Synthesis and Evaluation of Dolastatin 10 Analogues Containing Heteroatoms on the Amino Acid Side Chains

Article abstract of DOI:10.1021/acs.jnatprod.7b00359

Synthetic analogues of the natural occurring dolastatin 10 are of great interest in cancer due to their potent in vitro activity and their uses as payloads in antibody drug conjugates (ADCs). Modification of the dolastatin 10 core scaffold has mainly focused on modifications of the P1, N-terminus, and P5, C-terminus, with minimal attention to the P2 subunit. In this paper we discuss the introduction of heteroatoms to the P2 side chain, which results in potent activity in vitro. The most active compounds contained azides in the P2 unit and required a phenylalanine-derived P5 subunit.

Full text of DOI:10.1021/acs.jnatprod.7b00359

Article
pubs.acs.org/jnp
Synthesis and Evaluation of Dolastatin 10 Analogues Containing
Heteroatoms on the Amino Acid Side Chains
Julien Dugal-Tessier, Stuart D. Barnscher,† Akira Kanai, and Brian A. Mendelsohn*
Agensys Inc., an affiliate of Astellas Pharma Inc., 1800 Stewart Street, Santa Monica, California 90404, United States
S
* Supporting Information
ABSTRACT: Synthetic analogues of the natural occurring dolastatin 10 are
of great interest in cancer due to their potent in vitro activity and their uses as
payloads in antibody drug conjugates (ADCs). Modification of the dolastatin
10 core scaffold has mainly focused on modifications of the P1, N-terminus,
and P5, C-terminus, with minimal attention to the P2 subunit. In this paper
we discuss the introduction of heteroatoms to the P2 side chain, which results
in potent activity in vitro. The most active compounds contained azides in
the P2 unit and required a phenylalanine-derived P5 subunit.
ancer is a leading cause of death worldwide, and as a
demonstrated sub-nanomolar activity in vitro toward a variety of
Cgrowing burden on society it remains an active field of cancer cell lines but were found to show no clinical benefit in a
variety of indications.5−17 Potent natural products and their
derivatives, despite their promise in vitro, have not been as successful
in the clinic as their potency would suggest.3,18 As a consequence,
dolastatin 10 has been modified to be used as a cytotoxic molecule in
targeted therapies. One targeted therapy approach has used an
auristatin E derivative, monomethyl auristatin E (MMAE), the active
payload in Adcetris.19 Other synthetic dolastatin 10 derivatives,
auristatins, are being investigated as the cytotoxic molecule in
antibody drug conjugate (ADC) clinical trials.18 For this reason
derivatives of dolastatin 10 are still of great interest.
scientific research.1 One area that has been successful in the
advancement of cancer treatment has been the search and
isolation of natural products that have potent cell-based activity.2
Many of these natural products are highly cytotoxic against
in vitro models, but when administered in vivo these natural
products demonstrate systemic toxicity, which limits their use as
single-agent therapeutics.3 One such natural product is dolastatin 10
(D10) isolated from the sea hare Dolabella auricularia from the
Pettit lab (Figure 1).4 Dolastatin 10 and analogues (i.e., TZT-1027)
Dolastatin 10 is a pentapeptide; each amino acid subunit is
sequentially named P1 to P5 starting from the N-terminus
(Figure 1A). Figure 1B illustrates some of the modification of the
dolastatin 10 around the P5 subunit.20,21
The most prevalent areas of modification have centered
around the P1 and P5 subunits and the introduction of additional
groups to extend the pentapeptide motif on either side of the
linear peptide.20 Modifications of the P2−P4 subunits, the core
structure, have been underrepresented, as changes to these
subunits result in attenuated compound potency.21 In particular,
modification of the P2 subunit has received little attention
outside of substituting the natural valine residue with leucine or
isoleucine.22 In this paper we discuss modification of the P2 side
chain that allows the introduction of heteroatoms and other non-
natural amino acids while maintaining potent in vitro activity.
RESULTS AND DISCUSSION
■
In our recent report, we investigated the addition of heterocycles
in the P5 subunit based on the reports from Pettit.23,24 These P5
Received: April 24, 2017
Figure 1. Dolastatin derivatives. Dov, dolavaline; Dil, (3R,4S,5S)-
dolaisoleuine; Dap, (2R,3R,4S)-dolaisoleuine; Doe, (S)-dolaphenine.
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Article
Scheme 1. General Synthesis of Auristatins
a
b
c
d
e
f
g
CMPI, iPrNEt2, EtOAc. Piperidine, MeCN. Dolavaline, HATU, iPrNEt2, DMF. TFA, CH2Cl2. HATU, iPrNEt2, DMF. TFA, CH2Cl2. P4−P5,
HATU, iPrNEt2, DMF.
heterocycle modifications generated more hydrophilic ADCs
that had lower aggregation. As a follow-up to our P5 modifi-
cations we were interested in the introduction of heteroatoms in
the P2 side chain of the dolastatin 10 scaffold. The functional
groups chosen were amines, azides, oxygens, and thiols, which
could expand the utility in the P2 position.
P2 heteroatoms did not result in activity as potent as expected
with the pyridine P5, we decided to explore different P5s with
higher homology to the dolastatin 10 P5. We focused on
L-phenylalanine (Phe) methyl ester based P5s that would allow
the compounds to be more membrane permeable (4−6). These
compounds were more potent than the corresponding pair
(compounds 1−3) with a pyridine P5. Having obtained <1 nM
in vitro potency with a P2-modified auristatin, different ana-
logues were made with the phenylalanine methyl ester P5 to
explore allowable chemical modifications (compounds 7 to 15).
Compounds 7, 9, 12, and 13 all had GI50 values of ≤1.1 nM. Of
note, compound 13 contained a free branched amine in the P2
side chain that at physiological pH would have an additional
positive charge.
Despite this, 13 was still active compared to 8, 10, and 14, which
contain unbranched free amines in the P2 side chain. Compound
12 contained an alkyne in the P2 side chain and demonstrated
similar activity in vitro to the azide-containing compounds.
Compound 11, with a cysteine unit in the P2 position, was not as
active compared to other P2-substituted compounds due to the
presence of disulfide dimers that occur spontaneously upon
standing under conditions of purification and the cytotoxicity
assay. Compound 15, containing a carboxylic acid in the P2
position, was not active against any of the cell lines tested.
Compounds 16 to 19 were synthesized with a phenylalanine in
the P5 position. As anticipated these were less active compared to
their corresponding methyl ester counterparts probably due to
reduced membrane permeability. Despite the poor membrane
permeability, these compounds may still have utility as a targeted
ADC payload because antibodies have the ability to release
the payload inside the cells, such as MMAF (monomethyl
auristatin F)-derived ADCs.
