C8-Linked Pyrrolobenzodiazepine Monomers with Inverted Building Blocks Show Selective Activity against Multidrug Resistant Gram-Positive Bacteria

Article abstract of DOI:10.1021/acsinfecdis.7b00130

Antimicrobial resistance has become a major global concern. Development of novel antimicrobial agents for the treatment of infections caused by multidrug resistant (MDR) pathogens is an urgent priority. Pyrrolobenzodiazepines (PBDs) are a promising class of antibacterial agents initially discovered and isolated from natural sources. Recently, C8-linked PBD biaryl conjugates have been shown to be active against some MDR Gram-positive strains. To explore the role of building block orientations on antibacterial activity and obtain structure activity relationship (SAR) information, four novel structures were synthesized in which the building blocks of previously reported compounds were inverted, and their antibacterial activity was studied. The compounds showed minimum inhibitory concentrations (MICs) in the range of 0.125-32 μg/mL against MDR Gram-positive strains with a bactericidal mode of action. The results showed that a single inversion of amide bonds reduces the activity while the double inversion restores the activity against MDR pathogens. All inverted compounds did not stabilize DNA and lacked eukaryotic toxicity. The compounds inhibit DNA gyrase in vitro, and the most potent compound was equally active against both wild-Type and mutant DNA gyrase in a biochemical assay. The observed activity of the compounds against methicillin resistant S. aureus (MRSA) strains with equivalent gyrase mutations is consistent with gyrase inhibition being the mechanism of action in vivo, although this has not been definitively confirmed in whole cells. This conclusion is supported by a molecular modeling study showing interaction of the compounds with wild-Type and mutant gyrases. This study provides important SAR information about this new class of antibacterial agents.

Full text of DOI:10.1021/acsinfecdis.7b00130

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Article
C8-linked pyrrolobenzodiazepine monomers with inverted building
blocks show selective activity against MDR Gram-positive bacteria
Paolo Andriollo, Charlotte Hind, Pietro Picconi, Kazi S Nahar, Shirin Jamshidi,
Amrit Varsha, Melanie Clifford, J Mark Sutton, and Khondaker Miraz Rahman
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00130 • Publication Date (Web): 20 Dec 2017
Downloaded from http://pubs.acs.org on December 21, 2017
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C8ꢀlinked pyrrolobenzodiazepine monomers with inverted building blocks
show selective activity against MDR Gramꢀpositive bacteria
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⊥
2 ⊥
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Paolo Andriollo , Charlotte K. Hind Pietro Picconi , Kazi S Nahar , Shirin Jamshidi , Amrit
,
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2*
1*
Varsha , Melanie Clifford , J. Mark Sutton and Khondaker Miraz Rahman .
,
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Institute of Pharmaceutical Science, King’s College London, 150 Stamford Street, London SE1 9NH, UK.
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Public Health England, National Infections Service, Manor Farm Road, Porton Down, Salisbury SP4 0JG,
UK.
⊥
These authors contributed equally to the manuscript.
Corresponding authors
For KMR: eꢀmail k.miraz.rahman@kcl.ac.uk
For JMS: eꢀmail mark.sutton@phe.gov.uk
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Antimicrobial resistance has become a major global concern. Development of novel
antimicrobial agents for the treatment of infections caused by multidrug–resistant (MDR)
pathogens is an urgent priority. Pyrrolobenzodiazepines (PBDs) are a promising class of
antibacterial agents initially discovered and isolated from natural sources. Recently C8ꢀlinked
PBD biaryl conjugates have been shown to be active against some MDR Gramꢀpositive strains.
To explore the role of building block orientations on antibacterial activity and obtain SAR
information, four novel structures were synthesized in which the building blocks of previously
reported compounds were inverted, and their antibacterial activity studied. The compounds
showed MICs in the range of 0.125 µg/mL to 32 µg/mL against MDR Gramꢀpositive strains with
a bactericidal mode of action. The results showed that a single inversion of amide bonds reduces
the activity while the double inversion restores the activity against MDR pathogens. All inverted
compounds did not stabilize DNA and lacked eukaryotic toxicity. The compounds inhibit DNA
gyrase in vitro, and the most potent compound was equally active against both wildꢀtype and
mutant DNA gyrase in a biochemical assay. The observed activity of the compounds against
MRSA strains with equivalent gyrase mutations is consistent with gyrase inhibition being the
mechanism of action in vivo, although this has not been definitively confirmed in whole cells.
This conclusion is supported by a molecular modelling study showing interaction of the
compounds with wildꢀtype and mutant gyrases. This study provides important SAR information
about this new class of antibacterial agents.
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KEYWORDS
Antimicrobial Resistance, Medicinal Chemistry Optimization, Minimum Inhibitory
Concentration, Pyrrolobenzodiazepine, ESKAPE Pathogens
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The resistance of pathogens to antibacterial agents has become a major global concern. Multiple
drug resistant (MDR) bacteria cause many common and severe infections for which treatment is
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increasingly becoming difficult or in some cases impossible. The spread of healthcareꢀ
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associated infections and associated antimicrobial resistance is facilitated by interspecies gene
transmission, poor sanitation, and hygienic conditions, along with the increasing frequency of
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global travel, trade and disease transmission. Of great concern among all the bacteria that are
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developing resistance, is a group of pathogens termed the ESKAPE pathogens, which are
characterized by the rapid acquisition of resistance to multiple classes of antibiotic (with
resistance to 3 or more classes referred to as multidrug resistance). These species are primarily
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associated with nosocomial infections especially amongst immunocompromised patients and
1, 5
this also has an additional economic impact upon the healthcare system.
Although the
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measurement of additional costs is complicated the estimated economic impact for the U.S.
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economy is reported to be higher than $20 billion in direct annual healthcare costs.
In the last 30 years, no major classes of broad spectrum antibiotics have been introduced to the
market and recently approved agents like linezolid (2000), daptomycin (2003), and retapamutilin
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(
2007) are active only against Gramꢀpositive pathogens. A recent report from the World Health
Organisation has highlighted the limited number of drugs in the pipeline with only a few
molecules in phase 2 and 3 of clinical development against ESKAPE pathogens; these are
predominantly focused on Gramꢀpositive species or are iterations of existing drugs (notably betaꢀ
lactamase inhibitor combinations). This highlights the need to identify and evaluate new
therapeutic options and/or to modify existing chemical scaffolds to obtain antibiotics with
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improved properties and a number of agents developed through this approach are currently
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undergoing clinical trials.
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Applying the same principle, we selected the pyrrolobenzodiazepines (PBDs) as a relatively
unexplored chemical scaffold to generate new antibacterial compounds with improved
bioactivity, and elucidate the structure activity relationship (SAR) of C8ꢀlinked PBDs. PBDs are
naturally occurring molecules produced by Streptomyces bacteria whose family members include
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anthramycin and tomaymycin.
PBDs are a class of sequenceꢀspecific DNA minor groove
binding agents that are selective for GCꢀrich sequences, which have been evaluated as potential
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chemotherapeutic agents in recent years. PBDs have a chiral centre at their C11a(S)ꢀposition
which provides an appropriate 3D shape for them to fit securely within the DNA minorꢀgroove.
They also possess a “soft” electrophilic imine moiety at their N10ꢀC11 position which can form
an aminal linkage between their C11ꢀposition and the C2ꢀNH group of a guanine base only
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4, 15
when the molecule is secure within the minor groove.
