Total Synthesis and Biological Evaluation of Tubulysin Analogues

Article abstract of DOI:10.1021/acs.joc.6b01314

We report a second-generation synthesis of the exceedingly potent antimitotic agent N¹⁴-desacetoxytubulysin H (1) as well as the preparation of nine analogues of this lead structure. Highlights of our synthetic efforts include an efficient late

Full text of DOI:10.1021/acs.joc.6b01314

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Article
Total Synthesis and Biological Evaluation of Tubulysin Analogs
Raffaele Colombo, Zhiyong Wang, Junyan Han, Raghavan Balachandran, Hikmat N.
Daghestani, Daniel P. Camarco, Andreas Vogt, Billy W. Day, David Mendel, and Peter Wipf
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b01314 • Publication Date (Web): 22 Jul 2016
Downloaded from http://pubs.acs.org on July 26, 2016
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The Journal of Organic Chemistry
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Total Synthesis and Biological Evaluation of Tubulysin
Analogs
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Raffaele Colombo,1,4 Zhiyong Wang,1 Junyan Han,1 Raghavan Balachandran,2 Hikmat N. Daghestani,2,3
∆
Daniel P. Camarco,3 Andreas Vogt,3 Billy W. Day,2,3 David Mendel,4 and Peter Wipf1,2,
*
1Department of Chemistry, University of Pittsburgh, Pittsburgh PA 15260, United States
2Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh PA 15261, United States
3Department of Computational and Systems Biology and University of Pittsburgh Drug Discovery
Institute, University of Pittsburgh, Pittsburgh PA 15261, United States
4Lilly Research Laboratories, A Division of Eli Lilly and Company, Indianapolis, IN 46285, United
States
RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required
according to the journal that you are submitting your paper to)
Abstract. We report a second-generation synthesis of the exceedingly potent antimitotic agent, N14-
desacetoxytubulysin H (1), as well as the preparation of nine analogs of this lead structure. Highlights of
our synthetic efforts include an efficient late stage functionalization that allows for the preparation of
new side chain and backbone modified analogs. We also discovered C-terminal modifications that
preserve the exquisite biological activity of acid 1 and offer the opportunity for effective conjugation to
cell type-targeting moieties. All analogs had antiproliferative activities in the high picomolar to low
nanomolar range and caused apoptosis and mitotic arrest as measured in a high content nuclear
morphology assay. The ten synthetic agents described herein spanned a range of almost 4 orders of
magnitude in biological activity and illustrate the continued potential to discover extraordinarily potent
antiproliferative compounds based on natural product leads.
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Introduction
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Tubulysins are a family of natural tetrapeptides with attractive prospects in anti-cancer drug
development due to their exquisitely potent antiproliferative and antiangiogenic properties (Figure 1).1,2
Tubulysins can perturb microtubule polymerization and induce apoptosis of various cancer cell lines at
subnanomolar concentrations.3
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Mep
Ile
Tuv
R2
Tut/Tup
17
O
H
N
13
(R)
(R)
11
(R)
14
O
N
R1
N
N
(S)
(S)
O
R3
S
HN(R)
5
(S)
1 COOH
Natural tubulysins
Tubulysin
R1
R2
R3
A
B
C
D
E
F
OH
OH
OH
H
OAc
OAc
OAc
OAc
OAc
OAc
OAc
OAc
OAc
OAc
OAc
OAc
OAc
OAc
H
CH2OC(O)CH2CH(CH3)2
CH2OC(O)CH2CH2CH3
CH2OC(O)CH2CH3
CH2OC(O)CH2CH(CH3)2
CH2OC(O)CH2CH2CH3
CH2OC(O)CH2CH3
CH2OC(O)CH=C(CH3)2
CH2OC(O)CH3
CH2OC(O)CH3
H
H
H
G
H
I
OH
H
OH
H
U
V
W
X
Z
H
H
OH
OH
OH
OH
H
CH2OC(O)CH2CH2CH3
H
H
Pretubulysin A
Pretubulysin D
Me
H
Me
Desacetyl tubulysin D
Desacetyl tubulysin E
H
OH
CH2OC(O)CH2CH(CH3)2
CH2OC(O)CH2CH2CH3
H
OH
Synthetic tubulysins
N14-Desacetoxytubulysin H (1)
TUB-OMOM
H
H
F
OAc
OCH2OCH3
OAc
Me
Me
Bn
KEMTUB10
Figure 1. Structures of select natural and synthetic tubulysins.
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The Journal of Organic Chemistry
The remarkable ability of tubulysins to inhibit the growth of cancer cells, including those with multidrug
resistance,4 exceeds paclitaxel, epothilones and vinblastine by 10-2,000 fold.5 Besides inhibition of cell
proliferation, tubulysins show strong angiogenesis effects,6 display antivascular properties in vitro and
in vivo,7 and therefore represent promising novel agents for the treatment of metastatic and invasive
tumors, in particular in combination with drug targeting strategies.8,9 While tubulysins have previously
been isolated in low quantities (0.5-5 mg/L) from the myxobacterial strains Archangium gephyra and
Angiococcus disciformis,1 substantial efforts have also been directed toward the total synthesis of these
natural products and their analogs.3,10
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Shortly after the elucidation of the structure and configuration of tubulysin D by degradation studies,11
the preparation of two subunits was reported.12,13 Subsequently, several preparations of stereoisomers of
tubulysins U and V were published,14,15,16 including a total synthesis of tubulysins U and V.17 Two total
syntheses of tubulysin D, which contains the acid- and base-labile N,O-acetal functionality, were
reported in 2006 and 2010.16,18 Tubulysin B has also received considerable attention and a total synthesis
was first reported in 2009,19 followed by a more efficient synthesis which also allowed the preparation
of folate conjugates for tumor site-directed drug delivery.20
In 2009, pretubulysin D was isolated in minor amounts from cultures of A. disciformis.21 This putative
tubulysin biosynthesis precursor lacks the N,O-acetal and 11-OAc groups. Its structure was elucidated
by a combination of mass spectrometry, total synthesis and NMR.22 While pretubulysin D is ca. 10-fold
less active than tubulysins A and D, it is still effective at nano- to picomolar concentrations against
cancer cell lines and retains the high tubulin-degrading activity of some of the more complex tubulysins.
Other pretubulysins from Angiococcus disciformis An d48 and Cystobacter SBCb004 strains
demonstrate in fact that structural complexity can be reduced without a dramatic drop in potency.22
Pretubulysin D showed strong anti-angiogenic and antivascular properties in vitro and in vivo, which
bodes well for the treatment of metastatic cancer.6,7,8 Pretubulysins and their structural analogs might
prove to be more appealing for tumor therapy since preclinical studies have shown that the more potent
tubulysins are generally too toxic for use as chemotherapeutics and possess a very narrow therapeutic
window.20,23,24 Accordingly, drug-targeting approaches offer a more attractive treatment option.
Tubulysins can be conjugated to polymers, small ligands, or antibodies in order to increase their tumor
specificity. For example, tubulysin A was covalently attached to a linear β-cyclodextrin based polymer
through a disulfide linker (CDP-TubA), and this conjugate showed a 100-fold increase in the maximum
tolerated dose (MTD) in nude mice and an increased efficacy in two human cancer xenografts, whereas
tubulysin A at its MTD was completely inactive.23 The synthetic tubulysin analogue TUB-OMOM was
conjugated to trastuzumab (a humanized anti-HER2 monoclonal antibody) and the resulting construct
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was shown to have dose-dependent antitumor effects, including complete in vivo tumor eradication in
trastuzumab-sensitive tumors.25 Finally, encouraging results were obtained in animal experiments with
EC0305, a small molecule folate-tubulysin conjugate, taking advantage of the high expression of the
folate receptor in many cancer types.20,24,26 In contrast, natural tubulysin B alone again proved inactive
when administered at doses near to or greater than its MTD. EC305 also displayed superior antitumor
activity compared to a folate-desacetylvinblastine monohydrazide conjugate (EC145, currently in phase
II clinical trials).27 EC1456, a second generation folate-tubulysin B conjugate bearing a highly
hydrophilic linker is being evaluated in a Phase I study in patients with advanced solid tumors.28,29,30
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As part of our investigations of natural products targeting tubulin,31 32 33 our group reported in 2007 the
synthesis of N14-desacetoxytubulysin H (1),34,35 a simplified analog that was also later also synthesized
by Patterson et al.36 N14-Desacetoxytubulysin H (1) possesses all the structural features of tubulysin H,
except its labile N,O-acetal functionality, and maintains high cytotoxicity in vitro. Several other non-
natural tubulysin analogs have been prepared recently, reflecting an unabated interest in this drug
class.37 SAR studies have mainly focused on iterative replacements of the four amino acid fragments (N-
methyl-D-pipecolic acid (Mep), L-isoleucine (Ile), tubuvaline (Tuv), and tubuphenylalanine
(Tup)/tubutyrosine (Tut)) (Figure 2).
,
,
R-configuration
ester
tolerated
Tuv replacements
R-configuration required;
different ring size tolerated
required
optimal for
activity
O
S
amides/hydrazide tolerated
esters less active
Ar
O
O
O
O
O
OAc
O
O
N
N
N
H
N
R
N
N
11
11
S
OH
O
N
N
N
H
S
N
N
S
pretubulysin
ketotubulysin
R = H (tubulysin U)
Me
Bn (KEMTUB 10)
CH2C(O)NHCH(CH3)2 (tubugis)
CH2O-alkyl
CH2S-alkyl
O
O
Tup not essential;
SAR relatively flat
3° amine required
oxazole
equally active
O
R
N,O-acetal not required
O
O
O
Mep
Ile
Tuv
Tup/Tut
O
O
N
N
O
N
O
N
N
H
N
H
O
S
tolerated
Mep replacements
tolerated
Tup/Tut replacements
O
oxazole-tubulysin
O
TUB-MOM
cyclotubulysin
N
Ph
Ph
Ph
N
HN
HN
HN
O
not tolerated
O
O
S
O
S
O
O
COOH
COOH
COOH
O
O
11
N
N
O
O
O
N
N
N
O
N
R
N
R
N
13
13
11
13
11
O
R1
R
S
HN
HN
Me
HN
R1 = OH or H
Ph
R = H
not tolerated:
CH2OC(O)CH2CH(CH3)2
R2 = NH-alkyl
= NH-NH2
CO-R2
(THP-tubulysins)
O
O
O
N
N
R
OH
O
Ac
O
13
11
O
N
less tolerated
O
O
N
N
N
H
13
*
N
R
N
R
*
Ph
S
S
S
HN
Ph
N
H
OH
H
HN
R = cyclohexyl
phenyl
p-MeO-phenyl
R = H (tubulysin V)
CH2OC(O)CH2CH(CH3)2
R = H or Me
11,13 = R,R; R,S; S,R; or S,S
N
H
COOH
COOH
O
CO2-alkyl
O
Figure 2. Summary of key SAR features of tubulysins
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The Journal of Organic Chemistry
Replacement of the Mep residue with other tertiary amine-containig acids exhibited little change in
biological activity (Figure 2).36,38,39,40,41,42 In contrast, the (R)-configuration proved to be important at this
site.39,40 Moreover, demethylating Mep, acylating, or deleting this residue was also detrimental to
activity.36,38,39,40
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9
While the SAR of the Ile residue has been relatively neglected, extensive modifications were directed
toward modifications of the Tuv residue. Replacement of the labile N,O-acetal at the N-14 position with
H- or Me-groups left the biological activity substantially unaltered.21,35,36 The N,O-acetal was also
successfully simplified using an N-alkylamide in tubugis,43 or an N-benzyl group in KEMTUB10.44
Cyclotubulysin, an analog containing a cyclic N,O-acetal with the C-11 alcohol, showed similar activity
to tubulysin U.16 N,S-thioacetates demonstrated antiproliferative activities comparable to tubulysins A
and B.45 In contrast, replacing the isopropyl group with a cyclohexyl, phenyl or p-methoxyphenyl erased
activity (IC50 >1,000 nM).46 Substitution of the acetyl group of tubulysin U with a methoxymethyl or
methyl ether reduced activity more modestly by a factor of 5 or 60, respectively.46 The configuration at
C-11 and, particularly, at C-13 proved essential for cytotoxic potency. An evaluation of all 4
stereoisomers of tubuvaline showed that only the natural compound (i.e. (11R,13S)-tubulysin D) had a
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subnanomolar IC50 in a selection of cell lines.16, A C-11 epimer was 2-3 orders of magnitude less
active, and a C-13 epimer as well as a dual epimer at C-11 and C-13 dropped by 3-5 orders and >6
orders of magnitude, respectively. A similar pattern was observed for the C-13 and C-11 epimers of
tubulysin U and tubulysin V,15,38 and rigidified, tetrahydropyran-containing tubulysins.48,49 Deacetylation
of tubuvaline decreased activity by 1,000-fold, but a replacement of the acetate in Tuv with a ketone
apparently conserved activity.15 As a general trend, increased lipophilicity in the Tuv residue correlated
with more potent antiproliferative activity, perhaps due to a higher cellular uptake of the more lipophilic
analogs.2 An oxazole replacement of the thiazole in tubulysin U only reduced cytotoxicity ca. 2-fold, but
substitutions with phenyl or triazole rings were 2-4 orders of magnitude less potent than the parent
tubulysin D. 50,51,52
The C-terminal tubutyrosine (Tut) and tubuphenylalanine (Tup) residues are, in general, more readily
modified than other residues, and Tup-containing analogs were slightly more potent than tubulysins
with Tut residues.2 Tubulysin B and U and their C-2 methyl epimers have almost identical biological
activities.19,38 Inversion of configuration at the benzylated C-4 position in tubulysin U resulted in a 10-
fold less potent compound,38 and two orders of magnitude were lost when both benzyl and methyl
groups were removed.36 C-Terminal amides and hydrazides were almost as active as the parent acids,
and these functionalizations have therefore been used to prepare tubulysin conjugates.23,24,36 Ester
prodrugs proved to be similarly potent in vivo,20a but, not surprisingly, were generally less active in
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vitro.53,54 Some truncated versions of Tup/Tut residues in tubulysin D retained sub- or low-nanomolar
IC50 values.36 Similarly, the C-2 desmethyl pretubulysin was 10-fold less active than pretubulysin.21 An
analog of pretubulysin with a C-terminal L-Phe in place of the Tup residue was also less active.41
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Recent results support the merits of using synthetic tubulysins in targeted anticancer therapies; for
example, KEMTUB10, featuring a p-fluorophenyl residue and an N-14 benzyl tubuvaline, demonstrated
picomolar potency in breast cancer cells,44 and exceptional potency was also reported for Tb14, a novel
propellane-containing tubulysin.55 We now report a 2nd generation synthesis of N14-desacetoxytubulysin
H (1), the preparation of C-terminal hydrazide and methyl ester of 1, as well as late stage
functionalizations of the Tup residue and the synthesis of other non-natural analogs of tubulysins that
may serve as warheads in antibody-drug conjugates for applications in cancer chemotherapy.
