Design and Synthesis of Novel Spiro Derivatives as Potent and Reversible Monoacylglycerol Lipase (MAGL) Inhibitors: Bioisosteric Transformation from 3-Oxo-3,4-dihydro-2 H-benzo[ b][1,4]oxazin-6-yl Moiety

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Design and Synthesis of Novel Spiro Derivatives as Potent and

Reversible Monoacylglycerol Lipase (MAGL) Inhibitors: Bioisosteric

Transformation from 3‑Oxo-3,4-dihydro‑2H‑benzo[b][1,4]oxazin-6-yl

Moiety

Shuhei Ikeda,* Hideyuki Sugiyama, Hidekazu Tokuhara, Masataka Murakami, Minoru Nakamura,

Yuya Oguro, Jumpei Aida, Nao Morishita, Satoshi Sogabe, Douglas R. Dougan, Sean C. Gay, Ling Qin,

Naoto Arimura, Yasuko Takahashi, Masako Sasaki, Yusuke Kamada, Kazunobu Aoyama, Kouya Kimoto,

and Makoto Kamata

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Supporting Information

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ABSTRACT: The therapeutic potential of monoacylglycerol

lipase (MAGL) inhibitors in central nervous system-related

diseases has attracted attention worldwide. However, the

availability of reversible-type inhibitor is still limited to clarify the

pharmacological eﬀect. Herein, we report the discovery of novel

spiro chemical series as potent and reversible MAGL inhibitors

with a diﬀerent binding mode to MAGL using Arg57 and His121.

Starting from hit compound 1 and its co-crystal structure with

MAGL, structure-based drug discovery (SBDD) approach enabled

us to generate various spiro scaﬀolds like 2a (azetidine-lactam), 2b

(cyclobutane-lactam), and 2d (cyclobutane-carbamate) as novel

bioisosteres of 3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl

moiety in 1 with higher lipophilic ligand eﬃciency (LLE).

Optimization of the left hand side aﬀorded 4f as a promising reversible MAGL inhibitor, which showed potent in vitro MAGL

inhibitory activity (IC₅₀6.2 nM), good oral absorption, blood−brain barrier penetration, and signiﬁcant pharmacodynamic changes

(2-arachidonoylglycerol increase and arachidonic acid decrease) at 0.3−10 mg/kg, po. in mice.

potential for various related diseases such as Parkinson’s

and Alzheimer’s diseases, multiple sclerosis, depression, and

epilepsy.^15,16

INTRODUCTION

■

The endocannabinoid signaling system, naturally modulated

by two endocannabinoids (ECs), 2-arachidonoylglycerol (2-

AG) and N-arachidonoylethanolamine (anandamide), via the

activation of cannabinoid receptors (CB₁and CB₂), regulates

numerous physiological processes such as pain perception,

appetite, learning and memory, and reward behaviors.¹CB₁

receptor is highly expressed in the central nervous system

(CNS) and less so in peripheral tissues, whereas CB₂

receptor is predominantly expressed in immune cells,

including microglia in the brain. Among ECs, 2-AG, a

major contributor in the brain,²⁻⁴is degraded to arachidonic

acid (AA) and glycerol primarily by monoacylglycerol lipase

(MAGL),^5,6a serine hydrolase abundant in brain tissue

(Figure 1A).⁷⁻⁹Therefore, MAGL inhibition causes an

elevation in 2-AG levels as well as a depletion in AA levels

in the brain, which enhances endocannabinoid signaling and

causes antineuroinﬂammatory eﬀects.¹⁰Based on these

observations, intensive investigations on MAGL inhibition

have been conducted¹¹⁻¹⁴to determine its therapeutic

Two types of MAGL inhibitors have been reported to date

(Figure 1B). Among them, irreversible ones such as

compound a (JZL184) and compound b (ABX-1431) have

been extensively used to conﬁrm the beneﬁcial biological

eﬀects of MAGL inhibition on CNS disease pathologies and

ABX-1431 has been evaluated in human clinical trials.¹⁷⁻²⁰In

the investigations, it was reported that the chronic blockade

of MAGL using a genetic method or chemical one with

JZL184 caused undesired responses such as pharmacological

tolerance and CB₁receptor desensitization.²⁰Thus, reversible

Received: March 10, 2021

American Chemical Society

https://doi.org/10.1021/acs.jmedchem.1c00432

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A

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Figure 1. (A) Enzymatic reaction by MAGL. (B) Representative MAGL inhibitors, irreversible type: compounds a (JZL184)²⁰and b (ABX-

1431);¹⁷reversible type: compounds c (PDB ID 3PE6),²⁹d (PDB ID 5ZUN),²¹and e (predicted complex).^22aMajor amino acids in MAGL

catalytic site are illustrated in blue, which form polar interactions with compounds c−e.

Figure 2. HTS hit 1 and co-crystal structure in the active site of MAGL (yellow, PDB ID 7L4T).

inhibitors have been expected to circumvent these potential

disadvantages and some chemical series represented by

compounds c,²⁹d,²¹and e^22awere reported to show potent

in vitro MAGL inhibitory activities as well as eﬃcacy in

models of neuropathic and inﬂammatory pain.²¹⁻²⁵Regarding

compounds c and d, their co-crystal structures with human

MAGL well illustrate the key binding features for the potent

activities. That is, the central amide carbonyl group recruits

Met123 and Ala51, the right half of the molecules forms

hydrogen bonds with Arg57, His272, and Glu53 in a direct or

water-mediated manner, and the left half occupies the

binding pocket by their rather lipophilic structures. In spite

of these great inventions, as far as we know, none of the

reversible inhibitors entered the clinical trial. Therefore,

further expansion of the chemical space in reversible

inhibitors is considered necessary to clarify the positive

pharmacological eﬀects of MAGL inhibition.

Based on these backgrounds, we aimed at generating novel,

potent, and reversible MAGL inhibitors, which show diﬀerent

binding modes to the target utilizing structure-based drug

B

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Figure 3. LLE/SBDD-based ligand optimization strategy.

Table 1. First Stage of RHS Optimization

a

b

Inhibitory activity against human MAGL enzyme. IC₅₀values and 95% conﬁdence intervals (in parentheses) were calculated from duplicate

measurements using a four-parameter logistic model. Measured at pH 7.4.²⁷LLE = pIC₅₀− log D.²⁶MDR1 eﬄux ratios in P-gp-overexpressing

cells at 1 μM of substrate.

c

d

e

discovery (SBDD) approach. To obtain a high-quality MAGL

inhibitor considering its physicochemical properties, we also

used lipophilic ligand eﬃciency (LLE)²⁶as an indicator

throughout our research because this strategy could provide a

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Figure 4. (A) Spiro formation from compound 1h and pK_atuning strategy. (B) RHS in the co-crystal structure of compound 1h with MAGL

(yellow, PDB ID 7L4U).

selective ligand to MAGL without too much addition of

lipophilicity, which often causes nonspeciﬁc bindings to oﬀ-

targets. Herein, we report an LLE/SBDD-based lead

generation/optimization approach from hit compound 1,

which leads to the discovery of (2s,4s)-2-((3-((3-chloro-4-

methylbenzyl)oxy)azetidin-1-yl)carbonyl)-7-oxa-5-azaspiro-

[3.4]octan-6-one (4f) as a novel, potent, and reversible

MAGL inhibitor.

The analysis on the co-crystal structure inspired us to think

of the ligand optimization strategy as follows (Figure 3). The

essential central carbonyl group should be kept in the

designed structure to maintain MAGL inhibitory activity. In

addition, LLE should be monitored to see if we are moving

in the right direction toward the ﬁnal target range of 5−7,

which is considered as an optimal range using oral drug data

analysis.²⁶As a ﬁrst step, the RHS optimization was planned

by SBDD because the two hydrogen bonds with His121 and

Arg57 would be the key contributors to the potency and LLE

of compound 1, considering the strength of the polar

interactions.³⁰For the RHS, we designed a cyclic unit with

amide functionality and a linker (L¹) that can interact with

His121 and Arg57. It was assumed that a tactful linker (L¹)

design by SBDD would be the key to enhance LLE. In the

second stage, arrangement of the physicochemical properties

was planned via modiﬁcation of the LHS moiety while

maintaining LLE. As indicated in the co-crystal structure, the

wider space around the LHS moiety would allow us to

investigate various types of LHS design, including diﬀerent

ring sizes of the amine portion, various types of linkers (L²),

and substituents on the benzene ring.

Optimization of RHS Using SBDD. In the ﬁrst step of

RHS optimization, we tried to enhance LLE by incorporating

an N atom into the benzene ring and removing the benzene

moiety itself (Table 1). The structural modiﬁcation of the

benzene spacer was expected to be tolerable because no

beneﬁcial molecular interaction was observed in the co-crystal

structure of 1 with MAGL. The synthesized compounds were

narrowed down to a promising lead candidate in light of LLE

and MDR1 eﬄux ratio (MDR BA/AB, human) as a surrogate

of blood−brain barrier (BBB) permeability.³¹Compared to

that of compound 1 (IC₅₀= 15 nM, LLE = 4.5), pyridine

derivatives 1a−c showed a slight loss of MAGL inhibitory

activity (IC₅₀= 26−32 nM), but all showed the LLE

enhancement (4.6−5.0). Among them, 1a displayed the

highest LLE (5.0) as well as an acceptable MDR1 eﬄux ratio

(1.8) less than 2.³¹

RESULTS AND DISCUSSION

■

Using high-throughput screening (HTS), we identiﬁed

compound 1, with potent MAGL inhibitory activity (IC₅₀

=

15 nM) and moderate LLE (LLE²⁶= pIC₅₀− log D²⁷= 4.5;

Figure 2). The crystal structures of human MAGL have been

disclosed as apo form and in complex with reversible and

irreversible inhibitors.^21,28,29Our co-crystallization experi-

ments successfully yielded high-quality crystals and the

density maps contained clear features of 1 in the active site

of MAGL. Examination of the co-crystal structure revealed a

novel binding mode to MAGL and critical aspects of

interaction as follows. The central amide carbonyl oxygen

atom points into the oxyanion hole²⁹and forms hydrogen

bonds with the backbone amide NHs from residues Ala51

and Met123 adjacent to the catalytic Ser122, suggesting a

noncovalent reversible mode of inhibition. On the right-hand

side (RHS) moiety, 3-oxo-3,4-dihydro-2H-benzo[b][1,4]-

oxazin-6-yl, the carbonyl oxygen atom forms a hydrogen

bond with the side chain of Arg57 and the NH group forms a

hydrogen bond with the side chain of His121. These two

direct hydrogen bonds formed between our ligand and

MAGL protein are unique considering the other binding

modes of MAGL inhibitors reported so far. On the left-hand-

side (LHS) moiety, ((2-chloro-4-ﬂuorophenoxy)methyl)-

piperidine, it packs a relatively wider region with the

lipophilic substituents. These eﬀective binding interactions

well explain the strong inhibitory activity of compound 1

(IC₅₀= 15 nM).

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Figure 5. Docking models of the designed spiro compounds using co-crystal structure of 1h (PDB ID 7L4U) as a template. (A) Spiroazetidine

2a (gray), (B) (2r,4s)-spirocyclobutane 2b (magenta), and (C) (2s,4r)-spirocyclobutane 2c (cyan).

Table 2. Second Stage of RHS Optimization

a

b

Inhibitory activity against human MAGL enzyme. IC₅₀values and 95% conﬁdence intervals (in parentheses) were calculated from duplicate

measurements using the four-parameter logistic model. Measured at pH 7.4.²⁷LLE = pIC₅₀− log D.²⁶MDR1 eﬄux ratios in P-gp-

overexpressing cells at 1 μM of substrate. pK_avalues were calculated by ACD Labs. Not determined.

c

d

e

f

g

By removing the RHS benzene ring from 1, the resultant

morpholinone derivative 1d showed a 10-fold decrease in

potency (IC₅₀= 1100 nM), whereas the lactam derivative 1e

also showed a 4-fold decreased potency (IC₅₀= 63 nM) but

maintained the LLE (4.6) in comparison with that of 1.

These data suggested that the ﬁxation of the terminal amide

portion by the benzene spacer would be critical to form the

hydrogen bonds with His121 and Arg57 eﬀectively. Between

E

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Figure 6. (A) LHS optimization strategy from compound 2d. (B) Co-crystal structure of 2d with MAGL (yellow, PDB ID 7L4W).

1d and 1e, the more rigid ﬁve-membered ring conformation

of 1e could aﬀord less discrepancy between the active

binding conformation and the most stable ligand conforma-

tion. Replacing the ethylene spacer of 1e with oxymethylene

one decreased potency (1f, IC₅₀= 190 nM) and LLE (3.8).

One reason for this result would be a slight conformational

change by changing the spacer which aﬀects the formation of

the hydrogen bonds with His121 and Arg57. In addition, the

decreased electron density on the O atom in the central

carbonyl group might be another reason by the trans-

formation from amide 1e to carbamate 1f, where the

hydrogen bonds would be formed with Ala51 and Met123.

After the chiral separation of 1e, we obtained compound 1h

as the eutomer (S-form) which showed potent MAGL

inhibitory activity (IC₅₀= 38 nM) with a high LLE (4.8) and

an acceptable MDR1 eﬄux ratio (1.5). The co-crystal

structure of 1h with MAGL assured the formation of two

hydrogen bonds with His121 and Arg57 by its lactam portion

(Figure 4B).

Thus, we identiﬁed two types of lead candidates such as 1a

and 1h. Although 1a exhibited slightly better LLE (5.0 vs

4.8), we selected 1h as the lead compound for further

optimization because 1h had a greater potential to boost LLE

than 1a using the SBDD approach (Figure 4A). More

speciﬁcally, the co-crystal structure inspired us to design a

spiro formation at its ethylene spacer to enhance potency and

LLE by ﬁxing the active conformation of 1h (Figure 4B). In

addition, the acidity increase (pK_adecrease) in the NH group

was expected to strengthen the hydrogen bond with His121

by replacing amide with carbamate.

appropriate distances and angles (Figure 5A,B). However,

(2s,4r)-spirocyclobutane 2c, stereoisomer of 2b, was

suggested to lose the interaction with His121 in a plausible

active conformation (Figure 5C).

Next, we evaluated the synthesized compounds 2a−f

(Table 2). As simulated in the docking studies, compounds

2a (IC₅₀= 11 nM, LLE = 5.3) and 2b (IC₅₀= 6.5 nM, LLE

= 5.4) showed drastic increases in potency and LLE from 1h,

whereas 2c (IC₅₀= 890 nM) showed a sharp drop of

potency. The stereochemistries of 2b and 2d were conﬁrmed

by 2D NMR analyses. Between 2a and 2b, we selected 2b

because it showed acceptable MDR1 eﬄux ratio (1.8) and

then further modiﬁed its lactam portion to the cyclic

carbamate of 2d, following our pK_atuning strategy. As

expected, potency and LLE boosts were observed in 2d (IC₅₀

= 3.4 nM, LLE = 5.9) which was possibly due to a stronger

interaction with His121 formed by the more acidic hydrogen.

Actually, the pK_aof the carbamate NH group in 2d is

calculated as 12.2 which is lower than 16.1 in lactam 2b.

Finally, we concluded that the four-membered spiro spacer

was optimal (n = 1, 2a: IC₅₀= 11 nM) considering the

potencies of ﬁve-membered (n = 2, 2e: IC₅₀= 73 nM) and

six-membered (n = 3, 2f: IC₅₀> 10000 nM) compounds,

which indicated that the compact azetidine spacer ﬁxed the

active conformation of 1h in an eﬃcient manner to form the

two key interactions with His121 and Arg57.

Optimization of LHS to Arrange the Physicochem-

ical Properties. Through in vitro proﬁling of compound 2d,

human ether-a-go-go (hERG) liability was found to be

addressed (autopatch clamp, 91% inhibition at 10 μM

without bovine serum albumin [BSA]). Since the co-crystal

structure of 2d with MAGL also indicated a wider space

around the LHS portion like hit compound 1, we conducted

the optimization of amine portion, linker moiety (L), and the

substituents on the benzene ring as initially planned (Figure

6A,B). Regarding the terminal aryl ring, it was assumed that

Before taking on the synthesis, we conducted docking

studies of the designed spiro compounds (2a−c) using the

co-crystal structure of 1h (Figure 5). As a result, it was

suggested that spiroazetidine 2a and (2r,4s)-spirocyclobutane

2b could eﬃciently bind to the active site of MAGL by

forming two hydrogen bonds with His121 and Arg57 at

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Table 3. First Stage of LHS Optimization at the Spacer Portion

a

b

Inhibitory activity against human MAGL enzyme. IC₅₀values and 95% conﬁdence intervals (in parentheses) were calculated from duplicate

measurements using the four-parameter logistic model. Measured at pH 7.4.²⁷LLE = pIC₅₀− log D.²⁶MDR1 eﬄux ratios in P-gp-

overexpressing cells at 1 μM of substrate. Autopatch clamp of hERG inhibition at 10 μM of substrate.

c

d

e

f

the benzene one should be selected exclusively because our

ligand already included two amide moieties as well as one

hydrogen bond donor. That is, an extra hetero atom

incorporation was not appropriate in this chemical space to

obtain a potent MAGL inhibitor with good BBB perme-

ability.³²During the optimization phase, we primarily aimed

at the improvement of hERG liability while keeping high LLE

and good MDR1 eﬄux ratio.

The incorporation of a methyl group at the 4-position in

the piperidine ring increased the potency (3a, IC₅₀= 2.7

nM), but strong hERG inhibition remained (80.0% at 10

μM; Table 3). Interestingly, the transposition of the methyl

group to the adjacent methylene linker led to a signiﬁcant

decrease in hERG liability while maintaining the potency (3b,

eutomer, IC₅₀= 2.7 nM, hERG = 43.3% at 10 μM). This

tendency was also observed in the other branched

compounds with diﬀerent sizes of the substituent (3c:

ethyl, eutomer; and 3d: triﬂuoromethyl, eutomer).

Replacing oxymethylene linker in 2d with methyleneoxy

one caused a slight loss of potency, but the hERG liability

showed a sign of improvement (3e, IC₅₀= 11 nM, hERG =

66.4% at 10 μM). Eventually, we combined these two linkers

(oxymethylene and methyleneoxy) with the diﬀerent amine

rings (pyrrolidine and azetidine) to aﬀord compounds 3f−i.

As a result, a drastic improvement of hERG liability in 3f and

3i was observed (3f: 18.6% at 10 μM, 3i: 20.1% at 10 μM),

compared to 91.0% at 10 μM in lead compound 2d. As in

the case of 3e, methyleneoxy linker showed decreased hERG

liability in each pair of the molecules, pyrrolidines 3f (18.6%)

and 3g (68.1%), azetidines 3h (33.7%) and 3i (20.1%).

Between 3f and 3i, compound 3i was selected for the next

stage because of its better LLE (5.5) and MDR1 eﬄux ratio

(1.8).

In the second stage of LHS optimization (Table 4), the

eﬀect of the substituents at the 4-position of the benzene ring

with 2-Cl substituent was initially investigated (4a−c).

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Table 4. Second Stage of LHS Optimization at the Terminal Benzene Ring

a

b

Inhibitory activity against human MAGL enzyme. IC₅₀values and 95% conﬁdence intervals (in parentheses) were calculated from duplicate

measurements using the four-parameter logistic model. Measured at pH 7.4.²⁷LLE = pIC₅₀− log D.²⁶MDR1 eﬄux ratios in P-gp-

overexpressing cells at 1 μM of substrate. Autopatch clamp of hERG inhibition at 10 μM of substrate.

c

d

e

f

Compared to 3i (IC₅₀= 34 nM), potency enhancement was

observed as the lipophilicity of the substituent increased (Me

of 4a: IC₅₀= 18 nM < Cl of 4b: IC₅₀= 7.0 nM < CF₃of 4c:

IC₅₀= 5.4 nM), which suggested that the substituent at the

4-position was important for the modulation of MAGL

inhibitory activity. Therefore, we also evaluated the potency

H

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Table 5. In Vitro Proﬁle Summary of 4f

g

b

physicochemical property

selectivity

binding proﬁles to MAGL

b

M_w= 364.8

log D = 2.3 LLE = 5.9 for MAGL

MAGL IC₅₀(nM) = 6.2 (5.6−6.9)

K_D(nM) = 1.5 ( 0.33)

c

FAAH IC₅₀(nM) > 10000

k_on(M⁻¹s⁻¹) = 2.5 × 10⁶( 2.3×10⁵)

k_off(s⁻¹) = 0.0036 ( 0.0006)

t_1/2(min) = 3.3

d

ABHD6 IC₅₀(nM) > 10000

e

3

F_sp= 0.56

hERG inh. at 10 μM = 14.4%

CB₁inh. 19% at 10 μM CB₂inh. 5% at 10 μM

a

f

aqueous solubility = 330 μg/mL

a

b

Measured in JP2 (pH 6.8) at 37 °C, JP2: second dissolution ﬂuid in the Japanese Pharmacopoeia.³⁴Inhibitory activity against human

recombinant MAGL. IC₅₀values and 95% conﬁdence intervals (in parentheses) were calculated from duplicate measurements using the four-

parameter logistic model. Inhibitory activity against human recombinant FAAH.³⁵Inhibitory activity against human ABHD6. Manual patch

c

d

e

f

clamp of hERG inhibition at 10 μM of substrate. Inhibitory activity against human cannabinoid receptors at 10 μM of substrate. The assay was

performed by Euroﬁns Panlabs Taiwan, Ltd. (https://www.euro ﬁ nsdiscoveryservices.com/). K_D, k_on, and k_offvalues were derived from surface

g

plasmon resonance (SPR) experiments. The values were expressed as mean S.D. (n = 6).

changes with 3,4-disubstituted analogs. The translocation of

the Cl atom of 4c (IC₅₀= 5.4 nM, LLE = 5.6) to the 3-

(t_1/2) of 3.3 min, which indicated that 4f is a reversible

MAGL inhibitor. Furthermore, the reversibility of 4f from

MAGL was tested using the method of the dilution assay

previously reported.²¹Recovery of the enzymatic activity after

rapid dilution was measured after incubation of MAGL in the

absence (control) or presence of 4f. The activity recovery

rate was comparable to that without the inhibitor. On the

other hand, the activity recovery was much slower when

using an irreversible inhibitor, JZL184. The result also

supports that 4f is a reversible inhibitor. Finally, the co-

crystal structure of 4f was obtained successfully and it

displayed a similar binding mode to that of 2d (Supporting

Information, Figure S2).

In Vivo Proﬁles of 4f. The eﬀect of 4f on the increase in

the level of 2-AG and the decrease in that of AA in the

mouse brain was evaluated at 1 h after the oral administration

of 0.1−10 mg/kg doses (4f: mouse MAGL IC₅₀[nM] = 13).

Compound 4f displayed linear pharmacokinetics in plasma

and dose-dependent pharmacokinetics in the brain with good

BBB penetration, which would be supported by its excellent

solubility and acceptable MDR1 eﬄux ratio (Table 6).

position aﬀorded 4d with enhanced potency and LLE (IC₅₀

=

3.2 nM, LLE = 6.1). Encouraged by this positive result, other

3,4-disubstituted compounds were synthesized in the

combination with Me, Cl, and CF₃substituent. As a result,

all of the synthesized compounds 4e−k showed the higher

LLEs (5.8−6.0) in comparison with 3i (LLE = 5.5). Among

them, 4f (3-Cl, 4-Me) provided the most balanced proﬁles in

terms of potency (IC₅₀= 6.2 nM), LLE (5.9), MDR eﬄux

ratio (1.2), and hERG liability (18.1% at 10 μM). Thus,

compound 4f was selected for further detailed proﬁling.

In Vitro Proﬁles of 4f. The low hERG liability (18.1% at

10 μM) of 4f in the screening autopatch clamp assay was

supported by the manual patch clamp assay (14.4% inhibition

at 10 μM), which suggested a low risk of cardiovascular QTc

prolongation (Table 5). To conﬁrm the MAGL selectivity of

4f, the inhibitory activities of the most closely related serine

hydrolases, such as fatty acid amide hydrolase (FAAH)¹¹and

α,β-hydrolase domain 6 (ABHD6),⁶were also evaluated.

Compound 4f exhibited no inhibition of these enzymes (IC₅₀

> 10000 nM), which indicated that 4f would enhance

endocannabinoid signaling mostly by the increase in the level

of 2-AG via selective MAGL inhibition in the brain. In

addition, the oﬀ-target binding potential to cannabinoid

receptors (CB₁and CB₂) were evaluated at Euroﬁns Panlabs

Taiwan, Ltd. Compound 4f showed less than 50% inhibition

at 10 μM (CB₁: 19% and CB₂: 5%), which suggested that

compound 4f would not interfere the endocannabinoid

signaling in a direct manner.

Table 6. Plasma/Brain Concentration of 4f in Mice at 0.1−

a

10 mg/kg, po.

concentration (ng/mL or ng/g)

dose (mg/kg)

plasma

brain

0.1

0.3

1

3

10

17

62

163

457

1515

31

99

169

322

711

Regarding the physicochemical properties of 4f, remarkable

thermodynamic aqueous solubility (330 μg/mL, pH 6.8) was

observed, which would be beneﬁcial in terms of preclinical in

vivo evaluation and clinical development. This feature is well

a

Evaluated at 1 h after oral administration (n = 4).

explained by its F_sprich (F_sp= 0.56) structure³³and low

lipophilicity (log D = 2.3) which came from our LLE/SBDD-

oriented strategy. In particular, the removal of the extra

3

Consistent with the brain concentration, compound 4f

resulted in a signiﬁcant elevation in the level of 2-AG and

reduction in that of AA from 0.3 mg/kg (Figure 7). These in

vitro and in vivo results suggested that compound 4f could

be a suitable agent for the validation of diverse therapeutic

applications exerted by the reversible MAGL inhibition.

3

aromatic ring from hit 1 (F_sp= 0.33, log D = 3.3) and spiro

3

design for lead 2d (F_sp= 0.58, log D = 2.6) would be the key

steps to reach a chemical structure such as 4f.

The binding proﬁle of 4f with MAGL using surface

plasmon resonance (SPR) experiments showed a dissociation

rate constant (k_off) of 0.0036 s⁻¹and dissociation half-life

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Figure 7. PD dose−response of 4f in C57BL/6J mice (8W), 1 h after oral administration at 0.1−10 mg/kg, po. (A) 2-AG level in the mouse

#

brain. (B) AA level in the mouse brain. Data are presented as the mean standard error of the mean (SEM) (n = 4); p < 0.025 vs control by

Williams test.

a

Scheme 1. Synthesis of 1a−h/2a−f in Table 1 or Table 2

a

Reagents and conditions: (a) (1) RHS acids, EDCI, HOBt, DIPEA, DMF, or MeCN, rt, 2 hovernight, 46−96% for 1a−e, 2b, and 2d, 56% in

three steps from 19 for 2b; (2) chiral separation by preparative SFC, 49% for 1g, 48% for 1h; (b) 5-(hydroxymethyl)pyrrolidin-2-one or amine 32,

4-nitrophenyl chloroformate, Et₃N, THF, 0 °C to rt or 0 °C to reﬂux, overnight, 42% for 1f and 5% for 2f; (c) Amine 28 or 1,7-

diazaspiro[4.4]nonan-2-one hydrochloride, triphosgene, DIPEA, MeCN, −10 °C to rt, 2.5−4 h, 26% for 2a and 31% for 2e.

were obtained by preparative chiral high-performance liquid

chromatography (HPLC) or supercritical ﬂuid chromatog-

raphy (SFC).

CHEMISTRY

■

Scheme 1 shows the synthesis of compounds 1a−h and 2a−f

in Table 1 or Table 2. Compounds 1a−e, 2b, and 2d were

synthesized by amidation of amine 35 with the corresponding

RHS acids using EDCI and HOBt. Chiral compounds 1g and

1h were obtained by preparative chiral SFC of 1e.

