Heat shock protein 70 inhibitors. 1. 2,5′-thiodipyrimidine and 5-(phenylthio)pyrimidine acrylamides as irreversible binders to an allosteric site on heat shock protein 70

Article

pubs.acs.org/jmc

Heat Shock Protein 70 Inhibitors. 1. 2,5′-Thiodipyrimidine and

5‑(Phenylthio)pyrimidine Acrylamides as Irreversible Binders to an

Allosteric Site on Heat Shock Protein 70

Yanlong Kang,^†,⊥Tony Taldone,*^,†Hardik J. Patel,^†Pallav D. Patel,^†Anna Rodina,^†

Alexander Gozman,^†,#Ronnie Maharaj,^∥Cristina C. Clement,^†,∇Maulik R. Patel,^†Jeﬀrey L. Brodsky,^‡

Jason C. Young,^§and Gabriela Chiosis*^,†

^†Program in Molecular Pharmacology and Chemistry and Department of Medicine, Memorial Sloan-Kettering Cancer Center, New

York, New York 10021, United States

^‡Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, United States

^§Groupe de Recherche Axe

H3G 0B1

́ ́

sur la Structure des Proteines, Department of Biochemistry, McGill University, Montreal, Quebec, Canada

^∥Pharmacology Program, Weill Graduate School of Medical Sciences, Cornell University, New York, New York 10021, United States

S

* Supporting Information

ABSTRACT: Heat shock protein 70 (Hsp70) is an important

emerging cancer target whose inhibition may aﬀect multiple

cancer-associated signaling pathways and, moreover, result in

signiﬁcant cancer cell apoptosis. Despite considerable interest from

both academia and pharmaceutical companies in the discovery and

development of druglike Hsp70 inhibitors, little success has been

reported so far. Here we describe structure−activity relationship

studies in the ﬁrst rationally designed Hsp70 inhibitor class that

binds to a novel allosteric pocket located in the N-terminal domain

of the protein. These 2,5′-thiodipyrimidine and 5-(phenylthio)-

pyrimidine acrylamides take advantage of an active cysteine

embedded in the allosteric pocket to act as covalent protein

modiﬁers upon binding. The study identiﬁes derivatives 17a and

20a, which selectively bind to Hsp70 in cancer cells. Addition of high nanomolar to low micromolar concentrations of these

inhibitors to cancer cells leads to a reduction in the steady-state levels of Hsp70-sheltered oncoproteins, an eﬀect associated with

inhibition of cancer cell growth and apoptosis. In summary, the described scaﬀolds represent a viable starting point for the

development of druglike Hsp70 inhibitors as novel anticancer therapeutics.

pathways involved in tumorigenesis and metastasis. Indeed,

simultaneous silencing of Hsc70 or Hsp70 expression in human

colon cancer cell lines induced proteasome-dependent degrada-

tion of Hsp90 onco-client proteins, cell-cycle arrest, and tumor-

speciﬁc apoptosis.⁴Importantly, silencing of Hsp70 isoforms in

nontumorigenic cell lines did not result in comparable growth

arrest or induction of apoptosis, indicating a potential therapeutic

window for Hsp70 targeted therapies.

The Hsp70’s are a family of highly homologous proteins

composed of two functional domains: the N-terminal ATPase

domain and the C-terminal client protein-binding domain.^5,6

The unique interplay between the two domains creates a ligand-

activated, bidirectional molecular switch. For example, ATP

binding to the ATPase domain induces a conformational change

that is rapidly propagated to the C-terminal and that results in

INTRODUCTION

■

The heat shock protein 70 (Hsp70) family members are

powerful proteins with major roles in malignancy, such as

inhibition of apoptosis, induction of resistance to chemotherapy,

and regulation of the stability of oncoproteins.¹⁻³Speciﬁcally,

Hsp70 expression blocks apoptosis at several levels, and in this

respect the chaperone inhibits key eﬀectors of the apoptotic

machinery, and also facilitates proteasome-mediated degradation

of apoptosis-regulatory proteins. The contribution of Hsp70

isoforms to tumorigenesis is mainly through their role as

cochaperones of heat shock protein 90 (Hsp90), a heat shock

protein known to regulate the transforming activities of several

kinases and transcription factors. In this process, Hsp70 initiates

the association of the client protein with Hsp90 through a

bridging protein called HSP-organizing protein (HOP). These

biological functions propose Hsp70 as an important target whose

inhibition or downregulation may result in signiﬁcant apoptosis

in a wide range of cancer cells and also in inhibition of signaling

Received: October 4, 2013

Published: February 18, 2014

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Figure 1. Chemical structure of reported potential Hsp70 inhibitors.

accelerated client protein dissociation. Conversely, client protein

binding to the C-terminal domain of ATP-bound Hsp70 induces

a conformational change that is propagated to the ATPase

domain and that results in a stimulation of the ATP hydrolysis

rate. The chaperoning activity of Hsp70 is further regulated by

cochaperones (e.g., Hsp40s, BAG, and Hsp110) that catalyze the

interconversion between the ATP- and ADP-bound states and

thus regulate chaperone function. Such structural regulation

suggests that Hsp70 may be vulnerable to most strategies that

interfere with its ﬂexibility.

Much eﬀort has recently been dedicated toward the discovery

of Hsp70 inhibitors, and unsurprisingly, molecules from a

number of chemical classes have been reported to interact with

Hsp70 through a variety of modes (Figure 1).^7,8A few, such as

15-deoxyspergualin (1) and piﬁthrin-μ (2-phenylethynesulfona-

mide) (2), are believed to target the C-terminal of Hsp70,^9,10

whereas others, such as dihydropyrimidines (i.e., 3 (MAL3-

101)),¹¹are thought to block J-domain-stimulated ATPase

activity of Hsp70. Compounds such as myricetin (4)¹²and 5

(MKT-077)¹³are proposed to interact with a pocket outside the

nucleotide-binding domain, whereas apoptozole (6) may bind to

the ATP-binding pocket of Hsp70.¹⁴

design approach to develop Hsp70 inhibitors, nucleotide

mimetics such as the dibenzyl-8-aminoadenosine analogue 7

(VER-155008) were developed to bind into the N-terminal ATP

pocket of Hsp70.¹⁸While these molecules are reported to elicit

their eﬀects through an Hsp70 mechanism, it is likely that they

also act on multiple other unrelated and as yet unspeciﬁed

mechanisms. Furthermore, these molecules have been hindered

by a nontractable structure−activity relationship (SAR), with

subtle changes resulting in drastic changes in activity. While these

molecules have been of some value as tool molecules to oﬀer

insight into the consequences of pharmacological modulation of

Hsp70, they have limited potential to become useful drugs.

At this point in time it is fair to say that Hsp70 has proven to be

a very diﬃcult target to drug. In contrast, Hsp90 has proven

highly amenable with numerous small-molecule ATP-compet-

itive inhibitors entering into the clinic.¹⁹In the case of Hsp90,

potent small-molecule inhibitors such as geldanamycin and

radicicol were known even before their precise mode of action

was determined. When X-ray crystal structures showed that they

bound to a clearly speciﬁed pocket (i.e., ATP pocket) and

behaved as ATP-competitive inhibitors, structure-based drug

design became possible. Unfortunately, no such molecules that

could potentially guide and truly inspire the development of

Hsp70 inhibitors exist for Hsp70.

The majority of these compounds were discovered in library

screens that aimed to identify inhibitors of either the ATPase or

the folding capacity of yeast or bacterial Hsp70^2,7,8or in the case

of 6 a cell-based screen of compounds capable of inducing

apoptosis.¹⁵5 was discovered following optimization eﬀorts¹⁶

that had previously identiﬁed such rhodacyanine dyes as

possessing anticancer activity.¹⁷In the only reported rational

The experiences of scientists at Vernalis oﬀer some further

insight into the diﬃculty of targeting Hsp70 and perhaps as to

why no natural product ATP-competitive inhibitor is known.^18,20

In the only rational approach reported to date, they designed a

series of adenosine analogues to act as direct ATP-competitive

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Figure 2. Rational design of the allosteric pocket ligands. (a,b) The homology-modeled hHsp70 structure led to the identiﬁcation of a novel allosteric

pocket (shown in green) located in the N-terminal domain of human Hsp70.²²This pocket contains a potentially reactive cysteine residue (position

shown in red lettering in panel a) and is located outside the ATP-binding site (shown in red in panel b). (c) The geometry of the N-terminal allosteric

pocket of hHsp70 as predicted by SiteMap (shown in yellow) and residues lining the pocket and their relative location (depicted as colored circles) are

presented. Ligands based on the 2,5′-thiopyrimidine and 5-(phenylthio)pyrimidine chemical scaﬀolds were designed to ﬁt into the allosteric pocket.

They adopt the conformation required for a proper ﬁt and position functionalities toward the shown pocket residues. An acrylamide positioned toward

Cys267 was also incorporated into these scaﬀolds.

inhibitors. However, despite obtaining molecules that bound

Hsp70 with high aﬃnity (K_d= 50 nM), their cellular potency was

disappointing, and a number of reasons were given for this poor

correlation. Hsp70 has a high aﬃnity for ADP (K_d= 0.11−0.5

μM) and coupled with high intracellular ATP concentrations

makes the prospect of competitive inhibition a daunting task.

Furthermore, the binding mode of ATP is such that important

polar contacts are made with its β- and γ-phosphate groups that

lie buried within a polar cavity. Attempts to mimic these

interactions have resulted in highly polar nondruglike molecules

such as 7, which despite potent aﬃnity (K_d= 0.3 μM) possess

weak cellular activity. Combined, these factors make the prospect

of obtaining compounds with potent in vivo activity low and

make a strong case against the development of reversible ATP-

competitive inhibitors of Hsp70 as a viable therapeutic strategy.²¹

We had sought alternative strategies toward inhibiting Hsp70

which would have the potential for potent in vivo activity.

Speciﬁcally, we had sought and subsequently identiﬁed another

pocket, outside of the active site.²²This allosteric site, located in

the N-terminal domain, was not evident nor entirely predicted by

the available crystal structures of Hsp70 and has been recently

discovered by us through computational analyses.²²We used the

homology model to design ligands that could bind to this

allosteric pocket.²²Here we describe structure−activity inves-

tigations in this ﬁrst reported allosteric pocket inhibitor class.

These ligands are 2,5′-thiodipyrimidine and 5-(phenylthio)-

pyrimidine scaﬀold compounds that we show here and in the

accompanying paper²³to be amenable to extensive medicinal

chemistry and to act on cancer cells through an Hsp70-mediated

mechanism.

LIGAND DESIGN AND COMPUTATIONAL

ANALYSIS OF LIGAND−HSP70 INTERACTIONS

■

There are several available crystal structures of Hsp70, but most

capture a relatively closed nucleotide-binding domain.²⁴⁻²⁷In

contrast, recent NMR techniques and molecular dynamics

studies suggest a model in which subdomains of the N-terminus

are bridged and in a close proximity only in the ATP-binding

conformation, with residues of these subdomains rearranging

and the cleft opening upon ADP binding.^28,29To overcome these

limitations of the currently available crystal structures and to

investigate pockets other than the ATP-binding site, we recently

developed a theoretical model of human Hsp70 (hHsp70).²²

This nucleotide-free structure of hHsp70 both captured the N-

terminal binding cleft in a conformation that is more open than

that captured by X-ray and unveiled a pocket that contains a

potentially reactive cysteine residue (Cys267 in hHsp70)

embedded inside the cavity (Figure 2a).²²The newly identiﬁed

allosteric pocket (depicted by the green surface in Figure 2a,b) is

located outside the nucleotide-binding domain and is ﬂanked by

subregions IB and IIB of the nucleotide-binding domain (NBD)

(Figure 2b).

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a

Scheme 1

a

Reagents and conditions: (a) (PMB)Cl, NaH, DMF, 0 °C to rt; (b) NIS, MeCN, rt; (c) 4,6-diamino-2-mercaptopyrimidine or 4-amino-2-

mercaptopyrimidine, neocuproine, CuI, K₂CO₃, DMSO, 120−130 °C; (d) Ac₂O, DMAP, 110−120 °C; (e) TFA, CHCl₃, 62 °C; (f) HF/pyridine,

NaNO₂, 0 °C; (g) amine, DMF, 90 °C; (h) NaOH, H₂O, MeOH, 60 °C; (i) acryloyl chloride, Et₃N, dioxane, rt.

This cavity is larger and potentially more druglike than the

nucleotide-binding pocket (depicted by the red surface in Figure

2b), because it contains a balanced number of hydrophilic and

hydrophobic residues (Figure 2c). At the lower part of the

allosteric site lies mainly a hydrophilic cavity containing Lys271

and Glu268 and further apart lined by Thr13, Thr14, Tyr15,

Lys56, Asp234, Asn235, Arg264, Thr265, and Arg269 (number-

ing as in hHsp70) (site A, Figure 2c). Hydrophobic amino acids

such as Leu237 and Val238 also line site A. This subpocket also

contains the potentially reactive cysteine residue, Cys267, that

could covalently link to a ligand containing the appropriate Cys-

reactive functionality, such as an acrylamide.³⁰Adjacent to site A,

and placed in the middle, is a larger cavity comprised of both

nonpolar and polar amino acid residues, such as Tyr41, Val59,

Pro63, Thr66, Asp69, Phe68, Arg72, Glu231, Val260, and

Arg261 (site B, Figure 2c). Placed at the upper part of the binding

site, and also potentially providing interactions to a small-

molecule ligand, are Lys88, His89, Trp90, Pro91, and Phe92 (site

C, Figure 2c).

point for the rational design of ligands (Figure 2c). We

concluded that ligands built around the 2,5′-thiodipyrimidine

and 5-(phenylthio)pyrimidine scaﬀolds, both little explored

chemical spaces, would adapt the necessary conformation and

provide the required attachment sites for ligand functionalization

(Figure 2c). Each designed ligand used this pharmacophore

template to attach X₃₋₇functionalities pointed toward several

amino acids lining the Hsp70 pocket (Figure 2c).

Because our homology model may not entirely mimic the

native protein structure, we included an additional binding hook,

i.e., an acrylamide functionality, to probe possible covalent bond

formation between the inhibitor and Cys267 upon protein

binding (Figure 2c). Due to the location of Cys267 deep inside

the cavity, such bond formation would be possible only after the

ligand was inserted into the pocket and has achieved a proper ﬁt.

By gaining aﬃnity through a covalent linkage in addition to

enthalpy, we hoped to increase the ligand’s apparent aﬃnity for

the protein in the event the ﬁt would be less than optimal. We

reasoned that if the initial ligand would be of suﬃciently good ﬁt,

additional enthalpy could be gained by properly modifying the

We analyzed the geometry and the environment of the

computationally identiﬁed allosteric pocket to provide a starting

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a

Scheme 2

a

Reagents and conditions: (a) NaOMe/MeOH, 90 °C, then NaOH, 60 °C; (b) acryloyl chloride, Et₃N, dioxane, rt; (c) TFA, CHCl₃, 62 °C.

a

Scheme 3

a

Reagents and conditions: (a) N-methylpiperazine or morpholine, DMF, 90 °C; (b) NIS, TFA, CH₃CN, rt; (c) 3-aminothiophenol or 4-amino-2-

mercaptopyrimidine, neocuproine, CuI, K₂CO₃, DMF, 110−130 °C; (d) acid chloride, Et₃N, CH₂Cl₂, rt.

appended functionalities, such as we proceed to do here and in

the adjoining paper.²³

resulted in target compounds 17a−c. Derivatives 20a−c were

prepared following a similar route from 2-amino-4,6-diethox-

ypyrimidine (8b), which was prepared by reﬂuxing a mixture of

2-amino-4,6-dichloropyrimidine and NaH in ethanol in 89%

yield (Scheme 1).

CHEMISTRY

■

The synthesis of all designed compounds evaluated in this study

is shown in Schemes 1−3 and described below. Reaction of 2-

amino-4,6-dimethoxypyrimidine (8a) with p-methoxybenzyl

chloride ((PMB)Cl) resulted in PMB-protected pyrimidine 9a

in 95% yield (Scheme 1). This was iodinated with N-

iodosuccinimide (NIS) to give 10a in 98%, which was further

coupled to 4,6-diamino-2-mercaptopyrimidine using CuI/neo-

cuproine to give 2,5′-thiodipyrimidine 11a in 65% yield.

Acetylation of the amino groups was accomplished with Ac₂O/

DMAP at 110 °C for 2 h to give 12a in 91% yield. Selective

removal of the PMB groups occurred by heating 12a in a 1:1

mixture of TFA/CHCl₃at 62 °C for 24 h to give 13a in 95%.

Fluorodediazoniation was accomplished with NaNO₂/HF/

pyridine to give 14a in 54% yield, which was then reacted with

a variety of amines to yield 15a−c. Deacetylation of these

intermediates followed by reaction with acryloyl chloride

The chemistry used to prepare 27a−d is similar and shown in

Scheme 1. Derivative 10a was coupled with 4-amino-2-

mercaptopyrimidine to give 21 in 92% yield. Acetylation

followed by PMB removal resulted in 23. Reaction with

NaNO₂/HF/pyridine gave ﬂuoro derivative 24, which was

reacted with a variety of amines to give 25a−d. Deacetylation

followed by reaction with acryloyl chloride resulted in target

compounds 27a−d. Reaction of 27a with m-CPBA at −78 °C

resulted in N-oxide 28. Reaction of 24 with NaOMe/MeOH

followed by deacetylation resulted in 29, which was reacted with

acryloyl chloride to give 30 (Scheme 2). Reaction of 11a with

acryloyl chloride followed by treatment with TFA resulted in 31

(Scheme 2).

