Effects of Systematic Shortening of Noncovalent C8 Side Chain on the Cytotoxicity and NF-κB Inhibitory Capacity of Pyrrolobenzodiazepines (PBDs)

Article abstract of DOI:10.1021/acs.jmedchem.8b01849

The systematic shortening of the noncovalent element of a C8-linked pyrrolobenzodiazepine (PBD) conjugate (13) led to the synthesis of a 19-member library of C8-PBD monomers. The critical elements of 13, which were required to render the molecule cytotoxic, were elucidated by an annexin V assay. The effects of shortening the noncovalent element of the molecule on transcription factor inhibitory capacity were also explored through an enzyme-linked immunosorbent assay-based measurement of nuclear NF-κB upon exposure of JJN-3 cells to the synthesized molecules. Although shortening the noncovalent interactive element of 13 had a less than expected effect upon compound cytotoxicity due to reduced DNA interaction, the transcription factor inhibitory capacity of the molecule was notably altered. This study suggests that a relatively short noncovalent side chain at the C8 position of PBD is sufficient to confer cytotoxicity. The shortened PBD monomers provide a new ADC payload scaffold because of their potent cytotoxicity and drug-like properties.

Full text of DOI:10.1021/acs.jmedchem.8b01849

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Article
Effects of Systematic Shortening of Non-covalent C8-side Chain on the
Cytotoxicity and NF-#B Inhibitory Capacity of Pyrrolobenzodiazepines.
David Corcoran, Thomas Lewis, Kazi Sharmin Nahar, Shirin Jamshidi, Christopher
Fegan, Chris Pepper, David E. Thurston, and Khondaker Miraz Rahman
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01849 • Publication Date (Web): 28 Jan 2019
Downloaded from http://pubs.acs.org on January 29, 2019
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Effects of Systematic Shortening of Non-covalent
C8-side Chain on the Cytotoxicity and NF-κB
Inhibitory Capacity of Pyrrolobenzodiazepines.
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David B. Corcoran , Thomas Lewis , Kazi S. Nahar , Shirin Jamshidi , Christopher
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Fegan , Chris Pepper , David E. Thurston* , Khondaker Miraz. Rahman*
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†School of Cancer and Pharmaceutical Sciences, King’s College London, London SE1
9NH, U.K.
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‡ School of Medicine, Cardiff University, Cardiff CF14 4XN, U.K.
⁑ Brighton and Sussex Medical School, University of Sussex, BN1 9PX, U.K.
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Keywords: Pyrrolobenzodiazepines, minor groove binders, NF-κB, Transcription Factors,
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Structure Activity Relationships
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ABSTRACT: The systematic shortening of the non-covalent element of a C8-linked
pyrrolobenzodiazepine (PBD) conjugate (13) led to the synthesis of a 19-member library
of C8-PBD monomers. The critical elements of 13, which were required to render the
molecule cytotoxic, were elucidated by an annexin V assay. The effects of shortening the
non-covalent element of the molecule on transcription factor inhibitory capacity were also
explored through an ELISA-based measurement of nuclear NF-B upon exposure of
JJN3 cells to the synthesised molecules. While shortening the non-covalently interactive
10 element of 13 had a lesser than expected effect upon compound cytotoxicity due to
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reduced DNA interaction, the transcription factor inhibitory capacity of the molecule was
12 notably altered. This study suggests that a relatively short non-covalent side chain at the
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C8-position of PBD is sufficient to confer cytotoxicity. The shortened PBD monomers
14 provide a new ADC payload scaffold due to their potent cytotoxicity and drug-like
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properties.
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Introduction
Since their discovery in Streptomyces bacteria over half a century ago, pyrrolo[2, 1-
c][1, 4]benzodiazepine (PBD) structures have been of interest as possible
chemotherapeutic agents. This is due to their ability to sequence selectively bind
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covalently to DNA, cause DNA strand cleavage and inhibit DNA associated enzymes .
