Discovery of Potent and Brain-Penetrant Tau Tubulin Kinase 1 (TTBK1) Inhibitors that Lower Tau Phosphorylation in Vivo

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Discovery of Potent and Brain-Penetrant Tau Tubulin Kinase 1

(TTBK1) Inhibitors that Lower Tau Phosphorylation In Vivo

Tamara Halkina,* Jaclyn L. Henderson, Edward Y. Lin, Martin K. Himmelbauer, J. Howard Jones,

Marta Nevalainen, Jun Feng, Kristopher King, Michael Rooney, Joshua L. Johnson, Douglas J. Marcotte,

Jayanth V. Chodaparambil, P. Rajesh Kumar, Thomas A. Patterson, Paramasivam Murugan, Eli Schuman,

LaiYee Wong, Thomas Hesson, Sarah Lamore, Channa Bao, Michael Calhoun, Hannah Certo,

Brenda Amaral, Gregory M. Dillon, Rab Gil ﬁ llan, and Felix Gonzalez-Lopez de Turiso *

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Supporting Information

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ABSTRACT: Structural analysis of the known NIK inhibitor 3 bound to the kinase domain of TTBK1 led to the design and

synthesis of a novel class of azaindazole TTBK1 inhibitors exempliﬁed by 8 (cell IC₅₀: 571 nM). Systematic optimization of this

series of analogs led to the discovery of 31, a potent (cell IC₅₀: 315 nM) and selective TTBK inhibitor with suitable CNS penetration

(rat K_p,uu: 0.32) for in vivo proof of pharmacology studies. The ability of 31 to inhibit tau phosphorylation at the disease-relevant Ser

422 epitope was demonstrated in both a mouse hypothermia and a rat developmental model and provided evidence that modulation

of this target may be relevant in the treatment of Alzheimer’s disease and other tauopathies.

Despite the potential advantage of targeting TTBK1 with a

small molecule inhibitor, this approach is not without risk.

Speciﬁcally, a closely related isoform (TTBK2, 88% identity

and 96% similarity to TTBK1 in the kinase domain) with a

role in cellular processes such as ciliogenesis has been

identiﬁed and it is unclear what selectivity proﬁle would be

needed to develop a safe and eﬃcacious therapeutic for a

chronic indication such as AD.¹²⁻¹⁴In addition, the few

TTBK1 inhibitors reported in the literature are not suitable for

proof of pharmacology studies due to lack of potency and CNS

penetration. Furthermore, the premise that small molecule

inhibition of TTBK1 leads to reduction of tau phosphorylation

had not been demonstrated in preclinical animal models.¹⁵In

this report, we describe the discovery of potent and selective

TTBK1 inhibitors leading to 31, a tool compound with

INTRODUCTION

■

Tau tubulin kinase 1 (TTBK1) is a kinase that is speciﬁcally

expressed in the CNS^1,2and has been reported to

phosphorylate tau at a number of disease-related epitopes.^3,4

Hyperphosphorylated tau, which aggregates in the form of

neuroﬁbrillary tangles (NFTs), is a key pathological hallmark

in a number of neurodegenerative diseases, including

Alzheimer’s disease (AD),^5,6and is thought to play a signiﬁcant

role in the progression of disease. Consequently, reducing tau

phosphorylation has been proposed as a therapeutic strategy to

prevent NFT formation^7,8and slow neurodegeneration.⁹To

test this hypothesis, small molecule inhibitors that modulate

tau phosphorylation through multiple pathways have been

identiﬁed and advanced to the clinic. In particular, small

molecule GSK3 inhibitors have progressed to late stage clinical

trials but, after more than three decades, these studies have not

led to an FDA-approved therapeutic and an opportunity exists

to identify alternative targets to modulate tau phosphoryla-

tion.^10,11Within this context, modulation of TTBK1 activity

might be a superior strategy given that this kinase is only

expressed in the CNS and its upregulation has been linked to

AD pathology in humans.¹

Received: March 2, 2021

Published: May 4, 2021

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Figure 1. Proﬁle of TTBK1 inhibitors reported in the literature.

Figure 2. (a) Cocrystal structure of 3 (magenta) with the human kinase domain of TTBK1 highlighting key interactions in the active site (PDB

code: 7JXX). (b) Surface view illustrating key elements of tertiary kinase structure and showcasing the narrow entrance to the back pocket. A water

molecule in the lipophilic back pocket is displayed as a red sphere. (c) Schematic representation of the binding mode of 3 in the kinase domain of

TTBK1.

Figure 3. Initial simpliﬁcation of the TTBK1 pharmacophore.

suitable properties to interrogate the link between TTBK1

inhibition and tau phosphorylation in vivo.

compound with signiﬁcant TTBK1 activity, the NF-κB

inducing kinase (NIK) inhibitor 3 (Figure 1, this analog was

reported to have >50% TTBK1 inhibition at a 100 nM

concentration).²¹The potency of this compound was

conﬁrmed in both a biochemical assay (TTBK1 IC₅₀: 199

nM, Figure 1) and a cellular assay (IC₅₀: 1.85 μM for the

inhibition of tau phosphorylation at Ser 422; see Supporting

Information). Subsequent evaluation of 3 in an in vitro

MDCK−MDR1 assay revealed a high eﬄux ratio (ER) of 110,

predicting low unbound exposure in the brain.²²

To enable a structure-based design optimization approach,

the X-ray cocrystal structure of 3 in complex with the human

kinase domain of TTBK1 (at a 1.5 Å resolution, Figure 2) was

elucidated. This structure showed that the aminopyrimidine

ring made two hydrogen bond contacts with the backbone of

RESULTS AND DISCUSSION

■

At the outset, we anticipated that the identiﬁcation of potent

and brain-penetrant TTBK1 inhibitors might be challenging

given that staurosporine, a pan-kinase inhibitor, is inactive

against this kinase at a 100 μM concentration.¹⁶In addition,

compounds 1¹⁷and 2¹⁸(Figure 1), which had been reported

as TTBK1 inhibitors, were found to have very weak activity in

a TTBK1 biochemical assay (1, IC₅₀: 4.61 μM; 2, IC₅₀: 2.51

μM).¹⁹To identify a more suitable candidate for optimization,

a number of approaches were explored, including a systematic

examination of the oﬀ-target activity of known kinase

inhibitors.²⁰This strategy led to the identiﬁcation of a

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Figure 4. Identiﬁcation of azaindazole TTBK1 inhibitors.

Table 1. Systematic Optimization of the 5,6-Fused Ring System

a

b

Results represent the arithmetic mean of a minimum of two determinations. MDCK−MDR1 human P-gp transfected cell line. P_app(A−B)

:

c

apparent permeability apical-to-basolateral (10⁻⁶cm/s); ER = B−A/A−B eﬄux ratio. Cl_int.

the hinge residues Gln 108 and Gln 110. Notably, the alkyne

group displaced the catalytic lysine and projected the tertiary

alcohol through a narrow tunnel into the back pocket of the

protein, thus positioning this group in an ideal orientation to

interact with both the backbone N−H of Phe 177 and the

carboxylic acid of Glu 77 (Figure 2a−c). This structure also

suggested that the N−H bond of the indole was solvent

exposed and that the three methylene groups of the seven-

membered ring helped maintain the planarity of the molecule

rather than making any contacts with the protein.

Our initial optimization of analog 3 focused on truncating

the cycloheptadiene ring to reduce its structural complexity

and allow for quick analog synthesis and optimization. We

hypothesized that the resulting indole 4 (Figure 3) would

maintain the key interactions observed in the X-ray structure of

3 (Figure 2), although an increased entropy of 4 relative to 3

may lead to an initial reduction in potency. As predicted,

compound 4 was ﬁvefold less potent relative to its tetracyclic

precursor (4, IC₅₀: 1022 nM vs 3, IC₅₀: 199 nM). To induce

coplanarity of the indole and the aminopyrimidine rings, a

nitrogen atom at the 2-position of the indole was introduced,

and the resulting indazole 5 demonstrated an improvement in

potency (5, IC₅₀: 185 nM, Figure 3).²³The high ER of 5 was

mitigated by methylation of N1 of the indazole to aﬀord 6,

which had a similar level of cellular potency relative to 3 (6,

cell IC₅₀: 1.42 μM and ER: 2.0 vs 3, cell IC₅₀: 1.85 μM and ER:

11).

To improve the potency of these inhibitors, we sought to

engage the catalytic lysine (Lys 63, Figure 2a) in a direct

hydrogen bond with the indazole core by introducing a

hydrogen bond acceptor at the 5-position of indazole 6 (Figure

4). Gratifyingly, the resulting azaindazole 7 was nearly fourfold

more potent in the biochemical assay relative to 6 (7, IC₅₀: 96

nM vs 6, IC₅₀: 352 nM, Figure 4) although this compound also

had a high ER (19). However, subsequent synthesis of the

regioisomeric N1-linked azaindazole 8 led to an analog with

similar potency (cell IC₅₀: 571 nM) and signiﬁcantly lower ER

(5.5). The improvement in the ER of 8 was rationalized by its

lower predicted basic pKa relative to 7 (8, N5 pKa: 4.9 vs 7,

N5 pKa: 5.8).

Subsequent systematic modiﬁcation of the 5,6-fused hetero-

cycle led to the discovery of compounds with improved

potency (Table 1). Speciﬁcally, both azaindole 9 and

azaindoline 10 displayed improved potency relative to 8 (9,

cell IC₅₀: 114 nM and 10, cell IC₅₀: 177 nM vs 8, cell IC₅₀: 571

nM). However, despite the reduced polar surface area (PSA)

(9, PSA: 90 and 10, PSA: 88 vs 8, PSA: 103), these

compounds were found to be P-gp substrates (9, ER: 10; 10,

ER: 26).²⁴The observed increase in the ER of these analogs

was consistent with progressively increasing predicted basicity

of N5 from 8 (pKa: 4.9) to 9 (pKa: 5.7) to 10 (pKa: 7.4, Table

1), suggesting that the ER is correlated with the basicity of

these compounds rather than with their PSA. We therefore

hypothesized that replacing N5 of 9 with a non-basic

functional group may lower the ER without disrupting the

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Table 2. SAR of the C3 and C4 Substitution of the 5-Azaindole Core

a

b

Results represent the arithmetic mean of a minimum of two determinations. MDCK−MDR1 human P-gp transfected cell line. P_app(A−B)

:

c

apparent permeability apical-to-basolateral (10⁻⁶cm/s); ER = B−A/A−B eﬄux ratio. Cl_int.

Figure 5. Optimization of alkyne substitution in the back pocket.

Figure 6. (a) Overlay of the cocrystal structures of 3 (magenta) and 18 (cyan) in the human kinase domain of TTBK1 highlighting key interactions

of 18 with the hinge and Lys 63 (only cartoon for the protein from the X-ray cocrystal structure of 18 is shown for clarity, PDB code: 7JXY). (b)

Protein surface view of the cocrystal structure of 18 (cyan) with the human kinase domain of TTBK1 showing interactions in the back pocket. (c)

Schematic representation of the binding mode of 18 in the kinase domain of TTBK1.

putative interaction with the protein. Consistent with our

substitution, respectively) showed a signiﬁcantly reduced ER of

prediction, indole analogs 11 and 12 (with 5-OMe and 5-F

3.0 and 0.8, respectively, but this reduction of the ER was

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Table 3. SAR of the Substituent in the Back Pocket

a

b

Results represent the arithmetic mean of a minimum of two determinations. MDCK−MDR1 human P-gp transfected cell line. P_app(A−B)

:

apparent permeability apical-to-basolateral (10⁻⁶cm/s); ER = B−A/A−B eﬄux ratio.

accompanied by a greater than 15-fold loss in cellular potency

relative to 9 (11, cell IC₅₀: 2.50 μM; 12, cell IC₅₀: 1.85 μM vs

9, cell IC₅₀: 114 nM).

its absolute stereochemistry. As expected, 18 had a similar

binding mode to that of compound 3. The aminopyrimidine

hinge binder was coplanar with the azaindazole core, and the

tertiary alcohol reached into the back pocket through the

narrow tunnel ﬂanked by Met 107 and Lys 63. Consistent with

our hypothesis, the additional interaction of N5 was conﬁrmed

to be a direct hydrogen bond with Lys 63. The boost in

potency provided by the ethyl group can be explained by the

displacement of a water molecule (visualized as a red sphere in

Figure 2b) from the hydrophobic back pocket and the resulting

favorable van der Waals interactions of the ethyl group with

lipophilic protein residues (Leu 81 and Phe 177).

Attempts to replace the alkyne within the context of both

the azaindazole and the azaindole cores were largely

unsuccessful (Table 3). Although our computational modeling

using the X-ray cocrystal structure of 18 with TTBK1

suggested reasonable docking poses that could maintain the

key hydrogen bonds in the back pocket, most non-alkyne

analogs, including a known bioisostere bicyclo[1.1.1]pentane

27,²⁵were inactive. The only analogs that retained some

activity against TTBK1 were alkenes 20 and 24 (IC₅₀: 1.94 and

0.41 μM, respectively), and fully saturating the side chain led

to a greater than 10-fold loss in potency (21, 16.9 μM).

As a suitable isostere for the alkyne pharmacophore could

not be identiﬁed,^26,27the chemical reactivity of this functional

group was explored in glutathione (GSH) conjugation

experiments (see Supporting Information). A selection of

inhibitors was incubated with GSH for up to 4 h, and it was

Having explored the impact of modifying the central 5,6-

fused heterocyclic core, we pursued optimization of the

putative solvent-exposed substituents in 9 (Table 2). We

hypothesized that substitution at positions C3 or C4 could

impart desirable physicochemical properties without disrupting

the key hinge and back pocket hydrogen bonds. As expected, a

variety of aliphatic substituents were tolerated (cell IC₅₀: 98−

742 nM, Table 2); however, the ER remained generally high.

Two notable exceptions were the C4-methyl ether 13 (ER:

2.9) and the C3-piperidine 17 (ER: 1.2). Unfortunately, these

analogs were relatively weak inhibitors in the cellular assay (13,

cell IC₅₀: 742 nM; 17, cell IC₅₀: 737 nM).

In an attempt to improve the cellular potency of these

inhibitors, we looked extensively at the substituents reaching

into the back pocket within both the azaindazole and the

azaindole series. Both the vector and the immediate environ-

ment of the tertiary alcohol were found to be critical to

potency and only small changes to the alkyl substituents were

tolerated. Speciﬁcally, extending one of the methyl groups in 8

and 9 by one carbon provided a three- to ﬁvefold improvement

in biochemical potency compared to their parent compounds

(18, IC₅₀: 18 nM, and 19, IC₅₀: 2.3 nM, Figure 5).

Elucidation of an X-ray cocrystal structure of 18 with

TTBK1 (at a 2.2 Å resolution, Figure 6) helped understand the

interactions of this inhibitor with the active site and established

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Table 4. Cytotoxicity and GSH Stability Data

a

Results represent the arithmetic mean of a minimum of two determinations.

Table 5. Hinge Binder Optimization

a

b

Results represent the arithmetic mean of a minimum of two determinations except where indicated. MDCK−MDR1 human P-gp transfected cell

c

line. P_app(A−B): apparent permeability apical-to-basolateral (10⁻⁶cm/s); ER = B−A/A−B eﬄux ratio. Only one determination.

observed that all the inhibitors with fully aromatic cores (6, 12,

17, 18, and 19, Table 4) were >99% unmodiﬁed under the

experimental conditions. In contrast, compound 10, with a

partially saturated azaindoline core, was not stable (17% GSH

addition observed, Table 4).

Shifting the primary amine from C2 to C6 of the pyrimidine in

19 aﬀorded 29 with a greatly reduced cytotoxicity of over 20

μM and an improvement in potency relative to 28 (29, cell

IC₅₀: 37 nM vs 28, cell IC₅₀: 1646 nM, Table 5). Useful levels

of cellular potency and an acceptable cytotoxicity proﬁle were

ultimately achieved by further modifying the C6 substitution of

the 2-aminopyrimidine. Thus, incorporation of a methyl group

at the C6-position led to compound 30, which had good

cellular potency (IC₅₀: 224 nM) and a moderate level of

cytotoxicity in HepG2 cells (Glu IC₅₀/Gal IC₅₀: 3.2/3.8 μM).

Replacing the methyl group in 30 with a 6-methoxy substituent

led to the discovery of 31, an analog with low ER (3.7), a

satisfactory level of both biochemical and cellular potency

(IC₅₀: 2.7 and 315 nM, respectively), and low cytotoxicity

(Glu IC₅₀/Gal IC₅₀: 8.9/10 μM). A regioisomer of 31 with the

C5-methyl ether substitution was also synthesized, but despite

its good potency (32, cell IC₅₀: 239 nM), this analog had a

Despite the high chemical stability of many of the alkynes in

Table 4, all these inhibitors were found to be highly cytotoxic

in a HepG2 in vitro assay. It is important to note that the

cytotoxicity in this assay could not be due to on-target TTBK1

inhibition since these cells do not express TTBK1 (see

Supporting Information). Additional cytotoxicity data from a

larger set of analogs with diﬀerent core and alkyne substituents

were also found to not be favorable (data not shown), which

prompted us to investigate the impact of modifying the hinge

binder. Initially, monomethylation of the primary amine in 9

led to compound 28 that had a slightly improved cytotoxicity

proﬁle, albeit with a signiﬁcant reduction in biochemical

potency (28, IC₅₀: 124 nM vs 9, IC₅₀: 9.8 nM) (Table 5).

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a

Table 6. Kinase Selectivity Proﬁle and Cytotoxicity of Key Analogs

a

Dendrograms are shown for a compound test concentration of 1 μM (Image generated using TREEspot Software Tool and reprinted with

b

permission from KINOMEscan, a division of DiscoveRx Corporation, DISCOVERX CORPORATION 2010). Results represent the arithmetic

mean of a minimum of two determinations.

Table 7. Proﬁle of TTBK1 Inhibitors 18, 19, and 31

b

HLM/RLM (mL/

Sol. (pH 6.8)

(μg/mL)

MDCK−LE

hERG IC₅₀

(μM)

MDCK−MDR1

RPPB/

HPPB (f_u)

Rat/Mouse Cl (mL/

min/kg)

Rat

d

a

c

analog

min/kg)

P_A−B/P_B−A

P_app(A−B)/ER

K_p,uu

e

18

19

31

<5/36

11/155

16/219

46

44

45

25/16

32/19

27/21

>30

3.3/5.7

3.0/8.6

7.8/3.7

13/8.8

5.2/2.7

1.2/2.1

17 /−

0.08

0.10

0.32

f

64 /−

g

h

62 /67

a

b

Cl_int. MDCKII cell line with low expression of P-gp. P_A−B: permeability apical-to-basolateral (10⁻⁶cm/s). P_B−A: permeability basolateral-to-apical

c

(10⁻⁶cm/s). MDCK−MDR1 human P-gp transfected cell line. P_app(A−B): apparent permeability apical-to-basolateral (10⁻⁶cm/s); ER = B−A/A−

B eﬄux ratio. DMA/EtOH/PG/water in a 1:1:3:5 ratio was used as a vehicle. V_dss: 2480 mL/kg, t_1/2: 4.3 h. V_dss: 2170 mL/kg, t_1/2: 3.6 h. V_dss

4870 mL/kg, t_1/2: 3.6 h.

d

e

f

g

:

h

V_dss: 474 mL/kg, t_1/2: 0.1 h.

high ER (57) and was cytotoxic at sub-micromolar

concentrations.

