New Broad-Spectrum Antibiotics Containing a Pyrrolobenzodiazepine Ring with Activity against Multidrug-Resistant Gram-Negative Bacteria

Article abstract of DOI:10.1021/acs.jmedchem.0c00328

It is urgent to find new antibiotic classes with activity against multidrug-resistant (MDR) Gram-negative pathogens as the pipeline of antibiotics is essentially empty. Modified pyrrolobenzodiazepines with a C8-linked aliphatic heterocycle provide a new class of broad-spectrum antibacterial agents with activity against MDR Gram-negative bacteria, including WHO priority pathogens. The structure-activity relationship established that the third ring was particularly important for Gram-negative activity. Minimum inhibitory concentrations for the lead compounds ranged from 0.125 to 2 mg/L for MDR Gram-negative, excluding Pseudomonas aeruginosa, and between 0.03 and 1 mg/L for MDR Gram-positive species. The lead compounds were rapidly bactericidal with >5 log reduction in viable count within 4 h for Acinetobacter baumannii and Klebsiella pneumoniae. The lead compound inhibited DNA gyrase in gel-based assays, with an IC50 of 3.16 ± 1.36 mg/L. This study provides a new chemical scaffold for developing novel broad-spectrum antibiotics which can help replenish the pipeline of antibiotics.

Full text of DOI:10.1021/acs.jmedchem.0c00328

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Article
New broad-spectrum antibiotics containing a pyrrolobenzodiazepine
ring with activity against multidrug resistant Gram-negative bacteria.
Pietro Picconi, Charlotte Hind, Kazi Sharmin Nahar, Shirin Jamshidi, Lucia Di Maggio, Naima Saeed,
Bonnie Evans, Jessica Solomons, Matthew E. Wand, J Mark Sutton, and Khondaker Miraz Rahman
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00328 • Publication Date (Web): 09 Jun 2020
Downloaded from pubs.acs.org on June 10, 2020
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New broad-spectrum antibiotics containing a pyrrolobenzodiazepine ring with activity
against multidrug resistant Gram-negative bacteria.
1
†
2†
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Pietro Picconi , Charlotte K. Hind , Kazi S. Nahar , Shirin Jamshidi , Lucia Di Maggio , Naima Saeed ,
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Bonnie Evans , Jessica Solomons , Matthew E. Wand , J. Mark Sutton and Khondaker Miraz Rahman .
1Institute of Pharmaceutical Science, King’s College London, London, SE1 9NH, UK.
2Public Health England, National Infections Service, Porton Down, Salisbury, Wiltshire, SP4 0JG UK.
†
These authors contributed equally to the study.
*
To whom correspondence should be addressed
For KMR: e-mail k.miraz.rahman@kcl.ac.uk
For JMS: e-mail mark.sutton@phe.gov.uk
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Abstract
It is urgent to find new antibiotic classes with activity against multidrug resistant (MDR) Gram-negative
pathogens as the pipeline of antibiotics is essentially empty. Modified pyrrolobenzodiazepines (PBDs) with a
C8-linked aliphatic-heterocycle, provide a new class of broad-spectrum antibacterial agents with activity
against multidrug resistant Gram-negative bacteria, including WHO priority pathogens. The structure-
activity-relationship established that the third ring was particularly important for Gram-negative activity.
Minimum inhibitory concentrations for the lead compounds ranged from 0.125 – 2 mg/L for MDR Gram-
negative, excluding Pseudomonas aeruginosa, and between 0.03 - 1 mg/L for MDR Gram-positive species.
The lead compounds were rapidly bactericidal with >5-log reduction in viable count within 4 hours for
Acinetobacter baumannii and Klebsiella pneumoniae. The lead compound inhibited DNA gyrase in gel-
based assays, with an IC50 of 3.16 +/- 1.36 mg/L. This study provides a new chemical scaffold for
developing novel broad-spectrum antibiotics which can help replenish the pipeline of antibiotics.
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Introduction
The fight against bacterial infections is rapidly being lost as microbes develop multiple mechanisms
to evade antibiotics. Antimicrobial resistance is one of the most significant health concerns
worldwide. It is estimated that 25,000 people die each year in Europe and 23,000 in the United
States because of resistant bacterial infections, with more than 2 million infections caused by drug
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resistant bacteria . These values are expected to reach 10 million deaths globally by 2050,
overtaking cancer and heart disease as the principal cause of death if no effective measures can be
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developed . Of particular concern is the rapid emergence of multidrug resistant (MDR) Gram-
negative pathogens, defined as having resistance to at least 3 frontline antibiotic classes, and
increasing reports of essentially pan-drug resistant (PDR) isolates in the clinic. 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 (2007) are active only
3
against Gram-positive pathogens . This makes it imperative that we discover new antibacterial
drugs with a broad spectrum of activity or specificity for Gram-negative species, in order to help
solve the crisis and provide new treatment options for MDR and PDR strains.
Figure 1: Reported antibacterial PBDs and structural modification employed to obtain compounds with
activity against Gram-negative bacteria.
PBDs are naturally occurring molecules produced by Streptomyces bacteria whose family members
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include anthramycin and tomaymycin . PBDs have a soft N10-C11 imine electrophile that can
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covalently bond to guanine bases . They have been extensively studied as anticancer agents
and, more recently, a large number of PBDs are being clinically evaluated as payloads for antibody
drug conjugates (ADCs) demonstrating the broad therapeutic utility of this chemical class12, 13. We
recently described a series of C8-linked PBD monomers that showed activity against Gram-positive
MDR strains14, 15, which were able to inhibit Staphylococcus gyrase in a biochemical assay. It has
been reported in the literature that the reason PBDs are only active against Gram-positive bacteria is
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due to their inability to cross Gram-negative membranes . We designed and synthesized a new
generation of C8-PBD monomers with an aliphatic third ring that showed, for the first time, notable
activity against Gram-negative bacteria. Introduction of the aliphatic third ring improved the
prokaryotic selectivity of the molecules and reduced eukaryotic toxicity of the molecules, as the
third ring interfered with the DNA binding ability of these molecules.
The synthesized compounds showed broad spectrum activity against both MDR and PDR clinical
ESKAPE strains with MICs ranging from 0.03 to 1 mg/L against Gram-positive species and 0.125
to 32 mg/L against Gram-negative species. The compounds demonstrated a rapid bactericidal mode
of action against both Gram-positive and Gram-negative species. The mechanism of action of this
type of compound was explored both in silico and through a range of microbiological techniques.
These methods suggest that inhibition of DNA gyrase is one of the main mechanisms of action,
although the possibility of activity through other mechanisms cannot be completely ruled out. The
efflux liability and entry into Gram-negative species of the chemical series and the likelihood of the
emergence of resistance was also studied. Importantly, the relative lack of DNA binding, as
observed by FRET-based DNA melting, combined with the absence of eukaryotic toxicity,
highlights the potential therapeutic value of this new type of C8-linked pyrrolobenzodiazepine
monomers as antibacterial compounds.
