From Pyrazolones to Azaindoles: Evolution of Active-Site SHP2 Inhibitors Based on Scaffold Hopping and Bioisosteric Replacement

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From Pyrazolones to Azaindoles: Evolution of Active-Site SHP2

Inhibitors Based on Scaﬀold Hopping and Bioisosteric Replacement

́

Yelena Mostinski, Guus J. J. E. Heynen, Maria Pascual Lopez-Alberca, Jerome Paul, Sandra Miksche,

Silke Radetzki, David Schaller, Elena Shanina, Carola Sey ﬀ arth, Yuliya Kolomeets, Nandor Ziebart,

Judith de Schryver, Sylvia Oestreich, Martin Neuenschwander, Yvette Roske, Udo Heinemann,

Christoph Rademacher, Andrea Volkamer, Jens Peter von Kries, Walter Birchmeier, and Marc Nazare*

́

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Supporting Information

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ABSTRACT: The tyrosine phosphatase SHP2 controls the

activity of pivotal signaling pathways, including MAPK, JAK-

STAT, and PI3K-Akt. Aberrant SHP2 activity leads to uncon-

trolled cell proliferation, tumorigenesis, and metastasis. SHP2

signaling was recently linked to drug resistance against cancer

medications such as MEK and BRAF inhibitors. In this work, we

present the development of a novel class of azaindole SHP2

inhibitors. We applied scaﬀold hopping and bioisosteric replace-

ment concepts to eliminate unwanted structural motifs and to

improve the inhibitor characteristics of the previously reported

pyrazolone SHP2 inhibitors. The most potent azaindole 45 inhibits SHP2 with an IC₅₀= 0.031 μM in an enzymatic assay and with

an IC₅₀= 2.6 μM in human pancreas cells (HPAF-II). Evaluation in a series of cellular assays for metastasis and drug resistance

demonstrated eﬃcient SHP2 blockade. Finally, 45 inhibited proliferation of two cancer cell lines that are resistant to cancer drugs

and diminished ERK signaling.

Structurally, SHP2 consists of three domainsN-terminal

INTRODUCTION

■

and C-terminal SH2 recognition elements and a PTP catalytic

The protein tyrosine phosphatase SHP2 is a member of a

domain. When the enzyme is in a basal auto-inhibited state, the

human protein phosphatases family (PTPs) and encoded by

the PTPN11 proto-oncogene.^1,2This ubiquitously expressed

enzyme controls the activation of several intracellular signaling

SH2 domains are covering the catalytic cavity of the PTP site.

After activation by binding of the phosphorylated tyrosine-

bearing proteins and peptides to SH2 domains, SHP2

undergoes conformational changes disrupting the autoinhibi-

tory interaction between the SH2 domain and the PTP site,

thereby transforming into an open conformation and exposing

the substrate-binding catalytic site.^2,26The development of a

small molecule that will compete with the endogenous

substrates and block the catalytic site is one of the main

strategies of inhibition of SHP2 (Figure 1, compounds 1−

4).^2,19−22Despite several promising reports, none of the

described inhibitors proceeded to advanced clinical studies so

far. One of the major reasons for the low success rate in the

development of SHP2 inhibitors is the shallow, highly polar,

lysine and arginine-rich active site, which is highly conserved

pathways, including receptor tyrosine kinase (e.g., met-

receptor), MAPK, JAK-STAT, and PI3K-Akt.^3,4It has been

reported that mutations in PTPN11 lead to the development of

various diseases, such as Noonan syndrome, LEOPARD

syndrome, juvenile myelomonocytic leukemia, and several

types of solid tumors.^2,5,6The clear connection between

activating mutations of PTPN11 and these disorders qualify

SHP2 as a highly relevant biological target for the development

of anticancer therapeutics. Moreover, the regulatory function

of SHP2 in intracellular signaling pathways provides an

opportunity for controlling the activity of known oncogenes,

such as BRAF and RAS. Identifying alternative ways to block

these targets is of utmost importance, especially in view of the

emerging clinical threat of drug resistance and relapsing

tumors.⁷⁻⁹Furthermore, recent reports on previously unre-

vealed key functions of SHP2 in various intracellular pathways

are successively illustrating its key pathophysiological signiﬁ-

cance in several cancers and in development of drug

resistance.¹⁰⁻¹⁴Therefore, several studies were devoted to

the discovery of small-molecule inhibitors of SHP2 as a prime

phosphatase target for anticancer therapy.^3,15−25

Received: July 28, 2020

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Figure 1. Previously reported SHP2 inhibitors and their IC₅₀values. Compounds 1−4 represent active-site inhibitors and 5−6 are allosteric

binders.

among the PTPs.^27,28Therefore, the design and synthesis of

potent but selective and cell-permeable small-molecule

inhibitors is a complex and challenging endeavor. Recently,

in an attempt to overcome these obstacles of active-site

inhibition, allosteric modulation of SHP2 activity was

investigated. The allosteric mechanism is based on stabilization

of the auto-inhibited conformation of SHP2 by simultaneous

binding to all three domains and shifting the equilibrium from

the active state to the inactive state (the “inverse agonist

principle”). Several small-molecule inhibitors, acting as a so-

called “molecular glue”, that bind and inhibit SHP2 through an

allosteric mechanism were reported (Figure 1, compounds 5−

6).^17,29−31These small molecules demonstrated remarkable

phosphatase selectivity and potency, and several candidates

entered clinical trials.³However, some studies indicated that

the allosteric modulators may exhibit limited hERG

selectivity^15,29and exert a surprisingly low eﬃcacy against

distinct oncogenic SHP2 variants in advanced biological

studies.³²⁻³⁶This limitation is associated with the high

vulnerability of the allosteric three-domain-binding mode to

mutations and to the existing high degree of aberrant activation

of SHP2 in several cancers.³²⁻³⁶Therefore, although allosteric

inhibition of SHP2 seems to be an excellent mode of

suppression for some oncogenic SHP2 variants, these speciﬁc

compounds might not be clinically useful to inhibit frequently

encountered mutated SHP2 variants.²⁹Consequently, the

development of chemically diﬀerent SHP2 inhibitors, which

are less sensitive to oncogenic mutations and underlie a robust

mode of action, which address the activated state of SHP2 is

highly desirable.

inhibitors and achieved sustained inhibition of tumor cell

proliferation in vitro and in vivo.^7,9

Despite its potency, the identiﬁed compound class possesses

several unfavorable functionalities and liabilities, which

prevents it from being a suitable platform for further lead

optimization. A major concern is related to a chemically

unstable hydrazone fragment attached to the pyrazolone

scaﬀold that potentially releases toxic metabolites in vivo and

shares structural features belonging to promiscuous pan-assay

interference compounds (PAINS).^39,40The presence of a nitro

group in PHPS1, and in GS493, which incorporates two nitro

groups, may account for potential cytotoxicity in vivo,^41,42and

the sulfonic acid hampers the cell permeability of the

inhibitors. However, both the nitro and sulfonic groups are

thought to be engaged in key binding interactions with the

SHP2 catalytic site, and therefore, their replacement is a

challenging task.^37,38In the current work, we designed and

synthesized highly potent, selective, and drug-like inhibitors of

SHP2,⁴³⁻⁴⁵based on the substitution of these detrimental

structural features according to structure-activity relationship

(SAR) studies, which included scaﬀold hopping and

bioisosteric replacement approaches.

RESULTS AND DISCUSSION

■

Scaﬀold Hopping of the Pyrazolone Core. Our

optimization study started with the investigation of a

rescaﬀolding of the pyrazolone core and elimination of the

hydrazone moiety.^46,47Unfortunately, the cocrystal structure of

SHP2 and the respective pyrazolone inhibitor is not available.

Moreover, the determination of the endogenous substrates of

SHP2 is still limited to date; thus, it is a daunting endeavor to

successfully mimic their putative interactions. We therefore

resorted to a ligand-based alignment approach preserving the

vectorial information and structural features commonly known

to be essential to address the phosphatase catalytic mechanism.

For the initial attempts of scaﬀold hopping, we preserved the

monocyclic framework by incorporation of the stable

carboxamide-imidazole backbone. Unfortunately, the corre-

sponding imidazole analogue 8 of PHPS1 was inactive (Table

In our previous work, we described the development of

pyrazolone-based inhibitors of SHP2. The hit candidate

PHPS1 was identiﬁed through virtual high-throughput screen-

ing and optimized to GS493.^37,38These molecules are

selective, active-site-directed competitive inhibitors with

prominent activity in enzymatic and cellular assays. We

demonstrated that these active-site inhibitors have a

remarkable synergistic eﬃcacy with the MEK and BRAF

B

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1). Supported by comparative alignment studies with PHPS1,

we concluded that a more rigid bicyclic core might be crucial

for inhibitor binding, as the carboxamide-imidazole backbone

is too ﬂexible to provide optimal orientation of the substituents

for favorable interactions with the catalytic site. Hence, we

proceeded to a conformational lock/ring-closure strategy and

synthesized a bicyclic pyrazolo-triazole PHPS1 analogue 9,

Table 1. Heterocyclic Candidates for Pyrazolone Scaﬀold

Replacement

which gratifyingly exhibited inhibitory activity with an IC₅₀

=

12 μM (see Supporting Information, Figure S1). This

supported our hypothesis that a ring-closure to a bicyclic

aromatic system would be a viable strategy for further

variations and would not impose signiﬁcant detrimental eﬀects

on the molecular recognition of the peripheral substituents of

the small-molecule ligand with the active site of SHP2.

This ﬁnding led us to examine additional heterocycles, with

lower apparent similarity to the pyrazolone hit, but with higher

abundance among existing drugs or drug candidates. In

addition, we hypothesized that a 5,6-fused system would

provide optimal geometrical preorientation, preserving the

original trajectory for the sulfonic group.⁴⁸We focused our

eﬀorts on the replacement of the pyrazolone core by azaindole

and indazole analogues. These privileged heterocyclic cores

attracted our attention because of their high stability,

straightforward synthetic derivatization possibilities, and good

drug-like properties.⁴⁹⁻⁵³Satisfyingly, indazole 10 and

azaindole 11 showed higher inhibitory activity than the parent

triazolo-pyrazole analogue. Because of the highly privileged

nature of the azaindole candidate, we considered this

framework as the most promising starting point and focused

therefore our further optimization eﬀorts on this scaﬀold.

Chemistry. 1,3,5-Trisubstituted 7-azaindoles were synthe-

sized as outlined in Scheme 1. The synthetic route commenced

with the iodination of the 3-position of commercially available

5-bromo-7-azaindole 12 following a previously reported

procedure.⁵⁴The second step involved a Chan-Lam N-

arylation to generate intermediate compound 14. Unfortu-

nately, the yield of the products was low, despite considerable

a

To evaluate the IC₅₀of compounds 7−11, a DiFMUP concentration

of 20 μM was used. For full assay details see the Experimental Section.

b

Lit. value for IC₅₀of PHPS1 = 2.1 μM. See ref 34. Other reference

compounds values: IC₅₀PTP II-B08 = 30 μM; IC₅₀NSC-87877 = 4.5

μM.

a

Scheme 1. General Synthesis of Azaindoles

a

Reagents and conditions (a) NIS, acetone, rt; 2−4 h, 92% (b) Cu(OAc)₂, DIPEA, rt, 48 h; 18−21% (c) NaHCO₃, Pd(PPh₃)₄, 3:1 ACN/water,

100 °C, 12 h, 42−69% for the ﬁrst coupling, 2−98% for the second coupling (d) K₂CO₃, Pd(dppf)Cl₂, dioxane/water, 80 °C, 3−6 h, 22−71% for

the ﬁrst coupling, 2.5−38% for the second coupling.

C

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a

Table 2. SAR of R²and R³

excess of reagents used. To overcome these limitations, we

varied the solvent, base, temperature, and copper source.

Nonetheless, no signiﬁcant improvements in yield were

observed. This poor outcome is in line with other literature

precedents, where only a scarce number of examples for N-

arylation of substituted azaindoles with typically low yields are

reported.^55,56The poor outcome of the reaction can be

explained by the deactivating nature of the substituents and

“trapping” eﬀect of the neighboring nitrogen of the pyridine

unit. Subsequent Suzuki couplings required K₂CO₃/Pd(dppf)-

Cl₂or NaHCO₃/Pd(PPh₃)₄catalytic systems depending on

the nature of the employed boron species. In order to

overcome the limitations of the Chan-Lam coupling step, we

attempted ﬁrst to carry out Suzuki coupling; however, this

transformation failedno conversion to the desired product

was observed and the starting material slowly decomposed

over time. Seeking an alternative route, we protected the N1 of

the azaindole with a Boc moiety, and sequentially substituted

the 3- and 5-positions with corresponding aryl groups utilizing

Suzuki coupling, followed by removal of the Boc group. The

ﬁnal N-substitution was performed using Ullman conditions

(for the detailed scheme see Supporting Information, Scheme

S1). Overall, the synthesis of azaindoles 16 was successfully

accomplished by both Ullman and Chan-Lam protocols.

However, the Ullman protocol requires a protection−

deprotection strategy, thus this synthetic path is longer and

despite higher yields obtained in each step of this route, it has a

similar overall yield. Therefore, we resorted to the Chan-Lam

reaction-based approach as the main strategy for the synthesis

of the azaindole products. In summary, all compounds could

be obtained by the developed synthetic route, which is robust,

eﬃcient, and short, providing the ﬁnal molecules in only four

steps. For the preparation of the product with nonaromatic

substituents on the pyrrole ring, we replaced the Chan-Lam

arylation step by a classic NaH-assisted reaction (for details see

the Experimental Section for the compounds 28 and 30−32).

Bioisosteric Replacement of Nitro and Sulfonic Acid

Functionalities and SAR Evaluation of Azaindoles. After

rescaﬀolding of the pyrazolone core, the next step was to

investigate the potential for a bioisosteric replacement of the

highly charged permeability-hampering sulfonic acid

group.^46,47,57In stark contrast to previous replacement

attempts at the pyrazolone core,^38,58this structural fragment

could be successfully exchanged by a simple carboxylic acid

(Table 2).⁵⁹Compounds 17 and 18 bearing such a carboxylic

acid in para- or meta-position showed superior inhibitory

activity with IC₅₀of 0.13 and 0.41 μM, respectively. We

therefore concluded that the azaindoles incorporating a

benzoic acid functionality would provide a promising basis

for further SAR exploration. Moreover, besides the fact that the

carboxylic group represents a physiologically much more

benign functionality compared to the sulfonic acid, in

particular for intracellular targets, it is a convenient synthetic

platform for a potential pro-drug approach. Because meta- and

para-substituted benzoic acids were similarly active, we

continued the SAR exploration aiming for a replacement of

the nitro group by testing both benzoic acid isomers and

varying the R²substitution at the 3-position of the azaindole.

Finding an appropriate replacement of the nitro moiety has

proven to be challenging⁶⁰because systematic studies of a

general bioisosteric relationship of the nitro functionality with

other chemical groups are lacking. Intuitively, strong electron-

withdrawing groups, such as triﬂuoromethyl and −CN, are

a

b

Bioisosteric replacement of nitro and sulfonic acid moieties. To

evaluate the IC₅₀of compounds 17−27, a DiFMUP concentration of

10 μM was used. For full assay details, see the Experimental Section.

Reference cmpd values: PHPS1 IC₅₀= 0.06 μM; PTP II-B08 IC₅₀

12 μM; NSC-87877 IC₅₀= 0.41 μM; PTP1B inhibitor IC₅₀= 3.1 μM.

=

used for bioisosteric substitution of the nitro functionality.^61,62

In addition, the incorporation of oxygen- and nitrogen-

containing heterocycles such as oxazolines and oxadiazoles

was found to be beneﬁcial in several reports.⁶³⁻⁶⁷Guided by

these observations, we prepared several analogues of 17 and

18, replacing the nitro group (Table 2) and tested their ability

to inhibit SHP2. Several azaindoles demonstrated good activity

with IC₅₀values below 1 μM, among the analogues containing

a triﬂuoromethyl group 21 and 22, nitrile-bearing analogue 27,

and the benzoxadiazoles 19 and 20, the latter being particularly

promising, because of their lower lipophilicity. The good

inhibitory eﬀect of these derivatives aligns well with previous

ﬁndings, showing the ability of the small molecules bearing

nitro-benzoxadiazole to inhibit the closely related PTP1B

enzyme.⁶⁸Moreover, the benzoxadiazole moiety is highly

ﬂuorescent, and it provides an additional useful feature for the

intended cellular permeability studies. Compounds 23−26,

however, demonstrated only moderate activity, compared to

the nitro analogue 17. A dioxolane-bearing derivative 26

supported the assumption that a strong electron-withdrawing

group might be required for the successful replacement of the

nitro moiety. Along these lines, pyridines 23−24 and the

pyrimidine 25 analogue indicated that the presence of a

protruding substituent is indispensable to establish these

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important interactions with the binding cavity. At this point by

comparing the activity of several pairs of the meta−para

benzoic acid isomers, we had suﬃcient evidence to conclude

that the para-derivative outperforms the meta-analogue, and

we therefore decided to utilize para-19 as a platform for the

next optimization step exploring the N1-azaindole substitution.

Despite their good biological activity, the novel compounds

showed only limited aqueous solubility because of the ﬂat

aromatic, hence highly lipophilic structure. Consequently, we

introduced variations at R¹aiming for a higher solubility while

maintaining or improving the activity (Table 3). Our previous

the compound 29 showed considerably reduced inhibition of

SHP2. Even small changes such as introducing a methylene

spacer to enhance conformational ﬂexibility in compounds 30

and 31 resulted in a decrease of activity, indicating detrimental

interactions with the protein environment at the catalytic site.

The same trend was observed with the meta-methoxy analogue

32. Installation of a simple unsubstituted phenyl group

(compound 33) resulted in improved activity, but as expected,

the solubility was further reduced. In an additional eﬀort to

circumvent the solubility limitations, we synthesized azainda-

zole congener 34 of the most promising compound 19. We

speculated that an azaindazole with an auxiliary nitrogen on the

heterocyclic scaﬀold may enhance solubility and cellular

permeability. Unfortunately, the solubility of azaindazole 34

was low, which was reﬂected in the absence of activity because

of precipitation under assay conditions. We further tried to

increase the solubility of the azaindazole by incorporating the

tetrahydropyran (THP) ether instead of the R¹aromatic ring,

but the corresponding azaindazole 35 was only weakly active.

Overall, the ﬁrst iteration in this SAR study again conﬁrmed

compound 19 as the best template for the subsequent second

optimization round.

Table 3. Investigation of the Scope for a Broader R1

Variation toward Improved Solubility

Here, we focused our attention on the R³residue, bearing

the carboxylic group (Table 4). This functionality was found to

Table 4. Second Iteration of SAR Studies

a

To evaluate the IC₅₀of compounds 28-33, a DiFMUP concentration

of 10 μM was used. For full assay details, see the Experimental

Section. Reference cmpd values: PHPS1 IC₅₀= 0.06 μM; PTP II-B08

IC₅₀= 12 μM; NSC-87877 IC₅₀= 0.41 μM; PTP1B inhibitor IC₅₀

3.1 μM. To evaluate the IC₅₀of compounds 34−35, a DiFMUP

concentration of 20 μM was used.

=

b

docking studies of the parent pyrazolone³⁷indicated that this

aromatic ring is not engaged in hydrogen bond interactions

with the SHP2 enzyme, and thus, we assumed a higher degree

of freedom for the modiﬁcation of this moiety. For example,

we synthesized isopropyl-bearing derivative 28, a known

bioisostere for aromatic rings. However, despite this

assumption, analogue 28 was lacking any signiﬁcant activity.

We then attempted to outbalance aﬃnity versus solubility by

the introduction of a bulky but hydrophilic tosylate group, but

a

To evaluate the IC₅₀of the compounds 36−42 and 45, a DiFMUP

concentration of 10 μM was used. Reference cmpd values: PHPS1

IC₅₀= 0.062 μM; PTP II-B08 IC₅₀= 12 μM; NSC-87877 IC₅₀= 0.41

μM; PTP1B inhibitor IC₅₀= 3.1 μM. To evaluate the IC₅₀of the

b

compounds 43-44, a DiFMUP concentration of 20 μM was used.

c

PTP1B inhibitor IC₅₀reference value for 45 is 2.5 μM.

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Figure 2. Binding mode of cocrystalized ligand (II-B08) of 3O5X²⁴(A) and of compounds 41 (B) and 45 (C) observed in docking studies

targeting the catalytic domain of SHP2 (PDB entry 3O5X²⁴). Yellow spherehydrophobic interaction, red arrowhydrogen bond acceptor, red

asterisknegative ionizable, blue ringaromatic interaction. Figure produced using LigandScout 4.4.⁷²

be pivotal in previous and current studies.^24,25Therefore, we

hypothesized that ﬁnding the optimal position in the catalytic

pocket addressing ARG465 by salt-bridge formation may be

most beneﬁcial for enhanced activity. More ﬂexible and shorter

nonaromatic moieties were therefore tethered to provide

compounds 36−38. Here, we observed the strong trend of the

inhibitor activity improvement with the increase in length and

rigidity of the R³substituent. Thus, the aromatic ring as a

spacer was conﬁrmed to have favorable properties for achieving

the optimal position and interactions of the carboxylic acid. To

validate the importance of a negatively charged carboxylic acid

and the ﬂexibility to a further bioisosteric replacement, we

constructed ester 39 and sulfonamide 40. As expected, deletion

of the charged carboxylic acid by an ester moiety (39)

decreased the inhibitor activity signiﬁcantly. Intriguingly, the

sulfonamide acid analogue 40 was highly active, which

indicates that a sulfonamide moiety may serve as an

appropriate bioisostere of the sulfonic and carboxylic acid in

this context.^25,69Next, taking into consideration the pseudo

symmetrical “propeller” nature of the decorated azaindoles, we

anticipated that our small-molecule inhibitor may have various

possible orientations for occupying the catalytic pocket. To test

our hypothesis, we synthesized the analogue 41, in which the

critical aﬃnity generating moieties, that is, the carboxylic acid

and the benzoxadiazole at R²and R³were inverted. In line with

our expectation, 41 was able to inhibit SHP2 with a remarkable

IC₅₀= 0.065 μM. This supported our assumption that the

benzoxadiazole and carboxylic groups could be swapped. We

speculated that the azaindole bearing two carboxylic acids may

exhibit superior activity and may possess synthetic advantages

such as one-pot functionalization of 3- and 5-positions.

However, the corresponding analogue 42 exhibited surprisingly

low inhibitory activity. Replacement of the carboxylic acid at

R²by an aromatic lactone 43 restored the activity; however,

the compound was highly unstable and underwent fast

hydrolysis to its inactive open carboxylic acid analogue 44

under any storage and assay conditions. To overcome this

stability issue and in search for an isomorphic noncharged

hybrid moiety, resembling the benzoxadiazole and a hydrolysis-

resistant lactone group, we replaced the benzo lactone at

residue R²by a stable coumarine lactone. Gratifyingly, the

corresponding compound 45 was found to have the highest

inhibitory activity on SHP2 within the entire SAR study with

an IC₅₀= 0.031 μM. We therefore proceeded to investigate the

most active azaindole of the series with regard to phosphatase

and kinase selectivity and studies in advanced cellular models

of metastasis and drug resistance. We also performed docking

and molecular dynamic experiments to validate the SAR study

ﬁndings.

