Design, synthesis and evaluation against Mycobacterium tuberculosis of azole piperazine derivatives as dicyclotyrosine (cYY) mimics

Article abstract of DOI:10.1016/j.bmc.2017.11.030

Three series of azole piperazine derivatives that mimic dicyclotyrosine (cYY), the natural substrate of the essential Mycobacterium tuberculosis cytochrome P450 CYP121A1, were prepared and evaluated for binding affinity and inhibitory activity (MIC) against M. tuberculosis. Series A replaces one phenol group of cYY with a C3-imidazole moiety, series B includes a keto group on the hydrocarbon chain preceding the series A imidazole, whilst series C explores replacing the keto group of the piperidone ring of cYY with a CH₂-imidazole or CH₂-triazole moiety to enhance binding interaction with the heme of CYP121A1. The series displayed moderate to weak type II binding affinity for CYP121A1, with the exception of series B 10a, which displayed mixed type I binding. Of the three series, series C imidazole derivatives showed the best, although modest, inhibitory activity against M. tuberculosis (17d MIC = 12.5 μg/mL, 17a 50 μg/mL). Crystal structures were determined for CYP121A1 bound to series A compounds 6a and 6b that show the imidazole groups positioned directly above the haem iron with binding between the haem iron and imidazole nitrogen of both compounds at a distance of 2.2 ?. A model generated from a 1.5 ? crystal structure of CYP121A1 in complex with compound 10a showed different binding modes in agreement with the heterogeneous binding observed. Although the crystal structures of 6a and 6b would indicate binding with CYP121A1, the binding assays themselves did not allow confirmation of CYP121A1 as the target.

Full text of DOI:10.1016/j.bmc.2017.11.030

Accepted Manuscript
Design, synthesis and evaluation against Mycobacterium tuberculosis of azole
piperazine derivatives as dicyclotyrosine (cYY) mimics
Hend A.A. Abd El-wahab, Mauro Accietto, Leonardo B. Marino, Kirsty J.
McLean, Colin W. Levy, Hamdy M. Abdel-Rahman, Mahmoud A. El-Gendy,
Andrew W. Munro, Ahmed S. Aboraia, Claire Simons
PII:
S0968-0896(17)31920-X
https://doi.org/10.1016/j.bmc.2017.11.030
BMC 14082
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
28 September 2017
14 November 2017
18 November 2017
Please cite this article as: El-wahab, H.A.A., Accietto, M., Marino, L.B., McLean, K.J., Levy, C.W., Abdel-Rahman,
H.M., El-Gendy, M.A., Munro, A.W., Aboraia, A.S., Simons, C., Design, synthesis and evaluation against
Mycobacterium tuberculosis of azole piperazine derivatives as dicyclotyrosine (cYY) mimics, Bioorganic &
Medicinal Chemistry (2017), doi: https://doi.org/10.1016/j.bmc.2017.11.030
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Design, synthesis and evaluation against Mycobacterium tuberculosis
of azole piperazine derivatives as dicyclotyrosine (cYY) mimics
a b
,
a c
,
d
Hend A.A. Abd El-wahab, Mauro Accietto, Leonardo B. Marino, Kirsty J.
b
b
e
e
McLean, Colin W Levy, Hamdy M. Abdel-Rahman, Mahmoud A. El-Gendy,
b
a
e
*
Andrew W. Munro, Ahmed S. Aboraia, and Claire Simons
a
School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff CF10 3NB, Wales, UK
Department of Medicinal Chemistry, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt
b
c
Department of Life Sciences, University of Modena and Reggio Emilia, Via G. Campi 183, 41125
Modena, Italy
d
Faculty of Pharmaceutical Sciences, UNESP - Univ Estadual Paulista, Araraquara, São Paulo 14801-
902, Brazil.
eManchester Institute of Biotechnology, School of Chemistry, University of Manchester, Manchester
M1 7DN
*
Address for correspondence: Claire Simons, School of Pharmacy and
Pharmaceutical Sciences, Cardiff University, Redwood Building, King Edward VII
Avenue, Cardiff CF10 3NB, Wales, UK. Tel: 02920-876307. E-mail:
simonsc@cardiff.ac.uk
1

Abstract
Three series of azole piperazine derivatives that mimic dicyclotyrosine (cYY), the
natural substrate of the essential Mycobacterium tuberculosis cytochrome P450
CYP121A1, were prepared and evaluated for binding affinity and inhibitory activity
(
MIC) against M. tuberculosis. Series A replaces one phenol group of cYY with a C3-
imidazole moiety, series B includes a keto group on the hydrocarbon chain preceding
the series A imidazole, whilst series C explores replacing the keto group of the
2 2
piperidone ring of cYY with a CH -imidazole or CH -triazole moiety to enhance
binding interaction with the heme of CYP121A1. The series displayed moderate to
weak type II binding affinity for CYP121A1, with the exception of series B 10a,
which displayed mixed type I binding. Of the three series, series C imidazole
derivatives showed the best, although modest, inhibitory activity against M.
tuberculosis (17d MIC = 12.5 µg/mL, 17a 50 µg/mL). Crystal structures were
determined for CYP121A1 bound to series A compounds 6a and 6b that show the
imidazole groups positioned directly above the haem iron with binding between the
haem iron and imidazole nitrogen of both compounds at a distance of 2.2 Å. A model
generated from a 1.5 Å crystal structure of CYP121A1 in complex with compound
10a showed different binding modes in agreement with the heterogeneous binding
observed. Although the crystal structures of 6a and 6b would indicate binding with
CYP121A1, the binding assays themselves did not allow confirmation of CYP121A1
as the target.
Keywords
Dicyclotyrosine derivatives; CYP121A1; Mycobacterium tuberculosis; binding
affinity; molecular modeling; X-ray crystallography
2

1
. Introduction
Tuberculosis (TB) is an infectious, communicable bacterial disease, most
commonly of the respiratory tract, usually caused by Mycobacterium tuberculosis
1
(
Mtb). Despite the 47% mortality rate reduction (1990-2014), case numbers are
2
rising in developing countries with 1.8 million deaths from TB in 2015. An
2
estimated 480,000 people developed multi-drug resistant TB (MDR-TB) in 2015.
The rise in MDR-TB, a form of TB caused by bacteria that do not respond to
3
isoniazid and rifampicin, the two most effective first-line TB drugs, is alarming
and highlights the need for the identification of new drug targets.
Figure 1. Formation of mycocyclosin. M. tuberculosis CYP121A1 catalyses the oxidative
crosslinking of the aryl side chains of the cyclic dipeptide, cyclo-L-Tyr-L-Tyr (cYY), to
produce the novel secondary metabolite mycocyclosin.
A potential new target is the cytochrome P450 enzyme CYP121A1 that was shown to
be essential for mycobacterial growth through gene deletion and complementation
4
studies. The natural substrate of CYP121A1 is the cyclic dipeptide (CDP)
dicyclotyrosine (cYY) and it was found that CYP121A1 exhibits a novel
diketopiperazine modifying activity, catalysing the formation of a C-C bond between
the two tyrosyl side chains of cYY resulting in a novel chemical entity called
mycocyclosin (Figure 1). The role of the secondary metabolite mycocylosin in M.
tuberculosis remains unclear, although CDPs and their derivatives have important
biological effects, including (i) cFP in the bacterial pathogen Vibrio cholerae,
production of which inhibits production of bacterial virulence factors, (ii) cLY which
3

