAT1 of fragment hit 2.

The n-Pr and bromide substituents of 13 displaced all the three

conserved interior water molecules.

2.4. Ligand efﬁciency of the obtained key compounds

Table 6 summarizes the inhibition activity and the ligand efﬁciency

of the obtained key compounds. Inhibition activity of a commercially

available resorcinol, methyl 2,4-dihydroxybenzoate, was also measured

and displayed IC₅₀values of 192 μM for PDHK2 and 345 μM for PDHK4,

5

T. Akaki et al.

Bioorganic & Medicinal Chemistry 44 (2021) 116283

compound 7.

c

b

a

33 : R=H

30

31

32

34 : R=Me

2.5. Chemistry

d

Compounds 4–7 were synthesized as shown in Scheme 1. After

protecting NH moiety of 7-bromo isatin 19 with PMB, the Wolff–Kishner

reduction gave lactam 21. Methylation of 21 with methyl iodide pro-

duced the key intermediate 22. Palladium-catalyzed boration of 22 with

vinyl pinacol diborane gave 23. 23 was hydrogenated with palladium

hydroxide on carbon, and subsequent PMB deprotection gave compound

4. PMB deprotection of 22 followed by palladium-catalyzed boration

afforded compound 5. Palladium catalyzed carbonylation of 22 gave 27,

and subsequent Curtius rearrangement produced the Boc-protected 28.

Deprotection of PMB and Boc with HBr-AcOH produced compound 6,

and 7 was prepared in the same procedure after methylation.

35 : R = H

8 : R = H

36 : R = Me

9 : R = Me

g

f

e

37

39

40

38

j

h

i

41

42

10

Scheme 2.

Compounds 8–10 were synthesized as shown in Scheme 2. The

palladium-catalyzed Suzuki-Miyaura cross-coupling reaction of 2-bro-

moaniline 30 with cyclopropylboronic acid gave the cyclo-

propylbenzene 31, and conversion of the amino group to the iodine via

the Sandmeyer reaction provided the iodobenzene 32. The palladium-

mediated Buchwald-Hartwig type amination of the iodobenzene 32

and the aminopyridine 33 gave the compound 35, and the subsequent

intramolecular Heck-type cyclization provided the pyridoindole 8.

Pyridoindole 9 was prepared by the same procedure from 32 and 34.

The synthesis of compound 10 was started from 1-bromo-3-ﬂuoro-2-

nitrobenzene 37. The ﬂuorine in compound 38, which was prepared

from 37 and cyclopropylboronic acid by conducting the Suzuki-Miyaura

cross-coupling reaction, was substituted by ethyl cyanoacetate to obtain

39. Iron-mediated reduction of the nitro group in 39 was accompanied

with direct cyclization and gave the indole 40. The reaction of 40 and

acetonitrile, treated under acidic conditions of 4N-HCl/dioxane ﬁrst,

then under basic conditions of saturated NaHCO₃solution, gave the

pyrimidoindole intermediate 41. The hydroxyl group in 41 was con-

verted to the chlorine 42 by treating with thionyl chloride, and subse-

quent hydrogenation with palladium hydroxide on carbon gave

pyrimidoindole 10.

Compounds 11–15 were synthesized as shown in Scheme 3. For the

synthesis of 11, 4-chloro-2-methyl-7H-pyrrolo[2,3-d]pyrimidine 43 was

protected by SEM to give 44. Next, the 6-position of pyrrolopyrimidine

was propylated and converted into 45, and the chloro was removed

reductively to obtain 46. 46 was brominated with NBS to give 5-bromo

compound 47. Finally, cyanation of 47 and deprotection of SEM of 48

with BF₃-Et₂O yielded 11. In the synthesis of 12 and 13, compound 50

was synthesized by the Sonogashira reaction of 5-iodo-2-methylpyrimi-

din-4-amine 49 with 1-penthyne, and then pyrrole cyclization under

basic conditions gave 51. NCS-Chlorination and NBS-bromination of 51

gave 12 and 13, respectively. The synthesis of 14 and 15 was started

from 45 and bromination at the 5-position led to 52. Conversion of the

bromo of 52 to a methyl and then removal of SEM of 53 using TFA gave

compound 15. Subsequent reductive dehalogenation of chlorine gave

14.

Compounds 16–18 were synthesized as shown in Scheme 4. Meth-

ylamide 16 was synthesized from 4-chloro-2-methyl-7H-pyrrolo[2,3-d]

pyrimidine 43. Similar to the synthesis of 53, compound 43 was

brominated to 54 and subsequently methylated to 55. After Ts protec-

tion of the pyrrole nitrogen, introduction of the ethyl ester to the 6-po-

sition of 56 by anion reaction with ethyl carbonochloridate and n-BuLi

gave compound 57. After hydrolysis of the ester and deprotection of Ts

with lithium hydroxide at the same time, to obtain carboxylic acid 58

was obtained. Methylamine, WSC, and DMAP were used to give meth-

ylamide 16. Compounds 17 and 18 were both synthesized from 59 ob-

tained by formylation of 44. The Wittig reaction of the aldehyde 59

yielded trans-butene 60, which was converted to 18 by SEM depro-

tection and NCS-chlorination. Carboxylic acid intermediate 62 was ob-

tained from aldehyde 59 by the Pinnick oxidation. Subsequent

amidation of 62 gave methyl amide 63. NBS-Bromination gave 5-bromo

c

b

a

19

20

21

22

d

f

e

24

23

4

h

g

22

b

d

f

25

5

e

a

c

11

47 : R = Br

45 : R = Cl

46 : R = H

43 : R = H

48 : R = CN

i

j

k

44 : R = SEM

22

g

h

27

26

j

i

l

49

50

13 : R = Br

51 : R = H

12 : R = Cl

6

m

n

45

15 : R = Cl

14 : R = H

28

n

k

52 : R = Br

53 : R = Me

l

29

7

Scheme 1.

Scheme 3.

6

T. Akaki et al.

Bioorganic & Medicinal Chemistry 44 (2021) 116283

mass spectra (HRMS) analyses were performed on an LC-MS system

composed of Agilent 1290 Inﬁnity LC and Thermo Fisher Orbitrap ID-X.

e

c

4.1.1. Preparation of compound 1,2, and 3

1,2, and 3 were obtained from commercial suppliers and used

without further puriﬁcation.

56 : R = H

58 : R = COOH

43 : R = H

54 : R = Br

55 : R = Me

d

f

a

57 : R = COOEt

16 : R = CONHMe

b

4.1.2. Synthesis of compound 4

Step 1: 7-Bromo-1-(4-methoxybenzyl)indoline-2,3-dione (20)

7-Bromo isatin (19; 2.5 g, 11.1 mmol) in DMF (10 mL) was added by

drops, using a dropping funnel, into a solution of 60% sodium hydride

(66 8 mg, 16.7 mmol) in DMF (20 mL) at 0 ^◦C. Para- Methoxy benzyl

chloride (2.3 mL, 16.7 mmol) was added dropwise at 0 ^◦C, and the

mixture was stirred at room temperature for 1.5 h. The reaction mixture

was quenched with methanol (2 mL) at 0 ^◦C and was diluted with H₂O

and EtOAc. The organic layer was washed with H₂O and brine, and dried

over Na₂SO₄. After ﬁltration and concentration, the residue was puriﬁed

by ﬂash chromatography (solvent: EtOAc/CHCl₃= 1/30 to 1/20) to give

the title compound 20 (1.75 g, 46% yield).

i

h

61 : R = H

18 : R = Cl

60

44

j

g

k

59 : R = CHO

62 : R = COOH

l

63 : R = CONHMe

n

m

17

64

¹H NMR (400 MHz, CDCl₃) δ: 3.78 (s, 3H), 5.38 (s, 2H), 6.86 (d, J =

8.8 Hz, 2H), 6.99 (dd, J = 8.0, 7.2 Hz, 1H), 7.23 (d, J = 8.8 Hz, 2H), 7.61

(dd, J = 7.2, 1.2 Hz, 1H), 7.67 (dd, J = 8.0, 1.2 Hz, 1H)

Scheme 4.

compound 64 and SEM removal gave compound 17.

Step 2: 7-Bromo-1-(4-methoxybenzyl)indolin-2-one (21)

3. Conclusions

Hydrazine monohydrate (2 mL) was added to 20 (1.75 g, 5.06 mmol)

◦

in EtOH (20 mL) and stirred at 95 C for 12 h. After cooling to room

Three lead compounds with novel chemotypes that bound to the ATP

binding site of PDHKs were successfully discovered by a fragment-based

drug discovery approach. X-ray fragment screening toward PDHK4 was

carried out to ﬁnd ligands with novel chemotypes, and fragment 1 with

the characteristic interaction in a deep pocket was selected for further

optimization. Fragment hit 1 was bound more deeply in the binding

pocket than the preceding inhibitors, and the analysis of the X-ray

structure with WaterMap calculations provided the strategy to displace a

remaining water molecule with a hydrophobic group. This strategy led

to the discovery of compound 7 with a signiﬁcant improvement in in-

hibition potency. Compound 7 showed IC₅₀values in low micromolar

range against all PDHK isozymes with high ligand efﬁciency, and the X-

ray structure of this compound with PDHK4 conﬁrmed our design

strategy. All three conserved water molecules in the deep pocket were

displaced, and a hydrophobic group occupied the pocket interior.

Compound 7 was selected as the ﬁrst lead compound, and the selectivity

proﬁle was evaluated. The compound 7 showed little inhibition on

HSP90 and BCKDK in the same GHKL family proteins, and showed

decent selectivity proﬁle in the kinase panel assay. To generate new lead

compounds, we designed a tri-cyclic core as a potential new scaffold by

superposing the binding pose of another fragment hit 2 with that of

compound 7. This merging strategy led to identifying a second lead

compound 10 with a tri-cyclic core. SAR information was successfully

transferred into the original fragment 2 bi-cyclic core giving the third

lead compound 13. The characteristic interaction of these novel in-

hibitors in a deep pocket provides new structural insights into the

PDHKs ATP binding site and opens a novel direction for the develop-

ment of more PDHKs inhibitors.

temperature, H₂O and EtOAc were added to the reaction mixture. The

organic layer was washed with H₂O and brine, and dried over Na₂SO₄.

