Structure–Activity Relationships of Lysophosphatidylserine Analogs as

Article pubs.acs.org/jmc

Structure−Activity Relationships of Lysophosphatidylserine Analogs as Agonists of G‑Protein-Coupled Receptors GPR34, P2Y10, and GPR174 Masaya Ikubo,†,# Asuka Inoue,#,‡,§ Sho Nakamura,† Sejin Jung,†,∞ Misa Sayama,† Yuko Otani,† Akiharu Uwamizu,‡ Keisuke Suzuki,‡ Takayuki Kishi,‡ Akira Shuto,‡ Jun Ishiguro,‡ Michiyo Okudaira,‡ Kuniyuki Kano,‡ Kumiko Makide,‡,§ Junken Aoki,*,‡,⊥ and Tomohiko Ohwada*,† †

Laboratory of Organic and Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan ‡ Laboratory of Molecular and Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3, Aoba, Aramaki, Aoba-ku, Sendai, Miyagi 980-8578, Japan § PRESTO and ⊥CREST, Japan Science and Technology Agency (JST), 4-1-8, Honcho, Kawaguchi-shi, Saitama 332-0012, Japan S Supporting Information *

ABSTRACT: Lysophosphatidylserine (LysoPS) is an endogenous lipid mediator generated by hydrolysis of membrane phospholipid phosphatidylserine. Recent ligand screening of orphan G-proteincoupled receptors (GPCRs) identified two LysoPS-specific human GPCRs, namely, P2Y10 (LPS2) and GPR174 (LPS3), which, together with previously reported GPR34 (LPS1), comprise a LysoPS receptor family. Herein, we examined the structure−activity relationships of a series of synthetic LysoPS analogues toward these recently deorphanized LysoPS receptors, based on the idea that LysoPS can be regarded as consisting of distinct modules (fatty acid, glycerol, and L-serine) connected by phosphodiester and ester linkages. Starting from the endogenous ligand (1-oleoyl-LysoPS, 1), we optimized the structure of each module and the ester linkage. Accordingly, we identified some structural requirements of each module for potency and for receptor subtype selectivity. Further assembly of individually structure-optimized modules yielded a series of potent and LysoPS receptor subtype-selective agonists, particularly for P2Y10 and GPR174.

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INTRODUCTION

containing various fatty acids, including 1-oleoyl-LysoPS (1), were detected.11 GPR34 is highly expressed in immune cells such as mast cells and macrophages, immune system-derived cell lines, and

A recent deorphaning project for orphan G-protein-coupled receptors (GPCRs) identified two common lysophosphatidylserine (LysoPS)-specific GPCRs, namely, P2Y10 (also designated as LPS2) and GPR174 (LPS3), and one speciesspecific receptor, A630033H20 (LPS2L (LPS2-like)).1 While human A630033H20 was the product of a truncated nonfunctional pseudo gene, homologues of P2Y10 and GPR174 have been found in human, mouse, and rat, and they respond to endogenous LysoPS (1) with submicromolar EC50 values. Thus, including GPR34 (LPS1),2,3 which was the first LysoPS receptor to have been identified and which responds to LysoPS with similar affinity,4 at least three LysoPS receptors are present in mammals (human, mouse, and rat). LysoPS is a new class of lysophospholipid mediator generated by enzymatic hydrolysis of membrane phospholipid, phosphatidylserine (PS).5−10 LysoPS has a modular structure consisting of a single fatty acid, glycerol, and L-serine, connected by phosphodiester and ester linkages (Figure 1). In supernatant of activated rat platelets, LysoPS species © 2015 American Chemical Society

Figure 1. Lysophosphatidylserine (LysoPS) containing oleic acid. The modular structure consists of a single fatty acid, glycerol, and L-serine, connected by phosphodiester and ester linkages. Received: December 27, 2014 Published: May 13, 2015 4204

DOI: 10.1021/jm5020082 J. Med. Chem. 2015, 58, 4204−4219

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Journal of Medicinal Chemistry Table 1. Agonistic Activities of Module-Modified Analoguesa

compd

1 (lysoPS (18:1) (oleic acid))c,e 2 (2-deoxy-lysoPS)

3a (lysoPS 2S-OH)

3b (2R-OMe) 3c (di-Me)

3d (2R-F)

3e (2S-F) 3f (di-F)

4a (16:1, palmitoleic acid)

4b (16:0, palmitic acid)

4c (14:0, myristic acid)

4d (12:0, lauric acid)

4e (10:0, capric acid) 5a (deoxy-(16:1))

5b (deoxy-(18:0))

5c (deoxy-(16:0))

5d (deoxy-(14:0))

5e (deoxy-(12:0))

5f (deoxy-(18:1E), elaidic acid

5g (deoxy-(18:1;vac), vaccenic acid)

GPR34

P2Y10

GPR174

(LPS1)

(LPS2)

(LPS3)

EC50

EC50

EC50

log EC50 [M]

log EC50 [M]

log EC50 [M]

(95% CI)b

(95% CI)b

(95% CI)b

Modification of Glycerol Moiety 550 nM >1 μM

410 nMd −6.38 (−6.57 to −6.20) >1 μMd 320 nMc −6.50 (−6.77 to −6.22) Fluorination of Glycerol Moiety >1 μM

28 nM 25 nM (−7.66 to −7.54) 39 nM −7.41 (−7.57 to −7.25) >1 μM 58 nM −7.23 (−7.44 to −7.03)

150 nM −6.84 (−7.32 to >1 μM >1 μM inactive 650 nM −6.19 (−6.47 to Modification of Fatty Acid Moiety (Natural Fatty Acid) 1.4 μM 86 nM −5.87 −7.07 (−6.24 to −5.49) (−7.17 to 290 nM 26 nM −6.54 −7.59 (−6.73 to −6.35) (−7.70 to >1 μM 72 nM −7.14 (−7.26 to 840 nM 940 nM −6.08 −6.03 (−6.37 to −5.78) (−6.17 to inactive >1 μM Modification of Fatty Acid Moiety 3.1 μM 58 nM −5.50 −7.24 (−5.97 to −5.04) (−7.31 to 1.7 μM 87 nM −5.78 −7.06 (−6.25 to −5.30) (−7.19 to >1 μM 21 nM −7.67 (−7.80 to 700 nM 45 nM −6.15 −7.35 (−6.40 to −5.91) (−7.45 to >1 μM 520 nM −6.28 (−6.48 to >1 μM 400 nM −6.39 (−6.67 to >1 μM 170 nM −6.77 (−7.03 to 4205

520 nM inactive −7.60

−6.35)

1.9 μM −5.72 (−5.92 to −5.53) inactive inactive

2.0 μM −5.70 (−6.16 to −5.23) >1 μM inactive

−5.90)

−6.97)

−7.49)

−7.03)

−5.89)

290 nM −6.54 (−6.63 to 540 nM −6.27 (−6.41 to 770 nM −6.11 (−6.30 to 1.8 μM −5.75 (−5.99 to >1 μM

−6.44)

−6.13)

−5.92)

−5.51)

>1 μM −7.17) inactive −6.94) >1 μM −7.54) >1 μM −7.25) inactive −6.09) inactive −6.12) inactive −6.51) DOI: 10.1021/jm5020082 J. Med. Chem. 2015, 58, 4204−4219

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Journal of Medicinal Chemistry Table 1. continued

compd

6a (ether (18:1))

6b (NH-amide (18:1))

6c (deoxyether (18:1))

7a (Me ester)

7b (alcohol (CH2OH)) 7c (D-serine (lysoPDS)) 7d (lysoPT)

7e (lysoPalloT (18:1))

7f (lysoPalloT (16:1))

7g (lysoPalloT (16:0))

7h (lysoPalloT (14:0))

7i (lysoPalloT (12:0)) 8-o-C7 (C3-ph-o-O-C7)

8-m-C7 (C3-ph-m-O-C7)

8-p-C7 (C3-ph-p-O-C7)

8-o-C9 (C3-ph-o-O-C9)

8-o-C11 (C3-ph-o-O-C11)e 8-m-C11 (C3-ph-m-O-C11)e 8-p-C11 (C3-ph-p-O-C11)

8-o-C15 (C3-ph-o-O-C15)e 8-m-C15 (C3-ph-m-O-C15)

8-p-C15 (C3-ph-p-O-C15)

GPR34

P2Y10

GPR174

(LPS1)

(LPS2)

(LPS3)

EC50

EC50

EC50

log EC50 [M]

log EC50 [M]

log EC50 [M]

(95% CI)b

(95% CI)b

(95% CI)b

Modification of Fatty Acid Linkage >1 μM 4.5 μM −5.35 (−5.98 to >1 μM 1.2 μM −5.94 (−6.17 to inactive 330 nM −6.48 (−6.71 to Modification of Amino Acid Moiety inactive 140 nM −6.84 (−6.98 to inactive >1 μM inactive >1 μM Threonine-Type Analogues inactive 2.2 μM −5.65 (−5.88 to inactive >1 μM

>1 μM −4.72)

−5.71)

−6.25)

−6.70)

4206

1.1 μM −5.96 (−6.20 to −5.72) inactive inactive inactive

−5.42)

2.1 μM −5.69 (−6.13 to −5.25) inactive 210 nM −6.67 (−6.84 to −6.50) inactive 710 nM −6.15 (−6.50 to −5.81) inactive inactive Modification of Fatty Acid Moiety (Non-Lipid Analogues) >1 μM 120 nM −6.91 (−7.05 to −6.77) inactive 100 nM −6.98 (−7.09 to −6.87) inactive 460 nM −6.34 (−6.50 to −6.17) 930 nM 3.2 nM −6.03 −8.50 (−6.22 to −5.85) (−8.60 to −8.40) 460 nM 2.0 nM inactive 12 nM inactive 83 nM −7.08 (−7.21 to −6.95) 240 nM 2.7 nM 160 nM 13 nM −6.80 −7.89 (−7.11 to −6.50) (−7.98 to −7.80) >1 μM 53 nM inactive

63 nM −7.20 (−7.34 to −7.07) inactive

830 nM −6.08 (−6.19 to 580 nM −6.23 (−6.36 to 1.1 μM −5.98 (−6.20 to 1.6 μM −5.81 (−5.91 to >1 μM 1.4 μM −5.87 (−6.20 to 82 nM −7.09 (−7.18 to 490 nM −6.31 (−6.50 to 510 nM −6.29 (−6.62 to 44 nM 18 nM 76 nM −7.12 (−7.24 to 33 nM 25 nM −7.60 (−7.80 to 86 nM

−5.97)

−6.11)

−5.76)

−5.70)

−5.53)

−6.99)

−6.12)

−5.96)

−7.00)

−7.41)

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Journal of Medicinal Chemistry Table 1. continued

compd

GPR34

P2Y10

GPR174

(LPS1)

(LPS2)

(LPS3)

EC50

EC50

EC50

log EC50 [M]

log EC50 [M]

log EC50 [M]

(95% CI)b

(95% CI)b

(95% CI)b

Modification of Fatty Acid Moiety (Non-Lipid Analogues) −7.27 (−7.38 to −7.17)

−7.07 (−7.32 to −6.81)

a Activities are represented in terms of EC50 (see Experimental Section). “Inactive” means almost no activity. “(EC50) > 1 μM” means that the activity is weak or marginally present. b95% confidence interval of a log EC50 value. cReference 1. dReference 21. eMean and standard error of mean (SEM) of log EC50 values are shown in Table 3.

