Studies towards hypoxia-activated prodrugs of PARP inhibitors

Article abstract of DOI:10.3390/molecules24081559

Poly(ADP-ribose)polymerase (PARP) inhibitors (PARPi) have recently been approved for the treatment of breast and ovarian tumors with defects in homologous recombination repair (HRR). Although it has been demonstrated that PARPi also sensitize HRR competent tumors to cytotoxic chemotherapies or radiotherapy, normal cell toxicity has remained an obstacle to their use in this context. Hypoxia-activated prodrugs (HAPs) provide a means to limit exposure of normal cells to active drug, thus adding a layer of tumor selectivity. We have investigated potential HAPs of model PARPi in which we attach a bioreducible “trigger” to the amide nitrogen, thereby blocking key binding interactions. A representative example showed promise in abrogating PARPi enzymatic activity in a biochemical assay, with a ca. 160-fold higher potency of benzyl phthalazinone 4 than the corresponding model HAP 5, but these N-alkylated compounds did not release the PARPi upon one-electron reduction by radiolysis. Therefore, we extended our investigation to include NU1025, a PARPi that contains a phenol distal to the core binding motif. The resulting 2-nitroimidazolyl ether provided modest abrogation of PARPi activity with a ca. seven-fold decrease in potency, but released the PARPi efficiently upon reduction. This investigation of potential prodrug approaches for PARPi has identified a useful prodrug strategy for future exploration.

Full text of DOI:10.3390/molecules24081559

molecules
Article
Studies Towards Hypoxia-Activated Prodrugs of
PARP Inhibitors
Benjamin D. Dickson 1,2,
*
, Way Wua Wong 1, William R. Wilson 1,2 and Michael P. Hay 1,2
1
Auckland Cancer Society Research Centre, School of Medical Sciences, Faculty of Medical and Health
Sciences, University of Auckland, Private Bag 92019, Auckland 1010, New Zealand;
w.wong@auckland.ac.nz (W.-W.W.); wr.wilson@auckland.ac.nz (W.R.W.); m.hay@auckland.ac.nz (M.P.H.)
Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Symonds St,
Auckland 1010, New Zealand
2
*
Correspondence: b.dickson@auckland.ac.nz; Tel.: +64-9-923-6798
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 4 March 2019; Accepted: 16 April 2019; Published: 19 April 2019
Abstract: Poly(ADP-ribose)polymerase (PARP) inhibitors (PARPi) have recently been approved for
the treatment of breast and ovarian tumors with defects in homologous recombination repair (HRR).
Although it has been demonstrated that PARPi also sensitize HRR competent tumors to cytotoxic
chemotherapies or radiotherapy, normal cell toxicity has remained an obstacle to their use in this
context. Hypoxia-activated prodrugs (HAPs) provide a means to limit exposure of normal cells
to active drug, thus adding a layer of tumor selectivity. We have investigated potential HAPs of
model PARPi in which we attach a bioreducible “trigger” to the amide nitrogen, thereby blocking
key binding interactions. A representative example showed promise in abrogating PARPi enzymatic
activity in a biochemical assay, with a ca. 160-fold higher potency of benzyl phthalazinone
4 than
the corresponding model HAP , but these N-alkylated compounds did not release the PARPi upon
5
one-electron reduction by radiolysis. Therefore, we extended our investigation to include NU1025,
a PARPi that contains a phenol distal to the core binding motif. The resulting 2-nitroimidazolyl ether
provided modest abrogation of PARPi activity with a ca. seven-fold decrease in potency, but released
the PARPi efficiently upon reduction. This investigation of potential prodrug approaches for PARPi
has identified a useful prodrug strategy for future exploration.
Keywords: PARP inhibitor; NU1025; hypoxia-activated prodrugs; hypoxia; prodrug; anti-cancer
agents; tumor targeting; radiolytic reduction
1. Introduction
Poly(ADP-ribose)polymerases (PARP) are a family of enzymes involved in the synthesis of
poly(ADP-ribose) (PAR) chains from NAD+. Of the eighteen described members, three (PARP-1, -2,
-3) have defined roles in DNA damage repair [1–4]. The role of PARP-1 is the most clearly defined
and it contributes most significantly to PAR synthesis. In response to single strand breaks (SSBs) in
DNA, PARP-1 binds to DNA and attaches PAR chains to nuclear proteins (PARylation) including itself
(autoPARylation). The PAR chains recruit base excision repair (BER) enzymes to the SSB and ultimately
lead to dissociation of PARP [1–5]. This role in DNA damage repair lead to an early proposal that
PARP inhibitors (PARPi) could find utility in cancer therapy [2].
PARP inhibitor cytotoxicity derives from a variety of mechanisms which have been well described
elsewhere [
DNA lesions when the replications forks collide with unrepaired SSB [
also generated through the inability of inhibited PARP to dissociate from DNA, so-called “PARP
trapping” [ 11]. In normal tissues, these lesions are repaired with high fidelity by homologous
6–11]. Briefly, catalytic inhibition of PARP stalls the BER process resulting in downstream
7,12,13]. DNA lesions are
9–
Molecules 2019, 24, 1559; doi:10.3390/molecules24081559
www.mdpi.com/journal/molecules

