Discovery of AZD8154, a Dual PI3Kγδ Inhibitor for the Treatment of Asthma

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Drug Annotation

Discovery of AZD8154, a Dual PI3Kγδ Inhibitor for the Treatment of

Asthma

̋

Matthew W. D. Perry,* Karin Bj ö rhall, Peter Bold, Mikael Brulls, Ulf B ö rjesson, Johan Carlsson,

Hui-Fang Amy Chang, Yunhua Chen, Anders Eriksson, Britt-Marie Fihn, Rebecca Fransson,

Linda Fredlund, Hongbin Ge, Haijuan Huang, Kostas Karabelas, Eva Lamm Bergstr ö m, Sarah Lever,

Helena Lindmark, Mickael Mogemark, Antonios Nikitidis, Anna-Pia Palmgren, Nils Pemberton,

Jens Petersen, Mio Rodrigo Blomqvist, Reed W. Smith, Matthew J. Thomas, Victoria Ullah,

Christian Tyrchan, Tiiu Wennberg, Annika Westin Eriksson, Wenzhen Yang, Shuchun Zhao,

̈

and Linda Oster

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ABSTRACT: Starting from our previously described PI3Kγ inhib-

itors, we describe the exploration of structure−activity relationships

that led to the discovery of highly potent dual PI3Kγδ inhibitors. We

explored changes in two positions of the molecules, including

macrocyclization, but ultimately identiﬁed a simpler series with the

desired potency proﬁle that had suitable physicochemical properties

for inhalation. We were able to demonstrate eﬃcacy in a rat ovalbumin

challenge model of allergic asthma and in cells derived from asthmatic

patients. The optimized compound, AZD8154, has a long duration of

action in the lung and low systemic exposure coupled with high

selectivity against oﬀ-targets.

Unlike PI3Kα and β, which are ubiquitously or widely

distributed throughout the body,⁴⁻⁶PI3Kγ and δ have a much

more restricted distribution and are principally found in

leukocytes.^7,8Inhibitors of PI3Ks have been investigated for

the treatment of cancers, with four agents now on the market,

but the roles of PI3Kγ and δ in leukocytes have also resulted in

investigation of inhibitors of these isoforms for the treatment

of inﬂammatory diseases.^9,10PI3Kδ has been shown to be

responsible for cytokine release from Th2 and Th17 cells,¹¹

while PI3Kγ has been shown to be involved in the activation of

both eosinophils and neutrophils.¹²These mechanisms are

believed to be important in asthma, both for allergic (Type 2)

asthma but also to less deﬁned non-Th2 asthmatics with mixed

T cell subsets driving the inﬂammatory responses.¹³

INTRODUCTION

■

Phosphoinositol 3-kinases (PI3Ks) are a group of lipid kinases

that phosphorylate the 3′-hydroxyl of the inositol ring of

phosphatidyl inositol lipids. They have been divided into three

classes, I, II, and III, based on their preferred substrates. The

most intensively studied of these classes are the four class I

enzymes, known as PI3Kα, β, γ, and δ. These enzymes exist as

heterodimers consisting of a catalytic p110 subunit and a

regulatory subunit. PI3Kα, β, and δ, which share p85

regulatory subunits and principally act downstream of tyrosine

kinase receptors, are known as class IA. PI3Kγ, in contrast, has

its own regulatory subunits (p101 and p84) and is found

downstream of GPCRs through the agency of GTPases; it is

the only member of class IB PI3Ks. The class I PI3Ks

principally convert the membrane lipid phosphoinositol-4,5-bis

phosphate (PIP₂) to phosphoinositol-3,4,5-tris phosphate

(PIP₃). PIP₃is recognized by proteins containing a pleckstrin

homology domain which are recruited to and activated by

PIP₃. Pleckstrin homology domains are found in a variety of

proteins that activate multiple pathways leading to cell

proliferation, diﬀerentiation, and other eﬀects. PI3Ks have

been widely studied, and extensive reviews cover the biology in

detail.¹⁻³

The oral dual PI3Kδγ inhibitor Duvelisib has been

investigated in clinical trials for asthma¹⁴and rheumatoid

arthritis,¹⁵where it has shown positive eﬀects, but further

Received: March 10, 2021

Published: June 3, 2021

https://doi.org/10.1021/acs.jmedchem.1c00434

J. Med. Chem. 2021, 64, 8053−8075

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Drug Annotation

Figure 1. Marketed and investigational PI3Kδ and PI3Kδγ inhibitors referred to in the text.

development for these indications has not been pursued,

possibly because of side eﬀects. The inhaled selective PI3Kδ

inhibitor Nemiralisib has been evaluated in clinical trials with

asthmatics,¹⁶COPD patients,¹⁷and patients with activated

PI3Kδ syndrome,¹⁸though development of this compound

appears to have stopped, potentially based on a lack of clinical

eﬃcacy (Figure 1).

Oral PI3Kδ inhibitors, such as Idelalisib (Figure 1) and

Duvelisib, have been eﬀective in the treatment of B cell

lymphomas, but dose-limiting toxicity has been a problem,¹⁹

and it is notable that Duvelisib was not further investigated for

inﬂammatory diseases beyond two small studies. Inhaled

administration has been used to target drugs to the lung where

systemic side eﬀects might otherwise result in unacceptable

toxicity.²⁰We were thus interested to discover a dual PI3Kγδ

inhibitor with properties suitable for inhaled administration

which we hoped would be able to display profound immune

modulation while minimizing undesired systemic eﬀects.

We have previously described our earlier series of PI3Kδ

inhibitors^21,22and the optimization of one of them for

inhalation. We have also described the discovery of a series

of PI3Kγ inhibitors²³with an unprecedented mode of action

and selectivity²⁴that led to the identiﬁcation of 1 (Figure 2) as

amines 16 and 17. These could then be used to displace a

halogen from a heterocycle under palladium-catalyzed

conditions to give compounds 18−22 (Scheme 2).

Synthesis of the macrocycles 32−37 used the sulfonyl

chloride 4 and readily assembled pyridine amide moieties (25,

27−31) that were joined together through formation of a

sulfonamide. Deprotection of the aminothiazole enabled

macrocyclization via an intramolecular Buchwald-Hartwig

coupling to give the target compounds (Scheme 3).

The substituted pyridine derivatives 52−72 were synthe-

sized by palladium-catalyzed amination of 16, 17, or analogues

thereof. The required bromopyridine derivatives were either

commercial or synthesized simply. Heterocycles 38, 39, and 40

were built up from the available acid or acid chloride. Most of

the reversed amides were made by amination of 2,6-

dibromopyridine with the appropriate lactam, though in

some cases acylation of 2-amino-6-bromopyridine followed

by cyclization was used. Alcohols were protected as TBS ethers

and free amines with carbobenzyloxy groups (Scheme 4).

RESULTS AND DISCUSSION

■

Despite the very high selectivity displayed by (2) and the close

analogue (5), it is noticeable that there is more activity against

PI3Kδ than against either PI3Kα or β; thus, we wondered if we

could discover modiﬁcations that gave a dual PI3Kγδ

inhibitory proﬁle while retaining selectivity against PI3Kα

and β.

As part of the SAR exploration of this series of molecules

and, having access to the sulfonyl chloride (4), we made a

series of sulfonamides through reaction with a variety of

amines. Small aliphatic amines had been explored previously²³

and did not show any signiﬁcant changes in potency, but

heterocyclic sulfonamides and, particularly, sulfonanilides

exhibited a marked jump in inhibition of PI3Kδ, though with

considerable variation, depending on substitution. The most

interesting analogues are shown in Table 1. It should be noted

that the ceiling for activity in our enzyme assays was

approximately pIC₅₀9.1, and any activity greater than about

pIC₅₀8.9 may be an underestimate. This was particularly an

issue for the PI3Kγ activity, and we regarded the potency ﬁgure

from the cell assays as indicative of the true inhibition

achieved.

Figure 2. PI3Kγ selective inhibitor 1.

a potential development candidate. Given the roles of both

PI3Kγ and PI3Kδ in hematopoetic cells, it was of great interest

to us to try to identify compounds that combined these

activities in a single molecule. In this work, we describe our

exploration of the chemistry around our PI3Kγ selective series

that led to the identiﬁcation of features that gave inhibition of

PI3Kδ while retaining activity against PI3Kγ and the

optimization of the compounds for inhaled delivery.

The gain in activity at PI3Kδ was modest for the parent

sulfonanilide (6); however, two diﬀerent sulfonamide sub-

stitution patterns were found to be favored for increasing

potency at PI3Kδ, meta-electron withdrawing groups (sulfone

7, cyano 8); however, this could not be enhanced by 3,5-

disubstitution (10). Alternatively, para-amino methyl sub-

stitution gave an improvement in PI3Kδ potency but at a slight

cost to that at PI3Kγ (12). The speciﬁcity of these

substitutions was shown by the meta-dimethylaminomethyl

CHEMISTRY

■

Synthesis of the compounds reported began with preparation

of the sulfonyl chloride 4, via displacement of chlorine from

chloride 2²³by benzyl thiolate followed by oxidative

chlorination of thioether 3 with sulfuryl chloride. Condensa-

tion of 4 with amines gave sulfonamides 5−15 (Scheme 1).

In order to explore substitution on the aminothiazole, we

began by hydrolyzing the acetamide of 1 and 5 to give the

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Drug Annotation

a

Scheme 1. Synthesis of Sulfonamides 5−15

a

i, PhCH₂SH, NaOt-Am, DMF, 110 °C; ii, SO₂Cl₂, MeCN, AcOH, H₂O, 0−5 °C; iii, R1R2NH, pyridine, CH₂Cl₂.

a

Scheme 2. Synthesis of N-Heterocyclic Derivatives 18−22

a

i, HCl, EtOH; ii, R4X, Pd(OAc)₂, Xantphos, dioxane, 120 °C.

analogue (11) and the para-cyano and sulfone analogues (14,

15). N-Methylation of the sulfonamide was not beneﬁcial (9,

13). For all these compounds, an appreciable drop in potency

in the cell assay of about 10-fold was unsurprising but left us

needing to ﬁnd additional potency to be useful.

rotates slightly to optimize the interaction, though the

observed distance is slightly too long (∼5.4 Å) for a direct

interaction (Figure 4). Inspection of the crystal structure of 20

in comparison to that of 12 showed that the two amino

functions both pointed toward the same Asp897 carboxylate.

This raised the prospect that a macrocycle might be an

eﬀective way forward.

Our initial design of macrocycle hybridized between the

amines 12 and 20. These compounds (32, 33) showed very

good potency at PI3Kδ but lost activity against PI3Kγ (Table

3). We continued to investigate macrocyclic analogues, and

after a preliminary study using ring closing metathesis

established that the optimal linker length was ﬁve atoms

between the amide and sulfonamide (Supplementary Table 1),

we made a series with purely aliphatic linkers (34−37). These

compounds had excellent activity at both PI3Kγ and PI3Kδ,

with good cellular activity at both isoforms as well, though

activity against PI3Kα also increased, resulting in lower

selectivity against this isoform than we desired. Activity against

PI3Kβ (not shown) was weaker.

In parallel with our exploration of macrocycles, we

continued to explore SAR of compounds related to the pyridyl

amides. We synthesized isosteres to amides as a possible way

to further improve δ cell potency (Table 4). Heterocycles

(52−54) were similar to or poorer than earlier compounds,

and likewise reversing the amide (55) had little eﬀect until we

N-alkylated (56, 57), which gave a signiﬁcant improvement in

potency against PI3Kδ, in contrast to the eﬀect seen previously

with the alternate orientation (22).

We were able to obtain X-ray crystal structures of 7 and 12

in a PI3Kδ construct. The protein structure observed matched

closely to that seen previously for 1, with the anilide moiety

ﬁtting nicely into the space available. For the sulfone of 7, we

were unable to identify any speciﬁc interactions made that

could account for the improved potency of this group relative

to others. The para-dimethylaminomethyl substituent of 12,

however, was close to, though probably beyond direct contact

with, the side chain carboxylate group of Asp897 (Figure 3).

Among the changes that we made to the parent compounds

(1 and analogues) during SAR exploration was to replace the

acetamide group with a heterocycle (Table 2). Of the various

heterocyclic rings that we attached, it was noticeable that

pyridyl (18) particularly with an amide substituent (19) gave

signiﬁcantly increased activity against PI3Kδ. Changing the

position of the amide (21) appeared to have little eﬀect, while

disubstitution on the amide (22) appeared not to be favorable

for activity at PI3Kδ.

Crystallization of 20 in PI3Kδ showed that the pyridine ring

was coplanar with the thiazole and with the nitrogen of the

pyridine pointing inward; we interpreted this as a lone pair-

sulfur interaction²⁵stabilizing this conformation. The amide

side chain continued along the same axis, leaving the terminal

dimethylamino group in proximity to Asp897, which in turn

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Drug Annotation

a

Scheme 3. Macrocycle Synthesis via Buchwald−Hartwig Amination

a

i, diamine, CH₂Cl₂; ii, 4-acetamidobenzaldehyde, NaBH₃CN, AcOH, MeOH then aq CH₂O; iii, HCl (3 M aq), EtOH; iv,

H₂N(CH₂)₂N(Me)(CH₂)₂NHBOC, T3P, Et₃N, EtOAc; v, HCl, dioxane, CH₂Cl₂; vi, 30, BOCNHCH₂CHO, NaBH₃CN, AcOH, MeOH; vii,

TFA, CH₂Cl₂; viii amine (28, 29, 25, 30, 31, 27), Et₃N, CH₂Cl₂; ix, HCl (3 M aq), EtOH; x, second or third generation Xantphos precatalyst,

Na₂CO₃, DMF, 140 °C.

In following up on the results for the reversed amides, we

made a series of lactams, cyclic carbamates, and ureas. In this

exploration, we also investigated the combination of the

sulfone or sulfonamide substituent on the isoindolinone with

the pyridine substituent. Many of the resulting compounds

exhibited the levels of PI3Kδ cell activity that we had been

hoping to see without any loss of PI3Kγ activity (Table 5).

We were able to crystallize 58 in PI3Kδ (Figure 5;

Supplementary Figure S2); the molecule binds in the same

general orientation as 20. The lactam moiety is almost

coplanar with the pyridine ring and is not within contact

distance of any residues of the protein. The carbonyl group is

oriented antiparallel to the CN of the pyridine, as expected

from electrostatic eﬀects, and it appears that the carbonyl

group can make a water-mediated hydrogen bond to the

terminal amino group of the nonconserved asparagine 836

(PI3Kα Gln859, PI3Kβ Asp856, PI3Kγ Lys890). This

hydrogen bond is not possible for the earlier amide

compounds and may account for the improved potency

observed at PI3Kδ with this motif. Consistent with the crystal

structures, the SAR for PI3Kδ activity was ﬂat, with only subtle

changes apparent upon varying this group. Inhibition of

PI3Kα, however, showed larger variations, and some

compounds had quite high activity against that isoform.

Inhibition of PI3Kβ was always low (data not shown).

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Drug Annotation

a

Scheme 4. Synthesis of Amide Isosteres and Reversed Amides

a

i, acetamidine hydrochloride, HATU, DIPEA, DMF; ii, MeNHNH₂, AcOH, DMF, 80 °C; iii, ethyl 2-isocyanoacetate, Et₃N, DMAP, THF; iv,

NaBH₄, EtOH, 0 °C; v, Ac₂O, py, 120 °C; vi, second or third generation Xantphos precatalyst, Na₂CO₃, DMF, 140 °C; vii, deprotection, if

required; viii, ClCH₂CH₂NCO, toluene; ix, NaH, DMF, THF; x, Cl(CH₂)_nZCOCl, pyridine; xi, Cs₂CO₂, DMF; xii second or third generation

Xantphos precatalyst, Na₂CO₃, DMF, 120 °C.

Given the broad range of compounds with good primary

potency against PI3Kγ and PI3Kδ, we focused attention on the

margin of selectivity against PI3Kα, for which we hoped to

achieve two log units (based on the enzyme inhibition values)

and the PI3Kδ cell potency, which we wanted to be as potent

as possible, giving as a good dual PI3Kγδ proﬁle as we could

attain. On the basis of these criteria, compounds 58 and 68

were selected for further proﬁling, with compound 59,

synthesized slightly later in our program, as a back-up.

From the outset of this program, we had intended to

administer our molecule by inhalation, and thus we had not

concerned ourselves with metabolic stability, being hopeful

instead of achieving low systemic exposure. As our program

developed, we found ourselves with a set of compounds with

high lipophilicity and often high crystallinity as well. Indeed,

some compounds we made, principally those with amide NH

groups in the X substituent (Table 5), were extremely

problematic to work with as they precipitated from DMSO

stock solution.

Several approaches have been described by which extended

duration of action for inhaled molecules can be achieved.²⁶

Given the physicochemical properties that we had, we decided

that we would focus our attention on a low-solubility approach,

whereby slow dissolution of the drug substance in the lung is

used to maintain an eﬃcacious concentration over a prolonged

period, thus providing a long duration of action. To this end,

we were determined to carry out our pharmacokinetic studies

by nebulization of suspensions of crystalline material in order

that the eﬀect of the dissolution rate could be observed. In

order to achieve timely availability of suitable material for PK

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Drug Annotation

Table 1. Data for Sulfonanilides

a

b

c

d

All values are n ≥ 3 unless otherwise shown. n = 2. n = 1. Inhibition of the production of ADP by recombinant human PI3K detected by ADP-

e

f

Glo (Promega). Inhibition of phosphorylation of AKT (at Ser473) in RAW264 cells stimulated by C5a. Inhibition of phosphorylation of AKT (at

Ser473) in JeKo-1 cells stimulated anti-IgM.

experiments, we initiated a procedure whereby, as soon as

synthesis was complete and primary assays requested, all

compounds were investigated for crystallinity by X-ray powder

diﬀraction. Unless the material was already crystalline, we then

slurried it in multiple solvent systems to identify a crystalline

form that could be used for inhalation dosing, though at the

initial stage we did not explicitly seek a preferred polymorph.

With crystalline material available, we were able to determine

crystalline solubility and melting onset to give an indication of

the likely behavior of the material in the lung. Our hypothesis

was that solubility of 1 μM or less would give an extended

duration upon dosing of a nebulized suspension. Compounds

were selected for further study based on their in vitro proﬁle

and dosed by nebulization to rats to study the pharmacoki-

netics. We also ran conventional iv and po pharmacokinetic

studies in order to understand the behavior of compound once

it was absorbed into the systemic circulation and the fate of

any fraction of the dose that was swallowed (Table 6). We

were interested to understand the permeabilities of these

molecules and tested them in CaCO-2 permeability assays;

however, low recovery of the compounds meant that we were

unable to deﬁne precise values with conﬁdence (data not

shown). The results we did get suggested that the macrocycles

(35, 37) had low permeability and moderate eﬄux, while the

nonmacrocycles (58, 68) had moderate permeability and little

eﬄux.

We were pleased to ﬁnd that our design hypothesis was

borne out in reality for the compounds that we tested and that

we could obtain an extensive residence in the lung through

very low solubility. Of note, this apparent terminal t_1/2(inhaled

t

_1/2) was longer in every case than the plasma half-life

measured from iv dosing, which implies that a depot eﬀect was

accounting for the prolonged half-life, in accordance with our

hypothesis. Compounds 35, 37, and 68 had very high in vivo

clearance, being measured at or above liver blood ﬂow,

probably as a result of biliary clearance (observed for other

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Drug Annotation

Table 2. SAR of Heterocyclic Substituents

a c

,

R4

pIC₅₀PI3K

compound

R3

Me

NHMe

Me

NHMe

Het

X

γ

δ

1

5

Ac

9.1

9.0

9.1

8.5

8.9

6.5

6.8

7.3

8.3

8.4

8.2

6.8

18

19

20

21

22

A

B

A

Me

X1

X2

X3

b

8.5

a

b

c

all values are n ≥ 3 unless otherwise shown. n = 2. Inhibition of the

production of ADP by recombinant human PI3K detected by ADP-

Glo (Promega).

Figure 3. Crystal structures of compounds 7 and 12 bound in PI3Kδ.

(Top) 7 bound in mPI3Kδ (PDB 7oi4, 1.8 Å). (Bottom) 12 bound in

mPI3Kδ (PDB 7ois, 2.3 Å) Note that for each compound hydrogen

bonds can be seen from the acetylaminothiazole to the hinge Val828

and from the carbonyl of the isoindolinone to the terminal amino of

Lys779.

compounds in this series, data not shown) and, for 35 and 68,

undetectable oral bioavailability. In contrast, 58 had a more

modest, though still quite high, clearance; despite this

clearance, bioavailability of 58 was very low. For 58 biliary

clearance was only a minor factor (about 2% total clearance);

however, Caco-2 permeability (P_app≈ 0.4 but poor recovery of

the lipophilic compound makes the precise value uncertain)

was low, which, coupled with the very low solubility, places it

into BCS category 4.²⁷In combination with the moderately

high clearance, the low bioavailability is thus unsurprising.

