Synthesis and Investigation of Phthalazinones as Antitubercular Agents

Article abstract of DOI:10.1002/asia.201801805

A series of 2- and 7-substituted phthalazinones was synthesised and their potential as anti-tubercular drugs assessed via Mycobacterium tuberculosis (mc²6230) growth inhibition assays. All phthalazinones tested showed growth inhibitory activity (MIC <100 μm), and those compounds containing lipophilic and electron-withdrawing groups generally exhibited better anti-tubercular activity. Several lead compounds were identified, including 7-((2-amino-6-(4-fluorophenyl)pyrimidin-4-yl)amino)-2-heptylphthalazin-1(2H)-one (MIC=1.6 μm), 4-tertbutylphthalazin-2(1H)-one (MIC=3 μm), and 7-nitro-phthalazin-1(2H)-one (MIC=3 μm). Mode of action studies indicated that selected pyrimidinyl-phthalazinones may interfere with NADH oxidation, however, the mode of action of the lead compound is independent of this enzyme. MIC=minimum inhibitory concentration.

Full text of DOI:10.1002/asia.201801805

CHEMISTRY
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Accepted Article
Title: The synthesis and investigation of phthalazinones as anti-
tubercular agents
Authors: Mattie Timmer, Kristiana Tika Santoso, Chen-Yi Cheung, Kiel
Hards, Gregory Cook, and Bridget Stocker
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The synthesis and investigation of phthalazinones as anti-
tubercular agents
Kristiana T. Santoso,[a,b,d] Chen-Yi Cheung,[c] Kiel Hards,[c,d] Gregory M. Cook,[c,d] Bridget L.
Stocker,[a,b,d,*] and Mattie S. M. Timmer[a,b,d, ]
*
Abstract: A series of 2- and 7-substituted phthalazinones was
synthesised and their potential as anti-tubercular drugs assessed via
Mycobacterium tuberculosis (mc26230) growth inhibition assays. All
phthalazinones tested showed growth inhibitory activity (MIC < 100
M), and those compounds containing lipophilic and electron-
withdrawing groups generally exhibited better anti-tubercular activity.
Several lead compounds were identified, including 7-((2-amino-6-(4-
fluorophenyl)pyrimidin-4-yl)amino)-2-heptylphthalazin-1(2H)-one
(MIC = 1.6 M), 4-tertbutylphthalazin-2(1H)-one (MIC = 3 M), and 7-
nitro-phthalazin-1(2H)-one (MIC = 3 M). Mode of action studies
indicated that selected pyrimidinyl-phthalazinones may interfere with
NADH oxidation, however, the mode of action of the lead compound
is independent of this enzyme.
Salmonella typhi.14 Although the mode of action of anti-bacterial
phthalazinones has not been studied, we reasoned that
phthalazinones might exhibit anti-tubercular activity due to their
ability to mimic the benzoquinone moiety in menaquinone (MK), a
key intermediate in the mycobacterial respiratory chain of
Mycobacterium tuberculosis, the causative agent of tuberculosis
(TB).15
Introduction
Phthalazinones exhibit a diverse range of pharmaceutical
properties, such as anti-cancer,1-3 anti-inflammatory,4-6 anti-
bacterial,1,7 anti-oxidant,1 and anti-neurodegenerative8 properties,
with mechanisms of action that are as varied as the diseases they
are used against.9-12 For example, the clinically used anti-cancer
drug oliparib (1, Figure 1) inhibits the poly(ADP-
ribose)polymerase,9 while azelastine (2) is an antihistamine
preferentially taken up by lung and alveolar macrophages.10 Anti-
bacterial phthalazinones include amide 3, which inhibits
Francisella tularensis, a potential bioterrorism agent, with a
minimum bactericidal concentration (MBC) of 0.94 M,13 and
oxadiazole 4, which is anti-bacterial against Escherichia coli and
Figure 1. Representative phthalazinones.