After ﬁltration and concentration, the residue was puriﬁed by ﬂash

chromatography (solvent: EtOAc/Hexane = 1/5, 1/4 to 1/3) to give the

title compound 21 (876 mg, 52% yield).

¹H NMR (400 MHz, CDCl₃) δ: 3.77 (s, 3H), 5.34 (s, 2H), 6.84 (d, J =

8.8 Hz, 2H), 6.86–6.92 (m, 2H), 7.16–7.21 (m, 3H), 7.28–7.32 (m, 1H),

7.35 (dd, J = 8.4, 1.2 Hz, 1H)

Step 3: 7-Bromo-1-(4-methoxybenzyl)-3,3-dimethylindolin-2-one

(22)

Compound 21 (876 mg, 2.64 mmol) in DMF (6 mL) was dropped into

the solution of 60% sodium hydride (232 mg, 5.81 mmol) in DMF (6 mL)

at 0 ^◦C. Methyl iodide (489

μL, 7.92 mmol) was added dropwise, and the

mixture was stirred at room temperature for 2 h. The reaction mixture

was quenched with water at 0 ^◦C, and extracted with EtOAc. The organic

layer was washed with H₂O and brine, and dried over Na₂SO₄. After

ﬁltration and concentration, the residue was puriﬁed by ﬂash chroma-

tography (solvent: EtOAc/Hexane = 1/6 to 1/5) to give the title com-

pound 22 (889 mg, 93% yield).

¹H NMR (400 MHz, CDCl₃) δ: 1.42 (s, 6H), 3.77 (s, 3H), 5.34 (s, 2H),

6.83 (d, J = 8.8 Hz, 2H), 6.90 (dd, J = 8.4, 7.2 Hz, 1H), 7.12–7.16 (m,

3H), 7.31 (dd, J = 8.4, 1.2 Hz, 1H)

Step 4: 1-(4-methoxybenzyl)-3,3-dimethyl-7-vinylindolin-2-one (23)

A mixture of 22 (100 mg, 0.28 mmol), vinyl pinacol diborane (71 μL,

0.42 mmol) and Na₂CO₃(89.0 mg, 0.84 mmol) in DME (2 mL) and H₂O

(1 mL) was treated with PdCl₂(dppf)-CH₂Cl₂(11.0 mg, 0.014 mmol) and

◦

stirred at 95 C for 3 h. After cooling to room temperature, H₂O and

EtOAc were added to the reaction mixture. The organic layer was

washed with H₂O and brine, and dried over Na₂SO₄. After ﬁltration and

concentration, the residue was puriﬁed by ﬂash chromatography (sol-

vent: EtOAc/Hexane = 1/5) to give the title compound 23 (80.0 mg,

93% yield).

4. Experimental section

4.1. Chemistry

¹H NMR (400 MHz, CDCl₃) δ: 1.44 (s, 6H), 3.78 (s, 3H), 5.08 (s, 2H),

5.20 (dd, J = 10.8, 1.2 Hz, 1H),5.48 (dd, J = 17.2, 1.2 Hz, 1H),

6.81–6.88 (m, 3H), 7.03 (dd, J = 7.6, 7.6 Hz, 1H), 7.09 (d, J = 8.8 Hz,

2H), 7.15–7.18 (m, 2H)

Solvents and reagents were obtained from commercial suppliers and

used as received. Flash column chromatography was performed using

Merck 230–400 mesh silica gel 60. Proton nuclear magnetic resonance

(1H NMR) spectra were recorded on a Varian MERCURYplus-AS400,

JEOL RESONANCE Inc. JNM-AL400, Bruker BioSpin K.K. AV400 or

AVANCE III 400, or Agilent Technologies Inc. 400-MR spectrometer in

the indicated solvent. Chemical shifts (δ) are reported in parts per

million relative to internal standard tetramethylsilane. High-resolution

Step 5: 7-Ethyl-1-(4-methoxybenzyl)-3,3-dimethylindolin-2-one

(24)

To a solution of 23 (80.0 mg, 0.26 mmol) in MeOH (3 mL) was added

Pd(OH)₂(10 mg) and stirred under 1.0 atm of hydrogen at room tem-

perature for 12 h. After removal of the catalyst by Celite® ﬁltration, the

7

T. Akaki et al.

Bioorganic & Medicinal Chemistry 44 (2021) 116283

ﬁltrate was evaporated in vacuo to give the title compound 24 (73.0 mg,

91% yield).

to 0 ^◦C, aqueous 2 N HCl (1.1 mL, 2.12 mmol) was added. After evap-

orating in vacuo, H₂O and CHCl₃were added to the residue. The organic

layer was washed with H₂O and brine, and dried over Na₂SO₄. After

ﬁltration, the solvent was evaporated in vacuo to give the title compound

27 (360 mg, overweight) as a crude product. This was used in the next

step without further puriﬁcation.

¹H NMR (400 MHz, CDCl₃) δ: 1.10 (t, J = 7.6 Hz, 3H), 1.44 (s, 6H),

2.39 (q, J = 7.6 Hz, 2H), 3.77 (s, 3H), 5.10 (s, 2H), 6.83 (d, J = 8.8 Hz,

1H), 6.99–7.01 (m, 2H), 7.04 (d, J = 8.8 Hz, 2H), 7.10 (dd, J = 6.2, 2.2

Hz, 1H)

Step 6: 7-Ethyl-3,3-dimethylindolin-2-one (4)

¹H NMR (400 MHz, CDCl₃) δ: 1.47 (s, 6H), 3.71 (s, 3H), 5.28 (s, 2H),

6.73 (d, J = 8.6 Hz, 2H), 6.95 (d, J = 8.6 Hz, 2H), 7.08 (dd, J = 8.0, 7.2

Hz, 1H), 7.38 (dd, J = 7.2, 1.2 Hz, 1H), 7.56 (dd, J = 8.0, 1.2 Hz, 1H)

Step 3: tert-Butyl (1-(4-methoxybenzyl)-3,3-dimethyl-2-oxoindolin-

7-yl)carbamate (28)

TFA (2 mL) was added to 24 (73.0 mg, 0.24 mmol) and stirred at

60 ^◦C for 2 h. The reaction mixture was evaporated in vacuo and

quenched with saturated aqueous NaHCO₃. EtOAc was added, and the

organic layer was separated. The organic layer was washed with H₂O

and brine, and dried over Na₂SO₄. After ﬁltration and concentration, the

residue was puriﬁed by preparative TLC (solvent: EtOAc/Hexane = 1/3)

to give the title compound 4 (28.0 mg, 62% yield).

To a solution of 27 (360 mg, approx.1.06 mmol) in toluene (5 mL)

were added Et₃N (233 μL, 1.67 mmol) and DPPA (310 μL, 1.44 mmol)

and stirred at 85 ^◦C for 10 min. tBuOH (1 mL) was added at room

¹H NMR (400 MHz, CDCl₃) δ: 1.26 (t, J = 7.6 Hz, 3H), 1.40 (s, 6H),

2.60 (q, J = 7.6 Hz, 2H), 6.98–7.07 (m, 3H), 7.91 (brs, 1H)

HRMS (ESI, m/z) calced for C₁₂H₁₄ON (Mꢀ H)^-188.1081, found

188.1082

temperature and stirred at 60 C for 3 h. After cooling to room tem-

◦

perature, H₂O and EtOAc were added to the reaction mixture. The

organic layer was washed with H₂O and brine, and dried over Na₂SO₄.

After ﬁltration and concentration, the residue was puriﬁed by ﬂash

chromatography (solvent: EtOAc/Hexane = 1/6 to 1/4) to give the title

compound 28 (290 mg, 19% yield for 2 steps).

4.1.3. Synthesis of compound 5

Step 1: 7-Bromo-3,3-dimethylindolin-2-one (25)

¹H NMR (400 MHz, CDCl₃) δ: 1.47 (s, 6H), 1.50 (s, 9H), 3.78 (s, 3H),

5.04 (s, 2H), 6.87 (d, J = 8.8 Hz, 2H), 6.99–7.08 (m, 2H), 7.13–7.18 (m,

3H)

TFA (2 mL) was added to 22 (310 mg, 0.86 mmol) and stirred at

50 ^◦C for 2 h. The reaction mixture was evaporated in vacuo and

quenched with saturated aqueous NaHCO₃. EtOAc was added, and the

organic layer was separated. The organic layer was washed with H₂O

and brine, and dried over Na₂SO₄. After ﬁltration and concentration, the

residue was puriﬁed by preparative TLC (solvent: EtOAc/CHCl₃= 1/10)

to give the title compound 25 (155 mg, 75% yield).

Step 4: 7-Amino-3,3-dimethylindolin-2-one (6)

HBr-AcOH (1 mL) was added to 28 (50.0 mg, 0.169 mmol) and

stirred at r.t. for 16 h. After cooling to 0 ^◦C, the reactant mixture was

diluted with sat. aqueous NaHCO₃, and extracted with AcOEt. The

organic layer was washed with H₂O and brine, dried over Na₂SO₄. After

ﬁltration and concentration, the residue was puriﬁed by preparative TLC

(solvent: EtOAc/CHCl₃= 1/1) to give the title compound 6 (4.0 mg,

13% yield).

¹H NMR (400 MHz, CDCl₃) δ: 1.39 (s, 6H), 6.92 (dd, J = 8.2, 7.2 Hz,

1H), 7.10 (dd, J = 7.2, 1.0 Hz, 1H), 7.31 (dd, J = 8.2, 1.0 Hz), 7.47 (brs,

1H)

Step 2: 7-Cyclopropyl-3,3-dimethylindolin-2-one (5)

¹H NMR (400 MHz, CDCl₃) δ: 1.38 (s, 6H), 6.63 (dd, J = 8.2, 1.4 Hz,

1H), 6.68 (dd, J = 7.2, 1.4 Hz), 6.90 (dd, J = 8.2, 7.2 Hz, 1H), 9.81 (brs,

1H)

A mixture of 25 (80.0 mg, 0.33 mmol), cyclopropyl boronic acid

(43.0 mg, 0.50 mmol) and Na₂CO₃(140 mg, 1.32 mmol) in DME (3 mL)

and H₂O (1 mL) was treated with PdCl₂(dppf)-CH₂Cl₂(24.0 mg, 0.03

mmol) and stirred at 95 ^◦C for 7 h. After cooling to room temperature,

H₂O and EtOAc were added to the reaction mixture. The organic layer

was washed with H₂O and brine, and dried over Na₂SO₄. After ﬁltration

and concentration, the residue was puriﬁed by preparative TLC (solvent:

EtOAc/Hexane = 1/2) to give the title compound 5 (18.0 mg, 27%

yield).

