Diazaspirononane Nonsaccharide Inhibitors of O-GlcNAcase (OGA) for the Treatment of Neurodegenerative Disorders

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Diazaspirononane Nonsaccharide Inhibitors of O‑GlcNAcase (OGA)

for the Treatment of Neurodegenerative Disorders

́

Carlos M. Mart í nez-Viturro,* Andres A. Trabanco,* Jordi Royes, Elena Fernandez, Gary Tresadern,*

́

Juan A. Vega, Alcira del Cerro, Francisca Delgado, Aranzazu Garc í a Molina, Fulgencio Tovar,

Paul Sha ﬀ er, Andreas Ebneth, Alexis Bretteville, Liesbeth Mertens, Marijke Somers, Jose M. Alonso,

́

and Jose M. Bartolome-Nebreda *

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ABSTRACT: O-GlcNAcylation is a post-translational modiﬁca-

tion of tau understood to lower the speed and yield of its

aggregation, a pathological hallmark of Alzheimer’s disease (AD).

O-GlcNAcase (OGA) is the only enzyme that removes O-linked N-

acetyl-^D-glucosamine (O-GlcNAc) from target proteins. Therefore,

inhibition of OGA represents a potential approach for the

treatment of AD by preserving the O-GlcNAcylated tau protein.

Herein, we report the multifactorial optimization of high-

throughput screening hit 8 to a potent, metabolically stable, and

orally bioavailable diazaspirononane OGA inhibitor (+)-56. The

human OGA X-ray crystal structure has been recently solved, but

bacterial hydrolases are still widely used as structural homologues.

For the ﬁrst time, we reveal how a nonsaccharide series of inhibitors binds bacterial OGA and discuss the suitability of two diﬀerent

bacterial orthologues as surrogates for human OGA. These breakthroughs enabled structure−activity relationships to be understood

and provided context and boundaries for the optimization of druglike properties.

microtubules, subsequent aggregation, and ﬁnal neuronal

death.^14,15Both tau aggregation spreading and concentration

of NFTs have shown correlation with progression of

neurodegeneration and cognitive decline, suggesting a key

role for tau in the evolution of the disease.¹⁶Furthermore,

NFTs are common to another, less prevalent but still highly

relevant, group of neurodegenerative diseases, globally termed

as tauopathies, comprising progressive supranuclear palsy

(PSP), frontotemporal dementia (FD), or corticobasal

syndrome (CBS) among others.¹⁷

INTRODUCTION

■

Alzheimer’s disease (AD)¹⁻³is the most common form of

dementia,⁴characterized by a progressive decline of cognitive

function, behavioral changes, and eventually death due to

neuronal loss.^5,6An estimated 35 million people worldwide

suﬀer from AD, and this number is expected to increase up to

57 million by 2030 and to 106 million by 2050 if no treatment

becomes available.⁷The total estimated worldwide cost of

dementia in 2018 was US$1 trillion, and this ﬁgure is expected

to rise to US$ 2 trillion by 2030.⁸⁻¹⁰Unfortunately, there are

no eﬀective disease-modifying treatments available, and current

approved medications only provide modest and transient

symptomatic relief for memory and other cognitive changes.¹¹

The two major deﬁning pathological hallmarks of AD are the

presence in the brain of extracellular senile plaques formed by

β-amyloid (Aβ) protein aggregates and intracellular neuro-

ﬁbrillary tangles (NFTs), the latter comprising paired helical

ﬁlaments (PHFs) of the abnormally hyperphosphorylated tau

protein.¹²Tau is an unfolded highly soluble protein, which

plays a critical role in the tubulin assembly and stabilization of

microtubules in neurons, which in turn are essential structures

for distributing proteins and nutrients within these cells.¹³In

AD pathogenesis, tau is post-translationally hyperphosphory-

lated on serine and threonine residues, which is thought to be

one of the underlying causes for its detachment from

O-GlcNAcylation¹⁸is a reversible and dynamic post-

translational modiﬁcation of proteins, including tau, where

N-acetyl-^D-glucosamine (GlcNAc) is attached via β-O-

glycosidic linkage to the hydroxyl group of serine and

threonine residues, yielding O-GlcNAcylated proteins.¹⁹

Unlike phosphorylation, the O-GlcNAcylation cycle is

regulated only by two enzymes: an O-GlcNAc transferase

Received: September 7, 2020

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Figure 1. Representative OGA inhibitors 1−7.

(OGT) that catalyzes the addition of O-GlcNAc residues to

target proteins and the hydrolase O-GlcNAcase (OGA) that

removes the O-linked glycoside from proteins.²⁰⁻²²O-

GlcNAcylated proteins, OGT and OGA, are particularly

abundant in the brain,²³and this modiﬁcation has been

suggested to be a key regulatory mechanism contributing to

neuronal communication, memory formation, and neuro-

degenerative disease.²⁴⁻²⁸It has been shown that in vitro O-

GlcNAcylation of tau lowers the speed and yield of its

aggregation and, additionally, may compete with phosphor-

ylation on the same residues or hinder it on adjacent ones, thus

suggesting a protective role for this modiﬁcation.²⁹⁻⁴¹

Decreased levels of O-GlcNAc have been found in the brains

of AD subjects, and moreover, tau aggregates can be entirely

devoid of O-GlcNAc.⁴²Therefore, maintaining O-GlcNAcyla-

tion of tau by small-molecule inhibitors of OGA represents a

potential approach to interfere with aggregation,⁴³thereby

rendering tau less prone to detaching from microtubules,

reducing the formation of neurotoxic tangles, and thus

preventing or slowing neurotoxicity and neuronal cell death.

Over the last decade, several OGA inhibitors (1−5) have

been investigated in in vitro and in vivo models of

neurodegenerative diseases (Figure 1),⁴⁴⁻⁴⁶with Thiamet-G

(4),⁴⁷a highly potent, stable, selective, and brain-penetrant

sugar-based derivative, being the most broadly studied

inhibitor. Thiamet-G increases tau O-GlcNacylation levels,

decreases tau phosphorylation, and inhibits formation of tau

aggregates and neuronal cell loss along with decreased severity

of breathing and increased survival in transgenic mouse

models.⁴⁸⁻⁵⁴

OGA, is to consider the safety or reported adverse events. In

this regard, the fact that Phase 1 clinical studies have shown

that single doses up to 1200 mg of MK-8719 were generally

well-tolerated and no adverse events were observed is

reassuring. Meanwhile, chronic exposure to the OGA inhibitor

ASN-120290 of P301S tau transgenic mice for 3.5 months did

not manifest any abnormalities with respect to mortality or

body weight and a >2-fold elevation of O-GlcNAc tau and

resulted in a signiﬁcant reduction in abnormally phosphory-

lated tau. More recently, in 2018, ASN-120290, also in

development for PSP, completed Phase 1 clinical studies in

healthy young and elderly volunteers, with single oral doses up

to 1000 mg as well as multiple oral doses up to 500 mg of

BID⁶¹and was also found safe and well-tolerated. To support

dose selection for PSP eﬃcacy trials, Asceneuron has reported

also the start of an additional clinical trial aimed at evaluating

target engagement of ASN-120290 in the brain using a

proprietary positron emission tomography (PET) ligand of

undisclosed structure.⁶²In 2019, Eli Lilly claimed to have an

OGA inhibitor undergoing Phase 1 clinical trials for AD⁶³(LY-

3372689, undisclosed structure)⁶⁴although no data has yet

been published. In addition, two OGA PET radioligands have

been reported: [¹⁸F]-MK-8553 (undisclosed structure),⁶⁵used

to demonstrate target engagement of MK-8719 in clinical

studies;⁶⁶and [¹⁸F]-LSN3316612 (7, Figure 1),⁶⁷which has

been used to image O-GlcNAcase in knockout mice and rhesus

monkeys and is currently being investigated to conﬁrm target

engagement for LY-3372689 in the clinic, showing good results

in healthy human volunteers.^68,69

OGA is a 103 kDa multidomain protein with the longest

isoform comprising 916 amino acids. An N-terminal glycoside

hydrolase 84 (GH84) catalytic domain and a C-terminal

histone acetyltransferases (HAT)-like domain are separated by

a 300-amino acid stalk domain.⁷⁰The hydrolase activity, the

removal of O-GlcNAc, resides in the GH domain. Indeed, the

active site is characterized by a conserved pair of catalytic

aspartic acid residues, Asp174 and Asp175 in the human

enzyme.⁷¹The presence of a large 150 amino acid disordered

region within the stalk domain, combined with its large size,

The clinical study of OGA inhibitors is in its infancy, but

Merck/Alectos (6, MK-8719)⁵⁵⁻⁵⁷and Asceneuron (ASN-

120290, formerly ASN-561, structure not disclosed)⁵⁸⁻⁶⁰have

been reported on Phase I clinical studies with their OGA

inhibitors. MK-8719 is a potent and selective brain-penetrant

analogue of Thiamet-G that has shown a robust pharmacody-

namic response in rats and favorable preclinical toxicological

proﬁle and is currently in development for PSP. Of particular

relevance for this new target class, and given the unique role of

B

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contributed to the diﬃculties in obtaining the human OGA

(hOGA) X-ray crystal structure. While major eﬀorts have led

to the recent publication of the hOGA structure,⁷²⁻⁷⁵it

remains nontrivial to solve, and for some time,^76,77and still

today,^78,79bacterial homologues have provided an alternative

to understand structure, function, and substrate and inhibitor

binding. Among the bacterial forms, clostridium perfringens

CpOGA was the ﬁrst reported with 15 structures now

deposited in the protein databank (PDB). A single structure

of oceanicola granulosus OgOGA⁸⁰was subsequently pub-

lished, showing better sequence similarity beyond just the

active site, although there have been no further reports on this

form. To date, the X-ray structures of the four reported

bacterial forms have been solved with saccharide inhibitors

such as 1 and 4.

polar compound (cLogD (pH = 7.4) = −3.57),⁸³and despite

the lack of cellular activity, it was considered as an attractive

starting point for a medicinal chemistry exploration. In this

article, we describe the optimization of hit 8 toward a series of

potent nonsaccharide OGA inhibitors for the potential

treatment of tauopathies, including AD.⁸⁴We report two

protein−ligand X-ray crystal structures with diﬀerent repre-

sentatives from the series. These structures are the ﬁrst

disclosure of optimized nonsaccharide inhibitors with an

entirely diﬀerent binding mode from that previously seen.

Interestingly, two alternative bacterial homologues were used:

CpOGA and the less frequently studied OgOGA. We show

how the latter provides a much better template homologue for

human OGA, both in sequence alignment in the vicinity of the

active site and in secondary and tertiary structures.

Our eﬀorts began by high-throughput screening (HTS) of

the Janssen compound library leading to the identiﬁcation of 8

RESULTS AND DISCUSSION

■

as a weak OGA inhibitor in a biochemical hOGA assay (IC₅₀

=

Synthesis. As a general strategy to explore the chemical

space around our initial hit 8 (Figure 2), we ﬁrst targeted the

synthesis of a set of N′-heteroaryl, N-Boc-protected diazaspiro-

cycles (Scheme 1). Thus, coupling of N-Boc diazaspirocycles

9, 12, and 15 with the corresponding heteroaryl halides under

aromatic nucleophilic substitution (SNAr) or Pd-catalyzed

3236 nM) with no activity up to a concentration of 10 μM in a

hOGA cell-based assay (Figure 2). Nonetheless, compound 8

Bu

̈

chwald−Hartwig⁸⁵⁻⁸⁷reactions yielded the desired inter-

mediates 10a−h, 13, and 16. Intermediate 10i (R₁= 2-methyl-

6-(triﬂuoromethyl)pyridin-4-yl) was prepared via Pd-catalyzed

Suzuki−Miyaura^88,89cross-coupling between intermediate 10h

(R₁= 2-chloro-6-(triﬂuoromethyl)pyridin-4-yl) and trimethyl-

boroxine. Cleavage of the Boc protecting group under acidic

conditions in intermediates 10a−i, 13, and 16 rendered the

spirocyclic amines 8, 11b−i, 14, and 17.

The preparation of pyrrolidines where a 2,6-dimethylpyr-

idin-4-yl substituent (R₁) is directly linked to a spirofused

cyclopentane (21), tetrahydrofurane (25), or cyclobutane (29)

Figure 2. Structure of initial hit 8 and exploration strategy.

is a low-molecular-weight (MW = 258; ligand eﬃciency (LE)⁸¹

= 0.29; lipophilic ligand eﬃciency (LLE)⁸²= 5.33) and highly

a

Scheme 1. Syntheses of Intermediate Diazaspirocycles 8, 11b−i, 14, and 17

a

Reagents and conditions: for 10a,b,d,e: (i) R₁Cl, base (N,N-diisopropylethylamine (DIPEA) or Et₃N), solvent (dichloromethane (DCM),

acetonitrile (ACN), nBuOH, or 2-PrOH), temperature (rt or 120 or 150 °C, MW); for 10c,f−h: (ii) R₁X, base (tBuONa or Cs₂CO₃), catalyst

(Pd₂(dba)₃/BINAP or Pd(dppf)Cl₂.DCM), PhMe, 90 °C; (iii) trimethylboroxine, XPhos Pd G3, Cs₂CO₃, 1,4-dioxane, 120 °C, MW; (iv) acid

(HCl, triﬂuoroacetic acid (TFA), Amberlyst-15), solvent (2-PrOH, MeOH, 1,4-dioxane, EtOAc, Et₂O, or DCM), temperature (rt or 80 °C).

C

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a

Scheme 2. Syntheses of Intermediates 21, 25, and 29

a

Reagents and conditions: (i) 4-bromo-2,6-dimethylpyridine, n-BuLi, hexanes, tetrahydrofuran (THF), −78 °C, 2 h, 54%; (ii) DAST, DCM, 0 °C

to rt, 1 h; (iii) H₂(1 atm), Pd/C 10%, MeOH, rt, 18 h, 50% (two steps); (iv) acid (HCl, TFA, Amberlyst-15), solvent (2-PrOH, MeOH, 1,4-

dioxane, EtOAc, Et₂O, or DCM), temperature (rt or 80 °C); (v) I₂, PPh₃, imidazole, toluene, 80 °C, 16 h, 48−77%; (vi) R₁Bpin, NaHMDS, NiI₂,

(1R,2R)-trans-2-aminocyclohexanol hydrochloride, 2-PrOH, 80 °C, 90 min, MW, 9−18%. R₁= 2,6-dimethylpyridin-4-yl.

a

Scheme 3. Syntheses of Intermediates 35 and 41

a

Reagents and conditions: (i) 4-bromo-1-butene, K₂CO₃, tetra-n-butylammonium bromide (TBABr), acetone, 60 °C, 18 h, 91%; (ii) TFA, DCM,

rt, 2 h, 98%; (iii) NaH, BnBr, THF, 0 °C to rt, 16 h, 47%; (iv) LiAlH₄, THF, 0 °C to rt, 16 h, 96%; (v) I₂, PPh₃, imidazole, 2-MeTHF, 0−75 °C, 1

h, 71%; (vi) allyl bromide, LiHMDS, hexanes, 2-MeTHF, −78 °C to rt, 0.5 h, 51%; (vii) LiBH₄, THF, rt; 34 h, 97%; (viii) I₂, PPh₃, imidazole,

PhMe, 80 °C, 16 h, 42%; (ix) B₂(Pin)₂, Xantphos, tBuOK, CuCl, THF, 30 °C, 16−48 h, 59−75%; (x) HetBr, KOH, Pd(PPh₃)₄, 1,4-dioxane, 90

°C, 16 h, 54−89%. (xi) H₂(H-cube,⁹³full hydrogen), Pd(OH)₂/C 20%, MeOH, 80 °C, 73%; (xii) HCl, 1,4-dioxane, rt, 2 h, 89%; R₁= 2,6-

dimethylpyridin-4-yl.

ring is described in Scheme 2. Thus, reaction of ketone 18 with

4-bromo-2,6-dimethylpyridine upon treatment with n-BuLi

yielded the hydroxy intermediate 19 in moderate yield.

Dehydroﬂuorination of alcohol 19 with diethylaminosulfur

triﬂuoride (DAST) proved challenging, leading to an

inseparable mixture of the dehydration and deoxoﬂuorinated

compound. This mixture was hydrogenated using Pd/C as the

catalyst to yield N-Boc-protected 2-azaspiro[4.4]nonane 20,

D

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a

Scheme 4. Syntheses of Intermediates 43, 45, 48, and 51

a

Reagents and conditions: (i) NaH, 4-bromo-2,6-dimethylpyridine, dimethylformamide (DMF), 0−90 °C, 48 h, 70%; (ii) HCl, 1,4-dioxane, rt, 1−

2 h, 51−97%; (iii) NaH, 4-chloro-2,6-dimethylpyridine, 15-crown-5, DMF, 0−60 °C, 18 h, 91%; (iv) NaBO₃·H₂O, THF, H₂O, rt, 2 h, 91−100%;

(v) 4-chloro-2,6-dimethylpyridine, tBuOK, dimethyl sulfoxide (DMSO), rt to 70 °C, 5 h, 70−79%; (vi) H₂(H-cube, full hydrogen), Pd(OH)₂/C

20%, MeOH, 80 °C, 100%; R₁= 2,6-dimethylpyridin-4-yl.

a

Scheme 5. Syntheses of targets 52a−d, 53, and 54

a

Reagents and conditions: (i) HetCHO, reducing agent (NaBH₃CN or NaBH(OAc)₃), solvent (DCM or MeOH), rt, 27−92%. (ii) HetCH₂Cl,

DIPEA, dichloroethane (DCE), −78 °C to rt, 30 min, 16−44%.

which was converted into the target intermediate 21 under

acid conditions (Scheme 2A).

were synthesized as outlined in Scheme 3. Synthesis of 35

started from the alkylation of the commercially available

pyrrolidinone 30 with 4-bromo-1-butene under phase-transfer

catalysis to yield compound 31 in good yield, which was

deprotected in acidic conditions and reprotected with a benzyl

group. Subsequent reduction with LiAlH₄to the alcohol

followed by treatment with I₂in the presence of PPh₃aﬀorded

intermediate 32 (Scheme 3A). In a similar way, for the

preparation of intermediate 41, ester 36 was treated with allyl

bromide and LiHMDS to yield compound 37. This

intermediate was transformed to the iodo derivative 38 in a

two-step sequence by reduction of the ester function with

LiBH₄to the alcohol followed by the Appel reaction to give the

iodoalkene 38. Finally, iodoalkenes 32 and 38 were trans-

Syntheses of intermediates 1-oxa-7-azaspiro[4.4]nonane 25

and 6-azaspiro[3.4]octane 29 are shown in Scheme 2B,C,

respectively. Starting from commercially available alcohols 22

and 26, an Appel-type reaction⁹⁰with I₂/PPh₃followed by Pd-

catalyzed cross-coupling of the iodo-intermediates 23 and 27

with 2,6-dimethylpyridine-4-boronic acid pinacol ester in the

presence of NiI as the catalyst⁹¹provided the targeted

intermediates 24 and 28; Boc cleavage under standard reaction

conditions aﬀorded the target compounds 25 and 29.

Intermediates 2-azaspiro[4.4]nonane 35 and 6-

azaspiro[3.4]octane 41, containing a methylene spacer

between the northern heteroaromatic group and the spirocycle,

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a

Scheme 6. Syntheses of 55−70

a

Reagents and conditions: for 55−58, 60, and 62−70: (i) HetCHO, NaBH(OAc)₃, DCM, rt (Et₃N or DIPEA was added when the product was

previously isolated as HCl salt); for 59 and 61: (ii) HetCHO, NaBH₃CN, AcONa/AcOH, MeOH, rt.

formed toward the desired spirocyclic intermediates 33 and 39

through a copper-catalyzed borylative ring-closing C−C

coupling.⁹²The Csp²−Csp³Suzuki cross-coupling reaction

between 33 and 39 and 4-bromo-2,6-dimethylpyridine yielded

the spirocyclic amines 34 and 40, which were deprotected in a

ﬁnal step under standard Boc and Bn deprotection reaction

conditions (Scheme 3).

Intermediates 43 and 45, where the northern heteroaromatic

is linked to the spirobicyclic core by an oxygen atom, were

prepared from the corresponding alcohols 22 and 26 through

an O-alkylation reaction with 4-chloro- or 4-bromo-2,6-

dimethylpyridine using sodium hydride as the base followed

by the cleavage of the Bn and Boc protecting groups,

respectively (Scheme 4A,B).

Syntheses of 2-azaspiro[4.4]nonane 48 and azaspiro[3.4]-

octane 51, with a longer CH₂O linker, is shown in Scheme

4C,D. Boronates 33 and 39 (Scheme 3A,B) were transformed

into the corresponding hydroxy derivatives 46 and 49 by

treatment with NaBO₃,⁹⁴⁹⁴which were subsequently O-

alkylated with 4-chloro-2,6-dimethylpyridine using tBuOK as

the base to yield ethers 47 and 50 and deprotected toward the

targeted spirocycles 48 and 51.

Reaction of the HTS hit 8 with a series of heteroaryl

carbaldehydes under standard reductive amination reaction

conditions aﬀorded the target compounds 52a−d in moderate

to good yields. Spirocycles 14 and 17 were converted to the

ﬁnal derivatives 53 and 54 via N-alkylation with N-(5-

(chloromethyl)thiazol-2-yl)acetamide (Scheme 5).

Finally, the preparation of the target compounds from NH-

spirocycles 11b−i, 21, 25, 29, 35, 41, 43, 45, 48, and 51

toward the ﬁnal targets 55−70 was achieved via reductive

amination with N-(5-formylthiazol-2-yl)acetamide (Scheme

6).