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recombination repair (HRR), however, in HRR incompetent cell (e.g., BRCA1 and BRCA2 mutants)
repair occurs via low fidelity pathways, including non-homologous end joining (NHEJ), resulting in
an accumulation of errors and ultimately cell death [12–14]. This combination of a genetic defect and a
pharmacological treatment combining to cause cell death is a form of “synthetic lethality” and has
provided the context for clinical PARPi approvals to date [14–16].
In tandem with development of potent small molecule PARPi, increased investigation of PARP
biology has established involvement of the PARP family in the wider DNA damage response [
3,
4
]. In addition to involvement in BER, PARPs participate in HRR, canonical NHEJ (cNHEJ) and
alternate end joining (alt-EJ), and have numerous interactions with nuclear proteins of unknown
consequence [3,4,17,18]. Due to this widespread involvement, PARPi can sensitize cells to a variety of
DNA damaging agents, and therefore combination with cytotoxic chemotherapies or radiotherapy
has been proposed as an approach for treatment of HRR competent tumors [19,20]. However, studies
have shown that use of PARPi in combination therapies often lead to normal tissue toxicity requiring
reduction in the dose of either the PARPi or chemotherapeutic agent [21–28].
Hypoxia is a well-established feature of many solid tumors which contributes to both tumor
progression and resistance to therapy [29–34]. As tumors grow, an oxygen gradient develops as its
metabolic consumption outstrips the oxygen supply. Tumor vasculature lacks the organization of
normal tissue vasculature which leads to tumor hypoxia, with chronic hypoxia due to oxygen diffusion
limitations, and acute hypoxia caused by transient blockages or flow reversals [29,34].
We, and others, have demonstrated that hypoxia can be exploited to activate a prodrug selectively
within a tumor [29,32,35]. These hypoxia-activated prodrugs (HAPs) rely on the different metabolic
fates of a bioreducible functional group (i.e., a trigger) in oxygenated versus hypoxic environments.
One such trigger, the nitroaromatic group, is reduced by one-electron reductases to a nitro radical
anion [29,32]. Under normoxia, this radical anion is oxidized back to the parent nitro group, whereas
under hypoxia, direct fragmentation of the radical anion, or further reduction to electron-donating
hydroxylamino or amino groups leads to activated species [36]. This shift in electron density can
activate the drug via fragmentation of a frangible linker (e.g., evofosfamide) [37] or through activation
of a reactive centre (e.g., PR-104) [38].
We considered that tumor-selective delivery of a PARPi via a HAP would increase the therapeutic
index of PARPi in combination with radiotherapy or chemotherapy. To explore this proposition we
started with olaparib (Lynparza)
1 as an ideal “effector” for use in a HAP as it has nanomolar potency
as a PARP-1 inhibitor and recently gained first-in-class registration in an BRCA mutant advanced
ovarian cancer setting as a monotherapy [15,39].
The PARPi binding mode exemplified by olaparib
which mimics the natural substrate nicotinamide, Figure 1. The phthalazinone carbonyl interacts with both
Ser904- H and Gly863-N and the amide proton interacts with Gly863-C . Additional interactions are
1 relies on a tridentate hydrogen-bond network
O
H
O
formed by Tyr907 and Tyr986 forming π-stacking arrangements with bound inhibitor [40].
O
NH
N
O
N
N
F
O
Olaparib 1
Figure 1. Olaparib 1 bound in the PARP-2 binding site (4tvj) [41].

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We predicted that the addition of a 2-nitroimidazolyl trigger to the phthalazinone NH of
olaparib would disrupt the bonding interaction with Gly863-CO, resulting in a detrimental effect
on PARP inhibition. This concept has precedence in the work of Threadgill and co-workers who
installed nitroheterocyclic triggers on a series of isoquinolin-1-ones , Figure 2, and demonstrated
modest abrogation of PARP inhibition [42 43]. Fragmentation of 2-nitrofuryl prodrugs 3a,b and
1
2
,
2-nitroimidazolyl prodrug 3c released effectors 2a–c, respectively, following chemical reduction
(NaBH4, Pd/C; SnCl2; Zn/NH4Cl) [42,43].
O
O
Chemical
reduction
N
NH
Z
Y
NO2
X
X
3a
3b
3c
2a
2b
2c
X = H, Y = O, Z = C
X = Me, Y = O, Z = C
X = Br, Y = NMe, Z = N
X = H
X = Me
X = Br
Figure 2. Reduction of nitroheterocyclic isoquinolinones 3a–c [42,43].
To build on this initial observation and to explore the potential of this prodrug approach we
prepared a series of model compounds and related 2-nitroimidazolyl derivatives based on a series of
PARPi. We prepared phthalazinone
corresponding 2-nitroimidazolyl derivative
inhibition. To assess trigger fragmentation, we prepared derivatives (3c
model PARPi (2a 2c and , Figure 3) that represent the core of literature PARPi. We also considered
4
as a representative of the structural core of olaparib
as a model HAP in order to assess disruption of PARP
10 11 and 13, Figure 3) of
1 and the
5
,
7,
8,
,
,
,
6
9
other possible sites of the PARPi core for placement of a trigger. The PARPi NU1025 12, Figure 3,
contains a phenol distal to the core binding motif common to PARPi. This phenol may interact with
Glu988 via a hydrogen-bond bridge with water molecules (depicted as red spheres in Figure 1) [40].
We proposed that forming a 2-nitroimidazolyl methyl ether at this phenol would hinder this interaction
and could undergo fragmentation following reduction, providing an alternate prodrug strategy [44
,45].
We evaluated the potency of model HAPs and 13 and the associated PARPi and 12 in a
5
4
biochemical assay (Reaction Biology Corp, Malvern, PA) as representatives of these two approaches
and investigated the stability of all model HAPs in a radiolytic reduction assay.

