DNA-Model-Based Design and Execution of Some Fused Benzodiazepine Hybrid Payloads for Antibody-Drug Conjugate Modality

Article abstract of DOI:10.1021/acsmedchemlett.0c00578

A new series with the tetrahydroisoquinoline-fused benzodiazepine (TBD) ring system combined with the surrogates of (1-methyl-1H-pyrrol-3-yl)benzene ( MPB ) payloads were designed and executed for conjugation with a monoclonal antibody for anticancer therapeutics. DNA models helped in rationally identifying modifications of the MPB binding component and guided structure-activity relationship generation. This hybrid series of payloads exhibited excellent in vitro activity when tested against a panel of various cancer cell lines. One of the payloads was appended with a lysosome-cleavable peptide linker and conjugated with an anti-mesothelin antibody via a site-specific conjugation method mediated by the enzyme bacterial transglutaminase (BTGase). Antibody-drug conjugate (ADC) 50 demonstrated good plasma stability and lysosomal cleavage. A single intravenous dose of ADC 50 (5 or 10 nmol/kg) showed robust efficacy in an N87 gastric cancer xenograft model.

Full text of DOI:10.1021/acsmedchemlett.0c00578

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DNA-Model-Based Design and Execution of Some Fused
Benzodiazepine Hybrid Payloads for Antibody−Drug Conjugate
Modality
Prasanna Sivaprakasam,* Ivar McDonald, Christiana Iwuagwu, Naidu S. Chowdari, Kevin M. Peese,
David R. Langley, Heng Cheng, Michael R. Luzung, Michael A. Schmidt, Bin Zheng, Yichen Tan,
Patricia Cho, Souvik Rakshit, Thirumalai Lakshminarasimhan, Sivakrishna Guturi,
Kishorekumar Kanagavel, Umamaheswararao Kanusu, Ankita G. Niyogi, Somprabha Sidhar,
Rajappa Vaidyanathan, Martin D. Eastgate, Srikanth Kotapati, Madhura Deshpande, Chin Pan,
Pina M. Cardarelli, Chunshan Xie, Chetana Rao, Patrick Holder, Ganapathy Sarma, Gregory Vite,
and Sanjeev Gangwar
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ABSTRACT: A new series with the tetrahydroisoquinoline-fused
benzodiazepine (TBD) ring system combined with the surrogates
of (1-methyl-1H-pyrrol-3-yl)benzene (“MPB”) payloads were
designed and executed for conjugation with a monoclonal antibody
for anticancer therapeutics. DNA models helped in rationally
identifying modifications of the “MPB” binding component and
guided structure−activity relationship generation. This hybrid
series of payloads exhibited excellent in vitro activity when tested
against a panel of various cancer cell lines. One of the payloads was
appended with a lysosome-cleavable peptide linker and conjugated
with an anti-mesothelin antibody via a site-specific conjugation
method mediated by the enzyme bacterial transglutaminase
(BTGase). Antibody−drug conjugate (ADC) 50 demonstrated good plasma stability and lysosomal cleavage. A single intravenous
dose of ADC 50 (5 or 10 nmol/kg) showed robust efficacy in an N87 gastric cancer xenograft model.
KEYWORDS: PBD, DNA minor groove binders, DNA binding models, ADC, cytotoxicity
lead to intercalation, scission, alkylation, or cross-linking of
ntibody−drug conjugates (ADCs) have ascended as
Apowerful biologics that combine the high affinity and strands. Hence, they are also called DNA minor groove binders
(MGBs). Another important class of payloads used in ADCs is
camptothecins.8 Camptothecin and its derivatives bind to the
topoisomerase I/DNA complex as their mechanism of action.9
One of the MGBs that is highly pursued in the ADC
architecture is the pyrrolo[2,1-c][1,4]benzodiazepine (PBD)
class of compounds (Figure 1, example payload 1).10−17 PBD
dimers bind in the minor groove of DNA, where they form
interstrand cross-links with guanines. Rahman et al.18 reported
that some non-covalent heteroaryl pharmacophores have a
strong preference for GC-rich DNA sequences either alone or
specificity of monoclonal antibodies (mAbs) with the promising
antitumor efficacy of the payloads.1,2 Over the last two decades,
ADCs have continued to evolve as an attractive modality for the
