A new delivery system for auristatin in STxB-drug conjugate therapy

Article abstract of DOI:10.1016/j.ejmech.2015.03.047

A key challenge in anticancer therapy is to gain control over the biodistribution of cytotoxic drugs. The most promising strategy consists in conjugating drugs to tumor-targeting carriers, thereby combining high cytotoxic activity and specific delivery. To target Gb3-positive cancer cells, we exploit the non-toxic B-subunit of Shiga toxin (STxB). Here, we have conjugated STxB to highly potent auristatin derivatives (MMA). A former linker was optimized to ensure proper drug-release upon reaching reducing environments in target cells, followed by a self-immolation step. Two conjugates were successfully obtained, and in vitro assays demonstrated the potential of this targeting system for the selective elimination of Gb3-positive tumors.

Full text of DOI:10.1016/j.ejmech.2015.03.047

European Journal of Medicinal Chemistry 95 (2015) 483e491
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
A new delivery system for auristatin in STxB-drug conjugate therapy
Corn eꢀ lie Batisse a , Estelle Dransart , Rafik Ait Sarkouh , Laura Brulle , Siau-Kun Bai ,
,
b
a
a
a
a
b
a
a, *
Sylvie Godefroy , Ludger Johannes , Fr eꢀ d eꢀ ric Schmidt
Institut Curie, CNRS, UMR 3666/INSERM U1143, 26 rue d'Ulm, 75248 Cedex 05 Paris, France
a
b
Immuno Targets SAS, 116 bd du Montparnasse, 75014 Paris, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 18 December 2014
Received in revised form
A key challenge in anticancer therapy is to gain control over the biodistribution of cytotoxic drugs. The
most promising strategy consists in conjugating drugs to tumor-targeting carriers, thereby combining
high cytotoxic activity and specific delivery. To target Gb3-positive cancer cells, we exploit the non-toxic
B-subunit of Shiga toxin (STxB). Here, we have conjugated STxB to highly potent auristatin derivatives
18 March 2015
Accepted 19 March 2015
Available online 28 March 2015
(
MMA). A former linker was optimized to ensure proper drug-release upon reaching reducing envi-
ronments in target cells, followed by a self-immolation step. Two conjugates were successfully obtained,
and in vitro assays demonstrated the potential of this targeting system for the selective elimination of
Gb3-positive tumors.
Keywords:
Auristatin
Shiga toxin
Conjugate
Cancer
©
2015 Elsevier Masson SAS. All rights reserved.
Carbamate
Disulfide
1
. Introduction
market. Based on information available as of November 2014, more
than forty ADCs are currently being investigated in clinical studies
as treatments for a variety of solid and liquid tumors. The ADCs in
the clinical pipeline [7,8] are directed against a plethora of different
antigenic targets, but are based on a limited number of highly
potent drugs, such as calicheamycins, auristatins, maytansinoids
and more recently duocarmycins and pyrolobenzodiazepines, and a
limited number of linker strategies. The use of highly potent drugs
is needed, for one because of limited overexpression of tumor-
associated receptors, and second because only limited amounts of
cytotoxic payload can be coupled onto mAbs to prevent a loss of
antigen binding capacity [9]. Moreover, the linker between the
cytotoxic drug and the carrier is a critical piece in the design of an
ideal carrier-drug conjugate [10e12]. It must be relatively stable in
the circulation and prevent unspecific drug-release, and yet release
the drug when it reaches the tumor.
For the development of new anti-cancer treatment modalities,
the selective delivery of highly cytotoxic drugs to tumor cells while
sparing normal tissues is a continuous challenge. Among the stra-
tegies that can be addressed to achieve this goal, the coupling of
cytotoxic drugs to tumor-targeting carriers appears to be at first
sight the most promising. Current drug targeting strategies exploit
selective ligands of membrane receptors as carrier for a toxic-
payload [1,2]. Indeed, increasing knowledge on cell surface mole-
cules that are overexpressed by cancer cells, termed tumor-
associated receptors or antigens [3], allows the use of specific li-
gands. After conjugation to cytotoxic agents, it results in the cyto-
toxic drug targeting and accumulating in the tumor with minimal
accumulation in normal tissues, thereby increasing the effective-
ness and reducing the toxicity of these drugs. Various carriers were
developed, including small molecules, polymer and proteins. At
this stage, monoclonal antibodies (mAb) are the most widely used
carrier moieties for the tumor-specific targeting of cytotoxic drugs
Here we exploit a carrier derived from the non-toxic B-subunit
of Shiga toxin (STxB), termed STxB/Cys [13], a homopentameric
protein. Each monomer has a molecular mass of 7.7 kDa. STxB binds
specifically to the glycosphingolipid globotriaosylceramide (Gb3 or
[4], with two antibody-drug conjugates (ADC), brentuximab
9
ꢀ1
vedotin and ado-trastuzumab emtansyne [5,6], having reached the
CD77), with an apparent binding constant in the order of 10 M
that results from the capacity to interact with up to 15 Gb3 mole-
cules per homopentamer. After binding to Gb3 at the surface of
target cells, STxB is internalized by endocytosis to reach early and
recycling endosomes. STxB bypasses the late endocytic pathway
*
Corresponding author.
E-mail address: Frederic.Schmidt@curie.fr (F. Schmidt).
http://dx.doi.org/10.1016/j.ejmech.2015.03.047
223-5234/© 2015 Elsevier Masson SAS. All rights reserved.
0

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C. Batisse et al. / European Journal of Medicinal Chemistry 95 (2015) 483e491
and avoids the degrading environment of lysosomes. Then STxB
directly reaches the trans-Golgi network (TGN), the stacks of the
Golgi apparatus and eventually the endoplasmic reticulum (ER).
This unconventional intracellular trafficking is termed the retro-
grade route. Aberrant glycosylation is a general feature of carci-
nogenesis [14], and Gb3 overexpression has been described for
various cancer cell lines and human cancers [15]. Strikingly, cancer
8
cells have up to 10 STxB binding sites, whereas antibodies have
6
usually at most 10 binding sites per target cells. Therefore STxB
would be expected to be more efficient than antibodies as carrier
for tumor-specific targeting of cytotoxic drugs. STxB might be a
carrier of choice to target Gb3-positive cancers.