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Results and Discussion
Our 1st generation total synthesis provided N14-desacetoxytubulysin H (1) in 20 steps and 2.1% overall
yield.34 In order to improve the overall conversion, we developed a modified 2nd generation synthesis of
1 (Schemes 1-3). For the preparation of the tubuvaline fragment (Scheme 1), Boc-Val-OH (2) was N-
methylated and subjected to an Arndt-Eistert chain extension followed by reduction with DIBAL-H to
give aldehyde 5 in 5 steps. The commercially available thiazole ester 6 was reduced, silylated with
TIPS-Cl, and condensed with aldehyde 5 after using Knochel’s conditions56 on bromide 8 for a Grignard
exchange reaction. Tubuvaline alcohols 9 and 10 were obtained as a 1:1 mixture of diastereomers, and
the undesired diastereomer 10 was converted in 78% yield to the desired 9 by oxidation with Dess-
Martin periodinane and selective reduction with (S)-2-methyl-CBS-oxazaborolidine.57 Acetylation of 9
and desilylation of the primary alcohol followed by a 2-step oxidation to the acid completed the
preparation of Boc-Tuv-OH (12) in excellent yield.
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The Journal of Organic Chemistry
1
2
3
4
5
6
1. i-BuOCOCl, TEA
THF, 0 °C to rt, 1 h
O
DIBAL-H, THF
NaH, MeI, THF
0 °C to rt, 24 h
Boc
Boc
CO2Me
Boc
-78 °C, 105 min
96%
N
N
N
CO2H
2. CH2N2, THF
-15 °C to rt, 5 h
BocHN
CO2H
85%
3. PhCO2Ag, Et3N
MeOH, 0 °C to rt, 12 h
3
5
4
2
7
8
77%
9
10
11
12
13
14
15
16
17
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20
21
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O
TIPSCl, imidazole
THF, DMF
OH
sec-Butyl-MgCl, 0 °C, THF
then 5, 0 °C to rt, 3 h
N
N
LiBH4, MeOH, THF
N
S
OTIPS
OEt
Br
Br
Br
rt, 13 h
S
0 °C to rt, 13 h
83%
S
8
7
6
92%
O
O
O
O
1. Ac2O, TEA, DMAP
CH2Cl2, rt, 5 h
2. TBAF, THF, 0 °C
90 min
1. DMP, CH2Cl2
0 °C, 1 h
OH
Boc
Boc
N
N
Boc
N
N
N
N
CO2H
89%
2. NaClO2, NaH2PO4
t-BuOH, , THF, H2O
2-methyl-2-butene
rt, 4 h
S
OTIPS
S
S
OH
11
9 (34%)
Boc-Tuv-OH (12)
+
1. DMP, CH2Cl2, 0 °C, 1 h
2. (S)-CBS-BH3
86%
78%
BH3•DMS, THF, rt, 2 d
OH
Boc
N
N
10 (36%)
S
OTIPS
Scheme 1. Preparation of Boc-Tuv-OH (12) using a Grignard exchange reaction with bromide 8.
For the synthesis of tubuphenylalanine ester 21, we oxidized phenylalaninol 13 and subjected the
resulting aldehyde 14 to a Wittig chain extension to give alkenoate 15 (Scheme 2).21,38,46 Saponification
and coupling with L-menthol provided ester 17, which provided a 5:1 ratio in favor of the desired
diastereomer 19 on hydrogenation with Pd/C in THF. In contrast, in EtOAc or MeOH, only a 2:1 ratio
was achieved.21,38,41,46 Hydrolysis of 19 in a 5:1 mixture of 6 M HCl and acetic acid at 105 °C cleanly
provided acid 20, whereas the use of 6 M HCl at 130-140 °C38,46 in the absence of acetic acid was not
reproducible in our hands.
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O
LiOH, THF
MeOH/H2O
rt, 40 h
1
2
3
4
Ph3P
Ph
Ph
OH
Ph
Ph
OEt
DMP, CH2Cl2
O
Boc
Boc
Boc
Boc
N
H
CO2H
N
H
N
H
N
H
CO2Et
0 °C, 1 h
96%
CH2Cl2, 0 °C to rt
14 h
86%
13
14
15
16
98%
5
6
7
8
9
Ph
Ph
Ph
DCC, DMAP
L-Menthol, Et2O
H2, Pd/C
THF, rt, 13 h
Boc
O
Boc
O
Boc
N
O
+
N
H
N
H
0 °C to rt, 24 h
75%
H
O
10
11
12
13
14
15
16
17
18
19
20
21
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60
O
O
18
17
19
15%
77%
99%
OH
Ph
Ph
, AcCl
6 M HCl, acetic acid
105 °C, 90 min
O
OH
HCl•H2N
HCl•H2N
0 °C to 60 °C, 3 h
quant.
O
O
21
20
Scheme 2. Preparation of tubuphenylalanine ester 21.
A series of sequential amide couplings completed the synthesis of N14-desacetoxytubulysin H (1,
Scheme 3). The use of the HATU/HOAT coupling reagent provided Tuv-Tup dipeptide 22 in excellent
yield,58 but acid fluoride 23 proved to be superior for the coupling of the sterically hindered α-branched
secondary amine derived from 22.59 HATU/HOAT was used again in the acylation with Mep-OH 25 to
give allyl ester 26. C-terminal ester deprotection with Pd[PPh3]4 and morpholine afforded N14-
desacetoxytubulysin H (1) in 20 steps and 16% overall yield for the longest linear sequence starting with
2.
O
O
1. TFA/CH2Cl2
O
O
O
rt, 1.5 h
2. DIPEA, DMF
rt, 14 h
21, HATU
HOAT, DIPEA
DMF, rt, 12 h
O
O
Boc-HN
Boc
N
N
Ph
O
Ph
O
N
N
12
S
S
HN
HN
O
90%
O
O
22
24
F
NHBoc
90%
23
O
S
1. TFA/CH2Cl2
rt, 1.5 h
2. HATU, HOAT
DIPEA, DMF
rt, 12 h
O
S
Pd(Ph3P)4
morpholine
CH2Cl2, 0 °C
3 h
O
O
O
O
H
N
H
N
O
O
N
N
Ph
Ph
O
N
N
N
N
14
O
O
HN
HN
90%
O
CO2H
26
1
OH
N
25
O
98%
Scheme 3. N-Terminal chain extensions to give N14-desacetoxytubulysin H (1)
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This modular and high-yielding route was well suited for iterative analog preparations and SAR studies,
but we were also interested in exploring late stage functionalizations of the peptide backbone and amino
acid side chains. After failed attempts to prepare a phenylalanine boronate via a recently reported Ir-
catalyzed C-H activation,60 we turned our attention to the selective iodination conditions reported by
Barluenga et al.61 We were pleased to observe complete consumption of 1 after 5 h at room temperature
in the presence of I2PyBF4 in CH2Cl2 and TFA (Scheme 4). Analysis of the crude reaction mixture
showed that the main product was iodo-tubulysin 27, which was isolated in 73% yield after RP-HPLC
purification. A Suzuki coupling of 27 proceeded under standard conditions with phenyl boronate and
yielded 72% of the novel ortho-biphenyl tubulysin 28. Alternatively, Ullmann-type coupling conditions
effected an intramolecular amidation to give indoline 29 in 80% yield.62 2D 1H NMR analysis of 29 and
comparison to literature data confirmed the structural assignment.63 In addition to the opportunity to
broaden the chemical diversity of tubulysins with these late-stage transformations, we suggest that this
strategy for combined backbone and side-chain modifications could be applied to other peptides with
aromatic residues.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
I
I2PyBF4,
CH2Cl2/TFA (10:1)
O
O
H
N
O
N
N
N
1
O
S
HN
rt, 5 h
73%
27
CO2H
CuI, L-proline, NaOH, DMSO/H2O (50:1)
Sodium ascorbate, 60 °C, 4 h
PhB(OH)2, Pd(Ph3P)4, Na2CO3
Toluene/MeOH (3:2), 80 °C, 5 h
80%
73%
O
S
O
Ph
O
H
N
O
O
O
H
N
O
O
N
N
N
N
N
N
O
S
O
HN
N
CO2H
29
28
HO2C
Scheme 4. Late stage side chain (28) and backbone (29) modifications of 1 based on a highly selective
Barluenga iodination of the Tup residue
Other modifications of the terminal residue in 1 took advantage of available synthetic intermediates and
traditional functional group interconversions (Scheme 5). The hydrazide of tubulysin B was reported to
be active,24 and therefore we prepared the corresponding analog 31 by coupling of 1 and hydrazide 30
followed by cleavage of the Boc-group with TFA. Previously published methods for the formation of
related hydrazides were low-yielding due to multiple side reactions, as discussed by Vlahov et al.64
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O
1. BocNHNH2 (30), HATU, HOAT
DIPEA, DMF, 0 °C to rt, 4 h
2. TFA, CH2Cl2, rt, 1 h
1
2
3
4
5
6
O
O
H
N
O
N
Ph
O
N
N
1
O
S
91%
HN
NH
NH2
31
7
Scheme 5. Synthesis of hydrazide 31 from tubulysin 1 and Boc-hydrazine (30).
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The synthesis of enoate analogs 35 and 37 started from the unsaturated Tup building block 15 (Scheme
6).12 Hydrolysis of 15 followed by conversion of the acid to the allyl ester gave 32, which was
deprotected with TFA and then coupled with the mixed anhydride of segment 12 to afford dipeptide 33.
Attachment of the Ile-residue was effectively achieved by coupling of 33 with Fmoc-Ile-F,58 and the
resulting protected tripeptide 34 was converted to analog 35 as well as the N-terminal proline derivative
37. Deprotection of the Fmoc group in 34 and coupling with Mep-OPFP or the proline active ester 36,
followed by deprotection of the allyl ester provided 35 and 37, respectively. These analogs share the C-
terminal 2-methyl α,β-unsaturated acid moiety with the potent microtubule-targeting hemiasterlins.65
O
O
Ph
Ph
1. LiOH, THF/H2O
1. TFA, CH2Cl2
Ph
O
Boc
N
N
Boc
O
Boc
O
2. Allyl bromide
Cs2CO3, DMF
2. 12, i-BuOCOCl
Et3N
78%
N
H
N
H
S
HN
O
O
O
15
32
33
84%
O
O
S
O
S
1. N(CH2CH2NH2)3
2. Mep-OC6F5
DMF
O
O
O
O
H
N
Ph
Ph
O
O
Fmoc-HN
1. TFA, CH2Cl2
N
N
N
N
N
O
HN
HN
2. Fmoc-Ile-F
DIPEA
3. Pd(Ph3P)4
dimedone
O
CO2H
34
35
70%
40%
O
1. N(CH2CH2NH2)3
2.
OC6F5
N
61%
36, DMF
O
3. Pd(Ph3P)4, dimedone
O
O
O
H
N
Ph
O
N
N
N
O
S
HN
CO2H
37
Scheme 6. Preparation of C- and N-terminal analogs 35 and 37 from synthetic intermediates 12 and 15
obtained in route to 1
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The Ile-Tuv segment has previously been modified, but still offers opportunities for structural
simplification with minimal loss of activity, as demonstrated for pretubulysin. We were particularly
interested to compare the de-acetoxylated analog 49 (cLogP 4.0), containing an alkene at the C(2)-C(3)
Tup position with the corresponding Val-analog 50 (cLogP 3.5), since these combined substitutions
would decrease lipophilicity and simplify the synthesis.
1
2
3
4
5
6
7
8
The known imine 40 was prepared according to a literature protocol from amide 38 and aldehyde 39,66
and trimethylsilyl acetylide addition in the presence of trimethylaluminum67 provided 41 in 17:1 d.r.
and 94% yield (Scheme 7). Chromatographic purification removed the minor diastereomer of 41, and
the desired absolute configuration was confirmed by an X-ray crystallographic analysis. N-methylation
of 41, cleavage of the TMS group in the resulting 42, and removal of the chiral auxiliary followed by
Boc-protection afforded alkyne 43. A Sonogashira coupling of 43 and bromothiazole 6 gave
enantiomerically pure 44 (ee > 99.9% by chiral HPLC analysis). Hydrogenation of 44 yielded the de-
acetoxylated Tuv analog 45. Saponification of the ethyl ester and coupling with amine 46, obtained by
protective group removal from 15, led to key intermediate 47 in 10 linear steps and 30% overall yield
from tert-butanesulfinamide 39. Removal of the N-terminal protective group in 47 and coupling with
Fmoc-Ile fluoride led to tripeptide 48, which was converted to the desired tubulysin analog 49 in three
steps and high yield. An analogous deprotection and coupling sequence using Fmoc-protected valine
acylfluoride, followed by condensation with N-methyl pipecolic acid pentafluorophenyl ester, generated
tubulysin 50. The total number of synthetic transformations (18) from commercially available materials
required for the preparation of 49 and 50 compared very well to the 27 total steps required for the
synthesis of 1, but of course the utility of these simplified analogs had yet to be determined by
biological testing.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
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1. K2CO3, MeOH
2. HCl, MeOH
(Me)2CHCHO (39)
MgSO4, PPTs (cat.)
