Rational Design of Azastatin as a Potential ADC Payload with Reduced Bystander Killing

Article abstract of DOI:10.1002/cmdc.202000497

Auristatins are a class of ultrapotent microtubule inhibitors, whose growing clinical popularity in oncology is based upon their use as payloads in antibody-drug conjugates (ADCs). The most widely utilized auristatin, MMAE, has however been shown to cause apoptosis in non-pathological cells proximal to the tumour (“bystander killing”). Herein, we introduce azastatins, a new class of auristatin derivatives encompassing a side chain amine for antibody conjugation. The synthesis of Cbz-azastatin methyl ester, which included the C2-elongation and diastereoselective reduction of two proteinogenic amino acids as key transformations, was accomplished in 22 steps and 0.76 % overall yield. While Cbz-protected azastatin methyl ester (0.13–3.0 nM) inhibited proliferation more potently than MMAE (0.47–6.5 nM), removal of the Cbz-group yielded dramatically increased IC₅₀-values (9.8–170 nM). We attribute the reduced apparent cytotoxicity of the deprotected azastatin methyl esters to a lack of membrane permeability. These results clearly establish the azastatins as a novel class of cytotoxic payloads ideally suited for use in next-generation ADC development.

Full text of DOI:10.1002/cmdc.202000497

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Rational Design of Azastatin as a Potential ADC Payload
with Reduced Bystander Killing
Rafael W. Hartmann,[a] Raphael Fahrner,[b] Denys Shevshenko,[b] Mårten Fyrknäs,[c]
Rolf Larsson,[c] Fredrik Lehmann,[b] and Luke R. Odell*[a]
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Auristatins are a class of ultrapotent microtubule inhibitors,
whose growing clinical popularity in oncology is based upon
their use as payloads in antibody-drug conjugates (ADCs). The
most widely utilized auristatin, MMAE, has however been shown
to cause apoptosis in non-pathological cells proximal to the
tumour (“bystander killing”). Herein, we introduce azastatins, a
new class of auristatin derivatives encompassing a side chain
amine for antibody conjugation. The synthesis of Cbz-azastatin
methyl ester, which included the C2-elongation and diastereo-
selective reduction of two proteinogenic amino acids as key
transformations, was accomplished in 22 steps and 0.76%
overall yield. While Cbz-protected azastatin methyl ester (0.13–
3.0 nM) inhibited proliferation more potently than MMAE (0.47–
6.5 nM), removal of the Cbz-group yielded dramatically in-
creased IC50-values (9.8–170 nM). We attribute the reduced
apparent cytotoxicity of the deprotected azastatin methyl esters
to a lack of membrane permeability. These results clearly
establish the azastatins as a novel class of cytotoxic payloads
ideally suited for use in next-generation ADC development.
Introduction
termed auristatins. The second generation of analogues is
characterized by the replacement of the N-terminal dimeth-
ylamino group with a nucleophilic secondary[11–13] or primary
amine.[14] These synthetic handles have been widely exploited
to conjugate the cytotoxins to tumour-specific antibodies in
order to mitigate toxicity and widen the therapeutic window
substantially. Today, three antibody–drug conjugates (ADCs)
derived from MMAE (monomethylauristatin E, 5) have received
marketing authorization.[15–18] Together with ADCs encompass-
ing MMAF (monomethylauristatin F, 6), the two payloads are
represented in at least a dozen clinical studies,[19,20] while Pfizer’s
development of an ADC armed with Aur0101 (4) ended in
Phase I due to its severely limited therapeutic window.[21]
Despite the success of some antibody-auristatin conjugates,
none of the payloads are without flaw. Due to its pronounced
cellular permeability, MMAE (5) is able to diffuse out of the
neoplastic target cell upon antibody detachment and spread
into neighbouring tissues in a process called “bystander killing”.
While this behaviour may in some instances be desirable to
target antigen-negative cancer cells and can deliver cytotoxicity
to non-vascularised sections of solid tumours, it can also cause
harm to healthy tissues.[22,23] MMAF (6), which is zwitterionic and
therefore less permeable upon release, is harmless for by-
stander cells by comparison.[22,24–26]
The differences between 5 and 6 highlight a difficulty
frequently encountered when comparing non-conjugated ADC-
payloads in vitro: In the absence of an antibody capable of
transporting the small molecules across the lipid membrane,
impermeable payloads, such as 6, appear substantially less
cytotoxic than their permeable congeners. As proven by the
comparable clinical utility of ADCs derived from either 5 or 6,
this difference is largely due to differences in permeability,[26] as
opposed to the payloads’ abilities to inhibit their cellular target.
To accommodate this discrepancy, we therefore propose the
use of the terms apparent cytotoxicity and inherent potency. An
Dolastatin 10 is a natural product first isolated by Pettit et al
from the marine mollusc Dolabella auricularia in 1987 (see
Figure 1). Despite the discovery of even more potent inhibitors
of cell division since,[1,2] the pentapeptide remains among the
most potent antineoplastic agents known to man,[3] owing to its
inhibition of tubulin polymerisation and GTP hydrolysis.[4]
Unfortunately, clinical development soon revealed that the
compound could cause granulocytopenia,[5,6] which prohibited
its administration at doses sufficient to treat breast,[7] lung,[8]
and prostate cancers.[9]
Structural modification by the Pettit[10] and Miyazaki[11]
groups yielded the first generation of synthetic analogues
[a] R. W. Hartmann, Prof. L. R. Odell
Department of Medicinal Chemistry
Uppsala University
Box 574
75123 Uppsala (Sweden)
E-mail: luke.odell@ilk.uu.se
[b] R. Fahrner, Dr. D. Shevshenko, Dr. F. Lehmann
Synthesis Division
Recipharm OT Chemistry
Virdings allé 32b
75450 Uppsala (Sweden)
[c] Prof. M. Fyrknäs, Prof. R. Larsson
Department of Medical Sciences, Cancer Pharmacology and Computational
Medicine
Uppsala University
University Hospital
75185 Uppsala (Sweden)
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202000497
This article belongs to the Special Collection “Nordic Medicinal Chemistry
2019–2020”
© 2020 The Authors. Published by Wiley-VCH GmbH. This is an open access
article under the terms of the Creative Commons Attribution License, which
permits use, distribution and reproduction in any medium, provided the
original work is properly cited.
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cause ADC-aggregation is likely linked to its relative hydro-
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phobicity and thus goes hand-in-hand with its propensity to
cause bystander killing. This hypothesis also explains the
striking differences between MMAE and MMAF on both counts.
Lastly, MMAE has been found to be slightly less cytotoxic in
vitro than its auristatin analogue,[29] highlighting the importance
of the N-terminal dimethylamino-moiety as found in dolastatin
10 for inherent potency.
The majority of SAR-studies have examined the effects of
variations of the N- and C-terminal amino acids.[26] Out of the
five amino acids, the least SAR-data is available for modifica-
tions of the Dap residue, presumably due to the considerable
synthetic effort required to generate analogues thereof. At the
onset of our study, there were no literature examples exploring
Dap-SAR and since, only one literature account has been
published.[30]
Compound 7, which we have named azastatin methyl ester
(azastatin-OMe, see Figure 2), was designed to ameliorate the
pharmacokinetic and physicochemical issues associated with
auristatins. Notably, the target structure contains a primary
amine in the (4-pyrrolidine)-position of the Dap residue, offering
an additional diversification point and a synthetic handle for
bioconjugation and subsequent ADC synthesis. Furthermore,
retention of the important N-terminal Dov binding motif should
not only enhance potency relative to monomethylauristatins
but also substantially reduce ADC hydrophobicity and aggrega-
tion.
Lastly, to imitate the relatively low permeability associated
with MMAF, its C-terminal phenylalanine residue was accom-
modated in the target molecule. The C-terminal carboxylic acid
was masked as its corresponding methyl ester to enable in vitro
cytotoxicity evaluation without the need for prior antibody
conjugation. As the C-terminal methyl ester of MMAF (6-OMe)
has been found to display a pronounced apparent cytotoxicity
and is frequently used as a benchmark in auristatin-SAR,[14,26] we
hypothesized that this analogous modification would not
distort the inherent potency of azastatin.
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Figure 1. The structural evolution of auristatins. While dolastatin 10, their
natural predecessor, has failed to gain clinical relevance, three antibody–
drug conjugates armed with a second generation auristatin have received
marketing authorization and are used in cancer treatment.
inherently potent tubulin-inhibitor such as 5 will only appear
cytotoxic if it can reach its target; if it cannot, as in the case of
6, its IC50-value is uninformative and the concept of inherent
potency is more useful to predict its utility as an ADC payload.
In fact, a warhead incapable of causing bystander-killing must
be impermeable and therefore display a low degree of apparent
cytotoxicity while retaining high inherent potency.
Moreover, the deficiencies associated with the use of
auristatins as ADC-payloads are not confined to bystander-
killing: Conjugation of a relatively hydrophobic auristatin to a
hydrophilic antibody can cause the protein to aggregate,
diminishing its biological half-life and thereby therapeutic
utility.[27] When taking into account other, even more hydro-
phobic ADC-payloads of other classes,[28] MMAE’s tendency to
Figure 2. Azastatin-OMe (7), the target molecule of the work outlined herein.
The Val-Dil core common to dolastatin 10 and its derivatives is highlighted
in blue, two key structural elements at the N- and C-termini are marked
green. The additional amino moiety intended for antibody conjugation is
drawn in purple.
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In this work, we describe the synthesis of azastatin-OMe (7)
Synthesis of N-terminal tripeptide
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and two of its congeners as potential ADC-payloads. Their
apparent cytotoxicity towards three tumour cell lines was
subsequently assessed and the differences were used to
extrapolate the hypothetical, inherent potency of the azastatin
core to gain insight into its potential utility in ADC research.
Reaction of commercially obtained para-nitrophenyl ester 12
with tert-butyl acetate lithium enolate 13 resulted in β-keto
ester 9a in 84% yield (see Scheme 1). Sodium borohydride
mediated reduction in DCM/iPrOH (15:1) then gave a mixture
of alcohols in a ratio of 5:1 in favour of the desired (3R)-
diastereomer (83% yield of diastereomer mixture). In our hands,
the use of this solvent mixture was preferable to a previously
published method using methanol,[11,31] in which we found the
diastereoselectivity to be negatively impacted by scale-up. A
method involving the use of lithium borohydride in THF has
also been published.[32]
The diastereomeric mixture was subsequently dimethylated
using methyl triflate to yield N,O-dimethyl intermediate 8a,[31]
which could be separated from the (3S)-diastereomer by flash
chromatography (60% yield of pure diastereomer). In order to
assess the relative configuration of the newly formed methoxy-
substituted stereocenter in 8a, it was cyclized to give lactam
18. The pyrrolidinone was spectroscopically identical to pre-
vious literature reports.[11,33,34] The Cbz-group was removed by
cyclohexene-mediated transfer hydrogenation, followed by
Results and Discussion
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Retrosynthetic analysis
While three of the five amino acids encompassing the target
molecule were commercially available (L-valine, L-phenylala-
nine) or could be generated from a commercial source in a
trivial fashion (N,N-dimethyl-L-valine), the two central γ-amino
acids (dolaisoleucine, dolaproine) deserved retrosynthetic atten-
tion (see Figure 3). In keeping with their structural similarities,
we envisioned a forward synthesis starting from analogous α-
amino acid esters (10a and 10b). The stage 1 substrates derived
from L-isoleucine and hydroxyproline, respectively, were to be
C2-elongated to β-keto-γ-amino acid esters (9a and 9b) by
reaction with enolates derived from either tert-butyl acetate
(11a) or 2-methyl malonate 11b. These stage 2 ketones were
hypothesized to lend themselves to diastereoselective carbonyl
reduction followed by N/O-methylation to yield the desired
stage 3 analogues, i. e. β-methoxy-γ-amino acid esters 8a and
8b.