Anthramycin was the first naturally
derived PBD, initially discovered and isolated in 1963 from Streptomyces and Micrococci
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bacteria , and later many other natural and synthetic PDBs followed. Naturally occurring PBDs
such as anthramycin, tomaymycin, neothramycin and sibiromycin form adducts that span three
17, 18
base pairs, with guanine in the central position,
and their preferred binding site is 5'ꢀAGAꢀ3',
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although more recent data suggest that they have a kinetic preference for 5′ꢀPyꢀGꢀPyꢀ3′
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sequences. PBD monomers can recognize and bind to specific sequences of DNA and therefore
have the potential to act as competitive inhibitors of transcription factors. Considering their
mechanism of action, PBDs have been extensively studied as anticancer agents but relatively
2
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unexplored as antibacterial agents.
Different studies showed the possibility of synthesizing
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PBD analogues characterized by their high selectivity for particular DNA sequences, by
25, 26
modifying the C8 position substituent.
For example, PBDꢀbiaryl conjugates, a subclass of
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monomers with C8ꢀsubunits, have shown preferences for GC sequences. Molecules belonging
to this subclass have been demonstrated to be well tolerated in mice at high concentrations, and
molecular dynamics simulations indicate that they are easily accommodated within the minor
groove causing little distortion to DNA structure.
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We previously studied numerous PBDꢀbiaryl conjugated structures, and identified some
characteristics that are important for optimal antibacterial activity against Gramꢀpositive
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pathogens. Among the compounds studied, we selected two compounds which showed
excellent antibacterial activity against MDR Gramꢀpositive pathogens (MIC range: 0.003ꢀ0.125
ꢁ
g/mL) for our investigation. The first compound is a PBDꢀPyꢀBzf (Py = pyrrole,
Bzf = benzofuran) conjugate, while the second structure was PBDꢀPyꢀBzt (Bzt =
benzothiophene) (Figure 1) conjugate. Starting from these two molecules we designed four new
molecules with a different orientation of the building blocks constituting the C8 lateral chain. It
has been reported in the literature that the standard orientation of the amide bonds provide
additional hydrogen bond contact with the minor groove of DNA, and inversion of the amide
23, 25
linkage should result in loss of these contacts and reduced binding to DNA.
There is a
positive correlation between DNA binding and eukaryotic toxicity of PBD monomers and a
reduction in binding should result in reduction or loss of toxicity. Firstly, we wanted to
investigate the antibacterial activity of the derivatives characterized by the inversion of the amide
linkage between Nꢀmethylpyrrole and the alkyl spacer and later the derivatives with both the
reverse Nꢀmethylpyrrole and the amide bonds between the building blocks (Figure 2), and obtain
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compounds with reduced eukaryotic toxicity. To achieve the synthesis of these new PBDꢀC8ꢀ
benzofused conjugates we needed a new PBD core with a 4CꢀNH linker in place of traditional
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4CꢀCOOH linker. Tiberghien et al. reported the use of this functionality in 2008 for the
synthesis of PBD dimers, but in this work, for the first time, it was used for the synthesis of C8ꢀ
linked PBD monomers and the synthesized compounds are the first examples of C8ꢀlinked PBD
monomers with an inverted amide linkage between the 4Cꢀspacer and the heterocyclic building
blocks. Moreover, from a synthetic chemistry point of view, this modification allowed us to
avoid the protection of the hydroxyl group at C11, otherwise necessary to prevent the
racemization of (S) C11a observed in the previous synthesis during the basic methyl ester
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hydrolysis employed in C8ꢀPBD monomer synthesis
The newly synthesized compounds were tested against susceptible and MDR Gramꢀpositive
bacterial strains. The compounds were also tested in a FRET based DNA melting assay, to
evaluate the effect of inverse orientation on the DNA binding ability of the compounds, and
against mammalian cell lines to assess their selectivity for prokaryotic cells. The mechanism of
action of the compounds were studied initially by an in silico screening against bacterial targets,
followed by a biochemical assay against the wildꢀtype and mutant target identified by the in
silico study. Finally, molecular modelling experiments were performed to rationalize the
obtained biological results.
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RESULT AND DISCUSSION
Chemistry. Both the synthetic strategies followed for the synthesis of the core bearing the 4Cꢀ
COOH (Scheme 1) and the core bearing 4CꢀNH functionality are based on the previously
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23
reported Thurston/Rahman approach. The PBD core used to synthesize the final compounds
with standard orientation was obtained as AllocꢀTHPꢀprotected PBD unit (9). Briefly, vanillin
was alkylated with methyl 4ꢀbrombutyrate employing K CO in DMF providing the ester
2
3
derivative 1 after precipitation. Subsequently, 1 underwent a selective nitration reaction using
KNO /TFA to give 2 in good yield after an aqueous workꢀup. The nitrobenzaldehyde derivative
3
2 was then oxidized to the carboxylic acid intermediate 3 using KMnO . The key intermediate 4
4
was obtained by amide coupling between 3, which had been activated to the corresponding acid
chloride by treatment with oxalyl chloride, and optically pure (S)ꢀpyrrolidine methanol.
Reduction of the nitro derivative 4 to the corresponding amino derivative 5 was accomplished
with Pd/C in a Parr hydrogenator system at 40 psi, followed by Alloc protection gave 6 in good
yield after purification by column chromatography. The Allocꢀprotected PBD ring system 7 was
then obtained by oxidative cyclization using the BAIB/TEMPO oxidizing system. In order to
prevent racemization at the C11a position of the PBD ring system, which occurs under alkaline
conditions, the alcohol derivative 7 was protected with a THP ether giving 8. The final
AllocꢀTHPꢀprotected PBD capping unit 9 was obtained via hydrolysis of the methyl ester using
0.5 M NaOH aqueous solution.
The synthetic strategy to obtain the C8ꢀlinked PBDs with inverted amide bonds (Scheme 2)
involved protection of the reactive amino group on the 4Cꢀspacer to avoid side reactions, and the
reaction steps were adjusted taking this modification into account. Particularly, acidic conditions
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were avoided after the introduction of the Bocꢀprotected amine side chain. To obtain the type 2
core, vanillin was coupled with benzyl bromide under alkaline condition giving the ether
derivative 10 after recrystallization. This additional step allowed the selective nitration that led to
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1 with a good yield. The benzyl group was then removed with concentrated HBr in acetic acid
to afford the nitroꢀvanillin derivate 12. The amide coupling reaction with (S)ꢀpyrrolidine
methanol to obtain 15 was performed using HBTU and DIPEA rather than oxalyl chloride to
avoid the generation of acid. The final Allocꢀprotected PBD capping unit was obtained via the
deꢀprotection of the amino group by TFA in DCM, just before the coupling with the selected tail.
The synthesis of the C8ꢀside chains was achieved by amide coupling between the selected
building blocks (Scheme 3). An EDCI/DMAP reagents system was used for the activation of the
acid, leading to intermediate 19 to 24 with variable yield (20% to 93%). Nitrile groups of 20 and
23 were hydrolyzed by refluxing in dioxane in a strongly acidic environment (with the presence
of H SO ) to give 25 and 26 after purification by column chromatography.
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The last step in the synthesis of the C8ꢀlinked benzofusedꢀNꢀmethylpyrrole PBD derivatives
consisted of several sequential passages. At first, when it was necessary, the capping units were
deꢀprotected. Methyl esters of derivatives 21 and 24 were hydrolyzed under basic conditions (in
a NaOH aqueous solution), leading to the acid compounds, while the amino groups on 19 and 22
were deꢀprotected in an acidic environment (TFA in DCM). Subsequently, the PBD core and the
benzofusedꢀNꢀmethylpyrrole tails were connected through an amide coupling reaction. Once
again, EDCI/DMAP system was used to carry out acid activation and coupling. Finally, the
conjugated products were deꢀprotected with pyrrolidine and Pd(PPh ) in DCM, affording the
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final compounds 27, 28, 29, 30, 31 and 32 (Scheme 4). The final deꢀprotection was common to
all the products regardless of the presence of THP protection, and both the protected and the final
PBD imine conjugates were purified by column chromatography.