Structural Basis of InhibitionDocking and Molec-

ular Dynamics. To rationalize the SAR of the presented

ligand series, compounds were docked into the SHP2 catalytic

domain of PDB entry 3O5X²⁴using the OpenEye Python

Toolkit.⁷⁰The cocrystalized ligand of 3O5X was employed in

the hybrid docking method⁷¹implemented in OEDocking to

guide the ligand placement because it contains a benzoic acid

similar to the majority of compounds of the investigated ligand

series (17−45).

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Figure 3. Results from a 20 ns MD simulation of SHP2 in complex with the most active compound 45. (A) Depiction of 10 snapshots taken every

2 ns. (B) rmsd plot for ligand and protein atoms. Figures produced using PyMOL 1.8.6⁷⁵and Microsoft Excel.

Similar to the cocrystalized inhibitor of 3O5X, the most

consistent binding mode of docked compounds involves a

charge interaction with ARG465 and a π−cation interaction

with LYS366 as identiﬁed with LigandScout 4.4⁷²(see Figure

1). The hydrogen bonds with the backbone nitrogen of

ARG465 and the thiol of CYS459 are only observed in case of

the cocrystalized inhibitor of 3O5X, which is consistent with

the lack of a hydroxyl group at the benzoic acid moiety of the

investigated compound series. Additionally, the identiﬁed

binding mode involves a hydrophobic contact with TYR279,

a further π−cation interaction with LYS364 and hydrogen

bonds with ASN281 and GLN506. This binding mode could

be observed for compounds 17−21, 27, 33, 41, and 45, which

all show an IC₅₀below 1 μM (Supporting Information, Figure

S2). Interestingly, albeit the azaindole moiety of the two most

active compounds 41 and 45 is placed in two diﬀerent

orientations, the docking poses allow almost identical

interactions (see Figure 2). This symmetric behavior can also

be observed for compound 19, in which R²and R³are inverted

compared to compound 41, but compound 19 still shows

considerable inhibitory potency. The lower potency of

compound 19 could thereby be explained by less favorable

hydrogen bond interactions formed by the benzoxadiazole

moiety, which is perfectly embedded between ASN281 and

GLN506 in case of compound 41. The importance of

hydrogen bonds with these two residues is also supported by

the decreased activity of compounds 23, 24, and 25, which

contain either a pyridine or a pyrimidine group at R²that are

too limited in size to perform suitable hydrogen bonding

interactions. In line with that, docking poses indicate an

acceptor-like interaction formed by the ﬂuorine atoms of the

triﬂuoromethyl groups of active compounds 21 and 22 with

ASN281 and GLN506. However, the lower activity of

compound 26 cannot be easily explained with this binding

mode because it contains a benzodioxolane that would allow

for favorable hydrogen bond interactions similar to the

benzoxadiazole moiety of compound 19. Possibly, the more

ﬂexible nonplanar nature of the dioxolane ring prevents the

formation of the optimal interactions.

(see Figure 2, Supporting Information Figure S2). The absence

of suitable interactions with LYS366 and ARG465 could also

explain the reduced activity of compounds 36, 37, 38, and 39

that are either too limited in size, miss the aromatic character

needed for π−cation interactions, or cannot participate in a

charged interaction with ARG465. The high activity of

compound 40 can be attributed to the relatively low pK_aof

the aryl-sulfonamide moiety, making the sulfonamide proton

suﬃciently acidic for eﬀective hydrogen bond interaction.⁶⁹

The activity results from compounds 28−33 clearly indicate

a strong preference for rigid structures with an aromatic moiety

at R³. Compound 28 lacks any aromatic character on the R¹

position and was inactive at a testing concentration of 10 μM.

Compounds 29−31 introduce a rotatable bond that may result

in an entropic penalty when binding to the catalytic domain of

SHP2. Compound 32 was synthesized with the methoxy group

in meta-position that may either clash with the protein or point

into the solvent, which could explain its reduced activity

compared to compound 19 with a methoxy group in para-

position. Interestingly, compound 33 with an unsubstituted

benzene moiety shows improved inhibitory potency compared

to its para-methoxy-substituted counterpart 19, which is

supported by the observation that the methoxy group is not

involved in any critical interactions.

The docking poses of compounds 41 and 45 present the

most favorable interaction pattern observed within the

investigated ligand series, which is also reﬂected by their

superior inhibitory potency. To evaluate the identiﬁed binding

mode further, the docking pose of the most active compound

45 was subjected to unbiased MD Simulation using Desmond

6.1.⁷³An analysis of the root-mean-squared deviation (rmsd)

of ligand and protein atoms in VMD 1.9.3⁷⁴revealed a similar

behavior for protein and ligand motions (see Figure 3).

Although the ligand appears to leave the position after 4 ns, it

returns to the initial binding mode quickly. In conclusion, the

binding pose stayed stable over 20 ns of MD simulation, which

underlines the validity of the identiﬁed binding mode.

Phosphatase Selectivity. Selective active-site inhibition

of PTPs, including SHP2, has proven to be an extremely

challenging task, primarily because of the highly conserved and

positively charged nature of the PTP active site. We therefore

tested the most potent azaindole 45 against a panel of 10

representative PTPs. Selectivity was high and greater than 35-

fold for PP1B, PP1A, PTPRC/CD45, DUSP22/MKPX, and

Switching the carboxyl group of R³from para- (compounds

17, 19, 21, and 23) to meta-position (compounds 18, 20, 22,

and 24) results in all pairs in a drop of inhibitory potency,

which can be rationalized by a less favorable position of the

aromatic ring not allowing π−cation interactions with LYS366

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PTPN2/TC-PTP and satisfyingly good for PTP1B (13-fold)

but, however, only moderate with a 4-fold selectivity for the

closely related phosphatase SHP1 (Table 5).

Table 6. Structural Diﬀerences and Similarities of Kinase

and Phosphatase Inhibitors

a

Table 5. Phosphatase Selectivity Evaluation for Compound

a

45

target

IC₅₀(μM)

selectivity ratio

SHP2

PP1B

PP1A

0.031

>100

0.20

0.13

1.1

>1000

6

4.5

35

PP2A alpha/PP2R1A complex

PTPN6/SHP1

PTPRC/CD45

% enzyme inhibition

target

AKT1

c-Src

CDK1/cyclin B

EGFR

KDR/VEGFR2

PDGFRb

PKA

19 (100 μM)

46 (100 μM)

DUSP22/MKPX

PTPN2/TC-PTP

PTPN7/LC-PTP

PTPN12/PTP-PEST

PTPN1/PTP1B-CD

2.1

1.3

0.13

0.41

68

41

4.5

13

35

13

89

47

99

b

−35

−95

33

92

43

51

60

95

97

a

For full experimental conditions, see Supporting Information, Table

S1.

4

b

−43

RIPK2

RSK2

TRKA

−3

35

40

Evaluation of Kinase Activity. As the rescaﬀolding

studies of our SHP2 inhibitor yielded the azaindole core as a

highly abundant and privileged kinase inhibitor motif in drug

discovery,^52,53we wanted to exclude any oﬀ-target kinase

activity. The crucial feature for kinase inhibition, in particular

when decorated with aromatic moieties at the 3- and 5-

positions, is the hydrogen bond donor−acceptor pattern of the

unsubstituted NH donor at N1 and the pyrrolidine nitrogen at

the 7-position of azaindoles with the hinge region of kinases.⁷⁶

Albeit, in principle, the installation of any functional group at

this N1 nitrogen should abolish oﬀ-target kinase activity, we

wanted to examine and conﬁrm this against a representative

panel of kinases. For comparison, we used the benzofurazan-

bearing molecule 46, which is the truncated analogue of 19

without the crucial substituent at N1 of the azaindole.

Interestingly, several kinase inhibitors incorporate such a

benzofurazan moiety.⁷⁷⁻⁷⁹Both compounds were tested

against a general panel of ten house-keeping kinases at high

concentration (100 μM) and low ATP concentration (10 μM).

As expected, N-substituted azaindoles showed only negligible

inhibition of the kinases (Table 6). In contrast, the N1-

truncated derivative 46 when tested against the same kinase

panel was signiﬁcantly more prone to kinase recognition and

showed compelling kinase inhibition for CDK1, AKT1, KDR,

RSK1, and TRKA. Moreover, 46 was tested against SHP2 and

was 26-fold less active than parent 19 (IC₅₀= 6.1 μM),

highlighting again the signiﬁcance of the N1 aryl substitution.

Thus, despite apparent structural similarity between inhibitors

46 and 19, the N1 aryl moiety turned out to be a prominent

selectivity switch driving inhibition toward the desired enzyme

class of either phosphatase or kinase. The most active

compound 45 was tested against the same panel of kinases

and showed low interference (for detailed results see

Supporting Information, Table S3).

a

For full experimental conditions, see Supporting Information, Table

b

S2. Negative values might indicate precipitation under assay

conditions.

HPAF-II. This cellular system recapitulates the metastatic

process of cell spreading and outgrowth and has been shown to

be strongly dependent on SHP2 activation.^4,80,81Compound

45 was tested against HPAF-II pancreatic cancer cells in

diﬀerent concentrations and clearly showed a dose-dependent

ability of the azaindole 45 to block the HGF-induced scattering

phenotype (Figure 4A,B). Concentration-dependent quantiﬁ-

cation of the inhibitory eﬀect by analysis of minimum neighbor

distances of proximal Hoechst stained nuclei revealed an IC₅₀

= 2.6 μM, while the previous reference pyrazolone GS493

showed an IC₅₀of 3.4 μM. Blocking of the cell scattering and

reversal of this phenotype was further conﬁrmed by impedance

measurements as a second independent readout (Figure

4D,E). In addition, 45 showed good cell permeability and

intracellular enrichment in the absence of any signs of cell

toxicity over 24 h (see Supporting Information, Figure S3). To

further conﬁrm and preclude any interference of these results

with a perturbation of the cellular viability or any other

nonspeciﬁc eﬀect, we proceeded to investigate cytotoxicity of

45 in HPAF-II and HepG2 cells in a long-term incubation of

72 h at high concentration of up to 40 μM. Again, no

signiﬁcant cytotoxic eﬀects could be observed in the relevant

concentration range in both cell lines, even under these

prolonged incubation times (Figure 4C).

Compound 45 Blocks Proliferation in Two Drug-

Resistant Cancer Cell Lines and Inhibits ERK Signaling.

Previous investigations have shown that simultaneous inhib-

ition of SHP2 and MEK have a synergistic eﬀect and allow for

superior tumor growth control in RAS-mutant tumors.^8,9In

addition, cooperative inhibition of BRAF and SHP2 can

overcome resistance to BRAF inhibitors mediated by EGFR

activation in colon cancer cells.⁷We therefore investigated if

45 in combination with BRAF inhibitor PLX4032 could also

block proliferation of BRAF inhibitor-resistant VACO432

colon cancer cells in a colony formation assay. While both

Evaluation of Azaindole Inhibitor 45 in Cellular

Models of Metastasis and Tumor Resistance. We next

turned our attention to the evaluation of our most active

inhibitor 45 in more advanced cellular models of metastasis

and drug resistance. First, we investigated if 45 was able to

block HGF-induced cell scattering and branching morpho-

genesis of the human pancreatic adenocarcinoma cell line

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Figure 4. Azaindoles inhibit HGF-induced cell separation in scatter and impedance assays in HPAF-II pancreatic cancer cells. (A) The HPAF-II

cell colonies were dissociated because of stimulation with HGF in the control cells (−HGF/+HGF). Treatment with compound 45 causes

inhibition of cell scattering. (B) Quantiﬁcation of changes in cell scattering was done by analysis of minimum neighbor distances. The mean SD

values are shown, representing the relative activity compared to control conditions (+HGF/1, −HGF/0). (C) HPAF-II and HepG2 cells were

exposed with diﬀerent concentrations of compounds for 72 h. Cytotoxicity was determined by ﬂuorescence microscopy using TO-PRO-3 staining

for dead cell quantiﬁcation. Mean values SD of damaged cells are indicated. (D) Impedance assay displays the changes in cell attachment and

spreading because of HGF stimulation during the ﬁrst 4 h, where the maximum cell index value was reached for the positive control (+HGF). The

normalized cell index curves show a clear dose-dependent inhibition by the compound 45. (E) Impedance assay dose−response curve. The graph

shows the result of the end-point analysis after 4 h HGF exposure and treatment with compound 45, demonstrating the inhibitory eﬀect. The

relative activity of normalized cell index values compared to control conditions (+HGF/1, −HGF/0) are displayed.

PLX4032 and 45 exhibited only a small eﬀect when applied as

single compounds, VACO432 cell proliferation was markedly

inhibited by 45/PLX4032 cotreatment (Figure 5A). A similar

result was obtained in HCC1806 breast cancer cells, which are

resistant to PI3K inhibition by BYL719 (Figure 5B). In this

model, 45 sensitized HCC1806 cells considerably to BYL719

treatment, while monotreatment with these compounds was

not as eﬀective.

To evaluate the biochemical eﬀect on ERK signaling of 45

and PLX4032 treatment, we subjected treated VACO432 cell

lysates to western blotting. Cells treated with PLX4032 alone

retained considerable phosphorylated ERK levels after 24 and

48 h. This explains the BRAF inhibitor-resistant phenotype

because ERK activity is essential for the proliferation of these

cells. In contrast, cotreatment with 45 and PLX4032 led to a

substantial decrease in ERK phosphorylation compared to

PLX4032 monotreatment (Figure 5C). Taken together, these

results show that 45 is capable of preventing a BRAF inhibitor-

resistant phenotype in a colon cancer cell line model and a

PI3K inhibitor-resistant phenotype in a breast cancer cell line.

In VACO432 cells, this is explained by the ability of 45 to

eﬀectively inhibit ERK signaling, when used in combination

with PLX4032.

CONCLUSIONS

■

We identiﬁed a novel class of azaindole SHP2 inhibitors, based

on scaﬀold hopping of a previously reported pyrazolone

framework and subsequent reﬁnement by SAR optimization.

Successful replacement of all physiologically incompatible

functionalities provided a much more drug-like lead platform,

which incorporates a privileged azaindole core, as well as

coumarine and benzofurazan moieties, present in numerous

approved drugs. The novel azaindole inhibitors showed high

activity against SHP2, with an IC₅₀of 0.031 μM for 45.

Docking studies aligned well with the observed SAR trends and

provided a structural rationalization for the inhibitor binding

mode. This could be further supported by molecular dynamic

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The HPLC was equipped with an Agilent Poroshell 120, C18 column,

2.1 × 100 mm, 1.8 μm. Method: ESI⁺, ﬂux: 0.6 mL/min, 5−99%

CH₃CN in H₂O + 0.1% FA, total runtime: 4.5 min. Unless otherwise

stated, all compounds were puriﬁed using an Isolera one Biotage ﬂash

chromatography system utilizing silica gel-packed columns RediSep

Rf from Teledyne Isco. Purity and characterization of all ﬁnal

compounds was established by a combination of LC−MS, LC−

HRMS, and NMR analytical techniques. All compounds were found

to be >95% pure by LC−MS and LC−HRMS analysis unless

otherwise stated.

Synthesis of Carboxamido-indazole 8. Methyl 3-(4-Nitro-

phenyl)-2,3-dioxopropanoate (47). Methyl 3-(4-nitrophenyl)-2,3-

dioxopropanoate (47) was synthesized according to previously

reported procedure.⁸²Brieﬂy, methyl 3-(4-nitrophenyl)-3-oxo-2-

(triphenyl-l5-phosphaneylidene)propanoate (2.0 g, 4.14 mmol, 1.0

equiv) was dissolved in 10 mL dichloromethane (DCM) and added

to a stirred solution of DMSO in acetone (0.1 M, 103 mL). The

mixture was stirred for 1 h at room temperature after which the

solvent was evaporated. The crude product was puriﬁed by column

chromatography (SiO₂, cyclohexane/ethylacetate 100:0 → 40:60) to

obtain 47 in 83% yield (878 mg).

Methyl 4-(4-Nitrophenyl)-2-phenyl-1H-imidazole-5-carboxylate

(48). 47 (878 mg, 3.44 mmol, 1 equiv) and benzaldehyde (0.702

mL, 6.88 mmol, 2 equiv) were added to a slurry of ammonium acetate

(2.65 g) in acetic acid (10 mL). The mixture was stirred 2 h at 70 °C,

then cooled to room temperature, and concentrated in vacuo. The

crude mixture was redissolved in ethylacetate and washed with water,

NaHCO₃, and brine. The ethylacetate layer was dried with MgSO₄

and concentrated in vacuo. The crude was puriﬁed by ﬂash

chromatography using EtOAc in cyclohexane as the eluent to obtain

48 in 9% yield (110.2 mg, 0.340 mmol, yellow amorphous solid).

LCMS (pos. ESI-TOF): m/z calcd for C₁₇H₁₃N₃O₄323.30 (M + H)⁺;

found, 323.95 (M + H)⁺.

Figure 5. Synergistic combination of 45 with BYL719 and PLX4032

in VACO432 and HCC1806 cell lines. (A) In vitro coinhibition by

PLX4032 (vemurafenib) and SHP2 (45) in colony formation

experiments with the VACO432 human colorectal cancer cell line.

(B) In vitro coinhibition by BYL719 (alpelisib) and SHP2 (45) in

colony formation experiments with the HCC1806 human breast

cancer cell line. Three independently repeated experiments were

performed with similar results. (C) Blocking of ERK phosphorylation

by PLX4032 and 45 cotreatment. Inhibition of ERK feedback

reactivation is sustained after 48 h compared to PLX4032 monotreat-

ment.

studies for the most active azaindole 45 showing that the

identiﬁed binding poses were stable.

4-(4-Nitrophenyl)-2-phenyl-1H-imidazole-5-carboxylic Acid (49).

48 (110.2 mg, 0.34 mmol, 1 equiv) was dissolved in MeOH (4 mL),

and LiOH (2 mL, 1 M) was added. The mixture was stirred for 3 h

under microwave irradiation at 90 °C. The progress of the reaction

was monitored by LCMS (3.3 min; M + H⁺= 309.95). After reaction

completion, the mixture was diluted with water and acidiﬁed with

HCl to a pH = 2−3. The precipitated product was collected by

suction ﬁltration. The ﬁlter cake was washed with suﬃcient water and

dried in vacuum to yield 49 in 95% (102 mg, 0.329 mmol). The crude

product was taken to the next step without further puriﬁcation.

4-(4-(4-Nitrophenyl)-2-phenyl-1H-imidazole-5-carboxamido)-

benzenesulfonic Acid (8). 49 (100.0 mg, 0.32 mmol, 1 equiv), p-

sulfanilic acid (56.0 mg, 0.32 mmol, 1 equiv), EDCI (43.7 mg, 0.32

mmol, 1 equiv), HOBT (62.0 mg, 0.32 mmol, 1 equiv), and DMAP

(4.0 mg, 0.032 mmol, 0.1 equiv) were stirred in dimethylformamide

(DMF) (3 mL) at room temperature for 72 h. The solution was

poured to 1 M HCl (5 mL) and extracted with EtOAc 2 × 10 mL.

The product was precipitated, and the solids were collected by suction

ﬁltration. The ﬁlter cake was washed with little EtOAc and dried in

vacuo, yielding 8 in 27% (40 mg, 0.08 mmol, yellow amorphous

solid). ¹H NMR (300 MHz, DMSO-d₆): δ 10.23 (s, 1H), 8.35 (d, J =

8.8 Hz, 2H), 8.25−8.13 (m, 4H), 7.76 (d, J = 8.5 Hz, 2H), 7.65−7.38

(m, 5H).

Synthesis of Pyrazolo-triazole 9. tert-Butyl 2-Cyano-3-(4-

nitrophenyl)-3-oxopropanoate (50). To a stirred suspension of

sodium hydride (60%, 400 mg, 10.0 mmol) in tetrahydrofuran (THF)

tert-butyl-cyano acetate (0.77 mL, 5.39 mmol) was added dropwise at

0 °C, and the reaction mixture was stirred for 30 min. After addition

of 4-nitro-benzoic acid chloride (1.00 g, 5.39 mmol), the reaction

mixture was allowed to warm to room temperature over a period of 6

h and was brought to pH = 3 by treatment with concentrated HCl.

After extraction with CH₂Cl₂(3 × 50 mL), the combined organic

layers were dried (MgSO₄) and ﬁltered, and the solvent was removed

under reduced pressure. tert-Butyl 2-cyano-3-(4-nitrophenyl)-3-

oxopropanoate (1.56 g, 100%) was obtained as a yellow amorphous

solid and used in the next step without further puriﬁcation. The crude

product tert-butyl 2-cyano-3-(4-nitrophenyl)-3-oxopropanoate (1.55

SHP2 activity was further cross-validated in advanced

cellular assays recapitulating metastatic tumor outgrowth and

drug-resistant tumor cells. In impedance and scatter assay

treatment of HPAF-II pancreatic cancer cells with 45 led to

almost full reversion of an HGF-induced scattering phenotype.

Compound 45 exhibited synergistic activity in combination

with BYL719 and PLX4032 and was able to inhibit cell

proliferation in drug-resistant VACO432 colorectal cancer and

HCC1806 breast cancer cells. Remarkably, the azaindole-based

inhibitor showed no signs of unspeciﬁc cytotoxicity and was

not active against a panel of representative kinases, distinguish-

ing it from the parent pyrazolone. In view of recently increased

interest toward SHP2 and the urge of ﬁnding adequate

therapeutic approaches for drug resistance and tumor relapse,

we believe that our compounds can serve as a platform for

further lead optimization and as a valuable chemical tool for

unraveling the (patho-)physiological role of SHP2 in cancer.

EXPERIMENTAL SECTION

■

Chemistry. All chemicals were purchased from commercial

suppliers: Fluorochem, Sigma-Aldrich, and Alfa Aesar and used as

received unless otherwise speciﬁed. NMR spectra were recorded at

either 295 K (300 MHz) or 300 K (600 MHz) at either Bruker AV

300 (300, 75 MHz) or Bruker AV 600 (600, 151 MHz)

spectrometers. Chemical shifts are reported in ppm (δ) referenced

to TMS (δ = 0.00 ppm), dimethylsulfoxide (DMSO) (2.50 ppm), and

CHCl₃(7.26 ppm). LC/MS analysis was performed on an Agilent

LC/MS 1260 analytical HPLC with DAD coupled to an Agilent 6120

single quadrupole mass spectrometer (ESI-SQ) equipped with a

Thermo Fisher Scientiﬁc Accucore C18 column, 2.1 × 30 mm, 2.6

μm. Method: ESI⁺, ﬂux: 0.8 mL/min, 5−95% CH₃CN in H₂O + 0.1%

FA, total runtime: 2.5 min. High-resolution mass spectra were

recorded on an Agilent 6220A accurate-mass time-of-ﬂight mass

spectrometer (ESI-TOF) with Agilent 1200 HPLC/DAD front-end.

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g, 5.34 mmol) was dissolved in AcCN (10 mL), treated with

triﬂuoroacetic acid (TFA) (5 mL) and pyridine (3 mL), and heated

under reﬂux for 22 h. The reaction solution was concentrated in

vacuum, the residue was diluted with H₂O and extracted with CH₂Cl₂

(4 × 20 mL). The combined organic layers were dried (MgSO₄) and

ﬁltered, and the solvent was removed under reduced pressure to give

50 (635 mg, 3.34 mmol, 62% over 2 steps) as a yellow amorphous

solid. Compound 50 was used in the next step without further

puriﬁcation.

product was puriﬁed by column chromatography to provide title

compound 54 (20 mg, 14% for 2 steps, yellow amorphous solid).