HRMS (ESI, m/z) calced for C₁₀H₁₁ON₂(Mꢀ H)^-175.0877, found

175.0878

4.1.5. Synthesis of compound 7

Step 1: tert-Butyl (1-(4-methoxybenzyl)-3,3-dimethyl-2-oxoindolin-

7-yl)(methyl)carbamate (29)

28 (145 mg, 0.37 mmol) in DMF (2 mL) was dropped into a solution

¹H NMR (400 MHz, CDCl₃) δ: 0.61–0.73 (m, 2H), 0.88–1.00 (m, 2H),

1.40 (s, 6H), 1.73–1.80 (m, 1H), 6.95–7.04 (m, 3H), 7.75 (brs, 1H)

HRMS (ESI, m/z) calced for C₁₃H₁₄ON (Mꢀ H)^-200.1081, found

200.1081

of 60% sodium hydride (18.0 mg, 0.44 mmol) in DMF (1 mL) at 0 ^◦C.

Methyl iodide (35.0 μ

L, 0.56 mmol) was added dropwise at 0 ^◦C, and the

mixture was stirred at room temperature for 3 h⋅H₂O and AcOEt were

added to the reaction mixture, and the organic layer was separated. The

organic layer was washed with H₂O and brine, dried over Na₂SO₄. After

ﬁltration, the solvent was evaporated in vacuo to give the title compound

29 (172 mg, overweight) as a crude product. This was used in the next

step without further puriﬁcation.

4.1.4. Synthesis of compound 6

Step 1: Ethyl 1-(4-methoxybenzyl)-3,3-dimethyl-2-oxoindoline-7-

carboxylate (26)

To a solution of 22 (400 mg, 1.11 mmol) in EtOH (10 mL) was added

PdCl₂(dppf)-CH₂Cl₂(180 mg, 0.22 mmol) and Et₃N (1.5 mL, 11.1

mmol), and stirred under 1.0 atm of carbon monoxide at 80 ^◦C for 12 h.

After cooling to room temperature, H₂O and EtOAc were added to the

reaction mixture. The organic layer was washed with H₂O and brine, and

dried over Na₂SO₄. After ﬁltration and concentration, the residue was

puriﬁed by ﬂash chromatography (solvent: EtOAc/Hexane = 1/6 to 1/4)

to give the title compound 26 (374 mg, 95% yield).

Step 2: 3,3-Dimethyl-7-(methylamino)indolin-2-one (7)

TFA (2 mL) was added to 29 (172 mg, approx. 0.37 mmol) in CHCl₃

(0.5 mL) and stirred at 80 ^◦C for 24 h. The reaction mixture was evap-

orated in vacuo and quenched with saturated aqueous NaHCO₃. CHCl₃

was added, and the organic layer was separated. The organic layer was

washed with H₂O and brine, dried over Na₂SO₄. After ﬁltration and

concentration, the residue was puriﬁed by preparative TLC (solvent:

EtOAc/CHCl₃= 1/5) to give the title compound 7 (50.0 mg, 70% yield

for 2 steps).

¹H NMR (400 MHz, CDCl₃) δ: 1.20 (t, J = 7.2 Hz, 3H), 1.45 (s, 6H),

3.73 (s, 3H), 4.14 (q, J = 7.2 Hz, 2H), 5.22 (s, 2H), 6.75 (d, J = 8.8 Hz,

2H), 6.92 (d, J = 8.8 Hz, 2H), 7.02 (dd, J = 7.8, 7.2 Hz, 1H), 7.32 (dd, J

= 7.2, 1.4 Hz, 1H), 7.35 (dd, J = 7.8, 1.4 Hz, 1H)

¹H NMR (400 MHz, CDCl₃) δ: 1.38 (s, 6H), 2.92 (s, 3H), 3.94 (brs,

1H), 6.59 (d, J = 8.0 Hz, 1H), 6.64 (d, J = 7.2 Hz, 1H), 7.00 (dd, J = 8.0,

7.2 Hz, 1H)

Step 2: 1-(4-Methoxybenzyl)-3,3-dimethyl-2-oxoindoline-7-carbox-

ylic acid (27)

HRMS (ESI, m/z) calced for C₁₁H₁₅ON₂(M + H)⁺191.1179, found

191.1175

To a solution of 26 (374 mg, 1.06 mmol) in MeOH (4 mL) and THF (2

mL) was added aqueous 2 N NaOH (1.1 mL, 2.12 mmol) at room tem-

perature. The reaction mixture was stirred at 60 ^◦C for 2 h. After cooling

4.1.6. Synthesis of compound 8

Step 1: 2-Cyclopropylaniline (31)

8

T. Akaki et al.

Bioorganic & Medicinal Chemistry 44 (2021) 116283

A mixture of 2-bromoaniline (30; 32.0 g, 186 mmol), cyclo-

HRMS (ESI, m/z) calced for C₁₄H₁₃N₂(M + H)⁺209.1073, found

propylboronic acid (23.9 g, 278 mmol), and K₃PO₄(113 g, 532 mmol) in

toluene (350 mL) and H₂O (100 mL) was treated with PdCl₂(dppf)-

209.1072

◦

CH₂Cl₂(5.66 g, 6.93 mmol) and stirred at 90 C for 5 h under argon

4.1.7. Synthesis of compound 9

◦

atmosphere. The mixture was cooled to 0 C, ammonium pyrrolidine-

Step 1: 3-Bromo-N-(2-cyclopropylphenyl)-6-methylpyridin-2-amine

(36)

dithiocarbamate (APDTC, 5.06 g, 30.8 mmol) was added and the

mixture was stirred at room temperature for 1 h. The reactant mixture

was ﬁltered through Celite®, and the organic layer was separated and

washed with H₂O and brine. The resultant organic layer was loaded on

silica gel column chromatography (eluted with n-hexane/EtOAc = 80/

20 (v/v), approximately 1.6 L) for elimination of the polar component.

The eluate was acidiﬁed by adding 4 M HCl in AcOEt (50 mL) without

concentration. The precipitated crystals were collected by ﬁltration,

washed with EtOAc and n-hexane, and dried to give the hydrochloride

salt of the title compound 31 as a white crystalline (28.6 g, 90% yield).

¹H NMR (400 MHz, DMSO‑d₆) δ: 0.69–0.73 (m, 2H), 0.95–1.00 (m,

2H), 1.97–2.06 (br m, 1H), 7.09–7.05 (m, 1H), 7.22–7.26 (m, 1H),

7.31–7.39 (br m, 1H), 9.90 (br s, 3H).

A mixture of 32 (150 mg, 0.615 mmol), 3-bromo-6-methylpyridin-2-

amine (34; 138 mg, 0.738 mmol), and cesium carbonate (300 mg, 0.921

mmol) in toluene (1.5 mL) was treated with Pd(OAc)₂(28.0 mg, 0.125

mmol) and Xantphos (72.0 mg, 0.139 mmol) and stirred at 130 ^◦C for 3 h

under argon atmosphere. After cooling to room temperature, the

mixture was diluted with EtOAc and H₂O. The insoluble solid was

ﬁltered through Celite®. The organic layer was separated from the

ﬁltrate and concentrated. The residue was puriﬁed by ﬂash chroma-

tography (Biotage-SNAP Ultra 10 g, eluted with n-hexane/EtOAc = 99/2

to 85/25 (v/v)) to give the title compound 36 (64.0 g, 34% yield).

¹H NMR (400 MHz, CDCl₃) δ: 0.70–0.75 (m, 2H), 1.03–1.09 (m, 2H),

1.80–1.89 (m, 1H), 2.45 (s, 3H), 6.51 (d, J = 7.9 Hz, 1H), 6.94 (td, J =

7.6, 1.3 Hz, 1H), 7.19 (dt, J = 7.6, 1.3 Hz, 1H), 7.25 (td, J = 8.1, 1.3 Hz,

1H), 7.62 (d, J = 7.9 Hz, 1H), 7.82 (s, 1H), 8.58 (dd, J = 8.1, 1.3 Hz, 1H)

Step 2: 8-Cyclopropyl-2-methyl-9H-pyrido[2,3-b]indole (9)

Step 2: 1-Cyclopropyl-2-iodobenzene (32)

Sodium nitrate (11.5 g, 167 mmol) in H₂O (70 mL) was dropped into

the mixture of the hydrochloride salt of 31 (25.6 g, 151 mmol) and 1 M

aqueous HCl solution (20 mL) at 0 ^◦C. After stirring at 0 ^◦C for 15 min, to

the reaction mixture was added sodium iodide (24.9 g, 166 mmol) in

H₂O (70 mL) at 0 ^◦C. The mixture was stirred at 0 ^◦C for 30 min and then

room temperature for 2 h. The reactant mixture was quenched with

aqueous NaS₂O₃, and the mixture was extracted with EtOAc. The extract

was washed with H₂O and brine, and dried over Na₂SO₄. The solvent

was evaporated in vacuo. The residue was puriﬁed by ﬂash chromatog-

raphy (Biotage-SNAP Ultra 100 g, eluted with n-hexane/EtOAc = 100/

0 to 95/5 (v/v)) to give the title compound 32 (31.9 g, 86% yield).

¹H NMR (400 MHz, CDCl₃) δ: 0.63–0.68 (m, 2H), 0.99–1.05 (m, 2H),

1.98–2.06 (m, 1H), 6.88 (t, J = 7.7 Hz, 1H), 6.92 (d, J = 7.7 Hz, 1H),

7.24 (t, J = 7.7 Hz, 1H), 7.83 (d, J = 7.7 Hz, 1H)

A solution of 36 (64 mg, 0.211 mmol) and DBU (63.0 μL, 0.421

mmol) in DMA (1.0 mL) was treated with Pd(OAc)₂(14.0 mg, 6.24 ×

10v mmol) and CyJohnPhos (22.0 mg, 6.28 × 10^{ꢀ 2}mmol) under argon

atmosphere and stirred at 140 ^◦C for 20 h. After cooling to room tem-

perature, the mixture was diluted with H₂O and extracted with EtOAc.