Figure 3. Retrosynthetic analysis of the target molecule. Both of the key γ-
amino acids dolaisoleucine and 4-aminodolaproine were to be obtained
from their respective α-amino acid homologues (stage 1) in an analogous
fashion: C2-elongation was to yield β-keto esters (stage 2), which were to be
reduced diastereoselectively to furnish highly chiral β-methoxy esters (stage
3).
Scheme 1. Synthesis of Dov-Val-Dil-OtBu, 15. Reagents and conditions: a)
tBuOAc, HNiPr2, nBuLi, THF, 84%. b) NaBH4, SiO2, DCM/iPrOH 15:1, 83% (5:1
mixture of diastereoisomers). c) MeOTf, LiHMDS, DMEU, THF, 60%. d) 1.
Cyclohexene, Pd/C, MeOH. 2. HCl/Dioxane, 90%. e) Cbz-Val-OH, BEP, NEtiPr2,
DCM, 85%. f) H2, Pd/C, MeOH. g) 17, DECP, NEt3, DCM, 54% over two steps.
h) CH2O, H2, Pd/C, H2O/MeOH, 99%. i) H2, Pd/C, EtOAc/MeOH 3:1, 53%.
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precipitation of the free amine from ethereal HCl to obtain
dolaisoleucine hydrochloride in 90% yield.[34]
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2-Bromo-1-ethylpyridinium tetrafluoroborate (BEP)[35] was
used to couple the secondary amine to Cbz-L-valine to give
dipeptide 14 (85% yield).[36] Compound 14 was deprotected by
means of hydrogenation while dolavaline (17) was furnished by
reductive amination of aqueous formaldehyde with L-valine (17,
99% yield).[37] Finally, the two fragments were coupled to yield
Dov-Val-Dil-OtBu (15) in 54% yield.[34]
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Synthesis of C-terminal dipeptide
To enable C2-elongation, pentafluorophenyl ester 20 (see
Scheme 2A) was prepared from trans-4-hydroxy-L-proline 19 by
Boc-protection, acetylation and DDC-mediated ester formation
in 60% yield over three steps. Electrophile 20 was subsequently
treated with magnesium complex 21 to yield 9b as a 1:1
mixture of epimers (72% yield, 15% of unreacted 20). The
synthetic sequence toward ketone 9b is based on Genet and
coworkers’ use of carbonyldiimidazol to activate Boc-N-proline
in situ, followed by an analogous quench with reagent 21,
which yielded desacetoxy-9b in 82% yield.[38]
In the presence of manganese dichloride, the subsequent
sodium borohydride mediated reduction of the β-oxoester 9b
yielded a mixture of diastereoisomers of 22 in an approximate
ratio of 9:1:9:1. Pleasingly, the major diastereomers could be
separated by flash chromatography.
Numerous authors have demonstrated the reliability of
cyclisation of desacetoxy-22 when faced with the task to assign
the relative conformation of the stereocenters α and β to the
ester carbonyl group.[38–40] Reflective of this approach, each
diastereomerically enriched fraction of 22 was treated with
trifluoroacetic acid to cause Boc-cleavage and upon basification,
the corresponding dihydroxy-γ-lactams (23) were obtained (see
Scheme 3). Nuclear Overhauser effect (NOE) spectroscopy of
one of the lactams revealed the spacial proximity of the α-
carbonyl hydrogen atom (H2) with that bound to the bridge-
head (H4a), indicating that the α-carbon was in the (R)-
configuration. The methyl group bound at the same carbon
showed an NOE-interaction with the proton vicinal to the OH-
group in the pyrrolidine-ring (H3), suggesting that this stereo-
center was also in the (R)-configuration. Based on this data, the
parent γ-amino acid was assigned to be the desired (R,R)-
diastereomer of 22.
Scheme 2. A) Synthesis of key intermediate 22. B) Rationalization of the syn-
selectivity of the reduction of 9b by means of the Cram chelate model.
Reagents and conditions: a) Boc2O, NaOH, H2O/THF. b) Ac2O, pyridine, DCM.
c) C6F5OH, DCC, EtOAc, 60% over three steps. d) EtO2C-CH(Me)-CO2H,
iPrMgBr, THF, 72% (15% of unreacted 20). e) NaBH4, MnCl2, MeOH, 95%.
35% of (R,R)-22 upon purification by flash chromatography on silica.
An analogous transformation followed by NOE spectro-
scopic analysis of the other major reduction product revealed
the spacial proximity of the methyl group, H3, and H4a,
suggesting that the lactam was (S,S)-configured. On the basis of
these findings, the parent γ-amino acid was concluded to be
(S,S)-22.
The marked syn-selectivity of the reduction (i. e. preference
for the formation of (R,R)- and (S,S)-22) has been described in
literature,[41] and can be rationalized by applying a modification
of the Cram chelate model (see Scheme 2B).[42] Based on this
reasoning, it appears likely that the predominant factor
determining the stereochemical outcome of the reduction is
the configuration of the methyl-substituted α-carbon, while the
two stereocenters on the pyrrolidine ring do not exert a
substantial degree of stereochemical induction. This behaviour
markedly distinguishes ketones 9a and 9b (see Figure 3), which
can be considered functionally equivalent in light of their
analogous roles as prochiral stage 2 intermediates.
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ester furnished Boc-4-(Cbz-amino)-Dap-Phe-OMe (26) in 58%
yield over four steps.
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Endgame peptide assembly
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tert-Butyl deprotection of 15 and Boc-deprotection of 26 were
accomplished by treatment with trifluoroacetic acid in dichloro-
methane (see Scheme 5). DEPC-mediated amide coupling[25]
yielded Cbz-protected pentapeptide 27. While hydrogenation in
ethyl acetate failed to remove the Cbz-group, the same reaction
in methanol only yielded mono- and dimethylated products as
opposed to the desired azastatin-OMe (7). On a test scale,
hydrogenation in ethanol yielded 7 seemingly quantitatively,
but when the reaction was scaled up, a 3:2 mixture of 7 and its
N-ethyl analogue 28 was obtained in a combined yield of 98%.
Pleasingly, separation of 7 and 28 was accomplished by flash
chromatography.
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Scheme 3. Stereochemical characterization of both major diastereomers of
22 by means of 1H-NMR and 1H-1H nuclear Overhauser effect (NOE)
spectroscopy. Selected spacial interactions are symbolized by red arrows.
Reagents and conditions: a) TFA/DCM 1:1. b) Cs2CO3, MeOH.
The target diastereomer (R,R)-22 was methylated using
dimethyl sulfate and sodium hydride to yield 8b in 80% yield
(see Scheme 4). The acetyl protecting group was selectively
removed by basic hydrolysis in aqueous methanol in 97% yield,
followed by azide-Mitsunobu inversion to yield 24 (85% yield).
Reduction of the newly introduced azide was accomplished by
hydrogenation, followed by Cbz-protection of the resulting
amine to yield intermediate 25. Cleavage of the ethyl ester
without epimerization proved difficult but was achieved by
hydrolysis in a mixture of excess lithium hydroxide in aqueous
Biological evaluation
Three fundamentally different cell lines were chosen for the
assessment of cytotoxicity: HepG2 cells, which derive from a
human hepatoblastoma;[43] HCT116 cells, a common model for
the study of metastatic colorectal carcinoma;[44,45] and RPMI
8226 cells, which originate from multiple myeloma.[46]
The cells, upon overnight cultivation, were treated with
MMAE (5), MMAF (6), azastatin-OMe (7), N-ethyl azastatin-OMe
(28), N-Cbz azastatin-OMe (27) or doxorubicin[47,48] at concen-
trations ranging from 100 μM to 10 pM and after 72 hours of
incubation, the survival index relative to control was deter-
mined by The Fluorometric Microculture Cytotoxicity Assay
(FMCA).[49] Based upon the concentration dependent proportion
of surviving cells, the half maximal inhibitory concentration
(IC50) of each compound was calculated (see Table 1).
°
methanol at 0 C for two days. Finally, HATU-mediated amide
coupling with commercially obtained L-phenylalanine methyl
Scheme 4. Synthesis of C-terminal dipeptide 26. Reagents and conditions: a)
NaH, Me2SO4, THF/DMF 3:1, 80%. b) K2CO3, MeOH/H2O 40:1, 97%. c) DIAD,
DPPA, PPh3, THF, 85%. d) H2, Pd/C, EtOAc. e) CbzCl, NEt3, DCM. f) LiOH,
MeOH/H2O, 1:1. g) H2N-Phe-OMe x HCl, HATU, NEtiPr2, DCM, 58% over four
steps.
Scheme 5. Ligation of the N-terminal tripeptide 15 and C-terminal dipeptide
26 to yield Cbz-Azastatin-OMe, 27. The final deprotection yielded both
Azastatin-OMe, 7 and its N-ethyl analogue, 28. Reactions and conditions: a)
DCM/TFA 1:1. b) DCM/TFA 8:3. c) DECP, NEt3, DME, 75% over three steps. d)
H2, Pd/C, EtOH, 98% (mixture of 7 and 28).
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170 nM, IC50,RPMI-8226 =9.8 nM) and N-ethyl azastatin-OMe (28,
IC50,HepG2 =11 nM, IC50,HCT116 =100 nM, IC50,RPMI-8226 =4.8 nM),
Table 1. Structures and IC50-values of three new auristatins (7, 28, 27)
compared to well-established monomethyl-auristatins and doxorubicin in
three cancer cell lines. Image: Predominant charge states of all five
auristatins at physiological pH.
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9
which both appeared substantially less cytotoxic in all cell lines
than MMAE (5). Both compounds are expected to be predom-
inantly dicationic at physiological pH, which likely diminishes
their cellular permeability. It may therefore be hypothesized
that, like the zwitterionic MMAF (6), both azastatin-OMe (7) and
N-ethyl azastatin-OMe (28) are characterized by a high degree
of inherent potency, which differs from their apparent cytotox-
icity only due to their limited permeability. This interpretation
of the data is in agreement with the observation that the
structurally similar, monocationic N-Cbz azastatin-OMe (27), to
which the cellular membrane poses much less of an obstacle on
its way to the target protein, displays exquisite cytotoxicity.