The mycobacterial respiratory chain is a potentially rich source of
antimycobacterial drug targets,15 as is perhaps best illustrated by
the recent discovery of bedaquiline (BDQ, 5, Figure 2), the first
FDA-approved drug for the treatment of TB in over forty years.16-
18 BDQ targets the F1F0-ATP synthase, acting as an uncoupler by
dissociating the transmembrane proton transfer from
phosphorylation reactions responsible for ATP generation. Other
TB drug targets within the respiratory chain include the
cytochrome bcc complex (inhibited by Q203, 6),19 cytochrome bc1
(lansoprazole, LPZ, 7)20 and, of particular interest to us, the
respiratory type II NADH dehydrogenase (NDH-II).21 NDH-II is
absent from mammalian mitochondria yet it is essential for growth
in mycobacteria as it catalyses the transfer of electrons from
NADH to MK via the aid of a flavin co-factor (FAD).22,23 There are
three distinct binding sites in NDH-II: the first and second domains,
which are situated in the aqueous environment of the cytoplasm
and which bind NADH and FAD, respectively; and a hydrophobic
[a]
K. T. Santoso, Assoc. Prof. B. L. Stocker, Assoc. Prof. M. S. M.
Timmer
School of Chemical and Physical Sciences, Victoria University of
Wellington, PO Box 600, 6140 Wellington, New Zealand
E-mail: bridget.stocker@vuw.ac.nz
E-mail: mattie.timmer@vuw.ac.nz
K. T. Santoso, Assoc. Prof. B. L. Stocker, Assoc. Prof. M. S. M.
Timmer
[b]
Centre for Biodiscovery, Victoria University of Wellington, PO Box
600, 6140 Wellington, New Zealand
[c]
[d]
C.-Y. Cheung, Dr. K. Hards, Prof. G. M. Cook
Department of Microbiology and Immunology, School of Biomedical
Sciences, University of Otago, Dunedin, New Zealand
K. T. Santoso, Dr. K. Hards, Assoc. Prof. B. L. Stocker, Assoc. Prof.
M. S. M. Timmer, Prof. G. M. Cook
Maurice Wilkins Centre for Molecular Biodiscovery, University of
Auckland, Auckland, New Zealand
Supporting information for this article is given via a link at the end of
the document
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membrane-anchoring domain, which contains the quinone Results and Discussion
binding site (Q-site).24,25 To date, the antimycobacterial drug
clofazimine (8) and our recently identified quinolinequinones,
such as QQ8c (9), have been shown to target NDH-II thereby
leading to increased NADH oxidation activity and bactericidal
levels of ROS.21,26,27
Modelling:
To determine whether the phthalazinones could be
accommodated in the NDH-II Q-site, molecular docking of 2-
methylphthalazin-1(2H)-one (10) was undertaken using USCF
Chimera/DOCK6 (Figures 4).30 Different molecular orientations
with similar binding scores were observed (Figure 4B), however,
in all instances, phthalazinone 10 was wedged between the
hydrophobic residues Q317 and I379 with a similar binding
orientation to that reported for MK and HQNO.24,25 Hydrogen
bonding was also evident between phthalazinone 10 and
guanidine residue R382 (Figure 4B). A similar interaction was
observed for the docked structure of MK into the Q-site, although
the authors queried the plausibility of this interaction as R382 is
embedded in the cell membrane.24 A hydrogen bond between
phthalazinone 10 and the ring nitrogen in FAD was also observed
for certain binding modes (Figure 4C). Further analysis of the
NDH-II Q-site also indicated that the pocket should be able to
accommodate substituents at the 2- and 7-positions, thus
providing scope to potentially improve the anti-bacterial activity of
this class of compounds. As quinolinyl-pyrimidines have been
found to interact with NDH-II,31 we also reasoned that the addition
of a pyrimidine to the phthalazinone scaffold might assist with
NDH-II binding by way of a hydrogen bond between the
pyrimidine amine and a glutamic acid residue (E324) in the Q-
site.24,25
Figure 2. Representative drugs that target the mycobacterial respiratory chain.
