Discovery of SHP2-D26 as a First, Potent, and Effective PROTAC Degrader of SHP2 Protein

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Discovery of SHP2-D26 as a First, Potent, and Eﬀective PROTAC

Degrader of SHP2 Protein

Mingliang Wang,^∥Jianfeng Lu,^∥Mi Wang, Chao-Yie Yang, and Shaomeng Wang*

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ABSTRACT: Src homology 2 domain-containing phosphatase 2 (SHP2) is an attractive

therapeutic target for human cancers and other human diseases. Herein, we report our

discovery of potent small-molecule SHP2 degraders whose design is based upon the

proteolysis-targeting chimera (PROTAC) concept. This work has led to the discovery of

potent and eﬀective SHP2 degraders, exempliﬁed by SHP2-D26. SHP2-D26 achieves

DC₅₀values of 6.0 and 2.6 nM in esophageal cancer KYSE520 and acute myeloid

leukemia MV4;11 cells, respectively, and is capable of reducing SHP2 protein levels by

>95% in cancer cells. SHP2-D26 is >30-times more potent in inhibition of

phosphorylation of extracellular signal-regulated kinase (ERK) and of cell growth than

SHP099, a potent SHP2 inhibitor, in KYSE520 and MV4;11 cancer cell lines. This study

demonstrates that induced SHP2 degradation is a very eﬀective approach to inhibit the

function of SHP2. Further optimization of these SHP2 degraders may lead to the

development of a new class of therapies for cancers and other human diseases.

agents.¹⁹⁻²⁵A major breakthrough by Novartis scientists was

the discovery of SHP099, a potent and allosteric SHP2

inhibitor, which was shown to selectively block SHP2

phosphatase activity and inhibit cancer cell growth in vitro

and tumor growth in xenograft models in mice.^26,27

Subsequently, additional allosteric SHP2 inhibitors including

SHP389 were reported and several of them have been

advanced into clinical development for the treatment of

human cancers.²⁸⁻³¹

Although allosteric SHP2 inhibitors have been shown to be

eﬀective in preclinical models of Kirsten rat sarcoma (KRAS)

mutant human cancer, we hypothesized that eﬃcient depletion

of SHP2 protein may provide an alternative and perhaps even

more eﬀective strategy for inhibition of the SHP2 activity.

Mainardi et al.³²demonstrated that SHP2 inactivation by

CRISPR-Cas9 induces senescence and impairs tumor growth

in xenograft models of KRAS mutant tumors. Furthermore,

Ruess et al.³³revealed that knockout of the PTPN11 gene,

which encodes SHP2, in KRAS mutant human ductal

adenocarcinoma (PDAC) cells results in reduction in cell

proliferation and PTPN11-knockout cells are uniquely

susceptible to mitogen-activated protein kinase (MEK)

inhibitors. Such ﬁndings provide evidence that depletion of

INTRODUCTION

■

Src homology 2 domain-containing phosphatase 2 (SHP2) is a

protein tyrosine phosphatase. Mutations of SHP2 are prevalent

in Noonan syndrome (50%) and LEOPARD syndrome

(80%)^1,2and activated mutations of SHP2 have also been

identiﬁed in juvenile myelomonocytic leukemia (JMML, 35%),

myelodysplastic syndrome (10%), B-cell acute lymphoblastic

leukemia (7%), and acute myeloid leukemia (AML, 4%).³

Somatic activating mutations in SHP2 have been associated

with several types of solid tumors, including lung adenocarci-

noma, colon cancer, neuroblastoma, glioblastoma, melanoma,

hepatocellular carcinoma, prostate cancer, and breast can-

cer.⁴⁻⁸Accumulated evidence demonstrates that in cancer

cells, SHP2 is involved in multiple signaling processes, such as

RAS-ERK, JAK-STAT, PI3K-AKT, NF-κB, and mTOR

pathways.⁹⁻¹³In the RAS-ERK pathway, SHP2 acts as a

positive regulator at upstream to promote RAS-RAF-ERK

kinase cascade signaling transduction. Therefore, SHP2

inhibition leads to dephosphorylation of ERK and suppression

of the pro-oncogenic function of RAS-RAF-ERK pathway,

resulting in cell growth inhibition and apoptosis induction in

cancer cells.¹³Furthermore, SHP2 also participates in the

programmed cell death pathway (PD-1/PD-L1) and inhibits T

cell activation, thus contributing to immune evasion.¹⁴⁻¹⁶

Hence, SHP2 is a very attractive cancer therapeutic target.

Due to the highly conserved and positively charged nature of

its protein-tyrosine phosphatase (PTP) catalytic site, SHP2 has

proved to be a diﬃcult target in the discovery of small-

molecule inhibitors.^17,18Previously reported SHP2 inhibitors

have not shown satisfactory selectivity and/or cellular activity,

and this has prevented their development as useful therapeutic

Received: March 20, 2020

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Figure 1. Chemical structures of three previously reported SHP2 inhibitors SHP099 (1), SHP389 (2) and 3, and two designed SHP2 inhibitors (4,

5).

Figure 2. (A) Co-crystal structure of SHP2 in a complex with SHP099 (PDB ID: 5EHR), and (B and C) modeled structures of SHP2 in a complex

with 4 or 5.

the SHP2 protein in tumor cells could be an eﬀective

therapeutic strategy for human cancers, particularly those

carrying a KRAS-mutation.

tion of the cocrystal structures of SHP099 (PDB ID: 5EHR)

and compound 3 (PDB ID: 6MD7) showed that all the atoms

in these SHP2 inhibitors are in close contacts with the SHP2

protein. However, in the cocrystal structure of SHP389 (2)

(PDB ID: 6MDC) in complex with SHP2, the cyclopropyl

group of SHP389 is exposed to the solvent, suggesting that this

site can be used for a tether in potential SHP2 degraders.

We designed compound 4 as a potential SHP2 inhibitor by

combining a portion of SHP099 with a similar group found in

compound 3 to facilitate our subsequent design and synthesis

of PROTAC SHP2 degraders. The predicted binding model of

4 in a complex with SHP2 (Figure 2B) suggested that the

hydrogen bond interactions between SHP099 and T108, F113,

and E250 in SHP2 are preserved in the interactions of

compound 4 and SHP2. The sulfur atom in 4 reorients the 2-

chloroaniline group in the binding site, while the chlorine atom

in 4 interacts with the hydrophobic pocket formed by side

chain atoms of L254, Q257, and Q495 in SHP2. The cation-

aromatic interaction between SHP099 and R111 in SHP2 is

also maintained between 4 and SHP2. Of signiﬁcance, the

amino group in 4 is exposed to the solvent, providing a

potential tethering site for the design of PROTAC degraders.

We synthesized compound 4 and determined its inhibition

of SHP2 enzymatic activity (see Figure S1). Compound 4 was

found to be a potent SHP2 inhibitor with IC₅₀= 76.2 nM. In

In 2001, Deshaies and Crews formally demonstrated the

proteolysis-targeting chimera (PROTAC) strategy to achieve

targeted protein degradation.³⁴A PROTAC molecule is a

bifunctional small molecule consisting of a ligand that binds to

the target protein of interest and another ligand that recruits an

E3 ligase system. The ligands are tethered together through a

chemical linker.³⁵⁻³⁷In recent years, the PROTAC approach

has gained a momentum in the discovery and development of

completely new classes of small-molecule therapeutic

agents.³⁸⁻⁴⁵Two PROTAC molecules targeting the androgen

receptor and estrogen receptor, discovered by Arvinas

scientists, have been advanced into clinical development for

the treatment of human cancers.⁴⁶

In this paper, we report our design, synthesis, and evaluation

of PROTAC SHP2 degraders. This study has resulted in the

discovery of SHP2-D26, which induces rapid and eﬃcient

degradation of SHP2. This compound is also 10−100 times

more potent than an allosteric SHP2 inhibitor in inhibition of

ERK activity and cell growth in cancer cell lines. This study has

laid a foundation for the development of a new class of

therapeutics through targeted degradation of SHP2 protein.

the same assay, SHP099 is slightly less potent, with IC₅₀

136.2 nM.

To facilitate the convenient synthesis of PROTAC SHP2

degraders, we synthesized compound 5 by changing the amine

group in 4 to an amide group and evaluated its inhibition of

SHP2. Our data showed that 5 is a potent SHP2 inhibitor with

IC₅₀= 98.7 nM. The predicted binding model of compound 5

=

RESULTS AND DISCUSSION

■

In the design of PROTAC SHP2 degraders, it is important to

identify both an SHP2 inhibitor and an E3 ligase ligand as well

as suitable tethering sites to link them. A number of cocrystal

structures of the SHP2 protein in complexes with potent SHP2

allosteric inhibitors have been reported (Figure 1). Examina-

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a

Table 1. PROTAC SHP2 Degraders Designed Using SHP2 Inhibitor 5 and a Previously Published Potent VHL-1 Ligand

a

All the data are averages of three independent experiments with a treatment time of 6 h.

with SHP2 showed that the acetyl group extends further into

the solvent exposed region (Figure 2C), making it a suitable

site for tethering. We therefore employed compound 5 as the

SHP2 inhibitor and its amide group as the tethering site for the

design of PROTAC SHP2 degraders.

The VHL-1/cullin 2 E3 ligase complex has been successfully

used in the design of a large number of PROTAC degraders

active against various proteins.⁴⁷⁻⁵⁷Based upon the cocrystal

structures of VHL-1 ligands in a complex with VHL-1 and on

our previous studies of androgen receptor (AR) and estrogen

receptor (ER) degraders,^58,59we designed and synthesized a

series of potential PROTAC SHP2 degraders using compound

5 and a potent and previously reported VHL ligand 6⁶⁰(Table

1). Because the length of the linker plays a key role in the

potency of PROTAC degrader molecules, we systematically

varied the linker length in compounds 7−19 with the objective

of determining the optimal linker length (Table 1). We ﬁrst

evaluated these compounds by Western blotting in the

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KYSE520 esophageal cancer cell line, which was shown to be

responsive to SHP2 inhibitors,²⁶for their ability to induce

degradation of SHP2 protein at 0.1 μM and 1 μM and

obtained the data summarized in Table 1 and Figure S2.

Our Western blotting data showed that compounds 7−12

with diﬀerent linker lengths, at 0.1 or 1 μM, have little or no

eﬀect on the level of the SHP2 protein in the KYSE520 cell

line. However, compound 13, which has one additional

methylene group in the linker when compared to 12, reduces

the SHP2 protein level by ∼80% at 0.1 and 1 μM. Increasing

the linker length in 13 by one additional methylene yielded 14,

which reduces the SHP2 protein level by >90% at 0.1 and 1

μM. Compounds 15−19 with longer linkers than those in 14

are all potent and eﬀective SHP2 degraders, capable of

reducing the SHP2 protein level by >90% at 0.1 and 1 μM.

Our previous studies showed that the linker composition in

PROTAC molecules plays a key role in their degradative

potencies and, accordingly, we performed further modiﬁcations

of linker composition while keeping the linker length similar to

that in 16. The results are summarized in Table 2 and Figures

S2 and S4.

Table 2. Optimization of the Linker Composition of SHP2

Degrader 16

a

Western blotting results showed that compounds 20−22

with a polyethylene glycol (PEG) unit embedded in the linker

are less potent than compound 16. We replaced the amide

bond in the middle of the linker in 16 with a positively charged

piperazinyl group, which yielded compounds 23 and 24, both

of which reduce the SHP2 protein level by ∼80% at 0.1 μM

and >95% at 1 μM in the KYSE520 cell line. Thus, while both

23 and 24 are very eﬀective SHP2 degraders, they are less

potent than 16. To examine the eﬀect of the positively charged

piperazinyl group in 23 and 24, we synthesized compound 25

containing a linker of 14 methylene groups. Compound 25 is

less eﬀective than either 23 or 24 in reducing the SHP2 protein

level at both 0.1 μM and 1 μM in the KYSE520 cell line.

Retention of the amide bond in the middle of the linker and

introduction of a positively charged piperazinyl group resulted

in compounds 26 and 27, both of which achieve near-complete

SHP2 degradation at both 100 nM and 1 μM. Introduction of

a phenoxyl group in the middle of the linker led to compound

28, which reduces SHP2 protein by >95% at both 0.1 μM and

1 μM. Compound 29, containing a positively charged

piperazinyl group in the central part of the linker, fails to

induce any SHP2 degradation at 100 nM but reduces SHP2

protein by ∼80% at 1 μM.

The acute myeloid leukemia MV4;11 cell line was also

shown to be very responsive to SHP2 inhibitors.²⁶We next

tested these compounds for their ability to reduce the SHP2

protein in the acute myeloid leukemia MV4;11 cell line at 100

nM with 24 h treatment time, obtaining the data summarized

in Table 2. The data showed that 20, 22, 26, 27, and 28 all

eﬀectively reduce SHP2 protein by >95% at 100 nM,

indicating that these compounds are potent and eﬀective

SHP2 degraders in the MV4;11 cell line. These linker

modiﬁcations thus have led to a number of highly potent

SHP2 degraders.

Further Evaluation of Compound 26 (SHP2-D26). In

addition to its excellent degradation potency in both the

KYSE520 and MV4;11 cell lines, compound 26 (SHP2-D26)

has good aqueous solubility. We further investigated SHP2-

D26 for its potency and mechanism of action in these two cell

lines.

a

All of the data are the average of three independent experiments with

a treatment time of 6 h in KYSE520 cell lines and 24 h in MV4;11 cell

lines.

lines (Figure 3A−D). Western blotting results showed that

SHP2-D26 eﬀectively reduces SHP2 protein in a dose-

dependent manner. Quantiﬁcation of the Western blotting

data showed that the compound achieves DC₅₀values

(concentration needed to induce targeted protein degradation

by 50%) of 6.0 and 2.6 nM in the KYSE520 and MV4;11 cell

lines, respectively (Figure 3B,D).

We next evaluated the kinetics of SHP2-D26 in induction of

SHP2 degradation in the KYSE520 and MV4;11 cell lines

(Figure 3E,F). In KYSE520 cells, SHP2-D26 at 100 nM

eﬀectively reduces the SHP2 protein level within 4 h and

We examined the degradation of SHP2 by SHP2-D26 in a

wide range of concentrations in KYSE520 and MV4;11 cell

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Figure 3. Further evaluation of SHP2-D26 for its degradation activity in the KYSE520 and MV4;11 cell lines. (A−D) Dose-dependent SHP2

degradation in the KYSE520 and MV4;11 cell lines, with SHP099 included as the inhibitor control. Cells were treated with SHP2-D26 for 12 h.

SHP2 protein was examined by Western blotting, and the protein level was quantiﬁed by densitometry and normalized to the corresponding

density of the GAPDH protein. (E,F). Degradation kinetics of SHP2-D26 in the KYSE520 and MV4;11 cell lines. Cells were treated with 100 nM

of SHP2-D26 for diﬀerent times. SHP2 protein was examined by Western blotting, and the GAPDH protein was used as the loading control. (G)

KYSE520 cell line was treated with VHL-1 ligand 6 (10 μM), compound 4 (at 10 μM), MLN4924 (0.5 μM), or MG132 (3 μm) for 1 h and then

treated with SHP-D26 (0.1 μM) for 3 h. The protein level of SHP2 was examined by Western blotting, and the GAPDH protein was used as the

loading control.

Figure 4. Compound 26 (SHP2-D26) suppresses the p-ERK pathway. KYSE520 or MV4;11 cells were treated as indicated with SHP2-D26 or

SHP099 for 48 h. The protein levels of SHP2 (Bethyl Lab. A301−544), ERK (#9102, CST), and phospho-ERK (#4370, CST) determined by

Western blotting. GAPDH was used as a loading control.

achieves essentially complete SHP2 depletion with 8 h

treatment (Figure 3E). Similar kinetics was observed in the

MV4;11 cell line (Figure 3F). These data show that SHP2

degradation induced by SHP2-D26 is fairly rapid in both

KYSE520 and MV4;11 cells.