Lastly, it appears noteworthy to point out that the slightly less
polar N-ethyl azastatin-OMe (28) displays a marginally superior
cytotoxicity compared to azastatin-OMe (7). Assuming that the
decreased hydrophilicity of N-ethyl azastatin-OMe (28) accounts
for a somewhat increased propensity to penetrate the cell
membrane relative to azastatin-OMe, this lends additional
credibility to the hypothesis of permeability-limited potency.
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[a]
IC50
Compound
HepG2
HCT116
RPMI 8226
MMAE, 5
0.52
(0.42-0.65)
130
6.5
(5.4–7.8)
2700
0.47
(0.43–0.52)
200
MMAF, 6
(110–150)
20
(16–25)
11
(8–13)
0.13
(0.11–0.16)
400
(2100–3300)
170
(140–200)
100
(84–120)
3.0
(2.1-4.3)
440
(170–240)
9.8
(8.0–12)
4.8
(4.2-5.5)
0.15
(0.13–0.17)
49
Aza-OMe, 7
N-Et Aza-OMe, 28
Cbz-Aza-OMe, 27
Doxorubicin
Conclusion
(320–520)
(410–470)
(40–59)
Three novel auristatins were synthesized with the goal of
capturing the inherent potency of dolastatin 10 (1) and
decreased efflux permeability of monomethyl auristatin F (6).
Based on this rationale, the target molecules were designed to
encompass dolastatin’s N-terminal dolavaline, an additional
amino-moiety at the pyrrolidine core, and MMAF’s C-terminal
phenylalanine residue masked with a methyl group. The
synthesis of the two central γ-amino acids of the pentapeptides
was achieved by a chiral-pool approach starting from isoleucine
and hydroxyproline, respectively.
In addition to the novel payloads, the well-characterized,
structurally similar cytotoxins MMAE (5) and MMAF (6) were
tested for their tumoricidal effects. By confirming the stark
differences in the IC50-values of the permeable MMAE (0.47–
6.5 nM) and impermeable MMAF (130 nMÀ 2.7 μM) also re-
ported elsewhere,[26] this work serves as additional evidence in
favour of the hypothesis of permeability-controlled apparent
cytotoxicity of auristatins. In the context of antibody-mediated
drug delivery, intrinsic membrane permeability of the payload
is however not required for it to reach antigen-positive target
cells.[23] As we have argued, one may in fact want to suppress
intrinsic permeability so as to mitigate bystander-killing, the
process by which a cytotoxin diffuses out of the target cell to
which it was delivered and harms neighbouring tissues
indiscriminately. We have therefore chosen to differentiate
between an analyte’s apparent cytotoxicity (i. e. the antineo-
plastic activity of payload as measured in vitro) and inherent
potency (i. e. the effective, not directly measurable ability of a
payload to cause apoptosis once released inside the cancer
cell).
[a]Mean IC50-values and 95% confidence intervals [nM] based on three
independent experiments (see SI).
MMAE (5) was found to be active in all three cell lines but
proved to be an order of magnitude less potent in HCT116 cells
(IC50 =6.5 nM) than in HepG2 (IC50 =0.52 nM) and RPMI 8226
cells (IC50 =0.47 nM). The same trend was observed for MMAF
(6), which was more than 100-fold less potent in each of the
cell lines (IC50,HCT116 =2.7 μM, IC50,HepG2 =130 nM, IC50,RPMI-8226
=
200 nM). These findings are in line with literature reports and
suggest that the decreased potency of MMAF (6) can be
attributed to its decreased cellular permeability as opposed to a
lower inherent potency, i.e. a diminished ability to inhibit the
function of its target protein.[22,24–26] Strikingly, in all but one cell
line, MMAF (6) appeared less cytotoxic than even doxorubicin
(IC50,HCT116 =440 nM, IC50,RPMI-8226 =32 nM), which is generally
considered insufficiently potent to be an ADC-payload.[50,51] The
only cell line in which MMAF (6) was observed to be marginally
more active than doxorubicin (IC50 =400 nM) was HepG2.
Like MMAE (5) and MMAF (6), N-Cbz azastatin-OMe (27)
more potently inhibited cell division in HepG2 (IC50 =0.13 nM)
and RPMI 8226 (IC50 =0.15 nM) than in HCT116 cells (IC50 =
3.0 nM), but exceeded even MMAE (5) in cytotoxicity by a factor
of more than two in all cell lines. In agreement with the
biological data published for other auristatins, these findings
suggest that the N-terminal dimethylamino group increases
potency,[26,29] and that nitrogen-substitution at the 4-position of
dolaproine does not diminish it.[30] The increased potency of N-
Cbz azastatin-OMe (27) relative to MMAE (5) and MMAF (6) is in
Cbz-protected azastatin methyl ester (27), whose cellular
influx was not stalled by the presence of a charged ammonium
stark contrast to azastatin-OMe (7, IC50,HepG2 =20 nM, IC50,HCT116
=
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spectra at 376 MHz and 13C-spectra at 101 MHz. All NMR-experi-
ments were performed using commercially obtained, deuterated
solvents with no further purification (Sigma-Aldrich/Merck, Ger-
many). DMSO-d6 was stored over molecular sieves (4 Å, Sigma-
Aldrich/Merck, Germany). NMR spectra were processed and inter-
preted using MestreNova (Mestrelab, Spain). High-resolution mass
spectra were acquired using a Premiere Q-TOF mass spectrometer
(Waters, US) operating in ES+ mode. The analytes were introduced
into the mass spectrometer after chromatography on an Acquity
UPLC system equipped with an Acquity UPLC BEH C18 (2.1×
100 mm) column (both Waters, US), running a gradient of 0.1%
formic acid in acetonitrile (5!95%) in 0.1% aqueous formic acid
over 6 min. Mass spectra and chromatograms were processed using
MassLynx and UNIFI computer software (Waters, US). Optical
rotation measurements were performed using an Autopol II S2
polarimeter equipped with 2.0 mL cuvette (both Rudolph Research
Analytical, US) with pathlength l=1 dm.
residue, displayed exquisite potency in all tested cell lines
(0.13–3.0 nM). The model peptide’s cytostatic prowess is
illustrative of the activity enhancement associated with dolava-
line incorporation and indicative that 4-pyrrolidine substitution
does not diminish inherent potency. On the other hand,
azastatin methyl ester (7) and its N-ethyl analogue (28), both of
which are less membrane-permeable than either MMAE (5) or
their Cbz-protected congener, exhibited a substantially reduced
apparent cytotoxicity in all cell lines (9.8–170 nM and 4.8–
100 nM, respectively).
Most forms of cancer remain incurable. While antibody–
drug conjugates represent a cutting-edge approach to extend-
ing patients’ lifespans, they have yet to find their way into first-
line treatment. The biological data portrayed herein suggest
that azastatins, which represent a novel class of payload for
antibody conjugation, may be ideally suited for the job. The
example of Cbz-azastatin methyl ester (27) serves to highlight
their inherent potency while the substantially decreased
cytotoxicity of non-protected azastatins (7 and 28, respectively)
can be extrapolated to indicate minimal bystander killing in the
ADC-context. We hope that the altered physicochemical proper-
ties of azastatins may yield ADCs with a wider therapeutic
window and ultimately constitute a steppingstone towards
propelling targeted therapeutics into first-line cancer treatment.
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NCbz-β-keto-γ-amino acid tert-butyl ester 9a
A three-necked, 250 mL round-bottom flask equipped with a
nitrogen inlet, thermometer and stirring bar was charged with
diisopropylamine (9.0 mL, 64.2 mmol, 3.1 eq.) and THF (35 mL) and
°
the resulting solution was cooled to À 78 C. Dropwise, over the
period of 15 minutes, n-butyllithium (2.5 M solution in hexanes,
25 mL, 62.5 mmol, 3.0 eq.) was added while the temperature inside
°
the reaction mixture was kept below À 45 C. After 30 minutes of
stirring, tert-butyl acetate (8.4 mL, 62.6 mmol, 3.0 eq.) was added
dropwise over the period of 10 minutes while the temperature
°
inside the flask was kept below À 55 C. The reaction mixture was
stirred for one hour on dry ice. Over the period of one hour, a
solution of 12 (8.0 g, 20.7 mmol, 1.0 eq.) in THF (34 mL) was added
dropwise, while the temperature inside the flask was kept below
Experimental Section
Synthesis and purification: General
°
À 55 C. After two hours, the reaction mixture was allowed to warm
°
to 0 C and the remaining enolate was quenched by the addition of
All solvents used in the synthesis and purification of the
compounds described herein were purchased from Chemtronica
(Sweden) and used without further purification. Solvents used in
non-aqueous reactions were stored over molecular sieves (3 Å or
4 Å, Sigma-Aldrich/Merck, Germany) prior to use. Cbz-L–Isoleucine
para-nitrophenyl ester was obtained from abcr (Germany). All other
starting materials and reagents were purchased from Sigma-
Aldrich/Merck (Germany). Non-aqueous reactions were carried out
in an atmosphere of nitrogen, which was pre-dried using a Drierite
gas drying unit (W.A. Hammond Drierite Company, US). Unless
otherwise noted, reactions were monitored using an Agilent (US)
1100 series LC/MS (single quadrupole) system equipped with an
electrospray interface, a UV diode array detector and an ACE3 C8
(3.0×50 mm) column (ACE, UK) with a gradient of acetonitrile (10!
97%) in 0.1% aqueous trifluoracetic acid over 3 min and a flow of
1 mL/min or an XBridge C18 (3.0×50 mm) column (Waters, US)
with a gradient of acetonitrile (5!97%) in 10 mM aqueous
ammonium bicarbonate. When analytes were too polar to allow for
facile reaction monitoring by reverse phase chromatography, TLC
on aluminium supported silica plates (Merck, Germany) was used to
monitor reactions, instead. Flash chromatography was performed
either manually using 40–63 μm silica (Carlo Erba, Italy) or
automatically using a CombiFlash Rf+ Lumen flash machine (Tele-
dyne Isco, US) equipped with a wide-range UV and evaporative
light scattering (ELS) detector and prepacked silica columns
(SiliCycle, Canada).
°
saturated aqueous NH4Cl (10 mL, temperature below 5 C). The
mixture was allowed to attain room temperature and was diluted
with water (100 mL) and EtOAc (100 mL). The phases were
separated, and the aqueous phase was extracted with EtOAc (3×
100 mL). The combined organic layers were washed with half-
saturated brine (100 mL), dried over MgSO4, filtered and the solvent
was removed under reduced to yield a yellow syrup. The latter was
taken up in DCM (200 mL), silica (50 g) was added and the solvent
was gently removed to yield a dry, yellow powder. The latter was
washed with EtOAc/heptane (1:1, 400 mL) and the solvent was
removed in vacuo to yield a yellow oil, which was purified by flash
chromatography on silica (petroleum ether/EtOAc 9:1!3:1) to
yield 9a as a pale yellow oil (6.3 g, 17.4 mmol, 84% yield). Another
method of preparation makes use of CDI and Cbz-L-isoleucine.[31]
MS (ESI) m/z (%): 386 (18) [M+Na]+, 308 (39) [M-tBu+H]+, 264
(100) [M-CO2tBu+H]+. 1H NMR (400 MHz, DMSO-d6), mixture of
rotamers: δ=7.72 and 7.67 (d, J=8.4 Hz and d, J=9.0 Hz, 1H),
7.41–7.28 (m, 6H), 5.07 and 5.05 (s, 2H), 4.01 and 3.87 (dd, J=8.6,
6.6 Hz and d, J=8.5 Hz, 6.1 Hz, 1H), 3.69–3.59 and 3.51 (m and d,
J=16.2 Hz, 1H), 3.45 (d, J=16.2 Hz, 1H), 1.91–1.82 (m, 1H), 1.45 and
1.38 (s and s, 9H), 1.33–1.25 (m, 1H), 1.17–1.08 (m, 1H), 0.84 (d, J=
6.8 Hz, 3H), 0.80 (t, J=7.4 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ
203.3, 166.2, 156.4, 136.9, 128.4, 127.9, 127.8, 80.8, 65.7, 64.7, 47.7,
39.5, 34.7, 27.6, 24.1, 15.5, 11.1.