Two critical features of the PBD structure confer this covalent binding capacity on the
molecule. S stereochemistry at the C11a position of the molecule bestows a right-handed
longitudinal twist upon the structure, allowing an effective fit along the minor groove of the
DNA helix 4-5. Complementing this optimal fit is the presence of an interconvertible imine-
10 carbinolamine moiety at the N10-C11 position of the molecule. This electrophilic centre
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undergoes attack from the C2-NH of minor groove guanine bases, forming the covalent
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PBD structures have been found to form covalent interactions with some guanines of
the DNA minor groove more frequently than others, particularly those flanked by purine
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16 bases5, 9 although more recent data suggests kinetic preferences when guanines are
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flanked by pyrimidines . This potential for relative selectivity of binding is of interest,
given that the pathogenesis of many cancers are associated with the aberrant binding of
transcription factors, such as nuclear factor kappa B (NF-κB), to sequence-specific
promoter regions of DNA11-15. The conjugation of subunits, capable of non-covalent
interactions with DNA bases, to the C8 position of the PBD structure (via a carbon linker
of variable length) has led to the development of hybrid molecules that can interact
selectively with longer sequences of DNA than the typical purine-guanine-purine motifs
of unmodified PBD structures. Such molecules have shown a capacity to inhibit the
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binding of transcription factors, such as NF-Y and NF-B to their respective promoter
10 sequences. More recent C8-modified PBDs, such as KMR-28-39 (Fig. 1B) synthesised
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by Rahman et al. , have demonstrated the evidence of pre-clinical activity of such PBD
12 conjugates. In addition to the cytotoxic activity, PBD-heterocyclic hybrids have shown
significant antimicrobial activity19-21. More recently PBDs are being used as cytotoxic
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Although the addition of non-covalently interactive subunits to the C8 position of PBD
structures has been associated with the downregulation of genes downstream of
transcription factor activity17-18, 23-24, the relationship between the structural requirements
and the potent cytotoxicity observed for these molecules has not been fully elucidated.
The extent of C8 diversification required to confer this transcription factor activity (i.e. the
core non-covalently interactive pharmacophore) remains unclear. In this paper, we
investigated some of these uncertainties through the systematic shortening of a C8 linked
monomer (13, Fig. 1C) containing a pyrrole-benzofuran motif which has been previously
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explored in both non-covalent distamycin and covalent duocarmycin analogues , and
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reduction of the non-covalent element of the hybrid on both transcription factor activity
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Results and Discussion
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Synthesis of PBD hybrids
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The PBD core (N10-Alloc, C11-THP protected) of the hybrids was synthesised from
vanillin, as previously described in the literature18 (See Supplementary information fpr
scheme). A four carbon linker was used to connect the PBD component with the non-
covalently interactive subunits, as chains of this length had proven optimal for DNA
binding in previous hybrid SAR studies. The linker was located at the C8 position of the
molecule to allow an isohelical fit of the non-covalent component of the hybrid along the
minor groove upon covalent PBD binding2.
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After the synthesis of the PBD core, the C8 hybrid 13 was constructed using a
12 combination of a benzofuran and an N-methyl pyrrole moiety. This was based upon the
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molecular structures of the side chains of KMR-28-39 (Fig. 1B) and other molecules
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14 recently described by Basher and co-workers . These benzofuran/pyrrole components
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were linked via Steglich amide bond formation at positions that maintained the overall fit
16 of the hybrid for the DNA minor groove (C2/C5 for benzofuran, C1/C4 for N-methyl pyrrole
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components). The full synthesis of the non-covalently interactive hybrid component of 13
is summarised in the Supplementary Information, with the conjugation to the PBD moiety
and activation of the N10/C11 imine component of the molecule detailed in Scheme 1. A
total of 19 new PBDs were synthesised by the systematic shortening of the non-covalently
interactive element of 13 (Table 1). This process was analogous to the synthesis of 13,
with component 11, replaced with designed building blocks to afford progressively
shortened C8-PBDs.