We examined if SHP2-D26 functions as a bona fide

PROTAC degrader in the KYSE520 cell line. A VHL-1 ligand

6 (Table 1), compound 4 (an SHP2 inhibitor), MLN4924 (an

E1 inhibitor), and MG132 (a proteasome inhibitor) all

eﬀectively block degradation of the SHP2 protein in

KYSE520 cells (Figure 3G). Therefore, our data show that

SHP2 degradation induced by SHP2-D26 requires its binding

to VHL-1 and SHP2 proteins and is also neddylation- and

proteasome-dependent, demonstrating that SHP2-D26 is a

bona fide PROTAC SHP2 degrader.

Since SHP2 protein is known to play an important role in

the MAPK/ERK signaling pathway, we examined the impact of

SHP2 degradation on the MAPK/ERK signaling pathway in

the KYSE520 and MV4;11 cell lines, with SHP099 included as

a control (Figure 4). Western blotting showed that both

SHP2-D26 and SHP099 dose-dependently inhibit phosphor-

ylation of ERK in the KYSE520 and MV4;11 cell lines.

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Figure 5. Cell growth inhibition in KYSE520 and MV4;11 cells treated with SHP2 degrader 26 (SHP2-D26) and SHP2 inhibitors SHP099, 4 and

5. KYSE520 and MV4;11 cells were treated with indicated doses for 4 days, and cell viability was determined by a colorimetric WST-8 assay.

a

Scheme 1. Synthesis of the VHL Ligand (6)

a

Reagents and conditions: (a) Boc₂O, NaHCO₃, EtOAc/H₂O, 2 h; (b) 4-methylthiazole, Pd(OAc)₂, KOAc, DMA, 90 °C, 12 h; (c) 4 N HCl

dioxane/MeOH, rt, 4h; (d) HATU, DIPEA, DMF, 0 °C to RT, 12 h; and (e) TFA, DCM, rt, 4h.

a

Scheme 2. Synthesis of the Key Intermediate (40)

a

Reagents and conditions: (a) tert-butylthiol, Cs₂CO₃, DMF, 120 °C, 36 h; (b) conc. HCl, 80 °C, 6 h; (c) K₃PO₄, CuI, 1,10-phenanthroline,

dioxane, 90 °C, 16 h; and (d) DIPEA, DMSO, 100 °C, 3 h.

However, in both cell lines, SHP2-D26 is much more potent

than SHP099 in inhibition of pERK. Speciﬁcally, in the

KYSE520 cell line, while 100 nM of SHP2-D26 is eﬀective in

reducing the level of pERK, >3,000 nM of the SHP2 inhibitor

SHP099 is needed to do so. In the MV4;11 cell line, while 100

nM of SHP2-D26 completely inhibits phorsphorylated ERK

(pERK), >3,000 nM of the SHP2 inhibitor SHP099 is required

to achieve complete inhibition of pERK. Therefore, our data

indicate that SHP2-D26 is >30-times more potent than

SHP099 in inhibition of pERK in both cell lines.

KYSE520 and MV4;11 cell lines (Figure 5). In the KYSE520

cell line, SHP2-D26 achieves IC₅₀values of 0.66 μM (Figure

5A). In comparison, SHP099, compounds 4 and 5 have IC₅₀

values of 18.2, 42.3, and 39.4 μM, respectively. Hence, SHP2-

D26 is 28-, 64-, and 60-times more potent than SHP099 and

compounds 4 and 5 in inhibition of cell growth in the

KYSE520 cell line. In the MV4;11 cell line, SHP2-D26,

SHP099, 4 and 5 have IC₅₀values of 9.9 nM, 1.0 μM, 3.9 μM

and 6.6 μM, respectively, in inhibition of cell growth. Thus,

SHP2-D26 is 100-, > 400 and >600-times more potent than

SHP099, compounds 4 and 5, respectively in cell growth

inhibition in the MV4;11 cell line.

We evaluated the cell growth inhibition of SHP2-D26, and

three SHP2 inhibitors (SHP099, compounds 4 and 5) in the

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a

Scheme 3. Synthesis of Compounds 7−19

a

Reagents and conditions: (a) 6, HATU, DIPEA, DMF, 0 °C to RT, 1 h; (b) 4 N HCl dioxane/MeOH, rt; (c) 40, DIPEA, DCM, 0 °C, 2 h; (d)

LiOH·H₂O, THF, MeOH, H₂O, RT, 3 h; (e) HATU, DIPEA, DMF, 0 °C to RT, 1 h; and (f) TFA, DCM, rt, 2 h.

a

Scheme 4. Synthesis of Compounds 20, 21, and 22

a

Reagents and conditions: (a) 6, HATU, DIPEA, DMF, 0 °C to rt, 1 h; (b) 4 N HCl dioxane, 2h; (c) 46b, HATU, DIPEA, DMF, 0 °C to rt, 1 h;

and (d) TFA, DCM, rt, 2h.

coupling with compound 33 led to 34, which after acidic

deprotection aﬀorded the VHL ligand (6).

CHEMISTRY

■

The compounds in Table 1 were synthesized from the VHL

ligand (6) and the key intermediate (42). The synthesis of

VHL ligand 6 follows a published procedure⁶⁰and is outlined

in Scheme 1. The commercially available (S)-1-(4-

bromophenyl)ethan-1-amine (30) was protected by Boc₂O,

and a subsequent Heck coupling reaction with 4-methyl-

thiazole aﬀorded compound 31. Removal of the Boc group

under acidic conditions gave compound 32, and amide

The synthesis of the key intermediate (40) is shown in

Scheme 2. The commercially available 2-chloro-3-ﬂuoroaniline

(35) was reacted with tert-butylthiol under basic conditions to

produce compound 36, which was heated in concentrated

hydrochloric acid to remove the tert-butyl group, yielding

compound 37. A cross-coupling reaction of the intermediate

37 with 3-bromo-6-chloropyrazin-2-amine (38) was carried

out in the presence of CuI and 1,10-phenanthroline as

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a

Scheme 5. Synthesis of Compounds 23, 24, and 25

a

Reagents and conditions: (a) TCFH, NMI, MeCN/THF, rt, 6 h; (b) piperidine, MeCN, rt, 2 h; (c) K₂CO₃, KI, DMF, 60 °C, 8 h; (d) HATU,

DIPEA, DMF, 0 °C to rt, 1 h; (e) TFA, DCM, rt, 2h; and (f) NaOH, THF-H₂O.

catalysts, and potassium phosphate (K₃PO₄) as a base in 1,4-

dioxane at 90 °C for 16 h to obtain compound 39, which was

reacted with tert-butyl (4-methylpiperidin-4-yl)carbamate

using DIPEA as a base in DMSO at 100 °C for 3 h to aﬀord

the key intermediate (40).

The synthesis of compounds 23−25 is shown in Scheme 5.

Because the starting material 40 is a non-nucleophilic amine,

the formation of amide bonds in high yields with common

coupling agents is diﬃcult. After trying multiple reaction

conditions, a combination of N,N,N,N-tetramethylchlorofor-

mamide hexaﬂuorophosphate (TCFH) and N-methylimida-

zole (NMI) was found to be eﬀective as a coupling reagent.⁶¹

The intermediate (52) was obtained by amide condensation

and subsequent removal of the Fmoc group. Following the

substitution reaction of compound 52 with 11-bromoundeca-

noic acid or 12-bromododecanoic acid, (54a, 54b) were

obtained. Following amide condensation with VHL ligand 6

and Boc removal, the ﬁnal compounds (23, 24) were

produced. Compound 25 was synthesized using the procedure

described for the synthesis of compound 23 with 16-

(benzyloxy)-16-oxohexadecanoic acid (55) as the starting

material.

As shown in Scheme 6, compounds 26−29 were synthesized

using the following procedure. Intermediates 59a/59b were

synthesized by the substitution reaction of tert-butyl

piperazine-1-carboxylate with methyl 9-bromononanoate or

methyl 10-bromononanoate followed by hydrolysis with

lithium hydroxide. The intermediates (60a, 60b) were

obtained by an amide condensation reaction of 59a or 59b,

respectively, with VHL ligand 6 followed by removal of the

Boc group. Amide condensation of 60a or 60b with 46b

followed by removal of the Boc group aﬀorded compounds 26

and 27. Compounds 28 and 29 were synthesized using the

procedure described for the synthesis of compound 26 with

As shown in Scheme 3, compounds 7−19 were synthesized

according to the following procedure. Using Boc-protected

amine-terminated acids with diﬀerent carbon chain lengths

(41a−41j) as starting materials, the intermediates (43a−43j)

were obtained by an amide condensation reaction followed by

removal of the Boc protecting group under acidic conditions.

The intermediates (46a−46c) were synthesized by the

reaction of the key intermediate 40 with methyl-3-chloro-3-

oxoproanoate esters with diﬀerent carbon chain lengths (44a−

44c) and subsequent hydrolysis of the methyl ester. Amide

coupling between compound 43 and 46 followed by removal

of the Boc protecting group aﬀorded the ﬁnal compounds (7−

19) in high yields.

As shown in Scheme 4, compounds 20−22 were treated in a

similar way to produce compounds 7−19. With a polyethylene

glycol unit (PEG) embedded in their chains, Boc-protected

amine-terminated acids (47, 49) were used as the starting

material. The intermediates (48, 50) were obtained by amide

condensation with the VHL ligand (6), and this was followed

by removal of the Boc group. Employing compounds 48 and

50 as intermediates, compounds 20−22 were synthesized by

condensation with the corresponding acid (48b) and

subsequent removal of the Boc group.

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a

Scheme 6. Synthesis of Compounds 26, 27, 28, and 29

a

Reagents and conditions: (a) K₂CO₃, NaI, MeCN, 60 °C, 16 h; (b) LiOH.H₂O, THF, MeOH, H₂O, rt, 3 h (c) 6, HATU, DIPEA, DMF, 0 °C to

rt, 1 h; (d) 4 N HCl dioxane, 2 h; (e) 46b, HATU, DIPEA, DMF, 0 °C to rt, 1 h; (f) TFA, DCM, rt, 2 h.

tert-butyl (4-hydroxyphenyl)carbamate (61) and tert-butyl (4-

(piperazin-1-yl)butyl)carbamate (65), respectively, as the

starting materials.

concept that targeted degradation of SHP2 is a very eﬀective

strategy in inhibition of SHP2 activity. Further optimization of

SHP2-D26 may lead to the development of a potent and

eﬀective SHP2 degrader for the treatment of human cancers.

In addition, a potent and eﬀective SHP2 degrader may also

have a great therapeutic potential for the treatment of certain

human genetic disorders caused by SHP2 mutation and

activation, such as Noonan syndrome and LEOPARD

syndrome.

CONCLUSION

■

We have designed, synthesized, and evaluated a series of

PROTAC SHP2 degraders. Our study has resulted in the

discovery of a number of highly potent SHP2 degraders, we

obtained a number of highly potent SHP2 degraders,

exempliﬁed by compound 26 (SHP2-D26). SHP2-D26

achieves DC₅₀values of 6.0 nM and 2.6 nM in KYSE520

and MV4;11 cells, respectively, and >95% degradation at 30

nM in both cell lines. SHP2-D26 is much more potent and

eﬀective in inhibition of ERK phosphorylation and in

inhibition of cell growth in KYSE520 and MV4;11 cells than

three SHP2 inhibitors. This study provides the ﬁrst proof-of-

EXPERIMENTAL SECTION

■

Chemistry. General Information. All commercial reagents and

solvents were used as supplied without further puriﬁcation. Proton

nuclear magnetic resonance (¹H NMR) and carbon nuclear magnetic

resonance (¹³C NMR) spectroscopy were performed on Bruker

1

Advance 400 NMR spectrometers. H NMR spectra are reported in

parts per million (ppm) downﬁeld from tetramethylsilane (TMS). All

I

https://dx.doi.org/10.1021/acs.jmedchem.0c00471

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

¹³C NMR spectra are reported in ppm and obtained with ¹H

MS (ESI⁺): calculated for C₄₅H₅₉ClN₁₁O₆S₂[M + 1]⁺, 948.38; found,

948.31.

decoupling. In the spectral data reported, the format (δ) chemical

shift (multiplicity, J values in Hz, integration) was used with the

following abbreviations: s = singlet, brs = broad singlet, d = doublet, t

= triplet, q = quartet, and m = multiplet. Electrospray ionization (ESI)

mass spectral (MS) analysis was performed on a Thermo Scientiﬁc

LCQ Fleet mass spectrometer. The ﬁnal products were puriﬁed by

reverse-phase HPLC (RP-HPLC) with solvent A (0.1% of TFSA in

water) and solvent B (0.1% of TFA in CH₃CN) as eluents with a ﬂow

rate of 45 mL/min. All ﬁnal compounds have purity ≥95% as

determined by Waters ACQUITY ultraperformance liquid chroma-

tography (UPLC) using a reverse-phase column (SunFire, C18, 5 μm,

4.6 × 150 mm2) and a solvent gradient of solvent A (H₂O with 0.1%

of TFA) and solvent B (CH₃CN with 0.1% of TFA).

(2S,4R)-1-((S)-2-Amino-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-

1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

(6). HATU (4.56 g, 12.0 mmol, 1.2 equiv) was added to a solution of

compound 32 (2.55 g, 10.0 mmol, 1.0 equiv), which was synthesized

according to the reported procedure,⁶⁰using (2S,4R)-1-((S)-2-((tert-

butoxycarbonyl)amino)-3,3-dimethyl-butanoyl)-4-hydroxypyrroli-

dine-2-carboxylic acid (33) (3.45 g, 10.0 mmol, 1.0 equiv), and

DIPEA (6.95 mL, 40.0 mmol, 4.0 equiv) in DMF (20 mL) at 0 °C

under nitrogen. The mixture was stirred at ambient temperature for

12 h when LC-MS showed that the reaction was complete. The

reaction mixture was quenched with H₂O (100 mL) and extracted

with EtOAc (100 mL × 2). The combined organic layers were washed

with brine (200 mL) and dried over Na₂SO₄. The organic solution

was ﬁltered and concentrated under reduced pressure to give a residue

which was dissolved in DCM (30.0 mL), and TFA (15.0 mL) was

added at 0 °C under nitrogen. The mixture was stirred at ambient

temperature for 4 h, when LC-MS showed that the reaction was

complete. The volatile components were removed on a rotary

evaporator, and the residue was puriﬁed by reverse phase column

chromatography to aﬀord compound 6 as a TFA salt (3.12 g, 56%).

¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 9.01 (s, 1H), 8.61 (d, J =

N1-(3-((3-Amino-5-(4-amino-4-methylpiperidin-1-yl)pyrazin-2-

yl)thio)-2-chlorophenyl)-N3-(4-(((R)-1-((2R,4S)-4-hydroxy-2-(((R)-1-

(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-

3,3-dimethyl-1-oxobutan-2-yl)amino)-4-oxobutyl)malonamide (8).

This compound was prepared from 43b and 46a by a procedure

1

similar to that used for compound 7. H NMR (400 MHz, DMSO-

d₆): δ (ppm) 10.34 (s, 1H), 9.00 (s, 1H), 8.39 (d, J = 8.0 Hz, 1H),

8.29−8.27 (m, 1H), 7.96 (brs, 3H), 7.88−7.81 (m, 2H), 7.66 (s, 1H),

7.45−7.37 (m, 4H), 7.16 (t, J = 8.0 Hz, 1H), 6.40 (dd, J = 8.0 Hz, J =

1.2 Hz, 1H), 6.21 (brs, 1H), 4.95−4.88 (m, 1H), 4.52 (d, J = 8.8 Hz,

1H), 4.42 (t, J = 8.0 Hz, 1H), 4.28 (s, 1H), 4.06−4.02 (m, 2H),

3.64−3.57 (m, 2H), 3.39 (s, 2H), 3.32−3.28 (m, 2H), 3.13−3.08 (m,

2H), 2.45 (s, 3H), 2.30−2.27 (m, 1H), 2.18−2.16 (m, 1H), 2.04−

1.97 (m, 1H), 1.78−1.73 (m, 1H), 1.70−1.64 (m, 6H), 1.37 (s, 3H),

1.36 (s, 3H), 0.94 (s, 9H); UPLC-MS (ESI⁺): calculated for

C₄₆H₆₁ClN₁₁O₆S₂[M + 1]⁺, 962.39; found, 962.33.

