Switching Lysophosphatidylserine G Protein-Coupled Receptor Agonists to Antagonists by Acylation of the Hydrophilic Serine Amine

Article abstract of DOI:10.1021/acs.jmedchem.1c00347

Three human G protein-coupled receptors (GPCRs)—GPR34/LPS₁, P2Y10/LPS₂, and GPR174/LPS₃—are activated specifically by lysophosphatidylserine (LysoPS), an endogenous hydrolysis product of a cell membrane component, phosphatidylserine (PS). LysoPS consists of-serine, glycerol, and fatty acid moieties connected by phosphodiester and ester linkages. We previously generated potent and selective GPCR agonists by modification of the three modules and the ester linkage. Here, we show that a novel modification of the hydrophilic serine moiety, that is, N-acylations of the serine amine, converted a GPR174 agonist to potent GPR174 antagonists. Structural exploration of the amide functionality provided access to a range of activities from agonist to partial agonist to antagonist. The present study would provide a new strategy for the development of lysophospholipid receptor antagonists.

Full text of DOI:10.1021/acs.jmedchem.1c00347

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Article
Switching Lysophosphatidylserine G Protein-Coupled Receptor
Agonists to Antagonists by Acylation of the Hydrophilic Serine
Amine
Misa Sayama, Akiharu Uwamizu, Masaya Ikubo, Luying Chen, Ge Yan, Yuko Otani, Asuka Inoue,
Junken Aoki,* and Tomohiko Ohwada*
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ABSTRACT: Three human G protein-coupled receptors
(GPCRs)GPR34/LPS1, P2Y10/LPS2, and GPR174/LPS3are
activated specifically by lysophosphatidylserine (LysoPS), an
endogenous hydrolysis product of a cell membrane component,
phosphatidylserine (PS). LysoPS consists of L-serine, glycerol, and
fatty acid moieties connected by phosphodiester and ester linkages.
We previously generated potent and selective GPCR agonists by
modification of the three modules and the ester linkage. Here, we
show that a novel modification of the hydrophilic serine moiety, that
is, N-acylations of the serine amine, converted a GPR174 agonist to
potent GPR174 antagonists. Structural exploration of the amide
functionality provided access to a range of activities from agonist to
partial agonist to antagonist. The present study would provide a new
strategy for the development of lysophospholipid receptor antagonists.
expressed in several immune organs, including spleen and lymph
nodes. GPR34 is also highly expressed in dorsal striatum. Studies
using knock-out (KO) mice and single nucleotide poly-
morphism (SNP) analyses of these LysoPS receptors have
revealed their roles in immunoregulation. For example, GPR34
activates the immune system to induce residence to Cryptococcus
neoformans infection,3 to produce pro-inflammatory cyto-
kines,3,4 and to promote phagocytosis5 and cellular migration.3,6
GPR174 is highly expressed in T cells and B cells7,8 and
negatively regulates their functions. An SNP of GPR174 is seen
in autoimmune diseases such as Graves’ disease9−12 and
Addison’s disease,13 and an altered expression of GPR174 is
also associated with vasovagal syncope.14 Recently, Shinjo et al.
and later Barnes and Cyster showed that LysoPS acts via
GPR174 to suppress interleukin-2 (IL-2) production in T
cells.8,15 Barnes et al. also showed that GPR174 is expressed on
regulatory T cells (Tregs), and GPR174 signaling suppresses
accumulation and activity of Tregs.7 Indeed, GPR174 KO mice
were less susceptible to the experimental autoimmune
INTRODUCTION
■
Lysophospholipids (LPLs) are amphiphilic compounds that
serve as signaling molecules (lipid mediators). Various LPLs are
generated by enzymatic hydrolysis of an ester linkage of
membrane phospholipids and are classified mainly by their
hydrophilic moiety; for example, lysophosphatidic acid (LPA)
has phosphoric acid as the hydrophilic head, while lysophos-
phatidylcholine (LPC) has phosphocholine. Among the LPLs,
lysophosphatidylserine (LysoPS) is an emerging lysophospho-
lipid mediator generated from phosphatidylserine (PS).
LysoPS is composed of three modules, L-serine, glycerol, and
fatty acid, and two linkages, phosphodiester and ester, which
connect the modules (Figure 1). It is distinct among LPLs in the
fact that it has an amino acid, the serine moiety, as a hydrophilic
head group. LysoPS specifically activates three G protein-
coupled receptors (GPCRs) in humans: GPR34/LPS1,1 P2Y10/
LPS2,2 and GPR174/LPS3.2 According to the BioGPS gene
database (http://biogps.org/), these three receptors are highly
Received: February 25, 2021
Published: July 7, 2021
Figure 1. Structure of LysoPS (18:1).
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encephalomyelitis than wild-type mice, and they suggested that
antagonists of GPR174 might be promising therapeutics for
autoimmune diseases.7 Qiu et al. showed that GPR174
deficiency was protective against sepsis in mice by altering the
levels of inflammatory agents, such as cytokines.16 Thus,
compounds that can modulate GPR174 activity, especially
GPR174 antagonists, might be useful both in biological research
and as candidate drugs for the control of autoimmune diseases,
cancer, and chronic/acute inflammation. The function of
P2Y10, compared to those of GPR34 and GPR174, is less
elucidated.
Our group has focused on identifying specific agonists,
antagonists, and inverse agonists that would be useful for
elucidating the receptor functions, in addition to being potential
starting points for drug discovery. Our previous structure−
activity relationship (SAR) studies showed that these LysoPS
receptors strictly recognize the serine moiety.17,18 Some
modification of the serine moiety is tolerated by GPR174 but
less so by GPR34 or P2Y10. For example, LysoPalloT, in which
the serine moiety is replaced with allo-threonine, shows
diminished activity for the activation of GPR34 and P2Y10,
but retains activity toward GPR174, and thus shows increased
subtype selectivity for GPR174.18 The carboxylic acid
functionality of the serine moiety was shown to be essential
for the activation of all LysoPS receptors because methyl
esterification and elimination of the carboxylic acid functionality
to afford a lysophosphatidylethanolamine derivative, which is
also an endogenous LPL, resulted in a marked decrease or loss of
activity toward all the LysoPS receptors.18 However, the
structure requirements for the hydrophilic module, especially
regarding modification of the amino group, are not yet fully
understood.
Figure 2. Molecular design of GPR174 antagonists.
tetraisopropylphosphorodiamidite (compound 3) using the
phosphoramidite method. The detailed synthetic route of each
hydrophilic head moiety (structure I) is described in
Experimental Section. We employed the procedure reported
by Aurelio et al.19 for the synthesis of the hydrophilic head of 7c
(Table 1), in which the serine amine is mono-methylated.
Two routes (A and B in Scheme 1), which differ in the order
of P−O bond formation in the coupling with hydrophilic head
moiety I and compound 2, are possible, and we chose one of
these two routes by considering the feasibility of product
purification. Thus, in route A, compound 3 is subjected to react
first with alcohol 2 to yield compound 4, followed by coupling
with the hydrophilic head moiety I (Scheme 1), or in route B,
the hydrophilic head moiety I first reacts with 3 to yield
intermediate II, and then II is coupled with 2 to afford III (II
and III are general structures with a various R group, Scheme 1).
Compound III was finally deprotected by hydrogenation to
yield the target LysoPS analogue (general structure IV). We
showed the synthesis of the derivative 7d through route A as a
representative example in Scheme 1: the hydrophilic head part 5
was synthesized by acylation of the amino group in L-serine
benzyl ester hydrochloride. Compound 5 was coupled with
phosphoramidite 4 to yield compound 6. Global deprotection of
6 by means of hydrogenation in THF afforded the final product
7d. In this hydrogenation process, THF was a better solvent than
conventional alcohol such as methanol because methanol
generated the methyl ester of the final carboxylic acid product,
probably due to increased intrinsic acidity of the carboxylic acid
of the target LysoPS analogue, due to the lack of the basic amino
group.
Discovery of a GPR174 Antagonist by α-Substitution
of the Serine Moiety. We evaluated the activities of the α-
substituted serine analogues by means of the previously
described transforming growth factor-α (TGFα) shedding
assay (Tables 1−4 and Figures 3, 4, and S1−S3).2 In this
assay, the GPCR activation level is evaluated as the level of the
ectodomain shedding of a membrane-bound preform of alkaline
phosphatase (AP)-tagged TGFα (AP-TGFα). Taking into
account the use of mice in future in vivo studies, mouse
GPR174 was used for the evaluation of LysoPS analogues.
LysoPS analogues evaluated in this study showed almost no
agonistic activity or very low activity toward mouse GPR34 or
P2Y10, as compared with endogenous pan-agonist LysoPS
(18:1) (data not shown), and we focus here on the activity
toward mouse GPR174.
In this study, we aimed to elucidate the effect of the amino
acid moiety, particularly the amino group, on the activities of
LysoPS analogues toward GPR174. Notably, our results indicate
that structural modifications at this location can switch the
activity of these analogues from agonistic to partial agonistic to
antagonistic.
RESULTS AND DISCUSSION
■
Molecular Design for Structural Change of the
Hydrophilic Moiety on GPR174 Agonists. Since GPR174
is the least susceptible to modification of the hydrophilic serine
part of the prototype LysoPS among the three LysoPS receptors,
we focused on the effect of structural change of the hydrophilic
moiety on GPR174 activation. We previously identified some
structural requirements favorable for GPR174 activation:18 (1)
conversion of the ester linkage between the glycerol and the fatty
acid moiety to an amide bond and (2) use of the meta-
substituted benzene system as a non-fatty acid surrogate. Thus,
we studied the SAR of analogues containing these two
modifications (Figure 2).
The synthetic schemes of the LysoPS analogues studied here
are shown in Scheme 1 and in Experimental Section. Functional
groups were protected with benzyl groups and removed by
hydrogenation in the final step. Since all the LysoPS analogues
studied here contain the original 2R-glycerol moiety, we
synthesized compound 2, which is a common building block
of the fatty acid and glycerol moiety of LysoPS analogues, in four
steps from the benzene-containing fatty acid surrogate 118 as
described previously.6 The phosphodiester linkage between
compound 2 and various hydrophilic head moieties (general
structure I in Scheme 1) was generated with benzyl N,N,N′,N′-
Compound 7a, which has the (S)-amino group like
endogenous LysoPS, was a highly active and subtype-selective
agonist toward GPR174, as expected (EC50: 1.6 nM) (Table 1
and Figures 3a and S2a). However, 7a showed a slight reduction
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Scheme 1. Synthesis of LysoPS Analogues
in Emax, the maximum TGFα shedding response (defined in
Figure 3) (Emax = 71.7 5.4%) compared with LysoPS (18:1)
(Emax = 100 0%) (Table 1). Next, we introduced an (S)-
methyl group (7b) instead of the amino group as the R group.
Methyl-substituted 7b showed partial agonistic activity with
greatly reduced Emax (EC50: 380 nM and Emax = 29.9 7.1%)
toward GPR174. The analogues with a mono-methylated (7c)
or acetylated (7d) (S)-amino group showed impaired or no
agonistic activity (7c EC50: 51 nM, Emax = 20.1 2.1%; 7d EC50
ND). Instead, 7d inhibited the GPR174 activation level to below
the basal state (Figures 3a and S2a), and thus, 7d showed inverse
agonistic activity toward GPR174.
We also evaluated the effect of stereochemistry of α-amino
and α-methyl substituents on GPR174 agonistic ability (Table 1
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Table 1. Structure Expansion of α-Substitution of the Serine
Moiety of the GPR174 Agonist
Table 2. Exploration of the Effect of Amide Substituents on
Receptor Activation
a
a
a
Activities are represented in terms of EC50, IC50, pEC50, pIC50, Emax
,
or Imax: pEC50 and pIC50 are calculated from the sigmoidal
concentration−response curve and shown as mean SEM (standard
error of the mean) of the indicated numbers of independent
experiments (n); EC50 and IC50 values are calculated from the
mean value of pEC50 and pIC50, respectively; the Emax values of the
analogues are calculated as the percentage of the maximum response
obtained by LysoPS (18:1) (see Figure 3, frame); Imax values are
calculated as the percentage of the span of the sigmoidal
concentration−activity response curves divided by the maximum
response in the absence of the inhibitor (see Figure 4, frame). “NA”
means “not available” because of very low activity and “ND” means
“not determined” because of no agonistic or antagonistic activity.
a
Activities are represented in terms of EC50, IC50, pEC50, pIC50, Emax
,
or Imax: pEC50 and pIC50 are calculated from the sigmoidal
concentration−response curve and shown as mean SEM of the
indicated numbers of independent experiments (n); the EC50 and
IC50 values are calculated from the mean value of pEC50 and pIC50,
respectively; the Emax values of the analogues are calculated as the
percentage of the maximum response obtained by LysoPS (18:1) (see
Figure 3, frame); the Imax values are calculated as the percentage of the
span of the sigmoidal concentration−activity response curves divided
by the maximum response in the absence of the inhibitor (see Figure
4, frame). “NA” means “not available” because of very low activity and
“ND” means “not determined” because of no agonistic or antagonistic
activity.
and Figures 3a and S2a). (R)-α-Amino analogue 7e showed
diminished agonistic activity, with only about one-fiftieth of the
potency of (S)-α-amino 7a (7e EC50 = 81 nM and 7a EC50 = 1.6
nM). The (R)-α-methyl analogue 7f showed partial agonistic
activity toward GPR174 (EC50 0.9 nM, Emax = 28.5 3.2%). The
α,α-dimethyl analogue 7g also showed almost no agonistic
potency toward GPR174 (EC50 > 3 μM) (Table 1 and Figures
3a and S2a).
As small modifications of the substituent at the α-position
dramatically changed the GPR174 agonistic potency, we
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a
Table 3. Change of Antagonistic Activities upon Structural Perturbation
a
Activities are represented in terms of EC50, IC50, pEC50, pIC50, Emax, or Imax: pEC50 and pIC50 are calculated from the sigmoidal concentration−
response curve and shown as mean SEM of the indicated numbers of independent experiments (n); EC50 and IC50 values are calculated from the
mean value of pEC50 and pIC50, respectively; the Emax values of the analogues are calculated as the percentage of the maximum response obtained
by LysoPS (18:1) (see Figure 3, frame); the Imax values are calculated as the percentage of the span of the sigmoidal concentration−activity
response curves divided by the maximum response in the absence of the inhibitor (see Figure 4, frame). “NA” means “not available” because of very
low activity and “ND” means “not determined” because of no agonistic or antagonistic activity.
suspected that these changes altered the efficacy, not the affinity,
of the analogues for the receptor. Thus, we evaluated the
inhibitory activities of these analogues in the presence of a
previously reported GPR174 agonist, LysoPalloT-NH-amide-
C3-ph-m-O-C1118 (Table 1 and Figures 4a and S3a). The
results of TGFα shedding assay of selected analogues showed
that mono-α-methyl analogues, 7b and 7f, have moderate
inhibitory activity, whereas α,α-dimethyl analogue 7g has
decreased inhibitory activity: 7b IC50 = 4.7 nM; 7f IC50 = 12.5
nM; and 7g IC50 = 1100 nM. Compounds 7b and 7f, which have
partial agonistic activity toward GPR174, did not completely
inhibit the activation of GPR174 by the positive agonist even at
their maximum concentration (Figures 4a and S3a). We define
amine and explored amide analogues (Table 2 and Figures 3b,
4b, S2b, and S3b).
We elongated the alkyl chain at the α-serine amide of 7d (C2)
to obtain 8a (C3), 8b (C4), and 8c (C5) (in parentheses:
number of carbon atom in the acyl group, Table 2). Compounds
8a (C3) and 8b (C4) showed GPR174 antagonistic activity as
potent as that of 7d (C2) (7d IC50 = 5.6 nM; 8a IC50 = 3.4 nM;
and 8b IC50 = 5.4 nM; Table 2 and Figures 4b and S3b).
However, 8c (C5), which bears a pentanamide, showed partial
agonistic activity toward GPR174 (EC50 = 0.58 nM and Emax
=
38.7 2.2%) compared with the agonist 7a; the Emax value of 8c
was lower than that of the agonist 7a (7a Emax = 71.7 5.4% and
8c Emax = 38.7 2.2%, Table 2 and Figures 3b and S2b). Also,
the maximum inhibition by 8c was weaker than that by the full
antagonist 7d (7d Imax = 117.7 6.4% and 8c Imax = 41 0%)
(Table 2 and Figures 4b and S3b).
The pivalamide analogue 8d (C5) also showed partial
agonistic activity toward GPR174 (EC50 = 1.1 nM), but its
Emax value was lower than that of 8c; that is, 8d has a more
Imax as the percentage of the activation decrease by an inhibitor
with respect to the span of the activation by the positive agonist
(Figure 4). When the analogue completely inhibits the
activation by the positive agonist, its Imax is 100%. The Imax
values of 7b and 7f were 69.5 8.9 and 59.7 4.5%, respectively
(Table 1). The N-methylated analogue 7c showed almost no
antagonistic activity, but the N-acetylated analogue 7d potently
inhibited the activation by LysoPalloT-NH-amide-C3-ph-m-
O-C11 (7c IC50 ND; 7d IC50 = 5.6 nM, Imax = 117.7 6.4%)
(Table 1 and Figures 4a and S3a). Thus, simple N-acetylation of
the serine amino group of the GPR174 agonist 7a affords the
GPR174 antagonist 7d.
antagonistic characteristic than the partial agonist 8c (8c Emax
=
38.7 2.2% and 8d Emax = 20 4.6%, Table 2 and Figures 3b
and S2b). While the molecular weights of 8c and 8d are the
same, these different behaviors of 8c and 8d suggest that a more
extended amide structure is likely to favor the agonistic activity.
Therefore, we synthesized N-benzoyl derivatives (8e−8h).
Benzamide analogue 8e and non-aromatic cyclohexanecarbox-
yamide analogue 8f showed agonistic activity with Emax as high as
that of 7a (7a Emax = 71.7 5.4%; 8e Emax = 52.1 9.2%; and 8f
Extension of the α-Amide Moiety Switches Activity
toward GPR174 Antagonism. As the α-amide analogue 7d
showed potent inhibitory activity toward GPR174, we next
focused on the N-acyl (amide) group of the hydrophilic serine
E
max = 69.5 1.8%, Table 2 and Figures 3b and S2b), and no
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favorable for the GPR174 agonistic activity and that the
hydrophobic space around N-alkyl amide may be limited.
Change of Antagonistic Activities upon Structural
Perturbation. With potent GPR174 antagonists such as 7d in
hand, we next focused on the structural requirements for
antagonists. In our previous SAR studies, we had identified some
important structural elements that increase the GPR174
agonistic activity: the structure of the fatty acid part and the
presence of a hydroxyl group at the glycerol moiety and a
carboxyl acid group at the serine moiety.18 We examined the
change of biological activities of antagonist 7d in response to
changes of the fatty acid surrogate, elimination of the glycerol
hydroxyl group, methyl esterification, and O-methylation of the
phosphodiester (Table 3 and Figures 3c, 4c, S2c, and S3c). The
results indicate that the structural requirements for GPR174
antagonists and agonists are similar.
Table 4. Effect of Stereochemistry of the N-Acetyl Group and
β-Methylation
a
The agonistic activities of LysoPS analogues toward GPR174
are strongly influenced by the structure of the hydrophobic fatty
acid moiety,18,20 and the introduction of a fatty acid surrogate
having an ortho-substituted benzene ring and short alkoxy chain
(OC7H15) (named as fatty acid o-O-C7 here) dramatically
decreased the agonistic activity.18 When o-O-C7 was introduced
into the GPR174 antagonist 7d instead of the meta-substituted
fatty acid surrogate, the analogue 9a showed almost no
antagonistic activity toward GPR174 (IC50 > 3 μM) (Table 3
and Figures 4c and S3c). Thus, the fatty acid moiety of the
GPR174 antagonist 7d is recognized by the receptor, probably
in a similar manner to that of GPR174 agonists.
A hydroxyl group on the glycerol moiety is also important for
the GPR174 agonistic activity.18,20 Deletion of the glycerol
hydroxyl group in antagonist 7d dramatically reduced its
antagonistic activity (9b IC50 = 270 nM) (Table 3 and Figures
4c and S3c). Thus, the glycerol hydroxyl group should play an
important role in the interaction with GPR174 in the antagonist-
bound form, as in the case of agonists.
a
Activities are represented in terms of EC50, IC50, pEC50, pIC50, Emax
,
or Imax: pEC50 and pIC50 are calculated from the sigmoidal
concentration−response curve and shown as mean SEM of the
indicated numbers of independent experiments (n); the EC50 and
IC50 values are calculated from the mean value of pEC50 and pIC50,
respectively; the Emax values of the analogues are calculated as the
percentage of the maximum response obtained by LysoPS (18:1) (see
Figure 3, frame); the Imax values are calculated as the percentage of the
span of the sigmoidal concentration−activity response curves divided
by the maximum response in the absence of the inhibitor (see Figure
4, frame). “NA” means “not available” because of very low activity and
“ND” means “not determined” because of no agonistic or antagonistic
activity.
