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Development of a Highly Potent Analogue and a Long-Acting Analogue of Oxytocin for the Treatment of Social Impairment-Like Behaviors Wataru Ichinose,†,¶,∇ Stanislav M. Cherepanov,§,∇ Anna A. Shabalova,§ Shigeru Yokoyama,§

Teruko Yuhi,§ Hiroaki Yamaguchi,∥ Ayu Watanabe,∥ Yasuhiko Yamamoto,⊥ Hiroshi Okamoto,⊥

Shinichi Horike,# Junpei Terakawa,# Takiko Daikoku,# Mizuki Watanabe,† Nariyasu Mano,∥

Haruhiro Higashida,*,§ and Satoshi Shuto*,†,‡

†Faculty of Pharmaceutical Sciences and‡Center for Research and Education on Drug Discovery, Hokkaido University, Sapporo 060-0812, Japan §Department of Basic Research on Social Recognition, Research Center for Child Mental Development, and ⊥Department of Biochemistry and Molecular Vascular Biology, Graduate School of Medical Sciences, Kanazawa University, Kanazawa 920-8640, Japan ∥Faculty of Pharmaceutical Sciences, Tohoku University and Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai 980-8574, Japan #Kanazawa University Advanced Science Research Center, Kanazawa 920-8640, Japan

*S Supporting Information

ABSTRACT: The nonapeptide hormone oxytocin (OT) has pivotal brain roles in social recognition and interaction and is thus a promising therapeutic drug for social deficits. Because of its peptide structure, however, OT is rapidly eliminated from the bloodstream, which decreases its potential therapeutic effects in the brain. We found that newly synthesized OT analogues in which the Pro7 of OT was replaced with N-(p-fluorobenzyl)glycine (2) or N-(3- hydroxypropyl)glycine (5) exhibited highly potent binding affinities for OT receptors and Ca2+ mobilization effects by selectively activating OT receptors over vasopressin receptors in HEK cells, where 2 was identified as a superagonist (EMax = 131%) for OT receptors. Furthermore, the two OT analogues had a remarkably long-acting effect, up to 16−24 h, on recovery from impaired social behaviors in two strains of CD38 knockout mice that exhibit autism spectrum disorder-like social behavioral deficits, whereas the effect of OT itself rapidly diminished.

■ INTRODUCTION Oxytocin (OT) is a peptide hormone and neuromodulator that exists in peripheral organs and tissues such as the uterus and mammary glands as well as in the central nervous system, and is mainly synthesized in the neurons of the supraoptic and paraventricular nuclei of the hypothalamus.1 OT in peripheral organs induces uterine contractions and milk ejection during delivery and lactation in the female reproductive period.2 OT also acts as a key molecule for neurotropic functions in both sexes.3 The molecule enhances social communication, such as social behavior, recognition, and memory in many species of mammals, including humans.4

Autism spectrum disorder (ASD) is a congenital neuro- developmental disease characterized by impairments in social communication, social interactions, repetitive behaviors, and restricted interests.5 Several therapeutic strategies have been applied to the treatment of ASD to improve the social impairment,6,7 including some small-molecule drugs under clinical studies such as bumetanide,8 but there are still no

sufficiently effective medications in the market. Therefore, OT may be a valuable lead for the development of a new medicine that takes advantage of the biological basis of social behavior in healthy subjects and restores normal social behavior in patients with psychiatric diseases such as ASD, depression, and schizophrenia.9

As a central neuromodulator, defects in the OT system may be related to the abnormal social communication behavior of patients with ASD because the concentration of OT in the blood or salivary glands is low in ASD patients,10 and gene mutations of the OT receptor (OTR) coding and noncoding regions are detected at a higher rate in ASD patients.11,12 In addition, gene mutations of transmembrane proteins, such as CD38 and CD157, which are related to the cascade of events induced by OT secretion,13−16 lead to autism-like social memory defects and hyperactivity, and mice lacking these

Received: October 31, 2018 Published: March 21, 2019

Article

pubs.acs.org/jmcCite This: J. Med. Chem. 2019, 62, 3297−3310

© 2019 American Chemical Society 3297 DOI: 10.1021/acs.jmedchem.8b01691 J. Med. Chem. 2019, 62, 3297−3310

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genes provide a possible behavioral model of attention-deficit hyperactivity disorder.13 Single nucleotide polymorphisms of CD38 and CD157 genes are associated with ASD,17,18 which suggest the importance of OT signaling mediated by CD38 and CD157 in social behavior. According to these findings, we previously developed CD38 knockout (CD38KO) mice19 that exhibited social amnesia,20 which is a key deficiency in ASD patients. Other remarkable features of CD38KO mice are a deficit in paternal nurturing behavior21 and in reward activity in male mice in the sucrose-intake test.21 Male CD38KO mice exhibit a notable decrease in immobility time in the tail suspension test,22 which represents hypermobility as a type of neurodevelopmental disorder such as attention-deficit hyper- activity disorder-like behavior, rather than depression. Interestingly, administration of OT rescued these impairments in CD38KO mice.21,22 Because these behavioral modifications were an important point in studies on OT analogues as described below, we were interested in further validating the pharmacological effects of OT and its analogues on these phenotypes with CD38 null mutant mice newly created by using the CRISPR/CAS9 gene-editing method (CD38KOCC). Some recent clinical trials of single intranasal OT

administration in ASD patients restored emotional recognition and steady eye gaze.23,24 The optimal dosing regimen of repeated applications of OT in ASD patients has not yet been determined because repeated intranasal application of OT failed to produce positive effects on the primary endpoints in some clinical trials.25,26 Several reports have indicated that intranasal OT administration increases episodes of social interactions as scored by caregivers and family observations at home,27 which suggests that OT has therapeutic benefits for ASD patients.27−29

We previously reported three OT analogues, designated lipooxytocins (LOTs), in which a palmitoyl group was linked to the cysteine terminal amino group and/or the tyrosine aromatic hydroxyl group of OT.30,31 These OT analogues and OT displayed curative effects against social impairments in CD38 and CD157 knockout mice, which exhibit ASD-like social behavioral deficits,21 with a single i.p. injection at a dose of 1−3 ng/g of body weight .22,30,31 Unlike OT, the LOT analogues are not immediately effective for the treatment of ASD-related behavioral deficits in these knockout mice, consistent with the finding that LOT analogues fail to elicit cytosolic Ca2+ mobilization by activating OTRs immediately after application in OTR-expressing HEK 293 cells in the culture.31 In sharp contrast to OT, however, their efficiency lasted approximately 24 h longer than that of OT because OT, a natural peptide, is rapidly eliminated in the body. We previously reported that i.p. injections of a LOT analogue resulted in a long-lasting elevation of the OT concentration in the cerebrospinal fluid (CSF), and the concentration was higher than that produced by i.p. injections of OT,31 which suggested that the LOT analogue was easily transported into the CSF because of its lipophilic property. In the present paper, we describe highly potent OT

analogues 2 and 5; in particular, 2 seems to be a “superagonist” that expresses greater agonistic efficacy than the natural full agonist OT and the clinically applied 1-deamino-1-carba-2- tyrosine(O-methyl)-oxytocin (carbetocin).32 Analogues 2 and 5 ameliorated social behavioral deficits more effectively and for a remarkably longer time than OT, like LOTs, in spite of their lacking lipophilic chains unlike LOTs. We studied the effects of

these analogues on pup retrieval as a parental behavior, abnormal emotional deficits in the sucrose preference test, and hypermobility in tail suspension tests, with two types of CD38 knockout mouse models of a developmental disorder such as ASD, in which the behaviors are sensitive to OT.

■ RESULTS AND DISCUSSION Design of New OT Analogues. Many structure−activity

relationship (SAR) studies have focused on enhancing the binding potency of the ligand to OTRs, as well as on decreasing the binding affinity for vasopressin receptor (AVPR) subtypes (V1aR, V1bR, and V2R) to minimize nonspecific effects on peripheral organs.33−40 These SAR studies revealed that deamination at the terminal cysteine residue, Cys1, induces an increase in OTR selectivity over AVPR. The modification was adopted as the first option in recent SAR studies because deamination also improved the hydrophobicity and chemical stability of OT. The medicine used clinically to induce uterine contraction, carbetocin, is also a deamino-OT analogue.41,42

Recently, Wisńiewski and co-workers have reported an excellent study on OT analogues.43 They synthesized a series of deamino-OT derivatives in which Pro7 and the disulfide linkage (−S−S−) between Cys1 and Cys6 in OT were replaced with N-substituted glycine residues and a thioether linkage (−CH2−S−), respectively.

43 In their screening, some OT analogues, such as 1 and 4 (Table 1), exhibited high agonistic activity on hOTR (EC50: 0.025 nM for 1 and 0.27 nM for 4) and significantly lower activity on hAVPRs (EC50: >10 nM (hV1aR and hV1bR) for 1 and EC50: >10 nM (hV1aR), 7.7 nM (hV1aR) for 4). Analogue 1, named merotocin, is now in clinical trials for inducing milk ejection in premature labor.44

Table 1. Molecular Structures of OT and Its Analogues

aReported in ref 43.

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We were very interested in these results in relation to our previous studies on LOTs with long-acting anti-anxiety effects, as described above, in which we conjugated long acyl chains at the terminal amino group on Cys1 and the phenolic hydroxyl group on Tyr2 of OT.30,31 We postulated that, in LOTs, the hydrophobic acyl chains would enhance intracerebral transfer, which leads to slow release of OT as a prodrug. Therefore, we expected that, using peptides such as 1 and 4 instead of OT, development of efficient prodrugs without AVPR activity might be achieved by their lipidation. As OT-related compounds, merotocin and its derivatives

exhibit potent and selective OT agonistic activity. Thus, in the present study, we designed OT analogues 2, 3, 5, and 6 (Table 1) in which Pro7 was replaced with N-p-fluorobenzyl- or N-(3- hydroxypropyl)glycine, similar to Wisńiewski’s deamino-OT analogues 1 and 4, respectively,43 but the terminal amine at Cys1 of OT was preserved because of its potential exploitation for lipidation. Synthesis. OT analogues 2, 3, 5, and 6 were synthesized by

the Fmoc strategy45 with the Rink amide resin as summarized in Scheme 1. The standard amino acid coupling was conducted with a solution of Fmoc-Xaa-OH (3.0 equiv), diisopropylcar-

bodiimide (DIC) (3.0 equiv), and HOAt (3.0 equiv) in N- methyl-2-pyrrolidone (NMP) and the Fmoc group was removed with 20% piperidine in dimethylformamide (DMF). To introduce the N-(p-fluorobenzyl)- or N-(3-(tert-butoxy)- propyl) group at the glycine residue, the resin was successively treated with bromoacetic acid/DIC/HOAt in NMP and p- fluorobenzylamine or 3-(tert-butoxy)propylamine in DMF at room temperature. For the synthesis of 2 and 5, the terminal Fmoc group was

removed on the resin. After treating the resin with trifluoro- acetic acid (TFA)/triisopropylsilane (TIS)/H2O (96/2/2), the cleaved linear precursor peptide was subjected to a cyclization reaction under oxidative conditions with I2 in MeOH. The high-performance liquid chromatography (HPLC) purification gave the desired cyclic peptides 2 (total yield 38%) and 5 (total yield 26%). For the synthesis of 3 and 6, after completing the amino acid

coupling on the resin, the resulting resin was successively treated with Pd(PPh3)4 and piperidine to remove the allyl group and Fmoc group, respectively, which was then treated with PyBop, HOAt, and DIPEA to give the corresponding cyclized peptides. After the cleavage of the peptides from the