N1-(3-((3-Amino-5-(4-amino-4-methylpiperidin-1-yl)pyrazin-2-

yl)thio)-2-chlorophenyl)-N3-(5-(((R)-1-((2R,4S)-4-hydroxy-2-(((R)-1-

(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)-pyrrolidin-1-yl)-

3,3-dimethyl-1-oxobutan-2-yl)amino)-5-oxopentyl)malonamide

(9). This compound was prepared from 43c and 46a by a procedure

1

similar to that used for compound 7. H NMR (400 MHz, DMSO-

d₆): δ (ppm) 10.33 (s, 1H), 9.00 (s, 1H), 8.39 (d, J = 8.0 Hz, 1H),

8.27−8.25 (m, 1H), 7.99 (brs, 3H), 7.83−7.81 (m, 2H), 7.66 (s, 1H),

7.45−7.37 (m, 4H), 7.16 (t, J = 8.0 Hz, 1H), 6.40 (dd, J = 8.0 Hz, J =

1.2 Hz, 1H), 6.17 (brs, 1H), 4.95−4.88 (m, 1H), 4.51 (d, J = 8.8 Hz,

1H), 4.42 (t, J = 8.0 Hz, 1H), 4.28 (s, 1H), 4.05−4.02 (m, 2H),

3.64−3.56 (m, 2H), 3.39 (s, 2H), 3.33−3.28 (m, 2H), 3.13−3.08 (m,

2H), 2.45 (s, 3H), 2.29−2.23 (m, 1H), 2.16−2.13 (m, 1H), 2.01−

1.97 (m, 1H), 1.78−1.70 (m, 4H), 1.54−1.41 (m, 4H), 1.38 (s, 3H),

1.36 (s, 3H), 0.93 (s, 9H); UPLC-MS (ESI⁺): calculated for

C₄₇H₆₃ClN₁₁O₆S₂[M + 1]⁺, 976.41; found, 976.45.

N1-(3-((3-Amino-5-(4-amino-4-methylpiperidin-1-yl)pyrazin-2-

yl)thio)-2-chlorophenyl)-N3-(6-(((R)-1-((2R,4S)-4-hydroxy-2-(((R)-1-

(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)-pyrrolidin-1-yl)-

3,3-dimethyl-1-oxobutan-2-yl)amino)-6-oxohexyl)malonamide

(10). This compound was prepared from 43d and 46a by a procedure

8.0 Hz, 1H), 8.20 (brs, 3H), 7.45−7.37 (m, 4H), 4.95−4.90 (m, 1H),

4.54 (t, J = 8.0 Hz, 1H), 4.30 (brs, 1H), 3.90−3.87 (m, 1H), 3.76−

3.73 (m, 1H), 3.52−3.47 (m, 1H), 2.45 (s, 3H), 2.14−2.09 (m, 1H),

1.78−1.72 (m, 1H), 1.38 (d, J = 7.2 Hz, 3H), δ 1.02 (s, 9H); UPLC-

1

similar to that used for compound 7. H NMR (400 MHz, DMSO-

d₆): δ (ppm) 10.33 (s, 1H), 9.00 (s, 1H), 8.39 (d, J = 8.0 Hz, 1H),

8.26−8.23 (m, 1H), 7.96 (brs, 3H), 7.81 (d, J = 8.8 Hz, 2H), 7.66 (s,

1H), 7.45−7.36 (m, 4H), 7.16 (t, J = 8.0 Hz, 1H), 6.40 (dd, J = 8.0

Hz, J = 1.2 Hz, 1H), 6.20 (brs, 1H), 4.95−4.88 (m, 1H), 4.51 (d, J =

8.8 Hz, 1H), 4.42 (t, J = 8.0 Hz, 1H), 4.28 (s, 1H), 4.06−4.02 (m,

2H), 3.62−3.56 (m, 2H), 3.38 (s, 2H), 3.33−3.28 (m, 2H), 3.11−

3.06 (m, 2H), 2.45 (s, 3H), 2.26−2.23 (m, 1H), 2.14−2.10 (m, 1H),

2.01−1.98 (m, 1H), 1.78−1.67 (m, 5H), 1.54−1.41 (m, 4H), 1.38 (s,

3H), 1.36 (s, 3H), 1.27−1.23 (m, 2H), 0.93 (s, 9H); UPLC-MS

(ESI⁺): calculated for C₄₈H₆₅ClN₁₁O₆S₂[M + 1]⁺, 990.42; found,

990.39.

+

MS (ESI⁺): calculated for C₂₃H₃₃N₄O₃S [M + 1] , 445.23; found,

445.44.

N1-(3-((3-Amino-5-(4-amino-4-methylpiperidin-1-yl)pyrazin-2-

yl)thio)-2-chlorophenyl)-N3-(3-(((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-

(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)-pyrrolidin-1-yl)-

3,3-dimethyl-1-oxobutan-2-yl)amino)-3-oxopropyl)malonamide

(7). HATU (25 mg, 0.066 mmol, 1.1 equiv) was added to a mixture of

(2S,4R)-1-((S)-2-(3-aminopropanamido)-3,3-dimethylbutanoyl)-4-

hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-

2-carboxamide hydrochloride (43a) (33 mg, 0.06 mmol, 1.0 equiv), 3-

((3-((3-amino-5-(4-((tert-butoxycarbonyl)amino)-4-methylpiperidin-

1-yl)pyrazin-2-yl)-thio)-2-chlorophenyl)amino)-3-oxopropanoic acid

(46a) (33 mg, 0.06 mmol, 1.0 equiv), and DIPEA (39 mg, 0.30

mmol, 5.0 equiv) in DMF (2 mL) at 0 °C under N₂. The mixture was

stirred at ambient temperature for 1 h. After being quenched with

water (8 mL) and extracted with EtOAc (5 mL x 3), the organic

layers were washed with brine (15 mL), dried over anhydrous

Na₂SO₄, and concentrated under reduced pressure. The residue was

dissolved in DCM (3 mL), and triﬂuoracetic acid (1 mL) was added

at 0 °C. The reaction was stirred for 2 h, and the solvent was removed

in vacuo. The residue was puriﬁed by reverse-phase chromatography

N1-(3-((3-Amino-5-(4-amino-4-methylpiperidin-1-yl)pyrazin-2-

yl)thio)-2-chlorophenyl)-N3-(7-(((R)-1-((2R,4S)-4-hydroxy-2-(((R)-1-

(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)-pyrrolidin-1-yl)-

3,3-dimethyl-1-oxobutan-2-yl)amino)-7-oxoheptyl)malonamide

(11). This compound was prepared from 43e and 46a by a procedure

1

similar to that used for compound 7. H NMR (400 MHz, DMSO-

d₆): δ (ppm) 10.32 (s, 1H), 9.00 (s, 1H), 8.38 (d, J = 8.0 Hz, 1H),

8.25−8.22 (m, 1H), 7.96 (brs, 3H), 7.82−7.79 (m, 2H), 7.66 (s, 1H),

7.47−7.36 (m, 4H), 7.15 (t, J = 8.0 Hz, 1H), 6.40 (dd, J = 8.0 Hz, J =

1.2 Hz, 1H), 6.20 (brs, 1H), 4.95−4.88 (m, 1H), 4.52 (d, J = 8.8 Hz,

1H), 4.42 (t, J = 8.0 Hz, 1H), 4.28 (s, 1H), 4.07−4.02 (m, 2H),

3.63−3.57 (m, 2H), 3.38 (s, 2H), 3.33−3.26 (m, 2H), 3.12−3.07 (m,

2H), 2.45 (s, 3H), 2.29−2.21 (m, 1H), 2.15−2.08 (m, 1H), 2.04−

1.98 (m, 1H), 1.82−1.70 (m, 5H), 1.54−1.41 (m, 4H), 1.38 (s, 3H),

1.36 (s, 3H), 1.28−1.19 (m, 4H), 0.93 (s, 9H); UPLC-MS (ESI⁺):

calculated for C₄₉H₆₇ClN₁₁O₆S₂[M + 1]⁺, 1004.44; found, 1004.37.

N1-(3-((3-Amino-5-(4-amino-4-methylpiperidin-1-yl)pyrazin-2-

yl)thio)-2-chlorophenyl)-N3-(8-(((R)-1-((2R,4S)-4-hydroxy-2-(((R)-1-

(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)-pyrrolidin-1-yl)-

3,3-dimethyl-1-oxobutan-2-yl)amino)-8-oxooctyl)malonamide

(12). This compound was prepared from 43f and 46a by a procedure

1

over a C18 column to yield 7 (37 mg, 58%) as a white powder. H

NMR (400 MHz, DMSO-d₆): δ (ppm) 10.31 (s, 1H), 9.00 (s, 1H),

8.39 (d, J = 8.0 Hz, 1H), 8.32−8.29 (m, 1H), 7.97 (brs, 3H), 7.80 (d,

J = 7.2 Hz, 1H), 7.66 (s, 1H), 7.45−7.37 (m, 4H), 7.16 (t, J = 8.0 Hz,

1H), 6.40 (dd, J = 8.0 Hz, J = 1.2 Hz, 1H), 4.95−4.85 (m, 1H), 4.52

(d, J = 8.8 Hz, 1H), 4.43 (t, J = 8.0 Hz, 1H), 4.29 (s, 1H), 4.06−4.02

(m, 2H), 3.64−3.57 (m, 2H), 3.38 (s, 2H), 3.33−3.26 (m, 2H), 2.45

(s, 3H), 2.43−2.33 (m, 2H), 2.04−1.99 (m, 1H), 1.81−1.77 (m, 1H),

1.73−1.70 (m, 4H), 1.38 (s, 3H), 1.36 (s, 3H), 0.92 (s, 9H); UPLC-

J

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J. Med. Chem. XXXX, XXX, XXX−XXX

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1

similar to that used for compound 7. H NMR (400 MHz, DMSO-

3.12−3.07 (m, 2H), 2.45 (s, 3H), 2.29−2.21 (m, 1H), 2.13−1.98 (m,

2H), 1.83−1.70 (m, 5H), 1.50−1.40 (m, 4H), 1.38 (s, 3H), 1.36 (s,

3H), 1.28−1.16 (m, 14H), 0.93 (s, 9H); ¹³C NMR (100 MHz,

DMSO-d₆): δ (ppm) 172.03, 170.60, 169.59, 167.18, 165.73, 158.25,

157.90, 155.73, 153.52, 151.48, 147.72, 144.65, 137.25, 135.70,

131.11, 129.67, 128.81, 127.20, 126.37, 120.29, 119.71, 117.59,

114.67, 113.76, 68.74, 58.53, 56.31, 56.23, 52.13, 47.68, 43.05, 37.72,

35.19, 34.88, 34.10, 28.97, 28.88, 28.75, 28.66, 26.43, 26.37, 25.43,

22.43, 21.96, 15.97; UPLC-MS (ESI⁺): calculated for

C₅₄H₇₇ClN₁₁O₆S₂[M + 1]⁺, 1074.52; found, 1074.38.

d₆): δ (ppm) 10.33 (s, 1H), 9.00 (s, 1H), 8.38 (d, J = 8.0 Hz, 1H),

8.25−8.23 (m, 1H), 7.96 (brs, 3H), 7.82−7.78 (m, 2H), 7.66 (s, 1H),

7.45−7.35 (m, 4H), 7.16 (t, J = 8.0 Hz, 1H), 6.40 (dd, J = 8.0 Hz, J =

1.2 Hz, 1H), 6.20 (brs, 1H), 4.95−4.88 (m, 1H), 4.52 (d, J = 8.8 Hz,

1H), 4.41 (t, J = 8.0 Hz, 1H), 4.27 (s, 1H), 4.06−4.02 (m, 2H),

3.63−3.57 (m, 2H), 3.38 (s, 2H), 3.33−3.26 (m, 2H), 3.12−3.07 (m,

2H), 2.45 (s, 3H), 2.28−2.21 (m, 1H), 2.14−2.07 (m, 1H), 2.04−

1.98 (m, 1H), 1.82−1.70 (m, 5H), 1.51−1.42 (m, 4H), 1.38 (s, 3H),

1.36 (s, 3H), 1.25−1.14 (m, 6H), 0.93 (s, 9H); UPLC-MS (ESI⁺):

calculated for C₅₀H₆₉ClN₁₁O₆S₂[M + 1]⁺, 1018.46; found, 1018.41.

N1-(3-((3-Amino-5-(4-amino-4-methylpiperidin-1-yl)pyrazin-2-

yl)thio)-2-chlorophenyl)-N3-(9-(((R)-1-((2R,4S)-4-hydroxy-2-(((R)-1-

(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)-pyrrolidin-1-yl)-

3,3-dimethyl-1-oxobutan-2-yl)amino)-9-oxononyl)malonamide

(13). This compound was prepared from 43g and 46a by a procedure

N1-(3-((3-Amino-5-(4-amino-4-methylpiperidin-1-yl)pyrazin-2-

yl)thio)-2-chlorophenyl)-N4-(11-(((R)-1-((2R,4S)-4-hydroxy-2-(((R)-

1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)-pyrrolidin-1-

yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-11-oxoundecyl)-

succinimide (17). This compound was prepared from 43i and 46b by

1

a procedure similar to that used for compound 7. H NMR (400

1

similar to that used for compound 7. H NMR (400 MHz, DMSO-

MHz, DMSO-d₆): δ (ppm) 9.54 (s, 1H), 8.98 (s, 1H), 8.36 (d, J = 8.0

Hz, 1H), 7.97 (brs, 3H), 7.84−7.75 (m, 2H), 7.66 (s, 1H), 7.49−7.37

(m, 4H), 7.14 (t, J = 8.0 Hz, 1H), 6.43 (dd, J = 8.0 Hz, J = 1.2 Hz,

1H), 6.18 (brs, 2H), 4.95−4.88 (m, 1H), 4.52 (d, J = 8.8 Hz, 1H),

4.42 (t, J = 8.0 Hz, 1H), 4.27 (s, 1H), 4.05−4.02 (m, 2H), 3.64−3.57

(m, 2H), 3.33−3.26 (m, 2H), 3.05−3.00 (m, 2H), 2.63−2.59 (m,

2H), 2.45 (s, 3H), 2.41−2.37 (m, 2H), 2.28−2.21 (m, 1H), 2.13−

1.98 (m, 2H), 1.82−1.70 (m, 5H), 1.52−1.40 (m, 4H), 1.38 (s, 3H),

1.36 (s, 3H), 1.28−1.17 (m, 12H), 0.93 (s, 9H); UPLC-MS (ESI⁺):

calculated for C₅₄H₇₇ClN₁₁O₆S₂[M + 1]⁺, 1074.52; found, 1074.47.