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Biological Evaluation
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Cytotoxicity Screening
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previous (structurally similar) C8 linked PBD monomers, such as those described by
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14 Rahman and co-workers , it was hypothesized that compound 13 and its shortened
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derivatives could share such properties. Accordingly, the initial screening of the
16 compounds was carried out in primary tumour samples and a malignant cell line known
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to rely upon aberrant activity of NF-B. The pathogenesis of both chronic lymphocytic
leukaemia (CLL) and multiple myeloma has been linked to aberrant gene expression
mediated, at least in part, by NF-B 28-33. Primary tissues were obtained from CLL patients
(n = 3) with their informed consent and the authenticated multiple myeloma cell line, JJN-
3, was purchased from DSMZ (Germany). The compounds were added to cultures of the
primary CLL cells and JJN-3 cells at concentrations between 1 nM and 100 M in both
primary leukaemic cells and the cell line for 48 hours. An Annexin V binding assay was
used to measure cytotoxicity and LC values were calculated from the sigmoidal dose-
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response curves using Prism 6.0 software. A previously synthesized C8 linked PBD
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as a positive control. The results of this cytotoxicity evaluation are described in Table 1,
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Supplementary Information.
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Prior to the screening of the shortened compound library in the CLL and JJN-3 cell
cultures, it was expected that molecular shortening of the side chain of 13 would diminish
compound activity. This was based upon the hypothesis that both the overall affinity of
the PBD hybrid for DNA, as well as the length of DNA sequences bound by the compound,
would be reduced, due to the reduction in non-covalent interactions between the DNA
minor groove and the C8-side chain of the molecule. This concept has been reflected in
the cytotoxicity of previously synthesised PBD hybrids, which featured short C8
conjugated moieties. Such compounds either lacked notable cytotoxicity or were less
cytotoxic than their longer side chain counterparts. Examples of these compounds include
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The cytotoxicity of the compounds in the primary CLL cells and the JJN-3 cell line was
therefore unexpected. For several of the compounds tested, LC values were obtained
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16 in the low nanomolar range, with good concordance in cytotoxicity observed between the
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primary CLL cells and the myeloma cell line. In total, ten compounds maintained or
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improved activity with respect to 13 upon side chain shortening in primary CLL cells.
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The cellular screen provided some insight into the structural features of the non-
covalently interactive C8 side chain of 13 which may prove integral to its cytotoxic activity.
It appeared that the presence of the terminal pyrrole moiety of 13 is not-essential for its
activity, as cytotoxicity was largely preserved for the benzofuran-containing compounds
14 and 15. Aromaticity of the furan component of the benzofuran structure also appeared
be not crucial for its cytotoxicity. Indeed, the dihydrobenzofuran-containing compound,
10 16, was one of the most cytotoxic compounds compounds tested in primary CLL cells.
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Opening this furan ring also maintained activity at low nanomolar levels in both primary
12 CLL cells and the JJN3 cell line.
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The importance of the oxygen moiety of the benzofuran is less clear from this cellular
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evaluation. Replacement of the oxygen atom with a carbon (17) or its complete
16 elimination (22-24) had little effect upon compound activity. This indicates that the
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potential hydrogen bond acceptor role of the oxygen plays little part in the overall
cytotoxicity of 13. Interestingly, reduced cytotoxicity was observed for 21. In this case, the
benzofuran oxygen is present as part of a phenol, rather than an ether functional group.
The phenol moiety of 21 may act as a hydrogen bond donor, in contrast to its ether-
containing analogues. The latter of these characteristics appeared have an important
effect on overall cytotoxicity.