Scheme 1. Synthesis of OT Analogues 2, 3, 5, and 6

Table 2. Binding Affinity and Biological Activities of OT, AVP, and Analogsa,b,c

binding affinity Ki nM(95% CI) agonist potency EC50, nM(95% confidence interval) selectivity

agonist efficacy Emax %

compound hOTR hOTR hV1aR hV1bR hV1aR/hOTR hV1bR/hOTR hOTR

OT 0.58 (0.42−0.80) 0.01 (0.004−0.02)

0.19 (0.1−0.32) 0.24 (0.19−0.3) 18.8 24.2 100

AVP 2.0 (1.42−2.86) 0.1 (0.053−0.21) 0.004 (0.001−0.011)

0.0035 (0.0007−0.016)

0.03 0.03 39

carbetocin 1.8 (1.17−2.80) 0.01 (0.001−0.09)

2.3 (0.2−25) 4.8 (1.28−17.8) 226 479 57

1 1.1 (0.85−1.43) 0.025 (0.01−0.062)

>10 >10 >400 >400 61

2 0.51 (0.44−0.72) 0.028 (0.01−0.062)

2.2 (0.55−8.47) 0.50 (0.15−1.6) 75.3 17.5 131

3 0.90 (0.63−1.30) 0.089 (0.02−0.36)

4.0 (0.79−20.6) 0.80 (0.35−1.85) 45.0 9.0 82

4 1.4 (1.02−1.9) 0.27 (0.061−1.19)

>10 7.7 (3.58−16.49) >37 28.4 57

5 1.4 (0.92−2.10) 0.031 (0.004−0.26)

>10 >10 >323 >322 88

6 3.0 (2.10−4.23) 0.15 (0.021−1) >10 >10 >66.6 >66.6 50 aKi was determined by the radioactive binding displacement assay and experiments were performed twice in duplicate.

bEC50 values of agonist activities were determined at hOTR, hV1aR, and hV1bR using the [Ca

2+]i measurement with a microspectrofluorometric system, number of independent experiments: n = 3. Each experiment was performed in duplicate, number of measured cells in each cell dish: 20. cEmax values of agonist potencies were calculated from EC50 curves for hOTR, hV1aR, and hV1bR.

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resin with TFA/TIS/H2O (96/2/2), hydrogenation of the cleaved cyclic peptides and HPLC purification provided the cyclic peptides 3 (total yield 23%) and 6 (total yield 11%). Known 1 and 4 were also prepared as reference ligands for

biological evaluation by the previously reported method.43

Receptor Binding Assay. We first examined the [3H]OT binding of compounds to the human OTRs (hOTRs) in crude membrane fractions of HEK cells expressing hOTRs, and the data are summarized in Table 2. [3H]OT bounds specifically and reversibly to OTRs with a Kd of 0.3 ± 0.06 nM (data obtained from two experiments, each performed in duplicate; Figure S1), whereas nonspecific binding was defined in the presence of 1 μM unlabeled OT. The binding affinities of OT, AVP, carbetocin, and compounds 1−6 were determined by using a radioactive competitive assay. Displacement of bound [3H]OT was measured over a concentration range of 1 pM to 1 μM (Figure 1).

AVP and all-screened analogues showed strong binding affinity for hOTR (Ki ≤ 3.0 nM). Particularly, compound 2 exhibited high binding affinity for hOTR comparable to that of native OT (Ki = 0.58 nM vs 0.51 nM), whereas the affinities of other examined compounds slightly decreased relative to that of OT. Compounds 2 (Ki = 0.51 nM) and 3 (Ki = 0.90 nM) derived from analogue 1 with N-(p-fluorobenzyl)glycine had higher affinity than those of compounds 5 (Ki = 1.4 nM) and 6 (Ki = 3.0 nM) derived from analogue 4 with N-(3- hydroxypropyl)glycine. The affinity of carbetocin (Ki = 1.81 nM) was in accordance with the reported value.41

Agonist-Induced Increase in Intracellular Ca2+ Con- centrations. We examined receptor activation followed by Ca2+ mobilization from intracellular inositol-1,4,5-trisphos- phate-sensitive Ca2+ stores by measuring cytosolic-free Ca2+

concentrations ([Ca2+]i) in HEK-293 cells expressing hOTRs, human vasopressin 1a receptors (hV1aRs), and human vasopressin 1b receptors (hV1bRs). Overall data, including the half-maximal effective concentration (EC50), Emax, and thus selectivity, for OT, AVP, carbetocin, and OT analogues 1−6 are presented in Table 2. Their potency for hOTR is ranked with EC50 values as OT

(0.010 nM), carbetocin (0.010 nM) > 1 (0.025 nM) ≈ 2 (0.028 nM) ≈ 5 (0.031 nM) > 3 (0.089 nM) ≈ AVP (0.10 nM) ≈ 6 (0.15 nM) > 4 (0.27 nM). Compounds 1, 3, 4, and 6 maintained partial agonist properties for hOTR (Emax = 61% for 1, 82% for 3, 57% for 4, and 50% for 6), while 5 showed almost full agonistic potency (Emax = 88%), as summarized in Table 2. Surprisingly, 2 demonstrated “superagonistic” potency compared with OT (Emax = 131% vs 100%; Table 2, Figure 2A,C). Partial agonistic effects on both hV1aR and hV1bR were induced by 2 (Figure 2A), while almost no agonistic effect on either receptor was induced by 5 (Figure 2B). The hOTR selectivity versus hV1aR and hV1bR of

compounds was also summarized in Table 2. The hOTR selectivity versus hV1aR of peptides 1−6 was higher than that

Figure 1. Representative radioligand concentration displacement curves for OT, AVP, OT, and compounds 1−6. All curves were normalized to the percentage of displacement of [3H]OT. Data are means ± standard error of the mean from results obtained from two experiments, each performed in duplicate.

Figure 2. Agonistic activities of OT analogues: Agonistic activities of 2 (A) and 5 (B) for hOTR, hV1aR, and hV1bR. Activities of OT, AVP, 2, and 5 for hOTR (C) and hV1aR (D), determined by [Ca

2+]i microspectrofluorometric measurement. A value of 100% indicated the maximum Ca 2+-

release after OT application. Number of independent experiments: n = 3. Each experiment was performed in duplicate and number of measured cells in each cell dish: 20.

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of OT; particularly, 2 and 5 showed high selectivity of >400- fold and >323-fold, respectively. Also, 2 and 5 showed high hOTR selectivity versus hV1bR >400-fold and >323-fold, respectively. On the basis of these in vitro experimental results,

particularly with regard to their hOTR selectivity and agonistic potency, we focused on OT analogues 2 and 5, in which the Pro7 of OT was replaced with N-(p-fluorobenzyl)glycine or N- (3-hydroxypropyl)glycine, respectively, and the terminal amino group and S−S linkage of OT were preserved, different from carbetocin and its deamino-OT derivatives 1 and 4. Thereafter, we tested and compared the in vivo effects of compounds 2 and 5 with that of OT. Parental Retrieval Test. We examined the effects of OT,

2, and 5 on parental behavior by using the latency to retrieve pups by WT and CD38KO sires, a social behavior test that we previously developed.20−22 Thirty minutes after a single intraperitoneal injection of phosphate-buffered saline (PBS), WT sires showed retrieval behavior with a mean latency of 233 ± 68 s (n = 10), whereas CD38KO sires had a mean latency of 600 ± 0 (n = 13, Figure 3A), which replicated previous

results.22 The latencies to retrieve pups by CD38KO sires 30 min after a single intraperitoneal injection of 100 ng/100 g body weight of OT, 2, or 5 were 240 ± 69 s (n = 10), 297 ± 87 s (n = 10), and 346 ± 82 s (n = 10), respectively. One-way ANOVA revealed significant differences between groups (F4,45 = 8.45, p = 0.0001). Bonferroni’s post hoc tests showed significant differences between CD38KO sires treated with OT (p = 0.006), 2 (p = 0.032), and 5 (P = 0.048).

Next, we examined how long the effects of OT, 2, and 5 lasted. As shown in Figure 3B, the activity of OT was completely lost at 6 h. Treatment with both 2 and 5 significantly affected the latency to retrieve pups to the nest at 6 and 12 h; at 24 h, however, only 5 was still active. Compared with the PBS controls, the latencies to retrieve pups at 5, 12, and 24 h after injection of 2 (10 mice/group) were 381 ± 65 s (p = 0.045), 155 ± 65 s (p = 0.0001), and 413 ± 79 s (p = 0.16); and after injection of 5 were 293 ± 86 s (p = 0.0013), 386 ± 75 s (p = 0.0001), and 100 ± 94 s (p = 0.00013), respectively. Two-way ANOVA indicated significant effects of interaction (F12,180 = 4.31 and p = 0.0001), time (F4,180 = 3.74 and p = 0.006), and treatment conditions (F3,180 = 28.943 and p = 0.0001). To clarify the long-acting effects of 2 and 5 in comparison

with OT, we evaluated them with two additional mouse models described below.

Tail Suspension Test. The long-acting effects of 2 and 5 were next examined in a tail suspension test, which evaluates depression-like emotional behavior. Our previous findings22

revealed a shorter duration of immobility in male CD38KO mice than in WT mice. CD38KO mice exhibit hyperactivity rather than the decreased activity that is typical in this task.13,22

This was confirmed by newly created Cd38 gene-knockout mice using the CRISP-CAS9 gene-editing method,46 des- ignated as CD38KOCC mice. CD38KOCC mice exhibited a shorter duration of immobility than WT ICR mice and similar hyperactivity as CD38KO (Figure S2) mice. Therefore, we used this new mouse line as a mouse model of hyperactivity. The effects of OT, 2, and 5 were examined at 30 min and 24

h after injection (Figure 4). Immobility was enhanced in

CD38KOCC male mice at 30 min (74.2 ± 2.9 s and n = 5) and treatment with OT, 2, or 5 significantly increased the immobility to 171 ± 13.46 s (n = 5, p = 0.012), 149.5 ± 13.8 s (n = 8, p = 0.029), and 151.83 ± 21.3 s (n = 6), respectively. After 24 h, only compound 5 significantly increased immobility (157.3 ± 21.4 s, n = 5, p = 0.039) compared with that after PBS treatment (56.5 ± 18.8 s, n = 5; p = 0.0135). A two-way ANOVA revealed significance of time (F1,31 = 7.103, p = 0.0121) and treatment (F3,31 = 10.55, p = 0.0001). No statistically significant difference in effects was observed between CD38KOCC mice treated with OT or 2 and those treated with PBS at 24 h after the injection.

