Synthesis and evaluation of in vivo anti-hypothermic effect of all stereoisomers of the thyrotropin-releasing hormone mimetic: Rovatirelin Hydrate

Article abstract of DOI:10.1002/psc.3228

We discovered the orally active thyrotropin-releasing hormone (TRH) mimetic: (4S,5S)-5-methyl-N-{(2S)-1-[(2R)-2-methylpyrrolidin-1-yl]-1-oxo-3-(1,3-thiazol-4-yl)propan-2-yl}-2-oxo-1,3-oxazolidine-4-carboxamide 1 (rovatirelin). The central nervous system (CNS) effect of rovatirelin after intravenous (iv) administration is 100-fold higher than that of TRH. As 1 has four asymmetric carbons in its molecule, there are 16 stereoisomers. We synthesized and evaluated the anti-hypothermic effect of all stereoisomers of 1, which has the (4S),(5S),(2S),(2R) configuration from the N-terminus to the C-terminus, in order to clarify the structure?activity relationship (SAR) of stereoisomers. The (4R),(5R),(2R),(2S)-isomer 16 did not show any anti-hypothermic effect. Only the (4S),(5S),(2S),(2S)-isomer 10, which has the (2S)-2-methylpyrrolidine moiety at the C-terminus showed the anti-hypothermic effect similar to 1. Stereoisomers, which have the (5R) configuration of the oxazolidinone at the N-terminus and the (2R) configuration at the middle-part, showed a much lower anti-hypothermic effect than that of 1. On the other hand, stereoisomers, which have the (4R) configuration of the oxazolidinone at the N-terminus or the (2S) configuration of the C-terminus, have little influence on the anti-hypothermic effect.

Full text of DOI:10.1002/psc.3228

Received: 8 April 2019
DOI: 10.1002/psc.3228
Revised: 10 October 2019
Accepted: 15 October 2019
R E S E A R C H A R T I C L E
Synthesis and evaluation of in vivo anti‐hypothermic effect of
all stereoisomers of the thyrotropin‐releasing hormone
mimetic: Rovatirelin Hydrate
Naotake Kobayashi1
Norihito Sato2 Katsuji Sugita2 Kouji Takahashi3
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Tamio Sugawara1 Yukio Tada1 Takayoshi Yoshikawa4
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1 Medicinal chemistry research laboratory,
Shionogi & Co., Ltd. 3‐1‐1, Futaba‐cho,
Toyonaka‐shi, Osaka 561‐0825, Japan
We discovered the orally active thyrotropin‐releasing hormone (TRH) mimetic: (4S,5S)‐
5‐methyl‐N‐{(2S)‐1‐[(2R)‐2‐methylpyrrolidin‐1‐yl]‐1‐oxo‐3‐(1,3‐thiazol‐4‐yl)propan‐
2‐yl}‐2‐oxo‐1,3‐oxazolidine‐4‐carboxamide 1 (rovatirelin). The central nervous system
(CNS) effect of rovatirelin after intravenous (iv) administration is 100‐fold higher than
that of TRH. As 1 has four asymmetric carbons in its molecule, there are 16 stereoiso-
mers. We synthesized and evaluated the anti‐hypothermic effect of all stereoisomers of
1, which has the (4S),(5S),(2S),(2R) configuration from the N‐terminus to the C‐terminus,
in order to clarify the structure−activity relationship (SAR) of stereoisomers. The (4R),
(5R),(2R),(2S)‐isomer 16 did not show any anti‐hypothermic effect. Only the (4S),(5S),
(2S),(2S)‐isomer 10, which has the (2S)‐2‐methylpyrrolidine moiety at the C‐terminus
showed the anti‐hypothermic effect similar to 1. Stereoisomers, which have the (5R)
configuration of the oxazolidinone at the N‐terminus and the (2R) configuration at
the middle‐part, showed a much lower anti‐hypothermic effect than that of 1. On the
other hand, stereoisomers, which have the (4R) configuration of the oxazolidinone at
the N‐terminus or the (2S) configuration of the C‐terminus, have little influence on
the anti‐hypothermic effect.
2 Research Laboratory for Development,
Shionogi & Co., Ltd. 3‐1‐1, Futaba‐cho,
Toyonaka‐shi, Osaka 561‐0825, Japan
3 DMPK Services, Shionogi Techno Advance
Research Co., Ltd. 3‐1‐1, Futaba‐cho,
Toyonaka‐shi, Osaka 561‐0825, Japan
4 Pharmacovigilance Japan, Allergan Japan K.
K., 4‐20‐3‐35, Ebisu Shibuya‐ku, Tokyo 150‐
6035, Japan
Correspondence
Naotake Kobayashi, Shionogi Pharmaceutical
Research Center 3‐1‐1, Futaba‐cho, Toyonaka‐
shi, Osaka 561‐0825, Japan.
Email: naotake.kobayashi@shionogi.co.jp
KEYWORDS
TRH, TRH mimetic, rovatirelin, stereoisomers, anti‐hypothermic effect, intravenous administration
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1
INTRODUCTION
phase III clinical trial (NCT02889302) for the treatment of
spinocerebellar degeneration (SCD).1-5
We previously reported the structure−activity relationship (SAR) stud-
ies of thyrotropin‐releasing hormone (TRH) mimetic. We evaluated the
anti‐hypothermic effect, physicochemical properties, pharmacokinetic
(PK) properties of each compound and finally, we discovered the orally
effective TRH mimetic, rovatirelin hydrate. Rovatirelin hydrate is in a
TRH is a neuropeptide composed of three amino acids. The chem-
ical structure of TRH was revealed to be L‐pyroglutamyl−L‐histidyl−L‐
prolinamide (L‐pGlu−L‐His−L‐Pro‐NH2; Figure 1).6-8 TRH indicates
mainly two biological effects via the central nervous system (CNS)
and endocrine system. The effects on CNS are expressed by the action
Abbreviations: TRH, thyrotropin‐releasing hormone; CNS, central nervous system; SCD, spinocerebellar degeneration; TSH, thyroid stimulating hormone; SAR, structure−activity relationship; PK,
pharmacokinetic; CPPs, CNS‐permeable prodrugs; BBB, blood‐brain barrier; iv, intravenous; MeOH, methanol; EtOH, ethanol; THF, tetrahydrofuran; DMSO, dimethylsulfoxide; DMF, N,N’‐
dimethylformamide; Et3N, triethylamine; TFA, trifluoroacetic acid; TsOH·H2O, p‐toluenesulfonic acid monohydrate; TsOH, p‐toluenesulfonic acid; Ph2CHN2, diphenyldiazomethane; Ac2O,
acetic anhydride; Cbz, benzyloxycarbonyl; Boc, tert‐butoxycarbonyl; Ms, methanesulfony; Bz, benzoyl; DCC, 1,3‐dicyclohexylcarbodiimide; HOBt, N‐hydroxybenzotriazole; HOSu, N‐
hydroxysuccinimide; TLC, thin‐layer chromatography
J Pep Sci. 2019;e3228.
wileyonlinelibrary.com/journal/psc
© 2019 European Peptide Society and John Wiley & Sons, Ltd.
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https://doi.org/10.1002/psc.3228

