Synthesis of oxytocin derivatives lipidated via a carbonate or carbamate linkage as a long-acting therapeutic agent for social impairment-like behaviors

Article abstract of DOI:10.1016/j.bmc.2019.06.018

In the course of our studies of hydrophobic oxytocin (OT) analogues, we newly synthesized lipidated OT (LOT-4a-c and LOT-5a-c), in which a long alkyl chain (C14-C16) is conjugated via a carbonate or carbamate linkage at the Tyr-2 phenolic hydroxy group an

Full text of DOI:10.1016/j.bmc.2019.06.018

Accepted Manuscript
Synthesis of Oxytocin Derivatives Lipidated via a Carbonate or Carbamate
Linkage as a Long-Acting Therapeutic Agent for Social Impairment-Like Be-
haviors
Stanislav M. Cherepanov, Risako Miura, Anna A. Shabalova, Wataru Ichinose,
Shigeru Yokoyama, Hayato Fukuda, Mizuki Watanabe, Haruhiro Higashida,
Satoshi Shuto
PII:
S0968-0896(19)30808-9
https://doi.org/10.1016/j.bmc.2019.06.018
BMC 14955
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
15 May 2019
6 June 2019
9 June 2019
Please cite this article as: Cherepanov, S.M., Miura, R., Shabalova, A.A., Ichinose, W., Yokoyama, S., Fukuda, H.,
Watanabe, M., Higashida, H., Shuto, S., Synthesis of Oxytocin Derivatives Lipidated via a Carbonate or Carbamate
Linkage as a Long-Acting Therapeutic Agent for Social Impairment-Like Behaviors, Bioorganic & Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.06.018
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis of Oxytocin Derivatives Lipidated via a Carbonate or Carbamate
Linkage as a Long-Acting Therapeutic Agent for Social Impairment-Like
Behaviors
Stanislav M. Cherepanov,1# Risako Miura,2# Anna A. Shabalova,1 Wataru Ichinose,2 Shigeru
Yokoyama,1 Hayato Fukuda,2 Mizuki Watanabe,2 Haruhiro Higashida,1* and Satoshi Shuto2,3*
1Department of Basic Research on Social Recognition, Research Center for Child Mental
Development, Kanazawa University, Kanazawa 920-8640, Japan
2Faculty of Pharmaceutical Sciences and 3Center for Research and Education on Drug
Discovery, Hokkaido University, Sapporo 060-0812, Japan
#S. M. C. and R. M. contributed equally to this work.
Corresponding Authors
Haruhiro Higashida: haruhiro@med.kanazawa-u.ac.jp, Phone & Fax: +81-76-234-4213
Satoshi Shuto: shu@pharm.hokudai.ac.jp, Phone & Fax: +81-11-706-3769

Graphic Abstract
O-R2
O
R1-HN
O
N
H
oxytocin (OT): R1 = R2 = H
HN
HN
S
S
very long-acting OT analogue
LOT-4c : R1 = CO(CH2)14CH3
R2 = COO(CH2)15CH3
O
O
H
N
N
O
O
N
H
H
N
O
O
O
N
H
H2N
O
O
NH2
NH2
Keywords
Lipidation; Long-Acting; Oxytocin; Social Impairment-Like Behavior; Prodrug
Abstract
In the course of our studies of hydrophobic oxytocin (OT) analogues, we newly synthesized
lipidated OT (LOT-4a-c and LOT-5a-c), in which a long alkyl chain (C14-C16) is conjugated
via a carbonate or carbamate linkage at the Tyr-2 phenolic hydroxy group and a palmitoyl group
at the terminal amino group of Cys-1. These LOTs did not activate OT and vasopressin receptors.
Among the LOTs, however, LOT-4c, having a C16-chain via a carbonate linkage at the phenolic
hydroxyl group of the Tyr-2, showed very long-lasting action for the recovery of impaired social
behavior in CD38 knockout mice, a rodent model of autistic phenotypes, whereas the effect of
OT itself rapidly diminished. These results indicate that LOT-4c may serve as a potential prodrug
in mice.

1. Introduction
Oxytocin (OT) is a cyclic neuropeptide and hormone comprising nine amino acids. OT is
synthesized in the neurons of the supraoptic and paraventricular nuclei of the hypothalamus1,2
and secreted into the bloodstream from the posterior pituitary lobe.3 Binding of OT with OT
receptors (OTRs)1 in the peripheral organs induces uterine contractions during delivery and
lactation in the postpartum period in females.4 Thus, OT is used clinically to induce labor as well
as to prevent postpartum bleeding.5 In the central nervous system (CNS), OT is a neuromodulator
with a key role in social memory, parental behavior, and pair-bonding in mammals.6 In humans,
clinical studies7 suggest a role of OT in the modulation of trust8, recognition of facial
expressions,9 and mother to infant gaze interactions.10
Autism spectrum disorder (ASD) is a set of congenital neurodevelopmental conditions
characterized by impairments in social communication, social interactions, and the presence of
repetitive behaviors and restricted interests.11 Recent clinical trials demonstrated that single
intravenous12 or intranasal OT administration13 in ASD patients led to recovery of emotion
recognition and social processing.14 Repeated intranasal application of OT, however, failed to
have positive effects over the long-term condition in clinical trials.15,16 One possible reason for
the results of the clinical trials is the pharmacokinetic properties of OT, i.e., short half-life in the
blood17 and poor penetration of the blood–brain barrier, which is typical of peptides.18 Therefore,
OT may not be a suitable candidate for the treatment of ASD symptoms, but a valuable lead for
the development of new compounds with improved pharmacokinetic profiles.
Lipidation, the conjugation of a peptide with long-chain fatty acids, may extend the half-life
of peptides in the blood.19 Using this strategy, we previously developed three OT analogues,
named lipooxytocins (LOTs, Figure 1), in which a palmitoyl group is conjugated to the terminal
amino group of Cys-1 and the phenolic hydroxyl group of Tyr-2 (LOT-1), or to only one of the
amino or phenolic hydroxyl groups (LOT-2, LOT-3).20,21 Administration of these analogues and