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Interestingly, the entire removal of the furan ring of the benzofuran side chain
component (25) failed to appreciably reduce the cytotoxicity. However, alterations to the
10 benzene element of the benzofuran had more telling effects upon cytotoxicity. The
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introduction of a nitrogen atom into the ring, through use of aminopyridine building blocks,
12 eliminated compound activity at the dose range tested. Such a deleterious effect upon
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compound activity was unexpected. In a similar fashion to the introduction of the phenol
14 group in 21, the nitrogen of the pyridine ring may ionise depending upon the pH
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conditions. In a mildly acidic environment, the pyridine ring is protonated (pKa of the
16 conjugate acid is around 5.72), creating a formal positive charge. This charge may affect
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the ability of the molecule to penetrate the cell membrane and reach the minor groove of
DNA. Moreover, the charged conjugate acid also conferred another hydrogen bond donor
group on the molecule. One or both of these effects is likely to impact upon the activity of
the PBD hybrids 26 and 27, which showed approximately a three-log reduction in
cytotoxicity when compared to 13.
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Removal of the aromaticity of this ring notably reduced the cytotoxicity. The
cyclohexane and ring-opened derivatives (28-32) showed poor activity in both primary
CLL cells and the JJN3 cell line. It could be hypothesised that the spatial alteration of this
10 portion of the side chain from a planar sp2 hybridised structure to sp3 derivative was
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responsible for this loss of activity. The removal of the delocalised electrons of the
12 aromatic system may also have affected compound activity, due to the removal of
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potential pi-pi interactions with DNA bases.
14
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Given the surprisingly similar cytotoxicity of 13 and its shortened derivatives in the CLL
16 primary tissue and JJN-3 cell line evaluations, it was decided to conduct an evaluation of
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the shortened PBD hybrids versus their parent 13 in healthy age-matched B- and T-cell
cultures. This would establish whether the cytotoxicity observed in the malignant cells
was mirrored in non-malignant tissues or whether a notional therapeutic index was
apparent. Six shortened derivatives of 13, which showed potent cytotoxicity in the initial
CLL and JJN-3 cellular screens, were chosen for evaluation in the healthy cells: 14, 15,
18, 19, 20 and 28. Normal peripheral blood B-cells and T-cells, derived from three donors,
were incubated with the seven compounds for 48 hours at concentrations ranging from
1nM -100 M. Subsequently the cells were harvested by centrifugation and apoptosis
was quantified using an Annexin V assay. The cytotoxicity dose-response curves
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10 obtained are detailed in the Supplementary Information, with Table 2 listing the LC50
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values obtained in both normal B- and T-cells, as well as the relative toxicity in these cells
12 compared to primary CLL cells.
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All of the compounds tested showed cytotoxicity ranging from 0.39 M – 12.4 M in
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both cell types. The compounds showed differential toxicity in the primary CLL cells
16 compared to the normal B-cells and normal T-cells with notional therapeutic indices
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Page 15 of 72
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between 1 and 2 logs. The least potent compound tested, 28 showed no therapeutic
index. Overall, the compounds showed better therapeutic indices against T-cells
compared to B-cells with average therapeutic indices of 229.1 and 80.9, respectively. The
observed therapeutic indices are large enough to consider further development of these
C8-linked PBDs. However, there was no correlation between therapeutic indices and the
length of the molecules. Essentially, this healthy cell evaluation confirmed that extensive
shortening of the non-covalent component of 13 did little to affect either the cytotoxicity
or selectivity profile of the compounds.
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Evaluation of the Effects of 13 and Derivatives on nuclear levels of NF-B
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Given the lack of observable cytotoxicity differences between 13 and all but its most
12 shortened C8-side chain derivatives, it was decided to evaluate whether the reduction of
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the C8 linked non-covalent interactive side chain had any effect upon potential
14 transcription factor inhibition by compound 13.
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The potent cytotoxicity observed for 13 and its derivatives in a number of NF-B
16 dependent cancer cell types provided indirect evidence for their potential as possible
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inhibitors of this transcription factor. Moreover, the previously reported cellular inhibition
by similar PBD hybrids of NF-B 17-18, 36 provided further impetus to directly measure the
potential of these novel C8-linked PBD monomers to alter the nuclear levels of the
transcription factor.