Table 2. Agonistic Activities of Mix-and-Match Analoguesa

compd

9-o-C7 (deoxy-C3-ph-o-O-C7)

GPR34

P2Y10

GPR174

(LPS1)

(LPS2)

(LPS3)

EC50

EC50

EC50

log EC50 [M]

log EC50 [M]

log EC50 [M]

(95% CI)

(95% CI)

(95% CI)

Deoxy-Non-Lipid Combination >1 μM

9-o-C11 (deoxy-C3-ph-o-O-C11)b 9-m-C11 (deoxy-C3-ph-m-O-C11)

1.8 μM −5.74 (−6.08 to −5.40) >1 μM >1 μM

9-p-C11 (deoxy-C3-ph-p-O-C11)

>1 μM

9-o-C9 (deoxy-C3-ph-o-O-C9)

11a (LysoPalloT-C3-ph-m-O-C11)b 11b (LysoPalloT-C3-ph-m-O-C7)

Fluorinated Non-Lipid 940 nM >1 μM LysoPalloT Non-Lipid inactive inactive

12 (LysoPalloT-NH-amide-C3-ph-m-O-C11)b

inactive

10a (2R-F-C3-ph-o-O-C11)b 10b (diF-C3-ph-o-O-C11)b

86 nM −7.07 (−7.15 to 3.7 nM −8.43 (−8.52 to 1.7 nM 33 nM −7.49 (−7.69 to 56 nM −7.26 (−7.46 to

>1 μM −6.98) >1 μM −8.35) >1 μM >1 μM −7.28) >1 μM −7.05)

1.3 nM 3.3 nM

890 nM >1 μM

550 nM >1 μM

40 nM 720 nM −6.14 (−6.22 to −6.06) 31 nM

>1 μM

Activities are represented in terms of EC50 (see Experimental Section). “Inactive” means almost no activity. “(EC50) > 1 μM” means that the activity is weak or marginally present. bMean and SEM of log EC50 values are shown in Table 3. a

lymphoma cells.12 GPR34 is also preferentially expressed in microglia, and its mRNA was up-regulated in activated microglia upon treatment with a demyelinating toxin, suggesting that the receptor plays a role in neuroinflammation.3,12 Studies in GPR34-deficient (KO) mice indicate that GPR34 is involved in immune responses to antigen and pathogen challenge.12−14 P2Y10 and GPR174 are specifically expressed in lymphoid organs.15 Interestingly, single nucleotide polymorphisms of GPR174 are associated with Graves’ disease,16−18 Addison’s disease,19 and vasovagal syncope.20 The above findings suggest that these LysoPS receptors are involved in immune responses and that endogenous LysoPS species have an immune-regulatory role. Therefore, structure− activity studies of LysoPS analogues are likely to be helpful in the design of synthetic agonists of these receptors as candidate

therapeutic agents and also as research tools.21,22 To explore the structure−function relationships, we hypothesized that LysoPS could be regarded as a modular assembly of fatty acid, glycerol, and phosphoserine.22 This idea seemed reasonable because several LysoPS analogues containing simple modifications in individual modules activated the three LysoPS receptor subtypes differently.21 Therefore, starting from the endogenous ligand (1, 1-oleoyl-LysoPS), we optimized the structures of the individual modules, as well as their linkages. We focused on unsaturated fatty acid derivatives because, like other lysophospholipid receptors for lysophosphatidic acid (LPA) and lysophosphatidylinositol (LPI),23 LysoPS receptors favor LysoPS with an unsaturated fatty acid component rather than a saturated fatty acid moiety.4 We also developed a nonlipid surrogate of the unsaturated fatty acid moiety of LysoPS. 4207

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Journal of Medicinal Chemistry

Figure 2. Glycerol modified derivatives.

This work has enabled us to identify and generalize some of the structural requirements for activation of individual LysoPS receptors. On the basis of the structure−activity relationships obtained, we were able to assemble individually structureoptimized modules to afford potent LysoPS receptor agonists, some of which showed subtype selectivity.

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value were computed for active compounds with plateau or semiplateau responses by fitting data to a four-parameter sigmoid curve using GraphPad Prism 6 (GraphPad, USA) (Tables 1 and 2). EC50 values were converted from log EC50. All the biological data are compiled in Tables 1 and 2 and Supporting Information (Figures S1−S10 and Tables S1−S10). In Tables 1 and 2, log EC50 and 95% CI were described. For active key compounds, standard errors of the mean (SEMs) of log EC50 values are shown in Table 3. Concentration−response curves of some of the potent/selective compounds in the TGFα shedding assay are shown in Figure 9, and the original concentration−response data are shown in Figure S11. Modification of Glycerol Module. Our previous study using 1-oleoyl-2-deoxy-LysoPS (2) suggested the importance of a sn-2 hydroxyl group for GPR174 activation (Figure 2, Table 1).21 Other glycerol analogues, including 2S-OH-LysoPS (3a), bearing a diastereomeric (S)-hydroxyl group at the sn-2 position, and 2R-OMe-LysoPS (3b) bearing an sn-2-(R)methoxy group, have been examined (Figure 2), and the results support the idea that the glycerol moiety contributes to receptor activation.21 To further examine structural requirements with respect to the glycerol moiety, in particular the role of the sn-2 hydroxyl group, we synthesized various LysoPS derivatives (3c−f, Figure 2), in which the glycerol hydroxyl functionality was modified. We used oleic acid (18:1) as a standard fatty acid, unless otherwise mentioned. For convenience, relative activities of synthetic analogues are shown with respect to the activities of 1-oleoyl-LysoPS (1), a prototype pan-agonist of the three LysoPS receptors (Table 1). We synthesized derivatives with geminal-dimethyl (2-diMeLysoPS, 3c), fluorine (2R-(3d) and 2S-(3e)), and germinaldifluoro (2-di-F-LysoPS, 3f) substitution.24,25 These derivatives showed very weak or no activity toward GPR174 (Table 1, Table S2 and Figure S2). Two diastereoisomeric monofluorinated derivatives (3d and 3e) showed somewhat different activation potencies with respect to P2Y10 (3d EC50 = 150 nM; 3e EC50 > 1 μM for P2Y10, Table 1), while their activities toward GPR34 were decreased. Intriguingly, the geminaldifluoro analogue (3f) showed essentially no activity toward GPR34 and GPR174; thus, 3f is a selective activator of P2Y10, though it is weaker than LysoPS (1) (3f, EC50 for P2Y10 is 650 nM). Although electronegativity considerations suggest that C−F would behave similarly to the sn-2 C−O functionality and act as a good H-bond acceptor,26 the C−F unit is actually a poor H-bond acceptor: organic fluorine has a very low proton affinity and is weakly polarizable.27 Therefore, these results supported the idea that in GPR174 activation, the sn-2 hydroxyl group of the glycerol moiety is considered to serve as a hydrogen bond donor, probably as well as a hydrophilic functionality.21 Modification of the Fatty Acid Module. Glycerol-Type Analogues. We next focused on the fatty acid moiety because

RESULTS AND DISCUSSION

Synthesis of LysoPS Analogues. We previously established synthetic routes to LysoPS analogues containing saturated fatty acids or oleic acid.22 Herein we changed that synthetic approach to obtain unsaturated fatty acid analogues, using the Boc-protection−acid-catalyzed deprotection procedure instead of the formerly used benzyl-protection−hydrogenative debenzylation procedure.21,22 This change enabled us to access LysoPS analogues containing functional groups sensitive to hydrogenation and thus to explore a broader range of LysoPS analogues. The phosphoramidite method was used to construct the phosphate diester linkage in these analogues. The synthesis of 1-oleoyl-LysoPS 1 is shown in Figure S12 in Suporting Information. Boc-L-serine tert-butyl ester was phosphorylated with tert-butyl tetraisopropylphosphorodiamidite in the presence of 1H-tetrazole, followed by a second phosphorylation with a protected glycerol. In situ oxidation of the resultant phosphite triester intermediate with tert-butyl hydroperoxide (TBHP) afforded the fully protected phosphate triester. Deprotection of PMP ether on the terminal oxygen atom of the glycerol moiety was achieved by selective oxidation with CAN, followed by acylation with oleoyl chloride. Global deprotection with TFA furnished the product (1) as the TFA salt.22 The synthesis of 9-o-C11 and 12 are shown in Figures 8 and 9 (vide infra), which were identified in this work to be a potent and selective P2Y10 agonist and GPR174 agonist, respectively. Synthesis of nonaromatic LysoPS analogues (1 and 2a−7i) and that of aromatic LysoPS analogues (8−12) are described in Supporting Information. Transforming Growth Factor α (TGFα) Shedding Assay. TGFα shedding assay was performed as described previously.1,21 The TGFα shedding assay detects activation of GPCRs by measuring ectodomain shedding of a membranebound proform of alkaline phosphatase-tagged TGFα (APTGFα) into conditioned media. Since AP-TGFα shedding response occurs almost exclusively downstream of Gα12/13 and Gαq signaling,1 we additionally transfected a chimeric Gαq/i1 subunit to detect activation of Gi-coupled GPR34. We examined TGFα shedding response of synthetic compounds using mouse GPR34, P2Y10, and GPR174 LysoPS receptors.1 After subtraction of baseline responses in mock-transfected cells (also see data processing in Supporting Information Figure S12), agonist activity in each receptor is represented as follows: The log EC50 and 95% confidence interval (CI) of a log EC50 4208

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Figure 3. Fatty acid-modified derivatives of LysoPS-type analogues.