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KOBAYASHI ET AL.
FIGURE 1 Chemical structure of TRH, taltirelin hydrate, rovatirelin hydrate, NP‐647 and CPPs.
of TRH as a neurotransmitter or a neuromodulator.9-11 The effects on
the endocrine system are expressed by stimulating the secretion of
thyroid stimulating hormone (TSH) and prolactin from the anterior
pituitary.12 The effects on CNS of TRH have been applied the treat-
ment of CNS disorders in clinical setting.13,14 Prior to 2000, a large
number of TRH analogues have been reported.15-40 In particular,
taltirelin hydrate (Figure 1) has high CNS effects separate from endo-
crine effects in order to avoid side effects caused by TSH releasing.
Around the same time, we discovered rovatirelin hydrate (Figure 1).
Two subtypes of TRH receptor (TRH receptor type 1: TRH‐R141
and TRH receptor type 2: TRH‐R242,43) are reported. TRH‐R1 and
TRH‐R2 are relevant to endocrine effects and CNS effects, respec-
tively.44 In human, it has been reported that a single type of TRH
receptor is expressed and revealed the activities similar to TRH‐R1.45
After 2000, TRH analogue NP‐647 (Figure 1), which bound selectively
to the TRH receptor subtype, have been reported and showed only
CNS effects.46-50 Another line of research has been the application
of a prodrug approach toTRH analogues, for example, CNS‐permeable
prodrugs (CPPs) (Figure 1) have been synthesized in order to increase
the penetration of biological membranes including the blood‐brain
barrier (BBB), and successively obtained a high CNS effect.51-55
Regarding the biological activities of the stereoisomers of TRH and
TRH analogues, the configurations of the N‐terminus and the C‐
terminus were found to be important for the biological activities
resulting from evaluation of the TSH releasing activity or CNS
effect.15-19,56
(Figure 1), in order to clarify the SAR of stereoisomers by evaluating
the anti‐hypothermic effect. The chemical structures and their abso-
lute configurations of TRH mimetics 1−16 are shown in Table 1.
In this paper, we report the synthesis of TRH mimetics 1−16 and
the evaluation of their anti‐hypothermic effect, which is one of the
CNS effects of TRH, based on their reserpine‐induced hypothermia
in mice.15-19
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2
RESULTS AND DISCUSSION
Chemistry
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2.1
TRH mimetics 1−16 were synthesized by combining four N‐terminus
fragments (17−20), two middle‐part fragments (21, 22) and two C‐
terminus fragments (23, 24) (Figure 2).
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2.2
N‐Terminus fragments
Four N‐terminus fragments 17−20 were synthesized from readily
available L‐threonine 25 or D‐threonine 26. Among them, 19 and 20
were synthesized via allo‐L‐threonine hydrochloride 27 and allo‐D‐
threonine hydrochloride 28, respectively based on previous reports
(Scheme 1).57-61 The detailed synthetic method, yields and physical
data of all intermediates are shown in the Supporting Information.
Reagents and conditions: (a) CbzCl, NaHCO3, 1,4‐dioxane‐H2O, 0 °
C to rt; (b) (1) aq. NaOH, MeOH; (2) aq. HCl, MeOH, 29−71%; (c) (1)
SOCl2, MeOH, 0 °C; (2) BzCl, NaHCO3, 1,4‐dioxane‐H2O, 0 °C; (d)
SOCl2, 0 °C; (e) aq. HCl, reflux, 60−71%.
As rovatirelin: (4S,5S)‐5‐methyl‐N‐{(2S)‐1‐[(2R)‐2‐methylpyrrolidin‐
1‐yl]‐1‐oxo‐3‐(1,3‐thiazol‐4‐yl)propan‐2‐yl}‐2‐oxo‐1,3‐oxazolidine‐4‐
carboxamide 1 has four asymmetric carbons in its molecule, we
hypothesized that the existing stereochemistry of 1 was essential to
show the highest anti‐hypothermic effect and other stereoisomers
would not show the anti‐hypothermic effect compare with 1.
Then, we synthesized all stereoisomers of 1, which has the (4S),
(5S),(2S),(2R) configurations from the N‐terminus to the C‐terminus
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2.3
Middle‐part fragments
Two middle‐part fragments 21 and 22 were synthesized from (2S)‐2‐
amino‐3‐(thiazol‐4‐yl)propanoic acid 30 and (2R)‐2‐amino‐3‐(thiazol‐

KOBAYASHI ET AL.
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TABLE 1 Chemical structures and anti‐hypothermic effect of TRH, TRH mimetics 1−16 after iv administration to reserpine‐induced hypothermia
in mice. The absolute configurations are shown near the asymmetric carbons of the chemical structures.
compounds
TRH
chemical structures and absolute configurations
dose (iv) (μmol/kg)
ΔAUC0−7h (°C·h)
50
35.4 3.7
1
0.50
0.50
0.50
0.50
0.50
45.7 0.1
7.8 2.8
−1.8 1.6
6.0
(rovatirelin)
2
3
4
5
1.6 3.8
(Continues)

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KOBAYASHI ET AL.
TABLE 1 (Continued)
compounds
6
chemical structures and absolute configurations
dose (iv) (μmol/kg)
ΔAUC0−7h (°C·h)
0.50
11.1 16.4
7
8
9
0.50
0.50
0.50
9.0 2.5
14.4 11.0
7.2 9.6
10
0.50
39.1 4.5
11
0.50
6.8 6.1
(Continues)

KOBAYASHI ET AL.
5 of 16
TABLE 1 (Continued)
compounds
12
chemical structures and absolute configurations
dose (iv) (μmol/kg)
ΔAUC0−7h (°C·h)
0.50
23.4 2.5
13
0.50
17.7 2.3
14
15
16
0.50
0.50
0.50
22.8 0.9
9.2 0.4
−3.2 0.6
Each value represents the mean SD of at least three animals (except for two animals for compound 4).
The formula for computation is as follows:
AUC0−7h: area under the rectal temperature‐time curve for 7 h,
ΔAUC0−7h = ΔAUC0−7h of rectal temperature (compound) − ΔAUC0−7h of rectal temperature (saline).
Anti‐hypothermic effect: administration dose (50 or 0.50 μmol/kg) × 7 h/ΔAUC0−7h
.
The detailed calculation method is shown in the Supporting Information.

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KOBAYASHI ET AL.
4‐yl)propanoic acid 31, respectively. Optically pure amino acids 30 and
31 were synthesized from 2‐amino‐3‐(thiazol‐4‐yl)propanoic acid
dihydrochloride 29 based on previous reports.62-66 Treatment of the
amino acids 30 and 31 with p‐toluenesulfonic acid monohydrate
(TsOH·H2O) afforded amino acid p‐toluenesulfonate 32 and 33, which
were not isolated, respectively. The p‐toluenesulfonate 32 and 33
were esterified with diphenyldiazomethane (Ph2CHN2) to give the
middle‐part fragments 21 and 22, respectively (Scheme 2). The
detailed synthetic method, yields and physical data of all intermediates
are shown in the Supporting Information.
of the benzhydryl group of dipeptide esters 38−45 with trifluoroacetic
acid (TFA) in anisole gave dipeptide mimetics 46−53, respectively.
Finally, the dipeptide mimetics 46−53 were coupled with two
C‐terminus fragments 23 and 24 using DCC in the presence of
N‐hydroxysuccinimide (HOSu) and triethylamine to afford TRH
mimetics 1−16, respectively. The detailed physical data of dipeptide
benzhydryl esters, dipeptide mimetics are shown in the Supporting
Information.
Reagents and conditions: (a) HOBt, DCC, Et3N, THF, 0 °C to rt,
68%−quant; (b) TFA, anisole, 0 °C, 56−98%; (c) HOSu, DCC, Et3N,
THF‐DMF, 0 °C to rt, 32−71%.
Reagents and conditions: (a) TsOH·H2O, H2O, not isolated; (b)
Ph2CHN2, MeOH, 0 °C, 97%.
Anti‐hypothermic effect of TRH mimetics 1−16
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2.4
C‐Terminus fragments
The anti‐hypothermic effect of TRH and TRH mimetics 1−16 (Table 1)
were evaluated by in vivo antagonistic effect on reserpine‐induced
hypothermia in mice.15-19 Reserpine depletes catecholamine and sero-
tonin in the brain and causes hypothermia. On the other hand, TRH
and TRH analogues promote catecholamine secretion against reser-
pine and stimulate the sympathetic nervous system. As a result, the
body temperature increases.25 The mice used for the study had rectal
temperatures of 30 °C or lower about 18 h after subcutaneous admin-
istration of reserpine (3 mg/kg). Rectal temperature was measured by
thermistor before and after intravenous (iv) administration of TRH and
TRH mimetics up to 7 h at a dose of 50 and 0.50 μmol/kg, respec-
tively. The anti‐hypothermic effect of all compounds were evaluated
based on the area under the temperature‐time curve after dosing
(AUC0−7h).4 Also, the anti‐hypothermic effect of each compound,
which were measured by administration doses (iv) and ΔAUC0−7h of
TRH, TRH mimetics and saline, are shown in Table 1. The rectal
temperature‐time curves of each compound are shown in the
Supporting Information.
Two
C‐terminus
fragments,
2‐(2R)‐methylpyrrolidine
p‐
toluenesulfonate 23 and 2‐(2S)‐methylpyrrolidine p‐toluenesulfonate
24 were synthesized via N‐Boc‐2‐(2R)‐methylpyrrolidine 36 and
N‐Boc‐2‐(2S)‐methylpyrrolidine 37 from readily available N‐Boc‐L‐pro-
line 34 and N‐Boc‐D‐proline 35 as starting materials (Scheme 3).67-69
The detailed synthetic method, yields and physical data of all interme-
diates are shown in the Supporting Information.
Reagents and conditions: (a) BH3‐THF complex, THF, 0 °C to rt; (b)
MsCl, Et3N, THF, 0 °C to rt; (c) LiEt3BH, THF, 0 °C to rt, 42−74%; (d)
(1) HCO2H, rt; (2) TsOH·H2O, quant.
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2.5
TRH mimetics 1−16
All fragments were coupled from the N‐terminus to the C‐terminus in
the presence of peptide coupling agents (Scheme 4).70 Four N‐
terminus fragments 17−20 were coupled with two middle‐part frag-
ments 21 and 22 using 1,3‐dicyclohexylcarbodiimide (DCC) in the
presence of N‐hydroxybenzotriazole (HOBt) and triethylamine to
afford dipeptide benzhydryl esters 38−45, respectively. Deprotection
Although most of the stereoisomers were inactive, only the (4S),
(5S),(2S),(2S)‐isomer 10 showed high anti‐hypothermic effect. Five
FIGURE 2 All fragments and their absolute configurations.

