Stereoselective Synthesis of β-(5-Arylthiazolyl) α-Amino Acids and Use in Neurotensin Analogues

Article abstract of DOI:10.1002/ejoc.201501495

A series of new unnatural amino acids bearing a β-arylthiazole side chain was synthesized by exploiting a diastereoselective alkylation starting from glycine tert-butyl ester Schiff base with hydroxypinanone as the chiral inducer. This strategy afforded β-arylthiazole alanines in good chemical yields and with 98 % ee. Due to their aromatic properties, these newly generated amino acids were used to prepare neurotensin (NT)[8-13] analogues by serving as replacements for the native Tyr11 residue. Incorporation of the (L)-(+)-(β-phenylthiazol-4-yl)alanine residue at NT[8-13] position 11 improved plasma stability and selectivity towards NTS1, while also preserving native receptor binding affinity and biological activity. New β-arylthiazole alanines were synthesized in good chemical yields and with 98 % ee using a diastereoselective alkylation; these alanine derivatives were then used as Tyr11 replacements in the construction of neurotensin (NT)[8-13] analogues. The new NT analogues showed improved plasma stability and selectivity towards NTS1 thus preserving the hypotensive properties of the native peptide.

Full text of DOI:10.1002/ejoc.201501495

DOI: 10.1002/ejoc.201501495
Full Paper
Modified Neurotensin
Stereoselective Synthesis of ꢀ-(5-Arylthiazolyl) α-Amino Acids
and Use in Neurotensin Analogues
Denisa Hapău,[a] Emmanuelle Rémond,[b] Roberto Fanelli,[b] Mélanie Vivancos,[c]
Adeline René,[b] Jérôme Côté,[c] Élie Besserer-Offroy,[c] Jean-Michel Longpré,[c]
Jean Martinez,[b] Valentin Zaharia,[a] Philippe Sarret,[c] and Florine Cavelier*[b]
Abstract: A series of new unnatural amino acids bearing a ꢀ- newly generated amino acids were used to prepare neurotensin
arylthiazole side chain was synthesized by exploiting a dia- (NT)[8–13] analogues by serving as replacements for the native
stereoselective alkylation starting from glycine tert-butyl ester Tyr11 residue. Incorporation of the (L)-(+)-(ꢀ-phenylthiazol-4-
Schiff base with hydroxypinanone as the chiral inducer. This yl)alanine residue at NT[8–13] position 11 improved plasma sta-
strategy afforded ꢀ-arylthiazole alanines in good chemical bility and selectivity towards NTS1, while also preserving native
yields and with 98 % ee. Due to their aromatic properties, these receptor binding affinity and biological activity.
Introduction
ganocatalysts for intramolecular Stetter reactions[7] and α-thi-
azole alanines are often found in synthetic peptides as bio-
isosteres of natural occurring amino acids; such peptides have
had diverse applications such as in the preparation of integrin
inhibitors,[8] glutamate carboxypeptidase II inhibitors,[9] or pept-
idase IV inhibitors.[10] Furthermore, the metal complexation
properties of the thiazole rings were also exploited for the de-
velopment of molecules with promising anti-cancer activity.[11]
The field of new synthetic peptides and peptidomimetics is
experiencing renewed interest in recent years and several
peptides are currently used for therapeutic applications in the
treatment of cancer, diabetes, cardiovascular or renal insuffi-
ciency diseases.[1] The challenge is to synthesize more active
and more stable analogues by introducing non-proteinogenic
amino acids into the peptide sequence, thus allowing modula-
tion of properties such as steric hindrance, polarity, conforma-
tional capabilities and proteolytic stability.[2] Therefore, the de-
velopment of efficient sources for unnatural amino acids useful
in high-throughput synthesis of customized peptides is of cru-
cial interest. Among heteroaromatic amino acids, some contain-
ing a thiazole moiety are known to display antibacterial,[3] anti-
trypanosomal,[4] and antimalarial properties.[5] Many macrolac-
tams containing thiazole-derived amino acids isolated from ma-
rine organisms have generated a great deal of synthetic interest
because of their high cytotoxicity, as well as their metal binding
and transport properties.[6] Synthetic thiazole and aryl-substi-
tuted thiazole amino acids have a broad spectrum of applica-
tions. Functionalized thiazole alanines have been used as or-
The synthesis of (
ther by a stereoconservative procedure from hexafluoroace-
tone-protected ( )-aspartic and (
)-glutamic acids,[12] or from
L)-(2-arylthiazol-4-yl)alanines was described ei-
L
L
(S)-methyl 2-amino-5-bromo-4-oxopentanoate by a Hantzsch-
type condensation.[13]
We recently reported an efficient stereoselective synthesis
of silicon-containing and unsaturated amino acids exploiting
hydroxypinanone-induced diastereoselective alkylation chemis-
try.[14] We report herein the first efficient stereoselective synthe-
sis of (L)-(ꢀ-arylthiazol-4-yl)alanines. These new unnatural α-
amino acids are very promising by virtue of their interesting
properties resulting from their aromatic side chains. The diaro-
matic system can, indeed, play an important role in ligand–
receptor interactions as a result of hydrophobic properties and
π-stacking interactions. To demonstrate one valuable applica-
tion of these new entities, they have been introduced into the
structure of neurotensin (NT)[8–13] (H-Lys-Lys-Pro-Tyr-Ile-Leu-
OH) via replacement of the Tyr11 residue. NT is a neuropeptide
that elicits changes in blood pressure, variation in body temper-
ature, and naloxone-insensitive analgesic responses.[15] Struc-
ture–activity studies have demonstrated that the minimal se-
quence required for full biological activity is the C-terminal hex-
apeptide NT[8–13].[16] Among the known NT receptors, it has
been shown that NTS1 is overexpressed in various relevant tu-
mors, including ductal breast cancer[17] and pancreatic tu-
mors.[18] NTS2 recently emerged as an attractive target in pain
treatment.[19] From a drug design perspective, it is therefore
[a] Iuliu Haţieganu University of Medicine and Pharmacy, Department of
Organic Chemistry,
Victor Babeş 41, 400012 Cluj-Napoca, Romania
[b] Institut des Biomolécules Max Mousseron, IBMM, UMR-5247, CNRS,
Université Montpellier, ENSCM,
Place Eugène Bataillon, 34095 Montpellier cedex 5, France
E.mail: florine.cavelier@umontpellier.fr
http://www.umontpellier.fr/recherche/unites-de-recherche/institut-des-
biomolecules-max-mousseron-ibmm/
[c] Department of pharmacology and physiology, Institut de Pharmacologie
de Sherbrooke, Faculty of Medicine and Health Sciences, Université de
Sherbrooke,
Sherbrooke, Québec J1K2R1, Canada
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/ejoc.201501495.
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crucial to develop novel therapeutic agents that display select- amino acids were obtained by treatment with trifluoroacetic
ivity for one receptor subtype. In this respect, position 11 of NT acid (TFA) in the presence of triisopropylsilane. Finally, the
plays a key role in dictating receptor selectivity.[20] Based on amine functions were Boc-protected thus affording amino acids
the extended aromaticity of the ꢀ-arylthiazole moiety, relative 4a–d in 60–91 % yield over three steps and in > 98 % ee (as
to the Tyr11 aromatic side-chain, and comparable to a Trp side detected by chiral RP-HPLC) (Scheme 2). To evaluate ee values
chain, we have introduced these new unnatural amino acids racemic amino acids were prepared by alkylation of diethyl
into NT[8–13] and evaluated their influence on binding, stability acetamidomalonate with 2-aryl-4-(chloromethyl)thiazoles fol-
and activities of the resulting peptide analogues relative to the lowed by treatment with Boc2O.
native peptide.
