Generation of Highly Selective, Potent, and Covalent G Protein-Coupled Receptor Kinase 5 Inhibitors

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Generation of Highly Selective, Potent, and Covalent G Protein-

Coupled Receptor Kinase 5 Inhibitors

Rachel A. Rowlands,^∥Qiuyan Chen,^∥Renee A. Bouley, Larisa V. Avramova, John J. G. Tesmer,*

and Andrew D. White *

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ABSTRACT: The ability of G protein-coupled receptor (GPCR) kinases (GRKs) to regulate the desensitization of GPCRs has

made GRK2 and GRK5 attractive targets for treating diseases such as heart failure and cancer. Previously, our work showed that

Cys474, a GRK5 subfamily-speciﬁc residue located on a ﬂexible loop adjacent to the active site, can be used as a covalent handle to

achieve selective inhibition of GRK5 over GRK2 subfamily members. However, the potency of the most selective inhibitors

remained modest. Herein, we describe a successful campaign to adapt an indolinone scaﬀold with covalent warheads, resulting in a

series of 2-haloacetyl-containing compounds that react quickly and exhibit three orders of magnitude selectivity for GRK5 over

GRK2 and low nanomolar potency. They however retain a similar selectivity proﬁle across the kinome as the core scaﬀold, which

was based on Sunitinib.

studies where GRK5 was knocked down, cardiomyocytes were

protected from hypertrophic cardiomyopathy.⁷The inﬂuence

of GRK5 in progressive heart failure and hypertrophic

cardiomyopathy however remains unclear, in part because

GRK2 can also mediate hypertrophic responses⁸and there are

few compounds known to have clear GRK5 selectivity that

could be used to test mechanistic hypotheses in physiologically

relevant cells or animals.

INTRODUCTION

■

G protein-coupled receptor (GPCR) kinases (GRKs)

selectively recognize and phosphorylate activated GPCRs,

leading to their desensitization and internalization, a process

critical for maintaining cellular homeostasis. The seven human

GRKs (GRK1−GRK7) are classiﬁed via structural and

sequence similarity into three subfamilies: GRK1 (GRK1 and

GRK7), GRK2 (GRK2 and GRK3), and GRK4 (GRK4,

GRK5, and GRK6).¹GRK1 and GRK7 are found primarily in

the retina and GRK4 in the testes, whereas GRK2, GRK3,

GRK5, and GRK6 are more ubiquitously expressed. Of these

kinases, GRK2 and GRK5 are the two isoforms with the

highest concentration in cardiovascular tissue. Because they are

overexpressed in the diseased heart and their inhibition or

ablation has been shown to prevent heart failure and

hypertrophic cardiomyopathy, they have become important

therapeutic targets.^2,3GRK2 and GRK5 are also potential

targets for treatment of cancer^4,5and other pathophysiological

conditions.¹GRK5 is further unique among GRKs because it

undergoes Ca²⁺/calmodulin-dependent nuclear localization,

where it phosphorylates histone deacetylase 5 (HDAC5),

inducing an increase in transcription of associated genes.⁶In

RESULTS AND DISCUSSION

■

Previously, we developed a set of GRK5/6-selective

pyrrolopyrimidine inhibitors using a covalent strategy.⁹This

eﬀort established that Cys474, located on a loop known as the

active site tether (AST) that packs over the ATP binding site

in AGC kinases, can serve as a covalent handle to achieve

Received: August 31, 2020

Published: January 4, 2021

https://dx.doi.org/10.1021/acs.jmedchem.0c01522

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GRK5 selectivity. However, the aﬃnities of the best

compounds were low micromolar at best. Because the intrinsic

aﬃnity of the compound plays an important role in dictating

the concentration of the protein-inhibitor covalent complex,

which must persist long enough for a covalent reaction to

occur,¹⁰the discovery of a more intrinsically potent scaﬀold for

GRK5 inhibition was prioritized. To this end, we considered a

set of known GRK5 modulators derived from an indolinone

scaﬀold present in the FDA-approved receptor tyrosine kinase

(RTK) inhibitor Sunitinib (1) (Figure 1), a compound that

enantiomers of Ullrich-57. We found that the (S)-enantiomer

5b was over 1000-fold less potent (Table 1), indicating that

the (R)-enantiomer (5a) places the methyl and benzyl

pendants in a more ideal position. The structural rationale

for this based on the dock remains elusive because of

unknowns about the overall conformation of the GRK5 kinase

domain (see Figure 2). All other compounds were therefore

synthesized with the same stereoconﬁguration as 5a.

Initial diversiﬁcation tested a series of alkenyl or alkynyl

amines as covalent modiﬁers. The propargyl analogue, 5c,

demonstrated similar potency to 5a (GRK5 IC₅₀= 21 nM) but

exhibited over a magnitude higher selectivity over GRK2

(2100-fold). We speculate that this high level of selectivity is

due to a potential clash of the propargyl warhead with the

GRK2 AST and/or large lobe, given the predicted vector for

the alkynyl and alkenyl groups (Figure 2A). For all other amide

linked compounds with an alkenyl or alkynyl warhead (5d−

5f), the selectivity for GRK5 over GRK2 remained between

330 and 1400 fold, but in each case, the IC₅₀for GRK5 was

higher than that of 5c (Table 1). Linker length also

contributed to GRK5 aﬃnity, as demonstrated by a

comparison of 5c with 5e, and 5d with 5f, wherein the latter

compounds have homologated alkenyl and alkynyl warheads

and somewhat less potency. Thus, a single methylene linker to

the reactive center seems optimal for maintaining GRK5

activity among these trial compounds.

By reversing the warhead amide, the covalent warheads used

in our previous work⁹became synthetically accessible. To

accomplish this, Knoevenagel condensation with 3,5-dimethyl-

4-nitro-1-pyrrolocarbaldehyde yielded a common intermediate,

7a (Scheme 2). 7a was itself tested for inhibitory activity

because it possesses the ability to release nitric oxide, a known

vasodilator, rendering it a possible dual mechanism com-

pound.¹³The nitro group was however poorly tolerated

(GRK5 IC₅₀= 730 nM) and only 10-fold selective for GRK5.

A zinc-catalyzed reduction of the nitro group of 7a to the free

amine 8a allowed for rapid derivatization through amide

coupling to yield ﬁnal compounds 9a−9h (Scheme 2). 9a,

which features a 2-butynyl acid warhead that reacted with

Cys474 in our prior study,⁹exhibited low micromolar potency

(Table 1). The commonly used acrylamide and vinyl

sulfonamide variants, 9b and 9c, respectively, showed high

nanomolar activity against GRK5 and retained >100-fold

selectivity over GRK2. The 2-chloroacetylamido-containing

compound 9e showed moderate potency (GRK5 IC₅₀= 220

nM) but 1500-fold selectivity over GRK2. Because 5a had low

nanomolar potency, we introduced a similar reactive

appendage, dimethylaminobutenoic acid, in 9f. This com-

pound showed an increased IC₅₀(360 nM, thus 24-fold higher

than 5a), but only 50-fold selectivity over GRK2. The 2-

bromoacetylamido compound 9g was found to have a greater

potency against GRK5 (IC₅₀= 8.6 nM) than its chloro analog

9e, while retaining a high level of selectivity against GRK2

(1400-fold). Thus, we concluded that the bromo group of 9g

must be better at ﬁlling a lipophilic pocket than the chloro

group, but it was not possible to model this because the

structure of the AST loop is uncertain in our GRK5/6 overlay

model (Figure 2). It was also observed that 9g took longer to

fully engage Cys474 within the 30 min incubation time frame

(Figure 3). 9h was also potent against GRK5 (IC₅₀= 80 nM),

whereas compounds 10a and 10b exhibited only moderate

potency.

Figure 1. Lead compounds 1 and 5a with design strategy.

targets both GRK5 and multiple RTKs including the platelet-

derived growth factor receptor and vascular endothelial growth

factor receptor.^4,11We focused on Ullrich-57 (5a), which was

reported to have low nanomolar activity against GRK5 (GRK5

IC₅₀= <0.1 μM), although its selectivity was not reported.¹²

We independently synthesized 5a and also tested its parent

compound, Sunitinib, and showed that 5a has orders of

magnitude more potency against GRK5 than our previous

pyrrolopyrimidine scaﬀold and that both exhibit one to two

orders of magnitude selectivity for GRK5 over GRK2 (Figure 1

and Table 1). Modeling the 5a complex with GRK5 was

performed by docking in the program MOE. The highest

scoring pose predicted that 5a would bind in the active site of

GRK5 in a fashion that would allow the formation of three

hydrogen bonds with the hinge, which would consequently

project its diethylamine moiety toward the AST loop (Figure

2A). We hypothesized that replacing the diethylamine arm of

the scaﬀold with thiol reactive warheads could allow for

covalent attachment to Cys474 and generation of even more

selective and potent covalent GRK5 inhibitors. Thus, the goal

for our ﬁrst series of compounds was to identify a covalent

warhead that aﬀords the highest incorporation and the most

selectivity for GRK5 over GRK2. As a control, potency against

the canonical AGC kinase protein kinase A (PKA) was also

evaluated; however, all but one of our compounds had

negligible eﬀects against this enzyme.

Synthesis for this series began with an amide coupling to

give common intermediate 3 (Scheme 1). In a convergent line

of synthesis, a secondary amide coupling with the starting

material 6 gave intermediates 4a−4e. Combining the two lines

of synthesis, Knoevenagel condensation yielded compounds

5a−5f, wherein 5a and 5b represent the (R) and (S)

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a

Table 1. IC₅₀Values (μM SD) and Reactivity of Indolinone Compounds

a

All data were ﬁt to a log([inhibitor]) versus response model with variable slope and automatic outlier rejection in GraphPad Prism. Curves that

had R²values less than 0.8 after ﬁtting were omitted. ND, not determined. NA, not applicable. Values in parentheses indicate the number of

independent experimental curves. CCG215022 and paroxetine are positive controls for GRK5 and GRK2, respectively. Incubation time needed to

observe adduct formation with GRK5 by intact mass MS. 5a and 5b are the (R) and (S) enantiomers of Ullrich-57, respectively.

b

c

We evaluated adduct formation in this series at 30 min and 3

h by intact mass spectrometry (MS) (Table 1, Figure 3, and

Figure S1). Only a few compounds exhibited a signal for

GRK5 at the 3 h timepoint. 5c, which exhibited similar

potency to the parent compound 5a and over 2000-fold

selectivity against GRK2, was surprisingly not covalent under

our conditions. Neither were compounds 5d−5f. 9a and 9b

were likewise unreactive at the 30 min timepoint. The vinyl

sulfonamide 9c was able to react after a 3 h incubation (Figure

S1) despite its moderate aﬃnity (740 nM). The chloroacetyl

9e however had nearly complete covalent engagement by 30

min (Figure 3A). 9e was unreactive against GRK5-C474S,

consistent with Cys474 being the covalent handle (Figure 3D).

Its bromoacetyl analog 9g also rapidly reacted with GRK5

(Figure 3B), but 9h, 10a, and 10b were unreactive at 30 min.

