Synthesis and structure-activity relationship studies of novel tubulysin U analogues-effect on cytotoxicity of structural variations in the tubuvaline fragment

Article abstract of DOI:10.1039/c3ob27111k

Tubulysins are cytotoxic natural products with promising anti-cancer properties, originally isolated from myxobacterial cultures. Structurally, tubulysins are tetrapeptides, incorporating three unusual (Mep, Tuv and Tup) and one proteinogenic amino acid (Ile). Here we describe the synthesis and structure-activity relationship studies of novel tubulysin U and V analogues, with variations in the central Tuv fragment, which is known to be of paramount importance for tubulysins' potency and hence cytotoxicity, but has seldom been modified in previous studies. Specifically, we replaced the natural iso-propyl and acetoxy functionalities with other structurally related groups. In general, the new analogues showed much lower potency relative to native tubulysin U. However, one of the synthetic analogues (1f) having a MOM function replacing the acetyl group exhibited a 22 nM IC₅₀ on the HT-29 cell line which is comparable to the IC₅₀ displayed by tubulysin U (3.8 nM). Furthermore, the synthetic methodology reported herein was found to be flexible enough to deliver different core-modified tubulysin analogues and hence may be regarded as a scalable and convenient strategy for the chemical generation of novel tubulysin analogues.

Full text of DOI:10.1039/c3ob27111k

Organic &
Biomolecular Chemistry
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PAPER
Synthesis and structure–activity relationship studies of
novel tubulysin U analogues – effect on cytotoxicity of
structural variations in the tubuvaline fragment†
Cite this: Org. Biomol. Chem., 2013, 11,
2273
Sreejith P. Shankar,a Monika Jagodzinska,a,b Luciana Malpezzi,a Paolo Lazzari,c
Ilaria Manca,d Iain R. Greig,e Monica Sani*c,f and Matteo Zanda*c,e,f
Tubulysins are cytotoxic natural products with promising anti-cancer properties, originally isolated from
myxobacterial cultures. Structurally, tubulysins are tetrapeptides, incorporating three unusual (Mep, Tuv
and Tup) and one proteinogenic amino acid (Ile). Here we describe the synthesis and structure–activity
relationship studies of novel tubulysin U and V analogues, with variations in the central Tuv fragment,
which is known to be of paramount importance for tubulysins’ potency and hence cytotoxicity, but has
seldom been modified in previous studies. Specifically, we replaced the natural iso-propyl and acetoxy
functionalities with other structurally related groups. In general, the new analogues showed much lower
potency relative to native tubulysin U. However, one of the synthetic analogues (1f) having a MOM func-
tion replacing the acetyl group exhibited a 22 nM IC50 on the HT-29 cell line which is comparable to the
IC50 displayed by tubulysin U (3.8 nM). Furthermore, the synthetic methodology reported herein was
found to be flexible enough to deliver different core-modified tubulysin analogues and hence may be
regarded as a scalable and convenient strategy for the chemical generation of novel tubulysin analogues.
Received 29th October 2012,
Accepted 11th January 2013
DOI: 10.1039/c3ob27111k
www.rsc.org/obc
Introduction
Tubulysins 1 (Fig. 1) are a family of tetrapeptides produced in
rather small quantities (<4 mg L−1 culture broth) by two
different species of Myxobacteria.1 First isolated from the
culture supernatant of an Archangium gephyra strain, tubuly-
sins were later found in the fermentation broth of Angiococcus
disciformis,
which
preferentially
produced
tubulysin
D. Tubulysins are extremely toxic to mammalian cells, includ-
ing multidrug-resistant cell lines, with IC50 values between
0.01 and 10 nM.2 The cytotoxic activity of the tubulysins stems
Fig. 1 Structure of the tubulysins.
from their ability to bind tubulin and disintegrate micro-
tubules of dividing cells, thus inducing apoptosis and hence
the name.3
aDipartimento di Chimica, Materiali e Ingegneria Chimica “Giulio Natta”,
Politecnico di Milano, Via Mancinelli 7, 20131 Milan, Italy
bDepartment of Pharmaceutical Chemistry, Jagiellonian University,
Collegium Medicum, Medyczna 9 30-688 Krakow, Poland
cKemoTech s.r.l., Parco Scientifico della Sardegna, Edificio 3,
Loc. Piscinamanna, 09010 Pula, CA, Italy
From a structural point of view, tubulysins are linear tetra-
peptides incorporating L-isoleucine and three unnatural amino
acids. At the N-terminus, all the family members have
N-methyl pipecolic acid (Mep) and isoleucine (Ile, the only
proteinogenic amino acid). The central position is occupied by
the unusual amino acid tubuvaline (Tuv) containing a thiazole
heterocycle. The tetrapeptide is completed at the C-terminus
by either tubuphenylalanine (Tup, present in tubulysins D, E,
F, H, U and V) or tubutyrosine (Tut, present in tubulysins A, B,
C, G, I, Y and Z), which are γ-amino acid homologues of
dC.N.R. Istituto di Farmacologia Traslazionale, UOS Cagliari, Edificio 5,
Loc. Piscinamanna, 09010 Pula, CA, Italy
eKosterlitz Centre for Therapeutics, Institute of Medical Sciences,
University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, Scotland, UK.
http://www.abdn.ac.uk/kosterlitz/
fC.N.R. – Istituto di Chimica del Riconoscimento Molecolare, Sezione ‘A. Quilico’,
Via Mancinelli 7, 20131 Milano, Italy
†CCDC 853619 for 8a and 853618 for 15b. For crystallographic data in CIF or
other electronic format see DOI: 10.1039/c3ob27111k
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Table 1 The tubulysins family
Results and discussion
Tubulysin
R1
R2
R3
Back in 2007 we reported a scalable synthesis of tubulysins U
and V.6 With a reliable synthetic strategy in hand, we decided
to explore whether the same could be extended to analogues of
tubulysin U, preferably incorporating modified Tuv fragments.
We decided to explore two different structural variations,
namely (1) the replacement of the iso-propyl group by other
alkyl and aryl groups and (2) the replacement of the O-acetyl
moiety with alkoxy or benzoyl groups. This endeavour was
undertaken in order to determine the importance and role of
the central part of the tubulysins on their biological activity,
with the view of using this knowledge in future studies on the
key binding interactions with tubulin. So far the only available
information on the role of the Tuv fragment was published by
Wipf and co-workers,7 who demonstrated that the natural
A
B
C
D
E
F
G
H
I
U
V
Y
Z
p-OH-Ph
p-OH-Ph
p-OH-Ph
Ph
Ph
Ph
p-OH-Ph
Ph
p-OH-Ph
Ph
Ph
p-OH-Ph
p-OH-Ph
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
H
–CH2OCO-i-butyl
–CH2OCO-n-propyl
–CH2OCOEt
–CH2OCO-i-butyl
–CH2OCO-n-propyl
–CH2OCOEt
–CH2OCOCHvC(CH3)2
–CH2OCOMe
–CH2OCOMe
H
H
H
H
Ac
H
phenylalanine and tyrosine, respectively. Additionally, the (R)-configuration of the O-acetate is preferred and it influences
N-terminal moiety of Tuv may be further functionalized with tubulin assembly. Subsequently, Shibue et al. demonstrated
an unusual N,O-acetal substituent having different ester func- that tubulysin D stereoisomers having different configurations
tionalities (tubulysins A–I) (Table 1). Tubulysins U–Z are of the Tuv stereogenic centres are equivalent to the natural
devoid of such an N-Tuv substituent and as a consequence isomer in terms of inhibition of tubulin polymerisation, but
show lower cytotoxicity, albeit for tubulysin U this is still in the are considerably less cytotoxic, generally displaying an IC50 at
nanomolar range.
least three orders of magnitude higher.8 To the best of our
Although at first sight the tubulysins present a relatively knowledge, nothing has been published about the replace-
simple linear tetrapeptide structure, the presence of six stereo- ment of the Tuv iso-propyl group. However, a recent accurate
genic centres and several chemically and configurationally- NMR structural analysis of the tubulin-bound conformation of
sensitive functionalities render their total synthesis a challen- tubulysins suggested that the hydrophobic Tuv core of the
ging endeavour. Due to their considerable interest and potential tubulysins, which includes the iso-propyl group, plays an extre-
as powerful anticancer agents, several reports on the total syn- mely important role in the binding process.9
thesis of natural or modified tubulysins have been published.4
Satisfactorily, we were able to extend our existing synthetic
Among them, one should mention the structurally simplified strategy,6 with only slight modifications, to the generation of
“Tubugis” compounds,4m “pre-tubulysins”,4i,n–p oxo-tubuly- Tuv-analogues. Thus, the synthesis started from the conden-
sins,4c N-methyl-tubulysins,4d,f and tubulysins U and V.5,6 sation of L-cysteine hydrochloride ethyl ester 2 with methyl
These modified tubulysin analogues are generally less potent glyoxal in the presence of a base affording the thiazoline 3,
than the more synthetically challenging natural tubulysins A which was immediately oxidised using MnO2 to the corres-
and D, although they mostly display sub-nanomolar cyto- ponding 2-acetyl thiazole ethyl ester 4 in 52% overall yield
toxicity on several different cancer cell lines. Remarkably, only (Scheme 1).
a few papers describing scalable syntheses of the tubulysins
Meanwhile, a one-pot reaction involving Boc-carbamate,
have been published.5 Last but not least, little information is benzene sulfinic acid sodium salt and the corresponding alde-
available on the structural modification of the central Tuv frag- hyde in THF–H2O or MeOH–H2O afforded the α-amino sul-
ment, which would be essential for drawing a reliable picture fones 5a–d (Scheme 2). Enolisation of the thiazole 4 using
of the structure–activity relationship (SAR) features of the tubu- NaH followed by the addition of amino sulfones 5a–d gave the
lysins, with the view of developing more synthetically accessi- β-amino ketones 6a–d in satisfactory yields as racemic mix-
ble and chemically/metabolically stable tubulysin analogues tures. In order to obtain stereochemically pure Tuv precursors,
for pre-clinical studies.
we performed an oxazoborolidine (CBS) mediated reduction10
We have very recently reported the synthesis and cyto- of 6a–d to 7a–d using the (S)-CBS catalyst in the presence of a
toxicity evaluation of an oxazole analogue of tubulysin U that BH3·Me2S complex at 0 °C. The required alcohols (R,R)-7a–d,
showed slightly improved activity towards human promyelo-
genous leukaemic cells (HL-60),4l and this prompted us to
evaluate the significance of the Tuv moiety in deciding the
cytotoxicity of tubulysins. In this article we describe the total
synthesis of tubulysin U and V analogues bearing structural
modifications in the Tuv fragment, with the aim of carrying
out an SAR study designed to shed light on the role and impor-
Scheme 1 Synthesis of 2-acetyl-thiazole ethyl ester 4. Reagents and con-
tance of the Tuv amino acid on the cytotoxic activity of the
tubulysins.
ditions: (a) methyl glyoxal solution, NaHCO3, EtOH–H2O (1 : 1), overnight;
(b) MnO2, MeCN, 65 °C, overnight.
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Scheme 3 Synthesis of O-alkylated Tuv precursors. Reagents and conditions:
(a) MeI, tBuOK, THF, −78 °C; (b) MOMCl, NaH, THF, 0 °C.
Scheme 2 Synthesis of stereopure (R,R)-Tuv precursor. Reagents and con-
ditions: (a) formic acid, THF–H2O or MeOH–H2O, 24 h; (b) NaH, THF, 2–3 h;
(c) (S)-(−)-2-methyl-CBS-oxazaborolidine, BH3·Me2S, THF, 0 °C, 2–3 h.
which could be obtained in pure form by flash chromato-
graphy (FC), were produced along with the diastereoisomers
8a–d in a ca. 1 : 1 ratio (Scheme 2), as determined by NMR
analysis of the crude reaction mixtures.
Diastereomers 8, with the exception of 8a, could not be
obtained in pure form by FC because they were always recov-
ered as mixtures either with 7 or with unidentified by-
products, which were initially not observed in the reduction of
6a. Diastereomers 7a–d were obtained in 88–90% ee, as deter-
mined by chiral HPLC analysis, whereas among diastereomers
8, only 8a could be submitted to chiral HPLC analysis,
showing 96% ee.
The relative configuration of the two stereocenters in 8a was
confirmed by X-ray diffraction analysis (Fig. 2) of a suitable
racemic crystal and the absolute configuration was assessed by
chemical correlation with the enantiopure O-Ac-Cbz-Tuv-OEt
described by Wipf et al.11
The next step consisted of the O-alkylation of the free
hydroxy group in 7a, so as to obtain the O-alkylated N,C-pro-
tected tubuvalines 7e,f. This was achieved by reaction of
methyl iodide or MOM-Cl with the corresponding potassium
or sodium bases, respectively (Scheme 3).
Scheme 4 Synthesis of stereopure Tup-OMe. Reagents and conditions:
(a) Et3N, dry THF, 0 °C to rt, 2 h; (b) PPh3, THF, 2 h, 0.38 N NaOH, toluene, 3 h;
(c) MeI, CH2Cl2, 0 °C to rt, overnight; (d) Dess–Martin periodinane, CH2Cl2, 6 h;
(e) CH2Cl2, 0 °C to rt, 8 h; (f) H2, Pd/C, EtOAc, overnight; (g) 6 N HCl, 130 °C,
1.5 h; (h) 2,2-dimethoxypropane, conc. HCl, MeOH, 60 °C, overnight.
bromoacetyl bromide followed by reaction with triphenylphos-
phine gave rise to the ylide 12, which was reacted with methyl
iodide to yield 13, which was subjected to Wittig olefination
with the aldehyde 9. The latter was obtained by Dess–Martin
oxidation of Boc-phenylalaninol 9, affording the α,β unsatu-
rated compound 14 in good yield. Hydrogenolysis of 14 with
Pd/C provided a 2 : 1 diastereomeric mixture of Tup-derivatives
15, separable by FC. One-pot acid hydrolysis of all the protect-
ing groups gave stereochemically pure H-Tup-OH·HCl 16,
which was then esterified to yield H-Tup-OMe·HCl 17, ready
for subsequent couplings.
The synthesis of the Tup unit is shown in Scheme 4. The
key reaction for assembling the Tup fragment was a Wittig
reaction between N-Boc protected phenylalanine aldehyde 95
and the ylide 13, which was chosen because we knew that
N-Boc(−)-menthyl esters 15a and 15b could eventually be sepa-
rated by FC. Starting from (−)-menthol, acylation with
The relative stereochemistry of the two isomers was
assigned based on X-ray analysis of the undesired diastereo-
isomer 15b (Fig. 3).
Final assembly of tubulysins 1a–f was achieved by a stan-
dard stepwise peptide synthesis protocol, as shown in
Scheme 5. Boc deprotection of 7a–f with 20% TFA in CH2Cl2
and subsequent coupling of the resulting amines 18a–f with
Boc-Ile-OH afforded the corresponding dipeptides 19a–f in
Fig. 2 X-ray structure of racemic 8a.