STxB/Cys is a genetically engineered STxB possessing a cysteine
residue to the C-terminus of each monomer, enabling five defined
chemical coupling site. STxB/Cys was previously used as a Gb3-
targeting carrier of photosensitizer [16], mild cytotoxic drugs
Fig. 1. General design of STxB-drug conjugates.
[17,18], or various antigens that were delivered to dendritic cells in
immunotherapeutic strategies [19].
(
Boc) derivative. The hydroxyl function was then substituted in a
We have previously described the use of STxB/Cys as carrier of a
camptothecin drug [17], with the synthesis of a STxB-SN38 conju-
gate that showed a Gb3-dependant cytotoxic activity on cells in
culture. The linker that was used in this study was designed on a 2-
methylaminoethanethiol core, enabling drug-release due to disul-
fide bond reduction, followed by an intramolecular self-immolative
thioacetate through Mitsunobu reaction to afford 3. The sulfhydryl
function was activated in situ by dithiodipyridine, leading to a di-
sulfide bond (4). Deprotection of the amine was performed in acidic
medium and the formed chlorohydrate was kept as a salt (5). The
linker was then reacted with phosgene and triethylamine to give
the carbamoyl chloride 6 (Fig. 2). The hydroxyl function of N-Boc-
protected MMAE 12 was then coupled to the bifunctional linker 6
via the carbamoyl chloride in the presence of stochiometric amount
of 4-dimethylaminopyridine (DMPA) (Fig. 3). Thus we have suc-
cessfully synthesized a MMAE-linker intermediate 14 including a
carbamate involving hydroxyl function of MMAE. The number of
steps needed for the synthesis was reduced compared to the one
previously described. However the procedure remains long and
could limit the scale up for further development.
We designed and synthesized two new linkers 9a and 9b around
a mercaptoethanol core, as a structure optimization of previous
linker 6. Briefly, commercial mercaptoethanol 7 was first protected
by either 2,2-dipyridyl-disulfide (8a) or 3-nitro-2-pyridinesulfenyl
chloride (8b) leading to a disulfide bond. The synthesis of 8a was
performed in acidic medium and allow the formation of the same
leaving group thiopyridine (X ¼ H) than the one of the linker 6. On
the other hand, considering the instability of 3-nitro-2-
pyridinesulfenyl chloride (Npys-Cl), several solvents were evalu-
ated for the reaction with the mercaptoethanol. The cyclohexane, a
non-dissolving solvent was selected to avoid premature degrada-
tion of reactant. A leaving group including a nitro function
5
-ring cyclization. The conjugate was completely stable in several
media including pure fetal calf serum and the intracellular cleavage
to release the free SN38 was shown. The Gb3-specific cytotoxic
activity of the STxB-SN38 conjugate was also demonstrated in vitro.
However, the conjugate showed a moderate therapeutic potency in
a xenograft tumor model in mice, even at the maximum tolerated
dose of STxB (unpublished data). To optimize the use of STxB as a
therapeutic carrier, we choose to generate STxB conjugates with
highly potent cytotoxic drugs and to optimize the linker strategy
used (Fig. 1).
Monomethylauristatin E and F are potent derivatives of the
natural product dolastatin 10 that inhibits tubulin polymerization
in dividing cells and thereby induces apoptosis [20,21]. Dose-
limiting toxicities of MMAE have been reported [22] and this
drug may be more useful when selectively directed to cancer cells
[23]. An example of such targeted MMAE delivery is provided by
brentuximab vedotin, a marketed anti-CD30 based ADC. Mono-
methylauristatin F is another auristatin derivative with impaired
membrane translocation capabilities due to a negatively charged C-
terminal domain at physiological pH. Previous studies have shown
that the activity of MMAE is greatly potentiated through active
delivery via an antibody-drug conjugate (ADC), suggesting that
MMAF mild activity is due to its inability to cross cellular mem-
branes [10]. Here, we describe the synthesis of STxB-based conju-
gates with two highly potent MMA derivatives [24], MMAE and
MMAF pentapeptides (Fig. 3), that differ only in the last amino acid,
respectively ephedrine and phenylalanine, thus displaying a
different hydrophily. A rational optimization of both structure and
synthesis route was carried out to improve the conjugate efficacy.
(X ¼ NO
2
) was synthesized (8b). The free hydroxyl function of these
two protected disulfenyl ethanols were then reacted with 4-4-
nitrophenylchloroformate (PNPeCl) to give the heterobifunctional
linkers 9a and 9b (Fig. 2). Obviously this synthesis way eliminated
the use of highly toxic phosgene and allows the easy isolation of the
activated linkers 9a and 9b. The hydroxyl function of N-Boc-pro-
tected MMAE 12 was coupled to the linker 9b via the PNP carbonate
in the presence of stochiometric amount of hydroxybenzotriazole
(HOBt) allowing the formation of the MMAE-linker intermediate 19
including a carbonate bond (Fig. 3). In another hand the amine
function of commercial MMAE was coupled to the linkers 9a and
2
. Results and discussion
9
b, allowing the formation of two MMAE-linker intermediates 16a
and 16b including a “reverse” carbamate bond and two different
) (Fig. 3). Thus we have
2
.1. Chemistry
leaving group (16a X ¼ H, and 16b X ¼ NO
2
Three self-immolative linkers that includes disulfide bond were
successfully synthesized two heterobifunctional linkers allowing
the generation of three MMAE-linker intermediates. These linkers
allowed the covalent linking of the MMAE via either its hydroxyl or
its amine function, including either a carbonate (19) or a carbamate
bond (16a and 16b). The procedure used reduces significantly the
number of steps needed to synthesize the MMAE-linker
synthesized to build five MMA-linker intermediates, either via
alcohol terminus (MMAE) or the methylamine terminus function
(
both MMAE and MMAF).
The heterobifunctional linker 6 synthesis was optimized start-
ing from a procedure previously described [17]. Commercial ami-
noalcohol 1 was first N-monoprotected as a tert-butoxycarbonyl

C. Batisse et al. / European Journal of Medicinal Chemistry 95 (2015) 483e491
485
Fig. 2. Synthesis routes of respectively three heterobifunctionnal linkers.
