Synthesis and evaluation of novel dolastatin 10 derivatives for versatile conjugations

Article abstract of DOI:10.1016/j.bmc.2018.02.011

Dolastatin 10 (1) is a highly potent cytotoxic microtubule inhibitor (cytotoxicity IC₅₀ < 5.0 nM) and several of its analogs have recently been used as payloads in antibody drug conjugates. Herein, we describe the design and synthesis of a series of novel dolastatin 10 analogs useful as payloads for conjugated drugs. We explored analogs containing functional groups at the thiazole moiety at the C-terminal of dolastatin 10. The functional groups included amines, alcohols, and thiols, which are representative structures used in known conjugated drugs. These novel analogs showed excellent potency in a tumor cell proliferation assay, and thus this series of dolastatin 10 analogs is suitable as versatile payloads in conjugated drugs. Insights into the structure–activity relationships of the analogs are also discussed.

Full text of DOI:10.1016/j.bmc.2018.02.011

Accepted Manuscript
Synthesis and evaluation of novel dolastatin 10 derivatives for versatile conju-
gations
Shinya Yokosaka, Akiko Izawa, Chizuka Sakai, Eri Sakurada, Yasuhiro Morita,
Yukihiro Nishio
PII:
S0968-0896(17)32495-1
https://doi.org/10.1016/j.bmc.2018.02.011
BMC 14197
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 December 2017
9 February 2018
10 February 2018
Please cite this article as: Yokosaka, S., Izawa, A., Sakai, C., Sakurada, E., Morita, Y., Nishio, Y., Synthesis and
evaluation of novel dolastatin 10 derivatives for versatile conjugations, Bioorganic & Medicinal Chemistry (2018),
doi: https://doi.org/10.1016/j.bmc.2018.02.011
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Synthesis and evaluation of novel dolastatin
10 derivatives for versatile conjugations
Shinya Yokosaka, Akiko Izawa, Chizuka Sakai, Eri Sakurada, Yasuhiro Morita, Yukihiro Nishio
Toray Industries Inc., Pharmaceutical Research Laboratories

Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
Synthesis and evaluation of novel dolastatin 10 derivatives for versatile conjugations
Shinya Yokosaka, Akiko Izawa, Chizuka Sakai, Eri Sakurada, Yasuhiro Morita, Yukihiro Nishio*
Toray Industries Inc., Pharmaceutical Research Laboratories, 6-10-1 Tebiro, Kamakura, Kanagawa 248-8555, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
Dolastatin 10 (1) is a highly potent cytotoxic microtubule inhibitor (cytotoxicity IC50 < 5.0
nM) and several of its analogs have recently been used as payloads in antibody drug
conjugates. Herein, we describe the design and synthesis of a series of novel dolastatin 10
analogs useful as payloads for conjugated drugs. We explored analogs containing functional
groups at the thiazole moiety at the C-terminal of dolastatin 10. The functional groups included
amines, alcohols, and thiols, which are representative structures used in known conjugated
drugs. These novel analogs showed excellent potency in a tumor cell proliferation assay, and
thus this series of dolastatin 10 analogs is suitable as versatile payloads in conjugated drugs.
Insights into the structure-activity relationships of the analogs are also discussed.
Received in revised form
Accepted
Available online
Keywords:
Dolastatin 10
Tubulin inhibitor
cytotoxicity
2017 Elsevier Ltd. All rights reserved
.
1. Introduction
Dolastatin 10 (1) is an anti-microtubule agent that was isolated
from the sea hare Dolabella auricularia in the Indian Ocean in
1987 (Figure 1).1 Dolastatin 10 (1) was reported to exhibit
exceptionally potent anti-proliferative activity against various
cancer cell lines and is considered to be a promising anti-cancer
drug.2 However, neither dolastatin 10 (1) nor any of its analogs
have been approved as anti-cancer agents to date due to their
strong side-effects.3-6 Recently, however, these potent cytotoxic
compounds have attracted attention as payloads for conjugated
drugs.7 Monomethyl auristatin E (MMAE) (2) is a representative
example of a payload and is used in an approved antibody drug
conjugate (ADC), brentuximab vedotin.8 In this ADC, MMAE
(2) is attached to a monoclonal antibody which delivers the
cytotoxic agent to cancer cells expressing a specific antigen,
thereby reducing the side-effects of the cytotoxic agent. The
success of ADC suggests that conjugating dolastatin 10 analogs
to targeting units is a promising strategy for utilizing their
potential.
Figure 1. Dolastatin 10 (1) and selected derivatives.
conjugation with a linker, and the hydroxyl group of MMAE (2)
has also been used for linkage.12 Aliphatic amines and alcohols
are representative functional groups used for generating
conjugated drugs, and other functional groups such as thiols,
anilines, and phenols can be also used to link payloads to other
components.13-17 However, there are no reports investigating the
installation of these groups on the dolastatin 10 structure,
hampering the utility of dolastatin 10 analogs in conjugated drugs.
To expand the scope of the conjugation chemistry of dolastatin
10 derivatives, we explored new analogs with various functional
groups (thiols, aromatic amines, and phenols) in addition to
reported aliphatic amines and alcohols. Herein, we report novel
derivatives of dolastatin 10 that can be used for versatile
conjugations.
Conjugated drugs usually consist of a payload, a linker, and a
targeting unit. The combinations of these components are
critically important for the efficacy of the resulting conjugated
drug.7,9 Among the components, the linker unit must be
optimized to meet several requirements, such as stability in
plasma, the ability to release the payload at the target site, and
appropriate physicochemical properties. Various linker
technologies have been developed and the structure of the linker
is critically influenced by the functional group(s) in the payload.
Several dolastatin 10 derivatives have been designed for
conjugated drugs, including MMAE (2), monomethyl auristatin F
(MMAF) (3),10 and PF-0638101 (4).11 Typically, the primary or
secondary aliphatic amine on the N-terminal is used for
2. Compound design
Dolastatin 10 (1) is composed of the five amino acids
dolavaline (Dov), valine (Val), dolaisoleuine (Dil), dolaproine
(Dap), and dolaphenine (Doe). Previous structure-activity

relationship (SAR) studies suggested that the N-terminal Dov
moiety and C-terminal Doe moiety are relatively tolerant toward
chemical modifications.2 Maderna et al. reported the co-crystal
structure of the PF-06380101 (4) and (Tc) 2R tubulin
constructs.11 According to the reported structure, an amino group
at the N-terminal forms hydrogen bond networks with two amino
acid residues of tubulin. However, there appears to be space for
the installation of an additional functional group at the C-
terminal and thus we considered modification of the C-terminal
Doe moiety. We examined earlier reports of C-terminal
modifications, in particular the methyl ester derivative 5 and the
ethyl amide derivative 6 reported by Miyazaki et al. in 1993 in
which a carbonyl functionality was installed at the 2-position of
the thiazole of Doe.18 Both derivatives 5 and 6 were reported to
possess activity equipotent to that of dolastatin 10 (1), although
no specific description of their cytotoxicity value (IC50) was
provided. Given the tolerance of the Doe moiety against chemical
modification, we hypothesized that the installation of functional
groups at the termini of these functionalities would have little
impact on the cytotoxicity of dolastatin 10 (1). Thus, we planned
to synthesize and evaluate the derivatives shown in Figure 2.
This plan is advantageous from the synthetic point of view in that
peptide synthesis is usually performed in the C-terminal to N-
terminal direction. Typically, derivatization of the C-terminal
residue is less straightforward than that of the N-terminal residue,
but in the case of the planned derivatives shown in Figure 2,
functional groups at the C-terminal can be introduced more easily
by preparing the precursor with a carboxylic acid at the Doe
moiety, as they are selected in the final step of the synthetic route.
Figure 2. Plan for the exploration of novel dolastatin 10 derivatives.
reported. A Doe unit with an ethoxycarbonyl group at the 2-
position of the thiazole was prepared using a modification of
Hantzsch thiazole synthesis.22 The left-hand Dov-Val-Dil unit 9
was synthesized by two cycles of Cbz deprotection and
condensation of the corresponding amino acid units, followed by
deprotection of the t-Bu ester. The right-hand Dap-Doe-CO2Et
unit 11 was prepared by coupling of the Dap and Doe units and
Boc-deprotection. Next, these two units were coupled using
HATU with diisopropyl ethyl amine to provide the ethyl ester
derivative 12, yielding the equivalent of the methyl ester
derivative 5. Then, the ethyl ester of Doe was saponified to give
the carboxylic acid precursor 13.