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Figure 3. Poly(ADP-ribose)polymerase inhibitor (PARPi) (2a, 2c, 4, 6, 9 and 12) and corresponding
model hypoxia-activated prodrugs (HAPs) (3c, 5, 7, 8, 10, 11 and 13).
2. Results and Discussion
2.1. Synthesis
2.1.1. Synthesis of PARPi
Benzyl phthalazinone
Scheme 1 [39]. Benzamide
4
6
was prepared from isobenzofuranone 14 via addition of hydrazine hydrate,
was prepared by alkylation of hydroxybenzoic acid 15 in the presence of
potassium iodide and potassium carbonate [46]. The remaining amide PARPi scaffolds (2a, 2c, and 9,
Figure 3) were sourced commercially. The phenol containing PARPi NU1025 12 was prepared in four
steps from benzoic acid 16 as previously described by Griffin et al. [47].

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Scheme 1. Synthesis of PARPi
BnCl,
4
,
6, and 12. i) NaOH, H2NNH2
H2O, 70 ◦C 18 h, 75%; ii) K2CO3, KI,
·
∆
18 h, 21%; iii) 5% Pd/C, EtOH, H2 60 psi, 16 h, 99%; iv) CDI, DMF, 70 ◦C, NH3(aq), 16 h, 74%;
v) AcCl, pyridine, THF 16 h, then 0.5 M NaOH, 1 h, 48%; and vi) BBr3, DCM, 30 ◦C 72 h, 63%.
2.1.2. Synthesis of Model HAPs
The key nitroimidazole alcohol intermediate 18 was prepared following the previously described
route [35,48]. Conversion to chloride 19 was achieved by mesylation of 18 and in situ chloride
displacement, Scheme 2 [44]. Alcohol 18 was converted to amine 21 via reductive amination of
aldehyde 20, prepared by oxidation of 18. Aldehyde 20 was converted to secondary alcohol 22 by
addition of a methyl titanium species as described by Winn et al. [49]. Oxidation to ketone 23 was
followed by reductive amination to provide secondary amine 24. Both amines 21 and 24 were used
without isolation in subsequent reactions, due to their instability.

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Scheme 2. Synthesis of prodrug triggers. i) DIPEA, MsCl, 0.5 h, 91%; ii) MnO2,
45 ◦C 2h, then NaCNBH4, 48h, then used without purification, see Scheme 4; iv) MeMgBr, TiCl4,
to
30 ◦C, 20 30 ◦C to 20 ◦C 3 h, 53%; v) MnO2, 18h, 81%; vi) NH4OAc, 40 ◦C 1 h, then NaCNBH4,
18 h, then used without purification, see Scheme 4.
∆
18h, 76%; iii) NH4OAc,
−
78 ◦C
−
,
−
−
∆
Preparation of model prodrugs 3c
12, and 19 in the presence of a suitable base Scheme 3. LiHMDS furnished isoquinolin-1-one
prodrugs 3c and 11, and sodium hydride was used for phthalazinone prodrugs and 10. Formation of
the N-alkyl products was confirmed by comparison of the N
H2 13C NMR data to literature values.
In O-alkyl products the O H2 resonance appears between 50–65, whereas the N H2 resonance in
compounds 3c 10, and 11 occurs in the range 30–50 as characteristic of N-alkylated compounds [50].
Phenoxy prodrug 13 was prepared using K2CO3 to deliver selective phenol alkylation.
, 5, 10, 11, and 13 was achieved by combination of PARPi 2c, 4,
9, 2a,
5
C
C
δ
C
,
5
,
δ
Scheme 3. Synthesis of model HAPs 3c, 5, 10, 11, and 13. i) NaH, 0.5 h, 50%; ii) NaH, 0.5 h, 10%;
iii) LiHMDS, 2 h, NaI, 18 h, 49%; iv) LiHMDS, 18 h, 55%; v) K2CO3, 72 h, 47%.