KOBAYASHI ET AL.
7 of 16
As for the C‐terminus, the (4S),(5S),(2S),(2S)‐isomer 10 showed the
highest anti‐hypothermic effect among the stereoisomers 2−16. The
stereoisomers showed similar anti‐hypothermic effect between
the (4R),(5S),(2S),(2R)‐isomer 6 and the (4R),(5S),(2S),(2S)‐isomer 7,
between the (4R),(5R),(2S),(2R)‐isomer 13 and the (4R),(5R),(2S),(2S)‐
isomer 14. On the other hand, the (4S),(5R),(2S),(2R)‐isomer 2 and
the (4R),(5R),(2R),(2R)‐isomer 15 showed the anti‐hypothermic effect
but the (4S),(5R),(2S),(2S)‐isomer 3 and the (4R),(5R),(2R),(2S)‐isomer
16 did not show the anti‐hypothermic effect at all.
SCHEME 1 Synthesis of N‐terminus fragments 17−20.
Therefore, the (4S),(5S),(2S),(2R)‐isomer 1 (rovatirelin) showed the
best anti‐hypothermic effect among the stereoisomers 1−16 and
was selected as the candidate for clinical trials.
stereoisomers 6, 8 and 12−14 showed about 2‐ to 5‐fold loss of anti‐
hypothermic effect compared with that of 1. On the other hand, six
stereoisomers 2, 4, 7, 9, 11 and 15 showed little anti‐hypothermic
effect. Moreover, three stereoisomers 3, 5 and 16 showed no anti‐
hypothermic effect.
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3
SUMMARY AND CONCLUSIONS
As for the N‐terminus, the (4R),(5S),(2S),(2R)‐isomer 6 showed 4‐
fold loss of the anti‐hypothermic effect compared with that of 1. On
the other hand, the (4S),(5R),(2S),(2R)‐isomer 2 showed approximately
6‐fold loss of the anti‐hypothermic effect compared with that of 1.
Moreover, the stereoisomers 6−9, which have the (4R),(5S) configura-
tions of oxazolidinone tended to show higher anti‐hypothermic effect
than that of the stereoisomers 2−5, which have the (4S),(5R) configu-
rations of oxazolidinone. The stereoisomers 10−12, which have (4S),
(5S) configurations and 13−15, which have (4R),(5R) configurations of
the oxazolidinone tended to show higher anti‐hypothermic effect than
that of 2−5, which have (4S),(5R) configurations, and 6−9, which have
(4R),(5S) configurations. It seemed that the (5R) configuration of
oxazolidinone strongly affects the anti‐hypothermic effect rather than
the (4R) configuration of oxazolidinone.
The 16 stereoisomers of 1 were synthesized from all stereoisomers of
four N‐terminus fragments, two middle‐part fragments and two
C‐terminus fragments. Four N‐terminus fragments 17, 19 and 18, 20
were synthesized from readily available L‐ or D‐threonine. Two
middle‐part fragments 21 and 22 were synthesized from unnatural
amino acids 3‐(thiazol‐4‐yl)‐L‐ and D‐alanine 25 and 27, respectively.
Two C‐terminus fragments 23 and 24 were synthesized from readily
available N‐Boc‐L‐ and N‐Boc‐D‐proline, respectively. All fragments
were coupled from the N‐terminus to the C‐terminus by the usual
peptide coupling method to afford TRH mimetics 1−16.
The in vivo anti‐hypothermic effect of all stereoisomers was eval-
uated based on reserpine‐induced hypothermia in mice. It seemed
that no significant relationship between stereochemistry and the
anti‐hypothermic effect was indicated from the results of SAR
studies. However, the absolute configuration of the 5‐position of
the oxazolidinone at the N‐terminus and the middle‐part seemed
to be important for display of the anti‐hypothermic effect. In
fact, stereoisomers 4, 5, 15 and 16, which have both the (5R) config-
uration of the oxazolidinone at the N‐terminus and the (2R)
As for the middle‐part, the (4S),(5S),(2R),(2R)‐isomer 11 showed
6‐fold loss of the anti‐hypothermic effect compared with 1. And the ste-
reoisomers, which have the (5R) configuration of oxazolidinone and (2R)
of the middle‐part, in particular, the (4S),(5R),(2R),(2R)‐isomer 4 and the
(4S),(5R),(2R),(2S)‐isomer 5, showed little anti‐hypothermic effect.
SCHEME 2 Synthesis of the middle‐part
fragments 21 and 22.
SCHEME 3 Synthesis of C‐terminus
fragments 23 and 24.

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KOBAYASHI ET AL.
SCHEME 4 General synthetic method of TRH mimetics 1−16.
configuration at the middle‐part showed much lower anti‐
hypothermic effect than that of 1. On the other hand, it seemed that
the (4R) configuration of the oxazolidinone at the N‐terminus or the
(2S) configuration of the C‐terminus has little influence on the anti‐
hypothermic effect.
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4
EXPERIMENTAL SECTION
General
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4.1
All solvents and reagents were obtained from commercial sources and
were used as received. Melting points (mp) were measured with a
Yanagimoto melting point apparatus and are uncorrected. 1H and
13C NMR spectra were taken with a Varian VXR‐200 or Gemini‐200
300 FT‐NMR spectrometer using tetramethylsilane as an internal
standard. Infrared spectra were recorded on a Nicolet 20SXB FT‐IR
spectrometer. Elemental analyses were performed by Shionogi
Although it was considered that the absolute configurations of
TRH and TRH analogues were important to show high biological activ-
ities18,56, our SAR result was incompatible with that of previous
reports. As a result of the SAR study, our hypothesis that the (4S),
(5S),(2S),(2R) stereochemistry of 1 was correct. Thus, this result could
be useful when designing new CNS effective compounds around
rovatirelin.
Research Laboratories (Shionogi
& Co., Ltd. 3‐1‐1, Futaba‐cho,