OT by single intraperitoneal injection (1 ng/g of body weight) led to recovery of social behavior
in CD38 knockout (CD38KOCC) mice,21,22 a rodent model of autistic phenotypes developed in our
laboratory.23, 24 It is noteworthy that the recovery effects the LOTs lasted approximately 24 h –
remarkably longer than that of OT.21,22 Furthermore, elevated OT levels in the cerebrospinal fluid
were observed 24 h after a single intraperitoneal injection of LOT-1,21 suggesting that LOT-1 was
easily transported into the cerebrospinal fluid owing to its lipophilic properties. Because neither of
the three LOTs induces OTR-mediated cytosolic Ca2+-mobilization in OTR- expressing HEK 293
cells,21 they are likely to be OT prodrugs that release OT in the brain. It is also likely that their
long-acting effects result from the slow hydrolysis of their ester and/or amide linkage-conjugating
palmitoyl groups.
In the course of our studies on long-acting OT analogues,20-24 we newly synthesized additional
lipidated OT (LOT-4a-c and LOT-5a-c, Figure 1), which had a carbonate or a carbamate linkage
between the Tyr-2 phenolic hydroxy group and a long alkyl chain (C14-C16), unlike the ester
linkage in the previous LOTs, but had a palmitoyl group at the terminal amino group of Cys-1 like
the previous LOTs. In this paper, we describe the synthesis and results on the pharmacological
evaluation of these new lipidated OTs.

O-R2
O
R1-HN
O
N
H
HN
HN
S
S
O
O
H
N
N
O
O
N
H
O
H
N
O
O
O
H2N
N
H
O
NH2
NH2
oxytocin (OT): R1 = R2 = H
LOT-1 : R1 = R2 = CO(CH2)14CH3
LOT-2 : R1 = CO(CH2)14CH3, R2 = H
LOT-3 : R1 = H, R2 = CO(CH2)14CH3
LOT-4a : R1 = CO(CH2)14CH3, R2 = COO(CH2)13CH3
LOT-4b : R1 = CO(CH2)14CH3, R2 = COO(CH2)14CH3
LOT-4c : R1 = CO(CH2)14CH3, R2 = COO(CH2)15CH3
LOT-5a : R1 = CO(CH2)14CH3, R2 = CONH(CH2)13CH3
LOT-5b : R1 = CO(CH2)14CH3, R2 = CONH(CH2)14CH3
LOT-5c : R1 = CO(CH2)14CH3, R2 = CONH(CH2)15CH3
Figure 1. Chemical structure of OT and LOTs.
2. Results
2.1 Synthesis. All of the newly designed LOTs were synthesized from LOT-2 as shown in
Scheme 1. We previously prepared LOT-2 by treating OT with 1.3 equivalents of palmitic
anhydride (Pal2O) in the presence of Et3N in CH2Cl2/DMF. Because the removal of unreacted
Pal2O and its hydrolyzed product palmitic acid is troublesome using this procedure, the purity of
LOT-2 was often insufficient. Therefore, we reassessed the procedure and found that we could
reproducibly obtain high-purity LOT-2 in high yield when the reaction was carried out with
reduced Pal2O (1.0 equiv) and the product was purified by silica gel column chromatography
with a high-polar solvent system (CHCl3/MeOH = 3:1).
We next investigated the formation of the carbonate or carbamoyl linkage at the phenolic
hydroxy group of Tyr-2 in LOT-2 and found that the carbonate linkage was effectively formed
by using 4-nitrophenyl carbonates 1a–c. Thus, treatment of LOT-2 with the carbonates 1a–c
with a long alkyl-chain in the presence of DMAP and Et3N in CH2Cl2/DMF effectively provided
all of desired three LOT-4a–c. On the other hand, formation of the carbamate linkage was
unsuccessful by using the corresponded 4-nitrophenycarbamates. The carbamates LOT-5a–c

were effectively prepared, however, when LOT-2 was treated with carbamic chlorides 2a-c in
the presence of DMAP and Et3N in CH2Cl2/DMF.
O-R2
O
R1-HN
O
N
H
1a–c or 2a–c
Et3N, DMAP
HN
HN
LOT-4a (65%), LOT-4b (87%), LOT-4c (81%)
LOT-5a (91%), LOT-5b (55%), LOT-5c (53%)
S
S
CH2Cl2/DMF
O2N
O
O
H
N
N
O
O
N
H
O
O
H
N
O
O
O
O
(CH2)nCH3
H2N
N
Cl
N
H
(CH2)nCH3
H
O
O
O
NH2
NH2
2a : n = 13
2b : n = 14
2c : n = 15
1a : n = 13
1b : n = 14
1c : n = 15
Pal2O (1.0 eq.)
Et3N (3.0 eq)
DMF/CH2Cl2
OT: R1 = R2 = H
LOT-2 : R1 = CO(CH2)14CH3
96%
Scheme 1. Synthesis of LOT-4a–c and LOT-5a–c.