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In contrast alkylation of primary amide
6
did not proceed despite screening a number of bases.
In an alternative approach phenol 25 was first converted to acid 26 and then the corresponding acyl
chloride which was used directly to acylate amine 21 giving prodrug 7, Scheme 4.
The fragmentation rates of 2-nitroheteroaromatic triggers are influenced by the ability of
α
-methylene substituents to stabilize developing positive charge in the fragmentation reaction [45].
Although an -disubstituted trigger might be expected to provide maximal fragmentation rates these
are extremely difficult to prepare [45 49]. We elected to prepare the -substituted amide to explore the
α
,
α
8
influence of substitution on fragmentation rate. Formation of an acyl chloride from 26 and acylation of
amine 24 yielded prodrug 8, Scheme 4.
Scheme 4. Synthesis of primary amide PARPi prodrugs 7 and 8. i) K2CO3, BnBr, ∆ 18 h, then NaOH,
∆
2 h, 82%; ii) (COCl)2, 18 h, then 21, pyridine, 0 ◦C–rt, 4 h, 70%; iii) (COCl)2, 18 h, then 24, pyridine, 0
◦C–rt, 18 h, 29%.
2.2. Biochemical PARP-1 Inhibition
The PARP-1 inhibitory activity of the compounds was determined for the PARPi/model HAP pairs
and 12 13 in a radiometric PARP-1 inhibition assay, Table 1. Both compounds and 12 were potent
PARPi. Model HAP provided significant (ca. 160-fold) deactivation of PARP inhibition, consistent
with disruption of the key hydrogen-bonding network between the amide of PARPi and PARP-1,
4
/
5
/
4
5
4
Figure 1. In contrast, phenol prodrug 13 only showed ca. seven-fold disruption of PARP-1 inhibition,
consistent with the added 2-nitroimidazolyl ether interfering with a secondary interaction, such as
with Glu988, but not disrupting the key binding interactions.
Table 1. Summary of radiometric PARP-1 inhibition data.
Ratio b
PARPi
IC50 (µM) a
Model HAP
IC50 (µM) a
4
12
0.173 ± 0.068
5
13
27.4 ± 0.5
0.25 ± 0.07
b
160 ± 60
0.038 ± 0.014
6.8 ± 3.1
a
Values are mean, and errors are ranges for repeat experiments (n = 2). Values are IC50 ratios, and errors are root
mean square of the standard error.
2.3. Radiolytic Reduction
The ability of the model HAPs to release the effectors was assessed in a radiolytic reduction
assay that provides obligate one-electron reduction with well-defined stoichiometry. Radiolysis of
water generates both reducing (e-(aq)) and oxidizi•n-g (OH•, H•) radicals but the latter can be scavenged
by formate ions to generate the reducing CO2 radical to give a total of 0.62 µ
mol.J−1 reducing

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radicals [51
,52]. Compounds (10
µM) in anoxic 100 mM sodium formate/5 mM sodium phosphate
buffer, pH 7.0, were irradiated (40 Gy) using a cobalt-60 source and analyzed by HPLC with in-line
photodiode array absorbance and single-stage quadrupole mass spectrometry immediately after
irradiation and compared with unirradiated controls. Samples were also analyzed following incubation
for 5 h at 37 ◦C after irradiation to allow for slow fragmentation of hydroxylamine or amine intermediates
to be observed. However, incubation did not alter the quantitation or identity of the species produced.
Therefore, only immediate analysis results are reported. Typical results are illustrated for compounds
5, Figure 4; 3c, Figure 5; and 13, with a summary of prodrug loss and product formation in Table 2.
Table 2. Summary of radiolytic reduction results for immediate analysis following 40 Gy irradiation in
anoxic formate buffer.
Intermediate Species Detected b
Model HAP
% Loss Prodrug Candidate a
% PARPi Formed c
d
3c
5
7
65.1 ± 7.4
85.3 ± 2.1
39.2
73.9
56.3
H (349.1/351.0 m/z)
H (362.2 m/z)
H (353.2 m/z)
H (367.2 m/z)
H (272.1 m/z)
H (271.1 m/z)
-
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
39.9 ± 7.9
8
10
11
13
52.3 ± 3.6
55.0 ± 9.1
a
b
c
Values are mean, and errors are ranges for duplicate samples, quantified from the absorbance chromatograms.
Identified by mass spectrometry as H (hydroxylamino), reported m/z is (M + H)+ for positive mode ionization.
d
Yield of PARPi as a % of initial parent compound, quantified from the absorbance chromatograms.
n.d.,
not detected.
Figure 4. LC/MS analysis of the radiolytic reduction of model HAP
5. (
A) Diode array chromatograms
at 276 nm (bandwidth 16 nm), with 0 and 40 Gy. (
m/z 100–600), with 0 and 40 Gy. ( ) Mass spectrum of peak at retention time 4.701 min (identified as
hydroxylamine). ( ) Mass spectrum of peak at 7.932 min (identified as prodrug by comparison with
authentic 5). 5 Indicates the retention time of the parent PARPi 4.
B) Total ion chromatograms (positive mode, scanning
C
D