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Evaluation of DNA binding ability of the synthesized compounds
The ability of compounds 27, 28, 29, 30, 31 and 32 to bind and stabilize the DNA was evaluated
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using a FRETꢀbased DNA melting assay to understand the effect of building block orientation
on the DNA binding ability of the compounds. The compounds were tested using two
fluorophoreꢀlabelled oligonucleotide sequences (Sequence F1: 5’ꢀFAMꢀTATꢀATAꢀTAGꢀATAꢀ
TTTꢀTTTꢀTATꢀCTAꢀTATꢀATAꢀ3’ꢀTAMRA, and SequenceꢀF2: 5’ꢀFAMꢀTATꢀAGAꢀTATꢀ
AGAꢀTATꢀTTTꢀATAꢀTCTꢀATAꢀTCTꢀATAꢀ3’ꢀTAMRA). Netropsin, a known DNA minor
groove binder, was used as a positive control. The result of the assay is reported in Table 1.
Previously reported C8ꢀPBD conjugates 27 and 30, and control compound netropsin showed
notable stabilization of both DNA sequences at 1 µM, but the C8ꢀPBD compounds with inverted
building blocks, 28, 29, 31 and 32 did not stabilize the DNA sequence, with ꢂT values < 1 °C
m
at 1 µM concentration (drug:DNA ratio 5:1). The result suggests the inversion of single or both
amide bonds reduced the ability of these compounds to interact with and stabilize the DNA
sequences.
Evaluation of Eukaryotic Toxicity
The inability of the inverted C8ꢀPBD conjugates to stabilize DNA sequences suggested the
compounds may show reduced toxicity against eukaryotic cell lines, as DNA stabilization has
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been associated with cytotoxicity for PBDꢀtype covalent minor groove binders. The eukaryotic
cytotoxicity of the synthesized compounds was tested against the cervical cancer cell line HeLa
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and the nonꢀtumor lung fibroblast WI38 using an MTT assay. The original C8ꢀPBD
benzofused conjugates 27 and 30 showed very potent cytotoxicity against the HeLa cell line
(Figure 3) with only 25% and 32% cells viable after 24 hours. The compounds showed slightly
less toxicity against the nonꢀtumor cell line WI 38 with 42% and 47% cells viable after 24 hours.
However, the inverted compounds 28, 29, 31 and 32 showed notably less toxicity against both
HeLa and WI38 cell lines. In the case of the inverted compounds >70% of cells were viable after
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4 hours treatment with the molecules. This lack of toxicity was in line with the DNA binding
ability of the compounds, and this is consistent with previous reports that PBDs exert their
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cytotoxicity by alkylating and stabilizing DNA sequences.
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Microbiological Evaluation
The inverted amide compounds and the parent compounds were tested against a selected panel of
Gramꢀpositive bacteria to assess their antibacterial activity (Table 2). The Gramꢀpositive panel
was comprised of Staphylococcus aureus and Enterococcus spp. strains, which comprise the ‘S’
and the ‘E’ from the ESKAPE pathogens acronym. The strains used were; methicillin sensitive S.
aureus (MSSA) strain ATCC 9144, two methicillin resistant S. aureus (MRSA) strains,
EMRSAꢀ15 (strain HO 5096 0412) and EMRSAꢀ16 (strain MRSA 252), vancomycin sensitive
E. faecalis (VSE), strain NCTC 755, vancomycin resistant E. faecalis (VRE), strain NCTC
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2201 and vancomycin resistant E. faecium (VRE), strain NCTC 12204. As expected, compound
27 showed good antibacterial activity with MICs lower than 0.125 µg/mL against all
Gramꢀpositive strains tested. Surprisingly, the inversion of one amide linkage in compound 28
resulted in a considerable loss of activity with an MIC of 2 µg/mL against VRE strains and
16 µg/mL against VSE and MSSA strains. The drop in activity was particularly surprising
against MRSA strains, with an MIC higher than 32 µg/mL. Interestingly, compound 29,
characterized by the inversion of both the amide linkages and of the Nꢀmethylpyrrole building
block, presented a better antibacterial profile than 28. Although it was found to be less potent
than 27, compound 29 maintained moderate activity against the bacterial strains with MICs in
the range between 0.5 and 4 µg/mL. The observations made for the benzofuran series of C8ꢀPBD
conjugates were further confirmed by evaluating the C8ꢀbenzothiophene series of inverted and
parent PBD compounds (Table 2). Compound 32 showed antibacterial activity comparable to
previously reported compound 30 against Enterococci strains with MICs lower than 0.125
µg/mL. However, a drop in activity was observed against S. aureus strains with MICs higher
than 32 µg/mL against MRSA and of 16 µg/mL against MSSA. Compound 31 showed
antibacterial activity similar to 32 against S. aureus strains, but generally showed higher MICs
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against other strains. Overall, the inverted C8ꢀlinked benzothiophene PBD compounds showed
better antibacterial activity against Enterococcus strains compared to the benzofuran compounds,
while among the four newly synthesized compounds, the inverted benzofuran derivative 29 was
found to be most active against S. aureus strains.
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The mode of action of compound 29 was explored using timeꢀkill assays. One MRSA (EMRSAꢀ
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5) and one VRE (NCTC 12201) strain were treated with compound 29 at 4 × MIC for 24 hours
and cell counts elucidated at specified time points. The results show a rapidly bactericidal mode
of action in both strains for compound 29, with cell counts falling below the limit of detection
within 2 hours, whilst the control antibiotic ciprofloxacin displayed a bacteriostatic mode of
action (Figure 4). Although a small population of cells was detected after 24 hours of treatment
with compound 29 in EMRSAꢀ15 (in two of three replicate experiments), no resistance to the
compound was detected in this population (assessed by determining the MIC of surviving cells),
and as such they most likely represent a persistor population. This is observed in EMRSA15
(fluoroquinolone resistant) but not in strain NCTC12201 (fluoroquinolone sensitive), although
the role of fluoroquinolone resistance in persistor survival in these assays is unproven.
In silico DNA Interaction Study: In order to rationalize the obtained results, a molecular
modelling study was carried out using three random sequences of DNA. ATꢀrich Seqꢀ1 (5'ꢀ
TATATAAGATATATATAꢀ 3'), GCꢀrich Seqꢀ2 (5'ꢀTAGCTAGCTAGCTAGCGꢀ3') and mixed
AT/GC Seqꢀ3 (5'ꢀGCGCGCGCGCGGCGCGCꢀ3'). Covalent molecular docking between the N
atom of NH2 group of Guanine and the C11 atom of C=N imine bond in PBDs, showed
favorable complexes with good affinities between PBDs and DNA. As PBDs are known to work
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by interacting with the DNA minor groove, we calculated the relative binding free energy of the
DNAꢀcompound complex (Figure 5) using molecular dynamics simulations followed by
Molecular MechanicsꢀPoisson Boltzmann Surface Area (MMꢀPBSA) / Molecular Mechanicsꢀ
Generalized Born Surface Area (MMꢀGBSA) calculations (Table 3). These models showed
favorable energy values of ∆GGB and ∆GPB for the reference compounds in all three sequences
while the results for the inverted compounds (28, 29, 31 and 32) were considerably lower than
the parent compounds 27 and 30. The modelling study supported the lack of DNA stabilization
observed in the FRET melting study and the lack of eukaryotic toxicity. Overall, a positive
correlation between the free energy of binding and antibacterial activity was observed suggesting
DNA binding plays a role in the antibacterial potency of these compounds.