Figure 3. Pup retrieval behavior by sires: The latencies for pup retrieval by WT or CD38KO sires at 30 min (A) and the time-course (B) after a single intraperitoneal injection of PBS, OT, 2, or 5 (100 ng/100 g body weight); n = 10/group. One-way ANOVA followed by Bonferroni’s post hoc test was performed for 30 min (F4,45 = 8.45 and p = 0.0001), and two-way ANOVA followed by Bonferroni’s post hoc test was performed for the time course (time F4,180 = 3.74, p = 0.006; treatment F3,180 = 28.94, p = 0.0001; interaction F12,180 = 4.31, p = 0.0001). The significance levels of the Bonferroni post hoc test performed to compare the test treatments with the CD38KO treated by PBS were *p < 0.05 and **p < 0.01.

Figure 4. Tail suspension test: Immobility time of male CD38KOCC mice at 30 min and 24 h after a single intraperitoneal injection of PBS, OT, 2, and 5. n = 5−8 per group. Two-way ANOVA followed by Bonferroni’s post hoc test showed a significant effect of treatment (F3,31 = 10.55, p = 0.0001) and time (F1,31 = 7.103, p = 0.0121). *p < 0.05.

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Sucrose Preference. We previously reported that OT was associated with paternal behavior in the mouse.21,22 Therefore, to confirm the long-lasting effects of 2 and 5 compared with that of OT, we performed a sucrose preference test that allowed us to quantify the sensory system and anhedonia, which is an indicator of nucleus accumbens dysfunction.21 We used a 1% sucrose solution in this task in which mice were allowed to freely choose between water and sucrose solutions.21,22 Sucrose consumption was significantly lower in CD38KOCC male mice than in WT mice, similar to CD38KO mice (Figure S3). CD38KOCC male mice treated with PBS showed no sucrose preference (0.46 ± 0.04, n = 8), whereas CD38KOCC mice showed significant recovery of sucrose preference following administration of OT (0.80 ± 0.02, p = 0.0001, n = 5), 2 (0.79 ± 0.02, p = 0.0001, n = 5), and 5 (0.72 ± 0.05, p = 0.001, n = 5) at 60 min (Figure 5A). One-way ANOVA revealed significant differences between groups (F3,19 = 22.13, p = 0.0001). At 24 h after the injection, only 5 demonstrated a significant

elevation in sucrose consumption (0.67 ± 0.02, n = 5, p = 0.046), with a tendency toward a long-lasting effect (Figure 5B), whereas injection of OT or 2 had no effect. One-way ANOVA revealed significant differences between groups (F3,19 = 3.26, p = 0.044). Pharmacokinetics. To understand the pharmacological

potency and duration of action, we tested the pharmacoki- netics of OT, 2, and 5. Figure 6 shows the plasma concentration of OT, 2, and 5 after intravenous administration, measured by the LC−mass spectrometry (MS)/MS method. The plasma concentrations immediately after administration of OT, 2, and 5 were 334 ± 155 ng/mL (n = 5), 118 ± 60 ng/mL (n = 3), and 108 ± 35 ng/mL (n = 3), respectively. These three compounds rapidly disappeared from the plasma with a half-life (t1/2) of almost 10 min (Figure 6B,C; the t1/2 values in the fast phase for OT, 2, and 5 were 2.91, 2.84, and 3.1 min, respectively; the t1/2 values in the slow phase were 8.84, 2.86, and 4.23 min, respectively; Table 3), which suggested that 2 cleared from the blood more rapidly than OT and 5 did. Within 60 min after i.v. administration of compounds 2 and 5, OT levels were significantly elevated to 1.21 ± 0.86 and 0.6 ± 0.11 ng/mL, respectively. Table 3 shows the CSF concen- trations of 2 and 5 after intravenous administration. The CSF concentration of 2 at 30 min after the injection was 100 pg/mL (93 pM). Surprisingly, 12 h after injection, the concentration of compound 2 in the CSF increased to 380 pg/mL (350 pM). The presence of 5 was determined at both 30 min and 12 h,

but its exact concentration was not determined because the obtained values were below the detection limit.

Figure 5. Sucrose preference test: Preference of CD38KOCC males for 1% sucrose solution consumption at 1 (A) or 24 h (B) after treatment with PBS, OT, 2, or 5 (single intraperitoneal injection), n = 5−8. One-way ANOVA followed by Bonferroni’s post hoc test was performed for 60 min (F3,19 = 22.13 and p = 0.0001) and 24 h (time F3,19 = 3.26, p = 0.044) after and showed significant differences for *p < 0.05 and **p < 0.01.

Figure 6. Plasma concentrations of OT, and 2, or 5 after intravenous administration of OT (A), 2 (B), and 5 (C). ICR mice were administered with OT, 2, or 5 at a dose of 3 μg/mouse via the tail vein. Blood samples were collected at the indicated times after injection. Each point represents the mean ± standard error of three to five mice.

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The stability of the compounds in the serum was assessed by calculating the percent of the initial concentration in the fresh sample and was considered acceptable if it was 85−115% (Figure S4). All analyses were within the acceptable value and were stable in mouse plasma after 2 h at 37 °C. These data indicate that the disappearance of OT, 2, and 5 in the blood was not due to decomposition by enzymatic digestion but rather by clearance or adsorption to various tissues and organs. Kinetics of Resurrection. To calculate the kinetic

parameters (Kon, Koff≠, and t1/2) of the compounds, we used the equations of Motulsky and Mahan.47 Initially the kinetic parameters of [3H]OT were determined, which were obtained through construction of a family of association kinetic curves using a range of three different [3H]OT concentrations (Figure S5 and Table 4).

Figure 7 shows kinetic competition curves for OT (A), 2 (B), and 5 (C), which were assessed at three concentrations (near Ki, 5-fold Ki, or 10-fold Ki). The data indicated differences in both the Kon and Koff parameters for the compounds (Table 4). Compound 2 exhibited fast kinetics with a t1/2 that was approximately three times lower than that of [3H]OT and eight times lower than that of 5. Compound 5 demonstrated slower association and dissociation rates than [3H]OT. Discussion. The four newly synthesized OT analogues

showed their high affinity for hOTRs in the Ki range of 0.51− 3.0 nM, with the lowest values of 0.51 nM for analogue 2, whereas OT had a value of 0.58 nM, which suggested that 2 is equipotent to natural OT. Furthermore, the in vitro agonist potency of different concentrations of these compounds was in a range of 10−147 pM, as calculated based on the EC50 of [Ca2+]i changes in hOTR-expressing HEK cells, with the highest EC50 of 10 pM for OT and carbetocin and 28 pM for 2. These results indicated the very potent agonistic characteristics of these compounds. Interestingly, the dose−response curves revealed that 2 had 131% of the agonist potency of natural OT (100%).

Some reports have described “superagonists” as compounds that have efficacy in the assay greater than that of the natural full agonist for membrane receptors, such as nicotinic acetylcholine receptors or adrenergic β2-receptors.

48−53 There- fore, the above data showed that compound 2 possesses agonistic ability superior to natural agonist OT, which indicated that it is a superagonist. This is the first example of a superagonist for OTR. Historically, fluorination of ligands has improved their pharmacological and pharmacokinetic properties.54,55 Therefore, the unique transformation from the proline residue at position seven to an N-(p-fluorobenzyl)-

Table 3. Kinetic Parameters of OT, 2, and 5 after Intravenous Administrationa

t1/2 (min) CSF concentration (pg/mL)

compound plasma fast plasma slow 30 min 12 h

OT 2.91 (1.48−8.4) 8.84 (5.83−18.2) not determined not determined 2 2.84 (2.83−2.85) 2.86 (2.77−2.96) 100 380 5 3.1 (2.59−3.82) 4.23 (3.73−4.89) below lower limit of quantification below lower limit of quantification

aConcentrations were obtained by LC−MS/MS. Compounds (3 μg/mouse) were administered via the tail vein. Number of measurements in each time point n = 3−5. The t1/2 for the fast phase of decay calculated for 15 min and for the slow phase for 15−60 min.

Table 4. Comparison of the Receptor Kinetic Parameters of OT Agonistsa

compound Ki nM Kon

M−1 min−1 Koff min −1

t1/2 min

[3H]OT 0.3 (0.18−0.42) 1.737 × 108 0.05433 ± 0.007 11.85 OT 0.58 (0.42−0.8) 8.065 × 106 0.04222 ± 0.009 15.23 2 0.51

(0.44−0.72) 1.05 × 108 0.1003 ± 0.048 6.38

5 1.4 (0.92−2.1) 7.473 × 106 0.01746 ± 0.007 36.65 aKinetic parameters were determined by the radioactive binding competitive kinetics assay. Number of independent experiments, n = 2. Each experiment was performed in duplicate.

Figure 7. Competition kinetics curves for OT (A), 2 (B), and 5 (C). HEK-293 hOTR membranes were incubated with 500 pM [3H]OT and varying concentrations of the competitors. Plates were incubated for the indicated times, and nonspecific binding levels were determined in the presence of cold OT (1 μM).