can inhibit biofilm formation in the skin bacterium Staphylococcus epidermidis; and
5
-7
cHP which has reported neuroprotective effects in mammals.
Recent studies revealed that only cyclodipeptides with a diketopiperazine ring and
two aryl side chains with L-configuration bound well to CYP121A1, and that only
8,9
cYY itself was efficiently transformed to a product.
The binding of cYY to CYP121A1 includes a direct hydrogen bond interaction
between the carbonyl of cYY and the side chain nitrogen of Asn85, and a water-
mediated hydrogen bonding pattern. Hydrophobic interactions are demonstrated on
the other side chain (Figure 2). The CDP cYY is a small molecule that partially
3
occupies (300 Å) the large active site cavity (1350 Å ) of CYP121A1, and makes
several interactions with P450 amino acids, both directly and via interstitial active site
9
water molecules.
Figure 2. Crystal structure of cYY bound to CYP121A1. cYY is shown with carbon atoms in
yellow, and with interactions between cYY and active site amino acids and water molecules
shown with dotted lines. cYY shows indirect binding coordination of the CYP121A1 heme
iron through an axial water molecule (red) to the heme iron (cyan) (pdb code: 3G5H).
4

Three small series that mimic the natural substrate cYY have been developed (Figure
). Series A replaces one of the phenol rings of cYY, which is positioned directly
3
above the heme group in the crystal structure (Figure 2), for a C3-imidazole. Flexible
alignment showed that the three carbon chain allowed optimal overlap of the
imidazole ring with the phenol ring (Figure 3). Also in this series, the central
piperazine ring retains the planar conformation of cYY.
Series B flexible alignment
Series C flexible alignment
Series A flexible alignment
Figure 3. Designed cYY mimics from series A, B and C. Flexible alignment of cYY (yellow)
with series A (cyan), series B (orange) and series C (magenta).
Series B includes a keto group on the hydrocarbon chain preceding the series A
imidazole. The rationale for this modification of series B was to mimic the piperazine
keto group, and the shorter two carbon chain, attached to a piperidine ring with more
conformational flexibility, that was a sufficient length to allow overlap of the
imidazole with the phenol ring (Figure 3). Series 3 is perhaps the closest mimic
retaining two aromatic rings, although it has the piperazine ring with more
5

conformational flexibility, as with series B. In Series C the keto group of the
piperidone ring of cYY is replaced by a CH -imidazole or CH -triazole, to determine
2
2
whether a direct interaction between the inhibitor compound and the heme group
might place the inhibitor more optimally within the active site.
The choice of imidazole and triazole as the haem-binding heterocycles was based on
1
0,11
previous binding studies,
which showed that the imidazole interacts optimally
III
through a N:-Fe -haem coordinate link, through the N3 of imidazole, while
introducing an additional nitrogen in the heterocyclic ring (triazole) reduces the
coordination potential through the N: due to the electron withdrawing effect of the
additional electronegative nitrogen. This effect is further increased with two
additional nitrogens (tetrazole) drawing electron density from both sides of the
coordinating nitrogen. Although the triazole is expected to have a lower binding
affinity for the haem iron, the lower basicity of the triazole can confer greater
1
2
isoform-specific selectivity in various CYP enzymes.
2
. Results and discussion
2.1 Chemistry
Series A began with the preparation of the 4-(substituted)-3-oxo-piperazine-1-
1
3
carboxylic acid tert-butyl esters (3) according to the literature procedure by addition
of the substituted benzyl chlorides (1) to a solution of 1-Boc-3-oxopiperazine (2) in
anhydrous DMF in the presence of NaH (Scheme 1). Deprotection of the Boc
protecting group of (3) was achieved using 4 M HCl solution in dioxane to obtain the
piperazine (4) in very good yields. Treatment of 1-(substituted)-piperazin-2-one (4)
with K
2 3
CO and dibromopropane for 48 h gave the alkylated products (5) after
1
4
purification by flash column chromatography. Reaction of 4-(bromopropyl)-1-(4-
6

substituted-benzyl)-piperazin-2-ones (5) with the sodium salt of imidazole gave the
final imidazole products (6) in moderate yields (Scheme 1).
o
Scheme 1. Reagents and conditions: (i) NaH, DMF, 70 C, o/n (ii) 4 M HCl, dioxane, o/n (iii)
o
K
2
CO
3
, acetone, Br(CH
2
)
3
Br, 48 h (iv) imidazole, NaH, DMF, 45 C, o/n. [a, R = 4-F; b, R =
4
-Cl; c, R= 4-OCH
3
; d, R = 2,4-diCl]
Series B began with the preparation of 1-(substituted-benzyl)piperazine (8) by
addition of substituted benzyl chloride (1) to a solution of excess piperazine (7) in
anhydrous THF. Treatment of the formed 1-(substituted-benzyl)piperazine (8) with
15
triethylamine and 2-chloroacetyl chloride gave the acylated products (9), which
were pure enough to proceed for the next step. The final compounds (10) were
prepared by reaction of the acylated products (9) with 1,2,4-triazole or imidazole in
2 3
the presence of K CO (Scheme 2).
o
3
Scheme 2. Reagents and conditions: (i) THF, 70 C, 2.5 h (ii) 2-chloroacetylchloride, Et N,
o
o
o
0
C, 2 h then rt 5 h (iii) K
2
CO
3
, CH
3
CN, imidazole or triazole, 45 C, 1 h then 9, 70 C, o/n.
7

For Series C the substitutions were limited to unsubstituted and methoxy groups. The
methoxy substituents were included as H-bond acceptors to mimic the phenolic
hydroxyl of the tyrosine group of cYY. The imines (12) were prepared by reaction of
ethylenediamine and the desired benzaldehyde (11) in ethanol overnight at room
16
temperature (Scheme 3). Subsequent reduction of the imines (12) with NaBH gave
4
1
7
the diamines (13) , which on reaction with 2,3-dibromopropionic acid ethyl ester and
triethylamine in toluene at 80 °C resulted in cyclisation to the piperazine esters (14).
4
Ester reduction was performed using LiAlH giving the corresponding alcohols (15).
The unsubstituted and 4-methoxy derivatives 15a and 15b were then converted to the
chlorides (16a and 16b) on reaction with thionyl chloride at reflux overnight.
Attempts to convert the dimethoxy alcohol (15c) to the chloride were unsuccessful
with a complex reaction mixture obtained, and therefore the dimethoxy alcohol (15c)
was first converted to the mesylate intermediate by reaction with methanesulphonyl
chloride in the presence of base (Et N). Treatment of the chlorides and mesylate with
3
either the sodium salt of imidazole or triazole generated in situ on reaction of
imidazole and triazole, respectively, with NaH in acetonitrile or toluene, gave the
final imidazole and triazole compounds (17) (Scheme 3).
8

