Synthesis and in vitro evaluation of SG3227, a pyrrolobenzodiazepine dimer antibody-drug conjugate payload based on sibiromycin

Article abstract of DOI:10.1016/j.bmcl.2017.01.074

A novel pyrrolobenzodiazepine dimer payload, SG3227, was rationally designed based on the naturally occurring antitumour compound sibiromycin. SG3227 was synthesized from a dimeric core in an efficient fashion. An unexpected room temperature Diels-Alder reaction occurred during the final step of the synthesis and was circumvented by use of an iodoacetamide conjugation moiety in place of a maleimide. The payload was successfully conjugated to trastuzumab and the resulting ADC exhibited potent activity against a HER2-expressing human cancer cell line in vitro.

Full text of DOI:10.1016/j.bmcl.2017.01.074

Accepted Manuscript
Synthesis and in vitro evaluation of SG3227, a pyrrolobenzodiazepine dimer
antibody-drug conjugate payload based on sibiromycin
Gary C. Kemp, Arnaud C. Tiberghien, Neki V. Patel, Francois D'Hooge, Sanjay
M. Nilapwar, Lauren R. Adams, Simon Corbett, David G. Williams, John.A.
Hartley, Philip W. Howard
PII:
S0960-894X(17)30086-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.01.074
BMCL 24650
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 December 2016
23 January 2017
24 January 2017
Please cite this article as: Kemp, G.C., Tiberghien, A.C., Patel, N.V., D'Hooge, F., Nilapwar, S.M., Adams, L.R.,
Corbett, S., Williams, D.G., Hartley, John.A., Howard, P.W., Synthesis and in vitro evaluation of SG3227, a
pyrrolobenzodiazepine dimer antibody-drug conjugate payload based on sibiromycin, Bioorganic & Medicinal
Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.01.074
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Synthesis and in vitro evaluation of SG3227,
a pyrrolobenzodiazepine dimer antibody-drug conjugate
payload based on sibiromycin
Leave this area blank for abstract info.
Gary C. Kemp†, Arnaud C. Tiberghien†,*, Neki V. Patel, Francois D’Hooge, Sanjay M. Nilapwar,
Lauren R. Adams, Simon Corbett, David G. Williams, John. A. Hartley, Philip W. Howard*

Bioorganic & Medicinal Chemistry Letters
Synthesis and in vitro evaluation of SG3227, a pyrrolobenzodiazepine dimer
antibody-drug conjugate payload based on sibiromycin
Gary C. Kemp†, Arnaud C. Tiberghien†,*, Neki V. Patel, Francois D’Hooge, Sanjay M. Nilapwar, Lauren
R. Adams, Simon Corbett, David G. Williams, John. A. Hartley, Philip W. Howard*
Spirogen, QMB Innovation Centre, 42 New Road, London, E1 2AX, UK
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A novel pyrrolobenzodiazepine dimer payload, SG3227, was rationally designed based on the
naturally occurring antitumour compound sibiromycin. SG3227 was synthesised from a dimeric
core in an efficient fashion. An unexpected room temperature Diels-Alder reaction occurred
during the final step of the synthesis and was circumvented by use of an iodoacetamide
conjugation moiety in place of a maleimide. The payload was successfully conjugated to
trastuzumab and the resulting ADC exhibited potent activity against a HER2-expressing human
cancer cell line in vitro.
Keywords:
SG3227
Antibody-drug conjugate
Sibiromycin
Pyrrolobenzodiazepine dimer
DNA minor groove binding agent
The pyrrolobenzodiazepines (PBDs) are a family of naturally
occurring antitumour agents isolated from various Streptomyces
species.1-3 The PBD monomers target purine-guanine-purine
motifs in the minor groove of DNA. Once situated in the minor
groove, an aminal bond is formed between the C11 position of
the PBD and the N2 of guanine. PBD dimers can bind to both
opposing strands of DNA, creating covalent crosslinks at purine-
GATC-pyrimidine sequences. Their ability to form such DNA
interstrand crosslinks makes PBD dimers highly potent
anticancer agents.4-6
of a pro-drug like character, since as long as the peptide trigger
remains in place, the drug cannot crosslink DNA. Another
advantage is that there are fewer potentially reactive groups in
the compound that can cause complications during synthesis.
Due to their high potency, PBD dimers have been used as
8
warheads in antibody-drug conjugates (ADCs).7, Currently,
there are two PBD warheads being evaluated in various phase I,
II and III clinical trials.9-11
Sibiromycin is the most potent naturally occurring PBD (IC50
L1210, leukaemia = 2.9 nM).12 It contains an amino sugar
derivative at the C7 position and a trans-propenyl group at the
C2 position that interacts favourably with the minor groove of
DNA. We have previously synthesized a sibiromycin-like PBD
dimer13, SG2219, a more potent cytotoxic agent than stand-alone
clinical agent SG20006 (Figure 1, Table 1).
In this letter we report the synthesis, conjugation and in vitro
activity of an ADC with a sibiromycin-like PBD dimer payload,
SG3227. It was envisioned that the SG2219 warhead
(pharmacologically-active unit) would be linked to the antibody
by a cathepsin-cleavable “trigger” peptide via the N10 position of
the PBD, masking one of the imine functionalities. This strategy
imparts two important benefits - one is that the payload has more
Figure 1. Sibiromycin, a naturally-occurring PBD; SG2000, a clinical stand-
alone PBD dimer; SG2219, the PBD warhead released by SG3227 ADCs;
and SG3227, a PBD dimer payload for ADC synthesis.