When the carboxyl group of antagonist 7d was converted to
methyl ester (9c), the IC50 value for antagonistic activity was
decreased dramatically (7d IC50 = 5.6 nM and 9c IC50 = 610
nM) (Table 3 and Figures 4c and S3c). Deletion of the carboxyl
group in 7d also abolished the GPR174 antagonistic activity (9d
IC50 ND) (Table 3 and Figures 4c and S3c). On the other hand,
when the phosphodiester of 7d was O-methylated (9e), the
antagonistic activity was still about one-ninth of that of 7d (7d
IC50 = 5.6 nM and 9e IC50 = 51 nM) (Table 3 and Figures 4c and
S3c). Thus, although both the carboxylate and the phospho-
diester of the GPR174 antagonist 7d are recognized by the
receptor, the carboxylic acid functionality is more important for
the antagonist−receptor interaction. A similar trend was
previously observed for GPR174 agonists: methylation of the
carboxylic acid group or methylation of the phosphodiester
reduced the GPR174 agonistic activity.18 Thus, structural
perturbations such as changes of the fatty acid surrogate,
elimination of the glycerol hydroxyl group, methyl esterification,
and O-methylation of phosphodiester have similar effects on the
biological activities of antagonist 7d and agonists.18 These
results are consistent with the idea that the structural
requirements for the activity of both GPR174 agonists and
antagonists are similar.
antagonistic activity was detected in the presence of agonist
LysoPalloT-NH-amide-C3-ph-m-O-C11 (Table 2 and Figures
4b and S3b). Compound 8g, which has a p-methyl group on the
benzamide moiety of 8e, showed partial agonistic activity (Emax
= 31.6 3%) (Table 2 and Figures 3b and S2b). The agonistic
activity of 8g was lower than that of 8c in terms of EC50 (8c EC50
= 0.58 nM and 8g EC50 = 9 nM). Compound 8h, which has a p-t-
butyl substituent on the benzamide, was inactive toward
GPR174 in the agonist assay and its inhibitory activity was
greatly diminished compared to that of 7d (8h EC50 ND, IC50 =
700 nM) (Table 2 and Figures 3b, 4b, S2b, and S3b). As a
substituent on the benzamide moiety decreases the agonistic
activity, this region of 8e may be involved in recognition by the
receptor GPR174. Thus, the agonist, partial agonist, or
antagonist characteristic of these analogues appears to depend
on the structure of the α-amide moiety in the hydrophilic
structure. These results suggest that a linearly extended amide is
Effect of Stereochemistry of the N-Acetyl Group and β-
Methylation. Conversion of (S)-acetamide stereochemistry of
7d to (R)-acetamide greatly decreased the GPR174 antagonistic
activity (10a IC50 = 360 nM, Table 4 and Figures 4d and S3d).
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Figure 3. Agonistic activities of LysoPS analogues for GPR174. HEK293FT cells were transfected with mouse GPR174-coding plasmid or an empty
plasmid and an AP-TGFα-coding plasmid and treated with LysoPS analogues. Receptor-specific GPCR activation (% AP-TGFα release) was
determined by subtracting the background responses in the mock-transfected cells from the responses in the GPR174-transfected cells. Data are mean
and SEM for several independent experiments indicated in Tables 1−4. The definition of Emax is also shown in the upper frame.
Methylation at the β-position of the serine carbonyl group in
7d or 10a decreased the GPR174 antagonistic activity: 10b IC50
= 100 nM; 10c IC50 = 120 nM; 10d inactive; and 10e inactive
(Table 4 and Figures 4d and S3d). The agonistic activity was
retained when the serine moiety of LysoPS was converted to
allo-threonine.18 However, introduction of a methyl group at the
β-position of the serine carbonyl group in antagonist 7d with the
same stereochemistry as in allo-threonine (10b) greatly reduced
the antagonistic activity compared with 7d (7d IC50 = 5.6 nM
and 10b IC50 = 100 nM) (Table 4 and Figures 4d and S3d).
Thus, the interaction of the hydrophilic moiety with the receptor
appears to be different between agonists and antagonists.
Overall, our results suggest that all the fatty acid, glycerol, and
phosphoserine moieties in GPR174 antagonists are recognized
by the receptor, and therefore, agonists and antagonists may
share the same binding pocket in GPR174.
Amide Functionality Is Placed Near TM7 in GPR174−
7d Binding Models. In order to investigate the origin of the
antagonistic efficacy of α-amide derivatives at the atomic level,
we created binding models of 7d and GPR174. As the structure
of GPR174 has not been reported, we built a homology model of
GPR174 based on the crystal structure of the closely related
GPCR, LPA receptor 6 (LPA6) (PDBID: 5XSZ).21 As the LPA6
crystal structure lacks a bound ligand and its binding pocket is
supposed to take a wide-open conformation before activation by
an agonist, the structure should be suitable for antagonist
binding. Since our results (Table 3) suggest that agonists and
antagonists share the same binding pocket, we docked the
antagonist 7d to GPR174 with reference to the binding position
of the agonist in a GPR34 model that we reported previously.20
We obtained two binding models (models 1 and 2) of GPR174
and 7d after docking and optimization of the whole complex by
molecular dynamics simulation (Figure 5, model 1: blue protein
and yellow ligand; model 2: red protein and yellow ligand). The
two receptor protein structures are comparable, and the fatty
acid moiety of 7d was placed around transmembrane helices
(TMs) 4, 5, and 6 at the intracellular part of the binding pocket
in both models. This is consistent with the experimental result
that the fatty acid moiety in GPR174 is recognized by the
receptor. On the other hand, the hydrophilic part of 7d showed a
larger difference between the binding models: in model 1, the
acetamide part in 7d points toward TM2, being placed at a more
extracellular location than in model 2. In model 2, the acetamide
moiety faces TM7 (Figure 5, right, and see Figure 6).
GPR174 is related to the purinergic receptor family. In the
purinergic receptor P2Y12, for which both agonist- and
antagonist-bound crystal structures are available,22,23 it is
considered that the inward movement of the extracellular
parts of TMs 6 and 7 to the center of the binding pocket occurs
after binding of an agonist. Similarly, in GPR174, we suspect that
when the pocket around TM7 in model 2 (TM7 pocket, Figure
6a) is occupied by the α-amide moiety of LysoPS derivatives, the
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Figure 4. Antagonistic activities of LysoPS analogues for GPR174. The concentration−response curves of each LysoPS analogue were shown.
HEK293FT cells transiently expressing both AP-TGFα and mouse GPR174 were treated with varying concentrations of test compounds in the
presence of a GPR174 agonist, LysoPalloT-NH-amide-C3-ph-m-O-C11 (100 nM). Cells expressing only AP-TGFα (GPR174 (−)) were used as a
negative control. Receptor-specific AP-TGFα release was calculated by subtracting the responses in negative control cells from those in GPR174-
expressing cells. Data are means and SEM for three independent experiments. Definition of Imax is also shown in the upper frame.
antagonistic activity may arise due to inhibition of the inward
movement of TM7 (schematic view in Figure 6b). However, as
the pocket around TM7 is not large (Figure 6a), extended
amides such as those in 8c (pentamide), 8e (benzamide), and 8f
(cyclohexanecarboxyamide) are unlikely to be accommodated
effectively in the TM7 pocket. Indeed, 8c, 8e, and 8f showed
agonistic activity rather than antagonistic activity (Table 2).
From this perspective, model 2 seems more consistent with the
experimental SAR than model 1, although further studies are
necessary to better define the mechanism of agonist−antagonist
switching.
short-chain alkylamide at the serine α-position. Furthermore,
the agonist−antagonist characteristic of the analogues changed
depending on the length and shape of the α-amide, affording
partial agonists with varying values of Emax (Emax: 8d < 8c < 8e <
8f).
Every structural module of the obtained antagonists, that is,
fatty acid, glycerol, and phosphoserine, appears to be recognized
by the receptor, and our results suggest that agonists and
antagonists occupy essentially the same binding position in the
receptor. Modeling studies indicate that the efficacy of the
LysoPS analogues depends on the ability of the amide group to
block the inward movement of the extracellular part of the
receptor, especially TM7. To our knowledge, this is the first
report of potent GPR174 antagonists. We believe that these
antagonists will be useful as tools to elucidate the immunological
functions of GPR174, and they should also provide a good
starting point for the development of candidate therapeutics for
CONCLUSIONS
■
In this study, we explored the structural requirements at the α-
position of the serine moiety of LysoPS analogues for activity
toward the receptor GPR174. We obtained highly potent and
GPR174-selective antagonists (7d, 8a, and 8b) by attaching a
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autoimmune diseases. Further studies will be needed to establish
in detail the mechanism that underlies the switch from agonist to
antagonist activity in these analogues.
EXPERIMENTAL SECTION
■
Melting points were determined with a Yanaco micro melting point
1
apparatus without correction. H (400 MHz) and 13C (100 MHz)
NMR spectra were recorded on a Bruker AVANCE 400. The chemical
shifts were calibrated with tetramethylsilane as an internal standard or
with the solvent peak and are shown in ppm (δ) values; coupling
constants are shown in hertz (Hz). The following abbreviations are
used: s = singlet, d = doublet, t = triplet, q = quartet, quin = quintet, dd =
double doublet, m = multiplet, and br s = broad singlet. An electron
spray ionization time-of-flight (ESI-TOF) mass spectrometer (Bruker
micrOTOF-05) was used to obtain high-resolution mass spectra
(HRMS). All commercially available compounds and solvents were
used as received. Combustion analyses were carried out in the
Microanalytical Laboratory of the Graduate School of Pharmaceutical
Sciences, the University of Tokyo. The purity of final compounds was
examined by reverse-phase HPLC analysis: the purities of the final
compounds, 7a−7g, 8a−8d, 8f, 9b−9d, and 10a−10e, were ≥95%.
The compounds with the purity below 95% were 8e (88%), 8g (94%),
8h (86%), 9a (94%), and 9e (90%). The HPLC charts of the lead
compounds (7a, 7d, 8a, 8c, and 8f) are provided in the Supporting
Information (Figure S4). HPLC conditions: Imtakt Unison UK-C18
(Imtakt Corporation, Kyoto, Japan), 3 μm, 150 × 4.6 mm, acetonitrile/
H2O containing 0.1% HCO2H as an eluent, detection: 210 nm UV, and
flow rate 1.0 mL/min.
Synthesis of 7d. Compound 3.
Figure 5. Two docking models of the antagonist (7d)−GPR174
complex. Model 1 (GPR174: blue ribbon) and model 2 (GPR174: red
ribbon) were created on the basis on the crystal structure of LPA6
(PDBID: 5XSZ). Views from the side (left) and from the top (right) are
shown. The bound antagonist 7d is shown as spheres or sticks (carbon:
yellow, hydrogen: white, oxygen: red, nitrogen: blue, and phosphorus:
purple). In the top view (right), only the acetamide part of the ligand 7d
is emphasized as spheres to indicate its different position in models 1
and 2.
Bis(diisopropylamino)chlorophosphine (995.8 mg, 3.732
mmol) was dissolved in anhydrous Et2O (10 mL). A solution
of benzyl alcohol (405.9 mg, 3.753 mmol) and Et3N (525 μL)
was added dropwise at 0 °C, and the whole was stirred at 0 °C
under an Ar atmosphere for 4 h. The solution was filtered
through Celite, and the filtrate was evaporated to yield 3 as a
colorless oil (1149.4 mg, 3.3958 mmol), which was used without
further purification.
Compound 11.
(R)-3-Amino-1,2-propanediol (1255.4 mg, 13.779 mmol), fatty
acid surrogate 1 (4006.9 mg, 12.503 mmol), and 1-
hydroxybenzotriazole monohydrate (2024.6 mg, 13.220
mmol) were dissolved in anhydrous DMF (40 mL). EDCI·
HCl (2878.2 mg, 15.014 mmol) was added at 0 °C, and the
whole was stirred at room temperature under an Ar atmosphere
for 5.5 h. A saturated aqueous solution of NaHCO3 (70 mL) was
added. The mixture was filtered to obtain the precipitate 11, a
colorless powder (4734.3 mg, 12.029 mmol, 96%).
Figure 6. Putative inhibition of TM7 movement by the ligand α-amide
moiety placed in the receptor TM7 pocket. (a) Top view of GPR174−
7d complex model 2. The receptor surface is shown in addition to the
ribbon representation of the receptor model (colored red). The
antagonist 7d is shown as sticks and its acetamide part is emphasized as
spheres (carbon: yellow, hydrogen: white, oxygen: red, nitrogen: blue,
and phosphorus: purple). (b) Schematic view of the putative inhibition
of the inward movement of TM7 by antagonists. Top view of the
receptor 7 TMs and the bound ligand is shown to indicate the proposed
mechanism of agonist−antagonist switching.
1H NMR (400 MHz, CDCl3): 7.208−2.167 (1H, m), 6.770−6.733
(3H, m), 6.005 (1H, t, J = 5.7 Hz), 3.924 (2H, t, J = 6.6 Hz), 3.680 (1H,
quin, J = 5.1 Hz), 3.493−3.275 (4H, m), 2.927 (2H, t, J = 7.5 Hz), 2.766
(2H, br s), 2.515 (2H, t, J = 7.5 Hz), 1.761 (2H, quin, J = 7.0 Hz),
1.473−1.267 (16H, m), 0.881 (3H, t, J = 6.9 Hz). 13C NMR (100 MHz,
CDCl3): 174.15, 159.33, 141.96, 129.55, 120.46, 114.76, 112.21, 70.95,
67.96, 63.46, 42.16, 38.14, 31.89, 31.69, 29.60, 29.57, 29.41, 29.32,
29.29, 26.04, 22.67, 14.10. HRMS (ESI-TOF, [M + Na]+): calcd for
+
C23H39NNaO4 , 416.2771; found, 416.2742.
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Compound 12.
Compound 2.
Compound 13 (299.6 mg, 0.3963 mmol) was dissolved in
CH2Cl2 (1 mL) and MeOH (3 mL). p-TsOH·H2O (7.9 mg,
0.042 mmol) was added, and the mixture was stirred at room
temperature for 30 min. Et3N (10 μL) was added to the mixture
and the solution was washed with brine, dried over Na2SO4, and
filtered. The solvent was evaporated. The residue was column-
chromatographed on an open column with silica gel (n-hexane/
AcOEt = 1:1 to 1:6) to afford 2 as a colorless solid (160.2 mg,
0.3312 mmol, 84%).
Compound 11 (4725.9 mg, 12.008 mmol) and DIPEA (6.2 mL,
36 mmol) were dissolved in anhydrous THF (40 mL). MMTrCl
(4077.0 mg, 13.203 mmol) was added to the solution, and the
whole was stirred at room temperature under an Ar atmosphere
for 4.5 h. A saturated aqueous solution of NaHCO3 (50 mL) was
added. The water layer was separated and extracted twice with
AcOEt (50 mL × 2). The combined organic layer was washed
with brine, dried over Na2SO4 and filtered, and the solvent was
evaporated. The residue was column-chromatographed on an
open column with silica gel (n-hexane/AcOEt = 2:1, 1:1 to 2:3)
to afford 12 (6840.0 mg, 10.272 mmol, 86%) as a yellow sticky
oil.
1H NMR (400 MHz, CDCl3): 7.368−7.257 (5H, m), 7.194−7.153
(1H, m), 6.761−6.724 (3H, m), 5.740 (1H, t, J = 5.8 Hz), 4.558 (1H, d,
J = 11.8 Hz), 4.498 (1H, d, J = 11.8 Hz), 3.914 (2H, t, J = 6.6 Hz),
3.636−3.574 (1H, m), 3.553−3.505 (2H, m), 3.460−3.395 (1H, m),
3.337−3.278 (1H, m), 3.164−3.131 (1H, m), 2.909 (2H, t, J = 7.6 Hz),
2.467 (2H, t, J = 7.6 Hz), 1.757 (2H, quin, J = 7.0 Hz), 1.471−1.265
(16H, m), 0.880 (3H, t, J = 6.7 Hz). 13C NMR (100 MHz, CDCl3):
173.49, 159.32, 142.08, 137.96, 129.49, 128.56, 128.00, 127.81, 120.43,
114.64, 112.22, 77.54, 71.70, 67.89, 60.75, 39.37, 38.19, 31.89, 31.65,
29.59, 29.57, 29.40, 29.32, 29.30, 26.05, 22.67, 14.10. HRMS (ESI-
1H NMR (400 MHz, CDCl3): 7.415−7.394 (4H, m), 7.309−7.258
(7H, m), 7.244−7.202 (2H, m), 7.186−7.146 (1H, m), 6.828 (2H, d, J
= 8.9 Hz), 6.738−6.714 (3H, m), 5.579 (1H, t, J = 5.6 Hz), 3.913 (2H,
t, J = 6.6 Hz), 3.841−3.828 (1H, m), 3.770 (3H, s), 3.550−3.490 (1H,
m), 3.196−3.098 (3H, m), 3.027 (1H, d, J = 4.7 Hz), 2.850 (2H, t, J =
7.8 Hz), 2.371 (2H, t, J = 7.7 Hz), 1.755 (2H, quin, J = 7.1 Hz), 1.469−
1.264 (16H, m), 0.880 (3H, t, J = 6.9 Hz). 13C NMR (100 MHz,
CDCl3): 173.31, 159.30, 158.64, 144.11, 144.06, 142.22, 135.21,
130.30, 129.45, 128.28, 127.91, 127.03, 120.39, 114.63, 113.18, 112.12,
86.48, 70.25, 67.88, 64.62, 55.19, 42.82, 38.20, 31.89, 31.73, 30.92,
29.60, 29.57, 29.40, 29.32, 29.30, 26.05, 22.67, 14.11. HRMS (ESI-
+
TOF, [M + Na]+): calcd for C30H45NNaO4 , 506.3241; found,
506.3238. Anal. Calcd for C30H45NO4: C, 74.50; H, 9.38; N, 2.90.
Found: C, 74.30; H, 9.25; N, 2.81. mp 43.0−44.8 °C (colorless solids,
recrystallized from n-hexane/CH2Cl2).
Compound 4.
+
TOF, [M + Na]+): calcd for C43H55NNaO5 , 688.3972; found,
688.3989.
Compound 13.
Compound 2 (485.9 mg, 1.005 mmol) was dissolved in CH2Cl2
and toluene. To remove traces of water, the solution was
evaporated. Compound 3 (crude, 585.4 mg, 1.730 mmol) was
added, and the mixture was dissolved in anhydrous CH2Cl2 (4.0
mL). A solution of 1H-tetrazole (76.1 mg, 1.09 mmol) in
anhydrous THF (4.0 mL) was added at 0 °C. The mixture was
stirred at room temperature under an Ar atmosphere for 7.4 h. A
saturated aqueous solution of NaHCO3 (10 mL) was added.
The whole was extracted three times with CH2Cl2 (10 mL × 3).
The combined organic layer was washed with brine, dried over
Na2SO4, and filtered, and the solvent was evaporated. The
residue was column-chromatographed on an open column with
silica gel (n-hexane/AcOEt/Et3N = 40:1:1 to 40:8:1) to afford 4
(crude) as a colorless oil (586.1 mg, 0.8129 mmol, 81%).
HRMS (ESI-TOF, [M + H]+): calcd for C43H66N2O5P+, 721.4704;
found, 721.4696.
Compound 12 (6784.5 mg, 10.188 mmol) was dissolved in
anhydrous THF (40 mL). NaH (60% dispersion in oil, 617.7
mg, 15.44 mmol) was added to the solution at 0 °C, and the
mixture was stirred at 0 °C under an Ar atmosphere for 20 min.
Benzyl bromide (1.6 mL) was added to the mixture at 0 °C, and
the mixture was stirred at room temperature under an Ar
atmosphere for 4.3 h. A saturated aqueous solution of NaHCO3
(40 mL) was added. The water layer was separated and extracted
twice with AcOEt (40 mL × 2). The combined organic layer was
washed with brine, dried over Na2SO4, and filtered, and the
solvent was evaporated. The residue was column-chromato-
graphed on an open column with silica gel twice (first, n-hexane/
AcOEt = 4:1 to 2:1, second, n-hexane/AcOEt = 4:1, 2:1 to 1:1)
to afford 13 (6987.7 mg, 9.2425 mmol, 91%) as a colorless oil.
1H NMR (400 MHz, CDCl3): 7.447−7.427 (4H, m), 7.362−7.280
(11H, m), 7.234−7.198 (2H, m), 7.167−7.127 (1H, m), 6.816 (2H, d, J
= 8.9 Hz), 6.723−6.701 (3H, m), 5.616−5.601 (1H, m), 4.603 (1H, d, J
= 11.7 Hz), 4.436 (1H, d, J = 11.7 Hz), 3.899 (2H, t, J = 6.6 Hz), 3.770
(3H, s), 3.634−3.573 (2H, m), 3.302−3.157 (3H, m), 2.827 (2H, t, J =
7.9 Hz), 2.323−2.283 (2H, m), 1.750 (2H, quin, J = 7.0 Hz), 1.466−
1.258 (16H, m), 0.879 (3H, t, J = 6.9 Hz). 13C NMR (100 MHz,
CDCl3): 171.95, 156.26, 158.57, 144.18, 142.41, 138.19, 135.31,
130.30, 129.37, 128.48, 128.34, 127.90, 127.86, 127.82, 126.94, 120.37,
114.59, 113.13, 112.03, 86.54, 76.46, 71.86, 67.83, 63.70, 55.17, 40.60,
38.28, 31.88, 31.69, 30.91, 29.59, 29.56, 29.40, 29.31, 26.05, 22.66,
Compound 5.