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Mechanistic Evaluation
As the inverted C8ꢀanalogues did not stabilise DNA sequences and were nonꢀtoxic to eukaryotic
cells, the mechanism of action of these molecules is likely to be DNA independent. To explore
this further, we carried out an in silico docking campaign with known bacterial targets (ESI) to
get an indication of the mechanism of action of these inverted pyrrolobenzodiazepines. Among
the targets studied, the inverted compound 29 interacted with the ligand binding domain of DNA
gyrase and showed excellent affinity towards both subꢀunits of bacterial DNA gyrase. The best
pose of compound 29 with the bacterial gyrase from Staphylococcus aureus (PDB ID 2XCT)
which gave a CHEM Score of 21.68 and affinity of ꢀ29 (kcal/mol) for subunit 1 of Gyrase A, and
a CHEM Score of 23.29 and affinity of ꢀ30 (kcal/mol) for subunit 2 of Gyrase A (Figure 6 and
Table S2). The 2D models shown in Figure 6C suggests compound 29 forms three conventional
hydrogen bonds with serine 98, arginine 92 and glutamine 95 of the subunit 1 of DNA gyrase A.
Other key interactions are reported in Table S4. Similarly, compound 29 also forms three
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conventional hydrogen bonds with serine 85, arginine 92 and serine 98 of the subunit 2 of
Gyrase A (Figure 6D). A number of hydrophobic and electrostatic interactions with different
amino acids from subunits of Gyrase A were also observed (Table S5). The interaction of 29
with the ligand binding site of Gyrase A suggests the antimicrobial activity is due to the
inhibition of Gyrase A by directly interacting with the enzyme, and not due to its ability to
associate with DNA.
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The in silico observation was validated by carrying out a DNA gyrase inhibition assay using a
commercial S. aureus gyrase supercoiling high throughput plate assay (#SATRG01) kit obtained
from Inspiralis (Norwich, UK) (Figure 7 and S4). Compound 29 inhibited wildꢀtype gyrase
isolated from S. aureus with an IC50 of 0.95±0.30 µg/mL which is significantly (P<0.05) better
than ciprofloxacin which inhibited the wildꢀtype gyrase with an IC50 of 2.85 ±1.80 µg/mL.
Surprisingly, compound 27 and 30 also showed comparable IC s, 1.65±0.68 µg/mL and
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.28±0.98 µg/mL, respectively, against wild type gyrase (Figure 7). There was no statistically
significant difference between the IC of compounds 27, 29 and 30. This data does not provide a
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clear differential in gyrase inhibition between compounds that stabilise DNA (27 and 30) and
may inhibit gyrase, a DNA associated enzyme, and compounds that have not shown DNA
stabilisation (29 and ciprofloxacin, Table 1), where we would infer direct inhibition by gyrase
binding. Competition assays were used to try and define the basis of the interaction. However, no
clear answer could be obtained, as doubling the concentration of enzyme in the gyrase assays
reduced the inhibition caused by compound 29 and ciprofloxacin but not compound 30, which
supports our inference, but a reduction in inhibition was also observed for compounds 27 which
could not be explained (Figure S5).
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One of the most common mutations found in fluoroquinolone resistant S. aureus strains is the
S84L mutation in the gyrA subunit. We tested compound 29 against a gyrase enzyme complex
containing this mutation and found that at 20 ꢀg/ml, compound 29 inhibited the mutant enzyme
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by 60.0% ±11.2%, whilst ciprofloxacin inhibited the mutant enzyme by just 43.3% ±1.1 at the
higher concentration of 32 ꢀg/ml (Figure S9). This data suggests that compound 29 retains
better activity against the fluoroquinoloneꢀresistant enzyme than ciprofloxacin. The ability of
compound 29 to inhibit the mutated DNA gyrase led us to carry out a further molecular
modelling study to rationalize the biochemical assay result. The molecular model of gyrase A
with S84L mutation in the gyrA subunit was developed with PyMol using PDB ID 2XCT as the
template. The structure was minimized, equilibrated and subsequently validated by AMBER
package program. Interestingly, compound 29 interacted with the ligand binding site of mutant
Gyrase A (Figure S2) with greater affinity and higher CHEMSCORE (23.4 for subunit 1 and
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3.4 for subunit 2) compared to the wildꢀtype enzyme. However, ciprofloxacin showed a
significantly reduced affinity and CHEMSCORE (15.8 for subunit 1 and 15.9 for subunit 2) for
the mutant enzyme (Figure S3, Table S3), which is consistent with the literature. Compound 29
formed five conventional hydrogen bonds with both subunit 1 and subunit 2 of the mutant
enzyme (Tables S6 and S7). The antibacterial activity observed for compound 29 against
Staphylococcus strains (Table 2) appeared to support the biochemical assay and the in silico
observation as it showed similar activity (MIC 2ꢀ4 µg/mL) against the MSSA strain ATCC9144,
which doesn’t bear the S84L mutation in gyrase A and the MRSA strains, EMRSAꢀ15 and
EMRSAꢀ16, with the S84L mutation in gyrase A.
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Conclusions. This study was focused on understanding how the building blocks orientation of
the lateral C8 side chain of PBDs affect the antibacterial activity of C8ꢀPBD conjugates, and the
suitability of developing C8ꢀlinked inverted PBDs as antibacterial agents. Furthermore, we
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explored the suitability of using the novel 4CꢀNH linker on the C8ꢀposition of the PBDs, which
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provides some advantages for the synthesis of the C8ꢀlinked pyrrolobenzodiazepines by avoiding
the base catalyzed hydrolysis step that can result in C11a racemization. Four inverted C8ꢀlinked
benzofused PBDs were successfully synthesized employing both 4CꢀCOOH and 4CꢀNH linkers.
2
Two of them presented the inversion of the amide linkage between Nꢀmethylpyrrole and the PBD
core while the other two presented the inversion of both amide bonds between the three moieties
constituting the lateral chain and the heterocyclic building blocks. The single amide bond
inverted compounds showed notably inferior antibacterial activity owing to their inability to
effectively associate with the DNA minor groove. However, the double inverted compounds
showed comparable activity to the C8ꢀbenzofused PBDs with natural orientation. The inverted
C8ꢀanalogues did not stabilize DNA sequences and showed selective toxicity towards
prokaryotic cells. The differential sensitivity observed between S. aureus and Enterococcus spp.
for compounds 31, 32 and, to a lesser extent, 28, is intriguing and pointed towards a new
mechanism of action rather than solely DNAꢀbinding, as observed for traditional DNA minor
groove binders. In silico studies followed by biochemical assays suggested that the compounds
are capable of binding to DNA gyrase and showed inhibition of DNA gyrase in vitro. . The data
in this study shows that these molecules can inhibit both wildꢀtype and mutant DNA gyrase
(S84L mutation in gyrase A) enzymes in vitro and kill S. aureus strains, carrying this mutation,
that are resistant to fluoroquinolones. The observed IC is comparable to ciprofloxacin in the in
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vitro assay, and this was also the case for nonꢀinverted PBDs (27 and 30). Future studies are
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required to reꢀevaluate of the mechanism of bacterial killing by this class of molecules, both in
terms of the kinetics of the in vitro gyrase inhibition assay and by showing bacterial responses
which are consistent with DNAꢀgyrase inhibition in vivo. This deeper understanding of the
mechanism of action provides an opportunity to develop these inverted C8ꢀPBD analogues as
antibacterial agents due to their lack of eukaryotic toxicity and reasonable activity against MDR
Gramꢀpositive pathogens, potentially reviving PBD based antiꢀinfective research.
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METHODS
Chemistry. All solvents and reagents for the synthesis were obtained from commercially
available sources, including, among others, SigmaꢀAldrich, Fisher Scientific, Fluorochem and
Alfa Aesar. Thinꢀlayerꢀchromatography (TLC) analysis was performed on silica gel plates (E.