Fig. 3. Synthesis routes of four STxB-MMA conjugates.
intermediates. Moreover, the coupling of the linkers 9a and 9b via
simultaneity of both mechanisms [25,26].
the amine function of MMAE eliminates the need of protection/
deprotection steps (Fig. 3) and simplifies again the synthesis
procedure.
2.2. Conjugation to STxB
This procedure was used to successfully synthesize the MMAF-
linker intermediate 22, linking the amine function of the com-
mercial MMAF to the linker 9b (Fig. 3).
The five MMA-linker intermediates, either carbamate, “reverse”
carbamate or carbonate, were readily linked to the free sulfhydryl
residues of each STxB/Cys monomer by disulfide substitution. The
substitution levels of the coupling products were determined by
using mass spectrometry and HPLC.
After conjugation of STxB/Cys with the MMAE-linker interme-
diate 14, the mass spectrum displayed one major peak corre-
sponding to desired conjugate 15. After conjugation of STxB/Cys
with the MMAE-linker intermediate 16a, the mass spectrum dis-
played two peaks, the first one corresponding to the STxB-MMAE
conjugate 17 mass and a second peak consistent with a STxB-
pyridine by-product (Fig. 5). In contrast, after conjugation of
The self-immolative mechanism probably differed for the
various linkers, owing to the presence of oxygen and nitrogen
leaving groups with variable efficiency (Fig. 4). Intermediate 14,
based on linker 6, was predicted to spontaneously form a 5-ring 3-
methylthiazolidinone, as the SN38 phenol alcohol is a better leav-
ing group than methylamine [17]. On the contrary the driving force
of self-immolation of intermediates based on linker 9 would more
likely be the release of carbon dioxide, forming a 3-ring thiirane. In
similar linker strategies, mechanistic studies highlighted the

486
C. Batisse et al. / European Journal of Medicinal Chemistry 95 (2015) 483e491
Fig. 4. Possible mechanisms of self-immolative drug-release step for the two linkers 6 and 9.
STxB/Cys with the nitropyridine counterpart 16b, the mass spec-
trum displayed only the desired peak corresponding to the STxB-
MMAE conjugate 17. Similarly, after conjugation of STxB/Cys with
the MMAF-linker intermediate 21, the mass spectrum displayed
only the desired peak corresponding to STxB-MMAF conjugate 22.
The mass spectrum after conjugation of STxB/Cys with the car-
bonate MMAE-linker intermediate 19 displayed a major peak cor-
responding to a STxB-linker conjugate, and only a small peak
corresponding to the desired carbonate STxB-MMAE conjugate 20.
HPLC analysis confirmed these results providing an accurate
coupling yield. Conjugate STxB-MMAE 17, starting from MMAE-
linker intermediate 16a, was obtained impure (28% of STxB-
pyridine by-product) and in low yield (25%). This yield was
clearly improved by the use of MMAE-linker intermediate 16b,
reaching an almost quantitative yield (95%) without the by-product
presence. Similarly STxB-MMAF conjugate 22 was produced with
high yields (96%). No HPLC analyses were run on carbamate 15 or
carbonate conjugate 20.
better leaving group in the MMAE-linker intermediate 16b
improved drastically this yield by favoring the pyridine-2(1H)-
thione formation. Obviously, the carbonate bond of conjugate 20
was already cleaved in solution, mostly likely due to a low stability
in aqueous media. Lowering pH during coupling (MES buffer) and
storage did not improve these results, and the product was not
further investigated.
The three STxB-MMA conjugates 15, 17 and 22 were produced
with high substitution yield (5 molecules of MMA per STxB) and
only those three conjugates were investigated for their in vitro
characterization.
2.3. In vitro characterization of the STxB-MMA conjugates
Considering the unconventional intracellular trafficking that
characterizes STxB and allows the specific delivery of free drugs, we
evaluated by using immunofluorescence analysis whether the
conjugationprocess or presence of drug impaired the STxB trafficking
capacity. The three STxB-MMA conjugates 15, 17 and 22 were evalu-
ated for their intracellular trafficking characteristics. Data showed
that these three STxB-MMA conjugates accumulated as efficiently as
The low coupling yield of MMAE-linker intermediate 16a most
likely resulted from an unfavorable asymmetrical substitution of
the disulfide bond during conjugation step (Fig. 5). Presence of a
Fig. 5. STxB coupling mechanism and probable formation of by-products starting from MMAE-linker intermediate 16a.

C. Batisse et al. / European Journal of Medicinal Chemistry 95 (2015) 483e491
487
Table 1
STxB/Cys in the Golgi apparatus, demonstrating that despite MMA
loading, the functional properties of STxB were preserved.
IC50 values of three STxB-MMA conjugates on Gb3þ versus Gb3- (PPMP treated)
HT29 cells in comparison to non-vectorized MMA.
The in vitro cytotoxic activities (Table 1) of the conjugates 15, 17
and 22 were investigated by using a colorimetric assay with the
Gb3-expressing colorectal carcinoma cell line (HT29). To establish
specificity, Gb3-negative control situation was tested: HT29 cells
were treated with the glycosylceramide synthase inhibitor 1-
Cytotoxic activity on HT29 e IC50
(nM)
Gb3þ
Gb3e
MMAE
STxB-MMAE 15
MMAE
STxB-MMAE 17
MMAF
STxB-MMAF 22
3.0
e
6.2
e
phenyl-2-palmitoylamino-3-morpholino-1-propanol
leading to the inhibition of glycosphingolipid expression.
(PPMP),
0.3
0.4
3.1
395
1.0
199
398
400
The STxB-MMAE conjugate 17 including a “reverse” carbamate,
displayed a significant cytotoxic activity on Gb3-positive HT29
cells. Importantly, the cytotoxic activity of free MMAE was Gb3-
independent. Surprisingly, no cytotoxic activity was observed for
the STxB-MMAE conjugate 15, including a carbamate, neither on
Gb3-positive nor on Gb3-negative HT29 cells (Fig. 6).
þ
-
Fig. 6. Cytotoxic activity of the three STxB-MMA conjugates on Gb3 versus Gb3 (PPMP treated) HT29 cells. HT29 cells previously incubated with ppmp (Gb3-) or without (Gb3þ)
ꢁ
were plated in a 96-well plate. After overnight incubation, cells were incubated with STxB-MMA conjugate or commercial MMA alone for 6 h at 37 C. After additional 5 days
incubation with fresh culture medium, the percentage of living cells was quantified by colorimetric assay (MTT). All points were done in triplicate.