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because of steric reasons. Furthermore the benzoate moiety
proved to be labile even to the mild basic reaction conditions
used for hydrolysing the C-terminal ethyl ester. Thus, we
decided to modify the synthetic sequence starting from the
Tuv stereoisomer ent-8a, as portrayed in Scheme 6. The
racemic β-amino ketone 6a was subjected to reduction using
(R)-CBS catalyst, yielding a 1 : 1 diastereomeric mixture of alco-
hols ent-7,8a. Saponification of ent-8a with LiOH to 26 and
subsequent coupling with Tup-OMe 17 afforded the corres-
ponding dipeptide 27 in good yield. Next, a Mitsunobu reac-
tion using PPh3, benzoic acid and a 40% toluene solution of
DEAD afforded the O-benzoyl derivative 28, having the desired
stereochemistry, resulting from inversion of configuration of
the starting (S)-Tuv stereocenter. TFA mediated Boc deprotec-
tion of 28 to 29, and subsequent coupling with Boc-Ile-OH
gave the tripeptide 30. After further N-deprotection, 31 was
coupled with N-methyl pipecolic acid (23) affording the tetra-
peptide 32. Eventually, selective cleavage of the C-terminal
methyl ester was accomplished using Me3SnOH which was pre-
viously shown to be effective for a highly selective hydrolysis of
tubulysin methyl esters.8 Thus treatment of 32 with Me3SnOH
at 70 °C for 32 h delivered 1g, albeit in modest yields, due to
the formation of several unidentified by-products.
Fig. 3 X-ray structure of the N-boc-(−)-menthyl ester 15b.
The tubulysin analogues 1a–f were tested for their anti-
tumour activity on the human colon cancer cell line HT-29 and
the results are summarised in Table 2.
These data show that replacement of the acetyl group Tuv
fragment of tubulysin U by a methyl group (compound 1e)
caused a two-fold loss of potency, in line with the previously
observed decreased cytotoxicity of O-deacetylated tubulysin V,5
whereas the methoxymethyl group produced a minimal drop
of cytotoxicity and the resulting compound 1f showed remark-
able potency. These findings are very important because the
acetyl group of tubulysins is hydrolytically- and metabolically-
labile, and its replacement with a functionality less susceptible
to hydrolysis could produce more stable and easier-to-handle
tubulysin derivatives. Finally, replacement of the acetyl with
the benzoyl group in compound 1g also produced a drop of
potency.
Scheme 5 Final assembling of fragments. Reagents and conditions: (a) TFA–
CH2Cl2 (1 : 4), 0 °C to rt, 1 h; (b) HOBt, EDC·HCl, then Boc-Ile-OH, DIPEA, CH2Cl2,
0 °C to rt, 3 h; (c) LiOH·H2O, THF–H2O (4 : 1), 0 °C to rt, 5 h; (d) HOAt, HATU,
then H-Tup-OMe (17), Et3N, CH2Cl2, 0 °C to rt, 3 h; (e) TFA–CH2Cl2 (1 : 4), 0 °C to
rt, 1 h; (f) HOAt, HATU, then Mep-OH (23), Et3N, 0 °C to rt, 3 h; (g) 1 N LiOH,
THF, 0 °C to rt, 2–3 days; (h) Ac2O, pyridine, overnight.
satisfactory yields. Hydrolytic cleavage of ethyl ester under
mild alkaline conditions delivered the acids 20a–f, which, on
The inactivity of analogues 1b–d demonstrates the impor-
subsequent coupling with H-Tup-OMe, 17, afforded the tripep- tance of the iso-propyl group on the Tuv fragment – replacing
tides 21a–f. Coupling between N-methyl pipecolic acid 23 and
the tripeptides 22a–f, obtained by treatment of 21a–f with TFA,
the iPr group with other alkyl and aryl groups causes a dra-
matic drop of cytotoxicity. These finding are in line with the
proceeded smoothly in the presence of HATU, HOAt and Et3N, recently published SAR data analysis based on the tubulin-
affording the tetrapeptides 24a–f in good yields and without
any detectable loss of stereochemical purity. Saponification of
bound structure of the tubulysins determined by NMR struc-
tural analysis. Indeed, the Tuv iso-propyl belongs to the
the methyl ester function using 1 M aqueous LiOH in THF “hydrophobic core” of the tubulysins, that extends from the Ile
gave the final carboxylic acid tubulysin U analogues 1e,f and
25a–d in good yields. The latter compounds were eventually
acetylated by treatment with acetic anhydride in pyridine,
affording the corresponding tubulysin U derivatives 1a–d in
stereopure form.
Eventually we accomplished the synthesis of the O-benzoyl
derivative of tubulysin U, which turned out to be more challen-
ging than expected. In fact, attempts to achieve direct benzoy-
side chain to the thiazole ring, which was correctly deemed to
be essential by Carlomagno et al.9
Conclusions
We developed a scalable and efficient total synthesis of tubuly-
sins U and V,6 which is flexible enough to be used for the syn-
lation of the Tup hydroxy group were unsuccessful, possibly thesis of analogues incorporating different tubuvaline
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Scheme 6 Synthesis of O-benzoyl tubulysin U analogue 1g. Reagents and conditions: (a) (R)-(+)-2-methyl-CBS-oxazaborolidine, BH3·Me2S, THF, 0 °C, 2–3 h; (b)
LiOH·H2O, THF–H2O (4 : 1), 0 °C to rt; (c) HOAt, HATU, then H-Tup-OMe 17, Et3N, CH2Cl2, 0 °C to rt; (d) PPh3, PhCOOH, DEAD, benzene; (e) TFA–CH2Cl2 (1 : 4), 0 °C to
rt; (f ) HOBt, EDC·HCl, then Boc-Ile, sym-collidine, CH2Cl2, 0 °C to rt; (g) TFA–CH2Cl2 (1 : 4), 0 °C to rt, 1 h; (h) HOAt, HATU, then Mep 23, Et3N, 0 °C to rt, 3 h; (i)
Me3SnOH, DCE, 70 °C, 32 h.
set of novel tubulysin U analogues was performed. One of the
synthesised analogues 1f, containing an O-MOM replacement
of the natural O-acetyl group on the central Tuv fragment,
essentially retained the activity of the parent analogue 1a. All
the other replacements rendered the molecule biologically
inactive or remarkably less active than the natural analogue,
demonstrating the importance of the Tuv iso-propyl side-chain
for the biological activity of the tubulysins.
Table 2 Biological tests of the tubulysin analogues
Tubulysin analogue
IC50 (nM)
1a R1 = iPr, R2 = Ac
1b R1 = Ph, R2 = Ac
1c R1 = PMP, R2 = Ac
1d R1 = c-hex, R2 = Ac
1e R1 = iPr, R2 = Me
1f R1 = iPr, R2 = MOM
1g R1 = iPr, R2 = COPh
3.8
>1000
>1000
>1000
240
22
>1000
Experimental section
General methods
Commercially-available reagent-grade solvents were employed
without purification. All reactions where an organic solvent
was employed were performed under a nitrogen atmosphere,
after flame-drying of the glass apparatus. Melting points (m.p.)
fragments in quantities and purities sufficient to allow in vitro are uncorrected and were obtained on a capillary apparatus.
biological screenings for cytotoxicity. An SAR study on a small TLCs were run on silica gel 60 F254 Merck. Flash
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Chromatography (FC) purifications were performed with silica 4.03–3.91 (m, 1H), 3.38–3.17 (m, 2H), 1.37 (t, J = 6.9 Hz, 3H),
gel 60 (60–200 μm, Merck). 1H-, 13C-, and 19F-NMR spectra 1.32 (s, 9H), 0.92 (d, J = 6.6 Hz, 6H); 13C NMR (100.5 MHz,
were run at 250, 400 or 500 MHz. Chemical shifts are CDCl3) δ: 192.2, 167.3, 160.6, 155.4, 148.6, 133.0, 121.4, 61.6,
expressed in ppm (δ), using tetramethylsilane (TMS) as the 53.1, 41.4, 32.2, 28.2, 19.1, 14.1; LC/MS (ESI) m/z 393.4
internal standard for 1H and 13C nuclei (δH and δC = 0.00), [M + Na]+.
while C6F6 was used as the external standard (δF −162.90)
Ethyl 2-(3-(tert-butoxycarbonylamino)-3-phenylpropanoyl)-
thiazole-4-carboxylate, 6b. (Pale yellow solid, 43% yield); Rf =
for 19F.
Ethyl 2-acetylthiazole-4-carboxylate, 4. To
a
solution of 0.36 (3 : 7 AcOEt–hexane); FT-IR (film) νmax: 3363.4, 3110.1,
1H NMR
cysteine hydrochloride ethyl ester 2 (15 g, 80.79 mmol) in a 2979.4, 1706.3, 1495.9, 1366.7, 1218.6 cm−1
;
1 : 1 EtOH–H2O mixture (1.5 L) NaHCO3 (6.786 g, 80.79 mmol) (400 MHz, CDCl3) δ: 8.47 (s, 1H), 7.69–6.95 (m, 5H), 5.46–5.33
and pyruvic aldehyde (35% w in H2O, 17.5 mL, 114 mmol) (m, 2H), 4.51 (q, J = 7.2 Hz, 2H), 3.90–3.74 (m, 2H), 1.49 (t, J =
were added. The reaction mixture was stirred at rt for 18 h, 7.2 Hz, 3H), 1.46 (s, 9H); 13C NMR (100.5 MHz, CDCl3) δ:
then concentrated to half of its original volume (no heating). 191.6, 167.4, 161.1, 155.4, 149.1, 133.7, 129.0, 129.0, 127.9,
NaCl was added to saturate the aqueous phase. The aqueous 126.8, 126.4, 122.7, 62.2, 45.3, 30.0, 28.7, 14.7; LC/MS (ESI) m/z
layer was extracted with CHCl3 (2 × 300 mL). The combined 427.1 [M + Na]+.
organic phase was dried over Na2SO4, concentrated in vacuo
and the crude was used in the next step without any further propanoyl)thiazole-4-carboxylate, 6c. (White solid, 52% yield);
purification. Rf = 0.35 (35 : 65 AcOEt–hexane); FT-IR (film) νmax: 3445.8,
Ethyl-2-(3-(tert-butoxycarbonylamino)-3-(4-methoxyphenyl)-
To a solution of compound 3 (16.31 g, 80.74 mmol) in 3020.2, 1706.3, 1215.9 cm−1; 1H NMR (400 MHz, CDCl3) δ: 8.40
MeCN (500 mL) MnO2 (140 g, 1.615 mol) was added. The reac- (s, 1H), 7.28 (d, J = 8.7 Hz, 2H), 6.83 (d, J = 8.7 Hz, 2H),
tion mixture was heated at 65 °C overnight, then filtered over a 5.33–5.20 (m, 2H), 4.44 (q, J = 7.1 Hz, 2H), 3.82–3.62 (m, 2H),
celite pad and the residue washed with AcOEt (2 × 200 mL). 3.76 (s, 3H), 1.42 (t, J = 7.1 Hz, 3H), 1.38 (s, 9H). 13C NMR
The filtrate was concentrated in vacuo. The crude was purified (100.5 MHz, CDCl3) δ: 191.8, 167.5, 161.1, 159.3, 157.2, 155.4,
by FC (1 : 3 AcOEt–hexane) to give the acyl thiazole 4 (8.321 g, 149.0, 133.8, 128.0, 114.4, 80.0, 62.3, 55.7, 51.0, 45.2, 28.7,
52% in two steps) as a yellow solid. Rf = 0.33 (1 : 3 AcOEt– 14.7; LC/MS (ESI) m/z 457.0 [M + Na]+.
1
hexane); H NMR (400 MHz, CDCl3) δ: 8.40 (s, 1H), 4.43 (q, J =
Ethyl 2-(3-(tert-butoxycarbonylamino)-3-cyclohexylpropanoyl)-
7.1 Hz, 3H), 2.75 (s, 3H), 1.41 (t, J = 7.1 Hz, 3H). LC/MS (ESI) thiazole-4-carboxylate, 6d. (White solid, 62% yield); Rf = 0.41
m/z 199.8 [M + H]+, 221.8 [M + Na]+. For a complete set of spec- (3 : 7 AcOEt–hexane); FT-IR (film) νmax: 3364.6, 2976.4, 1734.7,
troscopic data, see ref. 12.
1709.2, 1505.6, 1368.2 cm−1; 1H NMR (400 MHz, CDCl3) δ: 8.37
General procedure for the synthesis of the tert-butyl (phe- (s, 1H), 4.79 (d, J = 8.9 Hz, 1H), 4.39 (q, J = 7.1 Hz, 2H),
nylsulfonyl)methyl carbamates 5a–d. To a solution of tert- 4.00–3.92 (m, 1H), 3.38–3.22 (m, 2H), 1.80–1.48 (m, 6H), 1.37
butyl carbamate (1 equiv.) in a H2O–MeOH 1 : 2 mixture or (t, J = 7.1 Hz, 3H), 1.32 (s, 9H), 1.22–0.96 (m, 5H); 13C NMR
H2O–THF 1 : 2 were added the aldehyde (2 equiv.), benzene (100.5 MHz, CDCl3) δ: 192.8, 167.8, 161.1, 155.8, 149.0, 133.5,
sulfinic acid sodium salt (2 equiv.) and formic acid (2 equiv.). 79.4, 62.0, 52.7, 42.4, 41.7, 30.2, 29.2, 28.6, 26.6, 26.4, 26.4,
The reaction mixture was stirred at rt for 24 h and cooled in an 14.6; LC/MS (ESI) m/z 433.1 [M + Na]+.
ice-bath. The white precipitate was filtered off, washed with
General procedure for the stereoselective reduction of 6a–d. To
water and hexane and dried to give the corresponding tert- a solution of (S)-(−)-2-methyl-CBS-oxazaborolidine (152 mg,
butyl(phenylsulfonyl)methylcarbamate as a white solid and 0.55 mmol) in dry THF (20 mL) cooled at 0 °C, BH3·Me2S
was used in the next step without further purification or (10 M solution in THF, 499 μL, 4.99 mmol) was added. The
characterisation.
solution was stirred for 10 min at 0 °C and a solution of 6a
General procedure for the preparation of β-amino carbonyl (1.846 g, 4.99 mmol) in dry THF (10 mL) was added. The reac-
compounds, 6a–d. To a suspension of NaH (60% dispersion tion was warmed to rt and stirred for 3 h. The reaction was
in mineral oil, 995 mg, 24.87 mmol) in dry THF (60 mL) acyl quenched with MeOH (2 mL), the solvent removed in vacuo
thiazole 4 (3 g, 15.07 mmol) was added. After stirring for and the crude purified by FC.
15 min a solution of sulfone 5a (3.067 g, 9.8 mmol) in dry
Ethyl 2-((1R,3R)-3-(tert-butoxycarbonylamino)-1-hydroxy-4-
THF (60 mL) was added over a period of 30 min. The reaction methylpentyl)thiazole-4-carboxylate, 7a. (White solid, 39%
was stirred for 2 h, quenched with an NH4Cl saturated yield) Rf = 0.51 (4 : 6 AcOEt–hexane); [α]2D3 = +4.5 (c = 1.74,
aqueous solution and extracted with AcOEt (2 × 50 mL). The CHCl3); FT-IR (film) νmax: 3375.1, 2965.9, 1734.3, 1660.4,
collected organic phases were dried over anhydrous Na2SO4, 1505.6, 1391.9 cm−1; 1H NMR (400 MHz, CDCl3) δ: 8.08 (s, 1H),
filtered and concentrated in vacuo. The crude residue was puri- 5.14 (br s, 1H), 4.98 (br d, J = 10.7 Hz, 1H), 4.58 (d, J = 9.4 Hz,
fied by FC.