Scheme 3. Reagents and conditions: (i) H
2
NCH
2
CH
2
NH
2
, EtOH, o/n (ii) NaBH
4
, MeOH, o/n
, Et O, o/n
CN, 80 C, o/n (vii)
, 4 h (vii) NaH, imidazole, toluene, 115 C, o/n. [a, R = H, X =CH; b, R =
H, X = N; c, R= 4-OCH , X = N; d, R = 3,5-diOCH , X = CH]
o
(
iii) 2,3-dibromopropionic acid ethyl ester, toluene, Et
3
N, 80 C, o/n (iv) LiAlH
4
2
o
o
(v) SOCl
2
, toluene, 115 C, o/n (vi) NaH, imidazole or triazole, CH
3
o
3 2 2
MsCl, Et N, CH Cl
3
3
2.2 Binding affinity
UV–visible absorption spectroscopy is an important technique for the characterization
and quantification of cytochrome P450 enzymes, including the assessment of whether
an applied ligand is a likely substrate or inhibitor, based on it causing a heme Soret
band blue or red shift, respectively. The oxidized form of pure CYP121A1 shows
spectral properties that are similar to those of many other predominantly low-spin
P450 enzymes, with the major (Soret or γ) ferric heme band located at 416 nm (most
P450s have the low-spin Soret feature at ~418-419 nm), and the smaller α and β
bands at 565 and 538 nm, respectively. On reduction of CYP121A1 with sodium
dithionite and binding of carbon monoxide at basic pH, the Soret band shifts to 448
9

nm with a small decrease in intensity, confirming that cysteine thiolate coordination is
II
18
retained in the Fe -CO state (Figure 4).
Figure 4: Spectroscopic characteristics of CYP121A1. CYP121A1 (solid line, 5.2 µM) in the
normal low-spin resting form with Soret peak at 416nm and α and β bands at 565 and 538
nm, respectively. CYP121A1 retains a small degree of high-spin with a charge transfer band
at 649 nm and small Soret shoulder at 394 nm. Reduction with sodium dithionite (dotted line)
shows a decrease in intensity of the Soret peak and shift to 406 nm with fusion of the α and β
bands to a new peak at 547nm. Addition of carbon monoxide to reduced CYP121A1 shows
the characteristic ‘P450’ peak at 448 nm.
Binding titrations were carried out for compounds 6 (series A), 10 (series B) and 17
(
series C). Results shown in Table 1 revealed that compounds from series A, B and C
produce small spectral changes and produce moderate to high dissociation constants
using either a hyperbolic fit or the Hill function in cases where the data were
sigmoidal in nature. Addition of all the compounds (with the exception of 10a)
showed a type II (inhibitor-like) red shift in the heme Soret peak to a longer
wavelength, indicating binding of the azole (or triazole) nitrogen to the heme iron
(
Figure 5). Series A displays the tightest binding with lowest K values determined in
d
1
0

particular for the halogenated compound 6b (4-Cl group, K
compound 6a (4-F group, K = 225.8 ± 2.6 µM). Interestingly, the triazole-containing
-4-dichloro derivative compound 10a displays a heterogeneity in binding mode with
d
= 81.3 ± 2.9 µM) and
d
2
the development of a blue-shifted high spin type I (substrate-like) shoulder at 393 nm
that is accompanied by the appearance of a small charge transfer species at 648 nm,
which is also associated with substrate-like binding. In contrast, the remaining Series
B and C compounds display small type II inhibitor like spectral changes upon binding
to CYP121A1 (Figure 5). The binding of compounds 6b, 6c and 10a to CYP121A1
was best fitted using the Hill function. The apparent cooperative nature of the binding
of these ligands may suggest that more than one molecule of these compounds can
enter the CYP121A1 active site, or e.g. that there are interactions between CYP121A1
proteins that influence binding affinity. Such phenomena are relatively common in
1
9
P450 enzymes, as described by Korzekwa et al.
A
B
1
1

Figure 5. CYP121A1 titrations with selected novel ligands. (A) Spectral titration data for
CYP121A1 with various ligands designed and synthesized in this study. Most ligands
produce a type II spectral shift indicative of direct or indirect (e.g. via H
interactions of these ligands with the P450 heme iron. The exception shown here is molecule
0a, which induces a type I spectral change indicative of substrate-like P450 behaviour. (B)
Data plots for each ligand, fitted using either hyperbolic (Michaelis-Menten) or sigmoidal
Hill) functions. Data for compound 6a were best fitted using the hyperbolic function, while
2
O molecule(s))
1
(
data for compounds 6b, 6c and 10a were fitted using the Hill function.
Azole antifungal drugs (clotrimazole, econazole, fluconazole, ketoconazole
and miconazole) were found to bind tightly to Mtb CYP121A1 inducing a shift of the
1
2

soret peak to 423.5 nm. The K
d
values for clotrimazole, econazole and miconazole are
<
0.2 ꢀM indicating tight binding. Fluconazole a triazole compound, showed weaker
binding with a K
d
value 8.6 ±0.2 μM, while the imidazole-containing clotrimazole
value of 0.07 ± 0.01 μM, as
showed much higher affinity than fluconazole with a K
d
18
determined from a fit to the Morrison equation (Table 1). The natural substrate cYY
has a K value of 5.82 ±0.16 μM.
d
Table 1. K
d
, MIC data and calculated LogP for series A and series B
a
Compound
R
X
K (µM)
d
MIC90
cLogP
(µg/mL)
6a
6b
6c
4-F
-
-
-
225.8 ± 2.6
81.3 ± 2.9
>100
1.19
1.59
0.91
4-Cl
>100
4-OCH
3
308.9 ± 21.8
>100
6
1
1
1
1
1
1
1
d
2,4-diCl
2,4-diCl
4-F
-
967.1 ± 41.3
417 ± 39
100
>100
>100
>100
50
2.15
1.63
1.33
1.88
3.56
3.06
2.81
3.05
0a
0b
0c
7a
7b
7c
7d
N
N
960 ± 62
4-F
CH
CH
N
1142 ± 144
1968 ± 137
1834 ± 62
1298 ± 124
1435 ± 94
H
H
>100
100
4-OCH
3
N
3,5-diOCH
3
CH
12.5
1
5
1
Fluconazole
-
-
-
-
-
-
8.6 ± 0.2
0.07 ± 0.01
5.82 ± 0.16
>100
0.1
-
0.87
5.97
1.26
5
Clotrimazole
cYY
2
1
a. Crippen's fragmentation
2.3 EPR spectroscopy of ligand binding to CYP121A1
X-band EPR spectroscopy provides important information on P450 heme iron
coordination environment and any heterogeneity therein. EPR studies were done for
1
3