The dimer core 6 was converted to its acyl chloride and coupled
to 7 (Scheme 2),4, 5 to form bis-secondary alcohol 8. Oxidation
in the presence of TCCA and TEMPO gave the bis-ketone 9 in
quantitative yield. Subsequent triflation with triflic anhydride in
Table 1. In vitro activity of PBD dimers SG2219 and SG2000
Cell lines K562 NCIN87 BT474 SKBR3 MDA-MB-468
SG2219
SG2000
1
5
641
121
520
6.4
78
the presence of 2,6-lutidine selectively led to the desired C2-C3
18
bis-enol triflate regioisomer.17,
Suzuki coupling with trans-
735
66
7000
propen-1-ylboronic acid, followed by reduction of the nitro
groups with zinc in formic acid/methanol, resulted in bis-aniline
12.
IC50 values in pM (mean of three independent experiments) were determined
using CellTiter96 (MTS) following 96 h incubation.
The symmetry of 12 was broken by Alloc protection of one of
the two aniline groups in a statistical manner using allyl
chloroformate. The remaining free aniline was then converted to
the isocyanate with triphosgene and subsequently quenched with
benzyl alcohol 5 to give carbamate 14. The TBS protecting
groups were then efficiently removed with TBAF. The resulting
unsymmetrical bis-alcohol 15 was subjected to Swern oxidation,
with concomitant ring closure of both B-rings present in the
dimer to give 16. The Alloc groups were then removed and the
resulting intermediate 17 was immediately coupled to Mal-PEG8-
acid.
We have previously reported a monomeric approach to the
synthesis of PBD payloads via TIPS-protected phenol building
blocks.14 We herein report an alternative synthesis with fewer
synthetic steps starting from dimeric core 6 (Scheme 2).
Scheme 1 shows the synthesis of the alloc-protected linker
building-block 5.15 L-valine was Alloc-protected, then activated
as the succinimide ester, reacted with L-alanine, followed by
coupling with p-aminobenzyl alcohol to yield 5. The use of
activating agent EEDQ avoided any undesired esterification and
potential epimerisation.16
Scheme 1. Synthesis of the peptide linker. Reagents and conditions: (a) Allyl chloroformate, K2CO3, H2O/THF 0 °C to rt; (b) N-hydroxysuccinimide, DCC,
DCM, rt; (c) L-Ala-OH, NaHCO3, THF/H2O, rt, 50% (3 steps); (d) p-Aminobenzyl alcohol, EEDQ, THF, rt, 87%.
Scheme 2. Synthesis of SG3227. Reagents and conditions: (a) Oxalyl chloride, DMF, DCM, -40 °C, 96%; (b) TCCA, TEMPO, DCM, 0 °C, 100%; (c)
(CF3SO2)2O, 2,6-lutidine, DCM, -50 °C, 99%; (d) Pd(PPh3)4, propenyl boronic acid, K3PO4, dioxane, rt, 54% (3 steps); (e) Zn, 5% HCO2H/MeOH, 64%; (f) Allyl
chloroformate, pyridine, DCM, 52%; (g) Triphosgene, TEA, THF, Alloc-Val-Ala-PABA 5, 67%; (h) TBAF (1M in THF), 66%; (i) DMSO, oxalyl chloride,
TEA,
THF,
56%;
(j)
Pd(PPh3)4,
pyrrolidine,
DCM;
(k)
23,
DIC,
DCM,
74%
(2
steps,
SG3227
only).