NCbz-Dolaisoleucine tert-butyl ester 8a
Analytical characterisation: General
A 500 mL round-bottom flask equipped with a stir bar was charged
with 9a (19.0 g, 52.3 mmol, 1.0 eq.), which was dissolved in a
mixture of isopropanol and DCM (1:15, 240 mL). Silica was added
NMR spectra were recorded using an Ascend 400 spectrometer
(Bruker, US) at 298 K. 1H-spectra were recorded at 400 MHz, 19F-
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(37.7 g, 627.5 mmol, 12.0 eq.) and the resulting suspension was
cooled on ice. Sodium borohydride (6.0 g, 158.6 mmol, 3.0 eq.) was
added incrementally over the period of one hour. The suspension
was stirred on ice for four hours. The remaining borohydride was
quenched by the addition of glacial acetic acid (20 mL) and the
mixture was allowed to attain room temperature. Once gas
evolution had seized, the reaction mixture was filtered, and the
solids were washed with EtOAc (300 mL). The solvent was removed
under reduced pressure and the resulting oil was co-evaporated
with diethyl ether several times and was subsequently purified by
flash chromatography on silica (petroleum ether/EtOAc 4:1) to
yield a mixture of the two diastereoisomers of the CbzN-β-hydroxy-
γ-amino acid tert-butyl esters (15.8 g, 43.2 mmol, 83% yield) in a
ratio of roughly 5:1 in favour of the desired (3R)-isomer. The
mixture was used directly in the next step without further
purification. Another method of preparation makes use of methanol
as solvent.[31] MS (ESI) m/z (%): 310 (100) [M-tBu+H]+.
Monocylic lactam 18
10 mL round-bottom flask was charged with 8a (40 mg,
1
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4
5
6
7
8
9
A
102 μmol), Pd/C (10% w/w, 5 mg) and a stir bar were added, and
the flask was sealed with a septum. The atmosphere was evacuated
and refilled with nitrogen three times. EtOAc/MeOH (3:1, 3 mL) was
injected via syringe and a hydrogen atmosphere was applied. The
mixture was stirred at room temperature for two days. The
suspension was passed through a syringe filter, the liquid layer was
concentrated in vacuo, and the residue was purified by flash
chromatography on silica (hexane/acetone 4:1) to yield 18 (10 mg,
54 μmol, 53% yield) as a colourless oil. The procedure is an exact
reproduction of the method originally published by Pettit and
coworkers.[11,33,34] MS (ESI) m/z (%): 186 (100) [M+H]+. 1H NMR
(400 MHz, CDCl3) δ=3.67 (ddd, J=7.0, 6.8 Hz, 1.7 Hz, 1H), 3.45 (dd,
J=3.6, 1.7 Hz, 1H), 3.28 (s, 3H), 2.80 (s, 3H), 2.54 (ddd, J=17.8, 7.0,
0.9 Hz, 1H), 2.38 (dd, J=17.8, 1.1 Hz, 1H), 1.82–1.71 (m, 1H), 1.51–
1.40 (m, 1H), 1.35–1.26 (m, 1H), 0.99 (t, J=7.4 Hz, 3H), 0.70 (d, J=
6.9 Hz, 3H). The unusually low trans-coupling constant between the
protons geminal to the methoxy- and isobutyl-groups (3.67 and
3.45 ppm, respectively, 3J=1.7 Hz) does not escape us, but
corresponds exactly to the value reported by Pettit,[34] whose
structural assignment is supported by NOESY.[33]
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A three-necked, 1 L round-bottom flask equipped with a thermom-
eter, septum, dropping funnel with nitrogen inlet and stirring bar
was charged with lithium bis(trimethylsilyl)amide (1 M in THF,
112 mL, 112 mmol, 2.6 eq.). The base was diluted with THF (70 mL),
N,N’-dimethylethyleneurea (18.7 mL, 173 mmol, 4.0 eq.) was added
°
via syringe and the resulting solution was cooled to À 78 C.
Dropwise, over the period of two hours, a solution of a mixture of
the 3R- and 3S-diastereomers of CbzN-β-hydroxy-γ-amino acid tert-
butyl esters (5:1 ratio in favour of the 3R-isomer, 15.8 g, 43.2 mmol,
1.0 eq.) in THF (141 mL) was added to the reaction mixture via the
dropping funnel. The temperature inside the flask was kept at
Dolaisoleucine tert-butyl ester hydrochloride (Dil-OtBu x HCl)
A two-necked, 50 mL round-bottom flask was charged with 8a
(1.63 g, 4.14 mmol), Pd/C (10% w/w, 1.6 g) and were methanol
(16 mL) were added and cyclohexene (8 mL) was added by syringe.
°
below À 60 C over the course of the addition. The mixture was
°
The flask was placed in an oil bath preheated to 80 C and the
°
stirred at À 78 C for another hour. Dropwise, over the period of
suspension was refluxed for exactly 7 minutes. The flask was
immersed into a dry ice bath to terminate the reaction. The mixture
was quickly filtered through celite and the solids were washed with
methanol (35 mL). The solvent was sporadically evaporated in
vacuo and the residue was taken up in diethyl ether (80 mL). The
15 minutes, methyl trifluoromethanesulfonate (29.4 mL, 260 mmol,
6.0 eq.) was added to the reaction mixture, which was kept at
°
°
below À 55 C. The resulting solution was warmed to À 47 C
°
(acetonitrile/dry ice) and after another 30 minutes, to À 20 C
(acetone/ice). After stirring for two hours at that temperature, the
mixture was cooled on ice and the reaction was quenched by the
addition of saturated aqueous NH4Cl (50 mL). The two-phasic
mixture was diluted with water (300 mL) and the phases were
separated. The aqueous phase was extracted with EtOAc (3×
200 mL), the combined organic layers were washed with half-
saturated brine (200 mL) and subsequently dried over MgSO4. Upon
filtration and evaporation of the solvent, a yellow oil with solid
components was obtained. To the latter were added silica (80 g)
and DCM (500 mL), the solvent was removed in vacuo, and the
resulting dispersion was washed with EtOAc/heptane (1:1, 500 mL)
to yield a yellow oil which still contained solid components. The oil
was taken up in EtOAc/heptane (1:1, 600 mL) and the solution was
washed with water (2×300 mL). The organic layer was then dried
over MgSO4, filtered and the solvent was removed in vacuo to yield
a homogenous, yellow oil, which was purified by flash chromatog-
raphy on silica (petroleum ether/ EtOAc 9:1) to yield 8a (10.1 g,
25.6 mmol, 60% yield, 72% yield with regard to diastereomeric
purity of starting material) as a colourless oil. The procedure is
based upon a previously published method.[31] HRMS (ESI) m/z [M+
H]+ calcd for C22H36NO5: 394.2588, found: 394.2525. 1H NMR
(400 MHz, CDCl3), miture of two rotamers: δ=7.36–7.27 (m, 5H),
5.15 and 5.10 (dd, J=12.4, 6.1 Hz and J=12.4, 3.0 Hz, 2H), 4.33-3.94
(m, 1H), 3.90 and 3.84 (ddd, J=9.3, 6.2, 3.4 Hz and td, J=7.4, 4.0 Hz,
1H), 3.39 and 3.28 (s, 3H), 2.79 and 2.78 (s, 3H), 2.48-2.29 (m, 2H),
1.80–1.68 (m, 1H), 1.54–1.46 (m, 1H), 1.45 and 1.44 (s, 9H), 1.13–1.00
(m, 1H), 0.97 and 0.92 (d, J=6.7 Hz, 3H), 0.88 and 0.85 (t, J=5.9 Hz,
3H); 13C NMR (100 MHz, CDCl3), mixture of two rotamers: δ=171.5
and 171.3, 157.3 and 157.2, 137.1 and 136.8, 128.5, 128.2 and 128.1,
127.9 and 127.7, 80.9 and 80.8, 78.6 and 78.3, 67.5 and 67.2, 58.1
and 58.0, 38.9 and 38.6, 34.7 and 34.1, 28.2, 26.1 and 26.0, 16.3 and
16.2, 11.4 and 11.3.
°
resulting solution was cooled to À 78 C and 4 M HCl in dioxane
(1.2 mL) was added dropwise while stirring, leading to the
formation of a white precipitate. After stirring for an additional
15 minutes, the solids were filtered off, washed with diethyl ether
(30 mL) and dried in vacuo to yield Dil-OtBuxHCl (1.10 g, 3.72 mmol,
90% yield) as a white solid. The compound was spectroscopically
identical to a commercially obtained sample (Aurum Pharmatech,
US) and in good agreement with literature reports.[33,34] MS (ESI) m/z
(%): 260 (100) [M+H]+.