Merck silica gel 60 F254 plates) and visualized by ultraꢀviolet (UV) radiation at 254 nm. Flash
chromatography for the purification of compound was performed with silica gel as a stationary
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phase (Merck 60, 230ꢀ400 mesh). H and C nuclear magnetic resonance (NMR) analyses were
performed on a Bruker Spectrosp in 400Hz spectrometer. IR spectra were collected with an
FT/IR IRAffinityꢀ1S IR spectrophotometer (Shimadzu). HRMS was performed on a Thermo
ScientificꢀExactive HCD Orbitrap Mass Spectrometer. LCꢀMS analyses were performed on a
Waters Alliance 2695 system, eluting in gradient with a flow rate of 0.5 mL/min using a solvent
gradient starting with 5% acetonitrile that was increased to 95% acetonitrile over a 7.5 minutes’
time period (ESI). The analyses were performed on a Monolithic C18 50×4.60 mm column
(made by Phenomenex). UV detection was performed on a Diode Array Detector. Mass spectra
were registered in both the ESI+ and ESIꢀ mode. The hydrogenation reaction was conducted
using a Parr hydrogenation system. Dynamic light scattering experiment was performed using a
Nanoseries machine (Malvern Instruments, UK).
Methyl 4ꢀ(4ꢀformylꢀ2ꢀmethoxyphenoxy)butanoate (1). Methyl 4ꢀbromobutanoate (17.7 mL,
140.2 mmol, 1.05 equiv.) and potassium carbonate (30.4 g, 210.3 mmol 1.5 equiv.) were added
to a solution of vanillin (20.0 g, 131.4 mmol, 0.9 equiv.) in DMF (80 mL). The suspension was
stirred at room temperature for 6 h, until TLC showed the completion of the reaction. At that
point H O (1 L) was added to the reaction, causing the formation of a precipitate that was filtered
2
and collected giving pure 1 (32.0 g, 95%) as a white solid (m.p. 65 °C ).
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R value (100% DCM): 0.62; H NMR (400 MHz, CDCl ) δ 9.84 (1H, s), 7.40ꢀ7.44 (2H, m),
f
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.98 (1H, d, J = 8 Hz), 4.16 (2H, t, J = 6.2 Hz), 3.92 (3H, s), 3.69 (3H, s), 2.56 (2H, t, J= 7.2
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Hz), 2.17ꢀ2.23 (2H, m). C NMR (100 MHz, CDCl ) δ 190.9, 173.4, 153.8, 149.9, 130.3, 126.8,
3
+
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11.5, 109.2, 67.8, 56.4, 51.7, 30.3, 24.2. m/z (+ESI) calc. for C H O (M) 252.2 found 253.1
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(
[M]+H) .
Methyl 4ꢀ(4ꢀformylꢀ2ꢀmethoxyꢀ5ꢀnitrophenoxy)butanoate (2). A solution of 1 (10.0 g, 39.6
mmol, 1.0 equiv.) in trifluoroacetic acid (12 mL) was added dropwise to a solution of KNO (5.0
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g, 49.5 mmol, 1.25 equiv.) in trifluoroacetic acid (12 mL) kept at 0°C under a magnetic stirrer.
After 40 min, TLC and LCMS monitoring showed completion of the reaction. The reaction
mixture was evaporated under reduced pressure using a rotary evaporator. The residue was
dissolved in EtOAc (50 mL), and the organic phase was washed with brine (3×50 mL). The
organic phases were dried over MgSO and concentrated by a rotary evaporator giving pure 2
4
(10.7 g, 92%) as an amber oil.
1
R value (100% DCM): 0.72; H NMR (400 MHz, CDCl ) δ 10.29 (1H, s), 7.46 (1H, s), 7.11
f
3
(
1H, s), 4.06 (2H, t, J = 6.2 Hz), 3.85 (3H, s), 3.56 (3H, s), 2.42 (2H, t, J= 7.2 Hz), 2.04ꢀ2.11
13
(
2H, m); C NMR (100 MHz, CDCl ) δ 188.5, 172.8, 152.7, 150.9, 143.5, 124.7, 110.5, 108.2,
3
+
+
6
7.8, 56.4, 51.3, 29.7, 23.2 m/z (+ESI) calc. for C H NO (M) 297.2 found 298.1 ([M]+H) .
13 15 7
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ꢀMethoxyꢀ4ꢀ(4ꢀmethoxyꢀ4ꢀoxobutoxy)ꢀ2ꢀnitrobenzoic acid (3). Compound 2 (10.0 g, 33.6
mmol, 1.0 equiv.) was dissolved in acetone (400 mL). A hot solution of 10% potassium
permanganate (275 mL) was added to the solution of 2 in a flask fitted with a condenser. The
reaction mixture was left under reflux until the reaction went to completion (according to TLC).
At that point, the reaction mixture was cooled down to room temperature. The brown residue
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
7
8
9
0
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9
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7
8
9
0
formed was filtered through a celite patch and washed with 600 mL hot H O. A solution of 16%
2
sodium bisulphite in 1N HCl (400 mL) was added to the filtrate, and the pH of the solution was
adjusted to 1 using concentrated HCl. This caused the precipitation of a yellow solid that was
filtered, collected and dried giving pure 3 (9.0 g, 82%) as a yellow solid (m.p. 114 °C).
1
R value (EtOAc:MeOH 50:50): 0.4; H NMR (400 MHz, CDCl ) δ 7.38 (1H, s), 7.21 (1H, s),
f
3
4
.15 (2H, t, J = 5.8 Hz), 3.97 (3H, s), 3.70 (3H, s), 2.56 (2H, t, J= 7.2 Hz), 2.17ꢀ2.24 (2H, m);
1
3
C NMR (100 MHz, CDCl ) δ 172.8, 166.0, 151.8, 149.1, 141.2, 121.3, 111.5, 107.2, 68.3, 56.4,
3
+
ꢀ
5
1.3, 29.7, 23.8. m/z (+ESI) calc. for C H NO (M) 313.2 found 312.1 ([M]ꢀH)
13 15 8
(S)ꢀMethyl
4ꢀ(4ꢀ(2ꢀ(hydroxymethyl)pyrrolidineꢀ1ꢀcarbonyl)ꢀ2ꢀmethoxyꢀ5ꢀ
nitrophenoxy)butanoate (4). A solution was prepared by dissolving 3 (7.9 g, 25.2 mmol, 1.0
equiv.) in dry DCM (50 mL) in a round bottom flask previously dried in an oven. Oxalyl
chloride (6.5 mL, 75.6 mmol, 3 equiv.) and a catalytic amount of DMF (2ꢀ3 drops) were added to
the solution that initiated the reaction. The solution was left under a magnetic stirrer for 1 h until
it ceased the formation of HCl. Dry toluene (15 mL) was added to the reaction mixture that was
evaporated under reduced pressure in a rotary evaporator to eliminate the excess of oxalyl
chloride. The reaction mixture was dissolved in dry DCM (50 mL and the solution was dropwise
added to a solution of triethylamine (10.5 mL, 75.6 mmol, 3 equiv.) and +(S)ꢀ
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pyrrolidinemethanol (3.7 mL, 37.8 mmol, 1.5 equiv.) in dry DCM (30 mL) kept at 0 °C under
N atmosphere. The reaction mixture was then allowed to stir overnight. After 15 h TLC showed
2
the completion of the reaction, and the reaction mixture was extracted using 1 N HCl (2×70 mL)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
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3
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4
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5
5
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5
5
5
6
0
1
2
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5
6
7
8
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0
1
2
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8
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0
1
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9
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5
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8
9
0
and brine (2×70 mL). The combined organic fractions were dried over MgSO and concentrated
4
by the rotary evaporator to give a yellow oil. The crude was purified by column chromatography
(
mobile phase: from EtOAc, 100, v/v to EtOAc /MeOH, 98/2, v/v) affording pure 4 (5.5 g, 55%)
as a pale yellow solid (m. p. 82 °C).