LCMS: m/z 440.1/442.0 (M + Na)⁺, R_t= 1.38 min. Alternatively,

collected tert-butyl 5-(4-((neopentyltrioxidaneyl)thio)phenyl)-3-(4-

nitrophenyl)-1H-indazole-1-carboxylate may be submitted to a Boc

deprotection with TFA in DCM for 2 h to provide 54 in quantitative

yield. ¹H NMR (300 MHz, CDCl₃): δ 8.50−8.36 (m, 2H), 8.29−8.14

(m, 3H), 8.06−7.98 (m, 2H), 7.90−7.67 (m, 4H), 3.77 (s, 2H), 0.96

(s, 11H). LCMS (ESI⁺): m/z 466.2 (M + H)⁺.

3-(4-Nitrophenyl)-1-phenyl-1H-pyrazol-5-amine (51). A mixture

of nitrile 50 (300 mg, 1.58 mmol) and phenylhydrazine hydrochloride

(228 mg, 1.58 mmol) in MeOH (2 mL) was heated at 120 °C for 45

min in a microwave. Filtering of the resulting precipitate and washing

with MeOH yield pyrazole 51 (484 mg, 1.53 mmol, 97%) as a

4-(1-(4-Methoxyphenyl)-3-(4-nitrophenyl)-1H-indazol-5-yl)-

benzenesulfonic acid (10) was prepared as follows: 54 (90 mg, 0.19

mmol, 1 equiv) was dissolved in dioxane (12 mL), and then trans-1,2-

cyclohexanediamine (21.6 mg, 0.096 mmol, 0.5 equiv), CuI (3.6 mg,

0.0096 mmol, 0.05 equiv), K₃PO₄(84 mg, 0.38 mmol, 2 equiv), and

4-methoxy-1-iodobenzene (9 mg, 0.038 mmol, 1.2 equiv) were added

sequentially. The reaction was heated to 100 °C and stirred for 24 h,

then solution was allowed to cool to room temperature, diluted with

EtOAc and H₂O, and extracted with EtOAc. The crude product was

dissolved in DMF (2 mL), TBAF (75 mg, 0.69 mmol, 4 equiv) was

added, and the reaction stirred for 2 h. The mixture was concentrated

in vacuo and puriﬁed by HPLC (30−100% ACN in water) to yield 4-

(1-(4-methoxyphenyl)-3-(4-nitrophenyl)-1H-indazol-5-yl)-

benzenesulfonic acid 10 (17 mg, 0.034 mmol, 18% for 2 steps, light-

1

colorless amorphous solid. H NMR (300 MHz, DMSO-d₆): δ 8.27

(d, J = 8.6 Hz, 2H, Ar−H), 8.05 (d, J = 8.7 Hz, 2H, Ar−H), 7.69 (d, J

= 7.9 Hz, 2H, Ar−H), 7.55 (t, J = 7.7 Hz, 2H, Ar−H), 7.41 (t, J = 7.3

Hz, 1H, Ar−H), 6.10 (s, 1H, Ar−H), 5.62 (s, 2H, −NH₂) ppm. ¹³C

NMR (75 MHz, DMSO-d₆): δ 149.3, 148.4, 146.8, 140.5, 139.4,

129.7, 127.3, 126.3, 124.5, 123.7, 88.3. HRMS (pos. ESI-TOF): m/z

calcd for C₁₅H₁₃N₄O₂281.1033 (M + H)⁺; found, 281.1032 (M +

H)⁺.

(E)-4-((5-Amino-3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)-

diazenyl)benzenesulfonic Acid (52). Phenylsulfonic acid (570 mg,

3.33 mmol) was dissolved in 2 M HCl (1 mL), and the solution was

cooled to 0 °C, followed by addition of NaNO₂(215 mg, 3.12 mmol)

in H₂O (2.5 mL). Amine 51 (583 mg, 2.0 mmol) and NaOAc (850

mg) were suspended in EtOH (11 mL) and added dropwise to the

reaction mixture. After warming to room temperature, the precipitate

was ﬁltered oﬀ and washed with H₂O. Drying in high vacuum gave 52

(366 mg, 0.75 mmol, 40%) as orange amorphous solid, which was

used directly in the next step without further puriﬁcation. LCMS (pos.

ESI-TOF): m/z calcd for C₂₁H₁₆N₆O₅S⁺465.10 (M + H)⁺; found,

465.03 (M + H)⁺.

4-(6-(4-Nitrophenyl)-4-phenylpyrazolo[3,4-d][1,2,3]triazol-

2(4H)-yl)benzenesulfonic Acid (9). Diazene 52 (200 mg, 0.39 mmol)

was dissolved in anhydrous DMF (8 mL), and CuSO₄·5H₂O (96 mg,

0.39 mmol) as well as K₂CO₃(320 mg, 2.32 mmol) was added

subsequently. After heating under reﬂux for 5 h, the solvent was

removed, and the solid was washed with H₂O and dried in high

vacuum to give 9 (140 mg, 0.30 mmol, 78%) as a brown oily solid. ¹H

NMR (600 MHz, DMSO-d₆): δ 8.51 (d, J = 8.9 Hz, 2H), 8.47 (d, J =

8.8 Hz, 2H), 8.30 (d, J = 8.6 Hz, 2H), 8.15 (d, J = 7.9 Hz, 2H), 7.91

(d, J = 8.7 Hz, 2H), 7.68 (t, J = 7.9 Hz, 2H), 7.39 (t, J = 7.4 Hz, 1H)

ppm. HRMS (neg. ESI-TOF): m/z calcd for C₂₁H₁₃N₆O₅S⁻461.0674

[M − H]⁻; found, 461.0682 [M − H]⁻.

Synthesis of Indazole 10. tert-Butyl 5-bromo-3-(4-nitrophenyl)-

1H-indazole-1-carboxylate (53) was prepared utilizing tert-butyl 5-

bromo-3-iodo-1H-indazole-1-carboxylate (29.1 mg, 0.305 mmol, 1

equiv), Pd(dppf)Cl₂·CH₂Cl₂(29 mg, 0.04 equiv), 4-nitrophenylbor-

onic acid pinacol ester (76.0 mg, 0.305 mmol, 1.0 equiv), and K₂CO₃

(1.22 mmol, 0168.9 g, 4 equiv) in degassed dioxane/water (1:3, 0.06

M) at 80 °C for 1 h. The solution was allowed to cool to room

temperature, diluted with EtOAc and H₂O, and extracted with EtOAc.

The combined organic layers were dried with MgSO₄and

concentrated in vacuo. The crude product was checked by LCMS

and contained product 53 (LCMS (ESI⁺): m/z 440.1/442.0 (M +

Na)⁺, R_t= 1.44 min), and boc-deprotected product (318.1/320.1 (M

+ H)⁺, R_t= 1.23 min), and the mixture was taken to the next step

without further puriﬁcation.

1

orange amorphous solid). H NMR (300 MHz, DMSO-d₆): δ 8.53−

8.37 (m, 4 H), 7.94−7.85 (m, 2H), 7.85−7.68 (m, 6H), 3.92−3.84

(m, 2H), 3.88 (s, 3H). HRMS (pos. ESI-TOF): m/z calcd for

C₂₆H₁₉N₃O₆S, 502.1067 [M + H]⁺; found, 502.1054 [M + H]⁺.

Synthesis of the Compounds 11 and 17−46. General

Procedure A: Cham−Lam Coupling of Azaindoles. To a stirred

suspension of 5-bromo-3-iodo-1H-pyrrolo[2,3-b]pyridine, boronic

acid (2.5−3.0 equiv) and Cu(OAc)₂(2.5−3.0 equiv) in DCM

(0.026 M), DIPEA (2.5−4.5 equiv) was added in one portion. After

stirring for 48 h at room temperature, the mixture was ﬁltered and

concentrated in vacuo. The crude product was puriﬁed by column

chromatography (SiO₂, cyclohexane/DCM) to yield N-arylated 5-

bromo-3-iodo azaindoles.

5-Bromo-3-iodo-1-(4-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridine

(55). General procedure A was applied using 5-bromo-3-iodo-1H-

pyrrolo[2,3-b]pyridine (1.0 g, 3.1 mmol), (4-methoxyphenyl)boronic

acid (1.18 g, 7.75 mmol), Cu(OAc)₂(1.41 g, 7.75 mmol) and DIPEA

(1.4 mL, 7.75 mmol). The crude product was puriﬁed by column

chromatography (SiO₂, cyclohexane/DCM, 26−50% DCM) to give

1

55 in 21% yield (270.0 mg, 0.63 mmol, white amorphous solid). H

NMR (300 MHz, CDCl₃): δ 8.39−8.32 (m, 1H), 7.94−7.88 (m, 1H),

7.56−7.47 (m, 3H), 7.08−7.00 (d, J = 8.9 Hz, 2H), 3.87 (s, 3H). ¹³C

NMR (75 MHz, CDCl₃): δ 158.9, 145.8, 145.4, 133.7, 131.8, 130.3,

125.9 (2C), 125.4, 114.9 (2C), 113.3, 55.8, 54.5. HRMS (pos. ESI-

TOF): m/z calcd for C₁₄H₁₁BrIN₂O, 428.9094/430.9075 [M + H]⁺;

found, 428.9072/430.9054 [M + H]⁺.

5-Bromo-3-iodo-1-isopropyl-1H-pyrrolo[2,3-b]pyridine (56).

NaH (60% in oil, 84 mg, 1.36 mmol) was added to a stirred solution

of 5-bromo-3-iodo-1H-pyrrolo[2,3-b]pyridine (400 mg, 1.24 mmol, 1

equiv) in DMF (50 mL) at 0 °C. After stirring of the reaction mixture

for 5 min, 2-bromopropane (174 μL, 1.86 mmol, 1.5 equiv) was

added. The mixture was stirred for 1 h at room temperature and then

quenched with 50 mL water. The water layer was extracted three

times with 50 mL of DCM, and the combined organic layers were

dried over MgSO₄and concentrated in vacuo. The crude product was

extracted two times with 150 mL of hot Et₂O. The organic layers were

combined, ﬁltered, and evaporated to dryness to give 56 in 88% yield

(400 mg, 1.096 mmol). The compound was used directly for the next

reaction step without further puriﬁcation. LCMS (pos. ESI-TOF): m/

z calcd for C₁₀H₁₁BrIN₂, 364.9/366.9 [M + H]⁺; found, 364.8/366.8

[M + H]⁺.

5-(4-((Neopentyltrioxidaneyl)thio)phenyl)-3-(4-nitrophenyl)-1H-

indazole (54) was prepared utilizing 53 (ca. 0.305 mmol, 1 equiv,

crude, mixture of boc-protected and deprotected azaindole), Pd-

(dppf)Cl₂·CH₂Cl₂(30 mg, 0.038 mmol, 0.12 equiv), neopentyl 4-

(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzenesulfonate (134

mg, 0.380 mmol, 1.24 equiv), and K₂CO₃(1.21 mmol, 210 mg, 5

equiv) in degassed dioxane/water (1:3, 0.06 M) at 80 °C for 1 h. The

solution was allowed to cool to room temperature, diluted with

EtOAc and H₂O, and extracted with EtOAc. The combined organic

layers were dried with MgSO₄and concentrated in vacuo. The crude

5-Bromo-3-iodo-1-tosyl-1H-pyrrolo[2,3-b]pyridine (57). To a

stirred solution of 5-bromo-3-iodo-1H-pyrrolo[2,3-b]pyridine (4.00

g, 12.4 mmol) in DMF was added NaH (60%, 573 mg, 14.3 mmol) in

small portions. Then, TosCl was added, and the solution was stirred

at room temperature for 3 h. After addition of H₂O, the precipitate

was ﬁltered oﬀ, the water layer was extracted three times with 50 mL

K

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of DCM, and the combined organic layers were dried over MgSO₄

and concentrated in vacuo. The crude product was puriﬁed by column

chromatography (SiO₂, cyclohexane/DCM, 26−50% DCM) to give

57 (3.23 g, 54%, white amorphous solid). ¹H NMR (300 MHz,

DMSO-d₆): δ 8.55−8.45 (1H, m), 8.22 (s, 1H), 8.04−7.95 (m, 3H),

7.46−7.34 (m, 2H), 2.34 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆):

δ 146.1, 146.0, 144.4, 133.9, 132.4, 131.9, 130.2 (2C), 127.7 (2C),

126.6, 115.7, 63.1, 21.1. HRMS (pos. ESI-TOF): m/z calcd for

C₁₄H₁₁BrIN₂O₂S, 476.8764/478.8744 [M + H]⁺; found, 476.8743/

478.8722 [M + H]⁺.

1-Benzyl-5-bromo-3-iodo-1H-pyrrolo[2,3-b]pyridine (58). NaH

(60% in oil, 180 mg, 4.5 mmol, 3 equiv) was added to a stirred

solution of 5-bromo-3-iodo-1H-pyrrolo[2,3-b]pyridine (500 mg, 1.5

mmol, 1 equiv) in DMF (50 mL). After stirring of the reaction

mixture for 30 min, benzyl bromide (267 μL, 2.25 mmol, 1.5 equiv)

was added. The mixture was stirred for 12 h at room temperature and

then quenched with water. The water layer was extracted three times

with DCM, and the combined organic layers were dried over Na₂SO₄

and concentrated in vacuo. The crude product 58 (650 mg) was pure

enough as judged by NMR and used for the next reaction step

without further puriﬁcation.

5-Bromo-3-iodo-1-(4-methoxybenzyl)-1H-pyrrolo[2,3-b]pyridine

(59). NaH (60% in oil, 84 mg, 1.36 mmol) was added to a stirred

solution of 5-bromo-3-iodo-1H-pyrrolo[2,3-b]pyridine (400 mg, 1.24

mmol, 1 equiv) in DMF (50 mL) at 0 °C. After stirring of the reaction

mixture for 5 min, 4-methoxylbenzyl bromide (267 μL, 1.86 mmol,

1.5 equiv) was added. The mixture was stirred for 1 h at room

temperature and then quenched with 50 mL water. The water layer

was extracted three times with 50 mL of DCM, and the combined

organic layers were dried over MgSO₄and concentrated in vacuo. The

crude was extracted two times with 150 mL of Et₂O under reﬂux. The

hot solutions were combined, ﬁltered, and evaporated to dryness. The

residue was triturated with 8 mL of Et₂O, sonicated for 5 min, ﬁltered,

and dried in vacuo to give 59 in 49% yield (292 mg, 0.659 mmol). The

compound was used directly for the next step without further

puriﬁcation LCMS (pos. ESI-TOF): m/z calcd for C₁₅H₁₃BrIN₂O;

442.9/444.9 [M + H]⁺; found, 442.9/444.8 [M + H]⁺.

portion. After stirring for 48 h at room temperature, the mixture was

ﬁltered and concentrated in vacuo. The crude product was puriﬁed by

column chromatography (SiO₂, cyclohexane/DCM, 26−50% DCM)

to give methyl 3-iodo-1-(4-methoxyphenyl)-1H-pyrrolo[2,3-b]-

pyridine-5-carboxylate 62 in 20% yield (588 mg, 1.44 mmol, white

1

amorphous solid). H NMR (300 MHz, CDCl₃): δ 9.02−8.95 (m,

1H), 8.45−8.37 (m, 1H), 7.59 (s, 1H), 7.58−7.50 (m, 2H), 7.13−

7.69 (m, 2H), 3.97 (s, 3H), 3.85 (s, 3H). LCMS (pos. ESI-TOF): m/

z calcd for C₁₆H₁₄IN₂O₃, 409.0 [M + H]⁺; found, 408.9 [M + H]⁺.

5-Bromo-3-iodo-1-(4-methoxyphenyl)-1H-pyrazolo[3,4-b]-

pyridine (63). 5-Bromo-3-iodo-1H-pyrazolo[3,4-b]pyridine (2.1 g, 6.5

mmol), (4-methoxyphenyl)boronic acid (1.10 g, 7.2 mmol, 1.2

equiv), and Cu(OAc)₂(1.17 g, 6.5 mmol, 1 equiv) were mixed in

MeOH (170 mL). After stirring for 48 h at room temperature, the

mixture was ﬁltered and concentrated in vacuo. The crude product

was puriﬁed by column chromatography (SiO₂, cyclohexane/DCM

74:26 → 50:50) to obtain 63 (590.0 mg, 0.13 mmol, 21%, yellowish

1

amorphous solid). H NMR (300 MHz, CDCl₃): δ 8.44−8.36 (m,

1H), 7.96−7.92 (m, 1H), 7.60−7.50 (m, 3H), 7.11−7.03 (d, J = 8.9

Hz, 2H), 3.88 (s, 3H). LCMS (pos. ESI-TOF): m/z calcd for

C₁₃H₁₀BrIN₃O, 429.9/431.9 [M + H]⁺; found, 429.8/431.8 [M +

H]⁺.

5-Bromo-3-iodo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-

b]pyridine (64). 5-Bromo-3-iodo-1H-pyrazolo[3,4-b]pyridine (500.0

mg, 1.54 mmol, 1 equiv) was dissolved in EtOAc (9 mL), and then

catalytic amounts of p-TSA and DHP (260.0 mg, 2.09 mmol, 2 equiv)

were added sequentially at room temperature. The mixture was

heated to 60 °C and stirred for 12 h. The solution was allowed to cool

and concentrated in vacuo. The crude product was puriﬁed by column

chromatography (SiO₂, cyclohexane/DCM 74:26 → 50:50) to give 5-

bromo-3-iodo-1H-pyrazolo[3,4-b]pyridine 64 in 80% yield (500 mg,

1.23 mmol, yellowish oily solid). ¹H NMR (300 MHz, CDCl₃): δ 8.62

(d, J = 2.1 Hz, 1H), 7.98 (d, J = 2.1 Hz, 1H), 6.05 (dd, J = 10.5, 2.5

Hz, 1H), 4.17−4.02 (m, 1H), 3.85−3.65 (m, 1H), 3.57−3.32 (m,

1H), 2.73−2.50 (m, 1H), 2.00 (ddd, J = 15.6, 5.0, 2.8 Hz, 1H), 1.67−

1.56 (m, 3H). LCMS (pos. ESI-TOF): m/z calcd for C₁₃H₁₀BrIN₃O,

407.9/409.9 [M + H]⁺; found, 407.9/409.9 [M + H]⁺.

General Procedure B: Suzuki Coupling Using Pd(PPh₃)₄. Pd-

(PPh₃)₄(0.075 equiv) was added to a stirred solution of heterocyclic

halide (1 equiv), boronic acid or boronate ester (1.2 equiv), and

NaHCO₃(3 equiv) in degassed H₂O/MeCN (1:3, 0.02 M) at 100

°C. The resulting reaction mixture was stirred for 12−16 h under N₂.

The solution was allowed to cool to room temperature, diluted with

DCM and H₂O, and extracted with DCM. The combined organic

layers were dried with Na₂SO₄or MgSO₄and concentrated in vacuo.

The crude product was puriﬁed by column chromatography to yield

the substituted product or recrystallization and silica ﬁltration to yield

the substituted product or used directly as a crude for the further step.

General Procedure C: Suzuki Coupling Using Pd(dppf)Cl₂.

Heterocyclic halide (1 equiv), Pd(dppf)Cl₂(0.025 equiv), boronic

acid or boronate ester (1.0−1.2 equiv), and K₂CO₃(3 equiv) were

dissolved in degassed H₂O/dioxane (1:3, 0.06 M), and the resulting

reaction mixture was stirred for 3−12 h at 80 °C under N₂. The

solution was allowed to cool to room temperature, diluted with DCM

and H₂O, and extracted with DCM. The combined organic layers

were dried with Na₂SO₄or MgSO₄and concentrated in vacuo. The

crude product was puriﬁed by column chromatography or

recrystallization and silica ﬁltration to yield the product. In some

cases, the product was pure enough and used without further

puriﬁcation in the subsequent reaction step.

5-Bromo-(1-(4-methoxyphenyl)-3-(4-nitrophenyl)-1H-pyrrolo-

[2,3-b]pyridine) (65). General B was applied using halide 55 (770 mg,

1.795 mmol), 4,4,5,5-tetramethyl-2-(4-nitrophenyl)-1,3-dioxolane

(541 mg, 2.153 mmol), NaHCO₃(453.7 mg, 5.38 mmol), and

Pd(PPh₃)₄(156 mg, 0.134 mmol) in 70 mL of 3:1 MeCN/H₂O. The

reaction mixture was diluted with DCM (25 mL) and water (25 mL).

The aqueous phase was extracted three times with DCM (25 mL).

The organic phases were combined, dried over MgSO₄, and

evaporated to dryness. The crude residue was boiled with 10 mL of

MeOH, sonicated, and ﬁltered to yield crude 5-bromo-1-(4-

5-Bromo-3-iodo-1-(3-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridine

(60). General procedure A was applied using 5-bromo-3-iodo-1H-

pyrrolo[2,3-b]pyridine (2.0 g, 6.2 mmol), (3-methoxyphenyl)boronic

acid (2.36 g, 15.5 mmol), Cu(OAc)₂(2.8 g, 15.5 mmol), and DIPEA

(2.72 mL, 15.5 mmol). The crude product was puriﬁed by column

chromatography (SiO₂, cyclohexane/DCM, 26−50% DCM) to give

5-bromo-3-iodo-1-(3-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridine 60

in 21% yield (535 mg, 1.2 mmol, white amorphous solid). ¹H

NMR (300 MHz, CDCl₃): δ 8.43−8.38 (m, 1H), 7.97−7.89 (m, 1H),

7.63 (s, 1H), 7.43 (d, J = 8.1 Hz, 1H), 7.28−7.16 (m, 2H), 6.94 (ddd,

J = 8.4, 2.5, 0.9 Hz, 1H), 3.89 (s, 3H). ¹³C NMR (75 MHz, CDCl₃): δ

160.3, 145.5, 145.3, 138.3, 133.2, 131.7, 130.2, 125.6, 116.0, 113.5,

112.7, 110.0, 55.5, 55.3. HRMS (pos. ESI): m/z calcd for

C₁₄H₁₁BrIN₂O, 428.9094/430.9075 [M + H]⁺; found, 428.9116/

430.9098 [M + H]⁺.

5-Bromo-3-iodo-1-phenyl-1H-pyrrolo[2,3-b]pyridine (61). Gen-

eral procedure A was adapted using 5-bromo-3-iodo-1H-pyrrolo[2,3-

b]pyridine (1.5 g, 4.6 mmol), phenylboronic acid (1.7 g, 13.8 mmol, 3

equiv), Cu(OAc)₂(2.5 g, 13.8 mmol, 3 equiv), and DIPEA (3.6 mL,

22.5 mmol, 4.5 equiv) that was added in one portion. After stirring for

48 h at room temperature, the mixture was ﬁltered and concentrated

in vacuo. The crude product was puriﬁed by column chromatography

(SiO₂, cyclohexane/DCM, 26−50% DCM) to give 61 in 18% yield

1

(340 mg, 0.85 mmol, beige amorphous solid). H NMR (300 MHz,

CDCl₃): δ 8.42−8.32 (m, 1H), 7.95−7.89 (m, 1H), 7.74−7.62 (m,

2H), 7.62 (s, 1H), 7.57−7.45 (m, 2H), 7.46−7.34 (m, 1H). LCMS

(pos. ESI-TOF): m/z calcd for C₁₃H₉BrIN₂, 398.9/400.9 [M + H]⁺;

found, 398.8/400.8 [M + H]⁺.