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Figure 2: Structure of control compound 1,2 and structures of final compounds 3-17
Results and Discussion
Design and Synthesis of PBD monomers
Three C8-linked biaryl-PBD monomers with reported activity against Gram-positive species were
selected as the starting point for the medicinal chemistry modification (Figure 1). These monomers
contain C8-substituents with two heteroaromatic or biaryl building blocks connected via amide
bonds. The side chains were elongated with a third aliphatic heterocycle resulting in an
unconventional class of PBD derivatives that have never been investigated before. Initially, three
nitrogen-containing aliphatic heterocycles, thiomorpholine, morpholine and piperidine, were
introduced and this was followed by coupling the heteroaromatic ring containing PBD monomers
with diethyl and dimethylamine. The structural modification aimed to modify the physicochemical
properties of the molecules and occupy a different chemical space as polar compounds have
1
7
traditionally shown better activity against Gram-negative bacteria . The secondary aim was to
reduce the DNA binding affinity of the compounds to minimise eukaryotic toxicity, as the
replacement of an aromatic group with an aliphatic ring was expected to reduce both interaction and
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fit of the molecules within the DNA minor groove. A total of 15 target compounds were designed
and successfully synthesised to explore the structure-activity relationship (SAR) (Figure 2) and
understand the role of the third ring in extending the antibacterial activity of the compounds. C8-
linked monomers 1 (GWL-78) and 2, in which the heteroaromatic pyrrole ring replaced the third
aliphatic ring, were used as control to compare their activity against Gram-negative bacteria.
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Figure 3: Synthetic approach to obtain antibacterial PBD monomers
The target compounds were synthesised using a convergent strategy based on literature reported
procedures. The synthetic process is based on the C8 derivatization of an alloc, THP-protected
pyrrolobenzodiazepine core with different polyamide side chains. The protected PBD core 26 was
synthesised in nine steps starting from vanillin accordingly to a reported procedure from our group.
The synthetic methods and characterization of intermediates that lead to 26 are included in the
supporting information file (chemistry section). The three-component side chains were synthesised
by sequential addition of the different constituents via amide coupling. Alternative strategies based
either on the use of protecting groups or functional groups interconversion, were employed to avoid
side reactions. For the synthesis of benzofused containing lateral chains (scheme 1) the initial step
involved the formation of an amide bond between Boc-protected N-methyl pyrrole carboxylic acid
and the corresponding 5-amino benzofused methyl ester to give intermediates 27 and 28. The esters
derivative underwent basic hydrolysis to give the carboxylic acids that were successfully coupled
with diethylamine, piperidine, morpholine or thiomorpholine to give the complete C8-side chains
31-38. The coupling system EDCI/DMAP was used as reagent for the formation of the amide bonds
with good yield with the only exception of diethylamine products. In this case the coupling between
the carboxylic acid derivatives of 27 and 28 and the secondary amine was performed using a
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propylphosphonic anhydride (T
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P) sustained dehydration reaction in the presence of DIPEA. The
synthesis of dimethylamine derivative 29 and 30 was accomplished with direct coupling between
Boc-protected N-methyl pyrrole carboxylic acid and corresponding commercially available 5-amino
benzofused dimethyl carboxamides. The coupling system EDCI/DMAP was used for formation of
the amide bond similarly to previous examples.
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Scheme 1: Synthesis of C8 benzofused lateral chains 29-38. Reagents and conditions: a) EDCI,
DMAP, DMF, r.t., overnight; b) NaOH, MeOH, H
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O, r.t., overnight then EDCI, DMAP, DMF, r.t.,
P, DIPEA, DMF, r.t., overnight
overnight; c) NaOH, MeOH, H 0, r.t., overnight then T
2
3
Two different approaches were employed, depending on the aliphatic substitution of the products,
for the synthesis of methylpyrrole benzenamine (MPB) containing lateral chains (scheme 2). In the
case of piperidine, morpholine and thiomorpholine substitution, the MPB central building block 39
was synthesized via Suzuki coupling between Br-methyl pyrrole carboxylic acid and the para-Boc-
protected benzenamine boronic acid. After acid catalysed Boc-deprotection, the aniline derivative
of 39 underwent amide coupling reaction with Boc-protected N-methyl pyrrole carboxylic acid. The
reaction was performed with EDCI/DMAP coupling system to give intermediate 40. The synthetic
process was completed by basic hydrolysis of the methyl ester moiety and successive EDCI/DMAP
sustained amide coupling reaction with piperidine, morpholine or thiomorpholine to give complete
side chain 41-43 respectively. This synthetic approach failed in the formation of dimethyl and
diethyl amide derivatives due to the poor yield of amide coupling reaction of the considered
secondary amines using traditional coupling reagents.
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For this reason, a reverse-direction synthetic process, with aliphatic derivatization as the first step,
was applied for the synthesis of dimethyl and diethyl substituted MPB containing side chains.
Specifically, the synthetic route was modified with the initial synthesis of dimethyl and diethyl N-
methyl Br pyrrole intermediates 44 and 45 via amide bond formation based on acyl chloride
formation. Commercially available 4-Br-N-methyl pyrrole methyl ester was hydrolysed and the
corresponding carboxylic acid was activated to acyl chloride using oxalyl chloride in the presence
of catalytic DMF. The reaction mixture was then treated with the corresponding secondary amine to
give the desired product.
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Intermediates 44 and 45 underwent Suzuki coupling reaction with para-Boc-protected benzenamine
boronic acid to give MPB modified derivatives 46 and 47. The obtained intermediates were
deprotected via acid treatment and then successfully coupled with Boc-protected N-methyl pyrrole
carboxylic acid to give final complete lateral chains 48-49.
Scheme 2: Synthesis of C8 MPB containing lateral chains 41-43, 49-49. Reagents and conditions: a)
Tetrakis(triphenylphospine)Pd, K
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CO
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DMAP, DMF, r.t., overnight; c) NaOH, MeOH, H
overnight; d) oxalyl chloride, DMF, dry DCM, Et
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r.t., 30 minutes then EDCI, DMAP, DMF, r.t., overnight.
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Finally, the convergent synthetic strategy was completed with the coupling of C8-side chains 29-38,
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1-43, 48-49 to the PBD core 26 using EDCI/DMAP mediated amide coupling. This reaction was
followed by palladium catalysed deprotection of allyl carbamate moiety and concerted THP
deprotection of the alchool moiety to generate the reactive N10-C11 imine bond, giving the final
products 3-17 (scheme 3).
Scheme 3: Synthesis of PBD monomer 3-17. Reagents and conditions: a) HCl 4M in dioxane, MeOH,
r.t., 30 minutes then EDCI, DMAP, DMF, r.t., overnight; b) Tetrakis(triphenylphospine)Pd,
piperidine, triphenylphospine, DCM, r.t., 30 minutes.
