N-Substituted pyrrolopyrimidines and purines as p90 ribosomal S6 protein kinase-2 (RSK2) inhibitors

Bioorg. Med. Chem. 41 (2021) 116220

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Bioorganic & Medicinal Chemistry

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

N-Substituted pyrrolopyrimidines and purines as p90 ribosomal S6 protein

kinase-2 (RSK2) inhibitors

Kimberly A. Casalvieri, Christopher J. Matheson, Becka M. Warﬁeld, Donald S. Backos,

Philip Reigan^*

Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, 12850 East

Montview Boulevard, Aurora, CO, 80045, USA

A R T I C L E I N F O

A B S T R A C T

Keywords:

The RSK2 kinase is the downstream effector of the Ras/Raf/MEK/ERK pathway, that is often aberrantly activated

in acute myeloid leukemia (AML). Recently, we reported a structure-activity study for BI-D1870, the pan-RSK

inhibitor, and identiﬁed pteridinones that inhibited cellular RSK2 activity that did not result in concomitant

cytotoxicity. In the current study, we developed a series of pyrrolopyrimidines and purines to replace the

pteridinone ring of BI-D1870, with a range of N-substituents that extend to the substrate binding site to probe

complementary interactions, while retaining the 2,6-diﬂuorophenol-4-amino group to maintain interactions with

the hinge domain and the DFG motif. Several compounds inhibited cellular RSK2 activity, and we identiﬁed

compounds that uncoupled cellular RSK2 inhibition from potent cytotoxicity in the MOLM-13 AML cell line.

These N-substituted probes have revealed an opportunity to further examine substituents that extend from the

ATP- to the substrate-binding site may confer improved RSK potency and selectivity.

RSK

Kinase

Inhibitor

Structure-activity relationship

1. Introduction

cycle progression, cell proliferation, cell growth, protein synthesis, nu-

clear signaling, cell migration, and cell survival.^1–3The RSKs share a

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

onine kinases 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.^1–3The NTKD is

homologous to that of AGC kinases, whereas the CTKD is closely related

to the calcium/calmodulin-dependent protein kinase (CAMK) family.

The C-terminal region contains the ^D-domain that allows the binding of

extracellular signal-regulated kinase (ERK) that initiates activation of

RSK. The activation of the RSKs involves phosphorylation of serine and

threonine residues in the CTKD and linker region by ERK, which pro-

motes the recruitment of phosphoinositide-dependent protein kinase 1

(PDK1) to phosphorylate the NTKD, leading to RSK activation.⁴The

classical activation of RSKs1-3 by ERK and PDK1 occurs via the Ras/Raf/

MEK/ERK pathway and RSK4 has an alternative mechanism of activa-

tion.⁵RSK2 can also be activated via the tyrosine kinase cell-surface

receptor ﬁbroblast growth factor receptor 3 (FGFR3) that has also

been reported to promote ERK-RSK2 interaction and phosphorylation of

RSK2,^6,7In general, the RSKs phosphorylate a range of cytoplasmic and

nuclear substrates that regulate diverse cellular process such as cell-

high-degree of structural homology (73–80% sequence identity) espe-

cially in the kinase domains (78–90%); therefore, it would be expected

that the isoforms have overlapping substrates although there is also

increasing evidence of isoform speciﬁcity in mediating diverse cellular

processes.^1–3The aberrant expression and/or activity of RSKs have been

associated with several cancer types, and the 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 sur-

vival, and RSK3 and RSK4 acting as tumor suppressors through multiple

cellular mechanisms.^8–10

The investigation of the role of the RSKs in cancer, and the identi-

ﬁcation of overlapping and isoform-selective substrates has been hin-

dered by the lack of optimal loss-of-function genetic tools and small

molecule RSK inhibitors.¹¹The pyrrolopyrimidine FMK (ﬂuo-

romethylketone) is an irreversible inhibitor that covalently binds a

cysteine residue in the ATP-binding site of the CTKD of RSK1, RSK2, and

RSK4 (the cysteine residue is absent in RSK3).¹²Although FMK is a

potent and surprisingly speciﬁc RSK inhibitor for a cysteine alkylating

agent, its application is limited since it is not effective once the kinase is

* Corresponding author.

E-mail address: philip.reigan@cuanschutz.edu (P. Reigan).

https://doi.org/10.1016/j.bmc.2021.116220

Received 26 March 2021; Received in revised form 11 May 2021; Accepted 16 May 2021

Available online 19 May 2021

K.A. Casalvieri et al.

Bioorganic & Medicinal Chemistry 41 (2021) 116220

activated and the CTKD is not always required for RSK activation at the

NTKD.^12,13The dihydropteridinone BI-D1870 (Fig. 1) is a reversible

ATP-competitive pan-RSK inhibitor that binds the ATP-binding site of

the NTKD. BI-D1870 is remarkably selective for the RSKs compared with

other AGC kinases,¹⁴its in vitro IC₅₀against the RSKs is 15–30 nM at 100

core heterocycle eliminates the interaction with Asp148 in the hinge

domain that is observed with the anilino amine in BI-D1870. Therefore,

building off this observation and our previous structure–activity study,

we describe a series of substituted purines and pyrrolopyrimidines that

maintain the diﬂuorophenol and extend substitutions from the N⁹of the

purines and N⁷of the pyrrolopyrimidines towards the RSK substrate

binding site to probe additional interactions that may improve RSK

inhibitory potency and could be targeted to improve RSK selectivity. The

series was evaluated for their ability to inhibit RSK2 activity and their

impact on cell viability in AML cells. Our studies support that the

pteridinone core of BI-D1870 can be replaced by purine and pyrrolo-

pyrimidine heterocycles while maintaining RSK inhibitory activity.

μ

M ATP,¹¹and ~10

μM is required to completely inhibit RSK activity in

cells.¹¹Although some of the cell signaling effects of BI-D1870 have

been attributed to non-speciﬁc interactions,^15,16it has narrow-band

activity within the kinome with PLK1, Aurora B, MELK, and MST2

among its other targets.^11,14,17BI-D1870 has displayed high clearance

and a short plasma half-life in in vivo models and this has limited its

evaluation in advanced preclinical in vivo cancer models.^18,19The

impressive potency and selectivity of BI-D1870 led to the development

of LJI308 (IC₅₀3–13 nM) and LJH685 (IC₅₀4–6 nM); however, these

compounds act more as chemical probes than therapeutic candidates

and also suffer from poor PK proﬁles.^20,21

2. Results and discussion

2.1. Computational-based inhibitor design

Previously, we reported a structure–activity study of BI-1870 to

determine the structural features that are required for RSK inhibi-

tion.^22,23Our studies revealed that halogen substitutions on the 4-

hydroxyanilino group improved RSK inhibitory potency with the

diﬂuorophenol conferring the most potent activity, the elimination of C⁷

chirality did not impact RSK inhibitory potency, removal of the N⁵-

methyl maintained RSK inhibitory activity, and the N⁸-alkyl substitution

was required for potent RSK inhibition. Importantly, we identiﬁed

compounds that demonstrated potent inhibition of cellular RSK2 activ-

ity, but this did not result in cytotoxicity in the MOLM-13 acute myeloid

leukemia (AML) cell line. In contrast, BI-D1870 inhibited cellular RSK2

and displayed potent cytotoxicity in MOLM-13 cells, which could be

attributed to interactions with additional molecular targets.²⁴The

removal of C⁷chirality in the pteridinone and/or the N⁵-methyl resulted

in compounds with equivalent RSK2 inhibitory potency as BI-D1870 and

support that alternative heterocyclic ring systems could be used to

replace the dihydropteridinone. A recent study presented a series of

azaindoles, based on LJI308 and LJH685, and displayed similar RSK

inhibitory potency (Fig. 1).²⁵In the azaindole series the diﬂuorophenol

was directly coupled to the C⁵of the azaindole ring, to adopt a similar

propeller-shaped conformation as reported for LJI308 and LJH685.^21,25

This restricted non-coplanar conformation of the azaindoles and LJI308

and LJH685 forms a C-shaped clamp around the DFG motif, and this

could contribute to the potency and impressive selectivity of these

compounds. However, the direct coupling of the diﬂuorophenol to the

The docked structure of BI-D1870 in the ATP-binding site of the

RSK2 NTKD was used as the basis for the design of candidate RSK in-

hibitors (PDB: 4NUS, Fig. 2). Based on the ﬁndings from our previous

SAR investigation of BI-D1870, the main objective of this study was to

develop a series of purine and pyrrolopyrimidines to replace the chiral

dihydropteridinone core and to examine N-substitutions that could be

accommodated and extend towards the RSK2 substrate domain. For

direct comparison, the isopentyl-, methylpiperazinylphenyl-, and mor-

pholinophenyl- substituents of BI-D1870, LJH685, and LJI308 were ﬁrst

added to both the purine (43 and 44) and pyrrolopyrimidine cores (10,

11, and 60). In all cases, the diﬂuorophenolamino was incorporated at

the C2 position of the purine and pyrrolopyrimidines. Although the

isolation of methylpiperazinylphenyl-purine was unsuccessful, we were

able to obtain the corresponding methylpiperazinyl-pyrrolopyrimidine

(11).²⁶The molecular docking of the methylpiperazinylphenyl-

substituted pyrrolopyrimidine 11 into the ATP-binding site of the

RSK2 NTKD indicated that the compound would maintain the H-bond

interaction between the anilino and Asp148 in the hinge domain and

form an additional interaction between the methylpiperazinyl and

Phe212 of the DFG motif (Fig. 3), which could improve both potency

and selectivity for the RSKs and is therefore an attractive compound for

evaluation. The purine and pyrrolopyrimidine analogs without an N⁹

and N⁷substituent were also synthesized as 64 and 65, respectively, in

order to demonstrate the requirement of an appropriate substituent

within the space bordering the RSK substrate domain. From this initial

series of purines and pyrrolopyrimidines with N-unsubstituted, N-iso-

pentyl-, N-methylpiperazinylphenyl-, and N-morpholinophenyl-

substituted side chains, the N-substituted purine-based compounds were

equivalent or more potent RSK inhibitors than the corresponding pyr-

rolopyrimidines (Tables 1 & 2, Supplemental Fig. S1); therefore, the

purine core was used for subsequent N-substituted probes.

F

OH

HO

F

N

O

N

F

N

H

To expand the purine series, a 2-carbon spacer was inserted between

the benzene and morpholino rings of the 4-morpholinophenyl side chain

of 44 to give 45 and examine if the introduction of ﬂexibility to the

morpholino allows a more favorable binding conformation. From the

computational-based docking, additional aromatic rings systems

substituted from the N⁹-position of the purine have the potential for

interaction with Phe212 of the DFG motif (Fig. 2). Furthermore,

extending an aromatic substitution from the N⁹-position of the purine

out from the ATP-binding site towards the substrate domain may be

favored as there is a hydrophobic region from Phe79 to Val82; therefore,

we aimed to probe this region by sequentially adding complexity to the

side chain. Beginning with a biphenyl ring system of 46, complexity

and/or ﬂexibility were added as the para-phenoxy- of 48 and para-

benzyloxy- of 50. It has previously been reported that ortho-substituted

moieties in similarly oriented RSK inhibitors were not well-tolerated

while meta- and para-substitutions were favored.²¹For this reason, we

also proposed the meta-substituted phenoxy- (47) and benzyloxy- (49)

phenylpurines. While the para- and meta- benzyloxyphenyl-purines were

N

BI-D1870 (1)

LJH685 (2)

F

OH

F

N

F

N

O

HN

LJI308 (3)

2,6-difluoro-4-(4-(4-(4-methylpiperazin

-1-yl)phenyl)-1H-pyrrolo[2,3-c]pyridin-5

-yl)phenol (4)

Fig. 1. Chemical structures of the known RSK inhibitors. BI-D1870 (1),

LJH685 (2), LJI308 (3), and the azaindole 2,6-diﬂuoro-4-(4-(4-(4-methyl-

piperazin-1-yl)phenyl)-1H-pyrrolo[2,3-c]pyridin-5-yl)phenol (4).

2

K.A. Casalvieri et al.

Bioorganic & Medicinal Chemistry 41 (2021) 116220

Fig. 2. BI-D1870 docked to the ATP-site of

the NTKD of RSK2. (A) Ribbon representation

of the RSK2 NTKD with amino acid residues of

the hinge domain and DFG motif displayed in

stick form (grey) and the docked R-isomer of BI-

D1870 (orange). (B) Interactions of BI-D1870

(orange) with residues (grey) in the ATP-

binding site and the potential to extend N-sub-

stitutions to the Lys195 residue. H-bonds shown

as yellow dashed lines and aromatic H-bonds

shown as cyan dashed lines. (C) Connelly sur-

face representation of the ATP-binding site to

show available space to extend N-substitutions.

Lys100

B

Leu150

Asp148

A

Asp211

Phe212

Gly213

Hinge

domain

Lys195

DFG

motif

Lys100

C

Asp148

Leu150

Asp211

Phe212

Lys195

A

B

C

Lys100

Asp148

Asp211

Phe212

Asp211

Phe212

Asp211

Phe212

Leu150

Gly213

Protonated N can

form a pi-cation

bond with Phe212

Lys195

Fig. 3. Predicted binding of N-substituted purines and pyrrolopyrimidines. Stick representation of critical residues (grey) in the ATP-binding site of the NTKD of

RSK2 with docked conformations of (A) 43 and 60, (B) 44 and 10, and (C) 11. H-bonds shown as yellow dashed lines, aromatic H-bonds shown as cyan dashed lines,

and pi-cation interactions shown as green dashed lines. Overall, there was no predicted difference in purine and pyrrolopyrimidine binding conformation, but an

additional pi-cation interaction was observed between the N-methylpiperazinylphenyl of 11 and Phe212 of the DFG motif.

subsequently deprotected to the phenols to give 54 and 53, respectively,

and examine the potential H-bond interaction with Asn198, the pro-

tected intermediates may also form a pi-pi interaction with Phe156.

Additionally, the amino acid residues adjacent to the DFG and hinge

region that border the ATP-binding site and the substrate domain

include Gln76, Asn198, and Lys195 could be targetable with N⁹sub-

stituents containing aromatic ring systems and acidic protons for H-bond

donor interactions. Terminal carboxylic acid and dimethylamino

groups, which are protonated at physiological pH, extending from the

N⁹-phenyl ring by a two- or three-carbon linker were proposed for their

potential to form anchoring H-bond interactions with charged/polar

amino acids; however, attempts to synthesize these compounds were

unsuccessful. The synthesis of the corresponding esters of the proposed

carboxylic acids as 51 and 52 was achieved.

2.2. Chemical synthesis of pyrrolopyrimidines and purines

From our previous SAR study of BI-D1870,^22,23we believe it will be

important to retain the 2,6-diﬂuorophenol-4-amino at the C²position of

all the proposed purines and pyrrolopyrimidines. Therefore, benzyl

alcohol was reacted with sodium hydride followed by addition of 1,2,3-

triﬂuoro-5-nitrobenzene (55) to afford benzyloxy-protected intermedi-

ate 56. The protected intermediate was subsequently treated with 25%

w/w Pd/C in order to deprotect the phenol and reduce the nitro group

yielding 4-amino-2,6-diﬂuorophenol (57) (Supplemental Scheme S1).

In general, pyrrolopyrimidines were synthesized via a Chan Lam

coupling of 2-chloro-7H-pyrrolo[2,3-d]pyrimidine ²⁶and the necessary

boronic acid to yield intermediates 8 and 9 (Scheme 1). Both in-

26

termediates as well as 2-chloro-7H-pyrrolo[2,3-d]pyrimidine

were

each coupled to 4-amino-2,6-diﬂuorophenol 57 using the TFA/TFE-

3

K.A. Casalvieri et al.

Bioorganic & Medicinal Chemistry 41 (2021) 116220

Table 1

Table 2

Inhibition of RSK2 activity by N-substituted purines. Inhibitory activity of syn-

thesized purine compounds in a recombinant kinase assay against RSK2 given as

the half-maximal inhibitory concentrations (IC₅₀); values are the mean ± S.D. (n

Inhibition of RSK2 activity by N-substituted pyrrolopyrimidines. Inhibitory ac-

tivity of synthesized pyrrolopyrimidine compounds in a recombinant kinase

assay against RSK2 given as the half-maximal inhibitory concentrations (IC₅₀);

= 3).

values are the mean ± S.D. (n = 3).

Compound

R

RSK2 IC₅₀(nM)

Compound

R

RSK2 IC₅₀(nM)

BI-D1870

–

H

22.6 ± 6.0

65

60

H

77% @ 10 μM

64

43

67% @ 10

μ

M

95.8 ± 11

55.5 ± 5.6

10

25.7 ± 2.4

44

45

46

24.6 ± 3.0

18.4 ± 4.0

49% @ 10

11

34.7 ± 1.3

μ

M

purines 49 and 50 underwent a subsequent Pd-mediated benzyl

deprotection to generate the phenol analogs 53 and 54. To synthesize

N⁹-unsubstituted purine, 2-amino-6-chloropurine (61) underwent a

Balz-Scheinmann reaction to generate the dihalogenated purine 62

(Supplemental Scheme S3). Palladium hydroxide was then utilized to

selectively remove the chlorine substituent to yield the ﬂuorinated

heterocycle 63, which can then be coupled to 4-amino-2,6-diﬂuorophe-

nol (57) using the TFA/TFE-mediated S_NAr reaction to generate 64.