O
S
CH3I, LDA
HMPA
1
2
3
4
TMS
H
N
S
HN
S
N
S
NH2
TMS
TMS
3. Boc2O, DIPEA
DMAP, CH2Cl2
CH2Cl2, rt, 36 h
96%
AlMe3, BuLi
THF, -78 °C
70%
O
O
O
toluene, -78 °C
38
42
40
94%
41
85%
5
6
7
8
9
O
N
1. NaOH, EtOH/H2O
2. EDC, HOBT
OEt
Br
S
DMAP, CH2Cl2
6
H2, Pd/C
84%
Boc
N
Boc
Boc
N
N
N
CO2Et
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N
Ph
S
H
Pd(PhCN)2, P(t-Bu)3
CuI, (i-Pr)2NH, dioxane
CO2Et
43
44
45
S
H2N
CO2Et
96%
70%
46
O
N
Ph
O
Ph
O
1. TFA, CH2Cl2
Boc
Fmoc-HN
N
N
S
N
2. Fmoc-Ile-F, DIPEA
90%
S
HN
HN
CO2Et
CO2Et
47
48
1. TFA, CH2Cl2
1. N(CH2CH2NH2)3
81%
O
2. Fmoc-Val-F, DIPEA
3. N(CH2CH2NH2)3
4. Mep-OC6F5, DMF
5. LiOH, EtOH, H2O
2. Mep-OC6F5, DMF
3. LiOH, EtOH, H2O
72%
H
N
Ph
O
N
N
N
O
H
Ph
O
O
S
HN
N
N
N
N
CO2H
49
O
S
HN
50
CO2H
Scheme 7. Total syntheses of tubulysins 49 and 50 using the structurally simplified Tuv-Tup analog 47
as a common intermediate
Biological Evaluation of Tubulysins
In the biological study of tubulysins, we used paclitaxel, vincristine, monomethyl auristatin D (MMAD)
and E (MMAE) as standards due to their related mechanism of action and potency range (Table 1).68
The effects of these compounds and tubulysin analogs on cell death and mitotic arrest were
benchmarked as described earlier.69 70 Briefly, HeLa human cervical carcinoma cells (10,000 per well)
,
were plated in collagen-1 coated 384-well microplates and treated with vehicle (DMSO) or ten two-fold
o
concentration gradients of test agents within 4-6 h of seeding. Cells were incubated for 24 h at 37 C,
fixed with formaldehyde, and labeled with 10 µg/mL Hoechst 33342. Plates were analyzed on an
ArrayScan high content reader (Thermo Fisher Cellomics) for cell numbers and nuclear condensation.
Cell growth inhibition was assessed by fitting of the resulting sigmoidal cell density curves to a four
parameter logistic equation, followed by extrapolating the concentration of drugs that caused 50% cell
loss (IC50). Nuclear condensation curves, which measure apoptosis and/or mitotic arrest, were based on
the percentage of cells with condensed nuclei. Because the shape of these curves was often not
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sigmoidal, we determined minimum detectable effective concentrations (MDEC) as previously
described.69
1
2
3
As expected, the highly potent MMAD and MMAE demonstrated subnanomolar IC50 values and
MDECs of 40 pM and 190 pM, respectively (Table 1). Paclitaxel was still very potent with a IC50 of 1.2
nM and an MDEC of 290 pM, whereas vincristine was 10-20 times less potent (IC50 15.7 nM, MDEC
5.1 nM). Compound 1 was the most potent of all analogs prepared and averaged an IC50 of 0.23 nM and
a MDEC of 110 pM, approximately in between MMAD and MMAE, and significantly more potent than
paclitaxel and vincristine. Not surprisingly, the allyl ester of 1, compound 26, lost about a factor of 30 in
IC50 potency in HeLa cells (7.5 nM), but, interestingly, the nuclear condensation assay showed an
MDEC of 630 pM for allyl ester 26, i.e. only ca. 6-fold higher than acid 1. The most surprising
biological response, however, was found for hydrazide 31, which, while ca. 9-fold less potent than 1 in
the growth inhibition assay (IC50 0.99 nM), was equivalent to 1 in the nuclear condensation assay
(MDEC 100 pM). These results bode well for optimizing the physicochemical properties of tubulysin
acids and enhance their cell-type specificity through C-terminal conjugation to antibodies or small
molecule receptor or enzyme ligands.
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Our discovery of the ability of Barluenga’s iodination protocol to selectively ortho-functionalize the
Tup residue in 1 also allowed us to test the antiproliferative profile of novel tubulysin analogs, such as
iodide 27, biphenyl 28, and indoline 29. Perhaps not surprisingly, given the relatively minor structural
modification, iodobenzene 27 was equipotent to the parent compound 1 in both growth inhibition and
nuclear condensation assays, and therefore 27 or an isotopically labeled variant could become useful as
a reference in target identification and PK/PD studies. For compound 28 and, in particular, the backbone
modified 29, both IC50 and MDEC values dropped off significantly by factors of 4-5 (for 28 vs 1) and 80
(for 29 vs 1). The ortho-phenyl substitution in 28 is likely to interfere sterically with tubulin binding;
the drop in activity is in qualitative agreement with a model positioning the Tup side chain into a
hydrophobic binding pocket near Q15 on β1-tubulin.68 For indoline 29, this interaction is similarly, but
more severely impacted, and the conformational constraint potentially disrupts hydrogen bonds of the
Tuv amide carbonyl group with the main chain NH of Tyr224 and Gly225 at the N-terminus of helix
H7.68 Finally, the dehydroanalog of 1, the α,β-unsaturated acid 35, the corresponding N-methyl proline
analog 37, and the synthetically streamlined deacetoxylated compounds 49 and 50, showed steep drop-
offs in potency, with the α,β-unsaturated acid 35 being the most potent derivative (IC50 81.3 nM, MDEC
12.6 nM) and the valine deacetoxy enoate 50 falling into the low micromolar range (IC50 1.5 µM,
MDEC 0.48 µM). The differential cytotoxicity range of these derivatives could make them attractive
candidates to test for synergistic activities with signaling pathway inhibitors or other mechanism-based
anticancer agents, as well as for cancer cell type specificities.
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1
2
Table 1. Antiproliferative activities and nuclear condensation effects of tubulysin analogs and reference
3
4
compounds in HeLa cells.
5
6
7
8
Compounds
Cell Density
IC50 [nM]
Nuclear Condensation
MDEC [nM]
9
Mean
1.21
15.7
0.14
0.32
0.23
7.5
SD
0.26
7.2
n
10
9
Mean
SD
n
6
9
3
3
3
3
2
2
2
3
4
3
3
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Paclitaxel
0.29
5.11
0.04
0.19
0.11
0.63
0.12
0.42
8.12
0.10
12.6
39.5
215
0.14
4.44
0.02
0.11
0.03
0.21
0.08
0.23
6.34
0.05
2.0
Vincristine
MMAD
MMAE
1
0.12
0.14
0.12
4.0
3
3
3
26
3
27
0.24
1.11
18.3
0.99
81.3
309
0.12
0.64
2.8
3
28
2
29
2
31
1.2
3
35
55.2
145
62
4
37
3
24.7
128
49
379
3
50
1,466
657
3
482
204
Conclusion
In summary, we devised a high-yielding second-generation route for the synthesis of N14-
desacetoxytubulysin H (1) and introduced efficient late-stage functionalizations that allow the
preparation of novel side chain and backbone modified analogs. We further expanded on the SAR of
tubulysins and discovered C-terminal modifications that preserve the exquisite biological activity of
acid 1 and offer the opportunity for conjugation to biomolecules and receptor ligands to improve target
selectivity. We also discovered moderately active analogs that might prove useful in synergistic
combination cancer drug treatment regimens. Overall, our ten biologically characterized synthetic
analogs span a range of almost 4 orders of magnitude in biological potency, illustrating the diverse
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biological response that can be achieved in structure-activity investigations of tubulysins, and natural
products in general.71
1
2
3
4
5
6
Experimental Section
7
8
9
General Information. All reactions involving moisture sensitive reagents were conducted in oven-dried
glassware under a nitrogen or argon atmosphere. Analytical thin-layer chromatography (TLC) was
preformed on SiO2 60 F-254 plates. Visualization was accomplished by UV irradiation at 254 nm, or by
staining with any one of the following reagents: iodine, 5% phosphomolybdic acid hydrate in ethanol,
ninhydrin (0.3% w/v in glacial acetic acid/n-butyl alcohol 3:97), Vaughn’s reagent (4.8 g of
(NH4)6Mo7O24•4H2O and 0.2 g of Ce(SO4)2•4H2O in 10 mL of conc. H2SO4 and 90 mL of H2O), or para-
anisaldehyde (7.5 mL of para-anisaldehyde, 25 mL of conc. H2SO4, and 7.5 mL of acetic acid in 675
mL of 95% ethanol). Flash chromatography was performed using SiO2 60 (particle size 0.040-0.055
mm, 230-400 mesh). Hydrogenation reactions were performed on an H-cube. Melting points were
obtained on a capillary melting point apparatus fitted with a digital thermometer and are not corrected.
Specific rotations of chiral compounds were obtained at the designated concentration and temperature
using a 1 dm cell. Infrared spectra were collected with neat material (oil or solid) on a FT-IR
spectrometer, and ATR correction was performed on the spectra obtained. Proton and carbon NMR
spectra were obtained on 300, 400, 500 and 600 MHz NMR spectrometers. Chemical shifts are reported
as δ values in parts per million (ppm) as referenced to residual solvent. 1H NMR spectra are tabulated as
follows: chemical shift, multiplicity (s = singlet, bs = broad singlet, d = doublet, t = triplet, q = quartet,
m = multiplet), number of protons, and coupling constant(s). LC-MS analysis was performed using an
analytical C18 column (100 × 4.6 mm, 3.5 µm, 0.4 mL/min). HRMS analyses used an Orbitrap
quadrupole instrument in positive (+) and negative (-) mass detection mode.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthetic Procedures and Spectroscopic Data.
(S)-2-((tert-Butoxycarbonyl)(methyl)amino)-3-
methylbutanoic acid (3).72 Sodium hydride (1.24 g, 46.0 mmol) was added in small portions over 45
min to a stirred solution of Boc-Val-OH (2, 1.00 g, 4.60 mmol) and methyl iodide (2.8 mL, 46 mmol) in
THF (30 mL) at 0 °C. The reaction mixture was stirred at rt for 24 h under a nitrogen atmosphere, then
quenched with water (15 mL). Ethyl acetate (15 mL) was added and the mixture was concentrated under
vacuum, diluted with water (100 mL) and washed with diethyl ether (2x). The aqueous solution was
acidified to pH 3 with solid citric acid and extracted with ethyl acetate (3x). The combined extracts were
washed with brine, dried (Na2SO4), and concentrated to afford 3 (0.91 g, 85%) as a pale yellow oil
20
1
which crystallized on standing: [α]D -106 (c 0.6 in EtOH); H NMR (mixture of rotamers, 400 MHz,
CDCl3) δ 4.00-3.97 (m, 1 H), 2.91 (s, 1.5 H), 2.90 (s, 1.5 H), 2.40-2.20 (m, 1 H), 1.50 (s, 9 H), 1.06 (d, J
= 6.6 Hz, 3 H), 0.95 (d, J = 6.6 Hz, 1.5 H), 0.93 (d, J = 6.5 Hz, 1.5 H).
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(R)-Methyl 3-((tert-butoxycarbonyl)(methyl)amino)-4-methylpentanoate (4).34 A solution of 3 (0.91 g,
3.9 mmol) in THF (60 mL) was treated at 0 °C with triethylamine (0.61 mL, 4.3 mmol) and isobutyl
chloroformate (0.58 mL, 4.3 mmol). The reaction mixture was stirred at rt for 1 h, cooled to 0 °C, and
filtered through a small plug of Celite®. The plug was washed with cold THF. The solution was
concentrated to afford (S)-2-((tert-butoxycarbonyl)(methyl)amino)-3-methylbutanoic (isobutyl
1
2
3
4
5
6
7
8
1
carbonic) anhydride 3a as yellow oil which was used in the next step without further purification: H
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
NMR (mixture of rotamers, 300 MHz, CDCl3) δ 4.57 (d, J = 10.1 Hz, 1 H), 4.08 (d, J = 6.7 Hz, 2 H),
2.93 (s, 1.5 H), 2.87 (s, 1.5 H), 2.38-2.25 (m, 1 H), 2.12-1.99 (m, 1 H), 1.49 (s, 9 H), 1.09 (d, J = 6.6 Hz,
3 H), 1.10-0.95 (m, 10 H).
Aqueous NaOH (15%, 6 mL) was added dropwise to a suspension of Diazald (4.0 g, 19 mmol) in EtOH
(20 mL). The formed diazomethane was transferred and bubbled with a flow of N2 into a solution of (S)-
2-((tert-butoxycarbonyl)(methyl)amino)-3-methylbutanoic (isobutyl carbonic) anhydride 3a (1.3 g, 3.9
mmol) in THF (30 mL) at -10 °C. After the complete addition of NaOH into the Diazald solution, the
reaction flask was kept under nitrogen and left without stirring at rt for 5 h. The reaction mixture was
quenched with 0.5 M AcOH in Et2O (excess), diluted with sat. NaHCO3 and Et2O, and extracted with
Et2O (2x). The combined organic layers were washed with brine, dried (Na2SO4), and concentrated. The
crude residue was purified by chromatography through a small plug of SiO2 (hexanes:EtOAc, 90:10 to
85:15) to afford (S)-tert-butyl (1-diazo-4-methyl-2-oxopentan-3-yl)(methyl)carbamate 3b (0.87 g, 87%)
1
as a yellow oil: H NMR (300 MHz, CD2Cl2) δ 5.47-5.35 (m, 1 H), 4.32-4.08 (m, 1 H), 2.74 (s, 3 H),
2.33-2.22 (m, 1 H), 1.49 (s, 9 H), 0.96 (d, J = 6.5 Hz, 3 H), 0.87 (d, J = 6.4 Hz, 3 H).
Silver benzoate (0.093 g, 0.41 mmol) in Et3N (4.3 mL) was added dropwise to a stirred solution of (S)-
tert-butyl (1-diazo-4-methyl-2-oxopentan-3-yl)(methyl)carbamate 3b (0.87 g 3.4 mmol) in MeOH (34
mL) at 0 °C under nitrogen in a flask protected from light. The reaction mixture was stirred at 0 °C for 1
h (bubbles were observed), 10 h at rt, then concentrated. The crude black residue was purified by
chromatography on SiO2 (hexanes:EtOAc, 90:10 to 85:15) to afford 4 (0.79 g, 89%) as a colorless oil:
1H NMR (mixture of rotamers, 300 MHz, CDCl3) δ 4.07 (td, J = 10.4, 4.3 Hz, 0.5 H), 3.98-3.91 (m, 0.5
H), 3.67 (s, 1.6 H), 3.66 (s, 1.4 H), 2.76 (s, 1.6 H), 2.72 (s, 1.4 H), 2.67-2.45 (m, 2 H), 1.88-1.76 (m, 1
H), 1.48 (s, 5 H), 1.46 (s, 4 H), 0.95 (d, J = 6.6 Hz, 3 H), 0.89 (d, J = 6.6 Hz, 3 H); 13CNMR (mixture of
rotamers, 76 MHz, CDCl3) δ 172.3, 172.1, 155.8, 79.5, 79.1, 60.3, 59.4, 51.6, 36.4, 36.0, 31.0, 30.5,
30.1, 28.4, 20.1, 20.0, 19.7, 19.6.