Figure 4. Deoxyglycerol-type analogues containing various fatty acid moieties.

Figure 5. Structures of ester linkage surrogates.

the previous study4,22 suggested that LysoPS receptors recognize the fatty acid moiety like other lysophospholipid receptors for lysophosphatidic acid and lysophosphatidylinositol. LysoPS receptors show preference for unsaturated fatty acid rather than saturated fatty acid as a component of LysoPS, in a similar manner to other lysophospholipid receptors for lysophosphatidic acid (LPA) and lysophosphatidylinositol (LPI).23,24 First of all, we examined the effect of the fatty acid chain length as well as degree of unsaturation on the activity of LysoPS-type (2-hydroxyglycerol) derivatives (4a−e) (Figure 3, Table 1, Table S3 and Figure S3). GPR34 and P2Y10 were activated by palmitoyl (16:0) LysoPS (4b) similarly to LysoPS (1) (4b, EC50 = 290 nM for GPR34; EC50 = 26 nM for P2Y10; EC50 = 540 nM for GPR174), and GPR174 was most highly activated by palmitoleoyl (16:1) LysoPS (4a) rather than oleoyl (18:1) LysoPS (1) (4a, EC50 = 1.4 μM for GPR34; EC50 = 86 nM for P2Y10; EC50 = 290 nM for GPR174). LysoPS analogues with a shorter fatty acid (4c and 4d) consistently showed decreased activation potencies toward the three receptors, while 4c (14:0) and 4d (12:0) still retained the activation potency toward P2Y10 and GPR174 (by 4c) (Table

1 and Figure S3). The analogue (4e) containing a fatty acid as short as C10 showed diminished activity toward all three receptors. These results suggest that, like other receptors for lysophospholipids, a minimum hydrophobic volume is essential for LysoPS receptor agonists and also indicate that subtype selectivity among LPS receptors can be achieved by modifying the fatty acid moiety (vide infra). Deoxyglycerol-Type Analogues. We next synthesized 2deoxyglycerol-type LysoPS analogues 5a−g (Figure 4) containing isomers of the oleoyl group, in which the olefin geometry (E) and the position of the Z-olefin are different. In biological evaluation, the geometrical (5f) and positional (5g) isomers of the oleoyl group exhibited reduced activity toward P2Y10 as compared with 1-oleoyl-2-deoxy-LysoPS (2) (Table 1 and Tables S4 and Figure S4). These observations indicate that the shape of the fatty acid moiety plays a significant role in interactions with the LysoPS receptors, in addition to the effect of hydrophobicity. In these 2-deoxy derivatives (5a−g), activation potency toward GPR34 was consistently reduced. The analogues containing a shorter fatty acid (5d and 5e) showed P2Y10 activation activity. These 2-deoxy analogues 4209

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Figure 6. Structures of amino acid derivatives.

GPR174 (7e for GPR174, EC50 = 830 nM; 7f for GPR174, EC50 = 580 nM, Table 1), but the potency was reduced in the saturated fatty acid substituted counterparts (16:0 (7g) and 14:0 (7h)) and almost abrogated in the case of short-chain fatty acid derivatives such as 12:0 (7i) (Figure 6 and Table 1; see also Table S6 and Figure S6). Thus, the physicochemical features of the fatty acid moiety required for activity of LysoPalloT analogues (7) toward GPR174 are very similar to those of the LysoPS analogues (3). Elimination of the 2hydroxyl group from the glycerol moiety diminished the GPR174 activation potential of LysoPalloT analogues (7) (see Figure S6(C)). Thus, allo-type methylation of the amino acid moiety is a GPR174-targeting modification. Aromatized Non-Lipid Surrogates of Fatty Acids. The structure−activity relationship for the fatty acid moiety indicated that carbon chain length, unsaturation, olefin geometry, and the position of the olefin functionality influence the activation potency toward LysoPS receptors (Figures 3 and 4). From the viewpoint of space-filling and to avoid the inherent chemical instability of the olefin functionality, we utilized 3-phenylpropanoic acid (8) (Figure 7A),28 and to provide sufficient hydrophobicity, we introduced an alkyl chain onto the benzene ring through an ether linkage. The alkyl ether chain lengths (C7, C9, C11, and C15) and the substitution positions on the phenyl ring (ortho, meta, and para) were varied. As the ether carbon chain was elongated in the LysoPS (2-hydroxyglycerol)-type analogues of ortho-ether derivatives 8-o-CX (C3-o-O-CX) (X = 7, 9 11, and 15), the activation potency toward all three LysoPS receptors dramatically increased. 8-o-C11 and 8-o-C15 showed potent activity toward all three receptors (8-o-C11 for GPR34, EC50 = 460 nM; for P2Y10, EC50 = 2.0 nM, for GPR174, EC50 = 44 nM; 8-o-C15 for GPR34, EC50 = 240 nM; for P2Y10, EC50 = 2.7 nM; for GPR174, EC50 = 33 nM, Table 1, Table S7 and Figure S7), though the potency leveled off with increasing alkyl chain length (see Figure 10A and Figure S11). The shorter ether carbon chain derivative (8-o-C7) showed significantly attenuated activity toward P2Y10 as compared with the longer

(5a−g) did not show activity toward GPR174 (see Figure S4(C)). Modification of the Ester Linkage. We next modified the ester linkage between the fatty acid and the glycerol moiety (Figure 5), replacing it with an ether (6a) or an amide bond (6b). The LysoPS ether analogue 6a showed markedly lower or diminished activation potency for all three receptors (Table 1 and Figure S5), suggesting an important role of the carbonyl group of the ester linkage. The amide linked compound 6b showed reduced potency toward GPR34 and P2Y10, but its potency toward GPR174 was enhanced over that of LysoPS 1 (6b for GPR174: EC50 = 63 nM, Table 1 and Figure S5). As expected, the 2-deoxy-type ether compound (deoxy ether 6c) showed weaker (for P2Y10) or almost no activity toward the two other receptors (Table 1 and Figure S5). Modification of the Amino Acid Module. Analogues modified at the serine moiety, such as commercially available and endogenous 1-oleoyl-lysophosphatidylethanolamine (LysoPE), and chemically synthesized derivatives, including LysoPS Me ester (7a), LysoPS CH2OH (7b), and lysoPDS (D-serine) (7c) (Figure 6 and Table 1), showed little activity toward the three receptors (except 7a toward P2Y10), suggesting that the amino acid functionality is critical for activation of LysoPS receptors.21 Next, we synthesized LysoPS analogues with a methyl group at the β-position of serine, corresponding to modification of Lserine to threonine or allothreonine (Figure 6).22 As already reported,21 when the stereochemistry of the methyl group was R (L-threonine), LysoPT (7d) showed no activation potency toward the three LysoPS receptors, though 7d was reported to be a strong inducer of mast cell degranulation.22 The (S)methyl analogue (i.e., allo-threonine), LysoPalloT (7e), showed reduced P2Y10 activation activity and apparent GPR174 activation activity, though interestingly, it showed no mast cell degranulation activity.22 7e diminished the activation potency toward GPR34.21 LysoPalloT analogues, particularly those containing unsaturated fatty acid chains (18:1 (7e) and 16:1 (7f)), activated 4210

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Figure 7. Aromatic fatty acid surrogates and mix-and-match design: (A) structures of aromatic ether-type surrogates of fatty acid; (B) combinations of module structures.

of GPR34, P2Y10, and GPR174 (Table 1; see Figure 10A, Figure 10B, and also Figure S11). In addition, we found that regioisomers of the alkyl ether derivatives exhibited distinct preferences for activation among the three LysoPS receptors (Tables S7 and S8 and Figures S7

ether analogues, and the activities toward GPR34 and GPR174 were very weak (Table 1 and Figure S7). Thus, among the compounds examined, 8-o-C11 and 8-o-C15 (Figure 7A) are superior to the endogenous LysoPS (18:1) (1) as a pan-agonist 4211

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Journal of Medicinal Chemistry Table 3. Parameters for the Active Key Compoundsa compd 1 (lysoPS (18:1) (oleic acid)) 8-o-C11 (C3-ph-o-O-C11) 8-m-C11 (C3-ph-m-O-C11) 8-o-C15 (C3-ph-o-O-C15) 9-o-C11 (deoxy-C3-ph-o-O-C11) 10a (2R-F-C3-ph-o-O-C11) 10b (di-F-C3-ph-o-O-C11) 11a (LysoPalloT-C3-ph-m-O-C11) 12 (LysoPalloT-NH-amide-C3-ph-m-O-C11) a

GPR34

P2Y10

(LPS1)

(LPS2)

(LPS3)

log EC50 [M]

log EC50 [M]

log EC50 [M]

−6.26 −6.34 NA −6.63 NA −6.03 NA NA NA

−7.55 −8.70 −7.91 −8.58 −8.77 −8.87 −8.48 −6.26 NA

−6.28 −7.36 −7.74 −7.48 NA −6.05 NA −7.40 −7.51

± 0.04 ± 0.10 ± 0.08 ± 0.07

± ± ± ± ± ± ± ±

0.06 0.08 0.09 0.06 0.05 0.06 0.11 0.13

GPR174

± ± ± ±

0.07 0.14 0.11 0.04

± 0.04 ± 0.07 ± 0.10

Mean ± SEM of 11 (1, 8-o-C15) and 5 (8-o-C11, 8-m-C11, 9-o-C11, 10a, 10b, 11a, 12) independent experiments. NA, not available.

