Switching on the activity of 1,5-diaryl-pyrrole derivatives against drug-resistant ESKAPE bacteria: Structure-activity relationships and mode of action studies

Article abstract of DOI:10.1016/j.ejmech.2019.05.087

Antibiotic resistance represents a major threat worldwide. Gram-positive and Gram-negative opportunistic pathogens are becoming resistant to all known drugs mainly because of the overuse and misuse of these medications and the lack of new antibiotic development by the pharmaceutical industry. There is an urgent need to discover structurally innovative antibacterial agents for which no pre-existing resistance is known. This work describes the identification, synthesis and biological evaluation of a novel series of 1,5-diphenylpyrrole compounds active against a panel of ESKAPE bacteria. The new compounds show high activity against both wild type and drug-resistant Gram + ve and Gram-ve pathogens at concentrations similar or lower than levofloxacin. Microbiology studies revealed that the plausible target of the pyrrole derivatives is the bacterial DNA gyrase, with the pyrrole derivatives displaying similar inhibitory activity to levofloxacin against the wild type enzyme and retaining activity against the fluoroquinolone-resistant enzyme.

Full text of DOI:10.1016/j.ejmech.2019.05.087

Accepted Manuscript
Switching on the activity of 1,5-diaryl-pyrrole derivatives against drug-resistant
ESKAPE bacteria: Structure-activity relationships and mode of action studies
Domiziana Masci, Charlotte Hind, Mohammad K. Islam, Anita Toscani, Melanie
Clifford, Antonio Coluccia, Irene Conforti, Meir Touitou, Siham Memdouh, Xumin Wei,
Giuseppe La Regina, Romano Silvestri, J. Mark Sutton, Daniele Castagnolo
PII:
S0223-5234(19)30505-7
DOI:
https://doi.org/10.1016/j.ejmech.2019.05.087
EJMECH 11397
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 March 2019
Revised Date: 29 May 2019
Accepted Date: 29 May 2019
Please cite this article as: D. Masci, C. Hind, M.K. Islam, A. Toscani, M. Clifford, A. Coluccia, I. Conforti,
M. Touitou, S. Memdouh, X. Wei, G. La Regina, R. Silvestri, J.M. Sutton, D. Castagnolo, Switching on
the activity of 1,5-diaryl-pyrrole derivatives against drug-resistant ESKAPE bacteria: Structure-activity
relationships and mode of action studies, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.05.087.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Switching on the activity of 1,5-diaryl-pyrrole
derivatives against drug-resistant ESKAPE bacteria:
structure-activity relationships and mode of action
studies
Domiziana Mascia,c,¶, Charlotte Hindb,¶, Mohammad K. Islama,1, Anita Toscania, Melanie
Cliffordb, Antonio Colucciac, Irene Confortia,c, Meir Touitoua, Siham Memdouha, Xumin Weia,
Giuseppe La Reginac, Romano Silvestric, J. Mark Suttonb,*, Daniele Castagnoloa,*
*Corresponding author: daniele.castagnolo@kcl.ac.uk; mark.sutton@phe.gov.uk
a School of Cancer and Pharmaceutical Sciences, King’s College London, London SE1 9NH,
United Kingdom
b Research and Development Institute, National Infections Service, Public Health England,
Porton Down, Salisbury SP4 0JG, Wiltshire, United Kingdom
c Department of Drug Chemistry and Technologies, Sapienza University of Rome, Laboratory
affiliated to Instituto Pasteur Italia – Fondazione Cenci Bolognetti, Piazzale Aldo Moro 5, I-
00185 Roma, Italy
Present address:
1 Present address: Department of Pharmaceutical Chemistry, University of Dhaka, Bangladesh.
ABSTRACT
Antibiotic resistance represents a major threat worldwide. Gram-positive and Gram-negative
opportunistic pathogens are becoming resistant to all known drugs mainly because of the overuse
and misuse of these medications and the lack of new antibiotic development by the
pharmaceutical industry. There is an urgent need to discover structurally innovative antibacterial
agents for which no pre-existing resistance is known. This work describes the identification,
synthesis and biological evaluation of a novel series of 1,5-diphenylpyrrole compounds active
against a panel of ESKAPE bacteria. The new compounds show high activity against both wild
type and drug-resistant Gram+ve and Gram-ve pathogens at concentrations similar or lower than
levofloxacin. Microbiology studies revealed that the plausible target of the pyrrole derivatives is
the bacterial DNA gyrase, with the pyrrole derivatives displaying similar inhibitory activity to
levofloxacin against the wild type enzyme and retaining activity against the fluoroquinolone-
resistant enzyme.
Keywords. Antimicrobial resistance, ESKAPE bacteria, drug resistance, pyrrole, DNA gyrase
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1. Introduction
The treatment of infectious diseases, especially those caused by drug-resistant bacterial
opportunistic pathogens, is becoming a major threat for the World Healthcare Systems [1,2]. For
decades, bacterial infections have been successfully treated with a plethora of antibiotics
developed mainly between the 1940s and the 1960s [3], so that at the end of the 1960s, many
experts were confident that the war against bacterial infections was definitely won and that it was
time to "close the book on infectious diseases" [4].
However, many decades after the first patients were treated with antibiotics, bacterial infections
have again become a global threat due to the rapid emergence of drug resistant bacteria, which
undermine the efficacy of available antibiotics worldwide. The antibiotic resistance crisis has
been attributed mainly to the overuse and misuse of these medications, as well as a lack of new
drug development by the pharmaceutical industry, due to poor economic incentives and
challenging regulatory requirements [5]. Antibiotic resistance is a serious threat to Global Public
Health and the treatments available to date are often ineffective, especially against infections
caused by Gram-negative bacteria.
Opportunistic pathogens such as the Gram-negative (Gram-ve) pathogens Pseudomonas
aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli or the Gram-
positive (Gram+ve) bacteria Staphylococcus aureus and Enterococcus faecalis/faecium are
widespread around the world and are becoming resistant to all known drugs. E. coli infections in
some parts of the world frequently resist antibiotic treatments with third generation
cephalosporins [6], making many urinary tract infections, usually regarded as merely a nuisance,
very difficult to treat. Many epidemic K. pneumoniae strains have become resistant to
carbapenem antibiotics [7] as well as to fluoroquinolones [8]. Countries worldwide reported that
half or more of Staphylococcus infections are resistant to methicillin (MRSA strains) [9,10]
whilst infections caused by P. aeruginosa represent a global emergency due to the ability to
rapidly develop resistance during the course of treating an infection [11,12].
In order to face up to the emergency of antibacterial resistance several simultaneous actions can
be taken: 1) to reduce the misuse of antibiotics, 2) to develop alternatives to antibiotics or use
antibiotics in combination, 3) to develop appropriate antibiotic dosing regimens that reduce the
selection of resistance [13-18]. However, the best solution to the bacterial drug-resistance
problem would be to discover and develop novel classes of antibiotics for which there is no pre-
existing resistance among human bacterial pathogens [19].
The discovery of new antibiotics is a major challenge, and the rate of successfully discovering
new and especially broad spectrum antibiotic hits is low [14,20]. With the aim to speed up the
drug discovery process and to reduce the limitations of target-based high-throughput screening
(HTS) protocols and compound collections, we recently adopted an alternative strategy, based on
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the molecular hybridization of known drugs, to design new antitubercular drugs [21]. The in-
silico hybridization of the old antitubercular pyrrole BM212 [22] with SQ109 [23], a new anti-
tubercular drug currently undergoing clinical phase II trials, led to the discovery of the pyrrole 1
which showed potent activity against various mycobacteria, including multi drug-resistant
(MDR) tuberculosis strains (Fig. 1). A structure-activity relationship (SAR) study revealed that
the 2,5-dimethylpyrrole derivatives of 1 possess high antitubercular activity, whilst the 1,5-
diphenylpyrrole analogues, such as 2, were poorly active against the same mycobacterial strains.
Fig. 1. Structure of pyrroles 1 and 2 and SAR evaluation plan.
Despite the phylogenetic and structural differences between mycobacteria and Gram+/-ve
bacteria, we explored the antimicrobial spectrum of 1 against a panel of Gram+/-ve pathogens.
Pyrrole 1 was screened against a selection of bacteria including MRSA strains, vancomycin
resistant Enterococcusstrains (VRE) and other Gram-ve microorganisms such as P. aeruginosa,
K. pneumoniae and E. coli, but, disappointingly, it showed no or moderate inhibition of growth
of various bacteria (Tables 2-3). On the other hand, the 1,5-diarylpyrrole 2 derivative, poorly
active against mycobacteria, showed a very good antibacterial profile, being able to inhibit
growth of a number of Gram+ve strains at micromolar concentrations. Initial studies [24] carried
out on previously synthesised pyrrole derivatives clearly indicated that the replacement of the
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methyl group on C5 of the pyrrole 1 with a phenyl ring was a key element to switch the
selectivity and the antimicrobial activity from mycobacteria to Gram+ve bacteria.
Herein, we describe the design, synthesis and discovery of a new series of 1,5-diarylpyrrole
derivatives with general structure A (Fig. 1), highly active against both susceptible and drug-
resistant Gram+/-ve bacteria. The chemical space around the pyrrole nucleus of 1-2 was explored
and the key SAR responsible for the activity are disclosed. The bactericidal/bacteriostatic
activity of the new compounds was also investigated. Finally, a mechanism of action for the new
compounds is proposed as the new pyrroles proved to inhibit the bacterial DNA gyrase at
concentrations similar to levofloxacin.
2. Results and discussion
2.1. Chemistry
A library of pyrrole derivatives 8 bearing different substituents on the N-phenyl rings at N1
and C5 positions as well as different amine-side chains at C3, was synthesized (Scheme 1 and
Table 1). The 2,5-hexandiones 5 were first obtained through Stetter reaction of different
aldehydes 3 with methylvinyl ketone 4 [25]. Paal-Knorr condensation of diketones 5 with
appropriate anilines led to pyrroles 6 [26] which were in turn formylated in the presence of
POCl3 and DMF to afford aldehydes 7. Finally, the reductive amination of 7 with different
amines in the presence of Na(OAc)3BH as reducing agent and stoichiometric AcOH led to
desired pyrrole derivatives 8a-8v in high yields. Derivatives 8w-aa were synthesized from
aldehydes 7 via a slightly modified reductive amination procedure using the Boc-protected
amines 9-11, followed by Boc cleavage with HCl/AcOEt. Pyrroles 1 and 2 were synthesized as
previously reported [19].
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Scheme 1. Synthesis of pyrroles 1, 2 and 8a-aa
Table 1.
Structures of pyrroles 1, 2 and 8a-aa.
Pyrrole
compounds
8
Diketones
5
Pyrroles 6 &
Aldehydes 7
R
R1
R2 or R3
Me
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
Cyclohexyl
Cyclohexyl
Bn
5a
5b
5a
6a, 7a
6b, 7b
6a,7a
1
4-Cl-Ph
Me
2
8a
8b
8c
4-Cl-Ph
4-Cl-Ph
Bn
5b
6b, 7b
Cycloheptyl
5