(R)-tert-Butyl methyl(4-methyl-1-oxopentan-3-yl)carbamate (5).34 A solution of 4 (0.6 g, 2.3 mmol) in
dry Et2O (30 mL) was treated at -78 °C dropwise over 30 min with a 1 M solution of DIBALH in
hexane (3.0 mL, 3.0 mmol). The reaction mixture was stirred at -78 °C for 105 min, quenched at -78 °C
with H2O (0.1 mL), 15% NaOH in H2O (0.1 mL) and water (0.4 mL), slowly warmed to 0 °C, dried
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(Na2SO4), and filtered through a small pad of Celite®. The residue was purified by chromatography on
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SiO2 (hexanes:EtOAc, 85:15 to 80:20) to afford 5 (0.51 g, 96%) as a colorless oil that was used
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immediately without further purification: H NMR (characteristic signals, mixture of rotamers, 400
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6
7
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MHz, CDCl3) δ 9.68 (t, J = 4.4 Hz, 1 H), 4.34 (td, J = 10.6, 4.1 Hz, 0.5 H), 4.11 (td, J = 9.1, 3.9 Hz, 0.5
H), 2.79-2.46 (m, 5 H), 1.85-1.78 (m, 1 H), 1.49 (s, 4 H), 1.46 (s, 5 H), 0.99-0.96 (m, 3 H), 0.92 (d, J =
6.6 Hz, 3 H).
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2-Bromothiazol-4-yl)methanol (7).34 To an ice-cooled solution of ethyl 2-bromothiazole-4-carboxylate 6
(2.5 g, 10.5 mmol) in anhydrous THF (25 mL) was added dropwise lithium borohydride (4.0 M solution
in THF, 4.0 mL, 15.9 mmol) over 20 min. A solution of methanol (0.64 mL, 15.9 mmol) in anhydrous
THF (2 mL) was then added over 30 min, and the mixture was stirred for 4h, allowing the temperature
to rise to rt. The reaction mixture was diluted with ethyl acetate, washed with 1 M KHSO4, sat.
NaHCO3, and brine, dried (Na2SO4), concentrated, and purified by chromatography on SiO2
(EtOAc:hexanes, 3:7) to afford 7 (1.3 g, 83%) as a colorless oil: 1H NMR (400 MHz, CDCl3) δ 7.20 (s,
1 H), 4.77 (d, J = 4.8 Hz, 2 H), 2.37 (t, J = 4.8 Hz, 1 H).
2-Bromo-4-(((triisopropylsilyl)oxy)methyl)thiazole (8).73 A solution of triisopropylchlorosilane (2.0 mL,
9.3 mmol) in anhydrous THF (8 mL) was added over 20 min to a solution of 7 (1.5 g, 7.7 mmol) and
imidazole (0.63 g, 9.3 mmol) in anhydrous DMF (10 mL). The reaction mixture was stirred overnight,
diluted with 1 M KHSO4, and extracted with Et2O (3x). The combined organic layers were washed with
1 M KHSO4, sat. NaHCO3, and brine, dried (Na2SO4), and concentrated. The crude residue was purified
by chromatography on SiO2 (hexanes:EtOAc, 95:05) to afford 8 (2.5 g, 92%) as a colorless oil: 1H NMR
(300 MHz, CDCl3) δ 7.12 (t, J = 1.4 Hz, 1 H), 4.85 (d, J = 1.4 Hz, 2 H), 1.14-1.05 (m, 3 H), 1.02-0.99
(m, 18 H).
tert-Butyl
((1R,3R)-1-hydroxy-4-methyl-1-(4-(((triisopropylsilyl)oxy)methyl)thiazol-2-yl)pentan-3-
(9) and tert-butyl ((1S,3R)-1-hydroxy-4-methyl-1-(4-
yl)(methyl)carbamate
(((triisopropylsilyl)oxy)methyl)thiazol-2-yl)pentan-3-yl)(methyl)carbamate (10). A 2 M solution of sec-
BuMgCl in THF (3.7 mL, 7.5 mmol) was added to a solution of bromide 8 (2.6 g, 7.5 mmol) in THF (50
mL) at 0 °C. The reaction mixture was stirred at 0 °C for 15 min, kept under vacuum (gentle bubbling)
at 0 °C for 5 min, stirred at 0 °C for 15 min, kept under vacuum at 0 °C for 5 min and stirred at 0 °C for
other 5 min. A solution of aldehyde 5 (0.90 g, 3.9 mmol) in dry THF (4 mL) was added dropwise and
the reaction mixture was stirred at 0 °C for 30 min, then warmed to rt and stirred for 3 h, quenched with
sat. NaHCO3, and extracted with EtOAC (3x). The combined organic layers were washed with brine,
dried (Na2SO4), and concentrated. The crude residue was purified by chromatography on SiO2
(hexanes:EtOAc, 9:1 then 85:15, then 8:2) to afford 9 (0.66 g, 34%) and epimer 10 (0.70 g, 36%). For
the conversion of 10 to 9, a solution of 10 (0.4 g, 0.80 mmol) in CH2Cl2 (10 mL) was treated at 0 °C
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with Dess-Martin periodinane (DMP, 0.37 g, 0.87 mmol), stirred for 45 min at 0 °C, then diluted with
CH2Cl2, 1 M sodium thiosulfate and sat NaHCO3. The aqueous layer was extracted with CH2Cl2 (2x)
and the combined organic layers were washed with brine, dried (Na2SO4), and concentrated. The crude
residue was purified through a small plug of SiO2 (hexanes:EtOAc, 8:2) to afford a colorless oil, which
was cooled to 0 °C and treated with a mixture of BH3-DMS complex (0.8 mL, 1.6 mmol) and (S)-CBS-
BH3 complex (0.17 g, 0.80 mmol) in dry THF (6 mL). The reaction mixture was stirred for 24 at rt.
Additional BH3-DMS complex (4.2 mL, 8.4 mmol) was added and stirring was continued at rt for 3
days. The mixture was quenched by dropwise, very slow addition of MeOH (2 mL) at 0 °C, followed by
water (2 mL) and ethanolamine (0.5 mL). The solution was stirred at rt for 1 h, concentrated, diluted
with NaHCO3 and extracted with EtOAc (3x). The combined organic phases were washed with brine,
dried (Na2SO4), and concentrated. The residue was purified by chromatography on SiO2
(hexanes:EtOAc, 90:10 to 85:15) to afford additional 9 (0.31 g, 78%) as a colorless oil.
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9: Rf 0.5 (hexanes:EtOAc, 8:2); [α]D -11.2 (c 1.0, CH2Cl2); IR 3386, 2957, 2942, 2864, 1689, 1663,
1462, 1137, 1100, 880, 682 cm-1; 1H NMR (400 MHz, CDCl3) δ 7.19 (s, 1 H), 4.98-4.95 (m, 3 H), 4.69
(d, J = 10.8 Hz, 1 H), 3.98 (td, J = 11.3, 2.7 Hz, 1 H), 2.78-2.74 (m, 3 H), 2.08-1.91 (m, 2 H), 1.80-1.71
(m, 1 H), 1.49 (s, 9 H), 1.23-1.15 (m, 3 H), 0.98-0.91 (m, 18 H), 0.97 (d, J = 6.5 Hz, 3 H), 0.92 (d, J =
13
6.5 Hz, 3 H); C NMR (101 MHz, CDCl3) δ 175.0, 158.5, 157.1, 112.9, 80.6, 69.2, 63.0, 57.9, 38.0,
29.9, 28.6, 28.5, 28.3, 20.3 (2C), 18.1, 12.1; HRMS (ESI+) m/z calcd for C25H49O4N2SSi 501.3177,
found 501.3176.
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10: Rf 0.25 (hexanes:EtOAc, 8:2); H NMR (mixture of rotamers, 300 MHz, CDCl3) δ 7.11 (s, 0.3),
7.04 (s, 0.7 H), 4.95-4.83 (m, 3.3 H), 4.69 (d, J = 8.3 Hz, 0.7 H), 3.77 (td, J = 10.4, 3.4 Hz, 0.8 H), 3.67-
3.58 (m, 0.2 H), 2.50 (s, 0.7 H), 2.32-2.11 (m, 4 H), 1.72-1.60 (m, 1 H), 1.34 (s, 2 H), 1.32 (s, 7 H),
1.14-1.05 (m, 4 H), 1.03-1.01 (m, 18 H), 0.93 (d, J = 6.6 Hz, 2.3 H), 0.88 (d, J = 6.6 Hz, 0.7 H), 0.76 (d,
J = 6.5 Hz, 3 H); HRMS (ESI+) m/z calcd for C25H49O4N2SSi 501.3177, found 501.3173.
(1R,3R)-3-((tert-Butoxycarbonyl)(methyl)amino)-1-(4-(hydroxymethyl)thiazol-2-yl)-4-methylpentyl
acetate (11).34 Ac2O (0.52 mL, 5.6 mmol) and DMAP (0.17 g, 1.4 mmol) were added to a solution of
alcohol 9 (1.4 g, 2.8 mmol) and Et3N (0.77 mL, 5.6 mmol) in CH2Cl2 (28 mL). The reaction mixture was
stirred at rt for 5 h, diluted with EtOAc, and washed with 1 M NaHSO4, sat. NaHCO3 and brine, dried
(Na2SO4), and concentrated. The crude residue was purified by chromatography on SiO2
(hexanes:EtOAc, 9:1) to afford (1R,3R)-3-((tert-butoxycarbonyl)(methyl)amino)-4-methyl-1-(4-
20
(((triisopropylsilyl)oxy)methyl)thiazol-2-yl)pentyl acetate 9a55 (1.42 g, 94%) as a colorless oil: [α]D
1
+10.6 (c 1.0, CH2Cl2); IR 2957, 2938, 2864, 1753, 1689, 1462, 1365, 1219, 1134, 880, 682 cm-1; H
NMR (mixture of rotamers, 400 MHz, CDCl3) δ 7.21 (s, 1 H), 5.93 (dd, J = 9.9, 2.7 Hz, 0.3 H), 5.86
(dd, J = 11.7, 2.8 Hz, 0.7 H), 4.98 (d, J = 1.1 Hz, 1.3 H), 4.97 (d, J = 1.0 Hz, 0.7 H), 4.14-4.07 (m, 0.7
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H), 3.89-3.67 (s, 0.3), 2.72 (s, 3 H), 2.41-2.29 (m, 1 H), 2.19 (s, 3 H), 2.10-2.03 (m, 1 H), 1.76-1.67 (m,
1 H), 1.46 (s, 9 H), 1.24-1.16 (m, 3 H), 1.12-1.08 (m, 18 H), 1.00 (d, J = 6.5 Hz, 2.3 H), 0.99 (d, J = 6.4
Hz, 0.7 H), 0.88 (d, J = 6.6 Hz, 3 H); 13C NMR (mixture of rotamers, 101 MHz, CDCl3) δ 170.2, 169.2,
157.4, 156.3, 128.5, 127.9, 113.5, 113.4, 79.6, 79.3, 70.1, 69.2, 62.8, 62.7, 56.4, 34.9, 30.4, 28.5, 28.4,
28.1, 21.1, 21.0, 20.0, 19.6, 18.0, 17.9, 12.0; HRMS (EI) m/z calcd for C27H51O5N2SSi 543.3282, found
543.3281.
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A 1 M solution of TBAF in THF (7.3 mL, 7.3 mmol) was added to a solution of 9a (1.6 g, 2.9 mmol) in
THF (20 mL) at 0 °C. The reaction mixture was stirred for 90 min at 0 °C, quenched with sat. NaHCO3
and extracted with EtOAc (3x). The combined organic phases were washed with brine, dried (Na2SO4),
and concentrated. The crude residue was purified by chromatography on SiO2 (hexanes:EtOAc, 4:6 to
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3:7) to afford 11 as a colorless oil (1.07 g, 95%): [α]D +14.7 (c 1.0, CH2Cl2); IR 3438, 2964, 2931,
2875, 1749, 1685, 1473, 1447, 1365, 1219, 1152, 1130, 1040, 772 cm-1; 1H NMR (mixture of rotamers,
300 MHz, CDCl3) δ 7.16 (s, 0.3 H), 7.15 (s, 0.7 H), 5.92 (dd, J = 9.0, 3.8 Hz, 0.3 H), 5.84 (dd, J = 11.6,
2.8 Hz, 0.7 H), 4.74 (s, 2 H), 4.08 (td, J = 11.1, 3.0 Hz, 0.7 H), 3.83-3.72 (m, 0.3), 3.08 (s, 1 H), 2.70 (s,
2 H), 2.64 (s, 1 H), 2.41-2.24 (m, J = 3.7 Hz, 1 H), 2.15 (s, 3 H), 2.08-1.99 (m, J = 2.8 Hz, 1 H), 1.75-
1.63 (m, 1 H), 1.45 (s, 9 H), 1.33-1.23 (m, 1 H), 0.98 (d, J = 6.6 Hz, 3 H), 0.87 (d, J = 6.6 Hz, 3 H); 13C
NMR (mixture of rotamers, 101 MHz, CDCl3) δ 170.9, 170.2, 169.9, 169.6, 156.6, 156.5, 156.3 (2C),
115.0, 114.7, 79.7, 79.3, 70.6, 69.4, 60.9, 60.8, 56.4, 53.9, 35.0, 30.8 (2C), 30.4, 28.5, 28.4, 28.1 (2C),
20.9, 20.8, 20.3, 20.0, 19.8, 19.6, 14.1; HRMS (ESI+) m/z calcd for C18H31O5N2S+ 387.1948, found
387.1943.