TTBK2 in a biochemical assay, indicating that inhibition of

this isoform was not responsible for the observed cytotoxicity.

In addition, compound 31 was also evaluated in a Euroﬁns

panel against 68 common target proteins which revealed that

only Adenosine A₁, A_2A, and A₃were potential oﬀ targets (61,

64, and 73% inhibition, respectively, at a 10 μM test

concentration).

As a further assessment of compound safety liabilities, we

progressed our top compounds into kinase selectivity screening

using the DiscoverX kinome panel (468 kinases). Compounds

19 and 31 (Table 6, bottom) both showed an excellent

selectivity proﬁle. The selectivity of 31 was conﬁrmed in

follow-up dose−response experiments, in which this inhibitor

was tested against the major oﬀ-target kinases observed in the

kinome panel (see Supporting Information). These assays

revealed that only CK1δ and PRKD1 were inhibited with less

than a 20-fold selectivity over TTBK1 and that 31 had very

weak activity against NIK (>400-fold selectivity vs TTBK1).

Notably, inhibitors 18, 19, and 31 were not selective against

Further characterization of compounds 18, 19, and 31

revealed that all three analogs had good solubility and

̀

permeability and did not inhibit the human Ether-a-go-go-

Related Gene (hERG) channel at a concentration up to 30 μM

in an in vitro assay (Table 7). Compound 18 also had low in

vitro intrinsic clearance in human liver microsomes (HLM)

and low in vivo rat clearance. In contrast, 19 and 31 had good

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HLM stability but a signiﬁcantly lower rat liver microsomal

(RLM) stability, which translated to high in vivo clearance.

The CNS penetration of these analogs was tested in rat K_p,uu

experiments (see Supporting Information). These studies

revealed that while 18 and 19 had low brain penetration

(K_p,uu< 0.1), compound 31 had an improved K_p,uuof 0.32,

making it a good candidate for in-depth in vivo proof of

pharmacology experiments.

In Vivo Pharmacology. To assess the ability of compound

31 to inhibit in vivo tau phosphorylation at disease-relevant

sites, two diﬀerent animal models were utilized: a mouse

isoﬂurane-induced hypothermia model (Figure 7) and a rat

Figure 8. Inhibitor 31 was administered at 80 mg/kg (I.P.) to post-

natal day 10 rats. Tau phosphorylation at Ser 422 was attenuated at

0.5, 1, 3, and 6 h timepoints. Phosphorylation signal at Ser 422 was

normalized to the level of total tau in each sample. Data are

represented as the mean +/− SEM. (p = 0.0525, t = 0.5 h; p = 0.004, t

= 1 h; p = 0.0019, t = 3 h; p = 0.0021, t = 6 h; p = 0.1552, t = 24 h). A

one-way ANOVA was used to look for signiﬁcance across timepoints.

A Tukey’s multiple comparisons test was used to look for eﬀects

between groups, using the level of phosphorylation at Ser 422 present

at the 0 h timepoint as the control.

were corrected to free concentration using an experimentally

derived unbound free fraction value of 31 in brain tissue. As

expected, we observed a substantial increase in tau

phosphorylation at the TTBK1 epitope Ser 422 following

isoﬂurane treatment when compared to non-hypothermic

control animals (Figure 7; p = 0.0236; unaltered western

blot images are available in Supporting Information, Figure

S1). Animals that received 180 mg/kg compound 31

demonstrated a signiﬁcant reduction in tau phosphorylation

at Ser 422 when compared to vehicle-treated animals (p =

0.0039). In addition, although not statistically signiﬁcant, there

was a trend indicating a dose-responsive eﬀect of compound

31 on tau phosphorylation levels at Ser 422 (20 mg/kg, 21%

decrease and 60 mg/kg, 60% decrease, respectively, in tau

phosphorylation at Ser 422 vs vehicle-treated control).

Due to the biological constraints of maintaining animals in a

hypothermic state for long periods of time (maximum time

under isoﬂurane was 1 h), a rat developmental model of tau

phosphorylation was also explored. This model relied on the

reported observation that, during brain development, tau

phosphorylation is signiﬁcantly elevated at multiple epitopes,

including Ser 422, for several weeks after birth (see Supporting

Information, Figure S2). To take advantage of this feature, we

designed an experiment to understand the temporal correlation

between brain drug exposure and inhibition of tau phosphor-

ylation. In this study, compound 31 was administered I.P. to

post-natal day 10 rats at an 80 mg/kg dose (this dose was

selected using the data from a previous hypothermia study, an

80 mg/kg dose was predicted to sustain coverage of the cellular

IC₉₀). Groups of animals were sacriﬁced at several timepoints

following treatment, and cortical samples from each hemi-

sphere were isolated to determine the tau phosphorylation

level and drug concentration in the brain (Figure 8, time: 0.5−

24 h; see also Supporting Information, Figure S3). Our results

demonstrate a signiﬁcant lowering of tau phosphorylation at

Ser 422 within 1 h after administration of compound 31 (p =

0.0525, t = 0.5 h; p = 0.004, t = 1 h; Figure 8). This eﬀect

lasted for longer than 6 h (p = 0.0019, t = 3 h; p = 0.0021, t = 6

h; p = 0.1552, t = 24 h; Figure 8), and tau phosphorylation

level returned to baseline 24 h after administration of the

TTBK1 inhibitor. Twenty-four hours following compound

administration, no detectable levels of 31 were found in the

Figure 7. Inhibitor 31 was administered at 2.5, 7, 20, 60, and 180 mg/

kg to 2 month-old, male C57Bl/6 mice (n = 4−6 per group) 5 min

prior to isoﬂurane-induced hypothermia. At the 20, 60, and 180 mg/

kg groups, tau phosphorylation at Ser 422 was attenuated relative to

non-hypothermic controls. Phosphorylation signal at Ser 422 was

normalized to the level of total tau in each sample. Data are

represented as the mean +/− SEM. (*p = 0.0236; **p = 0.0039

relative to vehicle). The level of tau phosphorylation at Ser 422 was

normalized to the amount of total tau within a sample. To determine

the eﬀect of hypothermia on tau phosphorylation levels, an unpaired

t-test was run directly comparing animals with or without isoﬂurane

treatment. To determine drug treatment eﬀects, an ordinary one-way

ANOVA was used across all groups that underwent hypothermia. A

Tukey’s multiple comparisons test was used to look for eﬀects

between groups using the vehicle-treated hypothermia group as the

control.

developmental model (Figure 8). In the ﬁrst model, the in vivo

tau phosphorylation was induced by subjecting the animals to

an isoﬂurane anesthesia, which leads to an increase in tau

phosphorylation by temperature-dependent inhibition of the

phosphatase PP2A.²⁸This study was performed in wild-type

C57Bl/6 mice to ensure that both tau and tau kinases were

present at physiological levels. Five minutes prior to the

isoﬂurane treatment, the animals were administered with

TTBK1 inhibitor 31 at a range of concentrations (2.5, 7, 20,

60, and 180 mg/kg; via intraperitoneal (I.P.) administration)

and the isoﬂurane treatment continued for a period of 1 h.

This treatment resulted in a 10 degree drop in the body

temperature of each animal (there was no diﬀerence in body

temperature between the compound and vehicle-treated

groups), and at the end of the isoﬂurane treatment, the

animals were euthanized and the cortex of each hemisphere

was dissected and ﬂash frozen. For each subject, one cortical

hemisphere was taken for western blot analysis of tau

phosphorylation and one cortical hemisphere was used to

determine the levels of compound 31 in the brain. Total drug

in the brain was determined by measuring total compound

exposure in cortical tissue via LC−MS/MS and these values

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Scheme 1. Synthesis of Compounds 4−7

a

Reagents and conditions: (a) 33, TsCl, NEt₃, DCM, 16 h, 68%; (b) 34, TsCl, NEt₃, DMAP, DCM, 2 h, 75%; (c) 34, NaOH, MeI, THF, 3 h, 91%;

(d) NaH, MeI, DMF, 30 °C, 2.5 h, 54%; (e) Tributyl(1-ethoxyvinyl)stannane, Pd(PPh₃)₄, DMF, 80 °C, 4 h, 90%; (f) DMF−DMA, 80−100 °C,

3−48 h; (g) Guanidine thiocyanate, NaOEt, EtOH, 80−90 °C, 3−40 h, 55−56% over two steps; (h) 2-Methylbut-3-yn-2-ol, Pd(PPh₃)₄, NaI, Zn,

NEt₃, DBU, DMSO, 90 or 100 °C, 16 h, 15−76%.

brain. At this time point, tau phosphorylation at Ser 422

returned to pre-treatment levels, indicating that the observed

lowering of tau phosphorylation was due to the eﬀect of

compound 31 and not due to a physiological lowering of tau

phosphorylation within the subjects over time.

proteins that were used as normalization controls (Supporting

Information, Figure S1, Tau5). Overall, these data demonstrate

that inhibition of TTBK1 with the selective inhibitor 31 led to

an acute lowering of tau phosphorylation at Ser 422 in two

preclinical animal models and that this eﬀect was due to

inhibition of the kinase activity of TTBK1. It is important to

note that the Ser 422 epitope was speciﬁcally chosen for these

studies because it is a direct phosphorylation site of TTBK1

and is not aﬀected by TTBK2 activity in neurons.⁴In addition,

signiﬁcant increases in the phosphorylation of tau at Ser 422

are detected early in the disease course of AD²⁹and the

number of tau-Ser 422 immuno-positive neurons correlates

with cholinergic neuron loss and cognitive impairment in

disease.³⁰The studies presented here demonstrate that the

eﬀects of compound 31 on tau phosphorylation are both dose-

and time-dependent and further validate TTBK1 as a potential

drug target for the treatment of AD and related tauopathies.

Chemistry. The synthesis of analogs 4−7 was enabled by

the preparation of intermediates 35−38 (Scheme 1).

Speciﬁcally, intermediates 35 and 36 were accessed from

commercially available indole 33 and indazole 34 following a

tosylation reaction with TsCl in DCM. Intermediate 37 was

synthesized from 34 by alkylation with MeI and NaOH in

THF. In contrast, intermediate 38 was synthesized from

iodoindazole 47, which was alkylated by reaction with MeI and

NaH in DMF and was subsequently reacted in a Stille reaction

with tributyl(1-ethoxyvinyl)stannane and Pd(PPh₃)₄. The

resulting intermediates 35−38 were reacted with DMF−

DMA at 80−100 °C to provide intermediates 39−42, which

were converted to the desired aminopyrimidines 43−46 by

treatment with guanidine thiocyanate and NaOEt in hot

EtOH. The desired TTBK1 inhibitors 4−7 were synthesized

To exclude the possibility that the eﬀects of compound 31

on tau phosphorylation could be the result of unseen neuronal

toxicity, we tested this inhibitor in vitro at a dose range from

0.0625 to 100 μM. In this experiment, primary mouse neuronal

cultures at DIV14 were placed in an Incucyte system (Essen

Biosciences) for live-cell imaging at 20× magniﬁcation and

analysis of neurite branching was used as a surrogate of neuron

health. For each dose concentration, a total of eight biological

replicates were used and, at each timepoint, ﬁve images were

captured for each well and values were averaged per well. For

all experiments, the addition of 10% triton solution was used as

a positive control to show the window of maximal eﬀect for

this assay. This experiment demonstrated that, at acute

timepoints (up to 1 h following administration), compound

31 had no eﬀect on neurite length when tested up to 100 μM

(see Supporting Information, Figure S4). To test if this

compound had chronic eﬀects on neuron health, we also

imaged every 6 h and up to 24 h following treatment. In this

second experiment, we observed that compound 31 did cause a

signiﬁcant decrease in neurite length at the 50 and 100 μM

concentrations beginning at 12−18 h following treatment

(Supporting Information, Figure S4). However, it must be

noted that, in our in vivo experiments, we saw signiﬁcant

lowering of tau phosphorylation at free drug levels much lower

than these neurotoxic values (mouse isoﬂurane-induced

hypothermia, 10 μM; rat developmental model, 5 μM). In

addition, in the western blots from the in vivo experiments, we

saw no eﬀect of our compound on lowering other neuronal

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Scheme 2. Synthesis of Compounds 8−15 and 18−19

a

Reagents and conditions: (a) 57−62, 4-Chloropyrimidin-2-amine, NaH, DMF, 60−80 °C, 1−18 h, 40−94%; (b) 63, 4-Chloropyrimidin-2-amine,

DBU, DMF, 140 °C, 2 h; (c) 65, 4-Chloropyrimidin-2-amine, Cs₂CO₃, DMF, 120 °C, 2 h; (d) Morpholine, DBU, DMF, 150 °C, 12 h, 31%; (e)

Cyclopropylboronic acid, Pd(dtbpf)Cl₂, K₃PO₄, dioxane, 140 °C, 1 h, 52%; (f) 2-Methylbut-3-yn-2-ol, Pd(PPh₃)₄, NaI, Zn, NEt₃, DBU, DMSO, 90

or 100 °C, 1−24 h, 6.5−47%; (g) 3-Methylpent-1-yn-3-ol, NaI, Pd(PPh₃)₄, Zn, DBU, Et₃N, DMSO, 90 or 100 °C; (h) Chiral separation, 34% from

57, 15% from 58.

from 43−46 under modiﬁed Sonogashira coupling conditions

(Scheme 1).

23 were synthesized from intermediate 57, which was treated

with 3-methylazetidin-3-ol and SPhos third-generation pre-

catalyst in the presence of t-BuONa to provide 22, or with (4-

hydroxyphenyl)boronic acid in the presence of Pd(dppf)Cl₂to

give 23.

Analogs 8−15 were synthesized from commercially available

building blocks 49−56 (Scheme 2; note that compound 54

can also be prepared in three steps from more readily available

4,6-dichloro-1H-pyrrolo[3,2-c]pyridine, see Supporting Infor-

mation). These starting materials were reacted with 4-

chloropyrimidin-2-amine in the presence of base to provide

intermediates 57−63 and 65. Intermediate 63 was subjected to

an S_NAr reaction with morpholine to provide intermediate 64,

and intermediate 66 was accessed by a Suzuki reaction of 64

with cyclopropylboronic acid. The desired analogs 8−15 were

prepared via a modiﬁed Sonogashira coupling between 2-

methylbut-3-yn-2-ol and intermediates 57−62, 64, and 66,

respectively. Analogs 18 and 19 were synthesized from

intermediates 57 and 58, respectively, using 3-methylpent-1-

yn-3-ol and similar coupling conditions.

The synthesis of analogs 16 and 17 started with azaindole

67, which was protected with SEM-Cl and reacted with the

corresponding boronic ester to provide intermediates 69 and

70. Subsequent reduction with PtO₂and deprotection

furnished intermediates 73 and 74, which were reacted with

4-chloropyrimidin-2-amine and NaH in hot DMF to provide

75 and 76. Compound 76 was deprotected with HCl and

methylated by reductive amination with formaldehyde and

NaCNBH₃. A subsequent modiﬁed Sonogashira reaction with

2-methylbut-3-yn-2-ol provided 16 and racemic 17, which was

puriﬁed by chiral SFC (Scheme 3).

The synthesis of compound 24 was carried out by treating

alkyne 9 with Red-Al to provide the desired trans-alkene

(Scheme 5). Analog 25 was synthesized starting with

intermediate 58, which was reacted under Pd-mediated

coupling conditions with ethyl 2-(4,4,5,5-tetramethyl-1,3,2-

dioxaborolan-2-yl)cyclopropane-1-carboxylate to furnish inter-

mediate 78. Subsequent reaction with excess MeLi provided

compound 25. The synthesis of 26 started with commercially

available ketone 79, which was reacted with 4-methylbeneze-

nesulfonohydrazide to provide the tosyl hydrazone 80. This

intermediate was then reacted with 58 under Pd-mediated

conditions to aﬀord cyclobutene intermediate 81. Grignard

reaction of 81 with excess MeMgBr provided 82, which was

hydrogenated to provide the desired analog 26. The synthesis

of bicyclopentane 27 started with commercially available

intermediate 83, which underwent S_NAr reaction with 4-

chloropyrimidin-2-amine after deprotonation with NaH in

DMF. Subsequent reaction of 84 with excess MeMgBr

provided compound 27.

The synthesis of compounds 28−32 followed the general

synthetic route described in Scheme 6. Indole 50 or indoline

51 was treated under S_NAr conditions with the corresponding

chloroheteroaryl electrophile to provide intermediates 85−89

(note that intermediate 85 was isolated as a productive side

product (forming via the oxidation of the azaindoline core to

the azaindole core) during the S_NAr reaction of 51 with 4-

The synthesis of analogs 20 and 21 started with compound

8, which was treated with Red-Al to furnish 20 or with H₂and

Pd/C to provide analog 21 (Scheme 4). Compounds 22 and

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Scheme 3. Synthesis of Compounds 16 and 17

a

Reagents and conditions: (a) SEM-Cl, NaH, DMF, 0 °C to rt, 4.5 h, 86%; (b) 2-(3,6-Dihydro-2H-pyran-4-yl)-4,4,5,5-tetramethyl-1,3,2-

dioxaborolane, Pd(dtbpf)Cl₂, K₂CO₃, dioxane/water, 110 °C, 1 h, 89%; (c) tert-Butyl 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-

dihydropyridine-1(2H)-carboxylate, Pd(dppf)Cl₂, K₂CO₃, dioxane/water, 100 °C, 2 h, 53%; (d) PtO₂, H₂(1 atm), EtOAc/EtOH, rt, 3−4 h, 91%;

(e) TBAF, ethylene diamine, THF, 60−65 °C, 3 h, 83−86%; (f) 73, 4-Chloropyrimidin-2-amine, NaH, DMF, 110 °C, 40 min, 73%; (g) 74, 4-

Chloropyrimidin-2-amine, Cs₂CO₃, DMF, 90 °C, 4 h, 80%; (h) HCl, EtOAc, rt, 2 h; (i) Formaldehyde, NaCNBH₃, MeOH, rt, 2 h, 35% over 2

steps; (j) 2-Methyl-3-yn-2-ol, Pd(PPh₃)₄, NaI, Zn, Et₃N, DBU, DMSO, 95 °C, 5−20 h, 29−45%; (k) Chiral separation.

Scheme 4. Synthesis of Compounds 18−21

a

Reagents and conditions: (a) Red-Al, THF, 0 °C to rt, 14 h, 16.7%; (b) H₂, Pd/C, MeOH, 1 h, 57%; (c) 3-Methylazetidin-3-ol, SPhos-Pd-G3, t-

BuONa, dioxane, 90 °C, 17 h, 8%; (d) (4-Hydroxyphenyl)boronic acid, Pd(dppf)Cl₂, K₂CO₃, dioxane/water, 90 °C, 17 h, 57%.

chloro-N-methylpyrimidin-2-amine). These intermediates were

coupled with the corresponding alkynes under modiﬁed

Sonogashira conditions to provide the desired analogs 28−32.

frontotemporal dementia (FTD). A systematic search of the

oﬀ-target activity of known kinase inhibitors led to the

identiﬁcation of the NIK inhibitor 3, which served as a suitable

starting point in our TTBK1 inhibitor program. An X-ray

cocrystal structure of 3 bound to the kinase domain of TTBK1

guided the initial optimization of this series of analogs.