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glycine residue resulted in compound 2, which could be considered a superagonist. However, for a stricter conclusion about superior agonistic

activities of compound 2, we should determine pharmaco- logical parameters in calcium assays by using cell cultures expressing mouse OTRs for explanation of actions of OT analogues in mouse behavior. Alternatively, different functional assays, such as BRET assay or ex vivo assays, such as myometrial contraction in human or mouse uterus, can be used. Parental behavior was examined by evaluating the retrieval

of biological pups by sires, as we reported previously.21,22,56

This is a clear defective phenotype in CD38KO sires because wild-type ICR sires typically display nurturing behavior that is essentially similar to dam’s parental behavior by retrieving pups placed outside the nest. As expected from their immediate binding to OTRs, these analogues exhibited effects on retrieval immediately or 30 min or 1 h post injection. This immediate action (similar to OT) and long-lasting action (unlike OT) could be beneficial for the therapeutic use of these analogues. The CD38KO sires retrieved almost no pups, as evidenced by a mean latency of 600 s to start retrieval, which was the maximum (ceiling) level allowed in the trial.31 The latency decreased approximately to 200−300 s by the administration of 2 or 5, which indicated that CD38KO sires began to nurture, and similar levels were exhibited by WT sires and CD38KO sires treated with OT or its analogues. Restoration of social/parental behavior continued for at least

16 h after intraperitoneal administration of 2 and 5 in CD38KO sires, at which time the effect by OT completely disappeared. After 24 h, significant remission was observed only in sires treated with 5, which suggested long-lasting action by both OT analogues with slight difference in the duration of the sustained effects between 2 and 5. LOT1 having two palmitoyl chains has been shown to have no immediate effects at 30 min because of its prodrug property30,31 but had effects lasting 24 h, whereas 2 and 5 not only had both immediate and long-lasting effects. In this respect, 2 and 5 appear to be better OT analogues. We used two additional models namely tail suspension test

and sucrose preference models to investigate that whether peptides 2 and 5 have more efficient effects in vivo than OT as OTR ligands from pharmacological and pharmacokinetic viewpoints. Thus, we used a new mouse line with Cd38 deletion,19 in

which the Cd38 gene was knocked out by using the CRISP- CAS9 method46 in the ICR strain. A significant effect of 5, but not 2, was observed at 24 h in a sucrose preference test evaluating the memory of a sweet drink as well as anhedonia in CD38KOCC male mice. Restoration from hyperactive behavior at the control (WT) level by treatment with 5, but not 2, also persisted for 24 h in CD38KOCC male mice. These experiments using this knockout line (CD38KOCC) demon- strated that the long-lasting effect of 5 is not mouse strain (CD38KO)-specific. The concentration profiles of OT, 2, and 5 in the plasma

after intravenous administration revealed that the levels of all three compounds decreased rapidly, with approximately the same half-life (2−4 min) of the rapid phase and slightly different half-lives for the slower phase. This difference in the half-life decay in the blood may not be sufficient evidence by itself for the long-acting effect of compounds 2 and 5. Compound 2 was detected in the CSF, however, even at 12 h

after intravenous administration, which may relate to its long- acting potency, while we could not detect 5 in the CSF in the present experiments. The fast decay in the plasma and the low levels of

compounds in the CSF are informative about the possible involvement of other mechanisms of action of OT, 2, and 5: peripheral activity of OT on the sympathetic nervous system,57

OT-induced endogenous OT release,58 prosocial activity of OT metabolites,59 and OT activation of V1AR.

60 Because the in vivo behavioral effects of compound 5, which has almost no V1AR activity, are similar to those of OT, we consider that V1AR activation has only a small role in the current behavioral experiments. In any event, we cannot exclude V1AR antagonism and this question should be addressed in future research that includes detailed studies of blood−brain barrier permeability. The data presented in Figure 6 indicate stable elevations of

plasma OT concentrations for at least 1 h after i.v. administration of 2 and 5. As reported previously, we found OT-induced OT release in the hypothalamus mediated by ADPR,58 which resulted in increased OT secretion from the pituitary into the blood. Compounds 2 and 5 may activate similar mechanisms of OT autoregulation. Here, we first time used the competitive kinetics assay for

hOTRs, which was previously used for muscarinic receptors61

suggesting a relation between the long-lasting effects of the compound and a long residence time on the receptor as well as a small Koff.

61 In our experiment, the receptor binding kinetics of [3H]OT were measured in the presence of OT, 2, and 5, and the Koff of 5 was smaller than those of OT and 2. This low kinetic parameter value seems to indicate a longer receptor residence time. The longer receptor residence time might be related to the long-acting effects of compound 5. We cannot directly compare the residence time and long-lasting effects on behavior, however, because different parameters, such as the buffers used and physiologic conditions, may affect the receptor kinetics.62

■ CONCLUSIONS We developed OT analogues 2 and 5, which are very active OTR ligands both in vitro and in vivo evaluation systems. Notably, only a subtle modification of OT, that is, replacement of Pro7 with N-substituted glycine residues, significantly changed the pharmacological effects to provide a superagonist, 2, and a very long-acting agonist, 5. The remarkable improvement in the effective duration, which overcomes the critical disadvantages of short-acting OT, suggests that these analogues could be superior alternatives to OT for the treatment of several diseases in which social impairments are prominent. OT itself is currently being applied in clinical trials in ASD,27,28 schizophrenia,63 and depression.64 Thus, OT analogues 2 and 5 have potential therapeutic merit. In addition, we are interested in lipidation of 2 and 5 as described above, which is now under investigation.

■ EXPERIMENTAL SECTION General Methods of Chemistry. 1H NMR spectra were

recorded in CDCl3 at ambient temperature unless otherwise noted, at 400 or 500 MHz, with TMS as an internal standard. 13C NMR spectra were recorded in CDCl3 at ambient temperature. Silica gel column chromatography was performed with silica gel 60 N (spherical, neutral, 63−210 μm, Kanto Chemical Co., Inc.). Flash column chromatography was performed with silica gel 60 N (spherical, neutral, 40−50 μm, Kanto Chemical Co., Inc.). Purities

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of final compounds were determined by HPLC: column, Kanto C18 GP, 250 × 4.6 (mm); column temperature, 30 °C; flow rate, 1.0 mL/ min; solvent A, water containing 0.1% TFA; solvent B, acetonitrile containing 0.1% TFA; and detection, 240 nm. The purities of all final compounds exceeded 95%. General Procedure for Peptide Coupling on Resins. The

peptides were assembled on the Rink amide resin by the Fmoc strategy. The fully protected peptide resin was synthesized manually, starting from 150 mg (0.10 mmol) of Rink Amide resin Nova Gel (200−400 mesh, loading 0.67 mmol/g, Novabiochem). The deprotection of the Fmoc group was conducted by adding 20% piperidine solution in DMF (1 mL) for 10 min twice at room temperature, and then the resin was washed with DMF (5 × 1 mL). The standard amino acid coupling was conducted with a solution of Fmoc-Xaa-OH (3.0 equiv), DIC (3.0 equiv), and HOAt (3.0 equiv) in NMP (0.7 mL) at room temperature for 2 h, and then the resin was washed with DMF (5 × 1 mL). Each process was held in a shaker. To introduce the N-(p-fluorobenzyl)- or N-(3-(tert-butoxy)propyl)- glycine residue, the resin was treated with bromoacetic acid (4.0 equiv), DIC (4.0 equiv), and HOAt (4.0 equiv) in NMP (1 mL) at room temperature for 2 h, and then treated with p-fluorobenzylamine (10 equiv) or 3-(tert-butoxy)propylamine (10 equiv) in DMF (1.0 M, 1 mL) at room temperature for 2 h. In the coupling just after the acylation, 4 equiv of amino acid was used. Peptides 2 and 5. The following amino acid derivatives were

used for the coupling: Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Cys- (Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ile-OH, and Fmoc-Tyr(tBu)-OH. Upon completion of the solid-phase synthesis, the resin was treated with TFA/TIS/H2O 96/2/2 (v/v/ v) solution (10 mL) at room temperature for 1.5 h and filtered off. The filtrate was poured into diethyl ether (40 mL), and the resulting mixture was centrifuged to give the crude peptide as the precipitate. After being dried over in vacuo, the precipitate was dissolved in H2O/ acetonitrile 1/4 (v/v, ca. 1 mg/mL). To the solution, a MeOH solution of I2 (0.1 M) was added slowly until the solution showed yellow color. After stirring the resulting mixture at room temperature for 6 h, solid sodium ascorbate was added until yellow color disappeared. The mixture was concentrated in vacuo, and then loaded onto a HPLC column and purified using H2O/acetonitrile gradient containing trifluoroacetic acid (0.1% v/v). The fractions containing the desired peptide was pooled and lyophilized. The peptide was obtained as white powder (2: 45 mg, 0.038 mmol, 38%; 5: 30 mg, 0.026 mmol, 26%, as a trifluoroacetic acid salt). 2; HPLC: Rt = 11.1 min, solvent A/B = 75/25; purity: 97.8%; LRMS (ESI) m/z: 1075.45 [(M + H)+], 1097.43 [(M + Na)+]; HRMS (ESI): calcd for C47H67FN12O12S2Na, 1097.4319 [(M + Na)

+]; found: 1097.4321. 5; HPLC: Rt = 5.61 min, solvent A/B = 80/20; purity: 98.8%; LRMS (ESI) m/z: 1025.45 [(M + H)+], 1047.44 [(M + Na)+]; HRMS (ESI): calcd for C43H69N12O13S2Na, 1025.4543 [(M + Na)

+]; found: 1025.4540. Peptides 3 and 6. The following amino acid derivatives were

used for the coupling: Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Cys- ((CH2)2CH(NH-pNZ)CO2Allyl)-OH,

43 Fmoc-Asn(Trt)-OH, Fmoc- Gln(Trt)-OH, Fmoc-Ile-OH, and Fmoc-Tyr(tBu)-OH. Upon com- pletion of the solid-phase peptide coupling, the resin was treated with Pd(PPh3)4 (347 mg, 0.3 mmol) in chloroform/N-methyl morphor- ine/acetic acid 37/1/2 (v/v/v, 3 mL) solution at room temperature for 2.5 h, and then washed with DMF, 0.5% DIEA in DMF, 0.5% sodium diethyldithiocarbamate in DMF, and finally DMF. After treating with 20% piperidine in DMF, the resin was treated with PyBOP (260 mg, 0.5 mmol), HOAt (68 mg, 0.5 mmol), and DIPEA (0.17 mL, 1.0 mmol) in DMF (1 mL). The resin was treated with cleaving cocktail, and the crude peptide was precipitated and centrifuged in diethyl ether, by the procedure described above for the coupling of 2 and 5. After being dried over in vacuo, the crude peptide was treated with Pd−C (10%, 45 mg, 0.42 equiv) in EtOH/ MeOH (1/1, 10 mL) under the hydrogen atmosphere for 7 h. After removal of the Pd catalyst by filtration through Celite, the filtrate was concentrated in vacuo and loaded onto a HPLC column and purified using H2O/acetonitrile gradient containing trifluoroacetic acid (0.1%

v/v). The fractions containing the desired peptide were pooled and lyophilized. The peptide was obtained as white powder (3: 27 mg, 0.023 mmol, 23%; 6: 12 mg, 0.011 mmol, 11%, as a trifluoroacetic acid salt). 3; HPLC, Rt = 5.4 min, solvent A/B = 70/30; purity: 97.4%; LRMS (ESI) m/z: 1057.50 [(M + H)+], 1079.48 [(M + Na)+]; HRMS (ESI): calcd for C48H69FN12O12SNa, 1079.4755 [(M + Na)+]; found: 1079.4773. 6; HPLC, Rt = 4.9 min, solvent A/B = 80/ 20; purity: 96.1%; LRMS (ESI): m/z 1007.50 [(M + H)+], 1029.48 [(M + Na)+]; HRMS (ESI): calcd for C44H70N12O13SNa, 1029.4798 [(M + Na)+]; found: 1029.4812.

