Development of Potent Pyrazolopyrimidinone-Based WEE1 Inhibitors with Limited Single-Agent Cytotoxicity for Cancer Therapy

Accepted Article

Title: Development of potent pyrazolopyrimidinone-based WEE1

inhibitors with limited single-agent cytotoxicity for cancer therapy

Authors: Christopher J Matheson, Kimberly A Casalvieri, Donald S

Backos, and Philip Reigan

This manuscript has been accepted after peer review and appears as an

Accepted Article online prior to editing, proofing, and formal publication

of the final Version of Record (VoR). This work is currently citable by

using the Digital Object Identifier (DOI) given below. The VoR will be

published online in Early View as soon as possible and may be different

to this Accepted Article as a result of editing. Readers should obtain

the VoR from the journal website shown below when it is published

to ensure accuracy of information. The authors are responsible for the

content of this Accepted Article.

To be cited as: ChemMedChem 10.1002/cmdc.201800188

Link to VoR: http://dx.doi.org/10.1002/cmdc.201800188

A Journal of

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Development of potent pyrazolopyrimidinone-based WEE1

inhibitors with limited single-agent cytotoxicity for cancer therapy

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

[a]

Dr. C. J. Matheson, K. A. Casalvieri, Dr. D. S. Backos, Dr. P. Reigan

Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences,

University of Colorado Anschutz Medical Campus

Address 112850 East Montview Boulevard, Aurora, CO, 80045 (USA)

E-mail: philip.reigan@ucdenver.edu

Supporting information for this article is given via a link at the end of the document.

Abstract: WEE1 kinase regulates the G2-M cell-cycle checkpoint, a

critical mechanism for DNA repair in cancer cells that can confer

resistance to DNA-damaging agents. We previously reported a series

of pyrazolopyrimidinones based on AZD1775, a known WEE1

inhibitor, as an initial investigation into the structural requirements for

WEE1 inhibition. Our lead inhibitor demonstrated WEE1 inhibition in

the same nanomolar range as AZD1775, and potentiated the effects

of cisplatin in medulloblastoma cells, but had reduced single-agent

cytotoxicity. These results prompted the development of a more

comprehensive series of WEE1 inhibitors. Here, we report a series of

pyrazolopyrimidinones and identify a more potent WEE1 inhibitor than

AZD1775 and additional compounds that demonstrate that WEE1

inhibition can be achieved with reduced single-agent cytotoxicity.

These studies support that WEE1 inhibition can be uncoupled from

the potent cytotoxic effects observed with AZD1775, and this may

have important ramifications in the clinical setting where WEE1

inhibitors are used as chemosensitizers for DNA-targeted

chemotherapy.

related (ATR) kinase mediate the repair of DNA double-strand

4]

and DNA single-strand breaks, respectively.^[1,

ATR

phosphorylates the checkpoint kinase CHK1 in response to

DANN damage, which in-turn phosphorylates and activates

WEE1 (Figure 1). The inhibitory phosphorylation of Tyr15 on

CDK1 by WEE1 impedes CDK1/cyclin B function, resulting in cell-

cycle arrest at G2-M and DNA-damage repair.^[1]WEE1 is

expressed at high levels in a number of cancers and is associated

with poor clinical outcome,^[5]suggesting that these cancers are

heavily dependent on this mechanism for survival.

Introduction

WEE1 is a nuclear kinase that regulates the G2-M cell-cycle

checkpoint by modulating the activity of cyclin-dependent kinase

1 (CDK1) and prevents entry to mitosis in response to DNA

damage.^[1]In normal cells, DNA damage can be repaired at either

the G1 or the G2 checkpoint to preserve genomic integrity;

however, cancer cells often have deficiencies in the G1

checkpoint as a means to acquire and propagate pro-oncogenic

mutations, which results in an overreliance of DNA repair at the

G2-M checkpoint.^[2]The G2-M cell-cycle checkpoint is a critical

mechanism for DNA repair in cancer cells and can confer

resistance to DNA-damaging agents; therefore, the molecular

components of the G2-M checkpoint, such as WEE1, are

attractive targets for anticancer drug development.^[3]Ataxia-

telangiectasia-mutated (ATM) kinase and ataxia-telangiectasia-

Figure 1. WEE1 and the G2-M cell-cycle checkpoint. Schematic

representation of the G2-M cell-cycle checkpoint, in which DNA damage

detected by ATR triggers phosphorylation of CHK1, leading to the inhibition of

the phosphatase Cdc25 and subsequent phosphorylation of WEE1 and the

CDK1-CyclinB complex, resulting in mitotic arrest. Inhibition of WEE1 is

sufficient to prevent cell cycle arrest, forcing the cell to undergo mitosis.

The pyrazolopyrimidinone AZD1775 (formerly MK1775) is a

potent, ATP-competitive inhibitor of WEE1 kinase activity.^[6]

Inhibition of WEE1 promotes unscheduled mitotic entry through

CDK1 activation, leading to loss of genome integrity, and

ultimately mitotic catastrophe and apoptosis.^[7]In addition, WEE1

inhibition has been shown to induce replication stress through

CDK1/2-dependent aberrant firing of replication origins and

subsequent nucleotide shortage.^[8]Therefore, due to the critical

role of WEE1 in regulating cell-cycle events, the incorporation of

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AZD1775 into radiation therapy and/or DNA-damaging agent

regimens have emerged as an attractive strategy for combination

therapy that is currently being evaluated in multiple clinical trials

in a range of cancer types (www.ClinicalTrials.gov). Although

AZD1775 has been reported to be well-tolerated in clinical trial,

there have been few preclinical and clinical studies examining the

effect of AZD1775 as a single-agent. A recent report from an

ongoing Phase I study evaluating the safety and maximum

tolerated dose (MTD) of AZD1775 in advanced refractory solid

AZD1775 to describe a more diverse, rationally designed series

of pyrazolopyrimidinones as small molecule inhibitors of WEE1

kinase activity to allow for a more comprehensive understanding

of the SAR for WEE1 inhibition with limited cytotoxicity.

A

B

tumors

(NCT01748825)

disclosed

supraventricular

tachyarrhythmia and myelosuppression as dose-limiting

toxicities.^[9]However, many of the ongoing Phase II trials using

various combination therapies are using dosing strategies below

the established MTD for AZD1775. In preclinical studies,

AZD1775 demonstrated potent cytotoxicity in a broad panel of

cancer cell lines and led to xenograft tumor growth inhibition;^[10]

however, these anticancer activities cannot be attributed to WEE1

inhibition alone since AZD1775 is known to inhibit other kinases

and may have off-target effects outside of the kinome.^{[6, 11]}The

combination of WEE1 inhibitors with DNA-damaging agents has

been proposed to be more effective in p53-mutant cancer cells

that rely on DNA repair at the G2-M checkpoint. However, several

preclinical studies have demonstrated that AZD1775 has

anticancer activity independent of p53 function, and this has also

been attributed to the alternative functions of WEE1 and/or the

off-target effects of AZD1775.^{[5d, 10]}

C

D

Figure 2. Structure of WEE1 inhibitors AZD1775 and CM-061 (1) and

predicted WEE1-AZD1775 interactions. (A) Ribbon representation (blue

ribbon, grey amino acid residues; with clipped pane molecular surface colored

dark grey) of the ATP-binding site of WEE1 displaying the predicted binding

mode of AZD1775 (cyan). Red dashed lines indicate H-bonds and yellow

dashed lines indicate π-π interactions. (B) A stick display style representation

of AZD1775 (cyan) and key interacting amino acid residues and target residues

(grey) for inhibitor design. Red dashed lines indicate H-bonds and yellow

dashed lines indicate π-π interactions. (C) Ligand interaction diagram of the

predicted binding mode of AZD1775 in the ATP-binding site of WEE1. Red

residues are charged negative, purple residues are charged positive,

hydrophobic residues are hydrophobic, and blue resiues are polar, purple

arrows indicate H-bonds, green lines indicate π-π interactions, and grey circles

indicate areas of solvent exposure. (D) The chemical structure of the WEE1

inhibitors AZD1775 and CM-061 (1).

In our previous study, we performed an initial examination

of the structural components of AZD1775 that are required for

WEE1 inhibition.^[12]We used a computational-based model of

AZD1775 docked into the WEE1 crystal structure to guide the

design of a series of 14 pyrazolopyrimidinones as candidate

WEE1 inhibitors. From this series, we identified a WEE1 inhibitor

(CM-061, 1, Figure 2) that demonstrated inhibition of in vitro

WEE1 kinase activity in the same nanomolar range as AZD1775,

maintained comparable synergistic activity with cisplatin in

medulloblastoma cells, but exhibited reduced single-agent

cytotoxicity.^[12]From our initial structure-activity relationship (SAR)

study we concluded that AZD1775 has off-target effects that result

in its potent cellular toxicity, side chain modifications on the

pyrazolopyrimidinone core of AZD1775 can have marked and

independent effects on WEE1 inhibition and cytotoxicity, and

WEE1 inhibition can be achieved without potent cytotoxicity. This

initial SAR study had limited scope, that focused on the sequential

increase in complexity of the N-methylpiperazine group of the lead

compound without systematically testing interactions within the

WEE1 ATP-binding domain. In this manuscript, we perform a

more extensive analysis of the predicted binding mode of

Results

Computational-based design of WEE1 inhibitors

To further develop our understanding of the SAR for WEE1

inhibition, and to improve the activity of this class of compounds,

we re-examined our computational model of the binding

conformation of AZD1775 in the ATP-binding site of WEE1. As

expected from an ATP-competitive inhibitor, the core

pyrazolopyrimidinone heterocycle forms H-bonding interactions

with the hinge residues C379 and N376 (Figure 2). Focusing on

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the aniline side chain, we observed that the aniline 3-position was

oriented adjacent to the backbone nitrogen of G382 (Figure 2A),

and the introduction of moieties capable of acting as H-bond

acceptors in this region could improve binding to WEE1. Thus, we

and allow additional flexibility for the hydroxyl to form an H-bond

interaction with D463. To evaluate this hypothesis, the

pyridylmethanol analog (30) was proposed, and a phenyl analog

of AZD1775 (31) as there were no apparent interactions involving

the pyridine nitrogen. The final region of the molecule we

investigated was the N²-allyl group, which does not appear to be

critical for the binding of AZD1775 to WEE1 beyond hydrophobic

interactions with V313, A326, and K328; however, the allyl group

may be a metabolic liability via CYP450-mediated conversion to

the diol or primary alcohol. Furthermore, we hypothesized that its

removal may allow deeper access into the ATP-binding domain

for tighter binding. To investigate the necessity for the allyl group,

both the N²-methyl (32) and unsubstituted (35) analogs of

AZD1775 were proposed. At the time of writing this manuscript,

the 1.9Å co-crystal structure of the human WEE1 kinase domain

in complex with AZD1775 was published (PDB: 5V5Y).^[13]The

proposed binding orientation of AZD1775 in the ATP-binding site

of WEE1 in our computational model was in good agreement with

the co-crystal structure, with only minor differences observed in

the orientation of the side chains. Importantly, both our

computational model and the WEE1-AZD1775 co-crystal

structure equally support the proposed structural modifications to

AZD1775 described above.

proposed to synthesize

a series of compounds bearing

dimethylamine and dimethylamide groups at the aniline 3-position

(25-27), as well as a 3-cyanoaniline analog, to vary both the

direction and distance of the H-bond acceptor in this region.

Interestingly, the N-methylpiperazine moiety of AZD1775 was not

predicted to make any substantive interactions (Figure 2A), due

to the rigidity of the ring system; however, both E303 and D386

are in close proximity to the N-methylpiperazine moiety (~5-6Å)

and have the potential for interaction. We hypothesized that

substitution with a more flexible side chain containing a terminal

basic group to interact with E303 and/or D386 could enhance

binding and potency (28 and 29). Finally, as an extension to our

previous studies into the structural complexity required to confer

potency at this position,^[12]the 6-amino (22) and unsubstituted

aniline (24) analogs of AZD1775 were included in the proposed

compound series.

From our previous study,^[12]the hydroxyl of the pyridyl side

chain was preferred for WEE1 binding, through interaction with

D463 or the Mg²⁺ion; however, the methyl groups may impede

binding due to steric hinderance with the protein surface. The

removal of the methyl groups may alleviate this potential clash

Scheme 3. Synthesis of analogs. Synthetic strategy for the preparation of proposed WEE1 inhibitor analogs of AZD1775 (22-35). Reagents and conditions: a) t-

butyl carbazate, toluene, reflux, 18 h; b) alkyl halide, BnNEt₃CI, K₂CO₃, MeCN, RT-50°C, 18-48 h or PMB-OH, DEAD, PPh₃, THF, RT, 16 h; c) MeNHNH₂, THF,

RT, 18 h; d) i) DIPEA, THF, reflux, 72 h, ii) TFA, RT-70°C, 2 h or TFA, DCM, RT, 18 h; e) N,N'-diethylethylenediamine, Cul, K₂CO₃, 1,4-dioxane, 95°C, 18 h; f) i)

mCPBA, toluene, RT, 1 h, ii) aniline /amine, DIPEA, toluene, RT, 18 h; g) TFA, reflux, 16 h; h) 4-ToISO₂Na, Pd(PPh₃)₄, MeOH/THF, RT, 2 h.