Speciﬁcally, we hypothesized that truncation of the heptadiene

CONCLUSIONS

■

TTBK1 is a CNS-speciﬁc kinase that has been implicated in

the pathological phosphorylation of tau in AD and

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Scheme 5. Synthesis of Compounds 24−27

a

Reagents and conditions: (a) Red-Al, THF, 0 °C to rt, 16 h, 92%; (b) Ethyl 2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclopropane-1-

carboxylate, Pd(dtbpf)Cl₂, K₂CO₃, DME/H₂O, 100 °C, 6 h, 18%; (c) MeLi, THF, −78 °C, 4 h, 37%; (d) 4-Methylbenzenesulfonohydrazide,

MeOH, 2 h, 86%; (e) 80, Pd(MeCN)₂Cl₂, dppp, Cs₂CO₃, dioxane, 95 °C, 20 h, 21%; (f) MeMgBr, THF, 1 h, 29%; (g) H₂, Pd/C, MeOH, EtOAc,

16 h, 75%; (h) 4-Chloropyrimidin-2-amine, NaH, DMF; (i) TMSCHN₂, MeOH, DCM, 1.5 h; (j) MeMgBr, THF, 1 h, 15% from 83.

a

Scheme 6. Synthesis of Compounds 28−32

a

Reagents and conditions: (a) NaH, DMF, rt to 70 °C, 13−87%; (b) NaI, Pd(PPh₃)₄, Zn, DBU, NEt₃, DMSO, 100 °C, 2−14 h; (c) Chiral

separation, 1.7−48%.

ring in 3 and introduction of a nitrogen atom instead of C5 of

the indole would lead to compounds with improved proﬁle

(Figure 9). This hypothesis was conﬁrmed by analog 7, which

had good TTBK1 activity in both biochemical and cellular

assays. The high ER (19) of compound 7 was partially

addressed by the synthesis of the regioisomeric azaindazole 8

(ER: 5.5). Subsequent optimization of both the 5,6-fused

heterocyclic core and the substituents projected to the back

pocket of the protein led to the discovery of the potent TTBK1

inhibitors 18 and 19, which showed high levels of cytotoxicity.

This liability was mitigated by C6 substitution of the

aminopyrimidine hinge binder leading to inhibitor 31, a

potent (cell IC₅₀: 315 nM) and brain-penetrant (rat K_p,uu

:

0.32) TTBK1 inhibitor with a suitable proﬁle for in vivo proof

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Figure 9. Summary of identiﬁcation and optimization of novel TTBK1 inhibitors.

TFA)/90% MeCN (containing 0.1% v/v of TFA) for 2 min and then

holding at 10% water (containing 0.1% v/v of TFA) and 90% MeCN

(containing 0.1% v/v of TFA) up to 3 min at a ﬂow rate of 3 mL/min

(injection volume 5 μL and using a Waters Sunﬁre C18 4.6 × 20 mm

× 3.5 μm IS column). MS mode: MS:ESI+ scan range 100−1000

daltons. PDA detection 210−400 nm. Method 2 (acidic condition):

mobile phase prepared with 1.5 mL of TFA in 4 L of water (solvent

A) and acetonitrile (solvent B), using the elution gradient 10−80%

(solvent B) over 1.35 min and holding at 80% for 0.9 min at a ﬂow

rate of 0.8 mL/min; column: Xtimate C18 2.1 × 30 mm × 3 μm;

detection wavelength: 220 nm and 254 nm; column temperature: 50

°C; MS mode: MS:ESI+. Method 3 (basic condition): mobile phase

prepared with 0.8 mL of aq NH₄OH in 4 L of water (solvent A) and

acetonitrile (solvent B), using the elution gradient 10−80% (solvent

B) over 2 min and holding at 80% for 0.48 min at a ﬂow rate of 1.2

mL/min; column: Xbridge Shield RP-18 2.1 × 50 mm × 5 μm;

detection wavelength: 220 and 254 nm; column temperature: 50 °C;

MS mode: MS:ESI+. Trithiocyanuric acid (TMT) solution for

sequestering palladium catalyst was prepared as follows: NaCl (14.0

g) was dissolved in water (250 mL), and then trithiocyanuric acid

(1.80 g, CAS #638-16-4), EtOH (200 mL), and aq NH₄OH (15 mL,

to improve the solubility of TMT) were added. The mixture was

sonicated for 15 min before further EtOH (240 mL) was added.

General Procedure 1 for the Preparation of Compounds 4−

19 and 28−32: Heteroaryl Halide−Alkyne Cross-Coupling

Reaction. Under an atmosphere of nitrogen, a mixture of heteroaryl

halide 43−46, 57−62, 64, 65, or 85−89 (1 equiv), alkyne (2-

methylbut-3-yn-2-ol or 3-methylpent-1-yn-3-ol, 1.00−10.0 equiv),

Pd(PPh₃)₄(20.0 mol %), NaI (20.0 mol %), DBU (2.00 equiv, unless

otherwise noted), triethylamine (2.00 equiv, unless otherwise noted),

and Zn powder (20.0 mol %) in DMSO was stirred at 90 or 100 °C

for an indicated period of time. The reaction suspension was cooled

to room temperature, ﬁltered, and puriﬁed by prep-HPLC or was

subjected to an aqueous workup followed by prep-HPLC to provide

the title compounds.

of pharmacology studies. The ability of 31 to inhibit tau

phosphorylation at a therapeutically relevant epitope (Ser 422)

was established in both a mouse isoﬂurane-induced hypo-

thermia model and in a rat developmental model. It is

important to note that the advancement of TTBK1 inhibitors

to clinical development may require some level of selectivity

against TTBK2 given that mutations in this gene have been

linked to the development of spinocerebellar ataxia type 11¹¹

and knockout of TTBK2 in the postnatal mouse brain leads to

progressive degeneration of the cerebellum.³¹Overall, we have

demonstrated that inhibition of TTBK1 with the selective

inhibitor 31 is a viable strategy to modulate the phosphor-

ylation of tau at disease-relevant epitopes although additional

studies are needed before a suitable TTBK inhibitor can be

progressed to the clinic as a potential treatment of Alzheimer’s

disease and related tauopathies.

EXPERIMENTAL SECTION

■

All solvents and chemicals used were reagent grade. Anhydrous

solvents were purchased from Sigma-Aldrich and used as received.

Analytical thin layer chromatography (TLC) and silica gel column

chromatography were performed on Merck silica gel 60 (230−400

mesh). Aqueous ammonium hydroxide (NH₄OH) used for

chromatography and TMT solution were purchased from Fisher

(supplier item A669S500, 28−30% w/w ammonia). Removal of

solvents was conducted by using a rotary evaporator, and residual

solvents were removed from non-volatile compounds using a vacuum

manifold maintained at approximately 1 Torr. NMR spectra were

recorded on a Bruker Avance 400 MHz and 500 MHz NMR

spectrometer. Chemical shifts (δ) are reported in parts per million

(ppm) relative to a residual undeuterated solvent as an internal

reference, and coupling constants (J) are reported in hertz (Hz).

Splitting patterns are indicated as follows: s = singlet; d = doublet; t =

triplet; q = quartet; qn = quintet; dd = doublet of doublets; dt =

doublet of triplets; tt = triplet of triplets; m = multiplet; br = broad

peak. All yields reported are isolated yields. All ﬁnal compounds were

puriﬁed to ≥95% purity as determined by LC/MS analysis using one

of the following three methods. Method 1: using a linear gradient of

elution: 90% water (containing 0.1% v/v of TFA)/10% MeCN

(containing 0.1% v/v of TFA) to 10% water (containing 0.1% v/v of

4-(3-(2-Aminopyrimidin-4-yl)-1H-indol-5-yl)-2-methylbut-3-yn-

2-ol (4). The reaction was carried out according to general procedure

1 using 43 (70.0 mg, 250 μmol) and 2-methylbut-3-yn-2-ol (48 μL,

500 μmol) in 3.0 mL of DMSO at 100 °C for 16 h. The reaction was

ﬁltered. Prep-HPLC puriﬁcation (column: Xtimate C18 150 × 30 mm

× 5 μm; gradient: MeCN/water (10 mM NaHCO₃), 29−59%, 25

mL/min) aﬀorded the title compound (45.0 mg, 64%) as a white

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1

solid. H NMR (400 MHz, CD₃OD) δ ppm 8.65 (s, 1H), 8.35 (s,

(m, 1H), 5.53 (s, 1H) 1.52 (s, 6H). MS (ESI): 294.2 [M + H]⁺.

HRMS (ESI): m/z calculated for C₁₆H₁₅N₅O + H⁺[M + H]⁺:

294.1350. Found: 294.1350.

1H), 8.01 (d, J = 6.6 Hz, 1H), 7.44 (d, J = 8.6 Hz, 1H), 7.30 (dd, J =

8.4, 1.5 Hz, 1H), 7.24 (br d, J = 6.2 Hz, 1H), 1.60 (s, 6H). MS (ESI):

293.1 [M + H]⁺. HRMS (ESI): m/z calculated for C₁₇H₁₆N₄O + H⁺

[M + H]⁺: 293.1397. Found: 293.1397.

4-(1-(2-Aminopyrimidin-4-yl)-2,3-dihydro-1H-pyrrolo[3,2-c]-

pyridin-6-yl)-2-methylbut-3-yn-2-ol (10). The reaction was carried

out according to general procedure 1 using 59 (100 mg, 410 μmol)

and 2-methylbut-3-yn-2-ol (78 μL, 810 μmol) in 2.0 mL of DMSO at

100 °C for 17 h. The reaction was ﬁltered. Prep-HPLC puriﬁcation

(column: Xtimate C18 150 × 25 mm × 5 μm; gradient: MeCN/water

(10 mM NH₄HCO₃), 13−43%, 25 mL/min) aﬀorded the title

4-(3-(2-Aminopyrimidin-4-yl)-1H-indazol-5-yl)-2-methylbut-3-

yn-2-ol (5). The reaction was carried out according to general

procedure 1 using 44 (100 mg, 330 μmol) and 2-methylbut-3-yn-2-ol

(64 μL, 660 μmol) in 3.0 mL of DMSO at 90 °C for 16 h. The

reaction was ﬁltered. Prep-HPLC puriﬁcation (column: Xtimate C18

150 × 25 mm × 5 μm; gradient: MeCN/water (10 mM NH₄HCO₃),

24−54%, 25 mL/min) aﬀorded the title compound (15.0 mg, 15%) as

1

compound (42.0 mg, 36%) as a white solid. H NMR (400 MHz,

DMSO-d₆) δ ppm 8.31 (s, 1H), 8.21 (s, 1H), 8.06 (d, J = 5.6 Hz,

1H), 6.58 (s, 2H), 6.10 (d, J = 5.6 Hz, 1H), 5.52 (s, 1H), 4.02 (t, J =

8.4 Hz, 2H), 3.19 (t, J = 8.0 Hz, 2H), 1.49 (s, 6H). MS (ESI): 295.9

[M + H]⁺. HRMS (ESI): m/z calculated for C₁₆H₁₇N₅O + H⁺[M +

H]⁺: 296.1506. Found: 296.1508.

1

a white solid. H NMR (400 MHz, DMSO-d₆) δ ppm 8.67 (dd, J =

1.4, 0.8 Hz, 1H), 8.29 (d, J = 5.1 Hz, 1H), 7.60 (dd, J = 8.7, 0.7 Hz,

1H), 7.39 (dd, J = 8.6, 1.5 Hz, 1H), 7.27 (d, J = 5.1 Hz, 1H), 6.79 (s,

2H), 6.05 (br s, 1H), 5.45 (br s, 1H), 1.52 (s, 6H). MS (ESI): 294.0

[M + H]⁺. HRMS (ESI): m/z calculated for C₁₆H₁₅N₅O + H⁺[M +

H]⁺: 294.1350. Found: 294.1352.

4-(1-(2-Aminopyrimidin-4-yl)-5-methoxy-1H-indol-6-yl)-2-meth-

ylbut-3-yn-2-ol (11). The reaction was carried out according to

general procedure 1 using 60 (60.0 mg, 188 μmol) and 2-methylbut-

3-yn-2-ol (37 μL, 380 μmol) in 2.0 mL of DMSO at 100 °C for 1 h.

The mixture was diluted with water (5 mL) and extracted with EtOAc

(10 mL × 3). The organic layers were combined and concentrated.

The crude product was puriﬁed by prep-HPLC (column: Xtimate

C18 150 × 25 mm × 5 μm; gradient: MeCN/water (10 mM

NH₄HCO₃), 35−65%, 25 mL/min) to aﬀord the title compound (7.3

mg, 12%) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ ppm 8.69

(s, 1H), 8.19 (d, J = 6.0 Hz, 1H), 7.88 (d, J = 3.5 Hz, 1H), 7.14 (s,

1H), 6.85 (d, J = 6.0 Hz, 1H), 6.68 (d, J = 3.5 Hz, 1H), 3.89 (s, 3H),

1.61 (s, 6H). MS (ESI): 323.1 [M + H]⁺. HRMS (ESI): m/z

calculated for C₁₈H₁₈N₄O₂+ H⁺[M + H]⁺: 323.1503. Found:

323.1506.

4-(3-(2-Aminopyrimidin-4-yl)-1-methyl-1H-indazol-5-yl)-2-meth-

ylbut-3-yn-2-ol (6). The reaction was carried out according to general

procedure 1 using 45 (100 mg, 330 μmol) and 2-methylbut-3-yn-2-ol

(64 μL, 660 μmol) in 3.0 mL of DMSO at 90 °C for 16 h. The

reaction was ﬁltered. Prep-HPLC puriﬁcation (column: Xtimate C18

150 × 25 mm × 5 μm; gradient: MeCN/water (10 mM NH₄HCO₃),

27−57%, 25 mL/min) aﬀorded the title compound (42.0 mg, 42%) as

a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.66 (d, J = 0.6

Hz, 1H), 8.28 (d, J = 5.1 Hz, 1H), 7.72 (dd, J = 8.7, 0.6 Hz, 1H), 7.44

(dd, J = 8.7, 1.6 Hz, 1H), 7.21 (d, J = 5.1 Hz, 1H), 6.79 (s, 2H), 6.05

(br s, 1H), 4.14 (s, 3H), 1.51 (s, 6H). MS (ESI): 308.1 [M + H]⁺.

HRMS (ESI): m/z calculated for C₁₇H₁₇N₅O + H⁺[M + H]⁺:

308.1506. Found: 308.1505.

4-(3-(2-Aminopyrimidin-4-yl)-1-methyl-1H-pyrazolo[3,4-c]-

pyridin-5-yl)-2-methylbut-3-yn-2-ol (7). The reaction was carried out

according to general procedure 1 using 46 (27.0 mg, 100 μmol) and

2-methylbut-3-yn-2-ol (20 μL, 210 μmol) in 3.0 mL of DMSO at 100

°C for 16 h. Reaction was ﬁltered. Prep-HPLC puriﬁcation (column:

Xtimate C18 150 × 25 mm × 5 μm; gradient: MeCN/water (10 mM

NH₄HCO₃), 15−45%, 25 mL/min) aﬀorded the title compound (8.0

mg, 76%) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ ppm 9.04

(d, J = 1.2 Hz, 1H), 8.71 (s, 1H), 8.28 (d, J = 5.2 Hz, 1H), 7.39 (d, J =

5.2 Hz, 1H), 4.28 (s, 3H), 1.62 (s, 6H). MS (ESI): 309.1 [M + H]⁺.

HRMS (ESI): m/z calculated for C₁₆H₁₆N₆O + H⁺[M + H]⁺:

309.1459. Found: 309.1456.

4-(1-(2-Aminopyrimidin-4-yl)-5-ﬂuoro-1H-indol-6-yl)-2-methyl-

but-3-yn-2-ol (12). The reaction was carried out according to general

procedure 1 using 61 (50.0 mg, 163 μmol) and 2-methylbut-3-yn-2-ol

(78 μL, 810 μmol) in 8.0 mL of DMSO at 100 °C for 5 h. The

mixture was diluted with water (20 mL) and extracted with EtOAc

(30 mL × 4). The combined organic layers were dried over Na₂SO₄

and concentrated under reduced pressure. The crude product was

puriﬁed by prep-HPLC (column: Xtimate C18 150 × 25 mm × 5 μm;

gradient: MeCN/water (10 mM NH₄HCO₃), 26−56%, 25 mL/min)

to aﬀord the title compound (6.0 mg, 12%) as a light yellow solid. ¹H

NMR (400 MHz, DMSO-d₆) δ ppm 8.77 (d, J = 6.8 Hz, 1H), 8.28 (d,

J = 5.6 Hz, 1H), 8.18 (d, J = 3.6 Hz, 1H), 7.48 (d, J = 9.6 Hz, 1H),

6.99 (s, 2H), 6.93 (d, J = 5.6 Hz, 1H), 6.77 (d, J = 3.6 Hz, 1H), 5.52

(s, 1H), 1.54 (s, 6H). MS (ESI): 311.0 [M + H]⁺. HRMS (ESI): m/z

calculated for C₁₇H₁₅FN₄O + H⁺[M + H]⁺: 311.1303. Found:

311.1306.

4-(1-(2-Aminopyrimidin-4-yl)-1H-pyrazolo[4,3-c]pyridin-6-yl)-2-

methylbut-3-yn-2-ol (8). The reaction was carried out according to

general procedure 1 using 57 (65.0 mg, 250 μmol) and 2-methylbut-

3-yn-2-ol (48 μL, 500 μmol) in 3.0 mL of DMSO at 100 °C for 16 h.

Reaction was ﬁltered. Prep-HPLC puriﬁcation (column: Waters

Xbridge Prep OBD C18 150 × 30 mm × 5 μm; gradient: MeCN/

water (0.050 M NH₄OH), 18−48%, 25 mL/min) aﬀorded the title

4-(1-(2-Aminopyrimidin-4-yl)-4-methoxy-1H-pyrrolo[3,2-c]-

pyridin-6-yl)-2-methylbut-3-yn-2-ol (13). The reaction was carried

out according to general procedure 1 using 62 (14.0 mg, 51.0 μmol)

and 2-methylbut-3-yn-2-ol (25 μL, 263 μmol) in 500 μL of DMSO at

90 °C for 16 h. The reaction was ﬁltered. Prep-HPLC puriﬁcation

(column: Waters XSelect CSH C18 OBD 100 × 50 mm × 5 μm;

gradient: MeCN/water (0.2% v/v NH₄OH), 5−70%, 25 mL/min)

1

compound as a white solid (9.0 mg, 12%). H NMR (400 MHz,

CD₃OD) δ ppm 9.06 (d, J = 1.1 Hz, 1H), 8.93 (s, 1H), 8.51 (d, J =

0.9 Hz, 1H), 8.31 (d, J = 5.7 Hz, 1H), 7.27 (d, J = 5.5 Hz, 1H), 1.63

(s, 6H). MS (ESI): 295.0 [M + H]⁺. HRMS (ESI): m/z calculated for

C₁₅H₁₄N₆O + H⁺[M + H]⁺: 295.1302. Found: 295.1305.