The general synthetic route is shown in Scheme 1, in which the
auristatins were made in two distinct pieces, P1−P2−P3 and
P4−P5. This synthesis was adapted depending on the desired
material, but the convergent approach and conditions shown
were general for all the compounds made. Dil-OtBu (P3) was
coupled with desired Fmoc-protected P2 with CMPI (2-chloro-
1-methylpyridinium iodide) followed by Fmoc deprotection
with piperidine. Dov (P1) was added with HATU (1-[bis(di-
methylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxid hexafluorophosphate), and the tert-butyl ester protecting
group was removed with trifluoroacetic acid (TFA) to yield P1−
P2−P3 carboxylic acid. Dipeptide P4−P5 was coupled using
HATU to form the Boc-Dap-OH (P4) amide bond with the
desired P5. The Boc group was removed with TFA to yield
P4−P5. These two subunits were coupled in a modular fashion to
yield the scaffold of all the compounds made. Compounds were
then converted or deprotected to yield compounds illustrated in
Table 1. For example, an azide in the P2 side chain could be used
as a final auristatin compound or could also be readily converted
to an amine using a Staudinger reduction with PMe3.
Table 1 highlights each compound’s in vitro potency with their
respective P2 and P5 units; the P3 and P4 units remained
unchanged. An in vitro cell proliferation assay was performed
using a mammary gland carcinoma line (HCC1954) and a
human prostate cancer line (PC3). Compounds demonstrated
similar GI50 values (within 1 log) in both HCC1954 and PC-3
cell lines; however, the PC3 cell line was less sensitive overall.
In our previous studies we showed that MMAPYE
(monomethyl auristatin PYE, Table 1) had high in vitro activity
and lower aggregation as an ADC.24 As a starting point for the
structure−activity relationship (SAR) exercise, the PYE P5 was
held constant and the P2 subunit was replaced with L-serine (1),
L-threonine (2), and 3S-azido-2S-aminobutyric acid [Abu(3-
N3)] (3). All three compounds lost potency compared to
MMAPYE (<1 nM), but the azide-containing compound still
had single-digit nanomolar GI50 values. Because the addition of
Having established that the Abu(3-N3) (6) and its corre-
sponding amine (13) demonstrate potent activity in vitro, these
P2 modifications were preserved while the P5 unit was modified.
It has been reported that phenylethylamine derivatives are
usually equipotent with phenylalanine and dolaphenine.21
In contrast, P2-modified auristatins with phenylethylamine
(20), norephedrine (21), 2-chlorophenylethylamine (22), and
4-chlorophenylethylamine (23) present in the P5 position
resulted in a loss of potency compared to phenylalanine-based
P5s (6).
B
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Table 1. Derivatives of P2 Side Chain Modification with GI50 Values and Properties
a
a
compound
R1
R2
R3
GI50 HCC1954 (nM)
GI50 PC3 (nM)
>1000
41.8
1
CH2OH
H
H
H
CH2-2Py
CH2-2Py
CH2-2Py
CH2Ph
>1000
21
2
(R)-CH(OH)CH3
(S)-CH(N3)CH3
CH2OH
3
3.7
5.1
4
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2Me
CO2H
CO2H
CO2H
CO2H
H
0.34
0.16
0.041
0.036
3.5
1.2
5
(R)-CH(OH)CH3
(S)-CH(N3)CH3
CH2N3
CH2Ph
0.38
0.093
0.11
6
CH2Ph
7
CH2Ph
8
CH2NH2
CH2Ph
20
1.1
>1000
9
CH2CH2N3
CH2Ph
0.43
>1000
3.2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
CH2CH2NH2
CH2SH
CH2Ph
CH2Ph
3.8
CH2CCH
CH2Ph
0.16
0.076
3.4 × 102
>1000
>1000
4.4
0.36
0.54
(S)-CH(NH2)CH3
−(CH2)4-NH2
CH2CO2H
CH2Ph
CH2Ph
NC
6.1 × 102
>1000
19
5.0
11
4.7
CH2Ph
(S)-CH(N3)CH3
CH2OH
CH2Ph
CH2Ph
(R)-CH(OH)CH3
(S)−CH(NH2)CH3
(S)-CH(N3)CH3
(S)-CH(N3)CH3
(S)-CH(N3)CH3
(S)-CH(N3)CH3
(S)-CH(N3)CH3
(S)-CH(N3)CH3
(S)-CH(N3)CH3
(S)-CH(N3)CH3
(S)-CH(N3)CH3
CH2Ph
3.5
CH2Ph
2.5
CH2Ph
3.1
Me
(S)-CH(OH)Ph
CH2-Ph(4-Cl)
CH2-Ph(2-Cl)
CH2Ph
34
50
40
34
H
33
H
13
CONH2
CONHtBu
CO2tBu
tetrazole
2-thiazole
0.4
1.7
CH2Ph
0.4
1.2
CH2Ph
0.089
27
0.045
CH2Ph
41
33
CH2Ph
18
a
Results are the average of two independent triplicate runs with compound purity above >90%.
Because the phenylalanine P5 was required for the P2 Abu(3-
N3) compounds to be active, we explored different phenylalanine
derivatives including Phe-carboxamide (24), Phe-tert-butyl
amide (25), Phe-tert-butyl ester (26), and two carboxylic acid
mimics, a triazole (27) and a thiazole (28). All compounds
showed potent activity in vitro except for the carboxylic acid
mimics 27 and 28. This suggests that the presence of an ester or
amide at P5 is important for the activity of these molecules
in vitro. Compounds 27 and 28 do inhibit tubulin polymerization
in a biochemical assay (Supporting Information), suggesting that
other factors for in vitro potency are at play other than simply
tubulin polymerization inhibition. The presence of bigger,
bulkier P5 groups Phe-tert-butyl amide (25) and Phe-tert-butyl
ester (26) gave similar results to the smaller amide and ester Phe-
carboxamide (24) and Phe-methyl ester (6).
To summarize, compounds with a P2-modified side chain can
be active in vitro; however, the modified P2 compounds follow a
different P5 activity trend than valine-based P2 compounds
(Figure 2). The replacement of the thiazole group with a proton
resulted in loss of potency, in contrast with P2 valine auristatins,
which are equipotent.21 The basis of this novel P2-modified
auristatin SAR is currently under investigation but could be due
to many factors such as binding to the tubulin dimer, con-
formation of the pentapeptide, or changes in active transport.25
Research conducted at Pfizer demonstrated that two
Figure 2. SAR conclusion with P2-modified auristatin.
conformations of PF-06380101 are found bound to tubulin,
one productive and one not productive, and could be a cause
of the differences observed.25 Tubulin polymerization assays
have been performed with many of the compounds synthesized,
but due to the high compound concentrations required, in
our screening assay, no major differences were observed to
C
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was dried over a pad of magnesium sulfate, filtered, and concentrated
in vacuo. Fmoc-Ser(Bzl)-Dil-OtBu was isolated by flash chromatog-
raphy on silica gel (40 μm, 60 Å, 3.0 × 17.0 cm) using 18% to 90%
EtOAc in hexanes as the eluent. A total of 3.75 g of Fmoc-Ser(Bzl)-Dil-
OtBu (5.69 mmol, 84%) was obtained.