Screening of PBDs to define SAR
Bacterial panels containing drug sensitive and MDR strains of Klebsiella pneumoniae,
Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus spp.
were used as an initial screen to determine the efficacy of the PBD compounds in this series and
establish a structure-activity relationship (SAR) (Tables 1 and 2) (resistance profiles for all strains
are in Table S1). All the designed PBD molecules had a 3rd aliphatic ring or a ring fragment and
contained pyrrole as the first unit followed by methylpyrrole benzenamine (MPB), benzofuran or
benzothiophene as the second unit. Against the Gram-negative species tested, the control PBD
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molecule 2 did not show any notable activity, with minimum inhibitory concentrations (MICs)
rd
ranging from 64 to >128 mg/L (Table 1). However, the aliphatic 3 ring containing C8-linked
PBDs (compounds 3 - 17) showed notable improvement in activity against Gram-negative bacteria,
except P. aeruginosa, with MICs as low as 0.5 mg/L against MDR A. baumannii and K.
pneumoniae strains.
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Table 1: Activity of the synthesized compounds against Gram-negative bacteria (mg/L)
A. baumannii
K. pneumoniae
P. aeruginosa
Resistance profile
Compound
S
MDR
S
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S
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ATCC
AYE
M6
NCTC
13368
>128
128
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PA01
NCTC
13437
>128
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>128
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>128
>32
>32
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64
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>32
>32
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0.5
2
1
2
9
2
8
64
10
8
8
128
64
>128
2
128
8
64
1
1
1
1
1
2
3
4
8
8
32
2
2
2
64
32
4
4
8
128
>128
>128
>128
>32
>128
>128
>128
>128
>32
64
2
32
2
>128
16
2
>128
>128
32
15
1
1
6
7
4
2
2
2
4
>32
Compounds bearing GC-targeting the methylphenylbenzenamine (MPB) substituent as a central
group (compounds 13, 15 to 17) showed good efficacy against A. baumannii and K. pneumoniae
but were inactive against P. aeruginosa. Compounds containing benzofuran as the second ring were
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found to be more active than the corresponding benzothiophene series. The structural modification
of the 3rd aliphatic ring showed a significant influence on the activity of the compounds. In all
cases compounds with thiomorpholine with the third ring (example compounds 7, 12 and 17) had
MICs of ≤ 2 mg/L against the A. baumannii strains tested. The presence of a second heteroatom in
the aliphatic 3rd ring improved activity, but intriguingly, shortening the 3rd ring to a dimethyl or
diethyl side chain (example compounds 3, 4, 8, 9 and 13) retained activity, with dimethyl analogues
showing better activity than their diethyl counterparts, except compounds containing the MPB ring
in which diethyl substitution, compound 14, resulted in loss of activity.
4
5
6
7
8
9
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
Against the Gram-positive species tested, all compounds, including the control PBD molecule,
showed excellent activity, with MICs ranging from 0.03 to 1 mg/L (Table 2).
Table 2: Activity of the synthesized compounds against MDR Gram-positive bacteria. MICs are
expressed in mg/L.
E. faecium
MDR (VRE)
E. faecalis
S (VSE)
S. aureus
MDR (MRSA)
Resistance profiles
Compound
S (MSSA)
NCTC
6571
NCTC
NCTC
12201
0.5
NCTC
775
NCTC
13616
1
NCTC
13277
2
1
2204
1
2
0.5
1
0.5
<0.125
<0.125
<0.125
<0.125
<0.125
<0.125
0.03
<0.125
0.5
<0.125
<0.125
0.25
0.5
1
<0.125
<0.125
<0.125
<0.125
<0.125
<0.125
0.03
3
<0.125
0.25
<0.125
0.5
4
<0.125
<0.125
<0.125
<0.125
0.03
5
0.25
0.25
0.5
6
<0.125
<0.125
0.03
<0.125
<0.125
0.03
0.25
0.25
0.06
1
7
8
9
1
0.5
1
1
0.5
10
<0.125
<0.125
<0.125
<0.12
0.25
<0.125
<0.125
<0.125
<0.12
0.25-0.5
<0.03
<0.12
<0.03
0.25
0.5
1
<0.125
<0.125
<0.125
<0.12
1
1
1
2
<0.125
<0.125
<0.12
0.5
<0.125
<0.125
<0.12
1
0.5
<0.125
<0.12
1
13
1
1
1
1
4
5
6
7
0.25-0.5
<0.03
<0.03
<0.12
<0.03
0.12
0.06
0.12
<0.12
0.06
<0.12
<0.03
<0.12
<0.03
<0.12
<0.03
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6
Two compounds (7 and 8) with promising activity against both Gram-negative and Gram-positive
strains were selected for further screening against a wider panel of bacteria including MDR Gram-
negative strains of E. coli, A. baumannii, K. pneumoniae and Burkholderia cepacia/cenocepacia
complex. The K. pneumoniae strains selected for the study were resistant against most marketed
antibiotics (Table 3 and Table S1) including one essentially PDR strain (51851). Similarly, the A.
0
1
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6
7
8
9
0
1
2
3
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7
8
9
0
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9
0
1
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8
9
0
1
2
3
4
5
6
7
8
9
0
baumannii strains and E. coli strains containing ESBL and carbapenemase genes were resistant to 6
to 8 clinically used antibiotics (Table S1). Both compounds were found to be active against almost
all strains tested except B. multivorans C1962 against which compound 8 was found to be inactive
(defined here as an MIC >32 mg/L). The levels of activity observed, considering the high levels of
intrinsic or acquired resistance in many of these strains, are exceptional, with low and in some cases
sub µM MICs. Notably, compound 7 exhibited a range of MICs from 0.25 to 2 mg/L for K.
pneumoniae, 0.125 to 1 mg/L for A. baumannii/baylyi, 0.5 to 1 mg/L for E. coli and 0.125 to 2
mg/L for B. cepacia complex. These values are exciting and suggest that this represents a promising
new chemical scaffold that can be further developed as broad-spectrum antibiotics.
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Table 3: Activity of compounds 7 and 8 against an extended panel of MDR Gram-negative bacteria
4
5
6
7
8
9
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
(MIC in mg/L).