The co-crystallisation of NDH-II with MK has proven challenging
and has only been achieved with the yeast equivalent of the
enzyme.28 Notwithstanding, docking studies of MK and other
quinones in the presence of FAD in NDH-II have demonstrated
that quinones fit well into the Q-site.24 Moreover, a hydrogen bond
between one of the carbonyl oxygen atoms of the quinones and
an amine proton on the FAD isoalloxazine ring, as well as a
distant edge-to-face π-π interaction between MK and the FAD
isoalloxazine ring, were observed. Co-crystallisation of
Caldalkalibacillus thermarum NDH-II with a known inhibitor, 2-
heptyl-4-hydroxyquinoline-N-oxide (HQNO), revealed similar
binding interactions.25 Accordingly, we proposed that the ketone-
containing bicyclic planar phthalazinone scaffold could be
accommodated between the hydrophobic residues Q317 and
I379 in the Q-site,24,25 with the ketone oxygen participating in
hydrogen bonding interactions with the ring nitrogen of the FAD
isoalloxazine ring. Moreover, with a pKa ≈ 8.88,29 phthalazinones
are able to carry charge through the cell membrane and thus act
as an uncoupler. Accordingly, we proposed to synthesise a series
of phthalazinones (I, Figure 3) and to test the ability of these
derivatives to inhibit the growth of M. tuberculosis (mc26230) and
in addition, target NDH-II.
Figure 4. Docking studies of phthalazinone 10 in C. thermarum NDH-II (PDB:
6BDO),25 calculated using USCF Chimera/DOCK6. A). Structure of 2-
methylphthalazin-1(2H)-one (10). B). Phthalazinone 10 can adopt many
different orientations in the binding tunnel, sandwiched between Q317 and I379.
C). Positioning of a highly ranked conformer of 10 with a hydrogen bond to FAD.
Retrosynthesis:
A retrosynthetic route was devised (Scheme 1), whereby
pyrimidinyl-phthalazinones functionalised at the 2- and 6-
positions (II) could be assembled from an appropriately
functionalised chloropyrimidine III and 7-aminophthalazinone I
(R2 = NH2). Here, phthalazinones bearing apolar alkyl and
aromatic groups were proposed so as to increase the lipophilicity
Figure 3. Target phthalazinone structures.
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of the phthalazinone scaffold and assist with binding to the
hydrophobic NDH-II binding site24,25 while electron-withdrawing
groups, such as fluorophenyl and sulfonamide, on the
phthalazinone scaffold were thought to improve the ability of
compounds to disrupt the mycobacterial respiratory chain.32 In
addition, the inclusion of a terminal alkyne substituent on the
phthalazinone scaffold was proposed to increase the anti-
tubercular activity of the compounds as several anti-tubercular
agents containing alkyne groups have previously been
reported.33,34 The phthalazinones I could be synthesised by
condensation of 3-bromo-6-nitro-isobenzofuranone IV with
various hydrazines, followed by reduction, while the
chloropyrimidines III, equipped with phenyl or methyl groups,
were envisioned to be available from the condensation of
functionalised beta-ketoesters (V) with guanidine. Furanone IV
(R2 = NO2) itself can be prepared via the reduction of phthalic
anhydride, followed by nitration and subsequent benzylic
bromination. Using this same route, a library of phthalazinones I
(where R2 = H) could also be synthesised from phthalic anhydride.
Scheme 2. Synthesis of first series of phthalazinones.
Next, the amino-phthalazinones were prepared (Scheme 3).