1H), 4.36 (q, J = 7.3 Hz, 2H), 3.75–3.64 (m, 1H), 2.04 (dt, J =
Ethyl 2-(3-(tert-butoxycarbonylamino)-4-methylpentanoyl)- 12.1, 2.2 Hz, 1H,), 1.81–1.63 (m, 2H), 1.40 (s, 9H), 1.37 (t, J =
thiazole-4-carboxylate, 6a. (White solid, 52% yield); Rf = 0.55 7.3 Hz, 3H), 0.93 (d, J = 7.0 Hz, 3H), 0.91 (d, J = 6.8 Hz, 3H);
(4 : 6 AcOEt–hexane); FT-IR (film) νmax: 3364.6, 2976.4, 1734.9, 13C NMR (100.5 MHz, CDCl3) δ: 176.6, 161.5, 157.9, 146.8,
1709.5, 1668.3, 1505.7, 1367.7 cm−1 1H NMR (400 MHz, 127.1, 80.2, 69.1, 61.2, 52.3, 41.8, 32.2, 28.3, 19.3, 18.3, 14.3;
;
CDCl3) δ: 8.37 (s, 1H), 4.77 (br s, 1H), 4.39 (q, J = 6.9 Hz, 2H), LC/MS (ESI) m/z 395.1 [M + Na]+.
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Ethyl 2-((1R,3R)-3-(tert-butoxycarbonylamino)-1-hydroxy-3- hexane); [α]2D3 = +13.6 (c = 0.95, CHCl3); FT-IR (film) νmax
:
phenylpropyl)thiazole-4-carboxylate, 7b. (Pale yellow solid, 3350.7, 2945.5, 1682.9, 1650.2, 1530.2, 1239.8, 1170.1 cm−1; 1H
36% yield); Rf = 0.26 (35 : 55 AcOEt–hexane); [α]2D3 = +72.38 (c = NMR (400 MHz, CDCl3) δ: 8.13 (s, 1H), 4.60 (br d, J = 9.0 Hz,
0.6, CHCl3); FT-IR (film) νmax: 3349.9, 3119.3, 2979.1, 2929.5, 1H), 4.42 (q, J = 7.1 Hz, 2H), 3.87 (m, 1H), 3.45 (s, 3H), 1.84
1716.6, 1497.1, 1391.8 cm−1; 1H NMR (400 MHz, CDCl3) δ: 8.09 (dd, J = 14.3, 2.7 Hz, 1H), 1.78 (dd, J = 6.8, 2.9 Hz, 1H),
(s, 1H), 7.34–7.16 (m, 5H), 5.25 (br s, 1H), 5.17 (d, J = 10.9 Hz, 1.76–1.65 (m, 2H), 1.45 (s, 9H), 1.39 (t, J = 7.07 Hz, 3H), 0.90
1H), 5.08–4.99 (m, 2H), 4.39 (q, J = 6.9 Hz, 2H), 2.60–2.46 (m, (d, J = 6.75 Hz, 3H), 0.86 (d, J = 7.07 Hz, 3H); 13C NMR
1H), 2.15–1.94 (m, 2H), 1.43 (s, 9H), 1.38 (t, J = 7.1 Hz, 3H); 13
C
(100.5 MHz, CDCl3) δ: 175.6, 161.3, 156.2, 147.6, 127.9, 79.2,
NMR (100.5 MHz, CDCl3) δ: 176.5, 161.9, 157.6, 147.3, 141.3, 61.8, 59.2, 52.1, 41.7, 33.2, 28.8, 19.4; LC/MS (ESI) m/z 409.1
129.3, 128.2, 127.6, 127.0, 81.0, 69.4, 61.7, 51.9, 45.8, 28.7, [M + Na]+.
14.7; LC/MS (ESI) m/z 407.0 [M + H]+, 429.1 [M + Na]+.
Ethyl
2-((1R,3S)-3-(tert-butoxycarbonylamino)-1-methoxy-
Ethyl 2-((1R,3R)-3-(tert-butoxycarbonylamino)-1-hydroxy-3- methyl-4-methylpentyl)thiazole-4-carboxylate, 7f. To a solu-
(4-methoxyphenyl)propyl)thiazole-4-carboxylate, 7c. (Pale yellow tion of alcohol 7a (1.1 g, 2.96 mmol) in dry THF (50 mL)
solid, 39% yield); Rf = 0.29 (4 : 6 AcOEt–hexane); [α]2D3 = +55.69 cooled at 0 °C, NaH (60% dispersion in mineral oil, 355 mg,
(c = 0.96, CHCl3); FT-IR (film) νmax: 3349.8, 3020.3, 1716.4, 8.87 mmol) was added. The resulting suspension was stirred
1
1497.2 cm−1; H NMR (400 MHz, CDCl3) δ: 8.07 (s, 1H), 7.21 at 0 °C for 15 min, then neat MOMCl (1.12 mL, 14.8 mmol)
(d, J = 8.5 Hz, 2H), 6.81 (d, J = 8.5 Hz, 2H), 5.31 (br s, 1H), was added. After stirring at rt for 3 h, the reaction was
5.21–5.01 (m, 2H), 4.97–4.92 (m, 1H), 4.37 (q, J = 7.1 Hz, 2H), quenched by adding a 1 N HCl aqueous solution (20 mL). The
3.74 (s, 3H), 2.53–2.47 (m, 1H), 2.03–1.97 (m, 1H), 1.40 (s, 9H), layers were separated and the organic layer was washed with
1.35 (t, J = 7.1 Hz, 3H); 13C NMR (100.5 MHz, CDCl3) δ: 176.7, brine, dried over anhydrous Na2SO4, filtered and concentrated
161.9, 159.4, 157.5, 147.3, 133.4, 128.1, 127.6, 114.6, 80.8, 69.4, in vacuo. The residue was purified by FC to give 7f (2 : 8 AcOEt–
61.7, 55.6, 51.3, 45.7, 28.7, 14.7; LC/MS (ESI) m/z 459.0 hexane) (428 mg, 35%) as a white foam. Rf = 0.43 (3 : 7 AcOEt–
[M + Na]+.
hexane); [α]2D3 = +12.6 (c = 0.75, CHCl3); FT-IR (film) νmax
:
Ethyl 2-((1R,3R)-3-(tert-butoxycarbonylamino)-1-hydroxy-3- 3342.6, 2932.5, 2879.9, 1680.8, 1650.2, 1528.2, 1235.8,
1
cyclohexylpropyl)thiazole-4-carboxylate, 7d. (White solid, 40% 1171.1 cm−1; H NMR (400 MHz, CDCl3) δ: 7.97 (s, 1H), 4.84
yield); Rf = 0.34 (45 : 55 AcOEt–hexane); [α]2D3 = +46.29 (c = 0.7, (br d, J = 9.0 Hz, 1H), 4.57 (d, J = 6.7 Hz, 1H), 4.55 (d, J =
CHCl3); FT-IR (film) νmax: 3375.1, 2965.9, 1734.3, 1505.6, 6.7 Hz, 1H), 4.48 (br d, J = 10.6 Hz, 1H), 4.22 (q, J = 7.06 Hz,
1
1391.0 cm−1; H NMR (400 MHz, CDCl3) δ: 8.06 (s, 1H), 5.10 2H), 3.63 (m, 1H), 3.20 (s, 3H), 1.74 (m, 1H), 1.64 (m, 1H), 1.56
(br s, 1H), 4.96–490 (m, 1H), 4.70–4.56 (m, 1H), 4.37 (q, J = 7.1 (m, 1H), 1.26 (s, 9H), 1.20 (t, J = 7.1 Hz, 3H), 0.72 (d, J = 6.7 Hz,
Hz, 2H), 3.67–3.60 (m, 1H), 2.36–2.33 (m, 1H), 1.96–1.88 (m, 3H), 0.70 (d, J = 7.1 Hz, 3H); 13C NMR (100.5 MHz, CDCl3) δ:
1H), 1.74–1.63 (m, 5H), 1.46 (br s, 1H), 1.37 (s, 9H), 1.35 (t, J = 175.8, 161.5, 155.9, 147.2, 127.7, 97.6, 61.5, 56.9, 51.9, 40.9,
7.1 Hz, 3H), 1.30–0.80 (m, 5H); 13C NMR (100.5 MHz, CDCl3) δ: 33.2, 28.5, 19.0, 17.9, 14.6; LC/MS (ESI) m/z 417.1 [M + H]+,
177.8, 161.9, 157.1, 146.9, 127.7, 80.3, 71.6, 61.6, 53.6, 43.1, 439.1 [M + Na]+.
41.6, 30.0, 28.7, 28.4, 26.7, 26.5, 26.5, 14.7; LC/MS (ESI) m/z
(1R,2S,5R)-2-Isopropyl-5-methylcyclohexyl 2-bromoacetate,
11. To a solution of menthol (5 g, 32 mmol) in dry THF
413.1 [M + H]+, 435.1 [M + Na]+.
Ethyl 2-((1R,3S)-3-(tert-butoxycarbonylamino)-1-hydroxy-4- (30 mL) Et3N (4.8 mL, 35 mmol) was added. After cooling at
methylpentyl)thiazole-4-carboxylate, 8a. Rf = 0.27 (4 : 6 AcOEt– 0 °C, bromoacetyl bromide (3 mL, 35 mmol) was added drop-
hexane); [α]2D3 = +57.7 (c = 1.2, CHCl3); FT-IR (film) νmax
:
wise. The temperature was allowed to warm to rt and the reac-
3370.7, 2966.5, 1737.8, 1717.3, 1648.9, 1506.4, 1368.3 cm−1; 1H tion mixture was stirred for 2 h. After cooling at 0 °C, the
NMR (400 MHz, CDCl3) δ: 8.07 (s, 1H), 5.18–5.04 (m, 1H), 4.82 reaction was quenched with a 1 N HCl aqueous solution
(br s, 1H), 4.56 (br d, J = 8.6 Hz, 1H), 4.38 (q, J = 6.9 Hz, 2H), (5 mL) and AcOEt was added (30 mL). The layers were sepa-
3.68–3.56 (m, 1H), 2.39–2.26 (m, 1H), 1.98–1.77 (m, 2H), 1.39 rated and the organic phase was dried over anhydrous Na2SO4,
(s, 9H), 1.37 (t, J = 6.9 Hz, 3H), 0.94 (d, J = 6.8 Hz, 3H), 0.90 (d, filtered and concentrated in vacuo. The crude was purified by
J = 6.7 Hz, 3H); 13C NMR (100.5 MHz, CDCl3) δ: 177.4, 161.4, FC (3 : 97 AcOEt–hexane) to give 11 (6.4 g, 72%) as a colorless
156.5, 146.7, 127.0, 79.6, 70.8, 61.1, 53.4, 40.7, 32.3, 28.2, 1.9, oil. Rf = 0.5 (2 : 98 AcOEt–hexane); [α]2D3 = −64.1 (c = 1.68,
17.3, 14.2; LC/MS (ESI) m/z 395.1 [M + Na]+.
Ethyl 2-((1R,3S)-3-(tert-butoxycarbonylamino)-1-methoxy-4- literature.13
CHCl3); spectral data matches with those reported in the
methylpentyl)thiazole-4-carboxylate, 7e. To a 1 M solution of
(1R,2S,5R)-2-Isopropyl-5-methylcyclohexyl 2-(triphenylphos-
t-BuOK (0.54 mL, 0.54 mmol) in dry THF (5 mL), cooled phoranylidene) acetate, 12. To
a solution of 11 (6.4 g,
at −78 °C, a solution of 7a (100 mg, 0.28 mmol) in dry THF 23 mmol) in dry THF (30 mL), under a nitrogen atmosphere,
(3 mL) was added. After 15 min, MeI (83 μL, 1.34 mmol) was PPh3 (6 g, 23 mmol) was added. After refluxing for 2.5 h, the
added and the resulting mixture was stirred at −78 °C for 1 h. reaction mixture was concentrated in vacuo. The resulting solid
H2O (6 mL) was added and the aqueous layer was extracted was washed with a 7 : 3 mixture of hexane–Et2O and filtered to
with EtOAc (3 × 10 mL). The solvent was removed in vacuo and give 11 g of phosphonium salt as a white solid, which was
the crude was purified by FC (2 : 8 AcOEt–hexane) affording 7e used in the next step without further purification. To a suspen-
(48 mg, 45%) as a yellowish foam. Rf = 0.54 (4 : 6 AcOEt– sion of phosphonium salt (11 g, 23 mmol) in toluene (150 mL)
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a 0.38 N NaOH aqueous solution (25 mL) was added dropwise (dd, J = 13.3, 4.0 Hz, 1H), 2.76 (dd, J = 13.3, 6.7 Hz, 1H),
over a period of 5 min. The reaction mixture was stirred for 3 h 1.99–1.95 (m, 1H), 1.87–1.80 (m, 2H), 1.68 (s, 3H), 1.39 (s, 9H),
and the layers were separated. The organic phase was dried 1.25–1.23 (m, 1H), 1.10–0.92 (m, 2H), 0.90–0.87 (m, 10H), 0.75
over anhydrous Na2SO4, filtered and concentrated in vacuo to (d, J = 6.9 Hz, 3H); 13C NMR (100.5 MHz, CDCl3) δ: 177.2,
give 12 (11 g, quantitative yield) as a white foam. Rf = 0.87 (3 : 7 155.2, 139.1, 134.3, 132.5, 129.6, 129.2, 128.9, 126.7, 66.8, 54.5,
MeOH–CHCl3); [α]D23 = −7.8 (c = 0.9, CHCl3); FT-IR (film) νmax
3156.2, 3066.4, 1777.5, 1717.5, 1611.9, 1497.5 cm−1 1H NMR (ESI) m/z 466.1 [M + Na]+, 442.2 [M + H]+.