Results and Discussion
The synthesis of (L)-(2-arylthiazol-4-yl)alanines was accom-
plished using as the key reaction the diastereoselective alkyl-
ation of (1R,2R,5R)-2-hydroxy-3-pinanone Schiff base of glycine
tert-butyl ester 2 (Scheme 1). The 2-aryl-4-(iodomethyl)thiazoles
derivatives 1a–d were obtained using Hantzsch condensation
of various thiobenzamides with 1,3-dichloroacetone, followed
by treatment with potassium iodide (Scheme 1).[21] The alkyl-
ation of 2 was performed by initial addition of LDA at –78 °C,
followed by addition of 4-iodomethyl-2-arylthiazoles 1a–d.
After stirring for 12 h at –78 °C, and then for 2 h at –10 °C,
corresponding alkylated Schiff bases 3a–d and 3a′–d′ were
each obtained as mixtures of two diastereomers in ratios up to
Scheme 2. Synthesis of Boc protected amino acids 4a–d.
It is well-known that, depending on the chirality of the
hydroxypinanone moiety, it is possible to control the stereo-
chemistry of the final product. The formation of the (
mer of 4d was confirmed by measuring the optical rotation of
the corresponding deprotected amino acid int-(
)-4d ([α]D20
–9.9 {c = 1.0, H2O} see Supporting Information), and comparing
with the literature value ([α]D20 = –9.5 {c = 1.0, H2O}).[12b] Thus,
starting from (1R,2R,5R)-hydroxypinan-3-one, S-4a–d were ob-
tained, in agreement with the ability of this chiral moiety to
dictate generation of the absolute S-configuration.[22] The enan-
tiomeric purities for 4a–d were determined by chiral RP-HPLC
analyses (≥ 98 % ee).
L)-enantio-
L
=
1
96:4 as determined by H NMR of crude products and isolated
in 60–82 % yield (Scheme 1, Table 1).
Table 1. , yield and chiral induction of the alkylation step.
To validate the potential of these novel unnatural amino
acids, they were introduced into the neurotensin (NT) peptide
to explore structure-activity relationships and to improve meta-
bolic peptide stabilities.[23] For this purpose, we synthesized
four new NT analogues, replacing the Tyr11 residue with (L)-(+)-
(ꢀ-arylthiazol-4-yl)alanines 4a–d (Figure 1).
Hexapeptide NT[8–13] analogues were synthesized in solu-
tion using a [3 + 3] fragment condensation strategy. This al-
lowed us to avoid racemisation during the coupling step owing
to the presence of a proline residue.[24] All coupling reactions
for the preparation of both tripeptides and hexapeptides were
performed using BOP as the condensation agent and diiso-
propylamine as base, in DMF.
After separation of major diastereoisomer 3 by chromatogra-
Boc-deprotection of the dipeptide Boc-Ile-Leu-OMe with TFA
phy, the chiral auxiliary was removed by hydrolysis and free followed by condensation with 4a–d afforded corresponding
Scheme 1. (+)-(1R,2R,5R)-2-hydroxy-3-pinanone-induced diastereoselective alkylation.
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3.46 1.345 nM for hNTS1 but displayed a binding profile with
hNTS2 (139 22.5 nM) similar to those noted with hNTS2 and
compounds 5–7. These results suggest that the presence of rich
electron-donating groups on the arylthiazoles in compounds 5
and 6 are deleterious to NTS1 interactions relative to cases in-
volving a basic arylthiazole group (compound 8). Furthermore,
incorporation of (L)-(+)-(ꢀ-arylthiazol-4-yl)alanine residues at po-
sition 11 only slightly improved peptide stabilities and resist-
ance to enzyme degradation (Table 2). This may be explained
by the fact that these chemical modifications do not appear to
prevent Lys8–Lys9 amino-terminal cleavage by the endopept-
idase EC 3.4.24.15.[25]
Based on the higher binding affinity of compounds 7 and 8
for hNTS1, we next investigated the impact of (L)-(+)-(2-arylthi-
azol-4-yl)alanine incorporation on biological activity. A growing
body of work has revealed that activation of NTS1 following
intravenous administration of NT compounds results in a drop
in blood pressure.[15,26] We thus injected compounds 7 and 8 to
Sprague Dawley rats and monitored variations in arterial blood
pressure via direct and continuous intra-carotid measurements
(Figure 2).
Figure 1. NT[8–13] analogues containing (L)-(+)-(2-arylthiazol-4-yl)alanines
4a–d.
tripeptides Boc-4a–d-Ile-Leu-OMe in very good yields (>83 %).
Further Boc-deprotection of these fragments was performed in
the presence of triisopropylsilane as a scavenger to avoid tert-
butylation of the 2-phenylthiazole moiety.[24] The condensation
of N-deprotected tripeptides with the Boc-Lys(Z)-Lys(Z)-Pro-OH
fragment afforded corresponding hexapeptides Boc-Lys(Z)-
Lys(Z)-Pro-4a–d-Ile-Leu-OMe in acceptable yields (26–35 %)
considering the steric hindrance of two tripeptides. Further sa-
ponification of the methyl ester in the presence of KOH, fol-
lowed by a final one-pot Z- and Boc-deprotection in TFA/
TFMSA/TIS, afforded the corresponding fully deprotected hexa-
peptides (5–8, Figure 1). Peptides 5–8 were then purified by
preparative RP-HPLC.
We first evaluated the ability of these NT[8–13] derivatives
5–8 carrying ꢀ-arylthiazole alanine residues to inhibit the bind-
ing of 125I-Tyr3-NT to membranes prepared from cells stably ex-
pressing either hNTS1 or hNTS2 receptors. As shown in Table 2,
insertion of the arylthiazole residues into position 11 decreased
peptide binding affinities, relative to native NT[8-13], to both
NTS1 and NTS2.
Figure 2. Effects of intravenous injection of compounds 7 (JMV5620) and 8
(JMV5621) on mean arterial pressure (ΔMAP) in anaesthetized rats. Tracings
depicting the hypotensive effects after administration of each compound at
1 mg/kg. Data are expressed as means SEM obtained with 4 animals.
Intravenous bolus administration of 1 mg/kg of compounds
7 and 8 led to a short-lasting depressor phase of nearly 40 Torr
followed by a pressor phase starting after 30 s. This return of
arterial blood pressure to baseline values within less than 1 min
is probably induced by the release of catecholamines from the
adrenal medulla.[27] Finally, compound 8 produced a final sus-
tained and pronounced hypotensive phase, as previously ob-
served with the native NT peptide.[23a] However, in the same
experimental paradigm, compound 7 failed to induce a sus-
tained hypotensive effect at the employed dosage.
Table 2. Binding and stability of neurotensin analogues.
