Substituted pteridinones as p90 ribosomal S6 protein kinase (RSK) inhibitors: A structure-activity study

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Substituted pteridinones as p90 ribosomal S6 protein kinase (RSK) inhibitors:

a structure-activity study

Kimberly A. Casalvieri, Christopher J. Matheson, Donald S. Backos, Philip

Reigan

PII:

S0968-0896(19)31117-4

https://doi.org/10.1016/j.bmc.2019.115303

BMC 115303

DOI:

Reference:

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Bioorganic & Medicinal Chemistry

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Revised Date:

Accepted Date:

13 September 2019

20 December 2019

31 December 2019

Please cite this article as: K.A. Casalvieri, C.J. Matheson, D.S. Backos, P. Reigan, Substituted pteridinones as

p90 ribosomal S6 protein kinase (RSK) inhibitors: a structure-activity study, Bioorganic & Medicinal Chemistry

(2020), doi: https://doi.org/10.1016/j.bmc.2019.115303

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Graphical Abstract

Substituted pteridinones as p90 ribosomal S6

protein kinase (RSK) inhibitors: a structure-

activity study

Leave this area blank for abstract info.

Kimberly A. Casalvieri, Christopher J. Matheson, Donald S. Backos, and Philip Reigan

Hinge

domain

Leu150

Asp148

33

Asp211

Gly213

DFG

motif

Phe212

Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier.com

Substituted pteridinones as p90 ribosomal S6 protein kinase (RSK) inhibitors: a

structure-activity study

Kimberly A. Casalvieri^a, Christopher J. Matheson^a, Donald S. Backos^a, and Philip Reigan^a

^aDepartment of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, 12850

East Montview Boulevard, Aurora, CO, 80045, USA.

ARTICLE INFO

ABSTRACT

Article history:

Received

Received in revised form

Accepted

The activity of p90 ribosomal S6 kinase 2 (RSK2) has emerged as an attractive target for cancer

therapy due to its role in the regulation of diverse cellular processes, such as cell transformation

and proliferation. Several pan-RSK inhibitors have been identified with BI-D1870 and the pseudo-

analogs LJH685 and LJI308 being the most selective, potent, and frequently used small molecule

inhibitors. We designed and synthesized a series of pteridinones and pyrimidines to evaluate the

structural features of BI-D1870 that are required for RSK2 inhibition. We have identified

inhibitors of RSK2 activity, evaluated their target engagement in cells, and measured their effect

on cell viability and cytotoxicity in the MOLM-13 acute myeloid leukemia (AML) cell line. The

results of our studies support that RSK2 inhibition can be achieved in MOLM-13 cells without

potent cytotoxicity. The structure-activity data from this study will be used as a platform to

develop novel RSK2 inhibitors.

Available online

Keywords:

RSK2

kinase

inhibitor

pteridinones

cancer

1. Introduction

squamous cell carcinoma, glioblastoma, leukemia, lung cancer,

malignant melanoma, multiple myeloma, ovarian carcinoma, and

The p90 ribosomal S6 kinases (RSK1-4) are a family of

serine/threonine kinases that are comprised of two functionally

distinct kinase domains, a catalytic N-terminal kinase domain

(NTKD) and an activating C-terminal kinase domain (CTKD)

connected by a linker region. The RSK1-3 family members act as

the downstream effectors of the Ras/Raf/MEK/ERK pathway,^1-4

while RSK4 has an alternative mechanism of activation.⁵

Extracellular signal-regulated kinase (ERK) binds the inactive

form of RSK1-3 at the C-terminal, and upon activation ERK

phosphorylates serine and threonine residues in the CTKD and

linker region, promoting recruitment of phosphoinositide-

prostate cancer.⁸

The Ras/Raf/MEK/ERK pathway is often activated in leukemia

and confers a poor prognosis and resistance to chemotherapy.^{9, 10}

Activating mutations of the surface receptor kinase FMS-like

tyrosine kinase 3 (FLT3) due to internal tandem duplication (ITD)

are the most common abnormality in acute myeloid leukemia

(AML)

leading

to

constitutive

activation

of

the

Ras/Raf/MEK/ERK pathway.¹¹More recent studies have shown

that RSK2 activity is required for the initiation and progression of

FLT3/ITD-induced AML.¹²Fibroblast growth factor receptor 3

(FGFR3), another tyrosine kinase cell-surface receptor, has also

been implicated in the progression of hematological malignancies,

including AML, by activating RSK2 via an alternative two-step

dependent protein kinase

1

(PDK1) that facilitates

phosphorylation of the NTKD, leading to full activation of RSK.⁶

The RSKs phosphorylate a range of cytoplasmic and nuclear

substrates involved in transcription, translation, cell-cycle

regulation, and cell survival.^1-4Although the RSKs share a high-

degree of structural homology (73–80% sequence identity), there

is increasing evidence of isoform specificity in mediating diverse

cellular processes.^1-4The overall functional differences of the RSK

isoforms are more apparent in cancer, with increased expression

and activity of RSK1 and/or RSK2 promoting tumor growth and

survival, whereas RSK3 and RSK4 act as tumor suppressors

through multiple cellular mechanisms.⁷The aberrant expression

and/or activity of RSKs has been associated with several cancer

types, including breast cancer, colorectal cancer, head and neck

mechanism

facilitating

ERK-RSK2

interaction

and

14

phosphorylation of RSK2.^13,

RSK1 and RSK2 are the

predominant isoforms expressed in AML,¹⁵and have been

reported to phosphorylate cAMP response-element binding

protein (CREB), a nuclear transcription factor critical for

hematopoietic cell proliferation, differentiation, and survival.¹⁶

Approximately 60% of patients with AML express CREB at high

levels and this is associated with an increased risk of relapse and

decreased survival.¹⁵The phosphorylation of CREB by RSK1 or

RSK2 promotes cell survival by increasing the transcription of

Bcl-2, Bcl-xL, and induced myeloid leukemia cell differentiation

protein (Mcl-1).¹⁷Therefore, targeting RSK1 and/or RSK2 with

small molecule inhibitors would prevent these pro-survival

signaling events and be an attractive therapeutic strategy for the

treatment of AML.

methyl maintains RSK inhibitory activity, and the N⁸-alkyl

substitution is required for potent RSK inhibition. We have

identified compounds that inhibit RSK2 activity in cells and our

cellular viability and toxicity studies suggest that RSK2 inhibition

is uncoupled from potent cytotoxicity in AML cells. Our studies

have provided important structural information for the

development of pteridinone-based RSK inhibitors and potential

insights into structural modifications that may limit metabolism

and improve RSK selectivity.

Several small molecule RSK inhibitors have been reported that

target either the NTKD or CTKD of the RSKs. The flavonol

rhamnoside SL0101 was the first pan-RSK inhibitor to be

identified acting at the NTKD and several other flavanol

rhamnosides have since been reported as RSK inhibitors; however,

poor pharmacokinetic (PK) properties and off-target effects have

limited their development.^{18, 19}The fluoromethyl ketone, FMK, is

a irreversible inhibitor that covalently binds to a cysteine residue

in the CTKD of RSK1, RSK2, and RSK4 (the cysteine residue is

absent in RSK3).²⁰Although FMK is a potent and surprisingly

specific RSK inhibitor, its application is limited since it is not

effective once the kinase is activated and the CTKD is not always

required for RSK activation.^{20, 21}The reversible pan-RSK inhibitor

BI-D1870 (Figure 1) is a dihydropteridinone that competes with

ATP at the NTKD, with an in vitro IC₅₀of 31nM for RSK1, 24nM

for RSK2, 18nM for RSK3, and 15nM for RSK4, and a

concentration of ~10μM is required to completely inhibit RSK

activity in cells.²²Although some of the cell signaling effects of

BI-D1870 have been attributed to non-specific interactions, it has

respectable narrow-band activity within the kinome with PLK1,

Aurora B, MELK, and MST2 among its other targets.^22-24

Unfortunately, BI-D1870 has a poor PK profile, displaying high

clearance and a short plasma half-life and this has prevented its

clinical evaluation.^{25, 26}The impressive potency and selectivity of

BI-D1870 led to the development of LJI308 (IC₅₀values of 3nM

for RSK1, 4nM for RSK2, and 13nM for RSK3), and LJH685 (IC₅₀

values of 6nM for RSK1, 5nM for RSK2, 4nM for RSK3);

however, these compounds also suffer from poor PK profiles,

which has limited their development and clinical utility.^{27, 28}

2. Results and Discussion

2.1 Computational-based RSK2 inhibitor design

Although the NTKD crystal structures for both RSK1 and RSK2

have been resolved, we utilized the RSK2 NTKD structure co-

crystallized with BI-D1870 for our computational-based docking

simulations and inhibitor design studies,²⁸as we could directly

compare the in silico predicted binding with the co-crystallized

conformation of BI-D1870. The RSKs share a high degree of

homology, particularly in the ATP-binding site, and the objective

of this study was to understand the structural properties required

for RSK inhibition and not to examine the potential for isoform

selectivity; therefore, the crystal structure of the NTKD of RSK2

was used for all computational-based docking simulations. The

preparation of the BI-D1870 structure for docking simulations

highlighted the chiral carbon at C⁷, an often-overlooked feature of

BI-D1870. Interestingly, while the R-isomer is the species co-

crystallized with RSK2, the modeling results suggested that both

isomers of BI-D1870 are capable of binding to RSK2. Overall, our

docked conformation for BI-D1870 was similar to that of the

RSK2 co-crystallized BI-D1870 structure and displayed few

binding interactions with the residues of the ATP-binding site of

the RSK2 NTKD (Figure 2), which is surprising for a potent,

nanomolar inhibitor. As is common to many ATP-competitive

kinase inhibitors, the modeling revealed a donor-acceptor motif of

H-bond interactions present between the core pteridinone

heterocycle of BI-D1870 and the kinase hinge backbone residues

Asp148 and Leu150. An additional H-bond interaction also occurs

between the phenol of BI-D1870 and the carbonyl of Asp211in the

DFG motif.

F

OH

F

OH

HO

F

N

O

N

F

N

H

N

O

BI-D1870 (1)

LJH685 (2)

LJI308 (3)

To confirm the necessity of the H-bond interaction with DFG

motif, we synthesized negative control compounds that either lack

the hydroxyl group (24) or substitute it for a methoxy group (25).

In addition, we examined whether a carbon extension of the phenol

to a benzyl alcohol could extend the position of the H-bond donor

further into the binding pocket and offer an improved bond angle

and orientation for the interaction to occur. A means for

synthesizing the difluoro-benzyl alcohol building block required

for this compound was not readily commercially available,

requiring alternative synthesis of the benzylic alcohol lacking the

fluorine atoms (26) to test this modification. In order to account

for more than one modification, the unsubstituted phenol analog

of BI-D1870 (27) was also proposed to allow for direct

comparison. The fluorine atoms present in BI-D1870 likely serve

to protect the compound from ortho- oxidative metabolism of the

phenol ring; however, the substitution pattern and identity of the

halogen atoms, and hence their electronic effects may also serve

to optimize the pKa of the phenol hydrogen to facilitate H-bonding

with Asp211 of the DFG motif. Therefore, both the mono- and di-

halogenated analogs containing fluorine, chlorine, and bromine

were proposed (28-32) in order to identify the ideal substitution

pattern and electron-withdrawing characteristics adorning the

phenol ring to facilitate this key H-bond interaction. The docked

conformation of BI-D1870 in the ATP-binding site of the RSK2

Figure 1. Chemical structures of the known RSK inhibitors BI-

D1870, LJH685, and LJI308.

Despite the shortcomings of BI-D1870, it is a relatively specific

and potent nanomolar inhibitor of the RSK isoforms and has been

used by a number of groups to identify the physiological substrates

and functional roles for RSK in cells.²²Considering the specific

and potent RSK inhibition demonstrated by BI-D1870 and the

emergence of RSK1 and RSK2 as anticancer targets in multiple

cancer types, it is surprising that there have been few structure-

activity relationship (SAR) studies of substituted pteridinones as

RSK inhibitors. The difluorophenolpyridine derivatives LJH685

and LJI308 are the only BI-D1870 analogs that have been reported

and these were not developed as part of an exhaustive structure-

activity study and do not interrogate substitution of the pteridinone

core of BI-D1870. Therefore, we performed a SAR study to

determine the necessary structural features of BI-D1870 required

for RSK inhibition. Herein, we describe a series of substituted

pteridinones and pyrimidines based on BI-D1870, the evaluation

of their inhibition of RSK activity, and their impact on cell

viability or cytotoxicity in AML cells. Our studies indicate that

halogen substitutions on the 4-hydroxyanilino can greatly impact

RSK inhibitory potency, elimination of C⁷chirality does not

appear to impact RSK inhibitory potency, removal of the N⁵-

NTKD suggested that the piperazinone ring of the pteridinone does

not make any key interactions with the kinase other than

hydrophobic contacts (Figure 2). Therefore, we proposed to

remove the piperazinone ring and sequentially add functional

complexity present in the parent compound to the pyrimidine core

(38), to determine the structural components required for binding

of BI-D1870 to RSK2. The compounds proposed included the

aminonitropyrimidine pseudocycle (43), the diaminopyrimidine

(44), and the pteridines lacking the N⁵-methyl (33), lacking the N⁸-

isopentyl chain (39), and devoid of C⁷chirality (34).

hydrogenation in the presence of HCl to generate the salt form of

amino acid ethyl esters 11 and 12. These, along with alanine ethyl

ester (13), were reacted with 2,4-dichloro-5-nitropyrimidine (14)

to generate the required 4-amino-5-nitropyrimidines, both

containing (15-16) and lacking (17) the N⁸-isopentyl group present

in BI-D1870. An iron/acetic acid reduction of the 5-nitro group

with concomitant pteridinone cyclization was utilized to form the

heterocyclic intermediates 18, 19, and 20, which were

subsequently methylated at the N⁵-position to generate the core

pteridinones 21, 22, and 23.

(1)

b

N

a

NH₂

+

N

H

O

Br

O

4

5

6

7

(2)

O

N

c

N

+

H

O

N

O

H

O

8

9

10

(3)

Cl

H₂N

R¹

NO₂

R²

NO₂

Cl

d

e

N

+

7 10

,

O

R²

Cl

N

R¹

Cl

N

O

; R¹=CH₂CH₂CH(CH₃

; R¹=H, R²=CH₃

)

₂, R²=CH₃

₂, R²=H

14

15

16

17

; R¹=CH₂CH₂CH(CH₃

)

₂, R²=CH₃

11

12

13

; R¹=CH₂CH₂CH(CH₃

; R¹=H, R²=CH₃

)

₂, R²=H

R³

N

O

f

h

N

R⁴

Cl

N

R¹

R²

N

R¹

R²

H

; R¹=CH₂CH₂CH(CH₃

)

₂, R²=CH₃, R³=H

24 25 31 39

)

18

See Table (

,

-

g

; R¹=CH₂CH₂CH(CH₃

)

₂, R²=CH₃, R³=CH₃

₂, R²=H, R³=H

21

19

g

; R¹=CH₂CH₂CH(CH₃

)

₂, R²=H, R³=CH₃

22

20

; R¹=H, R²=CH₃, R³=H

g

; R¹=H, R²=CH₃, R³=CH₃

23

Scheme 1. Synthesis substituted pteridinones. Reagents and

conditions: (a) Cs₂CO₃, DMF, rt, 48 h; (b) ethyl 2-

bromopropionate, K₂CO₃, DMF, 110°C, 4 h; (c) i) MeOH, 10%

AcOH, 50°C, 1 h; ii) NaBH₃CN, 50°C, 1 h; (d) 10% Pd/C, H₂,

EtOH, 10M HCl, rt, 2-16 h; (e) Et₂O/H₂O, K₂CO₃, -15°C, 2 h; (f)

Fe, AcOH, 70°C  100°C; (g) MeI, NaH, DMA, -15°C, 30 min;

(h) relevant aniline, TFA, TFE, MW, 140°C, 30 min.

Figure 2. Crystal structure of the NTKD of RSK2 (PDB: 5D9K)

with docked BI-D1870. (A) Ribbon representation of the RSK2

NTKD with amino acid residues of the hinge domain and the DFG

motif displayed in stick form (grey) with the docked R-isomer of

BI-D1870 (orange). (B) Stick display style representation of the

docked conformation of the S-isomer (cyan) and R-isomer

(orange) of BI-D1870 and key interacting amino acid residues

(grey), where green dashed lines indicate H-bonds. Ligand

interaction map of the predicted binding mode of (C) the S-isomer

and (D) the R-isomer of BI-D1870 in the ATP-binding site of the

RSK2 NTKD, where red residues are charged negative, purple

residues are charged positive, green residues are hydrophobic, and

blue residues are polar, and purple arrows indicate H-bonds.

While the dichlorino aniline was commercially available, the

difluoro- (41) and dibromoanilines (42) were generated from the

dihalonitrophenols using a tin(II) chloride nitro reduction. The N⁵-

unmethylated (18-20) and methylated (21-23) pteridinone cores

were then coupled in combination with the corresponding aniline

to generate final compounds 24, 25, and 31-39 (Scheme 1). In

following the TFA/TFE-mediated synthetic approach, the

proposed compounds investigating carbon extension of the phenol

underwent coupling via nucleophilic attack by both the aniline and

the phenol, and thus an alternative approach was required. 4-

Nitrophenol and 4-nitrobenzyl alcohol were alternatively

protected at the hydroxyl position with triisopropylsilylchloride,

reduced to the corresponding aniline, and coupled to the

2.2 Chemical synthesis of BI-D1870 analogs

A series of 19 analogs of BI-D1870 were synthesized in order to

investigate the structural components of BI-D1870 necessary for

potent RSK inhibition. In general, various anilines were coupled

to the heterocyclic core of BI-D1870 or analogs thereof using a

trifluoroacetic acid (TFA) and trifluoroethanol (TFE)-mediated

S_NAr reaction according to Carbain et al.²⁹To synthesize the

pteridinone heterocyclic core of BI-D1870 (Scheme 1), 1-bromo-

3-methylbutane (4) was coupled with benzylamine (5) to afford

the desired secondary amine (6), which was subsequently reacted

with ethyl 2-bromopropanoate to generate intermediate 7. To

synthesize the analogous intermediate lacking a chiral carbon (10),

pteridinone core 21 via

a Buchwald-Hartwig amination.