(400 MHz, CDCl3) δ: 7.67–7.62 (m, 6H), 7.53–7.50 (m, 3H), (2S,4R)-((1R,2S,5R)-2-Isopropyl-5-methylcyclohexyl)
:
47.5, 41.6, 41.4, 34.7, 31.6, 25.8, 23.3, 22.4, 21.4, 16.2; LC/MS
;
4-(tert-
7.44–7.41 (m, 7H), 4.57–4.55 (m, 1H), 2.84–2.81 (m, 1H), butoxycarbonylamino)-2-methyl-5-phenylpentanoate, 15a–b. To
1.94–1.90 (m, 1H), 1.57–1.54 (m, 1H), 1.43–1.39 (m, 2H), a solution of 14 (2.1 g, 4.85 mmol) in EtOAc (20 mL), a catalytic
0.98–0.88 (m, 2H), 0.81–0.65 (m, 10H); 13C NMR (100.5 MHz, amount of Pd/C was added. The reaction mixture was stirred
CDCl3) δ: 133.4, 133.3, 132.1, 129.0, 128.9, 71.2, 48.1, 42.3, under a hydrogen atmosphere overnight and then filtered
34.9, 31.7, 31.1, 29.8, 23.6, 22.5, 16.5; LC/MS (ESI) m/z 481.2 through celite. The filtrate was concentrated under reduced
[M + Na]+.
pressure and the two diastereomers were separated by FC
(1R,2S,5R)-2-Isopropyl-5-methylcyclohexyl 2-(triphenylphos- (iPrO2–hexane 3 : 7) to give 15a (950 mg) and 15b (910 mg,
phoranylidene)propanoate, 13. To a suspension of phosphor- 87% overall yield) as white solids; 15a: Rf = 0.62 (1 : 4 AcOEt–
ane 12 (11 g, 23 mmol) in toluene (150 mL) a 0.38 N NaOH hexane); [α]2D3 = −17.1 (c = 1.4, CHCl3); m.p. = 87–88 °C; FT-IR
aqueous solution (25 mL) was added dropwise over a period of (film) νmax = 2958, 2931, 2871, 1704, 1498, 1454, 1390, 1365,
5 min. The reaction mixture was stirred for 3 h and the layers 1253, 1173 cm−1 1H NMR (400 MHz, CDCl3) δ 7.29–7.26 (m,
;
were separated. The organic phase was dried over anhydrous 2H), 7.22–7.16 (m, 3H), 4.66 (dt, J = 10.8, 4.3 Hz, 1H), 4.34
Na2SO4, filtered and concentrated in vacuo to give 11 g of pure (br s, 1H), 3.92–3.80 (br m, 1H), 2.80–2.70 (m, 2H), 2.62–2.50
phosphonium salt as a white foam. To a solution of phos- (m, 1H), 2.02–1.96 (m, 1H), 1.91–1.81 (m, 2H), 1.71–1.64 (m,
phonium salt (11 g, 23 mmol) in DCM (60 mL), cooled at 0 °C, 2H), 1.54–1.35 (m, 4H), 1.39 (s, 9H), 1.15 (d, J = 7.0 Hz, 3H),
MeI (2.1 mL, 34 mmol) was added dropwise. After stirring for 1.10–1.00 (m, 1H), 0.92–0.80 (m, 1H), 0.90 (d, J = 6.9 Hz, 3H),
10 min the temperature was allowed to warm to rt. The reac- 0.89 (d, J = 6.9 Hz, 3H), 0.75 (d, J = 6.9 Hz, 3H); 13C NMR
tion mixture was stirred overnight and the solvent was evapor- (100.5 MHz, CDCl3) δ 175.7, 155.1, 137.9, 129.5, 128.3, 126.3,
ated. The crude was dissolved in toluene (100 mL) and a 0.38 74.1, 49.9, 47.1, 41.2, 40.8, 37.8, 36.8, 34.3, 31.4, 29.7, 28.4,
N NaOH aqueous solution (25 mL) was added. The mixture 26.3, 23.4, 22.0, 20.8, 17.7, 16.0; LC(MS (ESI) m/z 446.3
was stirred for 2 h and the layers were separated. The organic [M + H]+, 468.4 [M + Na]+. 15b: Rf = 0.7 (1 : 4 AcOEt–hexane);
phase was dried over anhydrous Na2SO4, filtered and concen- [α]2D3 = −31.6 (c = 0.7, CHCl3); FT-IR (film) νmax: 2957.9, 2929.2,
trated in vacuo to give pure 13 (10.4 g, 96%) as a yellow foam. 1870.8, 1715.6, 1496.8 cm−1 1H NMR (400 MHz, CDCl3) δ:
;
Rf = 0.6 (2 : 8 MeOH–CHCl3); [α]2D3 = −43.2 (c = 1.2, CHCl3); 7.30–7.26 (m, 2H), 7.22–7.15 (m, 3H), 4.64 (dt, J = 10.8, 4.3 Hz,
FT-IR (film) νmax: 3158.1, 3060.4, 1777.5, 1718.3, 1611.9, 1H), 4.34 (br s, 1H), 3.95–3.80 (br m, 1H), 2.83–2.73 (m, 2H),
1
1495.4 cm−1; H NMR (400 MHz, CDCl3) δ: 7.69–7.64 (m, 5H), 2.50–2.41 (m, 1H), 1.98–1.92 (m, 1H), 1.86–1.60 (m, 4H),
7.56–7.52 (m, 4H), 7.47–7.44 (m, 7H), 4.53–4.50 (m, 1H), 1.54–1.31 (m, 4H), 1.39 (s, 9H), 1.13 (d, J = 7.0 Hz, 3H),
2.09–2.02 (m, 1H), 1.92–1.86 (m, 1H), 1.72–1.65 (m, 1H), 1.59 1.10–1.00 (m, 1H), 0.93–0.80 (m, 1H), 0.89 (d, J = 6.4 Hz, 3H),
(s, 3H), 1.50–1.41 (m, 2H), 1.35–1.25 (m, 2H), 1.13–0.98 (m, 0.88 (d, J = 6.9 Hz, 3H), 0.74 (d, J = 6.8 Hz, 3H); 13C NMR
1H), 0.93–0.89 (m, 4H), 0.79–0.74 (m, 3H), 0.69–0.62 (m, 3H); (100.5 MHz, CDCl3) δ: 176.0, 155.3, 137.8, 129.4, 128.2, 126.2,
13C NMR (100.5 MHz, CDCl3) δ: 133.8, 133.6, 132.1, 131.8, 74.0, 49.7, 46.9, 42.0, 40.7, 37.2, 36.8, 34.1, 31.3, 29.6, 28.2,
128.6, 128.1, 46.1, 34.2, 31.2, 29.1, 26.2, 22.0, 21.1, 15.7; LC/MS 26.1, 23.3, 21.9, 20.8, 16.7, 16.1; LC/MS (ESI) m/z 446.3
(ESI) m/z 495.1 [M + Na]+.
[M + H]+, 468.3 [M + Na]+.
(1R,2S,5R)-2-Isopropyl-5-methylcyclohexyl 4-(tert-butoxycar-
(2S,4R)-Methyl 4-amino-2-methyl-5-phenylpentanoate hydro-
bonylamino)-2-methyl-5-phenylpent-2-enoate, 14. To a solu- chloride, 17. A suspension of N-Boc menthyl ester 15a
tion of 13 (4.53 g, 9.6 mmol) in DCM (80 mL), cooled at 0 °C, (950 mg, 2.13 mmol) in 6 N HCl (10 mL) was refluxed at
aldehyde 95 (1.6 g, 6.4 mmol) was added. After stirring for 145 °C for 3–5 h and then cooled to rt. AcOEt (10 mL) was
15 min at 0 °C, the temperature was allowed to warm to rt and added and the phases were separated. The aqueous layer was
the reaction mixture was stirred for 8 h. The reaction was concentrated under reduced pressure to obtain Tup·HCl 16
quenched with a 1 N NaHSO4 aqueous solution (50 mL) and (422 mg, 95%) as a white solid and was used in the next step
extracted with DCM (2 × 50 mL). The organic layer was washed without further purification. To a suspension of 16 (398 mg,
with brine (1 × 50 mL), dried over anhydrous Na2SO4, filtered 1.64 mmol) in MeOH (15 mL), 2,2-dimethoxypropane (405 μL,
and concentrated in vacuo. The crude was purified by FC (2 : 8 3.28 mmol) followed by conc. HCl (4.8 μl, 0.015 mmol) were
AcOEt–hexane) to give 14 (2.3 g, 78%) as a white foam; Rf = 0.5 added. The reaction mixture was heated to 50 °C and stirred
(3 : 7 AcOEt–hexane); [α]2D3 = −8.52 (c = 0.7, CHCl3); FT-IR (film) overnight. The solvent was removed in vacuo to give pure
νmax
:
3096.4, 2641.9, 1974.6, 1881.2, 1711.8, 1620.6, Tup-OMe 17 (383 mg, 91%) as a white foam; Rf = 0.3 (1 : 9
1
1459.8 cm−1; H NMR (400 MHz, CDCl3) δ: 7.25–7.18 (m, 3H), MeOH–CH2Cl2); [α]2D3 = +8.6 (c 1.0, MeOH); 1H NMR (400 MHz,
7.13–7.11 (m, 3H), 6.48–6.45 (m, 1H), 4.72–4.65 (m, 3H), 2.91 CD3OD) δ: 7.46–7.15 (m, 5H), 3.63 (s, 3H), 3.59–3.47 (m, 1H),
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3.04 (dd, J = 19.9, 6.2 Hz, 1H), 2.91 (dd, J = 13.7, 7.7 Hz, 1H), 1H), 5.24 (ddd, J = 11.3, 8.3, 2.9 Hz, 1H), 5.05–4.98 (m, 1H),
2.79–2.65 (m, 1H), 2.08–1.95 (m, 1H), 1.16 (d, J = 6.9 Hz, 3H); 4.91 (d, J = 8.1 Hz, 1H), 4.39 (q, J = 7.1 Hz, 2H), 3.99–3.92 (m,
13C NMR (100.5 MHz, CD3OD) δ: 178.1, 137.8, 131.3, 130.9, 1H), 3.77 (s, 3H), 2.65 (ddd, J = 14.1, 11.8, 2.6 Hz, 1H), 2.07
129.3, 53.3, 41.1, 37.9, 37.7, 18.7; LC/MS (ESI) m/z 221.9 (ddd, J = 14.1, 11.1, 2.7 Hz, 1H), 1.97–1.90 (m, 1H), 1.48 (br s,
[M + H]+.
1H), 1.38 (s, 9H), 1.38 (t, J = 7.1 Hz, 3H), 1.18–1.10 (m, 1H),
General procedure for coupling Tuv 7a–f with Boc-Ile. A 0.94 (d, J = 6.8 Hz, 3H), 0.89 (t, J = 7.4 Hz, 3H); 13C NMR
solution of 7a (150 mg, 0.40 mmol) in a 20% mixture of TFA in (100.5 MHz, CDCl3) δ: 176.4, 173.4, 161.9, 159.7, 147.4, 132.8,
DCM (5 mL) was stirred for 1 h. The solvent was removed 128.3, 127.7, 122.7, 114.7, 69.2, 61.7, 60.0, 55.7, 50.5, 44.2,
under reduced pressure to give 18a as the TFA salt and was 36.7, 30.0, 28.6, 25.1, 16.3, 14.7, 11.6; LC/MS (ESI) m/z 572.2
used in the next step without further purification. To a solu- [M + Na]+.
tion of Boc-isoleucine (93 mg, 0.40 mmol) in DCM (5 mL),
Ethyl 2-((1R,3R)-3-((2S,3S)-2-(tert-butoxycarbonylamino)-3-
HOBt (60 mg, 0.44 mmol), EDC·HCl (84 mg, 0.44 mmol), methylpentanamido)-1-hydroxy-3-cyclohexylpropyl)thiazole-4-
DIPEA (153 μL, 0.88 mmol) and Tuv-TFA salt 18a (160 mg, carboxylate, 19d. (Off-white foam, 97% yield); Rf = 0.52 (1 : 1
0.40 mmol) were added. After stirring for 3 h water (10 mL) AcOEt–hexane); [α]2D3 = +30.59 (c = 1.79, CHCl3); IR (film) νmax
:
was added and the layers separated. The organic phase was 3419.4, 3020.1, 1651.6, 1369.0, 1216.0, 1165.2, 1099.8 cm−1; 1H
washed with a 1 N HCl aqueous solution (10 mL), a saturated NMR (400 MHz, CDCl3) δ: 8.05 (s, 1H), 6.45 (d, J = 7.5 Hz, 1H),
NaHCO3 aqueous solution (10 mL) and brine (10 mL). After 5.23 (d, J = 8.2 Hz, 1H), 4.97 (d, J = 8.3 Hz, 1H), 4.37 (q, J =
drying over anhydrous Na2SO4 and concentration in vacuo the 7.1 Hz, 2H), 4.09–3.93 (m, 1H), 3.77–3.63 (m, 1H), 2.42 (dd, J =
crude dipeptide was purified by FC.
14.6, 2.4 Hz, 1H), 1.90–1.38 (m, 9H), 1.36 (s, 9H), 1.35 (t, J =
Ethyl 2-((1R,3R)-3-((2S,3S)-2-(tert-butoxycarbonylamino)-3- 7.1 Hz, 3H), 1.20–0.95 (m, 7H), 0.92 (d, J = 6.7 Hz, 3H), 0.86 (t,
methylpentanamido)-1-hydroxy-4-methylpentyl)thiazole-4-car- J = 7.9 Hz, 3H); 13C NMR (100.5 MHz, CDCl3) δ: 178.3, 172.7,
boxylate, 19a. (White foam, 80% yield); Rf = 0.47 (4 : 6 AcOEt– 161.9, 156.9, 147.4, 127.8, 80.8, 71.4, 61.6, 60.7, 53.6, 42.9,
hexane); [α]2D3 = −3.1 (c = 1.1, CHCl3); FT-IR (film) νmax: 3419.0, 41.2, 36.8, 30.1, 28.7, 28.6, 26.7, 26.5, 26.5, 19.6, 16.1, 14.7,
1651.6, 1497.9, 1216.0, 1165.2, 1099.8 cm−1
(400 MHz, CDCl3) δ: 8.08 (s, 1H), 6.35 (d, J = 8.1 Hz, 1H), 5.01
;
1H-NMR 14.4, 11.4; LC/MS (ESI): m/z 526.2 [M + H]+, 548.2 [M + Na]+.
Ethyl 2-((1R,3R)-3-((2S,3S)-2-(tert-butoxycarbonylamino)-3-
(d, J = 7.8 Hz, 1H), 4.86 (d, J = 10.0 Hz, 1H), 4.37 (q, J = 7.1 Hz, methylpentanamido)-1-methoxy-4-methylpentyl)thiazole-4-
2H), 4.15–3.88 (m, 2H), 2.17 (t, J = 10.1 Hz, 1H), 1.83–1.81 (m, carboxylate, 19e. (White foam, 58% yield); Rf = 0.28 (1 : 3
1H), 1.80–1.73 (m, 2H), 1.51 (s br, 1H), 1.41 (s, 9H), 1.35 (t, J = AcOEt–hexane); [α]2D3 = +1.9 (c = 0.62, CHCl3); FT-IR (film) νmax
:
7.1 Hz, 3H), 1.16–1.09 (m, 2H), 1.01 (d, J = 6.5 Hz, 3H), 3367.8, 2963.1, 1716.4, 1501.4, 1366.3, 1237.3, 1173.2; 1H NMR
0.97–0.83 (m, 9H); 13C NMR (100.5 MHz, CDCl3) δ: 176.8, (400 MHz, CDCl3) δ: 8.09 (s, 1H), 6.25 (d, J = 9.3 Hz, 1H), 4.98
174.3, 161.9, 156.3, 147.4, 127.6, 80.0, 69.2, 61.7, 52.1, 41.2, (br d, 1H), 4.53 (dd, J = 9.32, 3.54 Hz, 1H), 4.38 (q, J = 7.1 Hz,
36.0, 32.4, 28.6, 25.0, 19.8, 18.6, 16.3, 14.7, 11.4; LC/MS (ESI) 2H), 4.12 (m, 1H), 3.85 (t, J = 7.8 Hz, 1H), 3.41 (s, 3H), 1.91
m/z 486.2 [M + H]+, 508.0 [M + Na]+.