Methyl 3-Iodo-1-(4-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridine-

5-carboxylate (62). General procedure A was applied using methyl

3-iodo-1H-pyrrolo[2,3-b]pyridine-5-carboxylate (2.22 g, 7.3 mmol),

p-methoxyphenylboronic acid (2.8 g, 18.0 mmol), Cu(OAc)₂(3.34 g,

18 mmol), and DIPEA (3.6 mL, 18 mmol) that was added in one

L

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methoxyphenyl)-3-(4-nitrophenyl)-1H-pyrrolo[2,3-b]pyridine 65

(300 mg, 0.131 mmol, amorphous orange solid). The compound

was directly used in the next reaction step without further puriﬁcation.

LCMS (ESI): m/z calcd for C₂₀H₁₅BrN₃O₃, 424.0/426.0[M + H]⁺;

found, 423.9/425.9 [M + H]⁺.

CDCl₃): δ 158.6, 148.4, 146.7, 146.3, 144.5, 130.9, 130.3, 127.8,

126.3, 125.7 (2C), 121.0, 120.7, 116.0, 114.8 (2C), 112.8, 109.1,

107.9, 101.3, 55.7. LCMS (pos. ESI-TOF): m/z calcd for

C₂₁H₁₆BrN₂O₃, 423.0/425.0 [M + H]⁺; found, 423.07425.0 [M +

H]⁺.

5-(5-Bromo-1-(4-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-

benzo[c][1,2,5]oxadiazole (66). General procedure B was applied

using halide 55 (300 mg, 0.7 mmol), benzo[c][1,2,5]oxadiazole-5-

boronic acid pinacol ester (206 mg, 0.84 mmol), NaHCO₃(176 mg,

2.1 mmol), Pd(PPh₃)₄(61 mg, 0.05 mmol), and 35 mL of 3:1

MeCN/H₂O. Puriﬁcation of the crude by ﬂash column chromatog-

raphy (SiO₂, 0−30% ethyl acetate in cyclohexane) yielded 5-(5-

bromo-1-(4-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzo-

[c][1,2,5]oxadiazole 66 in 51% yield (150 mg, 0.36 mmol, yellow

4-((5-(4-Bromophenyl)-1-(4-methoxyphenyl)-1H-pyrrolo[2,3-b]-

pyridin-3-yl)benzonitrile) (71). General procedure C was applied

using halide 55 (300 mg, 0.69 mmol), 4-cyanophenylboronic acid

(112 mg, 0.77 mmol), K₂CO₃(290 mg, 2.1 mmol), and Pd(dppf)Cl₂

(13 mg, 0.017 mmol) in 13 mL of 3:1 dioxane/H₂O. After extraction

of the water layer with DCM and drying with Na₂SO₄, the solution

was concentrated under vacuum until the formation of a precipitate.

The precipitate was collected by ﬁltration and dried under vacuum to

give 4-(5-(4-bromophenyl)-1-(4-methoxyphenyl)-1H-pyrrolo[2,3-b]-

pyridin-3-yl)benzonitrile 71 in 28% yield (78 mg, 0.19 mmol, white

amorphous solid). This solid was taken to the next reaction step

without further puriﬁcation as NMR and LCMS analysis revealed that

the compound was pure enough.

5-(5-Bromo-1-isopropyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzo[c]-

[1,2,5]oxadiazole (72). General procedure C was applied using halide

56 (250 mg, 0.564 mmol), benzo[c][1,2,5]oxadiazole-5-boronic acid

(167 mg, 0.677 mmol), K₂CO₃(233 mg, 1.692 mmol), and

Pd(dppf)Cl₂(10.3 mg, 0.014 mmol) in 10 mL of 3:1 dioxane/

H₂O, and the reaction was stirred at 55 °C. The mixture was diluted

with DCM (30 mL) and extracted two times with 10% K₂CO₃

solution, followed by brine. The combined organic phases were dried

over MgSO₄, ﬁltered, and evaporated. The residue was stirred with 10

mL of Et₂O under sonication for 5 min. The supernatant was removed

(repeated twice), and the remaining solid was dried in vacuo.

Puriﬁcation of the crude product by ﬂash column chromatography

(SiO₂, 0% to 40% ethyl acetate in cyclohexane, slow gradient)

provided 5-(5-bromo-1-isopropyl-1H-pyrrolo[2,3-b]pyridin-3-yl)-

benzo[c][1,2,5]oxadiazole 72 in 71% yield (280 mg, 0.784 mmol,

yellow amorphous solid). LCMS (pos. ESI-TOF): m/z calcd for

C₁₆H₁₄BrN₄O, 357.03/359.03 [M + H]⁺; found, 357.0/359.0 [M +

H]⁺.

1

amorphous solid). H NMR (300 MHz, CDCl₃): δ 8.49−8.43 (m,

2H), 8.02 (s, 1H), 7.94 (dd, J = 9.5 Hz, 1.0, 1H), 7.83 (s, 1H), 7.74

(dd, J = 9.4, 1.4 Hz, 1H), 7.66−7.54 (m, 2H), 7.15−7.06 (m, 2H),

3.92 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆): δ 158.2, 149.7, 148.1,

146.1, 144.3, 136.9, 133.7, 131.2, 129.9, 129.5, 125.6 (2C), 119.7,

116.4, 114.4, 113.4 (2C), 112.6, 109.3, 55.5. HRMS (pos. ESI-TOF):

m/z calcd for C₂₀H₁₄BrN₄O₂, 421.0295/423.0277 [M + H]⁺; found,

421.0272/423.0252 [M + H]⁺.

5-Bromo-1-(4-methoxyphenyl)-3-(4-(triﬂuoromethyl)phenyl)-

1H-pyrrolo[2,3-b]pyridine (67). General procedure C was applied

using halide 55 (300 mg, 0.69 mmol), 4-triﬂuorohenylboronic acid

(146 mg, 0.77 mmol), K₂CO₃(290 mg, 2.1 mmol), and Pd(dppf)Cl₂

(13 mg, 0.017 mmol) in 13 mL of 3:1 dioxane/H₂O. Puriﬁcation of

the crude product by ﬂash column chromatography (SiO₂, 0−40%

ethyl acetate in cyclohexane, slow gradient) provided 5-bromo-1-(4-

methoxyphenyl)-3-(4-(triﬂuoromethyl)phenyl)-1H-pyrrolo[2,3-b]-

pyridine 67 in 39% yield (122 mg, 0.27 mmol, white amorphous

1

solid). H NMR (300 MHz, CDCl₃): δ 8.47−8.39 (m, 1H), 8.38−

8.29 (m, 1H), 7.76−7.70 (m, 4H), 7.62 (s, 1H), 7.60 (d, J = 8.5 Hz,

2H), 7.07 (d, J = 8.3 Hz, 2H), 3.88 (s, 3H). LCMS (pos. ESI-TOF):

m/z calcd for C₂₁H₁₅BrF₃N₂O, 447.0/449.0 [M + H]⁺; 447.1/449.1

[M + H]⁺.

5-Bromo-1-(4-methoxyphenyl)-3-(pyridin-4-yl)-1H-pyrrolo[2,3-

b]pyridine (68). General procedure C was applied using halide 55

(280 mg, 0.65 mmol), 3,4-pyridinylboronic acid (79 mg, 0.65 mmol),

K₂CO₃(269 mg, 1.95 mmol), and Pd(dppf)Cl₂(11 mg, 0.015 mmol)

in 8 mL of 3:1 dioxane/H₂O. Puriﬁcation of the crude product by

ﬂash column chromatography (SiO₂, 0−100% EtOAc in cyclohexane)

yielded 5-bromo-1-(4-methoxyphenyl)-3-(pyridin-4-yl)-1H-pyrrolo-

[2,3-b]pyridine 68 in 34% yield (85 mg, 0.22 mmol, white amorphous

5-(5-Bromo-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzo[c]-

[1,2,5]oxadiazole (73). General procedure B was applied using halide

57 (1.00 g, 2.10 mmol), benzo[c][1,2,5]oxadiazole-5-boronic acid

pinacol ester (619 mg, 2.520 mmol), NaHCO₃(528 mg, 6.29 mmol),

Pd(PPh₃)₄(174 mg, 0.151 mmol) in 100 mL of MeCN, and 33.3 mL

of H₂O. Puriﬁcation of the crude product by ﬂash column

chromatography (SiO₂, 0−100% ethyl acetate in cyclohexane) yielded

5-(5-bromo-1-tosyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzo[c][1,2,5]-

oxadiazole 73 in 69% yield (630 mg, 0.13 mmol, yellow amorphous

1

solid). H NMR (300 MHz, CDCl₃): δ 8.79−8.65 (m, 2H), 8.54−

1

8.37 (m, 2H), 7.82 (s, 1H), 7.69−7.53 (m, 4H), 7.20−7.01 (m, 2H),

3.89 (s, 3H). LCMS (ESI): m/z calcd for C₁₉H₁₅BrN₃O, 380.0/382.0

[M + H]⁺; found, 380.0/382.0 [M + H]⁺.

solid). H NMR (300 MHz, CDCl₃): δ 8.57−8.52 (m, 1H), 8.34−

8.27 (m, 1H), 8.18−8.05 (m, 3H), 7.98 (d, J = 9.6 Hz, 2H), 7.72−

7.61 (m, 1H), 7.34 (d, J = 8.2 Hz, 2H), 2.41 (s, 3H). ¹³C NMR (75

MHz, CDCl₃): δ 149.5, 148.5, 146.7, 146.3, 145.8, 135.5, 134.6,

132.2, 131.1, 129.9 (2C), 128.6 (2C), 126.0, 122.2, 117.8, 117.3,

116.1, 112.8, 21.8. HRMS (pos. ESI-TOF): m/z calcd for

C₂₀H₁₄BrN₄O₃S, 468.9965 [M + H]⁺; found, 468.9936 [M + H]⁺.

5-(1-Benzyl-5-bromo-1H-pyrrolo[2,3-b]pyridin-3-yl)benzo[c]-

[1,2,5]oxadiazole (74). General procedure C was applied using halide

58 (334 mg, 0.809 mmol), benzo[c][1,2,5]oxadiazole-5-boronic acid

(238 mg, 0.970 mmol), K₂CO₃(336 mg, 2.43 mmol), and

Pd(dppf)Cl₂(15 mg, 0.020 mmol) in 13 mL of 3:1 dioxane/H₂O.

The mixture was diluted with DCM and extracted two times with

water, followed by brine. The combined organic phases were dried

over MgSO₄, ﬁltered, and evaporated. The residue was stirred with

Et₂O under sonication for 5 min. The supernatant was removed, and

the remaining solid was dried in vacuo to give 5-(1-benzyl-5-bromo-

1H-pyrrolo[2,3-b]pyridin-3-yl)benzo[c][1,2,5]oxadiazole 74 in 57%

yield (188 mg, 0.464 mmol, brown oily solid). The product was used

in the next reaction step without further puriﬁcation. LCMS (pos.

ESI-TOF): m/z calcd for C₂₀H₁₄BrN₄O, 405.03/407.03 [M + H]⁺;

found, 404.9/406.9 [M + H]⁺.

5-Bromo-1-(4-methoxyphenyl)-3-(pyrimidin-5-yl)-1H-pyrrolo-

[2,3-b]pyridine (69). General procedure C was applied using halide

55 (200 mg, 0.46 mmol), 3-5,5-pyrimidinboronic acid (57 mg, 0.46

mmol), K₂CO₃(192 mg, 1.3 mmol), and Pd(dppf)Cl₂(9 mg, 0.012

mmol) in 6 mL of 6:2 dioxane/H₂O. Puriﬁcation of the crude product

by ﬂash column chromatography (SiO₂, 0−100% EtOAc in

cyclohexane) yielded 5-bromo-1-(4-methoxyphenyl)-3-(pyrimidin-5-

yl)-1H-pyrrolo[2,3-b]pyridine in an inseparable mixture with

triphenylphosphine oxide. The impure product 69 was used for the

next reaction step without further puriﬁcation.

3-(Benzo[d][1,3]dioxol-5-yl)-5-bromo-1-(4-methoxyphenyl)-1H-

pyrrolo[2,3-b]pyridine (70). General procedure B was applied using

halide 55 (300 mg, 0.69 mmol), benzo[d][1,3]dioxol-5-ylboronic acid

(138 mg, 0.82 mmol), NaHCO₃(174 mg, 2.0 mmol), and Pd(PPh₃)₄

(60 mg, 0.05 mmol) in 40 mL of 3:1 MeCN/H₂O. Puriﬁcation of the

crude product by ﬂash column chromatography (SiO₂, 0−40% ethyl

acetate in cyclohexane, slow gradient) yielded 3-(benzo[d][1,3]-

dioxol-5-yl)-5-bromo-1-(4-methoxyphenyl)-1H-pyrrolo[2,3-b]-

pyridine 70 in 42% yield (124 mg, 0.29 mmol, white amorphous

1

solid). H NMR (300 MHz, CDCl₃): δ 8.47−8.38 (m, 1H), 8.38−

5-(5-Bromo-1-(4-methoxybenzyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-

benzo[c][1,2,5]oxadiazole (75). General procedure C was applied

using halide 59 (250 mg, 0.564 mmol), benzo[c][1,2,5]oxadiazole-5-

8.27 (m, 1H), 7.68−7.60 (m, 2H), 7.53 (s, 1H), 7.16−7.03 (m, 4H),

7.01−6.89 (m, 1H), 6.02 (s, 2H), 3.87 (s, 3H), ¹³C NMR (75 MHz,

M

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boronic acid (167 mg, 0.677 mmol), K₂CO₃(233 mg, 1.692 mmol),

and Pd(dppf)Cl₂(10.3 mg, 0.014 mmol) in 10 mL of 3:1 dioxane/

H₂O. The mixture was diluted with DCM (30 mL) and extracted two

times with 10% K₂CO₃solution, followed by brine. The combined

organic phases were dried over MgSO₄, ﬁltered, and evaporated. The

residue was stirred with 10 mL of Et₂O under sonication for 5 min.

The supernatant was removed (repeated twice), and the remaining

solid was dried in vacuo to give 5-(5-bromo-1-(4-methoxybenzyl)-1H-

pyrrolo[2,3-b]pyridin-3-yl)benzo[c][1,2,5]oxadiazole 75 in quantita-

tive yield (272 mg, 0.625 mmol, brown amorphous solid). The

product was taken for the next step without further puriﬁcation.

LCMS (pos. ESI-TOF): m/z calcd for C₂₁H₁₆BrN₄O₂, 435.04/437.04

[M + H]⁺; found, 434.9/437.0 [M + H]⁺.

5-(5-Bromo-1-(3-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-

benzo[c][1,2,5]oxadiazole (76). General procedure C was applied

using halide 60 (535 mg, 1.24 mmol), benzo[c][1,2,5]oxadiazole-5-

boronic acid pinacol ester (307 mg, 1.24 mmol), K₂CO₃(513 mg, 2.1

mmol), and Pd(dppf)Cl₂(18 mg, 0.05 mmol) in 24 mL of 3:1

dioxane/H₂O. Puriﬁcation of the crude product by ﬂash column

chromatography (SiO₂, 0−40% ethyl acetate in hexane) yielded 5-(5-

bromo-1-(3-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzo-

[c][1,2,5]oxadiazole 76 in 43% yield (210 mg, 0.53 mmol, yellow

amorphous solid). ¹H NMR (300 MHz, CDCl₃): δ 8.49 (s, 1H), 8.45

(s, 1H), 8.02 (s, 1H), 7.95 (d, J = 9.3 Hz, 1H), 7.87 (s, 1H), 7.75 (d, J

= 9.3 Hz, 1H), 7.53−7.42 (m, 1H), 7.38−7.27 (m, 2H), 6.97 (d, J =

8.3 Hz, 1H), 3.90 (s, 3H). ¹³C NMR (75 MHz, CDCl₃): δ 160.6,

149.9, 148.5, 146.6, 145.5, 138.4, 136.9, 132.7, 130.6, 130.5, 128.3,

120.7, 117.4, 116.5, 114.4, 114.0, 113.1, 111.0, 110.7, 55.7. LCMS

(pos. ESI-TOF): m/z calcd for C₂₀H₁₄BrN₄O₂, 421.0/423.0 [M +

H]⁺; found, 420.9/423.1 [M + H]⁺.

acid (127 mg, 0.71 mmol), K₂CO₃(293 mg, 2.11 mmol), and 13 mg

(0.017 mmol) of Pd(dppf)Cl₂, 13 mL of 3:1 dioxane/H₂O.

Puriﬁcation of the crude product by ﬂash column chromatography

(SiO₂, 0−50% EtOAc in cyclohexane) yielded methyl 4-(5-bromo-1-

(4-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoate 80 in

1

22% yield (65 mg, 0.15 mmol, white amorphous solid). H NMR

(300 MHz, CDCl₃): δ 8.45−8.34 (m, 2H), 8.15 (d, J = 8.2 Hz, 2H),

7.77−7.66 (m, 3H), 7.61 (d, J = 8.4 Hz, 2H), 7.07 (d, J = 8.2 Hz,

2H), 3.96 (s, 3H), 3.88 (s, 3H). LCMS (ESI): m/z calcd for

C₂₂H₁₈BrN₂O₃, 437.1/439.1 [M + H]⁺; found, 436.9/438.9 [M +

H]⁺.

6-(5-Bromo-1-(4-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-

isobenzofuran-1(3H)-one (81). General procedure B coupling was

applied using 55 (200 mg, 0.47 mmol), 6-(4,4,5,5-tetramethyl-1,3,2-

dioxaborolan-2-yl)isobenzofuran-1(3H)-one (145 mg,0.56 mmol),

NaHCO₃(117 mg, 1.4 mmol) Pd(PPh₃)₄(38 mg, 0.034 mmol),

and 35 mL of 3:1 MeCN/H₂O. The crude mixture was cooled to

room temperature and ﬁltrated. The resulted ﬁltrate was dissolved in

DCM and extracted with water. The organic phase was concentrated

in vacuo to provide crude 6-(5-bromo-1-(4-methoxyphenyl)-1H-

pyrrolo[2,3-b]pyridin-3-yl)isobenzofuran-1(3H)-one 81 in 25%

yield. Because of a rapid hydrolysis of the lactone, the crude product

was taken to the next step without further puriﬁcation.

7-(5-Bromo-1-(4-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-

2H-chromen-2-one (82). General procedure C was applied using

halide 55 (159.6 mg, 0.373 mmol), 7-(4,4,5,5-tetramethyl-1,3,2-

dioxaborolan-2-yl)-2H-chromen-2-one (121.8 mg, 0.447 mmol),

K₂CO₃(154.6 mg, 1.119 mmol), and Pd(dppf)Cl₂(6.8 mg, 0.009

mmol) in 7.5 mL of 3:1 dioxane/H₂O, and the reaction was stirred at

55 °C. The mixture was diluted with DCM (30 mL) and extracted

two times with 5% K₂CO₃solution, followed by brine. The combined

organic phases were dried over MgSO₄, ﬁltered, and evaporated.

Puriﬁcation of the crude product by ﬂash column chromatography

(SiO₂, 0−50%) THF in DCM provided 7-(5-bromo-1-(4-methox-

yphenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-2H-chromen-2-one 82 in

30% yield (50 mg, 0.111 mmol, yellow amorphous solid). LCMS

(pos. ESI-TOF): m/z calcd for C₂₃H₁₆BrN₂O₃, 447.03/449.02 [M +

H]⁺; found, 446.9/449.0 [M + H]⁺.

5-(5-Bromo-1-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzo[c]-

[1,2,5]oxadiazole (77). General procedure C was applied using halide

61 (200 mg, 0.5 mmol), benzo[c][1,2,5]oxadiazole-5-boronic acid

pinacol ester (123 mg, 0.5 mmol), K₂CO₃(207 mg, 1.5 mmol), and

Pd(dppf)Cl₂(9.2 mg, 0.0125 mmol) in 10 mL of 3:1 dioxane/H₂O.

The obtained crude product (117 mg) was analyzed by LCMS and

NMR and was pure enough for the next reaction step without further

puriﬁcation.

4-(1-(4-Methoxyphenyl)-3-(4-nitrophenyl)-1H-pyrrolo[2,3-b]-

pyridin-5-yl)benzenesulfonic Acid (11). Compound 11 was synthe-

sized according to general procedure B using halide 65 (100 mg,

0.236 mmol), (4-((neopentyloxy)sulfonyl)phenyl)boronic acid

(354.3 mg, 0.377 mmol), NaHCO₃(79.3 mg, 0.944 mmol), and

Pd(PPh₃)₄(20.5 mg, 0.018 mmol) in 10 mL of 3:1 MeCN/H₂O. The

reaction mixture was diluted with DCM (30 mL), more water (25

mL) was added, and the solution was extracted four times with 30 mL

of 4:1 DCM/THF. The combined organic layers were dried over

MgSO₄, ﬁltered, and evaporated in vacuo. The crude residue was

sonicated with 10 mL of 9:1 Et₂O/DCM for 5 min, and centrifuged.

The supernatant was removed, and the remaining was solid dried in

vacuo. The compound was dissolved in 2.5 mL of DMF, 130 mg

(1.186 mmol) of tetramethylammonium chloride was added, and the

solution was reﬂuxed overnight. After cooling to room temperature, a

yellow precipitate was formed, isolated by ﬁltration, and reﬂuxed in 1

mL of DMF. After cooling to room temperature, the yellow

suspension was ﬁltered through a Buchner funnel. The solid was

washed with 5 mL of EtOH, 10 mL of EtOAc, and 10 mL of Et₂O and

dried in vacuo to give 4-(1-(4-methoxyphenyl)-3-(4-nitrophenyl)-1H-

pyrrolo[2,3-b]pyridin-5-yl)benzenesulfonic acid 11 in 10% yield (12

mg, 0.024 mmol, amorphous yellow solid). ¹H NMR (300 MHz,

DMSO-d₆): δ 8.90−8.76 (m, 2H), 8.73−8.64 (m, 1H), 8.46−8.28

(m, 4H), 8.24 (d, J = 8.8 Hz, 2H), 8.18 (d, J = 8.7 Hz, 2H), 7.87 (d, J

= 8.8 Hz, 2H), 7.18 (d, J = 8.9 Hz, 2H), 3.86 (s, 3H). HRMS (pos.

ESI-TOF): m/z calcd for C₂₆H₂₀N₃O₆S, 502.1067 [M + H]⁺; found,

502.1054 [M + H]⁺.