New derivatives with various functional groups were
synthesized from precursor 13 by condensation of the
corresponding amines and alcohols, followed by deprotection of
the protecting groups to provide functional groups at the terminal
when needed. For amide formation, HATU with diisopropyl
ethyl amine was used for the condensation of amines and 13. For
ester formation, the procedure reported by Siina et al.23 gave the
desired esters in good yields. Boc, THP, and Trt protecting
groups were used for terminal amines, alcohols, and thiols,
respectively. These protecting groups were removed by HCl,
TsOH, or TFA/Et4SiH at the final step, followed by typical
procedures. Thirteen novel derivatives (14a-g, 15a-d, and 16a-c)
with terminal functional groups were synthesized, and N-ethyl
amide derivative 6 was also prepared for comparison with the
other amide derivatives.
3. Results and discussion
3.1. Synthesis
The synthesis of the new derivatives commenced by preparing
carboxylic acid precursor 13 in a convergent manner (Scheme 1).
Dov,19 Dil20, and Dap21 units were synthesized as previously
Scheme 1. Synthesis of dolastatin 10 derivatives with C-terminal modifications. Reagents and conditions: (a) H2, Pd/C, MeOH, 71%; (b) Cbz-Val-OH,
COMU, DIPEA, DMF, 97%; (c) H2, Pd/C, MeOH, 93%, (d) Dov-OH, HATU, DIPEA, DMF, 75%; (e) HCl, AcOEt, 99%; (f) Boc-Dap-OH, HATU, DIPEA,
DMF, 85%; (g) TFA, CH2Cl2; (h) HATU, DIPEA, DMF, 83%, (i) NaOH, EtOH, quant. (j) amine, HATU, DIPEA, DMF, 43-100% (6, 14a-g, 15a-d, 16a) or
alcohol, 2-methyl-6-nitrobenzoic anhydride, Et3N, DMAP, CH2Cl2 73-92% (16b-c) (k) deprotection of protecting groups (Boc: 14a-b , 15a-b, 16a, THP:
16b, Trt: 14d-e, 15d, 16c) if needed

3.2. Biological evaluation
There is a tendency that the compounds having the secondary
amide (X = -NMe-: 15) on the linker moiety (X) showed stronger
potency than the compounds having the primary amide (X = -
NH-: 14). The representative example is the difference of the IC50
between 14a and 15a. The calculated logD7.4 value of 15a is
lower than that of 14a, whereas 15a have slightly higher
permeability than 14a. Substitution of amide proton by methyl
group was thought to increase lipophilicity and membrane
permeability of the compounds. However, conformational
changes of the peptide caused by the secondary amide structure
could affect permeability and potency of the derivatives.
The cytotoxicities of the compounds were evaluated against
SKOV-3 (human ovarian cancer), A549 (human adeno
carcinoma derived from lung cancer), and L1210 (mouse
lymphocytic leukemia) cell lines (Table 1). MMAE (2) and the
ethyl amide derivative 6 showed excellent potency for each cell
line. The ethyl ester derivative 12 which is comparable to the
methyl ester derivative 5 had stronger cytotoxicity against
SKOV-3 than those of the two compounds. However, the amine
derivatives with a primary amide linker (14a, 14b) showed
drastically decreased potency against all three cell lines. The
alcohol derivative 14c exhibited one order of magnitude higher
IC50 values compared to those of the ethyl amide derivative 6.
The thiol derivative 14d showed equivalent or higher potency
than the ethyl amide derivative 6 against SKOV-3 and A549 cells,
but 5 times weaker cytotoxicity against L1210. A thiol-
containing derivative with a longer linker, 14e, was not active
against any of the cell lines. From the results obtained using 14a,
14b, and 14e, we hypothesized that introduction of a highly polar
functional group (e.g., the amine moiety of 14a, 14b) or a long
and polar linker (e.g., the amide linker moiety of 14e) might
hamper the membrane permeability of the compounds due to an
increase of polar surface area. We therefore investigated the
aniline derivative 14f and phenol derivative 14g to increase
lipophilicity. Both derivatives showed excellent cytotoxicity
against all three cell lines. Lipophilicity was adjusted by
changing the linker moiety (X) to tertiary N-methyl amide (X = -
NMe-: 15) and ester (X = -O-: 16). The effect of introducing a
Me group to the amide linker was especially remarkable for the
amine derivatives (15a, 15b) and their potencies were 10-100
times higher than that of the corresponding secondary amide
derivatives (12a, 12b). The IC50 of the alcohol derivative 15c was
slightly lower than that of the secondary amide derivative
whereas the thiol derivative 15d maintained strong potency.
Changing the secondary amide linker to the ester linker provided
an effect similar to that of the N-methyl amide linker. Improved
potency was observed for the amine derivative (16a) and the
alcohol derivative (16b), and the thiol derivative (16c)
maintained high potency.
Interestingly, Figure 3 shows that ester derivatives (X = -O-)
tend to be more potent than amide derivatives (X = -NH- and –
NMe-) in the cell-based assay. The ester group can likely
undergo hydrolysis intracellularly to give the carboxylic acid
derivative 13 which showed only weak cytotoxicity (IC50 >10
nM). A similar phenomenon was reported for MMAF (3), which
has a free carboxylic acid, is reported to show only weak
cytotoxicity, whereas its methyl ester derivative has >1,000 times
stronger activity.10 These carboxylic acids might retain
intracellularly long time due to low cell permeation and enhance
cytotoxic activity of the parent ester derivatives. Another
possibility is that the carboxylic acids might have stronger anti-
microtubule activity than other tubulin inhibitos and show high
cytotoxic activity only when they are released inside the cell,
though it is difficult to differentiate affinity of compounds by the
tubulin polymerization assay. Therefore, 13 could also be used
for the payload in conjugate drugs by using its carboxylic acid as
the conjugation point.
High cytotoxicity is reported to be necessary for payloads
used in conjugated drugs because only a relatively low amount of
the payload is delivered to the targeted tumor cell.24 Compounds
14f and 15d have almost the same potency as MMAE (2) but
have different functional groups on the C-terminal. Brentuximab
vedotin has been conjugated with MMAE via a dipeptide linker,
Val-Cit, and a self-immolative linker, p-amino benzyl alcohol
(PABA). PABA is an important unit for converting the secondary
aliphatic amine at the N-terminal of MMAE to aniline, since the
bond between the dipeptide linker and the aniline can be cleaved
smoothly by the lysosomal protease cathepsin B.13 The aniline
NH2 of compound 14f can play the role of PABA and be directly
link with the dipeptide linker. A disulfide linker is another
powerful strategy for conjugated drugs, and direct conjugation
between Cys in THIOMAB technology and maytansinoid
derivatives, without the need for any linker, was recently
reported.14 MMAE (2) with a disulfide linkage is also mentioned
in the literature but its aliphatic amine must be converted to
carbamate for conjugation, resulting in a structural change that
appears to affect the release rate of the free payload. Compound
15d can be applied for disulfide linkage directly through the
sulfhydryl group on the C-terminal. Since conjugated drugs
consist of various units, reducing the number of components
would aid prediction of the behavior of each unit, decrease the
cost of synthesis, and aid quality control. Therefore, the novel
dolastatin 10 derivatives described here may be valuable
payloads because of their very high potency and the variety of
functional groups which can be used with a wide range of linker
technologies for conjugation chemistry.
We investigated the physicochemical properties and the
tubulin inhibition activities of the derivatives to obtain insights
into the mechanisms underlying the differences in cytotoxicity.
Figure 3 shows pIC50 of the dolastatin 10 derivatives against
SKOV-3 plotted versus membrane permeability recorded with
the Parallel Artificial Membrane Permeability Assay (PAMPA)
system. The plot shows a moderate linear relationship between
pIC50 and PAMPA permeability, suggesting that low membrane
permeability weakens the cytotoxicity of the compounds by
decreasing the amount of compound that enters the cells.
However, it is also possible that installation of a polar functional
group at the C-terminal attenuates the inhibitory activity of the
compound against tubulin polymerization. To confirm this, we
investigated the tubulin polymerization inhibition activities of the
dolastatin 10 derivatives. The results obtained using 14a and 14f
are shown in Figure 4. Although the IC50 values of these two
derivatives differ by >30 times against SKOV-3, their tubulin
polymerization inhibition potencies were similar. Other
derivatives also showed similar tubulin polymerization inhibition
potencies (data not shown). Taking these results together, it is
highly likely that the differences in cytotoxicity among the
derivatives are due to differences in their physicochemical
properties rather than to differences in their anti-microtubule
potencies.
4. Conclusion
In summary, we have described the design, synthesis, and
evaluation of a series of new dolastatin 10 derivatives that are
suitable for use in conjugated drugs. By installing the carbonyl
moiety at the 2-position of the thiazole at the C-terminal we
could efficiently explore novel derivatives with various

functional groups, resulting in the identification of highly potent
amine, aniline, alcohol, phenol and thiol group derivatives. These
findings might provide new designs and expand conjugation
methods for the synthesis of conjugated drugs. We also obtained
insights into the structure-activity relationships and structure-
property relationships of the compounds, which may be useful
for the further design and investigation of new dolastatin 10
derivatives. The utility of the new derivatives as payloads for
conjugated drugs is under investigation.