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Chemistry

the presence of an aniline did not appear to be critical for activity,

with the aminopyrimidine 22 (IC₅₀= 17.7 ± 7.8 nM) exhibiting

approximate equipotency when compared to the unsubstituted

aniline 24 (IC₅₀= 9.6 ± 2.6 nM) and only a modest loss in activity

with respect to AZD1775 (IC₅₀= 5.1 ± 0.9 nM). From our data, it

appears that the steric constraints in this region of the ATP-

binding domain limit the extent of substitution tolerated at the

aniline 3-position. This is supported by the sequential decrease in

activity from the dimethylaniline 25 (IC₅₀= 4.7 ± 1.7 nM) to the

dimethylbenzylamine 26 (IC₅₀= 9.1 ± 1.2 nM) and the 3-

dimethylamide analog 27 (IC₅₀= 27.3 ± 6.4 nM). The equipotency

of 25 with AZD1775 in the absence of the N-methylpiperazine

moiety, previously shown to be favorable for WEE1 binding,^[12]

would suggest that the 3-dimethylamino group improves binding

with WEE1, likely through the proposed H-bond with G382. The

inhibitory activity of the compounds designed to remove the

AZD1775 analogs were prepared as outlined in Scheme 3

and described in the Experimental Section and Supporting

Information. Briefly, tert-butylcarbazate was protected through

reaction with phthalic anhydride (2), after which the carbamate

nitrogen was functionalized with the necessary alkyl group

through either nucleophilic substitution or Mitsunobu reaction.

Removal of the phthalamide protecting group with methyl

hydrazine gave the key tert-butyl alkylcarbazates (7-9), which

were

reacted

with

ethyl

4-chloro-2-methylthio-5-

pyrimidinecarboxylate (10) in the presence of TFA to form the

core pyrazolopyrimidin-3-one scaffolds (11-13). An Ullman-type

aryl amination with the relevant functionalized pyridines (14-16)

gave the penultimate pyrazole products (17-21), after which

activation of the thioether with m-CPBA and reaction with relevant

anilines or amines afforded the desired AZD1775 analogs (22-33). conformational restraints imparted by the N-methylpiperazine ring

Surprisingly, a desired 3-cyanoaniline analog was inaccessible of AZD1775 appear to support our hypothesis that this would

via this route, with no reaction occurring under ambient conditions, improve interactions with either E303 or D386. However, the

and degradation evident under increased temperature, and so

this compound was omitted from the series.

distance between the aniline ring and the basic dimethylamine

plays a role, as evidenced by the reduction in inhibitory activity for

compound 29 (IC₅₀= 7.8 ± 0.4 nM) as opposed to the

improvement in activity for 28 (IC₅₀= 1.7 ± 0.9 nM) when

compared to AZD1775.

The proposed synthesis of the N²-unsubstituted analog of

AZD1775 (35) through TFA-mediated removal of the para-

methoxybenzyl (PMB) protecting group did not afford the desired

compound. In addition to removal of the PMB group, an acid-

mediated elimination of the 2-propanol hydroxyl moiety adorning

the pyridine ring resulted in formation of the isopropenyl analog

(34) observed by NMR and MS. This compound was included in

the kinase activity assay to further the scope of the SAR. As an

alternative approach to the desired compound the allyl group of

AZD1775 was directly removed through reaction with sodium

para-toluenesulfinate tetrahydrate. As it was anticipated that the

acid mediated elimination side reaction would occur during the

TFA-mediated conversion of the PMB-protected compound (23)

to the 6-aminopyrimidine analog (22), the desired unsubstituted

The removal of the methyl groups from the pyridyl side chain

resulted in a small reduction in activity (30; IC₅₀= 10.4 ± 2.9 nM),

with only negligible effects of lipophilic efficiency (LipE; AZD1775

= 6.11; 30 = 6.06). These methyl groups may act as a

conformational lock that optimizes the position of the hydroxyl

group for a distant H-bond interaction with D463 or the Mg²⁺ion,

mitigating an entropic penalty to binding that is incurred upon their

removal. The pyridine nitrogen was not necessary for activity as

equipotency with AZD1775 was obtained for compound 31 (IC₅₀

= 6.9 ± 1.0 nM).

The substitution of the N²-allyl group with a methyl group

resulted in a near 4-fold reduction in activity (32; IC₅₀= 19.9 ± 3.8

nM), while its removal in the desired compound 35 (IC₅₀= 118 ±

28 nM) and the synthetic side product 34 (IC₅₀= 190 ± 12 nM)

reduced activity. We hypothesize that these substitutions may

alter the electronics around the pyrazole carbonyl and thus the

ability to act as an H-bond acceptor with N376. To test this

hypothesis, we examined the electrostatic and hydrophobic field

properties of the three compounds and found that, compared to

AZD1775, both analogs exhibited an attenuated negative

electrostatic field in this region, with the alterations in the

unsubstituted species predicted to be particularly pronounced

(Supporting Information, Figure S1).

amine

was

introduced

through

the

reaction

of

pyrazolopyrimidinone (18) with ammonia. The PMB-protected

compound was also included in the kinase activity assay to gain

additional insight into the SAR for WEE1 inhibition.

In vitro WEE1 kinase activity assay

All of the synthesized compounds and relevant synthetic

intermediates were tested for their inhibitory activity against

recombinant WEE1 in the established TR-FRET activity assay,^[12]

and the resultant IC₅₀values are detailed in Table 1. For

modification of the pyrazolopyrimidinone 6-position (Table 1; R³),

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WEE1 inhibitor effects on cellular viability and cytotoxicity

Table 1. Structures and inhibitory activities of pyrazolopyrimidinone WEE1

inhibitors.

Previously, we utilized an MTS assay to determine the

single-agent effects of WEE1 inhibitors on the cell viability of

ONS-76 (p53 wild-type) and Daoy (p53 mutant) medulloblastoma

cell lines.^[12]All of the WEE1 inhibitors described in this

manuscript with potent WEE1 kinase inhibitory activity in the in

vitro TR-FRET assay (IC₅₀< 10 nM) were selected for evaluation

in the MTS assay. In ONS-76 cells only three compounds

exhibited an appreciable effect on cellular metabolic activity within

the concentration range of the assay, to the degree that EC₅₀

values could be determined (Figure 3A). These 3 compounds

included AZD1775, that has been previously shown to be a potent

inhibitor of cellular metabolic activity with an EC₅₀of 159 ± 31 nM

in this assay,^[12]28 (EC₅₀= 252 ± 9 nM), and 29 (EC₅₀= 203 ± 40

nM). Interestingly, 28 demonstrated improved recombinant WEE1

kinase inhibitory activity over AZD1775, but had a decreased

effect on cell viability. There was a significant lack of impact on

cell viability for 31 (57% effect at 1.16 µM) although inhibition of

WEE1 kinase activity was comparable to AZD1775, and the only

structural modification was removal of the pyridine ring nitrogen.

In Daoy cells, the disparity between WEE1 inhibtion and cell

viability was further pronounced (Figure 3B). The p53 mutant

status of Daoy cells would be anticipated to show greater

correlation between WEE1 inhibition and single-agent WEE1

Compound

R¹

R²

R³

IC₅₀(nM)

78.1 ± 36

18

22

23

24

25

26

27

28

17.7 ± 7.8

149 ± 51

9.6 ± 2.6

4.7 ± 1.7

9.1 ± 1.2

27.3 ± 6.4

1.7 ± 0.9

29

7.8 ± 0.4

5.1 ± 0.9

AZD1775

inhibitor effects on cell viability. However, only AZD1775 (EC₅₀

=

179 ± 16 nM) was sufficiently active to allow an EC₅₀to be

determined. The WEE1 inhibitors that demonstrated potent

inhibition of cell viability in ONS-76 cells were also the most active

inhibitors of Daoy cell viability (28 = 84% effect at 600 nM; 29 =

81% effect at 600 nM), but their effect was significantly diminished

with respect to AZD1775. There was no effect on cellular viability

with any of the additional WEE1 inhibitors within the concentration

range of the assay.

30

31

32

33

10.4 ± 2.9

6.9 ± 1.0

19.9 ± 3.8

89.7 ± 30.2

In order to determine the cytotoxic effect of WEE1 inhibitors

we used the CellTox^TMGreen Cytotoxicity Assay. This assay uses

an asymmetric cyanine dye that is non-toxic and excluded from

viable cells; however, changes in membrane integrity that occur

as a result of cell death allows the dye to enter the compromised

cells and bind DNA, enhancing the dyes flurorescent properties.

Using this assay we evaluated the cytotoxic effects of AZD1775

and compounds 28, 29, and 31 in Daoy cells over 72 hours

(Figure 3C and Supporting Information Figures S2 and S3).

Overall, the cytotoxicity of the WEE1 inhibitors were

concentration- and exposure time-dependent, but EC₅₀values

could only be determined for AZD1775 (EC₅₀= 188 ± 26 nM) and

28 (EC₅₀= 651 ± 16 nM) after 72 hours. The ranking of the potency

34

35

190 ± 12.0

118 ± 28.3

Inhibitory activity for the synthesized compounds from a recombinant WEE1 TR-

FRET binding assay are given as the half maximal inhibitory concentration (IC₅₀

in nM.

)

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= 651 ± 16 nM). Sigmoidal dose-response curves were not obtained for

compounds 29 and 31 up to 2400 nM inhibitor dose (n=3, error bars/± = S.D.).

of cytotoxicity for these compounds correlated with the cell

vaibility assay. AZD1775 had the most potent cytotoxic properties

and demonstrated cytotoxic effects within 24 hours at

concentrations >600 nM. After 72 hours AZD1775 demonstrated

cytotoxic effects at concentrations >75 nM. Although 28 showed

some cytotoxicty within 24 hours at 1.2 µM, it was significantly less

cytotoxic than AZD1775 over the treatment period. Compounds

29 and 31 did not display sufficient cytotoxicity for EC₅₀

determination and did not demonstrate any appreciable

cytotoxicity until 48 hours and only at concentrations >300 nM.

Effect of WEE1 inhibitors on cellular pCDK1

All of the WEE1 inhibitors that demonstrated activity in the

TR-FRET assay were advanced to determine their effect on the

cellular concentration of phosphorylated CDK1 at Tyr15, an

effective biomarker of cellular WEE1 activity.^[14]Initially, Daoy

cells were treated with all compounds at a single concentration of

220 nM, which we previously determined as the EC₅₀for AZD1775

in this assay (Figure 4A).^[12]Of the compounds tested, only

AZD1775 (p<0.001), 28 (p<0.001), and 29 (p<0.01) showed a

statistically significant decrease in pCDK1 levels when compared

to DMSO control, and were selected for further testing across a

concentration range. Compound 31 did not cause a significant

reduction in pCDK1 levels compared with DMSO, but the level of

pCDK1 was observed to decrease to a level not statistically

different from the AZD1775 treatment group. Interestingly, both

24 (p<0.05) and 25 (p<0.01) resulted in significant increases in

pCDK1 levels with respect to DMSO, suggesting an off-target

activation of the DNA damage response (DDR) pathway. The

selected compounds were administered to cells at concentrations

between 1 µM and 62 nM, and the resultant cell extracts were used

to establish a dose-response in the pCDK1 ELISA (Figure 4B).

Although the dose-response curves did not permit the

determination of EC₅₀values, AZD1775, 28, and 29 demonstrated

comparable effects on pCDK1 levels within the concentration

range of the assay. This is unsurprising given the similar IC₅₀

values for these compounds from the TR-FRET assay and that

both 28 and 29 were the most potent of the novel compounds in

the MTS assay in both ONS-76 and Daoy cells. However, in the

MTS and CellTox assays using Daoy cells, the effect of 28 and

29 on cell viability and cytotoxicity were significantly reduced

when compared to AZD1775 supporting that the inhibition of

cellular WEE1 is not directly responsible for the potent cytotoxic

effects of AZD1775. Although these compounds were not

designed to address this observation, these findings for

compounds 28, 29, and moreover 31 corroborate our previous

research, where we first proposed that WEE1 inhibtion is

uncoupled from the potent cytotoxic effects of AZD1775.^[12]

A

B

C

Figure 3. Effect of single-agent WEE1 inhibitors on cell viability and

cytotoxicity. (A) Dose-response for ONS-76 cells treated with a concentration

range of WEE1 inhibitors for 72 hours in an MTS assay. AZD1775 (red) inhibits

cellular metabolic viability most potently (EC₅₀= 159 ± 31 nM), with 29 (orange,

EC₅₀= 203 ± 40 nM) and 28 (green, EC₅₀= 252 ± 9 nM) displaying somewhat

comparable results. Limited effects were observed for other inhibitors within the

series (n=3, error bars/± = S.D.). (B) Dose-response for Daoy cells treated with

a concentration range of WEE1 inhibitors for 72 hours in an MTS assay.

AZD1775 (red, EC₅₀= 179 ± 16 nM) exhibits significantly more potent effects on

cellular metabolic viability than 29 (orange, 81% activity at 600 nM) and 28

(green, 84% activity at 600 nM). No other analogs displayed any reduction in

MTS signal up to 600 nM inhibitor dose (n=3, error bars/± = S.D.). (C) Dose-

response for Daoy cells treated with a concentration range of WEE1 inhibitors

for 72 hours in a CellTox green cytotoxicity assay. AZD1775 (red, EC₅₀= 188 ±

26 nM) exhibits significantly more potent cytotoxic effects than 28 (green, EC₅₀

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allyl group at the pyrazole N²position in order to engineer out

potential metabolic liabilities; however, substitution of this group

may be limited. A significant loss of WEE1 inhibitory potency was

observed with methyl N²substitution (32; IC₅₀= 19.9 ± 3.8 nM)

and complete loss of activity with removal of the N²substitution

altogether (35; IC₅₀= 118 ± 28 nM), likely due to the loss in

hydrophobic interactions and electronic effects on the pyrazole

ring (Supporting Information Figure S1). The WEE1-AZD1775 co-

crystal structure indicates that the allyl group may have a

hydrophobic contact with Lys328.^[11c]Further compounds may

need to be developed to more thoroughly investigate the scope of

replacing the allyl group to include substituents that maintain the

electronic distribution of negative electrostatic and hydrophobic

fields and that can be accommodated in the small hydrophobic

pocket in the ATP-binding site of WEE1.