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As a result, the effect of 13 and three of its shortened derivatives (15, 19, 20, 21 and
25), on nuclear levels of NF-B in JJN-3 cells was investigated. The derivative
compounds were chosen due to their potency, and to provide representative examples of
the various stages of the shortening of the non-covalently interactive component of the
10 compound. JJN-3 cell lines were chosen over the primary CLL cell samples for this
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analysis as the levels of nuclear NF-B were more consistent in the cell line compared to
12 primary tumour cells derived from different patients.
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14 The measurement of the effects of the four compounds on nuclear levels of NF-B
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involved the use of an adapted enzyme-linked immunosorbent assay (ELISA). Nuclear
16 extracts were prepared from treated and control cells using a nuclear extraction kit (Active
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Motif). Activated NF-B subunits (p65, RelB, p52, or p50) in the samples were measured
by oligonucleotide-based ELISA (Active Motif). Treated and control samples were
incubated in wells of a 96-well microtiter plate that were coated with the NF-B consensus
nucleotide sequence (5-GGGACTTTCC-3). NF-B from the cell samples attached to the
wells and was captured by specific antibody to either p65, RelB, p50, or p52. Binding of
the specific NF-B family member was then detected by an anti-rabbit-HRP-conjugated
antibody. Developing solution was then added to the plate for 5 minutes prior to the
addition of stop solution. The intensity of the developed colour was proportional to the
quantity of p65, RelB, p50, or p52 in each sample and the amount of bound NF-B was
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10 quantified (ng/g nuclear extract) from subunit recombinant protein standard curves. The
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specificity of the binding to the nucleotide sequence was ascertained by pre-mixing free
12 consensus nucleotide to the sample before adding the nuclear extract to the well. Sample
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determinations were normalised by the total cell protein, as determined by protein assay
14 kit (Pierce). Data were expressed as a percentage of the untreated controls.
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The quantified DNA binding activity for each NF-B subunit in cells exposed to 13, 15,
19, 20, 21 and 25 was then related to a control value obtained from cells that had not
been exposed to a PBD hybrid. The experiment was repeated in triplicate, and the effect
of the compounds upon NF-kB DNA binding of four subunits of the NF-ĸB are illustrated
in Figures 2 & 3 below. The c-Rel subunit not evaluated in these experiments.
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The evaluation of the effects on nuclear levels of NF-B by the shortened PBD hybrids
produced some interesting results. The significant knockdown of p65 by 15 aside, the
shortened compounds tested produced no significant reductions in nuclear levels of the
10 p65 subunit activity, in spite of their highly potent cytotoxicity in JJN-3 cells. When placed
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in the context of the role of NF-B subunit function in carcinogenesis, this was an
12 interesting outcome. p65 over-expression has been implicated in the progression of
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malignancy; a previous analysis of a cohort of CLL patient samples revealed significant
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14 upregulation of the p65 subunit and this was associated with disease progression .
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Page 19 of 72
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The nuclear activity of p52 was unaffected or increased (13 and 19) by the PBD hybrids,
while the RelB subunit was significantly decreased by 13, but not by the shortened
derivatives. Interestingly, all three of the shortened compounds tested significantly
reduced p50 levels. This was not the case for 13. However, the role of p50 in
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carcinogenesis is to date ill-defined , and in some cases increased levels of the subunit
39
may promote apoptotic pathways . Accordingly, the differential capacity of the PBD
hybrids to inhibit the p65 subunit was considered in greater detail.
An evaluation of the structures of the three compounds (Fig. 2) tested in this assay may
10 help explain the differences in effect upon nuclear levels of NF-B observed, especially
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the levels of p65. The parent 13 PBD hybrid molecule showed consistent inhibition of
12 nuclear p65 activity. Compound 15, which also significantly inhibited the p65 subunit,
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shares the greatest side chain similarity with 13 of the three compounds tested. The 15
14 side chain retains an intact benzofuran moiety common to its predecessors. This
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benzofuran is disrupted in 19 and 25, with both the aromaticity and rigid cyclic structure
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of the furan ring of the benzofuran removed. Both compounds showed no discernible
effect on nuclear p65 levels, despite their potency.