With compounds available that gave extended lung exposure

through inhaled dosing, we were in a position to explore

pharmacodynamic eﬀects. Disease models for asthma,

particularly severe asthma, are in their infancy, so we opted

to use a mechanistic model of allergic lung inﬂammation

driven by an allergic response to ovalbumin,²⁸even though we

realized that this simplistic model did not recapitulate the full

complexity of asthma. Rats were sensitized by ip administration

of ovalbumin, then primed by inhalation of ovalbumin,

Figure 4. Crystal structure of 20 in PI3Kδ. Figure 4 X-ray structure of

compound 20 bound in mPI3Kδ (PDB 7oij, 1.7 Å). Note how the

amine at the terminus of the side chain ends up close to the

sulfonamide on the isoindolinone suggesting the possibility for

macrocyclization.

expected to result in recruitment of antigen speciﬁc T cells

and granulocytes to the lung. On the day of challenge test,

compound was administered to the lung 1 h prior to

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Table 3. Activities of Macrocyclic Dual PI3Kγδ Inhibitors

ac

,

a

PI3K enzyme IC₅₀

PI3K cell IC₅₀

d

e

compound

core

linker

γ

δ

α

γ

δ

b

32

33

34

35

36

37

F

G

L1

L2

L3

L4

L5

8.1

8.0

8.9

8.8

9.1

8.6

9.1

9.0

9.3

9.2

8.4

8.3

8.8

9.0

9.1

8.8

9.0

8.9

8.5

8.7

b

7.5

7.4

7.3

7.4

L6

a

b

c

Al values are n ≥ 3 unless otherwise shown. n = 1. Inhibition of the production of ADP by recombinant human PI3K detected by ADP-Glo

d

e

(Promega). Inhibition of phosphorylation of AKT (at Ser473) in RAW264 cells stimulated by C5a Inhibition of phosphorylation of AKT (at

Ser473) in JeKo-1 cells stimulated anti-IgM.

administration of ovalbumin to the lung. Following drug

treatment and ovalbumin challenge we waited for 48 h until

the animals were terminated and the recruitment of

eosinophils to the lung was measured by bronchioalveolar

lavage (BAL) to give an indication of the anti-inﬂammatory

eﬀect of the compound. (Figure 6)

To further analyze the eﬀects of the compounds, we could

track the progress of target engagement by measurement of the

degree of phosphorylation of the downstream target S6

ribosomal protein in the lymphocyte population at 2 h post

dosing. We were further able to measure the levels of

inﬂammatory cytokines in lung lavage ﬂuid. In both cases,

the data were consistent with the picture obtained from the

eosinophil level (Supplementary Figures S3 and S4).

In order to probe the pharmacodynamic duration of the

compounds, we ran an experiment where the compound was

dosed at various time points prior to the ovalbumin challenge

(Figure 7).

dosing of compound 58 might be required to achieve a durable

eﬀect in man.

Comparison of the four compounds that we had evaluated in

vivo was based on a balance of factors. The weakly basic

compound 35 (pK_a6.4) had the least attractive proﬁle when

tested in a panel of 150 diverse targets, showing activity at 1

μM at 16 of these, consistent with the common experience that

lipophilic amines are more likely to exhibit oﬀ-target eﬀects. In

contrast, the neutral compounds 37 (8 targets inhibited at 1

μM), 58 (7), and 68 (5) were less promiscuous (Supple-

mentary Table S2).

Kinase selectivity for the compounds was evaluated in a

panel of 348 protein kinases at 1 μM; however, none of the

compounds showed >50% activity against any of the tested

kinases (full details in Supplementary Data Table S3 and

Figure S5). Compounds 37, 58, and 68 were also evaluated in

a panel of lipid kinases; beyond the activity at class I PI3Ks, we

also observed some inhibition of class 2 PI3Ks (Table 7). The

activity against class 2 PI3Ks, PIK3C2B, and PIK3C2G was

signiﬁcant; however, we decided to take a compound forward

with these activities as a potential risk.

In this experiment, we saw that we obtained statistically

signiﬁcant inhibition with dosing 7 h prior to challenge and a

weaker eﬀect with 12 h predosing. Dosing 24 h before

challenge had no eﬀect. These data suggested that twice-daily

The synthetic complexity of the macrocycles was an

additional factor in our prioritization since this was anticipated

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Drug Annotation

Table 4. Amide Replacements

Table 5. Lactams, Carbamates, and Ureas

ab

,

pIC50

PI3Kγ PI3Kδ PI3Kγ cell PI3Kδ cell

compound

X

R3

Me

52

53

54

55

56

57

H

I

J

K

L

9.0

9.2

9.0

9.1

8.8

7.6

8.7

7.8

8.9

9.1

NHMe

9.0

8.2

a

b

All values are n ≥ 3. Inhibition of the production of ADP by

recombinant human PI3K detected by ADP-Glo (Promega).

to result in delays and costs during the development process.

Compound 58 was selected as having the best overall proﬁle

and was advanced to preclinical studies.

PI3K enzyme

ab d

, ,

b

IC₅₀

PI3K cell IC₅₀

e

f

compound

X

R3

Me

NHOx

Me

NHMe

Me

NHMe

Me

NHMe

NHOx

Me

NHMe

δ

α

γ

δ

In order to build conﬁdence in our inhibitor for the

treatment of human disease, we carried out studies using blood

obtained from GINA 3 or 4 asthmatic patients. In peripheral

blood mononuclear cells (PBMCs), a PI3Kδ response was

obtained through stimulation of T cell antigen receptor with a

mixture of antibodies to CD2, 3, and 28 for 24 h. Inhibition of

this response was measured by detection of the impact of δ, γ

and dual γ δ inhibitors on the release of IL-5 and IL-17. In

contrast, eosinophils from the same patients’ blood was treated

with eotaxin-3 (CCL26) to produce a PI3Kγ-driven eﬀect,

observable by the appearance of CD11b expression, that can be

measured by ﬂow cytometry. Both these eﬀects could be

inhibited by the dual PI3Kγδ inhibitor 58, in contrast to a pure

PI3Kδ inhibitor (PI3Kδ pK_i9.9; PI3Kγ pIC₅₀5.2)²⁹or a pure

PI3Kγ inhibitor (PI3Kδ pIC₅₀< 6.1; PI3Kγ pIC₅₀9.1) (1),

each of which could only eﬀectively inhibit one of the

responses (Figure 8).

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

M

N

O

P

Q

R

S

9.2

9.1

9.2

9.1

9.3

8.9

9.0

9.2

9.0

8.9

9.2

9.1

8.8

7.2

6.9

7.5

7.4

7.8

7.4

7.5

7.7

7.3

7.4

7.0

7.7

7.4

9.1

9.0

8.7

9.0

8.6

8.7

8.8

9.0

9.1

9.0

8.9

9.0

8.4

8.9

8.4

8.3

8.5

8.4

8.1

7.9

8.2

8.1

8.4

8.6

8.2

S

T

U

V

c

a

All compounds reached the maximum inhibition in the PI3Kγ

b

enzyme inhibition assay. All values are n ≥ 3 unless otherwise shown.

c

d

CONCLUSION

n = 2. Inhibition of the production of ADP by recombinant human

■

e

PI3K detected by ADP-Glo (Promega). Inhibition of phosphor-

We have described how, through SAR exploration and paying

particular attention to cellular activity, we went from a highly

selective PI3Kγ inhibitor to a series of dual PI3Kγδ inhibitors.

On the basis of the physicochemical properties of the series

and our desire for inhaled administration, we focused on

making crystalline material and understanding solid state

properties. Administration of compounds to rats by nebu-

lization showed prolonged retention of compounds in the

lungs and extended pharmacodynamic eﬀects in a mechanistic

model, building conﬁdence in the suitability of the identiﬁed

ylation of AKT (at Ser473) in RAW264 cells stimulated by C5a.

Inhibition of phosphorylation of AKT (at Ser473) in JeKo-1 cells

f

stimulated anti-IgM.

compounds for investigation as inhaled agents. Studies using

human patient derived blood cells demonstrated a potential

beneﬁt of dual PI3Kγδ inhibition. On the basis of excellent

potency and good in vivo PK with long duration of action

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Drug Annotation

added, and the mixture was stirred overnight. Solvent was evaporated,

and the residue was puriﬁed by preparative reverse phase HPLC.

Typically a 5 μM Xbridge C-18 19 × 150 mm column was used

running a gradient of acetonitrile in 0.05% TFA in water at 20 mL/

min.

The following compounds were prepared according to general

procedure A and the appropriate aniline:

N-(5-{2-[(1S)-1-Cyclopropylethyl]-1-oxo-7-(phenylsulfamoyl)-

2,3-dihydro-1H-isoindol-5-yl}-4-methyl-1,3-thiazol-2-yl)acetamide

1

(6). H NMR (500 MHz, DMSO-d₆) δ 12.30 (s, 1H; exchangeable

NH and reduced to ∼60%), 10.14 (s, 1H; exchangeable NH and

reduced to ∼72%), 7.96 (d, J = 1.5 Hz, 1H), 7.81 (d, J = 1.5 Hz, 1H),

7.23 (dd, J = 8.5, 7.3 Hz, 2H), 7.12−7.07 (m, 2H), 7.07−7.01 (m,

1H), 4.80−4.67 (m, 2H), 3.70 (dq, J = 9.3, 6.8 Hz, 1H), 2.36 (s, 3H),

2.16 (s, 3H), 1.36 (d, J = 6.8 Hz, 3H), 1.24−1.13 (m, 1H), 0.67−0.58

(m, 1H), 0.50−0.40 (m, 2H), 0.39−0.30 (m, 1H).

¹³C NMR (126 MHz, DMSO-d₆) δ 168.8, 164.6, 156.4, 146.1,

144.9, 137.1, 136.1, 135.8, 129.4, 127.4, 126.9, 125.8, 125.0, 121.5,

120.5, 52.8, 46.5, 22.5, 18.1, 16.3, 15.7, 4.0, 3.4.

ES+ (M + H)⁺observed 511.1487 expected 511.1474.

N-[5-(2-[(1S)-1-Cyclopropylethyl]-7-{[3-(methylsulfonyl)phenyl]-

sulfamoyl}-1-oxo-2,3-dihydro-1H-isoindol-5-yl)-4-methyl-1,3-thia-

Figure 5. Crystal structure of 58 bound to PI3Kδ. X-ray view of 58

bound in PI3Kδ (PDB code 7oil, 2.0 Å). The binding mode is

essentially identical to earlier compounds. The lactam can be seen to

make a water-mediated hydrogen bond to the terminal NH₂of

Asp836, which itself is in a network of hydrogen bonds with Thr833.

Other residues shown are Trp760 and Met752 that form the other

side of the pocket around the lactam and Lys779 that forms H-bonds

with the carbonyl of the isoindolinone and the sulfone.

1

zol-2-yl]acetamide (7). H NMR (600 MHz, DMSO-d₆) δ 12.29 (s,

1H), 10.55 (s, 1H), 7.97 (d, J = 1.7 Hz, 1H), 7.90 (d, J = 1.7 Hz, 1H),

7.63 (t, J = 2.0 Hz, 1H), 7.59 (d, J = 7.8 Hz, 1H), 7.53 (t, J = 8.0 Hz,

1H), 7.48−7.43 (m, 1H), 4.80−4.65 (m, 2H), 3.69 (dq, J = 9.3, 6.8

Hz, 1H), 3.12 (s, 3H), 2.37 (s, 3H), 2.16 (s, 3H), 1.35 (d, J = 6.8 Hz,

3H), 1.22−1.12 (m, 1H), 0.65−0.55 (m, 1H), 0.52−0.38 (m, 2H),

0.38−0.29 (m, 1H).

¹³C NMR (151 MHz, DMSO-d₆) δ 168.74, 164.31, 156.44, 146.21,

144.93, 141.86, 138.14, 135.81, 135.59, 130.74, 127.71, 127.08,

125.82, 124.41, 122.90, 121.44, 117.69, 52.75, 46.47, 43.37, 22.45,

17.96, 16.33, 15.57, 3.95, 3.37.

coupled with an acceptable oﬀ-target proﬁle, compound 58

(AZD8154) was selected for development.

ES+ (M + H)⁺observed 589.1263 expected 589.1249.

EXPERIMENTAL SECTION

N-(5-{7-[(3-Cyanophenyl)sulfamoyl]-2-[(1S)-1-cyclopropylethyl]-

■

1-oxo-2,3-dihydro-1H-isoindol-5-yl}-4-methyl-1,3-thiazol-2-yl)-

All reagents obtained from commercial sources were used without

further puriﬁcation; anhydrous solvents were used without further

drying. All compounds were puriﬁed to ≥95% purity as judged by

HPLC with UV and MS analysis. When Cl or Br was present,

expected isotopic distribution patterns were observed. Proton (¹H)

and carbon (¹³C) NMR spectra were recorded at ambient

temperature. Solutions were typically prepared in deuterated dimethyl

sulfoxide (DMSO-d₆), deuteromethanol (CD₃OD), or deuterochloro-

form (CDCl₃) with chemical shifts referenced to solvent as an internal

standard. ¹H NMR data are reported indicating the chemical shift (δ),

the multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m,

multiplet; br, broad; dd, doublet of doublets; etc.), the coupling

constant (J) in Hz, and the integration (e.g., 1H). ¹³C NMR spectra

are reported with proton decoupling.

1

acetamide (8). H NMR (600 MHz, DMSO-d₆) δ 12.30 (s, 1H),

10.48 (s, 1H), 7.98 (d, J = 1.5 Hz, 1H), 7.89 (d, J = 1.6 Hz, 1H),

7.52−7.40 (m, 4H), 4.78−4.68 (m, 2H), 3.72−3.64 (m, 1H), 2.38 (s,

3H), 2.16 (s, 3H), 1.36 (d, J = 6.8 Hz, 3H), 1.22−1.14 (m, 1H),

0.65−0.58 (m, 1H), 0.49−0.40 (m, 2H), 0.40−0.32 (m, 1H).

¹³C NMR (151 MHz, DMSO-d₆) δ 168.8, 164.3, 156.5, 146.2,

144.9, 138.2, 135.8, 135.4, 130.9, 128.22, 127.8, 127.2, 125.9, 124.5,

122.8, 121.4, 118.0, 112.1, 52.7, 46.5, 22. 5, 18.0, 16.3, 15.7, 3.9, 3.3.

ES+ (M + H)⁺observed 536.1440 expected 536.1426.

N-(5-{7-[(3-Cyanophenyl)(methyl)sulfamoyl]-2-[(1S)-1-cyclopro-

pylethyl]-1-oxo-2,3-dihydro-1H-isoindol-5-yl}-4-methyl-1,3-thiazol-

2-yl)acetamide (9). ¹H NMR (600 MHz, DMSO-d₆) δ 12.27 (s, 1H),

7.99 (d, J = 1.5 Hz, 1H), 7.78 (d, J = 1.5 Hz, 1H), 7.73 (t, J = 1.9 Hz,

1H), 7.66 (dt, J = 7.8, 1.4 Hz, 1H), 7.63 (ddd, J = 8.3, 2.4, 1.1 Hz,

1H), 7.51 (t, J = 8.0 Hz, 1H), 4.67−4.59 (m, 2H), 3.58 (dq, J = 9.2,

6.8 Hz, 1H), 3.41 (s, 3H), 2.29 (s, 3H), 2.16 (s, 3H), 1.28 (d, J = 6.8

Hz, 3H), 1.15−1.08 (m, 1H), 0.62−0.55 (m, 1H), 0.47−0.36 (m,

2H), 0.28−0.21 (m, 1H).

General Procedure A − Synthesis of Sulfonamides from

Sulfonyl Chloride 4. (S)-6-(2-Acetamido-4-methylthiazol-5-yl)-2-

(1-cyclopropylethyl)-3-oxoisoindoline-4-sulfonyl chloride²³(4, 80

mg) was dissolved in a mixture of dichloromethane (3 mL) and

pyridine (42 μL, 2 equiv). The appropriate aniline (1 equiv) was

Table 6. Physicochemical Properties and PK Data for Advanced Compounds

b

rat PK

inhaled

iv

po

c

compound crystalline solubility (phosphate buﬀer pH 7.4) μM melting onset °C LogD pH 7.4 t_1/2

h

V_ss(L/kg) CL mL/min/kg t_1/2

h

F %

a

35

37

58

68

1.09

349

377

285

187

3.9

3.7

4.4

3.5

7.5

7.3

8.6

4.0

5.4

83

110

20

1.8

ND

NT

<4

0.084

0.054

0.337

6.9

7.1

4.2

1.8

2.9

0.7

136

ND

a

b

ND = not detected; NT = not tested. PK experiments are the mean of values obtained for at least two animals. Compound was dosed at 0.5 mg/

c

kg iv and 1.0 mg/kg po. Material of known crystallinity was suspended in pH 7.4 phosphate buﬀer at 1 mg/mL and stirred at 1100 rpm for 24 h.

Solid material was removed by centrifugation and the concentration of substrate was determined by HPLC MS.

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Drug Annotation

Figure 6. (A−D) Inhibition of eosinophil migration in response to an inhaled ovalbumin challenge. Rats (BN, male, 10 per group) were sensitized

to ovalbumin by ip administration on two separate occasions, then challenged with aerosolized administration of ovalbumin and, after a week dosed

with compound by nebulization before subsequent challenge with aerosolized ovalbumin. After 48 h the animals were terminated, the lungs

lavaged, and the numbers of eosinophils in the BAL ﬂuid were measured by ﬂow cytometry. Budesonide (300 μg, dosed i.t.) was used as positive

control. Individual animals are represented by points with the average eﬀect shown in bars. Compound amounts represent the administered dose of

compound.

¹³C NMR (151 MHz, DMSO-d₆) δ 168.7, 162.7, 156.3, 146.4,

144.5, 142.0, 135.1, 134.3, 130.3, 130.2, 129.9, 129.4, 128.3, 128.0,

127.8, 121.6, 118.1, 111.8, 51.9, 45.5, 37.9, 22.4, 18.0, 16.1, 15.6, 3.9,

3.3.

= 6.7 Hz, 3H), 1.17 (dd, J = 8.2, 4.4 Hz, 1H), 0.61 (dt, J = 8.6, 4.1 Hz,

1H), 0.38−0.48 (m, 2H), 0.3−0.37 (m, 1H).

¹³C NMR (151 MHz, DMSO-d₆) δ 169.2, 164.5, 156.9, 146.7,

145.1, 144.1, 143.5, 134.8, 127.9, 127.0, 126.0, 123.4, 121.9, 119.3,

115.4, 52.7, 43.4, 22.9, 18.4, 16.8, 16.1, 4.4, 3.8.

ES+ (M + H)⁺observed 550.1573 expected 550.1583.

N-[5-(7-{[3-Cyano-5-(methylsulfonyl)phenyl]sulfamoyl}-2-[(1S)-

1-cyclopropylethyl]-1-oxo-2,3-dihydro-1H-isoindol-5-yl)-4-methyl-

ES+ (M + H)⁺observed 614.1219 expected 614.1202.

N-(5-{2-[(1S)-1-Cyclopropylethyl]-7-({3-[(dimethylamino)-

methyl]phenyl}sulfamoyl)-1-oxo-2,3-dihydro-1H-isoindol-5-yl}-4-

methyl-1,3-thiazol-2-yl)acetamide (11). ¹H NMR (600 MHz,

DMSO-d₆) δ 12.28 (s, 1H), 10.01 (s, 1H), 7.95 (d, J = 1.2 Hz,

1

1,3-thiazol-2-yl]acetamide (10). H NMR (600 MHz, DMSO-d₆) δ

12.30 (s, 1H), 10.90 (s, 1H), 7.67−8.13 (m, 5H), 4.70 (s, 2H), 3.66

(p, J = 6.8 Hz, 1H), 3.22 (s, 3H), 2.38 (s, 3H), 2.17 (s, 3H), 1.34 (d, J

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Drug Annotation

Figure 7. Experiment showing duration of eﬀect of nebulized 58 in

rats. Compound 58 was dosed by nebulization (240 μg/kg

administered dose) to male rats (BN, 8−10 per group) that had

been sensitized to ovalbumin at varying intervals prior to ovalbumin

challenge. 48 h after challenge, the animals were terminated, the lungs

lavaged, and the numbers of eosinophils in the lung lavage ﬂuid were

measured by ﬂow cytometry, and the results were compared to time-

matched controls.

Table 7. Inhibition of Lipid Kinases

bd

,

pIC₅₀

example PIK3C2A PIK3C2B PIK3C2G PIK3C3 PI4KA PI4KB

c

a

c

37

58

68

6.5

5.4

5.5

9.1

8.5

9.0

NT

7.2

7.6

6.3

7.5

<5.0

7.7

5.5

6.3

NT

a

b

c

d

NT = not tested. n = 3 unless otherwise shown. n = 1. FRET

Adapta assays measuring inhibition of phosphorylation of a synthetic

substrate run by Thermo Fisher Scientiﬁc.

1H), 7.78 (d, J = 1.4 Hz, 1H), 7.17 (t, J = 7.8 Hz, 1H), 7.03−7.01 (m,

1H), 6.99 (d, J = 8.0 Hz, 1H), 6.92 (d, J = 7.6 Hz, 1H), 4.73 (s, 2H),

3.75−3. 67 (m, 1H), 3.23 (q, J = 13.2 Hz, 2H), 2.35 (s, 3H), 2.16 (s,

3H), 1.96 (s, 6H), 1.37 (d, J = 6.8 Hz, 3H), 1.23−1.15 (m,1H),

0.66−0.60 (m, 1H), 0.50−0.43 (m, 2H), 0.37−0.31 (m, 1H).