treatment of hematological malignancies and solid tumors.3
Currently, there are nine approved and marketed ADCs.4 A
cytotoxic agent, often called a payload, that is generally too toxic
for systemic administration is combined with mAbs with the
help of linkers to specifically target tumor cells. On the basis of
the mechanism of action, payloads fall into three categories:
antimitotic, DNA-interacting, and transcription-inhibiting. The
antimitotic class of payloads used in ADCs act by interacting
with tubulin and include maytansinoids, auristatins, tubulysins,
and many others.5 The DNA-interacting class includes
calicheamicin, duocarmycin, etc.6 The transcription inhibitors
include amatoxins that bind to RNA polymerase II.7 Antimitotic
and DNA-interacting payloads are most broadly used in ADCs.
DNA-interacting agents act by binding to the minor groove and
Received: October 31, 2020
Accepted: January 27, 2021
Published: February 10, 2021
https://dx.doi.org/10.1021/acsmedchemlett.0c00578
© 2021 American Chemical Society
ACS Med. Chem. Lett. 2021, 12, 404−412
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available anilines was evaluated to rationally replace the “MPB”
moiety. Among these, we envisioned a (methyltriazolo)benzene
replacement (compound 6) for the “MPB” moiety. We
hypothesized that the flat (methyltriazolo)benzene group
would nicely complement the minor groove, with one of the
ring nitrogens in the triazole ring being in close proximity to the
exocyclic amine of G7 in the DNA model to engage in a
hydrogen-bonding interaction dynamically. While the H226 cell
line activity was retained or improved in 6 compared with its
progenitor 4, the other three cell lines showed diminished
activity, with the worst being the OVCAR3 cell line. Compound
7 was made to explore the impact of removing the methyl group
in the triazole ring, and it turned out to be equipotent to its
methyl counterpart in the H226, N87, and HCT116 cell lines
and ∼3-fold more potent in the OVCAR3 cell line. As an N-
linked imidazole derivative, 8 lacks the hydrogen-bond acceptor
close to the exocyclic amine of G7 and showed a considerable
loss of activity across all four cell lines compared with 6. C-linked
imidazole 9 had further-diminished activities across all four cell
lines. The substantial drop in the activity is perhaps partly due to
the possibility of tautomerization where hydrogen bonding with
the exocyclic amine of G7 is absent in one set of tautomers. The
perturbed coplanarity between the two terminal rings may also
play a role in reducing the activity. For oxazole 10, which has the
ring nitrogen to complement the exocyclic amine of G7, the
activity was restored in all of the cell lines except H226
compared to its triazole counterpart 6.
Next, we kept the methyltriazolo ring constant and surveyed
the structure−activity relationship (SAR) around the N-linked
phenyl group. o-Fluoro analogue 11 showed comparable activity
in the H226 cell line and improved activities in the other three
cell lines relative to 6. Slightly bulky ortho substituents
negatively impacted the activities in all four cell lines
(compounds 12−16). o-Hydroxy analogue 17 showed activities
comparable to those of 6 in the H226 and HCT116 cell lines.
Pyridyl derivative 18 showed an enhancement in activity in the
N87 cell line relative to its phenyl counterpart 7. Compound 19
with a difluoromethyl group instead of the methyl group in 13
showed an improvement in the activities in the N87 and
OVCAR3 cell lines. Desmethyl analogue 20 showed a modest
drop in activity across all of the tested cell lines except N87
compared with its parent analogue 13. Dimethyl derivative 21
exhibited a dramatic loss in the activities across all four cell lines,
likely hinting at the need for coplanarity of the distal biaryl ring
system. We revisited N-linked imidazole (cf. compound 8) and
introduced substitutions in the linking phenyl and terminal
imidazole rings. Compounds 22−26 are the outcomes of this
effort, and the combination of chloroimidazole with either a