47

48

329 ± 37

148 ± 27

49

50

63% @ 10

42% @ 10

μ

M

2.3. Kinase inhibition assay

The synthesized compounds were evaluated for their effect on RSK2

activity in a recombinant TR-FRET kinase inhibition assay and their half-

maximal inhibitory potency values (IC₅₀

) were calculated using

nonlinear regression analysis of the log dose–response (Tables 1 & 2,

Supplemental Fig. S1). As positive controls, BI-D1870, LJH695, and

LJI308 were tested in the TR-FRET assay and resulted in IC₅₀values of

22.6 ± 6.0 nM, 8.0 ± 1.2 nM, and 7.1 ± 0.8 nM, respectively. The trend

in RSK2 inhibitory potency for the N-unsubstituted, N-isopentyl-, and N-

morpholinophenyl- for the purine and pyrrolopyrimidine heterocyclic

cores was similar, with the N-morpholinophenyl- being the most potent

and the N-unsubstituted being the least potent; however, the N-iso-

pentyl- side chain demonstrated improved potency for the purine over

the corresponding pyrrolopyrimidine. Interestingly, the pyrrolopyr-

imidine 11 containing the methylpiperazinylphenyl side chain of

LJH685 resulted in a reduction in RSK2 inhibitory potency in compar-

ison with the morpholinophenyl side chain. Overall, these ﬁndings

conﬁrmed that the synthesis of the N⁹-methylpiperazinylphenyl-purine

as well as pyrrolopyrimidines with the remaining proposed side chains

was unnecessary as the purine ring could serve as the core heterocycle to

probe N⁹substitutions.

53

54

51

79.8 ± 4.6

81.8 ± 0.7

15.7 ± 4.0

52

17.8 ± 1.0

mediated S_NAr reaction to yield the ﬁnal pyrrolopyrimidines 65, 10, 11.

Furthermore, the isopentyl compound was generated via coupling of the

2-chloro-7H-pyrrolo[2,3-d]pyrimidine ²⁶with 1-bromo-3-methylbutane

to yield intermediate 59, followed by 4-amino-2,6-diﬂuorophenol 57

coupling via the TFA/TFE-mediated S_NAr reaction to yield ﬁnal com-

pound 60 (Supplemental Scheme S2).

The introduction of ﬂexibility for the morpholino substituent via a

two-carbon linker in 45 resulted in a modest improvement in RSK2

inhibitory activity compared with the more restricted 44. Overall, the

efforts to improve RSK inhibitory potency through introduction of an

additional pi-pi interaction were unsuccessful. While adding ﬂexibility

to biphenyl ring of 46 through para-phenoxy in 48 and meta-phenoxy in

47 improved potency, this activity was still not comparable to that of BI-

D1870. Although the para- and meta- substituted benzyloxy purines

appear to be too rigid or conformationally restricted of a substituent for

the pi-pi interaction to occur, the corresponding free phenols 54 and 53

did show some improvement in inhibitory activity against RSK and may

warrant further investigation. Surprisingly, in this assay the esters 51

and 52 exhibited improvement in RSK2 inhibitory activity over BI-

The purines were generated by ﬁrst treating 2,4-dichloro-5-nitropyr-

imidine ²⁶with the necessary aniline in order to selectively couple the

aniline at the 4-position of the pyrimidine (13–22, Scheme 2). The

nitropyrimidines were then reduced to diamines 23–32 using tin(II)

chloride, which were subsequently reacted with triethyl orthoformate

under microwave irradiation to yield cyclized purines 33–42. 4-Amino-

2,6-diﬂuorophenol (57) was coupled to the purines using a triﬂuoro-

acetic acid (TFA) and triﬂuoroethanol-mediated S_NAr reaction accord-

ing to Carbain et al.,²⁷to generate ﬁnal compounds 43–52. Additionally,

4

K.A. Casalvieri et al.

Bioorganic & Medicinal Chemistry 41 (2021) 116220

F

R

HO

F

b

a

N

+

HO

B

N

Cl

N

Cl

N

H

OH

H

5

6; R= NCH₂CH₂O

7; R= NCH₂CH₂NCH₃

R

8; R= NCH₂CH₂O

10; R= NCH₂CH₂O

9; R= NCH₂CH₂NCH₃

11; R= NCH₂CH₂NCH₃

Scheme 1. Chemical synthesis of N-substituted pyrrolopyrimidines. Reagents and Conditions: (a) pyridine, Cu(OAc)₂, rt, 3 days; (b) 4-amino-2,6-diﬂuorophe-

nol, TFA, TFE, MW, 140 ^◦C, 1.5 h.

2.4. Cellular viability effects of RSK2 inhibitors

NO₂

Cl

NO₂

NH₂

a

b

N

The compounds that exhibited activity in the recombinant RSK2

kinase activity assay within the concentration range of BI-D1870 were

assessed in cell systems for effects on cell viability, as well as their ability

to inhibit cellular RSK2 activity. For direct comparison, the N-

substituted isopentyl and morpholinophenyl purines, 43 and 44, and

pyrrolopyrimidines, 60 and 10, as well as the methylpiperazinylphenyl

pyrrolopyrimidine 11 were tested in an MTS cell viability assay in

MOLM-13 cells (Fig. 4, Table 3). We have previously shown that the

MOLM-13 AML cell line expresses high level of RSK2 and activated RSK2

protein.^22,23

R

N

R

N

Cl

N

Cl

N

Cl

N

H

12

13; R=

14; R=

15; R=

16; R=

17; R=

18; R=

19; R=

20; R=

21; R=

22; R=

23; R=

24; R=

25; R=

26; R=

27; R=

28; R=

29; R=

30; R=

31; R=

32; R=

O

N

O

N

O

The morpholinophenyl purine containing an additional two-carbon

linker 45 as well as the two methyl ester purines, 51 and 52, that

demonstrated improved RSK2 inhibitory activity compared with BI-

D1870 were also evaluated in the cell viability assay. Following treat-

ment with inhibitors for 72 h, both isopentyl-substituted heterocycles

(43 and 60), the morpholinophenyl purine ²⁶and the morpholinophenyl

purine with an additional two-carbon linker (45) all demonstrated

similar effects on MOLM-13 cell viability as BI-D1870. Surprisingly,

morpholinophenyl and methylpiperazinylphenyl pyrrolopyrimidines 10

and 11 showed potent cytotoxic effects in the MTS assay. The potent RSK

inhibitors LJH685 and LJI308 demonstrated minimal effects on cell

viability. Two carbon-linked ester 51 showed no effect on cellular

O

F

HO

F

N

c

d

N

Cl

N

H

R

33; R=

34; R=

35; R=

36; R=

37; R=

38; R=

39; R=

40; R=

41; R=

42; R=

43; R=

44; R=

45; R=

46; R=

47; R=

48; R=

49; R=

50; R=

51; R=

52; R=

viability up to 60 μM while the three carbon-linked ester 52 displayed

minimal effects similar to that of LJH685 and LJI308.

O

N

O

N

O

2.5. Inhibition of cellular RSK2 activity

Inhibitors of puriﬁed RSK2 activity identiﬁed in the TR-FRET assay

were also assessed for their capacity to inhibit cellular RSK2 using the

NanoBRET™ intracellular kinase assay (Fig. 5). The cellular RSK2 ac-

tivity assay was performed by Reaction Biology Corporation using

HEK293 cells transfected with RSP6KA3-nanoLuc fusion vector and

O

e

O

53; R=

54; R=

OH

Scheme 2. Chemical synthesis of N-substituted purines. Reagents and

Conditions: (a) aniline, DCM, ꢀ 15 ^◦C, 10 min; (b) SnCl₂, EtOH, reﬂux, 2 h; (c)

triethyl orthoformate, TFA, TFE, MW, 140 ^◦C, 1.5 h; (d) 4-amino-2,6-diﬂuoro-

phenol, TFA, TFE, MW, 140 ^◦C, 1.5 h; (e) NH₄HCO₂, Pd/C (20%), DMF, MW,

80 ^◦C, 15 min.

D1870. We had intended to synthesize the corresponding carboxylic

acids but various attempts were unsuccessful and we had expected the

methyl esters to block anchoring H-bond interactions resulting in

decreased RSK2 inhibitory potency.

Fig. 4. Cellular effects of RSK inhibitors on viability 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 (n = 3, error bars: ± S.D.).

5

K.A. Casalvieri et al.

Bioorganic & Medicinal Chemistry 41 (2021) 116220

The pan-RSK inhibitor BI-D1870 and 52 were the most potent in-

Table 3

Cellular effects of RSK inhibitors on viability in MOLM-13

hibitors of cellular RSK2 activity and demonstrated dose-dependent

cells. Calculated EC₅₀for RSK inhibitors treated over a con-

centration range from 60 µM to 250 nM in an MTS assay (n =

3, error bars: ± S.D.).

inhibition from 0.2 μM. The N-isopentyl substituted purine 43 demon-

strates more potent inhibition of cellular RSK activity than the corre-

sponding pyrrolopyrimidine 60. The N⁷-substituted morpholinophenyl

and methylpiperazinylphenyl pyrrolopyrimidines 10 and 11 demon-

strated a concentration-dependent reduction of cellular RSK2 activity;

however, the N⁹-substituted morpholinophenyl purine 44 demonstrated

Compound

MTS 72 hr EC₅₀(µM)

BI-D1870 ²⁶

2.60 ± 0.6

LJH685 ²⁶

96% @ 60 µM

87% @ 60 µM

1.30 ± 0.06

0.23 ± 0.003

0.07 ± 0.003

0.97 ± 0.1

LJI308 ²⁶

60

more potent RSK2 inhibition at lower concentrations 3–6 μM and this

would suggest that 44 has improved RSK selectivity compared with 10.

Taken together, these results for 60 and 44 conﬁrmed the preference for

the purine over the pyrrolopyrimidine as the core heterocycle. The more

ﬂexible morpholino substituent of 45 did not result in improved cellular

RSK2 inhibition over the more rigid morpholinophenyl of 44. Surpris-

ingly divergent results for cellular RSK2 inhibition were obtained for the

methyl esters 51 and 52. The phenylethyl methyl ester 51 demonstrated

no cellular RSK2 inhibition over the concentration range and when

combined with the MTS data this would suggest that 51 has poor cell

penetration. In contrast, the phenylpropyl methyl ester 52 demonstrated

the most potent inhibition of cellular RSK2 activity of all the compounds

evaluated. This result with 52 supports the ﬁndings from our previous

study,²³that RSK2 inhibition can be achieved without concomitant

cytotoxicity. It is important to note that 52 is likely to be prone to ester

hydrolysis in cells resulting in the corresponding carboxylic acid, that

may also be a RSK2 inhibitor (Fig. 6); however, efforts to synthesize the

carboxylic acid derivative were unsuccessful.

10

11

43

44

53% @ 60 µM

1.9 ± 0.07

45

51

5% @ 60 µM

13.9 ± 0.7

52

A

3. Conclusions

The central discovery from our previous structure–activity study of

BI-D1870 was that LJH685, LJI308, and some pteridinones demon-

strated inhibition of cellular RSK2 activity, but this did not result in

potent and acute cytotoxicity in the MOLM-13 AML cell line.²³Although

it may be possible that some cells are more sensitive to RSK inhibition,

BI-D1870 also inhibited cellular RSK2 activity, but displayed potent and

acute cytotoxicity in MOLM-13 cells likely due to interaction with

additional molecular targets.²⁴This result with BI-D1870 is concerning

as it is commonly used as a chemical tool for pan-RSK inhibition; how-

ever, overall our study supported that there is further scope to develop

RSK inhibitors and that RSK inhibition alone may not be sufﬁcient to

result in cytotoxicity. The objective of our current study was to replace

the pteridinone ring to remove the puckered piperazine ring and

isomeric issues, to retain the anilino group for H-bond interactions with

Asp148, to explore the feasibility of N-substituents that extend to the

substrate domain, and to identify any inhibitors that had minimal

cytotoxicity. A total of 17 novel pyrrolpyrimidines and purines were

synthesized and isolated that explore N-substitutions that extend from

the ATP-binding pocket to the kinase domain. These compounds elimi-

nate the isomeric pteridinone core of BI-D1870 but retain the 2,6-

diﬂuorophenol-4-amino group the maintains the H-bond interactions

between the amino and Asp148 and the phenol and Asp211 of the DFG

motif. The results from recombinant and cellular RSK2 activity assays

supported a preference for the purine over the pyrrolopyrimidine core

when comparing the N-substituted isopentyl and morpholinophenyl

compounds. Of the 17 compounds generated, 7 demonstrated potent

inhibition of RSK2 activity and demonstrated cellular target engage-

ment; however, only 52 showed increased potency of cellular RSK2 in-

hibition compared to BI-D1870 without concomitant cytotoxic effects.

Effects on cell viability were only observed with 52 at high concentra-

B

Fig. 5. Inhibition of cellular RSK2 activity by nanoBRET assay. Representative

BRET ratios are compared to DMSO and reported as percentage DMSO control.

Inhibitors were treated over a concentration range of 195 nM to 100 µM for 2 h

(n = 4, error bars: ± S.D.). (A) All inhibitors were analyzed in relation to BI-

D1870 and (B) potent inhibitors BI-D1870, 44 and 52 were analyzed in mul-

tiple comparisons by two-way ANOVA, ***P < 0.001; ****P < 0.0001.

results were normalized to DMSO control. Results from our previous

work showed equipotency amongst BI-D1870, LJH685, and LJI308 up to

tions > 10 μM after 72 hr of exposure. The compounds in this study serve

as initial chemical probes to examine the accommodation of N-sub-

stituents at the boundary between the ATP-binding site and substrate

binding site. Our pharmacophore model supports that the 2,6-diﬂuoro-

phenol-4-amino group and the pyrimidine ring are needed RSK2

inhibitory activity but the optimal N-substituent has yet to be identiﬁed

(Fig. 6); therefore, there is scope for the further inhibitor development.

40 μM in this assay; therefore, compounds were compared only with BI-

D1870 as a positive control.²³All of the compounds tested displayed

dose-dependent inhibition of cellular RSK2 activity from 3 μM, with the

exception of 51, which demonstrated no RSK2 inhibition over the con-

centration range (0.4–100 M).

μ

6

K.A. Casalvieri et al.

Bioorganic & Medicinal Chemistry 41 (2021) 116220

Fig. 6. Predicted binding of N-substituted purine

ester 52 and its corresponding acid. Stick repre-

sentation of critical residues (grey) in the ATP-

binding site of the NTKD of RSK2 with docked

conformations of (A) 52 and (B) its corresponding

acid. H-bonds shown as yellow dashed lines and

aromatic H-bonds shown as cyan dashed lines. (C)

C

A

Lys100

Asp148

Asp211

Phe212

Leu150

A

pharmacophore model comprised of the N-

substituted purines and pyrrolopyrimidines from

this study that indicates that the pyrimidine ring

coupled to the diﬂuorophenol is essential for RSK2

inhibitory activity. Intersite distances between the

pharmacophoric points of model HHRR_1. Grey

spheres are excluded volume shells, green spheres

are hydrophobes (H), and orange rings are aromatic

rings (R).

Gly213

Ester has the potential

to form an aromatic

H-bond with Phe212

Lys195

B

Lys100

Asp148

Asp211

Leu150

Phe212

Gly213

Terminal carboxylic

acid has the potential

to H-bond Lys195

Lys195

Collectively, our data for LJH685, LJI308, the pteridinones from our

previous study, and compound 52 from our current study supports that

RSK2 inhibition can be uncoupled with the potent cytotoxic effects

observed with BI-D1870. Furthermore, there is scope to develop RSK

inhibitors with optimized N-substituents, and that by extending inhibi-

tor interactions into the substrate binding site further RSK selectivity

and potency, and potentially RSK isoform selectivity could be achieved.

LJI308 in the ATP-binding site of the RSK NTKD were used as the basis

for the design of candidate RSK inhibitors, with the aim of introducing

substitutions to test the importance of interactions with residues in the

ATP-binding site. The synthesized purines and pyrrolopyrimidines that

demonstrated potent RSK inhibition in the TR-FRET kinase activity

assay were prepared and docked in the RSK crystal structure in order to

identify the critical interactions that resulted in RSK inhibition.

The Phase module was used for pharmacophore modeling.^32,33Pre-

viously prepared and docked BI-D1870, LJH685, LJI308, purines, and

pyrrolopyrimidines were imported with their respective IC₅₀values,

determined by the recombinant kinase TR-FRET assay for pharmaco-

phore hypothesis generation. Compounds with IC₅₀values accurately

determined by the recombinant kinase assay were deﬁned as active.

Compounds for which the recombinant kinase assay could not accu-

rately determine IC₅₀values against RSK2 were deﬁned as inactive. The

highest-ranking docked conformation of each compound from the pre-

viously performed Glide docking was used to compose a set of pre-

aligned ligands. Default values and feature settings were used for hy-

pothesis generation and the Phase hypo scoring function was used to

score and rank compounds and hypotheses. Phase calculated vector,

volume, site scores, survival scores, and survival activities to score and

rank 20 possible feature combinations able to produce common phar-

macophores. The highest-ranking hypothesis (HHRR_1) used 52 (the

most active molecule in the recombinant kinase assay) as the reference

ligand for hypothesis generation.

4. Experimental

4.1. Computational-based modeling

BI-D1870, LJH685, LJI308, purines, and pyrrolopyrimidines were

docked into the ATP-binding site of the NTKD of RSK2 (PDB: 4NUS)

crystal structure,²⁰using the Glide module within Schrodinger (Release

¨

28–30

¨

2020-3, Schrodinger LLC, New York, NY).

Prior to docking, the

water molecules were removed, and the proteins were 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 protein structure was performed using

the default constraint of 0.30 Å RMSD and the OPLS_2005 force ﬁeld.³¹

The prepared proteins were subjected to SiteMap analysis,³⁰that iden-

tiﬁed the ATP-binding site in the NTKD and docking grids were gener-

ated using Receptor Grid Generation. BI-D1870 and analogs 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.³¹

Molecular docking simulations were performed using the Glide ligand

docking module in XP (extra precision) mode and included post-docking

minimization.²⁹The docked structures of BI-D1870, LJH685, and

4.2. Chemistry

All melting points (MP) were determined using a Mettler Toledo

7

K.A. Casalvieri et al.

Bioorganic & Medicinal Chemistry 41 (2021) 116220

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 and the spin-

multiplicity abbreviated as: s (singlet), d (doublet), t (triplet), q (quar-

tet), quin (quintet), m (multiplet), bs (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 4000 V and the fragmentor voltage

was 200 V. Data was acquired over a range of m/z 50–1700. Fourier

Transform Infrared (FTIR) spectra were obtained using a Bruker Alpha

Platinum-ATR as a neat sample.

chromatography to afford the desired compound.