N1-(3-((3-Amino-5-(4-amino-4-methylpiperidin-1-yl)pyrazin-2-

yl)thio)-2-chlorophenyl)-N4-(12-(((R)-1-((2R,4S)-4-hydroxy-2-(((R)-

1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)-pyrrolidin-1-

yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-12-oxododecyl)-

succinimide (18). This compound was prepared from 43j and 46b by

d₆): δ (ppm) 10.32 (s, 1H), 8.99 (s, 1H), 8.38 (d, J = 8.0 Hz, 1H),

8.26−8.23 (m, 1H), 7.97 (brs, 3H), 7.82−7.78 (m, 2H), 7.66 (s, 1H),

7.45−7.36 (m, 4H), 7.16 (t, J = 8.0 Hz, 1H), 6.40 (dd, J = 8.0 Hz, J =

0.8 Hz, 1H), 6.20 (brs, 1H), 4.95−4.88 (m, 1H), 4.51 (d, J = 8.8 Hz,

1H), 4.41 (t, J = 8.8 Hz, 1H), 4.27 (s, 1H), 4.05−4.02 (m, 2H),

3.63−3.57 (m, 2H), 3.38 (s, 2H), 3.33−3.26 (m, 2H), 3.12−3.07 (m,

2H), 2.45 (s, 3H), 2.28−2.21 (m, 1H), 2.13−1.98 (m, 2H), 1.82−

1.70 (m, 5H), 1.47−1.40 (m, 4H), 1.38 (s, 3H), 1.36 (s, 3H), 1.28−

1.17 (m, 8H), 0.93 (s, 9H); UPLC-MS (ESI⁺): calculated for

C₅₁H₇₁ClN₁₁O₆S₂[M + 1]⁺, 1032.47; found, 1032.42.

N1-(3-((3-Amino-5-(4-amino-4-methylpiperidin-1-yl)pyrazin-2-

yl)thio)-2-chlorophenyl)-N3-(10-(((R)-1-((2R,4S)-4-hydroxy-2-(((R)-

1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)-pyrrolidin-1-

yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-10-oxodecyl)-

malonamide (14). This compound was prepared from 43h and 46a

1

a procedure similar to that used for compound 7. H NMR (400

1

by a procedure similar to that used for compound 7. H NMR (400

MHz, DMSO-d₆): δ (ppm) 9.54 (s, 1H), 8.98 (s, 1H), 8.36 (d, J = 8.0

Hz, 1H), 7.95 (brs, 3H), 7.84−7.75 (m, 2H), 7.66 (s, 1H), 7.48−7.37

(m, 5H), 7.14 (t, J = 8.0 Hz, 1H), 6.43 (dd, J = 8.0 Hz, J = 1.2 Hz,

1H), 6.18 (brs, 2H), 4.95−4.88 (m, 1H), 4.51 (d, J = 8.8 Hz, 1H),

4.42 (t, J = 8.0 Hz, 1H), 4.28 (s, 1H), 4.05−4.02 (m, 2H), 3.63−3.57

(m, 2H), 3.33−3.26 (m, 2H), 3.05−3.00 (m, 2H), 2.63−2.59 (m,

2H), 2.45 (s, 3H), 2.41−2.37 (m, 2H), 2.28−2.21 (m, 1H), 2.13−

1.98 (m, 2H), 1.82−1.70 (m, 5H), 1.52−1.41 (m, 4H), 1.37 (s, 3H),

1.36 (s, 3H), 1.27−1.16 (m, 14H), 0.93 (s, 9H); UPLC-MS (ESI⁺):

calculated for C₅₅H₇₉ClN₁₁O₆S₂[M + 1]⁺, 1088.53; found, 1088.49.

N1-(3-((3-Amino-5-(4-amino-4-methylpiperidin-1-yl)pyrazin-2-

yl)thio)-2-chlorophenyl)-N5-(12-(((R)-1-((2R,4S)-4-hydroxy-2-(((R)-

1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)-pyrrolidin-1-

yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-12-oxododecyl)-

glutaramide (19). This compound was prepared from 43j and 46c by

MHz, DMSO-d₆): δ (ppm) 10.33 (s, 1H), 9.00 (s, 1H), 8.39 (d, J =

8.0 Hz, 1H), 8.26−8.23 (m, 1H), 7.98 (brs, 3H), 7.83−7.78 (m, 2H),

7.67 (s, 1H), 7.47−7.33 (m, 4H), 7.15 (t, J = 8.0 Hz, 1H), 6.41 (dd, J

= 8.0 Hz, J = 1.2 Hz, 1H), 6.21 (brs, 1H), 4.96−4.88 (m, 1H), 4.52

(d, J = 8.8 Hz, 1H), 4.42 (t, J = 8.0 Hz, 1H), 4.28 (s, 1H), 4.06−4.03

(m, 2H), 3.64−3.58 (m, 2H), 3.39 (s, 2H), 3.34−3.27 (m, 2H),

3.13−3.08 (m, 2H), 2.46 (s, 3H), 2.29−2.22 (m, 1H), 2.14−1.99 (m,

2H), 1.82−1.71 (m, 5H), 1.51−1.39 (m, 4H), 1.38 (s, 3H), 1.36 (s,

3H), 1.27−1.19 (m, 10H), 0.93 (s, 9H); UPLC-MS (ESI⁺):

calculated for C₅₂H₇₃ClN₁₁O₆S₂[M + 1]⁺, 1046.49; found, 1046.45.

N1-(3-((3-Amino-5-(4-amino-4-methylpiperidin-1-yl)pyrazin-2-

yl)thio)-2-chlorophenyl)-N3-(11-(((R)-1-((2R,4S)-4-hydroxy-2-(((R)-

1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)-pyrrolidin-1-

yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-11-oxoundecyl)-

malonamide (15). This compound was prepared from 43i and 46a

1

a procedure similar to that used for compound 7. H NMR (400

1

by a procedure similar to that used for compound 7. H NMR (400

MHz, DMSO-d₆): δ (ppm) 9.52 (s, 1H), 8.99 (s, 1H), 8.38 (d, J = 8.0

Hz, 1H), 7.96 (brs, 3H), 7.80−7.75 (m, 2H), 7.67 (s, 1H), 7.45−7.37

(m, 4H), 7.15 (t, J = 8.0 Hz, 1H), 6.43 (dd, J = 8.0 Hz, J = 1.2 Hz,

1H), 6.19 (brs, 2H), 4.95−4.89 (m, 1H), 4.51 (d, J = 8.8 Hz, 1H),

4.41 (t, J = 8.0 Hz, 1H), 4.27 (s, 1H), 4.06−4.02 (m, 2H), 3.64−3.57

(m, 2H), 3.33−3.26 (m, 2H), 3.04−2.99 (m, 2H), 2.45 (s, 3H),

2.39−2.35 (m, 2H), 2.28−2.21 (m, 1H), 2.14−1.98 (m, 4H), 1.82−

1.70 (m, 6H), 1.47−1.41 (m, 4H), 1.37 (s, 3H), 1.36 (s, 3H), 1.27−

1.18 (m, 16H), 0.93 (s, 9H); UPLC-MS (ESI⁺): calculated for

C₅₆H₈₁ClN₁₁O₆S₂[M + 1]⁺, 1102.55; found, 1102.48.

MHz, DMSO-d₆): δ (ppm) 10.31 (s, 1H), 8.99 (s, 1H), 8.36 (d, J =

8.0 Hz, 1H), 8.24−8.22 (m, 1H), 7.95 (brs, 3H), 7.82−7.75 (m, 2H),

7.66 (s, 1H), 7.44−7.37 (m, 4H), 7.16 (t, J = 8.0 Hz, 1H), 6.41 (dd, J

= 8.0 Hz, J = 1.2 Hz, 1H), 6.18 (brs, 2H), 4.95−4.88 (m, 1H), 4.51

(d, J = 8.8 Hz, 1H), 4.42 (t, J = 8.0 Hz, 1H), 4.28 (s, 1H), 4.05−4.02

(m, 2H), 3.64−3.57 (m, 2H), 3.38 (s, 2H), 3.33−3.26 (m, 2H),

3.12−2.97 (m, 2H), 2.45 (s, 3H), 2.28−2.21 (m, 1H), 2.13−1.98 (m,

2H), 1.82−1.70 (m, 5H), 1.50−1.40 (m, 4H), 1.38 (s, 3H), 1.36 (s,

3H), 1.29−1.15 (m, 12H), 0.93 (s, 9H); UPLC-MS (ESI⁺):

calculated for C₅₃H₇₅ClN₁₁O₆S₂[M + 1]⁺, 1060.50; found, 1060.51.

N1-(3-((3-Amino-5-(4-amino-4-methylpiperidin-1-yl)pyrazin-2-

yl)thio)-2-chlorophenyl)-N3-(12-(((R)-1-((2R,4S)-4-hydroxy-2-(((R)-

1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)-pyrrolidin-1-

yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-12-oxododecyl)-

malonamide (16). This compound was prepared from 43j and 46a

N1-(3-((3-Amino-5-(4-amino-4-methylpiperidin-1-yl)pyrazin-2-

yl)thio)-2-chlorophenyl)-N4-((R)-13-((2R,4S)-4-hydroxy-2-(((R)-1-(4-

(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine-1-car-

bonyl)-14,14-dimethyl-11-oxo-3,6,9-trioxa-12-azapentadecyl)-

succinamide (20). This compound was prepared from 48 and 46b by

1

a procedure similar to that used for compound 7. H NMR (400

1

by a procedure similar to that used for compound 7. H NMR (400

MHz, DMSO-d₆): δ (ppm) 10.29 (s, 1H), 9.00 (s, 1H), 8.45 (d, J =

8.0 Hz, 1H), 8.38 (t, J = 5.2 Hz, 1H), 8.07 (brs, 3H), 7.80 (dd, J = 8.0

Hz, J = 1.6 Hz, 1H), 7.66 (s, 1H), 7.45−7.33 (m, 5H), 7.15 (t, J = 8.0

Hz, 1H), 6.40 (dd, J = 8.0 Hz, J = 1.2 Hz, 1H), 6.21 (brs, 1H), 4.94−

4.87 (m, 1H), 4.54 (d, J = 8.8 Hz, 1H), 4.44 (t, J = 8.0 Hz, 1H), 4.28

(s, 1H), 4.05−3.92 (m, 4H), 3.63−3.54 (m, 10H), 3.48−3.42 (m,

4H), 3.35−3.27 (m, 4H), 2.45 (s, 3H), 2.39−2.35 (m, 2H),2.08−1.99

MHz, DMSO-d₆): δ (ppm) 10.30 (s, 1H), 8.98 (s, 1H), 8.36 (d, J =

8.0 Hz, 1H), 8.24−8.21 (m, 1H), 7.96 (brs, 3H), 7.83−7.75 (m, 2H),

7.66 (s, 1H), 7.44−7.37 (m, 4H), 7.16 (t, J = 8.0 Hz, 1H), 6.41 (dd, J

= 8.0 Hz, J = 1.2 Hz, 1H), 6.15 (brs, 2H), 4.95−4.88 (m, 1H), 4.51

(d, J = 8.8 Hz, 1H), 4.42 (t, J = 8.0 Hz, 1H), 4.28 (s, 1H), 4.05−4.02

(m, 2H), 3.64−3.57 (m, 2H), 3.38 (s, 2H), 3.33−3.26 (m, 2H),

K

https://dx.doi.org/10.1021/acs.jmedchem.0c00471

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

1

(m, 1H), 1.80−1.69 (m, 5H), 1.38 (s, 3H), 1.36 (s, 3H), 0.94 (s,

9H); UPLC-MS (ESI⁺): calculated for C₅₁H₇₁ClN₁₁O₉S₂[M + 1]⁺,

1080.46; found, 1080.21.

used for compound 7. H NMR (400 MHz, DMSO-d₆): δ (ppm)

9.49 (s, 1H), 8.99 (s, 1H), 8.38 (d, J = 8.0 Hz, 1H), 7.95 (brs, 3H),

7.81−7.78 (m, 2H), 7.66 (s, 1H), 7.45−7.34 (m, 4H), 7.15 (t, J = 8.0

Hz, 1H), 6.42 (dd, J = 8.0 Hz, J = 1.2 Hz, 1H), 6.18 (brs, 2H), 4.95−

4.88 (m, 1H), 4.51 (d, J = 8.8 Hz, 1H), 4.42 (t, J = 8.0 Hz, 1H), 4.27

(s, 1H), 4.05−4.02 (m, 2H), 3.63−3.57 (m, 2H), 3.33−3.26 (m, 2H),

2.45 (s, 3H), 2.38−2.34 (m, 2H), 2.28−2.17 (m, 1H), 2.16−1.98 (m,

2H), 1.80−1.68 (m, 5H), 1.60−1.56 (m, 2H), 1.53−1.41 (m, 2H),

1.38 (s, 3H), 1.36 (s, 3H), 1.34−1.18 (m, 20H), 0.93 (s, 9H); UPLC-

MS (ESI⁺): calculated for C₅₅H₈₀ClN₁₀O₅S₂[M + 1]⁺, 1059.54;

found, 1059.51.

N1-(3-((3-Amino-5-(4-amino-4-methylpiperidin-1-yl)pyrazin-2-

yl)thio)-2-chlorophenyl)-N4-((R)-14-((2R,4S)-4-hydroxy-2-(((R)-1-(4-

(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine-1-car-

bonyl)-15,15-dimethyl-12-oxo-3,6,9-trioxa-13-azahexadecyl)-

succinamide (21). This compound was prepared from 50a and 46b

1

by a procedure similar to that used for compound 7. H NMR (400

MHz, DMSO-d₆): δ (ppm) 10.28 (s, 1H), 9.00 (s, 1H), 8.40−8.36

(m, 2H), 8.06 (brs, 3H), 7.84 (d, J = 8.8 Hz, 1H), 7.80 (dd, J = 8.0

Hz, J = 1.6 Hz, 1H), 7.66 (s, 1H), 7.45−7.33 (m, 4H), 7.16 (t, J = 8.0

Hz, 1H), 6.40 (dd, J = 8.0 Hz, J = 1.2 Hz, 1H), 6.19 (brs, 1H), 4.95−

4.88 (m, 1H), 4.52 (d, J = 8.8 Hz, 1H), 4.42 (t, J = 8.0 Hz, 1H), 4.27

(s, 1H), 4.05−4.00 (m, 2H), 3.63−3.55 (m, 4H), 3.51−3.42 (m,

12H), 3.34−3.26 (m, 4H), 2.57−2.52 (m, 1H), 2.45 (s, 3H), 2.40−

2.32 (m, 3H), 2.07−1.99 (m, 1H), 1.80−1.69 (m, 5H), 1.38 (s, 3H),

1.36 (s, 3H), 0.94 (s, 9H); UPLC-MS (ESI⁺): calculated for

C₅₂H₇₃ClN₁₁O₉S₂[M + 1]⁺, 1094.47; found, 1094.35.

(2R,4S)-1-((R)-2-(9-(4-(4-((3-((3-Amino-5-(4-amino-4-methylpi-

peridin-1-yl)pyrazin-2-yl)thio)-2-chlorophenyl)amino)-4-

oxobutanoyl)piperazin-1-yl)nonanamido)-3,3-dimethylbutanoyl)-

4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)-

pyrrolidine-2-carboxamide (26). This compound was prepared from

60a and 46b by a procedure similar to that used for compound 7. ¹H

NMR (400 MHz, DMSO-d₆): δ (ppm) 9.67 (s, 1H), 9.57 (s, 1H),

8.99 (s, 1H), 8.38 (d, J = 8.0 Hz, 1H), 7.97 (brs, 3H), 7.79 (d, J = 8.8

Hz, 1H), 7.66 (s, 1H), 7.47−7.35 (m, 4H), 7.15 (t, J = 8.0 Hz, 1H),

6.41 (dd, J = 8.0 Hz, J = 1.2 Hz, 1H), 6.19 (brs, 1H), 4.95−4.89 (m,

1H), 4.51 (d, J = 8.8 Hz, 1H), 4.45−4.39 (m, 2H), 4.28 (s, 1H),

4.14−4.02 (m, 3H), 3.63−3.56 (m, 2H), 3.50−3.45 (m, 2H), 3.40−

3.26 (m, 3H), 3.10−3.05 (m, 3H), 2.96−2.83 (m, 2H), 2.72−2.63

(m, 4H), 2.45 (s, 3H), 2.29−2.20 (m, 1H), 2.13−1.99 (m, 2H),

1.82−1.70 (m, 5H), 1.69−1.58 (m, 2H), 1.53−1.42 (m, 2H), 1.38 (s,

3H), 1.36 (s, 3H), 1.30−1.19 (m, 8H), 0.93 (s, 9H); ¹³C NMR (100

MHz, DMSO-d₆): δ (ppm) 171.97, 170.87, 170.59, 170.18, 169.57,

158.28, 157.94, 155.78, 153.53, 151.51, 147.74, 144.63, 137.30,

135.95, 131.10, 129.69, 128.82, 126.95, 126.38, 122.27, 120.32,

117.80, 114.86, 113.77, 68.75, 58.54, 56.31, 55.55, 52.09, 50.92, 50.61,

47.69, 41.72, 38.15, 37.76, 35.23, 34.88, 34.12, 28.57, 28.51, 28.37,

27.30, 26.45, 25.90, 25.37, 23.13, 22.42, 21.98, 15.98; UPLC-MS

(ESI⁺): calculated for C₅₆H₈₀ClN₁₂O₆S₂[M + 1]⁺, 1115.54; found,

1115.29.