R value (EtOAc:MeOH 90:10): 0.46; H NMR (400 MHz, CDCl ) δ 7.69 (1H, s), 6.79 (1H, s),
1
f
3
4.14 (2H, t, J = 4.4 Hz), 3.96 (3H, s), 3.90 (1H, m), 3.78 (1H, m), 3.69 (3H, s), 3.16 (2H, t, J =
1
3
6
.8 Hz), 2.55 (2H, t, J = 4.8 Hz), 2.10ꢀ2.22 (3 H, m), 1.70ꢀ1.90 (4 H, m); C NMR (100 MHz,
CDCl ) δ 173.2, 154.8, 148.4, 109.2, 108.4, 68.4, 66.1, 61.5, 56.7, 51.7, 49.5, 30.3, 28.4, 24.4,
3
+
+
2
4.2. m/z (+ESI) calc. for C H N O (M) 396.3 found 397.0 ([M]+H) .
18 24 2 8
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(
S)ꢀMethyl
4ꢀ(5ꢀaminoꢀ4ꢀ(2ꢀ(hydroxymethyl)pyrrolidineꢀ1ꢀcarbonyl)ꢀ2ꢀ
methoxyphenoxy)butanoate (5). A catalytic amount of Pd/C (10% w/w) was added to a
solution of 4 (5.5 g, 13 mmol, 1 equiv.) in EtOH (100 mL). The reaction mixture was
hydrogenated in a Parr hydrogenator at 40 psi until for 4 h when TLC showed the completion of
the reaction. At that point, the reaction was filtered under vacuum through a patch of celite. The
resulting solution was evaporated using rotary evaporator giving pure 5 (4.5 g, 95 %) as a dark
yellow solid (m. p. 46 °C).
0
1
2
3
4
5
6
7
8
9
0
1
2
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8
9
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5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
R value (EtOAc:MeOH 90:10): 0.36; H NMR (400 MHz, CDCl ) δ 6.76 (1H, s), 6.39 (1H, s),
f
3
4
2
1
.39 (1H, bs), 4.03 (2H, t, J = 4.4 Hz), 3.78 (3H, s), 3.69 (3H, s), 3.62 (1H, m), 3.53 (1H, m),
13
.54 (2H, t, J = 4.8 Hz), 2.15 (3H, m), 1.65ꢀ1.87 (4H, m); C NMR (100 MHz, CDCl ) δ 172.5,
3
70.7, 150.3, 140.5, 140.1, 135.0, 112.3, 110.5, 101.2, 66.5, 59.9, 56.3, 52.4, 50.6, 29.4, 27.5,
+
+
23.9, 23.4. m/z (+ESI) calc. for C H N O (M) 366.4 found 367.2 ([M]+H) .
1
8
26
2
6
(S)ꢀMethyl 4ꢀ(5ꢀ(allyloxycarbonylamino)ꢀ4ꢀ(2ꢀ(hydroxymethyl)pyrrolidineꢀ1ꢀcarbonyl)ꢀ2ꢀ
methoxyphenoxy)butanoate (6). A solution was prepared by dissolving 5 (3.3 g, 9 mmol, 1.0
equiv.) in dry DCM (40 mL). Dry pyridine (1.7 mL, 21.1 mmol, 2.3 equiv.) and a solution of
allyl chloroformate (0.91 mL, 8.5 mmol, 0.95 equiv.) in anhydrous DCM (30 mL) were
sequentially added to this solution, which was kept at ꢀ10°C under N atmosphere. The reaction
2
mixture was left under a magnetic stirrer at room temperature for 2 h, until TLC showed the
completion of the reaction. At that point, the reaction mixture was extracted with a saturated
CuSO solution (70 mL), saturated aqueous NaHCO (100 mL) and brine (100 mL). The organic
4
3
phase was dried over MgSO and concentrated under reduced pressure using a rotary evaporator.
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The crude of the reaction was subsequently purified by column chromatography (mobile phase:
EtOAc, 100%) affording pure 6 (3.6 g, 88%) as a pale yellow solid (m. p. 49 °C)
1
R value (DCM:Acetone 60:40): 0.51; H NMR (400 MHz, CDCl ) δ 8.72 (1H, bs), 7.77 (1H, s),
f
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
.82 (1H, s), 5.95 (1H, m), 5.35 (1H, dd, J = 17.2, 1.2 Hz), 5.23 (1H, dd, J = 10.0, 0.8 Hz), 4.63
(2H, dd, J = 5.6, 1.2 Hz), 4.40 (1H, bs), 4.11 (2H, t, J = 4.4 Hz), 3.82 (3H, s), 3.61 (3H, s), 3.59
1
3
(1H, m), 3.50 (1H, m), 2.54 (2H, t, J = 4.8 Hz), 2.17 (3H, m), 1.72ꢀ1.92 (4H, m); C NMR (100
MHz, CDCl ) δ 173.4, 170.9, 153.6, 150.5, 144.0, 132.3, 131.9, 118.2, 115.7, 111.6, 105.6, 67.7,
3
+
6
6.6, 65.7, 61.6, 60.4, 56.6, 51.7, 30.7, 28.3, 25.1, 24.3. m/z (+ESI) calc. for C H N O (M)
22 30 2 8
+
450.4 found 451.2 ([M]+H) .
Allyl 11ꢀhydroxyꢀ7ꢀmethoxyꢀ8ꢀ(4ꢀmethoxyꢀ4ꢀoxobutoxy)ꢀ5ꢀoxoꢀ2,3,11,11aꢀhexahydroꢀ1H-
pyrrolo[2,1ꢀc][1,4]benzodiazepineꢀ10(5H)ꢀcarboxylate (7). BAIB (3.7 g, 11.6 mmol, 1.2
equiv.) and TEMPO (152.0 mg, 1.0 mmol, 0.1 equiv.) were sequentially added to a solution of 6
(4.41 g, 9.7 mmol, 1.0 equiv.) in DCM (200 mL). The reaction was left under a magnetic stirrer
for 6 h until TLC showed the completion of the reaction. At that point, the reaction mixture was
sequentially washed with saturated sodium metabisulphite (100 mL), saturated aqueous NaHCO3
(
2×100 mL) and brine (100 mL). The organic phase was dried over MgSO and concentrated
4
under reduced pressure using a rotary evaporator. The crude of the reaction was purified by
column chromatography (mobile phase: EtOAc/hexane, 50/50, v/v) affording pure 7 (3.3 g, 75%)
as a yellow solid (m.p. 74 °C).
1
R value (DCM:Acetone 60:40): 0.51; H NMR (400 MHz, CDCl ) δ 7.22 (1H, s), 6.69 (1H, s),
f
3
5.80 (1H, m), 5.62 (1H, d, J = 4.0 Hz), 5.07 (2H, d, J = 12.0 Hz), 4.61 (1H, dd, J = 13.2, 5.6 Hz),
4
.41 (1H, bs), 4.21 (2H, d, J = 12.0 Hz), 3.89 (3H, s), 3.67 (3H, s), 3.49 (1H, t, J = 8.0 Hz), 3.43
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5
5
5
5
6
13
(
1H, m), 2.475 (2H, t, J = 7.2 Hz), 2.12 (4H, m), 1.95 (2H, m); C NMR (100 MHz, CDCl ) δ
3
1
73.4, 167.0, 155.9, 149.9, 148.7, 131.8, 128.3, 126.0, 117.9, 114.2, 110.8, 85.9, 67.9, 66.7, 60.3,
60.1, 56.1, 51.6, 46.3, 30.3, 28.7, 24.2, 23.0, 20.9. m/z (+ESI) calc. for C H N O (M)+ 448.4
2
2
28
2
8
0
1
2
3
4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
found 449.2 ([M]+H)+.
