Scholarly article on [3-(4-methylphenoxy)phenyl]methanol 72714-64-8 from Journal of Medicinal Chemistry p. 3750

Article

pubs.acs.org/jmc

Conformational Constraint of the Glycerol Moiety of

Lysophosphatidylserine Aﬀords Compounds with Receptor Subtype

Selectivity

Sejin Jung,^†,§Asuka Inoue,^‡,∥Sho Nakamura,^†Takayuki Kishi,^‡Akiharu Uwamizu,^‡Misa Sayama,^†

Masaya Ikubo,^†Yuko Otani,^†Kuniyuki Kano,^‡Kumiko Makide,^‡,∥Junken Aoki,*^,‡,⊥

and Tomohiko Ohwada*^,†

^†Laboratory of Organic and Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1

Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

^‡Laboratory of Molecular and Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3, Aoba,

Aramaki, Aoba-ku, Sendai, Miyagi 980-8578, Japan

^§Research Foundation ITSUU Laboratory, C1232 Kanagawa Science Park R & D Building, 3-2-1 Sakado, Takatsu-ku, Kawasaki,

Kanagawa 213-0012, Japan

^∥PRESTO, Japan Science and Technology Agency (JST), 4-1-8, Honcho, Kawaguchi-shi, Saitama 332-0012, Japan

^⊥AMED-CREST, Japan Agency for Medical Research and Development, 1-7-1 Otemachi, Chiyoda-ku, Tokyo 100-0004, Japan

S

* Supporting Information

ABSTRACT: Lysophosphatidylserine (LysoPS) is an endog-

enous lipid mediator that speciﬁcally activates membrane

proteins of the P2Y and its related families of G protein-

coupled receptors (GPCR), GPR34 (LPS₁), P2Y10 (LPS₂),

and GPR174 (LPS₃). Here, in order to increase potency and

receptor selectivity, we designed and synthesized LysoPS

analogues containing the conformational constraints of the

glycerol moiety. These reduced structural ﬂexibility by ﬁxation

of the glycerol framework of LysoPS using a 2-hydroxymethyl-

3-hydroxytetrahydropyran skeleton, and related structures

identiﬁed compounds which exhibited high potency and

selectivity for activation of GPR34 or P2Y10. Morphing of the structural shape of the 2-hydroxymethyl-3-

hydroxytetrahydropyran skeleton into a planar benzene ring enhanced the P2Y10 activation potentcy rather than the GPR34

activation.

pharmaceutical signiﬁcance. These receptors are mainly ex-

pressed in immune-related tissues,^15,16and GPR174 was

reported to be abunduntly expressed in regulatory T cells.¹⁷

Thus, it is plausible that LysoPS is involved in the pathogenesis

of the immunity system. In order to provide useful tools for

elucidation of the physiological roles of LysoPS and its speciﬁc

receptors, we have conducted structure−activity relationship

(SAR) studies by means of optimization of the individual

structural modules of LysoPS (fatty acid, glycerol, and amino

acid), as well as their linkages (Figure 1). Our results support

the idea that LysoPS is assembled from modular components

that inﬂuence subtype selectivity.^18,19

INTRODUCTION

■

Lysophosphatidylserine (LysoPS), which is derived from

phosphatidylserine by enzymatic deacylation,¹has lipid

mediator-like actions, and induces multiple cellular responses

both in vitro and in vivo, including mast cell degranulation,²⁻⁵

neurite outgrowth in PC12 cells,⁶suppression of proliferation

of T lymphocytes,⁷migration of ﬁbroblasts and tumor cells,^8,9

and engulfment of apoptotic cells by macrophages.¹⁰

Recent studies to ﬁnd ligands of orphan G-protein-coupled

receptors (GPCRs) identiﬁed three LysoPS-speciﬁc receptors,

namely P2Y10 (also known as LPS₂), A630033H20 (LPS_2L,

(LPS₂-like)), and GPR174 (LPS₃) in addition to the previously

identiﬁed LysoPS receptor (GPR34 (LPS₁)).¹¹⁻¹³Orthologs of

GPR34, P2Y10, and GPR174 have been found in human,

mouse, rat, and zebraﬁsh, and they respond to endogenous

LysoPS with EC₅₀values at the submicromolar level.¹⁴The

human A630033H20 gene apparently encodes frame-shift

product and is regarded as a a nonfunctional pseudogene.

Therefore, there appear to be three LysoPS receptors of

Its modular structure and many rotatable single bonds make

LysoPS very ﬂexible, and it may adopt various active

conformations that could account for its pan-agonistic activity

toward GPR34, P2Y10, and GPR174. This idea led us to

Received: December 11, 2015

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containing aromatized nonlipid surrogates of fatty acids are

eﬀective for activation of our target receptors.^18,19

In addition, the hydroxyl group at the sn-2 position of the

glycerol backbone has been proposed to have a key role in

activation of GPR174, probably by acting as a hydrogen-

bonding donor to the receptor.^18,19Therefore, we introduced a

2-hydroxymethyl-3-hydroxytetrahydropyran skeleton (b) in

place of the glycerol moiety (a) with the aim of increasing

the receptor selectivity by deleting the hydrogen atom of the sn-

2 hydroxyl group of glycerol (Figure 2). The introduced 2-

hydroxymethyl-3-hydroxytetrahydropyran moiety (b, Figure 2)

completely reproduces the three carbon and three oxygen

atoms of the glycerol backbone (a, Figure 2).

In addition, its cyclic structure allows LysoPS analogues to

generate two diﬀerent regioisomers due to the nonequivalent

substituents, i.e., one containing the acyl chain at the primary

alcohol position, and the other containing the acyl chain at the

secondary alcohol position (Figure 3). Thus, introduction of

the tetrahydropyran moiety serves to restrict the ring

conformation as well as the space-ﬁlling of the phosphodiester

and acyl units. Advantages of 2-hydroxymethyl-3-hydroxyte-

trahydropyrans (b) include readily availability from commer-

cially available sugar compounds and their potential for

derivatization due to the nonequivalent primary and secondary

alcohols and two stereogenic centers at the ring junction.

We also focused on 2-hydroxymethyl-3,6-dihydro-2H-pyran-

3-ol (c, Figure 2) as an unsaturated cyclic LysoPS analogue.

This unsaturated ring moiety exists in a half-chair conformation

with the oxygen atom (O1 in Figure 2) on one side and C2 on

the other side of the plane formed by the remaining four

atoms.^20,21These conformational features may provide a more

restricted glycerol skeleton by removing the 1,3-diaxial

interaction, potentially leading to more stable structures and

limitations on conformational ﬂexibility with respect to the ring

structure. Finally, we tried replacing this unsaturated ring (c)

with a benzene ring (d, Figure 2), because the completely

planar structure and limited conformational ﬂexibility might

generate a limited range of conformations with potential for

increased selectivity and potency of ligands toward speciﬁc

receptors.

Figure 1. Structure of LysoPS (1-oleoyl-lysophosphatidylserine),

showing its structural modules and linkers in diﬀerent colors.

hypothesize that suitable conformational constraints could

generate structures with selective receptor-activating activity.

Therefore, in this study, we designed and synthesized a number

of conformationally constrained LysoPS analogues by embed-

ding the glycerol moiety into 2-hydroxymethyl-3-hydroxyte-

trahydropyran and related skeletons (see Figure 2), and we

examined the selective receptor-activating ability of the

resulting derivatives.

Figure 2. Generation of LysoPS analogues by replacing the glycerol

module with various conformationally restricted structures.

RESULTS AND DISCUSSIONS

■

Molecular Design of Conformationally Constrained

LysoPS Derivatives. LysoPS is composed of several

substructural modules (amino acid, glycerol, and fatty acid

chain) connected via linkers (phosphodiester and ester

linkages), as shown in Figure 1. Our previous SAR studies

indicated that ^L-serine is an essential module for the activities

toward all the three LysoPS receptors, and LysoPS derivatives

The detailed molecular design is shown in Figure 3. The

conformation of the glycerol moiety can be ﬁxed in two ways,

Figure 3. Design of cyclic LysoPS analogues by constraint of the glycerol moiety.

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Figure 4. Saturated cyclic LysoPS analogues.

Figure 5. Modiﬁcation of acyl chain (C): introducing substituents on the terminal benzene ring.

Figure 6. Investigation of isomers of acyl chain (C).

Figure 7. Unsaturated cyclic LysoPS analogues.

i.e., by linking carbon 3 to the hydroxyl group (i) or by linking

carbon 1 to the hydroxyl group (ii). The former method

generates 1°-acyl-2°-phosphoserine analogues and the latter

generates 2°-acyl-1°-phosphoserine analogues (Figure 3).

Moreover, the stereochemistry at the ring junction can take

trans and cis conﬁgurations and provides four diﬀerent

frameworks of cyclic LysoPS analogues (trans and cis-1°-acyl-

2°-phosphoserine; trans and cis-2°-acyl-1°-phosphoserine)

(Figure 3).

Synthesis of LysoPS Derivatives Based on the 2-

Hydroxymethyl-3-hydroxytetrahydropyran and Related

Scaﬀolds. LysoPS analogues (1−12) based on the 2-

hydroxymethyl-3-hydroxytetrahydropyran scaﬀold with all

possible combinations arising from the presence of primary

and secondary alcohols and cis and trans conﬁgurations were

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Figure 8. LysoPS analogues containing the aromatized glycerol moiety.

a

Scheme 1. Synthesis of 1°-Acyl-2°-phosphoserine-type Analogues (A) and 2°-Acyl-1°-phosphoserine-type Analogue (B)

a

Conditions for part A: (a) HSiEt₃, BF₃·OEt₂, CH₂Cl₂, r.t. (b) Pd/C, MeOH. (c) NaOMe, MeOH, r.t. (d) TBSCl, imidazole, CH₂Cl₂, r.t. (f)

Amberlyst15, MeOH, r.t., 99%. (g) Oleoyl chloride, DMAP, CH₂Cl₂, r.t. (h) R-CO₂H, diisopropylcarbodiimide, N,N-dimethylaminopyridine,

CH₂Cl₂, r.t. (i) TFA, r.t.

designed (Figure 4). In this paper, we focused on three kinds of

acyl chains, i. e., oleic acid (A), and its surrogates (B) and (C)

(Figure 4), which were previously identiﬁed as potentially

eﬀective to activate speciﬁc receptors.^17,18We also explored the

subsituent eﬀect of the acyl chain (C) (13−16, Figure 5) and

the eﬀects of the aromatic arrangemets toward GPR34 (17−20,

Figure 6).

We also designed LysoPS anlogues (21−32) containing the

unsaturated 2-hydroxymethyl-3,6-dihydro-2H-pyran-3-ol scaf-

fold (Figure 7). Finally aromatized glycerol mimics (33−35)

were designed (Figure 8). The synthesis of active derivatives

are shown in the Experimental Section, and the synthesis of

weak or no-active derivatives are shown in the Supporting

Information.

Synthesis of Designed Molecules. The 2-hydroxymethyl-

3-hydroxytetrahydropyran moiety was obtained through

established synthetic methods from sugar compounds with

appropriate stereochemistry (Scheme 1).^22,23Tri-O-acetyl-D-

glucal²²was used as a starting material for trans derivatives, and

tri-O-acetyl-^D-galactal²³was used for cis derivatives (Scheme 1).

The starting sugars (tri-O-acetyl-^D-glucal or tri-O-acetyl-D-

galactal) were subjected to ionic hydrogenation (HSiEt₃and

BF₃·OEt₂), followed by hydrogenation over Pd/C, and then

hydrolysis of two acetyl groups with NaOMe/MeOH (i.e., via

36 and 37) to obtain trans- and cis-2-hydroxymethyl-3-

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hydroxytetrahydropyrans (38a and 38b, respectively). The

primary alcohol was selectively protected with a TBS group

(39a and 39b), followed by formation of a phosphate diester

linkage using the phosphoramidite method with compound 40.

Acid-catalyzed deprotection of the silyl ether (41) was carried

out to free the primary alcohol 42a (trans) or 42b (cis),

followed by acylation with various fatty acid analogues, and

then complete deprotection of 43−45 in TFA furnished the

desired LysoPS analogues, trans-1°-acyl-2°-phosphoserines (1−

3) and cis-1°-acyl-2°-phosphoserines (4−6) (Scheme 1A). 2°-

Acyl-1°-phosphoserine compounds (trans-(7−9) and cis-(10−

12)) were also obtained simply by changing the reaction order

of esteriﬁcation and connection of the phosphoserine group

through the intermediates (58−66, Scheme S1). The analogues

containing a para-substituted benzene ring with an acyl chain

(C) (13−16, Figure 5, Scheme 1B and Scheme S2) and the

analogues containing a diﬁﬀerent arrangement of three

aromatic rings of the acyl chain (C) (17−20, Figure 6) were

also synthesized. The syntheses of the derivatives 17−20 were

shown in the Supporting Information (Schemes S3 and S4).

A 3,6-dihydro-2H-pyran moiety was obtained as an

intermediate by skipping the hydrogenation step to obtain

unsaturated cyclic moiety 47a or 47b (Scheme 2).

unsaturated primary acyl compounds (21−26). To obtain

secondary acyl compounds (27−32, Figure 7), the order of

connection of the phosphoserine unit and the ester was

reversed through the intermediates (67−75, Scheme S5).

Three kinds of benzene-type LysoPS analogues, ortho (33),

meta (34) and para (35), can be generated (Figure 6). To

obtain these benzene-type LysoPS analogues, salicylic alcohol

(o-hydroxybenzyl alcohol 53a, m-hydroxybenzyl alcohol 53b

and p-hydroxybenzyl alcohol 53c) were used (Scheme 4). The

synthesis was performed as shown in Scheme 4: the benzyl

alcohol was protected with a TBS group (54) and the

phosphate group was connected to the phenolic oxygen atom

(55), followed by deprotection of the TBS silylether (56), and

then esteriﬁcation of the benzyl alcohol to aﬀord the protected

benzene analogues (57). Deprotection in TFA gave the

aromatic LysoPS derivatives (33-35).

Eﬀect of Replacement of the Glycerol Backbone with

a Tetrahydropyran Ring Moiety and Inﬂuence of

Regioisomers. The agonistic activities of the synthesized

cyclic LysoPS analogues toward GPR34 and P2Y10 in the

TGFα shedding assay are summarized in Table 1 (see also

Figure S1). The original concentration−response data are

shown in Figure S2 (GPR34) and Figure S3 (P2Y10). All the

LysoPS analogues synthesized and tested in this study lack the

sn-2 hydroxy, which was found to be essential for the activation

of GPR174. In fact, all the LysoPS analogues did not show

signiﬁcant activity toward GPR174 (vide infra).

Scheme 2. Synthesis of Modiﬁed Unsaturated Cyclic

Glycerol Framework

Activity toward GPR34 1°-Acyl-2°-phosphoserine analogues

(1−6 except 6) (1: EC₅₀= 100 nM; 2: EC₅₀= 640 nM; 3: EC₅₀=

340 nM; 4: EC₅₀= 210 nM; 5: EC₅₀= 110 nM; 6: EC₅₀= 140

nM) were generally more eﬀective for GPR34 receptor

activation as compared with the corresponding regioisomeric

2°-acyl-1°-phosphoserine analogues (7-12 except 12) (7:

EC₅₀= NA (not available due to weak activity); 8: EC₅₀=

NA: 9: EC₅₀= >1.0 μM; 10: EC₅₀> 1.0 μM; 11: EC₅₀> 1 μM;

12: EC₅₀= 190 nM) regardless of cis or trans conﬁguration and

fatty acid chain (A), (B) or (C) (Figure 4 and Table 1).

Futhermore, the 1°-acyl-2°-phosphoserine analogues (1−6)

showed comparable or stronger activation potency toward

GPR34 receptors, as compared with the prototype LysoPS

(EC₅₀= 240 nM). This result suggested that the space-ﬁlling

47a and 47b were connected to phosphoserine by means of

the phosphoramidite method (to give 48) followed by

deprotection of silylether to aﬀord the free primary alcohol

(49) and then esteriﬁcation (50−52, Scheme 3). Finally,

deprotection was carried out with TFA to aﬀord the

a

Scheme 3. Synthesis of Oleﬁn-trans-1°-acyl-2°-phosphoserine

a

Conditions: (a) 1H-tetrazole, CH₂Cl₂, THF, r.t.; TBHP, CH₂Cl₂, r.t., Ar. (b) Amberlyst-15, MeOH, r.t., 99%. (c) Oleoyl chloride, DMAP, CH₂Cl₂,

r.t. (d) Diisopropylcarbodiimide, (B) or (C), N,N-dimethylaminopyridine, CH₂Cl₂, r.t. (e) TFA, r.t.

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Scheme 4. Synthesis of Benzene-LysoPS Analogues

characteristics of the substructures of the 1°-acyl-2°-phospho-

serine and 2°-acyl-1°-phosphoserine derivatives are diﬀerent

(Figure 9), and this diﬀerence may contribute to the receptor

activation.

1 μM for P2Y10 activation). The conclusion that acyl chain (B)

is the most favorable for P2Y10 activation is consistent with our

previous SAR study.^18,19

Activity toward GPR174. All the compounds (1-12)

showed no signiﬁcant activity toward GPR174, presumably

because of deletion of the hydroxyl group at the sn-2 position of

the glycerol backbone (Figure 4). As representative examples,

the null activities of the derivatives 1, 2, and 3 were shown in

Figure S4 (Supporting Information).

On the other hand, one set of the regioisomeric cis-

derivatives (6 and 12) was rather inconsistent with the above

trend: both 1°-acyl-2°-phosphoserine 6 and 2°-acyl-1°-

phosphoserine 12 showed similar reactivity toward GPR34

activation (6: EC₅₀= 140 nM; 12: EC₅₀= 190 nM for GPR34).

Moreover, cis- and trans-stereoisomerism inﬂuenced the

potency of GPR34 activation: in the case of 1°-acyl-2°-

phosphoserine analogues, cis isomers 5 and 6, bearing acyl

chains (B) and (C), respectively, exhibited more potent

agonistic activity toward GPR34 than the corresponding trans

isomers (2 and 3), while the derivatives bearing acyl chain (A),

i.e., trans 1 and cis 4 showed a reverse order of activity toward

GPR34.

It is noteworthy that the nonlipid type of acyl chain (C) is

the most eﬀective substructure for selectivity toward GPR34

over P2Y10 activation, as compared with the acyl chains (A)

and (B) (Figure 4) (vide infra). Among these compounds (3, 6,

9 and 12), 6 and 12 showed the comparable agonistic

selectivity toward GRR34, being approximately 100-fold more

potent than that toward P2Y10. This result may indicate that

the combination of cis conﬁguration with acyl chain (C) aﬀords

a favorable overall molecular shape for high subtype selectivity

(vide infra).

Activity toward P2Y10. All of the derivatives (1-12)

showed very weak potency toward P2Y10 in comparison with

endogenous LysoPS (EC₅₀= 25 nM). However, some of the

derivatives bearing the acyl chains (A) and (B) showed distinct

activity such as 1 (acyl chain (A)), 2, 5, and 8 (all bearing acyl

chain (B)) (1: EC₅₀= 450 nM; 2: EC₅₀= 610 nM; 5: EC₅₀= 470

nM; 8: EC₅₀= 290 nM for P2Y10 activation), even though the

acivity is weaker than the original LysoPS. Intriguingly as

pointed above, derivatives (3, 6, 9 and 12) bearing the acyl

chain (C) diminished the activity toward P2Y10 signiﬁcantly

Eﬀect of Fatty Acid C. In order to study further the reason

for the equivalent eﬀectiveness of 1°-acyl-type and 2°-acyl-type

derivatives containing fatty acid C, i.e., cis-1°-(C)-2°-

phosphoserine (6) and cis-2°-(C)-1°-phosphoserine (12)

analogues, toward GPR34, we synthesized additional LysoPS

analogues (13−16) bearing a substituent on the terminal

benzene ring of acyl chain (C) (Figure 5), i.e., we introduced

methyl and chlorine substituents on the terminal benzene ring

of acyl chain (C) (Figure 5). As shown in Table 1, the activities

toward GPR34 of the analogues containing a para-substituted

benzene ring with acyl chain (C), cis-2°-(C)-1°-phosphoserine

analogues 15 and 16, were retained, and were comparable to or

slightly weaker than those of cis-1°-(C)-2°-phosphate ana-

logues 13 and 14 (13: EC₅₀= 37 nM; 14: EC₅₀= 36 nM; 15:

EC₅₀= 120 nM; 16: EC₅₀= 77 nM for GPR34). These

activities are stronger than those of the unsubstituted acyl chain

(C) derivatives (6 and 12). These results suggest a consistent

trend of activity toward GPR34 with respect to regioisomerism

of the acyl chain (C)-substituted derivatives. Furthermore,

these derivatives did not show signiﬁcant activity toward P2Y10

(13: EC₅₀> 1 μM; 14: EC₅₀> 1 μM; 15: EC₅₀> 1 μM; 16:

inactive for P2Y10) and therefore derivatives 13 and 14 showed

the most potent and selective agonistic activity toward GRR34,

being approximately 10-fold more potent than LysoPS itself.

The above results encouraged us to examine the most

eﬀective shape of fatty acid (C) in cis-1°-(C)-2°-phosphoserine

(6) and cis-2°-(C)-1°-phosphoserine (12) analogues (Figure

6). According to the shedding assay results in Table 1, meta (17

and 19) and para isomers (18 and 20) of acyl chain (C) were

(3: EC₅₀> 1 μM; 6: EC₅₀> 1 μM; 9: EC₅₀> 1 μM; 12: EC₅₀

>

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a,b

Table 1. Agonistic Activities of Analogues Studied in This Work

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Table 1. continued

a

b

c

Activities are represented in terms of EC₅₀(see Experimental Section). “NA” means “not available” because of very low activity. Log EC₅₀(M)

and E_max(% AP-TGFα release) values are calculated from a sigmoidal concentration−response curve (see Experimental Section) and shown as Mean

d

SEM of indicated numbers of independent experiments (n). EC₅₀values are calculated from a mean value of LogEC₅₀. RIA (relative intrinsic

activity; a dimensionless parameter; ref 24) is an estimation of agonist activity and represented as a E_max/EC₅₀value relative to that of a reference

compound (6 for GPR34 and a compound 10b (diF-C3-ph-o-O-C11) in the previous work (ref 19) for P2Y10). By deﬁnition, the RIA value of a

reference compound is equal to 1. EC₅₀values are calculated from a mean value of LogEC₅₀.

Figure 10. LysoPS analogues diﬀerentially activate GPR34. HEK293

cells transiently transfected with GPR34-encoding expression vector or

an empty vector were treated with compounds and receptor-speciﬁc

AP-TGFα release responses were determined by subtracting back-

ground responses in the empty vector-transfected cells (actual signals

in the GPR34-expressing cells and the empty vector-transfected cells

are shown in Supporting Information Figure S2). Data are mean and

SEM (standard error of the mean) for seven to 11 independent

experiments.

Figure 9. Diﬀerent space-ﬁlling characteristics of substructures of

cyclic LysoPS analogues, trans 1°-acyl-2°-phosphoserine (1) vs trans

2°-acyl-1°-phosphoserine (7). Representative structures evaluated by

conformation search calculations.

inactive toward GPR34 (17: EC₅₀> 1 μM; 18: inactive; 19:

inactive; 20: EC₅₀> 1 μM for GPR34), while ortho compounds

6 and 12 are potent activators of GPR34 (Figure 6). This

suggests that an ortho-substituted propionic acid skeleton is

important for GPR34 activation. Furthermore, derivatives 17-

20 did not show signiﬁcant activity for P2Y10 at all (17: EC₅₀>

1 μM; 18: EC₅₀> 1 μM; 19: > 1 μM; 20: EC₅₀> 1 μM for

P2Y10).

In the present combinations of 1-acyl/2-acyl and cis/trans

isomerisms of the 2-hydroxymethyl-3-hydroxytetrahydropyran

ring system, the concentration−response data of the resultant

potent derivatives (6, 12, 13, 14) is shown in Figure 10,

together with LysoPS and some less active counterparts (3 and

9).

Eﬀect on Intracellular Calcium Mobilization in

HEK293-GPR34 Cells. We further examined whether the

active compounds found by the TGFα shedding assay (cis-1°-

acyl-2°-phosphate analogues 6, 13, and 14 and cis-2°-acyl-1°-

phosphate analogues 12, 15, and 16) induced a transient

increase in an intracellular Ca²⁺concentration ([Ca²⁺]_i)

through activation of GPR34 (Figure 11). The compounds

(6, 13, 14, 12, 15, and 16) induced robust Ca²⁺responses in

the cells expressing GPR34 and the chimeric Gα_q/i1subunit.

The responses were completely absent in cells expressing the

chimeric Gα_q/i1subunit alone (mock cells; data not shown).

The data clearly showed that the compounds 6 and 12−16

induced a transient increase in [Ca²⁺]_i, which is consistent with

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Figure 11. Eﬀects of shedding-assay-positive compounds on intracellular calcium mobilization ([Ca²⁺]_i) in HEK293-GPR34 cells. Calcium 5 dye-

loaded HEK293 cells transiently expressing GPR34 together with the chimeric Gα_q/i1subunit were stimulated with 1 μM compound (black symbol)

or 100 nM compound (gray symbol), and ([Ca²⁺]_i) was measured ﬂuorometrically using a FlexStation 3 (Molecular Devices) liquid handling

microplate reader (Molecular Devices). Dotted lines, SD of triplicate measurements of a single assay. A test compound was added at approximately

35 s from the initial measurement. Note that there was not signiﬁcant [Ca²⁺]_iin the negative-control cells expressing the Gα_q/i1subunit alone (not

shown).

activation of GPR34 (Figure 11). The eﬀects of other

compounds (1−35) on a transient increase in [Ca²⁺]_iwere

compiled in Supporting Information (Figures S5 and S6).

Analysis of Solution Conformations of Saturated 2-

Hydroxymethyl-3-hydroxytetrahydropyran Derivatives.

We examined the conformations of the saturated 2-hydrox-

ymethyl-3-hydroxytetrahydropyran derivatives in solution by

using NMR spectroscopy. Although it would be preferable to

examine the conformation of whole molecule including the acyl

chain and phosphoserine (Figure 4), this was not feasible due

1

to the broadness and overlapping of the H NMR signals.

Therefore, we studied the conformation of several model

compounds including saturated 76 (trans), 77 (cis), containing

bulky TBS protecting group on the primary alcohol, and 78

(trans), 79 (cis), composed of bulky protected phosphoserine

unit on the secondary alcohol, in two diﬀerent solvents, CDCl₃

and CD₃OD (Figure 12) (Supporting Information Figure S7).