Compounds 1f and 2f were synthesized via 4-nitrophenyl

carbamate of 35 followed by addition of 5-(hydroxymethyl)-

pyrrolidin-2-one or amine 32 in the presence of Et₃N.

Compounds 2a and 2e were synthesized via the formation of

a reactive carbamate of 35 with triphosgene and DIPEA,

followed by addition of amine 28 or 1,7-diazaspiro[4.4]-

nonan-2-one hydrochloride.

Scheme 3 shows the synthesis of RHS acids 6 and 11 for

compounds 1b and 1d in Table 1. Acid 6 was obtained via

oxidation of the known aldehyde 5.³⁶The synthesis of 11

started from N-α-carbobenzoxy-^DL-glutamic acid γ-t-butyl

ester 7. Reduction of the carboxylic acid in 7 by NaBH₄

via mixed acid anhydride gave alcohol 8. The removal of the

Cbz group aﬀorded amino alcohol 9, which was reacted with

chloroacetyl chloride to form morpholinone 10. Finally,

deprotection of the t-butyl ester furnished acid 11.

Scheme 4 shows the synthesis of RHS spirocyclobutane

acids 18, 21, and 24 for the compounds in Tables 2 and 4.

Starting from 3-oxocyclobutanecarboxylic acid derivatives 12a

(t-Bu ester) and 12b (Et ester), the oxo groups were ﬁrst

transformed into nitro groups via a two-step sequence: (1)

Scheme 2 shows the synthesis of compounds 3a−i and

4a−k in Table 3 or Table 4. Basically, they were synthesized

by amidation of the corresponding LHS amines with RHS

acid 24 using EDCI and HOBt. Chiral compounds 3b−d

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a

Scheme 2. Synthesis of 3a−i/4a−k in Table 3 or Table 4

a

Reagents and conditions: (a) Amine 41a, EDCI, HOBt, DIPEA, DMF, rt, 4 h; (2) chiral separation by preparative HPLC, 25% for 3b in three

steps from 23b; (b) (1) LHS amines, EDCI, HOBt, DIPEA, DMF, or MeCN, rt, 3−64 h, 32−89% for 3a, 3e−i, and 4a−k; (2) chiral separation by

preparative SFC or HPLC, 24% for 3c and 17% for 3d in two steps.

a

Scheme 3. Synthesis of RHS Acids 6 and 11

a

Reagents and conditions: (a) NaClO₂, 2-methyl-2-butene, NaH₂PO₄, t-BuOH/H₂O, rt, 3 h, 68%; (b) isobutyl chloroformate, Et₃N, 0 °C, 0.5 h,

then NaBH₄, 0 °C, 0.5 h, 88%; (c) H₂, 10% Pd-C, EtOAc, rt, 6 h, then H₂, 20% Pd(OH)₂-C, EtOAc, rt, 6 h, 100%; (d) chloroacetic chloride, Et₃N,

THF, −78 °C, 0.5 h, then t-BuOK, −78 °C to rt, 2 h, 40%; (e) TFA, rt, overnight, 93%.

oxime formation with hydroxylamine, (2) oxidation with urea

hydrogen peroxide (UHP) and triﬂuoroacetic anhydride

(TFAA), aﬀording nitro compounds 14, 15a, and 15b.

Compound 14 (t-Bu ester derivative) was used as a cis/trans

mixture for the synthesis of target acids 18 and 24. On the

other side, Et ester derivatives 15a (minor single isomer,

stereochemistry not determined) and 15b (major single

isomer, stereochemistry not determined) could be separated

by silica gel column chromatography and used in the next

steps to synthesize 21 and 24, respectively. In light of scale-

up synthesis for 24, t-Bu ester derivative 14 was preferentially

utilized. The spirolactam structures in 18 and 21 were

formed via a three-step sequence as follows: (1) Michael

addition of 14 and 15a to methyl acrylate with 1,8-

diazabicyclo[5.4.0]undec-7-ene (DBU), (2) reduction of the

nitro groups in 16 and 19 with NaBH₄/NiCl₂followed by

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a

Scheme 4. Synthesis of RHS Acids 18, 21, and 24

a

Reagents and conditions: (a) NH₂OH-HCl, NaOAc, NaH₂PO₄, EtOH, reﬂux, 4−18 h, 92% for 13a and 89% for 13b; (b) UHP, Na₂HPO₄,

TFAA, MeCN, −10 °C to 80 °C, 2.5 h, 64%; (c) UHP, Na₂HPO₄, TFAA, MeCN, 80 °C, 2 h, 18% for 15a and 25% for 15b; (d) methyl acrylate,

DBU, 0 °C, 20 min to 1 h, 30% for 16 and 25% for 19; (e) NaBH₄, NiCl₂·6H₂O, then aq. K₂CO₃, −10 °C to rt, 3−7 h, 78% for 17; (f) TFA, 0 °C

to rt, 4 h, 86%; (g) 2 M NaOH, THF/MeOH/H₂O, rt, overnight; (h) 37% aq. formaldehyde, Et₃N, MeCN, 0 °C to rt, 2 h, 52%; (i) (1) H₂(50

bar), Raney Ni, isopropanol, 70 °C, 3 h, 82%, (2) triphosgene, Et₃N, THF, −10 °C to rt, 2.5 h, 81%; (j) TFA, 0 °C to rt, 2 h, 92%; (k) 35% aq.

formaldehyde, Et₃N, MeCN, rt, 1 h, 47%; (l) (1) H₂, 10% Pd-C, MeOH/THF, rt, 72 h (2) triphosgene, DIPEA, THF, −10 °C, 0.5 h, 51% in two

steps; (m) 2 M NaOH, THF/MeOH, rt, 1 h.

intramolecular cyclization with K₂CO₃, and (3) cleavage of

the ester groups in 17 and 20. On the other side, the spiro

carbamate structure was formed via a four-step sequence as

follows: (1) Henry reaction of 14 and 15b to formaldehyde,

(2) reduction of the nitro groups in 22a and 22b under

hydrogen atmosphere in the presence of Raney Ni or Pd-C,

(3) reaction of the corresponding aminoalcohols with

triphosgene, (4) cleavage of the t-Bu ester groups in 23a

and 23b. It should be noted that both the Michael addition

and the Henry reaction gave the stereoisomers of 16, 19, 22a,

and 22b which were easily separable by silica gel column

chromatography, respectively.

via similar three-sequence: (1) Michael addition of 25 and 29

to methyl acrylate, (2) reduction of the nitro groups in 26

and 30 with NaBH₄/NiCl₂followed by intramolecular

cyclization with K₂CO₃, (3) cleavage of the Boc groups in

27 and 31.

Scheme 6 shows the synthesis of LHS amine 35 as the

common structure for the compounds in Tables 1 and 2.

Mitsunobu reaction of Boc-4-(hydroxymethyl)piperidine 33

followed by deprotection of Boc group gave 35.

Scheme 7 shows the synthesis of LHS amines 38, 41a−e,

and 44a−c with various linkers for the compounds in Table

3. Nucleophilic aromatic substitution of 1-chloro-2,5-diﬂuor-

obenzene by Boc-4-(hydroxymethyl)-4-methylpiperidine 36

occurred at 2-position in the presence of t-BuOK, followed

by deprotection of Boc group, furnished 38. Compounds

Scheme 5 shows the synthesis of RHS spiroamines 28 and

32 for the compounds in Table 2. They were synthesized

from Boc-3-nitroazetidine 25 and Boc-3-nitropiperidine 29

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Scheme 5. Synthesis of RHS Amines 28 and 32

a

Reagents and conditions: (a) methyl acrylate, K₂CO₃, MeOH, 0 °C or 0 °C to rt, 3−4 h, 53% for 26 and 62% for 30; (b) NaBH₄, NiCl₂·6H₂O,

then aq. K₂CO₃, −10 °C to rt, 3.5−4 h, 76% for 27 and 81% for 31; (c) p-TsOH-H₂O, EtOAc, reﬂux, 3−18 h, 90% for 28 and 99% for 32.

a

Scheme 6. Synthesis of LHS Amine 35

a

Reagents and conditions: (a) 2-chloro-4-ﬂuorophenol, ADDP, PBu₃, 0 °C to rt, 18 h, THF, 95%; (b) 4 M HCl/EtOAc, rt, 14 h, 67%.

41a−e with phenyloxymethylene/phenyloxymethine-type

linkers were synthesized from alcohols 39a−e via a three-

step sequence: (1) sulfonylation by MsCl, Tf₂O, or TsCl with

Et₃N or pyridine, (2) nucleophilic replacement by 2-chloro-4-

ﬂuorophenol with Cs₂CO₃, K₃PO₄, or K₂CO₃, (3) cleavage of

the Boc groups in 40a−e with p-TsOH-H₂O or 4 M HCl in

EtOAc. On the other side, compounds 44a−c with

phenylmethyleneoxy-type linkers were synthesized from

secondary alcohols 42a−c via a two-step sequence: (1)

alkylation with 1-(bromomethyl)-2-chloro-4-ﬂuorobenzene

with NaH, (2) cleavage of the Boc groups in 43a−c with

p-TsOH-H₂O.

Scheme 8 shows the synthesis of LHS amines 46a−g,

48a−c, and 50 for the compounds in Table 4. Compounds

46a−d were synthesized from alcohols 45a−d via a three-

step sequence: (1) sulfonylation with MsCl and Et₃N, (2)

nucleophilic displacement by t-butyl-3-hydroxyazetidine-1-

carboxylate with NaH, and (3) cleavage of Boc group with

p-TsOH-H₂O. Compounds 46e,f were synthesized from

alcohols 45e,f via a three-step sequence: (1) bromination

with PBr₃, (2) nucleophilic displacement by t-butyl-3-

hydroxyazetidine-1-carboxylate with NaH, and (3) cleavage

of Boc group with p-TsOH-H₂O. Compound 46g was

synthesized from alcohol 45b via four-step sequence: (1)

sulfonylation with MsCl and Et₃N, (2) nucleophilic displace-

ment by t-butyl-3-hydroxyazetidine-1-carboxylate with NaH,

(3) methylation by Suzuki coupling, (4) cleavage of Boc

group with p-TsOH-H₂O. Compounds 48a−c were synthe-

sized from chlorides 47a−c via a two-step sequence: (1)

nucleophilic displacement by t-butyl-3-hydroxyazetidine-1-

carboxylate with NaH, (2) cleavage of Boc group with p-

TsOH-H₂O. Compound 50 was synthesized from acid 49 via

four-step sequence: (1) reduction of the carboxylic acid by

BH₃−THF complex, (2) sulfonylation with MsCl and Et₃N,

(3) nucleophilic displacement by t-butyl-3-hydroxyazetidine-

1-carboxylate with NaH, (4) cleavage of Boc group with p-

TsOH-H₂O.

CONCLUSIONS

■

To generate a novel, potent, and reversible MAGL inhibitor,

the SBDD approach as well as monitoring of LLE values

were utilized for lead generation/optimization process. The

co-crystal structure of hit compound 1 (MAGL IC₅₀= 15

nM, LLE = 4.5) with MAGL revealed the unique polar

interactions with the side chains of Arg57 and His121 by the

amide group of 3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-

yl moiety. While maintaining these key interactions,

bioisosteric transformation of 3-oxo-3,4-dihydro-2H-benzo-

[b][1,4]oxazin-6-yl moiety based on SBDD led to the

discovery of novel spiro chemical series (azetidine-lactam

2a, cyclobutane-lactam 2b, and cyclobutane-carbamate 2d)

with higher LLE values than that of hit 1 (5.3, 5.4, and 5.9,

respectively). To improve the hERG liability of the lead

compound 2d, optimization was conducted for the remaining

molecular portion, 4-((2-chloro-4-ﬂuorophenoxy)methyl) pi-

peridine moiety. Modiﬁcation of the ring size of the amine

portion, the oxymethylene linker, and the substituents on the

benzene ring enabled us to identify (2s,4s)-2-((3-((3-chloro-

4-methylbenzyl)oxy)azetidin-1-yl)carbonyl)-7-oxa-5-azaspiro-

[3.4]octan-6-one (4f), which showed potent activity (MAGL

IC₅₀= 6.2 nM), a high LLE (5.9), good selectivity toward

the closely related serine hydrolases (FAAH IC₅₀> 10000

nM and ABHD6 IC₅₀> 10000 nM), no signiﬁcant binding

potentials to cannabinoid receptors (CB₁: 19% and CB₂: 5%

at 10 μM), and low hERG liability (14.4% inh. at 10 μM,

manual patch clamp, without BSA). In vivo proﬁling of 4f in

mice showed good pharmacokinetics, BBB penetration, and

signiﬁcant pharmacodynamic changes (2-AG increase and AA

decrease) at 0.3−10 mg/kg, po. that were consistent with the

dose-dependent brain concentrations of 4f in the brain,

indicating that 4f could be a promising reversible MAGL

inhibitory agent. Further detailed pharmacological character-

ization of 4f will be reported in due course.

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a

Scheme 7. Synthesis of LHS Amines 38, 41a−e, and 44a−c

a

Reagents and conditions: (a) 1-chloro-2,5-diﬂuorobenzene, t-BuOK, THF, 0 °C to rt, overnight, then reﬂux, 4 h, 57%; (b) 2 M HCl/MeOH, rt,

overnight, 92%; (c) (1) MsCl, Et₃N, THF/MeCN, 0 °C to rt, 3−6 days, toward 40a,b; Tf₂O, pyridine, 0 °C to rt, overnight, toward 40c; TsCl,

pyridine, 0 °C to rt, overnight, toward 40d,e, 54-97%; (2) 2-chloro-4-ﬂuorophenol, Cs₂CO₃, DMF, 80 °C, overnight, for 40a−c; 2-chloro-4-

ﬂuorophenol, K₃PO₄, DMF, 80 °C, 2 h, for 40d; 2-chloro-4-ﬂuorophenol, K₂CO₃, DMF, 80 °C, overnight, for 40e, 48−87% for 40a and 40d,e; (d)

p-TsOH-H₂O, EtOAc, 70 °C to reﬂux, 2 h, 73−92% for 41a, and 41d,e; 4 M HCl/EtOAc, rt, 2 h, 43% for 41b and 16% for 41c in two steps; (e) 1-

(bromomethyl)-2-chloro-4-ﬂuorobenzene, NaH, DMF, 0 °C to rt, 16 h, 78−100% for 43a−c; (f) p-TsOH-H₂O, EtOAc, reﬂux, 3 h, 25−76% for

44a−c.

analyte detector (NQAD), or a photodiode array detector. The

EXPERIMENTAL SECTION

■

column was a Capcell Pak C18AQ (50 mm × 3.0 mm I.D.,

General Chemistry Information. All solvents and reagents

were obtained from commercial suppliers and used without further

puriﬁcation. Melting points were determined using a DSC1 system

(Mettler-Toledo International Inc., Greifensee, Switzerland). Proton

Shiseido, Japan) or L-column 2 ODS (30 mm × 2.0 mm I.D., CERI,

Japan) with a temperature of 50 °C and a ﬂow rate of 0.5 mL/min.

Mobile phases A and B under a neutral condition were a mixture of

50 mmol/L ammonium acetate, water, and acetonitrile (1:8:1, v/v/

and carbon nuclear magnetic resonance (¹H NMR, ¹³C NMR)

v) and a mixture of 50 mmol/L ammonium acetate and acetonitrile

spectra were recorded on Bruker (300, 400, or 500 MHz)

(1:9, v/v), respectively. The ratio of mobile phase B was increased

instruments. Chemical shifts are given in parts per million (ppm)

linearly from 5% to 95% over 3 min, 95% over the next 1 min.

downﬁeld from tetramethylsilane (δ) as the internal standard in

deuterated solvent, and coupling constants (J) are in hertz (Hz).

Data are reported as follows: chemical shift, integration, multiplicity

Mobile phases A and B under an acidic condition were a mixture of

0.2% formic acid in 10 mmol/L ammonium formate and 0.2%

formic acid in acetonitrile, respectively. The ratio of mobile phase B

(s = singlet, d = doublet, t = triplet, q = quartet, quin = quintet, m

was increased linearly from 14% to 86% over 3 min, 86% over the

= multiplet, and brs = broad signal), and coupling constants.

next 1 min. The purities of all tested compounds were >95% as

Analytical thin-layer chromatography (TLC) was performed on silica

determined by elemental analyses within 0.4% of the calculated

gel 60 F₂₅₄plates (Merck) or NH TLC plates (Fuji Silysia Chemical

Ltd.). Chromatographic puriﬁcation was performed on silica gel

columns [(Merck Kieselgel 60, 70−230 mesh size or 230−400 mesh

size, Merck) or (Chromatorex NH-DM 1020, 100−200 mesh size)]

or on Purif-Pack (SI or NH, SHOKO SCIENTIFIC). LCMS

analysis was performed on Shimadzu UFLC/MS (Prominence

UFLC high-pressure gradient system/LCMS-2020) or Agilent LC/

MS system (Agilent 1200SL/Agilent 6130MS), operating in ESI (+

or −) or APCI (+ or −) ionization mode. Analytes were eluted

using a linear gradient of 0.05% TFA containing water/acetonitrile

or 5 mM ammonium acetate containing water/acetonitrile mobile

phase and detected at 220 nm. Analytical HPLC was performed

with a Corona Charged Aerosol Detector (CAD), a Nano quantity

values or analytical HPLC. Yields were not optimized.

7-(4-((2-Chloro-4-ﬂuorophenoxy)methyl)piperidine-1-carbonyl)-

1H-pyrido[2,3-b][1,4]oxazin-2(3H)-one (1a). A mixture of 35 (43.3

mg, 0.15 mmol), 2-oxo-2,3-dihydro-1H-pyrido[2,3-b][1,4]oxazine-7-

carboxylic acid (30 mg, 0.15 mmol), HOBt-H₂O (28.4 mg, 0.19

mmol), EDCI (35.5 mg, 0.19 mmol), DIPEA (0.067 mL, 0.39

mmol), and DMF (2 mL) was stirred at rt for 3 h. After addition of

water, the resultant precipitates were collected, washed with water

and EtOAc, dried in vacuo, and triturated with hot EtOAc to give

1a (48 mg, 0.114 mmol, 74%) as a white solid. ¹H NMR (300

MHz, DMSO-d₆) δ 1.30 (2H, qd, J = 12.2, 4.0 Hz), 1.73−2.20 (3H,

m), 2.74−3.22 (2H, m), 3.61−4.02 (3H, m), 4.30−4.61 (1H, m),

4.83 (2H, s), 7.14−7.25 (3H, m), 7.39−7.47 (1H, m), 7.83 (1H, d,

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a

Scheme 8. Synthesis of LHS amines 46a−g, 48a−c, and 50

a

Reagents and conditions: (a) (1) MsCl, Et₃N, THF, 0 °C to rt, 0.5−16 h, quant.; (2) t-butyl 3-hydroxyazetidine-1-carboxylate, NaH, DMF, 0 °C

to rt, 3−38 h, 87−93%; (3) p-TsOH-H₂O, EtOAc, reﬂux, 2 h, 69−88% for 46a−d; (b) (1) PBr₃, DME, 0 °C to rt, 2.5−3 h, 86-100%; (2) t-butyl 3-

hydroxyazetidine-1-carboxylate, NaH, DMF, 0 °C to rt, overnight, 89−99%; (3) p-TsOH-H₂O, EtOAc, reﬂux, 2−3 h, 83-92% for 46e,f; (c) (1)

MsCl, Et₃N, THF, 0 °C to rt, 100%; (2) t-butyl 3-hydroxyazetidine-1-carboxylate, NaH, DMF, 0 °C to rt, 87%; (3) trimethylboroxine, Pd(OAc)₂,

SPhos, K₃PO₄, toluene−H₂O, 100 °C, 16 h, 63%; (4) p-TsOH-H₂O, EtOAc, reﬂux, 2 h, 74% for 46 g; (d) (1) t-butyl 3-hydroxyazetidine-1-

carboxylate, NaH, DMF, 0 °C to rt, overnight, 79−94%; (2) p-TsOH-H₂O, EtOAc, reﬂux, 2−3 h, 84-86% for 48a−c; (e) (1) BH₃−THF complex,

THF, 0 °C to rt, 18 h, 100%; (2) MsCl, Et₃N, THF, 0 °C to rt, 2 h, quant.; (3) t-butyl 3-hydroxyazetidine-1-carboxylate, NaH, DMF, 0 °C to rt, 64

h, 76%; (4) p-TsOH-H₂O, EtOAc, reﬂux, 2 h, 93% for 50.

J = 2.1 Hz), 10.92 (1H, brs). ¹³C NMR (101 MHz, DMSO-d₆) δ

35.8, 67.5, 73.6, 115.0, 115.19, 115.22, 115.3, 117.4, 117.7, 122.4,

122.5, 127.5, 139.3, 151.19, 151.22, 151.6, 155.0, 157.3, 164.3,

166.6. LCMS (ESI/APCI) m/z 420.1 [M + H]⁺. Anal. Calcd for

C₂₀H₁₉ClFN₃O₄: C, 57.22; H, 4.56; N, 10.01. Found: C, 57.26; H,

4.77; N, 10.02. HPLC purity 99.2%. Mp 237 °C.

was added Et₃N (0.155 mL, 1.11 mmol) at rt, and the mixture was

stirred at rt overnight. The mixture was diluted with aq. NaHCO₃

and extracted with MeOH/EtOAc. The extract was washed with

water and brine, dried over Na₂SO₄, passed through a short pad of

silica gel eluting with MeOH/EtOAc (1:20), and concentrated in

vacuo. The residue was triturated with EtOAc/IPE to give 1c (105

1

mg, 0.250 mmol, 54%) as a pale brown solid. H NMR (300 MHz,

7-(4-((2-Chloro-4-ﬂuorophenoxy)methyl)piperidine-1-carbonyl)-

1H-pyrido[3,4-b][1,4]oxazin-2(3H)-one (1b). A mixture of 35 (70

mg, 0.25 mmol), 6 (48.5 mg, 0.25 mmol), HOBt (40.5 mg, 0.30

mmol), EDCI (57.5 mg, 0.30 mmol), DIPEA (0.104 mL, 0.60

mmol), and DMF (1.5 mL) was stirred at rt for 16 h. After addition

of water, the resultant precipitate was collected, washed with water,

and dried in vacuo. The residual solid was triturated with hot

EtOAc/IPA to give 1b (67.0 mg, 0.160 mmol, 64%) as a white

DMSO-d₆) δ 1.22−1.41 (2H, m), 1.82 (2H, brs), 2.08 (1H, d, J =

5.7 Hz), 2.74−3.24 (2H, m), 3.56−3.83 (1H, m), 3.94 (2H, d, J =

6.4 Hz), 4.26−4.58 (1H, m), 4.83 (2H, s), 7.15−7.17 (1H, m), 7.19

(1H, d, J = 1.9 Hz), 7.22 (1H, d, J = 2.3 Hz), 7.43 (1H, dt, J = 8.1,

1.6 Hz), 7.83 (1H, d, J = 2.3 Hz), 10.94 (1H, brs). ¹³C NMR (101

MHz, DMSO-d₆) δ 35.8, 67.5, 73.6, 114.9, 115.17, 115.20, 115.3,

117.4, 117.7, 122.39, 122.41, 122.5, 127.5, 139.3, 151.19, 151.22,

151.6, 155.0, 157.3, 164.3, 166.6. LCMS (ESI/APCI) m/z 420.2 [M

+ H]⁺. Anal. Calcd for C₂₀H₁₉ClFN₃O₄: C, 57.22; H, 4.56; N,

10.01. Found: C, 57.30; H, 4.82; N, 9.86. HPLC purity 100%. Mp

237 °C.

1

solid. H NMR (300 MHz, DMSO-d₆) δ 1.22−1.41 (2H, m), 1.69−

1.97 (2H, m), 2.00−2.20 (1H, m), 2.75−2.89 (1H, m), 2.99−3.16

(1H, m), 3.86−4.00 (3H, m), 4.42−4.55 (1H, m), 4.74 (2H, s),

7.06 (1H, s), 7.14−7.21 (2H, m), 7.38−7.47 (1H, m), 8.15 (1H, s),

11.19 (1H, s). ¹³C NMR (101 MHz, DMSO-d₆) δ 28.6, 29.4, 35.9,

42.0, 46.8, 67.3, 73.7, 110.6, 114.9, 115.15, 115.21, 115.3, 117.4,

117.6, 122.4, 122.5, 135.1, 136.1, 140.8, 149.2, 151.21, 151.24,

155.0, 157.3, 165.2, 166.5. LCMS (ESI/APCI) m/z 420.1 [M +

H]⁺. Anal. Calcd for C₂₀H₁₉ClFN₃O₄: C, 57.22; H, 4.56; N, 10.01.

Found: C, 57.33; H, 4.73; N, 9.94. HPLC purity 100%. Mp 226 °C.

6-(4-((2-Chloro-4-ﬂuorophenoxy)methyl)piperidine-1-carbonyl)-

2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one (1c). To a stirred mixture of

35 (130 mg, 0.46 mmol), 3-oxo-3,4-dihydro-2H-pyrido[3,2-b][1,4]-

oxazine-6-carboxylic acid (90 mg, 0.46 mmol), EDCI (107 mg, 0.56

mmol), HOBt-H₂O (71.1 mg, 0.46 mmol), and MeCN (4.00 mL)

5-(3-(4-((2-Chloro-4-ﬂuorophenoxy)methyl)piperidin-1-yl)-3-

oxopropyl)morpholin-3-one (1d). A mixture of 35 (146 mg, 0.52

mmol), 11 (90 mg, 0.52 mmol), EDCI (120 mg, 0.62 mmol),

HOBt-H₂O (80 mg, 0.52 mmol), Et₃N (0.173 mL, 1.25 mmol), and

MeCN (4.00 mL) was stirred at rt overnight. The mixture was

diluted with aq. NaHCO₃and extracted with EtOAc. The extract

was washed with water and brine, dried over Na₂SO₄, and

concentrated in vacuo. The residue was puriﬁed by column

chromatography on silica gel with MeOH/EtOAc (0−9%) to give

1

1d (200 mg, 0.501 mmol, 96%) as a colorless oil. H NMR (300

MHz, CDCl₃) δ 1.27−1.46 (2H, m), 1.82−1.95 (3H, m), 1.96−2.24

O

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(2H, m), 2.47 (2H, t, J = 7.0 Hz), 2.54−2.74 (1H, m), 3.10 (1H,

td, J = 13.1, 2.4 Hz), 3.46−3.58 (1H, m), 3.62 (1H, brs), 3.74−3.99

(4H, m), 4.05−4.26 (2H, m), 4.69 (1H, d, J = 13.6 Hz), 6.74 (1H,

brs), 6.80−7.00 (2H, m), 7.13 (1H, dd, J = 8.3, 3.0 Hz). LCMS

(ESI/APCI) m/z 399.1 [M + H]⁺. HPLC purity 100%.

0.29 mmol), and the mixture was stirred at rt for 2 h. The mixture

was diluted with water and extracted with EtOAc. The organic layer

was separated, washed with water and brine, dried over Na₂SO₄, and

concentrated in vacuo. The residual solid was recrystallized from

EtOAc-IPA to give 2a (29 mg, 0.073 mmol, 26%) as a white solid.

¹H NMR (300 MHz, DMSO-d₆) δ 1.10−1.29 (2H, m), 1.70−1.81

(2H, m), 1.88−2.05 (1H, m), 2.11−2.33 (4H, m), 2.67−2.85 (2H,

m), 3.73−3.84 (2H, m), 3.86−3.96 (6H, m), 7.13−7.20 (2H, m),

7.38−7.46 (1H, m), 8.19 (1H, s). LCMS (ESI/APCI) m/z 396.1

[M + H]⁺. Anal. Calcd for C₁₉H₂₃ClFN₃O₃: C, 57.65; H, 5.86; N,

10.62. Found: C, 57.29; H, 6.03; N, 10.50. HPLC purity 98.9%. Mp

197 °C.