CbzN-Val-Dil-OtBu 14
A 250 mL round-bottom flask was charged with Dil-OtBuxHCl
(2.77 g, 10.7 mmol, 1.0 eq.) and Cbz-Val (4.56 g, 18.1 mmol, 1.7 eq.),
the solids were dissolved in DCM (50 mL) and the flask was sealed
°
with a septum. The mixture was cooled to À 20 C (acetone/dry ice)
and
2-bromo-1-ethylpyridinium
tetrafluoroborate
(5.86 g,
21.4 mmol, 2.0 eq.) was added, followed by N,N-diisopropylethyl-
amine (7.44 mL, 42.7 mmol, 4.0 eq.). The mixture was kept at
°
À 20 C for one hour and was subsequently stirred at room
temperature for 20 hours. The reaction mixture was then diluted
with DCM (50 mL), washed with brine (2×50 mL), dried over MgSO4
and filtered. The yellow oil obtained after evaporation of the
solvent was taken up in DCM (100 mL), silica (50 g) was added and
the solvent was removed in vacuo to yield a dry, faintly yellow
powder. The latter was washed with EtOAc/heptane (2:3, 300 mL)
and the solvent was removed in vacuo to yield a yellow oil, which
was further purified by flash chromatography on silica (petroleum
ether/EtOAc 7:3) to yield 14 (4.48 g, 9.08 mmol, 85% yield) as a
transparent oil, which crystallized upon prolonged standing. The
procedure is based upon a previously published method.[36] MS
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(ESI) m/z (%): 493 (100) [M+H]+, 437 (34) [M-tBu+H]+. 1H NMR
(400 MHz, CDCl3), mixture of two rotamers: δ=7.33–7.27 (m, 5H),
5.63 and 5.50 (d, J=8.9 Hz and J=9.2 Hz, 1H), 5.07 (s and s, 2H),
4.78–4.56 (m, 1H), 4.49 (dd, J=9.2, 5.7 Hz, 1H), 3.98–3.92 and 3.91–
3.81 (m and m, 1H), 3.32 3.31 (s and s, 3H), 2.93 and 2.74 (s and s,
3H), 2.42 (dd, J=15.6, 2.8 Hz, 1H), 2.28 (dd, J=15.6, 9.2 Hz, 1H),
2.03–1.92 (m, 1H), 1.43 and 1.42 (br s and br s, 9H), 0.98 (d, J=
6.8 Hz, 3H), 0.94 (d, J=6.7 Hz, 3H), 0.89 (d, J=6.8 Hz, 3H), 0.81 (t,
J=7.4 Hz, 3H). 13C NMR (101 MHz, CDCl3), mixture of rotamers: δ=
173.7 and 173.2, 171.2 and 171.1, 156.5 and 156.2, 136.5, 128.5,
128.4, 128.0, 127.9, 81.1 and 80.9, 78.2, and 77.2, 66.8 and 66.7, 64.0
and 60.4, 58.2 and 57.9, 56.0 and 55.6, 38.5 and 37.8, 35.1 and 33.3,
31.5 and 31.1, 30.5, 28.1 and 28.0, 26.7 and 25.9, 21.1 and 19.7, 17.0,
16.2 and 15.8, 14.2, 12.2 and 11.0.
63.9, 58.1, 53.9, 43.1, 38.7, 33.2, 31.1, 28.3, 27.9, 25.9, 20.4, 19.9, 18.3,
18.0, 15.9, 10.9.
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5
6
7
8
9
NBoc-OAc-L-hydroxyproline pentafluorophenyl ester 20
A 500 mL round-bottom flask was charged with trans-4-hydroxy-L-
proline (19, 15.0 g, 114 mmol, 1.0 eq.) and a solution of sodium
hydroxide (5.5 g, 138 mmol, 1.2 eq.) in water (75 mL) was added.
The solids dissolved upon stirring. Over the course of 30 minutes, a
solution of di-(tert-butyl)-dicarbonate (29 mL, 126 mmol, 1.1 eq.) in
THF (150 mL) was added. The resulting two-phasic mixture was
stirred vigorously for 48 hours. Water was added (75 mL) to dissolve
the solids, the phases were separated, and the aqueous phase was
washed with diethyl ether (3×50 mL). It was then acidified to pH 2
with aqueous 1 M NaHSO4 and extracted with EtOAc (4×150 mL).
The combined organic layers were dried over MgSO4, the drying
agent was filtered off and the solvent was removed in vacuo to
yield crude (2S,4R)-1-(tert-butoxycarbonyl)-4-hydroxypyrrolidine-2-
carboxylic acid (16.7 g) as a thick syrup, which was used in the next
step without further purification. TLC (nBuOH/AcOH/H2O 4:1:1,
ninhydrin+Δ) Rf =0.6.
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N,N-Dimethylvaline 17
A 500 mL round-bottom flask was charged with L-valine (501 mg,
4.28 mmol, 1.0 eq.), Pd/C (10% w/w, 0.10 g) was added, the flask
was sealed with a septum, and the atmosphere was evacuated and
backfilled with nitrogen three times. MeOH (100 mL) was injected
by syringe, followed by aqueous formaldehyde (37% w/w, 0.71 mL,
9.39 mmol, 2.2 eq.) and a stream of hydrogen gas was bubbled
through the suspension for four hours. The mixture was then stirred
in a hydrogen atmosphere for 18 hours. The suspension was filtered
through celite, the plug was washed with MeOH (30 mL) and the
solvent was removed in vacuo to yield a colourless oil which
crystallized readily upon standing. 17 was obtained as a crystalline,
off-white solid (614 mg, 4.26 mmol, 99% yield). The procedure is an
exact reproduction of a published method and the products’
spectra were in good agreement with literature.[37] TLC (nBuOH/
AcOH/H2O 4:1:1, KMnO4) Rf = 0.31.
To a 250 mL round-bottom flask containing crude (2S,4R)-1-(tert-
butoxycarbonyl)-4-hydroxypyrrolidine-2-carboxylic acid (16.7 g,
72.2 mmol, 1.0 eq.) were added DCM (35.2 mL) and pyridine
(35.2 mL, 435 mmol, 6.0 eq.) were added and the resulting two-
phasic mixture was ultrasonicated for ten minutes. Then, while
stirring on ice, acetic anhydride (34.3 mL, 363 mmol, 5.0 eq.) was
added via syringe, and the resulting mixture was stirred on ice for
one hour and then at room temperature for five hours. The solvent
was removed in vacuo and the residue was co-evaporated with
pyridine several times. Water (50 mL) was added, the mixture was
ultrasonicated until nearly homogenous, and was then freeze-dried
to yield crude (2S,4R)-4-acetoxy-1-(tert-butoxycarbonyl)pyrrolidine-
2-carboxylic acid (20.9 g) as a thick, yellow syrup, which was used in
the next step without further purification despite containing
solvent residues.
Dov-Val-Dil-OtBu 15
A 25 mL round-bottom flask was charged with 14 (177 mg,
359 μmol, 1.0 eq.) and a stir bar was added. MeOH (4 mL) and Pd/C
(10% w/w, 76 mg) were added. The flask was sealed with a septum,
the atmosphere was evacuated and backfilled with nitrogen three
times. A stream of hydrogen gas was bubbled through the
suspension for two hours. The solids were filtered off over celite
and the filter plug was washed with water (5 mL) and MeOH
(50 mL). The solvents were removed in vacuo to yield crude Val-Dil-
OtBu (128 mg), which was used directly in the next step without
further purification.
To
a 500 mL round-bottom flask containing crude (2S,4R)-4-
acetoxy-1-(tert-butoxycarbonyl)pyrrolidine-2-carboxylic acid (20.9 g,
76.5 mmol, 1.0 eq.) was added pentafluorophenol (18.7 g,
102 mmol, 1.3 eq.), followed by EtOAc (150 mL). The mixture was
stirred until a solution had been obtained. Dicyclohexylcarbodii-
mide (21.1 g, 102.3 mmol, 1.3 eq.) was added and the mixture was
stirred at room temperature for 16 hours. The resulting suspension
was filtered, and the solids were washed with EtOAc (2×100 mL).
The filtrate was concentrated in vacuo and purified by flash
chromatography on silica (petroleum ether/EtOAc 1:1) to give 20
(30.1 g, 68.6 mmol, 60% yield over three steps) as a colourless oil.
HRMS (ESI) m/z [M+Na]+ calcd for C18H18F5NNaO6: 462.0946, found:
A
10 mL vial was charged with N,N-dimethylvaline (104 mg,
716 μmol, 2.0 eq.), DCM (2.4 mL) and triethylamine (98 μL,
717 μmol) were added, and the resulting suspension was stirred on
ice. Diethyl cyanophosphonate (108 μL, 712 μmol, 2.0 eq.) was
added and the reaction mixture was stirred on ice for five minutes
before being added to another 10 mL vial containing Val-Dil-OtBu
(128 mg, 357 μmol, 1.0 eq.). The reaction mixture was stirred on ice
for 20 minutes and then at room temperature for 40 minutes. The
solvent was removed in vacuo, and the residue was purified by
flash chromatography on silica (petroleum ether/ acetone 9:1) to
yield 17 (95 mg, 196 μmol, 55% yield over two steps) as a
crystalline, off-white solid. The products’ spectra were in good
agreement with literature.[52] HRMS (ESI) m/z [M+H]+ calcd for
1
462.0910. H NMR (400 MHz, CDCl3), mixture of two rotamers: δ=
5.38–5.31 (m, 1H), 4.75–4.66 and 3.82–3.70 (m and m, 2H), 3.64–3.56
(m, 1H), 2.67–2.52 (m, 1H), 2.46–2.33 (m, 1H), 2.09 and 2.08 (s and s,
3H), 1.48 and 1.46 (s and s, 9H). 13C NMR (101 MHz, CDCl3), mixture
of two rotamers: δ=170.5 and 170.4, 168.9 and 168.4, 154.2 and
153.5, 142.4 and 141.0, 140.0 and 139.3, 138.6 and 136.8, 124.9, 81.7
and 81.3, 72.7 and 71.7, 57.54 and 57.52, 52.4 and 52.2, 37.1 and
35.7, 28.4 and 28.2, 21.1. 19F NMR (376 MHz, CDCl3) δ=-152.2 and
À 152.9 (m and m, 2F), À 157.1 and À 157.7 (t, J=21.6 Hz and t, J=
21.7 Hz, 1F), À 161.7 and À 162.2 (m and m, 2F).
1
C26H52N3O5: 486.3901, found: 486.3891. H NMR (400 MHz, CDCl3) δ
6.87 (d, J=9.0 Hz, 1H), 4.86–4.62 (m, 2H), 3.92–3.83 (m, 1H), 3.34 (s,
3H), 2.99 (s, 3H), 2.49–2.41 (m, 2H), 2.33–2.29 (m, 1H), 2.24 (s, 6H),
2.10–1.99 (m, 2H), 1.71–1.61 (m, 1H), 1.45 (s, 9H), 1.36–1.25 (m, 2H),
1.02 (d, J=6.8 Hz, 3H), 0.99 (d, J=6.8 Hz, 3H), 0.96 (d, J=5.5 Hz,
3H), 0.94 (d, J=5.4 Hz, 3H), 0.91 (d, J=6.7 Hz, 3H), 0.80 (t, J=7.4 Hz,
3H). 13C NMR (101 MHz, CDCl3) δ 173.5, 171.9, 171.3, 81.0, 78.3, 76.7,
NBoc-OAc-β-keto-γ-amino acid ethyl ester 9b
A 250 mL round-bottom flask was charged with a stir bar and 3-
ethoxy-2-methyl-3-oxopropanoic acid (5.1 g, 34.9 mmol, 1.5 eq.).