Successive TIPS-deprotection was performed to generate

compounds 26 and 27 (Supplemental Scheme S1). In

investigating a means for the most efficient synthetic route for

generating BI-D1870 analogs, all mono-halogenated anilines were

prepared from the corresponding methoxy-pteridinone

intermediates via TFA/TFE coupling and subsequent BBr₃

demethylation to yield compounds 28-30 (Supplemental Scheme

S2). These compounds collectively investigate how amendable BI-

D1870 is to modification of the halogen substitution pattern,

removal of the N⁵-methyl, removal of the N⁸-isopentyl chain, and

a

reductive amination between isovaleraldehyde (8) and

benzylglycine ethyl ester (9) was performed in the presence of

sodium cyanoborohydride. The benzyl protecting groups of

intermediates 7 and 10 were removed via a palladium-mediated

elimination of C⁷chirality for binding with the ATP-binding

domain of RSK2. Finally, to examine modification to the

pteridinone core of BI-D1870, 2,4-dicholoro-5-nitropyrimidine

was treated with ammonia to selectively displace the 4-chloro

atom and yield intermediate 40 (Scheme 2). Difluoroaniline 41

was then coupled to afford aminonitropyrimidine pseudocycle 43,

the 5-nitro group of which was reduced with tin(II) chloride to give

the 4,5-diaminopyrimidine 44.

R³

N

O

N

R¹

N

R²

H

Compound

R¹

R²

R³

RSK2 Inhibition

F

NO₂

NH₂

NO₂

Cl

HO

F

NO₂

NH₂

HO

F

NH₂

a

N

b

c

HO

F

N

BI-D1870 (1)

CH₃

23.3 ± 8.2 nM

Cl

N

Cl

N

H

N

H

N

14

40

43

44

F

24

25

CH₃

71% @ 10 µM

43% @ 10 µM

F

Scheme 2. Synthesis substituted pyrimidines. Reagents and

conditions: (a) 2M NH₃/EtOH, DCM, 0°C  RT, 45 min; (b)

aniline 43, TFA, TFE, MW, 140°C, 30 min; (c) SnCl₂, EtOH,

reflux, 24 h.

F

O

F

HO

26

27

CH₃

739 ± 14.1 nM

54.8 ± 1.4 nM

2.3 Inhibition of RSK2 kinase activity

HO

The synthesized compounds were tested in a recombinant TR-

FRET kinase inhibition assay against RSK2 and their half-

maximal inhibitory potency values (IC₅₀) were calculated using

nonlinear regression analysis of the log dose-response (Table 1,

Table 2). Compounds found to have either improved or equipotent

activity when compared to BI-D1870 (RSK2 IC₅₀= 23.3 ± 8.2 nM)

were tested against the remaining three RSK isoforms

(Supplemental Table S1) to probe for isoform selectivity and

were selected for cellular testing. As anticipated, elimination of the

H-bond interaction between the kinase DFG-motif and the phenol

of BI-D1870 with 24 and 25 led to a complete loss in potency,

demonstrating the necessity of this interaction for RSK inhibition.

While the carbon extension of the phenol to the benzyl alcohol

caused a significant loss in potency, this may be attributed to the

loss of the two fluorine atoms, as a decrease in activity was

likewise observed for the accompanying unsubstituted phenol. The

two fluorine atoms of BI-D1870 may play a role in improving

metabolic stability, but also alter the pKa of the adjacent phenol

and, consequently, the ability of the phenol to H-bond with

Asp211 of the DFG motif. The stepwise improvement in inhibitory

activity between monobromo 30, monochloro 29, and monofluoro

28 analogs may be attributed to the electronic effects of the

halogen substitutions, and thus, the pKa of the phenol. However,

it is also possible that the steric bulk of the chlorine and bromine

substituents may not be well tolerated in comparison to smaller

hydrogen and fluorine atoms, as indicated by the improved activity

of the unsubstituted phenol in comparison to 29 and 30. Complete

loss of activity occurred with both the dichloro 31 and dibromo 32

analogs and is further indication that the steric bulk and/or

electronic effects of chloro- and bromo-substitutions are sub-

optimal for RSK2 inhibition.

F

28

29

30

31

32

33

34

35

36

37

CH₃

H

CH₃

H

25.4 ± 3.2 nM

71.5 ± 10.3 nM

141 ± 25.7 nM

83% @ 10 µM

78% @ 10 µM

18.2 ± 1.4 nM

17.6 ± 1.4 nM

38.3 ± 7.9 nM

23.4 ± 4.7 nM

24.7 ± 1.8 nM

Cl

Br

Cl

Br

F

HO

Cl

HO

Br

HO

F

HO

F

CH₃

H

F

HO

H

F

HO

F

H

F

HO

H

CH₃

Table 1. Inhibition of RSK2 activity by substituted pteridinones.

Inhibitory activity of compounds in the TR-FRET kinase assay

against RSK2 given as the half-maximal inhibitory concentrations

(IC₅₀) or percentage inhibition; values are the mean ± S.D. (n=3).

As presented in Table 2, replacement of the pterdinone core of BI-

D1870 with a pyrimidine resulted in complete loss of activity. In

addition, all the compounds that sequentially incorporated

components of the core pteridinone heterocycle resulted in

reduced RSK2 inhibitory activity. Interestingly, removal of the N⁸-

isopentyl chain alone caused a complete loss in activity against

RSK2. Removal of the N⁵-methyl group of BI-D1870 was not only

advantageous as a means to reduce the number of synthetic steps,

it also offered some improvement in activity over BI-D1870.

Additionally, eliminating chirality is attractive in order to avoid

enantiomer selectivity potentially attenuating the activity of the

racemate, and removal of the chiral C⁷also improved inhibitory

potency. After analyzing the initial TR-FRET results, several

additional compounds were synthesized combining modifications

that led to improved or equivalent RSK2 inhibitorypotency with

respect to BI-D1870 (35-37). These include monofluoro

halogenation of the phenol ring, removal of the N⁵-methyl from

the pteridinone, and elimination of C⁷-chirality. Interestingly,

combinations of these modifications did not result in any

substantial additive improvements in RSK2 inhibitory potency as

all three analogs demonstrated equivalent RSK2 inhibitory activity

as BI-D1870. For comparison, the previously described

compounds LJH685 and LJI308 were also tested against RSK2

activity and resulted in improved inhibitory potency over BI-

D1870 (LJH685 IC₅₀= 11.8 ± 0.5 nM; LJI308 IC₅₀= 12.9 ± 0.3

nM, Supplemental Table S2), which is in agreement with

published literature.²⁸

F

HO

R

F

N

H

Compound

38

R

RSK2 Inhibition

Figure 3. Cellular effects of RSK inhibitors on viability and

toxicity in MOLM-13 cells. Dose-response curves for RSK

inhibitors treated over a concentration range from 60 µM to 250

nM in an MTS assay (A) and CellTiter-Glo assay (B) after 72 h.

Dose-response curves for RSK inhibitors treated for 24 h (C) and

72 h (D) over a concentration range from 60 µM to 250 nM in a

CellTox Green assay (n = 3, error bars: ± S.D.).

N

45% @ 10 µM

N

O

N

39

83% @ 10 µM

N

H

NO₂

N

43

44

72% @ 10 µM

38% @ 10 µM

N

NH₂

To gain further insight into the potential mechanism of action of

RSK inhibitors, a CellTiter-Glo^®cell viability assay (Figure 3B)

and a CellTox^™Green cellular toxicity assay (Figure 3C, Figure

3D) were performed in MOLM-13 cells. The results suggest that

BI-D1870 and potent analogs thereof act as cytotoxic agents rather

than cytostatic, as indicated by the equivalent and potent EC₅₀

values determined for BI-D1870 in the viability and toxicity assays

after 72 h. Results in the CellTiter-Glo^®assay for all synthesized

analogs coincide with the trends seen in the MTS assay in which

compound 28 was the only analog to demonstrate improved

potency over BI-D1870. Again, compound 34 displayed

equipotency to BI-D1870 and 33, LJH685 and LJI308 exhibited

significantly diminished effects on MOLM-13 cell viability.

Additional evidence to suggest that BI-D1870 and its inhibitory

analogs act in a cytotoxic manner was the dramatic increase in

cellular toxicity at 72 h. Interestingly, BI-D1870 was the only

cytotoxic compound at 24 h, but all compounds exhibited

cytotoxicity after 72 h, with analogs 28, 34, and 35 demonstrating

equipotent cytotoxic effects to BI-D1870, while 33, LJH685, and

LJI308 exhibited some cytotoxicity but to a lesser degree than BI-

D1870 (Table 3).

NH₂

N

Table 2. Inhibition of RSK2 activity by substituted pyrimidines.

Inhibitory activity of synthesized compounds in the TF-FRET

kinase assay against RSK2 given as percentage inhibition; values

are the mean (n=3).

2.4 Growth inhibition and cytotoxic effects of RSK2 inhibitors

The compounds that exhibited activity in the kinase activity assay

within the concentration range of BI-D1870 were evaluated in cell

systems for effects on cell viability and toxicity. A series of

leukemic cell lines were analyzed for constitutive phospho- and

total-RSK1 and RSK2 protein via Western Blot (Supplemental

Figure S1) to ensure use of an appropriate cell line for these assays.

From these data, it was determined that MOLM-13 cells expressed

robust quantities of active RSK1 and RSK2 and these cells were

used for all cell viability and cytotoxicity assays. Results from an

MTS assay offered initial insights into the effects of inhibitors on

cellular viability (Figure 3A). After treatment with inhibitors for

72 h, the mono-fluoro analog 28 was the only compound to

demonstrate improved inhibition of cell growth over BI-D1870,

while the pteridinone lacking C⁷-chirality (34) displayed

equipotency to BI-D1870. Despite analog 34 showing no

improvement in activity in this assay, eliminating chirality while

retaining effects on cell viability may be a key finding for further

development of the BI-D1870 molecule. Interestingly, pteridinone

33 lacking the N⁵-methyl and the known RSK inhibitors LJH685

and LJI308 demonstrated minimal effects on cell viability despite

being potent RSK2 inhibitors.

Table 3. Cellular effects of RSK inhibitors on viability and

toxicity in MOLM-13 cells. Inhibitory activity of compounds in

the TR-FRET kinase assay against RSK2 as the half-maximal

inhibitory concentrations (IC₅₀) values are the mean ± S.D. (n=3).

EC₅₀values or percentage inhibition values from cellular assays

were determined for RSK inhibitors treated for over

concentration range from 60 µM to 250 nM.

a

2.5 Inhibition of cellular RSK2 activity

viability and 28 and 34 were also the most potent inducers of

cytotoxicity after 72 h of exposure.

Inhibitors of purified RSK2 activity identified in the TR-FRET

assay were evaluated for their capacity to inhibit intracellular

RSK2 using a NanoBRET^™intracellular kinase assay (Figure 4,

Supplemental Figure S2). Compound 28 had no direct effect on

cellular RSK2 activity compared with BI-D1870, despite being the

only analog to show an improvement in inhibitory potency over

BI-D1870 against MOLM-13 cellular viability and toxicity.

Conversely, 33, LJH685, and LJI308 had a minimal impact on cell

viability and toxicity but were the only compounds to demonstrate

inhibition of cellular RSK2 activity, albeit at a lower potency than

that of BI-D1870. Collectively, the data from the cell-based

experiments suggest that the effects and impact on cellular

viability and toxicity seen with BI-D1870 and the synthesized

RSK inhibitors are independent of their effects on cellular RSK2

inhibition alone and most likely result of interactions with both

RSK2 and alternative molecular targets. Collectively, the data

support that cellular RSK2 inhibition is possible with compound

33, LJH685, and LJI308 without potent and acute cytotoxic effects.

Figure 5. Compound 33 docked into the crystal structure of the

NTKD of RSK2 (PDB: 5D9K). (A) Molecular surface of the ATP-

binding site (colored grey) displaying the hydrophobic pocket

between the Phe79 and Val82 residues and the limited scope for

N⁵modification with the docked S-isomer (green) and R-isomer

(pink) of compound 33. (B) Stick display style representation of

the docked conformation of the S-isomer (green) and R-isomer

(pink) of compound 33 and key interacting amino acid residues

(grey), where green dashed lines indicate H-bonds. Ligand

interaction map of the predicted binding mode of (C) the S-isomer

and (D) the R-isomer of 33 in the ATP-binding site of the RSK2

NTKD, where red residues are charged negative, purple residues

are charged positive, green residues are hydrophobic, and blue

residues are polar, and purple arrows indicate H-bonds.

Figure 4. Inhibition of cellular RSK2 activity measured by

nanoBRET assay. Representative BRET ratio compared to DMSO

control. Inhibitors were treated over a concentration range of 40

µM to 1.25µM for 2 h (n = 3, error bars: ± S.D.). Each compound

was analyzed in relation to BI-D1870 *Pꢀ<ꢀ0.05; **Pꢀ<ꢀ0.005;

***Pꢀ<ꢀ0.0001, two-way ANOVA.

We have developed a series of substituted pteridinones and

pyrimidines to examine the structural features of BI-D1870 that

are required for potent inhibition of RSK2 kinase activity. The S-

and R-isomers of BI-D1870 were docked into the ATP-binding site

of the RSK2 NTKD crystal structure to guide the design of BI-

D1870 analogs. Overall, the BI-D1870 R- and S-isomers had

similar docked conformations and interactions with residues in the

hinge domain and DFG motif. We designed compounds to replace

the substituents of BI-D1870 that displayed interactions with the

DFG motif and those that did not appear to interact with amino

acid residues in the ATP-binding site. Our studies revealed that the

H-bond interaction between the phenol and Asp211 was critical

for RSK2 inhibition, the halogen substitution of the phenol has a

significant impact on binding affinity, removal of the N⁵methyl

maintains RSK2 inhibitory potency, but removal of the N⁸-

isopentyl or the piperazinone ring abolished RSK2 inhibitory

activity. The chiral C⁷of BI-D1870 has been an overlooked feature

of this commonly used pan-RSK inhibitor and the removal of the

chiral center in compound 34 demonstrated some modest

improvement in RSK2 inhibition in the TR-FRET assay. However,

in the nanoBRET assay, inhibition of cellular RSK2 activity was

observed with only BI-D1870, LJH685, LJI308, and 33, and to a

lesser extent with 35 and 36, but no appreciable inhibition was

achieved with 34 or 37. This reduction in RSK2 inhibitory potency

for 34-37 in cells may be due to interaction with other cellular

targets as supported by the cell viability and cytotoxicity data. The

compounds 28, 34, and 37 demonstrated potent effects on cell

Interestingly, we are the first to report inhibition of cellular RSK2

activity using the known RSK inhibitors BI-D1870, LJH685, and

LJI308 in the nanoBRET intracellular kinase assay. In fact, no

cellular engagement assessment of LJH685 and LJI308 has

previously been reported. The determination of direct inhibition of

cellular RSK activity by small molecules by ELISA or Western

blot can be confounding due to the numerous RSK substrates that

can be activated by other kinases.³⁰Overall, LJH685, LJI308, and

33 demonstrated potent inhibition of cellular RSK2 activity, but

did not display potent effects on cell viability or acute cytotoxicity,

with cytotoxic effects only observed in MOLM-13 cells at high

concentrations after 72 h of exposure. Although BI-D1870 is a

potent pan-RSK inhibitor and has narrow-spectrum kinome

activity, some non-specific interactions have been reported, and

combinations of these interactions may contribute to its potent

inhibition of cell growth and induction of cytotoxicity. BI-D1870

was originally developed in a campaign to identify PLK1

inhibitors and it is a nanomolar inhibitor of PLK1, but a more

potent inhibitor of the RSKs.^{22, 24}Therefore, the combination of

RSK and PLK1 inhibition could contribute to the potent

cytotoxicity observed in MOLM-13 cells. Additionally, BI-D1870

may also interact with proteins outside of the kinome and it has

been reported to interact with the bromodomain (BRD) containing

bromo and extra terminal (BET) BRD4 protein with a K_dof 3.5 ±

0.17 μM by isothermal titration calorimetry.³¹The lower affinity

of BI-D1870 for BRD4 compared with the dihydropteridinone BI-

D2536 was proposed to be due to the methyl group at C⁷reducing

hydrophobic contact with the BRD compared with the ethyl group

of BI-D2536. Therefore, in the case of compound 33 the methyl

group at C⁷and the lack of methyl substitution of N⁵not only

reduces hydrophobic contact but also alters the acetyl-lysine

mimetic group required for BRD binding which could explain its

reduced cytotoxicity. However, compounds 35 and 36 are

unsubstituted at C⁷and N⁵further reducing the potential for

hydrophobic contact wih BRD4 and altering the acetyl-lysine

mimetic group, but these compounds demonstrated potent

cytotoxicity in MOLM-13 cells. Collectively, these data suggest

that the potent cytotoxicity of compounds 35 and 36 was not a

direct result of interacting with BRD4 or there is an alternative

SAR for BRD binding. Our SAR study supports that N⁵

substitution is not required for RSK inhibition as demonstrated

with compounds 33, 35, and 36; however, the most potent RSK2

inhibition was recorded for 33 without the potent cytotoxicity

observed with 35 and 36. The docking of compound 33 to RSK2

reveals that removal of the N⁵methyl does not result in additional

interactions or alter the interactions with the hinge domain residues

or the DFG motif observed for BI-D1870 and comparing the

docked conformations of the two compounds there is limited space

to accommodate N⁵substitutions (Figure 5). Overall, the data

from our study indicate that the SAR for RSK inhibition without

potent cytotoxicity is narrow for pteridinone analogs of BI-D1870.

3. Conclusions

In summary, we demonstrate that prolonged exposure of cells to

high concentrations of RSK inhibitors LJH685, LJI308, and 33

was required to induce cytotoxicity in MOLM-13 cells. This

finding was surprising as the RSKs phosphorylate a number of

substrates involved in many diverse cellular processes critical for

cell survival. It is possible that some cell types may be more

sensitive to RSK inhibition; however, the MOLM-13 cells were

selected from a panel of leukemic cancers as it had high levels of

activated RSK1 and RSK2 (Supplemental Figure S1), supporting

that RSK1 and RSK2 are mediating functions in the cell.