Initially, it was envisaged that nitro-phthalide 25 could be
synthesised by subjecting bromo-benzofuranone 13 to nitric acid,
however, 13 was unstable under highly acidic conditions which
necessitated the need to first nitrate isobenzofuran-1(3H)-one
(12) before subjecting the intermediate, 6-nitrobenzofuran-1(3H)-
one, to Wohl-Ziegler conditions to yield the desired phthalide 25.38
Next, bromo-phthalide 25 was reacted with phenylhydrazinium
chloride to give phthalazinone 26, of which a crystal structure was
obtained to unequivocally confirm the configuration of the product
(Scheme 3). The iron-mediated reduction of 26 then yielded the
desired amino-phthalazinone 27.39 Similarly, treatment of nitro-
phthalide 25 with hydrazine hydrate gave phthalazinone 28, which
was then alkylated using propargyl bromide or 1-bromoheptane
to give propargyl-phthalazinone 29 and heptyl-phthalazinone 31,
respectively, in good to excellent yield. These two alkylated
phthalazinones were then reduced, with 7-amino-2-propargyl-
phthalazin-1(2H)-one (30) and 7-amino-2-heptylphthalazin-
1(2H)-one (32) being obtained in 88% and 98% yield from their
respective nitro-functionalised precursors.
Scheme 1. Retrosynthesis for the preparation of pyrimidinyl-phthalazinones (II)
and phthalazinones (I).
To synthesise the first library of phthalazinones, phthalic
anhydride (11, Scheme 2) was reduced using sodium borohydride
to give isobenzofuranone 12, which then underwent a Wohl-
Ziegler reaction35 to give bromo-lactone 13.36 Lactone 13 was
then treated with various hydrazines to afford phthalazinones 14-
1
22 in good yields (34-92%, Scheme 2A). Here, H and 13C NMR
spectroscopy confirmed the loss of resonances corresponding to
H-3 and C-3 of bromide 13 (7.41 ppm and 74.8 ppm, respectively,
in CDCl3) and the appearance of resonances associated with the
4-positions of the phthalazinones (8.00 – 8.30 ppm, 136.0 – 139.0
ppm, respectively). In addition, IR spectra of phthalazinones
showed absorptions between 1102–1120 cm-1, typical of N-N
stretches.37 Phthalazinone 14 was also alkylated using
heptylbromide and propargylbromide under basic conditions to
give alkyl-phthalazinones 23 and 24, respectively (Scheme 2B).
The connectivity of the alkyl groups to the ring nitrogens was
confirmed by HMBC, in which correlations between the N-CH2
and the carbonyl carbons were observed for both 23 and 24.
Scheme 3. Synthesis of the amino phthalazinones.
Finally, the assembly of pyrimidinyl-phthalazinones was
undertaken. First, the chloropyrimidines were synthesised via the
treatment of guanidine carbonate (33) with either ethyl
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acetoacetate (34),40 ethyl benzoylacetate (35),41 or ethyl-4-
fluorobenzoylacetate (36) to give hydroxypyrimidines 37, 38, and
39, respectively, in good yield (Scheme 4A). The
hydroxypyrimidines were then treated with POCl3 under heating
with reflux to yield the corresponding chloropyrimidines 40,40 4142
and 42. Condensation of the chloropyrimidines with amino-
phthalazinones 27, 30 and 32 under acidic conditions then gave
pyrimidinyl-phthalazinones 43-51 in moderate to good yields
(Scheme 4B). Successful formation of pyrimidinyl-phthalazinones
43-51 was confirmed via NMR spectroscopy, which included an
HMBC between the bridging amine NH and carbons at the 5'-, 8-
and 6-positions.
= 0.2 µM). Here, the unfunctionalised phthalazinone 14 exhibited
an MIC = 50 µM (entry 1, Table 1), while the incorporation of an
aliphatic chain at the 2-position led to an increase in inhibitory
activity of the phthalazinones, with tert-butyl- (15), heptyl- (23) and
propargyl- (24) phthalazinones all exhibiting low micromolar MIC
values (3 µM, 6 µM, 12.5 µM, respectively, entries 2-4). Although
the incorporation of lipophilic groups at this position significantly
increased the potency of the compounds, there was no direct
correlation between activity and the lipophilicity of the molecule
as a whole, as expressed in terms of cLogP, the calculated
logarithm of the n-octanol/water partition coefficient. Notably, the
cLogP for the most potent compound, tert-butyl-phthalazinone 15
(cLogP = 1.848), was in between those of heptyl compound 23
(cLogP = 3.785) and unsubstituted phthalazinone 14 (cLogP =
0.155). Phenyl-substituted phthalazinone 16 was a poorer
inhibitor (MIC = 100 µM, entry 5), although when electron-
withdrawing groups were incorporated onto the phenyl ring (e.g.