¹³C NMR (151 MHz, DMSO-d₆) δ 169.2, 165.1, 156.9, 146.5,

145.2, 140.7, 137.5, 136.3, 136.2, 129.5, 127.8, 127.6, 126.3, 125.8,

121.9, 121.1, 119.9, 63.4, 53.2, 46.9, 45.2, 22.9, 18.5, 16.7, 16.1, 4.5,

3.9.

ES+ (M + H)⁺observed 568.2058 expected 568.2052.

N-(5-{2-[(1S)-1-Cyclopropylethyl]-7-({4-[(dimethylamino)-

methyl]phenyl}sulfamoyl)-1-oxo-2,3-dihydro-1H-isoindol-5-yl}-4-

methyl-1,3-thiazol-2-yl)acetamide (12). ¹H NMR (600 MHz,

DMSO-d₆) δ 12.28 (s, 1H), 10.10 (s, 1H), 7.96 (d, J = 1.6 Hz,

1H), 7.79 (d, J = 1.6 Hz, 1H), 7.15−7.11 (m, 2H), 7.05−7.01 (m,

2H), 4.80−4.70 (m, 2H), 3.71 (dq, J = 9.4, 6.8 Hz, 1H), 3.21 (s, 2H),

2.35 (s, 3H), 2.16 (s, 3H), 2.03 (s, 6H), 1.37 (d, J = 6.8 Hz, 3H),

1.24−1.15 (m, 1H), 0.64−0.59 (m, 1H), 0.51−0.41 (m, 2H), 0.38−

0.31 (m, 1H).

Figure 8. PI3K isoform speciﬁc inhibition of T cell cytokine release

and eosinophil surface CD11b expression, using PBMC and puriﬁed

granulocytes from severe asthma patients (GINA 3 and 4). Eﬀects of

PI3Kγ (Cpd 1), PI3Kδ (Nemiralisib), or PI3Kγδ dual (Cpd 58)

selective inhibitors on (A) IL-5, (B) IL-17 release after anti-CD2/3/

28 stimulation of PBMCs, and (C) CD11b surface expression on

blood-derived eosinophils stimulated with eotaxin-3 measured by ﬂow

cytometry. The impact of treatment is visualized as %-inhibition.

Error bars depicts SEM, asthmatic donors N = 6−10.

¹³C NMR (151 MHz, DMSO-d₆) δ 168.7, 164.7, 156.4, 146.1,

144.8, 136.1, 135.8, 135.7, 135.7, 129.6, 127.4, 126.8, 125.8, 121.5,

120.5, 62.6, 52.7, 46.5, 44.9, 22. 5, 18.0, 16.3, 15.7, 4.0, 3.4.

ES+ (M + H)⁺observed 568.2070 expected 568.2052.

N-(5-{2-[(1S)-1-cyclopropylethyl]-7-[{4-[(dimethylamino)-

methyl]phenyl}(methyl)sulfamoyl]-1-oxo-2,3-dihydro-1H-isoindol-

5-yl}-4-methyl-1,3-thiazol-2-yl)acetamide (13). ¹H NMR (600

MHz, DMSO-d₆) δ 12.24 (s, 1H), 7.93 (d, J = 1.6 Hz, 1H), 7.65

(d, J = 1.6 Hz, 1H), 7.21−7.16 (m, 2H), 7.16−7.11 (m, 2H), 4.67−

4.58 (m, 2H), 3.63 (dq, J = 9.2, 6.8 Hz, 1H), 3.43 (s, 3H), 3.35 (s,

2H), 2.22 (s, 3H), 2.15 (s, 3H), 2.08 (s, 6H), 1.30 (d, J = 6.8 Hz,

3H), 1.18−1.09 (m, 1H), 0.63−0.56 (m, 1H), 0.47−0.37 (m, 2H),

0.32−0.24 (m, 1H).

¹³C NMR (151 MHz, DMSO-d₆) δ 168.7, 163.7, 162.9, 156.2,

146.2, 144.3, 139.9, 136.8, 135.2, 134.8, 129.4, 127.7, 127.4, 125.5,

121.7, 62.3, 51.9, 45.4, 44.6, 38.7, 22.4, 19.0, 16.1, 15.6, 4.0, 3.3.

ES+ (M + H)⁺observed 582.2244 expected 582.2209.

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Drug Annotation

N-(5-{7-[(4-cyanophenyl)sulfamoyl]-2-[(1S)-1-cyclopropylethyl]-

1-oxo-2,3-dihydro-1H-isoindol-5-yl}-4-methyl-1,3-thiazol-2-yl)-

¹³C NMR (151 MHz, DMSO-d₆) δ 164.7, 158.08, 155.3, 150.5,

145.9, 144.9, 138.3, 136.7, 136.0, 126.7, 126.3, 125.1, 119.7, 115.3,

107.8, 52.3, 46.3, 29.2, 23.4, 17.9, 16.7, 15.5, 3.9, 3.4.

1

acetamide (14). H NMR (500 MHz, DMSO-d₆) δ 12.32 (s, 1H),

ES+ (M + H)⁺observed 498.1617 expected 498.1633.

10.63 (br. s, 1H), 7.99 (d, J = 1.5 Hz, 1H), 7.93 (d, J = 1.6 Hz, 1H),

7.74−7.67 (m, 2H), 7.31−7.25 (m, 2H), 4.78−4.65 (m, 2H), 3.66

(dq, J = 9.2, 6.8 Hz, 1H), 2.38 (s, 3H), 2.17 (br. s, 3H), 1.33 (d, J =

6.8 Hz, 3H), 1.20−1.11 (m, 1H), 0.65−0.56 (m, 1H), 0.48−0.36 (m,

2H), 0.36−0.27 (m, 1H).

(S)-6-((5-(2-(1-Cyclopropylethyl)-7-(methylsulfonyl)-1-oxoisoin-

dolin-5-yl)-4-methylthiazol-2-yl)amino)-N-(2-methoxyethyl)-

1

picolinamide (19). H NMR (500 MHz, DMSO-d₆) δ 11.88 (s, 1H;

exchangeable NH and reduced to ∼57%), 8.15 (br. t, J = 5.6 Hz, 1H),

8.08 (s, 2H), 7.92 (t, J = 7.9 Hz, 1H), 7.61 (d, J = 7.4 Hz, 1H), 7.26

(d, J = 8.3 Hz, 1H), 4.83−4.57 (m, 2H), 3.67−3.59 (m, 4H), 3.59−

3.54 (m, 2H), 3.54−3.48 (m, 2H), 3.04 (s, 3H), 2.47 (s, 3H), 1.32 (d,

J = 6.8 Hz, 3H), 1.24−1.09 (m, 1H), 0.67−0.52 (m, 1H), 0.51−0.35

(m, 2H), 0.34−0.20 (m, 1H).

¹³C NMR (126 MHz, DMSO-d₆) δ 168.8, 164.1, 156.5, 146.3,

145.0, 141.8, 135.7, 135.6, 133.8, 127.8, 127.2, 126.0, 121.5, 119.2,

118.6, 106.2, 52.7, 46.4, 22.5, 18.0, 16.4, 15.7, 3.9, 3.4.

ES+ (M + H)⁺observed 536.1389 expected 536.1426.

N-[5-(2-[(1S)-1-Cyclopropylethyl]-7-{[4-(methylsulfonyl)phenyl]-

sulfamoyl}-1-oxo-2,3-dihydro-1H-isoindol-5-yl)-4-methyl-1,3-thia-

zol-2-yl]acetamide (15). ¹H NMR (600 MHz, DMSO-d₆) δ 12.31 (s,

1H), 10.69 (s, 1H), 8.00 (s, 1H), 7.95 (d, J = 1.2 Hz, 1H), 7.80 (d, J =

8.7 Hz, 2H), 7.35 (d, J = 8.7 Hz, 2H), 4.73 (s, 2H), 3.73−3. 65 (m,

1H), 3.12 (s, 3H), 2.40 (s, 3H), 2.18 (s, 3H), 1.35 (d, J = 6.8 Hz,

3H), 1.18−1.14 (m,1H), 0.65−0.59 (m, 1H), 0.48−0.40 (m, 2H),

0.37−0.31 (m, 1H).

¹³C NMR (151 MHz, DMSO-d₆) δ 163.7, 163.5, 157.2, 150.4,

147.1, 145.8, 145.2, 139.5, 137.7, 135.8, 127.4, 126.9, 126.6, 119.5,

114.7, 114.5, 70.4, 57.6, 52.1, 45.9, 43.1, 38.5, 18.0, 16.6, 15.6, 3.9,

3.4.

ES+ (M + H)⁺observed 570.1837 expected 570.1844.

(S)-6-((5-(2-(1-Cyclopropylethyl)-7-(N-methylsulfamoyl)-1-oxoi-

soindolin-5-yl)-4-methylthiazol-2-yl)amino)-N-(3-(dimethylamino)-

1

¹³C NMR (151 MHz, DMSO-d₆) δ 169.2, 164.7, 156.9, 151.8,

146.8, 145.4, 142.4, 136.4, 136.2, 129.2, 128.2, 127.6, 126.4, 122.0,

119.5, 53.1, 46.9, 44.0, 22.9, 18.4, 16.8, 16.1, 4.4, 3.9.

ES+ (M + H)⁺observed 589.1236 expected 589.1249.

(S)-5-(2-Amino-4-methylthiazol-5-yl)-2-(1-cyclopropylethyl)-7-

(methylsulfonyl)isoindolin-1-one (16). 1 (2.28 g, 5.26 mmol) was

suspended in EtOH (11.5 mL). Hydrochloric acid (3.8 M, 13.8 mL,

52.6 mmol) was added to the slurry. The reaction mixture was stirred

at reﬂux for 20 h. The reaction mixture was allowed to come to RT

and was then cooled to 0 °C. The mixture was neutralized with 2 M

NaOH and was then extracted with DCM (100 mL; 2 × 50 mL), and

the combined organic layers were dried through a phase separator and

concentrated to dryness to give the title compound (1.76 g, 86%) as a

yellow solid.

propyl)picolinamide (20). H NMR (600 MHz, DMSO-d₆) δ 11.90

(bs, 1H), 9.47 (bs, 1H), 8.24 (t, J = 6.3 Hz, 1H), 8.08 (d, J = 1.3 Hz,

1H), 7.96 (d, J = 1.5 Hz, 1H), 7.96−7.92 (m, 1H), 7.63 (q, J = 5.2

Hz, 1H), 7.62−7.59 (m, 1H), 7.30−7.27 (m, 1H), 4.76 (s, 2H),

3.69−3.63 (m, 1H), 3.51 (q, J = 6.7 Hz, 2H), 3.16−3.08 (m, 2H),

2.74 (s, 3H), 2.73 (s, 3H), 2.53 (d, J = 5.2 Hz, 3H), 2.51 (s, 3H),

2.00−1.92 (m, 2H), 1.33 (d, J = 6.8 Hz, 3H), 1.21−1.14 (m, 1H),

0.65−0. 59 (m, 1H), 0.48−0.4 (m, 2H), 0.31−0.26 (m, 1H).

¹³C NMR (151 MHz, DMSO-d₆) δ 165.1, 164.8, 157.7, 150.8,

147.8, 146.4, 145.9, 140.0, 136.6, 136.6, 127.3, 126.6, 125.8, 119.9,

115.4, 114.9, 55.2, 52.9, 46.9, 42.7, 36.7, 29.7, 25.2, 18.4, 17.2, 16.0,

4.4, 3.8.

ES+ (M + H)⁺observed 612.2409 expected 612.2427.

(S)-6-((5-(2-(1-Cyclopropylethyl)-7-(N-methylsulfamoyl)-1-oxoi-

soindolin-5-yl)-4-methylthiazol-2-yl)amino)-N-(3-(dimethylamino)-

¹H NMR (500 MHz, DMSO-d₆) δ 7.9−7.86 (m, 2H), 7.31 (s,

2H), 4.66 (s, 2H), 3.61 (m, 4H), 2.30 (s, 3H), 1.29 (d, J = 6.8 Hz,

3H), 1.17−1.09 (m, 1H), 0.62−0.56 (m, 1H), 0.46−0.36 (m, 2H),

0.29−0.23 (m, 1H).

1

propyl)nicotinamide (21). H NMR (600 MHz, DMSO-d₆) δ 11.81

(s, 1H), 8.84 (d, J = 2.3 Hz, 1H), 8.44 (t, J = 5.6 Hz, 1H), 8.12 (dd, J

= 8.7, 2.4 Hz, 1H), 8.01 (d, J = 1.6 Hz, 1H), 7.89 (d, J = 1.6 Hz, 1H),

7.61 (s, 1H), 7.09 (d, J = 8.6 Hz, 1H), 4.75 (s, 2H), 3.65 (dq, J = 9.2,

6.8 Hz, 1H), 3.27 (td, J = 7.2, 5.6 Hz, 2H), 2.52 (s, 3H), 2.47 (s, 3H),

2.25 (t, J = 7.1 Hz, 2H), 2.13 (s, 6H), 1.65 (p, J = 7.1 Hz, 2H), 1.32

(d, J = 6.8 Hz, 3H), 1.20−1.11 (m, 1H), 0.64−0.57 (m, 1H), 0.47−

0.37 (m, 2H), 0.32−0.25 (m, 1H).

¹³C NMR (126 MHz, DMSO-d₆) δ 166.9, 163.7, 146.8, 145.8,

137.5, 136.3, 126.3, 125.8, 125.7, 115.4, 51.9, 45.8, 43.0, 17.9, 16.8,

15.5, 3.8, 3.4.

ES+ (M + H)⁺392.

¹³C NMR (151 MHz, DMSO-d₆) δ 164.7, 164.3, 157.6, 152.7,

146.2, 145.9, 145.1, 137.0, 136.3, 136.0, 126.7, 126.3, 125.3, 122.7,

120.4, 110.3, 56.8, 52.4, 46.4, 45.2, 37.6, 29.2, 27.1, 18.0, 16.6, 15.5,

3.9, 3.4.

(S)-6-(2-Amino-4-methylthiazol-5-yl)-2-(1-cyclopropylethyl)-N-

methyl-3-oxoisoindoline-4-sulfonamide (17). Prepared from 5

analogously to 16.

¹H NMR (500 MHz, DMSO-d₆) δ 9.25 (s, 1H), 7.98 (d, J = 1.4

Hz, 1H), 7.80 (d, J = 1.5 Hz, 1H), 7.56 (q, J = 5.1 Hz, 1H), 4.75 (s,

2H), 3.69−3.61 (m, 1H), 2.51 (s, 3H), 2.34 (s, 3H), 1.32 (d, J = 6.8

Hz, 3H), 1.21−1.13 (m, 1H), 0.65−0.57 (m, 1H), 0.48−0.38 (m,

2H), 0.31−0.24 (m, 1H).

ES+ (M + H)⁺observed 612.2415 expected 612.2427.

(S)-6-((5-(2-(1-Cyclopropylethyl)-7-(N-methylsulfamoyl)-1-oxoi-

soindolin-5-yl)-4-methylthiazol-2-yl)amino)-N,N-diethylpicolina-

mide (22). ¹H NMR (500 MHz, DMSO-d₆) δ 11.75 (s, 1H), 7.98 (d,

J = 1.5 Hz, 1H), 7.86 (d, J = 1.5 Hz, 1H), 7.84 (t, J = 7.8 Hz, 1H),

7.59 (q, J = 5.2 Hz, 1H), 7.11 (d, J = 8.4 Hz, 1H), 7.02 (d, J = 7.4 Hz,

1H), 4.75 (s, 2H), 3.65 (dq, J = 9.2, 6.8 Hz, 1H), 3.49 (q, J = 7.0 Hz,

2H), 3.35−3.30 (m, 2H), 2.51 (s, 3H), 2.47 (s, 3H), 1.32 (d, J = 6.8

Hz, 3H), 1.22−1.12 (m, 4H), 1.03 (t, J = 7.1 Hz, 3H), 0.65−0.56 (m,

1H), 0.50−0.37 (m, 2H), 0.34−0.23 (m, 1H).

ES+ (M + H)⁺407.

General Procedure B, Arylations of Amines 16 and 17. (S)-

6-(2-Amino-4-methylthiazol-5-yl)-2-(1-cyclopropylethyl)-N-methyl-

3-oxoisoindoline-4-sulfonamide (150 mg, 0.37 mmol), halohetero-

cycle (0.44 mmol, 1.2 equiv), potassium phosphate (157 mg, 0.74

mmol, 2 equiv), PdOAc₂(41 mg, 0.18 mmol, 0.5 equiv), and

Xantphos (214 mg, 0.37 mmol. 1 equiv) were mixed in dioxane (10

mL) and sealed into a microwave tube. The reaction was heated to

120 °C for 60 min in a microwave reactor. The solvent was removed

under reduced pressure, and the crude product was puriﬁed by

preparative HPLC.

¹³C NMR (151 MHz, DMSO-d₆) δ 167.3, 164.7, 157.8, 152.3,

150.1, 145.9, 145.1, 139.1, 136.4, 136.1, 126.8, 126.0, 125.2, 120.1,

114.1, 111.1, 52.4, 46.3, 42.3, 38.5, 29.1, 17.9, 16.6, 15.5, 14.3, 12.8,

3.9, 3.4.

ES+ (M + H)⁺observed 583.2151 expected 583.2161.

N-(3-Aminopropyl)-6-chloropicolinamide (23). Propane-1,3-dia-

mine (1.6 g, 21.5 mmol) was added to ethyl 6-chloropicolinate (1.0 g,

5.4 mmol) in DCM (10 mL). The resulting mixture was stirred at RT

overnight, and then the solvent was evaporated. The residue was

puriﬁed by reverse phase C18-ﬂash chromatography, elution gradient

0 to 100% MeCN in water to give the title compound (1.1 g, 96%) as

a brown oil.

(S)-2-(1-Cyclopropylethyl)-N-methyl-6-(4-methyl-2-((6-methyl-

pyridin-2-yl)amino)thiazol-5-yl)-3-oxoisoindoline-4-sulfonamide

1

(18). H NMR (600 MHz, DMSO-d₆) δ 11.46 (s, 1H), 7.98 (d, J =

1.6 Hz, 1H), 7.89 (d, J = 1.6 Hz, 1H), 7.65−7.58 (m, 2H), 6.87 (d, J

= 8.2 Hz, 1H), 6.82 (d, J = 7.3 Hz, 1H), 4.76 (s, 2H), 3.65 (dq, J =

9.2, 6.8 Hz, 1H), 2.52 (d, J = 5.2 Hz, 3H), 2.48 (s, 3H), 2.44 (s, 3H),

1.32 (d, J = 6.8 Hz, 3H), 1.20−1.12 (m, 1H), 0.64−0.57 (m, 1H),

0.48−0.38 (m, 2H), 0.31−0.25 (m, 1H).

ES+ [M + H]⁺214/216.

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Journal of Medicinal Chemistry

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Drug Annotation

N-(4-Aminobutyl)-6-bromopicolinamide hydrochloride (24).

Step 1: tert-Butyl (4-(6-bromopicolinamido)butyl)carbamate

6-Bromopicolinic acid (2.0 g, 9.90 mmol) was dissolved in ethyl

acetate (20 mL). Triethylamine (3.0 mL, 21.8 mmol) was added

followed by tert-butyl (4-aminobutyl)carbamate (2.05 g, 10.9 mmol)

and then 1-propanephosphonic acid cyclic anhydride (50% in EtOAc)

(8.8 mL, 14.8 mmol). The resultant hazy solution was stirred

overnight at RT. The reaction was quenched with water, and the

layers were separated. The organic layer was washed with water and

then with a sat. solution of sodium bicarbonate and then ﬁnally with

dilute hydrochloric acid (<1 N). The organic layer was dried over

sodium sulfate, ﬁltered, and concentrated to obtain the subtitle

compound (3.50 g, 95%) as a light yellow colored gum which slowly

crystallized into a solid.

Step 2 N-(3-((4-acetamidobenzyl)(methyl)amino)propyl)-6-chlor-

opicolinamide (500 mg, 1.33 mmol) was dissolved in a mixture of

hydrochloric acid (3 M aq, 4.45 mL, 13.34 mmol) and ethanol (4.45

mL) to give a colorless solution. The resulting mixture was stirred at

85 °C for 3 h. The solvent was removed under reduced pressure. The

crude product was neutralized with saturated aqueous NaHCO₃, and

evaporated to dryness. The residue was puriﬁed by C18-ﬂash

chromatography, elution gradient 0 to 100% MeCN in water to

give the title compound (460 mg, 100%) as a pale yellow oil.

ES+ [M + H]⁺333/335.

N-(4-((4-Aminobenzyl)(methyl)amino)butyl)-6-chloropicolina-

mide (29). Prepared analogously to 28.

ES+ [M + H]⁺347/349.

N-(2-((2-Aminoethyl)(methyl)amino)ethyl)-6-bromopicolina-

mide (30). Methyl 6-bromopicolinate (2.0 g, 9.26 mmol) was

dissolved in THF (20 mL). N-1-(2-Aminoethyl)-N-1-methylethane-

1,2-diamine (2.17 g, 18.52 mmol) was added, and the mixture was

heated to 50 °C for 16 h. The mixture was allowed to cool, and the

volatiles were evaporated. The residue was puriﬁed using a Biotage

KP-SIL 100 g column eluting with a gradient of 0−8% methanolic

ammonia (2 M) in DCM to give the title compound (2.40 g, 86%) as

a light yellow colored viscous oil.