7
8
Species
Strain
MIC
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
NCTC 13368
M6
2
1
32
1
NCTC 13438
NCTC 13439
NCTC 13443
16
0.5
2
4-8
4-8
16
1
K. pneumoniae
2
0.25
1
4
5
6704
1851
4
2
4
MGH 78578
ATCC 17978
AYE
1
4-8
2
0.5
1
2
NCTC 13424
ADP1
0.5
0.125
0.25
0.5
0.5
1
1
1
A. baumannii
NCTC 13302
UKA2
4
4
UKA7
4
W1
4
CFI_017_VIM4
CFI_033_NDM
CFI_160_KPC2
CFI_161_NDM_5_OXA_18
1
1
2
0.5
0.5
0.5
1
4
1
E. coli
LEC001
1
0.5
0.5
1
2
1
3
19238/UR
NCTC 12923
C1576
2
16
>32
1
B. multivorans
B. cenocepacia
C1962
2
K56-2
2
CEP509
2
8
NCTC 17765
LMG 17997
NCTC 10743
0.125
0.125
1-2
1
B. cepacia
0.25
16
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0
1
2
3
4
5
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7
8
9
0
1
2
3
4
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6
7
8
9
0
Figure 4: Rapid bactericidal kill by synthesised compounds 7 and 8, with occasional breakthrough
resistance in some drug-strain combinations. The data are the average of three independent
experiments and error bars represent the standard deviation from the mean.
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Defining the mode of action of PBDs through time-kill assays
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9
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1
1
1
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2
2
2
2
2
2
2
2
2
2
3
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5
5
5
5
6
The mode of action of compounds 7 and 8 was explored using time-kill assays. Three K.
pneumoniae (NCTC 13368, 16 and NCTC 13438) and three A. baumannii (AYE, ATCC 17978 and
NCTC 13424) strains were selected for time-kill analysis (Figure 4). These strains were selected to
provide a range of drug resistance profiles from a relatively drug-sensitive strain (ATCC 17978),
through to MDR strains with multiple resistance mechanisms (NCTC 13438, 16 and NCTC 13424)
for each species. Compound 8 was rapidly bactericidal in all strains tested; the viable count was
below the limit of detection (LOD) within 4 hours and continued to be below the LOD for the full
assay for all K. pneumoniae strains challenged with 8, indicating that there was no resistance to this
compound within the 24 hour period of the assay. A small number of viable cells were observed at
the end of 24 hours for A. baumannii strains AYE and NCTC 13424 (Figure 4). Subsequent testing
with 8 on these populations showed no increase in MIC above wild-type levels, and these
populations were not considered to be resistant (results not shown). Compound 7 was also
bactericidal against the majority of strains tested, although the speed of killing was slower than
compound 8 in a number of cases (NCTC 13438, AYE, NCTC 13424). In the case of K.
pneumoniae NCTC 13368, which had a much higher intrinsic resistance to compound 7, only a
small reduction in viable count was observed at 6 hours, with bacterial numbers returning to the
levels of the untreated control by 24 hours, in two out of three replicate experiments. When these
bacteria were isolated, passaged 10x in the absence of selection and re-assayed, the MICs increased
for both compound 7 (32 to ≥128 mg/L) and compound 8 (2 to >32 mg/L). No increases in MIC
were observed for other antibiotics, including aminoglycosides, fluoroquinolones and -lactams,
except for an increase in colistin resistance of 4 to 32mg/L in one of the two isolates (Table S2).
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
Evaluation of the influence of efflux and influx on the activity of the PBD compounds
2
+
Two efflux pump inhibitors, PAβN (used in the presence of Mg to prevent non-specific effects on
membrane permeability) 18 which competitively inhibits specific RND efflux pumps and CCCP,
which inhibits the proton motive force, were used to assess the impact of efflux on the activity of
the lead compounds against wild-type bacterial strains, together with the resistant mutants isolated
from the time-kill experiments.
The result (Table 4) suggests that both compounds are substrates for RND-family efflux pumps, but
the presence and/or expression of specific efflux pumps differs between the strains tested. To
investigate this further, specifically in the P. aeruginosa strain PAO1, transposon mutants in
various RND efflux pumps were obtained from the Manoil collection (Univ. Washington; Table
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2
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2
2
2
2
2
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5
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S3). Transposon mutants in any component of the tripartite RND efflux system MexAB-OprM
reduced the MIC by ≥8-fold for both compounds 7 and 8. The MIC of compound 8 was also
decreased ≥4 fold by transposons in MexCD but not its outer membrane component OprJ; no such
effect was observed for compound 7 with transposon mutants in any component of this pump.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
6
7
8
9
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1
2
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
A membrane permeabiliser, polymyxin b nonapeptide (PMBN), used at a concentration which did
not affect bacterial growth in its own right, was also used to understand to what extent efficacy for
compounds with high MICs were impacted by entry of the compounds into the cell. Effects in P.
aeruginosa were particularly marked, with addition of PMBN reducing the MIC of 8 against the
MDR strain NCTC 13437 from >32 mg/L to 0.03 - 1.0 mg/L, a reduction of between 32- and 1000-
fold. Similarly, a reduction in MIC against PAO1 was observed between 4- and ≥16-fold for
compound 8. PMBN also reduced the MIC of compound 7 against an extended panel of P.
aeruginosa strains from >32 to 1 – 8 mg/L (8- to ≥64 fold). The activity of these compounds in P.
aeruginosa strains in the presence of a permeabilizer shows that these compounds are effective at
killing strains once they are able to cross the outer membrane barrier. This offers the opportunity to
optimise the active compounds using medicinal-chemistry modifications or to explore active
delivery mechanisms to allow the compounds to permeate the Pseudomonas membrane.
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Table 4: Inhibiting efflux and increasing influx potential reduces the MIC for selected compounds
in strains of K. pneumoniae, A. baumannii and P. aeruginosa. A four-fold change in MIC in the
presence of efflux inhibitors or membrane permeabilisers was defined as significant (highlighted by
shading). All values are given in mg/L. These data are representative of three or more independent
experiments. ND = Not done.
4
5
6
7
8
9
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
Efflux inhibition
Outer membrane
permeabilisation
PMBN
Strain
Compound
MIC
2
CCCP
2
PAβN
0.5
8
NCTC 13368
8
7
7
7
8
7
8
7
8
7
8
7
8
7
8
7
8
7
ND
8
32
128
>128
1
>32
128
>128
0.06-0.5
0.12-1
0.5
NCTC 13368-7-R1
NCTC 13368-7-R2
16
32
128
2
8
64
0.25
0.5
1
2
NCTC 13438
AYE
0.5
4
0.25
4
ND
ND
ND
ND
ND
ND
ND
ND
2-16
0.25-8
<0.03-1
2-32
4
1
0.125
4
0.25
2
4
ATCC 17978
NCTC 13424
PA01
0.5
2
0.25
2
0.25
2
0.5
2
0.25
1
0.25
2
>32
128
>32
>32
>32
>32
>32
>32
2
32
16
>32
NCTC 13437
Understanding the mechanism of action.
Effect of the aliphatic third ring substitution on DNA binding and eukaryotic toxicity of the
synthesized compounds.