Biological Activity. The targeted compounds⁹⁵were ﬁrst

evaluated for their in vitro inhibitory activity against

recombinant hOGA. In this biochemical assay, compound-

dependent inhibition of the hydrolysis of ﬂuorescein mono-β-

N-acetyl-^D-glucosamine by hOGA is measured. Compounds

showing a hOGA IC₅₀< 100 nM were progressed to a cell-

based assay (hOGA cell) in HEK293 cells inducible for P301L

mutant Tau, where hOGA inhibition was evaluated through

immunocytochemical detection of O-GlcNAcylated proteins

with a monoclonal antibody. Active compounds in cells were

further assayed for their in vitro metabolic stability in mouse

liver microsome preparations, permeability, eﬄux, and

interactions with cytochrome P450 (CYP) enzymes.⁹⁶

The introduction of (hetero)aromatic motifs connected by a

methylene spacer into our core template (8) resulted in a

pronounced increase in OGA inhibitory activity. Representa-

tive examples from this exploration (52a−d) are shown in

Table 1 (Table 1, entries 2−5). The acetamidothiazole⁹⁷⁻⁹⁹

derivative 52a showed the best activity in the biochemical

assay with an 800-fold increase in hOGA inhibitory activity

compared to hit 8 (52a, IC₅₀= 4 nM vs 8, IC₅₀= 3236 nM).

Benzodioxolane (52b), quinoxaline (52c), and benzothiazole

(52d) were also suitable aromatic substituents of the south

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Table 1. South-Side (R₂) and Spirodiamine Exploration

a

Compounds are racemic mixtures.

pyrrolidine nitrogen resulting in potent OGA inhibitors (IC₅₀

= 129, 74, and 22 nM, respectively). Interestingly, 52a was the

only example showing activity below 1 μM in the OGA cell-

based assay (52a, hOGA cell IC₅₀= 575 nM).

We next looked at the inﬂuence of the spirodiamine on the

OGA inhibitory activity of 52a. Ring contraction of the

diazaspirononane into a diazaspirooctane was tolerated in

terms of primary activity; however, both diazospiroctanes 53

and 54 were approximately 10-fold and 8-fold less potent than

52a in the hOGA biochemical assay (Table 1, entries 6 and 7).

This lower inhibitory activity was also observed in the hOGA

cell-based assay, with compounds showing activity in the 10

μM range.

asymmetric unit, comprising residues Asn39 to Ile624, each

with a similar conformation. The structure revealed the

catalytic GH domain in its TIM (triose-phosphate isomerase)

barrel fold, with the active site solvent accessible and located

on the C-terminal face of the barrel; the N-terminus and stalk

domains were also present; see Figure 3A. The electron density

showed an unambiguous binding mode for ligand 54, including

its orientation and conformation. The amino acid residues

forming the ligand binding site and the ligand were well

deﬁned. The thiazoloacetamide moiety entered the deepest

into the catalytic pocket in the region where hydrolysis occurs;

see Figure 3B. It sits favorably sandwiched between aromatic

residues Tyr335 and Trp394 above and below while making

dual hydrogen-bond interactions from either side of the amide:

the carbonyl acting as an acceptor to Asn396 and the NH as a

donor to the catalytic Asp297. The thiazolo nitrogen acts as a

hydrogen-bond acceptor with the donor Lys218. The

protonated basic nitrogen of the azetidine ring formed a

At this early stage, we set out to gain an experimental

understanding of the binding mode to guide molecular design.

We cocrystallized 54 with the bacterial homologue CpOGA.

The structure was solved and reﬁned to a ﬁnal resolution of

2.30 Å. The crystals contained six monomers in the

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Figure 3. X-ray crystal structure of 54 with CpOGA. (A) Overall structure of CpOGA shown as a ribbon diagram and showing ligand 54 as spheres.

The N-terminus is shown in pale pink, the GH catalytic domain in cyan, and the stalk domain in magenta. (B) Closeup view of the 54 binding

mode in the OGA active site showing key amino acids and the transparent protein binding site surface.

strong salt bridge interaction with its NH pointing directly

toward Asp401. Finally, the triazolopyrimidine is solvent-

exposed on the protein surface but forms an ideal oﬀset face-

to-face π−π interaction with Tyr189. The structure partially

explains the structure−activity relationships (SARs) in Table 1

with those compounds capable of forming a stronger hydrogen

bond to Asn396 showing better activity, suggesting this was a

key interaction in this subpocket.

for the sp²nitrogen, showed a 2-fold increase in the enzymatic

activity and a similar cellular activity when compared with the

pyrimidine matched pair 57 (Table 2, entries 4 and 5).

Unfortunately, similar to 52a, compound 58 showed poor

permeability. The pyridine analogues 59 and 60 (Table 2,

entries 6 and 7), bearing a methoxy and a triﬂuoromethyl

substituent, respectively, showed improved permeability

probably due to the reduced basicity of the sp²pyridyl

nitrogen compared to 58 and the increased LogD (cLogD =

0.67 and 2.73, respectively). The bulkier triﬂuoromethyl

substituent was detrimental for OGA inhibition activity, and

60 was 3-fold less potent than 58. The methoxypyridine

derivative 59 showed good activity, permeability, and micro-

somal stability data; however, it suﬀers from greater P-gp eﬄux

((BA⁻/AB⁻) = 27.7) when compared to the quinoline

derivative 56. The 2-pyridyl analogue 61 (Table 2, entry 8)

was equipotent to the triazolopyrimidine derivative 52a in the

biochemical assay and showed a ∼12-fold improved activity in

the cell-based assay, which could be explained by the higher

lipophilicity (cLogD = 1.53) and improved permeability ((AB

+ GF) = 11.3 × 10⁻⁶cm/s). Unfortunately, 61 suﬀered from

an increased metabolic turnover and high P-gp eﬄux.

From this exploration on the northern R₁substituent, we

concluded that compounds 56 and 58 showed a more

balanced proﬁle, combining good OGA inhibitory activity in

the cell-based assay with good microsomal stability. Due to its

lower molecular weight and higher ligand-lipophilic eﬃciency

(56, LLE = 4.89 vs 58, LLE = 5.88), we selected 58 as a lead to

explore modiﬁcations aimed at improving the suboptimal

permeability.

Prior to performing further X-ray crystallography, we

screened compounds for inhibition activity versus the

CpOGA construct used for structural biology. An identical

protocol was applied as reported for the hOGA enzymatic

assay. Among various compounds tested, 52a and 54 had

CpOGA inhibition IC₅₀values of 290 and 3300 nM,

respectively, both ∼2 orders of magnitude less active than

for hOGA (4 and 34 nM). The disparity suggested that the

binding mode in CpOGA may be unreliable as a model for

hOGA. Direct comparison with the recent hOGA X-ray

structures was not possible as they were unavailable at the

Considering the structural information and biological results

above, an exploration of the north heterocycle was initiated.

The importance of the π−π interaction with Tyr189 was clear;

therefore, the acetamidothiazole heterocycle (R₂) and the

diazaspirononate scaﬀold were kept constant, and alternative

heteroaromatic groups to probe the Tyr189 interaction were

explored (Table 2). Furthermore, 52a has a high total polar

surface area (TPSA = 120 Å²) and a low calculated LogD

(cLogD = −0.75); this polarity could contribute to its low

permeability (MDCK-MDR1 permeability (AB + GF) = 1.7 ×

10⁻⁶cm/s) and moderate cellular activity (hOGA cell IC₅₀

=

575 nM). Moreover, 52a was found to be a moderate P-gp

substrate (eﬄux (BA⁻/AB⁻) = 8.4) and presented low in vitro

clearance values in human (h) and mouse (m) liver

microsomes. A strategy to reduce the nitrogen count was

chosen to lower the TPSA. A ﬁrst example involved replacing

the distal fused triazo ring with an imidazo equivalent (Table 2,

entry 2); 55 showed high enzymatic activity, IC₅₀= 1 nM, but

a comparable drop in cellular activity, IC₅₀= 437, as seen for

52a. The change from triazolopyrimidine 52a to methylquino-

line 56 resulted in a ∼2-fold decrease in enzymatic potency

(Table 2, entries 1 and 3: 52a, IC₅₀= 4 vs 56, IC₅₀= 9 nM)

but a beneﬁcial 14-fold increase in the cellular activity (52a,

cell IC₅₀= 575 vs 56, IC₅₀= 39 nM), which correlates with the

decrease in TPSA and the increase in LogD.

Monocyclic heteroaromatics (57−61) were also found to be

suitable replacements for the triazolopyrimidine substituent.

Thus, the less polar (TPSA = 103 Å²; cLogD = −0.37)

dimethylpyrimidine 57 (Table 2, entry 4) showed a 3-fold

drop in the enzymatic activity (IC₅₀13 and 4 nM for 57 and

52a, respectively) but a 3-fold increase in the cellular activity

(170 vs 575 nM). The dimethylpyridine 58 (TPSA = 90 Å²;

cLogD = −0.83), with an experimental measured pK_aof 10.39

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Table 2. North-Side (R₁) Exploration

a

Compounds are racemic mixtures.

time, so instead we used sequence alignments of human and

bacterial forms to consider the diﬀerences around the binding

site. We identiﬁed that the α10−α11 loop is nine residues

longer in CpOGA compared with hOGA and furthermore that

OgOGA had the same loop size as hOGA;¹⁰⁰see Figure 4A.

This loop was a concern because in CpOGA it occupied the

periphery of the site and interacted with ligand 54, but this

would not be the case in hOGA. The published literature of

bacterial OGA homologues is dominated by BtOGA and

CpOGA forms, but both would suﬀer from this issue;

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Figure 4. Comparison of bacterial and human forms of OGA and the new structure of OgOGA solved with ligand 55. (A) Closeup view of the

α10−α11 loop (inside red-box) in CpOGA (cyan), OgOGA (magenta), and hOGA (yellow). (B) Similarity of the dimeric structure of OgOGA

solved with compound 55 (blue and red for each monomer) compared with hOGA (orange and magenta for each monomer). (C) Closeup of the

X-ray structure of the binding mode of 55 showing important binding site amino acids. The surface is color-coded to show the contribution from

the α-monomer (blue) and the β-monomer (red).

Figure 5. Comparison of X-ray structures of the OgOGA dimer solved with ligand 55 and representative published examples from CpOGA,

BtOGA, OgOGA, TtOGA, and hOGA (PDB ids 2CBJ, 5FKY, 2XSB, 5DIY, and 5M7U, respectively). Only two structures, the OgOGA dimer

presented in this work and hOGA, contain the β monomer in close proximity of the binding site.

therefore, we switched to using the much less studied OgOGA

for additional structural biology attempts.

Using the new OgOGA construct, we successfully solved an

X-ray crystal structure with ligand 55. The OgOGA structure

was reﬁned to a ﬁnal resolution of 1.79 Å and clearly showed

the ligand binding mode and adjacent amino acids. Pleasingly,

the binding conformation of the ligand was similar to 54 in

CpOGA, meaning our design workup to that point had not

been misguided. Furthermore, the new structure conﬁrmed the

shorter α10−α11 loop and showed that it no longer interacted

with the ligand and hence conﬁrmed the suspected diﬀerence

with CpOGA. However, there were other more substantial

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Table 3. Modiﬁcations in the Central Scaﬀold and the Linker (R₁= 2,6-Dimethyl-4-pyridine; R₂= N-(5-Methylenethiazol-2-

yl)acetamide)

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Table 3. continued

a

b

Compounds are racemic mixtures. Compounds are mixtures of diastereoisomers and enantiomers.

diﬀerences in the protein environment around the ligand

compared to CpOGA.

between the two monomers. The signiﬁcance of the dimer

conformation was later revealed when it was seen to overlay

well with the more recent hOGA structure; see Figure 4B.

Thus, this resulting bacterial crystal structure based on

OgOGA provided a near-identical model for hOGA not only

in the tertiary structure, but even for amino acids around the

binding site, including the proximity and interactions of the

In this case, the crystals contained four monomers in the

asymmetric unit, comprising residues Gly1 to Val447. Again,

the structure contained the catalytic GH and stalk domains.

However, in this crystal, the four monomers formed two

dimers with the C-terminal (residues 425−447) interchanged

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second β monomer. As mentioned, ligand 55 adopts a similar

conformation in the binding site as seen for CpOGA.

Furthermore, the thiazoloacetamide forms identical interac-

tions in the deepest part of the catalytic pocket. It is

sandwiched between Tyr160 and Trp219, and the carbonyl

oxygen forms a hydrogen bond with Asn221, while the NH is

oriented as a donor toward the catalytic Asp115, and ﬁnally the

Lys39 side chain forms a hydrogen bond to the thiazolo sp²

nitrogen. The protonated nitrogen basic center of the spiro

pyrrolidine ring system again interacts with Asp226, and the

distal bicycle sits well-aligned forming a π−π interaction with

Tyr10. However, the major diﬀerence with CpOGA is seen in

the distal part of the ligand that is no longer solvent-exposed

on the protein surface. Instead, the β monomer of OgOGA

forms several interactions on the topside of the ligand, notably

sandwiching the bicycle by stacking Trp387 parallel to and

above the ligand; see Figure 4C. Further amino acids from the

β monomer including His383 and Phe367 enclose the ligand in

a hydrophobic pocket.

At the time the dimeric form of the OgOGA structure solved

with 55 was unexpected. Now it is possible to perform a

comparison with more recently published hOGA structures,

and the dimeric bacterial OgOGA structure presented here is

the only one able to reproduce the proximity of the two α-

helices 15 and 16 from the β monomer as seen in hOGA; see

Figure 5. As the binding site resides on the surface of the TIM

barrel, the rest of the site is formed by the loops that link the α-

helices and the parallel β-strands within this conserved protein

fold. In these regions, such as the important β7−α7 loop,

which forms many interactions with the ligand, the structure is

well-conserved and similarly placed in all examples. Solving

structures with hOGA is still a challenging task due to

expression and stabilizing of the truncated or deletion

constructs. Therefore, structural biology with bacterial

homologues remains a valuable approach, particularly if greater

throughput is required for instance in the case of fragment

screening. In such cases, we recommend the approach here

based on using the less common OgOGA form, which was

chosen because of its closer similarity to hOGA around the

binding site, particularly the shorter α10−α11 loop.

As a strategy to improve the permeability of the compounds,

the exploration of the spirocyclic core was revisited focusing on

modiﬁcations that would result in compounds with increased

cLogD when compared to 58 (Table 2, entry 5: cLogD =

−0.83) targeting a range of 0.5 < cLogD < 3. Based on the

crystal structure of 55 with OgOGA, the northern pyrrolidine

nitrogen does not make any strong interactions in the catalytic

pocket; therefore, we hypothesized that the replacement of the

north pyrrolidine ring 58 by cycloalkyl or tetrahydrofuranyl

rings should be tolerated in terms of primary activity and have

the desired increase on the overall lipophilicity of molecules.

To this end, a set of spirocyclicmonoamine-containing

compounds was designed (62−70), and their biological data

along with TPSA and calculated LogD values are shown in

Table 3. All new compounds had similar TPSA values to 58

and were more lipophilic, with calculated logD values in the

targeted range. Unfortunately, most modiﬁcations resulted in

compounds with reduced activity in the biochemical assay.

Thus, compound 62 (Table 3, entry 2), where the northern

nitrogen atom in 58 is replaced by a carbon atom, was found to

be ∼10-fold less potent in the hOGA biochemical assay (62,

IC₅₀= 66 nM vs 58, IC₅₀= 6 nM). The eﬀect of this

modiﬁcation was less pronounced in the cell-based assay, with

62 being ∼2-fold less active than 58. The replacement of the

northern pyrrolidine by a tetrahydrofuran ring 63 (Table 3,

entry 3) was better tolerated, and compound 63 showed a ∼3-

fold decrease in primary activity when compared to 58 (63,

IC₅₀= 21 nM vs 58, IC₅₀= 6 nM). Unfortunately, although the

increase in cLogD translated in an increase in permeability (63,

MDCK-MDR1 permeability (AB + GF) = 12.6 × 10⁻⁶cm/s),

this did not result in increased cell-based activity (63, cell IC₅₀

= 603 nM). Ring contraction of the spirocyclopentane in 62 to

the spirocyclobutane 64 (Table 3, entries 2 and 4) led to ∼2-

fold potency increase in the enzymatic assay (64, IC₅₀= 36 nM

vs 62, IC₅₀= 66 nM) while keeping similar activity in the cell-

based assay (64, cell IC₅₀=182 nM vs 62, cell IC₅₀= 214 nM);

however, the compound was signiﬁcantly less active than 58.

The lower activity of the carbon-linked examples 62−64

compared to the nitrogen-linked aromatic such as 58 was

attributed to a loss of planarity between the distal aromatic and

the nitrogen in the crystal structure of 55 (Figure 4C), which

would not be the case for the increased sp³character of the

carbon-linked molecules.

The inﬂuence of a ﬂexible linker between the spirocyclic

scaﬀold and the distal pyridyl group was then explored. The

introduction of a CH₂spacer (Table 3, entries 5−7) was

clearly detrimental for the OGA inhibitory activity. Com-

pounds 65, cis-66, and trans-66 had hOGA IC₅₀values of 309,

209, and 204 nM, respectively, in the biochemical assay. This

was explainable in terms of a deterioration in the interaction of

the distal aromatic group of the ligand. The pyridine

heterocycle needs to be well-positioned to maintain the

aromatic sandwich interaction between Trp387 and Tyr10

above and below the ligand. A similar negative trend for

activity was observed for compounds having an oxygen linker

(Table 3, entries 8−11), with the isomer cis-67 (IC₅₀= 30

nM) being the most potent from this subseries. The use of the

longer −CH₂O− linker (Table 3, entries 12 and 13) did not

result in an activity improvement, and compounds 69 and 70

were more than 22-fold less potent than the starting lead 58.

Given the worse structure−activity relationships (SARs)

obtained with the initial exploration of lead 58, we turned our

attention to the potent, stable, and more permeable lead 56

(Table 2, entry 3) for an advanced characterization of this

novel series on nonsaccharide OGA inhibitors. In addition to

the previously discussed in vitro data, 56 showed no interaction

with any of the tested cytochrome P450 (CYP450) isoforms

(3A4, 2C9, 2D6, 1A2, C9) at the highest concentration tested

(IC₅₀> 10 μM) and presented good kinetic solubilities of 87

μM (pH = 2) and 71 μM (pH = 7.4). Compound 56 was

separated into the corresponding enantiomers (+)-56 and

(−)-56 by supercritical ﬂuid chromatography (SFC) puriﬁca-

tion. Activity data along with intrinsic clearance, permeability,

and eﬄux values are summarized in Table 4. Enantiomer

(+)-56 showed signiﬁcantly better activity in both the

Table 4. In Vitro Data for Compounds 56, (+)-56, and

(−)-56

PgP

cell

LM Clint h,

m

MDCK-MDR1

Perm (AB + GF)

10⁻⁶cm/s

eﬄux

(BA⁻/

AB⁻)

IC₅₀

cpnd

56

(+)-56

(−)-56

(nM) (nM) (μL/min/mg)

9

5

25

39

17

263

11, 23

<8, 15

5.3

2.3

8.4

16.3

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a

Table 5. Mouse PK Data for (+)-56

iv

po

Cl (mL/min/kg)

55.8 ( 6.6)

Vd_ss(L/kg)

17.8 ( 7.9)

AUC_last(ng h/mL)

1944 ( 961)

C_max(ng/mL)

331 ( 179)

F (%)

23.1 ( 11.4)

a

Study in male CD1 mice dosed at 1 mg/kg iv in saline 0.9% and 30 mg/kg po in water.

coupling constants in hertz. Splitting patterns are deﬁned by s

(singlet), d (doublet), dd (double doublet), t (triplet), q (quartet),

quin (quintet), sex (sextet), sep (septet), or m (multiplet). Solvents

used for the NMR experiments are reported per compound. Details of

the various high-performance liquid chromatography−mass spec-

trometry (HPLC−MS) methods used are provided in the Supporting

Information. Purities of all new compounds were determined by

analytical reverse-phase (RP) HPLC or reverse-phase ultraperform-

ance liquid chromatography (RP UPLC) coupled to a mass

spectrometry detector, using the area percentage method on the

UV trace scanning from 200 to 450 nm, and compounds were found

to have ≥95% purity unless otherwise speciﬁed. Optical rotations

measurements were carried out on a 341 PerkinElmer polarimeter in

the indicated solvents.

Experimental Section. Synthesis of Compounds 10a−h. tert-

Butyl 7-(5-Methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)-2,7-

diazaspiro[4.4]nonane-2-carboxylate (10a). A mixture of tert-butyl

2,7-diazaspiro[4.4]nonane-2-carboxylate (1.7 g, 7.51 mmol), 7-

chloro-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidine (1.266 g, 7.51

mmol), and Et₃N (2.088 mL, 15.0 mmol) in DCM (31 mL) was

stirred at rt for 1 h. The mixture was diluted with water and extracted

with DCM. The organic layer was washed with brine, dried (MgSO₄),

and ﬁltered, and the solvents were evaporated in vacuo. The crude

product was puriﬁed by ﬂash column chromatography (silica; 7 M

solution of ammonia in methanol in DCM 0/100 to 2/98). The

desired fractions were collected and evaporated in vacuo to yield 10a

as a white solid (2.1 g, 78%). ¹H NMR (400 MHz, DMSO-d₆) δ ppm

1.40 (d, J = 4.39 Hz, 9H), 1.75−2.13 (m, 4H), 2.39 (s, 3H), 3.18−

3.30 (m, 2H), 3.36 (br d, J = 6.24 Hz, 2H), 3.65−4.22 (m, 4H), 6.12

(s, 1H), 8.26 (s, 1H). Liquid chromatography−mass spectrometry

(LC-MS) m/z 359.3 [M + H]⁺.

tert-Butyl 7-(5-Methylimidazo[1,2-a]pyrimidin-7-yl)-2,7-

diazaspiro[4.4]nonane-2-carboxylate (10b). A mixture of 2-amino-

4-chloro-6-methylpyrimidine (0.5 g, 3.5 mmol), chloroacetaldehyde

(0.650 mL, 4.22 mmol) and EtOH (11 mL) was stirred at 90 °C for 5

h. The reaction was diluted with DCM and basiﬁed with sat K₂CO₃.