5-(3-(4-((2-Chloro-4-ﬂuorophenoxy)methyl)piperidin-1-yl)-3-

oxopropyl)pyrrolidin-2-one (1e). A mixture of 35 (143 mg, 0.51

mmol), 3-(5-oxopyrrolidin-2-yl)propanoic acid (80 mg, 0.51 mmol),

HOBt-H₂O (94 mg, 0.61 mmol), EDCI (117 mg, 0.61 mmol),

DIPEA (0.222 mL, 1.27 mmol), and DMF (2 mL) was stirred at rt

for 3 h. The mixture was diluted with water and extracted with

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

brine, dried over Na₂SO₄, and concentrated in vacuo. The residue

was puriﬁed by column chromatography (NH silica gel, eluted with

0−100% EtOAc in hexane; 5−30% MeOH in EtOAc) to give 1e

(2r,4s)-2-(4-((2-Chloro-4-ﬂuorophenoxy)methyl)piperidine-1-car-

bonyl)-5-azaspiro[3.4]octan-6-one (2b). A mixture of 18 (347 mg,

2.05 mmol), 35 (575 mg, 2.05 mmol), HOBt-H₂O (377 mg, 2.46

mmol), EDCI (472 mg, 2.46 mmol), DIPEA (1.79 mL, 10.3 mmol),

and DMF (12 mL) was stirred at rt for 2 h. The mixture was

diluted with water and extracted with EtOAc/IPA. The organic layer

was separated, washed with water and brine, dried over Na₂SO₄,

ﬁltered through a NH silica gel pad, and concentrated in vacuo. The

residual solid was triturated with hot EtOAc/IPA to give 2b (373

1

(174 mg, 0.454 mmol, 89%) as a white solid. H NMR (300 MHz,

CDCl₃) δ 1.22−1.43 (2H, m), 1.70−1.94 (4H, m), 1.96−2.46 (7H,

m), 2.54−2.72 (1H, m), 3.01−3.17 (1H, m), 3.62−3.99 (4H, m),

4.59−4.76 (1H, m), 6.22 (1H, brs), 6.79−6.97 (2H, m), 7.12 (1H,

dd, J = 8.0, 2.9 Hz). ¹³C NMR (101 MHz, CDCl₃) δ 27.3, 28.4,

29.6, 30.3, 31.9, 36.3, 41.6, 45.4, 54.2, 73.8, 113.9, 114.1, 114.15,

114.23, 117.4, 117.7, 123.6, 123.7, 150.96, 150.99, 155.3, 157.7,

170.36, 170.38, 178.3. LCMS (ESI/APCI) m/z 383.2 [M + H]⁺.

Anal. Calcd for C₁₉H₂₄ClFN₂O₃: C, 59.61; H, 6.32; N, 7.32. Found:

C, 59.55; H, 6.41; N, 7.29. HPLC purity 96.2%. Mp 103 °C.

(5-Oxopyrrolidin-2-yl)methyl 4-((2-chloro-4-ﬂuorophenoxy)-

methyl)piperidine-1-carboxylate (1f). To a stirred mixture of 5-

(hydroxymethyl)pyrrolidin-2-one (101 mg, 0.88 mmol), Et₃N (0.244

mL, 1.75 mmol), and THF (4.00 mL) was added 4-nitrophenyl

chloroformate (186 mg, 0.92 mmol) at 0 °C, and the mixture was

stirred at rt overnight. To this mixture were added Et₃N (0.366 mL,

2.63 mmol) and 35 (246 mg, 0.88 mmol) at rt, and the mixture was

stirred overnight. The mixture was diluted with water and extracted

with EtOAc. The extract was washed with sat. NaHCO₃and brine,

dried over Na₂SO₄, and concentrated in vacuo. The residue was

puriﬁed by column chromatography on silica gel with MeOH/

EtOAc (0−20%), and crystallized from EtOAc/IPE to give a crude

material. This material (171 mg) was puriﬁed by preparative HPLC

and the target fractions were concentrated in vacuo. The residue was

passed through the short pad of NH silica gel eluting with MeOH/

EtOAc (10%). The ﬁltrate was concentrated in vacuo, and the

residue was crystallized from EtOAc/IPE to give 1f (141 mg, 0.366

1

mg, 0.945 mmol, 46%) as an oﬀ-white solid. H NMR (300 MHz,

DMSO-d₆) δ 1.04−1.29 (2H, m), 1.69−1.89 (2H, m), 1.94−2.37

(9H, m), 2.52−2.66 (1H, m), 2.89−3.13 (2H, m), 3.75−3.99 (3H,

m), 4.31−4.47 (1H, m), 7.11−7.22 (2H, m), 7.36−7.46 (1H, m),

7.93 (1H, s). ¹³C NMR (101 MHz, DMSO-d₆) δ 28.6, 29.5, 30.5,

34.1, 36.0, 39.0, 41.5, 44.7, 55.9, 73.6, 114.9, 115.15, 115.23, 117.4,

117.6, 122.4, 122.5, 151.21, 151.24, 154.9, 157.3, 171.6, 176.0.

LCMS (ESI/APCI) m/z 395.1 [M + H]⁺. Anal. Calcd for

C₂₀H₂₄ClFN₂O₃0.3H₂O: C, 60.01; H, 6.19; N, 7.00. Found: C,

60.07; H, 6.03; N, 7.00. HPLC purity 96.8%. Mp 196 °C.

(2s,4r)-2-(4-((2-Chloro-4-ﬂuorophenoxy)methyl)piperidine-1-car-

bonyl)-5-azaspiro[3.4]octan-6-one (2c). A mixture of 21 (127 mg,

0.75 mmol), 35 (210 mg, 0.75 mmol), HOBt-H₂O (138 mg, 0.90

mmol), EDCI (173 mg, 0.90 mmol), DIPEA (0.654 mL, 3.75

mmol), and DMF (10 mL) was stirred at rt for 2 h. The mixture

was diluted with water and extracted with EtOAc. The organic layer

was separated, washed with water and brine, dried over Na₂SO₄,

ﬁltered through a NH silica gel pad, and concentrated in vacuo. The

residue was puriﬁed by column chromatography (NH silica gel,

eluted with 0−100% EtOAc in hexane; 3−25% MeOH in EtOAc)

and recrystallized from EtOAc/IPA/heptane to give 2c (165 mg,

0.418 mmol, 56%) as an oﬀ-white solid. ¹H NMR (300 MHz,

DMSO-d₆) δ 1.06−1.31 (2H, m), 1.71−1.86 (2H, m), 1.91−2.15

(5H, m), 2.25−2.40 (4H, m), 2.54−2.67 (1H, m), 2.91−3.07 (1H,

m), 3.12−3.26 (1H, m), 3.60−3.73 (1H, m), 3.83−3.99 (2H, m),

4.34−4.48 (1H, m), 7.12−7.21 (2H, m), 7.38−7.48 (1H, m), 8.12

(1H, s). ¹³C NMR (101 MHz, DMSO-d₆) δ 28.5, 29.2, 29.3, 30.5,

35.2, 36.0, 38.7, 38.7, 41.5, 44.7, 57.9, 73.6, 114.9, 115.1, 115.16,

115.22, 117.4, 117.6, 122.3, 122.5, 151.21, 151.23, 154.9, 157.3,

171.9, 176.0. LCMS (ESI/APCI) m/z 395.1 [M + H]⁺. Anal. Calcd

for C₂₀H₂₄ClFN₂O₃: C, 60.83; H, 6.13; N, 7.09. Found: C, 61.01;

H, 6.22; N, 7.08. HPLC purity 100%. Mp 162 °C.

1

mmol, 42%) as a white solid. H NMR (300 MHz, DMSO-d₆) δ

1.09−1.34 (2H, m), 1.66−1.86 (3H, m), 1.91−2.05 (1H, m), 2.05−

2.24 (3H, m), 2.81 (2H, brs), 3.68−3.79 (1H, m), 3.82−4.14 (6H,

m), 7.17 (2H, dd, J = 6.6, 1.7 Hz), 7.42 (1H, dt, J = 8.4, 1.6 Hz),

7.85 (1H, s). LCMS (ESI/APCI) m/z 385.1 [M + H]⁺. Anal. Calcd

for C₁₈H₂₂ClFN₂O₄: C, 56.18; H, 5.76; N, 7.28. Found: C, 56.09;

H, 5.72; N, 7.21. HPLC purity 100%.

(5R)-5-(3-{4-[(2-chloro-4-ﬂuorophenoxy)methyl]piperidin-1-yl}-

3-oxopropyl)pyrrolidin-2-one (1g) and (5S)-5-(3-{4-[(2-chloro-4-

ﬂuorophenoxy)methyl]piperidin-1-yl}-3-oxopropyl)pyrrolidin-2-

one (1h). Racemate 1e (1.26 g, 3.29 mmol) was optically resolved

by preparative SFC (CHIRALPAK IA, 20 mm I.D. × 250 mm L., 5

μm particle size, Daicel, Japan, eluent: CO₂/MeOH = 600:400 v/v,

ﬂow rate: 55 mL/min, temp.: 35 °C) to give 1g (0.611 g, 1.60

mmol, 49%) and 1h (0.609 g, 1.59 mmol, 48%) as a colorless oil.

¹H NMR (300 MHz, CDCl₃) δ 1.21−1.45 (2H, m), 1.72−1.81

(1H, m), 1.82−1.94 (3H, m), 2.01 (1H, d, J = 13.2 Hz), 2.06−2.19

(1H, m), 2.21−2.37 (3H, m), 2.38−2.47 (2H, m), 2.63 (1H, t, J =

12.2 Hz), 3.09 (1H, td, J = 13.1, 2.4 Hz), 3.64−4.03 (4H, m), 4.69

(1H, d, J = 13.6 Hz), 6.36 (1H, brs), 6.80−6.87 (1H, m), 6.88−

6.97 (1H, m), 7.13 (1H, dd, J = 8.1, 2.8 Hz). LCMS (ESI/APCI)

m/z 383.1 [M + H]⁺. HPLC purity 99.8% for 1g, 99.7% for 1h.

2-(4-((2-Chloro-4-ﬂuorophenoxy)methyl)piperidine-1-carbonyl)-

2,5-diazaspiro[3.4]octan-6-one (2a). To a cooled (−10 °C)

mixture of 35 (80 mg, 0.29 mmol) and MeCN (4 mL) were

added DIPEA (0.298 mL, 1.71 mmol) and Bis(trichloromethyl)-

carbonate (29.7 mg, 0.10 mmol). The mixture was stirred at the

same temperature for 2 h. To the mixture was added 28 (134 mg,

(2s,4s)-2-(4-((2-Chloro-4-ﬂuorophenoxy)methyl)piperidine-1-

carbonyl)-7-oxa-5-azaspiro[3.4]octan-6-one (2d). A mixture of 24

(43.0 mg, 0.25 mmol), 35 (70.4 mg, 0.25 mmol), HOBt-H₂O (46.2

mg, 0.30 mmol), EDCI (57.8 mg, 0.30 mmol), DIPEA (0.158 mL,

0.90 mmol), and DMF (1.5 mL) was stirred at rt for 18 h. After

addition of water, the resultant precipitate was collected, washed

with water, and dried in vacuo. The crude solid was recrystallized

from EtOH (8 mL)/H₂O (5 mL) to give 2d (69 mg, 0.174 mmol,

1

69%) as a white solid. H NMR (300 MHz, DMSO-d₆) δ 1.04−1.27

(2H, m), 1.70−1.87 (2H, m), 1.93−2.11 (1H, m), 2.23−2.66 (5H,

m), 2.90−3.08 (2H, m), 3.76−3.87 (1H, m), 3.91 (2H, d, J = 6.2

Hz), 4.32−4.42 (3H, m), 7.12−7.20 (2H, m), 7.38−7.46 (1H, m),

8.02 (1H, s). LCMS (ESI/APCI) m/z 397.0 [M + H]⁺. Anal. Calcd

for C₁₉H₂₂ClFN₂O₄H₂O: C, 55.01; H, 5.83; N, 6.75. Found: C,

55.31; H, 5.82; N, 6.76. HPLC purity 99.9%. Mp 170 °C.

7-(4-((2-Chloro-4-ﬂuorophenoxy)methyl)piperidine-1-carbonyl)-

1,7-diazaspiro[4.4]nonan-2-one (2e). To a cooled (−10 °C)

P

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mixture of 35 (140 mg, 0.5 mmol) and MeCN (4 mL) were added

DIPEA (0.523 mL, 3.00 mmol) and triphosgene (51.9 mg, 0.18

mmol). The mixture was stirred at the same temperature for 30 min.

To the mixture was added 1,7-diazaspiro[4.4]nonan-2-one hydro-

chloride (88 mg, 0.50 mmol), and the mixture was stirred at rt for 2

h. The mixture was diluted with water and extracted with EtOAc.

The organic layer was separated, washed with water and brine, dried

over MgSO₄, and concentrated in vacuo. The residue was puriﬁed

by column chromatography (NH silica gel, eluted with 50−100%

EtOAc in hexane to 20% MeOH in EtOAc). The residue was

triturated with EtOAc-hexane to give 2e (63.0 mg, 0.154 mmol,

mmol) at rt. The mixture was stirred at rt for 4 h. The mixture was

poured into sat.NaHCO_3.and extracted with EtOAc. The organic

layer was separated, washed with and brine, dried over MgSO₄, and

concentrated in vacuo. The residue was puriﬁed by column

chromatography (silica gel, eluted with 0−20% MeOH in EtOAc).

The residue was triturated with EtOAc-hexane to give rac-3b (100

mg). This was optically resolved by preparative HPLC (CHIR-

ALPAK ID, 50 mm I.D. × 500 mm L., 5 μm particle size, Daicel,

Japan, eluent:hexane/IPA = 150:850 v/v, ﬂow rate: 60 mL/min,

temp.: 30 °C). The fractions of tR1 were concentrated, and the

residual solid was recrystallized from IPA/EtOAc/hexane to give 3b

(31 mg, 0.075 mmol, >99.9% ee, 3 steps 25% from 23b) as a white

1

31%) as a white solid. H NMR (300 MHz, DMSO-d₆) δ 1.26 (2H,

1

d, J = 5.7 Hz), 1.66−1.87 (4H, m), 1.87−2.03 (3H, m), 2.13−2.38

(2H, m), 2.71 (2H, t, J = 12.6 Hz), 3.12−3.29 (2H, m), 3.39 (2H,

t, J = 6.7 Hz), 3.69 (2H, d, J = 12.1 Hz), 3.91 (2H, d, J = 6.4 Hz),

7.17 (2H, d, J = 5.4 Hz), 7.42 (1H, d, J = 8.3 Hz), 8.01 (1H, s).

¹³C NMR (101 MHz, DMSO-d₆) δ 28.7, 28.8, 30.5, 31.3, 36.1, 37.3,

45.8, 46.8, 58.5, 63.7, 73.9, 114.9, 115.17, 115.20, 115.3, 117.4,

117.7, 122.4, 122.5, 151.25, 151.27, 154.9, 157.3, 162.1, 176.2.

LCMS (ESI/APCI) m/z 410.1 [M + H]⁺. Anal. Calcd for

C₂₀H₂₅ClFN₃O₃: C, 58.61; H, 6.15; N, 10.25. Found: C, 58.43;

H, 6.24; N, 10.18. HPLC purity 99.9%. Mp 150 °C.

solid. H NMR (300 MHz, DMSO-d₆) δ 1.03−1.30 (5H, m), 1.67

(1H, d, J = 12.7 Hz), 1.82 (2H, d, J = 7.6 Hz), 2.19−2.43 (5H, m),

2.80−3.06 (2H, m), 3.83 (1H, d, J = 13.0 Hz), 4.21−4.57 (4H, m),

7.01−7.28 (2H, m), 7.40 (1H, dd, J = 8.3, 2.6 Hz), 8.02 (1H, s).

LCMS (ESI/APCI) m/z 411.2 [M + H]⁺. Anal. Calcd for

C₂₀H₂₄ClFN₂O₄: C, 58.47; H, 5.89; N, 6.82. Found: C, 58.07; H,

5.77; N, 6.80. HPLC purity 100%.

(2s,4s)-2-(4-((R or S)-1-(2-Chloro-4-ﬂuorophenoxy)propyl)-

piperidine-1-carbonyl)-7-oxa-5-azaspiro[3.4]octan-6-one (3c). A

mixture of 41b (127 mg, 0.47 mmol), 24 (80 mg, 0.47 mmol),

EDCI (134 mg, 0.70 mmol), HOBt (76 mg, 0.56 mmol), Et₃N

(0.326 mL, 2.34 mmol), and DMF (5 mL) was stirred at rt

overnight. The mixture was poured into water and extracted with

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

brine, dried over MgSO₄, and concentrated in vacuo. The residue

was puriﬁed by column chromatography (NH silica gel, eluted with

0−10% MeOH in EtOAc) to give rac-3c (145 mg). This was

optically resolved by preparative SFC (CHIRALPAK IA, 20 mm

I.D. × 250 mm L., 5 μm particle size, Daicel, Japan, eluent: CO₂/

EtOH = 700:300 v/v, ﬂow rate: 50 mL/min, temp.: 35 °C). The

fractions of tR1 were concentrated to give 3c (47 mg, 0.111 mmol,

7-(4-((2-Chloro-4-ﬂuorophenoxy)methyl)piperidine-1-carbonyl)-

1,7-diazaspiro[4.5]decan-2-one (2f). To a stirred suspension of 35

(140 mg, 0.50 mmol) in THF (5 mL) was added 4-nitrophenyl

chloroformate (111 mg, 0.55 mmol) at 0 °C, and then Et₃N (0.278

mL, 2.00 mmol) was added dropwise thereto at 0 °C. The mixture

was allowed to warm up to rt and stirred for 1h. To this mixture

were added 32 (249 mg, 0.5 mmol) and Et₃N (0.278 mL, 2.00

mmol) in THF (5 mL), and the mixture was heated to reﬂux

overnight. The mixture was diluted with water and extracted with

EtOAc. The extract was washed with sat.NaHCO₃and brine, dried

over MgSO₄, and concentrated in vacuo. The residue was puriﬁed

by column chromatography (NH silica gel, eluted with 50−100%

EtOAc in hexane to 30% MeOH in EtOAc). The residue was

triturated with EtOAc-hexane to give 2f (10.0 mg, 0.024 mmol, 5%)

1

>99.9% ee, 2 steps 24%) as a colorless amorphous solid. H NMR

(300 MHz, DMSO-d₆) δ 0.89 (3H, t, J = 7.3 Hz), 1.03−1.33 (2H,

m), 1.50−1.97 (5H, m), 2.21−2.59 (5H, m), 2.82−3.10 (2H, m),

3.81 (1H, d, J = 13.2 Hz), 4.23 (1H, d, J = 4.9 Hz), 4.36 (3H, s),

7.08−7.27 (2H, m), 7.40 (1H, dd, J = 8.3, 3.0 Hz), 8.04 (1H, s).

LCMS (ESI/APCI) m/z 425.2 [M + H]⁺. HPLC purity 100%.

(2s,4s)-2-(4-((R or S)-1-(2-Chloro-4-ﬂuorophenoxy)-2,2,2-

triﬂuoroethyl)piperidine-1-carbonyl)-7-oxa-5-azaspiro[3.4]octan-

6-one (3d). A mixture of 41c (146 mg, 0.47 mmol), 24 (80 mg,

0.47 mmol), EDCI (134 mg, 0.70 mmol), HOBt (76 mg, 0.56

mmol), Et₃N (0.326 mL, 2.34 mmol), and DMF (5 mL) was stirred

at rt overnight. The mixture was poured into water and extracted

with EtOAc. The organic layer was separated, washed with water

and brine, dried over MgSO₄, and concentrated in vacuo. The

residue was puriﬁed by column chromatography (NH silica gel,

eluted with 0−10% MeOH in EtOAc) to give rac-3d (133 mg).

This was optically resolved by preparative HPLC (CHIRALPAK

AD, 50 mm I.D. × 500 mm L., 5 μm particle size, Daicel, Japan,

eluent:hexane/IPA = 800/200 v/v, ﬂow rate: 80 mL/min, temp.: 30

°C). The fractions of tR1 were concentrated to give 3d (38 mg,

0.082 mmol, >99.9% ee, 2 steps 17%) as a colorless amorphous

1

as a pale yellow solid. H NMR (300 MHz, DMSO-d₆) δ 1.20−1.37

(2H, m), 1.45−1.61 (3H, m), 1.61−2.02 (6H, m), 2.06−2.30 (2H,

m), 2.66−2.82 (2H, m), 2.84−3.05 (3H, m), 3.06−3.18 (1H, m),

3.54−3.71 (2H, m), 3.92 (2H, d, J = 6.3 Hz), 7.16 (2H, dd, J = 6.7,

1.7 Hz), 7.42 (1H, dt, J = 8.2, 1.7 Hz), 7.92 (1H, s). LCMS (ESI/

APCI) m/z 424.1 [M + H]⁺. HPLC purity 99.1%.

(2s,4s)-2-(4-((2-Chloro-4-ﬂuorophenoxy)methyl)-4-methylpiperi-

dine-1-carbonyl)-7-oxa-5-azaspiro[3.4]octan-6-one (3a). To a

stirred mixture of 24 (46.5 mg, 0.27 mmol), 38 (80 mg, 0.27

mmol), EDCI (62.6 mg, 0.33 mmol), HOBt-H₂O (41.6 mg, 0.27

mmol), and MeCN (2 mL) was added Et₃N (0.076 mL, 0.54

mmol) at rt, and the mixture was stirred for 3h. The mixture was

diluted with aq. NaHCO₃, and extracted with EtOAc. The extract

was washed with water and brine, dried over Na₂SO₄, and

concentrated in vacuo. The residue was puriﬁed by column

chromatography on silica gel with MeOH/EtOAc (0−5%), and

crystallized from EtOAc/hexane to give 3a (80 mg, 0.195 mmol,

1

72%) as a white solid. H NMR (300 MHz, CDCl₃) δ 1.18 (3H, s),

1

solid. H NMR (300 MHz, DMSO-d₆) δ 1.10−1.53 (2H, m), 1.68−

1.42−1.57 (2H, m), 1.67 (2H, ddd, J = 13.8, 9.4, 4.5 Hz), 2.40−

2.57 (2H, m), 2.58−2.80 (2H, m), 3.02 (1H, quin, J = 7.9 Hz), 3.29

(2H, dtd, J = 13.5, 9.8, 3.5 Hz), 3.44−3.61 (1H, m), 3.70 (2H, d, J

= 0.9 Hz), 4.07 (1H, dt, J = 13.7, 4.7 Hz), 4.37 (2H, s), 5.80 (1H,

s), 6.77−6.87 (1H, m), 6.88−7.00 (1H, m), 7.13 (1H, dd, J = 8.0,

2.9 Hz). LCMS (ESI/APCI) m/z 411.2 [M + H]⁺. HPLC purity

99.0%.

(2s,4s)-2-(4-((R or S)-1-(2-Chloro-4-ﬂuorophenoxy)ethyl)-

piperidine-1-carbonyl)-7-oxa-5-azaspiro[3.4]octan-6-one (3b). To

a mixture of 23b (60 mg, 0.30 mmol), THF (1 mL), and MeOH

(0.5 mL) was added 2 M NaOH (0.181 mL, 0.36 mmol), and the

mixture was stirred at rt for 1 h. The mixture was neutralized with 2

M HCl (0.181 mL), and the mixture was concentrated in vacuo.

The residue was diluted with DMF (2 mL), followed by the

addition of 41a (129 mg, 0.30 mmol), HOBt-H₂O (55.4 mg, 0.36

mmol), EDCI (69.3 mg, 0.36 mmol) and DIPEA (0.189 mL, 1.08

1.86 (2H, m), 2.13−2.64 (6H, m), 2.90−3.09 (2H, m), 3.81 (1H,

brs), 4.27−4.48 (3H, m), 5.18 (1H, t, J = 5.1 Hz), 7.24 (1H, ddd, J

= 9.3, 8.1, 3.0 Hz), 7.40 (1H, dd, J = 9.3, 4.9 Hz), 7.49 (1H, dd, J =

8.3, 3.0 Hz), 8.03 (1H, d, J = 1.5 Hz). LCMS (ESI/APCI) m/z

465.1 [M + H]⁺. HPLC purity 96.7%.

(2s,4s)-2-(4-((2-Chloro-4-ﬂuorobenzyl)oxy)piperidine-1-carbon-

yl)-7-oxa-5-azaspiro[3.4]octan-6-one (3e). EDCI (240 mg, 1.25

mmol) was added to a solution of 44a (400 mg, 0.96 mmol), 24

(181 mg, 1.06 mmol), HOBt (169 mg, 1.25 mmol), and Et₃N

(0.201 mL, 1.44 mmol) in DMF (2.5 mL) at 0 °C, and the reaction

mixture was stirred at rt for 16 h. The mixture was diluted with

EtOAc and the solution was washed with 1 M HCl, 10% K₂CO₃,

and brine, and dried over Na₂SO₄. The solution was passed through

NH silica gel and concentrated in vacuo. The residue was solidiﬁed

with hexane−EtOAc (3:1 v/v) to give 3e (338 mg, 0.852 mmol,

Q

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J. Med. Chem. XXXX, XXX, XXX−XXX

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1

89%) as a white solid. H NMR (300 MHz, DMSO-d₆) δ 1.32−1.50

4.47 (1H, m), 4.50 (2H, s), 7.18−7.31 (1H, m), 7.47 (1H, dd, J =

8.9, 2.5 Hz), 7.57 (1H, dd, J = 8.3, 6.6 Hz), 8.08 (1H, s). ¹³C NMR

(101 MHz, DMSO-d₆) δ 26.4, 38.1, 54.9, 55.3, 57.0, 67.2, 67.8,

75.7, 114.7, 114.9, 116.9, 117.2, 131.90, 131.93, 132.2, 132.3, 133.9,

134.0, 158.0, 160.8, 163.3, 173.2. LCMS (ESI/APCI) m/z 369.1 [M

+ H]⁺. Anal. Calcd for C₁₇H₁₈ClFN₂O₄: C, 55.37; H, 4.92; N, 7.60.

Found: C, 55.45; H, 5.05; N, 7.63. HPLC purity 100%. Mp 128 °C.

(2s,4s)-2-(3-((2-Chloro-4-methylbenzyl)oxy)azetidine-1-carbon-

yl)-7-oxa-5-azaspiro[3.4]octan-6-one (4a). A mixture of 46a (500

mg, 1.30 mmol), 24 (223 mg, 1.30 mmol), EDCI (275 mg, 1.43

mmol), HOBt-H₂O (219 mg, 1.43 mmol), DIPEA (0.682 mL, 3.91

mmol), and DMF (4.34 mL) was stirred at rt for 3 h. The mixture

was partitioned between EtOAc and water. The organic layer was

washed with brine, dried over MgSO₄, passed through a short pad of

NH silica gel, and concentrated in vacuo. The resultant residue was

washed with IPE/EtOAc to give 4a (279 mg, 0.765 mmol, 59%) as

(2H, m), 1.76−1.92 (2H, m), 2.24−2.45 (4H, m), 2.94−3.23 (3H,

m), 3.52−3.73 (2H, m), 3.76−3.88 (1H, m), 4.36 (2H, s), 4.56

(2H, s), 7.18−7.27 (1H, m), 7.44 (1H, dd, J = 8.9, 2.6 Hz), 7.56

(1H, dd, J = 8.5, 6.4 Hz), 8.02 (1H, s). LCMS (ESI/APCI) m/z

397.1 [M + H]⁺. HPLC purity 100%.