1

aﬀorded the title compound (1.0 mg, 6.5%). H NMR (400 MHz,

4-(1-(2-Aminopyrimidin-4-yl)-1H-pyrrolo[3,2-c]pyridin-6-yl)-2-

methylbut-3-yn-2-ol (9). The reaction was carried out according to

general procedure 1 using 58 (50.0 mg, 204 μmol) and 2-methylbut-

3-yn-2-ol (79 μL, 816 μmol) in 2.0 mL of DMSO at 100 °C for 12 h.

EtOAc (20 mL) and water (20 mL) were added. The aqueous layer

was separated and extracted with EtOAc (20 mL × 2). The combined

organic layers were washed with brine (10 mL), dried over Na₂SO₄,

ﬁltered, and concentrated. The reaction was repeated (same scale),

and the crude residues from the two reactions were combined and

subjected to prep-HPLC puriﬁcation (column: Xtimate C18 150 × 25

mm × 5 μm; gradient: MeCN/water (10 mM NH₄HCO₃), 20−50%,

25 mL/min) to aﬀord the title compound (15.0 mg, 13%) as a white

CD₃OD) δ ppm 8.32 (s, 1H), 8.29 (d, J = 5.77 Hz, 1H), 7.87 (d, J =

3.51 Hz, 1H), 6.90 (d, J = 5.77 Hz, 1H), 6.79 (d, J = 3.51 Hz, 1H),

4.05 (s, 3H), 1.61 (s, 6H). MS (ESI): 324.2 [M + H]⁺. HRMS (ESI):

m/z calculated for C₁₇H₁₇N₅O₂+ H⁺[M + H]⁺: 324.1455. Found:

324.1457.

4-(1-(2-Aminopyrimidin-4-yl)-4-morpholino-1H-pyrrolo[3,2-c]-

pyridin-6-yl)-2-methylbut-3-yn-2-ol (14). The reaction was carried

out according to general procedure 1 using 64 (37.0 mg, 112 μmol),

2-methylbut-3-yn-2-ol (44 μL, 450 μmol), DBU (67 μL, 450 μmol),

and triethylamine (62 μL, 480 μmol) in 1.0 mL of DMSO at 90 °C

for 12 h. The mixture was ﬁltered through Celite and diluted with

DCM (5 mL). The organic phase was washed with water (2 mL) and

brine (2 mL), dried over Na₂SO₄, ﬁltered, and concentrated. The

crude product was puriﬁed by prep-HPLC (column: Waters XSelect

CSH Prep C18 OBD 100 × 50 mm × 5 μm; gradient: MeCN/water

1

solid. H NMR (400 MHz, DMSO-d₆) δ ppm 8.85 (d, J = 0.8 Hz,

1H), 8.64 (d, J = 0.8 Hz, 1H), 8.34 (d, J = 5.2 Hz, 1H), 8.21 (d, J =

3.6 Hz, 1H), 7.10−7.09 (m, 2H), 6.99 (d, J = 5.6 Hz, 1H), 6.95−6.93

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(0.2% v/v NH₄OH), 5−65%, 80 mL/min) to aﬀord the title

methylpent-1-yn-3-ol (1.3 mL, 11.4 mmol), and triethylamine (2.4

mL, 17.0 mmol) in 15.0 mL of DMSO at 100 °C for 5 h. The mixture

was diluted with water (50 mL) and extracted with EtOAc (15 mL ×

3). The combined organic layers were concentrated under reduced

pressure. The crude product was puriﬁed by column chromatography

(SiO₂, gradient: 20−50% EtOAc/petroleum ether) to aﬀord rac-1-(1-

(2-aminopyrimidin-4-yl)-1H-pyrazolo[4,3-c]pyridin-6-yl)-3-methyl-

pent-1-yn-3-ol as a yellow solid with some impurities (2.00 g). MS

(ESI): 309.1 [M + H]⁺. Step 2: Chiral separation of racemic product

from step 1 was performed using the following column: Phenomenex-

Cellulose-2 (250 × 30 mm × 5 μm); method: 40% IPA with 0.1%

NH₄OH in CO₂(ﬂow rate: 60 mL/min). The ﬁrst peak eluting oﬀ

the column provided (R)-1-(1-(2-aminopyrimidin-4-yl)-1H-pyrazolo-

[4,3-c]pyridin-6-yl)-3-methylpent-1-yn-3-ol (the enantiomer of 18) as

a yellow solid in 97.3% ee (Rt = 2.620 min, 656 mg, 37% over two

1

compound (20.0 mg, 47%) as a white solid. H NMR (400 MHz,

CD₃OD) δ ppm 8.28 (d, J = 6.1 Hz, 1H), 8.21 (s, 1H), 7.85 (d, J =

4.3 Hz, 1H), 6.87 (d, J = 6.1 Hz, 1H), 6.84 (d, J = 3.7 Hz, 1H), 3.90−

3.83 (m, 4H), 3.60−3.55 (m, 4H), 1.60 (s, 6H). MS (ESI): 379.2 [M

+ H]⁺. HRMS (ESI): m/z calculated for C₂₀H₂₂N₆O₂+ H⁺[M + H]⁺:

379.1877. Found: 379.1873.

4-(1-(2-Aminopyrimidin-4-yl)-3-cyclopropyl-1H-pyrrolo[3,2-c]-

pyridin-6-yl)-2-methylbut-3-yn-2-ol (15). The reaction was carried

out according to general procedure 1 using 66 (20.0 mg, 70.0 μmol),

2-methylbut-3-yn-2-ol (27 μL, 280 μmol), DBU (42 μL, 280 μmol),

and triethylamine (39 μL, 280 μmol) in 700 μL of DMSO at 90 °C

for 16 h. The reaction was ﬁltered. Prep-HPLC puriﬁcation (column:

Waters XSelect CSH Prep OBD C18 100 × 19 mm × 5 μm; gradient:

MeCN/water (0.2% v/v NH₄OH), 5−55%, 30 mL/min) aﬀorded the

title compound (8.0 mg, 34%) as a white solid. ¹H NMR (500 MHz,

CD₃OD) δ ppm 8.85 (s, 1H), 8.76 (s, 1H), 8.26 (d, J = 6.1 Hz, 1H),

7.73 (s, 1H), 6.89 (d, J = 6.1 Hz, 1H), 2.10−2.01 (m, 1H), 1.62 (s,

6H), 1.07−0.99 (m, 2H), 0.82−0.74 (m, 2H). MS (ESI): 334.3 [M +

H]⁺. HRMS (ESI): m/z calculated for C₁₉H₁₉N₅O + H⁺[M + H]⁺:

334.1663. Found: 334.1661.

1

steps. H NMR and MS identical to 18). The second peak furnished

(S)-1-(1-(2-aminopyrimidin-4-yl)-1H-pyrazolo[4,3-c]pyridin-6-yl)-3-

methylpent-1-yn-3-ol 18 as a yellow solid in 100% ee (Rt = 3.455, 595

1

mg, 34% over two steps). Stereochemistry arbitrarily assigned. H

NMR (400 MHz, CD₃OD) δ ppm 9.06 (d, J = 0.8 Hz, 1H), 8.92 (s,

1H), 8.51 (d, J = 0.8 Hz, 1H), 8.31 (d, J = 5.6 Hz, 1H), 7.27 (d, J =

5.6 Hz, 1H), 1.89−1.80 (m, 2H), 1.59 (s, 3H), 1.15 (t, J = 7.6 Hz,

3H). ¹³C NMR (126 MHz, CD₃OD) δ ppm 164.6, 161.8, 160.6,

146.3, 143.5, 140.4, 139.6, 123.9, 115.1, 99.6, 95.4, 83.3, 69.6, 37.5,

29.2, 9.4. LCMS (ESI, m/z): 309.1 [M + H]⁺. HRMS (ESI): m/z

calculated for C₁₆H₁₆N₆O + H⁺[M + H]⁺: 309.1459. Found:

309.1462.

4-(1-(2-Aminopyrimidin-4-yl)-3-(tetrahydro-2H-pyran-4-yl)-1H-

pyrrolo[3,2-c]pyridin-6-yl)-2-methylbut-3-yn-2-ol (16). The reaction

was carried out according to general procedure 1 using 75 (25.0 mg,

80.0 μmol), 2-methylbut-3-yn-2-ol (29 μL, 303 μmol), DBU (45 μL,

300 μmol), and triethylamine (29 μL, 300 μmol) in 760 μL of DMSO

at 95 °C for 5 h. The reaction was ﬁltered. Prep-HPLC puriﬁcation

(column: XSELECT CSH C18 100 × 50 mm × 5 μm; gradient:

MeCN/water (0.2% v/v NH₄OH), 5−60%, 25 mL/min) aﬀorded the

(S)-1-(1-(2-Aminopyrimidin-4-yl)-1H-pyrrolo[3,2-c]pyridin-6-yl)-

3-methylpent-1-yn-3-ol (19). Step 1: The reaction was carried out

according to general procedure 1 using 58 (100 mg, 407 μmol) and 3-

methylpent-1-yn-3-ol (184 μL, 1.63 mmol) in 2.0 mL of DMSO at

100 °C for 18 h. TMT solution (1 mL) was added, the mixture was

stirred at room temperature for 30 min and ﬁltered. EtOAc (30 mL)

and water (30 mL) were added. The aqueous layer was separated and

extracted with EtOAc (20 mL × 2). The combined organic layers

were concentrated under reduced pressure. The crude product was

puriﬁed by prep-HPLC (column: Xtimate C18 150 × 25 mm × 5 μm;

gradient: MeCN/water (10 mM NH₄HCO₃), 24−64%, 25 mL/min)

to aﬀord rac-1-(1-(2-aminopyrimidin-4-yl)-1H-pyrrolo[3,2-c]pyridin-

6-yl)-3-methylpent-1-yn-3-ol as a white solid (50.0 mg, 40%). MS

(ESI): 308.1 [M + H]⁺. Step 2: Chiral separation of racemic product

from step 1 was performed using the following column: C2 (250 × 30

mm × 10 μm); method: 40% EtOH with 0.1% NH₄OH in CO₂(ﬂow

rate: 80 mL/min, detection wavelength: 220 nm). The ﬁrst peak

eluting oﬀ the column provided (R)-1-(1-(2-aminopyrimidin-4-yl)-

1H-pyrrolo[3,2-c]pyridin-6-yl)-3-methylpent-1-yn-3-ol (the enan-

tiomer of 19) as a white solid in 100% ee (Rt = 6.637 min, 25.2

mg, 50%, ¹H NMR and MS identical to 19). The second peak

furnished (S)-1-(1-(2-aminopyrimidin-4-yl)-1H-pyrrolo[3,2-c]-

pyridin-6-yl)-3-methylpent-1-yn-3-ol 19 as a white solid in 99.5% ee

(Rt = 6.433 min, 18.7 mg, 37%). Stereochemistry arbitrarily assigned.

¹H NMR (400 MHz, CDCl₃) δ ppm 8.88 (d, J = 1.2 Hz, 1H), 8.44 (s,

1H), 8.37 (d, J = 5.6 Hz, 1H), 7.73 (d, J = 4.0 Hz, 1H), 6.80−6.78

(m, 1H), 6.74 (d, J = 5.6 Hz, 1H), 5.31 (s, 2H), 2.60 (s, 1H), 1.87−

1.83 (m, 2H), 1.64 (s, 3H), 1.15 (t, J = 7.2 Hz, 3H). ¹³C NMR (126

MHz, CDCl₃) δ ppm 163.0, 160.2, 158.7, 144.1, 139.0, 136.2, 126.8,

126.6, 113.7, 106.3, 100.1, 91.4, 84.0, 69.1, 36.6, 29.2, 9.2. LCMS

(ESI, m/z): 308.1 [M + H]⁺. HRMS (ESI): m/z calculated for

C₁₇H₁₇N₅O + H⁺[M + H]⁺: 308.1506. Found: 308.1508.

(E)-4-(1-(2-Aminopyrimidin-4-yl)-1H-pyrazolo[4,3-c]pyridin-6-

yl)-2-methylbut-3-en-2-ol (20). To a solution of 8 (30.0 mg, 102

μmol) in THF (4.0 mL) was added Red-Al (204 μL, 408 μmol, 60%

w/w in toluene) at 0 °C. The resulting mixture was stirred at 0 °C for

2 h and at 18 °C for 12 h. The mixture was quenched with sat. aq

NaHCO₃(10 mL). The aqueous layer was extracted with EtOAc (20

mL × 3). The combined organic layers were dried over Na₂SO₄,

ﬁltered, and concentrated. The crude residue was puriﬁed by prep-

HPLC (column: Waters Xbridge Prep OBD C18 150 × 30 mm × 5

μm; gradient: MeCN/water (0.05% v/v NH₄OH), 15−45%, 25 mL/

min) to aﬀord the title compound as a white solid (5.2 mg, 16.7%).

1

title compound (13.0 mg, 45%). H NMR (500 MHz, CD₃OD) δ

ppm 8.86 (s, 1H), 8.78 (s, 1H), 8.29 (d, J = 6.1 Hz, 1H), 7.86 (s, 1H),

6.94 (d, J = 6.1 Hz, 1H), 4.08 (dd, J = 3.7, 10.4 Hz, 2H), 3.67 (dt, J =

1.8, 11.9 Hz, 2H), 3.23 (tt, J = 3.7, 11.6 Hz, 1H), 2.05−1.99 (m, 2H),

1.95−1.84 (m, 2H), 1.62 (s, 6H). MS (ESI): 378.2 [M + H]⁺. HRMS

(ESI): m/z calculated for C₂₁H₂₃N₅O₂+ H⁺[M + H]⁺: 378.1925.

Found: 378.1922.

(S)-4-(1-(2-Aminopyrimidin-4-yl)-3-(1-methylpiperidin-3-yl)-1H-

pyrrolo[3,2-c]pyridin-6-yl)-2-methylbut-3-yn-2-ol (17). Step 1: The

reaction was carried out according to general procedure 1 using 77

(120 mg, 350 μmol), 2-methylbut-3-yn-2-ol (338 μL, 3.50 mmol),

and triethylamine (150 μL, 1.05 mmol) in 8.5 mL of DMSO at 95 °C

for 20 h. The mixture was diluted with water (10 mL) and extracted

with EtOAc (15 mL × 3). The combined organic layers were washed

with brine (40 mL × 3), dried over Na₂SO₄, ﬁltered, and

concentrated. The crude product was puriﬁed by prep-TLC (1:10

MeOH/DCM) to aﬀord rac-4-(1-(2-aminopyrimidin-4-yl)-3-(1-

methylpiperidin-3-yl)-1H-pyrrolo[3,2-c]pyridin-6-yl)-2-methylbut-3-

yn-2-ol as a brown oil (40.0 mg, 29%). MS (ESI): 391.0 [M + H]⁺.

Step 2: Chiral separation of racemic product from step 1 was

performed using the following column: DAICEL CHIRALPAK AD-H

(250 × 30 mm × 5 μm); method: 40% EtOH with 0.1% NH₄OH in

CO₂(ﬂow rate: 50 mL/min). The ﬁrst peak eluting oﬀ the column

provided (R)-4-(1-(2-aminopyrimidin-4-yl)-3-(1-methylpiperidin-3-

yl)-1H-pyrrolo[3,2-c]pyridin-6-yl)-2-methylbut-3-yn-2-ol (the enan-

tiomer of 17) as an oﬀ-white solid in 100% ee (Rt = 6.050 min, 18.8

mg, 29%, ¹H NMR and MS identical to 17). The second peak

furnished (S)-4-(1-(2-aminopyrimidin-4-yl)-3-(1-methylpiperidin-3-

yl)-1H-pyrrolo[3,2-c]pyridin-6-yl)-2-methylbut-3-yn-2-ol 17 as an

oﬀ-white solid in 99.5% ee (Rt = 6.749 min, 18.0 mg, 28%). The

stereochemistry was arbitrarily assigned. ¹H NMR (400 MHz,

CDCl₃) δ ppm 8.89 (d, J = 0.8 Hz, 1H), 8.39 (d, J = 1.2 Hz, 1H),

8.35 (d, J = 5.2 Hz, 1H), 7.52 (s, 1H), 6.71 (d, J = 6.0 Hz, 1H), 5.25

(s, 2H), 3.25−3.18 (m, 1H), 3.14−3.11 (m, 1H), 2.91−2.88 (m, 1H),

2.34 (s, 3H), 2.11−2.08 (m, 2H), 2.02−2.01 (m, 1H), 1.85−1.80 (m,

2H), 1.68 (s, 6H), 1.54−1.50 (m, 1H). LCMS (ESI, m/z): 391.0 [M

+ H]⁺. HRMS (ESI): m/z calculated for C₂₂H₂₆N₆O + H⁺[M + H]⁺:

391.2241. Found: 391.2244.

(S)-1-(1-(2-Aminopyrimidin-4-yl)-1H-pyrazolo[4,3-c]pyridin-6-

yl)-3-methylpent-1-yn-3-ol (18). Step 1: The reaction was carried out

according to general procedure 1 using 57 (1.40 g, 5.68 mmol), 3-

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¹H NMR (400 MHz, CDCl₃) δ ppm 9.05 (s, 1H), 8.49 (s, 1H), 8.37

rac-trans-2-(2-(1-(2-Aminopyrimidin-4-yl)-1H-pyrrolo[3,2-c]-

pyridin-6-yl)cyclopropyl)propan-2-ol (25). To a solution of 78 (28.0

mg, 86.6 μmol) in THF (2.0 mL) was added methyl lithium (1.1 mL,

1.70 mmol, 1.6 M in Et₂O), and the mixture was stirred at −78 °C for

4 h under a nitrogen atmosphere. After this time, the mixture was

diluted with water (30 mL) and extracted with EtOAc (20 mL × 3).

The combined organic layers were dried over Na₂SO₄, ﬁltered, and

concentrated. The crude residue was puriﬁed by prep-HPLC

(column: Xtimate C18 150 × 25 mm × 5 μm; gradient: MeCN/

water (10 mM NH₄HCO₃) as 17−47%, 25 mL/min) to aﬀord the

title compound (10.2 mg, 37%) as a light yellow solid. ¹H NMR (400

MHz, CDCl₃) δ ppm 8.68 (s, 1H), 8.57 (s, 1H), 8.29 (d, J = 5.5 Hz,

1H), 8.02 (d, J = 3.0 Hz, 1H), 7.03 (s, 2H), 6.96 (d, J = 5.5 Hz, 1H),

6.80 (d, J = 3.0 Hz, 1H), 4.20 (s, 1H), 2.28−2.25 (m, 1H), 1.56−1.53

(m, 1H), 1.19 (s, 3H), 1.16 (s, 3H), 1.04−1.02 (m, 1H), 0.95−0.93

(m, 1H). LCMS (ESI, m/z): 310.0 [M + H]⁺. HRMS (ESI): m/z

calculated for C₁₇H₁₉N₅O + H⁺[M + H]⁺: 310.1663. Found:

310.1664.

(d, J = 5.6 Hz, 1H), 8.27 (s, 1H), 7.32 (d, J = 5.6 Hz, 1H), 7.10 (d, J =

15.6 Hz, 1H), 6.87 (d, J = 15.6 Hz, 1H), 5.21 (s, 2H), 1.49 (s, 6H).

MS (ESI): 297.1 [M + H]⁺. HRMS (ESI): m/z calculated for

C₁₅H₁₆N₆O + H⁺[M + H]⁺: 297.1459. Found: 297.1461.

