Design, synthesis, and antimycobacterial activity of novel ciprofloxacin derivatives

Article abstract of DOI:10.1111/cbdd.13534

Tuberculosis is the deadliest infectious disease affecting humankind with a death toll of approximately 1.7 million people in 2016. The increasing prevalence of multidrug-resistant strains of the causative pathogen, Mycobacterium tuberculosis (Mtb) which

Full text of DOI:10.1111/cbdd.13534

PROFESSOR DAVID N'DA (Orcid ID : 0000-0002-2327-0551)
Article type : Research Article
Running title: Antimycobacterial novel ciprofloxacin derivatives
Design, synthesis and antimycobacterial activity of novel ciprofloxacin
derivatives
Pieter Cilliersa, Ronnett Seldonb, Frans J Smitc, Janine Aucampc, Audrey Jordaand, Digby F.
Warnerd,e, David D. N’Da*c
aPharmaceutical Chemistry, School of Pharmacy, North-West University, Potchefstroom 2520, South
Africa;
bSAMRC Drug Discovery and Development Research Unit, University of Cape Town, Cape Town
7700, South Africa
cCentre of Excellence for Pharmaceutical Sciences, North-West University, Potchefstroom 2520,
South Africa.
dSAMRC/NHLS/UCT Molecular Mycobacteriology Research Unit, Department of Pathology and
Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape
Town 7925, South Africa
eWellcome Centre for Clinical Infectious Diseases Research in Africa, University of Cape Town, Cape
Town 7925, South Africa
*Corresponding author: e-mail: david.nda@nwu.ac.za, Tel.: +27 18 299 2256; Fax: +27 18 299 4243
Abstract
Tuberculosis is the deadliest infectious disease affecting humankind with a death toll of
approximately 1.7 million people in 2016. The increasing prevalence of multidrug-resistant
strains of the causative pathogen, Mycobacterium tuberculosis (Mtb) which results in
reduced effectiveness of the current therapies, underscores the urgent need for the
development of new antitubercular drugs. In the search for such drugs, we investigate two
series of ciprofloxacin (CPX) derivatives (analogues and hybrids). We herein report the
design, synthesis and biological activity of these series against the human virulent Mtb
H37Rv strain in vitro. The small propionyl analogue 11 (MIC90 1.6 µM; SI>61) and the large
cholesteryl hybrid 32 (MIC90 2.0 µM; SI>6) were the most active derivatives, comparable to
ciprofloxacin (MIC90 1.8 µM). However, the slightly less active but non-cytotoxic para-
fluorobenzyl hybrid 28 (MIC90 3.7 µM; SI 27) was more selective towards bacteria than 32.
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/cbdd.13534
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Thus, the CPX derivatives 11 and 28 were identified as preferred antitubercular hits for
further investigation including DMPK properties determination and in vivo activity
assessment in animal models.
Keywords: Mycobacterium tuberculosis, ciprofloxacin, 1,2,3-triazole, click-chemistry,
hybrids
Introduction
Tuberculosis (TB) endures, to this day, to be the scourge of humankind (Goldberg et al.,
2012; Sharma & Mohan, 2013). TB, together with human immunodeficiency virus (HIV) and
malaria, constitute the three leading infectious diseases causing of numerous deaths
worldwide (Dixit et al., 2012; Dixit et al., 2016). TB is, in fact, the deadliest single disease
globally with 1.7 million people succumbing to the disease in 2016 (WHO, 2017).
Additionally, co-morbidity with TB and either of the other two diseases is widespread and
each increases the risk of the others (Gray & Cohn, 2013; Li & Zhou, 2013). TB was the
leading cause of HIV-related deaths in 2016 (WHO, 2017) as a result of its opportunistic
nature (Castro, 1995) and transmissibility (WHO, 2017).
Further complicating TB control, 490 000 people developed multidrug-resistant (MDR) TB in
2016 (WHO, 2017). MDR-TB is defined as strains of Mycobacterium tuberculosis (Mtb)
resistant to isoniazid and rifampicin (Jordao & Vieira, 2011), the two frontline drugs of
standard TB treatment regimens (CDC, 2011; Frieden et al., 2003). The rise of resistance
against these two primary drugs necessitates the use of at least five second-line TB-agents
including
pyrazinamide,
fluoroquinolones,
aminoglycosides,
thioamides,
and
cycloserine/terizidone (WHO, 2016). The inclusion of a fluoroquinolone drug is thus
compulsory as this greatly improves the treatment outcome for MDR-TB (WHO, 2016).
Treatment of MDR-TB is complicated as these second-line agents are usually more toxic,
expensive, subject to limited availability, and require a lengthy duration of administration
(Frieden et al., 2003). This leads to poor patient adherence (Frieden et al., 2003), enabling
the acquisition of additional resistance mutations which results in extensively drug resistant
(XDR-TB) disease (O’Donnell et al., 2014). XDR-TB is MDR-TB which is resistant to any
fluoroquinolone and at least one of three injectable second-line aminoglycoside drugs
(Jordao & Vieira, 2011). This form of the disease presents high mortality rates due to
ineffectiveness of drugs and subsequently treatment failure (Tabarsi & Mardani, 2012).
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Therefore, novel and more potent drugs are vital to shorten the treatment regimens for all
strains of Mtb, with less toxic effects.
The development of clinically useful drugs with new structures and novel mechanisms of
action (Hu et al., 2017) is challenging, time consuming, risky and expensive (Projan, 2003).
Therefore, a more practical approach that forms the cornerstone for drug discovery is the
exploitation of structure-activity relationships (SAR) of lead compounds such as existing
drugs to deliver analogues with improved therapeutic profiles (Guha, 2013; Hu et al., 2017).
The 4-quinolone scaffold forms part of a promising class of synthetic antibiotics (Emmerson
& Jones, 2003) which sparked great scientific and clinical interest (Andersson & MacGowan,
2003) on its discovery in the 1960s (Emmerson & Jones, 2003). The relative simplicity of this
pharmacophore creates an array of possibilities for structural changes to further increase its
antibacterial activity (Sharma et al., 2009).
Investigations of the SAR of fluoroquinolones indicate the carboxylic acid and the
keto group in position C-3 and C-4, respectively, to be essential for their biological activities
(Chu & Fernandes, 1989). Modifications in position C-3 usually produce compounds with
reduced activity. However, substituting the carboxylic acid with certain esters or an
isothiazole group affords derivatives with in vivo and potent in vitro antibacterial activity when
compared to ciprofloxacin (Sharma et al., 2009). On the other hand, modifications made in
position C-7 of the fluoroquinolone structure control its wide spectrum of pharmacological
activity, potency, and pharmacokinetic profile (Andersson & MacGowan, 2003; Rajulu et al.,
2013). Thus, the advancement of quinolones based on the prototypical fluoroquinolone,
ciprofloxacin (CPX), exhibiting a broad-spectrum of activity and excellent safety profile is
supported by CPX being the most widely prescribed antibiotic (Breda et al., 2009; King et al.,
2000). Newer-generation of fluoroquinolones such as sparfloxacin, gatifloxacin, moxifloxacin,
gemifloxacin, grepafloxacin, and caderofloxacin are built on CPX nucleus (Asif, 2014) due to
the presence of optimal groups (Figure 1) (Sharma et al., 2009). However, these drugs still
experience bacterial resistance which renders the search for the next-generation of
fluoroquinolones an imperative.
Consideration of the above SAR formed our basis to construct new fluoroquinolone from the
framework of CPX. Modifications in position C-11 with the introduction of ethyl ester and
piperazinyl amide groups in prodrug approach, and of various substituents on the piperazine
ring in position C-7, are explored to generate new analogues (Series 1) endowed with
enhanced antibacterial potency paired with improved hydrosolubility as depicted in Figure 1.
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Another popular and well-established strategy to assist in the swift development of novel
drugs is molecular hybridisation (Smit et al., 2015). Herein, a more effective hybrid molecule
or chemical entity is developed by covalently linking two or more different pharmacophores -
exploiting the potential synergism produced from each pharmacophore’s individual biological
activity and mode of action (Meunier, 2008; Smit et al., 2015).
1,2,3-Triazole (TZ) is a five membered heterocyclic motif (Dabak et al., 2003) which offers a
remarkably versatile scaffold (Kharb et al., 2011) and forms part of numerous drug
categories used to treat various human conditions including bacterial (e.g. TB), viral (e.g.
HIV), and parasitic (e.g. malaria) diseases, inflammation, diabetes, epilepsy, cancer,
hypertension, anxiety, depression, Parkinson′s disease, and obesity (Kharb et al., 2011).
Moreover, its favourable properties – such as high polarity and hydrogen bonding capability,
both of which improve its hydrosolubility, stability and rigidity – all contribute to enhancing the
biological activities of linked drugs (Agalave et al., 2011; Kant et al., 2016; Xu et al., 2017).
Drugs currently on the market containing the triazole moiety include cefatrizine, tazobactam,
cephalosporin (antibiotics) (Kant et al., 2016), TSAO (anti-HIV), carboxyamidotriazole (anti-
cancerous), and radezolid (Bhukya & Tangutur, 2017) while I-A09, another derivative, is
currently undergoing trials for use against TB (Figure 2). Other research that was also
inspired by both moieties (CPX and TZ) to find innovative and effective antibiotics were
explored and investigated against various Gram-negative and Gram-positive bacteria (Kant
et al., 2016; McPherson et al., 2012; Rajulu et al., 2013). Xu et al. ( Xu et al., 2018)
investigated a combination of these two moieties against Mtb H37Rv, and found a lipophilic
hybrid 8 that was eight times more active than CPX and with comparable activity to
rifampicin (MIC: 0.39 μg/ml). However, its cytotoxicity was more harmful to mammalian cells
than CPX, which precluded it for further development.
Based on the molecular hybridization strategy, and consistent with the aim to address the
drug-resistance issue through increased activity, 1,2,3-triazole was conjoined to the standard
to CPX, in position N-15 to generate hybrids (Series 2) as depicted in Figure 3. This add-on
is expected to have a synergistic effect owing to the activities of both pharmacophores
against Mtb. In fact, five- to six-membered heterocyclic substituents containing nitrogen have
been determined to be ideal substituents for increased fluoroquinolone potency (Domagala,
1994).
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Material and methods
Materials
Solvents were obtained from either ACE Chemicals (e.g. acetonitrile; DCM dichloromethane;
diethyl ether, ethyl acetate, methanol, monoglyme) or Sigma-Aldrich South Africa (e.g. DMF
dimethylformamide, DMSO dimethyl sulfoxide, THF tetrahydrofuran), respectively. For inert
reactions, DCM was distilled over calcium hydride and stored over 3 Å molecular sieves.
Reagents, such as absolute ethanol (abs EtOH), ammonium chloride (NH4Cl), benzyl
bromide, CPX, copper sulfate pentahydrate (CuSO4·5H2O), 4-dimethylaminopyridine
(DMAP), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N,N,N′,N′-Tetramethyl-O-
(1H-benzotriazol-1-yl)uronium hexafluorophosphate, propargyl bromide (HBTU), p-
bromobenzyl azide, p-fluorobenzyl azide, p-methylbenzyl azide, p-nitrobenzyl azide, p-
trifluorobenzyl azide, methylpiperazine (mPz), piperazine (Pz), sodium ascorbate (NaAsc),
sodium azide (NaN3), sodium bicarbonate (NaHCO₃), trimethylamine (TEA) were all
acquired from Sigma-Aldrich (South Africa). Cholesteryl chloride and cholesteryl
chloroformate were ordered from Merck (South Africa).