(2s,4s)-2-(3-((2-Chloro-4-ﬂuorobenzyl)oxy)pyrrolidine-1-carbon-

yl)-7-oxa-5-azaspiro[3.4]octan-6-one (3f). EDCI (496 mg, 2.59

mmol) was added to a solution of 44b (800 mg, 1.99 mmol), 24

(375 mg, 2.19 mmol), HOBt (350 mg, 2.59 mmol), and Et₃N

(0.416 mL, 2.99 mmol) in DMF (5 mL) at 0 °C, and the reaction

mixture was stirred at rt for 16 h. The mixture was diluted with

EtOAc and the solution was washed with 1 M HCl, 10% K₂CO₃,

and brine, and dried over Na₂SO₄. The solution was passed through

NH silica gel and concentrated in vacuo. The residue was solidiﬁed

with hexane−EtOAc (3:1 v/v) to give rac-3f (594 mg, 1.55 mmol,

1

a white solid. H NMR (300 MHz, DMSO-d₆) δ 2.18−2.40 (7H,

78%) as a white solid. H NMR (300 MHz, DMSO-d₆) δ 1.84−2.17

m), 2.64−2.80 (1H, m), 3.65 (1H, dd, J = 10.5, 3.9 Hz), 3.91 (1H,

dd, J = 9.3, 3.2 Hz), 4.01 (1H, dd, J = 10.3, 6.5 Hz), 4.19−4.29

(1H, m), 4.34 (2H, s), 4.36−4.45 (1H, m), 4.47 (2H, s), 7.17 (1H,

d, J = 7.6 Hz), 7.30 (1H, s), 7.39 (1H, d, J = 7.7 Hz), 8.08 (1H, s).

¹³C NMR (101 MHz, DMSO-d₆) δ 20.8, 26.3, 38.1, 54.9, 55.4, 57.1,

67.6, 67.7, 75.7, 128.4, 130.0, 130.6, 132.3, 132.8, 140.1, 158.0,

173.2. LCMS (ESI/APCI) m/z 365.1 [M + H]⁺. Anal. Calcd for

C₁₈H₂₁ClN₂O₄: C, 59.26; H, 5.80; N, 7.68. Found: C, 59.07; H,

5.77; N, 7.69. HPLC purity 99.6%. Mp 129 °C.

(2H, m), 2.24−2.44 (4H, m), 2.82−2.99 (1H, m), 3.23−3.55 (4H,

m), 4.14−4.29 (1H, m), 4.36 (2H, s), 4.46−4.58 (2H, m), 7.18−

7.28 (1H, m), 7.41−7.48 (1H, m), 7.48−7.56 (1H, m), 8.06 (1H,

s). LCMS (ESI/APCI) m/z 383.1 [M + H]⁺. HPLC purity 99.1%.

(2s,4s)-2-(3-((2-Chloro-4-ﬂuorophenoxy)methyl)pyrrolidine-1-

carbonyl)-7-oxa-5-azaspiro[3.4]octan-6-one (3g). EDCI (310 mg,

1.62 mmol) was added to a solution of 41d (500 mg, 1.24 mmol),

24 (234 mg, 1.37 mmol), HOBt (219 mg, 1.62 mmol), and Et₃N

(0.260 mL, 1.87 mmol) in DMF (4 mL) at 0 °C, and the reaction

mixture was stirred at rt for 16 h. The mixture was diluted with

EtOAc and the solution was washed with 1 M HCl, 10% K₂CO₃,

and brine, and dried over Na₂SO₄. The solution was passed through

NH silica gel and concentrated in vacuo. The residue was solidiﬁed

with hexane−EtOAc (3:1 v/v) to give rac-3g (407 mg, 1.06 mmol,

(2s,4s)-2-(3-((2,4-Dichlorobenzyl)oxy)azetidine-1-carbonyl)-7-

oxa-5-azaspiro[3.4]octan-6-one (4b). A mixture of 48a (564 mg,

1.39 mmol), 24 (239 mg, 1.39 mmol), EDCI (294 mg, 1.53 mmol),

HOBt-H₂O (235 mg, 1.53 mmol), DIPEA (0.731 mL, 4.18 mmol),

and DMF (4.65 mL) was stirred at rt for 16 h. The mixture was

partitioned between EtOAc and water. The organic layer was

washed with brine, dried over MgSO₄, passed through a short pad of

NH silica gel, and concentrated in vacuo. The resultant residue was

washed with IPE to give 4b (261 mg, 0.677 mmol, 49%) as a white

1

85%) as a white solid. H NMR (300 MHz, DMSO-d₆) δ 1.64−1.88

(1H, m), 1.93−2.15 (1H, m), 2.24−2.45 (4H, m), 2.56−2.77 (1H,

m), 2.82−2.98 (1H, m), 3.13−3.63 (4H, m), 3.96−4.09 (2H, m),

4.36 (2H, s), 7.14−7.21 (2H, m), 7.39−7.46 (1H, m), 8.06 (1H, s).

LCMS (ESI/APCI) m/z 383.1 [M + H]⁺. HPLC purity 100%.

(2s,4s)-2-(3-((2-Chloro-4-ﬂuorophenoxy)methyl)azetidine-1-car-

bonyl)-7-oxa-5-azaspiro[3.4]octan-6-one (3h). EDCI (163 mg,

0.85 mmol) was added to a mixture of 41e (300 mg, 0.77

mmol), 24 (132 mg, 0.77 mmol), DIPEA (0.446 mL, 2.55 mmol)

and HOBt-H₂O (130 mg, 0.85 mmol) in DMF (6 mL) at rt. The

mixture was stirred at rt overnight. EtOAc and water were added to

the reaction mixture at rt, then the organic layer was separated. The

aqueous layer was extracted with EtOAc. The combined organic

layer was washed with water and brine, dried over MgSO₄, and

concentrated in vacuo. The residue was collected, washed with

EtOAc and dried to give 3h (90 mg, 0.244 mmol, 32%) as an oﬀ-

1

solid. H NMR (300 MHz, DMSO-d₆) δ 2.16−2.40 (4H, m), 2.71

(1H, q, J = 9.1 Hz), 3.68 (1H, dd, J = 10.1, 3.5 Hz), 3.93 (1H, dd, J

= 9.6, 4.0 Hz), 4.03 (1H, dd, J = 10.5, 6.5 Hz), 4.26 (1H, dd, J =

8.6, 7.1 Hz), 4.34 (2H, s), 4.38−4.47 (1H, m), 4.50 (2H, s), 7.43−

7.50 (1H, m), 7.50−7.58 (1H, m), 7.64 (1H, d, J = 2.1 Hz), 8.09

(1H, s). ¹³C NMR (101 MHz, DMSO-d₆) δ 26.4, 38.1, 54.9, 55.3,

57.0, 67.1, 67.9, 75.7, 127.9, 129.2, 131.7, 133.7, 133.8, 134.7, 158.0,

173.2. LCMS (ESI/APCI) m/z 385.1 [M + H]⁺. Anal. Calcd for

C₁₇H₁₈Cl₂N₂O₄: C, 53.00; H, 4.71; N, 7.27. Found: C, 53.00; H,

4.68; N, 7.31. HPLC purity 100%. Mp 128 °C.

(2s,4s)-2-(3-((2-Chloro-4-(triﬂuoromethyl)benzyl)oxy)azetidine-

1-carbonyl)-7-oxa-5-azaspiro[3.4]octan-6-one (4c). To a stirred

mixture of 24 (78 mg, 0.46 mmol), 48b (200 mg, 0.46 mmol),

EDCI (105 mg, 0.55 mmol), HOBt-H₂O (69.9 mg, 0.46 mmol),

and MeCN (3 mL) was added dropwise Et₃N (0.152 mL, 1.10

mmol) at rt, and the mixture was stirred at rt overnight. The

mixture was diluted with EtOAc, washed with aq. NaHCO₃, water,

and brine, dried over Na₂SO₄, and concentrated in vacuo. The

residue was puriﬁed by column chromatography on silica gel with

MeOH/EtOAc (0−9%) to give crude amorphous powder (152.0

mg). This material was crystallized from EtOAc/hexane to give 4c

1

white solid. H NMR (300 MHz, DMSO-d₆) δ 2.21−2.41 (4H, m),

2.66−2.82 (1H, m), 3.02 (1H, tt, J = 8.4, 5.7 Hz), 3.71 (1H, dd, J =

9.5, 5.6 Hz), 3.88−3.99 (2H, m), 4.14−4.22 (3H, m), 4.35 (2H, s),

7.15−7.25 (2H, m), 7.39−7.47 (1H, m), 8.09 (1H, s). ¹³C NMR

(101 MHz, DMSO-d₆) δ 26.1, 28.2, 38.07, 38.12, 50.2, 52.2, 54.9,

71.0, 75.7, 115.0, 115.2, 115.75, 115.84, 117.4, 117.7, 122.6, 122.7,

151.08, 151.11, 155.2, 157.6, 158.0, 173.1. LCMS (ESI/APCI) m/z

369.1 [M + H]⁺. Anal. Calcd for C₁₇H₁₈ClFN₂O₄: C, 55.37; H,

4.92; N, 7.60. Found: C, 55.52; H, 5.12; N, 7.62. HPLC purity

100%. Mp 190 °C.

1

(136 mg, 0.325 mmol, 71%) as a white solid. H NMR (300 MHz,

(2s,4s)-2-(3-((2-Chloro-4-ﬂuorobenzyl)oxy)azetidine-1-carbon-

yl)-7-oxa-5-azaspiro[3.4]octan-6-one (3i). EDCI (257 mg, 1.34

mmol) was added to a solution of 44c (400 mg, 1.03 mmol), 24

(194 mg, 1.13 mmol), HOBt (181 mg, 1.34 mmol), and Et₃N

(0.216 mL, 1.55 mmol) in DMF (2.5 mL) at 0 °C, and the reaction

mixture was stirred at rt for 16 h. The mixture was diluted with

EtOAc and the solution was washed with 1 M HCl, 10% K₂CO₃,

and brine, and dried over Na₂SO₄. The solution was passed through

NH silica gel and concentrated in vacuo. The residue was solidiﬁed

with hexane−EtOAc (3:1 v/v) to give 3i (270 mg, 0.732 mmol,

CDCl₃) δ 2.39−2.52 (2H, m), 2.53−2.65 (2H, m), 2.65−2.81 (1H,

m), 3.95−4.13 (2H, m), 4.20−4.38 (4H, m), 4.47 (1H, tt, J = 6.4,

4.1 Hz), 4.60 (2H, s), 6.22 (1H, brs), 7.48−7.81 (3H, m). ¹³C

NMR (101 MHz, DMSO-d₆) δ 26.4, 38.1, 54.9, 55.3, 57.0, 67.1,

68.2, 75.7, 123.8, 124.6, 126.4, 130.3, 130.6, 133.2, 140.5, 158.0,

173.2. LCMS (ESI/APCI) m/z 419.1 [M + H]⁺. Anal. Calcd for

C₁₈H₁₈ClF₃N₂O₄: C, 51.62; H, 4.33; N, 6.69. Found: C, 51.49; H,

4.42; N, 6.79. HPLC purity 99.6%. Mp 114 °C.

(2s,4s)-2-(3-((3-Chloro-4-(triﬂuoromethyl)benzyl)oxy)azetidine-

1-carbonyl)-7-oxa-5-azaspiro[3.4]octan-6-one (4d). A mixture of

46b (351 mg, 0.80 mmol), 24 (137 mg, 0.80 mmol), EDCI (169

mg, 0.88 mmol), HOBt-H₂O (135 mg, 0.88 mmol), DIPEA (0.420

mL, 2.40 mmol), and DMF (2.67 mL) was stirred at rt for 3 h. The

1

71%) as a white solid. H NMR (300 MHz, DMSO-d₆) δ 2.18−2.41

(4H, m), 2.64−2.81 (1H, m), 3.61−3.73 (1H, m), 3.86−3.97 (1H,

m), 3.97−4.09 (1H, m), 4.19−4.30 (1H, m), 4.34 (2H, s), 4.37−

R

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mixture was partitioned between EtOAc and water. The organic

layer was washed with brine, dried over MgSO₄, passed through a

short pad of NH silica gel, and concentrated in vacuo. The resultant

residue was washed with IPE/EtOAc to give 4d (149 mg, 0.356

H]⁺. Anal. Calcd for C₁₉H₁₈F₆N₂O₄: C, 50.45; H, 4.01; N, 6.19.

Found: C, 50.47; H, 4.27; N, 6.29. HPLC purity 99.3%. Mp 141 °C.

(2s,4s)-2-(3-((4-Chloro-3-(triﬂuoromethyl)benzyl)oxy)azetidine-

1-carbonyl)-7-oxa-5-azaspiro[3.4]octan-6-one (4h). To a stirred

mixture of 24 (78 mg, 0.46 mmol), 46f (200 mg, 0.46 mmol),

EDCI (114 mg, 0.59 mmol), HOBt-H₂O (69.9 mg, 0.46 mmol),

and MeCN (4 mL) was added dropwise Et₃N (0.152 mL, 1.10

mmol) at rt, and the mixture was stirred at rt overnight. The

mixture was diluted with EtOAc, washed with aq. NaHCO₃, water,

and brine, dried over Na₂SO₄, and concentrated in vacuo. The

residue was puriﬁed by column chromatography on silica gel with

MeOH/EtOAc (0−9%) and crystallized from EtOAc/IPE to give 4h

1

mmol, 44%) as a white solid. H NMR (300 MHz, DMSO-d₆) δ

2.20−2.39 (4H, m), 2.69−2.79 (1H, m), 3.71 (1H, dd, J = 10.5, 3.7

Hz), 3.95 (1H, dd, J = 9.7, 3.7 Hz), 3.99−4.08 (1H, m), 4.21−4.30

(1H, m), 4.34 (2H, s), 4.38−4.46 (1H, m), 4.55 (2H, s), 7.52 (1H,

d, J = 8.7 Hz), 7.70 (1H, s), 7.86 (1H, d, J = 8.3 Hz), 8.09 (1H, s).

¹³C NMR (101 MHz, DMSO-d₆) δ 26.4, 38.1, 54.9, 55.3, 57.0, 67.9,

68.6, 75.7, 123.4, 126.1, 126.9, 128.4, 130.6, 131.18, 131.19, 145.3,

158.0, 173.2. LCMS (ESI/APCI) m/z 419.2 [M + H]⁺. Anal. Calcd

for C₁₈H₁₈ClF₃N₂O₄: C, 51.62; H, 4.33; N, 6.69. Found: C, 51.46;

H, 4.39; N, 6.65. HPLC purity 100%. Mp 126 °C.

1

(148 mg, 0.354 mmol, 78%) as a white solid. H NMR (300 MHz,

DMSO-d₆) δ 2.20−2.41 (4H, m), 2.64−2.83 (1H, m), 3.69 (1H,

dd, J = 10.4, 4.0 Hz), 3.94 (1H, dd, J = 9.0, 3.8 Hz), 4.02 (1H, dd, J

= 10.4, 6.6 Hz), 4.21−4.31 (1H, m), 4.34 (2H, s), 4.37−4.48 (1H,

m), 4.52 (2H, s), 7.62−7.78 (2H, m), 7.83 (1H, d, J = 1.5 Hz),

8.10 (1H, s). ¹³C NMR (101 MHz, DMSO-d₆) δ 26.4, 38.1, 54.9,

55.3, 57.0, 67.8, 68.7, 75.7, 119.2, 121.9, 124.7, 126.5, 126.8, 127.2,

127.4, 130.28, 130.30, 132.1, 133.8, 138.4, 158.0, 173.2. LCMS

(2s,4s)-2-(3-((3,4-Dichlorobenzyl)oxy)azetidine-1-carbonyl)-7-

oxa-5-azaspiro[3.4]octan-6-one (4e). A mixture of 48c (547 mg,

1.35 mmol), 24 (232 mg, 1.35 mmol), EDCI (285 mg, 1.49 mmol),

HOBt-H₂O (228 mg, 1.49 mmol), DIPEA (0.709 mL, 4.06 mmol),

and DMF (4.51 mL) was stirred at rt for 16 h. The mixture was

partitioned between EtOAc and water. The organic layer was

washed with brine, dried over MgSO₄, passed through a short pad of

NH silica gel, and concentrated in vacuo. The resultant residue was

washed with IPE to give 4e (248 mg, 0.644 mmol, 48%) as a white

(ESI/APCI) m/z 419.1 [M

+

H]⁺. Anal. Calcd for

C₁₈H₁₈ClF₃N₂O₄: C, 51.62; H, 4.33; N, 6.69. Found: C, 51.48;

H, 4.58; N, 6.74. HPLC purity 100%. Mp 149 °C.

1

(2s,4s)-2-(3-((4-Methyl-3-(triﬂuoromethyl)benzyl)oxy)azetidine-

1-carbonyl)-7-oxa-5-azaspiro[3.4]octan-6-one (4i). A mixture of

46c (500 mg, 1.20 mmol), 24 (205 mg, 1.20 mmol), EDCI (253

mg, 1.32 mmol), HOBt-H₂O (202 mg, 1.32 mmol), DIPEA (0.628

mL, 3.59 mmol), and DMF (3.99 mL) was stirred at rt for 64 h.

The mixture was partitioned between EtOAc and water. The organic

layer was washed with brine, dried over MgSO₄, passed through a

short pad of NH silica gel, and concentrated in vacuo. The resultant

residue was washed with IPE/EtOAc to give 4i (302 mg, 0.758

solid. H NMR (300 MHz, DMSO-d₆) δ 2.18−2.40 (4H, m), 2.72

(1H, t, J = 8.5 Hz), 3.68 (1H, dd, J = 10.3, 4.1 Hz), 3.92 (1H, dd, J

= 9.3, 3.9 Hz), 4.01 (1H, dd, J = 9.7, 7.1 Hz), 4.19−4.28 (1H, m),

4.34 (2H, s), 4.35−4.42 (1H, m), 4.44 (2H, s), 7.35 (1H, dd, J =

8.3, 1.9 Hz), 7.59−7.66 (2H, m), 8.09 (1H, s). ¹³C NMR (101

MHz, DMSO-d₆) δ 26.3, 38.1, 54.9, 55.4, 57.1, 67.7, 68.8, 75.7,

128.5, 130.1, 130.7, 131.0, 131.5, 139.4, 158.0, 173.1. LCMS (ESI/

APCI) m/z 385.1 [M + H]⁺. Anal. Calcd for C₁₇H₁₈Cl₂N₂O₄: C,

53.00; H, 4.71; N, 7.27. Found: C, 52.93; H, 4.69; N, 7.28. HPLC

purity 100%. Mp 154 °C.

1

mmol, 63%) as a white solid. H NMR (300 MHz, DMSO-d₆) δ

2.19−2.39 (4H, m), 2.44 (3H, d, J = 1.9 Hz), 2.64−2.80 (1H, m),

3.66 (1H, dd, J = 10.4, 4.0 Hz), 3.92 (1H, dd, J = 9.2, 3.6 Hz), 4.01

(1H, dd, J = 10.7, 6.2 Hz), 4.20−4.29 (1H, m), 4.31−4.43 (3H, m),

4.48 (2H, s), 7.40−7.47 (1H, m), 7.50−7.57 (1H, m), 7.63 (1H, s),

8.09 (1H, s). ¹³C NMR (101 MHz, DMSO-d₆) δ 18.92, 18.94, 26.3,

38.1, 54.9, 55.4, 57.1, 67.6, 69.4, 75.7, 125.0, 125.4, 127.8, 132.3,

132.7, 135.89, 135.90, 136.5, 158.0, 173.2. LCMS (ESI/APCI) m/z

399.2 [M + H]⁺. Anal. Calcd for C₁₉H₂₁F₃N₂O₄: C, 57.28; H, 5.31;

N, 7.03. Found: C, 56.89; H, 5.61; N, 7.01. HPLC purity 100%. Mp

133 °C.

(2s,4s)-2-(3-((3-Chloro-4-methylbenzyl)oxy)azetidine-1-carbon-

yl)-7-oxa-5-azaspiro[3.4]octan-6-one (4f). A mixture of 46e (14.0

g, 36.5 mmol), 24 (6.24 g, 36.5 mmol), EDCI (7.69 g, 40.1 mmol),

HOBt-H₂O (6.14 g, 40.1 mmol), DIPEA (19.1 mL, 109 mmol), and

DMF (91 mL) was stirred at rt for 16 h. The mixture was

partitioned between EtOAc and water. The organic layer was

washed with brine, dried over anhydrous MgSO₄, passed through a

short pad of NH silica gel, and concentrated in vacuo to give a

crude material (10.0 g). This material was dissolved in hot EtOH

(40 mL) and hexane/i-Pr₂O (1:1 v/v, 80 mL) was added to the

mixture. The obtained white crystal was collected by ﬁltration and

dried in vacuo to give 4f (8.72 g, 23.9 mmol, 66%) as a white solid.

¹H NMR (300 MHz, DMSO-d₆) δ 2.18−2.40 (7H, m), 2.72 (1H,

quin, J = 8.9 Hz), 3.65 (1H, dd, J = 10.1, 3.9 Hz), 3.90 (1H, dd, J =

9.4, 3.4 Hz), 4.00 (1H, dd, J = 10.5, 6.5 Hz), 4.18−4.28 (1H, m),

4.30−4.44 (5H, m), 7.21 (1H, dd, J = 7.7, 1.5 Hz), 7.33 (1H, d, J =

7.7 Hz), 7.39 (1H, d, J = 1.5 Hz), 8.08 (1H, s). ¹³C NMR (101

MHz, DMSO-d₆) δ 19.7, 26.3, 38.1, 54.9, 55.4, 57.1, 67.5, 69.4,

75.7, 127.1, 128.6, 131.6, 133.6, 135.2, 137.9, 158.0, 173.1. LCMS

(ESI/APCI) m/z 365.1 [M + H]⁺. Anal. Calcd for C₁₈H₂₁ClN₂O₄:

C, 59.26; H, 5.80; N, 7.68. Found: C, 59.18; H, 5.93; N, 7.54.

HPLC purity 99.7%. Mp 118 °C.

(2s,4s)-2-(3-((3-Methyl-4-(triﬂuoromethyl)benzyl)oxy)azetidine-

1-carbonyl)-7-oxa-5-azaspiro[3.4]octan-6-one (4j). A mixture of

46g (268 mg, 0.64 mmol), 24 (110 mg, 0.64 mmol), EDCI (135

mg, 0.71 mmol), HOBt-H₂O (108 mg, 0.71 mmol), DIPEA (0.336

mL, 1.93 mmol), and DMF (2.14 mL) was stirred at rt for 3 h. The

mixture was partitioned between EtOAc and water. The organic

layer was washed with brine, dried over MgSO₄, passed through a

short pad of NH silica gel, and concentrated in vacuo. The resultant

residue was washed with IPE/EtOAc to give 4j (149 mg, 0.374

1

mmol, 58%) as a white solid. H NMR (300 MHz, DMSO-d₆) δ

2.18−2.39 (4H, m), 2.44 (3H, d, J = 1.7 Hz), 2.68−2.78 (1H, m),

3.69 (1H, dd, J = 10.5, 3.7 Hz), 3.93 (1H, dd, J = 8.8, 4.1 Hz),

3.98−4.08 (1H, m), 4.20−4.30 (1H, m), 4.34 (2H, s), 4.36−4.44

(1H, m), 4.49 (2H, s), 7.35 (1H, d, J = 7.6 Hz), 7.40 (1H, s), 7.65

(1H, d, J = 8.1 Hz), 8.09 (1H, s). ¹³C NMR (101 MHz, DMSO-d₆)

δ 19.16, 19.17, 26.4, 38.1, 54.9, 55.4, 57.1, 67.8, 69.4, 75.7, 125.1,

125.7, 126.2, 127.1, 131.6, 136.60, 136.61, 142.7, 158.0, 173.2.

LCMS (ESI/APCI) m/z 399.2 [M + H]⁺. Anal. Calcd for

C₁₉H₂₁F₃N₂O₄: C, 57.28; H, 5.31; N, 7.03. Found: C, 57.04; H,

5.42; N, 7.01. HPLC purity 99.2%. Mp 138 °C.

(2s,4s)-2-(3-((4-Chloro-3-methylbenzyl)oxy)azetidine-1-carbon-

yl)-7-oxa-5-azaspiro[3.4]octan-6-one (4k). A mixture of 46d (500

mg, 1.30 mmol), 24 (223 mg, 1.30 mmol), EDCI (275 mg, 1.43

mmol), HOBt-H₂O (219 mg, 1.43 mmol), DIPEA (0.682 mL, 3.91

mmol), and DMF (4.34 mL) was stirred at rt for 3 h. The mixture

was partitioned between EtOAc and water. The organic layer was

washed with brine, dried over MgSO₄, passed through a short pad of

(2s,4s)-2-(3-((3,4-Bis(triﬂuoromethyl)benzyl)oxy)azetidine-1-car-

bonyl)-7-oxa-5-azaspiro[3.4]octan-6-one (4g). A mixture of 50

(500 mg, 1.06 mmol), 24 (182 mg, 1.06 mmol), EDCI (224 mg,

1.17 mmol), HOBt-H₂O (179 mg, 1.17 mmol), DIPEA (0.556 mL,

3.18 mmol), and DMF (3.54 mL) was stirred at rt for 16 h. The

mixture was partitioned between EtOAc and water. The organic

layer was washed with brine, dried over MgSO₄, passed through a

short pad of NH silica gel, and concentrated in vacuo. The resultant

residue was washed with IPE/EtOAc to give 4g (239 mg, 0.528

1

mmol, 50%) as a white solid. H NMR (300 MHz, DMSO-d₆) δ

2.20−2.40 (4H, m), 2.66−2.81 (1H, m), 3.73 (1H, dd, J = 10.6, 4.0

Hz), 3.92−4.10 (2H, m), 4.28 (1H, dd, J = 8.2, 6.5 Hz), 4.34 (2H,

s), 4.39−4.50 (1H, m), 4.64 (2H, s), 7.89 (1H, d, J = 8.1 Hz), 7.99

(1H, s), 8.02-8.14 (2H, m). LCMS (ESI/APCI) m/z 453.1 [M +

S

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NH silica gel, and concentrated in vacuo. The resultant residue was

DMSO-d₆) δ 1.40 (9H, s), 1.54−1.79 (2H, m), 2.15−2.42 (2H, m),

3.36 (1H, dt, J = 6.5, 3.0 Hz), 3.42−3.53 (1H, m), 3.76 (1H, dd, J

= 11.7, 3.8 Hz), 3.93 (2H, s), 8.11 (1H, brs). LCMS (ESI/APCI)

m/z 174.1 [M − t-Bu + H]⁺.

washed with IPE to give 4k (152 mg, 0.417 mmol, 32%) as a white

1

solid. H NMR (300 MHz, DMSO-d₆) δ 2.20−2.37 (7H, m), 2.67−

2.77 (1H, m), 3.65 (1H, dd, J = 10.2, 3.8 Hz), 3.90 (1H, dd, J =

9.6, 4.2 Hz), 4.00 (1H, dd, J = 10.3, 6.3 Hz), 4.19−4.27 (1H, m),

4.28−4.43 (5H, m), 7.18 (1H, dd, J = 8.3, 1.9 Hz), 7.32 (1H, s),

7.39 (1H, d, J = 8.1 Hz), 8.09 (1H, s). ¹³C NMR (101 MHz,

DMSO-d₆) δ 20.0, 26.3, 38.1, 54.9, 55.4, 57.1, 67.5, 69.6, 75.7,

127.6, 129.2, 131.2, 133.0, 135.8, 137.1, 158.0, 173.1. LCMS (ESI/

APCI) m/z 365.2 [M + H]⁺. Anal. Calcd for C₁₈H₂₁ClN₂O₄: C,

59.26; H, 5.80; N, 7.68. Found: C, 59.43; H, 5.94; N, 7.72. HPLC

purity 100%. Mp 146 °C.