Compound
hNTS1
hNTS2
Selectivity Plasma
hNTS1 vs. stability
IC50 [nM]
IC50 [nM]
hNTS2
(half-life min)
NT[8–13]
1.01 0.09
1377 312
1285 217
55.1 17.7
3.46 1.34
5.52 2.23
86.9 16.2
80.0 25.3
81.1 13.6
139 22.5
5.46
0.06
0.06
1.47
40.3
0.78 0.10
1.80 0.14
1.40 0.11
1.25 0.06
2.70 0.51
5 (JMV5618)
6 (JMV5619)
7 (JMV5620)
8 (JMV5621)
Conclusions
Compounds 5 and 6, incorporating either (
L
)-(+)-(ꢀ-p-tolylthi-
azol-4-yl)alanine or (
L
)-(+)-(ꢀ-m-tolylthiazol-4-yl)alanine residue, In conclusion, we have developed a synthesis of new ꢀ-arylthi-
respectively at position 11, displayed improved selectivity to azole-based α-amino acids using diastereoselective alkylation
NTS2 with respect to 7 and 8. This is in accordance with previ- of (1R,2R,5R)-2-hydroxy-3-pinanone Schiff base with good yields
ous studies demonstrating that replacement of Tyr11 leads to and 98 % ee. Introduction of these new amino acids at position
improved NTS2 receptor selectivity.[20a] However, we found that 11 of NT[8–13] was achieved yielding new NT analogs. Incorpo-
binding interactions with compound 8, in which the Tyr11 resi- ration of (
due was substituted by a ( )-(+)-(ꢀ-phenylthiazol-4-yl)alanine 11 of NT[8–13] resulted in NT analogs possessing good affinity
residue, remained in the low nanomolar range with an IC50 of for, and selectivity towards, NTS1, while also preserving the hy-
L)-(+)-(2-phenylthiazol-4-yl)alanine residue in position
L
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4-(Iodomethyl)-(2-m-tolyl)thiazole (1b): Compound 1b (64 %
yield) was obtained as a white solid following the described general
procedure, starting from 3-methylthiobenzamide (2.26 g, 15 mmol)
and 1,3-dichloroacetone (1.91 g, 15 mmol), m.p. 71 °C. 1H NMR
(CDCl3, 400 MHz): δ = 2.45 (s, 3 H, CH3), 4.60 (s, 2 H, CH2I), 7.29 (s,
2 H, Hthiazole and Harom), 7.33–7.35 (m, 1 H, Harom), 7.74 (d, J = 8.0 Hz,
1 H, Harom), 7.81 (s, 1 H, Harom) ppm. 13C NMR (CDCl3, 75 MHz):
δ = –0.9, 21.8, 116.4, 124.3, 127.6, 129.3, 131.6, 133.7, 139.3, 154.4,
potensive properties of native NT. Modifications to the absolute
configuration and aromatic portions of the arylthiazole-derived
amino acids are currently under investigation.
Experimental Section
General: All reactions involving air-sensitive reagents were per-
formed under argon. Thin layer chromatography was carried out
using Merck Kieselgel 60 F254 sheets. Spots were visualized by
treatment with iodine in the case of alkylated Schiff bases and by
treatment with an ethanolic solution of ninhydrin 10 %, followed
by heating in the case of amino acids and peptides. Purifications
were performed with column chromatography using silica gel
(Merck 60, 230–400 mesh) or with a Biotage Isolera 4 instrument
using SNAP KP-SIL flash cartridges. Melting points were determined
with a Buchi 510 melting point apparatus and are uncorrected. In-
frared spectra were recorded using a Perkin–Elmer spectrum 100
FT-IR spectrometer. Proton nuclear magnetic resonance 1H NMR and
carbon nuclear magnetic resonance 13C NMR spectra were recorded
with a Bruker Avance DPX-300 spectrometer at 300 and 75 MHz
respectively. All chemical shifts were recorded as values (ppm) rela-
tive to internal tetramethylsilane when CDCl3 was used as solvent.
Melting points were determined with a Büchi 510 melting point
apparatus and are uncorrected. Low-resolution electrospray ioniza-
tion (ESI) mass spectra were recorded using a micromass platform
electrospray mass spectrometer. Spectra were recorded in positive
mode (ESI+). Optical rotations values were measured with a Perkin–
Elmer 341 polarimeter (20 °C, sodium ray). High resolution mass
spectra (HRMS) were recorded using a Synapt G2-S (Waters) mass
detector operating at capillary tension of 1 kV and cone tension of
20 V. HPLC analyses were performed with a Beckman System Gold
126 instrument variable detector using column Chiracel OD-RH
(0.46 cm × 15 cm), flow: 1 mL/min, H2O (0.1 % TFA)/CH3CN
(0.1 % TFA).
169.2 ppm. FT-IR: ν = 3080, 1590, 1460, 1435, 1415, 1162, 1144,
˜
1002, 782, 761 cm–1. HRMS calcd. for [M + H]+ C11H11NSI: 315.9657,
found 315.9656.
4-(Iodomethyl)-2-(p-chlorophenyl)-thiazole (1c): Compound 1c
(63 % yield) was obtained as a white solid, following the described
general procedure, starting from 4-chlorothiobenzamide (2.57 g,
15 mmol) and 1,3-dichloroacetone (1.91 g, 15 mmol), m.p. 123 °C.
1H NMR (CDCl3, 400 MHz): δ = 4.56 (s, 2 H, CH2I), 7.25 (s, 1 H,
Hthiazole), 7.40 (d, J = 8.0 Hz, 2 H, Harom), 7.88 (d, J = 8.0 Hz, 2 H,
H
arom) ppm. 13C NMR (CDCl3, 75 MHz): δ = –1.9, 116.1, 127.6, 129.0,
131.6, 136.1, 154.1, 166.9 ppm. FT-IR: ν = 3080, 1370, 990, 920, 840,
˜
755, 704 cm–1. HRMS calcd. for [M + H]+ C10H8NSClI: 335.9111,
found 335.9112.
4-(Iodomethyl)-2-phenylthiazole (1d): Compound 1d (60 % yield)
was obtained as a white solid using thiobenzamide (2.05 g,
15 mmol) and 1,3-dichloroacetone (1.91 g, 15 mmol), m.p. 104 °C.
1H NMR (CDCl3, 400 MHz): δ = 4.58 (s, 2 H, CH2I), 7.25 (s, 1 H,
H
thiazole), 7.43–7.45 (m, 3 H, Harom), 7.93–7.96 (m, 2 H, Harom) ppm.
13C NMR (CDCl3, 75 MHz): δ = –1.3, 116.2, 126.8, 129.1, 130.4, 133.5,
154.2, 168.6 ppm. FT-IR: ν = 3080, 1509, 1497, 1460, 1144,
˜
1003 cm–1. HRMS calcd. for [M + H]+ C10H9NSI: 301.9500, found
301.9499.
Diasteroselective Synthesis of Compounds 3a–d: To a solution
of diisopropylamine (0.62 mL, 4.40 mmol) in dry THF (8.5 mL) was
added slowly n-butyllithium 2.5
M in n-hexane (1.69 mL, 4.22 mmol)
at –10 °C. After 30 min, the mixture was cooled to –78 °C and Schiff
base 2 (494 mg, 1.76 mmol) dissolved in THF (1.5 mL) was added.
After 30 min, one of the corresponding 4-(iodomethyl)-2-arylthia-
zoles 1a–d (3.17 mmol, 1.8 equiv.) dissolved in THF (1.5–2 mL) was
added slowly. The mixture was stirred at –78 °C for 12 h, and then at
–10 °C for 2 h. The reaction mixture was quenched with a saturated
solution of ammonium chloride (10 mL). THF was removed under
reduced pressure and the aqueous phase was extracted three times
with ethyl acetate (3 × 10 mL). The organic layer was dried with
anhydrous MgSO4, filtered and concentrated in vacuo. The dia-
stereomeric ratio was determined by 1H NMR of the crude product.