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The results of this assay provided for a more subtle insight into potential mechanisms
of activity of PBD hybrids than a reliance on cell cytotoxicity assays alone. Although all of
the compounds tested showed promising potency characteristics in a range of cancer cell
lines, their NF-fB transcription factor inhibitory effects differed depending upon the side
chain composition. Compound 13, containing the longest non-covalently interactive
element, displayed the most effective inhibition of NF-B and effects upon nuclear p65,
10 and RelB levels appeared to diminish in tandem with the reduction of this element in the
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derivatives 15, 19 and 25 (although some increased inhibition of p50 was observed).
12
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The clear differences in NF-B inhibitory capacity observed through the systematic
14 shortening of 13 contrasted with the relatively comparable cytotoxicity profiles of all but
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the most shortened library derivatives. Two possible explanations may help explain this
16 incongruity.
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Page 21 of 72
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Firstly, it is possible that the effects of the non-covalently interactive elements of the
PBD hybrids on nuclear NF-ĸB activity are unrelated to their cytotoxic mechanism of
activity. Such an assessment would regard the PBD monomers primarily as traditional
DNA alkylating agents, with the transcription factor inhibitory effects (such as the p65
inhibition), displayed by molecules such as 13 symptoms, rather than the causes of PBD
cytotoxicity. In this scenario, the non-covalent element of the molecules merely serves to
confer different physicochemical properties on the compound or alter the nuclear
penetration rate and extent of adduct formation with DNA.
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The second, more compelling explanation for differences in transcription factor
12 inhibitory activity between of PBD hybrids of varying non-covalently interactive elements
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is slightly more nuanced. While all PBD molecules feature an ability to bind covalently to
14 DNA, and act to some extent as traditional alkylating agents, some may do so in a
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relatively more selective manner than others. It may be argued that the shortened
16 derivatives of 13, such as 25, which feature short, low molecular weight side chains, bind
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to guanines along the minor groove indiscriminately. It is unlikely that such shortened
structures (which form adducts spanning c. 4-5 DNA bases) would feature either the
affinity or diameter to specifically disrupt conserved 8-9 base transcription factor promotor
sequences. On the other hand, the presence of longer non-covalently interactive
elements (such as those found in 13) conjugated to PBD structures may subtly alter their
binding distribution along the minor groove. By adding moieties capable of interacting
non-covalently with DNA sequences in a relatively selective manner, the covalent adducts
of PBD structures may be (to some extent) preferentially directed toward the longer, more
specific sequences of DNA that are bound by transcription factors. While the traditional
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10 effects of non-selective alkylation undoubtedly still occur, additional mechanisms of cell
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disruption (such as transcription factor inhibition) may also underpin the cytotoxicity of
12 molecules such as 13.
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2D QSAR Analysis
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A two dimensional quantitative QSAR study of all compounds was carried out using
16 Multiple Linear Regression (MLR) method to determine if there are any correlations
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Page 23 of 72
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between the biological activity (1/LC ) and physicochemical descriptors LogP, molecular
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weight, steric factors and electronic factors. Leave-one-out cross validation was the
regression method applied in the MLR model to study the correlation of the descriptors and
the LC50 of the compounds. The results are listed in Table 3. The study showed slightly
better correlation between the biological activity of the molecules in primary CLL cells
(0.63) compared to JJN3 cell lines (0.52). The correlation improved when only LogP
(0.74) or molecular weight (0.68) were considered in primary CLL cells. However, the
correlations between biological activity in JJN3 cell line and LogP (0.58) or molecular
weight (0.48) were moderate.
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12 Molecular Modelling with DNA sequences
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As all the compounds including the shortened compounds are expected to exert their
14 cytotoxicity by covalent bonding with DNA and subsequent inhibition of transcription
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factors, a molecular modelling study was carried with 4 compounds (13, 20, 23 and 32)
16 with different C8-side chain length and one AT-rich and one GC-rich DNA sequences.