L-Serine benzyl ester hydrochloride (693.8 mg, 2.995 mmol)
was dissolved in anhydrous CH2Cl2 (10 mL). Et3N (1040 μL)
and acetyl chloride (235 μL, 3.31 mmol) were added, and the
mixture was stirred at room temperature under an Ar
atmosphere for 1.5 h. MeOH (ca. 2 mL) was added, and the
solvent was evaporated with silica gel. The residue was column-
chromatographed on an open column with silica gel (n-hexane/
acetone = 2:1 to 1:1) to afford 5 as a colorless solid (637.9 mg,
2.689 mmol, 90%).
+
14.10. HRMS (ESI-TOF, [M + Na]+): calcd for C50H61NNaO5 ,
778.4442; found, 778.4427.
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Figure 7. Synthesis of 7a.
1H NMR (400 MHz, CDCl3): 7.401−7.314 (5H, m), 6.524 (1H, d, J
= 6.4 Hz), 5.217 (2H, s), 4.727−4.691 (1H, m), 4.007−3.980 (1H, m),
3.934−3.906 (1H, m), 2.670 (1H, br s), 2.054 (3H, s). 13C NMR (100
MHz, CDCl3): 170.64, 170.36, 135.06, 128.66, 128.56, 128.16, 67.54,
63.47, 54.87, 23.10. HRMS (ESI-TOF, [M + Na]+): calcd for
Synthesis of 7a (Figure 7). Compound 14.
+
C12H15NNaO4 , 260.0893; found, 260.0909. mp 82.0−82.5 °C
N-Carbobenzyloxy-L-serine benzyl ester (495.1 mg, 1.503
mmol) was dissolved in CH2Cl2 and toluene. To remove traces
of water, the solution was evaporated. 3 (crude, 931.7 mg, 2.753
mmol) was added, and the mixture was dissolved in anhydrous
CH2Cl2 (6.0 mL). A solution of 1H-tetrazole (111.0 mg, 1.584
mmol) in anhydrous THF (6.0 mL) was added at 0 °C. The
mixture was stirred at room temperature under an Ar
atmosphere for 2.2 h. A saturated aqueous solution of
NaHCO3 (15 mL) was added. The aqueous layer was separated
and extracted twice with CH2Cl2 (15 mL × 2). The combined
organic layer was washed with brine, dried over Na2SO4, and
filtered. The solvent was evaporated. The residue was column-
chromatographed on an open column with silica gel (n-hexane/
AcOEt/Et3N = 40:2:1, 40:3:1, 40:4:1, to 40:5:1) to afford 14 as
a colorless oil (724.8 mg, 1.279 mmol, 85%) (Figure 7).
1H NMR (400 MHz, CDCl3): 7.354−7.223 (15H, m), 5.898 (J = 8.6
Hz) and 5.669 (J = 8.5 Hz) (1H, 2d), 5.168−5.085 (4H, m), 4.721−
4.511 (3H, m), 4.173−4.123 (1H, m), 3.942−3.848 (1H, m), 3.610−
3.511 (2H, m), 1.150−1.099 (12H, m). 13C NMR (100 MHz, CDCl3):
169.91, 155.99, 155.85, 139.09, 138.89, 136.30, 136.23, 135.33, 135.21,
128.45, 128.41, 128.39, 128.23, 128.19, 128.12, 128.03, 127.98, 127.94,
127.34, 127.24, 127.03, 126.84, 67.20, 67.11, 66.88, 66.82, 65.44, 65.37,
65.26, 65.19, 64.16, 64.01, 63.66, 63.51, 55.37, 55.31, 55.24, 55.17,
43.09, 42.96, 30.83, 24.54, 24.48, 24.41, 24.34. HRMS (ESI-TOF, [M +
Na]+): calcd for C31H40N2O6P+, 567.2618; found, 567.2644.
(colorless needles, recrystallized from n-hexane/CH2Cl2).
Compounds 6 and 7d.
Compounds 5 (47.2 mg, 0.199 mmol) and 4 (233.4 mg, 0.3237
mmol) were dissolved in CH2Cl2 and toluene. To remove traces
of water, the solution was evaporated. The mixture was dissolved
in anhydrous CH2Cl2 (1.0 mL). A solution of 1H-tetrazole (27.8
mg, 0.397 mmol) in anhydrous THF (1.0 mL) was added at 0
°C. The mixture was stirred at room temperature under an Ar
atmosphere for 3 h. TBHP in decane (5.0−6.0 M) (73 μL) was
added, and the mixture was stirred at room temperature for 1.5
h. H2O (10 mL) was added, and the whole was extracted three
times with CH2Cl2 (10 mL × 3). The combined organic layer
was washed with brine, dried over Na2SO4, and filtered. The
solvent was evaporated to afford a colorless oil, which was
column-chromatographed on a flash column with silica gel
(CHCl3/AcOEt = 1:1 to n-hexane/acetone = 2:3) to afford
crude 6 as a colorless oil (109.7 mg).
Compound 15.
Crude 6 (106.4 mg) and Pd/C (18.2 mg) were dissolved in THF (5
mL). The mixture was stirred at room temperature under a H2
atmosphere for 5 h, then filtered through Celite, and the solvent was
evaporated. The residue was purified by reversed-phase medium-
pressure liquid chromatography (CH3CN/H2O = 3:7 to 7:3,
containing 0.1% HCOOH). The solid was vacuum-dried at 40 °C to
afford 7d (46.6 mg, 0.0773 mmol, 39%, two steps).
Compounds 14 (180.4 mg, 0.3184 mmol) and 2 (99.1 mg, 0.205
mmol) were dissolved in CH2Cl2 and toluene. To remove traces
of water, the solution was evaporated. The mixture was dissolved
in anhydrous CH2Cl2 (2.0 mL). A solution of 1H-tetrazole (29.3
mg, 0.418 mmol) in anhydrous THF (2.0 mL) was added. The
mixture was stirred at room temperature under an Ar
atmosphere for 2.5 h. TBHP in decane (5.0−6.0 M) (75 μL)
was added, and the mixture was stirred at room temperature for
2 h. H2O (10 mL) was added, and the whole was extracted three
times with CH2Cl2 (10 mL × 3). The combined organic layer
was washed with brine, dried over Na2SO4, and filtered. The
solvent was evaporated to afford a colorless oil. The residue was
column-chromatographed on a flash column with silica gel
1H NMR (400 MHz, DMSO-d6): 8.385 (1H, d, J = 7.8 Hz), 7.886
(1H, t, J = 5.6 Hz), 7.149 (1H, dd, J = 8.1 Hz, 8.1 Hz), 6.754−6.699
(3H, m), 4.480−4.439 (1H, m), 4.110−3.993 (2H, m), 3.910 (2H, t, J
= 6.5 Hz), 3.746−3.675 (2H, m), 3.657−3.595 (1H, m), 3.199−3.123
(1H, m), 3.054−2.990 (1H, m), 2.757 (2H, t, J = 7.9 Hz), 2.397−2.358
(2H, m), 1.869 (3H, s), 1.683 (2H, quin, J = 6.9 Hz), 1.392−1.356 (2H,
m), 1.247 (14H, br s), 0.854 (3H, t, J = 6.9 Hz). 31P NMR (161 MHz,
DMSO-d6): −0.92. HRMS (ESI-TOF, [M − H]−): calcd for
C28H46N2O10P−, 601.2896; found, 601.2914. HPLC: 97% (CH3CN/
H2O = 1:1 (0.1% HCO2H)).
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Figure 8. Synthesis of 7b.
Synthesis of 7b (Figure 8). Compound 16.
(CHCl3/AcOEt = 4:1) to afford 15 as a colorless oil (148.4 mg,
0.1538 mmol, 75%).
1H NMR (400 MHz, CDCl3): (mixtures of diastereomers) 7.309−
7.270 (20H, m), 7.165−7.120 (1H, m), 6.759−6.696 (3H, m), 6.073−
6.022 (1H, m), 5.857−5.813 (1H, m), 5.199−5.054 (4H, m), 5.026−
4.945 (2H, m), 4.612−4.544 (2H, m), 4.438−4.397 (2H, m), 4.340−
4.212 (1H, m), 4.038−3.799 (4H, m), 3.645−3.528 (1H, m), 3.372−
3.256 (2H, m), 2.838 (2H, t, J = 6.8 Hz), 2.437−2.270 (2H, m), 1.743
(2H, quin, J = 6.9 Hz), 1.462−1.237 (16H, m), 0.878 (3H, t, J = 6.8
Hz). 13C NMR (100 MHz, CDCl3): (mixtures of diastereomers)
172.31, 168.77, 159.25, 155.90, 142.31, 137.52, 136.00, 134.91, 129.37,
128.77, 128.64, 128.60, 128.51, 128.22, 128.09, 128.01, 120.43, 114.67,
112.03, 75.34, 71.90, 71.84, 69.80, 67.84, 67.71, 67.51, 67.17, 54.52,
39.13, 39.04, 38.01, 31.88, 31.53, 30.90, 29.59, 29.56, 29.40, 29.30,
26.04, 22.65, 14.09. HRMS (ESI-TOF, [M + NH4]+): calcd for
C55H73N3O11P+, 982.4977; found, 982.4998.
Methyl (S)-(+)-3-hydroxyisobutyrate (249.9 mg, 2.115 mmol)
was dissolved in MeOH (2 mL). An aqueous solution of KOH
(1.95 M, 2 mL) was added, and the whole was stirred at room
temperature for 2 h. The solvent was evaporated. The residue
was dissolved in anhydrous DMF (3 mL) and benzyl bromide
(245 μL) was added to the mixture. The mixture was heated to
80 °C under an Ar atmosphere for 22 h. An aqueous solution of
HCl (2 M, 5 mL) and H2O (10 mL) were added, and the whole
was extracted three times with AcOEt (10 mL × 3). The
combined organic layer was washed with brine, dried over
Na2SO4, and filtered, and the solvent was evaporated. The
residue was column-chromatographed on a flash column with
silica gel (n-hexane/AcOEt = 7:2) to afford 16 as a colorless oil
(226.2 mg, 1.165 mmol, 55%) (Figure 8).
Compound 7a.
1H NMR (400 MHz, CDCl3): 7.397−7.307 (5H, m), 5.162 (2H, s),
3.780−3.695 (2H, m), 2.774−2.690 (1H, m), 1.205 (3H, d, J = 7.2 Hz).
13C NMR (100 MHz, CDCl3): 175.44, 135.75, 128.59, 128.27, 128.02,
66.40, 64.52, 41.75, 13.39. HRMS (ESI-TOF, [M + Na]+): calcd for
Compound 15 (119.9 mg, 0.1242 mmol) and Pd/C (10.5 mg)
were dissolved in MeOH (4.0 mL) and AcOH (1.0 mL). The
mixture was stirred at room temperature under a H2 atmosphere
for 22.6 h. The mixture was filtered on Celite, and the solvent
was evaporated. The residue was purified by reversed-phase
medium-pressure liquid chromatography (CH3CN/H2O = 2:8
to 7:3, containing 0.1% HCOOH). The solid was vacuum-dried
at 40 °C to afford 7a as a colorless powder (42.3 mg, 0.0755
mmol, 61%).
+
C11H14NaO3 , 217.0835; found, 217.0847.
Compound 17.
Compound 16 (180.3 mg, 0.9289 mmol) was dissolved in
CH2Cl2 and toluene. To remove traces of water, the solution was
evaporated. 3 (678.0 mg, 2.003 mmol) was added, and the
mixture was dissolved in anhydrous CH2Cl2 (3.0 mL). A
solution of 1H-tetrazole (67.9 mg, 0.969 mmol) in anhydrous
THF (3.0 mL) was added at 0 °C. The mixture was stirred at
room temperature under an Ar atmosphere for 2 h. A saturated
aqueous solution of NaHCO3 (10 mL) was added and the
aqueous layer was extracted three times with CH2Cl2 (10 mL ×
3). The combined organic layer was washed with brine, dried
over Na2SO4, and filtered. The solvent was evaporated to afford
1H NMR (400 MHz, DMSO-d6): 8.725 (2H, br s), 7.938 (1H, t, J =
5.6 Hz), 7.147 (1H, dd, J = 8.0 Hz, 8.0 Hz), 6.752−6.697 (3H, m),
4.071−4.043 (3H, m), 3.911 (2H, t, J = 6.5 Hz), 3.599−3.549 (3H, m),
3.160−3.126 (1H, m), 3.042−2.994 (1H, m), 2.754 (2H, t, J = 7.8 Hz),
2.366 (2H, t, J = 7.9 Hz), 1.683 (2H, quin, J = 6.9 Hz), 1.428−1.247
(16H, m), 0.854 (3H, t, J = 6.8 Hz). HRMS (ESI-TOF, [M − H]−):
calcd for C26H44N2O9P−, 559.2790; found, 559.2791. HPLC: 98%
(CH3CN/H2O = 1:1 (0.1% HCO2H)).
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Figure 9. Synthesis of 7c.
(1H, m), 3.419−3.305 (2H, m), 2.892−2.777 (3H, m), 2.408−2.364
(2H, m), 1.778−1.725 (2H, m), 1.464−1.392 (2H, m), 1.346−1.263
(14H, m), 1.186−1.146 (3H, m), 0.879 (3H, t, J = 6.8 Hz). 13C NMR
(100 MHz, CDCl3): (mixtures of diastereomers) 173.17, 172.27,
172.25, 159.26, 142.36, 137.74, 135.62, 129.37, 128.66, 128.61, 128.60,
128.55, 128.48, 128.26, 128.05, 128.00, 127.96, 127.95, 120.42, 114.65,
112.05, 75.46, 75.40, 71.85, 69.58, 69.52, 68.73, 68.68, 67.84, 66.59,
66.57, 66.37, 40.43, 40.35, 38.96, 38.14, 31.88, 31.63, 29.59, 29.56,
29.40, 29.31, 26.05, 22.66, 14.10, 13.39, 13.36. HRMS (ESI-TOF, [M +
Na]+): calcd for C48H64NNaO9P+, 852.4211; found, 852.4239. Anal.
Calcd for C48H64NO9P·0.5H2O: C, 68.71; H, 7.81; N, 1.67. Found: C,
68.71; H, 7.79; N, 1.63.
a colorless oil. The residue was column-chromatographed on an
open column with silica gel (n-hexane/AcOEt/Et3N = 40:1:1)
to afford 17 as a colorless oil (265.8 mg, 0.6160 mmol, 66%).
1H NMR (400 MHz, CDCl3): 7.344−7.230 (10H, m), 5.156−5.076
(2H, m), 4.739−4.681 (1H, m), 4.657−4.604 (1H, m), 3.940−3.562
(4H, m), 2.863−2.776 (1H, m), 1.223−1.196 (3H, m), 1.173−1.153
(12H, m). 13C NMR (100 MHz, CDCl3): 174.42, 174.35, 139.48,
136.07, 128.47, 128.20, 128.05, 128.03, 127.99, 127.95, 127.18, 126.92,
66.17, 66.15, 65.46, 65.38, 65.28, 65.21, 65.11, 43.08, 42.95, 41.54,
41.47, 24.61, 24.58, 24.55, 24.51, 13.83, 13.79. HRMS (ESI-TOF, [M +
H]+): calcd for C24H35NO4P+, 432.2298; found, 432.2313.
Compound 18.
Compound 7b.
Compounds 17 (92.5 mg, 0.214 mmol) and 2 (51.6 mg, 0.137
mmol) were dissolved in CH2Cl2 and toluene. To remove traces
of water, the solution was evaporated. The residue was dissolved
in anhydrous CH2Cl2 (1.0 mL) and a solution of 1H-tetrazole
(19.3 mg, 0.275 mmol) in anhydrous THF (1.0 mL) was added.
The mixture was stirred at room temperature under an Ar
atmosphere for 3 h. TBHP in decane (5.0−6.0 M) (50 μL) was
added, and the mixture was stirred at room temperature for 1.6
h. H2O (10 mL) was added, and the whole was extracted three
times with CH2Cl2 (10 mL × 3). The combined organic layer
was washed with brine, dried over Na2SO4, and filtered. The
solvent was evaporated to afford a colorless oil. The residue was
column-chromatographed on a flash column with silica gel
(CHCl3/AcOEt = 4:1) to afford 18 as a colorless oil (87.4 mg,
0.105 mmol, 77%).
Compound 18 (70.9 mg, 0.0854 mmol) and Pd/C (8.0 mg)
were dissolved in MeOH (4 mL) and AcOH (1 mL). The
mixture was stirred at room temperature under a H2 atmosphere
for 24.3 h. The mixture was filtered on Celite, and the solvent
was evaporated. The residue was purified by reversed-phase
medium-pressure liquid chromatography (CH3CN/H2O = 1:1
to 7:3, containing 0.1% HCOOH). The residue was column-
chromatographed on a flash column with silica gel (CHCl3/
MeOH/AcOH = 8:1:1 to 7:1:2) and vacuum-dried at 40 °C to
afford 7b (18.8 mg, 0.0336 mmol, 39%).
1H NMR (400 MHz, DMSO-d6): 7.988 (1H, br s), 7.142 (1H, dd, J
= 8.1 Hz, 8.1 Hz), 6.753−6.698 (3H, m), 3.910 (2H, t, J = 6.5 Hz),
3.694 (2H, br s), 3.596−3.572 (2H, m), 3.516−3.507 (1H, m), 3.075−
3.025 (2H, m), 2.752 (2H, t, J = 7.8 Hz), 2.360 (2H, t, J = 7.9 Hz), 1.682
(2H, quin, J = 6.9 Hz), 1.393−1.248 (16H, m), 0.978 (3H, d, J = 7.0
Hz), 0.854 (3H, t, J = 6.8 Hz). HRMS (ESI-TOF, [M − H]−): calcd for
C27H45NO9P−, 558.2837; found, 558.2866. HPLC: 98% (CH3CN/
H2O = 3:2 (0.1% HCO2H)).
1H NMR (400 MHz, CDCl3): (mixtures of diastereomers) 7.354−
7.274 (15H, m), 7.171−7.124 (1H, m), 6.748−6.696 (3H, m), 6.022−
5.955 (1H, m), 5.143−5.073 (2H, m), 5.032−4.986 (2H, m), 4.588
(1H, d, J = 11.7 Hz), 4.477 (1H, dd, J = 11.6 Hz, 3.5 Hz), 4.229−4.135
(1H, m), 4.099−3.961 (2H, m), 3.930−3.872 (3H, m), 3.619−3.553
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Synthesis of 7c (Figure 9). Compound 19.
Compound 22.
L-Serine (525.8 mg, 5.003 mmol) was dissolved in 6 M aqueous
solution of HCl (1 mL) and AcOH (10 mL). The whole was
stirred at room temperature for 5 min. Acetyl chloride (10 mL)
was then added to the solution dropwise at 0 °C via 7 min, and
the mixture was stirred at 0 °C for 5 min. Et2O (25 mL) was
added. A colorless solid, 19, was obtained by filtration (750.4
mg, 4.087 mmol, 82%) (Figure 9).
Compound 21 (571.4 mg, 1.948 mmol) was dissolved in CHCl3
(10 mL). Et3SiH (0.93 mL) and TFA (10 mL) were added to
the solution, and the whole was stirred at room temperature for
96.2 h. Toluene (5 mL) was added and evaporated twice. The
residue was column-chromatographed on an open column with
silica gel (n-hexane/acetone = 2:1 to 0:1) to afford 22 as a
colorless oil (507.4 mg, 1.718 mmol, 88%).
1H NMR (400 MHz, DMSO-d6): 14.076 (1H, br s), 8.695 (3H, br
1H NMR (400 MHz, CDCl3): 7.365−7.291 (5H, m), 7.136 (2H, br
s), 5.225−5.077 (2H, m), 4.988−4.956 and 4.892−4.858 (1H, 2m),
4.581−4.348 (2H, m), 2.972 (3H, s), 2.014 and 1.977 (3H, 2s) (two
rotamers). 13C NMR (100 MHz, CDCl3): 172.52, 172.46, 170.72,
170.67, 156.94, 155.99, 136.10, 135.89, 128.48, 128.16, 128.12, 127.94,
127.77, 67.90, 67.85, 61.26, 58.46, 58.15, 32.78, 32.56, 20.64, 20.56
s), 4.488 (1H, dd, J = 11.9 Hz, 3.2 Hz), 4.346 (1H, dd, J = 11.9 Hz, 4.6
Hz), 4.288 (1H, t, J = 3.6 Hz), 2.049 (3H, s). 13C NMR (100 MHz,
DMSO-d6): 169.91, 168.33, 61.45, 51.24, 20.61. HRMS (ESI-TOF, [M
+
+ H]+): calcd for C5H10NO4 , 148.0604; found, 148.0632.
−
(two rotamers). HRMS (ESI-TOF, [M − H]−): calcd for C14H16NO6 ,
Compound 20.
294.0983; found, 294.0987.
Compound 23.
Compound 19 (740.2 mg, 4.032 mmol) was dissolved in THF
(10 mL) and a saturated solution of NaHCO3 (7.5 mL). Benzyl
chloroformate (828.4 mg, 4.856 mmol) in 5 mL of THF was
added to the solution, and the whole was stirred at room
temperature for 23.8 h. H2O (10 mL) and 2 M aqueous solution
of HCl (3 mL) were added, and the whole was extracted three
times with AcOEt (20 mL × 3). The combined organic layer was
washed with brine, dried over Na2SO4, and filtered. The solvent
was evaporated to afford a yellow oil. The residue was column-
chromatographed on an open column with silica gel (n-hexane/
acetone = 2:1 to 0:1) to afford 20 (865.4 mg, 3.077 mmol, 76%).