Thioethers shown in Scheme 3 were prepared from

chloropyrimidine 32 or 38. Reaction of 32 with N-methylpiper-

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Figure 3. Known Hsp70 functions used to design the biological testing of the Hsp70 inhibitors in cancer cells. Hsp90 in concert with Hsp70 maintains

the transforming capacity of several oncoproteins whose aberrant activity leads to increased cell proliferation and survival. The function of the Hsp90

protein complex requires the HSP-organizing protein (HOP), involved in the formation of the active chaperone complex. Altering the formation of the

Hsp90−HOP−Hsp70 complex leads to degradation of the oncoprotein via a proteasome-mediated pathway and is associated with cell growth inhibition

and/or cell death. To conﬁrm an Hsp70-mediated mechanism, it is expected that compounds will be active at similar concentrations in this battery of

assays.

Table 1

X₁=

X₂

IC₅₀(μM) growth

IC₅₀(μM) caspase-3,7

a

b

compd

X₃

X₅, X₆

2 × −OCH₃

X₇

inhibition

activation

17a

17b

17c

20a

20b

20c

27a

27b

27c

27d

28

N

C

N

−NH₂

−H

−NH₂

−H

methylpiperazine

morpholine

piperidine

0.90 0.2

12.5 0.7

17.1 2.2

0.89 0.2

2.2 1.2

4.5 0.2

1.25 0.7

2.0 0.5

3.15 1.5

46.1 15.5

>100

1.2 0.4

12.1 2.5

16.2 0.5

4.0 0.0

10.5 3.5

4.0 2.0

4.0 0.5

7.2 0.7

12.4 1.5

>100

2 × −OCH₃

2 × −OC₂H₅

2 × −OCH₃

2 × −CH₃

methylpiperazine

morpholine

piperidine

methylpiperazine

morpholine

piperidine

pyrrolidine

1-methylpiperazine 1-oxide

−OCH₃

>100

30

ND

7.5 0.3

4.5 0.5

ND

31

−NH₂

methylpiperazine

3.9 1.4

7.8 1.1

46.5 3.4

ND

36a

37

morpholine

27.5 2.5

6.0 2.1

15.4 0.7

14.3 1.5

42a

43

methylpiperazine

morpholine

−H

−NH₂

2 × −CH₃

2 × −OCH₃

14.4 2.7

3.8 1.2

44a

2-(2-(2-(2-(piperazin-1-yl)ethoxy)ethoxy)

ethoxy) ethanol

45a

N

−NH₂

−OCH₃, −(OC₂H₄O)₄H

methylpiperazine

76.5 21.5

100 10.5

a

b

Inhibition of growth measured in Kasumi-1 acute myeloid leukemia cells. Values are the mean SEM. Caspase-3,7 activation measured in

MOLM13 acute myeloid leukemia cells. Values are the mean SEM.

azine or morpholine resulted in 33a and 33b, respectively.

Iodination of these derivatives, followed by coupling with 3-

aminothiophenol resulted in 35a and 35b, respectively. These

compounds were then reacted with a variety of unsaturated acid

chlorides to give target compounds 36a,b and 37. Target

compounds 42a−c and 43 were prepared similarly starting from

chloropyrimidine 38 (Scheme 3).

BIOLOGICAL EVALUATION IN THE HSP70

INHIBITOR SERIES

■

Recent studies suggest that, unlike normal cells, cancer cells

express modiﬁed heat shock protein species characterized by

both enhanced cochaperone recruitment and distinct post-

translational modiﬁcations.³¹⁻³³Evidence also indicates that

certain small molecules show preferential binding to these

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Table 2

a

b

Inhibition of growth measured in Kasumi-1 acute myeloid leukemia cells. Values are the mean SEM. Caspase-3,7 activation measured in

MOLM13 acute myeloid leukemia cells. Values are the mean SEM.

tumor-modiﬁed heat shock proteins.³²Our goal being to develop

Hsp70 inhibitors for cancer treatment, we accordingly employed

here a testing strategy that used phenotypic assays to read

ﬁngerprints of Hsp70 inhibition in tumor cells rather than a

classical recombinant protein-based approach (Figure 3).

To design a tumor−Hsp70 tailored testing module, we took

advantage of known biological activities mediated by Hsp70 in

cancer. As mentioned, Hsp70 is both an antiapoptotic molecule⁴

and a transforming protein that acts together with Hsp90 to

regulate the altered activity of several cancer kinases.^1,2,34Thus,

our testing strategy incorporated assays that measured induction

of apoptosis (i.e., the ability to activate caspase-3,7, Tables 1 and

2 and induce PARP cleavage, Table 3) and those that read a

phenotypic outcome similar to that of Hsp90 inhibitors (i.e.,

degradation of Hsp90/Hsp70-sheltered oncoproteins such as

HER2 and Raf-1 in the proper genetic background, Table 3) (see

tests 1 and 2 in Figure 3). Because a compound that interferes

with nucleotide binding to the ATP-binding pocket of Hsp90,

either competitively or allosterically, could result in a phenotype

closely resembling Hsp70 inhibition, we also included an assay to

test for possible direct binding to Hsp90 (Table 3).

Table 3

a

growth

Hsp90

c

b

compd

HER2

Raf-1

cPARP

inhibition

binding

4

>100

2

>100

2.5

1.7

7.5

2.5

3.1

5

>100

NA

2.0

5

NA

2.9

0.8

9

13.5

d

PU24FCl

17a

17c

20a

20c

27a

27d

31

0.66

0.7

2.5

1

>500

>250

>500

1.5

10

1.1

7

2

7.5

20

5

7.5

25

1.2

5

17

3.5

20

7.5

10

3.2

16.5

1.8

16.7

7.5

50

37

15

4

42a

42c

44a

5

30

2.5

75

50

40

7.5

60

10

45a

100

a

b

All values are in micromolar units. HER2 and Raf-1 steady-state

levels, PARP cleavage and inhibition of growth measured in SKBr3

breast cancer cells. Binding to SKBr3 cell extracts. Structure and

activity reported in ref 38.

c

d

HER2 is a key transforming protein in the HER2-over-

expressing breast cancer cells SKBr3, and its degradation or

inhibition is suﬃcient to inhibit the growth of these cells. Thus, in

addition to testing the eﬀect of these derivatives on HER2

expression, we also measured their activity on cell growth (Table

3).^35,36In addition, the compounds were further tested for their

ability to inhibit the growth of cancer cells of distinct genetic

origin, including the P-gp/MDR-1-expressing Kasumi-1 acute

myeloid leukemia cells (Table 1) (see test 3 in Figure 3).

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Figure 4. Designed ligands interact speciﬁcally with Hsp70 in cancer cells. (a) Streptavidin beads were incubated with the indicated concentrations of

44b, 45b, and ^D-biotin, the unbound agent was washed oﬀ, and the resulting beads carrying 44b, 45b, or D-biotin were probed with SKBr3 cell extracts

(500 μg). Hsp70 isolated on the beads was probed by Western blot (WB). A representative blot is presented (top). Blots were quantiﬁed by

densitometry and values, in relative luminescence units, graphed against the concentration of added biotinylated Hsp70 inhibitor (bottom). Results

from three independent experiments were graphed to determine the relative binding aﬃnity (K_d) of 44b and 45b using equations as implemented in the

GraphPad Prism software. Key: points, mean; bars, SD. (b) Cells were treated with the indicated concentrations of 44b for 6 h prior to lysing and

precipitation of protein complexes on streptavidin beads. Beads were washed with high-salt (1 M NaCl) buﬀer before elution of proteins on a denaturing

gel and silver staining. BB70 is an antibody speciﬁc for Hsp70. This antibody also recognizes Grp78 and Grp75, the endoplasmic reticulum and the

mitochondrial Hsp70 paralogues, respectively. (c) Protein complexes were isolated as indicated in (b) in cells pretreated with 17a. CP = chemical

precipitation. (d) Analysis of the Hsp70−HOP complex. SKBr3 cells were treated for 24 h with vehicle or indicated concentrations of 20a. Upon cell

lysing, Hsp70 complexes were isolated with an anti-Hsp70 antibody (IP BB70) and analyzed by WB. Speciﬁcity of binding was tested with a control IgG.

Gels were quantiﬁed by densitometry, values normalized to the control (vehicle only treated cells), and data graphed against the concentration of 20a.

Error bars represent the SEM (n = 2).

For select derivatives we probed their ability to bind Hsp70

from tumor extracts (Figure 4a) and from live cancer cells

(Figure 4b,c) and investigated their ability to alter the Hsp70−

HOP complex in cancer cells (Figure 4d; see test 4 in Figure

3).^5,6,32

For a speciﬁc Hsp70-mediated biological eﬀect it is expected

that compounds should act with a similar potency in the above-

described battery of assays (for example, the IC₅₀measured in the

client degradation assay should be near the IC₅₀measured in

Hsp70−HOP complex alteration and so on).^2,35,36Inclusion in

the testing module of three diﬀerent Hsp70-addicted cell lines,

including one P-gp/MDR-1 expressing, increased our likelihood

to eliminate early on poorly permeable compounds and those

marred by potential for P-gp/MDR-1-induced resistance.

STRUCTURE−ACTIVITY RELATIONSHIP IN THE

HSP70 INHIBITOR SERIES

■

In addition to ligand design, we used the homology model to

investigate structure−activity relationships in the designed

Hsp70 inhibitor series (Figure 5). To study potential ligand−

protein interactions, we docked each derivative into the allosteric

site. This strategy, combined with the phenotypic, target-derived

biological investigation (Figure 3), as described above, was used

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Site A. At the bottom of the allosteric pocket lies Cys267,

which is predicted to form a covalent bond with the acrylamide

positioned at X₄on ring B (Figure 5). The contribution of the

acrylamide moiety toward covalent binding with the protein can

be seen by comparing compounds in which it is attached to either

a pyrimidine or phenyl ring. Since the two nitrogen atoms of the

pyrimidine ring render the ring more electron deﬁcient,

acrylamide functions attached to it would be expected to be

more active Michael acceptors than in the corresponding phenyl

compounds. Indeed, a trend for increased activity was seen for

the pyrimidine-based compounds when compared to the similar

phenyl derivatives (Table 1, 27a and 27b versus 36a and 37,

respectively).

In addition to covalent Cys modiﬁcation, the acrylamide

substituent at position X₄is poised to form several interactions

with the adjacent protein residues (Figure 5). Speciﬁcally, the

alkene group is positioned to establish hydrophobic interactions

with Leu237 and Val238. The carbonyl group is poised to form

electrostatic interactions with the guanidine group of Arg264, ε-

NH₂of Lys271, and backbone −NH of Glu268 and could

potentially also form hydrogen bonds with the surrounding

residues.

To further understand the contribution of the acrylamide to

binding, we prepared several methyl-substituted acrylamide

derivatives (Table 2). Each of these derivatives was found to be

less active than the unsubstituted acrylamide derivatives (36a

versus 36b; 42a versus 42b and 42c). This decrease in reactivity

can be explained by both steric and electronic eﬀects. The methyl

group could act to sterically hinder nucleophilic attack by the

reactive cysteine residue (Cys267). Furthermore, the methyl

group is a mild electron-donating group and would decrease the

electrophilicity of the Michael acceptor.

Ring B occupies the lower part of the binding site which also

contains Arg264. Thus, the aromatic rings that were designed to

compose ring B, such as phenyl and pyrimidine, are predicted to

be stabilized by cation−π interactions with the guanidine group

of this arginine residue (Figure 5). Ring B also orients

substituents toward several important pocket residues. Speciﬁ-

cally, at the lower part of the pocket are Asp234 and Glu268,

which have the potential to interact with substituents on ring B,

such as those located at position X₃(Figure 5). Accordingly,

derivatives for which X₃was NH₂(i.e., 17a) were more potent

than similar derivatives having a hydrogen at this position (i.e.,

27a) potentially because NH₂could form hydrogen bonds with

the carboxylate groups of Asp234 and Glu268.

Figure 5. Binding interactions of hHsp70 with derivative 17a, as

predicted by Glide (Schrodinger LLC, New York). Hydrogen bonds are

shown as dotted red lines, and the interaction distance is shown by

dotted purple lines.

to understand diﬀerences in tumor Hsp70 inhibitory activity

among the designed derivatives.

Site C. Attached to ring A is substituent X₇pointing toward

the exit of the binding site (Figure 2c). At this position we could

accommodate a variety of substituents such as piperazine,

morpholine, piperidine, pyrrolidine, methoxy, and amino (Table

1). Methylpiperazine, however, was preferred over all others

(17a versus 17b, 17c, and 31; 20a versus 20b and 20c; 27a versus

27b, 27c, 27d, and 30; 36a versus 37). At physiological pH,

methylpiperazine is likely to be protonated, and this H is

predicted by docking to form a hydrogen bond interaction with

the backbone carbonyl of His89 (Figure 5). Indeed, an

approximately 10-fold drop in activity was observed when the

N-Me of methylpiperazine was substituted with O (as in

morpholine) or C (as in piperidine). Additionally, when this

amine was oxidized to the corresponding N-oxide, as in derivative

28, activity was almost abolished, potentially due to the

unfavorable interaction of the N-oxide oxygen with the backbone

carbonyl of His89. The pyrrolidine-containing derivative 27d

had a poor aqueous solubility, which likely accounted for the

erratic cellular activity we observed for this compound.

Site B. Substituents X₅and X₆of ring A are placed in the

middle cavity of the binding site comprised of both nonpolar and

polar amino acid residues, as described above (Figure 2c).

Substituents at these positions, such as methoxy, ethoxy, and

methyl, were well tolerated (compare 17a (methoxy), 20a

(ethoxy), and 42a (methyl)). Modeling predicts for such

substituents a potential for hydrophobic (alkyl portion of these

substituents with Val59) and electrostatic (oxygen of methoxy

and ethoxy with Arg261) interactions (Figure 5), and all

derivatives containing these substituents had favorable biological

activity. Modeling also indicated the potential for steric clash for

X₅and X₆substituents of larger size, such as in 45a, and indeed,

we observed a substantial loss of activity for this derivative (Table

1).

EVALUATION OF SELECTIVITY AND

TARGET-MEDIATED CELLULAR ACTIVITY

■

From these studies, derivatives 17a and 20a emerged as most

active (Tables 1 and 3). Next, we attached 17a to biotin with two

aims in mind, ﬁrst, to demonstrate eﬀective and selective

isolation of Hsp70 from cancer cells by 17a and, second, to

further validate that the acrylamide was not the sole contributor

to ligand−protein binding. Speciﬁcally, we have created two

derivatives of 17a by attaching to it linkers that allow for biotin

modiﬁcation of 17a (Figure 4a). In derivative 44a, the linker was

attached from the piperazine ring and thus predicted to point

outside the allosteric pocket. Indeed, 44a retained biological

activity similar to that of 17a (Tables 1 and 3). In derivative 45a,

the linker was attached on ring A through substituent X₅, thus at a

position that would create steric clashes between the substituent

and Hsp70 (Figure 4a). Indeed, 45a was of much reduced activity

when compared to 17a (Tables 1 and 3). We next attached 44a

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and 45a to biotin and then immobilized, dose-dependently, the

resulting 44b and 45b biotin-labeled ligands onto streptavidin-

containing beads. We then used these beads to isolate Hsp70

from cancer cell extracts.

DnaJ (bacterial Hsp70−Hsp40) complex formation (IC₅₀= 14.5

μM).¹²We could however not measure Hsp70-mediated

biological eﬀects for 4 in the SKBr3 cancer cells at concentrations

as high as 100 μM, possibly due to the poor stability of 4 in the

cells. Indeed, LC/MS−MS analyses of cellular extracts

demonstrated rapid myricetin degradation with the agent

virtually undetectable after 10 min of incubation (not shown).

Interestingly, when tested for its potential to bind Hsp90, we

found 4 to alter geldanamycin’s binding to Hsp90 (Table 3).

Polyphenols such as 4 are recognized for their high propensity to

bind nonspeciﬁcally to proteins. As such, caution must be taken

when interpreting biological and biochemical results derived

through the use of such compounds.

Next, we tested select lead compounds in vitro for potential to

interfere with other proteins. At the physiologically relevant

concentration of 10 μM, derivative 17a was inert when tested

against the scanMAX 402 kinase panel (Figure 6). This panel

contains a set of kinases covering the AGC, CAMK, CMGC,

CK1, STE, TK, TKL, lipid, and atypical kinase families, plus

important mutant forms. Developed by Ambit Biosciences, it

employs proprietary active-site-dependent competition binding

assays to determine how compounds bind to kinases. It is based

on a competition binding assay that quantitatively measures the

ability of a compound to compete with an immobilized, active-

site-directed ligand and can be used in detection of multiple

inhibitor types (e.g., types I and II and non-ATP-compet-

itive).^40,41

As expected from a mode of binding in which enthalpy

substantially contributes to binding, eﬃcient isolation of Hsp70

from a cancer cell extract was observed for the beads containing

derivative 44b but not 45b (Figure 4a). If binding were solely

driven by covalent modiﬁcation through the acrylamide

functionality, both 44b and 45b would interact equally well

with Hsp70 and, thus, isolate similar amounts of Hsp70 from the

cell extract. In conclusion, a proper ﬁt into the protein pocket in

addition to covalent modiﬁcation was important for the ability of

these ligands to bind to and inhibit Hsp70.