2.2 In vitro study. We examined the ability of the compounds to activate OTRs. In the
presence of a compound, we measured [Ca2+]i, which is dependent on Ca2+-mobilization from
intracellular stores induced by OTR activation in HEK-293 cells expressing human OTRs
(hOTRs) or human vasopressin receptors 1a and 1b (hV1aRs and hV1bRs)24. The overall data,
including the half-maximal effective concentration (EC50) and the maximal effect (Emax) for OT
and the OT analogues are presented in Table 1.
Table 1. Biological activities of OT and analogues
Agonist
Agonist potency
Activity
Emax
EC50, nM (95% confidence interval)
%
hOTR
0.0046
hV1aR
0.18
(0.09-0.34)
hV1bR
0.2
(0.11-0.36)
hOTR
OT
100
(0.0013-0.015)
2.7
LOT-4a
>100000
>100000
>100000
>100000
>100000
>100000
>100000
>100000
>100000
>100000
>100000
>100000
15.3
(0.09-73)
>100000
LOT-4b
LOT-4c
LOT-5a
LOT-5b
LOT-5c
0
>100000
>100000
>10000
0
0
Ambiguous
0
>100000
EC50 values of agonist activities were determined for hOTR, hV1aR, and hV1bR using [Ca2+]i
measurement with a microspectrofluorometric system. Number of independent experiments: n = 3, each
c
experiment was performed in duplicate, number of measured cells in each cell-dish: 20. Emax values of
agonist potencies were calculated from EC50 curves for hOTR, hV1aR, and hV1bR.
In the Ca2+-assay, OT had an EC50 of 0.0046 nM (0.0013-0.015) for hOTRs and 100-fold lower
activity for hV1aRs and hV1bRs (Table 1). These data are similar to our previous results.24 Most
of the newly synthesized LOTs, however, exhibited no agonistic effect in this system. Only
LOT-4a exhibited very weak partial agonist activity with an EC50 of 2.7 nM (0.09-73) and an
Emax of 15.3%.

2.3. Effects on Parental Nurturing Behavior. CD38 is transmembrane protein, involved in
process of OT release in CNS.23,25 A single nucleotide polymorphisms of CD38 gene are
associated with ASD.26 According to these findings, we previously developed CD38KOCC
mice24 that display autistic phenotypes features such as deficit in social memory and
recognition.24, 28 One of the features of CD38KOCC mice is a deficit in paternal nurturing
behavior.29 OT administration recovers recovery of this impairment in CD38KOCC mice.,22, 29
Thus, these mice are useful for evaluating the effects of OT-like compounds to recover this
autistic phenotype.20-25
Thus, we examined the effects of OT, LOT4-a–c and LOT-5-a–c on parental nurturing
behavior, i.e., the latency to retrieve pups by CD38KOCC sires at 30 min and at 24 h after
treatment with the compounds.22 Thirty minutes after a single intraperitoneal (ip)- injection of
phosphate-buffered saline (PBS), the mean parental scores of CD38KOCC sires to retrieve pups
were 0 ± 0 s (n = 10, Table 2). The mean scores of CD38KOCC sires to retrieve pups within 30
min after a single ip-injection of OT (100 ng/100 g body weight) was 5.3 ± 1.14 (p= 0.03),
consistent with previous reports.22,24 At 30 min after the treatment, among the LOTs, only LOT-
4a induced a difference in pup retrieval latencies compared with mice treated with PBS (6.8 ±
1.5 vs 0 ± 0, respectively; p = 0.031). A one-way ANOVA revealed significant differences
between groups (F8,81 = 3.93, p = 0.0006). At 24 h after administration, however, LOT-4c
demonstrated clear difference from PBS treated group: 5.2 ± 1 (n =10) vs 0 ± 0 (n = 10),
(multiple comparisons with Bonferroni post hoc correction p = 0.011). All of LOTs other than
LOT-4a did not demonstrated difference compare with PBS treated mice. A one-way ANOVA
revealed significant differences between groups (F8,81 = 4.2, p = 0.0003)
Table 2 Pup retrieval behavior by sires.
Parental scores
30 min
p-value
Compound
PBS
24 h
30 min
-
24h
-
0 ± 0
0 ± 0

OT
5.3 ± 1.14
6.8 ± 1.5
3.9 ± 1.6
2 ± 1.33
0 ± 0
0.03
0.002
0.23
0.99
0.5
0.99
0.42
0.55
0.011
0.2
LOT-4a
LOT-4b
LOT-4c
LOT-5a
LOT-5b
LOT-5c
3.1 ± 1.32
2.9 ± 1.19
5.2 ± 1
3.3 ± 1.37
2 ± 0.85
3.6 ± 1.48
3.1 ± 1.01
3.4 ± 1.4
0.99
0.99
0.42
0.27
1.9 ± 1.27
The parental scores by CD38KOCC sires at 30 min and 24 h after a single intraperitoneal injection of PBS,
OT or LOTs (100 ng/100 g body weight); n = 10/group. One-way ANOVA followed by Bonferroni’s post
hoc test was performed for 30 min (F8,81 = 3.93, p = 0.0006), and 24 h (F8,81 = 4.2, p = 0.0003) P-values
indicated Bonferroni post hoc test compared with CD38KOCC treated by PBS.
10
PBS
OT
8
6
4
2
0
LOT-4c
**
*
0.08
0
20
40
60
Time (h)
Figure 2. Pup retrieval behavior by sires. The parental scores for pup retrieval CD38KOCC sires. The
time-course after a single intraperitoneal injection of PBS, OT, or LOT-4c (100 ng/100 g body weight); n
= 10/group. Two-way ANOVA followed by Bonferroni’s test for multiple comparisons was performed
for the time-course (time F5,162 = 1.867, p = 0.1 treatment F2,162= 5.19, p = 0.0065, interaction F10, 162
=
1.88, p = 0.05). * p < 0.05 and ** p < 0.01 compared to CD38KOCC PBS- treated mice with Bonferroni’s
correction for multiple comparisons.

We then examined the long-lasting activity of LOT-4c over time based on parental
scores.22 As shown in Figure 2, the activity of OT was lost at 6 h. These data are consistent with
previous findings.24 LOT-4c exhibited a tendency to increase paternal nurturing scores at 12 h
and significantly increased paternal nurturing scores at 24 h, but lost its activity at the 48h time-
point. Two-way ANOVA indicated significant effects of treatment conditions (F2, 162 = 5.191 P
= 0.0065).