(dd, J = 14.4, 3.53 Hz, 1H), 1.85 (dd, J = 8.9, 3.6 Hz, 1H),
Ethyl 2-((1R,3R)-3-((2S,3S)-2-(tert-butoxycarbonylamino)-3- 1.82–1.75 (m, 2H), 1.54 (m, 1H), 1.39 (s, 9H), 1.36 (t, J = 7.1 Hz,
methylpentanamido)-1-hydroxy-3-phenylpropyl)thiazole-4-car- 3H), 1.13 (m, 1H), 0.94 (d, J = 6.75 Hz, 3H), 0.90–0.86 (m, 9H);
boxylate, 19b. (Pale yellow foam, 75% yield); Rf = 0.42 (1 : 1 13C NMR (100.5 MHz, CDCl3) δ: 175.8, 171.9, 161.6, 156.2,
AcOEt–hexane); [α]D23 = +65.67 (c = 0.34, CHCl3); IR (film) νmax
3419.0, 3020.1, 1651.5, 1497.9, 1369.0, 1216.0 cm−1
(400 MHz, CDCl3) δ: 8.09 (s, 1H), 7.37–7.23 (m, 5H), 6.85 (d, J = [M + H]+, 522.1 [M + Na]+.
:
147.6, 127.9, 79.1, 61.7, 59.1, 51.2, 40.1, 36.8, 30.1, 32.5, 28.6,
;
1H NMR 25.1, 19.2, 18.4, 16.3, 14.7, 11.5; LC/MS (ESI) m/z 500.1
7.1 Hz, 1H), 5.37 (d, J = 3.5 Hz, 1H), 5.29 (ddd, J = 11.3, 8.3,
Ethyl 2-((1R,3R)-3-((2S,3S)-2-(tert-butoxycarbonylamino)-3-
3.0 Hz, 1H), 5.05–5.01 (m, 1H), 4.92 (d, J = 8.4 Hz, 1H), 4.39 (q, methylpentanamido)-1-methoxymethyloxy-4-methylpentyl)-
J = 7.1 Hz, 2H), 4.01–3.95 (m, 1H), 2.66 (ddd, J = 14.1, 11.8, thiazole-4-carboxylate, 19f. (White foam, 58%); Rf = 0.20
2.6 Hz, 1H), 2.10 (ddd, J = 14.0, 11.0, 3.0 Hz, 1H), 1.97–1.91 (3 : 7 AcOEt–hexane), [α]2D3 = +3.1 (c = 0.8, CHCl3); 1H NMR
(m, 1H), 1.49 (br s, 1H), 1.38 (t, J = 7.1 Hz, 3H), 1.37 (s, 9H), (400 MHz, CDCl3) δ: 8.06 (s, 1H), 6.25 (br d, J = 8.1 Hz, 1H),
1.19–1.05 (m, 1H), 0.95 (d, J = 6.8 Hz, 3H), 0.89 (t, J = 7.3 Hz, 5.03 (d, J = 8.7 Hz, 1H), 4.87 (t, J = 6.4 Hz, 1H), 4.70 (d, J = 6.5
3H); 13C NMR (100.5 MHz, CDCl3) δ: 176.3, 173.5 (2C), 161.9, Hz, 1H), 4.66 (d, J = 6.5 Hz, 1H), 4.35 (q, J = 7.0 Hz, 2H), 4.06
147.4, 140.7, 129.3, 128.3, 127.7, 127.1, 69.2, 61.7, 60.1, 51.1, (m, 1H), 3.81 (t, J = 8.0 Hz, 1H), 3.34 (s, 3H), 1.92 (t, J = 6.6 Hz,
44.3, 36.6, 30.0, 28.6, 25.1, 16.3, 14.7, 11.6; LC/MS (ESI) m/z 2H), 1.87–1.75 (m 2H), 1.51 (m, 1H), 1.35 (s, 9H), 1.33 (t, J =
520.2 [M + H]+, 542.2 [M + Na]+.
7.4 Hz, 3H), 1.10 (m, 1H), 0.90 (d, J = 6.7 Hz, 3H), 0.87–0.81
Ethyl 2-((1R,3R)-3-((2S,3S)-2-(tert-butoxycarbonylamino)-3- (m, 9H); 13C NMR (100.5 MHz, CDCl3) δ: 175.5, 172.1, 161.6,
methylpentanamido)-1-hydroxy-3-(4-methoxyphenyl)propyl)- 156.2, 147.5, 127.7, 97.5, 75.2, 61.6, 60.1, 57.1, 51.1, 39.7, 32.6,
thiazole-4-carboxylate, 19c. (Pale yellow foam, 63% yield); 28.6, 25.1, 18.9, 18.2, 16.1, 14.6, 11.5; LC/MS (ESI) m/z 530.2
Rf = 0.22 (1 : 1 AcOEt–hexane); [α]D23 = +78.95 (c = 0.57, CHCl3); [M + H]+, 552.2 [M + Na]+.
IR (film) νmax: 3410.0, 3020.2, 1651.6, 1497.2 cm−1
(400 MHz, CDCl3) δ 8.09: (s, 1H), 7.23 (d, J = 8.7 Hz, 2H), 6.84 Tup-OMe 17. To
(d, J = 8.7 Hz, 2H), 6.73 (d, J = 7.4 Hz, 1H), 5.41 (d, J = 4.3 Hz, 0.45 mmol) in a 4 : 1 THF–H2O mixture (5 mL), LiOH·H2O
;
1H NMR
General procedure for coupling Ile-Tuv-COOEt 19a–f with
solution of dipeptide 19a (210 mg,
a
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(28 mg, 0.67 mmol) was added. The reaction mixture was νmax: 3349.4, 3064.1, 1729.1, 1652.0, 1084.1 cm−1 1H NMR
;
stirred for 5 h. H2O (5 mL) and AcOEt (10 mL) were added and (400 MHz, CDCl3) δ 7.99 (s, 1H), 7.33–7.15 (m, 7H), 7.10 (d, J =
the layers were separated. The aqueous phase was acidified to 7.1 Hz, 1H), 6.90 (d, J = 8.1 Hz, 2H), 6.80 (d, J = 7.4 Hz, 1H),
pH 1–2 with a 1 N HCl aqueous solution and was extracted 5.41–5.19 (m, 2H), 4.96–4.91 (m, 2H), 4.39 (d, J = 4.6 Hz, 1H),
with AcOEt (2 × 10 mL). The combined organic extract was 3.98 (t, J = 7.1 Hz, 1H), 3.80 (s, 3H), 3.61 (s, 3H), 3.00–2.80 (m,
dried over anhydrous Na2SO4, filtered and concentrated 2H), 2.64–2.54 (m, 1H), 2.46 (t, J = 12.6 Hz, 1H), 2.14 (t, J =
in vacuo to give the corresponding acid 20a, which was used in 12.2 Hz, 1H), 2.04–1.88 (m, 3H), 1.61–1.44 (m, 2H), 1.38 (s,
the next step without further purification. To a solution of 20a 9H), 1.14 (d, J = 6.9 Hz, 3H), 0.96 (d, J = 6.6 Hz, 3H), 0.90 (t, J =
(180 mg, 0.40 mmol) in DCM (5 mL), HOAt (60 mg, 7.2 Hz, 3H); 13C NMR (100.5 MHz, CDCl3) δ: 176.9, 175.1,
0.44 mmol), HATU (167 mg, 0.44 mmol), Et3N (123 μL, 173.2, 161.1, 159.7, 156.6, 150.2, 137.9, 137.8, 132.0, 130.0,
0.88 mmol) and Tup-OMe 17 (103 mg, 0.40 mmol) were added. 128.7, 128.2, 126.8, 123.5, 114.8, 69.0, 55.7, 52.0, 50.3, 48.7,
After stirring for 3 h water (10 mL) was added and the layers 44.5, 41.5, 38.2, 36.9, 36.6, 30.0, 28.6, 25.2, 18.1, 17.2, 16.3,
separated. The organic phase was washed with a 1 N HCl 11.7; LC/MS (ESI) m/z 747.2 [M + Na]+.
aqueous solution (10 mL), a saturated NaHCO3 aqueous solu-
(2S,4R)-Methyl 4-(2-((6S,9R,11R)-((S)-sec-butyl)-9-cyclohexyl-
tion (10 mL) and brine (10 mL). After drying over anhydrous 2,2-dimethyl-4,7,13-trioxo-3,12-dioxa-5,8-diazatetradecan-11-yl)-
Na2SO4 and concentration in vacuo the crude tripeptide was thiazole-4-carboxamido)-2-methyl-5-phenylpentanoate, 21d. (Off-
purified by FC.
(2S,4R)-Methyl 4-(2-((6S,9R,11R)-6-((S)-sec-butyl)-9-isopropyl- [α]2D3 = +12.73 (c = 0.22, CHCl3); FT-IR (film) νmax: 3295.4,
2,2-dimethyl-4,7,13-trioxo-3,12-dioxa-5,8-diazatetradecan-11-yl)- 3025.2, 2960.5, 1729.7, 1645.6, 1541.7 cm−1 1H NMR
white foam, 75% yield). Rf = 0.47 (65 : 35 AcOEt–hexane);
;
thiazole-4-carboxamido)-2-methyl-5-phenylpentanoate, 21a. (Pale (400 MHz, CDCl3) δ: 7.94 (s, 1H), 7.35–7.11 (m, 5H), 6.44 (br s,
yellow foam, 63% yield); Rf = 0.42 (2 : 3 AcOEt–hexane); 1H), 5.41 (br s, 1H), 5.25 (d, J = 7.1 Hz, 1H), 4.74–4.84 (m, 1H),
[α]2D3 = +15.2 (c = 0.7, CHCl3); FT-IR (film) νmax: 3290.4, 3020.0, 4.42–4.31 (m, 1H), 4.16–4.05 (m, 1H), 3.82–3.58 (m, 2H), 3.61
2960.5, 1729.7, 1645.6, 1541.7 cm−1 1H NMR (400 MHz, (s, 3H), 2.98–2.86 (m, 2H), 2.66–2.54 (m, 1H), 2.26 (d, J =
;
CDCl3) δ: 8.00 (s, 1H), 7.31–7.13 (m, 5H), 7.08 (br s, 1H), 6.36 14.5 Hz, 1H), 2.11–1.96 (m, 2H), 1.95–1.41 (m, 9H), 1.36 (s,
(d, J = 8.9 Hz, 1H), 5.23 (br s, 1H), 4.88 (br d, J = 10.4 Hz, 1H), 9H), 1.30–0.95 (m, 6H), 1.15 (d, J = 7.1 Hz, 3H), 0.97 (d, J =
4.60 (d, J = 9.2 Hz, 1H), 4.45–4.32 (m, 1H), 3.89–3.68 (m, 2H), 6.6 Hz, 3H), 0.89 (t, J = 7.3 Hz, 3H); 13C NMR (100.5 MHz,
3.62 (s, 3H), 2.93 (dd, J = 10.3, 6.1 Hz, 1H), 2.85 (dd, J = 13.5, CDCl3) δ: 177.0, 176.6, 172.6, 161.3, 150.4, 138.2, 129.9, 128.7,
6.1 Hz, 1H), 2.66–2.54 (m, 1H), 2.07–1.89 (m, 3H), 1.88–1.71 126.8, 123.2, 80.8, 71.0, 60.9, 53.2, 52.0, 48.9, 43.3, 41.5, 41.1,
(m, 3H), 1.67–1.48 (m, 2H), 1.43 (s, 9H), 1.15 (d, J = 7.1 Hz, 38.4, 36.9, 30.1, 28.9, 28.6, 26.8, 26.6, 26.5, 25.5, 18.1, 16.2,
3H), 1.00–0.88 (m, 12H); 13C NMR (100.5 MHz, CDCl3) δ: 14.4, 11.4; LC/MS (ESI): m/z 723.2 [M + Na]+.
176.4, 175.5, 174.5, 160.7, 157.7, 149.6, 137.4, 129.5, 128.2,
(2S,4R)-Methyl 4-(2-((3R,5R,8S)-8-((S)-sec-butyl)-5-isopropyl-
126.3, 122.8, 80.2, 68.7, 52.3, 52.6, 48.2, 42.3, 41.4, 40.9, 37.7, 12,12-dimethyl-7,10-dioxo-2,11-dioxa-6,9-diazatridecan-3-yl)-
37.1, 36.4, 32.2, 28.2, 18.2, 17.6, 16.2, 14.5, 11.5; LC/MS (ESI) thiazole-4-carboxamido)-2-methyl-5-phenylpentanoate, 21e.
m/z 662.9 [M + H]+, 684.8 [M + Na]+.
(White foam, 78% yield); Rf = 0.35 (2 : 3 AcOEt–hexane);
(2S,4R)-Methyl 4-(2-((6S,9R,11R)-6-((S)-sec-butyl)-2,2-dimethyl- [α]2D3 = −6.8 (c = 0.9, CHCl3); FT-IR (film) νmax: 3318.6, 2964.7,
4,7,13-trioxo-9-phenyl-3,12-dioxa-5,8-diazatetradecan-11-yl)- 2933.4, 1651.8, 1539.8, 1495.4, 1249.4; 1H NMR (400 MHz,
thiazole-4-carboxamido)-2-methyl-5-phenylpentanoate, 21b. (Pale CDCl3) δ: 8.01 (s, 1H), 7.26–7.18 (m, 5H), 7.08 (d, J = 9.6 Hz,
yellow foam, 66% yield); Rf = 0.43 (65 : 35 AcOEt–hexane); 1H), 5.06 (bd, 1H), 4.44 (d, J = 7.7 Hz, 1H), 4.15 (m, 1H), 3.88
[α]2D3 = + 35.9 (c = 1.18, CHCl3); FT-IR (film) νmax: 3313.0, (t, J = 7.8 Hz, 1H), 3.52 (s, 3H), 3.42 (s, 3H), 2.95 (dd, J = 13.5,
3028.5, 2966.1, 2931.8, 1656.9, 1540.8, 1496.7, 1366.9, 5.8 Hz, 1H), 2.81 (dd, J = 13.5, 7.2 Hz, 1H), 2.5 (m, 1H),
1247.9 cm−1 1H NMR (400 MHz, CDCl3) δ: 7.97 (s, 1H), 2.01–1.86 (m, 3H), 1.81–1.75 (m, 2H), 1.67–1.60 (m, 1H), 1.57
;
7.33–7.05 (m, 12H), 5.66–5.25 (m, 2H), 5.22–5.09 (m, 1H), 4.93 (m, 1H), 1.41 (s, 9H), 1.14 (d, J = 6.74 Hz, 3H), 0.96 (d, J =
(d, J = 10.5 Hz, 1H), 4.51–4.34 (m, 1H), 4.07–3.94 (m, 1H), 3.49 6.74 Hz, 3H), 0.90 (m, 12H); 13C NMR (100.5 MHz, CDCl3) δ:
(s, 3H), 2.96–2.77 (m, 2H), 2.55–2.30 (m, 2H), 2.16 (t, J = 12.2 177.4, 174.5, 171.8, 171.8, 161.0, 156.4, 150.4, 138.0, 129.8,
Hz, 1H), 1.96–1.85 (m, 3H), 1.63–1.42 (m, 2H), 1.38 (s, 9H), 128.8, 126.8, 123.7, 78.9, 59.0, 51.8, 51.1, 48.7, 42.4, 40.7, 37.9,
1.13 (d, J = 6.4 Hz, 3H), 0.95 (d, J = 6.4 Hz, 3H), 0.87 (t, J = 37.1, 36.8, 30.1, 32.5, 28.8, 25.1, 19.5, 18.3, 17.2, 16.3, 11.6;
7.0 Hz, 3H); 13C NMR (100.5 MHz, CDCl3) δ: 177.5, 175.6, LC/MS (ESI) m/z 698.2 [M + Na]+, 714.2 [M + K]+.
173.2, 161.3, 150.0, 141.0, 137.8, 129.9, 129.3, 128.8, 128.2,
(2S,4R)-Methyl 4-(2-((5R,7R,10S)-10-((S)-sec-butyl)-7-isopro-
127.0, 126.9, 123.7, 122.7, 68.9, 51.9, 50.9, 48.5, 44.4, 42.2, pyl-14,14-dimethyl-9,12-dioxo-2,4,13-trioxa-8,11-diazapentade-
39.0, 37.9, 37.1, 30.0, 28.6, 25.2, 17.7, 17.3, 16.2, 11.6; LC/MS can-5-yl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoate,
(ESI) m/z 717.2 [M + Na]+.