The organic layer was washed twice with H₂O and brine, and dried over

Na₂SO₄. After ﬁltration and concentration, the residue was puriﬁed by

ﬂash chromatography (Biotage-SNAP Ultra 10 g, eluted with n-hexane/

EtOAc = 90/10 to 0/100 (v/v)). After concentration, for further puri-

ﬁcation, the residue was puriﬁed by preparative TLC (n-hexane/AcOEt

= 1/1 (v/v)) to give the title compound 9 (35 mg, 75% yield).

¹H NMR (400 MHz, CDCl₃) δ: 0.77–0.81 (m, 2H), 1.01–1.05 (m, 2H),

2.06–2.14 (m, 1H), 2.69 (s, 3H), 7.05 (d, J = 7.8 Hz, 1H), 7.16–7.22 (m,

2H), 7.85 (dd, J = 7.2, 1.8 Hz, 1H), 8.19 (d, J = 7.8 Hz, 1H), 8.67 (s, 1H)

HRMS (ESI, m/z) calced for C₁₅H₁₅N₂(M + H)⁺223.1230, found

223.1226

Step 3: 3-Bromo-N-(2-cyclopropylphenyl)pyridin-2-amine (35)

A mixture of 32 (1.00 g, 4.10 mmol), 3-bromopyridin-2-amine (33;

709 mg, 4.10 mmol), and sodium tert-pentoxide (902 mg, 8.19 mmol) in

toluene (10 mL) was treated with Pd(OAc)₂(92.0 mg, 0.41 mmol) and

◦

Xantphos (237 mg, 0.41 mmol) and stirred at 140 C for 4.5 h under

argon atmosphere. After cooling to room temperature, the mixture was

diluted with EtOAc and H₂O. The insoluble solid was ﬁltered through

Celite®. The organic layer was separated from the ﬁltrate and concen-

trated. The residue was puriﬁed by ﬂash chromatography (Biotage-

SNAP Ultra 25 g, eluted with n-hexane/EtOAc = 99/1 to 90/10 (v/v)) to

give the title compound 35 (945 mg, 80% yield).

4.1.8. Synthesis of compound 10

Step 1: 1-Cyclopropyl-3-ﬂuoro-2-nitrobenzene (38)

A mixture of 1-bromo-3-ﬂuoro-2-nitrobenzene (37, 49.8 g, 226

mmol), cyclopropylboronic acid (21.4 g, 249 mmol), and K₃PO₄(106 g,

499 mmol) in DME (300 mL) and H₂O (150 mL) was treated with

PdCl₂(dppf)-CH₂Cl₂(9.25 g, 11.3 mmol) and stirred under reﬂux for 1 h.

After cooling to room temperature, the mixture was diluted with AcOEt

and H₂O, ﬁltered through Celite®, and the organic layer was separated

and washed with H₂O and brine. After ﬁltration and concentration, the

residue was puriﬁed by ﬂash chromatography (solvent: EtOAc/Hexane

= 5/95 to 25/75) to give the title compound 38 (36.8 g, 89% yield).

¹H NMR (400 MHz, DMSO‑d₆) δ: 0.77–0.81 (m, 2H), 0.99–1.04 (m,

2H), 1.85–1.92 (m, 1H), 7.03–7.05 (m, 1H), 7.35–7.40 (m, 1H),

7.52–7.58 (m, 1H)

¹H NMR (400 MHz, CDCl₃) δ: 0.67–0.78 (m, 2H), 0.97–1.12 (m, 2H),

1.80–1.91 (m, 1H), 6.63 (dd, J = 7.7, 4.9 Hz, 1H), 6.97 (td, J = 7.7, 1.2

Hz, 1H), 7.19 (dt, J = 7.7, 1.2 Hz, 1H), 7.26 (td, J = 8.1, 1.2 Hz, 1H),

7.75 (dd, J = 7.7, 1.6 Hz, 1H), 7.78 (br s, 1H), 8.18 (dd, J = 4.9, 1.6 Hz,

1H), 8.39 (dd, J = 8.1, 1.2 Hz, 1H)

Step 4: 8-Cyclopropyl-9H-pyrido[2,3-b]indole (8)

A solution of 35 (945 mg, 3.27 mmol) and DBU (1.48 mL, 9.80

mmol) in DMA (19 mL) was treated with Pd(OAc)₂(73.4 mg, 0.327

mmol) and CyJohnPhos (229 mg, 0.65 mmol) under argon atmosphere

Step 2: Ethyl 2-cyano-2-(3-cyclopropyl-2-nitrophenyl)acetate (39)

Compound 38 (3.00 g, 16.6 mmol) and ethyl 2-cyanoacetate (3.50

mL, 33.1 mmol) was dissolved in DMF (15 mL). To the solution, K₂CO₃

(6.90 g, 49.7 mmol) was added, and the mixture was stirred at 90 ^◦C for

3 h. After cooling to 0 ^◦C, the reactant mixture was diluted with H₂O (50

mL), acidiﬁed by adding 6.0 M aqueous HCl solution (25 mL), and

extracted with 1:1n-hexane-AcOEt. The organic layer was washed with

H₂O and brine, and dried over Na₂SO₄. After ﬁltration, the solvent was

evaporated in vacuo to give the title compound 39 (5.0 g, overweight) as

a crude product which was used for the next step without further

puriﬁcation.

◦

and stirred at 140 C for 2 h. After cooling to room temperature, the

mixture was diluted with H₂O and extracted with EtOAc. The organic

layer was washed twice with H₂O and brine, and dried over Na₂SO₄.

After ﬁltration and concentration, the residue was puriﬁed by ﬂash

chromatography (Biotage-SNAP Ultra 25 g, eluted with n-hexane/

EtOAc = 92/8 to 34/66 (v/v)). After concentration, for further puriﬁ-

cation, the residue was slurried in n-hexane/AcOEt (2/1 (v/v)). The

precipitated solid was collected by ﬁltration, washed with n-hexane, and

dried to give the title compound 8 (251 mg, 37% yield).

¹H NMR (400 MHz, DMSO‑d₆) δ: 0.76–0.80 (m, 2H), 1.03–1.08 (m,

2H), 2.38–2.45 (m, 1H), 6.99 (d, J = 7.6 Hz, 1H), 7.13 (t, J = 7.6 Hz,

1H), 7.20 (dd, J = 7.8, 4.8 Hz, 1H), 7.95 (d, J = 7.6 Hz, 1H), 8.43 (dd, J

= 4.8, 1.5 Hz, 1H), 8.48 (dd, J = 7.8, 1.5 Hz, 1H), 11.92 (s, 1H)

¹H NMR (400 MHz, CDCl₃) δ: 0.64–0.77 (m, 2H), 0.96–1.06 (m, 2H),

1.30 (t, J = 7.3 Hz, 3H), 1.98–2.06 (m, 1H), 4.26 (q, J = 7.3 Hz, 2H),

5.00 (s, 1H), 7.21 (dd, J = 7.7, 0.8 Hz, 1H), 7.04 (t, J = 7.7 Hz, 1H), 7.54

9

T. Akaki et al.

Bioorganic & Medicinal Chemistry 44 (2021) 116283

(dd, J = 7.7,1.6 Hz, 1H)

4-Chloro-2-methyl-7H-pyrrolo[2,3-d]pyrimidine (43, 5.00 g, 29.8

mmol) was added to a suspension of sodium hydride 60% dispersion in

mineral oil (1.43 g, 35.8 mmol) and DMF (30 mL) at 0 ^◦C. After stirring

for 30 min at 0 ^◦C, 2-(chloromethoxy)ethyltrimethylsilane (6.79 mL,

38.7 mmol) was added to the solution at 0 ^◦C, and the mixture was

stirred at room temperature for 1.5 h. The mixture was diluted with H₂O

and extracted with EtOAc, and the organic layer was washed with H₂O

and brine, and dried over MgSO₄. After ﬁltration and concentration, the

residue was puriﬁed by silica gel column chromatography (eluted with

n-hexane/EtOAc = 100/0 to 90/10 (v/v)) to give the title compound 44

(7.80 g, 89% yield).

Step 3: Ethyl 2-amino-7-cyclopropyl-1H-indole-3-carboxylate (40)

To the solution of 39 (5.0 g, approx. 16 mmol) in AcOH (30 mL) was

added iron powder (5.09 g, 91.1 mmol), and the mixture was stirred at

90 ^◦C for 2 h. After cooling to room temperature, the reactant mixture

was diluted with 1:1 toluene-H₂O (60 mL), ﬁltered through Celite®, and

the organic layer was separated and washed with H₂O, saturated

NaHCO₃solution and brine sequentially, and dried over Na₂SO₄. After

ﬁltration and concentration, the residue was puriﬁed by ﬂash chroma-

tography (Yamazen-Universal Premium L, eluted with n-hexane/EtOAc

= 85/15 to 40/60 (v/v)) to give the title compound 40 (2.10 g, 52% two-

step yield from 38).

¹H NMR (400 MHz, DMSO‑d₆) δ: ꢀ 0.10 (s, 9H), 0.81–0.86 (m, 2H),

2.65 (s, 3H), 3.50–3.55 (m, 2H), 5.59 (s, 2H), 6.63 (d, J = 3.7 Hz, 1H),

7.75 (d, J = 3.7 Hz, 1H)

¹H NMR (400 MHz, CDCl₃) δ: 0.71 (br s, 2H), 0.93 (br s, 2H), 1.42 (t,

J = 7.1 Hz, 3H), 1.91 (br s, 1H), 4.36 (q, J = 7.1 Hz, 2H), 5.68 (br s, 1H),

6.79 (d, J = 7.4 Hz, 1H), 7.04 (t, J = 7.4 Hz, 1H), 7.64 (d, J = 7.4 Hz,

1H), 7.92 (br s, 2H)

Step 2: 4-Chloro-2-methyl-6-propyl-7-((2-(trimethylsilyl)ethoxy)

methyl)-7H-pyrrolo[2,3-d]pyrimidine (45)

Step 4: 8-Cyclopropyl-2-methyl-9H-pyrimido[4,5-b]indol-4-ol (41)

Compound 40 (5.00 g, 20.5 mmol) was dissolved in 4 M aqueous HCl

solution (25 mL). To the solution, MeCN (7.5 mL) was added, and the

mixture was stirred at room temperature for 20 h. The resultant sus-

pension was diluted with 1:1n-hexane-dioxane (50 mL), and the

precipitated solid was collected by ﬁltration, washed with n-hexane, and

dried in vacuo. The obtained solid was redissolved in MeOH (65 mL) and

H₂O (20 mL). Saturated NaHCO₃solution (40 mL) was added to the

solution, and the reactant mixture was stirred at 75 ^◦C for 2.5 h. After

cooling to 0 ^◦C, the reactant mixture was acidiﬁed by adding 6 M

aqueous HCl solution (7.5 mL), diluted with H₂O (100 mL), and then

stirred at 0 ^◦C for 15 min. The precipitated solid was collected by

ﬁltration, washed with H₂O, and dried to give the title compound 41

(4.88 mg, 99% yield).