3. Discussion
We evaluated the effects of OT and six lipidated OT analogues, LOT-4a-c and LOT-5a-c,
on a typical social behavior, male parental behavior (Table 2). The impairment in paternal
retrieval behavior displayed by male CD38KOCC mice induced by the CRISP-Cas9 gene-editing
method was identical to that reported in CD38KO mice generated by the classical homologous
recombination method.27 Thirty minutes after administering OT to CD38KOCC sires, OT elicited
recovery from the paternal behavior deficit exhibited by PBS-treated mice. Five lipidated
analogues, LOT-4a, LOT4b, LOT-5a, LOT-5b, and LOT-5c, exhibited essentially no recovery
of function, whereas another analogue, LOT4c, effectively reversed the behavioral deficit. LOT-
4c exhibited effects on parental behavior 24 h after injection (Figure 2). The use of novel
indicators of other social behaviors evaluated in our previous reports is needed to confirm the
benefit of the OT analogue LOT-4c over natural and/or synthetic OT as a long-acting analogue.
In the present study, we tested the analogues in only one social behavioral test, in contrast to
our previous papers. Nevertheless, we applied two different scales to measure paternal nurturing
behavior. We measured the parental score which reflects paternal behavior for all five pups and
the quality of the retrieval behavior. The current judgment on the basis of both retrieval latency
and parental score allows for the evaluation of OT, LOT-4, and LOT-5 analogues in the parental
behavior test.
The mechanism underlying the activity of LOT-4a to exhibit OT-like in vivo effects for a
short-time period (30 min after administration) is unclear. One possible mechanism may be its
high permeability through the blood–brain barrier due to lipidation,30 although permeability was
not examined in the present study.
A limitation of our study of LOT-4c is the absence of data other than the effects at 30 min
and 24 h for recovery of parental behavior in CD38KOCC mice. Experiments with chronic
treatment should be performed. On the basis of the beneficial effects of LOT-4a in short-term

activity and LOT-4c in long-term activity experiments, a combination of the two LOTs may be
advantageous to improve the behavior. In addition, we do not know where and when the
lipidated moieties are removed from LOTs and release OT that can be easily transported to the
brain by the receptor for advanced glycation end-products (RAGE), because RAGE is a binding
partner and carrier of OT on brain capillary endothelial cell membranes.31,32 Therefore, LOTs
could serve as prodrugs after cleaving the lipidated moieties, even at the peripheral and central
sites,33 where an intensified OT-induced OT release autoregulation mightoccur.34
4. Conclusion
We identified a potent lipidated OT analogue, LOT-4c having a C16-chain via a carbonate
linkage at the phenolic hydroxyl group of Tyr-2, which showed very long-lasting recovery of
parenting behavior in CD38KOCC mice, a rodent model of an autistic phenotype. Although the
precise mechanism is unknown, LOT-4c is likely to be a prodrug that slowly releases OT in the
bloodstream or in the brain. Based on these results together with previous findings, we conclude
that some LOTs are beneficial over native OT and potentially useful candidates for the treatment
of psychiatric diseases, including ASD.20-22
5.
Experimental Section
5.1 Compound Synthesis
1
5.1.1 General Methods of Chemistry. H-NMR spectra were recorded in CDCl3 at ambient
13
temperature unless otherwise noted, at 400 or 500 MHz, with TMS as an internal standard. C
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 of final compounds were
confirmed as >95% by HPLC: column, Kanto C18 GP, 250 × 4.6 (mm); column temperature,

25°C; flow rate, 1.0 mL/min; solvent, MeOH/ H2O = 95:5 containing 0.1% TFA; detection, 220
nm.
5.1.2 N-Palmitoyloxytocin (LOT-2). To a solution of oxytocin acetate (12.6 mg, 12.5 µmol)
and Et3N (5.2 µL, 37 µmol) in DMF (0.25 mL) was added a solution of Pal2O (6.2 mg, 12.5
µmol) in CH2Cl2 (0.80 mL), and the reaction mixture was stirred for 3 h. After the solvent was
evaporated, the residue was purified by silica gel column chromatography (MeOH/ CHCl3 = 1:5)
1
to give the title compound (14.9 mg, 12.0 µmol, 96%) as white powder: H NMR (500 MHz,
DMSO-d6)  9.13 (s, 2H), 8.62 (br, 1H), 8.22-8.08 (m, 4H), 7.92 (m, 2H), 7.62 (br, 1H), 7.35 (s,
1H), 7.28 (s, 1H), 7.09-7.05 (m, 4H), 6.88 (s, 1H), 6.79 (s, 1H), 6.61 (d, 2H, J = 8.0 Hz), 4.95
(br, 1H), 4.69 (m, 1H), 4.55 (m, 1H), 4.38 (m, 1H), 4.30 (m, 1H), 4.16 (m, 1H), 3.95 (br, 1H),
3.88 (br, 1H), 3.64-3.52 (m, 1H), 3.28 (m, 1H), 3.13 (d, J = 12 Hz, 1H), 3.05-2.97 (m, 2H), 2.76-
2.66 (m, 2H), 2.62-2.52 (m, 2H), 2.11 (t, J = 7.5 Hz, 2H), 2.03 (t, J = 7.0 Hz, 1H), 2.01-1.71 (m,
1
6H), 1.62 (m, 1H), 1.57-1.40 (m, 5H), 1.31-1.12 (m, 25H), 0.90-0.80 (m, 15H). The H NMR
data are in accord with those reported previously.20
5.1.3 Alkyl (4-nitrophenyl) carbonate (1a-c). To a solution of an alcohol (CH3(CH2)nOH, n =
13, 14, 15, 0.50 mmol) in CH2Cl2 (5.0 mL) was added p-nitrophenyl chloroformate (101 mg,
0.50 mmol) and pyridine (81 µL, 1.0 mmol) at 0 °C, and the reaction mixture was stirred at room
temperature for 11 h. The mixture was partitioned between hexane and H2O, and the organic
layer was dried (Na2SO4) and evaporated. The residue was purified by silica gel column
chromatography (EtOAc/ hexane = 1:50) to give the title compound as white powder. 1a (163
1
mg, 0.429 mmol, 86%): H NMR (400 MHz, CDCl3)  8.28 (d, J = 9.6 Hz, 2H, aromatic), 7.39
(d, J = 9.6 Hz, 2H, aromatic), 4.29 (t, J = 6.8 Hz, 2H, OCH2), 1.76 (m, 2H, OCH2CH2), 1.42 (m,
2H, OCH2CH2CH2), 1.22-1.38 (m, 20H, CH2×10), 0.88 (t, J = 6.8 Hz, 2H, CH3); 13C NMR
(CDCl3, 100 MHz)  155.6, 152.5, 145.3, 125,3, 121.8, 69.7, 31.9, 29.7, 29.6, 29.6, 29.6, 29.5,
1
29.4, 29.4, 29.2, 28.5, 25.6, 22.7, 14.1. 1b (72.6 mg, 0.184 mmol, 37%): H NMR (400 MHz,
CDCl3)  8.28 (d, J = 9.6 Hz, 2H, aromatic), 7.39 (d, J = 9.6 Hz, 2H, aromatic), 4.29 (t, J = 6.8