We successfully detected major conformation of each cyclic

scaﬀolds of model saturated 76 (trans) and 77 (cis) compounds

(Figure 12A) by means of NOESY spectra. That is, in the case

of a saturated trans-conﬁguration (like 76), the large

substituent on the primary position and the hydroxyl group

on the secondary position are located in diequatorial positions

(76-ee) rather than in diaxial positions (76-aa), reasonably

because the diequatorial conformation is the most favorable to

avoid steric hindrance generated by 1,3-diaxial interactions

between the methylene moiety at the primary oxygen and the

diaxial H₃and H₅protons (Figure 12A). In solution phase a

single conformation can be detected. In the case of cis-

conﬁguration (like 77), the NOESY experiments suggested that

Figure 12. Conformational equilibrium of the (A) trans isomer (76)

and (B) cis isomer (77) of 2-hydroxymethyl-3-hydroxytetrahydropyran

models substituted with a TBS group on the primary position; and of

the (C) trans isomer (78) and (D) cis isomer (79) of substituted

phosphorserine on the secondary position.

the primary substituent prefers to exist in the equatorial

position and the hydroxyl group on secondary position likely to

be on axial position (77-ea), rather than the conformation (77-

ae) in which the former in the axial position and the latter in

the equatorial position, due to avoiding 1,3-diaxial interaction

between the methylene moiety of the primary position and

diaxial H₃and H₅protons.

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Figure 13. Conformational equilibrium of the cis isomer of 2-hydroxymethyl-3-hydroxytetrahydropyran models (80), which has substituents on both

the primary and secondary positions.

Figure 14. Distributions of the dihedral angles of the tetrahydropyran ring in calculated ring structures which are obtained by molecular dynamic

simulation, indicating the eﬀect of increased ring planarity arising from replacement of tetrahydropyran with a dihydropyran moiety: (A) torsion

angle 1; (B) torsion angle 2.

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Figure 15. Morphing the glycerol moiety into a benzene ring.

We also studied the conformation of model saturated

compounds 78 (trans) and 79 (cis) which contain a protected

phosphserine moiety on the secondary alcohol, in CDCl₃and

CD₃OD (Figure 12C and D). The NMR analysis indicated that

a single conformation was favored in 78-ee and 79-ea, similar to

that of the former model compounds (76-ee and 77-ea), which

contain a TBS group on the primary position.

Finally, we studied the more close model compound 80 (cis)

via NMR spectroscopy, which has an acetyl group on the

primary alcohol and the protected phosphserine moiety on the

secondary alcohol (Figure 13). In this cis conﬁguration, a single

major conformation can be detected in the NMR spectra; that

is, the acyl subsituent on the primary position is favorable to be

in the equatorial position and the protected phosphserine

group on the secondary position is likely to be in the axial

position. This conformational preference probably resulted

from the steric hindrance between the methylene substituent

on the primary position and the H₃and H₅protons expected in

the alternative conformation (Figure 13). The observed

conformational preference of saturated model compounds 78

and 79 coincided with those found in saturated models 76 and

77, indicating the presence of conformational bias independent

of the O-subsitutents.

Introduction of a 2-hydroxymethyl-3-hydroxytetrahydropyr-

an skeleton (b, Figure 2) in place of the glycerol moiety (a,

Figure 2) indeed restricts the conformation of the glycerol

moiety, leading to restruction of the extension of fatty acid and

phosphoserine moieties. The overall shape of the molecules

may be suitable for GPR34 binding, rather than for P2Y10

binding.

Eﬀect of Unsaturation of the Tetrahydropyran Frame-

work on Receptor Activation. The synthesized dihydroyran-

type LysoPS analogues (c in Figure 2, 21−32 in Figure 7) were

evaluated for agonistic activity toward GPR34 and P2Y10

(Table 1). Interestingly, unsaturated cyclic LysoPS analogues

also have similar activity tendency to the saturated cyclic

LysoPS analogues case, that is, 1-acyl-2-phosphoserine

than the cis-unsaturated analogues (24−26): (21: EC₅₀= 450

nM; 22: EC₅₀= 17 nM; 23: EC₅₀= 330 nM; 24: EC₅₀> 1 μM;

25: EC₅₀= 42 nM; 26: EC₅₀> 1.0 μM for P2Y10) (Table 1).

On the other hand, the 2°-acyl-1°-phosphoserine analogues

(27−32) did not show signiﬁcant P2Y10 activity (27: EC₅₀> 1

μM; 28: EC₅₀> 1 μM; 29: EC₅₀> 1.0 μM; 30: EC₅₀> 1 μM;

31: EC₅₀> 1 μM; 32: EC₅₀> 1 μM for P2Y10). The

implication that acyl chain (B) is a favorable substructure for

P2Y10 activation is consistent with the previous SAR study,¹⁹

in which 1°-acyl-type analogues, trans-isomers 21 ((A)), 22

((B)) and 23 ((C)), and cis-isomers 24 ((A)), 25 ((B)) and 26

((C)), were compared. We hypothesized that these changes in

subtype selectivity can be interpreted in terms of increased ring

planarity of the dihydropyran ring moiety due to the

introduction of a double bond into the tetrahydropyran ring,

based on the distribution of accessible dihedral angles in the

calculated ring structures. That is, the respective disributions of

two dihedral angles (torsion 1 (A) amd torsion 2 (B)) of

unsaturated analogues 22 (trans) and 25 (cis) decreases in the

absolute magnitude as comapred with those of the saturated

counterparts (2 (trans) and 5 (cis), respectively), supporting

the above interpretaion (Figure 14). Consequently, we next

aimed to replace the dihydropyran moiety (c, Figure 2) with a

benzene ring (d, Figure 2) in order to further investigate the

eﬀect of ring planarity on the agonistic activity.

Aromatized Glycerol Mimics. We synthesized analogues

in which the tetrahydropyran framework was replaced with a

benzene ring and examined their agonistic activities (Figure

15).

Three kinds of benzene-type LysoPS analogues, ortho (33),

meta (34), and para (35), can be generated (Figure 8 and

Scheme 4). The agonistic activities of the synthesized aromatic

LysoPS analogues were evaluated, and the results are shown in

Table 1.

Replacement of glycerol (a) with a benzene ring (d)

increased the potency toward P2Y10, as compared with the

saturated (b) and unsaturated tetrahydropyran (c) analogues

(Figure 2). While the para-analogue (35) shows signiﬁcantly

diminished activities at any receptor (35: inactive for GPR34;

EC₅₀> 1 μM for P2Y10), the ortho (33) and meta isomers (34)

showed potent and selective activation of P2Y10 (33: EC₅₀700

nM for GPR34; EC₅₀: 45 nM for P2Y10; 34: inactive for

GPR34; EC₅₀= 6.7 nM for P2Y10). Comparison of saturated

(2) and unsaturated cyclic analogues (22) and the meta isomer

(34) showed that compounds 22 and 34, which include

increased planarity of the ring moiety, have more potent

agonistic activities toward P2Y10 than that of compound 2,

which means that ring planarity is probably involved in the

agonsitic activity for P2Y10 and GPR34 with potency and

receptor selectivity. Among the results of the agonistic activities

of benzene ring analogues, the meta isomer showed the most

potent agonistic activity against P2Y10 with high selectivity.

The results suggested that the positions of the substituents also

has an inﬂuence on the acitivity change. According to this

analogues (21−26 except 26) (21: EC₅₀> 1 μM; 22: EC₅₀

very weak; 23: EC₅₀= 460 nM; 24: EC₅₀> 1 μM; 25: EC₅₀

=

510 nM; 26: EC₅₀= 600 nM for GPR34) generally showed

higher potency toward GPR34 than their regioisomers, the 2-

acyl-1-phosphoserine analogues (27−32 except 32: 27:

inactive; 28: inactive; 29: EC₅₀> 1 μM; 30: EC₅₀560 nM:

31: EC₅₀> 1 μM; 32:EC₅₀= 180 nM for GPR34), with

improved selectivity. In addition, in the 1-acyl-2-phosphoserine

topology the potency of the unsaturated analogues (21-26) for

GPR34 activation was reduced as compared with the saturated

analogues (1−6). The trans isomers (21−23) of 1-acyl-2-

phosphoserine derivatives showed activity comparable to the cis

isomers (24−26) for GPR34 activation.

In particular, the dihydropyran analogues (c in Figure 2, 21−

26 in Figure 7) showed more potent agonistic activities against

P2Y10 than the corresponding saturated analogues (1−6), and

the trans-unsaturated analogues (21−23) were more potent

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consideration, the substituents on the meta position (34) are

the best framework of the benzene ring, which showed more

potent agonistic activity against P2Y10 than that of the ortho

and para isomers.

The present shape morphing the 2-hydroxymethyl-3-

hydroxytetrahydropyran ring system into a benzene ring

through the dihydropyran ring enhanced the P2Y10 activation

potency rather than GPR34 activation. The concentration−

response data of the resultant potent derivatives (22, 25, 34) is

shown in Figure 16, together with LysoPS and some less active

counterparts (33 and 35).

not show signiﬁcant activity toward GPR174. These results are

consistent with the previous ﬁnding that this hydroxyl group is

important for activation of GPR174.

Therefore, these cyclic LysoPS analogues are expected to be

useful tools for selective modulation of GPR34 and P2Y10 in

studies of the receptor functions. Further study of the

conﬁgurations and space-ﬁlling conformations of these

compounds may be useful in identifying recognition motifs

and pharmacophores, and may also be helpful in future

computational modeling and docking studies of GPR34 and

P2Y10.

EXPERIMENTAL SECTIONS

■

Bioassay Based on Ectodomain Shedding of a Membrane-

Bound Proform of Alkaline Phosphatase-Tagged TGFα. TGFα

shedding assay was performed as described previously^11,12,17,18with

minor modiﬁcations. The TGFα shedding assay detects activation of

GPCRs by measuring ectodomain shedding of a membrane-bound

proform of alkaline phosphatase (AP)-tagged TGFα (AP-TGFα) into

conditioned media. To monitor activation of G_i-coupled GPR34, we

additionally transfected a chimeric Gα_q/i1subunit to detect activation

of G_i-coupled GPR34. We examined TGFα shedding response of

synthetic compounds using mouse GPR34, P2Y10, and GPR174

LysoPS receptors.

Figure 16. LysoPS analogues diﬀerentially activate P2Y10. HEK293

cells transiently transfected with P2Y10-encoding expression vector or

an empty vector were treated with compounds, and receptor-speciﬁc

AP-TGFα release responses were determined by subtracting back-

ground responses in the empty vector-transfected cells (actual signals

in the P2Y10-expressing cells and the empty vector-transfected cells

are shown in Supporting Information Figure S3). Data are mean and

SEM (standard error of the mean) for three to ten independent

experiments.

We employed two diﬀerent HEK293 cell lines (HEK293A and

HEK293FT) because the responses induced by LysoPS in receptor-

expressing cells were diﬀerent among the LysoPS receptors.¹⁸That is,

for GPR34 and P2Y10, HEK293A showed better responses than

HEK293FT, and conversely, for GPR174, HEK293FT showed better

responses.¹⁸As for the usage of LPA receptor antagonist (Ki16425,

antagonist for LPA₁, LPA₂, and LPA₃), we have found that addition of

Ki16425 reduced the background response in mock-transfected cells.¹⁸

This is due to a possible contamination of a small amount of LPA or

LPA-like compounds in LysoPS analog preparation or a possible

conversion of LysoPS analogs to LPA-like compound after they are

added to the cells (removal of serine moieties from LysoPS-like

analogs by lysophospholipase D activity results in production of LPA-

like molecules). HEK293 cells endogenously express LPA₁, LPA₂and

LPA₃and presence of Ki16425 in the assay condition could diminish

eﬀects of LPA-like molecules on signals mediated by the endogenous

LPA receptors.

Brieﬂy, transfection was performed in a 100 mm culture dish using

the following combination of a cell line and expression vector

plasmids: for GPR34, HEK293A cells with a mixture of plasmids (AP-

TGFα (2.5 μg), mouse GPR34 (2.5 μg), and chimeric Gα_q/i1subunit

(0.5 μg)) using 20 μL of 1 mg/mL Polyethylenimine Max solution

(Polysciences); for P2Y10, HEK293A cells with a mixture of plasmids

(AP-TGFα (2.5 μg) and mouse P2Y10 (2.5 μg)) using Lipofectamine

2000 reagent (Thermo Fisher Scientiﬁc); for GPR174, HEK293FT

cells with a mixture of plasmids (AP-TGFα (2.5 μg) and mouse

GPR174 (2.5 μg)) using 20 μL of the Polyethylenimine Max solution.

Twenty-four h after transfection, cells were detached, suspended in 5

mM HEPES (pH 7.4)-containing Hanks’ Balanced Salt Solution

(HBSS) and seeded (80 μL per well) in a half-area 96-well plate in

which serial concentrations of compounds (10 μL per well; diluted

with 0.01% (w/v) bovine serum albumin-containing HBSS) and 100

μM Ki16425 (LPA₁and LPA₃inhibitor; 10 μL per well; diluted with

HBSS) were predispensed (total conditioned media volume of 100 μL

per well). After 1.5-h incubation, conditioned media (80 μL per well)

were transferred to a blank, half-area 96-well plate. p-nitrophenyl

phosphate solution was added and absorbance at wavelength of 405

nm (OD₄₀₅) was measured using a microplate reader (SpectraMax 340

PC384, Molecular Devices) before and after 1-h incubation at room

temperature.

Actin Stress Fiber Formation Assay. To further conﬁrm

the potency of the present LysoPS analogues at the cellular

level in vitro, we utilized another in vitro GPCR assay (actin

stress ﬁber formation assay), in which P2Y10-induced Gα_12/13

signaling is detectable as formation of actin stress ﬁbers.¹We

tested P2Y10-selective agonists 22, 33, and 34, the GPR34-

selective agonist 6 and 12 in addition to pan-agonist LysoPS,

GPR34 - P2Y10 dual agonist 25 and nonresponsive compound

35. LysoPS induced formation of actin stress ﬁbers in P2Y10-

expressing cells (Figure 17). Among the tested compounds, 22,

25, 33, and 34 induced formation of actin stress ﬁbers, but 6,

12, and 35 did not. Actin stress ﬁber formation was not

observed in stably mock-infected cells (Supporting Information,

Figure S8). These results further establish the potency of these

analogues and also support the generality of the characteristics

of these LysoPS analogues found in the TGFα shedding assay.

CONCLUSION

■

We have discovered lysophosphatidylserine analogues contain-

ing the conformationally constrained glycerol that show potent

and selective activity toward GPR34 and P2Y10. We

established a synthetic route to 1°-acyl-2°-phosphoserine and

2°-acyl-1°-phosphoserine analogues by introducing a tetrahy-

dropyran moiety, which has a nonequivalent hydroxyl group, to

obtain a set of cyclic LysoPS analogues. 1°-Acyl-2°-

phosphoserine analogues showed greater potency as receptor

activators. In these compounds, the acyl chain (B) was eﬀective

for activity toward P2Y10 and the acyl chain (C) for activity

toward GPR34, in accordance with our previous SAR study.

Futhermore, all the derivatives (1−35) studied in this paper did

To calculate GPCR-mediated AP-TGFα shedding responses of

compounds, raw absorbance data were processed as below (also see

the Supporting Information Figure S1): (1) calculate increase of

OD₄₀₅values before and after 1-h incubation, (2) calculate percentage

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Figure 17. Formation of actin stress ﬁbers in P2Y10-expressing cells induced by P2Y10 agonists. McA-RH7777 cells stably expressing mouse P2Y10

or stably infected with an empty retrovirus (control) were serum-starved and treated with or without 100 nM compounds for 30 min. The cells were

ﬁxed and stained with Alexa 594-labeled phalloidin (ﬁlamentous actin, red) and DAPI (nuclei, blue). Images were obtained with the IN Cell Analyzer

2000 (GE Healthcare) ﬂuorescent imaging system. An inlet in each panel shows an enlarged view of a single cell. Note that the P2Y10-dependent

ﬁber-like pattern of phalloidin staining was visible in an intracellular region in cells stimulated with LysoPS, 22, 25, 33, or 34. No signiﬁcant ﬁber-like

pattern of phalloidin staining was visible in mock cells (Figure S7).

of OD₄₀₅values in conditioned media (AP-TGFα_CM; relative amount

of cleaved AP-TGFα in conditioned media), (3) subtract a vehicle-

treated AP-TGFα_CMvalue form compound-treated AP-TGFα_CM

values (AP-TGFα release; compound-induced response from a

baseline (spontaneous) AP-TGFα shedding response), (4) subtract

AP-TGFα release in mock-transfected cells from that in GPCR-

expressing cells (Receptor-speciﬁc AP-TGFα release). A four-

parameter sigmoid curve was ﬁtted to concentration−response plots

(typically 10 concentration points) using GraphPad Prism 6

(GraphPad, USA) and LogEC₅₀and E_maxvalues were computed for

active compounds with plateau or semiplateau responses.

A detailed description of the compound preparation and

administration for the biological assay is as follows: we dissolved a

synthesized compound (a powder form) in 0.1% (w/v) bovine serum

albumin (BSA; protease- and fatty acid-free grade from SERVA)-

containing Dulbecco’s phosphatase-buﬀered saline (D-PBS) at a

concentration of 1 mM with repeated vortexes and water-bath

sonication. The dissolved compound solution was aliquoted and

stored at −25 °C. On the day of the TGFα shedding assay, we thawed

frozen aliquots of compound solutions and diluted the solution using

0.01% (w/v)-containing Hank’s balanced salt solution supplemented

with 5 mM HEPES (pH 7.4). The serially diluted solution (3.2-fold

dilution starting from 320 μM, which corresponds to a ﬁnal

concentration of 32 μM) was used as 10X concentration of test

compounds. We predispensed 10 μL (per well in a 96-well plate) of

100 μM Ki16425, antagonist for LPA₁, LPA₂and LPA₃, diluted with 5

mM HEPES (pH 7.4)-containing HEPES. We seeded 80 μL of cell

suspension and treated with 10 μL of 10X test compound (also see

below). For an initial experiment, we added test compounds to cells

transfected with the AP-TGFα plasmid and the pCAGGS empty

vector (mock-transfected cells) and measured nonspeciﬁc AP-TGFα

release signal. For each compound, we chose highest concentration

(32 μM, 10 μM, 3.2 μM or 1 μM at a ﬁnal concentration) that did not

show high nonspeciﬁc AP-TGFα release signal in the mock-transfected

cells, and used the highest concentration for the subsequent assay for

measuring LysoPS receptor activity.

In Figures 10 and 16, we did not include data points at 10 μM

concentration for LysoPS(18:1) and 25. We often observed that high

concentrations of compounds damaged cells owing to their detergent-

like eﬀects and caused artiﬁcial signals even in mock-transfected cells.

Thus, at these high concentrations, we could not correctly measure

receptor activation. The membrane-damaging eﬀects depend on

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physical property of each compound. In practice, we initially tested

eﬀects of test compounds on mock-transfected cells and determined

the concentration at which test compounds induced such artifacts. We

then chose the highest concentrations at which we would use for

following experiments using receptor-expressing cells. For example,

18:1 LysoPS and the compound 25 at 10 μM induced artiﬁcial signals

and thus we omitted testing at that concentration in the following

experiments. In the present work, we focused on development of

highly active agonists and, even in the absence of the data point at 10

μM, we could successfully determine saturable concentration−

response curves for them.

O-(Hydroxy(((2R,3S)-2-((oleoyloxy)methyl)tetrahydro-2H-pyran-

3-yl)oxy)phosphoryl)-L-serine (1). Compound 43a (111.2 mg, 0.1433

mmol) was dissolved in triﬂuoroacetic acid at 0 °C and stirred at 0 °C

for 30 min. After 30 min, solvent was evaporated under vacuum and

the residue was puriﬁed by column chromatography CHCl₃: MeOH:

AcOH = 8:1:1) to yield the compound which had one tert-butyl group

in its molecule (103.1 mg). The obtained compound was dissolved in

triﬂuoroacetic acid at 0 °C and stirred at room temperature for 2.5 h.

After 2.5 h, solvent was evaporated under vacuum and the residue was

puriﬁed by column chromatography (CHCl₃: MeOH: AcOH = 8:1:1

to 6:1:3) to yield 1 (75.9 mg, 0.1347 mmol, 94%, white solid).

¹H NMR (CDCl₃, 400 MHz): 5.398−5.306 (1H, m), 5.104−5.042

(1H, m), 4.605 (2H, brs), 4.493 (1H, brs), 4.397−4.396 (1H, m),

4.272 (1H, m), 4.120−4.095 (2H, m), 3.602−3.485 (2H, m), 2.390−

2.287 (3H, m), 2.035−1.966 (2H, m), 1.794−1.573 (7H, m), 1.261

(20H, m), 0.890−0.856 (3H, m). ³¹P NMR (CDCl₃, 161 MHz):

−3.10. HRMS (ESI-TOF: [M-H]⁻): Calcd for C₂₇H₄₉NO₉P⁻:

562.3150, Found 562.3178. Anal. Calcd for C₂₇H₅₀NO₉P

+0.8CF₃CO₂H; C, 52.46; H, 7.77; N, 2.14; Found C, 52.20; H,

7.72; N, 2.16. Mp. 122−130 °C.

Ca²⁺Mobilization Assay. HEK293A cells were seeded in culture

medium (DMEM supplemented 10% fetal bovine serum, 100 U/mL

penicillin and 100 μg/mL streptomycin) in 100 mm culture dishes and

incubated for 24 h. Then, mouse GPR34-expressing pCAGGS plasmid

(5 μg per one 100 mm dish) and Gα_q/i1-expressing pCAGGS plasmid

(2.5 μg) were transfected in HEK293A cells using Lipofectamine 2000

reagent (Thermo Fisher Scientiﬁc; 12.5 μL) according to manufac-

turer’s protocol. For negative-control (mock) cells, empty pCAGGS

plasmid (5 μg) and the Gα_q/i1-expressing pCAGGS plasmid (2.5 μg)

were transfected. After incubation for 24 h, the cells were detached

with 0.05% (w/v) trypsin containing 0.52 mM EDTA. After

centrifugation, cells were suspended at a cell concentration of 5 ×

10⁵cells/mL in serum-free DMEM, and seeded (50 μL per well) in

CELLSTAR Advanced TC half a rea 96-well black plates (Greiner Bio-

One). The plates were placed in an incubator for 24 h. A Ca²⁺

indicator (FLIPR Calsium 5 Assay Kit; Molecular Devices) was added

(50 μL per well) and loaded into cells with 1-h incubation in the

presence of 2.5 mM probenecid according to manufacturer’s protocol.

Then, the cell plate and a compound plate containing 5X test

compounds were positioned in a ﬂuorescence microplate reader

(FlexStation 3; Molecular Devices) and ﬂuorescent signal was

measured with automated pipetting (25 μL of 5X compounds per

well).

Actin Stress Fiber Formation Assay. Actin stress ﬁber formation

assay was performed as described previously (1). McA-RH7777 rat

hepatoma cells stably expressing mouse P2Y10 or mock-infected cells

were seeded in a collagen type I coated 96-well plate at 2.5 × 10⁴cells

per well and incubated for 24 h in culture medium (DMEM

supplemented with 10% (v/v) FBS). The cells were washed once with

PBS and serum-starved for 1 h in serum-free, 0.01% (w/v) BSA

(essentially fatty acid-free grade)-containing DMEM. The cells were

then stimulated with LysoPS (1) or its analogues at a concentration of

100 nM for 30 min. The cells were washed with PBS and ﬁxed with 4%

(w/v) paraformaldehyde-containing PBS for 20 min. After brief

washing with PBS, the cells were permeabilized with 0.1% (v/v)

Triton X-100-containing PBS and stained with Alexa Fluor 594-

conjugated phalloidin (for ﬁlamentous actin) at 1.5 μg/mL and 4′,6-

diamidino-2-phenylindole (DAPI; for nuclei) at 1 μg/mL in 3% (w/v)

BSA-PBS for 1 h. The cells were washed with PBS and ﬂuorescence

images were acquired using a ﬂuorescent microscopy (IN Cell

Analyzer 2000, GE Healthcare). The two channels were pseudocol-

ored (red for phalloidin and blue for DAPI) and shown as a merged

image.

O-(Hydroxy(((2R,3S)-2-(((3-(2-(undecyloxy)phenyl)propanoyl)-

oxy)methyl)tetrahydro-2H-pyran-3-yl)oxy)phosphoryl)-L-serine (2).

Compound 44a was dissolved in triﬂuoroacetic acid at 0 °C. This was

stirred at 0 °C for 10 min. After 10 min, the reaction mixture was

stirred at room temperature for 3 h. After 3 h, solvent was removed

and the residue was puriﬁed by column chromatography (CHCl₃:

MeOH: AcOH = 8:1:1 to 6:1:2) to yield 2 (43.6 mg, 0.0725 mmol,

94%, white soild).

¹H NMR (CDCl₃, 400 MHz): 7.227−7.171 (1H, m), 7.120−7.057

(1H, m), 6.877−6.805 (m, 2H), 4.597 (brs, 2H), 4.450−4.399 (m,

2H), 4.215−4.070 (3H, m), 3.979 (2H, t, J = 6.8 Hz), 3.575−3.454

(2H, m), 2.939−2.903 (2H, m), 2.751−2.733 (2H, m), 2.299−2.290

(1H, m), 1.816−1.650 (5H, m), 1.427−1.271 (20H, m), 0.878 (3H, t,

J = 6.8 Hz). ³¹P NMR (CDCl₃, 161 MHz): −2.90. HRMS (ESI-TOF:

[M-H]⁻): Calcd for C₂₉H₄₇NO₁₀P⁻: 600.2943. Found 600.2934. Anal.

Calcd for C₂₉H₄₈NO₁₀P + CF₃CO₂H; C, 52.02; H, 6.90; N, 1.96.

Found C, 52.06; H, 7.01; N, 1.91. Mp. 107−128 °C.

O-(Hydroxy(((2R,3S)-2-(((3-(2-((3-phenoxybenzyl)oxy)phenyl)-

propanoyl)oxy)methyl)tetrahydro-2H-pyran-3-yl)oxy)phosphoryl)-

L-serine (3). Compound 45a was dissolved in TFA at 0 °Ç and stirred

at 0 °C for 10 min. After 10 min, the reaction mixture was stirred at

room temperature for 2.5 h. After 2.5 h, the reaction mixture was

evaporated and the residue was puriﬁed by column chromatography

(CHCl₃: MeOH: AcOH = 8:1:1 to 6:1:3) to yield 3 (47.3 mg, 0.0751

mmol, 90%, white solid, 71% (2 steps)).

¹H NMR (CDCl₃, 400 MHz): 7.359−7.314 (3H, m), 7.203−6.879

(10H, m), 5.078 (2H, s), 4.264−4.598 (2H, m), 4.465−4.378 (2H,

m), 4.209−4.064 (3H, m), 3.565−3.443 (2H, m), 2.927−2.936 (2H,

m), 2.748−2.729 (2H, m), 2.308 (1H, brs), 1.781−1.694 (3H, m). ³¹P

NMR (CDCl₃, 161 MHz): −2.73. HRMS (ESI-TOF: [M-H]⁻): Calcd

for C₃₁H₃₅NO₁₁P⁻: 628.1953. Found 628.1968. Anal. Calcd for

C₃₁H₃₆NO₁₁P + CF₃CO₂H; C, 50.17; H, 4.58; N, 1.70. Found C,

50.07; H, 4.95; N, 1.60. Mp. 88−98 °C.

O-(Hydroxy(((2R,3R)-2-((oleoyloxy)methyl)tetrahydro-2H-pyran-

3-yl)oxy)phosphoryl)-L-serine (4). Compound 43b (113.6 mg, 0.1464

mmol) was dissolved in triﬂuoroacetic acid (2 mL) at 0 °C and stirred

at room temperature for 2 h. After 2 h, solvent was removed under

vacuum and the residue was puriﬁed by column chromatography

(CHCl₃: MeOH: AcOH = 8:1:1 to 6:1:3) to yield 4 (62.4 mg, 0.1107

mmol, 76%, white solid).