4.2.1. 6-Chloro-2-ﬂuoro-9H-purine (62)

2-Amino-6-cloropurine (61) (1.00 g, 5.90 mmol) was taken up in

HBF₄(20 mL, 48 wt% in H₂O, 0.3 M) and cooled to 0 ^◦C. A solution of

sodium nitrite (0.84 g, 11.8 mmol) in water (12 mL, 1 M) was added

dropwise over 30 min. Upon completion of addition the reaction mixture

was stirred at RT for 15 min, after which the solution was cooled to 0 ^◦C

and neutralized with 6 M NaOH. The product was then extracted with

EtOAc (3 × 15 mL) and the combined organic extracts were dried

(MgSO₄) and concentrated in vacuo to afford the desired compound as a

yellow solid (0.83 g, 4.81 mmol, 82%). Rf 0.53 (17:3 DCM: MeOH); M.p.

169–173 ^◦C (Lit. = 171–173 ^◦C) ³⁴; IR (cm^{ꢀ 1}) 3071, 2953, 2922, 2849,

2786, 1616, 1582; ¹H NMR (400 MHz, DMSO‑d₆) 8.71 (1H, s, H-8),

14.08 (1H, bs, NH); ¹³C NMR (100 MHz, DMSO‑d₆) 155.7 (Ar C), 157.8

–

General Procedure A: A solution of necessary aniline (1.1–2 equiv.,

as indicated) in DCM or EtOH (0.5–2 M) was added drop-wise to a so-

lution of 2,4-dichloro-5-nitropyrimidine (1.0 equiv.) in DCM (0.5–1 M)

stirring at ꢀ 15 ^◦C. The reaction was stirred for 15 min, concentrated in

vacuo and puriﬁed via gel chromatography, if necessary, to afford the

desired compound.

(Ar C). HRMS cal. C₅HClFN₄(ESꢀ ) m/z 170.987377 [Mꢀ H]^-, found

–

170.9845.

4.2.2. 2-Fluoro-9H-purine (63)

Palladium hydroxide (0.70 g, 20% wt./C) and ammonium formate

(0.77 g, 12.2 mmol) were added to a solution of chloro-purine 62 (0.70

g, 4.06 mmol) in MeOH (100 mL, 0.04 M). The reaction was heated

under reﬂux for 2 h before the catalyst was removed via ﬁltration over

Celite. The product was concentrated in vacuo and puriﬁed via silica gel

chromatography (9:1 DCM: MeOH) to afford the desired compound as a

as a white solid (0.33 g, 2.42 mmol, 60%). Rf 0.32 (9:1 DCM: MeOH); M.

p. 222–226 ^◦C (Lit. = 219 ^◦C³⁴); IR (cm^{ꢀ 1}) 3132, 3073, 3030, 2973,

2926, 2850, 2822, 2641, 2606, 1891, 1621, 1570; ¹H NMR (400 MHz,

General Procedure B: To a solution of nitropyrimidine precursor

(13–22) (1.0 equiv.) in EtOH (0.1 M) was added tin(II) chloride (4

equiv.). 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, 20 mL/mmol) and a saturated aqueous solution of NaHCO₃was

added until the aqueous phase reached pH 9–10. The resulting precip-

itate was removed via ﬁltration and the organic extracts were collected,

washed with brine (20 mL/mmol), and extracted with EtOAc:THF (1:1;

3 × 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.

DMSO‑d₆) 8.68 (1H, s, H-8), 9.03 (1H, s, H-6), 13.59 (1H, br s, NH); ¹³

C

–

NMR (100 MHz, DMSO‑d₆) 147.6 (Ar C), 148.4 (Ar C), 157.5 (Ar C),

159.6 (Ar C). HRMS cal. C₅H₄FN₄(ES+) m/z 139.041999 [M+H]⁺,

–

General Procedure C: The relevant diaminopyrimidine (23–32)

(1.0 equiv.), triethyl orthoformate (2.5 equiv.) and TFA (0.1 equiv.)

were taken up in TFE (0.2 M) and heated under microwave irradiation

conditions at 140 ^◦C for 1.5 h 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 × 15 mL/mmol). The

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

concentrated in vacuo. The resultant residue was puriﬁed via silica gel

chromatography to afford the desired compound.

found 139.0430.

4.2.3. 4-((9H-Purin-2-yl)amino)-2,6-diﬂuorophenol (64)

Purine 63 (79 mg, 0.543 mmol), aniline 57 (158 mg, 1.09 mmol) and

TFA (208 μL, 2.72 mmol) were taken up in TFE (5 mL) and reacted ac-

cording to the described General Procedure D. Puriﬁcation via silica gel

chromatography (9:1 DCM: MeOH) and subsequent trituration with

MeOH afforded the target compound as a brown solid (61.3 mg, 0.23

mmol, 43%). Rf 0.33 (9:1 DCM: MeOH); M. p. 250–251 ^◦C; IR (cm^{ꢀ 1}

)

3383, 3259, 1639, 1594, 1559, 1529, 1505; ¹H NMR (400 MHz,

DMSO‑d₆) 7.57 (2H, d, J = 10.4 Hz, H-3^′/5^′), 8.22 (bs, OH), 8.82 (H-6),

9.53 (C2-NH), 9.59 (H-8), 12.99 (H8-NH); ¹³C NMR (100 MHz,

General Procedure D: The relevant heterocycle (8,9, 33–42) (1.0

equiv.), amino-diﬂuorophenol 57 (2.0–2.5 equiv., as indicated) and TFA

(2.0–7.0 equiv.) were taken up in TFE (0.1 M) and heated under mi-

crowave irradiation conditions at 140 ^◦C for 1.5 h (unless otherwise

indicated) before being concentrated in vacuo. The residue was resus-

pended 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 × 15 mL/mmol). The combined

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

trated in vacuo. The resultant residue was puriﬁed via silica gel chro-

matography and/or triturated as speciﬁed to afford the desired

compound. ²⁷

–

DMSO‑d₆) 102.1 (d, J_CF= 28.5 Hz, Ar C), 127.4 (dd, J_CF= 17.1, 15.7

–

Hz, Ar C), 133.5 (dd, J_CF= 13.5,12.2 Hz, Ar C), 152.8 (dd, J_CF

=

–

237.5, 9.0 Hz, Ar C), 156.6 (Ar C). HRMS cal. C₁₁H₈F₂N₅O (ES+) m/z

264.069691 [M+H]⁺, found 264.0602.

4.2.4. 2-Chloro-N-isopentyl-5-nitropyrimidin-4-amine (13)

Isopentylamine (120 μL, 1.04 mmol, 2.0 equiv.) was taken up in

EtOH (0.5 mL, 2 M) and reacted with a solution of 2,4-dichloro-5-nitro-

pyrimidine (102 mg, 0.52 mmol) in DCM (0.8 mL, 0.7 M) according to

the described General Procedure A. Puriﬁcation via silica gel chro-

matography (9:1 Hexanes: EtOAc) afforded the target compound as a

yellow solid (0.35 g, 1.44 mmol, 80%). Rf 0.41 (4:1 Hexanes: EtOAc); M.

p. 55–57 ^◦C; IR (cm^{ꢀ 1}) 3349, 3046, 2953, 2867, 1604, 1567, 1509; ¹H

NMR (400 MHz, DMSO‑d₆) 0.92 (6H, d, J = 6.5 Hz, CH(CH₃)₂, 1.43–1.54

(2H, m, NHCH₂CH₂), 1.54–1.68 (1H, m, CH(CH₃)₂, 3.55 (2H, dt, J = 7.0,

6.9 Hz, NHCH₂CH₂), 9.00 (1H, s, H-6), 9.05–9.17 (1H, m, NH); ¹³C NMR

(100 MHz, DMSO‑d₆) 22.8 (CH(CH₃)₂), 25.7 (CH(CH₃)₂), 37.7

General Procedure E: The relevant benzyl-protected diﬂuorophenol

purine (49 and 50) (1.0 equiv.) was taken up in DMF (0.05 M) prior to

the addition of ammonium formate (5.0 equiv.) and Pd/C (20% w/w).

The reaction mixture was heated under microwave irradiation condi-

tions at 80 ^◦C for 15 min, ﬁltered, washed with MeOH (10 mL/mmol)

and the ﬁltrate was concentrated in vacuo. The resultant residue was

puriﬁed via silica gel chromatography and triturated with MeOH to

afford the desired compound.

–

(NHCH₂CH₂), 39.4 (NHCH₂CH₂), 127.6 (Ar C), 155.3 (Ar C), 157.7

–

General Procedure F: To a stirring solution containing 2-chloro-7H-

pyrrolo[2,3-d]pyrimidine (1 equiv.), the necessary boronic acid (1.3

equiv.) and copper acetate (1.1 equiv.) in CHCl₃(0.1 M) was added

pyridine (7.5 equiv.). The reaction was stirred for 3 days before being

concentrated in vacuo. The resultant residue was puriﬁed via column

(Ar C), 162.8 (Ar C). HRMS cal. C₉H₁₃ClN₄O₂(ES+) m/z 244.072704

[M+H]⁺, found 244.0788.

4.2.5. 2-Chloro-N4-isopentylpyrimidine-4,5-diamine (23)

Nitropyrimidine 13 (0.30 g, 1.23 mmol) and tin(II) chloride (0.96 g,

8

K.A. Casalvieri et al.

Bioorganic & Medicinal Chemistry 41 (2021) 116220

4.92 mmol) were taken up in EtOH (12 mL) and reacted according to

General Procedure B. Puriﬁcation via silica gel chromatography (24:1

DCM: MeOH) afforded the target compound as a brown solid (0.23 g,

1.07 mmol, 89%). Rf 0.2 (24:1 DCM: MeOH); M.p. 105–107 ^◦C; IR

(cm^{ꢀ 1}) 3433, 3357, 2950, 2869, 1650, 1585; ¹H NMR (400 MHz,

DMSO‑d₆) 0.91 (6H, d, J = 6.6 Hz, CH(CH₃)₂, 1.45 (2H, dt, J = 8.2, 7.0

Hz, NHCH₂CH₂), 1.57–1.73 (1H, m, CH(CH₃)₂, 3.26–3.47 (2H, m,

NHCH₂CH₂), 4.88 (2H, s, NH₂), 6.76 (1H, t, J = 5.6 Hz, NH), 7.36 (1H, s,

H-6); ¹³C NMR (100 MHz, DMSO‑d₆) 22.9 (CH(CH₃)₂), 25.7 (CH(CH₃)₂),

DCM: MeOH) afforded the target compound as a brown solid (88.1 mg,

0.29 mmol, 99%). Rf 0.19 (24:1 DCM: MeOH); M.p. 118–121 ^◦C; IR

(cm^{ꢀ 1}) 3324, 2916, 2855, 1597, 1570, 1508; ¹H NMR (400 MHz,

DMSO‑d₆) 3.08 (4H, m, N(CH₂CH₂)₂O), 3.74 (4H, m, N(CH₂CH₂)₂O),

5.19 (2H, s, NH₂), 6.95 (2H, d, J = 7.0 Hz, H-3^′′/5^′′), 7.50 (2H, d, J = 7.0

Hz, H-2^′′/6^′′), 7.57 (1H, s, H-6), 8.47 (1H, s, NH); ¹³C NMR (100 MHz,

–

DMSO‑d₆) 49.3 (NCH₂CH₂O), 66.6 (NCH₂CH₂O), 115.9 (Ar C), 122.6

–

(Ar C), 128.1 (Ar C), 131.5 (Ar C), 138.1 (Ar C), 146.6 (Ar C),

–

147.8 (Ar C), 151.2 (Ar C), 162.5 (Ar C). HRMS cal. C₁₄H₁₆ClN₅O

–

38.0 (NHCH₂CH₂), 39.1 (NHCH₂CH₂), 127.4 (Ar C), 136.0 (Ar C),

(ES+) m/z 305.104336 [M+H]⁺, found 305.1126.

–

147.5 (Ar C), 154.1 (Ar C). HRMS cal. C₉H₁₅ClN₄(ES+) m/z

214.098523 [M+H]⁺, found 214.1064.

4.2.10. 4-(4-(2-Chloro-9H-purin-9-yl)phenyl)morpholine (34)

Diaminopyrimidine 24 (0.11 g, 0.33 mmol), triethyl orthoformate

4.2.6. 2-chloro-9-isopentyl-9H-purine (33)

(0.14 mL, 0.82 mmol) and TFA (3 μL, 0.03 mmol) were taken up in TFE

Diaminopyrimidine 23 (0.22 g, 1.03 mmol), triethyl orthoformate

(1.6 mL) and reacted according to General Procedure C. Puriﬁcation

via silica gel chromatography (4:1 DCM: MeOH) afforded the target

compound as a brown solid (98.0 mg, 0.31 mmol, 95%). Rf 0.58 (19:1

DCM: MeOH); M.p. 195–197 ^◦C; IR (cm^{ꢀ 1}) 3107, 1613, 1595, 1560,

1520; ¹H NMR (400 MHz, DMSO‑d₆) 3.22 (4H, t, J = 4.7 Hz, N

(CH₂CH₂)₂O), 3.78 (4H, t, J = 4.7 Hz, N(CH₂CH₂)₂O), 7.17 (2H, d, J =

8.5 Hz, H-3^′′/5^′′), 7.64 (2H, d, J = 8.5 Hz, H-2^′′/6^′′), 8.94 (1H, s, H-8),

9.19 (1H, s, H-6); ¹³C NMR (100 MHz, DMSO‑d₆) 55.4 (NCH₂CH₂O),

(0.43 mL, 2.58 mmol) and TFA (8 μL, 0.10 mmol) were taken up in TFE

(5 mL) and reacted according to General Procedure C. Puriﬁcation via

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

compound as a yellow oil (0.20 g, 0.91 mmol, 88%). Rf 0.56 (19:1 DCM:

MeOH); IR (cm^{ꢀ 1}) 3083, 2955, 2873, 1582, 1506; ¹H NMR (400 MHz,

DMSO‑d₆) 0.93 (6H, d, J = 6.6 Hz, CH(CH₃)₂, 1.13–1.61 (1H, m, CH

(CH₃)₂, 1.75 (2H, dt, J = 8.1, 7.4 Hz, NHCH₂CH₂), 4.26 (2H, t, J = 8.1

Hz, NHCH₂CH₂), 8.71 (1H, s, H-8), 9.08 (1H, s, H-6); ¹³C NMR (100

MHz, DMSO‑d₆) 22.6 (CH(CH₃)₂), 25.6 (CH(CH₃)₂), 38.2 (NHCH₂CH₂),

–

66.4 (NCH₂CH₂O), 115.8 (Ar C), 125.3 (Ar C), 125.3 (Ar C), 133.9

–

(Ar C), 147.7 (Ar C), 150.8 (Ar C), 151.5 (Ar C), 153.2 (Ar C),

–

42.2 (NHCH₂CH₂), 133.5 (Ar C), 148.6 (Ar C), 150.2 (Ar C), 153.1

153.7 (Ar C). HRMS cal. C₁₅H₁₄ClN₅O (ES+) m/z 315.088686

(Ar C), 153.6 (Ar C). HRMS cal. C₁₀H₁₃ClN₄(ES+) m/z 224.082874

[M+H]⁺, found 315.0963.

–

[M+H]⁺, found 224.089.

4.2.11. 2,6-Diﬂuoro-4-((9-(4-morpholinophenyl)-9H-purin-2-yl)amino)

phenol (44)

4.2.7. 2,6-Diﬂuoro-4-((9-isopentyl-9H-purin-2-yl)amino)phenol (43)

Purine 33 (81.5 mg, 0.33 mmol), aniline 57 (98.5 mg, 0.67 mmol)

Purine 34 (76.9 mg, 0.24 mmol), aniline 57 (69.5 mg, 0.48 mmol)

and TFA (128

μL, 1.67 mmol) were taken up in TFE (3.3 mL) and reacted

and TFA (136 μL, 1.19 mmol) were taken up in TFE (2.4 mL) and reacted

according to General Procedure D. Puriﬁcation via silica gel chroma-

tography (4:1 DCM: MeOH) afforded the target compound as a brown

solid (56.4 mg, 0.17 mmol, 47%). Rf 0.23 (19:1 DCM: MeOH); M.p.

203–205 ^◦C; IR (cm^{ꢀ 1}) 3258, 3032, 2950, 1606, 1516; ¹H NMR (400

MHz, DMSO‑d₆) 0.97 (6H, d, J = 6.7 Hz, CH(CH₃)₂, 1.48–1.64 (1H, m,

CH(CH₃)₂, 1.78 (2H, dt, J = 7.6, 7.2 Hz, NHCH₂CH₂), 4.20 (2H, t, J =

7.6 Hz, NHCH₂CH₂), 7.60 (2H, d, J = 10.1 Hz, H-3^′/5^′), 8.29 (1H, s, H-

8), 8.82 (1H, s, H-6), 9.51 (1H, s, NH), 9.72 (1H, s, OH); ¹³C NMR (100

MHz, DMSO‑d₆) 22.6 (CH(CH₃)₂), 25.7 (CH(CH₃)₂), 38.4 (NHCH₂CH₂),

according to General Procedure D. Puriﬁcation via silica gel chroma-

tography (19:1 DCM: MeOH) afforded the target compound as a brown

solid (30.1 mg, 0.07 mmol, 29%). Rf 0.24 (19:1 DCM: MeOH); M.p.