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4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-iPr-Ph
4-F-Ph
4-Cl
4-Cl
2-adamantyl
Cyclohexylmethyl
iPr
8d
8e
8f
4-Cl
4-Cl
8g
8h
8i
α-methyl-Bn
PhNHCH2CH2
Cyclohexyl
4-Cl
4-F
6c, 7c
4-F
Cycloheptyl
Cycloheptyl
Cyclohexyl
8j
4-iPr
2,5-Me
3-F
6d, 7d
6e, 7e
6f, 7f
8k
8l
Cyclohexyl
8m
8n
8o
8p
8q
8r
8s
2,4-Cl
4-MeO
4-NO2
2-CF3
4-OH
4-Cl
Cyclohexyl
6g, 7g
6h, 7h
6i, 7i
Cyclohexyl
Cyclohexyl
Cyclohexyl
6j, 7j
Cyclohexyl
6k, 7k
6l, 7l
Cyclohexyl
5c
5d
5e
4-Cl
Cyclohexyl
6m, 7m
6n, 7n
8t
4-tBu-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl
Cyclohexyl
8u
8v
8w
8x
4-Cl
4-OH-cyclohexyl
4-NH2-cyclohexyl
Guanyl-(CH2)8-
4-Cl
6b, 7b
4-Cl
5b
5a
4-guanidine-
cyclohexyl
4-Cl-Ph
4-Cl
8y
4-guanidine-
cyclohexyl
4-Cl-Ph
Me
4-NO2
4-Cl
8z
6i, 7i
Guanyl-(CH2)8-
6a, 7a
8aa
6

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2.2. Biological evaluation and SAR analysis.
All the compounds were tested for their biological activity by determining the minimum
inhibitory concentrations (MIC) against a panel of susceptible and drug-resistant Gram+ve and
Gram-ve bacterial strains (ESKAPE) (Tables 2-3). The Gram+ve panel includes the methicillin-
susceptible (NCTC 6571) and the methicillin-resistant EMRSA15 (NCTC 13616, containing the
gyrA S84L and parC S80F mutations) and EMRSA16 (NCTC 13277, containing the gyrA S84F
and parC S80F mutations) S. aureus as well as the vancomycin-susceptible (VSE) (NCTC 775)
and vancomycin-resistant (VRE) (NCTC 12201 and NCTC 12204) Enterococcus spp. The
Gram-ve panel includes drug susceptible and drug resistant strains of K. pneumoniae (NCTC
13368 and M6), A. baumannii (AYE and ATCC 17978), P. aeruginosa (PA01 and NCTC
13437) and E. coli (NCTC 12923).
2.2.1. Antibacterial activity against Gram+ve strains.
We first analysed the data arising from the screening of pyrroles 8a-aa on Gram+ve bacteria.
The results are reported in Table 2.
Table 2.
Antibacterial activity of pyrroles 1, 2 and 8a-aa against Gram+ve bacteria (MSSA, MRSA, VSE
and VRE).
MIC µg/mL
MSSA
MRSA
VSE
VRE
NCTC
Cmpd
NCTC
6571
NCTC
13616
NCTC
13277
NCTC NCTC
775
12201
12204
64
64
64
32
32
16
2
1
4
4
4
>128
4
4
4
2
>128
>128
>128
>128
128
ND
2
8a
8b
8c
8d
8e
8f
8
4
4
4
8
8
4
4
4
8
4
4
4
2
8
4
2
2
4
8
4
4
2
4
2
8-16
4
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4
4-8
8
4
4-8
8
4
4-8
8
2-4
4
2-4
4
2
4-8
4
8g
8h
4
4
8i
4
8
4
4
4
2
8j
4
4
4
2
2
2
8k
4
4
8
4
4
2
8l
8
8
8
8
4
4
8m
4
4
4
4
4
2
8n
16
8
16
16
8
16
16
8
8
8
8
8o
8
8
4
8p
4
4
4
2
8q
32
4
8-64
4
32
4
64
4
32
4
32
2
8r
8s
8
8
8
8
4
4
8t
4
4
4
4
2
2
8u
16
8
16
8
16
8
16
8
16
8
8
8v
4
8w
4
4
4
8
8
4
8x
2
2-4
8
4
8
16
64
16
1
4
8y
8z
4
8
32
8-16
1
16
4-8
0.5
4
8
8
8aa
0.12
8
8
Levofloxacin
In agreement with preliminary results, the insertion of a second aryl ring at C5 on the pyrrole
nucleus proved to be fundamental for the antibacterial activity. In fact, the 2,5-dimethyl pyrroles
1 and 8a, both endowed with antitubercular activity, showed no or poor inhibition of growth of
Gram+ve strains, whilst their corresponding 1,5-diaryl-pyrroles 2 and 8b proved to be active
with MIC values of 4-8 µg/mL against both drug susceptible and drug resistant Staphylococcus
8

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and Enterococcus strains with a microbiology profile similar to levofloxacin. The presence of a
cyclohexylamine (2) rather than a benzyl (8b) side chain at C3 of the pyrrole nucleus appears to
be slightly beneficial to improve the activity against MSSA and MRSA strains. The replacement
of the cyclohexyl ring in 2 with bulkier aliphatic groups (cycloheptyl, adamantyl and
cyclohexylmethyl) led to 8c-e which showed an MIC = 2 µg/mL against VRE bacteria similar or
better than levofloxacin. A similar trend was observed with derivatives 8j-k bearing a
cycloheptylamine side chain. In general, the presence of a cyclic and bulky aliphatic amine side
chain is beneficial in improving the activity, especially against VRE strains. As further
corroboration of this observation, compound 8g, a branched and bulkier analogue of 8b, also
showed a slightly improved antibacterial profile against VRE. On the other hand, the presence of
a smaller or aromatic side chain on the pyrrole ring led to compounds 8f and 8h with lower
activity against MSSA and MRSA strains. The compounds 8i-r were synthesized with the aim to
explore the influence on the antibacterial activity of different substituents on the N-phenyl ring.
In general, all the compounds showed very good activity, especially against VRE strains. The
presence of a halogen substituent or an alkyl group on the N-phenyl ring favors the improvement
of the activity against MSSA and EMRSA (compounds 8j, 8k, 8n, MIC = 4 µg/mL), whilst
electron-withdrawing (8p) or electron-donating (8o, 8r) substituents led to a decrease of activity.
However, no electronic effect of the substituents on the phenyl ring was observed and
compounds bearing electron-withdrawing or electron-donating groups showed similar
antibacterial activity. Compounds 8s-u, synthesized to explore the chemical space around the
phenyl ring at C5, also showed good activity against VRE bacteria (MIC = 2 µg/mL). The
cyclohexylamine side chain of 2 was finally replaced with more hydrophilic and polar groups in
compounds 8v-aa. Compounds 8v-w bearing a diaminocyclohexyl or hydroxy-cyclohexylamine
side chain at C3 showed lower activity than 2 against all Gram+ve bacteria, whilst the guanidine
derivative 8y showed a good activity against MSSA and MRSA strains (MIC = 2-4 µg/mL).
Compound 8x bearing an amino-octylguanidine chain showed good activity against MSSA and
MRSA (MIC = 4 µg/mL), whilst the presence of a nitro substituent on the N-phenyl ring in 8z
proved to be detrimental. Finally, the 2,5-dimethylpyrrole 8aa showed worse antibacterial
activity than its 1,5-diaryl-pyrrole analogue 8w, further confirming the key role of an aryl
substituent on C5.
2.2.2. Antibacterial activity against Gram-ve strains
The pyrrole compounds 1, 2 and 8a-aa were then assayed against a panel of Gram-ve bacteria.
Data are reported in Table 3 [27].
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Table 3.
Antibacterial activity of pyrroles 1, 2 and 8 against Gram-ve bacteria.
MIC µg/mL
K. pneumoniae
A. baumannii
P. aeruginosa
E. coli
Cmpd
NCTC
M6
ATCC
NCTC
NCTC
12923
AYE
PA01
13368
17978
>64
64
4
13437
>64
>128
32
>64
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
32
>64
>128
64
>64
128
8
>64
>128
>128
>128
>128
>128
16
-
>128
-
1
2
2+PMBN
8b
>128
32
>128
16
>128
16
32
2
>128
>128
>128
16
>128
>128
>128
2
8b+PMBN
8k
>128
>128
>128
>128
128
>128
>128
16
>128
4
8k+PMBN
8k+PAβN
8s
4
4
128
>128
>128
32
8
>128
8
32
2
>128
8
>128
2
8s+PMBN
8s+PAβN
8v
8
8
>128
>128
32
>128
>128
32
16
128
32
64
16
8
>128
8
8w
32
16
32
128
64
8
8x
128
32
64-128
32
16
16
16
64
16
0.12
128
16
8
8y
8y+PMBN
8y+PAβN
8z
128
64
128
16
4
128
64
64
128
64
8
>128
>128
1
>128
128
0.12
>128
64
>128
>128
2
>128
64
64
16
8aa
8
64
<0.12
Levofloxacin
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The 2,5-dimethylpyrrole 1 and the 1,5-diaryl-pyrroles 2 and 8b proved to be inactive against the
whole panel of Gram-ve bacteria at the highest concentration tested (128 µg/mL). The low
activity of these derivatives could be ascribable to two major factors, namely 1) the chemical
structure of the pyrroles derivatives which prevents the binding to any bacterial target in Gram-
ve bacteria, or 2) the low permeability of the Gram-ve outer membrane (OM) which prevents the
pyrrole derivatives from reaching their molecular target. Thus, with the aim to understand if
pyrrole compounds were simply unable to cross the OM, 2 and 8b were assayed in combination
with polymyxin B nonapeptide (PMBN). PMBN is a modification of polymixin B which lack the
acyl chain important for antibiotic activity but is still able to permeabilize the outer membrane of
Gram-ve bacteria and to allow lipophilic substances to penetrate inside the bacterial cell. As
such, whilst it retains some antimicrobial activity, it is much less active than polymixin B. The
conditions used for these experiments are selected such that the single PMBN concentration used
for permeabilization is not antibacterial in its own right and indeed does not impact at all on the
growth of the strains being tested. Interestingly, both 2 and 8b showed improved antibacterial
activity when used in combination with PMBN. In particular pyrrole2 proved to be very active
against A. baumannii, with MIC = 4-8 µg/mL and promisingly active against the MDR P.
aeruginosa strain NCTC 13437 with MIC = 32 µg/mL. Pyrrole 8b proved to be less active than
2, also confirming the previous SAR observation that a cyclohexylamino side chain on the
pyrrole ring is preferable to a benzylamino group. The experiments carried out with PMBN
confirmed our initial hypothesis that the 1,5-diaryl-pyrroles 2 and 8b are active against some
Gram-ve species but cannot enter the cells due to the presence of the outer membrane barrier.
The OM in Gram-ve bacteria has a major impact on the susceptibility of the microorganism to
antibiotics which act on intracellular targets. Small hydrophilic antibiotics, such as β-lactams,
use the porins to gain access to the cell interior, while macrolides and other hydrophobic drugs
diffuse across the lipid bilayer [28]. Porins are large water-filled channels in the outer membrane
of Gram-ve bacteria and they act as filters for hydrophobic compounds [29]. In general, small
hydrophilic molecules and charged ions can be transported by diffusion through the porins [30].
Thus, we decided to decorate the pyrrole nucleus at C3 with different hydrophilic side chains
bearing protonatable amine and guanidine moieties. Compound 8w, designed as an analogue of 2
and containing an extra amine group on the cyclohexyl group, did show an improved
antibacterial activity against the whole panel of Gram-ve bacteria with MIC = 8-32 µg/mL.
Pyrrole 8y, bearing a guanidine group on the cyclohexyl group also showed a good antibacterial
profile especially against ATCC 17978 and NCTC 13437 (MIC = 16 µg/mL) and E. coli NCTC
12923 (MIC = 8 µg/mL). On the other hand, pyrrole 8v, bearing a hydroxyl moiety on the
cyclohexyl group, did not show any activity against Gram-ve bacteria, thus confirming the
observation that a protonatable amine or guanidine moiety is essential to allow the pyrrole
derivatives to cross the OM. Compound 8x showed the best profile especially against A.
baumannii and E. coli (MIC = 8 µg/mL). Finally, we compared the antibacterial activity of the
1,5-diaryl-pyrrole 8x with the corresponding 2,5-dimethyl-pyrrole analogue 8aa. The presence of
a second phenyl ring on the pyrrole scaffold in 8x proved again to be beneficial leading to an
11