However, a significant amount of by-products were formed
during the coupling reaction and pure 18 was not isolated. It was
hypothesized that a Diels-Alder [4+2] cycloaddition had occurred
between the maleimide conjugation moiety and the trans-
propenyl/C-ring diene. Test reactions on monomeric
intermediates19 confirmed this observation (Scheme 3). The
carbamate ring-closed intermediate 19 underwent full conversion
to the Diels-Alder product 20 within 6 hours at room
temperature. However, the ring-open intermediate 21 took one
week to fully react. The lower reactivity of 21 is thought to be
due to the electron-withdrawing A-ring nitro group remotely
conjugated to the diene.
4), which was coupled to amine 17 using DIC, to give ADC
payload SG3227 in 74% yield.
A number of precautions were necessary to avoid undesired
side-reactions of the highly labile iodoacetamide group. Firstly,
the use of DIC as a coupling agent in place of EDCI.HCl. Indeed,
it was found that chloride ions present in the reagent substitute
with the iodoacetamide, forming the less reactive
chloroacetamide. This also meant that brine (saturated sodium
chloride solution) had to be excluded during work-up. Other
precautions included protection from light and heat, as both can
result in iodoacetamide degradation.
In order to evaluate the biological potency of SG3227, the
payload was conjugated stochastically to trastuzumab (an anti-
HER2 monoclonal antibody), and in a site-specific manner to an
engineered trastuzumab variant. Stochastic ADC production
involves partial reduction of the trastuzumab inter-chain disulfide
bonds to release cysteine thiols, which can then react with the
iodoacetamide-containing payload. The resulting trastuzumab-
SG3227 conjugate had an average drug-to-antibody ratio (DAR)
of 2.22. In contrast, with the engineered variant, a full reduction
followed by re-oxidation of the inter-chain disulfide bonds is
required to selectively free the engineered cysteine thiols for
conjugation. Consequently, the drug distribution and DAR of
such ‘site-specific’ ADCs are much more homogeneous than that
for stochastic conjugates, and DAR 2 is usually the predominant
species generated. In this instance, the average DAR was 1.68.
Several process modifications, such as the use of propylene
glycol, avoidance of chloride salts and prolonged conjugation
times, were implemented to achieve conjugation (see supporting
information). Whilst conjugation with iodoacetamide was found
challenging, this moiety represents a viable alternative to
maleimides when they are incompatible with the ADC warhead.
It is thought that the Diels-Alder reaction is facilitated by the
electron-withdrawing carbonyl groups in the maleimide
dienophile, lowering the LUMO energy level. In addition, the
enamide nitrogen conjugated to the diene raises the energy level
of the HOMO sufficiently high to allow the reaction to proceed at
ambient temperature.20 Compound 20 was deprotected21 to
unmask imine monomer SG3225. In vitro cytotoxicity assay of
SG3225 showed significant lack of activity in K562 cells (>1000
nM). It is hypothesised that steric clashes of the maleimide ring
with the DNA minor groove walls prevent efficient binding (see
proposed 3D model in the supporting information).
In order to circumvent the Diels-Alder side-reactions, it was
decided that an iodoacetamide would be used in place of the
maleimide, in a modification of the approach suggested by Alley
et al.22 which discussed the stability of maleimide and
bromoacetamide linkers. Interestingly, this strategy was used
independently by Puthenveetil et al.23 when faced with a similar
challenge. An acetamide linkage has the additional advantage of
avoiding deconjugation via a retro-Michael reaction following
attachment to an antibody.24 Iodoacetic anhydride was coupled to
amino-PEG4-acid yielding iodoacetamide-PEG4-acid 23 (Scheme
Scheme 3. Diels-Alder reactions of N-methyl maleimide with PBD intermediates 19 and 21. Reagents and conditions: (a) DCM, rt, 60%, (b) Pd(PPh3)4,
pyrrolidine, DCM. (Tentative structure elucidation for Diels Alder products consistent with selective endo stereochemistry, see two dimensional COSY and
NOESY
NMRs
of
SG3225
in
supporting
information).

Scheme 4. Synthesis of the iodoacetamide PEG acid 23. Reagents and conditions: (a) DCM, rt, 77%.
Supplementary Material
Both the stochastic Her-SG3227 (trastuzumab-SG3227) and
engineered Her-SG3227 ADCs exhibited high cytotoxic potency
in the HER2-expressing gastric carcinoma cell line NCI-N87
(Figure 2), with IC50 values of 1.48 ng/mL and 4.29 ng/mL
respectively. The same ADCs were about 3 orders of magnitude
less potent in the HER2-negative cell line MDA-MB-468 with
IC50 values of 2130 ng/mL and 1220 ng/mL, respectively.
2D NMRs for SG3225. Experimental procedures for Iodoacetamide 23,
SG3227, and conjugation protocols. (PDF).
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synthesis (1.61% overall yield for the 13 steps sequence starting
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