To a stirred rt solution of Fmoc-Ser(Bzl)-Dil-OtBu (1.79 g,
2.72 mmol) in MeCN (5 mL) was added piperidine (4 mL). After
5 h, analysis by LCMS showed the reaction was complete. The crude
reaction mixture was extracted with hexanes, and the MeCN layer was
concentrated in vacuo to yield crude H-Ser(Bzl)-Dil-OtBu, which was
used without further purification.
To a stirred rt suspension of crude H-Ser(Bzl)-Dil-OtBu and Dov
(0.790 g, 5.44 mmol) in DMF (10 mL) was added DIEA (1.45 mL,
8.16 mmol), followed by HATU (2.07 g, 5.44 mmol). After 5 h, analysis
by LCMS showed that the reaction was complete. The crude reaction
mixture was diluted with saturated sodium bicarbonate (10 mL) and
extracted with EtOAc (40 mL × 2). The combined organic fractions
were washed with brine, dried over a pad of magnesium sulfate, filtered,
and concentrated in vacuo. The crude oil was purified by preparative
RP-HPLC with a Phenomenex Synergi 10 μ Max-RP 80 Å column
(150 × 30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as the
eluent. A total of 0.882 g of Dov-Ser(Bzl)-Dil-OtBu (1.30 mmol, 48%)
was obtained as the TFA salt.
To a rt solution of Dov-Ser(Bzl)-Dil-OtBu (0.288 g, 0.425 mmol) in
CH2Cl2 (5 mL) was added TFA (4 mL). After 10 h, analysis by LCMS
showed the reaction was complete. Volatile organics were evaporated
in vacuo to yield crude Dov-Ser(Bzl)-Dil-OH TFA salt, which was used
without further purification.
To a stirred rt suspension of crude Dov-Ser(Bzl)-Dil-OH TFA salt
and H-Dap-Phe-OMe TFA salt (0.163 g, 0.467 mmol) in DMF (10 mL)
was added DIEA (0.303 mL, 1.70 mmol), followed by HATU (0.323 g,
0.850 mmol). After 6 h, analysis by LCMS showed the reaction was
complete. The crude reaction mixture was diluted with saturated sodium
bicarbonate (10 mL) and extracted with EtOAc (40 mL × 2). The
combined organic fractions were washed with brine, dried over a pad of
magnesium sulfate, filtered, and concentrated in vacuo. The crude oil
was purified by preparative RP-HPLC with a Phenomenex Synergi 10 μ
Max-RP 80 Å column (150 × 30 mm) using 10% to 90% MeCN in
0.05% aqueous TFA as the eluent. A total of 0.217 g of Dov-Ser(Bzl)-
Dil-Dap-Phe-OMe (0.228 mmol, 54%) was obtained as the TFA salt.
A stirred rt suspension of Dov-Ser(Bzl)-Dil-Dap-Phe-OMe TFA salt
(0.217 g, 0.228 mmol) and palladium on activated charcoal (10% Pd
basis, 0.174 g) in MeOH (5 mL) was hydrogenated under refluxing
conditions. After 48 h, analysis by LCMS showed the reaction
was complete. The crude reaction mixture was filtered over a pad of
diatomaceous earth, and the filtrate was concentrated. The crude oil was
purified by preparative RP-HPLC with a Phenomenex Synergi 10 μ
Max-RP 80 Å column (150 × 30 mm) using 10% to 90% MeCN in
0.05% aqueous TFA as the eluent. A total of 0.104 g of the title
compound was obtained as the TFA salt (0.121 mmol, 47%) as a white,
amorphous solid: LCMS tR = 2.32 min (method A); ESIMS m/z 748.72
[M + H]+; HRESIMS m/z 748.4846 [M + H]+ (calcd for C39H66N5O9,
748.4855).
Compound 5. To a stirred rt solution of Fmoc-Thr(Bzl)-OH
(2.92 g, 6.76 mmol) and H-Dil-OtBu hydrochloride (2.00 g, 6.76 mmol)
in EtOAc (15 mL) was added DIEA (2.17 mL, 12.2 mmol). The solution
was cooled (0 °C) and stirred for 20 min. DIEA (2.17 mL, 12.2 mmol)
was added to the reaction mixture. The solution was cooled (0 °C) and
stirred for 20 min. CMPI (2.76 g, 10.8 mmol) was added to the reaction
mixture, which was allowed to reach rt. After 12 h, analysis by LCMS
showed the reaction was complete. The crude reaction was washed with
0.1 M HCl (150 mL × 2). The organic fraction was dried over a pad of
magnesium sulfate, filtered, and concentrated in vacuo. Fmoc-Thr(Bzl)-
Dil-OtBu was isolated by flash chromatography on silica gel (40 μm,
60 Å, 3.0 × 17.0 cm) using 18% to 90% EtOAc in hexanes as the eluent.
A total of 3.71 g of Fmoc-Thr(Bzl)-Dil-OtBu (5.51 mmol, 82%) was
obtained.
explain the differences in activity seen in vitro (Supporting
Information).
CONCLUSION
■
We have developed novel auristatins that have a P2 side chain
modification. These auristatins demonstrate activity in vitro
comparable to other compounds in this class, and they are
efficacious as a drug in ADCs. We determined that modification
of the P2 side chain required a specific aromatic P5 subunit (ester
or amide), and these P2 modifications did not follow previously
reported SAR trends for P2 valine-based auristatins.21 It is also
important to note when modifying the auristatin core structure
that different peripheral groups (i.e., P5) are necessary to deter-
mine if the changes were beneficial or not.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Reactions were carried out at
ambient temperature with exposure to air, unless otherwise noted. All
reagents and solvents were purchased from commercial sources and
used as received. NMR spectra were obtained on a Bruker AV 500 MHz
spectrometer at 25 °C. The NMR spectra were obtained from DMSO-d6
and were referenced to the residual solvent peak (1H: 2.50 ppm; 13C:
39.5 ppm). High-resolution mass spectrometry samples were injected
without column onto a Dionex 3000-Orbitrap Velos LC-MS. Flash
chromatography was carried out on a Yamazen purification system using
prepacked Yamazen Universal columns. Preparative HPLC was carried
out using a Phenomenex Gemini-NX 10 μm, C18 110 Å column (150 ×
30 mm) using a 5% to 100% gradient of MeCN/0.05% aqueous TFA
mixture over 13 min unless another column or solvent system is noted.
Drug compounds purified by preparative HPLC were assumed to be
salts containing one molecule of TFA or formic acid (FA).