17-22, entries 6-11), lower MICs of 50 µM were obtained for all
derivatives, except the one that contained a p-CF3 group (19,
entry 8), where an MIC = 12.5 µM was observed. Thus, these
results imply that increasing the electron-withdrawing nature of
the phthalazinones can increase the inhibitory activity of the
compounds towards M. tuberculosis.
Table 1. Inhibition of M. tuberculosis by phthalazinones
Entry
1
Compound
MIC (µM)[a,b]
50
2
3
4
3
6
12.5
Scheme 4: Synthesis of the pyrimidinyl-phthalazinones
5
100
Having completed the synthesis of phthalazinones and
pyrimidinyl-phthalazinones, the compounds were then screened
for their ability to inhibit the growth of M. tuberculosis, with the first
line TB drug isoniazid (INH) being used as a positive control (MIC
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16
100
6
50
17
18
50
7
50
100
8
9
12.5
19
50
50
20
21
22
23
24
100
10
50
50
11
12
50
100
12.5
100
3
13
14
6
100
25
12.5
15
100
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two of the pyrimidinyl-phthalazinones, as evidenced by the low
MIC values of fluorophenyl-phthalazinones 49 (MIC = 12.5 µM,
entry 25) and 51 (MIC = 1.6 µM, entry 27), with phthalazinone 51
exhibiting the highest anti-tubercular activity of the series. Thus,
while the incorporation of a pyrimidine group did not significantly
increase anti-tubercular activity of all phthalazinones, the lead
M. tuberculosis inhibitor did contain this functional group. Taken
together, these studies demonstrate that the inclusion of electron-
withdrawing substituents and lipophilic group at the 2-position can
increase the anti-tubercular activity of phthalazinones, however,
the size and position of functional groups on the phthalazinone
scaffold also influences inhibitory activity.
26
50
27
1.6
[a] Minimal inhibitory concentrations (MIC) were determined using a microtitre
plate assay. M. tb mc26230 cultures were incubated with the compounds for 5
days before resazurin was added and MICs determined.
In an attempt to better explain the anti-tubercular activity of the
phthalazinones, investigations into the ability of the compounds to
affect respiratory NDH-II-dependent NADH oxidation were
undertaken. Here, the rates of NADH oxidation in the absence
and presence of inhibitors were measured using inverted
membrane vesicles (IMVs) from M. smegmatis.43,44 The addition
of several phthalazinones to IMVs oxidising NADH showed a
trend whereby the rates of NADH oxidation were enhanced
compared to the basal rate (Figure 5), particularly pyrimidinyl-
phthalazinones 46, 49 and 50. These data suggest that these
[b] isoniazid (INH) was used as a positive control (MIC = 0.2 µM).
Next, the inhibitory activity of phthalazinones containing electron-
withdrawing and electron-donating groups at the 7-positions were
investigated. Here, the incorporation of a nitro group at the 7-
position led to a remarkable increase in anti-tubercular activity for
phthalazinone 28 (MIC = 3 µM, entry 12) compared to the
unfunctionalised phthalazinone 14 (MIC = 50 µM, entry 1) and a
two-fold improvement in activity for nitro-phthalazinone 29 (MIC =
6 µM, entry 13) compared to the corresponding propargyl-
phthalazinone 24 (MIC = 12.5 µM, entry 4). In addition, it is
noteworthy that phthalazinones 28 and 15 have appreciable anti-
tubercular activities despite their low lipophilicities (cLogP
= -0.102 and 1.848, respectively). For heptyl-phthalazinone 31
however, incorporation of the 6-nitro group led to a loss in anti-
tubercular activity (MIC = 100 µM, entry 14 vs. 6 µM for 23, entry
3), while no change in inhibitory activity was observed between
phenyl-phthalazinones 26 and 16 (entries 15 and 5, respectively).