N1-(3-((3-Amino-5-(4-amino-4-methylpiperidin-1-yl)pyrazin-2-

yl)thio)-2-chlorophenyl)-N4-((R)-16-((2R,4S)-4-hydroxy-2-(((R)-1-(4-

(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidine-1-car-

bonyl)-17,17-dimethyl-14-oxo-3,6,9,12-tetraoxa-15-azaoctadecyl)-

succinamide (22). This compound was prepared from 50b and 46b

1

by a procedure similar to that used for compound 7. H NMR (400

MHz, DMSO-d₆): δ (ppm) 10.29 (s, 1H), 9.00 (s, 1H), 8.45 (d, J =

8.0 Hz, 1H), 8.38−8.36 (m, 1H), 7.96 (brs, 3H), 7.80 (dd, J = 8.0 Hz,

J = 1.6 Hz, 1H), 7.66 (s, 1H), 7.46−7.33 (m, 5H), 7.16 (t, J = 8.0 Hz,

1H), 6.39 (dd, J = 8.0 Hz, J = 1.2 Hz, 1H), 6.21 (brs, 1H), 4.94−4.87

(m, 1H), 4.54 (d, J = 8.8 Hz, 1H), 4.43 (t, J = 8.0 Hz, 1H), 4.28 (s,

1H), 4.05−4.02 (m, 2H), 3.96 (s, 2H), 3.62−3.53 (m, 14H), 3.47−

3.42 (m, 4H), 3.33−3.26 (m, 4H), 2.45 (s, 3H), 2.38−2.33 (m, 2H),

2.07−2.02 (m, 1H), 1.87−1.70 (m, 5H), 1.38 (s, 3H), 1.36 (s, 3H),

0.94 (s, 9H); UPLC-MS (ESI⁺): calculated for C₅₃H₇₅ClN₁₁O₁₀S₂[M

+ 1]⁺, 1124.48; found, 1124.35.

(2R,4S)-1-((R)-2-(11-(4-(2-((3-((3-Amino-5-(4-amino-4-methylpi-

peridin-1-yl)pyrazin-2-yl)thio)-2-chlorophenyl)amino)-2-oxoethyl)-

piperazin-1-yl)undecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-

N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-car-

boxamide (23). This compound was prepared from 54a and 6 by a

procedure similar to that used for compound 7. ¹H NMR (400 MHz,

DMSO-d₆): δ (ppm) 9.79 (s, 1H), 8.99 (s, 1H), 8.38 (d, J = 8.0 Hz,

1H), 7.97 (brs, 3H), 7.83−7.78(m, 2H), 7.66 (s, 1H), 7.44−7.36 (m,

4H), 7.19 (t, J = 8.0 Hz, 1H), 6.42 (dd, J = 8.0 Hz, J = 1.2 Hz, 1H),

6.18 (brs, 1H), 4.93−4.89 (m, 1H), 4.51 (d, J = 8.8 Hz, 1H), 4.41 (t, J

= 8.0 Hz, 1H), 4.28 (s, 1H), 4.05−4.02 (m, 2H), 3.61−3.51 (m, 4H),

3.36−3.26 (m, 4H), 3.15−2.99 (m, 6H), 2.73−2.61 (m, 2H), 2.45 (s,

3H), 2.33−2.22 (m, 2H), 2.13−1.98 (m, 3H), 1.82−1.63 (m, 7H),

1.49−1.45 (m, 2H), 1.38 (s, 3H), 1.36 (s, 3H), 1.32−1.21 (m, 10H),

0.93 (s, 9H); UPLC-MS (ESI⁺): calculated for C₅₆H₈₂ClN₁₂O₅S₂[M

+ 1]⁺, 1101.57; found, 1101.62.

(2R,4S)-1-((R)-2-(12-(4-(2-((3-((3-Amino-5-(4-amino-4-methylpi-

peridin-1-yl)pyrazin-2-yl)thio)-2-chlorophenyl)amino)-2-oxoethyl)-

piperazin-1-yl)dodecanamido)-3,3-dimethylbutanoyl)-4-hydroxy-

N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-car-

boxamide (24). This compound was prepared from 54b and 6 by a

procedure similar to that used for compound 7. ¹H NMR (400 MHz,

DMSO-d₆): δ (ppm) 9.80 (s, 1H), 8.99 (s, 1H), 8.37 (d, J = 8.0 Hz,

1H), 7.97 (brs, 3H), 7.83−7.78(m, 2H), 7.66 (s, 1H), 7.45−7.36 (m,

4H), 7.20 (t, J = 8.0 Hz, 1H), 6.42 (dd, J = 8.0 Hz, J = 1.2 Hz, 1H),

6.18 (brs, 1H), 4.95−4.87 (m, 1H), 4.51 (d, J = 8.8 Hz, 1H), 4.41 (t, J

= 8.0 Hz, 1H), 4.28 (s, 1H), 4.05−4.02 (m, 2H), 3.63−3.52 (m, 4H),

3.38−3.26 (m, 4H), 3.15−2.99 (m, 6H), 2.73−2.63 (m, 2H), 2.45 (s,

3H), 2.29−2.22 (m, 2H), 2.13−1.98 (m, 3H), 1.82−1.63 (m, 7H),

1.52−1.43 (m, 2H),1.38 (s, 3H), 1.36 (s, 3H), 1.32−1.21 (m, 12H),

0.93 (s, 9H); UPLC-MS (ESI⁺): calculated for C₅₇H₈₄ClN₁₂O₅S₂[M

+ 1]⁺, 1115.58; found, 1115.53.

(2R,4S)-1-((R)-2-(10-(4-(4-((3-((3-Amino-5-(4-amino-4-methylpi-

peridin-1-yl)pyrazin-2-yl)thio)-2-chlorophenyl)amino)-4-

oxobutanoyl)piperazin-1-yl)decanamido)-3,3-dimethylbutanoyl)-

4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)-

pyrrolidine-2-carboxamide (27). This compound was prepared from

60b and 46b by a procedure similar to that used for compound 7. ¹H

NMR (400 MHz, DMSO-d₆): δ (ppm) 9.73 (s, 1H), 9.57 (s, 1H),

8.99 (s, 1H), 8.38 (d, J = 8.0 Hz, 1H), 7.99 (brs, 3H), 7.79 (d, J = 8.8

Hz, 1H), 7.66 (s, 1H), 7.47−7.35 (m, 4H), 7.14 (t, J = 8.0 Hz, 1H),

6.42 (dd, J = 8.0 Hz, J = 1.2 Hz, 1H), 6.19 (brs, 1H), 4.95−4.88 (m,

1H), 4.51 (d, J = 8.8 Hz, 1H), 4.46−4.39 (m, 2H), 4.28 (s, 1H),

4.14−4.02 (m, 3H), 3.63−3.56 (m, 2H), 3.50−3.45 (m, 2H), 3.37−

3.26 (m, 3H), 3.10−3.03 (m, 3H), 2.97−2.84 (m, 2H), 2.72−2.61

(m, 4H), 2.45 (s, 3H), 2.29−2.21 (m, 1H), 2.13−1.99 (m, 2H),

1.82−1.70 (m, 5H), 1.69−1.57 (m, 2H), 1.52−1.41 (m, 2H), 1.38 (s,

3H), 1.36 (s, 3H), 1.31−1.20 (m, 10H), 0.93 (s, 9H); UPLC-MS

(ESI⁺): calculated for C₅₇H₈₂ClN₁₂O₆S₂[M + 1]⁺, 1129.56; found,

1129.38.

N1-(3-((3-Amino-5-(4-amino-4-methylpiperidin-1-yl)pyrazin-2-

yl)thio)-2-chlorophenyl)-N4-(4-((8-(((R)-1-((2R,4S)-4-hydroxy-2-

(((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)-pyrroli-

din-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-8-oxooctyl)oxy)-

phenyl)succinimide (28). This compound was prepared from 64 and

1

46b by a procedure similar to that used for compound 7. H NMR

(400 MHz, DMSO-d₆): δ (ppm) 9.84 (s, 1H), 9.60 (s, 1H), 8.99 (s,

1H), 8.38 (d, J = 8.0 Hz, 1H), 7.95 (brs, 3H), 7.80 (d, J = 8.8 Hz,

1H), 7.66 (s, 1H), 7.49−7.34 (m, 7H), 7.14 (t, J = 8.0 Hz, 1H),

6.87−6.82 (m, 2H), 6.42 (dd, J = 8.0 Hz, J = 1.2 Hz, 1H), 6.19 (brs,

1H), 4.95−4.88 (m, 1H), 4.52 (d, J = 8.8 Hz, 1H), 4.45−4.40 (m,

1H), 4.27 (s, 1H), 4.05−4.02 (m, 2H), 3.91−3.88 (m, 2H), 3.63−

3.57 (m, 2H), 3.33−3.26 (m, 2H), 3.17−3.08 (m, 1H), 2.73−2.69

(m, 2H), 2.63−2.59 (m, 2H), 2.45 (s, 3H), 2.29−2.22 (m, 1H),

2.14−1.98 (m, 2H), 1.82−1.64 (m, 6H), 1.54−1.44 (m, 2H), 1.52−

1.41 (m, 2H), 1.38 (s, 3H), 1.36 (s, 3H), 1.31−1.15 (m, 6H), 0.93 (s,

9H); UPLC-MS (ESI⁺): calculated for C₅₇H₇₅ClN₁₁O₇S₂[M + 1]⁺,

1124.50; found, 1124.56.

N1-(3-((3-Amino-5-(4-amino-4-methylpiperidin-1-yl)pyrazin-2-

yl)thio)-2-chlorophenyl)-N16-((R)-1-((2R,4S)-4-hydroxy-2-(((R)-1-(4-

(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)-pyrrolidin-1-yl)-3,3-

dimethyl-1-oxobutan-2-yl)hexadecanediamide (25). This com-

pound was prepared from 56 and 6 by a procedure similar to that

L

https://dx.doi.org/10.1021/acs.jmedchem.0c00471

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

N1-(3-((3-Amino-5-(4-amino-4-methylpiperidin-1-yl)pyrazin-2-

yl)thio)-2-chlorophenyl)-N4-(4-(4-(5-(((R)-1-((2R,4S)-4-hydroxy-2-

(((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)carbamoyl)-pyrroli-

din-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-5-oxopentyl)-

piperazin-1-yl)butyl)-succinamide (29). This compound was pre-

pared from 68 and 46b by a procedure similar to that used for

compound 7. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 9.56 (s, 1H),

8.99 (s, 1H), 8.35 (d, J = 8.0 Hz, 1H), 8.02 (brs, 3H), 7.94−7.91 (m,

1H), 7.86 (d, J = 8.4 Hz, 1H), 7.80 (d, J = 8.8 Hz, 1H), 7.66 (s, 1H),

7.46−7.36 (m, 5H), 7.14 (t, J = 8.0 Hz, 1H), 6.87−6.82 (m, 2H),

6.43 (dd, J = 8.0 Hz, J = 1.2 Hz, 1H), 6.17 (brs, 2H), 4.95−4.87 (m,

1H), 4.52 (d, J = 8.8 Hz, 1H), 4.43−4.37 (m, 1H), 4.23 (s, 1H),

4.05−4.01 (m, 2H), 3.67−3.56 (m, 3H), 3.47−3.27 (m, 5H), 3.10−

3.05 (m, 8H), 2.65−2.61 (m, 2H), 2.45 (s, 3H), 2.42−2.39 (m, 2H),

2.30−2.27 (m, 1H), 2.22−2.15 (m, 1H), 2.07−1.99 (m, 1H), 1.83−

1.71 (m, 5H), 1.63−1.49 (m, 6H), 1.48−1.40 (m, 2H), 1.38 (s, 3H),

1.36 (s, 3H), 0.94 (s, 9H); UPLC-MS (ESI⁺): calculated for

C₅₆H₈₁ClN₁₃O₆S₂[M + 1]⁺, 1124.50; found, 1124.47.

3-(tert-Butylthio)-2-chloroaniline (36). Cesium carbonate (84.0 g,

257.8 mmol, 2.5 equiv) was added at rt to a solution of 2-chloro-3-

ﬂuoroaniline (15.0 g, 103.1 mmol) and tert-butylthiol (41.0 mL, 32.8

g, 360.9 mmol, 3.5 equiv) in anhydrous DMF (160 mL). The reaction

mixture was heated to 120 °C and stirred for 36 h under N₂. After

cooling, the reaction mixture was diluted with EtOAc (300 mL),

washed with H₂O, brine, dried over Na₂SO₄, and evaporated in vacuo

to aﬀord crude 3-(tert-butylthio)-2-chloroaniline (19.8 g, 91.8 mmol)

which was used directly in the next step. UPLC-MS (ESI⁺): calculated

for C₁₀H₁₅ClNS [M + 1]⁺, 216.06; found, 216.10.

= 1.6 Hz, 1H), 5.41 (s, 2H), 3.85−3.81 (m, 2H), 3.23−3.18 (m, 2H),

2.09−2.05 (m, 2H), 1.46−1.41 (m, 2H), 1.39 (s, 9H), 1.25 (s, 3H);

+

C₂₁H₃₀ClN₆O₂S [M + 1] , 465.18; found, 465.23.

(2S,4R)-1-((S)-2-(3-Aminopropanamido)-3,3-dimethylbutanoyl)-

4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)-

pyrrolidine-2-carboxamide Hydrochloride (43a). HATU (295 mg,

0.66 mmol, 1.1 equiv) was added to a solution of 6 (335 mg, 0. 60

mmol, 1 equiv), 41a (125 mg, 0.66 mmol, 1.1 equiv), and DIEA (0.42

mL, 2.40 mmol, 4.0 equiv) in DMF (6 mL), and the resulting mixture

was stirred at rt for 1 h. The solution was diluted with EtOAc and

washed with H₂O, saturated sodium bicarbonate aqueous solution,

and brine and then dried over sodium sulfate. After removal of the

solvent in vacuo, the residue was puriﬁed by HPLC and treated with 4

N HCl dioxane for 2 h to aﬀord 43a as a hydrochloride salt (235 mg

1

71%). H NMR (400 MHz, DMSO-d₆): δ (ppm) 9.04 (s, 1H), 8.43

(d, J = 8.0 Hz, 1H), 8.23 (d, J = 8.8 Hz, 1H), 7.90 (s, 3H), 7.45−7.37

(m, 4H), 4.95−4.88 (m, 1H), 4.52 (d, J = 8.8 Hz, 1H), 4.44−4.40 (m,

1H), 3.65−3.59 (m, 1H), 2.98−2.93 (m, 2H), 2.63−2.58 (m, 2H),

2.46 (s, 3H), 2.07−1.99 (m, 1H), 1.82−1.76 (m, 1H), 1.37 (d, J = 7.2

Hz, 3H), 1.30−1.26 (m, 1H), 0.95 (s, 9H); UPLC-MS (ESI⁺):

calculated for C₂₆H₃₈N₅O₄S [M + 1]⁺, 516.26; found, 516.19

(2S,4R)-1-((S)-2-(4-Aminobutanamido)-3,3-dimethylbutanoyl)-

4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)-

pyrrolidine-2-carboxamide Hydrochloride (43b). This compound

was prepared using a procedure similar to that used for compound

43a. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 9.09 (s, 1H), 8.43 (d,

J = 8.0 Hz, 1H), 8.01 (s, 3H),7.95 (d, J = 8.8 Hz, 1H), 7.46−7.37 (m,

4H), 4.95−4.88 (m, 1H), 4.50 (d, J = 8.8 Hz, 1H), 4.45−4.41 (m,

1H), 4.32−4.26 (m, 1H), 3.63−3.59 (m, 1H), 2.79−2.71 (m, 2H),

2.63−2.58 (m, 2H), 2.46 (s, 3H), 2.30−2.21 (m, 2H), 2.05−1.99 (m,

1H), 1.82−1.73 (m, 3H), 1.37 (d, J = 7.2 Hz, 3H), 1.31−1.26 (m,

1H), 0.94 (s, 9H); UPLC-MS (ESI⁺): calculated for C₂₇H₄₀N₅O₄S

[M + 1]⁺, 530.28; found, 530.19.