2-((1R,3R)-1-Acetoxy-3-((tert-butoxycarbonyl)(methyl)amino)-4-methylpentyl)thiazole-4-carboxylic
acid (12).34 A solution of 11 (0.54 g, 1.4 mmol) and Dess-Martin periodinane (DMP, 0.68 g, 1.6 mmol)
in CH2Cl2 (25 mL) was stirred at 0 °C for 1 h, then diluted with CH2Cl2 (100 mL), 1 M Na2S2O3 (30 mL)
and NaHCO3 (30 mL). The organic phase was washed with sat. NaHCO3, dried (Na2SO4) and
concentrated. The crude residue was purified by chromatography on SiO2 (EtOAc:hexanes, 3:7 to 4:6)
to afford (1R,3R)-3-((tert-butoxycarbonyl)(methyl)amino)-1-(4-formylthiazol-2-yl)-4-methylpentyl
acetate 11a (0.51 g, 95%) as colorless oil which was used immediately in the next step: IR 3102, 2068,
1749, 1685, 1480, 1387, 1365, 1216, 1152, 1130, 1044 cm-1; 1H NMR (mixture of rotamers, 500 MHz,
CDCl3) δ 10.03 (s, 0.3 H), 10.02 (s, 0.7 H), 8.16 (s, 0.3 H), 8.15 (s, 0.7 H), 5.97 (dd, J = 9.9, 2.8 Hz, 0.3
H), 5.89 (dd, J = 11.6, 2.9 Hz, 0.7 H), 4.13-4.08 (m, 0.7 H), 3.82 (br s, 0.3 H), 2.73 (s, 3 H), 2.41-2.34
(m, 1 H), 2.19 (s, 3 H), 2.18-2.12 (m, 1 H), 1.78-1.64 (m, 2 H), 1.46 (s, 9 H), 1.01-0.99 (m, 3 H), 0.89
13
(d, J = 6.6 Hz, 3 H); C NMR (mixture or rotamers, 101 MHz, CD2Cl2) δ 184.7, 184.6, 172.0, 171.0,
170.2, 169.6, 156.3, 156.1, 154.8, 127.7, 127.3, 79.7, 79.4, 70.1, 69.4, 56.4, 34.6, 30.7, 30.4, 29.7,
28.43, 28.37, 28.1, 21.0, 20.8, 20.2, 20.0, 19.8, 19.6. HRMS (ESI+) m/z calcd for C18H28O5N2SNa+
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407.1611, found 407.1610.
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A solution of aldehyde 11a (0.49 g, 1.3 mmol) in tert-butyl alcohol (20 mL) was treated with a solution
of 2-methyl-2-butene in THF (2 M, 5.1 mL, 10.2 mmol), followed by the dropwise addition of a mixture
of sodium chlorite (0.69 g, 7.6 mmol) and sodium dihydrogenphosphate monohydrate (2.3 g, 19 mmol)
in water (10 mL). The reaction mixture was stirred at room temperature for 4 h, diluted with 0.5 M
NaHSO4 (50 mL) and extracted with ethyl acetate (3x). The combined organic layers were washed with
brine, dried (Na2SO4), concentrated under vacuum, and purified by chromatography on SiO2
(CH2Cl2:MeOH, 97:3, then CH2Cl2:MeOH:AcOH, 95:5:0.5) to obtain 12 (0.48 g, 94%) as a colorless
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oil: IR 3468, 3121, 2968, 2938, 2882, 1738, 1685, 1480, 1447, 1368, 1216, 1160, 1044 cm-1; H NMR
(mixture of rotamers, 400 MHz, CDCl3) δ 9.18 (s, 1 H), 8.25 (s, 0.3 H), 8.23 (s, 0.7 H), 5.96 (dd, J =
9.2, 3.1 Hz, 0.3), 5.88 (dd, J = 11.4, 2.8 Hz, 0.7 H), 4.11-4.05 (m, 0.7 H), 3.76 (br s, 0.3 H), 2.69 (s, 2.1
H), 2.67 (s, 0.9 H), 2.37-2.29 (m, J = 3.5 Hz, 1 H), 2.18-2.10 (m, 4 H), 1.73-1.65 (m, 1 H), 1.43 (s, 9 H),
13
1.22-1.15 (m, 1 H), 0.96 (d, J = 6.5 Hz, 3 H), 0.88-0.84 (m, 4 H); C NMR (mixture of rotamers, 101
MHz; CDCl3) δ 171.7, 170.5, 170.3, 169.6, 164.1, 163.9, 156.4, 146.6, 146.4, 129.0, 80.1, 79.6, 70.3,
69.4, 56.3, 34.6, 31.8, 30.3, 29.0, 28.4, 28.3, 28.1, 22.6, 21.0, 20.8, 20.2, 19.9, 19.7, 19.5, 14.1. HRMS
(ESI-) m/z calcd for C18H27O6N2S 399.1595, found 399.1605.
(S)-tert-Butyl (1-oxo-3-phenylpropan-2-yl)carbamate (14).74 A solution of phenylalaninol 13 (2.5 g, 10
mmol) in CH2Cl2 (100 mL) was treated with Dess-Martin periodinane (4.6 g, 11 mmol), stirred at rt for
1 h, and diluted with 1 M Na2S2O3 (30 mL) and NaHCO3 (30 mL). The organic phase was separated and
washed with NaHCO3, dried (Na2SO4), and concentrated. The crude residue was purified by
1
chromatography on SiO2 (EtOAc:hexanes, 5:5 to 6:4) to afford 14 (2.4 g, 96%) as a colorless solid: H
NMR (300 MHz, CDCl3) δ 9.64 (s, 1 H), 7.37-7.28 (m, 3 H), 7.20-7.17 (m, 2 H), 5.04 (s, 1 H), 4.44 (d,
J = 6.4 Hz, 1 H), 3.13 (d, J = 6.4 Hz, 2 H), 1.45 (s, 9 H).
(S,E)-Ethyl
4-((tert-butoxycarbonyl)amino)-2-methyl-5-phenylpent-2-enoate
(15).17,41
(1-
[Ethoxycarbonyl]ethylidene)triphenylphosphorane (5.86 g, 16.2 mmol) was added as solid in a single
portion to a solution of 14 (2.88 g, 11.6 mmol) in CH2Cl2 (70 mL) at 0 °C. The reaction mixture was
stirred at rt for 14 h, concentrated to 10-15 mL and poured into a plug of SiO2 moistened with
EtOAc:hexanes, 4:6, and eluted with EtOAc:hexanes, 5:5, to afford 15 (3.8 g, 98%) as a colorless solid:
1H NMR (400 MHz. CDCl3) δ 7.32-7.31 (m, 2 H), 7.24 (t, J = 7.2 Hz, 1 H), 7.20-7.18 (m, 2 H), 6.54
(dq, J = 9.1, 1.2 Hz, 1 H), 4.68-4.61 (m, 2 H), 4.20 (q, J = 7.1 Hz, 2 H), 2.94 (dd, J = 13.3, 5.6 Hz, 1 H),
2.80 (dd, J = 13.4, 7.1 Hz, 1 H), 1.72 (d, J = 0.7 Hz, 3 H), 1.43 (s, 9 H), 1.30 (t, J = 7.1 Hz, 3 H).
(S,E)-4-((tert-Butoxycarbonyl)amino)-2-methyl-5-phenylpent-2-enoic acid (16).74 A solution of ester 15
(3.8 g, 11 mmol) in THF:H2O:MeOH, 1:0.5:1 (25 mL) was treated with 4 M LiOH (11 mL), stirred at rt
for 40 h, and concentrated. The aqueous layer was washed with Et2O (2x), then acidified to pH 2-3 with
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1 M KHSO4, and extracted with EtOAc (2x). The combined organic layers were washed with brine and
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concentrated to afford 16 (3.0 g, 86%) as a colorless solid: [α]D +44.0 (c 1, CHCl3, lit. +44.9); H
NMR (300 MHz, CDCl3) δ 10.25 (s, 1 H), 7.35-7.19 (m, 5 H), 6.68 (d, J = 8.7 Hz, 1 H), 4.67 (s, 2 H),
2.97 (dd, J = 13.1, 5.4 Hz, 1 H), 2.81 (dd, J = 13.3, 7.1 Hz, 1 H), 1.71 (s, 3 H), 1.44 (s, 9 H).
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(S,E)-(1R,2S,5R)-2-Isopropyl-5-methylcyclohexyl
4-((tert-butoxycarbonyl)amino)-2-methyl-5-
phenylpent-2-enoate (17).74 Dicyclohexylcarbodiimide (DCC, 0.45 g, 2.2 mmol) was added to a solution
of 16 (0.60 g, 1.8 g), menthol (0.42 g, 2.7 mmol) and DMAP (20 mg, 0.15 mmol) in Et2O (30 mL) at 0
°C. The reaction mixture was stirred at rt for 24 h, cooled to 0 °C, filtered through a small plug of
Celite® and concentrated. The residue was purified by chromatography on SiO2 (hexanes:EtOAc, 95:5 to
92.5:7.5) to afford 17 (0.6 g, 75%) as a colorless oil: 1H NMR (300 MHz, CDCl3) δ 7.33-7.25 (m, 4 H),
7.19-7.16 (m, 2 H), 6.49 (dd, J = 9.1, 0.9 Hz, 1 H), 4.77-4.51 (m, 3 H), 3.01-2.90 (m, 1 H), 2.89-2.77
(m, 1 H), 2.07-2.01 (m, 1 H), 1.93-1.83 (m, J = 2.7 Hz, 1 H), 1.74-1.69 (m, 5 H), 1.44-1.40 (m, 11 H),
1.34-1.29 (m, 1 H), 1.12-1.00 (m, 2 H), 0.95-0.89 (m, 8 H), 0.79 (d, J = 6.9 Hz, 3 H).
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(2R,4R)-(1R,2S,5R)-2-Isopropyl-5-methylcyclohexyl
4-((tert-butoxycarbonyl)amino)-2-methyl-5-
phenylpentanoate (18) and (2S,4R)-(1R,2S,5R)-2-isopropyl-5-methylcyclohexyl
4-((tert-
butoxycarbonyl)amino)-2-methyl-5-phenylpentanoate (19).74 A solution of 17 (2.6 g, 0.90 mmol) in
THF (100 mL) was treated with Pd/C (0.293 mg, 0.29 mmol) and stirred under an atmosphere of H2
overnight at rt, filtered on Celite® and concentrated. The crude residue was purified by chromatography
on SiO2 (hexanes:Et2O, 85:15) to afford 18 (0.39 g, 15%) and 19 (2.0 g, 77%) as well as a mixed
fraction (0.15 g, 6%).
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18: H NMR (400 MHz, CDCl3) δ 7.31 (t, J = 7.4 Hz, 2 H), 7.24 (d, J = 7.2 Hz, 1 H), 7.20 (t, J = 7.1
Hz, 2 H), 4.67 (td, J = 10.8, 4.2 Hz, 1 H), 4.40 (d, J = 8.7 Hz, 1 H), 3.92-3.92 (m, 1 H), 2.86-2.75 (m, 2
H), 2.49 (q, J = 6.2 Hz, 1 H), 1.98 (d, J = 11.6 Hz, 1 H), 1.87-1.67 (m, 4 H), 1.57-1.36 (m, 13 H), 1.16
(d, J = 7.0 Hz, 3 H), 1.11-1.02 (m, 1 H), 0.99-0.87 (m, 9 H), 0.77 (d, J = 6.9 Hz, 3 H).
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19: H NMR (400 MHz, CDCl3) δ 7.32 (d, J = 7.0 Hz, 2 H), 7.26-7.23 (m, 1 H), 7.21 (t, J = 6.4 Hz, 2
H), 4.70 (td, J = 10.9, 4.3 Hz, 1 H), 4.39-4.37 (m, 1 H), 3.90 (br s, 1 H), 2.87-2.75 (m, 2 H), 2.65-2.54
(m, 1 H), 2.02 (d, J = 11.9 Hz, 1 H), 1.93-1.83 (m, 2 H), 1.73-1.67 (m, 2 H), 1.51-1.37 (m, 14 H), 1.17
(t, J = 7.4 Hz, 3 H), 1.12-1.02 (m, 2 H), 0.98-0.84 (m, 9 H), 0.77 (d, J = 7.0 Hz, 3 H).
(2S,4R)-4-Amino-2-methyl-5-phenylpentanoic acid hydrochloride (20).74 A suspension of 19 (1.33 g,
2.98 mmol) in 6 M HCl (35 mL) and AcOH (10.5 mL) was heated at reflux (110 °C) for 1.5 h, diluted
with H2O, washed with Et2O and concentrated to afford 20 (0.72 g, 99%) as a colorless crystalline solid:
1H NMR (300 MHz, D2O) δ 7.38-7.24 (m, 5 H), 3.54 (qt, J = 6.9 Hz, 1 H), 2.98 (dd, J = 14.2, 6.5 Hz, 1
H), 2.85 (dd, J = 14.2, 7.8 Hz, 1 H), 2.61 (app sxt, J = 7.1 Hz, 1 H), 1.96 (ddd, J = 14.7, 8.5, 6.1 Hz, 1
H), 1.71-1.62 (m, 1 H), 1.11 (d, J = 7.0 Hz, 3 H).
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(2S,4R)-Allyl 4-amino-2-methyl-5-phenylpentanoate hydrochloride (21).34 Acetyl chloride (2.4 mL, 34
mmol) was slowly added to iced-cold allyl alcohol (12 mL). The reaction mixture was stirred for 5 min
at 0 °C, treated with acid 20 (0.36 g, 1.2 mmol), stirred at 60 °C for 3 h, and concentrated. The residue
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1
was dried under vacuum to afford the desired 21 (0.42 g, quant) as a wax: H NMR (300 MHz,
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CD3OD:CDCl3, 1:1) δ 7.39-7.22 (m, 5 H), 5.94-5.80 (m, 1 H), 5.32-5.22 (m, 2 H), 4.56 (d, J = 5.8 Hz, 2
H), 3.68-3.58 (m, 1 H), 3.52-3.41 (m, 1 H), 2.99 (dd, J = 13.9, 6.5 Hz, 1 H), 2.88 (dd, J = 14.0, 7.7 Hz, 1
H), 2.73-2.66 (m, J = 5.3 Hz, 1 H), 2.06-1.98 (m, 1 H), 1.67 (ddd, J = 14.3, 8.4, 5.5 Hz, 1 H), 1.21 (d, J
= 7.0 Hz, 3 H).
