Design and Synthesis of Tesirine, a Clinical Antibody-Drug Conjugate Pyrrolobenzodiazepine Dimer Payload

Article abstract of DOI:10.1021/acsmedchemlett.6b00062

Pyrrolobenzodiazepine dimers are an emerging class of warhead in the field of antibody-drug conjugates (ADCs). Tesirine (SG3249) was designed to combine potent antitumor activity with desirable physicochemical properties such as favorable hydrophobicity a

Full text of DOI:10.1021/acsmedchemlett.6b00062

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Letter
Design and synthesis of tesirine, a clinical antibody-
drug conjugate pyrrolobenzodiazepine dimer payload
Arnaud Charles Tiberghien, Jean-Noel Levy, Luke A. Masterson, Neki V. Patel, Lauren
R. Adams, Simon Corbett, David G. Williams, John A. Hartley, and Philip W. Howard
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00062 • Publication Date (Web): 24 May 2016
Downloaded from http://pubs.acs.org on May 30, 2016
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Design and synthesis of tesirine, a clinical antibody-drug conjugate
pyrrolobenzodiazepine dimer payload
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Arnaud C. Tiberghien*, Jean-Noel Levy†, Luke A. Masterson, Neki V. Patel, Lauren R. Adams, Simon
Corbett, David G. Williams, John A. Hartley, Philip W. Howard*
Spirogen, QMB Innovation Centre, 42 New Road, E1 2AX, London
Tesirine, Antibody-drug conjugates, Talirine, SG3249, Rova-T
ABSTRACT: Pyrrolobenzodiazepine dimers are an emerging class of warhead in the field of antibody-drug conjugates (ADCs).
Tesirine (SG3249) was designed to combine potent anti-tumor activity with desirable physico-chemical properties such as favorable
hydrophobicity, and improved conjugation characteristics. One of the reactive imines was capped with a cathepsin B-cleavable valine-
alanine linker. A robust synthetic route was developed to allow the production of tesirine on clinical scale, employing a flexible,
convergent strategy. Tesirine was evaluated in vitro both in stochastic and engineered ADC constructs, and was confirmed as a potent
and versatile payload. The conjugation of tesirine to anti-DLL3 rovalpituzumab has resulted in rovalpituzumab-tesirine (Rova-T),
currently under evaluation for the treatment of small cell lung cancer.
2
Since their discovery in 1963,1, pyrrolobenzodiazepines
(PBDs) have been studied for their anti-tumor properties. Both
naturally occurring monomer anthramycin, and synthetic dimer
SG2000 have undergone clinical evaluation as stand-alone
agents.3 Until recently,4 however, this more potent class of
molecule remained under-exploited as a source of warhead for
antibody-drug conjugates (ADCs).
Antibody-drug conjugates combine the potency of
a
cytotoxic warhead with the selectivity of an antibody to enable
targeted killing of tumor cells. Most ADCs currently in clinical
evaluation are based on anti-mitotic warheads such as auristatin
and maytansine.5
Given their potency and mode of action as sequence-selective
cross-linking agents, we hypothesized the ADC field would
benefit from the employment of PBDs as a new class of
warhead. A collaboration between Spirogen and Seattle
Genetics led to the development of SGN-CD33A, an ADC
under clinical investigation for the treatment of acute myeloid
leukemia, featuring the PBD payload SGD-1910 or talirine.6, 7
We aimed to improve on the design of SGD-1910 by lowering
hydrophobicity whilst maintaining potency. (Figure 1)
Figure 1. Structures of SG2202 and ADC warhead SG3199. LogD
values calculated at pH 7.4. Structures of payloads (drug-linker)
SG3249 (tesirine), and SGD-1910 (talirine). Red coding denotes
released warhead. Conventional ring numbering shown on
SG3199.
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We also investigated trapping one of the reactive imine groups at the phenolic position. TIPS was selected for its
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moieties in its carbinolamine form by incorporating the N10
nitrogen as part of a cathepsin B-cleavable valine-alanine
carbamate prodrug linker.8 Finally, a discrete PEG8 spacer was
inserted to further enhance the solubility properties of the
molecule. This design resulted in the payload SG3249
(tesirine).
relatively high resistance to acid and base, with the option of
selective phenolic silyl deprotection in the presence of aliphatic
silyl ethers. Clean and efficient Pinnick oxidation of
benzaldehyde 4 gave acid 5. Amide bond formation with
hydroxyproline derivative 611 gave alcohol
7
(see
supplementary information), which was oxidized using the
reactive TEMPO/TCCA combination. Thermodynamic
treatment of compound 817, 18 with triflic anhydride gave triflate
9 in 78% yield (Scheme 2). This was followed by a sp2-sp3
Suzuki coupling to install a methyl group at the C2 position.