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increase of the antibacterial activity especially against K. pneumoniae (MIC = 16-32 µg/mL for
8x versus MIC = >128 µg/mL for 8aa). Derivatives 8k, 8s and 8y were also tested in
combination with PMBN and with the Resistance-Nodulation Division (RND) efflux pump
inhibitor, phenylalanine-arginine β-naphthylamide (PAβN). Both 8k and 8s showed significantly
improved MICs (4 to 128 fold potentiation) in the presence of PMBN in all strains tested, except
the two K. pneumoniae strains where the MICs were unchanged. Both compounds also showed
potentiation with the efflux pump inhibitor PAβN, in A. baumannii and E. coli, suggesting that
they are efflux pump substrates in these species, but not in K. pneumoniae and P. aeruginosa.
Compound 8y, did not show any potentiation by PMBN or PAβN. This data further corroborated
the observation that a hydrophilic moiety on the pyrrole scaffold is essential to allow the
compound to penetrate the Gram-ve cells and dispatch their antibacterial activity.
2.3. Time kill assays and drug resistance
All the pyrroles 8 showed variable activity against Gram+/-ve strains. To determine if the
compounds were bacteriostatic or bactericidal, a time kill assay was performed. Three pyrrole
derivatives, namely 8k, 8s and 8y, were selected on the basis of their antibacterial activities and
chemical structures to carry out additional experiments. Compounds 8k, 8s and 8y were added at
4 x MIC concentration to cultures of bacteria, and aliquots were taken at 0, 2, 4, 6 and 24 hour
timepoints. Time kill assays demonstrated bacteriostatic modes of action at 4 x MIC
concentrations for 8k, 8s and 8y in VRE at 24 hours, whilst in MRSA, 8k and 8y were
bacteriostatic, but 8s was observed to be bactericidal at 24 hours (Fig. 2). No resistant
populations were generated during the 24 hour assay.
Fig. 2. Time kill analysis for 8k, 8s and 8y in MRSA and VRE strains. NT = no treatment
control.
MRSA and VRE strains were also passaged in the presence of increasing concentrations of
compounds to try to generate resistance or plated onto agar containing higher than MIC
concentrations of compounds, with the aim to obtain genomic DNA for studies on the
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mechanism of action of the new compounds. However, despite repeated attempts and the use of
several methods to generate resistance, including broth and agar-based methods, no stable
resistance emerged, whilst resistance was generated for the control antibiotic levofloxacin in
both strains, using all methods. In the standard agar-based method, this equates to a frequency of
<7.6x10-8 and <9.4x10-8 at 2xMIC for strains NCTC 13616 and NCTC 6521, respectively. In
comparison, levofloxacin at 2xMIC had mutation frequencies of 1.87x10-5 and 7.08x10-6 for the
same strains. Whilst this represents encouraging data, as it clearly shows the efficacy of 1,5-
diphenyl-pyrroles against drug-resistant strains and low propensity to induce resistance, it should
not be interpreted as being an indication that resistance will not emerge to these compounds. It
also makes it difficult to use resistance emergence to generate target mutations that would help to
define the mechanism of action.
2.4. Mechanism of action and computational studies
The pyrrole 2 is a structural analogue of the antitubercular agent BM212, which is known to
inhibit the MmpL3 mycolic acid transporter protein in M. tuberculosis. However, this protein is
not present in Gram+/-ve bacteria and thus a different mechanism of action had to be
hypothesised. Recent literature showed that pyrrole derivatives often act as antibacterial agents
through inhibition of bacterial DNA gyrase [31,32], and we were intrigued by the possibility that
1,5-diphenyl-pyrroles could act as DNA gyrase inhibitors as well.
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Fig. 3. Inhibition of the a) wild-type (S. aureus DNA Gyrase GyrA2B2 complex) enzyme and b)
fluoroquinolone-resistant gyrase enzyme (S. aureus DNA Gyrase S84L) by 8k, 8s and 8y.
-ve = no enzyme added, +ve = enzyme added.
A biochemical assay to measure inhibition of the wild-type DNA gyrase enzyme (GyrA2B2
complex) from S. aureus was performed. Compounds 8k, 8s and 8y displayed inhibitory activity
against the wild-type enzyme from S. aureus. (Fig. 3, Table 4). In particular, pyrrole8y had a
similar IC50 against the wild-type gyrase than levofloxacin, whilst 8k was slightly less active.
Because of the flat shape of the inhibitory curve for 8s, an IC50 could not be calculated for this
compound. The same assay was also repeated on the quinolone-resistant mutant (Ser84Leu)
gyrase from S. aureus, which is present in most MRSA isolates [33,34]. Interestingly, unlike
levofloxacin, which showed an increased IC50 of 196.9 µg/mL against the fluoroquinolone-
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resistant enzyme, the three compounds 8k, 8s and 8ymaintained the high inhibitory activity
observed against the wild type enzyme (Table 4). Fluoroquinolone antibiotics such as
levofloxacin cannot bind to the S84L mutated DNA gyrase due to the mutational loss of the
serine residue [35], but this does not appear to affect the ability of the pyrrole derivatives to bind
to the enzyme. Most fluoroquinolones also bind to topoisomerase IV, but whilst levofloxacin
maintained a very low IC50 against topoisomerase IV from S. aureus, compound 8y had a much
higher IC50 against this enzyme, suggesting a preference of the pyrrole derivatives to the gyrase
enzyme over topoisomerase IV.
Table 4.
IC50 values of 8k, 8s and 8yand levofloxacin (LVX) against DNA gyrase wild-type GyrA2B2
and mutant S84L, and topoisomerase IV. Students t-test analysis was performed for each
compound compared to the levofloxacin control and the P value expressed in the table.
DNA gyrase GyrA2B2
DNA gyrase S84L
Topoisomerase IV
IC50
IC50
IC50
P
value
SD
SD
P value
SD
P value
(µg/mL)
(µg/mL)
19.01
29.11
7.23
(µg/mL)
15.49
NC
1.99
-
0.006
1.64
11.97
1.26
0.0011
0.0016
0.009
-
ND
-
-
8k
8s
-
0.55
-
ND
-
-
7.48
8.71
3.13
0.95
28.8
1.47
0.19
<0.0001
-
8y
196.9
36.59
3.65
LVX
NC = not calculable, ND = Not done, SD = Standard deviation
Since the crystal structures of the wild-type gyrases of S. aureus are available, we decided to
further investigate and understand the exact mechanism of action of the novel pyrrole derivatives
8s-8y through modelling studies. The DNA gyrase of S. aureus possess three potential binding
sites for drugs, namely the quinolone binding site, to which levofloxacin binds, the Novel
Bacterial Topoisomerase Inhibitor (NBTI) site and the ATP binding sites.[36] By docking
studies, we decided to evaluate which site among the three would be more suitable for the
binding of compounds 8s-8y.
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To study the quinolone site, we carried out by PLANTS [37] the superimposition of the studied
compounds with moxifloxacin (PDB code 5CDQ) [36], QPT-1 (PDB code 5CDM) [38] and
etoposide (PDB code 5CDN) [38]. The latter shared with the reported compounds an unrelated
quinolone chemotype and a worthy activity against the quinolone-resistant GyrA S84L [39,40].
Fig. 4. PLANTS proposed binding mode of 8y (magenta) for PDB 5CDM. The GyrA and GyrB
subunits are reported in green and cyan cartoon, respectively. The DNA structure is depicted as
orange cartoon, the nucleotides are in white lines. Residues involved in contacts with 8y are
reported as cyan stick. Ser84 is also showed as green stick. H-bond is depicted as a yellow dot
line.
The reported compounds exhibited consistent binding modes for the studied crystal structures
and showed good overlapping with the reference compounds (Supplementary data Fig. S1).
Analyses of the docking results highlighted key interactions of derivative 8y: (i) the pyrrole
nucleus and the chlorophenyl ring on C5 made stacking interactions with the +1 and -1 bases at
the DNA cleavage site [37]; (ii) the N1-chlorophenyl formed hydrophobic contacts with Gly436
and Arg458 side chains of the TOPRIM domain of the GyrB; (iii) the secondary amine and the
cyclohexyl ring formed a H-bond and hydrophobic contacts, respectively, with the closest DNA
bases; (iv) the guanidine group formed polar contacts with the DNA-sugar moiety (Fig. 4).
Interestingly, the π−π interaction of the phenyl ring on C5 with the guanine nucleobase confirms
16