1H NMR (400 MHz, CDCl3): 7.389−7.302 (5H, m), 5.632 (1H, d, J
Compound 22 (504.2 mg, 1.707 mmol) was dissolved in
dioxane (9.0 mL) and 2 M aqueous solution of HCl (9.0 mL).
The mixture was stirred at 60 °C for 17.6 h. An aqueous solution
of HCl (1 M, 20 mL) was added, and the whole was extracted
three times with AcOEt (20 mL × 3). The combined organic
layer was washed with brine, dried over Na2SO4, and filtered.
The solvent was evaporated to afford a colorless oil. The residue
was column-chromatographed on an open column with silica gel
(n-hexane/acetone = 2:1 to 1:1) to afford 23 as a colorless oil
(335.9 mg, 1.326 mmol, 78%).
= 8.2 Hz), 5.163−5.099 (2H, m), 4.681−4.642 (1H, m), 4.493 (1H, dd,
J = 11.5 Hz, 3.8 Hz), 4.401 (1H, dd, J = 11.3 Hz, 3.5 Hz), 4.293 (4H, br
1H NMR (400 MHz, CDCl3): 7.398−7.310 (5H, m), 6.069 (2H, br
s), 5.150 (2H, s), 4.589 (1H, t, J = 6.0 Hz), 4.122−3.852 (2H, m), 3.002
(3H, s). 13C NMR (100 MHz, CDCl3): 173.87, 173.25, 157.42, 135.97,
128.52, 128.16, 127.93, 127.81, 67.96, 61.88, 61.00, 60.73, 33.69, 33.35
s), 2.046 (3H, s).
Compound 21.
−
(two rotamers). HRMS (ESI-TOF, [M − H]−): calcd for C12H14NO5 ,
252.0877; found, 252.0887.
Compound 24.
Compound 20 (834.7 mg, 2.968 mmol) was dissolved in
anhydrous toluene (35 mL). Paraformaldehyde (1004.8 mg)
and CSA (71.2 mg, 0.307 mmol) were added to the solution.
The whole was heated to reflux and stirred under an Ar
atmosphere for 2.5 h. The mixture was filtered on Celite. To the
filtrate, Et2O (15 mL) and a saturated solution of NaHCO3 (20
mL) were added. The organic layer was separated and washed
with brine, dried over Na2SO4, and filtered. The solvent was
evaporated to afford a yellow oil. The residue was column-
chromatographed on an open column with silica gel (n-hexane/
AcOEt = 3:1) to afford 21 as a colorless oil (600.9 mg, 2.049
mmol, 69%).
Compound 23 (310.1 mg, 1.224 mmol) was dissolved in
anhydrous THF (12 mL). Triethylamine (510 μL), TBAI
(136.2 mg, 0.3697 mmol), and BnBr (160 μL) were added at 0
°C, and the mixture was stirred at room temperature under an Ar
atmosphere for 20.4 h. H2O (20 mL) was added, and the whole
was extracted three times with AcOEt (15 mL × 3). The
combined organic layer was washed with brine, dried over
Na2SO4, and filtered. The solvent was evaporated to afford a
yellow oil. The residue was column-chromatographed on an
open column with silica gel (n-hexane/AcOEt = 3:2) to afford
24 as a colorless oil (285.2 mg, 0.8306 mmol, 68%).
1H NMR (400 MHz, CDCl3): 7.415−7.337 (5H, m), 5.583 (1H, br
1H NMR (400 MHz, CDCl3): 7.341−7.271 (5H, m), 5.232−4.995
(4H, m), 4.609 and 4.509 (1H, 2t, J = 5.0 Hz and 6.5 Hz), 4.133−4.063
(1H, m), 3.979−3.963 and 3.822 (1H, m and br s), 2.971 and 2.964
(3H, 2s), 2.643 and 2.260 (1H, 2br s), (two rotamers). 13C NMR (100
MHz, CDCl3): 170.03, 169.75, 157.01, 155.93, 136.21, 136.00, 135.20,
135.01, 128.51, 128.40, 128.30, 128.10, 128.07, 127.98, 127.88, 127.69,
s), 5.255 (1H, dd, J = 4.2 Hz, 0.9 Hz), 5.206 (2H, s), 4.648 (1H, br s),
4.445−4.362 (2H, m), 2.044 (3H, s). 13C NMR (100 MHz, CDCl3):
169.93, 152.36, 135.08, 128.70, 128.38, 78.61, 68.22, 62.34, 54.50,
a
+
20.56. HRMS (ESI-TOF, [M + Na]+): calcd for C14H15NNaO6 ,
316.0792; found, 316.0794.
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Figure 10. Synthesis of 7e.
67.54, 66.99, 61.93, 61.45, 60.77, 33.46, 33.22 (two rotamers). HRMS
mg, 0.236 mmol) in anhydrous THF (1.0 mL) was added. The
mixture was stirred at room temperature under an Ar
atmosphere for 3.1 h. TBHP in decane (5.0−6.0 M) (41 μL)
was added, and the mixture was stirred at room temperature for
2 h. H2O (10 mL) was added, and the whole was extracted three
times with CH2Cl2 (10 mL × 3). The combined organic layer
was washed with brine, dried over Na2SO4, and filtered. The
solvent was evaporated to afford a colorless oil. The residue was
column-chromatographed on a flash column with silica gel
(CHCl3/AcOEt = 4:1) to afford 26 as a colorless oil (67.0 mg,
0.0684 mmol, 61%).
+
(ESI-TOF, [M + H]+): calcd for C19H22NO5 , 344.1492; found,
344.1503.
Compound 25.
Compound 24 (124.0 mg, 0.3611 mmol) was dissolved in
CH2Cl2 and toluene. To remove traces of water, the solution was
evaporated. Compound 3 (crude, 260.0 mg, 0.7681 mmol) was
added, and the mixture was dissolved in anhydrous CH2Cl2
(1.25 mL). A solution of 1H-tetrazole (26.9 mg, 0.384 mmol) in
anhydrous THF (1.25 mL) was added at 0 °C. The mixture was
stirred at room temperature under an Ar atmosphere for 2 h. A
saturated aqueous solution of NaHCO3 (10 mL) was added, and
the whole was extracted three times with CH2Cl2 (10 mL × 3).
The combined organic layer was washed with brine, dried over
Na2SO4, and filtered. The solvent was evaporated to afford a
colorless oil. The residue was column-chromatographed on an
open column with silica gel (n-hexane/AcOEt/Et3N = 40:1:1 to
40:3:1) to afford 25 as a colorless oil (135.3 mg, 0.2330 mmol,
65%).
1H NMR (400 MHz, CDCl3): (mixture of diastereomers and two
rotamers) 7.305−7.114 (21H, m), 6.742−6.691 (3H, m), 6.111−5.999
(1H, m), 5.186−4.975 (6H, m), 4.889−4.829 and 4.654−4.606 (1H,
2m), 4.587−4.552 (1H, m), 4.517−4.307 (3H, m), 4.025−3.978 (1H,
m), 3.906−3.848 (3H, m), 3.587−3.551 (1H, m), 3.404−3.266 (2H,
m), 2.933−2.851 (5H, m), 2.392 (2H, t, J = 7.2 Hz), 1.741 (2H, quin, J
= 6.9 Hz), 1.427−1.391 (2H, m), 1.299−1.264 (14H, m), 0.879 (3H, t,
J = 6.7 Hz). 13C NMR (100 MHz, CDCl3): (mixture of diastereomers
and two rotamers) 172.33, 172.27, 167.94, 167.89, 159.21, 156.58,
156.55, 155.62, 142.29, 137.64, 136.14, 135.91, 135.37, 134.97, 134.82,
129.31, 128.66, 128.55, 128.42, 128.21, 128.18, 128.08, 128.01, 127.94,
127.91, 127.87, 127.62, 120.37, 114.62, 111.99, 75.34, 71.78, 69.67,
69.61, 67.78, 67.60, 67.56, 67.23, 67.19, 66.47, 64.74, 64.63, 64.57,
59.85, 38.88, 38.00, 33.75, 33.55, 32.90, 32.75, 31.82, 31.56, 29.53,
29.50, 29.34, 29.25, 25.99, 22.60, 14.04. HRMS (ESI-TOF, [M +
Na]+): calcd for C56H71KN2O11P+, 1017.4427; found, 1017.4450.
Compound 7c.
1H NMR (400 MHz, CDCl3): 7.343−7.240 (15H, m), 5.214−4.987
and 4.829−4.801 (5H, 2m), 4.731−4.563 (2H, m), 4.147−3.952 (2H,
m), 3.641−3.548 (2H, m), 3.015 and 3.010 (3H, 2s), 1.161−1.121
(12H, m) (two rotamers). 13C NMR (100 MHz, CDCl3): 169.28,
169.26, 169.20, 156.78, 156.72, 156.01, 155.97, 139.35, 139.28, 136.58,
136.37, 135.46, 135.30, 128.53, 128.50, 128.42, 128.38, 128.30, 128.22,
128.06, 127.91, 127.82, 127.80, 127.73, 127.72, 127.30, 127.26, 127.23,
126.92, 126.89, 126.86, 67.38, 66.85, 65.39, 65.33, 65.28, 65.22, 65.15,
65.09, 61.72, 61.55, 61.48, 61.42, 61.31, 61.26, 61.09, 60.93, 60.51,
60.43, 60.35, 60.25, 60.17, 43.13, 43.00, 32.73, 32.60, 32.46, 32.25,
24.57, 24.55, 24.51, 24.48, 24.45 (two rotamers). HRMS (ESI-TOF,
[M + Na]+): calcd for C32H41N2NaO6P+, 603.2594; found, 603.2586.
Compound 26.
Compound 26 (60.0 mg, 0.0613 mmol) and Pd/C (8.5 mg)
were dissolved in MeOH (2 mL) and AcOH (0.5 mL). The
mixture was stirred at room temperature under a H2 atmosphere
for 27.4 h. The mixture was filtered on Celite, and the solvent
was evaporated. The residue was purified by reversed-phase
medium-pressure liquid chromatography (CH3CN/H2O = 3:7
to 7:3, containing 0.1% HCOOH). The solid was vacuum-dried
at 40 °C to afford 7c as a colorless powder (14.4 mg, 0.0251
mmol, 41%).
1H NMR (400 MHz, DMSO-d6): 7.938 (1H, t, J = 5.7 Hz), 7.148
(1H, dd, J = 8.1 Hz, 8.1 Hz), 6.756−6.698 (3H, m), 4.177 (1H, t, J =
11.0 Hz), 4.073−3.994 (1H, m), 3.928−3.879 (3H, m), 3.623−3.529
(4H, m), 3.174−3.112 (1H, m), 3.046−2.984 (1H, m), 2.755 (2H, t, J
Compounds 25 (110.9 mg, 0.1910 mmol) and 2 (54.2 mg, 0.112
mmol) were dissolved in CH2Cl2 and toluene. To remove traces
of water, the solution was evaporated. The mixture was dissolved
in anhydrous CH2Cl2 (1.0 mL). A solution of 1H-tetrazole (16.5
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Figure 11. Synthesis of 7f.
= 7.9 Hz), 2.558 (3H, s), 2.388−2.349 (2H, m), 1.684 (2H, quin, J = 6.9
Hz), 1.408−1.358 (2H, m), 1.249 (14H, br s), 0.855 (3H, t, J = 6.9 Hz).
HRMS (ESI-TOF, [M − H]−): calcd for C27H46N2O9P−, 573.2946;
found, 573.2948. Anal. Calcd for C27H47N2O9P·2H2O: C, 53.10; H,
8.42; N, 4.59. Found: C, 53.28; H, 8.16; N, 4.81. HPLC: 98%
(CH3CN/H2O = 1:1 (0.1% HCO2H)).
Compound 29.
Compounds 28 (50.5 mg, 0.153 mmol) and 4 (161.1 mg, 0.2234
mmol) were dissolved in CH2Cl2 and toluene. To remove traces
of water, the solution was evaporated. The mixture was dissolved
in anhydrous CH2Cl2 (0.75 mL). A solution of 1H-tetrazole
(21.2 mg, 0.303 mmol) in anhydrous THF (0.75 mL) was
added. The mixture was stirred at room temperature under an Ar
atmosphere for 2 h. TBHP in decane (5.0−6.0 M) (55 μL) was
added, and the mixture was stirred at room temperature for 2 h.
H2O (10 mL) was added, and the whole was extracted three
times with CH2Cl2 (10 mL × 3). The combined organic layer
was washed with brine, dried over Na2SO4, and filtered. The
solvent was evaporated to afford a colorless oil. The residue was
column-chromatographed on a flash column with silica gel three
times (first, CHCl3/AcOEt = 5:1, second, CHCl3/AcOEt = 6:1,
third, CHCl3/AcOEt = 6:1) to afford 29 as a colorless oil (pure
fraction, 25.6 mg, 0.0265 mmol, 17%).
Synthesis of 7e (Figure 10). Compounds 27 and 28.
D-Serine (1051.2 mg, 10.003 mmol) was dissolved in THF (20
mL) and a saturated aqueous solution of NaHCO3 (15 mL). Z-
Cl (2050.1 mg, 12.018 mmol) and THF (10 mL) were added,
and the whole was stirred at room temperature under an Ar
atmosphere for 24.2 h. H2O (15 mL) and 2 M aqueous solution
of HCl (15 mL) were added, and the whole was extracted three
times with AcOEt (30 mL × 3). The combined organic layer was
washed with brine, dried over Na2SO4, and filtered. The solvent
was evaporated to afford a pale yellow solid. The residue was
column-chromatographed on an open column with silica gel (n-
hexane/acetone = 2:1 to 0:1) to afford 27 (1534.2 mg) (Figure
10).
1H NMR (400 MHz, CDCl3): (mixtures of diastereomers) 7.318−
7.257 (20H, m), 7.159−7.119 (1H, m), 6.733−6.695 (3H, m), 6.141 (J
= 8.2 Hz) and 5.940 (J = 8.3 Hz) (1H, 2d), 5.882−5.833 (1H, m),
5.208−4.954 (6H, m), 4.598−4.542 (2H, m), 4.473−4.360 (2H, m),
4.319−4.239 (1H, m), 4.064−3.850 (4H, m), 3.574−3.551 (1H, m),
3.407−3.240 (2H, m), 2.874−2.818 (2H, m), 2.378−2.296 (2H, m),
1.743 (2H, quin, J = 7.0 Hz), 1.475−1.391 (2H, m), 1.300−1.201
(14H, m), 0.880 (3H, t, J = 6.8 Hz). 13C NMR (100 MHz, CDCl3):
(mixtures of diastereomers) 172.25, 168.90, 168.81, 159.26, 155.90,
155.84, 142.33, 137.63, 137.47, 136.01, 135.37, 135.31, 134.89, 129.37,
128.74, 128.63, 128.60, 128.49, 128.26, 128.21, 128.08, 127.98, 127.96,
120.41, 114.66, 112.04, 75.48, 75.42, 71.91, 71.73, 69.82, 69.77, 67.84,
67.74, 67.68, 67.43, 67.17, 66.67, 66.61, 54.57, 54.51, 54.44, 39.05,
38.07, 37.98, 31.87, 31.56, 29.58, 29.55, 29.40, 29.30, 26.04, 22.65,
14.09. HRMS (ESI-TOF, [M + Na]+): calcd for C55H69N2NaO11P+,
987.4531; found, 987.4503.
Compound 27 (1521.8 mg) and TBAI (702.9 mg, 1.908 mmol) were
dissolved in anhydrous THF (40 mL). Et3N (2.65 mL) and BnBr (905
μL) were added at 0 °C. Then, the mixture was stirred at room
temperature under an Ar atmosphere for 18.4 h. H2O (40 mL) was
added, and the whole was extracted three times with AcOEt (40 mL ×
3). The combined organic layer was washed with brine, dried over
Na2SO4, and filtered. The solvent was evaporated to afford a pale yellow
oil. The residue was column-chromatographed on an open column with
silica gel (n-hexane/AcOEt = 3:2) to afford 28 as a colorless solid
(907.0 mg, 2.754 mmol, 43% from D-serine).
1H NMR (400 MHz, CDCl3): 7.346 (10H, br s), 5.741−5.726 (1H,
m), 5.210 (2H, s), 5.117−5.114 (2H, m), 4.487 (1H, br s), 3.997 (1H,
br s), 3.939 (1H, br s), 2.180−2.165 (1H, m). 13C NMR (100 MHz,
CDCl3): 170.33, 156.24, 135.99, 135.06, 128.62, 128.52, 128.50,
128.22, 128.16, 128.09, 67.49, 67.20, 63.27, 56.15.
Compound 7e.
+
HRMS (ESI-TOF, [M + Na]+): calcd for C18H19NNaO5 , 352.1155;
found, 352.1163. Anal. Calcd for C18H19NO5: C, 65.64; H, 5.82; N,
4.25. Found: C, 65.47; H, 5.90; N, 4.28.
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(ESI-TOF, [M + H]+): calcd for C24H35NO4P+, 432.2298; found,
432.2305.
Compound 32.
Compound 29 (23.4 mg, 0.0242 mmol) and Pd/C (10.2 mg)
were dissolved in MeOH (3.0 mL). The mixture was stirred at
room temperature under a H2 atmosphere for 22 h. The mixture
was filtered on Celite, and the solvent was evaporated. The
residue was purified by reversed-phase medium-pressure liquid
chromatography (CH3CN/H2O = 1:3 to 7:3, containing 0.1%
HCOOH) to afford 7e (4.6 mg, 0.0082 mmol, 34%). 1H NMR
(400 MHz, DMSO-d6): 8.670 (2H, br s), 7.950 (1H, t, J = 5.7
Hz), 7.148 (1H, dd, J = 8.1 Hz, 8.1 Hz), 6.756−6.699 (3H, m),
4.058−4.019 (2H, m), 3.929−3.897 (3H, m), 3.593−3.542
(3H, m), 3.138−2.993 (2H, m), 2.755 (2H, t, J = 7.8 Hz), 2.366
(2H, t, J = 8.0 Hz), 1.685 (2H, quin, J = 6.9 Hz), 1.394−1.250
(16H, m), 0.856 (3H, t, J = 6.8 Hz). HRMS (ESI-TOF, [M −
H]−): calcd for C26H44N2O9P−, 559.2790; found, 559.2776.
HPLC: 98% (CH3CN/H2O = 1:1 (0.1% HCO2H)).
Compounds 31 (106.0 mg, 0.2456 mmol) and 2 (84.3 mg, 0.174
mmol) were dissolved in CH2Cl2 and toluene. To remove traces
of water, the solution was evaporated. The residue was dissolved
in anhydrous CH2Cl2 (1.5 mL) and a solution of 1H-tetrazole
(24.7 mg, 0.353 mmol) in anhydrous THF (1.5 mL) was added.
The mixture was stirred at room temperature under an Ar
atmosphere for 2.5 h. TBHP in decane (5.0−6.0 M) (60 μL) was
added, and the mixture was stirred at room temperature for 1.5
h. H2O (10 mL) was added, and the whole was extracted three
times with CH2Cl2 (10 mL × 3). The combined organic layer
was washed with brine, dried over Na2SO4, and filtered. The
solvent was evaporated to afford a colorless oil. The residue was
column-chromatographed on a flash column with silica gel twice
(CHCl3/AcOEt = 4:1) to afford 32 as a colorless oil (123.4 mg,
0.1487 mmol, 61%).
Synthesis of 7f (Figure 11). Compound 30.
Methyl (R)-(−)-3-hydroxyisobutyrate (243.3 mg, 2.060 mmol)
was dissolved in MeOH (2 mL). An aqueous solution of KOH
(1.95 M, 2 mL) was added, and the whole was stirred at room
temperature for 75 min. The solvent was evaporated. The
residue was dissolved in anhydrous DMF (3 mL), and benzyl
bromide (245 μL) was added to the mixture. The mixture was
heated to 80 °C under an Ar atmosphere for 15.6 h. A 2 M
aqueous solution of HCl (5 mL) and H2O (10 mL) were added,
and the whole was extracted three times with AcOEt (10 mL ×
3). The combined organic layer was washed with brine, dried
over Na2SO4, and filtered, and the solvent was evaporated. The
residue was column-chromatographed on a flash column with
silica gel (n-hexane/AcOEt = 3:1) to afford 30 as a colorless oil
(192.1 mg, 0.9890 mmol, 48%) (Figure 11).