To demonstrate the selectivity of interaction, we incubated

cancer cells with 44b and then proceeded to isolate the captured

proteins on streptavidin beads. Upon washing the aﬃnity-

puriﬁed complexes and running them on a denaturing gel, the

selectivity of 44b toward Hsp70 was demonstrated by the

presence of a single band upon silver staining (Figure 4b). This

band as demonstrated by its size of 70 kDa, its ability to run at a

position identical to that of the protein isolated by an anti-Hsp70

antibody (Figure 4b, BB70), and its recognition by an Hsp70-

speciﬁc antibody (Figure 4a) and mass spectrum analysis²²

corresponds to Hsp70. Furthermore, preincubation of cells with

soluble 17a prior to aﬃnity puriﬁcation led to a dose-dependent

reduction of immobilized Hsp70, demonstrating the speciﬁcity

of the interaction (Figure 4c). Altogether, these ﬁndings conﬁrm

that the designed ligands act in cells by speciﬁcally binding to

Hsp70.

When cancer cells were incubated with select derivatives (i.e.,

20a), we observed a dose-dependent alteration in the formation

of Hsp70−HOP complexes in cancer cells (Figure 4d). As

mentioned above, HOP is a cochaperone that bridges Hsp70 and

Hsp90 to form a megachaperone complex (see test 4 in Figure

3). This chaperone machinery regulates the stability of several

onco-client proteins, such as HER2 and Raf-1, whose

degradation, as caused by chaperone inhibition, was tested

here (Table 3). Of importance, alteration in the Hsp70−HOP

complex formation occurred at the same low concentrations

(IC₅₀= 1.73 μM, Figure 4d) where we also observed degradation

of HER2 and Raf-1 by this inhibitor (1 and 2.5 μM, respectively,

Table 3), further supporting the Hsp70-mediated mechanism of

action of these ligands.

We noted no eﬀect on Hsp90 for the ligands reported here at

concentrations as high as 500 μM (Table 3). Speciﬁcally, unlike

the direct Hsp90 inhibitor PU24FCl,³⁸a compound with cellular

activity comparable to that of the most active Hsp70 inhibitors

described here, such as 17a and 20a (Table 3, HER2, Raf-1), the

Hsp70 inhibitors failed to compete with a ﬂuorescently labeled

geldanamycin derivative, GM-Cy3B³⁹for Hsp90 binding (Table

3). Geldanamycin is an Hsp90 inhibitor that binds to the Hsp90

regulatory pocket³⁷located in the N-terminal domain of Hsp90.

The ﬂuorescence polarization based Hsp90 assay that incorpo-

rates GM-Cy3B is designed in such a way that it probes not only

for direct binders to the ATP pocket but also for those

compounds that may allosterically interfere with the conforma-

tion of Hsp90 to block compound access to the ATP pocket.

We also tested myricetin (4; Figure 1) in these assays (Table

3). Using NMR techniques,¹²this compound was proposed to

interact with the bacterial Hsp70 homologue, DnaK, at a site

potentially close to that occupied by our designed Hsp70

inhibitors. 4 was also recently reported to inhibit the DnaK−

CONCLUSIONS

■

In summary, we describe here SAR studies in the ﬁrst rationally

designed scaﬀolds with binding potential to a novel allosteric

pocket in human Hsp70. This site is located in the N-terminal

domain of the chaperone and was recently identiﬁed by our

homology modeling studies.²²It is not revealed by reported

crystal structures of Hsp70, as these capture the protein in a

closed conformation in which the reactive Cys267 is unavailable

for ligand binding and the pocket is narrow.²⁴⁻²⁷The designed

ligands are of a yet unexplored chemical space based on the 2,5′-

thiodipyrimidine and 5-(phenylthio)pyrimidine scaﬀolds, and

thus, we are also ﬁrst to report here a chemical strategy that

allows for the assembly of such compounds.

The weight of the evidence in this paper and in our previous

report²²indicates that these compounds are acting on Hsp70

through binding to the allosteric pocket. Only if reasonably

correct could the homology model enable the rational design of a

ligand that, when incubated with the thousands of proteins

expressed in a cancer cell, substantially aﬃnity puriﬁes one,

Hsp70. Seconded by a biological investigation of these ligands

based on speciﬁc modulation of the target, Hsp70, in the context

of a cancer cell, our data clearly deﬁne the mode of action of these

ligands, in the cancer cell, through inhibition of Hsp70. From

these studies, we identify derivatives 17a and 20a as being the

most active of the series. Addition of high nanomolar to low

micromolar concentrations of these agents to several cancer cells

led to a reduction in the steady-state levels of Hsp70−HOP−

Hsp90 complex chaperoned oncoproteins, an eﬀect associated

with inhibition of cell growth and apoptosis.

Because Hsp70 contains an active cysteine embedded in the

allosteric pocket, the derivatives presented herein contain an

acrylamide functionality that may create a covalent bond with

such a residue upon Hsp70 protein binding. We have used

several techniques to demonstrate the potential formation of a

covalent bond between the inhibitor and the protein.²²First,

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conditions eluted preferentially the Hsp70-C267S mutant

protein over the WT form from preformed Hsp70−44b

complexes.

There is precedent for the use of an acrylamide “warhead” in

the development of several irreversible kinase inhibitors

currently in clinical trials for cancer.⁴²⁻⁴⁶There is, of course, a

concern that such an entity could indiscriminately react with

non-target-related proteins, resulting in unwanted biological

eﬀects. However, in spite of the presence of an acrylamide,

derivatives described here are not excessively reactive and, in

addition, have an appropriate ﬁt in the active site of the target,

anticipating a favorable enthalpic eﬀect. It is also important to

note that, for proteins with long half-lives, such as Hsp70,⁴⁷

irreversible inhibition is mechanistically advantageous as it

confers complete target inhibition until resynthesis of the target

protein⁴⁸and thus allows for less frequent dosing, which may also

tip the balance toward a better therapeutic index for such

compounds.

In addition to providing both a novel pharmacophore and

medicinal chemistry for its assembly, in this paper we describe a

testing battery for assessing Hsp70-mediated mechanisms in

cancer cells and for evaluating speciﬁc ligand action in cells

through Hsp70 inhibition. Altogether, these ﬁndings provide a

novel blueprint for a cancer-oriented development of Hsp70-

directed ligands.

To conclude, the series of novel chemical entities reported

herein interact with a yet unexplored pocket in Hsp70. These

compounds of tractable SAR favorably permeate cancer cells and

exhibit their biological activity through inhibition of the tumor

Hsp70 species, validating the scaﬀold as an important starting

point for the development of Hsp70 inhibitors with potential

therapeutic applications. The accompanying paper in this issue

describes the synthesis and structure−activity evaluation of

inhibitors of this series that target the same allosteric pocket of

Hsp70 described herein but act through a reversible mechanism

of action.²³

EXPERIMENTAL SECTION

■

Chemistry. All reagents were purchased from either Aldrich or Acros

Organics and used without puriﬁcation. All reactions were performed

under argon protection. NMR spectra were recorded on a Bruker AV-

III-500 MHz NMR spectrometer. Chemical shifts are reported in δ

values in parts per million downﬁeld from TMS as the internal standard.

¹H data are reported as follows: chemical shift, multiplicity (s = singlet, d

= doublet, t = triplet, q = quartet, br = broad, m = multiplet), coupling

constant (Hz), integration. ¹³C chemical shifts are reported in δ values in

parts per million downﬁeld from TMS as the internal standard. High-

resolution mass spectra were recorded on a Waters LCT Premier

system. Low-resolution mass spectra were obtained on a Waters Acquity

Ultra Performance LC instrument with electrospray ionization and an

SQ detector. Analytical HPLC was performed on a Waters

Autopuriﬁcation system with PDA, MicroMass ZQ, and ELSD

detectors. The purity of the title compounds used in pharmacology

testing was determined by HPLC−MS using the following method: 10−

12 min gradient on a Waters2525 binary gradient pump of increasing

concentrations of acetonitrile in water (5% → 95%) containing 0.1%

formic acid with a ﬂow rate of 1.2 mL/min and UV detection at λ = 220

and 254 nm on an XBridge C18 150 mm × 4.6 mm, 5 μm column. Title

compounds used in pharmacology testing were >95% pure. Analytical

thin-layer chromatography was performed on 250 μM silica gel F₂₅₄

plates. Preparative thin-layer chromatography was performed on 1000

μM silica gel F₂₅₄plates. Flash column chromatography was performed

employing 230−400 mesh silica gel. Solvents were HPLC grade.

Myricetin was purchased from Indoﬁne Chemical Co. (Hillsborough,

Figure 6. Derivative 17a (at 10 μM) was tested in the scanMAX screen

(Ambit) against 402 kinases. The TREEspot interaction map for 17a is

presented. Only c-Met (red dot on the kinase tree) appears as a potential

low-aﬃnity kinase hit of 17a. KINOMEscan’s selectivity score (S) is a

quantitative measure of compound selectivity. It is calculated by dividing

the number of kinases that bind to the compound by the total number of

distinct kinases tested, excluding mutant variants. S(35) = (number of

nonmutant kinases with %Ctrl < 35)/(number of nonmutant kinases

tested).

when the biotinylated analogue of 17a, 44b, was incubated with

cancer cells, we noted a time-dependent progressive increase in

the amount of immobilized Hsp70. This proﬁle is indicative of

time-dependent covalent modiﬁcation of the protein and is

speciﬁc for compounds such as 17a, where irreversible binding

plays a role. Second, mass spectrum analysis of the trypsin digest

of the 44b−Hsp70 complex identiﬁed a major m/z peak at

1867.915.²²This corresponds to 44b bound to LRTAC²⁶⁷ERAK

and conﬁrms that the site of interaction of 17a with Hsp70 is

within site 1, where Cys267 is located and, furthermore, that 17a

forms an irreversible bond with the protein upon binding. Third,

when we probed binding of 44b to both the WT Hsp70 protein

and the C267S mutant, we noted that high-salt, high-detergent

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NJ). The synthesis of compounds 44a,b and 45a,b is described in detail

in the Supporting Information.

and the residue was puriﬁed by column chromatography (CHCl₃/

MeOH−NH₃(7 N), 10:1) to yield 32 mg (90%) of 15a. ¹H NMR (500

MHz, CDCl₃): δ 8.36 (br s, 1H), 8.13 (br s, 2H), 3.88 (s, 6H), 3.87 (m,

4H), 2.46 (m, 4H), 2.35 (s, 3H), 1.99 (s, 6H). ¹³C NMR (125 MHz,

CDCl₃): δ 172.2, 169.4, 168.9, 160.4, 158.9, 96.1, 95.9, 54.7, 54.3, 46.1,

43.8, 24.7. MS (m/z): [M + H]⁺463.2.

2-((4,6-Dimethoxy-2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)-

thio)pyrimidine-4,6-diamine (16a). A mixture of 15a (50 mg, 0.108

mmol) and 1 N NaOH(aq) (2 mL) in 4 mL of methanol was stirred at

60 °C for 1 h. Solvents were removed under reduced pressure, and the

residue was puriﬁed by preparatory TLC to aﬀord 39 mg (95%) of 16a.

¹H NMR (500 MHz, CDCl₃): δ 5.19 (s, 1H), 4.48 (s, 4H), 3.88 (s, 6H),

4,6-Dimethoxy-N,N-bis(4-methoxybenzyl)pyrimidin-2-amine

(9a). To a solution of 2-amino-4,6-dimethoxypyrimidine (2.0 g, 12.9

mmol) (8a) in 20 mL of DMF at 0 °C was added NaH (1.24 g, 51.5

mmol), and the mixture was stirred at rt for 10 min. 4-Methoxybenzyl

chloride (4.03 g, 25.7 mmol) was added, and the mixture was stirred at rt

overnight. The reaction was quenched with methanol and solvent

removed under reduced pressure. The residue was dissolved in EtOAc,

washed with brine, and dried over MgSO₄. Solvent was evaporated

under reduced pressure, and the residue was puriﬁed by column

chromatography (hexane/EtOAc, 4:1) to aﬀord 4.8 g (95%) of 9a. ¹H

NMR (500 MHz, CDCl₃): δ 7.26 (d, J = 8.4 Hz, 4H), 6.89 (d, J = 8.4 Hz,

4H), 5.47 (s, 1H), 4.78 (s, 4H), 3.85 (s, 6H), 3.80 (s, 6H). ¹³C NMR

(125 MHz, CDCl₃): δ 171.9, 161.5, 158.7, 130.9, 129.1, 113.8, 78.7,

55.2, 53.4, 48.3. MS (m/z): [M + H]⁺396.3.

3.87 (m, 4H), 2.48 (m, 4H), 2.35 (s, 3H). MS (m/z): [M + H]⁺378.9.

N-(6-Amino-2-((4,6-dimethoxy-2-(4-methylpiperazin-1-yl)-

pyrimidin-5-yl)thio)pyrimidin-4-yl)acrylamide (17a). To a solution of

16a (0.370 g, 0.977 mmol) and Et₃N (0.988 g, 9.77 mmol) in 10 mL of

anhydrous dioxane was added acryloyl chloride (0.855 g, 9.77 mmol)

dropwise under a water bath. The resulting mixture was stirred at rt for

24 h. Solvent was removed under reduced pressure, and the residue was

puriﬁed by preparatory TLC (CHCl₃/MeOH−NH₃(7 N), 10:1) to

aﬀord 0.211 g (50%) of 17a. ¹H NMR (500 MHz, CDCl₃): δ 7.96 (br s,

1H), 7.04 (s, 1H), 6.41 (d, J = 16.8 Hz, 1H), 6.17 (dd, J = 16.8, 10.3 Hz,

1H), 5.78 (d, J = 10.3 Hz, 1H), 4.83 (br s, 2H), 3.88 (s, 6H), 3.87 (m,

4H), 2.47 (m, 4H), 2.35 (s, 3H). ¹³C NMR (125 MHz, CDCl₃): δ 169.3,

168.8, 162.6, 162.5, 158.3, 154.9, 128.9, 127.1, 86.7, 78.2, 53.1, 52.4,

44.5, 41.9. HRMS (m/z): [M + H]⁺calcd for C₁₈H₂₅N₈O₃S, 433.1770;

found, 433.1750. HPLC: (a) H₂O + 0.1% TFA, (b) ACN + 0.1% TFA

(5−95% ACN in 10 min), t_R= 6.28 min.

N,N′-(2-((4,6-Dimethoxy-2-morpholinopyrimidin-5-yl)thio)-

pyrimidine-4,6-diyl)diacetamide (15b). To a solution of 14a (60 mg,

0.157 mmol) in 2 mL of DMF was added morpholine (54 mg, 0.620

mmol), and the resulting solution was heated at 90 °C for 1 h. Solvent

and excess reagent were removed under reduced pressure, and the

residue was puriﬁed by preparatory TLC (CHCl₃/MeOH−NH₃(7 N),

10:1) to yield 56 mg (79%) of 15b. ¹H NMR (500 MHz, CDCl₃): δ 8.41

(br s, 1H), 7.82 (br s, 2H), 3.83 (s, 6H), 3.77 (m, 4H), 3.68 (m, 4H),

2.16 (s, 6H). HRMS (m/z): [M + H]⁺calcd for C₁₈H₂₄N₇O₅S,

450.1560; found, 450.1548. HPLC: (a) H₂O + 0.1% TFA, (b) ACN +

0.1% TFA (5−95% ACN in 10 min), t_R= 8.47 min.

2-((4,6-Dimethoxy-2-morpholinopyrimidin-5-yl)thio)pyrimidine-

4,6-diamine (16b). A mixture of 15b (45 mg, 0.100 mmol) and 1 N

NaOH(aq) (2 mL) in 4 mL of methanol was stirred at 60 °C for 1 h.

Solvents were removed under reduced pressure, and the residue was

puriﬁed by preparatory TLC to aﬀord 35 mg (95%) of 16b. ¹H NMR

(500 MHz, CDCl₃): δ 5.19 (s, 1H), 4.44 (s, 4H), 3.87 (s, 6H), 3.83 (m,

4H), 3.77 (m, 4H), 2.16 (s, 6H). HRMS (m/z): [M + H]⁺calcd for

C₁₄H₂₀N₇O₃S, 366.1348; found, 366.1361. HPLC: (a) H₂O + 0.1%

TFA, (b) ACN + 0.1% TFA (5−95% ACN in 10 min), t_R= 6.45 min.