Separation of diastereoisomers 3a–d was performed by flash chro-
matography on silica gel using (cyclohexane/ethyl acetate, 9:1, then
8:2, and 7:3 with 1 % Et3N) as eluent.
General Procedure for the Preparation of 4-(Iodomethyl)-2-aryl-
thiazole 1a–d: A mixture of thiobenzamide (2.05 g, 15 mmol) and
1,3-dichloroacetone (1.91 g, 15 mmol) dissolved in anhydrous acet-
one (20 mL) was stirred at room temperature for 24 h. The formed
precipitate was filtered, dried and treated with concentrated sulfuric
acid. After 3 h, the mixture was poured into ice and the formed
precipitate was filtered, washed with water until neutral pH and
dried. The resulting solid (2.1 g, 10 mmol) was dissolved in acetone
(20 mL) and treated with a solution of NaI (1.8 g, 12 mmol) in
acetone (20 mL). The reaction mixture was refluxed for 1 h and
filtered. The filtrate was partially concentrated under reduced pres-
sure, poured into ice and the formed precipitate of 4-(iodomethyl)-
2-phenylthiazole 1d was filtered and dried. The crude product was
recrystallized from ethanol/H2O. The 4-(iodomethyl)-2-aryl thiazoles
1a–c were obtained by the same procedure, starting from 1,3-di-
chloroacetone and the corresponding 4-methylthiobenzamide, 3-
methylthiobenzamide and 4-chlorothiobenzamide, respectively.
Compound 3a: Compound 3a (304 mg, 57 %, dr 95:5) was obtained
as a yellow oil following the general alkylation procedure of Schiff
base 2 (317 mg, 1.13 mmol) and 4-(iodomethyl)-2-(p-tolyl)thiazole
(1a) (640 mg, 2.03 mmol). Rf = 0.34 (cyclohexane/ethyl acetate, 7:3).
1H NMR (CDCl3, 300 MHz): δ = 0.27 (s, 3 H, CH3 bridge), 1.14 (s, 3 H,
CH3 bridge), 1.26 (s, 3 H, C(OH)CH3), 1.44 (s, 9 H, OtBu), 1.86–1.93 (m,
2 H, CH2CHC(OH)CH3), 2.18–2.37 (m, 5H (m, 2 H, CHC(CH3)2 over-
lapped with s, 3 H, Ar-CH3)), 2.50–2.56 (m, 2 H, CH2CN), 3.19–3.27
(m, 1 H, CH2CHα), 3.43–3.49 (m, 1 H, CH2CHα), 4.75–4.79 (m, 1 H,
CH2CHα), 6.86 (s, 1 H, CHthiazole), 7.22 (d, J = 7.81 Hz, 2 H, Harom),
7.74 (d, J = 7.98 Hz, 2 H, Harom) ppm. 13C NMR (CDCl3, 75 MHz): δ =
21.4, 22.3, 27.2, 28.1, 28.4, 33.3, 34.8, 38.1, 38.3, 49.9, 62.1, 81.3,
115.0, 126.3, 129.5, 131.2, 140.1, 154.4, 167.9, 170.1, 178.8 ppm.
4-(Iodomethyl)-(2-p-tolyl)thiazole (1a): Compound 1a (63 %
yield) was obtained as a white solid, following the described gen-
eral procedure, starting from 4-methylthiobenzamide (2.26 g,
15 mmol) and 1,3-dichloroacetone (1.91 g, 15 mmol), m.p. 128 °C.
1H NMR (CDCl3, 400 MHz): δ = 2.39 (s, 3 H, CH3), 4.57 (s, 2 H, CH2I),
7.21 (s, 1 H, Hthiazole), 7.24 (d, J = 8.0 Hz, 2 H, Harom), 7.83 (d, J =
8.0 Hz, 2 H, Harom) ppm. 13C NMR (CDCl3, 75 MHz): δ = –0.8, 21.9,
116.1 127.0, 130.1, 131.2, 141.0, 154.3, 169.1 ppm. FT-IR: ν = 3080,
˜
1540, 1450, 1410, 1390, 1160, 990, 950, 820 cm–1. HRMS calcd. for Compound 3b: Compound 3b (304 mg, 57 %, dr 96:4) was ob-
[M + H]+ C11H11NSI: 315.9657, found 315.9660.
tained as a yellow oil following the general alkylation procedure of
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Schiff base 2 (317 mg, 1.13 mmol) and 4-(iodomethyl)-2-(m-tolyl)thi-
Boc-(L)-(ꢀ-(p-tolyl)thiazole-4-yl)alanine (4a): Compound S-4a
azole 1b (640 mg, 2.03 mmol). Rf = 0.34 (cyclohexane/ethyl acetate, (198 mg, 91 % overall yield) were obtained as a yellow solid by
1
7:3). H NMR (CDCl3, 300 MHz): δ = 0.20 (s, 3 H, CH3 bridge), 1.06 (s,
treatment of mono-alkylated Schiff base 3a (283 mg, 0.60 mmol),
3 H, CH3 bridge), 1.18 (s, 3 H, C(OH)CH3), 1.44 (s, 9 H, OtBu), 1.86– m.p. 97 °C. MS ESI+ for C18H22N2O4S: [M + H]+ = 363.3. [α]D20 = +9
1.93 (m, 2 H, CH2CHC(OH)CH3), 2.21–2.38 (m, 5H (m, 2 H, CHC(CH3)2
1
(c = 1.0, CHCl3). H NMR (CDCl3, 300 MHz): δ = 1.63 (s, 9 H, OtBu),
overlapped with s, 3 H, Ar-CH3)), 2.45–2.55 (m, 2 H, CH2CN), 3.11– 2.58 (s, 3 H, CH3), 3.44–3.58 (m, 2 H, CH2), 4.73 (m, 1 H, CHα), 6.19
3.19 (m, 1 H, Het-CH2CHα), 3.35–3.41 (m, 1 H, Het-CH2CHα), 4.65– (br. s, 1 H, NH), 7.28 (s, 1 H, Hthiazole), 7.43 (d, J = 7.8 Hz, 2 H, Harom),
4.69 (m, 1 H, Het-CH2CHα), 6.80 (s, 1 H, CHthiazole), 7.09–7.21 (m, 2 7.94 (d, J = 7.9 Hz, 2 H, Harom), 10.39 (br. s, 1 H, OH) ppm. 13C NMR
H, Harom), 7.54–7.59 (m, 2 H, Harom) ppm. 13C NMR (CDCl3, 75 MHz):
δ = 21.1, 22.0, 26.9, 27.0, 27.8, 28.1, 33.0, 34.5, 37.8, 38.0, 49.6, 61.8,
81.1, 115.1, 123.2, 126.6, 128.5, 130.4, 133.4, 138.3, 154.2, 167.7,
169.8, 178.6 ppm.
(CDCl3, 75 MHz): δ = 21.5, 28.3, 33.2, 53.2, 80.1, 115.3, 126.5, 129.7,
129.9, 141.2, 151.9, 155.6, 169.2, 173.7 ppm. HRMS calcd. for [M +
H]+ C18H23N2O4S: 363.1379, found 363.1380. The enantiomeric ex-
cess ee = 98.1 % was determined by RP-HPLC on chiral column OD-
RH, ACN (0.1 % TFA)/H2O (0.1 % TFA) 35:65, 1 mL min–1, λ = 214 nm,
20 °C, tR (S) = 18.97 min, tR (R) = 20.67 min.