THF (15 mL) was added and the resulting mixture was cooled on
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ice. Dropwise, under a nitrogen atmosphere, a solution of iso-
propylmagnesium bromide in 2-Me-THF (24 mL, 2.9 M, 69.6 mmol,
3.0 eq.) was added via syringe. The mixture was allowed to attain
room temperature, where it was stirred for one hour. It was then
cooled back on ice and a solution of 20 (10.2 g, 23.2 mmol, 1.0 eq.)
in THF (30 mL) was added over the period of five minutes. The
reaction was stirred at room temperature for two days, whereupon
it was quenched by pouring the reaction mixture into 25 mL of ice-
cold methanol while stirring. The solvent was evaporated in vacuo,
the residues were suspended in ethyl acetate (200 mL) and the
The resulting solution was stirred for 90 minutes, whereupon the
solvent was removed in vacuo. Cesium carbonate (49 mg, 150 μmol,
2.2 eq.) was added, followed by non-dry MeOH (334 μL). The
mixture was stirred for one hour, during which it became a
homogenous solution. The solvent was removed in vacuo and the
oily residue was dried under fine vacuum for one day before NMR-
spectroscopic analyses were performed. HRMS (ESI) m/z [M+H]+
1
2
3
4
5
6
7
8
9
1
calcd for C8H14NO3: 172.0968, found: 172.0966. H NMR (400 MHz,
DMSO-d6) δ=5.50 (dt, J=5.5, 1.3 Hz, 1H), 5.06–5.03 (m, 1H), 4.40–
4.34 (m, 1H), 3.71 (ddd, J=9.3, 6.4, 6.0 Hz, 1H), 3.53–3.46 (m, 2H),
2.74 (d, J=11.9 Hz, 1H), 2.56–2.46 (m obscured by solvent peak,
1H), 1.96 (ddd, J=12.6, 6.0, 1.6 Hz, 1H), 1.51 (ddd, J=12.8, 9.4,
5.1 Hz, 1H), 1.00 (d, J=7.0 Hz, 2H). 13C NMR (101 MHz, DMSO-d6)
δ=173.6, 81.0, 71.9, 64.6, 50.7, 47.8, 39.3, 12.7.
suspension was transferred to
a separatory funnel. To the
separatory funnel were added more ethyl acetate (150 mL), hexanes
(50 mL), and half-saturated brine (100 mL) and the phases were
separated. The aqueous phase was diluted with more water
(100 mL) and extracted with ethyl acetate (2×250 mL). The pooled
organic layers were dried over MgSO4, the solids were removed by
filtration and the solvent was removed in vacuo. The residue was
purified by flash chromatography on silica (petroleum ether/EtOAc
17:3!4:1) to yield 9b (6.4 g, 16.7 mmol, 72% yield) as a colourless
oil. HRMS (ESI) m/z [M+H]+ calcd for C17H28NO7: 358.1866, found:
358.1851. 1H NMR (400 MHz, CD3CN), mixture of two rotamers of
each of two diastereoisomers: δ=5.23–5.12 (m, 1H), 4.73–4.45 (m,
1H), 4.13 and 4.12 (q, J=7.1 Hz, and q, J=7.1 Hz, 2H), 3.86 and
3.83–3.72 (q, J=7.1 Hz, and m, 1H), 3.67–3.42 (m, 1H), 2.46–2.25 (m,
1H), 2.22–2.06 (m, 1H), 2.00 and 2.00 and 1.99 (s, 3H), 1.43 and 1.40
and 1.39 and 1.37 (s, 9H), 1.31–1.18 (m, 6H). 13C NMR (101 MHz,
CD3CN), mixture of two rotamers of each of two diastereomers: δ
207.2 and 207.0 and 206.6 and 206.6, 171.6 and 171.6, 171.6 and
171.5 and 171.4 and 171.2, 156.1 and 155.5 and 155.2 and 154.8,
81.7 and 81.5 and 81.5 and 81.3, 74.4 and 74.1 and 73.4 and 73.2,
64.7 and 64.1 and 63.77, 62.59 and 62.53, 53.9 and 53.8 and 53.5
and 53.5, 52.36 and 51.60 and 50.62 and 50.39, 37.1 and 37.0 and
36.5 and 35.8, 28.9 and 28.8, 21.6, 14.8 and 14.8, 13.9 and 13.9 and
13.7 and 13.5.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
Bicyclic lactam (S,S)-23
A 4 mL vial was charged with (S,S)-22 (24 mg, 67 μmol 1.0 eq.), a
stirring bar was added, followed by DCM (334 μL) and TFA (334 μL).
The resulting solution was stirred for 90 minutes, whereupon the
solvent was removed in vacuo. Cesium carbonate (49 mg, 150 μmol,
2.2 eq.) was added, followed by non-dry MeOH (334 μL). The
mixture was stirred for one hour, during which it became a
homogenous solution. The solvent was removed in vacuo and the
oily residue was dried under fine vacuum for one day before NMR-
spectroscopic analyses were performed. HRMS (ESI) m/z [M+H]+
1
calcd for C8H14NO3: 172.0968, found: 172.0960. H NMR (400 MHz,
DMSO-d6) δ=5.11 (d, J=4.3 Hz, 1H), 5.05 (d, J=3.8 Hz, 1H), 4.42–
4.34 (m, 1H), 4.18 (ddd, J=9.5, 6.5, 4.4 Hz, 2H), 3.87 (dd, J=4.4,
4.3 Hz, 1H), 3.47 (dd, J=11.7, 5.2 Hz, 1H), 2.70 (dd, J=11.8, 2.4 Hz,
1H), 2.19 (q, J=7.7 Hz, 1H), 1.94 (ddd, J=12.8, 9.4, 5.4 Hz, 1H), 1.53
(ddd, J=12.8, 6.7, 2.4 Hz, 1H), 1.12 (d, J=7.7 Hz, 3H). 13C NMR
(101 MHz, DMSO-d6) δ=176.5, 72.4, 72.1, 62.3, 51.0, 50.4, 32.6, 13.7.
NBoc-OAc-β-methoxy-γ-amino acid ethyl ester 8b
NBoc-OAc-β-hydroxy-γ-amino acid ethyl ester (R,R)-22
A 10 mL round-bottom flask wash charged with (R,R)-22 (123 mg,
342 μmol, 1.0 eq.), THF (2.5 mL) and DMF (855 μL). Dimethylsulfate
(81 μL, 856 μmol, 2.5 eq.) was added, the flask was sealed with a
septum, a nitrogen atmosphere was applied, and the reaction
A 50 mL round-bottom flask was charged with 9b (845 mg,
2.36 mmol, 1.0 eq.), manganese(II)-chloride (596 mg, 4.74 mmol,
2.0 eq.) and a stir bar. MeOH (11.8 mL) was added, and the mixture
was cooled on ice. While stirring, sodium borohydride (180 mg,
4.76 mmol, 2.0 eq.) was added in increments over the period of ten
minutes. The mixture was stirred on ice for one hour, after which
the solvent was removed in vacuo. The residues were taken up in
EtOAc/MeOH 9:1 (50 mL), the resulting suspension was filtered
through a silica plug. Upon removal of the solvent, a mixture of
four diastereoisomers of 22 was obtained (807 mg, 2.25 mmol, 95%
yield). The isomers were separated by flash chromatography on
silica (petroleum ether/EtOAc 3:1!7:3). (R,R)-22 eluted after the
(S,S)-isomer and was obtained in 35% yield (297 mg, 0.83 mmol) as
°
mixture was cooled to À 15 C (NaCl/ice). Sodium hydride (16 mg,
60% dispersion over mineral oil, 400 μmol, 1.2 eq.) was added and
°
°
the mixture was stirred at À 15 C for 30 minutes and at 0 C for five
hours. EtOAc (5 mL) was added, followed by half-saturated aqueous
NH4Cl (2 mL). The resulting two-phasic mixture was stirred at room
temperature for 30 minutes, was the transferred to a separatory
funnel, diluted with water (10 mL), and the phases were separated.
The aqueous layer was extracted with DCM (4×10 mL), the organic
layers were pooled and dried over MgSO4. Upon filtration and
evaporation of the solvent in vacuo, a crude oil was obtained, which
was purified by flash chromatography on silica (petroleum ether/
EtOAc 9:1!3:1) to give 8b (102 mg, 273 μmol, 80% yield) as a
colourless oil. HRMS (ESI) m/z [M+Na]+ calcd for C18H31NO7Na:
396.1993, found: 396.1964. 1H NMR (400 MHz, CDCl3), mixture of
two rotamers: δ 5.28–5.16 (m, 1H), 4.24–4.06 (m, 2H), 4.06–3.93 (m,
1H), 3.93–3.85 and 3.62–3.52 (m and m, 1H), 3.84–3.70 (m, 1H),
3.48–3.36 (m, 1H), 3.42 (s, 3H), 2.46–2.33 (m, 1H), 2.33–2.18 (m, 1H),
2.05–1.95 (m, 1H), 2.01 (s, 3H), 1.51 and 1.45 (br s and br s, 9H), 1.25
(t, J=7.1 Hz, 3H), 1.28–1.20 (m, 6H). 13C NMR (101 MHz, CDCl3),
mixture of two rotamers: δ 174.3, 170.8 and 170.7, 154.5 and 154.4,
82.9 and 81.6, 80.3 and 79.8, 73.5 and 73.0, 61.5 and 61.1, 60.6, 58.8
and 58.5, 52.9 and 52.4, 43.3, 32.0 and 31.3, 29.8 and 28.6, 21.3,
14.3, 14.1.
a colourless oil. (R,R)-22. HRMS (ESI) m/z [M+H]+ calcd for
20
°
C17H30NO7: 360.2017, found: 360.2009. [α]D À 42.4 (c 0.03, MeOH).
1H NMR (400 MHz, CD3CN), mixture of two rotamers: δ=5.18–5.08
(m, 1H), 4.19–4.09 (m, 1H), 4.10–3.99 (m, 2H), 3.94–3.79 (m, 1H),
3.65–3.52 (m, 1H), 3.46–3.22 (m, 2H), 2.40–2.29 (m, 1H), 2.29–2.16
(m, 1H), 2.00–1.95 (m, 4H), 1.44 (br s, 9H), 1.22 (t, J=7.1 Hz, 3H),
1.16 (d, J=6.8 Hz, 3H). 13C NMR, mixture of two rotamers: (101 MHz,
CD3CN) δ 175.5 and 175.2, 171.3, 155.6 and 155.2, 80.2, 74.2 and
73.9, 73.2 and 72.5, 61.1 and 61.0, 60.0 and 58.8, 53.7 and 53.0, 44.6
and 44.2, 31.9 and 31.7, 28.6, 21.2, 14.5 and 14.4, 14.47 and 14.45.
Bicyclic lactam (R,R)-23
A 4 mL vial was charged with (R,R)-22 (24 mg, 67 μmol 1.0 eq.), a
stirring bar was added, followed by DCM (334 μL) and TFA (334 μL).
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A 10 mL vial was charged with crude tert-butyl (2S,4S)-4-amino-2-
((1R,2R)-3-ethoxy-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-
carboxylate (46 mg, 139μmol, 1.0 eq.), capped, cooled on ice, and
by syringe, a solution of benzyl chloroformate (26 μL, 182 μmol,
1.3 eq.) in DCM (0.5 mL) was added, followed by a solution of
triethylamine (38 μL, 278 μmol, 2.0 eq.) in DCM (0.5 mL). The
resulting solution was stirred on ice for 30 minutes and at room
temperature for two hours. The reaction mixture was subsequently
filtered through a silica plug, which was washed with more DCM
(5 mL) and the volatiles were removed in vacuo to yield crude tert-
butyl (2S,4S)-4-(((benzyloxy)carbonyl)amino)-2-((1R,2R)-3-ethoxy-1-
methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-carboxylate (42 mg) as
a colourless oil, which was used directly in the next step without
purification.