5-(5-Bromo-1-(4-methoxyphenyl)-1H-pyrazolo[3,4-b]pyridin-3-

yl)benzo[c][1,2,5]oxadiazole (78). General procedure B was applied

using halide 63 (300 mg, 0.70 mmol), benzo[c][1,2,5]oxadiazole-5-

boronic acid pinacol ester (205 mg, 0.84 mmol), NaHCO₃(180 mg,

2.14 mmol), and Pd(PPh₃)₄(59 mg, 0.052 mmol) in 40 mL of 3:1

MeCN/H₂O. Puriﬁcation of the crude product by ﬂash column

chromatography (0−50% ethyl acetate in cyclohexane) gave 5-(5-

bromo-1-(4-methoxyphenyl)-1H-pyrazolo[3,4-b]pyridin-3-yl)benzo-

[c][1,2,5]oxadiazole 78 in 38% yield (110 mg, 0.26 mmol, yellow

1

amorphous solid). H NMR (300 MHz, CDCl₃): δ 8.74−8.68 (m,

1H), 8.70−8.57 (m, 1H), 8.41−8.27 (m, 2H), 8.19−8.09 (m, 2H),

8.07−7.94 (m, 1H), 7.16−7.07 (m, 2H), 3.90 (s, 3H). ¹³C NMR (75

MHz, CDCl₃): δ 158.8, 150.6, 149.6, 149.5, 149.0, 140.2, 135.8,

132.1, 131.9, 131.8, 123.6 (2C), 117.4, 116.2, 114.7, 114.6 (2C),

112.9, 77.2, 55.8.

5-(5-Bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]-

pyridin-3-yl)benzo[c][1,2,5]oxadiazole (79). General procedure B

was applied using halide 64 (385 mg, 0.94 mmol), benzo[c][1,2,5]-

oxadiazole-5-boronic acid pinacol ester (280 mg, 1.1 mmol),

NaHCO₃(235 mg, 2.8 mmol), and Pd(PPh₃)₄(81 mg, 0.07

mmol) in 55 mL of 3:1 MeCN/H₂O. Puriﬁcation of the crude

product by ﬂash column chromatography (0−40% ethylacetate in

cyclohexane) gave 5-5-(5-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-

pyrazolo[3,4-b]pyridin-3-yl)benzo[c][1,2,5]oxadiazole 79 in 54%

1

yield (200 mg, 0.5 mmol, yellow amorphous solid). H NMR (300

MHz, CDCl₃): δ 8.67 (d, J = 2.1 Hz, 1H), 8.57 (d, J = 2.1 Hz, 1H),

8.37−8.20 (m, 2H), 7.96 (dd, J = 9.7, 0.8 Hz, 1H), 6.20 (dd, J = 10.4,

2.6 Hz, 1H), 4.28−4.09 (m, 1H), 4.00−3.78 (m, 1H), 2.85−2.62 (m,

1H), 2.30−2.14 (m, 1H), 2.11−1.98 (m, 1H), 1.93−1.57 (m, 3H).

Methyl 4-(5-Bromo-1-(4-methoxyphenyl)-1H-pyrrolo[2,3-b]-

pyridin-3-yl)benzoate (80). General procedure C was applied using

halide 55 (304 mg, 0.71 mmol), 4-methoxycarbonylphenylboronic

4-(1-(4-Methoxyphenyl)-3-(4-nitrophenyl)-1H-pyrrolo[2,3-b]-

pyridin-5-yl)benzoic Acid (17). Compound 17 was synthesized

according to general procedure B using 75 mg (0.177 mmol) of

halide 65 (75 mg, 0.177 mmol), 4-carboxyphenylboronic acid (35.2

mg, 0.212 mmol), NaHCO₃(44.6 mg, 0.531 mmol), and Pd(PPh₃)₄

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(25.3 mg, 0.013 mmol) in 7.5 mL of 3:1 MeCN/H₂O. The reaction

mixture was diluted with DCM (30 mL), more water (25 mL) was

added, and the solution was extracted once with dichlormethane.

Then, the water layer was acidiﬁed until the formation of an orange

precipitate and extracted four times with 30 mL of 4:1 DCM/THF.

The combined organic layers were dried over MgSO₄, ﬁltered, and

evaporated in vacuo. The crude product was puriﬁed by column

chromatography (SiO₂, THF in DCM, 0−50% + 0.01% AcOH). The

combined fractions were evaporated, and the residue was sonicated

with 1 mL of DCM for 5 min and centrifuged. The supernatant was

removed, and the remaining solid was dried in vacuo to give 4-(1-(4-

methoxyphenyl)-3-(4-nitrophenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-

benzoic acid 17 in 36% yield (30 mg, 0.064 mmol, orange amorphous

(SiO₂, 30−100% ethyl acetate in cyclohexane) yielded 3-(3-

(benzo[c][1,2,5]oxadiazol-5-yl)-1-(4-methoxyphenyl)-1H-pyrrolo-

[2,3-b]pyridin-5-yl)benzoic acid 20 in 30% yield (14 mg, 0.035 mmol,

1

orange amorphous solid). H NMR (300 MHz, DMSO-d₆): δ 8.87−

8.83 (m, 1H), 8.74−8.68 (m, 2H), 8.52 (s, 1H), 8.34 (s, 1H), 8.29−

8.25 (m, 1H), 8.16−8.11 (m, 2H), 8.02−7.97 (m, 1H), 7.90−7.86

(m, 2H), 7.67 (t, J = 7.7 Hz, 1H), 7.22−7.13 (m, 2H), 3.86 (s, 3H)

ppm. ¹³C NMR (151 MHz, DMSO-d₆): δ 167.3, 158.1, 149.8, 148.1,

147.3, 143.1, 138.7, 137.4, 134.0, 132.0, 131.6, 130.4, 130.3, 129.9,

129.3, 128.2, 128.1, 127.3, 125.5 (2C), 118.2, 116.3, 114.4 (2C),

113.5, 109.2, 55.5. HRMS (pos. ESI-TOF): m/z calcd for

C₂₇H₁₉N₄O₄, 463.1401 [M + H]⁺; found, 463.1378 [M + H]⁺.

4-(1-(4-Methoxyphenyl)-3-(4-(triﬂuoromethyl)phenyl)-1H-

pyrrolo[2,3-b]pyridin-5-yl)benzoic Acid (21). General procedure B

was applied using halide 67 (70 mg, 0.156 mmol), 4-carboxyphe-

nylboronic acid (31.6 mg, 0.190 mmol), NaHCO₃(39.5 mg, 0.470

mmol), Pd(PPh₃)₄(13.5 mg, 0.012 mmol) in 10 mL of 3:1 MeCN/

H₂O. The reaction mixture was diluted with DCM (50 mL), more

water (25 mL) was added, and the solution was extracted one time

with DCM. Then, the water layer was acidiﬁed until the formation of

a precipitate and then extracted four times with 30 mL of a mixture of

DCM/THF 4:1. The combined organic layer was dried over MgSO₄,

ﬁltered, and evaporated in vacuo. The crude residue was absorbed on

isolute and puriﬁed by column chromatography (SiO₂, THF in DCM,

0−50%) to obtain 4-(1-(4-methoxyphenyl)-3-(4-(triﬂuoromethyl)-

phenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzoic acid 21 in 84% yield

1

solid). H NMR (600 MHz, DMSO-d₆): δ 8.81−8.76 (m, 2H), 8.64

(s, 1H), 8.32 (d, J = 8.9 Hz, 2H), 8.23 (d, J = 8.9 Hz, 2H), 8.07 (d, J =

8.4 Hz, 2H), 7.99 (d, J = 8.3 Hz, 2H), 7.87 (d, J = 8.9 Hz, 2H), 7.17

(d, J = 8.9 Hz, 2H), 3.86 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆): δ

167.2, 158.2, 147.5, 145.2, 143.1, 142.5, 141.2, 130.4, 130.0, 130.0

(2C), 129.5, 127.4 (2C), 127.2 (2C), 126.9, 125.5 (2C), 124.3 (2C),

118.3, 114.4 (2C), 113.6, 55.6. HRMS (pos. ESI-TOF): m/z calcd for

C₂₇H₂₀N₃O₅, 465.1325 [M+]⁺; found, 465.1296 [M+]⁺.

3-(1-(4-Methoxyphenyl)-3-(4-nitrophenyl)-1H-pyrrolo[2,3-b]-

pyridin-5-yl)benzoic Acid (18). Compound 18 was synthesized

according to general procedure B using halide 65 (75 mg, 0.177

mmol), 4-carboxyphenylboronic acid (35.2 mg, 0.212 mmol),

NaHCO₃(44.6 mg, 0.531 mmol), and Pd(PPh₃)₄(25.3 mg, 0.013

mmol) in 7.5 mL of 3:1 MeCN/H₂O. The reaction mixture was

diluted with DCM (30 mL), more water (25 mL) was added, and the

solution was extracted with DCM. The water layer was acidiﬁed until

the formation of an orange precipitate and extracted four times with

30 mL of 4:1 DCM/THF. The combined organic layers were dried

over MgSO₄, ﬁltered, and evaporated in vacuo. The crude residue was

puriﬁed by column chromatography (THF in DCM, 0−50% + 0.01%

AcOH). The combined fractions were evaporated, and the residue

was sonicated with 1 mL of DCM for 5 min and centrifuged. The

supernatant was removed, and the remaining solid was dried in vacuo

to give 3-(1-(4-methoxyphenyl)-3-(4-nitrophenyl)-1H-pyrrolo[2,3-

b]pyridin-5-yl)benzoic acid 18 in 34% yield (28 mg, 0.060 mmol,

1

(64.3 mg, 0.131 mmol, pale pink amorphous solid). H NMR (600

MHz, DMSO-d₆): δ 8.80−8.74 (m, 1H), 8.74−8.68 (m, 1H), 8.49 (s,

1H), 8.15 (d, J = 8.1 Hz, 2H), 8.06 (d, J = 8.4 Hz, 2H), 7.97 (d, J =

8.4 Hz, 2H), 7.87 (d, J = 8.9 Hz, 2H), 7.83 (d, J = 8.2 Hz, 2H), 7.17

(d, J = 9.0 Hz, 2H), 3.85 (s, 3H). ¹⁹F NMR (564 MHz, DMSO-d₆): δ

−60.7. HRMS (pos. ESI-TOF): m/z calcd for C₂₈H₂₀F₃N₂O,

489.1421 [M + H]⁺; found, 489.1441[M + H]⁺.

3-(1-(4-Methoxyphenyl)-3-(4-(triﬂuoromethyl)phenyl)-1H-

pyrrolo[2,3-b]pyridin-5-yl)benzoic Acid (22). General procedure B

was applied using halide 67 (43 mg, 0.096 mmol), 3-carboxyphe-

nylboronic acid (19.4 mg, 0.117 mmol), NaHCO₃(24.2 mg, 0.288

mmol), and Pd(PPh₃)₄(9.6 mg, 0.008 mmol) in 6 mL of 3:1 MeCN/

H₂O. The reaction mixture was diluted with DCM (50 mL), more

water (25 mL) was added, and the solution was extracted one time

with DCM. Then, the water layer was acidiﬁed until the formation of

a pale yellow precipitate and extracted four times with 30 mL of a

mixture of DCM/THF 4:1. The combined organic layers were dried

over MgSO₄, ﬁltered, and evaporated in vacuo. The crude residue was

puriﬁed by column chromatography (SiO₂, THF in DCM, 0−50%) to

give 3-(1-(4-methoxyphenyl)-3-(4-(triﬂuoromethyl)phenyl)-1H-

pyrrolo[2,3-b]pyridin-5-yl)benzoic acid 22 in 98% yield (46.0 mg

1

amorphous orange solid). H NMR (600 MHz, DMSO-d₆): δ 8.75−

8.67 (m, 2H), 8.62 (s, 1H), 8.37−8.27 (m, 3H), 8.24−8.19 (m, 2H),

8.12−8.04 (m, 1H), 8.01−7.96 (m, 1H), 7.90−7.81 (m, 2H), 7.66 (t,

J = 7.7 Hz, 1H), 7.17 (d, J = 9.0 Hz, 2H), 3.86 (s, 3H). ¹³C NMR

(151 MHz, DMSO-d₆): δ 167.2, 158.0, 147.3, 145.1, 142.9, 141.2,

138.8, 132.0, 131.6, 130.3, 129.9, 129.8, 129.4, 128.2, 128.0, 127.1

(2C), 126.7, 125.5 (2C), 124.3 (2C), 118.2, 114.4 (2C), 113.5, 55.5.

HRMS (pos. ESI-TOF): m/z calcd for C₂₇H₂₀N₃O₅, 466.1397 [M +

H]⁺; found, 466.1376 [M + H]⁺.

1

0.094 mmol, pale yellow amorphous solid). H NMR (600 MHz,

4-(3-(Benzo[c][1,2,5]oxadiazol-5-yl)-1-(4-methoxyphenyl)-1H-

pyrrolo[2,3-b]pyridin-5-yl)benzoic Acid (19). General procedure B

was applied using halide 66 (100 mg, 0.24 mmol), 4-carboxyphe-

nylboronic acid (47 mg, 0.28 mmol), NaHCO₃(60 mg, 0.72 mmol),

and Pd(PPh₃)₄(21 mg, 0.018 mmol) in 10 mL of 3:1 MeCN/H₂O.

Puriﬁcation of the crude product by ﬂash column chromatography

(SiO₂, 0−10% methanol in DCM) yielded 4-(3-(benzo[c][1,2,5]-

oxadiazol-5-yl)-1-(4-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-

benzoic acid 19 in 30% yield (33 mg, 0.07 mmol, orange amorphous

solid). ¹H NMR (300 MHz, DMSO-d₆): δ 8.90−8.80 (m, 1H), 8.78−

8.66 (m, 2H), 8.52 (s, 1H), 8.30 (d, J = 9.0 Hz, 1H), 8.16 (m, 5H),

8.16−7.92 (m, 2H), 7.24−7.07 (m, 2H), 3.87 (s, 3H). ¹³C NMR (151

MHz, DMSO-d₆): δ 167.3, 158.1, 149.8, 148.1, 147.5, 143.2, 142.4,

137.4, 133.9, 130.5, 130.3, 129.9 (2C), 129.5, 128.0, 127.5 (2C),

127.3, 125.5 (2C), 118.2, 116.3, 114.4 (2C), 113.5, 109.2, 55.5.

HRMS (pos. ESI-TOF): m/z calcd for C₂₇H₁₉N₄O₄, 463.1401 [M +

H]⁺; found, 463.1421 [M + H]⁺.

DMSO-d₆): δ 8.74−8.69 (m, 1H), 8.66−8.63 (m, 1H), 8.50 (s, 1H),

8.37−8.27 (m, 1H), 8.14 (d, J = 8.0 Hz, 2H), 8.10−8.04 (m, 1H),

8.02.-7.98 (m, 1H), 7.92−7.87 (m, 2H), 7.83 (d, J = 8.1 Hz, 2H),

7.64 (t, J = 7.7 Hz, 1H), 7.17 (d, J = 8.9 Hz, 2H), 3.86 (s, 3H). ¹⁹F

NMR (564 MHz, DMSO-d₆): δ −60.7. HRMS (pos. ESI-TOF): m/z

calcd for C₂₈H₂₀F₃N₂O, 489.1421 [M + H]⁺; found, 489.1438 [M +

H]⁺.

4-(1-(4-Methoxyphenyl)-3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]-

pyridin-5-yl)benzoic Acid (23). Compound 23 was synthesized

according to general procedure B using halide 68 (122 mg, 0.321

mmol), 4-carboxyphenylboronic acid (63.9 mg, 0.385 mmol),

NaHCO₃(80.9 mg, 0.963 mmol), and Pd(PPh₃)₄(27.8 mg, 0.024

mmol) in 7.5 mL of 3:1 MeCN/H₂O. The reaction mixture was

diluted with DCM (30 mL), more water (25 mL) was added, and the

solution was extracted once with DCM. The organic layer was

discarded, and the water layer was acidiﬁed and extracted four times

with 30 mL of 4:1 DCM/THF. The combined organic layers were

dried over MgSO₄, ﬁltered, and evaporated in vacuo. The crude

residue was puriﬁed by column chromatography (SiO₂, THF in

DCM, 0−50%). The combined fractions were evaporated, and the

residue was sonicated with 5 mL of Et₂O for 5 min, and then

centrifuged. The supernatant was removed, and the remaining was

3-(3-(Benzo[c][1,2,5]oxadiazol-5-yl)-1-(4-methoxyphenyl)-1H-

pyrrolo[2,3-b]pyridin-5-yl)benzoic Acid (20). General procedure B

was applied using halide 66 (50 mg, 0.12 mmol), 3-carboxyphe-

nylboronic acid (24 mg, 0.14 mmol), NaHCO₃(30 mg, 0.36 mmol),

and Pd(PPh₃)₄(11 mg, 0.009 mmol) in 5 mL of 3:1 MeCN/H₂O.

Puriﬁcation of the crude product by ﬂash column chromatography

O

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solid dried in vacuo to give 4-(1-(4-methoxyphenyl)-3-(pyridin-4-yl)-

1H-pyrrolo[2,3-b]pyridin-5-yl)benzoic acid 23 in 23% yield (31 mg,

0.074 mmol, yellow amorphous solid). ¹H NMR (300 MHz, DMSO-

d₆): δ 8.80−8.71 (m, 2H), 8.71−8.55 (m, 3H), 8.10−8.03 (m, 2H),

8.03−7.95 (m, 4H), 7.91−7.80 (m, 2H), 7.23−7.10 (m, 2H), 3.85 (s,

3H). ¹³C NMR (151 MHz, DMSO-d₆): δ 167.1, 158.1, 149.6, 147.5,

143.0, 142.5, 141.8, 130.3, 129.9, 129.9 (3C), 129.5, 129.3, 127.4

(2C), 126.8, 125.5 (2C), 121.0 (2C), 118.2, 114.4 (2C), 112.6, 55.5.

HRMS (pos. ESI-TOF): m/z calcd for C₂₆H₁₉N₃O₃, 421.1426 [M]⁺;

found, 421.1459 [M]⁺.

benzoic acid 27 in 34% yield (27 mg, 0.035 mmol, white amorphous

solid). ¹H NMR (600 MHz, DMSO-d₆): δ 8.76−8.73 (m, 1H), 8.72−

8.69 (m, 1H), 8.57 (s, 1H), 8.16 (d, J = 8.3 Hz, 2H), 8.09 (d, J = 8.3

Hz, 2H), 7.99 (d, J = 8.0 Hz, 2H), 7.94 (d, J = 8.3 Hz, 2H), 7.88 (d, J

= 8.9 Hz, 2H), 7.18 (d, J = 8.9 Hz, 2H), 3.86 (s, 3H). ¹³C NMR (151

MHz, DMSO-d₆): δ 167.1, 158.0, 147.3, 142.9, 142.5, 138.9, 132.8

(2C), 130.4, 129.9 (2C), 129.5, 129.2, 128.9, 127.3 (2C), 127.1 (2C),

126.7, 125.4 (2C), 119.2, 118.2, 114.4 (2C), 113.9, 108.2, 55.5.

HRMS (pos. ESI-TOF): m/z calcd for C₂₈H₂₀N₃O₃, 446.1499 [M +

H]⁺; found, 446.1480 [M + H]⁺.

4-(3-(Benzo[c][1,2,5]oxadiazol-5-yl)-1-isopropyl-1H-pyrrolo[2,3-

b]pyridin-5-yl)benzoic Acid (28). Compound 28 was synthesized

according to general procedure B using halide 72 (114 mg, 0.319

mmol), 4-carboxyphenylboronic acid (80.0 mg, 0.383 mmol),

NaHCO₃(80 mg, 0.957 mmol), and Pd(PPh₃)₄(27.6 mg, 0.020

mmol) in 10 mL of 3:1 MeCN/H₂O. The reaction mixture was

diluted with DCM (30 mL), more water (25 mL) was added, and the

solution was extracted with DCM. The organic layer was discarded,

and the water layer was acidiﬁed and extracted four times with 30 mL

of 4:1 DCM/THF. The combined organic layers were dried over

MgSO₄, ﬁltered, and evaporated in vacuo. The crude residue was

puriﬁed by column chromatography (SiO₂, THF in DCM, 0−15%).

The combined fractions were evaporated, and the residue was

sonicated with 5 mL of n-pentane for 5 min and centrifuged. The

supernatant was removed, and the remaining solid was dried in vacuo

to give 4-(3-(benzo[c][1,2,5]oxadiazol-5-yl)-1-isopropyl-1H-pyrrolo-

[2,3-b]pyridin-5-yl)benzoic acid 28 in 28% yield (36 mg, 0.090 mmol,

3-(1-(4-Methoxyphenyl)-3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]-

pyridin-5-yl)benzoic Acid (24). General procedure C was applied

using halide 68 (85 mg, 0.22 mmol), 3-carboxyphenylboronic acid

(44 mg, 0.26 mmol), K₂CO₃(91 mg, 0.66 mmol), and Pd(dppf)Cl₂

(4 mg, 0.006 mmol) in 4 mL of 3:1 dioxane/H₂O. Puriﬁcation of the

crude product by ﬂash column chromatography (SiO₂, 0−25%

MeOH in DCM) yielded 4-(1-(4-methoxyphenyl)-3-(pyridin-4-yl)-

1H-pyrrolo[2,3-b]pyridin-5-yl)benzoic acid 24 in 10% yield (9.5 mg,

1

0.022 mmol, white amorphous solid). H NMR (600 MHz, DMSO-

d₆): δ 8.81−8.68 (m, 2H), 8.67−8.62 (m, 3H), 8.32 (s, 1H), 8.10 (d,

J = 7.7 Hz, 1H), 8.01 (d, J = 7.7 Hz, 1H), 7.96 (d, J = 5.2 Hz, 2H),

7.87 (d, J = 8.6 Hz, 2H), 7.68 (t, J = 7.7 Hz, 1H), 7.19 (d, J = 8.6 Hz,

2H), 3.88 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆): δ 167.4, 158.1,

150.1 (2C), 142.9, 142.9, 141.4, 138.7, 131.9, 130.4, 129.8, 129.5,

129.4, 128.2, 127.9, 126.8, 125.5 (2C), 120.9 (2C), 118.2, 114.5

(2C), 112.6, 55.5. HRMS (pos. ESI-TOF): m/z calcd for

C₂₆H₂₀N₃O₃, 422.1499 [M + H]⁺; found, 422.1489 [M + H]⁺.

4-(1-(4-Methoxyphenyl)-3-(pyrimidin-5-yl)-1H-pyrrolo[2,3-b]-

pyridin-5-yl)benzoic Acid (25). General procedure B was applied

using halide 69 (30 mg, 0.08 mmol), 4-carboxyphenylboronic acid

(17 mg, 0.09 mmol), NaHCO₃(21 mg, 0.24 mmol), and Pd(PPh3)₄

(6 mg, 0.005 mmol) in 7 mL of 3:1 MeCN/H₂O. Puriﬁcation of the

crude product by ﬂash column chromatography (SiO₂, 0−25%

MeOH in DCM) yielded 4-(1-(4-methoxyphenyl)-3-(pyrimidin-5-

yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzoic acid 25 in 25% yield (8.4

mg, 0.019 mmol, white amorphous solid). ¹H NMR (600 MHz,

DMSO-d₆): δ 9.43−9.34 (m, 2H), 9.17 (s, 1H), 8.81−8.73 (m, 2H),

8.63 (s, 1H), 8.12−7.99 (m, 4H), 7.88 (d, J = 8.9 Hz, 2H), 7.20 (d, J

= 8.9 Hz, 2H), 3.88 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆): δ

167.2, 158.0, 156.1, 154.3 (2C), 147.2, 143.0, 142.4, 130.4, 129.9

(2C), 129.6, 129.2, 128.8, 128.3, 127.4 (2C), 126.9, 125.4 (2C),

118.2, 114.5 (2C), 108.9, 55.5. HRMS (pos. ESI-TOF): m/z calcd for

C₂₅H₁₉N₄O₃, [M + H]⁺423.1463; found, [M + H]⁺423.1452.