Table 1. Structure-activity relationships and structure-property relationships for dolastatin 10 derivatives
IC50 (nM)
A549
1.3
PAMPA
Permeability
(10-6 cm/s)
0.079
a
compound
-X-
-Y
logD7.4
SKOV-3
0.66
L1210
2.1
6.5
-
MMAE (2)
-
-
-H
2.03
5.39
6.26
3.28
2.59
3.24
5.80
6
-NH-
-O-
0.55
0.74
-
1.7
3.2
12
-H
0.024
>10
14a
14b
14c
14d
-NH-
-NH-
-NH-
-NH-
-NH2
-NHMe
-OH
-SH
>10
9.9
>50
27
0.056
0.004
0.020
1.1
>10
4.7
1.7
28
0.44
1.9
31
14e
-NH-
>10
>10
>50
<0.001
5.98
14f
-NH-
-NH-
0.27
0.37
1.1
1.3
4.1
5.6
0.30
0.36
5.37
5.91
14g
15a
-NMe-
-NMe-
-NMe-
-NMe-
-O-
-NH2
-NHMe
-OH
0.56
0.19
1.5
0.81
1.9
1.6
1.2
0.83
1.8
6.9
12
14
0.072
<0.001
0.077
1.1
1.91
1.04
2.03
4.26
4.73
4.74
5.8
15b
15c
8.5
7.0
29
15d
-SH
1.1
16a
-NH2
-OH
0.85
0.086
0.13
0.007
0.04
16b
16c
-O-
4.7
14
-O-
-SH
0.005
a log D value calculated by ACD/Labs
Figure 4. Results of the tubulin polymerization assay. The effect of
compounds on tubulin polymerization was determined in a biochemical
fluorescence-based assay (Cytoskeleton, Denver, CO) conducted
according to manufacturer’s protocol. Fluorescence changes were
monitored kinetically for 1 h.
Figure 3. Plot of pIC50 against SKOV-3 versus PAMPA permeability (log scale).
The red, blue, and dashed lines represent the linear fits of the ester analogues, the
amide analogues, and all the analogues, respectively.

5. Experimental section
(ESI) m/z 486 (M+H+). LRMS (ESI) m/z calcd for C26H51N3O5 486.4
[M+Na]+, Found 486.4.
All reagents and solvents were obtained from commercial suppliers and
used without further drying or purification. All reactions sensitive to oxygen
or moisture were conducted under a nitrogen or argon atmosphere. Analytical
thin layer chromatography (TLC) was performed on Merck 60F254 analytical
plates. Flash chromatography separations were performed using Yamazen
Smart Flash W-Prep 2XY, Hi-Flash Column silica gel (40 µm, 60Ǻ). 1H
NMR and 13C NMR spectra were recorded on JEOL JNM-ECZ400 and JEOL
JNM-ECS400 instruments. All chemical shift (δ) values were referenced to
CDCl3 or DMSO as an internal standard and reported as shift (proton count,
multiplicity, coupling constant (J)). The following abbreviations are used for
spin multiplicity: s = singlet, d = doublet, t = triplet, q = quartet, m –
multiplet, br = broad. Low-resolution mass spectra (LRMS) were recorded on
an Agilent Technologies 6130 Quadrupole LC/MS (ESI) and high-resolution
Ethyl 2-((S)-1-((2R,3R)-3-((S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl)-3-
methoxy-2-methylpropanamido)-2-phenylethyl)thiazole-4-carboxylate
(11)
To a solution of 10 (18.9 mg, 0.0502 mmol) in AcOEt (1.0 mL) was added
HCl-AcOEt (4 N, 188 µL, 0.753 mmol) at room temperature. After stirring
for 3 h at room temperature, the reaction mixture was concentrated under
reduced pressure to afford the crude Boc-deprotected product. To a solution
of
(2R,3R)-3-((S)-1-(tert-butoxycarbonyl)pyrrolidin-2-yl)-3-methoxy-2-
methylpropanoic acid (Boc-Dap-OH) (15.0 mg, 0.0522 mmol) in DMF (0.50
mL) was added DIPEA (19.3 µL, 0.110 mmol), HATU (20.1 mg, 0.0529
mmol), and the above crude product (13.8 mg, 0.0499 mmol) in DMF (0.50
mL) at 0°C. After stirring for 1 h at room temperature, water was added to the
reaction mixture and the aqueous layer was extracted with AcOEt. The
combined organic layers were washed with saturated aqueous NaHCO3, and
then brine, dried over anhydrous Na2SO4, and concentrated under reduced
pressure. The residue was purified by column chromatography on silica gel
(n-hexane:AcOEt = 4:1 to 2:3) to afford 11 (17.9 mg, 0.0328 mmol, 65%
yield) as a colorless oil.
mass spectra (HRMS) were recorded on
instrument.
a Shimadzu LCMS-IT-TOF
tert-Butyl
(3R,4S,5S)-4-((S)-2-(((benzyloxy)carbonyl)amino)-N,3-
dimethylbutanamido)-3-methoxy-5-methylheptanoate (8)
To a solution of 7 (23.3 mg, 0.0592 mmol) in MeOH (1.0 mL) was added
10% Pd/C, 50%wet (2.3 mg) under an Ar atmosphere, and the reaction
mixture was stirred at room temperature under 1 atm of hydrogen for 2 h. The
reaction mixture was filtered through a pad of Celite and the filtrate was
concentrated under reduced pressure to afford the crude Cbz-deprotected
product. To a solution of ((benzyloxy)carbonyl)-L-valine (14.8 mg, 0.0589
mmol) in DMF (0.50 mL) was added DIPEA (19.5 µL, 0.112 mmol), COMU
(28.7 mg, 0.0670 mmol), and the above crude product (14.5 mg, 0.0559
mmol) in DMF (0.50 mL) at 0°C. After stirring for 24 h at room temperature,
water was added to the reaction mixture and the aqueous layer was extracted
with AcOEt. The combined organic layers were washed with 1 N aqueous
HCl, saturated aqueous NaHCO3, and then brine, dried over anhydrous
Na2SO4, and concentrated under reduced pressure. The residue was purified
by column chromatography on silica gel (n-hexane:AcOEt = 9:1 to 7:3) to
afford 8 (16.1 mg, 0.348 mmol, 59% yield) as a colorless oil.
1H-NMR (400 MHz, CDCl3) δ: 8.04 (1H, s), 7.30-7.13 (5H, m), 6.46-5.76
(1H, br m), 5.62-5.56 (1H, m), 4.43 (2H, qd, J = 7.1, 1.3 Hz), 3.90-3.15 (6H,
m), 3.35 (3H, s), 2.40-2.22 (1H, m), 1.85-1.60 (4H, m), 1.45 (9H, s), 1.41 (3H,
t, J = 7.1 Hz), 1.24-1.09 (3H, m). LRMS (ESI) m/z calcd for C28H39N3O6S
546.3 [M+Na]+, Found 546.3.
Ethyl
2-((S)-1-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-
(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-
methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-
methylpropanamido)-2-phenylethyl)thiazole-4-carboxylate (12)
To a solution of Dov-Val-Dil-Ot-Bu (9) (24.3 mg, 0.0501 mmol) in CH2Cl2
(0.50 mL) was added TFA (0.50 mL) at 0°C. After stirring for 1 h at room
temperature, the reaction mixture was concentrated under reduced pressure to
afford the crude t-Bu-deprotected product. To a solution of Boc-Dap-Doe-
CO2Et (11) (17.9 mg, 0.0328 mmol,) in CH2Cl2 (0.50 mL) was added TFA
(0.50 mL) at 0°C. After stirring for 20 min at room temperature, the reaction
mixture was concentrated under reduced pressure to afford the crude Boc-
deprotected product. To a solution of the above crude Dov-Val-Dil-OH in
DMF (0.50 mL) was added DIPEA (19.3 µL, 0.110 mmol), HATU (18.7 mg,
0.0491 mmol), and the above crude Dap-Doe-CO2Et in DMF (0.50 mL) at
0°C. After stirring for 4 h at room temperature, water was added to the
reaction mixture and the aqueous layer was extracted with AcOEt. The
combined organic layers were washed with saturated aqueous NaHCO3, and
then brine, dried over anhydrous Na2SO4, and concentrated under reduced
pressure. The residue was purified by column chromatography on silica gel
(CHCl3:MeOH = 1:0 to 97:3) to afford 5 (24.7 mg, 0.0288 mmol, 88% yield)
as a white solid.