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the key role of the second aromatic ring for the antibacterial activity. In fact, the dimethyl pyrrole
analogues like 1, 8a or 8aa, miss this extra binding, which may explain the loss of antibacterial
activity.
All the compounds were also docked at the GyrA S84L enzyme. It was already reported that
both QPT-1 and etoposide had better IC50 values for the mutated enzyme than for the wild type
[39,40]. Indeed, the S48L mutation did not affect the binding mode. Furthermore, the
hydrophobic Leu instead of the Ser was at bond distance to form further hydrophobic contacts.
Similarly, the 8y-8s binding modes were not affected by the mutation and also in these cases we
observed hydrophobic contacts with the Leu84 (Supplementary data Fig. S2-S3). These results
provide further confirmation of the proposed binding mode.
Docking studies of 8y-8s at the NBTI site (PDB code 5BS3) were then performed [41]. In this
case, the proposed binding modes were not consistent. Actually, the NBTI matched a well-
defined pharmacophore model featured by a bulkier aromatic group, a linker bearing a basic
nitrogen atom and an aromatic/hydrophobic moiety arranged in a linear molecular geometry
[42]. Compounds 8y-8s did not show any linear geometry and the most active derivative 8y had
the protonable nitrogen atom located far from the pharmacophore model. Based on these results,
we suggest that 8y-8s compounds do not bind the NBTI site.
Finally, the gyrase ATP site (PDB code 5D6Q) was investigated [43]. According to the reported
structural requirements for binding to this site [44,45], a compound should possess at least a
donor/acceptor pattern, a more or less sterically constrained linker and an acid or polar moiety
[46-49]. The proposed binding modes of 8y-8s did not match any feature of the pharmacophore
model. These results also suggested that compounds 8y-8s do not bind the ATP site.
As further confirmation to validate these results, we used the MM-GBSA method [50] to
calculate the binding free energy of the 8y best scored pose for each studied site (Supplementary
data). The free energy calculation yielded the most favourable binding energy at the quinolone
binding site, thus confirming, together with microbiology data, the mode of action of novel 1,5-
diaryl-pyrroles.
2.5. Toxicity studies
The three lead compounds, 8y, 8k and 8s, were tested against the well validated cell-lines HeLa
and HEK293 for toxicity to human cells. Compound 8y offered a significant therapeutic window
of around 5-15fold between the IC50 (11.67 - 18.9 µg/mL for the two cell-lines) and the peak
activity against S. aureus (2 µg/mL). This was significantly greater than some of the other lead
compounds (8k and 8s) which showed IC50 values very close to the MIC (1.95 – 2.83 µg/mL and
2.4 – 3.0 µg/mL respectively). This difference in the cytotoxicity profiles for the three
compounds, which have similar levels of activity against bacteria, suggests there is significant
opportunity to further expand the therapeutic window through targeted modifications.
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3. Conclusions
The identification of novel classes of antibacterial agents is a high priority in medicine due to the
emergence of multi drug-resistant microorganisms. The vast majority of the antibiotics currently
in use have been produced between 1930 and 1962 and since then almost exclusively analogues
of existing classes have reached the market. The development of novel antibacterials, possibly
with innovative chemical structures, with different modes of action and strongly active against
drug-resistant rather than the wild-type bacteria, is highly desirable. Within this work, we have
identified a new class of 1,5-diphenyl-pyrrole antibacterial compounds, designed using the
antitubercular agent BM212 as template, through appropriate molecular decorations. The new
compounds proved to be active against a wide panel of ESKAPE bacteria with activity similar or
better than levofloxacin, while they possess only moderate antitubercular activity. Microbiology
studies led to the identification of the plausible cellular target, namely the bacterial DNA gyrase.
Interestingly, the pyrrole derivatives inhibit the DNA gyrase of S. aureus at concentrations
similar than levofloxacin, and their activity is maintained against the mutated DNA gyrase form
S84L, responsible for most fluoroquinolone resistance in S. aureus. Modelling studies pointed
out that the 1,5-diphenyl-pyrroles bind the fluoroquinolones binding site in DNA gyrase, but the
different chemical structures allow them to not be affected by the S84L mutation responsible for
drug-resistance. Finally, when given at higher concentration than MIC (time kills and mutation
frequencies) or when cultured at sub-MIC to above-MIC levels (serial-passage), the new pyrroles
have not generated resistance in bacteria. This suggests that they are excellent scaffolds for new
antibacterial agents that could be used alone or as part of novel combination therapies with
existing antibiotics.
4. Experimental section
4.1. Chemistry
4.1.1. General
1H-NMR and 13C-NMR spectra were recorded with an Ascend 400 spectrometer Bruker, at room
temperature (rt) operating at the frequencies indicated. Chemical shifts (δ) are in ppm, referenced
to tetramethylsilane. Coupling constants (J) are reported in Hertz. Splitting patterns are
abbreviated as follows: singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), sextet (sxt),
broad (br) or some combination of them. Mass spectra (HRMS) were recorded at the EPSRC
National Mass Spectrometry Service Centre on a Thermo Scientific LTQ Orbitrap XL Mass
Spectrometer using low-resolution ESI or high-resolution nano ESI techniques. The purity of the
compounds was assessed by reverse-phase liquid chromatography coupled with a mass
spectrometer (Agilent series 1100 LC/MSD) with a UV detector at λ = 254 nm and an
electrospray ionization source (ESI). HPLC analysis was carried out using a Perkin-Elmer 1100
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HPLC system coupled with UV/Vis set to 254 nm. Mass spectra were acquired in positive mode
scanning over the mass range of 50–1500. The following ion source parameters were used:
drying gas flow, 9 mL/min; nebulize pressure, 40 psig; and drying gas temperature, 350 ºC. All
target compounds possessed a purity of ≥ 95% as verified by HPLC analyses. TLC was
performed using commercially available pre-coated plates and visualized with UV light at 254
nm; KMnO4 was used to reveal the products. Flash column chromatography was carried out
using Sigma Aldrich silica gel particle size, 40-63 µm particle size 60 Å. All reactions were
conducted under a nitrogen atmosphere in oven-dried glassware unless stated otherwise. All
solvents
and
commercially
available
reagents
were
used
as
received.
The compound 2,5-hexandione 5a was purchased from Sigma-Aldrich.
4.1.2. Synthesis of differently-substituted 1,4-diketones
Compounds 5b-e were synthesized as described in the literature [22,57,58,59].
4.1.3. General Procedure for the synthesis of pyrroles
5
6
Pyrroles 6a-d, 6g, 6i, 6l, 6m were synthesized according to a previously-described literature
procedure [22,26,60,61].
The novel pyrrole compounds 6e, 6f, 6h, 6j, 6k, 6n were prepared as followed.
The appropriate 1,4-pentanedione 5b, 5e (3.13 mmol) was dissolved in ethanol (5 mL) in a
sealed glass tube equipped with a magnetic stirring bar. Then the suitable aniline (3.13 mmol)
and p-toluensulfonic acid (0.22 mmol) were added. The tube was heated in the microwave
reactor for 40 minutes (200W, internal temperature 180 °C, and internal pressure 200psi). After
cooling down the reaction mixture was extracted with EtOAc (20 mL), washed once with a
saturated solution of NaHCO3 (10 mL) and twice with brine (20 mL). The organic layers were
combined, dried over MgSO4 and filtered. The filtrate was concentrated in vacuo and proved to
be pure enough to be used in the next step without any further purification.
4.1.3.1. 2-(4-chlorophenyl)-1-(2,5-dimethylphenyl)-5-methyl-1H-pyrrole (6e
)
Yield: 60 %. 1H NMR (400 MHz, CDCl3) δ 7.11 (s, 2H), 7.10 (s, 1H), 7.08 - 7.05 (m, 2H), 7.01
(s, 1H), 7.00 - 6.97 (m, 1H), 6.41 - 6.37 (m, 1H), 6.10 (d, 1H, J = 3.7 Hz), 2.35 (s, 3H), 2.00 (s,
3H), 1.79 (s, 3H) ppm. 13C NMR (100 MHz, CDCl3) δ 138.3, 136.5, 133.6, 132.7, 132.3, 131.8,
131.3, 130.8, 129.7, 129.4, 128.3, 128.0, 108.4, 107.4, 21.00, 16.9, 12.9 ppm. HRMS (m/z) [M +
H]+calcd for C19H18ClN, 296.1201; found, 296.1200.
4.1.3.2. 2-(4-chlorophenyl)-1-(3-fluorophenyl)-5-methyl-1H-pyrrole (6f
)
Yield: 79 %. 1H NMR (400 MHz, CDCl3) δ 7.40 - 7.30 (m, 1H), 7.17 - 7.11 (m, 2H), 7.11 - 7.04
(m, 1H), 6.98 (d, 2H, J = 8.6 Hz), 6.94 (br d, 1H, J = 7.9 Hz), 6.92 - 6.87 (m, 1H), 6.35 (d, 1H, J
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13
= 3.4 Hz), 6.10 (d, 1H, J = 2.6 Hz), 2.16 (s, 3H) ppm. C NMR (100 MHz, CDCl3)δ163.9,
133.0, 132.1, 131.7 (d), 130.2 (d), 128.8, 128.3, 124.3 (d), 115.8 (d), 114.7 (d), 109.4, 108.1,
13.3 ppm. HRMS (m/z) [M + H]+calcd for C17H13ClFN, 286.0793; found, 286.0786.
4.1.3.3. 2-(4-chlorophenyl)-1-(4-methoxyphenyl)-5-methyl-1H-pyrrole (6h
)
Yield: 68 %. 1H NMR (400 MHz, CDCl3) δ 7.10 (d, J = 8.8 Hz, 2H), 7.06 (d, J = 9 Hz, 2H), 6.99
(d, J = 8.8 Hz, 2H), 6.89 (d, J = 9 Hz, 2H), 6.33 (d, J = 3.4 Hz, 2H), 6.07 (d, J = 3.5 Hz, 2H),
3.84 (s, 3H), 2.11 (s, 3H) ppm. 13C NMR (100 MHz, CDCl3) δ 158.9, 133.1, 132.5, 132.2, 132.1,
131.5, 129.5, 128.9, 128.3, 114.4, 108.7, 107.5, 55.6, 13.3 ppm. HRMS (m/z) [M + H]+calcd for
C18H16ClNO, 298.0993; found, 298.0992.
4.1.3.4. 2-(4-chlorophenyl)-5-methyl-1-(2-(trifluoromethyl)phenyl)-1H-pyrrole (6j
)
Yield: 44 %.1H NMR (400 MHz, CDCl3) δ 7.93 - 7.90 (m, 1H), 7.55 (d, J = 8.6 Hz, 2H), 7.42 (d,
J = 8.6 Hz, 1H), 7.32 (d, J = 8.6 Hz, 2H), 7.08 - 6.97 (m, 2H), 6.53 (d, J = 3.3 Hz, 1H), 6.06 (dd,
13
J1 = 3.2 Hz, J2 = 1.0 Hz, 1H), 2.36 (s, 3H) ppm. C NMR (100 MHz, CDCl3) δ 152.3, 151.2,
144.7, 141.8, 139.6, 135.0, 129.5, 128.9, 128.8, 128.2, 124.5, 122.8, 107.9, 106.5, 13.7 ppm.
HRMS (m/z) [M + H]+calcd for C18H13ClF3N, 336.0761; found, 336.0760.
4.1.3.5. 4-(2-(4-chlorophenyl)-5-methyl-1H-pyrrol-1-yl) phenol (6k
)
Yield: 72 %. 1H NMR (400 MHz, CDCl3) δ 7.11 (d, J = 8.5 Hz, 2H), 7.02 – 6.97 (m, 4H), 6.82
(d, J = 8.6 Hz, 2H), 6.31 (d, J = 3.3 Hz, 1H), 6.07 (br. s, 1H), 4.93 (br. s, 1H), 2.11 (s, 3H) ppm.
13C NMR (100 MHz, CDCl3) δ 155.0, 133.1, 132.5, 132.3, 132.2, 131.5, 129.7, 128.9, 128.3,
116.0, 108.8, 107.5, 13.3 ppm. HRMS (m/z) [M + H]+calcd for C17H14ClNO, 284.0837; found,
284.0834.
4.1.3.6. 2-(4-(tert-butyl)phenyl)-1-(4-chlorophenyl)-5-methyl-1H-pyrrole (6n
)
1
Yield: 80 %. H NMR (400 MHz, CDCl3) δ 7.34 (d, J = 8.8 Hz, 2H), 7.18 (d, J = 8.7 Hz, 2H),
7.11 (d, J = 8.8 Hz, 2H), 6.98 (d, J = 8.7 Hz, 2H), 6.32 (d, J = 3.4 Hz, 1H), 6.09 (dd, J1 = 3.4 Hz,
13
J2 = 0.8 Hz, 1H), 2.13 (s, 3H), 1.27 (s, 9H) ppm. C NMR (100 MHz, CDCl3) δ 148.8, 138.2,
134.3, 133.2, 131.4, 130.4, 129.8, 129.3, 127.5, 125.1, 108.8, 107.9, 34.5, 31.4, 13.4 ppm.
HRMS (m/z) [M + H]+calcd for C21H22ClN, 324.1514; found, 324.1515.
4.1.4. General Procedure for the synthesis of Compounds 7 [21]
POCl3 (4 mmol) was added dropwise to a round-bottom flask containing ice cold DMF (5 mL)
under N2 atmosphere. After 15 min, a solution of the appropriate pyrrole 6a - n (1 mmol) was
added to the stirring solution. Then the reaction mixture was allowed to stir at 100 °C for 3 h.
The reaction was monitored by TLC. After completion, the reaction was quenched with 10% w/v
NaOH solution (20 mL). The reaction mixture was then diluted with EtOAc (10 mL) and washed
20