Therefore, the N⁵unsubstituted compound 33 may be a more

selective RSK inhibitor than BI-D1870, as it has equivalent RSK2

inhibitory potency, but has dramatically reduced cytotoxicity and

mirrors more the overall profiles for LJH685 and LJI308. Further

studies are required to determine the impact of RSK inhibition in

cancer cells as synergistic combination therapies may need to be

identified,³²or RSK inhibitors may be therapeutically more

effective in a different context, such as eliminating the cancer stem

cell population.³³Our findings indicate there is still further scope

for the development of novel RSK inhibitors and that RSK

inhibition alone is not sufficient to induce acute cytotoxicity in

MOLM-13 cells.

4. Experimental

4.1 Computational-based modeling

The primary goal of our study was to identify the structural

components of BI-D1870 that were required for RSK2 inhibition

and those that were amenable to modification to further support

the development of pteridinone-based RSK2 inhibitors (Figure 6).

Our study confirmed that the phenol H-bond with Asp211 of the

DFG motif was a critical interaction for RSK2 inhibition, but this

moiety may be prone to metabolism and protective prodrug

strategies may be necessary. The development of a prodrug of a

RSK2 inhibitor may be an interesting strategy as it would be more

feasible to develop a tumor-selective prodrug than an RSK-

isoform selective inhibitor. Furthermore, prodrug forms of RSK

inhibitors may facilitate the accumulation of RSK inhibitors in the

cell and, thus, prolonged RSK inhibition. Another important result

from our SAR study was that the removal of the N⁸-isopentyl chain

from the pteridinone ring of BI-D1870 abolished RSK2 inhibitory

activity (Table 2). From our computational models of the BI-

D1870 isomers docked into RSK2 and the co-crystal structures of

RSK2 with BI-D1870 or LJH685,^{27, 28}the isopentyl of BI-D1870

and phenylpiperazinyl of LJH685 extend out of the ATP-binding

site and into a hydrophobic pocket towards the kinase substrate

binding site. The extension and optimization of substituents from

the N⁸of the pteridinone ring into this hydrophobic region may

present the opportunity for improvement in inhibitory potency and

potentially RSK isoform selectivity.

The pteridinones and pyrimidines were docked into the ATP-

binding site of the NTKD of RSK2 (PDB: 5D9K) crystal

structure,²⁸using the Glide module within Schrödinger (Release

2018-1, Schrödinger LLC, New York, NY).^34-36The compounds

were prepared using LigPrep by generating possible states at the

target pH 7 .0 using Epik and minimized by applying the

OPLS_2005 force field.³⁷The crystal structure of the NTKD of

RSK2 co-crystallized with BI-D1870 (PDB: 5D9K) was obtained

from the Protein Data Bank (PDB) ²⁸. The water molecules were

removed from the RSK2 structure and the protein was prepared by

assigning bond orders, adding hydrogens, and repairing any side

chains or missing amino acid sequences. To complete protein

preparation a restrained minimization of the RSK2 structure was

performed using the default constraint of 0.30Å RMSD and the

OPLS_2005 force field.³⁷The prepared proteins were subjected to

SiteMap analysis,³⁶that identified the ATP-binding site in the

NTKD and docking grids were generated using Receptor Grid

Generation. Molecular docking simulations were performed using

the Glide ligand docking module in XP (extra precision) mode and

included post-docking minimization.³⁵The docked structures of

BI-D1870 in the ATP-binding site of the RSK NTKD were used

as the basis for the design of the pteridinones and pyrimidines,

with the aim of examining substitutions to test the importance of

interactions with residues in the ATP-binding site. The

pteridinones and pyrimidines were prepared and docked in the

RSK2 crystal structure in order to identify the critical interactions

that resulted in RSK inhibition.

Limited space for N⁵substitution

H-bond with Leu150

of hinge domain

and alkyl substitutions may promote

H

N

F

H-bond between phenol

and Asp211 of DFG motif

required for RSK2 inhibition

interaction with bromodomains

O

OH

F

N

H

R- and S-isomers do

not appear to impact

RSK2 inhibition

4.2 Chemistry

Halogen substitution impacts

the strength of the H-bond

between phenol and Asp211

H-bond with Asp148

of hinge domain

All melting points (M.p.) were determined using a Mettler Toledo

M540 melting point apparatus. ¹H and ¹³C nuclear magnetic

resonance (NMR) spectra were obtained as solutions in deuterated

solvents DMSO-d₆or CDCl₃using a 400 MHz Bruker Avance III

400 spectrometer. Chemical shifts (δ) are reported in parts per

million (ppm) and the spin-multiplicity abbreviated as: s (singlet),

d (doublet), t (triplet), q (quartet), quin (quintet), m (multiplet), bs

Substitution at N⁸appears to be critical

for anchoring in the hydrophobic pocket

Figure 6. General pteridinone pharmacophore model for RSK

inhibition.

(broad singlet), dd (doublet of doublets), or dt (doublet of triplets)

with coupling constants (J) given in Hertz (Hz).High-resolution

mass spectrometry (HRMS) was performed using an Agilent 6520

tandem quadrupole-time of flight (Q-TOF) mass spectrometer

coupled to an electrospray ionization source. Spray was induced

with a capillary voltage of 4000V and the fragmentor voltage was

200V. Data was acquired over a range of m/z 50-1700. Compound

purity was determined by NMR and mass spectrometry analysis.

Fourier Transform Infrared (FTIR) spectra were obtained using a

Bruker Alpha Platinum-ATR as a neat sample.

solution of BBr₃(4 equiv; 1M in DCM) was added dropwise. The

solution was held at 0°C for 30 minutes then allowed to warm to

RT and stirred for an additional 2.5 h. The reaction was quenched

via dropwise addition onto ice water (50 mL/mmol) before being

washed with saturated NaHCO₃solution (20 mL/mmol) and

extracted with EtOAc:THF (1:1, 3 x 15 mL/mmol). The combined

organic extracts were washed with brine, dried (MgSO₄) and

concentrated in vacuo. The crude residue was purified via silica

gel chromatography to give the desired compound.

4.2.1 N-Benzyl-3-methylbutan-1-amine (6). A solution of

cesium carbonate (0.43 g, 1.32 mmol) and benzyl amine (5) (290

μL, 2.65 mmol) in DMF (4.5 mL, 0.29 M) was stirred at RT for 30

min prior to the addition of alkyl halide 4 (159 μL, 1.32 mmol).

The reaction was stirred for 48 h before the solid material was

removed via filtration and washed with EtOAc (20 mL). The

organic filtrate was then washed thoroughly with H₂O (6 x 30 mL)

and the combined organic extracts were washed with brine (30

mL), dried (MgSO₄), and concentrated in vacuo.³⁸The crude

residue was purified via silica gel chromatography (19:1

DCM:MeOH) to afford the desired compound as a yellow oil (3.83

g, 21.6 mmol, 73%). Rf 0.24 (19:1 DCM:MeOH); IR (cm^-1) 3061,

General Procedure A. A solution of benzyl-protected amino acid

ethyl ester (7, 10) (1 equiv.) and Pd/C (10% w/w) in EtOH (0.3M)

containing concentrated HCl (10M) was hydrogenated for 2 h prior

to filtration of the catalyst. The material was concentrated in vacuo

to afford the desired compound.³⁸

General Procedure B. A solution of the amino acid ethyl ester

precursor (11-13) (1.2 equiv.) in H₂O (0.6M) was added to a

stirring solution of 2,4-dichloro-5-nitropyrimidine (1 equiv.) in

Et₂O (0.25M). The reaction was cooled to -15°C and K₂CO₃(4.8

equiv.) was added slowly before the reaction was warmed to RT

over 2 h. The phases were partitioned and the organic phase was

extracted with Et₂O (3 x 10 mL/mmol) and the combined organic

extracts were dried (MgSO₄), concentrated in vacuo, and purified

via gel chromatography, if necessary, to afford the desired

compound. ³⁸

1

3027, 2953, 2918, 2868, 2814, 1675, 1604, 1547; H NMR (400

MHz, DMSO-d₆) 0.84 (6H, d, J = 6.7 Hz, CH(CH₃)₂), 1.31 (2H,

dt, J = 7.4, 7.1 Hz, NHCH₂CH₂), 1.51-1.69 (1H, m, CH(CH₃)₂),

1.97 (1H, bs, NH), 2.47 (2H, t, J = 7.4 Hz, NHCH₂CH₂), 3.67 (2H,

s, benzyl CH₂), 7.16-7.25 (1H, m, H-4 ), 7.25-7.40 (4H, m, H-

2/3/5/6); ¹³C NMR (100 MHz, DMSO-d₆) 23.1 (CH(CH₃)₂), 26.0

(CH(CH₃)₂), 47.3 (NHCH₂CH₂), 53.6 (benzyl CH₂), 126.9 (Ar-C),

128.3 (Ar-C), 128.5 (Ar-C), 141.6 (Ar-C).

General Procedure C.

A

solution of the necessary

nitropyrimidine (15-17) (1 equiv.) in acetic acid (0.2M) was heated

to 70°C prior to the addition of iron powder (5 equiv.). The

solution was further heated to 100°C, stirred for 30 minutes, and

filtered hot. The filtrate was concentrated in vacuo before being

partitioned between EtOAc (20 mL/mmol) and a saturated

NaHCO₃solution (20 mL/mmol) and extracted with EtOAc (3 x

15 mL/mmol). The combined organic extracts were dried (MgSO₄)

and concentrated in vacuo, and the crude residue was purified via

silica gel chromatography to afford the desired compound.³⁸

4.2.2 Ethyl N-benzyl-N-isopentylalaninate (7). A solution of

amine 6 (52 mg, 0.28 mmol), ethyl 2-bromopropionate (37 µL,

0.28 mmol), and K₂CO₃(61 mg, 0.42 mmol) taken up in DMF

(0.35 mL, 0.8M) was heated to 110°C and stirred for 4 h prior to

filtration of the inorganic salts. The filtrate was concentrated in

vacuo, resuspended in diethyl ether (15 mL), washed with H₂O (10

mL) and extracted with ether (3 x 15 mL).³⁸The combined organic

extracts were dried (MgSO₄) and concentrated in vacuo to afford

the desired compound as a yellow oil (3.12 g, 11.2 mmol, 74%).

Rf 0.81 (19:1 DCM:MeOH); IR (cm^-1) 3061, 3028, 2954, 2867,

General Procedure D. To an oven dried flask cooled under N₂

was added a solution of pteridinone 18-20 (1 equiv.) in DMA

(0.37M). The solution was cooled to -15°C and NaH (1.1 equiv.,

60% dispersion in mineral oil) was added followed by subsequent

addition of MeI (1.2-4.0 equiv.). The solution was stirred for 30

minutes before being quenched dropwise onto ice. The organic

material was extracted with EtOAc (3 x 10 mL/mmol), the

combined organic extracts were dried (MgSO₄), and concentrated

in vacuo. The resultant residue was purified via column

chromatography, if necessary, to afford the desired compound.³⁸

1

1728; H NMR (400 MHz, DMSO-d₆) 0.75 (3H, d, J = 6.7 Hz,

CH(CH₃)₂), 0.77 (3H, d, J = 6.7 Hz, CH(CH₃)₂), 1.19 (3H, d, J =

7.1 Hz, NCHCH₃), 1.22 (3H, t, J = 7.1 Hz, OCH₂CH₃), 1.17- 1.30

(2H, m, NCH₂CH₂), 1.46-1.62 (1H, m, CH(CH₃)₂), 2.39-2.62 (2H,

m, NCH₂CH₂), 3.45 (1H, q, J = 7.1 Hz, NCHCH₃), 3.52 (0.5H, s,

benzyl CH₂), 3.56 (0.5H, s, benzyl CH₂), 3.74 (0.5H, s, benzyl

CH₂), 3.78 (0.5H, s, benzyl CH₂), 4.02-4.19 (2H, m, OCH₂CH₃),

7.18-7.26 (1H, m, H-4), 7.27-7.35 (4H, m, H-2/3/5/6). ¹³C NMR

(100 MHz, DMSO-d₆) 14.8, 15.2, 22.7 (CH(CH₃)₂), 23.2

(CH(CH₃)₂), 25.6 (CH(CH₃)₂), 37.4 (NCH₂CH₂), 48.7

(NCH₂CH₂), 54.9 (benzyl-CH₂), 57.4, 60.2, 127.2 (Ar-C), 128.5

(Ar-C), 128.7 (Ar-C), 140.8 (Ar-C), 173.4 (C=O). HRMS cal. for

C₁₆H₃₀NOSi (ES+) m/z 278.212004 [M+H]⁺, found 278.20918.

General Procedure E. The relevant halogenated heterocycle (1.0

equiv.), substituted aniline (2.0 equiv.) and TFA (5.0 equiv.) were

taken up in TFE (0.1M) and heated under microwave irradiation

conditions at 140°C for 30 minutes before being concentrated in

vacuo. The residue was resuspended in EtOAc:THF (1:1, 20

mL/mmol), washed with saturated NaHCO₃solution (20

mL/mmol), and the aqueous phase was further extracted with

EtOAc:THF (1:1, 3 x 15 mL/mmol). The combined organic

extracts were washed with brine, dried (MgSO₄) and concentrated

in vacuo. The resultant residue was purified via column

chromatography and/or triturated as specified to afford the desired

compound.²⁹

4.2.3 Ethyl N-benzyl-N-isopentylglycinate (10). A solution of

isovaleraldehyde (8) (1.4 mL, 13.0 mmol) and benzylglycine ethyl

ester (9) (0.49 mL, 2.59 mmol) in MeOH (8 mL, 0.3M) containing

AcOH (0.86 mL, 10% v/v) was heated at 50°C for 1 h before

NaBH₃CN (0.21 g, 3.11 mmol) was added. The solution was

stirred for an additional hour before being quenched with H₂O (10

mL). The material was washed with saturated NaHCO₃solution

(25 mL), extracted with EtOAc (3 x 15 mL), dried (MgSO₄), and

the combined organic extracts were concentrated in vacuo. The

crude residue was purified via silica gel chromatography (23:2

Hexanes:EtOAc) to give the desired compound as a colorless oil

General Procedure F. To an oven dried flask cooled under N₂

was added a solution of methoxyanilino pteridinone (51-53) (1

equiv.) in DCM (0.2M). The solution was cooled to 0°C before a

(0.49 g, 1.86 mmol, 78%). Rf 0.85 (9:1 Hexanes:EtOAc); IR (cm^-

solution of 2,4-dichloro-5-nitropyrimidine (26 mg, 0.13 mmol) in

of Et₂O (0.5 mL) and reacted according to the described General

Procedure B. The crude material was then purified via silica gel

chromatography (19:1 Hexanes:EtOAc) to afford the desired

compound as a yellow solid (0.32 g, 0.095 mmol, 73%). Rf 0.35

(9:1 Hexanes:EtOAc); M.p. 65-68°C; IR (cm^-1) 2956, 2871, 1721,

1581, 1530; ¹H NMR (400 MHz, DMSO-d₆) 0.87 (6H, d, J = 5.7

Hz, CH(CH₃)₂), 1.21 (3H, t, J = 7.1 Hz, OCH₂CH₃), 1.43-1.59

(3H, m, CH(CH₃)₂, NCH₂CH₂), 3.18-3.59 (2H, m, NCH₂CH₂),

4.16 (2H, q, J = 7.1, OCH₂CH₃), 4.31 (2H, s, NCH₂CO₂), 8.84

(1H, s, H-6); ¹³C NMR (100 MHz, DMSO-d₆) 14.5 (OCH₂CH₃),

22.7 (CH(CH₃)₂), 26.1 (CH(CH₃)₂), 34.6 (NCH₂CH₂), 51.1, 51.9,

61.3 (OCH₂CH₃), 131.6 (Ar-C), 154.5 (Ar-C), 157.3 (Ar-C), 159.6

(Ar-C), 168.5 (C=O). HRMS cal. for C₁₃H₂₀ClN₄O₄(ES+) m/z

331.117309 [M+H]⁺, found 331.121466.

1

¹) 2953, 2927, 2866, 1734; H NMR (400 MHz, DMSO-d₆) 0.80

(6H, d, J = 6.6 Hz, CH(CH₃)₂), 1.18 (3H, t, J = 7.1 Hz, OCH₂CH₃),

1.31 (2H, dt, J = 7.4, 7.0 Hz, NCH₂CH₂), 1.56 (1H, m, CH(CH₃)₂),

2.55 (2H, t, J = 7.4 Hz, NCH₂CH₂), 3.26 (2H, s, benzyl CH₂), 3.69

(2H, s, NCH₂CO₂), 4.07 (2H, q, J = 7.1 Hz, OCH₂CH₃), 7.19-7.27

(1H, m, H-4), 7.27-7.33 (4H, m, H-2/3/5/6).; ¹³C NMR (100 MHz,

DMSO-d₆) 14.6 (OCH₂CH₃), 23.0 (CH(CH₃)₂), 25.8 (CH(CH₃)₂),

36.4 (NCH₂CH₂), 51.6, 54.4, 58.2, 60.1, 127.4 (Ar-C), 128.6 (Ar-

C), 129.1 (Ar-C), 139.7 (Ar-C), 171.2 (C=O). HRMS cal. for

C₁₆H₂₆NO₂(ES+) m/z 264.196354 [M+H]⁺, found 264.192609.