1H-NMR (400MHz, CDCl3) δ: 7.39-7.28 (5H, m), 5.51 (1H, d, J = 8.3
Hz), 5.10 (2H, s), 4.77-4.65 (1H, m), 4.51 (1H, dd, J = 9.3, 5.9 Hz), 3.91-3.85
(1H, m), 3.34 (3H, s), 2.95 (3H, s), 2.44 (1H, d, J = 15.1 Hz), 2.30 (1H, dd, J
= 15.1, 9.4 Hz), 2.03-1.97 (1H, m), 1.70-1.60 (1H, m), 1.46 (9H, s), 1.10-0.90
(11H, m), 0.83 (3H, t, J = 7.4 Hz). LRMS (ESI) m/z 493[M+H]+.
tert-Butyl (3R,4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-
methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-
methylheptanoate (9)
To a solution of 8 (38.9 mg, 0.0788 mmol) in MeOH (1.0 mL) was added
10% Pd/C, 50%wet (2.3 mg) under an Ar atmosphere, and the reaction
mixture was stirred at room temperature under 1 atm of hydrogen for 4 h. The
reaction mixture was filtered through a pad of Celite and the filtrate was
concentrated under reduced pressure to afford the crude Cbz-deprotected
product. To a solution of dimethyl-L-valine (Dov-OH) (13.8 mg, 0.0950
mmol) in DMF (0.50 mL) was added DIPEA (19.5 µL, 0.112 mmol), HATU
(39.5 mg, 0.104 mmol), and the above crude product (31.0 mg, 0.0865 mmol)
in DMF (0.50 mL) at 0°C. After stirring for 3 h at room temperature, water
was added to the reaction mixture and the aqueous layer was extracted with
AcOEt. The combined organic layers were washed with 1 N aqueous HCl,
saturated aqueous NaHCO3, and then brine, dried over anhydrous Na2SO4,
and concentrated under reduced pressure. The residue was purified by column
chromatography on silica gel (n-hexane:AcOEt = 1:1 to 47: 3) to afford 9
(24.3 mg, 0.0501 mmol, 63% yield) as a colorless oil.
1H-NMR (400 MHz, CDCl3) δ: 8.03 (1H, s), 7.36-7.13 (5H, m), 6.91-6.84
(1H, m), 6.46-5.70 (1H, br m), 5.56-5.49 (1H, m), 4.90-4.70 (2H, m), 4.42
(2H, q, J = 7.1 Hz), 4.15-3.00 (13H, m), 3.03 (3H, s), 2.44-1.55 (17H, m),
1.41 (3H, td, J = 7.1, 1.8 Hz), 1.28-0.91 (20H, m), 0.81 (3H, t, J = 7.1 Hz).
13C-NMR (400 MHz, CDCl3) δ: 173.99, 172.95, 171.76, 171.64, 170.22,
161.34, 146.94, 136.80, 129.37, 128.45, 127.26, 126.84, 81.45, 77.13, 76.47,
61.52, 61.39, 60.39, 59.01, 57.96, 53.72, 53.02, 47.58, 43.70, 42.87, 40.62,
37.45, 32.97, 30.97, 30.93, 27.62, 25.64, 24.97, 24.48, 20.12, 17.71, 17.64,
14.35, 13.88, 10.67. HRMS (ESI) m/z calcd for C45H72N6O8S 857.5205
[M+H]+, Found 857.5184.
2-((S)-1-(((2S,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(Dimethylamino)-3-
methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-
methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropyl)amino)-2-
phenylethyl)thiazole-4-carboxylic acid (13)
1H-NMR (400 MHz, CDCl3) δ: 6.91-6.84 (1H, m), 4.82-4.70 (1H, m), 4.79
(1H, dd, J = 9.3, 6.3 Hz), 3.91-3.85 (1H, m), 3.34 (3H, s), 2.99 (3H, s), 2.48-
2.41 (2H, m), 2.33-2.26 (1H, m), 2.24 (6H, s), 2.12-1.98 (2H, m), 1.73-1.60
(1H, m), 1.45 (9H, s), 1.05-0.91 (17H, m), 0.80 (3H, t, J = 7.3 Hz). LRMS

To a solution of 12 (14.9 mg, 0.0174 mmol) in EtOH (1.0 mL) was added 1 N
aqueous NaOH (19.1 µL 0.019 mmol) at room temperature. After stirring for
3 h at room temperature, 1 N aqueous HCl (19.1 µL) and water were added to
the reaction mixture and the aqueous layer was extracted with CHCl3. The
combined organic layers were dried over anhydrous Na2SO4 and concentrated
under reduced pressure to afford the crude product 13 (14.3 mg, 0.172 mmol,
99% yield) as a white solid, which was used in the next reaction without
further purification.
33.54, 33.13, 33.09, 32.93, 31.77, 31.71, 28.58, 27.15, 27.09, 26.53, 25.88,
25.43, 24.52, 19.94, 19.81, 19.42, 19.30, 19.10, 16.63, 16.25, 15.86, 15.18,
10.92. HRMS (ESI) m/z calcd for C45H74N8O7S 893.5293 [M+Na]+, Found
893.5293.
2-((S)-1-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(Dimethylamino)-3-
methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-
methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-2-
phenylethyl)-N-(2-(methylamino)ethyl)thiazole-4-carboxamide
1H-NMR (400 MHz, CDCl3) δ: 7.97 (1H, s), 7.70-7.42 (1H, m), 7.30-7.15
(5H, m), 7.13-7.09 (1H, m), 5.62-5.55 (1H, m), 4.87-4.67 (2H, m), 4.11-3.10
(7H, m), 3.32 (3H×2, s), 3.04 (3H, s), 2.70-2.28 (4H, m), 2.53 (6H, s), 2.19-
2.06 (2H, m), 1.98-1.55 (4H, m), 1.40-1.20 (1H, m), 1.18-0.69 (23H, m). 13C-
NMR (400 MHz, CDCl3) δ: 173.75, 173.08, 170.20, 170.09, 168.52, 165.36,
150.17, 137.04, 129.43, 128.35, 126.69, 125.80, 81.31, 77.21, 77.15, 60.75,
59.68, 57.83, 57.39, 54.10, 52.77, 47.63, 44.59, 41.52, 41.02, 37.37, 32.71,
31.82, 30.91, 27.83, 25.73, 25.01, 24.42, 19.76, 18.51, 15.79, 14.55, 10.69 .
HRMS (ESI) m/z calcd for C43H68N6O8S 829.4892 [M+H]+, Found 829.4885.
(14b)
Step 1: N-Boc protected 14b was synthesized from 13 (25.0 mg, 0.0302
mmol) and tert-butyl (2-aminoethyl)carbamate (7.88 mg, 0.0452 mmol) in a
manner similar to that described for compound 6. White amorphous; Yield:
31.7 mg (quantitative yield). Step 2: Compound 14b was synthesized from N-
Boc protected 14b (29.7 mg, 0.0301 mmol) in a manner similar to that
described for compound 14a. A white amorphous; Yield: 25.3 mg (88%
yield)
1H-NMR (400 MHz, CD3OD) δ: 8.63 (1H, br s), 8.22-8.14 (1H, m), 7.35-7.15
(5H, m), 5.70-5.52 (1H, m), 4.83-4.63 (2H, m), 4.17-3.04 (20H, m), 2.91 (3H,
s), 2.90 (3H, s), 2.74 (3H, s), 2.51-1.98 (6H, m), 1.91-1.50 (4H, m), 1.42-0.80
(24H, m).13C-NMR (400 MHz, CD3OD) (presumed to be a mixture of
cis/trans rotamers) δ: 176.63, 176.37, 175.28, 174.80, 174.74, 174.49, 171.94,
171.59, 166.75, 166.71, 164.58, 164.55, 150.54, 150.39, 138.81, 138.63,
130.14, 129.94, 129.82, 129.67, 127.97, 127.83, 125.48, 125.29, 86.42, 83.13,
79.61, 79.50, 78.99, 73.94, 62.07, 61.48, 60.56, 60.50, 58.50, 58.28, 58.20,
58.02, 56.94, 56.74, 53.80, 52.69, 52.67, 50.68, 49.29, 48.04, 45.50, 45.38,
43.48, 43.38, 41.62, 41.29, 41.13, 40.72, 38.08, 37.10, 37.05, 36.87, 33.90,
33.64, 33.57, 33.05, 32.91, 31.79, 31.73, 28.59, 27.16, 27.10, 26.53, 25.91,
25.43, 24.52, 19.93, 19.82, 19.38, 19.26, 19.10, 16.57, 16.22, 15.87, 15.15,
10.92. HRMS (ESI) m/z calcd for C46H76N8O7S 907.5409 [M+Na]+, Found
907.5459.
2-((S)-1-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(Dimethylamino)-3-
methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-
methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-2-
phenylethyl)-N-ethylthiazole-4-carboxamide (6)
To a solution of 13 (14.3 mg, 0.0172 mmol) in DMF (0.50 mL) was added
DIPEA (4.52 µL, 0.0257 mmol), HATU (9.84 mg, 0.0258 mmol), and Et2NH
(2.0 M in THF, 25.9 µL, 0.0518 mmol) at 0°C. After stirring for 1 h at room
temperature, water was added to the reaction mixture and the aqueous layer
was extracted with AcOEt. The combined organic layers were washed with
saturated aqueous NaHCO3, and then brine, dried over anhydrous Na2SO4,
and concentrated under reduced pressure. The residue was purified by column
chromatography on silica gel (CHCl3:MeOH = 1:0 to 19:1) to afford 6 (6.3
mg, 0.074 mmol, 43% yield) as a white solid.