1H NMR (400 MHz, CDCl3): (mixtures of diastereomers) 7.350−
7.278 (15H, m), 7.173−7.125 (1H, m), 6.749−6.698 (3H, m), 6.033−
5.946 (1H, m), 5.142−5.074 (2H, m), 5.025−4.997 (2H, m), 4.590
(1H, d, J = 11.7 Hz), 4.478 (1H, d, J = 11.7 Hz), 4.232−4.165 (1H, m),
4.097−3.973 (2H, m), 3.935−3.881 (3H, m), 2.894−2.783 (3H, m),
2.407−2.368 (2H, m), 1.780−1.726 (2H, m), 1.693 (2H, br s), 1.464−
1.264 (16H, m), 1.183−1.160 (3H, m), 0.880 (3H, t, J = 6.9 Hz). 13
C
NMR (100 MHz, CDCl3): (mixtures of diastereomers) 173.19, 173.15,
172.27, 172.25, 159.26, 142.36, 137.74, 135.61, 129.37, 128.65, 128.60,
128.55, 128.48, 128.26, 128.05, 127.99, 127.97, 127.95, 120.42, 114.64,
112.05, 75.45, 75.39, 71.86, 69.55, 69.50, 68.74, 68.67, 67.84, 66.60,
66.56, 66.36, 40.45, 40.41, 40.37, 40.33, 38.99, 38.13, 31.88, 31.62,
29.59, 29.56, 29.40, 29.30, 26.05, 22.66, 14.09, 13.38. HRMS (ESI-
TOF, [M + Na]+): calcd for C48H64NNaO9P+, 852.4211; found,
852.4205. Anal. Calcd for C48H64NO9P·0.5H2O: C, 68.71; H, 7.81; N,
1.67. Found: C, 68.81; H, 7.79; N, 1.58.
1H NMR (400 MHz, CDCl3): 7.394−7.304 (5H, m), 5.519 (2H, s),
3.777−3.692 (2H, m), 2.771−2.687 (1H, m), 1.202 (3H, d, J = 7.2 Hz).
13C NMR (100 MHz, CDCl3): 175.44, 135.76, 128.59, 128.28, 128.03,
66.40, 64.53, 41.75, 13.40. HRMS (ESI-TOF, [M + Na]+): calcd for
+
C11H14NaO3 , 217.0835; found, 217.0840.
Compound 7f.
Compound 31.
Compound 32 (51.2 mg, 0.0617 mmol) and Pd/C (5.8 mg)
were dissolved in MeOH (4 mL) and AcOH (1 mL). The
mixture was stirred at room temperature under a H2 atmosphere
for 28.5 h. The mixture was filtered on Celite, and the solvent
was evaporated. The residue was purified by reversed-phase
medium-pressure liquid chromatography (CH3CN/H2O = 3:7
to 7:3, containing 0.1% HCOOH). After vacuum drying at 40
°C, the solid was washed with CH3CN (300 μL × 3). The solid
was vacuum-dried at 40 °C to afford 7f (18.2 mg, 0.0325 mmol,
53%).
Compound 30 (183.8 mg, 0.9470 mmol) was dissolved in
CH2Cl2 and toluene. To remove traces of water, the solution was
evaporated. Compound 3 (676.1 mg, 1.997 mmol) was added,
and the mixture was dissolved in anhydrous CH2Cl2 (3.0 mL). A
solution of 1H-tetrazole (67.5 mg, 0.964 mmol) in anhydrous
THF (3.0 mL) was added at 0 °C. The mixture was stirred at
room temperature under an Ar atmosphere for 2 h. A saturated
aqueous solution of NaHCO3 (10 mL) was added, and the
aqueous layer was extracted three times with CH2Cl2 (10 mL ×
3). The combined organic layer was washed with brine, dried
over Na2SO4, and filtered. The solvent was evaporated to afford
a colorless oil. The residue was column-chromatographed on an
open column with silica gel (first, n-hexane/AcOEt/Et3N =
40:2:1, second n-hexane/AcOEt/Et3N = 40:1:1) to afford 31 as
a colorless oil (252.5 mg, 0.5852 mmol, 62%).
1H NMR (400 MHz, DMSO-d6): 7.889 (1H, t, J = 5.8 Hz), 7.149
(1H, dd, J = 8.1 Hz, 8.1 Hz), 6.754−6.699 (3H, m), 3.980−3.834 (4H,
m), 3.752−3.608 (3H, m), 3.183−3.121 (1H, m), 3.055−2.992 (1H,
m), 2.757 (2H, t, J = 7.9 Hz), 2.692−2.642 (1H, m), 2.374 (2H, t, J =
7.9 Hz), 1.684 (2H, quin, J = 6.9 Hz), 1.407−1.248 (16H, m), 1.072
(3H, d, J = 7.1 Hz), 0.855 (3H, t, J = 6.8 Hz). HRMS (ESI-TOF, [M −
H]−): calcd for C27H45NO9P−, 558.2837; found, 558.2862. Anal. Calcd
for C27H46NO9P·H2O: C, 56.14; H, 8.38; N, 2.42. Found: C, 56.20; H,
8.15; N, 2.35. HPLC: 95% (CH3CN/H2O = 3:2 (0.1% HCO2H)).
1H NMR (400 MHz, CD2Cl2): 7.348−7.229 (10H, m), 5.133−
5.052 (2H, m), 4.703−4.647 (1H, m), 4.632−4.580 (1H, m), 3.895−
3.552 (4H, m), 2.833−2.742 (1H, m), 1.192−1.146 (15H, m). HRMS
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Figure 12. Synthesis of 7g.
Synthesis of 7g (Figure 12). Compound 33.
dd, J = 12.7 Hz, 8.0 Hz), 4.618 (1H, dd, J = 12.7 Hz, 8.2 Hz), 3.777 (3H,
s), 3.698 (1H, dd, J = 9.6 Hz, 5.8 Hz), 3.646−3.553 (3H, m), 1.206
(6H, d, J = 1.8 Hz), 1.165 (6H, d, J = 1.8 Hz), 1.148 (6H, d, J = 1.8 Hz).
13C NMR (100 MHz, CDCl3): 176.12, 159.33, 139.66, 139.58, 129.55,
128.43, 128.16, 127.10, 126.82, 113.76, 70.35, 70.19, 65.95, 65.11,
64.94, 55.19, 44.27, 44.19, 43.06, 42.94, 24.59, 24.53, 24.52, 24.47,
22.17. HRMS (ESI-TOF, [M + H]+): calcd for C26H39NO5P+,
476.2560; found, 476.2587.
Hydroxypivalic acid (1000.0 mg, 8.4651 mmol), p-methox-
ybenzoyl chloride (1590.9 mg, 10.158 mmol), and KHCO3
(1016.9 mg, 10.157 mmol) were dissolved in anhydrous DMF
(10 mL). The mixture was stirred at room temperature for 44.5
h. H2O (10 mL) was added, and the whole was extracted with
AcOEt. The combined organic layer was washed with brine,
dried over Na2SO4, and filtered. The solvent was evaporated.
The residue was column-chromatographed on an open column
with silica gel (n-hexane/AcOE = 3:1 to 1:1) to afford 33 as a
colorless oil (1590.2 mg, 6.6736 mmol, 79%) (Figure 12).
1H NMR (400 MHz, CDCl3): 7.272 (2H, d, J = 9.4 Hz), 6.889 (2H,
d, J = 8.8 Hz), 5.078 (2H, s), 3.811 (3H, s), 3.556 (2H, s), 2.386 (1H, br
s), 1.195 (6H, s). 13C NMR (100 MHz, CDCl3): 177.43, 159.57,
129.66, 128.62, 127.99, 113.95, 69.66, 66.20, 55.24, 44.20, 22.03.
Compound 35.
Compounds 34 (129.6 mg, 0.2725 mmol) and 2 (81.7 mg, 0.169
mmol) were dissolved in CH2Cl2 and toluene. To remove traces
of water, the solution was evaporated. The mixture was dissolved
in anhydrous CH2Cl2 (1.2 mL). A solution of 1H-tetrazole (23.7
mg, 0.338 mmol) in anhydrous THF (1.2 mL) was added at 0
°C. The mixture was stirred at room temperature under an Ar
atmosphere for 3.3 h. TBHP in decane (5.0−6.0 M) (60 μL) was
added, and the mixture was stirred at room temperature for 2.1
h. H2O (10 mL) was added, and the whole was extracted three
times with CH2Cl2 (10 mL × 3). The combined organic layer
was washed with brine, dried over Na2SO4, and filtered. The
solvent was evaporated to afford a colorless oil. The residue was
column-chromatographed on a flash column with silica gel
(CHCl3/AcOEt = 4:1) to afford 35 as a colorless oil (116.3 mg,
0.1331 mmol, 79%).
+
HRMS (ESI-TOF, [M + H]+): calcd for C13H18NaO4 , 261.1097;
found, 261.1085.
Compound 34.
Compound 33 (237.8 mg, 0.9980 mmol) was dissolved in
CH2Cl2 and toluene. To remove traces of water, the solution was
evaporated. Compound 3 (517.6 mg, 1.529 mmol) and
anhydrous CH2Cl2 (3.0 mL) were added. A solution of 1H-
tetrazole (70.8 mg, 1.01 mmol) in anhydrous THF (3.0 mL) was
added at room temperature. The mixture was stirred at room
temperature under an Ar atmosphere for 3 h. A saturated
aqueous solution of NaHCO3 (10 mL) was added, and the
whole was extracted three times with CH2Cl2 (10 mL × 3). The
combined organic layer was washed with brine, dried over
Na2SO4, and filtered. The solvent was evaporated to afford a
colorless oil. The residue was column-chromatographed on an
open column with silica gel (n-hexane/AcOEt/Et3N = 40:1:1)
to afford 34 as a colorless oil (232.8 mg, 0.4895 mmol, 49%).
1H NMR (400 MHz, CDCl3): 7.330−7.295 (4H, m), 7.275−7.246
(3H, m), 6.838 (2H, d, J = 8.8 Hz), 5.065−4.998 (2H, m), 4.695 (1H,
1H NMR (400 MHz, CDCl3): (mixtures of diastereomers) 7.346−
7.221 (12H, m), 7.173−7.124 (1H, m), 6.830 (2H, d, J = 8.4 Hz),
6.750−6.697 (3H, m), 6.057 and 6.003 (1H, 2t, J = 5.9 Hz), 5.027 (2H,
s), 5.009−4.969 (2H, m), 4.590 (1H, d, J = 11.6 Hz), 4.483 (1H, dd, J =
11.7 Hz, 2.2 Hz), 4.025−3.956 (3H, m), 3.934−3.880 (3H, m), 3.750
(3H, s), 3.606−3.569 (1H, m), 3.426−3.310 (2H, m), 2.877 (2H, t, J =
7.8 Hz), 2.414−2.371 (2H, m), 1.783−1.709 (2H, m), 1.447−1.264
(16H, m), 1.184−1.173 (6H, m), 0.880 (3H, t, J = 6.8 Hz). 13C NMR
(100 MHz, CDCl3): (mixtures of diastereomers) 174.99, 174.94,
172.27, 172.24, 159.49, 159.23, 142.35, 137.74, 135.63, 135.60, 135.57,
135.53, 129.70, 129.35, 128.60, 128.58, 128.45, 127.94, 127.92, 127.89,
120.40, 114.62, 113.85, 112.03, 75.48, 75.45, 75.41, 75.38, 73.26, 73.20,
71.82, 69.49, 69.45, 69.43, 69.40, 67.82, 66.42, 66.40, 66.34, 66.28,
55.17, 43.52, 43.50, 43.44, 43.41, 38.92, 38.10, 31.86, 31.61, 29.56,
29.54, 29.38, 29.28, 26.02, 22.63, 21.76, 14.07. HRMS (ESI-TOF, [M +
Na]+): calcd for C50H68NNaO10P+, 896.4473; found, 896.4480.
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Figure 13. Synthesis of 8a.
Compounds 36 and 7g.
Na2SO4, and filtered. The solvent was evaporated. The residue
was column-chromatographed on an open column with silica gel
twice (first, n-hexane/AcOEt = 1:1, second, n-hexane/AcOEt =
2:1) to afford 37 as a colorless oil (1408.4 mg, 4.7688 mmol,
68%) (Figure 13).
1H NMR (400 MHz, CDCl3): 7.372−7.328 (5H, m), 5.456 (1H, br
s), 5.247−5.180 (2H, m), 4.421 (1H, br s), 3.983 (1H, dd, J = 11.2 Hz,
3.8 Hz), 3.910 (1H, dd, J = 11.2 Hz, 3.4 Hz), 1.440 (9H, s). 13C NMR
(100 MHz, CDCl3): 170.64, 155.71, 135.18, 128.62, 128.48, 128.17,
80.33, 67.39, 63.62, 55.84, 28.26. HRMS (ESI-TOF, [M + Na]+): calcd
Compounds 35 (36.3 mg, 0.0415 mmol) and 1,3-dimethox-
ybenzene (58.1 mg, 0.421 mmol) were dissolved in anhydrous
CH2Cl2 (0.5 mL). TFA (0.1 mL) was added at 0 °C, and the
mixture was stirred at room temperature for 1.5 h. CH2Cl2 (ca. 2
mL) was added, and the solvent was removed by evaporation.
The residue (crude 36, colorless oil, 105.2 mg) and Pd/C (18.1
mg) were dissolved in MeOH (2.0 mL). The mixture was stirred
at room temperature under a H2 atmosphere for 20.6 h. Then,
the mixture was filtered on Celite, and the solvent was
evaporated. The residue was purified by a flash column with
silica gel (CHCl3/MeOH/AcOH = 8:1:1) to afford 7g as a pale
yellow powder (12.1 mg, 0.0211 mmol, 51%).
+
for C15H21NNaO5 , 318.1312; found, 318.1304.
Compound 38.
To the solution of 37 (1374.7 mg, 4.6547 mmol) in CH2Cl2
(17.5 mL), TFA (3.5 mL) was added at 0 °C. The mixture was
stirred at room temperature for 2.3 h. The solvent was
evaporated, and the residue was dried to afford 38 as a pale
yellow solid (1426.5 mg, 4.6129 mmol, 99%, calculated as TFA
salt), which was used without further purification.
1H NMR (400 MHz, DMSO-d6): 8.013 (1H, br s), 7.140 (1H, dd, J
= 8.1 Hz, 8.1 Hz), 6.752−6.690 (3H, m), 3.909 (2H, t, J = 6.5 Hz),
3.585−3.506 (4H, m), 3.043 (2H, br s), 2.750 (2H, t, J = 7.9 Hz), 2.357
(2H, t, J = 8.0 Hz), 1.681 (2H, quin, J = 6.9 Hz), 1.405−1.247 (16H,
m), 1.025 (6H, s), 0.853 (3H, t, J = 6.8 Hz). HRMS (ESI-TOF, [M −
H]−): calcd for C28H47NO9P−, 572.2994; found, 572.2993. HPLC:
99% (CH3CN/H2O = 3:2 (0.1% HCO2H)).
+
HRMS (ESI-TOF, [M + H]+): calcd for C10H14NO3 , 196.0968;
found, 196.0952.
Compound 39.
Synthesis of 8a (Figure 13). Compound 37.
Compound 38 (620.0 mg, 2.005 mmol) was dissolved in
anhydrous CH2Cl2 (8.0 mL). Et3N (560 μL) and propionyl
chloride (0.2 mL, 2.3 mmol) were added, and the mixture was
stirred at room temperature under an Ar atmosphere for 40 min.
MeOH (ca. 2 mL) was added, and the solvent was evaporated
with silica gel. The residue was column-chromatographed on an
open column with silica gel (n-hexane/acetone = 3:2) to afford
39 as a pale yellow solid (364.4 mg, 1.450 mmol, 72%).
(tert-Butoxycarbonyl)-L-serine (1443.2 mg, 7.0328 mmol) and
Cs2CO3 (2741.1 mg, 8.4130 mmol) were dissolved in
anhydrous DMF (40 mL). Benzyl bromide (1.0 mL) was
added, and the mixture was stirred at room temperature under
an Ar atmosphere for 20 h. H2O (70 mL) was added, and the
whole was extracted three times with Et2O (40 mL × 3). The
combined organic layer was washed with brine, dried over
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Figure 14. Synthesis of 8b.
1H NMR (400 MHz, CDCl3): 7.394−7.318 (5H, m), 6.476 (1H, d, J
= 6.6 Hz), 5.246−5.182 (2H, m), 4.735−4.698 (1H, m), 3.990 (1H, dd,
J = 11.2 Hz, 4.0 Hz), 3.923 (1H, dd, J = 11.2 Hz, 3.4 Hz), 2.518 (1H, br
s), 2.294 (2H, q, J = 7.6 Hz), 1.165 (3H, t, J = 7.6 Hz). 13C NMR (100
MHz, CDCl3): 174.39, 170.41, 135.05, 128.65, 128.54, 128.15, 67.53,
63.57, 54.78, 29.44, 9.55. HRMS (ESI-TOF, [M + Na]+): calcd for
C13H17NNaO4 , 274.1050; found, 274.1060. mp 51.5−53.0 °C
(colorless needles, recrystallized from n-hexane/CH2Cl2).
Compound 40.
Compounds 40 (383.2 mg, 0.7843 mmol) and 2 (223.8 mg,
0.4627 mmol) were dissolved in CH2Cl2 and toluene. To
remove traces of water, the solution was evaporated. The
mixture was dissolved in anhydrous CH2Cl2 (4.0 mL). A
solution of 1H-tetrazole (63.6 mg, 0.908 mmol) in anhydrous
THF (4.0 mL) was added at 0 °C. The mixture was stirred at
room temperature under an Ar atmosphere for 2.3 h. TBHP in
decane (5.0−6.0 M) (168 μL) was added, and the mixture was
stirred at room temperature for 2 h. H2O (10 mL) was added,
and the whole was extracted three times with CH2Cl2 (10 mL ×
3). The combined organic layer was washed with brine, dried
over Na2SO4, and filtered. The solvent was evaporated to afford
a colorless oil. The residue was column-chromatographed on a
flash column with silica gel (CHCl3/AcOEt = 3:2) to afford 41
as a colorless oil (pure fraction, 51.1 mg, 0.0576 mmol, 12%).
1H NMR (400 MHz, CDCl3): (mixtures of diastereomers) 7.354−
7.274 (15H, m), 7.199−7.128 (1H, m), 6.811−6.660 (3H, m), 5.980−
5.915 (1H, m), 5.223−5.118 (2H, m), 4.997 and 4.975 (2H, 2s), 4.831
(1H, br s), 4.611−4.566 (1H, m), 4.476−4.381 (2H, m), 4.296−4.247
(1H, m), 4.018−3.827 (4H, m), 3.597−3.573 (1H, m), 3.362−3.336
(2H, m), 2.976 (J = 7.9 Hz) and 2.877 (J = 7.7 Hz) (2H, 2t), 2.403 (2H,
t, J = 6.9 Hz), 2.313−2.177 (2H, m), 1.822 (2H, br s), 1.780−1.727
(2H, m), 1.465−1.263 (16H, m), 1.176 (J = 7.6 Hz) and 1.117 (J = 7.6
Hz) (3H, 2t), 0.879 (3H, t, J = 6.7 Hz). 13C NMR (100 MHz, CDCl3):
(mixtures of diastereomers) 173.96, 172.38, 168.88, 159.24, 142.24,
137.57, 137.54, 135.31, 135.25, 134.93, 129.36, 128.78, 128.75, 128.64,
128.56, 128.48, 128.45, 128.21, 128.15, 128.00, 127.94, 120.37, 114.65,
112.00, 75.45, 75.39, 71.94, 69.81, 69.75, 67.82, 67.58, 67.49, 67.43,
67.38, 67.31, 66.63, 66.57, 52.65, 52.58, 39.08, 38.03, 31.83, 31.55,
29.54, 29.52, 29.36, 29.26, 29.16, 26.01, 22.61, 14.06, 9.45. HRMS
(ESI-TOF, [M + Na]+): calcd for C50H67N2NaO10P+, 909.4426; found,
909.4418.
+
Compound 39 (252.3 mg, 1.004 mmol) was dissolved in
CH2Cl2 and toluene. To remove traces of water, the solution was
evaporated. Compound 3 (crude, 571.6 mg, 1.689 mmol) was
added, and the mixture was dissolved in anhydrous CH2Cl2 (4.0
mL). A solution of 1H-tetrazole (72.9 mg, 1.04 mmol) in
anhydrous THF (4.0 mL) was added at 0 °C. The mixture was
stirred at room temperature under an Ar atmosphere for 2.5 h. A
saturated aqueous solution of NaHCO3 (10 mL) was added, and
the whole was extracted three times with CH2Cl2 (15 mL × 3).
The combined organic layer was washed with brine, dried over
Na2SO4, and filtered. The solvent was evaporated to afford a
colorless oil. The residue was column-chromatographed on an
open column with silica gel (n-hexane/AcOEt/Et3N = 40:1:1 to
40:16:1) to afford 40 as a colorless oil (394.9 mg, 0.8083 mmol,
81%).
1H NMR (400 MHz, CDCl3): 7.380−7.272 (10H, m), 6.552 (J = 8.1
Hz) and 6.303 (J = 8.0 Hz) (1H, 2d), 5.170−5.146 (2H, m), 4.813−
4.555 (3H, m), 4.226−4.112 (1H, m), 3.924−3.821 (1H, m), 3.635−
3.533 (2H, m), 2.244−2.185 (1H, m), 1.996−1.870 (1H, m), 1.267−
1118 (13.5H, m), 1.105 (1.5H, t, J = 7.6 Hz). 13C NMR (100 MHz,
CDCl3): 173.65, 173.47, 170.08, 169.99, 139.20, 139.13, 138.83,
135.46, 135.29, 128.53, 128.51, 128.45, 128.32, 128.29, 128.25, 128.19,
128.03, 127.63, 127.37, 127.27, 126.97, 67.26, 67.12, 65.51, 65.33,
65.27, 65.10, 64.40, 64.25, 63.64, 63.49, 53.47, 53.42, 53.34, 43.20,
43.12, 43.07, 43.00, 29.47, 29.15, 24.65, 24.60, 24.58, 24.53, 24.46, 9.56.