The organic layer was separated, dried (MgSO₄), and ﬁltered, and the

solvent was evaporated in vacuo. The crude product was puriﬁed by

ﬂash column chromatography (silica; EtOAc in heptane 0/100 to 30/

70). The desired fractions were collected and concentrated in vacuo

to yield 7-chloro-5-methylimidazo[1,2-a]pyrimidine (210 mg, 36%)

as a yellow solid that was used directly in the next step.

DIPEA (0.180 mL, 1.0 mmol) and 7-chloro-5-methylimidazo[1,2-

a]pyrimidine (210 mg, 0.75 mmol) were added to a stirred solution of

tert-butyl 2,7-diazaspiro[4.4]nonane-2-carboxylate (155 mg, 0.68

mmol) in 1-butanol (9 mL) in a sealed tube and under N₂. The

mixture was stirred at 120 °C for 4 h. Sat. NaHCO₃was added, and

the mixture was extracted with EtOAc. The organic layer was

separated, dried (MgSO₄), and ﬁltered, and the solvents were

evaporated in vacuo. The crude was puriﬁed by ﬂash column

chromatography (silica; EtOAc in heptane 0/100 to 100/0). The

desired fractions were collected and concentrated in vacuo to yield

10b (155 mg, 60%) as a yellow foam. LC-MS m/z 358.3 [M + H]⁺.

tert-Butyl 7-(2-Methylquinolin-4-yl)-2,7-diazaspiro[4.4]nonane-

2-carboxylate (10c). Tris(dibenzylideneacetone)dipalladium (1.5 g,

4.41 mmol) was added to a stirred suspension of tert-butyl 2,7-

diazaspiro[4.4]nonane-2-carboxylate (10 g, 44.2 mmol), 2-(dicyclo-

hexylphosphino)-2′,4′,6′-triisopropylbiphenyl (2.1 g, 4.4 mmol),

Cs₂CO₃(43.1 g, 132.6 mmol), and 4-chloro-2-methylquinoline

(8.64 g, 48.6 mmol) in PhMe (50 mL) under nitrogen. The mixture

was stirred at 90 °C for 12 h. The mixture was treated with water and

extracted with DCM. The organic layer was separated, dried

biochemical and cell-based assays while maintaining good

metabolic stability, comparable permeability, and moderate

eﬄux values to those of the racemic compound 56. To

determine the druglike properties of the series, in vivo

pharmacokinetic studies (PKs) were conducted with com-

pound (+)-56. Relevant mouse PK parameters are shown in

Table 5. Pharmacokinetic (PK) studies of (+)-56 dosed at 1

mg/kg iv and 30 mg/kg PO revealed a moderate clearance

value (55.8 mL/min/kg) and an extensive volume of

distribution (Vd_ss= 17.8 L/kg). Moreover, (+)-56 showed a

moderate systemic exposure (AUC_last= 1944 ng h/mL) and

oral bioavailability (F_abs= 23%).

In light of its PK proﬁle, (+)-56 was selected as a

representative example to assess the central penetration of

the spirodiamine series in male C57BL/6J mice at 30 mg/kg

po. Interestingly, (+)-56 was found to cross the blood−brain

barrier, showing a modest total cortex-to-plasma ratio of 0.14.

This result suggests potential for this chemotype to reach the

central nervous system (CNS) in suﬃcient concentration after

further optimization.

CONCLUSIONS

■

In summary, we have reported on the medicinal chemistry

exploration of the weak HTS hit 8 leading to a series of

spirocyclic nonsaccharide OGA inhibitors. SAR studies

identiﬁed compound (+)-56 as a potent, stable, and orally

bioavailable OGA inhibitor with good permeability in line with

the ﬁrst pass metabolism and moderate eﬄux potential. Taken

together and despite the modest central penetration observed

for (+)-56, these results suggest promise for the spirodiamine

series. Our structural biology eﬀorts have led to new insights

into the bacterial OGA homologues. For cases where hOGA

crystallography may still be prohibitive, we recommend the use

of the less common OgOGA form that we have shown can

display the similar dimeric structure seen for hOGA and which

is crucial for protein−ligand interactions around the binding

site. For the ﬁrst time, the binding mode of potent nonsugar-

derived OGA inhibitors has been reported. These druglike

compounds occupy a similar site but form diﬀerent

interactions to the sugarlike counterparts; this opens up new

chemical space and promise for future drug discovery.

EXPERIMENTAL SECTION

■

Chemistry. Unless otherwise noted, all reagents and solvents were

obtained from commercial suppliers and used without further

puriﬁcation. Thin-layer chromatography (TLC) was carried out on

silica gel 60 F254 plates (Merck). Flash column chromatography was

performed on silica gel, particle size 60 Å, mesh of 230−400 (Merck)

under standard techniques. Microwave-assisted reactions were

performed in a single-mode reactor, Biotage Initiator Sixty microwave

reactor (Biotage), or in a multimode reactor, MicroSYNTH

Labstation (Milestone, Inc.). All ﬁnal compounds were characterized

by H NMR and high-resolution mass spectra (HRMS). H nuclear

magnetic resonance spectra were recorded with either a Bruker DPX-

400 or a Bruker AV-500 spectrometer (Bruker AG) with standard

pulse sequences, operating at 400 and 500 MHz, respectively.

Chemical shifts are reported in parts per million (δ) units and

1

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(Na₂SO₄), and ﬁltered, and the solvents were evaporated in vacuo.

The crude product was puriﬁed by ﬂash column chromatography

(silica; EtOAc in petroleum ether 0/100 to 50/50). The desired

fractions were collected and concentrated in a vacuum to yield 10c as

tert-Butyl 7-(2-Chloro-6-(triﬂuoromethyl)pyridin-4-yl)-2,7-

diazaspiro[4.4]nonane-2-carboxylate (10h). 1,1′-Bis-

(diphenylphosphino)ferrocene-palladium(II) dichloride dichlorome-

thane adduct (54 mg, 0.07 mmol) was added to a stirred suspension

of 2-chloro-4-iodo-6-(triﬂuoromethyl)pyridine (400 mg, 1.30 mmol),

tert-butyl 2,7-diazaspiro[4.4]nonane-2-carboxylate (295 mg, 1.30

mmol), and Cs₂CO₃(848 mg, 2.60 mmol) in toluene (4 mL) in a

sealed tube under N₂. The mixture was stirred at 100 °C for 16 h.

Then, the mixture was diluted with EtOAc and washed with water.

The organic layer was separated, dried (Na₂SO₄), ﬁltered, and

concentrated in vacuo. The residue was puriﬁed by ﬂash column

chromatography (silica; EtOAc in heptane 0/100 to 50/50). The

desired fractions were collected and concentrated in vacuo to yield

1

a yellow solid (9.66 g, 59%). H NMR (400 MHz, CDCl₃) δ ppm

1.44 (s, 9H), 1.82 (s, 1H), 1.87−1.97 (m, 2H), 2.01−2.11 (m, 1H),

2.59 (s, 3H), 3.30−3.41 (m, 2H), 3.41−3.54 (m, 2H), 3.55−3.66 (m,

2H), 3.76 (br d, J = 6.17 Hz, 2H), 6.36 (s, 1H), 7.26−7.32 (m, 1H),

7.55 (t, J = 7.61 Hz, 1H), 7.89 (d, J = 8.16 Hz, 1H), 8.08 (br t, J =

8.16 Hz, 1H). LC-MS m/z 368.1 [M + H]⁺.

tert-Butyl 7-(2,6-Dimethylpyrimidin-4-yl)-2,7-diazaspiro[4.4]-

nonane-2-carboxylate (10d). A mixture of tert-butyl 2,7-diazaspiro-

[4.4]nonane-2-carboxylate (250 mg, 1.11 mmol), 4-chloro-2,6-

dimethylpyrimidine (189 mg, 1.33 mmol) and DIPEA (0.381 mL,

2.21 mmol) in 2-PrOH (3.75 mL) in sealed tube was stirred at 120

°C for 30 min and then at 150 °C for 90 min under microwave

irradiation. Then, the solvent was evaporated in vacuo and the crude

product was dissolved in EtOAc and washed with a saturated solution

of NaHCO₃. The organic layer was separated, dried (MgSO₄), ﬁltered

and the solvents evaporated in vacuo. The crude product was puriﬁed

by ﬂash column chromatography (silica; MeOH in DCM 0/100 to

15/85). The desired fractions were collected and evaporated in vacuo

to yield 10d as a colorless syrup (265 mg, 72%). ¹H NMR (400 MHz,

CDCl₃) δ ppm 1.46 (d, J = 4.16 Hz, 9H), 1.75−1.98 (m, 4H), 2.33 (s,

3H), 2.49 (s, 3H), 3.09−3.95 (m, 8H), 5.96 (s, 1H). LC-MS m/z

333.2 [M + H]⁺.

1

10h as a pale-yellow solid (283 mg, 54%). H NMR (400 MHz,

CDCl₃) δ ppm 1.47 (br s, 9H), 1.93 (br t, J = 6.47 Hz, 2H), 1.99−

2.23 (m, 2H), 3.18−3.71 (m, 8H), 6.47 (s, 1H), 6.66 (br s, 1H). LC-

MS m/z 406.1 [M + H]⁺.

tert-Butyl 7-(2-Methyl-6-(triﬂuoromethyl)pyridin-4-yl)-2,7-

diazaspiro[4.4]nonane-2-carboxylate (10i). Trimethylboroxine

(0.197 mL, 1.40 mmol) was added to a stirred suspension of 10h

(283 mg, 0.70 mmol), XPhos Pd G3 (59 mg, 0.07 mmol), and

Cs₂CO₃(454 mg, 1.40 mmol) in 1,4-dioxane (4.8 mL) in a sealed

tube under N₂. The mixture was stirred at 120 °C for 10 min under

microwave irradiation. The mixture was diluted with EtOAc and

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

brine, dried (Na₂SO₄), ﬁltered, and concentrated in vacuo. The

residue was puriﬁed by ﬂash column chromatography (silica; EtOAc

in heptane 0/100 to 100/0). The desired fractions were collected and

concentrated in vacuo to yield 10i as a colorless oil (253 mg, 94%).

¹H NMR (400 MHz, CDCl₃) δ ppm 1.47 (br d, J = 3.70 Hz, 9H),

1.83−2.18 (m, 4H), 2.50 (s, 3H), 3.26−3.39 (m, 4H), 3.40−3.58 (m,

4H), 6.33 (s, 1H), 6.58 (br s, 1H). LC-MS m/z 386.1 [M + H]⁺.

Syntheses of Compounds 8, 11b−i. 5-Methyl-7-(2,7-diazaspiro-

[4.4]nonan-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidine (8). 10a (2.11 g,

5.89 mmol), 6 M HCl in 2-PrOH (3.92 mL, 23.6 mmol), and 2-PrOH

(264 mL) were stirred at 80 °C for 1 h. The solvent was evaporated in

vacuo, and the solid was triturated in Et₂O to yield 8 as a bis

hydrochloride salt and as a white solid (1.8 g, 92%). 8 was puriﬁed by

ion-exchange chromatography (Isolute SCX-2, MeOH, and then 7 N

solution of ammonia in methanol). The desired fraction was collected

and concentrated in vacuo to yield 8 as a free base and as a white solid

(1.4 g, 92%). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.92−2.07 (m,

3H), 2.11−2.20 (m, 1H), 2.39 (s, 3H), 3.13−3.23 (m, 2H), 3.24−

3.32 (m, 2H), 3.85−4.14 (m, 4H), 6.10 (s, 1H), 8.29 (s, 1H). HRMS

(ESI+) m/z [M + H]⁺calculated for C13H19N6: 259.1671; found:

259.1671.

tert-Butyl 7-(2,6-Dimethylpyridin-4-yl)-2,7-diazaspiro[4.4]-

nonane-2-carboxylate (10e). A mixture of tert-butyl 2,7-diazaspiro-

[4.4]nonane-2-carboxylate (100 mg, 0.44 mmol), 4-chloro-2,6-

dimethylpyridine (75 mg, 0.53 mmol), and DIPEA (0.152, 0.88

mmol) in 2-PrOH (1.5 mL) in a sealed tube was stirred at 120 °C for

30 min and then at 150 °C for 90 min under microwave irradiation.

Then, the solvent was evaporated in vacuo and the crude product was

dissolved in EtOAc and washed with a saturated NaHCO₃solution.

The organic layer was separated, dried (MgSO₄), and ﬁltered, and the

solvents were evaporated in vacuo. The crude product was puriﬁed by

ﬂash column chromatography (silica; MeOH in DCM 0/100 to 15/

85). The desired fractions were collected and evaporated in vacuo to

1

yield 10e as a colorless syrup (78 mg, 53%). H NMR (400 MHz,

CDCl₃) δ ppm 1.46 (br s, 9H), 1.71−2.20 (m, 4H), 2.42 (s, 6H),

3.02−3.65 (m, 8H), 6.09 (s, 2H). LC-MS m/z 332.3 [M + H]⁺.

tert-Butyl 7-(2-Methoxy-6-methylpyridin-4-yl)-2,7-

diazaspiro[4. 4]nonane-2-carboxylate (10f). Tris-

(dibenzylideneacetone)dipalladium (408 mg, 0.45 mmol), rac-

BINAP (277 mg, 0.45 mmol), and Cs₂CO₃(2.92 g, 8.91 mmol)

were added to a stirred mixture of tert-butyl 2,7-diazaspiro[4.4]-

nonane-2-carboxylate (1.109 g, 4.90 mmol) and 4-bromo-2-methoxy-

6-methylpyridine (900 mg, 4.45 mmol) in toluene (20 mL). The

reaction mixture was stirred at 90 °C for 16 h. The mixture was

treated with water; then, it was extracted with EtOAc, dried

(Na₂SO₄), and ﬁltered, and the solvent was concentrated in vacuo.

The residue was puriﬁed by ﬂash column chromatography (silica;

MeOH in DCM 0/100 to 8/92). The desired fractions were

collected, and the solvent was evaporated under a vacuum to yield 10f

as a yellow oil (1.3 g, 80%). LC-MS m/z 348.2 [M + H]⁺.

5-Methyl-7-(2,7-diazaspiro[4.4]nonan-2-yl)imidazo[1,2-a]-

pyrimidine (11b). 4 M HCl in 1,4-dioxane (1.63 mL, 6.51 mmol) was

added to 10b (155 mg, 0.43 mmol) at rt. The mixture was stirred to

room temperature for 1 h. The solvent was evaporated, and the crude

was basiﬁed with sat. K₂CO₃and extracted with DCM. The organic

layer was separated, and the solvent was evaporated in vacuo. The

crude product was puriﬁed by ﬂash column chromatography (silica; 7

N solution of NH₃in MeOH in DCM 0/100 to 10/90). The desired

fractions were collected and concentrated in vacuo to yield 11b as a

yellow solid (98 mg, 87%). LC-MS m/z 258.0 [M + H]⁺.

tert-Butyl 7-(4,6-Dimethylpyridin-2-yl)-2,7-diazaspiro[4.4]-

nonane-2-carboxylate (10g). Pd₂(dba)₃(39 mg, 0.04 mmol) and

rac-BINAP (42 mg, 0.07 mmol) were added to a stirred mixture of 2-

Boc-2,7-diazaspiro[4.4]nonane (186 mg, 0.82 mmol), 2-chloro-4,6-

dimethylpyridine (134 mg, 0.95 mmol), and Cs₂CO₃(450 mg, 1.38

mmol) in toluene (3.4 mL). The reaction mixture was stirred at 90 °C

into a sealed tube and under a N₂atmosphere for 16 h. The mixture

was cooled to rt and ﬁltered through Celite, and the Celite pad was

washed with EtOAc. The combined organic ﬁltrates were evaporated

in vacuo to yield 10g as an orange syrup that was used in the next

2-Methyl-4-(2,7-diazaspiro[4.4]nonan-2-yl)quinoline (11c). A

solution of 10c (4 g, 10.9 mmol) in MeOH (75 mL) was added

dropwise to a stirred suspension of Amberlyst-15 (13.4 g) in MeOH

(10 mL). The mixture was shaken in a solid phase reactor at rt for 16

h. The resin was washed with MeOH (this fraction was discarded)

and then with 3.5 N solution of NH₃in MeOH. The ﬁltrate was

1

concentrated in vacuo to yield 11c as a brown oil (2.38 g, 82%). H

NMR (400 MHz, CDCl₃) δ ppm 1.73−1.93 (m, 3H), 1.98−2.07 (m,

2H), 2.60 (s, 3H), 2.86−2.97 (m, 2H), 3.02−3.11 (m, 2H), 3.64 (s,

2H), 3.75 (t, J = 6.70 Hz, 2H), 6.36 (s, 1H), 7.27−7.33 (m, 1H), 7.55

(ddd, J = 8.38, 6.88, 1.39 Hz, 1H), 7.90 (dd, J = 8.55, 0.92 Hz, 1H),

8.13 (dd, J = 8.55, 0.92 Hz, 1H). HRMS (ESI+) m/z [M + H]⁺

calculated for C17H22N3: 268.1814; found: 268.1813.

1

reaction step without further puriﬁcation (431 mg, 76%). H NMR

(400 MHz, CDCl₃) δ ppm 1.46 (br s, 9H), 1.80−2.04 (m, 4H), 2.21

(s, 3H), 2.36 (s, 3H), 3.21−3.59 (m, 8H), 5.98 (br s, 1H), 6.28 (br s,

1H). LC-MS m/z 332.2 [M + H]⁺.

O

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2-(2,6-Dimethylpyrimidin-4-yl)-2,7-diazaspiro[4.4]nonane (11d).

4 M HCl in 1,4-dioxane (1.99 mL, 7.98 mmol) was added to a stirred

solution of 10d (265 mg, 0.80 mmol) in 1,4-dioxane (4.2 mL) at rt.

The reaction mixture was stirred at rt for 16 h. The solvent was

evaporated to yield 11d as a white solid that was used in the next step

without further puriﬁcation (310 mg, 145%). LC-MS m/z 233.2 [M +

H]⁺.

2-(2,6-Dimethylpyridin-4-yl)-2,7-diazaspiro[4.4]nonane (11e). 4

M HCl in 1,4-dioxane (0.588 mL, 2.35 mmol) was added to a stirred

solution of 10e in 1,4-dioxane (1.2 mL) at rt. The reaction mixture

was stirred at rt for 16 h. The solvent was evaporated and the residue

triturated with EtOAc to yield 11e as a brownish solid (57 mg, 80%).

HRMS (ESI+) m/z [M + H]⁺calculated for C14H22N3: 232.1814;

found: 232.1814.

2-(2-Methoxy-6-methylpyridin-4-yl)-2,7-diazaspiro[4.4]nonane

(11f). TFA (2.865 mL, 37.4 mmol) was added to a stirred mixture of

10f (1.3 g, 3.74 mmol) in DCM (10 mL). The reaction mixture was

stirred at rt for 2 h. The reaction mixture was concentrated to yield

11f as yellow solid (3.5 g, 75%). LC-MS m/z 248.0 [M + H]⁺.

2-(4,6-Dimethylpyridin-2-yl)-2,7-diazaspiro[4.4]nonane (11g).

TFA (1.5 mL, 19.6 mmol) was added to a stirred solution of 10g

(431 mg, 0.62 mmol) in DCM (4.5 mL) at rt. The mixture was stirred

at rt for 7 h, and then the volatiles were evaporated in vacuo. The

residue thus obtained was puriﬁed by ion-exchange chromatography

(ISOLUTE SCX-2, MeOH, and then 7 N solution of NH₃in

MeOH). The desired fractions were collected and concentrated in

vacuo to yield 11g as an orange oil that was used in the next reaction

step without further puriﬁcation (129 mg, 66%). LC-MS m/z 232.2

[M + H]⁺.

2-(2-Methyl-6-(triﬂuoromethyl)pyridin-4-yl)-2,7-diazaspiro[4.4]-

nonane (11i). 6 M HCl in 2-PrOH (2.188 mL, 13.1 mmol) was

added to a stirred solution of 10i (253 mg, 0.66 mmol) in MeOH (5.3

mL) at rt. The mixture was stirred at rt for 16 h. Then, the solvents

were evaporated in vacuo to yield 11i as a white solid (211 mg,

100%). ¹H NMR (400 MHz, DMSO-d₆) δ ppm 1.96 (t, J = 7.40 Hz,

2H), 2.01−2.10 (m, 1H), 2.11−2.22 (m, 1H), 2.48 (s, 3H), 3.04−

3.22 (m, 2H), 3.29 (br dd, J = 7.40, 4.62 Hz, 2H), 3.41−3.72 (m,

4H), 6.71 (br s, 1H), 6.91 (br s, 1H), 9.62 (br s, 2H). HRMS (ESI+)

m/z [M + H]⁺calculated for C14H19F3N3: 286.1531; found:

286.1534.

6H), 4.09-4.32 (m, 2H), 5.80 (s, 1H), 8.19 (s, 1H). LC-MS m/z

345.2 [M + H]⁺.