4.2.4

N-(1-Ethoxy-1-oxopropan-2-yl)-3-methylbutan-1-

aminium (11). A solution of amino acid ethyl ester 7 (0.52 g, 1.80

mmol) and Pd/C (55 mg, 0.18 mmol) in EtOH (6 mL)

supplemented with concentrated HCl (180 µL) was hydrogenated

according to General Procedure A to afford the desired

compound as a white solid (1.70 g, 7.65 mmol, 99%). M.p. 98-

103°C (Lit. = 105°C)³⁹; IR (cm^-1) 2964, 2921, 2870, 2796, 2667,

2623, 2443, 1741, 1561; ¹H NMR (400 MHz, DMSO-d₆) 0.83 (3H,

d, J = 6.7 Hz, CH(CH₃)₂), 0.84 (3H, d, J = 6.7 Hz, CH(CH₃)₂),

1.15 (3H, d, J = 6.9, NH₂CHCH₃), 1.19 (3H, t, J = 7.1, OCH₂CH₃),

1.26 (2H, dt, J = 7.2, 7.1 Hz, NH₂CH₂CH₂), 1.52-1.66 (1H, m,

CH(CH₃)₂), 1.77 (2H, bs, NH₂), 2.31-2.43 (1H, m, NCH₂CH₂),

2.43-2.58 (1H, m, NCH₂CH₂), 3.22 (1H, q, J = 6.9 Hz,

NH₂CHCH₃), 4.09 (2H, q, J = 7.1 Hz, OCH₂CH₃); ¹³C NMR (100

MHz, DMSO-d₆) 14.6 (OCH₂CH₃), 19.1 (NH₂CHCH₃), 22.8

(CH(CH₃)₂), 23.2 (CH(CH₃)₂), 25.8 (CH(CH₃)₂), 39.2

(NH₂CH₂CH₂), 45.6 (NHCH₂CH₂), 56.6, 60.3, 175.7 (C=O).

4.2.8 Ethyl (2-chloro-5-nitropyrimidin-4-yl)alaninate (17). A

solution of alanine ethyl ester 13 (50.8 mg, 0.33 mmol) in H₂O (0.6

mL) was added to a stirring solution of 2,4-dichloro-5-

nitropyrimidine (53 mg, 0.27 mmol) in Et₂O (1.1 mL) and reacted

according to the described General Procedure B. The crude

material was then purified via silica gel chromatography (19:1

Hexanes:EtOAc) to afford desired compound as a white solid

(0.52 g, 0.19 mmol, 70%). Rf 0.3 (19:1 DCM:MeOH); M. p. 58-

1

60°C; IR (cm^-1) 3337, 2989, 1731, 1610, 1573, 1517; H NMR

(400 MHz, DMSO-d₆) 1.20 (3H, t, J = 7.1 Hz, OCH₂CH₃), 1.51

(3H, d, J = 7.1 Hz, NHCHCH₃), 4.17 (2H, q, J = 7.1, OCH₂CH₃),

4.72-4.83 (1H, dq, J =7.1, 7.1 Hz, NHCHCH₃), 9.07 (1H, s, NH),

9.09 (1H, s, H-6); ¹³C NMR (100 MHz, DMSO-d₆) 14.5

(OCH₂CH₃), 17.0 (NHCHCH₃), 50.5 (NHCHCH₃), 61.6 (127.9

(Ar-C), 154.8 (Ar-C), 157.9 (Ar-C), 162.5 (Ar-C), 171.5 (C=O).

HRMS cal. for C₉H₁₂ClN₄O₄(ES+) m/z 275.054709 [M+H]⁺,

found 275.057639.

4.2.5 N-(2-Ethoxy-2-oxoethyl)-3-methylbutan-1-aminium (12).

A solution of glycine ethyl ester 10 (0.50 g, 0.19 mmol) and Pd/C

(9.4 mg, 20% w/w) in EtOH (0.63 mL) containing concentrated

HCl (19 µL) was hydrogenated according to General Procedure

A to afford the desired compound as a white solid (0.32 g, 1.52

mmol, 93%). M. p. 208-210°C; IR (cm^-1) 2945, 2869, 2770, 2720,

2616, 2454, 1756; ¹H NMR (400 MHz, DMSO-d₆) 0.88 (6H, d, J

= 6.4 Hz, CH(CH₃)₂), 1.24 (3H, t, J = 7.1 Hz, OCH₂CH₃), 1.47-

1.71 (3H, m, CH(CH₃)₂, NCH₂CH₂), 2.92 (2H, t, J = 8.0 Hz,

NCH₂CH₂), 3.95 (2H, s, NHCH₂CO₂), 4.21 (2H, q, J = 7.1 Hz,

OCH₂CH₃), 9.39 (2H, s, NH₂); ¹³C NMR (100 MHz, DMSO-d₆)

14.4 (OCH₂CH₃), 22.6 (CH(CH₃)₂), 25.8 (CH(CH₃)₂), 34.3

(NCH₂CH₂), 45.8, 47.1, 62.1 (OCH₂CH₃), 167.2 (C=O).

4.2.9

2-Chloro-8-isopentyl-7-methyl-7,8-dihydropteridin-

6(5H)-one (18). A solution of amino acid ethyl ester 15 (0.12 g,

0.29 mmol) in acetic acid (1.5 mL) was heated to 70°C prior to the

addition of iron powder (99 mg, 1.45 mmol,). The solution was

reacted according to General Procedure C to afford the desired

compound as a yellow solid (61 mg, 0.23 mmol, 79%). Rf 0.32

(19:1 DCM:MeOH); M. p. 182-184°C (Lit. = 182-185°C)³⁹; IR

(cm^-1) 3743, 3172, 3109, 3050, 2956, 2925, 2872, 1675, 1606; ¹H

NMR (400 MHz, DMSO-d₆) 0.91 (3H, d, J = 6.3 Hz, CH(CH₃)₂),

0.92 (3H, d, J = 6.3 Hz, CH(CH₃)₂), 1.36 (3H, d, J = 6.8 Hz,

NCHCH₃), 1.39-1.64 (3H, m, NCH₂CH₂, CH(CH₃)₂), 3.13-3.22

(1H, m, NCH₂CH₂), 3.81-3.93 (1H, m, NCH₂CH₂), 4.24 (1H, q, J

= 6.8 Hz, NCHCH₃), 7.55 (1H, s, H-4), 10.75 (1H, s, NH); ¹³C

NMR (100 MHz, DMSO-d₆) 18.1 (NCHCH₃), 22.7 (CH(CH₃)₂),

22.9 (CH(CH₃)₂), 25.9 (CH(CH₃)₂), 35.6 (NCH₂CH₂), 43.2

(NCH₂CH₂), 56.4 (NCHCH₃), 119.2 (Ar-C), 138.2 (Ar-C), 151.2

(Ar-C), 152.8 (Ar-C), 165.6 (C=O). HRMS cal. for C₁₂H₁₈ClN₄O

(ES+) m/z 269.116914 [M+H]⁺, found 269.111913.

4.2.6

Ethyl

N-(2-chloro-5-nitropyrimidin-4-yl)-N-

isopentylalaninate (15). A solution of amino acid ethyl ester salt

11 (0.51 g, 2.67 mmol) in H₂O (4.5 mL) was added to a stirring

solution of 2,4-dichloro-5-nitropyrimidine (0.44 g, 2.22 mmol) in

Et₂O (9 mL) and reacted according to the described General

Procedure B to afford the desired compound as a yellow oil (0.71

g, 2.06 mmol, 93%) Rf 0.79 (19:1 DCM:MeOH); IR (cm^-1) 2958,

1

2873, 1738, 1579, 1537; H NMR (400 MHz, DMSO-d₆) 0.80-

0.90 (6H, m, CH(CH₃)₂), 1.14 (3H, t, J = 7.1 Hz, OCH₂CH₃), 1.43-

1.59 (6H, m, CH(CH₃)₂, NCH₂CH₂, NCHCH₃), 3.10- 3.25 (1H, m,

NCH₂CH₂), 2.25-3.38 (1H, m, NCH₂CH₂), 4.01-4.22 (2H, m,

OCH₂CH₃), 4.57 (1H, q, J = 6.9 Hz, NCHCH₃), 8.84 (1H, s, H-6);

¹³C NMR (100 MHz, DMSO-d₆) 14.4 (OCH₂CH₃), 15.0

(NCHCH₃), 22.6 (CH(CH₃)₂), 22.7 (CH(CH₃)₂), 26.2 (CH(CH₃)₂),

35.1 (NCH₂CH₂), 50.0 (NCH₂CH₂), 59.2, 61.2, 131.8 (Ar-C),

153.7 (Ar-C), 157.4 (Ar-C), 159.0 (Ar-C), 170.4 (C=O). HRMS

cal. for C₁₄H₂₀ClN₄O₄(ES-) m/z 343.117309 [M-H]⁺, found

343.116834.

4.2.10

2-Chloro-8-isopentyl-7,8-dihydropteridin-6(5H)-one

(19). A solution of pteridinone 16 (0.24 g, 0.73 mmol) in acetic

acid (4 mL) was heated to 70°C before iron powder (0.22 g, 3.63

mmol) was added. The solution was then reacted according to

General Procedure C to afford the desired compound as a white

solid (0.14 g, 0.56 mmol, 76%). Rf 0.39 (19:1 DCM:MeOH); M.

p. 185-187°C; IR (cm^-1) 3110, 3061, 2953, 2922, 2865, 1693,

1598, 1565, 1532; ¹H NMR (400 MHz, DMSO-d₆) 0.92 (6H, d, J

= 6.3 Hz, CH(CH₃)₂), 1.37-1.65 (3H, m, CH(CH₃)₂, NCH₂CH₂),

3.49 (2H, t, J = 7.3 Hz, NCH₂CH₂), 4.13 (2H, s, NCH₂CO₂), 7.50

(1H, s, H-4), 10.74 (1H, s, NH); ¹³C NMR (100 MHz, DMSO-d₆)

22.8 (CH(CH₃)₂), 25.7 (CH(CH₃)₂), 34.2 (NCH₂CH₂), 44.9

(NCH₂CH₂), 50.1 (NCH₂CO), 119.5 (Ar-C), 137.7 (Ar-C), 151.5

4.2.7

Ethyl

N-(2-chloro-5-nitropyrimidin-4-yl)-N-

isopentylglycinate (16). A solution of amino acid ethyl ester 12

(33 mg, 0.16 mmol) in H₂O (0.5 mL) was added to a stirring

1

(Ar-C), 152.6 (Ar-C), 162.8 (C=O). HRMS cal. for C₁₁H₁₆ClN₄O

(ES+) m/z 255.101264 [M+H]⁺, found 255.096829.

3215, 3085, 3061, 2939, 1679, 1605, 1563; H NMR (400 MHz,

DMSO-d₆) 1.37 (3H, d, J = 6.8 Hz, NHCHCH₃), 3.20 (3H, s,

NCH₃), 4.32 (1H, q, J = 6.8 Hz, NHCHCH₃), 7.81 (1H, s, H-4),

8.59 (1H, s, NH). ¹³C NMR (100 MHz, DMSO-d₆) 20.3

(NHCHCH₃), 28.4 (NCH₃), 51.0 (NHCHCH₃), 120.7 (Ar-C),

138.7 (Ar-C), 153.0 (Ar-C), 153.1 (Ar-C), 165.3 (C=O). HRMS

cal. C₈H₁₀ClN₄O (ES+) m/z 213.054314 [M+H]⁺, found

213.056105.

4.2.11 2-Chloro-7-methyl-7,8-dihydropteridin-6(5H)-one (20).

A solution of pteridinone 17 (0.60 g, 2.19 mmol) in acetic acid (11

mL) was heated to 70°C prior to the addition of iron powder (0.64

g, 11.0 mmol). The solution was then reacted according to

General Procedure C to afford the desired compound as a white

solid (0.32 g, 1.16 mmol, 73%). Rf 0.25 (19:1 DCM:MeOH); M.

p. 250-265°C decomposed; IR (cm^-1) 3206, 3099, 2921, 1678,

4.2.15

2-((3,5-Difluorophenyl)amino)-8-isopentyl-5,7-

1

1609, 1554; H NMR (400 MHz, DMSO-d₆) 1.32-1.43 (3H, m,

dimethyl-7,8-dihydropteridin-6(5H)-one (24). Pteridinone

heterocycle 21 (53 mg, 0.18 mmol), 3,5-Difluoroaniline (47.5 mg,

0.354 mmol) and TFA (68 µL, 0.885 mmol) were taken up in TFE

(2 mL) and reacted according to the described General Procedure

E. Purification via silica gel chromatography (1:1

Hexanes:EtOAc) afforded the target compound as a white solid

(30 mg, 0.08 mmol, 43%). Rf 0.22 (1:1 Hex: EtOAc); M. p. 137-

139°C ; IR (cm^-1) 3275, 2957, 2871, 1670, 1629, 1602, 1575, 1531;

¹H NMR (400 MHz, DMSO-d₆) 0.92 (3H, d, J = 6.7 Hz,

CH(CH₃)₂), 0.93 (3H, d, J = 6.7 Hz, CH(CH₃)₂), 1.30 (3H, d, J =

6.8 Hz, NCHCH₃), 1.43-1.70 (3H, m, CH(CH₃)₂, NCH₂CH₂),

3.11-3.23 (1H, m, NCH₂CH₂), 3.24 (3H, s, NCH₃), 3.93-4.06 (1H,

m, NCH₂CH₂), 4.28 (1H, q, J = 6.8 Hz, NCHCH₃), 6.62 (1H, m,

H-4’), 7.49 (2H, d, J = 8.7 Hz, H-2’/6’), 7.85 (1H, s, H-4), 9.54

(1H, s, NH); ¹³C NMR (100 MHz, DMSO-d₆) 17.6 (NCHCH₃),

22.9 (CH(CH₃)₂), 22.9 (CH(CH₃)₂), 26.2 (CH(CH₃)₂), 28.2

(NCH₃), 36.0 (NCH₂CH₂), 43.5 (NCH₂CH₂), 56.6 (NCHCH₃),

95.4 (dd, J_CF= 26.6 Hz, Ar-C), 100.7 (d, J_CF= 29.6 Hz, Ar-C),

115.7 (Ar-C), 138.9 (Ar-C), 144.4 (dd, J_CF= 14.4, 14.2 Hz, Ar-C),

151.0 (Ar-C, 155.4 (Ar-C), 163 (dd, J_CF= 240.8, 16.1 Hz, Ar-C),

164.3 (C=O). HRMS cal. C₁₉H₂₄F₂N₅O (ES+) m/z 376.194891

[M+H]⁺, found 376.190797.

NHCHCH₃), 4.18-4.32 (1H, m, NHCHCH₃), 7.53 (1H, s, H-4),

7.54 (1H, s, NH), 8.45 (1H, s, NHCHCH₃), 10.6 (1H, s, NHCO).

¹³C NMR (100 MHz, DMSO-d₆) 20.0 (NHCHCH₃), 51.0

(NHCHCH₃), 118.8 (Ar-C), 138.1 (Ar-C), 152.6 (Ar-C), 152.7

(Ar-C), 166.3 (C=O). HRMS cal. for C₇H₈ClN₄O (ES+) m/z

199.038664 [M+H]⁺, found 199.040771.

4.2.12 2-Chloro-8-isopentyl-5,7-dimethyl-7,8-dihydropteridin-

6(5H)-one (21). To an oven dried flask cooled under N₂was added

a solution of pteridinone 18 (51 mg, 0.19 mmol) in DMA (0.5 mL).

The solution was cooled to -15°C and NaH (10 mg, 0.21 mmol)

followed by MeI (46 μL, 0.74 mmol) were added. The solution

was reacted according to General Procedure D to afford the

desired compound as a yellow oil (52 mg, 0.18 mmol, 99%). Rf

0.63 (9:1 DCM:MeOH); IR (cm^-1) 3057, 2955, 2928, 2869, 1679,

1579, 1519; ¹H NMR (400 MHz, DMSO-d₆) 0.91 (3H, d, J = 6.2

Hz, CH(CH₃)₂), 0.92 (3H, d, J = 6.2 Hz, CH(CH₃)₂), 1.34 (3H, d,

J = 6.8 Hz, NCHCH₃), 1.40-1.63 (3H, m, NCH₂CH₂, CH(CH₃)₂),

3.11-3.20 (1H, m, NCH₂CH₂), 3.22 (3H, s, NCH₃), 3.82-3.97 (1H,

m, NCH₂CH₂), 4.36 (1H, q, J = 6.8 Hz, NCHCH₃), 7.86 (1H, s, H-

4). ¹³C NMR (100 MHz, DMSO-d₆) 18.2 (NCHCH₃), 22.7

(CH(CH₃)₂), 22.9 (CH(CH₃)₂), 25.9 (CH(CH₃)₂), 28.5 (N-methyl),

35.6 (NCH₂CH₂), 43.3 (NCH₂CH₂), 56.4 (NCHCH₃), 121.1 (Ar-

C), 138.8 (Ar-C), 151.6 (Ar-C), 153.2 (Ar-C), 164.6 (C=O).

4.2.16 2-((3,5-Difluoro-4-methoxyphenyl)amino)-8-isopentyl-

5,7-dimethyl-7,8-dihydropteridin-6(5H)-one (24). Pteridinone

heterocycle 21 (51 mg, 0.177 mmol), 3,5-Difluoro-4-

methoxyaniline (57 mg, 0.354 mmol) and TFA (68 µL, 0.885

mmol) were taken up in TFE (2 mL) and reacted according to the

described General Procedure E. Purification by amine

chromatography (1:1 Hexanes:EtOAc) afforded the target

compound as a white solid (39.9 mg, 0.098 mmol, 27%). Rf 0.22

(1:1 Hexanes:EtOAc); M. p. 138-141°C; IR (cm^-1) 3272, 3194,

4.2.13

2-Chloro-8-isopentyl-5-methyl-7,8-dihydropteridin-

6(5H)-one (22). To an oven dried flask cooled under N₂was added

a solution of cyclized pteridinone 19 (31 mg, 0.12 mmol) in DMA

(0.5 mL). The solution was cooled to -15°C and NaH (51 mg, 0.13

mmol) was added. The reaction mixture was stirred for 10 min and

MeI (9 uL, 0.14 mmol) was added. The solution was reacted

according to General Procedure D and the crude residue was

purified via silica gel chromatography (97:3 DCM:MeOH) to

afford the desired compound as a white solid (0.25 g, 0.092 mmol,

76%). Rf 0.57 (19:1 DCM:MeOH); M. p. 132-135°C; IR (cm^-1)

3061, 2943, 2871, 1675, 1565, 1529; ¹H NMR (400 MHz, DMSO-

d₆) 0.92 (6H, d, J = 6.5 Hz, CH(CH₃)₂), 1.46 (2H, dt, J = 7.5, 6.8

Hz, NCH₂CH₂), 1.45-1.65 (1H, m, CH(CH₃)₂), 3.20 (3H, s, NCH₃),

3.51 (2H, t, J = 7.5 Hz, NCH₂CH₂), 4.23 (2H, s, NCH₂CO₂), 7.78

(1H, s, H-4). ¹³C NMR (100 MHz, DMSO-d₆) 22.8 (CH(CH₃)₂),

25.7 (CH(CH₃)₂), 28.0 (NCH₃), 34.1 (NCH₂CH₂), 44.9

(NCH₂CH₂), 50.1 (NCH₂CO), 121.3 (Ar-C), 138.1 (Ar-C), 151.9

(Ar-C), 153.1 (Ar-C), 161.8 (C=O). HRMS cal. C₁₂H₁₈ClN₄O

(ES+) m/z 269.116914 [M+H]⁺, found 269.116008.