284–288 ^◦C; IR (cm^{ꢀ 1}) 3260, 3095, 2963, 1610, 1524; ¹H NMR (400

MHz, DMSO‑d₆) 3.19 (4H, t, J = 4.8 Hz, N(CH₂CH₂)₂O), 3.78 (4H, t, J =

4.8 Hz, N(CH₂CH₂)₂O), 7.13 (2H, d, J = 8.4 Hz, H-3^′′/5^′′), 7.56 (2H, d, J

= 10.1 Hz, H-3^′/5^′), 7.70 (2H, d, J = 8.4 Hz, H-2^′′/6^′′), 8.55 (1H, s, H-8),

8.93 (1H, s, H-6), 9.52 (1H, s, NH), 9.77 (1H, s, OH); ¹³C NMR (100

MHz, DMSO‑d₆) 48.7 (NCH₂CH₂O), 66.5 (NCH₂CH₂O), 102.4 (d, J_CF

=

–

– – – –

27.7 Hz, Ar C), 115.8 (Ar C), 124.8 (Ar C), 126.4 (Ar C), 127.6

41.7 (NHCH₂CH₂), 102.2 (d, J_CF= 27.5 Hz, Ar C), 127.5 (dd, J_CF

=

–

– –

(dd, J_CF= 16.9, 16.4 Hz, Ar C), 128.8 (Ar C), 133.1 (dd, J_CF= 14.1,

17.0, 16.5 Hz, Ar C), 128.6 (Ar C), 133.3 (dd, J_CF= 12.9, 12.6 Hz,

–

– – – –

13.5 Hz, Ar C), 143.4 (Ar C), 150.0 (Ar C), 151.0 (Ar C), 152.2

Ar C), 144.6 (Ar C), 149.5 (Ar C), 152.5 (Ar C), 152.7 (dd, J_CF

=

–

238.2, 9.0 Hz, Ar C), 156.3 (Ar C). HRMS cal. C₁₆H₁₇F₂N₅O (ES+) m/

(Ar C), 152.7 (dd, J_CF= 238.8, 7.9 Hz, Ar C), 156.7 (Ar C). HRMS

z 333.140115 [M+H]⁺, found 333.1475.

cal. C₂₁H₁₈F₂N₆O₂(ES+) m/z 424.145930 [M+H]⁺, found 424.152.

4.2.8. 2-Chloro-N-(4-morpholinophenyl)-5-nitropyrimidin-4-amine (14)

4-Morpholinoaniline (0.89 g, 5.16 mmol, 2.0 equiv.) was taken up in

DCM (2.6 mL, 2 M) and reacted with a solution of 2,4-dichloro-5-nitro-

pyrimidine (0.52 g, 2.58 mmol) in DCM (3.7 mL, 0.7 M) according to the

described General Procedure A. Puriﬁcation via silica gel chromatog-

raphy (7:3 Hexanes: EtOAc) afforded the target compound as a brown

solid (0.77 g, 2.29 mmol, 89%). Rf 0.32 (2:1 Hexanes: EtOAc); M.p.

184–187 ^◦C; IR (cm^{ꢀ 1}) 3289, 2948, 2836, 1618, 1576, 1506; ¹H NMR

(400 MHz, DMSO‑d₆) 3.15 (4H, t, J = 4.8 Hz, N(CH₂CH₂)₂O), 3.75 (4H,

t, J = 4.8 Hz, N(CH₂CH₂)₂O), 7.00 (2H, d, J = 8.9 Hz, H-3^′′/5^′′), 7.38

(2H, d, J = 8.9 Hz, H-2^′′/6^′′), 9.11 (1H, s, H-6), 10.33 (1H, s, NH); ¹³C

NMR (100 MHz, DMSO‑d₆) 48.8 (NCH₂CH₂O), 66.5 (NCH₂CH₂O), 115.4

4.2.12. N-(4-(Benzyloxy)phenyl)-2-chloro-5-nitropyrimidin-4-amine (20)

4-(Benzyloxy)aniline (0.47 g, 2.33 mmol, 1.5 equiv.) was taken up in

DCM (2.3 mL, 1 M) and reacted with a solution of 2,4-dichloro-5-nitro-

pyrimidine (0.31 g, 1.55 mmol) in DCM (3.1 mL, 0.5 M) according to the

described General Procedure A. Puriﬁcation via silica gel chromatog-

raphy (9:1 Hexanes: EtOAc) afforded the target compound as an orange

solid (0.46 g, 1.29 mmol, 83%). Rf 0.37 (4:1 Hexanes: EtOAc); M.p.

134–136 ^◦C; IR (cm^{ꢀ 1}) 3322, 1575, 1499; ¹H NMR (400 MHz, DMSO‑d₆)

5.14 (2H, s, OCH₂), 7.08 (2H, d, J = 8.8 Hz, H-3^′′/5^′′), 7.29–7.54 (7H, m,

H-2^′′/6^′′, H-2^′′′/6^′′′, H-3^′′′/5^′′′, H-4^′′′), 9.12 (1H, s, H-6), 10.37 (1H, s,

NH); ¹³C NMR (100 MHz, DMSO‑d₆) 69.9 (OCH₂), 115.3 (Ar C), 126.8

–

(Ar C), 128.1 (Ar C), 128.2 (Ar C), 128.4 (Ar C), 128.9 (Ar C),

–

– – – –

129.6 (Ar C), 137.4 (Ar C), 154.4 (Ar C), 157.1 (Ar C), 158.0

(Ar C), 126.0 (Ar C), 128.0 (Ar C), 128.2 (Ar C), 149.6 (Ar C),

–

154.2 (Ar C), 158.1 (Ar C), 162.5 (Ar C). HRMS cal. C₁₄H₁₄ClN₅O₃

(Ar C), 162.5 (Ar C). HRMS cal. C₁₇H₁₃ClN₄O₃(ES+) m/z

(ES+) m/z 335.078515 [M+H]⁺, found 335.0837.

356.067618 [M+H]⁺, found 356.0740.

4.2.9. 2-Chloro-N4-(4-morpholinophenyl)pyrimidine-4,5-diamine (24)

Nitropyrimidine 14 (97.7 mg, 0.30 mmol) and tin(II) chloride (0.25

g, 1.20 mmol) were taken up in EtOH (3 mL) and reacted according to

General Procedure B. Puriﬁcation via silica gel chromatography (24:1

4.2.13. N4-(4-(Benzyloxy)phenyl)-2-chloropyrimidine-4,5-diamine (30)

Nitropyrimidine 20 (0.40 g, 1.12 mmol) and tin(II) chloride (0.85 g,

4.52 mmol) were taken up in EtOH (12 mL) and reacted according to

General Procedure B. Puriﬁcation via silica gel chromatography (10:1

9

K.A. Casalvieri et al.

Bioorganic & Medicinal Chemistry 41 (2021) 116220

DCM: MeOH) afforded the target compound as a purple oil (0.43 g, 1.32

DCM (2.3 mL, 1 M) and reacted with a solution of 2,4-dichloro-5-nitro-

pyrimidine (0.31 g, 1.55 mmol) in DCM (3.1 mL, 0.5 M) according to the

described General Procedure A. Puriﬁcation via silica gel chromatog-

raphy (9:1 Hexanes: EtOAc) afforded the target compound as an orange

solid (0.46 g, 1.29 mmol, 82%). Rf 0.38 (4:1 Hexanes: EtOAc); M.p.

124–126 ^◦C; IR (cm^{ꢀ 1}) 3305, 1616, 1567, 1508; ¹H NMR (400 MHz,

DMSO‑d₆) 5.13 (2H, s, OCH₂), 6.95 (1H, ddd, J = 8.4, 2.3, 0.92 Hz, H-

4^′′), 7.17 (1H, dd, J = 8.0, 1.5 Hz, H-4^′′′), 7.30 (1H, dd, J = 2.3, 2.2 Hz,

H-2^′′), 7.31–7.50 (6H, m, H-5^′′, H-6^′′, H-2^′′′/H-6^′′′, H3^′′′/H-5^′′′), 9.15

(1H, s, H-6), 10.39 (1H, s, NH); ¹³C NMR (100 MHz, DMSO‑d₆) 69.9

mmol, 93%). Rf 0.33 (97:3 DCM: MeOH); M.p. 142–145 ^◦C; IR (cm^{ꢀ 1}

)

3340, 3259, 3157, 1652, 1610, 1568, 1502; ¹H NMR (400 MHz,

DMSO‑d₆) 5.10 (2H, s, OCH₂), 5.21 (2H, bs, NH₂), 7.02 (2H, d, J = 8.9

Hz, H-3^′′/5^′′), 7.29–7.51 (5H, m, H-2^′′′/6^′′′, H-3^′′′/5^′′′, H-4^′′′), 7.54 (2H,

d, J = 8.9 Hz, H-2^′′/6^′′), 7.60 (1H, s, H-6), 8.52 (1H, s, NH); ¹³C NMR

–

(100 MHz, DMSO‑d₆) 69.9 (OCH₂), 115.4 (Ar C), 122.9 (Ar C), 128.2

–

(Ar C), 128.3 (Ar C), 128.9 (Ar C), 132.7 (Ar C), 137.7 (Ar C),

–

138.3 (Ar C), 146.5 (Ar C), 151.1 (Ar C), 154.5 (Ar C). HRMS cal.

C

₁₇H₁₅ClN₄O (ES+) m/z 326.093438 [M+H]⁺, found 326.1008.

–

(OCH₂), 111.7 (Ar C), 113.1 (Ar C), 117.4 (Ar C), 128.2 (Ar C),

– – – –

128.3 (Ar C), 128.4 (Ar C), 129.0 (Ar C), 130.0 (Ar C), 137.3

4.2.14. 9-(4-(Benzyloxy)phenyl)-2-chloro-9H-purine (40)

–

Diaminopyrimidine 30 (0.34 g, 1.05 mmol), triethyl orthoformate

(Ar C), 137.9 (Ar C), 154.2 (Ar C), 158.1 (Ar C), 159.0 (Ar C),

–

(0.44 mL, 2.63 mmol) and TFA (8

μ

L, 0.11 mmol) were taken up in TFE

162.4 (Ar C). HRMS cal. C₁₇H₁₃ClN₄O₃(ES+) m/z 356.0676181

(5 mL) and reacted according to General Procedure C. Puriﬁcation via

silica gel chromatography (3:2 Hexanes: EtOAc) afforded the target

compound as a brown solid (0.32 g, 0.96 mmol, 91%). Rf 0.60 (97:3

DCM: MeOH); M.p. 152–154 ^◦C; IR (cm^{ꢀ 1}) 3133, 3088, 3023, 2930,

2870, 1728, 1589, 1513; ¹H NMR (400 MHz, DMSO‑d₆) 5.22 (2H, s,

OCH₂), 7.27 (2H, d, J = 9.0 Hz, H-3^′′/5^′′), 7.32–7.55 (5H, m, H-2^′′′/6^′′′,

H-3^′′′/5^′′′, H-4^′′′), 7.73 (2H, d, J = 9.0 Hz, H-2^′′/6^′′), 8.96 (1H, s, H-8),

9.20 (1H, s, H-6); ¹³C NMR (100 MHz, DMSO‑d₆) 70.1 (OCH₂), 116.2

[M+H]⁺, found 356.0731.

4.2.18. N4-(3-(Benzyloxy)phenyl)-2-chloropyrimidine-4,5-diamine (29)

Nitropyrimidine 19 (0.42 g, 1.18 mmol) and tin(II) chloride (0.89 g,

4.72 mmol) were taken up in EtOH (12 mL) and reacted according to

General Procedure B. Puriﬁcation via silica gel chromatography (97:3

DCM: MeOH) afforded the target compound as a purple solid (0.34 g,

1.02 mmol, 86%). Rf 0.43 (97:3 DCM: MeOH); M.p. 132–134 ^◦C; IR

(cm^{ꢀ 1}) 3420, 3347, 3261, 3178, 1659, 1607, 1568; ¹H NMR (400 MHz,

DMSO‑d₆) 5.11 (2H, s, OCH₂), 5.32 (2H, s, NH₂), 6.68–6.78 (1H, m, H-

4^′′), 7.21–7.63 (8H, m, H-2^′′, H-5^′′, H-6^′′, H-2^′′′/H-6^′′′, H3^′′′/H-5^′′′), 7.68

(1H, s, H-6), 8.60 (1H, s, NH); ¹³C NMR (100 MHz, DMSO‑d₆) 69.7

–

(Ar C), 126.0 (Ar C), 127.1 (Ar C), 128.2 (Ar C), 128.4 (Ar C),

–

129.0 (Ar C), 133.9 (Ar C), 137.2 (Ar C), 147.7 (Ar C), 150.8

–

(Ar C), 153.2 (Ar C), 153.7 (Ar C), 158.7 (Ar C). HRMS cal.

C

₁₈H₁₃ClN₄O (ES+) m/z 336.0777887 [M+H]⁺, found 336.0831.

–

(OCH₂), 107.5 (Ar C), 109.7 (Ar C), 113.4 (Ar C), 128.2 (Ar C),

– – – –

128.3 (Ar C), 128.6 (Ar C), 128.9 (Ar C), 129.9 (Ar C), 137.5

4.2.15. 4-((9-(4-(Benzyloxy)phenyl)-9H-purin-2-yl)amino)-2,6-

diﬂuorophenol (50)

–

(Ar C), 139.1 (Ar C), 140.9 (Ar C), 146.2 (Ar C), 150.5 (Ar C),

–

Purine 40 (0.20 g, 0.59 mmol), aniline 57 (0.27 g, 1.78 mmol) and

TFA (0.2 mL, 2.97 mmol) were taken up in TFE (4.8 mL) and reacted

according to General Procedure D. Puriﬁcation via silica gel chroma-

tography (19:1 DCM: MeOH) afforded the target compound as a brown

solid (78 mg, 0.18 mmol, 30%). Rf 0.28 (19:1 DCM: MeOH); M.p.

228–230 ^◦C; IR (cm^{ꢀ 1}) 3124, 1610, 1589, 1516; ¹H NMR (400 MHz,

DMSO‑d₆) 5.22 (2H, s, OCH₂), 7.24 (2H, d, J = 8.8 Hz, H-3^′′/5^′′),

7.31–7.62 (7H, m, H3^′/5^′, H-2^′′′/6^′′′, H-3^′′′/5^′′′, H-4^′′′), 7.79 (2H, d, J =

8.8 Hz, H-2^′′/6^′′), 8.57 (1H, s, H-8), 8.94 (1H, s, H-6), 9.53 (1H, s, NH),

9.77 (1H, s, OH); ¹³C NMR (100 MHz, DMSO‑d₆) 70.0 (OCH₂), 102.4 (d,

159.1 (Ar C). HRMS cal. C₁₇H₁₅ClN₄O (ES+) m/z 326.0934373

[M+H]⁺, found 326.1013.

4.2.19. 9-(3-(Benzyloxy)phenyl)-2-chloro-9H-purine (39)

Diaminopyrimidine 29 (0.25 g, 0.78 mmol), triethyl orthoformate

(0.32 mL, 1.94 mmol) and TFA (6 μL, 0.08 mmol) were taken up in TFE

(4 mL) and reacted according to General Procedure C. Puriﬁcation via

silica gel chromatography (7:3 Hexanes: EtOAc) afforded the target

compound as a brown solid (0.22 g, 0.65 mmol, 84%). Rf 0.36 (97:3

DCM: MeOH); M.p. 127–128 ^◦C; IR (cm^{ꢀ 1}) 3094, 3043, 1739, 1595,

1583, 1495; ¹H NMR (400 MHz, DMSO‑d₆) 5.21 (2H, s, OCH₂), 7.19 (1H,

ddd, J = 8.2, 2.5, 2.5 Hz, H-4^′′), 7.30–7.67 (8H, m, H-2^′′, H-5^′′, H-6^′′, H-

2^′′′/H-6^′′′, H3^′′′/H-5^′′′, H-4^′′′), 9.07 (1H, s, H-8), 9.22 (1H, s, H-6); ¹³C

–

J_CF= 29.5 Hz, Ar C), 116.0 (Ar C), 125.5 (Ar C), 127.7 (Ar C),

–

128.1 (Ar C), 128.2 (Ar C), 128.4 (Ar C), 128.8 (Ar C), 129.0

–

(Ar C), 137.3 (Ar C), 143.5 (Ar C), 150.0 (Ar C), 152.3 (Ar C),

– –

NMR (100 MHz, DMSO‑d₆) 70.2 (OCH₂), 111.0 (Ar C), 115.1 (Ar C),

152.7 (dd, J_CF= 237.8, 9.1 Hz, Ar C), 156.7 (Ar C), 158.2 (Ar C).

HRMS cal. C₂₄H₁₇F₂N₅O₂(ES+) m/z 445.13503 [M+H]⁺, found

445.1408.

–

116.3 (Ar C), 128.4 (Ar C), 128.5 (Ar C), 129.0 (Ar C), 131.1

–

(Ar C), 134.2 (Ar C), 135.2 (Ar C), 137.0 (Ar C), 147.4 (Ar C),

–

151.0 (Ar C), 153.0 (Ar C), 153.8 (Ar C), 159.6 (Ar C). HRMS cal.

4.2.16. 2,6-Diﬂuoro-4-((9-(4-hydroxyphenyl)-9H-purin-2-yl)amino)

phenol (54)

C

₁₈H₁₅ClN₄O (ES+) m/z 336.0777870 [M+H]⁺, found 336.0837.

Benzyl-protected purine 50 (77.0 mg, 0.17 mmol), ammonium

formate (56.8 mg, 0.87 mmol) and Pd/C (18.0 mg, 20% w/w) were

reacted in DMF (3.5 mL) according to General Procedure E. Puriﬁca-

tion via silica gel chromatography (3:7 DCM: MeOH) followed by sub-

sequent trituration with MeOH afforded the target compound as a brown

solid (43 mg, 0.12 mmol, 70%). Rf 0.29 (9:1 DCM: MeOH); M.p. >

300 ^◦C; IR (cm^{ꢀ 1}) 3106, 1735, 1605, 1519; ¹H NMR (400 MHz,

DMSO‑d₆) 6.97 (2H, d, J = 8.8 Hz, H-3^′′/5^′′), 7.54 (2H, d, J = 10.1 Hz, H-

3^′/5^′), 7.65 (2H, d, J = 8.8 Hz, H-2^′′/6^′′), 8.53 (1H, s, H-8), 8.93 (1H, s,

H-6), 9.51 (1H, s, NH), 9.76 (1H, s, OH); 9.87 (1H, s, benzyl-OH); ¹³C

4.2.20. 4-((9-(3-(Benzyloxy)phenyl)-9H-purin-2-yl)amino)-2,6-

diﬂuorophenol (49)

Purine 39 (0.13 g, 0.40 mmol), aniline 57 (0.13 g, 0.80 mmol) and

TFA (61 μL, 0.80 mmol) were taken up in TFE (4 mL) and reacted ac-

cording to General Procedure D. Puriﬁcation via silica gel chroma-

tography (1:1 Hexanes: EtOAc) afforded the target compound as a

brown solid (0.13 g, 0.29 mmol, 52%). Rf 0.28 (1:1 Hexanes: EtOAc); M.

p. 216–219 ^◦C; IR (cm^{ꢀ 1}) 3074, 1593, 1513; ¹H NMR (400 MHz,

DMSO‑d₆) 5.20 (2H, s, OCH₂), 7.10–7.18 (1H, m, H-4^′′), 7.30–7.66 (8H,

m, H-3^′/5^′, H-2^′′, H-5^′′, H-6^′′, H-2^′′′/H-6^′′′, H3^′′′/H-5^′′′, H-4^′′′), 8.68 (1H,

s, H-8), 8.96 (1H, s, H-6); 9.55 (1H, s, NH), 9.82 (1H, s, OH); ¹³C NMR

–

NMR (100 MHz, DMSO‑d₆) 102.4 (d, J_CF= 28.1 Hz, Ar C), 116.3

–

(100 MHz, DMSO‑d₆) 70.2 (OCH₂), 102.4 (d, J_CF= 27.7 Hz, Ar C),

(Ar C), 125.5 (Ar C), 126.4 (Ar C), 127.7 (Ar C), 128.7 (Ar C),

–

– – –

110.2 (Ar C), 114.5 (Ar C), 115.8 (Ar C), 127.8 (dd, J_CF= 16.9,

133.0 (Ar C), 143.5 (Ar C), 150.0 (Ar C), 152.2 (Ar C), 152.7 (dd,

–

– – – –

16.7 Hz, Ar C), 128.3 (Ar C), 128.5 (Ar C), 129.0 (Ar C), 130.9

J_CF

=

237.6, 9.1 Hz, Ar C), 156.7 (Ar C), 157.5 (Ar C).