The cytotoxicity of C8-linked PBD monomers is linked with their ability to stabilise DNA
7
sequences. A FRET-based DNA melting assay was used to explore the effect of the aliphatic third
ring on the DNA stabilisation property of the newly synthesized molecules. Two fluorophore
labelled oligonucleotides (Sequence F1: 5’-FAM-TAT-ATA-TAG-ATA-TTT-TTT-TAT-CTA-
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1
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2
2
2
2
2
2
2
2
2
2
3
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TAT-ATA-3’-TAMRA, and Sequence-F2: 5’-FAM-TAT-AGA-TAT-AGA-TAT-TTT-ATA-TCT-
ATA-TCT-ATA-3’-TAMRA) with preferred PBD binding sites were used in the assay, and the
1
9
stabilisation was compared with a previously reported C8-PBD monomer; GWL-78 (compound
1
) and the control compound 2 with the third heteroaromatic ring. In all cases, compounds with
rd
0
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1
2
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0
1
2
3
4
5
6
7
8
9
0
aliphatic 3 moiety showed between 3- to 4-fold lower Tm values at both 5:1 and 1:1 ligand-DNA
rd
ratio compared to the control compounds, showing that the introduction of the aliphatic 3 moiety
reduced the ability of these compounds to interact with and stabilize the DNA sequences (Table
S4). The eukaryotic toxicity of the synthesised PBD derivatives was tested at a single concentration
(20 µM) using the MTT viability assay on WI-38 fibroblast cell line. The control compounds, 1
and 2, showed potent cytotoxicity with only 35% and 32% of cells viable after 24 hours. However,
the newly synthesized compounds showed notably less toxicity, with >85% of cells viable after 24
hours treatment with the molecules (Figure S1). Considering an average molecular weight of 700
Da for the synthesized PBD monomers, the concentration used for the test (20 µM) corresponds to a
concentration of 14 mg/L, which is approximately 100 times the average MIC reported for Gram-
positive strains and almost 10 times the average MIC reported for Gram-negative strains for active
compounds. The obtained results confirmed the presence of a selective toxicity profile towards
prokaryotic cells for the PBD-C8 conjugates, confirming the possibility of further development of
this class of compounds as antibacterial agents.
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Inhibition of DNA Gyrase
1
4
The C8-linked PBDs have been recently reported as DNA gyrase inhibitors . This was further
explored by docking compound 7 and 8 with the bacterial gyrase from S. aureus (PDB ID 2XCT)
and E. coli (PDB ID 6RKS) (Figure 5 and Figure S2). Both compounds bound to the ligand binding
domain of DNA gyrase A and showed interaction with several amino acid residues through
hydrogen bonds and hydrophobic interactions. The best pose of compound 7 gave a ChemScore of
0
1
2
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9
0
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0
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8
9
0
2
7.62 and a binding affinity of -31.67 kcal/mole, and the values for compound 8 was comparable
with a ChemScore of 27.35 and binding affinity of – 30.70 kcal/mole in the case of DNA gyrase A
from S. aureus. For the DNA gyrase A from E. coli the ChemScores were 25.16 and 22.01 while
the free energy of binding values were 30.46 kcal/mole and -25.51 kcal/mole for compounds 7 and
8
, respectively. The 2D models shown in Figure S3 suggests compound 7 forms six hydrogen
bonds within the ligand binding domain of DNA gyrase A from S. aureus, and five hydrogen bonds
within the ligand binding domain of DNA gyrase A from E. coli. Similarly, compound 8 forms
five hydrogen bonds within the ligand binding domain of DNA gyrase A from S. aureus, and four
hydrogen bonds within the ligand binding domain of DNA gyrase A from E. coli. Both compounds
show notable hydrophobic interaction within the binding site (Tables S5 to S8). The interaction of
compounds 7 and 8 with the ligand binding sites of Gyrase A suggests the inhibition of Gyrase A
plays a crucial role in the antimicrobial activity of this class of compound.
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Figure 5: Molecular model showing the location of the binding pocket and the interaction of
compound 7 with A) DNA gyrase A from S. aureus; B) DNA gyrase A from E. Coli.
We have recently shown that PBD monomers caused inhibition of DNA gyrase in a rapid assay
1
4
format . A gel based assay was used here to evaluate whether compounds 7 and 8 were also able to
inhibit DNA gyrase in vitro. In assays using purified S. aureus DNA gyrase, compound 8 showed
an IC50 of 3.16 ± 1.36 mg/L compared to ciprofloxacin IC50 of 13.05 ± 1.36 mg/L (Figure 6). It
was not possible to determine an IC50 for compound 7 as solubility issues prevented the use of high
enough concentrations of compound to achieve > 50% inhibition (at a concentration of 32 mg/L).
Against the E. coli DNA gyrase it was not possible to reach a concentration high enough to achieve
greater than 50% inhibition for either compound 7 or 8, again due to solubility issues. The relative
inhibition of the Gram-positive vs. Gram-negative gyrase correlates with the relatively higher MICs
of Gram-negative bacteria species and this supports the idea that the novel PBDs described here
work through inhibition of DNA gyrase.
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Figure 6: Inhibition of gyrase enzyme from A) S. aureus and B) E. coli by synthesized compounds
7 and 8 and the control, ciprofloxacin. Results represent the mean of three independent experiments,
the error bars are the standard deviation of the mean.
Whilst we have evidence of inhibition of DNA gyrase activity, for both Gram-positive and Gram-
negative DNA gyrase, this does not preclude the possibility that the PBDs cause inhibition via
binding to DNA and/or a DNA-Gyrase complex. To further define the mechanism of action, a
panel of fluorescent E. coli reporter strains were used to understand the profile of cellular responses
to PBDs and compare these with molecules with known mechanisms of action. A panel of strains
2
0
derived from a comprehensive library of E. coli promoter fusions were used . Specific reporters
2
1
for compounds affecting DNA metabolism were selected, in line with previous studies and
validated with DNA gyrase inhibitors (fluoroquinolones, novobiocin), compounds known to bind
DNA (mitomycin C, PBD control) and control compounds affecting other cellular targets
(doxycycline, ampicillin, rifampicin). The four promoter fusions showed the expected induction
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with fluoroquinolones (specifically levofloxacin, ciprofloxacin, norfloxacin and moxifloxacin) and
the quinolone antibiotic nalidixic acid, associated with activation of the bacterial SOS response as a
result of the poisoning of the DNA gyrase. The level of fold induction was similar between the
known gyrase inhibitors, with fold inductions ranging from 20-35 fold for recA, 5-13 fold for lexA,
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4-32 fold for recN and 17-41 fold for sulA (Figure 7). Whilst compound 7 and 8 both showed
activation of the same 4 promoter fusions, the levels were significantly lower for each of the genes
8-10 fold recA, 5-9 fold lexA, 6-11 fold recN and 6-7 fold sulA). By comparison, the three
(
antibiotics known not to induce the SOS response, doxycycline, rifampicin and ampicillin, showed
no induction of any of the four promoters above the level of the control strain. Interestingly, three
compounds showed similar levels of activation of the promoters to the two PBDs. Two of these are
2
2
known to be DNA binding molecules, mitomycin C and the control PBD dimer DSB-120 , whilst
the third, novobiocin, inhibits DNA gyrase by interaction with the ATP-binding site on the GyrB
subunit. These results suggest that the PBDs act by interfering with DNA metabolism, in a way that
is clearly different from fluoroquinolones. This is consistent with MIC data which shows no
significant differences between fluoroquinolone sensitive and resistant strains. Further studies will
be required to delineate the specific interactions with DNA and/or individual gyrase subunits and
the specific mechanisms of inhibition.