Allyl
,3,11,11aꢀhexahydroꢀ1H-pyrrolo[2,1ꢀc][1,4]benzodiazepineꢀ10(5H)ꢀcarboxylate (8). DHP
6.3 mL, 75.0 mmol, 10.0 equiv.) was added to a solution of 7 (3.3 g, 7.5 mmol, 1.0 equiv.) in
7ꢀmethoxyꢀ8ꢀ(4ꢀmethoxyꢀ4ꢀoxobutoxy)ꢀ5ꢀoxoꢀ11ꢀ(tetrahydroꢀ2H-pyran-2ꢀyloxy)ꢀ
2
(
the presence of a catalytic amount of PTSA (33.0 mg, 0.2 mmol, 0.03 equiv.) in EtOAc (50 mL).
The reaction mixture was left under a magnetic stirrer for 2 h until TLC showed the completion
of the reaction. At that point and the reaction mixture was extracted with saturated aqueous
NaHCO (2×50 mL) and brine (50 mL). The organic phase was dried over MgSO , and
3
4
evaporated using a rotary evaporator under reduced pressure. The crude of the reaction was
purified by column chromatography (mobile phase: DCM/acetone, 90/10, v/v) affording pure 8
(3.6 g, 85%) as a yellow solid (m. p. 53 °C).
1
R value (DCM:Acetone 60:40): 0.67; H NMR (400 MHz, CDCl ) δ 7.19 (1 H, s), 6.88 (1 H, s),
f
3
6
3
.60 (1 H, s), 5.68ꢀ5.90 (4 H, m), 5.00ꢀ5.20 (8 H, m), 4.30ꢀ4.70 (4 H, m), 4.05ꢀ4.15 (6 H, m),
.80ꢀ3.92 (8 H, m), 3.62ꢀ3.73 (8 H, m), 3.40ꢀ 3.55 (8 H, m), 2.50ꢀ2.64 (4 H, m), 2.90ꢀ2.10 (8H,
1
3
m), 1.65ꢀ1.84 (6 H, m), 1.42ꢀ1.62 (15 H, m); C NMR (100 MHz, CDCl ) δ 173.4, 167.4, 149.1,
3
1
5
5
32.0, 114.9, 100.0, 98.4, 96.1, 94.6, 91.7, 88.6, 68.0, 67.7, 66.5, 63.6, 62.9, 60.1, 56.1, 51.6,
+
1.2, 46.3, 30.9, 30.2, 29.0, 25.4, 24.2, 20.0. m/z (+ESI) calc. for C H N O (M) 532.5 found
2
7
36
2
9
+
33.2 ([M]+H) .
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ꢀ(10ꢀ(Allyloxycarbonyl)ꢀ7ꢀmethoxyꢀ5ꢀoxoꢀ11ꢀ(tetrahydroꢀ2H-pyran-2ꢀyloxy)ꢀ
2,3,5,10,11,11aꢀhexahydroꢀ1H- pyrrolo[2,1ꢀc][1,4]benzodiazepine-8ꢀyloxy)butanoic acid (9).
An excess of NaOH 1 M aqueous solution was added to a solution of 8 (3.8 g, 7.1 mmol, 1.0
equiv.) in MeOH (60 mL). The reaction mixture was left under a magnetic stirrer overnight until
TLC showed the completion of the reaction. MeOH was evaporated under reduced pressure
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
using a rotary evaporator and H O (30 mL) was added to the residue. Citric acid 1 M aqueous
2
solution was added until acidic pH was reached. The aqueous layer was then extracted with
EtOAc (2×50 mL). The combined organic layers were dried over MgSO and concentrated under
4
reduced pressure using a rotary evaporator, giving pure PBD protected acid core 9 (3.2 g, 87%)
as a light yellow solid (m. p. 72 °C).
1
R value (DCM:Acetone 60:40): 0.27; H NMR (400 MHz, CDCl ) δ 7.20 (2 H, s), 6.89 (1 H, s),
f
3
6.58 (1 H, s), 5.87 (2 H, d, J = 9.2 Hz), 5.72 (2 H, d, J = 9.2 Hz), 4.95ꢀ5.18 (5 H, m), 4.30ꢀ4.60
(5 H, m), 4.00ꢀ4.15 (7 H, m), 3.82ꢀ3.91 (7 H, m), 3.42ꢀ3.69 (9 H, m), 2.49ꢀ2.60 (4 H, m), 1.90ꢀ
1
3
2
.20 (12 H, m), 1.67ꢀ1.81 (4 H, m), 1.40ꢀ1.60 (8 H, m); C NMR (100 MHz, CDCl ) δ 177.6,
3
167.6, 149.8, 132.1, 131.9, 126.7, 117.3, 114.9, 110.8, 100.7, 96.0, 91.7, 88.5,67.9, 66.6, 63.6,
+
6
5
0.1, 56.1, 46.5, 31.1, 30.3, 28.8, 25.2, 24.1, 23.2, 20.0. m/z (+ESI) calc. for C H N O (M)
26 34 2 9
+
18.5 found 519.2 ([M]+H) .
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2
2
2
2
2
2
3
3
3
3
3
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3
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3
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4
ꢀ(Benzyloxy)ꢀ3ꢀmethoxybenzaldehyde (10). Benzylbromide (12.9 mL, 108.4 mmol, 1.1
equiv.) and potassium carbonate (6.8 g, 49.3 mmol, 0.5 equiv.) were added to a solution of
vanillin (15.0 g, 98.6 mmol, 1.0 equiv.) in acetone (225 mL). The suspension was stirred under
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
reflux overnight until TLC showed the completion of the reaction. At that point H O (5000 mL)
2
was added to the reaction, causing the formation of a precipitate that was filtered and
recrystallized from EtOH at 0 °C giving pure 10 (14.9 g, 62%) as a pale yellow solid (m. p. 61
°
C).
1
R value (100% DCM): 0.71; H NMR (400 MHz, CDCl ) δ 9.83 (1H, s), 7.29ꢀ7.46 (7H, m),
f
3
1
3
6
1
.98 (1H, d, J = 8.06 Hz), 5.25 (2H, s), 3.94 (3H, s); C NMR (100 MHz, CDCl ) δ 191.0,
3
53.6, 150.1, 136.0, 130.3, 128.7, 128.2, 127.2, 126.6, 112.3, 109.3, 70.9, 56.1. m/z (+ESI) calc.
+
+
For C H O (M) 242.3 found 242.9 ([M]+H) .
15 14
3
4
ꢀ(Benzyloxy)ꢀ5ꢀmethoxyꢀ2ꢀnitrobenzaldehyde (11). A solution of 10 (14.8 g, 61.1 mmol, 1.0
equiv.) in trifluoroacetic acid (18 mL) was added dropwise to a solution of KNO (7.7 g, 76.4
3
mmol, 1.25 equiv.) in trifluoroacetic acid (18 mL) kept at 0°C under magnetic stirrer. After 40
min, the reaction went to completion by TLC and LCMS. The reaction mixture was evaporated
under reduced pressure using a rotary evaporator. The residue was dissolved in EtOAc (500 mL),
and the organic phase was washed with brine (3×500 mL). The organic phases were dried over
MgSO and concentrated by rotary evaporator giving pure 11 (14.5 g, 98%) as a bright yellow
4
solid (m. p. 114 °C).
1
R value (100% DCM): 0.73; H NMR (400 MHz, CDCl ) δ 10.42 (1H, s), 7.66 (1H, s),
f
3
1
3
7
.34ꢀ7.46 (6H, m), 5.26 (2H, s), 4.0 (3H, s); C NMR (100 MHz, CDCl ) δ 187.8, 153.7, 151.4,
3
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34.85, 129.0, 128.9, 128.7, 127.6, 125.7, 110.0, 108.9, 71.6, 56.73. m/z (+ESI) calc. For
+
ꢀ
C H NO (M) 287.3 found 285.9 ([M]ꢀH) .