A

B

A

B

Figure 4. WEE1 inhibitor effects on cellular pCDK1(Tyr15) concentrations.

(A) Quantitative ELISA determination of pCDK1(Tyr15) levels in Daoy cell

lysates (0.05 mg/mL total protein) after treatment for 24 hours with a single 220

nM dose of all active WEE1 inhibitors. A decrease in pCDK1 concentration when

compared to DMSO control was observed for AZD1775, 28 and 29, whereas

the decrease in pCDK1 upon treatment with 31 was statistically the same as

that for AZD1775 (n=3, error bars = S.D., compared with DMSO; * = p<0.05, **

= p<0.01, *** = p<0.001; compared to AZD1775; n.s. = no significance). (B)

pCDK1(Tyr15) ELISA dose response for Daoy cell lysates (0.05 mg/mL total

protein) after treatment for 24 hours with AZD1775 (red), 28 (green), 29 (orange)

and 31 (blue) (n=3, error bars/± = S.D.).

Figure 5. Predicted WEE1-compound 28 interactions. (A) Ribbon

representation (blue ribbon, grey amino acid residues; with clipped pane

molecular surface colored dark grey) of the ATP-binding site of WEE1 displaying

the predicted binding mode of 28 (orange). Red dashed lines indicate H-bonds

and yellow dashed lines indicate π-π interactions. (B) Ligand interaction

diagram of the predicted binding mode of 28 in the ATP-binding site of WEE1.

Red residues are charged negative, purple residues are charged positive,

hydrophobic residues are hydrophobic, and blue resiues are polar, purple

arrows indicate H-bonds, green lines indicate π-π interactions, and grey circles

indicate areas of solvent exposure.

Discussion

In this manuscript, we have described

a series of

pyrazolopyrimidinones to further our understanding of the SAR for

WEE1 inhibition. The evaluation of this series of

pyrazolopyrimidinones against WEE1 kinase activity has allowed

us to evaluate our computational model as a basis for WEE1

inhibitor design. We have identified several compounds bearing

modification of the aniline substituent that resulted in equipotency

with AZD1775 in terms of inhibition of WEE1 kinase activity.

These data support our previous findings,^[12]that the N-

methylpiperazine of AZD1775 is amenable to modification.

Furthermore, we have identified compound 28 that is a more

potent inhibitor of WEE1 kinase activity compared to AZD1775

(IC₅₀= 1.7 ± 0.9 nM versus IC₅₀= 5.1 ± 0.9 nM). From the

computational-based molecular docking of compound 28 into the

ATP-binding site of WEE1 we found that the additional flexibility

in the alkylamino side chain allowed improved interaction with

D386, and this may be responsible for the observed improvement

in inhibitory activity (Figure 5). We proposed replacement of the

Following the testing protocol that we have previously

reported,^[12]compounds that showed activity in the in vitro WEE1

kinase activity assay were tested as single agents in both ONS-

76 (p53 wild-type) and Daoy (p53 mutant) cells to determine their

effect on cell viability. Similarly, to what we have previously

reported, there was a disparity between WEE1 inhibitory potency

and the effect on cell viability in an MTS assay. Only the WEE1

inhibitors 28 and 29 possessed an effect on cellular viability over

the concentration range examined, and even these effects were

less potent than AZD1775, particularly in Daoy cells. This is

interesting observation, as it would be assumed that the effect of

WEE1 inhibitors would not only be more potent in Daoy cells due

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to the mutant status of p53, but that the cellular effects of WEE1

inhibitors as single agents would more closely correlate with

inhibitory potency. We also examined the cytotoxic effects of the

WEE1 inhibitors on Daoy cells using the CellTox assay. The

cytotoxicity of the WEE1 inhibitors correlated with their effects on

cell viability by MTS assay. AZD1775 demonstrated the most

potent single-agent cytotoxic effects within 24 hours and over the

72 hour treatment period, supporting our previous data using

xCELLigence Real-Time Cell Analysis.^[12]The WEE1 inhibitor 28

displayed significantly reduced cytotoxicity compared with

AZD1775, and 29 and 31 had negligible cytotoxic efffects over the

72 hour treatment period.

less potent compared with WEE1 (IC₅₀= 5 nM), and 2- to 3-fold

less potent against the proto-oncogene Src family tyrosine kinase

YES1 (IC₅₀= 14 nM). The identity of the other kinases from this

profile screen have yet to be reported; however, it was stated in

the same study that AZD1775 showed >100-fold selectivity for

WEE1 over MYT1, but since WEE1 has only a ~30% sequence

identity with MYT1 this difference in selectivity was not surprising.

A kinome interaction network study identified ABL1, LCK, LRRK2,

TNK2, and SYK as targets of AZD1775 with Ki values below 1

μM.^[11a]Recently, it has been reported that AZD1775 has potent

nanomolar activity against PLK1, and that AZD1775 should now

be considered a dual WEE1 and PLK1 inhibitor.^{[11b, 11c]}A full

kinome profile for AZD1775 across a panel of 468 human kinases

at a single-agent concentration of 500 nM identified PLK1, FLT3

(D835V), JAK3, GCN2 (S808G), ABL1 (H396P), and JAK2 as

additional kinase targets.^[11c]Furthermore, the authors suggested

that WEE1 and PLK1 were the most relevant targets for mediating

In order to determine the effect of compounds on cellular

WEE1 activity, Daoy cell extracts were probed for pCDK1(Tyr15)

concentration in an ELISA assay. Compounds 28, 29, and 31

reduced cellular pCDK1 levels, and hence inhibited cellular WEE1

activity, to statistically comparable levels observed with AZD1775

treatment. Interestingly, both 24 and 25 increased cellular pCDK1, the anticancer effects of AZD1775; however, our studies support

suggesting that treatment with these compounds results in

activation of the DDR, either through direct compound mediated

DNA-damage or through effects on other members of the

CHK1/WEE1 pathway. This observation may warrant further

investigation to more thoroughly determine the significance of this

effect and the mode of action, as any compounds within this class

possessing such effects would be highly undesirable. The dose-

response effects of AZD1775 and compounds 28, 29, and 31 on

pCDK levels in Daoy cells were similar, despite less comparable

effects on cell viabilityand cytotoxicity. We have previously shown

that compounds, such as CM-061 (Figure 2, 1), have little effect

on cell viability as single agents, but can inhibit cellular WEE1

activity and this accompanies its ability to synergize with DNA-

damaging agents such as cisplatin.^[12]Therefore, despite

compounds 28, 29, and 31 exhibiting reduced inhibition of cell

viability and cytotoxicity as single agents, these compounds are

more suitable for use as potentiators of DNA-damaging agent

chemotherapy. These findings may be of critical importance in

evaluating WEE1 inhibition as a therapeutic strategy as the

toxicity of AZD1775 may exacerbate therapy-related adverse

effects in patients with medulloblastoma and other cancers.

Prior to our initial study,^[12]there was limited SAR data for

AZD1775 in the context of WEE1 inhibition. A screening program

conducted by Bayu Pharmaceutical Co., that focused on the

identification of WEE1 inhibitors from a small chemical compound

library led to the discovery of AZD1775.^[6]In a kinase profile of

223 kinases, AZD1775 demonstrated >80% inhibition of 8 other

kinases at 1.0 µM in an in vitro radiolabeled ATP kinase assay.^[6]

The IC₅₀values determined for 7 of these kinases were 10-fold

that WEE1 inhibition can be achieved with little impact on

cytotoxicity. Therefore, we examined the effect of AZD1775 and

the WEE1 inhibitors 28, 29, and 31 on PLK1 activity in a TR-FRET

assay (Supporting Information Table S1). AZD1775 was the most

potent PLK1 inhibitor in this assay (IC₅₀77.6 ± 9.3 nM), followed

by 29 (IC₅₀161 ± 10 nM), 28 (IC₅₀270 ± 29 nM), and then 31 (IC₅₀

394 ± 37 nM); however, the ranking of PLK1 inhibitory potency by

these compounds does not correlate with the rank order of

cytotoxicity and it is unknown, but unlikely that a 3.5 to 5-fold

difference in PLK1 inhibition would significantly impact cytotoxicity.

Overall, kinome profiling studies support that AZD1775 has

narrow-spectrum activity and considering it interacts with an ATP-

binding site its kinome selectivity profile is amirable; however,

what is concerning is the potent single-agent cytotoxicity of

AZD1775. A recent study presented data supporting our findings

that AZD1775 induces apoptosis independent of WEE1 inhibition,

and demonstrated potent single-agent effects of AZD1775 on cell

viability in a panel of NSCLC cells.^[11b]In order to identify the

molecular targets of AZD1775 they developed an immobilized

analog, termed i-AZD1775, to conduct a proteome-wide search of

H322 cells and confirmed PLK1 as a AZD1775 target. A short-

coming of this immobilized analog approach was that the effect of

i-AZD1775 on cell viability was not determined. i-AZD1775 has an

alkyl amino linker from the N-piperazine of AZD1775 and may not

interact with the molecular target(s) that results in the potent

cytotoxicity observed with AZD1775, as our previous work has

shown that substitution of the N-piperazine reduces

cytotoxicity.^[12]Based on the results from the kinome profiling

studies, we would propose that the potent cytotoxicity of AZD1775

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is due to a combination of interactions within the kinome and

molecular targets outside of the kinome and this is the focus of

on-going investigations.

carried out on an API 4000. Fourier Transform Infrared (FTIR)

spectra were obtained using a Bruker Alpha Platinum-ATR as a

neat sample.

General procedure for the synthesis of substituted

pyrazolopyrimidinones

Conclusions

In summary, we have developed WEE1 inhibitors based on

the pyrazolopyrimidinone core of AZD1775 and, combined with

our previous results, we definitively show, principally with

compound 31, that the potent cytotoxicity of AZD1775 can be

uncoupled from WEE1 inhibition. Furthermore, we have identified

compound 28 as a more potent inhibitor of WEE1 than AZD1775,

that demonstrates WEE1 inhibition both in vitro and in cells, but

has a reduced single-agent effect on cell viability and cytotoxicity

compared to AZD1775. We present potent WEE1 inhibitors,

compounds 28, 29, and 31, with limited cytotoxic activity that we

are transitioning into advanced preclinical evaluation. The

identification of potent WEE1 inhibitors with diminished single-

agent cytotoxic effects is an important feature when developing

chemosensitization agents, as this may allow for the safe

administration of WEE1 inhibitor and dose reductions of DNA-

damaging agents while maintaining clinical efficacy and reducing

therapy-related adverse effects.

Detailed synthetic methods and compound characterization for all

compounds are provided in Supporting Information.

General Procedure for the alkylation of tert-butyl (1,3-

dioxoisoindolin-2-yl)carbamate (4-6). To a suspension of the

carbamate (3) (1.0 equiv.) in MeCN (2 mL/mmol) was added

benzyltriethylammonium chloride (0.1-0.2 equiv.), K₂CO₃(4.0

equiv.) and the relevant alkylhalide (1.5-5.0 equiv.) sequentially.

The reaction mixture was stirred at RT or 50°C for 18-48 h, before

water (2 mL/mmol) was added and the organic phase was

extracted with Et₂O (2 x 5 mL/mmol). The combined organic

extracts were dried (MgSO₄) and evaporated to dryness and were

purified by chromatography on silica.

General procedure for the removal of phthlalimide protecting

groups (7-9). Methylhydrazine (1.25 equiv.) was added to an ice

cooled solution of phthalimide 4-6 (1.0 equiv.) in THF (2 mL/mmol).

The reaction mixture was allowed to warm to RT and was stirred

for 18 h. The resultant suspension was passed through a filter,

and the filtrate was concentrated in vacuo. A mixture of

Hexanes:EtOAc (3:1, 1 mL/mmol) was added, and the precipitate

formed was removed via filtration. This process was repeated a

further 2 times, and the final filtrate was concentrated to give the

target compound.

Experimental Section

Chemistry

General: Where appropriate, reactions were carried out under an

inert atmosphere of nitrogen with dry solvents, unless otherwise

stated. Dry solvents were obtained directly from manufacturer and

used

without

further

purification.

Yields

refer

to

General

procedure

for

the

synthesis

of

chromatographically and spectroscopically pure isolated yields.

Reagents were purchased at the highest commercial-quality and

used without further purification, unless otherwise stated.