₁₇H₁₁F₂N₅O₂(ES+) m/z 355.088080 [M+H]⁺, found 355.0964.

– – –

(Ar C), 132.9 (dd, J_CF= 12.8, 12.8 Hz, Ar C), 136.2 (Ar C), 137.1

C

–

(Ar C), 143.2 (Ar C), 150.3 (Ar C), 152.1 (Ar C), 153.8 (dd, J_CF

=

– – –

237.8, 8.7 Hz, Ar C), 156.8 (Ar C), 159.7 (Ar C). HRMS cal.

4.2.17. N-(3-(Benzyloxy)phenyl)-2-chloro-5-nitropyrimidin-4-amine (19)

3-(Benzyloxy)aniline (0.47 g, 2.33 mmol, 1.5 equiv.) was taken up in

C

₂₄H₁₇F₂N₅O₂(ES+) m/z 445.1350306 [M+H]⁺, found 445.1406.

10

K.A. Casalvieri et al.

Bioorganic & Medicinal Chemistry 41 (2021) 116220

4.2.21. 2,6-Diﬂuoro-4-((9-(3-hydroxyphenyl)-9H-purin-2-yl)amino)

phenol (53)

in TFE (4.8 mL) and reacted according to General Procedure D. Puri-

ﬁcation via silica gel chromatography (19:1 DCM: MeOH) afforded the

target compound as a brown solid (21.9 mg, 0.08 mmol, 16%). Rf 0.25

(19:1 DCM: MeOH); M.p. 244–248 ^◦C; IR (cm^{ꢀ 1}) 3397, 3377, 3114,

1606, 1510; ¹H NMR (400 MHz, DMSO‑d₆) 6.41 (1H, d, J = 2.7 Hz, H-5),

7.19 (1H, t, J = 2.7 Hz, H-6), 7.59 (2H, d, J = 10.4 Hz, H-3^′/5^′), 8.70

(1H, s, H-4), 9.39 (2H, bs, OH, NH), 11.59 (1H, s, NH); ¹³C NMR (100

Benzyl-protected purine 49 (71.7 mg, 0.16 mmol), ammonium

formate (54.3 mg, 0.80 mmol) and Pd/C (18.1 mg, 20% w/w) were

reacted in DMF (3.2 mL) according to General Procedure E. Puriﬁca-

tion via silica gel chromatography (9:1 DCM: MeOH) followed by sub-

sequent trituration with MeOH afforded the target compound as a purple

solid (9.8 mg, 0.03 mmol, 19%). Rf 0.34 (9:1 DCM: MeOH); M.p. >

300 ^◦C; IR (cm^{ꢀ 1}) 3429, 3279, 3111, 1615, 1524; ¹H NMR (400 MHz,

DMSO‑d₆) 6.90 (1H, ddd, J = 8.2, 2.4, 2.3 Hz, H-4^′′), 7.28 (1H, dd, J =

2.3, 2.2 Hz, H-2^′′) 7.30–7.35 (1H, m, H-6^′′), 7.40 (1H, dd, J = 8.0, 8.0 Hz,

H-5^′′), 7.56 (2H, d, J = 10.1 Hz, H-3^′/5^′), 8.61 (1H, s, H-8), 8.95 (1H, s,

H-6); 9.55 (1H, s, NH), 9.80 (1H, s, OH), 9.96 (1H, s, OH); ¹³C NMR (100

–

MHz, DMSO‑d₆) 100.22 (Ar C), 102.4 (d, J_CF= 27.2 Hz, Ar C), 112.6

–

(Ar C), 124.3 (Ar C), 126.9 (dd, J_CF= 17.2, 16.7 Hz, Ar C), 133.9

–

(dd, J_CF= 12.5, 13.1 Hz, Ar C), 150.6 (Ar C), 152.7 (dd, J_CF= 237.9,

–

9.4 Hz, Ar C), 152.8 (Ar C), 156.0 (Ar C). HRMS cal. C₁₂H₈F₂N₄O

(ES+) m/z 262.0666173 [M+H]⁺, found 262.0743.

–

MHz, DMSO‑d₆) 102.4 (d, J_CF= 27.7 Hz, Ar C), 110.8 (Ar C), 114.1

4.2.25. 4-(4-(2-Chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)phenyl)

morpholine (8)

–

(Ar C), 115.2 (Ar C), 127.8 (dd, J_CF= 16.8, 16.5 Hz, Ar C), 128.9

–

(Ar C), 130.7 (Ar C), 133.0 (dd, J_CF= 12.5, 12.4 Hz, Ar C), 136.0

2-Chloro-7H-pyrrolo[2,3-d]pyrimidine (0.25 g, 1.63 mmol), 4-mor-

pholinophenylboronic acid (0.44 g, 2.12 mmol) and copper acetate

(0.33 g, 1.80 mmol) in CHCl₃(16 mL) was added pyridine (1 mL) and

the solution was reacted according to General Procedure F. Puriﬁca-

tion via silica gel chromatography (13:7 Hexanes: EtOAc) afforded the

target compound as a white solid (0.15 g, 0.47 mmol, 28%). Rf 0.29 (1:1

Hexanes: EtOAc); M.p. 70–73 ^◦C; IR (cm^{ꢀ 1}) 2955, 2829, 1581, 1526; ¹H

NMR (400 MHz, DMSO‑d₆) 3.19 (4H. t, J = 5.0 Hz, N(CH₂CH₂)₂O), 3.78

(4H, t, J = 4.7 Hz, N(CH₂CH₂)₂O), 6.87 (1H, d, J = 3.7 Hz, H-5), 7.13

(2H, d, J = 8.9 Hz, H-2^′′/6^′′), 7.59 (2H, d, J = 8.9 Hz, H-3^′′/5^′′), 7.93 (1H,

d, J = 3.7 Hz, H-6), 9.02 (1H, s, H-4); ¹³C NMR (100 MHz, DMSO‑d₆)

–

(Ar C), 143.2 (Ar C), 150.2 (Ar C), 152.1 (Ar C), 153.8 (dd, J_CF

=

–

237.7, 8.7 Hz, Ar C), 156.7 (Ar C), 158.9 (Ar C). HRMS cal.

C

₁₇H₁₁F₂N₅O₂(ES+) m/z 355.088080 [M+H]⁺, found 355.0952.

4.2.22. 2-Chloro-7-isopentyl-7H-pyrrolo[2,3-d]pyrimidine (59)

To a stirring solution of 2-chloro-7H-pyrrolo[2,3-d]pyrimidine (52.7

mg, 0.33 mmol, 1.0 equiv.) in MeCN (0.65 mL, 0.5 M) was added NaH

(21.7 mg, 0.36 mmol, 1.1 equiv., 60% w/w). The reaction was allowed

to stir for 30 min prior to the addition of 1-bromo-3-methylbutane (43

μ

L, 0.36 mmol, 1.1 equiv.). The reaction was allowed to stir for 16 h, an

– –

48.7 (NCH₂CH₂O), 66.5 (NCH₂CH₂O), 101.5 (Ar C), 115.8 (Ar C),

additional 1.1 equivalents of alkyl halide was added, and the reaction

was stirred for an additional 24 h before being concentrated in vacuo.

The crude residue was resuspended in EtOAc (10 mL), washed with H₂O

(10 mL) and extracted with EtOAc (3 × 10 mL). The combined organic

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

silica gel chromatography (85:15 Hexanes: EtOAc) to afford the desired

yellow oil (71.2 mg, 0.32 mmol, 93%). Rf 0.27 (1:1 Hexanes: EtOAc); IR

(cm^{ꢀ 1}) 3103, 2954, 2872, 1587, 1552, 1512; ¹H NMR (400 MHz,

DMSO‑d₆) 0.90 (6H, d, J = 6.6 Hz, CH(CH₃)₂, 1.36–1.52 (1H, m, CH

(CH₃)₂), 1.75 (2H, dt, J = 7.3, 7.1 Hz, NCH₂CH₂), 4.22 (2H, t, J = 7.3 Hz,

NCH₂CH₂), 6.68 (1H, d, J = 3.6 Hz, H-5), 7.71 (1H, d, J = 3.6 Hz, H-6),

8.90 (1H, s, H-4); ¹³C NMR (100 MHz, DMSO‑d₆) 22.6 (CH(CH₃)₂), 25.6

–

118.8 (Ar C), 125.3 (Ar C), 128.3 (Ar C), 131.8 (Ar C), 150.7

–

(Ar C), 151.4 (Ar C), 152.4 (Ar C), 153.2 (Ar C). HRMS cal.

C

₁₆H₁₅ClN₄O (ES+) m/z 314.0934373 [M+H]⁺, found 314.1015.

4.2.26. 2,6-Diﬂuoro-4-((7-(4-morpholinophenyl)-7H-pyrrolo[2,3-d]

pyrimidin-2-yl)amino)phenol (10)

Pyrrolopyrimidine 8 (77 mg, 0.25 mmol), aniline 57 (73 mg, 0.51

mmol) and TFA (39 μL, 0.51 mmol) were taken up in TFE (2.5 mL) and

reacted according to General Procedure D. Puriﬁcation via silica gel

chromatography (49:1 DCM: MeOH) afforded the target compound as

an off-white solid (16.3 mg, 0.04 mmol, 15%). Rf 0.27 (97:3 DCM:

MeOH); M.p. 276–279 ^◦C; IR (cm^{ꢀ 1}) 3369, 2943, 1721, 1607, 1521; ¹H

NMR (400 MHz, DMSO‑d₆) 3.17 (4H. t, J = 4.7 Hz, N(CH₂CH₂)₂O), 3.79

(4H, t, J = 4.7 Hz, N(CH₂CH₂)₂O), 6.64 (1H, d, J = 3.7 Hz, H-5), 7.11

(2H, d, J = 9.0 Hz, H-2^′′/6^′′), 7.53–7.64 (3H, m, H-6, H-3^′/5^′), 7.68 (2H,

d, J = 8.9 Hz, H-3^′′/5^′′), 8.80 (1H, s, H-4), 9.43 (1H, s, NH), 9.56 (1H, s,

OH); ¹³C NMR (100 MHz, DMSO‑d₆) 49.0 (N(CH₂CH₂)₂O), 66.5 (N

–

(CH(CH₃)₂), 38.7 (NHCH₂CH₂), 42.7 (NHCH₂CH₂), 100.2 (Ar C),

–

118.1 (Ar C), 131.8 (Ar C), 151.7 (Ar C), 152.6 (Ar C). HRMS cal.

C

₁₁H₁₄ClN₃(ES+) m/z 223.0876259 [M+H]⁺, found 223.0952.

4.2.23. 2,6-Diﬂuoro-4-((7-isopentyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)

amino)phenol (60)

– –

(CH₂CH₂)₂O), 101.6 (Ar C), 102.0 (d, J_CF= 28.2 Hz, Ar C), 113.2

Pyrrolopyrimidine 59 (102 mg, 0.45 mmol), aniline 57 (138 mg,

– – –

(Ar C), 115.9 (Ar C), 124.8 (Ar C), 127.0 (dd, J_CF= 16.9, 16.7 Hz,

0.89 mmol) and TFA (171

μL, 2.24 mmol) were taken up in TFE (4.5 mL)

– – –

Ar C), 129.5 (Ar C), 133.8 (dd, J_CF= 12.9, 12.9 Hz, Ar C), 150.1

and reacted according to General Procedure D. Puriﬁcation via silica

gel chromatography (4:1 Hexanes: EtOAc) afforded the target com-

pound as a white solid (31.4 mg, 0.10 mmol, 21%). Rf 0.22 (3:1 Hex-

anes: EtOAc); M.p. 175–177 ^◦C; IR (cm^{ꢀ 1}) 3452, 2952, 2872, 1612,

1571, 1533; ¹H NMR (400 MHz, DMSO‑d₆) 0.95 (6H, d, J = 6.7 Hz, CH

(CH₃)₂, 1.48–1.60 (1H, m, CH(CH₃)₂), 1.71 (2H, dt, J = 7.2, 6.9 Hz,

NCH₂CH₂), 4.17 (2H, t, J = 7.2 Hz, NCH₂CH₂), 6.43 (1H, d, J = 3.5 Hz,

H-5), 7.30 (1H, d, J = 3.5 Hz, H-6), 7.62 (2H, d, J = 11.2 Hz, H-3^′/5^′),

8.68 (1H, s, H-4), 9.41 (1H, s, NH), 9.47 (1H, s, OH); ¹³C NMR (100

MHz, DMSO‑d₆) 22.7 (CH(CH₃)₂), 25.7 (CH(CH₃)₂), 38.8 (NHCH₂CH₂),

–

(Ar C), 151.2 (Ar C), 151.6 (Ar C), 152.7 (dd, J_CF= 237.7, 9.6 Hz,

–

Ar C), 156.2 (Ar C). HRMS cal. C₂₂H₁₉F₂N₅O₂(ES+) m/z

423.1506825 [M+H]⁺, found 423.1574.

4.2.27. 2-Chloro-7-(4-(4-methylpiperazin-1-yl)phenyl)-7H-pyrrolo[2,3-d]

pyrimidine (KAC-205)

2-Chloro-7H-pyrrolo[2,3-d]pyrimidine (0.30 g, 1.95 mmol), (4-(4-

methylpiperazin-1-yl)boronic acid (0.57 g, 2.54 mmol) and copper ac-

etate (0.39 g, 2.15 mmol) in CHCl₃(20 mL) was added pyridine (1.2 mL)

and the solution was reacted according to General Procedure F. Puri-

ﬁcation via silica gel chromatography (13:7 Hexanes: EtOAc) and sub-

sequent puriﬁcation via amine chromatography (9:1 Hexanes: EtOAc)

afforded the target compound as a white solid (0.16 g, 0.48 mmol, 24%).

Rf 0.23 (19:1 DCM: MeOH); M.p. 84–87 ^◦C; IR (cm^{ꢀ 1}) 2932, 2833, 2796,

1583, 1515; ¹H NMR (400 MHz, CDCl₃) 2.37 (3H, s, NCH₃), 2.60 (4H, t,

–

42.5 (NHCH₂CH₂), 99.9(Ar C), 102.2 (d, J_CF= 27.6 Hz, Ar C), 112.8

–

(Ar C), 127.0 (dd, J_CF= 16.9, 16.7 Hz, Ar C), 127.4 (Ar C), 133.8

–

(dd, J_CF= 12.9, 12.9 Hz, Ar C), 150.9 (Ar C), 151.4 (Ar C), 152.7

– –

= 237.7, 8.9 Hz, Ar C), 155.8 (Ar C). HRMS cal.

(dd, J_CF

C

₁₇H₁₈F₂N₄O (ES+) m/z 332.1448678 [M+H]⁺, found 332.1520.

4.2.24. 4-((7H-Pyrrolo[2,3-d]pyrimidin-2-yl)amino)-2,6-diﬂuorophenol

(65)

J = 5.0 Hz, N(CH₂CH₂)₂NCH₃), 3.28 (4H, t, J = 5.0 Hz, N

(CH₂CH₂)₂NCH₃), 6.68 (1H, d, J = 3.7 Hz, H-5), 7.04 (2H, d, J = 9.0 Hz,

H-3^′′/5^′′), 7.43 (1H, d, J = 3.7 Hz, H-6), 7.52 (2H, d, J = 9.0 Hz, H-2^′′/

6^′′), 8.86 (1H, s, H-4); ¹³C NMR (100 MHz, CDCl₃) 46.2 (NCH₃), 48.9 (N

2-Chloro-7H-pyrrolo[2,3-d]pyrimidine (79.1 mg, 0.45 mmol), ani-

line 57 (149 mg, 0.98 mmol) and TFA (75 μL, 0.98 mmol) were taken up

11

K.A. Casalvieri et al.

Bioorganic & Medicinal Chemistry 41 (2021) 116220

–

(CH₂CH₂)₂NCH₃), 55.0 (N(CH₂CH₂)₂NCH₃), 100.8 (Ar C), 116.4

H-4^′′′), 7.41–7.50 (2H, m, H-3^′′′/5^′′′), 7.62–7.71 (3H, m, H-4^′′, H-5^′′, H-

6^′′), 9.08 (1H, s, H-8), 9.21 (1H, s, H-6); ¹³C NMR (100 MHz, DMSO‑d₆)

–

(Ar C), 118.3 (Ar C), 125.0 (Ar C), 128.3 (Ar C), 130.1 (Ar C),

–

– – – –

114.2 (Ar C), 118.3 (Ar C), 118.6 (Ar C), 119.5 (Ar C), 124.6

150.6 (Ar C), 151.3 (Ar C), 151.6 (Ar C), 154.1 (Ar C). HRMS cal.