15
13
5
4ꢀHydroxyꢀ5ꢀmethoxyꢀ2ꢀnitrobenzaldehyde (12). A solution of 11 (14.4g, 50.1 mmol, 1
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equiv.) in acetic acid (120 mL) was heated up to 85°C, and a hydrobromic acid solution 48%
(40 mL) was added. The mixture was kept under a magnetic stirrer for 1 h. The solid product
obtained was filtered and recrystallized from hot EtOH. The solid obtained was dried in a
vacuum oven giving pure 12 (6.1 g 62%) as a yellow solid (m. p. 212 °C).
1
R value (100% DCM): 0.51; H NMR (400 MHz, DMSOꢀd) δ 11.12 (1H, br. s), 10.16 (1H, s),
f
1
3
7.50 (1H, s), 7.35 (1H, s), 3.94 (3H, s); C NMR (100 MHz, DMSOꢀd) δ 188.3, 151.8, 151.0,
+
+
1
43.7, 123.4, 111.0, 110.6, 56.31. m/z (+ESI) calc. C H NO (M) 197.2 found 198.0 ([M]+H) .
8 7 5
tertꢀButyl (3ꢀ(4ꢀformylꢀ2ꢀmethoxyꢀ5ꢀnitrophenoxy)propyl)carbamate (13). tertꢀButyl (3ꢀ
bromopropyl)carbamate (7.1 g, 29.6 mmol, 1.05 equiv.) and potassium carbonate (5.8 g, 42.3
mmol, 1.5 equiv.) were added to a solution of 12 (5.6 g, 28.2 mmol, 1.0 equiv.) in DMF (22 mL).
The suspension was stirred at 50 °C for 24 h, until TLC showed the completion of the reaction.
At that point, H O (500 mL) was added to the reaction, and the product was extracted in EtOAc.
2
The organic solvent was then evaporated using the rotary evaporator, and the compound was
purified by flash column chromatography (mobile phase hexane/EtOAc, 80:20, v/v) to give pure
13 (9.3 g, 93%) as a brown oil.
1
R value (100% DCM): 0.68; H NMR (400 MHz, CDCl ) δ 10.44 (1H, s), 7.59 (1H, s), 7.41
f
3
(1H, s), 4.23 (2H, t, J = 5.92), 4.01 (3H, s), 3.34ꢀ3.43 (2H, m), 2.05ꢀ2.13 (2H, m), 1.45 (9H, s);
1
3
C NMR (100 MHz, CDCl ) δ 188.5, 155.6, 152.6, 151.2, 143.5, 124.6, 110.0, 108.1, 77.6, 67.3,
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+
5
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6.4, 35.7, 28.8, 28.1; m/z (+ESI) calc. C H N O (M) 354.4 found 255.0 ([M]+HꢀBoc) ,
16 22 2 7
+
77.0 ([M]+Na) .
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ꢀ(3ꢀ((tertꢀButoxycarbonyl)amino)propoxy)ꢀ5ꢀmethoxyꢀ2ꢀnitrobenzoic acid (14). Compound
3 (9.2 g, 25.9 mmol, 1.0 equiv.) was dissolved in acetone (500 mL). A hot solution of 10%
potassium permanganate (250 mL) was added to the solution of 13 in a flask fitted with a
condenser. The reaction mixture was left under reflux until the reaction went to completion
(
according to TLC). At that point, the reaction mixture was cooled down to room temperature.
The brown residue formed was filtered through a celite path and washed with 600 mL hot H O.
2
A solution of sodium bisulphite 16% (400 mL) was added to the filtrate, and the pH of the
solution was adjusted to 3.5 using concentrated citric acid. The product was then extracted in
EtOAc. The organic solvent was removed using the rotary evaporator, giving 14 (8.1 g, 83%) as
a yellow solid (m. p. 107 °C).
1
R value (EtOAc:MeOH 50:50): 0.57; H NMR (400 MHz, CDCl ) δ 7.29 (1H, s), 7.08 (1H,
f
3
br.s.), 4.10 (2H, t, J = 5.29 Hz), 3.89 (3H, s), 3.31ꢀ3.38 (2H, m), 2.02 (2H, quin, J = 5.79 Hz),
1
3
1.44 (9H, s); C NMR (100 MHz, CDCl ) δ 191.1, 156.2, 152.7, 149.0, 128.7, 127.2, 110.8,
3
+
1
07.6, 79.3, 68.3, 56.5, 38.4, 28.5, 28.4. m/z (+ESI) calc. C H N O (M) 370.4 found 271.0
1
6
22
2
8
+
+
([M]+HꢀBoc) , 393.1 ([M]+Na) .
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tertꢀButyl
(S)ꢀ(3ꢀ(4ꢀ(2ꢀ(hydroxymethyl)pyrrolidineꢀcarbonyl)ꢀ2ꢀmethoxyꢀ5ꢀ
nitrophenoxy)propyl)carbamate (15). A solution was prepared by dissolving 14 (8.0 g, 21.6
mmol, 1.1 equiv.), HBTU (18.6 g, 49.0 mmol, 2.5 equiv.) and DIPEA (10.2 mL, 58.8 mmol, 3
equiv.) in DMF (250 mL). The solution was left under a magnetic stirrer for 20 min. Then
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(S)ꢀ(+)ꢀ2ꢀpyrrolidinemethanol (1.93 mL, 19.6 mmol, 1.0 equiv.) was added to the mixture which
was kept under magnetic stirring at 50 °C for 15 h until TLC showed the completion of the
reaction. At that point water was added to the reaction mixture, and the product was extracted in
EtOAc. The organic phase was then, washed with brine and a saturated NaHCO solution. The
3
organic solution was concentrated using a rotary evaporator, and the product was purified by
flash column chromatography (mobile phase: from 100% EtOAc to EtOAc/MeOH, 98:2, v/v) to
afford pure 15 (3.2 g, 35%) as brown solid (m. p. 68°C).
1
R value (EtOAc:MeOH 90:10): 0.42; H NMR (400 MHz, CDCl ) δ 7.67 (1H, s), 6.79 (1H, s),
f
3
4.16 (2H, t, J = 5.92 Hz), 3.97 (3H, s), 3.85ꢀ3.91 (1H, m), 3.75ꢀ3.82 (1H, m), 3.33ꢀ3.40 (2H, m),
13
3
.15 (2H, t, J = 6.55), 2.12ꢀ2.21 (2H, m), 2.03ꢀ2.09 (2H, m), 1.68ꢀ1.93(4H, m), 1.44 (9H, s); C
NMR (100 MHz, CDCl ) δ 200.7, 156.0, 154.7, 153.2, 148.3, 127.9, 109.0, 108.0, 79.2, 68.4,
3
+
61.6, 56.7, 49.5, 38.5, 29.1, 28.5, 24.4. m/z (+ESI) calc. C H N O (M) 453.5 found 454.1
2
1
31
3
8
+
(
[M]+H) .
tertꢀButyl
(S)ꢀ(3ꢀ(5ꢀaminoꢀ4ꢀ(2ꢀ(hydroxymethyl)pyrrolidineꢀ1ꢀcarbonyl)ꢀ2ꢀ
methoxyphenoxy)propyl)carbamate (16). A catalytic amount of Pd/C (10% w/w) was added to
a solution of 15 (3.2 g, 7.1 mmol, 1.0 equiv.) in EtOH (30 mL). The reaction mixture was
hydrogenated in a Parr hydrogenator at 40 psi overnight until TLC showed the completion of the
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