N-(6-Amino-2-((4,6-dimethoxy-2-morpholinopyrimidin-5-yl)-

thio)pyrimidin-4-yl)acrylamide (17b). To a solution of 16b (50 mg,

0.111 mmol) and Et₃N (56 mg, 0.555 mmol) in 2 mL of anhydrous

dioxane was added acryloyl chloride (50 mg, 0.555 mmol) under a water

bath. The resulting mixture was stirred at rt for 24 h. Solvent was

removed under reduced pressure, and the residue was puriﬁed by

preparatory TLC (CHCl₃/MeOH−NH₃(7 N), 10:1) to aﬀord 26 mg

(55%) of 17b. ¹H NMR (500 MHz, CDCl₃): δ 7.73 (br s, 1H), 7.05 (s,

1H), 6.42 (d, J = 16.9 Hz, 1H), 6.17 (dd, J = 16.7, 10.4 Hz, 1H), 5.80 (d, J

= 10.3 Hz, 1H), 4.78 (br s, 2H), 3.89 (s, 6H), 3.81 (m, 4H), 3.75 (m,

4H). HRMS (m/z): [M + H]⁺calcd for C₁₇H₂₂N₇O₄S, 420.1454; found,

420.1470.

N,N′-(2-((4,6-Dimethoxy-2-(piperidin-1-yl)pyrimidin-5-yl)thio)-

pyrimidine-4,6-diyl)diacetamide (15c). To a solution of 14a (60 mg,

0.157 mmol) in 2 mL of DMF was added piperidine (52 mg, 0.611

mmol), and the resulting solution was heated at 90 °C for 1 h. Solvent

and excess reagent were removed under reduced pressure, and the

residue was puriﬁed by preparatory TLC (CHCl₃/MeOH−NH₃(7 N),

10:1) to yield 55 mg (78%) of 15c. ¹H NMR (500 MHz, CDCl₃): δ 8.32

(br s, 1H), 7.77 (br s, 2H), 3.88 (s, 6H), 2.16 (m, 6H), 1.54−1.70 (m,

6H). HRMS (m/z): [M + H]⁺calcd for C₁₉H₂₆N₇O₄S, 448.1767; found,

5-Iodo-4,6-dimethoxy-N,N-bis(4-methoxybenzyl)pyrimidin-2-

amine (10a). To a solution of 9a (4.8 g, 12.3 mmol) in 50 mL of

acetonitrile was added NIS (4.13 g, 18.4 mmol), and the resulting

solution was stirred at rt for 1 h. Solvent was evaporated, and the residue

was puriﬁed by column chromatography (hexane/EtOAc, 4:1) to aﬀord

6.3 g (98%) of 10a. ¹H NMR (500 MHz, CDCl₃): δ 7.18 (d, J = 8.6 Hz,

4H), 6.83 (d, J = 8.6 Hz, 4H), 4.71 (s, 4H), 3.89 (s, 6H), 3.79 (s, 6H).

¹³C NMR (125 MHz, CDCl₃): δ 169.1, 160.9, 158.8, 130.5, 129.0, 113.9,

55.3, 54.7, 48.6, 43.9. MS (m/z): [M + H]⁺522.4.

2-((2-(Bis(4-methoxybenzyl)amino)-4,6-dimethoxypyrimidin-5-

yl)thio)pyrimidine-4,6-diamine (11a). A mixture of 10a (6.2 g, 11.9

mmol), 4,6-diamino-2-mercaptopyrimidine (1.7 g, 11.9 mmol), neo-

cuproine (0.538 g, 2.38 mmol), CuI (0.452 g, 2.38 mmol), and K₂CO₃

(3.3 g, 33.8 mmol) in 100 mL of DMSO was stirred at 120 °C for 16 h.

Solvent was removed under reduced pressure, and the residue was

puriﬁed by column chromatography (CH₂Cl₂/MeOH−NH₃(7 N),

20:1) to aﬀord 4.2 g (65%) of 11a. ¹H NMR (500 MHz, DMSO-d₆): δ

7.18 (d, J = 8.6 Hz, 4H), 6.80 (d, J = 8.6 Hz, 4H), 5.09 (s, 1H), 4.68 (s,

4H), 4.40 (s, 4H), 3.79 (s, 6H), 3.74 (s, 6H). MS (m/z): [M + H]⁺

536.5.

N,N′-(2-((2-(Bis(4-methoxybenzyl)amino)-4,6-dimethoxypyrimi-

din-5-yl)thio)pyrimidine-4,6-diyl)diacetamide (12a). A solution of

11a (3.2 g, 6.0 mmol) and DMAP (0.037 g, 0.3 mmol) in 20 mL of acetic

anhydride was stirred at 110 °C for 2 h. Solvent was removed under

reduced pressure, and the residue was puriﬁed by column chromatog-

raphy (hexane/EtOAc, 1:1) to aﬀord 3.4 g (91%) of 12a. ¹H NMR (500

MHz, CDCl₃/DMSO-d₆): δ 8.25 (br s, 1H), 7.70 (br s, 2H), 7.18 (d, J =

10.0 Hz, 4H), 6.81 (d, J = 10.0 Hz, 4H), 4.70 (s, 4H), 3.78 (s, 6H), 3.74

(s, 6H), 2.10 (s, 6H). MS (m/z): [M + H]⁺620.4.

N,N′-(2-((2-Amino-4,6-dimethoxypyrimidin-5-yl)thio)pyrimidine-

4,6-diyl)diacetamide (13a). A solution of 12a (0.950 g, 1.5 mmol) in 20

mL of TFA/CHCl₃(1:1) was heated at 62 °C for 24 h. Excess TFA and

solvent were removed under reduced pressure, and the residue was

puriﬁed by column chromatography (CH₂Cl₂/MeOH, 20:1) to aﬀord

0.550 g (95%) of 13a. ¹H NMR (500 MHz, DMSO-d₆): δ 10.51 (br s,

2H), 8.37 (br s, 1H), 6.98 (s, 2H), 3.78 (s, 6H), 2.06 (s, 6H). ¹³C NMR

(125 MHz, DMSO-d₆): δ 170.9, 170.1, 169.2, 162.5, 158.9, 92.8, 78.5,

53.9, 24.1. MS (m/z): [M + H]⁺380.2.

N,N′-(2-((2-Fluoro-4,6-dimethoxypyrimidin-5-yl)thio)pyrimidine-

4,6-diyl)diacetamide (14a). 13a (2.0 g, 5.3 mmol) was added to a

plastic tube ﬁtted with a stir bar and cooled to 0 °C followed by addition

of a solution of HF/pyridine (3.6 mL, 144 mmol). NaNO₂(0.545 g, 7.9

mmol) was added in portions over a period of 20 min with stirring. The

resulting solution was vigorously stirred for an additional 50 min at 0 °C

and 2 h at rt. CaCO₃(14.4 g, 144 mmol) was added to quench excess

HF. The mixture was extracted with CH₂Cl₂and puriﬁed by column

chromatography (CH₂Cl₂/MeOH−NH₃(7N), 20:1) to aﬀord 1.1 g

1

(54%) of 14a. H NMR (500 MHz, CDCl₃): δ 8.45 (s, 1H), 7.61 (s,

2H), 4.00 (s, 6H), 2.18 (s, 6H). MS (m/z): [M + H]⁺383.2.

N,N′-(2-((4,6-Dimethoxy-2-(4-methylpiperazin-1-yl)pyrimidin-5-

yl)thio)pyrimidine-4,6-diyl)diacetamide (15a). To a solution of 14a

(30 mg, 0.078 mmol) in 2 mL of DMF was added 1-methylpiperazine

(31 mg, 0.31 mmol), and the resulting solution was heated at 90 °C for 1

h. Solvent and excess reagent were removed under reduced pressure,

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448.1766. HPLC: (a) H₂O + 0.1% TFA, (b) ACN + 0.1% TFA (5−95%

ACN in 10 min), t_R= 10.13 min.

reduced pressure, and the residue was puriﬁed by column chromatog-

raphy (hexane/EtOAc, 1:1) to aﬀord 1.2 g (89%) of 12b. ¹H NMR (500

MHz, CDCl₃): δ 8.22 (s, 2H), 7.21 (d, J = 8.5 Hz, 4H), 6.86 (d, J = 8.5

Hz, 4H), 4.70 (s, 4H) 4.32 (q, J = 7.1 Hz, 4H), 3.80 (s, 6H), 2.16 (s, 6H),

1.20 (t, J = 7.1 Hz, 6H). MS (m/z): [M + H]⁺648.1.

N,N′-(2-((2-Amino-4,6-diethoxypyrimidin-5-yl)thio)pyrimidine-

4,6-diyl)diacetamide (13b). A solution of 12b (2.00 g, 3.09 mmol) in

20 mL of TFA/CHCl₃(1:1) was heated at 62 °C for 24 h. Excess TFA

and solvent were removed under reduced pressure, and the residue was

puriﬁed by column chromatography (CH₂Cl₂/MeOH, 20:1) to aﬀord

1.15 g (92%) of 13b. MS (m/z): [M + H]⁺407.8.

N,N′-(2-((2-Fluoro-4,6-diethoxypyrimidin-5-yl)thio)pyrimidine-

4,6-diyl)diacetamide (14b). 13b (1.5 g, 3.68 mmol) was added to a

plastic tube ﬁtted with a stir bar and cooled to 0 °C followed by addition

of a solution of HF/pyridine (3.0 mL, 120 mmol). After several minutes

NaNO₂(0.380 g, 5.52 mmol) was added in portions over a period of 20

min with stirring. The resulting solution was vigorously stirred for an

additional 50 min at 0 °C. CaCO₃(12.0 g, 120 mmol) was added to

quench excess HF. The mixture was extracted with CH₂Cl₂and puriﬁed

by column chromatography (CH₂Cl₂/MeOH−NH₃(7 N), 20:1) to

aﬀord 0.76 g (46%) of 14b. ¹H NMR (500 MHz, CDCl₃): δ 8.47 (br s,

1H), 7.85 (br s, 2H), 4.44 (q, J = 7.1 Hz, 4H), 2.17 (s, 6H), 1.31 (t, J =

7.1 Hz, 6H). MS (m/z): [M + H]⁺411.3.

N,N′-(2-((4,6-Diethoxy-2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)-

thio)pyrimidine-4,6-diyl)diacetamide (18a). To a solution of 14b

(0.165 g, 0.402 mmol) in 3 mL of DMF was added 1-methylpiperazine

(400 mg, 4.4 mmol), and the resulting solution was heated to 90 °C for 1

h. Solvent was removed under reduced pressure, and the residue was

puriﬁed by column chromatography (CH₂Cl₂/MeOH−NH₃(7 N),

10:1) to yield 0.180 g (91%) of 18a. ¹H NMR (500 MHz, CDCl₃): δ

8.35 (br s, 1H), 8.06 (br s, 2H), 4.35 (q, J = 7.1 Hz, 4H), 3.82 (m, 4H),

2.43 (m, 4H), 2.36 (s, 3H), 2.15 (s, 6H), 1.26 (t, J = 7.1 Hz, 6H). MS

(m/z): [M + H]⁺491.2.

2-((4,6-Diethoxy-2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)thio)-

pyrimidine-4,6-diamine (19a). A mixture of 18a (0.130 g, 0.265 mmol)

and 1 N NaOH(aq) (2 mL) in 7 mL of methanol was stirred at 60 °C for

1 h. Solvent was removed under reduced pressure, and the residue was

puriﬁed by preparatory TLC (CH₂Cl₂/MeOH, 10:1) to aﬀord 0.100 g

(93%) of 19a. ¹H NMR (500 MHz, CDCl₃): δ 5.17 (br s, 1H), 4.48 (s,

4H), 4.34 (q, J = 7.1 Hz, 4H), 3.83 (m, 4H), 2.47 (m, 4H), 2.35 (s, 3H),

1.27 (t, J = 7.1 Hz, 6H). MS (m/z): [M + H]⁺407.1.

N-(6-Amino-2-((4,6-diethoxy-2-(4-methylpiperazin-1-yl)-

pyrimidin-5-yl)thio)pyrimidin-4-yl)acrylamide (20a). To a solution of

19a (20 mg, 0.049 mmol) and Et₃N (49 mg, 0.49 mmol) in 1 mL of

anhydrous dioxane was added acryloyl chloride (44 mg, 0.49 mmol).

The resulting mixture was stirred at rt for 12 h. Solvent was removed

under reduced pressure, and the residue was puriﬁed by preparatory

TLC (CHCl₃/MeOH−NH₃(7 N), 10:1) to aﬀord 9 mg (40%) of 20a.

¹H NMR (500 MHz, CDCl₃): δ 8.14 (br s, 1H), 7.04 (s, 1H), 6.40 (d, J =

16.9 Hz, 1H), 6.19 (dd, J = 16.9, 10.4 Hz, 1H), 5.77 (d, J = 10.4 Hz, 1H),

4.83 (br s, 2H), 4.35 (q, J = 7.0 Hz, 4H), 3.83 (m, 4H), 2.46 (m, 4H),

2.35 (s, 3H), 1.28 (t, J = 7.0 Hz, 6H). ¹³C NMR (125 MHz, DMSO-d₆):

δ 170.1, 169.2, 164.8, 164.2, 159.4, 156.4, 131.3, 128.1, 87.5, 79.9, 62.0,

54.3, 46.6, 43.2, 14.4. HRMS (m/z): [M + H]⁺calcd for C₂₀H₂₉N₈O₃S,

461.2083; found, 461.2096. HPLC: (a) H₂O + 0.1% TFA, (b) ACN +

0.1% TFA (5−95% ACN in 10 min), t_R= 5.57 min.

N,N′-(2-((4,6-Diethoxy-2-morpholinopyrimidin-5-yl)thio)-

pyrimidine-4,6-diyl)diacetamide (18b). To a solution of 14b (165 mg,

0.402 mmol) in 3 mL of DMF was added morpholine (350 mg, 4.02

mmol), and the resulting solution was heated at 90 °C for 1 h. Solvent

and excess reagent were removed under reduced pressure, and the

residue was puriﬁed by preparatory TLC (CHCl₃/MeOH−NH₃(7 N),

10:1) to yield 192 mg (81%) of 18b. MS (m/z): [M + H]⁺478.1. ¹H

NMR (500 MHz, CDCl₃): δ 8.35 (br s, 1H), 8.06 (br s, 2H), 4.34 (q, J =

7.0 Hz, 4H), 3.82−3.84 (m, 4H), 2.43−2.45 (m, 4H), 2.15 (s, 6H), 1.26

(t, J = 7.0 Hz, 6H).

2-((4,6-Dimethoxy-2-(piperidin-1-yl)pyrimidin-5-yl)thio)-

pyrimidine-4,6-diamine (16c). A mixture of 15c (55 mg, 0.123 mmol)

and 1 N NaOH(aq) (2 mL) in 4 mL of methanol was stirred at 60 °C for

1 h. Solvents were removed under reduced pressure, and the residue was

puriﬁed by preparatory TLC to aﬀord 43 mg (96%) of 16c. ¹H NMR

(500 MHz, CDCl₃): δ 5.18 (s, 1H), 4.42 (s, 4H), 3.88 (s, 6H), 3.80 (m,

4H), 1.60−1.71 (m, 6H). HRMS (m/z): [M + H]⁺calcd for

C₁₅H₂₂N₇O₂S, 364.1556; found, 364.1544. HPLC: (a) H₂O + 0.1%

TFA, (b) ACN + 0.1% TFA (5−95% ACN in 10 min), t_R= 7.43 min.

N-(6-Amino-2-((4,6-dimethoxy-2-(piperidin-1-yl)pyrimidin-5-yl)-

thio)pyrimidin-4-yl)acrylamide (17c). To a solution of 16c (46 mg,

0.127 mmol) and Et₃N (130 mg, 1.30 mmol) in 4 mL of anhydrous

dioxane was added acryloyl chloride (119 mg, 1.30 mmol) dropwise

under a water bath. The resulting mixture was stirred at rt for 12 h.

Solvent was removed under reduced pressure, and the residue was

puriﬁed by preparatory TLC (CHCl₃/MeOH−NH₃(7 N), 10:1) to

aﬀord 28 mg (52%) of 17c. ¹H NMR (500 MHz, CDCl₃/DMSO-d₆): δ

9.33 (s, 1H), 7.09 (s, 1H), 6.47−6.35 (m, 2H), 5.71 (d, J = 9.3 Hz, 1H),

5.31 (br s, 2H), 3.88 (s, 6H), 3.81 (m, 4H), 1.69 (m, 2H), 1.62 (m, 4H).

HRMS (m/z): [M + H]⁺calcd for C₁₈H₂₄N₇O₃S, 418.1661; found,

418.1660. HPLC: (a) H₂O + 0.1% TFA, (b) ACN + 0.1% TFA (5−95%

ACN in 10 min), t_R= 8.06 min.

2-Amino-4,6-diethoxypyrimidine (8b). To a solution of 2-amino-

4,6-dichloropyrimidine (1.0 g, 6.09 mmol) in 20 mL of absolute ethanol

was added NaH (0.585 g, 24.39 mmol) at rt. The mixture was stirred

under reﬂux for 12 h. Solvent was removed under reduced pressure, and

the residue was dissolved in CH₂Cl₂and washed with brine. Solvent was

evaporated, and the resulting solid was puriﬁed by column

chromatography (hexane/EtOAc, 4:1) to aﬀord 1.0 g (89%) of 8b. ¹H

NMR (500 MHz, CDCl₃): δ 5.42 (s, 1H), 4.78 (br s, 2H), 4.24 (q, J = 7.1

Hz, 4H), 1.34 (t, J = 7.1 Hz, 6H). MS (m/z): [M + H]⁺183.9.

4,6-Diethoxy-N,N-bis(4-methoxybenzyl)pyrimidin-2-amine (9b).