5-Methyl-7-(2,6-diazaspiro[3.4]octan-6-yl)-[1,2,4]triazolo[1,5-a]-

pyrimidine (17). 1 N HCl in Et₂O (11.15 mL, 11.15 mmol) was

added to a stirred solution of 16 (768 mg, 2.23 mmol) in DCE (12

mL). The mixture was stirred at 50 °C for 5 h. Then, the solvent was

1

evaporated in vacuo to yield 17 as a white solid (690 mg, 98%). H

NMR (500 MHz, CD₃OD) δ ppm 2.28 (t, J = 7.1 Hz, 2H), 2.46 (s,

3H), 3.57 (d, J = 8.7 Hz, 2H), 3.68 (d, J = 9.0 Hz 2H), 3.91−4.43 (m,

4H), 6.09 (s, 1H), 8.17 (s, 1H). LC-MS m/z 245.0 [M + H]⁺.

tert-Butyl 7-(2,6-Dimethylpyridin-4-yl)-7-hydroxy-2-azaspiro-

[4.4]nonane-2-carboxylate (19). n-BuLi (4.47 mL, 7.15 mmol, 1.6

M in hexane) was added dropwise to a solution of 4-bromo-2,6-

dimethylpyridine (1.108 g, 5.96 mmol) in THF (10 mL) at −78 °C

under N₂. The mixture was stirred for 20 min; then, tert-butyl 7-oxo-

2-azaspiro[4.4]nonane-2-carboxylate (950 mg, 3.57 mmol) in THF

(10 mL) was added. The mixture was stirred at −78 °C for 2 h; then,

it was left to warm up to rt and stirred for 20 min. NH₄Cl saturated

solution was added and extracted with DCM. The organic layer was

separated, dried (Na₂SO₄), ﬁltered, and concentrated in vacuo. The

crude was puriﬁed by ﬂash column chromatography (silica; EtOAc in

heptane 0/100 to 30/70). The desired fractions were collected and

concentrated in vacuo to yield 19 as a white solid (740 mg, 54%). ¹H

NMR (500 MHz, CDCl₃) δ ppm 1.38−1.51 (m, 9H), 1.68−1.92 (m,

3H), 1.93−2.21 (m, 6H), 2.52 (s, 6H), 3.14−3.52 (m, 4H), 7.01−

7.05 (m, 2H). HRMS (ESI+) m/z [M + H]⁺calculated for

C20H31N2O3: 347.2335; found: 347.2335.

tert-Butyl 7-(2,6-Dimethylpyridin-4-yl)-2-azaspiro[4.4]nonane-2-

carboxylate (20). DAST (0.495 mL, 4.04 mmol) was added dropwise

to a stirred mixture of 18 (700 mg, 2.02 mmol) in DCM (15 mL) at 0

°C under N₂. The mixture was stirred at 0 °C for 1 h; then, it was

allowed to warm up to rt and stirred for 1 h. The mixture was treated

with water and sat. NaHCO₃and extracted with DCM. The organic

layer was separated, dried (Na₂SO₄), ﬁltered, and concentrated. The

crude was puriﬁed by ﬂash column chromatography (silica; EtOAc in

heptane 0/100 to 30/70). The desired fractions were collected and

concentrated in vacuo to yield a mixture of ﬂuorinated diastereomers

and impuriﬁed with the dehydroﬂuorinated product that was used in

the next step without further puriﬁcation (650 mg, 92%). LC-MS m/z

349.3 [M + H]⁺.

10% Pd on C (106 mg, 0.10 mmol) was added to a stirred solution

of the mixture of diastereoisomers (650 mg, 1.98 mmol) in MeOH

(25 mL). The mixture was hydrogenated under hydrogen

(atmospheric pressure) at room temperature for 18 h. The mixture

was ﬁltered through Celite, and the ﬁltrate was evaporated in vacuo to

yield 20 as a colorless oil (600 mg, 92%). ¹H NMR (500 MHz,

CDCl₃) δ ppm 1.47 (s, 9H), 1.53−1.91 (m, 5H), 2.00−2.22 (m, 2H),

2.53 (s, 6H), 3.09 (br d, J = 6.94 Hz, 1H), 3.18−3.47 (m, 4H), 3.48

(s, 1H), 6.86 (s, 2H). LC-MS m/z 331.4 [M + H]⁺.

7-(2,6-Dimethylpyridin-4-yl)-2-azaspiro[4.4]nonane (21). The

resin Amberlyst-15 (4.11 mmol/g) was added to 20 (600 mg, 1.82

mmol) in MeOH (90 mL). The reaction was shaken over 24 h in a

solid phase reactor. The solvent was removed and discarded, and the

resin was washed a few times with MeOH; then, 7 N NH₃in MeOH

solution was added to the resin and shaken over 2 h. The solvent was

removed and the resin was washed a few times with 7 N NH₃in

MeOH solution. The solvent was evaporated in vacuo to yield 21 as a

colorless oil (330 mg, 79%). LC-MS m/z 231.3 [M + H]⁺.

tert-Butyl 3-Iodo-1-oxa-7-azaspiro[4.4]nonane-7-carboxylate

(23). Iodine (357 mg, 1.41 mmol) was added portionwise to a

stirred mixture of tert-butyl 3-hydroxy-1-oxa-7-azaspiro[4.4]nonane-7-

carboxylate (311 mg, 1.28 mmol), triphenylphosphine (369 mg, 1.41

mmol), and imidazole (131 mg, 1.92 mmol) in toluene (4.6 mL).

Then, the mixture was stirred for 16 h at 80 °C. Then, the mixture

was cooled down to rt, water was added, and it was stirred for 10 min.

The organic layer was separated, and the aqueous phase was extracted

several times with EtOAc. The organic layer was separated, dried

(MgSO₄), and ﬁltered, and the solvents were evaporated in vacuo.

The crude was puriﬁed by ﬂash column chromatography (silica;

EtOAc in heptane 0/100 to 15/85). The desired fractions were

tert-Butyl 2-(5-Methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)-2,6-

diazaspiro[3.4]octane-6-carboxylate (13). tert-Butyl 2,6-

diazaspiro[3.4]octane-6-carboxylate (500 mg, 2.36 mmol), 7-chloro-

5-methyl-[1,2,4]triazolo[1,5-a]pyrimidine (397 mg, 2.36 mmol),

DIPEA (0.812 mL, 4.71 mmol), and ACN (8 mL) were stirred at

rt for 20 min. Then, the solvent was evaporated in vacuo. Water was

added, and the solid was ﬁltered oﬀ. The solid was dried in vacuo to

yield 13 as a white solid (751 mg, 93%). LC-MS m/z 345.3 [M + H]⁺.

5-Methyl-7-(2,6-diazaspiro[3.4]octan-2-yl)-[1,2,4]triazolo[1,5-a]-

pyrimidine (14). 6 N HCl in 2-PrOH (3.59 mL, 21.5 mmol) was

added to a stirred solution of 13 (742 mg, 2.15 mmol) in 2-PrOH (11

mL). The mixture was stirred at rt for 48 h. Then, the solvent was

1

evaporated in vacuo to yield 14 as a white solid (620 mg, 91%). H

NMR (400 MHz, DMSO-d₆) δ ppm 2.28 (t, J = 7.40 Hz, 2H), 2.50

(dt, J = 3.58, 1.91 Hz, 3H), 3.14−3.29 (m, 2H), 3.45 (t, J = 5.66 Hz,

2H), 4.03−5.29 (m, 4H), 6.03−6.67 (m, 1H), 8.33−8.96 (m, 1H),

9.76 (br s, 2H). LC-MS m/z 245.2 [M + H]⁺.

tert-Butyl 6-(5-Methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)-2,6-

diazaspiro[3.4]octane-2-carboxylate (16). tert-Butyl 2,6-

diazaspiro[3.4]octane-2-carboxylate (629 mg, 2.97 mmol), 7-chloro-

5-methyl-[1,2,4]triazolo[1,5-a]pyrimidine (500 mg, 2.97 mmol),

Et₃N (0.825 mL, 5.93 mmol), and DCM (12.5 mL) were stirred at

rt for 20 min. The mixture was diluted in water and extracted with

DCM. The organic layer was washed with brine, dried (MgSO₄), and

ﬁltered, and the solvents were evaporated in vacuo. The crude

product was puriﬁed by ﬂash column chromatography (silica; 7 M

solution of ammonia in methanol in DCM 0/100 to 2/98). The

desired fractions were collected and evaporated in vacuo to yield 16 as

1

a white solid (763 mg, 75%). H NMR (500 MHz, CDCl₃) δ ppm

1.46 (s, 9H), 2.27 (t, J = 6.94 Hz, 2H), 2.52 (s, 3H), 3.7−4.04 (m,

P

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collected and concentrated in vacuo to yield 23 as a white solid (349

mg, 77%). H NMR (400 MHz, CDCl₃) δ ppm 1.44 (br s, 9H),

1.87−2.02 (m, 1H), 2.19−2.29 (m, 1H), 2.29−2.38 (m, 1H), 2.55−

2.68 (m, 1H), 3.15−3.25 (m, 1H), 3.37−3.59 (m, 3H), 3.96−4.06

(m, 1H), 4.10−4.19 (m, 1H), 4.19−4.32 (m, 1H). HRMS (ESI+) m/

z [MM + H]⁺calculated for C24H41I2N2O6: 707.1054; found:

707.1053.

2.49 (s, 6H), 3.20−3.54 (m, 4H), 6.71−6.84 (m, 2H)H)H). HRMS

(ESI+) m/z [M + H]⁺calculated for C₁₉H₂₉N₂O₂: 317.2229; found:

317.2229.

2-(2,6-Dimethylpyridin-4-yl)-6-azaspiro[3.4]octane (29). 4 M

HCl in 1,4-dioxane (0.15 mL, 0.58 mmol) was added to a stirred

solution of 28 (31 mg, 0.10 mmol) in 1,4-dioxane (0.11 mL) under a

nitrogen atmosphere and was stirred at rt for 2 h. Then, the solvent

was evaporated in vacuo to yield 29 as a colorless oil (24 mg, 100%).

LC-MS m/z 217.2 [M + H]⁺.

1-(tert-Butyl) 3-Methyl 3-(but-3-en-1-yl)-2-oxopyrrolidine-1,3-

dicarboxylate (31). Potassium carbonate (6.784 g, 49.1 mmol),

tetrabutylammonium bromide (1.3 g, 4.1 mmol), and 4-bromo-1-

butene (3.11 mL, 30.7 mmol) were added to a stirred solution of 1-

(tert-butyl) 3-methyl 2-oxopyrrolidine-1,3-dicarboxylate (4.975 g,

20.5 mmol) in acetone (300 mL) at rt. The mixture was stirred at

60 °C for 2 h. Then, more potassium carbonate (3.39 g, 24.5 mmol)

and 4-bromo-1-butene (3.11 mL, 30.7 mmol) were added, and the

mixture was stirred at 60 °C for 2 h more. Then, more 4-bromo-1-

butene (6.23 mL, 61.4 mmol) was added, and the mixture was stirred

at 60 °C for 16 h more. The solvent was evaporated in vacuo. The

crude was puriﬁed by ﬂash column chromatography (silica; EtOAc in

heptane 0/100 to 14/86). The desired fractions were collected and

concentrated in vacuo to yield 31 as a viscous colorless oil (5.5 g,

91%). ¹H NMR (CDCl₃, 400 MHz) δ ppm 1.52 (s, 9H), 1.79 (ddd, J

= 13.6, 10.8, 5.3 Hz, 1H), 1.92 (dt, J = 13.1, 8.8 Hz, 1H), 1.97−2.15

(m, 2H), 2.23 (ddd, J = 13.5, 11.1, 5.4 Hz, 1H), 2.54 (ddd, J = 13.1,

6.4, 4.2 Hz, 1H), 3.71 (dt, J = 8.4, 2.3 Hz, 2H), 3.74 (s, 3H), 4.97 (dd,

J = 10.2, 1.4 Hz, 1H), 5.03 (dd, J = 17.1, 1.6 Hz, 1H), 5.77 (ddt, J =

16.8, 10.2, 6.5 Hz, 1H). HRMS (ESI+) m/z [M + Na]⁺calculated for

C15H23NNaO5: 320.1474; found: 320.1479.

1

tert-Butyl 3-(2,6-Dimethylpyridin-4-yl)-1-oxa-7-azaspiro[4.4]-

nonane-7-carboxylate (24). 2,6-Dimethyl-4-(4,4,5,5-tetramethyl-

1,3,2-dioxaborolan-2-yl)pyridine (264 mg, 0.95 mmol), nickel(II)

iodide (9 mg, 0.03 mmol), (1R,2R)-2-aminocyclohexanol hydro-

chloride (4 mg, 0.03 mmol), and sodium bis(trimethylsilyl)amide

(0.949 mL, 0.949 mmol, 1 M in THF) in 2-PrOH (7 mL) were

stirred for 10 min under a nitrogen atmosphere. Then, 23 (166 mg,

0.48 mmol) was added. The mixture was stirred at 80 °C for 1.5 h

under microwave irradiation. The mixture was cooled down, diluted

in EtOH, and ﬁltered through a Celite pad. The solvent was removed

in vacuo. The crude was puriﬁed by ﬂash column chromatography

(silica; MeOH in DCM 0/100 to 15/85). The desired fractions were

evaporated in vacuo and puriﬁed by HPLC (stationary phase: C18

XBridge 30 × 100 mm², 5 μm), mobile phase: gradient from 81%

0.1% NH₄CO₃H/NH₄OH pH 9 solution in water, 19% CH₃CN to

64% 0.1% NH₄CO₃H/NH₄OH pH 9 solution in water, 36% CH₃CN,

1

yielding 24 as a colorless oil (21 mg, 18%). H NMR (400 MHz,

CDCl₃) δ ppm 1.46 (s, 9H), 1.80−1.93 (m, 1H), 1.99−2.04 (m, 1H),

2.05−2.15 (m, 2H), 2.30−2.44 (m, 1H), 2.47−2.55 (m, 6H), 3.20−

3.67 (m, 4H), 3.73−3.87 (m, 1H), 4.16−4.27 (m, 1H), 6.77−6.88

(m, 2H). HRMS (ESI+) m/z [M + H]⁺calculated for

C19H29N2O3: 333.2178; found: 333.2177.

3-(2,6-Dimethylpyridin-4-yl)-1-oxa-7-azaspiro[4.4]nonane (25).

4 M HCl in 1,4-dioxane (0.13 mL, 0.52 mmol) was added to a

stirred solution of 24 (29 mg, 0.09 mmol) in 1,4-dioxane (0.1 mL)

under a nitrogen atmosphere and was stirred at rt for 2 h. Then, the

solvent was evaporated in vacuo to yield 25 as a colorless oil that was

used in the next step without further puriﬁcation (20 mg, 100%). LC-

MS m/z 233.1 [M + H]⁺.

1-Benzyl-3-(but-3-en-1-yl)-3-(iodomethyl)pyrrolidine (32). Meth-

yl 3-(but-3-en-1-yl)-2-oxopyrrolidine-3-carboxylate: TFA (27.4 mL,

358 mmol) was added to 31 (5.5 g, 18.5 mmol) in DCM (34 mL) at

0 °C. Then, the mixture was stirred at rt for 2 h. The crude was

diluted in DCM, and sat. NaHCO₃was added until basic pH. Then,

the organic layer was separated, dried (MgSO₄), ﬁltered, and

evaporated in vacuo to yield methyl 3-(but-3-en-1-yl)-2-oxopyrroli-

dine-3-carboxylate as a colorless oil that was used in the next step

tert-Butyl 2-Iodo-6-azaspiro[3.4]octane-6-carboxylate (27). Io-

dine (747 mg, 2.94 mmol) was added portionwise to a stirred solution

of triphenylphosphine (772 mg, 2.94 mmol) and imidazole (200 mg,

2.94 mmol) in toluene (14 mL) and stirred for 30 min at rt. Then,

tert-butyl 2-hydroxy-6-azaspiro[3.4]octane-6-carboxylate (608 mg,

2.68 mmol) in toluene (14 mL) was added to the mixture and

stirred at 80 °C for 16 h. The mixture was cooled down. Then, water

and EtOAc were added. The organic layer was separated, dried

(MgSO₄), and ﬁltered, and the solvents were evaporated in vacuo.

The crude was puriﬁed by ﬂash column chromatography (silica;

EtOAc in heptane 0/100 to 15/85). The desired fractions were

collected and evaporated in vacuo to yield 27 as a clear transparent oil

1

without further puriﬁcation (3.577 g, 98%). H NMR (CDCl₃, 400

MHz) δ ppm 1.69−1.83 (m, 1H), 2.00−2.10 (m, 4H), 2.11−2.20 (m,

1H), 2.64 (ddd, J = 13.0, 7.8, 3.4 Hz, 1H), 3.31 (tdd, J = 9.7, 3.4, 1.1

Hz, 1H), 3.43 (dt, J = 9.3, 7.6 Hz, 1H), 3.73 (s, 3H), 4.95 (dd, J =

10.3, 1.7 Hz, 1H), 5.02 (dd, J = 17.2, 1.7 Hz, 1H), 5.78 (ddt, J = 16.5,

10.2, 6.4 Hz, 1H). LC-MS m/z 198.1 [M + H]⁺.

Methyl 1-benzyl-3-(but-3-en-1-yl)-2-oxopyrrolidine-3-carboxylate:

Methyl 3-(but-3-en-1-yl)-2-oxopyrrolidine-3-carboxylate (3.058 g,

15.5 mmol) in THF (15 mL) was added to a stirred suspension of

NaH (733 mg, 18.3 mmol, 60% dispersion in mineral oil) in THF (5

mL) at 0 °C under a nitrogen atmosphere. The reaction mixture was

stirred for 5 min at 0 °C. Then, benzyl bromide (2.33 mL, 19.6

mmol) was added at 0 °C and stirred for 10 min. Then, it was warmed

naturally to rt and stirred for 16 h more. The mixture was treated with

water. The organic layer was separated, dried (MgSO₄), and ﬁltered,

and the solvents were evaporated in vacuo. The crude was puriﬁed by

ﬂash column chromatography (silica; EtOAc in heptane 0/100 to 15/

85). The desired fractions were collected and concentrated in vacuo

to yield methyl 1-benzyl-3-(but-3-en-1-yl)-2-oxopyrrolidine-3-carbox-

1

(429 mg, 48%). H NMR (400 MHz, CDCl₃) δ ppm 1.45 (s, 9H),

1.83−1.92 (m, 1H), 1.97 (q, J = 6.70 Hz, 1H), 2.52−2.85 (m, 4H),

3.20−3.47 (m, 4H), 4.36−4.56 (m, 1H). HRMS (ESI+) m/z [MM +

H]⁺calculated for C₂₄H₄₁I₂N₂O₄: 675.1155; found: 675.1152.

tert-Butyl 2-(2,6-Dimethylpyridin-4-yl)-6-azaspiro[3.4]octane-6-

carboxylate (28). 2,6-Dimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxabor-

olan-2-yl)pyridine (602 mg, 2.17 mmol), nickel(II) iodide (20 mg,

0.07 mmol), (1R,2R)-2-aminocyclohexanol hydrochloride (10 mg,

0.07 mmol), and sodium bis(trimethylsilyl)amide (2.17 mL, 2.17

mmol, 1 M in THF) in 2-PrOH (16 mL) were stirred for 10 min

under nitrogen. Then, 27 (365 mg, 1.08 mmol) was added. The

mixture was stirred at 80 °C for 1.5 h under microwave irradiation.

The mixture was cooled down, diluted in EtOH, and ﬁltered through

a Celite pad. The solvent was removed in vacuo. The crude was

puriﬁed by ﬂash column chromatography (silica; MeOH in DCM 0/

100 to 15/85). The desired fractions were evaporated in vacuo and

puriﬁed by HPLC (stationary phase: C18 XBridge 30 × 100 mm², 5

μm), mobile phase: gradient from 81% 0.1% NH₄CO₃H/NH₄OH pH

9 solution in water, 19% CH₃CN to 64% 0.1% NH₄CO₃H/NH₄OH

pH 9 solution in water, 36% CH₃CN, yielding 28 as a transparent oil

1

ylate as a clear colorless oil (2.113 g, 47%). H NMR (CDCl₃, 400

MHz) δ ppm 1.81−1.90 (m, 1H), 1.95 (ddd, J = 13.2, 8.9, 7.6 Hz,

1H), 2.00−2.13 (m, 2H), 2.13−2.23 (m, 1H), 2.41−2.57 (m, 1H),

3.15 (td, J = 9.2, 3.2 Hz, 1H), 3.32 (dt, J = 9.5, 7.8 Hz, 1H), 3.73 (s,

3H), 4.44 (d, J = 14.7 Hz, 1H), 4.50 (d, J = 14.7 Hz, 1H), 4.96 (dd, J

= 10.2, 1.7 Hz, 1H), 5.03 (dd, J = 17.2, 1.7 Hz, 1H), 5.73−5.88 (m,

1H), 7.17−7.24 (m, 2H), 7.24−7.41 (m, 3H). HRMS (ESI+) m/z

[M + H]⁺calculated for C17H22NO3: 288.1600; found: 288.1602.

(1-Benzyl-3-(but-3-en-1-yl)pyrrolidin-3-yl)methanol: Lithium alu-

minum hydride (10.45 mL, 10.45 mmol, 1 M solution in THF) was

added to a stirred solution of methyl 1-benzyl-3-(but-3-en-1-yl)-2-

oxopyrrolidine-3-carboxylate (2 g, 6.97 mmol) in THF (33 mL) at 0

°C. Then, it was warmed naturally at room temperature and stirred

1

(31 mg, 9%). H NMR (400 MHz, CDCl₃) δ ppm 1.42−1.55 (m,

9H), 1.76−1.87 (m, 1H), 1.96−2.22 (m, 4H), 2.26−2.43 (m, 2H),

Q

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for 2 h. Then, more lithium aluminum hydride (10.45 mL, 10.45

mmol, 1 M solution in THF) was added and stirred for 16 h. The

mixture was treated with water and extracted with EtOAc. The

organic layer was separated, dried (MgSO₄), and ﬁltered and the

solvents were evaporated in vacuo to yield (1-benzyl-3-(but-3-en-1-

yl)pyrrolidin-3-yl)methanol as a clear transparent oil (1.64 g, 96%).

¹H NMR (CDCl₃, 400 MHz) δ ppm 1.42−1.50 (m, 2H), 1.69 (ddd, J

= 12.95, 9.71 3.0 Hz, 1H), 1.93−2.04 (m, 2H), 2.03−2.08 (m, 1H),

2.13−2.19 (m, 1H), 2.20−2.30 (m, 1H), 2.87 (d, J = 9.0 Hz, 1H),

3.03 (td, J = 9.0, 3.0 Hz, 1H), 3.25 (br s, 1H), 3.43 (dd, J = 9.71, 1.85

Hz, 1H), 3.57 (d, J = 3.0 Hz, 2H), 3.59−3.63 (m, 1H), 4.88−5.08 (m,

2H), 5.79 (ddt, J = 17.0, 10.3, 6.5 Hz, 1H), 7.23−7.36 (m, 5H).

HRMS (ESI+) m/z [M + H]⁺calculated for C₁₆H₂₄NO: 246.1858;

found: 246.1863.