Chemical Synthesis. General Procedures. Melting points were

determined with a Yanaco micro melting point apparatus without

1

correction. H- (400 MHz) and ¹³C- (100 MHz) NMR spectra were

recorded on a Bruker Avance400. Chemical shifts were calibrated with

tetramethylsilane as an internal standard or with the solvent peak, and

are shown in ppm (δ) values, and coupling constants are shown in

hertz (Hz). The following abbreviations are used: s = singlet, d =

doublet, t = triplet, q = quartet, dd = double doublet, dt = double

triplet, dq = double quartet, h = hextet, m = multiplet, and brs = broad

singlet. Electron spray ionization time-of-ﬂight mass spectra (ESI-TOF

MS) were recorded on a Bruker micrOTOF-05 to give high-resolution

mass spectra (HRMS). All reagents were commercially available and

used without further puriﬁcation, unless otherwise noted. Flash

column chromatography was carried out on silica gel (silica gel (40−

63 μm)). The combustion analyses were carried out in the

microanalytical laboratory of this department.

¹H NMR (CDCl₃, 400 MHz): 5.398−5.255 (1H, m), 5.102−5.042

(1H, m), 4.637 (2H, brs), 4.510 (2H, brs), 4.333−4.312 (1H, m),

4.189−4.161 (2H, m), 3.885−3.871 (1H, m), 3.677−3.619 (1H, m),

2.389−2.352 (2H, m), 2.177−2.149 (1H, m), 2.059−1.969 (3H, m),

1.824−1.793 (1H, m), 1.646−1.573 (5H, m), 1.262 (20H, brs),

0.891−0.857 (3H, m). ³¹P NMR (CDCl₃, 161 MHz): −2.87. HRMS

(ESI-TOF: [M-H]⁻): Calcd for C₂₇H₄₉NO₉P⁻: 562.3150. Found

562.3154. Anal. Calcd for C₂₇H₅₀NO₉P + CF₃COOH; C, 51.40; H,

7.59; N, 2.07. Found C, 51.00; H, 7.47; N, 2.02. Mp. 121−146 °C.

N

DOI: 10.1021/acs.jmedchem.5b01925

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

O-(Hydroxy(((2R,3R)-2-(((3-(2-(undecyloxy)phenyl)propanoyl)-

oxy)methyl)tetrahydro-2H-pyran-3-yl)oxy)phosphoryl)-L-serine (5).

Compound 44b (126.7 mg, 0.1557 mmol) was dissolved in

triﬂuoroacetic acid (2 mL) at 0 °C and stirred at room temperature

for 3 h. After 3 h, solvent was removed under vacuum and the residue

was puriﬁed by column chromatography (CHCl₃: MeOH: AcOH =

8:1:1 to 6:1:3) to yield 5 (80.38 mg, 0.1336 mmol, 86%, white solid).

¹H NMR (CDCl₃, 400 MHz): 7.230−7.171 (1H, m), 7.070−7.053

(1H, m), 6.873−6.799 (2H, m), 4.607−4.583 (2H, m), 4.451 (2H,

brs), 4.334−4.285 (1H, m), 4.172−4.131 (2H, m), 3.980 (2H, t, J =

6.8 Hz), 3.767−3.751 (1H, m), 3.625−3.567 (1H, m), 2.933−2.900

(2H, m), 2.748−2.712 (2H, m), 2.163−2.133 (1H, m), 2.010−1.981

(1H, m), 1.827−1.757 (3H, m), 1.537−1.409 (3H, m), 1.365−1.278

(15H, m), 0.885 (3H, t, J = 6.8 Hz). ³¹P NMR (CDCl₃, 161 MHz):

−2.96. HRMS (ESI-TOF: [M-H]⁻): Calcd for C₂₉H₄₇NO₁₀P⁻:

600.2943. Found 600.2925. Anal. Calcd for C₂₉H₄₈NO₁₀P +

0.7CF₃CO₂H; C, 53.38; H, 7.15; N, 2.06. Found C, 53.75; H, 7.30;

N, 2.08. Mp. 160−168 °C.

O-(Hydroxy(((2R,3R)-2-(((3-(2-((3-phenoxybenzyl)oxy)phenyl)-

propanoyl)oxy)methyl)tetrahydro-2H-pyran-3-yl)oxy)phosphoryl)-

L-serine (6). Compound 45b (74.0 mg, 0.0879 mmol) was dissolved in

triﬂuoroacetic acid (2 mL) at 0 °C and stirred at room temperature for

3.5 h. After 3.5 h, solvent was removed under vacuum and the residue

was puriﬁed by column chromatography (CHCl₃: MeOH: AcOH =

8:1:1 to 6:1:3) to yield 6 (35.25 mg, 0.0560 mmol, 64%, white solid).

¹H NMR (CDCl₃, 400 MHz): 7.364−7.325 (3H, m), 7.214−7.085

(5H, m), 7.024−7.005 (2H, m), 6.970−6.945 (1H, m), 6.909−6.889

(2H, m), 5.082 (2H, s), 4.665 (2H, brs), 4.510−4.370 (2H, m), 4.306

(brs, 1H), 4.147−4.122 (m, 2H), 3.758 (brs, 1H), 3.602−3.550 (m,

1H), 2.958 (brs, 2H), 2.744−2.727 (2H, m), 2.199−2.156 (1H, m),

1.994−1.982 (1H, m), 1.733 (1H, brs), 1.534−1.509 (1H, m). ³¹P

NMR (CDCl₃, 161 MHz): 0.97. HRMS (ESI-TOF: [M-H]⁻): Calcd

for C₃₁H₃₅NO₁₁P⁻: 628.1953, Found 628.1934. Anal. Calcd for

C₃₁H₃₆NO₁₁P + 1.2CF₃CO₂H; C, 52.34; H, 4.89; N, 1.83. Found C,

52.28; H, 4.99; N, 1.71. Mp. 107−127 °C.

C₃₂H₃₈NO₁₁P + 3.4CF₃CO₂H; C, 45.18; H, 4.02; N, 1.36. Found C,

45.01; H, 4.27; N, 1.57. Mp. 170−178 °C.

O-((((2R,3R)-3-((3-(2-((3-(4-Chlorophenoxy)benzyl)oxy)phenyl)-

propanoyl)oxy)tetrahydro-2H-pyran-2-yl)methoxy)(hydroxy)-

phosphoryl)-L-serine (15). Compound 66c (37.2 mg, 0.0426 mmol)

was dissolved in TFA (1 mL) at 0 °C and stirred at 0 °C for 10 min

and at room temperature for 1 h. The reaction mixture was evaporated

and the residue was puriﬁed by column chromatography

(CHCl₃:MeOH:H₂O= 65:25:4 (360 mL)) to yield 15 (36.7 mg,

0.0553 mmol, 100%).

¹H NMR (CDCl₃, 400 MHz): 7.361 (t, 1H, J = 8.0 Hz), 7.323−

7.293 (m, 2H), 7.225−7.173 (m, 2H), 7.106−7.089 (m, 1H), 7.052

(m, 1H), 6.917−6.899 (m, 5H), 5.109 (s, 2H), 5.031(m, 1H), 4.558

(m, 2H), 4.468 (m, 1H), 4.144−4.117 (m, 1H), 3.981−3.932 (m, 2H),

3.819 (m, 1H), 3.634−3.582 (m, 1H), 3.060−2.945 (m, 2H), 2.847−

2.698 (m, 2H), 1.835−1.689 (m, 3H), 1.470−1.422 (m, 1H). ³¹P

NMR (CDCl₃, 400 MHz): −2.69. HRMS (ESI-TOF [M-H]⁻): Calcd

for C₃₁H₃₄ClNO₁₁P⁻662.1564, Found 662.1589. Anal. Calcd for

C₃₁H₃₅ClNO₁₁P + 6CF₃CO₂H + 2H₂O; C, 37.31; H, 3.28 ; N, 1.01;

Found C, 37.14; H, 3.42; N, 1.30. Mp. 178−189 °C.

O-(Hydroxy(((2R,3R)-3-((3-(2-((3-(p-tolyloxy)benzyl)oxy)phenyl)-

propanoyl)oxy)tetrahydro-2H-pyran-2-yl)methoxy)phosphoryl)-L-

serine (16). Compound 66d (14.9 mg, 0.0175 mmol) was dissolved in

TFA (1 mL) at 0 °C and stirred at 0 °C for 10 min and at room

temperature for 1.5 h. The reaction mixture was evaporated and the

residue w as puri ﬁ e d by c ol u m n c hr o ma t og ra ph y

(CHCl₃:MeOH:H₂O= 65:25:4(360 mL)) to yield 16 (46.9 mg,

0.0729 mmol, quantitative).

¹H NMR (CDCl₃, 400 MHz): 7.319−7.280 (m, 1H), 7.199−7.030

(m, 6H), 6.908−6.869 (m, 5H), 5.062 (s, 2H), 4.991(m, 1H), 4.521

(m, 2H), 4.447−4.412 (m, 1H), 4.096−4.071 (m, 1H), 3.979−3.900

(m, 2H), 3.779 (m, 1H), 3.592−3.540 (m, 1H), 3.073−2.917 (m, 2H),

2.873−2.723 (m, 2H), 2.329 (s, 3H), 1.802−1.652 (m, 3H), 1.432−

1.406 (m, 1H). ³¹P NMR (CDCl₃, 161 MHz): δ −2.58. HRMS (ESI-

TOF [M-H]⁻): Calcd for C₃₂H₃₇NO₁₁P⁻642.2110. Found 642.2121.

Anal. Calcd for C₃₂H₃₈NO₁₁P + 6CF₃CO₂H; C, 39.80; H, 3.34; N,

1.05. Found C, 39.72; H, 3.66; N, 1.06. Mp. 179−189 °C.

O-(Hydroxy(((2R,3S)-2-((oleoyloxy)methyl)-3,6-dihydro-2H-

pyran-3-yl)oxy)phosphoryl)-L-serine (21). Compound 50a (98.8 mg,

0.1277 mmol) was dissolved in triﬂuoroacetic acid (2 mL) at 0 °C,

stirred at 0 °C for 10 min and stirred at room temperature for 2 h.

After 2 h, the reaction mixture was evaporated to remove solvent and

the residue was puriﬁed by column chromatography (CHCl₃: MeOH:

AcOH = 7:1:1 to 6:1:4) to yield 21 (34.6 mg, 0.0616 mmol, 48%,

white solid).

O-((((2R,3R)-2-(((3-(2-((3-(4-Chlorophenoxy)benzyl)oxy)phenyl)-

propanoyl)oxy)methyl)tetrahydro-2H-pyran-3-yl)oxy) (hydroxy)-

phosphoryl)-L-serine (13). Compound 45c (18.3 mg, 0.0209 mmol)

was dissolved in TFA (1.5 mL) at 0 °C and stirred at 0 °C for 10 min

and at room temperature for 1 h. The reaction mixture was evaporated

and the residue was puriﬁed by column chromatography

(CHCl₃:MeOH:H₂O= 65:25:4 (190 mL)) to yield 13 (17.4 mg,

0.0262 mmol, quantitative, TFA salt).

¹H NMR (CDCl₃): 7.257−7.218 (1H, m), 7.185−7.144 (2H, m),

7.100−7.043 (2H, m), 6.985−6.931 (2H, m), 6.851−6.772 (5H, m),

4.976 (2H, s), 4.581 (2H, m), 4.433 (1H, m), 4.348 (1H, m), 4.186

(1H, m), 4.064−4.040 (2H, m), 3.666 (1H, m), 3.524−3.466 (1H, m),

2.845 (2H, m), 2.640−2.606 (2H, m), 2.034 (1H, m), 1.913−1.889

(1H, m), 1.653 (1H, m), 1.468−1.383 (1H, m). ³¹P NMR (CDCl₃):

−2.81. HRMS (ESI-TOF [M-H]⁻): Calcd for C₃₁H₃₄ClNO₁₁P⁻

662.1563. Found 662.1576. Anal. Calcd for C₃₁H₃₅ClNO₁₁P +

3.5CF₃CO₂H; C, 42.94 H, 3.63; N, 1.32. Found C, 42.74; H, 3.88 ;

N, 1.57. Mp. 171−182 °C.

O-(Hydroxy(((2R,3R)-2-(((3-(2-((3-(p-tolyloxy)benzyl)oxy)phenyl)-

propanoyl)oxy)-methyl)tetrahydro-2H-pyran-3-yl)oxy)phosphoryl)-

L-serine (14). Compound 45d 18.3 mg, 0.0209 mmol) was dissolved

in TFA (1.5 mL) at 0 °C and stirred at 0 °C for 10 min and at room

temperature for 1 h. The reaction mixture was evaporated and the

r es i d u e w a s p ur iﬁ ed by co lumn ch romato grap hy

(CHCl₃:MeOH:H₂O= 65:25:4 (180 mL)) to yield 14 (16.4 mg,

0.0255 mmol, 75%).

¹H NMR (CDCl₃, 400 MHz): 5.900−5.875 (1H, m), 5.803−5.718

(1H, m), 5.295−5.211 (1H, m), 5.003−4.942 (m, 1H), 4.570 (brs,

1H), 4.470−4.302 (m, 3H), 4.183−4.142 (m, 3H), 3.697−3.621 (1H,

m), 2.308−2.271 (2H, m), 1.928−1.878 (2H, m), 1.552−1.496 (4H,

m), 1.174 (19H, brs), 0.806−0.772 (3H, m). ³¹P NMR (CDCl₃): =

−2.72. HRMS (ESI-TOF: [M-H]⁻): Calcd for C₂₇H₄₇NO₉P⁻:

560.2994, Found 560.3027. Anal. Calcd for C₂₇H₄₈NO₉P +

CF₃CO₂H + H₂O; C, 48.84; H, 7.10; N, 1.92; Found C, 48.93; H,

7.28; N, 2.13. Mp. 140−151 °C.

O-(Hydroxy(((2R,3S)-2-(((3-(2-(undecyloxy)phenyl)propanoyl)-

oxy)methyl)-3,6-dihydro-2H-pyran-3-yl)oxy)phosphoryl)-L-serine

(22). Compound 51a (35.5 mg, 0.0437 mmol) was dissolved in

triﬂuoroacetic acid (2 mL) at 0 °C and stirred at room temperature for

1.5 h. After 1.5 h, solvent was removed under vacuum and the residue

was puriﬁed by column chromatography (CHCl₃: MeOH: AcOH =

6:1:2 to 4:1:3) to yield 22 (15.3 mg, 0.0255 mmol, 58%, white solid,

(21%, 2 steps)).

¹H NMR (CDCl₃, 400 MHz): 7.320 (t, 1H, J = 8.0 Hz), 7.218−

7.064 (m, 6H), 6.931−6.898 (m, 5H), 5.077 (s, 2H), 4.752−4.622 (m,

2H), 4.609−4.457 (m, 2H), 4.297−4.291 (m, 1H), 4.165−4.141 (m,

2H), 3.772−3.708 (m, 1H), 3.623−3.598 (m, 1H), 2.964−2.957 (m,

2H), 2.754−2.737 (m, 2H), 2.336 (s, 3H), 2.177−2.152 (m, 1H),

2.076−2.014 (m, 1H), 1.756−1.741 (m, 1H), 1.584−1.527 (m, 1H).

³¹P NMR (CDCl₃, 161 MHz): −2.77. HRMS (ESI-TOF [M-H]⁻):

¹H NMR (CDCl₃, 400 MHz): 7.214−7.176 (1H, m), 7.076−7.059

(1H, m), 6.895−6.829 (2H, m), 6.027−6.003 (1H, m), 5.876−5.809

(1H, m), 4.738−4.424 (5H, m), 4.310−4.254 (3H, m), 4.011−3.977

(2H, m), 3.864−3.861 (1H, m), 2.951−2.866 (2H, m), 2.773−2.756

(2H, m), 1.816−1.746 (2H, m), 1.427−1.267 (16H, m), 0.890−0.857

(3H, m). ³¹P NMR (CDCl₃, 161 MHz): −1.85. HRMS (ESI-TOF [M-

H]⁻): Calcd for C₂₉H₄₅NO₁₀P⁻: 598.2787. Found 598.2779. Anal.

Calcd for C₃₂H₃₇NO₁₁P⁻642.2110. Found 642.2126. Anal. Calcd for

O

DOI: 10.1021/acs.jmedchem.5b01925

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Calcd for C₂₉H₄₆NO₁₀P + 1.5CF₃CO₂H; C, 49.87; H, 6.17; N, 1.82.

Found C, 49.72; H, 6.10; N, 1.68. Mp.141−155 °C.

chromatography (CHCl₃: MeOH: AcOH = 6:1:2 (270 mL) to 6:1:3

(300 mL) to yield 33 (93.3 mg, 0.1572 mmol, 100%, white solid).

¹H NMR (CDCl₃, 400 MHz): 7.309−7.254 (m, 2H), 7.226−7.120

(m, 3H), 7.024−7.005 (m, 1H), 6.892−6.824 (m, 2H), 5.227 (s, 2H),

4.690 (m, 2H), 4.494 (m, 1H), 3.991 (t, 2H, J = 6.8 Hz), 2.972−2.935

(m, 2H), 2.796−2.759 (m, 2H), 1.820−1.749 (m, 2H), 1.456−1.402

(m, 2H), 1.322−1.253 (m, 14H), 0.880 (t, 3H, J = 6.8 Hz). ³¹P NMR

(CDCl₃, 161 MHz): −6.99.

O-(Hydroxy(((2R,3S)-2-(((3-(2-((3-phenoxybenzyl)oxy)phenyl)-

propanoyl)oxy)methyl)-3,6-dihydro-2H-pyran-3-yl)oxy)-

phosphoryl)-L-serine (23). Compound 52a (98.0 mg, 0.1167 mmol)

was dissolved in triﬂuoroacetic acid (2 mL) at 0 °C and stirred at room

temperature for 1 h. After 1 h, solvent was removed under vacuum and

the residue was puriﬁed by column chromatography (CHCl₃: MeOH:

AcOH = 6:1:2 to 4:1:3) to yield 23 (12.9 mg, 0.0206 mmol, 18%,

white solid (11%, 2 steps)).

HRMS (ESI-TOF [M-H]⁻): Calcd for C₃₀H₄₃NO₉P⁻592.2681.

Found 592.2664. Anal. Calcd for C₃₀H₄₄NO₉P + 1.1CF₃CO₂H; C,

53.76; H, 6.27; N, 1.95. Found C, 53.84; H, 6.29; N, 1.99. Mp. 145−

156 °C.

¹H NMR (CDCl₃, 400 MHz): 7.364−7.302 (3H, m), 7.209−

7.7.026 (5H, m), 7.009−6.863 (5H, m), 6.027−5.944 (m, 1H),

5.801−5.775 (m, 1H), 5.063 (s, 2H), 4.666−4.102 (m, 8H), 3.774−

3.757 (1H, m), 2.988−2.928 (2H, m), 2.747−2.666 (2H, m). ³¹P

NMR (CDCl₃, 161 MHz): −2.66. HRMS (ESI-TOF [M-H]⁻): Calcd

for C₃₁H₃₃NO₁₁P⁻626.1797. Found 626.1811. Anal. Calcd for

C₃₁H₃₄NO₁₁P + 2CF₃CO₂H + H₂O; C, 48.12; H, 4.38; N, 1.60.

Found C, 48.15; H, 4.26; N, 1.78. Mp. 134−150 °C.

O-(Hydroxy(3-(((3-(2-(undecyloxy)phenyl)propanoyl)oxy)-

methyl)phenoxy)phosphoryl)-L-serine (34). Compound 57b (23.5

mg, 0.0292 mmol) was dissolved in TFA (1.3 mL) at 0 °C and stirred

at 0 °C for 10 min and at room temperature for 1.5 h. The reaction

mixture was evaporated and the residue was puriﬁed by column

chromatography (CHCl₃: MeOH: H₂O= 65:25:4 (190 mL)) to yield

34 (16.2 mg, 0.0273 mol, 94%).

O-(Hydroxy(((2R,3R)-2-((oleoyloxy)methyl)-3,6-dihydro-2H-

pyran-3-yl)oxy)phosphoryl)-L-serine (24). Compoud 50b (114.8 mg,

0.1483 mmol) was dissolved in triﬂuoroacetic acid (2 mL) at 0 °C,

stirred at 0 °C for 10 min and stirred at room temperature for 2.5 h.

After 2.5 h, the reaction mixture was evaporated to remove solvent and

the residue was puriﬁed by column chromatography (CHCl₃: MeOH:

AcOH = 7:1:1 to 6:1:4) to yield 24 (74.8 mg, 0.1332 mmol, 90%,

white solid).

¹H NMR (CDCl₃, 400 MHz): 7.323−7.284 (m, 1H), 7.201−7.158

(m, 1H), 7.129−7.110 (m, 1H), 7.051−7.026 (m, 2H), 7.004−6.983

(m, 1H), 6.870−6.807 (m, 2H), 5.085 (s, 2H), 4.643 (m, 2H), 4.431

(m, 1H), 3.994−3.961 (m, 2H), 2.948−2.911 (m, 2H), 2.755−2.718

(m, 2H), 1.812−1.742 (m, 2H), 1.467−1.398 (m, 2H), 1.353−1.249

(m, 14H), 0.889−0.854 (m, 3H).

³¹P NMR (CDCl₃, 161 MHz): −7.26. HRMS (ESI-TOF [M-H]⁻):

Calcd for C₃₀H₄₃NO₉P⁻592.2681. Found 592.2680. Anal. Calcd for

C₃₀H₄₄NO₉P + 1.5CF₃CO₂H; C, 51.83; H, 5.96; N, 1.83. Found C,

52.04; H, 6.09; N, 1.95. Mp. 181−190 °C.

¹H NMR (CDCl₃, 400 MHz): 6.151−5.930 (2H, m), 5.396−5.306

(1H, m), 5.102−5.041 (1H, m), 4.644−4.255 (5H, m), 4.297−4.255

(2H, m), 3.987 (1H, brs), 2.434−2.371 (2H, m), 2.061−1.970 (2H,

m), 1.647−1.529 (4H, m), 1.264 (21H, brs), 0.892−0.858 (3H, m).

³¹P NMR (CDCl₃, 161 MHz): −2.66. HRMS (ESI-TOF: [M-H]⁻):

O-(Hydroxy(4-(((3-(2-(undecyloxy)phenyl)propanoyl)oxy)-

methyl)phenoxy)phosphoryl)-L-serine (35). Compound 57c (43.3

mg, 0.0537 mmol) was dissolved in TFA (1.5 mL) at 0 °C and stirred

at 0 °C for 10 min and at room temperature for 2 h. The reaction

mixture was evaporated and the residue was puriﬁed by column

chromatography (CHCl₃: MeOH: H₂O= 65:25:4) to yield 35 (36.6

mg, 0.0607 mmol, quatitative, TFA salt).

Calcd for C₂₇H₄₇NO₉P⁻: 560.2994. Found 560.2991. Anal. Calcd for

C₂₇H₄₈NO₉P + 0.9CF₃CO₂H + H₂O; C, 50.70; H, 7.52; N, 2.05.

Found C, 50.95; H, 7.33; N, 2.01. Mp. 130−140 °C.

O-(Hydroxy(((2R,3R)-2-(((3-(2-(undecyloxy)phenyl)propanoyl)-

oxy)methyl)-3,6-dihydro-2H-pyran-3-yl)oxy)phosphoryl)-L-serine

(25). Compound 51b (91.1 mg, 0.1122 mmol) was dissolved in

triﬂuoroacetic acid (2 mL) at 0 °C and stirred at room temperature for

2.5 h. After 2.5 h, solvent was removed under vacuum and the residue

was puriﬁed by column chromatography (CHCl₃: MeOH: AcOH =

6:1:2 to 5:1:3) to yield 25 (52.1 mg, 0.0869 mmol, 77%, white solid).

¹H NMR (CDCl₃, 400 MHz): 7.201−7.057 (2H, m), 6.866−6.829

(2H, m), 6.126−6.018 (2H, m), 4.603−4.189 (m, 8H), 3.976 (t, 2H, J

= 6.8 Hz), 3.877 (m, 1H), 2.940−2.907 (m, 2H), 2.759−2.723 (2H,

m), 1.824−1.754 (2H, m), 1.455−1.271 (16H, m), 0.897−0.863 (3H,

m). ³¹P NMR (CDCl₃, 161 MHz): −2.64. HRMS (ESI-TOF [M-

H]⁻): Calcd for C₂₉H₄₅NO₁₀P⁻598.2787. Found 598.2785. Anal.

Calcd for C₂₉H₄₆NO₁₀P + 0.8CF₃CO₂H; C, 53.20; H, 6.78; N, 2.03.

Found C, 53.47; H, 6.84; N, 2.12. Mp. 151−163 °C.

O-(Hydroxy(((2R,3R)-2-(((3-(2-((3-phenoxybenzyl)oxy)phenyl)-

propanoyl)oxy)methyl)-3,6-dihydro-2H-pyran-3-yl)oxy)-

phosphoryl)-L-serine (26). Compound 52b (43.1 mg, 0.0513 mmol)

was dissolved in triﬂuoroacetic acid (1 mL) at 0 °C and stirred at room

temperature for 2.5 h. After 2.5 h, solvent was removed under vacuum

and the residue was puriﬁed by column chromatography (CHCl₃:

MeOH: AcOH = 6:1:2 to 5:1:3) to yield 26 (13.7 mg, 0.0128 mmol,

43%, white solid (8%, 2 steps)).

¹H NMR (CDCl₃, 400 MHz): 7.263−7.254 (2H, m), 7.210−7.171

(1H, m), 7.044−7.026 (3H, m), 6.886−6.831 (2H, m), 5.110 (2H, s),

4.721 (2H, m), 4.531 (1H, m), 3.991 (2H, t, J = 6.4 Hz), 2.955 (2H, t,

J = 7.6 Hz), 2.762 (2H, t, J = 7.6 Hz), 1.822−1.752 (2H, m), 1.459−

1.405 (2H, m), 1.332−1.267 (14H, m), 0.871 (3H, t, J = 6.8 Hz). ³¹P

NMR (CDCl₃, 400 MHz): −6.77. HRMS (ESI-TOF [M-H]⁻): Calcd

for C₃₀H₄₃NO₉P⁻: 592.2681. Found 592.2680. Anal. Calcd for

C₃₀H₄₄NO₉P + 2CF₃CO₂H + H₂O; C, 48.63; H, 5.76; N, 1.67.

Found C, 48.55; H, 5.86; N, 1.88. Mp. 184−193 °C.