To a solution of 8b (1.00 g, 5.46 mmol) in 20 mL of DMF at 0 °C

was added NaH (0.524 g, 21.83 mmol), and the resulting solution was

stirred at rt for 10 min. 4-Methoxybenzyl chloride (1.88 g, 12.0 mmol)

was added, and the mixture was stirred at rt overnight. The reaction was

quenched with ethanol, and solvent was removed under reduced

pressure. The residue was dissolved in EtOAc, washed with brine, dried

over MgSO₄, and concentrated to give a residue that was puriﬁed by

column chromatography (hexane/EtOAc, 4:1) to aﬀord 2.25 g (97%) of

9b. ¹H NMR (500 MHz, CDCl₃): δ 7.18 (d, J = 8.1, 4H), 6.84 (d, J = 8.1,

4H), 5.38 (s, 1H), 4.70 (s, 4H), 4.27 (q, J = 7.1 Hz, 4H), 3.79 (s, 6H),

1.29 (t, J = 7.1 Hz, 6H). ¹³C NMR (125 MHz, CDCl₃): δ 171.5, 161.4,

158.6, 130.9, 129.0, 113.7, 78.6, 61.8, 55.2, 48.1, 14.6. MS (m/z): [M +

H]⁺424.2.

5-Iodo-4,6-diethoxy-N,N-bis(4-methoxybenzyl)pyrimidin-2-

amine (10b). To a solution of 9b (2.2 g, 5.2 mmol) in 50 mL of

acetonitrile was added NIS (1.7 g, 8 mmol), and the resulting solution

was stirred at rt for 1 h. Solvent was removed under reduced pressure,

and the residue was puriﬁed by column chromatography (hexane/

1

EtOAc, 4:1) to aﬀord 2.75 g (96%) of 10b. H NMR (500 MHz,

CDCl₃): δ 7.17 (m, 4H), 6.84 (m, 4H), 4.68 (s, 4H), 4.34 (q, J = 7.1 Hz,

4H), 3.80 (s, 6H), 1.32 (t, J = 7.1 Hz, 6H). ¹³C NMR (125 MHz,

CDCl₃/DMSO-d₆): δ 168.3, 160.4, 158.2, 130.1, 128.4, 113.2, 62.6,

54.8, 48.1, 44.2, 14.1. MS (m/z): [M + H]⁺550.1.

2-((2-(Bis(4-methoxybenzyl)amino)-4,6-diethoxypyrimidin-5-yl)-

thio)pyrimidine-4,6-diamine (11b). A mixture of 10b (2.75 g, 5.0

mmol), 4,6-diamino-2-mercaptopyrimidine (0.71 g, 5.0 mmol), neo-

cuproine (0.226 g, 1.0 mmol), CuI (0.190 g, 1.0 mmol), and K₂CO₃

(1.38 g, 10.0 mmol) in 60 mL of DMSO was stirred at 120 °C for 16 h.

Solvent was removed under reduced pressure, and the residue was

partially puriﬁed by column chromatography (CH₂Cl₂/MeOH−NH₃

(7 N), 20:1) to aﬀord 2.2 g (80%) of impure 11b [MS (m/z): [M + H]⁺

564.2], which was used without further puriﬁcation in the next step.

N,N′-(2-((2-(Bis(4-methoxybenzyl)amino)-4,6-diethoxypyrimidin-

5-yl)thio)pyrimidine-4,6-diyl)diacetamide (12b). A solution of 11b

(1.2 g, 2.19 mmol) and DMAP (0.013 g, 0.11 mmol) in 20 mL of acetic

anhydride was stirred at 110 °C for 2 h. Solvent was removed under

2-((4,6-Diethoxy-2-morpholinopyrimidin-5-yl)thio)pyrimidine-

4,6-diamine (19b). A mixture of 18b (0.200 g, 0.42 mmol) and 1 N

NaOH(aq) (2 mL) in 5 mL of methanol was stirred at 60 °C for 1 h.

Solvent was removed under reduced pressure, and the residue was

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puriﬁed by chromatography (CH₂Cl₂/MeOH−NH₃(7 N), 10:1) to

aﬀord 0.153 g (93%) of 19b. ¹H NMR (500 MHz, CDCl₃): δ 6.39 (br s,

1H), 4.31 (q, J = 6.2 Hz, 4H), 3.51−3.83 (m, 4H), 1.90−1.95 (m, 4H),

1.19 (t, J = 6.2 Hz, 6H). MS (m/z): [M + H]⁺394.1.

under reduced pressure, and the residue was puriﬁed by column

chromatography (CH₂Cl₂/MeOH−NH₃(7 N), 20:1) to aﬀord 4.16 g

1

(85%) of 23. H NMR (500 MHz, CDCl₃/MeOH-d₄): δ 10.23 (br s,

1H), 8.21 (s, 1H), 7.75 (s, 1H), 5.98 (br s, 2H), 3.88 (s, 6H), 2.14 (s,

3H). ¹³C NMR (125 MHz, CDCl₃/MeOH-d₄): δ 170.8, 170.2, 170.0,

161.8, 157.5, 157.4, 105.1, 80.2, 53.7, 23.8. HRMS (m/z): [M + H]⁺

calcd for C₁₂H₁₅N₆O₃S, 323.0926; found, 323.0913. HPLC: (a) H₂O +

0.1% TFA, (b) ACN + 0.1% TFA (5−95% ACN in 10 min), t_R= 5.56

min.

N-(2-((2-Fluoro-4,6-dimethoxypyrimidin-5-yl)thio)pyrimidin-4-

yl)acetamide (24). 23 (1.72 g, 5.32 mmol) was added to a plastic tube

ﬁtted with a stir bar and cooled to 0 °C followed by addition of a solution

of HF/pyridine (3.6 mL, 144 mmol). NaNO₂(0.545 g, 7.9 mmol) was

added in portions over a period of 20 min with stirring. The resulting

solution was vigorously stirred for an additional 50 min at 0 °C and 2 h at

rt. CaCO₃(14.4 g, 144 mmol) was added to destroy excess HF. The

mixture was extracted with CH₂Cl₂and puriﬁed by column

chromatography (CH₂Cl₂/MeOH, 20:1) to aﬀord 0.99 g (57%) of

24. ¹H NMR (500 MHz, CDCl₃): δ 8.34 (d, J = 5.6 Hz, 1H), 7.82 (d, J =

5.6 Hz, 1H), 4.00 (s, 6H), 2.21 (s, 3H). ¹³C NMR (125 MHz, CDCl₃): δ

173.2, 169.2, 162.8, 161.1, 159.0, 157.1, 105.9, 90.7, 55.8, 24.8. HRMS

(m/z): [M + H]⁺calcd for C₁₂H₁₃FN₅O₃S, 326.0723; found, 326.0732.

HPLC: (a) H₂O + 0.1% TFA, (b) ACN + 0.1% TFA (5−95% ACN in 10

min), t_R= 7.75 min.

N-(2-((4,6-Dimethoxy-2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)-

thio)pyrimidin-4-yl)acetamide (25a). To a solution of 24 (213 mg,

0.655 mmol) in DMF (10 mL) was added 1-methylpiperazine (0.291

mL, 262 mg, 2.62 mmol), and the resulting solution was heated at 90 °C

for 1 h. Solvent was removed under reduced pressure, and the residue

was puriﬁed by chromatography (CH₂Cl₂/MeOH−NH₃(7 N), 50:1 to

30:1) to aﬀord 0.253 g (95%) of 25a. ¹H NMR (500 MHz, CDCl₃): δ

8.34 (d, J = 5.6 Hz, 1H), 8.09 (br s, 1H), 7.75 (br s, 1H), 3.90 (s, 6H),

3.88 (m, 4H), 2.48 (m, 4H), 2.38 (s, 3H), 2.18 (s, 3H). HRMS (m/z):

[M + H]⁺calcd for C₁₇H₂₄N₇O₃S, 406.1661; found, 406.1661. HPLC:

(a) H₂O + 0.1% TFA, (b) ACN + 0.1% TFA (5−95% ACN in 10 min),

t_R= 5.75 min.

2-((4,6-Dimethoxy-2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)-

thio)pyrimidin-4-amine (26a). 25a (253 mg, 0.624 mmol) in MeOH

(2 mL) and 1 N NaOH(aq) (10 mL) was stirred at 60 °C for 80 min.

Solvent was removed under reduced pressure, and the residue was

puriﬁed by chromatography (CH₂Cl₂/MeOH−NH₃(7 N), 50:1 to

25:1) to aﬀord 221 mg (97%) of 26a. ¹H NMR (500 MHz, CDCl₃): δ

7.95 (d, J = 5.8 Hz, 1H), 6.04 (d, J = 5.8 Hz, 1H), 5.01 (s, 2H), 3.89 (br s,

10H), 2.48 (m, 4H), 2.36 (s, 3H). HRMS (m/z): [M + H]⁺calcd for

C₁₅H₂₂N₇O₂S, 364.1556; found, 364.1542. HPLC: (a) H₂O + 0.1%

TFA, (b) ACN + 0.1% TFA (5−95% ACN in 10 min), t_R= 4.90 min.

N-(2-((4,6-Dimethoxy-2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)-

thio)pyrimidin-4-yl)acrylamide (27a). To a solution of 26a (50 mg,

0.138 mmol) and Et₃N (69 mg, 0.688 mmol) in CH₂Cl₂(2 mL) was

added acryloyl chloride (62 mg, 0.688 mmol) under a water bath. The

resulting mixture was stirred at rt for 24 h. Solvent was removed under

reduced pressure, and the residue was puriﬁed by preparatory TLC

(CHCl₃/MeOH−NH₃(7 N), 10:1) to aﬀord 42 mg (72%) of 27a. ¹H

NMR (500 MHz, CDCl₃): δ 8.37 (d, J = 5.6 Hz, 1H), 8.07 (s, 1H), 7.85

(d, J = 5.6 Hz, 1H), 6.47 (d, J = 16.9 Hz, 1H), 6.23 (dd, J = 16.9, 10.3 Hz,

1H), 5.86 (d, J = 10.3 Hz, 1H), 3.89 (m, 10H), 2.49 (m, 4H), 2.37 (s,

3H). ¹³C NMR (125 MHz, CDCl₃): δ 171.5, 171.1, 164.0, 160.2, 159.0,

157.1, 130.3, 129.9, 105.8, 79.5, 54.9, 54.3, 46.2, 43.6. HRMS (m/z): [M

+ H]⁺calcd for C₁₈H₂₄N₇O₃S, 418.1661; found, 418.1666.

N-(6-Amino-2-((4,6-diethoxy-2-morpholinopyrimidin-5-yl)thio)-

pyrimidin-4-yl)acrylamide (20b). To a solution of 19b (20 mg, 0.051

mmol) and Et₃N (51 mg, 0.51 mmol) in 1 mL of anhydrous dioxane was

added acryloyl chloride (46 mg, 0.51 mmol). The resulting mixture was

stirred at rt for 12 h. Solvent was removed under reduced pressure, and

the residue was puriﬁed by preparatory TLC (CHCl₃/MeOH−NH₃(7

N), 10:1) to aﬀord 10 mg (42%) of 20b. ¹H NMR (500 MHz, CDCl₃): δ

8.14 (br s, 1H), 7.04 (s, 1H), 6.40 (d, J = 16.9 Hz, 1H), 6.18 (dd, J = 16.9,

10.4 Hz, 1H), 5.78 (d, J = 10.4 Hz, 1H), 4.82 (s, 2H), 4.35 (q, J = 7.1 Hz,

4H), 3.79 (m, 4H), 3.76 (m, 4H), 1.27 (t, J = 7.2 Hz, 6H). HRMS (m/

z): [M + H]⁺calcd for C₁₉H₂₆N₇O₄S, 448.1767; found, 448.1754.

HPLC: (a) H₂O + 0.1% TFA, (b) ACN + 0.1% TFA (5−95% ACN in 10

min), t_R= 7.97 min.

N,N′-(2-((4,6-Diethoxy-2-(piperidin-1-yl)pyrimidin-5-yl)thio)-

pyrimidine-4,6-diyl)diacetamide (18c). To a solution of 14b (165 mg,

0.402 mmol) in 3 mL of DMF was added piperidine (342 mg, 4.02

mmol), and the resulting solution was heated at 90 °C for 1 h. Solvent

and excess reagent were removed under reduced pressure, and the

residue was puriﬁed by preparatory TLC (CHCl₃/MeOH−NH₃(7 N),

10:1) to yield 167 mg (87%) of 18c. ¹H NMR (500 MHz, CDCl₃): δ

8.60 (br s, 2H), 8.35 (br s, 1H), 4.34 (q, J = 7.1 Hz, 4H), 3.75 (m, 4H),

2.17 (s, 6H), 1.67 (m, 2H), 1.58 (m, 4H), 1.27 (t, J = 7.1 Hz, 6H). MS

(m/z): [M + H]⁺476.2.

2-((4,6-Diethoxy-2-(piperidin-1-yl)pyrimidin-5-yl)thio)pyrimidine-

4,6-diamine (19c). A mixture of 18c (0.200 g, 0.42 mmol) and 1 N

NaOH(aq) (2 mL) in 5 mL of methanol was stirred at 60 °C for 1 h.

Solvent was removed under reduced pressure, and the residue was

puriﬁed by chromatography (CH₂Cl₂/MeOH−NH₃(7 N), 10:1) to

aﬀord 0.156 g (95%) of 19c. MS (m/z): [M + H]⁺392.2.

N-(6-Amino-2-((4,6-diethoxy-2-(piperidin-1-yl)pyrimidin-5-yl)-

thio)pyrimidin-4-yl)acrylamide (20c). To a solution of 19c (30 mg,

0.076 mmol) and Et₃N (76 mg, 0.76 mmol) in 1 mL of anhydrous

dioxane was added acryloyl chloride (69 mg, 0.76 mmol) dropwise. The

resulting mixture was stirred at rt for 12 h. Solvent was removed under

reduced pressure, and the residue was puriﬁed by preparatory TLC

(CHCl₃/MeOH−NH₃(7 N), 10:1) to aﬀord 16 mg (48%) of 20c. ¹H

NMR (500 MHz, CDCl₃): δ 8.09 (br s, 1H), 7.03 (s, 1H), 6.40 (dd, J =

16.9, 1.1 Hz, 1H), 6.19 (dd, J = 16.9, 10.3 Hz, 1H), 5.77 (dd, J = 10.3, 1.1

Hz, 1H), 4.82 (s, 2H), 4.35 (q, J = 7.1 Hz, 4H), 3.76 (m, 4H), 1.65−1.69

(m, 2H), 1.57−1.61 (m, 4H), 1.26 (t, J = 7.1 Hz, 6H). HRMS (m/z): [M

+ H]⁺calcd for C₂₀H₂₈N₇O₃S, 446.1974; found, 446.1974. HPLC: (a)

H₂O + 0.1% TFA, (b) ACN + 0.1% TFA (5−95% ACN in 10 min), t_R=

10.67 min.

5-((4-Aminopyrimidin-2-yl)thio)-4,6-dimethoxy-N,N-bis(4-

methoxybenzyl)pyrimidin-2-amine (21). A mixture of 10a (8.2 g, 15.8

mmol), 4-amino-2-mercaptopyrimidine (2.2 g, 17.3 mmol), neo-

cuproine (0.711 g, 3.16 mmol), CuI (0.616 g, 3.16 mmol), and

K₂CO₃(4.36 g, 31.6 mmol) in 100 mL of DMSO was stirred at 130 °C

for 16 h. Solvent was removed under reduced pressure, and the residue

was puriﬁed by column chromatography (CH₂Cl₂/MeOH, 100:0 to

90:10) to aﬀord 7.6 g (92%) of 21. ¹H NMR (500 MHz, CDCl₃): δ 7.97

(d, J = 5.7 Hz, 1H), 7.23 (d, J = 8.3 Hz, 4H), 6.86 (d, J = 8.3 Hz, 4H),

6.05 (d, J = 5.7 Hz, 1H), 4.94 (br s, 2H), 4.75 (s, 4H), 3.87 (s, 6H), 3.81

(s, 6H). MS (m/z): [M + H]⁺521.1.

N-(2-((2-(Bis(4-methoxybenzyl)amino)-4,6-dimethoxypyrimidin-

5-yl)thio)pyrimidin-4-yl)acetamide (22). A solution of 21 (7.6 g, 14.6

mmol) in 50 mL of acetic anhydride was stirred at 120 °C for 1 h.

Solvent was removed under reduced pressure, and the residue was

puriﬁed by column chromatography (CH₂Cl₂/MeOH, 100:0 to 90:10)

to aﬀord 7.37 g (93%) of 22. ¹H NMR (500 MHz, CDCl₃): δ 9.17 (s,

1H), 8.32 (d, J = 5.5 Hz, 1H), 7.76 (br s, 1H), 7.22 (d, J = 8.3 Hz, 4H),

6.85 (d, J = 8.3 Hz, 4H), 4.73 (s, 4H), 3.84 (s, 6H), 3.79 (s, 6H), 2.16 (s,

3H). MS (m/z): [M + H]⁺563.2.