To a solution of 44 (2.00 g, 6.71 mmol) in THF (20 mL), 1.63 M n-

butyllithium in hexane (5.34 mL, 8.70 mmol) was added at ꢀ 78 ^◦C. After

stirring for 3 h at ꢀ 78 ^◦C, 1-iodopropane (847

μL, 8.72 mmol) was added

to the reactant mixture, and then the mixture was stirred at room tem-

perature for 1.5 h. The mixture was diluted with H₂O and extracted with

AcOEt, and the organic layer was washed with H₂O and brine, and dried

over MgSO₄. After ﬁltration and concentration, the residue was loaded

on silica gel column chromatography (eluted with n-hexane/EtOAc =

100/0 to 95/5 (v/v)) for elimination of the polar component. The title

compound 45 (1.10 g) was obtained as a mixture with unknown com-

pounds, which was applied to the next step without further puriﬁcation.

Step 3: 2-Methyl-6-propyl-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-

pyrrolo[2,3-d]pyrimidine (46)

A solution of 45 (1.10 g, ~3.2 mmol) and potassium carbonate (450

mg, 3.26 mmol) in MeOH (11 mL) was treated with 10% palladium on

activated carbon (110 mg) and stirred under 1.0 atm of hydrogen

overnight at room temperature. After removal of the palladium catalyst

by Celite® ﬁltration, the ﬁltrate was diluted with H₂O and extracted

with AcOEt. The organic layer was washed with H₂O and brine, and

dried over MgSO₄. After ﬁltration and concentration, the title compound

46 (1.00 g) was obtained, which was applied to the next step without

further puriﬁcation.

¹H NMR (400 MHz, DMSO‑d₆) δ: 0.77–0.73 (m, 2H), 0.99–1.04 (m,

2H), 2.30–2.37 (m, 1H), 2.42 (s, 3H), 6.80 (d, J = 7.5 Hz, 1H), 7.10 (t, J

= 7.5 Hz, 1H), 7.73 (dd, J = 7.5, 0.9 Hz, 1H), 12.07 (s, 1H), 12.12 (s, 1H)

Step 5: 4-Chloro-8-cyclopropyl-2-methyl-9H-pyrimido [4,5-b] indole

(42)

To a suspension of 41 (4.88 g, 20.4 mmol) in CHCl₃(66 mL) was

added thionyl chloride (7.42 mL, 102 mmol) and DMF (33 mL), and the

mixture was stirred at 60 ^◦C for 2 h. The resultant solution was cooled to

◦

0 C and quenched with H₂O. The mixture was extracted with AcOEt,

Step 4: 5-Bromo-2-methyl-6-propyl-7-((2-(trimethylsilyl)ethoxy)

methyl)-7H-pyrrolo[2,3-d]pyrimidine (47)

and the organic layer was washed twice with H₂O and brine, and dried

over Na₂SO₄. After ﬁltration and concentration, the residue was puriﬁed

by ﬂash chromatography (Yamazen-Universal Premium 3L, eluted with

n-hexane/EtOAc = 90/10 to 20/80 (v/v)) to give the title compound 42

(2.44 mg, 46% yield).

To a solution of 46 (1.00 g, ~3.2 mmol) in DMF (20 mL) was added

N-bromosuccinimide (582 mg, 3.27 mmol). After stirring at room tem-

perature for 1 h, the reactant mixture was quenched with saturated

NaHCO₃solution. The mixture was extracted with AcOEt, and the

organic layer was washed with H₂O and brine, and dried over MgSO₄.

After ﬁltration and concentration, the residue was puriﬁed by silica gel

column chromatography (eluted with n-hexane/EtOAc = 95/5 to 90/10

(v/v)) to give the title compound 47 (770 mg, 30% yield, 3 steps from

44).

¹H NMR (400 MHz, CDCl₃) δ: 2.05–2.09 (m, 2H), 2.27–2.31 (m, 2H),

3.08–3.14 (m, 1H), 3.68 (s, 3H), 7.26–7.31 (m, 2H), 7.93–7.98 (m, 1H),

8.84 (s, 1H)

Step 6: 8-Cyclopropyl-2-methyl-9H-pyrimido[4,5-b]indole (10)

A solution of 42 (40.0 mg, 0.155 mmol) in THF (0.6 mL) and MeOH

(0.6 mL) was treated with 10% palladium hydroxide on activated carbon

(15.0 mg) and K₂CO₃(32.0 mg, 0.233 mmol), and the mixture was

stirred under 1.0 atm of hydrogen at room temperature for 7 h. After

removal of the palladium catalyst by Celite® ﬁltration, the ﬁltrate was

concentrated. The residue was suspended in 1:1n-hexane-AcOEt and

slurried for a while. The precipitated solid was collected by ﬁltration,

washed with 2:1n-hexane-AcOEt, and dried to give the title compound

10 (30.0 mg, 86% yield).

¹H NMR (400 MHz, DMSO‑d₆) δ: ꢀ 0.10 (s, 9H), 0.82–0.87 (m, 2H),

0.95 (t, J = 7.6 Hz, 3H), 1.69 (tq, J = 7.6, 7.6 Hz, 2H), 2.67 (s, 3H), 2.84

(t, J = 7.6 Hz, 2H), 3.48–3.54 (m, 2H), 5.64 (s, 2H), 8.72 (d, 1H)

Step 5: 2-Methyl-6-propyl-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-

pyrrolo[2,3-d]pyrimidine-5-carbonitrile (48)

To a solution of 47 (200 mg, 0.520 mmol) in THF (2.0 mL), 1.63 M n-

butyllithium in hexane (414

μ

L, 0.676 mmol) was added at ꢀ 78 ^◦C. After

stirring for 1 h at ꢀ 78 ^◦C, p-toluenesulfonyl cyanide (122 mg, 0.673

mmol) was added to the reactant mixture, and then the mixture was

stirred at room temperature for 3 h. The mixture was quenched with

saturated NH₄Cl solution and extracted with AcOEt. The organic layer

was washed with H₂O and brine, and dried over MgSO₄. After ﬁltration

and concentration, the residue was puriﬁed by preparative TLC (n-

hexane/AcOEt = 4/1 (v/v)) to give the title compound 48 (110 mg) as a

mixture with 2-methyl-6-propyl-7-((2-(trimethylsilyl)ethoxy)methyl)-

7H-pyrrolo[2,3-d]pyrimidine, which was applied to the next step

without further puriﬁcation.

¹H NMR (400 MHz, DMSO‑d₆) δ: 0.77–0.81 (m, 2H), 1.04–1.09 (m,

2H), 2.33–2.40 (m, 1H), 2.71 (s, 3H), 7.02 (d, J = 7.6 Hz, 1H), 7.20 (t, J

= 7.6 Hz, 1H), 7.97 (d, J = 7.6 Hz, 1H), 9.30 (s, 1H), 12.27 (s, 1H).

HRMS (ESI, m/z) calced for C₁₄H₁₄N₃(M + H)⁺224.1182, found

224.1183

4.1.9. Synthesis of compound 11

Step 1: 4-Chloro-2-methyl-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-

pyrrolo[2,3-d]pyrimidine (44)

10

T. Akaki et al.

Bioorganic & Medicinal Chemistry 44 (2021) 116283

Step 6: 2-Methyl-6-propyl-7H-pyrrolo[2,3-d]pyrimidine-5-carbon-

itrile (11)

To a solution of 48 (50.0 mg, ~0.150 mmol) in CH₂Cl₂(1.0 mL),

boron triﬂuoride diethyl etherate (57.0 L, 0.512 mmol) was added at

was slurried in n-hexane/AcOEt (2/1 (v/v)). The precipitated solid was

collected by ﬁltration, washed with n-hexane, and dried to give the title

compound 13 (41.0 mg, 63% yield).

μ

¹H NMR (400 MHz, DMSO‑d₆) δ: 0.90 (t, J = 7.4 Hz, 3H), 1.69 (qt, J

= 7.4, 7.4 Hz, 2H), 2.63 (s, 3H), 2.71 (t, J = 7.4 Hz, 2H), 8.65 (s, 1H),

12.23 (br s, 1H)

room temperature. After stirring overnight at room temperature, the

mixture was quenched with saturated NaHCO₃solution and extracted

with AcOEt. The organic layer was washed with H₂O and brine, and

dried over MgSO₄. After ﬁltration and concentration, for puriﬁcation,

the residue was slurried in n-hexane/AcOEt (1/1 (v/v)). The precipi-

tated solid was collected by ﬁltration, washed with n-hexane, and dried

to give the title compound 11 (13.0 mg, 28% yield, 2 steps from 47).

¹H NMR (400 MHz, DMSO‑d₆) δ: 0.92 (t, J = 7.4 Hz, 3H), 1.77 (tq, J

= 7.4, 7.4 Hz, 2H), 2.65 (s, 3H), 2.87 (t, J = 7.4 Hz, 2H), 8.93 (s, 1H),

12.89 (br s, 1H)

HRMS (ESI, m/z, MH+) Calcd for C₁₀H₁₃N₃Br: 254.0284, Found:

254.0287

4.1.12. Synthesis of compound 15

Step 1: 5-Bromo-4-chloro-2-methyl-6-propyl-7-((2-(trimethylsilyl)

ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidine (52)

To a solution of 45 (450 mg, 1.32 mmol) in DMF (5.0 mL) was added

N-bromosuccinimide (260 mg, 1.46 mmol). After stirring overnight at

room temperature, the reactant mixture was quenched with saturated

NaHCO₃solution. The mixture was extracted with AcOEt, and the

organic layer was washed with H₂O and brine, and dried over MgSO₄.