¹H NMR (500 MHz, CDCl₃) δ 8.14 (dd, J = 7.6, 1.0 Hz, 1H), 7.86

(d, J = 6.8 Hz, 1H), 7.70 (t, J = 7.7 Hz, 1H), 7.60 (dd, J = 7.9, 1.0 Hz,

1H), 4.60 (s, 0H), 4.11 (q, J = 7.1 Hz, 1H), 3.47 (q, J = 6.8 Hz, 2H),

3.15 (d, J = 7.1 Hz, 2H), 2.03 (s, 1H), 1.52−1.71 (m, 4H), 1.25 (t, J =

7.1 Hz, 2H).

ES+ [M + H]⁺372/374.

Step 2: N-(4-Aminobutyl)-6-bromopicolinamide hydrochloride

tert-Butyl (4-(6-bromopicolinamido)butyl)carbamate (3.5 g, 9.40

mmol) was dissolved in CH₂Cl₂(20 mL). Dioxane-HCl 4 M (5 mL,

20 mmol) was added and the solution was stirred overnight at RT.

The resulting precipitate was collected and was washed with CH₂Cl₂

to give the title compound (2.80 g, 97%) as a white colored solid.

¹H NMR (500 MHz, DMSO-d6) δ 8.72 (t, J = 6.2 Hz, 1H), 8.03

(dd, J = 7.6, 0.9 Hz, 1H), 7.94 (t, J = 7.7 Hz, 1H), 7.83−7.9 (m, 3H),

5.76 (s, 1H), 3.31 (d, J = 6.1 Hz, 2H), 2.79 (q, J = 6.2 Hz, 2H), 1.52−

1.61 (m, 4H).

¹H NMR (500 MHz, CDCl₃) δ 8.33 (br. s, 1H), 8.12 (dd, J = 7.6,

0.9 Hz, 1H), 7.68 (t, J = 7.7 Hz, 1H), 7.57 (dd, J = 7.9, 1.0 Hz, 1H),

3.52 (q, J = 5.82 Hz, 2H), 2.78 (dd, J = 6.4, 5.1 Hz, 2H), 2.61 (t, J =

6.1 Hz, 2H), 2.49 (dd, J = 6.4, 5.0 Hz, 2H), 2.28 (s, 3H).

ES+ [M + H]⁺301/303.

N-(3-((2-Aminoethyl)(methyl)amino)propyl)-6-chloropicolina-

mide (31). Step 1: 6-Chloro-N-(3-(methylamino)propyl)-

picolinamide (26, 1.0 g, 4.39 mmol), tert-butyl (2-oxoethyl)carbamate

(1.05 g, 6.59 mmol), and acetic acid (0.025 mL, 0.44 mmol) were

dissolved in MeOH (50 mL) to give a yellow solution which was

stirred at 25 °C for 30 min, and then sodium cyanoborohydride

(0.828 g, 13.18 mmol) was added, and the resulting mixture was

stirred at 25 °C for 12 h. The solvent was evaporated, and the residue

was puriﬁed by ﬂash silica chromatography, elution gradient 0 to 5%

MeOH in DCM to give tert-butyl (2-((3-(6-chloropicolinamido)-

propyl)(mmethyl)amino)ethyl)carbamate (1.00 g, 61%) as a yellow

liquid. ES+ [M + H]⁺371/373.

ES+ [M + H]⁺272/274.

N-(5-Aminopentyl)-6-bromopicolinamide (25). Prepared analo-

gously to 23 using pentane-1,5-diamine and methyl 6-bromopicoli-

nate.

¹H NMR (400 MHz, DMSO-d₆) δ 8.66 (t, J = 5.7 Hz, 1H), 8.03

(dd, J = 7.5, 1.0 Hz, 1H), 7.94 (t, J = 7.7 Hz, 1H), 7.85 (dd, J = 7.9,

0.9 Hz, 1H),3.32−3.23 (m, 2H), 2.55−2.51 (m, 1H), 1.53 (p, J = 7.4

Hz, 3H), 1.42−1.22 (m, 6H).

ES+ [M + H]⁺286/288.

6-Chloro-N-(3-(methylamino)propyl)picolinamide (26). Prepared

analogously to 23 using N-1-methylpropane-1,3-diamine.

¹H NMR (300 MHz, CDCl₃δ 8.46 (s, 1H), 8.14 (dd, J = 7.5, 1.1

Hz, 1H), 7.83 (td, J = 7.8, 1.2 Hz, 1H), 7.47 (dd, J = 7.8, 1.1 Hz, 1H),

3.57 (q, J = 6.0 Hz, 2H), 2.74 (td, J = 6.7, 1.2 Hz, 2H), 2.49 (s, 3H),

1.92−1.83 (m, 2H).

Step 2: tert-Butyl (2-((3-(6-chloropicolinamido)propyl)(methyl)-

amino)ethyl)carbamate (600 mg, 1.62 mmol) was dissolved in DCM

(10 mL) to give a colorless solution. TFA (5 mL, 64.9 mmol) was

added, and the resulting mixture was stirred at 25 °C for 1 h. The

volatiles were evaporated, and the residue was dissolved in DCM (150

mL) and washed with 2 M NaOH (50 mL) and saturated brine (100

mL × 2). The organic phase was dried over Na₂SO₄, ﬁltered, and

evaporated to give the title compound (400 mg, 91%) as a yellow

liquid.

ES+ [M + H]⁺228/230.

N-(2-(2-Aminoethoxy)ethyl)-6-bromopicolinamide (27). 2-(2-

Aminoethoxy)ethanamine (4.82 g, 46.3 mmol) was added to a

solution of methyl 6-bromopicolinate (5.0 g, 23.1 mmol) in THF

(100 mL) at 25 °C over a period of 15 min. The resulting solution

was heated to 50 °C for 45 h. The solvent was evaporated, and the

residue was puriﬁed by ﬂash silica chromatography, elution gradient

10 to 10% MeOH in DCM to give the title compound (5.0 g, 75%) as

a brown oil.

1H NMR (400 MHz, CDCl₃) δ 8.22 (s, 1H), 8.15 (dd, J = 7.6, 1.0

Hz, 1H), 7.72 (t, J = 7.7 Hz, 1H), 7.61 (dd, J = 8.0, 1.0 Hz, 1H),

3.73−3.62 (m, 4H), 3.55 (t, J = 5.1 Hz, 2H), 2.91 (t, J = 5.1 Hz, 2H).

ES+ [M + H]⁺288/290.

N-(3-((4-Aminobenzyl)(methyl)amino)propyl)-6-chloropicolina-

mide (28). Step 1 N-(3-aminopropyl)-6-chloropicolinamide (1.1 g,

5.15 mmol), N-(4-formylphenyl)acetamide (0.924 g, 5.66 mmol),

acetic acid (0.309 mL, 5.41 mmol) and sodium cyanotrihydroborate

(0.971 g, 15.44 mmol) were dissolved in MeOH (20 mL) to give a

brown solution. The resulting mixture was stirred at RT for 1 h.

Aqueous formaldehyde (4.73 mL, 51.48 mmol) was added. The

resulting mixture was stirred at RT for a further 30 min. The crude

product was added to a silica gel column and was eluted with

DCM:EA:MeOH 5:5:1 to give N-(3-((4-acetamidobenzyl)(methyl)-

amino)propyl)-6-chloropicolinamide (0.550 g, 28%) as a pale tan oil.

ES+ [M + H]⁺375/377.

¹H NMR (300 MHz, CDCl₃) δ 9.03 (bs, 1H), 8.12 (d, J = 7.6 Hz,

1H), 7.80 (t, J = 7.8 Hz, 1H), 7.43 (d, J = 7.9 Hz, 1H), 3.56 (q, J = 6.0

Hz, 2H), 2.86 (t, J = 5.9 Hz, 2H), 2.56−2.42 (m, 4H), 2.26 (d, J = 4.9

Hz, 3H), 1.90−1.77 (m, 4H).

ES+ [M + H]⁺271/273.

(S)-1²-(1-Cyclopropylethyl)-2⁴,12-dimethyl-1²,1³-dihydro-1¹H-16-

thia-3,6,12,15-tetraaza-2(5,2)-thiazola-1(6,4)-isoindolina-4(2,6)-

pyridina-14(1,4)-benzenacyclohexadecaphane-1³,5-dione 16,16-

dioxide (32). Step 1: (S)-6-(2-Acetamido-4-methylthiazol-5-yl)-2-(1-

cyclopropylethyl)-3-oxoisoindoline-4-sulfonyl chloride (4, 558 mg,

1.23 mmol), N-(3-((4-aminobenzyl)(methyl)amino)propyl)-6-chlor-

opicolinamide (28, 450 mg, 1.35 mmol), and triethylamine (373 mg,

3.69 mmol) were dissolved in DCM (10 mL) to give a yellow

solution. The resulting mixture was stirred at RT for 3 h, and then the

solvent was evaporated. The residue was puriﬁed by ﬂash C18-ﬂash

chromatography, elution gradient 0 to 100% MeCN in water to give

(S)-N-(3-((4-(6-(2-acetamido-4-methylthiazol-5-yl)-2-(1-cyclopropy-

lethyl)-3-oxoisoindoline-4-sulfonamido)benzyl)(methyl)amino)-

propyl)-6-chloropicolinamide (400 mg, 43%) as a yellow solid.

ES+ [M + H]⁺750/752.

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Journal of Medicinal Chemistry

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Drug Annotation

Step 2: (S)-N-(3-((4-(6-(2-Amino-4-methylthiazol-5-yl)-2-(1-cy-

clopropylethyl)-3-oxoisoindoline-4-sulfonamido)benzyl)(methyl)-

amino)propyl)-6-chloropicolinamide.

¹H NMR (600 MHz, DMSO-d₆) δ 11.89 (s, 1H), 8.07 (s, 1H),

7.94−7. 89 (m, 3H), 7.58 (d, J = 7.4 Hz, 1H), 7.24 (d, J = 8.3 Hz,

1H), 7.10 (t, J = 4.8 Hz, 1H), 4.75 (s, 2H), 3.68−3.61 (m, 1H),

3.45−3.37 (m, 2H), 2.93−2. 82 (m, 2H), 2.62 (t, J = 7.9 Hz, 2H),

2.50 (s, 3H), 2.49−2.44 (m, 2H), 2.21 (s, 3H), 1.32 (d, J = 6.8 Hz,

3H), 1.19−1.13 (m, 1H), 0.63−0.58 (m, 1H), 0.47−0.38 (m, 2H),

0.30−0.26 (m, 1H).

(S)-N-(3-((4-(6-(2-Acetamido-4-methylthiazol-5-yl)-2-(1-cyclo-

propylethyl)-3-oxoisoindoline-4-sulfonamido)benzyl)(methyl)-

amino)propyl)-6-chloropicolinamide (390 mg, 0.52 mmol) was

dissolved in ethanol (8.7 mL) to give a colorless solution. Hydrogen

chloride (3 M aq, 8.66 mL, 26 mmol) was added, and the reaction

was heated under reﬂux for 3 h. The solvent was evaporated, and the

residue was diluted with MeOH (5 mL), basiﬁed with saturated

NaHCO₃, and puriﬁed by ﬂash C18-ﬂash chromatography, elution

gradient 0 to 100% MeCN in water to give the subtitle compound

(320 mg, 87%) as a yellow solid.

¹³C NMR (151 MHz, DMSO-d₆) δ 164.6, 163.9, 157.6, 150.7,

147.7, 146.6, 144.5, 139.9, 136.3, 136.0, 129.2, 127.4, 126.8, 120.3,

115.2, 114.8, 56.9, 55.1, 52.6, 46.8, 43.5, 37.7, 18.4, 16.4, 16.0, 4.4,

3.8.

ES+ (M + H)⁺observed 596.2120 expected 596.2114.

(S)-1²-(1-cyclopropylethyl)-2⁴,10-dimethyl-1²,1³-dihydro-1¹H-14-

thia-3,6,10,13-tetraaza-2(5,2)-thiazola-1(6,4)-isoindolina-4(2,6)-

pyridinacyclotetradecaphane-1³,5-dione 14,14-dioxide (36). Pre-

pared analogously to compound 32 using 4 and 31.

Step 1: (S)-N-(3-((2-(6-(2-Acetamido-4-methylthiazol-5-yl)-2-(1-

cyclopropylethyl)-3-oxoisoindoline-4-sulfonamido)ethyl)(methyl)-

amino)propyl)-6-chloropicolinamide.

ES+ [M + H]⁺708/710.

Step 3: (S)-N-(3-((4-(6-(2-Amino-4-methylthiazol-5-yl)-2-(1-cy-

clopropylethyl)-3-oxoisoindoline-4-sulfonamido)benzyl)(methyl)-

amino)propyl)-6-chloropicolinamide (320 mg, 0.45 mmol), PdOAc₂

(51 mg, 0.23 mmol), 2-di-tert-butylphosphino-2′,4′,6′-triisopropylbi-

phenyl (211 mg, 0.50 mmol) and DIPEA (0.473 mL, 2.71 mmol)

were dissolved in DMF (40 mL) to give a brown solution. The

resulting mixture was stirred at 130 °C overnight. The solvent was

evaporated, and the residue was puriﬁed by preparative HPLC (X

Bridge RP18, 19 × 150 mm, 5 μm; mobile phase A: water/10 nmol

NH₄HCO₃, mobile phase B: MeCN; ﬂow rate: 30 mL/min; gradient:

48% B to 55% B over 8 min) to give the title compound (30 mg,

10%) as a yellow solid.

¹H NMR (300 MHz, CDCl₃) δ 9.57 (bs, 1H), 8.62 (bs, 1H), 8.11

(s, 1H), 8.08 (d, J = 7.6 Hz, 1H), 7.77 (t, J = 7.8 Hz, 2H), 7.65 (s,

1H), 7.38 (d, J = 7.9 Hz, 1H), 4.70−4. 45 (m, 2H), 3.87−3.70 (m,

1H), 3.47 (q, J = 6.1 Hz, 2H), 3.17 (bs, 2H), 2.66−2.44 (m, 3H),

2.42 (s, 3H), 2.30 (s, 3H), 2.18 (bs, 3H), 1.67 (bs, 3H), 1.35 (d, J =

6.8 Hz, 3H), 1.11−0.94 (m, 1H), 0.73−0.59 (m, 1H), 0.52−0.29 (m,

3H).

¹H NMR (600 MHz, DMSO-d₆) δ 11.82 (s, 1H), 10.08 (s, 1H),

8.00 (s, 1H), 7.91−7.85 (m, 2H), 7.59−7.56 (m, 1H), 7.51 (d, J = 7.4

Hz, 1H), 7.20 (t, J = 8.7 Hz, 3H), 7.04 (d, J = 8.4 Hz, 2H), 4.81 (s,

2H), 3.78−3.72 (m, 1H), 3.32 (s, 6H), 2.36 (s, 3H), 2.30 (s, 3H),

2.01−1.92 (m, 2H), 1.40 (d, J = 6.8 Hz, 3H), 1.26−1.19 (m,1H),

0.67−0.61 (m, 1H), 0.54−0.45 (m, 2H), 0.42−0.35 (m, 1H).

¹³C NMR (151 MHz, DMSO-d₆) δ 165.4, 163.8, 157.7, 150.6,

147.9, 146.4, 140.0, 137.7, 136.5, 136.3, 135.9, 130.6, 129.0, 128.3,

126.7, 122.1, 119.5, 114.8, 114.6, 61.6, 53.4, 53.3, 47.1, 44.0, 39.3,

27.5, 18.5, 16.1, 156.0, 4.5, 3.8.

ES+ (M + H)⁺688/690.

Step 2: (S)-N-(3-((2-(6-(2-Amino-4-methylthiazol-5-yl)-2-(1-cy-

clopropylethyl)-3-oxoisoindoline-4-sulfonamido)ethyl)(methyl)-

amino)propyl)-6-chloropicolinamide.

¹H NMR (400 MHz, CDCl₃) δ 8.59 (s, 1H), 8.07 (d, J = 7.6 Hz,

1H), 7.98 (s, 1H), 7.80−7.70 (m, 2H), 7.55 (s, 1H), 7.37 (d, J = 7.9

Hz, 1H), 5.21−5.06 (m, 2H), 4.45−4.68 (m, 2H), 3.81−3.71 (m,

1H), 3.50−3.41 (m, 2H), 3.21−3.10 (m, 2H), 2.57 (bs, 2H), 2.45

(bs, 2H), 2.35 (d, J = 1.4 Hz, 3H), 2.17 (s, 3H), 1.34 (d, J = 6.8 Hz,

3H), 1.25 (s, 1H), 1.06−0.96 (m, 1H), 0.90−0.79 (m, 1H), 0.69−

0.60 (m, 1H), 0.49−0.30 (m, 3H).

ES+ (M + H)⁺observed 672.2441 expected 672.2427.

(S)-1²-(1-Cyclopropylethyl)-2⁴,10-dimethyl-1²,1³-dihydro-1¹H-14-

thia-3,6,10,13-tetraaza-2(5,2)-thiazola-1(6,4)-isoindolina-4(2,6)-

pyridina-12(1,4)-benzenacyclotetradecaphane-1³,5-dione 14,14-

dioxide (33). Prepared analogously to compound 32 using 4 and 29.

¹H NMR (600 MHz, DMSO-d₆) δ 11.81 (s, 1H), 9.96 (s, 1H),

7.98 (s, 1H), 7.93−7. 87 (m, 1H), 7.84 (t, J = 5.8 Hz, 1H), 7.73−7.69

(m, 1H), 7.55 (d, J = 7.2 Hz, 1H), 7.21 (d, J = 8.2 Hz, 1H), 7.12 (d, J

= 8.4 Hz, 2H), 6.98 (d, J = 8.4 Hz, 2H), 4.73 (s, 2H), 3.76−3.73 (m,

1H), 3.21 (d, J = 5.6 Hz, 2H), 2.36 (s, 3H), 2.17 (s, 3H), 1.79 (t, J =

6.6 Hz, 2H), 1.40 (d, J = 6.8 Hz, 3H), 1.33−1.26 (m, 2H), 1.25−1.17

(m, 3H), 0.67−0.61 (m, 1H), 0.53−0.44 (m, 2H), 0.39−0.33 (m,

1H).

ES+ (M + H)⁺646/648.

Step 3: (S)-1²-(1-cyclopropylethyl)-2⁴,10-dimethyl-1²,1³-dihydro-

1¹H-14-thia-3,6,10,13-tetraaza-2(5,2)-thiazola-1(6,4)-isoindolina-4-

(2,6)-pyridinacyclotetradecaphane-1³,5-dione 14,14-dioxide.

¹H NMR (600 MHz, DMSO-d₆) δ 11.88 (s, 1H), 7.98 (s, 1H),

7.95−7. 89 (m, 2H), 7.79 (d, J = 1.2 Hz, 1H), 7.68 (t, J = 5.9 Hz,

1H), 7.56 (d, J = 7.3 Hz, 1H), 7.24 (d, J = 8.3 Hz, 1H), 4.76 (s, 2H),

3.71−3. 64 (m, 1H), 3.29−3.23 (m, 2H), 3.22−3.13 (m, 2H), 2.45 (s,

3H), 2.38−2.29 (m, 2H), 2.26−2.18 (m, 2H), 1.75 (s, 3H), 1.59 (p, J

= 7.8 Hz, 2H), 1.35 (d, J = 6.8 Hz, 3H), 1.21−1.14 (m, 1H), 0.65−

0.58 (m, 1H), 0.46−0.40 (m, 2H), 0.32−0.26 (m, 1H).

¹³C NMR (151 MHz, DMSO-d₆) δ 165.4, 163.9, 157.4, 150.7,

147.8, 146.0, 144.6, 140.4, 140.0, 136.1, 126.7, 126.3, 125.7, 120.1,

114.9, 114.7, 54.9, 52.79, 52.77, 46.7, 43.4, 41.8, 38.4, 26.1, 18.5, 16.2,

16.1, 4.5, 3.7.

¹³C NMR (151 MHz, DMSO-d₆) δ 165.3, 164.1, 157.7, 150.8,

148.1, 146.1, 145.6, 139.9, 136.5, 136.5, 136.1, 136.0, 130.1, 128.6,

128.4, 126.4, 121.9, 119.4, 115.0, 114.7, 62.0, 54.0, 53.3, 46.9, 43.2,

39.3, 26.9, 24.1, 18.6, 16.3, 16.1, 4.5, 3.8.

ES+ (M + H)⁺observed 686.257 expected 686.2583.

ES+ (M + H)⁺observed 610.2282 expected 610.2270.

(S)-1²-(1-Cyclopropylethyl)-2⁴-methyl-1²,1³-dihydro-1¹H-9-oxa-

13-thia-3,6,12-triaza-2(5,2)-thiazola-1(6,4)-isoindolina-4(2,6)-pyri-

dinacyclotridecaphane-1³,5-dione 13,13-dioxide (37). Prepared

analogously to compound 32 using 4 and 27.

Step 1: (S)-N-(2-(2-((6-(2-Acetamido-4-methylthiazol-5-yl)-2-(1-

cyclopropylethyl)-3-oxoisoindoline)-4-sulfonamido)ethoxy)ethyl)-6-

bromopicolinamide.