KOBAYASHI ET AL.
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Toyonaka‐shi, Osaka 561‐0825, Japan). Optical rotations were deter-
mined with a Perkin‐Elmer 430 polarimeter. For silica gel column chro-
matography, Kiesel gel 60 (0.063−0.20 mm, Merck) was employed for
the purification. For gel filtration chromatography, MCI GEL CHP‐20P
(75−150 gm, Mitsubishi Chemical Industries) was utilized with aqueous
MeOH as an eluent. Thin‐layer chromatography (TLC) was carried out
with Merck silica gel 603−254 plates mainly with use of the following
two solvent systems: (a) CHCl3/MeOH (9/1), (b) CHCl3/MeOH/H2O
(32/6/0.5) and (c) CHCl3/MeOH/H2O (6/4/1). The spots were detected
under ultraviolet irradiation at 254 nm and by the use of
phosphomolybdic acid in ethanol solution and ninhydrin sprays.
1H NMR (200 MHz, DMSO‐d6): δ 8.07 (brs, 1H), 4.58 (m, 1H), 3.96
(dd, J = 5.2, 0.8 Hz, 1H), 3.34 (brs, 1H), 1.38 (d, J = 6.2 Hz, 3H).
[α]2D2 −42.4° (c 1.0, H2O).
Anal. Calcd for C5H7NO4: C, 41.38; H, 4.86; N, 9.65. Found: C,
41.20; H, 4.82; N, 9.67.
lit.57 mp 131−133.5 °C., [α]D27 −37.7° (c 2.7, H2O).
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4.2.2
(4S,5S)‐5‐Methyl‐2‐oxooxazolidine‐4‐carbox-
ylic acid (19)
The cyclization of N‐benzyloxycarbonyl‐allo‐L‐threonine (60) (4.73 g,
18.0 mmol) in methanol (36.0 mL) with 1 M aqueous sodium hydrox-
ide solution (36.0 mL, 36.0 mmol) yielded the title compound 19
(1.92 g, 73%) as colorless crystals.
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4.2
General procedure for preparing the
N‐terminus fragments 17−20
mp 165−168 °C.
IR (KBr): 3363, 2632, 2550, 1746, 1685, 1412, 1224, 1156, 1060
(4S,5R)‐5‐Methyl‐2‐oxooxazolidine‐4‐carboxylic acid (17).
To an ice cooled solution of N‐benzyloxycarbonyl‐L‐threonine (54)
(20.0 g, 79.0 mmol) in methanol (160 mL), 1 M aqueous sodium
hydroxide solution (160 mL, 160 mmol) was added and the mixture
was stirred for 15 min at the same temperature. After the ice bath
was removed, the mixture was stirred continuously for 3.5 h. Ethyl
acetate (100 mL) was added to the mixture and extracted. The aque-
ous layer was cooled in an ice bath, then 1 M aqueous hydrochloric
acid solution (160 mL, 160 mmol) was added. The mixture was con-
centrated under reduced pressure. To the residue, chloroform (500
mL) was added and extracted. The organic layer was dried over anhy-
drous magnesium sulfate and concentrated in vacuo. The residue was
crystallized with chloroform to give the title compound 17 (3.29 g,
29%) as a colorless solid.
cm−1
.
1H NMR (200 MHz, DMSO‐d6): δ 7.89 (brs, 1H), 4.85 (m, 1H), 4.27
(d, J = 8.4 Hz, 1H), 1.24 (d, J = 6.4 Hz, 3H).
[α]2D5−20.7° (c 1.0, H2O).
Anal. Calcd for C5H7NO4: C, 41.38; H, 4.86; N, 9.65. Found: C,
41.23; H, 4.75; N, 9.63.
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4.2.3
(4R,5R)‐5‐Methyl‐2‐oxooxazolidine‐4‐carbox-
ylic acid (20)
The cyclization of N‐benzyloxycarbonyl‐allo‐D‐threonine (61) (35.5 g,
140 mmol) in methanol (280 mL) with 1 M aqueous sodium hydroxide
solution (280 mL, 280 mmol) yielded the title compound 20 (12.9 g,
64%) as colorless crystals.
mp 133−135 °C.
mp 168−171 °C.
IR (KBr): 3303, 3245, 2714, 2600, 2507, 1744, 1727, 1673, 1227,
IR (KBr): 3360, 2996, 2931, 1746, 1683, 1413, 1225, 1156, 1061 cm−1
.
1207 cm−1
.
1H NMR (200 MHz, DMSO‐d6): δ 7.87 (brs, 1H), 4.84 (m, 1H), 4.27
(d, J = 8.6 Hz, 1H), 1.24 (d, J = 6.2 Hz, 3H).
[α]2D5 +19.3° (c 1.0, H2O).
1H NMR (200 MHz, DMSO‐d6): δ 8.07 (brs, 1H), 4.58 (m, 1H), 3.95
(d, J = 5.2 Hz, 1H), 1.38 (d, J = 6.2 Hz, 3H).
[α]2D5 +40.9° (c 1.0, H2O).
Anal. Calcd for C5H7NO4: C, 41.38; H, 4.86; N, 9.65. Found: C,
41.54; H, 4.94; N, 9.75.
Anal. Calcd for C5H7NO4·0.1H2O: C, 40.88; H, 4.94; N, 9.53.
Found: C, 40.84; H, 4.90; N, 9.68.
lit.57 mp 139.8−140.2 °C., [α]D29:8 +41.8° (c 2.7, H2O).
In a similar manner, 5‐methyl‐2‐oxooxazolidine‐4‐carboxylic acid
18−20 were prepared.
|
4.3
General procedure for preparing the
middle‐part fragments 21 and 22
Benzhydryl
(2S)‐2‐amino‐3‐(thiazol‐4‐yl)propanoate
p‐toluene-
sulfonate (21).
|
4.2.1
(4R,5S)‐5‐Methyl‐2‐oxooxazolidine‐4‐carbox-
To a solution of (2S)‐2‐amino‐3‐(thiazol‐4‐yl)propanoic acid (30)
(13.8 g, 80.0 mmol) in water (150 mL), p‐toluenesulfonic acid
monohydrate (15.2 g, 80.0 mmol) was added. The mixture was con-
centrated in vacuo to give 29.8 g of crude (2S)‐2‐amino‐3‐(thiazol‐4‐
yl)propanoic acid p‐toluenesulfonate (32). To an ice cooled solution
of compound 32 in methanol (400 mL), diphenyldiazomethane
(54.3 g, 278 mmol) was added portionwise and the mixture was
stirred for 1 h at the same temperature. Acetic acid (100 mL) was
added and the mixture was concentrated in vacuo. To the residue,
ylic acid (18)
The cyclization of N‐benzyloxycarbonyl‐D‐threonine (55) (9.00 g, 35.5
mmol) in methanol (71.0 mL) with 1 M aqueous sodium hydroxide
solution (71.0 mL, 71.0 mmol) yielded the title compound 18 (3.51 g,
68%) as colorless crystals.
mp 135−137 °C.
IR (KBr): 3303, 3246, 2714, 2599, 2507, 1744, 1728, 1673, 1227,
1207 cm−1
.

10 of 16
KOBAYASHI ET AL.
diethyl ether (700 mL) was added and the precipitate was collected
by filtration to give the title compound 21 (39.5 g, 97%) as a color-
less solid.
Anal. Calcd for C10H19NO2: C, 64.83; H, 10.34; N, 7.56. Found: C,
64.61; H, 10.34; N, 7.47.
In a similar manner, (S)‐isomer 37 was prepared.
mp 139−140 °C.
IR (KBr): 1753, 1602, 1512, 1496, 1260, 1224, 1171, 1124, 1036,
|
4.4.2
N‐tert‐Butoxycarbonyl‐(2S)‐2‐
1012 cm−1
.
methylpyrrolidine (37)
1H NMR (200 MHz, CD3OD): δ 8.92 (d, J = 2.0 Hz, 1H), 7.70 (m,
2H), 7.40−7.20 (m, 13H), 6.91 (s, 1H), 4.62 (t, J = 5.8 Hz, 1H), 3.47
(d, J = 5.8 Hz, 2H), 2.36 (s, 3H).
The reduction of N‐tert‐butoxycarbonyl‐(2R)‐2‐[(methylsulfonyl)
oxymethyl]pyrrolidine (69) (2.50 g, 8.95 mmol) with 1 M lithium
triethylborohydride inTHF (18.0 mL, 18.0 mmol) yielded the title com-
pound 37 (1.24 g, 75%) as a colorless oil.
[α]2D3 −34.7° (c 1.0, CHCl3).
Anal. Calcd for C26H26N2O5S2: C, 61.16; H, 5.13; N, 5.49; S, 12.56.
Found: C, 61.14; H, 5.32; N, 5.41; S, 12.46.
bp 55−56 °C (3 mmHg).
IR (CHCl3): 1681, 1478, 1454, 1404, 1366, 1170 cm−1
1H NMR (300 MHz, CDCl3): δ 3.87 (brs, 1H), 3.35 (m, 2H), 2.05
−1.40 (m, 4H), 1.46 (s, 9H), 1.16 (d, J = 6.3 Hz, 3H).
[α]2D4 +33.6° (c 1.6, CHCl3).
.
In a similar manner, (R)‐isomer 22 was prepared.
Benzhydryl (2R)‐2‐amino‐3‐(thiazol‐4‐yl)propanoate p‐toluene-
sulfonate (22).
The esterification of (2R)‐2‐amino‐3‐(thiazol‐4‐yl)propanoic acid
(31) (13.6 g, 78.7 mmol) with p‐toluenesulfonic acid monohydrate
(15.0 g, 78.7 mmol) and diphenyldiazomethane (30.6 g, 157 mmol)
yielded the title compound 22 (39.1 g, 97%) as a colorless solid.
mp 139−140 °C.
Anal. Calcd for C10H19NO2: C, 64.83; H, 10.34; N, 7.56. Found: C,
64.65; H, 10.31; N, 7.52.
|
4.4.3
(2R)‐2‐Methylpyrrolidine p‐toluenesulfonate (23)
IR (KBr): 3437, 3236, 1753, 1603, 1512, 1496, 1260, 1223, 1171,
1124, 1036, 1011 cm−1
.
N‐tert‐Butoxycarbonyl‐(2R)‐2‐methylpyrrolidine (36) (8.79 g, 47.4
mmol) was dissolved in formic acid (48.0 mL, 1.27 mol) and stirred
for 20 h at room temperature. p‐Toluenesulfonic acid monohydrate
(9.01 g, 47.4 mmo) in water (50.0 mL) was added to the solution.
The mixture was concentrated in vacuo to give the title compound
23 (12.4 g, quant) as a colorless amorphous powder, which was used
without further purification.
1H NMR (200 MHz, CD3OD): δ 8.92 (d, J = 1.8 Hz, 1H), 7.70 (m,
2H), 7.40−7.20 (m, 13H), 6.91 (s, 1H), 4.62 (t, J = 6.0 Hz, 1H), 3.47
(d, J = 6.0 Hz, 2H), 2.36 (s, 3H).
[α]2D5 +33.1° (c 1.0, CHCl3).
Anal. Calcd for C26H26N2O5S2: C, 61.16; H, 5.13; N, 5.49; S, 12.56.
Found: C, 60.99; H, 5.19; N, 5.28; S, 12.29.
1H NMR (300 MHz, CD3OD): δ 7.70 (d, J = 8.7 Hz, 2H), 7.23 (d, J =
8.7 Hz, 2H), 3.63 (m, 1H), 3.28 (m, 2H), 2.37 (s, 3H), 2.30−1.90 (m, 3H),
1.62 (m, 1H), 1.37 (d, J = 6.9 Hz, 3H).
|
4.4
General procedure for preparing the
In a similar manner, (S)‐isomer 24 was prepared.
C‐terminus fragments 23 and 24
|
4.4.4
(2S)‐2‐Methylpyrrolidine p‐toluenesulfonate (24)
|
4.4.1
N‐tert‐Butoxycarbonyl‐(2R)‐2‐
methylpyrrolidine (36)
The deprotection of N‐tert‐butoxycarbonyl‐(2S)‐2‐methylpyrrolidine
(37) (0.371 g, 2.00 mmol) with formic acid (2.00 mL, 53.0 mmol) and
p‐toluenesulfonic acid monohydrate (0.380 g, 2.00 mmol) yielded the
title compound 24 (0.516 g, quant) as a colorless amorphous powder,
which was used without further purification.
1H NMR (300 MHz, CD3OD): δ 7.70 (d, J = 8.7 Hz, 2H), 7.23 (d, J =
8.7 Hz, 2H), 3.63 (m, 1H), 3.28 (m, 2H), 2.37 (s, 3H), 2.30−1.90 (m, 3H),
1.62 (m, 1H), 1.38 (d, J = 6.9 Hz, 3H).
To an ice cooled 1 M lithium triethylborohydride in THF (1.20 L, 1.20
mol), N‐tert‐butoxycarbonyl‐(2S)‐2‐[(methylsulfonyl)oxymethyl]pyrrol-
idine (68) (161 g, 580 mmol) in THF (380 mL) was added dropwise
for 2 h. The ice bath was removed, and the mixture was stirred for 2
h at room temperature. The mixture was poured into ice cooled water
(1.00 L) and stirred for 1 h. Toluene (1.50 L) was added and extracted.
The organic layer was washed with aqueous sodium hydrogen carbon-
ate solution (1.00 L) and water (1.00 L × 2). The organic layer was
dried over anhydrous magnesium sulfate and concentrated in vacuo.
The residue was distilled under reduced pressure to afford the title
compound 36 (90.7 g, 85%) as a colorless oil.
|
4.5
General procedure for preparing the TRH
mimetics 1−16
|
4.5.1
(4S,5S)‐5‐Methyl‐N‐{(2S)‐1‐[(2R)‐2‐
bp 56−58 °C (3 mmHg).
IR (CHCl3): 1681, 1477, 1454, 1403, 1366, 1170 cm−1
1H NMR (300 MHz, CDCl3): δ 3.87 (brs, 1H), 3.35 (brs, 2H), 2.10
methylpyrrolidin‐1‐yl]‐1‐oxo‐3‐(1,3‐thiazol‐4‐yl)
propan‐2‐yl}‐2‐oxo‐1,3‐oxazolidine‐4‐carboxamide (1)
.
−1.70 (m, 3H), 1.60−1.40 (m, 1H), 1.47 (s, 9H), 1.16 (d, J = 6.0 Hz, 3H).
To an ice cooled solution of (2S)‐2‐[(4S,5S)‐5‐methyl‐2‐
oxooxazolidine‐4‐carboxamido]‐3‐(thiazol‐4‐yl)propanoic acid (50)
25
[α]
−33.2° (c 1.7, CHCl3).
D