CYP121A1 in the ligand-free state and in complex with the compounds from series
A, B and C.
Table 2. X-band EPR spectra of CYP121A1 in its ligand-free form and for complexes with
ligands from series A, B and C.
CYP121A1
ligand/additive
High-spin (HS)
g-values
Low-spin (LS) g-values
% HS
No additive
DMSO
7.94, 3.59
7.94, 3.58
7.94, 3.48
7.94, 3.43
7.96
2.49, 2.25, 1.89
2.49, 2.25, 1.89
2.46, 2.26, 1.90
2.46, 2.26, 1.90
2.47, 2.26, 1.90
2.45, 2.26, 1.90
2.47 (sh. 2.41), 2.26, 1.90
2.47, 2.26, 1.90
2.47, 2.26, 1.89
2.47, 2.26, 1.89
2.47, 2.26, 1.89
2.47, 2.26, 1.90
2.47, 2.26, 1.90
2.46, 2.25, 1.90
2.45, 2.26, 1.90
2.5
2.5
1.2
1.3
1.2
1.2
22
6
6
a
b
6
c
6d
7.97
1
0a
0b
7.92, 3.61, 1.70
7.96, 3.51
7.96, 3.48
7.95, 3.58
7.96, 3.54
7.95, 3.52
7.95, 3.44
8.05, 3.44
7.94, 3.58
1
1.3
1.2
1.2
1.3
1.1
1.1
2.9
2.5
1
0c
7a
1
1
7b
7c
7d
1
1
cYY
Fluconazole
The g-values for the LS and HS forms of CYP121A1 are indicated in each case with the
percentage high-spin (HS) in each sample calculated from integration of the spectral peaks.
The g-values are given as g
too weak to be resolved clearly in most samples. The low intensity of the g
precludes its accurate positioning in the case of compounds 6c and 6d. The “sh.” in the case
of compound 10a indicates a shoulder feature at g = 2.41 on the main g value at 2.47.
/g /g
z y x
for both the LS and HS components. The HS g
x
signal is
y
signal also
z
z
Ligand-free CYP121A1 has a major rhombic LS spectrum with g-values at 2.49 (g
z
),
2
.25 (g ) and 1.85 (g ) (2.49/2.25/1.85), consistent with a dominant axial coordination
y
x
1
4

state from cysteine thiolate (proximal) and water (distal) ligands. In addition to the LS
signal, there is a small proportion of HS heme retained in CYP121A1, with g-values
at 7.94 and 3.59 (Table 2, Figure 6). This small HS signal is consistent with the native
CYP121A1 UV-Vis spectrum (Figure 4). EPR analyses of CYP121A1 in complex
with the compounds, with the exception of the series B compound 10a, revealed
subtle perturbations in the EPR spectrum, with ~50% decreases in the HS spectral
component and a slight narrowing of the LS signal with g-values of the
8
2
.46(2.47)/2.26/1.90 (c.f. cYY 2.46, 2.25, 1.90) .
Figure 6. X-band EPR spectra for CYP121A1 in the ligand-free form and when bound to
compounds 6a and 10a. The g-values are indicated for each peak with HS signals labelled
with an asterisk.
These results are not immediately indicative of direct coordination of the heme iron
2
by nitrogen atoms in these molecules, although indirect ligation (via H O
molecule(s)) cannot be ruled out. In contrast, the addition of compound 10a displayed
a significant perturbation of the EPR spectrum with a 9-fold increase in the amount of
HS ferric heme iron (Table 2). A small degree of heterogeneity in the LS signal was
z
also observed, with the formation of a shoulder in the LS g signal at 2.41 suggesting
1
5

a different binding mode for this ligand in the CYP121A1 active site. Selected EPR
spectra are shown in Figure 6.
2.4 Antimycobacterial assay
The derivatives of series A, B and C were screened against Mtb H37Rv by the REMA
2
0
(
Resazurin Microtiter Assay) method. In series A only compound 6d displayed
inhibitory activity against M. tuberculosis (Table 1) and compounds from Series B
were inactive (all >100 µg/mL). Series C was more promising with inhibitory activity
observed for three of the derivatives, and with notable activity for the imidazole
derivatives 17a and 17d (50 and 12.5 µg/mL respectively). There would appear to be
a correlation between lipophilicity (cLogP, Table 1) and antimycobacterial activity,
which might be explained by enhanced uptake across the lipid dense outer
mycobacterial cell wall.
2
.5 Crystallography and Molecular Modelling
The structures of CYP121A1 in complex with compounds 6a and 6b were solved to
.64 and 1.5 Å, respectively (PDB codes 5O4L and 5O4K) (Table 3). Compound 6a
1
and 6b both sit in a very similar position within the CYP121A1 active site with the
imidazole nitrogen coordinated to the heme iron at a distance of 2.2 Å in both cases
(
Figure 6). A direct H-bond interaction between the keto group of the piperazine ring
and Asn85 is observed. In addition, water-bridged H-bonds are formed between the
protonated piperazine amine and Met62, Val82 and Val83, and between the
piperazine ring and propyl chain linker and Ala233, Gln385 and Arg386. The
observed interactions of 6a and 6b closely mimic the interactions observed with cYY
binding (Figure 2).
1
6

Table 3. X-ray crystallography data collection and final structural refinement statistics for
CYP121A1 in complex with compounds 6a and 6b.
Data Collection and
Refinement
CYP121A1/6a complex
CYP121A1/6b complex
a
PDB ID 5O4L
PDB ID 5O4K
Data Collection
Wavelength (Å)
Space group
0.9795
0.9795
P6 22
5
P6
5
22
Cell dimensions
a, b, c (Å)
77.53, 77.53, 264.95
77.625, 77.625, 264.291
ꢁ
, ꢂ, ꢃ (°)
90.0, 90.0, 120.0
90.0, 90.0, 120.0
Resolution (Å)
Unique reflections
Multiplicity
Completeness (%)
Mean I/sigma(I)
Wilson B-factor (Å )
R-merge
65.09 - 1.64 (1.70 - 1.64)
47.12 - 1.5 (1.56 - 1.5)
58784 (5714)
19 (20)
100 (100)
12.5(1.7)
20.3
76451 (7498)
19 (20)
100 (100)
18.1 (3.0)
16.1
2
16.9
11.1
R-meas
17.7
11.5
R-pim
Refinement
4.1
2.6
CC1/2
0.99 (0.49)
58784 (5714)
2000 (195)
0.18 (0.33)
0.20 (0.34)
3588
0.99 (0.91)
76444 (7497)
No. Reflections (unique)
No. Reflections (R-free)
R-work
1993 (195)
0.16 (0.24)
0.19 (0.26)
3719
3128
81
R-free
No. non-hydrogen atoms
macromolecules
ligands
3115
71
solvent
Protein residues
RMS(bonds)
402
394
0.016
1.66
99
0.5
0
0.87
3.11
510
394
0.019
1.92
99
0.98
0
1.8
3.24
RMS(angles)
Ramachandran favored (%)
Ramachandran allowed (%)
Ramachandran outliers (%)
Rotamer outliers (%)
Clashscore
Average B-factor
macromolecules
ligands
23.93
22.66
25.95
33.39
1
20
18.32
21.99
30
solvent
No. TLS groups
1
a
Data for the highest resolution shell are shown in parentheses
1
7