Amino-phthalazinones 27, 30, and 32 (entries 16-18) resulted in
a loss of M. tuberculosis growth inhibition when compared to the
equivalent unsubstituted phthalazinones 16, 24, and 23,
respectively (entries 3-5), thus demonstrating that the instalment
of an electron-donating amino group appears to lead to a
reduction in potency of the drugs. This may be due to the ability
of the amine groups in the amino-phthalazinones to form
hydrogen bonds with amino acid residues away from the putative
NDH-II Q-site, e.g. glutamic acid E324, or the relatively high
polarity of the amino-phthalazinones could lower their binding
affinity for the hydrophobic NDH-II Q-site.
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
Compound number
Figure 5: Rates of NADH oxidation when IMVs are treated with phthalazinones
14-24, 26-32, and pyrimidinyl-phthalazinones 43-51. Negative control is vehicle
only, whereby IMV was not treated with compound or NADH. The basal rate is
the rate of NADH oxidation by IMVs without the addition of drug. Data are
expressed as average ± standard deviation of three independent experiments.
Finally, the ability of pyrimidinyl-phthalazinones 43-51 to inhibit
M. tuberculosis growth were determined (entries 19-27). In
general, compounds bearing a 6-methyl-pyrimidine moiety were
poor M. tuberculosis inhibitors, with phthalazinones 43-45 having
high MIC values (50 – 100 µM, entries 19-21). Similarly, while
phenyl-pyrimidinyl-alkyne 47 elicited good anti-tubercular activity
(MIC = 12.5 µM, entry 23), 6-phenyl-pyrimidines 46 and 48 had
low potency against M. tuberculosis with an MIC value of 100 µM
(entries 22 and 24). Aside from propargyl-phthalazinone 50 (entry
26), the incorporation of the more electron-withdrawing fluoro-
phenyl-pyrimidine led to an increase in anti-tubercular activity in
compounds might activate NDH-II activity probably via a redox
cycling mechanism.21,27 Conversely, many phthalazinones
appeared to cause a lower rate of NADH oxidation, notably
phenyl-phthalazinone 16, heptyl-phthalazinone 23, and nitro-
heptyl-phthalazinone 31, which could be a result of NDH-II
inhibition (direct or indirect). Accordingly, certain phthalazinones
and pyrimidinyl-phthalazinones appear to bind NDH-II, although
with different modes of action. Whereas representative
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pyrimidinyl-phthalazinones activate NDH-II, which could lead to
the formation of lethal levels of ROS,21,27 some phthalazinones
may exert their anti-tubercular activity through the inhibition of
NDH-II and/or acting as an uncoupler of oxidative phosphorylation
in mycobacteria.15 On the other hand, a lack of correlation was
observed between the MIC values of alkyne bearing compounds
and their ability to affect NDH-II. For example, the rates of NADH
oxidation upon treatment with alkyne-substituted phthalazinones
24 and 29 (MIC = 12.5 µM and 6 µM, respectively) were
comparable to the basal rate of NADH oxidation, while alkynes 30
and 50, with comparatively high MIC values (50 µM for both),
increased the rate of NADH oxidation in IMVs. As alkynes can
undergo covalent interactions with non-target enzymes, it is
possible that the observed anti-tubercular activities of propargyl-
phthalazinones 24 and 29 resulted from off-target effects.
Similarly, the mode of action of the lead phthalazinones (e.g. 15
and 28) and pyrimidinyl-phthalazinones (e.g. 51) appears to be
independent of NDH-II.
Keywords: Nitrogen Heterocycles • Medicinal Chemistry •
Phthalazinones • Tuberculosis
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a
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Acknowledgements
This work was funded by the Maurice Wilkins Centre for Molecular
Biodiscovery. KTS was supported by a Maurice Wilkins Centre
PhD Scholarship. The authors would like to thank Dr. Mathew
Anker for assistance with obtaining a crystal structure of
compound 26.
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