((2R,3S)-3-Acetoxy-3,6-dihydro-2H-pyran-2-yl)methyl acetate

(36a). Tri-O-acetyl-^D-glucal (1.0003 g, 3.6731 mmol) and triethylsi-

lane (512.53 mg, 4.41 mmol) were dissolved in CH₂Cl₂(5 mL) at

room temperature under Ar. The solution was cooled to 0 °C. BF₃·

OEt₂(521.32 mg, 3.67 mmol) was added dropwise to the solution

above at 0 °C under Ar atmosphere. The reaction mixture was stirred

0 °C under Ar for 15 min. The reaction mixture was quenched with

10% aqueous NaHCO₃solution (0.5 mL) and diluted with ether (5.5

mL). The whole was washed with H₂O (5 mLx2), brine (5 mL), and

then dried over MgSO₄. Combined organic layer was evaporated to

yield 36a (784.6 mg, 3.66 mmol, 100%, colorless oil). ¹H NMR

(CDCl₃, 400 MHz): 5.866−5.834 (1H, m), 5.666 (1H, ddd, J = 10.4

Hz, 4.4 Hz, 2.4 Hz), 5.177−5.133 (1H, m), 4.126−4.054 (4H, m),

3.628 (1H, sp, J = 8.4 Hz, 2.8 Hz), 1.997 (3H, s), 1.981 (3H, s). ¹³C

NMR (CDCl₃, 100 MHz): 170.68, 170.16, 129.42, 124.18, 73.76,

65.19, 65.00, 63.19, 20.91, 20.70. HRMS (ESI-TOF [M + Na]⁺):

¹H NMR (CDCl₃, 400 MHz): 7.361−7.311 (3H, m), 7.209−7.078

(5H, m), 7.015−6.871 (5H, m), 6.114−6.090 (1H, m), 5.980 (1H, m),

5.078 (2H, s), 4.571−4.502 (3H, m), 4.466−4.434 (1H, m), 4.386−

4.337 (2H, m), 4.260−4.165 (2H, m), 3.858−3.842 (1H, m), 3.016−

2.901 (2H, m), 2.811−2.722 (2H, m). ³¹P NMR (CDCl₃, 161 MHz):

−3.09. HRMS (ESI-TOF [M-H]⁻): Calcd for C₃₁H₃₃NO₁₁P⁻:

626.1797. Found 626.1801. Anal. Calcd for C₃₁H₃₄NO₁₁P +

2CF₃CO₂H; C, 49.13; H, 4.24; N, 1.64. Found C, 48.78; H, 4.24;

N, 1.61. Mp. 175−188 °C.

O-(Hydroxy(2-(((3-(2-(undecyloxy)phenyl)propanoyl)oxy)-

methyl)phenoxy)phosphoryl)-L-serine (33). Compound 57a (127.0

mg, 0.1576 mmol) was dissolved in TFA (2 mL) at 0 °C and stirred at

0 °C for 10 min and at room temperature for 1.5 h. The reaction

mixture was evaporated and the residue was puriﬁed by column

+

Calcd for C₁₀H₁₄NaO₅: 237.0733. Found: 237.0731. Anal. Calcd for

C₁₀H₁₄O₅+ 0.2H₂O: C, 55.14; H, 6.66; N, 0.00. Found: C, 55.17; H,

6.45; N, 0.00.

((2R,3R)-3-Acetoxy-3,6-dihydro-2H-pyran-2-yl)methyl acetate

(36b). Tri-O-acetyl-O-galactal (5.2392 mg, 19.2441 mmol) was

dissolved in CH₂Cl₂(100 mL). Triethylsilane (2.6852 g, 23.0929

mmol) was added to the solution above at 0 °C. BF₃·OEt₂(3.2776 g,

23.0929 mmol) was added dropwise to the solution above at 0 °C.

The reaction mixture was stirred at room temperature under Ar for 15

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min. Saturated aqueous NaHCO₃solution (50 mL) was added to the

reaction mixture and the whole was extracted with CH₂Cl₂(20 mLx3).

Combined organic layer was washed with water, brine, dried over

MgSO₄and evaporated. The residue was puriﬁed by column

chromatography (ether: petroleum ether =1:1) to yield 36b (3.9502

mg, 18.3499 mmol, 96%, colorless oil).

¹³C NMR (CDCl₃, 100 MHz): 79.11, 68.57, 66.34, 64.08, 30.36, 20.04.

+

HRMS (ESI-TOF [M + Na]⁺): Calcd for C₆H₁₂NaO₃155.0679.

Found 155.0664.

(2R,3S)-2-(((tert-Butyldimethylsilyl)oxy)methyl)tetrahydro-2H-

pyran-3-ol (39a). Compound 38a (24.0 mg, 0.1816 mmol) and

imidazole (27.2 mg, 0.3995 mmol) was dissolved in DMF (0.25 mL)

and cooled to 0 °C. t-Butyldimethylchloro silane (32.8 mg, 0.2179

mmol) was added to the solution above at 0 °C and the reaction

mixture was stirred at room temperature under Ar for 13 h. After 13 h,

H₂O (1.5 mL) and diethyl ether (1.5 mL) was added to the reaction

mixture and organic layer was separated. This organic layer was

washed with H₂O (1.5 mLx2), brine (2 mL) and dried over MgSO₄,

then evaporated. The residue was puriﬁed by column chromatography

to yield 39a (35.5 mg, 0.1441 mmol, 79%, colorless oil).

¹H NMR (CDCl₃, 400 MHz): 6.092−6.052 (1H, m), 5.996−5.947

(1H, m), 5.084−5.059 (1H, tt, J = 0.4 Hz, 2.4 Hz), 4.304 (1H, dddd, J

= 3.6 Hz, 2.0 Hz), 4.224−4.142 (3H, m), 3.850 (1H, sp, J = 2.4 Hz),

2.054 (3H, s), 2.047 (3H, s). ¹³C NMR (CDCl₃, 100 MHz): 170.73,

170.47, 132.33, 122.09, 73.72, 65.70, 64.26, 63.30, 20.89, 20.80. HRMS

+

(ESI-TOF [M + Na]⁺): Calcd for C₁₀H₁₄NaO₅: 237.0733. Found

237.0732. Anal. Calcd for C₁₀H₁₄O₅; C, 56.07; H, 6.59; N, 0.00.

Found: C, 55.90; H, 6.40; N, 0.00.

¹H NMR (CDCl₃, 400 MHz): 3.922−3.849 (2H, m), 3.692 (1H,

dd, J = 10.0, 8.0 Hz)), 3.571 (2H, dddd, J = 10.8 Hz, 8.8 Hz, 4.8 Hz,

2.0 Hz), 3.368−3.303 (1H, m), 3.193−3.139 (1H, m), 2.131−2.075

(1H, m), 1.684−1.616 (2H, m), 1.474−1.371 (1H, m), 0.902 (9H, s),

0.103 (1H, s), 0.097 (3H, s). ¹³C NMR (CDCl₃, 100 MHz): 79.33,

71.14, 67.61, 66.74, 31.59, 25.82, 24.91, 18.16, −5.57, −5.65. HRMS

(ESI-TOF [M + Na]⁺): Calcd For C₁₂H₂₆NaO₃Si⁺: 269.1543. Found

269.1554. Anal. Calcd for C₁₂H₂₆O₃Si + 0.15CH₂Cl₂; C, 56.31; H,

10.23; N, 0.00. Found: C, 56.50 H, 10.04; N, 0.00.

(2R,3R)-2-(((tert-Butyldimethylsilyl)oxy)methyl)tetrahydro-2H-

pyran-3-ol (39b). Compound 38b (32.5 mg, 0.2459 mmol) was

dissolved in CH₂Cl₂(0.7 mL) and imidazole (36.8 mg, 0.5410 mmol)

was added to the solution. This was cooled to 0 °C and tert-

butyldimethylsilyl chloride (44.5 mg, 0.2951 mmol) was added. The

reaction mixture was stirred at room temperature under Ar for 13 h.

After, 13 h, H₂O (5 mL) was added to the reaction mixture and the

whole was extracted CH₂Cl₂(5 mLx3). Combined organic layer was

washed with brine, dried over MgSO₄and evaporated. The residue was

puriﬁed by column chromatography (n-hexane: ethyl acetate =4:1) to

yield 39b (47.0 mg, 0.1907 mmol, 78%, colorless oil). ¹H NMR

(CDCl₃, 400 MHz): 4.028−3.979 (1H, m), 3.910−3.894 (1H, m),

3.822−3.741 (2H, m), 3.504−3.438 (1H, m), 3.336−3.308 (1H, m),

2.774 (1H, brs), 2.056−1.915 (2H, m), 1.664−1.576 (1H, m), 1.395−

1.330 (1H, m), 0.894 (9H, s), 0.082 (3H, s), 0.075 (3H, s). ¹³C NMR

(CDCl₃, 100 MHz): 79.04, 68.66, 65.71, 64.52, 30.32, 25.89, 20.19,

18.30, −5.43, −5.49. HRMS (ESI-TOF [M + Na]⁺): Calcd for

C₁₂H₂₆NaO₃Si⁺: 269.1543. Found 269.1554. Anal. Calcd for

C₁₂H₂₆O₃Si; C, 58.49; H, 10.63; N, 0.00. Found C, 58.19; H, 10.39;

N, 0.00.

(2R,3S)-2-(Acetoxymethyl)tetrahydro-2H-pyran-3-yl acetate

(37a). Compound 36a (2.1379 g, 9.9799 mmol) was dissolved in

MeOH (24 mL) and 10% Pd/C (220 mg) in MeOH (6 mL) was

added to the solution above. The reaction mixture was placed under

H₂atmosphere and stirred at room temperature for 3 h. After 3 h, the

reaction mixture was ﬁltered on Celite and the ﬁltrate was evaporated

under vacuum to yield 37a (2.0224 mg, 9.3530 mmol, 94%, colorless

oil).

¹H NMR (CDCl₃, 400 MHz): 4.676−4.613 (1H, m), 4.186−4.093

(1H, m), 3.970−3.926 (1H, m), 3.467−3.424 (1H, m), 3.403−3.338

(1H, dt, J = 2.8 Hz, 11.6 Hz), 2.216−2.177 (1H, m), 2.054 (3H, s),

2.007 (3H, s), 1.760−1.668 (2H, m), 1.489−1.419 (1H, m). ¹³C NMR

(CDCl₃, 100 MHz):170.90, 169.91, 77.53, 68.06, 67.90, 63.05, 29.20,

24.83, 21.03, 20.82. HRMS (ESI-TOF [M + Na]⁺): Calcd for

+

C₁₀H₁₆NaO₅: 239.0890. Found: 239.0919. Anal. Calcd for C₁₀H₁₆O₅

+ 0.1CH₂Cl₂: C, 53.98; H, 7.27; N, 0.00. Found: C, 54.14 H, 7.24; N,

0.00.

(2R,3R)-2-(Acetoxymethyl)tetrahydro-2H-pyran-3-yl acetate

(37b). Compound 36b (555.6 mg, 2.5936 mol) was dissolved in

MeOH (7 mL) and 10% Pd/C (56 mg) in MeOH (3 mL) was added

to the solution above. The reaction mixture was placed under H₂

atmosphere and stirred at room temperature for 3 h. After 3 h, the

reaction mixture was ﬁltered on Celite and the ﬁltrate was evaporated

to yield 37b (539.6 mg, 2.4955 mmol, 96%, colorless oil).

¹H NMR (CDCl₃, 400 MHz): 4.906 (1H, brs), 4.121−4.019 (3H,

m), 3.699−3.664 (1H, m), 3.502 (1H, dt, J = 12.0 Hz, 2.4 Hz), 2.090

(3H, s), 2.048−2.000 (4H, m), 1.956−1.836 (1H, m), 1.734−1.646

(1H, m), 1.456−1.400 (1H, m). ¹³C NMR (CDCl₃, 100 MHz):

170.77, 170.48, 75.76, 68.00, 67.03, 63.90, 27.51, 21.08, 20.84, 20.53.

+

HRMS (ESI-TOF [M + Na]⁺): Calcd for C₁₀H₁₆NaO₅: 239.0890.

Phosphoserine unit. 40 was prepared as described previously,

Found 239.0880.

starting from L-serine.¹⁹

(2R,3S)-2-(Hydroxymethyl)tetrahydro-2H-pyran-3-ol (38a). Com-

pound 37a (1.9824 g, 9.1680 mmol) was dissolved in MeOH (22 mL)

and sodium methoxide (247.6 mg, 4.5840 mmol) was added to the

solution at room temperature. The reaction mixture was stirred at

room temperature under Ar for 1.5 h. After 1.5 h, the solvent was

removed and the residue was dissolved in CHCl₃. This solution was

ﬁltered on Celite and the ﬁltrate was evaporated to yield 38a (1.2031

mg, 9.1034 mmol, 99%, colorless oil).

tert-Butyl O-(tert-butoxy(diisopropylamino)phosphanyl)-N-(tert-

butoxycarbonyl)-L-serinate (40). Bis(diisopropylamino)tert-butyl-

phosphine (1.0 g, 3.83 mmol) was dissolved in CH₂Cl₂(15 mL)

and toluene (1.5 mL). tert-Butyl (tert-butoxycarbonyl)-L-serinate

(1.301 g, 4.979 mmol) was added to the solution and the solvent

was evaporated to remove containing water. The residue was dissolved

in dry CH₂Cl₂(15 mL) under Ar atmosphere and 1H-tetrazole in

THF (15 mL) was added to the solution at room temperature. The

reaction mixture was stirred for 3.5 h. After 3.5 h, the reaction was

quenched with saturated aqueous NaHCO₃(20 mL) and the whole

was extracted with CH₂Cl₂(20 mL x 3). Combined organic layer was

washed with brine, dried over MgSO₄and evaporated. The residue was

puriﬁed by column chromatography (n-hexane: ethyl acetate: Et₃N =

35:4:1) to yield 40 (1.56 g, 3.36 mmol, 68%, yellow oil).

¹H NMR (CDCl₃, 400 MHz): 3.931−3.901 (1H, m), 3.853−3.751

(2H, m), 3.584−3.522(1H, m), 3.397−3.332 (1H, m), 3.150−3.105

(1H, m), 2.598 (2H, brs), 2.134−2.083 (1H, m), 1.710−1.643 (2H,

m), 1.483−1.381 (1H, m). ¹³C NMR (CDCl₃, 100 MHz): 81.73,

67.65, 67.50, 63.35, 32.51, 25.40. HRMS (ESI-TOF [M + Na]⁺):

+

Calcd for C₆H₁₂NaO₃: 155.0679. Found: 155.0677.

(2R,3R)-2-(Hydroxymethyl)tetrahydro-2H-pyran-3-ol (38b). Com-

pound 37b (508.9 mg, 2.3535 mmol) was dissolved in MeOH (7.5

mL) and sodium methoxide (63.6 mg, 1.1768 mmol) was added to the

solution at room temperature. The reaction mixture was stirred at

room temperature under Ar for 1.5 h. After 1.5 h, solvent was removed

and the residue was dissolved in CHCl₃. This solution was ﬁltered on

Celite and the ﬁltrate was evaporated to yield crude 38b (397.6 mg,

3.0085 mmol, quantitative, yellow oil).

¹H NMR (CDCl₃, 400 MHz): 5.431 (1/2H, d, J = 6.3 Hz), 5.260

(1/2H, d, J = 6.3 Hz), 3.844−3.795 (1H, m), 3.676−3.596 (1H, m),

3.539−3.446 (2H, m), 4.135−4.071 (1H, m), 3.844−3.795 (1H, m),

3.676−596, (1H, m), 3.539−3.446 (2H, m), 1.376−1.368 (9H, m),

1.347−1.340 (9H, m), 1.273−1.237 (9H, m), 1.089−1.052 (12H, m).

¹³C NMR (CD₂Cl₂, 100 MHz): 168.74, 168.72, 80.56, 80.39, 78.20,

78.14, 74.03, 73.92, 62.85, 62.71, 62.46, 62.32, 54.48, 54.43, 54.35,

42.32, 42.27, 42.20, 42.14, 29.87, 27.33, 27.30, 26.98, 23.70, 23.63,

23.58, 23.52, 23.24, 23.16. ³¹P NMR (CD₂Cl₂, 161 MHz): 138.92,

138.44. HRMS (ESI-TOF [M + Na]⁺): Calcd for C₂₂H₄₅N₂NaO₆P⁺:

¹H NMR (CDCl₃, 400 MHz): 4.028−4.008 (1H, m), 3.850−3.762

(3H, m), 3.521−3.465 (1H, m), 3.367 (1H, s), 3.113 (1H, brs),

2.003−1.902 (2H, m), 1.665−1.606 (1H, m), 1.398−1.367 (1H, m).

Q

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Journal of Medicinal Chemistry

Article

487.2907. Found: 487.2906. Anal. Calcd for C₂₂H₄₅N₂O₆P: C, 56.88;

H, 9.76; N, 6.03. Found: C, 55.94; H, 9.72; N, 5.86.

evaporated and the residue was puriﬁed by column chromatography

(ethyl acetate) to yield 42a (121.3 mg, 0.2371 mmol, 73%, white sticky

solid).

tert-Butyl O-(tert-butoxy(((2R,3S)-2-(((tert-butyldimethylsilyl)-

oxy)methyl)tetrahydro-2H-pyran-3-yl)oxy)phosphoryl)-N-(tert-bu-

toxycarbonyl)-L-serinate (41a). 40 (338.0 mg, 0.7486 mmol) was

dissolved in CH₂Cl₂(5 mL) and toluene (0.5 mL) and coevaporated

to remove containing water. 39a (276.7 mg, 1.123 mmol) was added

to the solution above and dissolved in CH₂Cl₂(5 mL) and toluene

(0.5 mL) then, coevaporated. The residue was dissolved in dry CH₂Cl₂

(5 mL) and 1H-tetrazole (209.7 mg, 2.994 mmol) in THF (5 mL) was

added to the solution at room temperature under Ar. The reaction

mixture was stirred at room temperature under Ar for 13 h. TBHP was

added to the reaction mixture and stirred at room temperature under

Ar for 1.5 h. After 1.5 h, the reaction mixture was quenched with H₂O

(10 mL) and extracted with CH₂Cl₂(5 mLx3). The organic layer was

washed with brine, dried over MgSO₄and evaporated. The residue was

puriﬁed by column chromatography (n-hexane: ethyl acetate =4:1 to

2:1) to yield 41a (397.1 mg, 0.6345 mmol, 85%, colorless oil (57%, 2

steps)).

¹H NMR (CDCl₃, 400 MHz): 5.524−5.505 (1H, m), 4.331−4.127

(4H, m), 3.927−3.888 (1H, m), 3.794−3.687 (2H, m), 3.322 (1H, dt,

J = 3.6 Hz, 11.2 Hz), 3.172−3.134 (1H, m), 2.876 (1H, brs), 2.277−

2.227 (1H, m), 1.734−1.542 (3H, m), 1.456−1.438 (18H, m), 1.408

(9H, s). ¹³C NMR (CDCl₃, 100 MHz): 168.26, 155.19, 84.46, 84.39,

82.78, 82.72, 80.79, 80.72, 79.97, 77.39, 77.07, 76.76, 71.84, 71.79,

67.68, 67.65, 67.56, 67.51, 61.77, 61.64, 54.41, 54.32, 30,78, 30.75,

30.72, 29.79, 29.77, 29.76, 29.73, 28.29, 27.93, 27.92, 25,25. ³¹P NMR

(CDCl₃, 161 MHz): −5.18, −5.33. HRMS (ESI-TOF [M + Na]⁺):

Calcd for C₂₂H₄₂NNaO₁₀P⁺: 534.2439. Found 534.2409. Anal. Calcd

for C₂₂H₄₂NO₁₀P + 0.2CH₂Cl₂; C, 50.47; H, 8.03; N, 2.65. Found C,

50.40; H, 8.17; N, 2.51.

tert-Butyl O-(tert-butoxy(((2R,3R)-2-(hydroxymethyl)tetrahydro-

2H-pyran-3-yl)oxy)-phosphoryl)-N-(tert-butoxycarbonyl)-L-serinate

(42b). Compound 41b (428.2 mg, 0.6842 mmol) was dissolved in

MeOH (17 mL) and cooled to 0 °C. Amberlyst15 (2.0021 g) was

added to the solution above, and stirred at 0 °C under Ar for 10 min.

After 10 min, the reaction mixture was stirred at room temperature

under Ar for 16 h. After 16 h, reaction mixture was ﬁltered on Celite

and the ﬁltrate was evaporated. The residue was puriﬁed by column

chromatography (ethyl acetate) to yield 42b (275.2 mg, 0.5380 mmol,

79%, colorless oil).

¹H NMR (CDCl₃, 400 MHz): 5.503−5.423 (1H, m), 4.337−4.216

(2H, m), 4.193−4.150 (1H, m), 4.106−4.028 (2H, m), 3.913−3.860

(1H, m), 3.696−3.641 (1H, m), 3.342−3.279 (1H, m), 3.239−3.197

(1H, m), 2.351−2.323 (1H, m), 1.656−1.562 (3H, m), 1.472−1.455

(18H, m), 1.427 (9H, s), 0.876 (9H, s), 0.057−0.045 (6H, m). ¹³C

NMR (CDCl₃, 100 MHz): 168.31, 155.20, 83.59, 83.52, 82.60, 82.55,

81.40, 81.30, 79.87, 72.42, 72.36, 67.24, 67.22, 63.40, 54.42, 54.33,

30.90, 30.72, 29.84, 29.79, 28.432, 27.96, 26.00, 25.95, 25.05, 25.01,

18.48, 18.45, −5.10, −5.13. ³¹P NMR (CDCl₃, 161 MHz): −6.379.

¹H NMR (CDCl₃, 400 MHz): 5.528−5.509 (0.4H, m), 5.388−

5.368 (0.6H, m), 4.455−4.411 (1H, m), 4.285−4.239 (2H, m),

4.199−4.103 (m, 1H), 3.898−3.863 (1H, m), 3.699 (1H, brs), 3.553−

3.470 (2H, m), 3.414−3.349 (2H, m), 2.094−1.981 (1H, m), 1.925−

1.771 (1H, m), 1.649−1.540 (1H, m), 1.430−1.382 (18H, m), 1.351−

1.307 (10H, m). ¹³C NMR (CDCl₃, 100 MHz): 168.36, 168.21,

155.23, 155.08, 84.64, 84.56, 83.84, 83.76, 82.72, 79.90, 79.82, 78.12,

78.08, 70.79, 70.74, 70.45, 70.39, 67.79, 67.76, 67.46, 67.40, 60.71,

60.50, 54.32, 54.24, 29.75, 29.71, 29.70, 29.65, 28.67, 28.64, 28.55,

28.53, 28.21, 27.84, 20.29, 20.27, 14.10. ³¹P NMR (CDCl₃, 161 MHz):

−3.49, −4.01. HRMS (ESI-TOF [M + Na]⁺): Calcd for

C₂₂H₄₂NNaO₁₀P⁺: 534.2439. Found 534.2414. Anal. Calcd for

C₂₂H₄₂NO₁₀P; C, 51.65; H, 8.28; N, 2.74. Found C, 51.58; H, 7.98;

N, 2.57.

((2R,3S)-3-((tert-Butoxy((S)-3-(tert-butoxy)-2-((tert-

butoxycarbonyl)amino)-3-oxopropoxy)phosphoryl)oxy)tetrahydro-

2H-pyran-2-yl)methyl oleate (43a). Compound 42a (122.6 mg,

0.2397 mmol) was dissolved in CH₂Cl₂(3 mL) and N, N-

dimethylaminopyridine (87.8 mg, 0.7190 mmol) was added to the

solution above. Oleoyl chloride (108.2 mg, 0.3595 mmol) was added

to the mixture at rt and stirred at rt under Ar atmosphere for 2.5 h.

The reaction mixture was quenched by H₂O (7 mL) and the whole

was extracted with CH₂Cl₂(5 mL × 2). Combined organic layer was

washed with brine, dried over MgSO₄and evaporated. The residue was

puriﬁed by column chromatography (n-hexane: ethyl acetate =2:1) to

yield 43a (120.9 mg, 0.1558 mmol, 65%, translucent oil).

−6.415. HRMS (ESI-TOF [M

+

Na]⁺): Calcd for

C₂₈H₅₆NNaO₁₀PSi⁺: 648.3303. Found 648.3312. Anal. Calcd for

C₂₈H₅₆NO₁₀PSi; C, 53.74; H, 9.02; N, 2.24. Found C, 53.71; H, 9.01;

N, 2.32.

tert-Butyl O-(tert-butoxy(((2R,3R)-2-(((tert-butyldimethylsilyl)-

oxy)methyl)tetrahydro-2H-pyran-3-yl)oxy)phosphoryl)-N-(tert-bu-

toxycarbonyl)-L-serinate (41b). 40 (439.2 mg, 0.9726 mmol) and

compound 39b (359.5 mg, 1.4589 mmol) were dissolved in CH₂Cl₂

(3 mL) and toluene (0.5 mL) and evaporated to remove containing

water. The residue was dissolved in dry CH₂Cl₂(6.5 mL) and 1H-

tetrazole (272.5 mg, 3.8904 mmol) in THF (6.5 mL) was added to the

solution. This reaction mixture was stirred at room temperature under

Ar for 19 h. After 17 h, tert-butylhydroperoxide (0.3890 mg, 1.9452

mmol) was added to the reaction mixture and stirred at room

temperature under Ar for 1.5 h. After 1.5 h, the reaction mixture was

quenched by water (15 mL) and the whole was extracted with CH₂Cl₂

(10 mLX3). Combined organic layer was washed with brine, dried

over MgSO₄and evaporated. The residue was puriﬁed by column

chromatography (n-hexane: ethyl acetate =4:1 to 2:1) to yield 41b

(456.6 mg, 0.7296 mmol, 50%, colorless oil, (75% (2 steps)).

¹H NMR (CDCl₃, 400 MHz): 5.416−5.364 (1H, m), 4.380−4.472

(1H, m), 4.277−4.223 (2H, m), 4.141−4.077 (1H, m), 3.903−3.864

(1H, m), 3.608−3.514 (2H, m), 3.386−3.327 (1H, m), 3.270−3.255

(1H, m), 2.224−2.120 (1H, m), 1.886−1.802 (1H, m), 1.581−1.513

(1H, m), 1.386−1.329 (28H, m), 0.773−0.768 (9H, m), −0.047 (3H,

s), −0.061 (3H, s). ¹³C NMR (CDCl₃, 100 MHz): 168.29, 168.27,

155.11, 83.36, 83.27, 83.20, 82.37, 79.63, 79.61, 79.52, 79.45, 71.66,

71.59, 71.54, 67.83, 67.77, 67.18, 67.12, 67.04, 63.10, 63.73, 54.36,

54.28, 29.76, 29.73, 29.71, 29.69, 28.71, 28.54, 28.21, 27.85, 27.02,

25.82, 25.75, 20.22, 18.20, 18.18, 14.08, −5.36, −5.40. ³¹P NMR

(CDCl₃, 161 MHz): −5.43, −5.98. HRMS (ESI-TOF [M + Na]⁺):

Calcd for C₂₈H₅₆NNaO₁₀PSi⁺: 648.3303. Found 648.3307. Anal. Calcd

for C₂₈H₅₆NO₁₀PSi; C, 53.74; H, 9.02; N, 2.24. Found C, 53.61; H,

9.08; N, 2.20.