(2S,4R)-1-((S)-2-(5-Aminopentanamido)-3,3-dimethylbutanoyl)-

4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)-

pyrrolidine-2-carboxamide Hydrochloride (43c). This compound

was prepared using a procedure similar to that used for compound

43a. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 9.17 (s, 1H), 8.45 (d,

J = 8.0 Hz, 1H), 8.02 (brs, 3H), 7.87 (d, J = 8.8 Hz, 1H), 7.49−7.35

(m, 4H), 4.93−4.89 (m, 1H), 4.50 (d, J = 8.8 Hz, 1H), 4.44−4.39 (m,

1H), 4.28−4.26 (m, 1H), 3.65−3.59 (m, 1H), 2.80−2.68 (m, 2H),

2.47 (s, 3H), 2.32−2.17 (m, 2H), 2.09−1.97 (m, 1H), 1.82−1.75 (m,

1H), 1.59−1.48 (m, 4H), 1.43−1.41 (m, 1H), 1.37 (d, J = 7.2 Hz,

3H), 0.93 (s, 9H); UPLC-MS (ESI⁺): calculated for C₂₈H₄₂N₅O₄S

[M + 1]⁺, 544.30; found, 544.25.

(2R,4S)-1-((R)-2-(6-Aminohexanamido)-3,3-dimethylbutanoyl)-

4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)-

pyrrolidine-2-carboxamide Hydrochloride (43d). This compound

was prepared using a procedure similar to that used for compound

43a. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 9.07 (s, 1H), 8.42 (d,

J = 8.0 Hz, 1H), 7.93 (brs, 3H), 7.83 (d, J = 8.8 Hz, 1H), 7.53−7.35

(m, 4H), 4.95−4.89 (m, 1H), 4.51 (d, J = 8.8 Hz, 1H), 4.44−4.40 (m,

1H), 4.29−4.27 (m, 1H), 3.65−3.59 (m, 1H), 2.76−2.70 (m, 2H),

2.47 (s, 3H), 2.28−2.09 (m, 2H), 2.07−1.99 (m, 1H), 1.82−1.75 (m,

1H), 1.59−1.43 (m, 5H), 1.37 (d, J = 7.2 Hz, 3H), 1.30−1.22 (m,

2H), 0.93 (s, 9H); UPLC-MS (ESI⁺): calculated for C₂₉H₄₄N₅O₄S

[M + 1]⁺, 558.31; found, 558.27

(2R,4S)-1-((R)-2-(7-Aminoheptanamido)-3,3-dimethylbutanoyl)-

4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)-

pyrrolidine-2-carboxamide Hydrochloride (43e). This compound

was prepared using a procedure similar to that used for compound

43a. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 9.09 (s, 1H), 8.45 (d,

J = 8.0 Hz, 1H), 7.96 (brs, 3H), 7.81 (d, J = 8.8 Hz, 1H), 7.49−7.34

(m, 4H), 4.95−4.88 (m, 1H), 4.52 (d, J = 8.8 Hz, 1H), 4.44−4.40 (m,

1H), 4.30−4.26 (m, 1H), 3.61−3.56 (m, 1H), 2.75−2.70 (m, 2H),

2.46 (s, 3H), 2.28−2.09 (m, 2H), 2.05−2.00 (m, 1H), 1.81−1.74 (m,

1H), 1.54−1.42 (m, 5H), 1.37 (d, J = 7.2 Hz, 3H), 1.31−1.21 (m,

4H), 0.93 (s, 9H); UPLC-MS (ESI⁺): calculated for C₃₀H₄₆N₅O₄S

[M + 1]⁺, 572.33; found, 572.31.

3-Amino-2-chlorobenzenethiol Hydrochloride (37). 3-(tert-Bu-

tylthio)-2-chloroaniline (19.5 g, 90.4 mmol) was suspended in conc.

HCl (170 mL), and the reaction mixture was vigorously stirred for 6 h

at 80 °C. After cooling down, the suspension was ﬁltered, the white

solids were washed with cold conc. HCl (15 mL) and hexane (30

mL), and dried under reduced pressure to give 3-amino-2-

chlorobenzenethiol hydrochloride (13.8 g, 78%). UPLC-MS (ESI⁺):

calculated for C₆H₇ClNS [M + 1]⁺, 160.00; found, 160.10.

3-((3-Amino-2-chlorophenyl)thio)-6-chloropyrazin-2-amine

(39). Potassium phosphate (K₃PO₄, 20.7 g, 97.6 mmol, 2.6 equiv) was

added to a solution of 3-bromo-6-chloropyrazin-2-amine (7.8 g, 37.5

mmol, 1.0 equiv) and 3-amino-2-chlorobenzenethiol hydrochloride

(9.6 g, 48.8 mmol, 1.3 equiv) in dioxane (120 mL). The reaction

mixture was degassed and stirred at rt for 15 min, then CuI (1.4 g, 7.5

mmol, 0.2 equiv) and 1,10-phenanthroline (2.7 g, 15.0 mmol, 0.4

equiv) were added. After being degassed three times, the mixture was

stirred at 90 °C under dry N₂for 16 h. The reaction mixture was

cooled to rt, diluted with EtOAc (150 mL), and ﬁltered through a pad

of Celite followed by an EtOAc wash. The volatiles were removed

under reduced pressure, and the residue was puriﬁed by silica gel

chromatography eluting with 0−10% MeOH/DCM to aﬀord 3-((3-

amino-2-chlorophenyl)thio)-6-chloropyrazin-2-amine (6.6 g, 61%) as

1

a yellow solid. H NMR (400 MHz, DMSO-d₆): δ (ppm) 7.70 (s,

1H), 7.01−6.97 (m, 3H), 6.79 (dd, J = 8.0 Hz, J = 1.2 Hz, 1H), 6.49

(dd, J = 7.6 Hz, J = 1.6 Hz, 1H), 5.51 (s, 2H); UPLC-MS (ESI⁺):

calculated for C₁₀H₉Cl₂N₄S [M + 1]⁺, 286.99; found, 287.05.

tert-Butyl (1-(6-amino-5-((3-amino-2-chlorophenyl)thio)pyrazin-

2-yl)-4-methylpiperidin-4-yl (40). DIPEA (6.5 mL, 4.9 g, 37.5 mmol,

3.0 equiv) (1.0 mL) was added at rt to a solution of 3-((3-amino-2-

chlorophenyl)thio)-6-chloropyrazin-2-amine (3.6 g, 12.5 mmol, 1.0

equiv) and tert-butyl (4-methylpiperidin-4-yl)carbamate (5.4 g, 25.0

mmol, 2.0 equiv) in DMSO (50 mL). The reaction mixture was

warmed to 100 °C and stirred for 3 h. After cooling, it was poured

onto ice-cold water (200 mL). After extraction with EtOAc (100 mL

× 2), the combined organic layers were washed with water, brine,

dried over Na₂SO₄, and evaporated under vacuum. The residue was

puriﬁed by silica gelﬂash column chromatography with hexane:EtOAc

(4:1−1:3) to aﬀord tert-butyl (1-(6-amino-5-((3-amino-2-

chlorophenyl)thio)pyrazin-2-yl)-4-methylpiperidin-4-yl as a white

1

solid (4.4 g, 76% yield). H NMR (400 MHz, DMSO-d₆): δ (ppm)

7.59 (s, 1H), 6.83 (t, J = 8.0 Hz, J = 1.2 Hz, 1H), 6.62 (s, 1H), 6.55

(dd, J = 8.0 Hz, J = 1.2 Hz, 1H), 5.99 (s, 2H); 5.81 (dd, J = 8.0 Hz, J

(2R,4S)-1-((R)-2-(8-Aminooctanamido)-3,3-dimethylbutanoyl)-4-

hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-

M

https://dx.doi.org/10.1021/acs.jmedchem.0c00471

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

2-carboxamide Hydrochloride (43f). This compound was prepared

using a procedure similar to that used for compound 43a. H NMR

3-((3-((3-Amino-5-(4-((tert-butoxycarbonyl)amino)-4-methylpi-

peridin-1-yl)pyrazin-2-yl)thio)-2-chlorophenyl)amino)-3-oxopropa-

noic Acid (46a). Lithium hydroxide monohydrate (932 mg, 22.2

mmol) was added at 0 °C to a solution of residue obtained from up

and was dissolved in THF/MeOH/H₂O (15 mL/10 mL/5 mL). The

reaction mixture was allowed to warm to rt and stirred for 2 h. After

quenching with 1 N HCl to pH ∼ 3, the resulting mixture was

extracted with EtOAc (40 mL × 2). The organic layer was washed

with brine, dried over anhydrous Na₂SO₄, and then concentrated

under reduced pressure. The residue was then puriﬁed by silica gel

column chromatography eluting with 0−5% MeOH/DCM to give 3-

((3-((3-amino-5-(4-((tert-butoxycarbonyl)amino)-4-methylpiperidin-

1-yl)pyrazin-2-yl)thio)-2-chlorophenyl)amino)-3-oxopropanoic acid

(2.5 g, 61%, two steps) as a white-yellow solid. ¹H NMR (400

MHz, DMSO-d₆): δ (ppm) 12.72 (s, 1H), 9.85 (s, 1H), 7.62 (s, 1H),

7.58 (d, J = 7.6 Hz, 1H), 7.17 (t, J = 8.0 Hz, J = 1.2 Hz, 1H), 6.63 (s,

1H), 6.42 (dd, J = 8.0 Hz, J = 1.2 Hz, 1H), 6.11 (s, 2H); 5.81 (dd, J =

8.0 Hz, J = 1.6 Hz, 1H), 3.86−3.84 (m, 2H), 3.49 (s, 2H), 3.24−3.19

(m, 2H), 2.10−2.06 (m, 2H), 1.46−1.41 (m, 2H), 1.39 (s, 9H), 1.25

(s, 3H); UPLC-MS (ESI⁺): calculated for C₂₄H₃₂ClN₆O₅S [M + 1] ⁺,

551.18; found, 551.25.

4-((3-((3-Amino-5-(4-((tert-butoxycarbonyl)amino)-4-methylpi-

peridin-1-yl)pyrazin-2-yl)thio)-2-chlorophenyl)amino)-4-oxobuta-

noic Acid (46b). This compound was prepared using a procedure

similar to that used for compound 46a using methyl 4-chloro-4-

oxobutanoate as a starting material. ¹H NMR (400 MHz, DMSO-d₆):

δ (ppm) 12.14 (s, 1H), 9.55 (s, 1H), 7.62 (s, 1H), 7.44 (d, J = 7.6 Hz,

1H), 7.14 (t, J = 8.0 Hz,), 6.63 (s, 1H), 6.42 (dd, J = 8.0 Hz, J = 1.2

Hz, 1H), 6.10 (s, 2H); 3.87−3.83 (m, 2H), 3.36 (s, 2H), 3.24−3.19

(m, 2H), 2.64−2.62 (m, 2H), 2.10−2.05 (m, 2H), 1.46−1.41 (m,

2H), 1.39 (s, 9H), 1.25 (s, 3H); UPLC-MS (ESI⁺): calculated for

C₂₅H₃₄ClN₆O₅S [M + 1]⁺, 565.20; found, 565.11.

1

(400 MHz, DMSO-d₆): δ (ppm) 9.07 (s, 1H), 8.43 (d, J = 8.0 Hz,

1H), 7.94 (brs, 3H), 7.80 (d, J = 8.8 Hz, 1H), 7.46−7.37 (m, 4H),

4.95−4.88 (m, 1H), 4.51 (d, J = 8.8 Hz, 1H), 4.44−4.40 (m, 1H),

4.29−4.25 (m, 1H), 3.61−3.56 (m, 1H), 2.76−2.71 (m, 2H), 2.46 (s,

3H), 2.28−2.08 (m, 2H), 2.04−1.99 (m, 1H), 1.81−1.75 (m, 1H),

1.53−1.42 (m, 5H), 1.37 (d, J = 7.2 Hz, 3H), 1.31−1.26 (m, 6H),

0.93 (s, 9H); UPLC-MS (ESI⁺): calculated for C₃₁H₄₈N₅O₄S [M +

1]⁺, 586.34; found, 586.37.

(2R,4S)-1-((R)-2-(9-Aminononanamido)-3,3-dimethylbutanoyl)-

4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)-

pyrrolidine-2-carboxamide Hydrochloride (43g). This compound

was prepared using a procedure similar to that used for compound

43a. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 9.10 (s, 1H), 8.42 (d,

J = 8.0 Hz, 1H), 7.95 (brs, 3H), 7.80 (d, J = 8.8 Hz, 1H), 7.49−7.36

(m, 4H), 4.95−4.87 (m, 1H), 4.51 (d, J = 8.8 Hz, 1H), 4.46−4.39 (m,

1H), 4.30−4.24 (m, 1H), 3.61−3.56 (m, 1H), 3.12−3.05 (m,

1H),2.75−2.70 (m, 2H), 2.46 (s, 3H), 2.28−2.07 (m, 2H), 2.04−1.99

(m, 1H), 1.82−1.76 (m, 1H), 1.55−1.41 (m, 5H), 1.37 (d, J = 7.2 Hz,

3H), 1.31−1.25 (m, 8H), 0.93 (s, 9H); UPLC-MS (ESI⁺): calculated

for C₃₂H₅₀N₅O₄S [M + 1]⁺, 600.36; found, 600.33.

(2R,4S)-1-((R)-2-(10-Aminodecanamido)-3,3-dimethylbutanoyl)-

4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)-

pyrrolidine-2-carboxamide Hydrochloride (43h). This compound

was prepared using a procedure similar to that used for compound

43a. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 9.07 (s, 1H), 8.42 (d,

J = 8.0 Hz, 1H), 7.92 (brs, 3H), 7.79 (d, J = 8.8 Hz, 1H), 7.48−7.37

(m, 4H), 4.95−4.87 (m, 1H), 4.51 (d, J = 8.8 Hz, 1H), 4.42−4.39 (m,

1H), 4.29−4.25 (m, 1H), 3.63−3.56 (m, 1H), 3.14−3.08 (m, 1H),

2.76−2.69 (m, 2H), 2.46 (s, 3H), 2.28−2.06 (m, 2H), 2.03−1.98 (m,

1H), 1.81−1.75 (m, 1H), 1.55−1.42 (m, 5H), 1.37 (d, J = 7.2 Hz,

3H), 1.31−1.24 (m, 10H), 0.93 (s, 9H); UPLC-MS (ESI⁺):

calculated for C₃₃H₅₂N₅O₄S [M + 1]⁺, 614.37; found, 614.35.