21f. (White foam, 84%); Rf = 0.32 (3 : 7 AcOEt–hexane);
(2S,4R)-Methyl 4-(2-((6S,9R,11R)-6-((S)-sec-butyl)-9-(4-meth- [α]2D3 = −7.1 (c = 1.28, CHCl3); 1H NMR (400 MHz, CDCl3) δ:
oxyphenyl)-2,2-dimethyl-4,7,13-trioxo-3,12-dioxa-5,8-diazatetra- 8.01 (s, 1H), 7.28–7.20 (m, 5H), 7.11 (d, J = 9.6 Hz, 1H), 6.07
decan-11-yl)thiazole-4-carboxamido)-2-methyl-5-phenylpen- (br s, 1H), 4.97 (m, 1H), 4.84 (dd, J = 9.9, 2.2 Hz, 1H), 4.76 (d,
tanoate, 21c. (Pale yellow foam, 97% yield); Rf = 0.41 (65 : 35 J = 6.7 Hz, 1H), 4.71 (d, J = 6.7 Hz, 1H), 4.45 (m, 1H), 4.14 (m,
AcOEt–hexane); [α]2D3 = +67.33 (c = 0.9, CHCl3); FT-IR (film) 1H), 3.86 (t, J = 8.0 Hz, 1H), 3.54 (s, 3H), 3.38 (s, 3H), 2.98 (dd,
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J = 13.8, 5.6 Hz, 1H), 2.84 (dd, J = 13.5, 7.4 Hz, 1H), 2.54–2.48 CDCl3) δ: 7.96 (s, 1H), 7.49 (br d, 1H), 7.24–7.20 (m, 5H), 5.02
(m, 1H), 2.04–1.82 (m, 6H), 1.69–1.57 (m, 2H), 1.43 (s, 9H), (d, J = 5.4 Hz, 1H), 4.80 (br d, J = 10.1 Hz, 1H), 4.67 (d, J =
1.16 (d, J = 6.7 Hz, 3H), 0.97 (d, J = 6.7 Hz, 3H), 0.93–0.82 9.2 Hz, 1H), 4.38–4.30 (m, 1H), 3.77–3.71 (m, 1H), 3.58 (s, 3H),
(m, 9H); 13C NMR (100.5 MHz, CDCl3) δ: 177.3, 174.2, 171.8, 2.97 (dd, J = 13.3, 4.9 Hz, 1H), 2.80 (dd, J = 13.3, 7.0 Hz, 1H),
161.0, 156.4, 150.3, 138.1, 129.8, 128.8, 126.9, 123.6, 97.5, 74.7, 2.66–2.54 (m, 1H), 2.15–2.11 (m, 1H), 2.00–1.88 (m, 2H),
60.2, 57.0, 51.9, 51.1, 48.7, 42.4, 40.5, 37.9, 37.1, 32.6, 30.0, 1.80–1.74 (m, 1H), 1.66 (m, 1H), 1.39 (s, 9H), 1.13 (d, J = 6.9 Hz,
25.2, 19.3, 18.2, 17.3, 16.2, 11.5; LC/MS (ESI) m/z 727.2 3H), 0.92 (d, J = 7.9 Hz, 3H), 0.90 (d, J = 8.6 Hz, 3H); 13C NMR
[M + Na]+.
(100.5 MHz, CDCl3) δ: 177.1, 176.2, 161.3, 156.9, 150.2, 138.2,
Ethyl
2-((1S,3S)-3-(tert-butoxycarbonylamino)-1-hydroxy-4- 129.8, 128.8, 126.8, 123.2, 80.0, 70.9, 53.4, 52.0, 49.2, 41.7,
methylpentyl)thiazole-4-carboxylate, ent-7a. The reduction 41.2, 37.7, 36.9, 33.2, 28.7, 19.5, 18.0; LC/MS (ESI) m/z 548.1
was carried out with (R)-CBS catalyst according to the same [M + H]+, 570.2 [M + Na]+.
procedure as described for the reduction of 6a–d to 7a–d
(1R,3R)-3-(tert-Butoxycarbonylamino)-1-(4-((2R,4S)-5-methoxy-
(white solid, 35% yield); Rf 0.50 (2 : 3 AcOEt–hexane); 4-methyl-5-oxo-1-phenylpentan-2-ylcarbamoyl)thiazol-2-yl)-4-
[α]2D3 = −5.0 (c = 1.70, CHCl3); 1H NMR (400 MHz, CDCl3) δ: methylpentyl benzoate, 28. To a solution of 27 (120 mg,
8.08 (s, 1H), 5.14 (br s, 1H), 4.98 (br d, J = 10.7 Hz, 1H), 4.58 0.21 mmol) in benzene (5 mL), PPh3 (144 mg, 0.54 mmol) and
(d, J = 9.4 Hz, 1H), 4.36 (q, J = 7.3 Hz, 2H), 3.75–3.64 (m, 1H), benzoic acid (66 mg, 0.54 mmol) were added followed by
2.04 (dt, J = 12.1, 2.2 Hz, 1H), 1.81–1.63 (m, 2H), 1.40 (s, 9H), DEAD (40% solution in toluene, 249 μL, 0.54 mmol). The reac-
1.37 (t, J = 7.3 Hz, 3H), 0.93 (d, J = 7.0 Hz, 3H), 0.91 (d, J = tion mixture was stirred at rt for 45 min and then a NaHCO3
6.8 Hz, 3H); 13C NMR (100.5 MHz, CDCl3) δ: 176.6, 161.5, saturated aqueous solution was added (10 mL). The layers
157.9, 146.8, 127.1, 80.2, 69.1, 61.2, 52.3, 41.8, 32.2, 28.3, 19.3, were separated and the organic layer was concentrated
18.3, 14.3; LC/MS (ESI): m/z 395.1 [M + Na]+.
in vacuo. The crude was purified via FC (3 : 7 AcOEt–hexane)
Ethyl 2-((1S,3R)-3-(tert-butoxycarbonylamino)-1-hydroxy-4- affording 28 (129 mg, 95%) as a white foam. Rf = 0.47 (2 : 3
methylpentyl)thiazole-4-carboxylate, ent-8a. (White foam, 35% AcOEt–hexane); [α]2D3 = −3.84 (c = 0.26, CHCl3); 1H NMR
yield); Rf 0.24 (3 : 2 hexane–AcOEt); ee = 96% (determined (400 MHz, CDCl3) δ: 8.07 (d, J = 7.3 Hz, 2H), 8.01 (s, 1H),
by HPLC analysis with CHIRACEL OD stationary phase, 7.58–7.45 (m, 3H), 7.23–7.18 (m, 5H), 6.50 (br s, 2H), 6.35
=
n-Hex–i-PrOH 9 : 1, 1.0 mL min−1); [α]D23
−64.3 (c = 0.10, (br s, 1H), 4.54 (d, J = 9.2 Hz, 1H), 4.40–4.34 (m, 1H), 3.85–3.73
1
CHCl3); m.p. = 76–78 °C; H NMR (400 MHz, CDCl3) δ: 8.07 (s, (m, 1H), 3.61 (s, 3H), 2.66–2.32 (m, 2H), 2.03–1.97 (m, 2H),
1H), 5.18–5.04 (m, 1H), 4.82 (br s, 1H), 4.56 (br m, 1H), 4.38 1.88–1.70 (m, 2H), 1.61 (m, 1H), 1.36 (s, 9H), 1.14 (d, J =
(q, J = 6.9 Hz, 2H), 3.68–3.56 (m, 1H), 2.49–2.41 (m, 1H), 7.0 Hz, 3H), 0.95 (d, J = 6.6 Hz, 3H), 0.93 (d, J = 7.3 Hz, 3H);
2.39–1.77 (m, 2H), 1.39 (s, 9H), 1.37 (t, J = 7.3 Hz, 3H), 0.94 (d, 13C NMR (100.5 MHz, CDCl3) δ: 177.0, 176.1, 170.4, 160.8,
J = 7.0 Hz, 3H), 0.90 (d, J = 6.8 Hz, 3H); 13C NMR (100.5 MHz, 157.1, 155.7, 150.3, 138.0, 136.5, 133.8, 130.2, 129.8, 128.8,
CDCl3) δ 177.4, 161.4, 156.5, 146.7, 127.0, 79.6, 70.8, 61.1, 53.4, 126.8, 123.8, 80.9, 70.2, 52.9, 52.0, 49.1, 41.4, 41.2, 37.6, 36.9,
40.7, 32.3, 28.2, 19.2, 17.3, 14.2; LC/MS (ESI): m/z 395.1 33.3, 28.2, 19.0, 18.1; LC/MS (ESI) m/z 674.1 [M + Na]+.
[M + Na]+.
4-{[2-(3-tert-Butoxycarbonylamino-1-hydroxy-4-methyl-pentyl)- tanamido)-1-(4-((2R,4S)-5-methoxy-4-methyl-5-oxo-1-phenylpen-
thiazole-4-carbonyl]-amino}-2-methyl-5-phenylpentanoic acid tan-2-ylcarbamoyl)thiazol-2-yl)-4-methylpentyl benzoate, 30. A
(1R,3R)-3-((2S,3S)-2-(tert-Butoxycarbonylamino)-3-methylpen-
methyl ester, 27. To a solution of ent-8a (212 mg, 0.57 mmol) solution of 28 (120 mg, 0.18 mmol) in a 20% mixture of TFA in
in a 4 : 1 THF–H2O mixture (5 mL), LiOH·H2O (34 mg, DCM (5 mL) was stirred for 1 h. The solvent was removed
0.85 mmol) was added. The reaction mixture was stirred for in vacuo to give 29, which was used in the next step without
5 h. H2O (5 mL) and AcOEt (10 mL) were added and the layers further purification. To a solution of 29 (120 mg, 0.18 mmol)
were separated. The aqueous phase was acidified to pH 1–2 in DCM (5 mL), HOAt (27 mg, 0.20 mmol), HATU (70 mg,
with a 1 N HCl aqueous solution and was extracted with AcOEt 0.20 mmol), Et3N (54 μL, 0.40 mmol) and Boc-Ile (46 mg,
(2 × 10 mL). The combined organic extract was dried over 0.20 mmol) were added. After stirring for 3 h water (10 mL)
anhydrous Na2SO4, filtered and concentrated in vacuo to give was added and the layers separated. The organic phase was
the corresponding acid 26, which was used in the next step washed with a 1 N HCl aqueous solution (10 mL), a saturated
without further purification. To a solution of 26 (138 mg, NaHCO3 aqueous solution (10 mL) and brine (10 mL). After
0.40 mmol) in DCM (5 mL), HOAt (60 mg, 0.44 mmol), HATU drying over anhydrous Na2SO4 and concentration in vacuo, the
(167 mg, 0.44 mmol), Et3N (123 μL, 0.88 mmol) and Tup-OMe crude was purified by FC to give the tripeptide 30 (white foam,
17 (103 mg, 0.40 mmol) were added. After stirring for 3 h 82%). Rf = 0.67 (1 : 1 AcOEt–hexane); [α]2D3 = −13.3 (c = 0.59,
1
water (10 mL) was added and the layers separated. The organic CHCl3); H NMR (400 MHz, CDCl3) δ 8.05 (d, J = 7.9 Hz, 2H),
phase was washed with a 1 N HCl aqueous solution (10 mL), a 8.01 (s, 1H), 7.57 (t, J = 7.4 Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H),
saturated NaHCO3 aqueous solution (10 mL) and brine 7.30–7.16 (m, 6H), 6.90 (br s, 1H), 6.32 (d, J = 9.2 Hz, 1H), 6.20
(10 mL). After drying over anhydrous Na2SO4 and concen- (dd, J = 9.5, 3.5 Hz, 1H), 5.93 (br d, J = 10.4 Hz, 1H), 5.07 (d, J =
tration in vacuo the crude was purified by FC to give the dipep- 8.9 Hz, 1H), 4.15 (d, J = 9.2 Hz, 1H), 4.37 (m, 1H), 3.85–3.75
tide 27 (186 mg, 85%) as a white foam. Rf = 0.35 (1 : 1 AcOEt– (m, 1H), 3.60 (s. 3H), 2.94 (dd, J = 13.6, 6.0 Hz, 1H), 2.85 (dd,
hexane); [α]2D3 = −32.8 (c = 0.51, CHCl3); 1H NMR (400 MHz, J = 13.6, 7.0 Hz, 1H), 2.64–2.56 (m, 1H), 2.37–2.30 (m, 1H),
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2.18–2.11 (m, 1H), 2.03–1.96 (m, 1H), 1.91–1.79 (m, 2H), 14.9, 14.4, 11.3; LC/MS (ESI): m/z 720.2 [M + H]+, 742.2
1.65–1.58 (m, 1H), 1.56–1.48 (m, 1H), 1.40 (s, 9H), 1.14 (d, J = [M + Na]+.
7.0 Hz, 3H), 0.95 (d, J = 5.7 Hz, 3H), 0.93 (d, J = 6.3 Hz, 3H),
(2S,4R)-Methyl 4-(2-((1R,3R)-1-hydroxy-3-((2S,3S)-3-methyl-2-
0.89 (d, J = 6.9 Hz, 3H), 0.85 (d, J = 7.3 Hz, 3H); 13C NMR ((R)-1-methylpiperidine-2-carboxamido)pentanamido)-3-(4-meth-
(100.5 MHz, CDCl3) δ: 176.9, 172.2, 170.3, 165.8, 160.8, 156.5, oxyphenyl)propyl)thiazole-4-carboxamido)-2-methyl-5-phenyl-
150.3, 138.1, 133.8, 130.2, 129.8, 129.7, 128.8, 126.8, 125.4, pentanoate, 24c. (Pale yellow foam, 75% yield); Rf = 0.40 (1 : 9
123.9, 79.1, 70.8, 62.4, 52.0, 51.0, 49.0, 41.6, 40.9, 38.1, 36.8, MeOH–CH2Cl2); [α]2D3 = +72.29 (c = 0.83, CHCl3); FT-IR (film)
32.2, 30.0, 28.6, 25.2, 19.3, 18.1, 16.0, 14.7; EI MS m/z: 787.2 νmax: 3340.2, 3064.1, 1729.1, 1652.0, 1084.1 cm−1
;
1H NMR
[M + Na]+.