Compound 3c: Compound 3c (364 mg, 44 %, dr 93:7) was obtained
as colorless oil following the general alkylation procedure of Schiff
base 2 (494 mg, 1.76 mmol) and 4-(iodomethyl)-2-(p-chloro-
phenyl)thiazole (1c) (1.06 g, 3.16 mmol). Rf = 0.34 (cyclohexane/
ethyl acetate, 7:3). 1H NMR (CDCl3, 300 MHz): δ = 0.21 (s, 3 H,
CH3 bridge), 1.10 (s, 3 H, CH3 bridge), 1.19 (s, 3 H, C(OH)CH3), 1.39 (s, 9
H, OtBu), 1.81–1.87 (m, 2 H, CH2CHC(OH)CH3), 2.20–2.36 (m, 2 H,
CHC(CH3)2), 2.48–2.62 (m, 2 H, CH2CN), 3.19–3.24 (m, 1 H, CH2CHα),
3.37–3.38 (m, 1 H, CH2CHα), 4.68–4.72 (m, 1 H, CH2CHα), 6.87 (s, 1
H, CHthiazole), 7.32 (d, J = 8.6 Hz, 2 H, Harom), 7.75 (d, J = 8.6 Hz, 2 H,
Harom) ppm. 13C NMR (CDCl3, 75 MHz): δ = 22.2, 27.1, 28.0, 28.3,
33.3, 34.7, 38.0, 38.2, 49.9, 62.0, 81.5, 115.7, 127.5, 129.1, 132.2,
135.8, 154.8, 166.3, 169.9, 179.1 ppm.
Boc-(L)-(ꢀ-(m-tolyl)thiazole-4-yl)alanine (4b): Compound S-4b
(150 mg, 64 % overall yield) were obtained as a yellow solid by
treatment of mono-alkylated Schiff base 3b (300 mg, 0.65 mmol),
m.p. 53–54 °C. MS ESI+ for C18H22N2O4S: [M + H]+ = 363.2. [α]D20
=
+13 (c = 1.0, CHCl3). 1H NMR (CDCl3, 300 MHz): δ = 1.37 (s, 9 H,
OtBu), 2.34 (s, 3 H, CH3), 3.14–3.32 (m, 2 H, CH2), 4.47 (m, 1 H, CHα),
5.94 (d, J = 5.8 Hz, 1 H, NH), 7.03 (s, 1 H, Hthiazole), 7.19–7.26 (m, 2
H, Harom), 7.57–7.60 (m, 2 H, Harom), 10.42 (br. s, 1 H, OH) ppm. 13C
NMR (CDCl3, 75 MHz): δ = 21.4, 28.3, 33.2, 53.2, 80.2, 115.7, 123.8,
127.1, 129.1, 131.7, 132.0, 139.1, 151.9, 155.7, 169.4, 173.6 ppm.
HRMS calcd. for [M + H]+ C18H23N2O4S: 363.1379, found 363.1375.
The enantiomeric excess ee = 99.1 % was determined by RP-HPLC
on chiral column OD-RH, ACN (0.1 % TFA)/H2O (0.1 % TFA) 35:65,
1 mL min–1, λ = 280 nm, 20 °C, tR (S) = 19.25 min, tR (R) = 21.53 min.
Compound 3d: Compound 3d (315 mg, 44 %, dr 96:4) was ob-
tained as a yellow oil following the general alkylation procedure of
Schiff base 2 (466 mg, 1.56 mmol) and 4-(iodomethyl)-2-phenylthia-
zole 1d (860 mg, 2.86 mmol). Rf = 0.3 (cyclohexane/ethyl acetate,
Boc-(L)-(ꢀ-(p-chlorophenyl)thiazole-4-yl)alanine (4c): Compound
S-4c (238 mg, 82 % overall yield) were obtained as a yellow solid
by treatment of mono-alkylated Schiff base 3c (371 mg, 0.76 mmol),
1
7:3). H NMR (CDCl3, 300 MHz): δ = 0.27 (s, 3 H, CH3 bridge), 1.14 (s,
m.p. 143 °C. MS ESI+ for C17H19ClN2O4S: [M + H]+ = 383.2. [α]D20
=
3 H, CH3 bridge), 1.26 (s, 3 H, C(OH)CH3), 1.44 (s, 9 H, OtBu), 1.86–1.94
(m, 2 H, CH2CHC(OH)CH3), 2.18–2.37 (m, 2 H, CHC(CH3)2), 2.50–2.62
(m, 2 H, CH2CN), 3.21–3.26 (m, 1 H, Het-CH2CHα), 3.44–3.50 (m, 1 H,
Het-CH2CHα), 4.75–4.80 (m, 1 H, Het-CH2CHα), 6.91 (s, 1 H, CHthiazole),
7.39–7.40 (m, 3 H, Harom), 7.85–7.87 (m, 2 H, Harom) ppm. 13C NMR
(CDCl3, 75 MHz): δ = 22.2, 27.1, 28.0, 28.4, 33.3, 34.8, 37.8, 38.2,
49.9, 62.0, 81.3, 115.4, 126.3, 128.8, 129.8, 133.7, 154.5, 167.6, 170.1,
178.8 ppm.
+14 (c = 1.0, CHCl3). 1H NMR (CDCl3, 300 MHz): δ = 1.60 (s, 9 H,
OtBu), 3.46–3.51 (m, 2 H, CH2), 4.77–4.78 (m, 1 H, CHα), 6.04 (d, J =
5.6 Hz, 1 H, NH), 7.43 (s, 1 H, Hthiazole), 7.54 (d, J = 8.3 Hz, 2 H, Harom),
7.95 (d, J = 8.5 Hz, 2 H, H arom), 9.89 (br. s, 1 H, OH) ppm. 13C NMR
(CDCl3, 75 MHz): δ = 28.3, 33.1, 53.1, 80.3, 116.1, 127.7, 129.3, 131.1,
136.5, 152.4, 155.7, 167.4, 174.5 ppm. HRMS calcd. for [M + H]+
C17H20N2O4SCl: 383.0832, found 383.0834. The enantiomeric excess
ee = 98 % was determined by RP-HPLC on chiral column OD-RH,
ACN (0.1 % TFA)/H2O (0.1 % TFA) 35:65, 1 mL min–1, λ = 214 nm,
20 °C, tR (S) = 19.75 min, tR (R) = 22.25 min.
Synthesis of Boc-(
L)-(ꢀ-arylthiazole-4-yl)alanines 4a–d: To a solu-
tion of alkylated Schiff base 3a–d (0.2
M) in THF, was added a solu-
tion of citric acid 15 % (10 mL for 1 mmol of 3a–d). The mixture
was stirred at room temperature for 3 d. After removing THF in
vacuo, the aqueous layer was extracted with diethyl ether (ratio
organic layer/aqueous layer = 1:1), in order to remove the hydroxy-
pinanone. The pH was then adjusted to 8–9 by addition of K2CO3.