Liquid chromatography mass spectrometry (LCMS) retention times
were acquired on an Acquity UPLC BEH C8 1.7 μm 2.1 × 50 mm
column, 40 °C. Method A: 0−0.5 min: isocratic 80:10:10 H2O/
MeCN/1% FA in H2O; 0.5−3.5 min: 80:10:10 H2O/MeCN/1% FA in
H2O to 90:10 MeCN/1% FA; 3.50−3.99 min: isocratic 90:10 MeCN/
1% FA in H2O; 3.99−4.00 min: linear gradient 90:10 MeCN/1% FA in
H2O to 80:10:10 H2O/MeCN/1% FA in H2O. Method B: 0−0.5 min:
isocratic 85:5:10 H2O/MeCN/1% FA in H2O; 0.5−1.6 min: linear
gradient 85:5:10 H2O/MeCN/1% FA in H2O to 98:2 MeCN/1% FA in
H2O; 1.60−1.9 min: linear gradient 98:2 MeCN/1% FA in H2O to
85:5:10 H2O/MeCN/1% FA in H2O; 1.9−2.0 min: isocratic 85:5:10
H2O/MeCN/1% FA in H2O.
Compound 4. To a stirred room-temperature (rt) suspension of
Boc-Dap-OH dicyclohexylamine salt (8.00 g, 17.1 mmol) and H-Phe-
OMe HCl salt (4.42 g, 20.5 mmol) in CH2Cl2 (20 mL) was added
diisopropylethylamine (DIEA; 9.13 mL, 51.3 mmol), followed by
diethylpyrocarbonate (DEPC; 5.15 mL, 34.2 mmol). After 10 h, analysis
by LCMS showed the reaction was complete. Boc-Dap-Phe-OMe was
isolated by flash chromatography on silica gel (40 μm, 60 Å, 3.0 ×
17.0 cm) using 2% to 10% MeOH in CH2Cl2 as the eluent. A total of
7.45 g of Boc-Dap-Phe-OMe (16.61 mmol, 97%) was obtained.
To a stirred rt solution of Boc-Dap-Phe-OMe (4.67 g, 10.4 mmol) in
CH2Cl2 (10 mL) was added TFA (10 mL). After 10 h, analysis by LCMS
showed the reaction was complete. The crude oil was purified by
preparative RP-HPLC with a Phenomenex Synergi 10 μ Max-RP 80 Å
column (150 × 30 mm) using 10% to 90% MeCN in 0.05% aqueous
TFA as the eluent. A total of 2.52 g of H-Dap-Phe-OMe (6.23 mmol,
59%) was obtained as the TFA salt.
To a stirred rt solution of Fmoc-Ser(Bzl)-OH (2.82 g, 6.76 mmol)
and H-Dil-OtBu hydrochloride (2.00 g, 6.76 mmol) in EtOAc (15 mL)
was added DIEA (2.17 mL, 12.2 mmol). The solution was cooled to
0 °C and stirred for 20 min. DIEA (2.17 mL, 12.2 mmol) was added to
the reaction mixture. The solution was cooled to 0 °C and stirred for
20 min. 2-Chloro-1-methylpyridinium iodide (2.76 g, 10.8 mmol) was
added to the reaction mixture, which was allowed to reach rt. After 12 h,
analysis by LCMS showed the reaction was complete. The crude
reaction was washed with 0.1 M HCl (150 mL × 2). The organic fraction
To a stirred rt solution of Fmoc-Thr(Bzl)-Dil-OtBu (3.71 g,
5.51 mmol) in MeCN (5 mL) was added piperidine (4 mL). After
5 h, analysis by LCMS showed the reaction was complete. The crude
D
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reaction mixture was extracted with hexanes, and the MeCN layer was
concentrated in vacuo to yield crude H-Thr(Bzl)-Dil-OtBu, which was
used without further purification.
The organic fraction was dried over a pad of magnesium sulfate, filtered,
and concentrated in vacuo to yield crude Fmoc-Abu(3-N3)-Dil-OtBu
(1.12 g, 1.84 mmol), which was used without further purification.
To a stirred rt solution of Fmoc-Abu(3-N3)-Dil-OtBu (1.00 g,
1.65 mmol) in MeCN (10 mL) was added piperidine (2 mL). After 5 h,
analysis by LCMS showed the reaction was complete. The crude
reaction mixture was extracted with hexanes, and the MeCN layer was
concentrated in vacuo to yield crude H-Abu(3-N3)-Dil-OtBu, which was
used without further purification.
To a stirred rt suspension of crude H-Abu(3-N3)-Dil-OtBu and Dov
(0.478 g, 3.29 mmol) in DMF (10 mL) was added DIEA (0.880 mL,
4.94 mmol), followed by HATU (1.25 g, 3.29 mmol). After 6 h, analysis
by LCMS showed that the reaction was complete. The crude reaction
mixture was diluted with saturated sodium bicarbonate (10 mL) and
extracted with EtOAc (40 mL × 2). The combined organic fractions
were washed with brine, dried over a pad of magnesium sulfate, filtered,
and concentrated in vacuo. The crude oil was purified by preparative
RP-HPLC with a Phenomenex Synergi 10 μ Max-RP 80 Å column
(150 × 30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as the
eluent. A total of 0.670 g of Dov-Abu(3-N3)-Dil-OtBu (1.07 mmol,
65%) was obtained as the TFA salt.
To a stirred rt suspension of crude H-Thr(Bzl)-Dil-OtBu and Dov
(1.60 g, 11.0 mmol) in DMF (20 mL) was added DIEA (2.95 mL,
16.5 mmol), followed by HATU (4.19 g, 11.0 mmol). After 6 h, analysis
by LCMS showed that the reaction was complete. The crude reaction
mixture was diluted with saturated sodium bicarbonate (10 mL) and
extracted with EtOAc (40 mL × 2). The combined organic fractions
were washed with brine, dried over a pad of magnesium sulfate, filtered,
and concentrated in vacuo. The crude oil was purified by preparative
RP-HPLC with a Phenomenex Synergi 10 μ Max-RP 80 Å column
(150 × 30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as the
eluent. A total of 1.09 g of Dov-Thr(Bzl)-Dil-OtBu (1.58 mmol, 29%)
was obtained as the TFA salt.
To a rt solution of Dov-Thr(Bzl)-Dil-OtBu TFA salt (0.331 g,
0.478 mmol) in CH2Cl2 (5 mL) was added TFA (4 mL). After 10 h,
analysis by LCMS showed the reaction was complete. Volatile organics
were evaporated in vacuo to yield crude Dov-Thr(Bzl)-Dil-OH TFA
salt, which was used without further purification.