7-((2,6-Dimethylpyridin-4-yl)methyl)-2-azaspiro[4.4]nonane

(35). A solution of 34 (137 mg, 0.26 mmol) in MeOH (5.2 mL) was

hydrogenated in an H-Cube reactor (1 mL/min, 30 mm Pd(OH)₂/C

20% cartridge, full H₂mode, 80 °C, three cycles). The organic solvent

was concentrated in vacuo and was puriﬁed by RP HPLC (stationary

phase: C18 XBridge 30 × 100 mm², 5 μm), mobile phase: gradient

from 81% 0.1% NH₄CO₃H/NH₄OH pH 9 solution in water, 19%

CH₃CN to 64% 0.1% NH₄CO₃H/NH₄OH pH 9 solution in water,

36% CH₃CN, yielding 35 (46 mg, 73%). ¹H NMR (400 MHz,

CDCl₃) δ ppm 1.22−1.40 (m, 2H), 1.50−1.61 (m, 1H), 1.68 (br d, J

= 8.32 Hz, 1H), 1.73−1.90 (m, 5H), 2.13−2.29 (m, 1H), 2.47 (s,

6H), 2.50−2.66 (m, 2H), 2.94 (s, 1H), 2.96−3.10 (m, 1H), 3.17−

3.33 (m, 2H), 6.73 (s, 2H). HRMS (ESI+) m/z [M + H]⁺calculated

for C16H25N2: 245.2018; found: 245.2016.

1-(tert-Butyl) 3-Methyl 3-allylpyrrolidine-1,3-dicarboxylate (37).

LiHMDS (65.8 mL, 65.8 mmol, 1 M in hexane) was added dropwise

to a solution of methyl 1-Boc-3-pyrrolidinecarboxylate (10.057 g, 43.9

mmol) in 2-methyltetrahydrofurane (200 mL) at −78 °C under N₂

and stirred for 10 min. Then, allyl bromide (7.58 mL, 87.7 mmol) was

added at −78 °C and stirred for 30 min. Then, it was warmed up to

room temperature. The mixture was quenched with sat. NH₄Cl at 0

°C and diluted with EtOAc. The organic layer was separated, dried

(MgSO₄), and ﬁltered, and the solvents were evaporated in vacuo.

The crude was puriﬁed by ﬂash column chromatography (silica;

EtOAc in heptane 0/100 to 15/85). The desired fractions were

collected and concentrated in vacuo to yield 37 (5.977 g, 51%) as a

1-Benzyl-3-(but-3-en-1-yl)-3-(iodomethyl)pyrrolidine: Iodine (586

mg, 2.30 mmol) was added portionwise to a stirred solution of (1-

benzyl-3-(but-3-en-1-yl)pyrrolidin-3-yl)methanol (515 mg, 2.09

mmol), triphenylphosphine (605 mg, 2.30 mmol), and imidazole

(214 mg, 3.14 mmol) in 2-methyltetrahydrofurane (4.3 mL) at 0 °C.

The mixture was stirred for 1 h at 75 °C. The mixture was treated

with 10% Na₂SO₃and extracted with EtOAc. The organic layer was

separated, dried (Na₂SO₄), and ﬁltered, and the solvent was

evaporated. The residue was puriﬁed by column chromatography

(silica; EtOAc in heptane 0/100 to 15/85). The desired fractions

were collected and the solvent was evaporated to yield 32 as a clear

1

transparent oil (530 mg, 71%). H NMR (500 MHz, CDCl₃) δ ppm

1

yellow oil. H NMR (CDCl₃, 400 MHz) δ ppm 1.38−1.50 (s, 9H),

1.60−1.66 (m, 2H), 1.68−1.80 (m, 2H), 1.92−2.03 (m, 2H), 2.35 (d,

J = 9.83 Hz, 1H), 2.46−2.53 (m, 1H), 2.56 (d, J = 9.54 Hz, 1H), 2.70

(td, J = 8.38, 5.78 Hz, 1H), 3.32 (d, J = 9.54 Hz, 1H), 3.40 (d, J =

9.54 Hz, 1H), 3.52−3.60 (m, 2H), 4.95 (dd, J = 10.11, 1.16 Hz, 1H),

5.00−5.06 (m, 1H), 5.82 (ddt, J = 17.05, 10.26, 6.57, 6.57 Hz, 1H),

7.21−7.27 (m, 1H), 7.28−7.34 (m, 4H). LC-MS m/z 356.1 [M +

H]⁺.

1.79−1.91 (m, 1H), 2.24−2.37 (m, 1H), 2.38−2.49 (m, 2H), 3.17−

3.30 (m, 1H), 3.33−3.50 (m, 2H), 3.70 (s, 3H), 3.79 (br d, J = 11.3

Hz, 1H), 5.02−5.16 (m, 2H), 5.59-5.78 (m, 1H). LC-MS m/z 214.1

[M − tBu]⁺.

tert-Butyl 3-Allyl-3-(iodomethyl)pyrrolidine-1-carboxylate (38).

tert-Butyl 3-allyl-3-(hydroxymethyl)pyrrolidine-1-carboxylate: Lith-

ium borohydride (1.45 g, 66.6 mmol) was added to a stirred solution

of 37 (5.977 g, 22.2 mmol) in THF (103 mL) at 0 °C and stirred for

2 h at rt. Then, more lithium borohydride (725 mg, 33.3 mmol) was

added and the mixture was stirred for 16 h at rt. Then, more lithium

borohydride (725 mg, 33.3 mmol) was added and the mixture was

stirred for 16 h more. The mixture was treated with water and

extracted with EtOAc. The organic layer was separated, dried

(MgSO₄), and ﬁltered and the solvents were evaporated in vacuo to

yield tert-butyl 3-allyl-3-(hydroxymethyl)pyrrolidine-1-carboxylate as

a clear transparent oil (5.285 g, 99%). ¹H NMR (CDCl₃, 400 MHz) δ

ppm 1.44 (s, 9H), 1.62−1.73 (m, 1H), 1.74−1.87 (m, 1H), 2.14−

2.30 (m, 2H), 3.06−3.17 (m, 1H), 3.20−3.31 (m, 1H), 3.31−3.45

(m, 2H), 3.43−3.54 (m, 2H), 5.05−5.18 (m, 2H), 5.72−5.90 (m,

1H). HRMS (ESI+) m/z [M − H]⁺calculated for C13H22NO3:

240.1600; found: 240.1600.

2-Benzyl-7-((4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-

methyl)-2-azaspiro[4.4]nonane (33). Copper(I) chloride (5 mg,

0.05 mmol), bis(pinacolato)diboron (315 mg, 1.24 mmol), and 4,5-

bis(diphenylphosphino)-9,9-dimethylxanthene (30 mg, 0.05 mmol)

were mixed together under a nitrogen atmosphere in THF (1.34 mL).

Then, potassium tert-butoxide (1.24 mL, 1.24 mmol, 1 M in THF)

was added to the mixture and stirred for 1 min. Then, 32 (367 mg,

1.03 mmol) in THF (2.28 mL) was added dropwise at 30 °C and

stirred for 16 h. The mixture was ﬁltered through Celite and washed

with DCM, and the solvents were evaporated in vacuo. The crude was

puriﬁed by ﬂash column chromatography (silica; EtOAc in heptane 0/

100 to 15/85). The desired fractions were collected and concentrated

1

in vacuo to yield 33 as a colorless oil (306 mg, 90%). H NMR (500

MHz, CDCl₃) δ ppm 0.78−0.90 (m, 2H), 1.11−1.20 (m, 2H), 1.23

(s, 12H), 1.58−1.64 (m, 2H), 1.72−1.83 (m, 3H), 1.89 (dd, J =

12.43, 7.22 Hz, 1H), 1.94−2.05 (m, 1H), 2.32−2.39 (m, 2H), 2.59 (t,

J = 7.08 Hz, 2H), 3.59 (s, 2H), 7.19−7.25 (m, 1H), 7.27−7.34 (m,

4H). LC-MS m/z 356.3 [M + H]⁺.

tert-Butyl 3-allyl-3-(iodomethyl)pyrrolidine-1-carboxylate: Iodine

(31 g, 122 mmol) was added portionwise to triphenylphosphine

(32 g, 122 mmol) and imidazole (8.32 g, 122 mmol) in toluene (100

mL) at rt and stirred for 30 min. Then, tert-butyl 3-allyl-3-

(hydroxymethyl)pyrrolidine-1-carboxylate (9.923 g, 41.1 mmol) in

toluene (60 mL) was added to the stirred solution and stirred at 80

°C for 16 h. The mixture was cooled down at rt, treated with water

and sat. Na₂S₂O₃, and extracted with EtOAc. The organic layer was

separated, dried (MgSO₄), and ﬁltered, and the solvents were

evaporated in vacuo. The residue was puriﬁed by ﬂash column

chromatography (silica; EtOAc in heptane 0/100 to 15/85 for the

ﬁrst puriﬁcation and EtOAc in DCM 0/100 to 2/98 for the second

puriﬁcation). The desired fractions were collected and concentrated

2-Benzyl-7-((2,6-dimethylpyridin-4-yl)methyl)-2-azaspiro[4.4]-

nonane (34). Tetrakis(triphenylphosphine)palladium (0) (10 mg,

0.01 mmol) was added to a sealed vial under a nitrogen atmosphere.

Then, 33 (104 mg, 0.29 mmol) in 1,4-dioxane (0.76 mL) followed by

4-bromo-2,6-dimethylpyridine (163 mg, 0.88 mmol) and potassium

hydroxide (0.585 mL, 1.76 mmol, 3 M in water) were added and

stirred for 16 h at 90 °C. The mixture was cooled and diluted in

DCM. The mixture was ﬁltered through Celite and anhydrous

MgSO₄. The solvent was evaporated in vacuo. The crude was puriﬁed

by ﬂash column chromatography (silica; MeOH in DCM 0/100 to 5/

95). The desired fractions were collected and concentrated in vacuo

1

in vacuo to yield 38 as a clear colorless oil (6.025 g, 42%). H NMR

(CDCl₃, 400 MHz) δ ppm 1.45 (s, 9H), 1.80−1.89 (m, 2H), 2.21−

2.34 (m, 2H), 3.17−3.35 (m, 4H), 3.36−3.48 (m, 2H), 5.12−5.26

(m, 2H), 5.62−5.79 (m, 1H). HRMS (ESI+) m/z [MM + H]⁺

calculated for C₂₆H₄₅I₂N₂O₄: 703.1469; found: 703.0930.

tert-Butyl 2-((4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-

methyl)-6-azaspiro[3.4]octane-6-carboxylate (39). Copper(I)

chloride (15 mg, 0.15 mmol), bis(pinacolato)diboron (895 mg,

3.52 mmol), and 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene

1

to yield 34 as a colorless oil (137 mg, 89%). H NMR (CDCl₃, 400

MHz) δ ppm 1.08−1.36 (m, 2H + 2H′), 1.49−1.59 (m, 2H), 1.61−

1.70 (m, 2H + 2H′), 1.70−1.74 (m, 2H′), 1.75−1.97 (m, 2H + 2H′),

2.09 (m, 1H′), 2.20 (m, 1H), 2.35 (m, 2H), 2.47 (m, 6H + 10H′),

2.62 (m, 4H), 3.60 (s, 2H + 2H′), 6.74 (s, 2H + 2H′), 7.29 (m, 5H +

5H′). HRMS (ESI+) m/z [M + H]⁺calculated for C₂₃H₃₁N₂:

335.2487; found: 335.2581.

R

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(85 mg, 0.15 mmol) were mixed together under a nitrogen

atmosphere in THF (3 mL). Then, potassium tert-butoxide (3.523

mL, 3.523 mmol, 1 M solution in THF) was added to the mixture and

stirred for 1 min at 30 °C. Then, 38 (1.031 g, 2.94 mmol) in THF (4

mL) was added dropwise to the mixture at 30 °C and stirred for 48 h.

The solvents were evaporated in vacuo. The crude product was

puriﬁed by ﬂash column chromatography (silica; EtOAc in heptane 0/

100 to 10/90). The desired fractions were collected and concentrated

3-((2,6-Dimethylpyridin-4-yl)oxy)-1-oxa-7-azaspiro[4.4]nonane

(43). 4 M HCl in 1,4-dioxane (1.15 mL, 4.61 mmol) was added to a

stirred solution of 42 (268 mg, 0.77 mmol) in 1,4-dioxane (0.9 mL)

under a nitrogen atmosphere. The mixture was stirred at rt for 2 h.

The solvent was evaporated in vacuo to yield 43 as a red sticky oil

(112 mg, 51%). ¹H NMR (400 MHz, CD₃OD) δ ppm 0.41−0.55 (m,

1H), 0.56−0.67 (m, 1H), 0.67−0.84 (m, 1H), 0.96−1.07 (m, 7H),

1.62−1.67 (m, 1H), 1.70−1.90 (m, 3H), 2.43−2.50 (m, 1H), 2.58

(td, J = 10.75, 3.93 Hz, 1H), 3.74−3.87 (m, 1H), 5.59 (d, J = 4.16 Hz,

2H). HRMS (ESI+) m/z [M + H]⁺calculated for C14H21N2O2:

249.1603; found: 249.1602.

tert-Butyl 2-((2,6-Dimethylpyridin-4-yl)oxy)-6-azaspiro[3.4]-

octane-6-carboxylate (44). tert-Butyl 2-hydroxy-6-azaspiro[3.4]-

octane-6-carboxylate (98 mg, 0.43 mmol) and 15-Crown-5 (0.103

mL, 0.52 mmol) were added to a stirred mixture of sodium hydride

(52 mg, 0.30 mmol, 60% dispersion in mineral oil) in DMF (2.2 mL)

at 0 °C. The mixture was stirred at this temperature for 30 min, and 4-

chloro-2,6-dimethylpyridine (0.055 mL, 0.43 mmol) was added. The

mixture was stirred at 60 °C for 18 h. The reaction was quenched with

water at 0 °C and concentrated under a vacuum. The residue was

puriﬁed by ﬂash column chromatography (silica; MeOH in DCM 0/

100 to 5/95). The desired fractions were concentrated in vacuo to

yield 44 as an oil (130 mg, 91%). LC-MS m/z 333.2 [M + H]⁺.

2-((2,6-Dimethylpyridin-4-yl)oxy)-6-azaspiro[3.4]octane (45). 4

M HCl in 1,4-dioxane (0.94 mL, 3.76 mmol) was added to 44 (125

mg, 0.38 mmol), and the reaction mixture was stirred at rt for 1 h.

The solvent was evaporated in vacuo. The residue was puriﬁed by ion-

exchange chromatography (ISOLUTE SCX-2, MeOH, and then 7 M

solution of ammonia in MeOH). The desired fractions were collected

and concentrated in vacuo to yield 45 as a white solid (85 mg, 97%).

LC-MS m/z 233.2 [M + H]⁺.

1

in vacuo to yield 39 as a colorless oil (751 mg, 59%). H NMR

(CDCl₃, 400 MHz) δ ppm 0.94 (d, J = 7.7 Hz, 2H + 2H′), 1.22 (s,

12H + 12H′), 1.44 (s, 9H + 9H′), 1.62 (m, 2H + 2H′), 1.83 (d, J =

6.8 Hz, 2H′), 1.85 (d, J = 6.8 Hz, 2H), 2.03 (d, J = 8.5 Hz, 2H′), 2.07

(d, J = 10.8 Hz, 2H), 2.40 (m, 1H + 1H′), 3.13 (s, 2H′), 3.18 (s, 2H),

3.27 (t, J = 6.6 Hz, 2H′), 3.33 (t, J = 6.7 Hz, 2H). LC-MS m/z 703.6

[MM + H]⁺.

tert-Butyl 2-((2,6-Dimethylpyridin-4-yl)methyl)-6-azaspiro[3.4]-

octane-6-carboxylate (40). Tetrakis(triphenylphosphine)palladium

(0) (24 mg, 0.02 mmol) was added to a sealed vial under a nitrogen

atmosphere. Then, 39 (246 mg, 0.70 mmol) in 1,4-dioxane (1.8 mL)

followed by 4-bromo-2,6-dimethylpyridine (391 mg, 2.10 mmol) and

potassium hydroxide (1.4 mL, 4.2 mmol, 3 M in water) were added

and stirred for 16 h at 90 °C. The mixture was cooled and diluted in

DCM. The mixture was ﬁltered through Celite and anhydrous

MgSO₄. The solvent was evaporated in vacuo. The crude product was

puriﬁed by ﬂash column chromatography (silica; MeOH in DCM 0/

100 to 5/95). The desired fractions were collected and concentrated

in vacuo. The residue was puriﬁed by RP HPLC (stationary phase:

C18 XBridge 30 × 100 mm², 5 μm), mobile phase: gradient from 54%

0.1% NH₄CO₃H/NH₄OH pH 9 solution in water, 46% CH₃CN to

64% 0.1% NH₄CO₃H/NH₄OH pH 9 solution in water, 36% CH₃CN,

1

yielding 40 as a clear transparent oil (107 mg, 46%). H NMR (400

(2-Benzyl-2-azaspiro[4.4]nonan-7-yl)methanol (46). Sodium per-

borate monohydrate (661 mg, 6.61 mmol) was added to a stirred

solution of 33 (336 mg, 0.36 mmol) in THF (9.9 mL) and water (6.6

mL). The mixture was stirred at rt for 2 h. The mixture was extracted

with EtOAc. The organic layer was separated, dried (MgSO₄), and

ﬁltered, and the solvents were removed in vacuo. The crude was

puriﬁed by ﬂash column chromatography (silica; EtOAc in heptane 0/

100 to 100/0). The desired fractions were collected and evaporated in

vacuo to yield 46 as a transparent oil (182 mg, 112%). ¹H NMR (400

MHz, CDCl₃) δ ppm 1.27−1.39 (m, 2H + 2H′), 1.53−1.67 (m, 2H +

2H′), 1.67−1.73 (m, 2H + 1H′), 1.78 (m, 2H + 2H′), 1.81−1.89 (m,

1H′), 2.09 (m, 1H′), 2.19 (m, 1H), 2.40 (d, J = 2.0 Hz, 2H′), 2.47 (q,

J = 9.2 Hz, 2H), 2.61 (m, 3H + 3H′), 3.45 (d, J = 6.9 Hz, 2H′), 3.47

(d, J = 6.9 Hz, 2H), 3.60 (s, 2H′), 3.60 (s, 2H), 7.19−7.26 (m, 1H +

1H′), 7.28−7.35 (m, 4H + 4H′). HRMS (ESI+) m/z [M + H]⁺

calculated for C₁₆H₂₄NO: 246.1858; found: 246.1918.

MHz, CDCl₃) δ ppm 1.45 (s, 9H), 1.59−1.80 (m, 6H), 1.81−1.92

(m, 1H), 1.97−2.15 (m, 2H), 2.48 (s, 6H), 2.58−2.67 (m, 2H),

3.16−3.26 (m, 1H), 3.28−3.40 (m, 3H), 6.66−6.80 (m, 2H). HRMS

(ESI+) m/z [M + H]⁺calculated for C20H31N2O2: 331.2386;

found: 331.2386.

2-((2,6-Dimethylpyridin-4-yl)methyl)-6-azaspiro[3.4]octane (41).

4 M HCl in 1,4-dioxane (0.604 mL, 2.41 mmol) was added to a

stirred solution of 40 (133 mg, 0.40 mmol) in 1,4-dioxane (0.47 mL)

under a nitrogen atmosphere and was stirred at rt for 2 h. Then, the

solvent was evaporated in vacuo to yield 41 as a colorless oil (96 mg,

1

89%). H NMR (400 MHz, CD₃OD) δ ppm 0.31−0.41 (m, 2H),

0.41−0.48 (m, 1H), 0.49−0.55 (m, 1H), 0.55−0.63 (m, 1H), 0.64−

0.72 (m, 1H), 1.11−1.14 (m, 7H), 1.34−1.41 (m, 2H), 1.61 (t, J =

2.66 Hz, 1H), 1.66−1.73 (m, 3H), 1.77−1.85 (m, 1H), 5.94−6.02

(m, 2H). HRMS (ESI+) m/z [M + H]⁺calculated for C15H23N2:

231.1861; found: 231.1862.

tert-Butyl 3-((2,6-Dimethylpyridin-4-yl)oxy)-1-oxa-7-

azaspiro[4.4]nonane-7-carboxylate (42). Sodium hydride (132

mg, 3.29 mmol, 60% dispersion in mineral oil) was added to a

stirred solution of tert-butyl 3-hydroxy-1-oxa-7-azaspiro[4.4]nonane-

7-carboxylate (267 mg, 1.10 mmol) in DMF (4 mL) in a microwave

vial under nitrogen at rt. The mixture was stirred at rt for 45 min.

Then, 4-bromo-2,6-dimethylpyridine (245 mg, 1.32 mmol) was added

at 0 °C and stirred at 90 °C for 48 h. The solvent was evaporated,

dissolved in EtOAc, and treated with water. The organic layer was

separated, dried (MgSO₄), and ﬁltered, and the solvents were

evaporated in vacuo. The crude product was puriﬁed by ﬂash column

chromatography (silica; MeOH in DCM 0/100 to 5/95) and by RP

HPLC (stationary phase: C18 XBridge 30 × 100 mm², 5 μm), mobile

phase: gradient from 67% 0.1% NH₄CO₃H/NH₄OH pH 9 solution in

water, 33% CH₃CN to 50% 0.1% NH₄CO₃H/NH₄OH pH 9 solution

in water, 50% CH₃CN. The desired fractions were collected and

concentrated in vacuo to yield 42 as a colorless oil (268 mg, 70%). ¹H

NMR (CDCl₃, 400 MHz) δ ppm 1.43 (s, 9H + 9H′), 1.86 (m, 2H′),

1.93−2.05 (m, 2H), 2.08−2.20 (m, 2H′), 2.20−2.37 (m, 2H), 2.45 (s,

6H + 6H′), 3.26 (m, 1H + 1H′), 3.44 (m, 2H + 2H′), 3.52 (m, 1H +

1H′), 3.97−4.02 (m, 1H + 1H′), 4.08−4.21 (m, 1H + 1H′), 4.96 (m,

1H + 1H′), 6.42 (s, 2H + 2H′). HRMS (ESI+) m/z [M + H]⁺

calculated for C19H29N2O4: 349.2127; found: 349.2127.