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twice with EtOAc (10 mL) and once with brine (20 mL). The organic extracts were collected,
dried over MgSO4, filtered and concentrated in vacuo to give a crude that was then filtered
through a pad of silica gel and evaporated under reduced pressure. The residue was then used in
the next step without further purification.
4.1.5. Synthesis of Pyrrole Derivatives 1-2, 8a-b
Pyrroles 1, 2 and 8a-b were synthesized according to literature [21].
4.1.6. General Procedure for the synthesis of Pyrrole Derivatives 8c-v [21]
The appropriate aldehyde 7a-n (1 mmol) was dissolved in 5 mL of THF in a round-bottom flask.
AcOH (1 mmol) and the appropriate amine (1.2 mmol) were added to the mixture and allowed to
stir at room temperature for 30 minutes before the addition of NaB(AcO)3H (3 mmol). The
mixture was then allowed to stir at room temperature for 24 h. After completion, the reaction was
quenched with 1 M NaOH solution (25 mL). The mixture was allowed to stir for a further 30
minutes, before the mixture was diluted with EtOAc (10 mL) and washed twice with EtOAc (10
mL) and once with brine (20 mL). The organic extracts were collected, dried over MgSO4,
filtered and concentrated under reduced pressure. The crude residue was purified with flash
chromatography (EtOAc/MeOH 9:1 v/v), affording the desired compounds 8c-v.
4.1.7. General Procedure for the synthesis of Pyrrole Derivatives 8w-aa [21]
The synthesis of the Boc-protected 8w-aa compounds was performed according to General
Procedure for the synthesis of Pyrrole Derivatives 8c-v. Subsequently, the appropriate Boc-
protected 8w-aa (0.2 mmol) was placed in a sealed vial to which 3 mL of freshly prepared
HCl/AcOEt solution was added. The mixture was stirred at room temperature for 24 h and then
the solvent was removed under reduced pressure. The residue was washed several times with
small portions of cold Et2O affording the desired compounds 8w-aa as HCl salts in quantitative
yields.
4.1.7.1. N-((1,5-bis(4-chlorophenyl)-2-methyl-1H-pyrrol-3-yl)methyl)cycloheptanamine (8c)
1
Yield: 67 %. H NMR (400 MHz, CDCl3) δ 7.35 – 7.31 (m, 2H), 7.12 – 7.09 (m, 2H), 7.06 –
7.03 (m, 2H), 6.95 – 6.91 (m, 2H), 6.39 (s, 1H), 3.67 (s, 2H), 2.82 – 2.75 (m, 1H), 2.07 (s, 3H),
13
1.96 – 1.89 (m, 2H), 1.73 – 1.66 (m, 2H), 1.56 – 1.47 (m, 6H), 1.32 – 1.26 (m, 2H) ppm. C
NMR (100 MHz, CDCl3) δ 137.79, 133.53, 132.26, 131.75, 131.56, 129.80, 129.45, 129.18,
128.94, 128.40, 110.35, 58.92, 43.15, 34.65, 28.41, 24.59, 11.09 ppm. HRMS (m/z) [M +
H]+calcd for C25H28Cl2N2, 427.1702; found, 427.1689.
4.1.7.2. N-((1,5-bis(4-chlorophenyl)-2-methyl-1H-pyrrol-3-yl)methyl)adamantan-2-amine (8d
)
21

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1
Yield: 51 %. H NMR (400 MHz, CDCl3) δ 7.34 (d, J = 8.7 Hz, 2H), 7.11 (d, J = 8.5 Hz, 2H),
7.06 (d, J = 8.7 Hz, 2H), 6.94 (d, J = 8.5 Hz, 2H), 6.40 (s, 1H), 3.67 (s, 2H), 2.87 (br s, 1H), 2.09
(s, 3H), 2.05 (br s, 1H), 1.98 (br s, 2H), 1.89 – 1.86 (m, 3H), 1.80 – 1.73 (m, 6H), 1.54 – 1.51 (m,
2H) ppm. 13C NMR (100 MHz, CDCl3) δ 137.83, 133.51, 132.17, 131.73, 131.63, 129.82,
129.46, 128.97, 128.41, 110.51, 61.76, 42.83, 38.09, 37.72, 31.98, 31.52, 29.85, 27.99, 27.78,
11.14 ppm. HRMS (m/z) [M + H]+calcd for C28H30Cl2N2, 465.1859; found, 465.1847.
4.1.7.3. 1-(1,5-bis(4-chlorophenyl)-2-methyl-1H-pyrrol-3-yl)-N-(cyclohexylmethyl)-methanamine
(8e)
1
Yield: 82 %. H NMR (400 MHz, CDCl3) δ 7.32 (d, J = 8.6 Hz, 2H), 7.09 (d, J = 8.5 Hz, 2H),
7.03 (d, J = 8.6 Hz, 2H), 6.91 (d, J = 8.5 Hz, 2H), 6.50 (s, 1H), 4.79 (br. s, 1H), 3.80 (s, 2H),
2.62 (d, 2H, J = 6.8 Hz), 2.08 (s, 3H), 1.87 – 1.84 (m, 2H), 1.74 – 1.63 (m, 6H), 1.30 – 1.23 (m,
3H) ppm. 13C NMR (100 MHz, CDCl3) δ 137.50, 133.71, 132.56, 131.93, 131.25, 130.40,
129.76, 129.49, 128.99, 128.38, 110.74, 54.64, 44.85, 36.61, 31.39, 26.46, 25.91, 11.21 ppm.
HRMS (m/z) [M + H]+calcd for C25H28Cl2N2, 427.1702; found, 427.1688.
4.1.7.4. N-((1,5-bis(4-chlorophenyl)-2-methyl-1H-pyrrol-3-yl)methyl)propan-2-amine (8f)
1
Yield: 37 %. H NMR (400 MHz, CDCl3) δ 7.32 (d, 2H, J = 8.6 Hz), 7.09 (d, 2H, J = 8.7 Hz),
7.04 (d, J = 8.7 Hz, 2H), 6.92 (d, J = 8.6 Hz, 2H), 6.39 (s, 1H), 3.68 (s, 2H), 2.99 – 2.93 (m, 1H),
2.78 (br s, 1H), 2.07 (s, 3H), 1.15 (d, J = 6.3 Hz, 6H) ppm. 13C NMR (100 MHz, CDCl3) δ 137.5,
133.7, 132.5, 131.9, 131.2, 130.4, 129.8, 129.5, 128.9, 128.4, 110.8, 48.4, 41.6, 21.1, 11.2 ppm.
HRMS (m/z) [M + H]+calcd for C21H22Cl2N2, 373.1233; found, 373.1039.
4.1.7.4. N-((1,5-bis(4-chlorophenyl)-2-methyl-1H-pyrrol-3-yl)methyl)-1-phenylethan-1-amine
(8g)
Yield: 22 %. 1H NMR (400 MHz, CDCl3) δ 7.42 – 7.28 (m, 7H), 7.10 (d, J = 8.5 Hz, 2H), 7.03
(d, J = 8.6 Hz, 2H), 6.92 (d, J = 8.4, 2H), 6.38 (s, 1H), 3.96 – 3.90 (m, 1H), 3.53 (s, 2H), 1.94 (s,
3H), 1.44 – 1.43 (m, 3H) ppm. 13C NMR (100 MHz, CDCl3) δ 145.7, 137.8, 133.5, 132.2, 131.7,
131.6, 129.8, 129.4, 129.2, 128.9, 128.6, 128.4, 127.1, 126.9, 119.9, 110.3, 58.2, 43.6, 24.7, 11.0
ppm. HRMS (m/z) [M + H]+calcd for C26H24Cl2N2, 435.1389; found, 435.1192.
4.1.7.5.
N-((1,5-bis(4-chlorophenyl)-2-methyl-1H-pyrrol-3-yl)methyl)-N2-phenylethane-1,2-
diamine (8h)
Yield: 26 %. 1H NMR (400 MHz, CDCl3) δ 7.30 (d, J = 8.6 Hz, 2H), 7.16 – 7.08 (m, 4H), 6.95
(d, J = 8.6 Hz, 2H), 6.86 (d, J = 8.6 Hz, 2H), 6.68 (t, J = 7.3 Hz, 1H), 6.60 (d, J = 7.7 Hz, 2H),
6.43 (s, 1H), 3.86 (s, 2H), 3.42 – 3.39 (m, 2H), 3.09 – 3.06 (m, 2H), 2.02 (s, 3H) ppm. 13C NMR
(100 MHz, CDCl3) δ 147.6, 137.3, 133.9, 132.9, 132.2, 131.0, 129.7, 129.5, 129.4, 129.0, 128.5,
22