¹H NMR (CDCl₃, 400 MHz): 5.670−5.650 (0.3H, m), 5.486−

5.466 (0.7H, m), 5.356−5.273 (2H, m), 4.423 (1H, dd, J = 12.0 Hz,

2.0 Hz), 4.358−4.283 (2H, m), 4.246−4.089 (2H, m), 4.061 (1H, dd,

J = 12.0 Hz, 6.0 Hz), 3.935−3.902 (1H, m), 3.409−3.304 (2H, m),

2.373−2.312 (3H, m), 2.031−1.957 (4H, m), 1.727−1.561 (5H, m),

1.477−1.427 (27H, m), 1.294−1.242 (20H, m), 0.875−0.837 (3H,

m). ¹³C NMR (CDCl₃, 100 MHz): 173.62, 173.60, 168.28, 155.19,

130.18, 130.03, 129.94, 129.74, 128.00, 127.89, 84.03, 83.96, 82.65,

82.59, 79.89, 79.79, 78.44, 78.32, 78.23, 72.67, 72.61, 72.16, 72.10,

67.62, 67.55, 67.44, 67.38, 63.56, 54.39, 54.31, 34.08, 31.90, 30.81,

29.82, 29.79, 29.78, 29.75, 29.70, 29.69, 29.66, 29.63, 29.59, 29.49,

29.33, 29.32, 29.30, 29.27, 29.18, 29.13, 29.12, 29.00, 28.96, 28.32,

27.95, 27.94, 27.19, 27.17, 24.96, 24.94, 24.84, 22.66, 22.55, 14.09,

14.05. ³¹P NMR (CDCl₃, 161 MHz): −6.27, −6.32. HRMS (ESI-TOF

[M + Na]⁺): Calcd for C₄₀H₇₄NNaO₁₁P⁺: 798.4892. Found 798.4891.

Anal. Calcd for C₄₀H₇₄NO₁₁P + 0.2CH₂Cl₂; C, 60.92; H, 9.40; N, 1.77.

Found C, 61.14; H, 9.12; N, 1.82.

tert-Butyl O-(tert-butoxy(((2R,3S)-2-(hydroxymethyl)tetrahydro-

2H-pyran-3-yl)oxy)-phosphoryl)-N-(tert-butoxycarbonyl)-L-serinate

(42a). Compound 41a (203.1 mg, 0.3245 mmol) was dissolved in

MeOH (8 mL) and cooled to 0 °C Amberlyst15 (1.0155 g) was added

to the solution above and the reaction mixture was stirred at 0 °C

under Ar atmosphere for 10 min. After 15 min, the reaction mixture

was stirred at room temperature under Ar atmosphere for 13 h. After

13 h, the reaction mixture was ﬁltered on Celite and the ﬁltrate was

((2R,3R)-3-((tert-Butoxy((S)-3-(tert-butoxy)-2-((tert-

butoxycarbonyl)amino)-3-oxopropoxy)phosphoryl)oxy)tetrahydro-

R

DOI: 10.1021/acs.jmedchem.5b01925

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Journal of Medicinal Chemistry

Article

2H-pyran-2-yl)methyl oleate (43b). Compound 42b (99.4 mg,

0.1943 mmol) was dissolved in CH₂Cl₂(2.5 mL) and N,N-

dimethylaminopyridine (71.2 mg, 0.5829 mmol) was added to the

solution. The whole was cooled to 0 °C and oleoyl chloride (87.7 mg,

0.2915 mmol) was added under Ar. The reaction mixture was stirred at

room temperature under Ar for 3 h. After 3 h, methanol (2 mL) was

added and stirred at room temperature for 16 h. After 16 h, solvent

was removed under vacuum and the residue was puriﬁed by column

chromatography (n-hexane: ethyl acetate =4:1 to 2:1) to yield 43b

(115.6 mg, 0.1490 mmol, 77%, colorless oil).

hexane: ethyl acetate =2:1 to 1:1) to yield 44b (120.1 mg, 0.1475

mmol, 64%, colorless oil).

¹H NMR (CDCl₃, 400 MHz): 7.152−7.100 (2H, m), 6.835−6.779

(2H, m), 5.637−5.617 (0.3H, m), 5.508−5.487 (0.5H, m), 4.408−

4.315 (3H, m), 4.233−4.103 (3H, m), 4.016−3.976 (1H, m), 3.926

(2H, t, J = 6.4 Hz), 3.556−3.520 (1H, m), 3.439 (1H, dt, J = 12.0 Hz,

2.0 Hz), 2.942−2.897 (2H, m), 2.663−2.610 (2H, m), 2.264−2.170

(1H, m), 2.047−1.907 (1H, m), 1.802−1.732 (2H, m), 1.688−1.603

(1H, m), 1.477−1.417 (30H, m), 1.378−1.250 (14H, m), 0.862 (3H,

t, J = 6.4 Hz). ¹³C NMR (CDCl₃, 100 MHz): 173.16, 168.35, 156.96,

155.29, 155.23, 129.93, 128.84, 128.79, 127.48, 127.44, 120.10, 110.95,

83.84, 83.77, 82.64, 82.61, 79.87, 79.80, 76.50, 76.42, 76.38, 76.31,

71.92, 71.86, 71.61, 71.56, 67.83, 67.73, 67.41, 64.41, 64.37, 54.52,

54.44, 54.35, 33.94, 33.92, 31.89, 29.85, 29.81, 29.76, 29.61, 29.58,

29.37, 29.33, 29.30, 28.76, 28.66, 28.31, 27.94, 27.93, 26.11, 26.06,

26.02, 22.66, 20.08, 20.06, 14.10.

¹H NMR (CDCl₃, 400 MHz): 5.618−5.598 (0.3H, m), 5.492−

5.471 (0.5H, m), 5.388−5.266 (2H, m), 4.444−4.405 (1H, m),

4.358−4.313 (2H, m), 4.299−4.172 (1H, m), 4.161−4.068 (2H, m),

4.023−3.984 (m, 1H), 3.612−3.574 (m, 1H), 3.487−3.422 (m, 1H),

2.333−2.172 (m, 3H), 2.027−1.911 (5H, m), 1.707−1.552 (3H, m),

1.480−1.419 (28H, m), 1.293−1.233 (20H, m), 0.872−0.837 (3H,

m). ¹³C NMR (CDCl₃, 100 MHz): 173.58, 168.35, 155.22, 129.97,

129.95, 129.72, 83.88, 83.81, 82.64, 82.61, 79.88, 79.80, 76.36, 76.29,

71.86, 71.80, 71.59, 71.54, 67.83, 67.75, 64.37, 64.21, 54.43, 54.35,

34.09, 31.88, 29.85, 29.81, 29.77, 29.74, 29.69, 29.68, 29.49, 29.33,

29.32, 29.30, 29.17, 29.15, 29.11, 29.09, 29.07, 28.96, 28.31, 27.95,

27.93, 27.20, 27.15, 24.87, 24.84, 22.66, 22.55, 20.08, 20.06, 14.09. ³¹P

NMR (CDCl₃, 161 MHz): −5.29, −5.82. HRMS (ESI-TOF [M +

Na]⁺): Calcd for C₄₀H₇₄NNaO₁₁P⁺: 798.4892. Found 798.4891. Anal.

Calcd for C₄₀H₇₄NO₁₁P+0.1CH₂Cl₂; C, 61.43; H, 9.46; N, 1.79.

Found C, 61.46; H, 9.36; N, 1.77.

tert-Butyl O-(tert-butoxy(((2R,3S)-2-(((3-(2-(undecyloxy)phenyl)-

propanoyl)oxy)methyl)-tetrahydro-2H-pyran-3-yl)oxy)phosphoryl)-

N-(tert-butoxycarbonyl)-L-serinate (44a). Compound 42a (94.0 mg,

0.1838 mmol) and carboxylic acid derivative (B) (76.6 mg, 0.2389

mmol) were dissolved in CH₂Cl₂(1 mL). EDCI (45.8 mg, 0.2389

mmol) and DMAP (2.9 mg, 0.0239 mmol) were added to the solution

above at rt. Reaction mixture was stirred at room temperature under

Ar atmosphere for 48 h. EDCI (1.3 equiv) and MeOH (1 mL) were

added to the reaction mixture and stirred at room temperature for 2 h.

H₂O (5 mL) was added to the reaction mixture and extracted with

CH₂Cl₂(5 mL × 3). Organic layer was washed with brine, dried over

MgSO₄and evaporated. The residue was puriﬁed by column

chromatography (n-hexane: ethyl acetate =2:1) to yield 44a (53.2

mg, 0.0654 mmol, 36%, colorless oil).

³¹P NMR (CDCl₃, 161 MHz): −5.29, −5.85. HRMS (ESI-TOF [M

+ Na]⁺): Calcd for C₄₂H₇₂NNaO₁₂P⁺: 836.4684. Found 836.4685.

Anal. Calcd for C₄₂H₇₂NO₁₂P+0.4H₂O; C, 61.45; H, 8.88; N, 1.71.

Found C, 61.09; H, 8.66; N, 1.73.

tert-Butyl O-(tert-butoxy(((2R,3S)-2-(((3-(2-((3-phenoxybenzyl)-

oxy)phenyl)propanoyl)-oxy)methyl)tetrahydro-2H-pyran-3-yl)oxy)-

phosphoryl)-N-(tert-butoxycarbonyl)-L-serinate (45a). Compound

42a (54.4 mg, 0.1063 mmol) and carboxylic acid derivative (C) (48.2

mg, 0.1382 mmol) were dissolved in CH₂Cl₂(2 mL). EDCI (26.5 mg,

0.1382 mmol) and DMAP (1.7 mg, 0.0138 mmol) were added to the

solution above at rt. Reaction mixture was stirred at room temperature

under Ar atmosphere for 24 h. EDCI (0.3 equiv) and MeOH (1 mL)

were added to the reaction mixture and stirred at room temperature

for 1 h. H₂O (5 mL) was added to the reaction mixture and extracted

with CH₂Cl₂(3 mLx 3). Organic layer was washed with brine, dried

over MgSO₄and evaporated. The residue was puriﬁed by column

chromatography (n-hexane: ethyl acetate =2:1 to 1:1) to yield crude

45a (69.1 mg, 0.0821 mmol, 77%, colorless oil).

¹H NMR (CDCl₃, 400 MHz): 7.356−7.312 (3H, m), 7.198−7.086

(4H, m), 7.046−7.000 (3H, m), 6.954−6.925 (1H, m), 6.930 (1H, dd,

J = 1.2 Hz, 2.4 Hz), 6.873 (1H, dt, J = 0.8 Hz, 7.6 Hz), 6.844−6.824

(1H, m), 5.708−5.688 (0.3H, m), 5.508−5.487 (0.7H, m), 5.050 (2H,

s), 4.458 (1H, dd, J = 2.0 Hz, 1.2 Hz), 4.379−4.302 (2H, m), 4.265−

4.164 (1H, m), 4.152−4.047 (2H, m), 3.928−3.895 (1H, m), 3.390−

3.296 (2H, m), 3.006−2.960 (2H, m), 2.689−2.652 (2H, m), 2.373−

2.343 (1H, m), 1.726−1.565 (3H, m), 1.489−1.438 (27H, m). ¹³C

NMR (CDCl₃, 100 MHz): 173.09, 168.30, 157.64, 156.89, 156.39,

155.22, 139.36, 139.35, 130.17, 129.94, 129.80, 129.26, 129.20, 127.47,

127.45, 123.48, 121.56, 120.80, 119.19, 117.87, 117.05, 111.54, 84.12,

84.04, 82.68, 82.63, 79.92, 78.33, 78.23, 72.24, 72.19, 69.32, 67.56,

67.47, 63.79, 54.41, 54.33, 33.89, 30.94, 29.83, 29.79, 29.75, 28.34,

27.97, 27.96, 25.87, 24.96, 24.93.

¹H NMR (CDCl₃, 400 MHz): 7.158−7.119 (2H, m), 6.844−6.785

(2H, m), 5.705−5.684 (0.3H, m), 5.503−5.483 (0.7H, m), 4.458 (1H,

dd, J = 2.0 Hz, 12.0 Hz), 4.373−4.305 (2H, m), 4.255−4.170 (1H, m),

4.154−4.047 (2H, m), 3.950−3.904 (3H, m), 3.393−3.307 (2H, m),

2.957−2.918 (2H, m), 2.376−2.345 (2H, m), 1.798−1.568 (5H, m),

1.488−1.259 (43H, m), 0.887−0.853 (3H, m). ¹³C NMR (CDCl₃, 100

MHz): 173.22, 173.20, 168.30, 156.96, 155.32, 155.21, 129.95, 129.01,

128.96, 127.40, 127.38, 120.12, 110.92, 84.05, 83.98, 82.66, 82.61,

79.91, 79.81, 78.47, 78.38, 78.35, 78.26, 77.25, 72.77, 72.70, 72.22,

72.16, 67.75, 67.58, 67.49, 67.40, 63.75, 54.41, 54.32, 33.92, 31.91,

30.95, 30.81, 29.83, 29.80, 29.76, 29.63, 29.61, 29.58, 29.38, 29.33,

29.31, 28.33, 27.97, 27.95, 26.10, 25.91, 25.75, 24.97, 24.94, 22.67,

14.11. ³¹P NMR (CDCl₃, 161 MHz): −6.26, −6.29. HRMS (ESI-TOF

[M + Na]⁺): Calcd for C₄₂H₇₂NNaO₁₂P⁺: 836.4684. Found 836.4686.

Anal. Calcd for C₄₂H₇₂NO₁₂P; C, 61.97; H, 8.92; N, 1.72. Found C,

61.70; H, 8.68; N, 1.74.

tert-Butyl O-(tert-butoxy(((2R,3R)-2-(((3-(2-(undecyloxy)phenyl)-

propanoyl)oxy)methyl) tetrahydro-2H-pyran-3-yl)oxy)phosphoryl)-

N-(tert-butoxycarbonyl)-L-serinate (44b). Compound 42b (117.8

mg, 0.2303 mmol) and carboxylic acid derivative (B) (95.9 mg, 0.2994

mmol) were dissolved in CH₂Cl₂(4.3 mL) EDCI (57.4 mg, 0.2994

mmol) and N,N-dimethylaminopyridine (7.4 mg, 0.0606 mmol) were

added to the reaction mixture. This was stirred at room temperature

under Ar for 3 days. MeOH (1.5 mL) and EDCI (48.6 mg, 0.2533

mmol) were added to the reaction mixture and stirred at room

temperature under Ar for 1.5 h. H₂O (15 mL) was added to the

reaction mixture, the whole was washed with 5% aqueous KHSO₄

solution (15 mL) and extracted with CH₂Cl₂(8 mLX3). Combined

organic layer was washed with brine, dried over MgSO₄and

evaporated. The residue was puriﬁed by column chromatography (n-

³¹P NMR (CDCl₃, 161 MHz): −6.28, −6.33. HRMS (ESI-TOF [M

+ Na]⁺): Calcd for C₄₄H₆₀NNaO₁₃P⁺: 864.3694. Found 864.3689.

tert-Butyl O-(tert-butoxy(((2R,3R)-2-(((3-(2-((3-phenoxybenzyl)-

oxy)phenyl)propanoyl)-oxy)methyl)tetrahydro-2H-pyran-3-yl)oxy)-

phosphoryl)-N-(tert-butoxycarbonyl)-L-serinate (45b). Compound

42b (98.1 mg, 0.1918 mmol) and carboxylic acid derivative (C) (86.9

mg, 0.2493 mmol) were dissolved in CH₂Cl₂(43.6 mL) EDCI (47.8

mg, 0.2493 mmol) and N,N-dimethylaminopyridine (3.0 mg, 0.0249

mmol) were added to the reaction mixture. This was stirred at room

temperature under Ar for 3 days. MeOH (2 mL) and EDCI (40.4 mg,

0.2110 mmol) were added to the reaction mixture and stirred at room

temperature under Ar for 1.5 h. H₂O (15 mL) was added to the

reaction mixture, the whole was washed with 5% aqueous KHSO₄

solution (15 mL) and extracted with CH₂Cl₂(8 mLX3). Combined

organic layer was washed with brine, dried over MgSO₄and

evaporated. The residue was puriﬁed by column chromatography (n-

hexane: ethyl acetate =2:1 to 1:1) to yield 45b (79.8 mg, 0.0948 mmol,

49%, colorless oil).

¹H NMR (CDCl₃, 400 MHz): 7.359−7.308 (3H, m), 7.179−7.085

(4H, m), 7.056−7.003 (3H, m), 6.937 (1H, dd, J = 8.0 Hz, 2.0 Hz),

6.892−6.831 (2H, m), 5.644−5.624 (0.3H, m), 5.517−5.496 (0.5H,

m), 5.049 (2H, s), 4.373−4.326 (3H, m), 4.247−4.192 (1H, m),

S

DOI: 10.1021/acs.jmedchem.5b01925

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

4.174−4.086 (2H, m), 4.013−3.973 (1H, m), 3.547−3.512 (1H, m),

3.460−3.396 (1H, m), 2.992−2.944 (2H, m), 2.675−2.620 (2H, m),

2.263−2.166 (1H, m), 2.025−1.899 (1H, m), 1.677−1.578 (1H, m),

1.487−1.375 (28H, m). ¹³C NMR (CDCl₃, 100 MHz): 173.04,

168.37, 157.66, 157.64, 156.89, 156.39, 155.25, 139.34, 139.32, 130.18,

130.16, 129.93, 129.92, 129.81, 129.12, 129.06, 127.56, 127.52, 123.49,

121.57, 120.80, 119.18, 117.88, 117.09, 111.57, 83.80, 82.67, 82.64,

79.90, 79.84, 76.47, 76.39, 76.34, 76.27, 71.94, 71.87, 71.63, 71.57,

69.31, 67.80, 67.72, 67.44, 64.49, 64.44, 54.45, 54.36, 33.94, 33.92,

29.87, 29.82, 29.77, 28.76, 28.65, 28.33, 27.95, 26.04, 26.00, 20.00,

14.20. ³¹P NMR (CDCl₃, 161 MHz): −5.27, −5.85. HRMS (ESI-TOF

[M + Na]⁺): Calcd for C₄₄H₆₀NNaO₁₃P⁺: 864.3694. Found 864.3690.

Anal. Calcd for C₄₄H₆₀NO₁₃P + 2.5H₂O; C, 59.58; H, 7.39; N, 1.58.

Found C, 59.53; H, 7.38; N, 1.71.

tert-Butyl O-(tert-butoxy(((2R,3R)-2-(((3-(2-((3-(4-

chlorophenoxy)benzyl)oxy)phenyl)-propanoyl)oxy)methyl)-

tetrahydro-2H-pyran-3-yl)oxy)phosphoryl)-N-(tert-butoxycarbon-

yl)-L-serinate (45c). Compound (42b) (29.4 mg, 0.0594 mmol) and

79b (21.4 mg, 0.0594 mmol) was dissolved in CH₂Cl₂(0.7 mL) and

DIC (9.2 mg, 0.687 8 mol) and DMAP (2.1 mg, 0.0168 mmol) was

added to the solution above. The reaction mixture was stirred at r.t.

under Ar for 17 h. After 17 h, solvent was removed under vacuum and

puriﬁed by column chromatography (n-hexane: ethyl acetate =4:1

(120 mL) to 2:1 (300 mL) to 1:1 (200 mL)) to yield 45c (18.5 mg,

0.0211 mmol, 36%, colorless oil).

(2R,3S)-2-(Hydroxymethyl)-3,6-dihydro-2H-pyran-3-ol (46a).

Compound 36a (784.5 mg, 3.6621 mmol) was dissolved in MeOH

(10 mL) and sodium methoxide (98.9 mg, 1.8311 mmol) was added

to the solution above. The reaction mixture was stirred at room

temperature under Ar atmosphere for 10 min. After 10 min, solvent

was evaporated and the residue was dissolved in chloroform. This

solution was ﬁltered on Celite and the ﬁltrate was evaporated to yield

46a (414.6 mg, 3.1858 mmol, 87%, yellow oil).

¹H NMR (CDCl₃, 400 MHz): 5.848−5.781 (2H, m), 4.190−4.101

(3H, m), 3.874 (1H, dd, J = 11.6 Hz, 3.6 Hz), 3.785 (1H, dd, J = 11.6

Hz, 5.6 Hz), 3.354−3.306 (1H, m), 2.622 (1H, s). ¹³C NMR (CDCl₃,

100 MHz): 128.54, 127.62, 78.52, 65.35, 64.15, 63.03. HRMS (ESI-

+

TOF [M + Na]⁺): Calcd for C₆H₁₀NaO₃: 153.0522. Found 153.0549.

(2R,3R)-2-(Hydroxymethyl)-3,6-dihydro-2H-pyran-3-ol (46b).

Compound 36b (543.0 mg, 2.5348 mmol) was dissolved in methanol

(5 mL), sodium methoxide (68.5 mg, 1.2674 mmol) was added to the

solution above. The reaction mixture was stirred at room temperature

under Ar for 1.5 h. After 1.5 h, the reaction mixture was evaporated to

remove solvent and the residue was dissolved in chloroform. The

solution was ﬁltered on Celite and ﬁltrate was evaporated to yield

crude 46b (329.3 mg, 2.5301 mmol, 100%, colorless sticky oil).

¹H NMR (CDCl₃, 400 MHz): 6.057−6.009 (1H, m), 5.987−5.949

(1H, m), 4,281 (1H, dddd, J = 16.8 Hz, 11.6 Hz 3.6 Hz, 1.6 Hz),

4.198−4.142 (1H, m), 3.949−3.882 (2H, m), 3.811 (1H, dd, J = 11.6

Hz, 4.4 Hz), 3.577 (1H, spt, J = 2.0 Hz, 2.4 Hz, 6.4 Hz), 2.454−2.233

(2H, m). ¹³C NMR (CDCl₃, 100 MHz): 130.48, 126.42, 77.97, 66.15,

¹H NMR (CDCl₃, 400 MHz): 7.344 (t, 1H, J = 8.0 Hz), 7.308−

7.266 (m, 2H), 7.182−7.129 (m, 3H), 7.039−7.035 (m, 1H), 6.967−

6.829 (m, 5H), 5.642−5.496 (m, 1H), 5.057 (s, 2H), 4.409−4.333 (m,

3H), 4.247−4.100 (m, 3H), 4.018−3.979 (m, 1H), 3.556−3.520 (m,

1H), 3.461−3.402 (m, 1H), 2.995−2.947 (M, 2H), 2.651 (dd, 2H, J =

15.2 and 7.2 Hz), 2.265−2.168 (m, 1H), 2.041−1.900 (m, 1H),

1.687−1.600 (m, 1H), 1.488−1.422 (m, 27H), 1.392−1.387 (m, 1H).

¹³C NMR (CDCl₃, 100 MHz): 173.03, 168.38, 157.29, 156.34, 155.62,

+

63.38. 62.98. HRMS (ESI-TOF [M + Na]⁺): Calcd for C₆H₁₀NaO₃:

153.0522. Found 153.0541.

(2R,3S)-2-(((tert-Butyldimethylsilyl)oxy)methyl)-3,6-dihydro-2H-

pyran-3-ol (47a). Compound 46a (445.6 mg, 3.4340 mmol) was

dissolved in CH₂Cl₂(10 mL) and imidiazole was added to the

solution. This was cooled to 0 °C and tert-butyldimethylsilyl chloride

(567.7 mg, 3.7667 mmol) was added. This reaction mixture was stirred

at 0 °C under Ar atmosphere for 10 min and stirred at room

temperature for 15 h. After 15 h, water (20 mL) was added to the

reaction mixture and the whole was extracted with CH₂Cl₂(15

mLX3). Combined organic layer was washed with brine, dried over

MgSO₄and evaporated. The residue was puriﬁed by column

chromatography to yield 47a (594.1 mg, 2.4309 mmol, 71%, colorless

oil).

155.26, 139.52, 139.50, 130.17, 130.06, 129.79, 129.12, 129.07, 128.48,

127.56, 127.52, 121.99, 120.87, 120.35, 117.94, 117.16, 111.57, 83.92,

82.69, 82.67, 79.93, 76.46, 76.39, 76.36, 76.28, 71.64, 71.59, 69.25,

67.81, 67.72, 67.43, 64.54, 64.46, 54.45, 54.36, 33.92, 29.87, 29.82,

29.77, 28.76. 28.65, 28.32, 27.95, 26.00, 25.97, 20.05. ³¹P NMR

(CDCl₃, 161 MHz): −5.28, −5.87.

HRMS (ESI-TOF [M + Na]⁺): Calcd for C₄₄H₅₉ClNNaO₁₃P⁺

898.3305. Found 898.3282. Anal. Calcd for C₄₄H₅₉ClNO₁₃P; C, 58.64;

H, 6.58; N, 1.54. Found C, 58.84; H, 6.67; N, 1.65.

¹H NMR (CDCl₃, 400 MHz): 5.839−5.773 (2H, m), 4.209−4.186

(1H, m), 4.158−4.082 (2H, m), 3.964−3.921 (1H, m), 3.750−3.705

(1H, m), 3.395−3.338 (1H, m), 3.049 (1H, brs), 0.908 (9H, s), 0.108

(6H, s). ¹³C NMR (CDCl₃, 100 MHz): 127.92, 127.08, 76.70, 67.33,

65.92, 65.29, 25.86, 18.24, −5.51, −5.57. HRMS (ESI-TOF [M +

Na]⁺): Calcd for C₁₂H₂₄NaO₃Si⁺: 267.1387, Found 267.1370. Anal.

Calcd for C₁₂H₂₄O₃Si; C, 58.97; H, 9.90; N, 0.00. Found C, 58.89; H,

9.79; N, 0.00.

tert-Butyl O-(tert-butoxy(((2R,3R)-2-(((3-(2-((3-(p-tolyloxy)-

benzyl)oxy)phenyl)-propanoyl)oxy)methyl)tetrahydro-2H-pyran-3-

yl)oxy)phosphoryl)-N-(tert-butoxycarbonyl)-L-serinate (45d). Com-

pound 42b (37.0 mg, 0.0704 mmol) and 79c (25.5 mg, 0.0704 mmol)

was dissolved in CH₂Cl₂(1 mL) and DIC (11.5 mg, 0.0915 mol) and

DMAP (2.6 mg, 0.0211 mmol) was added to the solution above. The

reaction mixture was stirred at r.t. under Ar for 15 h. After 15 h,

solvent was removed under vacuum and puriﬁed by column

chromatography (n-hexane: ethyl acetate =4:1 (100 mL) to 2:1 (300

mL) to 1:1 (200 mL)) to yield 45d (31.9 mg, 0.0373 mmol, 53%,

colorless oil).

(2R,3R)-2-(((tert-Butyldimethylsilyl)oxy)methyl)-3,6-dihydro-2H-

pyran-3-ol (47b). Compound 46b (460.6 mg, 3.5347 mmol) was

dissolved in CH₂Cl₂(10 mL) and imidazole (529.4 mg, 7.7762 mmol)

was added to the solution above. This solution was cooled to 0 °C and

tert-butyldimethylsilyl chloride (639.3 mg, 4.2416 mmol) was added.

The reaction mixture was stirred at 0 °C under Ar for 10 min and

stirred at room temperature under Ar for 21 h. The reaction mixture

was quenched by water (20 mL) and the whole was extracted with

CH₂Cl₂(15 mL X3). Combined organic layer was washed with brine,

dried over MgSO₄, and evaporated. The residue was puriﬁed by

column chromatography (n-hexane: ethyl acetate =1:1 to ethyl

acetate) to yield 47b (275.2 mg, 1.1260 mmol, 79%, colorless oil).