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These compounds represent a mix of high-molecular weight to the shortest compounds
synthesized with a range of cytotoxicity. The molecular docking study suggested that all
4 compounds are able to snugly bind with the DNA minor groove and retains the shape
characteristics required for the DNA minor groove binding molecules (Fig. 4, and Figs. S3
and S4). The energy of binding calculation showed highest value for compound 13 with -
11 KJ/mole for AT-rich DNA and -10.7 KJ/mole for the GC-rich DNA. The values obtained
for 20 and 23 were comparable, and the value for compound 32 was slightly lower (Table
S1). The findings of the study broadly supports the experimental observation as the
shortened molecules retained their ability to interact with DNA sequences. However, the
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10 relative lack of activity of compound 32 cannot be explained by modelling alone as the
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physicochemical property of the molecule likely to have played an important role in the
12 overall activity of the molecule.
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Conclusion
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The systemic shortening of C8-side chain of PBD monomers show that transcription
16 factor inhibitory effects may not necessarily translate into increased overall cytotoxicity or
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tolerability in vitro for PBD type molecules. This is an interesting observation that would
be useful in the design of mono-alkylating PBD monomers that are currently being
explored as standalone anticancer agents and payloads for antibody drug conjugates. It
is possible, however, that differential tolerability effects may only manifest themselves in
in vivo evaluations. Indeed, the high therapeutic index of historical PBD hybrids (featuring
longer non-covalently interactive elements of similar structure to that of 13) versus
unsubstituted PBD cores40 provides the rationale for in vivo investigations of the novel
PBD structures described in this paper. Such evaluations are currently underway.
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Experimental
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Synthesis: General Material and Methods: Synthetic building blocks, reagents, solvents and
chemicals were purchased from Sigma-Aldrich, Fluorochem, Fisher Scientific, Alfa Aesar and
Bachem. All reactions carried out were monitored by thin layer chromatography carried out on E.
14 Merck silica gel 60 F254 plates (0.25 mm). The plates were visualised using UV light at 254nm
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and potassium permanganate staining where required. Purification using Flash Column
Chromatography was carried out using standard glass columns and silica gel as a stationary phase
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17 (Merck 60, 230-400 mesh ASTM). Where freeze drying of intermediates/final compounds was
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carried out, this was done using a Heto-Lyolab 3000 freeze dryer. High Performance Liquid
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Chromatography coupled to Mass Spectrometry was carried out on reactions mixtures,
intermediates and final products. This analysis was performed on a Waters Alliance 2695
separation system using water (solvent A) and acetonitrile (solvent B) as mobile phases using a
Monolithic C18 50 X 4.60 mm column by Phenomenex. UV detection was performed on a Diode
Array Detector. Formic acid was used to ensure acidic conditions throughout the analysis and was
used at concentrations of 0.1%.
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All the compounds tested for their biological activity are >95% pure, confirmed with two different
HPLC analysis methods. A 10 minute (method A) and 5 minute (method B) gradient was used to
analyse submitted samples. The flow rate for both was 0.5ml/min; 200 µl was split over a zero
10 dead volume T piece which passed into the mass spectrometer. The wavelength range of the UV
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detector of the HPLC system was 220-400 nm. The function type used was a diode array (535
scans). The column type was a monolithic C18 50 X 4.60 mm column.
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Method A (10min run): 95% A 5% B for 2 minutes increasing to 50% B over 3 minutes. The
14 gradient was then held at 50% B for 1 minute and then increased to 95% B over 1.5 minutes. The
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quantity of B was then returned to 5% B over 1.5minutes and held for 1 minute.
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Method B (5min run): 95% A 5% B at 0 minutes increasing to 90% B over 3 minutes. The gradient
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was then increased to 95% B over 0.5 minutes and then held for 1 minute. The quantity of B was
18 then returned to 5% B over 0.5 minutes.