ACCEPTED MANUSCRIPT
128.4, 117.6, 112.9, 110.6, 46.0, 43.8, 41.0, 11.1 ppm. HRMS (m/z) [M + H]+calcd for
C26H25Cl2N3, 450.1498; found, 450.1489.
4.1.7.6.
N-((5-(4-chlorophenyl)-1-(4-fluorophenyl)-2-methyl-1H-pyrrol-3-
yl)methyl)cyclohexanamine (8i)
Yield: 68 %. 1H NMR (400 MHz, CDCl3) δ 7.09 – 7.04 (m, 6H), 6.91 (d, J = 8.6 Hz, 2H), 6.48
(s, 1H), 3.78 (s, 2H), 2.74 – 2.69 (m, 1H), 2.05 (s, 3H), 1.79 – 1.76 (m, 2H), 1.66 – 1.62 (m, 2H),
13
1.37 – 1.22 (m, 6H) ppm. C NMR (100 MHz, CDCl3) δ 163.1, 160.6, 135.1 (d), 132.6, 131.8,
131.4, 130.2 (d), 128.9, 128.3, 116.3, 116.1, 110.4, 56.2, 41.7, 32.0, 25.8, 25.0, 11.1 ppm.
HRMS (m/z) [M + H]+calcd for C24H26ClFN2, 397.1841; found, 397.1832.
4.1.7.7.
N-((5-(4-chlorophenyl)-1-(4-fluorophenyl)-2-methyl-1H-pyrrol-3-
yl)methyl)cycloheptanamine (8j
)
Yield: 65 %. 1H NMR (400 MHz, CDCl3) δ 7.09 – 7.04 (m, 6H), 6.90 (d, J = 8.7 Hz, 2H), 6.54
(s, 1H), 3.80 (s, 2H), 2.98- 2.92 (m, 1H), 2.06 (s, 3H), 1.77 – 1.63 (m, 5H), 1.56 – 1.54 (m, 4H),
13
1.48 – 1.39 (m, 3H) ppm. C NMR (100 MHz, CDCl3) δ 163.1, 160.6, 135.0 (d), 132.7, 131.9,
131.3, 130.2 (d), 128.9, 128.3, 116.4, 116.1, 110.6, 58.1, 41.7, 33.0, 28.1, 24.4, 11.2 ppm.
HRMS (m/z) [M + H]+calcd for C25H28ClFN2, 411.1998; found, 411.1989.
4.1.7.8.
N-((5-(4-chlorophenyl)-1-(4-isopropylphenyl)-2-methyl-1H-pyrrol-3-
yl)methyl)cycloheptanamine (8k
)
1
Yield: 72 %. H NMR (400 MHz, CDCl3) δ 7.18 (d, J = 8.4 Hz, 2H), 7.04 (d, J = 8.9 Hz, 2H),
6.99 (d, J = 8.4 Hz, 2H), 6.91 (d, J = 8.9 Hz, 2H), 6.56 (s, 1H), 3.81 (s, 2H), 3.00 – 2.86 (m, 2H),
2.07 (s, 3H), 1.79 – 1.64 (m, 5H), 1.57 – 1.52 (m, 4H), 1.48 – 1.37 (m, 3H), 1.26 (s, 3H), 1.24 (s,
3H) ppm. 13C NMR (100 MHz, CDCl3) δ 148.7, 136.5, 132.6, 131.6, 131.5, 131.0, 128.8, 128.3,
128.2, 127.2, 110.4, 58.0, 33.8, 32.8, 29.8, 28.1, 24.4, 24.1, 11.3 ppm. HRMS (m/z) [M +
H]+calcd for C28H35ClN2, 435.2562; found, 435.2368.
4.1.7.9.
N-((5-(4-chlorophenyl)-1-(2,5-dimethylphenyl)-2-methyl-1H-pyrrol-3-
yl)methyl)cyclohexanamine (8l
)
Yield: 40 %. 1H NMR (400 MHz, CDCl3) δ 7.11 - 7.07 (m, 2 H), 7.04 - 7.09 (m, 3 H), 6.95 (d, J
= 8.6, 2 H), 6.81 (s, 1 H), 4.01 (d, J = 3.2 Hz, 2 H), 2.99 - 2.94 (m, 1 H), 2.30 (s, 3 H), 2.25 -
2.17 (m, 2 H), 1.96 (s, 3 H), 1.84 - 1.77 (m, 2 H), 1.74 (s, 3 H), 1.70 - 1.60 (m, 4 H), 1.22 - 1.16
13
(m, 2H) ppm. C NMR (100 MHz, CDCl3) δ137.6, 136.7, 133.3, 132.7, 131.6, 131.5, 131.2,
130.8, 129.6, 129.5, 128.2, 128.0, 127.0, 111.0, 77.4, 55.0, 39.8, 29.4, 29.0, 24.9, 24.7, 20.8,
16.8, 10.7 ppm. HRMS (m/z) [M + H]+calcd for C26H31ClN2, 407.2249; found, 407.2056.
4.1.7.10.
yl)methyl)cyclohexanamine (8m
N-((5-(4-chlorophenyl)-1-(3-fluorophenyl)-2-methyl-1H-pyrrol-3-
)
23

ACCEPTED MANUSCRIPT
Yield: 64 %. 1H NMR (400 MHz, CDCl3) δ 7.34 - 7.29 (m, 1H), 7.09 - 7.03 (m, 3H), 6.93 - 6.88
(m, 3H), 6.82 (dt, J1 = 9.3 Hz, J2 = 2.1 Hz, 1H), 6.58 (s, 1H), 3.81 (s, 2H), 2.82 - 2.75 (m, 1H),
2.12 (br. s, 1H), 2.09 (s, 3H), 1.82 - 1.76 (m, 2H), 1.65 - 1.60 (m, 1H), 1.50 - 1.40 (m, 2H), 1.30 -
13
1.20 (m, 4H) ppm. C NMR (100 MHz, CDCl3) δ 164.0, 161.5, 140.5 (d), 132.7, 132.0, 131.2,
130.4 (d), 128.9, 128.4, 124.6 (d), 116.1, 115.1, 111.0, 55.8, 40.9, 31.1, 25.6, 24.9, 11.2 ppm.
HRMS (m/z) [M + H]+calcd for C24H26ClFN2, 397.1841; found, 397.1828.
4.1.7.11.
N-((5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-2-methyl-1H-pyrrol-3-
yl)methyl)cyclohexanamine (8n
)
1
Yield: 57 %. H NMR (400 MHz, CDCl3) δ 7.54 (d, J = 2.1 Hz, 1H), 7.28 (d, J = 2.2 Hz, 1H),
7.20 - 7.15 (dd, J1 = 11.8 Hz, J2 =8.6 Hz, 3H), 7.00 (d, J = 8.6 Hz, 2H), 6.72 (s, 1H), 4.03 - 3.95
(m, 2H), 2.93 (t, J = 11.2 Hz, 1H), 2.21 - 2.18 (m, 2H), 2.04 (s, 3H), 1.91 - 1.82 (m, 2H), 1.69 -
1.58 (m, 3H), 1.33 - 1.29 (m, 4H) ppm. 13C NMR (100 MHz, CDCl3) δ137.6, 135.3, 134.6,
132.7, 132.1, 131.9, 130.7, 130.5, 128.8, 128.6, 128.3, 123.2, 117.3, 110.9, 55.3, 39.9, 29.1, 25.0,
24.8, 10.7 ppm. HRMS (m/z) [M + H]+calcd for C24H25Cl3N2, 447.1156; found, 447.1157.
4.1.7.12.
N-((5-(4-chlorophenyl)-1-(4-methoxyphenyl)-2-methyl-1H-pyrrol-3-
yl)methyl)cyclohexanamine (8o
)
1
Yield: 60 %. H NMR (400 MHz, CDCl3) δ 7.08 (d, J = 8.5 Hz, 2H), 7.01 (d, J = 8. Hz, 2H),
6.93 (d, J = 8.5 Hz, 2H), 6.86 (d, J = 8.7 Hz, 2H), 6.46 (s, 1H), 3.96 (s, 2H), 3.82 (s, 3H), 2.96 -
2.91 (m, 1H), 2.03 (s, 3H), 1.85 - 1.71 (m, 4H), 1.65 - 1.59 (m, 2H), 1.52 - 1.45 (m, 2H), 1.21 -
1.11 (m, 2H) ppm. 13C NMR (100 MHz, CDCl3) δ159.2, 133.1, 132.5, 131.8, 131.4, 131.2,
129.6, 128.9, 128.3, 114.5, 110.6, 55.6, 55.1, 39.9, 29.2, 25.0, 24.7, 11.3 ppm. HRMS (m/z) [M
+ H]+calcd for C25H29ClN2O, 409.2041; found, 409.1849.
4.1.7.13.
N-((5-(4-chlorophenyl)-2-methyl-1-(4-nitrophenyl)-1H-pyrrol-3-
yl)methyl)cyclohexanamine (8p
)
1
Yield: 68 %. H NMR (400 MHz, CDCl3) δ 8.22 (d, J = 8.9 Hz, 2H), 7.25 (d, J = 8.8 Hz, 2H),
7.11 (d, J = 8.4 Hz, 2H), 6.89 (d, J = 8.4 Hz, 2H), 6.56 (s, 1H), 3.79 (s, 2H), 2.72 (t, J =
unresolved, 1H), 2.13 (s, 3H), 2.10 - 2.05 (m, 2H), 1.82 - 1.75 (m, 2H), 1.67 - 1.60 (m, 1H), 1.42
13
- 1.15 (m, 5H) ppm. C NMR (100 MHz, CDCl3) δ 146.6, 144.6, 132.6, 132.4, 130.9, 129.9,
129.2, 129.1, 128.6, 124.6, 111.9, 56.2, 41.5, 31.9, 25.8, 25.0, 11.4 ppm. HRMS (m/z) [M +
H]+calcd for C24H26ClN3O2, 424.1786; found, 424.1776.
4.1.7.14.
N-((5-(4-chlorophenyl)-2-methyl-1-(2-(trifluoromethyl)phenyl)-1H-pyrrol-3-
yl)methyl)cyclohexanamine (8q).
1
Yield: 52 %. H NMR (400 MHz, CDCl3) δ 7.73 (d, J = 7.7 Hz, 1H), 7.64 (t, J = 7.5 Hz, 1H),
7.55 (t, J = 7.5 Hz, 1H), 7.38 (d, J = 7.8 Hz, 1H), 7.05 (d, J = 8.7 Hz, 2H), 6.93 (d, J = 8.7 Hz,
24