1

3116, 2956, 2931, 2870, 1667, 1610, 1574, 1510; H NMR (400

MHz, DMSO-d₆) 0.91 (3H, d, J = 6.9 Hz, CH(CH₃)₂), 0.93 (3H, d,

J = 6.9 Hz, CH(CH₃)₂), 1.28 (3H, d, J = 6.8 Hz, NCHCH₃), 1.43-

1.69 (3H, m, CH(CH₃)₂, NCH₂CH₂), 3.09-3.22 (1H, m,

NCH₂CH₂), 3.22 (3H, s, NCH₃), 3.82 (3H, s, OCH₃), 3.93-4.04

(1H, m, NCH₂CH₂), 4.27 (1H, q, J = 6.8 Hz, NCHCH₃), 7.52 (2H,

d, J = 11.4 Hz, H-2’/6’), 7.83 (1H, s, H-4), 9.35 (1H, s, NH); ¹³

C

NMR (100 MHz, DMSO-d₆) 17.5 (NCHCH₃), 22.9 (CH(CH₃)₂),

22.9 (CH(CH₃)₂), 26.2 (CH(CH₃)₂), 28.2 (NCH₃), 36.0

(NCH₂CH₂), 43.4 (NCH₂CH₂), 56.6 (NCHCH₃), 62.5 (OCH₃),

101.9 (d, J_CF= 27.5 Hz, Ar-C), 115.5 (Ar-C), 129.2 (dd, J_CF= 15.3

Hz, Ar-C), 137.8 (dd, J_CF= 13.4 Hz, Ar-C), 138.9 (Ar-C), 151.0

(Ar-C), 155.5 (Ar-C), 156.8 (dd, , J_CF= 241.9, 8.3, Ar-C), 164.3

(C=O). HRMS cal. C₂₀H₂₆F₂N₅O₂(ES+) m/z 406.205456 [M+H]⁺,

found 406.199354.

4.2.14 2-Chloro-5,7-dimethyl-7,8-dihydropteridin-6(5H)-one

(23). To an oven dried flask cooled under N₂was added a solution

of pteridinone precursor 20 (0.15 g, 0.76 mmol) in DMA (2 mL).

The solution was cooled to -15°C and NaH (31 mg, 0.76 mmol)

was added. The reaction mixture was stirred for 10 min and MeI

(56.4 µL, 1.51 mmol) added. The solution was reacted according

to General Procedure D and the crude residue was purified via

silica gel chromatography (9:1 DCM:MeOH) to afford the desired

compound as a. off white-solid (0.076 g, 0.36 mmol, 46%). Rf 0.57

(19:1 DCM:MeOH); M. p. 257-267°C decomposed; IR (cm^-1)

4.2.17 2-((4-(Hydroxymethyl)phenyl)amino)-8-isopentyl-5,7-

dimethyl-7,8-dihydropteridin-6(5H)-one (26). To a solution of

TIPS-protected pteridinone intermediate 44 (96 mg, 0.182 mmol)

in THF (1.8 mL, 0.1M) stirring at -15°C was added a 1M solution

of TBAF (0.22 ml, 0.219 mmol) dropwise. The reaction was

reacted for 2 h before the solvent was removed in vacuo. The crude

material was purified via silica gel chromatography (19:1

DCM:MeOH) to afford the desired compound as a white solid (41

mg, 0.11 mmol, 61%). Rf 0.25 (19:1 DCM:MeOH); M. p. 177-

178°C; IR (cm^-1) 3337, 3256, 3187, 2930, 2850, 1638, 1610, 1572,

1532; H NMR (400 MHz, DMSO-d₆) 0.93 (3H, d, J = 6.4 Hz,

4.2.20 2-((3-Chloro-4-hydroxyphenyl)amino)-8-isopentyl-5,7-

dimethyl-7,8-dihydropteridin-6(5H)-one (29). Monochloro-

methoxy pteridinone 52 (80.7 mg, 0.198 mmol) in DCM (1 mL)

was reacted with 1M BBr₃according to General Procedure F.

The crude residue was purified via silica gel chromatography (19:1

DCM:MeOH) to give the desired compound as a gray solid (19.8

mg, 0.051 mmol, 26%). Rf 0.33 (19:1 DCM:MeOH); M. p. 220-

230°C decomposed; IR (cm^-1) 3183, 3126, 2952, 2865, 1638,

1611, 1577, 1513; ¹H NMR (400 MHz, DMSO-d₆) 0.91 (3H, d, J

= 6.7 Hz, CH(CH₃)₂), 0.93 (3H, d, J = 6.7 Hz, CH(CH₃)₂), 1.26

(3H, d, J = 6.7 Hz, NCHCH₃), 1.43-1.69 (3H, m, CH(CH₃)₂,

NCH₂CH₂), 3.05-3.16 (1H, m, NCH₂CH₂), 3.21 (3H, s, NCH₃),

3.96-4.07 (1H, m, NCH₂CH₂), 4.22 (1H, q, J = 6.7 Hz, NCHCH₃),

6.83 (1H, d, J = 8.8 Hz, H-5’), 7.39 (1H, dd, J = 8.8, 2.6 Hz, H-

6’), 7.78 (1H, s, H-4), 7.81 (1H, d, J = 2.6 Hz, H-2’), 8.92 (1H, s,

NH), 9.54 (1H, s, OH); ¹³C NMR (100 MHz, DMSO-d₆) 17.3

(NCHCH₃), 22.9 (CH(CH₃)₂), 23.0 (CH(CH₃)₂), 26.1 (CH(CH₃)₂),

28.2 (NCH₃), 36.1 (NCH₂CH₂), 43.0 (NCH₂CH₂), 56.5

(NCHCH₃), 114.7 (Ar-C), 116.8 (Ar-C), 118.8 (Ar-C), 119.5 (Ar-

C), 120.0 (Ar-C), 134.5 (Ar-C), 139.2 (Ar-C), 147.4 (Ar-C), 151.1

(Ar-C), 156.1 (Ar-C), 164.2 (C=O). HRMS cal. C₁₉H₂₅ClN₅O₂

(ES+) m/z 390.169678 [M+H]⁺, found 390.161688.

1

CH(CH₃)₂), 0.96 (3H, d, J = 6.4 Hz, CH(CH₃)₂), 1.27 (3H, d, J =

6.8 Hz, NCHCH₃), 1.45-1.69 (3H, m, CH(CH₃)₂, NCH₂CH₂),

3.07-3.18 (1H, m, NCH₂CH₂), 3.22 (3H, s, NCH₃), 3.96-4.09 (1H,

m, NCH₂CH₂), 4.25 (1H, q, J = 6.8 Hz, NCHCH₃), 4.40 (2H, d, J

= 5.6 Hz, CH₂OH), 4.99 (1H, t, J = 5.6 Hz, CH₂OH), 7.15 (2H, d,

J = 8.5 Hz, H-3’/5’), 7.68 (2H, d, J = 8.5 Hz, H-2’/6’), 7.79 (1H,

s, H-4), 9.03 (1H, s, NH); ¹³C NMR (100 MHz, DMSO-d₆) 17.4

(NCHCH₃), 23.0 (CH(CH₃)₂), 23.0 (CH(CH₃)₂), 26.3 (CH(CH₃)₂),

28.2 (NCH₃), 36.1 (NCH₂CH₂), 43.3 (NCH₂CH₂), 56.6

(NCHCH₃), 63.3 (CH₂OH), 114.7 (Ar-C), 118.1 (Ar-C), 127.3

(Ar-C), 134.9 (Ar-C), 139.2 (Ar-C), 140.5 (Ar-C), 151.0 (Ar-C),

156.2 (Ar-C), 164.3 (C=O). HRMS cal. C₂₀H₂₈N₅O₂(ES+) m/z

370.224299 [M+H]⁺, found 370.218594.

4.2.18 2-((4-Hydroxyphenyl)amino)-8-isopentyl-5,7-dimethyl-

7,8-dihydropteridin-6(5H)-one (27). To a solution of TIPS-

protected pteridinone intermediate 50 (87 mg, 0.17 mmol) in THF

(1.7 mL, 0.1M) stirring at -15°C was added a 1M solution of TBAF

in THF (0.2 mL, 0.204 mmol) dropwise. The solution was reacted

for 2 h before the solvent was concentrated in vacuo. The crude

material was purified via silica gel chromatography (19:1

DCM:MeOH) to afford the desired compound as a brown solid

(36.9 mg, 0.104 mmol, 61%). Rf 0.23 (97:3 DCM:MeOH); M. p.

260-261°C; IR (cm^-1) 3278, 2955, 2865, 1646, 1606, 1539, 1505;

¹H NMR (400 MHz, DMSO-d₆) 0.91 (3H, d, J = 6.4 Hz,

CH(CH₃)₂), 0.94 (3H, d, J = 6.4 Hz, CH(CH₃)₂), 1.25 (3H, d, J =

6.8 Hz, NCHCH₃), 1.40-1.67 (3H, m, CH(CH₃)₂, NCH₂CH₂),

3.01-3.13 (1H, m, NCH₂CH₂), 3.21 (3H, s, NCH₃), 3.92-4.05 (1H,

m, NCH₂CH₂), 4.22 (1H, q, J = 6.8 Hz, NCHCH₃), 6.63 (2H, d, J

= 8.9 Hz, H-3’/5’), 7.46 (2H, d, J = 8.9 Hz, H-2’/6’), 7.74 (1H, s,

H-4), 8.72 (1H, s, NH), 8.89 (1H, s, OH); ¹³C NMR (100 MHz,

DMSO-d₆) 17.3 (NCHCH₃), 22.9 (CH(CH₃)₂), 23.0 (CH(CH₃)₂),

26.3 (CH(CH₃)₂), 28.2 (NCH₃), 36.1 (NCH₂CH₂), 43.2

(NCH₂CH₂), 56.6 (NCHCH₃), 114.2 (Ar-C), 115.2 (Ar-C), 120.6

(Ar-C), 133.5 (Ar-C), 139.4 (Ar-C), 140.5 (Ar-C), 151.0 (Ar-C),

156.5 (Ar-C), 164.32(C=O). HRMS cal. C₁₉H₂₆N₅O₂(ES+) m/z

356.20865 [M+H]⁺, found 356.202489.

4.2.21 2-((3-Bromo-4-hydroxyphenyl)amino)-8-isopentyl-5,7-

dimethyl-7,8-dihydropteridin-6(5H)-one (30). Monobromo-

methoxy pteridinone 53 (84.8 mg, 0.178 mmol) in DCM (1 mL)

was reacted with 1M BBr₃according to General Procedure F.

The crude residue was purified via silica gel chromatography (47:3

DCM:MeOH) to give the desired compound as a gray solid (28.5

mg, 0.066 mmol, 35%). Rf 0.42 (47:3 DCM:MeOH); M. p. 220-

235°C decomposed; IR (cm^-1) 3168, 3122, 2948, 1639, 1607,

1574, 1509; ¹H NMR (400 MHz, DMSO-d₆) 0.91 (3H, d, J = 6.6

Hz, CH(CH₃)₂), 0.93 (3H, d, J = 6.6 Hz, CH(CH₃)₂), 1.26 (3H, d,

J = 6.7 Hz, NCHCH₃), 1.42-1.70 (3H, m, CH(CH₃)₂, NCH₂CH₂),

3.02-3.15 (1H, m, NCH₂CH₂), 3.21 (3H, s, NCH₃), 3.96-4.08 (1H,

m, NCH₂CH₂), 4.22 (1H, q, J = 6.7 Hz, NCHCH₃), 6.82 (1H, d, J

= 8.8 Hz, H-5’), 7.44 (1H, dd, J = 8.8, 2.5 Hz, H-6’), 7.77 (1H, s,

H-4), 7.95 (1H, d, J = 2.5 Hz, H-2’), 8.91 (1H, s, NH), 9.62 (1H,

s, OH); ¹³C NMR (100 MHz, DMSO-d₆) 17.3 (NCHCH₃), 22.9

(CH(CH₃)₂), 23.1 (CH(CH₃)₂), 26.1 (CH(CH₃)₂), 28.2 (NCH₃),

36.1 (NCH₂CH₂), 43.0 (NCH₂CH₂), 56.5 (NCHCH₃), 109.1 (Ar-

C), 114.7 (Ar-C), 116.4 (Ar-C), 119.5 (Ar-C), 123.0 (Ar-C), 134.7

(Ar-C), 139.1 (Ar-C), 148.5 (Ar-C), 151.1 (Ar-C), 156.1 (Ar-C),

164.2 (C=O). HRMS cal. C₁₉H₂₅BrN₅O₂(ES+) m/z 434.119161

[M+H]⁺, found 434.112483.

4.2.19 2-((3-Fluoro-4-hydroxyphenyl)amino)-8-isopentyl-5,7-

dimethyl-7,8-dihydropteridin-6(5H)-one (28). Monofluoro-

methoxy pteridinone 51 (82 mg, 0.206 mmol) in DCM (1 mL) was

reacted with 1M BBr₃according to General Procedure F. The

crude residue was purified via silica gel chromatography (19:1

DCM:MeOH) to give the desired compound as a white solid (24.5

mg, 0.048 mmol, 32%). Rf 0.4 (19:1 DCM:MeOH); M. p. 225-

4.2.22 2-((3,5-Dichloro-4-hydroxyphenyl)amino)-8-isopentyl-

5,7-dimethyl-7,8-dihydropteridin-6(5H)-one (31). Pteridinone

heterocycle 21 (104 mg, 0.368 mmol), 4-Amino-2,6-

dichlorophenol (X) (131 mg, 0.735 mmol) and TFA (141 µL, 1.84

mmol) were taken up in TFE (3.8 mL) and reacted according to

the described General Procedure E. Purification via silica gel

chromatography (17:3 DCM:MeOH) and subsequent trituration

with MeOH afforded the desired compound as a brown solid (61

mg, 0.143 mmol, 39%). Rf 0.46 (1:2 Hexanes:EtOAc); M. p. 185-

187°C decomposed; IR (cm^-1) 3373, 2847, 2865, 1683, 1612,

1593, 1563, 1526; ¹H NMR (400 MHz, DMSO-d₆) 0.90 (3H, d, J

= 6.9 Hz, CH(CH₃)₂), 0.92 (3H, d, J = 6.9 Hz, CH(CH₃)₂), 1.27

(3H, d, J = 6.7 Hz, NCHCH₃), 1.43-1.70 (3H, m, CH(CH₃)₂,

NCH₂CH₂), 3.06-3.19 (1H, m, NCH₂CH₂), 3.22 (3H, s, NCH₃),

3.97-4.11 (1H, m, NCH₂CH₂), 4.24 (1H, q, J = 6.7 Hz, NCHCH₃),

7.78 (2H, s, H-2’/6’), 7.82 (1H, s, H-4), 9.12 (1H, s NH), 9.45 (1H,

s, OH); ¹³C NMR (100 MHz, DMSO-d₆) 17.3 (NCHCH₃), 22.8

(CH(CH₃)₂), 23.1 (CH(CH₃)₂), 26.0 (CH(CH₃)₂), 28.2 (NCH₃),

36.0 (NCH₂CH₂), 43.0 (NCH₂CH₂), 56.4 (NCHCH₃), 115.2 (Ar-

C), 118.3 (Ar-C), 122.8 (Ar-C), 135.3 (Ar-C), 139.0 (Ar-C), 142.9

1

227°C; IR (cm^-1) 3271, 2956, 2928, 1646, 1606, 1584, 1539; H

NMR (400 MHz, DMSO-d₆) 0.90 (3H, d, J = 6.7 Hz, CH(CH₃)₂),

0.92 (3H, d, J = 6.7 Hz, CH(CH₃)₂), 1.25 (3H, d, J = 6.7 Hz,

NCHCH₃), 1.42-1.70 (3H, m, CH(CH₃)₂, NCH₂CH₂), 3.06-3.17

(1H, m, NCH₂CH₂), 3.21 (3H, s, NCH₃), 3.91-4.04 (1H, m,

NCH₂CH₂), 4.26 (1H, q, J = 6.7 Hz, NCHCH₃), 6.81 (1H, dd, J =

9.3, 9.3 Hz, H-5’), 7.13-7.26 (1H, m, H-6’), 7.63 (1H, dd, J = 14.1,

2.0 Hz, H-2’), 7.75 (1H, s, H-4), 9.04 (1H, s, NH), 9.26 (1H, s,

OH); ¹³C NMR (100 MHz, DMSO-d₆) 17.4 (NCHCH₃), 22.9

(CH(CH₃)₂), 26.2 (CH(CH₃)₂), 28.2 (NCH₃), 36.1 (NCH₂CH₂),

43.3 (NCH₂CH₂), 56.6 (NCHCH₃), 107.2 (d, J_CF= 23.3 Hz, Ar-C),

114.8 (d, J_CF= 2.8 Hz, Ar-C), 114.9 (Ar-C), 117.8 (d, J_CF= 4.0

Hz, Ar-C), 134.1 (d, J_CF= 9.4 Hz, Ar-C), 138.8 (d, J_CF= 12.7 Hz,

Ar-C), 138.9 (Ar-C), 150.9 (d, J_CF= 237.2 Hz, Ar-C), 151.0 (Ar-

C), 156.0 (Ar-C), 164.2 (C=O). HRMS cal. C₁₉H₂₅FN₅O₂(ES+)

m/z 374.199228 [M+H]⁺, found 374.192346.

(Ar-C), 151.1 (Ar-C), 155.7 (Ar-C), 164.3 (C=O). HRMS cal.

C₁₉H₂₄Cl₂N₅O₂(ES+) m/z 424.130706 [M+H]⁺, found

424.122146.

34.2 (NCH₂CH₂), 45.2 (NCH₂CH₂), 50.3 (NCH₂CO), 101.8 (d, J_CF

= 27.4 Hz, Ar-C), 115.5 (Ar-C), 127.1 (dd, J_CF= 16.7, 16.5 Hz,

Ar-C), 133.7 (dd, J_CF= 12.9, 12.9 Hz, Ar-C), 138.4 (Ar-C), 151.1

(Ar-C), 152.7 (dd, J_CF= 237.6, 9.1 Hz, Ar-C), 155.6 (Ar-C), 164.3

(C=O). HRMS cal. C₁₈H₂₂F₂N₅O₂(ES+) m/z 378.174156 [M+H]⁺,

found 378.171721.

4.2.23 2-((3,5-Dibromo-4-hydroxyphenyl)amino)-8-isopentyl-

5,7-dimethyl-7,8-dihydropteridin-6(5H)-one (32). Pteridinone

heterocycle 21 (81 mg, 0.287 mmol), aniline 42 (157 mg, 0.574

mmol) and TFA (110 µL, 1.43 mmol) were taken up in TFE (3

mL) and reacted according to the described General Procedure

E. Purification via silica gel chromatography (9:1 DCM:MeOH)

and subsequent trituration with MeOH afforded the target

compound as a brown solid (81 mg, 0.16 mmol, 61%). Rf 0.42 (1:2

Hexanes:EtOAc); M. p. 184-186°C; IR (cm^-1) 3375, 2957, 2865,

4.2.26 2-((3-Fluoro-4-hydroxyphenyl)amino)-8-isopentyl-7,8-

dihydropteridin-6(5H)-one (35). Pteridinone heterocycle 19 (75

mg, 0.294 mmol), 4-Amino-2-fluorophenol (78 mg, 0.59 mmol)

and TFA (112 µL, 1.47 mmol) were taken up in TFE (3 mL) and

reacted according to the described General Procedure E.

Purification via silica gel chromatography (93:7 DCM:MeOH)

and subsequent trituration with MeOH afforded the target

compound as a purple solid (85 mg, 0.25 mmol, 83%). Rf 0.33

(93:7 DCM:MeOH); M. p. 210-247°C decomposed; IR (cm^-1)

3290, 2954, 1678, 1621, 1581, 1503; ¹H NMR (400 MHz, DMSO-

d₆) 0.93 (6H, d, J = 6.6 Hz, CH(CH₃)₂), 1.44-1.70 (3H, m,

CH(CH₃)₂, NCH₂CH₂), 3.52 (2H, t, J = 7.9 Hz, NCH₂CH₂), 4.04

(2H, s, NCH₂CO), 6.78 (1H, dd, J = 9.9, 9.0 Hz, H-5’), 7.14-7.22

(1H, m, H-6’), 7.46 (1H, s, H-4), 7.65 (1H, dd, J = 14.3, 2.4 Hz,

H-2’), 8.80 (1H, s, NH), 9.20 (1H, s, OH), 10.37 (1H, s,

NHCOCH₂); ¹³C NMR (100 MHz, DMSO-d₆) 22.9 (CH(CH₃)₂),

26.3 (CH(CH₃)₂), 34.2 (NCH₂CH₂), 45.1 (NCH₂CH₂), 50.4

(NCH₂CO), 106.8 (d, J_CF= 23.1 Hz, Ar-C), 112.4 (Ar-C), 114.6

(d, J_CF= 2.8 Hz, Ar-C), 117.8 (d, J_CF= 3.9 Hz, Ar-C), 134.4 (d,

J_CF= 9.5 Hz, Ar-C), 138.5 (d, J_CF= 3.3 Hz, Ar-C), 138.6 (Ar-C),

150.6 (Ar-C), 152.1 (d, J_CF= 237.5 Hz, Ar-C), 156.1 (Ar-C), 162.4

(C=O). HRMS cal. C₁₇H₂₁FN₅O₂(ES+) m/z 346.167927 [M+H]⁺,

found 346.162472.

1

1683, 1612, 1591, 1560, 1521; H NMR (400 MHz, DMSO-d₆)

0.90 (3H, d, J = 6.9 Hz, CH(CH₃)₂), 0.92 (3H, d, J = 6.9 Hz,

CH(CH₃)₂), 1.27 (3H, d, J = 6.7 Hz, NHCHCH₃), 1.41-1.73 (3H,

m, CH(CH₃)₂, NCH₂CH₂), 3.02-3.18 (1H, m, NCH₂CH₂), 3.22

(3H, s, NCH₃), 3.97-4.13 (1H, m, NCH₂CH₂), 4.23 (1H, q, J = 6.7

Hz, NCHCH₃), 7.82 (1H, s, H-4), 7.98 (2H, s, H-2’/6’), 9.10 (1H,

s, NH) 1.30 (1H, bs, OH); ¹³C NMR (100 MHz, DMSO-d₆) 17.2

(NCHCH₃), 22.9 (CH(CH₃)₂), 23.2 (CH(CH₃)₂), 26.0 (CH(CH₃)₂),

28.2 (NCH₃), 36.0 (NCH₂CH₂), 42.8 (NCH₂CH₂), 56.4

(NCHCH₃), 112.6 (Ar-C), 115.2 (Ar-C), 121.9 (Ar-C), 136.4 (Ar-

C), 139.1 (Ar-C), 144.6 (Ar-C), 151.1 (Ar-C), 155.6 (Ar-C), 164.3

(C=O). HRMS cal. C₁₉H₂₄Br₂N₅O₂(ES+) m/z 512.029671

[M+H]⁺, found 512.025409.

4.2.24 2-((3,5-Difluoro-4-hydroxyphenyl)amino)-8-isopentyl-

7-methyl-7,8-dihydropteridin-6(5H)-one (33). Pteridinone

heterocycle 18 (83 mg, 0.298 mmol), aniline 41 (88 mg, 0.752

mmol) and TFA (114 µL, 1.49 mmol) were taken up in TFE (3

mL) and reacted according to the described General Procedure

E. Purification via silica gel chromatography (97:3 DCM:MeOH)

afforded the target compound as a brown solid (77.5 mg, 0.205

mmol, 67%). Rf 0.26 (1:2 Hexanes:EtOAc); M. p. 170-210°C

4.2.27 2-((3,5-Difluoro-4-hydroxyphenyl)amino)-8-isopentyl-

7,8-dihydropteridin-6(5H)-one (36). Pteridinone heterocycle 19

(77 mg, 0.29 mmol), aniline 41 (87 mg, 0.59 mmol) and TFA (112

µL, 1.47 mmol) were taken up in TFE (3 mL) and reacted

according to the described General Procedure E. Purification via

silica gel chromatography (93:7 DCM:MeOH) and subsequent

trituration with MeOH afforded the target compound as a brown

solid (73 mg, 0.20 mmol, 67%). Rf 0.2 (1:2 Hexanes:EtOAc); M.

p. 222-262°C decomposed; IR (cm^-1) 3294, 3114, 2956, 2869,

1681, 1621, 1583, 1517; ¹H NMR (400 MHz, DMSO-d₆) 0.93 (6H,

d, J = 6.6 Hz, CH(CH₃)₂), 1.42-1.70 (3H, m, CH(CH₃)₂,

NCH₂CH₂), 3.52 (2H, t, J = 7.9 Hz, NCH₂CH₂), 4.06 (2H, s,

NCH₂CO), 7.41 (2H, d, J = 10.7 Hz, H-2’/6’), 7.48 (1H, s, H-4),

1

decomposed; IR (cm^-1) 2951, 1677, 1621, 1581, 1513; H NMR

(400 MHz, DMSO-d₆) 0.91 (3H, d, J = 6.8 Hz, CH(CH₃)₂), 0.93

(3H, d, J = 6.8 Hz, CH(CH₃)₂), 1.30 (3H, d, J = 6.7 Hz,

NHCHCH₃), 1.41-1.72 (3H, m, CH(CH₃)₂, NCH₂CH₂), 3.07-3.20

(1H, m, NCH₂CH₂), 3.89-4.01 (1H, m, NCH₂CH₂), 4.13 (1H, q, J

= 6.7 Hz, NCHCH₃), 7.35-7.47 (2H, d, J = 10.6 Hz, H-2’/6’), 7.53

(1H, s, H-4), 9.05 (1H, s, NH), 9.39 (1H, s, OH), 10.39 (1H, s,

NHCO); ¹³C NMR (100 MHz, DMSO-d₆) 17.5 (NCHCH₃), 22.9

(CH(CH₃)₂), 26.2 (CH(CH₃)₂), 36.0 (NCH₂CH₂), 43.3

(NCH₂CH₂), 56.6 (NCHCH₃), 101.8 (d, J_CF= 27.2 Hz, Ar-C),

112.8 (Ar-C), 126.9 (dd, J_CF= 17.3, 17.2 Hz, Ar-C), 133.8 (dd, J_CF

= 12.8, 12.6 Hz, Ar-C), 138.7 (Ar-C), 150.4 (Ar-C), 152.7 (dd, J_CF

= 237.2, 9.0 Hz, Ar-C), 155.9 (Ar-C), 164.3 (C=O). HRMS cal.

C₁₈H₂₂F₂N₅O₂(ES+) m/z 378.174156 [M+H]⁺, found 378.167664.

8.99 (1H, s, NH), 9.39 (1H, s, OH), 10.41 (1H, s, NHCOCH₂); ¹³

C

NMR (100 MHz, DMSO-d₆) 22.9 (CH(CH₃)₂), 26.2 (CH(CH₃)₂),

34.2 (NCH₂CH₂), 45.2 (NCH₂CH₂), 50.4 (NCH₂CO), 101.7 (d, J_CF

= 27.3 Hz, Ar-C), 112.9 (Ar-C), 126.9 (dd, J_CF= 16.7, 16.7 Hz,

Ar-C), 133.8 (dd, J_CF= 12.8, 12.5 Hz, Ar-C), 138.3 (Ar-C), 150.5

(Ar-C), 152.7 (dd, J_CF= 237.5, 9.9 Hz, Ar-C), 155.7 (Ar-C), 162.4

(C=O). HRMS cal. C₁₇H₂₀F₂N₅O₂(ES+) m/z 364.158506 [M+H]⁺,

found 364.150723.

4.2.25 2-((3,5-Difluoro-4-hydroxyphenyl)amino)-8-isopentyl-

5-methyl-7,8-dihydropteridin-6(5H)-one (34). Pteridinone

heterocycle 22 (94 mg, 0.345 mmol), aniline 41 (102 mg, 0.689

mmol) and TFA (86 µL, 1.73 mmol) were taken up in TFE (3.5

mL) and reacted according to the described General Procedure

E. Purification via silica gel chromatography (24:1 DCM:MeOH)

and subsequent trituration with MeOH afforded the target

compound as a white solid (64.4 mg, 0.17 mmol, 49%). Rf 0.39

(19:1 DCM:MeOH); M. p. 240-248°C decomposed; IR (cm^-1)

3281, 3096, 2960, 2916, 2872, 1649, 1618, 1562, 1524; ¹H NMR

(400 MHz, DMSO-d₆) 0.93 (3H, d, J = 6.5 Hz, CH(CH₃)₂), 1.44-

1.57 (2H, m, NCH₂CH₂), 1.57-1.70 (1H, m, CH(CH₃)₂), 3.20 (3H,

s, NCH₃), 3.49-3.60 (2H, m, NCH₂CH₂), 4.15 (2H, s, NCH₂CO),

7.43 (2H, d, J = 10.7 Hz, H-2’/6’), 7.73 (1H, s, H-4), 9.07 (1H, s,

NH), 9.38 (1H, s, OH); ¹³C NMR (100 MHz, DMSO-d₆) 22.5

(CH(CH₃)₂), 22.9 (CH(CH₃)₂), 26.2 (CH(CH₃)₂), 27.8 (NCH₃),

4.2.28

2-((3-Fluoro-4-hydroxyphenyl)amino)-8-isopentyl-5-

Pteridinone

methyl-7,8-dihydropteridin-6(5H)-one (37).

heterocycle 22 (75 mg, 0.28 mmol), 4-Amino-2-fluorophenol (75

mg, 0.56 mmol) and TFA (107 µL, 1.4 mmol) were taken up in

TFE (3 mL) and reacted according to the described General

Procedure E. Purification via silica gel chromatography (19:1

DCM:MeOH) and subsequent trituration with MeOH afforded the

target compound as a purple solid (54.2 mg, 0.151 mmol, 54%).

Rf 0.32 (19:1 DCM:MeOH); M. p. 247-252°C decomposed; IR

1

(cm^-1) 3281, 2919, 1654, 1599; H NMR (400 MHz, DMSO-d₆)

0.93 (6H, d, J = 6.5 Hz, CH(CH₃)₂), 1.45-1.56 (2H, m, NCH₂CH₂),

1.56-1.69 (1H, m, CH(CH₃)₂), 3.20 (3H, s, NCH₃), 3.53 (2H, t, J =

7.9 Hz, NCH₂CH₂), 4.13 (2H, s, NCH₂CO), 6.79 (1H, dd, J = 9.9,

9.3 Hz, H-5’), 7.16-7.25 (1H, m, H-6’), 7.62-7.75 (2H, m, H-4, H-

2’), 8.89 (1H, s, NH), 9.18 (1H, s, OH); ¹³C NMR (100 MHz,

DMSO-d₆) 22.9 (CH(CH₃)₂), 26.2 (CH(CH₃)₂), 27.7 (NCH₃), 34.2

mL) and a saturated aqueous solution of NaHCO₃was added until

the aqueous phase reached pH 9-10. The resulting precipitate was

removed via filtration and the organic extracts were collected,

washed with brine (20 mL), evaporated to dryness, and purified

via silica gel chromatography (19:1 DCM:MeOH) to afford the

desired compound as a brown solid (0.37 g, 2.56 mmol, 77%). Rf

0.4 (19:1 DCM:MeOH); M.p. 140-150°C decomposed; IR (cm^-1)

3376, 3311, 2532, 1618, 1598, 1520; ¹H NMR (400 MHz, DMSO-

d₆) 5.03 (2H, s, NH₂), 6.13-6.24 (2H, d, J = 9.9 Hz, H-3/5), 8.65

(1H, s, OH). ¹³C NMR (100 MHz, DMSO-d₆) 97.7 (d, J_CF= 25.0

(NCH₂CH₂), 45.1 (NCH₂CH₂), 50.3 (NCH₂CO), 106.9 (d, J_CF

=

23.2 Hz, Ar-C), 114.6 (d, J_CF= 3.1 Hz, Ar-C), 114.7 (Ar-C), 117.8

(d, J_CF= 4.0 Hz, Ar-C), 134.3 (d, J_CF= 9.6 Hz, Ar-C), 138.6 (d,

J_CF= 2.6 Hz, Ar-C), 138.7 (Ar-C), 150.8 (d, J_CF= 237.1 Hz, Ar-

C), 151.2 (Ar-C), 156.0 (Ar-C), 161.3 (C=O). HRMS cal.

C₁₈H₂₃FN₅O₂(ES+) m/z 360.183578 [M+H]⁺, found 360.179437.

4.2.29 2,6-Difluoro-4-(pyrimidin-2-ylamino)phenol (38). 2-

Chloropyrimidine (32 mg, 0.262 mmol), aniline 41 (76 mg, 0.524

mmol) and TFA (100 µL, 1.31 mmol) were taken up in TFE (2.7

mL) and reacted according to the described General Procedure

E. Purification by amine chromatography (1:1 DCM:MeOH)

afforded the target compound as a brown solid (36.9 mg, 0.165

mmol, 63%). Rf 0.15 (1:1 DCM:MeOH); M. p. 200-203°C; IR

(cm^-1) 3406, 2920, 2849, 1590, 1520; ¹H NMR (400 MHz, DMSO-

d₆) 6.86 (1H, t, J = 4.8 Hz, H-5), 7.49 (2H, d, J = 10.9 Hz, H-

3’/5’), 8.49 (2H, d, J = 4.8 Hz, H-4/6), 9.58 (1H, bs, OH), 9.67

(1H, s, NH); ¹³C NMR (100 MHz, DMSO-d₆) 102.8 (d, J_CF= 27.1

Hz, Ar-C), 113.1 (Ar-C), 128.0 (dd, J_CF= 17.7, 15.2 Hz, Ar-C),

132.6 (dd, J_CF= 13.1, 12.5 Hz, Ar-C), 152.6 (dd, J_CF= 238.0, 8.9

Hz, Ar-C), 158.5 (Ar-C), 106.1 (Ar-C). HRMS cal. C₁₀H₈F₂N₃O

(ES+) m/z 224.063543 [M+H]⁺, found 224.066029.

Hz, Ar-C), 123.3 (dd, J_CF= 17.1, 16.5 Hz, Ar-C), 142.1 (dd, J_CF=

12.8, 12.4 Hz, Ar-C), 154.0 (dd, J_CF= 238.1, 9.1 Hz, Ar-C). HRMS

cal. C₆H₄F₂NO (ES-) m/z 144.026095 [M-H]^-, found 144.027107.

4.2.33 4-Amino-2,6-dibromophenol (42). To a solution of 2,6-

dibromo-4-nitrophenol (0.41 g, 1.35 mmol) in EtOH (13.5 mL,

0.1M) was added SnCl₂(1.32 g, 6.74 mmol). The solution was

heated under reflux for 1 h before the solvent was removed in

vacuo. The sample was resuspended in EtOAc (1:1, 20 mL) and

saturated NaHCO₃solution was added until the aqueous phase

reached a pH 9-10. The resulting precipitate was removed via

filtration and the organic extracts were collected, washed with

brine (30 mL), evaporated to dryness, and purified via silica gel

chromatography (1:1 Hexanes:EtOAc) to afford the desired

compound as a yellow solid (0.31 g, 1.16 mmol, 85%). Rf 0.69

(19:1 DCM:MeOH); M. p. 175-188°C decomposed; IR (cm^-1)

1

4.2.30

2-((3,5-Difluoro-4-hydroxyphenyl)amino)-5,7-

3365, 3265, 3158, 1602, 1561; H NMR (400 MHz, DMSO-d₆)

dimethyl-7,8-dihydropteridin-6(5H)-one (39). Pteridinone

heterocycle 23 (82 mg, 0.376 mmol), aniline 41 (110 mg, 0.752

mmol) and TFA (144 µL, 1.88 mmol) were taken up in TFE (4

mL) and reacted according to the described General Procedure

E. Purification via silica gel chromatography (97:3 DCM:MeOH)

and subsequent trituration with MeOH afforded the target

compound as a brown solid (40 mg, 0.12 mmol, 32%). Rf 0.26 (1:2

Hexanes:EtOAc); M. p. 216-218°C; IR (cm^-1) 3455, 3420, 3330,

1661, 1608, 1522; ¹H NMR (400 MHz, DMSO-d₆) 1.36 (3H, d, J

= 6.8 Hz, NHCHCH₃), 3.20 (3H, s, NCH₃), 4.20 (1H, q, J = 6.8

Hz, NHCHCH₃), 7.49 (2H, d, J = 11.0 Hz, H-2’/6’), 7.77 (2H, m,

H-4, NHCHCH₃), 9.03 (1H, s, NH), 9.35 (1H, s, OH); ¹³C NMR

(100 MHz, DMSO-d₆) 20.2 (NCHCH₃), 28.2 (NCH₃), 51.1

(NCHCH₃), 101.9 (d, J_CF= 27.9 Hz, Ar-C), 114.8 (Ar-C), 126.9

(dd, J_CF= 17.3, 16.9 Hz, Ar-C), 133.8 (dd, J_CF= 12.9, 12.8 Hz, Ar-

C),133.8 (Ar-C), 152.1 (Ar-C), 152.7 (dd, J_CF= 238.0., 9.2 Hz, Ar-

C), 155.8 (Ar-C), 165.0 (C=O). HRMS cal. C₁₄H₁₄F₂N₅O₂(ES+)

m/z 322.111556 [M+H]⁺, found 322.103014.

5.01 (2H, s, NH₂), 6.76 (2H, s, H-3/5), 8.70 (1H, s, OH); ¹³C NMR

(100 MHz, DMSO-d₆) 114.0 (Ar-C), 117.6 (Ar-C), 140.9 (Ar-C),

144.7 (Ar-C). HRMS cal. C₆H₄Br₂NO (ES-) m/z 263.86596 [M-

H]^-, found 263.86705.

4.2.34

4-((4-Amino-5-nitropyrimidin-2-yl)amino)-2,6-

difluorophenol (43). Pyrimidine 40 (71 mg, 0.396 mmol), aniline

41 (115 mg, 0.792 mmol) and TFA (152 µL, 1.98 mmol) were

taken up in TFE (4 mL) and reacted according to the described

General Procedure E. The crude material was resuspended in

THF (20 mL), filtered over Celite, and the filtrate was

concentrated in vacuo. Trituration of the crude material with

MeOH afforded the target compound as a brown solid (66 mg, 0.23

mmol, 58%). Rf 0.47 (9:1 DCM:MeOH); M. p. 253-277°C

decomposed; IR (cm^-1) 3481, 3375, 3251, 1628, 1599, 1552, 1521;

¹H NMR (400 MHz, DMSO-d₆) 7.49-7.77 (2H, m, H-3’/5’), 8.24

(1H, bs, NH₂), 8.74 (1H, bs, NH₂), 8.97 (1H, s, H-4), 9.80 (1H, s,

NH), 10.24 (1H, bs, OH); ¹³C NMR (100 MHz, DMSO-d₆) 104.1

(d, J_CF= 27.2 Hz, Ar-C), 129.4 (dd, J_CF= 16.8, 16.6 Hz, Ar-C),

131.2 (dd, J_CF= 12.6, 12.6 Hz, Ar-C), 152.5 (dd, J_CF= 238.2, 8.9

Hz, Ar-C), 157.6 (Ar-C), 157.7 (Ar-C), 159.8 (Ar-C). HRMS cal.

C₁₀H₈F₂N₅O₃(ES+) m/z 284.059521 [M+H]⁺, found 284.06317.

4.2.31 2-Chloro-5-nitropyrimidin-4-amine (40). A solution of

2,4-dichloro-5-nitropyrimidine (14) (0.90 g, 4.64 mmol) in DCM

(6.7 mL, 0.7 M) was added dropwise to a solution of 2M Ammonia

in EtOH (6 mL) stirring at 0°C. The reaction was stirred for 30 min

at 0°C, after which the mixture was warmed to RT and the solvent

removed in vacuo.⁴⁰The crude residue was purified via silica gel

chromatography (4:1 Hexanes:EtOAc) to afford the desired

compound as a yellow solid (2.40 g, 13.8 mmol, 89%). Rf 0.24

(4:1 Hexanes:EtOAc); M.p. 212-213°C (Lit. = 220-221)⁴¹; IR (cm^-

¹) 3430, 3056, 1642, 1579, 1534; ¹H NMR (400 MHz, DMSO-d₆)

8.58 (1H, s, NH₂), 9.02 (1H, s, H-6), 9.19 (1H, s, NH₂); ¹³C NMR

(100 MHz, DMSO-d₆) δ 127.0 (Ar-C), 157.5 (Ar-C), 158.0 (Ar-

C), 162.6 (Ar-C); HRMS cal. C₄H₂ClN₄O₂(ES-) m/z 172.986629

[M-H]^-, found 172.988897.

4.2.35

4-((4,5-Diaminopyrimidin-2-yl)amino)-2,6-

difluorophenol (44). To a solution of 5-nitropyrimidine 43 (58.7

mg, 0.205 mmol, 1 equiv.) in EtOH (2.5 mL, 0.1M) was added tin

(II) chloride (159 mg, 0.82 mmol, 4 equiv.). The reaction was

heated at reflux for 24 h before the reaction was cooled to RT and

concentrated in vacuo. The sample was resuspended in

EtOAc:THF (1:1, 20 mL) and a saturated aqueous solution of

NaHCO₃was added until the aqueous phase reached pH 9-10. The

resulting precipitate was removed via filtration and the organic

phase was collected, washed with brine (20 mL), dried (MgSO₄),

and concentrated in vacuo. The crude residue was purified by silica

gel chromatography (1:1 DCM:MeOH) to give the desired

compound as a brown solid (21.1 mg, 0.083 mmol, 41%). Rf 0.11

(9:1 DCM:MeOH); M. p. 218-227°C decomposed; IR (cm^-1) 3442,

3208, 1641, 1580, 1523; ¹H NMR (400 MHz, DMSO-d₆) 4.20 (2H,

s, NH₂), 6.29 (2H, s, NH₂), 7.40 (1H, s, H-4), 7.44 (2H, d, J = 11.1

4.2.32 4-Amino-2,6-difluorophenol (41). To a solution of 2,6-

difluoro-4-nitrophenol (50 mg, 0.29 mmol) in EtOH (3 mL, 0.1 M)

was added tin(II) chloride (0.22 g, 1.14 mmol). The reaction was

heated under reflux for 1.5 h before the solvent was removed in

vacuo. The resulting residue was dissolved in EtOAc:THF (1:1, 15

Hz, H-3’/5’), 8.54 (1H, s, NH), 9.16 (1H, s, OH); ¹³C NMR (100

MHz, DMSO-d₆) 100.9 (d, J_CF= 27.3 Hz, Ar-C), 120.9 (Ar-C),

125.9 (Ar-C), 134.9 (dd, J_CF= 12.8, 12.3 Hz, Ar-C), 138.3 (dd, J_CF

= 17.7, 13.2 Hz, Ar-C), 152.8 (dd, J_CF= 227.6, 8.9 Hz, Ar-C),

153.0 (Ar-C), 155.2 (Ar-C), 156.4 (Ar-C). HRMS cal.

C₁₀H₁₀F₂N₅O (ES+) m/z 254.085341 [M+H]⁺, found 254.086226.

prior to filtration of the palladium. The filtrate was removed in

vacuo and the crude residue was purified via silica gel

chromatography (4:1 Hexanes:EtOAc) to afford the desired

compound as a colorless oil (0.50 g, 1.87 mmol, 95%). Rf 0.25

(4:1 Hexanes:EtOAc); IR (cm^-1) 3430, 3351, 2941, 2864, 1615,

1

1505; H NMR (400 MHz, DMSO-d₆) 1.04 (18H, d, J = 7.1 Hz,

Si(CH(CH₃)₂)₃), 1.11- 1.26 (3H, m, Si(CH(CH₃)₂)₃), 4.59 (2H, s,

NH₂), 6.45 (2H, d, J = 8.7 Hz, H-2/6), 6.56 (2H, d, J = 8.7 Hz, H-

3/5); ¹³C NMR (100 MHz, DMSO-d₆) 12.5 (Si(CH(CH₃)₂), 18.0

(Si(CH(CH₃)₂), 115.4 (Ar-C), 120.2 (Ar-C), 143.1 (Ar-C), 146.4

(Ar-C).

4.2.36 Triisopropyl((4-nitrobenzyl)oxy)silane (45). To an oven

dried flask cooled under N₂was added a solution of benzyl alcohol

(0.10 g, 0.65 mmol) in anhydrous DMF (1.3 mL, 0.5M). To the

stirring solution was added imidazole (0.11 g, 1.63 mmol)

followed by TIPS-Cl (168 µL, 0.78 mmol). The solution was

stirred at RT for 2 h prior to neutralization with 2M HCl. The crude

material was then extracted with DCM (3 x 15 mL), dried

(MgSO₄), and the combined organic extracts were concentrated in

vacuo. The crude material was purified via silica gel

chromatography (19:1 Hexanes:EtOAc) to afford the desired

compound as a colorless oil (0.59 g, 1.92 mmol, 95% yield). Rf

0.58 (9:1 Hexanes:EtOAc); IR (cm^-1) 2942, 2864, 1603, 1520; ¹H

NMR (400 MHz, DMSO-d₆) 1.06 (18H, d, J = 7.1 Hz,

Si(CH(CH₃)₂)₃), 1.12-1.16 (3H, m, Si(CH(CH₃)₂)₃), 4.95 (2H, s,

OCH₂), 7.62 (2H, d, J = 8.4 Hz, H-2/6), 8.24 (2H, d, J = 8.4 Hz,

H-3/5). ¹³C NMR (100 MHz, DMSO-d₆) 11.8 (Si(CH(CH₃)₂), 18.3

(Si(CH(CH₃)₂), 64.2 (benzyl-CH₂), 123.9 (Ar-C), 127.0 (Ar-C),

146.9 (Ar-C), 149.8 (Ar-C). HRMS cal. C₁₆H₂₈NO₃Si (ES+) m/z

310.183847 [M+H]⁺, found 310.189782.

HRMS cal. C₁₅H₂₈NOSi (ES+) m/z 266.194017 [M+H]⁺, found

266.187327.

4.2.40

8-Isopentyl-5,7-dimethyl-2-((4-

(((triisopropylsilyl)oxy)methyl)phenyl)amino)-7,8-

dihydropteridin-6(5H)-one (49). Pteridinone heterocycle 21 (81

mg, 0.28 mmol), TIPS-protected aniline 47 (96 mg, 0.057 mmol)

and K₂CO₃(103 mg, 0.71 mmol) were taken up in MeCN (3 mL

0.1M), degassed under a stream of N₂, and heated to 110°C for 2

h. The reaction was then cooled, filtered over Celite and the filtrate

concentrated in vacuo. Purification via silica gel chromatography

(1:2 Hexanes:EtOAc) afforded the desired compound as an impure

yellow oil (46.7 mg, 0.09 mmol, 31%). Rf 0.32 (1:2

Hexanes:EtOAc). Crude product was taken through for

deprotection as 26 without further characterization of intermediate

49.

4.2.37 Triisopropyl(4-nitrophenoxy)silane (46). To an oven

dried flask cooled under N₂was added a solution of nitrophenol

(0.11 g, 0.72 mmol) in anhydrous DMF (1.5 mL, 0.5M). To the

stirring solution was added imidazole (0.12 g, 1.80 mmol)

followed by TIPS-Cl (185 ul, 0.86 mmol). The solution was stirred

at RT for 2 h prior to neutralization with 2M HCl. The crude

material was then extracted with DCM (3 x 15 mL), dried

(MgSO₄), and the combined organic extracts were concentrated in

vacuo. The crude material was purified via silica gel

chromatography (19:1 Hexanes:EtOAc) to afford the desired

compound as a colorless oil (0.60 g, 2.02 mmol, 90% yield). Rf

0.63 (9:1 Hexanes:EtOAc); IR (cm^-1) 2945, 2866, 1682, 1590; ¹H

NMR (400 MHz, DMSO-d₆) 1.07 (18H, d, J = 7.5 Hz,

Si(CH(CH₃)₂)₃), 1.32 (3H, m, Si(CH(CH₃)₂)₃), 7.07 (2H, d, J = 9.1

Hz, H-2/6), 8.18 (2H, d, J = 9.1 Hz, H-3/5). ¹³C NMR (100 MHz,

DMSO-d₆) 12.5 (Si(CH(CH₃)₂), 18.3 (Si(CH(CH₃)₂), 116.3 (Ar-

C), 126.6 (Ar-C), 140.1 (Ar-C), 164.4 (Ar-C). HRMS cal.

C₁₅H₂₆NO₃Si (ES+) m/z 296.168197 [M+H]⁺, found 296.170894.

4.2.41

8-Isopentyl-5,7-dimethyl-2-((4-

((triisopropylsilyl)oxy)phenyl)amino)-7,8-dihydropteridin-

6(5H)-one (50). Pteridinone heterocycle 21 (81 mg, 0.28 mmol),

TIPS-protected aniline 48 (92 mg, 0.34 mmol) and K₂CO₃(103

mg, 0.71 mmol) were taken up in MeCN (3 mL, 0.1M), degassed

under a stream of N₂, and heated to 110°C for 2 h. The reaction

was then cooled, filtered over Celite and the filtrate concentrated

in vacuo. Purification via silica gel chromatography (1:2

Hexanes:EtOAc) afforded the desired compound as an impure

yellow oil (43.2 mg, 0.08 mmol, 30%). Crude product was taken

through for deprotection as 27 without further characterization of

intermediate 50.

4.2.42 2-((3-Fluoro-4-methoxyphenyl)amino)-8-isopentyl-5,7-

dimethyl-7,8-dihydropteridin-6(5H)-one (51). Pteridinone

heterocycle 21 (120 mg, 0.42 mmol), monofluoro-methoxyaniline

(124 mg, 0.85 mmol) and TFA (162 µL, 2.12 mmol) were taken

up in TFE (4 mL) and reacted according to the described General

Procedure E. Purification via silica gel chromatography (97:3

DCM:MeOH) afforded the target compound as a pink solid (0.15

g, 0.38 mmol, 89%). Rf 0.32 (97:3 DCM:MeOH); M. p. 135-

173°C decomposed; IR (cm^-1) 3259, 2958, 1667, 1608, 1575,

4.2.38 4-(((Triisopropylsilyl)oxy)methyl)aniline (47).

A

solution of nitrobenzyl alcohol 45 (0.58 g, 1.84 mmol) and Pd/C

(60.4 mg, 10% w/w) in EtOH (18 mL, 0.1M) was hydrogenated

for 3 h prior to filtration of the palladium. The filtrate was

concentrated in vacuo and the crude residue was purified via silica

gel chromatography (4:1 Hexanes:EtOAc) to afford the desired

compound as a colorless oil (0.43 g, 1.54 mmol, 83%). Rf 0.22

(9:1 Hexanes:EtOAc); IR (cm^-1) 3447, 3363, 2939, 2862, 1622,

1516; ¹H NMR (400 MHz, DMSO-d₆) 0.91-1.21 (3H, m,

Si(CH(CH₃)₂)₃), 1.03 (18H, d, J = 6.4 Hz, Si(CH(CH₃)₂)₃), 4.59

(2H, s, OCH₂), 4.93 (2H, s, NH₂), 6.51 (2H, d, J = 8.2 Hz, H-2/6),

6.97 (2H, d, J = 8.2 Hz, H-3/5); ¹³C NMR (100 MHz, DMSO-d₆)

12.0 (Si(CH(CH₃)₂), 18.4 (Si(CH(CH₃)₂), 65.2 (benzyl-CH₂),

114.1 (Ar-C), 127.7 (Ar-C), 128.8 (Ar-C), 148.1 (Ar-C). HRMS

cal. C₁₆H₃₀NOSi (ES+) m/z 280.209666 [M+H]⁺, found

280.208982.

1

1515; H NMR (400 MHz, DMSO-d₆) 0.87 (3H, d, J = 6.4 Hz,

CH(CH₃)₂), 0.89 (3H, d, J = 6.5 Hz, CH(CH₃)₂), 1.36 (3H, d, J =

6.8 Hz, NCHCH₃), 1.40-1.52 (1H, m, CH(CH₃)₂), 1.52-1.65 (2H,

m, NCH₂CH₂), 3.14-3.27 (1H, m, NCH₂CH₂), 3.20 (1H, s, NCH₃),

3.80 (3H, s, OCH₃), 3.86-3.98 (1H, m, NCH₂CH₂), 4.36 (1H, q, J

= 6.8 Hz, NCHCH₃), 7.09 (1H, dd, J = 9.3, 9.3 Hz, H-5’), 7.21-

7.29 (1H, m, H-6’), 7.64 (1H, dd, J = 13.9, 2.4 Hz, H-2’), 7.73

(1H, s, H-4), 9.68 (1H, s, NH); ¹³C NMR (100 MHz, DMSO-d₆)

18.3 (NCHCH₃), 22.7 (CH(CH₃)₂), 22.8 (CH(CH₃)₂), 26.2

(CH(CH₃)₂), 28.4 (NCH₃), 35.7 (NCH₂CH₂), 44.4 (NCH₂CH₂),

56.7, 56.8, 108.9 (d, J_CF= 23.1 Hz, Ar-C), 114.6 (d, J_CF= 2.5 Hz,

Ar-C), 115.3 (Ar-C), 116.5 (Ar-C), 133.3 (d, J_CF= 9.8 Hz, Ar-C),

143.0 (d, J_CF= 10.3 Hz, Ar-C), 150.3 (Ar-C), 151.5 (Ar-C), 152.7

(Ar-C), 152.9 (Ar-C), 164.0 (C=O). HRMS cal. C₂₀H₂₇FN₅O₂

(ES+) m/z 388.214878 [M+H]⁺, found 388.210712.

4.2.39 4-((Triisopropylsilyl)oxy)aniline (48). A solution of

TIPS-protected nitrophenol 46 (0.58 g, 1.96 mmol) and Pd/C (64.2

mg, 10% w/w) in EtOH (19 mL, 0.1M) was hydrogenated for 3 h

4.2.43 2-((3-Chloro-4-methoxyphenyl)amino)-8-isopentyl-5,7-

dimethyl-7,8-dihydropteridin-6(5H)-one (52). Pteridinone

heterocycle 21 (123 mg, 0.42 mmol), monochloro-methoxyaniline

(138 mg, 0.85 mmol) and TFA (162 µL, 2.12 mmol) were taken

up in TFE (4 mL) and reacted according to the described General

Procedure E. Purification via silica gel chromatography (49:1

DCM:MeOH) afforded the target compound as a brown solid (0.13

g, 0.33 mmol, 76%). Rf 0.38 (97:3 DCM:MeOH); M. p. 173-

RSK3 and RSK4 respectively. Assays were performed at 25°C in

a reaction mixture consisting of 2 μL serially diluted inhibitor

solution, 4 μL kinase, 2 μL substrate, and 2 μL ATP. Reagents

were incubated at room temperature for 1 h before the reaction was

stopped through the addition of 5 μL of EDTA at a final

concentration of 10 mM. After a 5 min incubation period, 5 μL of

Eu anti-phospho-40S Ribosomal Protein S6 (Ser235/236)

antibody (PerkinElmer) at a final concentration of 2 nM was

added. The plate was read using a Biotek Synergy H1 Hybrid plate

reader (Excitation=340 nm; Substrate emission=665 nm;

Antibody emission=615 nm; Delay=100 μs; Integration=200 μs).

Emission ratios (665 nm/615 nm) were calculated for each well

and half-maximal inhibitory concentrations (IC₅₀) were

determined for each inhibitor through non-linear regression

analysis of the log dose-response curves.

1

188°C decomposed; IR (cm^-1) 2948, 1669, 1606; H NMR (400

MHz, DMSO-d₆) 0.84 (3H, d, J = 5.9 Hz, CH(CH₃)₂), 0.86 (3H,

d, J = 5.9 Hz, CH(CH₃)₂), 1.34-1.49 (1H, m, CH(CH₃)₂), 1.39 (3H,

d, J = 6.8 Hz, NCHCH₃), 1.49-1.63 (2H, m, NCH₂CH₂), 3.17-3.28

(1H, m, NCH₂CH₂), 3.20 (3H, s, NCH₃), 3.78-3.95 (1H, m,

NCH₂CH₂), 3.83 (3H, s, OCH₃), 4.39 (1H, q, J = 6.8 Hz,

NCHCH₃), 7.12 (1H, d, J = 8.9 Hz, H-5’), 7.41 (1H, dd, J = 8.9,

2.4 Hz, H-6’), 7.72 (1H, s, H-4), 7.74 (1H, d, J = 2.4 Hz, H-2’),

9.93 (1H, s, NH); ¹³C NMR (100 MHz, DMSO-d₆) 18.8

(NCHCH₃), 22.7 (CH(CH₃)₂), 22.8 (CH(CH₃)₂), 26.2 (CH(CH₃)₂),

28.6 (NCH₃), 35.5 (NCH₂CH₂), 45.0 (NCH₂CH₂), 56.7, 56.9,

113.4 (Ar-C), 115.5 (Ar-C), 121.3 (Ar-C), 121.7 (Ar-C), 123.4

(Ar-C), 132.1 (Ar-C), 151.1 (Ar-C), 151.6 (Ar-C), 151.8 (Ar-C),

163.9 (C=O). HRMS cal. C₂₀H₂₇ClN₅O₂(ES+) m/z 404.185328

[M+H]⁺, found 404.178289.

4.4 Cell culture

MOLM-13 (DSMZ), MOLM-14 (DSMZ), THP-1 (ATCC), and

U937 cells were cultured in RPMI1640 (Gibco) supplemented

with 10% FBS (Sigma Aldrich) and 1% penicillin/streptomycin

solution (Cellgro), K562 and MV4-11 were cultured in Iscove

Modified Dulbecco Medium (IMDM; Gibco) supplemented with

10% FBS (Sigma Aldrich) and 1% penicillin/streptomycin

solution (Cellgro), and HL-60 cells were cultured in Iscove

Modified Dulbecco Medium (IMDM; Gibco) supplemented with

20% FBS (Sigma Aldrich) and 1% penicillin/streptomycin

solution (Cellgro) at 37^°C in an incubator humidifier with 95% air

and 5% CO₂. HEK293 cells (ATCC) were cultured in Dulbecco’s

Modified Eagle Medium (DMEM; Gibco) supplemented with

10% FBS (Sigma Aldrich) and 1% penicillin/streptomycin

solution (Cellgro) at 37^°C in an incubator humidifier with 95% air

and 5% CO₂. HEK293 cells were allowed to adhere for 24 h prior

to use in assays. In all assays, the final DMSO concentration did

not exceed 0.5% for all treatment conditions.

4.2.44 2-((3-Bromo-4-methoxyphenyl)amino)-8-isopentyl-5,7-

dimethyl-7,8-dihydropteridin-6(5H)-one (53). Pteridinone

heterocycle 21 (123 mg, 0.42 mmol), monobromo-methoxyaniline

(174 mg, 0.85 mmol) and TFA (162 µL, 2.12 mmol) were taken

up in TFE (4 mL) and reacted according to the described General

Procedure E. Purification via silica gel chromatography (97:3

DCM:MeOH) afforded the target compound as a purple solid (0.19

g, 0.43 mmol, 98%). Rf 0.28 (97:3 DCM:MeOH); M. p. 143-

1

158°C decomposed; IR (cm^-1) 2962, 1674, 1565, 1528; H NMR

(400 MHz, DMSO-d₆) 0.84 (3H, d, J = 5.9 Hz, CH(CH₃)₂), 0.86

(3H, d, J = 5.9 Hz, CH(CH₃)₂), 1.35-1.49 (1H, m, CH(CH₃)₂), 1.39

(3H, d, J = 6.8 Hz, NCHCH₃), 1.49-1.63 (2H, m, NCH₂CH₂), 3.15-

3.29 (1H, m, NCH₂CH₂), 3.20 (1H, s, NCH₃), 3.82 (3H, s, OCH₃),

3.85-3.95 (1H, m, NCH₂CH₂), 4.40 (1H, q, J = 6.8 Hz, NCHCH₃),

7.09 (1H, d, J = 8.9 Hz, H-5’), 7.45 (1H, dd, J = 8.9, 2.5 Hz, H-

6’), 7.71 (1H, s, H-4), 7.88 (1H, d, J = 2.5 Hz, H-2’), 9.96 (1H, s,

NH); ¹³C NMR (100 MHz, DMSO-d₆) 18.7 (NCHCH₃), 22.7

(CH(CH₃)₂), 22.8 (CH(CH₃)₂), 26.2 (CH(CH₃)₂), 28.6 (NCH₃),

35.5 (NCH₂CH₂), 44.8 (NCH₂CH₂), 56.8, 56.9, 110.7 (Ar-C),

113.2 (Ar-C), 115.5 (Ar-C), 122.4 (Ar-C), 126.4 (Ar-C), 132.6

(Ar-C), 151.5 (Ar-C), 151.8 (Ar-C), 152.4 (Ar-C), 163.9 (C=O).

HRMS cal. C₂₀H₂₇BrN₅O₂(ES+) m/z 448.134811 [M+H]⁺, found

448.131605.

4.5 Western blot

Cell lines were maintained according to cell culture protocol prior

to preparation of untreated cell lysates for Western blot analysis.

Cells were trypsinized (if adherent), collected and centrifuged at

1000 rpm for 10 minutes at 4°C. Media was aspirated and cells

were washed with phosphate buffered saline (PBS) and

centrifuged again. PBS was aspirated and cell pellets were

resuspended in TES/SB buffer supplemented with protease

inhibitors and kept on ice for the duration of lysate preparation.

Samples were sonicated (3 x 5 s) and centrifuged at 12000 rpm for

10 minutes at 4°C. Protein concentrations of isolated lysates were

determined for each cell line via a Lowry Protein Assay (according

to supplier protocol) and diluted such that each sample contains 25

μg of total protein in 40 μL of TES/SB buffer supplemented with

SDS Loading Buffer and 20% BME. Lysates were centrifuged and

boiled for 10 minutes prior to loading into a 10% acrylamide gel.

The gel was run and the proteins transferred to a nitrocellulose

membrane, blocked with 5% milk in TBS-T for 1 h, and incubated

with the necessary primary antibody at the indicated dilution in

TBS-T containing 5% BSA overnight at 4°C. Membranes were

washed in TBS-T, and incubated with the appropriate secondary

antibody for 45 minutes. Gels were developed on a Biorad

ChemiDoc™ MP Imaging System according to the manufacturer’s

protocol. Antibodies used included phosphor-RSK1(Ser221),

total-RSK1, phosphor-RSK2 (Ser227), and total-RSK2 and were

purchased from Cell Signaling Technologies.

4.3 Inhibition of RSK activity

LANCE (Lanthanide Chelate Excite)® Eu time-resolved

fluorescence resonance energy transfer (TR-FRET) kinase assay

(PerkinElmer) was performed in 384-well OptiPlates (Corning)

using recombinant RSK1, RSK2, RSK3 and RSK4 kinases

(CarnaBio), ULight™-phospho-40S ribosomal protein S6

(Ser235/236) peptide substrate (PerkinElmer), and ATP (Sigma)

according to the supplier protocols. All reagents were prepared in

kinase buffer containing 2mM DTT, 50 mM HEPES, 1mM EGTA,

10 mM MgCl₂, 0.01% Tween 20, pH 7.5. Inhibitor solutions were

prepared such that the final DMSO concentration did not exceed

0.5%, which was shown to have no impact on kinase activity.

RSK1 was used at a final concentration of 1 nM while RSK2,

RSK3 and RSK4 were used at a final concentration of 500 pM.

ULight^™-rpS6 substrate was used at a final concentration of 250

nM for all isoforms and ATP was administered at a final

concentration of 5 μM, 3 μM, 1 μM and 6 μM for RSK1, RSK2,

4.6 Cellular Metabolic Viability and Cytotoxicity Assays.

4.6.1 MTS Cellular Viability Assay. MOLM-13 cells were

seeded into sterile 96-well plates (Corning) at 60,000 cells per well

in 100 μL of media. Inhibitor solutions were administered in 50 μL

over a concentration range from 60 μM to 247 nM and cells were

incubated at 37°C for 24 to 72 h. Following 3 h incubation with 30

μL CellTiter 96^®Aqueous One Cell Proliferation Reagent

(Promega), cell viability was measured by formazan concentration

assessment through colorimetric analysis using a Biotek Synergy

H1 Hybrid plate reader (Absorption=490).

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Carriere, A.; Ray, H.; Blenis, J.; Roux, P. P., The RSK

4.6.2 CellTiter-Glo Viability Assay. MOLM-13 cells were

seeded into sterile opaque 96-well plates (Thermo Scientific) at

30,000 cells per well in 50 μL of media. Inhibitor solutions were

administered in 50 μL over a concentration range from 60 μM to

247 nM and cells were incubated at 37°C for 24 to 72 h. Cells were

equilibrated at RT for 30 min, dosed with 100 μL CellTiter-Glo^®

Reagent, and mixed on an orbital shaker for 2 min. Luminescence

was measured following 10 min incubation at rt using a BioTek

Synergy H1 plate reader.

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4.6.3 CellTox Green Cytotoxicity Assay. MOLM-13 cells were

seeded into sterile black 96-well plates (Corning) at 60,000 cells

per well in 50 μL of media containing 1:500 dilution of CellTox^™

Green dye (Promega). Inhibitor solutions were administered in 50

μL over a concentration range from 60 μM to 247 nM. Cells were

incubated at 37^°C and fluorescence was measured at 0, 24, 48 and

72 h using a BioTek Synergy H1 plate reader (Excitation=485 nm;

Emission=520 nm).

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RSK in cancer. Expert Opin Ther Targets 2011, 15 (1), 5-9.

8. Houles, T.; Roux, P. P., Defining the role of the RSK

isoforms in cancer. Semin Cancer Biol 2018, 48, 53-61.

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4.7 Intracellular RSK2 Kinase Assay

HEK293 cells were cultured in DMEM, as indicated above, prior

to transfection. Cells were pelleted and resuspended in DMEM to

a density of 200,000 cells per mL. A lipid:DNA complex

containing a 9:1 ratio of Transfection Carrier DNA to NanoLuc^®-

RPS6KA3 fusion DNA was prepared according to Promega

NanoBRET^™Intracellular Kinase Assay protocol (K-5 kit). A

solution containing 1 part lipid:DNA complex with 20 parts

HEK293 cells was dispensed into an appropriate sterile tissue

culture flask such that a cell density of 55,000-80,000 cells per cm²

is reached and incubated for 24 h. Following transfection, media

was aspirated and cells were readjusted to a density of 200,000

cells per mL in Opti-MEM I Reduced Serum Medium (no phenol

red; Gibco). Cell solution was administered at 85 μL per well into

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Steelman, L. S.; Franklin, R. A.; Abrams, S. L.;

Chappell, W.; Kempf, C. R.; Basecke, J.; Stivala, F.; Donia, M.;

Fagone, P.; Nicoletti, F.; Libra, M.; Ruvolo, P.; Ruvolo, V.;

Evangelisti, C.; Martelli, A. M.; McCubrey, J. A., Roles of the

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25 (7), 1080-94.

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duplication mutation prevents apoptosis in interleukin-3-deprived

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sterile white 96-well plates (Thermo Scientific).

A

20X

12.

Elf, S.; Blevins, D.; Jin, L.; Chung, T. W.; Williams,

NanoBRET^®Tracer K-5 reagent was prepared according to

protocol and administered at 5 μL per well. Inhibitor solutions

were administered in 10 μL over a concentration range from 40

μM to 1.25 μM and cells were incubated for 2 h at 37^°C. The plates

were equilibrated at rt for 15 min prior to administration of 50 μL

3X Complete Substrate plus Extracellular Inhibitor solution

containing Nano-Glo^®Substrate and Extracellular NanoLuc^®

Inhibitor, prepared according to assay protocol. Cells were

incubated at rt for 2-3 min and fluorescence was measured using a

BioTek Synergy H1 plate reader (Donor = 450 nm; Acceptor =

610 nm). BRET ratios were calculated by dividing the acceptor

emission by the donor emission, correcting for background by

subtracting the ‘no tracer’ BRET ratio from each sample.

I. R.; Lee, B. H.; Lin, J. X.; Leonard, W. J.; Taunton, J.; Khoury,

H. J.; Kang, S., p90RSK2 is essential for FLT3-ITD- but

dispensable for BCR-ABL-induced myeloid leukemia. Blood

2011, 117 (25), 6885-94.

13.

Kang, S.; Dong, S.; Gu, T. L.; Guo, A.; Cohen, M. S.;

Lonial, S.; Khoury, H. J.; Fabbro, D.; Gilliland, D. G.;

Bergsagel, P. L.; Taunton, J.; Polakiewicz, R. D.; Chen, J.,

FGFR3 activates RSK2 to mediate hematopoietic transformation

through tyrosine phosphorylation of RSK2 and activation of the

MEK/ERK pathway. Cancer Cell 2007, 12 (3), 201-14.

14.

Kang, S.; Elf, S.; Dong, S.; Hitosugi, T.; Lythgoe, K.;

Guo, A.; Ruan, H.; Lonial, S.; Khoury, H. J.; Williams, I. R.;

Lee, B. H.; Roesel, J. L.; Karsenty, G.; Hanauer, A.; Taunton,

J.; Boggon, T. J.; Gu, T. L.; Chen, J., Fibroblast growth factor

receptor 3 associates with and tyrosine phosphorylates p90 RSK2,

leading to RSK2 activation that mediates hematopoietic

transformation. Mol Cell Biol 2009, 29 (8), 2105-17.

Acknowledgement

Research reported in this publication was supported by an ALSAM

Therapeutics Innovation grant.

15.

Mitton, B.; Dutta, R.; Hsu, Y.-C.; Sakamoto, K., The

Role of pp90rsk-Mediated CREB Phosphorylation in Acute

Myelogenous Leukemia. Blood 2014, 124 (21), 2229-2229.

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Supplementary Material

Schemes for alternative synthetic routes and mono-halogenated

pteridinone analogs; tables of IC₅₀values for RSK inhibitors

against the RSK1, 3, and 4 isoforms and for LJH685 and LJI308

against RSK2; Western blot of total and phosphor-RSK1 and

RSK2 expression in leukemic cell lines; and inhibition of cellular

RSK2 activity measured by nanoBRET assay.

Declaration of interests

☒ 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.

☐The authors declare the following financial

interests/personal relationships which may be

considered as potential competing interests:

Article Doi

Substituted pteridinones as p90 ribosomal S6 protein kinase (RSK) inhibitors: A structure-activity study

DOI: 10.1016/j.bmc.2019.115303

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Article abstract of DOI:10.1016/j.bmc.2019.115303

Full text of DOI:10.1016/j.bmc.2019.115303

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