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Figure 7: Reporter assay to explore the mechanism of action of compounds 7 and 8. Data is the
mean of three independent experiments and error bars represent the standard deviation of the mean.
2
-way ANOVA and Dunnett’s multiple comparisons test were performed and included here for
reporter strain recA, all compounds compared to ciprofloxacin **** p < 0.0001.
Emergence of resistance – Mutation Frequency
The mutation frequency of the most active compound 8 was tested against representative strains of
the three Gram-negative pathogens in comparison with ciprofloxacin. At 4 X MIC, mutation
-8
-8
frequencies of <4.3 x 10 and <1.5 x 10 were measured across three replicate experiments for
NCTC 12923 and ATCC 17978 respectively. In both cases, the values were significantly lower than
-7
-7
the control antibiotic, ciprofloxacin (4.4 x 10 for NCTC 12923 and 8.4 x 10 for ATCC17978).
This was not the case in the K. pneumoniae isolate M6, where the mutation frequency was 1.0 x 10-
6
-7
for compound 8, compared to 8.9 x 10 for ciprofloxacin. This higher mutation frequency is
reflected in the time-kill experiments, where resistance was only seen in a single K. pneumoniae
strain (NCTC 13368), following exposure to concentrations above the MIC and not in A.
baumannii.
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The mutations associated with resistance in NCTC 13368 were identified by whole genome
sequence analysis of stable resistant isolates. In one of the time-kill repeats, the resistant strains
showed the deletion of a single base in position 50 of a gene encoding a predicted nucleoside-
specific channel-forming protein (KPN_RS01945), termed Tsx. The mutation caused a frameshift
in the tsx gene and was predicted to result in a truncated 42 amino acid protein. A second SNP was
also identified in another uncharacterised permease gene, leading to an amino acid change M137K.
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A clean transposon mutant (tnkp1_lr150124p14q189) in tsx, in K. pneumoniae strain MKP103, also
showed elevated resistance to compounds 7 and 8, with respect to their parental strain (compound 7
from 32 mg/L in wild-type to >128 mg/L for tsx transposon, and for compound 8 from 16 to >32
mg/L) (Table S3). The Tsx protein has previously been described as mediating resistance to the
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antibiotic albicidin in K. oxytoca. . A second resistant NCTC 13368 isolate showed a SNP,
leading to an amino acid change H50N in a predicted MerR family regulator (KPN_RS12075).
Conclusions:
The lack of new antibiotics, especially for rapidly emerging MDR Gram-negative pathogens, is a
significant cause for concern. Several studies have suggested that there may be no new
antimicrobials, with distinct modes of action, available for treatment of these pathogens for a
2
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considerable time . Against this background, we report the identification of a new scaffold with
significant activity against both Gram-positive and MDR Gram-negative pathogens, which do not
appear to be impacted by common resistance mechanisms to other broad-spectrum antibiotics and
with a low mutation frequency leading to resistance in key species such as A. baumannii and E. coli.
Mechanistic evaluation shows a sub-micromolar IC50 in a gel-based S. aureus DNA Gyrase assay,
significantly lower than ciprofloxacin and not affected by GyrA mutations associated with
fluoroquinolone resistance. Using a panel of fluorescent reporters, it was possible to show
activation of genes which are associated with the SOS response in Gram-negative bacteria, but
again the magnitude of promoter activation differed from fluoroquinolones and was consistent with
both known DNA binders (e.g. mitomycin C) and DNA gyrase inhibitors that act via alternative
binding sites to fluoroquinolones (e.g. novobiocin).
The compounds show excellent activity against WHO priority pathogens with an in vitro selective
index which would allow their continued development. The developability assessment of the lead
compounds will be carried out to fully explore the clinical utility of this chemical class. There is
renewed interest in developing bacteria specific antibody drug conjugates. We intend to exploit
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well-understood and clinically validated routes to develop antibody drug conjugates (ADCs), which
has been successfully employed for PBD-type compounds in oncology, to further improve the
prokaryotic selectivity of these compounds and to provide selective targeting of such compounds to
bacteria. This will form the basis of broad-spectrum PBD antibiotics which may have significant
utility in the clinic.
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Experimental Section
General chemistry
All reagents and solvents employed in the synthetic processes were obtained from commercially
available sources including, among others, Sigma-Aldrich, Fisher Scientific, Fluorochem and Alfa
1
13
Aesar. H and C nuclear magnetic resonance (NMR) analyses were performed on a Bruker
Spectrospin 400Hz spectrometer. LC-MS analyses were performed on a Waters Alliance 2695
system, eluting in gradient. The analyses were performed on a Monolithic C18 50 X 4.60 mm
column by Phenomenex. UV detection was performed on a Diode Array Detector. Mass spectra
were registered in both ESI+ and ESI- modes. Melting points were determined using a Stuart
SMP30 melting point apparatus. All the compounds tested for their biological activity are >95%
pure, confirmed with two different HPLC analysis methods The HRMS analyses were performed on
a Thermo Scientific Exactive HCD Orbitrap Mass Spectrometer. The hydrogenation reaction was
conducted using a Parr hydrogenation system. Synthetic protocols and compounds characterization
for the previously reported PBD core are reported in supporting information file.
Synthesis of pyrrole benzofused intermediate 27 and 28
Commercially available 4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2-carboxylic acid
(200 mg, 0.8 mmol, 1.2 equiv.) was dissolved in DMF (5 mL). EDCI (2.5 equiv.) and DMAP (3
equiv.) were added to the solution that was left under magnetic stirrer in N atmosphere for 20
2
minutes. At that point, the corresponding 5-amino-2-methyl ester benzofused (1 equiv.) was added
to the reaction mixture and left under magnetic stirrer overnight. The reaction did not go to
completion. The reaction was quenched by addition of water (15 mL) that was then extracted with
ethyl acetate (3 x 10 mL). The organic phase was then sequentially washed with brine (15 mL),
NaHCO saturated aqueous solution (15 mL) and citric acid aqueous solution 0.1 M (15 mL). The
3
collected organic phase was dried on MgSO4 and subsequently evaporated using a rotary
evaporator giving crude of reaction that was subsequently purified by column chromatography
(mobile phase: from 100 DCM to 85/15, v/v, DCM/EtOAc) to give the final products 27 and 28.