3-(5-Oxomorpholin-3-yl)propanoic Acid (11). Compound 10

(236 mg, 1.03 mmol) was dissolved in TFA (4.00 mL) at rt, and

the mixture was stirred at rt overnight. The mixture was

concentrated in vacuo, and the residue was azeotroped with toluene

(2 times). The residue was crystallized from EtOAc/IPE/hexane to

1

give 11 (165 mg, 0.953 mmol, 93%) as a white solid. H NMR (300

MHz, DMSO-d₆) δ 1.56−1.82 (2H, m), 2.19−2.40 (2H, m), 3.35−

3.42 (1H, m), 3.42−3.51 (1H, m), 3.77 (1H, dd, J = 11.3, 3.4 Hz),

3.93 (2H, s), 8.13 (1H, s), 12.11 (1H, s). LCMS (ESI/APCI) m/z

174.2 [M + HM + H]⁺.

2-Oxo-2,3-dihydro-1H-pyrido[3,4-b][1,4]oxazine-7-carboxylic

Acid (6). To a solution of 5 (0.15 g, 0.83 mmol) in t-BuOH/H₂O

(10/10 mL) were added 2-methyl-2-butene (0.5 mL), NaClO₂(0.45

g, 5.0 mmol) and NaH₂PO₄(0.21 g, 1.3 mmol), the mixture was

stirred at rt for 3 h. After being concentrated in vacuo, the mixture

was puriﬁed by Pre-HPLC [Gilson-GX281; Column: Waters X-

Bridge C18: 100 mm×30 mm 5 μm; Mobile Phase: from 70%

[water + 10 mM TFA] and 30% [MeCN] to 50% [water + 10 mM

TFA] and 50% [MeCN] in 8 min, then changed to 5% [water + 10

mM TFA] and 95% [MeCN] in 0.2 min and under this condition

for 3.8 min; ﬂow rate: 20 mL/min; Column temperature: rt] to give

tert-Butyl-3-(hydroxyimino)cyclobutanecarboxylate (13a). To a

stirred solution of 12a (500 g, 2.94 mol) in EtOH (9 L) was added

NaOAc (976 g, 11.8 mol) and hydroxylamine hydrochloride (409 g,

5.88 mol) at rt, and the reaction mixture was reﬂuxed for 4 h. The

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

concentrated in vacuo. The residue was poured into water and

extracted with EtOAc. The organic layer was washed with brine,

dried over Na₂SO₄, ﬁltered, and concentrated in vacuo to give 13a

1

(500 g, 2.70 mmol, 92%) as an oﬀ-white solid. H NMR (400 MHz,

1

6 (0.11 g, 0.57 mmol, 68%) as a white solid. H NMR (400 MHz,

CDCl₃) δ 1.46 (9H, s), 3.09−3.16 (5H, m), 6.83 (1H, brs).

Ethyl 3-(Hydroxyimino)cyclobutanecarboxylate (13b). A mix-

ture of 12b (25.6 g, 180 mmol), hydroxylamine hydrochloride (15.0

g, 216 mmol), NaOAc (17.7 g, 216 mmol), and EtOH (200 mL)

was reﬂuxed for 18 h. After being cooled to rt, the precipitates were

ﬁltered, and the ﬁltrate was concentrated in vacuo. The residue was

diluted with EtOAc and water, and then extracted with EtOAc. The

organic layer was separated, washed with water and brine, dried over

Na₂SO₄, ﬁltered through a silica gel pad, and concentrated in vacuo.

The residue was puriﬁed by column chromatography (silica gel,

eluted with 0−60% EtOAc in hexane) to give 13b (25.1 g, 160

DMSO-d₆) δ 4.79 (2H, s), 7.57 (1H, s), 8.24 (1H, s), 11.26 (1H,

s). LCMS (ESI/APCI) m/z 195.1 [M + H]⁺.

tert-Butyl 4-(((Benzyloxy)carbonyl)amino)-5-hydroxypenta-

noate (8). To a stirred mixture of N-α-Carbobenzoxy-DL-glutamic

acid γ-t-butyl ester 7 (1.00 g, 2.96 mmol) and Et₃N (0.618 mL, 4.45

mmol) in THF (10.0 mL) was added dropwise isobutyl

chloroformate (0.461 mL, 3.56 mmol) at 0 °C, and the mixture

was stirred at the same temperature for 30 min. To this mixture was

added dropwise a solution of NaBH₄(336 mg, 8.89 mmol) in water

(3.00 mL), and the mixture was stirred at 0 °C for 30 min. The

reaction was quenched with sat. NH₄Cl at 0 °C, and the mixture

was stirred at rt for 30 min. The mixture was extracted with EtOAc,

washed with brine, dried over Na₂SO₄, and concentrated in vacuo.

The residue was puriﬁed by column chromatography on silica gel

with EtOAc/hexane (10−80%) to give 8 (841 mg, 2.60 mmol, 88%)

1

mmol, 89%) as a colorless oil. H NMR (300 MHz, CDCl₃) δ 1.28

(3H, t, J = 7.2 Hz), 3.04−3.32 (5H, m), 4.19 (2H, q, J = 7.2 Hz),

7.73 (1H, s). LCMS (ESI/APCI) m/z 158.1 [M + H]⁺.

tert-Butyl 3-Nitrocyclobutanecarboxylate (14). To a stirred

suspension of UHP (305 g, 3.24 mol) in MeCN (2 L) was added

dropwise a solution of TFAA (454 mL, 3.24 mol) in acetonitrile (1

L) at −10 °C over 1 h, and the reaction mixture was allowed to

warm up to rt for 1 h. The reaction mixture was added to a solution

of 13a (200 g, 1.08 mol) and Na₂HPO₄(1.08 kg, 7.57 mol) in

MeCN (2 L) at 80 °C over 30 min. The reaction mixture was

stirred at the same temperature for 30 min. After that, the reaction

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

poured into water, and extracted with EtOAc. The organic layer was

washed with brine, dried over Na₂SO₄, ﬁltered, and concentrated in

vacuo. The residue was puriﬁed by column chromatography (silica

gel, eluted with 6−10% EtOAc/petroleum ether) to aﬀord 14 (140

1

as a colorless oil. H NMR (300 MHz, DMSO-d₆) δ 1.38 (9H, s),

1.42−1.56 (1H, m), 1.69−1.87 (1H, m), 2.11−2.26 (2H, m), 3.18−

3.29 (1H, m), 3.33−3.38 (1H, m), 3.44 (1H, d, J = 5.3 Hz), 4.66

(1H, t, J = 5.7 Hz), 4.91−5.10 (2H, m), 6.97 (1H, d, J = 8.7 Hz),

7.25−7.44 (5H, m).

tert-Butyl 4-Amino-5-hydroxypentanoate (9). A mixture of 8

(840 mg, 2.60 mmol) and 10% Pd-C (50.0 mg, 0.47 mmol) in

EtOAc (8.00 mL) was stirred at rt for 6 h under H₂atmosphere.

The mixture was ﬁltered through a Celite pad, and the ﬁltrate was

concentrated in vacuo. The residue was dissolved in EtOAc (8.00

mL), and 20% Pd(OH)₂-C (50.0 mg, 0.36 mmol) was added

thereto under N₂atmosphere. The mixture was stirred at rt under

H₂atmosphere for 6 h. The mixture was ﬁltered through a pad of

Celite, and the ﬁltrate was concentrated in vacuo to give 9 (490 mg,

2.59 mmol, 100%) as a pale brown oil. ¹H NMR (300 MHz,

DMSO-d₆) δ 1.22−1.37 (1H, m), 1.39 (9H, s), 1.43−1.69 (3H, m),

2.12−2.38 (2H, m), 2.53−2.61 (1H, m), 3.06−3.27 (2H, m), 4.49

(1H, brs). LCMS (ESI/APCI) m/z 134.2 [M − t-Bu + H]⁺.

tert-Butyl 3-(5-Ooxomorpholin-3-yl)propanoate (10). To a

stirred solution of 9 (488 mg, 2.58 mmol) and Et₃N (0.430 mL,

3.09 mmol) in THF (8.00 mL) was added dropwise chloroacetyl

chloride (0.205 mL, 2.58 mmol) at −78 °C, and the mixture was

stirred at the same temperature for 30 min. To this mixture was

added t-BuOK (817 mg, 6.19 mmol) at −78 °C, and the mixture

was allowed to warm up to rt. After being stirred at the same

temperature for 2 h, the mixture was diluted with aq.NH₄Cl and

extracted with EtOAc. The extract was washed with water and brine,

dried over Na₂SO₄, and concentrated in vacuo. The residue was

puriﬁed by column chromatography on silica gel with EtOAc/

hexane (50−100%) and MeOH/EtOAc (5−9%) to give 10 (237

mg, 1.03 mmol, 40%) as a white solid. ¹H NMR (300 MHz,

1

g, 0.696 mol, cis/trans mixture, 64%) as a colorless oil. H NMR

(400 MHz, CDCl₃) δ 1.47 (9H, s), 2.71−2.91 (4H, m), 3.12−3.27

(1H, m), 4.78−4.82 (1H, m).

Ethyl 3-Nitrocyclobutanecarboxylate (15a and 15b). To a

mixture of 13b (5.00 g, 31.8 mmol), Na₂HPO₄(45.2 g, 318 mmol),

UHP (8.98 g, 95.4 mmol), and MeCN (60 mL) was added TFAA

(27.0 mL, 191 mmol) in MeCN (40 mL) dropwise over 20 min at

80 °C, and the mixture was stirred at the same temperature for 1.8

h. After being cooled to rt, the mixture was diluted with EtOAc and

water, and then extracted with EtOAc. The organic layer was

separated, washed with brine, dried over Na₂SO₄, ﬁltered through

silica gel pad, and concentrated in vacuo. The residue was puriﬁed

by column chromatography (silica gel, eluted with 0−40% EtOAc in

hexane) to give 15a (1.00 g, 5.77 mmol, 18%, minor single isomer,

less polar on Si-TLC) and 15b (1.38 g, 7.97 mmol, 25%, major

1

single isomer, more polar on Si-TLC) as a pale yellow oil. 15a: H

NMR (300 MHz, CDCl₃) δ 1.29 (3H, t, J = 7.2 Hz), 2.73−3.01

(4H, m), 3.21−3.34 (1H, m), 4.19 (2H, q, J = 7.2 Hz), 5.03−5.18

1

(1H, m); 15b: H NMR (300 MHz, CDCl₃) δ 1.28 (3H, t, J = 7.2

T

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Hz), 2.72−3.04 (5H, m), 4.19 (2H, q, J = 7.1 Hz), 4.77−4.92 (1H,

m).

(1s,3s)-tert-Butyl-3-(hydroxymethyl)-3-nitrocyclobutanecarboxy-

late (22a). To a stirred solution of 14 (300 g, 1.49 mol) in MeCN

(4 L) was added 37% (w/v) formaldehyde solution (243 mL, 2.99

mol) at rt, followed by the dropwise addition of Et₃N (211 mL, 1.57

mol) at 0 °C, and the reaction mixture was stirred at rt for 2 h.

After concentration in vacuo, the crude compound was puriﬁed by

column chromatography (silica gel, eluted with 15−35% EtOAc/

petroleum ether) to aﬀord 22a (180 g, 0.778 mol, 52%) as a white

(1r,3s)-tert-Butyl-3-(3-methoxy-3-oxopropyl)-3-nitrocyclobuta-

necarboxylate (16). To a stirred solution of 14 (150 g, 0.750 mol)

in MeCN (1.5 L) was added DBU (133 mL, 0.900 mol) at 0 °C,

followed by the dropwise addition of methyl acrylate (77.3 mL,

0.900 mol) at 0 °C, and the reaction mixture was stirred at the same

temperature for 1 h. The reaction mixture was quenched with water

and extracted with EtOAc. The organic layer was washed with brine,

dried over Na₂SO₄, ﬁltered, and concentrated in vacuo. The residue

was puriﬁed by column chromatography (silica gel, eluted with 6−

8% EtOAc/petroleum ether) to aﬀord 16 (65 g, 0.226 mol, 30%, cis

1

solid. H NMR (500 MHz, CDCl₃) δ 1.45 (9H, s), 2.57−2.62 (m,

3H), 2.79−2.86 (1H, m), 2.92−2.96 (2H, m), 4.02 (2H, d, J = 4.5

Hz). LCMS (ESI/APCI) m/z 174.1 [M − t-Bu − H]⁻.

(1s,3s)-Ethyl 3-(Hydroxymethyl)-3-nitrocyclobutane-1-carboxy-

late (22b). To a mixture of 15b (820 mg, 4.74 mmol), Et₃N

(0.726 mL, 5.21 mmol), and MeCN (8 mL) was added 35% (w/v)

formaldehyde solution (0.750 mL, 9.47 mmol), and the mixture was

stirred at rt for 1 h. The mixture was concentrated in vacuo, and the

residue was puriﬁed by column chromatography (silica gel, eluted

with 0−50% EtOAc in hexane) to give 22b (452 mg, 2.22 mmol,

47%) and its stereoisomer (244 mg, 1.20 mmol, 25%) as a pale

1

isomer) as a pale yellow solid. H NMR (500 MHz, CDCl₃) δ 1.44

(9H, s), 2.29−2.39 (4H, m), 2.49−2.53 (2H, m), 2.80 (1H, quin, J

= 8.8 Hz), 2.92−2.97 (2H, m), 3.69 (3H, s). LCMS (ESI/APCI)

m/z 230.1 [M − t-Bu − H]⁻.

(2r,4s)-tert-Butyl 6-Oxo-5-azaspiro[3.4]octane-2-carboxylate

(17). To a stirred solution of 16 (65.0 g, 0.230 mol) in MeOH

(650 mL) was added NiCl₂·6H₂O (53.7 g, 0.230 mol) at rt,

followed by portionwise addition of NaBH₄(43.0 g, 1.13 mol) at

−10 °C, and the reaction mixture was stirred at the same

temperature for 1 h. After that, K₂CO₃(125 g, 0.910 mol) in

water was added thereto at below 10 °C and stirred at rt for 2 h.

The reaction mixture was quenched with water and extracted with

EtOAc. The organic layer was washed with brine, dried over

Na₂SO₄, ﬁltered, and concentrated in vacuo. The crude residue was

washed with n-pentane to aﬀord 17 (40 g, 0.18 mmol, 78%) as a

1

yellow oil. H NMR (300 MHz, CDCl₃) δ 1.28 (3H, t, J = 7.1 Hz),

2.34 (1H, brs), 2.58−2.71 (2H, m), 2.84−3.11 (3H, m), 4.05 (2H,

brs), 4.18 (2H, q, J = 7.2 Hz). LCMS (ESI/APCI) m/z 174.1 [M −

t-Bu − H]⁻.

(2s,4s)-tert-Butyl-6-oxo-7-oxa-5-azaspiro[3.4]octane-2-carboxy-

late (23a). A mixture of 22a (210 g, 0.908 mol) and Raney nickel

(20.0 g) in isopropyl alcohol (2 L) was hydrogenated (50 bar) in an

autoclave at 70 °C for 3 h. The reaction mixture was ﬁltered

through a Celite pad. The ﬁltrate was concentrated in vacuo and

washed with n-pentane to aﬀord the corresponding amine (150 g,

0.745 mol, 82%) as a crude material (white solid), which was used

in the next step without further puriﬁcation.

1

white solid. H NMR (400 MHz, CDCl₃) δ 1.44 (9H, s), 2.17−2.21

(2H, m), 2.33−2.39 (6H, m), 2.76 (1H, quin, J = 8.40 Hz), 5.98

(1H, brs). LCMS (ESI/APCI) m/z 226.2 [M + H]⁺.

(2r,4s)-6-Oxo-5-azaspiro[3.4]octane-2-carboxylic Acid (18).

Compound 17 (80.0 g, 0.36 mol) was added to TFA (800 mL)

at 0 °C, and the reaction mixture was stirred at rt for 4 h. After that,

the reaction mixture was concentrated in vacuo. The residue was

azeotroped with toluene (three times) to give crude compound. The

crude material was stirred with methanol, ﬁltered, and dried in

To a stirred solution of the crude amine (120 g, 0.596 mol) in

THF (3 L) was added Et₃N (177 mL, 1.31 mol) at rt followed by

the dropwise addition of triphosgene (62.0 g, 0.210 mol) in THF

(0.5 L) at −10 °C, and the reaction mixture was stirred at the same

temperature for 30 min. Then, the reaction mixture was stirred at rt

for 2 h. The reaction was quenched with sat. NaHCO₃and

extracted with EtOAc. The organic layer was washed with brine,

dried over Na₂SO₄, ﬁltered, and concentrated in vacuo. The crude

compound was puriﬁed by column chromatography (silica gel,

eluted with 50% EtOAc/petroleum ether) to aﬀord 23a (110 g,

1

vacuo to aﬀord 18 (52.0 g, 0.307 mol, 86%) as a white solid. H

NMR (500 MHz, DMSO-d₆) δ 2.06−2.10 (2H, m), 2.12−2.15 (2H,

m), 2.19−2.23 (2H, m), 2.27−2.31 (2H, m), 2.71−2.78 (1H, m),

7.97 (1H, s), 12.14 (1H, brs). LCMS (ESI/APCI) m/z 170.2 [M +

H]⁺.

1

0.484 mol, 81%) as a white solid. H NMR (500 MHz, CDCl₃) δ

(1s,3r)- Ethyl 3-(3-Methoxy-3-oxopropyl)-3-nitrocyclobutane-1-

carboxylate (19). To a cooled (0 °C) solution of 15a (518 mg,

2.99 mmol) and methyl acrylate (0.323 mL, 3.59 mmol) in MeCN

(5 mL) was added DBU (0.224 mL, 1.50 mmol), and the mixture

was stirred at the same temperature for 20 min. The reaction was

quenched with aq. NH₄Cl and extracted with EtOAc. The organic

layer was separated, washed with water and brine, dried over

Na₂SO₄, and concentrated in vacuo. The residue was puriﬁed by

column chromatography (silica gel, eluted with 0−40% EtOAc in

hexane) to give 19 (196 mg, 0.756 mmol, 25%) and its stereoisomer

1.45 (9H, s), 2.46−2.53 (4H, m), 2.72 (1H, quin, J = 8.0 Hz), 4.34

(2H, s), 5.80 (1H, brs). LCMS (ESI/APCI) m/z 228.2 [M + H]⁺.

(2s,4s)-Ethyl 6-Oxo-7-oxa-5-azaspiro[3.4]octane-2-carboxylate

(23b). A mixture of 22b (770 mg, 3.79 mmol) and 10% Pd-C

(145 mg, 1.22 mmol) in MeOH (10 mL)/THF (10 mL) was

hydrogenated under balloon pressure at rt for 72 h. The catalyst was

removed by ﬁltration, and the ﬁltrate was concentrated in vacuo to

give (1s,3s)-ethyl 3-amino-3-(hydroxymethyl)cyclobutene-1-carbox-

ylate (659 mg, 3.80 mmol, 100%) as a pale yellow oil. This product

was subjected to the next reaction without further puriﬁcation.

To a cooled (−10 °C) solution of the crude amine (656 mg, 3.79

mmol), DIPEA (1.45 mL, 8.33 mmol), and THF (8 mL) was added

triphosgene (393 mg, 1.33 mmol), and the mixture was stirred at

the same temperature for 30 min. The mixture was diluted with

water and extracted with EtOAc. The organic layer was separated,

washed with brine, dried over Na₂SO₄, and concentrated in vacuo.

The residue was puriﬁed by column chromatography (silica gel,

eluted with 0−90% EtOAc in hexane) to give 23b (381 mg, 1.91

1

(533 mg, 2.056 mmol, 68%) as a colorless oil. H NMR (300 MHz,

CDCl₃) δ 1.28 (3H, t, J = 7.2 Hz), 2.24−2.33 (2H, m), 2.41−2.49

(2H, m), 2.55−2.66 (2H, m), 2.99−3.12 (2H, m), 3.15−3.29 (1H,

m), 3.68 (3H, s), 4.18 (2H, q, J = 7.2 Hz).

(2s,4r)-6-Oxo-5-azaspiro[3.4]octane-2-carboxylic acid (21). To

a cooled (−10 °C) mixture of 19 (195 mg, 0.75 mmol), NiCl₂·

6H₂O (179 mg, 0.75 mmol), and MeOH (2 mL) was added NaBH₄

(142 mg, 3.76 mmol) portionwise (×3). After being stirred at the

same temperature for 2 h, the reaction was quenched with aq.

K₂CO₃(416 mg in water (1 mL)) at 0 °C. The mixture was stirred

at 0 °C for 3 h and at rt for 2 h, and then ﬁltered through a Celite

pad. The ﬁltrate was concentrated in vacuo, and the residue was

dissolved in THF (2 mL)/MeOH (2 mL)/water (1 mL). To the

mixture was added 2 M NaOH (0.752 mL, 1.50 mmol), and the

mixture was stirred at rt overnight. The mixture was acidiﬁed with 6

M HCl to pH 4 and concentrated in vacuo. The crude residue was

subjected to the next reaction without further puriﬁcation. LCMS

(ESI/APCI) m/z 170.1[M + H]⁺.

1

mmol, 2 steps 51% from 22b) as a white solid. H NMR (300 MHz,

CDCl₃) δ 1.27 (3H, t, J = 7.2 Hz), 2.47−2.64 (4H, m), 2.74−2.88

(1H, m), 4.17 (2H, q, J = 7.1 Hz), 4.37 (2H, s), 5.80 (1H, brs).

LCMS (ESI/APCI) m/z 200.0 [M + H]⁺.

(2s,4s)-6-Oxo-7-oxa-5-azaspiro[3.4]octane-2-carboxylic Acid

(24). To 23a (110 g, 0.484 mol) was added precooled TFA (1

L) at 0 °C, and the reaction mixture was stirred at rt for 2 h. After

concentration in vacuo, the residue was azeotroped with toluene

(×3) to give a crude compound. The crude compound was stirred

with n-pentane, collected, and dried in vacuo to aﬀord 24 (76.0 g,

U

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1

0.444 mol, 92%) as a white solid. H NMR (500 MHz, DMSO-d₆)

3.65 (2H, m), 7.86 (1H, brs). LCMS (ESI/APCI) m/z 199.1 [M +

H]⁺.

δ 2.33−2.39 (4H, m), 2.73 (1H, quin, J = 9.08 Hz), 4.34 (2H, s),

8.09 (1H, s), 12.24 (1H, brs). LCMS (ESI/APCI) m/z 170.1 [M-

H]⁻.

1,7-Diazaspiro[4.5]decan-2-one bis(4-methylbenzenesulfonate)

(32). A mixture of 31 (603 mg, 2.37 mmol) and p-TsOH-H₂O (992

mg, 5.22 mmol) in EtOAc (15 mL) was reﬂuxed for 18 h. After

being cooled to rt, the mixture was concentrated in vacuo, and the

residual solid was collected, washed with EtOAc, and dried in vacuo

to give 32 (1.17 g, 2.35 mmol, 99%) as a white solid. ¹H NMR

(300 MHz, DMSO-d₆) δ 1.56−1.89 (4H, m), 1.97−2.10 (1H, m),

2.12−2.36 (8H, m), 2.80−3.19 (4H, m), 4.77−5.14 (1H, m), 7.12

(4H, d, J = 7.8 Hz), 7.48 (4H, d, J = 8.1 Hz), 7.75 (1H, s), 8.12-

8.92 (2H, m). LCMS (ESI/APCI) m/z 155.1 [M + H]⁺.

tert-Butyl 4-((2-Chloro-4-ﬂuorophenoxy)methyl)piperidine-1-

carboxylate (34). To a cooled (0 °C) solution of 33 (2.50 g,

11.6 mmol), 2-chloro-4-ﬂuorophenol (1.49 mL, 13.9 mmol), and

PBu₃(3.47 mL, 13.9 mmol) in THF (50 mL) was added ADDP

(3.52 g, 13.9 mmol), and the mixture was stirred at rt for 18 h.

After ﬁltration through a silica gel/NH silica gel pad, the ﬁltrate was

concentrated and puriﬁed by column chromatography (silica gel,

eluted with 0−50% EtOAc in hexane) to give 34 (3.81 g, 11.1

mmol, 95%) as a colorless oil. ¹H NMR (300 MHz, CDCl₃) δ

1.21−1.38 (2H, m), 1.42−1.51 (9H, m), 1.78−2.11 (3H, m), 2.64−

2.85 (2H, m), 3.75−3.97 (2H, m), 4.04−4.37 (2H, m), 6.80−7.00

(2H, m), 7.05−7.16 (1H, m). LCMS (ESI/APCI) m/z 288.1 [M −

t-Bu + H]⁺.

tert-Butyl 3-(3-Methoxy-3-oxopropyl)-3-nitroazetidine-1-car-

boxylate (26). To a stirred solution of 25 (65 g, 0.32 mol) in

MeOH (650 mL) were added methyl acrylate (34.7 mL, 0.385 mol)

and K₂CO₃(53.1 g, 0.385 mol) at 0 °C and the resultant reaction

mixture was stirred at the same temperature for 3 h. After

concentration in vacuo, the residue was quenched with sat. NH₄Cl

and extracted with EtOAc. The organic layer was washed with brine,

dried over Na₂SO₄, ﬁltered, and concentrated in vacuo. The crude

compound was puriﬁed by column chromatography (silica gel,

eluted with 16−18% EtOAc/petroleum ether) to aﬀord 26 (49 g,

1

0.17 mol, 53%) as a colorless oil. H NMR (400 MHz, CDCl₃) δ

1.47 (9H, s), 2.34−2.38 (2H, m), 2.52−2.56 (2H, m), 3.70 (3H, s),

4.03 (2H, d, J = 10.80 Hz), 4.45 (2H, d, J = 10.00 Hz). LCMS

(ESI/APCI) m/z 233.1 [M − t-Bu + H]⁺.

tert-Butyl-6-oxo-2,5-diazaspiro[3.4]octane-2-carboxylate (27).

To a stirred mixture of 26 (49 g, 0.17 mol) and NiCl₂·6H₂O

(40.3 g, 0.169 mol) in MeOH (500 mL) was added NaBH₄(32.1 g,

0.849 mol) at −10 °C, and the reaction mixture was stirred at the

same temperature for 2 h. The reaction was quenched with aq.

K₂CO₃(100 mL), and the reaction mixture was stirred at rt for 2 h.

The reaction mixture was ﬁltered through a Celite pad, and the

ﬁltrate was concentrated in vacuo. The residue was diluted with

water and extracted with EtOAc. The organic layer was dried over

anhydrous Na₂SO₄, ﬁltered, and concentrated in vacuo. The crude

compound was diluted with Et₂O and stirred for 1 h, collected, and

dried in vacuo to aﬀord 27 (29 g, 0.13 mol, 76%) as a white solid.

¹H NMR (400 MHz, CDCl₃) δ 1.46 (9H, s), 2.37−2.40 (4H, m),

4-((2-Chloro-4-ﬂuorophenoxy)methyl)piperidine Hydrochloride

(35). To a solution of 34 (3.81 g, 11.1 mmol) in EtOAc (30

mL) was added 4 M HCl-EtOAc (22.2 mL, 88.7 mmol), and the

mixture was stirred at rt for 14 h. The resultant precipitates were

collected, washed with EtOAc, and dried in vacuo to give 35 (2.09

1

g, 7.46 mmol, 67%) as a white solid. H NMR (300 MHz, DMSO-

d₆) δ 1.43−1.62 (2H, m), 1.84−1.98 (2H, m), 2.01−2.17 (1H, m),

2.83−2.97 (2H, m), 3.23−3.36 (2H, m), 3.93 (2H, d, J = 6.2 Hz),

7.13−7.26 (2H, m), 7.39−7.48 (1H, m), 8.81 (2H, brs). LCMS

(ESI/APCI) m/z 244.1 [M + H]⁺.

4.00−4.02 (4H, m), 6.30 (1H, brs). LCMS (ESI/APCI) m/z 171.3

[M − t-Bu + H]⁺.

2,5-Diazaspiro[3.4]octan-6-one bis(4-methylbenzenesulfonate)

(28). A mixture of 27 (28 g, 0.12 mol) and p-TsOH-H₂O (51.8

g, 0.272 mol) in EtOAc (450 mL) was heated to 75 °C for 3 h.