HRMS (ESI-TOF, [M + Na]+): calcd for C26H37N2NaO5P+, 511.2332;
found, 511.2337.
Compound 8a.
Compound 41 (47.9 mg, 0.0540 mmol) and Pd/C (5.7 mg)
were dissolved in MeOH (10 mL). The mixture was stirred at
room temperature under a H2 atmosphere for 24 h. The mixture
was filtered on Celite, and the solvent was evaporated. The
residue was purified by reversed-phase medium-pressure liquid
chromatography (CH3CN/H2O = 3:7 to 7:3, containing 0.1%
HCOOH) to afford 8a as a colorless powder (24.5 mg, 0.0397
mmol, 74%).
Compound 41.
1H NMR (400 MHz, DMSO-d6): 12.786 (1H, br s), 8.267 (1H, d, J
= 8.0 Hz), 7.878 (1H, t, J = 5.6 Hz), 7.149 (1H, dd, J = 8.0 Hz, 8.0 Hz),
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Figure 15. Synthesis of 8c.
6.751−6.700 (3H, m), 4.491−4.449 (1H, m), 4.124−4.013 (2H, m),
3.911 (2H, t, J = 6.5 Hz), 3.758−3.614 (3H, m), 3.184−3.123 (1H, m),
3.054−2.991 (1H, m), 2.757 (2H, t, J = 7.9 Hz), 2.376 (2H, t, J = 7.9
Hz), 2.159 (2H, q, J = 7.6 Hz), 1.683 (2H, quin, J = 6.9 Hz), 1.425−
1.248 (16H, m), 0.987 (3H, t, J = 7.6 Hz), 0.854 (3H, t, J = 6.8 Hz).
HRMS (ESI-TOF, [M − H]−): calcd for C29H48N2O10P−, 615.3052;
found, 615.3053. Anal. Calcd for C20H49N2O10P·0.7H2O: C, 55.35; H,
8.07; N, 4.45. Found: C, 55.28; H, 7.87; N, 4.47. HPLC: 96%
(CH3CN/H2O = 1:1 (0.1% HCO2H)).
in anhydrous CH2Cl2 (0.75 mL). A solution of 1H-tetrazole
(22.1 mg, 0.315 mmol) in anhydrous THF (0.75 mL) was added
at 0 °C. The mixture was stirred at room temperature under an
Ar atmosphere for 3 h. TBHP in decane (5.0−6.0 M) (58 μL)
was added, and the mixture was stirred at room temperature for
2 h. H2O (10 mL) was added, and the whole was extracted three
times with CH2Cl2 (10 mL × 3). The combined organic layer
was washed with brine, dried over Na2SO4, and filtered. The
solvent was evaporated to afford a colorless oil. The residue was
column-chromatographed on a flash column with silica gel three
times (first, n-hexane/acetone = 2:1, second, n-hexane/acetone
= 3:1, third, n-hexane/acetone = 3:1) to afford 43 as a colorless
oil (49.5 mg, 0.0549 mmol, 36%).
Synthesis of 8b (Figure 14). Compound 42.
1H NMR (400 MHz, CDCl3): (mixtures of diastereomers) 7.353−
7.272 (15H, m), 7.172−7.125 (1H, m), 6.852−6.699 (4H, m), 5.946
and 5.882 (1H, 2t, J = 5.9 Hz), 5.196−5.115 (2H, m), 4.996−4.974
(2H, m), 4.861−4.808 (1H, m), 4.581 (1H, d, J = 11.7 Hz), 4.464−
4.381 (2H, m), 4.288−4.215 (1H, m), 4.024−3.835 (4H, m), 3.636−
3.545 (1H, m), 3.365−3.329 (2H, m), 2.871 (2H, t, J = 7.8 Hz), 2.437−
2.347 (2H, m), 2.204−2.135 (2H, m), 1.782−1.712 (2H, m), 1.673−
1.572 (2H, m), 1.464−1.392 (2H, m), 1.347−1.263 (14H, m), 0.927−
0.861 (6H, m). 13C NMR (100 MHz, CDCl3): (mixtures of
diastereomers) 173.17, 173.14, 172.34, 172.33, 168.89, 168.86,
159.26, 142.27, 137.60, 137.57, 135.34, 135.28, 134.96, 134.95,
129.38, 128.79, 128.76, 128.66, 128.58, 128.52, 128.50, 128.48,
128.25, 128.18, 128.03, 128.01, 127.95, 120.39, 114.67, 112.03,
75.53, 75.49, 75.47, 75.42, 71.95, 69.83, 69.79, 69.73, 67.84, 67.62,
67.61, 67.52, 67.47, 67.43, 67.37, 66.65, 66.59, 66.54, 66.48, 52.63,
52.56, 39.13, 39.11, 38.07, 38.04, 31.85, 31.57, 29.56, 29.54, 29.38,
29.28, 26.02, 22.63, 18.84, 14.07, 13.64. HRMS (ESI-TOF, [M +
NH4]+): calcd for C51H73N3O10P+, 918.5028; found, 918.5020.
Compound 8b.
L-Serine benzyl ester hydrochloride (694.0 mg, 2.996 mmol)
was dissolved in anhydrous CH2Cl2 (10 mL). Et3N (1040 μL)
and butyryl chloride (375 μL, 3.59 mmol) were added, and the
mixture was stirred at room temperature under an Ar
atmosphere for 30 min. MeOH (ca. 2 mL) was added, and the
solvent was evaporated with silica gel. The residue was column-
chromatographed on an open column with silica gel (n-hexane/
acetone = 2:1 to 3:2) to afford 42 as a colorless solid (675.8 mg,
2.547 mmol, 85%) (Figure 14).
1H NMR (400 MHz, CDCl3): 7.396−7.320 (5H, m), 6.446 (1H, d, J
= 6.8 Hz), 5.249−5.183 (2H, m), 4.745−4.708 (1H, m), 3.993 (1H,
ddd, J = 11.1 Hz, 5.7 Hz, 4.0 Hz), 3.922 (1H, ddd, J = 11.1 Hz, 6.1 Hz,
3.3 Hz), 2.630 (1H, t, J = 5.9 Hz), 2.241 (2H, t, J = 7.5 Hz), 1.721−
1.628 (2H, m), 0.951 (3H, t, J = 7.4 Hz). 13C NMR (100 MHz,
CDCl3): 173.57, 170.41, 135.06, 128.65, 128.55, 128.18, 67.53, 63.67,
54.76, 38.34, 18.96, 13.65. HRMS (ESI-TOF, [M + Na]+): calcd for
+
C14H19NNaO4 , 288.1206; found, 288.1217. Anal. Calcd for
C14H19NO4·0.5H2O: C, 61.30; H, 7.35; N, 5.11. Found: C, 61.66; H,
7.20; N, 5.17. mp 41.0−42.0 °C (colorless powder, recrystallized from
n-hexane/CH2Cl2).
Compound 43.
Compound 43 (46.0 mg, 0.0510 mmol) and Pd/C (15.8 mg)
were dissolved in THF (5 mL). The mixture was stirred at room
temperature under a H2 atmosphere for 3.5 h. The mixture was
filtered on Celite, and the solvent was evaporated. The residue
was purified by reversed-phase medium-pressure liquid
chromatography (CH3CN/H2O = 3:7 to 7:3, containing 0.1%
HCOOH) to afford 8b as a colorless powder (11.7 mg, 0.0186
mmol, 36%).
Compounds 42 (40.8 mg, 0.154 mmol) and 4 (173.4 mg, 0.2405
mmol) were dissolved in CH2Cl2 and toluene. To remove traces
of water, the solution was evaporated. The mixture was dissolved
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1H NMR (400 MHz, DMSO-d6): 8.284 (1H, d, J = 7.8 Hz), 7.876
(1H, t, J = 5.7 Hz), 7.149 (1H, dd, J = 8.1 Hz, 8.1 Hz), 6.754−6.700
(3H, m), 4.495−4.453 (1H, m), 4.123−4.017 (2H, m), 3.911 (2H, t, J
= 6.5 Hz), 3.772−3.601 (4H, m), 3.185−3.123 (1H, m), 3.055−2.991
(1H, m), 2.758 (2H, t, J = 7.9 Hz), 2.377 (2H, t, J = 7.9 Hz), 2.124 (2H,
t, J = 7.3 Hz), 1.683 (2H, quin, J = 6.9 Hz), 1.509 (2H, sextet, J = 7.4
Hz), 1.407−1.356 (2H, m), 1.248 (14H, br s), 0.872−0.835 (6H, m).
HRMS (ESI-TOF, [M − H]−): calcd for C30H50N2O10P−, 629.3209;
found, 629.3228. HPLC: >99% (CH3CN/H2O = 1:1 (0.1% HCO2H)).
Synthesis of 8c (Figure 15). Compound 44.
0.843 (1.5H, J = 7.3 Hz) (3H, 2t). 13C NMR (100 MHz, CDCl3):
173.02, 172.88, 170.05, 169.97, 139.20, 139.12, 138.92, 138.84, 135.44,
135.28, 128.51, 128.50, 128.43, 128.30, 128.28, 128.24, 128.19, 128.05,
127.61, 127.37, 127.17, 126.95, 67.25, 67.12, 65.49, 65.31, 65.26, 65.08,
64.38, 64.22, 63.65, 63.50, 53.46, 53.40, 53.31, 43.19, 43.11, 43.07,
42.99, 36.23, 35.92, 27.60, 27.53, 24.65, 24.59, 24.57, 24.52, 24.45,
22.30, 22.21, 13.75. HRMS (ESI-TOF, [M + Na]+): calcd for
C28H41N2NaO5P+, 539.2645; found, 539.2655.
Compound 46.
Compounds 45 (165.1 mg, 0.3196 mmol) and 2 (97.6 mg, 0.202
mmol) were dissolved in CH2Cl2 and toluene. To remove traces
of water, the solution was evaporated. The mixture was dissolved
in anhydrous CH2Cl2 (1.0 mL). A solution of 1H-tetrazole (27.8
mg, 0.397 mmol) in anhydrous THF (1.0 mL) was added at 0
°C. The mixture was stirred at room temperature under an Ar
atmosphere for 6.5 h. TBHP in decane (5.0−6.0 M) (73 μL) was
added, and the mixture was stirred at room temperature for 1.5
h. H2O (10 mL) was added, and the whole was extracted three
times with CH2Cl2 (10 mL × 3). The combined organic layer
was washed with brine, dried over Na2SO4, and filtered. The
solvent was evaporated to afford a colorless oil. The residue was
column-chromatographed on a flash column with silica gel
(CHCl3/AcOEt = 10:1) to afford 46 as a colorless oil (128.5 mg,
0.1404 mmol, 70%).
L-Serine benzyl ester hydrochloride (693.6 mg, 2.994 mmol)
was dissolved in anhydrous CH2Cl2 (10 mL). Et3N (1040 μL)
and valeryl chloride (400 μL, 3.32 mmol) were added, and the
mixture was stirred at room temperature under an Ar
atmosphere for 30 min. MeOH (ca. 2 mL) was added, and the
solvent was evaporated with silica gel. The residue was column-
chromatographed on an open column with silica gel (n-hexane/
acetone = 2:1 to 3:2) to afford 44 as a colorless solid (794.5 mg,
2.844 mmol, 95%) (Figure 15).
1H NMR (400 MHz, CDCl3): 7.400−7.314 (5H, m), 6.436 (1H, d, J
= 6.8 Hz), 5.248−5.182 (2H, m), 4.740−4.703 (1H, m), 4.016−3.965
(1H, m), 3.921 (1H, ddd, J = 11.1 Hz, 5.8 Hz, 3.4 Hz), 2.611 (1H, t, J =
7.6 Hz), 2.621 (2H, t, J = 7.6 Hz), 1.662−1.586 (2H, m), 1.395−1.302
(2H, m), 0.908 (3H, t, J = 7.3 Hz). 13C NMR (100 MHz, CDCl3):
173.73, 170.41, 135.06, 128.65, 128.55, 128.17, 67.53, 63.68, 54.77,
36.21, 27.59, 22.30, 13.74. HRMS (ESI-TOF, [M + H]+): calcd for
1H NMR (400 MHz, CDCl3): (mixtures of diastereomers) 7.356−
7.270 (15H, m), 7.175−7.128 (1H, m), 6.817−6.702 (4H, m), 5.917 (J
= 6.1 Hz) and 5.850 (J = 5.9 Hz) (1H, 2t), 5.217−5.118 (2H, m),
5.031−4.945 (2H, m), 4.861−4.808 (1H, m), 4.585 (1H, d, J = 11.7
Hz), 4.466−4.386 (2H, m), 4.286−4.216 (1H, m), 4.026−3.838 (4H,
m), 3.615−3.547 (1H, m), 3.404−3.294 (2H, m), 2.872 (2H, t, J = 7.8
Hz), 2.456−2.327 (2H, m), 2.227−2.160 (2H, m), 1.784−1.711 (2H,
m), 1.619−1.535 (2H, m), 1.466−1.394 (2H, m), 1.355−1.265 (16H,
m), 0.895−0.858 (6H, m). 13C NMR (100 MHz, CDCl3): (mixtures of
diastereomers) 173.32, 173.30, 172.34, 172.33, 168.91, 168.89, 159.28,
142.28, 137.61, 137.57, 135.36, 135.29, 134.97, 129.39, 128.81, 128.78,
128.67, 128.60, 128.54, 128.52, 128.50, 128.27, 128.20, 128.06, 128.02,
127.97, 120.41, 114.68, 112.05, 75.54, 75.47, 75.43, 71.97, 69.84, 69.81,
69.79, 69.75, 67.86, 67.65, 67.54, 67.49, 67.44, 67.39, 66.64, 66.54,
66.47, 52.64, 52.58, 39.14, 38.10, 35.94, 31.87, 31.59, 29.58, 29.55,
29.40, 29.30, 27.47, 26.04, 22.65, 22.28, 14.08, 13.74. HRMS (ESI-
TOF, [M + Na]+): calcd for C52H71N2NaO10P+, 937.4739; found,
937.4756.
+
C15H22NO4 : 280.1543; found, 280.1545. Anal. Calcd for C15H21NO4:
C, 64.50; H, 7.58; N, 5.01. Found: C, 64.84; H, 7.60; N, 5.05. mp 57.0−
58.1 °C (colorless solids, recrystallized from n-hexane/CH2Cl2).
Compound 45.
Compound 44 (282.8 mg, 1.012 mmol) was dissolved in
CH2Cl2 and toluene. To remove traces of water, the solution was
evaporated. Compound 3 (507.5 mg, 1.499 mmol) and
anhydrous CH2Cl2 (4.0 mL) were added. A solution of 1H-
tetrazole (73.5 mg, 1.05 mmol) in anhydrous THF (4.0 mL) was
added at 0 °C. The mixture was stirred at room temperature
under an Ar atmosphere for 3 h. A saturated aqueous solution of
NaHCO3 (15 mL) was added, and the whole was extracted three
times with CH2Cl2 (15 mL × 3). The combined organic layer
was washed with brine, dried over Na2SO4, and filtered. The
solvent was evaporated to afford a colorless oil. The residue was
column-chromatographed on an open column with silica gel (n-
hexane/AcOEt/Et3N = 40:1:1 to 40:5:1) to afford 45 as a
colorless oil (393.0 mg, 0.7607 mmol, 75%).
Compound 8c.
Compound 46 (112.2 mg, 0.1226 mmol) and Pd/C (22.9 mg)
were dissolved in THF (5 mL). The mixture was stirred at room
temperature under a H2 atmosphere for 3 h. The mixture was
filtered on Celite, and the solvent was evaporated. The residue
was purified by reversed-phase medium-pressure liquid
chromatography (CH3CN/H2O = 3:7 to 7:3, containing 0.1%
HCOOH) to afford 8c as a colorless powder (51.7 mg, 0.0802
mmol, 65%).
1H NMR (400 MHz, CDCl3): 7.343−7.273 (10H, m), 6.561 (0.5H,
J = 8.2 Hz) and 6.306 (0.5H, J = 8.0 Hz) (1H, 2d), 5.203−5.134 (2H,
m), 4.814−4.557 (3H, m), 4.225−4.113 (1H, m), 3.924−3.815 (1H,
m), 3.638−3.536 (2H, m), 2.183 (1H, J = 7.6 Hz) and 1.928 (1H, J =
7.6 Hz) (2H, 2t), 1.615−1.557 (1H, m), 1.527−1.448 (1H, m), 1.379−
1.286 (1H, m), 1.267−1.125 (13H, m), 0.895 (1.5H, J = 7.3 Hz) and
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Figure 16. Synthesis of 8d.
1H NMR (400 MHz, DMSO-d6): 8.267 (1H, d, J = 7.8 Hz), 7.874
(1H, t, J = 5.7 Hz), 7.149 (1H, dd, J = 8.1 Hz, 8.1 Hz), 6.753−6.700
(3H, m), 4.494−4.451 (1H, m), 4.123−4.017 (2H, m), 3.911 (2H, t, J
= 6.5 Hz), 3.775−3.605 (3H, m), 3.188−3.127 (1H, m), 3.055−2.992
(1H, m), 2.758 (2H, t, J = 7.9 Hz), 2.378 (2H, t, J = 7.9 Hz), 2.147 (2H,
t, J = 7.3 Hz), 1.683 (2H, quin, J = 6.9 Hz), 1.470 (2H, quin, J = 7.5 Hz),
1.405−1.357 (2H, m), 1.309−1.217 (16H, m), 0.869−0.832 (6H, m).
HRMS (ESI-TOF, [M − H]−): calcd for C31H52N2O10P−, 643.3365;
found, 643.3393. HPLC: 99% (CH3CN/H2O = 3:2 (0.1% HCO2H)).
Synthesis of 8d (Figure 16). Compound 47.
Na2SO4, and filtered. The solvent was evaporated to afford a
colorless oil. The residue was column-chromatographed on an
open column with silica gel (n-hexane/AcOEt/Et3N = 40:1:1 to
40:4:1) to afford 48 as a colorless oil (380.8 mg, 0.7371 mmol,
74%).
1H NMR (400 MHz, CDCl3): 7.382−7.273 (10H, m), 6.650 (J = 8.0
Hz) and 6.534 (J = 7.8 Hz) (1H, 2d), 5.215−5.099 (2H, m), 4.777−
4.547 (3H, m), 4.189−4.119 (1H, m), 3.939−3.836 (1H, m), 3.633−
3.531 (2H, m), 1.191−1.124 (21H, m). 13C NMR (100 MHz, CDCl3):
178.34, 178.26, 170.16, 170.14, 139.21, 139.14, 139.08, 135.43, 135.35,
128.51, 128.35, 128.29, 128.27, 128.14, 128.09, 127.46, 127.37, 127.00,
126.95, 67.20, 67.17, 65.36, 65.33, 65.17, 63.82, 63.69, 63.54, 53.59,
53.53, 53.49, 53.42, 43.18, 43.16, 43.06, 43.03, 38.64, 38.57, 27.35,
27.28, 24.73, 24.65, 24.56, 24.50, 24.43. 31P NMR (161 MHz, CDCl3):
148.46, 148.41. HRMS (ESI-TOF, [M + H]+): calcd for
C28H42N2O5P+, 517.2826; found, 517.2811.
Compound 49.
Compound 38 (620.8 mg, 2.007 mmol as TFA salt) and Et3N
(840 μL) were dissolved in anhydrous CH2Cl2 (8.0 mL).
Pivaloyl chloride (0.27 mL) was added, and the mixture was
stirred at room temperature under an Ar atmosphere for 40 min.
MeOH (2 mL) was added, and the solvent was evaporated. The
residue was column-chromatographed on an open column with
silica gel (n-hexane/acetone = 3:1 to 2:1) to afford 47 as a yellow
oil (415.1 mg, 1.486 mmol, 74%) (Figure 16).
Compounds 48 (366.0 mg, 0.7085 mmol) and 2 (202.2 mg,
0.4180 mmol) were dissolved in CH2Cl2 and toluene. To
remove traces of water, the solution was evaporated. The
mixture was dissolved in anhydrous CH2Cl2 (4.0 mL). A
solution of 1H-tetrazole (60.1 mg, 0.858 mmol) in anhydrous
THF (4.0 mL) was added. The mixture was stirred at room
temperature under an Ar atmosphere for 2 h. TBHP in decane
(5.0−6.0 M) (153 μL) was added, and the mixture was stirred at
room temperature for 2 h. H2O (10 mL) was added, and the
whole was extracted three times with CH2Cl2 (10 mL × 3). The
combined organic layer was washed with brine, dried over
Na2SO4, and filtered. The solvent was evaporated to afford a
colorless oil. The residue was column-chromatographed on a
flash column with silica gel twice (first, CHCl3/AcOEt = 3:2,
second, CHCl3/AcOEt = 3:1) to afford 49 as a colorless oil
(pure fraction, 78.4 mg, 0.0857 mmol, 20%).
1H NMR (400 MHz, CDCl3): 7.397−7.321 (5H, m), 6.618 (1H, d, J
= 6.4 Hz), 5.255−5.179 (2H, m), 4.702−4.655 (1H, m), 3.985 (1H, dd,
J = 11.1 Hz, 4.1 Hz), 3.927 (1H, dd, J = 11.1 Hz, 3.4 Hz), 2.364 (1H, br
s), 1.226 (9H, s). 13C NMR (100 MHz, CDCl3): 179.23, 170.52,
135.07, 128.65, 128.54, 128.16, 67.52, 63.74, 54.90, 38.78, 27.40.