Reactions were monitored by thin-layer chromatography carried

out on 0.25 mm E. Merck silica gel plates (60 F254) using

ultraviolet light as visualizing agent. All melting points were

determined using a Stuart Scientific SMP40 melting point

apparatus and are uncorrected. ¹H and ¹³C nuclear magnetic

resonance (NMR) spectra were obtained as solutions in

deuterated solvents DMSO-d₆or CDCl₃using a Bruker Avance III

400 spectrometer recording at 400 MHz. 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), sept (septet), m (multiplet), or br (broad), with coupling

constants (J) given in Hertz (Hz). Mass Spectrometry (MS) was

pyrazolopyrimidinones (11-13). DIPEA (2.5 equiv.) and the

relevant hydrazine 7-9 (1.05 equiv.) were added to a solution of

ethyl 4-chloro-2-methylthio-5-pyrimidinecarboxylate (10; 1.0

equiv.) in THF (3 mL/mmol). The reaction mixture was heated at

reflux for 72 h, before being concentrated in vacuo. Et₂O (1

mL/mmol) was added to the residue, and the resultant precipitate

was collected by filtration. The filtrate was evaporated to dryness,

and the residue was cooled in an ice bath, after which TFA (1

mL/mmol) was added. The resultant solution was stirred at RT for

1 h, followed by 70°C for 1 h. The solvent was removed in vacuo

and the residue was dissolved in EtOH (1 mL/mmol) and cooled

in an ice bath, after which 6M NaOH (2 mL/mmol) was added. The

resultant solution was stirred at RT for 15 min, before being

acidified (pH 3) via the addition of conc. HCl. The solution was

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evaporated to dryness and the resultant residue was partitioned Hexanes:EtOAc); M.p. 118-120°C (Lit. = 123°C);^[15]IR (cm^-1) 2972,

between chloroform (2 mL/mmol) and water (2 mL/mmol), and the 2934, 1791, 1723, 1609; ¹H NMR (400 MHz, CDCl₃) 1.34 (5.1H,

organic phase was washed with brine (1 mL/mmol), dried s, C(CH₃)_3-major), 1.53 (3.9H, s, C(CH₃)_3-minor), 3.29 (1.7H, s, N-CH_3-

(MgSO₄), and concentrated in vacuo to afford the target _major), 3.32 (1.3H, s, N-CH_3-minor), 7.74-7.93 (4H, m, H-4/5/6/7); ¹³

compound. NMR (100 MHz, CDCl₃) 27.9 (C(CH₃)_3-major), 28.1(C(CH₃)_3-minor),

36.5 (N-CH_3-major), 38.1 (N-CH_3-minor), 82.2 (C(CH₃)_3-major), 82.9

C

General procedure for the preparation of pyridyl (C(CH₃)_3-minor), 123.8 (Ar-C), 129.9 (Ar-C), 130.1 (Ar-C), 134.6 (Ar-

pyrazolopyrimidinones (17-21). N,N’-Dimethylethylenediamine C), 134.7 (Ar-C), 153.6 (C=O_-major), 153.8 (C=O_-minor), 165.0 (C=O_-

(2.0 equiv.) was added to

a

solution of the relevant _major), 165.3 (C=O_-minor); MS [M+H]⁺m/z 276.8.

pyrazolopyrimidinone 11-13 (1.0 equiv.), the relevant

bromopyridine (14-16, 1.3 equiv.), copper iodide (1.0 equiv.) and Synthesis

of

tert-butyl

allyl(1,3-dioxoisoindolin-2-

K₂CO₃(1.4 equiv.) in 1,4-dioxane (2 mL/mmol) at 80°C. The yl)carbamate (5). The desired product was obtained as a white

resultant suspension was heated at 95°C for 18 h, over which time crystalline solid (15.7 g, 52.1 mmol, 85%) with no further

a color change of orange to dark green occurred. The reaction purification needed. Rf 0.52 (4:1 Hexane:EtOAc); M.p. 72-75°C

mixture was cooled to RT and diluted with NH₄OH (10 mL/mmol) (Lit. = 76-78°C);^[15]IR (cm^-1) 2978, 2936, 1792, 1719, 1641; ¹H

before being extracted with EtOAc (2 x 10 mL/mmol). The NMR (400 MHz, DMSO-d₆) 1.25 & 1.46 (9H, s, C(CH₃)₃), 4.19 (2H,

combined organic extracts were washed with brine (10 mL/mmol), d_app, J = 6.1 Hz, N-CH₂), 5.10-5.17 (1H, m, allyl C-H^trans), 5.27 (1H,

dried (MgSO₄) and evaporated to dryness before the crude dd, J = 17.3, 1.3 Hz, allyl C-H^cis), 5.78-5.93 (1H, m, allyl C-H),

material was purified via chromatography on silica.

7.93-8.02 (4H, m, H-4/5/6/7); ¹³C NMR (100 MHz, DMSO-d₆) 27.9

(C(CH₃)_3-major), 28.1 (C(CH₃)_3-minor), 51.7 (N-CH_2-major), 53.7 (N-

General procedure for the preparation of aniline pyridyl CH_2-minor), 82.1 (C(CH₃)_3-major), 82.8 (C(CH₃)_3-minor), 119.1 (allyl-

pyrazolopyrimidinones (22-33). mCPBA (1.1 equiv.) was added CH_2-major), 119.7 (allyl-CH_2-minor), 124.3, 124.4, 129.5, 129.6, 132.8

to a solution of pyrazolopyrimidinones 17-21 (1.0 equiv.) in (Ar-C), 133.3 (Ar-C), 135.9 (Ar-C), 136.0 (Ar-C), 153.0 (C=O_-major),

toluene (10 mL/mmol) and the resulting mixture was stirred at RT 153.1 (C=O_-minor), 165.3 (C=O_-major), 165.5 (C=O_-minor).

for 1 h. DIPEA (5.2 equiv.) and the relevant substituted aniline 39-

43 or amine (1.3 equiv.) were added, and the reaction mixture was Synthesis

of

tert-butyl

(1,3-dioxoisoindolin-2-yl)(4-

stirred at RT for 18 h. Saturated NaHCO₃solution (15 mL/mmol) methoxybenzyl)carbamate (6). The desired product was

was added, and the mixture was extracted with EtOAc (2 x 20 obtained as a pale orange solid observed to be a pair of rotamers

mL/mmol). The combined organic extracts were washed with by NMR (65 mg, 0.17 mmol, 89%). Rf 0.42 (3:1 Hexanes:EtOAc);

brine (5 mL/mmol), dried (MgSO₄) and concentrated in vacuo. M.p. 106-108°C; IR (cm^-1) 3003, 2979, 2962, 2934, 2836, 1793,

The resultant residues were purified via chromatography on silica 1737, 1715, 1610, 1511; ¹H NMR (400 MHz, CDCl₃) 1.37 (5.4H,

to give the target compounds (12-89%).

s, C(CH₃)_3-major), 1.55 (3.6H, s, C(CH₃)_3-minor), 3.77 (1.8H, s, OCH_3-

_major), 3.78 (1.2H, s, OCH_3-minor), 4.80 (0.8H, s, benzyl CH_2-minor),

4.83 (1.2H, s, benzyl CH_2-major), 6.82 (2H, dd, J = 10.0, 8.5 Hz, H-

4/7), 7.31 (2H, dd, J = 10.0, 8.5 Hz, H-5/6), 7.72-7.77 (2H, m,

Compound characterization

Synthesis of tert-butyl (1,3-dioxoisoindolin-2-yl)carbamate benzyl H-3/5), 7.80-7.86 (2H, m, benzyl H-2/6); ¹³C NMR (100

(3). The desired product was obtained as a white crystalline solid MHz, CDCl₃) 27.9 (C(CH₃)_3-major), 28.2 (C(CH₃)_3-minor), 52.0

(16.1 g, 61.4 mmol, 91%). Rf 0.68 (1:1 Hexane:EtOAc); M.p. 191- (benzyl-CH_2-major), 53.9 (benzyl-CH_2-minor), 55.2 (OCH₃), 82.4

194°C (Lit. = 186°C);^[15]IR (cm^-1) 3316, 2979, 1796, 1730, 1614, (C(CH₃)_3-major), 83.2 (C(CH₃)_3-minor), 113.7 (Ar-C), 123.7 (Ar-C),

1490; ¹H NMR (400 MHz, DMSO-d₆) 1.45 (9H, s, -OC(CH₃)₃), 127.1 (Ar-C), 129.8 (Ar-C), 130.0 (Ar-C), 130.5 (Ar-C), 134.5 (Ar-

7.87-8.04 (4H, m, H-4/5/6/7), 9.86 (1H, s, NH); ¹³C NMR (100 C), 153.5 (C=O_minor), 159.3 (C=O_major), 165.0 (C=O_major), 165.4

MHz, DMSO-d₆) 28.3 (C(CH₃)₃), 81.6 (C(CH₃)₃), 124.2 (Ar-C), (C=O_minor); MS [M+NH₄]⁺m/z 400.2.

129.8 (Ar-C), 135.8 (Ar-C), 154.4 (C=O), 165.9 (C=O).

Synthesis of tert-butyl 1-methylhydrazine-1-carboxylate (7).

Synthesis

of

tert-butyl

(1,3-dioxoisoindolin-2- The desired product was obtained as a pale-yellow oil (0.338 g,

yl)(methyl)carbamate (4). The desired product was obtained as 2.31 mmol, 77%). Rf 0.20 (1:1 Hexanes:EtOAc); IR (cm^-1) 3247,

a white crystalline solid (93 mg, 0.34 mmol, 89%). Rf 0.38 (1:1

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2924, 2854, 1697, 1640, 1568; ¹H NMR (400 MHz, CDCl₃) 1.47

1609, 1577, 1510;¹H NMR (400 MHz, CDCl₃) 2.53 (3H, s, SCH₃),

3.78 (3H, s, OCH₃), 5.03 (2H, s, N²-CH₂), 6.84 (2H, d, J = 8.5 Hz,

benzyl H-3/5), 7.28 (2H, d, J = 8.5 Hz, benzyl H-2/6), 8.66 (1H, s,

H-4); ¹³C NMR (100 MHz, DMSO-d₆) 13.8 (SCH₃), 46.8 (N²-CH₂),

55.5 (OCH₃), 103.7, 114.0, 114.1, 114.3, 129.5, 130.5, 158.3,

159.1; MS [M+H]⁺m/z 303.2.

(9H, s, C(CH₃)₃), 3.05 (3H, s, N-CH₃), 4.10 (2H, br s, NH₂).

Synthesis of tert-butyl 1-allylhydrazine-1-carboxylate (8). The

desired product was obtained as a pale-yellow oil (8.47 g, 49.2

mmol, 96%). Rf 0.22 (4:1 Hexane:EtOAc); IR (cm^-1) 3336, 2977,

2932, 1690;¹H NMR (400 MHz, DMSO-d₆) 1.40 (9H, s, -C(CH₃)₃),

3.85 (2H, ddd, J = 5.5, 1.4, 1.4 Hz, N-CH₂), 4.46 (2H, s, NH₂),

5.06-5.09 (1H, m, allyl C-H^trans), 5.11 (1H, br, allyl C-H^cis), 5.74-

5.86 (1H, m, allyl C-H); ¹³C NMR (125 MHz, DMSO-d₆) 28.5

(C(CH₃)₃), 53.6 (N-CH₂), 79.4 (C(CH₃)₃), 116.2 (allyl-CH₂), 134.6

(allyl-CH), 156.5 (C=O).

Synthesis of 2-(6-bromopyridin-2-yl)propan-2-ol (14). The

desired product was obtained as a yellow oil (0.365 g, 1.69 mmol,

85%). Rf 0.60 (1:1 Hexane:EtOAc); IR (cm^-1) 3420, 2975, 2930,

1731, 1701, 1580, 1553;¹H NMR (400 MHz, DMSO-d₆) 1.42 (6H,

s, C(CH₂)₂), 5.33 (1H, s, OH), 7.47 (1H, dd, J = 7.7, 0.9 Hz, H-5),

7.67 (1H, dd, J = 7.7, 0.9 Hz, H-3), 7.73 (1H, dd, J = 7.7, 7.7 Hz,

H-4); ¹³C NMR (100 MHz, DMSO-d₆) 30.9 (C(CH₂)₂), 72.6

(C(CH₂)₂), 118.5 (Ar-C), 126.0 (Ar-C), 140.4 (Ar-C), 140.5 (Ar-C),

170.8 (Ar-C).

Synthesis of tert-butyl 1-(4-methoxybenzyl)hydrazine-1-

carboxylate (9). The desired product was obtained as a pale

yellow oil (0.275 g, 1.09 mmol, 76%). Rf 0.24 (4:1

Hexane:EtOAc); IR (cm^-1) 3336, 2975, 2933, 2836, 1688, 1612,

1511;¹H NMR (400 MHz, CDCl₃) 1.51 (9H, s, C(CH₃)₃), 3.81 (3H,

s, OCH₃), 4.04 (2H, br s, NH₂), 4.50 (2H, s, N-CH₂), 6.88 (2H, d,

J = 8.4 Hz, H-3/5), 7.24 (2H, d, J = 8.4 Hz, H-2/6); ¹³C NMR (100

MHz, CDCl₃) 28.5 (C(CH₃)₃), 53.7 (OCH₃), 55.3 (NCH₂), 80.7

(C(CH₃)₃), 113.9 (Ar-C), 129.3 (Ar-C), 130.0 (Ar-C), 156.8 (Ar-C),

159.0 (C=O); MS [M+H]⁺m/z 253.2.