ACCEPTED MANUSCRIPT
2H), 6.42 (s, 1H), 3.72 (s, 2H), 2.59 - 2.53 (m, 1H), 1.98 - 1.95 (m, 1H), 1.93 (s, 3H), 1.79 - 1.72
13
(m, 2H), 1.66 - 1.60 (m, 1H), 1.34 - 1.11 (m, 6H) ppm. C NMR (100 MHz, CDCl3) δ 137.7,
133.3, 132.8, 132.4, 131.9, 131.6, 130.2, 129.2, 128.4, 128.3, 127.7 (d), 124.2, 121.5, 119.6,
109.9, 56.3, 42.6, 33.6, 33.3, 26.3, 25.2 (d), 10.6 ppm. HRMS (m/z) [M + H]+calcd for
C25H26ClF3N2, 447.1809; found, 447.1807.
4.1.7.15.
8r
4-(5-(4-chlorophenyl)-3-((cyclohexylamino)methyl)-2-methyl-1H-pyrrol-1-yl)phenol
(
)
1
Yield: 64 %. H NMR (400 MHz, CDCl3) δ 6.98 (d, J = 8.5 Hz, 2H), 6.89 (d, J = 8.6 Hz, 2H),
6.86 - 6.80 (m, 4H), 6.65 (s, 1H), 3.96 (s, 2H), 2.99 (t, J = 10.7 Hz, 1H), 2.26 - 2.19 (m, 2H),
1.99 (s, 3H), 1.83 - 1.77 (m, 2H), 1.68 - 1.50 (m, 4H), 1.19 - 1.08 (m, 2H) ppm. 13C NMR (100
MHz, CDCl3) δ 156.8, 133.1, 132.5, 131.8, 131.2, 130.5, 129.4, 128.9, 128.2, 116.2, 109.9, 60.6,
48.5, 33.3, 25.6, 25.0, 11.2 ppm. HRMS (m/z) [M + H]+calcd for C24H27ClN2O, 395.1885;
found, 395.1884.
4.1.7.16.
N-((1-(4-chlorophenyl)-5-(4-isopropylphenyl)-2-methyl-1H-pyrrol-3-
yl)methyl)cyclohexanamine (8s
)
1
Yield 34 %. H NMR (400 MHz, CDCl3) δ 7.30 (d, J = 8.5 Hz, 2H), 7.06 (d, J = 8.5 Hz, 2H),
6.99 (d, J = 8.5 Hz, 2H), 6.92 (d, J = 8.3 Hz, 2H), 6.46 (s, 1H), 3.79 (s, 2H), 2.85 - 2.77 (m, 1H),
2.75 - 2.67 (m, 1H), 2.08 (s, 3H), 1.81 - 1.74 (m, 2H), 1.71 - 1.48 (m, 2H), 1.42 - 1.27 (m, 4H),
13
1.24 - 1.22 (m, 1H), 1.18 (d, J = 6.9 Hz, 6H), 1.12 - 1.00 (m, 1H) ppm. C NMR (100 MHz,
CDCl3) δ 146.7, 138.1, 133.8, 133.3, 130.4, 130.0, 129.3, 127.8, 126.3, 110.0, 56.1, 41.7, 33.8,
29.8, 25.9, 25.1, 24.0, 11.2 ppm. HRMS (m/z) [M + H]+calcd for C27H33ClN2, 421.2405; found,
421.2391.
4.1.7.17.
N-((1-(4-chlorophenyl)-5-(4-fluorophenyl)-2-methyl-1H-pyrrol-3-
yl)methyl)cyclohexanamine (8t
)
1
Yield 53 %. H NMR (400 MHz, CDCl3) δ 7.32 (d, J = 8.6 Hz, 2H), 7.04 (d, J = 8.5 Hz, 2H),
6.97 (dd, J1 = 8.7 Hz, J2 = 5.4 Hz, 2H), 6.83 (t, J1 = 8.7 Hz, 2H), 6.36 (s, 1H), 3.73 (s, 2H), 2.66 -
2.57 (m, 1H), 2.07 (s, 3H), 2.03 - 1.95 (m, 2H), 1.85 (t, J1 = 6.5 Hz, 1H), 1.80 - 1.74 (m, 2H),
1.67 - 1.60 (m, 1H), 1.38 - 1.29 (m, 2H), 1.21 - 1.14 (m, 2H) ppm. 13C NMR (100 MHz, CDCl3)
δ 162.6, 160.2, 137.9, 133.5, 132.6, 129.9, 29.6, 129.5, 129.4, 115.3, 115.1, 110.0, 56.6, 42.5,
33.1, 25.8, 25.2, 11.1 ppm. HRMS (m/z) [M + H]+calcd for C24H26ClFN2, 397.1841; found,
397.1651.
4.1.7.18.
N-((5-(4-(tert-butyl)phenyl)-1-(4-chlorophenyl)-2-methyl-1H-pyrrol-3-
yl)methyl)cyclohexanamine (8u
)
25

ACCEPTED MANUSCRIPT
1
Yield 86 %. H NMR (400 MHz, CDCl3) δ 7.31 (d, J = 8.7 Hz, 2H), 7.15 (d, J = 8.5 Hz, 2H),
7.07 (d, J = 8.7 Hz, 2H), 6.94 (d, J = 8.5 Hz, 2H), 6.43 (s, 1H), 3.77 (s, 2H), 2.72 - 2.64 (m,
1H), 2.07 (s, 3H), 2.05 - 2.00 (m, 2H), 1.81 - 1.73 (m, 2H), 1.67 - 1.58 (m, 2H), 1.42 - 1.31 (m,
2H), 1.25 (s, 9H), 1.19 - 1.05 (m, 2H) ppm. 13C NMR (100 MHz, CDCl3) δ 156.4, 149.2, 137.8,
134.1, 133.6, 130.0, 129.6, 129.4, 127.5, 125.2, 110.6, 55.3, 34.5, 31.4, 29.8, 25.2, 24.8, 11.5
ppm. HRMS (m/z) [M + H]+calcd for C28H35ClN2, 435.2562; found, 435.2553.
4.1.7.19. 4-(((1,5-bis(4-chlorophenyl)-2-methyl-1H-pyrrol-3-yl)methyl)amino)cyclohexan-1-ol
(8v)
1
Yield: 30 %. H NMR (400 MHz, CDCl3) δ 7.33 (d, J = 8.7 Hz, 2H), 7.09 (d, J = 8.7 Hz, 2H),
7.03 (d, J = 8.6 Hz, 2H), 6.91 (d, J = 8.6 Hz, 2H), 6.36 (s, 1H), 3.68 (s, 2H), 3.66 - 3.60 (m, 1H),
2.65 - 2.56 (m, 1H), 2.05 (s, 3H), 2.03 - 1.96 (m, 4H), 1.38 - 1.27 (m, 4H) ppm. 13C NMR (100
MHz, CDCl3) δ 137.6, 133.6, 132.4, 131.8, 131.4, 129.7, 129.5, 128.9, 128.4, 119.0, 110.1, 55.7,
50.6, 42.9, 34.0, 30.8, 11.0 ppm. HRMS (m/z) [M + H]+calcd for C24H26Cl2N2O, 429.1495;
found, 429.1488.
4.1.7.20. Boc-Protected 8w
1
Yield: 25 %. H NMR (400 MHz, CDCl3) δ 7.33 (d, J = 8.5 Hz, 2H), 7.10 (d, J = 8.5 Hz, 2H),
7.04 (d, J = 8.6 Hz, 2H), 6.92 (d, J = 8.6 Hz, 2H), 6.42 (s, 1H), 3.72 (s, 2H), 2.07 (s, 3H), 2.04 -
2.01 (m, 2H), 1.44 (s, 9H), 1.33 - 1.22 (m, 4H), 1.20 - 1.10 (m, 2H) ppm. 13C NMR (100 MHz,
CDCl3) δ 153.1, 144.2, 137.2, 133.4, 132.2, 129.4, 129.2, 128.8, 128.7, 128.2, 128.1, 120.5,
109.7, 79.1, 64.5, 57.6, 48.7, 31.8, 30.1, 28.2, 10.7 ppm. HRMS (m/z) [M + H]+calcd for
C29H35Cl2N3O2, 528.2179; found, 528.2178.
4.1.7.21. N1-((1,5-bis(4-chlorophenyl)-2-methyl-1H-pyrrol-3-yl)methyl)cyclohexane-1,4-diamine
(8w)
Yield: 100 %. 1H NMR (400 MHz, CD3OD) δ 7.47 (d, J = 8.5 Hz, 2H), 7.19 (d, J1 = 8.4 Hz, 2H),
7.15 (d, J1 = 8.5 Hz, 2H), 7.04 (d, J1 = 8.4 Hz, 2H), 6.55 (s, 1H), 4.19 (s, 2H), 2.42 - 2.35 (m,
2H), 2.24 - 2.18 (m, 2H), 2.16 (s, 3H), 1.72 - 1.60 (m, 6H) ppm. 13C NMR (100 MHz, CD3OD) δ
138.6, 135.3, 134.8, 133.6, 133.2, 132.5, 131.2, 130.7, 130.4, 129.4, 112.3, 111.3, 56.0, 49.9,
42.3, 29.7, 28.1, 11.1 ppm. HRMS (m/z) [M + H]+calcd for C24H27Cl2N3, 428.1655; found,
428.1649.
4.1.7.22. Boc-Protected 8x
Yield: 52 %. 1H NMR (400 MHz, CDCl3) δ 11.5 (br. s, 1H), 8.30 - 8.26 (m, 1H), 7.33 (d, J = 8.6
Hz, 2H), 7.10 (d, J = 8.5 Hz, 2H), 7.04 (d, J = 8.5 Hz, 2H), 6.92 (d, J = 8.6 Hz, 2H), 6.40 (s, 1H),
3.70 (s, 2H), 3.41 - 3.36 (m, 2H), 2.71 (t, J = 7.3 Hz, 2H), 2.07 (s, 3H), 1.61 - 1.52 (m, 4H), 1.49
(d, J = 5 Hz, 18H), 1.35 - 1.30 (m, 8H) ppm. 13C NMR (100 MHz, CDCl3) δ 163.8, 156.2, 153.5,
26