4-(3-(Benzo[d][1,3]dioxol-5-yl)-1-(4-methoxyphenyl)-1H-pyrrolo-

[2,3-b]pyridin-5-yl)benzoic Acid (26). General procedure B was

applied using halide 70 (50 mg, 0.12 mmol), 4-carboxyphenylboronic

acid (23 mg, 0.14 mmol), NaHCO₃(30 mg, 0.30 mmol), and

Pd(PPh₃)₄(10 mg, 0.009 mmol) in 5 mL of 3:1 MeCN/H₂O.

Puriﬁcation of the crude product by ﬂash column chromatography

(30−100% ethyl acetate in cyclohexane) yielded 4-(3-(benzo[d]-

[1,3]dioxol-5-yl)-1-(4-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-

yl)benzoic acid 26 in 22% yield (12 mg, 0.026 mmol, white

amorphous solid). ¹H NMR (600 MHz, DMSO-d₆): δ 8.74−8.67 (m,

1H), 8.57−8.52 (m, 1H), 8.24 (s, 1H), 8.06 (d, J = 8.3 Hz, 2H), 7.96

(d, J = 8.3 Hz, 2H), 7.89−7.83 (m, 2H), 7.49−7.44 (m, 1H), 7.41−

7.33 (m, 1H), 7.19−7.13 (m, 2H), 7.05 (d, J = 8.0 Hz, 1H), 6.09 (s,

2H), 3.86 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆): δ 167.2, 157.6,

147.8, 147.0, 146.0, 142.8, 142.4, 130.8, 130.0 (2C), 129.4, 128.5,

127.8, 127.2 (2C), 126.7, 126.4, 125.0 (2C), 120.3, 118.6, 115.7,

114.3 (2C), 108.8, 107.5, 100.9, 55.4. HRMS (pos. ESI-TOF): m/z

calcd for C₂₈H₂₁N₂O₅, 465.1445 [M + H]⁺; found, 465.1449 [M +

H]⁺.

1

amorphous yellow solid). H NMR (600 MHz, DMSO-d₆): δ 8.84−

8.77 (m, 1H), 8.74−8.72 (m, 1H), 8.61 (s, 1H), 8.45−8.40 (m, 1H),

8.22 (dd, J = 9.4, 1.5 Hz, 1H), 8.11 (dd, J = 9.4, 0.9 Hz, 1H), 8.08−

8.04 (m, 2H), 8.04−7.99 (m, 2H), 5.22 (p, J = 6.8 Hz, 1H), 1.59 (d, J

= 6.7 Hz, 6H). ¹³C NMR (151 MHz, DMSO-d₆): δ 167.3, 149.9,

148.1, 147.1, 142.4, 139.1, 138.0, 133.9, 132.0, 131.5, 129.3, 129.2,

128.2, 128.1, 128.0, 127.0, 117.5, 116.1, 112.2, 107.9, 45.9, 22.4 (2C).

HRMS (pos. ESI-TOF): m/z calcd for C₂₃H₁₉N₄O₃, 399.1480 [M +

H]⁺; found, 399.1452 [M + H]⁺.

4-(3-(Benzo[c][1,2,5]oxadiazol-5-yl)-1-tosyl-1H-pyrrolo[2,3-b]-

pyridin-5-yl)benzoic Acid (29). General procedure B was applied

using halide 73 (58 mg, 0.12 mmol), 4-carboxyphenylboronic acid

(24 mg, 0.15 mmol), NaHCO₃(30 mg, 0.36 mmol), and Pd(PPh₃)₄

(10 mg, 0.008 mmol) in 7 mL of 3:1 MeCN/H₂O. Puriﬁcation of the

crude product by ﬂash column chromatography (SiO₂, 30−100%

ethyl acetate in cyclohexane, with 5% MeOH for complete elution of

the product) yielded 4-(3-(benzo[c][1,2,5]oxadiazol-5-yl)-1-tosyl-1H-

pyrrolo[2,3-b]pyridin-5-yl)benzoic acid 29 in 11% yield (7 mg, 0.014

1

mmol, white amorphous solid). H NMR (300 MHz, DMSO-d₆): δ

8.88−8.81 (m, 1H), 8.81−8.74 (m, 1H), 8.74−8.65 (m, 1H), 8.65−

8.57 (m, 1H), 8.28−8.16 (m, 2H), 8.16−8.10 (m, 2H), 8.08−8.02

(m, 2H), 8.03−7.93 (m, 2H), 7.51−7.42 (m, 2H), 2.36 (s, 3H).

HRMS (pos. ESI-TOF): m/z calcd for C₂₇H₁₉N₄O₅S, 511.1071 [M +

H]⁺; found, 511.1070 [M + H]⁺.

4-(3-(Benzo[c][1,2,5]oxadiazol-5-yl)-1-benzyl-1H-pyrrolo[2,3-b]-

pyridin-5-yl)benzoic Acid (30). Compound 30 was synthesized

according to general procedure B using halide 74 (80 mg, 0.197

mmol), 4-carboxyphenylboronic acid (40.0 mg, 0.236 mmol),

NaHCO₃(50.0 mg, 0.591 mmol), and Pd(PPh₃)₄(16.5 mg, 0.014

mmol) in 10 mL of 3:1 MeCN/H₂O. The reaction mixture was

diluted with DCM (30 mL), more water (25 mL) was added, and the

solution was extracted once with DCM. Then, the water layer was

acidiﬁed and extracted four times with 30 mL of 4:1 DCM/THF. The

organic layer was dried over MgSO₄, ﬁltered, and evaporated in vacuo.

The crude residue was puriﬁed by column chromatography (SiO₂,

THF in DCM, 0−50%). The combined fractions were evaporated,

and the residue was sonicated with 5 mL of Et₂O for 5 min, and then

centrifuged. The supernatant was removed, and the remaining solid

was dried in vacuo to give 4-(3-(benzo[c][1,2,5]oxadiazol-5-yl)-1-

benzyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzoic acid 30 in 65% yield

4-(3-(4-Cyanophenyl)-1-(4-methoxyphenyl)-1H-pyrrolo[2,3-b]-

pyridin-5-yl)benzoic Acid (27). General procedure B was applied

using halide 71 (73 mg, 0.18 mmol), 4-carboxyphenylboronic acid

(33 mg, 0.2 mmol), NaHCO₃(45 mg, 0.54 mmol), and Pd(PPh₃)₄

(16 mg, 0.014 mmol) in 8 mL of 3:1 MeCN/H₂O. Puriﬁcation of the

crude product by recrystallization from cold DCM, redissolving in

EtOAc, and ﬁltration on short plug of silica yielded 4-(3-(4-

cyanophenyl)-1-(4-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-

1

(57 mg, 0.127 mmol yellow amorphous solid). H NMR (600 MHz,

DMSO-d₆): δ 8.85 (d, J = 2.0 Hz, 1H), 8.76 (d, J = 2.0 Hz, 1H), 8.60

(s, 1H), 8.45 (t, J = 1.2 Hz, 1H), 8.16−8.08 (m, 2H), 8.07 (d, J = 8.4

P

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Hz, 2H), 8.02 (d, J = 8.4 Hz, 2H), 7.39−7.32 (m, 4H), 7.31−7.25 (m,

1H), 5.61 (s, 2H). ¹³C NMR (151 MHz, DMSO-d₆): δ 170.4, 153.0,

151.3, 150.9, 146.2, 145.9, 140.8, 140.8, 136.9, 134.4, 133.1 (2C),

132.6, 132.2, 131.9 (2C), 130.8, 130.7 (2C), 130.6 (2C), 130.5,

120.6, 119.5, 115.7, 111.7, 70.2. HRMS (pos. ESI-TOF): m/z calcd

for C₂₇H₁₉N₄O₃, 447.1452 [M + H]⁺; found, 447.1481 [M + H]⁺.

4-(3-(Benzo[c][1,2,5]oxadiazol-5-yl)-1-(4-methoxybenzyl)-1H-

pyrrolo[2,3-b]pyridin-5-yl)benzoic Acid (31). Compound 31 was

synthesized according to general procedure B using halide 75 (117

mg, 0.245 mmol), 4-carboxyphenylboronic acid (50.0 mg, 0.295

mmol), NaHCO₃(61.7 mg, 0.735 mmol), and Pd(PPh₃)₄(21.0 mg,

0.018 mmol) in 10 mL of 3:1 MeCN/H₂O. The reaction mixture was

diluted with DCM (30 mL), more water (25 mL) was added, and the

solution was extracted with DCM. The organic layer was discarded,

and the water layer was acidiﬁed and extracted four times with 30 mL

of 4:1 DCM/THF. The combined organic layers were dried over

MgSO₄, ﬁltered, and evaporated in vacuo. The crude residue was

puriﬁed by column chromatography (SiO₂, THF in DCM, 0−50%).

The combined fractions were evaporated, and the residue was

sonicated with 5 mL of Et₂O for 5 min and then centrifuged. The

supernatant was removed, and the remaining solid was dried in vacuo

to give 4-(3-(benzo[c][1,2,5]oxadiazol-5-yl)-1-(4-methoxybenzyl)-

1H-pyrrolo[2,3-b]pyridin-5-yl)benzoic acid 31 in 2% yield (3 mg,

0.006 mmol, yellow amorphous solid). ¹H NMR (600 MHz, DMSO-

d₆): δ 8.84−8.81 (m, 1H), 8.78−8.75 (m, 1H), 8.57 (s, 1H), 8.46−

8.42 (m, 1H), 8.13−8.09 (m, 2H), 8.07 (d, J = 8.3 Hz, 2H), 8.02 (d, J

= 8.4 Hz, 2H), 7.36 (d, J = 8.7 Hz, 2H), 6.90 (d, J = 8.7 Hz, 2H), 5.52

(s, 2H), 3.71 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆): δ 167.2,

158.8, 149.8, 148.1, 147.6, 142.9, 142.7, 137.6, 133.7, 131.1, 129.9

(2C), 129.6, 129.4, 129.1, 129.0 (2C), 127.5 (2C), 127.2, 117.4,

116.3, 114.0 (2C), 112.4, 108.4, 55.1, 47.3. HRMS (pos. ESI-TOF):

m/z calcd for C₂₈H₂₁N₄O₄, 477.1564 [M + H]⁺; found, 477.1557 [M

+ H]⁺.

4-(3-(Benzo[c][1,2,5]oxadiazol-5-yl)-1-(3-methoxyphenyl)-1H-

pyrrolo[2,3-b]pyridin-5-yl)benzoic Acid (32). General procedure C

was applied using halide 76 (210 mg, 0.50 mmol), 4-carboxyphe-

nylboronic acid (90 mg, 0.55 mmol), K₂CO₃(206 mg, 0.54 mmol),

and Pd(dppf)Cl₂(9 mg, 0.014 mmol) in 8 mL of 3:1 dioxane/H₂O.

The reaction was stirred for 5 h and then allowed to cool down to

room temperature. The mixture was acidiﬁed until the formation of a

yellow precipitate (pH = 4.5) and extracted three times with DCM.

The combined organic layers were dried with Na₂SO₄and

concentrated in vacuo. The crude product was puriﬁed by column

chromatography to 4-(3-(benzo[c][1,2,5]oxadiazol-5-yl)-1-(3-me-

thoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzoic acid 32 in 2.5%

yield (6 mg, 0.015 mmol, yellow solid). The solid residue in the

aqueous layer was boiled with addition of acetic acid, ﬁltrated, and

lyophilized. The NMR of the solid showed that the product

coprecipitated with acetic acid. The yield of 4-(3-(benzo[c][1,2,5]-

oxadiazol-5-yl)-1-(3-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-

benzoic acid acetate is 48% yield (125 mg, 0.24 mmol, yellow

amorphous solid). ¹H NMR (600 MHz, DMSO-d₆): δ 8.91−8.88 (m,

1H), 8.85−8.76 (m, 2H), 8.57 (s, 1H), 8.30 (dd, J = 9.4, 1.5 Hz, 1H),

8.17 (d, J = 9.4, 1H), 8.11−8.06 (m, 2H), 8.04−7.98 (m, 2H), 7.68−

7.63 (m, 2H), 7.54 (t, J = 8.1 Hz, 1H), 7.05−7.02 (m, 1H), 3.91 (s,

3H). ¹³C NMR (151 MHz, DMSO-d₆): δ 167.1, 159.9, 149.7, 148.1,

147.4, 143.2, 142.3, 138.3, 137.2, 134.0, 130.2 (2C), 130.0, 129.9,

129.7, 127.5 (2C), 127.4, 118.6, 116.3, 115.9, 114.1, 112.2, 109.7,

109.6, 55.5. HRMS (pos. ESI-TOF): m/z calcd for C₂₇H₁₉N₄O₄,

463.1401 [M + H]⁺; found, 463.1404 [M + H]⁺.

precipitate was lyophilized, and the dry product was dissolved in

ACN/MeOH, absorbed on isolute, and puriﬁed by column

chromatography (SiO₂, MeOH in DCM, 0−25%) to give 4-(3-

(benzo[c][1,2,5]oxadiazol-5-yl)-1-phenyl-1H-pyrrolo[2,3-b]pyridin-

5-yl)benzoic acid 33 in 14% yield (5.3 mg, 0.011 mmol, yellow

amorphous solid). ¹H NMR (600 MHz, DMSO-d₆): δ 8.91−8.85 (m,

1H), 8.84−8.80 (m, 1H), 8.81−8.77 (m, 1H), 8.57 (s, 1H), 8.30 (d, J

= 9.4 Hz, 1H), 8.17 (d, J = 9.4 Hz, 1H), 8.12−8.06 (m, 2H), 8.06−

8.01 (m, 2H), 8.01−7.94 (m, 2H), 7.70−7.60 (m, 2H), 7.46 (t, J =

7.5 Hz, 1H). HRMS (pos. ESI-TOF): m/z calcd for C₂₆H₁₇N₄O₃,

434.1326 [M + H]⁺; found, 434.1341 [M + H]⁺.

Methyl 4-(3-(Benzo[c][1,2,5]oxadiazol-5-yl)-1-(4-methoxyphen-

yl)-1H-pyrazolo[3,4-b]pyridin-5-yl)benzoate (83). General proce-

dure B was applied using halide 78 (66 mg, 0.16 mmol), 4-

methoxycarbonylphenylboronic acid (36 mg, 0.20 mmol), NaHCO₃

(41 mg, 0.48 mmol), and Pd(PPh₃)₄(14 mg, 0.012 mmol) in 10 mL

of 3:1 MeCN/H₂O. Puriﬁcation of the crude product by ﬂash column

chromatography (0−100% DCM in cyclohexane) yielded 4-(3-

(benzo[c][1,2,5]oxadiazol-5-yl)-1-(4-methoxyphenyl)-1H-pyrazolo-

[3,4-b]pyridin-5-yl)benzoate 83 in 37% (28 mg, 0.058 mmol, yellow

1

amorphous solid). H NMR (300 MHz, CDCl₃): δ 9.08−8.87 (m,

1H), 8.74−8.61 (m, 1H), 8.50−8.35 (m, 2H), 8.30−8.15 (m, 4H),

8.11−7.94 (m, 1H), 7.89−7.73 (m, 2H), 7.23−7.07 (m, 2H), 3.99 (s,

3H), 3.91 (s, 3H). HRMS (pos. ESI-TOF): m/z calcd for

C₂₇H₂₀N₅O₄, [M + H]⁺, 478.1510; found, 478.1522.

4-(3-(Benzo[c][1,2,5]oxadiazol-5-yl)-1-(4-methoxyphenyl)-1H-

pyrazolo[3,4-b]pyridin-5-yl)benzoic Acid (34). Corresponding in-

dazol ester 83 (26 mg, 0.06 mmol, 1 equiv) was dissolved in dioxane

(1.5 mL), and an aqueous solution of NaOH (5 equiv, 2 M) was

added. The mixture was stirred at 60 °C for 12 h. The crude product

was concentrated in vacuo, more water was added, and the solution

was extracted one time with DCM. Then, the water layer was acidiﬁed

until the formation of a yellow precipitate and extracted one more

time with DCM. The organic layer was discarded, and the major

portion of the water was decanted. A yellow solid was washed with

DCM and acetone and lyophilized to give 4-(3-(benzo[c][1,2,5]-

oxadiazol-5-yl)-1-(4-methoxyphenyl)-1H-pyrazolo[3,4-b]pyridin-5-

yl)benzoic acid 34 in 68% yield (18 mg, 0.04 mmol, yellow

amorphous solid). ¹H NMR (600 MHz, DMSO-d₆): δ 9.22−9.11 (m,

1H), 9.10−8.97 (m, 1H), 8.94 (m, 1H), 8.54−8.37 (m, 1H), 8.29−

8.14 (m, 3H), 8.14−7.87 (m, 4H), 7.23−7.07 (m, 2H), 3.87 (s, 3H).

HRMS (pos. ESI-TOF): m/z calcd for C₂₆H₁₈N₅O₄[M + H]⁺,

464.1353; found, 464.1379. IC₅₀> 100 μM (DiFMUP assay,

DiFMUP concentration = 20 μM).

Methyl 4-(3-(Benzo[c][1,2,5]oxadiazol-5-yl)-1-(tetrahydro-2H-

pyran-2-yl)-1H-pyrazolo[3,4-b]pyridin-5-yl)benzoate (84). General

procedure B coupling was applied using halide 79 (100 mg, 0.25

mmol), 4-methoxycarbonylphenylboronic acid (54 mg, 0.30 mmol),

NaHCO₃(63 mg, 0.75 mmol), and Pd(PPh₃)₄(21 mg, 0.012 mmol)

in 10 mL of 3:1 MeCN/H₂O. Puriﬁcation of the crude product by

ﬂash column chromatography (0−50% ethylacetate in cyclohexane)

gave methyl 4-(3-(benzo[c][1,2,5]oxadiazol-5-yl)-1-(tetrahydro-2H-

pyran-2-yl)-1H-pyrazolo[3,4-b]pyridin-5-yl)benzoate 84 in 40% yield

1

(45 mg, 0.10 mmol, white amorphous solid). H NMR (300 MHz,

CDCl₃): δ 8.94−8.79 (m, 1H), 8.61−8.48 (m, 1H), 8.42−8.26 (m,

2H), 8.24−8.10 (m, 2H), 8.01−7.86 (m, 1H), 7.78−7.67 (m, 2H),

6.33−6.18 (m, 1H), 4.28−4.07 (m, 1H), 4.05−3.78 (m, 4H), 2.88−

2.65 (m, 1H), 2.29−2.14 (m, 1H), 2.14−1.99 (m, 1H), 1.98−1.78

(m, 2H), 1.78−1.59 (m, 1H). HRMS (pos. ESI-TOF): m/z calcd for

C₂₅H₂₂N₅O₄[M + H]⁺, 456.2; found, 456.0.

4-(3-(Benzo[c][1,2,5]oxadiazol-5-yl)-1-(tetrahydro-2H-pyran-2-

yl)-1H-pyrazolo[3,4-b]pyridin-5-yl)benzoic Acid (35). The corre-

sponding azaindazole ester 84 (45 mg, 0.01 mmol, 1 equiv) was

dissolved in dioxane (1.5 mL) and an aqueous solution of NaOH (5

equiv, 2 M) was added. The mixture was stirred at 60 °C for 12 h.

The crude was concentrated in vacuo, more water was added, and the

solution was extracted one time with DCM. Then, the water layer was

acidiﬁed until the formation of a white precipitate and extracted one

more time with DCM. The organic layer was discarded, and the major

portion of the water was decanted. White solid was washed with

4-(3-(Benzo[c][1,2,5]oxadiazol-5-yl)-1-phenyl-1H-pyrrolo[2,3-b]-

pyridin-5-yl)benzoic Acid (33). General procedure B was applied

using halide 77 (32 mg, 0.08 mmol), 4-carboxyphenylboronic acid

(16 mg, 0.1 mmol), NaHCO₃(20 mg, 0.24 mmol), and Pd(PPh₃)₄(7

mg, 0.006 mmol) in 10 mL of 3:1 MeCN/H₂O. The crude product

was concentrated in vacuo, more water was added, and the solution

was extracted with DCM. Then, the water layer was acidiﬁed until the

formation of a yellow precipitate and extracted one more time with

DCM. The organic layer was discarded (albeit containing traces of

product), and the major portion of the water was decanted. The crude

Q

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DCM and acetone and lyophilized to give 4-(3-(benzo[c][1,2,5]-

oxadiazol-5-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazolo[3,4-b]-

pyridin-5-yl)benzoic acid 35 in 54% yield (24 mg, 0.054 mmol, white

solution of LiOH (5 equiv, 2 M) was added. The mixture was stirred

at room temperature for 24 h, then an additional portion of LiOH (5

equiv, 2 M) was added. The reaction was stirred for additional 24 h,

then the crude product was concentrated in vacuo, more water was

added, and the solution was extracted one time with DCM. The water

layer was acidiﬁed until the formation of a yellow precipitate (pH =

4.0−4.5). The solid was collected by vacuum ﬁltration, washed with

DCM and ether, and lyophilized to give 3-(3-(benzo[c][1,2,5]-

oxadiazol-5-yl)-1-(4-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)-

acrylic acid 37 in 44% yield (8 mg, 0.02 mmol, yellow amorphous

solid). ¹H NMR (300 MHz, DMSO-d₆): δ 9.06−8.96 (m, 1H), 8.80−

8.68 (m, 2H), 8.52 (s, 1H), 8.34−8.19 (m, 1H), 8.19−8.06 (m, 1H),

7.92−7.78 (m, 3H), 7.24−7.10 (m, 2H), 6.90 (d, J = 16.1 Hz, 1H),

3.87 (s, 3H). ¹³C NMR (75 MHz, DMSO-d₆): δ 167.8, 158.1, 149.8,

148.1, 144.9, 141.9, 137.1, 133.8, 130.9, 130.1, 128.5, 125.5 (3C),

124.8, 119.0, 118.2, 116.2, 114.4 (2C), 113.6, 109.2, 55.5. HRMS

(pos. ESI-TOF): m/z calcd for C₂₃H₁₇N₄O₄[M + H]⁺, 414.1275;

found, [M + H]⁺414.1286.

1

amorphous solid). H NMR (300 MHz, CDCl₃): δ 9.22−9.08 (m,

1H), 9.07−8.99 (m, 1H), 8.90 (s, 1H), 8.45−8.30 (m, 1H), 8.23−

8.14 (m, 1H), 8.14−7.97 (m, 4H), 6.27−6.03 (m, 1H), 4.08−3.88

(m, 1H), 3.85−3.64 (m, 1H), 2.70−2.53 (m, 1H), 2.18−1.95 (m,

2H), 1.95−1.74 (m, 1H), 1.74−1.55 (m, 2H). ¹³C NMR (75 MHz,

DMSO-d₆): δ 167.2, 150.7, 149.5, 149.0, 148.6, 141.5, 140.7, 135.4,

132.1, 130.6, 130.1, 129.9 (2C), 129.3, 127.8 (2C), 116.7, 113.3,

113.2, 82.1, 67.3, 39.5, 28.8, 24.7, 22.3. HRMS (pos. ESI-TOF): m/z

calcd for C₂₄H₂₀N₅O₄, [M + H]⁺, 443.1540; found, 443.1542. IC₅₀

>

100 μM (DiFMUP assay, DiFMUP concentration = 20 μM).