The aqueous layer was extracted three times with dichloromethane
(ratio organic layer/aqueous layer: 2:1). After concentration, the cor-
responding solution of (ꢀ-arylthiazol-4-yl)alanine tert-butyl ester
was treated with a mixture of TFA/triisopropylsilane (10:1) (4 mL of
TFA and 0.4 mL of triisopropylsilane for 1 mmol). The mixture was
stirred at room temperature for 4 h. Then the solvent was evapo-
rated and the acid excess was removed by coevaporation with cy-
clohexane. The crude product was treated with Boc2O (1.2 equiv.),
in THF/H2O (1:1) at pH 8–9, adjusted with NaHCO3. The reaction
mixture was stirred at room temperature overnight. After comple-
tion of the reaction, THF was removed under reduced pressure and
the pH was adjusted to 3 with citric acid. The aqueous layer was
Boc-(L)-(ꢀ-phenylthiazole-4-yl)alanine (4d): Compound S-4d
(142 mg, 60 % overall yield) were obtained as a yellow solid by
treatment of mono-alkylated Schiff base 3d (310 mg, 0.68 mmol),
m.p. 57 °C. MS ESI+ for C17H20N2O4S: [M + H]+ = 349.2. [α]D20 = +17
1
(c = 1.0, CHCl3). H NMR (CDCl3, 300 MHz): δ = 1.57 (s, 9 H, OtBu),
3.33–3.54 (m, 2 H, CH2), 4.64–4.65 (m, 1 H, CHα), 6.11 (d, J = 2.1 Hz,
1 H, NH), 7.38 (s, 1 H, CHthiazole), 7.58–7.59 (m, 3 H, Harom), 7.98–7.99
(m, 2 H, Harom), 9.06 (br. s, 1 H, OH) ppm. 13C NMR (CDCl3, 75 MHz):
δ = 28.3, 33.2, 53.2, 80.3, 115.9, 126.5, 129.3, 130.9, 132.0, 133.0,
152.0, 155.7, 169.2, 173.2 ppm. HRMS calcd. for [M
+
H]+
C17H21N2O4S: 349.1222, found 349.1225. The enantiomeric excess
ee = 97 % was determined by RP-HPLC on chiral column (OD-RH,
ACN (0.1 % TFA)/H2O (0.1 % TFA) 30:70, 1 mL min–1, λ = 214 nm,
20 °C, tR (S) = 27.65 min, tR (R) = 30.38 min.
General Procedure for Coupling N-Protected Amino Acid with
C-Protected Amino Acid: To a solution of a C-protected amino acid
extracted three times with ethyl acetate (ratio organic layer/aque- (1 equiv.) in DMF (10 mL/mmol) were added BOP (1 equiv.), the
ous layer = 2:1). The organic layer was dried with anhydrous MgSO4 N-protected amino acid (1 equiv.) and then diisopropylethylamine
and filtered. The solvent was removed to afford the corresponding (2.5 equiv.). The reaction mixture was stirred at room temperature
Boc-( )-(2-arylthiazole-4-yl)alanines 4a–d. for 1.5 h. After removing DMF, the crude product was diluted in
L
Eur. J. Org. Chem. 2016, 1017–1024
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ethyl acetate (20 mL/mmol). The organic layer was washed with a Boc-(L)-4b-Ile-Leu-OMe: 143 mg of Boc-Ile-Leu-OMe (0.4 mmol)
solution of potassium hydrogen sulfate 1 (3 × 20 mL), a saturated
M
and 145 mg of (L)-3b (0.4 mmol) were used to afford 200 mg of
solution of sodium hydrogen carbonate (3 × 20 mL) and then a
saturated solution of sodium chloride (3 × 20 mL). The organic layer
was then dried with anhydrous MgSO4, filtered and concentrated.
Boc-(L)-4b-Ile-Leu-OMe as a colorless oil in 83 % yield; Rf = 0.55
(cyclohexane/ethyl acetate, 8:2); tR = 2.8 min. ESI-MS: [M + H]+
=
603.8.
General Procedure for Deprotecting Boc Group: A solution of a
N-protected peptide in TFA (4 mL/mmol) was stirred at room tem-
perature for 1 h. In the case of peptides containing amino acids
4a–d, which are sensitive to Friedel and Crafts alkylation, 0.1 mol-
% of triisopropylsilane was added to TFA. Excess TFA was then re-
moved in vacuo using cyclohexane as co-solvent.
Boc-(
and 153 mg of (
L
)-4c-Ile-Leu-OMe: 143 mg of Boc-Ile-Leu-OMe (0.4 mmol)
L)-3c (0.4 mmol) were used to afford 219 mg of
Boc-(L)-4c-Ile-Leu-OMe as a colorless oil in 88 % yield; Rf = 0.55
(cyclohexane/ethyl acetate, 8:2); tR = 2.8 min. ESI-MS: [M + H]+
=
624.3.
Boc-(
L
)-4d-Ile-Leu-OMe: 143 mg of Boc-Ile-Leu-OMe (0.4 mmol)
Boc-Lys(Z)-Pro-OMe: The dipeptide was obtained as described in
protocol 2.3.1 starting from H-Pro-OMe and Boc-Lys(Z)-OH. The di-
peptide was purified by flash chromatography on silica gel (cyclo-
hexane/ethyl acetate, 4:6) and isolated with 61 % yield. Analytical
RP-HPLC: tR = 2.3 min. Rf = 0.35 (cyclohexane/ethyl acetate, 4:6).
ESI-MS: [M + H]+ = 492.2.
and 139 mg of (
L
)-3d (0.4 mmol) were used to afford 229 mg of
Boc-(L)-3c-Ile-Leu-OMe as a colorless oil in 97 % yield; Rf = 0.55
(cyclohexane/ethyl acetate, 8:2). tR = 2.7 min. ESI-MS: [M + H]+
=
589.8.
General Procedure for Synthesis of Boc-Lys(Z)-Lys(Z)-Pro-(L)-4a–
d-Ile-Leu-OMe: A solution of Boc-( )-4a–d-Ile-Leu-OMe was stirred
L
Boc-Lys(Z)-Lys(Z)-Pro-OMe: Boc-Lys(Z)-Pro-OMe (2.65 g, 5.4 mmol)
was N-deprotected according to procedure 2.3.2. with 20 mL of TFA.
The obtained TFA·H-Lys(Z)-Pro-OMe was coupled in DMF (50 mL)
with Boc-Lys(Z)-OH·DCHA (3.02 g, 1 equiv.) according to protocol
3.2.1 with BOP (2.4 g, 1 equiv.) and diisopropylamine (2.35 mL,
2.5 equiv.). The tripeptide was purified by silica flash chromatogra-
phy (cyclohexane/ethyl acetate, 1:9 v/v) and isolated with 62 %
yield. Analytical RP-HPLC: tR = 2.6 min. ESI-MS: [M + H]+ = 754.4.
in TFA (4 mL/mmol) and 0.1 mol-% of triisopropylsilane at room
temperature for 1 h. Excess TFA was then removed in vacuo using
cyclohexane as a co-solvent. To a solution of the corresponding salt
TFA·H-(L)-4a–d-Ile-Leu-OMe in DMF (2 mL for 0.5 mmol) was added
BOP (1.5 equiv.), then Boc-Lys(Z)-Lys(Z)-Pro-OH (1 equiv.) and diiso-
propylamine (2.5 equiv.). The mixture was stirred overnight at room
temperature, DMF was removed and the crude product was diluted
in ethyl acetate (20 mL/mmol). The organic layer was successively
washed with a solution of potassium hydrogen sulfate 1
M (3 ×
Boc-Lys(Z)-Lys(Z)-Pro-OH: Boc-Lys(Z)-Lys(Z)-Pro-OMe (4.25 g,
20 mL), a saturated solution of sodium hydrogen carbonate (3 ×
20 mL) and then a saturated solution of sodium chloride (3 ×
20 mL). The organic layer was dried with anhydrous MgSO4, filtered
and concentrated. The crude product was purified by column chro-
matography on silica gel, using a gradient separation procedure
with a mixture of chloroform/methanol (99:1, then 95:5).