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Figure 5. LC/MS analysis of the radiolytic reduction of model HAP 3c. (
at 276 nm (bandwidth 16 nm), with 0 and 40 Gy. ( ) Total ion chromatograms (positive mode, scanning
m/z 100–600), with 0 and 40 Gy. ( ) Mass spectrum of peak at 3.891 min (identified as hydroxylamine).
) Mass spectrum of peak at 7.159 min (identified as prodrug by comparison with authentic 3c
Indicates the retention time of the parent PARPi 2c.
A) Diode array chromatograms
B
C
(
D
)
5
Radiolytic reduction of
detect any released PARPi
) or the extracted ion chromatogram at the 237.1 m/z base peak, Figure 4, Table 2. Inspection of
the mass spectrum revealed the hydroxylamine derivative of (362.2 m/z) in the irradiated solution
eluting at an earlier retention time, Figure 4. Previous studies have shown facile fragmentation of
nitroheteroaromatic ethers after reduction to a hydroxylamine intermediate [45 53 55]. In this instance
5
resulted in an 85.3% decrease in diode-array signal, but we did not
4
in either the diode-array chromatogram (with comparison to authentic
4
5
,
–
the stability of the hydroxylamine indicates that this system is too stable to release PARPi 4 [36,56].
We expanded our study to include analogous prodrugs based on the core scaffolds for other
reported PARPi to see if the observed stability of the phthalazinone N-nitroimidazole framework was
general [40]. We included bromoisoquinolinone 3c as it has previously been shown to fragment in a
chemical reduction system [43]. We assessed all the model prodrugs in our radiolytic reduction assay,
Table 2. Irradiation of amide model HAPs 3c, 7, 8, 10, and 11 resulted in loss of prodrug that was broadly
consistent with the expected four-electron stoichiometry. The major products were identified as the
corresponding hydroxylamines, Table 2. No evidence for the release of effectors was detected, although
authentic standards were detected with high sensitivity. Solutions of the prodrugs incubated for 5 h
at 37 ◦C post irradiation provided no evidence of effector release, suggesting slow fragmentation is
unlikely. This suggests that directly-linked model HAPs of the amides, including bromoisoquinolinone
3c, Figure 5, do not readily fragment on reduction, in contrast to previous reports [42,43].
This discrepancy may be caused by the choice of reduction system. The formate radiolytic reduction
system produces 4-electron reduction to the hydroxylamine species which we expected to fragment.
However, we did not see evidence of 6-electron reduction to the more strongly electron-donating amino
species. To address this we carried out reduction of 3c with Zn/NH4Cl and assessed the resulting
solution by LC/MS. After one hour exposure to Zn/NH4Cl in acetonitrile the solution was filtered and
analysed by LC/MS. Comparison to a control solution allowed estimated loss of prodrug at 45% based

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on the diode-array signal. At the extracted m/z 333.0 signal we detected the corresponding amino
species, Figure 6A, confirmed by its mass spectrum, Figure 6B. Re-analysis of the same sample after
standing for 3 h showed no change in the ion count for the amine (data not shown) and, importantly,
the expected fragmentation product 2c was not detected despite ready detection of the authentic
compound, Figure 6C,D. This result strengthens our observation that this type of prodrug does not
release the desired PARP inhibitor, even when reduced to the corresponding amine. We are unable to
account for observation of fragmentation in previous reports.
Figure 6. LC/MS analysis of the chemical reduction of model HAP 3c. (
at m/z 333 corresponding to the Br-79 isotopologue of the amino derivative of 3c. (
peak at 5.580 min. (
) Extracted ion chromatogram at m/z 224 corresponding to the 79Br isotopologue
A
) Extracted ion chromatogram
B) Mass spectrum of
C
of 2c from injection of a 1.8 µM solution. (D) Mass spectrum of peak at 6.839 min.
This disappointing result led us to explore a new prodrug approach. We prepared a
2-nitroimidazolyl ether linked to the phenol of NU1025 (12). Related prodrugs based on nitroheterocyclic
ethers have been demonstrated to release effectors efficiently [44,45,49]. Reduction of the PARPi
prodrug 13 did result in release of effector 12, Table 2. Formation of 12 was detected in the diode-array
chromatogram, Figure 7A, and confirmed by analysis of the mass spectrum in negative ionisation
mode (12 [M-H]- 175.1 m/z; 13 M-H 314.0 m/z, Figure 7C,D) with an efficient yield of 12 at ca. 55–60%
reduction of prodrug, Table 2.
In conclusion, the N-alkylated nitroimidazolyl prodrug system did provide deactivation of the
PARPi as intended. However, this model prodrug system does not fragment upon reduction to the
corresponding hydroxylamine or amine. In contrast, the phenol prodrug 13 shows only ca. seven-fold
reduction in PARP inhibition. Importantly, the 2-nitroimidazolyl ether does fragment upon reduction
and this provides a lead towards identification of novel prodrugs which can combine the efficient
fragmentation of the ether linker with a larger deactivation of the PARPi.