(2S,4R)-1-((S)-2-(11-Aminoundecanamido)-3,3-dimethylbutano-

yl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)-

pyrrolidine-2-carboxamide Hydrochloride (43i). This compound

was prepared using a procedure similar to that used for compound

43a. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 9.02 (s, 1H), 8.39 (d,

J = 8.0 Hz, 1H), 7.87 (brs, 3H), 7.79 (d, J = 8.8 Hz, 1H), 7.48−7.35

(m, 4H), 4.95−4.87 (m, 1H), 4.51 (d, J = 8.8 Hz, 1H), 4.41−4.38 (m,

1H), 4.27−4.24 (m, 1H), 3.63−3.56 (m, 1H), 3.16−3.09 (m, 1H),

2.77−2.72 (m, 2H), 2.46 (s, 3H), 2.26−2.09 (m, 2H), 2.02−1.96 (m,

1H), 1.82−1.78 (m, 1H), 1.52−1.43 (m, 5H), 1.37 (d, J = 7.2 Hz,

3H), 1.29−1.24 (m, 12H), 0.93 (s, 9H); UPLC-MS (ESI⁺):

calculated for C₃₄H₅₄N₅O₄S [M + 1]⁺, 628.39; found, 628.31.

(2R,4S)-1-((R)-2-(12-Aminododecanamido)-3,3-dimethylbutano-

yl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)-

pyrrolidine-2-carboxamide Hydrochloride (43j). This compound

was prepared using a procedure similar to that used for compound

43a. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 8.99 (s, 1H), 8.37 (d,

J = 8.0 Hz, 1H), 7.79 (d, J = 8.8 Hz, 1H), 7.64 (brs, 3H), 7.45−7.37

(m, 4H), 4.95−4.89 (m, 1H), 4.51 (d, J = 8.8 Hz, 1H), 4.43−4.39 (m,

1H), 4.29−4.26 (m, 1H), 3.63−3.57 (m, 1H), 3.16−3.09 (m, 1H),

2.80−2.72 (m, 2H), 2.45 (s, 3H), 2.33−1.98 (m, 3H), 1.82−1.75 (m,

1H), 1.51−1.45 (m, 5H), 1.37 (d, J = 7.2 Hz, 3H), 1.30−1.24 (m,

14H), 0.93 (s, 9H); UPLC-MS (ESI⁺): calculated for C₃₅H₅₆N₅O₄S

[M + 1]⁺, 642.40; found, 642.36.

5-((3-((3-Amino-5-(4-((tert-butoxycarbonyl)amino)-4-methylpi-

peridin-1-yl)pyrazin-2-yl)thio)-2-chlorophenyl)amino)-5-oxopenta-

noic Acid (46c). This compound was prepared using a procedure

similar to that used for compound 46a using methyl 5-chloro-5-

oxopentanoate as the starting material. ¹H NMR (400 MHz, DMSO-

d₆): δ (ppm) 12.15 (s, 1H), 9.53 (s, 1H), 7.62 (s, 1H), 7.40 (d, J =

7.6 Hz, 1H), 7.14 (t, J = 8.0 Hz,), 6.63 (s, 1H), 6.43 (dd, J = 8.0 Hz, J

= 1.2 Hz, 1H), 6.13 (s, 2H); 3.88−3.84 (m, 2H), 3.25−3.19 (m, 2H),

2.43−2.39 (m, 2H), 2.31−2.27 (m, 2H), 2.10−2.05 (m, 2H), 1.84−

1.77 (m, 2H), 1.73−1.68 (m, 1H), 1.45−1.40 (m, 2H), 1.39 (s, 9H),

1.25 (s, 3H); UPLC-MS (ESI⁺): calculated for C₂₆H₃₆ClN₆O₅S [M +

1]⁺, 579.22; found, 579.16.

(2R,4S)-1-((R)-14-Amino-2-(tert-butyl)-4-oxo-6,9,12-trioxa-3-

azatetradecanoyl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)-

phenyl)ethyl)pyrrolidine-2-carboxamide Hydrochloride (48). This

compound was prepared using a procedure similar to that used for

1

compound 43a. H NMR (400 MHz, DMSO-d₆): δ (ppm) 9.05 (s,

1H), 8.51 (d, J = 8.0 Hz, 1H), 7.97 (brs, 3H), 7.47−7.34 (m, 4H),

6.58 (brs, 1H), 4.93−4.87 (m, 1H), 4.55 (d, J = 8.8 Hz, 1H), 4.47−

4.42 (m, 1H), 4.31−4.27 (m, 1H), 3.98 (s, 2H), 3.63−3.56 (m, 12H),

2.98−2.94 (m, 1H), 2.46 (s, 3H), 2.09−2.04 (m, 1H), 1.37 (d, J = 7.2

Hz, 3H), 0.94 (s, 9H); UPLC-MS (ESI⁺): calculated for

C₃₁H₄₈N₅O₇S [M + 1]⁺, 634.33; found, 634.31.

(2R,4S)-1-((R)-1-Amino-14-(tert-butyl)-12-oxo-3,6,9-trioxa-13-

azapentadecan-15-oyl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-

yl)phenyl)ethyl)pyrrolidine-2-carboxamide Hydrochloride (50a).

This compound was prepared using a procedure similar to that

Methyl 3-((3-((3-Amino-5-(4-((tert-butoxycarbonyl)amino)-4-

methylpiperidin-1-yl)pyrazin-2-yl)thio)-2-chlorophenyl)amino)-3-

oxopropanoate (45a). Methyl 3-chloro-3-oxopropanoate (1.2 g, 8.9

mmol, 1.2 equiv) was added dropwise at 0 °C to a solution of tert-

butyl (1-(6-amino-5-((3-amino-2-chlorophenyl)thio)pyrazin-2-yl)-4-

methylpiperidin-4-yl (40) (3.4 g, 7.4 mmol, 1.0 equiv) and DIPEA

(3.9 mL, 2.9 g, 22.2 mmol, 3.0 equiv) in DCM (50 mL). The reaction

mixture was allowed to warm to rt and stirred for 1 h. It was poured

into aq NaHCO₃solution (50 mL). After extraction with DCM (30

mL × 2), the combined organic layers were washed with brine, dried

over Na₂SO₄, and evaporated under vacuum. The residue was used in

the next step without further puriﬁcation. UPLC-MS (ESI⁺):

calculated for C₂₅H₃₄ClN₆O₅S [M + 1]⁺, 565.20; found, 565.33.

1

used for compound 43a. H NMR (400 MHz, DMSO-d₆): δ (ppm)

9.11 (s, 1H), 8.44 (d, J = 8.0 Hz, 1H), 8.02 (brs, 3H), 7.89 (d, J = 8.0

Hz, 1H), 7.46−7.36 (m, 4H), 4.95−4.87 (m, 1H), 4.52 (d, J = 8.8 Hz,

1H), 4.44−4.40 (m, 1H), 4.29−4.26 (m, 1H), 3.62−3.46 (m, 15H),

2.97−2.93 (m, 1H), 2.46 (s, 3H), 2.40−2.33 (m, 1H), 2.05−2.00 (m,

1H), 1.81−1.74 (m, 1H), 1.37 (d, J = 7.2 Hz, 3H), 0.94 (s, 9H);

UPLC-MS (ESI⁺): calculated for C₃₂H₅₀N₅O₇S [M + 1]⁺, 648.34;

found, 648.25.

(2R,4S)-1-((R)-17-Amino-2-(tert-butyl)-4-oxo-6,9,12,15-tetraoxa-

3-azaheptadecanoyl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)-

phenyl)ethyl)pyrrolidine-2-carboxamide Hydrochloride (50b). This

compound was prepared using a procedure similar to that used for

N

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1

compound 43a. H NMR (400 MHz, DMSO-d₆): δ (ppm) 9.05 (s,

and concentrated; the residue was dissolved in THF (4 mL), MeOH

(2 mL), and water (2 mL), and NaOH (192 mg, 4.8 mmol, 4.0 equiv)

was added and stirred at rt for 8 h; the pH was adjusted to 6 and

diluted with EtOAc (30 mL) and water (30 mL). The layers were

separated and washed with water, dried with Na₂SO₄, ﬁltered and

concentrated, and the residue was puriﬁed by pre-HPLC to aﬀord 56

1H), 8.47 (d, J = 8.0 Hz, 1H), 7.96 (brs, 3H), 7.45−7.38 (m, 4H),

6.98 (brs, 1H), 4.94−4.87 (m, 1H), 4.54 (d, J = 8.8 Hz, 1H), 4.46−

4.41 (m, 1H), 4.31−4.27 (m, 1H), 3.98 (s, 1H), 3.63−3.53 (m, 16H),

2.98−2.94 (m, 2H), 2.46 (s, 3H), 2.08−2.04 (m, 1H), 1.37 (d, J = 7.2

Hz, 3H), 0.94 (s, 9H); UPLC-MS (ESI⁺): calculated for

+

1

C₃₃H₅₂N₅O₈S [M + 1] , 678.35; found, 678.31.

as a white solid (228 mg, 26% yield). H NMR (400 MHz, DMSO-

tert-Butyl (1-(6-Amino-5-((2-chloro-3-(2-(piperazin-1-yl)-

acetamido)phenyl)thio)pyrazin-2-yl)-4-methylpiperidin-4-yl)-

carbamate (52). tert-Butyl (1-(6-amino-5-((3-amino-2-

chlorophenyl)thio)-pyrazin-2-yl)-4-methylpiperidin-4-yl)carbamate

(40) (930 mg, 2.0 mmol, 1.0 equiv), 2-(4-(((9H-ﬂuoren-9-

yl)methoxy)carbonyl)piperazin-1-yl)acetic acid (51) (806 mg, 2.2

mmol, 1.1 equiv), and N-methylimidazole (574 mg, 7.0 mmol, 3.5

equiv) were dissolved in MeCN (8.0 mL) and THF (8.0 mL), and

then TCFH (730 mg, 2.6 mmol, 1.3 equiv) was added in a single

portion. The reaction was stirred until complete, judged by LC-MS.

The reaction was then diluted with EtOAc (30 mL) and water (30

mL). The layers were separated and washed with water, dried with

Na₂SO₄, ﬁltered and concentrated, the residue was dissolved in

piperidine (2 mL) and MeCN (10 mL) after stirring at rt for 2 h, and

the solvent was removed under vacuum and the residue was puriﬁed

d₆): δ (ppm) 11.96 (s, 1H), 9.47 (s, 1H), 7.62 (s, 1H), 7.40 (d, J =

8.0 Hz, 1H), 7.62 (s, 1H), 7.14 (t, J = 8.0 Hz, 1H), 6.63 (brs, 1H),

6.42 (dd, J = 8.0 Hz, J = 1.2 Hz, 1H), 6.09 (brs, 2H), 3.88−3.83 (m,

2H), 3.25−3.19 (m, 2H), 2.38−2.32 (m, 2H), 2.18 (t, J = 7.6 Hz,

2H), 2.11−2.03 (m, 2H), 1.63−1.54 (m, 2H), 1.52−1.42 (m, 4H),

1.39 (s, 9H), 1.35−1.19 (m, 23H); UPLC-MS (ESI⁺): calculated for

C₃₇H₅₈ClN₆O₅S [M + 1]⁺, 733.39; found, 733.41.

9-(4-(tert-Butoxycarbonyl)piperazin-1-yl)nonanoic Acid (59a). A

mixture of tert-butyl piperazine-1-carboxylate (57) (1.86 g, 10.0

mmol, 1 equiv), sodium iodide (1.49 g, 10.0 mmol, 1 equiv),

potassium carbonate (4.14 g, 30.0 mmol, 3 equiv), and methyl 9-

bromononanoate (3.02 g, 12.0 mmol, 1.2 equiv) in MeCN (60 mL)

was stirred at 60 °C for 16 h. After removal of solvent by rotary

evaporation, the resulting residue was diluted with DCM and

insoluble inorganics were ﬁltered oﬀ, and the liquid ﬁltrate was

concentrated in vacuo. The residue was dissolved in THF/MeOH/

H₂O (15 mL/9 mL/6 mL), and lithium hydroxide monohydrate

(1.68 g, 40.0 mmol, 2 equiv) was added at 0 °C. The reaction mixture

was allowed to warm to rt and stirred for 2 h. After quenching with 1

N HCl to pH ∼ 3, the resulting mixture was extracted with EtOAc

(100 mL × 2). The organic layer was washed with brine, dried over

anhydrous Na₂SO₄, and then concentrated under reduced pressure,

and the residue was puriﬁed by Biotage silica gel column

1

by pre-HPLC to aﬀord 52 as a white solid (566 mg, 48% yield). H

NMR (400 MHz, DMSO-d₆): δ (ppm) 10.00 (s, 1H), 8.01 (dd, J =

8.0 Hz, J = 1.2 Hz, 1H), 7.62 (s, 1H), 7.19 (t, J = 8.0 Hz, 1H), 6.63

(brs, 1H), 6.39 (dd, J = 8.0 Hz, J = 1.2 Hz, 1H), 6.10 (brs, 1H), 3.37−

3.29 (m, 2H), 3.27−3.17 (m, 2H), 3.16 (s, 1H), 2.87 (t, J = 4.8 Hz,

2H), 2.58−2.51 (m, 2H), 2.13−2.09 (m, 2H), 1.47−1.42 (m, 2H),

1.39 (s, 9H), 1.25 (s, 9H); UPLC-MS (ESI⁺): calculated for

+

C₂₇H₄₀ClN₈O₃S [M + 1] , 591.26; found, 591.21.

11-(4-(2-((3-((3-Amino-5-(4-((tert-butoxycarbonyl)amino)-4-

methylpiperidin-1-yl)pyrazin-2-yl)thio)-2-chlorophenyl)amino)-2-

oxoethyl)piperazin-1-yl)undecanoic Acid (54a). K₂CO₃(3.0 equiv)

and KI (1.2 equiv) were added to a solution of the intermediate 52

(118 mg, 0.2 mmol) and 11-bromoundecanoic acid (1.2 equiv) in

DMF (5.0 mL). After the mixture was stirred for 12 h at 60 °C, the

reaction was diluted with EtOAc (30 mL) and water (30 mL). The

layers were separated and washed with water, dried with Na₂SO₄,

ﬁltered and concentrated, and the residue was puriﬁed by pre-HPLC

1

chromatography to aﬀord 59a as a white solid (1.43 g, 42%): H

NMR (400 MHz, DMSO-d₆): δ (ppm) 11.99 (s, 1H), 4.06−3.90 (m,

2H), 3.43−3.20 (m, 4H), 3.09−2.86 (m, 4H), 2.19 (t, J = 7.6 Hz,

2H), 1.73−1.65 (m, 2H), 1.52−1.47 (m, 2H), 1.41 (s, 9H), 1.27 (s,

+

8H); UPLC-MS (ESI⁺): calculated for C₁₈H₃₅N₂O₄[M + 1]

343.26; found, 343.11.

,

10-(4-(tert-Butoxycarbonyl)piperazin-1-yl)decanoic Acid (59b).

This compound was prepared using a procedure similar to that used

for compound 59a using methyl 10-bromodecanoate as the starting

1

to aﬀord 54a as a white solid (71 mg, 46% yield). H NMR (400

1

MHz, DMSO-d₆): δ (ppm) 9.80 (s, 1H), 9.44 (brs, 1H), 7.81 (d, J =

8.0 Hz, 1H), 7.62 (s, 1H), 7.20 (t, J = 8.0 Hz, 1H), 6.64 (brs, 1H),

6.44 (dd, J = 8.0 Hz, J = 1.2 Hz, 1H), 6.09 (brs, 1H), 3.87−3.83 (m,

2H), 3.55−3.50 (m, 2H), 3.38 (s, 1H), 3.25−3.20 (m, 2H), 3.16−

3.03 (m, 6H), 2.73−2.65 (m, 2H), 2.19 (t, J = 7.6 Hz, 2H), 2.13−

2.06 (m, 2H), 1.66−1.58 (m, 2H), 1.53−1.45 (m, 4H), 1.39 (s, 3H),

1.32−1.24 (m, 15H); UPLC-MS (ESI⁺): calculated for

C₃₈H₆₀ClN₈O₅S [M + 1]⁺, 775.41; found, 775.32.