OTR Expression Plasmid Construction. The plasmid for hOTRs (pcDNAHOXTR) was constructed as reported previously.65

AVP Receptors Plasmid Construction. Human brain total RNA (1 μg; Clontech, Mountain View, CA, USA) was reverse-transcribed using a kit (Transcriptor First Strand cDNA Synthesis Kit; Roche Applied Science, Penzberg, Germany), according to the manufac- turer’s instruction. The resulting cDNA was subjected to the nested polymerase chain reaction (PCR) using KOD FX DNA polymerase (Toyobo, Osaka, Japan) essentially as described previously.65 The primers for the initial PCR were as follows: 5′-CCGAAGAGGGCC- GAGTAGGA-3′ (sense) and 5′-CTAGCTCAATTCAGCTAAT- GAGC-3′ (antisense) for HAVP1AR and 5′-TCCACTTGCATCCA- CACCCTC-3′ (sense) and 5′-GAGAACCTCCACTAGTCCTGG-3′ (antisense) for HAVP1BR. The aliquots (2 μL) were subjected to the secondary PCR as described above using another set of primers: 5′- CCGAAGAGGGCCGAGTAGGA-3′ (sense) and 5′-GCAAGGCT- CAAGTTGAAACAGGA-3′ (antisense) for HAVP1AR and 5′- CCTGCCACTCCATTTTATCCATC-3′ (sense) and 5′- CCTAAAAGATGATGGTCTCAGC-3′ (antisense) for HAVP1BR. The resulting products, carrying entire protein-coding sequences of human AVPR1AR and AVP1BR, were treated with Ex Taq (Takara, Otsu, Japan) for 10 min at 72 °C and cloned into a pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA, USA) to gain pCHV1AR and pCHV1BR, respectively. Then, the 1.4-kb EcoRV (vector)/SpeI (vector) fragment obtained from pCHV1AR and the 1.4-kb HindIII (vector)/NotI (vector) fragment obtained from pCHV1BR were ligated to the XbaI/EcoRV-cleaved pcDNA3 (+) (Invitrogen) and NotI/HindIII-cleaved pcDNA3 (+), respectively, to yield respective expression plasmids designated as pcDNAHV1AR and pcDNAHV1BR.

Cell Culture and Transfection. Human embryonic kidney HEK- 293 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere of 95% air and 5% CO2.

31 Cells were grown in culture dishes to 80−90% confluence and transfected with pcDNAHV1AR or pcDNAHV1BR using GeneJuice Transfection Reagent (EMD Millipore, Temecula, CA, USA), following the manufacturer’s instruction. Cells were selected in DMEM supple- mented with 5% FBS and 800 μg/mL G418 (geneticin; Sigma Chemical Co., St Louis, MO, USA). The resulting transformed cells, as well as HEK-293 cells stably expressing human OXTRs31 and those transfected with pcDNA3 (+) (mock-transfected cells31), were maintained in DMEM supplemented with 10% FBS and 100 μg/ mL G418.

Preparation of the Membrane Fraction. Frozen hOTR- expressed HEK cells were thawed and suspended in 20 mL of a cold lysis buffer containing 1 mM EDTA, 50 mM HEPES, 10 mM MgCl2, pH 7.4, with the addition of complete cocktail of protease inhibitor (Roche Diagnostics) as described previously.66 The cells allowed to swell for 20 min and were homogenized with a Teflon-glass homogenizer.67 Intact cells and nuclei pellets were removed by centrifugation at 500g for 10 min. at 4 °C. The supernatant was then centrifugated at 19 000 rpm for 60 min at 4 °C, and the pellet was resuspended in the cold lysis buffer and sucrose (10%). This procedure was repeated twice. The final pellet was kept frozen at −80 °C with no loss of binding.

Binding Affinity Measurement. Binding for hOTRs was measured by filtration binding using 500 pM [3H]-OT final concentration in binding buffer 50 mM Tris, 10 mM MgCl2, and 0.1% BSA (pH 7.4) containing membranes (5 μg per well). A

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multiscreen-FC glass filter containing 96-well plates (Millipore) was used. For total binding measurement, 25 μL of binding buffer was added to the respective wells, for nonspecific binding, 25 μL of 12 μM cold OT was added. For compound testing, 25 μL of a serial dilution of each compound in binding buffer was added. After compound addition and 1 h of incubation at room temperature, the binding was terminated by rapid filtration under vacuum through GF/C filters66

and washed five times with ice-cold binding buffer before counting. Nonspecific binding counts were subtracted from each well and data normalized to the maximum specific binding set at 100%.66 Ki values were calculated using the Cheng−Prusoff equation.68 Saturation binding experiments were performed in the presence of 3H-OT in the concentration range from 50 pM up to 4 nM. Nonlinear regression analysis by the GraphPad prism was performed to obtain the Kd value. Additionally, the Rosenthal−Scatchard plot was constructed and did not indicate positive or negative cooperativity. Saturation binding experiments indicated that a single homogenous population of binding sites was being labeled. In all experiments, total binding never exceeded 10% of that added, limiting complications associated with depletion of the free radioligand concentration. Saturation and competitive binding experiments, for each compound, were performed two times, each experiment in duplicate. OTR Kinetics. To determine the Kon, Koff and Kobs were calculated

at three different concentrations of 3H-OT (200 pM, 500 pM, 1 nM). The experiment was initiated by the addition of 3H-OT to membranes in binding buffer. Free 3H-OT was separated at multiple time points to construct association kinetic curves. After incubation, bound was separated from free by rapid filtration. Washing and counting were performed as described previously. The hill equation was used to determine Kon and Koff. The t1/2 was calculated by equation Koff = ln 2/t1/2. For evaluation of kinetic parameters of tested compounds, competitive kinetics experiments were obtained.62,68 Similar proce- dure as described above was performed with the addition of test compounds in three concentrations (app. Ki, 5Ki, and 10Ki). All experiments were performed two times, each in duplicate. [Ca2+]i Measurement. The method was previously described.

69

Briefly, transfected HEK-293 cells were loaded with fura-2/AM to a final concentration of 1 μM in complete medium and incubated at 37 °C. After 30 min loading, the cells were washed three times with HEPES-buffered saline (HBS) solution (145 mM NaCl, 5 mM KCl, 1 mM MgCl2, 20 mM HEPES−NaOH, 2 mM CaCl2, 20 mM glucose, and pH 7.4). The fluorescence of the cells loaded with fura-2/AM was then measured at 37 °C, at the determined sites, through a pinhole (10−20 μm in diameter). We used alternating excitation wavelengths of 340 and 380 nm in a Ca2+ microspectrofluorometric system (IX-73 Model; Olympus, Tokyo, Japan) and MetaFluor software (Molecular Devices, Sunnyvale, CA). The Ca2+ emission was detected every 3 s for 5 min after the application of PBS, OT, AVP, or analogues. The ratio of fluorescence at 340 and 380 nm (F340/F380) was used to determine [Ca2+]i. All data were normalized to the baseline fluorescence (F0) recorded 10 s before application of compounds and given as percentage from the maximum response obtained by OT for hOTRs and AVP for hV1ARs and hV1BRs. OT, AVP, and analogues for experiments were diluted in 50% ethanol to a concentration of 10−3 M and then diluted in distilled water to obtain the required concentrations. Each experiment was performed three times, each triplicated. Animals. Male and female Slc:ICR mice (Institute of Cancer

Research of the Charles River Laboratories, Inc., Wilmington, MA, USA) were obtained from Japan SLC, Inc. (Hamamatsu, Japan) through a local distributor (Sankyo Laboratory Service Corporation, Toyama, Japan). The procedure to produce the CD38 KO mice was described previously.20 CD38KOCC was produced as follows: the candidate sgRNA was designed to target exon 2 of the Cd38 locus by using the CRISPR Design. The sgRNA nucleotides were then inserted to pX330 (addgene), which is the human codon-optimized SpCas9 and chimeric gRNA expression plasmid. For RNA synthesis, the human codon-optimized SpCas946 and chimeric sgRNA were excised from pX330, and then placed downstream of the T7 promoter in the pCMV TNT vector (Promega). Then, the hCas9 mRNA and chimeric

sgRNA were synthesized using an in vitro RNA transcription kit (mMESSAGE mMACHINE T7 Transcription Kit), according to the manufacturer’s instructions. The synthesized mRNA and sgRNA were dissolved in Opti-MEM I at 1−2 μg/μL and introduced them into eggs using a super electroporator NEPA 21 (NEPA GENE). After DNA isolation from mouse tails in the F0 generation, we amplified the genomic regions spanning the sgRNA binding site using PCR. Then, we screened more than 30 pups by using the Guide-it Mutation Detection Kit (TAKARA). Finally, we identified a pup which possessed a mutation with an insertion of two base pairs. This insertion leads to premature stop codons within the open reading frame of the Cd38 gene. The ideal end result is a loss-of-function mutation within the Cd38 gene. The offspring of wild-type, CD38KO, and CD38KOCC mice were born in our laboratory colony. The pups were weaned at 21−28 days of age and housed in same-sex groups of five animals until pairing. A male and female of each genotype were paired and housed in a nursing cage in our laboratory under standard conditions (24 °C; 12 h light/dark cycle, lights on at 08:00) with food and water provided ad libitum. All animal experiments were performed in accordance with the Fundamental Guidelines for the Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science, and Technology of Japan and were approved by the Committee on Animal Experimentation of Kanazawa University (ethics approval code AP-173824).

Parental Retrieval Test. The design of the experiments for parental retrieval behavior was described previously.21,22 Virgin males and females of identical genotypes were paired at 56−64 days. A single male and a single female were continuously housed together in a standard mouse maternity cage from the mating period to the delivery of pups and then to postnatal days (3−5). All family units consisted of a new sire and dam, and their first litter of each genotype was used. All mice were experimentally naive. Thirty minutes before starting the experiment, the cages with the families were placed in the experimental room for habituation. The sire and dam were placed in a new clean cage with new woodchip bedding for 10 min, while the pups were left in the nest in the original cage. Five pups were randomly selected from the litter and placed individually at a site remote from the nest in the original cage. The sires were returned to the original home cage in the presence of their five biological pups to assess parental behavior. Parental retrieval behavior (latency to retrieve the first pup to the nest) was measured by observing the parent behavior for 10 min following the reunion. The sire received a single intraperitoneal injection of 0.3 mL of PBS or 0.3 mL of OT (1 mL per 100 g of body weight), compounds 2 or 5 at a concentration of 100 ng/mL dissolved in PBS. Thirty minutes, 6, 12, 24 or 48 h after the injection, paternal retrieval behavior was examined only once. The behavioral tests were carried out in a randomly mixed sequence of the experimental groups. For each time-point and each compound, 10 mice were used. The experiments were performed between 10:00 and 15:00 for each time-point. One-way ANOVA test was performed for comparison compound effects for 30 min. If the ANOVA test showed significance, Bonferroni’s post hoc test was performed. Two-way ANOVA followed by Bonferroni’s post hoc test was performed for time-course.