fluorophenyl or pyridine linking ring yielded acceptable activity
profiles in all four cell lines. N-Phenylpyrazole 27 showed
double-digit picomolar activities in three of the four cell lines.
Tricyclic analogues 28 and 29 showed activity profiles
comparable to that of 6. Tricyclic derivative 30 is not as flat as
28 and 29 and showed a marked drop in activities across all four
cell lines. Tricyclic analogue 31 exhibited further-diminished
activities in all four cell lines. While all three rings in the tricyclic
ring system in 30 fit well in the minor groove of the DNA model,
the morpholinotriazole portion of the tricyclic ring system in 31
projected outside the minor groove. Aza-N-methylbenzimida-
zole 32 had activities in the single-digit nanomolar range in all
four cell lines, which once again may partly be due to the putative
suboptimal fit of the bicyclic ring in the minor groove of the
DNA.
Figure 1. Example structures of PBD dimer,17 “PBD-py-MPB”,18 and
TBD dimer.19−21
when combined with PBDs. Compounds 2a and 2b are the two
most advanced candidates from Rahman et al.18 Notably, 2b
demonstrated significant antitumor efficacy in breast (MDA-
MB-231) and pancreatic (MIA PaCa-2) xenograft models.18
As part of our ADC discovery efforts19−21 and inspired by the
report by Rahman et al.,18 we sought to pursue PBD−4-(1-
methyl-1H-pyrrol-3-yl)benzeneamine (“MPB” as termed by
Rahman et al.18) hybrid payloads for the ADC modality (Figure
1, example payloads 2a and 2b). The inherent toxicity of these
payloads to normal cells motivated us to prosecute these agents
for a targeted therapeutic approach like ADC. Our prior
experience with PBD molecules led us to hypothesize that some
examples in this class might be too potent to achieve an
acceptable therapeutic index. An appealing aspect of these
hybrid molecules was the potential to tune the properties by
preparing a diverse set of analogues through variation of the
MPB portion of the molecules. We pursued the reported “PBD−
MPB” hybrid payloads in the context of our fused
benzodiazepine with a tetrahydroisoquinoline ring system,
herein called TBD (Figure 1, example payload 3).
For benchmarking purposes, we made 4 and 5, which are the
TBD versions of 2a and 2b, respectively, from the original
publication by Rahman et al.18 Initial characterization of the
payloads was based on an in vitro evaluation against a suite of
cellular proliferation assays for lung (H226), gastric (N87),
ovarian (OVCAR3), and colon (HCT116) cancer cell lines as
described in our recent publication.22 Compounds 4 and 5
showed quite potent (single- to double-digit and subpicomolar)
activities in these cell lines (Table 1). A double-stranded B-DNA
binding model for 4 was generated using the sequence 5′-
AAGAAGGCAA-3′, as reported by Rahman et al.18 We
hypothesized that 4, which is a TBD variant of 2a, would also
bind to the 5′-AAGAAGGCAA-3′ sequence that was demon-
strated to be the preferred binding sequence for 2b.18 Figure
2A,B illustrates the binding mode of 4 in the DNA minor groove.
The C11−N10 imine of TBD makes a covalent bond with the
exocyclic amine of G3 of the DNA, while the “py-MPB” portion
of the payload makes non-covalent interactions and nicely
follows the curvature of the DNA minor groove.
The DNA-binding model of 4 revealed opportunities to
modify the “MPB” portion of the payloads. A set of internally
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https://dx.doi.org/10.1021/acsmedchemlett.0c00578
ACS Med. Chem. Lett. 2021, 12, 404−412

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Table 1. Compounds and Cytotoxicity Data (IC50 in nM)
406
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ACS Med. Chem. Lett. 2021, 12, 404−412

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Table 1. continued
a
b
The IC50 value was the result of multiple determinations (n ≥ 2). The IC50 value was obtained after single determination (n = 1).
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https://dx.doi.org/10.1021/acsmedchemlett.0c00578
ACS Med. Chem. Lett. 2021, 12, 404−412