4-(1-(2-Aminopyrimidin-4-yl)-1H-pyrazolo[4,3-c]pyridin-6-yl)-2-

methylbutan-2-ol (21). To a solution of 8 (70.0 mg, 238 μmol) in

MeOH (40 mL) was added Pd/C (25.0 mg, 23.8 μmol, 10% w/w).

The resulting mixture was stirred under an atmosphere of hydrogen at

18 °C for 1 h. The mixture was ﬁltered, and the ﬁltrate was

concentrated under reduced pressure. The reaction was repeated

(same scale), and the crude residues from the two reactions were

combined and puriﬁed by prep-HPLC (column: Waters Xbridge Prep

OBD C18 150 × 30 mm × 5 μm; gradient: MeCN/water (0.05% v/v

NH₄OH), 20−50%, 30 mL/min) to aﬀord the title compound as a

1

white solid (40.0 mg, 57%). H NMR (400 MHz, CD₃OD) δ ppm

9.02 (d, J = 0.8 Hz, 1H), 8.74 (s, 1H), 8.45 (d, J = 0.8 Hz, 1H), 8.29

(d, J = 6.0 Hz, 1H), 7.26 (d, J = 5.6 Hz, 1H), 3.09−3.05 (m, 2H),

1.96−1.91 (m, 2H), 1.31 (s, 6H). MS (ESI): 299.1 [M + H]⁺. HRMS

(ESI): m/z calculated for C₁₅H₁₈N₆O + H⁺[M + H]⁺: 299.1615.

Found: 299.1618.

2-(3-(1-(2-Aminopyrimidin-4-yl)-1H-pyrrolo[3,2-c]pyridin-6-yl)-

cyclobutyl)propan-2-ol (26). To a solution of 82 (30.0 mg, 93.0

μmol) in MeOH/EtOAc (12.0 mL/3.0 mL) was added Pd/C (20.0

mg, 19.0 μmol, 10% w/w), and the reaction mixture was stirred at 16

°C under an atmosphere of hydrogen (15 psi) for 4.5 h. After this

time, the mixture was ﬁltered through a pad of Celite and the ﬁltrate

was concentrated. The crude product was puriﬁed by prep-TLC (1:15

MeOH/DCM) to give the title compound (25.0 mg, 75%) as a white

1-(1-(2-Aminopyrimidin-4-yl)-1H-pyrazolo[4,3-c]pyridin-6-yl)-3-

methylazetidin-3-ol (22). To a solution of 57 (50.0 mg, 203 μmol) in

dioxane (2.0 mL) was added 3-methylazetidin-3-ol (50.1 mg, 405

μmol), SPhos-Pd-G3·DCM (15.8 mg, 20.3 μmol), and t-BuONa

(58.4 mg, 608 μmol). The mixture was stirred at 90 °C for 17 h under

an atmosphere of nitrogen. The mixture was ﬁltered, and the ﬁltrate

was concentrated under reduced pressure. The reaction was repeated

(same scale), and the crude residues from the two reactions were

combined and puriﬁed by prep-HPLC (column: Xtimate C18 150 ×

25 mm × 5 μm; gradient: MeCN/water (10 mM NH₄HCO₃), 11−

41%, 25 mL/min) to aﬀord the title compound as a white solid (9.7

mg, 8.1%). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.70 (s, 1H), 8.37

(d, J = 0.8 Hz, 1H), 8.27 (d, J = 5.2 Hz, 1H), 7.46 (s, 1H), 7.01−6.98

(m, 3H), 5.63 (s, 1H), 3.97−3.95 (m, 2H), 3.89−3.87 (m, 2H), 1.48

(s, 3H). MS (ESI): 298.1 [M + H]⁺. HRMS (ESI): m/z calculated for

C₁₄H₁₅N₇O + H⁺[M + H]⁺: 298.1411. Found: 298.1412.

1

solid. H NMR (400 MHz, CD₃OD) δ ppm 8.72 (s, 1H), 8.62 (s,

1H), 8.29 (d, J = 6.0 Hz, 1H), 7.96 (d, J = 3.6 Hz, 1H), 6.92 (d, J =

6.0 Hz, 1H), 6.86 (d, J = 3.2 Hz, 1H), 3.58−3.53 (m, 1H), 2.45−2.39

(m, 4H), 2.31−2.26 (m, 2H), 1.16 (s, 6H). LCMS (ESI, m/z): 324.0

[M + H]⁺. HRMS (ESI): m/z calculated for C₁₈H₂₁N₅O + H⁺[M +

H]⁺: 324.1819. Found: 324.1822. Purity: 92.5%.

2-(3-(1-(2-Aminopyrimidin-4-yl)-1H-pyrrolo[3,2-c]pyridin-6-yl)-

bicyclo[1.1.1]pentan-1-yl)propan-2-ol (27). To a solution of 84

(59.0 mg of equimolar mixture with 83, ∼100 μmol of 84) in THF

(2.0 mL) was added dropwise via a syringe a solution of MeMgBr (3.0

M Et₂O, 0.34 mL) at room temperature, and the resulting mixture

was stirred for 1 h. After this time, the volatiles were removed under

reduced pressure. The crude residue was puriﬁed by mass-directed

HPLC (column: Waters XSelect CSH Prep C18 OBD 50 × 100 mm

× 5 μm; gradient: MeCN/water (0.2% v/v NH₄OH), 5−65%, 80

mL/min) to give the title compound (5.0 mg, 15%) as a pale yellow

solid. ¹H NMR (500 MHz, CD₃OD) δ ppm 8.77−8.73 (m, 1H), 8.38

(s, 1H), 8.30 (d, J = 5.5 Hz, 1H), 7.95 (d, J = 3.7 Hz, 1H), 6.91 (d, J =

6.1 Hz, 1H), 6.86 (d, J = 3.7 Hz, 1H), 2.10 (s, 6H), 1.24 (s, 6H). MS

(ESI): 336.2 [M + H]⁺. HRMS (ESI): m/z calculated for C₁₉H₂₁N₅O

+ H⁺[M + H]⁺: 336.1819. Found: 336.1822.

4-(1-(2-Aminopyrimidin-4-yl)-1H-pyrazolo[4,3-c]pyridin-6-yl)-

phenol (23). To a solution of 57 (50.0 mg, 203 μmol) in dioxane/

water (2.0 mL/0.5 mL) was added (4-hydroxyphenyl)boronic acid

(41.9 mg, 304 μmol), K₂CO₃(56.0 mg, 405 μmol), and Pd(dppf)Cl₂

(14.8 mg, 20.3 μmol). The mixture was stirred at 90 °C for 17 h

under an atmosphere of nitrogen. The mixture was concentrated

under reduced pressure. The crude residue was puriﬁed by prep-

HPLC (column: Phenomenex Synergi C18 150 × 30 mm × 4 μm;

gradient: MeCN/water (0.225% formic acid), 11−31%, 25 mL/min)

1

to aﬀord the title compound as a white solid (35.4 mg, 57%). H

2-Methyl-4-(1-(2-(methylamino)pyrimidin-4-yl)-1H-pyrrolo[3,2-

c]pyridin-6-yl)but-3-yn-2-ol (28). The reaction was carried out

according to general procedure 1 using a mixture of 4-(6-chloro-

2,3-dihydro-1H-pyrrolo[3,2-c]pyridin-1-yl)-N-methylpyrimidin-2-

amine and 85 (30.0 mg, 115 μmol), 2-methylbut-3-yn-2-ol (45 μL,

460 μmol), and triethylamine (48 μL, 345 μmol) in 2.0 mL of DMSO

at 100 °C for 2 h. EtOAc (20 mL) and water (20 mL) were added,

and the aqueous layer was separated and extracted with EtOAc (20

mL × 2). The combined organic layers were washed with brine (10

mL), dried over Na₂SO₄, ﬁltered, and concentrated under reduced

pressure. The crude residue was puriﬁed by column chromatography

(SiO₂, gradient: 0−100% EtOAc/petroleum ether) to aﬀord the title

compound (15.0 mg, with impurities). The product was further

puriﬁed by prep-HPLC (column: Phenomenex Synergy C18 150 × 30

mm × 4 μm; MeCN/water (0.225% formic acid), 15−35%, 25 mL/

min) to aﬀord the title compound as a white solid (2.7 mg, 1.7% over

NMR (400 MHz, DMSO-d₆) δ ppm 9.83 (br s, 1H), 9.23 (s, 1H),

9.05 (s, 1H), 8.68 (s, 1H), 8.37 (d, J = 6.0 Hz, 1H), 8.19−8.17 (m,

2H), 7.53 (br s, 2H), 7.18 (d, J = 6.0 Hz, 1H), 6.92−6.91 (m, 2H).

MS (ESI): 305.0 [M + H]⁺. HRMS (ESI): m/z calculated for

C₁₆H₁₂N₆O + H⁺[M + H]⁺: 305.1146. Found: 305.1146.

(E)-4-(1-(2-Aminopyrimidin-4-yl)-1H-pyrrolo[3,2-c]pyridin-6-yl)-

2-methylbut-3-en-2-ol (24). To a solution of 9 (50.0 mg, 170 μmol)

in THF (5.0 mL) was slowly added Red-Al (188 μL, 680 μmol, 70%

w/w in toluene) at 0 °C, and the mixture was stirred at 20 °C for 16

h. After this time, the reaction was quenched by addition of sat. aq

NaHCO₃(5 mL) and diluted with water (10 mL). The aqueous layer

was separated and extracted with EtOAc (20 mL × 3). The combined

organic layers were dried over Na₂SO₄, ﬁltered, and concentrated.

The crude was puriﬁed by prep-HPLC (column: Xtimate C18 150 ×

30 mm × 5 μm; gradient: MeCN/water (10 mM NH₄HCO₃), 12−

42%, 25 mL/min) to give the title compound (46.0 mg, 92%) as a

1

two steps). H NMR (400 MHz, CD₃OD) δ ppm 8.99 (s, 1H), 8.95

1

light yellow solid. H NMR (400 MHz, CDCl₃) δ ppm 8.86 (s, 1H),

(s, 1H), 8.40−8.38 (m, 1H), 8.25 (d, J = 4.0 Hz, 1H), 7.08 (d, J = 3.2

Hz, 1H), 6.97 (d, J = 2.4 Hz, 1H), 3.06 (s, 3H), 1.63 (s, 6 H). MS

(ESI): 308.1 [M + H]⁺. HRMS (ESI): m/z calculated for C₁₇H₁₇N₅O

+ H⁺[M + H]⁺: 308.1506. Found: 308.1507.

(S)-1-(1-(6-Aminopyrimidin-4-yl)-1H-pyrrolo[3,2-c]pyridin-6-yl)-

3-methylpent-1-yn-3-ol (29). Step 1: The reaction was carried out

8.37 (s, 1H), 8.22 (s, 1H), 7.65 (d, J = 3.5 Hz, 1H), 6.95 (d, J = 16.0

Hz, 1H), 6.81 (d, J = 16.0 Hz, 1H), 6.75−6.72 (m, 2H), 5.32 (s, 2H),

1.47 (m, 6H). LCMS (ESI, m/z): 295.9 [M + H]⁺. HRMS (ESI): m/

z calculated for C₁₆H₁₇N₅O + H⁺[M + H]⁺: 296.1506. Found:

296.1507.

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according to general procedure 1 using 86 (100 mg, 407 μmol) and 3-

methylpent-1-yn-3-ol (184 μL, 1.63 mmol) in 10.0 mL of DMSO at

100 °C for 10 h. The mixture was diluted with water (40 mL) and

extracted with EtOAc (30 mL × 4). The combined organic layers

were dried over Na₂SO₄, ﬁltered, and concentrated. The crude residue

was puriﬁed by prep-HPLC (column: Xtimate C18 150 × 25 mm × 5

μm; gradient: MeCN/water (10 mM NH₄HCO₃), 15−55%, 25 mL/

min) to aﬀord the title compound as a yellow solid (41.6 mg, 26%).

MS (ESI): 308.1 [M + H]⁺. Step 2: Chiral separation of racemic

product from step 1 was performed using the following column: AD

(250 × 30 mm × 5 μm); method: 40% IPA with 0.1% NH₄OH in

CO₂(ﬂow rate: 50 mL/min). The ﬁrst peak eluting oﬀ the column

provided (S)-1-(1-(6-aminopyrimidin-4-yl)-1H-pyrrolo[3,2-c]pyridin-

6-yl)-3-methylpent-1-yn-3-ol 29 as a light yellow solid in 99.2% ee (Rt

pyridin-6-yl)-3-methylpent-1-yn-3-ol (the enantiomer of 31) as a

white solid in 98% ee (Rt = 6.727 min, 6.1 mg, 17%, H NMR and

1

MS identical to 31). The second peak furnished (S)-1-(1-(2-amino-6-

methoxypyrimidin-4-yl)-1H-pyrrolo[3,2-c]pyridin-6-yl)-3-methyl-

pent-1-yn-3-ol 31 as a white solid in 98% ee (Rt = 7.333 min, 6.3 mg,

18%). Stereochemistry arbitrarily assigned. ¹H NMR (400 MHz,

CDCl₃) δ ppm 8.87−8.86 (m, 1H), 8.35 (s, 1H), 7.72 (d, J = 3.6 Hz,

1H), 6.75 (d, J = 2.8 Hz, 1H), 6.17 (s, 1H), 5.11 (s, 2H), 3.97 (s,

3H), 1.85 (q, J = 7.6 Hz, 2H), 1.63 (s, 3H), 1.14 (t, J = 7.6 Hz, 3H).

¹³C NMR (126 MHz, CDCl₃) δ ppm 172.3, 162.7, 159.3, 143.9,

138.9, 135.7, 127.3, 126.5, 113.2, 105.4, 91.4, 86.0, 84.0, 69.1, 54.0,

36.6, 29.2, 9.2. MS (ESI): 338.1 [M + H]⁺. HRMS (ESI): m/z

calculated for C₁₈H₁₉N₅O₂+ H⁺[M + H]⁺: 338.1612. Found:

338.1614.

1

= 6.738 min, 8.2 mg, 20%). Stereochemistry arbitrarily assigned. H

(S)-1-(1-(2-Amino-5-methoxypyrimidin-4-yl)-1H-pyrrolo[3,2-c]-

pyridin-6-yl)-3-methylpent-1-yn-3-ol (32). Step 1: The reaction was

carried out according to general procedure 1 using 89 (15.0 mg, 43.5

μmol), 3-methylpent-1-yn-3-ol (5 μL, 43.5 μmol), and triethylamine

(18 μL, 131 μmol) in 4.0 mL of DMSO at 100 °C for 12 h. The

mixture was diluted with water (8 mL) and extracted with EtOAc (15

mL × 3). The combined organic layers were washed with brine (30

mL × 3), dried over Na₂SO₄, ﬁltered, and concentrated. The reaction

was repeated three more times. The combined crude residue was

puriﬁed by prep-TLC (1:10 MeOH/DCM), followed by prep-HPLC

(column: YMC-Actus Pro C18 150 × 30 mm × 5 μm; gradient:

MeCN/water (0.1% TFA), 15−45%, 40 mL/min) to aﬀord rac-1-(1-

(2-amino-5-methoxypyrimidin-4-yl)-1H-pyrrolo[3,2-c]pyridin-6-yl)-

3-methylpent-1-yn-3-ol as a light brown oil (triﬂuoroacetate salt, 17.0

mg, 21%). MS (ESI): 338.1 [M + H]⁺. Step 2: Chiral separation of

racemic product from step 1 was performed using the following

column: DAICEL CHIRALCEL OJ-H (250 × 30 mm × 5 μm);

method: 30% EtOH with 0.1% NH₄OH in CO₂(ﬂow rate: 60 mL/

min). The ﬁrst peak eluting oﬀ the column provided (R)-1-(1-(2-

amino-5-methoxypyrimidin-4-yl)-1H-pyrrolo[3,2-c]pyridin-6-yl)-3-

methylpent-1-yn-3-ol (the enantiomer of 32) as a white solid in 100%

ee (Rt = 4.715 min, 1.8 mg, 11%, ¹H NMR and MS identical to 32).

The second peak furnished (S)-1-(1-(2-amino-5-methoxypyrimidin-4-

yl)-1H-pyrrolo[3,2-c]pyridin-6-yl)-3-methylpent-1-yn-3-ol 32 as a

white solid in 100% ee (Rt = 5.205 min, 1.8 mg, 11%).

Stereochemistry arbitrarily assigned. ¹H NMR (400 MHz,

CDC₃OD) δ ppm 8.79 (s, 1H), 8.28−8.27 (m, 2H), 8.09 (d, J =

3.6 Hz, 1H), 6.88−6.86 (m, 1H), 3.86 (s, 3H), 1.84−1.79 (m, 2H),

1.56 (s, 3H), 1.13 (t, J = 7.2 Hz, 3H). MS (ESI): 338.2 [M + H]⁺.

1-(5-Bromo-1-tosyl-1H-indol-3-yl)ethan-1-one (int-35). To a

solution of 1-(5-bromo-1H-indol-3-yl)ethan-1-one 33 (2.00 g, 10.0

mmol) in DCM (10.0 mL) was added TsCl (730 mg, 3.80 mmol) and

triethylamine (970 μL, 7.0 mmol) at 26 °C and stirred at 26 °C for 16

h. The reaction solution was diluted with DCM (50 mL), washed

with sat. aq NH₄Cl solution, sat. aq NaHCO₃solution, water, and

brine. The organic layer was dried over Na₂SO₄. Evaporation of

solvents aﬀorded the title compound as a red solid which was used

without puriﬁcation (950 mg, 68%).

1-(5-Bromo-1-tosyl-1H-indazol-3-yl)ethan-1-one (int-36). To a

solution of 1-(5-bromo-1H-indazol-3-yl)ethan-1-one 34 (240 mg,

1.00 mmol), DMAP (30.0 mg, 250 μmol), triethylamine (513 μL,

3.70 mmol) in DCM (15.0 mL) was added TsCl (270 mg, 1.50

mmol) at 16 °C and stirred at 16 °C for 2 h. The reaction solution

was diluted with DCM (50 mL), washed with sat. aq NaHCO₃, water,

and brine. The organic layer was dried over Na₂SO4. Evaporation of

solvents aﬀorded the title compound (300 mg, 75%) as a red solid

which was used without puriﬁcation.

1-(5-Bromo-1-methyl-1H-indazol-3-yl)ethan-1-one (37). To a

suspension of 1-(5-bromo-1H-indazol-3-yl)ethan-1-one 34 (260 mg,

1.10 mmol) and NaOH (132 mg, 3.30 mmol) in THF (10.0 mL) was

added MeI (137 μL, 2.20 mmol) at 16 °C and stirred at 16 °C for 3 h.