After ﬁltration and concentration, the residue was loaded on silica gel

column chromatography (eluted with n-hexane/EtOAc = 90/10 (v/v))

for elimination of the polar component. The title compound 52 (500 mg,

90% yield) was obtained, which was proceeded to the next step without

further puriﬁcation.

HRMS (ESI, m/z, MH+ ) Calcd for C₁₁H₁₃N₄: 201.1135, Found:

201.1138

4.1.10. Synthesis of compound 12

Step 1: 2-Methyl-5-(pent-1-yn-1-yl)pyrimidin-4-amine (50)

A solution of 5-iodo-2-methylpyrimidin-4-amine (49, 640 mg, 2.72

mmol), 1-penthyne (805 μ

L, 8.17 mmol), [1,1^′-bis(diphenylphosphino)

ferrocene]dichloropalladium(II) (220 mg, 0.269 mmol), copper(I) io-

dide (51.0 mg, 0.268 mmol) and triethylamine (569

μ

L, 4.08 mmol) in

Step 2: 4-Chloro-2,5-dimethyl-6-propyl-7-((2-(trimethylsilyl)ethoxy)

methyl)-7H-pyrrolo[2,3-d]pyrimidine (53)

DMF (10 mL) was stirred at 70 ^◦C for 2 h under argon atmosphere. The

mixture was cooled to room temperature, diluted with H₂O and

extracted with AcOEt. The organic layer was washed with H₂O and

brine, and dried over MgSO₄. After ﬁltration and concentration, the

residue was loaded on silica gel column chromatography (eluted with n-

hexane/EtOAc = 50/50 (v/v)) for elimination of the polar component.

The title compound 50 (305 mg, 64% yield) was obtained, which was

advanced to the next step without further puriﬁcation.

To a solution of 52 (500 mg, 1.19 mmol) in THF (5.0 mL), 1.63 M n-

butyllithium in hexane (950

μ

L, 1.55 mmol) was added at ꢀ 78 ^◦C. After

stirring for 1 h at ꢀ 78 ^◦C, methyl iodide (96.0

μL, 1.54 mmol) was added

to the reactant mixture, and then the mixture was stirred at room tem-

perature for 2 h. The mixture was diluted with H₂O and extracted with

AcOEt. The organic layer was washed with H₂O and brine, and dried

over MgSO₄. After ﬁltration and concentration, the residue was puriﬁed

by silica gel column chromatography (eluted with n-hexane/EtOAc =

90/10 (v/v)) to give the title compound 53 (337 mg, 80% yield).

¹H NMR (400 MHz, DMSO‑d₆) δ: ꢀ 0.10 (s, 9H), 0.81–0.87 (m, 2H),

0.93 (t, J = 7.4 Hz, 3H), 1.61 (qt, J = 7.4, 7.4 Hz, 2H), 2.35 (s, 3H), 2.59

(s, 3H), 2.78 (t, J = 7.4 Hz, 2H), 3.45–3.50 (m, 2H), 5.58 (s, 2H)

Step 3: 4-Chloro-2,5-dimethyl-6-propyl-7H-pyrrolo[2,3-d]pyrimi-

dine (15)

Step 2: 2-Methyl-6-propyl-7H-pyrrolo[2,3-d]pyrimidine (51)

To a solution of 50 (300 mg, 1.71 mmol) in NMP (3.0 mL), potassium

tert-butoxide (400 mg, 3.56 mmol) was added at room temperature and

◦

then stirred at 100 C for 1 h. The mixture was cooled to room tem-

perature, quenched with 1 N HCl solution and extracted with AcOEt. The

organic layer was washed with H₂O and brine, and dried over MgSO₄.

After ﬁltration and concentration, the residue was puriﬁed by silica gel

column chromatography (eluted with EtOAc) to give the title compound

51 (155 mg, 52% yield).

Compound 53 (150 mg, 0.424 mmol) was dissolved in TFA (1.5 mL),

and the mixture was stirred at room temperature for 1 h. After evapo-

ration of TFA, the residue was diluted with THF and neutralized with 2 N

NaOH solution, and then extracted with AcOEt. The organic layer was

washed with H₂O, 1 N HCl solution and brine, and dried over MgSO₄.

After ﬁltration and concentration, for puriﬁcation, the residue was

slurried in n-hexane/AcOEt (10/1 (v/v)). The precipitated solid was

collected by ﬁltration, washed with n-hexane, and dried to give the title

compound 15 (35.0 mg, 37% yield).

¹H NMR (400 MHz, DMSO‑d₆) δ: 0.92 (t, J = 7.4 Hz, 3H), 1.70 (qt, J

= 7.4, 7.4 Hz, 2H), 2.58 (s, 3H), 2.68 (t, J = 7.4 Hz, 2H), 6.19 (br s, 1H),

8.69 (s, 1H), 11.67 (br s, 1H)

Step 3: 5-Chloro-2-methyl-6-propyl-7H-pyrrolo[2,3-d]pyrimidine

(12)

To a solution of 51 (30.0 mg, 0.171 mmol) in DMF (1.0 mL) was

added N-chlorosuccinimide (34.0 mg, 0.255 mmol). After stirring at

room temperature for 5 h, the reactant mixture was quenched with

saturated NaHCO₃solution. The mixture was extracted with AcOEt, and

the organic layer was washed with H₂O and brine, and dried over

MgSO₄. After ﬁltration and concentration, the residue was puriﬁed by

preparative TLC (n-hexane/AcOEt = 1/1 (v/v)) to give the title com-

pound 12 (18.0 mg, 50% yield).

¹H NMR (400 MHz, DMSO‑d₆) δ: 0.88 (t, J = 7.4 Hz, 3H), 1.63 (qt, J

= 7.4, 7.4 Hz, 2H), 2.31 (s, 3H), 2.55 (s, 3H), 2.65 (t, J = 7.4 Hz, 2H),

11.88 (br s, 1H)

HRMS (ESI, m/z, MH+ ) Calcd for C₁₁H₁₅N₃Cl₁: 224.0949, Found:

224.0950

¹H NMR (400 MHz, DMSO‑d₆) δ: 0.90 (t, J = 7.4 Hz, 3H), 1.69 (qt, J

= 7.4, 7.4 Hz, 2H), 2.62 (s, 3H), 2.72 (t, J = 7.4 Hz, 2H), 8.73 (s, 1H),

12.11 (br s, 1H)

4.1.13. Synthesis of compound 14

A solution of 15 (25.0 mg, 0.112 mmol) and potassium carbonate

(15.0 mg, 0.109 mmol) in MeOH (1.0 mL) was treated with 10%

palladium on activated carbon (10 mg) and stirred under 1.0 atm of

hydrogen at room temperature for 2 h. After removal of the palladium

catalyst by Celite® ﬁltration, the ﬁltrate was diluted with H₂O and

extracted with AcOEt. The organic layer was washed with H₂O and

brine, and dried over MgSO₄. After ﬁltration and concentration, for

puriﬁcation, the residue was slurried in n-hexane/AcOEt (1/1 (v/v)).

The precipitated solid was collected by ﬁltration, washed with n-hexane,

and dried to give the title compound 14 (17.0 mg, 81% yield).

¹H NMR (400 MHz, DMSO‑d₆) δ: 0.88 (t, J = 7.4 Hz, 3H), 1.64 (qt, J

= 7.4, 7.4 Hz, 2H), 2.18 (s, 3H), 2.57 (s, 3H), 2.64 (t, J = 7.4 Hz, 2H),

HRMS (ESI, m/z, MH+) Calcd for C₁₀H₁₃N₃Cl: 210.0793, Found:

210.0788

4.1.11. Synthesis of compound 13

To a solution of 51 (45.0 mg, 0.256 mmol) in CHCl₃(1.0 mL) was

added N-bromosuccinimide (55.0 mg, 0.308 mmol). After stirring

overnight at room temperature, the reactant mixture was quenched with

saturated NaHCO₃solution. The mixture was extracted with AcOEt, and

the organic layer was washed with H₂O and brine, and dried over

MgSO₄. After ﬁltration and concentration, for puriﬁcation, the residue

11

T. Akaki et al.

Bioorganic & Medicinal Chemistry 44 (2021) 116283

8.67 (s, 1H), 11.40 (br s, 1H)

stirring overnight at 50 ^◦C, the mixture was diluted with H₂O. And then,

the mixture was neutralized with 6.0 M aqueous HCl solution. Precipi-

tated solid was collected by ﬁltration, washed with H₂O, and dried to

give the title compound 58 (300 mg, 97% yield) which was advanced to

the next step without further puriﬁcation.

HRMS (ESI, m/z, MH+) Calcd for C₁₁H₁₆N₃: 190.1339, Found:

190.1342

4.1.14. Synthesis of compound 16

Step1:

5-Bromo-4-chloro-2-methyl-7H-pyrrolo[2,3-d]pyrimidine

Step 6: 4-Chloro-N,2,5-trimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-

carboxamide (16)

(54)

To a suspension of 4-chloro-2-methyl-7H-pyrrolo[2,3-d]pyrimidine

(43, 2.00 g, 11.9 mmol) in CHCl₃(20 mL) was added N-bromosuccini-

mide (2.55 mg, 14.3 mmol). After stirring overnight at room tempera-

ture, the reactant mixture was quenched with saturated NaHCO₃

solution. The mixture was extracted with CHCl₃and AcOEt, and the

organic layer was washed with H₂O and brine, and dried over MgSO₄.

After ﬁltration and concentration, for puriﬁcation, the residue was

slurried in n-hexane/AcOEt (1/1 (v/v)). The precipitated solid was

collected by ﬁltration, washed with n-hexane, and dried to give the title

compound 54 (2.50 g, approx. 70% yield) as a mixture of 15% of 5,6-

dibromo-4-chloro-2-methyl-7H-pyrrolo[2,3-d]pyrimidine.

To a solution of 58 (100 mg, 0.443 mmol) in 1:1 CHCl₃-THF (2.0 mL)

was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochlo-

ride (169 mg, 0.886 mmol), N,N-dimethyl-4-aminopyridine (5.00 mg,

0.0409 mmol) and 40% methylamine in methanol (34.0

μg, 0.493

mmol), and the mixture was stirred overnight at room temperature. The

mixture was diluted with H₂O and extracted with AcOEt. The organic

layer was washed with H₂O, saturated aqueous NaHCO₃solution and

brine, and dried over MgSO₄. After ﬁltration and concentration, for

puriﬁcation, the residue was slurried in AcOEt. The precipitated solid

was collected by ﬁltration, washed with a small portion of AcOEt, and

dried to give the title compound 16 (73.0 mg, 69% yield).