To a stirred rt suspension of crude Dov-Thr(Bzl)-Dil-OH TFA salt
and H-Dap-Phe-OMe TFA salt (0.183 g, 0.526 mmol) in DMF (10 mL)
was added DIEA (0.341 mL, 1.92 mmol), followed by HATU (0.364 g,
0.957 mmol). After 24 h, analysis by LCMS showed the reaction was
complete. The crude reaction mixture was diluted with saturated sodium
bicarbonate (10 mL) and extracted with EtOAc (40 mL × 2). The
combined organic fractions were washed with brine, dried over a pad of
magnesium sulfate, filtered, and concentrated in vacuo. The crude oil
was purified by preparative RP-HPLC with a Phenomenex Synergi 10 μ
Max-RP 80 Å column (150 × 30 mm) using 10% to 90% MeCN in
0.05% aqueous TFA as the eluent. A total of 0.290 g of Dov-Thr(Bzl)-
Dil-Dap-Phe-OMe (0.300 mmol, 63%) was obtained as the TFA salt.
A stirred rt suspension of Dov-Thr(Bzl)-Dil-Dap-Phe-OMe TFA salt
(0.290 g, 0.300 mmol) and palladium on activated charcoal (10% Pd
basis, 0.232 g) in MeOH (5 mL) was hydrogenated. After 24 h, analysis
by LCMS showed the reaction was complete. The crude reaction
mixture was filtered over a pad of diatomaceous earth, and the filtrate
was concentrated. The crude oil was purified by preparative RP-HPLC
with a Phenomenex Synergi 10 μ Max-RP 80 Å column (150 × 30 mm)
using 10% to 90% MeCN in 0.05% aqueous TFA as the eluent. A total
of 0.129 g of the title compound was obtained as the TFA salt
(0.147 mmol, 43%) as a white, amorphous solid. 1H NMR (500 MHz,
DMSO-d6, a complex spectrum was observed, presumably due to
cis/trans conformational isomers) δ [9.04 (d, J = 7.8 Hz), 9.02 (d, J =
7.8 Hz) 1H], [8.50 (d, J = 8.4 Hz), 8.26 (d, J = 8.0 Hz) 1H], 7.27−7.11
(m, 5H), [4.74 (t, J = 8.2 Hz), 4.68 (t, J = 8.2 Hz) 1H], [4.66−4.57 (m),
4.48−4.43 (m) 2H], 3.98−3.92 (m, 1H), 3.87−3.78 (m, 1H), [3.76−
3.66 (m), 3.56−3.42 (m) 3H], [3.65 (s), 3.61 (s) 3H], 3.30−3.11 (m,
8H), 3.10−2.98 (m, 1H), [3.06 (s), 2.99 (s) 3H], 2.89−2.71 (m, 7H),
2.46−2.13 (m, 4H), 1.83−1.58 (m, 3H), 1.49−1.35 (m, 2H), 1.34−1.23
(m, 1H), [1.20 (d, J = 6.2 Hz), 1.11 (d, J = 6.2 Hz) 3H], [1.04 (d, J =
6.7 Hz), 1.01 (d, J = 6.7 Hz) 3H], 0.98−0.78 (m, 10H), 0.74 (td, J = 7.4,
2.5 Hz, 3H); 13C NMR (126 MHz, DMSO-d6) δ 174.06, 173.92, 172.65,
172.48, 171.52, 169.27, 169.13, 165.61, 165.59, 137.80, 137.61, 129.39,
129.17, 128.58, 126.88, 126.80, 85.67, 81.79, 78.07, 77.79, 71.77, 71.72,
67.53, 67.49, 61.39, 60.66, 58.96, 58.62, 57.58, 57.52, 56.11, 56.05, 53.52,
52.53, 52.33, 47.55, 46.62, 43.58, 43.35, 42.08, 42.04, 41.50, 37.48, 36.78,
36.68, 35.56, 32.38, 32.11, 31.83, 26.92, 25.78, 25.70, 24.75, 24.71, 23.56,
20.16, 19.93, 19.39, 16.88, 15.95, 15.70, 15.66, 15.28, 10.84, 10.74;
LCMS tR = 2.40 min (method A); ESIMS m/z 762.75 [M + H]+;
HRESIMS m/z 762.5009 [M + H]+ (calcd for C40H68N5O9, 762.5012).
Compound 6. To a stirred rt solution of (2S,3S)-Fmoc-Abu(3-N3)-
OH (1.00 g, 2.74 mmol) and H-Dil-OtBu hydrochloride (0.810 g,
2.74 mmol) in EtOAc (10 mL) was added DIEA (0.880 mL,
4.93 mmol). The solution was cooled (0 °C) and stirred for 20 min.
Additional DIEA (0.880 mL, 4.93 mmol) was added to the reaction
mixture and stirred for 20 min. CMPI (1.12 g, 4.38 mmol) was added to
the reaction mixture, which was allowed to reach rt. After 12 h, analysis
by LCMS showed the reaction was complete. The crude reaction was
washed with 0.1 M HCl (100 mL × 2), followed by brine (20 mL × 2).
To a rt solution of Dov-Abu(3-N3)-Dil-OtBu TFA salt (0.289 g,
0.425 mmol) in CH2Cl2 (5 mL) was added TFA (5 mL). After 12 h,
analysis by LCMS showed the reaction was complete. Volatile organics
were evaporated in vacuo to yield crude Dov-Abu(3-N3)-Dil-OH TFA
salt, which was used without further purification.
To a stirred rt suspension of crude Dov-Abu(3-N3)-Dil-OH TFA salt
(1.04 g, 1.82 mmol) and H-Dap-Phe-OMe TFA salt (1.59 g, 3.44 mmol)
in DMF (10 mL) was added DIEA (1.18 g, 1.60 mL, 9.11 mmol),
followed by the addition of HATU (1.74 g, 4.56 mmol). After 10 h,
analysis by LCMS showed the reaction was complete. The crude
reaction mixture was diluted with saturated sodium bicarbonate
(10 mL) and extracted with EtOAc (20 mL × 3). The combined
organic fractions were washed with brine, dried over a pad of magnesium
sulfate, filtered, and concentrated in vacuo. The crude oil was purified by
preparative RP-HPLC with a Phenomenex Gemini NX-C18 10 μ 110 Å
column (150 × 30 mm) using 10% to 90% MeCN in 0.1% aqueous
formic acid. A total of 935 mg of the title compound was obtained as the
formic acid salt (1.12 mmol, 49%) as a white, amorphous solid. 1H NMR
(500 MHz, DMSO-d6, a complex spectrum was observed, presumably
due to cis/trans conformational isomers) δ 9.18 (br s, 1H), [8.65−8.42
(m) 8.29 (dd, J = 8.1, 4.3 Hz) 1H], 7.20 (m, 5H), [5.05 (q, J = 7.0 Hz),
4.87 (dd, J = 9.4, 6.0 Hz) 1H], [4.71−4.63 (m), 4.56−4.43 (m), 2H],
4.10−3.93 (m, 2H), 3.76−3.70 (m, 2H), [3.66 (d, J = 4.0 Hz), 3.63 (d,
J = 4.0 Hz), 3H], 3.59−3.24 (m, 3H), 3.24−3.12 (m, 6H), [3.07 (s), 2.98
(s) 3H], 2.93−2.83 (m, 2H), [2.80 (s), 2.78 (s), 6H], 2.46−2.18 (m,
3H), 1.87−1.32 (m, 7H), 1.27 (t, J = 7.2 Hz, 3H), 1.08−0.89 (m, 10H),
0.88−0.82 (m, 3H), 0.78 (q, J = 6.5, 5.9 Hz, 3H); 13C NMR (126 MHz,
DMSO) δ 173.84, 173.53, 173.32, 173.11, 172.10, 171.96, 171.87,
170.57, 168.95, 168.56, 137.23, 137.04, 128.85, 128.82, 128.54, 128.02,
128.00, 126.30, 126.21, 85.23, 82.17, 81.28, 79.15, 76.93, 68.60, 61.02,
60.83, 60.31, 60.15, 58.37, 58.14, 57.82, 57.45, 57.27, 57.18, 53.32, 52.91,
52.00, 51.92, 51.81, 51.78, 46.92, 46.01, 43.00, 42.81, 42.74, 41.13, 37.18,
36.11, 34.09, 32.66, 30.61, 25.31, 25.25, 25.12, 24.20, 24.14, 23.05, 18.93,
18.60, 18.57, 17.23, 17.01, 15.71, 15.65, 15.32, 15.21, 15.12, 15.04, 14.72,
10.88, 10.65, 10.17; LCMS tR = 1.09 min (method B); ESIMS m/z
787.53 [M + H]+; HRESIMS m/z 787.5072 [M + H]+ (calcd for
C40H67N8O8, 787.5076).