After being cooled to rt, the resulting precipitates were collected,

washed with Et₂O, and dried in vacuo to aﬀord 28 (52.5 g, 0.112

mol, 90%) as a white solid. ¹H NMR (400 MHz, DMSO-d₆) δ

2.18−2.21 (2H, m), 2.29−2.35 (8H, m), 3.93−4.09 (4H, m), 7.13

(4H, d, J = 8.0 Hz), 7.49 (4H, d, J = 8.0 Hz), 8.28 (1H, brs), 8.46

(1H, brs), 8.83 (1H, brs). LCMS (ESI/APCI) m/z 127.2 [M + H]⁺.

tert-Butyl 3-(3-Methoxy-3-oxopropyl)-3-nitropiperidine-1-car-

boxylate (30). To a cooled (0 °C) solution of 29 (1.09g, 4.73

mmol) and methyl acrylate (0.192 mL, 4.73 mmol) in MeOH (10

mL) was added K₂CO₃(0.785 g, 5.68 mmol), and the mixture was

stirred at rt for 4 h. The mixture was poured into sat. NaHCO₃and

extracted with EtOAc. The organic layer was washed with and brine,

dried over MgSO₄, and concentrated in vacuo. The residue was

puriﬁed by column chromatography (silica gel, eluted with 0−60%

EtOAc in hexane) to give 30 (0.923 g, 2.92 mmol, 62%) as a

tert-Butyl 4-((2-Chloro-4-ﬂuorophenoxy)methyl)-4-methylpiper-

idine-1-carboxylate (37). To a stirred mixture of 36 (1.00 g, 4.36

mmol) and 1-chloro-2,5-diﬂuorobenzene (0.503 mL, 4.58 mmol) in

THF (10 mL) was added t-BuOK (587 mg, 5.23 mmol) at 0 °C,

and the mixture was stirred at rt overnight. The mixture was heated

to reﬂux for 2 h, and then 1-chloro-2,5-diﬂuorobenzene (0.503 mL,

4.58 mmol) and t-BuOK (587 mg, 5.23 mmol) were added to the

mixture at rt. The mixture was heated to reﬂux for further 2 h, and

then partitioned between water and EtOAc. The organic layer was

washed with water and brine, dried over Na₂SO₄, and concentrated

in vacuo. The residue was puriﬁed by column chromatography on

silica gel with EtOAc/hexane (0−15%) to give 37 (881 mg, 2.46

1

mmol, 57%) as a colorless oil. H NMR (300 MHz, CDCl₃) δ 1.16

(3H, s), 1.46 (11H, s), 1.59−1.70 (2H, m), 3.21 (2H, ddd, J = 13.7,

9.9, 3.6 Hz), 3.64−3.81 (4H, m), 6.79−6.97 (2H, m), 7.12 (1H, dd,

J = 7.9, 3.0 Hz). LCMS (ESI/APCI) m/z 302.1 [M − t-Bu + H]⁺.

4-((2-Chloro-4-ﬂuorophenoxy)methyl)-4-methylpiperidine Hy-

drochloride (38). A mixture of 37 (300 mg, 0.838 mmol) and 2

M HCl in MeOH (419 μL, 0.838 mmol) was stirred at rt overnight.

The mixture was concentrated in vacuo to give 38 (226 mg, 0.768

1

colorless oil. H NMR (300 MHz, DMSO-d₆) δ 1.38 (9H, s), 1.40−

1.49 (1H, m), 1.52−1.66 (1H, m), 1.77−1.93 (1H, m), 2.00−2.16

(2H, m), 2.23−2.42 (3H, m), 2.86−3.09 (1H, m), 3.32−3.43 (1H,

m), 3.59 (3H, s), 3.59−3.69 (1H, m), 4.30 (1H, d, J = 14.2 Hz).

LCMS (ESI/APCI) m/z 261.0 [M − t-Bu + H]⁺.

1

mmol, 92%) as a white solid. H NMR (300 MHz, DMSO-d₆) δ

1.12 (3H, s), 1.49−1.68 (2H, m), 1.73−1.91 (2H, m), 3.01−3.23

(4H, m), 3.86 (2H, s), 7.20 (2H, dd, J = 6.8, 1.9 Hz), 7.45 (1H, dt,

J = 8.5, 1.4 Hz), 8.35-8.85 (2H, m). LCMS (ESI/APCI) m/z 258.1

[M + H]⁺.

tert-Butyl 4-(1-(2-Chloro-4-ﬂuorophenoxy)ethyl)piperidine-1-

carboxylate (40a). To a mixture of 39a (3.02 g, 13.2 mmol) and

Et₃N (3.67 mL, 26.3 mmol) in THF (30 mL) at 0 °C was added

MsCl (1.22 mL, 15.8 mmol). After being stirred at rt for 3 days,

MsCl (0.306 mL, 3.95 mmol) and Et₃N (1.83 mL, 13.2 mmol) were

added thereto. The mixture was stirred for 3 days, and then MsCl

(0.306 mL, 3.95 mmol) and Et₃N (1.83 mL, 13.2 mmol) were

added. The mixture was stirred at rt for 6 h, and then EtOAc and

water were added to the reaction mixture. The EtOAc layer was

separated, washed with water and brine, dried over Na₂SO₄,

tert-Butyl 2-Oxo-1,7-diazaspiro[4.5]decane-7-carboxylate (31).

To a cooled (0 °C) mixture of 30 (923 mg, 2.92 mmol), NiCl₂·

6H₂O (694 mg, 2.92 mmol), and MeOH (20 mL) was added

NaBH₄(552 mg, 14.59 mmol) portionwise. After being stirred at

the same temperature for 2 h, the reaction was quenched with aq.

K₂CO₃[0.416 g in water (8 mL)]. The mixture was stirred at rt for

1.5 h and ﬁltered through a Celite pad. The ﬁltrate was partitioned

between EtOAc and water. The organic layer was washed with

brine, dried over MgSO₄, ﬁltered through a silica gel pad eluting

with EtOAc/MeOH (4:1 v/v), and concentrated in vacuo to give 31

1

(603 mg, 2.37 mmol, 81%) as a colorless oil. H NMR (300 MHz,

DMSO-d₆) δ 1.39 (9H, s), 1.40−1.48 (1H, m), 1.53−1.74 (4H, m),

1.75−1.88 (1H, m), 2.12−2.25 (2H, m), 2.79−3.06 (2H, m), 3.38−

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concentrated in vacuo. The residue was dissolved in MeCN (30.0

mL), and Et₃N (1.83 mL, 13.2 mmol) and MsCl (0.612 mL, 7.90

mmol) were successively added at 0 °C. The mixture was stirred for

3 h. The mixture was diluted with sat. NH₄Cl, and extracted with

EtOAc. The extract was washed with water and brine, dried over

Na₂SO₄, and concentrated in vacuo. The residue was puriﬁed by

column chromatography on silica gel with EtOAc/hexane (50%) to

give tert-butyl 4-(1-((methylsulfonyl)oxy)ethyl)piperidine-1-carbox-

¹H NMR (300 MHz, DMSO-d₆) δ 1.38 (9H, s), 2.90−3.01 (1H,

m), 3.73 (2H, brs), 3.88−4.00 (2H, m), 4.15 (2H, d, J = 6.0 Hz),

7.16−7.23 (2H, m), 7.40−7.46 (1H, m). LCMS (ESI/APCI) m/z

260.0 [M − t-Bu + H]⁺.

4-(1-(2-Chloro-4-ﬂuorophenoxy)ethyl)piperidine 4-Methylben-

zenesulfonate (41a). A mixture of 40a (0.340 g, 0.950 mmol)

and p-TsOH-H₂O (0.207 g, 1.09 mmol) in EtOAc (4 mL) was

reﬂuxed for 2 h. After being cooled to rt, the mixture was

concentrated in vacuo, and the residual semisolid was triturated with

EtOAc/heptane to give 41a (0.376 g, 0.875 mmol, 92%) as a white

1

ylate (3.56 g, 11.6 mmol, 88%) as a yellow oil. H NMR (300 MHz,

CDCl₃) δ 1.18−1.35 (2H, m), 1.41 (3H, d, J = 6.4 Hz), 1.46 (9H,

s), 1.61−1.84 (3H, m), 2.67 (2H, brs), 3.01 (3H, s), 4.09−4.35

(2H, m), 4.56−4.72 (1H, m).

1

solid. H NMR (300 MHz, DMSO-d₆) δ 1.19 (3H, d, J = 6.1 Hz),

1.39−1.64 (2H, m), 1.77−2.05 (3H, m), 2.29 (3H, s), 2.78−3.01

(2H, m), 3.24−3.45 (2H, m), 4.30−4.45 (1H, m), 7.05−7.29 (4H,

m), 7.37−7.55 (3H, m), 8.17 (1H, brs), 8.48 (1H, brs). LCMS

(ESI/APCI) m/z 258.0 [M + H]⁺.

The mesylate (0.617 g, 2.01 mmol), 2-chloro-4-ﬂuorophenol

(0.428 mL, 4.01 mmol), Cs₂CO₃(1.31 g, 4.01 mmol), and DMF (7

mL) was stirred at 80 °C overnight. The mixture was diluted with

EtOAc and water, and extracted with EtOAc. The organic layer was

washed with water and brine, dried over Na₂SO₄, and concentrated

in vacuo. The residue was puriﬁed by column chromatography

(silica gel, eluted with 0−20% EtOAc in hexane) to 40a (0.342 g,

4-(1-(2-Chloro-4-ﬂuorophenoxy)propyl)piperidine (41b). MsCl

(2.00 mL, 25.9 mmol) was added to a solution of 39b (1.80 g, 7.40

mmol) and Et₃N (5.15 mL, 37.0 mmol) in MeCN (30 mL) at 0 °C.

The mixture was stirred at rt over weekend. MsCl (2.00 mL, 25.9

mmol) and Et₃N (5.15 mL, 37.0 mmol) was added to the mixture

and the resulting mixture was stirred at rt overnight. The mixture

was poured into water and extracted with EtOAc. The organic layer

was separated, washed with water and brine, dried over MgSO₄, and

concentrated in vacuo. The residue was puriﬁed by column

chromatography (silica gel, eluted with 25−50% EtOAc in hexane)

to give tert-butyl 4-(1-((methylsulfonyl)oxy)propyl)piperidine-1-

1

0.956 mmol, 48%) as a colorless oil. H NMR (300 MHz, CDCl₃) δ

1.21−1.42 (5H, m), 1.46 (9H, s), 1.66−1.99 (3H, m), 2.61−2.80

(2H, m), 4.04−4.29 (3H, m), 6.80−6.96 (2H, m), 7.11 (1H, dd, J =

8.2, 2.7 Hz). LCMS (ESI/APCI) m/z 302.0 [M − t-Bu + H]⁺.

tert-Butyl 3-((2-Chloro-4-ﬂuorophenoxy)methyl)pyrrolidine-1-

carboxylate (40d). p-TsCl (837 mg, 4.39 mmol) was added to a

solution of 39d (803 mg, 3.99 mmol) in pyridine (10 mL) at 0 °C

and the mixture was stirred at rt for 16 h. The mixture was diluted

with EtOAc and the solution was washed with 1 M HCl, 10%

K₂CO₃, and brine, and dried over Na₂SO₄. The solution was passed

through NH silica gel and concentrated in vacuo to aﬀord tert-butyl

3-((tosyloxy)methyl)pyrrolidine-1-carboxylate (1.38 g, 3.88 mmol,

97%) as a colorless oil. This was used in the next step without

further puriﬁcation.

1

carboxylate (1.37 g, 4.26 mmol, 58%) as a colorless oil. H NMR

(300 MHz, DMSO-d₆) δ 0.91 (3H, t, J = 7.4 Hz), 1.02−1.25 (2H,

m), 1.39 (9H, s), 1.50−1.92 (5H, m), 2.67 (2H, brs), 3.16 (3H, s),

3.90−4.06 (2H, m), 4.45 (1H, q, J = 5.7 Hz). LCMS (ESI/APCI)

m/z 266.2 [M − t-Bu + H]⁺.

A mixture of the mesylate (650 mg, 2.02 mmol), 2-chloro-4-

ﬂuorophenol (0.432 mL, 4.04 mmol), Cs₂CO₃(1.32 g, 4.04 mmol),

and DMF (30 mL) was stirred at 80 °C overnight. The mixture was

diluted with EtOAc and water, and extracted with EtOAc. The

organic layer was washed with water and brine, dried over Na₂SO₄,

and concentrated in vacuo. The residue was puriﬁed by column

chromatography (silica gel, eluted with 0−20% EtOAc in hexane,

inject column: NH to remove the phenol) to give 40b. This was

dissolved in EtOAc (10 mL) and 4 M HCl in EtOAc (10 mL) was

added. The mixture was stirred at rt for 2 h. The mixture was

concentrated, and the residue was puriﬁed by column chromatog-

raphy (NH silica gel, eluted with 0−25% MeOH in EtOAc) to give

A mixture of the tosylate (1.38 g, 3.88 mmol), 2-chloro-4-

ﬂuorophenol (0.508 mL, 4.66 mmol), and K₃PO₄(1.24 g, 5.82

mmol) in DMF (10 mL) was heated at 80 °C for 2 h under N₂

atmosphere. The mixture was diluted with EtOAc and the solution

was washed with water and brine, dried over Na₂SO₄. The solution

was passed through NH silica gel, and the ﬁltrate was concentrated

in vacuo. The residue was chromatographed on silica gel (hexane/

EtOAc = 4:1 to 1:1) to aﬀord 40d (1.11 g, 3.37 mmol, 87%) as a

1

colorless oil. H NMR (300 MHz, DMSO-d₆) δ 1.39 (9H, s), 1.63−

1.82 (1H, m), 1.93−2.09 (1H, m), 2.55−2.69 (1H, m), 3.06−3.30

(2H, m), 3.33−3.50 (2H, m), 3.96−4.05 (2H, m), 7.15−7.21 (2H,

m), 7.39−7.45 (1H, m).

1

41b (235 mg, 0.865 mmol, 43%) as a colorless oil. H NMR (300

MHz, DMSO-d₆) δ 0.88 (3H, t, J = 7.4 Hz), 1.11−1.30 (2H, m),

1.45−1.75 (5H, m), 1.91 (1H, brs), 2.40 (2H, td, J = 12.1, 2.3 Hz),

2.86−2.99 (2H, m), 4.12−4.24 (1H, m), 7.07−7.25 (2H, m), 7.38

(1H, dd, J = 8.3, 3.0 Hz). LCMS (ESI/APCI) m/z 272.1 [M + H]⁺.

4-(1-(Chloro-4-ﬂuorophenoxy)-2,2,2-triﬂuoroethyl)piperidine

(41c). Tf₂O (2.00 mL, 11.9 mmol) was added to a solution of 39c

(2.24 g, 7.91 mmol) in pyridine (25 mL) at 0 °C. The mixture was

stirred at rt overnight. The mixture was concentrated in vacuo, and

the residue was dissolved in EtOAc and 1 M HCl. The organic layer

was separated, washed with water and brine, dried over MgSO₄, and

concentrated in vacuo. The residue was puriﬁed by column

chromatography (silica gel, eluted with 0−50% EtOAc in hexane)

to give tert-butyl 4-(2,2,2-triﬂuoro-1-(((triﬂuoromethyl)sulfonyl)-

oxy)ethyl)piperidine-1-carboxylate (3.00 g, 7.22 mmol, 91%) as a

tert-Butyl 3-((2-Chloro-4-ﬂuorophenoxy)methyl)azetidine-1-car-

boxylate (40e). To a solution of 39e (25.0 g, 134 mmol) in

pyridine (40 mL) was added p-TsCl (28.0 g, 147 mmol) at rt. After

being stirred overnight, the mixture was diluted with EtOAc (250

mL) and water (250 mL). The organic layer was washed with 0.5 M

HCl (100 mL × 3) and brine (150 mL), dried over MgSO₄, and

concentrated in vacuo to give 43g of crude product as pale brown

gum. A seed crystal was to the residue and settled overnight at rt.

The resultant crystal was triturated with hexane to give tert-butyl 3-

((tosyloxy)methyl)azetidine-1-carboxylate (28.0 g, 82.0 mmol, 61%)

1

as a white solid. H NMR (300 MHz, DMSO-d₆) δ 1.28 (2H, td, J

= 12.3, 4.1 Hz), 1.39 (9H, s), 1.72 (2H, t, J = 10.9 Hz), 2.30 (1H,

ddt, J = 11.9, 8.0, 4.2 Hz), 2.77 (2H, brs), 3.91−4.08 (2H, m),

5.85−5.99 (1H, m). LCMS (ESI/APCI) m/z 286.0 [M − t-Bu +

H]⁺.

The tosylate (2.33 g, 6.82 mmol) was added to a mixture of 2-

chloro-4-ﬂuorophenol (0.719 mL, 6.82 mmol) and K₂CO₃(1.89 g,

13.7 mmol) in DMF (10 mL) at rt. The mixture was stirred at 80

°C for 7 h and then stirred at rt overnight. EtOAc and water were

added to the reaction mixture at rt, then the organic layer was

separated. The aqueous layer was extracted with EtOAc. The

combined organic layer was washed with water and brine, dried over

MgSO₄, and concentrated in vacuo. The residue was puriﬁed by

column chromatography (silica gel, eluted with 5−26% EtOAc in

hexane) to give 40e (1.17 g, 3.69 mmol, 54%) as an oﬀ-white solid.

1

colorless oil. H NMR (300 MHz, DMSO-d₆) δ 1.28 (2H, td, J =

12.3, 4.1 Hz), 1.39 (9H, s), 1.72 (2H, t, J = 10.9 Hz), 2.30 (1H,

ddt, J = 11.9, 8.0, 4.2 Hz), 2.77 (2H, brs), 3.91−4.08 (2H, m),

5.85−5.99 (1H, m). LCMS (ESI/APCI) m/z 360.0 [M − t-Bu +

H]⁺.

A mixture of the mesylate (1.45 g, 3.49 mmol), 2-chloro-4-

ﬂuorophenol (0.745 mL, 6.98 mmol), Cs₂CO₃(2.28 g, 6.98 mmol),

and DMF (20 mL) was stirred at 80 °C overnight. The mixture was

diluted with EtOAc and water, and extracted with EtOAc. The

organic layer was washed with water and brine, dried over Na₂SO₄,

and concentrated in vacuo. The residue was puriﬁed by column

chromatography (silica gel, eluted with 0−20% EtOAc in hexane,

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inject column: NH to remove the phenol) to give 40c. This was

dissolved in EtOAc (10 mL) and 4 M HCl in EtOAc (10 mL) was

added. The mixture was stirred at rt for 2 h. The mixture was

concentrated in vacuo, and the residue was puriﬁed by column

chromatography (NH silica gel, eluted with 0−25% MeOH in

EtOAc) to give 41c (0.172 g, 0.552 mmol, 16%) as a colorless oil.

¹H NMR (300 MHz, DMSO-d₆) δ 1.22−1.52 (2H, m), 1.57−1.73

(2H, m), 1.83−2.27 (2H, m), 2.35−2.49 (2H, m), 2.84−3.03 (2H,

m), 5.02−5.19 (1H, m), 7.22 (1H, ddd, J = 9.3, 8.1, 3.0 Hz), 7.35−

7.54 (2H, m). LCMS (ESI/APCI) m/z 312.2 [M + H]⁺.

graphed on silica gel (hexane/EtOAc = 3:1 to 1:1) to aﬀord 43c

(1.82 g, 5.76 mmol, 100%) as a colorless oil. H NMR (300 MHz,

1

DMSO-d₆) δ 1.37 (9H, s), 3.66−3.74 (2H, m), 3.98−4.07 (2H, m),

4.33−4.41 (1H, m), 4.48 (2H, s), 7.24 (1H, td, J = 8.5, 2.6 Hz),

7.46 (1H, dd, J = 9.0, 2.6 Hz), 7.57 (1H, dd, J = 8.5, 6.2 Hz).

4-((2-Chloro-4-ﬂuorobenzyl)oxy)piperidine 4-Methylbenzenesul-

fonate (44a). A mixture of 43a (1.70 g, 4.94 mmol) and p-TsOH-

H₂O (1.04 g, 5.44 mmol) in EtOAc (30 mL) was reﬂuxed for 3 h

and cooled to rt. The resultant precipitate was collected, washed

with EtOAc, and dried in vacuo to give 44a (0.520 g, 1.25 mmol,

1

25%) as a white solid. H NMR (300 MHz, DMSO-d₆) δ 1.65−1.82

3-((2-Chloro-4-ﬂuorophenoxy)methyl)pyrrolidine 4-Methylben-

zenesulfonate (41d). A mixture of 40d (1.11 g, 3.37 mmol) and

p-TsOH-H₂O (0.704 g, 3.70 mmol) in EtOAc (20 mL) was reﬂuxed

for 2 h and cooled to rt. The resultant precipitate was collected,

washed with EtOAc, and dried in vacuo to give 41d (0.983 g, 2.45

(2H, m), 1.91−2.06 (2H, m), 2.29 (3H, s), 2.93−3.07 (2H, m),

3.12−3.25 (2H, m), 3.66−3.79 (1H, m), 4.55 (2H, s), 7.11 (2H, d,

J = 7.9 Hz), 7.20−7.29 (1H, m), 7.42−7.51 (3H, m), 7.57 (1H, dd,

J = 8.7, 6.4 Hz), 8.32 (2H, br s). LCMS (ESI/APCI) m/z 244.0 [M

+ H]⁺.

1

mmol, 73%) as a white solid. H NMR (300 MHz, DMSO-d₆) δ

3-((2-Chloro-4-ﬂuorobenzyl)oxy)pyrrolidine 4-Methylbenzene-

sulfonate (44b). A mixture of 43b (1.76 g, 5.34 mmol) and p-

TsOH-H₂O (1.12 g, 5.87 mmol) in EtOAc (30 mL) was reﬂuxed

for 3 h and cooled to rt. The resultant precipitate was collected,

washed with EtOAc, and dried in vacuo to give 44b (1.64 g, 4.08

1.70−1.86 (1H, m), 2.02−2.18 (1H, m), 2.29 (3H, s), 2.65−2.83

(1H, m), 2.98−3.45 (4H, m), 3.97−4.15 (2H, m), 7.12 (2H, d, J =

7.7 Hz), 7.16−7.25 (2H, m), 7.41−7.53 (3H, m), 8.62-8.80 (2H,

m).

3-((2-Chloro-4-ﬂuorophenoxy)methyl)azetidine 4-Methylbenze-

nesulfonate (41e). p-TsOH-H₂O (0.769 g, 4.04 mmol) was added

to a mixture of 40e (1.16 g, 3.67 mmol) in EtOAc (25 mL) at rt.

The mixture was stirred at 70 °C for 2 h and cooled at rt. The

resulting solid was collected, washed with EtOAc, and dried in

vacuo to give 41e (1.21 g, 3.11 mmol, 85%) as an oﬀ-white solid.

¹H NMR (300 MHz, DMSO-d₆) δ 2.29 (3H, s), 3.86−3.97 (2H,

1

mmol, 76%) as a white solid. H NMR (300 MHz, DMSO-d₆) δ

1.65−1.82 (2H, m), 1.90−2.07 (1H, m), 2.09−2.24 (1H, m), 2.29

(3H, s), 3.10−3.42 (4H, m), 4.30−4.38 (1H, m), 4.49−4.61 (2H,

m), 7.11 (2H, d, J = 7.9 Hz), 7.21−7.31 (1H, m), 7.43−7.52 (3H,

m), 7.58 (1H, dd, J = 8.5, 6.2 Hz), 8.81 (2H, br s). LCMS (ESI/

APCI) m/z 230.0 [M + H]⁺.

3-((2-Chloro-4-ﬂuorobenzyl)oxy)azetidine 4-Methylbenzenesul-

fonate (44c). A mixture of 43c (1.82 g, 5.76 mmol) and p-

TsOH-H₂O (1.21 g, 6.34 mmol) in EtOAc (30 mL) was reﬂuxed

for 3 h and cooled to rt. The resultant precipitate was collected,

washed with EtOAc, and dried in vacuo to give 44c (1.64 g, 4.23

m), 3.99−4.10 (2H, m), 4.18 (2H, d, J = 5.9 Hz), 7.11 (2H, d, J =

7.9 Hz), 7.19−7.24 (2H, m), 7.44−7.51 (3H, m), 8.60 (2H, brs).

tert-Butyl 4-((2-Chloro-4-ﬂuorobenzyl)oxy)piperidine-1-carboxy-

late (43a). To a solution of 42a (1.00 g, 4.97 mmol) in DMF (15

mL) was added 60% NaH (0.238 g, 5.96 mmol) at 0 °C. The

mixture was stirred at the same temperature for 30 min. Then, 1-

(bromomethyl)-2-chloro-4-ﬂuorobenzene (1.22 g, 5.47 mmol) was

added dropwise to the reaction mixture and the resulting white

suspension was stirred at rt for 16 h. The mixture was diluted with

EtOAc and the solution was washed with water and brine, dried

over Na₂SO₄. The solution was passed through NH silica gel, and

the ﬁltrate was concentrated in vacuo. The residue was chromato-

graphed on silica gel (hexane/EtOAc = 3:1 to 1:1) to aﬀord 43a

1

mmol, 73%) as a white solid. H NMR (300 MHz, DMSO-d₆) δ

2.29 (3H, s), 3.82−3.92 (2H, m), 4.12−4.22 (2H, m), 4.43−4.56

(3H, m), 7.11 (2H, d, J = 7.9 Hz), 7.22−7.33 (1H, m), 7.44−7.53

(3H, m), 7.59 (1H, dd, J = 8.7, 6.4 Hz), 8.65 (2H, br s). LCMS

(ESI/APCI) m/z 216.0 [M + H]⁺.

3-((2-Chloro-4-methylbenzyl)oxy)azetidine 4-Methylbenzenesul-

fonate (46a). To a solution of 45a (0.783 g, 5.00 mmol) and Et₃N

(1.53 mL, 11.0 mmol) in THF (16.7 mL) was added MsCl (0.774

mL, 10.0 mmol) dropwise at 0 °C. The resultant pale yellow

suspension was stirred at rt for 3 h. The mixture was partitioned

between water and EtOAc. The organic layer was washed with water

and brine, dried over MgSO₄, ﬁltered through a short pad of silica

gel, and concentrated in vacuo to give 2-chloro-4-methylbenzyl

methanesulfonate (1.17 g, 4.99 mmol, 100%), which was used in the

next step without further puriﬁcation.

1

(1.34 g, 3.90 mmol, 78%) as a colorless oil. H NMR (300 MHz,

DMSO-d₆) δ 1.35−1.49 (11H, s), 1.78−1.89 (2H, m), 3.00−3.13

(2H, m), 3.57−3.68 (3H, m), 4.55 (2H, s), 7.23 (1H, td, J = 8.6,

2.4 Hz), 7.43 (1H, dd, J = 8.9, 2.4 Hz), 7.56 (1H, dd, J = 8.7, 6.4

Hz).

tert-Butyl 3-((2-Chloro-4-ﬂuorobenzyl)oxy)pyrrolidine-1-carbox-

ylate (43b). To a solution of 43a (1.00 g, 5.34 mmol) in DMF (15

mL) was added 60% NaH (0.256 g, 6.41 mmol) at 0 °C. The

mixture was stirred at the same temperature for 30 min. Then, 1-

(bromomethyl)-2-chloro-4-ﬂuorobenzene (1.31 g, 5.87 mmol) was

added dropwise to the reaction mixture and the resulting white

suspension was stirred at rt for 16 h. The mixture was diluted with

EtOAc and the solution was washed with water and brine, dried

over Na₂SO₄. The solution was passed through NH silica gel, and

the ﬁltrate was concentrated in vacuo. The residue was chromato-

graphed on silica gel (hexane/EtOAc = 3:1 to 1:1) to aﬀord 43b

To a solution of tert-butyl 3-hydroxyazetidine-1-carboxylate

(0.786 g, 4.54 mmol) in DMF (8.51 mL) was added 60% NaH

(0.218 g, 5.45 mmol) at 0 °C. The mixture was stirred at the same

temperature for 5 min. Then, a solution of 2-chloro-4-methylbenzyl

methanesulfonate (1.17 g, 4.99 mmol) in DMF (2.84 mL) was

added dropwise to the reaction mixture. The pale yellow suspension

was stirred at rt for 38 h. The mixture was quenched with sat.