2-Benzyl-7-(((2,6-dimethylpyridin-4-yl)oxy)methyl)-2-azaspiro-

[4.4]nonane (47). Potassium tert-butoxide was added portionwise (97

mg, 0.86 mmol) to a stirred solution of 46 (106 mg, 0.43 mmol) in

DMSO (0.9 mL). The mixture was stirred at rt for 50 min. Then, 4-

chloro-2,6-dimethylpyridine (0.088 mL, 0.69 mmol) was added and

the resulting mixture was stirred at 70 °C for 5 h. The mixture was

cooled to rt; then, water and EtOAc were added. The organic layer

was separated, dried (Na₂SO₄), and ﬁltered, and the volatiles were

evaporated in vacuo. The residue was puriﬁed by ﬂash column

chromatography (silica; EtOAc in heptane 0/100 to 100/0). The

desired fractions were evaporated in vacuo to yield 47 as a brown oil

1

(120 mg, 79%). H NMR (500 MHz, CDCl₃) δ ppm 1.33−1.39 (m,

1H), 1.40−1.44 (m, 1H), 1.44−1.50 (m, 1H), 1.58−1.65 (m, 1H),

1.66−1.71 (m, 1H), 1.71−1.76 (m, 1H), 1.77−1.82 (m, 1H), 1.84 (br

d, J = 8.96 Hz, 1H), 1.89 (dd, J = 12.86, 7.95 Hz, 1H), 1.92−1.98 (m,

1H), 2.41 (d, J = 2.02 Hz, 1H), 2.46 (s, 6H), 2.47−2.55 (m, 2H),

2.59−2.69 (m, 2H), 3.61 (d, J = 2.60 Hz, 2H), 3.77−3.89 (m, 2H),

6.37−6.53 (m, 1H), 7.20−7.26 (m, 1H), 7.27−7.36 (m, 4H). HRMS

(ESI+) m/z [M + H]⁺calculated for C23H31N2O: 351.2436; found:

351.2454.

7-(((2,6-Dimethylpyridin-4-yl)oxy)methyl)-2-azaspiro[4.4]-

nonane (48). A solution of 47 (109 mg, 0.31 mmol) in MeOH (6.2

mL) was hydrogenated in an H-Cube reactor (1.2 mL/min, (30 mm)

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Pd(OH)₂/C 20% cartridge, full H₂mode, 80 °C, three cycles). The

solvent was concentrated in vacuo to yield 48 as a colorless oil (90

mg, 100%). LC-MS m/z 261.1 [M + H]⁺.

water and evaporated in vacuo. The crude was puriﬁed by ﬂash

column chromatography (silica; MeOH in DCM 0/100 to 10/90)

and by RP HPLC (stationary phase: C18 XBridge 30 × 100 mm², 5

μm; mobile phase: gradient from 90% 10 mM NH₄CO₃H pH 9

solution in water, 10% CH₃CN to 0% 10 mM NH₄CO₃H pH 9

solution in water, 100% CH₃CN). The desired fractions were

collected and concentrated in vacuo to yield 52b as a white solid (140

tert-Butyl 2-(Hydroxymethyl)-6-azaspiro[3.4]octane-6-carboxy-

late (49). Sodium perborate monohydrate (722 mg, 7.23 mmol)

was added to a solution of 39 (254 mg, 0.72 mmol) in THF (10.9

mL) and water (7.2 mL). The mixture was stirred at rt for 2 h. The

mixture was extracted with EtOAc. The organic layer was separated,

dried (MgSO₄), and ﬁltered, and the solvents were removed in vacuo.

The crude was puriﬁed by ﬂash column chromatography (silica;

EtOAc in heptane 0/100 to 100/0). The desired fractions were

collected and evaporated in vacuo to yield 49 as a transparent oil (159

1

mg, 92%). H NMR (500 MHz, CDCl₃) δ ppm 1.87−2.12 (m, 4H),

2.50 (s, 4H), 2.57−2.83 (m, 3H), 3.51−3.60 (m, 2H), 3.67−4.27 (m,

4H), 5.77 (s, 1H), 5.94 (s, 2H), 6.75 (s, 2H), 6.86 (s, 1H), 8.16 (s,

1H). HRMS (ESI+) m/z [M + H]⁺calculated for C21H25N6O2:

393.2039; found: 393.2032.

1

mg, 91%). H NMR (500 MHz, CDCl₃) δ ppm 0.79−0.88 (m, 2H),

6-((7-(5-Methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)-2,7-

diazaspiro[4.4]nonan-2-yl)methyl)quinoxaline (52c). DIPEA (0.3

mL, 1.74 mmol) was added to a stirred suspension of 8 (52 mg, 0.18

mmol) in DCM (1.8 mL). The mixture was stirred at rt for 5 min.

Then, quinoxaline-6-carbaldehyde (37 mg, 0.23 mmol) and sodium

triacetoxyborohydride (56 mg, 0.26 mmol) were added and the

mixture was stirred at rt for 16 h. The mixture was treated with 10%

Na₂CO₃and extracted with DCM. The organic layer was separated,

dried (MgSO₄), and ﬁltered, and the solvents were evaporated in

vacuo. The crude product was puriﬁed by ﬂash column chromatog-

raphy (silica; 7 N solution of ammonia in MeOH in DCM 0/100 to

10/90). The desired fractions were collected and concentrated in

vacuo to yield 52c as a yellow oil (19 mg, 27%). ¹H NMR (500 MHz,

CDCl₃) δ ppm 1.87−2.16 (m, 5H), 2.49 (s, 3H), 2.58 (d, J = 9.25 Hz,

1H), 2.67−2.76 (m, 2H), 2.81 (td, J = 8.60, 5.92 Hz, 1H), 3.82−3.94

(m, 3H), 4.00 (br s, 2H), 5.77 (s, 1H), 7.82 (dd, J = 8.67, 2.02 Hz,

1H), 8.03 (d, J = 1.16 Hz, 1H), 8.07 (d, J = 8.38 Hz, 1H), 8.16 (s,

1H), 8.81−8.85 (m, 2H). HRMS (ESI+) m/z [M + H]⁺calculated

for C22H25N8: 401.2202; found: 401.2203.

2-Methyl-5-((7-(5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)-

2,7-diazaspiro[4.4]nonan-2-yl)methyl)benzo[d]thiazole (52d).

AcOH (0.024 mL, 0.43 mmol) was added to a stirred mixture of 8

(55 mg, 0.21 mmol) and 2-methylbenzo[d]thiazole-5-carbaldehyde

(49 mg, 0.27 mmol) in MeOH (1 mL). The mixture was stirred at rt

for 4 h. Then, sodium cyanoborohydride (40 mg, 0.64 mmol) was

added, and the mixture was stirred at rt for 15 h. Then, the solvent

was concentrated in vacuo and the crude was treated with a saturated

solution of NaHCO₃and extracted with DCM. The organic layer was

separated, dried (MgSO₄), and ﬁltered, and the solvents were

evaporated in vacuo. The crude product was puriﬁed by RP HPLC

(stationary phase: C18 XBridge 30 × 100 mm², 5 μm; mobile phase:

gradient from 81% 10 mM NH₄CO₃H pH 9 solution in water, 19%

CH₃CN to 64% 10 mM NH₄CO₃H pH 9 solution in water, 36%

CH₃CN). The desired fractions were collected and the solvents

evaporated in vacuo to yield 52d as a pale-yellow solid (24 mg, 27%).

¹H NMR (500 MHz, CDCl₃) δ ppm 1.85−1.97 (m, 2H), 2.00−2.10

1.19−1.28 (m, 3H), 1.37−1.43 (m, 11H), 1.46−1.64 (m, 1H), 1.67−

1.80 (m, 3H), 1.80−1.87 (m, 1H), 1.89−1.97 (m, 1H), 1.97−2.05

(m, 1H), 2.35−2.47 (m, 1H), 3.11−3.19 (m, 1H), 3.19−3.36 (m,

3H), 3.50−3.61 (m, 2H). LC-MS m/z 186.1 [M − tBu]⁺.

tert-Butyl 2-(((2,6-Dimethylpyridin-4-yl)oxy)methyl)-6-azaspiro-

[3.4]octane-6-carboxylate (50). Potassium tert-butoxide (142 mg,

1.27 mmol) was added portionwise to a stirred solution of 49 (253

mg, 0.63 mmol) in DMSO (1.3 mL). The mixture was stirred at rt for

50 min. Then, 4-chloro-2,6-dimethylpyridine (0.130 mL, 1.02 mmol)

was added and the resulting mixture was stirred at 70 °C for 5 h. The

mixture was cooled to rt; then, water and EtOAc were added. The

organic layer was separated, dried (MgSO₄), and ﬁltered, and the

solvents were evaporated in vacuo. The crude product was puriﬁed by

ﬂash column chromatography (silica; EtOAc in heptane 0/100 to

100/0). The desired fractions were collected and evaporated in vacuo

1

to yield 50 as a brown oil (153 mg, 70%). H NMR (500 MHz,

CDCl₃) δ ppm 1.43 (br s, 9H), 1.75−1.95 (m, 4H), 2.02−2.18 (m,

2H), 2.39−2.48 (m, 6H), 2.69 (br d, J = 5.49 Hz, 1H), 2.91−3.16 (m,

1H), 3.19−3.28 (m, 1H), 3.28−3.41 (m, 2H), 3.91 (br d, J = 2.89 Hz,

1H), 6.41−6.52 (m, 1H). HRMS (ESI+) m/z [M + H]⁺calculated

for C20H31N2O3: 347.2335; found: 347.2335.

2-(((2,6-Dimethylpyridin-4-yl)oxy)methyl)-6-azaspiro[3.4]octane

(51). 4 M HCl in 1,4-dioxane (0.515 mL, 2.06 mmol) was added to a

stirred solution of 50 (119 mg, 0.24 mmol) in 1,4-dioxane (0.4 mL).

The mixture was stirred at rt for 10 min. Then, the solvent was

1

evaporated in vacuo to yield 51 as a colorless oil (85 mg, 87%). H

NMR (400 MHz, CD₃OD) δ ppm 0.09−0.16 (m, 1H), 0.21−0.41

(m, 1H), 0.48−0.71 (m, 4H), 0.73−0.80 (m, 1H), 1.11 (s, 6H),

1.22−1.41 (m, 1H), 1.64−1.83 (m, 4H), 2.74 (dd, J = 6.13, 4.28 Hz,

2H), 5.61−5.75 (m, 1H). HRMS (ESI+) m/z [M + H]⁺calculated

for C15H23N2O: 247.1810; found: 247.1810.

Syntheses of Compounds 52a−d. N-(5-((7-(5-Methyl-[1,2,4]-

triazolo[1,5-a]pyrimidin-7-yl)-2,7-diazaspiro[4.4]nonan-2-yl)-

methyl)thiazol-2-yl)acetamide (52a). N-(5-formylthiazol-2-yl)-

acetamide (138 mg, 0.81 mmol) and sodium cyanoborohydride (51

mg, 0.81 mmol) were sequentially added to a stirred mixture of 8

(200 mg, 0.67 mmol) and Et₃N (0.123 mL, 0.88 mmol) in DCM (9.2

mL). The mixture was stirred at rt for 18 h. The mixture was

quenched with water (0.5 mL) and ﬁltered through Celite, and the

ﬁltrate was concentrated in vacuo. The residue was puriﬁed by ﬂash

column chromatography (silica; 7 N solution of ammonia in

methanol in DCM 0/100 to 3/97). The desired fractions were

collected and concentrated in vacuo. The product was redissolved in

DCM (two drops) and triturated with Et₂O. The solid was ﬁltered

and dried to give 52a as a white solid (121 mg, 43%). ¹H NMR (400

MHz, DMSO-d₆) δ ppm 1.74−1.88 (m, 2H), 1.89−2.04 (m, 2H),

2.10 (s, 3), 2.38 (s, 3H), 2.44 (d, J = 9.25 Hz, 1H), 2.53−2.61 (m,

2H), 2.63−2.72 (m, 1H), 3.73 (s, 6H), 6.10 (s, 1H), 7.25 (s, 1H),

8.25 (s, 1H), 11.94 (br s, 1H). HRMS (ESI+) m/z [M + H]⁺

calculated for C19H25N8OS: 413.1872; found: 413.1865.

(m, 2H), 2.49 (s, 3H), 2.51 (d, J = 8.96 Hz, 1H), 2.64 (br d, J = 9.25

Hz, 2H), 2.75−2.81 (m, 1H), 2.83 (s, 3H), 3.72−3.81 (m, 3H),

3.85−4.05 (m, 3H), 5.76 (s, 1H), 7.34 (dd, J = 8.24, 1.59 Hz, 1H),

7.75 (d, J = 8.09 Hz, 1H), 7.90 (d, J = 0.87 Hz, 1H), 8.16 (s, 1H).

HRMS (ESI+) m/z [M + H]⁺calculated for C22H26N7S: 420.1970;

found: 420.1969.

N-(5-((2-(5-Methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)-2,6-

diazaspiro[3.4]octan-6-yl)methyl)thiazol-2-yl)acetamide (53). N-

(5-(chloromethyl)thiazol-2-yl)acetamide (144 mg, 0.76 mmol) was

added to a stirred solution of 14 (238 mg, 0.75 mmol) and DIPEA

(0.647 mL, 3.76 mmol) in DCE (4 mL) at −78 °C. The mixture was

warmed to rt and stirred for 30 min. Then, water was added. The

organic layer was separated, dried (MgSO₄), and ﬁltered, and the

solvents were evaporated in vacuo. The crude product was puriﬁed by

RP HPLC (stationary phase: C18 XBridge 30 × 100 mm², 5 μm;

mobile phase: gradient from 81% 0.1% NH₄CO₃H/NH₄OH pH 9

solution in water, 19% CH₃CN to 64% 0.1% NH₄CO₃H/NH₄OH pH

9 solution in water, 36% CH₃CN) and by chiral SFC (stationary

phase: CHIRALPAK AD-H 5 μm 250 × 20 mm²; mobile phase: 55%

CO₂, 45% EtOH (0.3% iPrNH₂)). The desired fractions were

collected and concentrated in vacuo to yield 53 as a white solid (49

7-(7-(Benzo[d][1,3]dioxol-5-ylmethyl)-2,7-diazaspiro[4.4]nonan-

2-yl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidine (52b). Benzo[d]-

[1,3]dioxole-5-carbaldehyde (64 mg, 0.43 mmol) was added to a

stirred suspension of 8 (100 mg, 0.39 mmol) in DCM (2.48 mL). The

mixture was stirred at rt for 3 h. Sodium cyanoborohydride (73 mg,

1.16 equiv) and MeOH (0.047 mL, 1.16 equiv) were added, and the

mixture was stirred at rt for 16 h. The mixture was quenched with

1

mg, 16%). H NMR (400 MHz, DMSO-d₆) δ ppm 2.02−2.20 (m,

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5H), 2.32−2.40 (m, 3H), 2.58 (t, J = 7.05 Hz, 2H), 2.76 (s, 2H), 3.73

(s, 2H), 4.37 (br s, 4H), 5.97 (s, 1H), 7.26 (s, 1H), 8.27 (s, 1H),

11.93 (br s, 1H). HRMS (ESI+) m/z [M + H]⁺calculated for

C18H23N8OS: 399.1716; found: 399.1713.

sodium triacetoxyborohydride (59 mg, 0.28 mmol) were added. The

reaction mixture was stirred for 16 h more, and then, a saturated

NaHCO₃solution was added. The organic layer was separated, dried

(MgSO₄), and ﬁltered, and the solvents were evaporated in vacuo.

The crude product was puriﬁed by ﬂash column chromatography

(silica; MeOH in DCM 0/100 to 25/75). The desired fractions were

collected and evaporated in vacuo and the residue was triturated with

N-(5-((6-(5-Methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)-2,6-

diazaspiro[3.4]octan-2-yl)methyl)thiazol-2-yl)acetamide (54). N-

(5-(chloromethyl)thiazol-2-yl)acetamide (144 mg, 0.75 mmol) was

added to a stirred solution of 17 (199 mg, 0.63 mmol) and DIPEA

(0.757 mL, 4.39 mmol) in DCE (3.3 mL) at −78 °C. The mixture

was stirred at rt for 30 min. Then, water was added. The organic layer

was separated, dried (MgSO₄), and ﬁltered, and the solvents were

evaporated in vacuo. The crude product was puriﬁed by RP HPLC

(stationary phase: C18 XBridge 70 × 100 mm², 5 μm; mobile phase:

gradient from 84% 0.1% NH₄CO₃H/NH₄OH pH 9 solution in water,

16% CH₃CN to 60% 0.1% NH₄CO₃H/NH₄OH pH 9 solution in

water, 40% CH₃CN). The desired fractions were collected and

concentrated in vacuo to yield 54 as a white solid (109 mg, 44%). ¹H

NMR (400 MHz, DMSO-d₆) δ ppm 2.05−2.19 (m, 5H), 2.38 (s,

3H), 2.58 (br t, J = 7.05 Hz, 2H), 2.76 (s, 2H), 3.74 (s, 2H), 4.37 (br

s, 4H), 5.97 (s, 1H), 7.26 (s, 1H), 8.27 (s, 1H), 11.96 (s, 1H). HRMS

(ESI−) m/z [M − H]⁺calculated for C18H21N8OS: 397.1559;

found: 397.1553.

N-(5-((7-(5-Methylimidazo[1,2-a]pyrimidin-7-yl)-2,7-diazaspiro-

[4.4]nonan-2-yl)methyl)thiazol-2-yl)acetamide (55). N-(5-For-

mylthiazol-2-yl)acetamide (78 mg, 0.46 mmol) and sodium

triacetoxyborohydride (145 mg, 0.49 mmol) were added to a stirred

solution of 11b (98 mg, 0.38 mmol) in DCM (5 mL). The reaction

mixture was stirred at rt for 16 h. Then, the mixture was diluted with

more DCM and basiﬁed with sat Na₂CO₃. The organic layer was

separated and concentrated in vacuo. The crude product was puriﬁed

by ﬂash column chromatography (silica; 7 N solution of NH₃in

MeOH in DCM 0/100 to 10/90). The desired fraction was collected

and concentrated in vacuo, and the residue was triturated with

diisopropyl ether (DIPE) to yield 55 as a yellow solid. ¹H NMR (300

MHz, CDCl₃) δ ppm 1.76−2.13 (m, 8H), 2.33 (s, 3H), 2.51 (s, 3H),

3.43−3.72 (m, 4H), 3.80 (s, 2H), 6.03 (s, 1H), 7.10 (s, 1H), 7.21 (s,

1H), 7.42 (s, 1H), 11.90 (br s, 1H). HRMS (ESI+) m/z [M + H]⁺

calculated for C20H26N7OS: 412.1920; found: 412.1932.

1

DIPE to yield 57 as a yellow solid (36 mg, 50%). H NMR (400

MHz, CDCl₃) δ ppm 1.50−2.08 (m, 4H), 2.29 (s, 3H), 2.33 (s, 3H),

2.40−2.94 (m, 4H), 2.49 (s, 3H), 3.01−3.73 (m, 4H), 3.78 (s, 2H),

5.95 (s, 1H), 7.19 (s, 1H), 11.11 (br s, 1H). HRMS (ESI+) m/z [M +

H]⁺calculated for C19H27N6OS: 387.1967; found: 387.1970.

N-(5-((7-(2,6-Dimethylpyridin-4-yl)-2,7-diazaspiro[4.4]nonan-2-

yl)methyl)thiazol-2-yl)acetamide (58). DIPEA (0.212 mL, 1.23

mmol) was added to a stirred suspension of 11e (57 mg, 0.25

mmol) in DCM (1.3 mL) at rt. The mixture was stirred at rt for 5

min, and then, N-(5-formylthiazol-2-yl)acetamide (50 mg, 0.30

mmol) and sodium triacetoxyborohydride (78 mg, 0.37 mmol)

were added. The reaction mixture was stirred for 16 h more, and then,

a saturated NaHCO₃solution was added. The organic layer was

separated, dried (MgSO₄), and ﬁltered, and the solvents were

evaporated in vacuo. The crude product was puriﬁed by ﬂash column

chromatography (silica; MeOH in DCM 0/100 to 25/75). The

desired fractions were collected and evaporated in vacuo and the

residue was triturated with DIPE to yield 58 as a white solid (38 mg,

40%). ¹H NMR (400 MHz, CDCl₃) δ ppm 1.72−2.07 (m, 4H), 2.30

(s, 3H), 2.40 (s, 6H), 2.53 (dd, J = 49.48, 9.02 Hz, 2H), 2.62−2.84

(m, 2H), 3.11−3.41 (m, 4H), 3.78 (s, 2H), 6.07 (s, 2H), 7.19 (s, 1H).

HRMS (ESI+) m/z [M + H]⁺calculated for C20H28N5OS:

386.2015; found: 386.2014.

N-(5-((7-(2-Methoxy-6-methylpyridin-4-yl)-2,7-diazaspiro[4.4]-

nonan-2-yl)methyl)thiazol-2-yl)acetamide (59). N-(5-formylthiazol-

2-yl)acetamide (1.378 g, 8.1 mmol), sodium cyanoborohydride

(1.388 g, 22.1 mmol), sodium acetate (1.812 g, 22.1 mmol), and

AcOH (0.042 mL, 0.74 mmol) were added to a mixture of 11f (3.5 g,

7.36 mmol) in MeOH (15 mL). The reaction mixture was stirred at rt

for 16 h. The reaction mixture was concentrated. The crude product

was puriﬁed HPLC (Phenomenex Gemini C18 150 × 50 mm², 10

μm, acetonitrile in water (0.05% ammonia hydroxide v/v) 35/65 to

65/35). The pure fractions were collected and the solvent was

evaporated to yield 59 as a yellow solid (82 mg, 3%). ¹H NMR (400

MHz, CDCl₃) δ ppm 1.91−2.04 (m, 3H), 2.28 (s, 3H), 2.35 (s, 3H),

2.44 (br d, J = 9.04 Hz, 1H), 2.57 (d, J = 9.04 Hz, 1H), 2.60−2.68 (m,

1H), 2.72−2.81 (m, 1H), 3.19 (d, J = 9.70 Hz, 1H), 3.25−3.40 (m,

4H), 3.76 (s, 2H), 3.88 (s, 3H), 5.57 (s, 1H), 5.96 (s, 1H), 7.18 (s,

1H), 11.15 (br s, 1H). HRMS (ESI+) m/z [M + H]⁺calculated for

C20H28N5O2S: 402.1964; found: 402.1966.