N-(2-((4,6-Dimethoxy-2-morpholinopyrimidin-5-yl)thio)-

pyrimidin-4-yl)acetamide (25b). To a solution of 24 (100 mg, 0.307

mmol) in DMF (4 mL) was added morpholine (107 mg, 1.23 mmol),

and the resulting solution was heated at 90 °C for 1 h. Solvent and excess

reagent were removed under reduced pressure, and the residue was

puriﬁed by preparatory TLC (CHCl₃/MeOH−NH₃(7 N), 10:1) to

yield 102 mg (85%) of 25b. ¹H NMR (500 MHz, CDCl₃): δ 8.35 (d, J =

5.6 Hz, 1H), 8.10 (s, 1H), 7.75 (br s, 1H), 3.89 (s, 6H), 3.84 (m, 4H),

3.77 (m, 4H), 2.18 (s, 3H). ¹³C NMR (125 MHz, CDCl₃): δ 168.6,

161.0, 159.2, 158.7, 130.6, 129.3, 113.8, 82.3, 71.5, 55.2, 47.9, 29.5.

HRMS (m/z): [M + H]⁺calcd for C₁₆H₂₁N₆O₄S, 393.1345; found,

N-(2-((2-Amino-4,6-dimethoxypyrimidin-5-yl)thio)pyrimidin-4-

yl)acetamide (23). A solution of 22 (7.0 g, 12.9 mmol) in 30 mL of TFA

was heated at 60 °C for 12 h. Excess TFA and solvent were removed

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393.1332. HPLC: (a) H₂O + 0.1% TFA, (b) ACN + 0.1% TFA (5−95%

ACN in 10 min), t_R= 7.57 min.

puriﬁed by preparatory TLC (CHCl₃/MeOH−NH₃(7 N), 10:1) to

yield 95 mg (82%) of 25d. ¹H NMR (500 MHz, CDCl₃): δ 8.34 (d, J =

5.6 Hz, 1H), 8.10 (s, 1H), 7.72 (br s, 1H), 3.89 (s, 6H), 3.59 (m, 4H),

2.17 (s, 3H), 1.96 (m, 4H). ¹³C NMR (125 MHz, CDCl₃): δ 171.7,

170.8, 159.0, 158.9, 156.9, 105.3, 78.3, 54.0, 46.6, 25.4, 24.7. HRMS (m/

z): [M + H]⁺calcd for C₁₆H₂₁N₆O₃S, 377.1396; found, 377.1383.

HPLC: (a) H₂O + 0.1% TFA, (b) ACN + 0.1% TFA (5−95% ACN in 10

min), t_R= 8.85 min.

2-((4,6-Dimethoxy-2-morpholinopyrimidin-5-yl)thio)pyrimidin-4-

amine (26b). A mixture of 25b (100 mg, 0.255 mmol) and 1 N

NaOH(aq) (4 mL) in 8 mL of methanol was stirred at 60 °C for 1 h.

Solvents were removed under reduced pressure, and the residue was

puriﬁed by preparatory TLC to aﬀord 84 mg (94%) of 26b. ¹H NMR

(500 MHz, CDCl₃): δ 7.95 (d, J = 5.7 Hz, 1H), 6.05 (d, J = 5.7 Hz, 1H),

4.96 (s, 2H), 3.90 (s, 6H), 3.82 (m, 4H), 3.76 (m, 4H). ¹³C NMR (125

MHz, CDCl₃): δ 171.3, 171.1, 162.5, 160.2, 156.2, 101.1, 80.5, 66.8,

54.2, 44.2. HRMS (m/z): [M + H]⁺calcd for C₁₄H₁₉N₆O₃S, 351.1239;

found, 351.1248. HPLC: (a) H₂O + 0.1% TFA, (b) ACN + 0.1% TFA

(5−95% ACN in 10 min), t_R= 6.30 min.

N-(2-((4,6-Dimethoxy-2-morpholinopyrimidin-5-yl)thio)-

pyrimidin-4-yl)acrylamide (27b). To a solution of 26b (50 mg, 0.143

mmol) and Et₃N (72 mg, 0.713 mmol) in CH₂Cl₂(2 mL) was added

acryloyl chloride (65 mg, 0.713 mmol) under a water bath. The resulting

mixture was stirred at rt for 24 h. Solvent was removed under reduced

pressure, and the residue was puriﬁed by preparatory TLC (CHCl₃/

MeOH−NH₃(7 N), 10:1) to aﬀord 43 mg (74%) of 27b. ¹H NMR (500

MHz, CDCl₃): δ 8.37 (d, J = 5.6 Hz, 1H), 8.03 (s, 1H), 7.85 (d, J = 5.6

Hz, 1H), 6.47 (d, J = 16.9 Hz, 1H), 6.22 (dd, J = 16.9, 10.3 Hz, 1H), 5.86

(d, J = 10.3 Hz, 1H), 3.89 (s, 6H), 3.85 (m, 4H), 3.77 (m, 4H). HRMS

(m/z): [M + H]⁺calcd for C₁₇H₂₁N₆O₄S, 405.1345; found, 405.1338.

HPLC: (a) H₂O + 0.1% TFA, (b) ACN + 0.1% TFA (5−95% ACN in 10

min), t_R= 8.07 min.

N-(2-((4,6-Dimethoxy-2-(piperidin-1-yl)pyrimidin-5-yl)thio)-

pyrimidin-4-yl)acetamide (25c). To a solution of 24 (100 mg, 0.307

mmol) in DMF (4 mL) was added piperidine (105 mg, 1.23 mmol), and

the resulting solution was heated at 90 °C for 1 h. Solvent and excess

reagent were removed under reduced pressure, and the residue was

puriﬁed by preparatory TLC (CHCl₃/MeOH−NH₃(7 N), 10:1) to

yield 104 mg (87%) of 25c. ¹H NMR (500 MHz, CDCl₃): δ 8.35 (d, J =

5.6 Hz, 1H), 8.00 (br s, 1H), 7.73 (br s, 1H), 3.88 (s, 6H), 3.80 (m, 4H),

2.18 (s, 3H), 1.66 (m, 2H), 1.59 (m, 4H). ¹³C NMR (125 MHz,

CDCl₃): δ 171.6, 171.0, 160.1, 158.9, 156.9, 105.3, 82.5, 54.1, 44.8, 29.7,

25.8, 24.8. HRMS (m/z): [M + H]⁺calcd for C₁₇H₂₃N₆O₃S, 391.1552;

found, 391.1549. HPLC: (a) H₂O + 0.1% TFA, (b) ACN + 0.1% TFA

(5−95% ACN in 10 min), t_R= 9.18 min.

2-((4,6-Dimethoxy-2-(pyrrolidin-1-yl)pyrimidin-5-yl)thio)-

pyrimidin-4-amine (26d). A mixture of 25d (90 mg, 0.239 mmol) and 1

N NaOH(aq) (4 mL) in 8 mL of methanol was stirred at 60 °C for 1 h.

Solvents were removed under reduced pressure, and the residue was

puriﬁed by preparatory TLC to aﬀord 77 mg (96%) of 26d. ¹H NMR

(500 MHz, CDCl₃): δ 7.96 (d, J = 5.7 Hz, 1H), 6.03 (d, J = 5.7 Hz, 1H),

4.88 (s, 2H), 3.90 (s, 6H), 3.58 (t, J = 6.0 Hz, 4H), 1.96 (t, J = 6.0 Hz,

4H). ¹³C NMR (125 MHz, CDCl₃): δ 171.7, 170.9, 162.4, 159.0, 156.3,

100.9, 79.0, 54.0, 46.5, 25.4. HRMS (m/z): [M + H]⁺calcd for

C₁₄H₁₉N₆O₂S, 335.1290; found, 335.1291. HPLC: (a) H₂O + 0.1%

TFA, (b) ACN + 0.1% TFA (5−95% ACN in 10 min), t_R= 7.32 min.

N-(2-((4,6-Dimethoxy-2-(pyrrolidin-1-yl)pyrimidin-5-yl)thio)-

pyrimidin-4-yl)acrylamide (27d). To a solution of 26d (50 mg, 0.150

mmol) and Et₃N (76 mg, 0.748 mmol) in CH₂Cl₂(2 mL) was added

acryloyl chloride (68 mg, 0.748 mmol) under a water bath. The resulting

mixture was stirred at rt for 24 h. Solvent was removed under reduced

pressure, and the residue was puriﬁed by preparatory TLC (CHCl₃/

MeOH−NH₃(7 N), 10:1) to aﬀord 43 mg (73%) of 27d. ¹H NMR (500

MHz, CDCl₃): δ 8.37 (d, J = 5.6 Hz, 1H), 7.94 (s, 1H), 7.83 (d, J = 5.6

Hz, 1H), 6.47 (dd, J = 16.9, 0.8 Hz, 1H), 6.22 (dd, J = 16.9, 10.4 Hz, 1H),

5.85 (dd, J = 10.4, 0.8 Hz, 1H), 3.91 (s, 6H), 3.61 (m, 4H), 1.98 (m, 4H).

¹³C NMR (125 MHz, CDCl₃): δ 171.6, 171.0, 164.7, 159.2, 158.8, 130.4,

129.7, 105.9, 54.2, 46.7, 25.5. HRMS (m/z): [M + H]⁺calcd for

C₁₇H₂₁N₆O₃S, 389.1396; found, 389.1398. HPLC: (a) H₂O + 0.1%

TFA, (b) ACN + 0.1% TFA (5−95% ACN in 10 min), t_R= 9.15 min.

4-(5-((4-Acrylamidopyrimidin-2-yl)thio)-4,6-dimethoxypyrimidin-

2-yl)-1-methylpiperazine 1-Oxide (28). To a mixture of 27a (20 mg,

0.0479 mmol) and NaHCO₃(40 mg, 0.479 mmol) in CH₂Cl₂(2 mL)

cooled to −78 °C was added m-CPBA (9.1 mg, 0.0527 mmol). The

reaction mixture was stirred for 4 h at −78 °C. Then Na₂S₂O₃was added

in one portion. Solvent was removed under reduced pressure, and the

residue was puriﬁed by preparatory TLC (CHCl₃/MeOH−NH₃(7 N),

20:1) to aﬀord 17 mg (83%) of 28. ¹H NMR (500 MHz, CDCl₃): δ 9.47

(br s, 1H), 8.33 (d, J = 5.7 Hz, 1H), 7.89 (d, J = 5.7 Hz, 1H), 6.43−6.49

(m, 2H), 5.81 (dd, J = 7.7, 3.8 Hz, 1H), 4.67−4.75 (m, 2H), 3.85−3.95

(m, 8H), 3.41−3.51 (m, 7H). ¹³C NMR (125 MHz, CDCl₃): δ 171.2,

170.8, 164.8, 159.6, 158.8, 157.7, 130.6, 129.7, 106.2, 81.1, 65.3, 60.2,

54.5, 38.6. HRMS (m/z): [M + H]⁺calcd for C₁₈H₂₄N₇O₄S, 434.1610;

found, 434.1592. HPLC: (a) H₂O + 0.1% TFA, (b) ACN + 0.1% TFA

(40−95% ACN in 10 min), t_R= 6.35 min.

2-((4,6-Dimethoxy-2-(piperidin-1-yl)pyrimidin-5-yl)thio)-

pyrimidin-4-amine (26c). A mixture of 25c (100 mg, 0.256 mmol) and

1 N NaOH(aq) (4 mL) in 8 mL of methanol was stirred at 60 °C for 1 h.

Solvents were removed under reduced pressure, and the residue was

puriﬁed by preparatory TLC to aﬀord 83 mg (93%) of 26c. ¹H NMR

(500 MHz, CDCl₃): δ 7.95 (d, J = 5.6 Hz, 1H), 6.03 (d, J = 5.6 Hz, 1H),

4.95 (s, 2H), 3.88 (s, 6H), 3.79 (m, 4H), 1.66 (m, 2H), 1.60 (m, 4H).

¹³C NMR (125 MHz, CDCl₃): δ 171.5, 171.0, 162.5, 160.0, 156.2, 101.0,

54.1, 44.8, 25.7, 24.8. HRMS (m/z): [M + H]⁺calcd for C₁₅H₂₁N₆O₂S,

349.1447; found, 349.1434. HPLC: (a) H₂O + 0.1% TFA, (b) ACN +

0.1% TFA (5−95% ACN in 10 min), t_R= 7.60 min.

2-((2,4,6-Trimethoxypyrimidin-5-yl)thio)pyrimidin-4-amine (29).

To a solution of 24 (100 mg, 0.307 mmol) in MeOH (5 mL) was

added NaOCH₃(36 mg, 0.675 mmol), and the resulting solution was

heated at 90 °C for 1 h. Then 1 N NaOH(aq) (2 mL) was added and

heating continued at 60 °C for 1 h. Solvent and excess reagent were

removed under reduced pressure, and the residue was puriﬁed by

preparatory TLC (CHCl₃/MeOH−NH₃(7 N), 10:1) to yield 68 mg

N-(2-((4,6-Dimethoxy-2-(piperidin-1-yl)pyrimidin-5-yl)thio)-

pyrimidin-4-yl)acrylamide (27c). To a solution of 26c (50 mg, 0.144

mmol) and Et₃N (72 mg, 0.718 mmol) in CH₂Cl₂(2 mL) was added

acryloyl chloride (65 mg, 0.718 mmol) under a water bath. The resulting

mixture was stirred at rt for 24 h. Solvent was removed under reduced

pressure, and the residue was puriﬁed by preparatory TLC (CHCl₃/

MeOH−NH₃(7 N), 10:1) to aﬀord 45 mg (78%) of 27c. ¹H NMR (500

MHz, CDCl₃): δ 8.37 (d, J = 5.6 Hz, 1H), 7.95 (s, 1H), 7.83 (d, J = 5.6

Hz, 1H), 6.48 (dd, J = 16.9, 0.8 Hz, 1H), 6.23 (dd, J = 16.9, 10.4 Hz, 1H),

5.85 (dd, J = 10.3, 0.8 Hz, 1H), 3.88 (s, 6H), 3.81 (m, 4H), 1.69 (m, 2H),

1.62 (m, 4H). ¹³C NMR (125 MHz, CDCl₃): δ 171.7, 171.0, 164.0,

160.1, 158.9, 157.0, 130.3, 105.6, 54.1, 44.8, 25.8, 24.8. HRMS (m/z):

[M + H]⁺calcd for C₁₈H₂₃N₆O₃S, 403.1552; found, 403.1541. HPLC:

(a) H₂O + 0.1% TFA, (b) ACN + 0.1% TFA (5−95% ACN in 10 min),

t_R= 9.23 min.

1

(75%) of 29. H NMR (500 MHz, DMSO-d₆): δ 7.77 (d, J = 5.8 Hz,

1H), 6.86 (br s, 2H), 6.11 (d, J = 5.8 Hz, 1H), 3.95 (s, 3H), 3.87 (s, 6H).

MS (m/z): [M + H]⁺295.9.

N-(2-((2,4,6-Trimethoxypyrimidin-5-yl)thio)pyrimidin-4-yl)-

acrylamide (30). To a solution of 29 (50 mg, 0.169 mmol) and Et₃N

(26 mg, 0.254 mmol) in CH₂Cl₂(2 mL) was added acryloyl chloride (23

mg, 0.254 mmol) under a water bath. The resulting mixture was stirred

at rt for 2 h. Solvent was removed under reduced pressure, and the

residue was puriﬁed by preparatory TLC (CHCl₃/MeOH−NH₃(7 N),

20:1) to aﬀord 40 mg (68%) of 30. ¹H NMR (500 MHz, CDCl₃): δ 8.29

(d, J = 5.6 Hz, 1H), 7.81 (d, J = 5.6 Hz, 1H), 7.78 (s, 1H), 6.40 (d, J =

16.9 Hz, 1H), 6.15 (dd, J = 16.9, 10.3 Hz, 1H), 5.80 (d, J = 10.3 Hz, 1H),

3.97 (s, 3H), 3.95 (s, 6H). HRMS (m/z): [M + H]⁺calcd for

C₁₄H₁₆N₅O₄S, 350.0923; found, 350.0936.

N-(2-((4,6-Dimethoxy-2-(pyrrolidin-1-yl)pyrimidin-5-yl)thio)-

pyrimidin-4-yl)acetamide (25d). To a solution of 24 (100 mg, 0.307

mmol) in DMF (4 mL) was added pyrrolidine (87 mg, 1.23 mmol), and

the resulting solution was heated at 90 °C for 1 h. Solvent and excess

reagent were removed under reduced pressure, and the residue was

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N-(6-Amino-2-((2-amino-4,6-dimethoxypyrimidin-5-yl)thio)-

pyrimidin-4-yl)acrylamide (31). To a solution of 11a (50 mg, 0.093

mmol) and Et₃N (14 mg, 0.140 mmol) in CH₂Cl₂(2 mL) was added

acryloyl chloride (12.7 mg, 0.140 mmol) under a water bath. The

resulting mixture was stirred at rt for 2 h. Solvent was removed under

reduced pressure, the residue was dissolved in 1 mL of TFA/CHCl₃

(1:1), and the resulting solution was heated at 62 °C for 12 h. TFA and

solvent were removed under reduced pressure, and the residue was

puriﬁed by preparatory TLC (CH₂Cl₂/MeOH−NH₃(7 N), 10:1) to

aﬀord 18 mg (55%) of 31. ¹H NMR (500 MHz, CDCl₃/MeOH-d₄): δ

7.04 (s, 1H), 6.32−6.41 (m, 2H), 5.80 (dd, J = 10.3, 0.8 Hz, 1H), 3.88 (s,

6H). HRMS (m/z): [M + H]⁺calcd for C₁₃H₁₆N₇O₃S, 350.1035; found,

350.1025. HPLC: (a) H₂O + 0.1% TFA, (b) ACN + 0.1% TFA (5−95%

ACN in 12 min), t_R= 5.92 min.