Activated conditions developed by Mu and Gibbs,19 employing
silver oxide and triphenylarsine, were required for efficient
coupling. Optimization of the reaction conditions was necessary
to avoid competing triflate reduction; the resulting impurity 10
having similar chromatographic retention properties to the
desired compound 11. When the same conditions were
investigated with homologous ethyl boronic acid, a much larger
proportion of reduced material 10 was found to contaminate the
mixture. The nitro group was reduced with zinc and dilute
formic acid in ethanol. These conditions were mild enough to
leave both the primary TBS ether and the internal unsaturation
intact.
The warhead component of tesirine, SG3199 (Figure 1), was
based on rational design principles. The C2 aryls, responsible
both for the high potency and poor aqueous solubility of
SG2202 and analogues9, 10, were replaced with simpler methyl
groups. The expected drop in potency was offset by the
introduction of a 5-carbon tether between the two monomers.
Both 3-carbon and 5-carbon tether linked PBDs are isohelical
with the DNA helix, but the 5-carbon tether is more flexible and
has greater opportunities for contact with the minor groove.11, 12
SG3199 was tested in a panel of cancer cell lines and retained
picomolar activity in vitro. (Table 1).
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Table 1. In vitro activity of warhead SG3199.
Cell line IC50
Compound
SG3199
K562
NCIN87
20 pM
BT474
1 nM
SKBR3
320 pM
Scheme 2: Synthesis of monomeric phenol 20 and 21
150 pM
IC50 values (mean of three independent experiments) were
determined using CellTiter96 (MTS) following 96h incubation.
Rather than following a linear route as used in the synthesis of
SG2202 or SG2000 (SJG-136),13 we decided to follow a
convergent synthetic route, allowing both the left-hand (linker)
and right-hand (imine) sides of the unsymmetrical dimer to be
made in parallel, followed by late-stage dimerization.
Monomeric routes have been developed previously by Howard
et al,12 and Kamal et al.14 Recently Kolakowski et al15 have
reported the application of a convergent strategy to the synthesis
of an unsymmetrical C2-aryl substituted PBD. In contrast to
these other strategies, we selected N-alloc and O-TBS as the
N10-imine and C11-hydroxy protecting groups for mild and
efficient deprotection in the final stages of the synthesis. Both
groups are resistant to base and mild acid, but can be
orthogonally removed when required.
Scheme 1: Synthesis and coupling of A and C rings
Reagents and conditions: (a) Tf2O, 2,6-lutidine, DCM, -45 ˚C, 78%, (b)
MeB(OH)2, Ag2O, AsPh3, Pd(PhCN)2Cl2, 70 ˚C, 70% (c) Zn (30 eq.),
HCOOH/ EtOH 5/95, 30 ˚C, 80% (d) Alloc-Cl, pyridine, DCM, -78 ˚C to
rt, 100% (e) triphosgene, Et3N, THF, 5 ˚C, then Alloc-Val-Ala-PAB-OH,
Et3N, THF, 40 ˚C, 50%, (f) AcOH/MeOH/THF/water 7/1/1/2, rt, 71%, 15,
80%, 16, (g) DMSO, (COCl)2, Et3N, DCM, -78˚C to rt, 66%, 17, 60%, 18
(h) TBS-OTf, 2,6-lutidine, DCM, 0 ˚C, 85%, 19, 65%, 20, (i) LiOAc,
DMF/water, 95/5, rt, 100%, 21, 100%, 22.
Reagents and conditions: (a) HNO3, 12 ˚C, 95%, (b) TFA, 85 ˚C, 50%,
(c) TIPSCl, imidazole, 100 ˚C, 88%, (d) NaClO2, NaH2PO4, H2O2, THF, rt
-78 ˚C to rt, 100%, (e) DCC, HOBt, Et3N, DCM, -10 ˚C to rt, 90%, (f)
TEMPO, TCCA, DCM, 100%.
At this key juncture in the synthesis, carbamates 13 and 14
were formed from 12, either by treatment with alloc
chloroformate, or through the initial formation of an
Controlled nitration of benzylvanillin afforded intermediate
2 (Scheme 1).16 This was followed by an exchange of protecting
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isocyanate20 followed by addition of alloc-Val-Ala-para-
15.88
UV 223 nm
-16000
-18000
-20000
-22000
-24000
-26000
-28000
-30000
aminobenzylalcohol.21 The two resulting monomeric
intermediates 13 and 14 were subjected to the same sequence of
reactions beginning with mild acid hydrolysis of the primary
TBS and ring-closing oxidation under Swern conditions.