Compound 7. To a stirred rt solution of Boc-β-azido-alanine
dicyclohexylamine salt (1.02 g, 2.47 mmol) and H-Dil-OtBu hydro-
chloride (0.730 g, 2.74 mmol) in EtOAc (10 mL) was added DIEA
(0.792 mL, 4.44 mmol). The solution was cooled (0 °C) and stirred for
20 min. Additional DIEA (0.792 mL, 4.44 mmol) was added to the
reaction mixture and stirred for 20 min. CMPI (1.01 g, 3.95 mmol) was
added to the reaction mixture, which was allowed to reach rt. After 12 h,
analysis by LCMS showed the reaction was complete. The crude reaction
was washed with 0.1 M HCl (100 mL × 2), followed by brine (20 mL × 2).
The organic fraction was dried over a pad of magnesium sulfate, filtered,
and concentrated in vacuo to yield crude Boc-β-azido-Ala-Dil-OtBu
(1.08 g, 2.29 mmol), which was used without further purification.
E
DOI: 10.1021/acs.jnatprod.7b00359
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Journal of Natural Products
Article
To a rt solution of Boc-β-azido-Ala-Dil-OtBu (1.08 g, 2.29 mmol) in
CH2Cl2 (5 mL) was added TFA (3 mL). After 10 h, analysis by LCMS
showed the reaction was complete. Volatile organics were evaporated
in vacuo to yield crude H-β-azido-Ala-Dil-OH TFA salt that was used
without further purification.
To a stirred rt suspension of Dov (0.739 g, 5.09 mmol) in DMF
(10 mL) was added DIEA (1.36 mL, 7.63 mmol), followed by HATU
(1.95 g, 5.09 mmol). After 10 min crude H-β-azido-Ala-Dil-OH was
added to the reaction mixture. After 6 h, analysis by LCMS showed that
the reaction was complete. The crude oil was purified by preparative
RP-HPLC with a Phenomenex Synergi 10 μ Max-RP 80 Å column
(150 × 30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as the
eluent. A total of 0.130 g of Dov-β-azido-Ala-Dil-OH (0.261 mmol,
10%) was obtained as the TFA salt.
To a stirred rt suspension of Dov-β-azido-Ala-Dil-OH TFA salt
(0.130 g, 0.261 mmol) and H-Dap-Phe-OMe TFA salt (0.205 g,
0.588 mmol) in DMF (10 mL) was added DIEA (0.140 mL,
0.783 mmol), followed by HATU (0.198 g, 0.522 mmol). After 6 h,
analysis by LCMS showed the reaction was complete. The crude reac-
tion mixture was diluted with saturated sodium bicarbonate (10 mL)
and extracted with EtOAc (40 mL × 2). The combined organic fractions
were washed with brine, dried over a pad of magnesium sulfate, filtered,
and concentrated in vacuo. The crude oil was purified by preparative
RP-HPLC with a Phenomenex Synergi 10 μ Max-RP 80 Å column
(150 × 30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as the
eluent. A total of 0.015 g of the title compound was obtained as the TFA
salt (0.017 mmol, 7%) as a white, amorphous solid: LCMS tR = 2.36 min
(method A); ESIMS m/z 773.48 [M + H]+; HRESIMS m/z 773.4916
(calcd for C39H65N8O8, 773.4920).
Compound 9. To a stirred rt solution of Boc-4-azido-homoalanine
dicyclohexylamine salt (1.01 g, 2.37 mmol) and H-Dil-OtBu hydro-
chloride (0.700 g, 2.37 mmol) in EtOAc (10 mL) was added DIEA
(0.759 mL, 4.26 mmol). The solution was cooled (0 °C) and stirred for
20 min. Additional DIEA (0.759 mL, 4.26 mmol) was added to the
reaction mixture and stirred for 20 min. CMPI (0.967 g, 3.79 mmol) was
added to the reaction mixture, which was allowed to reach rt. After 12 h,
analysis by LCMS showed the reaction was complete. The crude
reaction was washed with 0.1 M HCl (100 mL × 2), followed by brine
(20 mL × 2). The organic fraction was dried over a pad of magnesium
sulfate, filtered, and concentrated in vacuo to yield crude Boc-4-azido-
homoAla-Dil-OtBu (1.09 g, 2.25 mmol), which was used without further
purification.
To a rt solution of Boc-4-azido-homoAla-Dil-OtBu (1.09 g,
2.25 mmol) in CH2Cl2 (5 mL) was added TFA (3 mL). After 10 h,
analysis by LCMS showed the reaction was complete. Volatile organics
were evaporated in vacuo to yield crude H-4-azido-homoAla-Dil-OH
TFA salt, which was used without further purification.
To a stirred rt suspension of Dov (0.652 g, 4.49 mmol) in DMF
(10 mL) was added DIEA (1.20 mL, 6.73 mmol), followed by HATU
(1.71 g, 4.49 mmol). After 10 min crude H-4-azido-homoAla-Dil-OH
TFA salt was added to the reaction mixture. After 6 h, analysis by LCMS
showed that the reaction was complete. The crude oil was purified by
preparative RP-HPLC with a Phenomenex Synergi 10 μ Max-RP 80 Å
column (150 × 30 mm) using 10% to 90% MeCN in 0.05% aqueous
TFA as the eluent. A total of 0.127 g of Dov-4-azido-homoAla-Dil-OH
(0.223 mmol, 10%) was obtained as the TFA salt.