– – – – –

(Ar C), 130.7 (Ar C), 131.6 (Ar C), 134.2 (Ar C), 135.6 (Ar C),

C

₁₇H₁₈ClN₅(ES+) m/z 327.125073 [M+H]⁺, found 327.1331.

–

147.3 (Ar C), 151.0 (Ar C), 153.0 (Ar C), 153.8 (Ar C), 156.3

–

4.2.28. 2,6-Diﬂuoro-4-((7-(4-(4-methylpiperazin-1-yl)phenyl)-7H-pyrrolo

[2,3-d]pyrimidin-2-yl)amino)phenol (11)

(Ar C), 158.0 (Ar C). HRMS cal. C₁₇H₁₁ClN₄O (ES+) m/z

322.0621367 [M+H]⁺, found 322.0686.

Pyrrolopyrimidine 9 (101 mg, 0.31 mmol), aniline 57 (95 mg, 0.61

mmol) and TFA (47

μ

L, 0.61 mmol) were taken up in TFE (3 mL) and

4.2.32. 2,6-Diﬂuoro-4-((9-(3-phenoxyphenyl)-9H-purin-2-yl)amino)

phenol (47)

reacted according to General Procedure D. Puriﬁcation via silica gel

chromatography (93:7 DCM: MeOH) afforded the target compound as a

brown solid (14 mg, 0.03 mmol, 10%). Rf 0.3 (9:1 DCM: MeOH); ¹H

NMR (400 MHz, DMSO‑d₆) 2.25 (3H, s, NCH₃), 2.47–2.52 (4H, m, N

(CH₂CH₂)₂NCH₃), 320 (4H, t, J = 5.0 Hz, N(CH₂CH₂)₂NCH₃), 6.64 (1H,

d, J = 3.8 Hz, H-5), 7.09 (2H, d, J = 8.8 Hz, H-3^′′/5^′′), 7.55 (1H, d, J =

3.8 Hz, H-6), 7.56–7.74 (4H, m, H-3^′/5^′, H-2^′′/6^′′), 8.79 (1H, s, H-4),

9.44 (1H, bs, NH), 9.55 (1H, s, OH); ¹³C NMR (100 MHz, DMSO‑d₆) 46.2

(NCH₃), 48.6 [(NCH₂CH₂N)CH₃], 55.0 [(NCH₂CH₂N)CH₃], 101.6

Purine 37 (76.2 mg, 0.23 mmol), aniline 57 (68.6 mg, 0.47 mmol)

and TFA (36 μL, 0.46 mmol) were taken up in TFE (2.3 mL) and reacted

according to General Procedure D. Puriﬁcation via silica gel chroma-

tography (19:1 DCM: MeOH) afforded the target compound as a brown

solid (32.5 mg, 0.08 mmol, 33%). Rf 0.25 (19:1 DCM: MeOH); M.p.

163–167 ^◦C; IR (cm^{ꢀ 1}) 1587, 1517; ¹H NMR (400 MHz, DMSO‑d₆) 7.03

(1H, dd, J = 8.9, 2.2 Hz, H-2^′′), 7.10–7.16 (2H, m, H-2^′′′/6^′′′), 7.16–7.23

(1H, m, H-4^′′′), 7.42 (2H, dt, J = 8.3, 7.7 Hz, H-3^′′′/5^′′′), 7.49–7.65 (3H,

m, H-3^′/5^′, H-5^′′), 8.70 (1H, s, H-8), 8.95 (1H, s, H-6), 9.55 (1H, s, NH),

9.82 (1H, s, OH); ¹³C NMR (100 MHz, DMSO‑d₆) 102.4 (d, J_CF= 28.1 Hz,

–

(Ar C), 102.4 (d, J_CF= 28.6 Hz, Ar C), 113.2 (Ar C), 116.1 (Ar C),

–

124.8 (Ar C), 127.1 (dd, J_CF= 17.2, 13.8 Hz, Ar C), 129.2 (Ar C),

–

– – – – –

Ar C), 113.7 (Ar C), 117.3 (Ar C), 118.1 (Ar C), 119.6 (Ar C),

133.5 (dd, J_CF= 12.9, 12.0 Hz, Ar C), 150.1 (Ar C), 151.22 (Ar C),

–

– – –

124.6 (Ar C), 127.8 (dd, J_CF= 18.7, 16.2 Hz, Ar C), 130.7 (Ar C),

151.6 (Ar C), 152.7 (dd, J_CF= 236.5, 9.5 Hz, Ar C), 156.2 (Ar C).

HRMS cal. C₂₃H₂₂F₂N₆O (ES+) m/z 436.182317 [M+H]⁺, found

436.1898.

–

131.4 (Ar C), 132.9 (dd, J_CF= 12.8, 12.8 Hz, Ar C), 136.5 (Ar C),

–

143.1 (Ar C), 150.3 (Ar C), 152.0 (Ar C), 152.7 (dd, J_CF= 236.3,

–

8.7 Hz, Ar C), 156.4 (Ar C), 156.8 (Ar C), 158.2 (Ar C).

4.2.29. 2-Chloro-5-nitro-N-(3-phenoxyphenyl)pyrimidin-4-amine (17)

3-Phenoxyaniline (0.35 g, 1.86 mmol, 1.2 equiv.) was taken up in

DCM (2.0 mL, 1 M) and reacted with a solution of 2,4-dichloro-5-nitro-

pyrimidine (0.31 g, 1.55 mmol) in DCM (3.0 mL, 0.5 M) according to the

described General Procedure A. Puriﬁcation via silica gel chromatog-

raphy (9:1 Hexanes: EtOAc) afforded the target compound as an orange

solid (0.28 g, 0.80 mmol, 51%). Rf 0.31 (17:3 Hexanes: EtOAc); M.p.

236–237 ^◦C; IR (cm^{ꢀ 1}) 3300, 3055, 1610, 1567, 1518; ¹H NMR (400

MHz, DMSO‑d₆) 6.89–6.98 (1H, m, H-4^′′), 7.04–7.12 (2H, m, H-2^′′′/6^′′′),

7.17 (1H, t, J = 7.9 Hz, H-2^′′), 7.24–7.34 (2H, m, H-5^′′, H-4^′′′), 7.36–7.48

(3H, m, H-6^′′, H-3^′′′/5^′′′), 9.14 (1H, s, H-6), 10.43 (1H, s, NH); ¹³C NMR

4.2.33. 2-Chloro-5-nitro-N-(4-phenoxyphenyl)pyrimidin-4-amine (18)

4-Phenoxyaniline (0.29 g, 1.55 mmol, 1.2 equiv.) was taken up in

DCM (1.6 mL, 1 M) and reacted with a solution of 2,4-dichloro-5-nitro-

pyrimidine (0.25 g, 1.29 mmol) in DCM (2.6 mL, 0.5 M) according to the

described General Procedure A. Puriﬁcation via silica gel chromatog-

raphy (9:1 Hexanes: EtOAc) afforded the target compound as an orange

solid (0.32 g, 0.94 mmol, 72%). Rf 0.4 (17:3 Hexanes: EtOAc); M.p.

103–104 ^◦C; IR (cm^{ꢀ 1}) 3318, 1616, 1577; ¹H NMR (400 MHz, DMSO‑d₆)

7.02–7.10 (4H, m, H-3^′′/5^′′, H-2^′′′/6^′′′), 7.17 (1H, dd, J = 7.4, 7.2 Hz, H-

4^′′′), 7.42 (2H, dt, J = 7.4, 6.3 Hz, H-3^′′′/5^′′′), 7.50–7.57 (2H, m, H-2^′′/

6^′′), 9.14 (1H, s, H-6), 10.43 (1H, s, NH); ¹³C NMR (100 MHz, DMSO‑d₆)

–

(100 MHz, DMSO‑d₆) 115.1 (Ar C), 116.7 (Ar C), 119.1 (Ar C),

–

– – – –

119.0 (Ar C), 119.3 (Ar C), 124.2 (Ar C), 127.1 (Ar C), 130.6

119.4 (Ar C), 119.9 (Ar C), 124.3 (Ar C), 130.5 (Ar C), 130.6

–

– – – – –

(Ar C), 131.9 (Ar C), 154.4 (Ar C), 155.4 (Ar C), 156.9 (Ar C),

(Ar C), 138.1 (Ar C), 154.2 (Ar C), 156.6 (Ar C), 157.2 (Ar C),

–

158.1 (Ar C), 162.3 (Ar C). HRMS cal. C₁₆H₁₁ClN₄O₃(ES+) m/z

158.1 (Ar C), 162.5 (Ar C). HRMS cal. C₁₆H₁₁ClN₄O₃(ES+) m/z

342.051968 [M+H]⁺, found 342.0582.

342.0519689 [M+H]⁺, found 342.0578.

4.2.30. 2-Chloro-N4-(3-phenoxyphenyl)pyrimidine-4,5-diamine (27)

Nitropyrimidine 17 (0.24 g, 0.71 mmol) and tin(II) chloride (0.54 g,

2.84 mmol) were taken up in EtOH (8 mL) and reacted according to

General Procedure B. Puriﬁcation via silica gel chromatography (85:15

DCM: MeOH) afforded the target compound as a brown solid (0.18 g,

0.57 mmol, 80%). Rf 0.21 (97:3 DCM: MeOH); M.p. 146–147 ^◦C; IR

(cm^{ꢀ 1}) 3419, 3363, 3271, 3210, 1670, 1608, 1566; ¹H NMR (400 MHz,

DMSO‑d₆) 5.30 (2H, bs, NH₂), 6.67–6.75 (1H, m, H-4^′′), 7.04–7.12 (2H,

m, H-2^′′′/6^′′′), 7.16 (1H, t, J = 7.2 Hz, H-2^′′), 7.31–7.48 (5H, m, H-5^′′, H-

6^′′, H-4^′′′, H-3^′′′/5^′′′), 7.67 (1H, s, H-6), 8.67 (1H, bs, NH); ¹³C NMR (100

4.2.34. 2-Chloro-N4-(4-phenoxyphenyl)pyrimidine-4,5-diamine (28)

Nitropyrimidine 18 (0.27 g, 0.79 mmol) and tin(II) chloride (0.60 g,

3.15 mmol) were taken up in EtOH (8 mL) and reacted according to

General Procedure B. Puriﬁcation via silica gel chromatography (19:1

DCM: MeOH) afforded the target compound as a brown solid (0.26 g,

0.82 mmol, >99%). Rf 0.26 (19:1 DCM: MeOH); M.p. 66–71 ^◦C; IR

(cm^{ꢀ 1}) 3350, 3045, 1568; ¹H NMR (400 MHz, DMSO‑d₆) 5.27 (2H, bs,

NH₂), 6.95–7.08 (4H, m, H-3^′′/5^′′, H-2^′′′/6^′′′), 7.12 (1H, dd, J = 7.3, 7.3

Hz, H-4^′′′), 7.42 (2H, dt, J = 7.7, 7.4 Hz, H-3^′′′/5^′′′), 7.62–7.73 (3H, m, H-

6, H-2^′′/6^′′), 8.65 (1H, s, NH); ¹³C NMR (100 MHz, DMSO‑d₆) 118.5

–

– – – – –

(Ar C), 119.8 (Ar C), 122.6 (Ar C), 123.5 (Ar C), 128.4 (Ar C),

MHz, DMSO‑d₆) 110.4 (Ar C), 113.1 (Ar C), 115.4 (Ar C), 119.5

–

– – – –

130.5 (Ar C), 135.5 (Ar C), 138.8 (Ar C), 146.3 (Ar C), 150.8

(Ar C), 124.1 (Ar C), 128.7 (Ar C), 130.4 (Ar C), 130.6 (Ar C),

–

139.3 (Ar C), 141.3 (Ar C), 146.0 (Ar C), 150.3 (Ar C), 156.7

(Ar C), 152.3 (Ar C), 157.8 (Ar C). HRMS cal. C₁₆H₁₃ClN₄O (ES+)

(Ar C), 157.6 (Ar C). HRMS cal. C₁₆H₁₁ClN₄O₃(ES+) m/z

m/z 312.0777896 [M+H]⁺, found 312.0846.

–

342.051968 [M+H]⁺, found 342.0582.

4.2.35. 2-Chloro-9-(4-phenoxyphenyl)-9H-purine (38)

4.2.31. 2-Chloro-9-(3-phenoxyphenyl)-9H-purine (37)

Diaminopyrimidine 28 (0.23 g, 0.74 mmol), triethyl orthoformate

Diaminopyrimidine 27 (0.15 g, 0.49 mmol), triethyl orthoformate

(0.31 mL, 1.84 mmol) and TFA (6 μL, 0.07 mmol) were taken up in TFE

(0.20 mL, 1.22 mmol) and TFA (4

μ

L, 0.05 mmol) were taken up in TFE

(3.7 mL) and reacted according to General Procedure C. Puriﬁcation

via silica gel chromatography (85:15 DCM: MeOH) afforded the target

compound as a white solid (0.23 g, 0.72 mmol, 98%). Rf 0.3 (97:3 DCM:

MeOH); M.p. 146–147 ^◦C; IR (cm^{ꢀ 1}) 3069, 1583, 1503; ¹H NMR (400

MHz, DMSO‑d₆) 7.10–7.16 (2H, m, H-3^′′/5^′′), 7.18–7.30 (3H, m, H-4^′′′,

H-2^′′′/6^′′′), 7.42–7.51 (2H, m, H-3^′′′/5^′′′), 7.80–7.89 (3H, m, H-6, H-2^′′/

(2.5 mL) and reacted according to General Procedure C. Puriﬁcation

via silica gel chromatography (85:15 DCM: MeOH) afforded the target

compound as a white solid (0.15 g, 0.47 mmol, 97%). Rf 0.37 (97:3

DCM: MeOH);M.p. 149–151 ^◦C; IR (cm^{ꢀ 1}) 3047, 1577; ¹H NMR (400

MHz, DMSO‑d₆) 7.08–7.17 (3H, m, H-2^′′, H2^′′′/6^′′′), 7.18–7.24 (1H, m,

12

K.A. Casalvieri et al.

Bioorganic & Medicinal Chemistry 41 (2021) 116220

6^′′), 9.01 (1H, s, H-8), 9.22 (1H, s, H-6); ¹³C NMR (100 MHz, DMSO‑d₆)

(81 μL, 0.49 mmol) and TFA (2 μL, 0.02 mmol) were taken up in TFE

–

119.6 (Ar C), 119.8 (Ar C), 124.6 (Ar C), 126.4 (Ar C), 129.3

(1.2 mL) and reacted according to General Procedure C. Puriﬁcation

via silica gel chromatography (19:1 DCM: MeOH) afforded the target

compound as a white solid (59.6 mg, 0.17 mmol, 89%). Rf 0.26 (19:1

DCM: MeOH); M.p. 181–183 ^◦C; IR (cm^{ꢀ 1}) 3049, 2943, 2854, 2807,

1588, 1517; ¹H NMR (400 MHz, DMSO‑d₆) 2.40–2.48 (4H, m, N

(CH₂CH₂)₂)O, 3.84 (2H, t, J = 7.8 Hz, (CH₂CH₂N(CH₂CH₂)₂)O),

3.27–3.37 (2H, m, (CH₂CH₂N(CH₂CH₂)₂)O), 3.58 (4H, t, J = 4.5 Hz, N

(CH₂CH₂)₂)O, 7.51 (2H, d, J = 8.1 Hz, H-3^′′/5^′′), 7.74 (2H, d, J = 8.1 Hz,

H-2^′′/6^′′), 9.02 (1H, s, H-8), 9.22 (1H, s, H-6); ¹³C NMR (100 MHz,

DMSO‑d₆) 32.3 (CH₂CH₂N(CH₂CH₂)₂)O), 53.7 (CH₂CH₂N(CH₂CH₂)₂)

O), 60.2 (CH₂CH₂N(CH₂CH₂)₂)O), 66.7 (CH₂CH₂N(CH₂CH₂)₂)O), 124.1

–

(Ar C), 130.7 (Ar C), 134.0 (Ar C), 147.7 (Ar C), 150.9 (Ar C),

–

153.2 (Ar C), 153.8 (Ar C), 156.6 (Ar C), 157.3 (Ar C). HRMS cal.

C₁₇H₁₁ClN₄O (ES+) m/z 322.0621386 [M+H]⁺, found 322.0670.

4.2.36. 2,6-Diﬂuoro-4-((9-(4-phenoxyphenyl)-9H-purin-2-yl)amino)

phenol (48)

Purine 38 (83.4 mg, 0.25 mmol), aniline 57 (76.7 mg, 0.50 mmol)

and TFA (38 μL, 0.50 mmol) were taken up in TFE (2.5 mL) and reacted

according to General Procedure D. Puriﬁcation via silica gel chroma-

tography (19:1 DCM; MeOH) afforded the target compound as a brown

solid (47.7 mg, 0.11 mmol, 43%). Rf 0.25 (19:1 DCM: MeOH); M.p.

228–230 ^◦C; IR (cm^{ꢀ 1}) 1618, 1590, 1517; ¹H NMR (400 MHz, DMSO‑d₆)

7.08–7.15 (2H, m, H-3^′′/5^′′), 7.17–7.27 (3H, m, H-4^′′′, H-2^′′′/6^′′′),

7.41–7.51 (2H, m, H-3^′′′/5^′′′), 7.49–7.65 (2H, m, H-3^′/5^′), 7.85–7.95

(2H, m, H-2^′′/6^′′), 8.63 (1H, s, H-8), 8.96 (1H, s, H-6), 9.52 (1H, s, NH),

9.80 (1H, s, OH); ¹³C NMR (100 MHz, DMSO‑d₆) 102.4 (d, J_CF= 28.5 Hz,

–

(Ar C), 130.4 (Ar C), 132.1 (Ar C), 134.1 (Ar C), 141.6 (Ar C),

–

147.6 (Ar C), 150.9 (Ar C), 153.1 (Ar C), 153.8 (Ar C). HRMS cal.

C

₁₇H₁₈ClN₅O (ES+) m/z 343.1199889 [M+H]⁺, found 343.1288.