¹H NMR (CDCl₃, 400 MHz): 6.073−6.024 (1H, m), 5.941 (1H,

dddd, J = 10.0 Hz, 3.6 Hz, 2.0 Hz, 1.6 Hz), 4.242 (1H, ddd, J = 16.8

Hz, 3.6 Hz, 1.6 Hz), 4.138 (1H, ddd, J = 16.8 Hz, 4.0 Hz, 2.0 Hz),

3.982−3.947 (1H, m), 3.859 (1H, dd, J = 10.4 Hz, 6.4 Hz), 3.807 (1H,

dd, J = 10.8 Hz, 6.0 Hz), 3.534 (1H, dt, J = 6.4 Hz, 2.0 Hz), 1.978−

1.965 (1H, m), 0.900 (9H, s), 0.089−0.084 (6H, m). ¹³C NMR

(CDCl₃, 100 MHz): 130.31, 126.62, 78.11, 66.18, 62.87, 62.60, 25.90,

18.31, −5.33, −5.38. HRMS (ESI-TOF [M + Na]⁺): Calcd for

C₁₂H₂₄NaO₃Si⁺: 267.1387, Found 267.1383. Anal. Calcd for

¹H NMR (CDCl₃, 400 MHz): 7.326−7.287 (m, 1H), 7.177−7.110

(m, 5H), 7.016 (m, 1H), 6.940−6.830 (m, 5H), 5.646−5.497 (m, 1H),

5.040 (s, 2H), 4.401−4.277 (m, 3H), 4.241−4.196 (m, 1H), 4.177−

4.091 (m, 2H), 4.015−3.976 (m, 1H), 3.552−3.516 (m, 1H), 3.462−

3.398 (m, 1H), 2.993−2.946 (m, 2H), 2.678−2.623, 2.335 (s, 3H),

2.263−2.166 (m, 1H), 2.026−1.898 (m, 1H), 1.679−1.579 (m, 1H),

1.488−1.423 (m, 27H), 1.393−1.376 (m, 1H).

¹³C NMR (CDCl₃, 100 MHz): 173.07, 158.22, 156.42, 154.35,

139.21, 133.19, 130.30, 130.15, 129.83, 129.06, 127.55, 127.52, 121.16,

120.77, 119.41, 117.33, 116.58, 111.56, 82.68, 79.91, 76.36, 76.28,

71.89, 71.64, 69.35, 67.80, 67.72, 67.42, 64.50, 54.45, 33.94, 29.86,

29.82, 29.77, 28.76, 28.32, 27.95, 26.05, 26.01, 20.72, 20.06. ³¹P NMR

(CDCl₃, 161 MHz): −5.28, −5.87. HRMS (ESI-TOF [M + Na]⁺):

Calcd for C₄₅H₆₂NNaO₁₃P⁺878.3851. Found 878.3831. Anal. Calcd

for C₄₅H₆₂NO₁₃P + CH₂Cl₂; C, 58.72; H, 6.86; N, 1.49. Found C,

58.76; H, 6.86; N, 1.65.

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C₁₂H₂₄O₃Si; C, 58.97; H, 9.90; N, 0.00. Found C, 58.88; H, 9.60; N,

0.00.

brs), 1.441−1.435 (9H, m), 1.413 (9H, s), 1.380 (9H, s). ¹³C NMR

(CDCl₃, 100 MHz): 168.27, 168.20, 155.16, 129.28, 129.23, 125.40,

125.37, 84.39, 84.32, 84.28, 84.20, 82.69, 82.64, 68.66, 68.61, 68.55,

67.50, 65.32, 65.30, 61.56, 61.47, 54.37, 54.2829.72, 29.70, 29.68,

29.65, 28.20, 27.85, 27.84. ³¹P NMR (CDCl₃, 161 MHz): −5.13,

−5.31. HRMS (ESI-TOF [M + Na]⁺): Calcd for C₂₂H₄₀NNaO₁₀P⁺:

532.2282. Found 532.2283. Anal. Calcd for C₂₂H₄₀NO₁₀P +

0.2CH₂Cl₂; C, 50.64; H, 7.73; N, 2.66. Found C, 50.78; H, 7.74; N,

2.67.

tert-Butyl O-(tert-butoxy(((2R,3R)-2-(hydroxymethyl)-3,6-dihy-

dro-2H-pyran-3-yl)oxy)-phosphoryl)-N-(tert-butoxycarbonyl)-L-seri-

nate (49b). Compound 48b (285.4 mg, 0.4575 mmol) was dissolved

in methanol (7 mL) and this solution was cooled to 0 °C Amberlyst15

(1.4110 g) was added to the solution above, stirred at 0 °C under Ar

for 5 min and stirred at room temperature under Ar for 9 h. After 9 h,

reaction mixture was ﬁltered on Celite and the ﬁltrate was evaporated.

The residue was puriﬁed by column chromatography (ethyl acetate) to

yield crude 49b (191.6 mg, 0.3769 mmol, 82%, colorless oil).

¹H NMR (CDCl₃, 400 MHz): 6.112−6.074 (1H, m), 6.025−5.940

(1H, m), 5.506−5.408 (1H, m), 4.701−4.637 (1H, m), 4.396−4.308

(2H, m), 4.303−4.082 (4H, m), 3.785−3.636 (3H, m), 1.515−1.486

(18H, m), 1.444−1.436 (9H, m). ¹³C NMR (CDCl₃, 100 MHz):

168.31, 168.24, 155.23, 155.14, 133.03, 132.77, 122.50, 122.47, 122.32,

122.29, 84.93, 84.85, 84.23, 84.15, 82.84, 79.99, 79.92, 76.55, 76.53,

76.50, 76.47, 67.87, 67.81, 67.60, 67.55, 67.45, 67.39, 65.70, 65.61,

60.66, 60.30, 54.33, 54.24, 29.77, 29.75, 29.73, 29.71, 28.26, 27.89,

27.87.

tert-Butyl O-(tert-butoxy(((2R,3S)-2-(((tert-butyldimethylsilyl)-

oxy)methyl)-3,6-dihydro-2H-pyran-3-yl)oxy)phosphoryl)-N-(tert-bu-

toxycarbonyl)-L-serinate (48a). 40 (724.1 mg 1.6034 mmol) and

compound 47a (587.8 mg, 2.4051 mmol) was dissolved in dry CH₂Cl₂

(10 mL) and dry toluene (2 mL) and coevaporated to remove

containing water. The residue was dissolved in CH₂Cl₂(10 mL) and

1H-tetrazole (505.4 mg, 7.2152 mmol) in THF (10 mL) was added to

the solution above. This reaction mixture was stirred at room

temperature under Ar atmosphere for 21 h. After 21 h, tert-

butylhydroperoxide 5 M in decane (0.641 mg, 3.2068 mmol) was

added to the reaction mixture and stirred at room temperature under

Ar atmosphere for 7 h. After 7 h, water (20 mL) was added and the

whole was extracted with CH₂Cl₂(15 mLX2). Organic layer was

washed with brine, dried over MgSO₄and evaporated. The residue was

puriﬁed by column chromatography (n-hexane: ethyl acetate =4:1 to

2:1) to yield 48a (713.3 mg, 1.1435 mmol, colorless oil, 71% 2 steps).

¹H NMR (CDCl₃, 400 MHz): 5.980−5.881 (2H, m), 5.495−5.445

(1H, m), 4.711−4.647 (1H, m), 4.364−4.154 (4H, m), 3.927 (1H,

dddd, J = 11.6 Hz, 4.8 Hz, 4.4 Hz, 2.4 Hz), 3.734 (1H, dd, J = 11.6 Hz,

6.4 Hz), 3.516−3.475 (1H, m), 1.646 (1H, brs), 1.548−1.442 (27H,

m), 0.899 (9H, s), 0.083−0.077 (6H, m). ¹³C NMR (CDCl₃, 100

MHz): 168.37, 168.32, 155.24, 129.86, 125.15, 125.01. 83.88, 83.81,

82.68, 79/92, 77.88, 77.78, 69.26, 69.20, 68.93, 68.87, 67.97, 67.66,

67.47, 64.95, 64.93, 63.08, 54.53, 54.45, 54.36, 30.91, 29.86, 29.84,

29.81, 29.80, 29.14, 28.33, 27.97, 25.98, 25.93, 23.91, 18.46, 18.45,

14.19. ³¹P NMR (CDCl₃, 161 MHz): −5.79, −5.86. HRMS (ESI-TOF

[M + Na]⁺): Calcd for C₂₈H₅₄NNaO₁₀PSi⁺: 646.3147, Found

646.3151. Anal. Calcd for C₂₈H₅₄NO₁₀PSi + 0.1CH₂Cl₂; C, 53.38;

H, 8.64; N, 2.22; Found C, 53.11; H, 8.56; N, 2.24.

tert-Butyl O-(tert-butoxy(((2R,3R)-2-(((tert-butyldimethylsilyl)-

oxy)methyl)-3,6-dihydro-2H-pyran-3-yl)oxy)phosphoryl)-N-(tert-bu-

toxycarbonyl)-L-serinate (48b). 40 (319.8 mg, 0.7081 mmol) and

compound 47b (259.6 mg, 1.0622 mmol) were dissolved in CH₂Cl₂

(2 mL) and toluene (0.5 mL) and evaporated to remove containing

water. The residue was dissolved in dry CH₂Cl₂(2 mL) and 1H-

tetrazole (198.4 mg, 2.8327 mmol) in THF (2 mL) was added to the

solution. This reaction mixture was stirred at room temperature under

Ar for 2 h. After 2 h, tert-butylhydroperoxide (0.2832 mg, 1.4162

mmol) was added to the reaction mixture and stirred at room

temperature under Ar for 1.5 h. After 1.5 h, the reaction mixture was

quenched by water (20 mL) and the whole was extracted with CH₂Cl₂

(10 mL × 2). Combined organic layer was washed with brine, dried

over MgSO₄and evaporated. The residue was puriﬁed by column

chromatography (n-hexane: ethyl acetate =2:1) to yield 48b (349.6

mg, 0.5604 mmol, 79% (2 steps), colorless oil).

³¹P NMR (CDCl₃, 161 MHz): - 3.39, −3.69. HRMS (ESI-TOF [M

+ Na]⁺): Calcd for C₂₂H₄₀NNaO₁₀P⁺: 532.2282. Found: 532.2298.

((2R,3S)-3-((tert-Butoxy((S)-3-(tert-butoxy)-2-((tert-

butoxycarbonyl)amino)-3-oxopropoxy)phosphoryl)oxy)-3,6-dihy-

dro-2H-pyran-2-yl)methyl oleate (50a). Compound 49a (92.9 mg,

0.1823 mmol) was dissolved in CH₂Cl₂(10 mL) and N,N-

dimethylaminopyridine (66.8 mg, 0.5470 mmol) was added to the

solution. The whole was cooled to 0 °C and oleoyl chloride (82.3 mg,

0.2735 mmol) was added under Ar. The reaction mixture was stirred at

room temperature under Ar for 1.5 h. After 1.5 h, solvent was removed

under vacuum and the residue was puriﬁed by column chromatog-

raphy (n-hexane: ethyl acetate =4:1 to 2:1) to yield 50a (101.1 mg,

0.1306 mmol, 72%, colorless oil).

¹H NMR (CDCl₃, 400 MHz): 5.896−5.840 (2H, m), 5.569−5.549

(0.3H, m), 5.442−5.422 (0.6H, m), 5.311−5.227 (2H, m), 4.711−

4.619 (1H, m), 4.411 (1H, dd, J = 12.0 Hz, 2.0 Hz), 4.299−255 (2H,

m), 4.225−4.506 (4H, m), 3.612 (1H, m), 2.293 (2H, t, J = 7.6 Hz),

1.985−1.838 (4H, m), 1.582−1.547 (2H, m), 1.440−1.409 (18H, m),

1.378 (9H, s), 1.229−1.197 (20H, m), 0.826−0.792 (3H, m). ¹³C

NMR (CDCl₃, 100 MHz): 173.54, 173.52, 168.31, 168.26, 155.26,

155.20, 130,18, 130.02, 129.95, 129.73, 129.52, 129.49, 128.01, 127.89,

125.14, 124.97, 84.24, 84.17, 82.69, 79.90, 79.84, 77.24, 74.96, 74.88,

74.79, 69.12, 69.06, 68.72, 68.67, 67.58, 67.53, 65.20, 65.16, 63.04,

54.42, 54.33, 34.07, 31.89, 31.87, 31.49, 29.81, 29.76, 29.74, 29.68,

29.66, 29.65, 29.62, 29.58, 29.49, 29.33, 29.31, 29.29, 29.28, 29.26,

29.17.29.16, 29.11, 29.10, 29.07, 28.95, 28.31, 27.95, 27.94, 27.19,

27.1625.60, 24.84, 22.64, 22.62, 22.54, 14.07, 14.04. ³¹P NMR (CDCl₃,

161 MHz): - 5.70, −5.78. HRMS (ESI-TOF [M + Na]⁺): Calcd for

C₄₀H₇₂NNaO₁₁P⁺796.4735. Found 796.4730. Anal. Calcd for

C₄₀H₇₂NO₁₁P + 0.2CH₂Cl₂; C, 61.04; H, 9.23; N, 1.77. Found C,

61.02; H, 8.95; N, 1.82.

((2R,3R)-3-((tert-Butoxy((S)-3-(tert-butoxy)-2-((tert-

butoxycarbonyl)amino)-3-oxopropoxy)phosphoryl)oxy)-3,6-dihy-

dro-2H-pyran-2-yl)methyl oleate (50b). Compound 49b (100.6 mg,

0.1974 mmol) was dissolved in CH₂Cl₂(3 mL) and N,N-

dimethylaminopyridine (72.4 mg, 0.5923 mmol) was added to the

solution. The whole was cooled to 0 °C and oleoyl chloride (89.1 mg,

0.2962 mmol) was added under Ar. The reaction mixture was stirred at

0 °C under Ar for 10 min and stirred at room temperature under Ar

for 1 h. After 1 h, solvent was removed under vacuum and the residue

was puriﬁed by column chromatography (n-hexane: ethyl acetate

=2:1) to yield 50b (125.4 mg, 0.1620 mmol, 82%, colorless oil).

¹H NMR (CDCl₃, 400 MHz): 6.125−6.053 (2H, m), 5.513−5.442

(1H, m), 4.629−4.590 (1H, m), 4.381−4.277 (3H, m), 4.218−4.166

(2H, m), 3.858−3.741 (2H, m), 3.597−3.558 (1H, m), 1.490−1.468

(18H, m), 1.441 (9H, s), 0.901−0.883 (9H, m), 0.081−0.060 (6H,

m). ¹³C NMR (CDCl₃, 100 MHz): 168.45, 155.26, 132.82, 123.10,

82.58, 82.56, 79.82, 77.66, 77.58, 68.09, 65.80, 65.72, 63.01, 62.67,

54.40, 29.88, 29.87, 29.82, 28.34, 27.98, 27.94, 25.92, 18.36, 18.33,

−5.19, −5.20, −5.25, −5.28. ³¹P NMR (CDCl₃, 161 MHz): −5.56,

−5.82. HRMS (ESI-TOF [M + Na]⁺): Calcd for C₂₈H₅₄NNaO₁₀PSi⁺:

646.3147. Found 646.3160. Anal. Calcd for C₂₈H₅₄NO₁₀PSi; C, 53.91;

H, 8.73; N, 2.25. Found C, 53.64; H, 8.44; N, 2.35.

tert-Butyl O-(tert-butoxy(((2R,3S)-2-(hydroxymethyl)-3,6-dihydro-

2H-pyran-3-yl)oxy)-phosphoryl)-N-(tert-butoxycarbonyl)-L-serinate

(49a). Compound 48a (660.5 mg, 1.0588 mmol) was dissolved in

MeOH (16 mL). Amberlyst15 (3.3025 g) was added to the solution

above and the reaction mixture was stirred at room temperature under

Ar atmosphere for 11 h. After 11 h, the reaction mixture was ﬁltered

on Celite and the ﬁltrate was evaporated and the residue was puriﬁed

by column chromatography (ethyl acetate) to yield 49a (322.2 mg,

0.6324 mmol, 61%, white sticky solid).

¹H NMR (CDCl₃, 400 MHz): 5.873−5.803 (2H, m), 5.581−5.562

(1H, m), 4.823−4.776 (1H, m), 4.313−4.254 (m, 2H), 4.209−4.135

(m, 3H), 3.810−3.705 (m, 2H), 3.407−3.359 (m, 1H), 3.271 (1H,

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¹H NMR (CDCl₃, 400 MHz): 6.084−6.015 (2H, m), 5.600−5.447

(1H, m), 5.360−5.262 (2H, m), 4.622−4.590 (1H, m), 4.351−4.057

(6H, m), 3.783−3.759 (1H, m), 2.337−2.288 (2H, m), 2.006−1.948

(4H, m), 1.627−1.554 (2H, m), 1.472−1.410 (27H, m), 1.309−1.204

(21H, m), 0.861−0.827 (3H, m). ¹³C NMR (CDCl₃, 100 MHz):

173.58, 173.55, 173.53, 168.34, 168.32, 155.26, 155.21, 132.59, 132.33,

130.16, 129.99, 129.95, 129.71, 129.70, 128.02, 128.00, 127.87, 84.10,

84.06, 84.03, 83.98, 82.61, 82.59, 79.82, 79.79, 74.42, 74.35, 74.27,

68.17, 68.10, 67.87, 67.82, 67.56, 67.50, 67.43, 65.57, 65.47, 63.71,

63.48, 54.49, 54.38, 54.29, 34.08, 34.07, 31.89. 31.87, 31.49, 29.84,

29.82, 29.79, 29.78, 29.73, 29.70, 29.68, 29.66, 29.48, 29.26, 29.16,

29.14, 29.09, 29.07, 29.0628.30, 27.93, 27.91, 27.18, 27.17, 27.14,

25.60, 24.86, 24.83, 22.64, 22.53, 14.07, 14.04. ³¹P NMR (CDCl₃, 161

MHz): δ - 5.53, −5.67.

31.91, 29.87, 29.84, 29.83, 29.80, 29.64, 29.63, 29.60, 29.39, 29.35,

29.32, 28.33, 27.97, 27.94, 26.13, 26.08, 26.05, 25.93, 22.68, 14.12. ³¹P

NMR (CDCl₃, 161 MHz): −5.54, −5.72. HRMS (ESI-TOF [M +

Na]⁺): Calcd for C₄₂H₇₀NNaO₁₂P⁺: 834.4533. Found 834.4530.

tert-Butyl O-(tert-butoxy(((2R,3S)-2-(((3-(2-((3-phenoxybenzyl)-

oxy)phenyl)propanoyl)-oxy)methyl)-3,6-dihydro-2H-pyran-3-yl)-

oxy)phosphoryl)-N-(tert-butoxycarbonyl)-L-serinate (52a). Carbox-

ylic acid derivative (C) (68.2 mg, 0.1958 mmol), compound 49a (99.7

mg, 0.1958 mmol) were dissolved in CH₂Cl₂(1 mL) and

diisopropylcarbodiimide (32.1 mg, 0.2545 mmol) and N,N-dimethy-

laminopyridine (2.4 mg, 0.0196 mmol) were added to the solution

above. The mixture was stirred at room temperature under Ar

atmosphere for 18 h. After 18 h, the reaction mixture was quenched by

water (8 mL) and extracted with CH₂Cl₂(8 mLX2). Combined

organic layer was washed with brine, dried over MgSO₄and

evaporated. The residue was puriﬁed by column chromatography (n-

hexane: acetone =3:1) to yield 52a (98.9 mg, 0.1178 mmol, 60%,

colorless oil).

HRMS (ESI-TOF [M + Na]⁺): Calcd for C₄₀H₇₂NNaO₁₁P⁺:

796.4735. Found 796.4733.

Anal. Calcd for C₄₀H₇₂NO₁₁P; C, 62.07; H, 9.38; N, 1.80. Found: C,

61.88; H, 9.35; N, 1.79.

¹H NMR (CDCl₃, 400 MHz): 7.358−7.313 (3H, m), 7.191−7.084

(4H, m), 7.041−6.997 (3H, m), 6.951−6.926 (1H, m), 6.891−6.826

(2H, m), 5.920−5.910 (2H, m), 5.669−5.500 (1H, m), 5.048 (2H, s),

4.749−4.648 (1H, m), 4.492−4.456 (1H, m), 4.394−4.322 (2H, m),

4.289−4.185 (1H, m), 4.159−4.107 (3H, m), 3.662−3.619 (1H, m),

3.008−2.965 (2H, m), 2.696−2.658 (2H, m), 1.493−1.436 (27H, m).

¹³C NMR (CDCl₃, 100 MHz): 173.01, 168.34, 168.29, 157.65, 156.88,

tert-Butyl O-(tert-butoxy(((2R,3S)-2-(((3-(2-(undecyloxy)phenyl)-

propanoyl)oxy)methyl)-3,6-dihydro-2H-pyran-3-yl)oxy)-

phosphoryl)-N-(tert-butoxycarbonyl)-L-serinate (51a). Carboxylic

acid derivatives (B) (39.1 mg, 0.1219 mmol), compound 49a (62.1

mg, 0.1219 mmol) were dissolved in CH₂Cl₂(1 mL) and

diisopropylcarbodiimide (20.0 mg, 0.1585 mmol) and N,N-dimethy-

laminopyridine (1.5 mg, 0.0122 mmol) were added to the solution

above. The mixture was stirred at room temperature under Ar

atmosphere for 16 h. After 16 h, the reaction mixture was quenched by

water (7 mL) and extracted with CH₂Cl₂(7 mL × 2). Combined

organic layer was washed with brine, dried over MgSO₄and

evaporated. The residue was puriﬁed by column chromatography (n-

hexane: acetone =3:1) to yield crude 51a (39.2 mg, 0.0483 mmol,

40%, colorless oil).

156.38, 155.31, 155.23, 139.33, 130.19, 129.94, 129.81, 129.62, 129.58,

129.17, 129.12, 127.52, 127.50, 125.08, 124.93, 123.49, 121.56, 120.82,

119.19, 117.89, 117,04, 111.55, 84.42, 84.34, 84.29, 84.22, 82.72,

79.93, 29.87, 74.98, 74.88, 74.79, 69.31, 69.18, 69.12, 68.76, 68.70,

67.62, 67.56, 65.14, 65.10, 63.30, 63.26, 54.53, 54.44, 54.35,33.89,

29.83, 29.81, 29.76, 28.33, 27.97, 27.96, 25.92. ³¹P NMR (CDCl₃, 161

MHz): 5.64, −5.73.

¹H NMR (CDCl₃, 400 MHz): 7.178−7.127 (2H, m), 6.862−6.793

(2H, m), 5.928−5.900 (2H, m), 5.658−5.489 (1H, m), 4.752−4.652

(1H, m), 4.498−4.463 (1H, m), 4.396−4.114 (6H, m), 3.964−3.924

(2H, m), 3.669−3.628 (1H, m), 2.969−2.930 (2H, m), 2.698−2.621

(2H, m), 1.820−1.750 (2H, m), 1.500−1.262 (43H, m), 0.899−0.857

(3H, m). ¹³C NMR (CDCl₃, 100 MHz): 173.16, 168.29, 156.97,

155.24, 129.96, 129.58, 129.54, 128.94, 128.88, 128.84, 127.53, 127.45,

127.43, 125.13, 124.95, 120.13, 110.94, 84.21, 82.72, 79.93, 29.87,

74.91, 74.82, 69.19, 69.14, 68.76, 68.70, 67.75, 67.62, 65.16, 65.11,

63.22, 54.42, 33.92, 31.91, 29.81, 29.79, 29.77, 29.64, 29.62, 29.59,

29.38, 29.34, 29.31, 28.33, 27.97, 27.96, 26.11, 26.05, 25.97, 25.95,

22.68, 14.12. ³¹P NMR (CDCl₃, 161 MHz): −5.48, −5.68. HRMS

(ESI-TOF [M + Na]⁺): Calcd for C₄₂H₇₀NNaO₁₂P⁺: 834.4533. Found

834.4522.

tert-Butyl O-(tert-butoxy(((2R,3R)-2-(((3-(2-(undecyloxy)phenyl)-

propanoyl)oxy)methyl)-3,6-dihydro-2H-pyran-3-yl)oxy)-

phosphoryl)-N-(tert-butoxycarbonyl)-L-serinate (51b). Carboxylic

acid derivatives (B) (67.7 mg, 0.2111 mmol), compound 49b (107.5

mg, 0.2111 mmol) were dissolved in CH₂Cl₂(2 mL) and

diisopropylcarbodiimide (58.6 mg, 0.4644 mmol) and N,N-dimethy-

laminopyridine (8.0 mg, 0.0633 mmol) were added to the solution

above. The mixture was stirred at room temperature under Ar

atmosphere for 18 h. After 18 h, the reaction mixture was quenched by

water (10 mL) and extracted with CH₂Cl₂(10 mL × 2). Combined

organic layer was washed with brine, dried over MgSO₄and

evaporated. The residue was puriﬁed by column chromatography

(1st: CH₂Cl₂: MeOH = 12:1, second: n-hexane: acetone =5:1, third: n-

hexane: ethyl acetate =2:1) to yield crude 51b (101.0 mg, 0.1244

mmol, 59%, colorless oil).

HRMS (ESI-TOF [M + Na]⁺): Calcd for C₄₄H₅₈NNaO₁₃P⁺:

862.3538. Found 862.3521. Anal. Calcd for C₄₄H₅₈NO₁₃P; C, 60.33;

H, 7.13; N, 1.60. Found C, 60.12; H, 6.91; N, 1.51.

tert-Butyl O-(tert-butoxy(((2R,3R)-2-(((3-(2-((3-phenoxybenzyl)-

oxy)phenyl)propanoyl)-oxy)methyl)-3,6-dihydro-2H-pyran-3-yl)-

oxy)phosphoryl)-N-(tert-butoxycarbonyl)-L-serinate (52b). Carbox-

ylic acid derivative (C) (59.0 mg, 0.1693 mmol), compound 49b (86.2

mg, 0.1693 mmol) were dissolved in CH₂Cl₂(2 mL) and

diisopropylcarbodiimide (47.0 mg, 0.3724 mmol) and N,N-dimethy-

laminopyridine (2.1 mg, 0.0169 mmol) were added to the above

solution. The mixture was stirred at room temperature under Ar

atmosphere for 20 h. After 20 h, the reaction mixture was quenched by

water (7 mL) and extracted with CH₂Cl₂(7 mLδ2). Combined

organic layer was washed with brine, dried over MgSO₄and

evaporated. The residue was puriﬁed by column chromatography (n-

hexane: acetone =3:1) to yield crude 52b (43.4 mg, 0.0517 mmol,

31%, colorless oil).