To a stirred rt suspension of Dov-4-azido-homoAla-Dil-OH TFA salt
(0.127 g, 0.223 mmol) and H-Dap-Phe-OMe TFA salt (0.198 g,
0.568 mmol) in DMF (10 mL) was added DIEA (0.140 mL,
0.783 mmol), followed by HATU (0.198 g, 0.522 mmol). After 6 h,
analysis by LCMS showed the reaction was complete. The crude reac-
tion mixture was diluted with saturated sodium bicarbonate (10 mL)
and extracted with EtOAc (40 mL × 2). The combined organic fractions
were washed with brine, dried over a pad of magnesium sulfate, filtered,
and concentrated in vacuo. The crude oil was purified by preparative
RP-HPLC with a Phenomenex Synergi 10 μ Max-RP 80 Å column
(150 × 30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as the
eluent. A total of 0.015 g of the title compound was obtained as the TFA
salt (0.017 mmol, 7%) as a white, amorphous solid: LCMS tR = 2.38 min
(method A); ESIMS m/z 787.49 [M + H]+; HRESIMS m/z 787.5078
[M + H]+ (calcd for C40H67N8O8, 787.5076).
Compound 12. To a stirred 25 °C solution of Boc-propargylGly-
OH (1.00 g, 4.69 mmol) and H-Dil-OtBu HCl salt (1.15 g, 3.90 mmol)
in EtOAc (10 mL) was added DIEA (1.49 mL, 9.38 mmol). The solution
was cooled (0 °C) and stirred for 20 min. Additional DIEA (1.49 mL,
9.38 mmol) was added to the reaction mixture and stirred for 20 min.
CMPI (1.80 g, 7.04 mmol) was added to the reaction mixture, which was
allowed to reach rt. After 12 h, analysis by LCMS showed the reaction
was complete. The crude reaction was washed with 0.1 M HCl (20 mL × 2),
followed by brine (20 mL × 2). The organic fraction was dried using
magnesium sulfate, filtered, and concentrated in vacuo to give crude
product. A total of 2.05 g of Boc-propargylGly-Dil-OtBu was obtained
(4.50 mmol, 96%): LCMS tR = 3.46 min (method A); ESIMS m/z
455.42 [M + H]+.
To a stirred 25 °C solution of Boc-propargylGly-Dil-OtBu (2.05 g,
4.50 mmol) in CH2Cl2 (6 mL) was added TFA (6 mL). After 14 h,
analysis by LCMS showed the reaction was complete. The volatile
organics were concentrated in vacuo to give a crude product that was
used without further purification. A total of 1.30 g of H-propargylGly-
Dil-OH was obtained as the TFA salt (3.16 mmol, 70%).
To a stirred 25 °C solution of Dov (1.27 g, 8.71 mmol) in DMF
(10 mL) was added DIEA (2.33 mL, 13.1 mmol), followed by HATU
(3.32 g, 8.71 mmol). After 10 min, H-propargylGly-Dil-OH TFA salt
(1.30 g, 3.16 mmol) in a DMF solution was added to the reaction. After
8 h, analysis by LCMS showed the reaction was complete. The crude oil
was purified by preparative RP-HPLC with a Phenomenex Synergi 10 μ
Max-RP 80 Å column (150 × 30 mm) using 10% to 90% MeCN in
0.05% aqueous TFA as the eluent. A total of 455 mg of Dov-
propargyGly-Dil-OH was obtained as the TFA salt (0.844 mmol, 27%)
as a white, amorphous solid: LCMS tR = 1.65 min (method A); ESIMS
m/z 425.95 [M + H]+.
To a stirred rt solution of Dov-propargylGly-Dil-OH TFA salt
(0.198 g, 0.367 mmol) and H-Dap-Phe-OMe TFA salt (0.156 g, 0.338
mmol) in DMF (10 mL) was added DIEA (0.222 mL, 1.40 mmol),
followed by the addition of HATU (0.355 g, 0.931 mmol). After 10 h,
analysis by LCMS showed the reaction was complete. The crude
reaction was diluted with saturated sodium bicarbonate (10 mL) and
extracted with EtOAc (20 mL × 2). The combined organic fractions
were washed with brine, dried using magnesium sulfate, filtered, and
concentrated in vacuo. The crude oil was purified by preparative
RP-HPLC with a Phenomenex Synergi 10 μ Max-RP 80 Å column
(150 × 30 mm) using 10% to 90% MeCN in 0.05% aqueous TFA as the
eluent. A total of 10 mg of the title compound was obtained as the TFA
salt (0.012 mmol, 3%) as a white, amorphous solid: LCMS tR = 2.50 min
(method A); ESIMS m/z 756.44 [M + H]+; HRESIMS m/z 756.4902
[M + H]+ (calcd for C41H66N5O8, 756.4906).
Compound 13. To a stirred rt solution of Dov-Abu(3-N3)-Dil-Dap-
Phe-OMe TFA salt (10 mg, 0.011 mmol) in THF (0.10 mL) was added
trimethyl phosphine in THF (1 M, 0.022 mL, 0.022 mmol). After 4 h,
analysis by LCMS showed the reaction was complete and H2O
(0.05 mL) was added to the reaction mixture. The crude reaction
mixture was purified by preparative RP-HPLC with a Phenomenex
Synergi 10 μ Max-RP 80 Å column (150 × 30 mm) using 10% to 90%
MeCN in 0.05% aqueous TFA as the eluent. A total of 6.00 mg of the
title compound was obtained as the TFA salt (0.007 mmol, 62%) as a
white, amorphous solid: LCMS tR = 2.12 min (method A); ESIMS m/z
761.63 [M + H]+; HRESIMS m/z 761.5159 [M + H]+ (calcd for
C40H69N6O8, 761.5171).