488
C. Batisse et al. / European Journal of Medicinal Chemistry 95 (2015) 483e491
Table 2
and exhibited a receptor-dependent cytotoxic activity, as opposed
to free MMA compounds. Thus these data validate the conjugates
for further in vivo investigations in mouse tumor models.
IC50 values of two STxB-MMA conjugates on Gb3þ versus Gb3- (PPMP treated)
HT29 cells with or without pre-incubation in pure serum.
Cytotoxic activity on HT29 e IC50 (nM)
No pre-incubation
Pre-incubation in
serum
4. Experimental
4.1. General chemistry methods
Gb3þ
Gb3-
Gb3þ
Gb3-
MMAE
STxB-MMAE 17
MMAF
2.1
0.4
38.4
0.1
2.4
66
23.4
8.9
All chemistry reactions were performed under argon atmo-
0.6
0.3
87
sphere in dry glassware. THF was distilled from sodium and
benzophenone. MeOH and ACN were dried over molecular sieves
and used as such. Dichloromethane was distilled over phosphorous
hemipentoxyde. Chemicals were purchased and used without
additional purification. Solvent mixture for Rf and purification on
silica gel were termed as follows: (polar solvent/apolar solvent % of
polar in mixture). Reactions were followed by TLC (0.25 mm silica
gel 60-F plates). Visualization was accomplished with 254 nm UV
STxB-MMAF 22
11.0
Similarly to the STxB-MMAE conjugate 17, the STxB-MMAF
conjugate 22 showed a Gb3-dependant cytotoxic activity. When
conjugated to STxB/Cys, the cytotoxic activity of free MMAF was
even 100 fold increased on Gb3 positive cells. Moreover the cyto-
toxic activity of STxB-MMAF conjugate 22 was as low as free MMAF
in the absence of Gb3 expression.
The incompliant evaluation of conjugate 15 can be explained by
its structure: the low electrophily of the terminal oxygen of MMAE
makes it a poor leaving group in comparison to the methylamine
group of the linker 6. We hypothesize that during the self-
immolative addition/elimination step, the electron pair is casted
off on the methylamine linker side, and not on the oxygen of MMAE.
The conjugates 17 and 22 including a “reverse” carbamate were
potently cytotoxic to Gb3-expressing cells with an IC50 value
1
13
light. H NMR and C NMR spectra were recorded at room tem-
perature with a BRUCKER ACP 300 at respectively 300 MHz and
75 MHz. Chemical shifts are reported in ppm (part per million)
relative to the residual solvent peak as the internal reference.
Coupling constants J are given in Hertz. Spin multiplicities are re-
ported using the following abbreviations: s ¼ singulet, d ¼ doublet,
dd ¼ doublet doublet, t ¼ triplet, q ¼ quadruplet, m ¼ multiplet. IR
spectra were recorded with a Fourier transform infrared PERKIN-
ELMER 1710 spectrometer or a Nicolet Magna-IR 550 spectrom-
eter equipped with an ATR-diamond. Melting points were deter-
mined by capillary method and are uncorrected. Low-resolution
mass spectra were determined using a ThermoScientific DSQ2, a PE
Sciex API3000 or an ADVION Expression Compact mass
spectrometer.
(
define as the MMA concentration that causes 50% cell killing) of
the nanomolar range, while these conjugates were 100 fold less
toxic on Gb3-negative HT29 cells. These data documents the effi-
ciency of the Gb3-specific intracellular delivery using STxB. The
IC50 value of the STxB-MMAE conjugate 17 is close to this observed
with free MMAE demonstrating the efficient intracellular release of
the active principle thanks to our optimized “reverse” carbamate
linker. In contrast to MMAE that cross cell membranes easily,
MMAF impairs passive redistribution that can also be deduced by
the high IC50 value of free MMAF compared to the one of MMAE,
whose IC50 were independent of Gb3 expression. STxB conjugation
clearly allows a Gb3-specific cell penetration of MMAF, a hydro-
philic drug. All these results show that the conjugation of MMA
drugs to STxB markedly allows their specific delivery to Gb3-
expressing tumor cells, while maintaining or even increasing
their cytotoxic activity.
High molecular mass spectroscopy was performed on a
Voyager-DE PRO Maldi-TOF mass spectrometer (Applied Bio-
systems, Framingham, USA) operated in the delayed extraction and
linear mode. A solution of sinapinnic acid in acetonitrile/TFA was
used as the matrix. Samples were prepared by mixing with the
matrix at a ratio of 1/1. The mixture was spotted onto a Maldi-TOF
plate and allowed to dry.
4.2. Synthesis and characterization of described compounds
4.2.1. MMAE-linker intermediate 14 synthesis
4.2.1.1. 2-[N-(tert-butyloxycarbonyl)-N-methylamino]ethanol,
N-methylethanol (6.0 g, 0.08 mol) was added to alumina (12.4 g, 1.5
eq), followed by (Boc) O (9.6 g, 1.1 eq) in DCM (150 mL). The re-
2.
To anticipate the stability of our linker strategy for further in vivo
tests, we determined the cytotoxic activity of both STxB-MMAE 17
and STxB-MMAF 22 conjugates after prolonged pre-incubation at
2
action was stirred for 10 min at room temperature. The residue was
diluted in EtOAc, filtered and evaporated. Mono-protected com-
ꢁ
3
7 C in pure serum. The data showed that the cytotoxic activities of
the conjugates were similar to that obtained for the conjugates that
were not pre-incubated in serum (Table 2). These data clearly
established that the conjugates were stable in serum. Even if the
in vivo environment in the organism is more complex, these pre-
liminary results constitute a solid basis to further investigation in
mouse cancer models.
pound (13.6 g,
r
¼ 97%) was obtained as an oil.
Rf (EtOAc/Cyclohexane 20%) ¼ 0.25
1
H NMR (300 MHz, CDCl
3
): 3.72 (t, 2H, J ¼ 5.2 Hz), 3.37 (d, 2H,
J ¼ 5.4 Hz), 2.90 (s, 3H), 2.59 (bs, 1H), 1.44 (s, 9H).
1
3
C NMR (75 MHz, CDCl3): 157.1, 79.7, 61.1, 51.2, 35.3, 28.0.
þ
þ
MS (IC ): m/z ¼ 176 [M þ H ], calculated 176.
4
.2.1.2. 2-[N-(tert-butyloxycarbonyl)-N-methylamino]acetylthio-
3
. Conclusions
ethyl, 3. To a solution of N-methylethanol 2 (3.5 g, 20 mmol) in
ꢁ
1
00 ml of THF at 0 C was added thiolacetic acid (2.0 ml, 1.3 eq) and
ꢁ
In summary, the direct application of former linker 6 to a STxB-
triphenylphosphine (7.0 g, 1.3 eq) under argon. After stirring at 0 C
MMAE synthesis provided an inefficient conjugate. Optimization of
the linker strategy was carried out by “reversing” the carbamate,
thus linking MMA through its methylamine function. Two STxB-
MMA conjugates were reproducibly obtained in a potentially scal-
able four synthetic steps route, and showed a Gb3-dependant
cytotoxic activity in the lower nanomolar range. The newly
described conjugates were stable under physiological conditions
for 15 min, DIAD (6.0 ml,1.4 eq) was added. The mixture was stirred
at 0 C for 2 h then 5 h at rt. The mixture was concentrated, diluted
with EtOAc/Hexane (1/2), filtered through celite. The solution was
ꢁ
3
washed with saturated NaHCO and brine respectively, dried over
MgSO , filtered, evaporated and purified on column chromatog-
4
raphy on silica gel (EtOAc/hexane 10e20%) to afford (4.0 g,
of title compound.
r
¼ 86%)

C. Batisse et al. / European Journal of Medicinal Chemistry 95 (2015) 483e491
489
Rf (acetone/DCM 20%) ¼ 0.3
4.2.1.7. O-(pyridin-2-yldisulfanyl)ethyl-N-methylamino)-N-tert-
butoxycarbonylmonomethylauristatin E carbamate, 13. To a solution
of carbamoyl chloride (36.2 mg, 1.0 eq) and Boc-MMAE 12 (45 mg,
0.055 mmol) in 4 mL of anhydrous THF, DMAP (9.6 mg, 2.0 eq) and
1
H NMR (300 MHz, CDCl3): 3.34 (t, 2H, J ¼ 6.8 Hz), 3.00 (t, 2H,
J ¼ 6.8 Hz), 2.90 (s, 3H), 2.33 (s, 3H), 1.45 (s, 9H).
13
C NMR (75 MHz, CDCl
3
): 195.2, 155.4, 79.5, 48.2, 34.4, 30.5,
2
8.4, 27.1.
DIPEA (25
mL, 3.0 eq) were added. The mixture was stirred at room
þ
þ
MS (IC ): m/z ¼ 234 [M þ H ], calculated 234.
temperature overnight. After evaporation, the residue was purified
over silica gel (MeOH/DCM 4%). The carbamate 13 (27 mg,
was obtained as a yellow solid.
r
¼ 43%)
4
.2.1.3. 2-[N-(tert-butyloxycarbonyl)-N-methylamino]1-ethyl 2-
Rf (MeOH/DCM 4%) ¼ 0.35
pyridyldisulfide, 4. To a solution of thioacetate 3 (3 g, 12.8 mmol)
and dithiodipyridine (2.8 g, 1.0 eq) in 90 mL of anhydrous MeOH,
were added at 0 C, 4 mL of MeONa 1 M in MeOH. The mixture was
stirred during 30 min at room temperature, and quenched with
ꢁ
þ
Mp: 146 C.
þ
MS (ESI ): m/z ¼ 1066 [M þ Na ], calculated 1067.
ꢁ
þ
þ
MS (Maldi-TOF ): m/z ¼ 1044 [M þ H ], calculated 1045.
3
0 mL of water, extracted with EtOAc, washed with brine, dried
over MgSO and evaporated. The residue was purified by column
chromatography on silica gel (acetone/DCM 2%) in order to isolate
4
.2.1.8. O-((pyridin-2-yldisulfanyl) ethyl-N-methylamino)mono-
4
methylauristatin E carbamate, 14. To a solution of 13 (27 mg,
.026 mmol) in 0.5 mL dry DCM was added 0.5 mL of TFA. The
0
(
2.7 g,
r
¼ 70%) disulfide 4 as a brown oil.
yellow solution was stirred at room temperature over 2 h. After
completion of the reaction, the mixture was reduced under vacuum
and the residue was purified by chromatography (MeOH/DCM 7%),
Rf (acetone/DCM, 2%) ¼ 0.54
1
H NMR (300 MHz, CDCl3): 8.48 (d, 1H, J ¼ 4.8 Hz), 7.67e7.60
0
(
m, 2H), 7.10 (dd, 1H, J ¼ 6.6 Hz and J ¼ 4.8 Hz), 3.61 (t, 2H,
affording (22 mg,
r
¼ 93%) of the compound 14 as a white solid.
J ¼ 6.9 Hz), 2.92 (t, 2H, J ¼ 4.2 Hz), 2.87 (s, 3H), 1.45 (s, 9H).
Rf (MeOH/DCM 10%) ¼ 0.4
13
C NMR (75 MHz, CDCl
3
): 159.7, 155.3, 149.6, 137.3, 121.1, 120.7,
þ
þ
MS (ESI ): m/z ¼ 967 [M þ Na ], calculated 967.
7
9.7, 48.1, 36.2, 34.5, 28.3.
þ
þ
MS (Maldi-TOF ): m/z ¼ 945 [M þ H ], calculated 945.
þ
þ
MS (IC ): m/z ¼ 301 [M þ H ], calculated 301.
4.2.2. MMAE-linker intermediates 16 synthesis
4
.2.1.4. 2-[N-Methylamino]1-ethyl
2-pyridyl
disulfide,
5.
4.2.2.1. 2-(Pyridin-2-yldisulfanyl)ethanol, 8a. To a suspension of
dipyridyldisulfide (2.2 g, 1.0 eq) in MeOH (99 mL) and pyridine
(1 mL), was added dropwise mercaptoethanol (0.7 mL,10 mmol). As
the mixture turned yellow, the stirring was kept for 72 h at rt. After
concentration in vacuo, the residue was purified by column chro-
matography on silica gel (EtOAc/DCM 0%e5%) to afford 8a as a light
Disulfide 4 (2.5 g, 8.3 mmol) was suspended in 150 mL 1.5 M HCl in
EtOAc. After 2 h at room temperature, no more starting material
could be seen by TLC. The mixture was then evaporated to dryness
and the quantitatively obtained chlorohydrate was directly used in
the next step.
Rf (acetone/DCM 5%) ¼ 0.0
oil (1.09 g,
r
¼ 58%)
1
H NMR (300 MHz, MeOD): 8.71 (d, 1H, J ¼ 4.7 Hz), 8.33e7.97
Rf (EtOAc/DCM 40%) ¼ 0.35
0
1
(
2
m, 2H), 7.68 (dd,1H, J ¼ 6.5 Hz and J ¼ 4.7 Hz), 3.38 (m, 2H), 3.23 (t,
H NMR (300 MHz, CDCl
3
):
d
8.31 (d, 1H, J ¼ 4.8 Hz), 7.44 (t, 1H,
H, J ¼ 6.9 Hz), 2.77 (s, 3H).
J ¼ 7.8 Hz), 7.31 (d, 1H, J ¼ 8.1 Hz), 6.98 (t, 1H, J ¼ 5.6 Hz), 5.65 (m,
þ
þ
MS (IC ): m/z ¼ 201 [M þ H ], calculated 201.
1H) 3.64 (t, 2H, J ¼ 6.5 Hz), 2.80 (t, 2H, J ¼ 6.5 Hz).
13
3
C NMR (75 MHz, CDCl ): d 169.2, 156.9, 135.1, 119.6, 119.0, 56.7,
4
0.6.
IR,
4
.2.1.5. 2-[N-(Chlorocarbonyl)-N-methylamino]1-ethyl 2-
¡
1
n
(cm ): 3318, 2865, 1574, 1416, 1115.
pyridyldisulfide, 6. To a suspension of chlorohydrate 5 (2.2 g,
8
solution in toluene were added at 0 C, followed by Et
2
at room temperature. After evaporation, the residue was purified
over silica gel (acetone/DCM 5%). Carbamoyl chloride 6 (1.6 g,
r
-
þ
MS (ESI ): m/z ¼ 186 [M-H ], calculated 186.
.0 mmol) in 150 mL of DCM, 22 mL (excess) of a 20% phosgene
ꢁ
3
N (2.2 mL,
4
9
.2.2.2. 4-nitrophenyl (2-(pyridin-2-yldisulfanyl)ethyl) carbonate,
a. A solution of 8a (50 mg, 0.367 mmol), Et
.0 eq). The ice-bath was removed and the stirring pursued for 3 h
3
N (40
mL,1.1 eq) in ACN
ꢁ
(2 mL) was cooled to 0 C. The solution turned yellow as 4-
nitrophenylchloroformate (62 mg, 1.1 eq) was added, and was
stirred for 16 h at rt. After concentration in vacuo, the residue was
purified by column chromatography on silica gel (EtOAc/Cyclo-
hexane 10%e30%) to afford the title compound as a pale yellow oil
¼ 78%) was obtained as a colorless oil.
Rf (acetone/DCM 5%) ¼ 0.60
1
H NMR (300 MHz, CDCl3): 8.49 (d, 1H, J ¼ 3.2 Hz), 7.70e7.65
(
(
m, 2H), 7.13 (m, 1H), 3.79 and 3.70 (t, 2H, J ¼ 7.1 Hz), 3.17 and 3.11
s, 3H), 3.08 and 3.01 (t, 2H, J ¼ 7.4 Hz).
(80 mg,
r
¼ 85%).
Rf (EtOAc/Cyclohexane 40%) ¼ 0.30
13
C NMR (75 MHz, CDCl3): 159.9, 149.9, 137.8, 121.4, 120.7, 79.7,
1
H NMR (300 MHz, CDCl
3
):
d
8.46 (d, 1H, J ¼ 4.7 Hz), 8.27 (d, 2H,
4
8.8, 36.6, 34.2, 28.1.
MS (IC ): m/z ¼ 263 [M þ H ], calculated 263.
J ¼ 9.2 Hz), 7.76 (m, 2H), 7.37 (d, 2H, J ¼ 9.1 Hz), 7.17 (m, 1H), 4.54 (t,
þ
þ
2H, J ¼ 6.4 Hz), 3.15 (t, 2H, J ¼ 6.4 Hz).
13
C NMR (75 MHz, CDCl
3
): d 162.7, 154.9, 150.4, 149.4, 145.5,
4
.2.1.6. N-tert-butoxycarbonylmonomethylauristatin E, 12. A solu-
137.5, 125.5, 122.0, 121.7, 120.4, 66.6, 36.8.
¡1
tion of di-tert-butyl dicarbonate (1.2 mg, 1.0 eq) in DCM (1 ml) was
added dropwise to a stirred solution of MMAE (41.8 mg,
IR, n (cm ): 1766, 1522, 1203, 1111.
þ
þ
MS (ESI ): m/z ¼ 353 [M þ H ], calculated 353.
0
.058 mmol) and Et
The reaction mixture was stirred 2 h and was then washed with
NaHCO solution, water and then brine. The organic layer was dried
over MgSO and the volatiles were removed by evaporation to give
the title product 12 (45 mg,
3
N (8 mL, 1.0 eq) in DCM (1 ml).
4.2.2.3. N-(2-(pyridin-2-yldisulfanyl)-ethanoxycarbonyl)mono-
methylauristatin E, 16a. To a solution of MMAE (10 mg, 0.014 mmol)
in DCM (1 mL) was added 9a (10 mg, 2.0 eq), HOBt (2 mg, 1.0 eq)
and Et N (2 mL, 1.0 eq). After stirring 72 h at rt, the mixture was
3
evaporated and the residue was purified by column chromatog-
raphy on silica gel (MeOH/DCM 0%e15%) to afford 16a (11 mg,
3
4
r
¼ 95%) as a colorless oil.
Rf (MeOH/DCM 4%) ¼ 0.35
þ
þ
MS (ESI ): m/z ¼ 840 [M þ Na ], calculated 840.
þ
þ
MS (Maldi-TOF ): m/z ¼ 818 [M þ H ], calculated 818.
r
¼ 85%).

490
C. Batisse et al. / European Journal of Medicinal Chemistry 95 (2015) 483e491
Rf (MeOH/DCM 10%) ¼ 0.28
salt.
þ
þ
MS (Maldi-TOF ): m/z ¼ 932 [M þ H ], calculated 932.
Rf (MeOH/DCM 10%) ¼ 0.0
þ
þ
MS (Maldi-TOF ): m/z ¼ 999 [M þ Na ], calculated 998.
4
.2.2.4. 2-((3-nitropyridin-2-yl)disulfanyl)ethanol, 8b. To a suspen-
sion of (3-nitropyridin-2-yl)sulfanyl chloride (150 mg, 1.0 eq) in
cyclohexane (6 mL) was added a solution of mercaptoethanol
4.3. Synthesis of STxB conjugates
(
57
m
L, 0.803 mmol) in cyclohexane/DCM (1/1, 6 mL). After stirring
Genetically engineered STxB/Cys, possessing five C-terminal
cysteine residues was expressed and purified according to estab-
lished procedures [27]. MALDI-TOF mass spectrometry was used to
evaluate the formation of STxB-based conjugates. The mass error in
the range of around 8000 Da (size of the STxB monomer)
was ± 5 Da. Coupling yields were determined by HPLC analysis.
To determine coupling conditions, prodrug compounds were
dissolved in DMSO, and STxB/Cys diluted in a PBS buffer to a final
for 16 h at rt, the yellow mixture was concentrated in vacuo, and
the residue was purified by column chromatography on silica gel
(
EtOAc/DCM 0%e5%) to afford 8b as a yellow oil (77 mg,
r
¼ 42%).
Rf (MeOH/DCM 2%) ¼ 0.25
1
H NMR (300 MHz, CDCl
3
):
d
8.82 (d, 1H, J ¼ 4.5 Hz), 8.54 (d, 1H,
J ¼ 8.2 Hz), 7.41 (q, 1H, J ¼ 4.3 Hz), 3.71 (t, 2H, J ¼ 5.1 Hz), 3.03 (t, 2H,
J ¼ 5.1 Hz).
1
3
C NMR (75 MHz, CDCl
3
):
d
158.7, 153.9, 142.3, 134.6, 121.7, 58.8,
concentration of 1 mg/mL, to afford 20 mg of STxB/Cys per reaction.
4
3.1.
IR,
Three parallel incubations were carried out using respectively a
1, 3 and 9-fold molar excess of prodrug per STxB monomer. Each
mixture was diluted in DMSO and PBS buffer pH ¼ 7.5, to afford a
ꢀ
þ
1
n
(cm ): 3376, 2924, 1720, 1514, 1339, 1257.
þ
MS (ESI ): m/z ¼ 233 [M þ H ], calculated 233.
maximal volume of DMSO equal to 1
m
L in a total volume of 10
mL.
ꢁ
4.2.2.5. 4-nitrophenyl (2-((3-nitropyridin-2-yl)disulfanyl)ethyl) car-
Coupling reactions were carried out for 16 h at 21 C with
stirring, and the conjugates were dialyzed (10 kDa cut-off) against
water for three hours at rt for mass spectrometry analysis.
For upscaling, reactions were carried out with up to 30 mg of
STxB/Cys under conditions as determined in the pilot experiments.
bonate, 9b. A solution of 8b (60 mg, 0.258 mmol), Et
3
N (104
mL, 3.0
ꢁ
eq) in ACN (2 mL) was cooled to 0 C. The solution turned from
orange to yellow as 4-nitrophenylchloroformate (156 mg, 3.0 eq)
was added, and was stirred for 16 h at rt. After concentration in
vacuo, the residue was purified by column chromatography on
silica gel (EtOAc/Cyclohexane 10%e30%) to afford the title com-
ꢁ
Dialysis was in this case for 16 h at 4 C against PBS for later use on
cells.
pound as a yellow oil (57 mg,
r
¼ 56%).
STxB-MMAE, 15: m/z ¼ 8626 (calculated), 8628 (found).
Rf (EtOAc/Cyclohexane 40%) ¼ 0.39
STxB: m/z ¼ 7793 (calculated).
1
H NMR (300 MHz, CDCl
3
): 8.87 (m, 1H), 8.54 (d, 1H,
d
STxB-MMAE, 17: m/z ¼ 8613 (calculated), 8612 (found).
STxB: m/z ¼ 7793 (calculated), 7793 (found).
J ¼ 7.8 Hz), 8.29 (d, 2H, J ¼ 9.3 Hz), 7.40 (d, 3H, J ¼ 9.0 Hz), 4.59 (t,
2
H, J ¼ 6.6 Hz), 3.23 (t, 2H, J ¼ 6.5 Hz).
r
¼ 95%, n ¼ 4.8.
13
C NMR (75 MHz, CDCl
3
):
d
157.1, 155.8, 154.2, 153.6, 152.6,
STxB-MMAF, 20: m/z ¼ 8627 (calculated), 8627 (found).
STxB: m/z ¼ 7793 (calculated), 7794 (found).
1
45.9, 134.3, 125.8, 122.2, 121.6, 67.5, 36.6.
¡1
IR,
n
(cm ): 2924, 1767, 1522, 1341, 1212.
r
¼ 96%, n ¼ 4.8.
þ
þ
MS (ESI ): m/z ¼ 398 [M þ H ], calculated 381.
STxB-MMAE, 22: m/z ¼ 8613 (calculated), 8614 (found).
STxB: m/z ¼ 7793 (calculated), 7792 (found).
4
.2.2.6. N-(2-((3-nitropyridin-2-yl)disulfanyl)ethanoxycarbonyl)
STxB-mercaptoethanol: m/z ¼ 7869 (calculated), 7869 (found).
monomethylauristatin E, 16b. To a solution of MMAE (4.8 mg,
.007 mmol) in THF (2 mL) was added 9b (4.8 mg, 2.0 eq), HOBt
1 mg, 1.0 eq) and Et N (1 L, 1.0 eq). After stirring for 72 h at rt, the
0
4.4. Intracellular trafficking evaluation by immunofluorescence
(
3
m
ꢁ
mixture was evaporated and the residue was purified by column
chromatography on silica gel (MeOH/DCM 0%e15%) to afford 16b
Cells in DMEM/FCS were incubated for 30 min at 4 C with
conjugates or STxB/Cys at final concentrations of 0.2
m
M, washed,
ꢁ
(
4.2 mg,
r
¼ 62%).
incubated for 45 min at 37 C, fixed with 4% para-formaldehyde,
and permeabilized with saponine. Immunodetection was carried
out using a primary mouse-monoclonal anti-STxB antibody (13C4),
and a home-made rabbit polyclonal antibody against the Golgi
marker giantine, followed by detection thanks to appropriate flu-
orescently labeled secondary antibody.