19 High Resolution Mass Spectrometry was undertaken on all final compounds synthesised. This was
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carried out using a Waters Micromass QTOF Global system in positive W-mode. Compounds were
dissolved in DMSO at concentrations of 5mM and introduced into the instrument using metal
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22 coated borosilicate glass tips. HNMR and CNMR were carried out on a Bruker Advance NMR
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spectrometer. The proton NMR was run at 400 Hz while the carbon samples were run at 100Hz.
The temperature for all samples was 300 K. The spectra obtained for each sample were processed
and analysed using Topspin 3.0.
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Synthesis of PBD core, C8-tails and C8-linked PBD-imines
PBD core 10 was synthesized following literature procedure in nine steps in good yield.
The intermediates were purified and fully characterized before progressing to the next
step. (ESI).
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Synthesis of Methyl 4-(5-aminobenzofuran-2-carboxamido)-1-methyl-1H-pyrrole-2-
carboxylate (11):
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5-((tert-butoxycarbonyl)amino)benzofuran-2-carboxylic acid (0.5 g, 1.80 mmol) was dissolved
in DMF (3 ml). EDCI (0.70 g, 2 equiv.) and DMAP (0.69 g, 2.5 equiv.) were added to the reaction
10 mixture and the solution was stirred for 30 minutes. Methyl 4-amino-1-methyl-1H-pyrrole-2-
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carboxylate (0.40 g, 1.5 equiv.) was then added to the flask and the reaction mixture left overnight.
After TLC (20% ethyl acetate/DCM) and LC-MS showed completion of reaction, the reaction
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acetate layers were combined and washed with citric acid solution (50 ml), bicarbonate solution
(50 ml), water (50 ml) and brine (50 ml). The solution was concentrated in a rotary evaporator to
yield a brown oil. The crude material was briefly characterised by LC-MS before the Boc protected
17 material was dissolved in methanol (1 ml for every mg of starting material), and 4M HCl in dioxane
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(2 ml for every 25 mg of starting material). Gas evolution was observed. After one hour, the
reaction was found to be complete via LC-MS and TLC (20% ethyl acetate/DCM). The solution
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20 was concentrated using a rotary evaporator to yield the deprotected compound 11 as the
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hydrochloric acid salt in good yield (64% over two steps). HNMR (400 MHz, (CD ) SO): δ
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3 2
10.87 (s, 1H), 10.14 (bs, 2H), 7.81 (d, 1H, J= 8.8 Hz), 7.76-7.74 (m, 2H) 7.55 (d, 1H, J=
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1.2Hz), 7.36 (dd, 1H, J=1.6 Hz, 8.8 Hz), 7.03 (d, 1H, J= 1.2 Hz), 3.87 (s, 3H), 3.76 (s,
3H). EIMS (m/z): 314.0 (M+H)+.
Synthesis of allyl (11aS)-7-methoxy-8-(4-((2-((5-(methoxycarbonyl)-1-methyl-1H-pyrrol-
3-yl)carbamoyl)benzofuran-5-yl)amino)-4-oxobutoxy)-5-oxo-11-((tetrahydro-2H-pyran-2-
yl)oxy)-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-
carboxylate (12), and (S)-methyl 4-(5-(4-((7-methoxy-5-oxo-2,3,5,11a-tetrahydro-1H-
benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)butanamido)benzofuran-2-carboxamido)-1-
10 methyl-1H-pyrrole-2-carboxylate (13):
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10 (0.132 g, 1.1 equiv.) was dissolved in DMF (3 ml). EDCI (0.085 g, 2 equiv.) and
12 DMAP (0.071 g, 2.5 equiv.) were added to the reaction mixture and the solution was
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stirred for 30 minutes. 11 (0.07 g, 0.233 mmol) was then added to the flask and the
14 reaction mixture left overnight. After TLC (20% acetone/DCM)/LC-MS showed completion
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of reaction, the reaction mixture was placed in ice water (30 ml) and extracted with ethyl
16 acetate (3 x 50 ml). The ethyl acetate layers were combined and washed with saturated
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