General procedures
1
The H and 13C nuclear magnetic resonance (NMR) spectra were recorded on a
Bruker Advance™ III 600 spectrometer at a frequency of 600 MHz and 150.913 MHz,
respectively, in DMSO-d6 or CDCl3-d. Chemical shifts are reported in parts per million 
(ppm), with the residual protons of the solvent as reference. The splitting pattern
abbreviations are as follows: singlet (s), doublet (d), doublet of doublet (dd), doublet of
doublet of doublets (ddd), doublet of triplets (dt), doublet of quartets (dq), triplet (t), triplet of
doublets (td), triplet of triplets (tt), quartet of doublets (qd) and multiplet (m).
High resolution mass spectrometry (HRMS) was recorded on a Bruker MicroTOF Q II
mass spectrometer, equipped with an APCI or an ESI source, set at 200°C or 180°C,
respectively, using Bruker Compass DataAnalysis 4.0 software. A full scan from 50 -
1500 m/z was performed at a capillary voltage of 4500 V, an end plate offset voltage of -500
V, with the nebulizer set at 1.6 Bar and 0.4 Bar, respectively, and a collision cell RF voltage
of 100 Vpp.
Infrared (IR) spectra were recorded on a Bruker Alpha-P FTIR instrument. Thin layer
chromatography (TLC) was utilized to monitor the progress of reactions using silica gel
plates (60F254) purchased from Merck (Johannesburg, South Africa), and a UV-light (254
nm) as well as iodine to visualize spots. Column chromatography was performed using silica
gel (230 – 400 mesh, Sigma-Aldrich).
High performance liquid chromatography (HPLC) analysis of most final compounds
were performed, to determine purity. An Agilent 1100 HPLC system equipped with a
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quaternary pump and an Agilent 1100 series diode array detector were utilized. HPLC grade
acetonitrile (Merck) and Milli-Q water (Millipore) were used for chromatography. A Venusil
XBP C18 column (4.60 x 150 mm, 5 μm) with an initial mobile phase (70% MilliQ water: 30%
acetonitrile) was employed at a flow rate of 1ml/min. The concentration of acetonitrile in the
mobile phase was linearly increased over a period of 5 minutes to a final concentration of
85%. The time allowed for equilibration between runs was 5 minutes and the duration of
each HPLC run was 15 minutes. The concentration of the test compounds injected varied
(20 μl of 1 mM to 20 μl of 0.25 mM). The eluent was monitored at wavelengths of 210, 254,
and 300 nm.
Syntheses
CPX analogues 9 – 23
Acids 9 – 13 were prepared in accordance with the general procedure depicted in Scheme 1
and described as follows:
To a flask containing CPX, 1 (9.05 mmol, 3.0 g, 1 eq.), a mixture of THF (100 ml) and TEA
(2.6 eq.) were combined and subsequently the appropriate alkyl/acetyl halide/cholesteryl
chloroformate (1.3 eq.) was added. The reaction mixture was stirred at room temperature for
4 – 5 hours and progress of reaction was monitored by TLC. After completion, the solvent
was evaporated and the residue was suspended in DCM (250 ml) then washed successively
with brine (50 ml). The resulting organic layer was then dried over magnesium sulphate
(MgSO4), evaporated under reduced pressure and recrystallized from acetonitrile.
1-Cyclopropyl-6-fluoro-7-(4-(3-(methylsulfonyl)-2-oxoimidazolidine-1-carbonyl)piperazin-1-
yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (9).
The reaction of CPX (9.05 mmol, 3.0 g, 1 eq.), TEA (23.54 mmol, 3.28 ml) and 3-
chlorocarbonyl-1-methanesulfonyl-2-imidazolidinone (10.86 mmol, 2.46 g, 1.2 eq.) yielded
compound 9. Light orange crystals; yield: 3.19 g (68%); m.p. 266.1 – 267.8 ; IR vmax: 2928,
1729, 1668, 1629, 1583 cm-1; 1H NMR (600 MHz, DMSO) δ (ppm): 15.14 (s, 1H, C-11), 8.62
(s, 1H, H-2), 7.86 (d, J = 13.1 Hz, 1H, H-5), 7.55 (d, J = 7.1 Hz, 1H, H-8), 3.84 (t, J = 7.7 Hz,
2H, H-20), 3.80 – 3.78 (m, 1H, H-9), 3.76 – 3.70 (m, J = 7.7 Hz, 2H, H-21), 3.63 (t, J = 4.3
Hz, 4H, H-14), 3.37 (t, J = 4.2 Hz, 4H, H-13), 2.47 (s, 3H, H-22); 13C NMR (151 MHz, DMSO)
δ (ppm): 176.37 (C-4), 165.95 (C-11), 153.79 (C-16), 152.30, 151.45 (C-18), 148.07 (C-2),
144.85 (C-7), 111.03 (C-5), 106.71 (C-8), 39.80 (C-22), 35.93 (C-9), 34.34 (C-14), 7.64 (C-
10); HRMS (ESI) m/z: [M+H]+ 522.1460 (Calculated for C22H25FN5O7S: 522.1459); Purity
(HPLC): 97%.
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1-Cyclopropyl-6-fluoro-4-oxo-7-(4-((1-(4-(trifluoromethyl)benzyl)-1H-1,2,3-triazol-4-
yl)methyl)piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid (10).
Fluffy off-white crystals; yield: 3.5 g (87%); m.p 266.6 – 266.7 . IR vmax: 2842, 1720, 1648,
1627 cm-1; 1H NMR (600 MHz, DMSO) δ (ppm): 15.17 (s, 1H, C-11), 8.64 (s, 1H, H-2), 7.89
(d, J = 13.1 Hz, 1H, H-5), 7.56 (d, J = 7.4 Hz, 1H, H-8), 3.83 – 3.76 (m, 1H, H-9), 3.57 (t, J =
4.2 Hz, 4H), 3.38 (t, J = 5.4 Hz, 4H, H-19), 3.30 (t, J = 5.0 Hz, 4H, H-18), 3.18 (t, J = 4.6 Hz,
4H), 1.30 (q, J = 7.0 Hz, 2H, H-10b), 1.16 (q, J = 4.2 Hz, 2H, H-10a); 13C NMR (151 MHz,
DMSO) δ (ppm): 176.40 (C-4), 165.95 (C-11), 162.86 (C-16), 153.04 (C-6), 148.07 (C-2),
145.13 (C-7), 118.86 (C-3), 110.99 (C-5), 106.72 (C-8), 65.94 (C-13), 49.26 (C-18), 46.90
(C-19), 35.92 (C-9), 7.62 (C-10); HRMS (ESI) m/z: [M+H]+ 445.1868 (calculated for
C22H26FN4O5: 445.1887); Purity (HPLC): 98%.
1-Cyclopropyl-6-fluoro-4-oxo-7-(4-propionylpiperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic
acid (11).
Light yellow crystals; yield: 3.51 g (100%); m.p. 226.1 – 228.7 ; IR vmax: 2886, 1723, 1664,
1627, 1613 cm-1; 1H NMR (600 MHz, DMSO) δ (ppm): 15.16 (s, 1H, H-11), 8.63 (s, 1H, H-2),
7.88 (d, J = 13.1 Hz, 1H, H-5), 7.54 (d, J = 7.4 Hz, 1H, H-8), 3.81 – 3.75 (m, 1H, H-9), 3.64
(t, J = 7.8 Hz, 4H, H-13), 2.47 (t, J = 1.7 Hz, 4H, H-14), 2.36 (q, J = 7.4 Hz, 2H, H-17), 1.29
(q, J = 6.0 Hz, 2H, C-10b), 1.15 (q, J = 4.9 Hz, 2H, C-10a), 0.99 (t, J = 7.4 Hz, 3H, H-18); 13C
NMR (151 MHz, DMSO) δ (ppm): 176.40 (C-4), 171.58 (C-16), 165.96 (C-11), 148.10 (C-2),
145.03 (C-7), 111.03 (C-5), 106.72 (C-8), 49.65 (C-13), 44.45 (C-14), 35.92 (C-9), 25.52 (C-
17), 9.39 (C-18), 7.62 (C-10); HRMS (ESI) m/z: [M+H]+ 388.1645 (calculated for
C20H23FN3O4: 388.1673).
1-Cyclopropyl-7-(4-((((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-
2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-
yl)oxy)carbonyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (12).
The reaction of CPX (6.04 mmol, 2.0 g, 1eq.), TEA (6.64 mmol, 0.93 ml, 1.1 eq.) and
cholesteryl chloroformate (6.64 mmol, 2.98 g, 1.1 eq.) yielded compound 12. Light yellow
crystals; yield: 1.97 g (44%); IR vmax: 2934, 2867, 1701, 1626, 1242 cm-1; 1H NMR (600 MHz,
CDCl3) δ (ppm): 14.99 (s, 1H, H-11), 8.77 (s, 1H, H-2), 8.14 (d, J = 2.4 Hz, 1H, H-5), 7.31 (d,
J = 7.0 Hz, 1H, H-8), 5.39 (t, J = 2.7 Hz, 1H, H-6’), 4.59 – 4.53 (m, 1H, H-3’), 3.71 (t, J = 6.6
Hz, 4H, H-14), 3.43 – 3.39 (m, 1H, H-9), 3.28 (t, J = 4.7 Hz, 4H, H-13), 1.03 (s, 3H, H-19’),
0.86 (dd, J = 6.6, 2.8 Hz, 6H, H-26’, H-27’), 0.67 (s, 3H, H-18); 13C NMR (151 MHz, CDCl3) δ
(ppm): 177.28 (C-4), 167.15 (C-11), 166.14 (C-16), 139.20 (C-5’), 122.84 (C-6’), 113.37 (C-
5), 104.99 (C-8), 56.80 (C-14’), 56.25 (C-17’), 50.12 (C-9’), 42.44 (C-13), 22.70 (C-26’, C-
27’), 19.50 (C-19’), 11.99 (C-18’), 8.45 (C-10); HRMS (ESI) m/z: [M]+ 744.4733 (calculated
for C45H62FN3O5: 744.4746).
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1-Cyclopropyl-6-fluoro-4-oxo-7-(4-(prop-2-yn-1-yl)piperazin-1-yl)-1,4-dihydroquinoline-3-
carboxylic acid (13).
CPX (9.05 mmol, 3.0 g, 1 eq.) and the solvent mixture, monoglyme/DMSO (120 ml, 5:1, v/v)
were combined in a 150-ml flask, then left to stir a few minutes before adding TEA (27.15
mmol, 3.8 ml, 3 eq.), DMAP (0.91 mmol, 0.11 g, 0.1 eq.) and lastly propargyl bromide (18.1
mmol, 2.7 ml, 2eq.). The reaction mixture was refluxed overnight and filtered after cooling to
room temperature. The solvent, monoglyme, was removed under reduced pressure to give
the DMSO residue. This was suspended in DCM (200 ml), washed with NH4Cl (4 x 50 ml)
and then water (4 x 50 ml). The resulting organic layer was dried over Mg2SO4, evaporated
and crystallised from acetonitrile.
1
Gold crystals, yield: 1.98 g (66%); IR vmax: 3250, 2848, 2101, 1732, 1675, 1626 cm-1; H
NMR (600 MHz, CDCl3) δ (ppm): 15.01 (s, 1H, C-11), 8.74 (s, 1H, H-2), 7.98 (d, J = 13.1 Hz,
1H, H-5), 7.36 (d, J = 7.1 Hz, 1H, H-8), 3.57 – 3.53 (m, 1H, H-9), 3.41 (d, J = 2.5 Hz, 2H, H-
16), 3.39 (t, J = 4.6 Hz, 4H, H-14), 2.81 (t, J = 4.9 Hz, 4H, H-13), 2.31 (t, J = 2.4 Hz, 1H, H-
18), 1.39 (q, J = 6.6 Hz, 2H, H-10b), 1.20 (q, J = 6.5 Hz, 2H, H-10a); 13C NMR (151 MHz,
CDCl3) δ (ppm): 177.23 (C-4), 167.17 (C-11), 153.80 (C-6), 147.55 (C-2), 145.91 (C-7),
112.55 (C-5), 104.98 (C-8), 78.32 (C-17), 73.85 (C-18), 51.57 (C-13), 49.81 (C-14), 46.98
(C-16), 35.42 (C-9), 8.36 (C-10); HRMS (ESI) m/z: [M+H]+ 370.1529 (calculated for
C20H21FN3O3: 370.1567).
Esters 14 – 18 were prepared in accordance with the general procedure, as depicted in
Scheme 1 and described as follows:
Each acid analogues 9 – 13 (1 eq.) was dissolved in anhydrous DCM (35 ml) and stirred on
an ice bath. DMAP (0.1 eq.) and absolute EtOH (4 eq.) were added soon after, whilst a
solution of EDC. HCl (in 6 ml of DCM) was added slowly under N2 over 20 min. Stirring was
continued for 24 – 48 hours until room temperature was reached. The reaction mixture was
diluted (DCM, 250 ml), washed with NaHCO3 (3 x 25 ml), dried over MgSO4, filtered and the
organic solvent removed under reduced pressure. The resulting residue was purified by
column chromatography on silica gel eluting with DCM/MeOH (19:1, v/v).
Ethyl-1-cyclopropyl-6-fluoro-7-(4-(3-(methylsulfonyl)-2-oxoimidazolidine-1-
carbonyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (14).
Light yellow powder; yield: 0.05 g (4%); m.p.: 161.6 – 161.7 ; IR vmax: 2981, 2906, 1740,
1
1688, 1656 cm-1; H NMR (600 MHz, DMSO) δ (ppm): 8.40 (s, 1H, H-2), 7.73 (d, J = 13.2
Hz, 1H, H-5), 7.43 (d, J = 7.4 Hz, 1H, H-8), 4.19 (q, J = 7.1 Hz, 2H, C-11-CH2-), 3.84 (t, J =
7.7 Hz, 2H, H-20), 3.74 (t, J = 7.7 Hz, 2H, H-21), 3.63 (t, J = 10.1, 6.0 Hz, 5H, H-9, H-14),
3.30 (t, J = 4.8 Hz, 4H, H-13), 2.48 (s, 3H, H-22), 1.25 (t, J = 7.1 Hz, 3H, C-11-CH2-), 1.23
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(q, J = 1.3 Hz, 1H, H-10b), 1.08 (q, J = 3.9 Hz, 1H, H-10a); 13C NMR (151 MHz, DMSO) δ
(ppm): 171.58 (C-4), 164.45 (C-11), 152.45 (C-6), 152.25 (C-16), 151.43 (C-18), 148.13 (C-
2), 111.66 (C-5), 109.28 (C-3), 106.59 (C-8), 59.79 (C-11-O-CH2-), 49.43 (C-13), 41.37 (C-
20), 41.18 (C-21), 34.77 (C-9), 14.31 (C-11-O-CH2-CH3), 7.58 (C-10). HRMS (ESI) m/z:
[M+H]+ 550.1741 (calculated for C24H29FN5O7S: 550.1772); Purity (HPLC): 92%.
Ethyl-1-cyclopropyl-6-fluoro-7-(4-(morpholine-4-carbonyl)piperazin-1-yl)-4-oxo-1,4-
dihydroquinoline-3-carboxylate (15).
Cream coloured powder; yield: 0.24 g (23%); m.p.: 218.8 – 221.7 ; IR vmax: 2978, 2902,
2849, 1727, 1654, 1618 cm-1; 1H NMR (600 MHz, CDCl3) δ (ppm): 8.50 (s, 1H, H-2), 8.01 (d,
J = 13.1 Hz, 1H, H-5), 7.25 (d, J = 7.7 Hz, 1H, H-8), 4.37 (q, J = 7.1 Hz, 2H, C-11-CH2), 3.70
(t, J = 4.6 Hz, 4H, H-13), 3.50 (t, J = 4.7 Hz, 4H, H-19), 3.42 – 3.38 (m, 1H, H-9), 3.32 (t, J =
4.8 Hz, 4H, H-18), 3.24 (t, J = 5.0 Hz, 4H, H-13), 1.39 (t, J = 7.1 Hz, 3H, C-11-CH2-CH3),
1.31 (q, J = 6.9 Hz, 2H, H-10b), 1.13 (q, J = 6.6 Hz, 2H, H-10a); 13C NMR (151 MHz, CDCl3)
δ (ppm): 173.17 (C-4), 165.90 (C-11), 163.70 (C-16) 153.48 (C-6), 148.32 (C-2), 113.52 (C-
5), 105.16 (C-8), 61.02 (C-11-O-CH2-), 34.62 (C-9), 14.56 (C-11-O-CH2-CH3), 8.30 (C-10);
HRMS (ESI) m/z: [M+H]+ 473.2186 (calculated for C24H30FN4O5: 473.2195); Purity (HPLC):
95%.
Ethyl-1-cyclopropyl-6-fluoro-4-oxo-7-(4-propionylpiperazin-1-yl)-1,4-dihydroquinoline-3-
carboxylate (16).
White powder; yield: 0.68 g (6%); m.p.: 180.6 – 183.3 ; IR vmax: 3092, 2940, 1713, 1652,
1617, 1591 cm-1; 1H NMR (600 MHz, CDCl3) δ (ppm): 8.49 (s, 1H, H-2), 7.99 (d, J = 13.1 Hz,
1H, H-5), 7.24 (d, J = 7.0 Hz, 1H, H-8), 4.36 (q, J = 7.1 Hz, 2H, C-11-CH2-), 3.44 – 3.38 (m,
1H, H-9), 2.41 (q, J = 7.4 Hz, 2H, H-17), 1.39 (t, J = 7.1 Hz, 3H, H-18), 1.31 (q, J = 6.8 Hz,
2H, H-10b), 1.18 (t, J = 7.4 Hz, 3H, C-11-CH2-CH3), 1.13 (q, J = 6.6 Hz, 1H, H-10a); 13C
NMR (151 MHz, CDCl3) δ (ppm): 173.12 (C-4), 172.53 (C-16), 165.81 (C-11), 153.41 (C-6),
148.33 (C-2), 113.53 (C-5), 110.62 (C-3), 105.22 (C-8), 61.00 (C-17), 34.61 (C-9), 26.57 (C-
11-O-CH2), 14.55 (C-18), 9.56 (C-11-O-CH2-CH3), 8.28 (C-10); HRMS (ESI) m/z: [M+H]+
416.2020 (calculated for C22H27FN3O4: 416.1986); Purity (HPLC): 87%.
Ethyl-1-cyclopropyl-7-(4-((((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-
methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-
cyclopenta[a]phenanthren-3-yl)oxy)carbonyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylate (17).
1
Light yellow solid; yield: 0.04 g (4%); IR vmax: 3425, 2930, 2866, 1725, 1055 cm-1; H NMR
(600 MHz, CDCl3) δ (ppm): 8.50 (s, 1H, H-2), 8.07 (d, J = 13.0 Hz, 1H, H-5), 7.27 (d, J = 5.2
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Hz, 1H, H-8), 5.35 (t, J = 2.5 Hz, 1H, H-6’), 4.59 – 4.52 (m, 1H, H-9), 4.22 (q, J = 11.3, 5.9
Hz, 2H, C-11-O-CH2-), 3.69 (t, J = 4.7 Hz, 4H, H-14), 3.55 – 3.49 (m, 1H, H-3’), 3.22 (t, J =
3.9 Hz, 4H, H-13), 1.01 (s, 3H, H-19’), 0.86 (dd, J = 6.6, 2.8 Hz, 6H, H-26’, H-27’), 0.67 (s,
1H, H-18’). 13C NMR (151 MHz, CDCl3) δ (ppm): 140.91 (C-5’), 121.87 (C-6’), 71.95 (C-3’),
56.91 (C-14’), 56.29 (C-17’), 50.27 (C-9’), 42.46 (C-13’), 22.71 (C-26’, C-27’), 19.55 (C-19’),
12.01 (C-18’). HRMS (ESI) m/z: [M]+ 772.5026 (calculated for C47H67FN3O5: 772.5065);
Purity (HPLC): 85%.
Ethyl-1-cyclopropyl-6-fluoro-4-oxo-7-(4-(prop-2-yn-1-yl)piperazin-1-yl)-1,4-dihydroquinoline-
3-carboxylate (18).
Gold powder; yield: 0.04 g (4%); m.p.: 218.5 – 220.5 ; 1H NMR (600 MHz, CDCl3) δ (ppm):
8.52 (s, 1H, H-2), 7.98 (d, J = 13.2 Hz, 1H, H-5), 7.24 (d, J = 4.3 Hz, 1H, H-8), 4.90 (d, J =
2.4 Hz, 2H, C-11-CH2-), 3.45 – 3.42 (m, 1H, H-9), 3.39 (d, J = 2.4 Hz, 2H, H-16), 3.31 (t, J =
4.6 Hz, 4H, H-13), 2.79 (t, J = 4.9 Hz, 4H, H-14), 2.48 (t, J = 2.4 Hz, 1H, H-18), 1.31 (q, J =
7.1 Hz, 2H, H-10b), 1.14 (q, J = 6.6 Hz, 2H, H-10a); 13C NMR (151 MHz, CDCl3) δ (ppm):
175.84 (C-4), 172.98 (C-11), 153.54 (C-6), 148.62 (C-2), 113.43 (C-5), 105.01 (C-8), 74.85
(C-18), 73.74 (C-11-O-CH2-CH3), 52.21 (C-11-O-CH2-), 51.71 (C-14), 49.95 (C-13), 42.12
(C-16), 34.73 (C-9), 8.27 (C-10); Purity (HPLC): 87%.
Amide analogues 19 – 23 were prepared in accordance with the general procedure depicted
in Scheme 1 and described as follows:
Acids 9 – 13 (1g, 1eq.) were dissolved in dry DCM (30 ml) and then treated in an ice bath
with HBTU (1.2 eq.), TEA (3 eq.). The reaction mixture was stirred under N2 for 30 min
before the addition of mPz (2.4 eq.). The reaction was continued for 4 hours upon monitoring
with thin layer chromatography (TLC). Thereafter, the reaction mixture was diluted with
further DCM (150 ml), washed with NaHCO3 (50 ml), H2O (2 x 25 ml) and subsequently dried
over MgSO4. The solvent was removed in vacuo and the residue subjected to column
chromatography with DCM/MeOH (4:1 v/v) as eluent to isolate the target compound
(Bandyopadhyay et al., 2010).
1-Cyclopropyl-6-fluoro-3-(4-methylpiperazine-1-carbonyl)-7-(4-(3-(methylsulfonyl)-2-
oxoimidazolidine-1-carbonyl)piperazin-1-yl)quinolin-4(1H)-one (19).
Mustard powder; yield: 1.1 g (95%); m.p. 61.7 – 61.8 ; IR vmax: 2921, 2851, 1739, 1673,
1623, 1579 cm-1; 1H NMR (600 MHz, DMSO) δ (ppm): 8.01 (s, 1H, H-2), 7.77 – 7.73 (m, 1H,
H-5), 7.44 (d, J = 7.4 Hz, 1H, H-8), 3.84 (t, J = 7.7 Hz, 2H, H-20), 3.74 (t, J = 7.6 Hz, 2H, H-
21), 3.63 (t, J = 6.6 Hz, H-14); 3.60 – 3.57 (m, 1H, H-9), 3.27 (t, J = 4.3 Hz, 4H, H-13), 3.21
(s, 3H, H-22), 2.48 (t, J = 3.5, 1.7 Hz, 4H, mPz: -N-CH2-CH2-), 2.33 (d, J = 29.3 Hz, 4H,
mPz: -N-CH2-), 2.20 (s, 3H, Pz-CH3), 1.20 (q, J = 7.3 Hz, 2H, H-10b), 1.07 (q, J = 6.6, 2H, H-
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10a); 13C NMR (151 MHz, DMSO) δ (ppm): 171.27 (C-4), 164.91 (C-11), 152.44 (C-16),
152.25 (C-6), 151.43 (C-18), 143.70 (C-2), 111.18 (C-5), 106.33 (C-8), 54.79 (C-22), 41.45
(C-21), 41.29 (C-20), 34.23 (C-9), 7.66 (C-10); HRMS (ESI) m/z: [M+H]+ 604.2343
(C27H35FN7O6S: 604.2354).
1-Cyclopropyl-6-fluoro-3-(4-methylpiperazine-1-carbonyl)-7-(4-(morpholine-4-
carbonyl)piperazin-1-yl)quinolin-4(1H)-one (20).
Beige-powder; yield: 0.45 g (39%); m.p. 113.6 – 113.2 ; IR vmax: 2920, 2850, 1622, 1584,
1
1544 cm-1. H NMR (600 MHz, CDCl3) δ (ppm): 8.05 (s, 1H, H-2), 8.00 (d, J = 13.1 Hz, 1H,
H-5), 7.27 (d, J = 7.1 Hz, 1H, H-8), 3.70 (t, J = 4.6 Hz, 4H, H-13), 3.50 (t, J = 4.7 Hz, 4H, H-
19), 3.37 – 3.34 (m, 1H, H-9), 3.31 (t, J = 4.6 Hz, 4H, H-18), 3.24 (t, J = 4.5 Hz, 4H, H-14),
2.49 (m, J = 11.4, 5.9 Hz, 4H, mPz: -N-CH2-CH2-), 2.32 (s, 3H, Pz-CH3), 1.27 (q, J = 6.8 Hz,
2H, H-10b), 1.11 (q, J = 6.6 Hz, 2H, H-10a); 13C NMR (151 MHz, CDCl3) δ (ppm): 172.36 (C-
4), 165.80 (C-16), 163.69 (C-11), 153.36 (C-6), 144.94 (C-2), 113.05 (C-5), 104.91 (C-8),
66.74 (C-13), 49.95 (C-14), 47.58 (C-18), 46.72 (C-19), 46.14 (Pz-CH3), 34.40 (C-9), 8.23
(C-10); HRMS (ESI) m/z: [M+H]+ 527.2733 (calculated for C27H36FN6O4: 527.2782).
1-Cyclopropyl-6-fluoro-3-(4-methylpiperazine-1-carbonyl)-7-(4-propionylpiperazin-1-
yl)quinolin-4(1H)-one (21).
Tan powder; yield: 0.71 g (59%); m.p. 181.8 -185.0 ,. IR vmax: 2922, 2850, 1643, 1622,
1592 cm-1; 1H NMR (600 MHz, CDCl3) δ (ppm): 8.05 (s, 1H, H-2), 8.01 (d, J = 13.0 Hz,1H, H-
5), 7.27 (d, J = 6.4 Hz, 1H, H-8), 3.39 – 3.35 (m, J = 10.9, 7.1, 4.0 Hz, 1H, H-9), 2.49 (t, J =
4.9 Hz, 4H, mPz: -N-CH2-CH2-), 2.40 (q, J = 7.4 Hz, 2H, H-17), 2.32 (s, 3H, -CH2-N-CH3),
1.27 (q, J = 6.9 Hz, 2H), 1.18 (t, J = 7.4 Hz, 3H, H-18), 1.12 (q, J = 6.6 Hz, 2H); 13C NMR
(151 MHz, CDCl3) δ (ppm): 172.53 (C-4), 172.33 (C-16), 165.77 (C-11), 153.32 (C-6), 144.96
(C-2), 117.82 (C-3), 113.12 (C-5), 104.94 (C-8), 46.15 (Pz-CH3), 42.50 (-N-CH2-), 34.39 (C-
9), 9.56 (C-18), 8.21 (C-10); HRMS (ESI) m/z: [M+H]+ 470.2591 (calculated for C25H33FN5O3:
470.2567).
(3S,8S,9S,10R,13R,14S,17R)-10,13-Dimethyl-17-((R)-6-methylheptan-2-yl)-
2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 4-(1-
cyclopropyl-6-fluoro-3-(4-methylpiperazine-1-carbonyl)-4-oxo-1,4-dihydroquinolin-7-
yl)piperazine-1-carboxylate (22).
Beige powder; yield: 0.90 g (81%); m.p. 161.6 – 161.7 ; IR vmax: 2930, 2862, 1701, 1626,
1588 cm-1; 1H NMR (600 MHz, CDCl3) δ (ppm): 8.12 (s, 1H, H-2), 8.05 (d, J = 31 Hz, 1H, H-
5), 7.30 (d, J = 4.1 Hz, 1H, H-8), 5.38 (t, J = 4.7 Hz, 1H, H-6’), 4.58 – 4.52 (m, 1H, H-3’),
3.69 (t, J = 2.6 Hz, 2H, H-14), 3.41 (t, J = 4.1 Hz, 4H, H-13), 3.38 – 3.35 (m, J = 6.8, 3.9 Hz,
1H, H-9), 3.22 (d, J = 4.6 Hz, 4H, mPz: -N-CH2-), 2.49 (t, J = 5.8 Hz, 4H, mPz: -N-CH2-CH2-),
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2.32 (s, 3H, Pz-CH3), 1.02 (s, 3H, H-19’), 0.85 (dd, J = 6.6, 2.8 Hz, 6H, H-26’, H-27’), 0.67 (s,
3H, H-18’); 13C NMR (151 MHz, CDCl3) δ (ppm): 172.38 (C-4), 166.08 (C-11), 122.80 (C-6’),
117.28 (C-3), 112.91 (C-5), 105.04 (C-8), 75.45 (C-3’), 54.82 (mPz: -N-CH2-CH2-), 46.16
(Pz-CH3), 42.47 (C-14), 34.46 (C-9), 11.97 (C-18’), 8.26 (C-10); HRMS (ESI) m/z: [M]+
826.5665 (calculated for C50H73FN5O4); Purity (HPLC): 96%.
1-Cyclopropyl-6-fluoro-3-(4-methylpiperazine-1-carbonyl)-7-(4-(prop-2-yn-1-yl)piperazin-1-
yl)quinolin-4(1H)-one (23).
After above mentioned work-up, the product was precipitated out of ethyl acetate to yield
(85%) a fine white powder; m.p. 214.9 – 216.7 ; IR vmax: 3092, 2834, 2801, 2088,
1626 cm1; 1H NMR (600 MHz, CDCl3) δ (ppm): 8.04 (s, 1H, H-2), 7.98 (d, J = 13.2 Hz, 1H, H-
5), 7.27 (d, J = 8.1 Hz, 1H, H-8), 3.38 (d, J = 2.4 Hz, 3H, H-16), 3.37 – 3.35 (m, 1H, H-9),
3.33 – 3.27 (t, 4H, -N-CH2-), 2.79 (t, J = 4.8 Hz, 4H, H-14), 2.56 – 2.41 (t, J = 5.1 Hz, 4H,
mPz: -N-CH2-CH2-), 2.31 (s, 3H, Pz-CH3), 2.29 (t, J = 2.4 Hz, 1H, H-18), 1.26 (q, J = 6.8 Hz,
2H, H-10b), 1.11 (q, J = 6.6 Hz, 2H, H-10a); 13C NMR (151 MHz, CDCl3) δ (ppm): 172.39 (C-
4), 165.88 (C-11), 153.38 (C-6), 144.85 (C-2), 117.64 (C-3), 112.90 (C-5), 104.68 (C-8),
78.46 (C-17), 73.72 (C-18), 55.23 (mPz: -N-CH2-CH2-), 51.70 (C-14), 49.99 (mPz: -N-CH2-),
46.99 (C-16), 46.14 (Pz-CH3), 34.36 (C-9), 8.17 (C-10); HRMS (ESI) m/z: [M+H]+ 452.2466
(calculated for C25H31FN5O2: 452.2462); Purity (HPLC): 98%.
Azides 25a – g
Azides 25a – f, were prepared in accordance with the general procedure shown in Scheme 2
and described as follows:
Benzyl bromide 24a and benzyl bromide para-substituted 24b – f (1.0 eq.) were dissolved in
DMSO (15 ml) together with sodium azide (NaN3, 1.5 eq.). The reaction mixture was stirred
at room temperature overnight. The resulting reaction mixture was diluted with water (30 ml)
and the aqueous phase extracted with diethyl ether (3 x 40 ml). The combined organic layers
were washed with brine (3 x 50 ml) and dried over MgSO4. Removing the solvent left a clear
oil (Howson & Peter, 2010).
(Azidomethyl)benzene (25a).
Yield: 70%; 1H NMR (600 MHz, CDCl3) δ (ppm): 7.42 - 7.32 (m, 5H, H-3, H-4, H-5), 4.35 (s,
2H, H-1); 13C NMR (151 MHz, CDCl3) δ (ppm): 135.49 (C-2), 128.96 (C-4), 128.43 (C-5),
128.34 (C-3), 54.93 (C-1).
1-(Azidomethyl)-4-methylbenzene (25b).
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1
Yield: 95%; H NMR (600 MHz, CDCl3) δ (ppm): 7.23 – 7.18 (m, 4H, H-3, H-4), 4.30 (s, 2H,
H-1’), 2.37 (s, 3H, H-6); 13C NMR (151 MHz, CDCl3) δ (ppm): 138.28 (C-2), 132.41 (C-5),
129.63 (C-4), 128.40 (C-3), 54.75 (C-1), 21.31 (C-6).
1-(Azidomethyl)-4-fluorobenzene (25c).
1
Yield: 70%; H NMR (600 MHz, CDCl3) δ (ppm): 7.32 – 7.28 (m, 2H, H-3), 7.10 – 7.05 (m,
2H, H-4), 4.32 (s, 2H, H-1); 13C NMR (151 MHz, CDCl3) δ (ppm): 162.78 (C-5), 131.32 (C-2),
130.15 (C-3), 115.93 (C-4’), 54.20 (C-1).
1-(Azidomethyl)-4-bromobenzene (25d).
1
Yield: 100%; H NMR (600 MHz, CDCl3) δ (ppm): 7.51 (d, J = 8.4 Hz, 2H, H-4), 7.19 (d, J =
8.4 Hz, 2H, 3), 4.30 (s, 2H, H-1); 13C NMR (151 MHz, CDCl3) δ (ppm): 134.52 (C-2), 132.13
(C-3), 129.94 (C-4), 122.48 (C-5), 54.23 (C-1).
1-(Azidomethyl)-4-nitrobenzene (25e).
1
Yield: 100%; H NMR (600 MHz, CDCl3) δ (ppm): 8.24 (d, J = 8.6 Hz, 2H, H-4), 7.50 (d, J =
8.5 Hz, 2H, H-3), 4.50 (s, 2H, H-1); 13C NMR (151 MHz, CDCl3) δ (ppm): 147.88 (C-5),
142.83 (C-2), 128.72 (C-3), 124.18 (C-4), 53.86 (C-1).
1-(Azidomethyl)-4-(trifluoromethyl)benzene (25f).
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Yield: 73%, 1H NMR (600 MHz, CDCl3) δ (ppm): 7.15 (d, J = 7.7 Hz, 2H, H-4), 6.95 (d, J =
7.8 Hz, 2H, H-3), 4.5 (s, 2H, H-1); 13C NMR (151 MHz, CDCl3) δ (ppm): 125.92 (C-5), 54.20
(C-1.
Cholesteryl azide (25g).
Cholesteryl chloride (3.57 mmol, 1.66 g, 1eq) and NaN3 (17.73 mmol, 1.16 g, 5 eq.)
dissolved in DMF (25 ml) and heated overnight at 100 . Afterwards, the reaction mixture
was left to cool at room temperature then hexane (150 ml) and water (25 ml) were added.
The organic layer was then further washed with NaHCO3, brine and dried over MgSO4. The
crude organic layer was concentrated and subjected to flash column chromatography with
DCM as eluent (Sugandhi et al., 2007).
White solid; yield: 83%; IR vmax: 2934, 2866, 2649, 2084 cm-1 (in accordance to literature
1
(Sugandhi et al., 2007)); H NMR (600 MHz, CDCl3) δ (ppm): 5.37 (t,1H, H-6), 3.80 – 3.73
(m, J = 12.0, 4.5 Hz, H-3), 1.03 (s, 3H, H-19), 0.86 (dd, J = 6.6, 2.7 Hz, H, H-26, H-27), 0.67
(s, H-18); 13C NMR (151 MHz, CDCl3) δ (ppm): 140.96 (C-5), 122.64 (C-6), 60.51 (C-3),
56.85 (C-14), 56.29 (C-17), 50.22 (C-9), 43.56, 42.47 (C-13), 22.72 (C-26’, C-27), 19.41 (C-
19), 12.01 (C-18).
CPX-triazole hybrids 26 – 39
Hybrids 26 – 39 were prepared in accordance with the general procedure depicted in
Scheme 2.
Alkyne (13 or 23) (500 mg, 1.2 eq.) was dissolved in a mixture of DMF-water (15 ml, 9:1,
v/v). Whilst stirring, azide (25 a-g) (2.0 eq.), NaAsc (120 mg, 0.8 eq.), and CuSO4·5H2O (30
mg, 0.2 eq.) were consecutively added. The resulting reaction mixture was stirred vigorously
for a period of 24 – 36 hr at 60 °C monitored by TLC. Upon completion, NH4Cl (25 ml) was
added to quench the reaction mixture followed by extraction with DCM (3 x 40 ml). The
organic phase was dried over MgSO4 to afford hybrids 26 – 32 after purification by column
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chromatography (eluent: DCM/MeOH, 9:1 v/v), while compounds 33 – 39 were isolated by
precipitation with diethyl ether after column chromatography (eluent: DCM/MeOH, 4:1 v/v).
7-(4-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (26).
Beige powder; yield: 0.15 g (27%); m.p. 211.8 – 213.6 ; IR vmax: 2957, 2926, 2857, 1724,
1628 cm-1; 1H NMR (600 MHz, CDCl3) δ (ppm): 15.02 (s, 1H, H-11), 8.72 (s, 1H, H-2), 7.96
(d, J = 13.0 Hz, 1H, H-5), 7.46 (s, 1H, H-18), 7.40 – 7.35 (m, 3H, H-21, H-22), 7.32 (d, J =
7.1 Hz, 1H, H-8), 7.30 – 7.27 (m, 2H, H-23), 5.52 (s, 2H, H-19), 3.75 (s, 2H, H-16), 3.54 –
3.50 (m, 1H, H-9), 3.32 (t, J = 31.1 Hz, 4H, H-13), 2.74 (t, J = 22.2 Hz, 4H, H-14), 1.37 (q, J
= 10.5 Hz, 2H, H-10b), 1.17 (q, J = 6.5 Hz, 2H, H-10a); 13C NMR (151 MHz, CDCl3) δ (ppm):
177.19 (C-4), 167.88 (C-11), 153.75 (C-6), 147.51 (C-2), 112.49 (C-5), 104.96 (C-8), 54.34
(C-19), 53.15 (C-16), 52.53 (C-13), 49.66 (C-14), 35.41 (C-9), 8.35 (C-10); HRMS (ESI) m/z:
[M+H]+ 503.2201 (calculated for C27H28FN6O3: 503.2207); Purity (HPLC): 83%.
1-Cyclopropyl-6-fluoro-7-(4-((1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)piperazin-1-yl)-
4-oxo-1,4-dihydroquinoline-3-carboxylic acid (27).
Brown solid; yield: 0.17 g (28%); m.p. 203.7 – 205.5 ; IR vmax: 2958, 2927, 2859, 1722,
1
1628 cm-1; H NMR (600 MHz, CDCl3) δ (ppm): 15.11 (s, 1H, H-11), 8.80 (s, 1H, H-2), 8.04
(d, J = 13.0 Hz, 1H, H-5), 7.51 (s, 1H, H-18), 7.40 (d, J = 7.0 Hz, 1H, H-8), 7.26 (s, 5H, H-21,
H-22), 5.56 (s, 2H, H-19), 3.84 (s, 2H, H-16), 3.63 – 3.57 (m, 1H, H-9), 2.43 (s, 3H, H-24),
1.42 (q, J = 10.5 Hz, 2H, H-10b), 1.25 (q, J = 6.5 Hz, 2H, H-10a); 13C NMR (151 MHz,
CDCl3) δ (ppm): 177.21 (C-4), 167.19 (C-11), 153.76 (C-6), 147.54 (C-2), 129.93 (C-21),
128.33 (C-22), 112.51 (C-5), 108.19 (C-3), 104.99 (C-8), 54.16 (C-19), 53.11 (C-16), 35.42
(C-9), 21.31 (C-24), 8.36 (C-10); HRMS (ESI) m/z: [M+H]+ 517.2358 (calculated for
C28H30FN6O3: 517.2363); Purity (HPLC): 91%.
1-Cyclopropyl-6-fluoro-7-(4-((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)piperazin-1-yl)-
4-oxo-1,4-dihydroquinoline-3-carboxylic acid (28).
1
Beige powder; yield: (67%); m.p. 297.0 – 298.1 , IR vmax: 2929, 2828 cm-1. H NMR (600
MHz, DMSO) δ (ppm): 15.17 (s, 1H, H-11), 8.63 (s, 1H, H-2), 8.14 (s, 1H, H-18), 7.87 (d, J =
13.1 Hz, 1H, H-5), 7.52 (d, J = 7.4 Hz, 1H, H-8), 7.38 (t, J = 8.2, 5.7 Hz, 2H, H-21), 7.19 (t, J
= 8.8 Hz, 2H, H-22), 5.58 (s, 2H, H-19), 3.80 – 3.76 (m, 1H, H-9), 2.86 (s, 2H, H-16), 1.28 (q,
J = 6.8 Hz, 2H, H-10b), 1.14 (q, J = 6.5 Hz, 2H, H-10a); 13C NMR (151 MHz, DMSO) δ
(ppm): 176.37 (C-4), 165.95 (C-11), 148.07 (C-2), 130.31 (C-21), 115.63 (C-22), 111.10 (C-
18), (C-19), 35.89 (C-16), 35.80 (C-9), 30.79 (C-13, C-14), 7.58 (C-10); HRMS (ESI) m/z:
[M+H]+ 521.2107 (calculated for C27H26F2N6O3: 512.2113); Purity (HPLC): 89%.
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7-(4-((1-(4-Bromobenzyl)-1H-1,2,3-triazol-4-yl)methyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-
4-oxo-1,4-dihydroquinoline-3-carboxylic acid (29).
Brown powder; yield: 0.21 g (65%); m.p. 130.4 – 130.5 ; IR vmax: 2955, 2922, 2853, 1717,
1625, 1261 cm-1; 1H NMR (600 MHz, DMSO) δ (ppm): 15.11 (s, 1H, H-11), 8.65 (s, 1H, H-2),
8.37 (s, 1H, H-18), 7.94 – 7.90 (m, 1H, H-5), 7.66 – 7.46 (m, 4H, H-21, H-22), 7.29 (d, J =
8.1 Hz, 1H, H-8), 5.64 (s, 2H, H-19), 4.19 – 4.04 (m, 1H, H-9), 3.80 (s, 2H, H-16), 2.48 –
2.47 (m, 8H, H-13, H-14), 1.28 (q, J = 6.5 Hz, 2H, H-10b), 1.15 (q, J = 6.6 Hz, 2H, H-10a);
HRMS (ESI) m/z: [M+H]+ 581.1307 (calculated for C27H27BrFN6O3: 581.1321).
1-Cyclopropyl-6-fluoro-7-(4-((1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl)piperazin-1-yl)-4-
oxo-1,4-dihydroquinoline-3-carboxylic acid (30).
Dark yellow solid, yield: 0.09 g (15%); m.p. 118 – 119 ,. IR vmax: 2957, 2924, 2858, 1725,
1
1625 cm-1; H NMR (600 MHz, CDCl3) δ (ppm): 14.95 (s, 1H, H-11), 8.72 (s, 1H, H-2), 8.23
(d, J = 8.7 Hz, 3H, H-18, H-22), 7.95 (d, J = 12.9 Hz, 1H, H-5), 7.44 (d, J = 8.7 Hz, 2H, H-
21), 7.34 (d, J = 7.0 Hz, 1H, H-8), 5.67 (s, 2H, H-19), 3.94 (s, 2H, H-16), 3.55 – 3.51 (m, 1H,
H-9), 3.45 (t, J = 19.7 Hz, 4H, H-14), 2.93 (t, J = 15.3, 4H, H-13), 1.34 (q, J = 6.9 Hz, 2H, H-
10b), 1.19 (q, J = 6.5 Hz, 2H, H-10a); 13C NMR (151 MHz, CDCl3) δ (ppm): 177.14 (C-4),
167.90 (C-11), 153.94 (C-6), 150.34 (C-23), 148.26 (C-2), 148.14 (C-17), 147.64 (C-20),
128.92 (C-21), 124.49 (C-22), 119.81 (C-18), 112.66 (C-5), 104.01 (C-8), 53.37 (C-19),
52.77 (C-13, C-14), 52.35 (C-16), 35.46 (C-9), 8.40 (C-10); HRMS (ESI) m/z: [M+H]+
548.2052 (calculated for C27H27FN7O5: 548.2058); Purity (HPLC): 81%.
1-Cyclopropyl-6-fluoro-4-oxo-7-(4-((1-(4-(trifluoromethyl)benzyl)-1H-1,2,3-triazol-4-
yl)methyl)piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid (31).
Beige solid; yield: 0.11 g (49%); m.p. 122.3 – 123.7 ; IR vmax: 2956, 2923, 2853, 1725,
1
1625 cm-1; H NMR (600 MHz, CDCl3) δ (ppm): 15.00 (s, 1H, H-11), 8.71 (s, 1H, H-2), 7.94
(d, J = 13.0 Hz, 1H, H-5), 7.64 (d, J = 8.2 Hz, 2H, H-21), 7.57 (s, 1H, H-18), 7.40 (d, J = 8.1
Hz, 2H, H-22), 7.33 (d, J = 7.1 Hz, 1H, H-8), 5.61 (s, 2H, H-19), 3.82 (s, 2H, H-16), 3.54 –
3.51 (m, 1H, H-9), 1.36 (q, J = 6.5 Hz, 2H, H-10b), 1.18 (q, J = 6.5 Hz, 2H, H-10a); 13C NMR
(151 MHz, CDCl3) δ (ppm): 177.06 (C-4), 167.80 (C-11), 153.64 (C-6), 147.45 (C-2), 131.12
(C-24), 128.81 (C-21), 126.18 (C-22), 123.24 (C-18), 112.39 (C-5), 108.08 (C-3), 105.03 (C-
8), 53.67 (C-19), 53.08 (C-16), 52.50 (C-14), 49.45 (C-13), 35.33 (C-9), 8.26 (C-10); HRMS
(ESI) m/z: [M+H]+ 571.2019 (calculated for C28H26F4N6O3: 571.2081); Purity (HPLC): 85%.
1-Cyclopropyl-7-(4-((1-((3R,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-
methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-
cyclopenta[a]phenanthren-3-yl)-1H-1,2,3-triazol-4-yl)methyl)piperazin-1-yl)-6-fluoro-4-oxo-
1,4-dihydroquinoline-3-carboxylic acid (32).
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Light tan solid; yield: 0.32 g (36%); m.p. 150-155.2 ; IR vmax: 3250, 2935, 2849, 1735, 1628
cm-1; 1H NMR (600 MHz, CDCl3) δ (ppm): 15.04 (s, 1H, H-11), 8.75 (s, 1H, H-2), 7.99 (d, J =
13.0 Hz, 1H, H-5), 7.69 (t, 1H, H-6’), 7.52 (s, 1H, H-18), 7.36 (d, J = 7.1 Hz, 1H, H-8), 4.25 –
4.16 (m, 1H, H-3’), 3.56 – 3.53 (m, 1H, H-9), 3.40 (t, J = 4.8 Hz, 4H, H-13), 2.82 (t, J = 4.9
Hz, 4H, H-14), 0.86 (dd, J = 7.0 Hz, 6H, H-26’, H-27’), 0.67 (s, 1H, H-18’); 13C NMR (151
MHz, CDCl3) δ (ppm): 177.21 (C-4), 167.90 (C-11), 153.79 (C-6), 147.56 (C-2), 112.56 (C-
5), 108.21 (C-3), 105.00 (C-8), 68.28 (C-3’), 35.44 (C-9), 23.49 (C-26’, C-27’), 11.09 (18’),
8.37 (C-10); HRMS (ESI) m/z: [M+H]+ 781.5185 (calculated for C47H66FN6O3: 781.5180);
Purity (HPLC): 95%.
7-(4-((1-Benzyl-1H-1,2,3-triazol-4-yl)methyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-3-(4-
methylpiperazine-1-carbonyl)quinolin-4(1H)-one (33).
Light orange powder; yield: 0.6 g (24%); m.p. 282.9 – 283.0 ; IR vmax: 2957, 2927, 2857,
1
1726, 1624, 1580 cm-1; H NMR (600 MHz, CDCl3) δ (ppm): 8.03 (s, 1H, H-2), 7.95 (d, J =
13.2 Hz, 1H, H-5), 7.42 (s, 1H, H-18), 7.39 – 7.33 (m, 3H, H-22, H-23), 7.28 – 7.26 (m, 2H,
H-21), 7.24 (d, J = 7.1 Hz, 1H, H-8), 5.52 (s, 2H, H-19), 3.73 (s, 2H, H-16), 3.39 – 3.32 (m,
1H, H-9), 3.26 (t, J = 4.9 Hz, 4H, mPz: -N-CH2-), 2.72 (t, J = 4.5 Hz, 4H, H-14), 2.37 (s, 3H,
Pz-CH3), 1.25 (q, J = 12.9, 6.1 Hz, 2H, C-10b), 1.09 (q, J = 6.5 Hz, 2H, C-10a); 13C NMR
(151 MHz, CDCl3) δ (ppm): 172.43 (C-4), 165.86 (C-11), 153.39 (C-6), 144.68 (C-2), 129.25
(C-22), 128.90 (C-21), 128.24 (C-23), 122.69 (C-18), 117.41 (C-3), 112.82 (C-5), 104.65 (C-
8), 54.29 (C-19), 45.83 (Pz-CH3), 42.10 (C-13), 34.39 (C-9), 8.18 (C-10); HRMS (ESI) m/z:
[M+H]+ 585.3096 (calculated for C23H38FN8O2: 585.3102), Purity (HPLC): 99%.
1-Cyclopropyl-6-fluoro-7-(4-((1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)piperazin-1-yl)-
3-(4-methylpiperazine-1-carbonyl)quinolin-4(1H)-one (34).
Light orange powder; yield: 0.26 g (60%); m.p. 215.4 – 216.5 ; IR vmax: 2957, 2923, 2856,
1
1728, 1621, 1605 cm-1; H NMR (600 MHz, CDCl3) δ (ppm): 8.04 (s, 1H, H-2), 7.96 (d, J =
13.2 Hz, 1H, H-5), 7.39 (s, 1H, H-18), 7.25 (d, J = 7.1 Hz, 1H, H-8), 7.17 (s, 4H, H-21, H-22),
5.47 (s, 2H, H-19), 3.72 (s, 2H, H-16), 3.39 - 3.34 (m, 1H, H-9), 3.26 (t, 4H, mPz: -CH2-CH2-
N-), 2.72 (t, J = 4.5 Hz, 4H, H-14), 2.34 (s, 3H, H-24), 1.26 (q, J = 7.9, 5.7 Hz, 2H, H-10b),
13
1.10 (q, J = 6.6 Hz, 2H, H-10a); C NMR (151 MHz, CDCl3) δ (ppm): 172.45 (C-4), 165.87
(C-11), 153.41 (C-6), 144.97 (C-2), 138.87 (C-20), 138.50 (C-23), 131.65 (C-17), 129.91 (C-
21), 128.32 (C-22), 122.59 (C-18), 117.38 (C-3), 112.84 (C-5), 104.66 (C-8), 55.45 (C-14),
54.12 (mPz: -CH2-CH2-N-), 53.27 (C-19), 52.69 (C-16), 49.94 (mPz: -N-CH2-), 45.78 (Pz-
CH3), 34.41 (C-9), 21.30 (C-24), 8.19 (C-10); HRMS (ESI) m/z: [M+H]+ 599.3253 (calculated
for C33H40FN8O2: 599.3258); Purity (HPLC): 97%.
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1-Cyclopropyl-6-fluoro-7-(4-((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)piperazin-1-yl)-
3-(4-methylpiperazine-1-carbonyl)quinolin-4(1H)-one (35).
Ivory powder; yield: 0.21 g (63%); m.p. 261.6 – 261.7 ; IR vmax: 2935, 2832, 2800, 1681,
1622, 1590 cm-1; 1H NMR (600 MHz, CDCl3) δ (ppm): 8.04 (s, 1H, H-2), 7.95 (d, J = 13.2 Hz,
1H, H-5), 7.43 (s, 1H, H-18), 7.31 – 7.21 (m, 4H, H-21, H-22), 7.06 (d, J = 8.5 Hz, 1H, H-8),
5.49 (2H, H-19), 3.74 (s, 2H, H-16), 3.39 – 3.36 (m, 1H, H-9), 2.42 (s, 3H, Pz-CH3), 1.26 (q,
J = 6.7 Hz, 2H, H-10b), 1.10 (q, J = 6.5 Hz, 2H, H-10a); 13C NMR (151 MHz, CDCl3) δ (ppm):
172.46 (C-4), 165.86 (C-11), 162.99 (C-23), 153.41 (C-6), 145.00 (C-2), 138.50 (C-17),
130.14 (C-21), 121.71 (C-18), 117.31 (C-3), 116.27 (C-8), 112.82 (C-5), 104.69 (C-22),
53.23 (C-16), 52.71 (C-19), 45.63 (Pz-CH3), 8.19 (C-10); HRMS (ESI) m/z: [M+H]+ 603.3002
(calculated for C32H37F2N8O2: 603.3008).
7-(4-((1-(4-Bromobenzyl)-1H-1,2,3-triazol-4-yl)methyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-
3-(4-methylpiperazine-1-carbonyl)quinolin-4(1H)-one (36).
Gold powder; yield: 0.05 g (18%); m.p. 254.7 – 255.6 ; IR vmax: 2918, 2831, 1626, 1581,
1
1256 cm-1; H NMR (600 MHz, CDCl3) δ (ppm): 8.08 (s, 1H, H-2), 7.89 (d, J = 13.0 Hz, 1H,
H-5), 7.68 (s, 1H, H-18), 7.50 (d, J = 8.4 Hz, 2H, H-22), 7.28 (d, J = 7.0 Hz, 1H, H-8), 7.17
(d, J = 8.3 Hz, 2H, H-21), 5.49 (s, 2H, H-19), 3.71 (s, 2H, H-16), 3.42 – 3.36 (m, 9H, H-9, H-
14, mPz: -N-CH2-), 2.91 (t, J = 22.3 Hz, 4H, mPz: -CH2-CH2-N-), 2.78 (s, 3H, Pz-CH3),1.30
(q, J = 6.7 Hz, 2H, H-10b), 1.12 (q, J = 6.3 Hz, 2H, H-10a); 13C NMR (151 MHz, CDCl3) δ
(ppm): 166.79 (C-4), 165.76 (C-11), 152.63 (C-6), 145.69 (C-2), 132.48 (C-22), 129.89 (C-
21), 123.19 (C-18), 112.74 (C-5), 105.11 (C-8), 44.83 (Pz-CH3), 34.71 (C-9), 8.28 (C-10);
HRMS (ESI) m/z: [M+H]+ 663.2201 (calculated for C32H37BrFN8O2: 663.2207).
1-Cyclopropyl-6-fluoro-3-(4-methylpiperazine-1-carbonyl)-7-(4-((1-(4-nitrobenzyl)-1H-1,2,3-
triazol-4-yl)methyl)piperazin-1-yl)quinolin-4(1H)-one (37).
Gold coloured powder; yield: 0.19 g (55%); m.p. 281.5 – 281.6 ; IR vmax: 2957, 2928, 2857,
1
1727, 1622, 1589 cm-1. H NMR (600 MHz, CDCl3) δ (ppm): 8.22 (d, J = 8.8 Hz, 2H, H-22),
8.04 (s, 1H, H-2), 7.95 (d, J = 13.2 Hz, 1H, H-5), 7.52 (s, 1H, H-18), 7.42 (d, J = 8.8 Hz, 2H,
H-21), 7.25 (d, J = 7.1 Hz, 1H, H-8), 5.65 (s, 2H, H-19), 3.77 (s, 2H, H-16), 3.38 – 3.34 (m, J
= 4.0 Hz, 1H, H-9), 2.74 (t, J = 4.5 Hz, 4H, H-14), 2.40 (s, 3H, Pz-CH3), 1.26 (q, J = 4.6 Hz,
2H, H-10b), 1.10 (q, J = 6.6 Hz, 2H, H-10a); 13C NMR (151 MHz, CDCl3) δ (ppm): 172.45 (C-
4), 165.85 (C-11), 153.39 (C-6), 148.21 (C-23), 145.08 (C-2), 144.97 (C-20), 141.77 (C-17),
128.76 (C-21), 124.44 (C-22), 123.00 (C-18), 117.38 (C-3), 112.85 (C-5), 104.68 (C-8),
53.24 (C-16), 53.20 (C-14), 52.76 (C-19), 49.94 (mPz: -N-CH2-), 45.73 (Pz-CH3), 34.42 (C-
9), 8.21 (C-10); HRMS (ESI) m/z: [M+H]+ 630.2929 (calculated for C32H37FN9O4: 630.2953);
Purity (HPLC): 97%.
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1-Cyclopropyl-6-fluoro-3-(4-methylpiperazine-1-carbonyl)-7-(4-((1-(4-(trifluoromethyl)benzyl)-
1H-1,2,3-triazol-4-yl)methyl)piperazin-1-yl)quinolin-4(1H)-one (38).
Light pink powder; yield: 0.32 g (67%); m.p. 231.9 – 242.4 ,. IR vmax: 2958, 2922, 2850,
1
1727, 1621, 1589 cm-1; H NMR (600 MHz, CDCl3) δ (ppm): 8.05 (s, 1H, H-2), 7.96 (d, J =
13.2 Hz, 1H, H-5), 7.64 (d, J = 8.1 Hz, 2H, H-22), 7.47 (s, 1H, H-19), 7.38 (d, J = 8.1 Hz, 2H,
H-21), 7.25 (d, J = 7.2 Hz, 1H, H-8), 5.60 (s, 2H, H-19), 3.76 (s, 2H, H-16), 3.38 – 3.35 (m,
1H, H-9), 3.27 (t, 4H, mPz: -N-CH2-), 2.74 (t, J = 4.5 Hz, 4H, H-14), 2.41 (s, 3H, Pz-CH3),
1.26 (q, J = 8.2, 5.3 Hz, 2H, H-10b), 1.10 (q, J = 6.6 Hz, 2H, H-10a); 13C NMR (151 MHz,
CDCl3) δ (ppm): 172.46 (C-4), 165.86 (C-11), 153.41 (C-6), 145.00 (C-2), 128.38 (C-21),
126.26 (C-22), 122.84 (C-18), 117.36 (C-3), 112.85 (C-5), 104.68 (C-8), 53.23 (C-19), 52.75
(C-16), 49.96 (mPz: -N-CH2-), 34.42 (C-9), 8.20 (C-10); HRMS (ESI) m/z: [M+H]+ 653.2980
(calculated for C33H37F4N8O2: 653.2976); Purity (HPLC): 98%.
1-Cyclopropyl-7-(4-((1-((3R,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-
methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-
cyclopenta[a]phenanthren-3-yl)-1H-1,2,3-triazol-4-yl)methyl)piperazin-1-yl)-6-fluoro-3-(4-
methylpiperazine-1-carbonyl)quinolin-4(1H)-one (39).
Yellow solid; yield (12%); IR vmax: 2929, 2865, 1724, 1626 cm-1; 1H NMR (600 MHz, CDCl3) δ
(ppm): 8.04 (s, 1H, H-2), 7.97 (d, J = 13.2 Hz, 1H, H-5), 7.72 (s, 1H, H-18), 7.26 (d, J = 3.4
Hz, 1H, H-8), 5.49 (t, J = 4.5 Hz, 1H, H-6’), 4.20 (t, J = 6.0 Hz, 1H, H-3’), 3.76 (s, 2H, H-16),
3.37 (s, 1H, H-9), 2.37 (s, 3H, Pz-CH3), 1.09 (s, 3H, H-19’), 0.85 (dd, J = 2.6 Hz, 6H, H-26’,
H-27’), 0.67 (s, 3H, H-18’); 13C NMR (151 MHz, CDCl3) δ (ppm): 172.42 (C-4), 165.89 (C-
11), 153.41 (C-6), 144.93 (C-2), 124.41 (C-6’), 112.87 (C-5), 104.64 (C-8), 68.27 (C-3’),
50.26 (C-13), 45.89 (Pz-CH3), 42.42 (C-14), 34.40 (C-9), 11.98 (C-18’), 8.20 (C-10); HRMS
(ESI) m/z: [M+H]+ 863.6111 (calculated for C52H76FN8O5: 863.6075); Purity (HPLC): 99%.
In-vitro biological evaluation
Antimycobacterial activity assessment
The Broth Microdilution method was used to determine the antimycobacterial activities
(MIC90 values) of all compounds, as previously described (Wilson et al., 2017).
Cytotoxicity assays
WI-38 cell line. Human fetal lung fibroblast (HFLF) cell line from ECACC was routinely
maintained as a monolayer cell culture at 37ºC, 5% CO2, 95% air and 100% relative humidity
in EMEM containing 10% fetal bovine serum, 2 mM L-glutamine and 50 µg/ml gentamicin.
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For screening experiment, the cells (21-50 passages) were inoculated in a 96-well microtiter
plates at plating densities of 10 000 cells/well and were incubated for 24 h. After 24 h the
cells were treated with the experimental drugs which were previously dissolved in DMSO
and diluted in medium to produce 5 concentrations. Cells without drug served as control.
The blank contains complete medium without cells. Emetine was used as a standard. The
plates were incubated for 48 h after addition of the compounds. Viable cells were fixed to the
bottom of each well with cold 50% trichloroacetic acid, washed, dried and dyed by SRB.
Unbound dye was removed and protein-bound dye was extracted with 10mM Tris base for
optical density determination at the wavelength 540 nm using a multiwell spectrophotometer.
Data analysis was performed using GraphPad Prism software. 50% of cell growth inhibition
(IC50) was determined by non-linear regression.
CHO and HEK 293 cell lines. cells were cultured in Hyclone Ham’s F12 growth medium,
Kaighn’s modification supplemented with 10 % fetal bovine serum, 1 % Penicillin-
Streptomycin mixture and Amphotericin B. HEK-293 cells were cultured in Hyclone
Dulbecco’s modified Eagle’s medium with high glucose supplemented with 10 % fetal bovine
serum and 1 % L-glutamine, Penicillin-Streptomycin, Amphotericin B and non-essential
amino acids. Both cell lines are maintained in a humidified atmosphere at 37 °C and 5 %
CO2. For the MTT assay, 96 well plates were prepared with 200 μL of cell suspension (75
000 cells/mL for both cell lines) and incubated for 24 hours. The cells were then treated with:
(1) 100 μL of emetine dihydrochloride solution diluted with growth medium to the necessary
concentrations (positive control); (2) 80 μL of growth medium and 20 μL of solvent (negative
control to compensate for possible solvent effects); (3) 80 μL of growth medium and 20 μL of
experimental compound solutions. Blanks contained growth medium without cells. The
treated plates were incubated for 24 hours.
To initiate the MTT assay, 20 μL of sterile-filtered MTT solution (5 mg/mL in PBS) was
added and the plates incubated for 4 hours. The growth medium-MTT mixture was then
aspirated and 100 μL of 2-propanol added to dissolve purple formazan crystals. Absorbance
was measured at 560 and 650 nm using the Thermofisher Scientific GO Multiscan plate
reader. Due to light sensitivity of MTT reagent, the assay was performed in the dark. Thus,
the plates were covered with aluminium foil and the contents gently mixed for 15 minutes at
room temperature. Data analysis was performed for each biological replicate using SkanIt
4.0 Research Edition software. Background absorbance (650 nm) was subtracted from
absorbance values (560 nm), the mean absorbance calculated and the percentage cell
viability was determined by the following equation:
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Cell viability % = (Δ Abs sample – Δ Abs blank) / (Δ Abs neg control – Δ Abs blank) X 100
The IC50 and Z-score were determined for each compound’s biological replicate using the
SkanIt 4.0 software and four-parameter logistic (sigmoidal) regression. For the final IC50 of
each compound, the mean IC50 of the biological replicates were calculated and the mean
dose-response curve, with standard error of the means (SEM), were prepared in GraphPad
Prism 5.
Results and discussion
Chemistry
Two series of CPX derivatives were synthesized using different synthetic routes. Each series
was further divided into sub-series to highlight the differences in functional groups namely,
carboxylic acid, ethyl ester and 4-methylpiperazinyl amide in position 11 of CPX scaffold
The analogs (Series 1) were synthesized as shown in Scheme 1 starting from the
commercially available, CPX. The carboxylic acid sub-series was obtained in single
amidation reaction of CPX at its N-15 with acyl/carbamoyl/sulfonyl chloride or cholesteryl
chloroformate utilizing literature reported method (Qandil et al., 2014) to afford analogues 9
– 12. The nucleophile substitution SN2 using propargyl bromide at the N-15 resulted in 13.
All analogues were isolated in moderate to excellent yields (44 – 100%) after
recrystallization from acetonitrile. These acids were converted to ethyl esters 14 – 18 in poor
yields (4 – 23%) attributed to the purification process, and to the piperazinyl amides 19 – 23
more successfully in 39 – 95% yields using reported methods (Bandyopadhyay et al., 2010;
Soto-Castro et al., 2010). The compounds were isolated following purification by silica gel
chromatography. It is noteworthy indicating that following the procedure described by
Quandil et al. (Qandil et al., 2014), synthesis of the alkyne intermediate 13 was optimized by
changing the solvent system from dry dichloromethane to monoglyme-DMSO mixture (5:1)
upon reflux to improve solubility of the reagents which beneficially resulted in an increase of
its yield from 54 to 66%.
Scheme 1
Series 2 compounds were formed by hybridization of CPX and triazole scaffolds, and were
obtained in a two-step process. Firstly, the nucleophile substitution SN2 of benzyl/cholesteryl
bromides with sodium azide afforded the azido intermediates 25a - g in good yields (70 –
100%) (Howson & Peter, 2010). Secondly, the intermediates were subjected to the
regioselective Huisgen copper alkyne-azide cycloaddition ‘‘click’’ reaction (Dixit et al., 2012)
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with either alkynyl acid 13 to give the 1,4-disubstituted-1,2,3-triazole acids 26 - 32 or with
alkyne 18 to afford the amides 33 - 39 as depicted in Scheme 2. All hybrids were isolated in
poor to moderate yields (12 – 67%) after purification by precipitation from diethyl ether
followed by column chromatography on silica gel.
Scheme 2
The formation of the target compounds was confirmed by routine structural characterization
techniques such as FTIR, NMR and HRMS. The 1H NMR spectra of all title compounds were
thoroughly examined for characteristic peaks of the CPX and triazole scaffolds. Two quartets
found in the 1.3 – 1.0 ppm region and the multiplet in the region of 3.80 – 3.0 ppm were
assigned to the cyclopropyl protons H-10 and H-9, respectively. The doublets ca. 8.0 and 7.5
ppm were attributed to the resonance of H-5, and H-8, respectively, while the resonance of
H-2 results in a singlet at 8.6 ppm. These were evidence of the presence of the
fluoroquinolone pharmacophore. The former doublet is ascribed to the coupling of H-5 with F
in position 6 in the aromatic system (Qandil et al., 2014). This distinctive splitting pattern was
also seen in the 13C NMR spectra where C-F coupling constants (J) for C-5, C-6, and C-7
were 23, 248, and 10 Hz which is in accordance with previous findings (Qandil et al., 2014)
and further confirms the presence of the fluoroquinolone pharmacophore in the synthesized
compounds.
Evidence of the carboxylic acid functionality was obtained in the structures of compounds 9
– 13 and 26 – 32 with the presence a singlet ca. 15.00 ppm in 1H spectra which is assigned
to the acidic proton H-11. The disappearance of this peak in the spectra of the esters 14 –
18, and amides 19 -23 and 33 - 39 corroborated effectiveness of derivatization.
In the spectra of the hybrids, resonance of the aromatic proton of the triazole ring resulted in
a singlet at ca. 5.5 ppm while the singlet due to the two methylene protons in the linker
appeared in the 3.8 – 2.8 ppm region.
The FTIR spectra of all compounds were also inspected for the presence of characteristic
absorption peaks, allowing for the identification of functional groups. Evidence of carbonyl
C=O was confirmed with strong stretch ca. 1620 – 1700 cm-1 and variable broad O-H
stretching ca. 2842 cm-1 for the acids.
Physiochemical properties
The n-octanol/water partition coefficient (LogP) is a key parameter used for the
measurement of the balance between hydrophilicity and lipophilicity; giving insight into the
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transport characteristics of a chemical across biomembranes through passive diffusion
(Gombar & Enslein, 1996). LogP values between 1 and 5 are usually targeted while values
between 1 and 3 are ideal (Smit et al., 2015). However, it is well known that high or above-
average lipophilicity, is a hallmark for every antitubercular agents either in early and
advanced drug discovery process as can be seen from the most recent drugs introduced in
the market, bedaquiline (cLogP 7.25) and delamanid (cLogP 6.14), which have clogP values
above the safe threshold of 5 (Machado et al., 2018).
In Series 1, all amides 19 – 23 exhibited cLogP values lower than those of corresponding
acids 9 – 13 which in turn possessed lower clogP than the esters 14 – 18. The lipophilicity
order was as follows: amides< acids<esters. Conversely, the acids 26 – 32 in Series 2
exhibit cLogP values lower than the amides 33 – 39. Over both series, the cholesteryl
containing derivatives, 12, 17, 22, 32, and 39 showed poor drug-like properties with cLogP
values ca. 9, way above the safety threshold but couldn’t be disregarded in consideration of
the aforementioned facts (active transport).