Synthesis

of

1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-2-

methyl-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-

d]pyrimidin-3-one (17). The desired product was obtained as a

white solid (0.215 g, 0.65 mmol, 72%). Rf 0.34 (19:1

DCM:MeOH); M.p. 155-158°C; IR (cm^-1) 3432, 2973, 2928, 1683,

1604, 1562; ¹H NMR (400 MHz, DMSO-d₆) 1.46 (6H, s, C(CH₃)₂),

2.56 (3H, s, SCH₃), 3.49 (3H, s, N²-CH₃), 5.35 (1H, s, OH), 7.67

(1H, d_app, J = 7.7 Hz, H-5’), 7.79 (1H, d_app, J = 8.2 Hz, H-3’), 8.06

(1H, dd_app, J = 8.2, 7.7 Hz, H-4’), 9.00 (1H, s, H-4); ¹³C NMR (100

MHz, DMSO-d₆) 14.4 (SCH₃), 30.9 (C(CH₃)₂), 32.8 (N²-CH₃), 72.8

(C(CH₃)₂), 104.8, 116.6, 117.5, 139.7, 146.8, 154.7, 158.3, 160.4,

168.4, 175.9; MS [M+H]⁺m/z 332.6.

Synthesis

of

1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-2-

methyl-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-

d]pyrimidin-3-one (11). The desired product was obtained as a

yellow solid (0.302 g, 1.54 mmol, 75%). Rf 0.23 (4:1 DCM:MeOH);

M.p. 256-265°C (decomposed); IR (cm^-1) 3336, 3024, 2940, 1683,

1638, 1587; ¹H NMR (400 MHz, DMSO-d₆) 2.53 (3H, s, SCH₃),

3.36 (3H, s, N²-CH₃), 8.68 (1H, s, H-4), 12.60 (1H, br s, N¹-H); ¹³

C

Synthesis of 2-allyl-1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-

6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-

one (18). The desired product was obtained as a white solid

(0.653 g, 1.82 mmol, 81%). Rf 0.63 (9:1 DCM:MeOH); M.p. 108-

111°C; IR (cm^-1) 3337, 3081, 2966, 2924, 1663, 1601, 1559; ¹H

NMR (400 MHz, CDCl₃) 1.61 (6H, s, C(CH₃)₂), 2.61 (3H, s, S-CH₃),

3.77 (1H, s, OH), 4.82 (2H, d_app, J = 5.9 Hz, N²-CH₂), 4.95 (1H,

d_app, J = 16.9 Hz, alkene C-H^trans), 5.08 (1H, d_app, J = 10.3 Hz,

alkene C-H^cis), 5.72 (1H, ddt, J = 16.9, 10.3, 5.9 Hz, alkene C-H),

7.42 (1H, d_app, J = 7.7 Hz, H-5'), 7.78 (1H, d_app, J = 8.0 Hz, H-3'),

7.93 (1H, dd, J = 8.0, 7.7 Hz, H-4'), 8.96 (1H, s, H-4); ¹³C NMR

(100 MHz, CDCl₃) 14.5 (SCH₃), 30.5 (C(CH₃)₂), 47.5 (N²-CH₂),

72.5 (C(CH₃)₂), 116.4 (Ar-C), 116.6 (Ar-C), 119.3 (allyl-CH₂),

131.2, 139.2, 147.0 (Ar-C), 154.3 (Ar-C), 159.2 (C=O), 161.0 (Ar-

C), 166.1 (Ar-C), 177.0 (Ar-C); MS [M+H]⁺m/z 359.3.

NMR (100 MHz, DMSO-d₆) 13.9 (SCH₃), 31.0 (N²-CH₃), 103.8,

158.1; MS [M+H]⁺m/z 196.8.

Synthesis

of

2-allyl-6-(methylthio)-1,2-dihydro-3H-

pyrazolo[3,4-d]pyrimidin-3-one (12). The desired product was

obtained as a yellow solid (5.44 g, 24.5 mmol, 51%). Rf 0.45 (9:1

DCM:MeOH); M.p. 125-128°C; IR (cm^-1) 3032, 2979, 2926, 2659,

1656, 1615, 1566, 1514;¹H NMR (400 MHz, DMSO-d₆) 2.53 (3H,

s, SCH₃), 4.38 (2H, d_app, J = 5.2 Hz, N²-CH₂), 5.06-5.20 (2H, m,

allyl C-H^cis/trans), 5.87 (1H, ddt, J = 17.2, 10.5, 5.3 Hz, alkene C-H),

8.67 (1H, s, H-4), 12.65 (1H, br, N¹-H); MS [M+H]⁺m/z 223.1.

Synthesis

of

2-(4-methoxybenzyl)-6-(methylthio)-1,2-

dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one (13). The desired

product was obtained as a yellow solid (0.142 g, 0.47 mmol, 50%).

Rf 0.41 (9:1 DCM:MeOH); M.p. 209-212°C; IR (cm^-1) 3034, 2930,

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Synthesis of 2-allyl-1-(3-(2-hydroxypropan-2-yl)phenyl)-6-

(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one

(19). The desired product was obtained as a colorless oil (0.245

g, 0.68 mmol, 76%). Rf 0.26 (19:1 DCM:MeOH); IR (cm^-1) 3400,

3077, 2973, 2928, 2871, 1676, 1594, 1561; ¹H NMR (400 MHz,

(6H, s, N(CH₃)₂), 3.53 (2H, s, ArCH₂), 7.51 (1H, dd, J = 8.0, 7.9

Hz, H-5), 7.68 (1H, d_app, J = 7.9 Hz, H-6), 8.13 (1H, dd, J = 8.0,

2.0 Hz, H-4), 8.21 (1H, s_app, H-2); ¹³C NMR (100 MHz, CDCl₃) 45.4

(N(CH₃)₂), 63.4 (NCH₂), 122.2 (Ar-C), 123.7 (Ar-C), 129.2 (Ar-C),

135.0 (Ar-C), 141.4 (Ar-C), 148.4 (Ar-C).

CDCl₃) 1.64 (6H, s, C(CH₃)₂), 2.51 (3H, s, SCH₃), 4.45 (2H, d_app

,

J = 6.0 Hz, N2-CH₂), 4.99 (1H, d_app, J = 17.0, allyl C-H^trans), 5.14

(1H, d_app, J = 10.2 Hz, allyl C-H^cis), 5.71 (1H, ddt, J = 17.0, 10.2,

6.0 Hz, allyl C-H), 7.29 (1H, d_app, J = 7.2 Hz, H-6’), 7.47-7.56 (2H,

m, H-4’/5’), 7.58 (1H, s_app, H-2’), 8.92 (1H, s, H-4); ¹³C NMR (100

MHz, CDCl₃) 14.3 (SCH₃), 31.9 (C(CH₃)₂), 46.2 (N²-CH₂), 72.3

(C(CH₃)₂), 104.1, 119.4, 121.5, 123.2, 124.3, 129.3, 130.8, 135.4,

151.1, 154.3, 160.4, 161.6, 177.0; MS [M+H]⁺m/z 357.2.

Synthesis of N,N-dimethyl-3-nitrobenzamide (37). The desired

product was obtained as a pale-yellow oil/low-melting solid (0.468

g, 2.41 mmol, 81%). Rf 0.23 (1:1 Hexanes:EtOAc); IR (cm^-1) 3081,

3027, 2929, 2869, 1625, 1527; ¹H NMR (400 MHz, CDCl₃) 3.01

(3H, s, NCH₃), 3.14 (3H, s, NCH₃), 7.62 (1H, dd_app, J = 8.0, 7.8 Hz,

H-5), 7.77 (1H, ddd, J = 7.8, 1.3, 1.2 Hz, H-6), 8.24-8.29 (2H, m,

H-2/4); ¹³C NMR (100 MHz, CDCl₃) 35.5 (NCH₃), 39.5 (NCH₃),

112.3 (Ar-C), 124.4 (Ar-C), 129.7 (Ar-C), 133.1 (Ar-C), 137.9 (Ar-

C), 148.0 (Ar-C), 168.9 (C=O).

Synthesis

of

2-allyl-1-(6-(hydroxymethyl)pyridin-2-yl)-6-

(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one

(20). The desired product was obtained as a white solid (0.201 g,

0.61 mmol, 68%). Rf 0.23 (19:1 DCM:MeOH); M.p. 105-107°C; IR

(cm^-1) 3361, 3239, 2924, 2838, 1695, 1666, 1590, 1559; ¹H NMR

(400 MHz, CDCl₃) 2.60 (3H, s, SCH₃), 3.04 (1H, br, OH), 4.76-

4.86 (4H, m, N²-CH₂/CH₂OH), 4.97 (1H, d_app, J = 17.1, allyl C-

H^trans), 5.09 (1H, d_app, J = 10.3 Hz, allyl C-H^cis), 5.73 (1H, ddt, J =

17.1, 10.3, 6.2 Hz, allyl C-H), 7.30 (1H, d_app, J = 8.0 Hz, H-5’), 7.80

(1H, d_app, J = 8.1 Hz, H-3’), 7.92 (1H, dd_app, J = 8.1, 8.0 Hz, H-4’),

8.96 (1H, s, H-4); ¹³C NMR (100 MHz, CDCl₃) 14.5 (SCH₃), 47.5

(N²-CH₂), 64.4 (CH₂OH), 104.5, 117.0, 118.4, 119.3, 131.2, 138.9,

147.8, 154.3, 159.2, 161.0, 177.0; MS [M+H]⁺m/z 330.0.

Synthesis of N,N-dimethyl-2-(4-nitrophenoxy)ethan-1-amine

(38). The desired product was obtained as a yellow oil (0.860 g,

4.09, 100%). Rf 0.27 (19:1 DCM:MeOH); IR (cm^-1) 3114, 3084,

2945, 2824, 2774, 1737, 1591, 1508; ¹H NMR (400 MHz, CDCl₃)

2.36 (6H, s, N(CH₃)₂), 2.78 (2H, t, J = 5.6 Hz, OCH₂CH₂), 4.17

(2H, t, J = 5.6 Hz, OCH₂CH₂), 6.99 (2H, d, J = 9.2 Hz, H-2/6), 8.20

(2H, d, J = 9.2 Hz, H-3/5); ¹³C NMR (100 MHz, CDCl₃) 45.9

(N(CH₃)₂), 57.9 (OCH₂CH₂), 66.8 (OCH₂CH₂), 114.5 (Ar-C), 125.9

(Ar-C), 141.6 (Ar-C), 163.8 (Ar-C).

Synthesis of N¹,N¹-dimethylbenzene-1,3-diamine (39). The

desired product was obtained as a red oil (0.328 g, 2.40 mmol,

80%). Rf 0.44 (1:1 Hexanes:EtOAc); IR (cm^-1) 3343, 3220, 2878,

2800, 1606, 1579, 1501; ¹H NMR (400 MHz, CDCl₃) 2.94 (6H, s,

N(CH₃)₂), 3.62 (2H, br s, NH₂), 6.10-6.16 (2H, m, H-2/6), 6.24 (1H,

Synthesis of 1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-2-(4-

methoxybenzyl)-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-

d]pyrimidin-3-one (21). The desired product was obtained as an

off-white solid (84 mg, 0.19 mmol, 74%). Rf 0.26 (1:1

Hexanes:EtOAc); M.p. 143-145°C; IR (cm^-1) 3349, 2972, 2929,

2829, 1691, 1601, 1560; ¹H NMR (400 MHz, CDCl₃) 1.65 (6H, s,

C(CH₃)₂), 2.55 (3H, s, SCH₃), 3.73 (3H, s, OCH₃), 5.34 (2H, s, N²-

CH₂), 6.68 (2H, d, J = 8.4 Hz, benzyl H-3/5), 6.83 (2H, d, J = 8.4

dd, J = 8.1, 2.3 Hz, H-4), 7.07 (1H, dd, J = 8.1, 7.9 Hz, H-5); ¹³

C

NMR (100 MHz, CDCl₃) 40.6 (N(CH₃)₂), 99.6 (Ar-C), 103.8 (Ar-C),

104.3 (Ar-C), 129.9 (Ar-C), 147.3 (Ar-C), 151.9 (Ar-C).

Synthesis of 3-((dimethylamino)methyl)aniline (40). The

desired product was obtained as a pale red oil (0.347 g, 2.31

mmol, 72%). Rf 0.16 (19:1 DCM:MeOH); IR (cm^-1) 3270, 3147,

3079, 2974, 2942, 2858, 2816, 2774, 1666, 1610, 1552; ¹H NMR

(400 MHz, CDCl₃) 2.26 (6H, s, N(CH₃)₂), 3.35 (2H, s, ArCH₂), 3.65

(2H, br s, NH₂), 6.59-6.62 (1H, m, H-4), 6.69-6.72 (2H, m, H-2/6),

7.12 (1H, dd, J = 8.0, 7.9 Hz, H-5); ¹³C NMR (100 MHz, CDCl₃)

45.3 (N(CH₃)₂), 64.1 (ArCH₂), 118.9 (Ar-C), 120.5 (Ar-C), 125.0

(Ar-C), 128.9 (Ar-C), 138.1 (Ar-C), 139.6 (Ar-C).

Hz, benzyl H2/6), 7.44 (1H, d_app, J = 7.5 Hz, H-5’), 7.56 (1H, d_app

,

J = 8.0 Hz, H-3’), 7.87 (1H, dd_app, J = 8.0, 7.5 Hz, H-4’), 8.95 (1H,

s, H-4); ¹³C NMR (100 MHz, CDCl₃) 14.4 (SCH₃), 30.6 (C(CH₃)₂),

47.9 (N²-CH₂), 55.2 (OCH₃), 72.6 (C(CH₃)₂), 104.5, 114.0, 116.6,

127.3, 129.4, 139.2, 146.9, 154.3, 158.9, 159.4, 161.3, 166.1,

176.9; MS [M+H]⁺m/z 438.2.