+
HRMS (ESI-TOF, [M + H]+): calcd for C15H22NO4 , 280.1543; found,
280.1535.
Compound 48.
Compound 47 (278.0 mg, 0.9952 mmol) was dissolved in
CH2Cl2 and toluene. To remove traces of water, the solution was
evaporated. 3 (crude, 581.8 mg, 1.719 mmol) was added, and
the mixture was dissolved in anhydrous CH2Cl2 (4.0 mL). A
solution of 1H-tetrazole (74.4 mg, 1.06 mmol) in anhydrous
THF (4.0 mL) was added at 0 °C. The mixture was stirred at
room temperature under an Ar atmosphere for 2.4 h. A saturated
aqueous solution of NaHCO3 (10 mL) was added, and the
whole was extracted three times with CH2Cl2 (10 mL × 3). The
combined organic layer was washed with brine, dried over
1H NMR (400 MHz, CDCl3): (mixtures of diastereomers) 7.351−
7.276 (15H, m), 7.204−7.126 (1H, m), 7.011−6.936 (1H, m), 6.770−
6.683 (3H, m), 5.976−5.864 (1H, m), 5.225−5.133 (2H, m), 5.016−
4.922 (2H, m), 4.770−4.757 (1H, m), 4.703−4.667 (0.1H, m, rotamer
of another peak?), 4.578 (1H, dd, J = 11.7 Hz, 2.2 Hz), 4.471−4.369
(2H, m), 4.321−4.237 (1H, m), 3.578 (1H, br s), 3.410−3.290 (2H,
m), 2.987−2.949 and 2.887−2.849 (2H, 2m), 2.413−2.363 (2H, m),
1.744 (2H, quin, J = 6.7 Hz), 1.457−1.409 (2H, m), 1.346−1.262
(14H, m), 1.226 and 1.190 and 1.183 (9H, 3s), 0.879 (3H, t, J = 6.8
Hz). 13C NMR (100 MHz, CDCl3): (mixtures of diastereomers)
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Figure 17. Synthesis of 8e.
178.75, 178.72, 172.35, 168.98, 159.29, 142.29, 142.26, 137.66, 137.59,
135.30, 135.01, 129.41, 128.84, 128.79, 128.69, 128.67, 128.60, 128.55,
128.52, 128.50, 128.24, 128.19, 128.05, 128.02, 127.98, 120.42, 114.69,
112.07, 75.41, 71.98, 69.92, 69.86, 69.80, 67.87, 67.65, 67.62, 67.32,
66.78, 52.99, 39.12, 38.70, 38.09, 31.89, 31.61, 29.60, 29.57, 29.41,
29.31, 27.31, 26.06, 22.66, 14.10. HRMS (ESI-TOF, [M + Na]+): calcd
Synthesis of 8e (Figure 17). Compound 50.
for C52H71N2NaO10P+, 937.4739; found, 937.4729.
Compound 8d.
L-Serine (1051.8 mg, 10.008 mmol) and K2CO3 (4145.5 mg,
29.995 mmol) were dissolved in H2O (34 mL). Benzyl chloride
(1.7 mL) was added, and the whole was stirred at room
temperature from 18 h. A 2 M aqueous solution of HCl was
added to pH 3−4. The aqueous layer was extracted three times
with AcOEt (40 mL × 3). The combined organic layer was
washed with brine, dried over Na2SO4, and filtered. The solvent
was evaporated to afford 50 as a colorless powder (1024.1 mg,
4.8953 mmol, 49%) (Figure 17).
Compound 49 (76.7 mg, 0.0838 mmol) and Pd/C (12.9 mg)
were dissolved in MeOH (2 mL). The mixture was stirred at
room temperature under a H2 atmosphere for 23 h. The mixture
was filtered on Celite, and the solvent was evaporated. The
residue was purified by reversed-phase medium-pressure liquid
chromatography (CH3CN/H2O = 3:7 to 7:3, containing 0.1%
HCOOH) to afford 8d as a colorless powder (34.7 mg, 0.0538
mmol, 64%).
1H NMR (400 MHz, DMSO-d6): 12.669 (1H, br s), 8.393 (1H, d, J
= 7.7 Hz), 7.904−7.880 (2H, m), 7.575−7.532 (1H, m), 7.509−7.464
(2H, m), 4.981 (1H, br s), 4.501−4.456 (1H, m), 3.800 (2H, d, J = 5.1
Hz). 13C NMR (100 MHz, DMSO-d6): 171.95, 166.38, 133.98, 131.42,
128.30, 127.39, 61.20, 55.64. HRMS (ESI-TOF, [M − H]−): calcd for
−
C10H10NO4 , 208.0615; found, 208.0615.
Compound 51.
1H NMR (400 MHz, DMSO-d6): 12.690 (1H, br s), 7.921−7.860
(2H, m), 7.149 (1H, dd, J = 8.1 Hz, 8.1 Hz), 6.753−6.700 (3H, m),
4.420−4.377 (1H, m), 4.164−4.126 (2H, m), 3.911 (2H, t, J = 6.5 Hz),
3.775−3.555 (3H, m), 3.184−3.123 (1H, m), 3.053−2.990 (1H, m),
2.757 (2H, t, J = 7.9 Hz), 2.375 (2H, t, J = 7.9 Hz), 1.684 (2H, quin, J =
7.0 Hz), 1.407−1.248 (16H, m), 1.150−1.112 (9H, m), 0.855 (3H, t, J
= 6.9 Hz). HRMS (ESI-TOF, [M − H]−): calcd for C31H52N2O10P−,
643.3365; found, 643.3344. Anal. Calcd for C31H53N2O10P·0.5H2O: C,
56.95; H, 8.33; N, 4.29. Found: C, 56.97; H, 8.14; N, 4.07. HPLC: 97%
(CH3CN/H2O = 3:2 (0.1% HCO2H)).
Compound 50 (1008.1 mg, 4.8188 mmol) was dissolved in
anhydrous THF (40 mL). Triethylamine (2.0 mL), TBAI
(531.6 mg, 1.443 mmol), and BnBr (630 μL) were added at 0
°C, and the mixture was stirred at room temperature under an Ar
atmosphere for 17.1 h. H2O (40 mL) was added, and the whole
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Figure 18. Synthesis of 8f.
Compound 53.
was extracted three times with AcOEt (40 mL × 3). The
combined organic layer was washed with brine, dried over
Na2SO4, and filtered. The solvent was evaporated to afford a
yellow oil. The residue was column-chromatographed on an
open column with silica gel (n-hexane/acetone = 1:1) to afford
51 as a colorless solid (698.1 mg, 2.332 mmol, 48%).
1H NMR (400 MHz, CDCl3): 7.822−7.801 (2H, m), 7.535−7.491
(1H, m), 7.445−7.406 (2H, m), 7.373−7.328 (5H, m), 7.139 (1H, d, J
= 6.9 Hz), 5.278−5.213 (2H, m), 4.920−4.885 (1H, m), 4.108−4.025
(2H, m), 2.159 (1H, br s). 13C NMR (100 MHz, CDCl3): 170.44,
167.69, 135.05, 133.46, 131.99, 128.68, 128.63, 128.58, 128.19, 127.16,
67.66, 63.60, 55.32. HRMS (ESI-TOF, [M + H]+): calcd for
Compounds 52 (118.7 mg, 0.2212 mmol) and 2 (63.8 mg, 0.132
mmol) were dissolved in CH2Cl2 and toluene. To remove traces
of water, the solution was evaporated. The mixture was dissolved
in anhydrous CH2Cl2 (1.0 mL). A solution of 1H-tetrazole (18.7
mg, 0.267 mmol) in anhydrous THF (1.0 mL) was added at 0
°C. The mixture was stirred at room temperature under an Ar
atmosphere for 2 h. TBHP in decane (5.0−6.0 M) (47 μL) was
added, and the mixture was stirred at room temperature for 2 h.
H2O (10 mL) was added, and the whole was extracted three
times with CH2Cl2 (10 mL × 3). The combined organic layer
was washed with brine, dried over Na2SO4, and filtered. The
solvent was evaporated to afford a colorless oil. The residue was
column-chromatographed on a flash column with silica gel
(CHCl3/AcOEt = 8:1) to afford 53 as a colorless oil (90.1 mg,
0.0964 mmol, 73%).
+
C17H18NO4 , 300.1230; found, 300.1239. mp 105.8−107.0 °C
(colorless needles, recrystallized from n-hexane/CH2Cl2).
Compound 52.
Compound 51 (150.9 mg, 0.5041 mmol) was dissolved in
CH2Cl2 and toluene. To remove traces of water, the solution was
evaporated. Compound 3 (crude, 292.4 mg, 0.8639 mmol) was
added, and the mixture was dissolved in anhydrous CH2Cl2 (2.0
mL). A solution of 1H-tetrazole (37.2 mg, 0.531 mmol) in
anhydrous THF (2.0 mL) was added at 0 °C. The mixture was
stirred at room temperature under an Ar atmosphere for 3.3 h. A
saturated aqueous solution of NaHCO3 (10 mL) was added, and
the whole was extracted three times with CH2Cl2 (10 mL × 3).
The combined organic layer was washed with brine, dried over
Na2SO4, and filtered. The solvent was evaporated to afford a
colorless oil. The residue was column-chromatographed on an
open column with silica gel (n-hexane/AcOEt/Et3N = 40:2:1 to
40:6:1) to afford 52 as a colorless oil (140.4 mg, 0.2616 mmol,
52%).
1H NMR (400 MHz, CDCl3): (mixtures of diastereomers) 7.877−
7.839 (2H, m), 7.759 (J = 7.4 Hz) and 7.674 (J = 7.6 Hz) (1H, 2d),
7.495 (1H, dd, J = 7.4 Hz, 7.4 Hz), 7.420−7.382 (2H, m), 7.337−7.226
(15H, m), 7.164−7.114 (1H, m), 6.720−6.698 (3H, m), 5.881 (J = 5.7
Hz) and 5.787 (J = 5.7 Hz) (1H, 2t), 5.249−5.169 (2H, m), 5.020−
4.921 (3H, m), 4.569−4.464 (2H, m), 4.431−4.370 (2H, m), 4.047−
3.952 (1H, m), 3.908−3.820 (3H, m), 3.576−3.515 (1H, m), 3.344−
3.232 (2H, m), 2.959−2.820 (2H, m), 2.490−2.303 (2H, m), 1.742
(2H, quin, J = 6.9 Hz), 1.439−1.262 (16H, m), 0.878 (3H, t, J = 6.8
Hz). 13C NMR (100 MHz, CDCl3): (mixtures of diastereomers)
172.32, 168.85, 168.77, 167.18, 159.26, 142.26, 137.64, 137.53, 135.25,
135.20, 134.99, 134.97, 133.22, 133.18, 131.92, 129.39, 128.82, 128.74,
128.66, 128.63, 128.61, 128.58, 128.52, 128.51, 128.24, 128.19, 128.02,
127.98, 127.95, 127.33, 127.30, 120.41, 114.68, 114.66, 112.04, 75.50,
75.43, 75.34, 71.94, 70.01, 69.95, 69.90, 69.84, 67.85, 67.74, 67.72,
67.34, 66.85, 66.79, 66.71, 66.65, 53.54, 53.49, 53.43, 39.11, 39.07,
38.06, 38.02, 31.88, 31.57, 29.59, 29.56, 29.40, 29.30, 26.05, 22.66,
14.10. HRMS (ESI-TOF, [M + NH4]+): calcd for C54H71N3O10P+,
952.4872; found, 952.4855.
1H NMR (400 MHz, CDCl3): 7.788−7.705 (2H, m), 7.523−7.376
(2H, m), 7.367−7.200 (11.5H, m), 7.039 (0.5H, d, J = 7.8 Hz), 5.220−
5.216 (1H, m), 5.191 (1H, s), 5.105−4.948 (1H, m), 4.747−4.565
(2H, m), 4.303−4.204 (1H, m), 4.064−3.969 (1H, m), 3.644−3.529
(2H, m), 1.293−1.220 (1H, m, impurity), 1.150−1.108 (12H, m).
HRMS (ESI-TOF, [M + H]+): calcd for C30H38N2O5P+, 537.2513;
found, 537.2500.
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Compound 8e.
anhydrous CH2Cl2 (4.0 mL) were added. A solution of 1H-
tetrazole (72.6 mg, 1.04 mmol) in anhydrous THF (4.0 mL) was
added at 0 °C. The mixture was stirred at room temperature
under an Ar atmosphere for 3 h. A saturated aqueous solution of
NaHCO3 (15 mL) was added, and the whole was extracted three
times with CH2Cl2 (15 mL × 3). The combined organic layer
was washed with brine, dried over Na2SO4, and filtered. The
solvent was evaporated to afford a colorless oil. The residue was
column-chromatographed on an open column with silica gel (n-
hexane/AcOEt/Et3N = 40:1:1 to 40:5:1) to afford 55 as a
colorless oil (478.1 mg, 0.8810 mmol, 88%).
Compound 53 (81.0 mg, 0.0866 mmol) and Pd/C (8.0 mg)
were dissolved in MeOH (4 mL) and AcOH (1 mL). The
mixture was stirred at room temperature under a H2 atmosphere
for 31.4 h. The mixture was filtered on Celite, and the solvent
was evaporated. The residue was purified by reversed-phase
medium-pressure liquid chromatography (CH3CN/H2O = 3:7
to 7:3, containing 0.1% HCOOH) to afford 8e as a colorless
powder (36.7 mg, 0.0552 mmol, 64%).
1H NMR (400 MHz, CDCl3): 7.341−7.263 (10H, m), 6.525 (0.5H,
J = 8.2 Hz) and 6.314 (0.5H, J = 8.1 Hz) (1H, 2d), 5.202−5.105 (2H,
m), 4.797−4.554 (3H, m), 4.201−4.109 (1H, m), 3.925−3.801 (1H,
m), 3.632−3.533 (2H, m), 2.121−2.041 and 1.916−1.580 (5H, m),
1.462−1.121 (17H, m). 13C NMR (100 MHz, CDCl3): 175.90, 175.79,
170.12, 170.06, 139.23, 139.16, 139.04, 135.43, 135.30, 128.49, 128.39,
128.27, 128.24, 128.19, 128.08, 127.51, 127.35, 126.92, 67.22, 67.13,
65.47, 65.29, 65.26, 65.08, 64.22, 64.06, 63.64, 63.49, 53.33, 53.27,
53.22, 53.15, 45.17, 44.90, 43.19, 43.12, 43.07, 43.00, 29.45, 29.42,
29.21, 25.70, 25.66, 25.61, 25.54, 25.48, 24.66, 24.60, 24.58, 24.52,
24.45. HRMS (ESI-TOF, [M + H]+): calcd for C30H44N2O5P+,
543.2982; found, 543.2967.
1H NMR (400 MHz, CDCl3/TFA-d): 7.758 (2H, d, J = 7.4 Hz),
7.625 (1H, dd, J = 7.6 Hz, 7.6 Hz), 7.481 (2H, dd, J = 7.5 Hz, 7.5 Hz),
7.219 (1H, dd, J = 8.0 Hz, 8.0 Hz), 6.842−6.821 (1H, m), 6.763 (2H, br
s), 5.132 (1H, br s), 4.508 (2H, br s), 4.142 (1H, br s), 4.041 (2H, t, J =
6.7 Hz), 3.932 (2H, br s), 3.637 (1H, br s), 3.187−3.155 (1H, m),
2.877 (2H, br s), 2.602 (2H, br s), 1.769 (2H, quin, J = 7.1 Hz), 1.450−
1.264 (16H, m), 0.878 (3H, t, J = 6.8 Hz). HRMS (ESI-TOF, [M −
H]−): calcd for C33H48N2O10P−, 663.3052; found, 663.3058. HPLC:
88% (CH3CN/H2O = 3:2 (0.1% HCO2H)) (after purification by flash-
column-chromatography (CHCl3/acetone/AcOH = 2:1:1)).
Synthesis of 8f (Figure 18). Compound 54.
Compound 56.
Compounds 55 (185.0 mg, 0.3409 mmol) and 2 (97.2 mg, 0.201
mmol) were dissolved in CH2Cl2 and toluene. To remove traces
of water, the solution was evaporated. The mixture was dissolved
in anhydrous CH2Cl2 (2.0 mL). A solution of 1H-tetrazole (27.9
mg, 0.398 mmol) in anhydrous THF (2.0 mL) was added at 0
°C. The mixture was stirred at room temperature under an Ar
atmosphere for 2.5 h. TBHP in decane (5.0−6.0 M) (73 μL) was
added, and the mixture was stirred at room temperature for 2 h.
H2O (10 mL) was added, and the whole was extracted three
times with CH2Cl2 (10 mL × 3). The combined organic layer
was washed with brine, dried over Na2SO4, and filtered. The
solvent was evaporated to afford a colorless oil. The residue was
column-chromatographed on a flash column with silica gel twice
(CHCl3/AcOEt = 8:1) to afford 56 as a colorless oil (66.8 mg,
0.0710 mmol, 35%).
L-Serine benzyl ester hydrochloride (349.5 mg, 1.509 mmol)
was dissolved in anhydrous CH2Cl2 (6 mL). Et3N (520 μL) and
cyclohexanecarbonyl chloride (225 μL, 1.66 mmol) were added,
and the mixture was stirred at room temperature under an Ar
atmosphere for 30 min. MeOH (ca. 2 mL) was added, and the
solvent was evaporated with silica gel. The residue was column-
chromatographed on an open column with silica gel (n-hexane/
acetone = 3:1 to 2:1) to afford 54 as a colorless solid (424.8 mg,
1.391 mmol, 92%) (Figure 18).
1H NMR (400 MHz, CDCl3): 7.396−7.320 (5H, m), 6.431 (1H, d, J
= 6.9 Hz), 5.248−5.180 (2H, m), 4.723−4.687 (1H, m), 4.008−3.893
(2H, m), 2.614 (1H, t, J = 5.8 Hz), 2.213−2.137 (1H, m), 1.896−1.857
(2H, m), 1.801−1.769 (2H, m), 1.686−1.673 (1H, m), 1.496−1.380
(2H, m), 1.324−1.153 (3H, m). 13C NMR (100 MHz, CDCl3): 176.68,
170.46, 135.06, 128.64, 128.53, 128.17, 67.52, 63.78, 54.68, 45.19,
29.48, 25.64, 25.59, 25.57. HRMS (ESI-TOF, [M + H]+): calcd for
1H NMR (400 MHz, CDCl3): (mixtures of diastereomers) 7.350−
7.266 (15H, m), 7.170−7.123 (1H, m), 6.761−6.679 (4H, m), 5.927
and 5.860 (1H, 2t, J = 5.9 Hz), 5.203−5.115 (2H, m), 5.022−4.935
(2H, m), 4.838−4.788 (1H, m), 4.581 (1H, d, J = 11.6 Hz), 4.465−
4.375 (2H, m), 4.275−4.216 (1H, m), 4.023−3.836 (4H, m), 3.595−
3.545 (1H, m), 3.388−3.300 (2H, m), 2.869 (2H, t, J = 7.8 Hz), 2.446−
2.341 (2H, m), 2.174−2.064 (1H, m), 1.832−1.619 (7H, m), 1.425−
1.179 (21H, m), 0.876 (3H, t, J = 6.8 Hz). 13C NMR (100 MHz,
CDCl3): (mixtures of diastereomers) 176.20, 176.18, 172.28, 168.97,
168.95, 159.27, 142.29, 137.63, 137.59, 135.36, 135.29, 134.97, 129.38,
128.79, 128.75, 128.66, 128.58, 128.53, 128.51, 128.48, 128.26, 128.20,
128.03, 128.00, 127.96, 127.93, 120.40, 114.67, 112.04, 75.53, 75.46,
75.42, 71.96, 69.82, 69.78, 69.73, 67.85, 67.62, 67.52, 67.47, 66.68,
66.61, 66.54, 52.55, 52.48, 44.89, 39.11, 39.08, 38.11, 31.87, 31.59,
29.57, 29.55, 29.39, 29.29, 26.04, 25.62, 25.56, 25.52, 22.64, 14.08.
HRMS (ESI-TOF, [M + Na]+): calcd for C54H73N2NaO10P+,
963.4895; found, 963.4880.
+
C17H24NO4 , 306.1700; found, 306.1680. Anal. Calcd for C17H23NO4:
C, 66.86; H, 7.59; N, 4.59. Found: C, 61.69; H, 7.47; N, 4.58. mp
106.4−108.5 °C (colorless powder, recrystallized from n-hexane/
CH2Cl2).
Compound 55.
Compound 54 (305.1 mg, 0.9991 mmol) was dissolved in
CH2Cl2 and toluene. To remove traces of water, the solution was
evaporated. Compound 3 (540.7 mg, 1.597 mmol) and
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Figure 19. Synthesis of 8g.
Compound 8f.
(1H, br s), 2.402 (3H, s). 13C NMR (100 MHz, CDCl3): 170.50,
167.65, 142.51, 135.08, 130.60, 129.28, 128.67, 128.55, 128.17, 127.17,
67.62, 63.73, 55.33, 21.47. HRMS (ESI-TOF, [M + H]+): calcd for
C18H20NO4 , 314.1387; found, 314.1396. mp 122.0−123.4 °C
(colorless needles, recrystallized from n-hexane/CH2Cl2).