The reaction solution was quenched with water (10 mL) and stirred

at 16 °C for 2 h, then evaporated under reduced pressure. The

aqueous layer was extracted with DCM (20 mL × 3). The combined

organic phase was washed with water and brine and dried over

Na₂SO₄. Evaporation of solvents aﬀorded the title compound (250

NMR (400 MHz, DMSO-d₆) δ ppm 8.88 (s, 1H), 8.44 (s, 1H), 8.40

(s, 1H), 8.08 (d, J = 3.6 Hz, 1H), 7.17 (s, 2H), 6.95−6.94 (m, 1H),

6.66 (d, J = 1.2 Hz, 1H), 5.45 (s, 1H), 1.72−1.65 (m, 2H), 1.45 (s,

3H), 1.02 (t, J = 7.2 Hz, 3H). MS (ESI): 308.1 [M + H]⁺. HRMS

(ESI): m/z calculated for C₁₇H₁₇N₅O + H⁺[M + H]⁺: 308.1506.

Found: 308.1514. The second peak furnished (R)-1-(1-(6-amino-

pyrimidin-4-yl)-1H-pyrrolo[3,2-c]pyridin-6-yl)-3-methylpent-1-yn-3-

ol (the enantiomer of 29) as a light yellow solid in 98.9% ee (Rt =

1

7.045 min, 9.8 mg, 24%, H NMR and MS identical to 29).

(S)-1-(1-(2-Amino-6-methylpyrimidin-4-yl)-1H-pyrrolo[3,2-c]-

pyridin-6-yl)-3-methylpent-1-yn-3-ol (30). Step 1: The reaction was

carried out according to general procedure 1 using 87 (150 mg, 575

μmol), 3-methylpent-1-yn-3-ol (259 μL, 2.30 mmol), and triethyl-

amine (240 μL, 1.73 mmol) in 5.0 mL of DMSO at 100 °C for 3 h.

TMT solution (1 mL) was added to the reaction mixture, and the

mixture was ﬁltered. The ﬁltrate was concentrated under reduced

pressure and puriﬁed by prep-HPLC (column: Agela ASB 150 × 25

mm × 5 μm; gradient: MeCN/water (0.1% TFA), 8−28%, 25 mL/

min) to aﬀord rac-1-(1-(2-amino-6-methylpyrimidin-4-yl)-1H-

pyrrolo[3,2-c]pyridin-6-yl)-3-methylpent-1-yn-3-ol as a yellow solid

(79.0 mg, 43%). MS (ESI): 322.1 [M + H]⁺. Step 2: Chiral separation

of racemic product from step 1 was performed using the following

column: C2 (250 × 30 mm × 10 μm); method: 40% EtOH with 0.1%

NH₄OH in CO₂(ﬂow rate: 70 mL/min). The ﬁrst peak eluting oﬀ

the column provided (R)-1-(1-(2-amino-6-methylpyrimidin-4-yl)-1H-

pyrrolo[3,2-c]pyridin-6-yl)-3-methylpent-1-yn-3-ol (the enantiomer

of 30) as a light yellow solid in 100% ee (Rt = 6.136 min, 27.8 mg,

1

35%, H NMR and MS identical to 30). The second peak furnished

(S)-1-(1-(2-amino-6-methylpyrimidin-4-yl)-1H-pyrrolo[3,2-c]pyridin-

6-yl)-3-methylpent-1-yn-3-ol 30 as a white solid in 90% ee (Rt =

1

6.502 min, 37.7 mg, 48%). Stereochemistry arbitrarily assigned. H

NMR (400 MHz, CD₃OD) δ ppm 9.02 (s, 1H), 8.97 (s, 1H), 8.29 (d,

J = 3.6 Hz, 1H), 7.13 (d, J = 3.6 Hz, 1H), 7.04 (s, 1H), 2.47 (s, 3H),

1.88−1.82 (m, 2H), 1.60 (s, 3H), 1.14 (t, J = 7.6 Hz, 3H). MS (ESI):

322.0 [M + H]⁺. HRMS (ESI): m/z calculated for C₁₈H₁₉N₅O + H⁺

[M + H]⁺: 322.1663. Found: 322.1668.

(S)-1-(1-(2-Amino-6-methoxypyrimidin-4-yl)-1H-pyrrolo[3,2-c]-

pyridin-6-yl)-3-methylpent-1-yn-3-ol (31). Step 1: The reaction was

carried out according to general procedure 1 using 88 (120 mg, 435

μmol), 3-methylpent-1-yn-3-ol (196 μL, 1.74 mmol), and triethyl-

amine (302 μL, 2.18 mmol) in 3.0 mL of DMSO at 100 °C for 14 h.

The reaction was diluted with EtOAc (20 mL) and water (20 mL).

The aqueous layer was separated and extracted with ethyl acetate (10

mL × 3). The combined organic layers were washed with brine (10

mL), dried over Na₂SO₄, and concentrated under reduced pressure.

The crude residue was puriﬁed by prep-HPLC (column: Xtimate C18

150 × 25 mm × 5 μm; gradient: MeCN/water (10 mM NH₄HCO₃),

25−55%, 25 mL/min) to aﬀord rac-1-(1-(2-amino-6-methoxypyr-

imidin-4-yl)-1H-pyrrolo[3,2-c]pyridin-6-yl)-3-methylpent-1-yn-3-ol as

a white solid (35.0 mg, 24%). MS (ESI): 338.1 [M + H]⁺. Step 2:

Chiral separation of racemic product from step 1 was performed using

the following column: C2 (250 × 30 mm × 10 μm); method: 45%

IPA with 0.1% NH₄OH in CO₂(ﬂow rate: 60 mL/min; detection

wavelength: 220 nm). The ﬁrst peak eluting oﬀ the column provided

(R)-1-(1-(2-amino-6-methoxypyrimidin-4-yl)-1H-pyrrolo[3,2-c]-

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mg, 91%) as a red solid. The product structure was conﬁrmed by

NOE. ¹H NMR (400 MHz, CDCl₃) δ ppm 8.54 (d, J = 1.6 Hz, 1H),

7.53 (dd, J = 8.8, 1.6 Hz, 1H), 7.32 (d, J = 8.8 Hz, 1H), 4.14 (s, 3H),

2.70 (s, 3H).

and guanidine thiocyanate (400 mg, 4.00 mmol). The resulting

mixture was stirred at 80 °C for 3 h. Then the reaction suspension was

evaporated under reduced pressure, and the product was puriﬁed by

column chromatography (SiO₂, gradient: from 1:1 EtOAc/petroleum

ether to 1:20 MeOH/DCM) to aﬀord the title compound (170 mg,

56% over 2 steps) as a red solid. MS (ESI): 303.9 [M + H]⁺(⁷⁹Br).

4-(5-Chloro-1-methyl-1H-pyrazolo[3,4-c]pyridin-3-yl)pyrimidin-

2-amine (int-46). Na (526 mg, 22.9 mmol) was dissolved into EtOH

(25 mL). To this EtONa/EtOH solution were added 42 (404 mg,

1.53 mmol) and guanidine thiocyanate (367 mg, 6.10 mmol) at 30

°C. The resulting mixture was stirred at 80 °C for 3 h. The mixture

was concentrated under reduced pressure, and then it was dissolved in

EtOAc (30 mL) and washed with water (30 mL × 3). The organic

layer was dried over Na₂SO₄, ﬁltered, and concentrated. The crude

product was puriﬁed by prep-HPLC (column: Phenomenex Synergi

C18 150 × 30 mm × 4 μm; gradient: MeCN/water (0.05% HCl), 4−

24%, 25 mL/min) to aﬀord the title compound (120 mg, 30%) as a

yellow oil. MS (ESI): 261.0 [M + H]⁺.

1-(5-Chloro-1-methyl-1H-pyrazolo[3,4-c]pyridin-3-yl)ethan-1-

one (38). To a solution of compound 48 (500 mg, 1.70 mmol) in

DMF (15.0 mL) was added tributyl(1-ethoxyvinyl)stannane (677 mg,

1.87 mmol) and Pd(PPh₃)₄(197 mg, 170 μmol) with stirring at 30

°C under a nitrogen atmosphere. The mixture was heated to 80 °C

with stirring for 4 h under a nitrogen atmosphere. The mixture was

quenched with aq KF. HCl (aq, 3 M, 10 mL) was poured into the

mixture with stirring at 30 °C for 30 min. The mixture was extracted

with EtOAc (30 mL × 3), the combined organic layers were dried

over Na₂SO₄, ﬁltered, and concentrated. The crude product was

puriﬁed by column chromatography (SiO₂, gradient: 10−20%

EtOAc/petroleum ether) to aﬀord the title compound (320 mg,

90%) as a white solid. ¹H NMR (400 MHz, CD₃OD) δ ppm 8.97 (s,

1H), 8.13 (s, 1H), 4.29 (s, 3H), 2.67 (s, 3H). MS (ESI): 210.0 [M +

H]⁺.

(E)-1-(5-Bromo-1-tosyl-1H-indol-3-yl)-3-(dimethylamino)prop-2-

en-1-one (int-39). A mixture of 35 (950 mg, 2.42 mmol) and DMF−

DMA (10.0 mL) was stirred at 100 °C for 3 h. The reaction solution

was cooled to room temperature and evaporated under reduced

pressure to aﬀord the title compound (1.20 g, crude), which was used

without puriﬁcation. MS (ESI): 449.0 [M + H]⁺(⁸¹Br).

(E)-1-(5-Bromo-1-tosyl-1H-indazol-3-yl)-3-(dimethylamino)-

prop-2-en-1-one (int-40). A solution of 36 (300 mg, 750 μmol) in

DMF−DMA (5.0 mL) was stirred at 80 °C for 16 h. The reaction

solution was evaporated under reduced pressure to aﬀord the title

compound (450 mg, crude) as a yellow solid, which was used without

puriﬁcation. MS (ESI): 448.0 [M + H]⁺(⁷⁹Br).

(E)-1-(5-Bromo-1-methyl-1H-indazol-3-yl)-3-(dimethylamino)-

prop-2-en-1-one (int-41). A solution of 37 (250 mg, 1.00 mmol) in

DMF−DMA (5.0 mL) was stirred at 90 °C for 16 h. The reaction

solution was evaporated under reduced pressure to aﬀord the title

compound as a brown solid (450 mg, crude), which was used without

puriﬁcation. MS (ESI): 307.9 [M + H]⁺(⁷⁹Br).

(E)-1-(5-Chloro-1-methyl-1H-pyrazolo[3,4-c]pyridin-3-yl)-3-

(dimethylamino)prop-2-en-1-one (int-42). A solution of 38 (320

mg, 1.53 mmol) in DMF−DMA (20.0 mL) was stirred at 30 °C

under a nitrogen atmosphere. The mixture was heated to 90 °C for 48

h. The mixture was concentrated to provide the title compound (404

mg, crude), which was used without puriﬁcation. MS (ESI): 265.0 [M

+ H]⁺.

4-(5-Bromo-1H-indol-3-yl)pyrimidin-2-amine (int-43). Na (276

mg, 12.0 mmol) was dissolved in EtOH (10.0 mL). To this EtONa/

EtOH solution was added 39 (1.20 g, 2.40 mmol) followed by

addition of guanidine thiocyanate (700 mg, 7.50 mmol). The resulting

mixture was heated to 90 °C for 16 h. The solvent was removed under

reduced pressure, diluted with DCM (100 mL) and water (50 mL),

and ﬁltered through Celite. The organic phase was separated, washed

with water and brine, and dried over Na₂SO₄. Evaporation of solvents

aﬀorded crude product, which was puriﬁed by column chromatog-

raphy (SiO₂, 50% EtOAc/petroleum ether) to aﬀord the title

compound (380 mg, 55% over 2 steps) as an orange oil. MS (ESI):

289.0 [M + H]⁺(⁷⁹Br).

4-(5-Bromo-1H-indazol-3-yl)pyrimidin-2-amine (int-44). Na

(86.0 mg, 3.75 mmol) was dissolved in EtOH (10.0 mL). To this

EtONa/EtOH solution was added 40 (330 mg, 750 μmol) and

guanidine thiocyanate (220 mg, 2.25 mmol) at 16 °C and stirred at 80

°C for 20 h. Further guanidine thiocyanate (220 mg, 2.25 mmol) and

EtONa (680 mg, 10.0 mmol) were added, and the reaction

suspension was stirred at 90 °C for 20 h. The reaction suspension

was evaporated under reduced pressure, and the product was puriﬁed

by column chromatography (SiO₂, 1:20 MeOH/DCM) to aﬀord the

title compound (20.0 mg, 56% over 2 steps) as a yellow solid. MS

(ESI): 289.9 [M + H]⁺(⁷⁹Br).

5-Chloro-3-iodo-1-methyl-1H-pyrazolo[3,4-c]pyridine (48). To a

solution of 47 (880 mg, 3.15 mmol) in DMF (8.0 mL) was added

NaH (189 mg, 4.72 mmol), and the resulting mixture was stirred at

30 °C for 30 min after which time MeI (294 μL, 4.72 mmol) was

added. The mixture was stirred for 2 h at 30 °C, quenched with water

(10 mL), and concentrated. The product was puriﬁed by prep-HPLC

(column: Waters Xbridge Prep OBD C18 150 × 30 × 5 μm; gradient:

MeCN/water (0.05% v/v NH₄OH), 26−56%, 25 mL/min) to aﬀord

the title compound (500 mg, 54%) as white solid and isomer 5-

chloro-3-iodo-2-methyl-2H-pyrazolo[3,4-c]pyridine (100 mg, 10%).

¹H NMR (400 MHz, CD₃OD) δ ppm 8.86 (s, 1H), 7.46 (s, 1H), 4.21

(s, 3H). MS (ESI): 293.8 [M + H]⁺.

4-(6-Chloro-1H-pyrazolo[4,3-c]pyridin-1-yl)pyrimidin-2-amine

(int-57). To a solution of 49 (118 mg, 770 μmol) in DMF (5.0 mL)

at 10 °C was added NaH (63.7 mg, 1.55 mmol, 60% in mineral oil),

and the resulting mixture was stirred at 10 °C for 30 min. After this

time, 4-chloropyrimidin-2-amine (100 mg, 770 μmol) was added and

the reaction suspension was stirred at 60 °C for 3 h. After this time,

the reaction was concentrated, and the product was puriﬁed by

column chromatography (SiO₂, gradient: 0−100% EtOAc/petroleum

ether) to aﬀord the title compound (75.0 mg, 40%) as a yellow solid.

MS (ESI): 247.1 [M + H]⁺.

4-(6-Chloro-1H-pyrrolo[3,2-c]pyridin-1-yl)pyrimidin-2-amine

(int-58). To a suspension of NaH (197 mg, 4.92 mmol, 60% in

mineral oil) in DMF (2.0 mL) was added 50 (500 mg, 3.28 mmol)

dropwise in DMF (1.5 mL) at 0 °C under a nitrogen atmosphere, and

the mixture was stirred at room temperature for 1.5 h. After this time,

4-chloropyrimidin-2-amine (637 mg, 4.92 mmol) was added and the

reaction was stirred at 60 °C for 12 h. After this time, the mixture was

quenched with water (3 mL), which led to the product precipitating

out of solution. An additional amount of water (25 mL) was added,

and the resulting mixture was ﬁltered. The ﬁlter cake was then dried

to give the title compound (850 mg, crude) as a yellow solid, which

was used without further puriﬁcation. MS (ESI): 246.1 [M + H]⁺.

4-(6-Chloro-2,3-dihydro-1H-pyrrolo[3,2-c]pyridin-1-yl)pyrimidin-

2-amine (int-59). To 51 (500 mg, 3.23 mmol) in DMF (9.0 mL) was

added NaH (194 mg, 4.85 mmol, 60% in mineral oil), and the mixture

was stirred at 20 °C for 30 min. After this time, 4-chloropyrimidin-2-

amine (419 mg, 3.23 mmol) was added to the mixture and the

reaction was heated to 70 °C for 1 h. After this time, the mixture was

poured into water (20 mL) and extracted with EtOAc (50 mL × 6).

The combined the organic layers were concentrated, and the crude

product was dissolved in petroleum ether/EtOAc (1:1, 10 mL) and

ﬁltered. The ﬁltrate cake was dried to aﬀord the title compound as a

yellow solid (450 mg, 56%).

4-(6-Bromo-5-methoxy-1H-indol-1-yl)pyrimidin-2-amine (int-

60). To a solution of 52 (150 mg, 660 μmol) in DMF (5.0 mL)

was added NaH (26.0 mg, 660 μmol, 60% in mineral oil), and the

mixture was stirred at 15 °C for 30 min. After this time, 4-

chloropyrimidin-2-amine (103 mg, 796 μmol) was added and the

reaction was heated to 80 °C for 18 h. After this time, the mixture was

quenched with water (10 mL), stirred for 10 min, and ﬁltered. The

4-(5-Bromo-1-methyl-1H-indazol-3-yl)pyrimidin-2-amine (int-

45). Na (345 mg, 15.0 mmol) was dissolved in EtOH (10.0 mL).

To this EtONa/EtOH solution were added 41 (450 mg, 1.00 mmol)

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ﬁlter cake was washed with petroleum ether/EtOAc (10:1) to aﬀord

the title compound (200 mg, 94%) as a yellow solid. MS (ESI): 320.9

[M + H]⁺(⁸¹Br).

C18 OBD 100 × 50 mm × 5 μm; gradient: MeCN/water (0.1% v/v

TFA), 20−60%) to aﬀord the title compound (80.0 mg, 52%). MS

(ESI): 286.0 [M + H]⁺.

4-(6-Bromo-5-ﬂuoro-1H-indol-1-yl)pyrimidin-2-amine (61). To

53 (100 mg, 467 μmol) in DMF (5.0 mL) was added NaH (37.4 mg,

934 μmol, 60% in mineral oil) slowly at 0 °C, and the mixture was

stirred at 30 °C for 30 min. After this time, 4-chloropyrimidin-2-

amine (121 mg, 934 μmol) was added and the mixture was stirred at

60 °C for 16 h. After this time, the reaction was quenched with water

(1 mL) and concentrated. The crude residue was triturated with

petroleum ether/EtOAc (5:1, 20 mL) to give a yellow solid, which

was puriﬁed by prep-HPLC (column: Xtimate C18 150 × 25 mm × 5

μm; gradient: MeCN/water (10 mM NH₄HCO₃), 36−66%, 25 mL/

min) to aﬀord the title compound as a white solid (72.0 mg, 50%). ¹H

NMR (400 MHz, DMSO-d₆) δ ppm 9.11 (d, J = 6.4 Hz, 1H), 8.27 (d,

J = 5.6 Hz, 1H), 8.18 (d, J = 3.6 Hz, 1H), 7.61 (d, J = 9.2 Hz, 1H),

7.03 (s, 2H), 6.95 (d, J = 6.0 Hz, 1H), 6.79 (d, J = 3.6 Hz, 1H). MS

(ESI): 307.0 [M + H]⁺(⁷⁹Br).

3-Bromo-6-chloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-

pyrrolo[3,2-c]pyridine (68). To 67 (1.50 g, 6.50 mmol) in DMF

(16.2 mL) at 0 °C, NaH (363 mg, 9.10 mmol, 60% in mineral oil) was

added portionwise. The reaction was stirred at 0 °C for 20 min and at

room temperature for 40 min. After this time, the reaction was cooled

to 0 °C and 2-(chloromethoxy)ethyl-trimethyl-silane (1.19 g, 7.10

mmol, 1.3 mL) was added dropwise. The reaction was stirred at 0 °C

for 20 min and then at room temperature for 3 h. After this time, the

reaction was diluted with water and Et₂O. The aqueous layer was

separated and extracted with Et₂O (×2). The combined organic layers

were washed with brine, dried over MgSO₄, ﬁltered, and concentrated.