Hz, 2H, OCH2), 1.76 (m, 2H, OCH2CH2), 1.42 (m, 2H, OCH2CH2CH2), 1.22-1.38 (m, 22H,
13
CH2×11), 0.88 (t, J = 6.8 Hz, 2H, CH3); C NMR (CDCl3, 100 MHz)  155.6, 152.5, 145.3,
125,3, 121.8, 69.7, 31.9, 29.7, 29.7, 29.6, 29.6, 29.5, 29.4, 29.3, 29.2, 28.5, 25.6, 22.7, 14.1. 1c
(163 mg, 0.400 mmol, 80%): 1H NMR (400 MHz, CDCl3)  8.28 (d, J = 9.2 Hz, 2H, aromatic),
7.39 (d, J = 9.2 Hz, 2H, aromatic), 4.29 (t, J = 6.8 Hz, 2H, OCH2), 1.76 (m, 2H, OCH2CH2), 1.42
13
(m, 2H, OCH2CH2CH2), 1.21-1.38 (m, 24H, CH2×12), 0.88 (t, J = 6.8 Hz, 2H, CH3); C NMR
(CDCl3, 100 MHz)  155.6, 152.5, 145.3, 125,3, 121.8, 69.7, 31.9, 29.7, 29.6, 29.6, 29.5, 29.4,
29.3, 29.2, 28.5, 25.6, 22.7, 14.1.
5.1.4 LOT-4a–c. To a solution of LOT-2 (12.5 mg, 10.0 µmol) and Et3N (4.2 µL, 30.1 µmol)
and DMAP (1.2 mg, 10 µmol) in DMF (0.25 mL) was added a solution of alkyl (4-nitrophenyl)
carbonate (1a–c, 20 µmol) in CH2Cl2 (0.25 mL), and the reaction mixture was stirred at room
temperature for 24 h. After evaporation of the solvent, the residue was purified by HPLC
(column, Kanto RP-8 GP150-4.6; 0.1% TFA-containing MeOH／ H2O (95: 5); 220 nm; 1.00
mL/min; 25 °C). The eluent was evaporated and lyophilized to give the title compound as white
powder. LOT-4a (9.7 mg, 65%): 1H NMR (500 MHz, DMSO-d6)  8.80 (br, 1H), 8.36-8.17 (m,
3H), 8.11 (d, 1H, J = 9.0 Hz), 7.93 (m, 2H), 7.47 (br, 1H), 7.41-7.26 (m, 4H), 7.10-7.04 (m, 4H),
6.87 (s, 1H), 6.80 (s, 1H), 5.04 (br, 1H), 4.69 (m, 2H), 4.40 (m, 1H), 4.31 (d, J = 7.5 Hz, 1H),
4.15 (m, 3H), 3.97 (br, 1H), 3.84 (br, 1H), 3.65-3.51 (m, 5H), 3.38-3.27 (m, 2H), 3.02-2.80 (m,
3H), 2.65-2.54 (m, 3H), 2.10 (t, J = 7.5 Hz, 2H), 1.96 (m, 4H), 1.93-1.71 (m, 5H), 1.64-1.52 (m,
3H), 1.49-1.38 (m, 4H), 1.31-1.14 (m, 47H), 0.92-0.78 (m, 18H); LRMS (ESI) m/z 1507.86
[(M+Na)+]; HRMS (ESI) calcd for C74H124N12O15S2Na: 1507.8643 [(M+Na)+], found:
1
1507.8615. LOT-4b (13.1 mg, 87%): H NMR (500 MHz, DMSO-d6)  8.78 (br, 1H), 8.36-
8.17 (m, 3H), 8.10 (d, 1H, J = 9.0 Hz), 7.93 (m, 2H), 7.47 (br, 1H), 7.40-7.24 (m, 4H), 7.09-7.04
(m, 4H), 6.87 (s, 1H), 6.80 (s, 1H), 5.03 (br, 1H), 4.69 (m, 2H), 4.39 (m, 1H), 4.31 (d, J = 8.0
Hz, 1H), 4.16 (m, 3H), 3.96 (br, 1H), 3.84 (br, 1H), 3.70-3.56 (m, 5H), 3.36-3.24 (m, 2H), 3.05-
2.82 (m, 3H), 2.67-2.54 (m, 3H), 2.11 (t, J = 7.0 Hz, 2H), 1.96 (m, 4H), 1.92-1.71 (m, 5H), 1.68-