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Figure 7. LC/MS analysis of the radiolytic reduction of model HAP 13. (
at 276 nm (bandwidth 16 nm), with 0 and 40 Gy. ( ) Total ion chromatograms (negative mode, scanning
m/z 100–600), with 0 and 40 Gy. ( ) Mass spectrum of peak at 8.849 min (identified as effector by
comparison with authentic 12). ( ) Mass spectrum of peak at 10.529 min (identified as prodrug by
A) Diode array chromatograms
B
C
D
comparison with authentic compound 13).
3. Materials and Methods
3.1. Synthesis
3.1.1. General
All non-aqueous reactions were carried out under a dry nitrogen atmosphere unless otherwise
noted. DMF, DCM, and THF were purchased pre-dried and stored over molecular sieves from Acros
Organics. All commercial reagents were used without purification. Flash column chromatography
was carried out on a silica gel solid phase (Merck 230–400 mesh). Thin layer chromatography was
carried out using Merck 60 F254 aluminium plates pre-coated with silica. Compounds were identified
using UV fluorescence and/or staining with either vanillin in ethanolic sulphuric acid (with heating), 3,
5-dinotrophenylhydrazine in ethanolic sulfuric acid (with heating), ninhydrin in ethanol/glacial acetic
acid (95:5) (with heating), or iodine on silica gel. Melting points were determined on an Electrothermal
2300 melting point apparatus. High resolution mass spectra (HRMS) were measured on an Agilent
Technologies 6530 Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) LC/MS interfaced with an
Agilent Jet Stream electrospray ionization (ESI) source allowing positive or negative ions detection.
Low resolution mass spectra (LRMS) were measured on a Surveyor MSQ mass spectrometer using an
atmospheric pressure chemical ionization (APCI) mode with a corona voltage of 50 V and a source
temperature of 400 ◦C. NMR spectra data were recorded on a Bruker Avance 400 spectrometer (400 MHz,
1H nuclei, 100 MHz, 13C nuclei). All chemical shift (
relative to tetramethylsilane (0.0 ppm) as an internal reference, coupling constants were reported in
Hertz (Hz). Final products were analyzed by reverse-phase HPLC (Altima C18 5 m column, 150 mm
3.2 mm; Alltech Associated, Inc., Deerfield, IL) using an Agilent HP1100 equipped with a photodiode
δ) values were reported in parts per million (ppm)
µ
×
array detector. Mobile phases were gradients of 80% CH3CN/20% H2O (v/v) in 45 mM ammonium
formate at pH 3.5 and 0.5 mL/min. Purity was determined by monitoring at 330 ± 50 nm.

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3.1.2. Procedures
5-Bromo-2-((1-methyl-2-nitro-1H-imidazol-5-yl)methyl)isoquinolin-1(2H)-one (3c).
To
bromoisoquinolinone 2c (0.1 g, 0.69 mmol) in DMF (2 mL) was added a 1.0 M THF solution of
LiHMDS (0.6 mL, 0.6 mmol) and the mixture was stirred for 2 h. Nitroimidazole 19 (0.08 g, 0.45 mmol)
in DMF (2 mL) was added dropwise and the reaction was stirred for 2 h. NaI (0.002 g, 0.01 mmol)
was added and the mixture was stirred overnight. A second addition of 19 (0.08 g, 0.45 mmol) was
made and the mixture stirred a further 24 h. Water (2 mL) was added and the resulting mixture
extracted with EtOAc (2 × 5 mL). The organic fractions were washed with water (5 mL), brine (5 mL)
and dried over Na2SO4. Solvent was removed in vacuo and the crude product was purified by column
chromatography (2:1, DCM, EtOAc) to yield 3c (0.08 g, 49%) as an orange solid, mp 204–207 ◦C (lit. [43
]
◦
mp 208–210 C). δH (CDCl3) 8.41 (1H, dt, J = 7.8, 0.9 Hz, H-6), 7.94 (1H, dd, J = 7.7, 1.2 Hz, H-8),
7.39 (1H, t, J = 7.9 Hz, H-7), 7.23 (1H, s, H-40), 7.14 (1H, d, J = 7.7 Hz, H-3), 6.94 (1H, dd, J = 7.7,
0.6 Hz, H-4), 5.30 (2H, s, CH2), 4.06 (3H, s, CH3). δC ((CD3)2SO) 160.3 (C = O), 145.8 (C), 136.4 (CH),
135.8 (C), 134.2 (C), 133.9 (CH), 128.0 (CH), 127.9 (CH), 127.3 (CH), 126.9 (CH), 120.0 (C), 104.0 (CH),
41.3 (CH2), 34.3 (CH3). HRMS calcd for C14H1279BrN4O3 (M + H) m/z 363.0093, found 363.0087; for
C14H1281BrN4O3 (M + H) m/z 365.0073, found 365.0071. LRMS m/z 363.0 (100%, M79 + H), 365.0 (95%,
M81 + H). HPLC purity: 95.5% (effector 2c: not detected).
4-Benzylphthalazin-1(2H)-one (4). To a stirred suspension of benzalphthalide 14 (10 g, 45 mmol)
◦
in water (60 mL) was added aqueous NaOH (13 M, 15 mL) and the mixture was heated to 70 C.
Hydrazine hydrate (31 mL, 630 mmol) was added and the mixture stirred for 18 h. The reaction was
cooled to room temperature and acidified with 8 M aqueous HCl to pH 4. After 10 min the resulting
suspension was filtered and washed with water (2
×
50 mL) and diethyl ether (3
×
50 mL) to yield 4
(8.0 g, 75%) as a white solid, mp 198–200 ◦C (lit. [57] mp 200–201 ◦C). δH (CDCl3) 9.95 (1H, br s, NH),
8.47–8.43 (1H, m, H-8), 7.79–7.71 (3H, m, H-5, H-6, H-7), 7.33–7.00 (5H, m, Ar-H), 4.30 (2H, s, CH2Ph).
δC (CDCl3) 161.1 (C = O), 146.5 (C), 137.8 (C), 133.6 (CH), 131.5 (CH), 130.0 (C), 128.9 (2
(2 CH), 128.5 (C), 127.2 (CH), 127.0 (CH), 125.6 (CH), 39.1 (CH2). LRMS m/z 237.2 (100%, M + H).
HPLC purity: 99.2%. These data are in good agreement with literature values [57].
×
CH), 128.7
×
4-Benzyl-2-((1-methyl-2-nitro-1H-imidazol-5-yl)methyl)phthalazin-1(2H)-one (5). To phthalazinone
4
(0.33 g, 1.40 mmol) in DMF (2 mL) was added NaH (60% dispersion in mineral oil) (0.084 g, 2.10 mmol)
and the resulting solution stirred for 30 min at room temperature. Nitroimidazole 19 (0.34 g, 1.94 mmol) was
added and the solution was stirred for 30 min, then quenched on ice. The mixture was extracted with EtOAc
(10 mL), washed with water (3
product which was purified by column chromatography (1:1 X4, EtOAc) to yield
solid, mp 180–182 ◦C. δH ((CD3)2SO) 8.31 (1H, dd, J = 7.6, 1.4 Hz, H-8), 8.00 (1H, dd, J = 8.2, 1.0 Hz, H-5),
7.91–7.83 (2H, m, H-6, H-7), 7.30–7.24 (4H, m, Ar-H), 7.21–7.17 (2H, m, H-40, Ar-H), 5.45 (2H, s, CH2N), 4.31
(2H, s, CH2Ph), 3.93 (3H, s CH3). δC ((CD3)2SO) 158.0 (C = O), 145.9 (C), 145.6 (C), 137.7 (C), 134.0 (C), 133.6
×
10 mL), dried over MgSO4 and solvent removed in vacuo. The crude
(0.10 g, 50%) as a yellow
5
(CH), 132.0 (CH), 128.7 (2
×
CH), 128.4 (2
×
CH), 128.36 (CH), 128.36 (CH), 127.3 (C), 126.5 (2 CH), 125.8
×
(CH), 43.4 (CH2), 37.5 (CH2) 34.4 (CH3). HRMS calcd for C20H18N5O3 (M + H) m/z 376.1404, found 376.1412.
LRMS m/z 376.2 (100%, M + H). HPLC purity: 96.1% (effector 4: 0.3%).
2-(Benzyloxy)benzamide (6). To amide 15 (0.80 g, 5.83 mmol), K2CO3 (0.97 g, 7.02 mmol) and KI
(1.17 g, 7.02 mmol) in acetone (30 mL) was added benzyl chloride (0.81 mL, 7.02 mmol), and the reaction
stirred at reflux for 18 h. The solution was cooled and filtered and solvent removed in vacuo. The crude
product was recrystallised from acetone to yield 6 ]
(0.28 g, 21%) as a cream solid, mp 112–114 ◦C (lit. [58
mp 115–116 ◦C). δH (CDCl3) 8.25 (1H, dd, J = 7.8, 1.8 Hz, H-6), 7.73 (1H, br s, NH), 7.50–7.37 (6H, m,
Ar-H), 7.13–7.06 (2H, m, Ar-H), 5.81 (1H, br s, NH), 5.20 (2H, s, CH2). δC (CDCl3) 167.1 (C = O), 157.3
(C), 136.8 (C), 133.6 (CH), 132.9 (CH), 129.2 (2
112.9 (CH), 71.5 (CH2). LRMS m/z 228.2 (100%, M + H). HPLC purity: 100%. These data are in good
×
CH), 128.9 (CH), 128.1 (2
×
CH), 121.7 (CH), 121.3 (C),
agreement with literature values [59].
2-(Benzyloxy)-N-((1-methyl-2-nitro-1H-imidazol-5-yl)methyl)benzamide (7).
(1-Methyl-2-
nitro-1H-imidazol-5-yl)methanamine (21). To aldehyde 20 (0.60 g, 3.87 mmol) in MeOH (16 mL)