¹H NMR (400 MHz, DMSO‑d₆) δ: 2.60 (s, 3H), 2.61 (s, 3H), 2.81 (d, J

= 4.6 Hz, 3H), 8.11 (br d, J = 4.6 Hz, 1H), 12.32 (br s, 1H)

HRMS (ESI, m/z, MH+ ) Calcd for C₁₀H₁₂N₄Cl₁: 239.0694, Found:

239.0688

¹H NMR (400 MHz, DMSO‑d₆) δ: 2.62 (s, 3H), 7.82 (d, J = 2.5 Hz,

1H), 12.69 (br s, 1H)

Step 2: 4-Chloro-2,5-dimethyl-7H-pyrrolo[2,3-d]pyrimidine (55)

To a solution of 54 (85% purity, 2.50 g, 8.70 mmol) in THF (45 mL),

1.63 M n-butyllithium in hexane (15.0 mL, 23.3 mmol) was added at

◦

ꢀ 78 C. After stirring for 1 h at ꢀ 78 C, methyl iodide (662

μL, 10.6

4.1.15. Synthesis of compound 18

mmol) was added to the reactant mixture, and then the mixture was

stirred at 0 ^◦C for 1 h. The mixture was diluted with H₂O and extracted

with AcOEt. The organic layer was washed with H₂O and brine, and

dried over MgSO₄. After ﬁltration and concentration, for puriﬁcation,

the residue was slurried in n-hexane/AcOEt (2/1 (v/v)). The precipi-

tated solid was collected by ﬁltration, washed with n-hexane, and dried

to give the title compound 55 (660 mg, approx. 35% yield) as a mixture

of 10% of 4-chloro-2-methyl-7H-pyrrolo[2,3-d]pyrimidine.

¹H NMR (400 MHz, DMSO‑d₆) δ: 2.38 (br s, 3H), 2.58 (s, 3H), 7.30

(br s, 1H), 11.94 (br s, 1H)

Step1: 4-Chloro-2-methyl-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-

pyrrolo[2,3-d]pyrimidine-6-carbaldehyde (59)

To a solution of 44 (1.50 g, 5.03 mmol) in THF (15 mL), 1.63 M n-

butyllithium in hexane (4.01 mL, 6.54 mmol) was added at ꢀ 78 ^◦C. After

stirring for 3 h at ꢀ 78 ^◦C, N,N-dimethylformamide (503

μL, 6.54 mmol)

was added to the reactant mixture, and then the mixture was stirred at

room temperature for 1 h. The mixture was diluted with H₂O and

saturated aqueous NH₄Cl solution, and extracted with AcOEt. The

organic layer was washed with H₂O and brine, and dried over MgSO₄.

After ﬁltration and concentration, the residue was loaded on silica gel

column chromatography (eluted with n-hexane/EtOAc = 90/10 (v/v))

for elimination of the polar component. The title compound 59 (1.08 g,

66% yield) was obtained, which was applied to the next step without

further puriﬁcation.

Step 3: 4-Chloro-2,5-dimethyl-7-tosyl-7H-pyrrolo[2,3-d]pyrimidine

(56)

Compound 55 (90% purity, 660 mg, 3.27 mmol) was added to a

suspension of sodium hydride 60% dispersion in mineral oil (160 mg,

4.00 mmol) was dissolved in DMF (6.0 mL) at 0 ^◦C. After stirring for 30

min at 0 ^◦C, p-toluenesulfonyl chloride (762 mg, 3.99 mmol) was added

to the solution at 0 ^◦C, and the mixture was stirred at room temperature

for 30 min. The mixture was diluted with H₂O and extracted with AcOEt,

and the organic layer was washed with H₂O and brine, and dried over

MgSO₄. After ﬁltration and concentration, for puriﬁcation, the residue

was slurried in n-hexane. The precipitated solid was collected by

ﬁltration, washed with n-hexane, and dried to give the title compound

56 (1.00 g, 91% yield) which was applied to the next step without

further puriﬁcation.

Step

2:

(E)-4-Chloro-2-methyl-6-(prop-1-en-1-yl)-7-((2-(trime-

thylsilyl)ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidine (60)

Phenyllithium (2.1 M) in dibutyl ether (613 μL, 1.28 mmol) was

added at 0 ^◦C to a solution of ethyltriphenylphosphonium bromide (250

mg, 0.673 mmol) in THF (2.5 mL) and stirred for 15 min at 0 ^◦C. The

mixture was cooled to ꢀ 78 ^◦C and a solution 59 (200 mg, 0.613 mmol)

in THF (2.5 mL) was added. After stirring at ꢀ 78 ^◦C for 30 min, addi-

tional 2.1 M phenyllithium in dibutyl ether (292 μL, 0.613 mmol) was

added to the mixture, and then stirred for another 1.5 h at ꢀ 78 ^◦C. The

reactant mixture was diluted with H₂O and saturated aqueous NH₄Cl

solution, and extracted with AcOEt. The organic layer was washed with

H₂O and brine, and dried over MgSO₄. After ﬁltration and concentra-

tion, the residue was puriﬁed by preparative TLC (n-hexane/AcOEt = 4/

1 (v/v)) to give the title compound 60 (50.0 mg, 24% yield).

¹H NMR (400 MHz, DMSO‑d₆) δ: ꢀ 0.11 (s, 9H), 0.80–0.88 (m. 2H),

1.93 (d, J = 4.6 Hz, 3H), 2.63 (s, 3H), 3.46–3.52 (m, 2H), 5.65 (s, 2H),

6.64–6.66 (m, 2H), 6.77 (s, 1H)

Step 4: Ethyl 4-chloro-2,5-dimethyl-7-tosyl-7H-pyrrolo[2,3-d]py-

rimidine-6-carboxylate (57)

To a solution of 56 (1.00 g, 2.98 mmol) in THF (10 mL), 1.55 M n-

butyllithium in hexane (2.88 mL, 4.46 mmol) was added at ꢀ 78 ^◦C. After

stirring for 3 h at ꢀ 78 ^◦C, ethyl chloroformate (426

μL, 4.46 mmol) was

added to the reactant mixture, and then the mixture was stirred over-

night at room temperature. The mixture was diluted with H₂O and 1 N

HCl solution, and extracted with AcOEt. The organic layer was washed

with H₂O and brine, and dried over MgSO₄. After ﬁltration and con-

centration, the residue was puriﬁed by silica gel column chromatog-

raphy (eluted with n-hexane/EtOAc = 90/10 to 80/20 (v/v)) to give the

title compound 57 (560 mg, 46% yield) as a mixture containing a small

amount of impurities. This was advanced to the next step without

further puriﬁcation.

Step 3: (E)-4-Chloro-2-methyl-6-(prop-1-en-1-yl)-7H-pyrrolo[2,3-d]

pyrimidine (61)

Compound 60 (50 mg, 0.153 mmol) was dissolved in 1:1 CHCl₃-TFA

(2.0 mL), and the mixture was stirred at room temperature for 1 h. After

evaporation of CHCl₃and TFA, the residue was diluted with THF and

neutralized with 2 N NaOH solution, and then extracted with AcOEt. The

organic layer was washed with H₂O, 1 N HCl solution and brine, and

dried over MgSO₄. After ﬁltration and concentration, for puriﬁcation,

the residue was slurried in n-hexane/AcOEt (10/1 (v/v)). The precipi-

tated solid was collected by ﬁltration, washed with n-hexane, and dried

to give the title compound 61 (20.0 mg, 65% yield).

Step 5: 4-Chloro-2,5-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-car-

boxylic acid (58)

To a solution of 57 (560 mg, 1.37 mmol) in THF (5.0 mL), 1.0 M

lithium hydroxide solution (6.00 mL, 6.00 mmol) was added. After

12

T. Akaki et al.

Bioorganic & Medicinal Chemistry 44 (2021) 116283

¹H NMR (400 MHz, DMSO‑d₆) δ: 1.89 (d, J = 5.5 Hz, 3H), 2.59 (s,

slurried in AcOEt. The precipitated solid was collected by ﬁltration,

washed with n-hexane, and dried to give the title compound 17 (49.0

mg, 77% yield).

3H), 6.38–6.58 (m, 3H), 12.37 (br s, 1H)

Step 4: (E)-4,5-Dichloro-2-methyl-6-(prop-1-en-1-yl)-7H-pyrrolo

[2,3-d]pyrimidine (18)

¹H NMR (400 MHz, DMSO‑d₆) δ: 2.63 (s, 3H), 2.84 (d, J = 4.4 Hz,

3H), 8.29 (q, J = 4.4 Hz, 1H), 13.17 (br s, 1H)

HRMS (ESI, m/z, MH+) Calcd for C₉H₉O₁N₄Br₁Cl₂: 302.9643,

Found: 302.9636

To a solution of 61 (10.0 mg, 4.82 × 10^{ꢀ 2}mmol) in DMF (1.0 mL)

was added N-chlorosuccinimide (10.0 mg, 7.49 × 10^{ꢀ 2}mmol). After

stirring overnight at room temperature, the reactant mixture was

quenched with saturated NaHCO₃solution. The mixture was extracted

with AcOEt, and the organic layer was washed with H₂O and brine, and

dried over MgSO₄. After ﬁltration and concentration, the residue was

puriﬁed by preparative TLC (n-hexane/AcOEt = 1/1 (v/v)) to give the

title compound 18 (5.0 mg, 43% yield).

4.2. Crystallographic methods

Protein production and puriﬁcation of PDHK4 kinase domain (resi-

dues 10–411) and PDHK2 kinase domain (residues 16–407) were carried

out as previously reported.^16–17Brieﬂy, these PDHK kinase domains

were produced in Escherichia coli and puriﬁed by Ni-NTA afﬁnity col-

umn, protease digestion and gel ﬁltration. The puriﬁed PDHK4 proteins

were crystallized and crystals were obtained at 22 ^◦C using the hanging-

drop vapor diffusion method with reservoir containing 50 mM KHPO₄

pH 7.5, 1.7 M ammonium sulfate and 4% (v/v) PEG400, 5 mM ADP. X-

ray fragment screening was carried out using in-house fragment library.