Figure 8. Synthesis of 9-o-C11.

p-OC11 (8-p-C11), the activation potency toward GPR174 was stronger than that of LysoPS (1), but its activity toward P2Y10 was attenuated compared with the o-OC11 and m-OC11 derivatives, and it was almost inactive toward GPR34 (Table 1) (8-p-C11 for P2Y10, EC50 = 83 nM; for GPR174, EC50 = 76 nM). For the p-OC15 analogue (8-p-C15), the activity trend was similar to that of 8-p-C11: the activity of 8-p-C15 toward GPR174 was greater than that of LysoPS (1) (8-p-C15 for P2Y10, EC50 = 53 nM; for GPR174, EC50 = 86 nM, Table 1 and Figure S8). Mix-and-Match Compounds. Modification of the four modules or linkages of LysoPS 1, i.e., glycerol, fatty acid, serine, and the ester linkage (Figure 1), revealed that the optimal module structures for activation of the three LysoPS receptors are different and rather orthogonal. To examine the robustness of the structural requirements of the respective modules for agonist activity toward individual receptors and to obtain highly potent agonists, we synthesized hybrid analogues in which individual optimal modules are combined into a single molecule in various ways (Figure 7B). We confirmed that the performance of the independent optimal structure modules

and S8). In the case of the meta derivatives, elongation of the ethereal alkyl chain from m-OC7 (8-m-C7) to m-OC11 (8-mC11) increased the activation potency toward P2Y10 and GPR174 (8-m-C11 for P2Y10, EC50 = 12 nM; for GPR174, EC50 = 18 nM, Table 1 and Figure S7). In the case of meta derivatives (8-m-C7 and 8-m-C11), activation potency toward GPR34 almost disappeared (see Figure S7(A)). The metaOC11 derivative (8-m-C11) is therefore a dual agonist of P2Y10 and GPR174, with greater potency than LysoPS (1) (see Figure 10A, Figure 10C, and also Figure S11). The longeralkyl-chain analogue (8-m-C15) showed a similar trend, and the activities against P2Y10 and GPR174 were almost saturated (8-m-C15 for GPR34, EC50 = 160 nM; for P2Y10, EC50 = 13 nM; for GPR174, EC50 = 46 nM, Table 1 and Figure S11). Intriguingly, the activation activity toward GPR34 was restored in the meta (8-m-C15) containing a long alkyl chain (8-m-C15 for GPR34, EC50 = 160 nM, Table 1 and Figure S8). On the other hand, the para ether derivatives p-OC7 (8-p-C7), pOC11 (8-p-C11), and p-OC15 (8-p-C15) did not show significantly higher activities than the ortho and meta derivatives (Table 1 and Figures S7 and S8): in the case of 4212

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Figure 9. Synthesis of 12.

C9 and 9-o-C11 (1-o-OC11-2-deoxy-LPS) were the most potent and selective agonists for P2Y10. The corresponding regioisomers, meta derivative 9-m-C11 (2-deoxy-m-OC11) and para derivative 9-p-C11 (2-deoxy-p-OC11), were also synthesized because these combinations should also lead to virtual P2Y10 selectivity. In fact, the meta derivative (9-m-C11) and the para derivative (9-p-C11) exhibited selective activity toward P2Y10, but the activation magnitude was decreased in these regioisomers as compared with the ortho analogue (9-o-C11) (for P2Y10, 9-m-C11 EC50 = 33 nM; 9-p-C11 EC50 = 56 nM, Table 2) (Figure 7B). Therefore, 9-o-C11 (1-o-OC11-2-deoxyLPS) appears to be a selective strong agonist for P2Y10 (Figure 10A and Figure 10C; see also Figure S11). The synthesis of 9o-C11 is shown in Figure 8. Combinations of fluorine-substituted glycerol mimics and oOC11 fatty acid surrogate (10a and 10b) also showed selective and potent activity toward P2Y10 as expected (Figure 10B) because fluorine substitution at the sn-2 position of glycerol favors P2Y10 activation (Figure 7B(b)). The activity toward GPR34 was significantly reduced, and these compounds showed very weak or no activity toward GPR174 (Table 2). Monofluorine substitution (10a) and geminal-difluorine substitution (10b) increased selectivity for P2Y10 (10a for P2Y10, EC50 = 1.3 nM; for GPR34, EC50 = 940 nM; for GPR174, EC50

remained consistent even in hybrid molecules and afforded a synergistic increase in receptor-activation potential/selectivity, providing potent selective agonists particularly for P2Y10 and GPR174 (Table 2 and Figure 10, see also Figure S11). The mean and SEM values of log EC50 values for the key potent compounds, shown in Figure 10, were obtained from four independent experiments, and the values are shown in Table 3. P2Y10-Selective Agonists. Using the combination of 2deoxyglycerol and 3-(o-alkoxyphenyl)propanoic acid, we changed the length of the ether alkyl chain and synthesized several 2-deoxy-LysoPS-type derivatives, o-C7 (9-o-C7), o-C9 (9-o-C9), and o-C11 (9-o-C11) (Figure 7B(a)). The 2deoxyglycerol module in LysoPS analogues activated particularly P2Y10 but not GPR34 and GPR174. The 3-(oalkoxyphenyl)propanoic acid module strongly activates all three receptors and is thus a pan-agonistic element. The hybrid analogues (9-o-C7, 9-o-C9, and 9-o-C11) showed clear selectivity for P2Y10 over GPR34 (Table 2), whereas they showed very weak or no activity toward GPR174 (Figure 10A and Figure 10C; see also Figures S9 and S11). As the aromatic ether chain length was increased, the activation potency increased (for P2Y10, 9-o-C7 EC50 = 86 nM: 9-o-C9 EC50 = 3.7 nM: 9-o-C11 EC50 = 1.7 nM, Table 2) and those of 9-o-C9 and 9-o-C11 seemed to plateau. Among these analogues, 9-o4213

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Journal of Medicinal Chemistry 890 nM; 10b for P2Y10, EC50 = 3.3 nM; for GPR34, EC50 > 1.0 μM; for GPR174, EC50 > 1.0 μM, Table 2, see also Figures S10 and S11). GPR174-Selective Agonists. The combination of allothreonine and m-OC11 fatty acid surrogate (11a) afforded a GPR174 agonist much more potent than the allo-threonine (18:1) compound (7e) (Figure 7B(c)). These compounds (7e and 11a) showed similar activation profiles: GPR174 activation was favored, but P2Y10-activating ability remained (11a for GPR174, EC50 = 40 nM; P2Y10, EC50 = 550 nM, Table 2 and Figure 10C; 7e for GPR174, EC50 = 830 nM; P2Y10, EC50 > 1 μM, Table 1 and Figure S7). A shorter aromatic ether carbon chain (m-OC7) analogue, 11b, had weaker activation ability toward GPR174 (11b for GPR174, EC50 = 720 nM). Furthermore, the combination (12) of allo-threonine, mOC11 fatty acid surrogate, and an amide linkage afforded a GPR174-selective agonist (Table 2). The activity of 12 was more potent than that of LysoPS (1) (12 for GPR174, EC50 = 31 nM, Table 2 and Figure 7B(d)), but 12 is virtually selective for GPR174 and did not activate either GPR34 or P2Y10 efficiently (Figure 10B and Figure 10C; see also Figures S10 and S11). The synthesis of 12 is shown in Figure 9. These results support the postulate that LysoPS is composed of several distinct modules and that the optimal module structures are different and at least to some extent orthogonal for activation of different LysoPS receptors. We demonstrated here that selection and combination of appropriate modules could afford potent and subtype-selective agonists to the newly deorphanized LysoPS receptors, P2Y10 and GPR174. Validation of Potent LysoPS Analogues in Actin Stress Fiber Formation Assay. To further confirm the potency of our LysoPS analogues at the cellular level in vitro, we utilized another in vitro GPCR assay (actin stress fiber formation assay) in which P2Y10 activation via Gα12/13 is detectable as formation of actin stress fibers.1 We tested P2Y10-selective mix-and-match agonists 9-o-C11, 10b, and the GPR174-selective agonist 12 in addition to pan-agonist LysoPS (1). LysoPS (1) induces formation of actin stress fibers in P2Y10-expressing cells (Figure 11). Among the tested compounds, 9-o-C11 and 10b induced formation of actin stress fibers but 12 did not. Actin stress fiber formation was not observed in stably mock-infected cells (Figure 11, control). These results further establish the potency of these analogues and also support the generality of the characteristics of these LysoPS analogues found in the TGFα shedding assay.

Figure 10. LysoPS analogues differentially activate P2Y10 and GPR174. HEK293 cells transiently transfected with P2Y10-encoding expression vector, GPR174-encoding expression vector, or an empty vector were treated with compounds, and receptor-specific AP-TGFα release responses were determined by subtracting background responses in the empty-vector-transfected cells. Data are representative of four experiments. Error bars are SD (standard deviation) for three to six assay replicates from one experiment. For most data points, error bars are smaller than the symbols.