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(2S,4R)-Allyl 4-(2-((1R,3R)-1-acetoxy-3-((tert-butoxycarbonyl)(methyl)amino)-4-methylpentyl)thiazole-
4-carboxamido)-2-methyl-5-phenylpentanoate (22).34 Diisopropylethylamine (0.87 mL, 5.0 mmol) was
added to a solution of 12 (0.5 g, 1.2 mmol), HATU (0.62 g, 1.6 mmol), and HOAT (0.23 g, 1.6 mmol)
in DMF (10 mL) at 0 °C. After 30 min at 0 °C, the reaction mixture was treated with 21 (0.42 g, 1.5
mmol), stirred at 0 °C for 1 h and at rt for 14 h, diluted with EtOAc and 1 M KHSO4, washed with
KHSO4, sat. NaHCO3 (2x) and brine, dried (Na2SO4), and concentrated. The crude residue was purified
by chromatography on SiO2 (hexanes:EtOAc, 8:2 to 6:4) to afford 22 (0.71 g, 90%) as a colorless oil:
20
[α]D +9.3 (c 1.0, CH2Cl2); IR 3270, 3121, 3087, 2968, 2934, 2875, 1745, 1724, 1674, 1644, 1219,
1
1148, 1037 cm-1; H NMR (mixture of rotamers 6:4, 400 MHz, CD2Cl2) δ 7.97 (s, 0.6 H), 7.97 (s, 0.4
H), 7.30-7.19 (m, 5.4 H), 7.11 (d, J = 9.4 Hz, 0.6 H), 5.92-5.85 (m, 1.4 H), 5.77 (dd, J = 11.4, 2.9 Hz,
0.6 H), 5.27 (dt, J = 17.2, 1.4 Hz, 1 H), 5.18 (dq, J = 10.5, 1.2 Hz, 1 H), 4.56-4.47 (m, 2 H), 4.40-4.33
(m, 1 H), 4.05 (td, J = 11.1, 2.7 Hz, 0.6 H), 3.81 (bs, 0.4 H), 2.94-2.84 (m, 2 H), 2.70 (s, 1.8 H) 2.68-
2.57 (m, 2.2 H), 2.33-2.19 (m, 1 H), 2.15 (s, 1.2 H), 2.12 (s, 1.8 H), 2.06-1.98 (m, 2 H), 1.76-1.58 (m, 2
H), 1.43 (s, 5.4 H), 1.43 (s, 3.6 H), 1.16 (d, J = 7.1 Hz, 3 H), 0.99 (d, J = 6.9 Hz, 1.7 H), 0.98 (d, J = 6.9
13
Hz, 1.3 H), 0.89-0.86 (m, 3 H); C-NMR (mixture or rotamers, 101 MHz, CD2Cl2) δ 175.5, 170.64,
170.44, 170.0, 169.5, 160.1, 156.0, 150.21, 150.04, 138.09, 137.93, 132.6, 129.50, 129.44, 128.29,
128.25, 126.40, 126.34, 123.07, 122.93, 117.3, 79.4, 79.0, 70.7, 69.4, 65.0, 56.5, 48.49, 48.35, 41.43,
41.32, 38.06, 37.93, 36.5, 35.6, 35.0, 30.6, 30.4, 29.7, 28.18, 28.12, 20.88, 20.70, 20.0, 19.8, 19.59,
19.45, 17.70, 17.58; HRMS (ESI+) m/z calcd for C33H47O7N3SNa 652.3027, found 652.3024.
tert-Butyl ((2S,3S)-1-fluoro-3-methyl-1-oxopentan-2-yl)carbamate (23).76 A solution of DAST (0.77
mL, 4.8 mmol) in dry CH2Cl2 (5 mL) was added dropwise to a solution of Boc-Ile-OH (0.93 g, 4.0
mmol) and pyridine (0.33 mL, 4.0 mmol) in dry CH2Cl2 (20 mL). The reaction mixture was stirred at rt
for 45 min, diluted with CH2Cl2, washed with ice-cold water, dried (Na2SO4), filtered, concentrated. The
crude residue (0.87 g, 93%) was used in the next step without purification: 1H NMR (400 MHz, CDCl3)
δ 4.98 (d, J = 7.6 Hz, 1 H), 4.45 (dd, J = 8.3, 4.7 Hz, 1 H), 1.99-1.94 (m, 1 H), 1.52-1.48 (m, 10 H),
1.32-1.16 (m, 1 H), 1.05 (t, J = 6.9 Hz, 3 H), 0.98 (t, J = 7.4 Hz, 3 H); 13C NMR (101 MHz, CDCl3) δ
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162 (d, JC-F = 373 Hz), 155.3, 80.7, 57.1 (d, JC-F = 56.6 Hz), 37.1, 28.2, 25.1, 15.5, 11.5.
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(2S,4R)-Allyl 4-(2-((6S,9R,11R)-6-((S)-sec-butyl)-9-isopropyl-2,2,8-trimethyl-4,7,13-trioxo-3,12-dioxa-
5,8-diazatetradecan-11-yl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoate (24). A solution of
TFA (3 mL) in CH2Cl2 (3 mL) was added to a solution of 22 (0.445 g, 0.709 mmol) in CH2Cl2 (7 mL).
The reaction mixture was stirred at rt for 90 min, then concentrated. The resulting oil was diluted with
Et2O and concentrated 3 times to afford (2S,4R)-allyl 4-(2-((1R,3R)-1-acetoxy-4-methyl-3-
(methylamino)pentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoate 2,2,2-trifluoroacetate as a
colorless film which was used in the next step without further purification: HRMS (ESI+) m/z calcd for
C28H40O5N3S 530.2683, found 530.2679. Diisopropylethylamine (0.52 mL, 0.13 mmol) was added to a
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solution
of
(2S,4R)-allyl
4-(2-((1R,3R)-1-acetoxy-4-methyl-3-(methylamino)pentyl)thiazole-4-
carboxamido)-2-methyl-5-phenylpentanoate 2,2,2-trifluoroacetate (0.48 g, 0.75 mmol), and 23 (0.53 g,
2.0 mmol) in DMF (8 mL) at rt. The reaction mixture was stirred for 14 h, then diluted with EtOAc and
0.5 M KHSO4. The organic layer was washed with sat. NaHCO3 (2x) and brine, dried (Na2SO4), and
concentrated. The crude residue was purified by chromatography on SiO2 (hexanes:EtOAc, 65:35 to
20
6:4) to afford 24 (0.501 g, 90%) as a colorless foam: [α]D -3.7 (c 1.0, CH2Cl2); IR 3389, 3303, 2964,
1
2931, 2875, 1733, 1704, 1637, 1492, 1219, 1167, 1044, cm-1; H NMR (500 MHz, CDCl3) δ 8.02 (s, 1
H), 7.30-7.27 (m, 2 H), 7.23-7.20 (m, 3 H), 7.13 (d, J = 9.3 Hz, 1 H), 5.87 (ddt, J = 17.2, 10.5, 5.6 Hz, 1
H), 5.64 (dd, J = 11.4, 2.3 Hz, 1 H), 5.27 (dq, J = 17.2, 1.5 Hz, 1 H), 5.19 (dq, J = 10.5, 1.3 Hz, 1 H),
5.14 (d, J = 9.8 Hz, 1 H), 4.58-4.50 (m, 3 H), 4.46-4.39 (m, 2 H), 3.01 (s, 3 H), 2.97 (dd, J = 13.7, 5.8
Hz, 1 H), 2.89 (dd, J = 13.8, 6.7 Hz, 1 H), 2.68-2.61 (m, 1 H), 2.32 (ddd, J = 14.9, 11.6, 3.1 Hz, 1 H),
2.17 (s, 3 H), 2.08-2.01 (m, 2 H), 1.80-1.61 (m, 4 H), 1.45-1.41 (m, 10H), 1.19 (d, J = 7.1 Hz, 3 H),
1.15-1.09 (m, 1 H), 1.04 (d, J = 6.5 Hz, 3 H), 0.97 (d, J = 6.7 Hz, 3 H), 0.91 (t, J = 7.4 Hz, 3 H), 0.85 (d,
13
J = 6.6 Hz, 3 H); C NMR (mixture of rotamers, 126 MHz, CDCl3) δ 175.7, 174.3, 170.04, 170.00,
160.3, 156.0, 150.0, 137.5, 132.3, 129.6, 128.4, 126.5, 123.4, 117.9, 79.5, 69.5, 65.1, 55,6, 55.1, 48.4,
41.0, 37.6, 37.0, 36.6, 34.7, 30.0, 29.5, 28.3, 24.1, 20.8, 20.1, 19.5, 17.7, 16.0, 11.1; HRMS (ESI+) m/z
calcd for C39H59O8N4S 743.4048, found 743.4049.
(R)-1-Methylpiperidine-2-carboxylic acid (25).14 A suspension of 10% Pd/C (0.20 g, 0.19 mmol), 37%
aqueous formaldehyde (2.9 mL, 39 mmol) and D-pipecolic acid (1.0 g, 7.7 mmol) in MeOH (10 mL)
was stirred for 24 h under a hydrogen atmosphere, filtered through Celite® and concentrated to afford 25
1
as an off-white solid (1.0 g, 90%): H NMR (300 MHz, D2O) δ 3.44-3.36 (m, 2 H), 2.96 (td, J = 12.6,
2.9 Hz, 1 H), 2.78 (s, 3 H), 2.16-2.11 (m, 1 H), 1.88-1.77 (m, 2 H), 1.71-1.57 (m, 2 H), 1.53-1.45 (m, 1
H).
(2S,4R)-Allyl
4-(2-((1R,3R)-1-acetoxy-3-((2S,3S)-N,3-dimethyl-2-((R)-1-methylpiperidine-2-
carboxamido)pentanamido)-4-methylpentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoate
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(26).34 A solution of TFA (4 mL) in CH2Cl2 (4 mL) was added to a solution of 24 (0.66 g, 0.88 mmol) in
CH2Cl2 (8 mL). The reaction mixture was stirred at rt for 60 min, then concentrated. The resulting oil
was diluted with CH2Cl2 and concentrated 3 times to afford (2S,4R)-allyl 4-(2-((1R,3R)-1-acetoxy-3-
((2S,3S)-2-amino-N,3-dimethylpentanamido)-4-methylpentyl)thiazole-4-carboxamido)-2-methyl-5-
phenylpentanoate 2,2,2-trifluoroacetate as a colorless film that was used in the next step without further
purification: HRMS (ESI+) m/z calcd for C34H51O6N4S 643.3524, found 643.3525.
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Diisopropylethylamine (0.70 mL, 4.0 mmol) was added to a solution of 25 (0.19 g, 1.3 mmol), HATU
(0.51 g, 1.3 mmol), and HOAT (0.18 g, 1.3 mmol) in DMF (5 mL) at 0 °C. After 30 min at 0 °C, the
reaction mixture was treated with to (2S,4R)-allyl 4-(2-((1R,3R)-1-acetoxy-3-((2S,3S)-2-amino-N,3-
dimethylpentanamido)-4-methylpentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoate
2,2,2-
trifluoroacetate (0.67 g, 0.89 mmol), stirred at 0 °C for 30 min and at rt for 5 h, then diluted with
EtOAc, washed with sat. NaHCO3 (2x) and brine, dried (Na2SO4) and concentrated. The crude residue
was purified by chromatography on SiO2 (CH2Cl2:MeOH, 100:2 to 100:8) to afford 26 (0.67 g, 98%) as
a colorless oil: [α]D20 +20.6 (c 0.5, CH2Cl2); IR 3386, 3270, 3076, 2934, 2893, 2856, 1733, 1641, 1540,
1495, 1372, 1219, 843, 738 cm-1; 1H NMR (601 MHz, MeOD) δ 8.10 (s, 1 H), 7.28-7.24 (m, 4 H), 7.20-
7.18 (m, 1 H), 5.88 (ddt, J = 17.2, 10.5, 5.6 Hz, 1 H), 5.72 (dd, J = 11.2, 2.4 Hz, 1 H), 5.26 (dq, J =
17.2, 1.6 Hz, 1 H), 5.17 (dq, J = 10.5, 1.4 Hz, 1 H), 4.77 (d, J = 8.1 Hz, 1 H), 4.55-4.47 (m, 3 H), 4.41-
4.37 (m, J = 3.6 Hz, 1 H), 3.13 (s, 3 H), 3.01-2.98 (m, 1 H), 2.94 (dd, J = 13.7, 7.1 Hz, 1 H), 2.88 (dd, J
= 13.6, 6.8 Hz, 1 H), 2.69 (d, J = 10.5 Hz, 1 H), 2.66-2.63 (m, 1 H), 2.39 (ddd, J = 14.9, 11.5, 3.1 Hz, 1
H), 2.29-2.24 (m, 4 H), 2.22-2.15 (m, 4 H), 2.01 (ddd, J = 14.0, 10.0, 3.8 Hz, 1 H), 1.91-1.74 (m, 5 H),
1.70-1.55 (m, 5 H), 1.38-1.28 (m, 2 H), 1.22-1.17 (m, 4 H), 1.05 (d, J = 6.6 Hz, 3 H), 1.00 (d, J = 6.8
13
Hz, 3 H), 0.94 (t, J = 7.4 Hz, 3 H), 0.83 (d, J = 6.6 Hz, 3 H); C NMR (151 MHz, MeOD) δ 176.0,
173.7, 170.4, 170.3, 161.3, 149.4, 138.0, 132.3, 129.1, 128.0, 126.1, 123.8, 116.8, 69.8, 68.9, 64.8, 55.2,
53.5, 48.9, 43.2, 41.0, 37.5, 37.4, 36.5, 36.3, 34.3, 30.1, 29.6, 29.0, 24.6, 24.1, 22.7, 19.4, 19.1, 18.9,
16.7, 15.0, 9.8; HRMS (ESI+) m/z calcd for C41H62O7N5S 768.4364, found 768.4364.