N-(5-((7-(2-Methyl-6-(triﬂuoromethyl)pyridin-4-yl)-2,7-

diazaspiro[4.4]nonan-2-yl)methyl)thiazol-2-yl)acetamide (60). So-

dium triacetoxyborohydride (148 mg, 0.70 mmol) was added to a

stirred mixture of 11i (100 mg, 0.28 mmol), N-(5-formylthiazol-2-

yl)acetamide (71 mg, 0.42 mmol), and Et₃N (0.058 mL, 0.42 mmol)

in DCM (1 mL) and MeOH (1 mL) at rt. The mixture was stirred at

rt for 16 h. Then, the mixture was concentrated in vacuo and the

residue was puriﬁed by ﬂash column chromatography (silica; 7 N

ammonia solution in MeOH in DCM 0/100 to 5/95). The desired

fractions were collected and concentrated in vacuo to yield 60 as a

pale-yellow solid (78 mg, 64%). ¹H NMR (400 MHz, CDCl₃) δ ppm

1.88 (br d, J = 6.47 Hz, 2H), 1.96−2.14 (m, 2H), 2.30 (s, 3H), 2.48

(s, 4H), 2.56−2.92 (m, 3H), 3.24 (d, J = 9.71 Hz, 1H), 3.30−3.43

(m, 3H), 3.80 (br s, 2H), 6.31 (d, J = 1.85 Hz, 1H), 6.56 (d, J = 2.31

Hz, 1H), 7.21 (br s, 1H), 11.41 (br s, 1H). HRMS (ESI+) m/z [M +

H]⁺calculated for C20H25F3N5OS: 440.1732; found: 440.1734.

N-(5-((7-(4,6-Dimethylpyridin-2-yl)-2,7-diazaspiro[4.4]nonan-2-

yl)methyl)thiazol-2-yl)acetamide (61). A mixture of 11g (129 mg,

0.41 mmol), N-(5-formylthiazol-2-yl)acetamide (91 mg, 0.54 mmol),

and sodium acetate (101 mg, 1.24 mmol) in MeOH (7.3 mL) was

stirred at rt for 90 min. Then, sodium cyanoborohydride (36 mg, 0.58

mmol) and AcOH (0.056 mL, 0.99 mmol) were added and the

mixture was further stirred at rt for 16 h. The volatiles were

N-(5-((7-(2-Methylquinolin-4-yl)-2,7-diazaspiro[4.4]nonan-2-yl)-

methyl)thiazol-2-yl)acetamide (56). Sodium triacetoxyborohydride

(256 mg, 1.21 mmol) was added to a stirred suspension of 11c (214

mg, 0.80 mmol) and N-(5-formylthiazol-2-yl)acetamide (153 mg,

0.90 mmol) in DCM (8 mL). The mixture was stirred at rt for 60 h.

The mixture was basiﬁed with a saturated solution of NaHCO₃and

extracted with DCM/2-PrOH (9/1). The organic layer was separated,

dried (MgSO₄), and ﬁltered, and the solvents were evaporated in

vacuo. The crude product was puriﬁed by ﬂash column chromatog-

raphy (silica; 7 N solution of ammonia in MeOH in DCM 1/99 to

10/90) and by RP HPLC (stationary phase: C18 XBridge 30 × 100

mm², 5 μm; mobile phase: gradient from 85% NH₄HCO₃0.25%

solution in water, 15% CH₃CN to 55% NH₄HCO₃0.25% solution in

water, 45% CH₃CN). The desired fractions were collected and

concentrated in vacuo to yield 56 (197 mg, 58%) as a pale-yellow

foam. A puriﬁcation was performed via chiral SFC (stationary phase:

CHIRALPAK AD-H 5 μm 250 × 21.2 mm²; mobile phase: 55% CO₂,

45% MeOH (0.3% iPrNH₂)), yielding (+)-56 (81 mg, 24%) and

1

(−)-56 (70 mg, 21%) as pale-yellow solids. H NMR (400 MHz,

CDCl₃) δ ppm 1.84−2.14 (m, 5H), 2.29 (s, 3H), 2.53−2.61 (m, 2H),

2.55−2.67 (m, 1H), 2.64−2.86 (m, 3H), 3.56−3.76 (m, 4H), 3.79 (s,

2H), 6.35 (s, 1H), 7.20 (s, 1H), 7.27−7.34 (m, 1H), 7.56 (ddd, J =

8.32, 6.94, 1.16 Hz, 1H), 7.91 (d, J = 8.32 Hz, 1H), 8.13 (dd, J = 8.55,

0.92 Hz, 1H), 10.63−10.93 (m, 1H). HRMS (ESI+) m/z [M + H]⁺

20

calculated for C23H28N5OS: 422.2015; found: 422.2014. [α]

=

D

−32.75° (589 nm, c = 0.8 w/v %, MeOH, 20 °C). [α] _D²⁰= +34.88°

(589 nm, c 0.553 w/v %, MeOH, 20 °C).

N-(5-((7-(2,6-Dimethylpyrimidin-4-yl)-2,7-diazaspiro[4.4]nonan-

2-yl)methyl)thiazol-2-yl)acetamide (57). DIPEA (0.160 mL, 0.93

mmol) was added to a stirred suspension of 11d (50 mg, 0.19 mmol)

in DCM (1 mL) at rt. The mixture was stirred at rt for 5 min, and

then, N-(5-formylthiazol-2-yl)acetamide (38 mg, 0.22 mmol) and

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evaporated in vacuo. The residue thus obtained was puriﬁed by RP

HPLC (stationary phase: C18 XBridge 30 × 100 mm², 5 μm; mobile

phase: gradient from 80% NH₄HCO₃0.25% solution in water, 20%

CH₃CN to 0% NH₄HCO₃0.25% solution in water, 100% CH₃CN).

The desired fractions were collected and the volatiles were evaporated

CH₃CN to 56% NH₄HCO₃0.25% solution in water, 44% CH₃CN).

The desired fractions were collected and extracted with EtOAc. The

organic layer was separated, dried (MgSO₄), and ﬁltered and the

solvents were removed in vacuo to yield 64 as a colorless oil (26 mg,

42%). ¹H NMR (CDCl₃, 400 MHz) δ ppm 1.85 (t, J = 7.1 Hz, 2H′),

2.06 (t, J = 5.3 Hz, 2H), 2.17−2.08 (m, 2H + 2H′), 2.32 (s, 3H + 3

H′), 2.35−2.43 (m, 2H + 2H′), 2.47 (s, 6H + 6H′), 2.59 (s, 2H′),

2.62 (t, J = 7.2 Hz, 2H′), 2.68 (t, J = 6.8 Hz, 2H), 2.74 (s, 2H), 3.20−

3.29 (m, 1H′), 3.31−3.40 (m, 1H), 3.75 (s, 2H′), 3.80 (s, 2H), 6.74

(s, 2H + 2H′), 7.20 (s, 1H′), 7.23 (s, 1H), 12.42 (s, 1H + 1H′).

HRMS (ESI+) m/z [M + H]⁺calculated for C₂₀H₂₇N₄OS: 371.1906;

found: 371.1902.

1

in vacuo to yield 61 as a yellow foam (40 mg, 25%). H NMR (400

MHz, CDCl₃) δ ppm 1.76−2.06 (m, 7H), 2.19 (s, 3H), 2.31 (s, 3H),

2.35 (s, 3H), 2.51−2.61 (m, 2H), 2.65−2.77 (m, 2H), 3.31−3.55 (m,

4H), 3.77 (d, J = 1.16 Hz, 2H), 5.96 (s, 1H), 6.24 (s, 1H), 7.20 (s,

1H), 12.23 (br s, 1H). HRMS (ESI+) m/z [M + H]⁺calculated for

C20H28N5OS: 386.2015; found: 386.2051.

N-(5-((7-(2,6-Dimethylpyridin-4-yl)-2-azaspiro[4.4]nonan-2-yl)-

methyl)thiazol-2-yl)acetamide (62). N-(5-formylthiazol-2-yl)-

acetamide (59 mg, 0.35 mmol) and sodium triacetoxyborohydride

(183 mg, 0.87 mmol) were added to a stirred solution of 21 (66 mg,

0.29 mmol) in DCM (4.4 mL), and the reaction mixture was stirred at

room temperature for 18 h. Then, a saturated solution of NaHCO₃

was added and the product was extracted with DCM. The organic

layer was separated, dried (Na₂SO₄), and ﬁltered, and the solvents

were evaporated in vacuo. The residue was puriﬁed by RP HPLC

(stationary phase: XBridge C18 50 × 100 mm², 5 μm; mobile phase:

gradient from 73% NH₄HCO₃0.25% solution in water, 27% CH₃CN

to 56% NH₄HCO₃0.25% solution in water, 44% CH₃CN). The

desired fractions were collected and extracted with EtOAc. The

organic layer was separated, dried (MgSO₄), and ﬁltered and the

solvents were removed in vacuo to yield 62 as a colorless oil (70 mg,

63%). ¹H NMR (400 MHz, CDCl₃) δ ppm 1.60 (m, 2H + 2H′), 1.64

(m, 2H′), 1.68 (m, 2H), 1.72−1.79 (m, 2H + 2H′), 1.79−1.88 (m,

2H + 2H′), 2.05 (m, 2H + 2H′), 2.29 (s, 3H + 3H′), 2.44 (s, 6H +

6H′), 2.47 (m, 2H′), 2.53 (m, 2H), 2.67 (m, 2H + 2H′), 2.90 (m,

1H′), 3.00 (m, 1H′), 3.76 (s, 2H + 2H′), 6.77 (s, 2H + 2H′), 7.18 (s,

1H + 1H′), 12.80 (br s, 1H + 1H′). HRMS (ESI+) m/z [M + H]⁺

calculated for C21H29N4OS: 385.2062; found: 385.2061.

N-(5-((3-(2,6-Dimethylpyridin-4-yl)-1-oxa-7-azaspiro[4.4]nonan-

7-yl)methyl)thiazol-2-yl)acetamide (63). Et₃N (0.056 mL, 0.40

mmol) was added to a stirred solution of 25 (23 mg, 0.10 mmol)

in DCM (1.5 mL), and the mixture was stirred at rt for 10 min. Then,

N-(5-formylthiazol-2-yl)acetamide (20 mg, 0.12 mmol) and sodium

triacetoxyborohydride (64 mg, 0.30 mmol) were added and the

reaction mixture was stirred at rt for 18 h. Then, a saturated solution

of NaHCO₃was added and the product was extracted with DCM.

The organic layer was separated, dried (Na₂SO₄), and ﬁltered, and the

solvents were evaporated in vacuo. The residue was puriﬁed by RP

HPLC (stationary phase: XBridge C18 50 × 100 mm², 5 μm; mobile

phase: gradient from 73% NH₄HCO₃0.25% solution in water, 27%

CH₃CN to 56% NH₄HCO₃0.25% solution in water, 44% CH₃CN).

The desired fractions were collected and extracted with EtOAc. The

organic layer was separated, dried (MgSO₄), and ﬁltered and the

solvents were removed in vacuo to yield 63 as a colorless oil (21 mg,

55%). ¹H NMR (400 MHz, CDCl3) δ ppm 1.87 (m, 1H′), 1.94−2.10

(m, 2H + 2H′), 2.10−2.21 (m, 1H), 2.30 (s, 3H′), 2.31 (s, 3H), 2.33

(dd, J = 9.9, 5.1 Hz, 1H′), 2.42 (dd, J = 12.6, 7.8 Hz, 1H), 2.47 (s,

6H′), 2.48 (s, 6H), 2.65 (d, J = 9.6 Hz, 1H′), 2.69 (m, 2H′), 2.75 (dd,

J = 9.9, 4.3 Hz, 1H + 1H′), 2.77−2.86 (m, 2H), 2.89 (d, J = 10.1 Hz,

1H), 3.28 (m, 1H′), 3.37 (m, 1H), 3.66−3.77 (m, 1H + 1H′), 3.82 (s,

2H + 2H′), 4.13 (m, 1H + 1H′), 6.80 (s, 2H + 2H′), 7.23 (s, 1H +

1H′), 12.50 (s, 1H + 1H′). HRMS (ESI+) m/z [M − H]⁺calculated

for C₂₀H₂₅N₄O₂: 385.1698; found: 385.1699.

N-(5-((7-((2,6-Dimethylpyridin-4-yl)methyl)-2-azaspiro[4.4]-

nonan-2-yl)methyl)thiazol-2-yl)acetamide (65). To a solution of 35

(28 mg, 0.11 mmol) in DCM (1.6 mL) under a N₂atmosphere was

added N-(5-formylthiazol-2-yl)acetamide (23 mg, 0.13 mmol) and

stirred at room temperature for 30 min. Then, sodium triacetoxybor-

ohydride (36 mg, 0.17 mmol) was added and stirred at rt for 16 h.

The mixture was treated with sat NaHCO₃and extracted with DCM.

The organic layer was separated, dried (MgSO₄), and ﬁltered and the

solvents were evaporated in vacuo to yield 65 as a colorless oil (24

1

mg, 53%). H NMR (500 MHz, CDCl₃) δ ppm 1.14−1.43 (m, 2H),

1.59−1.90 (m, 6H), 2.02−2.20 (m, 1H), 2.30 (s, 3H), 2.43−2.65 (m,

10H), 2.80 (td, J = 7.08, 2.89 Hz, 2H), 3.88 (d, J = 2.31 Hz, 2H),

6.26−6.46 (m, 1H), 6.76 (s, 2H), 7.26 (s, 1H), 7.35−7.35 (m, 1H),

8.40 (s, 1H), 10.33−13.93 (m, 1H). HRMS (ESI−) m/z [M − H]⁺

calculated for C22H29N4OS: 397.2062; found: 397.2063.

N-(5-((2-((2,6-Dimethylpyridin-4-yl)methyl)-6-azaspiro[3.4]-

octan-6-yl)methyl)thiazol-2-yl)acetamide (Cis-66 and Trans-66).

Et₃N (0.199 mL, 1.43 mmol) was added to a stirred solution of 41

(95 mg, 0.36 mmol) in DCM (5.4 mL), and the mixture was stirred at

rt for 10 min. Then, N-(5-formylthiazol-2-yl)acetamide (73 mg, 0.43

mmol) and sodium triacetoxyborohydride (227 mg, 1.07 mmol) were

added and the reaction mixture was stirred at rt for 18 h. Then, a

saturated solution of NaHCO₃was added and the product was

extracted with DCM. The organic layer was separated, dried

(Na₂SO₄), and ﬁltered, and the solvents were evaporated in vacuo.

The residue was puriﬁed by RP HPLC (stationary phase: XBridge

C18 50 × 100 mm², 5 μm; mobile phase: gradient from 73%

NH₄HCO₃0.25% solution in water, 27% CH₃CN to 56% NH₄HCO₃

0.25% solution in water, 44% CH₃CN). The desired fractions were

collected and extracted with EtOAc. The organic layer was separated,

dried (MgSO₄), and ﬁltered and the solvents were removed in vacuo

to yield cis-66 (19 mg, 14%) and trans-66 (18 mg, 13%) as

1

transparent oils that precipitate upon standing. H NMR (500 MHz,

CDCl₃) δ ppm 1.71−1.77 (m, 2H), 1.92 (t, J = 6.79 Hz, 2H), 2.02−

2.09 (m, 2H), 2.31 (s, 3H), 2.38−2.48 (m, 1H), 2.47 (s, 6H), 2.53−

2.59 (m, 2H), 2.54−2.61 (m, 2H), 2.63 (t, J = 6.94 Hz, 2H), 3.75 (s,

2H), 6.70 (s, 2H), 7.20 (s, 1H), 12.44 (br s, 1H). HRMS (ESI+) m/z

[M − H]⁺calculated for C21H27N4OS: 383.1906; found: 383.1910.

¹H NMR (400 MHz, CDCl₃) δ ppm 1.64−1.75 (m, 2H), 1.83 (t, J =

7.05 Hz, 2H), 2.07−2.16 (m, 2H), 2.31 (s, 3H), 2.26−2.39 (m, 1H),

2.47 (s, 6H), 2.53−2.65 (m, 6H), 3.75 (s, 2H), 6.70 (s, 2H), 7.20 (s,

1H), 12.52 (br s, 1H). HRMS (ESI+) m/z [M − H]⁺calculated for

C21H27N4OS: 383.1906; found: 383.1906.

N-(5-((3-((2,6-Dimethylpyridin-4-yl)oxy)-1-oxa-7-azaspiro[4.4]-

nonan-7-yl)methyl)thiazol-2-yl)acetamide (cis-67 and trans-67).

Et₃N (0.25 mL, 1.80 mmol) was added to a stirred solution of 43

(112 mg, 0.45 mmol) in DCM (6.8 mL), and the mixture was stirred

at rt for 10 min. Then, N-(5-formylthiazol-2-yl)acetamide (92 mg,

0.54 mmol) and sodium triacetoxyborohydride (287 mg, 1.35 mmol)

were added and the reaction mixture was stirred at rt for 18 h. Then, a

saturated solution of NaHCO₃was added and the product was

extracted with DCM. The organic layer was separated, dried

(Na₂SO₄), and ﬁltered, and the solvents were evaporated in vacuo.

The residue was puriﬁed by RP HPLC (stationary phase: XBridge

C18 50 × 100 mm², 5 μm; mobile phase: gradient from 73%

NH₄HCO₃0.25% solution in water, 27% CH₃CN to 56% NH₄HCO₃

0.25% solution in water, 44% CH₃CN). The desired fractions were

collected and extracted with EtOAc. The organic layer was separated,

N-(5-((2-(2,6-Dimethylpyridin-4-yl)-6-azaspiro[3.4]octan-6-yl)-

methyl)thiazol-2-yl)acetamide (64). Et₃N (0.063 mL, 0.45 mmol)

was added to a stirred solution of 29 (24 mg, 0.11 mmol) in DCM

(1.7 mL), and the mixture was stirred at rt for 10 min. Then, N-(5-

formylthiazol-2-yl)acetamide (23 mg, 0.14 mmol) and sodium

triacetoxyborohydride (72 mg, 0.34 mmol) were added and the

reaction mixture was stirred at rt for 18 h. Then, a saturated solution

of NaHCO₃was added and the product was extracted with DCM.

The organic layer was separated, dried (Na₂SO₄), and ﬁltered, and the

solvents were evaporated in vacuo. The residue was puriﬁed by RP

HPLC (stationary phase: XBridge C18 50 × 100 mm², 5 μm; mobile

phase: gradient from 73% NH₄HCO₃0.25% solution in water, 27%

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dried (MgSO₄), and ﬁltered and the solvents were evaporated in

vacuo to yield cis-67 (30 mg, 16%) and trans-67 (24 mg, 13%) as

clear transparent oils that precipitate upon standing. H NMR (500

water, 19% CH₃CN to 64% 0.1% NH₄CO₃H/NH₄OH pH 9 solution

in water, 36% CH₃CN). The desired fractions were collected and

concentrated in vacuo to yield 70 as a colorless oil (75 mg, 35%). ¹H

NMR (400 MHz, CDCl₃) δ ppm 1.76−1.92 (m, 3H), 1.97 (t, J = 6.94

Hz, 1H), 2.05−2.19 (m, 2H), 2.30 (d, J = 2.31 Hz, 3H), 2.42 (d, J =

1.39 Hz, 6H), 2.55−2.68 (m, 5H), 3.74 (d, J = 8.09 Hz, 2H), 3.86 (t,

J = 6.13 Hz, 2H), 6.44 (d, J = 3.47 Hz, 2H), 7.16−7.22 (m, 1H),

12.77 (br s, 1H). HRMS (ESI+) m/z [M + H]⁺calculated for

C21H29N4O2S: 401.2011; found: 401.2011.

1

MHz, CDCl₃) δ ppm 1.93 (dt, J = 13.29, 7.80 Hz, 1H), 2.06−2.14

(m, 1H), 2.19−2.29 (m, 2H), 2.30 (s, 3H), 2.44 (s, 6H), 2.65−2.74

(m, 2H), 2.74−2.81 (m, 2H), 3.80 (s, 2H), 3.93 (dd, J = 10.40, 1.73

Hz, 1H), 4.14 (dd, J = 10.40, 4.91 Hz, 1H), 4.90−4.95 (m, 1H), 6.40

(s, 2H), 7.21 (s, 1H), 12.53 (br s, 1H). HRMS (ESI+) m/z [M + H]⁺

calculated for C20H27N4O3S: 403.1804; found: 403.1803. ¹H NMR

(500 MHz, CDCl₃) δ ppm 1.98 (dt, J = 13.29, 7.66 Hz, 1H), 2.11−

2.20 (m, 2H), 2.26−2.31 (m, 1H), 2.31 (s, 3H), 2.46 (s, 6H), 2.65−

2.70 (m, 1H), 2.70 (d, J = 9.83 Hz, 1H), 2.76−2.83 (m, 2H), 3.77−

3.86 (m, 2H), 3.98 (dd, J = 10.40, 1.73 Hz, 1H), 4.10 (dd, J = 10.40,

5.20 Hz, 1H), 4.90−4.95 (m, 1H), 6.42 (s, 2H), 7.23 (s, 1H), 12.42

(br s, 1H). LC-MS m/z 401.1 [M − H]⁺.

OGA Biochemical Assay. The assay is based on the inhibition of

the hydrolysis of ﬂuorescein mono-β-^D-N5 Acetyl-Glucosamine (FM-

GlcNAc) (Mariappa et al., 2015. Biochem. J. 470:255) by the

recombinant human Meningioma Expressed Antigen 5 (MGEA5),

also referred to as O-GlcNAcase (OGA). The hydrolysis FM-GlcNAc

(Marker Gene technologies, cat # WO 2018/141984 PCT/EP2018/

052901 5 10 15 20 25 30 35 -99- M1485) results in the formation of

β-^D-N-glucosamineacetate and ﬂuorescein. The ﬂuorescence of the

latter can be measured at excitation wavelength 485 nm and emission

wavelength 538 nm. An increase in enzyme activity results in an

increase in the ﬂuorescence signal. Full-length OGA enzyme was

purchased at OriGene (cat #TP322411). The enzyme was stored in

25 mM Tris-HCI, pH 7.3, 100 mM glycine, 10% glycerol at −20 °C.

Thiamet-G and GlcNAcStatin were tested as reference compounds

(Yuzwa et al., 2008. Nature Chemical Biology 4:483; Yuzwa et al.,

2012. Nature Chemical Biology 8:393). The assay was performed in

200 mM citrate/phosphate buﬀer supplemented with 0.005% Tween-

20. Then, 35.6 g of Na₂HPO₄·2H₂O (Sigma, #C0759) was dissolved

in 1 L of water to obtain a 200 mM solution. Next, 19.2 g of citric acid

(Merck, #1.06580) was dissolved in 1 L of water to obtain a 100 mM

solution; the pH of the sodium phosphate solution was adjusted with

the citric acid solution to 7.2. The buﬀer to stop the reaction consists

of a 500 mM carbonate buﬀer, pH 11.0. Then, 734 mg of FM-GlcNAc

was dissolved in 5.48 mL of DMSO to obtain a 250 mM solution,

which was stored at −20 °C. OGA was used at 2 nM concentration

and FM-GlcNAc at a 100 μM ﬁnal concentration. Dilutions were

prepared in an assay buﬀer. Then, 50 nL of a compound dissolved in

DMSO was dispensed on Black Proxiplate TM 384 Plus Assay plates

(Perkin Elmer, #6008269), and 3 μL of the ﬂ-OGA enzyme mix was

added subsequently. Plates were preincubated for 60 min at room

temperature, and then, 2 μL of the FM-GlcNAc substrate mix was

added. Final DMSO concentrations did not exceed 1%. Plates were

brieﬂy centrifuged for 1 min at 1000 rpm and incubated at room

temperature for 6 h. To stop the reaction, 5 μL of the STOP buﬀer

was added and plates were centrifuged again for 1 min at 1000 rpm.

Fluorescence was quantiﬁed in the Thermo Scientiﬁc Fluoroskan

Ascent or the PerkinElmer EnVision with excitation wavelength 485

nm and emission wavelength 538 nm. For analysis, a best-ﬁt curve is

ﬁtted by a minimum sum of squares method. From this, an IC₅₀value

and a Hill coeﬃcient were obtained. High control (no inhibitor) and

low control (saturating concentrations of the standard inhibitor) were

used to deﬁne the minimum and maximum values.

OGA: Cellular Assay. HEK293 cells inducible for P301L mutant

human Tau (isoform 2N4R) were established at Janssen. Thiamet-G

was used both for plate validation (high control) and as a reference

compound (reference EC₅₀assay validation). OGA inhibition is

evaluated through the immunocytochemical (ICC) detection of O-

GlcNAcylated proteins by the use of a monoclonal antibody

(CTD110.6; Cell Signaling, #9875) detecting O-GlcNAcylated

residues as previously described (Dorfmueller et al., 2010. Chem.

Biol. 17:1250). Inhibition of OGA will result in an increase of O-

GlcNAcylated protein levels resulting in an increased signal in the

experiment. Cell nuclei are stained with Hoechst to give a cell culture

quality control and a rough estimate of immediate compound toxicity,

if any. ICC pictures are imaged with a Perkin Elmer Opera Phenix

plate microscope and quantiﬁed with the provided software Perkin

Elmer Harmony 4.1. Cells were propagated in Dulbecco’s modiﬁed

Eagle’s medium (DMEM) high glucose (Sigma, #D5796) following

standard procedures. Two days before the cell assay, cells were split,

counted, and seeded in poly-^D-Lysine (PDL)-coated 96-well (Greiner,

#655946) plates at a cell density of 12 000 cells per cm²(4000 cells

Rac-N-(5-((2-((2,6-dimethylpyridin-4-yl)oxy)-6-azaspiro[3.4]-

octan-6-yl)methyl)thiazol-2-yl)acetamide (Cis-68 and Trans-68).

N-(5-Formylthiazol-2-yl)acetamide (75 mg, 0.44 mmol) and sodium

triacetoxyborohydride (232 mg, 1.10 mmol) were added to a stirred

solution of 45 (85 mg, 0.37 mmol) in DCM (1.7 mL). The mixture

was stirred at rt for 18 h. Then, a saturated solution of NaHCO₃was

added and the product was extracted with DCM. The organic layer

was separated, dried (MgSO₄), and ﬁltered, and the solvents were

evaporated in vacuo. The product was puriﬁed by ﬂash chromatog-

raphy (silica; MeOH in DCM 0/100 to 5/95). The desired fractions

were collected and the solvents were removed in vacuo to yield cis-68

1

(34 mg, 24%) and trans-68 (50 mg, 35%) as light-yellow solids. H

NMR (400 MHz, CDCl₃) δ ppm 1.97 (t, J = 6.82 Hz, 2H), 2.14−2.23

(m, 2H), 2.30 (s, 3H), 2.46 (s, 6H), 2.45−2.54 (m, 2H), 2.68 (t, J =

6.80 Hz, 2H), 2.69 (s, 2H), 3.76 (s, 2H), 4.65 (quin, J = 6.82 Hz,

1H), 6.38 (s, 2H), 7.20 (s, 1H), 11.78 (br s, 1H). HRMS (ESI+) m/z

[M + H]⁺calculated for C20H27N4O2S: 387.18547; found:

1

387.1857. H NMR (400 MHz, CDCl₃) δ ppm 1.95 (t, J = 7.17

Hz, 2H), 2.11−2.21 (m, 2H), 2.32 (s, 3H), 2.46 (s, 6H), 2.55 (ddd, J

= 9.94, 7.05, 2.89 Hz, 2H), 2.60−2.68 (m, 4H), 3.80 (s, 2H), 4.54

(quin, J = 6.94 Hz, 1H), 6.37 (s, 2H), 7.23 (s, 1H), 11.77 (br s, 1H).

HRMS (ESI+) m/z [M + H]⁺calculated for C20H27N4O2S:

387.1855; found: 387.1858.

N-(5-((7-(((2,6-Dimethylpyridin-4-yl)oxy)methyl)-2-azaspiro[4.4]-

nonan-2-yl)methyl)thiazol-2-yl)acetamide (69). N-(5-Formylthia-

zol-2-yl)acetamide (53 mg, 0.31 mmol) and sodium triacetoxybor-

ohydride (166 mg, 0.78 mmol) were added to 48 (68 mg, 0.26 mmol)

in DCM (4 mL), and the reaction mixture was stirred at rt for 18 h.

Then, a saturated solution of NaHCO₃was added and the mixture

was extracted with DCM. The organic layer was separated, dried

(Na₂SO₄), ﬁltered, and the solvents were evaporated in vacuo. The

crude was puriﬁed by ﬂash column chromatography (silica; MeOH in

DCM 0/100 to 6/94). The desired fractions were collected and

1

evaporated in vacuo to yield 69 as a colorless oil (56 mg, 96%). H

NMR (400 MHz, CDCl₃) δ ppm 1.30−1.50 (m, 2H + 2H′), 1.54−

1.68 (m, 2H + 2H′), 1.71 (t, J = 7.0 Hz, 2H′), 1.78 (t, J = 7.0 Hz,

2H), 1.84 (m, 1H′), 1.92 (m, 1H), 2.29 (s, 3H′), 2.30 (s, 3H), 2.36

(m, 2H + 2H′), 2.43 (s, 6H′), 2.44 (s, 6H + 2H′), 2.51 (d, J = 3.1 Hz,

2H), 2.64 (s, 1H + 1H′), 2.65 (m, 2H + 2H′), 3.75 (s, 2H + 2H′),

3.79−3.85 (m, 2H + 2H′), 6.44 (s, 2H′), 6.46 (s, 2H), 7.19 (s, 1H +

1H′), 12.64 (s, 1H + 1H′). HRMS (ESI+) m/z [M + H]⁺calculated

for C22H31N4O2S: 415.2168; found: 415.2166.

N-(5-((2-(((2,6-Dimethylpyridin-4-yl)oxy)methyl)-6-azaspiro[3.4]-

octan-6-yl)methyl)thiazol-2-yl)acetamide (70). Et₃N (0.3 mL, 2.16

mmol) was added to a stirred solution of 50 (153 mg, 0.54 mmol) in

DCM (8.2 mL), and the mixture was stirred at rt for 10 min. Then, N-

(5-formylthiazol-2-yl)acetamide (110 mg, 0.65 mmol) and sodium

triacetoxyborohydride (343 mg, 1.62 mmol) were added and the

reaction mixture was stirred at rt for 18 h. Then, a saturated solution

of NaHCO₃was added and extracted with DCM. The organic layer

was separated, dried (Na₂SO₄), and ﬁltered, and the solvents were

evaporated in vacuo. The crude was puriﬁed by ﬂash column

chromatography (silica; MeOH in DCM 0/100 to 6/94) and by RP

HPLC (stationary phase: C18 XBridge 30 × 100 mm², 5 μm; mobile

phase: gradient from 81% 0.1% NH₄CO₃H/NH₄OH pH 9 solution in

W

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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Article

per well) in 100 μL of assay medium (low-glucose medium is used to

reduce basal levels of GlcNAcylation) (Park et al., 2014. J. Biol.

Chem. 289:13519). On the day of compound test medium from assay

plates was removed and replenished with 90 μL of fresh assay

medium. Then, 10 μL of compounds at a 10-fold ﬁnal concentration

was added to the wells. Plates were centrifuged shortly before

incubation in the cell incubator for 6 h. The DMSO concentration

was set to 0.2%. The medium was discarded by applying a vacuum.

For staining of cells, the medium was removed and cells were washed

once with 100 μL of Dulbecco’s phosphate-buﬀered saline (D-PBS;

Sigma, #D8537). From the next step onward, unless otherwise stated,

the assay volume was always 50 μL and incubation was performed

without agitation and at room temperature. Cells were ﬁxed in 50 μL

of a 4% paraformaldehyde (PFA, Alpha Aesar, #043368) PBS solution

for 15 min at room temperature. The PFA PBS solution was then

discarded and cells were washed once in 10 mM Tris Buﬀer

(LifeTechnologies, #15567-027), 150 mM NaCl (LifeTechnologies,

#24740-0110), 0.1% Triton X (Alpha Aesar, #Al6046), pH 7.5 (ICC

buﬀer) before being permeabilized in the same buﬀer for 10 min.

Samples were subsequently blocked in ICC containing 5% goat serum

(Sigma, #G9023) for 45−60 min at room temperature. Samples were

then incubated with a primary antibody (1/1000 from a commercial

provider; see above) at 4 °C overnight and subsequently washed three

times for 5 min in an ICC buﬀer. Samples were incubated with a

secondary ﬂuorescent antibody (1/500 dilution, Lifetechnologies, #A-

21042), and nuclei were stained with Hoechst 33342 at a ﬁnal

concentration of 1 μg/mL in ICC (Lifetechnologies, #H3570) for 1 h.

Before analysis, samples were washed two times manually for 5 min in

an ICC base buﬀer. Imaging was performed using Perkin Elmer

Phenix Opera using a water 20× objective and recording nine ﬁelds

per well. Intensity readout at 488 nm was used as a measure of the O-

GlcNAcylation level of total proteins in wells. To assess the potential

toxicity of compounds, nuclei were counted using Hoechst staining.

IC₅₀values were calculated using parametric nonlinear regression

model ﬁtting. As a maximum inhibition, Thiamet-G at a 200 μM

concentration was present on each plate. In addition, a concentration

response of Thiamet-G was calculated on each plate.

In Vitro Permeability/P-gp Eﬄux Assay. The in vitro passive

permeability and the ability to be a transported substrate of P-

glycoprotein (P-gp) were tested at Cyprotex using MDCK cells stably

transduced with MDR1. Permeability experiments were conducted in

duplicate at a single concentration (5 μM) in a transwell system with

an incubation of 120 min. The apical-to-basolateral (A to B) transport

in the presence and absence of the P-gp inhibitor GF120918 and the

basolateral-to-apical (B to A) transport in the absence of the P-gp

inhibitor were measured, and permeation rates (apparent perme-

ability) of the test compounds (P_app× 10⁻⁶cm/s) were calculated.

Microsomal Metabolic Stability Assay. The metabolic stability

of a test compound was tested at Cyprotex using liver microsomes

(0.5 mg/mL protein) from human and preclinical species incubated

up to 60 min at 37 °C with a 1 μM test compound. The in vitro

metabolic half-life (t_1/2) was calculated using the slope of the log-

linear regression from the percentage parent compound remaining

versus time relationship. The in vitro intrinsic clearance (Cl_int) (mL/

min/mg microsomal protein) was derived from the half-life taking

into account the incubation volume and the weight of the microsomal

protein in the incubation.

PK Assay. All in vivo experimental procedures were performed

according to the applicable Directive 2010/63/EU of the European

Parliament and of the Council of September 22, 2010, on the

protection of animals used for scientiﬁc purposes and approved by the

local ethical committee. The pharmacokinetics (PK) and oral

bioavailability of (+)-56 were investigated in male CD1 mice dosed

at 1 mg/kg iv and 30 mg/kg PO in water. For each arm, three animals

were used. Animals had free access to food and water through each

study. For iv dosing, (+)-56 was dissolved in saline 0.9%, and for po, it

was dissolved in water adjusted to approximately pH 4 and

administered as a solution. Blood samples were taken at 0.117,

0.333, 1, 2, 4, 7, and 24 h for iv dosing and at 0.5, 1, 2, 4, 7, and 24 h

for po. Plasma was prepared by protein precipitation with acetonitrile,

and the supernatant was analyzed for concentrations of (+)-56 using a

qualiﬁed liquid chromatography−tandem mass spectrometry (LC-

MS/MS) method.

ASSOCIATED CONTENT

■

sı

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01479.

Details of the HPLC methods and purity determination;

HPLC chromatograms of all ﬁnal compounds; NMR

spectra for cis and trans-66, 67, and 68; X-ray

crystallography of compound 54 with CpOGA and of

55 with OgOGA; protein production, crystallization,

data processing, and structure solution; crystal structures

of CpOGA with 54 and of OgOGA with 55 have been

deposited in the protein databank with accession codes

7KHV and 7KHS, respectively (PDF)

Molecular formula strings (CSV)

AUTHOR INFORMATION

■

Corresponding Authors

Carlos M. Martínez-Viturro − Discovery Chemistry, Janssen

Research & Development, 45007 Toledo, Spain;

orcid.org/0000-0002-0241-6689;

Phone: +34925245763; Email: cmmartin@its.jnj.com

́

Andres A. Trabanco − Discovery Chemistry, Janssen Research

& Development, 45007 Toledo, Spain; orcid.org/0000-

0002-4225-758X; Phone: +34925245779;

Email: atrabanc@its.jnj.com

Gary Tresadern − Computational Chemistry, Janssen

Research & Development, Janssen Pharmaceutica NV, 2440

Beerse, Belgium; orcid.org/0000-0002-4801-1644;

Phone: +3214641569; Email: gtresade@its.jnj.com

́

Jose M. Bartolome-Nebreda − Discovery Chemistry, Janssen

Research & Development, 45007 Toledo, Spain;

Phone: +34925245778; Email: jbartolo@its.jnj.com

Authors

Jordi Royes − Department Química Física i Inorgànica,

University Rovira i Virgili, 43007 Tarragona, Spain;

orcid.org/0000-0001-6129-7344

CYP450 Inhibition Assay. The potential to reversibly inhibit the

major human P450 isoforms (CYPs 1A2, 2C9, 2C19, 2D6, and 3A4)

was determined in the CYP inhibition cocktail assay. Test compounds

were incubated across a concentration range (ﬁve concentrations)

with human liver microsomes and probe substrates for each of the six

isoforms (CYP1A2, 2C8, 2C9, 2C19, and 2D6 in the cocktail format

and 3A4 in the single probe format) to estimate the IC₅₀for inhibition

of the probe substrate by the test compound.

After the deﬁned incubation time, the reaction was quenched and

samples were centrifuged and prepared for analysis by LC/MS. The

percentage inhibition of the probe substrate metabolite formation was

plotted against inhibitor concentration and an IC₅₀calculated by

curve ﬁtting.

́

Elena Fernandez − Department Química Física i Inorgànica,

University Rovira i Virgili, 43007 Tarragona, Spain;

orcid.org/0000-0001-9025-1791

Juan A. Vega − Discovery Chemistry, Janssen Research &

Development, 45007 Toledo, Spain

Alcira del Cerro − Discovery Chemistry, Janssen Research &

Development, 45007 Toledo, Spain

Francisca Delgado − Discovery Chemistry, Janssen Research

& Development, 45007 Toledo, Spain

X

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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(3) Scheltens, P.; Blennow, K.; Breteler, M. M. B.; de Strooper, B.;

Frisoni, G. B.; Salloway, S.; Van der Flier, W. M. Alzheimer ’s disease.

Lancet 2016, 388, 505−517.

́

Aranzazu García Molina − Discovery Chemistry, Janssen

Research & Development, 45007 Toledo, Spain

Fulgencio Tovar − Villapharma Research S.L., Parque

Tecnológico de Fuente Álamo, 30320 Fuente Álamo de

Murcia, Spain

(4) What is Alzheimer’s? https://www.alz.org/alzheimers-dementia/

what-is-alzheimers. (accessed May 26, 2020).

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J. Med. Chem. 2012, 55, 8977−8978.

Paul Shaﬀer − X-Ray Crystallography, Janssen Pharmaceutical

Research & Development, Spring House, Pennsylvania

19477, United States

Andreas Ebneth − Neuroscience Discovery, Janssen Research

& Development, Janssen Pharmaceutica NV, 2440 Beerse,

Belgium

Alexis Bretteville − Neuroscience Discovery, Janssen Research

& Development, Janssen Pharmaceutica NV, 2440 Beerse,

Belgium

Liesbeth Mertens − Neuroscience Discovery, Janssen Research

& Development, Janssen Pharmaceutica NV, 2440 Beerse,

Belgium

Marijke Somers − Discovery DMPK, Janssen Research &

Development, Janssen Pharmaceutica NV, 2440 Beerse,

Belgium

Jose M. Alonso − Analytical Sciences, Janssen Research &

Development, 45007 Toledo, Spain

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Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c01479

Author Contributions

The manuscript was written through contributions of all

authors. All authors have given approval to the ﬁnal version of

the manuscript.

(14) Ksiezak-Reding, H.; Liu, W. K.; Yen, S. H. Phosphate analysis

and dephosphorylation of modified tau associated with paired helical

filaments. Brain Res. 1992, 597, 209−219.

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Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

We thank the analytical department in Toledo for helping with

compound characterization and puriﬁcation. We also thank

Jose M. Cid for his helpful discussions during the manuscript

writing.

(17) Lee, V. M.; Goedert, M.; Trojanowski, J. Q. Neurodegenerative

tauopathies. Annu. Rev. Neurosci. 2001, 24, 1121−1159.

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ABBREVIATIONS

■

BID, “bis in die” (twice a day); B₂(Pin)₂, bis(pinacolato)-

diboron; Boc, tert-butyloxycarbonyl; DAST, diethylaminosul-

fur triﬂuoride; DIPEA, N,N-diisopropylethylamine; dppf,

(diphenylphosphino)ferrocene; ESI, electrospray ionization;

HAT, histone acetyltransferases; Het, heteroaryl; HMDS,

hexamethyldisilazane; HRMS, high-resolution mass spectra;

iv, intravenous; MDCK-MDR1, Madin Darby canine kidney-

multidrug resistance-1; LM Clint, liver microsomes intrinsic

clearance; O-GlcNAcase (OGA), O-linked N-acetyl-^D-gluco-

(19) Wani, W. Y.; Chatham, J. C.; Darley-Usmar, V.; McMahon, L.

L.; Zhang, J. O-GlcNAcylation and neurodegeneration. Brain Res. Bull.

2017, 133, 80−87.

(20) Lombard, V.; Golaconda Ramulu, H.; Drula, E.; Coutinho, P.

M.; Henrissat, B. The carbohydrate-active enzymes database (CAZy)

in 2013. Nucleic Acids Res. 2014, 42, 490−495.

(21) Vocadlo, D. J. O-GlcNAc processing enzymes: catalytic

mechanisms, substrate specificity, and enzyme regulation. Curr.

Opin. Chem. Biol. 2012, 16, 488−497.

(22) Hart, G. W.; Housley, M. P.; Slawson, C. Cycling of O-linked

β -N-acetylglucosamine on nucleocytoplasmic proteins. Nature 2007,

446, 1017−1022.

sam i na se; r ac-BI N AP,

( ) - B I N A P , 2 , 2 ′- b i s -

(diphenylphosphino)-1,1′-binaphthalene; PgP, permeability

glycoprotein; PO, from the Latin “per os”, by mouth; RP

HPLC, reverse-phase high-performance liquid chromatogra-

phy; SNAr, aromatic nucleophilic substitution; TBABr, tetra-n-

butylammonium bromide; TFA, triﬂuoroacetic acid

(23) Gao, Y.; Wells, L.; Comer, F. I.; Parker, G. J.; Hart, G. W.

Dynamic O-glycosylation of nuclear and cytosolic proteins: cloning

and characterization of a neutral, cytosolic β -N-acetylglucosaminidase

from human brain. J. Biol. Chem. 2001, 276, 9838−9845.

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neurodegeneration. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 17319−

17320.

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Diazaspirononane Nonsaccharide Inhibitors of O-GlcNAcase (OGA) for the Treatment of Neurodegenerative Disorders

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