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CONCLUSIONS Endogenous LysoPS 1 is a pan agonist for LysoPS receptors GPR34, P2Y10, and GPR174. In the present study, we individually optimized substructure modules of LysoPS 1, that is, glycerol, fatty acid, phosphoserine, and acyl ester linkage, in order to increase the activation potency and receptor subtype selectivity. The fundamental structure−activity subtype-selectivity relationships thus obtained provided a basis for the design of novel LysoPS analogues. Various individual modifications resulted in subtype selectivity, compared with 1: that is, elimination of the hydroxyl group at the sn-2 position of glycerol, introduction of fluorine atoms into the glycerol moiety, introduction of a (S)-methyl group at the β position of serine, introduction of amide linkage, use of smaller fatty acid chains, and use of 3-(alkoxyphenyl)propionic acid as a fatty acid surrogate. The optimal structures for activation of the three LysoPS receptors are different and rather orthogonal. We found

that various combinations of the optimal substructures increased the activation potency and receptor subtype selectivity as compared with those of the endogenous ligand (1). The present results supported the hypothesis that the LysoPS structure is modularized in terms of its interactions with these receptors, that is the three receptors recognize the individual modules (substructures) in a rather independent manner. Since GPR34, P2Y10, and GPR174 are abundantly expressed in immune tissues, the subtype-selective and potent 4214

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solvents were used as received. The combustion analyses were carried out in the microanalytical laboratory of Graduate School of Pharmaceutical Sciences, the University of Tokyo. All the tested compounds are ≥95% purity on the basis of combustion analysis. We obtain unsaturated fatty acid analogues, using the Bocprotection−acid-catalyzed deprotection procedure in the final stage of the synthesis. The phosphoramidite method was used to construct the phosphate diester linkage in these analogues. The synthesis of 1-oleoyl-LysoPS 1 is shown in Figure S12. Boc-Lserine tert-butyl ester was phosphorylated with tert-butyl tetraisopropylphosphorodiamidite in the presence of 1H-tetrazole, followed by a second phosphorylation with a protected glycerol. In situ oxidation of the resultant phosphite triester intermediate with tertbutyl hydroperoxide (TBHP) afforded the fully protected phosphate triester. Deprotection of PMP ether on the terminal oxygen atom of the glycerol moiety was achieved by selective oxidation with CAN, followed by acylation with oleoyl chloride. Global deprotection with TFA furnished the product (1) as the TFA salt.22 Synthesis of nonaromatic LysoPS analogues (3a−7i) is described in Supporting Information, as well as that of aromatic LysoPS analogues (8−12). Methyl 3-(2-(Undecyloxy)phenyl)propanoate (14). To a solution of phenol 13, methyl 3-(2-hydroxyphenyl)propanoate (150.1 mg, 0.833 mmol), Cs2CO3 (338.7 mg, 1.040 mmol), and TBAI (92.7 mg, 0.251 mmol) in DMF (3 mL) was added 11undecylbromide (245.5 mg, 1.044 mmol). The whole was heated at 70 °C with stirring for 10 h. The reaction mixture was filtered, and the solvent was evaporated. The residue was column-chromatographed (hexane/Et2O = 30:1) to yield crude ester 14 (methyl 3-(2(undecyloxy)phenyl)propanoate). The crude was dissolved in MeOH (5 mL), and to this solution was added NaOMe (9.1 mg, 0.168 mmol). The whole was heated at 80 °C with stirring for 2 h. The reaction mixture was filtered, and the solvent was evaporated. The residue was chromatographed (hexane/AcOEt = 6:1) to yield methyl ester 14 (221.2 mg, 0.661 mmol, 79%, colorless oil). 1H NMR (CDCl3): δ = 7.194−7.134 (2H, m), 6.875−6.814 (2H, m), 3.958 (2H, t, J = 6.44 Hz), 3.667 (3H, s), 2.946 (2H, t, J = 7.80 Hz), 2.627 (2H, t, J = 7.80 Hz), 1.797 (2H, m), 1.470 (2H, m), 1.274 (14H, m), 0.886 (3H, t, J = 6.86 Hz). 3-(2-(Undecyloxy)phenyl)propanoic Acid (15). Methyl 3-(2(undecyloxy)phenyl)propanoate 14 (203.1 mg, 0.607 mmol), obtained above, was dissolved in MeOH (2 mL) and THF (2 mL). To this solution was added 2 N aqueous NaOH (2 mL). The resultant solution was stirred for 2 h at room temperature. Aqueous 1 N HCl was added to the aqueous layer to adjust pH to 2. The mixture was extracted three times with AcOEt (20 mL × 3). The combined organic layer was washed with brine, dried over Na2SO4, and evaporated. The residue was column-chromatographed (hexane/AcOEt = 1:1) to yield acid 15 (3-(2-(undecyloxy)phenyl)propanoic acid) (188.2 mg, 0.587 mmol, 97%, white solid). Mp: 50.5−51.5 °C. 1H NMR (CDCl3): δ = 11.277 (1H, brs), 7.211−7.159 (2H, m), 6.890−6.827 (2H, m), 3.971 (2H, t, J = 6.48 Hz), 2.963 (2H, t, J = 7.74 Hz), 2.684 (2H, t, J = 7.74 Hz), 1.810 (2H, m), 1.481 (2H, m), 1.283 (14H, m), 0.893 (3H, t, J = 6.84 Hz). 13C NMR (CDCl3): δ = 179.64, 156.95, 129.94, 128.57, 127.62, 120.18, 110.97, 67.74, 33.96, 31.91, 29.61, 29.58, 29.36, 29.34, 29.30, 26.15, 22.97, 22.68, 14.11. HRMS (ESI, [M − H]−) calcd for C20H31O3−: 319.2279. Found: 319.2269. Anal. Calcd for C20H32O3: C, 74.96; H, 10.06; N, 0.00. Found: C, 74.70; H, 9.82; N, 0.00. tert-Butyl O-(tert-Butoxy(diisopropylamino)phosphanyl)-N(tert-butoxycarbonyl)-L-serinate (17). Bis(diisopropylamino)-tertbutylphoshine (643.1 mg, 2.112 mmol) was dissolved in a mixture of CH2Cl2 (6 mL) and toluene (0.5 mL). To this solution, N-Boc-Lserine tert-butyl ester 16 (430.1 mg, 1.646 mmol) was added at room temperature. In order to eliminate traces of water, the resultant solution was evaporated under vacuum. Under an argon atmosphere, the residue was dissolved into CH2Cl2 (6 mL) and a solution of 1Htetrazole (137.2 mg, 1.959 mmol) in THF (6 mL) was added at room temperature. In a few minutes, white solids were precipitated. The whole was stirred for 3 h at room temperature, and the reaction was quenched with saturated aqueous NaHCO3 (20 mL), and the whole

Figure 11. Formation of actin stress fibers in P2Y10-expressing cells arising from P2Y10 agonists. McA-RH7777 cells stably expressing mouse P2Y10 or stably infected with an empty retrovirus (control) were serum-starved and treated with or without 1 μM compounds for 30 min. Stimulated cells were fixed and stained with Alexa 594-labeled phalloidin (F-actin, red) and DAPI (nuclei, blue). Images were obtained with a confocal laser microscope. An inlet in each panel shows an enlarged view of a single cell. Note that when P2Y10 was activated, fiber-like pattern of phalloidin staining was visible in an intracellular region (1, 9-o-C11, and 10b).

LysoPS analogues identified in this work are expected to be useful for testing the idea that dysfunction of these receptors impairs immune functions and is relevant to autoimmune disorders. The present work also provides a molecular basis for design of more potent and selective agonists for individual/ multiple LysoPS receptors, which should be helpful for drug development and validation of drug targets.

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EXPERIMENTAL SECTION

General Procedures of Synthesis. Melting points were determined with a Yanaco micro melting point apparatus without correction. 1H (400 MHz) and 13C (100 MHz) NMR spectra were recorded on a Bruker Avance 400. Chemical shifts were calibrated with tetramethylsilane as an internal standard or with the solvent peak and are shown in ppm (δ) values, and coupling constants are shown in hertz (Hz). The following abbreviations are used: s = singlet, d = doublet, t = triplet, q = quartet, dd = double doublet, dt = double triplet, dq = double quartet, h = hextet, m = multiplet, and brs = broad singlet. Electron spray ionization time-of-flight mass spectra (ESI-TOF MS) were recorded on a Bruker micrOTOF-05 to give high-resolution mass spectra (HRMS). All commercially available compounds and 4215