ACCEPTED MANUSCRIPT
137.7, 133.6, 132.3, 131.8, 131.5, 129.8, 129.5, 128.9, 128.4, 110.5, 83.1, 79.4, 41.1, 29.5, 29.4,
29.1, 28.4, 28.2, 27.4, 26.9, 11.1 ppm. HRMS (m/z) [M + H]+calcd for C37H51Cl2N5O4,
700.3391; found, 700.3386.
4.1.7.23. 1-(8-(((1,5-bis(4-chlorophenyl)-2-methyl-1H-pyrrol-3-yl)methyl)amino)octyl)guanidine
(8x)
Yield: 100 %. 1H NMR (400 MHz, CD3OD) δ 7.46 (d, J = 8.5 Hz, 2H), 7.20 - 7.14 (m, 4H), 7.03
(d, J = 8.7 Hz, 2H), 6.51 (s, 1H), 4.13 (s, 2H), 3.17 (t, J = 7.0 Hz, 2H), 3.10 - 3.05 (m, 2H), 2.14
(s, 3H), 1.79 - 1.70 (m, 2H), 1.62 - 1.55 (m, 2H), 1.43 - 1.38 (m, 8H) ppm. 13C NMR (100 MHz,
CD3OD) δ 158.5, 138.5, 135.2, 134.6, 133.4, 133.2, 132.4, 131.3, 130.6, 130.5, 129.3, 112.1,
45.0, 42.6, 30.6, 30.3, 30.1, 29.9, 27.7, 27.6, 27.3, 11.5 ppm. HRMS (m/z) [M + H]+calcd for
C27H35Cl2N5, 500.2342; found, 500.2341.
4.1.7.24. Boc-Protected 8y
Yield: 43 %.1H NMR (400 MHz, CDCl3) δ 11.5 (br. s, 1H), 8.22 (d, J = 8.3 Hz, 1H), 7.33 (d, J =
8.6 Hz, 2H), 7.10 (d, J = 8.6 Hz, 2H), 7.04 (d, J = 8.6 Hz, 2H), 6.93 (d, J = 8.6 Hz, 2H), 6.34 (s,
1H), 4.07 - 3.92 (m, 1H), 3.67 (s, 2H), 2.59 - 2.51 (m, 1H), 2.13 - 2.07 (m, 2H), 2.06 (s, 3H),
13
2.02 - 1.97 (m, 2H), 1.48 (d, J = 5.6 Hz, 18H), 1.37 - 1.27 (m, 4H) ppm. C NMR (100 MHz,
CDCl3) δ 163.9, 155.5, 153.4, 137.8, 133.5, 132.3, 131.7, 131.6, 131.3, 129.8, 129.4, 128.9,
128.4, 110.2, 83.0, 79.2, 55.8, 48.9, 43.0, 31.8, 31.6, 28.4, 28.2, 11.0 ppm. HRMS (m/z) [M +
H]+calcd for C35H45Cl2N5O4, 670.2921; found, 670.2920.
4.1.7.25.
1-(4-(((1,5-bis(4-chlorophenyl)-2-methyl-1H-pyrrol-3-
yl)methyl)amino)cyclohexyl)guanidine (8y
)
Yield: 100 %. 1H NMR (400 MHz, CD3OD) δ 7.46 (d, J = 8.5 Hz, 2H), 7.17 (d, J = 8.5 Hz, 2H),
7.16 (d, J = 8.4 Hz, 2H), 7.04 (d, J = 8.4 Hz, 2H), 6.58 (s, 1H), 4.18 (s, 2H), 3.56 - 3.45 (m, 1H),
3.29 - 3.21 (m, 1H), 2.37 - 2.29 (m, 2H), 2.16 (s, 3H), 2.14 -2.10 (m, 2H), 1.77 - 1.65 (m, 2H),
13
1.53 - 1.43 (m, 2H) ppm. C NMR (100 MHz, CD3OD) δ 157.8, 138.6, 135.2, 134.6, 133.5,
133.2, 132.5, 131.3, 130.6, 130.4, 129.4, 111.4, 56.4, 50.6, 42.1, 31.4, 28.5, 11.1 ppm. HRMS
(m/z) [M + H]+calcd for C25H29Cl2N5, 470.1873; found, 470.1864.
4.1.7.26. Boc-Protected 8z
Yield: 56 %. 1H NMR (400 MHz, CDCl3) δ 11.47 (br. s, 1H), 8.22 (d, J = 8.9 Hz, 2H), 7.26 (d, J
= 8.9 Hz, 2H), 7.10 (d, J = 8.6 Hz, 2H), 6.87 (d, J = 8.6 Hz, 2H), 6.60 (s, 1H), 4.07 - 3.98 (m,
1H), 3.89 (s, 2H), 2.91 - 2.82 (m, 1H), 2.21 - 2.15 (m, 2H), 2.11 (s, 3H), 1.79 - 1.62 (m, 2H),
13
1.47 (d, J = 6.2 Hz, 18H), 1.25 - 1.15 (m, 4H) ppm. C NMR (100 MHz, CDCl3) δ 163.82,
155.47, 153.44, 146.86, 144.36, 133.05, 132.71, 131.45, 131.13, 130.56, 129.27, 129.18, 128.69,
27

ACCEPTED MANUSCRIPT
124.72, 112.03, 83.25, 79.34, 54.31, 47.93, 40.20, 31.04, 30.42, 29.79, 28.40, 28.34, 28.19, 11.40
ppm. HRMS (m/z) [M + H]+calcd for C35H45ClN6O6, 681.3162; found, 681.3141.
4.1.7.27.
1-(4-(((5-(4-chlorophenyl)-2-methyl-1-(4-nitrophenyl)-1H-pyrrol-3-
yl)methyl)amino)cyclohexyl)guanidine (8z
)
Yield: 100 %. 1H NMR (400 MHz, CD3OD) δ 8.31 (d, J = 8.9 Hz, 2H), 7.42 (d, J = 8.9 Hz, 2H),
7.19 (d, J = 8.5 Hz, 2H), 7.04 (d, J = 8.5 Hz, 2H), 6.63 (s, 1H), 4.20 (s, 2H), 3.56 - 3.46 (m, 1H),
3.29 - 3.22 (m, 1H), 2.38 - 2.30 (m, 2H), 2.21 (s, 3H), 2.149 - 2.12 (m, 2H), 1.78 - 1.66 (m, 2H),
13
1.56 - 1.43 (m, 2H) ppm. C NMR (100 MHz, CD3OD) δ 157.8, 148.5, 145.4, 134.8, 133.8,
133.2, 132.2, 130.8, 130.6, 129.5, 125.7, 112.2, 56.5, 50.6, 42.1, 31.4, 28.6, 11.2 ppm. HRMS
(m/z) [M + H]+calcd for C25H29ClN6O2, 481.2113; found, 481.2098.
4.1.7.28. Boc-Protected 8aa
Yield: 24 %.1H NMR (400 MHz, CDCl3) δ 11.48 (br. s, 1H), 8.27 (br. s, 1H), 7.41 (d, J = 8.6
Hz, 2H), 7.09 (d, J = 8.5 Hz, 2H), 6.18 (s, 1H), 3.91 (s, 2H), 3.36 (q, J = 7 Hz, 2H), 2.83 (t, J = 8
Hz, 2H), 2.01 (s, 3H), 1.95 (s, 3H), 1.89 - 1.80 (m, 2H), 1.58 - 1.51 (m, 2H), 1.48 (d, J = 4.3 Hz,
18H), 1.35 - 1.19 (m, 8H) ppm. 13C NMR (100 MHz, CDCl3) δ 163.8, 156.2, 153.4, 137.0,
134.1, 129.7, 129.6, 129.2, 128.9, 108.3, 83.1, 79.3, 45.6, 42.9, 41.0, 29.2, 29.1, 28.4, 28.2, 27.0,
26.9, 12.8, 11.1 ppm. HRMS (m/z) [M + H]+calcd for C32H50ClN5O4, 604.3624; found,
604.3625.
4.1.7.29. 1-(8-(((1-(4-chlorophenyl)-2,5-dimethyl-1H-pyrrol-3-yl)methyl)amino)octyl)guanidine
(8aa)
Yield: 99 %. 1H NMR (400 MHz, CD3OD) δ 7.55 (d, J = 8.6 Hz, 2H), 7.21 (d, J = 8.6 Hz, 2H),
6.05 (s, 1H), 4.03 (s, 2H), 3.18 (t, J = 7 Hz, 2H), 3.03 - 2.99 (m, 2H), 2.03 (s, 3H), 2.00 (s, 3H),
1.77 - 1.68 (m, 2H), 1.64 - 1.57 (m, 2H), 1.44 - 1.37 (m, 8H) ppm. 13C NMR (100 MHz,
CD3OD) δ 158.6, 138.4, 135.4, 131.0, 130.7, 130.5, 130.0, 110.3, 47.7, 44.7, 42.5, 30.1, 30.0,
29.8, 27.6, 27.2, 12.7, 10.7 ppm. HRMS (m/z) [M + H]+calcd for C22H34ClN5, 500.2342; found,
500.2340.
4.1.8. Synthesis of the (trans-4-amino-cyclohexyl)-carbamic acid tert-butyl ester
9
Trans-1,4-diaminocyclohexane (1.09 mmol) was added to a solution of Et3N (1.09 mmol) in
THF (5 mL) and the resulting mixture was cooled to 0 °C. Then, a solution of di-tert-butyl
dicarbonate (4.38 mmol) in THF (5 mL) was added dropwise and the reaction was allowed to
warm up to room temperature and subsequently stirred for 24 h. The reaction mixture was then
diluted with water (10 mL) and washed twice with EtOAc (20 mL) and once with brine (10 mL).
The organic extracts were collected and then dried over MgSO4, filtered and concentrated under
28