NH₄Cl and partitioned between EtOAc and water. The organic layer

was washed with water and brine, dried over MgSO₄, and

concentrated in vacuo. The residue was puriﬁed by column

chromatography (silica gel, eluted with 0−30% EtOAc in hexane)

to give tert-butyl 3-((2-chloro-4-methylbenzyl)oxy)azetidine-1-car-

1

(1.76 g, 5.34 mmol, 100%) as a colorless oil. H NMR (300 MHz,

DMSO-d₆) δ 1.39 (9H, s), 1.88−2.03 (2H, s), 3.21−3.39 (4H, m),

4.12−4.21 (1H, m), 4.52 (2H, s), 7.22 (1H, td, J = 8.5, 2.6 Hz),

7.44 (1H, dd, J = 9.0, 2.6 Hz), 7.53 (1H, dd, J = 8.7, 6.4 Hz).

tert-Butyl 3-((2-Chloro-4-ﬂuorobenzyl)oxy)azetidine-1-carboxy-

late (43c). To a solution of 43c (1.00 g, 5.77 mmol) in DMF

(15 mL) was added 60% NaH (0.277 g, 6.93 mmol) at 0 °C. The

mixture was stirred at the same temperature for 30 min. Then, 1-

(bromomethyl)-2-chloro-4-ﬂuorobenzene (1.42 g, 6.35 mmol) was

added dropwise to the reaction mixture and the resulting white

suspension was stirred at rt for 16 h. The mixture was diluted with

EtOAc and the solution was washed with water and brine, dried

over Na₂SO₄. The solution was passed through NH silica gel, and

the ﬁltrate was concentrated in vacuo. The residue was chromato-

1

boxylate (1.32 g, 4.23 mmol, 93%) as a pale yellow oil. H NMR

(300 MHz, DMSO-d₆) δ 1.37 (9H, s), 2.30 (3H, s), 3.68 (2H, dd, J

= 8.8, 3.3 Hz), 4.01 (2H, dd, J = 9.1, 6.8 Hz), 4.35 (1H, tt, J = 6.4,

4.0 Hz), 4.46 (2H, s), 7.16 (1H, dd, J = 7.7, 0.9 Hz), 7.29 (1H, s),

7.39 (1H, d, J = 7.7 Hz).

A solution of tert-butyl 3-((2-chloro-4-methylbenzyl)oxy)-

azetidine-1-carboxylate (1.32 g, 4.23 mmol) and p-TsOH-H₂O

(0.886 g, 4.66 mmol) in EtOAc (21.2 mL) was reﬂuxed for 2 h.

After being cooled to rt, the mixture was diluted with hexane to

aﬀord white precipitates. The resultant precipitates were collected,

washed with EtOAc/hexane and dried in vacuo to give 46a (1.12 g,

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2.92 mmol, 69%) as a white solid. H NMR (300 MHz, DMSO-d₆)

A solution of tert-butyl 3-((4-methyl-3-(triﬂuoromethyl)benzyl)-

oxy)azetidine-1-carboxylate (2.86 g, 8.28 mmol) and p-TsOH-H₂O

(1.73 g, 9.11 mmol) in EtOAc (41.4 mL) was reﬂuxed for 2 h. After

being cooled to rt, the resultant precipitates were collected, washed

with EtOAc and dried in vacuo to give 46c (3.05 g, 7.31 mmol,

δ 2.21−2.36 (6H, m), 3.85 (2H, dd, J = 12.1, 5.3 Hz), 4.15 (2H,

dd, J = 12.1, 6.6 Hz), 4.39−4.56 (3H, m), 7.11 (2H, d, J = 7.7 Hz),

7.18 (1H, d, J = 7.0 Hz), 7.31 (1H, s), 7.41 (1H, d, J = 7.7 Hz),

7.44−7.50 (2H, m), 8.60 (2H, brs). LCMS (ESI/APCI) m/z 212.1

[M + H]⁺.

1

88%) as a white solid. H NMR (300 MHz, DMSO-d₆) δ 2.29 (3H,

3-((3-Chloro-4-(triﬂuoromethyl)benzyl)oxy)azetidine 4-Methyl-

benzenesulfonate (46b). To a solution of 45b (1.84 g, 8.74

mmol) and Et₃N (2.82 mL, 20.2 mmol) in THF (30.7 mL) was

added MsCl (1.43 mL, 18.5 mmol) dropwise at 0 °C. The resultant

pale yellow suspension was stirred at rt for 3 h. The mixture was

partitioned between water and EtOAc. The organic layer was

washed with water and brine, dried over MgSO₄, ﬁltered through a

short pad of silica gel, and concentrated in vacuo to give 3-chloro-4-

(triﬂuoromethyl)benzyl methanesulfonate (2.53 g, 8.76 mmol,

100%), which was used in the next step without further puriﬁcation.

To a solution of tert-butyl 3-hydroxyazetidine-1-carboxylate (1.45

g, 8.37 mmol) in DMF (15.7 mL) was added 60% NaH (0.402 g,

10.1 mmol) at 0 °C. The mixture was stirred at the same

s), 2.44 (3H, d, J = 1.7 Hz), 3.81−3.91 (2H, m), 4.09−4.19 (2H,

m), 4.44 (1H, tt, J = 6.5, 5.2 Hz), 4.52 (2H, s), 7.11 (2H, d, J = 8.3

Hz), 7.42−7.50 (3H, m), 7.51−7.58 (1H, m), 7.66 (1H, s), 8.59

(2H, brs). LCMS (ESI/APCI) m/z 246.1 [M + H]⁺.

3-((4-Chloro-3-methylbenzyl)oxy)azetidine 4-Methylbenzenesul-

fonate (46d). To a solution of 45d (1.40 g, 8.94 mmol) and Et₃N

(2.74 mL, 19.7 mmol) in THF (29.8 mL) was added MsCl (1.38

mL, 17.9 mmol) dropwise at 0 °C. The resultant pale-orange

suspension was stirred at rt for 16 h. The mixture was partitioned

between water and EtOAc. The organic layer was washed with water

and brine, dried over MgSO₄, ﬁltered through a short pad of silica

gel, and concentrated in vacuo to give 4-chloro-3-methylbenzyl

methanesulfonate (2.20 g, 9.37 mmol, quant.), which was used in

the next step without further puriﬁcation.

temperature for

5 min. Then, a solution of 3-chloro-4-

(triﬂuoromethyl)benzyl methanesulfonate (2.53 g, 8.76 mmol) in

DMF (5.23 mL) was added dropwise to the reaction mixture. The

pale yellow suspension was stirred at rt for 3 h. The mixture was

quenched with sat. NH₄Cl and partitioned between EtOAc and

water. The organic layer was washed with water and brine, dried

over MgSO₄, and concentrated in vacuo. The residue was puriﬁed

by column chromatography (silica gel, eluted with 0−30% EtOAc in

hexane) to give tert-butyl 3-((3-chloro-4-(triﬂuoromethyl)benzyl)-

oxy)azetidine-1-carboxylate (2.66 g, 7.27 mmol, 87%) as a colorless

To a solution of tert-butyl 3-hydroxyazetidine-1-carboxylate (1.41

g, 8.13 mmol) in DMF (15.2 mL) was added 60% NaH (0.390 g,

9.76 mmol) at 0 °C. The mixture was stirred at the same

temperature for 5 min. Then, a solution of 4-chloro-3-methylbenzyl

methanesulfonate (2.10 g, 8.94 mmol) in DMF (5.08 mL) was

added dropwise to the reaction mixture. The pale yellow suspension

was stirred at rt for 3 h. The mixture was quenched with sat. NH₄Cl

and partitioned between EtOAc and water. The organic layer was

washed with water and brine, dried over MgSO₄, and concentrated

in vacuo. The residue was puriﬁed by column chromatography

(silica gel, eluted with 0−30% EtOAc in hexane) to give tert-butyl 3-

((4-chloro-3-methylbenzyl)oxy)azetidine-1-carboxylate (2.21 g, 7.09

1

oil. H NMR (300 MHz, DMSO-d₆) δ 1.38 (9H, s), 3.73 (2H, dd, J

= 9.4, 3.4 Hz), 3.94−4.10 (2H, m), 4.30−4.41 (1H, m), 4.53 (2H,

s), 7.52 (1H, d, J = 7.6 Hz), 7.69 (1H, s), 7.85 (1H, d, J = 8.1 Hz).

A solution of tert-butyl 3-((3-chloro-4-(triﬂuoromethyl)benzyl)-

oxy)azetidine-1-carboxylate (340 mg, 0.930 mmol) and p-TsOH-

H₂O (194 mg, 1.02 mmol) in EtOAc (4.65 mL) was reﬂuxed for 2

h. After being cooled to rt, the resultant precipitates were collected,

washed with EtOAc and dried in vacuo to give 46b (351 mg, 0.802

1

mmol, 87%) as a yellow oil. H NMR (300 MHz, DMSO-d₆) δ 1.37

(9H, s), 2.33 (3H, s), 3.60−3.75 (2H, m), 3.94−4.08 (2H, m), 4.31

(1H, ddd, J = 6.4, 4.1, 2.4 Hz), 4.38 (2H, s), 7.18 (1H, dd, J = 8.1,

1.9 Hz), 7.33 (1H, d, J = 1.7 Hz), 7.38 (1H, d, J = 8.1 Hz).

A solution of tert-butyl 3-((4-chloro-3-methylbenzyl)oxy)-

azetidine-1-carboxylate (2.21 g, 7.09 mmol) and p-TsOH-H₂O

(1.48 g, 7.80 mmol) in EtOAc (35.4 mL) was reﬂuxed for 2 h. After

being cooled to rt, the resultant precipitates were collected, washed

with EtOAc and dried in vacuo to give 46d (2.37 g, 6.17 mmol,

1

mmol, 86%) as a white solid. H NMR (300 MHz, DMSO-d₆) δ

2.29 (3H, s), 3.89 (2H, dd, J = 12.1, 4.9 Hz), 4.10−4.22 (2H, m),

4.38−4.52 (1H, m), 4.58 (2H, s), 7.11 (2H, d, J = 7.7 Hz), 7.42−

7.49 (2H, m), 7.53 (1H, d, J = 8.1 Hz), 7.73 (1H, s), 7.88 (1H, d, J

= 8.3 Hz), 8.58 (2H, brs). LCMS (ESI/APCI) m/z 266.1 [M +

H]⁺.

1

87%) as a pale yellow solid. H NMR (300 MHz, DMSO-d₆) δ 2.29

(3H, s), 2.33 (3H, s), 3.83 (2H, dd, J = 12.1, 5.3 Hz), 4.05−4.19

(2H, m), 4.34−4.50 (3H, m), 7.11 (2H, d, J = 7.7 Hz), 7.20 (1H,

dd, J = 8.2, 1.8 Hz), 7.33 (1H, s), 7.41 (1H, d, J = 8.1 Hz), 7.44−

7.51 (2H, m), 8.57 (2H, brs). LCMS (ESI/APCI) m/z 212.1 [M +

H]⁺.

3-((3-Chloro-4-methylbenzyl)oxy)azetidine 4-Methylbenzenesul-

fonate (46e). To a stirred solution of 45e (15.5 g, 99.0 mmol) in

DME (200 mL) was added dropwise phosphorus tribromide (5.58

mL, 59.4 mmol) at 0 °C, and the mixture was stirred at rt for 3 h.

The reaction was quenched with ice water and then extracted with

EtOAc/hexane. The extract was washed with water, aq. NaHCO₃,

and brine, dried over MgSO₄, and concentrated in vacuo. The

residue was puriﬁed by column chromatography (silica gel, eluted

with 0−40% EtOAc in hexane) to give 4-(bromomethyl)-2-chloro-1-

methylbenzene (21.7 g, 99.0 mmol, 100%) as a colorless oil. ¹H

NMR (300 MHz, DMSO-d₆) δ 2.32 (3H, s), 4.68 (2H, s), 7.27−

7.40 (2H, m), 7.45−7.58 (1H, m).

To a solution of tert-butyl 3-hydroxyazetidine-1-carboxylate (8.89

g, 51.3 mmol) in DMF (96 mL) was added 60% NaH (2.46 g, 61.6

mmol) at 0 °C. The mixture was stirred at the same temperature for

10 min. Then, a solution of 4-(bromomethyl)-2-chloro-1-methyl-

benzene (12.4 g, 56.4 mmol) in DMF (32.1 mL) was added

dropwise to the reaction mixture. The white suspension was stirred

at rt for 16 h. The mixture was quenched with sat. NH₄Cl and

partitioned between EtOAc and water. The organic layer was

washed with water and brine, dried over MgSO₄, and concentrated

in vacuo. The residue was puriﬁed by column chromatography

(silica gel, eluted with 0−30% EtOAc in hexane) to give tert-butyl 3-

3-((4-Methyl-3-(triﬂuoromethyl)benzyl)oxy)azetidine 4-Methyl-

benzenesulfonate (46c). To a solution of 45c (1.89 g, 9.94

mmol) and Et₃N (3.05 mL, 21.9 mmol) in THF (33.1 mL) was

added MsCl (1.54 mL, 19.9 mmol) dropwise at 0 °C. The resultant

pale yellow suspension was stirred at rt for 30 min. The mixture was

partitioned between water and EtOAc. The organic layer was

washed with water and brine, dried over MgSO₄, ﬁltered through a

short pad of silica gel, and concentrated in vacuo to give 4-methyl-3-

(triﬂuoromethyl)benzyl methanesulfonate (2.73 g, 10.2 mmol,

quant.), which was used in the next step without further puriﬁcation.

To a solution of tert-butyl 3-hydroxyazetidine-1-carboxylate (1.57

g, 9.04 mmol) in DMF (17.0 mL) was added 60% NaH (0.434 g,

10.9 mmol) at 0 °C. The mixture was stirred at the same

temperature for

5 min. Then, a solution of 4-methyl-3-

(triﬂuoromethyl)benzyl methanesulfonate (2.67 g, 9.94 mmol) in

DMF (5.65 mL) was added dropwise to the reaction mixture. The

pale yellow suspension was stirred at rt for 3 h. The mixture was

quenched with sat. NH₄Cl and partitioned between EtOAc and

water. The organic layer was washed with water and brine, dried

over MgSO₄, and concentrated in vacuo. The residue was puriﬁed

by column chromatography (silica gel, eluted with 0−30% EtOAc in

hexane) to give tert-butyl 3-((4-methyl-3-(triﬂuoromethyl)benzyl)-

oxy)azetidine-1-carboxylate (2.86 g, 8.28 mmol, 92%) as a colorless

1

oil. H NMR (300 MHz, DMSO-d₆) δ 1.37 (9H, s), 2.44 (3H, d, J

= 1.7 Hz), 3.69 (2H, dd, J = 9.0, 3.3 Hz), 3.95−4.09 (2H, m),

4.28−4.38 (1H, m), 4.46 (2H, s), 7.39−7.47 (1H, m), 7.49−7.57

(1H, m), 7.63 (1H, s).

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((3-chloro-4-methylbenzyl)oxy)azetidine-1-carboxylate (15.9 g, 50.8

mmol, 99%) as a colorless oil. H NMR (300 MHz, DMSO-d₆) δ

1.37 (9H, s), 2.32 (3H, s), 3.67 (2H, dd, J = 9.1, 3.8 Hz), 3.92−

4.09 (2H, m), 4.25−4.35 (1H, m), 4.39 (2H, s), 7.21 (1H, dd, J =

8.0, 1.5 Hz), 7.33 (1H, d, J = 8.0 Hz), 7.39 (1H, d, J = 1.5 Hz).

A solution of tert-butyl 3-((3-methyl-4-(triﬂuoromethyl)benzyl)-

oxy)azetidine-1-carboxylate (299 mg, 0.866 mmol) and p-TsOH-

H₂O (181 mg, 0.950 mmol) in EtOAc (4.33 mL) was reﬂuxed for 2

h. After being cooled to rt, the resultant precipitates were collected,

washed with EtOAc/hexane, and dried in vacuo to give 46g (268

mg, 0.642 mmol, 74%) as a white solid. ¹H NMR (300 MHz,

DMSO-d₆) δ 2.29 (3H, s), 2.45 (3H, d, J = 1.7 Hz), 3.80−3.94

(2H, m), 4.09−4.22 (2H, m), 4.45 (1H, quin, J = 5.9 Hz), 4.53

(2H, s), 7.11 (2H, d, J = 7.9 Hz), 7.36 (1H, d, J = 7.7 Hz), 7.39−

7.44 (1H, m), 7.45−7.51 (2H, m), 7.67 (1H, d, J = 7.9 Hz), 8.26-

8.98 (2H, m). LCMS (ESI/APCI) m/z 246.1 [M + H]⁺.

1

A

solution of tert-butyl 3-((3-chloro-4-methylbenzyl)oxy)-

azetidine-1-carboxylate (39.3 g, 126 mmol) and p-TsOH-H₂O

(26.4 g, 139 mmol) in EtOAc (420 mL) was reﬂuxed for 2 h.

After being cooled to rt, the resultant precipitates were collected,

washed with EtOAc and dried in vacuo to give 46e (44.8 g, 117

1

mmol, 92%) as a white solid. H NMR (300 MHz, DMSO-d₆) δ

2.29 (3H, s), 2.32 (3H, s), 3.84 (2H, dd, J = 11.5, 4.7 Hz), 4.13

(2H, dd, J = 11.7, 6.8 Hz), 4.34−4.50 (3H, m), 7.11 (2H, d, J = 8.0

Hz), 7.22 (1H, dd, J = 7.6, 1.5 Hz), 7.35 (1H, d, J = 8.0 Hz), 7.42

(1H, d, J = 1.5 Hz), 7.45−7.52 (2H, m), 8.58 (2H, brs). LCMS

(ESI/APCI) m/z 212.1 [M + H]⁺.

3-((2,4-Dichlorobenzyl)oxy)azetidine 4-Methylbenzenesulfonate

(48a). To a solution of tert-butyl 3-hydroxyazetidine-1-carboxylate

(300 mg, 1.73 mmol) in DMF (3.25 mL) was added 60% NaH (83

mg, 2.1 mmol) at 0 °C. The mixture was stirred at the same

temperature for 5 min. Then, a solution of 47a (372 mg, 1.91

mmol) in DMF (1.08 mL) was added dropwise to the reaction

mixture. The white suspension was stirred at rt for 16 h. The

mixture was quenched with sat. NH₄Cl and partitioned between

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

brine, dried over MgSO₄, and concentrated in vacuo. The residue

was puriﬁed by column chromatography (silica gel, eluted with 0−

30% EtOAc in hexane) to give tert-butyl 3-((2,4-dichlorobenzyl)-

oxy)azetidine-1-carboxylate (540 mg, 1.63 mmol, 94%) as a colorless

3-((4-Chloro-3-(triﬂuoromethyl)benzyl)oxy)azetidine 4-Methyl-

benzenesulfonate (46f). To a stirred solution of 45f (2.08 g, 9.88

mmol) in DME (25 mL) was added dropwise phosphorus

tribromide (0.557 mL, 5.93 mmol) at 0 °C, and the mixture was

stirred at rt for 2.5 h. The reaction was quenched with ice water and

then extracted with EtOAc/hexane. The extract was washed with

water, aq. NaHCO₃, and brine, dried over Na₂SO₄, ﬁltered through

a silica gel/NH silica gel pad, and concentrated in vacuo to give 4-

(bromomethyl)-1-chloro-2-(triﬂuoromethyl)benzene (2.33 g, 8.50

1

oil. H NMR (300 MHz, DMSO-d₆) δ 1.37 (9H, s), 3.71 (2H, dd, J

1

mmol, 86%) as a colorless oil. H NMR (300 MHz, CDCl₃) δ 4.47

= 8.9, 3.2 Hz), 3.95−4.09 (2H, m), 4.33−4.43 (1H, m), 4.49 (2H,

s), 7.43−7.48 (1H, m), 7.52−7.58 (1H, m), 7.64 (1H, d, J = 1.9

Hz).

(2H, s), 7.43−7.59 (2H, m), 7.71 (1H, s).

To a solution of tert-butyl 3-hydroxyazetidine-1-carboxylate

(0.950 g, 5.48 mmol) in DMF (12 mL) was added 60% NaH

(0.285 g, 7.13 mmol) at 0 °C and the mixture was stirred at the

same temperature for 30 min. Then, a solution of 4-(bromomethyl)-

1-chloro-2-(triﬂuoromethyl)benzene (1.50 g, 5.48 mmol) in DMF

(6 mL) was added dropwise to the reaction mixture at 0 °C and the

mixture was stirred at rt overnight. The mixture was quenched with

ice water and extracted with EtOAc. The organic layer was washed

with water and brine, dried over Na₂SO₄, and concentrated in

vacuo. The residue was puriﬁed by column chromatography (silica

gel, eluted with 0−33% EtOAc in hexane) to give tert-butyl 3-((4-

chloro-3-(triﬂuoromethyl)benzyl)oxy)azetidine-1-carboxylate (1.78

g, 4.86 mmol, 89%) as a colorless oil. ¹H NMR (300 MHz,

CDCl₃) δ 1.44 (9H, s), 3.83−3.93 (2H, m), 4.05−4.16 (2H, m),

4.33 (1H, tt, J = 6.4, 4.3 Hz), 4.45 (2H, s), 7.40−7.54 (2H, m),

7.65 (1H, s).

A solution of tert-butyl 3-((2,4-dichlorobenzyl)oxy)azetidine-1-

carboxylate (540 mg, 1.63 mmol) and p-TsOH-H₂O (340 mg, 1.79

mmol) in EtOAc (8.13 mL) was reﬂuxed for 2 h. After being cooled

to rt, the resultant precipitates were collected, washed with EtOAc

and dried in vacuo to give 48a (564 mg, 1.40 mmol, 86%) as a

1

white solid. H NMR (300 MHz, DMSO-d₆) δ 2.29 (3H, s), 3.88

(2H, dd, J = 12.1, 5.1 Hz), 4.17 (2H, dd, J = 12.3, 6.6 Hz), 4.40−

4.53 (1H, m), 4.55 (2H, s), 7.11 (2H, d, J = 8.5 Hz), 7.43−7.51

(3H, m), 7.53−7.59 (1H, m), 7.66 (1H, d, J = 2.1 Hz), 8.63 (2H,

brs). LCMS (ESI/APCI) m/z 232.1 [M + H]⁺.

3-((2-Chloro-4-(triﬂuoromethyl)benzyl)oxy)azetidine 4-Methyl-

benzenesulfonate (48b). To a solution of tert-butyl 3-hydroxyaze-

tidine-1-carboxylate (0.600 g, 3.46 mmol) in DMF (6 mL) was

added 60% NaH (0.180 g, 4.50 mmol) at 0 °C and the mixture was

stirred at the same temperature for 10 min. Then, a solution of 47b

(0.873 g, 3.81 mmol) in DMF (2 mL) was added dropwise to the

reaction mixture at 0 °C and the mixture was stirred at rt overnight.

The mixture was diluted with EtOAc, washed with water and brine,

dried over Na₂SO₄, and concentrated in vacuo. The residue was

puriﬁed by column chromatography (silica gel, eluted with 0−25%

EtOAc in hexane) to give tert-butyl 3-((2-chloro-4-(triﬂuoromethyl)-

benzyl)oxy)azetidine-1-carboxylate (1.01 g, 2.75 mmol, 79%) as a

colorless oil. ¹H NMR (300 MHz, CDCl₃) δ 1.45 (9H, s), 3.93

(2H, dd, J = 9.8, 4.5 Hz), 4.09−4.21 (2H, m), 4.39 (1H, tt, J = 6.4,

4.3 Hz), 4.57 (2H, s), 7.51−7.58 (1H, m), 7.60−7.71 (2H, m).

A mixture of tert-butyl 3-((2-chloro-4-(triﬂuoromethyl)benzyl)-

oxy)azetidine-1-carboxylate (1.00 g, 2.74 mmol), p-TsOH-H₂O

(0.548 g, 2.88 mmol), and EtOAc (10 mL) was reﬂuxed for 3 h.

After being cooled to rt, the mixture was stirred for 1 h. Then, the

resultant precipitates were collected, washed with EtOAc and dried

A mixture of tert-butyl 3-((4-chloro-3-(triﬂuoromethyl)benzyl)-

oxy)azetidine-1-carboxylate (1.77 g, 4.85 mmol), p-TsOH-H₂O

(0.969 g, 5.09 mmol), and EtOAc (16 mL) was reﬂuxed for 3 h.

After being cooled to rt, the resultant precipitates were collected,

washed with EtOAc and dried in vacuo to give 46f (1.77 g, 4.04

1

mmol, 83%) as a white solid. H NMR (300 MHz, DMSO-d₆) δ

2.29 (3H, s), 3.82−3.96 (2H, m), 4.11−4.21 (2H, m), 4.39−4.51

(1H, m), 4.56 (2H, s), 7.11 (2H, d, J = 7.9 Hz), 7.41−7.55 (2H,

m), 7.61−7.79 (2H, m), 7.85 (1H, d, J = 1.5 Hz), 8.62 (2H, brs).

LCMS (ESI/APCI) m/z 266.1 [M + H]⁺.

3-((3-Methyl-4-(triﬂuoromethyl)benzyl)oxy)azetidine 4-Methyl-

benzenesulfonate (46g). To a mixture of tert-butyl 3-((3-chloro-

4-(triﬂuoromethyl)benzyl)oxy)azetidine-1-carboxylate (500 mg, 1.37

mmol), trimethylboroxine (191 μL, 1.37 mmol), SPhos (56.1 mg,

0.140 mmol) and K₃PO₄(870 mg, 4.10 mmol) in toluene (6.15

mL) and water (0.683 mL) was added Pd(OAc)₂(15.3 mg, 70.0

μmol). The mixture was stirred at 100 °C for 16 h. After being

cooled to rt, the mixture was partitioned between EtOAc and water.

The organic layer was washed with brine, dried over MgSO₄, and

concentrated in vacuo. The residue was puriﬁed by column

chromatography (silica gel, eluted with 0−30% EtOAc in hexane)

to give tert-butyl 3-((3-methyl-4-(triﬂuoromethyl)benzyl)oxy)-

azetidine-1-carboxylate (299 mg, 0.866 mmol, 63%) as a colorless

1

in vacuo to give 48b (1.01 g, 2.31 mmol, 84%) as a white solid. H

NMR (300 MHz, DMSO-d₆) δ 2.29 (3H, s), 3.92 (2H, dd, J = 12.1,

5.3 Hz), 4.19 (2H, dd, J = 12.1, 6.4 Hz), 4.48−4.60 (1H, m), 4.65

(2H, s), 7.11 (2H, d, J = 7.9 Hz), 7.47 (2H, d, J = 8.3 Hz), 7.74−

7.85 (2H, m), 7.91−7.97 (1H, m), 8.62 (2H, brs). LCMS (ESI/

APCI) m/z 266.0 [M + H]⁺.