The crude product was puriﬁed by column chromatography (40 g

SiO₂, gradient: 5−20% EtOAc/heptane) to aﬀord the title compound

1

as a pale yellow oil (2.02 g, 86%). H NMR (500 MHz, CDCl₃) δ

ppm 8.65 (s, 1H), 7.44 (s, 1H), 7.30 (s, 1H), 5.43 (s, 2H), 3.51 (t, J =

7.9 Hz, 2H), 0.94 (t, J = 7.9 Hz, 2H), 0.00 (s, 9H). MS (ESI): 361.1

[M + H]⁺(⁷⁹Br).

4-(6-Chloro-4-methoxy-1H-pyrrolo[3,2-c]pyridin-1-yl)pyrimidin-

2-amine (62). To a solution of 54 (19.0 mg, 100 μmol) in DMF (300

μL) was added NaH (7.0 mg, 180 μmol, 60% in mineral oil), and the

mixture was stirred at 25 °C for 30 min. After this time, 4-

chloropyrimidin-2-amine (16.0 mg, 124 μmol) was added and the

reaction mixture was stirred at 60 °C overnight. After this time, the

mixture was quenched with water and concentrated under reduced

pressure. The crude product was dissolved in DMSO, ﬁltered, and

puriﬁed by prep-HPLC (column: Waters XSelect CSH C18 OBD 100

× 50 mm × 5 μm; gradient: MeCN/water (0.2% v/v NH₄OH), 5−

6-Chloro-3-(3,6-dihydro-2H-pyran-4-yl)-1-((2-(trimethylsilyl)-

ethoxy)methyl)-1H-pyrrolo[3,2-c] Pyridine (Intermediate-69). To

68 (499 mg, 1.38 mmol), 2-(3,6-dihydro-2H-pyran-4-yl)-4,4,5,5-

tetramethyl-1,3,2-dioxaborolane (290 mg, 1.40 mmol), K₂CO₃(668

mg, 4.80 mmol), Pd(dtbpf)Cl₂(63.0 mg, 97.0 μmol) were added

dioxane (6.8 mL) and water (2.4 mL). The reaction vial was sealed

and heated to 110 °C for 1 h in a microwave. After this time, the

reaction was concentrated, then diluted with water and EtOAc. The

organic layer was removed via pipette, and the aqueous layer was

separated and extracted with EtOAc (×2). The combined organic

layers were concentrated, and the crude product was puriﬁed by

column chromatography (24 g SiO₂, gradient: 10−50% EtOAc/

heptane) to aﬀord the title compound as a pale yellow solid (450 mg,

89%). MS (ESI): 365.1 [M + H]⁺.

tert-Butyl 5-(6-Chloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-

pyrrolo[3,2-c]pyridin-3-yl)-3,6-di-hydropyridine-1(2H)-carboxylate

(70). To a solution of 68 (5.00 g, 13.8 mmol) and tert-butyl 5-

(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-

1(2H)-carboxylate (4.30 g, 13.8 mmol) in water (14.0 mL) and

DMSO (140 mL) was added K₂CO₃(4.20 g, 30.4 mmol) and

Pd(dppf)Cl₂(607 mg, 830 μmol). The mixture was stirred at 100 °C

for 2 h under a nitrogen atmosphere. The mixture was diluted with

water (400 mL) and extracted with EtOAc (160 mL × 5). The

combined organic layers were washed with brine (120 mL × 3), dried

over Na₂SO₄, ﬁltered, and concentrated under reduced pressure. The

crude product was puriﬁed by column chromatography (SiO₂,

gradient: 0−6% EtOAc/petroleum ether) to aﬀord the title

compound as a white solid (3.37 g, 53%). ¹H NMR (500 MHz,

CD₃OD) δ ppm 8.80 (s, 1H), 7.63 (s, 1H), 7.53 (s, 1H), 6.38 (t, J =

4.0 Hz, 1H), 5.54 (s, 2H), 4.26 (s, 2H), 3.63−3.59 (m, 2H), 3.52 (t, J

= 8.0 Hz, 2H), 2.39−2.37 (m, 2H), 1.51 (s, 9H), 0.87 (t, J = 8.0 Hz,

2H), −0.07 (s, 9H). MS (ESI): 464.2 [M + H]⁺.

1

60%, 25 mL/min) to aﬀord the title compound (14.0 mg, 49%). H

NMR (400 MHz, CD₃OD) δ ppm 8.29−8.27 (m, 2H), 7.82 (d, J =

3.76 Hz, 1H), 6.88 (d, J = 5.77 Hz, 1H), 6.78 (d, J = 3.26 Hz, 1H),

4.05 (s, 3H). MS (ESI): 276.0 [M + H]⁺.

4-(4,6-Dichloro-1H-pyrrolo[3,2-c]pyridin-1-yl)pyrimidin-2-amine

(int-63). A solution of 4-chloropyrimidin-2-amine (692 mg, 5.34

mmol), 55 (500 mg, 2.67 mmol), and DBU (800 μL, 5.34 mmol) in

DMF (20.0 mL) was heated at 140 °C in a sealed microwave tube for

2 h. The volatiles were removed under reduced pressure, and the

crude product was subjected to puriﬁcation by column chromatog-

raphy (24 g SiO₂, gradient: 0−15% MeOH (5% v/v NH₄OH)/

DCM). The title compound was obtained as an equimolar mixture

(1.13 g) with unreacted 4-chloropyrimidine-2-amine and was used as

such in the next step. MS (ESI): 280.1 [M + H]⁺.

4-(6-Chloro-4-morpholino-1H-pyrrolo[3,2-c]pyridin-1-yl)-

pyrimidin-2-amine (64). A solution of morpholine (38 μL, 430

μmol), 63 (100 mg, 360 μmol), and DBU (110 μL, 710 μmol) in

DMF (2.0 mL) was heated in a microwave at 150 °C for 12 h. The

resulting mixture was cooled to room temperature and puriﬁed by

prep-HPLC (column: Waters XSelect CSH Prep C18 OBD 100 × 50

mm × 5 μm; gradient: MeCN/water (0.2% v/v NH₄OH), 5−65%, 80

mL/min) to aﬀord the title compound (37.0 mg, 31%) as a pale

1

yellow solid. H NMR (400 MHz, CD₃OD) δ ppm 8.26 (d, J = 5.5

Hz, 1H), 8.10 (s, 1H), 7.76 (d, J = 4.3 Hz, 1H), 6.88−6.80 (m, 2H),

3.87−3.82 (m, 4H), 3.66−3.59 (m, 4H). MS (ESI): 331.1 [M + H]⁺.

4-(6-Chloro-3-iodo-1H-pyrrolo[3,2-c]pyridin-1-yl)pyrimidin-2-

amine (int-65). 4-Chloropyrimidin-2-amine (245 mg, 1.90 mmol), 56

(501 mg, 1.80 mmol), Cs₂CO₃(704 mg, 2.20 mmol), and DMF (6.0

mL) were combined in a 2−5 mL microwave vial. The reaction

mixture was heated to 120 °C for 2 h in a microwave reactor. After

this time, the reaction was diluted with water and the solid was

collected by ﬁltration. The ﬁlter cake was washed with water and ether

and dried under reduced pressure to aﬀord the title compound (528

mg, crude), which was used without puriﬁcation. MS (ESI): 372.0 [M

+ H]⁺.

6-Chloro-3-(tetrahydro-2H-pyran-4-yl)-1-((2-(trimethylsilyl)-

ethoxy)methyl)-1H-pyrrolo[3,2-c]pyridine (71). To a solution of 69

in EtOAc/EtOH (10.0 mL/10.0 mL) was added platinum dioxide

(82.0 mg, 360 μmol). The headspace was purged with nitrogen, then

hydrogen was bubbled through reaction solution for 10 min. The

reaction was stirred under a balloon of hydrogen at room temperature

for 3 h. After this time, the reaction was ﬁltered through Celite, and

the Celite plug was washed with EtOAc. The ﬁltrate was concentrated

and the crude product was puriﬁed by column chromatography (12 g

SiO₂, gradient: 10−50% EtOAc/heptane) to aﬀord the title

1

compound (405 mg, 91%) as a white solid. H NMR (500 MHz,

4-(6-Chloro-3-cyclopropyl-1H-pyrrolo[3,2-c]pyridin-1-yl)-

pyrimidin-2-amine (int-66). To a mixture of 65 (200 mg, 540 μmol),

cyclopropylboronic acid (69.0 mg, 810 μmol), Pd(dtbpf)Cl₂(35.0

mg, 50.0 μmol), and K₃PO₄(343 mg, 1.61 mmol) was added dioxane

(4.1 mL). The reaction mixture was sparged with nitrogen for 10 min,

then heated to 140 °C for 1 h in a microwave reactor. The reaction

was ﬁltered and puriﬁed by prep- HPLC (column: Waters Sunﬁre

CDCl₃) δ ppm 8.67 (s, 1H), 7.37 (s, 1H), 6.95 (s, 1H), 5.38 (s, 2H),

4.09 (dd, J = 3.4, 10.7 Hz, 2H), 3.61 (dt, J = 2.1, 11.8 Hz, 2H), 3.46

(t, J = 7.9 Hz, 2H), 3.08 (tt, J = 3.7, 11.8 Hz, 1H), 1.97 (br dd, J = 1.8,

13.4 Hz, 2H), 1.90−1.78 (m, 2H), 0.89 (t, J = 7.9 Hz, 2H), −0.05 (s,

9H). MS (ESI): 367.2 [M + H]⁺.

tert-Butyl 3-(6-Chloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-

pyrrolo[3,2-c]pyridin-3-yl)pi-peridine-1-carboxylate (72). To a

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solution of 70 (2.00 g, 4.30 mmol) in EtOH/EtOAc (100 mL/100

mL) was added platinum dioxide (294 mg, 1.30 mmol). The mixture

was stirred at 25 °C for 4 h under an atmosphere of hydrogen (15

psi). After this time, the mixture was ﬁltered and the ﬁltrate was

concentrated to aﬀord the title compound (2.10 g, crude), which was

used without puriﬁcation. ¹H NMR (400 MHz, CD₃OD) δ ppm 8.68

(s, 1H), 7.61 (s, 1H), 7.36 (s, 1H), 5.52 (s, 2H), 4.24 (d, J = 11.2 Hz,

1H), 4.03 (d, J = 12.4 Hz, 1H), 3.51 (t, J = 8.0 Hz, 2H), 3.07−2.96

(m, 3H), 2.19−2.17 (m, 1H), 1.82−1.79 (m, 2H), 1.66−1.62 (m,

1H), 1.48 (s, 9H), 0.85 (t, J = 8.0 Hz, 2H), −0.08 (s, 9H). MS (ESI):

466.3 [M + H]⁺.

6-Chloro-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrrolo[3,2-c]pyridine

(int-73). A solution of 71 (405 mg, 1.10 mmol) in TBAF (1.0 M in

THF, 3.8 mL) and ethylenediamine (450 μL, 6.70 mmol) was heated

to 60 °C for 3 h. After this time, the reaction was diluted with brine

and water and extracted with EtOAc (5 mL × 3). The combined

organic layers were concentrated under reduced pressure. The crude

product was puriﬁed by column chromatography (12 g SiO₂, gradient:

25−100% EtOAc/heptane) to aﬀord the title compound as a white

solid (215 mg, 83%). MS (ESI): 237.1 [M + H]⁺.

tert-Butyl 3-(6-Chloro-1H-pyrrolo[3,2-c]pyridin-3-yl)piperidine-1-

carboxylate (int-74). To a solution of 72 (2.10 g, 4.50 mmol) in

THF (40.0 mL) was added TBAF (1.0 M, 13.5 mL) and

ethylenediamine (1.8 mL, 27.1 mmol). The mixture was heated to

65 °C for 10 h. After this time, the mixture was diluted with water

(100 mL) and extracted with EtOAc (40 mL x 5). The combined

organic layers were dried over Na₂SO4, ﬁltered, and concentrated

under reduced pressure to aﬀord the title compound as a colorless oil

(1.55 g, 86%), which was used without puriﬁcation. MS (ESI): 336.0

[M + H]⁺.

4-(6-Chloro-3-(tetrahydro-2H-pyran-4-yl)-1H-pyrrolo[3,2-c]-

pyridin-1-yl)pyrimidin-2-amine (int-75). 4-Chloropyrimidin-2-amine

(60.0 mg, 460 μmol), 73 (100 mg, 420 μmol), NaH (22.0 mg, 550

μmol, 60% in mineral oil), and DMF (1.4 mL) were combined in a

2−5 mL microwave vial. The reaction mixture was heated to 110 °C

for 40 min in a microwave reactor. After this time, the reaction was

quenched with water (0.1 mL), diluted with DMSO until all solids

dissolved (3.0 mL), ﬁltered, and puriﬁed by prep-HPLC (column:

Waters Sunﬁre C18 OBD 100 × 50 mm × 5 μm; gradient: MeCN/

water (0.1 vol % TFA), 5−45%) to aﬀord the title compound, and it

was a white solid (137 mg, 73%, triﬂuoroacetate salt). MS (ESI):

330.1 [M + H]⁺.

in the next step without puriﬁcation. ¹H NMR (400 MHz, DMSO-d₆)

δ ppm 9.46−9.43 (m, 1H), 9.16 (d, J = 8.4 Hz, 1H), 8.92 (s, 1H),

8.88 (s, 1H), 8.52 (d, J = 6.8 Hz, 1H), 8.31 (s, 1H), 7.47 (d, J = 6.8

Hz, 1H), 3.52−3.48 (m, 2H), 3.35−3.32 (m, 2H), 3.10−3.05 (m,

1H), 3.04−2.92 (m, 1H), 2.15−2.11 (m, 1H), 1.92−1.82 (m, 2H),

1.79−1.71 (m, 1H). MS (ESI): 329.0 [M + H]⁺. Step 2: To solution

of the product from step 1 (441 mg, 1.34 mmol) in MeOH (15.0 mL)

was added aq formaldehyde (678 μL, 8.04 mmol, 37% w/w) and

NaCNBH₃(421 mg, 6.70 mmol). The reaction was stirred at room

temperature (26−29 °C) for 2 h. Sat. aq NaHCO₃(25 mL) was

added dropwise to the reaction mixture. The aqueous layer was

separated and extracted with DCM (30 mL × 3). The combined

organic layers were concentrated under reduced pressure. The crude

product was puriﬁed by prep-TLC (1:8 MeOH/DCM) to aﬀord the

title compound (160 mg, 35%) as a white solid. MS (ESI): 343.1 [M

+ H]⁺.

rac-trans-Ethyl 2-(1-(2-Aminopyrimidin-4-yl)-1H-pyrrolo[3,2-c]-

pyridin-6-yl)cyclopropane-1-carboxylate (78). To a solution of 58

(50.0 mg, 200 μmol) and ethyl 2-(4,4,5,5-tetramethyl-1,3,2-

dioxaborolan-2-yl)cyclopropane-1-carboxylate (489 mg, 2.04 mmol)

in DME (8.0 mL) and water (800 μL) was added K₂CO₃(84.0 mg,

610 μmol) and Pd(dtbpf)Cl₂(14.0 mg, 20 μmol), and the mixture

was stirred at 100 °C for 6 h. The mixture was ﬁltered, and the ﬁltrate

was concentrated. The crude residue was puriﬁed by prep-HPLC

(column: Xtimate C18 150 × 25 mm × 5 μm; gradient: MeCN/water

(10 mM NH₄HCO₃), 15−45%, 25 mL/min) to give in order of

elution: Isomer 1 (cis, racemic) (12.0 mg, 18%) as a light yellow solid.

¹H NMR (400 MHz, CDCl₃) δ ppm 8.71 (d, J = 1.0 Hz, 1H), 8.54 (d,

J = 1.0 Hz, 1H), 8.29 (d, J = 5.5 Hz, 1H), 7.97 (d, J = 4.0 Hz, 1H),

6.93 (d, J = 6.0 Hz, 1H), 6.85 (dd, J = 0.5, 3.5 Hz, 1H), 3.86−3.81

(m, 2H), 2.93−2.87 (m, 1H), 2.27−2.23 (m, 1H), 1.93−1.89 (m,

1H), 1.55−1.51 (m, 1H), 0.91 (t, J = 7.0 Hz, 3H). LCMS (ESI, m/z):

324.1 [M + H]⁺.

Isomer 2 (trans, racemic − desired diastereomer which was carried

1

forward) (8.0 mg, 12%) as a light yellow solid. H NMR (400 MHz,

DMSO-d₆) δ ppm 8.70 (d, J = 1.0 Hz, 1H), 8.67 (s, 1H), 8.28 (d, J =

5.5 Hz, 1H), 7.95 (d, J = 4.0 Hz, 1H), 6.92 (d, J = 5.5 Hz, 1H), 6.85−

6.84 (m, 1H), 4.20−4.15 (m, 2H), 2.84−2.80 (m, 1H), 2.24−2.20

(m, 1H), 1.69−1.67 (m, 1H), 1.61−1.58 (m, 1H), 1.28 (t, J = 7.0 Hz,

3H). LCMS (ESI, m/z): 324.1 [M + H]⁺.

Relative stereochemistry was conﬁrmed by 2D NMR.

Methyl 3-(2-Tosylhydrazineylidene)cyclobutane-1-carboxylate

(80). To a solution of methyl 3-oxocyclobutane-1-carboxylate 79

(60.0 g, 468 mmol) in MeOH (600 mL) was added p-toluenesulfonyl

hydrazide (87.2 g, 468 mmol). The reaction mixture was stirred at 25

°C for 2 h, and a white suspension was formed. The mixture was

ﬁltered, and the solid was washed with MeOH (200 mL) to give the

tert-Butyl 3-(1-(2-Aminopyrimidin-4-yl)-6-chloro-1H-pyrrolo[3,2-

c]pyridin-3-yl)piperidine-1-carboxylate (76). To a solution of 74

(900 mg, 2.70 mmol) in DMF (30.0 mL) was added Cs₂CO₃(1.31 g,

4.02 mmol) and 4-chloropyrimidin-2-amine (347 mg, 2.68 mmol).