KOBAYASHI ET AL.
11 of 16
(9.00 g, 30.1 mmol) and HOSu (3.63 g, 31.6 mmol) in THF (320 mL)‐
DMF (40.0 mL), DCC (6.51 g, 31.6 mmol) was added and the mixture
was stirred for 4 h at the same temperature. To this solution, (2R)‐2‐
methylpyrrolidine p‐toluenesulfonate (23) (7.75 g, 30.1 mmol) and
triethylamine (8.40 mL, 60.1 mmol) were added and the mixture was
stirred for 1 h at 0 °C. The ice bath was removed, and the mixture
was stirred for 24 h. The precipitate was filtered off and the filtrate
was concentrated in vacuo. The residue was purified by gel filtration
chromatography (MCI GEL CHP‐20P, 600 mL, eluent: H2O/MeOH)
and by silica gel column chromatography (eluent: CHCl3/MeOH). The
purified fractions were concentrated in vacuo and the residue was
lyophilized to afford the title compound 1 (6.22 g, 57%) as a white
amorphous powder.
mmol) yielded the title compound 3 (0.489 g, 67%) as a white amor-
phous powder.
IR (KBr): 3338, 3051, 2971, 1754, 1661, 1638, 1537, 1438, 1372,
1329, 1224, 1110, 1072 cm−1
.
1H NMR (300 MHz, CD3OD): δ 8.94 (d, J = 2.1 Hz, 1H), 7.33 (d, J =
2.1 Hz, 1H), 4.99 (m, 1H), 4.50 and 4.05 (m each, total 1H), 4.45 (m,
1H), 3.93 and 3.91 (d each, J = 5.1 Hz, total 1H), 3.72 and 3.42 (m
each, 2H), 3.40−3.10 (m, 2H), 2.10−1.50 (m, 4H), 1.45 and 1.44 (d
each, J = 6.3 Hz, total 3H), 1.16 and 1.08 (d each, J = 6.3 Hz, total 3H).
[α]2D5 +44.3° (c 1.0, H2O).
Anal. Calcd for C16H22N4O4S·0.7H2O: C, 50.70; H, 6.22; N, 14.78;
S, 8.46. Found: C, 50.75; H, 6.26; N, 15.05; S, 8.29.
IR (KBr): 3279, 2972, 2877, 1763, 1655, 1626, 1548, 1456, 1384,
|
4.5.4
(4S,5R)‐5‐Methyl‐N‐{(2R)‐1‐[(2R)‐2‐
1231 cm−1
.
methylpyrrolidin‐1‐yl]‐1‐oxo‐3‐(1,3‐thiazol‐4‐yl)
propan‐2‐yl}‐2‐oxo‐1,3‐oxazolidine‐4‐carboxamide (4)
1H NMR (300 MHz, CD3OD): δ 8.97 and 8.96 (d each, J = 2.1 Hz,
total 1H), 7.34 and 7.33 (d each, J = 2.1 Hz, total 1H), 5.19 and 5.05 (t
each, J = 7.5 Hz, total 1H), 4.92 (dq, J = 8.7, 6.6 Hz, 1H), 4.37 and 4.35
(d each, J = 8.7 Hz, total 1H), 4.07 and 3.92 (m each, total 1H), 3.75 (m,
1H), 3.41 (m, 1H), 3.22 (m, 2H), 2.00−1.50 (m, 4H), 1.28 and 1.22 (d
each, J = 6.6 Hz, total 3H), 1.21 and 1.02 (d each, J = 6.6 Hz, total 3H).
[α]2D2 −4.4° (c 1.0, MeOH).
The condensation of (2R)‐2‐[(4S,5R)‐5‐Methyl‐2‐oxooxazolidine‐4‐
carboxamido]‐3‐(thiazol‐4‐yl)propanoic acid (47) (0.800 g, 2.67 mmol)
and (2R)‐2‐methylpyrrolidine p‐toluenesulfonate (23) (0.688 g, 2.67
mmol) yielded the title compound 4 (0.577 g, 59%) as a white amor-
phous powder.
Anal. Calcd for C16H22N4O4S·H2O: C, 49.99; H, 6.29; N, 14.57; S,
8.34. Found: C, 49.79; H, 6.22; N, 14.55; S, 8.25.
IR (KBr): 3272, 3087, 2972, 2931, 2875, 1752, 1683, 1625, 1542,
1517, 1442, 1229, 1068 cm−1
.
In a similar manner, other stereoisomers 2−16 were prepared.
1H NMR (300 MHz, CD3OD): δ 8.95 and 8.94 (d each, J = 2.1 Hz,
total 1H), 7.33 (d, J = 2.1 Hz, 1H), 5.08 and 5.02 (dd each, J = 8.1, 6.6
Hz, total 1H), 4.52 and 4.06 (m each, total 1H), 4.37 (m, 1H), 3.92 and
3.89 (d each, J = 5.4 Hz, total 1H), 3.73 and 3.43 (m each, total 2H),
3.30−3.10 (m, 2H), 2.10−1.50 (m, 4H), 1.41 and 1.39 (d each, J = 6.3
Hz, total 3H), 1.16 and 1.11 (d each, J = 6.3 Hz, total 3H).
[α]2D4 +10.4° (c 1.0, H2O).
|
4.5.2
(4S,5R)‐5‐Methyl‐N‐{(2S)‐1‐[(2R)‐2‐
methylpyrrolidin‐1‐yl]‐1‐oxo‐3‐(1,3‐thiazol‐4‐yl)
propan‐2‐yl}‐2‐oxo‐1,3‐oxazolidine‐4‐carboxamide (2)
The condensation of (2S)‐2‐[(4S,5R)‐5‐methyl‐2‐oxooxazolidine‐4‐
carboxamido]‐3‐(thiazol‐4‐yl)propanoic acid (46) (0.600 g, 2.00 mmol)
and (2R)‐2‐methylpyrrolidine p‐toluenesulfonate (23) (0.515 g, 2.00
mmol) yielded the title compound 2 (0.500 g, 68%) as a white amor-
phous powder.
Anal. Calcd for C16H22N4O4S·0.2H2O: C, 51.93; H, 6.10; N, 15.14;
S, 8.66. Found: C, 52.04; H, 6.12; N, 15.21; S, 8.58.
|
IR (KBr): 3276, 3093, 2973, 2031, 2875, 1762, 1677, 1625, 1540,
4.5.5
(4S,5R)‐5‐Methyl‐N‐{(2R)‐1‐[(2S)‐2‐
1516, 1447, 1384, 1227, 1067 cm−1
.
methylpyrrolidin‐1‐yl]‐1‐oxo‐3‐(1,3‐thiazol‐4‐yl)
1H NMR (300 MHz, CD3OD): δ 8.97 and 8.96 (d each, J = 2.1 Hz,
total 1H), 7.33 (m, 1H), 5.11 and 4.97 (t each, J = 7.5 Hz, total 1H),
4.50 (m, 1H), 4.09 and 3.94 (m each, total 1H), 3.94 (d, J = 5.4 Hz,
1H), 3.70 and 3.42 (m each, 2H), 3.22 (m, 2H), 2.00−1.50 (m, 4H),
1.47 (d, J = 6.6 Hz, 3H), 1.26 and 1.04 (d each, J = 6.3 Hz, total 3H).
[α]2D5 +34.0° (c 1.0, H2O).
propan‐2‐yl}‐2‐oxo‐1,3‐oxazolidine‐4‐carboxamide (5)
The condensation of (2R)‐2‐[(4S,5R)‐5‐methyl‐2‐oxooxazolidine‐4‐
carboxamido]‐3‐(thiazol‐4‐yl)propanoic acid
(47) (0.800 g, 2.67 mmol) and (2S)‐2‐methylpyrrolidine p‐
toluenesulfonate (24) (0.688 g, 2.67 mmol) yielded the title compound
5 (0.391 g, 40%) as a white amorphous powder.
Anal. Calcd for C16H22N4O4S·0.9H2O: C, 50.22; H, 6.27; N, 14.64;
S, 8.38. Found: C, 50.23; H, 6.34; N, 14.88; S, 8.45.
IR (KBr): 3273, 3089, 2973, 2931, 2875, 1752, 1683, 1626, 1542,
1517, 1447, 1229, 1068 cm−1
.
1H NMR (300 MHz, CD3OD): δ 8.98 and 8.97 (d each, J = 1.8 Hz,
total 1H), 7.34 (d, J = 1.8 Hz, 1H), 5.13 and 5.00 (t each, J = 7.5 Hz,
total 1H), 4.44 (m, 1H), 4.09 and 3.97 (m each, total 1H), 3.94 (d, J =
5.4 Hz, 1H), 3.71 and 3.43 (m each, total 2H), 3.30−3.10 (m, 2H),
2.00−1.50 (m, 4H), 1.43 (d, J = 6.3 Hz, 3H), 1.28 and 1.04 (d each, J
= 6.6 Hz, total 3H).
|
4.5.3
(4S,5R)‐5‐Methyl‐N‐{(2S)‐1‐[(2S)‐2‐
methylpyrrolidin‐1‐yl]‐1‐oxo‐3‐(1,3‐thiazol‐4‐yl)
propan‐2‐yl}‐2‐oxo‐1,3‐oxazolidine‐4‐carboxamide (3)
The condensation of (2S)‐2‐[(4S,5R)‐5‐methyl‐2‐oxooxazolidine‐4‐
carboxamido]‐3‐(thiazol‐4‐yl)propanoic acid (46) (0.600 g, 2.00 mmol)
and (2S)‐2‐methylpyrrolidine p‐toluenesulfonate (24) (0.515 g, 2.00
24
[α] +20.7° (c 1.0, H2O).
D