¹H NMR (CDCl₃, 400 MHz): 7.362−7.313 (3H, m), 7.188−7.090

(4H, m), 7.058 (1H, m), 7.029−7.007 (2H, m), 6.952−6.927 (1H, m),

6.896−6.839 (2H, m), 6.090−6.024 (2H, m), 5.629−5.609 (1H, m),

5.058 (2H, s), 4.582−4.556 (1H, m), 4.375−4.075 (7H, m), 3.740−

3.708 (1H, m), 3.004−2.958 (2H, m), 2.689−2.637 (2H, m), 1.482−

1.230 (27H, m). ¹³C NMR (CDCl₃, 100 MHz): 173.05, 168.38,

157.66, 156.89, 156.40, 155.25, 139.34, 139.31, 132.62, 132.36, 130.19,

130.16, 129.92, 129.81, 129.10, 129.04, 127.59, 127.55, 123.49, 122.74,

122.69, 121.57, 120.82, 119.18, 117.89, 117.10, 111.57, 84.08, 82.65,

79.87, 74.47, 74.39, 74.35, 74.27, 69.32, 68.25, 67.91, 67.86, 67.61,

65.57, 65.46, 63.87, 63.72, 54.40, 54.32,33.95, 29.87, 29.83, 29.80,

29.34, 27.96, 27.94, 26.07, 26.04. ³¹P NMR (CDCl₃, 161 MHz):

−5.52, −5.72. HRMS (ESI-TOF [M + Na]⁺): Calcd for

C₄₄H₅₈NNaO₁₃P⁺: 862.3538. Found 862.3520.

Benzene LysoPS analogues. 3-(((tert-Butyldimethylsilyl)oxy)-

methyl)phenol (54b). m-Hydroxybenzylalcohol 53b was dissolved in

CH₂Cl₂(2 mL) and THF (1 mL) and imidazole (60.3 mg, 0.8861

mmol) was added to the solution at 0 °C. TBSCl (133.6 mg, 0.8861

mmol) in THF (3 mL) was added dropwise for 30 min. The reaction

mixture was stirred at 0 °C to rt under Ar for 2.5 h. After 2.5 h, the

reaction was quenched by water (10 mL) and extracted with ethyl

¹H NMR (CDCl₃, 400 MHz): 7.174−7.127 (2H, m), 6.854−6.800

(2H, m), 6.104−6.039 (2H, m), 5.630−5.475 (1H, m), 4.590−4.579

(1H, m), 4.381−4.106 (7H, m), 3.947 (2H, t, J = 6.4 Hz), 3.765−

3.732 (1H, m), 2.967−2.923 (2H, m), 2.690−2.642 (2H, m), 1.823−

1.753 (2H, m), 1.494−1.264 (43H, m), 0.897−0.860 (3H, m). ¹³C

NMR (CDCl₃, 100 MHz): 173.20, 173.19, 168.38, 156.98, 155.26,

132.61, 132.35, 129.95, 128.85, 128.80, 127.52, 127.48, 122.77, 122.73,

120.13, 110.98, 84.08, 82.65, 79.88, 74.51, 74.44, 74.40, 74.32, 68.25,

68.19, 67.86, 67.77, 67.52, 65.60, 65.49, 63.84, 63.69, 54.42, 33.96,

V

DOI: 10.1021/acs.jmedchem.5b01925

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Journal of Medicinal Chemistry

Article

acetate (10 mL × 2), washed with brine, dried over MgSO₄and

evaporated. The residue was puriﬁed by column chromatography (n-

hexane: ethyl acetate =2:1 (240 mL) to 1:1 (200 mL) to 1:2 (140 mL)

to yield 54b (136.8 mg, 0.5738 mmol, 71%).

and 1H-tetrazole (38.9 mg, 0.5547 mmol) in THF (0.6 mL) was

added to the solution above. The whole was stirred at room

temperature under Ar atmosphere for 20 h. After 20 h, the reaction

mixture was quenched by water (7 mL) and extracted with CH₂Cl₂(6

mL × 2). Combined organic layer was washed with brine, dried over

MgSO₄and evaporated. The residue was puriﬁed by column

chromatography (n-hexane: ethyl acetate =4:1) to yield crude trivalent

phosphodiester compound (141.8 mg, 0.2356 mmol, quatititative,

colorless oil).

The trivalent phosphodiester compound (141.8 mg, 0.2356 mmol)

was dissolved in CH₂Cl₂(1.5 mL) and added tert-butylhydroperoxide

in decane (0.0943 mL). The reaction mixture was stirred at room

temperature under Ar atmosphere for 70 min. After 70 min, solvent

was removed under vacuum and the residue was puriﬁed by column

chromatography (n-hexane: ethyl acetate =4:1 to 2:1) to yield 55c

(114.2 mg, 0.1849 mmol, 78%, white solid, 83% (2 steps)).

¹H NMR (CDCl₃, 400 MHz): 7.253−7.224 (2H, m), 7.135−7.097

(2H, m), 5.452−5.372 (1H, m), 4.668 (2H, s), 4.452−4.269 (3H, m),

1.487−2.469 (9H, m), 1.422−1.408 (18H, m), 0.902 (9H, s), 0.058

(6H, s). ¹³C NMR (CDCl₃, 100 MHz): 168.18, 155.16, 149.71,

149.63, 138.00, 137.99, 127.20, 119.74, 119.72, 119.70, 119,67, 84.90,

84.86, 84.82, 84.78, 82.74, 82.73, 79.89, 68.01, 67.95, 67.91, 64.32,

54.43, 54.39, 54.35, 54.30, 29.78, 29.77, 29.74, 29.73, 28.29, 27.90,

27.89, 25.90, 18.35, −5.29. ³¹P NMR (CDCl₃, 161 MHz): −11.04,

−11.35. HRMS (ESI-TOF [M + Na]⁺): Calcd for C₂₉H₅₂NNaO₉PSi⁺:

640.3041. Found 640.3016.

¹H NMR (CDCl₃, 400 MHz): 7.067−7.028 (m, 1H), 6.753−

6.711(m, 2H), 6.589−6.564 (m, 1H), 5.836 (bs, 1H), 4.584 (s, 2H),

0.835 (s, 9H), 0.000 (s, 6H). ¹³C NMR (CDCl₃, 100 MHz): 155.67,

143.06, 129.50, 118.41, 114.08, 113.22, 64.92, 26.02, 18.50, −5.19.

HRMS (ESI-TOF [M-H]⁻): Calcd for C₁₃H₂₁O₂Si⁻237.1316. Found

237.1343. Anal. Calcd for C₁₃H₂₂O₂Si + 0.1H₂O; C, 65.05; H, 9.26; N,

0.00. Found C, 64.96; H, 9.03; N, 0.00.

4-(((tert-Butyldimethylsilyl)oxy)methyl)phenol (54c). Imidazole

(258.8 mg, 3.8014 mmol) was added to the solution of 4-

hydroxybenzyl alcohol 53c (214.5 mg, 1.7279 mmol) in THF (4

mL) and TBSCl (286.5 mg, 1.9007 mmol) in THF (2 mL) was added

to the reaction mixture at 0 °C under Ar. The whole was stirred at 0

°C for 10 min, and stirred at room temperature for 2 h. After 2 h, the

reaction mixture was quenched by water (7 mL) and the whole was

extracted with ethyl acetate (7 mL × 3), washed with brine, dried over

MgSO₄and evaporated under vacuum. The residue was puriﬁed by

column chromatography (n-hexane: ethyl acetate =4:1 to ethyl

acetate) to yield 54c (357.0 mg, 1.4975 mmol, colorless oil, 87%,

sm recovery 7.9 mg).

¹H NMR (CDCl₃, 400 MHz): 7.191−7.169 (2H, m), 6.794−6.759

(2H, m), 6.363 (1H, brs), 4.682 (2H, s), 0.952 (9H, s), 0.118 (6H, s).

¹³C NMR (CDCl₃, 100 MHz): 154.95, 133.03, 127.98, 115.25, 65.02,

26.02, 18.49, −5.12. HRMS (ESI-TOF [M + Na]⁺): Calcd for

C₁₃H₂₂NaO₂Si⁺: 261.1281. Found 261.1282. Anal. Calcd for

C₁₃H₂₂O₂Si; C, 65.50; H,9.30; N, 0.00. Found C, 65.39; H, 9.25; N,

0.00.

Anal. Calcd for C₂₉H₅₂NO₉PSi; C, 56.38; H, 8.48;N, 2.27. Found C,

56.14; H, 8.29; N, 2.22.

2-Hydroxybenzyl 3-(2-(undecyloxy)phenyl)propanoate (56a).

DIC (204.2 mg, 1.6111 mmol) and DMAP (19.7 mg, 0.1611 mmol)

were added to the solution of salicylic alcohol 53a (100.0 mg, 0.8055

mmol) and acid derivative (B) (258.2 mg, 0.8055 mmol) in CH₂Cl₂(2

mL) and stirred at room temperature under Ar for 2 h. After 2 h, the

reaction mixture was quenched by water (10 mL) and the whole was

extracted with CH₂Cl₂(8 mL × 3). Combined organic layer was

washed with brine, dried over MgSO₄and evaporated. The residue was

puriﬁed by column chromatography (n-hexane: ethyl acetate =20:1

(420 mL) to 10:1 (220 mL) to 4:1 (250 mL)) to yield 56a (130.7 mg,

0.3064 mmol, 38%, colorless oil).

tert-Butyl O-(tert-butoxyl)3-(((tert-butyldimethylsilyl)oxy)-

methyl)phenoxy)phosphoryl)-N-(tert-butoxylcarbonyl)-L-serinate)

(55b). Compound 40 (258.56 mg, 0.5726 mmol) and 54b (105.0 mg,

0.4404 mmol) was dissolved in CH₂Cl₂(1 mL) and toluene (0.1 mL)

and coevaporated. The residue was dissolved in CH₂Cl₂(1 mL) and

1H-tetrazole (77.1 mg, 1.1011 mmol) in THF (1 mL) was added to

the solution above. The whole was stirred at room temperature under

Ar atmosphere for 20 h. After 20 h, the reaction mixture was quenched

by saturated NaHCO₃(aq) (10 mL) and extracted with CH₂Cl₂(8

mL × 3). Combined organic layer was washed with brine, dried over

MgSO₄and evaporated. The residue was puriﬁed by column

chromatography (n-hexane: ethyl acetate =4:1 (200 mL)) to yield

crude trivalent phosphodiester compound (246.0 mg, 0.4088 mmol,

93%, colorless oil).

¹H NMR (CDCl₃, 400 MHz): 7.357−7.283 (m, 2H), 7.217 (dt, 1H,

J = 8.0 Hz, 7.6 Hz, 1.6 Hz), 7.127 (dd, 1H, J = 7.6 Hz, 1.6 Hz), 7.011

(dd, 1H, J = 8.0 Hz, 1.2 Hz), 6.963 (dt, 1H, J = 7.6 Hz, 7.2 Hz, 1.2

Hz), 6.897−6.842 (m, 2H), 4.001 (t, 2H, J = 6.4 Hz), 3.025−2.987

(m, 2H), 2.758−2.720 (m, 2H) 1.882−1.812 (m, 2H), 1.557−1.485

(m, 2H), 1.387−1.355 (m, 14H), 0.966 (t, 3H, J = 6.8). ¹³C NMR

(CDCl₃, 100 MHz): 176.04, 156.94, 155.64, 132.15, 131.08, 129.99,

128.38, 127.72, 121.86, 120.52, 120.26, 117.84, 111.05, 67.79, 63.22,

34.20, 32.00, 29.70, 29.67, 29.43, 29.36, 26.23, 22.77, 14.20. HRMS

The trivalent phosphodiester compound (245.9 mg, 0.4086 mmol)

was dissolved in CH₂Cl₂(2.5 mL) and added tert-butylhydroperoxide

in decane (0.1634 mL). The reaction mixture was stirred at room

temperature under Ar atmosphere for 1.5 h. After 1.5 h, solvent was

removed under vacuum and the residue was puriﬁed by column

chromatography (n-hexane: ethyl acetate =4:1 (200 mL) to 2:1(180

mL)) to yield 55b (185.9 mg, 0.3009 mmol, 74%, colorless oil, 68% (2

steps)).

+

(ESI-TOF [M + Na]⁺): Calcd for C₂₇H₃₈NaO₄449.2662. Found

449.2635. Anal. Calcd for C₂₇H₃₈O₄; C, 76.02; H, 8.98; N, 0.00. Found

C, 75.74; H, 8.82; N, 0.00.

¹H NMR (CDCl₃, 400 MHz): 7.208−7.148 (m, 1H), 7.054−7.014

(m, 2H), 6.993−6.947 (m, 1H), 5.388−5.291 (m, 1H), 4.625 (s, 2H),

4.384−4.322 (m, 1H), 4.297−4.252 (m, 1H), 4.244−4.196 (m, 1H),

1.420−1.404 (m, 9H), 1.350−1.334 (m, 18H), 0.842 (s, 9H), 0.000 (s,

6H).

O-(tert-Butoxy)3-hydroxymethyl)phosphporyl-N-tert-butoxylcar-

bonyl)-^L-serine (56b). 55b (185.4 mg, 0.3001 mmol) in THF (2 mL)

was added HF-Py (168.7 μL) in pyridine (415.6 μL) and stirred at

room temperature under Ar for 2 h. After 2 h, the reaction was

quenched by water (10 mL), extracted with CH₂Cl₂(10 mL × 3).

Combined organic layer was washed with brine, dried over MgSO₄and

evaporated. The residue was puriﬁed by column chromatography (n-

hexane: ethyl acetate =2:1 (150 mL) to 1:1 (180 mL) to 1:2 (120 mL)

to yield 56b (57.7 mg, 0.1146 mmol, 38%).

¹³C NMR (CDCl₃, 100 MHz): 168.14, 155.15, 150.85, 150.78,

143.56, 143.55, 129.33, 129.31, 122.27, 118.28, 118.24, 117,51, 117.48,

117.46, 84.89, 84.81, 84.77, 82.68, 82.67, 79.84, 68.04, 67.98, 67.92,

64.33, 54.42, 54.37. 54.29, 29.76, 29.75, 29.72, 28.26, 27.88, 27.86,

15.89, 18.34, 18.01, −5.33. ³¹P NMR (CDCl₃, 161 MHz): −11.15,

−11.47. HRMS (ESI-TOF [M + Na]⁺): Calcd for C₂₉H₅₂NNaO₉PSi⁺

640.3041. Found 640.3039. Anal. Calcd for C₂₉H₅₂NO₉PSi; C, 56.38;

H, 8.48; N, 2.27. Found C, 56.14; H, 8.35; N, 2.28.

¹H NMR (CDCl₃, 400 MHz): 7.240−7.188 (m, 1H), 7.148 (m,

1H), 7.085−7.054 (m, 1H), 7.024−6.978 (m, 1H), 5.404−5.276 (m,

1H), 4.587 (s, 2H), 4.370−4.330 (m, 1H), 4.331−4.233 (m, 2H),

2.739 (bs, 1H), 1.546−1.422 (m, 9H), 1.372−1.322 (m, 18H). ¹³C

NMR (CDCl₃, 100 MHz): 168.31, 155.19, 150.92, 150.85, 143.42,

129.64, 129.59, 123.30, 123.23, 118.93, 118.43, 85.20, 82.98, 82.93,

80.08, 68.03, 64.41, 54.42, 54.32, 29.81, 29.78, 28.28, 27.91, 27.87. ³¹P

NMR (CDCl₃, 161 MHz): −11.30, −11.37. HRMS (ESI-TOF [M +

Na]⁺): Calcd for C₂₃H₃₆NNaO₉P⁺526.2176. Found 526.2149. Anal.

tert-Butyl O-(tert-butoxy(4-(((tert-butyldimethylsilyl)oxy)methyl)-

phenoxy)phosphoryl)-N-(tert-butoxycarbonyl)-L-serinate (55c).

Compound 40 (130.3 mg, 0.2885 mmol) and compound 54c (52.9

mg, 0.2219 mmol) was dissolved in CH₂Cl₂(1 mL) and toluene (0.1

mL) and coevaporated. The residue was dissolved in CH₂Cl₂(0.5 mL)

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Calcd for C₁₉H₃₀NO₉P: C, 54.86; H, 7.61; N, 2.78. Found C, 54.68; H,

7.45 ; N, 2.78.

mLX2), washed with brine, dried over Na₂SO₄and evaporated. The

residue was puriﬁed by column chromatography (acetone: n-hexane

=1:5/n-hexane: ethyl acetate =2:1) to yield 57b (27.8 mg, 0.0346

tert-Butyl O-(tert-butoxy(4-(hydroxymethyl)phenoxy)-

phosphoryl)-N-(tert-butoxy-carbonyl)-L-serinate (56c). Compound

tert-butyl O-(tert-butoxy(4-(((tert-butyldimethylsilyl)oxy)methyl)-

phenoxy)- phosphoryl)-N-(tert-butoxycarbonyl)-L-serinate 54c

(107.2 mg, 0.1735 mmol) was dissolved in THF (1 mL) and

hydrogen ﬂuoride-pyridine complex (119.7 μL) in pyridine (294.7 μL)

was added to the solution above. This reaction mixture was stirred at

room temperature under Ar atmosphere for 100 min. After 100 min,

the reaction mixture was quenched by water (10 mL). Water layer was

extracted with CH₂Cl₂(8 mLX2), combined organic layer was washed

with brine, dried over MgSO₄and solvent was evaporated. The residue

was puriﬁed by column chromatography (n-hexane: ethyl acetate =2:1

to 1:4) to yield 55c (67.3 mg, 0.1337 mmol, 77%, colorless oil).

¹H NMR (CDCl₃, 400 MHz): 7.293−7.266 (2H, m), 7.141−7.103

(2H, m), 5.462−5.388 (1H, m), 4.603 (2H, s), 4.447−4.266 (3H, m),

2.641 (1H, brs), 1.488−1.471 (9H, m), 1.426−1.380 (18H, m). ¹³C

NMR (CDCl₃, 100 MHz): 168.18, 155.20,1150.06, 129.99, 137.86,

128.20, 119.99, 119.94, 85.15, 85.09, 85.02, 82.84, 82.82, 80.00, 68.12,

68.07, 68.02, 64.35, 54.38, 54.34, 54.29, 30.89, 29.79, 29.78, 29.75,

29.73, 28.29, 27.91, 27.89. ³¹P NMR (CDCl₃, 161 MHz): −11.18,

−11.45. HRMS (ESI-TOF [M + Na]⁺): Calcd for C₂₃H₃₈NNaO₉P⁺:

526.2176. Found 526.2189. Anal. Calcd for C₂₃H₃₈NO₉P; C, 54.86;

H,7.61; N, 2.78. Found C, 54.91; H, 7.42; N, 2.84.

tert-Butyl O-(tert-butoxy(2-(((3-(2-(undecyloxy)phenyl)-

propanoyl)oxy)methyl)phenoxy)-phosphoryl)-N-(tert-butoxycar-

bonyl)-L-serinate (57a). Compound 40 (177.4 mg, 0.3928 mmol) and

56a (128.9 mg, 0.3022 mmol) was dissolved in CH₂Cl₂(2 mL) and

toluene (0.2 mL) and coevaporated. The residue was dissolved in

CH₂Cl₂(1 mL) and 1H-tetrazole (63.5 mg, 0.9065 mmol) in THF (1

mL) was added to the solution above. The whole was stirred at room

temperature under Ar atmosphere for 12 h. After 12 h, the reaction

mixture was quenched by saturated NaHCO₃(aq) (10 mL) and

extracted with CH₂Cl₂(8 mL × 3). Combined organic layer was

washed with brine, dried over MgSO₄and evaporated. The residue was

puriﬁed by column chromatography (n-hexane: ethyl acetate =4:1 to

yield crude trivalent phosphodiester compound (200.2 mg, 0.2534

mmol, 84%, colorless oil).

1

mmol, 30%/sm recovery 6.2 mg, 15%/ mixture 1.9 mg). H NMR

(CDCl₃, 400 MHz): 7.247−7.206 (m, 1H), 7.115−7.019 (m, 5H),

6.783−6.736 (m, 2H), 5.400−5.304 (m, 1H), 5.013−5.008 (m, 2H),

4.410−4.208 (m, 3H), 3.894−3.861 (m, 2H), 2.910−2.871 (m, 2H),

2.628−2.590 (m, 2H), 1.771−1.680 (m, 2H), 1.449−1.433 (m, 9H),

1.374−1.361 (m, 20H), 1.308−1.191 (m, 14H), 0.824−0.789 (m,

3H). ¹³C NMR (CDCl₃, 100 MHz): 173.08, 168.17, 156.96, 155.20,

150.90, 150.84, 138.07, 129.95, 129.75, 128.75, 127.55, 124.37, 120.18,

119.70, 119.65, 119.60, 111.0085.10, 82.81, 79.99, 68.07, 67.97, 67.76,

65.36, 54.39, 34.11, 31.91, 29.81, 29.77, 29.62, 29.59, 29.36, 29.34,

29.31, 28.30, 27.93, 27.90, 26.16, 26.13, 25.61, 22.68, 14.12. ³¹P NMR

(CDCl₃, 161 MHz): −11.20, −11.50. HRMS (ESI-TOF [M + Na]⁺):

Calcd for C₄₃H₆₈NNaO₁₁P⁺828.4422. Found 828.4431. Anal. Calcd

for C₄₃H₆₈NO₁₁P; C, 64.08; H, 8.50; N, 1.74. Found: C, 64.01; H,

8.44; N, 1.72.

tert-Butyl O-(tert-butoxy(4-(((3-(2-(undecyloxy)phenyl)-

propanoyl)oxy)methyl) phenoxy)phosphoryl)-N-(tert-butoxycar-

bonyl)-L-serinate (57c). Compound 56c (66.7 mg, 0.1325 mmol)

and carboxylic acid (B) (55.1 mg, 0.1722 mmol) were dissolved in

CH₂Cl₂(0.8 mL) and EDCI (38.1 mg, 0.1987 mmol) and N,N-

dimthylaminopyridine (8.1 mg, 0.0662 mmol) were added to the

solution above. The reaction mixture was stirred at room temperature

under Ar atmosphere for 1.5 h. After 1.5 h, the reaction mixture was

quenched by water (7 mL). Water layer was extracted with CH₂Cl₂(8

mL × 2), combined organic layer was washed with brine, dried over

MgSO₄and solvent was evaporated. The residue was puriﬁed by

column chromatography (n-hexane: ethyl acetate =4:1 to 2:1) to yield

57c (42.1 mg, 0.0552 mmol, 39%, colorless oil, 24.7 mg (mixture)).

¹H NMR (CDCl₃, 400 MHz): 7.271−7.242 (2H, m), 7.18407.098

(4H, m), 6.848−6.802 (2H, m), 5.460−5.384 (1H, m), 5.053 (2H, s),

4.478−4.299 (3H, m), 3.943 (2H, t, J = 6.4 Hz), 2.950 (2H, t, J = 7.6

Hz), 2.660 (2H, t, J = 7.6 Hz), 1.816−1.746 (2H, m), 1.520−1.503

(9H, m), 1.483−1.432 (20H, m), 1.354−1.260 (14H, m), 0.891−

0.857 (3H, m). ¹³C NMR (CDCl₃, 100 MHz): 173.15, 168.17, 156.97,

155.18, 150.66, 150.58, 132.75, 129.96, 129.60, 128.74, 127.56, 120.17,

120.07,10.04, 119.99, 111.00, 85.17, 82.83, 79.98, 68.10, 67.74, 65.34,

54.40, 43.16, 31.91, 29.82, 29.81, 29.78, 29.76, 29.61, 29.59, 29.35,

29.34, 29.31, 28.31, 27.93, 27.91, 26.23, 26.13, 22.68, 14.12. ³¹P NMR

(CDCl₃, 161 MHz): −11.14, −11.46. HRMS (ESI-TOF [M + Na]⁺):

Calcd for C₄₃H₆₈NNaO₁₁P⁺: 828.4422. Found 828.4418. Anal. Calcd

for C₄₃H₆₈NNaO₁₁P; C, 62.83; H, 8.50; N, 1.70. Found C, 62.93; H,

8.15; N, 1.73.

The trivalent phosphodiester compound (200.1 mg, 0.2533 mmol)

was dissolved in CH₂Cl₂(2 mL) and added tert-butylhydroperoxide in

decane (0.1013 mL). The reaction mixture was stirred at room

temperature under Ar atmosphere for 1.5 h. After 1.5 h, solvent was

removed under vacuum and the residue was puriﬁed by column

chromatography (n-hexane: ethyl acetate =4:1 (200 mL) to 2:1(240

mL)) to yield 57a (132.7 mg, 0.1671 mmol, 66%, colorless oil, 55%

(2steps)).

(2R,3R)-2-(((tert-Butyldimethylsilyl)oxy)methyl)tetrahydro-2H-

pyran-3-yl-3-(2-((3-(4-chlorophenoxy)benzyl)oxy)phenyl)-

propanoate (60c). Compound 39b (61.5 mg, 0.2497 mmol) and 79b

(95.6 mg, 0.2497 mmol) were dissolved in CH₂Cl₂(1 mL) and

diisopropylcarbodiimide (41.0 mg, 0.3246 mmol) and N,N-dimthyla-

minopyridine (9.1 mg, 0.0749 mmol) were added to the solution

above. The reaction mixture was stirred at room temperature under Ar

atmosphere for 22 h. After 22 h, the reaction mixture was evaporated

under vacuum to remove solvent. The residue was puriﬁed by column

chromatography (n-hexane: ethyl acetate =10:1 (220 mL) to 8:1 (90

mL) to 4:1 (100 mL) to 2:1 (150 mL)) to yield 60c (108.4 mg, 0.1773

mmol, 71%, colorless oil, and recovered starting material 39b: 23.0

mg).

¹H NMR (CDCl₃, 400 MHz): 7.306−7.169 (m, 3H), 7.102−7.027

(m, 3H), 6.767−6.726 (m, 2H), 5.458−5.320 (m, 1H), 5.144−5.135

(m, 2H), 4.408−4.201 (m, 3H), 3.886−3.853 (m, 2H), 2.916−2.877

(m, 2H), 2.633−2.594 (m, 2H), 1.744−1.674 (m, 2H), 1.433−1.416

(m, 9H), 1.371−1.345 (m, 19H), 1.255−1.155 (m, 15H), 0.799 (t,

3H, J = 6.8 Hz). ¹³C NMR (CDCl₃, 100 MHz): 172.99, 172.98,

168.16, 168.13, 156.95, 155.18, 148.69, 148.63, 129.96, 129.54, 129.50,

129.22, 128.79, 127.52, 127.43, 127.36, 124.93, 120.19, 119.88, 110.98,

85.28, 85.21, 82.77, 79 90, 77.41, 77.09, 76.77, 68.20, 68.14, 67.73,

60.90, 54.45, 54.39, 54.31, 34.12, 31.90, 29.79, 29.75, 29.61, 29.58,

29.36, 29.34, 29.32, 28.29, 27.92, 27.88, 26.16, 26.13, 22.67, 14.12. ³¹P

NMR (CDCl₃, 161 MHz): −11.03, −11.34. HRMS (ESI-TOF [M +

Na]⁺): Calcd for C₄₃H₆₈NNaO₁₁P⁺828.4422. Found 828.4421. Anal.

Calcd for C₄₃H₆₈NO₁₁P + 0.1CH₂Cl₂; C, 63.58; H, 8.38; N, 1.72.

Found C, 63.80; H, 8.40; N, 1.64.

tert-Butyl O-(tert-butoxy(3-(((3-(2-(undecyloxy)phenyl)-

propanoyl)oxy)methyl)phenoxy)-phosphoryl)-N-(tert-butoxycar-

bonyl)-L-serinate (57b). Compound 56b (57.6 mg, 0.1144 mmol)

and carboxylic acid derivative (B) (40.3 mg, 0.1258 mmol) in CH₂Cl₂

(1 mL) was added EDCI (26.3 mg, 0.1373 mmol) and DMAP (2.8

mg, 0.0229 mmol) and stirred at room temperature under Ar for 6 h.