1H-NMR (400 MHz, CDCl3) δ: 7.95 (1H, s), 7.50-7.13 (6H, m), 6.90 (1H, d, J
= 7.6 Hz), 5.56-5.48 (1H, m), 4.83-4.75 (2H, m), 4.15-3.10 (15H, m), 3.03
(3H, s), 2.50-2.30 (4H, m), 2.25 (6H, s), 2.11-1.92 (2H, m), 1.85-1.55 (4H,
m), 1.40-1.30 (1H, m), 1.27-1.23 (3H, m), 1.15-0.80 (23H, m). 13C-NMR
(400 MHz, CDCl3) δ: 173.96, 172.54, 171.76, 170.43, 169.95, 160.97, 149.91,
136.74, 129.30, 128.45, 126.90, 122.63, 85.36, 81.31, 76.45, 61.56, 60.53,
59.33, 57.97, 53.76, 52.83, 47.63, 43.97, 42.88, 40.82, 37.41, 34.16, 32.90,
31.02, 27.63, 25.67, 25.00, 24.53, 23.60, 20.13, 17.65, 15.77, 14.94, 14.17,
10.63. HRMS (ESI) m/z calcd for C45H75N7O6S 857.5365 [M+H]+, Found
857.5386.
2-((S)-1-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(Dimethylamino)-3-
methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-
methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-2-
phenylethyl)-N-(2-hydroxyethyl)thiazole-4-carboxamide (14c)
Compound 14c was synthesized from 13 (25.0 mg, 0.0301 mmol) and 2-
aminoethan-1-ol (2.21 mg, 0.0362 mmol) in a manner similar to that
described for compound 6. A white amorphous; Yield: 28.6 mg (quantitative
yield).
1H-NMR (400 MHz, CDCl3) δ: 7.96 (1H, s), 7.85-7.75 (1H, m), 7.60 (1H, br
s), 7.30-7.07 (5H, m), 6.91 (1H, d, J = 8.7 Hz), 5.50-5.43 (1H, m), 4.83-4.72
(2H, m), 4.16-4.09 (1H, m), 4.05-4.01 (1H, m), 3.84-3.78 (3H, m), 3.65-3.20
(6H, m), 3.32 (3H, s), 3.31 (3H, s), 3.04 (3H, s), 2.48-2.32 (4H, m), 2.23 (6H,
s), 2.16-1.63 (6H, m), 1.40-1.25 (1H, m), 1.08 (3H, d, J = 6.9 Hz), 1.04-0.88
(17H, m), 0.80 (3H, t, J = 6.4 Hz). 13C-NMR (400 MHz, CDCl3) δ: 174.05,
172.30, 171.79, 170.52, 169.95, 162.02, 149.31, 136.74, 129.29, 128.45,
126.90, 123.29, 85.49, 81.38, 76.41, 62.15, 61.54, 60.61, 59.54, 57.94, 53.80,
52.77, 47.66, 44.16, 42.86, 42.30, 40.67, 37.58, 32.84, 31.17, 27.62, 25.67,
24.95, 24.64, 23.56, 20.12, 17.65, 15.80, 14.22, 10.61. HRMS (ESI) m/z calcd
for C45H73N7O8S 872.5314 [M+H]+, Found 872.5323.
N-(2-Aminoethyl)-2-((S)-1-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-
(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-
methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-
methylpropanamido)-2-phenylethyl)thiazole-4-carboxamide (14a)
Step 1: N-Boc protected 14a was synthesized from 13 (48.0 mg, 0.0578
mmol) and tert-butyl (2-aminoethyl)carbamate (13.9 mg, 0.0868 mmol) in a
manner similar to that described for compound 6. White amorphous; Yield:
54.9 mg (98% yield) Step 2: To a solution of N-Boc protected 14a (44.5 mg,
0.056 mmol) in MeOH (1.0 mL) was added HCl-1,4-dioxane (4.0 N, 210 µL,
0.842 mmol) at room temperature. After stirring for 18 h at room temperature,
the reaction mixture was concentrated under reduced pressure to afford the
HCl salt of 14a (41.0 mg, 0.0434 mmol, 96% yield) as a white amorphous.
2-((S)-1-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(Dimethylamino)-3-
methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-
methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-2-
phenylethyl)-N-(2-mercaptoethyl)thiazole-4-carboxamide (14d)
1H-NMR (400 MHz, CD3OD) δ: 8.65 (1H, br s), [8.19 (0.5H, s), 8.16 (0.5H,
s)], 7.32-7.16 (5H, m), 5.69-5.56 (1H, m), 4.80-4.63 (2H, m), 4.15-3.05 (14H,
m), 3.33 (6H, s), 2.91 (6H, s), 2.51-1.38 (10H, m), 1.30-0.91 (21H, m), 0.85
(3H, t, J = 7.1 Hz). 13C-NMR (400 MHz, CD3OD) (presumed to be a mixture
of cis/trans rotamers) δ: 176.61, 176.36, 175.21, 174.81, 174.75, 174.49,
171.94, 171.69, 166.73, 166.71, 164.42, 164.41, 150.57, 150.37, 138.82,
138.64, 130.15, 129.96, 129.80, 129.66, 127.93, 127.82, 125.36, 125.22,
86.42, 83.11, 79.64, 78.96, 73.85, 73.77, 62.06, 61.50, 60.70, 60.50, 58.49,
58.28, 58.18, 57.96, 56.95, 56.74, 53.76, 52.71, 49.29, 48.04, 45.49, 45.35,
43.45, 43.34, 41.66, 41.09, 40.99, 40.72, 38.23, 37.99, 37.84, 36.82, 33.60,
Step 1: S-Trt protected 14d was synthesized from 13 (25.0 mg, 0.0301 mmol)
and 2-(tritylthio)ethan-1-amine (14.5 mg, 0.0454 mmol) in a manner similar
to that described for compound 6. White amorphous; Yield: 29.9 mg (88%
yield). Step 2: Compound 14d (25.3 mg, 0.0224 mmol) was treated with 0.02
M TES in 1:1 TFA/CH2Cl2 (4.5 mL) for 30 min at room temperature and
concentrated under reduced pressure. AcOEt was added to the resulting
residue, and the organic layers were washed with saturated aqueous NaHCO3,
and then brine, dried over anhydrous Na2SO4, and concentrated under reduced

pressure. The residue was purified by column chromatography on silica gel
(CHCl3:MeOH = 1:0 to 9:1) to afford 14d (18.3 mg, 0.0206 mmol, 92%
yield) as a white amorphous.
Compound 14g was synthesized from 13 (29.0 mg, 0.0349 mmol) and 4-(2-
aminoethyl)phenol (5.76 mg, 0.0420 mmol) in a manner similar to that
described for compound 6. A white solid; Yield: 33.2 mg (quantitative yield).
1H-NMR (400 MHz, CDCl3) δ: 7.97 (1H, s), 7.70 (1H, t, J = 6.1 Hz), 7.49
(1H, br s), 7.29-7.12 (5H, m), 6.90 (1H, d, J = 8.2 Hz), 5.54-5.48 (1H, m),
4.82-4.74 (2H, m), 4.14-4.03 (2H, m), 3.86 (1H, d, J = 7.7 Hz), 3.63 (2H, q, J
= 6.5 Hz), 3.51-3.16 (4H, m), 3.33 (3H, s), 3.32 (3H, s), 3.04 (3H, s), 2.81-
2.74 (2H, m), 2.48-2.33 (4H, m), 2.25 (6H, s), 2.11-1.92 (2H, m), 1.85-1.60
(4H, m), 1.46 (1H, t, J = 8.6 Hz), 1.40-1.22 (1H, m), 1.10 (3H, d, J = 7.2 Hz),
1.05-0.90 (17H, m), 0.81 (3H, t, J = 7.5 Hz). 13C-NMR (400 MHz, CDCl3) δ:
173.98, 172.69, 171.76, 170.46, 169.96, 161.17, 149.45, 136.71, 129.32,
128.47, 126.93, 123.16, 81.32, 78.50, 76.46, 61.57, 60.56, 59.33, 57.97, 53.77,
52.86, 47.65, 43.99, 42.89, 42.33, 40.78, 37.44, 32.90, 31.05, 30.99, 27.64,
25.68, 25.02, 24.63, 24.54, 20.14, 17.67, 15.79, 14.21, 10.63. HRMS (ESI)
m/z calcd for C45H73N7O7S2 888.5086 [M+H]+, Found 888.5074.