(400 MHz, CD3OD) δ: 8.02 (s, 1H), 7.29–7.11 (m, 7H), 6.84 (d,
General procedure for coupling the tripeptide Ile-Tuv-Tup- J = 8.6 Hz, 2H), 5.28 (dd, J = 9.6, 3.8 Hz, 1H), 4.90 (dd, J = 9.5,
OMe with Mep. A solution of tripeptide 21a (150 mg, 3.4 Hz, 1H), 4.36–4.31 (m, 1H), 4.28–4.22 (m, 1H), 3.74 (s, 3H),
0.27 mmol ) in a 20% mixture of TFA in DCM (5 mL) was 3.56 (s, 3H), 2.96–2.83 (m, 2H), 2.65–2.39 (m, 3H), 2.21–2.12
stirred for 1 h. The solvent was removed in vacuo to give 22a, (m, 1H), 2.10 (s, 3H), 2.05–1.80 (m, 3H), 1.73–1.42 (m, 7H),
which was used in the next step without further purification. 1.32–1.16 (m, 3H), 1.12 (d, J = 7.1 Hz, 3H), 0.97 (d, J = 6.7 Hz,
To a solution of 22a (100 mg, 0.18 mmol) in DCM (5 mL), 3H), 0.90 (t, J = 7.3 Hz, 3H); 13C NMR (100.5 MHz, CD3OD) δ:
HOAt (27 mg, 0.2 mmol), HATU (76 mg, 0.2 mmol), Mep 179.3, 178.8, 176.4, 173.6, 163.6, 161.0, 151.4, 139.9, 136.1,
(N-methyl-(R)-pipecolic acid (28 mg, 0.18 mmol) and Et3N 131.1, 130.0, 129.4, 128.1, 125.3, 115.6, 71.2, 70.3, 59.8, 59.6,
(50 μL, 0.36 mmol) were added. The reaction mixture was 57.2, 56.5, 52.9, 51.6, 50.8, 45.8, 45.4, 43.0, 39.6, 38.4, 38.1,
stirred for 3 h and then washed with a 1 N HCl aqueous 32.1, 26.7, 24.9, 18.8, 16.9, 11.7; LC/MS (ESI): m/z 750.3
solution (10 mL), a saturated NaHCO3 aqueous solution [M + H]+, 772.3 [M + Na]+.
(10 mL) and brine (10 mL). The combined organic extracts
(2S,4R)-Methyl 4-(2-((1R,3R)-1-hydroxy-3-((2S,3S)-3-methyl-2-
were dried over Na2SO4 and concentrated in vacuo. The crude ((R)-1-methylpiperidine-2-carboxamido)pentanamido)-3-cyclo-
tetrapeptide 35a–e thus obtained was purified using silica gel hexylpropyl)thiazole-4-carboxamido)-2-methyl-5-phenylpen-
chromatography.
(2S,4R)-Methyl 4-(2-((1R,3R)-1-hydroxy-4-methyl-3-((2S,3S)-3- MeOH–CH2Cl2); [α]2D3 = +19.23 (c = 0.52, CHCl3); FT-IR (film)
methyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido)- νmax: 3291.6, 3021.1, 2960.5, 1729.7, 1645.6 cm−1 1H NMR
tanoate, 24d. (Pale yellow foam, 97% yield); Rf = 0.47 (1 : 9
;
pentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoate, (400 MHz, CD3OD) δ: 7.89 (s, 1H), 7.33–7.07 (m, 5H), 4.85–4.81
24a. (White foam, 60% yield) Rf = 0.39 (1 : 9 MeOH–CH2Cl2); (m, 1H), 4.34–4.26 (m, 1H), 4.24–4.14 (m, 1H), 4.16–4.10 (m,
[α]2D3 = +35.3 (c 0.6, CDCl3); FT-IR (film) νmax: 3290.4, 2960.5, 1H), 3.61 (s, 3H), 3.01–2.80 (m, 3H), 2.71–2.59 (m, 1H),
1
1729.7, 1645.6, 1541.7 cm−1; H NMR (400 MHz, [D6]DMSO) δ: 2.55–2.43 (m, 1H), 2.14 (s, 3H), 2.07–1.83 (m, 6H), 1.81–1.50
8.08 (s, 1H), 7.75 (d, J = 8.9 Hz, 1H), 7.66 (d, J = 9.6 Hz, 1H), (m, 11H), 1.48–0.71 (m, 8H), 1.17 (d, J = 7.1 Hz, 3H), 0.98 (dd,
7.51 (br d, J = 8.9 Hz, 1H), 7.30–7.10 (m, 5H), 6.15 (d, J = 5.6 J = 6.6 Hz, 3H), 0.90 (t, J = 7.4 Hz, 3H); 13C NMR (100.5 MHz,
Hz, 1H), 4.73–4.65 (m, 1H), 4.24–4.12 (m, 2H), 4.03–3.91 (m, CD3OD) δ: 181.6, 180.7, 178.9, 175.5, 166.1, 165.9, 142.5, 133.5,
1H), 3.52 (s, 3H), 2.92–2.74 (m, 3H), 2.57–2.50 (m, 1H), 132.2, 130.4, 122.7, 73.6, 73.3, 62.5, 59.6, 55.6, 55.2, 53.4, 48.2,
2.47–2.38 (m, 1H), 2.11 (s, 3H), 2.03–1.69 (m, 6H), 1.68–1.26 47.0, 45.3, 44.0, 42.5, 40.9, 40.7, 34.8, 34.5, 34.0, 32.7, 30.6,
(m, 7H), 1.28–1.08 (m, 2H), 1.06 (d, J = 6.8 Hz, 3H), 0.93–0.76 30.4, 29.2, 26.8, 25.0, 19.4, 17.3, 12.1; LC/MS (ESI) m/z 727.3
(m, 12H); 13C NMR (100.5 MHz, [D6]DMSO) δ: 177.6, 175.4, [M + H]+, 749.3 [M + Na]+.
170.9, 159.8, 14.2, 138.1, 128.8, 127.8, 125.8, 122.8, 68.7, 67.5,
(2S,4R)-Methyl 4-(2-((1R,3R)-1-methoxy-4-methyl-3-((2S,3S)-3-
56.5, 50.9, 49.9, 47.8, 43.6, 40.3, 37.2, 35.9, 35.5, 31.4, 29.4, methyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido)-
24.5, 24.2, 22.6, 18.7, 17.7, 17.2, 15.2, 10.1; LC/MS (ESI) m/z pentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoate,
686.3 [M + H]+.
24e. (White foam, 82%); Rf = 0.60 (8 : 92 MeOH–CHCl3), [α]D23
=
(2S,4R)-Methyl 4-(2-((1R,3R)-1-hydroxy-3-((2S,3S)-3-methyl-2- −2.1 (c = 0.62, CHCl3); FT-IR (film) νmax: 3275.1, 2935.8,
((R)-1-methylpiperidine-2-carboxamido)pentanamido)-3-phe- 1735.5, 1642.1, 1541.8, 1494.6 cm−1 1H NMR (400 MHz,
;
nylpropyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoate, CDCl3) δ: 8.01 (s, 1H), 7.28–7.19 (m, 5H), 7.11 (d, J = 9.6 Hz,
24b. (Pale yellow foam, 72% yield); Rf = 0.33 (5 : 95 MeOH– 1H), 7.04 (d, J = 8.7 Hz, 1H), 6.19 (d, J = 9.9 Hz, 1H), 4.47–4.39
CH2Cl2); [α]2D3 = +30.4 (c = 0.5, CHCl3); FT-IR (film) νmax
:
(m, 2H), 4.20–4.13 (m, 2H), 3.53 (s, 3H), 3.43 (s, 3H), 2.97 (dd,
3304.5, 3064.0, 2925.5, 1729.2, 1651.5, 1540.3, 1216.0 cm−1; 1H J = 13.7, 5.6 Hz, 1H), 2.94–2.88 (m, 1H), 2.83 (dd, J = 13.8,
NMR (400 MHz, CDCl3) δ: 8.00 (s, 1H), 7.42–7.18 (m, 10H), 7.4 Hz, 1H), 2.56–2.51 (m, 2H), 2.22 (s, 3H), 2.10–2.04 (m, 2H),
7.08 (d, J = 9.7 Hz, 1H), 5.36–5.26 (m, 1H), 4.91 (d, J = 10.6 Hz, 1.99–1.87 (m, 2H), 1.80–1.49 (m, 6H), 1.43–1.33 (m, 1H),
1H), 4.54–4.25 (m, 2H), 3.51 (s, 3H), 2.99–2.80 (m, 3H), 1.22–1.17 (m, 1H), 1.15 (d, J = 7.1 Hz, 3H), 1.12 (d, J = 6.4 Hz,
2.55–2.44 (m, 2H), 2.37–1.84 (m, 5H), 2.03 (s, 3H), 1.82–1.16 3H), 1.00 (d, J = 6.7 Hz, 3H), 0.98–0.82 (m, 9H); 13C NMR
(m, 8H), 1.16 (d, J = 6.8 Hz, 3H), 1.00 (d, J = 5.6 Hz, 3H), 0.89 (100.6 MHz, CDCl3) δ: 177.0, 175.0, 174.3, 170.8, 160.7, 150.1,
(t, J = 7.2 Hz, 3H); 13C NMR (100.5 MHz, CDCl3) δ: 175.4, 172.8, 137.7, 129.5, 128.4, 126.5, 123.3, 78.6, 69.6, 58.7, 57.8, 55.4,
161.2, 150.1, 140.9, 137.9, 131.2, 130.0, 129.3, 128.8, 128.3, 51.5, 50.7, 48.3, 44.9, 42.0, 40.4, 37.5, 36.7, 35.3, 32.2, 30.8,
127.0, 126.9, 123.6, 122.7, 69.0, 55.7, 51.9, 51.2, 48.4, 44.7, 25.1, 24.9, 23.3, 19.0, 17.9, 16.9, 16.1, 10.8; LC/MS (ESI) m/z
42.2, 37.9, 37.2, 32.3, 30.1, 29.7, 25.3, 23.0, 17.4, 16.6, 16.4, 700.3 [M + H]+, 722.3 [M + Na]+.
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(2S,4R)-Methyl 4-(2-((1R,3R)-1-methoxymethyloxy-4-methyl- 2.28–2.21 (m, 1H), 2.14 (s, 3H), 2.13–2.07 (m, 1H), 2.02–1.96
3-((2S,3S)-3-methyl-2-((R)-1-methylpiperidine-2-carboxamido)- (m, 1H), 1.93–1.84 (m, 2H), 1.83–1.76 (m, 2H), 1.71–1.53 (m,
pentanamido)pentyl)thiazole-4-carboxamido)-2-methyl-5-phe- 5H), 1.45–1.35 (m, 1H), 1.23–1.12 (m, 1H), 1.14 (d, J = 7.0 Hz,
nylpentanoate, 24f. (White foam, 74%); Rf = 0.71 (8 : 92 3H), 0.97 (d, J = 6.8 Hz, 3H), 0.94–0.87 (m, 9H); 13C NMR
MeOH–CHCl3); [α]D23 = −11.2 (c = 0.6, CHCl3); 1H NMR (100.5 MHz, CD3OD) δ: 181.9, 173.7, 173.3, 171.7, 162.7, 151.1,
(400 MHz, CDCl3) δ: 7.99 (s, 1H), 7.25–7.14 (m, 5H), 7.02 139.6, 130.5, 129.3, 127.3, 125.0, 71.3, 69.6, 59.6, 56.3, 52.0,
(d, J = 8.7 Hz, 1H), 6.20 (d, J = 9.6 Hz, 1H), 4.80 (dd, J = 9.9, 51.0, 44.0, 41.9, 39.2, 38.9, 38.1, 37.6, 33.7, 31.0, 25.9, 25.4,
2.6 Hz, 1H), 4.74 (d, J = 6.7 Hz, 1H), 4.69 (d, J = 6.7 Hz, 1H), 23.5, 20.7, 19.5, 18.8, 18.6, 16.2, 11.1; LC/MS (ESI Ion Trap):
4.43–4.36 (m, 1H), 4.16–4.10 (m, 2H), 3.61 (s, 3H), 3.37 (s, 3H), m/z 720.3 [M + H]+.
2.95–2.80 (m, 3H), 2.63–2.57 (m, 1H), 2.49 (dd, J = 11.2, 3.2 Hz,
(2S,4R)-4-(2-((1R,3R)-1-Acetoxy-3-((2S,3S)-3-methyl-2-((R)-1-
1H), 2.20 (s, 3H), 2.04–1.95 (m, 4H), 1.88–1.74 (m, 3H), methylpiperidine-2-carboxamido)pentanamido)-3-phenylpro-
1.70–1.35 (m, 5H), 1.28–1.17 (m, 2H), 1.14 (d, J = 7.0 Hz, 3H,), pyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoic acid,
0.96 (d, J = 6.7 Hz, 3H), 0.92–0.84 (m, 9H); 13C NMR 1b. (White foam, 80% yield); Rf = 0.28 (1 : 9 MeOH–CH2Cl2);
1
(100.5 MHz, CDCl3) δ: 176.8, 175.4, 174.1, 171.3, 160.9, 150.5, [α]2D3 = −14.05 (c = 0.19, CHCl3); H NMR (400 MHz, CD3OD) δ
138.1, 129.8, 128.4, 126.8, 123.5, 97.4, 74.6, 70.0, 58.2, 57.0, 8.16 (s, 1H), 7.45–7.16 (m, 10H), 6.11 (dd, J = 9.6, 3.9 Hz, 1H),
55.7, 52.0, 51.0, 48.9, 45.3, 41.7, 40.5, 38.2, 36.8, 35.6, 32.6, 5.22 (dd, J = 9.8, 4.6 Hz, 1H), 4.50–4.38 (m, 1H), 4.32 (dd, J =
31.1, 25.5, 23.6, 19.2, 18.1, 16.3, 14.4, 11.1; LC/MS (ESI) m/z 14.3, 8.6 Hz, 1H), 3.22–3.10 (m, 1H), 3.05–2.89 (m, 2H), 2.70
752.2 [M + Na]+.
(ddd, J = 14.2, 10.3, 3.8 Hz, 1H), 2.62–2.40 (m, 3H), 2.35 (s,
(2S,4R)-Methyl-4-(2-((1R,3R)-1-benzoyloxy-4-methyl-3-((2S,3S)- 3H), 2.21 (s, 3H), 2.16–1.31 (m, 12H), 1.20 (d, J = 6.9 Hz, 3H),
3-methyl-2-((R)-1-methylpiperidine-2-carboxamido)pentana- 1.02 (d, J = 6.7 Hz, 3H), 0.97 (t, J = 7.3 Hz, 3H); 13C NMR
mido)pentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentano- (100.5 MHz, CD3OD) δ: 182.5, 173.2, 171.8, 170.9, 163.0, 151.1,
ate, 32. (White foam, 82%); Rf = 0.41 (1 : 9 MeOH–CHCl3); 143.1, 143.1, 139.7, 130.7, 129.9, 129.5, 128.8, 127.9, 127.6,
1
[α]2D3 = −20.5 (c = 0.39, CHCl3); H NMR (400 MHz, CDCl3) δ: 125.6, 72.5, 71.6, 70.9, 60.9, 57.8, 52.8, 52.1, 45.2, 44.0, 43.2,
8.08 (d, J = 8.1 Hz, 2H), 8.01 (s, 1H), 7.59 (t, J = 7.3 Hz, 1H), 40.6, 39.1, 32.2, 27.5, 26.6, 24.8, 22.2, 19.3, 17.7, 12.6; LC/MS
7.46 (t, J = 7.7 Hz, 2H), 7.30–7.16 (m, 5H), 6.90 (br s, 1H), 6.21 (ESI) m/z 748.3 [M + H]+, 770.3 [M + Na]+.