3-((3,4-Dichlorobenzyl)oxy)azetidine 4-Methylbenzenesulfonate

(48c). To a solution of tert-butyl 3-hydroxyazetidine-1-carboxylate

(300 mg, 1.73 mmol) in DMF (3.25 mL) was added 60% NaH (83

mg, 2.1 mmol) at 0 °C. The mixture was stirred at the same

temperature for 5 min. Then, a solution of 47c (372 mg, 1.91

mmol) in DMF (1.08 mL) was added dropwise to the reaction

1

oil. H NMR (300 MHz, DMSO-d₆) δ 1.37 (9H, s), 2.44 (3H, d, J

= 1.7 Hz), 3.71 (2H, dd, J = 9.1, 3.2 Hz), 3.97−4.09 (2H, m),

4.30−4.39 (1H, m), 4.48 (2H, s), 7.35 (1H, d, J = 8.1 Hz), 7.40

(1H, s), 7.65 (1H, d, J = 8.1 Hz).

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mixture. The yellow suspension was stirred at rt for 16 h. The

mixture was quenched with sat. NH₄Cl and partitioned between

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

brine, dried over MgSO₄, and concentrated in vacuo. The residue

was puriﬁed by column chromatography (silica gel, eluted with 0−

30% EtOAc in hexane) to give tert-butyl 3-((3,4-dichlorobenzyl)-

oxy)azetidine-1-carboxylate (527 mg, 1.59 mmol, 92%) as a colorless

oil. H NMR (300 MHz, DMSO-d₆) δ 1.37 (9H, s), 3.70 (2H, dd, J

= 9.1, 3.2 Hz), 3.95−4.08 (2H, m), 4.28−4.37 (1H, m), 4.43 (2H,

s), 7.35 (1H, dd, J = 8.2, 2.0 Hz), 7.58−7.66 (2H, m).

8.1 Hz), 8.33-8.96 (2H, m). LCMS (ESI/APCI) m/z 300.1 [M +

H]⁺.

Docking of the Designed Compounds, 2a−c. Docking

calculation was performed using the co-crystal structure of 1h

with MAGL (PDB ID: 7L4U) as the template using the Schrodinger

suite of software (Release 2016-3). The protein structure was

prepared in Protein Preparation Wizard. All water molecules were

removed before docking. The compound structure was prepared for

docking by LigPrep module and docked using Glide in SP mode.

The glide setting was the following, a core constraint by SMARTS

patterns, C1CN(CO)CCC1COc2c(Cl)cc(F)cc2, and the toler-

ance is 0.50 Å, unchecking canonicalize input conformation and

checking enhanced sampling. The docked poses were then

minimized using the local optimization feature using

MOE2016.0802.

1

A solution of tert-butyl 3-((3,4-dichlorobenzyl)oxy)azetidine-1-

carboxylate (527 mg, 1.59 mmol) and p-TsOH-H₂O (332 mg, 1.74

mmol) in EtOAc (7.93 mL) was reﬂuxed for 2 h. After being cooled

to rt, the resultant precipitates were collected, washed with EtOAc

and dried in vacuo to give 48c (547 mg, 1.35 mmol, 85%) as a

1

white solid. H NMR (300 MHz, DMSO-d₆) δ 2.29 (3H, s), 3.86

Measurement of MAGL Inhibitory Activity by Mass

Spectrometry. His-hMAGL or His-mMAGL was diluted with

enzyme reaction buﬀer (10 mM Tris−HCl, pH 7.5, 1 mM EDTA,

0.025% (w/v) Triton X-100, 0.01% bovine serum albumin) to a

concentration of 7.5 or 22.5 ng/mL, respectively. A solution (5 μL)

of a test compound dissolved in DMSO and enzyme reaction buﬀer

was added to each well of a 384-well assay plate (Greiner 781280),

then 5 μL of 150 μM 2-AG (Tocris Bioscience) was added to each

well, and 7.5 ng/mL His-hMAGL or 22.5 ng/mL His-mMAGL

solution (5 μL) was added and the mixture and incubated at r.t. for

10 min. Then, 2% formic acid (Wako Pure Chemical Industries,

Ltd.) was added by 10 μL to stop the reaction. Furthermore, MeCN

(50 μL) containing 3 μM AA-d8 (Cayman Chemical Company) was

added, and the mixture was stirred. The amount of AA in the

obtained enzyme reaction mixture was calculated by measuring by

RapidFire mass spectrometry and correcting by the amount of AA-

d8. High-throughput online solid-phase extraction was performed

using RapidFire 300 system (Agilent Technologies, Inc.) as

described previously.²¹IC₅₀values were calculated by XLﬁt

(IDBS) from the data expressed as inhibition (%) using 4 Parameter

Logistic Model. The response of vehicle control was set as the 0%

inhibition and the response without enzyme was set as the 100%

inhibition. The hMAGL/mMAGL IC₅₀data of 4f were obtained

from ﬁve experiments in duplicate, respectively. The hMAGL IC₅₀

data of 1b were obtained from two experiments in duplicate. The

hMAGL IC₅₀data of 1, 1a, 1c, 1d, 1e, 1f, 1g, 1h, 2a, 2b, 2c, 2d, 2e,

2f, 3a, 3b, 3c, 3d, 3e, 3f, 3g, 3h, 3i, 4a, 4b, 4c, 4d, 4e, 4g, 4h, 4i,

4j, and 4k were obtained from a single experiment in duplicate.

Measurement of ABHD6 Activity. Human ABHD6 was

expressed in Expi293 (Thermo Fisher Scientiﬁc). The cells were

sonicated, and the lysate was ultracentrifuged at 120000 g for 45

min at 4 °C. The microsome fraction was used as an ABHD6

enzyme. Enzyme and substrate were dissolved in ABHD6 assay

buﬀer [50 mM Tris−HCl, pH 7.5, 1 mM EDTA, 5 mM MgCl₂, 100

mM NaCl, and 0.1% (w/v) Triton X-100]. Compounds were

dissolved in DMSO and then diluted in the ABHD6 assay buﬀer.

Five microliters of compound solution was added to a 384-well clear

plate followed by 5 μL of ABHD6 solution (30 μg/mL). Then, 5 μL

of substrate solution (3 mM p-nitrophenyl acetate) was added and

mixed. After the incubation at rt for 30 min, the absorbance at 405

nm was measured using a SpectraMax multilabel plate reader

(Molecular Devices). The response of vehicle control was set as the

0% inhibition and the response without enzyme was set as the 100%

inhibition.

(2H, dd, J = 12.2, 5.0 Hz), 4.15 (2H, dd, J = 12.1, 6.6 Hz), 4.38−

4.46 (1H, m), 4.48 (2H, s), 7.11 (2H, d, J = 7.7 Hz), 7.36 (1H, dd,

J = 8.2, 2.0 Hz), 7.44−7.51 (2H, m), 7.62−7.69 (2H, m), 8.60 (2H,

brs). LCMS (ESI/APCI) m/z 232.1 [M + H]⁺.

3-((3,4-Bis(triﬂuoromethyl)benzyl)oxy)azetidine 4-Methylbenze-

nesulfonate (50). A solution of 49 (1.00 g, 3.87 mmol) in THF

(12.9 mL) was cooled to 0 °C. BH₃−THF (7.75 mL, 7.75 mmol,

1.0 M) was added thereto dropwise over 3 min at the same

temperature. The resultant mixture was stirred at rt for 18 h. The

reaction was quenched with MeOH at 0 °C and the mixture was

stirred at rt for 1 h. After removal of organic solvents in vacuo, the

mixture was partitioned between water and EtOAc. The organic

layer was washed with brine, dried over MgSO₄, ﬁltered through a

short pad of silica gel/NH silica gel (eluting with EtOAc), and

concentrated in vacuo to give (3,4-bis(triﬂuoromethyl)phenyl)-

1

methanol (0.945 g, 3.87 mmol, 100%) as a colorless oil. H NMR

(300 MHz, DMSO-d₆) δ 4.68 (2H, d, J = 5.7 Hz), 5.61 (1H, t, J =

5.7 Hz), 7.84 (1H, d, J = 8.1 Hz), 7.96 (1H, s), 8.02 (1H, d, J = 8.1

Hz).

To a solution of (3,4-bis(triﬂuoromethyl)phenyl)methanol (0.945

g, 3.87 mmol) and Et₃N (1.19 mL, 8.52 mmol) in THF (12.9 mL)

was added MsCl (0.599 mL, 7.74 mmol) dropwise at 0 °C. The

resultant pale yellow suspension was stirred at rt for 2 h. The

mixture was partitioned between water and EtOAc. The organic

layer was washed with water and brine, dried over MgSO₄, ﬁltered

through a short pad of silica gel, and concentrated in vacuo to give

3,4-bis(triﬂuoromethyl)benzyl methanesulfonate as a crude material

(1.32 g, quant.), which was used in the next step without further

1

puriﬁcation. H NMR (300 MHz, DMSO-d₆) δ 3.33 (3H, s), 5.48

(2H, s), 8.00 (1H, d, J = 8.1 Hz), 8.09-8.18 (2H, m).

To a solution of tert-butyl 3-hydroxyazetidine-1-carboxylate

(0.610 g, 3.52 mmol) in DMF (6.60 mL) was added 60% NaH

(0.169 g, 4.22 mmol) at 0 °C. The mixture was stirred at the same

temperature for 5 min. Then, a solution of the crude mesylate in

DMF (2.20 mL) was added dropwise to the reaction mixture. The

pale yellow suspension was stirred at rt for 64 h. The mixture was

quenched with sat. NH₄Cl and partitioned between EtOAc and

water. The organic layer was washed with water and brine, dried

over MgSO₄, and concentrated in vacuo. The residue was puriﬁed

by column chromatography (silica gel, eluted with 0−30% EtOAc in

hexane) to give tert-butyl 3-((3,4-bis(triﬂuoromethyl)benzyl)oxy)-

azetidine-1-carboxylate (1.07 g, 2.68 mmol, 76%) as a colorless oil.

¹H NMR (300 MHz, DMSO-d₆) δ 1.38 (9H, s), 3.75 (2H, dd, J =

9.2, 3.5 Hz), 3.98−4.10 (2H, m), 4.34−4.44 (1H, m), 4.63 (2H, s),

7.89 (1H, d, J = 8.1 Hz), 7.99 (1H, s), 8.05 (1H, d, J = 8.1 Hz).

A solution of tert-butyl 3-((3,4-bis(triﬂuoromethyl)benzyl)oxy)-

azetidine-1-carboxylate (1.07 g, 2.68 mmol) and p-TsOH-H₂O

(0.561 g, 2.95 mmol) in EtOAc (13.4 mL) was reﬂuxed for 2 h.

After being cooled to rt, the resultant precipitates were collected,

washed with EtOAc and dried in vacuo to give 50 (1.17 g, 2.48

Surface Plasmon Resonance (SPR)-Based Binding Kinetics.

SPR biosensing experiments were performed on a Biacore S200

instrument (GE Healthcare)at 20 °C. The puriﬁed human his-

MAGL was immobilized on a CM5 sensor chip in an HBS-EP

buﬀer [10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA,

0.05% (v/v) Surfactant P20] containing 1 mM DTT by the standard

amine-coupling procedure according to the manufacturer’s in-

structions. One μM human MAGL diluted with 10 mM acetate

buﬀer (pH 5.5) was contacted the surface of the activated chip for 5

min. The immobilization level with approximately 2000 RU was

captured onto the sensor chip. The binding kinetics and aﬃnity

1

mmol, 93%) as a white solid. H NMR (300 MHz, DMSO-d₆) δ

2.29 (3H, s), 3.84−3.99 (2H, m), 4.11−4.26 (2H, m), 4.50 (1H,

quin, J = 5.8 Hz), 4.68 (2H, s), 7.11 (2H, d, J = 7.9 Hz), 7.41−7.55

(2H, m), 7.90 (1H, d, J = 8.1 Hz), 8.02 (1H, s), 8.08 (1H, d, J =

AA

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assay was performed in HBS-EP buﬀer containing 0.2 mM TCEP

and 0.5% DMSO. Data processing and analysis were performed

using the Biacore S200 evaluation software (GE healthcare).

Sensorgrams were double-referenced prior to global ﬁtting analysis.

The curve data were ﬁtted to a 1:1 binding model for the

determination of the binding rate constants of association and

dissociation (k_onand k_off). Dissociation constant (K_D) was calculated

using the following equation: K_D= k_off/k_on. Dissociative half-lives t_1/2

were calculated from the dissociation rate constants k_off(t_1/2= ln 2/

k_off).

log D_{pH 7.4}(log D). log D, which is a partition coeﬃcient between

1-octanol and aqueous buﬀer pH 7.4, of the compounds was

measured by the chromatographic method.²⁷log D values of

compounds were calculated using a calibration curve based on the

retention time of standards. The retention time of the compounds

was determined by a UHPLC system of Prominence UFLC with a

photodiode array detector (Shimadzu Corp., Kyoto, Japan). A YMC-

PackPro C18 packaged column (3 μm, 2.0 mm × 75 mm; YMC

Co., Ltd., Kyoto, Japan) was used at 40 °C with an isocratic

method. The mobile phase was adjusted methanol/pH 6.5 Britton-

Robinson buﬀer (5:2, v/v) with phosphoric acid to pH 7.4 and a

ﬂow rate was 0.2 mL/min.

bovine serum and 500 μg/mL G418, centrifuged for 3 min at 900

rpm, washed with Dulbecco’s phosphate-buﬀered saline containing

MgCl₂and CaCl₂(PBS+), and resuspended in 3 mL of PBS+. The

hERG inhibition assay was performed on the IonWorks Quattro

(Molecular Devices) system in population patch clamp (PPC)

mode. The extracellular solution was PBS with calcium and

magnesium (catalog number 14040, Invitrogen). The intracellular

solution contained 140 mM KCl, 2 mM MgCl₂, 1 mM EGTA, and

20 mM HEPES, pH 7.3, with KOH. After perforation using 100 μg/

mL amphotericin B (Sigma-Aldrich), hERG current was measured

under the potential-clamp protocol (holding potential −80 mV, the

ﬁrst voltage 40 mV: 2 s, the second voltage −50 mV, 2 s). The peak

tail current before addition of the compounds was measured as the

pre hERG current. Test compounds were incubated on the cells for

a period of 5 min. The peak tail current after addition of the

compounds was measured as the post-hERG current. % hERG

inhibition was calculated (n = 3 or 4) according to the following

equation.

%hERG inhibition = 100 − (post hERG current

/pre hERG current) × 100

Aqueous Solubility. Aqueous solubility was measured by the

shake ﬂask method.³⁴Sample was weighed into Thomson ﬁlter vial

(Chrom Tech, Inc., Minnesota) containing a 0.45 μm polyvinylidene

diﬂuoride ﬁlter membrane. A total of 400 μL of second dissolution

ﬂuid in the Japanese Pharmacopoeia (JP2, pH 6.8) was added to the

vial. The vial was incubated at 37 °C with shaking at 500 rpm for 18

h and ﬁltrated by compressing. Aqueous solubility was determined

by a UHPLC system of Prominence UFLC (Shimadzu Corp.,

Kyoto, Japan) with a photodiode array detector. A YMC-UltraHT

Pro C18 packaged column (2 μm, 2.0 mm × 30 mm; YMC Co.,

Ltd., Kyoto, Japan) was used at 40 °C with the gradient programs.

The mobile phase was distilled water/50 mM ammonium acetate

buﬀer/MeCN (8:1:1, v/v/v) and 50 mM ammonium acetate buﬀer/

MeCN (1:9, v/v) and a ﬂow rate was 0.7 mL/min. The starting

mobile phase consisted of 95% A and 5% B for 0.1 min, changed

gradually to 5% A 210 and 95% B from 0.1 to 1.6 min, and then

returned to the initial condition after 1.3 min and was reequilibrated

for an additional 0.6 min.

Transcellular Transport Study Using Transporter-Expres-

sion System. Human MDR1-expressing LLC-PK1 cells were

cultured, and the transcellular transport study was performed with

minor modiﬁcations to the method reported previously.³⁷In brief,

the cells were grown in Transwell 96-well permeable support (pore

size 0.4 μm, 0.143 cm²surface area) with polycarbonate membrane

(Corning Life Sciences, Lowell, MA). The cells were preincubated

with M199 at 37 °C. Subsequently, transcellular transport was

initiated by the addition of M199 either to apical compartments (75

μL) or to basolateral compartments (250 μL) containing 1 μM test

compounds. The assay was terminated by the removal of each assay

plate after 1 h. Aliquots (25 μL) from the opposite compartments

were mixed with acetonitrile, and then centrifuged. The compound

concentrations in the supernatant were measured by LC/MS/MS.

The apparent permeability (P_app) of test compounds in the receiver

wells was determined and the eﬄux ratio (ER, MDR BA/AB) for

the MDR1 membrane permeability test was calculated using the

following equation

Protocol for hERG Electrophysiological Assay Using

Manual Patch Clamp. Experiments were performed using HEK

293 cells stably transfected with hERG at room temperature. Whole

cell voltage clamp recordings of hERG currents were made using an

Axopatch 200B ampliﬁer. Membrane currents were low pass ﬁltered

at 1 kHz and sampled at 2.5 kHz. Borosilicate glass pipettes

(Harvard Conﬁdential 37 Apparatus, Kent, U.K.) were pulled and

ﬁre polished to get ﬁnal resistances of 2−3 MΩ. Series resistances

were less than 6 MΩ and were compensated by 60−85%. The

pipette solution contained, KCl 130 mmol/L, NaCl 7 mmol/L,

MgCl₂1 mmol/L, ATP−2Na 5 mmol/L, HEPES 5 mmol/L, EGTA

5 mmol/L, pH 7.2. Cells were perfused with extracellular Tyrode’s

solution containing, NaCl 137 mmol/L, KCl 4 mmol/L, MgCl₂1

mmol/L, CaCl₂1.8 mmol/L, glucose 11 mmol/L, HEPES 10

mmol/L, pH 7.4. The standard voltage protocol to measure the

eﬀects of compounds on hERG current amplitudes was to hold the

membrane potential at −75 mV and apply 0.5 s depolarizations to

10 mV. Tail currents were evoked with 0.5 s pulses to −40 mV. The

protocol was repeated every 5 s, allowing complete current

deactivation between test pulses. Experiments were performed at

room temperature. The compound was dissolved in DMSO (10

μmol/L) and the solution was added to an external solution to

make ﬁnal compound solution at the concentration of 10 μmol/mL.

The peak tail current after addition of the compounds was measured

as the post-hERG current. % hERG inhibition was calculated (n =

2) according to the following equation.

%hERG inhibition = 100 − (post hERG current

/pre hERG current) × 100

Ethics Statement. The care and use of the animals and the

experimental protocols used in this research were approved by the

Experimental Animal Care and Use Committee of Takeda

Pharmaceutical Company Limited.

Animals. Seven-week-old male C57BL/6J Jcl mice were

purchased from CLEA Japan, Inc. (Tokyo, Japan). After acclimation

for a week in our laboratory, eight-week-old mice were used for

experiments. The animals were housed in a light-controlled room

(12-h light/dark cycle with lights on from 7:00 AM). Food and

water were provided ad libitum.

Drug Administration. Compound 4f was suspended in 0.5%

(w/v) methylcellulose in distilled water; oral administration (p.o.)

was conducted at a volume of 10 mL/kg in mice. A solution of 0.5%

methylcellulose was administered as the vehicle control.

In Vivo PK Study of 4f in Mice. These mice were sacriﬁced 60

min after administration of 4f, and the brain hemispheres and the

plasma were isolated (n = 4 in each group). The brains were rapidly

removed and rinsed in ice-cold saline. The hemispheres were then

ER(MDR BA/AB) = P_app,BtoA/P

app,AtoB

where P_app,AtoBand P_app,BtoArepresent the apparent permeability in

the apical-to-basal direction and the basal-to-apical direction,

respectively.

Protocol for hERG Electrophysiological Assay Using

IonWorks (Autopatch Clamp). The recombinant CHO-K1 cell

line expressing the human ether-a-go-go-related gene (CY3038) was

purchased from Millipore Corp. and cultured in large batches,

frozen, and stored at a density of 1.5 × 10⁷cells per vial to make

frozen cells ready to use immediately after thawing. For use, the

frozen cells were thawed in Ham’s F12 supplemented with 10% fetal

AB

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immediately isolated and frozen on dry ice. The isolated samples

were stored at −80 °C until use. The frozen samples were

homogenized in saline at 4 mL/g tissue except plasma. The plasma

and brain concentrations of compounds were measured by high-

performance liquid chromatography/tandem mass spectrometry

(LC/MS/MS).

Jumpei Aida − Research, Takeda Pharmaceutical Co., Ltd.,

Fujisawa, Kanagawa 251-8555, Japan; orcid.org/0000-

0002-7546-1915

Nao Morishita − Research, Takeda Pharmaceutical Co., Ltd.,

Fujisawa, Kanagawa 251-8555, Japan

Satoshi Sogabe − Research, Takeda Pharmaceutical Co.,

Ltd., Fujisawa, Kanagawa 251-8555, Japan; Present

Address: Axcelead Drug Discovery Partners, Inc., 26-1,

Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa 251-

0012, Japan; orcid.org/0000-0003-2393-9582

Douglas R. Dougan − Structural Biology and Biophysics,

Takeda California, Inc., San Diego, California 92121,

United States

Sean C. Gay − Structural Biology and Biophysics, Takeda

California, Inc., San Diego, California 92121, United

States; Present Address: Art Robbins Instruments, 1293,

Mountain View Alviso Rd, Suite D, Sunnyvale, California

94089, United States.

Ling Qin − Structural Biology and Biophysics, Takeda

California, Inc., San Diego, California 92121, United States

Naoto Arimura − Research, Takeda Pharmaceutical Co.,

Ltd., Fujisawa, Kanagawa 251-8555, Japan

Yasuko Takahashi − Research, Takeda Pharmaceutical Co.,

Ltd., Fujisawa, Kanagawa 251-8555, Japan

Measurement of Arachidonic Acid (AA) and 2-Arachido-

noylglycerol (2-AG) in Brain. These mice were sacriﬁced 60 min

after administration of 4f, and the brain hemispheres were isolated

(n = 4 in each group). The brains were rapidly removed and rinsed

in ice-cold saline. The hemispheres were then immediately dissected

and frozen on dry ice. The samples were homogenized with a shake

master (Bio Medical Science, Japan), and added 400 uL of

isopropanol to 100 mg of tissue weight and mixed well. The

suspension (50 uL) was transferred to another tube, and added an

equal volume of isopropanol, mixed well, and centrifuged at 15000

rpm for 5 min. The supernatant (5 uL) was transferred to a new

tube. The samples were dried up for 10 min under nitrogen spray.

The samples were diluted with 100 uL of diluent containing

isopropanol. The supernatant (1 uL) was analyzed by LC/MS/MS.

The value was calculated from a calibration curve using isopropanol

as a matrix. Analysis with LC/MS/MS was outsourced to LSI

Medience (Tokyo, Japan).

ASSOCIATED CONTENT

■

sı

* Supporting Information

Masako Sasaki − Research, Takeda Pharmaceutical Co., Ltd.,

Fujisawa, Kanagawa 251-8555, Japan

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00432.

Yusuke Kamada − Research, Takeda Pharmaceutical Co.,

Ltd., Fujisawa, Kanagawa 251-8555, Japan; Present

Address: Central Pharmaceutical Research Institute,

Japan Tobacco Inc., 1-1, Murasaki-cho, Takatsuki, Osaka

569-1125, Japan.; orcid.org/0000-0003-3701-2075

Kazunobu Aoyama − Research, Takeda Pharmaceutical Co.,

Ltd., Fujisawa, Kanagawa 251-8555, Japan; Present

Address: Axcelead Drug Discovery Partners, Inc., 26-1,

Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa 251-

0012, Japan

Kouya Kimoto − Pharmaceutical Sciences, Takeda

Pharmaceutical Co., Ltd., Fujisawa, Kanagawa 251-8555,

Japan

Makoto Kamata − Research, Takeda Pharmaceutical Co.,

Ltd., Fujisawa, Kanagawa 251-8555, Japan

Molecular formula strings (CSV)

Analytical HPLC traces for all of the tested

compounds, ¹H NMR and ¹³C NMR charts of

compound 4f, co-crystal structure of compound 4f

with MAGL, and X-ray data collection and reﬁnement

statistics for the crystal structures of MAGL in complex

with compounds 1, 1h, 2d, and 4f (PDF)

Accession Codes

The coordinates of the crystal structures of MAGL in

complex with compounds 1 (7L4T), 1h (7L4U), 2d

(7L4W), and 4f (7L50) have been deposited in the Protein

Data Bank. The authors will release the atomic coordinates

and experimental data upon article publication.

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00432

AUTHOR INFORMATION

■

Corresponding Author

Author Contributions

Shuhei Ikeda − Research, Takeda Pharmaceutical Co., Ltd.,

Fujisawa, Kanagawa 251-8555, Japan; orcid.org/0000-

0002-0121-2983; Phone: +81 466 32 1198;

Email: shuhei.ikeda@takeda.com; Fax: +81 466 29 4450

The manuscript was written through contributions of all

authors. All authors have given approval to the ﬁnal version

of the manuscript.

Notes

Authors

The authors declare no competing ﬁnancial interest.

Hideyuki Sugiyama − Research, Takeda Pharmaceutical Co.,

Ltd., Fujisawa, Kanagawa 251-8555, Japan

Hidekazu Tokuhara − Research, Takeda Pharmaceutical

Co., Ltd., Fujisawa, Kanagawa 251-8555, Japan; Present

Address: Axcelead Drug Discovery Partners, Inc., 26-1,

Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa 251-

0012, Japan

Masataka Murakami − Research, Takeda Pharmaceutical

Co., Ltd., Fujisawa, Kanagawa 251-8555, Japan

Minoru Nakamura − Research, Takeda Pharmaceutical Co.,

Ltd., Fujisawa, Kanagawa 251-8555, Japan

Yuya Oguro − Research, Takeda Pharmaceutical Co., Ltd.,

Fujisawa, Kanagawa 251-8555, Japan

ACKNOWLEDGMENTS

■

The authors acknowledge Dr. Tomohiro Kaku for fruitful

discussions on ligand design, Dr. Tatsuki Koike for valuable

suggestions about research plans, and Dr. Takanobu Kuroita

for substantial supports to progress this project. We are also

grateful to Dr. Robert Skene for depositing the coordinates of

the co-crystal structures.

ABBREVIATIONS USED

AA, arachidonic acid; ABHD6, α,β-hydrolase domain 6;

ADDP, 1,1′-(azodicarbonyl)dipiperidine; 2-AG, 2-arachido-

■

AC

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noylglycerol; Anandamide, N-arachidonoylethanolamine;

BBB, blood−brain barrier; BSA, bovine serum albumin;

CNS, central nervous system; DBU, 1,8-diazabicyclo[5.4.0]-

undec-7-ene; DIPEA, N,N-diisopropylethylamine; DME, 1,2-

dimethoxyethane; DMF, N,N-dimethylformamide; DMSO,

dimethyl sulfoxide; EC, endocannabinoid; EDCI, 1-(3-

dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride;

Et₃N, triethylamine; EtOAc, ethyl acetate; EtOH, ethanol;

FAAH, fatty acid amide hydrolase; hERG, human ether-a-go-

go-related gene; HOBt, 1-hydroxybenzotriazole; IPE, diiso-

propyl ether; k_off, dissociation rate constant; LLE, lipophilic

ligand eﬃciency; MAGL, monoacylglycerol lipase; MDR1,

multidrug resistance protein 1; MeCN, acetonitrile; MeOH,

methanol; SBDD, structure-based drug discovery; SPR,

surface plasmon resonance; TFAA, triﬂuoroacetic anhydride;

THF, tetrahydrofuran; UHP, urea hydrogen peroxide

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J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Design and Synthesis of Novel Spiro Derivatives as Potent and Reversible Monoacylglycerol Lipase (MAGL) Inhibitors: Bioisosteric Transformation from 3-Oxo-3,4-dihydro-2 H-benzo[ b][1,4]oxazin-6-yl Moiety

DOI: 10.1021/acs.jmedchem.1c00432

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Article abstract of DOI:10.1021/acs.jmedchem.1c00432

Full text of DOI:10.1021/acs.jmedchem.1c00432

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