After 6 h, the reaction mixture was diluted with CH₂Cl₂(8 mL) and

quenched by water (10 mL). The whole was extracted with CH₂Cl₂(8

¹H NMR (CDCl₃, 400 MHz): 7.362 (t, 1H, J = 8.0 Hz), 7.316−

7.276 (m, 2H), 7.221−7.183 (m, 2H), 7.168−7.144 (m, 1H), 7.061

(m, 1H), 6.978−6.846 (m, 5H), 5.076 (s, 2H), 4.995 (m, 1H), 4.007−

3.969 (m, 1H), 3.620−3.526 (m, 2H), 3.516−3.446 (m, 2H), 3.034−

2.995 (m, 2H), 2.751−2.625 (m, 2H), 2.049−1.995 (m, 1H), 1.816−

1.699 (m, 1H), 1.681−1.597 (m, 1H), 1.353−1.319 (m, 1H), 0.866 (s,

9H), 0.016, −0.003 (m, 6H). ¹³C NMR (CDCl₃, 100 MHz): 172.66,

157.27, 156.35, 155.69, 139.55, 130.15, 130.10, 129.79, 129.14, 128.45,

127.57, 122.00, 121.58, 120.90, 120.32, 117.98, 117.32, 117.23, 111.59,

78.65, 69.27, 68.06, 66.70, 62.11, 34.25, 27.50, 26.18, 25.86, 20.86,

18.22, 17.89. HRMS (ESI-TOF [M + Na]⁺): Calcd for

C₃₄H₄₃ClNaO₆Si⁺633.2410, Found 633.2418. Anal. Calcd for

X

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Article

C₃₄H₄₃ClO₆Si; C, 66.81; H, 7.09; N, 0.00; Found C, 66.63; H, 7.05; N,

0.00.

Hz), 2.447 (bs, 1H), 2.347 (s, 3H), 1.952−1.799 (m, 2H), 1.727−

1.638 (m, 1H), 1.423−1.370 (m, 1H).

(2R,3R)-2-(((tert-Butyldimethylsilyl)oxy)methyl)tetrahydro-2H-

pyran-3-yl- 3-(2-((3-(p-tolyloxy)benzyl)oxy)phenyl)propanoate

(60d). Compound 39b (73.4 mg, 0.2979 mmol) and 79c (108.0

mg, 0.2979 mmol) were dissolved in CH₂Cl₂(1.3 mL) and

diisopropylcarbodiimide (48.9 mg, 0.3872 mmol) and N,N-dimthyla-

minopyridine (10.9 mg, 0.0894 mmol) were added to the solution

above. The reaction mixture was stirred at room temperature under Ar

atmosphere for 24 h. After 24 h, the reaction mixture was evaporated

under vacuum to remove solvent. The residue was puriﬁed by column

chromatography (n-hexane: ethyl acetate =10:1 (220 mL) to 8:1 (90

mL) to 6:1 (70 mL) to 4:1 (100 mL) to 2:1 (120 mL)) to yield 60d

(116.2 mg, 0.1967 mmol, 71%, colorless oil) and the recovered starting

material 39b (18.8 mg).

¹³C NMR (CDCl₃, 400 MHz): 173.76, 158.22, 156.45, 154.41,

139.19, 133.18, 130.32, 130.14, 129.87, 128.84, 127.72, 121.19, 120.82,

119.39, 117.41, 116.65, 111.64, 78.50, 69.40, 68.07, 67.04, 61.97,

34.19, 27.83, 26.29, 20.84, 20.74. HRMS (ESI-TOF [M + Na]⁺):

+

Calcd for C₂₉H₃₂NaO₆499.2091. Found 499.2089. Anal. Calcd for

C₂₉H₃₂O₆+ 0.2CH₂Cl₂; C, 71.10; H, 6.57; N, 0.00. Found C, 70.71;

H, 6.76; N, 0.00.

tert-Butyl O-(tert-butoxy(((2R,3R)-3-((3-(2-((3-(4-chlorophenoxy)-

benzyl)oxy)phenyl)- propanoyl)oxy)tetrahydro-2H-pyran-2-yl)-

methoxy)phosphoryl)-N-(tert-butoxycarbonyl)-L-serinate (66c).

Compound 40 (52.3 mg, 0.1158 mmol) and 63c (86.3 mg, 0.1736

mmol) were dissolved in CH₂Cl₂(1 mL) and toluene (0.1 mL) and

coevaporated. The residue was dissolved in CH₂Cl₂(1 mL) and 1H-

tetrazole (16.2 mg, 0.2315 mmol) in THF (1 mL) was added to the

above solution. The whole was stirred at room temperature under Ar

atmosphere for 23 h. After 23 h, the reaction mixture was quenched by

saturated aqueous NaHCO₃(7 mL) and extracted with CH₂Cl₂(8

mLX2). Combined organic layer was washed with brine, dried over

MgSO₄and evaporated. The residue was puriﬁed by column

chromatography (n-hexane: ethyl acetate =4:1 (150 mL) to 2:1 (120

mL) to 1:1 (140 mL)) to yield crude trivalent phosphodiester

compound (37.5 mg, 0.0438 mmol, 38%, colorless oil).

The trivalent phosphodiester compound (37.4 mg, 0.0437 mmol)

was dissolved in CH₂Cl₂(0.8 mL) and added tert-butylhydroperoxide

in decane (0.0873 mL). The reaction mixture was stirred at room

temperature under Ar atmosphere for 1 h. After 1 h, solvent was

removed under vacuum and the residue was puriﬁed by column

chromatography (n-hexane: ethyl acetate =2:1 (270 mL) to 1:1 (160

mL)) to yield 66c (30.5 mg, 0.0350 mmol, 80%, colorless oil, 30%

(2steps)).

¹H NMR (CDCl₃, 400 MHz): 7.336 (t, 1H, J = 8.0 Hz), 7.219−

7.151 (m, 5H), 7.053−7.048 (m, 1H), 6.969−6.855 (m, 5H), 5.067 (s,

2H), 5.005 (m, 1H), 4.016−3.977 (m, 1H), 3.630−3.546 (m, 2H),

3.524−3.452 (m, 2H), 3.022 (t, 2H, J = 7.6 Hz), 2.762−2.634 (m,

2H), 2.357 (s, 3H), 2.059−2.006 (M, 1H), 1.813−1.707 (m, 1H),

1.687−1.602 (m, 1H), 1.358−1.325 (m, 1H), 0.878 (s, 9H), 0.028−

0.009 (m, 6H). ¹³C NMR (CDCl₃, 100 MHz): 172.72, 158.21, 156.45,

154.44, 139.25, 133.13, 130.31, 130.13, 129.89, 129.14, 127.57, 121.19,

120.81, 119.40, 117.39, 116.63, 111.59, 78.66, 69.39, 68.07, 66.68,

62.12, 34.26, 27.51, 26.23, 25.87, 20.86, 20.75, 18.23. HRMS (ESI-

TOF [M + Na]⁺): Calcd for C₃₅H₄₆NaO₆Si⁺613.2956. Found

613.2958. Anal. Calcd for C₃₅H₄₆O₆Si; C, 71.15; H, 7.85; N, 0.00;

Found C, 70.86; H, 7.90; N, 0.00.

(2R,3R)-2-(Hydroxymethyl)tetrahydro-2H-pyran-3-yl-3-(2-((3-(4-

chlorophenoxy)benzyl) oxy)phenyl)propanoate (63c). Compound

60c (103.6 mg, 0.1695 mmol) was dissolved in THF (1 mL) and

hydrogen ﬂuoride-pyridine complex (100.5 μL) in pyridine (247.9 μL)

was added to the solution above. This reaction mixture was stirred at

room temperature under Ar atmosphere for 6.5 h. After 6.5 h, the

reaction mixture was quenched by water (10 mL). Water layer was

extracted with CH₂Cl₂(10 mLX2), combined organic layer was

washed with brine, dried over MgSO₄and solvent was evaporated. The

residue was puriﬁed by column chromatography (n-hexane: ethyl

acetate =2:1 (180 mL) to 1:1 (100 mL) to 1:2 (180 mL)) to yield 63c

(84.0 mg, 0.1690 mmol, 99%, colorless oil).

¹H NMR (CDCl₃, 400 MHz): 7.287 (t, 1H, J = 8.0 Hz), 7.236−

7.192 (m, 2H), 7.136−7.064 (m, 3H), 6.972 (m, 1H), 6.895−6.835

(m, 3H), 6.816−6.767 (m, 2H), 5.483−5.464 (m, 1H), 5.005 (s, 2H),

4.829−4.823 (m, 1H), 4.282−4.207 (m, 2H), 4.156−4.108 (m, 1H),

3.943−3.897 (m, 1H), 3.877−3.785 (m, 2H), 3.638−3.582 (m, 1H),

3.432−3.364 (m, 1H), 2.939−2.862 (m, 2H), 2.671−2.540 (m, 2H),

1.900−1.865 (m, 1H), 1.722−1.524 (m, 2H), 1.391−1.366 (m, 27H),

1.281−1.230 (m, 1H). ¹³C NMR (CDCl₃, 100 MHz): 172.63, 168.39,

157.24, 156.31, 155.66, 155.32, 139.53, 130.10, 129.77, 128.92, 128.43,

127.61, 121.96, 120.89, 129.30, 117.94, 117.18, 111.62, 83.93, 82.60,

82.58, 78.88, 76.50, 69.26, 67.82, 67.75, 67.52, 66.84, 66.74, 54.41,

34.10, 29.79, 29.75, 29.71, 28.33, 27.95, 27,48, 27.42, 26.11, 20.47. ³¹P

NMR (CDCl₃, 161 MHz): −5.50, −5.78 HRMS (ESI-TOF [M +

Na]⁺): Calcd for C₄₄H₅₉ClNNaO₁₃P⁺898.3305, Found 898.3312.

Anal. Calcd for C₄₄H₅₉ClNO₁₃P + 0.6CH₂Cl₂; C, 57.83; H, 6.51; N,

1.51. Found: C, 57.80; H, 6.49; N, 1.57.

tert-Butyl O-(tert-butoxy(((2R,3R)-3-((3-(2-((3-(p-tolyloxy)benzyl)-

oxy)phenyl)-propanoyl)oxy)tetrahydro-2H-pyran-2-yl)methoxy)-

phosphoryl)-N-(tert-butoxycarbonyl)-L-serinate (66d). Compound

40 (46.5 mg, 0.1030 mmol) and 63d (73.6 mg, 0.1545 mmol) was

dissolved in CH₂Cl₂(1 mL) and toluene (0.1 mL) and coevaporated.

The residue was dissolved in CH₂Cl₂(0.5 mL) and 1H-tetrazole(14.4

mg, 0.2059 mmol) in THF (1 mL) was added to the solution above.

The whole was stirred at room temperature under Ar atmosphere for

24 h. After 24 h, the reaction mixture was quenched by saturated

NaHCO₃(aq) (6 mL) and extracted with CH₂Cl₂(8 mL × 3).

Combined organic layer was washed with brine, dried over MgSO₄and

evaporated. The residue was puriﬁed by column chromatography

(hexane: ethyl acetate =4:1 (150 mL) to 2:1 (150 mL) to 1:1 (160

mL)) to yield crude trivalent phosphodiester compound (17.7 mg,

0.0212 mmol, 21%, colorless oil).

¹H NMR (CDCl₃, 400 MHz): 7.359 (t, 1H, J = 8.0 Hz), 7.309−

7.259 (m, 2H), 7.206−7.153 (m, 3H), 7.069−7.064 (m, 1H), 6.973−

6.852 (m, 5H), 5.067 (s, 2H), 4.944−4.936 (m, 1H), 4.027−3.987 (m,

1H), 3.532−3.461 (m, 3H), 3.314−3.290 (m, 1H), 3.003 (t, 2H, J =

7.6 Hz), 2.704 (t, 2H, J = 8.0 Hz), 2.474 (bs, 1H), 1.946−1.791 (m,

2H), 1.724−1.634 (m, 1H), 1.416−1.366 (m, 1H). ¹³C NMR (CDCl₃,

400 MHz): 173.68, 157.28, 156.35, 155.66, 139.49, 130.15, 130.10,

129.80, 128.83, 128.46, 127.73, 122.00, 120.91, 120.33, 118.00, 117.22,

111.63, 78.51, 69.26, 68.06, 67.06, 61.9834.18, 27.82, 26.24, 20.84.

+

HRMS (ESI-TOF [M + Na]⁺): Calcd for C₂₈H₂₉FNaO₆519.1545,

Found 519.1534. Anal. Calcd for C₂₈H₂₉ClO₆+ 0.25CH₂Cl₂; C, 65.57;

H, 5.71; N, 0.00. Found C, 65.57; H, 5.83; N, 0.00.

(2R,3R)-2-(Hydroxymethyl)tetrahydro-2H-pyran-3-yl- 3-(2-((3-(p-

tolyloxy)benzyl)oxy)phenyl)propanoate (63d). Compound 60d

(110.9 mg, 0.1877 mmol) was dissolved in THF (1.) and hydrogen

ﬂuoride-pyridine complex (110.7 μL) in pyridine (274.5 μL) was

added to the solution above. This reaction mixture was stirred at room

temperature under Ar atmosphere for 19 h. After 19 h, the reaction

mixture was quenched by water (10 mL). Water layer was extracted

with CH₂Cl₂(10 mLX2), combined organic layer was washed with

brine, dried over MgSO₄and solvent was evaporated. The residue was

puriﬁed by column chromatography (n-hexane: ethyl acetate =2:1

(180 mL) to 1:1 (120 mL) to 1:2 (120 mL)) to yield 63d (57.0 mg,

0.1196 mmol, 64%, colorless oil/29.2 mg (small amount of impurity

was remained), 0.0613 mmol, 33%, colorless oil).

The trivalent phosphodiester compound (17.7 mg, 0.0212 mmol)

was dissolved in CH₂Cl₂(0.4 mL) and added tert-butylhydroperoxide

in decane (0.0423 mL). The reaction mixture was stirred at room

temperature under Ar atmosphere for 1.5 h. After 1.5 h, solvent was

removed under vacuum and the residue was puriﬁed by column

chromatography (hexane:ethyl acetate =2:1 (180 mL) to 1:1 (200

¹H NMR (CDCl₃, 400 MHz): 7.349−7.310 (m, 1H), 7.201−7.142

(m, 5H), 7.056−7,051 (m, 1H), 6.959−6.862 (m, 5H), 5.058 (s, 2H),

4.951−4.944 (m, 1H), 4.033−3.994 (m, 1H), 3.574−3.465 (m, 3H),

3.349−3.264 (m, 1H), 3.007 (t, 2H, J = 7.6 Hz), 2.710 (t, 2H, J = 7.6

Y

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mL) to 1:2 (120 mL)) to yield 66d (15.5 mg, 0.0182 mmol, 86%,

colorless oil, 29%(2steps)).

DAPI, 4′,6-diamidino-2-phenylindole; Mp, melting point;

EDCI, N-(3-(dimethylamino)propyl)-N-ethylcarbodiimide hy-

drochloride; HOBt·H₂O, 1-hydroxybenzotriazole hydrate;

TBAI, tetrabutylammonium iodide; DIPEA, N,N-diisopropyle-

thylamine

¹H NMR (CDCl₃, 400 MHz): 7.316 (t, 1H, J = 8.0 Hz), 7.177−

7.130 (m, 5H), 7.018 (m, 1H), 6.939−6.837 (m, 5H), 5.553−5.524

(m, 1H), 5.057 (s, 2H), 4.901−4.895 (m, 1H), 4.352−4.225 (m, 2H),

4.215−4.177 (m, 1H), 4.011−3.964 (m, 1H), 3.945−3.854 (m, 2H),

3.706−3.663 (m, 1H), 3.497−3.430 (m, 1H), 3.022−2.930 (m, 2H),

2.743−2.608 (m, 2H), 2.336 (s, 3H), 1.970−1.936 (m, 1H), 1.791−

1.593 (m, 2H), 1.560−1.436 (m, 27H), 1.387−1.274 (m, 1H).

¹³C NMR (CDCl₃, 100 MHz): 172.67, 168.40, 158.16, 156.40,

155.22, 154.40, 139.23, 133.14, 130.29, 130.07, 129.93, 127.60, 121.14,

120.80, 119.35, 117.35, 116.59, 111.62, 82.60, 82.57, 79.89, 76.50,

69.38, 67.82, 67.75, 67.51, 66.82, 66.71, 66.45, 54.42, 34.10, 29.79,

29.75, 29.71, 28.33, 27.95, 27.49, 27.43, 26.15, 20.72, 20.47. ³¹P NMR

(CDCl₃, 161 MHz): δ −5.50, −5.79. HRMS (ESI-TOF [M + Na]⁺):

Calcd for C₄₅H₆₂NNaO₁₃P⁺878.3851. Found 878.3847. Anal. Calcd

for C₄₅H₆₂NO₁₃P + 1.2H₂O; C, 61.59 ; H, 7.40 ; N, 1.60. Found C,

61.22; H, 7.05; N, 1.58.

REFERENCES

■

(1) Aoki, J.; Sato, T.; Nagai, Y.; Dohmae, N.; Takio, K.; Doi, T.; Arai,

H.; Inoue, K. Serine phospholipid-specific phospholipase A that is

secreted from activated platelets: a new member of the lipase family. J.

Biol. Chem. 1997, 272, 2192−2198.

(2) Martin, T. W.; Lagunoff, D. Interactions of lysophospholipids and

mast cells. Nature 1979, 279, 250−252.

(3) Smith, G. A.; Hesketh, T. R.; Plumb, R. W.; Metcalfe, J. C. The

exogenous lipid requirement for histamine relaease from rat peritoneal

mast cells stimulated by concanavalin A. FEBS Lett. 1979, 105, 58−62.

(4) Horigome, K.; Tamori-Natori, Y.; Inoue, K.; Nojima, S. Effect of

serine phospholipid structure on the enhancement of concanavalin A-

induced degranulation in rat mast cells. J. Biochem. 1986, 100, 571−

579.

(5) Bruni, A.; Bigon, E.; Battistella, A.; Boarato, E.l; Mietto, L.;

Toffano, G. Lysophosphatidylserine as histamine releaser in mice and

rats. Agents Actions 1984, 14, 619−625.

(6) Lourenssen, S.; Blennerhassett, M. G. Lysophosphatidylserine

potentiates nerve growth factor-induced differentiation of PC12 cells.

Neurosci. Lett. 1998, 248, 77−80.

(7) Bellini, F.; Bruni, A. Role of a serum phospholipase A₁in the

phosphatidylserine-induced T cell inhibition. FEBS Lett. 1993, 316, 1−

4.

(8) Lee, S. Y.; Lee, H. Y.; Kim, S. D.; Jo, S. H.; Shim, J. W.; Lee, H. J.;

Yun, J.; Bae, Y. S. Lysophosphatidylserine stimulates chemotactic

migration in U87 human glioma cells. Biochem. Biophys. Res. Commun.

2008, 374, 147−151.

(9) Park, K. S.; Lee, H. Y.; Kim, M. K.; Shin, E. H.; Jo, S. H.; Kim, S.

D.; Im, D. S.; Bae, Y. S. Lysophosphatidylserine stimulates L2071

mouse fibroblast chemotactic migration via a process involving

pertussis toxin-sensitive trimeric G-proteins. Mol. Pharmacol. 2006,

69, 1066−1073.

(10) Frasch, S. C.; Zemski-Berry, K.; Murphy, R. C.; Borregaard, N.;

Henson, P. M.; Bratton, D. L. Lysophospholipids of different classes

mobilize neutrophil secretory vesicles and induce redundant signaling

through G2A. J. Immunol. 2007, 178, 6540−6548.

(11) Inoue, A.; Ishiguro, J.; Kitamura, H.; Arima, N.; Okutani, M.;

Shuto, A.; Higashiyama, S.; Ohwada, T.; Arai, H.; Makide, K.; Aoki, J.

TGF alpha shedding assay: an accurate and versatile method for

detecting GPCR activation. Nat. Methods 2012, 9, 1021−1029.

(12) Kitamura, H.; Makide, K.; Shuto, A.; Ikubo, M.; Inoue, A.;

Suzuki, K.; Sato, Y.; Nakamura, S.; Otani, Y.; Ohwada, T.; Aoki, J.

GPR34 is a receptor for lysophosphatidylserine with a fatty acid at the

sn-2 position. J. Biochem. 2012, 151, 511−518.

ASSOCIATED CONTENT

* Supporting Information

■

S

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.5b01925.

Details of synthesis of nonaromatic LysoPS analogues

and that of aromatic LysoPS analogues, and Figures S1−

S8. (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Design and Chemical study: For T.O.: phone, +81-3-5841-

4730; fax, +81-3-5841-4735; E-mail, ohwada@mol.f.u-tokyo.ac.

■

jp.

*Biological study: For J.A.: phone, +81-22-795-6860; fax, +81-

22-795-6859; E-mail, jaoki@m.tohoku.ac.jp. Tohoku Univer-

sity.

Author Contributions

S.J. and A.I. contributed equally to this work. S. J., S. N., M. S.,

M. I., Y. O. and T. O. (The University of Tokyo) performed

chemical studies including design and synthesis of compounds.

S. J. studied solution structures of the model compounds and

performed calculational studies. A. I., A. U., T. K., T. K., K. K.,

K. M. and J. A. (Tohoku University) performed biological

studies including TGFα shedding assay, Ca²⁺assay, and actin

stress ﬁber formation assay.

Notes

The authors declare no competing ﬁnancial interest.

(13) Sugo, T.; Tachimoto, H.; Chikatsu, T.; Murakami, Y.; Kikukawa,

Y.; Sato, S.; Kikuchi, K.; Nagi, T.; Harada, M.; Ogi, K.; Ebisawa, M.;

Mori, M. Identification of a lysophosphatidylserine receptor on mast

cells. Biochem. Biophys. Res. Commun. 2006, 341, 1078−1087.

(14) (Review) Makide, K.; Uwamizu, A.; Shinjo, Y.; Ishiguro, J.;

Okutani, M.; Inoue, A.; Aoki, J. Lysophospholipids and their receptors:

novel lysophosphoplipid receptors: their structure and function. J.

Lipid Res. 2014, 55, 1986−1995.

ACKNOWLEDGMENTS

■

This research was supported by grants from the Ministry of

Education, Science, Sports, and Culture of Japan to J.A. and

T.O. A.I. and K.M were funded by PRESTO (Precursory

Research for Embryonic Science and Technology) from Japan

Science and Technology Agency (JST), and J.A. was funded by

AMED-CREST, AMED.

(15) Liebscher, I.; Muller, U.; Teupser, D.; Engemaier, E.; Engel, K.

̈

M. Y.; Ritscher, L.; Thor, D.; Sangkuhl, K.; Ricken, A.; Wurm, A.;

Piehler, D.; Schmutzler, S.; Fuhrmann, H.; Alber, F. W.; Reichenbach,

ABBREVIATIONS USED

■

A.; Thiery, J.; Schoneberg, T.; Schulz, A. Altered immune response in

̈

LysoPS, lysophosphatidylserine; LPS, lysophosphatidylserine

receptor; PS, phosphatidylserine; TGFα, transforming growth

factor-alpha; AP-TGFα, alkaline phosphatase-tagged TGFα;

HBSS, Hanks’ balanced salt solution; HEPES, 4-(2-

hydroxyethyl)piperazine-1-ethanesulfonic acid; CM, condi-

tioned media; DMEM, Dulbecco’s modiﬁed Eagle’s medium;

mice deficient for the G protein-coupled reaceptor GPR34. J. Biol.

Chem. 2011, 286, 2101−2110.

(16) Chu, X.; Shen, M.; Xie, F.; Miao, X. J.; Shou, W. H.; Liu, L.;

Yang, P. P.; Bai, Y. N.; Zhang, K. Y.; Yang, L.; Hua, Q.; Liu, W. D.;

Dong, Y.; Wang, H. F.; Shi, J. X.; Wang, Y.; Song, H. D.; Chen, S. J.;

Chen, Z.; Huang, W. An X chromosome-wide association analysis

Z

DOI: 10.1021/acs.jmedchem.5b01925

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

identifies variants in GPR174 as a risk factor for Graves’ disease. J.

Med. Genet. 2013, 50, 479−485.

(17) Barnes, M. J.; Li, C.-M.; Xu, Y.; An, J.; Huang, Y.; Cyster, J. G.

The lysophosphatidylserine receptpr GPR174 constrains regulatory T

cell development and function. J. Exp. Med. 2015, 212, 1011−1020.

(18) Uwamizu, A.; Inoue, A.; Suzuki, K.; Okudaira, M.; Shuto, A.;

Shinjo, Y.; Ishiguro, J.; Makide, K.; Ikubo, M.; Nakamura, S.; Jung, S.;

Sayama, M.; Otani, Y.; Ohwada, T.; Aoki, J. Lysophosphatidylserine

analogs differentially activate three LysoPS receptors. J. Biochem. 2015,

157, 151−160.

(19) Ikubo, M.; Inoue, A.; Nakamura, S.; Jung, S.; Sayama, M.; Otani,

Y.; Uwamizu, A.; Suzuki, K.; Kishi, T.; Shuto, A.; Ishiguro, J.;

Okudaira, M.; Kano, K.; Makide, K.; Aoki, J.; Ohwada, T. Structure-

activity relationships of lysophosphatidylserine analogs as agonists of

G-protein-coupled receptors GPR34, P2Y10, and GPR174. J. Med.

Chem. 2015, 58, 4204−4219.

(20) Dixon, E. A.; King, G. S. S.; Smithson, T. L.; Wieser, H. The

out-of plane ring deformations of 1,3-and 1,4-dioxene and 3,6-dihydro-

2h-pyran: comparison of a series of cyclohexenes. J. Mol. Struct. 1981,

71, 97−108.

(21) Wells, J. A.; Malloy, T. B., Jr. Microwave spectrum and dipole

moment of 3,6-dihydro-2H-pyran. J. Chem. Phys. 1974, 60, 3987−

3993.

(22) Nicolaou, K. C.; Hwang, C. K.; Marron, B. E.; DeFrees, S. A.;

Couladouros, E. A.; Abe, Y.; Carroll, P. J.; Snyder, J. P. Bridging of

macrodithionolactones to bicyclic systems. Synthesis and modeling of

oxapolycyclic frameworks. J. Am. Chem. Soc. 1990, 112, 3040−3054.

̌

̌ ́

(23) Snatzke, G.; Raza, Z.; Habus, I.; Sunjic, V. Chiropical properties

of tetrahydropyran-3,4-diols and 2-hydroxymethyltetrahydropyran-2-

ols derived fromL-arabinose, D-galactose, D-glucose, and D-xylose,

and enantioselectivity in reduction with their complexes. Carbohydr.

Res. 1988, 182, 179−196.

(24) Ehlert, F. J.; Griffin, M. T.; Sawyer, G. W.; Bailon, R. A simple

method for estimation of agonist activity at receptor subtypes:

comparison of native and cloned M3 muscarinic receptors in guinea

pig ileum and transfected cells. J. Pharmacol. Exp. Ther. 1999, 289,

981−992.

AA

DOI: 10.1021/acs.jmedchem.5b01925

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