a
b
Figure 6. X-ray crystal structures and electron density maps of (a) 6a and (b) 6b binding to
CYP121A1.
A 1.5 Å crystal structure of CYP121A1 in complex with compound 10a was obtained.
However, incomplete ligand occupancy prevented adequate refinement of the
structure. Analysis of the ligand density of compound 10a suggests that it likely
acquires different binding orientations with two major conformations being rotated
around the substituted piperazine central moiety of the ligand. In both orientations the
imidazole groups are located pointing away from the heme towards the top of the
active site cavity. In one orientation the 4-chloro group is situated 3.1 Å from the
heme iron and is sandwiched between the active site residues Ser237 and Ala233
(
Figure 7). In contrast, the same 4-chloro group in the second ligand orientation is
1.6 Å away from the iron and rotated 35.2 degrees from the former orientation with
1

respect to the heme iron (Figure 7). For this second orientation, the 2-4-
dichlorobenzene ring stacks with the aromatic Phe168 residue that interacts with the
one of the tyrosyls of the natural substrate cYY. The different orientations of
compounds 10a and the proximity of the halogen (4-Cl group) to the heme iron are in
agreement with the UV-vis spectral data observed that reflect different binding modes
and a degree of high-spin (HS) species present.
Binding mode 1 (cyan structure)
Binding mode 2 (magenta structure)
Figure 7. Multiple binding modes for compound 10a in its complex with CYP121A1.
Flexible alignment of series C with cYY as shown in Table 4 (17 (grey), natural
substrate cYY (yellow)), indicated the overlap of the cYY tyrosyl groups with the two
aromatic moieties. The cYY ketopiperazine ring is overlapped with the piperazine,
1
9

while the imidazole and triazole rings are placed in the same position of the cYY
carbonyl group, but at a longer distance where they act as hydrogen bond acceptors.
The score of alignment is shown in Table 4 with compound 17c showing the best
score (Table 4).
Table 4. Score of flexible alignment of series C compounds
Compound
R
H
X
CH
N
Score (Kcal/mol)
-21.99
1
7a
7b
1
H
-36.39
1
7c
OCH
3
N
-38.30
1
7d
3,5-dimethoxy
CH
-19.44
2
2
From docking studies using MOE (Molecular Operating Environment) software it
appeared that the orientation of the Series C piperazine derivatives (S- and R-
enantiomers) in the CYP121A1 active site may allow the formation of a transition
metal interaction directly between the nitrogen atom of the imidazole or triazole and
the heme iron, as the distance was less than 3 Å. Hydrophobic interactions made by
the series C compounds included those with Phe280, Ser237, Ala233, Pro285 and
Thr77, which are key amino acids that interact with the natural substrate cYY (Figure
8
). However, binding studies would suggest indirect binding to the heme via a water
molecule might be the more likely interaction mode.

Figure 8. Docking of the Series C piperazine derivative (S)-17c into the CYP121A1 active
site showing the distance (2.55 Å) between the triazole nitrogen and the haem iron.
3
. Conclusions
A series of novel ligands were developed as potential inhibitors of the Mtb P450
enzyme CYP121A1 (Figure 3). These molecules are structural mimics of the natural
cYY substrate, but to which a heme iron-coordinating imidazole or triazole group are
appended in different positions on the scaffold, with other functional groups also
introduced in place of the hydroxyl group(s) in cYY. For series A, crystallography
would indicate a direct binding interaction with the haem iron. However, this does not
translate to strong binding affinity. It could be that the structures are just locked in a
‘
bound’ orientation that favors their structural solution, whereas in solution they are
more mobile as indicated by the small spectral changes observed in the UV-Vis
spectrum. In series B compound 10a showed a distinct type I binding mode (Figure 5)
further supported by a significant perturbation of the EPR spectrum with a 9-fold
increase in the amount of HS ferric heme iron (Table 2, Figure 6). Further
2
1

development of series C molecules is warranted based on their inhibitory activity
against Mtb (Table 1). While CYP121A1 optical binding assays do not show
significant perturbation of the heme spectrum, this may be due to the series C
inhibitors binding in an orientation that does not influence the heme axial
coordination environment. However, at present we cannot conclude definitively that
the activity against Mtb results from inhibition of CYP121A1. Binding affinity in the
9
low µM range against CYP121A1 has been reported by Fonvielle et al for cYY
2
3,24
derivatives and by Hudson et al
for small fragment heterocyclic inhibitors.
However, no inhibitory activity (i.e. MIC values) against Mtb has been reported, and
so it is unknown whether the binding affinity would translate to a potent MIC value.
Similarly, the antifungal azole fluconazole shows good CYP121A1 binding affinity,
2
5
but no Mtb inhibitory activity (although other azole drugs, e.g. econazole, do
1
8
possess anti-Mtb activity). There are 20 different P450 isoforms encoded within the
2
6
M. tuberculosis genome, a number of which are considered important for
pathogenicity and survival in the host, and these P450s could provide novel drug
targets against pathogenic mycobacteria. The synthesis of CYP121A1 inhibitors is
still in its infancy and, with the exact physiological role of this enzyme still to be
determined, the development of selective inhibitors will be important for the analysis
of CYP121A1 mechanism, as well as being potential leads for new antibiotics that
target cytochrome P450 monooxygenase chemistry in Mtb.
4
. Experimental
4.1 General Experimental
All chemicals, reagents and solvents were purchased from Sigma-Aldrich and Fisher
Scientific companies and the required solvents were dried and stored over
2
2

4ꢀÅꢀmolecular sieves under nitrogen. Flash column chromatography was performed
with silica gel 60 (Merck 40–60 nm, 230–400 mesh) and TLC was performed on
precoated silica gel plates (Merck Kiesegel 60 F254) with visualization via UV light
(
254 nm) and / or vanillin stain. Melting points were determined on an electrothermal
instrument (Gallenkamp) and are uncorrected. Compounds were visualized by
1
13
irradiation with UV light at 250 nm and 366 nm. H and C-NMR spectra were
recorded on a Bruker Advance DPX500 spectrometer operating at 500 MHz and 125
MHz. Mass spectra (LRMS and HRMS) were determined under EI (Electron Impact)
or CI (Chemical Ionization) conditions at the EPSRC National Mass Spectrometry
Service Centre, University of Wales, Swansea, or at the Department of Chemistry,
University of Wales, Cardiff. Microanalysis data were performed by Medac Ltd.,
Brunel Science Centre, Surrey. The chloroacetyl derivatives (9) imines (12) and
1
5-17
diamines (13) were prepared as previously described.
4.2 Chemistry
4.2.1. General method for the synthesis of 1-Boc- 4-(substituted)-3-oxopiperazine
(
3)
To a solution of 1-Boc-3-oxopiperazine (2) (0.5 g, 2.49 mmol) in anhydrous DMF (10
mL) was added NaH (60% dispersion in mineral oil) (0.08 g, 3.7 mmol). The reaction
was stirred at room temperature for 2 h then substituted benzyl chloride (1) (2.99
mmol) was added and the reaction heated at 70 °C overnight. The reaction mixture
was cooled in an ice bath, quenched with water (5 mL) and then evaporated under
reduced pressure. The product was extracted with ethyl acetate (100 mL) and washed
with water (3 x 50 mL). The organic layer was dried (MgSO
reduced pressure and the residue purified by flash chromatography using
CH Cl :CH OH gradient elution unless stated otherwise.
4
), evaporated under
2
2
3
2
3