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Journal of Medicinal Chemistry was extracted three times with CH2Cl2 (10 mL × 3). The combined organic layer was washed with brine, dried over Na2SO4, and evaporated. The residue was column-chromatographed (n-hexane/ ethyl acetate/Et3N = 35:4:1) to yield 17 (704.1 mg, 1.516 mmol, 92%, yellow oil). In the purification with column chromatography, Et3Ndeactivated silica gel was used, that is, eluents containing 3% (v/v) Et3N. 1H NMR (CD2Cl2): δ = 5.500 (1/2H, d, J = 8.28 Hz), 5.332 (1/ 2H, d, J = 8.28 Hz), 4.218−4.138 (1H, m), 3.895 (1H, m), 3.747− 3.650 (1H, m), 3.569 (2H, m), 1.447 (9/2H, s), 1.439 (9/2H, s), 1.419 (9/2H, s), 1.412 (9/2H, s), 1.344 (9/2H, s), 1.308 (9/2H, s), 1.140 (12H, m). 13C NMR (CD2Cl2): δ = 169.61, 155.25, 155.16, 81.46, 81.31, 79.10, 79.05, 74.97, 74.89, 74.78, 63.67, 63.53, 63.26, 63.12, 55.26, 55.20, 55.13, 43.12, 43.06, 42.99, 42.93, 30.65, 30.62, 30.56, 30.54, 28.07, 28.05, 27.74, 24.46, 24.38, 24.34, 24.27, 23.97, 23.89, 23.86, 23.79. 31P NMR (CD2Cl2): δ = 138.847, 138.325. HRMS (ESI, [M + Na]+) calcd for C22H45N2NaO6P+: 487.2907. Found: 487.2927. 3-Hydroxypropyl 3-(2-(Undecyloxy)phenyl)propanoate (18). To a solution of 1,3-propandiol (287.1 mg, 3.773 mmol), acid 15 (301.9 mg, 0.942 mmol), and DMAP (10.9 mg, 0.0907 mmol) in CH2Cl2 (5 mL), EDCI (199.5 mg, 1.041 mmol) was added at 0 °C. Then the whole was stirred for 16 h at room temperature. After quenching with H2O (10 mL), the whole was extracted three times with CH2Cl2 (10 mL × 3), washed with brine, and dried over Na2SO4 and evaporated. The residue was column-chromatographed (hexane/ AcOEt = 4:1) to yield 18 (322.7 mg, 0.852 mmol, 90%, colorless oil). 1 H NMR (CDCl3): δ = 7.173−7.126 (2H, m), 6.871−6.815 (2H, m), 4.221 (2H, t, J = 6.10 Hz), 3.961 (2H, t, J = 6.48 Hz), 3.610 (2H, t, J = 6.02 Hz), 2.946 (2H, t, J = 7.68 Hz), 2.638 (2H, t, J = 7.66 Hz), 1.827 (2H, m), 1.470 (2H, m), 1.272 (14H, m), 0.884 (3H, t, J = 6.84 Hz) (no −OH peak). 13C NMR (CDCl3): δ = 174.86, 157.00, 129.95, 128.80, 127.57, 120.16, 111.06, 67.80, 61.14, 59.17, 34.19, 31.91, 31.78, 29.62, 29.37, 29.33, 26.32, 26.16, 22.67, 14.09. HRMS (ESI, [M + Na]+) calcd for C23H38NaO4+: 401.2662. Found: 401.2639. tert-Butyl O-(tert-Butoxy(3-((3-(2-(undecyloxy)phenyl)propanoyl)oxy)propoxy)phosphoryl)-N-(tert-butoxycarbonyl)L-serinate (19). In order to eliminate traces of water, phosphoamidite 17 (204.1 mg, 0.440 mmol) was dissolved in CH2Cl2 (5 mL) and toluene (0.5 mL), and the solvent was evaporated under vacuum. To this residue, alcohol 18 (201.5 mg, 0.532 mmol), CH2Cl2 (5 mL), and toluene (0.5 mL) were added, and the solvent was evaporated under vacuum. This residue was dissolved in CH2Cl2 (2 mL) under an argon atmosphere, and the solution of 1H-tetrazole (92.3 mg, 1.318 mmol) in THF (2 mL) was added at room temperature. In a few minutes, a white solid was precipitated. The reaction mixture was stirred for 11 h at room temperature, and a solution of TBHP in decane (5.0−6.0 M) (0.1761 mL, 0.881 mmol) was added at room temperature, and the whole was stirred for 5 h at room temperature. The solution was diluted with water (10 mL), and the whole was extracted three times with CH2Cl2 (10 mL × 3). The combined organic layer was washed with brine, dried over Na2SO4, and evaporated. The residue was column-chromatographed (hexane/AcOEt = 4:1) to yield 19 (307.0 mg, 0.405 mmol, 92%, colorless oil). 1H NMR (CDCl3): δ = 7.178− 7.112 (2H, m), 6.854−6.801 (2H, m), 5.486 (1H, m), 4.347 (1H, m), 4.212 (1H, m), 4.158 (2H, m), 4.019 (2H, m), 3.948 (2H, t, J = 6.54 Hz), 2.928 (2H, t, J = 7.88 Hz), 2.611 (2H, dt, J = 7.70, 1.04 Hz), 1.956 (2H, m), 1.788 (2H, m), 1.482−1.467 (18H, m), 1.436 (9H, s), 1.262 (14H, m), 0.874 (3H, t, J = 6.84 Hz). 13C NMR (CDCl3): δ = 173.20, 168.33, 156.93, 155.22, 129.89, 128.79, 127.50, 120.13, 110.99, 83.64, 83.57, 82.64, 82.61, 79.89, 67.74, 67.39, 64.06, 64.00, 60.36, 54.47, 54.39, 34.03, 31.87, 29.78, 29.76, 29.74, 29.72, 29.58, 29.56, 29.52, 29.33, 29.30, 28.28, 27.93, 26.13, 26.09, 22.64, 14.07. 31P NMR (CDCl3):δ = −5.547, −5.693. HRMS (ESI, [M + Na]+) calcd for C39H68NNaO11P+: 780.4422. Found: 780.4432. O-(Hydroxy(3-((3-(2-(undecyloxy)phenyl)propanoyl)oxy)propoxy)phosphoryl)-L-serine (9-o-C11). 19 (24.3 mg, 0.0274 mmol) was dissolved in TFA (1.0 mL) at 0 °C, and the mixture was stirred at room temperature for 1 h. The mixture was evaporated. The residue was column-chromatographed (CHCl3/MeOH/AcOH = 8:1:1) to yield 9-o-C11 as the AcOH-salt (14.4 mg, 0.0210 mmol,

77%, white powder). The AcOH salt was dissolved in TFA and evaporated to yield 9-o-C11 as the TFA salt (white powder). Mp: 151.5−153.0 °C. 1H NMR (CDCl3/TFA-d = 4:1): δ = 7.201 (1H, t, J = 7.54 Hz), 7.078 (1H, m), 6.887 (2H, m), 4.641 (2H, m), 4.533 (1H, m), 4.239 (2H, m), 4.061 (2H, m), 4.013 (2H, t, J = 6.68 Hz), 2.951 (2H, t, J = 6.96 Hz), 2.747 (2H, t, J = 7.00 Hz), 1.993 (2H, m), 1.798 (2H, m), 1.448 (2H, m), 1.274 (14H, m), 0.878 (3H, t, J = 6.74 Hz). 31 P NMR (CDCl3/TFA-d = 4:1): δ = −1.404. HRMS (ESI, [M − H]−) calcd for C26H43NO9P−: 544.2681. Found: 544.2700. Anal. Calcd For C26H44NO9P·0.7CF3COOH: C, 52.62; H, 7.20; N, 2.24. Found: C, 52.66; H, 7.43; N, 2.29. tert-Butyl (tert-Butoxycarbonyl)-L-allothreoninate (22). L-alloThreonine 20 (997.3 mg, 8.372 mmol) was dissolved in 1 N aqueous NaOH (10 mL), H2O (10 mL) and dioxane (20 mL) at 0 °C with stirring. After di-tert-butyl dicarbonate (2.763 g, 12.66 mmol) was added to the solution at 0 °C with stirring over 10 min, the reaction mixture was stirred at room temperature for 24 h. Then 5% aqueous KHSO4 was added to the reaction mixture to adjust to pH 3. The solution was extracted three times with AcOEt (40 mL × 3). The combined organic layer was washed with brine, dried with Na2SO4, and evaporated to yield the crude N-Boc-L-allo-threonine (21, 2.016 g, colorless oil). The crude N-Boc-L-allo-threonine was dissolved in anhydrous dichloromethane (150 mL), and O-tert-butyl-N,N′diisopropylisourea (5.872 g, 29.31 mmol) was added. The solution was stirred for 18 h at room temperature. Hexane was added in the mixture and stirred for 10 min. The mixture was filtered through Celite, and the filtrate was evaporated. The residue was columnchromatographed (hexane/AcOEt = 4:1) to yield 22 (1.581 g, 5.741 mmol, 69%, white solid). Mp: 62.1−63.0 °C. 1H NMR (CDCl3): δ = 5.226 (1H, m), 4.224 (1H, s), 4.123 (1H, m), 1.960 (1H, d, J = 6.00 Hz), 1.486 (9H, s), 1.455 (9H, s), 1.234 (3H, d, J = 6.40 Hz). 13C NMR (CDCl3): δ = 169.29, 156.49, 82.72, 80.28, 69.22, 59.45, 28.16, 27.88, 18.15. HRMS (ESI, [M + Na]+) calcd for C13H25NNaO5+: 298.1630. Found: 298.1609. Anal. Calcd for C13H25NO5: C, 56.66; H, 9.01; N, 5.11. Found: C, 56.76; H, 9.05; N, 5.39. tert-Butyl O-(tert-Butoxy(diisopropylamino)phosphanyl)-N(tert-butoxycarbonyl)-L-allothreoninate (23). Bis(diisopropylamino)-tert-butylphosphine (303.1 mg, 1.101 mmol) was dissolved in CH2Cl2 (5 mL) and toluene (0.5 mL). To this solution, N-Boc-Lallo-threonine tert-butyl ester 22 (508.2 mg, 1.669 mmol) was added. In order to eliminate traces of water, the resultant solution was evaporated under vacuum. The residue was dissolved in CH2Cl2 (5 mL) under an argon atmosphere, and a solution of 1H-tetrazole (105.9 mg, 1.512 mmol) in THF (5 mL) was added at room temperature. In a few minutes, a white solid was precipitated. The whole was stirred for 3 h at room temperature. The reaction was quenched with the addition of saturated aqueous NaHCO3 (10 mL), and the whole was extracted three times with CH2Cl2 (10 mL × 3). The combined organic layer was washed with brine and dried over Na2SO4. The solvent was evaporated, and the residue was chromatographed (hexane/AcOEt/ Et3N = 35:4:1) to yield 23 (445.0 mg, 0.930 mmol, 84%, yellow oil). In the purification with column chromatography, Et3N deactivated silica gel was used, that is, eluents containing 3% (v/v) Et3N. 1H NMR (CD2Cl2): δ = 5.825 (1/2H, m), 5.300 (1/2H, m), 4.085−3.959 (2H, m), 3.576 (2H, m), 1.436−1.431 (9H, m), 1.405−1.396 (9H, m), 1.360−1.308 (9H, m), 1.270 (3H, d, J = 6.56 Hz), 1.143 (12H, m). 13 C NMR (CD2Cl2): δ = 169.12, 169.06, 155.51, 155.26, 81.59, 81.22, 79.19, 79.06, 75.23, 75.13, 74.98, 74.87, 70.86, 70.70, 70.67, 70.53, 59.35, 59.31, 59.10, 43.32, 43.26, 43.20, 43.13, 30.83, 30.76,30.75, 30.68, 28.24, 28.18, 27.98, 27.95, 24.49, 24.46, 24.41, 24.37, 24.12, 24.03, 23.91, 23.85, 19.73, 19.71, 19.07. 31P NMR (CD2Cl2): δ = 138.879, 137.547. HRMS (ESI, [M + Na] + ) calcd for C23H47N2NaO6P+: 501.3064. Found: 501.3053. 3-(3-(Undecyloxy)phenyl)propanoic Acid (26). To a solution of phenol 24 (315.4 mg, 1.750 mmol), 11-bromoundecane (501.2 mg, 2.131 mmol), and TBAI (190.7 mg, 0.516 mmol) in DMF (5 mL) was added Cs2CO3 (695.6 mg, 2.135 mmol), and the whole was heated at 70 °C with stirring for 27 h. To this solution was added MeOH (2 mL), and the whole was heated at 70 °C with stirring for 15 h. The reaction mixture was diluted with H2O (10 mL) and AcOEt (20 mL), 4216