5.86 mmol) was dissolved in methanol (55 mL) and was treated
with a solution of KOH (4 N) (23.45 mmol, 5.86 mL). The reaction
mixture was stirred overnight at room temperature. After the evap-
oration of methanol, the pH of the aqueous solution was adjusted
to 2–3 with citric acid. The product was extracted three times with
ethyl acetate (ratio organic layer: aqueous layer = 2:1). The organic
layer was dried with anhydrous MgSO4, filtered and concentrated,
affording the tripeptide in quantitative yield. Analytical RP-HPLC:
tR = 2.3 min. ESI-MS: [M + H]+ = 740.4.
Boc-Lys(Z)-Lys(Z)-Pro-(
Lys(Z)-Pro·OH (0.16 mmol) and 98 mg of TFA·H-(
(0.16 mmol) were used to afford 102 mg of Boc-Lys(Z)-Lys(Z)-Pro-
)-4a-Ile-Leu-OMe as a colorless oil. 35 % yield; tR = 3 min. ESI-MS:
[M + H]+ = 1224.7; [M + Na]+ = 1246.8.
L)-4a-Ile-Leu-OMe: 118 mg of Boc-Lys(Z)-
L)-4a-Ile-Leu-OMe
(
L
Boc-Ile-Leu-OMe: H-Leu-OMe (3 g, 7.62 mmol) was coupled in DMF
(80 mL) with Boc-Ile-OH (1.76 g, 1 equiv.) according to protocol
2.3.1 with BOP (3.37 g, 1 equiv.) and diisopropylamine (3.31 mL,
2.5 equiv.). The dipeptide was purified by silica gel chromatography
(cyclohexane/ethyl acetate, 7:3) and isolated with 93 % yield. Rf =
0.5 (cyclohexane/ethyl acetate, 7:3). ESI-MS: [M + H]+ = 435.0.
Boc-Lys(Z)-Lys(Z)-Pro-( )-4b-Ile-Leu-OMe: 244 mg of Boc-Lys(Z)-
Lys(Z)-Pro·OH (0.33 mmol) and 203 mg of TFA·H-( )-4b-Ile-Leu-OMe
(0.33 mmol) were used to afford 143 mg of Boc-Lys(Z)-Lys(Z)-Pro-
L
L
(L)-4b-Ile-Leu-OMe as a colorless oil. 30 % yield; tR = 3 min. ESI-MS:
[M + H]+ = 1224.8.
General Procedure for Synthesis of Boc-(L)-4a–d-Ile-Leu-OMe: A
solution of Boc-Ile-Leu-OMe was stirred in TFA (4 mL/mmol) and
0.1 mol-% of triisopropylsilane at room temperature for 1 h. Excess
TFA was then removed in vacuo using cyclohexane as a co-solvent.
To a solution of the corresponding salt TFA·H-Ile-Leu-OMe in DMF
(10 mL/mmol) were added successively, BOP (1 equiv.), then N-pro-
Boc-Lys(Z)-Lys(Z)-Pro-(
Lys(Z)-Pro·OH (0.35 mmol) and 223 mg of TFA·H-(
L
)-4c-Ile-Leu-OMe: 240 mg of Boc-Lys(Z)-
)-4c-Ile-Leu-OMe
(0.35 mmol) were used to afford 130 mg of Boc-Lys(Z)-Lys(Z)-Pro-
L
(L)-4c-Ile-Leu-OMe as a colorless oil. 30 % yield; tR = 3 min. ESI-MS:
[M + H]+ = 1244.7.
tected amino acid (L)-4a–d (1 equiv.) and diisopropylethylamine
Boc-Lys(Z)-Lys(Z)-Pro-(L)-4d-Ile-Leu-OMe: 288 mg of Boc-Lys(Z)-
Lys(Z)-Pro·OH (0.4 mmol) and 246 mg of TFA·H-(L)-4d-Ile-Leu-OMe
(0.4 mmol) were used to afford 122 mg of Boc-Lys(Z)-Lys(Z)-Pro-( )-
(2.5 equiv.). The reaction mixture was stirred at room temperature
for 1.5 h. After removing DMF, the crude product was diluted in
ethyl acetate (20 mL/mmol). The organic layer was washed with a
solution of potassium hydrogen sulfate 1M (3 × 20 mL), a saturated
solution of sodium hydrogen carbonate (3 × 20 mL) and then a
saturated solution of sodium chloride (3 × 20 mL). The organic layer
was then dried with anhydrous MgSO4, filtered and concentrated.
The tripeptides were isolated in 82–97 % yield.
L
4d-Ile-Leu-OMe as a colorless oil. 26 % yield tR = 2.9 min. ESI-MS:
[M + H]+ = 1210.8; [M + Na]+ = 1232.7.
Boc-Lys(Z)-Lys(Z)-Pro-(
)-4a–d-Ile-Leu-OMe was dissolved in methanol (2 mL/0.1 mmol)
and was treated with a solution of KOH (4 ) (5 equiv.). The reaction
L)-4a–d-Ile-Leu-OH: Boc-Lys(Z)-Lys(Z)-Pro-
(L
N
Boc-(
and 145 mg of (
L
)-4a-Ile-Leu-OMe: 143 mg of Boc-Ile-Leu-OMe (0.4 mmol)
mixture was stirred overnight at room temperature. After the evap-
oration of methanol, the aqueous solution was treated with citric
acid to pH = 2–3. The product was extracted three times with ethyl
acetate (ratio organic layer: aqueous layer = 2:1). The organic layer
was dried with anhydrous MgSO4, filtered and concentrated.
L)-3a (0.4 mmol) were used to afford 198 mg of
Boc-(L)-4a-Ile-Leu-OMe as a colorless oil in 82 % yield; Rf = 0.55
(cyclohexane/ethyl acetate, 8:2); tR = 2.8 min. ESI-MS: [M + H]+
=
603.8.
Eur. J. Org. Chem. 2016, 1017–1024
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1022
© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
Boc-Lys(Z)-Lys(Z)-Pro-(
Lys(Z)-Pro-(L)-4a-Ile-Leu-OH (0.056 mmol) were used to afford
L
)-4a-Ile-Leu-OH: 69 mg of Boc-Lys(Z)-
half of the [125I][Tyr3]Neurotensin-specific binding. Data were plot-
ted using GraphPad Prism 6 using the One-site – Fit log (IC50) and
represent the mean SEM of at least three separate experiments.
55 mg of Boc-Lys(Z)-Lys(Z)-Pro-(L)-4a-Ile-Leu-OH as a colorless oil
in 81 % yield; tR = 2.8 min. ESI-MS: [M + H]+ = 1210.7; [M + Na]+
=
Animal Procedures: All animal procedures were approved by the
Ethical and Animal Care Committee of the Université de Sherbrooke
and were in accordance with policies and directives of the Canadian
Council on Animal Care. Adult male (250–300 g) Sprague-Dawley
rats (Charles River Laboratories, St-Constant, Québec, Canada) were
maintained on a 12 h light/dark cycle with free access to food and
water. Rats were acclimatized for 4 d to the animal facility and for
2 d to manipulations and devices prior to behavioral studies.