4
.2.1.1. tert-Butyl 4-(4-fluorobenzyl)-3-oxopiperazine-1-carboxylate (3a)13
Synthesised using 1-chloromethyl-4-fluorobenzene (0.43 g, 2.9 mmol). Yield: 63% as
1
a white solid, m.p: 86-92 °C. H-NMR (CDCl
3
): ꢄ 7.20 (m, 2H, Ar), 7.03 (m, 2H, Ar),
), 3.60 (t, J = 5.5 Hz, 2H, CH ), 3.27 (t, J = 5.5 Hz,
), 1.17 (s, 9H, t-Bu). C-NMR (CDCl ): ꢄ 165.8 (C=O, piperazine), 163.4,
61.4 (C-F), 153.8 (C=O), 132.1 (C), 132.05 (2 x CH, Ar), 115.7 (2 x CH, Ar), 80.9
), 45.6 (2 x CH ), 28.6 (3 x CH , t-Bu).
.2.1.2. tert-Butyl 4-(4-chlorobenzyl)-3-oxopiperazine-1-carboxylate (3b)
4
2
1
.59 (s, 2H, CH
2
), 4.10 (s, 2H, CH
2
2
1
3
H, CH
2
3
(
C, t-Bu), 49.3 (2 x CH
2
2
3
4
Synthesised according to the general procedure using 4-chlorobenzylchloride (0.48 g,
.9 mmol) the residue was purified by flash chromatography using petroleum ether:
2
EtOAc, the product was collected at 40:60 v/v. Yield: 41% as a white solid, m.p: 80-
1
8
2 °C. H-NMR (CDCl
3
): ꢄ 7.32 (d, J = 8.4 Hz, 2H, Ar), 7.22 (d, J = 8.4 Hz, 2H,
), 4.16 (s, 2H, CH ), 3.60 (t, J = 5.4 Hz, 2H, CH ), 3.26 (t, J =
): ꢄ 164.8 (C=O, piperazine),
53.8 (C=O), 134.8 (C), 133.7 (C), 129.6 (2 x CH, Ar), 129.0 (2 x CH), 80.9 (C, t-
), 45.7 (2 x CH ), 28.3 (3 x CH , t-Bu). Anal. Calcd for
(324.80): C, 59.17; H, 6.52; N, 8.62. Found: C, 59.15; H, 6.43; N,
Ar), 4.59 (s, 2H, CH
2
2
2
1
3
5
.2 Hz, 2H, CH
2
), 1.48 (s, 9H, t-Bu). C-NMR (CDCl
3
1
Bu), 49.5 (2 x CH
2
2
3
16 2 3
C H21ClN O
8.38.
4.2.1.3. tert-Butyl 4-(4-methoxybenzyl)-3-oxopiperazine-1-carboxylate (3c)
Synthesised using 4-methoxybenzylchloride (0.46 g, 2.9 mmol). Yield: 74% as a
1
white solid, m.p: 96-98 °C. H-NMR (CDCl
3
): ꢄ 7.2 (d, J = 8.6 Hz, 2H, Ar), 6.87 (d, J
), 4.14 (s, 2H, CH ), 3.8 (s, 3H, CH ), 3.57 (t, J =
), 3.24 (t, J = 5.4 Hz, 2H, CH ), 1.40 (s, 9H, t-Bu). C-NMR
): ꢄ 165.6 (C=O, piperazine), 162.5 (C), 153.8 (C=O), 129.7 (2 x CH, Ar),
28.6 (C), 114.1 (2 x CH, Ar), 80.7 (C, t-Bu), 55.3 (CH ), 49.3 (2 x CH ), 45.3 (2 x
=
8.5 Hz, 2H, Ar), 4.56 (s, 2H, CH
.4 Hz, 2H, CH
CDCl
2
2
3
13
5
2
2
(
3
1
3
2
2
4

CH), 28.3 (3 x CH
N, 8.70. Found: C, 63.48; H, 7.60; N, 8.60.
.2.1.4. tert-Butyl-4-(2,4-dichlorobenzyl)-3-oxopiperazine-1-carboxylate (3d)
Synthesised according to the general procedure using 2,4-dichloro-1-
chloromethyl)benzene (0.58 g, 2.9 mmol), the residue was purified by flash
3 24 2 4
, t-Bu). Anal. Calcd for C17H N O (320.38): C, 63.73; H, 7.55;
4
(
chromatography using petroleum ether: EtOAc, the product was collected at 40:60
1
v/v. Yield: 85 % as a cream solid, m.p: 98-106 °C. H-NMR (CDCl
3
): ꢄ 7.42 (d, J =
), 4.15 (s, 2H, CH ), 3.64 (t, J =
), 1.49 (s, 9H, t-Bu). C-NMR
): ꢄ 166.1 (C=O, piperazine), 153.8 (C=O), 134.4 (C), 134.2 (C), 132.4 (C),
30.7 (CH, Ar), 129.5 (CH, Ar), 127.66 (CH, Ar), 81.0 (C, t-Bu), 46.3 (2 x CH ), 46.9
), 28.3 (3x CH , t-Bu). Anal. Calcd for C16 (359.25): C, 53.49; H,
.61; N, 7.99. Found: C, 53.62; H, 5.59; N, 7.70.
.2.2. General method for the synthesis of 1-(substituted)-piperazin-2-ones (4)
1
.7 Hz, 1H, Ar), 7.26 (m, 2H, Ar), 4.75 (s, 2H, CH
2
2
13
5
.4 Hz, 2H, CH ), 3.33 (t, J = 5.4 Hz, 2H, CH
2
2
(
CDCl
3
1
2
(
2 x CH
2
3
2 2 3
H20Cl N O
5
4
To the N-Boc-protected amine (3) (2 mmol) was added to a HCl solution (4M in
dioxane, 33 mL). The reaction was stirred overnight at room temperature then 4M
aqueous NaOH was added until alkaline. The solution was extracted with ethyl
acetate (4 x 50 mL) then the organic layers combined, dried (MgSO
under reduced pressure. The product was pure enough for use in the next step.
.2.2.1. 1-(4-Fluorobenzyl)-piperazin-2-one (4a)
4
) and evaporated
4
Synthesised according to the general procedure using tert-butyl 4-(4-fluorobenzyl)-3-
oxopiperazine-1-carboxylate 3a (0.47 g, 1.52 mmol). Yield: 74% as a pale yellow oil.
1
H-NMR (CDCl
3
): ꢄ 7.21 (m, 2H, Ar), 7.02 (m, 2H, Ar), 4.57 (s, 2H, CH
2
), 3.50 (s,
2
H, CH ), 3.22 (t, J = 5.5 Hz, 2H, CH
2
2
), 3.04 (t. J = 5.5 Hz, 2H, CH ), 1.92 (br.s, 1H,
2
2
5