The mixture was heated to 90 °C for 4 h. The mixture was cooled to

room temperature, diluted with water (90 mL) and extracted with

EtOAc (40 mL × 3). The combined organic layers were washed with

brine (40 mL × 3), dried over Na₂SO₄, ﬁltered, and concentrated

under reduced pressure. The crude product was suspended in EtOAc/

EtOH (30 mL/10 mL). The solid was collected by ﬁltration. The

ﬁlter cake was dried to aﬀord the ﬁrst batch of the title compound as

white solid (625 mg, 54%). The ﬁltrate was concentrated under

reduced pressure and puriﬁed by column chromatography (SiO₂,

gradient: 0−75% EtOAc/petroleum ether) to aﬀord the second batch

of the title compound (300 mg, 26%) as a white solid (total amount

1

title compound as a white solid (120 g, 86%). H NMR (400 MHz,

DMSO-d₆) δ ppm 10.41 (s, 1H), 7.68 (d, J = 8.4 Hz, 2H), 7.38 (d, J =

8.0 Hz, 2H), 3.60 (s, 3H), 3.16−2.93 (m, 5H), 2.37 (s, 3H).

Methyl 3-(1-(2-Aminopyrimidin-4-yl)-1H-pyrrolo[3,2-c]pyridin-6-

yl)cyclobut-2-ene-1-carboxylate (81). To a mixture of 58 (1.00 g,

4.07 mmol) and 80 (1.81 g, 6.11 mmol) in dioxane (160 mL) was

added dppp (302 mg, 730 μmol), Cs₂CO₃(2.65 g, 8.14 mmol) and

Pd(MeCN)₂Cl₂(95.0 mg, 370 μmol), and the reaction mixture was

stirred at 95 °C under a nitrogen atmosphere for 20 h. The reaction

was repeated two more times using 50.0 and 500 mg of 58. The three

reaction mixtures were combined and diluted with EtOAc (200 mL)

and water (150 mL) and stirred vigorously for 5 min. The organic

layer was separated, washed with brine (150 mL × 2), dried over

anhydrous Na₂SO₄, ﬁltered, and concentrated. The crude residue was

puriﬁed by column chromatography (SiO₂, gradient: 50−85%

EtOAc/petroleum ether) to aﬀord the title compound as a yellow

1

isolated: 925 mg, 80%). H NMR (400 MHz, DMSO-d₆) δ ppm

8.78−8.76 (m, 2H), 8.31 (d, J = 5.6 Hz, 1H), 8.02 (s, 1H), 7.08 (s,

2H), 7.00 (d, J = 5.2 Hz, 1H), 4.13−4.10 (m, 1H), 3.96−3.89 (m,

1H), 3.01−2.99 (m, 2H), 2.89−2.86 (m, 1H), 2.12−2.09 (m, 1H),

1.78−1.72 (m, 2H), 1.55−1.52 (m, 1H), 1.40 (s, 9H). MS (ESI):

429.0 [M + H]⁺.

4-(6-Chloro-3-(1-methylpiperidin-3-yl)-1H-pyrrolo[3,2-c]pyridin-

1-yl)pyrimidin-2-amine (77). Step 1: To a solution of 76 (575 mg,

1.34 mmol) in MeOH (10.0 mL) was added HCl (4.0 M in EtOAc,

10.0 mL), and the mixture was stirred at room temperature for 2 h.

The mixture was concentrated under reduced pressure to aﬀord 4-(6-

chloro-3-(piperidin-3-yl)-1H-pyrrolo[3,2-c]pyridin-1-yl)pyrimidin-2-

amine hydrochloride as a white solid (441 mg, crude), which was used

1

solid (430 mg, 21%). H NMR (400 MHz, CD₃OD) δ ppm 8.79 (s,

1H), 8.60 (s, 1H), 8.29 (d, J = 6.0 Hz, 1H), 8.01 (d, J = 3.6 Hz, 1H),

6.92 (d, J = 5.6 Hz, 1H), 6.89 (d, J = 3.6 Hz, 1H), 6.61 (d, J = 1.2 Hz,

1H), 3.74 (s, 3H), 3.30−3.23 (m, 2H), 3.15−3.11 (m, 1H). LCMS

(ESI, m/z): 322.0 [M + H]⁺.

2-(3-(1-(2-Aminopyrimidin-4-yl)-1H-pyrrolo[3,2-c]pyridin-6-yl)-

cyclobut-2-en-1-yl)propan-2-ol (82). To a solution of 81 (150 mg,

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470 μmol) in THF (8.0 mL) at 0 °C was added MeMgBr (1.56 mL,

4.67 mmol, 3.0 M in Et₂O) dropwise over 3 min under a nitrogen

atmosphere. After the addition was completed, the cooling bath was

removed and the mixture was stirred at 17 °C for 1 h. The mixture

was quenched with water (8 mL) and extracted with EtOAc (15 mL

× 3). The combined organic layers were dried over Na₂SO₄, ﬁltered,

and concentrated. The crude was puriﬁed by prep-TLC (1:15

MeOH/DCM) to give the title compound as a white solid (43.0 mg,

and extracted with EtOAc (25 mL × 3). The combined organic layers

were dried over Na₂SO₄, ﬁltered, and concentrated under reduced

pressure. The crude residue was suspended in DCM (3 mL) and

petroleum ether (45 mL), the resulting slurry was stirred at 30 °C for

1 h and ﬁltered. The ﬁlter cake was washed with petroleum ether (15

mL × 3) and dried under reduced pressure to aﬀord the title

1

compound as a yellow solid (410 mg, crude). H NMR (400 MHz,

DMSO-d₆) δ ppm 8.73(s, 1H), 8.71(s, 1H), 8.19 (d, J = 2.8 Hz, 1H),

6.99 (br s, 2H), 6.93 (s, 2H), 2.31 (s, 3H). MS (ESI): 260.0 [M +

H]⁺.

1

29%). H NMR (400 MHz, CD₃OD) δ ppm 8.78 (s, 1H), 8.57 (s,

1H), 8.29 (d, J = 6.0 Hz, 1H), 8.00 (d, J = 3.6 Hz, 1H), 6.92 (d, J =

6.0 Hz, 1H), 6.89−6.88 (m, 1H), 6.69 (s, 1H), 2.95−2.89 (m, 2H),

2.80−2.76 (m, 1H), 1.29−1.27 (m, 6H). LCMS (ESI, m/z): 322.0

[M + H]⁺.

4-(6-Chloro-1H-pyrrolo[3,2-c]pyridin-1-yl)-6-methoxypyrimidin-

2-amine (int-88). To a solution of 50 (300 mg, 1.97 mmol) in DMF

(2.0 mL) was added to NaH (118 mg, 2.95 mmol, 60% in mineral oil)

in DMF (2.0 mL) at room temperature. The resulting mixture was

stirred at room temperature for 1 h. 4-Chloro-6-methoxypyrimidin-2-

amine (377 mg, 2.36 mmol) was added. The reaction was stirred at 60

°C for 12 h, then at 90 °C for 12 h. The reaction was cooled to room

temperature and quenched with water (3 mL). EtOAc (30 mL) and

water (25 mL) were added, and the aqueous layer was separated and

extracted with EtOAc (10 mL × 2). The combined organic layers

were washed with brine (10 mL), dried over Na₂SO₄, and

concentrated under reduced pressure. The crude residue was puriﬁed

by prep-HPLC (column: Phenomenex Synergi C18 150 × 30 mm × 4

μm; gradient: MeCN/water (0.05% HCl), 26−46%, 25 mL/min) to

aﬀord the title compound as a white solid (180 mg, 33%). MS (ESI):

276.0 [M + H]⁺.

4-(6-Chloro-1H-pyrrolo[3,2-c]pyridin-1-yl)-5-methoxypyrimidin-

2-amine (int-89). To a suspension of NaH (39.3 mg, 983 μmol, 60%

in mineral oil) in DMF (6.0 mL) was added 50 (100 mg, 655 μmol).

The resulting mixture was stirred at 18 °C for 30 min. 4-Chloro-5-

methoxypyrimidin-2-amine (116 mg, 655 μmol) was added, and the

reaction mixture was heated at 70 °C for 5 h. The mixture was cooled

to room temperature and quenched with water (10 mL). The aqueous

layer was separated and extracted with EtOAc (15 mL × 5). The

combined organic layers were washed with brine (50 mL × 3), dried

over Na₂SO₄, ﬁltered, and concentrated. The crude residue was

puriﬁed by prep-TLC (1:10 MeOH/DCM) to aﬀord the title

compound (30.0 mg, 80% purity) and recovered 50 (20.0 mg). MS

(ESI): 276.0 [M + H]⁺.

Methyl 3-(1-(2-Aminopyrimidin-4-yl)-1H-pyrrolo[3,2-c]pyridin-6-

yl)bicyclo[1.1.1]pentane-1-carboxylate (int-84). Step 1: To a

solution of 83 (100 mg, 410 μmol) in DMF (4.0 mL) and THF

(2.0 mL) was added NaH (50.0 mg, 1.24 mmol, 60% dispersion in

oil), and the reaction mixture was stirred at room temperature for 10

min. 4-Chloropyrimidin-2-amine (80.0 mg, 620 μmol) was then

added, and the resulting suspension was heated to 140 °C in a

microwave reactor for 30 min. After this time, the reaction mixture

was cooled to room temperature, ﬁltered through Celite and

concentrated to give an equimolar mixture of 1-[1-(2-amino-

pyrimidin-4-yl)pyrrolo[3,2-c]pyridin-6-yl]bicyclo[1.1.1]pentane-3-

carboxylic acid and 1-(1H-pyrrolo[3,2-c]pyridin-6-yl)bicyclo[1.1.1]-

pentane-3-carboxylic acid as determined by ¹H NMR (75.0 mg). The

mixture was used in the next step without puriﬁcation. LCMS (ESI,

m/z): 322.1 [M + H]⁺. Step 2: The mixture from step 1 (75.0 mg) in

MeOH (2.0 mL) and DCM (1.0 mL) was added dropwise via a

syringe trimethylsilyl diazomethane (0.68 mL, 2.0 M in hexanes) at

room temperature, and the mixture was stirred for 1.5 h. After this

time, the reaction mixture was diluted with DCM (3 mL) and washed

with sat. aq NaHCO₃(3 mL). The organic layer was dried over

Na₂SO₄, ﬁltered, and concentrated to aﬀord the title compound (59.0

mg, equimolar mixture with 83 as determined by NMR) which was

used in the next step without puriﬁcation. LCMS (ESI, m/z): 336.2

[M + H]⁺.

4-(6-Chloro-1H-pyrrolo[3,2-c]pyridin-1-yl)-N-methylpyrimidin-2-

amine (int-85). To a suspension of NaH (29.0 mg, 724 μmol, 60% in

mineral oil) in DMF (2.0 mL) was added 51 (80.0 mg, 517 μmol) in

DMF (1.0 mL). The mixture was stirred at room temperature (25−31

°C) for 40 min. Then 4-chloro-N-methylpyrimidin-2-amine (74.3 mg,

517 μmol) was added. The reaction was stirred at 60 °C for 4 h. The

reaction was quenched with water (5 mL), the precipitated solid was

collected by ﬁltration. The ﬁlter cake was dried under vacuum to

aﬀord a mixture of 4-(6-chloro-2,3-dihydro-1H-pyrrolo[3,2-c]pyridin-

1-yl)-N-methylpyrimidin-2-amine and title compound 85 (40.0 mg,

crude) as a white solid, which was used in the next step without

further puriﬁcation. MS (ESI): 262.0 [M + H]⁺and 260.0 [M + H]⁺.

6-(6-Chloro-1H-pyrrolo[3,2-c]pyridin-1-yl)pyrimidin-4-amine

(int-86). To a solution of 50 (500 mg, 3.28 mmol) in DMF (10.0

mL) was added NaH (393 mg, 9.83 mmol, 60% in mineral oil)

portionwise. The resulting mixture was stirred for 30 min. 6-

Chloropyrimidin-4-amine (849 mg, 6.55 mmol) was then added, and

the reaction was stirred at 60 °C for 16 h. The reaction mixture was

concentrated, and the residue was triturated with petroleum ether/

EtOAc (5:1, 40 mL) to give the crude residue. The reaction was

repeated with 100 mg of 50, and the crude residues from the two

reactions were combined and subjected to prep-HPLC puriﬁcation

(column: Xtimate C18 150 × 30 mm × 5 μm; gradient: MeCN/water

(10 mM NH₄HCO₃), 20−50%, 25 mL/min) to aﬀord the title

compound as a white solid (130 mg, 13%). MS (ESI): 246.0 [M +

H]⁺.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00382.

■

sı

Description of biochemical and cellular assays; descrip-

tion of glutathione (GSH) experiments; experimental

conditions for crystallization, collection, and reﬁnement

statistics for compounds 3 and 18; description of

cytotoxicity assay; selectivity proﬁle of compounds 18,

19, and 31; description of K_p,uustudies; (Figure S1)

unaltered western blots supporting quantiﬁcation of tau

phosphorylation summarized in Figure 7; (Figure S2)

basal Ser 422 levels in the developing rat brain; (Figure

S3) unaltered western blots supporting quantiﬁcation of

tau phosphorylation summarized in Figure 8; (Figure

S4) neurotoxicity of lead compound 31 in primary

mouse neurons; methods supporting in vivo pharmacol-

ogy; general description of in vitro DMPK assays; and

HPLC data for compounds 18, 19, and 31 (PDF)

Molecular formula strings (CSV)

4-(6-Chloro-1H-pyrrolo[3,2-c]pyridin-1-yl)-6-methylpyrimidin-2-

amine (87). To a solution of 50 (300 mg, 1.97 mmol) in DMF (5.0

mL) was added NaH (118 mg, 2.95 mmol, 60% in mineral oil). The

reaction mixture was stirred at 30 °C for 30 min. Then, 4-chloro-6-

methylpyrimidin-2-amine (565 mg, 3.93 mmol) was added. The

reaction mixture was stirred at 60 °C for 12 h. The reaction was

quenched with water (25 mL), and the aqueous layer was separated

AUTHOR INFORMATION

Corresponding Authors

■

Tamara Halkina − Department of Medicinal Chemistry,

Biogen, Cambridge, Massachusetts 02142, United States;

Phone: 617-679-3382; Email: toma.halkina@biogen.com

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Felix Gonzalez-Lopez de Turiso − Department of Medicinal

Chemistry, Biogen, Cambridge, Massachusetts 02142, United

States; orcid.org/0000-0001-9736-0907; Phone: 617-

914-1295; Email: felix.gonzalezlopezdeturiso@

biogen.com

Brenda Amaral − Department of Emerging Neurosciences

Research Unit, Biogen, Cambridge, Massachusetts 02142,

United States

Gregory M. Dillon − Department of Emerging Neurosciences

Research Unit, Biogen, Cambridge, Massachusetts 02142,

United States

Rab Gilﬁllan − Department of Medicinal Chemistry, Biogen,

Cambridge, Massachusetts 02142, United States

Authors

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00382

Jaclyn L. Henderson − Department of Medicinal Chemistry,

Biogen, Cambridge, Massachusetts 02142, United States;

orcid.org/0000-0002-7097-1350

Notes

Edward Y. Lin − Department of Medicinal Chemistry, Biogen,

Cambridge, Massachusetts 02142, United States

Martin K. Himmelbauer − Department of Medicinal

Chemistry, Biogen, Cambridge, Massachusetts 02142, United

States; orcid.org/0000-0003-4960-4457

J. Howard Jones − Department of Medicinal Chemistry,

Biogen, Cambridge, Massachusetts 02142, United States

Marta Nevalainen − Department of Medicinal Chemistry,

Biogen, Cambridge, Massachusetts 02142, United States

Jun Feng − Department of Medicinal Chemistry, Biogen,

Cambridge, Massachusetts 02142, United States

Kristopher King − Department of Drug Metabolism and

Pharmacokinetics, Biogen, Cambridge, Massachusetts 02142,

United States

Michael Rooney − Department of Drug Metabolism and

Pharmacokinetics, Biogen, Cambridge, Massachusetts 02142,

United States

Joshua L. Johnson − Department of Drug Metabolism and

Pharmacokinetics, Biogen, Cambridge, Massachusetts 02142,

United States

Douglas J. Marcotte − Department of Physical Biochemistry

and Molecular Design, Biogen, Cambridge, Massachusetts

02142, United States

Jayanth V. Chodaparambil − Department of Physical

Biochemistry and Molecular Design, Biogen, Cambridge,

Massachusetts 02142, United States

P. Rajesh Kumar − Department of Physical Biochemistry and

Molecular Design, Biogen, Cambridge, Massachusetts 02142,

United States

Thomas A. Patterson − Department of Physical Biochemistry

and Molecular Design, Biogen, Cambridge, Massachusetts

02142, United States

Paramasivam Murugan − Department of Bioassays, Biogen,

Cambridge, Massachusetts 02142, United States

Eli Schuman − Department of Bioassays, Biogen, Cambridge,

Massachusetts 02142, United States

LaiYee Wong − Department of Bioassays, Biogen, Cambridge,

Massachusetts 02142, United States

Thomas Hesson − Department of Bioassays, Biogen,

Cambridge, Massachusetts 02142, United States

Sarah Lamore − Department of Preclinical Safety, Biogen,

Cambridge, Massachusetts 02142, United States

Channa Bao − Department of Emerging Neurosciences

Research Unit, Biogen, Cambridge, Massachusetts 02142,

United States

Michael Calhoun − Department of Emerging Neurosciences

Research Unit, Biogen, Cambridge, Massachusetts 02142,

United States

The authors declare no competing ﬁnancial interest.

Compound 3: 7JXX; Compound 18: 7JXX. Authors will

release the atomic coordinates and experimental data upon

article publication.

All in vivo studies were conducted in accordance with Biogen

Institutional Animal Care and Use Committee (IACUC)

guidelines.

ACKNOWLEDGMENTS

■

We thank Kevin Barry for chiral separation support, Michael

Dechantsreiter for parallel synthesis support, Sam Cziria for

compound handling, and Jiasheng Huang for assistance with

the collection of analytical data. We thank Xiaoping Wang,

Hushan Zhang, and Wenkui Zhao for eﬀorts coordinating the

synthesis of some of the compounds included in this

manuscript and Simone Sciabola and Claire Metrick for

helpful discussions.

ABBREVIATIONS USED

■

AD, Alzheimer’s disease; aq, aqueous; Cl, clearance; CNS,

central nervous system; compd, compound; DCM, dichloro-

methane; DMAP, 4-dimethylaminopyridine; DMF, dimethyl-

formamide; DMSO, dimethyl sulfoxide; dppf, 1,1′-bis-

(diphenylphosphino)ferrocene; dtbpf, 1,1′-bis(di-tert-

butylphosphino)ferrocene; equiv., equivalent; ER, eﬄux ratio;

ESI, electrospray ionization; EtOAc, ethyl acetate; HLM,

human liver microsomes; HPLC, high-performance liquid

chromatography; HPPB, human plasma protein binding; Int,

intermediate; IP, intraperitoneal; MDCK, Madin−Darby

canine kidney cells; MDR1, multidrug resistance 1; MeOH,

methanol; NIK, NF-κB inducing kinase; NMR, nuclear

magnetic resonance; P-gp, P-glycoprotein; PK, pharmacoki-

netic; PSA, polar surface area; RLM, rat liver microsomes;

RPPB, rat plasma protein binding; SAR, structure−activity

relationship; satd., saturated; SEM, standard error of the mean;

Ser, serine; SFC, supercritical ﬂuid chromatography; SPhos, 2-

dicyclohexylphosphino-2′,6′-dimethoxybiphenyl; TFA, tri-

ﬂuoroacetic acid; TLC, thin layer chromatography; TTBK1,

tau tubulin kinase 1; TTBK2, tau tubulin kinase 2; XPhos, 2-

dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl

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6380

https://doi.org/10.1021/acs.jmedchem.1c00382

J. Med. Chem. 2021, 64, 6358−6380

Article Doi

Discovery of Potent and Brain-Penetrant Tau Tubulin Kinase 1 (TTBK1) Inhibitors that Lower Tau Phosphorylation in Vivo

DOI: 10.1021/acs.jmedchem.1c00382

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Article abstract of DOI:10.1021/acs.jmedchem.1c00382

Full text of DOI:10.1021/acs.jmedchem.1c00382

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