NBoc-hydroxy-β-methoxy-γ-amino acid ethyl ester
1
2
3
4
5
6
7
8
9
A 100 mL round-bottom flask was charged with 8b (500 mg,
1.34 mmol, 1.0 eq.), MeOH (48 mL) was added, followed by a
solution of potassium carbonate (185 mg, 1.34 mmol, 1.0 eq.) in
water (1.2 mL). The resulting solution was stirred on ice for four
hours. While continuing to stir on ice, HCl in EtOH (2.14 mL, 1.25 M,
2.68 mmol, 2.0 eq.) was added and the solvent was removed in
vacuo. The resulting semi-solid was co-evaporated with EtOAc
twice, taken up in EtOAc/MeOH 10:1 (27.5 mL), and filtered
through a silica plug, which was subsequently washed with
hexanes/EtOAc 1:1 (50 mL), and the solvent was evaporated in
vacuo. tert-butyl (2S,4R)-2-((1R,2R)-3-ethoxy-1-methoxy-2-methyl-3-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
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41
42
43
44
45
46
47
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49
50
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oxopropyl)-4-hydroxypyrrolidine-1-carboxylate
(NBoc-hydroxy-β-
methoxy-γ-amino acid ethyl ester, 430 mg, 1.30 mmol, 97% yield)
was obtained as a colourless oil. HRMS (ESI) m/z [M+Na]+ calcd for
C16H29NO6: 354.1918, found: 354.1893. 1H NMR (400 MHz, CDCl3),
mixture of two rotamers: δ 4.47–4.36 (m, 1H), 4.21–4.06 (m, 2H),
4.06–3.79 (m, 2H), 3.70–3.47 (m, 1H), 3.44 and 3.42 (s and s, 3H),
3.37–3.27 (m, 1H), 2.48–2.31 (m, 1H), 2.21–2.11 (m, 1H), 2.08–1.96
(m, 1H), 1.96–1.86 (m, 1H), 1.49 and 1.46 (br s and br s, 9H), 1.25 (t,
J=7.1 Hz, 3H), 1.23–1.18 (m, 3H). 13C NMR (101 MHz, CDCl3),
mixture of two rotamers: δ=174.4, 154.8, 82.9 and 81.6, 80.2 and
77.4, 70.6 and 70.1, 61.4, 60.7, 58.4 and 58.3, 55.7 and 55.2, 43.4,
34.5, 28.7 and 28.5, 14.3, 14.1.
A 10 mL round-bottom flask was charged with crude tert-butyl
(2S,4S)-4-(((benzyloxy)carbonyl)amino)-2-((1R,2R)-3-ethoxy-1-meth-
oxy-2-methyl-3-oxopropyl)pyrrolidine-1-carboxylate
(42 mg,
90 μmol, 1.0 eq.) and a stir bar. MeOH (2 mL) was added, the
°
resulting solution was cooled to 0 C (refrigerator) and a solution of
lithium hydroxide (65 mg, 2.71 mmol, 30 eq.) in water (2 mL) was
°
added dropwise. The reaction mixture was stirred at 0 C for
62 hours, whereupon it was diluted with water (20 mL) and
acidified to pH 2 using aqueous 1 M NaHSO4. The mixture was
subsequently extracted with EtOAc (3×15 mL), the organic layers
were dried over Na2SO4, the drying agent was removed by filtration
and the solvent was removed in vacuo to yield crude (2R,3R)-3-
((2S,4S)-4-(((benzyloxy)carbonyl)amino)-1-(tert-butoxycarbonyl)pyr-
rolidin-2-yl)-3-methoxy-2-methylpropanoic acid (33 mg), which was
used directly in the next step without purification. HRMS (ESI) m/z
[M-tBu+H]+ calcd for C17H25N2O5: 337.1758, found: 337.1718.
NBoc-azido-β-methoxy-γ-amino acid ethyl ester 24
A
10 mL vial, equipped with a stir bar, was charged with
triphenylphosphine (79 mg, 300 μmol, 2.0 eq.), the vial was capped,
and the atmosphere was evacuated and refilled with nitrogen. THF
(0.75 mL) was added by syringe, the vial was cooled on ice and
diisopropyl azodicarboxylate (59 μL, 300 μmol, 2.0 eq.) was added.
After one hour on ice, a suspension had formed. A solution of tert-
butyl (2S,4R)-2-((1R,2R)-3-ethoxy-1-methoxy-2-methyl-3-oxopropyl)-
4-hydroxypyrrolidine-1-carboxylate (49 mg, 148 μmol, 1.0 eq.) in
THF (0.75 mL) was added, followed 15 minutes later by diphenyl
phosphoryl azide (65 μL, 302 μmol, 2.0 eq.). The mixture was stirred
on ice for one hour and then at room temperature for 24 hours.
The reaction mixture was transferred to a round-bottom flask, the
solvent was evaporated and the residue was purified by flash
chromatography on silica (petroleum ether/EtOAc, 9:1!17:3) to
A 5 mL round-bottom flask was charged with crude (2R,3R)-3-
((2S,4S)-4-(((benzyloxy)carbonyl)amino)-1-(tert-butoxycarbonyl)pyr-
rolidin-2-yl)-3-methoxy-2-methylpropanoic acid (33 mg, 76 μmol,
1.0 eq.) and a stir bar was added, followed by L-phenylalanine
methyl ester hydrochloride (24 mg, 113 μmol, 1.5 eq.). The flask was
sealed with a septum and a solution of diisopropylethylamine
(40 μL, 227 μmol, 3.0 eq) in DCM (2.5 mL) was added, the solution
was cooled on ice, and HATU (43 mg, 113 μmol, 1.5 eq.) was added
in once increment. The resulting suspension was stirred vigorously
on ice for 30 minutes and then at room temperature for two hours.
The solvent was removed in vacuo, the residue was taken up in
MeOH (5 mL) and was dispersed over silica (1 g). The dispersion
was purified by flash chromatography on silica (petroleum ether/
EtOAc 1:1!0:1) to afford 26 as a colourless oil (44 mg, 74 μmol,
yield 24 (45 mg, 126 μmol, 85% yield) as
a colourless oil
contaminated with traces of triphenylphosphine oxide (according
to 13C-NMR). HRMS (ESI) m/z [M+Na]+ calcd for C16H28N4NaO5:
379.1952, found: 379.1830. 1H NMR (400 MHz, CD3CN) δ=4.19–3.98
(m, 1H), 3.93–3.83 (m, 1H), 3.85–3.76 (m, 1H), 3.40 (s, 3H), 3.00–2.85
(m, 1H), 2.42 (p, J=7.0 Hz, 1H), 2.30–2.20 (m, 1H), 2.00–1.95 (m, 1H),
1.44 (s, 9H), 1.23 (t, J=7.1 Hz, 3H), 1.17 (d, J=6.9 Hz, 3H). 13C NMR
(101 MHz, CD3CN) δ=175.2, 154.2, 83.4, 80.5, 61.3, 59.7, 59.0, 52.2,
51.2 43.6, 31.6, 28.6, 14.5, 13.5.
58% yield over four steps). HRMS (ESI) m/z [M+H]+ calcd for
20
°
C32H44N3O8: 598.3123, found: 598.3113. [α]D À 29.9 (c 0.40, MeOH).
1H NMR (400 MHz, CDCl3), mixture of two rotamers: δ=7.39–7.04
(m, 10H, obscured by CHCl3), 6.39 and 6.09 (br s and br s, 2H), 5.97
(s, 1H), 5.18–5.04 (m, 2H), 4.90–4.77 (m, 1H), 4.19–4.06 (m, 1H), 3.98–
3.91 and 3.91–3.83 (m and m, 1H), 3.79–3.66 (m, 4H), 3.61–3.48 (m,
1H), 3.41 (s, 3H), 3.23–3.17 (m, 1H), 3.17–3.11 (m, 1H), 3.05–2.96 (m,
1H), 2.36–2.24 and 2.24–2.11 (m and m, 1H), 2.02–1.92 and 1.92–
1.79 (m and m, 1H), 1.64–1.54 (m, 1H), 1.46 (s, 9H), 1.18 (d, J=
6.9 Hz, 3H). 13C NMR (101 MHz, CDCl3), mixture of two rotamers: δ
173.5 and 173.1, 172.4, 155.9, 155.0 and 154.4, 136.8, 136.3, 129.3,
128.8, 128.6, 128.2, 128.1, 127.3, 83.6 and 82.4, 80.4 and 80.0, 66.6,
60.8 and 58.6, 54.5 and 53.8, 53.3 and 52.8, 52.5, 49.5 and 48.6, 44.7
and 44.3, 37.8, 32.1 and 30.9, 28.6, 24.0, 14.8.
NBoc-4-(Cbz-amino)Dap-Phe-OMe 26
A 10 mL vial, equipped with a stir bar, was charged with 24 (45 mg,
126 μmol, 1.0 eq.), followed by Pd/C (10% w/w, 5 mg) and the vial
was capped. The atmosphere was evaporated and backfilled with
nitrogen three times. EtOAc (2 mL) was added by syringe, a
hydrogen balloon was installed, and the reaction mixture was
stirred at room temperature for 18 hours. It was then filtered
through a 450 nm syringe filter, which was subsequently washed
with more EtOAc (2 mL), and the resulting solution was evaporated
to dryness to yield crude tert-butyl (2S,4S)-4-amino-2-((1R,2R)-3-
ethoxy-1-methoxy-2-methyl-3-oxopropyl)pyrrolidine-1-carboxylate
(46 mg) as a colourless oil, which was used directly in the next step
without purification.
Cbz-azastatin methyl ester 27
A 10 mL round-bottom flask was charged with Dov-Val-Dil-OtBu
(15, 26.8 mg, 55.2 μmol), a stir bar and DCM (3 mL) were added and
the resulting solution was cooled on ice. While stirring, trifluoro-
acetic acid (1.5 mL) was added, followed after one hour by more
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°
trifluoroacetic acid (1.5 mL). The solution was stirred at 0 C
(refrigerator) for 16 hours. The volatiles were removed in vacuo and
the oily residue was co-evaporated with DCM three times to yield
crude Dov-Val-Dil-OH x TFA (31.0 mg), which was used directly in
the next step without purification.
DCM/MeOH 9:1!4:1+NH3, Note: the eluent for all columns was
prepared by extracting 1 mL of 28% aqueous NH4OH with DCM
and diluting the organic layer with the appropriate volume of
MeOH) to yield 8.5 mg of 7, 1.5 mg of 28 and 13.7 mg of a mixture
of the two.
1
2
3
4
5
6
7
8
A 10 mL round-bottom flask was charged with NBoc-4-(Cbz-amino)
Dap-Phe-OMe (26, 32.0 mg, 53.5 μmol), a stir bar and DCM (4 mL)
were added and the resulting solution was cooled on ice. While
stirring, trifluoroacetic acid (1 mL) was added, followed after two
hours by more trifluoroacetic acid (0.5 mL). After one more hour of
stirring, the solvent was removed in vacuo and the oily residue was
co-evaporated with DCM three times to yield crude 4-(Cbz-amino)
Dap-Phe-OMe x TFA (32.7 mg), which was used directly in the next
step without purification.