(S)-1²-(1-Cyclopropylethyl)-2⁴-methyl-1²,1³-dihydro-1¹H-13-thia-

3,6,12-triaza-2(5,2)-thiazola-1(6,4)-isoindolina-4(2,6)-pyridinacy-

clotridecaphane-1³,5-dione 13,13-dioxide (34). Prepared analo-

gously to compound 32 using 4 and 25.

¹H NMR (600 MHz, DMSO-d₆) δ 11.90 (s, 1H), 8.06 (s, 1H),

7.92 (t, J = 7.8 Hz, 1H), 7.90−7.85 (m, 2H), 7.57 (d, J = 7.3 Hz, 1H),

7.23 (d, J = 8.3 Hz, 1H), 7.15−7.11 (m, 1H), 4.75 (s, 2H), 3.67−3.60

(m, 1H), 3.26 (dt, J = 11.4, 5.8 Hz, 2H), 2.80−2.73 (m, 2H), 2.48 (s,

3H), 1.61−1.52 (m, 2H), 1.51−1.43 (m, 2H), 1.32 (d, J = 6.8 Hz,

3H), 1.27−1.20 (m, 2H), 1.20−1.12 (m, 1H), 0.64−0.58 (m, 1H),

0.47−0.38 (m, 2H), 0.30−0.25 (m, 1H).

¹H NMR (300 MHz, CDCl₃) δ 9.69 (s, 1H), 8.26−8.17 (m, 1H),

8.14 (d, J = 1.4 Hz, 1H), 8.12 (dd, J = 7.5, 1.0 Hz, 1H), 8.05−7.97

(m, 1H), 7.95 (t, J = 5.9 Hz, 1H), 7.74−7.65 (m, 2H), 7.58 (dd, J =

7.9, 1.0 Hz, 1H), 4.72−4.49 (m, 2H), 3.87−3.73 (m, 1H), 3.60−3.41

(m, 6H), 3.22 (q, J = 5.6 Hz, 2H), 2.43 (s, 3H), 2.30 (s, 3H), 1.35 (d,

J = 6.8 Hz, 3H), 1.11−0.95 (m, 1H), 0.70−0.60 (m, 1H), 0.51−0.40

(m, 1H), 0.4−0.31 (m, 2H).

ES+ (M + H)⁺observed 581.2052 expected 581.2004.

(S)-1²-(1-cyclopropylethyl)-2⁴,9-dimethyl-1²,1³-dihydro-1¹H-13-

thia-3,6,9,12-tetraaza-2(5,2)-thiazola-1(6,4)-isoindolina-4(2,6)-pyr-

idinacyclotridecaphane-1³,5-dione 13,13-dioxide (35). Prepared

analogously to compound 32 using 4 and 30.

ES+ (M + H)⁺705/707.

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Drug Annotation

Step 2: (S)-N-(2-(2-((6-(2-Amino-4-methylthiazol-5-yl)-2-(1-cy-

clopropylethyl)-3-oxoisoindoline)-4-sulfonamido)ethoxy)ethyl)-6-

bromopicolinamide.

¹H NMR (300 MHz, DMSO-d₆) δ 8.67 (s, 1H), 8.17 (d, J = 7.6

Hz, 1H), 7.94 (t, J = 7.8 Hz, 1H), 7.78 (d, J = 7.9 Hz, 1H), 4.29 (q, J

= 7.1 Hz, 2H), 1.26 (t, J = 7.1 Hz, 3H).

ES+ (M + H)⁺297/299.

¹H NMR (300 MHz, CDCl₃) δ 8.49 (s, 1H), 7.99 (dd, J = 7.4, 1.1

Hz, 1H), 7.91 (t, J = 7.6 Hz, 2H), 7.83 (dd, J = 7.8, 1.1 Hz, 1H), 7.78

(d, J = 1.4 Hz, 1H), 7.71 (d, J = 1.5 Hz, 1H), 4.70 (s, 2H), 3.67−3. 53

(m, 1H), 3.37 (t, J = 5.1 Hz, 2H), 3.26 (m, 4H), 3.03 (s, 2H), 2.28 (s,

3H), 1.33−1.20 (m, 4H), 1.13−1. 06 (m, 1H), 0.89−0.81 (m, 1H),

0.59−0.47 (m, 1H), 0.46−0.29 (m, 2H), 0.28−0.17 (m, 1H).

ES+ (M + H)⁺663/665.

Step 2 (5-(6-Bromopyridin-2-yl)oxazol-4-yl)methanol

Ethyl 5-(6-bromopyridin-2-yl)oxazole-4-carboxylate (200 mg, 0.67

mmol) was dissolved in EtOH (20 mL), and the solution was cooled

to 0 °C. NaBH₄(153 mg, 4.04 mmol) was added, and the resulting

mixture was heated to 70 °C for 2 h. Water (15 mL) was added to the

cooled reaction mixture which was then extracted with EtOAc (3 ×

15 mL), and the organic phases were combined, dried over Na₂SO₄,

ﬁltered, and evaporated. The residue was puriﬁed by ﬂash alumina

chromatography, elution gradient 0 to 80% EtOAc in petroleum ether

to give the title compound (110 mg, 64%) as a yellow solid.

¹H NMR (300 MHz, DMSO-d₆) δ 8.50 (s, 1H), 7.89 (t, J = 7.8 Hz,

1H), 7.82−7.75 (m, 1H), 7.63 (dd, J = 7.9, 0.7 Hz, 1H), 5.21 (t, J =

5.9 Hz, 1H), 4.76 (d, J = 5.8 Hz, 2H).

Step 3: (S)-1²-(1-Cyclopropylethyl)-2⁴-methyl-1²,1³-dihydro-1¹H-

9-oxa-13-thia-3,6,12-triaza-2(5,2)-thiazola-1(6,4)-isoindolina-4(2,6)-

pyridinacyclotridecaphane-1³,5-dione 13,13-dioxide.

¹H NMR (600 MHz, DMSO-d₆) δ 11.84 (s, 1H), 8.07−8.03 (m,

2H), 7.94−7.90 (m, 2H), 7.59−7.62 (m, 1H), 7.43−7.39 (m, 1H),

7.24 (d, J = 8.3 Hz, 1H), 4.72 (s, 2H), 3.66−3.60 (m, 1H), 3.60−3.

51 (m, 6H), 3.17−3. 08 (m, 2H), 1.31 (d, J = 6.8 Hz, 3H), 1.19−1.12

(m, 1H), 0.63−0.58 (m, 1H), 0.47−0.38 (m, 2H), 0.31−0.25 (m,

1H). 3H obscured.

ES+ (M + H)⁺255/257.

N-(6-Bromopyridin-2-yl)-N-methylacetamide (41). 6-Bromo-N-

methylpyridin-2-amine (200 mg, 1.07 mmol) was dissolved in acetic

anhydride (303 μL, 3.2 mmol) and pyridine (259 μL, 3.2 mmol). The

resulting solution was heated to 120 °C for 2 h. The reaction mixture

was concentrated, and then EtOAc (100 mL) was added. The

solution was washed with saturated NaHCO₃(75 mL × 2), 0.1 M

HCl (75 mL × 2) and saturated brine (75 mL × 2). The organic layer

was dried over Na₂SO₄, ﬁltered, and evaporated to give the title

compound (180 mg, 73%) as a yellow solid.

¹³C NMR (151 MHz, DMSO-d₆) δ 164.1, 163.9, 157.7, 150.9,

147.8, 146.4, 145.3, 139.9, 137.5, 136.0, 127.7, 127.0, 126.8, 120.5,

115.3, 114.9, 68.8, 68.6, 52.4, 46.4, 42.0, 39.0, 18.4, 16.9, 16.0, 4.4,

3.8.

ES+ (M + H)⁺observed 583.1800 expected 583.1797.

2-Bromo-6-(1,3-dimethyl-1H-1,2,4-triazol-5-yl)pyridine (38). 2-

Bromo-6-(1,5-dimethyl-1H-1,2,4-triazol-3-yl)pyridine (39). Step 1:

6-Bromopicolinic acid (200 mg, 0.99 mmol), acetimidamide hydro-

chloride (140 mg, 1.49 mmol) and DIPEA (0.692 mL, 3.96 mmol)

were dissolved in DMF (5 mL) at 25 °C under nitrogen. HATU (565

mg, 1.49 mmol) was added, and the resulting mixture was stirred for

12 h. The reaction mixture was poured onto water and was then

extracted with ethyl acetate (3 × 10 mL), and the organic phases were

combined, dried, ﬁltered, and evaporated to give a product that was

used without puriﬁcation (240 mg).

¹H NMR (300 MHz, CDCl₃) δ 7.58 (t, J = 7.8 Hz, 1H), 7.45−7.31

(m, 2H), 3.40 (s, 3H), 2.19 (s, 3H).

ES+ (M + H)⁺229/231.

1-(6-Bromopyridin-2-yl)pyrrolidin-2-one (42). Pyrrolidin-2-one

(1.61 mL, 21.1 mmol) was added to a solution of 2,6-

dibromopyridine (5.0 g, 21.1 mmol) in dioxane (5 mL). Xantphos

(3.66 g, 6.3 mmol) and Cs₂CO₃(20.63 g, 63.3 mmol), second

Generation XantPhos precatalyst (3.75 g, 4.2 mmol) were added. The

resulting mixture was stirred at 105 °C for 5 h. The crude product was

puriﬁed by ﬂash silica chromatography, elution gradient 0 to 1:20

EtOAc in petroleum ether to give the title compound (4.00 g, 79%) as

a white solid.

Step 2: Methylhydrazine sulfate (0.536 g, 3.72 mmol) was added to

(Z)-N-(1-aminoethylidene)-6-bromopicolinamide (previous step,

0.60 g, 2.48 mmol) and acetic acid (1.49 g, 24.8 mmol) in DMF

(20 mL) at 25 °C over a period of 15 min. The resulting mixture was

heated to 80 °C for 12 h. The reaction mixture was quenched with

water (25 mL) and extracted with DCM (3 × 20 mL), and the

organic layer was dried over Na₂SO₄, ﬁltered, and evaporated. The

residue was puriﬁed by ﬂash silica chromatography, eluting with 30%

EtOAc in petroleum ether, to give the title compounds (0.060 g,

9.6%) and (0.180 g, 29%) as yellow solids.

¹H NMR (400 MHz, DMSO-d₆) δ 8.33−8.26 (m, 1H), 7.79−7.71

(m, 1H), 7.4−7.33 (m, 1H), 3.97−3.89 (m, 2H), 2.58 (t, J = 8.1 Hz,

2H), 2.09−1.97 (m, 2H).

ES+ (M + H)⁺241/243.

1-(6-Bromopyridin-2-yl)imidazolidin-2-one (43). Step 1: 1-(6-

Bromopyridin-2-yl)-3-(2-chloroethyl)urea 6-bromopyridin-2-amine

(3.0 g, 17.3 mmol) was added to a solution of 1-chloro-2-

isocyanatoethane (2.20 g, 20.8 mmol) in toluene (20 mL. The

resulting mixture was heated to 70 °C for 2 h. The reaction mixture

was ﬁltered through a Celite pad, and the crude product was puriﬁed

by crystallization from EtOAc/petroleum ether = 1/10 to give the

subtitle compound (3.10 g, 64%) as a white solid.

1

38 H NMR (400 MHz, CDCl₃) δ 8.12 (d, J = 7.7 Hz, 1H), 7.65

(t, J = 7.8 Hz, 1H), 7.49 (d, J = 7.9 Hz, 1H), 4.27 (s, 3H), 2.40 (s,

3H).

ES+ (M + H)⁺253/255.

1

39 H NMR (400 MHz, DMSO-d₆) δ 7.77 (d, J = 7.6 Hz, 1H),

7.60 (t, J = 7.8 Hz, 1H), 7.41 (d, J = 7.8 Hz, 1H), 3.63 (s, 3H), 2.23

¹H NMR (300 MHz, DMSO-d₆) δ 9.59 (s, 1H), 7.70−7.57 (m,

2H), 7.30 (t, J = 5.9 Hz, 1H), 7.16 (dd, J = 6.7, 1.7 Hz, 1H), 3.68 (t, J

= 5.9 Hz, 2H), 3.48 (q, J = 5.9 Hz, 2H).

(s, 3H).

ES+ (M + H)⁺253/255.

(5-(6-Bromopyridin-2-yl)oxazol-4-yl)methanol (40). Step 1: Ethyl

5-(6-bromopyridin-2-yl)oxazole-4-carboxylate

ES+ (M + H])⁺= 278/280/282.

Oxalyl chloride (0.942 g, 7.43 mmol) was added to 6-

bromopicolinic acid (1 g, 4.95 mmol) and N,N-dimethylformamide

(0.036 g, 0.50 mmol) in DCM (15 mL) at 0 °C. The resulting

mixture was stirred at r.t. for 5 h. The volatiles were evaporated to

leave a brown solid (1.10 g, 100%) that was used without puriﬁcation.

6-Bromopicolinoyl chloride (500 mg, 2.27 mmol), ethyl 2-

isocyanoacetate (308 mg, 2.72 mmol), DMAP (14 mg, 0.11 mmol),

and TEA (1.31 g, 12.9 mmol) were dissolved in THF (20 mL), and

the mixture was heated to 60 °C for 12 h. The reaction mixture was

allowed to cool and was ﬁltered. The ﬁltrate was diluted with EtOAc,

and the resultant solution was washed with water and brine. The

organic layer was dried over Na₂SO₄, ﬁltered, and evaporated. The

residue was puriﬁed by ﬂash silica chromatography, elution gradient 0

to 20% EtOAc in petroleum ether to give the title compound (270

mg, 40%) as a brown solid.

Step 2:1-(6-Bromopyridin-2-yl)imidazolidin-2-one

A solution of 1-(6-bromopyridin-2-yl)-3-(2-chloroethyl)urea (3.0

g, 10.8 mmol) in DMF (25 mL) was added to a stirred suspension of

60% sodium hydride (0.775 g, 19.39 mmol) in THF (25 mL) at 0 °C

under nitrogen. The resulting mixture was stirred at 25 °C for 1 h.

The reaction mixture was quenched by the addition of water (10 mL),

ﬁltered, and evaporated to give the title compound (2.10 g, 81%)

white solid.

¹H NMR (300 MHz, DMSO-d₆) δ 8.16 (d, J = 8.3 Hz, 1H), 7.62

(t, J = 8.0 Hz, 1H), 7.33−7.39 (m, 1H), 7.18 (d, J = 7.5 Hz, 1H), 3.93

(dd, J = 8.9, 7.1 Hz, 2H), 3.40 (t, J = 8.0 Hz, 2H).

ES+ (M + H])⁺= 242/244.

1-(6-Bromopyridin-2-yl)-3-methylimidazolidin-2-one (44). Pre-

pared following the method of example 42 using 1-methylimidazo-

lidin-2-one.

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Drug Annotation

¹H NMR (400 MHz, CDCl₃) δ 8.27−8.17 (m, 1H), 7.48−7. 37

(m, 1H), 7.08−6.98 (m, 1H), 4.06−3.96 (m, 2H), 3.51−3.40 (m,

2H), 2.92−2.85 (m, 3H).

¹H NMR (300 MHz, DMSO-d₆) δ 8.09 (d, J = 8.1 Hz, 1H), 7.78

(t, J = 8.0 Hz, 1H), 7.46 (d, J = 7.7 Hz, 1H), 4.27 (s, 2H), 3.98 (dd, J

= 6.0, 3.6 Hz, 2H), 3.91 (dd, J = 6.0, 3.6 Hz, 2H).

ES+ (M + H])⁺= 256/258.

ES+ (M + H])⁺= 257/259.

(R)-1-(6-bromopyridin-2-yl)-3-((tert-butyldimethylsilyl)oxy)-

pyrrolidin-2-one (45). Step 1: (R)-3-((tert-butyldimethylsilyl)oxy)-

pyrrolidin-2-one

3-(6-Bromopyridin-2-yl)-1,3-oxazinan-2-one (49). Step 1: 3-

Chloropropyl (6-bromopyridin-2-yl)carbamate

Pyridine (3.51 mL, 43.4 mmol) was added dropwise to a solution

of 6-bromopyridin-2-amine (5.0 g, 28.9 mmol) and 3-chloropropyl

carbonochloridate (5.44 g, 34.7 mmol) in acetonitrile (50 mL) cooled

in ice over a period of 15 min. The resulting solution was stirred at

RT for 4 h. The reaction mixture was diluted with EtOAc(150 mL)

and was then washed with 0.1 M HCl (3 × 100 mL) and brine (2 ×

100 mL). The organic layer was dried over Na₂SO₄, ﬁltered, and

evaporated to give the subtitle compound (8.20 g, 97%) as a pale

yellow oil which solidiﬁed on standing.

tert-Butylchlorodimethylsilane (2.24 g, 14.8 mmol) was added to a

solution of (R)-3-hydroxypyrrolidin-2-one (1.0 g, 9.89 mmol) and

imidazole (1.35 g, 19.8 mmol) in DMF (15 mL). The resulting

mixture was stirred at RT for 2 h. The reaction mixture was diluted

with EtOAc (50 mL) and washed sequentially with water (20 mL ×

3), saturated brine (20 mL × 2). The aqueous layer was dried over

Na₂SO₄, ﬁltered, and evaporated. The crude product was puriﬁed by

ﬂash silica chromatography, elution gradient 0 to 2% DCM in MeOH

to give the subtitle compound (1.90 g, 89%) as a pale yellow solid.

¹H NMR (300 MHz, DMSO-d₆) δ 7.72 (s, 1H), 4.19 (t, J = 8.0 Hz,

1H), 3−3.22 (m, 2H), 2.21−2.37 (m, 1H), 1.77 (dq, J = 12.4, 8.4 Hz,

1H), 0.86 (s, 9H), 0.08 (d, J = 1.4 Hz, 6H).

¹H NMR (500 MHz, DMSO-d₆) δ 10.50 (s, 1H), 7.83 (dd, J = 8.3,

0.7 Hz, 1H), 7.71 (t, J = 7.9 Hz, 1H), 7.25−7.3 (m, 1H), 4.20 (t, J =

6.2 Hz, 2H), 3.66−3.77 (m, 2H), 2.06 (p, J = 6.3 Hz, 2H).

ES+ (M + H)⁺= 293/295/297.

ES+ (M-t-Bu)⁺= 158.

Step 2:3-(6-Bromopyridin-2-yl)-1,3-oxazinan-2-one

Step 2: (R)-1-(6-bromopyridin-2-yl)-3-((tert-butyldimethylsilyl)-

oxy)pyrrolidin-2-one

Cs₂CO₃(1.89 g, 5.79 mmol) was added to a solution of 3-

chloropropyl (6-bromopyridin-2-yl)carbamate (1.70 g, 5.79 mmol)

and KI (0.96 g, 5.79 mmol) in DMF (20 mL). The resulting

suspension was stirred at 60 °C for 1 h. The reaction mixture was

ﬁltered through Celite and then diluted with EtOAc (100 mL). The

resulting suspension was washed sequentially with water (100 mL ×

3) and saturated brine (100 mL × 2). The organic layer was dried

over Na₂SO₄, ﬁltered, and evaporated. The residue was puriﬁed by

ﬂash silica chromatography, elution gradient 0 to 50% EtOAc in

petroleum ether to give the title compound (1.20 g, 81%) as a white

solid.

Prepared following the method of example 42 using (R)-3-((tert-

butyldimethylsilyl)oxy)pyrrolidin-2-one

¹H NMR (300 MHz, DMSO-d₆) δ 8.31 (dd, J = 8.2, 0.7 Hz, 1H),

7.81−7. 73 (m, 1H), 7.40 (dd, J = 7.6, 0.7 Hz, 1H), 4.62 (dd, J = 9.7,

8.0 Hz, 1H), 4.02−3.89 (m, 1H), 3.73−3.58 (m, 1H), 2.48−2.37 (m,

1H), 1.95−1.77 (m, 1H), 0.90 (s, 9H), 0.14 (s, 3H), 0.13 (s, 3H).

ES+ (M + H)⁺= 371/373.

(R)-1-(6-Bromopyridin-2-yl)-4-((tert-butyldimethylsilyl)oxy)-

pyrrolidin-2-one (46). Step 1: (R)-4-((tert-butyldimethylsilyl)oxy)-

pyrrolidin-2-one.

¹H NMR (300 MHz, DMSO-d₆) δ 7.87 (dd, J = 8.2, 0.7 Hz, 1H),

7.74 (dd, J = 8.2, 7.6 Hz, 1H), 7.42 (dd, J = 7.6, 0.7 Hz, 1H), 4.35 (t, J

= 5.4 Hz, 2H), 3.87 (t, J = 6.2 Hz, 2H), 2.11 (qd, J = 6.1, 4.7 Hz, 2H).

ES+ (M + H)⁺= 257/259.

Prepared from (R)-4-hydroxypyrrolidin-2-one following the

method of example 45 Step 1.

ES+ (M + H])⁺= 216.

Step 2: (R)-1-(6-bromopyridin-2-yl)-4-((tert-butyldimethylsilyl)-

oxy)pyrrolidin-2-one.

Prepared from (R)-4-((tert-butyldimethylsilyl)oxy)pyrrolidin-2-one

Benzyl 4-(6-bromopyridin-2-yl)-3-oxopiperazine-1-carboxylate

(50). Prepared from benzyl 3-oxopiperazine-1-carboxylate following

the method of example 42.

¹H NMR (500 MHz, CDCl₃) δ 8.04 (s, 1H), 7.53−7.6 (m, 1H),

7.31−7.42 (m, 5H), 7.29 (d, J = 7.6 Hz, 1H), 5.18 (s, 2H), 4.35 (s,

2H), 4.08−4.18 (m, 2H), 3.79−3.85 (m, 2H), 1.23−1.34 (m, 1H).

ES+ (M + H)⁺= 390/392.

following the method of example 45 Step 2.

¹H NMR (400 MHz, DMSO-d₆) δ 8.30 (d, J = 8.3 Hz, 1H), 7.76

(t, J = 8.0 Hz, 1H), 7.38 (d, J = 7.7 Hz, 1H), 4.65−4.52 (m, 1H), 4.11

(dd, J = 11.6, 5.1 Hz, 1H), 3.80 (d, J = 11.6 Hz, 1H), 3.00 (dd, J =

17.1, 6.0 Hz, 1H), 2.39 (d, J = 17.1 Hz, 1H), 0.85 (s, 9H), 0.09 (s,

6H).

1-(6-Bromopyridin-2-yl)-4-methylpiperazin-2-one (51). Prepared

from 4-methylpiperazine-2-one following the method of Example 42.

¹H NMR (300 MHz, DMSO-d₆) δ 7.95 (d, J = 8.2 Hz, 1H), 7.77

(t, J = 7.9 Hz, 1H), 7.47 (d, J = 7.7 Hz, 1H), 3.83 (t, J = 5.6 Hz, 2H),

3.19 (s, 2H), 2.73 (t, J = 5.5 Hz, 2H), 2.27 (s, 3H).

ES+ (M + H])⁺= 371/373.

1-(6-Bromopyridin-2-yl)piperidin-2-one (47). Step 1: N-(6-

Bromopyridin-2-yl)-5-chloropentanamide

ES+ (M + H)⁺= 270/272.

5-Chloropentanoyl chloride (1.08 g, 6.94 mmol) was added to a

solution of 6-bromopyridin-2-amine (1.0 g, 5.78 mmol) and pyridine

(1,37 g, 17.3 mmol) in MeCN (10 mL). The resulting mixture was

stirred at RT for 5 h. The solvent was removed under reduced

pressure to give the subtitle compound (1.68 g, 100%) as a colorless

oil which crystallized on standing. Used without further puriﬁcation.

ES+ (M + H])⁺= 291/293/295.

(S)-2-(1-Cyclopropylethyl)-5-(2-((6-(1,3-dimethyl-1H-1,2,4-tria-

zol-5-yl)pyridin-2-yl)amino)-4-methylthiazol-5-yl)-7-

(methylsulfonyl)isoindolin-1-one (52). (S)-5-(2-Amino-4-methyl-

thiazol-5-yl)-2-(1-cyclopropylethyl)-7-(methylsulfonyl)isoindolin-1-

one (16, 100 mg, 0.26 mmol), 2-bromo-6-(1,3-dimethyl-1H-1,2,4-

triazol-5-yl)pyridine (38, 64 mg, 0.26 mmol), Xantphos (44 mg, 0.08

mmol), second Generation Xantphos precatalyst (45 mg, 0.05 mmol),

and Na₂CO₃(81 mg, 0.77 mmol) were dissolved in DMF (5 mL) and

sealed into a microwave tube. The reaction was heated to 120 °C for

1 h in a microwave reactor and cooled to RT. The reaction mixture

was poured into water (10 mL) and extracted with DCM (3 × 10

mL), and the organic layer was dried over Na₂SO₄, ﬁltered, and

evaporated. The residue was puriﬁed by preparative HPLC (XBridge

Prep Phenyl OBD column, 5 μ silica, 19 mm diameter, 150 mm

length) using decreasingly polar mixtures of water (containing 0.5%

NH₄HCO₃) and MeCN as eluents to give the title compound (44 mg,

30%) as a yellow solid.

Step 2:1-(6-Bromopyridin-2-yl)piperidin-2-one.

Cs₂CO₃(11.26 g, 34.6 mmol) was added to a solution of N-(6-

bromopyridin-2-yl)-5-chloropentanamide (1.68 g, 5.76 mmol) in

DMF (20 mL). The resulting suspension was stirred at 80 °C for 2 h.

The reaction mixture was allowed to cool and was then diluted with

DCM (75 mL) and washed sequentially with water (50 mL × 2) and

saturated brine (75 mL × 2). The organic layer was dried over

Na₂SO₄, ﬁltered and evaporated to give the title compound (1.48 g,

100%) as a colorless oil.

¹H NMR (300 MHz, DMSO-d₆) δ 7.79 (dd, J = 8.1, 1.1 Hz, 1H),

7.72 (dd, J = 8.1, 7.3 Hz, 1H), 7.42 (dd, J = 7.4, 1.0 Hz, 1H), 3.80 (t, J

= 5.7 Hz, 2H), 2.53−2.46 (m, 2H), 1.91−1.75 (m, 4H).

ES+ (M + H])⁺= 255/257.

4-(6-Bromopyridin-2-yl)morpholin-3-one (48). Prepared follow-

ing the method of example 42 using morpholin-3-one.

¹H NMR (600 MHz, DMSO-d₆) δ 11.75 (s, 1H), 8.03 (d, J = 1.6

Hz, 1H), 8.01 (d, J = 1.7 Hz, 1H), 7.90 (dd, J = 8.4, 7.4 Hz, 1H), 7.51

(dd, J = 7.3, 0.8 Hz, 1H), 7.24 (d, J = 8.3 Hz, 1H), 4.75−4.65 (m,

2H), 4.19 (s, 3H), 3.68−3.58 (m, 4H), 2.47 (s, 3H), 2.30 (s, 3H),

8069

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Drug Annotation

1.30 (d, J = 6.9 Hz, 3H), 1.18−1.11 (m, 1H), 0.64−0.56 (m, 1H),

0.48−0.37 (m, 2H), 0.31−0.23 (m, 1H).

126.24, 119.83, 112.00, 108.55, 52.01, 45.95, 43.08, 34.98, 22.98,

17.96, 16.58, 15.56, 3.87, 3.40.

¹³C NMR (151 MHz, DMSO-d₆) δ 163.65, 158.72, 157.46, 152.31,

151.15, 145.98, 145.31, 144.68, 139.12, 137.65, 135.75, 126.89,

126.68, 126.21, 119.83, 117.25, 112.48, 52.00, 45.95, 43.05, 37.57,

17.95, 16.61, 15.55, 13.45, 3.86, 3.40.

ES+ (M + H)⁺observed 540.1755 expected 540.1739.

(S)-N-(6-((5-(2-(1-Cyclopropylethyl)-7-(N-methylsulfamoyl)-1-ox-

oisoindolin-5-yl)-4-methylthiazol-2-yl)amino)pyridin-2-yl)-N-meth-

ylacetamide (57). Prepared from (S)-6-(2-amino-4-methylthiazol-5-

yl)-2-(1-cyclopropylethyl)-N-methyl-3-oxoisoindoline-4-sulfonamide

(17), and N-(6-bromopyridin-2-yl)-N-methylacetamide (41), follow-

ing the method used for 52.

ES+ (M + H)⁺observed 564.1871 expected 564.1851.

(S)-2-(1-Cyclopropylethyl)-5-(2-((6-(1,5-dimethyl-1H-1,2,4-tria-

zol-3-yl)pyridin-2-yl)amino)-4-methylthiazol-5-yl)-7-

(methylsulfonyl)isoindolin-1-one (53). Prepared from (S)-5-(2-

amino-4-methylthiazol-5-yl)-2-(1-cyclopropylethyl)-7-

(methylsulfonyl)isoindolin-1-one (16) and 2-bromo-6-(1,5-dimethyl-

1H-1,2,4-triazol-3-yl)pyridine (39) following the method used for 52.

¹H NMR (600 MHz, DMSO-d₆) δ 11.62 (s, 1H), 8.15 (d, J = 1.6

Hz, 1H), 8.06 (d, J = 1.6 Hz, 1H), 7.81 (t, J = 7.8 Hz, 1H), 7.57 (d, J

= 7.4 Hz, 1H), 7.07 (d, J = 8.2 Hz, 1H), 4.76−4.66 (m, 2H), 3.90 (s,

3H), 3.69−3.60 (m, 4H), 2.50 (s, 3H), 2.48 (s, 3H), 1.31 (d, J = 6.8

Hz, 3H), 1.20−1.12 (m, 1H), 0.64−0.56 (m, 1H), 0.48−0.38 (m,

2H), 0.31−0.25 (m, 1H).

¹H NMR (600 MHz, DMSO-d₆) δ 11.69 (s, 1H), 7.96 (d, J = 1.6

Hz, 1H), 7.89 (d, J = 1.6 Hz, 1H), 7.79 (t, J = 7.9 Hz, 1H), 7.61 (q, J

= 5.2 Hz, 1H), 7.05 (d, J = 7.6 Hz, 1H), 6.96 (d, J = 8.1 Hz, 1H),),

4.85−4.69 (m, 2H), 3.65 (dq, J = 9.2, 6.8 Hz, 1H), 3.37 (s, 3H), 2.51

(d, J = 5.3 Hz, 3H), 2.44 (s, 3H), 2.09 (s, 3H), 1.32 (d, J = 6.8 Hz,

3H), 1.19−1.11 (m, 1H), 0.64−0.57 (m, 1H), 0.48−0.38 (m, 2H),

0.31−0.25 (m, 1H).

¹³C NMR (151 MHz, DMSO-d₆) δ 169.70, 164.65, 157.60, 153.26,

150.51, 145.89, 145.09, 139.91, 136.25, 136.03, 126.74, 126.37,

125.30, 119.87, 112.07, 108.41, 52.33, 46.35, 34.97, 29.16, 22.99,

17.91, 16.56, 15.53, 3.89, 3.34.

¹³C NMR (151 MHz, DMSO-d₆) δ 163.77, 158.70, 158.40, 153.31,

150.83, 147.26, 145.90, 145.04, 138.69, 137.75, 136.74, 126.24,

126.19, 125.93, 120.58, 113.47, 110.61, 52.00, 45.93, 43.11, 35.33,

17.96, 17.02, 15.55, 11.49, 3.85, 3.42.

ES+ (M + H)⁺observed 564.1866 expected 564.1848.

(S)-2-(1-Cyclopropylethyl)-5-(4-methyl-2-((6-(2-oxopyrrolidin-1-

yl)pyridin-2-yl)amino)thiazol-5-yl)-7-(methylsulfonyl)isoindolin-1-

one (58). Prepared from (S)-5-(2-amino-4-methylthiazol-5-yl)-2-(1-

cyclopropylethyl)-7-(methylsulfonyl)isoindolin-1-one (16) and 1-(6-

bromopyridin-2-yl)pyrrolidin-2-one (42), following the method used

for 52.

ES+ (M + H)⁺observed 564.1898 expected 564.1851.

(S)-2-(1-Cyclopropylethyl)-5-(2-((6-(4-(hydroxymethyl)oxazol-5-

yl)pyridin-2-yl)amino)-4-methylthiazol-5-yl)-7-(methylsulfonyl)-

isoindolin-1-one (54). Prepared from (S)-5-(2-amino-4-methylthia-

zol-5-yl)-2-(1-cyclopropylethyl)-7-(methylsulfonyl)isoindolin-1-one

(16) and (5-(6-bromopyridin-2-yl)oxazol-4-yl)methanol (40) follow-

ing the method used for 52.

¹H NMR (600 MHz, DMSO-d₆) δ 11.54 (s, 1H), 8.06 (d, J = 1.6

Hz, 1H), 8.01 (d, J = 1.6 Hz, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.71 (t, J

= 8.0 Hz, 1H), 6.76 (d, J = 8.0 Hz, 1H), 4.70 (s, 2H), 4.23 (t, J = 7.1

Hz, 2H), 3.64 (s, 4H), 2.59 (t, J = 8.0 Hz, 2H), 2.45 (s, 3H), 2.10 (p,

J = 7.7 Hz, 2H), 1.31 (d, J = 6.8 Hz, 3H), 1.15 (tt, J = 8.1, 3.9 Hz,

1H), 0.56−0.64 (m, 1H), 0.37−0.48 (m, 2H), 0.24−0.31 (m, 1H).

¹³C NMR (151 MHz, DMSO-d₆) δ 174.6 (s), 163.7 (s), 157.5 (s),

149.9 (d, J = 11.4 Hz), 146.0 (s), 145.3 (s), 139.6 (s), 137.7 (s),

136.1 (s), 126.5 (d, J = 11.2 Hz), 126.1 (s), 119.6 (s), 105.7 (s),

105.3 (s), 52.0 (s), 48.5 (s), 46.0 (s), 43.1 (s), 33.0 (s), 18.0 (s), 17.3

(s), 16.8 (s), 15.6 (s), 3.9 (s), 3.4 (s).

¹H NMR (600 MHz, DMSO-d₆) δ 11.67 (s, 1H), 8.46 (s, 1H),

8.11 (d, J = 1.6 Hz, 1H), 8.08 (d, J = 1.6 Hz, 1H), 7.86 (t, J = 7.9 Hz,

1H), 7.36 (d, J = 7.4 Hz, 1H), 7.10 (d, J = 8.2 Hz, 1H), 5.25 (t, J = 5.9

Hz, 1H), 4.84 (d, J = 5.3 Hz, 2H), 4.75−4.65 (m, 2H), 3.67−3.60 (m,

4H), 2.49 (s, 3H), 1.32 (d, J = 6.8 Hz, 3H), 1.19−1.11 (m, 1H),

0.65−0.55 (m, 1H), 0.49−0.36 (m, 2H), 0.30−0.22 (m, 1H).

¹³C NMR (151 MHz, DMSO-d₆) δ 163.70, 157.94, 151.35, 151.32,

145.92, 145.13, 144.42, 139.00, 138.05, 137.65, 136.14, 126.79,

126.44, 125.86, 120.11, 114.08, 110.89, 55.13, 51.99, 45.88, 43.07,

18.00, 16.82, 15.58, 3.89, 3.41. (one signal obscured).

ES+ (M + H)⁺observed 552.1739 expected 552.1739.

ES+ (M + H)⁺observed 566.1558 expected 566.1532.

(S)-2-(1-Cyclopropylethyl)-6-(4-methyl-2-((6-(2-oxopyrrolidin-1-

yl)pyridin-2-yl)amino)thiazol-5-yl)-N-(oxetan-3-yl)-3-oxoisoindo-

line-4-sulfonamide (59). Step 1: (S)-N-(5-(2-(1-Cyclopropylethyl)-

7-(N-(oxetan-3-yl)sulfamoyl)-1-oxoisoindolin-5-yl)-4-methylthiazol-

2-yl)acetamide

(S)-N-(6-((5-(2-(1-Cyclopropylethyl)-7-(methylsulfonyl)-1-oxoi-

soindolin-5-yl)-4-methylthiazol-2-yl)amino)pyridin-2-yl)acetamide

(55). Prepared from (S)-5-(2-amino-4-methylthiazol-5-yl)-2-(1-cyclo-

propylethyl)-7-(methylsulfonyl)isoindolin-1-one (16) and N-(6-bro-

mopyridin-2-yl)acetamide following the method used for 52.

¹H NMR (600 MHz, DMSO-d₆) δ 11.42 (s, 1H), 10.06 (s, 1H),

8.08 (d, J = 1.6 Hz, 1H), 8.03 (d, J = 1.7 Hz, 1H), 7.68−7.62 (m,

2H), 6.75 (dd, J = 7.4, 1.4 Hz, 1H), 4.76−4.66 (m, 2H), 3.69−3.59

(m, 4H), 2.42 (s, 3H), 2.17 (s, 3H), 1.32 (d, J = 6.8 Hz, 3H), 1.21−

1.13 (m, 1H), 0.63−0.57 (m, 1H), 0.47−0.38 (m, 2H), 0.3−0.25 (m,

1H).

(S)-6-(2-Acetamido-4-methylthiazol-5-yl)-2-(1-cyclopropylethyl)-

3-oxoisoindoline-4-sulfonyl chloride (1.80 g, 4.0 mmol) was dissolved

in DCM (30 mL). DMAP (1.45 g, 11.9 mmol) and MgSO₄(0.48 g,

4.0 mmol) were added followed by oxetan-3-amine (0.435 g, 6.0

mmol). The resulting mixture was stirred at r.t. for 2 h. The reaction

mixture was ﬁltered through Celite. The solvent was evaporated, and

the residue was puriﬁed by ﬂash silica chromatography, elution

gradient 0 to 20% MeOH in DCM to give the subtitle compound

(1.80 g, 93%) as a yellow solid.

¹³C NMR (151 MHz, DMSO-d₆) δ 169.38, 163.73, 158.56, 150.42,

149.71, 145.70, 145.07, 139.66, 137.55, 136.39, 127.47, 126.60,

126.52, 119.82, 106.08, 105.11, 52.07, 45.89, 43.17, 24.26, 17.95,

16.52, 15.53, 3.85, 3.44.

¹H NMR (400 MHz, DMSO-d₆) δ 12.33 (s, 1H), 8.57 (d, J = 7.0

Hz, 1H), 8.02 (d, J = 1.6 Hz, 1H), 7.84 (d, J = 1.6 Hz, 1H), 4.75 (s,

2H), 4.53 (q, J = 6.4 Hz, 2H), 4.44 (dt, J = 13.7, 6.8 Hz, 1H), 4.35 (q,

J = 6.0 Hz, 2H), 3.72−3.61 (m, 1H), 2.44 (s, 3H), 2.17 (s, 3H), 1.33

(d, J = 6.8 Hz, 3H), 1.22−1.12 (m, 1H), 0.65−0.56 (m, 1H), 0.48−

0.38 (m, 2H), 0.32−0.25 (m, 1H).ES+ (M + H)⁺491.

ES+ (M + H)⁺observed 526.1610 expected 526.1583.

(S)-N-(6-((5-(2-(1-Cyclopropylethyl)-7-(methylsulfonyl)-1-oxoi-

soindolin-5-yl)-4-methylthiazol-2-yl)amino)pyridin-2-yl)-N-methyl-

acetamide (56). Prepared from (S)-5-(2-amino-4-methylthiazol-5-

yl)-2-(1-cyclopropylethyl)-7-(methylsulfonyl)isoindolin-1-one (16),

and N-(6-bromopyridin-2-yl)-N-methylacetamide (41), following

the method used for 52.

Step 2: (S)-6-(2-Amino-4-methylthiazol-5-yl)-2-(1-cyclopropyleth-

yl)-N-(oxetan-3-yl)-3-oxoisoindoline-4-sulfonamide

¹H NMR (600 MHz, DMSO-d₆) δ 11.68 (br. s, 1H), 8.03 (d, J =

1.6 Hz, 1H), 8.00 (d, J = 1.6 Hz, 1H), 7.78 (t, J = 7.9 Hz, 1H), 7.04

(d, J = 7.6 Hz, 1H), 6.95 (d, J = 8.1 Hz, 1H), 4.75−4.67 (m, 2H),

3.67−3.57 (m, 4H), 3.36 (s, 3H), 2.44 (s, 3H), 2.09 (s, 3H), 1.31 (d,

J = 6.9 Hz, 3H), 1.18−1.10 (m, 1H), 0.63−0.56 (m, 1H), 0.48−0.37

(m, 2H), 0.31−0.24 (m, 1H).

(S)-N-(5-(2-(1-Cyclopropylethyl)-7-(N-(oxetan-3-yl)sulfamoyl)-1-

oxoisoindolin-5-yl)-4-methylthiazol-2-yl)acetamide (1.80 g, 3.67

mmol) was dissolved in MeOH (20 mL). A solution of LiOH

(0.879 g, 36.7 mmol) in water (0.2 mL) was added, and the resulting

solution was heated to 80 °C for 10 h. The reaction mixture was

allowed to cool, and then the solvent was evaporated. The residue was

dissolved in DCM (100 mL) and washed sequentially with water (50

mL × 2) and saturated brine (50 mL × 2). The organic layer was

¹³C NMR (151 MHz, DMSO-d₆) δ 169.71, 163.68, 157.68, 153.27,

150.67, 145.95, 145.04, 139.89, 137.66, 136.00, 127.09, 126.69,

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Drug Annotation

dried over Na₂SO₄, ﬁltered, and evaporated to give the subtitle

compound (1.50 g, 91%) as a yellow solid.

((6-((S)-3-((tert-butyldimethylsilyl)oxy)-2-oxopyrrolidin-1-yl)-

pyridin-2-yl)amino)-4-methylthiazol-5-yl)-2-((S)-1-cyclopropyleth-

yl)-N-methyl-3-oxoisoindoline-4-sulfonamide (95 mg, 0.14 mmol) in

THF (6 mL) at 25 °C over 15 min. The resulting solution was stirred

at 25 °C for 3 h. The solvent was evaporated, and the residue was

puriﬁed by preparative HPLC.

¹H NMR (400 MHz, DMSO-d₆) δ 8.59 (s, 1H), 7.83 (d, J = 1.6

Hz, 1H), 7.70 (d, J = 1.6 Hz, 1H), 7.37 (s, 2H), 4.71 (s, 2H), 4.52 (q,

J = 6.8 Hz, 2H), 4.49−4.37 (m, 1H), 4.37−4.30 (m, 2H), 3.71−3.59

(m, 1H), 2.30 (s, 3H), 1.32 (d, J = 6.8 Hz, 3H), 1.20−1.10 (m, 1H),

0.66−0.55 (m, 1H), 0.48−0.36 (m, 2H), 0.32−0.22 (m, 1H).

ES+ (M + H)⁺449.

¹H NMR (600 MHz, DMSO-d₆) δ 11.58 (s, 1H), 7.98 (s, 1H),

7.92 (s, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.75 (t, J = 8.0 Hz, 1H), 7.61−

7.56 (m, 1H), 6.80 (d, J = 8.0 Hz, 1H), 4.76 (s, 2H), 4.44−4.39 (m,

1H), 4.31−4.25 (m, 1H), 4.02−3.94 (m, 1H), 3.69−3.62 (m, 1H),

2.53 (d, J = 5.2 Hz, 3H), 2.48−2.41 (m, 4H), 1.94−1.85 (m, 1H),

1.33 (d, J = 6.8 Hz, 3H), 1.23 (bs, 1H), 1.21−1.13 (m, 1H), 0.65−

0.58 (m, 1H), 0.49−0.39 (m, 2H), 0.32−0.26 (m, 1H).

¹³C NMR (151 MHz, DMSO-d₆) δ 175.2, 165.1, 158.0, 150.6,

150.2, 146.4, 145.7, 140.2, 136.8, 136.5, 127.2, 126.5, 125.7, 120.1,

106.5, 105.7, 70.8, 52.8, 46.9, 44.4, 29.6, 28.2, 18.4, 17.1, 16.0, 4.4,

3.8.

Step 3: (S)-2-(1-Cyclopropylethyl)-6-(4-methyl-2-((6-(2-oxopyr-

rolidin-1-yl)pyridin-2-yl)amino)thiazol-5-yl)-N-(oxetan-3-yl)-3-oxoi-

soindoline-4-sulfonamide Prepared from (S)-6-(2-amino-4-methyl-

thiazol-5-yl)-2-(1-cyclopropylethyl)-N-(oxetan-3-yl)-3-oxoisoindo-

line-4-sulfonamide, and 1-(6-bromopyridin-2-yl)pyrrolidin-2-one

(42), following the method used for 52.

¹H NMR (500 MHz, DMSO-d₆) δ 11.56 (s, 1H), 8.56 (d, J = 7.1

Hz, 1H), 7.98 (d, J = 1.4 Hz, 1H), 7.90 (d, J = 1.5 Hz, 1H), 7.88−

7.84 (m, 1H), 7.72 (t, J = 8.0 Hz, 1H), 6.79−6.76 (m, 1H), 4.75 (s,

2H), 4.57−4.50 (m, 2H), 4.48−4.39 (m, 1H), 4.40−4. 34 (m, 2H),

4.25 (t, J = 7.1 Hz, 2H), 3.72−3. 63 (m, 1H), 2.60 (t, J = 8.1 Hz, 2H),

2.45 (s, 3H), 2.10 (p, J = 7.9 Hz, 2H), 1.34 (d, J = 6.8 Hz, 3H), 1.21−

1.12 (m, 1H), 0.65−0.56 (m, 1H), 0.49−0.39 (m, 2H), 0.33−0.25

(m, 1H).

ES+ (M + H)⁺observed 583.1816 expected 583.1797.

2-((S)-1-Cyclopropylethyl)-6-(2-((6-((R)-4-hydroxy-2-oxopyrroli-

din-1-yl)pyridin-2-yl)amino)-4-methylthiazol-5-yl)-N-methyl-3-ox-

oisoindoline-4-sulfonamide (63). Prepared from (S)-6-(2-amino-4-

methylthiazol-5-yl)-2-(1-cyclopropylethyl)-N-methyl-3-oxoisoindo-

line-4-sulfonamide (17), and (R)-4-((tert-butyldimethylsilyl)oxy)-

pyrrolidin-2-one (46), following the method used for 62.

¹H NMR (500 MHz, DMSO-d₆) δ 7.97 (s, 1H), 7.92 (d, J = 1.5

Hz, 1H), 7.87 (d, J = 8.1 Hz, 1H), 7.73 (t, J = 8.0 Hz, 1H), 7.57 (s,

1H), 6.78 (d, J = 8.0 Hz, 1H), 5.30 (d, J = 3.1 Hz, 1H), 4.75 (s, 2H),

4.41 (bs, 1H), 4.34−4.27 (m, 1H), 4.17 (d, J = 11.6 Hz, 1H), 3.69−

3.60 (m, 1H), 3.00−2.61 (m, 1H), 2.52 (s, H), 2.44 (s, 3H), 2.36 (d, J

= 17.1 Hz, 1H), 1.33 (d, J = 6.8 Hz, 3H), 1.21−1.19 (m, 1H), 0.66−

0. 57 (m, 1H), 0.49−0.38 (m, 2H), 0.32−0.24 (m, 1H).

¹³C NMR (126 MHz, DMSO-d₆) δ 173.3, 164.7, 157.6, 150.0,

149.9, 145.9, 145.2, 139.7, 136.3, 136.1, 126.9, 126.4, 125.3, 119.6,

105.7, 105.3, 62.4, 57.5, 52.4, 46.4, 43.0, 29.2, 18.0, 16.6, 15.6, 4.0,

3.4.

¹³C NMR (126 MHz, DMSO-d₆) δ 174.6, 164.7, 157.6, 149.9,

149.8, 146.1, 145.4, 139.6, 137.4, 136.4, 126.1, 125.9, 124.8, 119.5,

105.7, 105.4, 76.9, 76.8, 52.5, 48.5, 47.2, 46.5, 33.0, 18.0, 17.3, 16.7,

15.6, 3.9, 3.3.

ES+ (M + H)⁺observed 609.1938 expected 609.1954.

(S)-2-(1-Cyclopropylethyl)-6-(4-methyl-2-((6-(2-oxoimidazolidin-

1-yl)pyridin-2-yl)amino)thiazol-5-yl)-N-(oxetan-3-yl)-3-oxoisoindo-

line-4-sulfonamide (60). Prepared from (S)-6-(2-amino-4-methyl-

thiazol-5-yl)-2-(1-cyclopropylethyl)-N-(oxetan-3-yl)-3-oxoisoindo-

line-4-sulfonamide (59, step 2), and 1-(6-bromopyridin-2-yl)-

imidazolidin-2-one (43), following the method used for 52.

¹H NMR (500 MHz, DMSO-d₆) δ 11.45 (s, 1H), 8.54 (d, J = 7.2

Hz, 1H), 7.97 (s, 1H), 7.89 (d, J = 1.3 Hz, 1H), 7.71 (d, J = 8.1 Hz,

1H), 7.62 (t, J = 8.0 Hz, 1H), 7.22 (s, 1H), 6.62 (d, J = 7.8 Hz, 1H),

4.74 (s, 2H), 4.57−4.50 (m, 2H), 4.44 (h, J = 6.6 Hz, 1H), 4.39−4.

32 (m, 2H), 4.31−4.23 (m, 2H), 3.72−3. 62 (m, 1H), 3.46 (t, J = 8.1

Hz, 2H), 2.44 (s, 3H), 1.34 (d, J = 6.8 Hz, 3H), 1.22−1. Thirteen (m,

1H), 0.66−0.57 (m, 1H), 0.47−0.39 (m, 2H), 0.33−0.25 (m, 1H).

¹³C NMR (126 MHz, DMSO-d₆) δ 164.7, 158.1, 157.8, 151.1,

149.9, 146.1, 145.3, 139.1, 137.4, 136.4, 126.2, 125.9, 124.8, 119.3,

103.5, 103.2, 76.9, 76.8, 52.4, 47.2, 46.4, 45.0, 36.6, 18.0, 16.7, 15.6,

3.9, 3.3.

ES+ (M + H)⁺observed 583.1812 expected 583.1797.

(S)-2-(1-Cyclopropylethyl)-5-(4-methyl-2-((6-(2-oxopiperidin-1-

yl)pyridin-2-yl)amino)thiazol-5-yl)-7-(methylsulfonyl)isoindolin-1-

one (64). Prepared from (S)-5-(2-amino-4-methylthiazol-5-yl)-2-(1-

cyclopropylethyl)-7-(methylsulfonyl)isoindolin-1-one (16), and 1-(6-

bromopyridin-2-yl)piperidin-2-one (47), following the method used

for 52.

¹H NMR (500 MHz, DMSO-d₆) δ 11.62 (s, 1H), 8.10−8. 02 (m,

2H), 7.72 (t, J = 8.0 Hz, 1H), 7.31 (d, J = 7.9 Hz, 1H), 6.85 (d, J = 8.0

Hz, 1H), 4.71 (s, 2H), 4.11−4.02 (m, 2H), 3.69−3.58 (m, 4H), 2.48

(s, 3H), 2.00−1.92 (m, 2H), 1.90−1.81 (m, 2H), 1.32 (d, J = 6.8 Hz,

3H), 1.21−1.12 (m, 1H), 0.64−0.54 (m, 1H), 0.48−0.37 (m, 2H),

0.31−0.23 (m, 1H).

ES+ (M + H)⁺observed 610.1912 expected 610.1906.

(S)-2-(1-Cyclopropylethyl)-5-(4-methyl-2-((6-(3-methyl-2-oxoimi-

dazolidin-1-yl)pyridin-2-yl)amino)thiazol-5-yl)-7-(methylsulfonyl)-

isoindolin-1-one (61). Prepared from (S)-5-(2-amino-4-methylthia-

zol-5-yl)-2-(1-cyclopropylethyl)-7-(methylsulfonyl)isoindolin-1-one

(16), and 1-(6-bromopyridin-2-yl)-3-methylimidazolidin-2-one (44),

following the method used for 52.

¹³C NMR (126 MHz, DMSO-d₆) δ 170.1, 163.7, 157.6, 151.9,

150.1, 146.0, 145.3, 138.9, 137.7, 136.1, 126.5, 126.1, 119.5, 112.0,

107.0, 76.9, 52.0, 48.0, 46.0, 43.1, 33.3, 22.6, 20.4, 18.0, 16.9, 15.6,

3.9, 3.4.

¹H NMR (600 MHz, DMSO-d₆)) δ 11.44 (s, 1H), 8.07 (d, J = 1.6

Hz, 1H), 8.01 (d, J = 1.4 Hz, 1H), 7.74−7.71 (m, 1H), 7.63 (t, J = 8.0

Hz, 1H), 6.61 (d, J = 7.9 Hz, 1H), 4.70 (s, 2H), 4.20 (dd, J = 8.9, 7.2

Hz, 2H), 3.64 (s, 4H), 3.48−3.52 (m, 2H), 2.81 (s, 3H), 2.45 (s, 3H),

1.32 (d, J = 6.8 Hz, 3H), 1.19−1.12 (m, 1H), 0.64−0.58 (m, 1H),

0.47−0.39 (m, 2H), 0.31−0.26 (m, 1H).

ES+ (M + H)⁺observed 566.1915 expected 566.1896.

(S)-2-(1-Cyclopropylethyl)-N-methyl-6-(4-methyl-2-((6-(2-oxopi-

peridin-1-yl)pyridin-2-yl)amino)thiazol-5-yl)-3-oxoisoindoline-4-

sulfonamide (65). Prepared from (S)-6-(2-amino-4-methylthiazol-5-

yl)-2-(1-cyclopropylethyl)-N-methyl-3-oxoisoindoline-4-sulfonamide

(17) and 1-(6-bromopyridin-2-yl)piperidin-2-one (47), following the

method used for 52.

¹³C NMR (151 MHz, DMSO-d₆) δ 164.2, 158.1, 157.1, 151.5,

150.4, 146.4, 145.7, 139.7, 138.1, 136.6, 127.0, 126.9, 126.6, 119.9,

103.7, 103.7, 52.5, 46.4, 43.9, 43.5, 42.9, 31.1, 18.5, 17.2, 16.1, 4.4,

3.9.

¹H NMR (600 MHz, DMSO-d₆) δ 11.61 (s, 1H), 8.00 (d, J = 1.3

Hz, 1H), 7.92 (d, J = 1.5 Hz, 1H), 7.72 (t, J = 8.0 Hz, 1H), 7.57 (q, J

= 5.2 Hz, 1H), 7.32−7.29 (m, 1H), 6.86 (d, J = 8.0 Hz, 1H), 4.76 (s,

2H), 4.09−4.04 (m, 2H), 3.69−3.62 (m, 1H), 2.52 (d, J = 5.3 Hz,

3H), 2.48 (s, 3H), 1.99−1.93 (m, 2H), 1.89−1.83 (m, 2H), 1.33 (d, J

= 6.8 Hz, 3H), 1.21−1.14 (m, 1H), 0.64−0.58 (m, 1H), 0.48−0.39

(m, 2H), 0.31−0.26 (m, 1H).

ES+ (M + H)⁺observed 567.1859 expected 567.1848.

2-((S)-1-Cyclopropylethyl)-6-(2-((6-((R)-3-hydroxy-2-oxopyrroli-

din-1-yl)pyridin-2-yl)amino)-4-methylthiazol-5-yl)-N-methyl-3-ox-

oisoindoline-4-sulfonamide (62). Prepared from (S)-6-(2-amino-4-

methylthiazol-5-yl)-2-(1-cyclopropylethyl)-N-methyl-3-oxoisoindo-

line-4-sulfonamide (17) and (R)-1-(6-bromopyridin-2-yl)-3-((tert-

butyldimethylsilyl)oxy)pyrrolidin-2-one (45), following the method

used for 52, followed by deprotection of the alcohol: (triethylamine

trihydroﬂuoride (2 mL, 12.28 mmol) was added dropwise to 6-(2-

¹³C NMR (151 MHz, DMSO-d₆) δ 170.6, 165.2, 158.1, 152.3,

150.5, 146.4, 145.8, 139.4, 136.8, 136.5, 127.1, 126.3, 125.6, 120.0,

112.5, 107.4, 52.8, 48.4, 46.9, 33.7, 29.6, 23.1, 20.8, 18.4, 17.2, 16.0,

4.4, 3.8.

8071

https://doi.org/10.1021/acs.jmedchem.1c00434

J. Med. Chem. 2021, 64, 8053−8075

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Drug Annotation

ES+ (M + H)⁺observed 581.1995 expected 581.2004.

(S)-4-(6-((5-(2-(1-Cyclopropylethyl)-7-(methylsulfonyl)-1-oxoi-

soindolin-5-yl)-4-methylthiazol-2-yl)amino)pyridin-2-yl)-

morpholin-3-one (66). Prepared from (S)-5-(2-amino-4-methylthia-

zol-5-yl)-2-(1-cyclopropylethyl)-7-(methylsulfonyl)isoindolin-1-one

(16) and 4-(6-bromopyridin-2-yl)morpholin-3-one (48), following

the method used for 52.

ES+ (M + H)⁺observed 568.1713 expected 568.1688.

(S)-2-(1-Cyclopropylethyl)-N-methyl-6-(4-methyl-2-((6-(2-oxo-

1,3-oxazinan-3-yl)pyridin-2-yl)amino)thiazol-5-yl)-3-oxoisoindo-

line-4-sulfonamide (70). Prepared from (S)-6-(2-amino-4-methyl-

thiazol-5-yl)-2-(1-cyclopropylethyl)-N-methyl-3-oxoisoindoline-4-sul-

fonamide (17) and 3-(6-bromopyridin-2-yl)-1,3-oxazinan-2-one (49),

following the method used for 52.

¹H NMR (600 MHz, DMSO-d₆) δ 11.64 (s, 1H), 8.07−8.03 (m,

2H), 7.76 (t, J = 8.0 Hz, 1H), 7.60 (d, J = 7.9 Hz, 1H), 6.88 (d, J = 8.0

Hz, 1H), 4.71 (s, 2H), 4.29 (s, 2H), 4.19−4.15 (m, 2H), 4.09−4.05

(m, 2H), 3.64 (s, 3H), 3.6−3.63 (m, 1H), 2.47 (s, 3H), 1.31 (d, J =

6.8 Hz, 3H), 1.19−1.12 (m, 1H), 0.63−0.57 (m, 1H), 0.46−0.38 (m,

2H), 0.30−0.25 (m, 1H).

¹H NMR (600 MHz, DMSO-d₆) δ 11.61 (s, 1H), 8.00 (d, J = 1.6

Hz, 1H), 7.92 (d, J = 1.6 Hz, 1H), 7.73 (t, J = 8.0 Hz, 1H), 7.56 (q, J

= 5.2 Hz, 1H), 7.37 (d, J = 7.8 Hz, 1H), 6.85 (d, J = 7.9 Hz, 1H),

4.80−4.68 (m, 2H), 4.39 (t, J = 5.4 Hz, 2H), 4.13 (t, J = 6.2 Hz, 2H),

3.65 (dq, J = 9.2, 6.8 Hz, 1H), 2.51 (d, J = 5.2 Hz, 3H), 2.47 (s, 3H),

2.22−2.15 (m, 2H), 1.32 (d, J = 6.9 Hz, 3H), 1.20−1.12 (m, 1H),

0.64−0.57 (m, 1H), 0.48−0.39 (m, 2H), 0.32−0.24 (m, 1H).

¹³C NMR (151 MHz, DMSO-d₆) δ 165.1, 157.6, 152.5, 151.5,

149.9, 145.9, 145.3, 139.6, 136.3, 136.0, 126.7, 125.9, 124.4, 120.8,

113.3, 107.7, 67.0, 52.8, 46.4, 44.6, 29.2, 21.9, 18.0, 16.7, 15.0, 4.6,

2.8.

¹³C NMR (151 MHz, DMSO-d₆) δ 167.0, 163.7, 157.4, 150.4,

150.1, 146.0, 145.3, 139.4, 137.7, 136.0, 126.5, 126.5, 126.1, 119.6,

110.4, 107.4, 67.9, 63.4, 52.0, 46.7, 46.0, 43.1, 18.0, 16.8, 15.6, 3.9,

3.4.

ES+ (M + H)⁺observed 568.1710 expected 568.1688.

ES+ (M + H)⁺583.1803 expected 583.1792.

(S)-2-(1-Cyclopropylethyl)-N-methyl-6-(4-methyl-2-((6-(3-

oxomorpholino)pyridin-2-yl)amino)thiazol-5-yl)-3-oxoisoindoline-

4-sulfonamide (67). Prepared from (S)-6-(2-amino-4-methylthiazol-

5-yl)-2-(1-cyclopropylethyl)-N-methyl-3-oxoisoindoline-4-sulfona-

mide (17), and 4-(6-bromopyridin-2-yl)morpholin-3-one (48),

following the method used for 52.

(S)-2-(1-Cyclopropylethyl)-N-methyl-6-(4-methyl-2-((6-(2-oxopi-

perazin-1-yl)pyridin-2-yl)amino)thiazol-5-yl)-3-oxoisoindoline-4-

sulfonamide (71). Prepared from (S)-6-(2-amino-4-methylthiazol-5-

yl)-2-(1-cyclopropylethyl)-N-methyl-3-oxoisoindoline-4-sulfonamide

(17) and benzyl 4-(6-bromopyridin-2-yl)-3-oxopiperazine-1-carbox-

ylate (50), following the method used for 52, followed by

deprotection of the carbamate: (BBr₃(0.185 mL, 1.96 mmol) was

added to (S)-benzyl 4-(6-((5-(2-(1-cyclopropylethyl)-7-(N-methyl-

sulfamoyl)-1-oxoisoindolin-5-yl)-4-methylthiazol-2-yl)amino)pyridin-

2-yl)-3-oxopiperazine-1-carboxylate (140 mg, 0.20 mmol) in DCM

(10 mL) at 0 °C over 0.5 h. The resulting mixture was stirred at RT

for 4 h. The reaction mixture was poured onto ice (20 mL), extracted

with DCM (2 × 20 mL), and the aqueous layer was adjusted to pH =

9 with saturated Na₂CO₃and extracted with DCM (3 × 50 mL). The

crude product was puriﬁed by preparative HPLC.

¹H NMR (600 MHz, DMSO-d₆) δ 11.64 (s, 1H), 7.98 (d, J = 1.6

Hz, 1H), 7.91 (d, J = 1.6 Hz, 1H), 7.75 (t, J = 8.0 Hz, 1H), 7.63−7.54

(m, 2H), 6.88 (d, J = 8.0 Hz, 1H), 4.80−4.70 (m, 2H), 4.29 (s, 2H),

4.21−4.14 (m, 2H), 4.09−4.04 (m, 2H), 3.65 (dq, J = 9.2, 6.8 Hz,

1H), 2.51 (d, J = 4.9 Hz, 3H), 2.46 (s, 3H), 1.32 (d, J = 6.8 Hz, 3H),

1.19−1.12 (m, 1H), 0.64−0.56 (m, 1H), 0.47−0. 38 (m, 2H), 0.32−

0.25 (m, 1H).

¹³C NMR (151 MHz, DMSO-d₆) δ 167.55, 165.5, 158.0, 150.9,

150.6, 146.4, 145.8, 139.9, 136.8, 136.5, 127.1, 126.3, 125.6, 120.0,

110.9, 108.0, 68.4, 63.9, 52.8, 47.2, 46.9, 29.6, 18.4, 17.2, 16.0, 4.4,

3.8.