Tail Suspension. The tail suspension test method was previously described.22 After 30 min of habituation in the experimental room, the WT and CD38KOCC mice were injected i.p. with 0.3 mL of PBS or OT at a concentration of 100 ng/mL dissolved in PBS. Thirty minutes after the injection, the mice were hanged by fixing their tails by tape to the suspension bar in a plastic suspension box (55 cm height × 60 cm width × 11.5 cm depth). To prevent observing or interacting with each mouse, the mouse was separated with walls but was not able contact or touch to the walls. Behavior was recorded for 6 min on a video source and was analyzed using ANY-Maze behavioral tracking software (Stoelting Co, USA). Total immobility time during the last 4 of the 6 min was used. Number of animals for test: n = 8 for compound 2, 30 min; n = 6 for compound 5, 30 min; n = 5 for all other groups. Two-way ANOVA test was performed. If the

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ANOVA test showed significance, Bonferroni’s post hoc test was performed. Sucrose Preference. Experimentally naiv̈e, young male adult

mice (6−8 weeks old) of WT and CD38KOCC strains were given a two-bottle choice between distilled water and sucrose solution at a 1% concentration based on our previous results,21 which were both available ad libitum. The bottle positions remained constant. Fresh sucrose solution was prepared each day. Cumulative water and sucrose intakes during 24 h were calculated by weighing. Food was provided ad libitum, but food intake was not recorded in this experiment. Number of animals: n = 8 for PBS-treated group; n = 5 for all other groups. The one-way ANOVA test was performed for 60 min and 24 h comparison independently. If ANOVA tests showed significance, Bonferroni’s post hoc test was performed. Dosing and Sampling. Male ICR mice weighing 30 g were used.

OT, 2, or 5 was injected intravenously at a dose of 100 ng/g of body weight. Mice were sacrificed using i.p. injection of 10% somnopenthyl (0.6 mL) immediately at 5, 15, and 30 min after the end of the injection, and blood samples were collected by cardiac puncture. CSF was collected 30 min or 12 h after injection. Plasma and CSF levels of OT and compound 2 and 5 were determined by LC−MS/MS. The OT level after administration of not only OT but also compound 2 and 5 was determined. Analysis of the Compounds in Plasma and CSF. 100 μL of

plasma was deproteinized by adding 400 μL of acetonitrile/methanol = 1:1 (v/v) containing the internal standard [13C6,

15N]OT. The mixture was vortexed and centrifuged at 15 000g for 10 min at room temperature. The resultant supernatant was transferred to another 1.5 mL polypropylene tube and evaporated to dryness under a nitrogen gas stream at 40 °C. The residue was reconstituted in 50 μL of mobile phase, and 2 μL aliquots were injected into the LC−MS/MS system. Chromatographic separation was performed using a Shimadzu Nexera high-performance LC system (Shimadzu, Kyoto) with a CAPCELL PAK C8DD column (50 mm × 2.0 mm i.d., 5 mm; Shiseido) and the analytical column was kept at 40 °C. The mobile phase consisted of solution A (10 mM ammonium formate in water) and solution B (acetonitrile), which formed the following gradient: 15−30% B (0−3 min); 30−90% B (3−5 min); 90% B (5−7 min); and 15% B (7−12 min). The flow rate was 0.3 mL/min. Positive-ion electrospray (ESI) tandem mass spectrometric analysis was performed using a TSQ Vantage EMR LC−MS/MS System (Thermo Scientific, Waltham, MA) at unit resolution with selected reaction monitoring. The transitions by selected reaction monitoring were m/z 1007.5 → 723.2 for OT, m/z 1075.2 → 723.2 for compound 2, m/z 1025.2 → 723.2 for compound 5, and m/z 1014.4 → 730.1 for internal standard. Data were acquired and analyzed using Xcalibur software (version 2.1; Thermo Scientific). The calibration curves were constructed by spiking the ten different concentrations of analytes with the mouse plasma pool. The calibration curves for OT and compound 5 and compound 2 were all linear in the ranges 0.005−150 and 0.015−150 ng/mL, respectively. The calibration curves were obtained by linear regression for all analytes with a weighting factor of 1/x and linearity (R) of the method in surrogate is greater than 0.998. The detection limits of OT, compound 2, and compound 5 were 0.05, 0.15, and 0.05 pg/mL, respectively. Stability Assay. One hundred microliters of pooled mouse plasma

containing OT, compound 2, or compound 5 (final concentration: 50 ng/mL) was incubated for 1, 5, 15, 30, 60, and 120 min at 37 °C. After designated time, 400 μL of acetonitrile/methanol = 1:1 (v/v) containing the internal standard was added and analyzed by LC−MS/ MS.

■ ASSOCIATED CONTENT *S Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmed- chem.8b01691.

Data on [3H]OT binding to membranes from hOTR- expressing HEK293 cells, comparison of wild type,

CD38KO, and CD38KOCC mice in tail suspended and sucrose preference tests and characterization of the kinetic parameters of [3H]OT (PDF) Molecular formula strings (CSV)

■ AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. Phone/Fax: +81-76- 234-4213 (H.H.). *E-mail: [email protected]. Phone/Fax: +81-11-706- 3769 (S.S.). ORCID Mizuki Watanabe: 0000-0001-7209-5713 Satoshi Shuto: 0000-0001-7850-8064 Present Address ¶Graduate School of Pharmaceutical Sciences, Tohoku University, Aramakiaza Aoba 6-3, Aoba-ku, Sendai 980-8578, Japan (W.I.). Author Contributions ∇W.I. and S.M.C. contributed equally to this work. The manuscript was written with contributions of all the authors. All the authors gave approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS This work was supported by the Industry-Academia Collab- orative R & D Programs (COI) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and partly by Platform Project for Supporting Drug Discovery and Life Science Research (BINDS) from AMED under grant number JP18am0101093. W.I. acknowledged the support of the JSPS program (15J02083).

■ ABBREVIATIONS ANOVA, analysis of variance; ASD, autism spectrum disorder; CD, cluster of differentiation; CRISPR, clustered regularly interspaced short palindromic repeats; CSFsss, cerebrospinal fluid; DIC, diisopropylcarbodiimide; HEK, human embryonic kidney; HOAt, 1-hydroxy-7-azabenzotriazole; 5-HT3, 5- hydroxytryptamine type 3 receptor; KO, knock out; LOT, lipo-oxytocin; OT, oxytocin; OTR, oxytocin receptor; pNz, para-nitrobenzyloxycarbonyl; PyBOP, benzotriazol-1-yl-oxy- tripyrrolidinophosphosphate hexafluorophosphate; RP-HPLC, reverse phase high-performance liquid chromatography; TIS, triisopropylsilane; Trt, trityl; VR, vasopressin receptor; V1aR, vasopressin type 1a receptor; V1bR, vasopressin type 1b receptor; V2R, vasopressin type 2 receptor; WT, wild type

■ REFERENCES (1) Kirsch, P. Oxytocin in the socioemotional brain: implications for psychiatric disorders. Dialogues Clin Neurosci 2015, 17, 463−476. (2) Bealer, S. L.; Armstrong, W. E.; Crowley, W. R. Oxytocin release in magnocellular nuclei: neurochemical mediators and functional significance during gestation. Am. J. Physiol.: Regul., Integr. Comp. Physiol. 2010, 299, R452−R458. (3) Rilling, J. K.; Young, L. J. The biology of mammalian parenting and its effect on offspring social development. Science 2014, 345, 771−776. (4) Marlin, B. J.; Froemke, R. C. Oxytocin modulation of neural circuits for social behavior. Dev. Neurobiol. 2017, 77, 169−189.