12 of 16
KOBAYASHI ET AL.
Anal. Calcd for C16H22N4O4S·0.2H2O: C, 51.93; H, 6.10; N, 15.14;
and (2R)‐2‐methylpyrrolidine p‐toluenesulfonate (23) (0.688 g, 2.67
mmol) yielded the title compound 8 (0.595 g, 61%) as a white amor-
phous powder.
S, 8.66. Found: C, 51.80; H, 6.10; N, 15.35; S, 8.69.
|
IR (KBr): 3274, 3108, 2973, 2931, 2875, 1763, 1676, 1626, 1540,
4.5.6
(4R,5S)‐5‐Methyl‐N‐{(2S)‐1‐[(2R)‐2‐
1517, 1442, 1384, 1227, 1067 cm−1
.
methylpyrrolidin‐1‐yl]‐1‐oxo‐3‐(1,3‐thiazol‐4‐yl)
1H NMR (300 MHz, CD3OD): δ 8.94 and 8.93 (d each, J = 2.1 Hz,
total 1H), 7.32 (d, J = 2.1 Hz, 1H), 5.02 (m, 1H), 4.45 (m, 1H), 4.44 and
4.05 (m each, total 1H), 3.93 and 3.91 (d each, J = 5.4 Hz, total 1H),
3.72 and 3.43 (m each, total 2H), 3.30−3.10 (m, 2H), 2.10−1.50 (m,
4H), 1.45 and 1.44 (d each, J = 6.6 Hz, total 3H), 1.16 and 1.08 (d each,
J = 6.6 Hz, total 3H).
propan‐2‐yl}‐2‐oxo‐1,3‐oxazolidine‐4‐carboxamide (6)
The condensation of (2S)‐2‐[(4R,5S)‐5‐methyl‐2‐oxooxazolidine‐4‐
carboxamido]‐3‐(thiazol‐4‐yl)propanoic acid (48) (0.500 g, 1.67 mmol)
and (2R)‐2‐methylpyrrolidine p‐toluenesulfonate (23) (0.430 g, 1.67
mmol) yielded the title compound 6 (0.240 g, 39%) as colorless
crystals.
[α]2D4 −45.3° (c 1.0, H2O).
mp 88−90 °C.
Anal. Calcd for C16H22N4O4S·0.2H2O: C, 51.93; H, 6.10; N, 15.14;
S, 8.66. Found: C, 51.76; H, 6.10; N, 15.40; S, 8.64.
IR (KBr): 3271, 3088, 2973, 2931, 2875, 1751, 1676, 1626, 1543,
1518, 1449, 1230, 1068 cm−1
.
1H NMR (300 MHz, CD3OD): δ 8.98 and 8.97 (d each, J = 1.8 Hz,
total 1H), 7.34 (d, J = 1.8 Hz, 1H), 5.13 and 5.00 (t each, J = 7.2 Hz,
total 1H), 4.45 (m, 1H), 4.09 and 3.97 (m each, total 1H), 3.94 (d, J =
4.8 Hz, 1H), 3.70 and 3.43 (m each, total 2H), 3.29 (m, 2H), 2.10
−1.50 (m, 4H), 1.43 (d, J = 6.3 Hz, 3H), 1.28 and 1.04 (d each, J =
6.6 Hz, total 3H).
|
4.5.9
(4R,5S)‐5‐Methyl‐N‐{(2R)‐1‐[(2S)‐2‐
methylpyrrolidin‐1‐yl]‐1‐oxo‐3‐(1,3‐thiazol‐4‐yl)
propan‐2‐yl}‐2‐oxo‐1,3‐oxazolidine‐4‐carboxamide (9)
The condensation of (2R)‐2‐[(4R,5S)‐5‐methyl‐2‐oxooxazolidine‐4‐
carboxamido]‐3‐(thiazol‐4‐yl)propanoic acid (49) (0.800 g, 2.67 mmol)
and (2S)‐2‐methylpyrrolidine p‐toluenesulfonate (24) (0.688 g, 2.67
mmol) yielded the title compound 9 (0.510 g, 52%) as a white amor-
phous powder.
[α]2D4 −20.2° (c 1.0, H2O).
Anal. Calcd for C16H22N4O4S·0.5H2O: C, 51.19; H, 6.17; N, 14.92;
S, 8.54. Found: C, 51.22; H, 6.17; N, 15.11; S, 8.72.
IR (KBr): 3282, 3101, 2973, 2931, 2875, 1762, 1676, 1625, 1540.
|
4.5.7
(4R,5S)‐5‐Methyl‐N‐{(2S)‐1‐[(2S)‐2‐
1516, 1447, 1384, 1227, 1067 cm−1
.
methylpyrrolidin‐1‐yl]‐1‐oxo‐3‐(1,3‐thiazol‐4‐yl)
propan‐2‐yl}‐2‐oxo‐1,3‐oxazolidine‐4‐carboxamide (7)
1H NMR (300 MHz, CD3OD): δ 8.97 and 8.96 (d each, J = 2.1 Hz,
total 1H), 7.33 (d, J = 2.1 Hz, 1H), 5.11 and 4.98 (d each, J = 7.2 Hz,
total 1H), 4.49 (m, 1H), 4.10 and 3.94 (m each, total 1H), 3.95 (d, J =
5.4 Hz, 1H), 3.69 and 3.41 (m each, total 2H), 3.30−3.10 (m, 2H),
2.00−1.50 (m, 4H), 1.47 (d, J = 6.3 Hz, 3H), 1.26 and 1.04 (d each, J
= 6.6 Hz, total 3H).
The condensation of (2S)‐2‐[(4R,5S)‐5‐methyl‐2‐oxooxazolidine‐4‐
carboxamido]‐3‐(thiazol‐4‐yl)propanoic acid (48) (0.660 g, 2.21 mmol)
and (2S)‐2‐methylpyrrolidine p‐toluenesulfonate (24) (0.569 g, 2.21
mmol) yielded the title compound 7 (0.505 g, 62%) as a white amor-
phous powder.
[α]2D4 −35.9° (c 1.0, H2O).
Anal. Calcd for C16H22N4O4S·0.2H2O: C, 51.93; H, 6.10; N, 15.14;
S, 8.66. Found: C, 51.92; H, 6.12; N, 15.34; S, 8.66.
IR (KBr): 3272, 3086, 2973, 2875, 1751, 1677, 1624, 1542, 1517,
1442, 1407, 1384, 1345, 1229, 1067 cm−1
.
1H NMR (300 MHz, CD3OD): δ 8.95 and 8.94 (d each, J = 2.1
Hz, total 1H), 7.33 (d, J = 2.1 Hz, 1H), 5.08 and 5.02 (dd each, J =
8.1, 6.6 Hz, total 1H), 4.52 and 4.06 (m each, total 1H), 4.36 (m,
1H), 3.93 and 3.89 (d each, J = 5.4 Hz, total 1H), 3.74 and 3.42
(m each, total 2H), 3.40−3.10 (m, 2H), 2.10−1.50 (m, 4H), 1.41 and
1.39 (d each, J = 6.6 Hz, total 3H), 1.16 and 1.11 (d each, J = 6.6
Hz, total 3H).
|
4.5.10
(4S,5S)‐5‐Methyl‐N‐{(2S)‐1‐[(2S)‐2‐
methylpyrrolidin‐1‐yl]‐1‐oxo‐3‐(1,3‐thiazol‐4‐yl)propan‐2‐
yl}‐2‐oxo‐1,3‐oxazolidine‐4‐carboxamide (10)
The condensation of (2S)‐2‐[(4S,5S)‐5‐methyl‐2‐oxooxazolidine‐4‐
carboxamido]‐3‐(thiazol‐4‐yl)propanoic acid (50) (0.599 g, 2.00 mmol)
and (2S)‐2‐methylpyrrolidine p‐toluenesulfonate (24) (0.515 g, 2.00
mmol) yielded the title compound 10 (0.288 g, 39%) as colorless
crystals.
[α]2D5 −10.4° (c 1.0, H2O).
Anal. Calcd for C16H22N4O4S·H2O: C, 49.99; H, 6.29; N, 14.57; S,
8.34. Found: C, 50.03; H, 6.35; N, 14.86; S, 8.37.
mp 231 °C (decomp.)
IR (KBr): 3326, 3053, 2969, 1746, 1709, 1654, 1635, 1538, 1436,
|
4.5.8
(4R,5S)‐5‐Methyl‐N‐{(2R)‐1‐[(2R)‐2‐
1384, 1339, 1241 cm−1
.
methylpyrrolidin‐1‐yl]‐1‐oxo‐3‐(1,3‐thiazol‐4‐yl)
propan‐2‐yl}‐2‐oxo‐1,3‐oxazolidine‐4‐carboxamide (8)
1H NMR (300 MHz, CD3OD): δ 8.95 and 8.93 (d each, J = 2.1 Hz,
total 1H), 7.32 (d, J = 2.1 Hz, 1H), 5.08 (m, 1H), 4.91 (m, 1H), 4.60 and
4.03 (m each, total 1H), 4.34 and 4.30 (d each, J = 8.4 Hz, total 1H),
3.83 (m, 1H), 3.42 (m, 2H), 3.40−3.10 (m, 2H), 2.10−1.50 (m, 4H),
The condensation of (2R)‐2‐[(4R,5S)‐5‐methyl‐2‐oxooxazolidine‐4‐
carboxamido]‐3‐(thiazol‐4‐yl)propanoic acid (49) (0.800 g, 2.67 mmol)