Protection of the 11-hydroxy group is critical at this point to
allow further chemistry to proceed under basic or acidic
conditions whilst preserving the 11a stereochemistry.22 The use
of highly reactive TBS triflate23 was necessary to achieve
efficient secondary alcohol protection. Finally, mild and
orthogonal deprotection of the phenolic TIPS in the presence of
aliphatic TBS was achieved with LiOAc in wet DMF24 to
provide both the right-hand and left-hand side monomers 21 and
22.
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15.69
a)
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ii
i
-32000
-12000
-14000
-16000
-18000
-20000
-22000
-24000
-26000
-28000
-30000
-32000
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15.88
UV 223 nm
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b)
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i
Williamson ether chemistry, proceeding via iodopentane
derivatization of alloc-protected monomer 21 and subsequent
reaction with monomer 22, provided dimer 24 in 86% yield
(Scheme 3). Removal of 11-hydroxy TBS with TBAF resulted
in partial racemization at C11a. (Detectable by LC and optical
rotation analyses of SG3249: Figure 2, Table 2). Buffering the
fluoride solution with acetic acid was found to prevent
racemisation.25, 26
13.5
14.0
14.5
15.0
15.5
16.0
16.5
17.0
17.5
18.0
18.5
19.0
19.5
Time (min)
Figure 2. UPLC of SG3249: a) unbuffered TBAF used in Scheme
3, step c; b) buffered TBAF. Ace Excel 2 C18-AR (2 µm, 3.0 mm
x 100 mm), 40 °C, 20 mM ammonium formate (pH = 4)
/acetonitrile, 25/75 to 55/45 over 30 min, 0.6 mL/min. Peaks i and
ii in a) Diastereoisomers of SG3249. Peak i in b): pure SG3249.
Table 2. SG3249 optical rotation
Scheme 3: Final steps, synthesis of SG3249 (tesirine)
SG3249 Batch
Unbuffered
Buffered
Optical rotation
+118° (c = 0.43)
+262° (c = 0.56)
TBS Cleavage
TBAF
TBAF/AcOH 1/1
SG3249 batches made with either an unbuffered or buffered TBS
cleavage step (Scheme 3, step c). Measurement in chloroform at
20 °C.
Both alloc groups were rapidly and efficiently removed with
Pd(0) and pyrrolidine as a an allyl scavenger27, in a Tsuji-Trost
reaction.28 The absence of pyrrolidine scavenging during the
deprotection resulted in the formation of N10-allylated
impurities. The resulting compound 26 was immediately used
in the next step without further purification. Traces of
palladium were effectively removed, both by chromatography
of the payload SG3249, and purification of the final ADC.
Interestingly, reversible macrocycle 27 was found to form upon
standing in solution with the valine amino group adding across
the PBD imine of 26 (NMR and structure in supporting
information). However, we found that both 26 and 27 could be
fully consumed under final coupling conditions with EDCI and
Mal-dPEG® -Acid to form SG3249 in 73% yield. The overall
yield for the synthesis was 0.54% from benzylvanillin over 30
steps.
Despite its length, the synthetic route described has shown a
number of advantages such as robustness on scale, modularity
of monomeric building blocks, as well as mild and efficient
final steps. The same route was used to provide clinical grade
material and is continuously being optimized.
8
To explore its potential as a payload, SG3249 was conjugated
to trastuzumab (Herceptin®) in a stochastic fashion, and to an
engineered version featuring two reactive cysteine positions
(site-specific Herceptin-SG3249) (Table 3). Finally, SG3249
was conjugated to a control, non-binding, engineered antibody.
SG3249 conjugation to all antibodies was highly efficient, as
illustrated by a readily tuneable stochastic ADC average drug-
Reagents and conditions: (a) 1,5-Diiodopentane, K2CO3, acetone, 60 ˚C,
90%, (b) 22, K2CO3, acetone, 65 ˚C, 86%, (c) TBAF/AcOH, THF, rt, 80%,
(d) Pd(PPh3)4, pyrrolidine, DCM, rt, 100%, (e) Mal-dPEG®8-acid, EDCI,
DCM, rt, 73%.
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to-antibody ratio (DAR of 2.5 in this instance; the DAR can be In conclusion, SG3249 possesses an attractive set of properties:
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tuned by varying the amount of TCEP reducing agent) and site-
specific ADC DAR of 1.8 (90% conjugation efficiency).
SG3249 was found readily soluble in the 10% DMSO aqueous
conjugation buffer (50 mg/L, see supporting information,
solubility limit not determined). In contrast, SGD-1910, bearing
C2-aryls and devoid of PEG linker, had to be conjugated in 50%
propylene glycol6. SG3249 low hydrophobicity (logD7.4 = 2.11)
resulted in low levels of aggregation (usually < 5%). As a result,
the conjugation process reproducibly delivered high monomeric
purity ADCs in high yields, on microgram to gram scale.
potency, synthetic route scalability, low hydrophobicity
contributing to low aggregation, cathepsin B-cleavable linker,
and efficient maleimide conjugation.