(br s, 1H), 6.15 (dd, J = 10.6, 3.1 Hz, 1H), 4.45–4.38 (m, 1H),
(2S,4R)-4-(2-((1R,3R)-1-Acetoxy-3-((2S,3S)-3-methyl-2-((R)-1-
4.20–4.14 (m, 1H), 4.11–4.05 (m, 1H), 3.62 (s, 3H), 2.96 (dd, J = methylpiperidine-2-carboxamido)pentanamido)-3-(4-methoxy-
13.6, 6.2 Hz, 1H), 2.87 (dd, J = 13.6, 6.9 Hz, 1H), 2.65–2.57 (m, phenyl)propyl)thiazole-4-carboxamido)-2-methyl-5-phenylpen-
1H), 2.52–2.44 (m, 1H), 2.37–2.29 (m, 1H), 2.12 (s, 3H), tanoic acid, 1c. (White foam, 95% yield); Rf = 0.37 (1 : 9
2.05–1.93 (m, 6H), 1.88–1.85 (m, 2H), 1.70–1.60 (m, 5H), MeOH–CH2Cl2); [α]2D3 = +21.63 (c = 0.59, CHCl3); 1H NMR
1.55–1.47 (m, 3H), 1.17 (d, J = 6.9 Hz, 3H), 0.95–0.86 (m, 12H); (400 MHz, CD3OD) δ: 8.13 (s, 1H), 7.29–7.03 (m, 7H), 6.85 (d,
13C NMR (63 MHz, CDCl3) δ: 176.6, 175.1, 170.8, 169.9, 165.4, J = 8.6 Hz, 2H), 6.04 (dd, J = 9.7, 3.8 Hz, 1H), 5.12 (dd, J = 10.0,
160.3, 150.1, 137.7, 133.5, 129.8, 129.4, 128.5, 128.3, 126.4, 4.8 Hz, 1H), 4.44–4.41 (m, 1H), 4.30–4.22 (m, 1H), 3.74 (s, 3H),
123.4, 70.4, 69.7, 57.8, 51.7, 50.4, 48.5, 44.9, 41.2, 39.9, 37.8, 3.23 (t, J = 12.8 Hz, 1H), 2.86 (d, J = 6.1 Hz, 2H), 2.72–2.42
35.6, 35.5, 34.9, 31.8, 29.7, 25.2, 24.8, 23.2, 18.8, 17.7, 15.8, (m, 3H), 2.41 (s, 3H), 2.11 (s, 3H), 2.05–1.14 (m, 13H), 1.11
10.7; LC/MS (ESI) m/z 790.3 [M + H]+, 812.3 [M + Na]+.
(d, J = 6.7 Hz, 3H), 0.94 (d, J = 6.7 Hz, 3H), 0.89 (t, J = 7.1 Hz,
General procedure for the hydrolysis of methyl ester 24a–f 3H); 13C NMR (100.5 MHz, CD3OD) δ: 180.5, 179.3, 173.7,
and acetylation of 25a–d. To a solution of compound 24a 172.4, 171.8, 164.0, 161.4, 151.4, 140.3, 135.8, 131.3, 130.1,
(120 mg, 0.17 mmol) in THF (5 mL), a 1 N aqueous solution of 129.7, 128.3, 120.6, 115.9, 71.9, 69.8, 61.5, 57.1, 56.7, 51.7,
LiOH (510 μL, 0.51 mmol) was added. After stirring for 3 days, 51.4, 44.4, 43.1, 42.6, 41.4, 40.7, 39.4, 31.3, 26.8, 25.6, 23.8,
TFA (53 μL, 0.68 mmol) was then added and the solvent was 21.6, 20.0, 17.0, 12.0; LC/MS (ESI) m/z 778.2 [M + H]+, 800.2
removed in vacuo to give 25a, which was used in the next step [M + Na]+.
without further purification. To a solution of 25a (126 mg,
(2S,4R)-4-(2-((1R,3R)-1-Acetoxy-3-((2S,3S)-3-methyl-2-((R)-1-
0.16 mmol) in pyridine (3 mL), Ac2O (1 mL) was added. The methylpiperidine-2-carboxamido)pentanamido)-3-cyclohexyl-
resulting mixture was stirred overnight. The mixture was propyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoic
concentrated under reduced pressure and the crude purified acid, 1d. (White foam, 75% yield); Rf = 0.38 (1 : 9 MeOH–
by FC.
CH2Cl2); [α]2D3 = +13.6 (c = 0.32, CHCl3); 1H NMR (400 MHz,
(2S,4R)-4-(2-((1R,3R)-1-Acetoxy-4-methyl-3-((2S,3S)-3-methyl- CD3OD) δ: 7.99 (s, 1H), 7.26–7.09 (m, 5H), 6.00 (dd, J = 9.5,
2-((R)-1-methylpiperidine-2-carboxamido)pentanamido)pentyl)- 4.9 Hz, 1H), 4.38 (d, J = 6.9 Hz, 1H), 4.24 (d, J = 8.2 Hz, 1H),
thiazole-4-carboxamido)-2-methyl-5-phenylpentanoic
acid, 4.09–3.95 (m, 1H), 3.23–3.12 (m, 1H), 3.02–2.86 (m, 2H),
1a. (White foam, 89% yield); Rf = 0.28 (1 : 9 MeOH–CH2Cl2); 2.77–2.54 (m, 1H), 2.53–2.41 (m, 1H), 2.48 (s, 3H), 2.12 (s, 3H),
[α]2D3 = −1.42 (c 1.55, MeOH); 1H NMR (400 MHz, CD3OD) δ: 2.08–0.87 (m, 25H), 1.19 (d, J = 6.9 Hz, 3H), 0.99 (d, J = 6.9 Hz,
8.07 (s, 1H), 7.21 (d, J = 4.2 Hz, 4H), 7.17–7.11 (m, 1H), 5.90 3H), 0.91 (t, J = 7.2 Hz, 3H); 13C NMR (100.5 MHz, CD3OD) δ:
(dd, J = 10.8, 3.0 Hz, 1H), 4.39–4.32 (m, 1H), 4.21 (d, J = 8.2 Hz, 173.9, 173.6, 172.2, 171.6, 163.9, 163.7, 151.9, 140.9, 131.5,
1H), 3.99–3.94 (m, 1H), 3.13–3.05 (m, 2H), 2.91(d, J = 6.8 Hz, 130.1, 128.1, 125.5, 73.3, 70.6, 61.8, 57.3, 52.4, 52.0, 45.0, 44.9,
2H), 2.57–2.49 (m, 1H), 2.48–2.42 (m, 1H), 2.40 (s, 3H), 43.5, 42.6, 40.0, 39.1, 38.7, 31.8, 31.6, 30.8, 29.7, 28.4, 28.2,
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26.9, 26.0, 20.7, 20.4, 19.5, 17.1, 12.0; LC/MS (ESI) m/z 754.7 Biological tests
[M + H]+, 776.7 [M + Na]+.
(2S,4R)-4-(2-((1R,3R)-1-Methoxy-4-methyl-3-((2S,3S)-3-methyl-
2-((R)-1-methylpiperidine-2-carboxamido)pentanamido)pentyl)-
Potential anti-tumor activity was evaluated through in vitro
assays based on the determination of cytotoxicity of the com-
pounds towards the HT29 (Human Caucasian Colon Adeno-
carcinoma) cell line. HT29 cells from ECACC (European
Collection of Cell Cultures) were purchased from Sigma
Aldrich, Milan, Italy.
thiazole-4-carboxamido)-2-methyl-5-phenylpentanoic
acid,
1e. (White foam, 40% yield); Rf = 0.47 (8 : 92 MeOH–CHCl3);
[α]2D3 = −7.3 (c = 0.24, CHCl3); 1H NMR (400 MHz, CD3OD)
δ: 8.08 (s, 1H), 7.21–7.14 (m, 5H), 4.48–4.42 (m, 1H), 4.37–4.33
(m, 1H), 4.22 (d, J = 8.5 Hz, 1H), 4.10–4.01 (m, 1H), 3.45
(s, 3H), 3.30–3.20 (m, 1H), 3.02–2.99 (m, 1H), 2.89 (d,
J = 6.5 Hz, 2H), 2.42–2.34 (m, 2H), 2.37 (s, 3H), 2.01–1.87
(m, 5H), 1.80–1.56 (m, 7H), 1.43–1.37 (m, 1H), 1.26–1.20
(m, 1H), 1.12 (d, J = 6.9 Hz, 3H), 1.01 (d, J = 6.6 Hz, 3H),
0.91 (m, 9H); 13C NMR (100.5 MHz, CD3OD) δ: 182.1, 175.8,
173.7, 173.6, 163.1, 151.1, 139.7, 130.6, 129.5, 127.6, 124.9,
79.9, 69.8, 59.8, 59.1, 56.4, 52.6, 50.7, 44.3, 42.7, 40.6, 39.3,
37.7, 33.8, 31.2, 26.1, 25.5, 23.6, 19.6, 18.7, 17.8, 16.5, 11.2;
LC/MS (ESI) m/z 686.3 [M + H]+, 708.3 [M + Na]+.
(2S,4R)-4-(2-((1R,3R)-1-Methoxymethyloxy-4-methyl-3-((2S,3S)-
3-methyl-2-((R)-1-methylpiperidine-2-carboxamido)pentana-
mido)pentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpenta-
noic acid, 1f. (White foam, 57% yield); Rf = 0.43 (8 : 92 MeOH–
CHCl3); [α]2D3 = −18.4 (c = 0.20, CHCl3); 1H NMR (400 MHz,
CD3OD) δ: 8.13 (s, 1H), 7.18–7.09 (m, 5H), 4.83 (d, J = 9.7 Hz,
1H), 4.70 (d, J = 6.9 Hz), 1H, 4.65 (d, J = 6.9 Hz, 1H), 4.46–4.40
(m, 1H), 4.20 (d, J = 8.3 Hz, 1H), 4.06–4.00 (m, 1H), 3.11
(s, 3H), 3.26–3.23 (m, 1H), 2.89–2.84 (m, 2H), 2.69–2.63
(m, 1H), 2.53 (s, 3H), 2.45–2.41 (m, 1H), 2.20–1.89 (m, 5H),
1.84–1.45 (m, 9H), 1.27–1.19 (m, 1H), 1.11 (d, J = 6.6 Hz, 3H),
1.00 (d, J = 6.6 Hz, 3H), 0.94–0.89 (m, 9H); 13C NMR
(100.5 MHz, CD3OD) δ: 178.3, 176.5, 176.4, 174.8, 165.8, 153.5,
142.5, 133.3, 132.2, 130.3, 128.4, 100.6, 78.1, 72.0, 64.5, 62.8,
59.9, 59.2, 55.2, 46.6, 45.2, 40.4, 36.7, 35.7, 33.6, 28.8, 27.8,
26.6, 26.0, 22.3, 21.5, 19.2, 17.4, 14.1; LC/MS (ESI) m/z 716.2
[M + H]+, 738.2 [M + Na]+.
The assays were performed according to the previously
reported procedure. The cell line was grown in 75 cm2 flasks
with culture medium DMEM (Dulbecco’s Medium Eagle Modi-
fied, Sigma Aldrich) and the following additives (Sigma
Aldrich): L-glutamine 2 mM, 10% fetal bovine serum, penicil-
lin/streptomycin, fungizone, gentamycin. The incubation was
carried out in a modified atmosphere incubator (37 °C, 5%
CO2). When cell confluence was obtained, the cells were propa-
gated by dilution in a ratio 1 : 3/1 : 10 by using a 0.25% trypsin
solution and then transferred into 96 well plates in the suit-
able culture medium. The cells were then treated for 72 hours
with different doses of the compounds under examination. For
these assays, the compounds were solubilised in DMSO. All
the tests were carried out at a constant DMSO concentration
equal to 0.1% by weight.
To control the cellular viability, the ATPlite test (Perkin
Elmer) was used, based on the determination of the ATP pro-
duction. ATP is in fact a marker of cellular viability as it is
present in all the metabolically active cells. The test is based
on the chemiluminescence due to the ATP reaction with the
luciferase and the D-luciferin. The emitted light is proportional
to the ATP concentration. The tests were carried out by a Victor
3 instrument (Perkin Elmer).
IC50 values were obtained from four different experiments.
Notes and references
(2S,4R)-4-(2-((1R,3R)-1-Benzoyloxy-4-methyl-3-((2S,3S)-3-methyl-
2-((R)-1-methylpiperidine-2-carboxamido)pentanamido)pentyl)-
thiazole-4-carboxamido)-2-methyl-5-phenylpentanoic acid,
1g. To a solution of ester 32 (73 mg, 0.095 mmol) in DCE
(3 mL), Me3SnOH (11 mg, 0.58 mmol) was added and the reac-
tion mixture was stirred under reflux for 32 h. The solvent was
removed in vacuo and the residue was purified by FC (5 : 95
MeOH–CH2Cl2) to give 1g (15 mg, 20%) as a white foam. Rf =
0.41 (1 : 9 MeOH–CH2Cl2); [α]2D3 = −26.6 (c = 0.33, CHCl3);
1H NMR (400 MHz, CD3OD) δ: 8.11 (s, 1H), 8.09–7.68 (m, 5H),
7.24–7.20 (m, 5H), 6.21–6.18 (m, 1H), 4.40–4.34 (m, 1H),
4.09–4.06 (m, 1H), 3.16–3.10 (m, 1H), 2.98–2.91 (m, 3H),
2.60–2.56 (m, 1H), 2.48–2.43 (m, 2H), 2.39 (s, 3H), 2.30–2.24
(m, 1H), 2.05–1.98 (m, 1H), 1.93–1.85 (m, 3H), 1.75–1.53
(m, 7H), 1.41–1.30 (m, 2H), 1.18 (d, J = 7.2 Hz, 3H), 1.00–0.91
(m, 12H); 13C NMR (100.5 MHz, CD3OD) δ: 176.4, 174.5, 169.9,
165.8, 153.9, 142.4, 137.2, 133.8, 133.7, 133.5, 133.3, 132.7,
135.5, 132.2, 130.2, 127.8, 74.9, 72.8, 62.3, 59.3, 55.2, 53.9,
46.9, 44.8, 42.1, 41.4, 40.5, 36.7, 34.0, 28.7, 28.4, 26.5, 22.3,
21.6, 21.4, 19.2, 14.0; LC/MS (ESI) m/z 776.3 [M + H]+, 798.3
[M + Na]+.
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