Based on the sum of the data, we rationalize that compounds

in the 5c−5f series were unreactive because of constraints

placed on the warhead by increased hydrogen bond

interactions with the GRK5 hinge relative to 9c, 9e, and 9g

where the attaching amide was ﬂipped, aﬀording a diﬀerent

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Figure 2. Docking models of lead compound 5a and the most potent derivative 9j and their comparison with the GRK5·CCG215022 complex. (A)

5a, with pink carbons, red oxygens, and blue nitrogens docked using the program MOE into the structure of GRK5 (gray, PDB ID 4WNK), with

the addition of the AST loop from GRK6 (purple, PDB ID 3NYN) because this element was disordered in the 4WNK structure and contains the

target Cys474 residue. The diamine moiety extends toward the AST loop where Cys474 is located. Three hydrogen bonds, shown as dashed lines,

are formed with the hinge of the kinase domain. None of the compounds in the 5a−5f series were able to form adducts despite some that

demonstrated high potency. (B) 9j (yellow carbons) wherein the amide functionality in the covalent warhead was ﬂipped relative to 5a and

therefore forms one less hydrogen bond with the hinge. Being less constrained, we hypothesize that this modiﬁcation allows the warhead to leave

the hinge region along a diﬀerent vector that leads to less steric collisions with the AST and to closer proximity with Cys474. Consequently,

compounds in the 9a series show adduct formation. Bromine is colored green. (C) Comparison of the ﬂuorophenyl groups of 9j and CCG215022

(from 4WNK). The ﬂuorine atom is colored cyan. Both ligands are modeled to form a hydrogen bond with Asp329, an invariant catalytic residue in

protein kinases. However, it is unclear whether this region of the scaﬀold is docked correctly because the conformation of the GRK5 kinase domain,

and in particular its P loop and AST, is not known. The SAR in Table 2 is best explained with the ﬂuorophenyl packing under the P loop as it does

in the CCG215022 complex.

a

Scheme 1. Convergent Route to 5a−5f

a

5a and 5b are enantiomers derived from 3 and 3a, respectively.

vector for the attached warhead and less conformational

constraints (Figure 2A,B).

site. In fact, most known GRK inhibitors, including

CCG215022, tend to have benzyl groups that pack in this

site (Figure 2C).¹⁴The enhancement in potency of 3-Me (9i)

is large compared to 9e, suggesting that a 3-Me, 4-F analog

would be even more potent. However, a lack of appropriate

chemical precursors prevented us from studying such

combinations. We note that we took advantage of the meta-

position of the ﬂuorobenzyl group of paroxetine to make a

highly successful series of GRK2 selective inhibitors.¹⁵The

position of the nitrogen in pyridyl pendants also appeared to

make a small diﬀerence. In 9k, the potency was 3.5-fold higher

than that of 9l with an ortho-nitrogen, suggesting once again

that the para-nitrogen in 9k fulﬁlls an electronic deﬁciency. 9j,

At this point, 9e and 9g provided the most reactivity

combined with the most selectivity for GRK5 over GRK2

(Table 1). Therefore, we initialized structure−activity relation-

ships (SAR) around indolinone scaﬀolds bearing 2-haloacety-

lamido warheads (Schemes 3 and 4 and Table 2). Benzyl and

pyridyl pendants in the R₁position (Table 2) were explored

ﬁrst. Overall, in terms of potency, GRK5 seemed to have a

strong preference for smaller electron-withdrawing substituents

in the para-position of the benzyl groups: F (9j) > Cl (9o) > H

(9e) > CH₃(9n). The MOE docked models do not explain

this preference, but such behavior would be expected if the

benzyl pendant packs instead under the P loop of the active

with a 4-ﬂuoro substituent, had 55-fold more potency (IC₅₀

=

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Scheme 2. General Synthetic Route to 9a−9h and 10a and 10b

Figure 3. Intact protein MS for compounds 9e, 9g, and 9j. (A) GRK5 (blue) and GRK5+9e (black) incubated for 30 min. 9e demonstrated full

labeling of GRK5. (B) GRK5 and GRK5+9g (pink) incubated for 30 min. 9g only labeled 50% of GRK5. (C) GRK5 and GRK5+9j (red)

incubated for 30 min. 9j fully labeled GRK5 in this timeframe. (D) GRK5-C474S mutant (purple) and GRK5-C474S+9e (teal) incubated for 30

min. 9e did not label GRK5-C474S, indicating that 9e and related compounds are engaging Cys474.

4 nM) relative to the parent compound 9e. It was also able to

rapidly form a covalent interaction with Cys474 within 30 min

(Figure 3C), and 9j was thus the most intriguing lead of the

second series (Table 2 and Figure 2C). When the 4-ﬂuoro

substituent was maintained and a 2-bromoacetylamido war-

head was used, the resulting compound, 9p, showed slightly

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Scheme 3. General Synthetic Route to 9i−9p

Scheme 4. General Synthetic Route to 9q−9t

lower potency (IC₅₀= 15 nM) than 9j or the des-ﬂuoro

compound 9g (Table 1). This is diﬀerent than expected from

comparison of 9g and 9e (Table 1), where the bromo

substitution rendered much higher potency. The structural

explanation for this is unclear.

Given the profound eﬀects of chirality on activity exhibited

by compounds 5a and 5b, we also expanded SAR around the

benzylic position of the scaﬀold (R₂in Table 2). When the

stereocenter was removed (9q), there was a small increase in

potency (IC₅₀= 130 nM) relative to the parent compound 9e

(less than 2-fold). However, there were also similar increases in

potency for 9s (GRK5 IC₅₀= 87 nM) and 9t (GRK5 IC₅₀= 95

nM). The geminal dimethyl of 9r however had greatly reduced

potency (IC₅₀= 12 μM), but the poor solubility of this

compound under our assay conditions may have artifactually

increased IC₅₀measurements. Therefore, we concluded that

the benzylic position (R₂in Table 2) is fairly insensitive to

modiﬁcation, at least when the groups concerned are the size

of isopropyl or smaller. Accordingly, when 9q, 9r, and 9s were

incubated for 8 h, covalent interaction with GRK5 was

observed as in 9e (Figure S2). It is not clear why 9t did not

also react.

Finally, although the indolinone scaﬀold oﬀers high potency

and selectivity for GRK5 over GRK2, there were a few

anticipated drawbacks in terms of its pharmacokinetic

properties. First, 9a−9t are less soluble due to the number

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a

Table 2. IC₅₀Values (μM SD) and Reactivity of Indolinone Variants with a Haloacetylamido Warhead

a

Data were ﬁt to a log([inhibitor]) versus response model with variable slope and automatic outlier rejection in GraphPad Prism. Curves with R²

values < 0.8 after ﬁtting were omitted. ND, not determined. Numbers in parentheses indicate the number of independent experimental curves.

b

c

d

Incubation time needed to observe adduct formation with GRK5 by intact mass MS. Same as 9j but with a 2-bromoacteylamido warhead. IC₅₀

estimates for this compound for GRK5 and GRK2 are likely high due to its poor solubility in our assay system.

a

Table 3. Impact and Kinetics of Covalent Modiﬁcation at GRK5-Cys474

compound

CCG273220

CCG273463

CCG273441

CCG215022

GRK5 IC₅₀(μM)

GRK5-C474S IC₅₀(μM)

GRK5-C474S/GRK5

K_I(μM)

k_inact(min⁻¹

)

9e

9g

9j

0.22 0.04 (3)

0.60 0.2 (5)

3

5

0.8

1.0 0.3 (3)

0.11 0.03 (3)

0.019 0.01 (3)

n/a

>1.0 0.05 (3)

>1.0 0.03 (3)

>0.9 0.02 (3)

n/a

0.0086 0.003 (7)

0.0038 0.001 (7)

0.28 0.01 (6)

0.044 0.02 (5)

0.019 0.007 (5)

0.22 0.05 (5)

a

Errors correspond to standard deviation, with the number of replicates given in parentheses. n/a, not applicable.

of aromatic rings and their rigid, linear conformation. The

pyridyl pendants of 9k and 9l were a ﬁrst attempt to address

this issue. 9l was found to improve solubility 3.5-fold over 9e

to 125 μg/mL (Table S1), which is still ∼3-fold less than

reported for Sunitinib (350 μg/mL). Metabolic liability was

also a concern because of the high lipophilicity of our

compounds and thus their potential metabolism by CYP3A4.

Pyridyl pendants are also known to limit metabolic liability

relative to benzyl rings, as are ﬂuorine-containing pendants, as

in 9j (Figure S4). We indeed found that 9j indeed had a longer

half-life (HLM t_1/2= 18.9 min) compared to the des-ﬂuoro

compound 9e (HLM t_1/2= 13.3 min). However, there was an

opposite trend in MLMs. Limited commercial availability of

ﬂuorine-substituted benzylic amines from the chiral pool

however limited our ability to explore the additional ortho- and

meta-substitution patterns.

Ullrich-57 (5a) is already a potent (15 nM) and moderately

selective GRK5 inhibitor relative to GRK2 (74-fold). Our

overarching goal here was to ascertain whether potency and

selectivity could be improved by covalent modiﬁcation of a

residue unique to the GRK5 subfamily of kinases. Interestingly,

although 9e, 9g, and 9i−9t all had 2-haloacetylamido

warheads, only a few were able to label Cys474 within a 30

min time period. The absence of reactivity could be explained

by the higher IC₅₀exhibited by many of these analogs. Those

compounds with lower aﬃnity for GRK5, for example, 9c, will

bind in the active site of GRK5 more transiently than 9e,

resulting in a longer incubation period being needed to detect

covalent engagement (Figure S1). 9e, 9g, and 9j were not only

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Figure 4. Kinetic analysis for (A, B) 9e, (C, D) 9g, and (E, F) 9j. Extracted values for K_Iand k_inactfrom ﬁtting the plots on the right are given in

Table 3. Data points represent three measurements with error bars indicating standard deviation. Because the time courses (left panels) were not

linear, only the 1 min time point was used to estimate k_obs

.

Figure 5. Kinome-wide selectivity panel for 9g at concentrations of (a) 1 μM, (b) 0.1 μM, and (c) 0.01 μM. Locations of GRK5 and GRK6 are

denoted by arrows. A clear dose response is evident, with fewer kinases inhibited at the lowest concentration of 9g. Illustration reproduced courtesy

of Cell Signaling Technology, Inc. (www.cellsignal.com).

the most eﬃcacious at modifying GRK5 (Figure 3), but also

they spanned a range of IC₅₀values (220 to 4 nM) and

exhibited selectivity for GRK5 over GRK2 by over three orders

of magnitude, 20-fold more than that exhibited by the

noncovalent compound 5a. We therefore performed a detailed

kinetic analysis on these compounds to gauge the impact of

their reactivity on measured potency and selectivity. We ﬁrst

showed that all three compounds exhibited 3−5-fold less

potency against GRK5-C474S (Table 3), whereas our control

compound CCG215022 was essentially unchanged. We then

determined K_Iand k_inactfor these compounds (Figure 4 and

Table 3), which showed that 9e, 9g, and 9j fully reacted with

GRK5 before the ﬁrst time point (hence k_inact> 1 min⁻¹) and

that K_Iscaled with the observed IC₅₀values reported for these

compounds (Tables 1 and 2). This shared reactivity is

consistent with the fact that they all contain haloacetyl

warheads. The results further suggest that a direct comparison

of the SAR among compounds in Table 2 along with 9e and 9g

from Table 1 is reasonable. It also implies that in this situation

where the reactive cysteine is on a ﬂexible loop near the ligand

binding site, K_Idominates the IC₅₀measurements. More

reactive warheads like 2-haloacetyls may also be required to

take advantage of a more transiently associated cysteine side

chain given the anticipated conformational variability of the

AST loop.

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thus our compounds represent a 70:30 mix of active versus inactive

isomers. All ﬁgures in the paper depict the active isomer.

Having conﬁrmed that 5c and 9g were potent and highly

GRK5 selective relative to GRK2, we explored their kinome-

wide selectivity. When tested at 1 μM (∼1000-fold higher than

their IC₅₀values), 5c inhibits GRK5 at 92% and GRK6 at 94%,

whereas 9g inhibits GRK5 at 93% and GRK6 at 97%. GRK3, a

close homolog of GRK2, was inhibited at only 21%. Both

compounds had many oﬀ-target eﬀects across the kinome

(Figure 5 and Figure S3), which is not surprising because the

indolinone series is derived from Sunitinib, which can similarly

inhibit the activity of many RTKs. We conﬁrmed that

selectivity improved at lower concentrations by testing 9g at

100 and 10 nM (Figure 4). 9g however continued to inhibit a

small number of tyrosine kinases and Ca²⁺/calmodulin-

dependent protein kinases at the lowest dose, at which

GRK5 was 50% inhibited, consistent with the IC₅₀value

measured in our assays (Table 1).

Intact Protein MS and Tandem MS/MS. Intact protein MS was

acquired with a Phenomenex C4 column paired with an Agilent 6545

Q-TOF LC/MS. For intact MS and tandem MS, all samples were

prepared with 20 μM GRK in assay buﬀer (see below) and 1 mM

compound and incubated at 4 °C for 3 h before being quenched with

1.0 μL of formic acid. In tandem MS/MS, we chose Glu-C as the

restricting enzyme to avoid small fragments with mass-to-charge ratios

below the limit of detection. All samples were digested with Glu-C

sequencing enzyme, procured from Sigma Aldrich (Roche Life

Sciences subsidiary), and used without further puriﬁcation. MS/MS

experiments were run on a nano-LC (Dionex RSLC-nano) with an

Orbitrap Fusion Tribrid ETD mass spectrometer. This work was

conducted by the Proteomics Resource Facility at the University of

Michigan.

Structural Models and Docking. GRK5 (PDB ID 4WNK)¹⁸

and GRK6 (PDB ID 3NYN)¹⁹were loaded into Molecular Operating

Environment 2018.01 (Molecular Operating Environment (MOE),

2018.01; Chemical Computing Group ULC, 1010 Sherbrooke St.

West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2018), and the

proteins were prepared using the QuickPrep function. The sequences

of both proteins were aligned and used to create a superposition of

the two proteins and a hybrid structure with the kinase domain of

GRK5 and the AST region of GRK6 was created for docking analysis

(Figure 1A). The highest scoring docked pose for each compound in

Tables 1 and 2 are provided in the Supporting Information.

Inhibition Assays. For compounds 5a−9t, IC₅₀values for human

GRK5 and bovine GRK2 were determined using a radiometric assay,

described as follows. GRK (50 nM) was incubated for 3−5 min with

500 nM porcine brain tubulin (PurSolutions) and 0.01−50 μM

inhibitor in 20 mM HEPES (pH 7.0), 2 mM MgCl₂, 0.025%

dodecylmaltoside (DDM), and 1% DMSO prior to initiation with the

addition of 5 μM ATP supplemented with radioactive [γ-³²P]-ATP

(PerkinElmer Life Sciences). Reactions were quenched at 8 min by

addition of 5 μL of 4X SDS gel loading dye to the 10 μL reactions.

Samples (12 μL) were separated on a 4−15% Criterion TGX precast

gel (Bio-Rad). For potent inhibitors with low nanomolar IC₅₀, the

inhibitor concentration was adjusted to approximately 0−50× [IC₅₀],

which was estimated from the ﬁrst run to get more accurate

measurements. Gels were dried, exposed to a storage phosphor screen

overnight, and scanned using a Personal Molecular Imager (Bio-Rad).

Bands corresponding to phosphorylated tubulin were quantiﬁed using

ImageQuant, plotted as a function of log[inhibitor], and ﬁt to the

four-parameter log(inhibitor) vs response model in GraphPad Prism

7.03 to determine the IC₅₀, mean, and standard deviation values.

Outliers were eliminated automatically at a 1% Q value. Experiments

were performed at least three times.

PKA inhibition assays were performed with the ADP-Glo system

(Promega Corporation) according to the manufacturer’s instructions.

PKA (500 nM) was incubated with 1 μg of CREBtide

(KRREILSRRPSYR) (Genscript Corporation) substrate, 50 μM

ATP, and inhibitor for 30 min in 20 mM HEPES (pH 7.0), 2 mM

MgCl₂, 0.025% dodecylmaltoside (DDM), and 4% DMSO. The

concentration range of each inhibitor varies depending on its

solubility at 4% DMSO with the highest concentration from 100 to

500 μM. After the initial reaction, ADP-Glo reagent was added to the

reaction and allowed to incubate for an additional 40 min. Last, the

kinase detection reagent was added and allowed to incubate for 30

min, and luminescence was measured with a FlexStation 3 Multi-

mode Microplate Reader (Molecular Devices). All data was analyzed

in the same way as in the GRK inhibition assay. Experiments were

performed three times in duplicate.

CONCLUSIONS

■

In summary, we have developed a potent series of covalent

inhibitors based on the indolinone scaﬀold that show higher

potency and three orders of magnitude selectivity for GRK5

over the GRK2 subfamily, improving on the potency and

selectivity of the parent compound Ullrich-57 (4- and 74-fold,

respectively). Our MS and kinetic results further suggested that

their ability to covalently engage a cysteine unique to the

GRK5 subfamily, which contributes 3−5-fold to their IC₅₀

values under our assay conditions, is responsible for these

improved characteristics. The compounds thus set the stage for

cell-based assays that would allow one to tease apart the roles

of GRK5 versus GRK2 in cellular processes linked to

cardiovascular disease and cancer. They also set the stage for

a future generation of GRK5 subfamily selective therapeutics

that would address potential toxicity issues associated with the

use of strongly reactive 2-haloacetyls and further improve

selectivity versus other subfamilies of protein kinases as well as

their moderate solubility and metabolic stability. Such would

facilitate their transition into in vivo trials. While this paper was

under review, a new GRK5 inhibitor, KR-39038, with 20 nM

IC₅₀was reported, although experiments addressing its

selectivity versus GRK2 and other kinases and its toxicology

were not provided.¹⁶This compound showed mild positive

eﬀects in a rodent model of hypertrophy but exhibited low

bioavailability and a short half-life (<1 h). These results

reinforce the potential utility of covalent GRK5 inhibitors that

could have long-lasting eﬀects even if the circulating

compound is rapidly cleared, which would in turn mitigate

oﬀ-target toxicity.¹⁰

EXPERIMENTAL SECTION

General Chemistry. All reagents from commercial sources were

used without further puriﬁcation unless otherwise noted. H NMR

■

1

spectra were taken in DMSO-d₆, MeOD, or CDCl₃at room

temperature on Varian MR 400 MHz, Varian Vnmrs 500 MHz, and

Varian Vnmrs 700 MHz instruments. The reported chemical shifts for

the ¹H NMR spectra were recorded in parts per million (ppm) on the

δ scale from an internal tetramethylsilane standard (0.0 ppm). Small-

molecule mass spectrometry data was measured using a Waters

Corporation Micromass LCT or Agilent6230 Q-TOF instrument.

HPLC was used to determine purity of compounds on an Agilent

1100 series with an Agilent Zorbax Eclipse Plus-C18 column. A

gradient of 10−90% acetonitrile/water over 6 min followed by 90%

acetonitrile/water for 7 min was used with detection at 254 nm.

Purity of all compounds was >95% as determined by HPLC. The

Sunitinib scaﬀold is well known to photoisomerize in solution,¹⁷and

Standard control compounds are run during each assay to assess

consistency across time, experimenters, and subtle changes in assay

conditions that are sometimes required to keep compounds soluble

and dispersed (e.g., through addition of DDM or 3% DMSO).

Paroxetine was used as a control for GRK2²⁰and PKA and

CCG215022 for GRK5.¹⁸

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Article

Covalent Inhibition Kinetic Analysis. For the covalent

inhibitors 9e, 9g, and 9i, K_Iand k_inactwere determined using a

radiometric assay as follows. The reactions contained 50 nM GRK5

and 5 μM porcine brain tubulin (PurSolutions) in 20 mM HEPES

(pH 7.0), 2 mM MgCl₂, 0.025% DDM, and 1% DMSO. Inhibitors at

diﬀerent concentrations (0.02−1 μM for 9j and 9g, and 0.3−20 μM

for 9e) were incubated with the reaction mix for 30 s prior to

initiation with the addition of 5 μM ATP supplemented with

radioactive [γ-³²P]-ATP (PerkinElmer Life Sciences). Reactions were

quenched at 1, 2, and 5 min by addition of 10 μL of 4X SDS gel

loading dye to the 10 μL reactions. Samples (12 μL) were separated

on a 4−15% Criterion TGX precast gel (Bio-Rad). Gels were dried,

exposed to a storage phosphor screen overnight, and scanned using a

Personal Molecular Imager (Bio-Rad). Bands corresponding to

phosphorylated tubulin were quantiﬁed using ImageQuant, back-

ground-corrected, normalized to the intensity level of phosphorylation

tubulin in the absence of any inhibitor, and plotted as a function of

time. Because the inactivation process became nonlinear after the ﬁrst

time point, the observed inactivation rate (k_obs) was estimated using

the 1 min time point. The k_obsvalues were replotted against inhibitor

concentration and ﬁtted to the equation, k_obs= k_inact[I]/(K_I+ [I]), to

obtain k_inactand K_Iin GraphPad Prism 7.03. Experiments were

performed three times.

Thermodynamic Solubility and Microsomal Stability.

Thermodynamic solubility for compounds was determined by Analiza

Inc. (Cleveland OH, analiza.com) using a miniaturized shake-ﬂask

solubility assay. Microsomal stability was determined by the

Pharmacokinetics and Mass Spectrometry Core at the University of

Michigan. Compounds 9c, 9e, and 9j were dissolved in DMSO (1

mM) and then further diluted to 100 μM with 0.1 M phosphate buﬀer

(with 3.3 mM MgCl₂). Microsomes (20 mg/mL) in 0.1 M phosphate

buﬀer (with 3.3 mM MgCl) were dosed with 20 μL of NADPH (4 mg

in 240 μL of 0.1 M phosphate buﬀer) and incubated at 37 °C for 3

min. Microsomes were then dosed with 4 μL of 100 μM of 9c, 9e, and

9j. At the following time points (0, 5, 10, 15, 30, 45, and 60 min), the

reaction solutions were stopped with cold acetonitrile containing 25

nM CE302 as an internal standard. The incubation solution was

centrifuged at 3500 rpm for 10 min to precipitate protein. The

supernatant was used for LC/MS/MS analysis.

Chemical Synthesis and Validation. (R)-2-Oxo-N-(1-

phenylethyl)indoline-5-carboxamide (3). To a round-bottom ﬂask

was added 297.7 mg (1.69 mmol) of 2-oxoindoline-5-carboxylic acid,

dissolved in 7 mL of dry DMF. To this dark red solution were added

0.244 mL (1.88 mmol) of (S)-1-phenylethan-1-amine, 0.300 mL

(1.69 mmol) of DIPEA, and 746.1 mg (1.95 mmol) of HATU. The

resultant dark red solution was allowed to stir at room temperature for

12 h, then added 200 mL of sat. Na₂CO₃, and extracted with EtOAc

(3 × 100 mL). The combined organic layer was washed with brine (2

× 50 mL) and then dried over MgSO_4.Puriﬁed by column

chromatography (0−15% DCM/MeOH) to give the desired product

as a strawberry pink solid. Yield: 401.9 mg, 84%. Molecular formula:

C₁₇H₁₆N₂O₂ESI-MS calc: 280.12 ESI-MS found: 281.1283 [M + 1]

HPLC: 5.198. ¹H NMR (400 MHz, DMSO-d₆) δ 10.62 (s, 1H), 8.63

(d, J = 8.0 Hz, 1H), 7.78 (d, J = 7.0 Hz, 2H), 7.39−7.35 (m, 2H),

7.31 (dd, J = 8.4, 6.8 Hz, 2H), 7.23−7.18 (m, 1H), 6.88−6.83 (m,

1H), 5.15 (p, J = 7.2 Hz, 1H), 3.53 (s, 2H), 1.46 (d, J = 7.1 Hz, 3H).

¹³C NMR (100 MHz, DMSO) δ 177.17, 165.76, 146.85, 145.65,

found: 281.0903 [M + 1] HPLC: 5.249. ¹H NMR (500 MHz,

DMSO-d₆) δ 10.67 (s, 1H), 8.66 (d, J = 8.0 Hz, 1H), 7.78 (d, J = 7.2

Hz, 2H), 7.38 (d, J = 8.4 Hz, 2H), 7.31 (t, J = 7.6 Hz, 2H), 7.20 (t, J =

7.3 Hz, 1H), 6.86 (d, J = 8.2 Hz, 1H), 5.15 (p, J = 7.2 Hz, 1H), 3.53

(s, 2H), 1.46 (d, J = 7.1 Hz, 3H). ¹³C NMR (176 MHz, DMSO) δ

176.83, 165.50, 146.49, 145.22, 128.27, 127.77, 127.54, 126.60,

126.14, 125.64, 123.69, 108.62, 48.52, 39.86, 39.74, 39.62, 39.50,

39.38, 39.26, 39.14, 35.70, 22.40.

(R)-2-Oxo-N-(1-(m-tolyl)ethyl)indoline-5-carboxamide (3b). To a

round-bottom ﬂask was added 236.1 mg (1.33 mmol) of 2-

oxoindoline-5-carboxylic acid, dissolved in 7 mL of dry DMF. To

this dark red solution were added 0.200 mL (1.47 mmol) of (R)-1-

(m-tolyl)ethan-1-amine, 0.300 mL (1.73 mmol) of DIPEA, and 510.2

mg (1.33 mmol) of HATU. The resultant dark red solution was

allowed to stir at room temperature for 12 h, then added 200 mL of

sat. Na₂CO₃, and extracted with EtOAc (3 × 100 mL). The combined

organic layer was washed with brine (2 × 50 mL) and then dried over

MgSO_4.Puriﬁed by column chromatography (0−15% DCM/MeOH)

to give the desired product as a strawberry pink solid. Yield: 125.6 mg,

31%. Molecular formula: C₁₈H₁₈N₂O₂ESI-MS calc: 294.14 ESI-MS

found: 295.0167 [M + 1] HPLC: 5.395. ¹H NMR (700 MHz,

DMSO-d₆) δ 10.61 (s, 1H), 8.58 (d, J = 8.1 Hz, 1H), 7.77 (d, J = 8.1

Hz, 2H), 7.18 (p, J = 7.6 Hz, 4H), 7.02 (d, J = 7.3 Hz, 1H), 6.85 (d, J

= 7.9 Hz, 1H), 5.11 (p, J = 7.3 Hz, 1H), 2.28 (s, 3H), 1.44 (d, J = 7.0

Hz, 3H). ¹³C NMR (176 MHz, DMSO) δ 176.62, 165.16, 146.32,

145.07, 137.11, 128.06, 127.65, 127.48, 127.10, 126.66, 125.53,

123.51, 123.09, 108.36, 48.29, 39.86, 39.74, 39.62, 39.50, 39.38, 39.26,

39.14, 38.22, 35.57, 22.34, 21.11.

(R)-N-(1-(4-Fluorophenyl)ethyl)-2-oxoindoline-5-carboxamide

(3c). To a round-bottom ﬂask was added 246.6 mg (1.41 mmol) of 2-

oxoindoline-5-carboxylic acid, dissolved in 7 mL of dry DMF. To this

dark red solution were added 0.200 mL (1.55 mmol) of (R)-1-(4-

ﬂuorophenyl)ethan-1-amine, 0.25 mL (1.41 mmol) of DIPEA, and

667.1 mg (1.61 mmol) of HATU. The resultant dark red solution was

allowed to stir at room temperature for 12 h, then added 200 mL of

sat. Na₂CO₃, and extracted with EtOAc (3 × 100 mL). The combined

organic layer was washed with brine (2 × 50 mL) and then dried over

MgSO_4.Puriﬁed by column chromatography (0−15% DCM/MeOH)

to give the desired product as a strawberry pink solid. Yield: 315 mg,

71%. Molecular formula: C₁₇H₁₅FN₂O₂ESI-MS calc: 298.11 ESI-MS

found: 299.1216 [M + 1] HPLC: 5.414. ¹H NMR (500 MHz,

DMSO-d₆) δ 10.61 (s, 1H), 8.62 (d, J = 8.0 Hz, 1H), 7.76 (d, J = 7.5

Hz, 2H), 7.43−7.38 (m, 2H), 7.16−7.08 (m, 2H), 6.87−6.82 (m,

1H), 5.14 (p, J = 7.2 Hz, 1H), 3.53 (s, 2H), 1.45 (d, J = 7.1 Hz, 3H).

¹³C NMR (176 MHz, DMSO) δ 176.84, 165.47, 160.34, 146.46,

141.34, 128.06, 128.01, 127.77, 127.49, 125.69, 123.63, 114.99,

114.87, 108.55, 47.90, 39.86, 39.74, 39.62, 39.50, 39.38, 39.26, 39.14,

38.33, 35.67, 22.35.

(R)-2-Oxo-N-(1-(pyridin-4-yl)ethyl)indoline-5-carboxamide (3d).

To a round-bottom ﬂask was added 248.1 mg (1.41 mmol) of 2-

oxoindoline-5-carboxylic acid dissolved in 7 mL of dry DMF. To this

dark red solution were added 0.200 mL (1.55 mmol) of (R)-1-

(pyridin-4-yl)ethan-1-amine, 0.25 mL (1.41 mmol) of DIPEA, and

652.4 mg (1.61 mmol) of HATU. The resultant dark red solution was

allowed to stir at room temperature for 12 h, then added 200 mL of

sat. Na₂CO₃, and extracted with EtOAc (3 × 100 mL). The combined

organic layer was washed with brine (2 × 50 mL) and then dried over

MgSO_4.Puriﬁed by column chromatography (0−15% DCM/MeOH)

to give the desired product as a strawberry pink solid. Yield: 200 mg,

48%. Molecular formula: C₁₆H₁₅N₃O₂ESI-MS calc: 281.12 ESI-MS

found: 282.1236 [M + 1] HPLC: 2.378. ¹H NMR (700 MHz,

DMSO-d₆) δ 10.64 (s, 1H), 8.74 (dd, J = 7.6, 2.1 Hz, 1H), 8.55−8.52

(m, 2H), 7.79 (dd, J = 7.5, 2.2 Hz, 2H), 7.45−7.42 (m, 2H), 7.08 (d,

J = 2.3 Hz, 1H), 7.01 (s, 1H), 6.89−6.85 (m, 1H), 5.14 (td, J = 7.3,

2.2 Hz, 1H), 3.55 (s, 2H), 3.46−3.41 (m, 3H), 1.47 (dd, J = 7.2, 2.3

Hz, 3H), 1.07−1.03 (m, 3H). ¹³C NMR (176 MHz, DMSO) δ

176.63, 165.65, 155.16, 148.63, 146.56, 127.76, 127.03, 125.62,

123.57, 121.58, 108.43, 56.00, 47.92, 39.86, 39.74, 39.62, 39.50, 39.38,

39.26, 39.14, 35.56, 21.45, 18.54.

129.29, 128.67, 126.52, 126.06, 108.90, 48.85, 36.09, 22.84.

(S)-2-Oxo-N-(1-phenylethyl)indoline-5-carboxamide (3a). To a

round-bottom ﬂask was added 201.8 mg (1.13 mmol) of 2-

oxoindoline-5-carboxylic acid dissolved in 7 mL of dry DMF. To

this dark red solution were added 0.160 mL (1.24 mmol) of (S)-1-

phenylethan-1-amine, 0.200 mL (1.13 mmol) of DIPEA, and 492.4

mg (1.30 mmol) of HATU. The resultant dark red solution was

allowed to stir at room temperature for 12 h, then added 200 mL of

sat. Na₂CO₃, and extracted with EtOAc (3 × 100 mL). The combined

organic layer was washed with brine (2 × 50 mL) and then dried over

MgSO_4.Puriﬁed by column chromatography (0−15% DCM/MeOH)

to give the desired product as a strawberry pink solid. Yield: 280 mg,

84%. Molecular formula: C₁₇H₁₆N₂O₂ESI-MS calc: 280.12 ESI-MS

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Article

(R)-2-Oxo-N-(1-(pyridin-2-yl)ethyl)indoline-5-carboxamide (3e).

To a round-bottom ﬂask was added 251.1 mg (1.41 mmol) of 2-

oxoindoline-5-carboxylic acid dissolved in 7 mL of dry DMF. To this

dark red solution were added 0.200 mL (1.55 mmol) of (R)-1-

(pyridin-2-yl)ethan-1-amine, 0.25 mL (1.41 mmol) of DIPEA, and

625.1 mg (1.62 mmol) of HATU. The resultant dark red solution was

allowed to stir at room temperature for 12 h, then added 200 mL of

sat. Na₂CO₃, and extracted with EtOAc (3 × 100 mL). The combined

organic layer was washed with brine (2 × 50 mL) and then dried over

MgSO_4.Puriﬁed by column chromatography (0−15% DCM/MeOH)

to give the desired product as a strawberry pink solid. Yield: 165 mg,

39%. Molecular formula: C₁₆H₁₅N₃O₂ESI-MS calc: 281.12 ESI-MS

found: 282.1236 [M + 1] HPLC: 2.111. ¹H NMR (700 MHz,

DMSO-d₆) δ 10.62 (d, J = 5.2 Hz, 1H), 8.63 (t, J = 6.6 Hz, 1H), 8.51

(t, J = 5.3 Hz, 1H), 7.80 (d, J = 5.6 Hz, 2H), 7.74 (q, J = 7.0 Hz, 1H),

7.38 (t, J = 6.8 Hz, 1H), 7.24 (q, J = 6.0 Hz, 1H), 6.86 (t, J = 6.7 Hz,

1H), 5.17 (p, J = 7.2 Hz, 1H), 3.54 (d, J = 5.2 Hz, 3H), 1.49 (t, J = 6.4

Hz, 3H). ¹³C NMR (176 MHz, DMSO) δ 177.27, 166.16, 163.50,

149.12, 137.26, 128.18, 127.79, 126.11, 124.08, 122.46, 120.59,

108.98, 50.76, 40.22, 40.10, 39.98, 39.86, 39.74, 39.62, 39.50, 36.07,

21.45.

(R)-N-(1-(3-Chlorophenyl)ethyl)-2-oxoindoline-5-carboxamide

(3f). To a round-bottom ﬂask was added 251.1 mg (1.41 mmol) of 2-

oxoindoline-5-carboxylic acid dissolved in 7 mL of dry DMF. To this

dark red solution were added 0.200 mL (1.55 mmol) of (R)-1-(3-

chlorophenyl)ethan-1-amine, 0.25 mL (1.41 mmol) of DIPEA, and

717.7 mg (1.91 mmol) of HATU. The resultant dark red solution was

allowed to stir at room temperature for 12 h, then added 200 mL of

sat. Na₂CO₃, and extracted with EtOAc (3 × 100 mL). The combined

organic layer was washed with brine (2 × 50 mL) and then dried over

MgSO_4.Puriﬁed by column chromatography (0−15% DCM/MeOH)

to give the desired product as a strawberry pink solid. Yield: 423.3 mg,

91%. Molecular formula: C₁₇H₁₅ClN₂O₂ESI-MS calc: 314.08 ESI-MS

found: 315.0438 [M + 1] HPLC: 5.730. ¹H NMR (700 MHz,

DMSO-d₆) δ 10.64−10.57 (m, 1H), 8.65 (dd, J = 7.5, 2.2 Hz, 1H),

7.78−7.74 (m, 2H), 7.41 (t, J = 2.1 Hz, 1H), 7.33 (dt, J = 6.7, 1.8 Hz,

2H), 7.26 (dp, J = 6.4, 2.2 Hz, 1H), 6.85 (dd, J = 8.1, 2.5 Hz, 1H),

5.15−5.06 (m, 1H), 3.53 (s, 2H), 1.45−1.42 (m, 3H). ¹³C NMR (176

MHz, DMSO) δ 176.91, 165.61, 148.09, 146.73, 133.15, 130.39,

127.97, 126.74, 126.14, 125.88, 125.13, 123.78, 108.70, 56.27, 48.40,

40.12, 40.00, 39.88, 39.76, 39.64, 39.52, 39.40, 35.83, 22.43, 18.80.

(R)-2-Oxo-N-(1-(p-tolyl)ethyl)indoline-5-carboxamide (3g). To a

round-bottom ﬂask was added 244.2 mg (1.41 mmol) of 2-

oxoindoline-5-carboxylic acid dissolved in 7 mL of dry DMF. To

this dark red solution were added 0.220 mL (1.55 mmol) of (R)-1-(3-

chlorophenyl)ethan-1-amine, 0.25 mL (1.41 mmol) of DIPEA, and

622.1 mg (1.62 mmol) of HATU. The resultant dark red solution was

allowed to stir at room temperature for 12 h, then added 200 mL of

sat. Na₂CO₃, and extracted with EtOAc (3 × 100 mL). The combined

organic layer was washed with brine (2 × 50 mL) and then dried over

MgSO_4.Puriﬁed by column chromatography (0−15% DCM/MeOH)

to give the desired product as a strawberry pink solid. Yield: 300.8 mg,

69%. Molecular formula: C₁₈H₁₈N₂O₂ESI-MS calc: 294.14 ESI-MS

found: 317 [M + Na] HPLC: 5.556. ¹H NMR (700 MHz, DMSO-d₆)

δ 10.61 (s, 1H), 8.57 (d, J = 8.1 Hz, 1H), 7.77 (d, J = 8.1 Hz, 2H),

7.26 (d, J = 7.8 Hz, 2H), 7.11 (d, J = 7.8 Hz, 2H), 6.85 (d, J = 8.0 Hz,

1H), 5.12 (p, J = 7.3 Hz, 1H), 3.53 (s, 2H), 2.26 (s, 3H), 1.44 (d, J =

7.0 Hz, 3H). ¹³C NMR (176 MHz, DMSO) δ 176.63, 165.17, 146.30,

142.10, 135.44, 128.66, 127.64, 127.54, 125.93, 125.51, 123.51,

108.36, 48.03, 39.86, 39.74, 39.62, 39.50, 39.38, 39.26, 39.14, 35.57,

22.29, 20.59.

125.55, 123.49, 108.37, 47.87, 40.00, 39.83, 39.67, 39.50, 39.33, 39.17,

39.00, 35.55, 22.09.

N-Benzyl-2-oxoindoline-5-carboxamide (3i). Prepared with pro-

tocol described for 3. Yields a strawberry pink solid, 174.5 mg, 56%.

Molecular formula: C₁₆H₁₄N₂O₂ESI-MS calc: 266.11 ESI-MS found:

1

267.2180 [M + 1] HPLC: 4.822. H NMR (700 MHz, DMSO-d₆) δ

10.61 (s, 1H), 8.87 (t, J = 6.1 Hz, 1H), 7.79−7.75 (m, 2H), 7.34−

7.28 (m, 5H), 7.23 (dt, J = 7.2, 4.3 Hz, 2H), 6.86 (d, J = 8.0 Hz, 1H),

4.46 (d, J = 5.9 Hz, 2H), 3.53 (s, 2H). ¹³C NMR (176 MHz, DMSO)

δ 176.60, 165.95, 146.42, 139.88, 128.65, 128.19, 128.12, 127.70,

127.59, 127.47, 127.29, 127.11, 126.61, 125.66, 123.45, 108.44, 47.80,

45.00, 42.52, 39.86, 39.74, 39.62, 39.50, 39.38, 39.26, 39.14, 38.22,

35.57.

2-Oxo-N-(2-phenylpropan-2-yl)indoline-5-carboxamide (3j).

Prepared using the protocol described for 3. Yields a strawberry

pink solid, 210.8 mg, 48%. Molecular formula: C₁₈H₁₈N₂O₂ESI-MS

calc: 294.14 ESI-MS found: 295.1456 [M + 1], 317.1275 [M + Na]

HPLC: 5.199. ¹H NMR (700 MHz, DMSO-d₆) δ 10.60 (s, 1H), 8.23

(s, 1H), 7.74 (s, 1H), 7.74−7.71 (m, 1H), 7.35 (d, J = 7.8 Hz, 2H),

7.26 (t, J = 7.7 Hz, 2H), 7.15 (t, J = 7.2 Hz, 1H), 6.84 (d, J = 8.1 Hz,

1H), 3.53 (s, 2H), 1.65 (s, 6H). ¹³C NMR (176 MHz, DMSO) δ

176.64, 165.50, 148.19, 146.15, 128.51, 128.35, 127.82, 1s27.67,

125.59, 125.37, 124.81, 124.61, 123.65, 108.26, 55.22, 39.86, 39.74,

39.62, 39.50, 39.38, 39.26, 39.14, 35.59, 29.66.

(R)-N-(2-Methyl-1-phenylpropyl)-2-oxoindoline-5-carboxamide

(3k). Prepared using the protocol described for 3. Yields a strawberry

pink solid, 389.9 mg, quantitative yield. Molecular formula:

C₁₉H₂₀N₂O₂ESI-MS calc: 308.15 ESI-MS found: 309.2172 HPLC:

5.793. ¹H NMR (700 MHz, DMSO-d₆) δ 10.44 (s, 1H), 8.37 (d, J =

8.9 Hz, 1H), 7.58 (dd, J = 10.7, 2.9 Hz, 2H), 7.22 (d, J = 7.6 Hz, 2H),

7.12 (t, J = 7.5 Hz, 2H), 7.03 (t, J = 7.3 Hz, 1H), 6.68 (d, J = 8.0 Hz,

1H), 4.48 (t, J = 9.2 Hz, 1H), 3.36 (s, 2H), 1.06 (d, J = 6.4 Hz, 2H),

0.83 (d, J = 6.5 Hz, 3H), 0.53 (d, J = 6.7 Hz, 3H). ¹³C NMR (176

MHz, DMSO) δ 176.69, 165.65, 164.62, 162.32, 146.32, 143.33,

128.02, 127.75, 127.70, 127.41, 126.61, 125.54, 123.53, 108.43, 59.92,

56.08, 41.67, 39.86, 39.74, 39.62, 39.50, 39.38, 39.26, 39.14, 38.23,

35.78, 35.62, 32.55, 30.77, 20.09, 19.90, 18.56.

2-Oxo-N-(3-phenyloxetan-3-yl)indoline-5-carboxamide (3l). Pre-

pared using the protocol described for 3. Yields a strawberry pink

solid, 62.1 mg, 31%. Molecular formula: C₁₈H₁₆N₂O₃ESI-MS calc:

308.12 ESI-MS found: 309.1235 HPLC: 4.601. ¹H NMR (700 MHz,

DMSO-d₆) δ 10.49 (d, J = 4.6 Hz, 1H), 9.16 (d, J = 4.6 Hz, 1H),

7.65−7.60 (m, 2H), 7.38−7.34 (m, 2H), 7.22−7.18 (m, 2H), 7.10

(tdd, J = 7.3, 4.9, 2.2 Hz, 1H), 6.74−6.70 (m, 1H), 4.82 (dd, J = 6.7,

4.8 Hz, 2H), 4.59 (dd, J = 6.7, 4.8 Hz, 2H), 3.38 (d, J = 4.7 Hz, 2H).

¹³C NMR (176 MHz, DMSO) δ 176.66, 165.18, 146.78, 143.10,

129.97, 128.28, 128.17, 127.72, 126.91, 126.72, 126.06, 125.80,

125.41, 124.81, 123.53, 120.11, 108.72, 108.56, 81.89, 58.27, 53.49,

39.86, 39.74, 39.62, 39.50, 39.38, 39.26, 39.14, 38.23, 35.58, 35.52,

30.66, 14.07.

N-(2-(Diethylamino)ethyl)-5-formyl-2,4-dimethyl-1H-pyrrole-3-

carboxamide (4a). To a round-bottom ﬂask were added 200.9 mg

(1.20 mmol) of 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid,

365.8 mg (1.79 mmol) of EDCI, 277.7 mg (1.79 mmol) of HOBt, 7

mL of dry DMF, 0.200 mL (1.44 mmol) of N1,N1-diethylethane-1,2-

diamine, and 0.34 mL (2.39 mmol) of TEA. The dark red solution

was allowed to stir at room temperature for 12 h before being

quenched with water and extracted with DCM (3 × 30 mL). The

combined organic layer was washed with brine (2 × 20 mL) and then

with 10% citric acid (3 × 50 mL), drawing the desired product into

the water layer. The aqueous layer was basiﬁed with Na₂CO₃, bringing

the pH up to 10, and then extracted with DCM (4 × 50 mL). The

combined organic layers were dried over MgSO₄and then evaporated

onto a silica gel and puriﬁed by column chromatography (5−15%

MeOH/DCM). The solvent was removed under pressure to give a

yellow solid. Result: light yellow solid, 90 mg, 27%. Molecular

formula: C₁₄H₂₃N₃O₂, ESI-MS calc: 265.18 ESI-MS found: 266.1749

HPLC: 2.681. ¹H NMR (400 MHz, DMSO-d₆) δ 11.85 (s, 1H), 9.54

(s, 1H), 7.95 (s, 2H), 2.34 (d, J = 20.8 Hz, 7H), 0.99 (s, 6H).

(R)-N-(1-(4-Chlorophenyl)ethyl)-2-oxoindoline-5-carboxamide

(3h). Prepared using the protocol described for 3. Yields a strawberry

pink solid, 471.0 mg, quantitative yield. Molecular formula:

C₁₇H₁₅ClN₂O₂ESI-MS calc: 314.08 ESI-MS found: 337.0717 [M +

Na] HPLC: 5.682. ¹H NMR (500 MHz, DMSO-d₆) δ 10.61 (s, 1H),

8.64 (d, J = 7.9 Hz, 1H), 7.76 (d, J = 7.9 Hz, 2H), 7.42−7.34 (m,

4H), 6.85 (d, J = 8.1 Hz, 1H), 5.13 (p, J = 7.2 Hz, 1H), 3.53 (s, 2H),

1.45 (d, J = 7.0 Hz, 3H). ¹³C NMR (126 MHz, DMSO) δ 176.61,

165.29, 146.40, 144.17, 130.98, 128.09, 127.92, 127.67, 127.28,

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Article

5-Formyl-2,4-dimethyl-N-(prop-2-yn-1-yl)-1H-pyrrole-3-carbox-

amide (4b). To a round-bottom ﬂask were added 199.4 mg (1.20

mmol) of 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid, 688.0

mg (1.79 mmol) of HATU, 6 mL of dry DMF, 0.100 mL (1.44

mmol) of propargylamine, and 0.34 mL (2.39 mmol) of TEA. The

dark red solution was allowed to stir at room temperature for 12 h

before being quenched with water and extracted with DCM (3 × 30

mL). The combined organic layers were taken and washed with brine

(2 × 20 mL) and then with 10% citric acid (3 × 50 mL), taking the

desired product into the aqueous layer. The aqueous layer was

basiﬁed with Na₂CO₃, bringing the pH up to 10, and then extracted

with DCM (4 × 50 mL). The combined organic layer was dried over

MgSO₄, and then the solvent was removed to give the ﬁnal product as

a light orange oil. Result: light orange oil, 61 mg, 25%. Molecular

formula: C₁₁H₁₂N₂O₂ESI-MS calc: 204.09 ESI-MS found: 205.0873

The combined organic layers were washed with brine (2 × 20 mL)

and then with 10% citric acid (3 × 50 mL), drawing the desired

material in to the aqueous layer. The aqueous layer was basiﬁed with

Na₂CO₃, bringing the pH up to 10, and then extracted with DCM (4

× 50 mL). The combined organic layer was dried over MgSO₄.

Result: light orange oil, 106.1 mg, 37.2%. Molecular formula:

1

C₁₂H₁₆N₂O₂ESI-MS calc: 220.12 MS: 221.1304 HPLC: 5.554. H

NMR (500 MHz, DMSO-d₆) δ 9.77 (s, 1H), 8.84 (d, J = 4.5 Hz, 1H),

8.73 (d, J = 8.4 Hz, 1H), 7.95 (s, 4H), 7.66 (dd, J = 8.7, 4.6 Hz, 1H),

5.12−4.99 (m, 1H), 2.59 (s, 7H). ¹³C NMR (126 MHz, DMSO) δ

179.37, 165.02, 162.73, 152.99, 146.27, 140.66, 134.88, 130.23,

122.09, 36.21, 31.20, 14.39, 10.98.

(R,Z)-3-((4-((2-(Diethylamino)ethyl)carbamoyl)-3,5-dimethyl-1H-

pyrrol-2-yl)methylene)-2-oxo-N-(1-phenylethyl)indoline-5-carbox-

amide (5a). To a dried sealed tube were added 97.9 mg (0.371

mmol) of (3) and 107.0 mg (0.357 mmol) of (4a), all of which were

dissolved in abs. EtOH (3.5 mL). To this solution were added two

drops of piperidine and heated to reﬂux (95 °C) for 4 h. Once

complete, the reaction was cooled to room temperature and then the

product was ﬁltered oﬀ as an orange solid. Yield: orange solid, 38.3

mg, 22%. Molecular formula: C₃₁H₃₇N₅O₃ESI-MS calc: 527.29 ESI-

MS found: 528.2955 HPLC: 5.621. ¹H NMR (500 MHz, DMSO-d₆)

δ 13.59 (s, 1H), 11.13 (s, 1H), 8.59 (d, J = 8.1 Hz, 1H), 8.25 (s, 1H),

7.71 (d, J = 6.8 Hz, 2H), 7.44 (d, J = 6.0 Hz, 1H), 7.41 (d, J = 7.9 Hz,

2H), 7.33 (t, J = 7.7 Hz, 2H), 7.22 (t, J = 7.7 Hz, 1H), 6.93 (d, J = 8.3

Hz, 1H), 5.19 (t, J = 7.5 Hz, 1H), 3.28 (d, J = 7.0 Hz, 2H), 2.44 (d, J

= 6.7 Hz, 6H), 1.51 (d, J = 7.1 Hz, 3H), 0.97 (t, J = 7.1 Hz, 7H). ¹³C

NMR (176 MHz, DMSO) δ 169.90, 165.95, 164.71, 145.11, 140.64,

136.48, 129.93, 128.30, 127.78, 126.64, 126.37, 126.18, 125.86,

125.28, 124.09, 120.76, 117.69, 114.37, 108.94, 51.72, 48.55, 46.59,

45.44, 39.86, 39.74, 39.62, 39.50, 39.38, 39.26, 39.14, 37.06, 22.35,

13.40, 11.92, 10.79.

(S,Z)-3-((4-((2-(Diethylamino)ethyl)carbamoyl)-3,5-dimethyl-1H-

pyrrol-2-yl)methylene)-2-oxo-N-(1-phenylethyl)indoline-5-carbox-

amide (5b). To a dried sealed tube were added 91.2 mg (0.325

mmol) of (3a) and 79.9 mg (0.299 mmol) of (4a), all of which were

dissolved in abs. EtOH (2.5 mL). To this solution were added two

drops of piperidine and heated to reﬂux (95 °C) for 4 h. Once

complete, the reaction was cooled to room temperature and then the

product was ﬁltered oﬀ as an orange solid. Yield: orange solid, 38.3

mg, 22%. Molecular formula: C₃₁H₃₇N₅O₃ESI-MS calc: 527.29 ESI

MS found: 528.2043 HPLC: 5.753. ¹H NMR (500 MHz, DMSO-d₆)

δ 13.59 (s, 1H), 11.16 (s, 1H), 8.67 (s, 1H), 8.32 (d, J = 5.5 Hz, 1H),

7.75−7.68 (m, 2H), 7.48 (s, 1H), 7.42 (d, J = 7.7 Hz, 3H), 7.32 (t, J =

7.6 Hz, 3H), 7.22 (t, J = 7.3 Hz, 1H), 6.94 (d, J = 8.1 Hz, 1H), 5.19

(p, J = 7.2 Hz, 1H), 2.45 (d, J = 3.4 Hz, 8H), 2.37 (s, 1H), 2.32 (s,

1H), 2.08 (s, 1H), 1.51 (d, J = 7.1 Hz, 4H), 0.99 (t, J = 7.5 Hz, 8H).

¹³C NMR (126 MHz, DMSO) δ 170.28, 147.82, 141.01, 130.32,

128.66, 127.23, 126.98, 126.61, 126.26, 125.66, 124.60, 118.30,

106.84, 102.36, 48.92, 47.02, 22.80, 17.46, 13.83, 11.24, 9.18.

(R,Z)-3-((3,5-Dimethyl-4-(prop-2-yn-1-ylcarbamoyl)-1H-pyrrol-2-

yl)methylene)-2-oxo-N-(1-phenylethyl)indoline-5-carboxamide

(5c). To a dried sealed tube were added 75.1 mg (0.27 mmol) of (3)

and 50 mg (0.28 mmol) of (4b), all of which were dissolved in abs.

EtOH (1.8 mL). To this solution were added 0.05 mL of piperidine

and heated to reﬂux (95 °C) for 4 h. Once complete, the reaction was

cooled to room temperature and an orange solid was ﬁltered oﬀ. The

solid was rinsed with cold EtOH to give the ﬁnal product. Yield:

bright orange solid, 58.6 mg, 46%. Molecular formula: C₂₈H₂₆N₄O₃

ESI-MS calc: 466.20 ESI-MS found: 467.2037 HPLC: 6.360. ¹H

NMR (700 MHz, DMSO-d₆) δ 13.61 (s, 1H), 11.15 (s, 1H), 8.61 (d,

J = 8.0 Hz, 1H), 8.26 (s, 1H), 8.06 (t, J = 5.5 Hz, 1H), 7.71 (d, J = 9.0

Hz, 2H), 7.41 (d, J = 7.8 Hz, 2H), 7.33 (q, J = 5.8, 3.9 Hz, 2H), 7.23

(t, J = 7.2 Hz, 1H), 6.94 (d, J = 8.2 Hz, 1H), 5.20 (t, J = 7.7 Hz, 1H),

4.02 (d, J = 5.5 Hz, 2H), 3.14−3.08 (m, 1H), 2.44 (d, J = 9.2 Hz,

7H), 1.51 (d, J = 7.1 Hz, 3H). ¹³C NMR (176 MHz, DMSO) δ

170.30, 166.26, 145.52, 141.06, 136.92, 130.46, 128.67, 128.29,

127.00, 126.82, 126.58, 126.28, 124.49, 120.45, 118.23, 115.03,

109.28, 82.17, 73.04, 48.87, 28.49, 22.77, 13.76, 11.13.

1

[M + 1], 242.1167 [M + K] HPLC: 3.625. H NMR (700 MHz,

DMSO-d₆) δ 11.87 (s, 1H), 9.55 (s, 1H), 7.95 (s, 16H), 3.97 (ddd, J

= 12.7, 5.7, 2.5 Hz, 3H), 3.08 (t, J = 2.4 Hz, 1H), 2.36 (s, 3H), 2.31

(s, 3H). ¹³C NMR (176 MHz, DMSO) δ 177.82, 164.77, 139.11,

138.51, 128.26, 119.20, 89.92, 82.16, 72.95, 55.38, 40.24, 38.71, 28.42,

12.90, 10.00.

N-Allyl-5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxamide (4c).

To a round-bottom ﬂask were added 204.4 mg (1.20 mmol) of 5-

formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid, 350.3 mg of EDCI

(1.79 mmol), 275.3 mg (1.79 mmol) of HOBt, 6 mL of dry DMF,

0.110 mL (1.44 mmol) of allylamine, and 0.34 mL (2.39 mmol) of

TEA. The dark red solution was allowed to stir at room temperature

for 12 h before being quenched with water and extracted with DCM

(3 × 30 mL). Combined organic layers were taken and washed with

LiCl (3 × 20 mL) and then with 10% citric acid (3 × 50 mL),

drawing the desired product into the aqueous layer. The aqueous

layer was basiﬁed with Na₂CO₃, bringing the pH up to 8, and then

extracted with DCM (4 × 50 mL). The combined organic layer was

dried over MgSO₄, and then the solvent was removed to give a yellow

solid. Result: light yellow solid, 64 mg, 25%. Molecular formula:

C₁₁H₁₄N₂O₂ESI-MS calc: 206.11 ESI-MS: 246.1621 [M+ MeCN]

HPLC: 3.885. ¹H NMR (500 MHz, DMSO-d₆) δ 11.38 (s, 1H), 9.54

(s, 1H),8.17 (s, 1H), 6.01 (tt, J = 10.8, 5.4 Hz, 1H), 5.87 (ddt, J =

16.4, 10.6, 5.3 Hz, 2H), 5.18 (t, J = 15.7 Hz, 3H), 5.08 (dd, J = 16.5,

10.2 Hz, 3H), 4.14 (d, J = 5.5 Hz, 2H), 3.82 (t, J = 5.7 Hz, 3H), 2.31

(s, 5H), 2.23 (s, 3H). ¹³C NMR (126 MHz, DMSO) δ 161.78,

150.29, 135.45, 135.21, 115.01, 114.35, 114.20, 40.54, 40.50, 39.50,

39.33, 39.17, 39.00, 38.83, 38.67, 38.50, 35.26, 30.25, 11.95, 9.27.

N-(But-3-yn-1-yl)-5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxa-

mide (4d). To a round-bottom ﬂask were added 199.6 mg (1.20

mmol) of 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid, 684.3

mg (1.79 mmol) of HATU, 6 mL of dry DMF, 0.100 mL (1.44

mmol) of 1-amino-3-butyne, and 0.30 mL (2.39 mmol) of DIPEA.

The dark red solution was allowed to stir at room temperature for 12

h before being quenched with water and extracted with DCM (3 × 30

mL). The combined organic layers were washed with LiCl (2 × 20

mL) and then with 10% citric acid (3 × 50 mL), drawing the desired

product into the water layer. The aqueous layer was basiﬁed with

Na₂CO₃, bringing the pH up to 10, and then extracted with DCM (4

× 50 mL). The combined organic layer was dried over MgSO₄, and

then the solvent was removed under pressure to give the ﬁnal product

as a yellow solid. Result: light yellow solid, 99.6 mg, 38.1%. Molecular

formula: C₁₂H₁₄N₂O₂ESI-MS calc: 218.11 ESI-MS found: 219.1129

HPLC: 3.997. ¹H NMR (700 MHz, DMSO-d₆) δ 11.83 (s, 1H), 2.83

(s, 1H), 2.69 (d, J = 1.0 Hz, 1H), 2.60 (d, J = 2.4 Hz, 3H), 2.37 (s,

4H), 2.32 (s, 3H). ¹³C NMR (176 MHz, DMSO) δ 178.94, 164.54,

160.23, 152.56, 147.04, 146.58, 126.55, 115.31, 88.48, 82.43, 72.02,

55.77, 37.85, 18.85, 13.97, 10.55.

N-(But-3-en-1-yl)-5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxa-

mide (4e). To a round-bottom ﬂask were added 197.8 mg (1.20

mmol) of 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid, 685.8

mg (1.79 mmol) of HATU, 6 mL of dry DMF, 107.1 mg (1.44 mmol)

of but-3-en-1-amine, and 0.45 mL (2.40 mmol) of DIPEA. The dark

red solution was allowed to stir at room temperature for 12 h before

being quenched with water and extracted with DCM (3 × 30 mL).

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(R,Z)-3-((4-(Allylcarbamoyl)-3,5-dimethyl-1H-pyrrol-2-yl)-

methylene)-2-oxo-N-(1-phenylethyl)indoline-5-carboxamide (5d).

To a dried sealed tube were added 53.6 mg (0.27 mmol) of (3)

and 50 mg (0.28 mmol) of (4c), all of which were dissolved in abs.

EtOH (3 mL). To this solution were added two drops (0.05 mL) of

piperidine and heated to reﬂux (95 °C) for 4 h. Once complete, the

reaction was cooled to room temperature and then an orange solid

was ﬁltered oﬀ. The solid was washed with cold EtOH to give the ﬁnal

product. Result: orange solid, 79.5 mg, 63%. Molecular formula:

C₂₈H₂₈N₄O₃ESI-MS calc: 468.22 ESI-MS found: 469.2224 HPLC:

(s, 1H), 7.76 (dd, J = 8.1, 1.7 Hz, 1H), 7.41 (d, J = 7.5 Hz, 2H), 7.33

(t, J = 7.6 Hz, 2H), 7.22 (t, J = 7.3 Hz, 1H), 6.97 (d, J = 8.1 Hz, 1H),

5.19 (p, J = 7.2 Hz, 1H), 2.08 (d, J = 1.0 Hz, 1H), 1.50 (d, J = 7.1 Hz,

3H). ¹³C NMR (176 MHz, DMSO) δ 169.90, 165.69, 144.98, 142.47,

136.80, 132.12, 130.48, 129.11, 128.20, 127.74, 126.10, 125.56,

123.33, 121.76, 115.38, 53.66, 22.28, 18.23, 10.50.

(R,Z)-3-((3,5-Dimethyl-4-nitro-1H-pyrrol-2-yl)methylene)-2-oxo-

N-(1-(m-tolyl)ethyl)indoline-5-carboxamide (7b). To a dried sealed

tube were added 200 mg (0.679 mmol) of (3b) and 139.6 mg (0.815

mmol) of 3,5-dimethyl-4-nitro-1H-pyrrole-2-carbaldehyde, all of

which were dissolved in abs. EtOH (6.0 mL). To this solution were

added 0.05 mL of piperidine and heated to reﬂux (95 °C) for 4 h.

Once complete, the reaction was cooled to room temperature and

then the product was ﬁltered oﬀ as a lemon yellow solid. Yield: lemon

yellow solid, 100 mg, 29.8%. Molecular formula: C₂₅H₂₄N₄O₄ESI-MS

1

6.450. H NMR (700 MHz, DMSO-d₆) δ 13.60 (s, 1H), 11.14 (s,

1H), 8.60 (d, J = 8.0 Hz, 1H), 8.25 (d, J = 1.6 Hz, 1H), 7.83 (t, J = 5.8

Hz, 1H), 7.73−7.68 (m, 2H), 7.41 (d, J = 7.6 Hz, 2H), 7.33 (t, J = 7.6

Hz, 2H), 7.22 (t, J = 7.3 Hz, 1H), 6.94 (d, J = 8.1 Hz, 1H), 5.90 (ddt,

J = 15.7, 10.3, 5.2 Hz, 1H), 5.22−5.18 (m, 2H), 5.10 (dd, J = 10.3, 1.8

Hz, 1H), 3.87 (t, J = 5.6 Hz, 2H), 2.44 (d, J = 7.8 Hz, 6H), 1.51 (d, J

= 7.1 Hz, 3H). ¹³C NMR (176 MHz, DMSO) δ 170.50, 145.29,

135.97, 130.84, 129.65, 128.44, 128.02, 126.34, 115.20, 108.42,

103.80, 91.16, 59.93, 22.53, 13.55, 10.93.

(R,Z)-3-((4-(But-3-yn-1-ylcarbamoyl)-3,5-dimethyl-1H-pyrrol-2-

yl)methylene)-2-oxo-N-(1-phenylethyl)indoline-5-carboxamide

(5e). To a dried sealed tube were added 99.9 mg (0.357 mmol) of (3)

and 82 mg of (4d), all of which were dissolved in abs. EtOH (3.5

mL). To this solution were added two drops (0.05 mL) of piperidine

and heated to reﬂux (95 °C) for 4 h. Once complete, the solution was

cooled to room temperature and then an orange solid was ﬁltered oﬀ.

The solid was washed with cold EtOH to give the ﬁnal product. Yield:

orange solid, 19 mg, 10%. Molecular formula: C₂₉H₂₈₁N₄O₃ESI-MS

calc: 480.22 ESI-MS found: 481.2226 HPLC: 6.444. H NMR (700

MHz, DMSO-d₆) δ 13.60 (s, 1H), 11.15 (s, 1H), 8.62 (d, J = 8.0 Hz,

1H), 8.25 (s, 1H), 7.79 (t, J = 5.8 Hz, 1H), 7.71 (d, J = 6.3 Hz, 2H),

7.41 (d, J = 7.6 Hz, 2H), 7.33 (t, J = 7.6 Hz, 2H), 7.23 (t, J = 7.3 Hz,

1H), 6.94 (d, J = 8.2 Hz, 1H), 5.19 (p, J = 7.3 Hz, 1H), 2.86 (t, J = 2.5

Hz, 1H), 2.45 (d, J = 12.0 Hz, 6H), 2.42 (td, J = 7.1, 2.4 Hz, 2H),

1.51 (d, J = 7.0 Hz, 3H). ¹³C NMR (176 MHz, DMSO) δ 169.83,

165.81, 164.71, 145.08, 140.56, 136.34, 130.00, 128.23, 127.80,

126.55, 126.12, 125.79, 125.18, 124.08, 120.61, 117.76, 82.50, 72.12,

48.42, 37.95, 22.33, 13.33, 10.70.

1

calc: 444.18 ESI-MS found: 445.1862 [M + 1] HPLC: 7.732. H

NMR (700 MHz, DMSO-d₆) δ 11.34 (s, 1H), 8.58 (d, J = 8.0 Hz,

1H), 8.33 (d, J = 1.6 Hz, 1H), 7.78 (s, 1H), 7.76 (dd, J = 8.2, 1.6 Hz,

1H), 7.23−7.18 (m, 3H), 7.03 (d, J = 6.1 Hz, 1H), 6.94 (d, J = 8.1

Hz, 1H), 5.16 (p, J = 7.2 Hz, 1H), 2.62 (s, 3H), 2.57 (s, 3H), 2.30 (s,

3H), 1.49 (d, J = 7.0 Hz, 3H). ¹³C NMR (176 MHz, DMSO) δ

169.83, 165.49, 144.90, 141.26, 137.14, 136.75, 133.66, 128.34,

128.09, 127.51, 127.15, 126.74, 125.07, 124.71, 124.39, 123.59,

123.17, 119.39, 118.96, 109.09, 48.36, 39.86, 39.74, 39.62, 39.50,

39.38, 39.26, 39.14, 22.26, 21.11, 14.76, 11.21.

(R,Z)-3-((3,5-Dimethyl-4-nitro-1H-pyrrol-2-yl)methylene)-N-(1-

(4-ﬂuorophenyl)ethyl)-2-oxoindoline-5-carboxamide (7c). To a

dried sealed tube were added 128 mg (0.429 mmol) of (3c) and

101.3 mg (0.60 mmol) of 3,5-dimethyl-4-nitro-1H-pyrrole-2-carbal-

dehyde, all of which were dissolved in abs. EtOH (3.0 mL). To this

solution were added 0.05 mL of piperidine and heated to reﬂux (95

°C) for 4 h. Once complete, the reaction was cooled to room

temperature and then the product was ﬁltered oﬀ as an orange solid.

Yield: orange solid, 96.3 mg, 50%. Molecular formula: C₂₄H₂₁FN₄O₄

ESI-MS calc: 448.15 ESI-MS found: 449.1619 [M + 1] HPLC: 7.422.

¹H NMR (700 MHz, DMSO-d₆) δ 11.37 (s, 1H), 8.64 (d, J = 7.8 Hz,

1H), 8.35 (d, J = 1.7 Hz, 1H), 7.83 (s, 1H), 7.76 (dd, J = 8.1, 1.7 Hz,

1H), 7.47−7.44 (m, 2H), 7.18−7.14 (m, 2H), 6.97 (d, J = 8.1 Hz,

1H), 5.19 (p, J = 7.2 Hz, 1H), 2.65 (s, 3H), 2.60 (s, 3H), 1.51 (d, J =

7.0 Hz, 3H). ¹³C NMR (176 MHz, DMSO) δ 169.85, 165.62, 136.79,

133.69, 128.31, 128.02, 127.97, 124.71, 123.71, 119.03, 114.88,

114.76, 50.22, 47.86, 39.86, 39.74, 39.62, 39.50, 39.38, 39.26, 39.14,

22.26, 14.78, 11.22.

(R,Z)-3-((4-(But-3-en-1-ylcarbamoyl)-3,5-dimethyl-1H-pyrrol-2-

yl)methylene)-2-oxo-N-(1-phenylethyl)indoline-5-carboxamide

(5f). To a dried sealed tube were added 94.8 mg (0.27 mmol) of (3)

and 87.2 mg (0.28 mmol) of (4e), all of which were dissolved in abs.

EtOH (2.2 mL). To this solution were added two drops (0.07 mL) of

piperidine and heated to reﬂux (95 °C) for 4 h. Once complete, the

solution was cooled to room temperature and then puriﬁed by column

chromatography. Fractions were collected, and the solvent was

removed under pressure to give the desired product as an orange

solid. Yield: orange solid, 103 mg, 59%. Molecular formula:

C₂₉H₃₀N₄O₃ESI-MS calc: 482.23 ESI-MS found: 483.2382 [M +

(R,Z)-3-((3,5-Dimethyl-4-nitro-1H-pyrrol-2-yl)methylene)-2-oxo-

N-(1-(pyridin-4-yl)ethyl)indoline-5-carboxamide (7d). To a dried

sealed tube were added 100 mg (0.455 mmol) of (3d) and 77.1 mg

(0.459 mmol) of 3,5-dimethyl-4-nitro-1H-pyrrole-2-carbaldehyde, all

of which were dissolved in abs. EtOH (3.0 mL). To this solution were

added 0.05 mL of piperidine and heated to reﬂux (95 °C) for 4 h.

Once complete, the reaction was cooled to room temperature and

then the product was ﬁltered oﬀ as a yellow solid. Yield: yellow solid,

48.0 mg, 31.3%. Molecular formula: C₂₃H₂₁N₅O₄ESI-MS calc: 431.16

ESI-MS found: 432.1656 [M + 1] HPLC: 5.496. ¹H NMR (700 MHz,

DMSO-d₆) δ 11.36 (s, 1H), 8.73 (d, J = 7.6 Hz, 1H), 8.52−8.50 (m,

2H), 8.36 (d, J = 1.6 Hz, 1H), 7.82 (s, 1H), 7.77 (dd, J = 8.1, 1.6 Hz,

1H), 7.40 (d, J = 5.2 Hz, 2H), 6.97 (d, J = 8.0 Hz, 1H), 5.15 (p, J =

7.3 Hz, 1H), 2.64 (s, 3H), 2.59 (s, 3H), 1.51 (d, J = 7.1 Hz, 3H). ¹³C

NMR (176 MHz, DMSO) δ 170.11, 166.24, 153.97, 149.80, 137.07,

135.63, 128.31, 127.80, 125.45, 124.98, 124.02, 121.55, 119.37,

109.42, 48.18, 40.12, 40.00, 39.88, 39.76, 39.64, 39.52, 39.40, 21.78,

15.04, 11.48.

(R,Z)-3-((3,5-Dimethyl-4-nitro-1H-pyrrol-2-yl)methylene)-2-oxo-

N-(1-(pyridin-2-yl)ethyl)indoline-5-carboxamide (7e). To a dried

sealed tube were added 165 mg (0.587 mmol) of (3e) and 122.8 mg

(0.762 mmol) of 3,5-dimethyl-4-nitro-1H-pyrrole-2-carbaldehyde, all

of which were dissolved in abs. EtOH (3.0 mL). To this solution were

added 0.05 mL of piperidine and heated to reﬂux (95 °C) for 4 h.

Once complete, the reaction was cooled to room temperature and

then the product was ﬁltered oﬀ as a yellow solid. Yield: yellow solid,

116 mg, 34%. Molecular formula: C₂₃H₂₁N₅O₄ESI-MS calc: 431.16

1

1] HPLC: 6.720. H NMR (700 MHz, DMSO-d₆) δ 13.54 (s, 1H),

11.13 (s, 1H), 8.61 (d, J = 8.2 Hz, 2H), 8.27−8.18 (m, 2H), 7.73−

7.71 (m, 1H), 7.68 (s, 1H), 7.42 (d, J = 8.3 Hz, 3H), 7.35−7.31 (m,

4H), 7.25−7.21 (m, 2H), 6.94 (d, J = 8.1 Hz, 1H), 5.21 (p, J = 7.3

Hz, 2H), 2.43 (d, J = 9.0 Hz, 2H), 2.29 (d, J = 11.4 Hz, 7H), 1.63−

1.59 (m, 3H), 1.51 (d, J = 7.1 Hz, 6H). ¹³C NMR (176 MHz,

DMSO) δ 169.79, 165.81, 165.04, 145.07, 140.57, 133.90, 128.74,

128.19, 127.78, 126.52, 126.11, 125.22, 123.96, 120.71, 117.70,

116.21, 114.09, 108.74, 48.41, 35.77, 33.74, 22.26, 12.40, 10.15.

(R,Z)-3-((3,5-Dimethyl-4-nitro-1H-pyrrol-2-yl)methylene)-2-oxo-

N-(1-phenylethyl)indoline-5-carboxamide (7a). To a dried sealed

tube were added 91.2 mg (0.892 mmol) of (3) and 79.9 mg (1.39

mmol) of 3,5-dimethyl-4-nitro-1H-pyrrole-2-carbaldehyde, all of

which were dissolved in abs. EtOH (2.5 mL). To this solution were

added two drops (0.05 mL) of piperidine and heated to reﬂux (95

°C) for 4 h. Once complete, the reaction was cooled to room

temperature and an orange solid was ﬁltered oﬀ. The solid was

washed with cold EtOH to give the desired product. Result: orange

solid, 269.3 mg, 69%. Molecular formula: C₂₄H₂₂N₄O₄ESI-MS calc:

1

430.16 ESI-MS found: 431.1715 HPLC: 7.400. H NMR (700 MHz,

DMSO-d₆) δ 8.63 (d, J = 8.0 Hz, 1H), 8.36 (d, J = 1.6 Hz, 1H), 7.83

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ESI-MS found: 432.0558 [M + 1] HPLC: 5.49. ¹H NMR (700 MHz,

DMSO-d₆) δ 11.36 (d, J = 7.2 Hz, 1H), 8.65−8.58 (m, 1H), 8.55−

8.47 (m, 1H), 8.39 (t, J = 7.7 Hz, 1H), 7.78 (ddd, J = 33.0, 15.8, 7.2

Hz, 3H), 7.42 (q, J = 10.5, 9.0 Hz, 1H), 7.25 (dd, J = 12.7, 6.8 Hz,

1H), 6.95 (d, J = 7.0 Hz, 1H), 5.22 (q, J = 8.8, 8.0 Hz, 1H), 2.66−

2.60 (m, 3H), 2.59 (s, 3H), 1.56−1.49 (m, 3H). ¹³C NMR (176

MHz, DMSO) δ 169.21, 165.90, 148.79, 136.83, 128.23, 124.86,

123.81, 120.44, 119.09, 109.32, 58.91, 50.44, 40.00, 39.88, 39.76,

39.74, 39.64, 39.62, 39.52, 39.40, 39.28, 21.09, 11.38.

(R,Z)-N-(1-(3-Chlorophenyl)ethyl)-3-((3,5-dimethyl-4-nitro-1H-

pyrrol-2-yl)methylene)-2-oxoindoline-5-carboxamide (7f). To a

dried sealed tube were added 242.2 mg (0.769 mmol) of (3f) and

150.1 mg (0.892 mmol) of 3,5-dimethyl-4-nitro-1H-pyrrole-2-

carbaldehyde, all of which were dissolved in abs. EtOH (6.0 mL).

To this solution were added 0.05 mL of piperidine and heated to

reﬂux (95 °C) for 4 h. Once complete, the reaction was cooled to

room temperature and then the product was ﬁltered oﬀ as an orange

solid. Yield: orange solid, 46 mg, 16%. Molecular formula:

C₂₄H₂₁ClN₄O_{4 1}ESI-MS calc: 464.13 ESI-MS found: 465.1318

HPLC: 7.699. H NMR (700 MHz, DMSO-d₆) δ 11.38 (s, 1H),

8.68 (d, J = 7.8 Hz, 1H), 8.36 (d, J = 1.6 Hz, 1H), 7.84 (s, 1H), 7.76

(dd, J = 8.2, 1.7 Hz, 1H), 7.46 (d, J = 2.1 Hz, 1H), 7.37 (d, J = 6.6 Hz,

2H), 7.29 (dt, J = 6.7, 2.3 Hz, 1H), 6.97 (d, J = 8.1 Hz, 1H), 5.17 (p, J

= 7.2 Hz, 1H), 2.65 (s, 3H), 2.60 (s, 3H), 1.50 (d, J = 7.1 Hz, 3H).

¹³C NMR (176 MHz, DMSO) δ 165.88, 147.85, 144.31, 138.20,

108.96, 55.31, 39.86, 39.74, 39.62, 39.50, 39.38, 39.26, 39.14, 29.65,

14.76, 11.21.

(R,Z)-3-((3,5-Dimethyl-4-nitro-1H-pyrrol-2-yl)methylene)-N-(2-

methyl-1-phenylpropyl)-2-oxoindoline-5-carboxamide (7k). Pre-

pared using the protocol described for 7a. Yields an orange solid,

144.6 mg, 64.8%. Molecular formula: C₂₆H₂₆N₄O₄ESI-MS calc:

458.20 ESI-MS found: 459.2109 HPLC: 7.896. ¹H NMR (700 MHz,

DMSO-d₆) δ 11.32 (s, 1H), 8.56 (p, J = 9.3, 8.3 Hz, 1H), 8.29 (dq, J

= 13.4, 7.5 Hz, 1H), 7.88−7.63 (m, 2H), 7.48−7.28 (m, 4H), 7.22

(qd, J = 14.5, 9.4, 7.5 Hz, 1H), 6.94 (tt, J = 14.2, 7.5 Hz, 1H), 4.79−

4.64 (m, 1H), 2.64−2.58 (m, 3H), 2.58 (s, 3H), 1.05 (tt, J = 13.4, 8.0

Hz, 3H), 0.75 (dp, J = 25.8, 6.7 Hz, 3H). ¹³C NMR (176 MHz,

DMSO) δ 169.69, 165.86, 143.12, 141.05, 127.89, 127.29, 127.17,

126.47, 125.00, 124.61, 124.32, 123.62, 119.24, 119.05, 108.85, 59.80,

39.74, 39.62, 39.50, 39.38, 39.26, 39.15, 39.03, 32.40, 19.97, 19.82,

14.64, 11.05.

(Z)-3-((3,5-Dimethyl-4-nitro-1H-pyrrol-2-yl)methylene)-2-oxo-N-

(3-phenyloxetan-3-yl)indoline-5-carboxamide (7l). Prepared using

the protocol described for 7a. Yields an orange solid, 47 mg, 42%.

Molecular formula: C₂₅H₂₂N₄O₅ESI-MS calc: 458.16 ESI-MS found:

459.1656 HPLC: 6.923. ¹H NMR (700 MHz, DMSO-d₆) δ 11.39 (s,

1H), 9.36 (s, 1H), 8.40 (d, J = 2.0 Hz, 1H), 7.83 (d, J = 2.2 Hz, 1H),

7.81−7.77 (m, 1H), 7.59−7.55 (m, 2H), 7.40 (t, J = 7.8 Hz, 2H),

7.29 (t, J = 7.4 Hz, 1H), 6.99 (dd, J = 8.4, 2.0 Hz, 1H), 5.04 (d, J =

6.7 Hz, 2H), 4.81 (d, J = 6.7 Hz, 2H), 2.63 (d, J = 2.0 Hz, 3H), 2.58

(d, J = 2.1 Hz, 3H). ¹³C NMR (176 MHz, DMSO) δ 169.85, 165.50,

142.99, 141.58, 136.84, 133.69, 127.65, 127.44, 126.92, 125.26,

124.89, 124.72, 124.58, 123.78, 119.24, 109.26, 81.88, 58.33, 14.77,

11.15.

130.27, 126.64, 126.10, 125.34, 125.04, 123.98, 119.28, 109.31,

102.54, 48.41, 40.00, 39.88, 39.76, 39.64, 39.52, 39.40, 39.28, 22.27,

14.93.

(R,Z)-3-((3,5-Dimethyl-4-nitro-1H-pyrrol-2-yl)methylene)-2-oxo-

N-(1-(p-tolyl)ethyl)indoline-5-carboxamide (7g). To a dried sealed

tube were added 253.9 mg (0.862 mmol) of (3g) and 140.9 mg

(0.837 mmol) of 3,5-dimethyl-4-nitro-1H-pyrrole-2-carbaldehyde, all

of which were dissolved in abs. EtOH (3.0 mL). To this solution was

added 0.05 mL of piperidine and heated to reﬂux (95 °C) for 4 h.

Once complete, the reaction was cooled to room temperature and

then the product was ﬁltered oﬀ as an orange solid. Yield: orange

solid, 76.1 mg, 25%. Molecular formula: C₂₅H₂₄N₄O₄ESI-MS calc:

(R,Z)-N-(1-(4-Chlorophenyl)ethyl)-3-((3,5-dimethyl-4-nitro-1H-

pyrrol-2-yl)methylene)-2-oxoindoline-5-carboxamide (7k). Pre-

pared using the protocol described for 7a. Yields an orange solid,

213.3 mg, 96%. Molecular formula: C₂₄H₂₁ClN₄O₄ESI-MS calc:

1

464.13 ESI-MS found: 465.1318 [M + 1] HPLC: 7.795. H NMR

(500 MHz, DMSO-d₆) δ 11.34 (s, 1H), 8.65 (d, J = 7.8 Hz, 1H),

8.35−8.30 (m, 1H), 7.79 (s, 1H), 7.75 (dd, J = 8.1, 1.7 Hz, 1H), 7.43

(d, J = 8.3 Hz, 2H), 7.39 (d, J = 8.5 Hz, 3H), 6.94 (d, J = 8.1 Hz, 1H),

5.17 (p, J = 7.2 Hz, 1H), 2.62 (s, 3H), 2.57 (s, 3H), 1.49 (d, J = 7.1

Hz, 3H). ¹³C NMR (126 MHz, DMSO) δ 169.83, 165.67, 144.05,

141.32, 136.76, 133.66, 131.02, 128.18, 128.10, 128.00, 127.49,

125.11, 124.70, 124.41, 123.63, 119.35, 118.98, 109.10, 48.00, 40.00,

39.83, 39.67, 39.50, 39.34, 39.17, 39.00, 22.07, 14.76, 11.21.

(R,Z)-3-((4-Amino-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-2-

oxo-N-(1-phenylethyl)indoline-5-carboxamide (8a). To a ﬂask were

added 90.7 mg of (7a) and 5 mL of 2:1 EtOH/EtOAc. To this slurry

were added 226.8 mg (14 equiv) of Zn powder and 2 mL (150 equiv)

of AcOH. The turbid orange solution was allowed to stir at 50 °C for

2 h. Once complete, the reaction was cooled to room temperature and

then EtOAc was added before basifying with sat. Na₂CO₃. The

basiﬁed aqueous layer was extracted with EtOAc (3 × 30 mL) and

then washed with water and brine (1 × 30 mL). The organic layer was

dried over MgSO₄, and then solvent was removed under pressure to

give the desired product as a red solid. Because the free amine is very

reactive, it was moved forward without further characterization.

Result: orange solid, 66.7 mg, 79.0%. Molecular formula: C₂₄H₂₄N₄O₂

ESI-MS calc: 400.19 ESI-MS found: 401.1955 HPLC: 5.177. ¹H

NMR (700 MHz, DMSO-d₆) δ 13.45 (s, 1H), 10.88 (s, 1H), 8.56 (d,

J = 8.0 Hz, 1H), 8.12 (d, J = 1.6 Hz, 1H), 7.62 (dd, J = 8.1, 1.6 Hz,

1H), 7.46 (s, 1H), 7.41 (d, J = 7.6 Hz, 3H), 7.33 (t, J = 7.6 Hz, 3H),

7.22 (t, J = 7.3 Hz, 1H), 6.89 (d, J = 8.1 Hz, 1H), 5.20 (q, J = 7.4 Hz,

1H), 4.02 (d, J = 14.5 Hz, 2H), 2.26 (s, 3H), 2.17 (s, 3H), 1.51 (d, J =

7.1 Hz, 3H).

1

444.18 ESI-MS found: 445.1864 [M + 1] HPLC: 7.662. H NMR

(700 MHz, DMSO-d₆) δ 9.73 (s, 1H), 8.57 (d, J = 8.2 Hz, 2H), 8.35

(d, J = 1.7 Hz, 1H), 7.83 (s, 1H), 7.76 (dd, J = 8.1, 1.7 Hz, 2H), 7.30

(d, J = 7.8 Hz, 3H), 7.14 (d, J = 7.9 Hz, 3H), 6.96 (d, J = 8.0 Hz, 2H),

5.17 (p, J = 7.2 Hz, 1H), 2.55 (d, J = 8.7 Hz, 6H), 2.28 (s, 5H). ¹³C

NMR (176 MHz, DMSO) δ 170.11, 165.77, 155.70, 142.17, 141.51,

137.04, 135.76, 135.76, 128.94, 126.27, 125.36, 119.23, 109.39, 52.91,

40.12, 40.00, 39.88, 39.76, 39.64, 39.52, 39.40, 22.49, 20.86, 15.04,

11.49, 10.19.

(Z)-N-Benzyl-3-((3,5-dimethyl-4-nitro-1H-pyrrol-2-yl)methylene)-

2-oxoindoline-5-carboxamide (7i). Prepared using the protocol

described for 7a. Yields an orange solid, 150 mg, 55%. Molecular

formula: C₂₃H₂₁₀N₄O₄ESI-MS calc: 416.15 ESI-MS found: 417.2934,

HPLC: 7.158. H NMR (700 MHz, DMSO-d₆) δ 11.44−11.06 (m,

1H), 8.84 (t, J = 6.1 Hz, 1H), 8.34 (s, 1H), 7.76 (d, J = 8.3 Hz, 1H),

7.69 (s, 1H), 7.34 (d, J = 5.6 Hz, 4H), 7.25 (d, J = 6.6 Hz, 1H), 6.92

(d, J = 8.2 Hz, 1H), 4.52 (d, J = 5.7 Hz, 2H). ¹³C NMR (176 MHz,

DMSO) δ 169.75, 166.10, 141.33, 139.82, 136.69, 133.57, 128.23,

127.89, 127.37, 127.18, 126.67, 124.95, 124.69, 124.44, 123.25,

119.29, 118.57, 109.16, 42.62, 39.86, 39.74, 39.62, 39.50, 39.38, 39.26,

39.14, 14.73, 11.11.

(Z)-3-((3,5-Dimethyl-4-nitro-1H-pyrrol-2-yl)methylene)-2-oxo-N-

(2-phenylpropan-2-yl)indoline-5-carboxamide (7j). Prepared using

the protocol described for 7a. Yields an orange solid, 76.1 mg, 32.1%.

Molecular formula: C₂₅H₂₄N₄O₄ESI-MS calc: 444.18 ESI-MS found:

(R,Z)-3-((4-Amino-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-2-

oxo-N-(1-(m-tolyl)ethyl)indoline-5-carboxamide (8b). To a ﬂask

were added 86.4 mg of (7b) and 5 mL of 2:1 EtOH/EtOAc. To this

slurry were added 210.9 mg (14 equiv) of Zn powder and 2 mL (150

equiv) of AcOH. The turbid orange solution was allowed to stir at 50

°C for 2 h. Once complete, the reaction was cooled to room

temperature and then EtOAc was added before basifying with sat.

Na₂CO₃. The basiﬁed aqueous layer was extracted with EtOAc (3 ×

1

445.2680 [M + 1] HPLC: 7.713. H NMR (700 MHz, DMSO-d₆) δ

11.32 (s, 1H), 8.31 (d, J = 1.7 Hz, 1H), 8.25 (s, 1H), 7.80 (s, 1H),

7.72 (dd, J = 8.0, 1.7 Hz, 1H), 7.42−7.39 (m, 2H), 7.28 (t, J = 7.8 Hz,

2H), 7.17 (t, J = 7.3 Hz, 1H), 6.92 (d, J = 8.1 Hz, 1H), 2.61 (s, 3H),

2.57 (s, 3H), 1.70 (s, 6H). ¹³C NMR (176 MHz, DMSO) δ 169.84,

165.70, 148.16, 141.11, 136.71, 133.63, 129.19, 127.83, 127.58,

125.62, 125.05, 124.73, 124.69, 124.29, 123.61, 119.44, 119.02,

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30 mL) and then washed with water and brine (1 × 30 mL). The

organic layer was dried over MgSO₄, and then the solvent was

removed under pressure to give the desired product as a red solid.

Note that the free amine is very reactive, so it was moved forward

without further characterization. Result: red solid, 86.6 mg, 94%.

Molecular formula: C₂₅H₂₆N₄O₂ESI-MS calc: 414.21 ESI-MS found:

415.2120 [M + 1] HPLC: 5.627.

(R,Z)-3-((4-Amino-3,5-dimethyl-1H-pyrrol-2-yl)methylene)-N-(1-

(4-ﬂuorophenyl)ethyl)-2-oxoindoline-5-carboxamide (8c). To a

ﬂask were added 96.3 mg of (7c) and 5 mL of 2:1 EtOH/EtOAc.

To this slurry were added 295 mg (14 equiv) of Zn powder and 2 mL

(150 equiv) of AcOH. The turbid orange solution was allowed to stir

at 50 °C for 2 h. Once complete, the reaction was cooled to room

temperature and then EtOAc was added before basifying with sat.

Na₂CO₃. The basiﬁed aqueous layer was extracted with EtOAc (3 ×

30 mL) and then washed with water and brine (1 x 30 mL). The

organic layer was dried over MgSO₄, and then the solvent was

removed under pressure to give the desired product as a red solid.

The free amine is very reactive, so it was moved forward without

further characterization. Result: red solid, 96 mg, 100%. Molecular

formula: C₂₄H₂₃FN₄O₂ESI-MS calc: 418.18 ESI-MS found: 419.1938

[M + 1] HPLC: 5.713.

this slurry were added 314.4 mg (14 equiv) of Zn powder and 2.0 mL