1.52 (m, 3H), 1.52-1.38 (m, 4H), 1.38-1.14 (m, 49H), 0.92-0.78 (m, 18H); LRMS (ESI) m/z
1521.88 [(M+Na)+]; HRMS (ESI) calcd for C75H126N12O15S2Na: 1521.8799 [(M+Na)+], found:
1
1522.8769. LOT-4c (12.3 mg, 81%): H NMR (500 MHz, DMSO-d6)  8.79 (br, 1H), 8.32-
8.17 (m, 3H), 8.11 (d, 1H, J = 8.5 Hz), 7.93 (m, 2H), 7.47 (br, 1H), 7.40-7.27 (m, 4H), 7.10-7.04
(m, 4H), 6.87 (s, 1H), 6.80 (s, 1H), 5.04 (br, 1H), 4.69 (m, 2H), 4.40 (m, 1H), 4.31 (d, J = 8.0
Hz, 1H), 4.15 (m, 3H), 3.96 (br, 1H), 3.84 (br, 1H), 3.66-3.53 (m, 5H), 3.36-3.26 (m, 2H), 3.05-
2.82 (m, 3H), 2.68-2.56 (m, 3H), 2.10 (t, J = 7.5 Hz, 2H), 1.96 (m, 4H), 1.95-1.73 (m, 5H), 1.70-
1.53 (m, 3H), 1.51-1.38 (m, 4H), 1.38-1.14 (m, 51H), 0.92-0.78 (m, 18H); LRMS (ESI) m/z
1535.89 [(M+Na)+]; HRMS (ESI) calcd for C76H128N12O15S2Na: 1535.8956 [(M+Na)+], found:
1535.8941.
5.1.5 Alkylcarbamic chloride (2a–c). To a solution of triphosgene (bis(trichloromethyl)
carbonate, 74.2 mg, 0.25 mmol) in CH2Cl2 (1.0 mL) was added an amine (0.50 mmol) and Et3N
(112 µL, 0.80 mmol) in CH2Cl2 (3.0 mL) at −20 °C, and the reaction mixture was stirred at room
temperature for 3 h. The mixture was filtrated with silica gel and the filtrate was partitioned with
saturated aqueous NH4Cl, H2O, and brine. The organic layer was dried (Na2SO4) and evaporated
to give the corresponding alkylcarbamic chloride 2a-c as an oil, respectively, which were
unstable and therefore immediately subjected to the next reaction without further purification.
5.1.6 LOT-5a–c. To a solution of LOT-2 (12.5 mg, 10 µmol) and Et3N (4.2 µL, 30 µmol) and
DMAP (1.2 mg, 10 µmol) in DMF (0.25 mL) was added a solution of alkylcarbamic chloride
(2a-c, 20 µmol) in CH2Cl2 (0.25 mL), and the reaction mixture was stirred at room temperature
for 1.5 h. After evaporation of the solvent, the residue was purified by HPLC (column, Kanto
RP-8 GP150-4.6; 0.1%TFA-containing MeOH/H2O (95:5); 220 nm; 1.0 mL/min; 25 °C). The
eluent was evaporated and lyophilized to give the title compound as white powder. LOT-5a
(13.5 mg, 9.09 µmol, 91%): 1H NMR (500 MHz, DMSO-d6)  8.79 (br, 1H), 8.31-8.19 (m, 3H),
8.12 (d, 1H, J = 8.5 Hz), 7.93 (m, 2H), 7.65 (t, J = 6.0 Hz, 1H), 7.48 (br, 1H),

7.36 (s, 1H), 7.28 (m, 4H), 7.12-7.04 (m, 2H), 6.97-6.79 (m, 4H), 5.05 (br, 1H), 4.73-4.62 (m,
2H), 4.39 (m, 1H), 4.31 (d, J = 8.0 Hz, 1H), 4.16 (m, 1H), 3.96 (br, 1H), 3.84 (br, 1H), 3.66-3.53
(m, 5H), 3.33-3.28 (m, 2H), 3.04-2.80 (m, 5H), 2.71-2.55 (m, 3H), 2.11 (t, J = 7.5 Hz, 2H), 2.07-
1.72 (m, 9H), 1.69-1.41 (m, 8H), 1.38-1.10 (m, 45H), 0.92-0.78 (m, 18H); LRMS (ESI) m/z
1506.88 [(M+Na)+]; HRMS (ESI) calcd for C74H125N13O14S2Na: 1506.8803 [(M+Na)+], found:
1506.8774. LOT-5b (8.2 mg, 55%): 1H NMR (500 MHz, DMSO-d6)  8.77 (br, 1H), 8.31-8.19
(m, 3H), 8.12 (d, 1H, J = 8.5 Hz), 7.92 (m, 2H), 7.65 (t, J = 6.0 Hz, 1H), 7.49 (br, 1H), 7.36 (s,
1H), 7.28 (m, 4H), 7.10-7.04 (m, 2H), 6.94-6.78 (m, 4H), 5.04 (br, 1H), 4.75-4.62 (m, 2H), 4.39
(m, 1H), 4.31 (d, J = 8.0 Hz, 1H), 4.16 (m, 1H), 3.97 (br, 1H), 3.84 (br, 1H), 3.65-3.53 (m, 5H),
3.33-3.26 (m, 2H), 3.03-2.79 (m, 5H), 2.68-2.56 (m, 3H), 2.11 (t, J = 7.0 Hz, 2H), 2.05-1.74 (m,
9H), 1.69-1.41 (m, 8H), 1.32-1.10 (m, 47H), 0.92-0.78 (m, 18H); LRMS (ESI) m/z 1520.89
[(M+Na)+]; HRMS (ESI) calcd for C75H127N13O14S2Na: 1520.8959 [(M+Na)+], found:
1
1520.8940. LOT-5c (10.1 mg, 6.68 µmol, 53%): H NMR (500 MHz, DMSO-d6)  8.78 (br,
1H), 8.29-8.19 (m, 3H), 8.12 (d, 1H, J = 8.5 Hz), 7.92 (m, 2H), 7.65 (t, J = 5.5 Hz, 1H), 7.49 (br,
1H), 7.36 (s, 1H), 7.28 (m, 4H), 7.10-7.06 (m, 2H), 6.94-6.78 (m, 4H), 5.05 (br, 1H), 4.76-4.64
(m, 2H), 4.39 (m, 1H), 4.31 (d, J = 8.0 Hz, 1H), 4.16 (m, 1H), 3.96 (br, 1H), 3.84 (br, 1H), 3.68-
3.52 (m, 5H), 3.34-3.28 (m, 2H), 3.02-2.81 (m, 5H), 2.69-2.56 (m, 3H), 2.11 (t, J = 7.5 Hz, 2H),
2.02-1.76 (m, 9H), 1.69-1.40 (m, 8H), 1.30-1.11 (m, 49H), 0.92-0.78 (m, 18H); LRMS (ESI) m/z
1534.91 [(M+Na)+]; HRMS (ESI) calcd for C76H129N13O14S2Na: 1534.9116 [(M+Na)+], found:
1534.9098.
5.2 Expression Plasmid Construction, Cell Culture and Transfection.
Plasmid constructs for hOTRs (pcDNAHOXTR)20, pcDNAHV1AR and pcDNAHV1BR24
were described previously.
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. Cells were grown in culture dishes to 80 to 90% confluence