12-(4-(2-((3-((3-Amino-5-(4-((tert-butoxycarbonyl)amino)-4-

methylpiperidin-1-yl)pyrazin-2-yl)thio)-2-chlorophenyl)amino)-2-

oxoethyl)piperazin-1-yl)dodecanoic Acid (54b). This compound

was prepared using a procedure similar to that used for compound

54a using 12-bromododecanoic acid as the starting material. ¹H NMR

(400 MHz, DMSO-d₆): δ (ppm) 9.78 (s, 1H), 9.36 (brs, 1H), 7.82

(d, J = 8.0 Hz, 1H), 7.62 (s, 1H), 7.20 (t, J = 8.0 Hz, 1H), 6.64 (brs,

1H), 6.44 (dd, J = 8.0 Hz, J = 1.2 Hz, 1H), 6.09 (brs, 1H), 3.87−3.83

(m, 2H), 3.54−3.49 (m, 2H), 3.36 (s, 1H), 3.25−3.20 (m, 2H),

3.16−3.02 (m, 6H), 2.72−2.64 (m, 2H), 2.19 (t, J = 7.6 Hz, 2H),

2.12−2.05 (m, 2H), 1.68−1.59 (m, 2H), 1.52−1.42 (m, 4H), 1.39 (s,

9H), 1.33−1.21 (m, 17H); UPLC-MS (ESI⁺): calculated for

C₃₉H₆₂ClN₈O₅S [M + 1]⁺, 789.42; found, 789.37.

16-((3-((3-Amino-5-(4-((tert-butoxycarbonyl)amino)-4-methylpi-

peridin-1-yl)pyrazin-2-yl)thio)-2-chlorophenyl)amino)-16-oxohexa-

decanoic Acid (56). Compound 40 (558 mg, 1.20 mmol, 1.0 equiv),

16-(benzyloxy)-16-oxohexadecanoic acid (55) (542 mg, 1.44 mmol,

1.2 equiv), and N-methylimidazole (345 mg, 4.2 mmol, 3.5 equiv)

were dissolved in MeCN (6.0 mL) and THF (6.0 mL), then TCFH

(437 mg, 1.56 mmol, 1.3 equiv) was added in a single portion. The

reaction was stirred until complete, judged by LC-MS. The reaction

was then diluted with EtOAc (30 mL) and water (30 mL). The layers

were separated and washed with water, dried with Na₂SO₄, ﬁltered,

material. H NMR (400 MHz, DMSO-d₆): δ (ppm) 11.98 (s, 1H),

4.07−3.92 (m, 2H), 3.47−3.34 (m, 2H), 3.26−3.13 (m, 2H), 3.09−

2.98 (m, 2H), 2.96−2.85 (m, 2H), 2.18 (t, J = 7.6 Hz, 2H), 1.70−

1.59 (m, 2H), 1.53−1.45 (m, 2H), 1.42 (s, 9H), 1.26 (s, 10H);

UPLC-MS (ESI⁺): calculated for C₁₉H₃₇N₂O₄[M + 1] ⁺, 357.27;

found, 357.16.

(2R,4S)-1-((R)-3,3-Dimethyl-2-(9-(piperazin-1-yl)nonanamido)-

butanoyl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)-

ethyl)pyrrolidine-2-carboxamide Hydrochloride (60a). This com-

pound was prepared using a procedure similar to that used for

compound 43a. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 9.18 (brs,

2H), 8.99 (s, 1H), 8.35 (d, J = 8.0 Hz, 1H), 7.76 (d, J = 8.8 Hz, 1H),

7.48−7.37 (m, 4H), 4.93−4.90 (m, 1H), 4.52 (d, J = 8.8 Hz, 1H),

4.43−4.39 (m, 1H), 4.30−4.26 (m, 1H), 3.64−3.56 (m, 3H), 3.51−

3.21 (m, 6H), 3.16−3.09 (m, 3H), 2.45 (s, 3H), 2.33−1.99 (m, 3H),

1.84−1.77 (m, 1H), 1.66−1.57 (m, 2H), 1.51−1.45 (m, 2H), 1.37 (d,

J = 7.2 Hz, 3H), 1.30−1.24 (m, 8H), 0.93 (s, 9H); UPLC-MS (ESI⁺):

calculated for C₃₆H₅₇N₆O₄S [M + 1]⁺, 669.42; found, 669.37.

(2R,4S)-1-((R)-3,3-Dimethyl-2-(10-(piperazin-1-yl)decanamido)-

butanoyl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)-

ethyl)pyrrolidine-2-carboxamide Hydrochloride (60b). This com-

pound was prepared using a procedure similar to that used for

compound 43a. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 9.24 (brs,

2H), 8.99 (s, 1H), 8.35 (d, J = 8.0 Hz, 1H), 7.76 (d, J = 8.8 Hz, 1H),

7.48−7.37 (m, 4H), 4.95−4.90 (m, 1H), 4.51 (d, J = 8.8 Hz, 1H),

4.43−4.39 (m, 1H), 4.31−4.27 (m, 1H), 3.63−3.55 (m, 3H), 3.52−

3.22 (m, 6H), 3.16−3.10 (m, 3H), 2.45 (s, 3H), 2.33−1.99 (m, 3H),

1.83−1.76 (m, 1H), 1.65−1.56 (m, 2H), 1.50−1.44 (m, 2H), 1.37 (d,

J = 7.2 Hz, 3H), 1.31−1.25 (m, 10H), 0.93 (s, 9H); UPLC-MS

(ESI⁺): calculated for C₃₇H₅₉N₆O₄S [M + 1]⁺, 683.43; found, 683.41.

O

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Article

8-(4-((tert-Butoxycarbonyl)amino)phenoxy)octanoic Acid (63).

This compound was prepared using a procedure similar to that used

for compound 59a using tert-butyl (4-hydroxyphenyl)carbamate and

methyl 8-bromooctanoate as the starting material. UPLC-MS (ESI⁺):

the level of SHP2 protein was examined by blot analysis. GAPDH was

used as a loading control.

Molecular Modeling. The crystal structures of SHP2 with

SHP099 (PDBID: 5EHR),²⁶9b (PDBID: 5XZR),²⁸

SHP099+SHP244 (PDBID: 6BMU),⁶²SHP504 (PDBID:

6BMV),⁶²and SHP099+SHP844 (PDBID: 6BMY)⁶²were used to

model the binding pose of compounds 4 and 5 with SHP2. SHP2

protein corrdinates from these crystal structures were extracted and

protonated at pH 7.0 using the “protonate 3D” module in the MOE

program (Molecular Operating Environment (MOE). Compounds 4

and 5 were created and optimized using MOE before performing an

ensemble docking calculation using the GOLD program (version 5.2).

Default parameters in the GOLD program were used in the docking

calculation, and the PLP scoring function was used as the ﬁtness

function to rank the docked poses. The top ranked pose was used as

the binding model.

+

calculated for C₁₉H₃₀NO₅[M + 1] , 352.21; found, 352.10.

(2R,4S)-1-((R)-2-(8-(4-Aminophenoxy)octanamido)-3,3-dime-

thylbutanoyl)-4-hydroxy-N-((R)-1-(4-(4-methylthiazol-5-yl)phenyl)-

ethyl)pyrrolidine-2-carboxamide Hydrochloride (64). This com-

pound was prepared using a procedure similar to that used for

compound 43a. ¹H NMR (400 MHz, DMSO-d₆): δ (ppm) 9.92 (brs,

2H), 9.00 (s, 1H), 8.36 (d, J = 8.0 Hz, 1H), 7.77 (d, J = 8.8 Hz, 1H),

7.48−7.37 (m, 4H), 7.29−7.26 (m, 2H), 7.05−7.02 (m, 2H), 4.95−

4.88 (m, 1H), 4.51 (d, J = 8.8 Hz, 1H), 4.44−4.40 (m, 1H), 4.31−

4.28 (m, 1H), 3.98−3.94 (m, 2H), 3.64−3.57 (m, 2H), 2.45 (s, 3H),

2.33−1.98 (m, 3H), 1.83−1.76 (m, 1H), 1.73−1.66 (m, 2H), 1.54−

1.42 (m, 2H), 1.37 (d, J = 7.2 Hz, 3H), 1.30−1.22 (m, 6H), 0.93 (s,

9H); UPLC-MS (ESI⁺): calculated for C₃₇H₅₂N₅O₅S [M + 1]⁺,

678.37; found, 678.41.

5-(4-(3-((tert-Butoxycarbonyl)amino)propyl)piperazin-1-yl)-

pentanoic Acid (67). This compound was prepared using a procedure

similar to that used for compound 59a using tert-butyl (3-(piperazin-

1-yl)propyl)carbamate and ethyl 5-bromopentanoate as starting

material. UPLC-MS (ESI⁺): calculated for C₁₇H₃₄N₃O₄[M + 1]⁺,

344.25; found, 344.17.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c00471.

■

sı

Western blotting analysis of SHP2 proteins in KYSE520

and MV4;11 cells treated with the SHP2 degraders and

¹H NMR, ¹³C NMR, and UPLC-MS spectra of

representative compounds (PDF)

Molecular string ﬁles for all of the ﬁnal target

compounds (CSV)

(2R,4S)-1-((R)-2-(5-(4-(3-Aminopropyl)piperazin-1-yl)-

pentanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((R)-1-(4-(4-

methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide Hydro-

chloride (68). This compound was prepared using a procedure similar

1

to that used for compound 43a. H NMR (400 MHz, DMSO-d₆): δ

(ppm) 9.08 (brs, 1H), 8.40 (d, J = 8.0 Hz, 1H), 7.96 (brs, 3H), 7.73

(d, J = 8.8 Hz, 1H), 7.43−7.36 (m, 4H), 4.93−4.87 (m, 1H), 4.50 (d,

J = 8.8 Hz, 1H), 4.42−4.38 (m, 1H), 4.30−4.26 (m, 1H), 3.62−3.54

(m, 1H), 3.51−3.21 (m, 6H), 3.16−3.10 (m, 3H), 2.45 (s, 3H),

2.38−1.96 (m, 7H), 1.83−1.76 (m, 1H), 1.65−1.56 (m, 2H), 1.52−

1.41 (m, 6H), 1.37 (d, J = 7.2 Hz, 3H), 0.93 (s, 9H); UPLC-MS

(ESI⁺): calculated for C₃₅H₅₆N₇O₄S [M + 1]⁺: 670.41; found, 670.27.

Cell Lines and Cell Culture. The MV4;11 cell line was purchased

from the American Type Culture Collection (ATCC), cultured in

Iscove’s modiﬁed Dulbecco’s media (IMDM). Esophageal cancer cell

lines KYSE70 and KYSE520 were purchased from DSMZ

(Braunschweig, Germany), grown in RPMI 1640 (Invitrogen). All

of the cells were supplemented with 10% fetal bovine serum

(Invitrogen) at 37 °C in a humidiﬁed 5% CO2 incubator.

Modeled structures of compound 4 in complex with

SHP2 (PDB)

Modeled structures of compound 5 in complex with

SHP2 (PDB)

AUTHOR INFORMATION

Corresponding Author

■

Shaomeng Wang − Departments of Internal Medicine,

Pharmacology, and Medicinal Chemistry, University of

Michigan, Ann Arbor, Michigan 48109, United States;

orcid.org/0000-0002-8782-6950; Email: shaomeng@

umich.edu

Cell Growth Assay. Cell viability was evaluated with a WST-8

assay (Dojindo) following the manufacturer’s instructions. Brieﬂy,

cells were seeded in 96-well cell culture plates at a density of 10,000−

20,000 cells/well in 200 μL for MV4;11 cell line or 2,000−3,000 for

KYSE-520 cell line of culture medium containing serial dilution of

testing compounds. After 4 days of treatment, cell growth was

measured by a lactate dehydrogenase-based WST-8 assay (Dojindo

Molecular Technologies) using a Tecan Inﬁnite M-1000 multimode

microplate reader (Tecan US, Morrisville, NC). The WST-8 reagent

was added to each well, and cells were incubated for an additional 1−

2 h and read at 450 nm. The readings were normalized to the vehicle-

treated cells, and the IC₅₀was calculated by nonlinear regression

analysis using the GraphPad Prism 6 software.

Western Blot Analysis. Western blotting and quantiﬁcation were

performed with regular Western blot method or LI-COR Odyssey

system. Treated cells were lysed by RIPA buﬀer supplemented with

protease and phosphatase inhibitors. The cell lysates were separated

by 4−12% SDS−PAGE gels and blotted into PVDF (polyvinylidene

diﬂuoride) membranes. Antibodies used in the study are indicated in

the ﬁgure legends. The net protein bands and loading controls are

calculated by deducting the background from the inverted band value.

The ﬁnal relative quantiﬁcation values are the ratio of the net band to

net loading control.

Authors

Mingliang Wang − Departments of Internal Medicine,

University of Michigan, Ann Arbor, Michigan 48109, United

States

Jianfeng Lu − Departments of Internal Medicine, University of

Michigan, Ann Arbor, Michigan 48109, United States

Mi Wang − Departments of Internal Medicine, University of

Michigan, Ann Arbor, Michigan 48109, United States

Chao-Yie Yang − Departments of Internal Medicine, University

of Michigan, Ann Arbor, Michigan 48109, United States;

orcid.org/0000-0002-5445-0109

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c00471

Author Contributions

^∥M.W. and J.L. contributed equally.

Notes

The authors declare the following competing ﬁnancial

interest(s): The University of Michigan has ﬁled a patent

application on these SHP2 degraders, which has been licensed

by Oncopia Therapeutics Inc. S. Wang, M. Wang, J. Lu, M.

Wang are co-inventors on the patent application. The

University of Michigan has received a research contract from

For the in vitro kinetics studies of SHP2 expression, cancer cells

seeded in a 6-well plate overnight were treated with the compounds

for another 2, 4, 8, 12, and 24 h. The treated cells were harvested, and

P

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ACKNOWLEDGMENTS

■

This study was supported in part by a research contract from

Oncopia Therapeutics Inc. and the Michigan Comprehensive

Cancer Center (now Rogel Cancer Center) Core grant (P30

CA046592) from the National Cancer Institute (NCI), NIH.

ABBREVIATIONS USED

■

SHP2, Src homology 2 domain-containing phosphatase 2;

PTP, protein tyrosine phosphatase; NS, Noonan Syndrome;

JMML, Juvenile myelomonocytic leukemia; ERK, extracellular

signal-regulated kinase; PROTAC, proteolysis targeting

chimera; HATU, 1-[bis(dimethylamino)-methylene]-1H-

1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexaﬂuorophosphate;

VHL, von Hippel−Lindau; TCFH, N,N,N,N-tetramethylchlor-

oformamidinium hexaﬂuorophosphate; NMI, N-methylimida-

zole; DIPEA, N,N-diisopropylethylamine; DMF, dimethylfor-

mamide; DCM, dichloromethane; HATU, 1-[bis-

(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]-pyridi-

nium 3-oxid hexaﬂuorophosphate; TFA, triﬂuoroacetic acid

(17) Scott, L. M.; Lawrence, H. R.; Sebti, S. M.; Lawrence, N. J.;

Wu, J. Targeting protein tyrosine phosphatases for anticancer drug

discovery. Curr. Pharm. Des. 2010, 16, 1843−1862.

(18) Butterworth, S.; Overduin, M.; Barr, A. J. Targeting protein

tyrosine phosphatase SHP2 for therapeutic intervention. Future Med.

Chem. 2014, 6, 1423−1437.

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https://dx.doi.org/10.1021/acs.jmedchem.0c00471

J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Discovery of SHP2-D26 as a First, Potent, and Effective PROTAC Degrader of SHP2 Protein

DOI: 10.1021/acs.jmedchem.0c00471

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Article abstract of DOI:10.1021/acs.jmedchem.0c00471

Full text of DOI:10.1021/acs.jmedchem.0c00471

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