(2S,4R)-4-(2-((1R,3R)-1-acetoxy-3-((2S,3S)-N,3-dimethyl-2-((R)-1-methylpiperidine-2-
carboxamido)pentanamido)-4-methylpentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoic acid
trifluoroacetate (1). Morpholine (0.068 mL, 0.78 mmol) was added to a solution of 26 (0.148 g, 0.20
mmol) in CH2Cl2 (6 mL) at 0 ºC under an atmosphere of N2, then Pd(Ph3P)4 (15 mg, 0.015 mmol) in
CH2Cl2 (1 mL) was added. The reaction mixture was stirred for 3 h at 0 ºC and purified by
chromatography on SiO2 (the reaction mixture was directly charged onto the column, prepacked with
CH2Cl2:MeOH, 99:1, using CH2Cl2:MeOH, 99:1 to 90:1 as the eluent) to afford 1 as a pale yellow film
that was further purified by RP chromatography (12-g C18 cartridge, 0.1% TFA:H2O:CH3CN, 85:15 to
1
40:60). Lyophilization afforded 1 (0.148 g, 90%) as a colorless fluffy solid: H NMR (500 MHz,
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MeOD) δ 8.10 (s, 1 H, Tuv thiazole CH), 7.27-7.24 (m, 4 H, Tup o-,o’-, m-, and m’-phenyl CHs), 7.20-
7.17 (m, 1 H, Tup p-phenyl CH), 5.74 (d, J = 11.0, 1 H Tuv CHOAc), 4.74 (d, J = 7.5, 1 H, Ile CH-
NH), 4.42-4.38 (m, 2 H, Tuv CH-N, Tup CH-NH), 3.77 (dd, J = 12.2, 2.1 Hz, 1 H, Mep CH-N), 3.49 (d,
J = 12.7 Hz, 1 H, Mep CH2-N a), 3.14 (s, 3 H, Tuv N-CH3), 3.09 (td, J = 12.9, 2.1 Hz, 1 H, Mep CH2-
N b), 2.95-2.89 (m, 2 H Tup CH2-Ph), 2.76 (s, 3 H, Mep CH3), 2.59-2.54 (m, 1 H, Tup α CH), 2.41
(ddd, J = 14.7, 11.6, 2.9 Hz, 1 H, Tuv CH2 a), 2.35-2.30 (m, 1 H, Tuv CH2 b), 2.19-2.17 (m, 4 H, Tuv
COCH3, Mep β CH2 a), 2.02 (ddd, J = 13.9, 9.9, 4.0 Hz, 1 H, Tup β CH2 a), 1.97-1.87 (m, 4 H, Tuv
CH-CH3, Ile CH-CH3, Mep γ CH2 a, Mep δ CH2 a), 1.84-1.75 (m, 2 H, Mep β CH2 b, Mep δ CH2 b),
1.69 (ddd, J = 14.2, 10.2, 4.1 Hz, 1 H, Tup β CH2 b), 1.65-1.58 (m, 2 H, Mep γ CH2 b, Ile CH2 a),
1.24-1.19 (m, 4 H, Ile CH2 b, Tup CH3), 1.06 (d, J = 6.6 Hz, 3 H, Tuv CH-CH3 a), 1.04 (d, J = 6.8 Hz,
3 H, Ile CH-CH3), 0.96 (t, J = 7.4 Hz, 3 H, Ile CH2-CH3), 0.87 (d, J = 6.6 Hz, 3 H, Tuv CH-CH3 b);
13C NMR (101 MHz, MeOD) δ 178.5 (Tup CO2H), 173.2 (Ile CO-N), 170.37 (Tuv thiazole C2), 170.18
(Tuv COCH3), 167.7 (Mep CO-NH), 161.4 (Tuv CO-NH), 149.4 (Tuv thiazole C4, 138.1 (Tup phenyl
C1), 129.1 (Tup m,m’-phenyl CHs), 127.9 (Tup o,o’-phenyl CHs), 126.0 (Tup p-phenyl CH), 123.7
(Tuv thiazole CH), 69.8 (Tuv CH-OAc), 66.8 (Mep CH-N), 56.9 (Tuv CH-N), 54.8 (Mep CH2-N), 54.6
(Ile CH-NH), 49.3 (Tup CH-NH), 41.5 (Mep CH3), 40.8 (Tup CH2-Ph), 37.7 (Tup β CH2), 36.4 (Tup α
CH), 36.0 (Ile CH-CH3), 34.2 (Tuv CH2), 29.5 (Tuv CH-CH3, Tuv N-CH3), 28.9 (Mep β CH2), 23.8
(Ile CH2), 22.6 (Mep δ CH2), 21.0 (Mep γ CH2), 19.4 (Tuv COCH3), 19.1 (Tuv CH-CH3 a), 18.9 (Tuv
CH-CH3 b), 17.1 (Tup CH3), 14.8 (Ile CH-CH3), 9.9 (Ile CH2-CH3); HRMS (ESI+) m/z calcd for
C38H58O7N5S 728.4051, found 728.4058.
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(2S,4R)-4-(2-((1R,3R)-1-Acetoxy-4-methyl-3-((2S,3S)-3-methyl-2-((R)-1-methylpiperidine-2-
carboxamido)pentanamido)pentyl)thiazole-4-carboxamido)-5-(2-iodophenyl)-2-methylpentanoic acid
2,2,2-trifluoroacetate (27). A solution of 1 (7.0 mg, 0.0083 mmol) in CH2Cl2 (0.6 mL) and TFA (0.06
mL) was treated with IPy2BF4 (5.6 mg, 0.014 mmol) and the maroon reaction mixture was stirred for 3 h
at rt, concentrated, dissolved in acetonitrile:water (1:1) and purified immediately (less than 5 min after
the sample was redissolved) by RP-HPLC (water + 0.1% TFA/acetonitrile + 0.1% TFA, 70:30 to
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35:60). Lyophilization afforded 27 (5.9 mg, 73%) as a colorless fluffy solid: H-NMR (mixture of
rotamers, 400 MHz, MeOD) δ, 8.05 (s, 1 H, Tuv thiazole CH), 7.82 (d, J = 7.9 Hz, 1 H, Tup phenyl CH
ortho to I and meta to CH2), 7.32 (d, J = 7.4, 1 H, Tup phenyl CH meta to I and ortho to CH2), 7.24 (t, J
= 7.4 Hz, 1 H, Tup phenyl CH para to I and meta to CH2), 6.91 (t, J = 7.6 Hz, 1 H, Tup phenyl CH meta
to I and para to CH2), 5.77-5.74 (m, 1 H, Tuv CH-OAc), 4.75 (d, J = 7.5 Hz, 1 H, Ile CH-NH), 4.58-
4.51 (m, 1 H, Tup CH-NH), 4.39 (bs, 1 H, Tuv CH-N), 3.75-3.72 (m, 1 H, Mep CH-N), 3.51-3.47 (m, 1
H, Mep ε CH2-N a), 3.16-3.02 (m, 6 H, Tuv CH3, Mep ε CH2-N b, Tup δ CH2), 2.75 (s, 3 H, Mep
CH3), 2.61 (br s, 1 H, Tup α CH), 2.42-2.37 (m, 2 H, Tuv CH2), 2.18-2.09 (m, 5 H, OCOCH3, Tup β
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CH2 a, Mep β CH2 a), 1.96-1.90 (m, 4 H, Tuv CH-CH3, Ile CH-CH3, Mep γ CH2 a, Mep δ CH2 a),
1.86-1.77 (m, 3 H, Mep β CH2 b, Mep γ CH2 b, Tup β CH2 b), 1.66-1.60 (m, 2 H, Mep δ CH2 b, Ile
CH2 a), 1.25 (m, 4 H, Tup CH3, Ile CH2 b), 1.06 (m, 6 H, Tuv CH-CH3 a, Ile CH-CH3), 0.97 (t, J =
7.3 Hz, 3 H, Ile CH2-CH3), 0.88 (d, J = 6.6 Hz, 3 H, Tuv CH-CH3 b); 13C NMR (101 MHz, MeOD) δ
178.4, 173.2, 170.4, 170.1, 167.7, 161.0, 149.0, 141.1, 139.2, 130.4, 128.0, 127.8, 123.9, 100.4, 70.0,
66.8, 57.1, 54.9, 54.6, 48.6, 45.2, 41.5, 38.2, 36.5, 36.0, 34.2, 29.5, 29.4, 28.9, 23.9, 22.6, 21.0, 19.4,
19.1, 18.9, 17.2, 14.9, 9.9; HRMS (ESI+) m/z calcd for C38H57O7N5SI 854.3018, found 854.3003.
(2S,4R)-5-([1,1'-Biphenyl]-2-yl)-4-(2-((1R,3R)-1-acetoxy-4-methyl-3-((2S,3S)-3-methyl-2-((R)-1-
methylpiperidine-2-carboxamido)pentanamido)pentyl)thiazole-4-carboxamido)-2-methylpentanoic acid
2,2,2-trifluoroacetate (28). Deoxygenated toluene (0.2 mL) was added under argon to a mixture of 27
(4.5 mg, 0.0046 mmol), PhB(OH)2 (1.1 mg, 0.0093 mmol) and Na2CO3 (2.0 mg, 0.019 mmol) in a MW
vial. Then Pd(PPh3)4 (0.1 mL of a toluene solution, containing 0.25 mg of Pd, 0.00023 mmol) was
added, followed by MeOH (0.2 mL). The reaction flask was sealed and the mixture was stirred at 80 °C
for 5 h. After cooling to room temperature, the reaction mixture was filtered through a 0.45 µm HPLC
filter and concentrated. The residue was redissolved in acetonitrile and water and purified by RP-HPLC
(C-18, water + 0.1% TFA/acetonitrile + 0.1% TFA, 35:75 to 60:40) to obtain powdery 28 (3.1 mg,
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73%): H NMR (400 MHz, MeOD) δ 8.04 (s, 1 H Tuv thiazole CH), 7.45-7.41 (m, 2 H Tup biphenyl
CHs), 7.38-7.32 (m, 4 H, Tup biphenyl CHs), 7.23-7.21 (m, 2 H, Tup biphenyl CHs), 7.16-7.14 (m, 1 H,
Tup biphenyl CH), 5.73 (d, J = 11.0 Hz, 1 H, Tuv CH-OAc), 4.74 (d, J = 7.5 Hz, 1 H, Ile CH-NH), 4.39
(bs, 1 H, Tuv CH-N), 4.28-4.21 (m, 1 H, Tup CH-NH), 3.77 (dd, J = 12.2, 2.8 Hz, 1 H, Mep CH-N),
3.49 (d, J = 12.2 Hz, 1 H, Mep CH2-N a), 3.14-3.05 (m, 4 H, Tuv N-CH3, Mep CH2-N b), 3.01 (dd, J =
13.9, 5.4 Hz, 1 H, Tup CH2-biphenyl a), 2.83-2.74 (m, 4 H, Tup CH2-biphenyl b, Mep N-CH3), 2.46-
2.28 (m, 3 H, Tuv CH2 a, Tuv CH2 b, Tup α CH), 2.19-2.17 (m, 4 H, Tuv COCH3, Mep β CH2 a),
1.96-1.90 (m, 4 H, Ile CH-CH3, Tuv CH-CH3, Mep δ CH2 a, Mep γ CH2 a), 1.84-1.77 (m, 3 H, Tup β
CH2 a, Mep β CH2 b, Mep δ CH2 b), 1.64-1.60 (m, 2 H, Mep γ CH2 b, Ile CH2 a), 1.51 (ddd, J = 14.0,
9.1, 4.9 Hz, 1 H, Tup β CH2 b), 1.25-1.19 (m, 1 H, Ile CH2 b), 1.07 (m, 9 H, Tup CH3, Tuv CH-CH3
a, Ile CH-CH3), 0.96 (t, J = 7.4 Hz, 3 H, Ile CH2-CH3), 0.87 (d, J = 6.5 Hz, 3 H, Tuv CH-CH3 b); 13C
NMR (101 MHz, MeOD) δ 178.4, 173.2, 170.4, 170.0, 167.7, 161.1, 149.4, 142.6, 141.8, 135.6, 130.0,
129.7, 129.1, 127.9, 126.8, 126.6, 125.9, 123.6, 69.7, 66.8, 56.1, 54.8, 54.6, 49.0, 41.5, 38.1, 37.7, 36.2,
36.0, 34.1, 29.5, 28.9, 23.8, 22.6, 21.0, 19.4, 19.2, 19.0, 16.7, 14.9, 9.9; HRMS (ESI+) m/z calcd for
C44H62O7N5S 804.4364, found 804.4385.
(S)-3-((R)-1-(2-((1R,3R)-1-Acetoxy-4-methyl-3-((2S,3S)-3-methyl-2-((R)-1-methylpiperidine-2-
carboxamido)pentanamido)pentyl)thiazole-4-carbonyl)indolin-2-yl)-2-methylpropanoic acid 2,2,2-
trifluoroacetate (29). A solution of CuI (0.00089 mmol, 0.17 mg) and proline (0.00096 mmol, 0.11 mg)
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in deoxygenated DMSO (0.2 mL) was added to a solution of 27 (0.0044 mmol, 4.3 mg) in deoxygenated
DMSO (0.5 mL), followed by addition of NaOH (0.010 mmol, 0.43 mg) and sodium ascorbate (0.00096
mmol, 0.19 mg) in H2O (0.010 mL). The reaction flask was sealed and heated at 60 °C for 5 h. After
cooling to room temperature, the mixture was diluted with water (2 mL) and purified by RP-HPLC (C-
18, water + 0.1% TFA/acetonitrile + 0.1% TFA, 35:75 to 60:40). Lyophilization afforded 29 (3.0 mg,
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80%): H NMR (600 MHz, MeOD) δ 8.20 (bs, 2 H, Tuv thiazole CH, Tup phenyl CH ortho to N and
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meta to CH2), 7.32 (d, J = 7.3 Hz, 1 H, Tup phenyl CH ortho to CH2 and meta to N), 7.22 (bs, 1 H, Tup
phenyl CH meta to N and para to CH2), 7.13 (t, J = 5.7 Hz, 1 H, Tup phenyl CH para to N and meta to
CH2), 5.76 (d, J = 10.5 Hz, 1 H, Tuv CHOAc), 5.34 (bs, 1 H, Tup CH-N), 4.73 (d, J = 7.4 Hz, 1 H, Ile
CH-NH), 4.39 (bs, 1 H, Tuv CH-N), 3.77 (dd, J = 12.2, 2.5 Hz, 1 H, Mep CH-N), 3.48 (d, J = 12 Hz, 1
H, Mep CH2-N a), 3.46-3.42 (m, 1 H, Tup CH2-Ar a), 3.13-3.07 (m, 4 H, Tuv N-CH3, Mep CH2-N b),
2.91 (d, J = 15.7 Hz, 1 H, Tup CH2-Ar b), 2.76 (s, 3 H, Mep N-CH3), 2.46-2.43 (m, 2 H, Tup α CH,
Tuv CH2 a), 2.35-2.29 (m, 1 H, Tuv CH2 b), 2.18-2.15 (m, 4 H, Tuv COCH3, Mep β CH2 a), 1.94-1.91
(m, 5 H, Ile CH-CH3, Tuv CH-CH3, Mep γ CH2 a, Mep δ CH2 a, Tup β CH2 a), 1.84-1.75 (m, 2 H,
Mep β CH2 b, Mep δ CH2 b), 1.68-1.59 (m, 3 H, Tup β CH2 b, Mep γ CH2 b, Ile CH2 a), 1.24-1.19
(m, 1 H, Ile CH2 b), 1.05-1.03 (m, 9 H, Tuv CH-CH3 a, Tup CH3, Ile CH-CH3), 0.95 (t, J = 7.4 Hz, 3
H, Ile CH2-CH3), 0.86 (d, J = 6.5 Hz, 3 H, Tuv CH-CH3 b); HRMS (ESI+) m/z calcd for C38H56O7N5S
726.3895, found 726.3892.