Synthesis of N,N-dimethyl-1-(3-nitrophenyl)methanamine

(36). The desired product was obtained as a yellow oil (0.601 g,

3.34 mmol, 72%). Rf 0.28 (1:1 Hexanes:EtOAc); IR (cm^-1) 2976,

2944, 2859, 2820, 2774, 1523; ¹H NMR (400 MHz, CDCl₃) 2.28

Synthesis of 3-amino-N,N-dimethylbenzamide (41). The

desired product was obtained as an off-white solid (0.327 g, 1.99

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mmol, 85%). Rf 0.26 (19:1 DCM:MeOH); M.p. 87-89°C; IR (cm^-1)

3419, 3345, 3240, 2928, 2850, 1649, 1579; ¹H NMR (400 MHz,

CDCl₃) 2.99 (3H, s, NCH₃), 3.11 (3H, s, NCH₃), 3.76 (2H, br s,

NH₂), 6.68-6.80 (3H, m, H-2/4/6), 7.18 (1H, dd, J = 7.7, 7.6 Hz, H-

5); ¹³C NMR (100 MHz, CDCl₃) 35.2 (NCH₃), 39.5 (NCH₃), 113.5

(Ar-C), 116.0 (Ar-C), 116.9 (Ar-C), 129.2 (Ar-C), 137.5 (Ar-C),

146.6 (Ar-C), 171.8 (C=O).

pyrazolo[3,4-d]pyrimidin-3-one (AZD-1775). The desired target

compound was obtained as a yellow solid (87 mg, 0.17 mmol,

55%).Rf 0.25 (9:1 DCM:MeOH); M.p. 170-174°C; IR (cm^-1) 3420,

2969, 2810, 2364, 1639, 1602, 1541, 1512; ¹H NMR (400 MHz,

DMSO-d₆) 1.47 (6H, s, C(CH₃)₂), 2.23 (3H, s, N-CH₃), 2.42-2.50

(4H, m, N-(CH₂CH₂)₂-NMe), 3.05-3.14 (4H, m, N-CH₂CH₂-NMe),

4.69 (2H, d_app, J = 5.9 Hz, N²-CH₂), 4.83 (1H, dd, J = 17.1, 1.3 Hz,

alkene C-H^trans), 5.00 (1H, dd, J = 10.3, 1.3 Hz, alkene C-H^cis),

5.32 (1H, s, OH), 5.67 (1H, ddt, J = 17.1, 10.3, 5.9 Hz, alkene C-

H), 6.93 (2H, d, J = 9.1 Hz, H-3''/5''), 7.54-7.60 (1H, m, H-5'), 7.61

(2H, d, J = 9.1 Hz, H-2''/6''), 7.76 (1H, d_app, J = 8.1 Hz, H-3'), 8.06

(1H, dd, J = 8.1, 7.3 Hz, H-4'), 8.83 (1H, s, H-4), 10.1 (1H, br, C⁶-

NH); ¹³C NMR (100 MHz, DMSO-d₆) 30.9 (C(CH₃)₂), 46.2 (N-CH₃),

47.1 (N²-CH₂), 48.9 (piperazine-CH₂), 55.1 (piperazine-CH₂), 72.8

(C(CH₃)₂), 116.0, 116.7, 118.7, 121.6, 131.3, 132.7, 139.3, 147.6,

156.5, 161.0, 161.6, 168.0; MS [M+H]⁺m/z 501.4.

Synthesis of 4-(2-(dimethylamino)ethyl)aniline (42). The

desired product was obtained as a yellow oil (0.422 g, 2.57 mmol,

59% - 2 steps). Rf 0.16 (9:1 DCM:MeOH); IR (cm^-1) 3317, 3018,

2771, 2705, 2448, 1612, 1518; ¹H NMR (400 MHz, CDCl₃) 2.33

(6H, s, N(CH₃)₂), 2.50-2.55 (2H, m, ArCH₂CH₂), 2.68-2.73 (2H, m,

ArCH₂CH₂), 3.55 (2H, br s, NH₂), 6.65 (2H, d, J = 8.5 Hz, H-2/6),

7.01 (2H, d, J = 8.5 Hz, H-3/5); ¹³C NMR (100 MHz, CDCl₃) 33.2

(ArCH₂CH₂), 45.3 (N(CH₃)₂), 61.8 (ArCH₂CH₂), 115.3 (Ar-C),

129.4 (Ar-C), 130.0 (Ar-C), 144.2 (Ar-C).

Synthesis of 1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-2-(4-

methoxybenzyl)-6-((4-(4-methylpiperazin-1-

Synthesis of 4-(2-(dimethylamino)ethoxy)aniline (43). The

desired product was obtained as a brown oil (0.673 g, 3.74 mmol,

92%). Rf 0.38 (9:1 DCM:MeOH); IR (cm^-1) 3335, 3216, 2943,

2867, 2822, 2774, 1627, 1508; ¹H NMR (400 MHz, CDCl₃) 2.35

(6H, s, N(CH₃)₂), 2.70 (2H, t, J = 5.8 Hz, OCH₂CH₂), 3.47 (2H, br

s, NH₂), 4.00 (2H, t, J = 5.8 Hz, OCH₂CH₂), 6.64 (2H, d, J = 8.8

Hz, H-3/5), 6.78 (2H, d, J = 8.8 Hz, H-2/6); ¹³C NMR (100 MHz,

CDCl₃) 45.8 (N(CH₃)₂), 58.4 (OCH₂CH₂), 66.6 (OCH₂CH₂), 115.8

(Ar-C), 116.4 (Ar-C), 140.1 (Ar-C), 152.0 (Ar-C).

yl)phenyl)amino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-

one (33). The desired target compound was obtained as a yellow

solid (0.188 g, 0.32 mmol, 89%). Rf 0.46 (9:1 DCM:MeOH); M.p.

196-199°C; IR (cm^-1) 3275, 3186, 2963, 2936, 2838, 1684, 1603,

1536, 1512; ¹H NMR (400 MHz, CDCl₃) 1.64 (6H, s, C(CH₃)₂),

2.40 (3H, s, N-CH₃), 2.62-2.67 (4H, m, N-(CH₂CH₂)₂-NMe), 3.20-

3.25 (4H, m, N-CH₂CH₂-NMe), 3.72 (3H, s, OCH₃), 5.29 (2H, s,

N²-CH₂), 6.67 (2H, d, J = 8.6 Hz, benzyl H-2/6), 6.85 (2H, d, J =

8.6 Hz, benzyl H-3/5), 6.91 (2H, d, J = 8.7 Hz, H-3’’/5’’), 7.37 (1H,

d_app, J = 7.8 Hz, H-5’), 7.43 (2H, d, J = 8.7 Hz, H-2’’/6’’), 7.57 (1H,

d_app, J = 8.1 Hz, H-3’), 7.82 (1H, dd, J = 8.1, 7.8 Hz, H-4’), 8.83

(1H, s, H-4); ¹³C NMR (100 MHz, CDCl₃) 30.6 (C(CH₃)₂), 46.0 (N-

CH₃), 48.1 (N²-CH₂), 49.4 (piperazine-CH₂), 55.0 (OCH₃), 55.2

(piperazine-CH₂), 72.5 (C(CH₃)₂), 113.9, 116.0, 116.3, 116.5,

122.1, 127.7, 129.5, 130.4, 138.8, 147.4, 148.1, 156.3, 159.2,

161.0, 162.5, 165.8; MS [M+H]⁺m/z 581.4.

Synthesis

of

1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-2-

methyl-6-((4-(4-methylpiperazin-1-yl)phenyl)amino)-1,2-

dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one (32). The desired

target compound was obtained as a yellow solid (0.16 mmol,

56%). Rf 0.39 (9:1 DCM:MeOH); M.p. 192-195°C; IR (cm^-1) 3265,

3184, 3090, 2972, 2928, 2810, 1668, 1619, 1536, 1512; ¹H NMR

(400 MHz, DMSO-d₆) 1.46 (6H, s, C(CH₃)₂), 2.23 (3H, s,

N(CH₂CH₂)₂NCH₃), 2.44-2.49 (4H, m, N(CH₂CH₂)₂NMe), 3.08-

3.13 (4H, m, N(CH₂CH₂)₂NMe), 3.42 (3H, s, N²-CH₃), 5.32 (1H, s,

OH), 6.93 (2H, d, J = 9.0 Hz, H-3’’/5’’), 7.59 (1H, d_app, J = 7.9 Hz,

H-5’), 7.62 (2H, d, J = 9.0 Hz, H-2’’/6’’), 7.80 (1H, d_app, J = 7.5 Hz,

H-3’), 8.08 (1H, dd, J = 7.9, 7.5 Hz, H-4’), 8.81 (1H, s, H-4). 10.11

(1H, br s, C⁶-NH); ¹³C NMR (100 MHz, DMSO-d₆) 30.9 (C(CH₃)₂),

33.0 (N²-CH₃), 46.3 (piperazine N-CH₃), 49.0 (piperazine-CH₂),

55.1 (piperazine-CH₂), 72.8 (C(CH₃)₂), 116.0, 116.7, 121.5, 131.4,

139.3, 147.6, 156.2, 160.9, 161.8, 168.1; MS [M+H]⁺m/z 475.2.

Synthesis of 2-allyl-1-(3-(2-hydroxypropan-2-yl)phenyl)-6-((4-

(4-methylpiperazin-1-yl)phenyl)amino)-1,2-dihydro-3H-

pyrazolo[3,4-d]pyrimidin-3-one (31). The desired target

compound was obtained as a pale yellow solid (53 mg, 0.11 mmol,

64%). Rf 0.39 (9:1 DCM:MeOH); M.p. 164-167°C; IR (cm^-1) 3287,

2972, 2935, 2838, 2798, 1701, 1668, 1606, 1542, 1512; ¹H NMR

(400 MHz, CDCl₃) 1.64 (6H, s, C(CH₃)₂), 2.39 (3H, s, N-CH₃),

2.60-2.64 (4H, m, N-(CH₂CH₂)₂-NMe), 3.18-3.23 (4H, m, N-

CH₂CH₂-NMe), 4.40 (2H, d_app, J = 6.1 Hz, N²-CH₂), 5.00 (1H, d_app

,

Synthesis of 2-allyl-1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-

6-((4-(4-methylpiperazin-1-yl)phenyl)amino)-1,2-dihydro-3H-

J = 17.2, alkene C-H^trans), 5.12 (1H, d_app, J = 10.1 Hz, alkene C-

H^cis), 5.73 (1H, ddt, J = 17.2, 10.1, 6.1 Hz, alkene C-H), 6.90 (2H,

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d, J = 8.8 Hz, H-3’’/5’’), 7.29-7.34 (1H, m, H-5’), 7.45 (2H, d, J =

solid (13 mg, 0.04 mmol, 29%). Rf 0.53 (KP-NH – 19:1

DCM:MeOH); M.p. 195-197°C; IR (cm^-1) 3325, 3187, 2979, 2924,

2856, 1667, 1649, 1616, 1563; ¹H NMR (400 MHz, DMSO-d₆)

1.46 (6H, s, C(CH₃)₂), 4.62 (2H, d_app, J = 5.6 Hz, N²-CH₂), 4.81

(1H, d_app, J = 17.1 Hz, alkene C-H^trans), 4.98 (1H, d_app, J = 10.0 Hz,

alkene C-H^cis), 5.32 (1H, s, OH), 5.64 (1H, ddt, J = 17.1, 10.0, 5.6

Hz, alkene C-H), 7.53 (2H, br s, NH₂), 7.59 (1H, d_app, J = 7.7 Hz,

H-5’), 7.71 (1H, d_app, J = 8.1 Hz, H-3’), 7.95 (1H, d_app, J = 8.1, 7.7

Hz, H-4’), 8.70 (1H, s, H-4); ¹³C NMR (100 MHz, DMSO-d₆) 30.9

(C(CH₃)₂), 47.1 (N²-CH₂), 72.8 (C(CH₃)₂), 98.9, 116.4, 116.6,

118.6, 132.7, 139.2, 147.8, 156.8, 161.9, 162.0, 165.4, 168.0; MS

[M+H]⁺m/z 327.2.

8.8 Hz, H-2’’/6’’), 7.49 (2H, d_app, J = 4.8 Hz, H-4’/6’), 7.60 (1H, s_app

,

H-2’), 8.83 (1H, s, H-4); ¹³C NMR (100 MHz, CDCl₃) 31.9

(C(CH₃)₂), 46.1 (N-CH₃), 46.5 (N²-CH₂), 49.4 (piperazine-CH₂),

55.0 (piperazine-CH₂), 72.4 (C(CH₃)₂), 116.6, 119.2, 123.7, 129.1,

130.7, 131.1, 136.2, 150.9, 156.3, 162.7; MS [M+H]⁺m/z 500.2.