4.2.40. 2,6-Diﬂuoro-4-((9-(4-(2-morpholinoethyl)phenyl)-9H-purin-2-yl)

amino)phenol (45)

–

Ar C), 119.4 (Ar C), 119.7 (Ar C), 124.5 (Ar C), 125.8 (Ar C),

Purine 35 (102.1 mg, 0.29 mmol), aniline 57 (88.7 mg, 0.58 mmol)

–

127.7 (dd, J_CF= 16.5, 15.9 Hz, Ar C), 128.8 (Ar C), 130.2 (Ar C),

and TFA (44 μL, 0.58 mmol) were taken up in TFE (2.9 mL) and reacted

–

130.7 (Ar C), 133.0 (dd, J_CF= 13.7, 13.7 Hz, Ar C), 143.3 (Ar C),

according to General Procedure D. Puriﬁcation via silica gel chroma-

tography (19:1 DCM; MeOH) afforded the target compound as a purple

solid (26.6 mg, 0.06 mmol, 20%). Rf 0.26 (9:1 DCM: MeOH); M.p.

216–219 ^◦C; IR (cm^{ꢀ 1}) 3255, 3093, 2954, 2862, 2817, 1606, 1518; ¹H

NMR (400 MHz, DMSO‑d₆) 2.45 (4H, t, J = 4.5 Hz, N(CH₂CH₂)₂)O, 2.58

(2H, t, J = 7.8 Hz, (CH₂CH₂N(CH₂CH₂)₂)O), 2.85 (2H, t, J = 7.8 Hz,

(CH₂CH₂N(CH₂CH₂)₂)O), 3.59 (4H, t, J = 4.5 Hz, N(CH₂CH₂)₂)O, 7.47

(2H, d, J = 8.4 Hz, H-3^′′/5^′′), 7.55 (2H, d, J = 10.2 Hz, H-3^′/5^′), 7.81

(2H, d, J = 8.4 Hz, H-2^′′/6^′′), 8.64 (1H, s, H-8), 8.95 (1H, s, H-6), 9.54

(1H, s, NH), 9.80 (1H, s, OH); ¹³C NMR (100 MHz, DMSO‑d₆) 32.3

–

150.2 (Ar C), 152.2 (Ar C), 152.6 (dd, J_CF= 238.0, 8.8 Hz, Ar C),

–

156.6 (Ar C), 156.8 (Ar C), 156.8 (Ar C). HRMS cal. C₂₃H₁₅F₂N₅O₂

(ES+) m/z 431.1193801 [M+H]⁺, found 431.1253.

4.2.37. 2-Chloro-N-(4-(2-morpholinoethyl)phenyl)-5-nitropyrimidin-4-

amine (15)

4-[2-(Morpholin-4-yl)ethyl]aniline (0.35 g, 1.71 mmol, 1.1 equiv.)

was taken up in EtOH (1.7 mL, 0.5 M) and reacted with a solution of 2,4-

dichloro-5-nitropyrimidine (0.30 g, 1.55 mmol) in DCM (3.0 mL, 0.5 M)

according to the described General Procedure A. Puriﬁcation via silica

gel chromatography (19:1 DCM: MeOH) afforded the target compound

as an orange solid (0.63 g, >99%). Rf 0.36 (19:1 DCM: MeOH); M.p.

193–220 ^◦C decomposed; IR (cm^{ꢀ 1}) 3311, 2525, 2416, 1704, 1615,

1577, 1516; ¹H NMR (400 MHz, DMSO‑d₆) 3.01–3.16 (4H, m, N

(CH₂CH₂)₂)O, 3.31–3.41 (4H, m, N(CH₂CH₂)₂)O, 3.84 (2H, t, J = 12.1

Hz, (CH₂CH₂N(CH₂CH₂)₂)O), 3.98 (2H, t, J = 12.1 Hz, (CH₂CH₂N

(CH₂CH₂)₂)O), 7.35 (2H, d, J = 8.5 Hz, H-3^′′/5^′′), 7.52 (2H, d, J = 8.5

Hz, H-2^′′/6^′′), 9.16 (1H, s, H-6), 10.44 (1H, s, NH); ¹³C NMR (100 MHz,

DMSO‑d₆) 29.0 (CH₂CH₂N(CH₂CH₂)₂)O), 51.4 (CH₂CH₂N(CH₂CH₂)₂)

O), 56.8 (CH₂CH₂N(CH₂CH₂)₂)O), 63.7 (CH₂CH₂N(CH₂CH₂)₂)O), 125.5

(CH₂CH₂N(CH₂CH₂)₂)O),

53.7

(CH₂CH₂N(CH₂CH₂)₂)O),

60.3

(CH₂CH₂N(CH₂CH₂)₂)O), 66.7 (CH₂CH₂N(CH₂CH₂)₂)O), 102.6 (d, J_CF

–

= 28.8 Hz, Ar C), 123.5 (Ar C), 127.7 (dd, J_CF= 17.8, 17.3 Hz,

–

Ar C), 128.9 (Ar C), 130.2 (Ar C), 132.9 (Ar C), 140.8 (Ar C),

–

143.3 (Ar C), 150.2 (Ar C), 151.8 (dd, J_CF= 238.0, 8.8 Hz, Ar C),

–

156.7 (Ar C). HRMS cal. C₂₃H₂₂F₂N₆O₂(ES+) m/z 452.17723067

[M+H]⁺, found 452.1859.

4.2.41. Methyl 3-(4-((2-chloro-5-nitropyrimidin-4-yl)amino)phenyl)

propanoate (21)

Methyl 3-(4-aminophenyl)propanoate (0.42 g, 2.2 mmol, 1.1 equiv.)

was taken up in DCM (4.5 mL, 0.5 M) and reacted with a solution of 2,4-

dichloro-5-nitropyrimidine (0.4 g, 2.1 mmol) in DCM (4.1 mL, 0.5 M)

according to the described General Procedure A. Puriﬁcation via silica

gel chromatography (19:1 DCM: MeOH) afforded the target compound

as a yellow solid (0.25 g, 0.74 mmol, 36%). Rf 0.29 (4:1 Hexanes:

EtOAc); M.p. 113–116 ^◦C; IR (cm^{ꢀ 1}) 3306, 3262, 2949, 1744, 1614,

1575, 1513; ¹H NMR (400 MHz, DMSO‑d₆) 2.68 (2H, t, J = 7.6 Hz,

CH₂CH₂COOCH₃), 2.89 (2H, t, J = 7.6 Hz, CH₂CH₂COOCH₃), 3.60 (3H,

s, COOCH₃), 7.30 (2H, d, J = 8.5 Hz, H-3^′′/5^′′), 7.45 (2H, d, J = 8.5 Hz,

H-2^′′/6^′′), 9.14 (1H, s, H-6), 10.40 (1H, s, NH); ¹³C NMR (100 MHz,

DMSO‑d₆) 30.2 (CH₂CH₂COOCH₃), 35.1 (CH₂CH₂COOCH₃), 51.8

–

(Ar C), 128.3 (Ar C), 129.4 (Ar C), 135.4 (Ar C), 135.6 (Ar C),

–

154.3 (Ar C), 158.1 (Ar C), 162.4 (Ar C). HRMS cal. C₁₆H₁₈ClN₅O₃

(ES+) m/z 363.10981688 [M+H]⁺, found 363.12.

4.2.38. 2-Chloro-N4-(4-(2-morpholinoethyl)phenyl)pyrimidine-4,5-

diamine (25)

Nitropyrimidine 15 (0.20 g, 0.55 mmol) and tin(II) chloride (0.43 g,

2.20 mmol) were taken up in EtOH (5.5 mL) and reacted according to

General Procedure B. Puriﬁcation via silica gel chromatography (9:1

DCM: MeOH) afforded the target compound as a brown oil (71.5 mg,

0.21 mmol, 39%). Rf 0.09 (19:1 DCM: MeOH); IR (cm^{ꢀ 1}) 3349, 3264,

2934, 2855, 2813, 1602, 1565, 1508; ¹H NMR (400 MHz, DMSO‑d₆)

2.38–2.45 (4H, m, N(CH₂CH₂)₂)O, 2.58 (2H, t, J = 8.4 Hz, (CH₂CH₂N

(CH₂CH₂)₂)O), 2.85 (2H, t, J = 8.4 Hz, (CH₂CH₂N(CH₂CH₂)₂)O), 3.59

(4H, t, J = 4.5 Hz, N(CH₂CH₂)₂)O, 5.26 (2H, s, NH₂), 7.20 (2H, d, J =

8.5 Hz, H-3^′′/5^′′), 7.54 (2H, d, J = 8.5 Hz, H-2^′′/6^′′), 7.63 (1H, s, H-6),

8.56 (1H, s, NH); ¹³C NMR (100 MHz, DMSO‑d₆) 32.3 (CH₂CH₂N

(CH₂CH₂)₂)O), 53.8 (CH₂CH₂N(CH₂CH₂)₂)O), 60.6 (CH₂CH₂N

–

(CH₂CH₂COOCH₃), 125.2 (Ar C), 128.2 (Ar C), 129.0 (Ar C), 134.7

–

(Ar C), 139.2 (Ar C), 154.3 (Ar C), 158.1 (Ar C), 162.5 (Ar C),

173.1 (CH₂CH₂COOCH₃). HRMS cal. C₁₄H₁₃ClN₆O₄(ES+) m/z

336.0625306 [M+H]⁺, found 336.0691.

4.2.42. Methyl 3-(4-((5-amino-2-chloropyrimidin-4-yl)amino)phenyl)

propanoate (31)

–

(CH₂CH₂)₂)O), 66.7 (CH₂CH₂N(CH₂CH₂)₂)O), 121.1 (Ar C), 128.4

Nitropyrimidine 21 (0.50 g, 1.48 mmol) and tin(II) chloride (1.12 g,

5.92 mmol) were taken up in EtOH (15 mL) and reacted according to

General Procedure B. Puriﬁcation via silica gel chromatography (19:1

DCM: MeOH) afforded the target compound as a brown solid (135 mg,

0.39 mmol, 53%). Rf 0.31 (19:1 DCM: MeOH); M.p. 132–135 ^◦C; IR

(cm^{ꢀ 1}) 3391, 3220, 2945, 1712, 1607, 1561, 1510; ¹H NMR (400 MHz,

DMSO‑d₆) 2.63 (2H, t, J = 7.7 Hz, CH₂CH₂COOCH₃), 2.83 (2H, t, J = 7.7

–

(Ar C), 129.3 (Ar C), 135.6 (Ar C), 137.5 (Ar C), 138.7 (Ar C),

–

146.4 (Ar C), 150.8 (Ar C). HRMS cal. C₁₆H₂₀ClN₅O (ES+) m/z

333.1356369 [M+H]⁺, found 333.1441.

4.2.39. 4-(4-(2-Chloro-9H-purin-9-yl)phenethyl)morpholine (35)

Diaminopyrimidine 25 (65.0 mg, 0.20 mmol), triethyl orthoformate

13

K.A. Casalvieri et al.

Bioorganic & Medicinal Chemistry 41 (2021) 116220

Hz, CH₂CH₂COOCH₃), 3.59 (3H, s, COOCH₃), 5.27 (2H, s, NH₂), 7.21

(2H, d, J = 8.6 Hz, H-3^′′/5^′′), 7.57 (2H, d, J = 8.6 Hz, H-2^′′/6^′′), 7.65 (1H,

s, H-6), 8.58 (1H, s, NH); ¹³C NMR (100 MHz, DMSO‑d₆) 30.2

(CH₂CH₂COOCH₃), 35.4 (CH₂CH₂COOCH₃), 51.8 (CH₂CH₂COOCH₃),

4.2.46. Methyl 4-(4-((5-amino-2-chloropyrimidin-4-yl)amino)phenyl)

butanoate (32)

Nitropyrimidine 22 (0.14 g, 0.41 mmol) and tin(II) chloride (0.31 g,

1.62 mmol) were taken up in EtOH (4 mL) and reacted according to

General Procedure B. Puriﬁcation via silica gel chromatography (97:3

DCM: MeOH) afforded the target compound as a brown oil (101 mg,

0.32 mmol, 78%). Rf 0.25 (97:3 DCM: MeOH); IR (cm^{ꢀ 1}) 3358, 3268,

3025, 2942, 2857, 1717, 1599, 1564, 1506; ¹H NMR (400 MHz,

DMSO‑d₆) 1.83 (2H, tt, J = 7.4, 7.4 Hz, CH₂CH₂CH₂COOCH₃), 2.32 (2H,

t, J = 7.4 Hz, CH₂CH₂CH₂COOCH₃), 2.57 (2H, t, J = 7.4 Hz,

CH₂CH₂CH₂COOCH₃), 3.59 (3H, s, OCH₃), 5.27 (2H, s, NH₂), 7.18 (2H,

d, J = 8.3 Hz, H-3^′′/5^′′), 7.57 (2H, d, J = 8.3 Hz, H-2^′′/6^′′), 7.64 (1H, s, H-

6), 8.57 (1H, s, NH); ¹³C NMR (100 MHz, DMSO‑d₆) 26.7

–

121.1 (Ar C), 128.4 (Ar C), 128.9 (Ar C), 135.7 (Ar C), 137.7

–

(Ar C), 138.8 (Ar C), 146.3 (Ar C), 150.8 (Ar C), 173.2

(CH₂CH₂COOCH₃). HRMS cal. C₁₅H₁₇ClN₄O₂(ES+) m/z 320.10400382

[M+H]⁺, found 320.1113.

4.2.43. Methyl 3-(4-(2-chloro-9H-purin-9-yl)phenyl)propanoate (41)

Diaminopyrimidine 31 (0.32 g, 1.04 mmol), triethyl orthoformate

(0.44 mL, 2.61 mmol) and TFA (8 μL, 0.11 mmol) were taken up in TFE

(5 mL) and reacted according to General Procedure C. Puriﬁcation via

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

compound as an orange solid (269 mg, 0.85 mmol, 81%). Rf (0.35 (19:1

DCM: MeOH); M.p. 126–129 ^◦C; IR (cm^{ꢀ 1}) 1724, 1581, 1515; ¹H NMR

(400 MHz, DMSO‑d₆) 2.72 (2H, t, J = 7.6 Hz, CH₂CH₂COOCH₃), 2.96

(2H, t, J = 7.6 Hz, CH₂CH₂COOCH₃), 3.61 (3H, s, COOCH₃), 7.51 (2H, d,

J = 8.5 Hz, H-3^′′/5^′′), 7.75 (2H, d, J = 8.5 Hz, H-2^′′/6^′′), 9.02 (1H, s, H-

8), 9.21 (1H, s, H-6); ¹³C NMR (100 MHz, DMSO‑d₆) 30.2

(CH₂CH₂COOCH₃), 35.1 (CH₂CH₂COOCH₃), 51.8 (CH₂CH₂COOCH₃),

(CH₂CH₂CH₂COOCH₃),

33.2

(CH₂CH₂CH₂COOCH₃),

34.2

–

(CH₂CH₂CH₂COOCH₃), 51.7 (CH₂CH₂CH₂COOCH₃), 121.2 (Ar C),

–

128.4 (Ar C), 129.0 (Ar C), 136.6 (Ar C), 137.5 (Ar C), 138.7

–

(Ar C), 146.4 (Ar C), 150.8 (Ar C), 173.7 (CH₂CH₂CH₂COOCH₃).

4.2.47. Methyl 4-(4-(2-chloro-9H-purin-9-yl)phenyl)butanoate (42)

Diaminopyrimidine 32 (0.18 g, 0.56 mmol), triethyl orthoformate

(0.23 mL, 1.39 mmol) and TFA (4.3 μL, 0.06 mmol) were taken up in TFE

–

124.2 (Ar C), 130.0 (Ar C), 132.2 (Ar C), 134.1 (Ar C), 141.7

(2.8 mL) and reacted according to General Procedure C. Puriﬁcation

via silica gel chromatography (49:1 DCM: MeOH) afforded the target

compound as a brown solid (166 mg, 0.50 mmol, 90%). Rf 0.46 (19:1

DCM: MeOH); M.p. 85–88 ^◦C; IR (cm^{ꢀ 1}) 3104, 2951, 1723, 1576, 1517;

¹H NMR (400 MHz, DMSO‑d₆) 1.90 (2H, tt, J = 7.5, 7.5 Hz,

CH₂CH₂CH₂COOCH₃), 2.36 (2H, t, J = 7.5 Hz, CH₂CH₂CH₂COOCH₃),

2.71 (2H, t, J = 7.5 Hz, CH₂CH₂CH₂COOCH₃), 3.61 (3H, s, OCH₃), 7.47

(2H, d, J = 8.5 Hz, H-3^′′/5^′′), 7.76 (2H, d, J = 8.5 Hz, H-2^′′/6^′′), 9.02 (1H,

s, H-8), 9.21 (1H, s, H-6); ¹³C NMR (100 MHz, DMSO‑d₆) 26.6

–

(Ar C), 147.5 (Ar C), 150.9 (Ar C), 153.1 (Ar C), 153.7 (Ar C),

173.0 (CH₂CH₂COOCH₃); HRMS cal. C₁₅H₁₃ClN₄O₂(ES+) m/z

316.07270227 [M+H]⁺, found 316.0793.

4.2.44. Methyl 3-(4-(2-((3,5-diﬂuoro-4-hydroxyphenyl)amino)-9H-purin-

9-yl)phenyl)propanoate (51)

Purine 41 (83.3 mg, 0.25 mmol), aniline 57 (74.7 mg, 0.51 mmol)

and TFA (39 μL, 0.51 mmol) were taken up in TFE (2.5 mL) and reacted

according to General Procedure D. Puriﬁcation via silica gel chroma-

tography (19:1 DCM; MeOH) afforded the target compound as a brown

solid (46.6 mg, 0.11 mmol, 42%). Rf 0.15 (19:1 DCM: MeOH); M.p.