Journal of Medicinal Chemistry Article

DOI: 10.1021/acs.jmedchem.8b01691 J. Med. Chem. 2019, 62, 3297−3310

3307

(5) Lai, M.-C.; Lombardo, M. V.; Baron-Cohen, S. Autism. Lancet 2014, 383, 896−910. (6) Peñagarikano, O. New therapeutic options for autism spectrum disorder: experimental evidences. Exp. Neurobiol. 2015, 24, 301−311. (7) Lacivita, E.; Perrone, R.; Margari, L.; Leopoldo, M. Targets for drug therapy for autism spectrum disorder: challenges and future directions. J. Med. Chem. 2017, 60, 9114−9141. (8) James, B. J.; Gales, M. A.; Gales, B. J. Bumetanide for Autism Spectrum Disorder in Children: A Review of Randomized Controlled Trials. Ann. Pharmacother. 2018, 52, 106002801881730. (9) Shamay-Tsoory, S.; Young, L. J. Understanding the oxytocin system and its relevance to psychiatry. Biol. Psychiatry 2016, 79, 150− 152. (10) Alabdali, A.; Al-Ayadhi, A.; El-Ansary, A. Association of social and cognitive impairment and biomarkers in autism spectrum disorders. J. Neuroinflammation 2014, 11, 4. (11) Feldman, R.; Zagoory-Sharon, O.; Weisman, O.; Schneiderman, I.; Gordon, I.; Maoz, R.; Shalev, I.; Ebstein, R. P. Sensitive parenting is associated with plasma oxytocin and polymorphisms in the OXTR and CD38 genes. Biol. Psychiatry 2012, 72, 175−181. (12) LoParo, D.; Waldman, I. D. The oxytocin receptor gene (OXTR) is associated with autism spectrum disorder: a meta-analysis. Mol. Psychiatr. 2015, 20, 640−646. (13) Jin, D.; Liu, H.-X.; Hirai, H.; Torashima, T.; Nagai, T.; Lopatina, O.; Shnayder, N. A.; Yamada, K.; Noda, M.; Seike, T.; Fujita, K.; Takasawa, S.; Yokoyama, S.; Koizumi, K.; Shiraishi, Y.; Tanaka, S.; Hashii, M.; Yoshihara, T.; Higashida, K.; Islam, M. S.; Yamada, N.; Hayashi, K.; Noguchi, N.; Kato, I.; Okamoto, H.; Matsushima, A.; Salmina, A.; Munesue, T.; Shimizu, N.; Mochida, S.; Asano, M.; Higashida, H. CD38 is critical for social behaviour by regulating oxytocin secretion. Nature 2007, 446, 41−45. (14) Lopatina, O.; Yoshihara, T.; Nishimura, T.; Zhong, J.; Akther, S.; Fakhrul, A. A.; Liang, M.; Higashida, C.; Sumi, K.; Furuhara, K.; Inahata, Y.; Huang, J. J.; Koizumi, K.; Yokoyama, S.; Tsuji, T.; Petugina, Y.; Sumarokov, A.; Salmina, A. B.; Hashida, K.; Kitao, Y.; Hori, O.; Asano, M.; Kitamura, Y.; Kozaka, T.; Shiba, K.; Zhong, F.; Xie, M. J.; Sato, M.; Ishihara, K.; Higashida, H. Anxiety- and depression-like behavior in mice lacking the CD157/BST1 gene, a risk factor for Parkinson’s disease. Front. Behav. Neurosci. 2014, 8, 133. (15) Higashida, H.; Yuhi, T.; Akther, S.; Amina, S.; Zhong, J.; Liang, M.; Nishimura, T.; Liu, H.-X.; Lopatina, O. Oxytocin release via activation of TRPM2 and CD38 in the hypothalamus during hyperthermia in mice: implication for autism spectrum disorder. Neurochem. Int. 2017, 119, 42−48. (16) Higashida, H.; Liang, M.; Yoshihara, T.; Akther, S.; Fakhrul, A.; Cherepanov, S.; Nam, T. S.; Kim, U. H.; Kasai, S.; Nishimura, T.; Mahmuda, N.; Yokoyama, S.; Ishihara, K.; Gerasimenko, M.; Salmina, A.; Zhong, J.; Tsuji, T.; Tsuji, C.; Lopatina, O. An immunohis- tochemical, enzymatic, and behavioral study of CD157/BST-1 as a neuroregulator. BMC Neurosci. 2017, 18, 35. (17) Munesue, T.; Yokoyama, S.; Nakamura, K.; Anitha, A.; Yamada, K.; Hayashi, K.; Asaka, T.; Liu, H.-X.; Jin, D.; Koizumi, K.; Islam, M. S.; Huang, J.-J.; Ma, W.-J.; Kim, U.-H.; Kim, S.-J.; Park, K.; Kim, D.; Kikuchi, M.; Ono, Y.; Nakatani, H.; Suda, S.; Miyachi, T.; Hirai, H.; Salmina, A.; Pichugina, Y. A.; Soumarokov, A. A.; Takei, N.; Mori, N.; Tsujii, M.; Sugiyama, T.; Yagi, K.; Yamagishi, M.; Sasaki, T.; Yamasue, H.; Kato, N.; Hashimoto, R.; Taniike, M.; Hayashi, Y.; Hamada, J.; Suzuki, S.; Ooi, A.; Noda, M.; Kamiyama, Y.; Kido, M. A.; Lopatina, O.; Hashii, M.; Amina, S.; Malavasi, F.; Huang, E. J.; Zhang, J.; Shimizu, N.; Yoshikawa, T.; Matsushima, A.; Minabe, Y.; Higashida, H. Two genetic variants of CD38 in subjects with autism spectrum disorder and controls. Neurosci. Res. 2010, 67, 181−191. (18) Yokoyama, S.; Al Mahmuda, N.; Munesue, T.; Hayashi, K.; Yagi, K.; Yamagishi, M.; Higashida, H. Association study between the CD157/BST1 gene and autism spectrum disorders in a Japanese population. Brain Sci. 2015, 5, 188−200. (19) Kato, I.; Yamamoto, Y.; Fujimura, M.; Noguchi, N.; Takasawa, S.; Okamoto, H. CD38 Disruption Impairs Glucose-induced Increases

in Cyclic ADP-ribose, [Ca2+]i, and Insulin Secretion. J. Biol. Chem. 1999, 274, 1869−1872. (20) Higashida, H.; Yokoyama, S.; Munesue, T.; Kikuchi, M.; Minabe, Y.; Lopatina, O. CD38 gene knockout juvenile mice: a model of oxytocin signal defects in autism. Biol. Pharm. Bull. 2011, 34, 1369−1372. (21) Akther, S.; Korshnova, N.; Zhong, J.; Liang, M.; Cherepanov, S. M.; Lopatina, O.; Komleva, Y. K.; Salmina, A. B.; Nishimura, T.; Fakhrul, A. A.; Hirai, H.; Kato, I.; Yamamoto, Y.; Takasawa, S.; Okamoto, H.; Higashida, H. CD38 in the nucleus accumbens and oxytocin are related to paternal behavior in mice. Mol. Brain 2013, 6, 41. (22) Cherepanov, S.; Akther, S.; Nishimura, T.; Shabalova, A.; Mizuno, A.; Ichinose, W.; Shuto, S.; Yamamoto, Y.; Yokoyama, S.; Higashida, H. Effects of three lipidated oxytocin analogs on behavioral deficits in CD38 knockout mice. Brain Sci. 2017, 7, 132. (23) Domes, G.; Heinrichs, M.; Kumbier, E.; Grossmann, A.; Hauenstein, K.; Herpertz, S. C. Effects of intranasal oxytocin on the neural basis of face processing in autism spectrum disorder. Biol. Psychiatry 2013, 74, 164−171. (24) Auyeung, B.; Lombardo, M. V.; Heinrichs, M.; Chakrabarti, B.; Sule, A.; Deakin, J. B.; Bethlehem, R. A. I.; Dickens, L.; Mooney, N.; Sipple, J. A. N.; Thiemann, P.; Baron-Cohen, S. Oxytocin increases eye contact during a real-time, naturalistic social interaction in males with and without autism. Transl. Psychiatry 2015, 5, No. e507. (25) Dadds, M. R.; MacDonald, E.; Cauchi, A.; Williams, K.; Levy, F.; Brennan, J. Nasal oxytocin for social deficits in childhood autism: a randomized controlled trial. J. Autism Dev. Disord. 2014, 44, 521−531. (26) Guastella, A. J.; Gray, K. M.; Rinehart, N. J.; Alvares, G. A.; Tonge, B. J.; Hickie, I. B.; Keating, C. M.; Cacciotti-Saija, C.; Einfeld, S. L. The effects of a course of intranasal oxytocin on social behaviors in youth diagnosed with autism spectrum disorders: a randomized controlled trial. J. Child Psychol. Psychiatry 2015, 56, 444−452. (27) Munesue, T.; Nakamura, H.; Kikuchi, M.; Miura, Y.; Takeuchi, N.; Anme, T.; Nanba, E.; Adachi, K.; Tsubouchi, K.; Sai, Y.; Miyamoto, K.; Horike, S.; Yokoyama, S.; Nakatani, H.; Niida, Y.; Kosaka, H.; Minabe, Y.; Higashida, H. Oxytocin for male subjects with autism spectrum disorder and comorbid intellectual disabilities: a randomized pilot study. Front. Psychiatry 2016, 7, 2. (28) Parker, K. J.; Oztan, O.; Libove, R. A.; Sumiyoshi, R. D.; Jackson, L. P.; Karhson, D. S.; Summers, J. E.; Hinman, K. E.; Motonaga, K. S.; Phillips, J. M.; Carson, D. S.; Garner, J. P.; Hardan, A. Y. Intranasal oxytocin treatment for social deficits and biomarkers of response in children with autism. Proc. Natl. Acad. Sci. U.S.A. 2017, 114, 8119−8124. (29) Yatawara, C. J.; Einfeld, S. L.; Hickie, I. B.; Davenport, T. A.; Guastella, A. J. The effect of oxytocin nasal spray on social interaction deficits observed in young children with autism: a randomized clinical crossover trial. Mol. Psychiatr. 2016, 21, 1225−1231. (30) Mizuno, A.; Cherepanov, S.; Kikuchi, Y.; Fakhrul, A.; Akther, S.; Deguchi, K.; Yoshihara, T.; Ishihara, K.; Shuto, S.; Higashida, H. Lipo-oxytocin-1, a novel oxytocin Analog conjugated with two palmitoyl groups, has long-lasting effects on anxiety-related behavior and social avoidance in CD157 knockout mice. Brain Sci. 2015, 5, 3− 13. (31) Cherepanov, S. M.; Yokoyama, S.; Mizuno, A.; Ichinose, W.; Lopatina, O.; Shabalova, A. A.; Salmina, A. B.; Yamamoto, Y.; Okamoto, H.; Shuto, S.; Higashida, H. Structure-specific effects of lipidated oxytocin analogs on intracellular calcium levels, parental behavior, and oxytocin concentrations in the plasma and cerebrospi- nal fluid in mice. Pharmacol. Res. Perspect. 2017, 5, No. e00290. (32) Widmer, M.; Piaggio, G.; Nguyen, T. M. H.; Osoti, A.; Owa, O. O.; Misra, S.; Coomarasamy, A.; Abdel-Aleem, H.; Mallapur, A. A.; Qureshi, Z.; Lumbiganon, P.; Patel, A. B.; Carroli, G.; Fawole, B.; Goudar, S. S.; Pujar, Y. V.; Neilson, J.; Hofmeyr, G. J.; Su, L. L.; Ferreira de Carvalho, J.; Pandey, U.; Mugerwa, K.; Shiragur, S. S.; Byamugisha, J.; Giordano, D.; Giordano, D.; Gülmezoglu, A. M. Heat- stable carbetocin versus oxytocin to prevent hemorrhage after vaginal birth. N. Engl. J. Med. 2018, 379, 743−752.