(1R,3R)-3-((2S,3S)-N,3-Dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido)-1-(4-
(((2R,4S)-5-hydrazinyl-4-methyl-5-oxo-1-phenylpentan-2-yl)carbamoyl)thiazol-2-yl)-4-methylpentyl
acetate trifluoroacetate (31). Diisopropylethylamine (0.138 mL, 0.79 mmol) was added to a solution of
1 (0.148 g, 0.18 mmol), HATU (0.082 g, 0.21 mmol), and HOAT (0.029 g, 1.2 mmol) in DMF (2 mL)
at 0 °C. After 30 min at 0 °C, Boc-hydrazine (30, 0.035 g, 0.26 mmol) was added and the reaction
mixture was stirred at 0 °C for 4 h, then diluted with EtOAc and sat. NaHCO3, washed with sat.
NaHCO3 (2x) and brine, dried (Na2SO4) and concentrated. The crude residue was purified by
chromatography on SiO2 (CH2Cl2:MeOH, 99:1 to 9:1) to afford tert-butyl 2-((2S,4R)-4-(2-((1R,3R)-1-
acetoxy-3-((2S,3S)-N,3-dimethyl-2-((R)-1-methylpiperidine-2-carboxamido)pentanamido)-4-
methylpentyl)thiazole-4-carboxamido)-2-methyl-5-phenylpentanoyl)hydrazinecarboxylate 30a (0.135 g,
91%) as a colorless glass: 1H NMR (mixture of rotamers, 500 MHz, CDCl3) δ 9.65 (bs, 1 H), 8.04 (s, 1
H), 7.32 (d, J = 9.6 Hz, 2 H), 7.28-7.22 (m, 6 H), 6.55 (bs, 1 H), 5.67 (dd, J = 11.2, 2.6 Hz, 1 H), 4.78 (t,
J = 8.4 Hz, 2 H), 4.54 (br s, 1 H), 3.07 (s, 3 H), 3.01-2.91 (m, 3 H), 2.36-2.32 (m, 5 H), 2.20 (s, 3 H),
2.07-1.99 (m, 2 H), 1.88-1.61 (m, 14 H), 1.51-1.48 (m, 13 H), 1.20-1.17 (m, 1 H), 1.14 (d, J = 6.8 Hz, 3
H), 1.06 (d, J = 6.6 Hz, 3 H), 1.00 (d, J = 6.7 Hz, 3 H), 0.94 (t, J = 7.4 Hz, 3 H), 0.83 (d, J = 6.6 Hz, 3
H); HRMS (ESI+) m/z calcd for C43H68O8N7S 842.4845, found 842.4847. A solution of TFA (1 mL) in
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CH2Cl2 (1 mL) was added to a solution of 30a (0.107 g, 0.13 mmol) in CH2Cl2 (2 mL). The reaction
mixture was stirred at rt for 60 min, then concentrated. The resulting oil was diluted with CH2Cl2 and
concentrated 3 times, then diluted with water and lyophilized to afford colorless fluffy 31 (0.120 g,
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quant.): H NMR (500 MHz, MeOD) δ 8.12 (s, 1 H, Tuv thiazole CH), 7.26-7.17 (m, 5 H, Tup phenyl
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CHs), 5.72 (dd, J = 11.3, 1.7 Hz, 1 H, Tuv CH-OAc), 4.75 (d, J = 7.4 Hz, 1 H, Ile CH-NH), 4.50 (br t, J
= 9.6 Hz, 1 H, Tup CH-NH), 4.29-4.27 (m, 1 H, Tuv CH-N), 3.77 (dd, J = 12.1, 2.0 Hz, 1 H, Mep CH-
N), 3.50 (d, J = 12.5 Hz, 1 H, Mep CH2-N a), 3.15-3.07 (m, 4 H, Tuv N-CH3, Mep CH2-N b), 2.91-
2.90 (m, 2 H, Tup CH2-Ph), 2.77 (s, 3 H, Mep CH3), 2.53 (bs, 1 H, Tup α CH), 2.40 (ddd, J = 14.7,
11.8, 2.7 Hz, 1 H, Tuv CH2 a), 2.28-2.19 (m, 2 H, Tuv CH2 b, Mep β CH2 a), 2.17 (s, 3 H, Tuv
COCH3), 2.09 (t, J = 12.1 Hz, 1 H, Tup β CH2 a), 1.98-1.85 (m, 4 H, Ile CH-CH3, Tuv CH-CH3, Mep
γ CH2 a, Mep δ CH2 a), 1.83-1.75 (m, 2 H, Tup β CH2 b, Mep δ CH2 b), 1.71-1.60 (m, 3 H, Tup β
CH2 b, Mep γ CH2 b, Ile CH2 a), 1.26-1.17 (m, 4 H, Ile CH2 b, Tup CH3), 1.06 (d, J = 6.9 Hz, 3 H,
Tuv CH-CH3 a), 1.04 (d, J = 7.0 Hz, 3 H, Ile CH-CH3), 0.97 (t, J = 7.4 Hz, 3 H, Ile CH2-CH3), 0.86 (d,
J = 6.6 Hz, 3 H, Tuv CH-CH3 b); 13C NMR (101 MHz, MeOD) δ 175.6, 173.2, 170.53, 170.35, 167.7,
161.8, 149.3, 137.9, 129.0, 127.9, 126.1, 124.1, 69.8, 66.8, 54.9, 54.6, 49.1, 41.5, 41.2, 38.3, 36.0, 35.4,
34.3, 29.5, 28.9, 23.9, 22.6, 21.0, 19.4, 19.1, 18.9, 17.6, 14.9, 9.9; HRMS (ESI+) m/z calcd for
C38H60O6N7S 742.4320, found 742.4296.
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(S,E)-Allyl 4-(tert-butoxycarbonylamino)-2-methyl-5-phenylpent-2-enoate (32). A solution of 15 (0.36
o
g, 1.1 mmol) and LiOH (1.0 N, 4.0 mL, 4.0 mmol) in THF (8 mL) was heated at 60 C for 8 h. The
reaction mixture was quenched by addition of 1 N HCl (5.0 mL, 5.0 mmol), and the aqueous layer was
extracted with ethyl acetate (25 mL x 3). The combinded organic layers were dried (Na2SO4) and
concentrated to give crude acid as a colorless solid. After addition of anhydrous DMF (3 mL), cesium
carbonate (0.98 g, 3.0 mmol) and allyl bromide (2.0 mL, 23.1 mmol), mixture was stirred at room
temperature for 24 h, diluted with ethyl acetate, washed with saturated sodium bicarbonate and brine,
dried (Na2SO4), concentrated and purified by chromatography on SiO2 (EtOAc:hexanes, 1:5) to give 32
(0.32 g, 84% for two steps) as a colorless solid: [α]D23 +34.4 (c 3.83, CH2Cl2); Mp 56.5-58.0 oC; IR (cm-
1) 3366, 2977, 2931, 1712, 1515, 1366, 1250, 1169; 1H NMR (CDCl3, 600 MHz) δ 7.34 (m, 2 H), 7.28
(t, 1 H, J = 7.2 Hz), 7.24 (d, 2 H, J = 7.2 Hz), 6.66 (d, 1 H, J = 9.0 Hz), 6.03-5.97 (m, 1 H), 5.38 (dd, 1
H, J = 16.5, 1.5 Hz), 5.30 (dd, 1 H, J = 10.5, 0.9 Hz), 4.97 (bs, 1 H), 4.76-4.73 (m, 1 H), 4.72-4.70 (m, 2
H), 3.03-2.99 (m, 1 H), 2.84 (dd, 1 H, J = 12.3, 7.5 Hz), 1.77 (s, 3 H), 1.48 (s, 9 H); 13C NMR (CDCl3,
150 MHz) δ 167.2, 154.9, 140.6, 136.6, 132.1, 129.4, 128.9, 128.2, 126.5, 117.7, 79.3, 65.2, 50.0, 41.0,
28.2, 12.4; HRMS (ESI) m/z calcd for C20H27NO4Na (M+Na) 368.1838, found 368.1823.
(S,E)-Allyl 4-(2-((1R,3R)-1-acetoxy-3-(tert-butoxycarbonyl(methyl)amino)-4-methylpentyl)thiazole-4-
carboxamido)-2-methyl-5-phenylpent-2-enoate (33). A solution of 32 (74 mg, 0.21 mmol) in CH2Cl2
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(1.0 mL) and trifluoroacetic acid (0.4 mL, 4.2 mmol) was stirred at room temperature for 2 h. The
solution was partitioned between ethyl acetate and water, and the organic layer was washed with
saturated sodium bicarbonate, dried (Na2SO4) and concentrated to give crude amine (52 mg, 99%) as a
colorless oil: MS (ESI) m/z 268 ([M+Na]+), 246 ([M+H]+). To a solution of 12 (52 mg, 0.13 mmol) and
triethylamine (0.05 mL, 0.38 mmol) in anhydrous THF (1 mL) cooled to -20 ºC was added dropwise
isobutyl chloroformate (0.03 mL, 0.22 mmol), and the resulting colorless suspension was stirred for 20
min. A solution of the crude amine (52 mg, 0.21 mmol) in anhydrous THF (1 mL) was then added via
cannula, and the mixture was stirred overnight, allowing the temperature to gradually rise to room
temperature. The reaction mixture was diluted with ethyl acetate, washed with brine, dried (Na2SO4),
concentrated, and purified by chromatography on SiO2 (EtOAc:hexanes, 1:2) to give 33 (64 mg, 78%
for two steps) as a colorless oil: [α]D23 +22.7 (c 0.71, CH2Cl2); IR (cm-1) 3367, 2971, 2930, 1753, 1713,
1689, 1538, 1390, 1224; 1H NMR (2:1 mixture of rotamers, 600 MHz, CDCl3) for major rotamer δ 8.03
(s, 1 H), 7.38 (d, 1 H, J = 8.4 Hz), 7.30-7.26 (m, 2 H), 7.23-7.21 (m, 3 H), 6.68 (dd, 1 H, J = 9.0, 1.5
Hz), 5.97-5.90 (m, 1 H), 5.81 (dd, 1 H, J = 11.4 Hz, 3.0 Hz), 5.33-5.29 (m, 1 H), 5.25-5.22 (m, 1 H),
5.19-5.13 (m, 1 H), 4.67-4.59 (m, 2 H), 4.12-4.05 (m, 1 H), 3.11 (dd, 1 H, J = 13.2, 6.0 Hz), 2.93 (dd, 1
H, J = 13.2, 8.4 Hz), 2.72 (s, 3 H), 2.32 (ddd, 1 H, J = 15.2, 11.7, 4.2 Hz), 2.26-2.21 (m, 1 H), 2.16 (s, 3
H), 2.05-2.00 (m, 1 H), 1.77 (d, 3 H, J = 1.2 Hz), 1.45 (s, 9 H), 1.02 (d, 3 H, J = 6.6 Hz), 0.89 (d, 3 H, J
= 6.0 Hz); 13C NMR (CDCl3, 150 MHz) δ 170.5, 170.2, 167.3, 160.1, 156.2, 149.5, 139.4, 136.5, 132.2,
130.2, 129.5, 128.5, 126.8, 123.7, 118.0, 79.4, 69.1, 65.4, 56.3, 48.7, 40.8, 34.8, 30.4, 28.3, 20.8, 20.0,
19.6, 12.7; Characteristic peaks of minor rotamer: 1H NMR (CDCl3, 600 MHz) δ 8.03 (s, 1 H), 7.62 (d,
1 H, J = 8.4 Hz), 6.72 (d, 1 H, J = 10.2 Hz), 2.65 (s, 3 H), 2.17 (s, 3 H), 1.76 (d, 3 H, J = 1.2 Hz), 0.99
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(d, 3 H, J = 6.6 Hz), 0.88 (d, 3 H, J = 5.4 Hz); C NMR (CDCl3, 150 MHz) δ 169.4, 167.3, 160.0,
149.7, 139.5, 136.6, 132.2, 130.0, 129.5, 128.4, 126.7, 123.5, 117.9, 79.8, 70.7, 65.4, 48.8, 40.8, 28.4,
21.0, 20.3, 19.7; HRMS (ESI) calcd for C33H45N3O7NaS (M+Na) 650.2876, found 650.2906.
(S,E)-Allyl 4-(2-((5S,8R,10R)-5-((S)-sec-butyl)-1-(9H-fluoren-9-yl)-8-isopropyl-7-methyl-3,6,12-trioxo-
2,11-dioxa-4,7-diazatridecan-10-yl)thiazole-4-carboxamido)-2-methyl-5-phenylpent-2-enoate (34). To a
solution of 33 (49 mg, 78 µmol) in dichloromethane (1 mL) was added trifluoroacetic acid (0.4 mL, 5.2
mmol), and the reaction mixture was stirred at room temperature for 4 h. The solution was partitioned
between ethyl acetate and water, and the organic layer was washed with saturated sodium bicarbonate,
dried (Na2SO4) and concentrated to give the crude amine as a colorless oil: MS (ESI) m/z 528 ([M+H]+).
To a solution of the amine in anhydrous DMF (0.3 mL) was added N,N-diisopropylethylamine (0.03
mL, 0.17 mmol) and Fmoc-Ile-F (60 mg, 0.17 mmol), and the mixture was stirred at room temperature
for 18 h. The solution was diluted with ethyl acetate, washed with saturated sodium bicarbonate and
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