DOI: 10.1021/jm5020082 J. Med. Chem. 2015, 58, 4204−4219

Article

Journal of Medicinal Chemistry and the whole was extracted three times with AcOEt (20 mL × 3). The organic layer was washed with brine, dried over Na2SO4, and evaporated. The crude mixture was column-chromatographed (hexane/Et2O = 30:1) to yield methyl ester 25 (572.8 mg, 1.712 mmol, 98%, colorless oil). 1H NMR (CDCl3): δ = 7.183 (1H, m), 6.775−6.724 (2H, m), 3.932 (2H, t, J = 6.58 Hz), 3.676 (3H, s), 2.920 (2H, t, J = 7.78 Hz), 2.593 (2H, t, J = 7.88 Hz), 1.767 (2H, m), 1.447 (2H, m), 1.271 (14H, m), 0.884 (3H, t, J = 6.88 Hz). Methyl ester 25 (572.0 mg, 1.710 mmol) was dissolved in MeOH (1 mL) and THF (1 mL). To this solution was added aqueous 2 N NaOH (1 mL). The resultant solution was stirred for 2 h at room temperature. Aqueous 2 N HCl was added to the aqueous layer to adjust the pH to 2. The solution was extracted three times with AcOEt (20 mL × 3). The combined organic layer was washed with brine, dried over Na2SO4, and evaporated. The residue was columnchromatographed (hexane/AcOEt = 1:1) to yield acid 26 (531.9 mg, 1.660 mmol, 97%, white solid). Mp: 67.0−68.0 °C. 1H NMR (CDCl3): δ =7.195 (1H, m), 6.791−6.737 (3H, m), 3.938 (2H, t, J = 6.58 Hz), 2.933 (2H, t, J = 7.82 Hz), 2.682 (1H, t, J = 7.80 Hz), 1.773 (2H, m), 1.452 (2H, m), 1.273 (14H, m), 0.886 (3H, t, J = 6.88 Hz). 13 C NMR (CDCl3): δ = 178.29, 159.35, 141.68, 129.50, 120.36, 114.68, 112.28, 67.93, 35.37, 31.90, 30.64, 29.61, 29.60, 29.58, 29.40, 29.32, 26.06, 22.67, 14.09. HRMS (ESI, [M − H]−) calcd for C20H31O3−: 319.2279. Found: 319.2296. Anal. Calcd for C20H32O3: C, 74.96; H, 10.06; N, 0.00. Found: C, 74.66; H, 9.95; N, 0.00. (R)-N-(2,3-Dihydroxypropyl)-3-(3-(undecyloxy)phenyl)propanamide (27). To a solution of acid 26 (387.7 mg, 1.210 mmol), EDCI (256.0 mg, 1.335 mmol), and HOBt·H2O (180.8 mg, 1.338 mmol) in CH2Cl2 (2 mL) was added a solution of (R)-1-amino2,3-propanediol (101.7 mg, 1.116 mol) in DMF (2 mL), and the mixture was stirred for 12 h at room temperature. To this mixture was added DIPEA (171.6 mg, 1.328 mmol), and stirring was continued for additional 8 h. The reaction mixture was diluted with H2O (10 mL), and the whole was extracted three times with CH2Cl2 (10 mL × 3). The combined organic layer was washed with brine and dried over Na2SO4 and evaporated. The residue was column-chromatographed (hexane/ethyl acetate = 1:1 to CHCl3/MeOH = 9:1) to yield 27 (335.7 mg, 0.853 mmol, 76%, white solid). Mp: 96.5−97.5 °C. 1H NMR (CDCl3): δ = 7.184 (1H, dd, J = 8.92, 7.48 Hz), 6.769−6.732 (3H, m), 6.071 (1H, m), 3.923 (2H, t, J = 6.58 Hz), 3.685 (1H, m), 3.454 (2H, m), 3.346 (2H, m), 3.083 (2H, brs), 2.928 (2H, t, J = 7.52 Hz), 2.525 (2H, t, J = 7.52 Hz), 1.759 (2H, m), 1.439 (2H, m), 1.267 (14H, m), 0.881 (3H, t, J = 6.86 Hz). 13C NMR (CDCl3): δ = 174.16, 159.37, 141.96, 129.55, 120.47, 114.79, 112.25, 70.96, 67.79, 63.50, 42.23, 38.14, 31.89, 31.70, 29.60, 29.57, 29.41, 29.32, 29.31, 26.05, 22.67, 14.09. HRMS (ESI, [M + Na]+) calcd for C23H39NNaO4+: 416.2771. Found: 416.2772. (R)-N-(2-((tert-Butyldimethylsilyl)oxy)-3-hydroxypropyl)-3(3-(undecyloxy)phenyl)propanamide (28). 27 (250.1 mg, 0.635 mmol) and imidazole (134.0 mg, 1.968 mmol) were dissolved in anhydrous DMF (4 mL), and a solution of TBSCl (247.5 mg, 1.642 mmol) in DMF (2 mL) was added at room temperature. The mixture was stirred for 18 h, then diluted with water (10 mL) and AcOEt (20 mL). The aqueous layer was separated, and the whole was extracted three times with AcOEt (10 mL × 3). The combined organic layer was dried over Na2SO4 and evaporated. The residue was columnchromatographed (hexane/AcOEt = 8:1) to yield the diprotected product (376.2 mg, 0.605 mmol, 95%, colorless oil). 1H NMR (CDCl3): δ = 7.184 (1H, dt, J = 7.48, 1.04 Hz), 6.771−6.719 (3H, m), 5.799 (1H, t, J = 5.12 Hz), 3.921 (2H, t, J = 6.56 Hz), 3.749 (1H, m), 3.533 (1H, dd, J = 10.24, 4.84 Hz), 3.533 (1H, dd, J = 10.24, 6.48 Hz), 3.363 (2H, m), 2.926 (2H, t, J = 7.90 Hz), 2.446 (2H, t, J = 7.92 Hz), 1.764 (2H, m), 1.446 (2H, m), 1.269 (14H, m), 0.885 (21H, m), 0.068 (3/2H, s), 0.058 (3/2H, s), 0.048 (3/2H, s), 0.043 (3/2H, s). HRMS (ESI, [M + Na]+) calcd for C35H67NNaO4Si2+: 644.4501. Found: 644.4515. To a solution of the product (211.8 mg, 0.340 mmol) in MeOH (5 mL) was added CSA (3.6 mg, 0.0155 mmol) at 0 °C. The solution was stirred for 1 h at 0 °C. Then the reaction was quenched with Et3N (1 mL), and the mixture was evaporated. The residue was column-

chromatographed (hexane/AcOEt = 2:1) to yield 28 (122.1 mg, 0.240 mmol, 71%, colorless oil). (Recovery of starting material was 32.4 mg, 0.0521 mmol, 15%.) 1H NMR (CDCl3): δ = 7.182 (1H, dd, J = 8.84, 7.48 Hz), 6.776−6.723 (3H, m), 5.707 (1H, t, J = 5.50 Hz), 3.924 (2H, t, J = 6.58 Hz), 3.783 (1H, m), 3.693 (1H, m), 3.572 (1H, dd, J = 11.68, 4.72 Hz), 3.231 (1H, dd, J = 11.68, 7.20 Hz), 3.065 (1H, m), 2.936 (3H, m), 2.505 (2H, m), 1.764 (2H, m), 1.444 (2H, m), 1.268 (14H, m), 0.878 (12H, m), 0.060 (3H, s), 0.058 (3H, s). 13C NMR (CDCl3): δ = 173.51, 159.37, 142.05, 129.51, 120.39, 114.66, 112.24, 70.96, 67.91, 62.85, 41.99, 38.21, 31.89, 31.66, 29.60, 29.57, 29.40, 29.32, 26.06, 25.77, 18.02, 14.09, −4.74, −4.83. HRMS (ESI, [M + Na]+) calcd for C29H53NNaO4Si+: 530.3636. Found: 530.3626. tert-Butyl O-(tert-Butoxy((R)-2-((tert-butyldimethylsilyl)oxy)3-(3-(3-(undecyloxy)phenyl)propanamido)propoxy)phosphoryl)-N-(tert-butoxycarbonyl)-L-allothreoninate (29). In order to eliminate traces of water, phosphoamidite 23 (80.1 mg, 0.167 mmol) was dissolved in CH2Cl2 (5 mL) and toluene (0.5 mL), and the solvent was evaporated under vacuum. To this residue, alcohol 28 (93.0 mg, 0.183 mmol), CH2Cl2 (5 mL), and toluene (0.5 mL) were added, and the solvent was evaporated under vacuum. This residue was dissolved in CH2Cl2 (2 mL) under an argon atmosphere, and a solution of 1H-tetrazole (39.4 mg, 0.562 mmol) in THF (2 mL) was added at room temperature. In a few minutes, a white solid was precipitated. The reaction mixture was stirred for 12 h at room temperature, and a solution of tert-butyl hydrogen peroxide (TBHP) in decane (5.0−6.0 M) (0.0675 mL, 0.338 mmol) was added at room temperature. The whole was stirred for 2 h at room temperature, then diluted with water (10 mL) and extracted three times with CH2Cl2 (10 mL × 3). The combined organic layer was washed with brine, dried over Na2SO4, and evaporated. The residue was column-chromatographed (hexane/ethyl acetate = 4:1) to yield 29 (96.9 mg, 0.108 mmol, 64%, colorless oil). 1H NMR (CDCl3): δ = 7.168 (2H, t, J = 7.72 Hz), 6.774−6.708 (3H, m), 6.528 (2/3H, m), 6.191 (1/3H), 5.591 (1H, m), 4.666 (1H, m), 4.308 (1H, m), 3.914 (5H, m), 3.498− 3.395 (1H, m), 3.294−3.163 (1H, m), 2.928 (2H, m), 2.493 (2H, m), 1.758 (2H, m), 1.512−1.420 (32H, m), 1.268 (14H, m), 0.884 (12H, m), 0.101 (6H, m). 31P NMR (CDCl3): δ = −5.111, −5.412. 13C NMR (CDCl3): δ = 172.21, 168.22, 167.99, 159.31, 159.28, 155.38, 142.55, 142.46, 129.40, 129.35, 120.44, 114.71, 114.66, 112.08, 82.81, 82.62, 79.95, 75.45, 69.26, 69.03, 68.33, 67.87, 58.50, 41.71, 41.48, 38.32, 38.13, 31.90, 31.76, 31.71, 29.83, 29.79, 29.61, 29.58, 29.43, 29.33, 28.31, 28.04, 26.07, 25.74, 25.73, 22.67, 18.01, 14.10. HRMS (ESI, [M + Na]+) calcd for C46H85N2NaO11PSi+: 923.5552. Found: 923.5538. O-(Hydroxy((R)-2-hydroxy-3-(3-(3-(undecyloxy)phenyl)propanamido)propoxy)phosphoryl)-L-allothreonine (12). 29 (48.6 mg, 0.0539 mmol) was dissolved in TFA (1.0 mL) and the mixture was stirred at room temperature for 1 h and then evaporated. The residue was chromatographed (CHCl3/MeOH/AcOH = 8:1:1 to 7:1:2) to yield 12 as the AcOH salt (16.4 mg, 0.0285 mmol, 53%, white powder). The AcOH salt was dissolved in TFA and evaporated to yield 12 as the TFA salt (white powder). Mp: 160.5−161.5 °C. 1H NMR (CDCl3/TFA-d = 4:1): δ = 7.231 (1H, m), 6.853−6.786 (3H, m), 5.016 (1H, m), 4.057 (5H, m), 3.436 (2H, m), 2.926 (2H, m), 2.690 (2H, m), 1.761 (2H, m), 1.576 (2H, m), 1.418 (3H, m), 1.268 (14H, m), 0.875 (3H, t, J = 6.52 Hz). 31P NMR (CDCl3/TFA-d = 4:1): δ = −1.696. HRMS (ESI, [M − H]−) calcd for C27H46N2O9P−: 573.2946. Found: 573.2976. Anal. Calcd For C 27H47FNO9P· 0.5CF3COOH: C, 53.24; H, 7.58; N, 4.43. Found: C, 53.31; H, 7.64; N, 4.69. Compound Administration. We used 0.1% (w/v) BSA (bovine serum albumin; fatty acid-free grade)-containing PBS (phosphate buffered saline) to dissolve all the synthesized compounds. We prepared small aliquots of dissolved solutions to avoid freeze-and-thaw cycles and stocked them at −20 °C. We confirmed that at least 100 μM solution, all the compounds became soluble and the solutions were clear. We used a concentration of 4.6 μM as the highest one on cells, except for the positive compound 8-o-C15, which was used at a concentration of 46.4 μM on GPR34-expressing cells. To dilute stock 4217