Journal of Medicinal Chemistry Article

DOI: 10.1021/acs.jmedchem.8b01691 J. Med. Chem. 2019, 62, 3297−3310

3308

(33) Adachi, Y.; Sakimura, K.; Shimizu, Y.; Nakayama, M.; Terao, Y.; Yano, T.; Asami, T. Potent and selective oxytocin receptor agonists without disulfide bridges. Bioorg. Med. Chem. Lett. 2017, 27, 2331− 2335. (34) Muttenthaler has reported that the transformation at the disulfide linker was also important for modulating the OT potency and deamination at Cys1 residue to earn the chemical stability and hydrophobicity: see ref 35. (35) Muttenthaler, M.; Andersson, A.; De Araujo, A. D.; Dekan, Z.; Lewis, R. J.; Alewood, P. F. Modulating oxytocin activity and plasma stability by disulfide bond engineering. J. Med. Chem. 2010, 53, 8585− 8596. (36) De Araujo, A. D.; Mobli, M.; Castro, J.; Harrington, A. M.; Vetter, I.; Dekan, Z.; Muttenthaler, M.; Wan, J.; Lewis, R. J.; King, G. F.; Brierley, S. M.; Alewood, P. F. Selenoether oxytocin analogues have analgesic properties in a mouse model of chronic abdominal pain. Nat. Commun. 2014, 5, 3165. (37) Muttenthaler, M.; Andersson, Å.; Vetter, I.; Menon, R.; Busnelli, M.; Ragnarsson, L.; Bergmayr, C.; Arrowsmith, S.; Deuis, J. R.; Chiu, H. S.; Palpant, N. J.; O’Brien, M.; Smith, T. J.; Wray, S.; Neumann, I. D.; Gruber, C. W.; Lewis, R. J.; Alewood, P. F. Subtle modifications to oxytocin produce ligands that retain potency and improved selectivity across species. Sci. Signal. 2017, 10, No. eaan3398. (38) Scibola, S.; Goetz, G. H.; Bai, G.; Rogers, B. N.; Gray, D. L.; Duplantier, A.; Fonseca, K. R.; Vanase-Frawley, M. A.; Kablaoui, N. M. Systematic N-methylation of oxytocin: Impact on pharmacology and intramolecular hydrogen bonding network. Bioorg. Med. Chem. 2016, 24, 3513−3520. (39) Modi, M. E.; Majchrzak, M. J.; Fonseca, K. R.; Doran, A.; Osgood, S.; Vanase-Frawley, M.; Feyfant, E.; McInnes, H.; Darvari, R.; Buhl, D. L.; Kablaoui, N. M. Peripheral administration of a long- acting peptide oxytocin receptor agonist inhibits fear-induced freezing. J. Pharmacol. Exp. Ther. 2016, 358, 164−172. (40) Ring, R. H.; Schechter, L. E.; Leonard, S. K.; Dwyer, J. M.; Platt, B. J.; Graf, R.; Grauer, S.; Pulicicchio, C.; Resnick, L.; Rahman, Z.; Sukoff Rizzo, S. J.; Luo, B.; Beyer, C. E.; Logue, S. F.; Marquis, K. L.; Hughes, Z. A.; Rosenzweig-Lipson, S. Receptor and behavioral pharmacology of WAY-267464, a non-peptide oxytocin receptor agonist. Neuropharmacology 2010, 58, 69−77. (41) Engstøm, T.; Barth, T.; Melin, P.; Vilhardt, H. Oxytocin receptor binding and uterotonic activity of carbetocin and its metabolites following enzymatic degradation. Eur. J. Pharmacol. 1998, 355, 203−210. (42) Rath, W. Prevention of postpartum haemorrhage with the oxytocin analogue carbetocin. Eur. J. Obstet. Gynecol. Reprod. Biol. 2009, 147, 15−20. (43) Wisńiewski, K.; Alagarsamy, S.; Galyean, R.; Tariga, H.; Thompson, D.; Ly, B.; Wisńiewski, H.; Qi, S.; Croston, G.; Laporte, R.; Rivier̀e, P. J.-M.; Schteingart, C. D. New, potent, and selective peptidic oxytocin receptor agonists. J. Med. Chem. 2014, 57, 5306− 5317. (44) ClinicalTrials.gov, NIH, https://www.clinicaltrials.gov/ct2/ show/NCT02545127?lead=ferring&gndr=Female&phase=01&rank= 5 (accessed on February 26, 2019). (45) Wellings, D. A.; Atherton, E. [4] Standard Fmoc protocols. Methods Enzymol. 1997, 289, 44−67. (46) Hsu, P. D.; Scott, D. A.; Weinstein, J. A.; Ran, F. A.; Konermann, S.; Agarwala, V.; Li, Y.; Fine, E. J.; Wu, X.; Shalem, O.; Cradick, T. J.; Marraffini, L. A.; Bao, G.; Zhang, F. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 2013, 31, 827−832. (47) Motulsky, H. J.; Mahan, L. C. The kinetics of competitive radioligand binding predicted by the law of mass action. Mol. Pharmacol. 1984, 25, 1−9. (48) Ihara, M.; Matsuda, K.; Shimomura, M.; Sattelle, D. B.; Komai, K. Super Agonist Actions of Clothianidin and Related Compounds on the SADβ2 Nicotinic Acetylcholine Receptor Expressed inXenopus laevisOocytes. Biosci. Biotechnol. Biochem. 2004, 68, 761−763.

(49) Brown, L. A.; Ihara, M.; Buckingham, S. D.; Matsuda, K.; Sattelle, D. B. Neonicotinoid insecticides display partial and super agonist actions on native insect nicotinic acetylcholine receptors. J. Neurochem. 2006, 99, 608−615. (50) Engel, S.; Neumann, S.; Kaur, N.; Monga, V.; Jain, R.; Northup, J.; Gershengorn, M. C. Low Affinity Analogs of Thyrotropin-releasing Hormone Are Super-agonists. J. Biol. Chem. 2006, 281, 13103−13109. (51) Langmead, C. J.; Christopoulos, A. Supra-physiological efficacy at GPCRs: superstition or super agonists? Br. J. Pharmacol. 2013, 169, 353−356. (52) Alix, K.; Khatri, S.; Mosier, P. D.; Casterlow, S.; Yan, D.; Nyce, H. L.; White, M. M.; Schulte, M. K.; Dukat, M. Superagonist, full agonist, partial agonist, and antagonist actions of arylguanidines at 5- hydroxytryptamine-3 (5-HT3) subunit A receptors. ACS Chem. Neurosci. 2016, 7, 1565−1574. (53) Rosethorne, E. M.; Bradley, M. E.; Gherbi, K.; Sykes, D. A.; Sattikar, A.; Wright, J. D.; Renard, E.; Trifilieff, A.; Fairhurst, R. A.; Charlton, S. J. Long receptor residence time of C26 contributes to super agonist activity at the human β2-receptor. Mol. Pharmacol. 2016, 89, 467−475. (54) Muller, K.; Faeh, C.; Diederich, F. Fluorine in pharmaceuticals: looking beyond intuition. Science 2007, 317, 1881−1886. (55) Vulpetti, A.; Dalvit, C. Fluorine local environment: from screening to drug design. Drug Discov. Today 2012, 17, 890−897. (56) Liu, H.-X.; Lopatina, O.; Higashida, C.; Fujimoto, H.; Akther, S.; Inzhutova, A.; Liang, M.; Zhong, J.; Tsuji, T.; Yoshihara, T.; Sumi, K.; Ishiyama, M.; Ma, W. J.; Ozaki, M.; Yagitani, S.; Yokoyama, S.; Mukaida, N.; Sakurai, T.; Hori, O.; Yoshioka, K.; Hirao, A.; Kato, Y.; Ishihara, K.; Kato, I.; Okamoto, H.; Cherepanov, S. M.; Salmina, A. B.; Hirai, H.; Asano, M.; Brown, D. A.; Nagano, I.; Higashida, H. Displays of paternal mouse pup retrieval following communicative interaction with maternal mates. Nat. Commun. 2013, 4, 1346. (57) Gutkowska, J.; Jankowski, M. Oxytocin revisited: its role in cardiovascular regulation. J. Neuroendocrinol. 2012, 24, 599−608. (58) Lopatina, O.; Liu, H.-X.; Amina, S.; Hashii, M.; Higashida, H. Oxytocin-induced elevation of ADP-ribosyl cyclase activity, cyclic ADP-riboseor Ca2+ concentrations is involved in autoregulation of oxytocin secretionin the hypothalamus and posterior pituitary in male mice. Neuropharmacology 2010, 58, 50−55. (59) Moy, S. S.; Teng, B. L.; Nikolova, V. D.; Riddick, N. V.; Simpson, C. D.; Van Deusen, A.; Janzen, W. P.; Sassano, M. F.; Pedersen, C. A.; Jarstfer, M. B. Prosocial effects of an oxytocin metabolite, but not synthetic oxytocin receptor agonists, in a mouse model of autism. Neuropharmacology 2019, 144, 301−311. (60) Grinevich, V.; Knobloch-Bollmann, H. S.; Eliava, M.; Busnelli, M.; Chini, B. Assembling the puzzle: Pathways of oxytocin signaling in the brain. Biol. Psychiatry 2016, 79, 155−164. (61) Dowling, M. R.; Charlton, S. J.; Charlton, S. J. Quantifying the association and dissociation rates of unlabelled antagonists at the muscarinic M3 receptor. Br. J. Pharmacol. 2006, 148, 927−937. (62) Sykes, D. A.; Dowling, M. R.; Leighton-Davies, J.; Kent, T. C.; Fawcett, L.; Renard, E.; Trifilieff, A.; Charlton, S. J. The influence of receptor kinetics on the onset and duration of action and the therapeutic index of NVA237 and tiotropium. J. Pharmacol. Exp. Ther. 2012, 343, 520−528. (63) Dagani, J.; Sisti, D.; Abelli, M.; Di Paolo, L.; Pini, S.; Raimondi, S.; Rocchi, M. B.; Saviotti, F. M.; Scocco, P.; Totaro, S.; Balestrieri, M.; de Girolamo, G. Do we need oxytocin to treat schizophrenia? A randomized clinical trial. Schizophr. Res. 2016, 172, 158−164. (64) Scantamburlo, G.; Hansenne, M.; Geenen, V.; Legros, J. J.; Ansseau, M. Additional intranasal oxytocin to escitalopram improves depressive symptoms in resistant depression: an open trial. Eur. Psychiatry 2015, 30, 65−68. (65) Amina, S.; Hashii, M.; Ma, W.-J.; Yokoyama, S.; Lopatina, O.; Liu, H.-X.; Islam, M. S.; Higashida, H. Intracellular calcium elevation induced by extracellular application of cyclic-ADP-ribose or oxytocin is temperature-sensitive in rodent NG108-15 neuronal cells with or without exogenous expression of human oxytocin receptors. J. Neuroendocrinol. 2010, 22, 460−466.

Journal of Medicinal Chemistry Article

DOI: 10.1021/acs.jmedchem.8b01691 J. Med. Chem. 2019, 62, 3297−3310

3309

(66) Beard, R.; Stucki, A.; Schmitt, M.; Py, G.; Grundschober, C.; Gee, A. D.; Tate, E. W. Building bridges for highly selective, potent and stable oxytocin and vasopressin analogs. Bioorg. Med. Chem. 2018, 26, 3039−3045. (67) Nakagawa, Y.; Higashida, H.; Miki, N. A single class of neurotensin receptors with high affinity in neuroblastoma X glioma NG108-15 hybrid cells that mediate facilitation of synaptic trans- mission. J. Neurosci. 1984, 4, 1653−1661. (68) Yung-Chi, C.; Prusoff, W. H. Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 1973, 22, 3099−3108. (69) Ma, W.-J.; Hashii, M.; Munesue, T.; Hayashi, K.; Yagi, K.; Yamagishi, M.; Higashida, H.; Yokoyama, S. Non-synonymous single- nucleotide variations of the human oxytocin receptor gene and autism spectrum disorders: a case-control study in a Japanese population and functional analysis. Mol. Autism. 2013, 4, 22.

Journal of Medicinal Chemistry Article

DOI: 10.1021/acs.jmedchem.8b01691 J. Med. Chem. 2019, 62, 3297−3310

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