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-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2-carboxamido)benzofuran-2-
carboxylate (27)
1
Obtained 0.200 g (reaction yield: 70%) as a yellow solid. H NMR (400 MHz, CHLOROFORM-d)
δ 8.09 (d, J = 2.01 Hz, 1H), 7.85 (s, 1H), 7.51 (d, J = 9.06 Hz, 1H), 7.47 (s, 1H), 7.40 (dd, J = 9.06
Hz, 2.27 Hz, 1H), 6.86 (br. s., 1H), 6.70 (br. s., 1H), 6.43 (br. s., 1H), 3.97 (s, 3 H), 3.91 (s, 3 H),
0
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1.51 (s, 9H). C NMR (101 MHz, CHLOROFORM-d) δ159.9, 159.7, 152.5, 146.2, 134.0, 127.4,
1
23.3, 121.9, 121.3, 118.8, 114.2, 113.8, 112.6, 103.9, 80.4, 52.5, 36.8, 28.4. m/z (+ESI) calc. for
+
+
C
21
H
23
N
3
O (M) 413.1 found 414.0 ([M]+H) .
6
Methyl 5-(4-((tert-butoxycarbonyl)amino)-1-methyl-1H-pyrrole-2-carboxamido)
benzo[b]thiophene-2-carboxylate (28)
1
Obtained 0.192 g (reaction yield: 65%) as a yellow-orange solid. H NMR (400 MHz,
CHLOROFORM-d) δ8.24 (d, J = 1.76 Hz, 1H), 7.97 (s, 1H), 7.78-7.76 (m, 2H), 7.49 (dd, J = 8.69
Hz, 2.14 Hz, 1H), 6.86 (br. s., 1H), 6.70 (br. s., 1H), 6.33 (br. s., 1H), 3.94 (s, 3H), 3.91 (s, 3H),
1
3
1
.52 (s, 9H). C NMR (100 MHz, CHLOROFORM-d) δ 163.2, 159.7, 153.5, 139.3, 137.7, 135.5,
1
34.4, 130.5, 123.3, 123.0, 122.0, 120.6, 119.0, 115.9, 104.1, 80.4, 52.5, 36.7, 28.3. m/z (+ESI)
+
+
calc. for C21
H
23
N
3
O S (M) 429.1 found 430.0 ([M]+H) .
5
Tert-Butyl (5-((2-(dimethylcarbamoyl)benzofuran-5-yl)carbamoyl)-1-methyl-1H-pyrrol-3-
yl)carbamate (29)
1
Obtained 0.190 g (reaction yield: 72%), as a yellow solid. H NMR (400 MHz, CHLOROFORM-d)
δ7.99 (d, J = 2.01 Hz, 1H), 7.70 (s, 1H), 7.48 (d, J = 8.81 Hz, 1H), 7.38 (dd, J = 8.94 Hz, 2.14
Hz,1H), 7.25 (d, J = 0.76 Hz, 1H), 6.88 (s, 1H), 6.68 (s, 1H), 6.32 (s, 1H), 3.92 (s, 3H), 3.35 (br. s.,
1
3
3
1
2
H), 3.15 (br. s., 3H), 1.52 (s, 9H). C NMR (101 MHz, CHLOROFORM-d) δ160.9, 159.8, 153.4,
51.5, 150.0, 133.8, 127.5, 123.4, 121.9, 120.0, 118.7, 113.5, 112.1, 111.9, 103.8, 80.4, 36.8,
+
+
8.4.m/z (+ESI) calc. for C22
H
26
N
4
O (M) 426.1 found 427.1 ([M]+H) .
5
Tert-butyl (5-((2-(dimethylcarbamoyl)benzo[b]thiophen-5-yl)carbamoyl)-1-methyl-1H-pyrrol-
-yl)carbamate (30)
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Obtained 0.130 g (reaction yield: 60%), as a brown solid. H NMR (400 MHz, CHLOROFORM-d)
δ8.25 (d, J = 2.01 Hz, 1H), 7.70 - 7.81 (m, 2H), 7.46 (s, 1H), 7.36 (dd, J = 8.69 Hz, 2.14 Hz, 1H),
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
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1
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6
.88 (br. s., 1H), 6.70 (br. s., 1H), 6.33 (br. s., 1H), 3.93 (s, 3H), 3.22 (br. s., 6H), 1.52 (s, 9H). C
NMR (101 MHz, CHLOROFORM-d) δ164.8, 159.7, 153.5, 139.4, 138.7, 135.8, 135.2, 125.5,
0
1
2
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4
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8
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23.3, 122.7, 122.0, 119.3, 118.8, 115.3, 103.9, 80.4, 36.8, 28.4. m/z (+ESI) calc. for C22
H
26
N
4
O S
4
(M) 442.1 found 443.1 ([M]+H)+
+
Synthesis of benzofused side chains 31 to 38
The selected benzofused methyl ester intermediate 27 or 28 (1 equiv.) was dissolved in MeOH (8
mL), and an excess of NaOH 1M aqueous solution was added to the solution. The reaction mixture
was left under magnetic stirrer overnight at room temperature until TLC showed total disappearance
of the starting material. The solvent was evaporated under vacuum using a rotary evaporator, and
citric acid 1M aqueous solution was added to the crude of reaction until acid pH was reached
causing the simultaneous precipitation of a white solid. The precipitate was collected by filtration
under vacuum and dissolved in DMF (4 mL). EDCI (2.5 equiv.) and DMAP (3 equiv.) were added
to the solution and the reaction mixture was left under magnetic stirrer for 20 minutes at room
temperature under N atmosphere. Change in colour of the solution was observed from light yellow
2
to dark brown. The correspondent amine (1.5 equiv.) was added to the solution and left under
magnetic stirrer overnight. The reaction did not go to completion. The reaction was quenched by the
addition of H
organic phases were washed sequentially with brine (10 mL), NaHCO
10 mL) and citric acid aqueous solution 0.1 M (10 mL). The organic phase was dried on MgSO4
2
O (10 mL) that was subsequently extracted with EtOAc (3 x 8 mL). The collected
3
saturated aqueous solution
(
and evaporated under vacuum using a rotary evaporator giving the correspondent crude of reaction
that was purified by column chromatography on silica gel (mobile phase: from 100 DCM to 70/30,
v/v, DCM/EA, depending on the substrate) to give the final products 33-38.
In the case of diethyl derivatives 31 and 32 a different procedure was applied. After the hydrolysis
of the methyl ester benzofused intermediates 27 and 28, the obtained acid (1 equiv.) was dissolved
in DMF (4 mL) and diethyl amine (2 equiv.) and DIPEA (2 equiv.) were added to the solution that
O
was kept at 0 C in an ice bath. At that point T
3
P (50% solution in DMF, 2 equiv.) was added the
reaction mixture that was allowed to reach room temperature and left under magnetic stirrer
overnight. The reaction didn’t go to completion and was quenched by addition of water (10 mL)
and then extracted with ethyl acetate (3 x 8 mL). The organic phase was washed with brine (10 mL)
and NaHCO saturated aqueous solution (10 mL). The collected organic phase was dried on MgSO4
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and subsequently evaporated under vacuum using a rotary evaporator. The crude of the reaction was
subsequently purified by column chromatography (mobile phase: from 100 DCM to 70/30, v/v,
DCM/EA) to give the final products 31 and 32.