Compound 24. To a stirred rt solution of Dov-Abu(3-N3)-Dil-Dap-
Phe-OH formic acid salt (42.1 mg, 0.051 mmol), ammonium chloride
(7.9 mg, 0.148 mmol), and TBTU (52.5 mg, 0.163 mmol) in DMF
(0.2 mL) was added DIEA (0.045 mL, 0.258 mmol). After 16 h, analysis
by LCMS showed the reaction was complete. The crude reaction
mixture was purified by preparative RP-HPLC with a Phenomenex
Gemini-NX 10 μ C-18 110 Å column (150 × 30 mm) using 5% to 95%
MeCN in 0.1% aqueous ammonium hydroxide as the eluent. A total of
13.4 mg of the title compound was obtained (0.017 mmol, 33%) as a
white, amorphous solid: 1H NMR (500 MHz, DMSO-d6) δ 9.17 (br s,
1H), [8.35−8.23 (m), 8.05−7.95 (m), 1H], [7.45−7.40 (m), 7.40−7.27
F
DOI: 10.1021/acs.jnatprod.7b00359
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(m), 1H], 7.25−7.17 (m,, 4H), 7.13 (t, J = 6.3 Hz, 1H), 7.04 (br s, 1H),
[5.05−4.97 (m), 4.87 (t, J = 8.4 Hz), 1H], [4.68−4.51 (m), 4.50−4.40
(m), 2H], 4.05−3.92 (m, 2H), 3.87−3.72 (m, 2H), 3.55−3.38 (m, 2H),
3.31−3.13 (m, 7H), 3.11−2.96 (m, 3H), 2.81−2.70 (m, 8H), 2.44 (d, J =
16.1 Hz, 1H), 2.35−2.17 (m, 2H), 1.88−1.60 (m, 1H), 1.47−1.40 (m,
1H), 1.37−1.29 (m, 3H), 1.28 (dd, J = 9.6, 6.5 Hz, 3H), 1.08−0.82 (m,
15H), 0.78 (t, J = 7.2 Hz, 3H); 13C NMR (126 MHz, DMSO) δ 173.35,
173.23, 173.05, 169.89, 169.50, 168.64, 168.58, 165.45, 138.05, 137.88,
128.88, 128.64, 127.91, 127.83, 127.77, 126.00, 125.87, 85.21, 81.31,
77.04, 71.11, 60.75, 60.11, 58.43, 58.02, 57.70, 57.48, 57.23, 57.16, 56.75,
53.46, 52.51, 52.14, 52.03, 46.93, 46.02, 43.44, 42.86, 41.09, 37.37, 37.24,
37.08, 35.25, 32.40, 31.49, 31.15, 26.48, 25.28, 25.13, 24.21, 24.05, 23.01,
18.98, 16.52, 15.64, 15.48, 15.29, 15.16, 14.60, 14.29, 10.54, 10.19;
LCMS tR = 1.05 min (method B); ESIMS m/z 772.61 [M + H]+;
HRESIMS m/z 772.5078 [M + H]+ (calcd for C39H66N9O7, 772.5080).
Compound 25. To a stirred rt solution of Dov-Abu(3-N3)-Dil-Dap-
Phe-OH formic acid salt (24.0 mg, 0.029 mmol), tert-butyl amine hydro-
chloride (7.9 mg, 0.072 mmol), and HATU (24.5 mg, 0.064 mmol) in
DMF (0.2 mL) was added DIEA (0.022 mL, 0.124 mmol). After 18 h,
analysis by LCMS showed the reaction was complete. The crude
reaction mixture was purified by preparative RP-HPLC with a
Phenomenex Gemini-NX 10 μ c-18 110 Å column (150 × 30 mm)
using 5% to 95% MeCN in 0.1% aqueous ammonium hydroxide
as the eluent. A total of 15.4 mg of the title compound was obtained
(0.017 mmol, 59%) as a white, amorphous solid: 1H NMR (500 MHz,
DMSO-d6, a complex spectrum was observed, presumably due to
cis/trans conformational isomers) δ 8.50 (br s, 1H), [8.34 (d, J = 8.3 Hz),
8.13 (d, J = 8.3 Hz) 1H], 7.27−7.10 (m, 5H), [4.97−4.91 (m), 4.76 (t,
J = 8.9 Hz) 1H], [4.67−4.48 (m), 4.33 (ddd, J = 10.2, 7.9, 5.2 Hz) 2H],
4.02−3.74 (m, 4H), 3.58−3.48 (m, 1H), 3.43−3.22 (m, 9H), 3.10−2.78
(m, 2H), [3.05 (s), 2.94 (s) 3H], 2.43−2.18 (m, 9H), 2.05−1.89 (m,
1H), 1.85−1.60 (m, 3H), 1.52−1.21 (m, 5H), [1.37 (s), 1.34 (s) 9H],
1.18−0.95 (m, 1H), [1.06 (d, J = 6.8 Hz), 1.02 (d, J = 6.8 Hz) 3H],
0.93−0.82 (m, 7H), 0.78−0.69 (m, 6H); 13C NMR (126 MHz,
DMSO-d6) δ 173.89, 173.68, 171.23, 171.17, 169.53, 169.14, 137.95,
137.68, 129.42, 129.13, 128.53, 128.51, 126.77, 126.70, 85.77, 81.85,
81.20, 81.01, 78.15, 77.46, 72.97, 61.38, 60.74, 59.01, 58.80, 58.76, 58.49,
57.84, 57.79, 54.09, 52.78, 52.23, 52.17, 47.49, 46.58, 43.61, 43.34, 41.76,
37.84, 37.00, 36.85, 35.75, 33.34, 32.20, 31.77, 28.02, 27.18, 25.80, 25.70,
24.79, 24.72, 23.61, 19.95, 16.65, 16.24, 15.92, 15.41, 11.32, 10.79;
LCMS tR = 1.27 min (method B); ESIMS m/z 828.8 [M + H]+;
HRESIMS m/z 828.5671 [M + H]+ (calcd for C43H74N9O7, 828.5706).
Compound 26. To a stirred 25 °C solution of Boc-Dap-OH
dicyclohexylamine salt (6.47 g, 13.8 mmol) and H-Phe-OtBu (3.91 g,
15.2 mmol) in CH2Cl2 (20 mL) was added DIEA (8.76 mL, 55.2 mmol),
followed by the addition of DEPC (3.12 mL, 20.7 mmol). After 8 h,
analysis by LCMS showed the reaction was complete. The volatile
organics were evaporated in vacuo to give a crude product that was used
without further purification. A total of 5.35 g of Boc-Dap-Phe-OtBu was
obtained (10.9 mmol, 79%): LCMS tR = 2.89 min (method A); ESIMS
m/z 491.48 [M + H]+.
1.42 min (method B); ESIMS m/z 828.94 [M + H]+; HRESIMS m/z
829.5536 [M + H]+ (calcd for C43H73N8O8, 829.5546).
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.jnatprod.7b00359.
1
Detailed experimental procedures, H and 13C NMR
spectra for 3, 5, 6, 14, 16, 18, 21, 22, 24, 25, and tubulin
assay results (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: bmendelsohn@agensys.com. Phone: 424-280-5607.
ORCID
Brian A. Mendelsohn: 0000-0002-6339-2377
Present Address
†Zymeworks, 540-1385 West 8th Avenue, Vancouver, BC,
Canada, V6H 3V9.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We would like to acknowledge the work of D. Jackson,
M. Shiwalker, and J. Yin for in vitro work and T. Martin and
A. Soohoo for technical assistance. The authors would like to
acknowledge K. Morrison and D. R. Stover for manuscript review
and helpful discussions.
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To a stirred rt solution of Boc-Dap-Phe-OtBu (5.25 g, 10.7 mmol) in
CH2Cl2 (10.0 mL) was added TFA (10.0 mL). After 12 h, analysis by
LCMS showed the reaction was complete. The crude oil was purified by
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acid salt (0.518 mmol, 72%) as a white, amorphous solid: LCMS tR =
G
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J. Nat. Prod. XXXX, XXX, XXX−XXX