KOBAYASHI ET AL.
13 of 16
1.21 and 1.15 (d each, J = 6.6 Hz, total 3H), 1.13 and 1.04 (d each, J =
6.6 Hz, total 3H).
|
4.5.13
(4R,5R)‐5‐Methyl‐N‐{(2S)‐1‐[(2R)‐2‐
methylpyrrolidin‐1‐yl]‐1‐oxo‐3‐(1,3‐thiazol‐4‐yl)
propan‐2‐yl}‐2‐oxo‐1,3‐oxazolidine‐4‐carboxamide (13)
[α]2D4 +8.2° (c 1.0, MeOH).
Anal. Calcd for C16H22N4O4S: C, 52.44; H, 6.05; N, 15.29; S, 8.75.
Found: C, 52.17; H, 6.06; N, 15.30; S, 8.82.
The condensation of (2S)‐2‐[(4R,5R)‐5‐methyl‐2‐oxooxazolidine‐4‐
carboxamido]‐3‐(thiazol‐4‐yl)propanoic acid (52) (0.535 g, 1.79 mmol)
and (2R)‐2‐methylpyrrolidine p‐toluenesulfonate (23) (0.460 g, 1.79
mmol) yielded the title compound 13 (0.462 g, 71%) as a white amor-
phous powder.
|
4.5.11
(4S,5S)‐5‐Methyl‐N‐{(2R)‐1‐[(2R)‐2‐
IR (KBr): 3274, 3089, 2972, 2875, 1749, 1684, 1626, 1543, 1517,
methylpyrrolidin‐1‐yl]‐1‐oxo‐3‐(1,3‐thiazol‐4‐yl)
propan‐2‐yl}‐2‐oxo‐1,3‐oxazolidine‐4‐carboxamide (11)
1448, 1383, 1343, 1230, 1099 cm−1
.
1H NMR (300 MHz, CD3OD): δ 8.97 and 8.96 (d each, J = 2.1 Hz,
total 1H), 7.35 (d, J = 2.1 Hz, 1H), 5.21 and 5.06 (dd each, J = 8.1, 6.6
Hz, total 1H), 4.90−4.70 (m, 1H), 4.33 and 4.31 (d each, J = 8.7 Hz,
total 1H), 4.20−3.99 (m, 1H), 3.77 and 3.45 (m each, total 2H), 3.20
(m, 2H), 2.10−1.50 (m, 4H), 1.30 and 1.10 (d each, J = 6.6 Hz, total
3H), 1.10 and 1.04 (d each, J = 6.6 Hz, total 3H).
The condensation of (2R)‐2‐[(4S,5S)‐5‐methyl‐2‐oxooxazolidine‐4‐
carboxamido]‐3‐(thiazol‐4‐yl)propanoic acid (51) (0.500 g, 1.67 mmol)
and (2R)‐2‐methylpyrrolidine p‐toluenesulfonate (23) (0.430 g, 1.67
mmol) yielded the title compound 11 (0.197 g, 32%) as colorless
crystals.
mp 170−172 °C.
[α]2D6 +1.2° (c 1.0, H2O).
IR (KBr): 3340, 3130, 3094, 2985, 2967, 2873, 1749, 1661, 1637,
Anal. Calcd for C16H22N4O4S·H2O: C, 49.99; H, 6.29; N, 14.57; S,
8.34. Found: C, 49.83; H, 6.17; N, 14.71; S, 8.53.
1538, 1442, 1382, 1340, 1235, 1098 cm−1
.
1H NMR (300 MHz, CD3OD): δ 8.95 and 8.94 (d each, J = 2.1 Hz,
total 1H), 7.35 and 7.34 (d each, J = 2.1 Hz, total 1H), 5.15 and 5.08
(dd each, J = 8.1, 6.6 Hz, total 1H), 4.90−4.70 (m, 1H), 4.62 and 4.05
(m each, total 1H), 4.31 and 4.26 (d each, J = 8.7 Hz, total 1H), 3.81
and 3.40 (m each, total 2H), 3.30−3.10 (m, 2H), 2.10−1.50 (m, 4H),
1.14 and 1.12 (d each, J = 6.3 Hz, total 3H), 1.07 and 0.99 (d each, J
= 6.6 Hz, total 3H).
|
4.5.14
(4R,5R)‐5‐Methyl‐N‐{(2S)‐1‐[(2S)‐2‐
methylpyrrolidin‐1‐yl]‐1‐oxo‐3‐(1,3‐thiazol‐4‐yl)
propan‐2‐yl}‐2‐oxo‐1,3‐oxazolidine‐4‐carboxamide (14)
The condensation of (2S)‐2‐[(4R,5R)‐5‐methyl‐2‐oxooxazolidine‐4‐
carboxamido]‐3‐(thiazol‐4‐yl)propanoic acid (52) (0.535 g, 1.79 mmol)
and (2S)‐2‐methylpyrrolidine p‐toluenesulfonate (24) (0.460 g, 1.79
mmol) yielded the title compound 14 (0.455 g, 70%) as a white amor-
phous powder.
[α]2D2 −20.1° (c 1.0, H2O).
Anal. Calcd for C16H22N4O4S: C, 52.44; H, 6.05; N, 15.29; S, 8.75.
Found: C, 52.40; H, 5.98; N, 15.19; S, 8.77.
IR (KBr): 3273, 3086, 2972, 2875, 1749, 1682, 1625, 1542, 1518,
1442, 1384, 1342, 1230, 1099 cm−1
.
1H NMR (200 MHz, CD3OD): δ 8.96 and 8.94 (d each, J = 2.0 Hz,
total 1H), 7.35 (d, J = 2.0 Hz, 1H), 5.10 (m, 1H), 4.90−4.70 (m, 1H),
4.61 and 4.05 (m each, total 1H), 4.31 and 4.26 (d each, J = 8.6 Hz,
total 1H), 3.81 and 3.40 (m each, total 2H), 3.27 and 3.13 (dd each, J
= 14.2, 8.4 Hz, total 2H), 2.10−1.50 (m, 4H), 1.14 and 1.13 (d each, J
= 6.4 Hz, total 3H), 1.06 and 0.99 (d each, J = 6.4 Hz, total 3H).
[α]2D6 +18.4° (c 1.0, H2O).
|
4.5.12
(4S,5S)‐5‐Methyl‐N‐{(2R)‐1‐[(2S)‐2‐
methylpyrrolidin‐1‐yl]‐1‐oxo‐3‐(1,3‐thiazol‐4‐yl)
propan‐2‐yl}‐2‐oxo‐1,3‐oxazolidine‐4‐carboxamide (12)
The condensation of (2R)‐2‐[(4S,5S)‐5‐Methyl‐2‐oxooxazolidine‐4‐
carboxamido]‐3‐(thiazol‐4‐yl)propanoic acid (51) (0.599 g, 2.00 mmol)
and (2S)‐2‐methylpyrrolidine p‐toluenesulfonate (23) (0.515 g, 2.00
mmol) yielded the title compound 12 (0.306 g, 42%) as a white amor-
phous powder.
Anal. Calcd for C16H22N4O4S·0.9H2O: C, 50.22; H, 6.27; N, 14.64;
S, 8.38. Found: C, 50.06; H, 6.21; N, 14.84; S, 8.31.
IR (KBr): 3398, 3274, 3090, 2972, 2875, 1748, 1683, 1626, 1542,
1517, 1446, 1383, 1342, 1230, 1099 cm−1
.
|
4.5.15
(4R,5R)‐5‐Methyl‐N‐{(2R)‐1‐[(2R)‐2‐
1H NMR (300 MHz, CD3OD): δ 8.97 and 8.96 (d each, J = 2.1 Hz,
total 1H), 7.35 (d, J = 2.1 Hz, 1H), 5.08 and 5.02 (dd each, J = 8.1, 6.6
Hz, total 1H), 4.86 (m, 1H), 4.33 and 4.32 (d each, J = 8.7 Hz, total 1H),
4.08 and 3.98 (m each, total 1H), 3.78 and 3.45 (m each, total 2H),
3.18 (m, 2H), 2.00−1.50 (m, 4H), 1.30 and 1.10 (d each, J = 6.6 Hz,
total 3H), 1.10 and 1.04 (d each, J = 6.6 Hz, total 3H).
methylpyrrolidin‐1‐yl]‐1‐oxo‐3‐(1,3‐thiazol‐4‐yl)propan‐2‐
yl}‐2‐oxo‐1,3‐oxazolidine‐4‐carboxamide (15)
The condensation of (2R)‐2‐[(4R,5R)‐5‐methyl‐2‐oxooxazolidine‐4‐
carboxamido]‐3‐(thiazol‐4‐yl)propanoic acid (53) (0.800 g, 2.67 mmol)
and (2R)‐2‐methylpyrrolidine p‐toluenesulfonate (23) (0.688 g, 2.67
mmol) yielded the title compound 15 (0.452 g, 46%) as colorless
needles.
[α]2D5 −2.5° (c 1.0, H2O).
Anal. Calcd for C16H22N4O4S·H2O: C, 49.99; H, 6.29; N, 14.57; S,
8.34. Found: C, 49.82; H, 6.31; N, 14.84; S, 8.26.
mp 232 °C (decomp.)