A soaking drop was prepared by adding the cocktail mixture of four

fragments to achieve a ﬁnal concentration of 12.5 mM. Crystals were

then transferred to the soaking drop and soaked for a period of 24 h. For

SBDD studies of PDHK4 in complex with fragment hit derivatives,

structures of these complexes with compounds 1, 3, and 7 were obtained

by soaking in a similar manner. Regarding PDHK2, the puriﬁed PDHK2

proteins were crystallized in complex with compound 13. Co-crystals

¹H NMR (400 MHz, CDCl₃) δ: 2.00 (dd, J = 6.7, 1.7 Hz, 3H), 2.73 (s,

3H), 6.23 (dq, J = 16.1, 6.7 Hz, 1H), 6.58 (dq, J = 16.1, 1.7 Hz, 1H), 9.57

(br s, 1H)

HRMS (ESI, m/z, MH+) Calcd for C₁₀H₁₀N₃Cl₂: 242.0246, Found:

242.0527

4.1.16. Synthesis of compound 17

Step 1: 4-Chloro-2-methyl-7-((2-(trimethylsilyl)ethoxy)methyl)-7H-

pyrrolo[2,3-d]pyrimidine-6-carboxylic acid (62)

Sodium dihydrogen phosphate dihydrate (114 mg, 0.734 mmol), 2-

methylbut-2-ene (650 μg, 6.14 mmol) and sodium chlorite (68.0 mg,

0.736 mmol) were sequentially added to a solution of 59 (200 mg, 0.613

mmol) in 2:2:1 t-BuOH-H₂O-THF (2.5 mL) and stirred overnight at room

temperature. The reactant mixture was diluted with H₂O and extracted

with AcOEt. The organic layer was washed with H₂O and brine, and

dried over MgSO₄. After ﬁltration and concentration, for puriﬁcation,

the residue was slurried in n-hexane. The precipitated solid was

collected by ﬁltration, washed with n-hexane, and dried to give the title

compound 62 (120 mg, 57% yield) as a mixture with a small amount of

impurities. This was used for the next step without further puriﬁcation.

Step 2: 4-Chloro-N,2-dimethyl-7-((2-(trimethylsilyl)ethoxy)methyl)-

7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide (63)

◦

were obtained at 4 C using the hanging-drop vapor diffusion method

with reservoir containing 50 mM Na acetate pH 5.5, 100 mM magne-

sium chloride and 8% (v/v) isopropanol.

Diffraction data of the soaked crystals and co-crystals were collected

at Photn Factory (Japan), SPring-8 (Japan), Canadian Light Source

(Canada) and Swiss Light Source (Switzerland). The data were inte-

grated with DIALS and scaled using aimless.¹⁸The structures of the

PDHK-compound complex were solved by molecular replacement with

MOLREP in CCP4 suite^19–20using the coordinates of the PDHK4 kinase

To a solution of 62 (120 mg, 0.350 mmol) in CHCl₃(2.0 mL) was

added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

(74.0 mg, 0.386 mmol), N,N-dimethyl-4-aminopyridine (2.00 mg,

domain (PDB ID 2ZKJ¹⁶) or the PDHK2 kinase domain (PDB ID 2BTZ¹⁷

)

as the model. The structural models were built in Coot²¹and reﬁned

using REFMAC5 in CCP4 suite²²and Phenix²³. Figures were created

0.0164 mmol) and 40% methylamine in methanol (39.0

μL, 0.386

mmol), and the mixture was stirred overnight at room temperature. The

mixture was diluted with H₂O and extracted with AcOEt. The organic

layer was washed with H₂O, saturated aqueous NaHCO₃solution and

brine, and dried over MgSO₄. After ﬁltration and concentration, the

residue was loaded on silica gel column chromatography (eluted with n-

hexane/EtOAc = 66/34 (v/v)) for elimination of the polar component.

The title compound 63 (90.0 mg, 72% yield) was obtained, which was

used for the next step without further puriﬁcation.

using PyMOL²⁴

.

4.3. Spectrophotometry assay to measure the inhibitory activity on

PDHKs

PDHK activity was assessed indirectly by measuring the residual PDH

activity after PDHK reaction essentially as described previously.²⁵

Brieﬂy, a porcine PDH complex (Sigma-Aldrich) and each of the re-

combinant human PDHK (1, 2, 3, or 4) enzymes were mixed and incu-

bated overnight at 4 ^◦C to obtain PDH/PDHK complex. The PDH/PDHK

complex and PDHK inhibitors were incubated for 45 min at room tem-

Step

3:

5-Bromo-4-chloro-N-2-dimethyl-7-((2-(trimethylsilyl)

ethoxy)methyl)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide (64)

To a suspension of 63 (90.0 mg, 0.253 mmol) in CHCl₃(2.0 mL) was

added N-bromosuccinimide (54.0 mg, 0.304 mmol). After stirring at

room temperature for 2.5 h, the reactant mixture was quenched with

saturated NaHCO₃solution. The mixture was extracted with AcOEt, and

the organic layer was washed with H₂O and brine, and dried over

MgSO₄. After ﬁltration and concentration, the residue was puriﬁed by

preparative TLC (n-hexane/AcOEt = 2/1 (v/v)) to give the title com-

pound 64 (92.0 mg, 84% yield) as a mixture with a small amount of

impurities. This mixture was used for the next step without further

puriﬁcation.

perature after adding 0.3 or 10 μM ATP to start the PDHK reaction. Then,

the substrates for PDH (5.0 mM coenzyme A, 5.0 mM sodium pyruvate

and 12 mM β-nicotinamide adenine dinucleotide) were added to start

the PDH reaction and incubated for 90 min at room temperature. The

PDH reaction was analyzed by measuring the reaction product, NADH.

The amount of NADH was determined measuring the absorbance at 340

nm before and after the PDH reaction. Concentration ꢀ response data

were ﬁtted to the following equation using Spotﬁre (TIBCO):

%inhibition = (m ax - m in)/ 1 + 10 ^{(log IC50 - log [com p]) x Hill}+ m in

Step 4: 5-Bromo-4-chloro-N-2-dimethyl-7H-pyrrolo[2,3-d]pyrimi-

dine-6-carboxamide (17)

where min is the 100% enzymatic activity control, max is the 0%

enzymatic activity control.

To a solution of 64 (90.0 mg, 0.207 mmol) in CH₂Cl₂(1.0 mL), boron

triﬂuoride diethyl etherate (77.0 μL, 0.624 mmol) was added at room

temperature. After stirring overnight at room temperature, the mixture

was quenched with 2 N NaOH solution and extracted with AcOEt. The

organic layer was washed with H₂O and brine, and dried over MgSO₄.

After ﬁltration and concentration, for puriﬁcation, the residue was

13

T. Akaki et al.

Bioorganic & Medicinal Chemistry 44 (2021) 116283

4.4. RapidFire MS assay to measure the inhibitory activity on PDHK2 and

PDHK4

Protein Data Bank with codes 7EAT for 1/PDHK4, 7EBB for 2/PDHK4,

7EA0 for 1/PDHK2, 7EAS for 2/PDHK2, 7EBG for 7/PDHK4 and 7EBH

for 13/PDHK2 respectively.

The PDH reaction mixtures were diluted 25 fold with 0.1% (v/v)

formic acid. The PDH reaction product acetyl-coenzyme A was subse-

quently analyzed using RapidFire300 (Agilent TechnologiShes), an in-

tegrated high throughput autosampler/solid-phase extraction system,

coupled to a 3200QTRAP triple-quadrupole mass spectrometer (Sciex).

The samples were aspirated under vacuum directly from 384-well assay

plates for 0.25 s and loaded onto a graphitic carbon (Type D) solid-phase

extraction cartridge (Agilent Technologies) with 5 mM ammonium ac-

etate (pH 10.0) at a ﬂow rate of 1.25 mL/min for 3 s. Acetyl-coenzyme A

was eluted with a 5 mM ammonium acetate (pH 10.0) /acetonitrile/

acetone mixture (50:25:25, v/v/v) at a rate of 1.5 mL/min for 5 s. The

cartridge was then re-equilibrated with 5 mM ammonium acetate (pH

10.0) at a rate of 1.25 mL/min for 0.5 s. The eluate was analyzed in the

mass spectrometer in the positive ionization mode using a gas temper-

ature of 350 ^◦C, a nebulizer pressure of 50 psi, and a spray voltage of

5500 V. The mass spectrometer was operated in the multiple reaction

monitoring (MRM) mode, with transitions (Q1/Q3) of analyte as fol-

lows: 808/79 and 808/408. The area under the curve (AUC) of the

extracted ion counts was calculated using RapidFire Integrator version

3.6 (Agilent Technologies). MS/MS signal of ATP was used to conﬁrm

every introduction of a sample into the mass spectrometer.

Declaration of Competing Interest

The authors declare that they have no known competing ﬁnancial

interests or personal relationships that could have appeared to inﬂuence

the work reported in this paper.

Acknowledgments

The authors thank Mr. Eita Nagao for analytical support, and Dr. Jiro

Kataoka for helpful discussion. We also thank Prof. Toshiya Senda for

the data collection at the Photon Factory, KEK.

Appendix A. Supplementary material

Supplementary data to this article can be found online at https://doi.

org/10.1016/j.bmc.2021.116283.

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4.7. Kinase selectivity assay

The kinase panel assay was conducted using the DiscoveRx KINOME

scan™ assay service toward 59 kinases.

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4.8. Computational study

¨

WaterMap (Schrodinger Release 2018–1: Schrodinger, LLC, New-

York, NY, 2018) calculations were performed on the co-crystal structure

of fragment hit 1 bound to PDHK4. Input structure was prepared by

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¨

Protein Preparation Wizard in Maestro (Schrodinger Release 2018–1)

using default options. WaterMap was run in default mode with OPLS3e

force ﬁelds, existing waters were deleted, and a 2 ns MD simulation. The

fragment hit 1 was used to deﬁne the binding site.

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Atomic coordinates and structure factors have been deposited in the

14

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15

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Fragment-based lead discovery to identify novel inhibitors that target the ATP binding site of pyruvate dehydrogenase kinases

DOI: 10.1016/j.bmc.2021.116283

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