The in vivo evaluation of SG3249 ADCs will be described
elsewhere. SG3249 has been conjugated with rovalpituzumab.
The resulting ADC has recently successfully completed a phase
I clinical trial for the treatment of small cell lung cancer.29, 30
Initial data from rovalpituzumab-tesirine (Rova-T) is
encouraging,31 and should pave the way for a phase II trial. In
addition, SG3249 forms the payload component of ADCT-301,
which is currently in a phase I trial for CD25-positive
hematological malignancies.32, 33
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Table 3. ADC conjugation properties and GI50 in SKBR3.
The pyrrolobenzodiazepine dimer class of warheads is finding
increasing utility in the ADC arena based on their versatility and
complementary mode of action to anti-mitotic tubulin binders.
Process
aggregation
(% dimer)
Yield
(%)
GI50
(ng/mL)
ADC
DAR
ASSOCIATED CONTENT
Supporting Information
Her-SG3249
2.51
1.81
86
89
2.81
3.93
1.74
2.62
Site-specific
Her-SG3249
The Supporting Information is available free of charge on the ACS
Publications website.
Detailed experimental procedures with analytical data for all
intermediates. Graphical 1H NMR and 13C NMR for compounds of
interest. Interpreted, graphical 1H NMR for SG3249 and
macrocycle 27. (PDF)
IgG control mAb-
SG3249
1.79
87
3.11
1101
DAR assessed by RP-HPLC. Process aggregation assessed by
SEC. GI50 in SKBR3 (Figure 3). Yields calculated based on
concentration measurements.
AUTHOR INFORMATION
Corresponding Authors
Gratifyingly, both the stochastic and site-specific Herceptin-
SG3249 ADCs were active in the ng/mL range against HER2-
expressing human breast cancer cell line SKBR3 (Figure 3,
Table 3). Both HER2-targeted ADCs were greater than two
orders of magnitude more potent than the control non-HER2-
binding ADC, indicating target specificity. The residual activity
of the non-targeted control ADC is likely due to non-specific
uptake (such as clathrin-mediated endocytosis) at high assay
concentrations. Linker stability studies will be undertaken to
further address this point and will appear in subsequent
publications. Preliminary evidence tends to show good linker
stability when SG3249 is conjugated at buried positions within
the antibody structure.
AT, e-mail: tiberghiena@medimmune.com
PH, e-mail: philip.howard@spirogen.com
Present Addresses
†Former employee of Spirogen. Present address: Stemcentrx, Inc.,
450 East Jamie Court, South San Francisco, CA 94080
Funding
This study was supported by Spirogen, a member of AstraZeneca
group of companies.
Notes
AT, LM, NP, LA, DW, JH and PH are Spirogen employees.
JL is a Stemcentrx employee.
ACKNOWLEDGMENT
The authors wish to thank Stephen Gregson for editing the
manuscript and providing valuable scientific feedback. The authors
also wish to thank Krisztina Radi for providing SG3249 UPLC
data, and Gyoung Dong Kang and Conor Barry for providing
HRMS data. Christian Noti and colleagues are acknowledged for
their NMR work on SG3249 and macrocycle 27. Michael Torgov
is acknowledged for his contribution to the development of SG3249
analytical conditions.
ABBREVIATIONS
ADC, antibody drug conjugate; PBD, pyrrolobenzodiazepine;
DLL3, Delta-like 3; Her, Herceptin; HER2, human epidermal
growth factor receptor 2; PABC; para-aminobenzylcarbamate;
DAR, drug-to-antibody ratio; TBAF, Tetrabutylammonium
fluoride; DCM, dichloromethane; Mal, Maleimide; dPEG, discrete
poly-ethylene glycol; EDCI, N-(3-Dimethylaminopropyl)-N′-
ethylcarbodiimide hydrochloride; TFA, Trifluoroacetic acid; TIPS,
Triisopropyl silyl; DCC, Dicyclohexylcarbodiimide; HOBt, 1-
Figure 3. Growth inhibition curves in human SKBR3 cells for
SG3249 ADCs.
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17.
Howard, P. W. Preparation of pyrrolobenzodiazepine dimers
Hydroxybenzotriazole; TEMPO, 2,2,6,6-Tetramethylpiperidine 1-
oxyl; TCCA, Trichloroisocyanuric acid; TBS, tert-
Butyldimethylsilyl; alloc, allyloxycarbonyl.
and their conjugates with cell binding agents, especially
pyrrolobenzodiazepine dimer drug linker antibody conjugates containing
dipeptide linkers, and their use for treating proliferative diseases,
particularly cancer. WO2014057074A1.
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