¹H NMR (600 MHz, DMSO-d₆) δ 11.60 (s, 1H), 7.98 (d, J = 1.6

Hz, 1H), 7.91 (d, J = 1.6 Hz, 1H), 7.72 (t, J = 8.0 Hz, 1H), 7.60−7.54

(m, 1H), 7.48 (d, J = 7.9 Hz, 1H), 6.85 (d, J = 8.0 Hz, 1H), 4.80−

4.71 (m, 2H), 4.06 (t, J = 5.5 Hz, 2H), 3.65 (dq, J = 9.1, 6.8 Hz, 1H),

3.48 (s, 2H), 3.10 (t, J = 5.5 Hz, 2H), 2.52 (d, J = 4.0 Hz, 3H), 2.46

(s, 3H), 1.32 (d, J = 6.8 Hz, 3H), 1.20−1.13 (m, 1H), 0.64−0.57 (m,

1H), 0.49−0.39 (m, 2H), 0.31−0.25 (m, 1H).

ES+ (M + H)⁺observed 583.1825 expected 583.1797.

(S)-2-(1-Cyclopropylethyl)-6-(4-methyl-2-((6-(3-oxomorpholino)-

pyridin-2-yl)amino)thiazol-5-yl)-N-(oxetan-3-yl)-3-oxoisoindoline-

4-sulfonamide (68). Prepared from (S)-6-(2-amino-4-methylthiazol-

5-yl)-2-(1-cyclopropylethyl)-N-(oxetan-3-yl)-3-oxoisoindoline-4-sul-

fonamide (59, step 2) and 4-(6-bromopyridin-2-yl)morpholin-3-one

(48), following the method used for 52.

¹³C NMR (151 MHz, DMSO-d₆) δ 168.8, 164.7, 157.5, 150.9,

150.0, 145.9, 145.3, 139.1, 136.3, 136.1, 126.7, 125.9, 125.1, 119.5,

111.2, 107.1, 52.4, 51.2, 48.5, 46.4, 42.7, 29.2, 18.3, 16.8, 16.0, 4.7,

3.4.

¹H NMR (600 MHz, DMSO-d₆) δ 11.64 (s, 1H), 8.56 (d, J = 6.3

Hz, 1H), 7.99 (d, J = 1.6 Hz, 1H), 7.89 (d, J = 1.6 Hz, 1H), 7.76 (t, J

= 8.0 Hz, 1H), 7.59 (d, J = 7.9 Hz, 1H), 6.88 (d, J = 8.0 Hz, 1H),

4.81−4.70 (m, 2H), 4.54 (dt, J = 11.1, 6.7 Hz, 2H), 4.48−4.41 (m,

1H), 4.37 (dt, J = 9.9, 6.3 Hz, 2H), 4.30 (s, 2H), 4.20−4.15 (m, 2H),

4.09−4.04 (m, 2H), 3.67 (dq, J = 9.4, 6.8 Hz, 1H), 2.46 (s, 3H), 1.34

(d, J = 6.8 Hz, 3H), 1.14−1.22 (m, 1H), 0. 64−0.58 (m, 1H), 0.47−

0.40 (m, 2H), 0.32−0.26 (m, 1H).

ES+ (M + H)⁺observed 582.1983 expected 582.1957.

(S)-2-(1-Cyclopropylethyl)-N-methyl-6-(4-methyl-2-((6-(4-meth-

yl-2-oxopiperazin-1-yl)pyridin-2-yl)amino)thiazol-5-yl)-3-oxoisoin-

doline-4-sulfonamide (72). Prepared from (S)-6-(2-amino-4-meth-

ylthiazol-5-yl)-2-(1-cyclopropylethyl)-N-methyl-3-oxoisoindoline-4-

sulfonamide (17),and 1-(6-bromopyridin-2-yl)-4-methylpiperazin-2-

one (51), following the method used for 52.

¹³C NMR (151 MHz, DMSO-d₆) δ 167.0, 164.7, 157.5, 150.4,

150.1, 146.1, 145.5, 139.4, 137.4, 136.3, 126.0, 125.9, 124.8, 119.5,

110.5, 107.5, 76.9, 76.8, 67.9, 63.4, 52.5, 47.7, 46.7, 46.5, 17.9, 16.8,

14.5, 4.8, 2.7.

¹H NMR (600 MHz, DMSO-d₆) δ 11.58 (s, 1H), 8.00 (d, J = 1.7

Hz, 1H), 7.90 (d, J = 1.7 Hz, 1H), 7.75 (t, J = 8.0 Hz, 1H), 7.58 (br.

q, J = 5.2 Hz, 1H), 7.44 (d, J = 8.0 Hz, 1H), 6.88 (d, J = 8.0 Hz, 1H),

4.80−4.72 (m, 2H), 4.08 (dd, J = 6.7, 4.6 Hz, 2H), 3.65 (dq, J = 9.2,

6.8 Hz, 1H), 3.21 (s, 2H), 2.77−2.85 (m, 2H), 2.52 (d, J = 4.3 Hz,

3H), 2.46 (s, 3H), 2.29 (s, 3H), 1.33 (d, J = 6.8 Hz, 3H), 1.20−1.13

(m, 1H), 0.64−0.58 (m, 1H), 0.48−0.38 (m, 2H), 0.32−0.26 (m,

1H).

ES+ (M + H)⁺observed 625.1906 expected 625.1903.

(S)-3-(6-((5-(2-(1-Cyclopropylethyl)-7-(methylsulfonyl)-1-oxoi-

soindolin-5-yl)-4-methylthiazol-2-yl)amino)pyridin-2-yl)-1,3-oxa-

zinan-2-one (69). Prepared from (S)-5-(2-amino-4-methylthiazol-5-

yl)-2-(1-cyclopropylethyl)-7-(methylsulfonyl)isoindolin-1-one (16)

and 3-(6-bromopyridin-2-yl)-1,3-oxazinan-2-one (49), following the

method used for 52.

¹³C NMR (126 MHz, DMSO-d₆) δ 166.9, 164.7, 157.5, 151.0,

150.1, 146.0, 145.3, 139.3, 136.3, 136.1, 126.8, 126.0, 125.2, 119.6,

110.8, 107.4, 59.8, 52.4, 51.3, 47.3, 46.4, 44.3, 29.2, 18.0, 16.7, 15.6,

4.0, 3.4.

¹H NMR (600 MHz, DMSO-d₆) δ 11.59 (s, 1H), 8.06 (d, J = 1.6

Hz, 1H), 8.03 (d, J = 1.6 Hz, 1H), 7.72 (t, J = 8.0 Hz, 1H), 7.37 (d, J

= 8.0 Hz, 1H), 6.84 (d, J = 8.0 Hz, 1H), 4.66−4.76 (m, 2H), 4.39 (t, J

= 5.4 Hz, 2H), 4.12 (t, J = 6.2 Hz, 2H), 3.66−3.59 (m, 4H), 2.47 (s,

3H), 2.23−2.17 (m, 2H), 1.31 (d, J = 6.8 Hz, 3H), 1.19−1.11 (m,

1H), 0.63−0.56 (m, 1H), 0.47−0.37 (m, 2H), 0.31−0.24 (m, 1H).

¹³C NMR (151 MHz, DMSO-d₆) δ 164.5, 157.5, 152.5, 151.5,

149.9, 146.4, 145.3, 139.3, 137.7, 136.0, 126.4, 126.1, 119.5, 110.7,

106.7, 67.0, 51.1, 46.0, 45.9, 43.1, 21.9, 18.0, 16.8, 15.6, 3.9, 3.4. One

C obscuredprobably overlap of two peaks at 126.4.

ES+ (M + H)⁺observed 596.2106 expected 596.2114.

Biological Assays. All animal procedures were approved by the

ethical committee in Gothenburg, Sweden and were carried out under

the appropriate license.

Enzymatic inhibition assays for PI3K isoforms were run as

previously described.²²

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Drug Annotation

Cellular Assays. Inhibition of activity in JeKo-1 cells (PI3Kδ) was

run as previously described.²²

conjugated antihuman CD16 (Clone 3G8, BD Biosciences) antibod-

ies diluted in Streck cell preservative (Streck) for 30 min, in the dark

at RT. Thereafter, residual red blood cells were lysed by addition of

Optilyse B (Beckman Coulter), and samples were washed and

resuspended in DPBS (Gibco), 2 mM EDTA (Invitrogen), 2% FBS

(Gibco) prior to analysis on an Accuri C6 Cytometer (BD

Biosciences). Data were analyzed with FlowJo Software (TreeStar,

Ashland, OR). Eosinophils were gated by CD16-CD11b+ staining

and SSC-A properties versus CD16+CD11b+ neutrophils, and

median ﬂuorescence intensity (MFI) values for CD11b were collected

for each population.

Inhibition of p-Akt in RAW-264 cells was run as previously

described.²³

Pharmacokinetic Studies. Pharmacokinetic studies were carried

out as described previously.³⁰

Rat Ovalbumin Challenge Study. All rat experiments were

approved by the by the regional ethics committee in Gothenburg with

approval number 94−2014 and the approved site number was 31−

5373/11.

Rats (male BN, Charles River Laboratories; male, 200−230 g; 8−

10 per group) were sensitized by subcutaneous injection of a mixture

of ovalbumin (100 μg) and aluminum hydroxide (100 mg) in saline

(1 mL total volume) on days 1 and 8. On day 15 the animals were

placed into a closed plexiglass box and were exposed to an aerosolized

solution of 1% ovalbumin for 20 min. Compounds were administered

by nebulization (compounds under investigation) or intratracheal

instillation (budesonide positive control) on day 22. After a gap,

normally 1 h, but of variable length for the time-course study, the

animals were again exposed to aerosolized 1% ovalbumin. Rats were

terminated either 2 h post challenge (for determination of

phosphorylation of S6 ribosomal protein) or 48 h post challenge

(for measurement of eosinophils and cytokines). Bronchioalveolar

lavage (BAL) was collected using 4 mL PBS (without Ca2⁺or Mg2⁺)

in a twice in, twice out manner. BAL ﬂuid was centrifuged and the

supernatant was divided into aliquots and stored at −70 °C until

analysis. Eosinophil levels were determined using a Sysmex ﬂow

cytometer.

ASSOCIATED CONTENT

■

sı

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00434.

Details of the preliminary macrocyclization experiments

and results; a ligand interaction diagram for the crystal

structure of 58; additional in vivo experiment results; oﬀ-

target and kinase screening results, copies of ¹H and ¹³

C

NMR spectra and HPLC traces for ﬁnal compounds

(PDF)

Table of molecular formula strings (CSV)

Accession Codes

Crystal structures of PI3Kδ small molecule complexes have

been deposited in the PDB under the following accession

codes 7 (7oi4); 12 (7ois); 20 (7oij); 58 (7oil). Authors will

release the atomic coordinates and experimental data upon

article publication. PDB IDs have been provided in ﬁgure

legends.

Cytokine and mediator levels were determined by a MSD v-plex

proinﬂammatory rat panel (Meso Scale Discovery), except for IL-17

which was determined using an ELISA with AbCam antibody 119536.

S6 ribosomal protein levels were determined by immunoﬂuor-

escence of pS6RP+ lymphocytes which were measured using

XPRabbit mAb (Alexa Fluor 488 Conjugate) #4803. Samples were

processed by ﬂow cytometry on a LSR Fortessa (BD Biosciences),

and the lymphocyte population was selected based on FSC-A and

SSC-A properties. Collected data were analyzed with FlowJo Software

(TreeStar).

AUTHOR INFORMATION

■

Corresponding Author

Human Peripheral Blood Studies. Patients and Sample

Collection. Fresh venous blood samples were obtained from asthmatic

blood donors in the West Sweden Asthma Study (WSAS).³¹The

study was approved by the Regional Ethical Approval Committee in

Gothenburg, Sweden, and all subjects had signed informed consents.

PBMC T Cell Cytokine Responses. Human PBMCs were isolated

from six asthmatic donors classiﬁed as moderate to severe asthma. All

asthmatic donors except one were treated with inhaled corticosteroids

and β2 agonists. PBMCs were isolated by density gradient

centrifugation using Polymorphoprep (Axis-Shield) and cultured in

U-shaped 96-well plates at 200 000 cells/well in RPMI1640 media

(Gibco) supplemented with 10% heat-inactivated FBS (hiFBS;

Gibco) and penicillin-streptomycin (100 U/mL; Gibco). Cells were

preincubated for 1 h with compounds in 3-fold serial dilutions

(duplicates) at ﬁnal concentrations of 1.84 μM to 0.13 pM at 37 °C,

5% CO₂and incubated for 24 h with anti-CD2/CD3/CD28 T cell

activation beads (100 000 beads/well; Miltenyi Biotec). Supernatants

were collected by centrifugation and analyzed for cytokines by a V-

PLEX custom IL-5 and IL-17A assay (Meso Scale Discovery).

Eosinophil CD11b Assay. Primary human eosinophils were

isolated from fresh venous blood samples from asthmatic donors

classiﬁed moderate to severe asthma (N = 6−10 depending on

compound). All asthmatic donors were treated with inhaled

corticosteroids and β2 agonists.

Matthew W. D. Perry − Medicinal Chemistry, Research and

Early Development, Respiratory and Immunology (R&I),

BioPharmaceuticals R&D, AstraZeneca, Gothenburg SE-431

83, Sweden; orcid.org/0000-0002-9799-0746;

Phone: +46725827070; Email: matthew.perry@

astrazeneca.com

Authors

Karin Björhall − Bioscience COPD/IPF, Research and Early

Development, Respiratory and Immunology (R&I),

BioPharmaceuticals R&D, AstraZeneca, Gothenburg SE-431

83, Sweden

Peter Bold − DMPK, Research and Early Development,

Respiratory and Immunology (R&I), BioPharmaceuticals

R&D, AstraZeneca, Gothenburg SE-431 83, Sweden

̋

Mikael Brulls − Early Product Development &

Manufacturing, Pharmaceutical Sciences R&D, AstraZeneca,

Gothenburg SE-431 83, Sweden

Ulf Börjesson − Computational Chemistry, Discovery Sciences,

R&D, AstraZeneca, Gothenburg SE-431 83, Sweden

Johan Carlsson − Discovery Biology, Discovery Sciences, R&D,

AstraZeneca, Gothenburg SE-431 83, Sweden; Present

Address: (J.C.) AstraZeneca Operations, Sweden

Granulocytes were isolated from whole blood by polymorphprep

(Axis-Shield), seeded in U-shaped 96-well plates at 1 × 10⁵cells/well

in Hank’s Balanced Salt solution media (HBSS, Gibco) with 0.1%

bovine serum albumin (BSA, Sigma-Aldrich), and preincubated with

inhibitors for 30 min at 37 °C, 5% CO₂followed by stimulation with 3

nM eotaxin-3 (Peprotech) for 30 min at RT.

̈

Biomanufacturing Center (SBC), Gartunavagen 14,

̈

Gartuna, SE-15257 Sodertalje, Sweden.

Hui-Fang Amy Chang − Medicinal Chemistry, Research and

Early Development, Respiratory and Immunology (R&I),

BioPharmaceuticals R&D, AstraZeneca, Gothenburg SE-431

83, Sweden

Stimulations were stopped by staining with APC-conjugated

antihuman CD11b (Clone Vim12, Life technologies) and PE-

8073

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J. Med. Chem. 2021, 64, 8053−8075

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Drug Annotation

Yunhua Chen − Pharmaron Beijing Co., Ltd., Beijing 100176,

P.R. China

Anders Eriksson − Medicinal Chemistry, Research and Early

Development, Respiratory and Immunology (R&I),

BioPharmaceuticals R&D, AstraZeneca, Gothenburg SE-431

83, Sweden

Ingelheim Pharma, Birkendorf Strasse, Biberach 88397,

Germany.

Victoria Ullah − Medicinal Chemistry, Research and Early

Development, Respiratory and Immunology (R&I),

BioPharmaceuticals R&D, AstraZeneca, Gothenburg SE-431

83, Sweden

Britt-Marie Fihn − DMPK, Research and Early Development,

Respiratory and Immunology (R&I), BioPharmaceuticals

R&D, AstraZeneca, Gothenburg SE-431 83, Sweden

Rebecca Fransson − Advanced Drug Delivery, Pharmaceutical

Sciences R&D, AstraZeneca, Gothenburg SE-431 83, Sweden

Linda Fredlund − Discovery Biology, Discovery Sciences,

R&D, AstraZeneca, Gothenburg SE-431 83, Sweden;

orcid.org/0000-0002-2104-5617

Christian Tyrchan − Medicinal Chemistry, Research and

Early Development, Respiratory and Immunology (R&I),

BioPharmaceuticals R&D, AstraZeneca, Gothenburg SE-431

83, Sweden; orcid.org/0000-0002-6470-984X

Tiiu Wennberg − Bioscience COPD/IPF, Research and Early

Development, Respiratory and Immunology (R&I),

BioPharmaceuticals R&D, AstraZeneca, Gothenburg SE-431

83, Sweden

Hongbin Ge − Pharmaron Beijing Co., Ltd., Beijing 100176,

P.R. China

Annika Westin Eriksson − Clinical Pharmacology and Safety

Sciences, R&D, AstraZeneca, Gothenburg SE-431 83, Sweden

Wenzhen Yang − Pharmaron Beijing Co., Ltd., Beijing

100176, P.R. China; orcid.org/0000-0003-2226-0869

Shuchun Zhao − Pharmaron Beijing Co., Ltd., Beijing

100176, P.R. China

Haijuan Huang − Pharmaron Beijing Co., Ltd., Beijing

100176, P.R. China

Kostas Karabelas − Projects, Research and Early Development,

Respiratory and Immunology (R&I), BioPharmaceuticals

R&D, AstraZeneca, Gothenburg SE-431 83, Sweden;

Present Address: (K.K.) Bioglan AB, Borrgatan 31, SE-

̈

Linda Oster − Structure & Biophysics, Discovery Sciences,

R&D, AstraZeneca, Gothenburg SE-431 83, Sweden

̈

202 13 Malmo, Sweden.

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00434

Eva Lamm Bergström − DMPK, Research and Early

Development, Respiratory and Immunology (R&I),

BioPharmaceuticals R&D, AstraZeneca, Gothenburg SE-431

83, Sweden

Notes

The authors declare the following competing ﬁnancial

interest(s): All authors were employees and/or shareholders

of AstraZeneca or Pharmaron when this work was carried out.

M.W.D.P., P.B., U.B., K.K:, M.M., A.N., J.P, N.P. and C.T. are

inventors of patents assigned to AstraZeneca covering

inhibitors of PI3Ks.

Sarah Lever − Medicinal Chemistry, Research and Early

Development, Respiratory and Immunology (R&I),

BioPharmaceuticals R&D, AstraZeneca, Gothenburg SE-431

83, Sweden

Helena Lindmark − Discovery Biology, Discovery Sciences,

R&D, AstraZeneca, Gothenburg SE-431 83, Sweden

Mickael Mogemark − Medicinal Chemistry, Research and

Early Development, Respiratory and Immunology (R&I),

BioPharmaceuticals R&D, AstraZeneca, Gothenburg SE-431

83, Sweden

Antonios Nikitidis − Medicinal Chemistry, Research and

Early Development, Respiratory and Immunology (R&I),

BioPharmaceuticals R&D, AstraZeneca, Gothenburg SE-431

83, Sweden

Anna-Pia Palmgren − Medicinal Chemistry, Research and

Early Development, Respiratory and Immunology (R&I),

BioPharmaceuticals R&D, AstraZeneca, Gothenburg SE-431

83, Sweden

Nils Pemberton − Medicinal Chemistry, Research and Early

Development, Respiratory and Immunology (R&I),

BioPharmaceuticals R&D, AstraZeneca, Gothenburg SE-431

83, Sweden; orcid.org/0000-0002-8875-7221

Jens Petersen − Structure & Biophysics, Discovery Sciences,

R&D, AstraZeneca, Gothenburg SE-431 83, Sweden

Mio Rodrigo Blomqvist − Bioscience Cough and In Vivo,

Research and Early Development, Respiratory and

Immunology (R&I), BioPharmaceuticals R&D, AstraZeneca,

Gothenburg SE-431 83, Sweden

ACKNOWLEDGMENTS

■

We acknowledge Annika Borde for help with drawing plots of

ovalbumin challenge results and our colleagues in the analytical

and puriﬁcation departments for their help with puriﬁcation

and analysis of the compounds described.

ABBREVIATIONS USED

■

BSA, bovine serum albumin; CCR2, CC chemokine receptor

2; CCL2, CC chemokine ligand 2; CCR5, CC chemokine

receptor 5; DCM, dichloromethane; DIPEA, diisopropylethyl-

amine; DPBS, Dulbecco’s phosphate-buﬀered saline; HBSS,

Hank’s balanced salt solution media; PBMC, peripheral blood

mononuclear cells; T3P, 1-propanephosphonic anhydride

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https://doi.org/10.1021/acs.jmedchem.1c00434

J. Med. Chem. 2021, 64, 8053−8075

Article Doi

Discovery of AZD8154, a Dual PI3Kγδ Inhibitor for the Treatment of Asthma

DOI: 10.1021/acs.jmedchem.1c00434

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Article abstract of DOI:10.1021/acs.jmedchem.1c00434

Full text of DOI:10.1021/acs.jmedchem.1c00434

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