1
3
NH). C-NMR (CDCl
3
): ꢄ 167.8 (C=O), 163.3 (C-F), 161.3 (C-F), 132.5 (C), 129.9
2 x CH, Ar), 115.6 (2 x CH, Ar), 50.3 (CH ), 49.2 (CH ), 47.2 (CH ), 43.2 (CH ).
.2.2.2. 1-(4-Chlorobenzyl)-piperazin-2-one (4b)
(
2
2
2
2
4
Synthesised according to the general procedure using 4-(4-chlorobenzyl)-3-oxo-
piperazine-1-carboxylic acid tert-butyl ester 3b (0.33 g, 1.02 mmol). Yield: 74% as a
1
pale yellow solid, m.p: 49-50 °C. H-NMR (CDCl
3
): ꢄ 7.29 (d, J = 6.9 Hz, 2H, Ar),
), 3.58 (s, 2H, CH ), 3.21 (t, J = 5.2 Hz,
), 1.97 (br.s, 1H, NH). C-NMR (CDCl ): ꢄ 167.9
),
7
.20 (d, J = 7.1 Hz, 2H, Ar), 4.56 (s, 2H, CH
2
2
1
3
2
H, CH ), 3.03 (t, J = 5.2, 2H, CH
2
2
3
(
C=O), 135.2 (C), 133.4 (C), 129.6 (2 x CH, Ar), 128.8 (2 x CH, Ar), 50.3 (CH
2
2 2 2
49.3 (CH ), 47.3 (CH ), 43.2 (CH ).
4.2.2.3. 1-(4-Methoxybenzyl)-piperazin-2-one (4c)
Synthesised according to the general procedure using 4-(4-methoxybenzyl)-3-oxo-
piperazine-1-carboxylic acid tert-butyl ester 3c (0.46 g, 2.08 mmol). Yield: 70% as
1
yellow crystals, m.p: 60-64 °C. H-NMR (CDCl
3
): ꢄ 7.20 (d, J = 8.7 Hz, 2H, Ar), 6.86
), 3.80 (s, 3H, CH ), 3.58 (s, 2H, CH ), 3.20
), 3.02 (t, J = 5.5 Hz, 2H, CH ), 1.85 (s, 1H, NH). C-NMR
): ꢄ 167.7 (C=O), 159.1 (C), 129.6 (2 x CH, Ar), 129.4 (C), 114.0 (2 x CH,
), 50.4 (CH ), 49.2 (CH ), 47.0 (CH ), 43.2 (CH ).
.2.2.4. 1-(2,4-Dichlorobenzyl)-piperazin-2-one (4d)
(
(
(
d, J = 8.7 Hz, 2H, Ar), 4.54 (s, 2H, CH
t, J = 5.5 Hz, 2H, CH
CDCl
2
3
2
1
3
2
2
3
Ar), 55.3 (CH
3
2
2
2
2
4
Synthesised according to the general procedure using 4-(2,4-dichlorobenzyl)-3-oxo-
piperazine-1-carboxylic acid tert-butyl ester 3d (0.76 g, 2.1 mmol). Yield: 72% as an
1
orange coloured solid, m.p: 96-102 °C. H-NMR (CDCl
3
): ꢄ 7.39 (d, J = 1.95 Hz, 1H,
), 3.57 (s, 2H, CH ), 3.27 (t, J = 5.5Hz, 2H,
): ꢄ 168.1 (C=O), 134.4 (C),
Ar), 7.25 (m, 2H, Ar), 4.71 (s, 2H, CH
2
2
1
3
CH
2
), 3.08 (t, J = 5.2 Hz, 2H, CH
2
). C-NMR (CDCl
3
2
6

1
4
4
33.9 (C), 132.8 (C), 130.4 (CH, Ar), 129.3 (CH, Ar), 127.5 (CH. Ar), 50.3 (CH
7.9 (CH ), 46.7 (CH ), 43.2 (CH ).
.2.3. General methods for synthesis of 4-(bromopropyl)-1-(4-substituted-benzyl)-
2
),
2
2
2
piperazin-2-one (5)
Method 1: To a solution of 1-(4-substituted-benzyl)-piperazin-2-one 4 (1 eq) in
anhydrous acetone (10 mL/1.5 mmol) were added K CO (3 eq) and dibromopropane
4 eq). The reaction mixture was stirred at room temperature for 4 days. The reaction
mixture was filtered and the filtrate was evaporated. The formed residue was extract
with CH Cl (100 mL) and washed with water (50 mL). The organic layer was dried
MgSO ), evaporated under reduced pressure and purified by flash chromatography
using CH
2
3
(
2
2
(
4
2
Cl
2
:CH
3
OH gradient elution.
o
Method 2: To a cooled (0 C) solution of 1-(4-substituted-benzyl)-piperazin-2-one 4
(
1 eq) in anhydrous acetone (10 mL /1.5 mmol) was added LiHMDS (1.2 eq) and the
reaction stirred at 0 °C for 1 h. Dibromopropane (2 eq) was added to the mixture and
the reaction stirred at room temperature for 48 h. The solvent was evaporated and the
residue extracted with CH
and water (50 mL). The organic layer was dried (MgSO
pressure and purified by flash chromatography using CH
elution.
.2.3.1. 4-(3-Bromopropyl)-1-(4-fluorobenzyl)-piperazin-2-one (5a)
2
Cl
2
(100 mL), washed with 2M aqueous NaHCO
), evaporated under reduced
Cl :CH OH gradient
3
(50 mL)
4
2
2
3
4
Synthesised according to method 2 using 1-(4-fluorobenzyl)-piperazin-2-one 4a (0.21
1
g, 1.05 mmol). Yield: 75% as an orange liquid. H-NMR (CDCl
3
): ꢄ 7.24 (m, 2H, Ar),
), 3.22 (t, J = 5.9
), 2.54 (t, J = 6.8 Hz,
): ꢄ 167.0 (C=O), 163.2, 132.3 (C-F),
7
.00 (m, 2H, Ar), 4.55 (s, 2H, CH
Hz, 2H, CH ), 3.21 (s, 2H, CH ), 2.65 (t, J = 5.4 Hz, 2H, CH
H, CH ), 2.01 (m, 2H, CH
2 2
), 3.46 (t, J = 6 .4 Hz, 2H, CH
2
2
2
13
2
2
2
). C-NMR (CDCl
3
2
7