1H-NMR (400 MHz, CDCl3) δ: 7.93 (1H, s), 7.41-7.30 (2H, m), 7.27-7.10
(5H, m), 7.04 (2H, d, J = 8.2 Hz), 6.95-6.85 (1H, m), 6.82 (2H, d, J = 8.2 Hz),
5.49-5.44 (1H, m), 4.85-4.72 (2H, m), 4.14-3.15 (9H, m), 3.33 (3H, s), 3.26
(3H, s), 3.03 (3H, s), 2.82 (2H, t, J = 6.9 Hz), 2.51-2.30 (4H, m), 2.23 (6H, s),
2.17-1.94 (2H, m), 1.90-1.60 (4H, m), 1.40-1.20 (1H, m), 1.15-0.88 (20H, m),
0.80 (3H, t, J = 7.3 Hz). 13C-NMR (400 MHz, CDCl3) δ: 174.05, 171.88,
171.19, 170.62, 169.77, 161.03, 155.49, 149.57, 136.61, 129.79, 129.74,
129.28, 128.46, 126.89, 122.93, 115.68, 85.29, 81.29, 76.40, 61.62, 60.48,
59.69, 57.98, 53.82, 52.61, 47.68, 43.85, 42.84, 40.76, 37.38, 36.50, 34.83,
32.85, 31.42, 30.93, 27.62, 25.63, 24.87, 23.54, 20.11, 17.64, 14.15, 13.60,
10.61. HRMS (ESI) m/z calcd for C51H77N7O8S 948.5627 [M+H]+, Found
948.5645.
2-((S)-1-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(Dimethylamino)-3-
methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-
methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-2-
phenylethyl)-N-(2-(3-mercaptopropanamido)ethyl)thiazole-4-
carboxamide (14e)
N-(2-Aminoethyl)-2-((S)-1-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-
(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-
methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-
methylpropanamido)-2-phenylethyl)-N-methylthiazole-4-carboxamide
(15a)
Step 1: S-Trt protected 14e was synthesized from 13 (20.0 mg, 0.0241 mmol)
and N-(2-aminoethyl)-3-(tritylthio)propanamide (11.7 mg, 0.0300 mmol) in a
manner similar to that described for compound 6. White amorphous; Yield:
19.7 mg (77% yield). Step 2: Compound 14e was synthesized from S-Trt
protected 14e (10.3 mg, 0.00857 mmol) in a manner similar to that described
for compound 14d. A white amorphous; Yield: 7.3 mg (89% yield).
Step 1: N-Boc protected 15a was synthesized from 13 (29.0 mg, 0.0350
mmol) and tert-butyl (2-(methylamino)ethyl)carbamate (7.31 mg, 0.0419
mmol) in a manner similar to that described for compound 6. A white
amorphous; Yield: 33.4 mg (96% yield). Step 2: Compound 15a was
synthesized from N-Boc protected 15a (33.4 mg, 0.0339 mmol) in a manner
similar to that described for compound 14a. A white amorphous; Yield: 29.0
mg (90% yield).
1H-NMR (400 MHz, CDCl3) δ: 7.97 (1H, s), 7.78 (1H, br s), 7.51 (1H, br s),
7.32-7.10 (5H, m), 7.00-6.85 (1H, m), 6.58 (1H, br s), 5.53-5.45 (1H, m),
4.82-4.74 (2H, m), 4.15-4.08 (1H, m), 4.06-4.00 (1H, m), 3.86 (1H, d, J = 7.7
Hz), 3.64-3.18 (8H, m), 3.33 (3H, s), 3.32 (3H, s), 3.04 (3H, s), 2.82-2.75 (2H,
m), 2.50 (2H, t, J = 6.8 Hz), 2.48-2.37 (4H, m), 2.26 (6H, s), 2.12-1.92 (2H,
m), 1.83-1.57 (4H, m), 1.27-1.19 (1H, m), 1.11-0.86 (20H, m), 0.81 (3H, t, J
= 7.2 Hz). 13C-NMR (400 MHz, CDCl3) δ: 174.03, 172.66, 171.76, 171.39,
170.42, 169.89, 162.37, 149.14, 136.66, 129.30, 128.47, 126.92, 123.29,
85.46, 81.39, 76.41, 61.62, 60.57, 59.37, 57.94, 53.79, 52.82, 47.68, 44.08,
42.86, 40.77, 40.48, 40.31, 39.16, 37.45, 32.86, 30.96, 27.61, 25.66, 24.98,
24.53, 23.57, 20.48, 20.11, 17.65, 15.78, 14.16, 10.60. HRMS (ESI) m/z calcd
for C48H78N8O8S2 959.5457 [M+H]+, Found 959.5437.
1H-NMR (400 MHz, CD3OD) δ: 8.98-8.60 (1H, m), 8.14-7.92 (1H, m), 7.37-
7.18 (5H, m), 5.66-5.57 (1H, m), 4.85-4.63 (2H, m), 4.20-3.07 (20H, m), 3.16
(3H, s), 2.92 (3H, s), 2.91 (3H, s), 2.48-1.39 (10H, m), 1.30-0.98 (21H, m),
0.87 (3H, t, J = 7.1 Hz). 13C-NMR (400 MHz, CDCl3) (salt free) δ: 174.03,
172.31, 171.76, 171.66, 170.35, 161.38, 149.65, 136.75, 129.31, 128.42,
126.84, 122.91, 85.40, 81.46, 76.43, 60.50, 59.40, 58.24, 57.95, 53.77, 52.73,
50.87, 47.62, 43.94, 42.84, 40.81, 38.77, 37.42, 36.08, 32.88, 30.97, 27.60,
25.64, 24.95, 24.58, 23.56, 20.09, 18.37, 17.64, 15.76, 10.61. HRMS (ESI)
m/z calcd for C46H76N8O7S 907.5450 [M+Na]+, Found 907.5470.
2-((S)-1-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(Dimethylamino)-3-
methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-
methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-2-
phenylethyl)-N-methyl-N-(2-(methylamino)ethyl)thiazole-4-carboxamide
(15b)
N-(4-Aminophenethyl)-2-((S)-1-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-
((S)-2-(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-
3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-
methylpropanamido)-2-phenylethyl)thiazole-4-carboxamide (14f)
Step 1: N-Boc protected 15b was synthesized from 13 (34.7 mg, 0.0418
mmol) and tert-butyl methyl(2-(methylamino)ethyl)carbamate (9.46 mg,
0.0502 mmol) in a manner similar to that described for compound 6. A white
amorphous; Yield: 38.3 mg (quantitative yield). Step 2: Compound 15b was
synthesized from N-Boc protected 15b (38.3 mg, 0.0383 mmol) in a manner
similar to that described for compound 14a. A white amorphous; Yield: 34.1
mg (92% yield).
Compound 14f was synthesized from 13 (43.6 mg, 0.0529 mmol) and 4-(2-
aminoethyl)aniline (10.7 mg, 0.0786 mmol) in a manner similar to that
described for compound 6. A white solid; Yield: 36.6 mg (74% yield).
1H-NMR (400 MHz, CDCl3) δ: 7.95 (1H, s), 7.43-7.36 (2H, m), 7.30-7.17
(5H, m), 7.04 (2H, d, J = 8.2 Hz), 6.89 (1H, d, J = 9.1 Hz), 6.65 (2H, d, J =
8.2 Hz), 5.53-5.46 (1H, m), 4.83-4.72 (2H, m), 4.15-4.09 (1H, m), 4.08-4.03
(1H, m), 3.87 (1H, d, J = 8.2 Hz), 3.70-3.18 (6H, m), 3.33 (3H, s), 3.31 (3H,
s), 3.03 (3H, s), 2.82 (2H, t, J = 7.1 Hz), 2.45-2.35 (4H, m), 2.24 (6H, s),
2.18-1.89 (2H, m), 1.84-1.64 (4H, m), 1.40-1.20 (1H, m), 1.10 (3H, d, J = 7.3
Hz), 1.03-0.90 (17H, m), 0.81 (3H, t, J = 7.3 Hz). 13C-NMR (400 MHz,
CDCl3) δ: 173.95, 173.49, 172.35, 171.74, 170.35, 160.98, 149.81, 144.88,
136.73, 129.59, 129.29, 128.61, 128.43, 126.87, 122.70, 115.32, 85.35, 81.32,
76.42, 61.55, 60.48, 59.28, 57.94, 53.75, 52.64, 47.60, 43.93, 42.84, 40.86,
40.63, 37.38, 34.97, 32.87, 30.97, 27.60, 25.64, 24.95, 24.54, 23.58, 20.10,
17.63, 15.74, 14.05, 10.60. HRMS (ESI) m/z calcd for C51H78N8O7S 947.5787
[M+H]+, Found 947.5819.