Tert-butyl (5-((2-(diethylcarbamoyl)benzofuran-5-yl)carbamoyl)-1-methyl-1H-pyrrol-3-
yl)carbamate (31)
0
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1
Obtained 0.036 g (reaction yield: 40%) as a white solid. H NMR (400 MHz, CHLOROFORM-d)
δ7.95 (d, J = 2.01 Hz, 1H), 7.80 (s, 1H), 7.34 - 7.47 (m, 2H), 7.23 (s, 1H), 6.88 (br. s., 1H), 6.68 (br.
1
3
s., 1H), 6.43 (br. s., 1H), 3.91 (s, 3H), 3.59 (br. s., 4H), 1.51 (s, 9H), 1.29 (br. s., 6H). C NMR
100 MHz, CHLOROFORM-d) δ160.3, 159.8, 153.5, 151.5, 150.5, 133.8, 127.5, 123.3, 121.9,
19.9, 118.7, 113.6, 111.9, 111.3, 103.9, 80.3, 36.7, 28.4, 14.2. m/z (+ESI) calc. for C24
(
1
H
30
N
4
O
5
+
+
(
M) 454.2 found 455.1([M]+H) .
Tert-butyl (5-((2-(diethylcarbamoyl)benzo[b]thiophen-5-yl)carbamoyl)-1-methyl-1H-pyrrol-3-
yl)carbamate (32)
1
Obtained 0.035 g (reaction yield: 41%) as a white solid. H NMR (400 MHz, CHLOROFORM-d)
δ8.20 (d, J = 2.01 Hz, 1H), 7.92 (br. s., 1H), 7.69 (d, J = 8.81 Hz, 1H), 7.37 (s, 1H), 7.31 (d, J =
8.81 Hz, 1H), 6.90 (br. s., 1H), 6.70 (br. s., 1H), 6.55 (br. s., 1H), 3.89 (s, 3H), 3.55 (q, J = 7.22 Hz,
1
3
4
1
2
H), 1.51 (s, 9H), 1.26 (t, J = 7.18 Hz, 6H). C NMR (100 MHz, CHLOROFORM-d)  164.2,
59.8, 153.5, 139.4, 138.8, 135.4, 135.3, 124.2, 123.2, 122.5, 122.0, 119.3, 118.9, 115.4, 80.2, 36.8,
+
+
8.4, 14.2. m/z (+ESI) calc. for C24
H
30
N
4
O S (M) 470.2 found 471.1 ([M]+H) .
4
Tert-butyl (1-methyl-5-((2-(piperidine-1-carbonyl)benzofuran-5-yl)carbamoyl)-1H-pyrrol-3-
yl)carbamate (33)
2
8
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Page 29 of 60
Journal of Medicinal Chemistry
1
2
3
1
Obtained 0.031 g (reaction yield: 34%) as a white solid. H NMR (400 MHz, CHLOROFORM-d)
δ7.91-7.93 (m, 2H), 7.42 (d, 1H), 7.34 - 7.39 (m, 1H), 7.13 (d, J = 0.76 Hz, 1H), 6.88 (s, 1H), 6.68
4
5
6
7
8
9
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
1
3
(
s, 1H), 6.55 (s, 1H), 3.88 (s, 3H), 3.72 (br. s., 4H), 1.62 - 1.75 (m, 6H), 1.50 (s, 9H). C NMR
(
100 MHz, CHLOROFORM-d) δ159.9, 159.8, 153.5, 151.3, 150.0, 133.9, 127.4, 123.3, 122.0,
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
1
20.0, 118.7, 113.5, 111.9, 111.2, 103.9, 80.2, 36.7, 28.4, 24.7, 24.6. m/z (+ESI) calc. for
+
+
C
25
H
30
N
4
O (M) 466.2 found 467.1 ([M]+H) .
5
Tert-butyl (1-methyl-5-((2-(piperidine-1-carbonyl)benzo[b]thiophen-5-yl)carbamoyl)-1H-
pyrrol-3-yl)carbamate (34)
1
Obtained 0.032 g (reaction yield: 33%) as a yellow solid. H NMR (400 MHz, CHLOROFORM-d)
δ8.20 (s, 1H), 7.86 (s, 1H), 7.72 (d, J = 9.06 Hz, 1H), 7.30 - 7.37 (m, 2H), 6.90 (br. s., 1H), 6.70 (br.
1
3
s., 1H), 6.48 (br. s., 1H), 3.91 (s, 3H), 3.68 (br. s., 4H), 1.59 - 1.75 (m, 6H), 1.52 (s, 9H). C NMR
100 MHz, CHLOROFORM-d)  163.7, 159.8, 153.5, 139.3, 138.2, 135.5, 135.3, 124.6, 123.3,
22.6, 122.0, 119.2, 118.8, 115.3, 104.0, 80.3, 36.8, 28.4, 24.6. m/z (+ESI) calc. for C25
(
1
H
30
N
4
O S
4
+
+
(M) 482.2 found 483.0 ([M]+H) .
Tert-butyl (1-methyl-5-((2-(morpholine-4-carbonyl)benzofuran-5-yl)carbamoyl)-1H-pyrrol-3-
yl)carbamate (35)
1
Obtained 0.038 g (reaction yield: 40%) as a grey solid. H NMR (400 MHz, CHLOROFORM-d)
δ8.01 (d, J = 2.01 Hz, 1H), 7.69 (s, 1H), 7.48 (d, J = 8.81 Hz, 1H), 7.41 (dd, J = 8.94 Hz, 2.14 Hz,
1H), 7.31 (d, J = 0.76 Hz, 1H), 6.87 (s, 1H), 6.69 (s, 1H), 6.29 (br. s., 1H), 3.93 (s, 3H), 3.89 (br.
1
3
s.,4H), 3.80 (br. s., 4H), 1.52 (s, 9H). C NMR (100 MHz, CHLOROFORM-d) δ159.6, 159.5,
1
3
51.4, 149.5, 133.9, 127.2, 123.2, 121.9, 120.0, 118.6, 116.3, 113.4, 112.6, 111.9, 103.7, 80.2, 66.9,
+
+
6.7, 28.3. m/z (+ESI) calc. for C24
H
28
N
4
O (M) 468.2 found 469.2 ([M]+H) .
6
Tert-butyl (1-methyl-5-((2-(morpholine-4-carbonyl)benzo[b]thiophen-5-yl)carbamoyl)-1H-
pyrrol-3-yl)carbamate (36)
2
9
ACS Paragon Plus Environment