Biological activities
For a novel drug to advance to clinical evaluation in humans, a chain of preclinical studies
including in vitro assays and in vivo testing need to be concluded.
Mycobacterial growth inhibitory potential of the synthesized compounds was assessed in the
standard Microplate Alamar Blue Assay (MABA) against Mtb H37Rv using a reporter mutant
constitutively expressing Green Fluorescent Protein (GFP) as described previously (Wilson
et al., 2017). All assays used the Middlebrook 7H9 Broth base supplemented with glucose
(GLU), enriched with ADC (albumin-dextrose-catalyse) and Tween 80 as surfactant. The
minimum concentrations that inhibit the growth of 90% of mycobacteria, expressed as MIC90
are summarized in Tables 1 for Series 1 and Table 2 for Series 2 alongside isoniazid (INH)
and rifampicin (RIF) as antitubercular standards.
Furthermore, three mammalian cell lines viz. normal human fetal lung fibroblasts HFLF WI-
38, the human embryonic kidney (HEK-293) and the Chinese Hamster Ovarian (CHO) cells
were used to determine the cytotoxicity of the compounds alongside the known cytotoxic
drug, emetine, as reference.
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It has been established over years of SAR investigations of quinolones that the carboxylic
group is crucial to the antimycobacterial activity of fluoroquinolone drugs (Figure 1).
However, choice for the synthesis of ester and amide derivatives in this study was motivated
by the prodrug approach wherein the derivatives would act as substrates for the acids.
Effectiveness of TB treatment is hindered by the unique structure, chemical composition and
lipophilic nature of Mtb’s cell wall which obstructs drug entry into the bacterium thereby
nullifying its activity (Brennan & Nikaido, 1995). Thus, lipophilicity is an inescapable property
to be mindful of when designing novel anti-TB drugs (Hu et al., 2017; Suresh et al., 2014).
Also, it has been reported that increasing the lipophilic character of fluoroquinolone through
derivatization at position C-7 leads to enhanced antimycobacterial activity et al., 2018), due
to increasing penetration and concentration in macrophages of the novel derivative
(Ginsburg et al., 2003; Shindikar & Viswanathan, 2005).
Persuaded by these previous findings, the highly lipophilic cholesteryl moiety was
conjugated to CPX or the CPX-TZ hybrid, anticipating a good penetration of mycobacteria
cell wall through passive diffusion which would result in enhanced activity.
Furthermore, an active transporter, mce4 has been indicated to transport cholesterol (Figure
5) into Mtb (Ouellet et al., 2011) where it is used as carbon source, thus making cholesterol
the driving factor behind Mtb’s virulence (Brzostek et al., 2007). In this latter design, the
cholesteryl moiety would be a hypothetical targeting moiety, facilitating the intracellular
uptake of the novel cholesteryl-CPX containing agent through active transportation mediated
by mce4. This would have the advantage of evading the obstruction of the bacterium cell
wall, thus enhancing the intake of the active drug, CPX or CPX-TZ inside the bacterial cell.
Series 1 analogues were brought about by introducing substituents at C-11 and N-15
positions of CPX structure in an effort to assess the influence of chemical functionality and
lipophilic character on the biological activity. The hybrids (Series 2) were mostly para-benzyl
substituted with either electron donating group (EDGs) or electron withdrawing groups
(EWGs). The choice of these groups was motivated by the need to judge of the impact the
electronic effect might have on the antimycobacterial activity.
All the acids evidently, except the cholesteryl derivative 12, possessed
antimycobacterial activity while the ethyl esters were partially active (2 out 5) and the amides
were completely inactive. Derivatives 10 and 11 with MIC90 1.6 µM were the most active
analogues with activity commensurable with that of the parent CPX. In Series 2, the
cholesteryl containing hybrid 32 was the most active with MIC90 2 µM comparable to CPX.
The acid analogues were more active than their hybrid acid counterparts. Dejectedly, no
derivative was found to be more potent than any of the reference drugs.
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The methyl piperazine amide analogues 19 – 23 and hybrids 33 – 39, which were
inactive against Mtb despite showing improved calculated drug-like properties.
Comparatively, the corresponding acids 9 – 13 and 26 – 32 were active. The ethyl esters
sub-series showed partial activity with half members being inactive. Thus, the C-3 bound
carboxylic acid group is a structural prerequisite for the fluoroquinolone scaffold to possess
anti-Mtb activity which is in accordance with previous findings (Andersson & MacGowan,
2003; Appelbaum & Hunter, 2000; Chu & Fernandes, 1989; Emmerson & Jones, 2003;
Tillotson, 1996).
Derivatives 12, 17, 22, 32, 39 which contain bulky cholesteryl moiety had off target
range (1-5) cLogP values. Therefore, these highly lipophilic derivatives were expected to
efficiently permeate through the lipophilic mycolic acid Mtb cell wall or be transported into the
bacterium through mce4 system which would ultimately result in high activities. However, the
inactivity of most of these derivatives (exception of 32, cLogP 8.91; MIC90 ~ 2.06 µM),
suggest that neither lipophilicity nor mce4 transporter controlled the intracellular uptake of
these compounds, with their high molecular weights, MW 780 on average, being possibly an
additional limiting factor.
CPX has a poor aqueous solubility which requires improvement through the
derivatization strategy. The cLogP values of synthesized compounds were mostly found to
be in the 2 – 5 range of favourable drug-likeness. Of note, these values are theoretical (i.e.,
calculated using MarvinSketch) and do not necessarily represent the actual physical
property. Indeed, CPX has in theory a cLogP value of 1.55, though in practice it is known for
its lowest in-class solubility (Firestone et al., 1998).
In both series, the functional group at C-11 controls the solubility of the derivative.
Indeed, in the analogues, the lipophilicity was in the ascending order, amide<acid<ester with
the efficacy in the increasing order, amide<ester<acid. In the hybrids, the amides were more
lipophilic than the acids which were more anti-Mtb active. Therefore, although, more
lipophilic derivative was expected to be more antimycobacterial active (Fan et al., 2018), the
opposite was observed which suggests that structural specificity rather than physical feature
(lipophilicity) governed the activity of these derivatives.
In Series 2, the active hybrids 27 possessed EDG (CH3), 28 – 31 had EWGs and 26
bore a neutral group (H). The antimycobacterial activities showed 26 to be more active than
27, which indicates that the presence of the lesser EDG increased the activity. The EWGs
were F, Br, NO2, CF3 with the increasing order of electronegativity being: F (28) <Br (29)
<NO2 (30) <CF3 (31). The increasing order of activity was 31, 30, 29 and 28 clearly indicates
that the activity increased with the decrease in electronic effect. Thus, the SAR of these acid
non-cholesteryl hybrids suggest that the activity is influenced by the electronic effect of the
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substituent on the phenyl ring. The stronger the EWG, the lesser antimycobacterial active is
the hybrid.
Overall, analogues 9, 10, 11 and 13, and hybrids 28, 29, 30 and 32 had promising
antimycobacterial activities (MIC90<10 µM) and were found to be nontoxic to the mammalian
cells, as can be gleaned from the IC50 values which were greater than 100 µM compared to
emetine. These derivatives were also selective in their antibacterial actions (SI>10) in
presence of the mammalian cells. Therefore, they all stand as validated hits based on
cellular potency and cytotoxicity criteria (Katsuno et al., 2015). However, expression of
biological activity of a drug which is consecutive to its permeation through biomembranes,
requires a multifunctional interaction of factors such as molecular size, lipophilicity, polar van
der Waals surface, and molecular flexibility (Martinez & Amidon, 2002). On the basis of
these structural criteria, and safety profile, compounds 11 among the analogues and 28
within the hybrid series, were identified as preferred hits into lead investigation.
Conclusion
Two series of ciprofloxacin derivatives namely fifteen analogues and fourteen hybrids
featuring carboxylic acid, ester or amide functional groups at carbon C-3 of the
fluoroquinolone scaffold, were synthesized through various synthetic routes including
amidation, esterification, nucleophilic substitution SN2 and Click chemistry. The derivatives
were evaluated in vitro for antimycobacterial activity against human virulent Mtb H37Rv
strain. The cytotoxicity was also assessed using three mammalian cell lines, normal human
fetal lung fibroblasts (HFLF WI-38), the human embryonic kidney (HEK-293) and the
Chinese hamster ovarian (CHO). The acid derivatives were confirmed to be active while the
amides were inactive. The esters showed mixed activity profile. Structure-activity relationship
also revealed the activity to be structure-specific in general, and governed by electronic
effect for the non-cholesteryl hybrids.
Low molecular weights analogue 11 featuring propyl side-chain, and hybrid 28
presenting para-fluorobenzyl moiety, possessed antimycobacterial activity MIC90<10 µM and
selective action towards bacteria (SI>10). Both compounds fulfilled criteria for validated
antimycobacterial hits for further development. This will entail optimization of these hits
through the investigation of derivatives bearing substituent(s) with reduced electron
withdrawing effect on the aryl ring. It will also be of interest to determine the DMPK
parameters and in vivo activities of these hits and their derivatives. This study could also be
extended to other fluoroquinolones such as moxifloxacin and gatifloxacin, which are
currently used for the treatment of tuberculosis, and possess chemical functionalities
suitable for derivatizations.
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Acknowledgments
This work was funded by grants from the South African National Research Foundation
(NRF) (to DD N’Da; UID 76443; 98937), and the Strategic Health Innovations Partnerships
(SHIP) Initiative of the South African Medical Research Council (to D.F.W). The authors
thank Dr Otto for NMR analysis and Dr Jordaan for MS analysis. Isoniazid was generously
donated by Aspen Pharmacare (Port Elizabeth, South Africa).
Disclaimer
Any opinions, findings and conclusions, or recommendations expressed in this material are
those of the authors and therefore the NRF does not accept any liability in regard thereto.
Conflict of Interest Statement
The authors have no conflict of interest to declare.
FIGURE LEGENDS
Figure 1. Illustration of new CPX analogues Series 1 design
Figure 2. Structures of 1,2,3-triazole scaffold and clinically important hybrids
Figure 3. Illustration of target CPX-triazole hybrids -Series 2
Scheme 1. Synthesis of CPX analogues - Series 1
Reagents and conditions: (a) 9 – 12: acyl/carbamoyl chloride or cholesteryl chloroforamate (1.3 eq.),
THF, TEA (2.6 eq.), 5 hr, rt ;13: propargyl bromide (2 eq), monoglyme:DMSO (5:1, v/v), DMAP (0.1
eq.), TEA (3 eq.), reflux, overnight; (b) 14 – 18: absolute EtOH, DMAP (0.1 eq.), EDC (1 eq.),
anhydrous DCM, 0 °C – rt, overnight; (c) 19 – 23: 4-methylpiperazine (2.4 eq.), HBTU (1.2 eq.), TEA
(3 eq.), anhydrous DCM, 0 °C – rt, 4 hr.
Scheme 2. Synthesis of CPX-triazole hybrids - Series 2
Reagents and conditions: (a) 25a – f: benzyl bromide, NaN3 (1.5 eq.), DMSO, 40 °C, overnight; 25 g:
cholesteryl halide (1 eq.), NaN3 (1.5 eq.), DMF, 100 °C, overnight; (b) 13 (1 eq.), benzyl/cholesteryl
azide (2 eq.), DMF:H2O (9:1, v/v), NaAsc (0.8 eq.), CuSO4·5H2O (0.2 eq.), rt, 24 – 30 hr; (c) 23 (1
eq.), benzyl/cholesteryl azide (2 eq.), DMF:H2O (9:1, v/v), NaAsc (0.8 eq.), CuSO4·5H2O (0.2 eq.), rt,
24 – 30 hr.
Table 1. In vitro antimycobacterial activities of CPX analogues 14 – 23.
Table 2. In vitro antimycobacterial activities of CPX hybrids 26 – 39.
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