DOI: 10.1021/jm5020082 J. Med. Chem. 2015, 58, 4204−4219

Article


Actin Stress Fiber Formation Assay. Actin stress fiber formation assay was performed as described previously.1 McA-RH7777 cells stably expressing P2Y10 and mock retrovirus-infected cells were seeded in a collagen type I coated 24-well plate at 2.5 × 104 cells per well and incubated for 24 h in culture medium (DMEM supplemented with 10% (v/v) FBS). Cells were washed with PBS and serum-starved for 1 h in serum-free DMEM. Cells were then stimulated with LysoPS (1) or its analogues at a concentration of 1 μM for 1 h in serum-free DMEM containing 0.01% (w/v) BSA (essentially fatty acid-free grade). Cells were fixed with 3.7% (w/v) formaldehyde-containing PBS. After brief washing with PBS, cells were permeabilized with 0.5% (v/v) Triton X-100-containing PBS and stained with Alexa Fluor 594conjugated phalloidin (for filamentous actin) at 1.5 μg/mL and 4′,6diamidino-2-phenylindole (DAPI; for nuclei) at 1 μg/mL in 3% (w/v) BSA-PBS. Fluorescence images were captured with a Zeiss LSM 7000 confocal microscope (Carl Zeiss).

solutions for cells, we used HBSS containing 5 mM HEPES (pH 7.4; HBSS assay solution) and 0.01% (w/v) BSA. TGFα Shedding Assay. TGFα shedding assay was performed as described previously.1,21 Briefly, agonistic activities of the LysoPS receptors for all synthesized compounds were measured as follows. HEK293A cells (GPR34/LPS1 and P2Y10/LPS2) or HEK293FT cells (GPR174/LPS3) were seeded in culture dishes (ϕ 100 mm) with a cell suspension volume of 10 mL at a density of 2 × 105 cells/mL in 10% (v/v) fetal bovine serum (FBS)-supplemented Dulbecco’s modified Eagle medium (DMEM) and cultured in a CO2 incubator. After 24 h, the cells were transfected with a mixture of plasmids encoding APTGFα (2.5 μg per dish) and an LysoPS receptor (2.5 μg per dish) using a using a Lipofectamine 2000 reagent (Life Technologies; 12.5 μL per dish). As a control (mock transfection; for measuring receptorindependent responses), cells were transfected with a mixture of plasmids encoding AP-TGFα (2.5 μg per dish) and an empty vector (2.5 μg per dish). For GPR34 and its control, a plasmid encoding a chimeric Gαq/i1 subunit (0.5 μg per dish) was additionally introduced. After 24 h, cells were harvested with a trypsin−EDTA solution, washed with PBS, and suspended in Hank’s balanced salt solution (HBSS; 35 mL per dish) containing 5 mM HEPES (pH 7.4). For GPR34-expressing and P2Y10-expressing cells and their corresponding control cells, cell suspension was seeded in a 96-well plate (80 μL per well) and mixed with an LPA receptor antagonist, Ki16425, at a final concentration of 10 μM (10 μL per well). For GPR174-expressing and its corresponding control cells, cell suspension was seeded in a 96-well plate (90 μL per well). After a 30 min incubation, cells were treated with compounds diluted in 0.01% (w/v) bovine serum albumin (BSA; essentially fatty acid-free grade; Sigma-Aldrich)-containing HBSS (10 μL per well; total volume of 100 μL per well) and incubated for 1 h at 37 °C. The cell plate was centrifuged for 2 min at 190g and conditioned medium (CM) was transferred into a blank 96-well plate (80 μL per well). AP solution (10 mM p-nitrophenylphosphate (pNPP), 40 mM Tris-HCl (pH 9.5), 40 mM NaCl, and 10 mM MgCl2) was added to both the CM plate and the cell plate (80 μL per well). Absorbance at 405 nm (OD405) of the two plates was measured before and after incubation for 1 h at 37 °C, using a microplate reader. APTGFα release was calculated by application of the following formula (also see Supporting Information Figure S12 for detailed calculation processes):

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ASSOCIATED CONTENT

S Supporting Information *

Details of synthesis of nonaromatic LysoPS analogues and that of aromatic LysoPS analogues; full biological data, Tables S1− S10 and Figures S1−S11; additional data of Tables 1 and 2; details of data processing of biological assay (Figure S12). The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jm5020082.

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AUTHOR INFORMATION

Corresponding Authors

*J.A. (for biological study): phone, +81-22-795-6860; fax, +8122-795-6859; e-mail, [email protected]. *T.O. (for chemical study): phone, +81-3-5841-4730; fax, +813-5841-4735; e-mail, [email protected]. Present Address ∞

S.J.: Research Foundation ITSUU Laboratory, 2-28-10 Tamagawa, Setagaya-ku, Tokyo 158-0094, Japan. Author Contributions #

M.I. and A.I. contributed equally to this work. M.I., S.N., S.J., M.S., Y.O., and T.O. (The University of Tokyo) performed chemical studies including design and synthesis of compounds. A.I., A.U., K.S., T.K., A.S., J.I., M.O., K.K., K.M., and J.A. (Tohoku University) performed biological studies including TGFα shedding assay and actin stress fiber formation assay.

OD405CM AP‐TGFαCM (%) = × 1.25 × 100 OD405CM + OD405Cell where 1.25 was used to normalize AP-TGFα in total CM (100 μL) from measured AP-TGFα in transferred CM (80 μL). AP‐TGFα release =

Notes

The authors declare no competing financial interest.

[AP‐TGFαCM under stimulated conditions (%)] −

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[AP‐TGFαCM under vehicle‐treated conditions (%)]

ACKNOWLEDGMENTS This research was supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan to J.A. and T.O. A.I. and K.M were funded by PRESTO (Precursory Research for Embryonic Science and Technology) from Japan Science and Technology Agency (JST), and J.A. was funded by CREST from JST.

Typically, AP-TGFαCM under a vehicle-treated condition ranged from 10% to 25% depending on transfected plasmids and assay conditions. Data are shown as receptor-specific responses as calculated below:

receptor‐specific AP‐TGFα release (%) = [AP‐TGFα release in receptor‐transfected cells (%)] −

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[AP‐TGFα release in mock‐transfected cells (%)]

ABBREVIATIONS USED LysoPS, lysophosphatidylserine; LPS, lysophosphatidylserine receptor; PS, phosphatidylserine; KO, knockout; LPA, lysophosphatidic acid; LPI, lysophosphatidylinositol; TGF, transforming growth factor α; AP-TGFα, alkaline phosphatase-tagged transforming growth factor α; CI, confidence interval; SEM, standard error of the mean; Mp, melting point; EDCI, N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride; HOBt·H2O, 1-hydroxybenzotriazole hydrate;

The log EC50 and Emax values were calculated for active compounds with plateau or semiplateau responses by fitting data to a fourparameter sigmoid curve using a GraphPad Prism 6 (GraphPad, USA) (Tables 1 and 2). EC50 values can be calculated from log EC50. All the biological data are compiled in Tables 1 and 2 and in Supporting Information (Figures S1−S10 and Tables S1−S10). Concentration−response curves of some of the potent/selective compounds in the TGFα shedding assay are shown in Figure 10, and the original concentration−response data are shown in Figure S11. 4218

DOI: 10.1021/jm5020082 J. Med. Chem. 2015, 58, 4204−4219

Article


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TBAI, tetrabutylammonium iodide; DIPEA, N,N-diisopropylethylamine; HBSS, Hanks’ balanced salt solution; HEPES, 4-(2hydroxyethyl)piperazine-1-ethanesulfonic acid; CM, conditioned media; DMEM, Dulbecco’s modified Eagle medium; DAPI, 4′,6-diamidino-2-phenylindole

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Structure–Activity Relationships of Lysophosphatidylserine Analogs as

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