4,6-Dimethoxy-2-(4-methylpiperazin-1-yl)pyrimidine (33a). A 5 g

(0.0286 mol) portion of 2-chloro-4,6-dimethoxypyrimidine (32) and

7.93 mL (7.16 g, 0.0715 mol) of N-methylpiperazine in 22 mL of DMF

were heated at 90 °C for 2.5 h. The reaction mixture was concentrated

under reduced pressure, and the residue was taken up into 200 mL of

CH₂Cl₂. This was washed with brine (3 × 50 mL), dried over MgSO₄,

ﬁltered, and concentrated to give 6.51 g (95%) of 33a. ¹H NMR (500

MHz, CDCl₃): δ 5.37 (s, 1H), 3.85 (s, 6H), 3.82 (m, 4H), 2.44 (m, 4H),

2.33 (s, 3H). ¹³C NMR (125 MHz, CDCl₃): δ 172.0, 160.8, 77.8, 55.0,

53.4, 46.3, 43.7. MS (m/z): [M + H]⁺239.2.

5-Iodo-4,6-dimethoxy-2-(4-methylpiperazin-1-yl)pyrimidine

(34a). To 1.59 g (6.67 mmol) of 33a in 40 mL of acetonitrile were added

1.80 g (8.01 mmol) of NIS and 0.771 mL (1.14 g, 10.0 mmol) of TFA,

and the resulting solution was stirred at rt for 1.5 h. The reaction mixture

was concentrated to dryness, and the residue was taken up into 100 mL

of CH₂Cl₂, washed with 10% sodium thiosulfate (50 mL) and 5%

NaHCO₃(3 × 50 mL), dried over MgSO₄, ﬁltered, and concentrated to

give a residue which was puriﬁed by column chromatography (CH₂Cl₂/

MeOH−NH₃(7 N), 1:0 to 20:1) to yield 2.06 g (86%) of 34a. ¹H NMR

(500 MHz, CDCl₃): δ 3.93 (s, 6H), 3.82 (m, 4H), 2.47 (m, 4H), 2.36 (s,

3H). MS (m/z): [M + H]⁺365.1.

138.4, 129.4, 121.9, 119.9, 117.1, 116.9, 81.3, 54.72, 54.67, 45.9, 43.3,

19.0. HRMS (m/z): [M + H]⁺calcd for C₂₁H₂₈N₅O₃S, 430.1913; found,

430.1902. HPLC: (a) H₂O + 0.1% TFA, (b) ACN + 0.1% TFA (40−

95% ACN in 10 min), t_R= 3.02 min.

4-(4,6-Dimethoxypyrimidin-2-yl)morpholine (33b). A 5 g (0.0286

mol) portion of 32 and 6.25 mL (6.23 g, 0.0715 mol) of morpholine in

22 mL of DMF were heated at 90 °C for 2.5 h. The reaction mixture was

concentrated under reduced pressure, and the residue was taken up into

200 mL of CH₂Cl₂. This was washed with brine (3 × 50 mL), dried over

MgSO₄, ﬁltered, and concentrated to give 6.00 g (93%) of 33b. ¹H NMR

(500 MHz, CDCl₃): δ 5.40 (s, 1H), 3.86 (s, 6H), 3.77 (m, 4H), 3.75 (m,

4H). ¹³C NMR (125 MHz, CDCl₃): δ 172.0, 160.9, 78.2, 66.8, 53.5,

44.2. MS (m/z): [M + H]⁺226.2.

4-(5-Iodo-4,6-dimethoxypyrimidin-2-yl)morpholine (34b). To 1.5

g (6.66 mmol) of 33b in 40 mL of acetonitrile was added 1.80 g (8.00

mmol) of NIS, and the resulting solution was stirred at rt for 2 h. The

reaction mixture was concentrated to dryness, and the residue was taken

up into 100 mL of CH₂Cl₂, washed with 10% sodium thiosulfate (50

mL) and 5% NaHCO₃(3 × 50 mL), dried over MgSO₄, ﬁltered, and

concentrated to give a residue which was puriﬁed by column

chromatography (hexane/EtOAc, 80:20) to yield 1.99 g (85%) of

34b. ¹H NMR (500 MHz, CDCl₃): δ 3.92 (s, 6H), 3.71−3.79 (m, 8H).

MS (m/z): 352.1 [M + H]⁺.

3-((4,6-Dimethoxy-2-morpholinopyrimidin-5-yl)thio)aniline

(35b). A mixture of 34b (1.33 g, 3.8 mmol), 3-aminothiophenol (0.48

mL, 0.571 g, 4.56 mmol), neocuproine (0.172 g, 0.76 mmol), CuI (0.144

g, 0.76 mmol), and K₂CO₃(1.57 g, 11.3 mmol) in 30 mL of DMF was

stirred at 120 °C for 16 h. Solvent was removed under reduced pressure,

and the residue was puriﬁed by column chromatography (hexane/

1

EtOAc, 1:1) to aﬀord 0.92 g (70%) of 35b. H NMR (500 MHz,

CDCl₃): δ 6.97 (m, 1H), 6.49 (d, J = 8.0 Hz, 1H), 6.37−6.40 (m, 2H),

3.90 (s, 6H), 3.83−3.86 (m, 4H), 3.77−3.80 (m, 4H). HRMS (m/z):

[M + H]⁺calcd for C₁₆H₂₁N₄O₃S, 349.1334; found, 349.1341. HPLC:

(a) H₂O + 0.1% TFA, (b) ACN + 0.1% TFA (40−95% ACN in 10 min),

t_R= 3.16 min.

3-((4,6-Dimethoxy-2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)-

thio)aniline (35a). A mixture of 34a (0.200 g, 0.549 mmol), 3-

aminothiophenol (70 μL, 0.082 g, 0.659 mmol), neocuproine (0.023 g,

0.11 mmol), CuI (0.038 g, 0.011 mmol), and K₂CO₃(0.152 g, 1.10

mmol) in DMF (7 mL) was stirred at 130 °C for 16 h. Solvent was

removed under reduced pressure, and the residue was puriﬁed by

column chromatography (CH₂Cl₂/MeOH−NH₃(7 N), 200:1 to 40:1)

to aﬀord 0.133 g (67%) of 35a. ¹H NMR (500 MHz, CDCl₃): δ 6.97 (t, J

= 8.0 Hz, 1H), 6.46−6.51 (m, 1H), 6.36−6.40 (m, 2H), 3.88−4.03 (m,

10H), 3.58 (br s, 2H), 2.52 (m, 4H), 2.38 (s, 3H). ¹³C NMR (125 MHz,

CDCl₃): δ 171.8, 160.1, 146.9, 139.5, 129.6, 116.2, 112.2, 112.1, 81.1,

55.0, 54.6, 46.3, 43.8. HRMS (m/z): [M + H]⁺calcd for C₁₇H₂₄N₅O₂S,

362.1651; found, 362.1649.

N-(3-((4,6-Dimethoxy-2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)-

thio)phenyl)acrylamide (36a). To a solution of 35a (50 mg, 0.138

mmol) and Et₃N (21 mg, 0.207 mmol) in CH₂Cl₂(2 mL) was added

acryloyl chloride (19 mg, 0.207 mmol) under a water bath. The resulting

mixture was stirred at rt for 2 h. Solvent was removed under reduced

pressure, and the residue was puriﬁed by preparatory TLC (CHCl₃/

MeOH−NH₃(7 N), 20:1) to aﬀord 44 mg (77%) of 36a. ¹H NMR (500

MHz, CDCl₃): δ 7.49 (m, 1H), 7.36 (m, 1H), 7.14 (m, 2H), 6.84 (d, J =

7.5 Hz, 1H), 6.36 (m, 1H), 6.22 (m, 1H), 5.73 (m, 1H), 4.02 (m, 4H),

3.90 (s, 6H), 2.70 (m, 4H), 2.50 (s, 3H). HRMS (m/z): [M + H]⁺calcd

for C₂₀H₂₆N₅O₃S, 416.1756; found, 416.1742.

N-(3-((4,6-Dimethoxy-2-morpholinopyrimidin-5-yl)thio)phenyl)-

acrylamide (37). To a solution of 35b (50 mg, 0.144 mmol) and Et₃N

(22 mg, 0.215 mmol) in CH₂Cl₂(2 mL) was added acryloyl chloride (19

mg, 0.215 mmol) under a water bath. The resulting mixture was stirred

at rt for 2 h. Solvent was removed under reduced pressure, and the

residue was puriﬁed by preparatory TLC (CHCl₃/MeOH−NH₃(7 N),

1

20:1) to aﬀord 36 mg (63%) of 37. H NMR (500 MHz, CDCl₃): δ

7.46−7.50 (m, 2H), 7.12−7.16 (m, 2H), 6.81 (d, J = 7.4 Hz, 1H), 6.38

(d, J = 16.9 Hz, 1H), 6.20 (dd, J = 16.9, 10.3 Hz, 1H), 5.71 (d, J = 10.3

Hz, 1H), 3.90 (s, 6H), 3.82−3.86 (m, 4H), 3.75−3.79 (m, 4H). ¹³C

NMR (125 MHz, CDCl₃): δ 171.5, 163.5, 160.1, 139.3, 138.2, 135.3,

131.2, 129.2, 127.7, 121.7, 116.7, 80.9, 66.7, 54.4, 44.2. HRMS (m/z):

[M + Na]⁺calcd for C₁₉H₂₂N₄O₄SNa, 425.1259; found, 425.1260.

HPLC: (a) H₂O + 0.1% TFA, (b) ACN + 0.1% TFA (40−95% ACN in

10 min), t_R= 7.15 min.

4,6-Dimethyl-2-(4-methylpiperazin-1-yl)pyrimidine (39a). A 1.43 g

(10 mmol) portion of 2-chloro-4,6-dimethylpyrimidine (38) and 2.50 g

(25 mmol) of N-methylpiperazine in 20 mL of DMF were heated at 90

°C for 2.5 h. The reaction mixture was concentrated under reduced

pressure, and the residue was taken up into 100 mL of CH₂Cl₂. This was

washed with brine (3 × 25 mL), dried over MgSO₄, ﬁltered, and

concentrated to give 1.92 g (93%) of 39a. MS (m/z): [M + H]⁺207.0.

5-Iodo-4,6-dimethyl-2-(4-methylpiperazin-1-yl)pyrimidine (40a).

To 1.61 g (7.8 mmol) of 39a in 40 mL of acetonitrile were added 3.7

g (16.4 mmol) of NIS and 2.4 mL (3.56 g, 31.2 mmol) of TFA, and the

resulting solution was stirred at rt for 17 h. The reaction mixture was

concentrated to dryness, and the residue was taken up into 150 mL of

CH₂Cl₂, washed with 10% sodium thiosulfate (50 mL) and 5%

NaHCO₃(3 × 50 mL), dried over MgSO₄, ﬁltered, and concentrated to

give a residue which was puriﬁed by column chromatography (CH₂Cl₂/

MeOH, 1:0 to 10:1) to yield 2.32 g (90%) of 40a. ¹H NMR (500 MHz,

CDCl₃): δ 3.82 (m, 4H), 2.52 (s, 6H), 2.43 (m, 4H), 2.33 (s, 3H). MS

(m/z): [M + H]⁺332.9.

N-(3-((4,6-Dimethoxy-2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)-

thio)phenyl)methacrylamide (36b). To a solution of 35a (50 mg,

0.138 mmol) and Et₃N (140 mg, 1.38 mmol) in CH₂Cl₂(2 mL) was

added methacryloyl chloride (144 mg, 1.38 mmol) dropwise under a

water bath. The resulting mixture was stirred at rt for 16 h. Solvent was

removed under reduced pressure, and the residue was puriﬁed by

preparatory TLC (CH₂Cl₂/MeOH, 20:1) to aﬀord 38 mg (65%) of 36b.

¹H NMR (500 MHz, CDCl₃): δ 7.40−7.47 (m, 2H), 7.12−7.18 (m,

2H), 6.79−6.83 (m, 1H), 5.74 (d, J = 1.2 Hz, 1H), 5.43 (d, J = 1.2 Hz,

1H), 3.99 (m, 4H), 3.91 (s, 6H), 2.65 (m, 4H), 2.47 (s, 3H), 2.03 (s,

3H). ¹³C NMR (125 MHz, CDCl₃): δ 171.8, 166.7, 160.1, 141.2, 139.5,

2-((4,6-Dimethyl-2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)thio)-

pyrimidin-4-amine (41a). A mixture of 40a (350 mg, 1.05 mmol), 4-

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amino-2-mercaptopyrimidine (147 mg, 1.15 mmol), neocuproine (47

mg, 0.21 mmol), CuI (40 mg, 0.21 mmol), and K₂CO₃(1.46 g, 10.5

mmol) in 20 mL of DMF was stirred at 110 °C for 16 h. Solvent was

removed under reduced pressure, and the residue was puriﬁed by

column chromatography (CH₂Cl₂/MeOH, 100:0 to 90:10) to aﬀord

261 mg (75%) of 41a. MS (m/z): [M + H]⁺332.4.

N-(2-((4,6-Dimethyl-2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)-

thio)pyrimidin-4-yl)acrylamide (42a). To a solution of 41a (50 mg,

0.151 mmol) and Et₃N (76 mg, 0.755 mmol) in CH₂Cl₂(2 mL) was

added acryloyl chloride (68 mg, 0.755 mmol) dropwise under a water

bath. The resulting mixture was stirred at rt for 16 h. Solvent was

removed under reduced pressure, and the residue was puriﬁed by

preparatory TLC (CH₂Cl₂/MeOH−NH₃(7 N), 10:1) to aﬀord 44 mg

(75%) of 42a. ¹H NMR (500 MHz, CDCl₃): δ 8.40 (d, J = 5.6 Hz, 1H),

7.90 (d, J = 5.6 Hz, 1H), 7.79 (br s, 1H), 6.49 (d, J = 16.9 Hz, 1H), 6.22

(dd, J = 16.9, 10.4 Hz, 1H), 5.89 (d, J = 10.4 Hz, 1H), 3.92 (m, 4H), 2.48

(m, 4H), 2.45 (s, 6H), 2.36 (s, 3H). HRMS (m/z): [M + H]⁺calcd for

C₁₈H₂₄N₇OS, 386.1763; found, 386.1762.

(E)-N-(2-((4,6-Dimethyl-2-(4-methylpiperazin-1-yl)pyrimidin-5-

yl)thio)pyrimidin-4-yl)but-2-enamide (42b). To a solution of 41a (50

mg, 0.151 mmol) and Et₃N (152 mg, 1.51 mmol) in CH₂Cl₂(2 mL) was

added crotonyl chloride (158 mg, 1.51 mmol) dropwise under a water

bath. The resulting mixture was stirred at rt for 16 h. Solvent was

removed under reduced pressure, and the residue was puriﬁed by

preparatory TLC (CH₂Cl₂/MeOH, 20:1) to aﬀord 41 mg (67%) of 42b.

¹H NMR (500 MHz, CDCl₃): δ 8.37 (d, J = 5.7 Hz, 1H), 7.88 (d, J = 5.7

Hz, 1H), 7.67 (s, 1H), 7.01−7.10 (m, 1H), 5.92 (dd, J = 15.2, 1.7 Hz,

1H), 3.96 (m, 4H), 2.52 (m, 4H), 2.45 (s, 6H), 2.43 (s, 3H), 1.94 (dd, J =

7.0, 1.7 Hz, 3H). HRMS (m/z): [M + H]⁺calcd for C₁₉H₂₆N₇OS,

400.1920; found, 400.1920.

Et₃N (79 mg, 0.786 mmol) in CH₂Cl₂(2 mL) was added acryloyl

chloride (71 mg, 0.786 mmol) dropwise under a water bath. The

resulting mixture was stirred at rt for 16 h. Solvent was removed under

reduced pressure, and the residue was puriﬁed by preparatory TLC

(CH₂Cl₂/MeOH−NH₃(7 N), 10:1) to aﬀord 58 mg (68%) of 43. ¹H

NMR (500 MHz, CDCl₃/MeOH-d₄): δ 8.39 (d, J = 5.7 Hz, 1H), 6.49

(d, J = 16.9 Hz, 1H), 6.27 (dd, J = 16.9, 10.3 Hz, 1H), 5.86 (d, J = 10.3

Hz, 1H), 3.89 (m, 4H), 3.78 (m, 4H), 2.46 (s, 6H). ¹³C NMR (125

MHz, CDCl₃): δ 172.2, 170.4, 164.6, 160.4, 159.0, 157.8, 130.1, 130.0,

109.2, 106.2, 66.8, 44.1, 23.7. MS (m/z): [M + H]⁺373.0.

Biological Testing. Cell Lines. SKBr3 cells were a gift from Dr. Neal

Rosen (Memorial Sloan-Kettering Cancer Center, MSKCC) and

Kasumi-1 and MOLM-13 from Dr. S. Nimer (MSKCC). Cells were

cultured routinely in DME/F12 (SKBr3) or in RPMI (Kasumi-1,

MOML-13) supplemented with 10% fetal bovine serum, 1% ^L-

glutamine, 1% penicillin, and streptomycin.