Synthesis of 2-allyl-1-(6-(hydroxymethyl)pyridin-2-yl)-6-((4-

(4-methylpiperazin-1-yl)phenyl)amino)-1,2-dihydro-3H-

pyrazolo[3,4-d]pyrimidin-3-one (30). The desired target

compound was obtained as a yellow solid (0.134 g, 0.29 mmol,

61%). Rf 0.34 (9:1 DCM:MeOH); M.p. 197-200°C; IR (cm^-1) 3253,

3176, 3065, 2939, 2818, 1687, 1674, 1610; ¹H NMR (400 MHz,

CDCl₃) 2.39 (3H, s, N-CH₃), 2.59-2.64 (4H, m, N-(CH₂CH₂)₂-NMe), Synthesis of 2-allyl-1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-

3.19-3.25 (4H, m, N-CH₂CH₂-NMe), 4.73 (2H, d_app, J = 6.0 Hz, N²-

CH₂), 4.82 (2H, s, CH₂OH), 4.98 (1H, d_app, J = 17.2, alkene C-

H^trans), 5.07 (1H, d_app, J = 10.2 Hz, alkene C-H^cis), 5.73 (1H, ddt, J

6-(phenylamino)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-

one (24). The desired target compound was obtained as a white

solid (35 mg, 0.09 mmol, 31%). Rf 0.50 (19:1 DCM:MeOH); M.p.

= 17.2, 10.2, 6.0 Hz, alkene C-H), 6.94 (2H, d, J = 8.6 Hz, H-3’’/5’’), 153-155°C; IR (cm^-1) 3245, 3191, 3080, 3056, 2975, 2929, 1671,

7.24 (1H, dapp, J = 7.5 Hz, H-5’), 7.47 (2H, d, J = 8.6 Hz, H-2’’/6’’),

7.77 (1H, d_app, J = 8.1 Hz, H-3’), 7.87 (1H, dd, J = 8.1, 7.5 Hz, H-

4’), 8.84 (1H, s, H-4); ¹³C NMR (100 MHz, CDCl₃) 46.1 (N-CH₃),

47.8 (N2-CH₂), 49.5 (piperazine-CH₂), 55.1 (piperazine-CH₂),

64.3 (CH₂OH), 116.4, 116.7, 117.8, 119.1, 122.0, 130.4, 131.6,

138.6, 148.2, 148.4, 156.3, 158.9, 161.4, 162.4; MS [M+H]⁺m/z

473.2.

1615, 1540; ¹H NMR (400 MHz, CDCl₃) 1.61 (6H, s, C(CH₃)₂),

3.97 (1H, s, OH), 4.78 (2H, d_app, J = 6.2 Hz, N²-CH₂), 4.96 (1H,

dd, J = 17.0, 1.1 Hz, alkene C-H^trans), 5.07 (1H, dd, J = 10.2, 1.1

Hz, alkene C-H^cis), 5.73 (1H, ddt, J = 17.0, 10.2, 6.2 Hz, alkene

C-H), 7.15 (1H, dd, J = 7.4, 7.3 Hz, H-4’’), 7.36-7.41 (3H, m, H-

5’/3’’/5’’), 7.63 (2H, d_app, J = 7.8 Hz, H-2’’/6’’), 7.79 (1H, d_app, J =

7.9 Hz, H-3’), 7.91 (1H, dd_app, J = 7.9, 7.8 Hz, H-4’), 8.90 (1H, s,

H-4); ¹³C NMR (100 MHz, CDCl₃) 30.6 (C(CH₃)₂), 47.6 (N²-CH₂),

72.5 (C(CH₃)₂), 101.1, 116.2, 116.3, 119.1, 120.6, 124.0, 128.9,

131.5, 138.2, 138.9, 147.4, 156.3, 161.0, 161.3, 162.0, 165.9; MS

[M+H]⁺m/z 403.4.

Synthesis of 2-allyl-1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-

6-((4-methoxybenzyl)amino)-1,2-dihydro-3H-pyrazolo[3,4-

d]pyrimidin-3-one (23). The desired target compound was

obtained as an off-white solid (55 mg, 0.12 mmol, 22%). Rf 0.38

(19:1 DCM:MeOH); M.p. 136-138°C; IR (cm^-1) 3442, 3219, 2972,

2922, 1667, 1614, 1593, 1546; ¹H NMR (400 MHz, CDCl₃) 1.60

(6H, s, C(CH₃)₂), 3.82 (3H, s, OCH₃), 3.98 (1H, s, OH), 4.55-4.61

(2H, m, NHCH₂), 4.71-4.78 (2H, m, N²-CH₂), 4.96 (1H, d_app, J =

16.4 Hz, alkene C-H^trans), 5.06 (1H, d_app, J = 9.9 Hz, alkene C-H^cis),

5.72 (1H, ddt, J = 16.4, 9.9, 6.2 Hz, alkene C-H), 6.89 (2H, d, J =

8.5 Hz, benzyl H-3/5), 7.26 (2H, d, J = 8.5 Hz, benzyl H-2/6), 7.33

(1H, d_app, J = 7.7 Hz, H-5’), 7.73 (1H, d_app, J = 8.1 Hz, H-3’), 7.85

(1H, dd_app, J = 8.1, 7.7 Hz, H-4’), 8.73 (1H, s, H-4); ¹³C NMR (100

MHz, CDCl₃) 30.5 (C(CH₃)₂), 42.3 (benzyl CH₂), 47.8 (N²-CH₂),

55.3 (OCH₃), 72.4 (C(CH₃)₂), 114.1, 115.7, 115.9, 119.0, 129.0,

130.0, 131.7, 138.8, 147.6, 156.3, 159.2, 161.6, 162.7, 163.4,

166.6; MS [M+H]⁺m/z 447.4.

Synthesis of 2-allyl-6-((3-(dimethylamino)phenyl)amino)-1-

(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-1,2-dihydro-3H-

pyrazolo[3,4-d]pyrimidin-3-one (25). The desired target

compound was obtained as a pale yellow/green solid (29 mg, 0.06

mmol, 23%). Rf 0.30 (19:1 DCM:MeOH); M.p. 81-84°C; IR (cm^-1)

3407, 3219, 3080, 2963, 2926, 1694, 1605, 1572, 1548; ¹H NMR

(400 MHz, CDCl₃) 1.61 (6H, s, C(CH₃)₂), 2.95 (6H, s, N(CH₃)₂,

3.92 (1H, s, OH), 4.76 (2H, d_app, J = 6.0 Hz, N²-CH₂), 4.95 (1H,

d_app, J = 17.2 Hz, alkene C-H^trans), 5.06 (1H, d_app, J = 10.1 Hz,

alkene C-H^cis), 5.73 (1H, ddt, J = 17.2, 10.1, 6.0 Hz, alkene C-H),

6.54 (1H, dd, J = 8.4, 2.0 Hz, H-4’’), 6.87 (1H, br s, H-2’’), 7.05

(1H, d_app, J = 7.9 Hz, H-6’’), 7.23 (1H, dd, J = 8.4, 7.9 Hz, H-5’’),

7.37 (1H, d_app, J = 7.6 Hz, H-5’), 7.50 (1H, br s, N-H), 7.81 (1H,

d_app, J = 7.9 Hz, H-3’), 7.87 (1H, dd_app, J = 7.9, 7.6 Hz, H-4’), 8.88

(1H, s, H-4); ¹³C NMR (100 MHz, CDCl₃) 30.5 (C(CH₃)₂), 40.6

(N(CH₃)₂), 47.6 (N²-CH₂), 72.5 (C(CH₃)₂), 101.0, 104.8, 108.7,

Synthesis

of

2-allyl-6-amino-1-(6-(2-hydroxypropan-2-

yl)pyridin-2-yl)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-

one (22). The desired target compound was obtained as a white

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109.2, 116.1, 116.5, 119.0, 129.4, 131.6, 138.9, 139.0, 147.5,

2827, 2782, 1745, 1672, 1603, 1568; ¹H NMR (400 MHz, CDCl₃)

1.61 (6H, s, C(CH₃)₂), 2.33 (6H, s, N(CH₃)₂), 2.53-2.59 (2H, m,

ArCH₂CH₂), 2.76-2.82 (2H, m, ArCH₂CH₂), 4.77 (2H, d_app, J = 5.8

Hz, N²-CH₂), 4.96 (1H, d_app, J = 17.2 Hz, alkene C-H^trans), 5.06 (1H,

d_app, J = 10.3 Hz, alkene C-H^cis), 5.73 (1H, ddt, J = 17.2, 10.3, 5.8

151.3, 156.3, 161.2, 161.4, 162.1, 165.9; MS [M+H]⁺m/z 446.2.

Synthesis

of

2-allyl-6-((3-

((dimethylamino)methyl)phenyl)amino)-1-(6-(2-

hydroxypropan-2-yl)pyridin-2-yl)-1,2-dihydro-3H-

Hz, alkene C-H), 7.21 (2H, d, J = 8.2 Hz, H-3’’/5’’), 7.39 (1H, d_app

,

J = 7.7 Hz, H-5’), 7.53 (2H, d, J = 8.2 Hz, H-2’’/6’’), 7.78 (1H, d_app

,

pyrazolo[3,4-d]pyrimidin-3-one (26). The desired target

compound was obtained as an off-white solid (35 mg, 0.08 mmol,

27%). Rf 0.18 (EtOAc); M.p. 121-123°C; IR (cm^-1) 3407, 3230,

2975, 2927, 2772, 1665, 1610, 1542; ¹H NMR (400 MHz, CDCl₃)

1.61 (6H, s, C(CH₃)₂), 2.29 (6H, s, N(CH₃)₂), 3.47 (2H, s, ArCH₂),

4.78 (2H, d_app, J = 6.1 Hz, N²-CH₂), 4.96 (1H, d_app, J = 17.1 Hz,

alkene C-H^trans), 5.07 (1H, d_app, J = 10.1 Hz, alkene C-H^cis), 5.73

(1H, ddt, J = 17.1, 10.1, 6.1 Hz, alkene C-H), 7.10 (1H, d_app, J =

7.5 Hz, H-4’’), 7.33 (1H, dd_app, J = 7.9, 7.5 Hz, H-5’’), 7.39 (1H,

J = 7.9 Hz, H-3’), 7.90 (1H, dd, J = 7.9, 7.7 Hz, H-4’), 8.87 (1H, s,

H-4); ¹³C NMR (100 MHz, CDCl₃) 30.6 (C(CH₃)₂), 33.8

(ArCH₂CH₂), 45.5 (N(CH₃)₂), 47.6 (N²-CH₂), 61.5 (ArCH₂CH₂),

72.5 (C(CH₃)₂), 101.1, 116.2, 116.3, 119.1, 120.6, 129.0, 131.6,

136.1, 136.2, 138.9, 147.5, 156.4, 161.1, 161.3, 162.1, 165.9; MS

[M+H]⁺m/z 474.4.

Synthesis

of

2-allyl-6-((4-(2-

(dimethylamino)ethoxy)phenyl)amino)-1-(6-(2-

d_app, J = 7.6 Hz, H-5’), 7.54-7.62 (2H, m, H-2’’/6’’), 7.82 (1H, d_app

,

hydroxypropan-2-yl)pyridin-2-yl)-1,2-dihydro-3H-

J = 7.9 Hz, H-3’), 7.92 (1H, dd_app, J = 7.9, 7.6 Hz, H-4’), 8.89 (1H,

s, H-4); ¹³C NMR (100 MHz, CDCl₃) 30.5 (C(CH₃)₂), 45.4

(N(CH₃)₂), 47.6 (N²-CH₂), 64.3 (Ar-CH₂), 72.5 (C(CH₃)₂), 101.2,

116.1, 116.4, 119.1, 119.2, 120.8, 124.6, 128.8, 131.6, 138.3,

138.9, 139.7, 147.4, 156.3, 161.1, 161.2, 162.0, 165.9; MS

[M+H]⁺m/z 460.0.

pyrazolo[3,4-d]pyrimidin-3-one (29). The desired target

compound was obtained as an off-white solid (47 mg, 0.11 mmol,

38%). Rf 0.28 (9:1 DCM:MeOH); M.p. 123-126°C; IR (cm^-1) 3248,

3081, 2976, 2937, 2870, 2821, 2773, 1680, 1614, 1512; ¹H NMR

(400 MHz, DMSO-d₆) 1.47 (6H, s, C(CH₃)₂), 2.28 (6H, s, N(CH₃)₂),

2.69 (2H, t, J = 5.2 Hz, OCH₂CH₂), 4.06 (2H, t, J = 5.2 Hz,

OCH₂CH₂), 4.69 (2H, d_app, J = 5.4 Hz, N²-CH₂), 4.83 (1H, d_app, J

= 17.1 Hz, alkene C-H^trans), 5.00 (1H, d_app, J = 10.3 Hz, alkene C-

H^cis), 5.33 (1H, s, OH), 5.67 (1H, ddt, J = 17.1, 10.3, 5.4 Hz, alkene

C-H), 6.94 (2H, d, J = 8.4 Hz, H-3’’/5’’), 7.59-7.66 (3H, m, H-

5’/2’’/6’’), 7.75 (1H, d_app, J = 7.4 Hz, H-3’), 8.05 (1H, dd, J = 7.8,

7.4 Hz, H-4’), 8.85 (1H, s, H-4), 10.19 (1H, br s, C⁶-NH); ¹³C NMR

(100 MHz, DMSO-d₆) 30.9 (C(CH₃)₂, 45.8 (N(CH₃)₂), 47.0 (N²-

CH₂), 58.1 (OCH₂CH₂), 66.2 (OCH₂CH₂), 72.8 (C(CH₃)₂), 114.8,

116.8, 118.7, 132.7, 139.3, 147.5, 154.9, 156.6, 161.6, 168.1; MS

[M+H]⁺m/z 490.4.

Synthesis of 3-((2-allyl-1-(6-(2-hydroxypropan-2-yl)pyridin-2-

yl)-3-oxo-2,3-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-

yl)amino)-N,N-dimethylbenzamide (27). The desired target

compound was obtained as a white solid (16 mg, 0.03 mmol,

12%). Rf 0.34 (19:1 DCM:MeOH); M.p. 93-96°C; IR (cm^-1) 3405,

3270, 2972, 2929, 1673, 1615, 1541; ¹H NMR (400 MHz, CDCl₃)

1.60 (6H, s, C(CH₃)₂), 2.97 (3H, s, NCH₃), 3.16 (3H, s, NCH₃),

3.97 (1H, s, OH), 4.79 (2H, d_app, J = 6.0 Hz, N²-CH₂), 4.95 (1H,

d_app, J = 17.0 Hz, alkene C-H^trans), 5.06 (1H, d_app, J = 10.3 Hz,

alkene C-H^cis), 5.72 (1H, ddt, J = 17.0, 10.3, 6.0 Hz, alkene C-H),

7.14 (1H, d_app, J = 7.5 Hz, H-4’’), 7.35-7.41 (2H, m, H-5’/5’’), 7.48

(1H, d_app, J = 8.1 Hz, H-6’’), 7.82 (1H, d_app, J = 8.1 Hz, H-3’), 7.94

Synthesis of 6-((4-(4-methylpiperazin-1-yl)phenyl)amino)-1-

(6-(prop-1-en-2-yl)pyridin-2-yl)-1,2-dihydro-3H-pyrazolo[3,4-

d]pyrimidin-3-one (34). The desired target compound was

obtained as a pale yellow solid (39 mg, 0.09 mmol, 91%). Rf 0.36

(9:1 DCM:MeOH); M.p. 260-270°C (decomposed); IR (cm^-1) 3245,

3175, 2933, 2836, 2791, 1691, 1611; ¹H NMR (400 MHz, DMSO-

d₆) 2.20 (3H, s, CCH₃), 2.25 (3H, s, N-CH₃), 2.47-2.51 (4H, m, N-

(CH₂CH₂)₂-NMe), 3.09-3.13 (4H, m, N-CH₂CH₂-NMe), 5.37 (1H,

s, alkene C-H), 6.12 (1H, s, alkene C-H), 6.93 (2H, d, J = 8.9 Hz,

H-3’’/5’’), 7.45 (1H, d_app, J = 7.5 Hz, H-3’), 7.67 (2H, d, J = 8.9 Hz,

H-2’’/6’’), 7.98 (1H, dd, J = 7.8, 7.5 Hz, H-4’), 8.09-8.16 (1H, m,

H-5’), 8.83 (1H, s, H-4), 9.88 (1H, s, C⁶-NH), 11.95 (1H, br s, N²-

H); ¹³C NMR (100 MHz, DMSO-d₆) 20.7 (C(CH₂)CH₃), 46.1 (N-

(1H, br s, N-H), 7.98-8.05 (2H, m, H-4’/2’’), 8.89 (1H, s, H-4); ¹³

C

NMR (100 MHz, CDCl₃) 30.5 ((C(CH₃)₂), 35.4 (NCH₃), 39.6

(NCH₃), 47.6 N²-CH₂), 72.5 (C(CH₃)₂), 101.5, 116.3, 116.6, 118.8,

119.1, 120.9, 121.9, 128.9, 131.5, 137.2, 138.6, 139.6, 147.2,

156.4, 160.8, 161.0, 161.8, 165.8, 171.2; MS [M+H]⁺m/z 474.2.

Synthesis

of

2-allyl-6-((4-(2-

(dimethylamino)ethyl)phenyl)amino)-1-(6-(2-hydroxypropan-

2-yl)pyridin-2-yl)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-

one (28). The desired target compound was obtained as an off-

white solid (36 mg, 0.08 mmol, 18%). Rf 0.14 (9:1 DCM:MeOH);

M.p. 116-119°C; IR (cm^-1) 3252, 3191, 3099, 2968, 2929, 2855,

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FULL PAPER

CH₃), 49.1 (piperazine-CH₂), 55.1 (piperazine-CH₂), 113.4, 116.2,

116.6, 117.3, 121.5, 132.1, 139.4, 142.5, 147.2, 154.7, 156.7,

157.4, 160.3; MS [M+H]⁺m/z 443.4.

effect on kinase activity. Inhibitors were assayed in the final

concentration range of 0.04 nM to 10 µM. Kinase Tracer 178 was

used at a final concentration of 150 nM and the antibody and

kinase were used at final concentrations of 3 nM and 5 nM,

respectively. All reagents were incubated together for 1 h at room

temperature and read using a PerkinElmer Envision 2104

Multilabel Reader enabled for TR-FRET (Excitation = 340 nm;

Tracer emission = 665 nm; Antibody emission = 615 nm; Delay =

100 µs; Integration = 200 µs). Emission ratios (665 nm/615 nm)

were determined for each inhibitor concentration and the data

analyzed using a non-linear regression analysis of the log dose-

Synthesis of 1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-6-((4-

(4-methylpiperazin-1-yl)phenyl)amino)-1,2-dihydro-3H-

pyrazolo[3,4-d]pyrimidin-3-one (35). The desired target

compound was obtained as an off-white solid (36 mg, 0.08 mmol,

80%). Rf 0.21 (1:1 DCM:MeOH); M.p. >350°C; IR (cm^-1) 3248,

3168, 2974, 2936, 2791, 1612, 1535; ¹H NMR (400 MHz, DMSO-

d₆) 1.52 (6H, s, C(CH₃)₂), 2.23 (3H, s, N-CH₃), 2.45-2.49 (4H, m,

N-(CH₂CH₂)₂-NMe), 3.05-3.09 (4H, m, N-CH₂CH₂-NMe), 5.83 (1H, response curve to determine IC₅₀values.

s, OH), 6.91 (2H, d, J = 9.1 Hz, H-3’’/5’’), 7.19 (1H, d_app, J = 7.5

Hz, H-5’), 7.69 (2H, d, J = 9.1 Hz, H-2’’/6’’), 7.80 (1H, dd, J = 7.9,

7.5 Hz, H-4’), 8.13 (1H, d_app, J = 7.9 Hz, H-3’), 8.45 (1H, s, H-4),

9.23 (1H, s, C⁶-NH); ¹³C NMR (100 MHz, DMSO-d₆) 31.2

(C(CH₃)₂), 46.3 (N-CH₃), 49.5 (piperazine-CH₂), 55.2 (piperazine-

CH₂), 72.3 (C(CH₃)₂), 116.4, 120.5, 133.5, 138.6, 146.4, 150.5,

152.5, 159.1, 166.4; MS [M+H]⁺m/z 461.2.

PLK1 Recombinant Kinase Inhibition Assay. LANCE^®Eu time-

resolved fluorescence resonance energy transfer (TR-FRET)

kinase assay (PerkinElmer) was performed in 384-well OptiPlates

(Corning) using recombinant PLK1 kinase (Carna), ULight™-p70

S6K (Thr389) substrate (PerkinElmer) and ATP (Sigma)

according to supplier protocols. All reagents were prepared in

kinase buffer (2 mM DTT, 50 mM HEPES, 1 mM EGTA, 10 mM

MgCl₂, 0.01% Tween20, pH 7.5) and inhibitor solutions were

prepared such that the final DMSO concentration did not exceed

0.5%, which was shown to have no effect on kinase activity. PLK1

was used at a final concentration of 50 nM, ULight™-CREBtide

substrate was used at a final concentration of 50 nM and ATP was

used at a final concentration of 5 µM. Assays were performed at

25°C in a reaction mixture consisting of 2.5 µL serially diluted

inhibitor solution, 2.5 µL kinase, 2.5 µL ATP and 2.5 µL substrate.

Reagents were incubated for 1 h before the reaction was halted

through the addition of EDTA (10 mM) after which Eu anti-

phospho-p70 S6K (Thr389) antibody (PerkinElmer) was added at

a final concentration of 2 nM for 1 h. The plate was read using a

Biotek Synergy H1 Hybrid plate reader enabled for TR-FRET

(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 concentration (IC50) values were

determined for each inhibitor through non-linear regression

analysis of the log dose-response curve.

Biological Assays

Cells and Reagents: Daoy and ONS-76 cells (Medulloblastoma)

were obtained from ATCC and were passaged for <6 months

following resuscitation. Cells were cultured in DMEM (Gibco)

supplemented with 10% FBS (Sigma Aldrich), 1 mM sodium

pyruvate (Gibco), 1X penicillin/streptomycin solution (Cellgro) and

1X non-essential amino acids (Sigma Aldrich) at 37°C in an

incubator humidifier with 95% air and 5% CO₂. Seeded cells were

allowed to adhere for 24 h prior to use in all assays. In all

treatment conditions, the final DMSO concentration did not

exceed 0.5%. All chemical reagents were purchased from the

Aldrich Chemical Company, Fisher Chemicals or Alfa Aesar

Chemicals and were of the highest available purity.

WEE1 Recombinant Kinase Inhibition Assay: LanthaScreen™

Eu time-resolved fluorescence resonance energy transfer (TR-

FRET) kinase binding assays (Invitrogen) were performed in 384-

well, low-volume plates (Corning) using recombinant WEE1

kinase, Kinase Tracer 178 and LanthaScreen™ Eu-anti-GST

antibody (Invitrogen). Assays were performed at 25°C in a

reaction mixture consisting of 5 µL serially diluted inhibitor

Cellular Metabolic Viability Assay: Daoy and ONS-76 cells

were seeded into sterile 96-well plates (Corning Inc.) at 2000

cells/well in 100 μL media. Inhibitors were administered at the

solution,

kinase/antibody solution. All reagents were prepared as solutions

in 1X kinase buffer A (Invitrogen) at 3X final desired concentration. 290 nM) and 2-fold concentrations above and below the EC₅₀.

5 µL Kinase Tracer 178 solution, and 5 µL

MTS EC₅₀of AZD1775 (Daoy; EC₅₀= 150 nM, ONS-76; EC₅₀

=

Inhibitor solutions were prepared such that final DMSO

concentrations did not exceed 0.5%, which was shown to have no

Cells were incubated for 72 h with 50 μL of each diluted inhibitor

solution. Cell viability was measured by 2 h incubation with 30 μL

16

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FULL PAPER

CellTiter 96^®AQueous One Cell Proliferation reagent (Promega)

and formazan concentration assessed through colorimetric

analysis using a BioTek Synergy H1 plate reader (Absorption =

490 nm).

conducted using Forge™ 10.4.2 (Cresset, Litlington,

Cambridgeshire, UK; http://www.cresset-group.com/forge/)^[18]

.

Acknowledgements

CellTox Green Cytotoxicity Assay: Daoy cells were seeded into

sterile 96-well plates (Corning Inc.) at 5000 cells/well in 100 μL

media containing 1:500 dilution of CellTox^TMGreen dye

(Promega). Inhibitor solutions (50 μL) were administered over a

concentration range from 4800 nM to 18.75 nM. Cells were

incubated for a total of 72 h and fluorescence was measured at 1,

24, 48, and 72 h using a BioTek Synergy H1 plate reader

(Excitation = 485 nm; Emission = 520 nm).

Research reported in this publication was supported by the

National Institute of Neurological Disorders and Stroke (NINDS)

of the National Institutes of Health (NIH) under Award Number

R21NS084084. The content is solely the responsibility of the

authors and does not necessarily represent the official views of

the NIH. Molecular modeling studies were conducted at the

University of Colorado Computational Chemistry and Biology

Core Facility, which is funded in part by NIH/NCATS Colorado

CTSA Grant Number UL1 TR001082.

Quantification of p-CDK1 (Tyr15) Levels: Daoy cells were

plated in sterile 6-well plates at 200,000 cells/well and treated with

active inhibitors at a dose of 220 nM and incubated for 24 h prior

to preparing cell lysates. Any compounds that were found to

inhibit cellular p-CDK1 levels at this concentration when

compared to DMSO control were tested across a concentration

range from 1000 nM to 62.5 nM. Following drug incubation, the

media was aspirated from cells before cells were trypsinized and

resuspended in TES/SB buffer containing protease inhibitors.

Cells were lysed on ice through sonication, and cell lysates were

diluted with ELISA Pathscan^®sample diluent to a final volume of

100 µL and protein concentration of 0.05 mg/ml prior to use. The

relative concentration of p-CDK1 Tyr15 was determined using an

enzyme-linked immunosorbent assay according to recommended

protocol (Cell Signaling, ELISA Pathscan^®phosphor-Cdc2

(Tyr15)).

Keywords: DNA damage • inhibitor • kinase • structure-activity

relationship • WEE1

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Entry for the Table of Contents

Wee changes impact activity profiles: The activity of WEE1 kinase is critical for DNA repair in cancer cells and can confer

resistance to DNA-targeted agents. Here, we report that structural modifications to the WEE1 inhibitor AZD1775 can result in

improved inhibitory potency against WEE1 kinase activity with limited cytotoxicity. These studies may have important implications in

the clinical setting where WEE1 inhibitors are used as chemosensitizers for DNA-targeted chemotherapy.

19

Article Doi

Development of Potent Pyrazolopyrimidinone-Based WEE1 Inhibitors with Limited Single-Agent Cytotoxicity for Cancer Therapy

DOI: 10.1002/cmdc.201800188

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Full text of DOI:10.1002/cmdc.201800188

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