and transfected with pcDNAHOXTR, pcDNAHV1AR or pcDNAHV1BR using GeneJuice
Transfection Reagent (EMD Millipore, Temecula, CA, USA) following the manufacturer’s
instruction. Cells were selected in DMEM supplemented with 5% FBS and 800 µg/mL G418
(geneticin; Sigma Chemical Co., St Louis, MO, USA). The resulting transformed cells, stably
expressing human OXTRs, human V1ARs and V1BRs and those transfected with pcDNA3 (+)
(mock-transfected cells)20 were maintained in DMEM supplemented with 10% FBS and 100
µg/mL G418.
5.3 Intracellular free calcium concentration measurement.
Transfected HEK-293 cells were loaded with fura-2/AM to a final concentration of 1 µmol/mL
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 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L
MgCl2, 20 mmol/L HEPES-NaOH, 2 mmol/L CaCl2, 20 mmol/L glucose, 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 sec for 5 min after application of PBS, OT, AVP or analogues. The ratio of
fluorescence at 340 nm and 380 nm (F340/F380) was used to determine intracellular free
calcium concentrations ([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, AVP for hV1AR and hV1BRs. OT, AVP and analogues for
experiments were diluted in 50% ethanol to a concentration of 10-3 mol/L and then diluted in
distilled water to obtain the required concentrations. Each experiment was performed 3 times,
each triplicated.
5.4 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 CD38KOCC was described previously24. 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).
5.5 Parental Retrieval Test.
The design of the experiments for parental retrieval behavior was described previously21, 24
.
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
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 phosphate-buffered saline (PBS) or 0.3

mL of OT (1 mL per 100 g of body weight), compounds at a concentration of 100 ng/mL
dissolved in PBS.
For screening experiments parental behavior was examined. Thirty minutes and 24 h after the
injection, only once. Paternal retrieval behavior was examined by measuring parental scores for
each pup. Each sire or received 2 points for each pup returned completely to the nest, 1 point if
the parent had contact with the pup or moved the pup to another place, and 0 if the pup was
intact.
For time-course experiment 30 min, 6, 12, 24 or 48 h after the injection, 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 and 24 h in screening experiment. If ANOVA- test shown significance
followed Bonferroni’s post-hoc test was performed. Two-way ANOVA followed by
Bonferroni’s post hoc test was performed for time-course.

REFERENCES
1. Gimpl, G.; Fahrenholz, F. The oxytocin receptor system: structure, function, and
regulation. Physiol Rev. 2001; 81:, 629–683.
2. Kirsch, P. Oxytocin in the socioemotional brain: implications for psychiatric disorders.
Dialogues Clin. Neurosci. 2015; 17: 463–476.
3. Ludwig, M.; Leng, G. Dendritic peptide release and peptide-dependent behaviours. Nat.
Rev. Neurosci. 2006; 7: 126–136
4. Bealer, S. L.; Armstrong, W. E.; Crowley, W. R. Oxytocin release in magnocellular
nuclei: neurochemical mediators and functional significance during gestation. Am. J.
Physio.l Regul. Integr. Comp. Physiol. 2010; 299: 452–458.
5. Weeks, A. The prevention and treatment of postpartum haemorrhage: what do we know,
and where do we go to next?. BJOG: An International Journal of Obstetrics and
Gynaecology. 2014; 122: 202–210.
6. Marlin, B. J.; Froemke, R. C. Oxytocin modulation of neural circuits for social behavior.
Dev. Neurobiol. 2017; 77:169-189.
7. Neumann, I. D. Brain oxytocin: A key regulator of emotional and social behaviours in
both females and males. J. Neuroendocrinol. 2008; 20: 858–865.
8. Kosfeld, M.; Heinrichs, M.; Zak, P. J.; Fischbacher, U.; Fehr, E. Oxytocin increases trust
in humans. Nature 2005; 435: 673–676
9. Hirosawa, T.; Kikuchi, M.; Okumura, E.; Yoshimura, Y.; Hiraishi, H.; Munesue, T.;
Takesaki, N.; Furutani, N.; Ono, Y.; Higashida, H.; Minabe, Y. Attentional control and
interpretation of facial expression after oxytocin administration to typically developed
male adults. PLoS ONE 2015; 10: e0116918.
10. Kim, S.; Fonagy, P.; Koos, O.; Dorsett, K.; Strathearn, L. Maternal oxytocin response
predicts mother-to-infant gaze. Brain Res. 2014; 1580: 133–142.