Western Blotting. Cells were grown to 60−70% conﬂuence and

treated with inhibitor or DMSO vehicle for the indicated times. Protein

lysates were prepared in 50 mM Tris, pH 7.4, 150 mM NaCl, and 1%

NP-40 lysis buﬀer. Protein concentrations were measured using the

BCA kit (Pierce) according to the manufacturer’s instructions. Protein

lysates (10−50 μg) were resolved by SDS−PAGE, transferred onto a

nitrocellulose membrane, and incubated with the indicated primary

antibodies: anti-HER2 from rabbit (1:250, 28-0004, Zymed), anti-

Hsp70 from mouse (1:500, SPA-810, Stressgen), anti-Raf-1 from rabbit

(1:500, sc-133, Santa Cruz), anti-PARP (p85 fragment) from rabbit

(1:500, G7341, Promega), anti-HOP from mouse (1:500, SRA-1500,

Enzo) and anti-β-actin from mouse (1:2500, A1978, Sigma-Aldrich).

Membranes were then incubated with a corresponding peroxidase-

conjugated secondary antibody (1:3000 dilution).

Hsp90 Binding Assay. For the competition studies, ﬂuorescence

polarization (FP) assays were performed as previously reported.⁴⁹

Brieﬂy, FP measurements were performed on an Analyst GT instrument

(Molecular Devices, Sunnyvale, CA). Measurements were taken in black

96-well microtiter plates (Corning no. 3650) where both the excitation

and the emission occurred from the top of the wells. A stock of 10 μM

GM-cy3B was prepared in DMSO and diluted with Felts buﬀer (20 mM

HEPES (K), pH 7.3, 50 mM KCl, 2 mM DTT, 5 mM MgCl₂, 20 mM

Na₂MoO₄, and 0.01% NP40 with 0.1 mg/mL BGG). To each 96-well

plate were added 6 nM ﬂuorescent GM (GM-cy3B), 3 μg of SKBr3

lysate (total protein), and tested inhibitor (initial stock in DMSO) in a

ﬁnal volume of 100 μL of HFB buﬀer. Drugs were added in triplicate

wells. For each assay, background wells (buﬀer only), tracer controls

(free, ﬂuorescent GM only), and bound GM controls (ﬂuorescent GM

in the presence of SKBr3 lysate) were included on each assay plate. GM

was used as a positive control. The assay plate was incubated on a shaker

at 4 °C for 24 h, and the FP values (mP) were measured. The fraction of

tracer bound to Hsp90 was correlated to the FP value and plotted

against the values of competitor concentrations. The inhibitor

concentration at which 50% of bound GM was displaced was obtained

by ﬁtting the data. All experimental data were analyzed using SOFTmax

Pro 4.3.1 and plotted using Prism 4.0 (Graphpad Software Inc., San

Diego, CA).

N-(2-((4,6-Dimethyl-2-(4-methylpiperazin-1-yl)pyrimidin-5-yl)-

thio)pyrimidin-4-yl)methacrylamide (42c). To a solution of 41a (50

mg, 0.151 mmol) and Et₃N (152 mg, 1.51 mmol) in CH₂Cl₂(2 mL) was

added methacryloyl chloride (158 mg, 1.51 mmol) dropwise under a

water bath. The resulting mixture was stirred at rt for 16 h. Solvent was

removed under reduced pressure, and the residue was puriﬁed by

preparatory TLC (CH₂Cl₂/MeOH, 20:1) to aﬀord 60 mg (75%) of 42c.

¹H NMR (500 MHz, CDCl₃): δ 8.37 (d, J = 5.7 Hz, 1H), 8.08 (br s, 1H),

7.87 (d, J = 5.7 Hz, 1H), 5.85 (s, 1H), 5.60 (s, 1H), 3.95 (m, 4H), 2.52

(m, 4H), 2.45 (s, 6H), 2.38 (s, 3H), 2.04 (s, 3H). HRMS (m/z): [M +

H]⁺calcd for C₁₉H₂₆N₇OS, 400.1920; found, 400.1909.

4-(4,6-Dimethylpyrimidin-2-yl)morpholine (39b). A 1.43 g (10

mmol) portion of 38 and 2.18 g (25 mmol) of morpholine in 20 mL of

DMF were heated at 90 °C for 2.5 h. The reaction mixture was

concentrated under reduced pressure, and the residue was taken up into

100 mL of CH₂Cl₂. This was washed with brine (3 × 25 mL), dried over

MgSO₄, ﬁltered, and concentrated to give 1.84 g (95%) of 39b. ¹H NMR

(500 MHz, CDCl₃): δ 6.30 (s, 1H), 3.79 (m, 4H), 3.75 (m, 4H), 2.29 (s,

6H). ¹³C NMR (125 MHz, CDCl₃): δ 167.1, 161.9, 109.4, 67.0, 44.3,

24.0. MS (m/z): [M + Na]⁺216.1.

4-(5-Iodo-4,6-dimethylpyrimidin-2-yl)morpholine (40b). To 1.50 g

(7.8 mmol) of 39b in 40 mL of acetonitrile were added 2.1 g (9.4 mmol)

of NIS and 0.898 mL (1.33 g, 11.7 mmol) of TFA, and the resulting

solution was stirred at rt for 2 h. The reaction mixture was concentrated

to dryness, and the residue was taken up into 100 mL of CH₂Cl₂, washed

with 10% sodium thiosulfate (50 mL) and 5% NaHCO₃(3 × 50 mL),

dried over MgSO₄, ﬁltered, and concentrated to give a residue which was

puriﬁed by column chromatography (CH₂Cl₂/MeOH, 1:0 to 20:1) to

Chemical Precipitation in Cell Extracts. Protein lysates were

prepared using 20 mM Tris, pH 7.4, 25 mM NaCl, 0.1% NP-40 lysis

buﬀer. Streptavidin agarose beads (50 μL) (Thermo Scientiﬁc) were

washed three times with lysis buﬀer, 44b and 45b were added at the

indicated concentrations, and the complexes were incubated at 4 °C for

1 h. Upon a three-time wash with buﬀer, the beads were added to the

indicated total cellular protein in the binding buﬀer. Samples were

incubated at 4 °C overnight, washed ﬁve times with the lysis buﬀer, and

applied to SDS−PAGE (see Figure 4a).

Chemical Precipitation in a Live Cell. Cancer cells were treated with

44b (25 μM) or ^D-biotin for 6 h and then lysed in a buﬀer containing 20

mM Tris, pH 7.4, 25 mM NaCl, and 0.1% NP-40. Aliquots (500 μg total

protein) were incubated with streptavidin beads for 1 h at 4 °C. Puriﬁed

protein complexes were washed with 20 mM Tris, pH 7.4, 1 M NaCl,

0.1% NP-40 (high salt) buﬀer and applied to SDS−PAGE. The gel was

stained with the SilverQuest staining kit (Invitrogen) (see Figure 4b).

1

yield 2.09 g (84%) of 40b. H NMR (500 MHz, CDCl₃): δ 3.77 (m,

4H), 3.73 (m, 4H), 2.53 (s, 6H). MS (m/z): [M + Na]⁺341.9.

2-((4,6-Dimethyl-2-morpholinopyrimidin-5-yl)thio)pyrimidin-4-

amine (41b). A mixture of 40b (1.0 g, 3.13 mmol), 4-amino-2-

mercaptopyrimidine (0.477 g, 3.76 mmol), neocuproine (0.140 g, 0.626

mmol), CuI (0.122 g, 0.626 mmol), and K₂CO₃(0.864 g, 6.26 mmol) in

60 mL of DMF was stirred at 110 °C for 16 h. Solvent was removed

under reduced pressure, and the residue was puriﬁed by column

chromatography (CH₂Cl₂/MeOH, 100:0 to 90:10) to aﬀord 0.690 g

(69%) of 41b. MS (m/z): [M + H]⁺319.1.

N-(2-((4,6-Dimethyl-2-morpholinopyrimidin-5-yl)thio)pyrimidin-

4-yl)acrylamide (43). To a solution of 41b (50 mg, 0.157 mmol) and

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Immunoprecipitation. Cancer cells were collected and lysed in a

buﬀer containing 20 mM Tris, pH 7.4, 25 mM NaCl, and 0.1% NP-40.

The anti-Hsp70 antibody (BB70; 5 μL) was added to 500 μg of extract

together with protein G agarose beads (30 μL) (Upstate), and the

mixture was incubated at 4 °C overnight. Samples were washed with lysis

buﬀer and applied to SDS−PAGE. The gel was stained with the

SilverQuest staining kit (Invitrogen) (see Figure 4b).

Competition Assay. Cancer cells were pretreated for 2 h with

increasing concentrations of 17a and then lysed in a buﬀer containing 20

mM Tris, pH 7.4, 25 mM NaCl, and 0.1% NP-40. Meanwhile, to prepare

the 44b−streptavidin bead conjugates, 44b (50 μM) was added to high-

capacity streptavidin agarose beads (Thermo Scientiﬁc) (30 μL per

experimental sample), incubated for 1 h at 4 °C, and then washed with

lysis buﬀer. Protein extracts (500 μg of total protein) were then

incubated overnight with the 44b−streptavidin bead conjugate. The

complexes were washed in high-salt buﬀer, applied to SDS−PAGE, and

stained with the SilverQuest staining kit (see Figure 4c).

Hsp70−HOP Complex Analysis. SKBr3 cells were treated with the

indicated concentrations of the inhibitor for 24 h. Samples were

collected and lysed in 20 mM Tris, pH 7.4, 25 mM NaCl, 0.1% NP-40

buﬀer with protease inhibitors added. Aliquots of 500 μg of total protein

adjusted to 100 μL with the lysis buﬀer were prepared. Samples were

incubated with 5 μL of BB70 antibody (Stressmarq) or normal IgG (as a

negative control) and 20 μL of protein G agarose beads (Upstate) at 4

°C overnight. Samples were washed ﬁve times with the lysis buﬀer and

applied to SDS−PAGE followed by a standard Western blotting

procedure to detect levels of HOP protein in the Hsp70 complexes upon

treatment.

Growth Inhibition Assay. We evaluated the antiproliferative eﬀects

of inhibitors using the dye Alamar Blue. This reagent oﬀers a rapid

objective measure of cell viability in cell culture, and it uses the indicator

dye resazurin to measure the metabolic capacity of cells, an indicator of

cell viability. Brieﬂy, cells were plated on Costar 96-well plates. For

attached cells (such as SKBr3), 8000 cells/well were used. For

suspension cells (such as Kasumi-1), 20000 cells/well were plated.

Cells were allowed to incubate for 24 h at 37 °C before drug treatment.

Drugs were added in triplicate at the indicated concentrations, and the

plate was incubated for 72 h. Alamar Blue (50 μM) was added and the

plate read 6 h later using the Analyst GT (ﬂuorescence intensity mode,

excitation 530 nm, emission 580 nm, with a 560 nm dichroic mirror).

Results were analyzed using the Softmax Pro software. The percentage

cell growth inhibition was calculated by comparing ﬂuorescence

readings obtained from treated versus control cells, accounting for the

initial cell population (time zero). IC₅₀was calculated as the drug

concentration that inhibits cell growth by 50%.

20, 1 mM DTT) to remove unbound ligand and to reduce nonspeciﬁc

phage binding. Binding reactions were assembled by combining kinases,

liganded aﬃnity beads, and test compounds in 1× binding buﬀer (20%

SeaBlock, 0.17× PBS, 0.05% Tween 20, 6 mM DTT). Test compounds

were prepared as 40× stocks in 100% DMSO and directly diluted into

the assay. All reactions were performed in polypropylene 384-well plates

in a ﬁnal volume of 0.04 mL. The assay plates were incubated at room

temperature with shaking for 1 h, and the aﬃnity beads were washed

with wash buﬀer (1× PBS, 0.05% Tween 20). The beads were then

resuspended in elution buﬀer (1× PBS, 0.05% Tween 20, 0.5 μM

nonbiotinylated aﬃnity ligand) and incubated at room temperature with

shaking for 30 min. The kinase concentration in the eluates was

measured by qPCR. KINOMEscan’s selectivity score (S) is a

quantitative measure of compound selectivity. It is calculated by

dividing the number of kinases that bind to the compound by the total

number of distinct kinases tested, excluding mutant variants. TREEspot

is a proprietary data visualization software tool developed by

KINOMEscan. Kinases found to bind are marked with red circles,

where larger circles indicate higher aﬃnity binding. The kinase

dendrogram was adapted and is reproduced with permission from

Science and Cell Signaling Technology, Inc.

Computational Studies. All computations were carried out on an

HP workstation xw8200 with the Ubuntu 8.10 operating system using

Maestro v8.5 (Schrodinger LLC, New York). Grids were prepared using

the Receptor Grid Generation tool in Glidev4.0. Docking calculations

were run in the standard precision (SP) mode of Glide v4.0. The

maxkeep variable, which sets the maximum number of poses generated

during the initial phase of the docking calculation, was set to 5000, and

the keep best variable, which sets the number of poses per ligand that

enter the energy minimization, was set to 1000. The energy

minimization protocol includes a dielectric constant of 4.0 and 1000

steps of conjugate gradient. Upon completion of each docking

calculation, at most 100 poses per ligand were allowed to generate.

The best docked conformation was chosen considering the orientation

and Glidescore (G-score).²²

ASSOCIATED CONTENT

* Supporting Information

■

S

Description of the synthesis of compounds 44a,b and 45a,b. This

material is available free of charge via the Internet at http://pubs.

acs.org.

Accession Codes

The PDB ID codes of crystal structures used as a starting point

for building the homology model are 1S3X, 2KHO, and 2P32.

Caspase-3,7 Activation.⁵⁰MOLM-13 cells (30000 cells/well) were

plated in black 96-well plates (Corning no. 3603) in 40 μL of RPMI

medium and left in an incubator (37 °C, 5% CO₂) for up to 24 h. Cells

were treated for 16 h with compounds or DMSO (control) at the

desired concentrations in 50 μL of medium. Drugs were added in

triplicate wells. Following exposure of cells to Hsp70 inhibitors, 50 μL of

buﬀer containing 10 mM HEPES (pH 7.5), 2 mM EDTA, 0.1% CHAPS,

and the caspase substrate Z-DEVD-R110 at 25 μM was added to each

well. Plates were incubated until the signal stabilized, and then the

ﬂuorescence signal of each well was measured in an Analyst GT

microplate reader. The percentage increase in apoptotic cells was

calculated by comparison of the ﬂuorescence reading obtained from

treated versus control cells.

Kinase Screen. For most assays, kinase-tagged T7 phage strains were

grown in parallel in 24-well blocks in an Escherichia coli host derived

from the BL21 strain. E. coli were grown to log phase and infected with

T7 phage from a frozen stock (multiplicity of infection 0.4) and

incubated with shaking at 32 °C until lysis (90−150 min). The lysates

were centrifuged (6000g) and ﬁltered (0.2 μm) to remove cell debris.

The remaining kinases were produced in HEK-293 cells and

subsequently tagged with DNA for qPCR detection. Streptavidin-

coated magnetic beads were treated with biotinylated small-molecule

ligands for 30 min at room temperature to generate aﬃnity resins for

kinase assays. The liganded beads were blocked with excess biotin and

washed with blocking buﬀer (SeaBlock (Pierce), 1% BSA, 0.05% Tween

AUTHOR INFORMATION

Corresponding Authors

*Phone: 646-888-2238. E-mail: taldonet@mskcc.org.

*Phone: 646-888-2235. E-mail: chiosisg@mskcc.org

■

Present Addresses

^⊥Y.K.: BioZone Pharmaceuticals Inc., 710 Fox Run Dr.,

Plainsboro, NJ 08536.

^#A.G.: Albany Medical Center, 43 New Scotland Ave., Albany,

NY 12208.

^∇C.C.C.: Albert Einstein College of Medicine, 1300 Morris Park

Ave., Bronx, NY 10461.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We are supported in part by MSKCC’s Technology Transfer

Fund (G.C., A.R., Y.K.), Department of Defense Grant

W81XWH-10-1-0490 (T.T.), Susan G. Komen for the Cure

(T.T., G.C.), the SPORE Pilot Award and Research &

Therapeutics Program in Prostate Cancer (G.C.), NIH Grant

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Journal of Medicinal Chemistry

Article

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133, 20267−20276.

R01 CA119001 (G.C.), Clinical and Translational Science

Center (CTSC) Grant UL1 RR024996 (A.G.), the Breast

Cancer Research Fund (G.C.), the Leukemia and Lymphoma

Society (G.C.), the Hirshberg Foundation for Pancreatic Cancer

Research (G.C.), and NIH Grants 1U01 AG032969-01A1

(G.C.), 1R01 CA172546-01A1 (G.C.), and 1R01 CA155226-

01 (G.C.). We thank Dr. George Sukenick and Dr. Hui Liu of the

NMR Analytical Core Facility at MSKCC for expert mass

spectral analysis.

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Article Doi

Heat shock protein 70 inhibitors. 1. 2,5′-thiodipyrimidine and 5-(phenylthio)pyrimidine acrylamides as irreversible binders to an allosteric site on heat shock protein 70

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