1232.7.
Boc-Lys(Z)-Lys(Z)-Pro-(
Lys(Z)-Pro-( )-4b-Ile-Leu-OMe (0.1 mmol) were used to afford
121 mg of Boc-Lys(Z)-Lys(Z)-Pro-( )-4b-Ile-Leu-OMe 99 % yield; tR =
2.8 min. ESI-MS: [M + H]+ = 1210.7.
L)-4b-Ile-Leu-OH: 122 mg of Boc-Lys(Z)-
L
L
Boc-Lys(Z)-Lys(Z)-Pro-( )-4c-Ile-Leu-OH: 130 mg of Boc-Lys(Z)-
L
Lys(Z)-Pro-( )-4c-Ile-Leu-OMe (0.1 mmol) were used to afford
L
Plasma Stability: Rat plasma was obtained from rat blood by keep-
ing the translucent phase after centrifugation at 15,000 g over
5 min. Plasma stability assay was carried out by incubating each
127 mg of Boc-Lys(Z)-Lys(Z)-Pro-(L)-4c-Ile-Leu-OH as a colorless oil
in 99 % yield; tR = 2.8 min. ESI-MS: [M + H]+ = 1230.7; [M + Na]+
=
1252.6.
analog in rat plasma at final concentration of 0.156 m
15, and 30 min at 37 °C before adding 50 μL of CH3CN containing
0.1 m Fmoc-Glycine as an internal standard. After centrifugation
M for 0, 2, 5,
Boc-Lys(Z)-Lys(Z)-Pro-(
Lys(Z)-Pro-( )-4d-Ile-Leu-OMe (0.1 mmol) were used to afford
117 mg of Boc-Lys(Z)-Lys(Z)-Pro-( )-4d-Ile-Leu-OH as a colorless oil.
98 % yield; tR = 2.7 min. ESI-MS: [M + H]+ = 1196.7.
L)-4d-Ile-Leu-OH: 123 mg of Boc-Lys(Z)-
L
M
L
at 15,000 g for 30 min, supernatant was filtered through 0.22 μm
filter and analyzed by UPLC/MS (Water H Class Acquity UPLC,
mounted with Acquity UPLC BEH C18 column, 1.7 μm, 2.1 × 50 mm
and paired to a SQ Detector 2). Quantification was done by deter-
mining the AUC (area under the curve) ratio of tested compound
over AUC of Fmoc-Glycine. Remaining compound over time was
plotted into GraphPad Prism 6 and the half-life of each compound
was calculated using one-phase decay fit and represented the mean
SEM of at least three separate experiments.
Synthesis of H-Lys-Lys-Pro-( )-4a–d-Ile-Leu-OH: A solution of
L
Boc-Lys(Z)-Lys(Z)-Pro-( )-4a–d-Ile-Leu-OH in a mixture of TFA/triiso-
L
propylsilane (2:1) was stirred at room temperature for 10 min. TFA
(5 equiv.) was then added and the reaction was stirred for 30 min.
After total conversion, TFA was removed by co-evaporation with
cyclohexane and the crude product was purified by preparative
HPLC, using a gradient separation procedure: 10 min from 0 to 25 %
acetonitrile; then 30 min from 25–35 % acetonitrile, then 10 min
from 35–100 % acetonitrile.
Blood Pressure Measurement: Male Sprague-Dawley rats were an-
esthetized with a mixture of ketamine/xylaxine (87 mg/kg: 13 mg/
kg, i.m.) and placed in supine position on a heating pad. Mean,
systolic and diastolic arterial blood pressure as well a heart rate
were measured through a catheter (PE 50 filled with heparinized
saline) inserted in the right carotid artery and connected to a Micro-
Med transducer (model TDX-300, USA) linked to a blood pressure
Micro-Med analyzer (model BPA-100c). Another catheter was in-
serted in the left jugular vein for bolus injections (1 mL/kg, 5–10 s)
of vehicle (isotonic saline) or compounds 7 (JMV5620) and 8
(JMV5621) at 1 mg/kg. Rats were given vehicle first, then only one
dose of one analogue before being euthanized. For relative potency
evaluation, changes in blood pressure from baseline to post-injec-
tion in individual animals were determined. Data represents the
mean SEM of at least four different experiments.
H-Lys-Lys-Pro-(L)-4a-Ile-Leu-OH (JMV5618): tR = 1.7 min; [M +
Na]+ = 864.47. HRMS: calcd. for C42H68N9O7S: [M + H]+ = 842.4962,
found 842.4962.
H-Lys-Lys-Pro-(L)-4b-Ile-Leu-OH (JMV5619): tR = 1.7 min; [M +
Na]+ = 864.48. HRMS: calcd. for C42H68N9O7S: [M + H]+ = 842.4962,
found 842.4959.
H-Lys-Lys-Pro-(L)-4c-Ile-Leu-OH (JMV5620): tR = 1.7 min; [M +
Na]+ = 884.42. HRMS calcd. for C41H65ClN9O7S: [M + H]+ = 862.4416,
found 862.4417.
H-Lys-Lys-Pro-(L)-4d-Ile-Leu-OH (JMV5621): tR = 1.6 min; [M +
Na]+ = 850.46. HRMS: calcd. for C41H66N9O7S: [M + H]+ = 828.4806,
found 828.4806.
Biological Results and Methods
Acknowledgments
Competitive Radioligand Binding Assay: CHO-K1 cells stably ex-
pressing hNTS1 or 1321N1 cells stably expressing hNTS2 were cul-
tured respectively in Ham's F12 or DMEM. Culture media were sup-
plemented with 10 % FBS, 100 U/mL penicillin, 100 μg/mL strepto-
This work was supported by research funding from Montpellier
University, France (F. C. for postdoctoral fellowship to R. F.) and
the Canadian Institutes of Health Research (P. S.). P. S. is the
recipient of the Canada Research Chair in Neurophysiopharma-
cology of Chronic Pain. J. C. holds a Réseau de Recherche en
Santé Buccodentaire et Osseuse (RSBO) postdoctoral fellowship.
The authors thank Prof. Éric Marsault for allowing them to use
the UPLC/MS instrument for plasma stability assays.
mycin, 20 mM HEPES and 0.4 mg/mL G418 at 37 °C in a humidified
chamber at 5 % CO2. Competitive radioligand binding experiments
were performed by incubating 50 μg of freshly prepared cells mem-
branes, expressing either hNTS1 or hNTS2 with 0.1 nM (for hNTS1)
or 0.4 nM (for hNTS2) 125I-Tyr3-NT (2200 Ci/mmol) and increasing
concentrations of the analogues ranging from 10–11 to 10–5
M
for
30 min at 25 °C in binding buffer (50 m
M Tris-HCl, pH 7.5, 0.2 %
Keywords: Medicinal chemistry · Receptors · Neurotensin
analogues · Amino acids · Peptides · Alkylation
BSA). After incubation, the binding reaction mixture was transferred
in polyethylenimine-coated 96 well filter plates (Millipore, Billerica,
MA). Reaction was terminated by filtration and plate was washed
three times with 200 μL of ice-cold binding buffer. Glass filters were
then counted in a γ-counter. Non-specific binding was measured in
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Received: December 2, 2015
Published Online: January 18, 2016
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© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim