Spacer length shapes drug release and therapeutic efficacy of traceless disulfide-linked ADCs ...

67 Pages·2012·5.29 MB·English

by  Gonçalo Bernardes

Electronic Supplementary Material (ESI) for Chemical Science
This journal is © The Royal Society of Chemistry 2012
Supporting Information
Spacer length shapes drug release and therapeutic efficacy
of traceless disulfide-linked ADCs targeting the tumor
neovasculature

Martina Steiner,a Isabelle Hartmann,a Elena Perrino,a Giulio Casi,b Samatanga
Brighton,c, d Ilian Jelesarov,c Gonçalo J. L. Bernardes*a and Dario Neria

aDepartment of Chemistry and Applied Biosciences, Swiss Federal Institute of
Technology (ETH Zürich), Wolfgang-Pauli Str. 10, 8093 Zurich, Switzerland.

bPhilochem AG, Libernstrasse 3, 8112 Otelfingen, Switzerland.

cDepartment of Biochemistry, University of Zürich, Winterthurerstr. 190, 8057
Zurich, Switzerland

dDepartment of Biology, Institute of Molecular Biology and Biophysics, Swiss
Federal Institute of Technology (ETH Zürich), Schafmattstrasse 20, 8093 Zürich,
Switzerland.

To whom correspondence should be addressed:
[email protected]; [email protected]

S1
Electronic Supplementary Material (ESI) for Chemical Science
This journal is © The Royal Society of Chemistry 2012
Supporting Information
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Electronic Supplementary Material (ESI) for Chemical Science
This journal is © The Royal Society of Chemistry 2012
Supporting Information
1 Synthesis of Cem, CemCH -SH and CemCH -SSPy
2 2
The synthesis of cemadotin and its derivatives is based on previously reported work
from our group.1, 2 In addition, the data for CemCH -SH has been previously
2
reported.1
Material and Methods: Analytical and preparative HPLC were performed on an
Alliance HT RP-HPLC with PDA UV detector, using either a Synergi 4 !m, Polar-RP
150*10 mm at a flow rate of 4 mL/min with linear gradients of solvents A and B
(A=millipore water with 0.1% trifluoroacetic acid (TFA), B=acetonitrile), or an X-
Terra Prep RP-18, 5 !m 150*10 mm at a flow rate of 4 mL/min with linear gradients
of A’ and B (A’= 0.1 M Triethylammonium acetate pH 7).
Synthesis of S-4(aminoethyl)benzyl ethanethioate
CH3
OH LiAlH4 OH Boc2O OH thioacetic acid S O
reflux, THF 1M NaOH, t-BuOH PPh3, DIAD, THF
N 96% NH2 98% NHBoc 46% NHBoc
TFA/DCM (1:1)
quantitative
CH3
S O
NH2
Synthesis of Cem, CemCH2-SH, CemCH2-SSPy
H O NH2 H O H
N N N N N OH HATU, DIEA, DMF N N N N N N
O O O O 67% O O O O
P5
(assembled by stepwise solid
phase peptide synthesis)
CH3
S O
NH2
HATU, DIEA, DMF
83%
O
N HN ON N N HN S CH3 1M HC74l %/MeOH N HN ON N N HN SH
O O O O O O O O CemCH2-SH
PySSPy, AcOH
CH3CN/H2O (9:1)
40%
S N
H O H S
N N N N
N N
O O O O CemCH2-SSPy
Scheme 1. Synthetic scheme for the synthesis of Cem, CemCH -SH and CemCH -SSPy.
2 2

S3
Electronic Supplementary Material (ESI) for Chemical Science
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Supporting Information
(4-(aminomethyl)phenyl)methanol
2-Cyanobenzaldehyde was converted into the corresponding amino-
OH
alcohol derivative as previously described.3, 4 A solution of 2-
NH
2
cyanobenzaldehyde (1 g, 7.6 mmol) in anhydrous THF (10 mL) was
added dropwise to a stirred suspension of LiAlH (1.74 g, 45.8 mmol) in anhydrous
4
THF (30 mL) at 0 ºC. The reaction mixture was heated to reflux and stirred for
5 hours before cooling down again to 0 ºC and quenching with H O (2 mL), 15%
2
NaOH (4 mL) and H O (6 mL). After stirring for 10 min at room temperature, the
2
mixture was filtered on a pad of celite (washed with EtOAc). Concentration in vacuo
gave (4-(aminomethyl)phenyl)methanol as a white solid (1.0 g, 96% yield), which
was used without further purification. Spectroscopic data was identical to that
previously reported.4 1H-NMR (300 MHz, CDCl ) (cid:1) 3.80 (s, 2H), 4.60 (s, 2H), 7.25
3
(m, 4H); ESI-MS: calcd. for C H NO ([M+H]+) m/z 138.08, found 137.79.
8 12
tert-butyl 4-(hydroxymethyl)benzylcarbamate
(4-(Aminomethyl)phenyl)methanol was converted to the Boc
OH
derivative as previously described.4 Briefly di-tert-butyl dicarbonate
NHBoc (1.99 g, 9.12 mmol) was added to a stirred solution of -
(aminomethyl)phenyl)methanol (1.0 g, 7.3 mmol) in tBuOH / 1M NaOH (2/1, 50 mL)
at 0 °C. The reaction was allowed to warm up to room temperature, and stirred for 2
additional hours. The reaction was then washed with a NaHCO saturated solution
3
and the aqueous phase extracted with EtOAc. The combined organic layers were dried
over Na SO , and concentrated in vacuo to give a white solid (1.70 g, 98% yield),
2 4
which was used in the next step without further purification. Spectroscopic data was
identical to that previously reported.4 1H-NMR (300 MHz, CDCl ) (cid:1) 1.36 (s, 9H),
3
4.23 (s, 2H), 4.61 (s, 2H), 7.23 (m, 4H); ESI-MS: calcd. for C H NO ([M+H]+) m/z
13 20 3
238.29, found 237.92.

S4
Electronic Supplementary Material (ESI) for Chemical Science
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Supporting Information
S-4-((tert-Butoxycarbonyl)amino)methyl)benzyl ethanethioate
tert-Butyl 4-(hydroxymethyl)benzylcarbamate was converted to
CH
3
the corresponding thioester derivative using Mitsunobu conditions.
S O
A two-neck round bottomed flask was charged with tert-butyl 4-
NHBoc
(hydroxymethyl)benzylcarbamate (1.90 g, 8.0 mmol),
triphenylphospine (32 mmol, 4 equiv.) and thioacetic acid (32 mmol, 4 equiv.) in
anhydrous THF (100 mL). The flask was immersed in an ice bath and the mixture was
stirred under an atmosphere of argon. Diisopropyl azodicarboxylate (DIAD)
(32 mmol, 4 equiv.) was then added dropwise. Upon completion of addition, the
mixture was allowed to warm to room temperature and was stirred overnight. The
reaction mixture was diluted with diethyl ether (100 mL) and washed twice with a
NaHCO saturated solution. The aqueous layers were combined and re-extracted with
3
diethyl ether (100 mL). The organics were combined, dried with Na SO , filtered, and
2 4
concentrated in vacuo. The resulting residue was diluted in diethyl ether (40 mL) and
hexanes were added (20 mL). The mixture was left standing overnight. The resulting
white solid was removed under vacuum and the filter cake washed with a 50% (v/v)
solution of hexanes/diethyl ether (100 mL). The solvent was removed under vacuum,
and the resulting yellow oil was purified by flash column chromatography
(hexanes/EtOAc, 8/1, R 0.15). Solvent evaporation resulted in an oil that upon
f
addition of hexanes resulted in a white solid. The product was filtered and washed
with cold hexanes to give the desired product as a white solid (1.15 g, 46% yield). 1H-
NMR (300 MHz, CDCl ) (cid:1) 1.43 (s, 9H), 2.32 (s, 3H), 4.08 (s, 2H), 4.26 (m, 2H),
3
7.21 (m, 4H); ESI-MS: calcd. for C H NO S ([M+H]+) m/z 296.40, found 295.90.
15 22 3
S-4-(Aminomethyl)benzyl ethanethioate
In a 10 mL flask S-4-((tert-butoxycarbonyl)amino)methyl)benzyl
CH
3
ethanethioate (0.4 g, 1.35 mmol) was dissolved in DCM (6 mL).
S O
TFA (2 mL) was added and the mixture stirred at room
NH
2 temperature for 15 minutes. After the mixture was evaporated and
lyophilized from dioxane, the desired product was isolated as a white powder
(260 mg, quantitative). 1H-NMR (300 MHz, CDCl ) (cid:1) 2.33 (s, 3H), 3.71 (s, 2H),
3
4.09 (m, 2H), 7.25 (m, 4H); ESI-MS: calcd. for C H NOS ([M+H]+) m/z 196.28,
10 14
found 195.83.
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Supporting Information
N,N-dimethylvalyl-valyl-N-methylvalyl-prolyl-proline (P5)
The pentapeptide P5 was assembled by step-
O
H
N N N OH wise solid phase peptide synthesis.
N N
O O O O Commercially available pre-loaded Fmoc-Pro
on Tentagel resin (3.88 g, 0.66 mmol, RAPP Polymere) was swollen first in DCM,
then in DMF (2 x 3 min x 15 mL, in both cases). The Fmoc group was removed with
20 % piperidine in DMF (2 x 5 min x 10 mL). The resin was washed with DMF.
Prior to coupling to the resin, amino acids were activated: 3 equiv. of each amino acid
were pre-activated for 10 minutes with equimolar amounts of a coupling agent in the
presence of 6 equiv. N,N-diisopropylethylamine (DIEA) at a concentration of 0.15 M
in DMF. In the case of Fmoc-Pro-OH and Fmoc-N(Me)-Val-OH single couplings
were performed using HBTU/HOBt; in the case of Fmoc-Val-OH and (Me) -Val-OH
2
prolonged triple and double couplings with HATU, respectively, were required for
complete reaction. The pentapeptide was cleaved from the resin after 2 hours
incubation with 60 mL of DCM/TFE/AcOH (3/1/1). The resin was filtered, the
solvent evaporated and the desired product lyophilized from dioxane (310 mg, 85%
yield). ESI-MS: calcd. for C H N O ([M+H]+) m/z 552.7, found 552.1.
28 50 5 6
Cem
In a 25 mL round bottom flask
H O H
N N N N containing P5 (40 mg, 0.073 mmol)
N N
O O O O
dissolved in DMF (2 mL), HATU
(28 mg, 0.073 mmol, 1 equiv.) and DIEA (51 !L, 0.290 mmol, 4 equiv.) were added.
After 10 minutes pre-activation, benzylamine (10 !L, 0.087 mmol, 1.2 equiv.) was
added dropwise. The reaction was judged completed after 2 hours by HPLC
chromatography. DMF was removed under vacuum, the crude product re-suspended
in 50% CH CN / H O, and purified over HPLC. The collected fractions were
3 2
lyophilized, and the desired peptide was isolated as a white powder (32 mg, 67 %
yield, HPLC retention time: 10.36 minutes, Synergi column, 20-80% B in 15 minutes,
4 mL/min). ESI-MS: calcd. for C H N O ([M+H]+) m/z 641.44, found 641.14.
35 57 6 5
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Supporting Information