255–259 ^◦C; IR (cm^{ꢀ 1}) 3361, 2966, 1723, 1613, 1579, 1521; ¹H NMR

(400 MHz, DMSO‑d₆) 2.72 (2H, t, J = 7.6 Hz, CH₂CH₂COOCH₃), 2.97

(2H, t, J = 7.6 Hz, CH₂CH₂COOCH₃), 3.61 (3H, s, COOCH₃), 7.47 (2H, d,

J = 8.4 Hz, H-3^′′/5^′′), 7.55 (2H, d, J = 10.8 Hz, H-3^′/5^′), 7.82 (2H, d, J =

8.4 Hz, H-2^′′/6^′′), 8.64 (1H, s, H-8), 8.95 (1H, s, H-6), 9.53 (1H, s, NH),

9.80 (1H, s, OH); ¹³C NMR (100 MHz, DMSO‑d₆) 30.2

(CH₂CH₂COOCH₃), 35.2 (CH₂CH₂COOCH₃), 51.8 (CH₂CH₂COOCH₃),

(CH₂CH₂CH₂COOCH₃),

33.1

(CH₂CH₂CH₂COOCH₃),

34.3

–

(CH₂CH₂CH₂COOCH₃), 51.8 (CH₂CH₂CH₂COOCH₃), 124.3 (Ar C),

–

130.0 (Ar C), 132.1 (Ar C), 134.1 (Ar C), 142.5 (Ar C), 147.5

–

(Ar C), 150.9 (Ar C), 153.1 (Ar C), 153.8 (Ar C), 173.6

(CH₂CH₂CH₂COOCH₃). HRMS cal. C₁₆H₁₅ClN₄O₂(ES+) m/z

330.0883546 [M+H]⁺, found 330.0951.

4.2.48. Methyl 4-(4-(2-((3,5-diﬂuoro-4-hydroxyphenyl)amino)-9H-purin-

9-yl)phenyl)butanoate (52)

Purine 42 (61.8 mg, 0.18 mmol), aniline 57 (54.2 mg, 0.36 mmol)

–

102.5 (d, J_CF= 27.8 Hz, Ar C), 128.9 (Ar C), 129.8 (Ar C), 127.7

and TFA (28 μL, 0.36 mmol) were taken up in TFE (1.8 mL) and reacted

–

(dd, J_CF= 17.1, 17.1 Hz, Ar C), 128.9 (Ar C), 129.8 (Ar C), 140.8

according to General Procedure D. Puriﬁcation via silica gel chroma-

tography (19:1 DCM; MeOH) afforded the target compound as a brown

solid (21.5 mg, 0.05 mmol, 26%). Rf 0.28 (19:1 DCM:MeOH); M.p.

193–196 ^◦C; IR (cm^{ꢀ 1}) 3359, 2948, 1716, 1612, 1580, 1520; ¹H NMR

(400 MHz, DMSO‑d₆) 1.83–1.95 (2H, m, CH₂CH₂CH₂COOCH₃), 2.37

(2H, t, J = 7.5 Hz, CH₂CH₂CH₂COOCH₃), 2.71 (2H, t, J = 7.5 Hz,

CH₂CH₂CH₂COOCH₃), 3.61 (3H, s, OCH₃), 7.44 (2H, d, J = 8.2 Hz, H-

3^′′/5^′′), 7.55 (2H, d, J = 10.5 Hz, H-3^′/5^′), 7.83 (2H, d, J = 8.4 Hz, H-2^′′/

6^′′), 8.65 (1H, s, H-8), 8.95 (1H, s, H-6), 9.52 (1H, s, NH), 9.80 (1H, s,

OH); ¹³C NMR (100 MHz, DMSO‑d₆) 26.7 (CH₂CH₂CH₂COOCH₃), 33.1

–

(Ar C), 143.2 (Ar C), 150.1 (Ar C), 152.1 (Ar C), 152.6 (dd, J_CF

=

238.0, 8.8 Hz, Ar C), 156.7 (Ar C), 173.1 (CH₂CH₂COOCH₃). HRMS

cal. C₂₁H₁₇F₂N₅O₃(ES+) m/z 425.12994429 [M+H]⁺, found 425.1372.

4.2.45. Methyl 4-(4-((2-chloro-5-nitropyrimidin-4-yl)amino)phenyl)

butanoate (22)

Methyl 4-(4-aminophenyl)butanoate (0.15 g, 0.78 mmol, 1.1 equiv.)

was taken up in DCM (1.6 mL, 0.5 M) and reacted with a solution of 2,4-

dichloro-5-nitropyrimidine (0.14 g, 0.71 mmol) in DCM (1.4 mL, 0.5 M)

according to the described General Procedure A. Puriﬁcation via silica

gel chromatography (4:1 Hexanes: EtOAc) afforded the target com-

pound as an orange solid (135 mg, 0.38 mmol, 53%). Rf 0.31 (4:1

Hexanes: EtOAc); M.p. 78–79 ^◦C; IR (cm^{ꢀ 1}) 3309, 3043, 2935, 2857,

1728, 1617,1573, 1510; ¹H NMR (400 MHz, DMSO‑d₆) 1.86 (2H, tt, J =

(CH₂CH₂CH₂COOCH₃),

34.3

(CH₂CH₂CH₂COOCH₃),

51.8

–

(CH₂CH₂CH₂COOCH₃), 102.4 (d, J_CF= 27.3 Hz, Ar C), 123.7 (Ar C),

–

127.7 (dd, J_CF= 17.0, 17.0 Hz, Ar C), 128.9 (Ar C), 129.8 (Ar C),

–

133.0 (Ar C), 141.7 (Ar C), 143.2 (Ar C), 150.2 (Ar C), 152.1

–

(Ar C), 152.6 (dd, J_CF= 239.1, 9.0 Hz, Ar C), 156.7 (Ar C), 173.6

(CH₂CH₂COOCH₃). HRMS cal. C₂₂H₁₉F₂N₅O₃(ES+) m/z 439.1455939

[M+H]⁺, found 439.1519.

7.5, 7.5 Hz, CH₂CH₂CH₂COOCH₃), 2.34 (2H, t,

J = 7.5 Hz,

CH₂CH₂CH₂COOCH₃), 2.63 (2H, t, J = 7.5 Hz, CH₂CH₂CH₂COOCH₃),

3.60 (3H, s, OCH₃), 7.27 (2H, d, J = 8.0 Hz, H-3^′′/5^′′), 7.45 (2H, d, J =

8.0 Hz, H-2^′′/6^′′), 9.15 (1H, s, H-6), 10.40 (1H, s, NH); ¹³C NMR (100

4.2.49. N-([1,1^′-Biphenyl]-4-yl)-2-chloro-5-nitropyrimidin-4-amine (16)

4-Aminobiphenyl (0.39 g, 2.32 mmol, 1.5 equiv.) was taken up in

DCM (2.3 mL, 0.5 M) and reacted with a solution of 2,4-dichloro-5-nitro-

pyrimidine (0.30 g, 1.55 mmol) in DCM (1.6 mL, 1 M) according to the

described General Procedure A. Puriﬁcation via silica gel chromatog-

raphy (9:1 Hexanes: EtOAc) afforded the target compound as an orange

solid (390 mg, 1.19 mmol, 77%). Rf 0.2 (9:1 Hexanes: EtOAc); M.p.

MHz,

DMSO‑d₆)

26.6

34.3

(CH₂CH₂CH₂COOCH₃),

33.2

51.7

(CH₂CH₂CH₂COOCH₃),

–

(CH₂CH₂CH₂COOCH₃), 125.2 (Ar C), 128.2 (Ar C), 129.1 (Ar C),

–

134.5 (Ar C), 140.1 (Ar C), 154.3 (Ar C), 158.1 (Ar C), 162.5

–

(Ar C), 173.6 (CH₂CH₂CH₂COOCH₃). HRMS cal. C₁₅H₁₅ClN₄O₄(ES+)

m/z 350.07818314 [M+H]⁺, found 350.0843.

14

K.A. Casalvieri et al.

Bioorganic & Medicinal Chemistry 41 (2021) 116220

177–179 ^◦C; IR (cm^{ꢀ 1}) 3295, 2918, 1614, 1569; ¹H NMR (400 MHz,

DMSO‑d₆) 7.39 (1H, dd, J = 7.4, 7.2 Hz, H-4^′′′), 7.49 (2H, dd, J = 7.4,

7.4 Hz, H-3^′′′/5^′′′), 7.66 (2H, d, J = 8.4 Hz, H-3^′′/5^′′), 7.69–7.74 (2H, m,

(Ser235/236) peptide substrate (PerkinElmer), and ATP (Sigma) ac-

cording to the supplier protocols. All reagents were prepared in kinase

buffer containing 2 mM DTT, 50 mM HEPES, 1 mM 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. RSK2 was used at a ﬁnal

concentration of 500 pM. ULight™-rpS6 substrate was used at a ﬁnal

concentration of 250 nM and ATP was administered at a ﬁnal concen-

H-2^′′′/6^′′′), 7.76 (2H, d, J = 8.4 Hz, H-2^′′/6^′′), 9.18 (1H, s, H-6), 10.51

13

–

(1H, s, NH); C NMR (100 MHz, DMSO‑d₆) 119.7 (Ar C), 124.4

–

(Ar C), 126.9 (Ar C), 127.2 (Ar C), 127.8 (Ar C), 129.4 (Ar C),

–

136.9 (Ar C), 137.4 (Ar C), 139.9 (Ar C), 150.5 (Ar C), 153.6

–

(Ar C), 154.7 (Ar C). HRMS cal. C₁₆H₁₁ClN₄O₂(ES+) m/z

326.05705365 [M+H]⁺, found 326.0623.

tration of 3

consisting of 2

substrate, and 2

for 1 h before the reaction was stopped through the addition of 5

EDTA at a ﬁnal concentration of 10 mM. After a 5 min incubation period,

μ

M. Assays were performed at 25 ^◦C in a reaction mixture

μ

L serially diluted inhibitor solution, 4

L ATP. Reagents were incubated at room temperature

L of

μL kinase, 2 μL

4.2.50. N4-([1,1^′-Biphenyl]-4-yl)-2-chloropyrimidine-4,5-diamine (26)

Nitropyrimidine 16 (0.39 g, 1.19 mmol) and tin(II) chloride (0.91 g,

4.76 mmol) were taken up in EtOH (6.6 mL) and reacted according to

General Procedure B. Puriﬁcation via silica gel chromatography (97:3

DCM: MeOH) afforded the target compound as a white solid (254 mg,

0.86 mmol, 72%). Rf 0.22 (97:3 DCM: MeOH); M.p. 111–114 ^◦C; IR

(cm^{ꢀ 1}) 3355, 3030, 1590, 1562; ¹H NMR (400 MHz, DMSO‑d₆) 5.35

(2H, s, NH₂), 7.34 (1H, dd, J = 7.4, 7.2 Hz, H-4^′′′), 7.46 (2H, dd, J = 7.6,

7.4 Hz, H-3^′′′/5^′′′), 7.66–7.71 (5H, m, H-6, H-3^′′/5^′′, H-2^′′′/6^′′′), 7.80

(2H, d, J = 8.6 Hz, H-2^′′/6^′′), 8.74 (1H, s, NH); ¹³C NMR (100 MHz,

μ

5 μL of Eu anti-phospho-40S Ribosomal Protein S6 (Ser235/236) anti-

body (PerkinElmer) at a ﬁnal concentration of 2 nM was added. The

plate was read using a Biotek Synergy H1 Hybrid plate reader (Excita-

tion = 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.

–

DMSO‑d₆) 121.2 (Ar C), 126.7 (Ar C), 127.3 (Ar C), 127.5 (Ar C),

–

128.6 (Ar C), 129.4 (Ar C), 135.0 (Ar C), 139.0 (Ar C), 139.2

–

(Ar C), 140.2 (Ar C), 146.2 (Ar C), 150.5 (Ar C). HRMS cal.

4.4. Cell culture

C

₁₆H₁₃ClN₄(ES+) m/z 296.0828753 [M+H]⁺, found 296.0891.

MOLM-13 (DSMZ) cells were cultured in RPMI1640 (Gibco) sup-

plemented with 10% FBS (Sigma Aldrich) and 1% penicillin/strepto-

mycin 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.

4.2.51. 9-([1,1^′-Biphenyl]-4-yl)-2-chloro-9H-purine (36)

Diaminopyrimidine 26 (0.25 g, 0.84 mmol), triethyl orthoformate

(0.35 mL, 2.11 mmol) and TFA (6.5 μL, 0.08 mmol) were taken up in TFE

(4.2 mL) and reacted according to General Procedure C. Puriﬁcation

via silica gel chromatography (1:1 Hexanes: EtOAc) afforded the target

compound as a white solid (225 mg, 0.73 mmol, 86%). Rf 0.41 (97:3

Hexanes: EtOAc); M.p. 169–171 ^◦C; IR (cm^{ꢀ 1}) 1724, 1578, ; ¹H NMR

(400 MHz, DMSO‑d₆) 7.43 (1H, dd, J = 7.4, 7.3 Hz, H-4^′′′), 7.53 (2H, dd,

J = 7.9, 7.4 Hz, H-3^′′′/5^′′′), 7.72 (2H, d, J = 7.4, H-3^′′/5^′′), 7.91–8.00

(4H, m, H-2^′′/6^′′, H-2^′′′/6^′′′), 9.11 (1H, s, H-8), 9.24 (1H, s, H-6); ¹³C

–

NMR (100 MHz, DMSO‑d₆) 124.5 (Ar C), 127.3 (Ar C), 128.4 (Ar C),

–

128.4 (Ar C), 129.6 (Ar C), 133.4 (Ar C), 134.2 (Ar C), 139.4

4.5. MTS cellular viability assay

–

(Ar C), 140.7 (Ar C), 147.4 (Ar C), 151.0 (Ar C), 153.1 (Ar C),

–

153.8 (Ar C). HRMS cal. C₁₇H₁₁ClN₄(ES+) m/z 306.06722495

MOLM-13 cells were seeded into sterile 96-well plates (Corning) at

[M+H]⁺, found 306.0733.

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

4.2.52. 4-((9-([1,1^′-Biphenyl]-4-yl)-9H-purin-2-yl)amino)-2,6-

diﬂuorophenol (46)

and cells were incubated at 37 ^◦C for 24 to 72 h. Following 3 h incu-

bation with 30 μL CellTiter 96® Aqueous One Cell Proliferation Reagent

Purine 36 (80.5 mg, 0.26 mmol), aniline 57 (76.5 mg, 0.52 mmol)

(Promega), cell viability was measured by formazan concentration

assessment through colorimetric analysis using a BioTek Synergy H1

Hybrid plate reader (Absorption = 490).

and TFA (49 μL, 0.52 mmol) were taken up in TFE (2.6 mL) and reacted

according to General Procedure D. Puriﬁcation via silica gel chroma-

tography (19:1 DCM; MeOH) afforded the target compound as a brown

solid (26.7 mg, 0.06 mmol, 25%). Rf 0.15 (19:1 DCM:MeOH); M.p.

270–271 ^◦C; IR (cm^{ꢀ 1}) 3073, 1603, 1512; ¹H NMR (400 MHz, DMSO‑d₆)

7.43 (1H, dd, J = 7.4, 7.3 Hz, H-4^′′′), 7.48–7.61 (4H, m, H-3^′′′/5^′′′, H-3^′/

5^′), 7.76 (2H, d, J = 7.6, H-3^′′/5^′′), 7.90 (2H, d, J = 8.6 Hz, H-2^′′′/6^′′′),

8.04 (2H, d, J = 8.6 Hz, H-2^′′/6^′′), 8.74 (1H, s, H-8), 8.98 (1H, s, H-6),

9.54 (1H, s, NH), 9.84 (1H, s, OH); ¹³C NMR (100 MHz, DMSO‑d₆) 102.5

4.6. Intracellular RSK2 kinase activity

The nanoBRET intracellular kinase assay was performed by Reaction

Biology Corporation. HEK293 cells were cultured according to their

speciﬁed protocol and transfected with NanoLuc®-RPS6KA3 fusion

vector containing 1

μg RPS6KA3 fusion DNA and 9 μg transfection

–

(d, J_CF= 26.8 Hz, Ar C), 124.0 (Ar C), 127.8 (dd, J_CF= 17.0, 17.0 Hz,

carrier DNA. Transfected cells were treated with supplied compounds

–

Ar C), 128.1 (Ar C), 128.4 (Ar C), 128.9 (Ar C), 129.6 (Ar C),

(10 mM stock in DMSO) at a starting concentration of 100 μM using 2-

–

132.9 (Ar C), 134.3 (Ar C), 139.5 (Ar C), 143.1 (Ar C), 150.3

fold dilutions. BRET ratios were calculated by dividing the acceptor

(Acceptor = 610 nm) emission by the donor (Donor = 450 nm) emission,

correcting for background by subtracting the ‘no tracer’ BRET ratio from

each sample.

–

(Ar C), 152.1 (Ar C), 152.4 (dd, J_CF= 237.9, 9.0 Hz, Ar C), 156.8

(Ar C). HRMS cal. C₂₃H₁₅F₂N₅O (ES+) m/z 415.124465 [M+H]⁺,

found 415.1300.

4.3. Inhibition of RSK activity

Declaration of Competing Interest

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 RSK2

kinase (CarnaBio), ULight™-phospho-40S ribosomal protein S6

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.

15

K.A. Casalvieri et al.

Bioorganic & Medicinal Chemistry 41 (2021) 116220

18 Pambid MR, Berns R, Adomat HH, et al. Overcoming resistance to Sonic Hedgehog

inhibition by targeting p90 ribosomal S6 kinase in pediatric medulloblastoma.

Pediatr Blood Cancer . 2014;61:107 –115 .

Acknowledgements

Research reported in this publication was supported by an ALSAM

Therapeutics Innovation grant.

19 Hammoud L, Adams JR, Loch AJ, et al. Identiﬁcation of RSK and TTK as Modulators

of Blood Vessel Morphogenesis Using an Embryonic Stem Cell-Based Vascular

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Appendix A. Supplementary material

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22 Casalvieri KA, Matheson CJ, Backos DS, Reigan P. Molecular docking of substituted

pteridinones and pyrimidines to the ATP-binding site of the N-terminal domain of

RSK2 and associated MM/GBSA and molecular ﬁeld datasets. Data Brief . 2020;29,

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16

Article Doi

N-Substituted pyrrolopyrimidines and purines as p90 ribosomal S6 protein kinase-2 (RSK2) inhibitors

DOI: 10.1016/j.bmc.2021.116220

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