Azastatin-OMe (7). HRMS (ESI) m/z [M+Na]+ calcd for
C41H70N6NaO8: 797.5147, found: 797.5112. 1H NMR (400 MHz,
CD3OD), mixture of two rotamers: δ=7.38–7.14 (m, 5H), 4.82–4.72
(m obscured by solvent peak, 2H), 4.65 (d, J=8.7 Hz, 1H), 4.28–4.00
(m, 2H), 3.96–3.85 (m, 1H), 3.75 and 3.72 (s and s, 3H), 3.69–3.47 (m,
2H), 3.45 and 3.39 (s and s, 3H), 3.33 and 3.29 (s and s obscured by
solvent peak, 3H), 3.29–3.25 (m, 1H), 3.24 and 3.15 (s and s, 3H),
3.09–2.87 (m, 2H), 2.71 and 2.69 (d and d, J=8.4 Hz and J=8.5 Hz,
1H), 2.56–2.40 (m, 2H), 2.34 and 2.32 (s and s, 6H), 2.30–2.20 (m,
1H), 2.14–2.00 (m, 2H), 1.96–1.87 (m, 1H), 1.84–1.71 (m, 2H), 1.39–
1.27 (m, 4H), 1.24 and 1.18 (d, J=6.7 Hz and J=6.8 Hz, 3H), 1.11–
0.94 (m, 12H), 0.90–0.83 (m, 6H). 13C NMR (101 MHz, CD3OD),
mixture of two rotamers: δ=176.3 and 176.0, 175.5 and 175.3,
173.6 and 173.5, 173.1 and 173.0, 172.2 and 171.8, 138.6, 138.5,
130.2, 130.0, 129.7, 129.6, 127.9, 127.8, 86.2, 82.7, 79.5 and 79.2,
75.8 and 75.7, 62.3 and 61.7, 60.3, 58.5 and 58.3, 57.7 and 57.6, 56.2
and 56.0, 54.5 and 54.0, 52.9 and 52.8, 45.4 and 45.1, 42.51 and
42.45, 38.1 and 37.9, 36.4, 33.5 and 33.0, 31.7, 30.7, 28.8, 27.0, 20.22
and 20.19, 20.0 and 19.5, 19.35 and 19.32, 16.2 and 15.9, 15.6, 15.2,
10.9 and 10.8. For MS-fragmentation pattern please refer to SI.
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23
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26
27
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A 10 mL round-bottom flask was charged with solutions of crude 4-
(Cbz-amino)Dap-Phe-OMe x TFA (32.7 mg, 53.5 μmol, 1.0 eq.) and
crude Dov-Val-Dil-OH x TFA (31.0 mg, 57.0 μmol, 1.1 eq.) in DCM
(2 mL each) and the solvent was removed in vacuo. A stir bar was
added, the flask was sealed with a septum, and 1,2-dimeth-
oxyethane (2 mL) was added by syringe. The resulting solution was
stirred on ice, triethylamine (36.6 μL, 267 μmol, 5.0 eq.) and diethyl
cyanophophonate (12.1 μL, 80.2 μmol, 1.5 eq.) were added, and the
reaction mixture was stirred on ice for two hours. The solvent was
then removed in vacuo, and the oily residue was purified by flash
chromatography on silica (DCM/MeOH 99:1!97:3+NH3. Note: the
eluent was prepared by extracting 1 mL of 28% aqueous NH4OH
with DCM and diluting the organic layer with the appropriate
volume of MeOH) to yield 27 (36.5 mg, 40.2 μmol, 75% yield over
N-Ethyl azastatin-OMe (28). HRMS (ESI) m/z [M+Na]+ calcd for
C43H74N6NaO8: 825.5460, found: 825.5413. 1H NMR (400 MHz,
CD3OD), mixture of rotamers: δ=1H NMR (400 MHz, CD3OD),
mixture of rotamers; δ=7.36–7.16 (m, 5H), 4.80–4.75 (m obscured
by solvent peak, 1H), 4.62–4.55 (m, 2H), 4.29–4.21 (m, 1H), 4.18–4.10
(m, 1H), 4.10–4.03 (m, 1H), 3.99–3.92 (m, 1H), 3.76 and 3.73 (s and s,
3H), 3.69–3.64 (m, 1H), 3.62–3.54 (m, 1H), 3.54–3.49 (m, 1H), 3.43
and 3.38 (s and s, 3H), 3.36–3.33 and 3.29–3.26 (m obscured by
solvent peak, 3H), 3.25 and 3.15 (s and s, 3H), 3.07–2.99 (m, 1H),
2.99–2.86 (m, 3H), 2.86–2.72 (m, 2H), 2.58–2.47 (m, 2H), 2.40 and
2.37 (s and s, 6H), 2.23–2.11 (m, 2H), 2.11–2.00 (m, 2H), 1.92–1.82
(m, 2H), 1.41–1.37 (m, 2H), 1.28–1.22 (m, 5H), 1.20–1.15 (m, 2H),
1.14–1.08 (m, 2H), 1.05–0.97 (m, 8H), 0.92–0.85 (m, 6H). Note: The
quantity of N-ethyl azastatin obtained was insufficient for character-
ization by 13C-NMR. The MS-fragmentation pattern of the peptide
(see SI) unambiguously established the regiochemistry of the
ethylation observed in the final deprotection step.
three steps) as a white solid. HRMS (ESI) m/z [M+H]+ calcd for
20
°
C49H77N6O10: 909.5696, found: 909.5624. [α]D À 36.2 (c 0.19,
MeOH). 1H NMR (400 MHz, CD2Cl2), mixture of two rotamers: δ=
7.39–7.12 (m, 1H), 6.87 and 6.81 (d and d, J=8.9 Hz and J=9.0 Hz,
1H), 6.48 and 6.44 (d and d, J=7.6 Hz and J=7.6 Hz, 1H), 6.12 and
5.88 (d and d, J=8.4 Hz and J=9.3 Hz, 1H), 5.13–5.02 (m, 2H), 4.96–
4.78 (m, 1H), 4.78–4.69 (m, 2H), 4.23–4.14 (m, 1H), 4.14–4.04 (m, 2H),
4.03–3.88 (m, 1H), 3.76 and 3.70 (s and s, 3H), 3.69–3.64 (m, 1H),
3.40 and 3.38 (s and s, 3H), 3.36–3.32 (m, 1H), 3.31 and 3.29 (s, 3H),
3.27–3.10 (m, 2H), 3.03–2.95 (m, 3H), 2.42–2.25 (m, 4H), 2.25 and
2.23 (s and s, 6H), 2.10–1.95 (m, 3H), 1.76–1.59 (m, 2H), 1.43–1.26
(m, 2H), 1.22 and 1.16 (d and d, J=6.8 Hz and J=6.9 Hz, 3H), 1.00–
0.89 (m, 15H), 0.82 (t, J=7.3 Hz, 3H). 13C NMR (101 MHz, CD2Cl2) δ=
173.7, 172.6, 172.4, 171.5, 171.1, 156.0, 137.4, 137.0, 129.6, 129.3,
129.1, 128.9, 128.8, 128.4, 128.3, 128.2, 127.3, 82.1, 78.6, 76.9, 66.7,
60.9, 59.4, 58.1, 55.1, 53.7, 52.6, 50.0, 44.9, 43.0, 37.93, 37.87, 33.3,
31.4, 30.7, 30.1, 28.0, 26.2, 20.3, 18.0, 14.9, 10.8.
In vitro cytotoxicity evaluation
MMAE and MMAF were purchased from Combi-Blocks (US).
Doxorubicin hydrochloride was obtained from Sigma-Aldrich/Merck
(Germany). HepG2 cells (human hepatocellular carcinoma cells,
ATCC), were grown in Eagle’s Minimum Essential Medium (ATCC).
RPMI 8226 (multiple myeloma cells, ATCC) were grown in complete
RPMI medium (ATCC). HCT116 cells (colon carcinoma cells, Anti-
cancer Inc.) were cultured in McCoy’s 5A Modified Medium (Sigma-
Aldrich). All media were supplemented with 10% foetal bovine
serum, penicillin/streptomycin (100 U/100 μg/mL) and 2 mM L-
glutamine. Cells were seeded in 384-well assay plates at a density
Azastatin methyl ester 7 and N-ethyl azastatin methyl ester
28
A 10 mL round-bottom flask was charged with a solution of 27
(28.0 mg, 30.8 μmol) in EtOH (1 mL) and the solvent was removed
in vacuo to yield a glassy solid. A stir bar and Pd/C (10% w/w,
5.6 mg) were added, the flask was sealed, and the atmosphere was
evacuated and backfilled with nitrogen three times. EtOH (2 mL)
was added by syringe and hydrogen was bubbled through the
resulting solution for two hours, whereupon the reaction mixture
was stirred under hydrogen for an additional 16 hours. It was then
filtered through a 450 nm syringe filter, which was subsequently
washed with more EtOH (2 mL), and the resulting solution was
evaporated to dryness to yield 23.7 mg of an oily residue, which
consisted of 7 and 28 (approximately 3:2 ratio, 18.1 μmol and
12.1 μmol, respectively, 30.2 μmol combined yield, 98%) according
to HPLC. A portion of each pure analyte was obtained by a series of
purifications by flash chromatography on silica (first and second
column: DCM/MeOH 97:3!93:7+NH3, third and fourth column:
°
of 1000 cells/well. The cells were incubated over night at 37 C and
test compounds were added, followed incubation for an additional
°
72 h at 37 C. The fraction of surviving cells was analysed by a
fluorometric microculture cytotoxicity assay, based on hydrolysis of
fluorescein diacetate to fluorescein by cells with intact plasma
membranes, as recently described.[49] IC50-values were calculated
from three independent experiments (see SI, mean�SEM). All
tested concentrations were assessed in multiple wells (n=2-4) in
each experiment. The IC50-values were determined with nonlinear
regression analysis using GraphPad Prism 8 Software Package (US).
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(Uppsala University, Sweden) for his advice on the spectroscopic
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Conflict of Interest
The authors declare no conflict of interest.
Keywords: Total synthesis · Medicinal chemistry · Cytotoxicity ·
Diastereoselectivity · Antibodies
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Manuscript received: July 8, 2020
Version of record online: ■■■, ■■■■
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R. W. Hartmann, R. Fahrner, Dr. D.
Shevshenko, Prof. M. Fyrknäs, Prof. R.
Larsson, Dr. F. Lehmann, Prof. L. R.
Odell*
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Rational Design of Azastatin as a
Potential ADC Payload with
Reduced Bystander Killing
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Another golden bullet: Antibody–
drug conjugates (ADCs) are becoming
increasingly common in clinical che-
motherapy. Unfortunately, some of
the cytotoxic drugs used to arm anti-
bodies can leak into surrounding
tissues after being delivered to the
tumour and cause “bystander killing”.
Herein, we report the design,
synthesis, and evaluation of azastatin,
a novel, more “bystander-friendly” mi-
crotubule inhibitor than those
preceding it.