1H-NMR (400 MHz, CD3OD) δ: 8.79-8.62 (1H, m), 8.15-8.02 (1H, m), 7.37-
7.18 (5H, m), 5.66-5.57 (1H, m), 4.85-4.63 (2H, m), 4.20-3.07 (20H, m), 3.27
(3H, s), 3.15 (3H, s), 2.91 (3H, s), 2.89 (3H, s), 2.51-1.39 (10H, m), 1.30-0.87
(21H, m), 0.86 (3H, t, J = 7.3 Hz). δ: 8.98-8.60 (1H, m), 8.14-7.92 (1H, m),
7.37-7.18 (5H, m), 5.66-5.57 (1H, m), 4.85-4.63 (2H, m), 4.20-3.07 (20H, m),
3.16 (3H, s), 2.92 (3H, s), 2.91 (3H, s), 2.48-1.39 (10H, m), 1.30-0.98 (21H,
m), 0.87 (3H, t, J = 7.1 Hz). 13C-NMR (400 MHz, CDCl3) (salt free) δ:
173.97, 173.57, 171.74, 171.65, 170.30, 164.27, 150.04, 136.89, 129.30,
128.40, 126.77, 124.45, 85.45, 81.56, 76.46, 60.51, 59.44, 58.86, 57.97, 53.76,
52.35, 50.53, 48.95, 47.61, 44.16, 42.84, 40.47, 37.41, 36.40, 34.50, 32.93,
30.97, 27.61, 25.66, 24.95, 24.57, 23.56, 20.10, 17.66, 15.78, 13.96, 10.63.
HRMS (ESI) m/z calcd for C47H78N8O7S 899.5787 [M+H]+, Found 899.5783.
2-((S)-1-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(Dimethylamino)-3-
methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-
methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-2-
phenylethyl)-N-(4-hydroxyphenethyl)thiazole-4-carboxamide (14g)

2-((S)-1-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(Dimethylamino)-3-
methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-
2-Hydroxyethyl
2-((S)-1-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-
(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-
methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-
methylpropanamido)-2-phenylethyl)thiazole-4-carboxylate (16b)
methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-2-
phenylethyl)-N-(2-hydroxyethyl)-N-methylthiazole-4-carboxamide (15c)
Compound 15c was synthesized from 13 (38.0 mg, 0.0458 mmol) and 2-
(methylamino)ethan-1-ol (3.70 mg, 0.0492 mmol) in a manner similar to that
described for compound 6. A white amorphous; Yield: 31.0 mg (85% yield).
Step 1: To a solution of 13 (43.0 mg, 0.0519 mmol) in CH2Cl2 (1.0 mL) was
added DMAP (1.27 mg, 10.4 µmol), Et3N (15.9 µL, 0.114 mmol), MNBA
(21.4 mg, 0.0622 mmol) and 2-((tetrahydro-2H-pyran-2-yl)oxy)ethan-1-ol
(9.10 mg, 0.0625 mmol) in CH2Cl2 (0.50 mL) at room temperature. After
stirring for 17 h at room temperature, water was added to the reaction mixture
and the aqueous layer was extracted with CHCl3. The combined organic
layers were washed with brine, dried over anhydrous Na2SO4, and
concentrated under reduced pressure. The residue was purified by column
chromatography on silica gel (CHCl3:MeOH = 1:0 to 9:1) to afford O-THP
protected 16b (36.3 mg, 0.0379 mmol, 73% yield) as a white amorphous.
Step 2: To a solution of O-THP protected 16b (36.3 mg, 0.0379 mmol) in
EtOH (1.0 mL) was added p-toluenesulfonic acid monohydrate (7.21 mg,
0.0379 mmol) at room temperature. After stirring for 18 h at room
temperature, the reaction mixture was concentrated under reduced pressure.
Saturated aqueous NaHCO3 was added to the resulting residue and the
aqueous layer was extracted with CHCl3. The combined organic layers were
washed with brine, dried over anhydrous Na2SO4, and concentrated under
reduced pressure. The residue was purified by column chromatography on
silica gel (CHCl3:MeOH = 1:0 to 9:1) to afford 16b (31.3 mg, 0.0358 mmol,
95% yield) as a white amorphous.
1H-NMR (400 MHz, CDCl3) δ: 7.84 (1H, s), 7.82-7.76 (1H, m), 7.30-7.00
(5H, m), 6.92 (1H, d, J = 9.1 Hz), 5.56-5.48 (1H, m), 5.07 (1H, br s), 4.87-
4.70 (2H, m), 4.18-3.20 (17H, m), 3.10 (3H, s), 3.02 (3H, s), 2.45-2.20 (4H,
m), 2.23 (6H, s), 2.11-1.89 (2H, m), 1.87-1.47 (4H, m), 1.40-1.30 (1H, m),
1.09 (3H, d, J = 6.9 Hz), 1.05-0.86 (17H, m), 0.80 (3H, t, J = 7.3 Hz). 13C-
NMR (400 MHz, CDCl3) δ: 174.05, 172.30, 171.79, 170.52, 169.95, 162.02,
149.31, 136.74, 129.29, 128.45, 126.90, 123.29, 85.49, 81.38, 76.41, 62.15,
61.54, 60.61, 59.54, 57.94, 53.80, 52.77, 47.66, 44.16, 42.86, 42.30, 40.67,
37.58, 32.84, 31.17, 27.62, 25.67, 24.95, 24.64, 23.56, 20.12, 17.65, 15.80,
14.22, 10.61. HRMS (ESI) m/z calcd for C46H75N7O8S 872.5314 [M+H]+,
Found 872.5323.
2-((S)-1-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(Dimethylamino)-3-
methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-
methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-2-
phenylethyl)-N-(2-mercaptoethyl)-N-methylthiazole-4-carboxamide (15d)
Step 1: S-Trt protected 15d was synthesized from 13 (39.0 mg, 0.0470 mmol)
and 2-(methylamino)ethan-1-ol (18.8 mg, 0.0564 mmol) in a manner similar
to that described for compound 6. A white amorphous; Yield: 44.5 mg (85%
yield). Step 2: Compound 15d was synthesized from S-Trt protected 15d
(44.5 mg, 0.0389 mmol) in a manner similar to that described for compound
14d. A white amorphous; Yield: 26.9 mg (77% yield).
1H-NMR (400 MHz, CDCl3) δ: 8.10 (1H, s), 7.49-7.44 (1H, m), 7.30-7.13
(5H, m), 6.90 (1H, d, J = 9.1 Hz), 5.55-5.48 (1H, m), 4.82-4.74 (2H, m), 4.50
(2H, t, J = 4.6 Hz), 4.20-3.26 (9H, m), 3.33 (3H, s), 3.32 (3H, s), 3.02 (3H, s),
2.46-2.34 (4H, m), 2.24 (6H, s), 2.11-1.90 (2H, m), 1.84-1.60 (4H, m), 1.43-
1.30 (1H, m), 1.09 (3H, d, J = 7.3 Hz), 1.03-0.89 (17H, m), 0.81 (3H, t, J =
7.5 Hz). 13C-NMR (400 MHz, CDCl3) δ: 174.07, 173.55, 172.97, 171.76,
170.47, 161.43, 146.29, 136.74, 129.32, 128.45, 127.98, 126.86, 85.42, 81.36,
76.39, 67.16, 67.07, 60.81, 60.52, 59.37, 57.93, 53.74, 52.91, 47.62, 43.99,
42.83, 40.46, 37.42, 32.97, 31.00, 30.95, 27.61, 25.64, 24.97, 24.49, 20.10,
17.66, 15.78, 14.10, 10.66. HRMS (ESI) m/z calcd for C45H72N6O9S 873.5154
[M+H]+, Found 873.5172.
1H-NMR (400 MHz, CDCl3) δ: 7.90 (1H, s), 7.60-7.45 (1H, m), 7.28-7.02
(5H, m), 6.90 (1H, d, J = 9.1 Hz), 5.58-5.51 (1H, m), 4.82-4.74 (2H, m),
4.20-3.20 (9H, m), 3.33 (3H, s), 3.32 (3H, s), 3.11 (3H, s), 3.03 (3H, s), 2.91-
2.77 (2H, m), 2.45-2.31 (4H, m), 2.24 (6H, s), 2.10-1.89 (2H, m), 1.86-1.60
(4H, m), 1.43-1.20 (1H, m), 1.11 (3H, d, J = 6.9 Hz), 1.05-0.90 (17H, m),
0.81 (3H, t, J = 7.3 Hz). 13C-NMR (400 MHz, CDCl3) δ: 173.93, 173.44,
171.72, 171.05, 170.34, 163.60, 150.02, 136.73, 129.30, 128.43, 126.83,
125.33, 85.46, 81.36, 76.42, 60.58, 59.31, 57.98, 54.16, 53.76, 53.40, 52.65,
47.61, 42.83, 42.79, 40.72, 37.38, 34.72, 32.91, 30.96, 27.61, 25.64, 24.98,
24.52, 23.59, 23.19, 20.10, 17.65, 15.77, 14.25, 10.63. HRMS (ESI) m/z calcd
for C46H75N7O7S2 902.5242 [M+H]+, Found 902.5233.