11. Lai, M. -C.; Lombardo, M. V. Baron-Cohen, S. Autism. Lancet 2014; 383: 896–910.
12. Hollander, E.; Bartz, J.;, Chaplin, W.; Phillips, A.; Sumner, J.; Soorya, L.; Anagnostou,
E.; Wasserman, S. Oxytocin increases retention of social cognition in autism. Biol
Psychiatry 2007; 61: 498–503
13. 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: article 2.
14. 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. Aca. Sci. U S A 2017;114: 8119–8124.
15. 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.
16. 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.
17. De Groot, A. N.; Vree, T. B.; Hekster, Y. A.; Pesman, G. J.; Sweep, F. C.; van Dongen, P.
J.; van Roosmalen, J. Bioavailability and pharmacokinetics of sublingual oxytocin in male
volunteers. J. Pharm. Pharmacol. 1995; 47: 571–575.
18. Schramme, A. R.; Pinto, C. R.; Davis, J.; Whisnant, C. S.; Whitacre, M. D.
Pharmacokinetics of carbetocin, a long-acting oxytocin analogue, following intravenous
administration in horses. Equine Vet. J. 2008; 40: 658–661.

19. Varamini, P.; Toth, I. Lipid- and sugar-modified endorphins 4. Novel targets for the
treatment of neuropathic pain. Front. Pharmacol. 2013; 4: Article 155
20. Mizuno, A.; Cherepanov, S. M.; 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.
21. 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 cerebrospinal fluid in mice.
Pharm. Res. Per. 2017; 5: e00290.
22. Cherepanov, S. M.; Akther, S.; Nishimura T.; Shabalova, A. 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: pii:
E132
23. 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.
24. Ichinose, W.; Cherepanov S. M., Shabalova, A. A.; Yokoyama, S.; Yuhi, T.; Yamaguchi,
H.; Watanabe A.; Yamamoto, Y.; Okamoto, H.; Horike, S.; Terakawa, J.; Daikoku, T.;
Watanabe, M.; Mano, N.; Higashida, H.; Shuto, S. Development of a highly potent

analogue and a long-acting analogue of oxytocin for the treatment of social impairment-
like behaviors. J. Med. Chem. 2019; 62; 3297-3310.
25. 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; 113: 42–48.
26. 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.; Tsuji,
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.
27. Kato, I.; Yamamoto, Y.; Fujiwara, M.; Noguchi, N.; Takasawa, S.; Okamoto, H. CD38
disruption impairs glucose-induced increases in cyclic ADP-ribose, [Ca2+], and insulin
secretion. J. Biol. Chem. 1999; 274: 1869–1872.
28. 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.
29. 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.
30. Egleton, R. D.; Davis, R. D. Development of neuropeptide drugs that cross the blood-
brain barrier. NeuroRx. 2005; 2: 44–53
31. Higashida, H.; Furuhara, K.; Yamauchi, A.M; Deguchi, K.; Harashima, A.; Munesue, S.;
Lopatina, O.; Gerasimenko, M.; Salmina, A.B.; Zhang. J.S.; Kodama, H.; Kuroda, H.;
Tsuji, C.; Shuto, S.; Yamamoto, H.; Yamamoto, Y. Intestinal transepithelial permeability
of oxytocin into the blood is dependent on the receptor for advanced glycation end
products in mice. Sci Rep. 2017; 7: 7883.
32. Yamamoto, Y.; Liang, M.; Munesue, S.; Deguchi, K.; Harashima, A.; Furuhara, K.; Yuhi,
T.; Zhong, J.; Akther, S.; Goto, H.; Eguchi, Y.; Kitao, Y.; Hori, O.; Shiraishi, Y.; Ozaki,
N.; Shimizu, Y.; Kamide, T.; Yoshikawa, A. Hayashi, Y.; Nakada, M.; Lopatina, O.;
Gerasimenko, M.; Komleva, Y.; Malinovskaya, N.; Salmina, A. B.; Asano, M.;
Nishimori, K.; Shoelson, S. E.; Yamamoto, H.; Higashida, H. Vascular RAGE transports
oxytocin into the brain to elicit its maternal bonding behaviour in mice. Commun Biol.
2019; 2: 76.
33. From the structure–activity relationship viewpoint, it is strange that only LOT-4a had
agonistic effect to hOTR (Table 1), because the structure of LOT-4a is very similar to
those of other LOT-4b,c and LOT-5a-c. A possibility that only a trace of OT
contaminating to LOT-4a sample might affect the agonistic effect in vitro due to the
remarkably strong agonistic potency of OT (EC50 = 4.6 pM) might not be eliminated.
34. Zhong, J.; Amina, S.; Liang, M.; Akther, S.; Yuhi, T.; Nishimura, T.; Tsuji, C.; Tsuji, T.;
Liu, H. X.; Hashii, M.; Furuhara, K.; Yokoyama, S.; Yamamoto, Y.; Okamoto, H.; Zhao,
Y. J.; Lee H. C.; Tominaga, M.; Lopatina, O.; Higashida, H. Cyclic ADP-ribose and heat
regulate oxytocin release via CD38 and TRPM2 in the hypothalamus during social or
psychological stress in mice. Front. Neurosci. 2016; 10: 304.