2
129.9 (2 x CH-Ar) 119.0 (C), 115.4 (CH, Ar), 115.4 (CH, Ar), 57.3 (CH ), 55.3
(
CH
calculated for C14
.2.3.2. 4-(3-Bromopropyl)-1-(4-chlorobenzyl)-piperazin-2-one (5b)
2
), 49.9 (CH
2
), 49.1 (CH
2
), 45.9 (CH
2
), 31.2 (CH
2
), 29.7 (CH
2
). [ES-HRMS]
+
+
H18BrFN
2
O: 330.0566 [M+H] . Found: 330.0566 [M+H] .
4
Synthesised according to method 1 using 1-(4-chlorobenzyl)-piperazin-2-one 4b (0.17
1
g, 0.76 mmol). Yield: 42% as a yellow oil. H-NMR (CDCl
3
): ꢄ 7.29 (d, J = 8.45 Hz,
), 3.46 (t, J = 6.5 Hz, 2H,
), 2.65 (t, J = 5.5 Hz, 2H, CH ),
): ꢄ 166.9 (C=O),
35.1 (C), 133.4 (C), 128.8 (2 x CH, Ar), 129.6 (2 x CH, Ar), 57.3 (CH ), 55.3 (CH ),
9.9 (CH ), 48.8 (CH ), 46.0 (CH ), 31.2 (CH ), 29.7 (CH ). [ES-HRMS] calculated
O: 344.0291 [M] . Found: 344.0283 [M] .
.2.3.3. 4-(3-Bromopropyl)-1-(4-methoxybenzyl)-piperazin-2-one (5c)
Synthesised according to method 1 using 1-(4-methoxybenzyl)-piperazin-2-one 4c
2
H, Ar), 7.20 (d, J = 8.45 Hz, 2H, Ar), 4.55 (s, 2H, CH
CH ), 3.22 (s, 2H, CH ), 3.21 (t, J = 5.5 Hz, 2H, CH
.55 (t, J = 6.8 Hz, 2H, CH
2
2
2
2
2
1
3
2
1
4
2
), 2.01 (m, 2H, CH
2
). C-NMR (CDCl
3
2
2
2
2
2
2
2
+
+
for C14H18BrClN
2
4
(
0.37 g, 1.67 mmol). The residue was purified by flash chromatography with
1
EtOAc:CH
3
OH: gradient elution. Yield: 69% as a colourless oil. H-NMR (CDCl
3
): ꢄ
7
3
2
.20 (d, J = 8.7 Hz, 2H, Ar), 6.87 (d, J = 8.7 Hz, 2H, Ar), 4.54 (s, 2H, CH
H, OCH ), 3.81 (s, 2H, CH ), 3.23 (m, 4H, 2 x CH ), 2.66 (t, J = 5.5 Hz, 2H, CH
.56 (t, J = 6.8 Hz, 2H, CH ), 2.03 (dt, J = 6.6, 13.2 Hz, 2H, CH
2
), 3.81 (s,
3
2
2
2
),
):
1
3
2
2
). C-NMR (CDCl
3
ꢄ 166.6 ( C=O ), 159.1 (C), 129.6 (2 x CH, Ar), 128.6 (C), 114.1 (2 x CH, Ar), 57.3
CH ), 55.4 (CH ), 55.3 (OCH ), 50.0 (CH ), 48.9 (CH ), 45.6 (CH ), 31.2 (CH ),
: 340.0786 [M] . Found:
(
2
2
3
2
2
2
2
+
2
3
4
9.7 (CH
2
2 2
). [ES-HRMS] calculated for C15H21BrN O
+
40.0786 [M] .
.2.3.4. 4-(3-Bromopropyl)-1-(2,4-dichlorobenzyl)-piperazin-2-one (5d)
2
8

Synthesised according to method 1 using 1-(2,4-dichlorobenzyl)-piperazin-2-one 4d
1
(
0.27 g, 1.04 mmol). Yield: 61% as a yellow viscous oil. H-NMR (CDCl
3
): ꢄ 7.41 (t,
), 3.49 (t, J = 6.5 Hz, 2H,
), 2.72 (t, J = 5.5 Hz, 2H, CH ),
):
ꢄ 167.2 (C=O), 134.4 (C), 133.9 (C), 132.7 (C), 130.4 (CH, Ar), 129.4 (CH, Ar),
27.6 (CH, Ar), 57.4 (CH ), 55.4 (CH ), 50.0 (CH ), 46.6 (CH ), 46.4 (CH ), 31.1
CH ), 29.7 (CH ).
.2.4. General methods for synthesis of 4-(3-imidazol-1-yl-propyl)-1-(substituted)-
J = 1.1 Hz, 1H, Ar), 7.25 (m, 2H, Ar), 4.71 (s, 2H, CH
CH ), 3.30 (t, J = 5.5 Hz, 2H, CH ), 3.25 (s, 2H, CH
.59 (t, J = 6.8 Hz, 2H, CH ), 2.05 (dt, J = 6.6, 13.3 Hz, 2H, CH
2
2
2
2
2
1
3
2
2
2
). C-NMR (CDCl
3
1
2
2
2
2
2
(
2
2
4
piperazin-2-one (6)
To a solution of NaH (2 eq) in dry DMF (10 mL/2 mmol of 5), imidazole (2 eq) was
added, the reaction was stirred for 1 h at 30 °C then cooled to room temperature. 4-
(
Bromoalkyl)-1-(4-substituted-benzyl)-piperazin-2-one (5) (1 eq) was added to the
reaction mixture and left stirring overnight at 45 °C. Water (3 mL) was added to
quench excess NaH and the reaction was evaporated under reduced pressure. Water
(
50 mL) was added to the formed residue and the product was extracted with ethyl
acetate (3 x 50 mL). The organic layers were combined and washed with water (50
mL). The organic layer was then dried (MgSO ), evaporated under reduced pressure
and purified by preparative TLC, using EtOAc : CH OH 5:1 v/v as eluent.
.2.4.1. 4-(3-(1H-imidazol-1-yl)propyl)-1-(4-fluorobenzyl)piperazin-2-one (6a)
4
3
4
Synthesised according to the general procedure using 5a (0.15 g, 0.45 mmol) Yield:
1
4
7
4
8% as a yellow oil. H-NMR (CDCl
3
): ꢄ 7.47 (s, 1H, imidazole), 7.24 (m, 2H, Ar),
),
), 2.61 (t,
.06 (s, 1H, imidazole), 7.02 (m, 2H, Ar), 6.90 (s, 1H, imidazole), 4.5 (s, 2H, CH
.04 (t, J = 6.7 Hz, 2H, CH ), 3.23 (t, J = 5.4 Hz, 2H, CH ), 3.19 (s, 2H, CH
), 2.34 (t, J = 6.6 Hz, 2H, CH ), 1.94 (m, 2H, CH ). C-NMR
2
2
2
2
1
3
J = 5.4 Hz, 2H, CH
2
2
2
2
9