Figure 1. HPLC trace (wavelength of 210 nm) and ESI-MS spectrum of Cem.
CemCH -SAc
2
In a 25 mL round bottom
O
S CH flask containing P5 (100 mg,
H O H 3
N N N N N N 0.181 mmol) dissolved in
O O O O
DMF (4 mL), HATU
(68.9 mg, 0.181 mmol, 1 equiv.) and DIEA (127 !L, 0.725 mmol, 4 equiv.) were
added. After 10 minutes pre-activation, S-4-(aminomethyl)benzyl ethanethioate
(42.5 mg, 0.218 mmol, 1.2 equiv.) was added as a solid. The reaction was judged
completed after 1 hour by HPLC chromatography. DMF was removed under vacuum,
the crude product re-suspended in 50% CH CN / H O, and purified over HPLC. The
3 2
collected fractions were lyophilized, and the desired peptide was isolated as a white
powder (110 mg, 83 % yield, HPLC retention time: 11.52 minutes, Synergi column,
20-80% B in 15 minutes, 4 mL/min). ESI-MS: calcd. for C H N O S ([M+H]+) m/z
38 61 6 6
730.0, found 728.9.
S7
Electronic Supplementary Material (ESI) for Chemical Science
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Supporting Information
CemCH -SH
2
Cem-CH -SAc (32 mg, 0.044 mmol) was dissolved in MeOH (1.5 mL, degassed).
2
Subsequently NaOH (1.5 mL, 200 mm in water, degassed) was added and the reaction
incubated for 30 minutes at room temperature. The reaction was judged to be over by
ESI-MS analysis. The reaction was quenched with Tris buffer (3 mL, 1 M, pH=7) and
the thiol reduced for 30 minutes with DTT (100 mm final concentration). The crude
mixture was purified by HPLC. The collected fractions were lyophilized, and the
desired peptide was isolated as a white powder (22 mg, 74% yield, HPLC retention
time: 11.22 minutes, Synergi, 20-80% B in 15 minutes). 1H-NMR (500 MHz, DMSO)
(cid:1) 0.69 (d, 3H, j= 6.8 Hz), 0.78-0.9 (m, 12H), 0.9-1 (m, 3H), 1.65-2.3 (m, 11H), 2.75
(s, 6H), 3.07 (s, 3H), 3.5-3.6 (m, 2H), 3.6-3.8 (m, 4H), 4.15 (dd, 1H, J= 14.3, 5.6 Hz),
4.25-4.35 (m, 3H), 4.48-4.6 (m, 2H), 4.97 (d, 1H, J= 11.9 Hz), 7.1-7.3 (m, 4H), 8.23-
8.29 (m, 1H), 8.9-9.0 (m, 1H), 9.8 (m, 1H); ESI-MS: calcd. for C H N O S:
36 59 6 5
([M+H]+) m/z 687.8, found 687.0.

Figure 2. HPLC trace (wavelength of 210 nm) and ESI-MS spectrum of CemCH -SH.
2
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Supporting Information

Figure 3. 1H-NMR spectrum of CemCH -SH.
2
CemCH -SSPy
2
CemCH -SH (40 mg,
S N 2
S
H O H 0.058 mmol) dissolved in
N N N N
N N
O O O O 9/1 CH3CN/H2O was added
dropwise to an AcOH (0.35 mL) solution of pyridyl-disulfide (103 mg, 0.466 mmol,
8 equiv.). After 1 h the reaction was evaporated, the crude resuspended in 1/9
CH CN/H O: the solid (pyridyl-disulfide) was removed by centrifugation, the cleared
3 2
solution was purified over HPLC and the desired product isolated as a white powder
(18.5 mg, 40% yield, HPLC retention time: 12.85 minutes X-Terra, 20-80% B in
15 minutes). 1H-NMR (500 MHz, DMSO) (cid:1) 0.63-0.73 (m, 6H), 0.76-1.07 (m, 12H),
1.65-2.17 (m, 10H), 2.17-2.23 (s, 7H), 2.62-2.64 (m, 1H), 3.06 (s, 3H), 3.46-3.62 (m,
2H), 3.62-3.82 (m, 2H), 4.08 (s, 2H), 4.11-4.2 (m, 1H), 4.25-4.36 (m, 2H), 4.44-4.56
(m, 2H), 4.97 (d, 1H, J= 8.5 Hz), 7.1-7.3 (m, 4H), 7.59-7.63 (m, 1H), 7.71-7.78 (m,
1H), 8.06-8.08 (m, 1H), 8.6-8.3 (m, 1H), 8.4-8.5 (m, 1H); ESI/MS: calcd for
C H N O S ([M+H]+) m/z 796.43 Da, found 796.08 Da.
41 62 7 5 2

S9
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Supporting Information

Figure 4. HPLC trace (wavelength of 210 nm) and ESI-MS spectrum of CemCH -SSPy.
2

Figure 5. 1H-NMR spectrum of CemCH -SSPy.
2
S10