Compound 58.
+
Compound 56 (58.5 mg, 0.0622 mmol) and Pd/C (20.9 mg)
were dissolved in THF (5 mL). The mixture was stirred at room
temperature under a H2 atmosphere for 3 h. The mixture was
filtered on Celite, and the solvent was evaporated. The residue
was purified by reversed-phase medium-pressure liquid
chromatography (CH3CN/H2O = 3:7 to 7:3, containing 0.1%
HCOOH) to afford 8f as a colorless powder (15.1 mg, 0.0225
mmol, 36%).
Compound 57 (312.5 mg, 0.9973 mmol) was dissolved in
CH2Cl2 and toluene. To remove traces of water, the solution was
evaporated. Compound 3 (540.7 mg, 1.597 mmol) and
anhydrous CH2Cl2 (4.0 mL) were added. A solution of 1H-
tetrazole (73.9 mg, 1.05 mmol) in anhydrous THF (4.0 mL) was
added at 0 °C. The mixture was stirred at room temperature
under an Ar atmosphere for 3.5 h. A saturated aqueous solution
of NaHCO3 (15 mL) was added, and the whole was extracted
three times with CH2Cl2 (15 mL × 3). The combined organic
layer was washed with brine, dried over Na2SO4, and filtered.
The solvent was evaporated to afford a colorless oil. The residue
was column-chromatographed on an open column with silica gel
(n-hexane/AcOEt/Et3N = 40:1:1, 40:4:1 to 40:5:1) to afford 58
as a colorless oil (417.4 mg, 0.7580 mmol, 76%).
1H NMR (400 MHz, DMSO-d6): 8.213 (1H, d, J = 7.8 Hz), 7.881
(1H, t, J = 5.6 Hz), 7.147 (1H, dd, J = 8.1 Hz, 8.1 Hz), 6.752−6.698
(3H, m), 4.446−4.403 (1H, m), 4.106−4.002 (2H, m), 3.909 (2H, t, J
= 6.5 Hz), 3.758−3.590 (3H, m), 3.179−3.117 (1H, m), 3.056−2.992
(1H, m), 2.757 (2H, t, J = 7.9 Hz), 2.375 (2H, t, J = 7.9 Hz), 2.229−
2.173 (1H, m), 1.699−1.581 (7H, m), 1.406−1.108 (21H, m), 0.853
(3H, t, J = 6.8 Hz). HRMS (ESI-TOF, [M − H]−): calcd for
C33H54N2O10P−, 669.3522; found, 669.3506. HPLC: >99% (CH3CN/
H2O = 3:2 (0.1% HCO2H)).
Synthesis of 8g (Figure 19). Compound 57.
1H NMR (400 MHz, CDCl3): 7.677 and 7.618 (2H, 2d, J = 8.2 Hz),
7.356−7.197 (11.5 H, m), 7.122 (1H, d, J = 8.0 Hz), 7.010 (0.5H, d, J =
7.8 Hz), 5.211 and 5.184 (2H, 2s), 5.010−4.942 (1H, m), 4.740−4.561
(2H, m), 4.288−4.196 (1H, m), 4.052−3.954 (1H, m), 3.640−3.527
(2H, m), 2.390 and 2.364 (3H, 2s), 1.148−1.106 (12H, m). HRMS
(ESI-TOF, [M + H]+): calcd for C31H40N2O5P+, 551.2669; found,
551.2664.
L-Serine benzyl ester hydrochloride (693.5 mg, 2.993 mmol)
was dissolved in anhydrous CH2Cl2 (10 mL). Et3N (1040 μL)
and p-toluoyl chloride (440 μL, 3.33 mmol) were added, and the
mixture was stirred at room temperature under an Ar
atmosphere for 30 min. MeOH (ca. 2 mL) was added, and the
solvent was evaporated with silica gel. The residue was column-
chromatographed on an open column with silica gel (n-hexane/
acetone = 3:1 to 2:1) to afford 57 as a colorless solid (794.4 mg,
2.535 mmol, 85%) (Figure 19).
Compound 59.
1H NMR (400 MHz, CDCl3): 7.722 (2H, d, J = 8.2 Hz), 7.384−
7.318 (5H, m), 7.242 (2H, d, J = 7.9 Hz), 7.072 (1H, d, J = 6.7 Hz),
5.294−5.227 (2H, m), 4.927−4.891 (1H, m), 4.076 (2H, br s), 2.518
Compounds 58 (186.2 mg, 0.3382 mmol) and 2 (96.9 mg, 0.200
mmol) were dissolved in CH2Cl2 and toluene. To remove traces
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Figure 20. Synthesis of 8h.
Compound 8g.
of water, the solution was evaporated. The mixture was dissolved
in anhydrous CH2Cl2 (1.0 mL). A solution of 1H-tetrazole (27.5
mg, 0.393 mmol) in anhydrous THF (1.0 mL) was added at 0
°C. The mixture was stirred at room temperature under an Ar
atmosphere for 6 h. TBHP in decane (5.0−6.0 M) (73 μL) was
added, and the mixture was stirred at room temperature for 2 h.
H2O (10 mL) was added, and the whole was extracted three
times with CH2Cl2 (10 mL × 3). The combined organic layer
was washed with brine, dried over Na2SO4, and filtered. The
solvent was evaporated to afford a colorless oil. The residue was
column-chromatographed on a flash column with silica gel twice
(CHCl3/AcOEt = 10:1) to afford 59 as a colorless oil (142.5 mg,
0.1501 mmol, 75%).
Compound 59 (106.6 mg, 0.1123 mmol) and Pd/C (24.3 mg)
were dissolved in THF (5 mL). The mixture was stirred at room
temperature under a H2 atmosphere for 5 h. The mixture was
filtered on Celite, and the solvent was evaporated. The residue
was purified by reversed-phase medium-pressure liquid
chromatography (CH3CN/H2O = 3:7 to 7:3, containing 0.1%
HCOOH). Because the HRMS spectrum showed partial
decomposition of the product at the phosphodiester part, the
product was further purified by a flash column with silica gel
(CHCl3/acetone/AcOH = 4:1:1 to 2:1:1) to afford 8g as a
colorless powder (33.2 mg, 0.0489 mmol, 44%).
1H NMR (400 MHz, CDCl3): (mixtures of diastereomers) 7.766−
7.729 (2H, m), 7.661 (J = 7.4 Hz) and 7.563 (J = 7.6 Hz) (1H, 2d),
7.360−7.226 (15H, m), 7.193 (2H, d, J = 8.0 Hz), 7.163−7.114 (1H,
m), 6.720−6.693 (3H, m), 5.882 (J = 5.9 Hz) and 5.781 (J = 5.9 Hz)
(1H, 2t), 5.236−5.164 (2H, m), 5.017−4.918 (3H, m), 4.566−4.456
(2H, m), 4.431−4.363 (2H, m), 4.041−3.950 (1H, m), 3.908−3.818
(3H, m), 3.589−3.450 (1H, m), 3.341−3.245 (2H, m), 2.838 (2H, t, J
= 7.6 Hz), 2.370−2.314 (5H, m), 1.740 (2H, quin, J = 6.8 Hz), 1.459−
1.388 (2H, m), 1.342−1.261 (14H, m), 0.877 (3H, t, J = 6.9 Hz). 13C
NMR (100 MHz, CDCl3): (mixtures of diastereomers) 172.31, 172.26,
168.96, 168.88, 167.14, 159.28, 142.43, 142.33, 142.32, 137.69, 137.58,
135.31, 135.25, 135.03, 135.01, 130.42, 130.39, 129.39, 129.26, 128.81,
128.74, 128.67, 128.62, 128.53, 128.50, 128.25, 128.19, 128.03, 128.00,
127.97, 127.33, 127.31, 120.42, 114.70, 114.68, 112.05, 75.52, 75.45,
75.37, 71.94, 69.99, 69.93, 69.88, 69.82, 67.87, 67.73, 67.71, 67.42,
66.85, 66.79, 66.64, 53.50, 53.43, 53.37, 39.12, 39.06, 38.08, 38.05,
31.89, 31.58, 29.60, 29.57, 29.42, 29.32, 26.06, 22.67, 21.47, 14.10.
HRMS (ESI-TOF, [M + NH4]+): calcd for C55H73N3O10P+, 966.5028;
found, 966.5032.
1H NMR (400 MHz, CDCl3/TFA-d): 7.650 (2H, d, J = 7.8 Hz),
7.286−7.198 (3H, m), 6.839−6.819 (1H, m), 6.766 (2H, br s), 5.112
(1H, br s), 4.519 (2H, br s), 4.139 (1H, br s), 4.040 (2H, t, J = 6.8 Hz),
3.937 (2H, br s), 3.591 (1H, br s), 3.223 (1H, d, J = 13.7 Hz), 2.881
(2H, t, J = 6.9 Hz), 2.610 (2H, t, J = 6.9 Hz), 2.416 (3H, s), 1.767 (2H,
quin, J = 7.1 Hz), 1.471−1.398 (2H, m), 1.300−1.266 (14H, m), 0.878
(3H, t, J = 6.8 Hz). HRMS (ESI-TOF, [M − H]−): calcd for
C34H50N2O10P−, 677.3209; found, 677.3229. HPLC: 94% (CH3CN/
H2O = 3:2 (0.1% HCO2H)).
Synthesis of 8h (Figure 20). Compound 60.
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Compound 62.
4-tert-Butylbenzoic acid (589.0 mg, 3.305 mmol) was dissolved
in anhydrous CH2Cl2 (7.0 mL). SOCl2 (480 μL, 6.62 mmol)
was added to the solution, and the whole was stirred at room
temperature under an Ar atmosphere for 24 h. The solvent was
evaporated to afford crude 4-(tert-butyl)benzoyl chloride as a
colorless oil and powder (648.6 mg), which was used without
further purification (Figure 20).
L-Serine benzyl ester hydrochloride (693.3 mg, 2.993 mmol) was
dissolved in anhydrous CH2Cl2 (5 mL). Et3N (1040 μL), crude 4-(tert-
butyl)benzoyl chloride (648.6 mg), and CH2Cl2 (5 mL) were added,
and the mixture was stirred at room temperature under an Ar
atmosphere for 30 min. MeOH (ca. 2 mL) was added, and the solvent
was evaporated with silica gel. The residue was column-chromato-
graphed on an open column with silica gel twice (first, n-hexane/
acetone = 2:1, second, n-hexane/acetone = 3:1 to 0:1) to afford 60 as a
colorless solid (821.7 mg, 2.312 mmol, 77%).
Compounds 61 (201.3 mg, 0.3396 mmol) and 2 (97.9 mg, 0.202
mmol) were dissolved in CH2Cl2 and toluene. To remove traces
of water, the solution was evaporated. The mixture was dissolved
in anhydrous CH2Cl2 (1.0 mL). A solution of 1H-tetrazole (27.5
mg, 0.393 mmol) in anhydrous THF (1.0 mL) was added at 0
°C. The mixture was stirred at room temperature under an Ar
atmosphere for 6 h. TBHP in decane (5.0−6.0 M) (73 μL) was
added, and the mixture was stirred at room temperature for 2 h.
H2O (10 mL) was added, and the whole was extracted three
times with CH2Cl2 (10 mL × 3). The combined organic layer
was washed with brine, dried over Na2SO4, and filtered. The
solvent was evaporated to afford a colorless oil. The residue was
column-chromatographed on a flash column with silica gel
(CHCl3/AcOEt = 10:1) to afford 62 as a colorless oil (139.1 mg,
0.1403 mmol, 69%).
1H NMR (400 MHz, CDCl3): 7.764 (2H, d, J = 8.6 Hz), 7.453 (2H,
d, J = 8.6 Hz), 7.398−7.317 (5H, m), 7.101 (1H, d, J = 6.9 Hz), 5.290−
5.223 (2H, m), 4.931−4.895 (1H, m), 4.106−4.035 (2H, m), 2.601
(1H, br s), 1.333 (9H, s). 13C NMR (100 MHz, CDCl3): 170.48,
167.62, 155.58, 135.08, 130.56, 128.67, 128.55, 128.16, 127.02, 125.56,
67.62, 63.77, 55.33, 34.96, 31.12. HRMS (ESI-TOF, [M + Na]+): calcd
+
for C21H25NNaO4 , 378.1676; found, 378.1674. Anal. Calcd for
C21H25NO4: C, 70.96; H, 7.09; N, 3.94. Found: C, 71.28; H, 7.20; N,
4.03. mp 124.5−125.1 °C (colorless plates, recrystallized from n-
hexane/CH2Cl2).
1H NMR (400 MHz, CDCl3): (mixtures of diastereomers) 7.819−
7.780 (2H, m), 7.694 (0.5H, J = 7.4 Hz) and 7.601 (0.5H, J = 7.6 Hz)
(1H, 2d), 7.432−7.405 (2H, m), 7.335−7.225 (15H, m), 7.160−7.109
(1H, m), 6.720−6.686 (3H, m), 5.896 and 5.811 (1H, 2t, J = 5.9 Hz),
5.237−5.165 (2H, m), 5.022−4.919 (3H, m), 4.574−4.458 (2H, m),
4.435−4.366 (2H, m), 4.043−3.960 (1H, m), 3.905−3.825 (3H, m),
3.597−3.512 (1H, m), 3.310−3.281 (2H, m), 2.863−2.818 (2H, m),
2.375−2.328 (2H, m), 1.774−1.721 (2H, m), 1.467−1.387 (2H, m),
1.342−1.261 (24H, m), 0.878 (3H, t, J = 6.9 Hz). 13C NMR (100 MHz,
CDCl3): (mixtures of diastereomers) 172.31, 172.27, 168.95, 168.87,
167.12, 167.10, 159.28, 155.47, 155.45, 142.32, 142.30, 137.69, 137.57,
135.30, 135.25, 135.04, 135.01, 130.37, 130.33, 129.39, 128.79, 128.71,
128.66, 128.62, 128.52, 128.49, 128.24, 128.18, 128.02, 127.97, 127.16,
125.53, 120.42, 114.69, 114.67, 112.05, 75.49, 75.43, 75.38, 71.94,
69.98, 69.92, 69.88, 69.82, 67.86, 67.71, 67.69, 67.46, 67.41, 66.86,
66.80, 66.73, 66.67, 53.50, 53.44, 53.38, 39.11, 39.08, 38.10, 38.05,
34.93, 31.88, 31.58, 31.11, 29.59, 29.57, 29.41, 29.31, 26.05, 22.66,
14.09. HRMS (ESI-TOF, [M + NH4]+): calcd for C58H79N3O10P+,
1008.5498; found, 1008.5515.
Compound 61.
Compound 60 (356.3 mg, 1.002 mmol) was dissolved in
CH2Cl2 and toluene. To remove traces of water, the solution was
evaporated. 3 (541.4 mg, 1.600 mmol) and anhydrous CH2Cl2
(4.0 mL) were added. A solution of 1H-tetrazole (72.9 mg, 1.04
mmol) in anhydrous THF (4.0 mL) was added at 0 °C. The
mixture was stirred at room temperature under an Ar
atmosphere for 3 h. A saturated aqueous solution of NaHCO3
(15 mL) was added, and the whole was extracted three times
with CH2Cl2 (15 mL × 3). The combined organic layer was
washed with brine, dried over Na2SO4, and filtered. The solvent
was evaporated to afford a colorless oil. The residue was column-
chromatographed on an open column with silica gel (n-hexane/
AcOEt/Et3N = 40:1:1 to 40:4:1) to afford 61 as a colorless oil
(466.9 mg, 0.7877 mmol, 79%).
Compound 8h.
1H NMR (400 MHz, CDCl3): 7.714 and 7.651 (2H, 2d, J = 8.5 Hz),
7.417 (1H, d, J = 8.6 Hz), 7.359−7.211 (11.5H, m), 7.015 (0.5H, d, J =
7.9 Hz), 5.212−5.210 (1H, m), 5.183 (1H, s), 5.014−4.951 (1H, m),
4.748−4.586 (2H, m), 4.299−4.197 (1H, m), 4.058−3.953 (1H, m),
3.648−3.533 (2H, m), 1.332 and 1.310 (9H, 2s), 1.154−1.117 (12H,
m). 13C NMR (100 MHz, CDCl3): 170.08, 169.99, 167.00, 166.98,
155.17, 154.97, 139.12, 138.98, 138.91, 135.45, 135.31, 131.00, 130.96,
128.52, 128.30, 128.29, 128.26, 128.23, 128.17, 128.03, 127.38, 127.32,
126.95, 126.93, 125.42, 125.35, 67.29, 67.19, 65.52, 65.38, 65.34, 65.20,
64.32, 64.16, 63.65, 63.50, 53.99, 53.93, 53.86, 43.24, 43.14, 43.12,
43.02, 34.90, 34.85, 31.12, 24.66, 24.62, 24.58, 24.54, 24.51, 24.47,
24.44, 24.40. HRMS (ESI-TOF, [M + Na]+): calcd for
C34H45N2NaO5P+, 615.2958; found, 615.2987.
Compound 62 (113.1 mg, 0.1141 mmol) and Pd/C (26.5 mg)
were dissolved in THF (5 mL). The mixture was stirred at room
temperature under a H2 atmosphere for 23 h. The mixture was
filtered on Celite, and the solvent was evaporated. The residue
was purified by reversed-phase medium-pressure liquid
chromatography (CH3CN/H2O = 35:65 to 7:3, containing
0.1% HCOOH). Because the HRMS spectrum showed partial
decomposition of the product at the phosphodiester part, the
product was further purified by a flash column with silica gel
(CHCl3/acetone/AcOH = 2:1:1) to afford 5l as a colorless
powder (35.2 mg, 0.0488 mmol, 43%).
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Figure 21. Synthesis of 9a.
1H NMR (400 MHz, CDCl3/TFA-d): 7.691 (2H, d, J = 7.8 Hz),
7.499 (2H, d, J = 8.2 Hz), 7.216 (1H, dd, J = 8.0 Hz, 8.0 Hz), 6.839−
6.767 (3H, m), 5.124 (1H, br s), 4.522 (2H, br s), 4.153 (1H, br s),
4.042 (2H, t, J = 6.7 Hz), 3.947 (2H, br s), 3.612 (1H, br s), 3.439 (br s)
and 3.215 (d, J = 13.4 Hz), 2.880 (2H, t, J = 7.0 Hz), 2.628−2.611 (2H,
m), 1.766 (2H, quin, J = 7.1 Hz), 1.424−1.263 (25H, m), 0.876 (3H, t, J
= 6.8 Hz). HRMS (ESI-TOF, [M − H]−): calcd for C37H56N2O10P−,
719.3678; found, 719.3656. HPLC: 86% (CH3CN/H2O = 3:2 (0.1%
HCO2H)).
1H NMR (400 MHz, DMSO-d6): 7.779 (1H, t, J = 5.7 Hz), 7.158−
7.095 (2H, m), 6.906 (1H, d, J = 7.5 Hz), 6.820 (1H, ddd, J = 7.4 Hz,
7.4 Hz, 1.0 Hz), 4.732 (1H, br s), 4.517 (1H, br s), 3.950 (2H, t, J = 6.4
Hz), 3.479−3.423 (1H, m), 3.257−3.240 (2H, m), 3.207−3.146 (1H,
m), 3.002−2.938 (1H, m), 2.760 (2H, t, J = 7.8 Hz), 2.347 (2H, t, J =
7.9 Hz), 1.730 (2H, quin, J = 6.9 Hz), 1.469−1.397 (2H, m), 1.369−
1.257 (6H, m), 0.868 (3H, t, J = 6.9 Hz). 13C NMR (100 MHz, DMSO-
d6): 172.09, 156.38, 129.28, 127.18, 120.03, 111.34, 70.53, 67.34, 63.59,
42.08, 35.16, 31.23, 28.77, 28.43, 25.67, 25.54, 22.04, 13.95. HRMS
+
(ESI-TOF, [M + H]+): calcd for C19H32NO4 , 338.2326; found,
Synthesis of 9a (Figure 21). Compound 64.
338.2325.
Compound 65.
Compounds 63 (1270.5 mg, 4.8059 mmol) and 1-hydrox-
ybenzotriazole monohydrate (777.6 mg, 5.754 mmol) were
dissolved in anhydrous DMF (5 mL). (R)-3-Amino-1,2-
propanediol (481.0 mg, 5.279 mmol) was added to the mixture
with anhydrous DMF (10 mL). Then, EDCI·HCl (1102.5 mg,
5.7511 mmol) was added at 0 °C, and the whole was stirred at
room temperature under an Ar atmosphere for 3.5 h. A saturated
aqueous solution of NaHCO3 (30 mL) was added. The mixture
was filtered to obtain the precipitate, 64, as a colorless solid
(664.1 mg, 1.968 mmol, 73%) (Figure 21).
Compound 64 (1101.6 mg, 3.2644 mmol) was dissolved in
anhydrous THF (10 mL). DIPEA (1.7 mL) and MMTrCl
(1306.4 mg, 4.2305 mmol) were added to the solution, and the
whole was stirred at room temperature under an Ar atmosphere
for 3 h. A saturated aqueous solution of NaHCO3 (20 mL) was
added to the solution, and the whole was extracted three times
with AcOEt (20 mL × 3). The combined organic layer was
washed with brine, dried over Na2SO4, and filtered. The solvent
was evaporated to afford a yellow oil. The residue was column-
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