Rf (MeOH/DCM 10%) ¼ 0.55
þ
þ
MS (Maldi-TOF ): m/z ¼ 999 [M þ Na ], calculated 998.
4
4
.2.3. MMAF-linker intermediate 19 synthesis
.2.3.1. N-(2-((3-nitropyridin-2-yl)disulfanyl)ethanoxycarbonyl)
monomethylauristatin F, 19. Similarly to 16b, from MMAF (5 mg,
0
.007 mmol), the title compound was obtained (6.0 mg,
r
¼ 87%).
4.5. Inhibition of Gb3 synthase
Rf (MeOH/DCM 5%) ¼ 0.0.
þ
þ
MS (Maldi-TOF ): m/z ¼ 1013 [M þ Na ], calculated 1012.
Cells were incubated for 6 days with 5
amide synthase inhibitor 1-phenyl-2-palmitoylamino-3-
morpholino-1-propanol (PPMP). Gb3 expression was determined
by FACS analysis after incubation with STxB-AlexaFluor . PPMP
treatment was scored successful when STxB signal were below 2%
of those observed on non-PPMP treated control cells.
mM of the glycosylcer-
4
4
.2.4. Carbonate MMAE-linker intermediate 21 synthesis
4
88
.2.4.1. (2-((3-nitropyridin-2-yl)disulfanyl)ethanol)-N-tert-butox-
ycarbonylmonomethylauristatin E carbonate, 20. Similarly to 16b,
from 12 (5.5 mg, 0.007 mmol), the title compound was obtained
(
3.9 mg,
r
¼ 52%).
Rf (MeOH/DCM 10%) ¼ 0.53
4.6. Antiproliferative activity evaluation
þ
þ
MS (Maldi-TOF ): m/z ¼ 1099 [M þ Na ], calculated 1099.
3000 Gb3 positive or negative cells per well were seeded in
4
.2.4.2. (2-((3-nitropyridin-2-yl)disulfanyl)ethanol)mono-
200 mL in 96-well dishes and cultured overnight before adding
methylauristatin E carbonate, 21. 20 (2 mg, 0.002 mmol) was sus-
pended in a mixture TFA/DCM (1/9, 1 mL) for 2 h at rt. The mixture
was evaporated and the residue was afforded 21 as a trifluoroacetic
conjugates or free compounds at the indicated concentrations in
the culture medium and further incubation for 6 h at 37 C. After
extensive washes, cells were further incubated for 5 days. The
ꢁ

C. Batisse et al. / European Journal of Medicinal Chemistry 95 (2015) 483e491
491
percentage of living cells was quantified using a colorimetric assay
MTT) based on mitochondrial metabolism. All points were deter-
mined in triplicate. IC50 values were calculated as the concentra-
tion of compounds inducing a 50% inhibition of cell proliferation.
R.H. Scheller, P. Polakis, W. Mallet, Nat. Biotechnol. 26 (2008) 925e932.
[
10] S.O. Doronina, B.A. Mendelsohn, T.D. Bovee, C.G. Cerveny, S.C. Alley,
D.L. Meyer, E. Oflazoglu, B.E. Toki, R.J. Sanderson, R.F. Zabinski, A.F. Wahl,
P.D. Senter, Bioconjug. Chem. 17 (2005) 114e124.
[11] R.Y. Zhao, S.D. Wilhelm, C. Audette, G. Jones, B.A. Leece, A.C. Lazar,
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J. Med. Chem. 54 (2011) 3606e3623.
12] F. Dosio, P. Brusa, L. Cattel, Toxins 3 (2011) 848e883.
13] L. Johannes, W. Romer, Nat. Rev. Microbiol. 8 (2012) 105e116.
14] S.-I. Hakomori, Cancer Res. 45 (1985) 2405e2414.
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[16] M. Amessou, D.L. Carrez, D. Patin, M. Sarr, D.S. Grierson, A. Croisy,
A.C. Tedesco, P. Maillard, L. Johannes, Bioconjug. Chem. 19 (2008) 532e538.
17] A. El Alaoui, F. Schmidt, M. Amessou, M. Sarr, D. Decaudin, J.-C. Florent,
(
Acknowledgments
[
[
[
[
ꢂImmunoTargets.
ꢂANRT (CIFRE fellowship).
ꢂPeptide CSB platform, Institut Pasteur de Lille (protein HPLC
analyses).
[
ꢂChimie-ParisTech Laboratoire de spectrom eꢀ trie de masse.
L. Johannes, Angew. Chem. Int. Ed. 46 (2007) 6469e6472.
[18] A. El Alaoui, F. Schmidt, M. Sarr, D. Decaudin, J.-C. Florent, L. Johannes,
ChemMedChem 3 (2008) 1687e1695.
19] N. Haicheur, F. Benchetrit, M. Amessou, C. Leclerc, T. Falgui eꢁ res, C. Fayolle,
ꢂInstitut Curie Protein Mass Spectrometry Laboratory.
[
Appendix A. Supplementary data
E. Bismuth, W.H. Fridman, L. Johannes, E. Tartour, Int. Immunol. 15 (2003)
1161e1171.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.03.047.
[20] W. Herz, G.W. Kirby, R.E. Moore, W. Steglich, C. Tamm, G.R. Pettit (Eds.),
Fortschritte der Chemie organischer Naturstoffe Progress in the Chemistry of
Organic Natural Products vol. 70, Springer, Vienna, 1997, p. 1.
[
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D.F. Chace, R.L. DeBlanc, R.P. Gearing, T.D. Bovee, C.B. Siegall, J.A. Francisco,
A.F. Wahl, D.L. Meyer, P.D. Senter, Nat. Biotech. 21 (2003) 941.
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