Methyl 3-(Benzo[c][1,2,5]oxadiazol-5-yl)-1-(4-methoxyphenyl)-

1H-pyrrolo[2,3-b]pyridine-5-carboxylate (85). General procedure B

was adapted using halide 62 (200 mg, 0.49 mmol), benzo[c][1,2,5]-

oxadiazole-5-boronic acid pinacol ester (258 mg, 0.10 mmol), K₂CO₃

(430 mg, 3.14 mmol), and Pd(PPh₃)₄(14 mg, 0.012 mmol) in 9 mL

of 3:1 MeCN/H₂O. Puriﬁcation of the crude product by ﬂash column

chromatography (SiO₂, 30−100% ethyl acetate in cyclohexane)

yielded methyl 3-(benzo[c][1,2,5]oxadiazol-5-yl)-1-(4-methoxyphen-

yl)-1H-pyrrolo[2,3-b]pyridine-5-carboxylate 60 in 18% yield (33 mg,

0.08 mmol, yellow amorphous solid). ¹H NMR (300 MHz, CDCl₃): δ

9.17−9.03 (m, 1H), 9.03−8.94 (m, 1H), 816−8.09 (m, 1H), 8.01−

7.92 (m, 1H), 7.90 (s, 1H), 7.86−7.72 (m, 1H), 7.73−7.57 (m, 2H),

7.22−7.14 (m, 2H), 4.00 (s, 3H),3.89 (s, 3H). LCMS (pos. ESI-

TOF): m/z calcd for C₂₂H₁₇N₄O₄, 401.1 [M + H]⁺; found, 401.0 [M

+ H]⁺.

4-(3-(Benzo[c][1,2,5]oxadiazol-5-yl)-1-(4-methoxyphenyl)-1H-

pyrrolo[2,3-b]pyridin-5-yl)cyclohex-3-ene-1-carboxylic Acid (38).

Compound 38 was synthesized according to general procedure B

using halide 66 (43 mg, 0.102 mmol), 4-(4,4,5,5-tetramethyl-1,3,2-

dioxaborolan-2-yl)cyclohex-3-ene-1-carboxylic acid (30.9 mg, 0.122

mmol), NaHCO₃(25.7 mg, 0.306 mmol), and Pd(PPh₃)₄(8.8 mg,

0.008 mmol) in 5 mL of 3:1 MeCN/H₂O. The reaction mixture was

diluted with DCM (30 mL), more water (25 mL) was added, and the

solution was extracted one time with DCM. The organic layer was

discarded, and the water layer was acidiﬁed and extracted four times

with 30 mL of 4:1 DCM/THF. The combined organic layers were

dried over MgSO₄, ﬁltered, and evaporated in vacuo. The crude

residue was puriﬁed by column chromatography (SiO₂, THF in

DCM, 0−25%). The combined fractions were evaporated, and the

residue was sonicated with 5 mL of 1:1 n-pentane/Et₂O for 5 min and

then centrifuged. The supernatant was removed, and the remaining

solid was dried in vacuo to give 4-(3-(benzo[c][1,2,5]oxadiazol-5-yl)-

1-(4-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)cyclohex-3-ene-

1-carboxylic acid 38 in 38% yield (18 mg, 0.036 mmol, amorphous

yellow solid). ¹H NMR (600 MHz, DMSO-d₆): δ 8.65 (s, 1H), 8.58−

8.56 (m, 1H), 8.56−8.53 (m, 1H), 8.47−8.44 (m, 1H), 8.22 (dd, J =

9.4, 1.5 Hz, 1H), 8.13 (dd, J = 9.4, 0.9 Hz, 1H), 7.85 (d, J = 8.9 Hz,

2H), 7.16 (d, J = 8.9 Hz, 2H), 6.39−6.34 (m, 1H), 3.85 (s, 3H),

2.80−2.59 (m, 4H), 2.54−2.47 (m, 1H), 2.25−2.15 (m, 1H), 1.93−

1.75 (m, 1H). ¹³C NMR (151 MHz, DMSO-d₆): δ 176.4, 157.9,

149.7, 148.1, 146.9, 142.1, 141.7, 137.6, 133.9, 131.7, 130.4, 129.9,

125.3 (2C), 124.7, 123.4, 117.7, 116.3, 114.3 (2C), 113.2, 108.7, 55.5,

38.0, 27.9, 26.5, 25.3. HRMS (pos. ESI-TOF): m/z calcd for

C₂₇H₂₃N₄O₄, 467.1750 [M + H]⁺; found, 467.1714 [M + H]⁺.

Ethyl-4-(3-(benzo[c][1,2,5]oxadiazol-5-yl)-1-(4-methoxyphenyl)-

1H-pyrrolo[2,3-b]pyridin-5-yl)benzoate (39). Compound 39 was

synthesized according to general procedure B using halide 66 (100

mg, 0.237 mmol), 4-(ethoxycarbonyl)phenyl)boronic acid (55.3 mg,

0.284 mmol), NaHCO₃(59.8 mg, 0.712 mmol), and Pd(PPh₃)₄(20.6

mg, 0.018 mmol) in 10 mL of 3:1 MeCN/H₂O. The reaction mixture

was diluted with DCM (30 mL), more water (25 mL) was added, and

the solution was extracted four times with 30 mL of 4:1 DCM/THF.

The organic layer was dried over MgSO₄, ﬁltered, and evaporated in

vacuo. The crude residue was dissolved in 5 mL of EtOH abs and

cooled to 0 °C, and 18 μL of thionyl chloride was added. The mixture

was reﬂuxed for 4 h, concentrated in vacuo, and afterward submitted

for ﬂash column chromatography without further workup. The

product was eluted with THF in DCM (SiO₂, 0−25%) to give impure

ethyl-4-(3-(benzo[c][1,2,5]oxadiazol-5-yl)-1-(4-methoxyphenyl)-1H-

pyrrolo[2,3-b]pyridin-5-yl)benzoate 39 in 43% yield over two steps

(50 mg, 0.102 mmol, yellow amorphous solid, 70% purity, the

byproduct is 5,5′-(1-(4-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridine-

3,5-diyl)bis(benzo[c][1,2,5]oxadiazole). ¹H NMR (600 MHz,

DMSO-d₆): δ 8.88 (dd, J = 5.1, 2.2 Hz, 1H), 8.78−8.74 (m, 1H),

8.72 (s, 1H), 8.54−8.50 (m, 1H), 8.30−8.22 (m, 1H), 8.13 (dd, J =

9.4, 1.0 Hz, 1H), 8.08 (d, J = 8.4 Hz, 2H), 8.06−8.03 (m, 2H), 7.90−

7.83 (m, 2H), 7.17 (dd, J = 9.1, 2.3 Hz, 2H), 4.36 (q, J = 7.1 Hz, 2H),

3-(Benzo[c][1,2,5]oxadiazol-5-yl)-1-(4-methoxyphenyl)-1H-

pyrrolo[2,3-b]pyridine-5-carboxylic Acid (36). Ester 85 (30 mg,

0.075 mmol, 1 equiv) was dissolved in dioxane (3 mL) and aqueous

NaOH (5 equiv, 2 M) was added. The mixture was stirred at 60 °C

for 12 h. The crude product was concentrated in vacuo, more water

was added, and the solution was extracted one time with DCM. Then,

the water layer was acidiﬁed until the formation of a yellow precipitate

(pH = 4.0−4.5). The solid was collected by vacuum ﬁltration, washed

with DCM and ether, and lyophilized to give 3-(benzo[c][1,2,5]-

oxadiazol-5-yl)-1-(4-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridine-5-

carboxylic acid 36 in 77% yield (24 mg, 0.057 mmol, yellow

1

amorphous solid). H NMR (600 MHz, DMSO-d₆): δ = 9.02−8.98

(m, 1H), 8.98−8.89 (m, 1H), 8.76 (s, 1H), 8.39−8.35 (m, 1H),

8.23−8.15 (m, 2H), 7.88−7.80 (m, 2H), 7.22−7.14 (m, 2H), 3.87 (s,

3H). ¹³C NMR (151 MHz, DMSO-d₆): δ 166.9, 158.3, 149.6, 149.2,

148.2, 145.6, 137.2, 133.8, 131.2, 130.3, 129.9, 125.8 (2C), 117.5,

117.0, 116.7, 114.5 (2C), 114.0, 109.5, 55.5. HRMS (pos. ESI-TOF):

m/z calcd for C₂₁H₁₅N₄O₄, 388.1118 [M + H]⁺; found, [M + H]⁺

388.1121.

Ethyl (E)-3-(3-(Benzo[c][1,2,5]oxadiazol-5-yl)-1-(4-methoxy-

phenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)acrylate (86). 66 (117 mg,

0.28 mmol, 1 equiv), ethyl acrylate (83 mg, 0.83 mmol, 3.0 equiv),

Pd(OAc)₂(6.24 mg, 0.03 mmol, 0.1 equiv), and tris(2-

methylphenyl)phosphine (16.9 mg, 0.06 mmol, 0.2 equiv) were

dissolved in a mixture of TEA/DMF 2:5 (7 mL), and purged with N₂

prior to use. The mixture was heated to 120 °C and stirred for 12 h

under N₂. The mixture was cooled to room temperature and water

was added. The water layer was extracted three times with DCM,

dried with Na₂SO₄, and evaporated under vacuum. The crude product

was puriﬁed by silica gel chromatography (SiO₂, DCM in cyclo-

hexane, 0−50%) to give ethyl (E)-3-(3-(benzo[c][1,2,5]oxadiazol-5-

yl)-1-(4-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)acrylate 86

in 17% yield (21 mg, 0.05 mmol, yellow amorphous solid). ¹H

NMR (300 MHz, CDCl₃): δ 8.66−8.60 (m, 1H), 8.57−8.44 (m, 1H),

8.11−8.03 (m, 1H), 7.99−7.84 (m, 3H), 7.84−7.74 (m, 1H), 7.70−

7.60 (m, 2H), 7.17−7.05 (m, 2H), 6.60 (d, J = 16.0 Hz, 1H), 4.32 (q,

J = 7.1 Hz, 2H), 3.89 (s, 3H), 1.39 (t, J = 7.1 Hz, 3H) ppm. LCMS

(pos. ESI-TOF): m/z calcd for C₂₅H₂₁N₄O₄, 441.2 [M + H]⁺; found,

441.1 [M + H]⁺.

3-(3-(Benzo[c][1,2,5]oxadiazol-5-yl)-1-(4-methoxyphenyl)-1H-

pyrrolo[2,3-b]pyridin-5-yl)acrylic Acid (37). Ester 86 (20 mg, 0.045

mmol, 1 equiv) was dissolved in THF (1.5 mL), and an aqueous

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3.86 (s, 3H), 1.36 (t, J = 7.1 Hz, 3H). ¹³C NMR (600 MHz, DMSO-

d₆, representative peaks): δ 165.6, 158.1, 149.8, 148.1, 147.5, 143.2,

142.8, 137.4, 134.0, 130.6, 130.2, 129.7 (2C), 129.3, 127.6 (2C),

127.3, 125.5 (2C), 118.2, 116.3, 114.4 (2C), 113.6, 109.2, 60.8, 55.5,

14.2. HRMS (pos. ESI-TOF): m/z calcd for C₂₉H₂₃N₅O₄S, [M + H]⁺

491.1714; found, 491.1743.

[1,2,5]oxadiazol-5-yl)-1-(4-methoxyphenyl)-1H-pyrrolo[2,3-b]-

pyridin-3-yl)benzoic acid 41 in 4% yield (2.6 mg, 0.056 mmol, yellow

amorphous solid) for two steps. The remaining solid in the aqueous

layer was reﬂuxed upon addition of acetic acid, ﬁltrated, and

lyophilized. The NMR of the solid showed a pure compound

identical to the product obtained by column chromatography. The

yield of combined products was 37% (24 mg, 0.051 mmol) over 2

steps. ¹H NMR (600 MHz, DMSO-d₆): δ 8.92−8.81 (m, 2H), 8.57−

8.40 (m, 2H), 8.25−8.15 (m, 2H), 8.14−7.98 (m, 4H), 7.91−7.83

(m, 2H), 7.21−7.11 (m, 2H), 3.87 (s, 3H). ¹³C NMR (151 MHz,

DMSO-d₆): δ 167.2, 157.9, 149.6, 148.3, 147.6, 143.0, 142.1, 138.2,

134.0, 130.4, 130.0 (2C), 128.8, 128.3, 127.8, 127.4, 126.6 (2C),

125.4 (2C), 118.3, 116.5, 114.8, 114.4 (2C), 112.3, 55.5. HRMS (pos.

ESI-TOF): m/z calcd for C₂₇H₁₉N₄O₄, 463.1401 [M + H]⁺; found,

463.1401 [M + H]⁺.

N-(4-(3-(Benzo[c][1,2,5]oxadiazol-5-yl)-1-(4-methoxyphenyl)-

1H-pyrrolo[2,3-b]pyridin-5-yl)phenyl)methanesulfonamide (40).

General procedure B was applied using halide 66 (150 mg, 0.35

mmol), 4-aminophenylboronic pinacol ester (93 mg, 0.42 mmol),

NaHCO₃(87 mg, 1.05 mmol), and Pd(PPh₃)₄(27 mg, 0.026 mmol)

in 20 mL of 3:1 MeCN/H₂O. After stirring the reaction overnight, the

mixture was cooled to room temperature, and water was added. The

water layer was extracted three times with DCM, and the combined

organic layers were concentrated in vacuo to give 180 mg of crude

amine intermediate. After analysis of the ¹H NMR, the isolated

product was taken to the next reaction step without further

puriﬁcation. Crude 4-(3-(benzo[c][1,2,5]oxadiazol-5-yl)-1-(4-me-

thoxyphenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)aniline (60 mg) was

dissolved in 4 mL of DCM, and 28 μL of pyridine (2.5 equiv, 0.34

mmol) and 26 μL of methanesulfonyl chloride (2.5 equiv, 0.34 mmol)

were added at room temperature. The mixture was stirred for 16 h,

concentrated in vacuo, and then submitted to ﬂash column

chromatography without further workup. The product was eluted

with ethyl acetate in cyclohexane (SiO₂, 0−100%) to give pure N-(4-

(3-(benzo[c][1,2,5]oxadiazol-5-yl)-1-(4-methoxyphenyl)-1H-pyrrolo-

[2,3-b]pyridin-5-yl)phenyl)methanesulfonamide 40 in 40% yield over

4,4′-(1-(4-Methoxyphenyl)-1H-pyrrolo[2,3-b]pyridine-3,5-diyl)-

dibenzoic Acid (42). General procedure C was applied using halide

55 (100 mg, 0.233 mmol), 4-methoxycarbonyl-phenylboronic acid

(125 mg, 0.694 mmol), K₂CO₃(300 mg, 2.170 mmol), and

Pd(dppf)Cl₂(6 mg, 0.008 mmol) in 10 mL of 3:1 dioxane/H₂O.

The reaction mixture was diluted with DCM (50 mL), more water

(25 mL) was added, and the solution was extracted four times with 30

mL of DCM and THF 4:1. The combined organic layers were dried

over MgSO₄, ﬁltered, and evaporated in vacuo. The crude residue was

puriﬁed by column chromatography (SiO₂, THF in DCM, 0−10%) to

yield dimethyl 4,4′-(1-(4-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridine-

3,5-diyl)dibenzoate contaminated with 4-methoxycarbonyl-phenyl-

boronic acid. The residue was dissolved in DCM (100 mL) and

extracted three times with 10% K₂CO₃solution (15 mL). The organic

layer was dried over MgSO₄, ﬁltered, and evaporated in vacuo to yield

monosubstituted intermediate in 38% yield (44.5 mg, 0.090 mmol,

white solid), which was used directly for the next reaction step.

Dimethyl 4,4′-(1-(4-methoxyphenyl)-1H-pyrrolo [2,3-b] pyridine-

3,5-diyl)dibenzoate (44.5 mg, 0.090 mmol) was dissolved in 6 mL

of THF and 2 mL of MeOH after which a solution of 73 mg (1.8

mmol, 2 mL, 0.9 M) of NaOH in water was added, and the solution

was stirred at room temperature for 8 h. The organic solvents were

removed in vacuo. The remaining aqueous solution was diluted with

DCM (10 mL), more water (15 mL) was added, and the solution was

extracted one time with DCM. Then, the water layer was acidiﬁed

until the formation of a white precipitate and extracted four times

with 25 mL of DCM and THF 4:1. The combined organic layers were

dried over MgSO₄, ﬁltered, and evaporated in vacuo. The residue was

boiled with DCM (3 mL) and after that sonicated. The supernatant

was removed (repeated twice), and the solid was dried in vacuo to

obtain 4,4′-(1-(4-methoxyphenyl)-1H-pyrrolo[2,3-b]pyridine-3,5-

diyl)dibenzoic acid 42 in 10% yield (11 mg, 0.023 mmol) as a

white amorphous solid. ¹H NMR (300 MHz, DMSO-d₆): δ 8.75 (d, J

= 2.1 Hz, 1H), 8.70 (d, J = 2.1 Hz, 1H), 8.49 (s, 1H), 8.09−8.03 (m,

6H), 7.98 (d, J = 8.4 Hz, 2H), 7.87 (d, J = 8.9 Hz, 2H), 7.17 (d, J =

8.9 Hz, 2H), 3.86 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆): δ 167.2,

167.1, 157.9, 147.3, 142.8, 142.6, 138.4, 130.5, 130.1 (2C), 130.0

(2C), 129.4, 129.1, 128.6, 128.2, 127.3 (2C), 126.7, 126.6 (2C),

125.4 (2C), 118.4, 114.7, 114.4 (2C), 55.5. HRMS (pos. ESI-TOF):

m/z calcd for C₂₈H₂₁N₂O₅, 465.1478 [M + H]⁺; found, 465.1445 [M

+ H]⁺.

1

two steps (24 mg, 0.046 mmol, yellow amorphous solid). H NMR

(300 MHz, DMSO-d₆): δ 9.97−9.81 (bs, 1H), 8.80−8.75 (m, 1H),

8.75−8.70 (m, 1H), 8.70−8.66 (m, 1H), 8.50 (s, 1H), 8.30−8.23 (m,

1H), 8.18−8.10 (m, 1H), 7.93−7.81 (m, 4H), 7.40−7.31 (m, 2H),

7.24−7.10 (m, 2H), 3.88 (s, 3H), 3.06 (s, 3H). ¹³C NMR (151 MHz,

DMSO-d₆): δ 158.0, 149.8, 148.1, 147.1, 142.9, 137.5, 134.0, 133.6,

130.4, 130.3, 130.1, 128.3 (2C), 126.5, 125.4 (2C), 120.2 (3C),

118.2, 116.3, 114.4 (2C), 113.3, 109.0, 55.5, 40.1. HRMS (pos. ESI-

TOF): m/z calcd for C₂₇H₂₂N₅O₄S, [M + H]⁺513.1416; found, [M +

H]⁺513.1419.

Methyl 4-(5-(Benzo[c][1,2,5]oxadiazol-5-yl)-1-(4-methoxyphen-

yl)-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoate (87). General procedure

B was applied using halide 80 (62 mg, 0.14 mmol), benzo[c][1,2,5]-

oxadiazole-5-boronic acid pinacol ester (41 mg, 0.17 mmol),

NaHCO₃(35 mg, 0.42 mmol), and Pd(PPh₃)₄(12 mg, 0.011

mmol) in 10 mL of 3:1 MeCN/H₂O. After the standard workup

procedure, 65 mg of the crude mixture was obtained. NMR of the

crude mixture showed clean product, 52 mg was taken for the next

step without further puriﬁcation, and the remaining 13 mg was

subjected to further puriﬁcation. Recrystallization from cold DCM,

redissolving in EtOAc, and ﬁltration over short plug of silica yielded

methyl 4-(5-(benzo[c][1,2,5]oxadiazol-5-yl)-1-(4-methoxyphenyl)-

1H-pyrrolo[2,3-b]pyridin-3-yl)benzoate 87 in 60% yield (8 mg,

1

0.017 mmol, yellow-orange amorphous solid). H NMR (300 MHz,

DMSO-d₆): δ 8.76−8.71 (m, 1H), 8.56−8.50 (m, 1H), 8.24−8.17

(m, 2H), 8.07−7.96 (m, 2H), 7.86−7.76 (m, 4H), 7.75−7.66 (m,

2H), 7.16−7.09 (m, 2H), 3.96 (s, 3H), 3.91 (s, 3H). ¹³C NMR (75

MHz, DMSO-d₆): δ 167.0, 158.9, 149.8, 148.6, 148.4, 143.4, 142.5,

138.8, 133.2 (2C), 130.6, 130.6 (2C), 128.8, 128.4, 127.8, 127.1,

126.9 (2C), 126.0 (2C), 119.3, 117.3, 116.1, 114.9, 113.0, 55.8, 52.3.

HRMS (pos. ESI-TOF): m/z calcd for C₂₈H₂₁N₄O₄477.1557 [M +

H]⁺; found, 477.1556 [M + H]⁺.

4-(5-(Benzo[c][1,2,5]oxadiazol-5-yl)-1-(4-methoxyphenyl)-1H-

pyrrolo[2,3-b]pyridin-3-yl)benzoic Acid (41). Methylester 87 (52

mg, 0.11 mmol, 1 equiv) was dissolved in dioxane (2.6 mL) and

MeOH (2.6 mL), and an aqueous solution of NaOH (0.35 mL, 12

equiv, 4 M) was added. The mixture was stirred at 60 °C for 1 h. The

crude product was concentrated in vacuo, and more water was added.

Then, the water layer was acidiﬁed until the formation of a yellow

precipitate (pH = 4.5) and extracted three times with DCM. The

combined organic layers were dried with Na₂SO₄and concentrated in

vacuo. The crude product was puriﬁed by column chromatography

(SiO₂, 50−100% EtOAc in cyclohexane) to give 4-(5-(benzo[c]-

4-(1-(4-Methoxyphenyl)-3-(3-oxo-1,3-dihydroisobenzofuran-5-

yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzoic Acid (43). Compound 43

was synthesized according to general procedure B using halide 81 (45

mg, 0.103 mmol), 4-cabroxyphenyl boronic acid (20.59 mg, 0.124

mmol), NaHCO₃(26.06 mg, 0.310 mmol), and Pd(PPh₃)₄(8.60 mg,

0.0074 mmol) in 6 mL of 3:1 MeCN/H₂O. The crude mixture was

cooled to room temperature and ﬁltrated. The resulted ﬁltrate was

dissolved in ethyl acetate and extracted with water. The organic phase

was concentrated in vacuo and puriﬁed by ﬂash column chromatog-

raphy (0−100% DCM in cyclohexane) to yield 43 in 6% (3 mg, 0.003

mmol, yellow amorphous solid). The product contained 10% of open

1

form 44 and undergoes spontaneous further hydrolysis over time. H

NMR (600 MHz, DMSO-d₆): δ 8.75 (q, J = 2.2 Hz, 2H), 8.58 (s,

1H), 8.25 (s, 1H), 8.15 (d, J = 9.0 Hz, 1H), 8.08 (d, J = 12.0 Hz, 2H),

S

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Journal of Medicinal Chemistry

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Article

7.98 (d, J = 6.0 Hz, 2H), 7.93 (d, J = 6.0 Hz, 1H), 7.89−7.86 (m,

2H), 7.18 (d, J = 6.0 Hz, 2H), 5.50 (s, 2H), 3.86 (s, 3H₃). HRMS

(pos. ESI-TOF): m/z calcd for C₂₉H₂₀N₂O₅, 477.1445 [M + H]⁺;

found, 477.1417 [M + H]⁺. IC₅₀= 0.58 μM (DiFMUP assay,

DiFMUP concentration = 20 μM).