2-Mercaptoethyl
2-((S)-1-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-
(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-
methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-
methylpropanamido)-2-phenylethyl)thiazole-4-carboxylate (16c)
Step 1: S-Trt protected 16c was synthesized from 13 (39.0 mg, 0.0470 mmol)
and 2-(tritylthio)ethan-1-ol (18.1 mg, 0.0564 mmol) in a manner similar to
that described for compound O-THP protected 16b. A white amorphous;
Yield: 49.0 mg (92% yield). Step 2: Compound 16c was synthesized from S-
Trt protected 16c (49.0 mg, 0.0433 mmol) in a manner similar to that
described for compound 14d. A white amorphous; Yield: 31.8 mg (83%
yield).
2-Aminoethyl
2-((S)-1-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-
(dimethylamino)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-
methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-
methylpropanamido)-2-phenylethyl)thiazole-4-carboxylate (16a)
Step 1: N-Boc protected 16a was synthesized from 13 (38.0 mg, 0.0458
mmol) and tert-butyl (2-hydroxyethyl)carbamate (8.87 mg, 0.0552 mmol) in
a manner similar to that described for compound 6. A white amorphous;
Yield: 14.3 mg (32% yield). Step 2: Compound 16a was synthesized from N-
Boc protected 16a (14.3 mg, 0.0147 mmol) in a manner similar to that
described for compound 14a. A white amorphous; Yield: 6.8 mg (50% yield).
1H-NMR (400 MHz, CDCl3) δ: 8.07 (1H, s), 7.41 (1H, br s), 7.30-7.11 (5H,
m), 6.90 (1H, d, J = 9.1 Hz), 5.55-5.49 (1H, m), 4.83-4.74 (2H, m), 4.47 (2H,
t, J = 6.9 Hz), 4.13-3.11 (7H, m), 3.32 (6H, s), 3.03 (3H, s), 2.88 (2H, t, J =
6.9 Hz), 2.47-2.28 (4H, m), 2.24 (6H, s), 2.11-1.98 (2H, m), 1.98-1.55 (4H,
m), 1.41-1.30 (1H, m), 1.10 (3H, d, J = 6.9 Hz), 1.05-0.91 (17H, m), 0.81 (3H,
t, J = 7.3 Hz). 13C-NMR (400 MHz, CDCl3) δ: 173.95, 173.26, 171.66,
170.20, 169.83, 160.83, 146.21, 136.69, 129.29, 128.39, 127.78, 126.80,
85.36, 81.34, 76.35, 66.29, 61.48, 60.33, 58.95, 57.89, 53.67, 53.05, 47.51,
43.63, 42.78, 40.48, 37.40, 32.91, 30.92, 30.40, 27.56, 25.59, 24.94, 24.38,
23.13, 20.06, 17.61, 15.71, 13.90, 10.61. HRMS (ESI) m/z calcd for
C45H72N6O8S2 889.4926 [M+H]+, Found 889.4954.
1H-NMR (400MHz, CDCl3) δ: 7.98 (1H, s), 7.78 (2H, br s), 7.54 (1H, br s),
7.32-7.00 (5H, m), 6.90 (1H, d, J = 9.1 Hz), 5.51-5.45 (1H, m), 4.80-4.70 (2H,
m), 4.18-4.00 (2H, m), 3.82 (2H, t, J = 4.8 Hz), 3.80-3.10 (7H, m), 3.33 (3H,
s), 3.32 (3H, s), 3.05 (3H, s), 2.52-2.30 (4H, m), 2.24 (6H, s), 2.14-1.92 (2H,
m), 1.86-1.60 (4H, m), 1.40-1.19 (1H, m), 1.15-0.91 (20H, m), 0.82 (3H, t, J
= 6.4 Hz). 13C-NMR (400 MHz, CDCl3) δ: 174.05, 172.27, 171.81, 170.53,
169.97, 162.05, 149.30, 136.74, 129.30, 128.46, 126.92, 123.31, 85.49, 81.39,
76.44, 70.50, 62.21, 60.62, 59.56, 57.95, 53.80, 52.77, 47.67, 44.18, 42.88,
42.32, 40.70, 37.59, 32.83, 31.18, 30.98, 27.62, 25.67, 24.95, 24.65, 20.13,
17.65, 15.80, 14.22, 10.62. HRMS (ESI) m/z calcd for C45H73N7O8S 872.5314
[M+H]+, Found 872.5331.
PAMPA Studies25
The Corning Gentest Pre-coated PAMPA Plate System was used to perform
permeability assays. Compound solutions were prepared by diluting 10
mmol/L DMSO stock solution with 20% MeOH in PBS (final concentration:
200 µmol/L). The compound solutions were added to the wells (320 µL/well)

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of the receiver plate and 20% MeOH in PBS was added to the wells (200
µL/well) of the pre-coated filter plate. The filter plate was then coupled with
the receiver plate and the plate assembly was incubated at room temperature
for 5 h. At the end of the incubation, the plates were separated and 150 µL
solution from each well of both the filter plate and the receiver plate was
transferred to clean plates. The final concentrations of compounds in both the
donor wells and acceptor wells were analyzed by LC-MS (Waters Acquity
UPLC-SQD using a Sigma-Aldrich 2.7 µm, 20 × 2.1 mm Ascentis Express
C18 column and a 0.6 mL/min flow rate). The permeabilities of the
compounds were calculated using the following formula:
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where A = filter area (0.3 cm2), VD = donor well volume (0.32 mL), VA
=
acceptor well volume (0.2 mL), t = incubation time (18,000 s), CA(t) =
compound concentration in the acceptor well at time t, CD(t) = compound
concentration in the donor well at time t, and Ceq
[CD(t)*VD+CA(t)*VA]/(VD+VA).
=
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(human Caucasian ovary adenocarcinoma), A549 (human Caucasian lung
carcinoma), and L1210 (mouse lymphocyte leukemia) cancer cell lines. Cells
were seeded in clear tissue-culture treated 96-well plates at approximately
5000−50000 cells per well in 200 µL of growth media (DMEM for the
SKOV-3 and A549 cells and RPMI-1640 for the L1210 cells, + 10% heat-
inactivated fetal bovine serum) and incubated overnight in a humidified
incubator at 37°C with 5% CO2. Stock solutions of the test compounds in
DMSO were diluted with growth media (DMEM or RPMI-1640 + 1% heat-
inactivated fetal bovine serum, without folic acid). The next day, 200 µL of a
300× stock of vehicle control (DMSO) or compound at various
concentrations was added to each well in triplicate. The plates were then
incubated in the humidified tissue culture incubator with 5% CO2 at 37°C for
2 days to measure cytotoxicity, and then 40 µL of Cell Titer 96 Aqueous One
Solution Reagent (Promega, Catalog No.G358B) was added to each well.
Plates were incubated at 37°C for 1 to 2 h to allow Cell Titer uptake, then the
absorbance at 490 nm was measured using a Microplate Reader (Bio-Rad,
Model 680). Data for compounds tested in this assay are graphed as percent
survival compared to untreated control wells.
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Tubulin Inhibition.
The inhibition of tubulin polymerization was evaluated using an HTS-
Tubulin Polymerization Assay Kit (Cytoskeleton Inc.; Catalog No. BK011P)
for porcine brain tubulin. Tubulin was seeded at approximately 100 µg per
well in 50 µL of general tubulin buffer, and then treated with 3, 1, or 0.3 µM
final concentration of compound in duplicate at the start of the assay. Tubulin
polymerization assays were carried out at 37°C for 60 min after addition of
the test compounds. Tubulin polymerization was determined by absorbance
spectroscopy using the optical density value at 360 nm (fluorescence
emission at 450 nm) each minute after addition of the test compounds. The
extent of tubulin polymerization by the compound-treated tubulin was
compared to that of buffer-treated tubulin.
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Acknowledgements
We are thankful to Mr. K. Goto (Toray Research Center, Inc.) for
recording HRMS spectra.
Funding: This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
16. Kolakowski RV, Haelsig KT, Emmerton KK, Leiske CI,
Miyamoto JB, Cochran JH, Lyon RP, Senter PD, Jeffrey SC. The
Methylene Alkoxy Carbamate Self-Immolative Unit: Utilization
for the Targeted Delivery of Alcohol-Containing Payloads with
Antibody–Drug Conjugates. Angew Chem Int Ed. 2016;55:1.
17. Kern JC, Cancilla M, Dooney D, Kwasnjuk K, Zhang R,
Beaumont M, Figueroa I, Hsieh S, Liang L, Tomazela D, Zhang J,
Brandish PE, Palmieri A, Stivers P, Cheng M, Feng G, Geda P,
Shah S, Beck A, Bresson D, Firdos J, Gately D, Knudsen N,
Declaration of interests: None.
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