14 of 16
KOBAYASHI ET AL.
IR (KBr): 3431, 3327, 3054, 2969, 1746, 1654, 1636, 1538, 1437,
1384, 1241 cm−1
were dissolved in saline. Rectal temperature was measured with a
thermistor (MGA‐III, Nihon Kohden) before and after iv administration
of TRH and TRH mimetics up to 7 h after a dose of 50 and 0.5
μmol/kg/mL, respectively. The antagonistic effect of the test drugs
on reserpine‐induced hypothermia was evaluated based on the area
under the temperature−time curve after dosing (AUC0−7h). All of the
mice used in the experiments were killed immediately after the last
measurement.
.
1H NMR (300 MHz, CD3OD): δ 8.95 and 8.93 (d each, J = 2.1 Hz,
total 1H), 7.32 (d, J = 2.1 Hz, 1H), 5.08 (m, 1H), 4.91 (m, 1H), 4.34 and
4.31 (d each, J = 8.7 Hz, total 1H), 3.92 (m, 1H), 3.42 (m, 2H), 3.30
−3.10 (m, 2H), 2.10−1.50 (m, 4H), 1.21 and 1.15 (d each, J = 6.6 Hz,
total 3H), 1.13 and 1.04 (d each, J = 6.6 Hz, total 3H).
[α]2D5 −10.0° (c 1.0, MeOH).
Supporting Information
Anal. Calcd for C16H22N4O4S: C, 52.44; H, 6.05; N, 15.29; S, 8.75.
Found: C, 52.44; H, 5.99; N, 15.34; S, 8.78.
Details are given of the synthetic procedure and analytical data for
the intermediates (N‐terminus: 27, 28 and 54−61; middle‐part: 30, 31
and 63−65; C‐terminus: 66−69; dipeptide benzhydryl esters: 38−45,
dipeptide mimetics: 46−53).
|
4.5.16
(4R,5R)‐5‐Methyl‐N‐{(2R)‐1‐[(2S)‐2‐
methylpyrrolidin‐1‐yl]‐1‐oxo‐3‐(1,3‐thiazol‐4‐yl)
propan‐2‐yl}‐2‐oxo‐1,3‐oxazolidine‐4‐carboxamide (16)
FUNDING
This research did not receive any specific grant from funding agencies
The condensation of (2R)‐2‐[(4R,5R)‐5‐methyl‐2‐oxooxazolidine‐4‐
carboxamido]‐3‐(thiazol‐4‐yl)propanoic acid (53) (0.800 g, 2.67 mmol)
and (2S)‐2‐methylpyrrolidine p‐toluenesulfonate (24) (0.460 g, 1.79
mmol) yielded the title compound 16 (0.542 g, 55%) as a white amor-
phous powder.
in the public, commercial, or not‐for‐profit sectors.
ACKNOWLEDGEMENTS
We thank R. Nishimura, K. Yoshino, K. Kuwano and R. Ikenishi for
the elemental analysis, M. Sugita for the IR spectroscopy and optical
rotation data, Y. Takayama and J. Kikuchi for the NMR spectroscopy
data. T. Kihara, Y. Fujimura and H. Nakai for discussions concerning
this work.
IR (KBr): 3274, 2971, 2875, 1760, 1676, 1627, 1540, 1516, 1448,
1232, 1097 cm−1
.
1H NMR (300 MHz, CD3OD): δ 8.97 and 8.96 (d each, J = 2.1 Hz,
total 1H), 7.34 (m, 1H), 5.19 and 5.04 (t each, J = 7.5 Hz, total 1H),
4.92 (m, 1H), 4.37 and 4.35 (d each, J = 8.7 Hz, total 1H), 4.07 (m,
1H), 3.77 (m, 2H), 3.30−3.10 (m, 2H), 2.00−1.50 (m, 4H), 1.28 and
1.22 (d each, J = 6.3 Hz, total 3H), 1.22 and 1.02 (d each, J = 6.3 Hz,
total 3H).
ORCID
Naotake Kobayashi https://orcid.org/0000-0001-8631-9124
[α]2D4 +2.3° (c 1.0, H2O).
Anal. Calcd for C16H22N4O4S·0.2H2O: C, 51.93; H, 6.10; N, 15.14;
S, 8.66. Found: C, 51.81; H, 6.10; N, 15.40; S, 8.66.
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How to cite this article: Kobayashi N, Sato N, Sugita K, et al.
Synthesis and evaluation of in vivo anti‐hypothermic effect of
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