Tween 20, 0.1% bovine serum albumin (BSA), 1 mM dithiothreitol

(DTT), freshly added prior to each measurement, and between 0.12

and 0.8 ng/μL SHP2 (ﬁnal concentration, applied concentration

adjusted to the activity of the enzyme batch used). The ﬁnal assay

volume was 30 μL. Enzyme and the test compound in buﬀer solution

were incubated for 1 h at RT. The reaction was started by adding 20

μM DiFMUP. Measurements were performed on a plate reader

(SAFIRE II, Tecan) with the following settings: ﬂuorescence reading

from top; excitation wavelength: 360 nm (bandwidth 20 nm);

emission wavelength 460 nm (bandwidth 20 nm); and ﬁve readings

with a time interval of 135 s. Measurements were performed in

triplicate. IC₅₀values were calculated with Prism 5 (for Windows,

Version 5.01, Graph Pad Software, Inc.).

Biochemical assay for the determination of IC₅₀values for

compounds 17−42 and 45−46 was carried out by Reaction Biology

Corp utilizing the DiFMUP assay protocol. Recombinant SHP2

(amino acids 246−593) was utilized. Brieﬂy, test compounds were

dissolved in DMSO at a concentration of 10 mM, and the assay was

carried out at a ﬁnal DMSO concentration <1%. The DiFMUP assay

buﬀer contained a ﬁnal concentration of 25 mM HEPES (pH 7.5), 5

mM MgCl₂, 0.01% Brij-35, 1 mM DTT, and 1% DMSO. Enzyme and

the test compound were incubated in buﬀer solution for 20 min at

room temperature. The reaction was started by adding DiFMUP with

a ﬁnal concentration of 10 μM. Measurements were performed in

duplicate using a 10-dose response protocol (concentration 100 to

0.005 μM). The following compounds were used as a reference

PHPS1, NSC87877, PTP II-B08, PTP1B inhibitor. The enzyme

activities were monitored (Ex/Em 355/460) as a time-course

measurement of the increase in the ﬂuorescence signal from the

ﬂuorescence substrate for 120 min at room temperature. Shown data

represent the average of duplicate measurement.

Docking and Binding Mode Investigations. The catalytic

domain of SHP2 from the protein data bank (PDB) entry 3O5X was

employed for docking studies with the OpenEye Python toolkit

[OpenEye Toolkits 2019.Oct.2; OpenEye Scientiﬁc Software: Santa

Fe, NM, 2019].⁷⁰The OEChem and OESpruce modules were used to

add hydrogens and to prepare protein and cocrystalized ligand for

docking. Docking of the investigated ligand series was performed with

the hybrid method, which uses the goodness of shape and interaction

overlap between the docked molecule and the cocrystalized ligand to

bias the exhaustive search of the docking algorithm. Reasonable

tautomeric states were generated for each ligand at pH 7.4 using the

OEQuacpac module. Conformations were generated with OEOmega,

and 20 docking poses were produced with high search resolution

using the OEDocking module. Chemgauss4 was used as a scoring

function. Finally, docking poses were analyzed in terms of

pharmacophoric interaction patterns using LigandScout 4.4 [Li-

gandScout 4.4, Inteligand: Vienna, Austria, 2019].⁷²

5-(5-(4-Carboxyphenyl)-1-(4-methoxyphenyl)-1H-pyrrolo[2,3-b]-

pyridin-3-yl)-2-(hydroxymethyl)benzoic Acid (44). Compound 44

was synthesized according to general procedure B using halide 81 (45

mg, 0.103 mmol), 4-cabroxyphenyl boronic acid (20.59 mg, 0.124

mmol), NaHCO₃(26.06 mg, 0.310 mmol), and Pd(PPh₃)₄(8.60 mg,

0.0074 mmol) in 6 mL of 3:1 MeCN/H₂O. The crude mixture was

cooled to room temperature and ﬁltrated. The resulted ﬁltrate was

dissolved in ethylacetate and extracted with water. The organic phase

was concentrated in vacuo and puriﬁed by ﬂash column chromatog-

raphy (0−100% DCM in cyclohexane) to give 44 in 20% yield (10

mg, 0.01 mmol, yellow amorphous solid). ¹H NMR (600 MHz,

DMSO-d₆): δ 8.74 (s, 1H), 8.67 (s, 1H), 8.42 (s, 1H), 8.19 (s, 1H),

8.06 (d, J = 6.0 Hz, 2H), 8.01−7.98 (m, 1H), 7.93 (d, J = 6.0 Hz,

2H), 7.91−7.84 (m, 3H), 7.17 (d, J = 6.0 Hz, 2H), 4.94 (s, 2H), 3.85

(s, 3H) ppm. HRMS (pos. ESI-TOF): m/z calcd for C₂₉H₂₃N₂O₆,

495.1551 [M + H]⁺; found, 495.1525 [M + H]⁺. IC₅₀= 17.0 μM

(DiFMUP assay, DiFMUP concentration = 20 μM).

4-(1-(4-Methoxyphenyl)-3-(2-oxo-2H-chromen-7-yl)-1H-pyrrolo-

[2,3-b]pyridin-5-yl)benzoic Acid (45). Compound 45 was synthesized

according to general procedure B using halide 82 (50 mg, 0.112

mmol), 4-carboxyphenylboronic acid (22 mg, 0.134 mmol), NaHCO₃

(28.2, 0.336 mmol), and Pd(PPh₃)₄(3 mg, 0.003 mmol) in 8 mL of

3:1 MeCN/H₂O. The reaction mixture was diluted with DCM (30

mL), and more water (25 mL) was added, and the solution was

extracted with DCM. Then, the water layer was acidiﬁed and

extracted four times with 30 mL of 4:1 DCM/THF. The combined

organic layer was dried over MgSO₄, ﬁltered, and evaporated in vacuo.

The crude residue was puriﬁed by column chromatography (SiO₂,

THF in DCM, 0−35%). The combined fractions were evaporated,

and the residue was sonicated with 5 mL of Et₂O for 5 min,

centrifuged, and the supernatant was removed. This procedure was

repeated with n-pentane. The remaining solid was dried in vacuo to

obtain 4-(1-(4-methoxyphenyl)-3-(2-oxo-2H-chromen-7-yl)-1H-

pyrrolo[2,3-b]pyridin-5-yl)benzoic acid 45 in 11% yield (6 mg,

0.012 mmol, yellow amorphous solid). ¹H NMR (600 MHz, DMSO-

d₆): δ 8.76−8.70 (m, 2H), 8.56 (s, 1H), 8.11 (d, J = 9.3 Hz, 1H), 8.07

(d, J = 8.3 Hz, 2H), 8.00−7.97 (m, 2H), 7.97−7.93 (m, 2H), 7.87 (d,

J = 7.9, 1.0 Hz, 2H), 7.83−7.80 (m, 1H), 7.21−7.14 (m, 2H), 6.47 (d,

J = 9.6, 0.9 Hz, 1H), 3.86 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆):

δ 167.2, 160.2, 157.9, 154.3, 147.3, 144.1, 142.9, 142.5, 137.9, 130.4,

129.9 (2C), 129.2, 129.1, 129.0, 127.4, 126.8, 125.3 (2C), 123.0

(2C), 118.3, 116.9, 115.0, 114.4 (2C), 114.2, 113.4, 55.5 ppm. HRMS

(pos. ESI-TOF): m/z calcd for C₃₀H₂₀N₂O₅, [M + H]⁺489.1445;

found, 489.1459.

Molecular Dynamic Simulation. The previously prepared

catalytic domain of SHP2 in complex with the identiﬁed docking

pose of the most active compound (45) was solvated in a cubic box

with 10 Å padding with the SPC water model and 0.15 NaCl using

4-(3-(Benzo[c][1,2,5]oxadiazol-5-yl)-1H-pyrrolo[2,3-b]pyridin-5-

yl)benzoic Acid (46). Compound 46 was obtained as a byproduct in

the preparation of 29 in 13% yield. Alternatively, 46 can be prepared

from methyl 4-(3-(benzo[c][1,2,5]oxadiazol-5-yl)-1-tosyl-1H-pyrrolo-

[2,3-b]pyridin-5-yl)benzoate by hydrolysis with 2 M NaOH. ¹H NMR

(300 MHz, DMSO-d₆): δ 12.55−12.42 (bs, 1H), 8.84−8.76 (m, 1H),

8.76−8.66 (m, 1H), 8.46−8.35 (m, 2H), 8.24−8.15 (m, 1H), 8.11−

8.03 (m, 3H), 8.01−7.94 (m, 2H). HRMS (pos. ESI-TOF): m/z

calcd for C₂₀H₁₃N₄O, 357.0987 [M + H]⁺; found, 357.0992 [M +

H]⁺.

̈

Maestro 12.3 [Maestro 12.3, Schrodinger Release 2020-1; Schro-

dinger, LLC: New York, NY, 2020]. The solvated system was

subjected to 20 ns of unbiased MD simulation with Desmond 6.1⁷³

̈

[Desmond 6.1, Schrodinger Release 2020-1; Schrodinger, LLC: New

York, NY, 2020] using the default equilibration and simulation

settings. Coordinates were saved every 100 ps and analyzed using the

molecular visualization program VMD 1.9.3 [VMD 1.9.3; University

of Illinois at Urbana−Champaign: Urbana, IL, 2016].⁷⁴

Reference compounds PHPS1, GS493, II-B08, and NSC87877

were purchased from commercial suppliers Merck (Merck Millipore)

and Merck (Sigma-Aldrich).

Phosphatase Proﬁling. Biochemical assay for the determination

of IC₅₀values was carried out by Reaction Biology Corp utilizing the

DiFMUP assay protocol. Brieﬂy, test compounds were dissolved in

DMSO at a concentration of 10 mM, and the assay was carried out at

a ﬁnal DMSO concentration <1%. The DiFMUP assay buﬀer

contained a ﬁnal concentration of 25 mM HEPES (pH 7.5), 5 mM

MgCl₂, 0.01% Brij-35, 1 mM DTT, and 1% DMSO. For PP2A alpha/

PPP2R1A complex, PP1A, and PP1B, 1 mM MnCl₂was added to

reaction buﬀer. Enzymes and test compound were incubated in buﬀer

solution for 20 min at room temperature. The reaction was started by

Biochemical SHP2 Assay. Biochemical assay for the determi-

nation of IC₅₀values for compounds 1−5 and 43−44 was carried out

according to the following DiFMUP protocol. Recombinant SHP2

(amino acids 262−532, produced in house) was utilized. Test

compounds were dissolved in (DMSO) at a concentration of 10 or

100 mM, and the assay was carried out at a ﬁnal concentration of

below 1% DMSO. DiFMUP assay buﬀer contains a ﬁnal

concentration of 25 mM MOPSO (pH 7.0), 50 mM NaCl, 0.05%

T

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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Article

adding DiFMUP with a ﬁnal concentration of 2 μM for PTPN1/

PTP1B-CD, 30 μM for PP1B, and 10 μM for all other PTPs.

Measurements were performed in duplicate using a 10-dose response

protocol (concentration 100 to 0.005 μM). Following compounds

were used as a referencePTP1B inhibitor and canthardic acid. The

enzyme activities were monitored (Ex/Em 355/460) as a time-course

measurement of the increase in the ﬂuorescence signal from the

ﬂuorescence substrate for 120 min at room temperature. Shown data

represent the average of duplicate measurement.

Kinase Proﬁling. Kinase activity assay was carried out by Reaction

Biology Corp. Brieﬂy, test compounds were dissolved in DMSO at a

concentration of 10 mM, and the assay was carried out at a ﬁnal

DMSO concentration <1%. The reaction assay buﬀer contained a ﬁnal

concentration of 20 mM Hepes (pH 7.5), 10 mM MgCl₂, 1 mM

EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mM Na₃VO₄, 2 mM

DTT, and 1% DMSO. Required cofactors were added individually to

each kinase reaction. Enzyme and the test compound (ﬁnal

concentration 100 μM) were incubated in buﬀer solution for 20

min at room temperature. The reaction was started by adding ³³P-γ-

ATP with a ﬁnal concentration of 10 μM. Kinase reaction was

incubated for 2 h at room temperature, and reactions were spotted

onto P81 ion-exchange paper. Kinase activity was detected by the

ﬁlter-binding method. Measurements were performed in duplicate

using a single dose response protocol, and percentage of kinase

activity was calculated. Shown data represent the average of duplicate

measurement. Staurosporine was used as a reference.

Cell Culture. The human pancreatic adenocarcinoma cell line

HPAF-II (ATCC CRL-1997) obtained from ATCC (American Type

Culture Collection) was cultured in Dulbecco’s modiﬁed Eagle’s

medium (Gibco BRL, Gaithersburg, MD, USA) containing 4.5 g/L

glucose, supplemented with 10% (v/v) fetal bovine serum (FBS,

Gibco BRL), and 100 U/mL penicillin−streptomycin (Gibco BRL).

The human hepatocellular carcinoma cell line HepG2, purchased

from ATCC (HB-8065) was maintained in RPMI-1640 medium

(Gibco BRL), supplemented with 10% (v/v) FBS (Gibco BRL), and

100 U/mL penicillin−streptomycin (Gibco BRL).

Scatter Assay. HPAF-II cells were plated at a density of 1000 cells

per well in medium supplemented with 5% FBS into 384-well cell-

bind microplates (Corning Life Sciences, Acton, MA, USA) and

incubated for 24 h prior to treatment. The test compounds were

dissolved and stored in DMSO at a concentration of 10 mM, and a 2-

fold serial predilution was prepared yielding a ﬁnal DMSO

concentration of 0.4% (v/v). Compound medium solutions were

added to the assay plates and incubated for 1 h. Subsequently,

recombinant human hepatocyte growth factor (HGF, provided by W.

Birchmeier) was added to a ﬁnal concentration of 300 pM. The cells

were further incubated for 20 h, ﬁxed with 4% formaldehyde/

phosphate-buﬀered saline, and the nuclei were stained with Hoechst

33342 (10 μM, Sigma-Aldrich).

fold concentrated compound medium solutions, followed by

stimulation with recombinant human HGF by adding a predilution

of 5 μL per well to achieve a ﬁnal concentration of 300 pM. Controls

received medium plus DMSO with a ﬁnal concentration of 0.2%. The

impedance was monitored every 5 min for 4 h.

Cell Growth and Cytotoxicity. The cells (HPAF-II and HepG2)

were seeded in 384-well microplates at 750 cells in 40 μL medium per

well (CellCarrier-384 ultra, PerkinElmer) and incubated for 20 h.

Then, 10 μL per well of 5-fold concentrated serial compound

dilutions were added into the plates (ﬁnal concentration 40 −0.6 μM,

0.4% DMSO). Following further incubation for 72 h, a live-cell

staining of nuclei was performed using Hoechst 33342 (1 μM, Sigma-

Aldrich) and TO-PRO-3 (1 μM, Invitrogen) as the dead cell

indicator. The Array-Scan XTI Reader (Thermo Fisher Scientiﬁc Inc.)

was used to acquire images with a 10× objective in the Hoechst

33342-associated ﬁlter channel (BGRFR 386_23) and the TO-PRO-

3-associated far-red ﬁlter channel (BGRFR 650_13). Cell prolifer-

ation was determined by counting of valid Hoechst 33342 ﬂuorescent

cell nuclei, whereas the number of TO-PRO-3 ﬂuorescent nuclei was

used for the cytotoxicity analysis.

Colony Formation Assay. VACO432 and HCC1806 cells were

seeded at 5000 cells/well in 12-well plates (Sarstedt) and allowed to

adhere overnight. The next day, compounds were added as indicated

and treatments were refreshed every 3−4 days. When vehicle

(DMSO)-treated cells (indicated as “UT” in all ﬁgures) reached

conﬂuence, all wells were ﬁxed in 3.7% formaldehyde, stained with

0.1% crystal violet and subsequently scanned.

Western Blotting. Cell samples were lysed in RIPA buﬀer [50

mM Tris pH 7.4, 150 mM NaCl, 1% NP40, 0.1% sodium dodecyl

sulfate (SDS) and 0.5% sodium deoxycholate] supplemented with

protease inhibitor (Roche, #11836153001) and phosphatase inhibitor

cocktails 2 and 3 (Sigma-Aldrich, #P57261 and #P0044). Protein

concentrations were determined by Pierce BCA protein assay kit

(Thermo Scientiﬁc, #23225). Proteins were separated by SDS-PAGE

in Laemmli buﬀer (0.25 M Tris, 1.92 M glycine, 1% SDS), transferred

to polyvinylidene diﬂuoride membranes (Carl Roth, pore size 0.45

μM) in transfer buﬀer (25 mM Tris, 192 mM glycine, 20% methanol),

and subsequently incubated overnight at 4 °C with indicated

antibodies in 5% BSA in PBST. ECL (PerkinElmer,

NEL104001EA) was used to detect antibodies in a Vilber Fusion

FX. Antibodies against HSP90 (sc-13119) and p-ERK Y204 (sc-7383)

were purchased from Santa Cruz Biotechnology..

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01265.

■

sı

Docking complexes in PDB format (ZIP)

An Array-Scan XTI Reader (Thermo Fisher Scientiﬁc Inc.) was

used to acquire images in the Hoechst 33342-associated ﬁlter channel

(BGRFR 386_23) with a 10× objective. For each well, four image

ﬁelds were acquired, ensuring that typically 1000 cells or more could

be analyzed using the morphology analysis bioapplication of the HCS

Studio software. The cell scattering was quantiﬁed based on the

analysis of nuclei minimum distances. Valid nuclei were identiﬁed

applying thresholds for size, shape, and intensity. The percentage of

nuclei showing a minimum neighbor distance above a deﬁned

threshold value was calculated.

Impedance. The scatterassay was additionally applied to label-free

kinetic impedance measurements using the xCeLLigence RTCA SP

System (ACEA Biosciences Inc., San Diego, CA, USA). This live-cell

assay was performed with HPAF-II cells in a 96-well E-plate format.

The electrical impedance was measured by the RTCA-integrated

software as a dimensionless parameter termed CI.

Ligand alignment studies, alternative synthetic pathway

for the preparation of the azaindoles, docking poses of

the representative compounds, full-detailed phosphatase

and kinase proﬁling, cell permeability experiments, and

HRMS data of the key compounds (PDF)

Molecular formula strings of compounds 8−46 (CSV)

AUTHOR INFORMATION

Corresponding Author

■

́

Marc Nazare − Leibniz-Forschungsinstitut für Molekulare

Pharmakologie (FMP), Campus Berlin-Buch, 13125 Berlin,

Germany; orcid.org/0000-0002-1602-2330; Phone: +49

30 9406-3083; Email: nazare@fmp-berlin.de

After doing the background measurement of 50 μL medium per

well, 12,000 cells were added in 40 μL of medium to each well of the

E-plate 96. The attachment and growth of the cells was recorded by

the xCELLigence system every 30 min for 20 h. Then, the cells were

preincubated with the compounds for 1 h by transferring 5 μL of 20-

Authors

Yelena Mostinski − Leibniz-Forschungsinstitut für Molekulare

Pharmakologie (FMP), Campus Berlin-Buch, 13125 Berlin,

Germany

U

https://dx.doi.org/10.1021/acs.jmedchem.0c01265

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Notes

Guus J. J. E. Heynen − Max Delbrück Center for Molecular

Medicine in the Helmholtz Association (MDC), Campus

Berlin-Buch, 13125 Berlin, Germany

The authors declare no competing ﬁnancial interest.

́

Maria Pascual Lopez-Alberca − Leibniz-Forschungsinstitut

ACKNOWLEDGMENTS

■

für Molekulare Pharmakologie (FMP), Campus Berlin-Buch,

13125 Berlin, Germany

M.N., Y.M., M.P.L.-A., J.d.S., S.M., and J.P. would like to thank

Dr. Edgar Specker (FMP) and the NMR core facility of the

FMP for their excellent support on compound character-

ization. Y.M. thanks the Minerva-Stiftung of the Max Planck

Society for a postdoctoral fellowship. A.V. and D.S. thank Prof.

Dr. Gerhard Wolber (Freie Universitat Berlin) for providing a

license for LigandScout 4.4.

Jerome Paul − Leibniz-Forschungsinstitut für Molekulare

Pharmakologie (FMP), Campus Berlin-Buch, 13125 Berlin,

Germany

Sandra Miksche − Leibniz-Forschungsinstitut für Molekulare

Pharmakologie (FMP), Campus Berlin-Buch, 13125 Berlin,

Germany

̈

Silke Radetzki − Leibniz-Forschungsinstitut für Molekulare

Pharmakologie (FMP), Campus Berlin-Buch, 13125 Berlin,

Germany

David Schaller − Charité−Universitätsmedizin Berlin, 10117

Berlin, Germany

ABBREVIATIONS

■

SHP2, Src homology-2 domain containing protein tyrosine

phosphatase-2; PTP, protein tyrosine phosphatase; MAPK,

mitogen-activated protein kinase; JAK-STAT, Janus kinase/

signal transducer and activator of transcription; PI3K-akt,

phosphatidylinositol-3-kinase-Akt; MEK, MAPK/ERK kinase;

BRAF, v-raf murine sarcoma viral oncogene homolog B1;

STAT3, signal transducer and activator of transcription 3;

PAINS, pan-assay interference compounds; SAR, structure−

activity relationship; Boc, tert-butyloxycarbonyl; NIS, N-

iodosuccinimide; DIPEA, N,N-diisopropylethylamine; DiFM-

UP, diﬂuoromethylumbelliferyl phosphate; THP, tetrahydro-

pyran; EGFR, epidermal growth factor receptor; ERK,

extracellular signal-regulated protein kinase; DMSO, dimethyl-

sulfoxide; DCM, dichloromethane; ACN, acetonitrile; BSA,

bovine serum albumin; SDS, sodium dodecyl sulfate

Elena Shanina − Max-Planck-Institut für Kolloid- und

Grenzﬂächenforschung, 14476 Potsdam, Germany

Carola Seyﬀarth − Leibniz-Forschungsinstitut für Molekulare

Pharmakologie (FMP), Campus Berlin-Buch, 13125 Berlin,

Germany

Yuliya Kolomeets − Leibniz-Forschungsinstitut für Molekulare

Pharmakologie (FMP), Campus Berlin-Buch, 13125 Berlin,

Germany

Nandor Ziebart − Leibniz-Forschungsinstitut für Molekulare

Pharmakologie (FMP), Campus Berlin-Buch, 13125 Berlin,

Germany

Judith de Schryver − Leibniz-Forschungsinstitut für

Molekulare Pharmakologie (FMP), Campus Berlin-Buch,

13125 Berlin, Germany

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́

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authors. All authors have given approval to the ﬁnal version of

the manuscript.

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From Pyrazolones to Azaindoles: Evolution of Active-Site SHP2 Inhibitors Based on Scaffold Hopping and Bioisosteric Replacement

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