Molecules 2019, 24, 1559
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◦
was added ammonium acetate (5.96 g, 77.4 mmol) and the mixture was stirred at 45 C for 2 h.
NaCNBH4 (0.17 g, 2.37 mmol) was added and the mixture stirred for 48 h, acidified to pH 1 with 6 M
aqueous HCl and solvent removed in vacuo. The aqueous residue was filtered and the filtrate basified to
pH 14 with 6 M aqueous KOH. The aqueous was extracted with DCM (3
×
50 mL), then saturated with
NaCl and further extracted with DCM (3 50 mL). The combined organic fractions were concentrated
×
and filtered through a plug of silica (9:1, DCM, MeOH) to give a crude residue 21 (0.15 g) which was
used without further purification. δH ((CD3)2SO) 7.07 (1H, s, H-4), 3.90 (3H, s, CH3), 3.77 (2H, s, CH2),
3.17 (2H, br s, NH2).
To benzoic acid 26 (0.16 g, 0.704 mmol) in DCM (3 mL) was added oxalyl chloride (0.30 mL,
3.54 mmol) and the mixture was stirred overnight at room temperature. Solvent was removed in vacuo.
The crude residue was taken up in DCM (2 mL) and added to a solution of amine 21 (0.10 g, 0.64 mmol)
in pyridine (2 mL) at 0 ◦C. The mixture was allowed to come to room temperature and stirred for 4 h.
Solvent was removed in vacuo and the crude residue was purified by column chromatography (2:1,
EtOAc, X4) to yield 7
(0.16 g, 70%) as yellow solid, mp 120–122 ◦C. δH ((CD3)2SO) 8.62 (1H, t, J = 5.5 Hz,
NH), 7.67 (1H, dd, J = 7.6, 1.7 Hz, H-6), 7.49–7.43 (1H, m, H-4), 7.42–7.38 (2H, m, Ar-H), 7.33–7.26 (3H,
m, Ar-H), 7.22 (1H, d, J = 7.8 Hz, H-3), 7.07–7.02 (2H, m, H-5, H-40), 5.19 (2H, s, OCH2), 4.55 (2H, d, J =
5.6 Hz, NCH2), 3.75 (3H, s, NCH3). δC ((CD3)2SO) 165.5 (C = O), 155.8 (C), 145.4 (C), 136.4 (C), 136.0
(C), 132.1 (CH), 130.0 (CH), 128.3 (2
×
CH), 127.9 (CH), 127.6 (2
×
CH), 127.2 (CH), 123.8 (C), 120.7 (CH),
113.3 (CH), 70.0 (CH2), 33.9 (CH3), 33.1 (CH2). HRMS calcd for C19H19N4O4 (M + H) m/z 367.1401,
found 367.1407. LRMS m/z 367.2 (100%, M + H). HPLC purity: 99.3% (effector 6: not detected).
2-(Benzyloxy)-N-(1-(1-methyl-2-nitro-1H-imidazol-5-yl)ethyl)benzamide (8). 1-(1-Methyl-2-
nitro-1H-imidazol-5-yl)ethan-1-amine (24). To ketone 23 (0.20 g, 1.2 mmol) in MeOH (4 mL) was
added ammonium acetate (1.8 g, 23.6 mmol) and the resulting solution was stirred at 40 ◦C for 1 h.
Activated 4 Å molecular sieves were added and the mixture was stirred a further 1 h. The mixture
was cooled to 0 ◦C and NaCNBH4 (0.05 g, 0.84 mmol) added, then the mixture was warmed to room
temperature and stirred overnight. Solvent was removed in vacuo and the resulting residue filtered
through a plug of silica (9:1, DCM, MeOH) to give a crude residue 24 (0.04 g) which was used without
further purification. δH ((CD3)2SO) 7.10 (1H, s, H-4), 4.05 (1H, q, J = 6.7 Hz, CH), 3.94 (3H, s, NCH3),
1.38 (3H, d, J = 6.7 Hz, CH3).
To benzoic acid 26 (0.06 g, 0.26 mmol) in DCM (1 mL) added oxalyl chloride (0.11 mL, 3.54 mmol)
and the mixture was stirred overnight at room temperature. Solvent was removed in vacuo. The crude
residue was taken up in DCM (1 mL) and added to a solution of amine 24 (0.04 g, 0.24 mmol) in
pyridine (0.5 mL) at 0 ◦C. The mixture was allowed to come to room temperature and stirred overnight.
Solvent was removed in vacuo and the crude residue was purified by column chromatography (2:1,
X4, EtOAc) to yield 8
(0.026 g, 29%) as a white solid, mp 151–153 ◦C. δH (CDCl3) 8.22 (1H, dd, J = 7.8,
1.8 Hz, H-6), 8.08 (1H, d, J = 8.2 Hz, NH), 7.54–7.50 (1H, m, H-4), 7.43–7.28 (5H, m, Ar-H), 7.15 (1H,
td, J = 7.2, 1.0 Hz, H-5), 7.10 (1H, d, J = 8.3 Hz, H-3), 6.81 (1H, d, J = 0.6 Hz, H-40), 5.36 (1H, p, J =
8.0 Hz, CH), 5.08 (2H, ddd, J = 10.8, 9.8, 9.8 Hz, OCH2), 3.84 (3H, s, NCH3), 1.44 (3H, d, J = 6.9 Hz,
CH3). δC (CDCl3) 164.5 (C = O), 157.1 (C), 139.3 (C), 135.0 (C), 133.7 (CH), 132.7 (CH), 129.5 (CH),
129.2 (2
×
CH), 128.4 (2
×
CH), 126.0 (CH), 122.2 (CH), 120.8 (C), 112.5 (CH), 71.9 (CH2), 39.8 (CH),
34.4 (CH3), 19.5 (CH3); C-NO2 was not observed. HRMS calcd for C20H21N4O4 (M + H) m/z 381.1571,
found 381.1568. LRMS m/z 381.2 (100%, M + H). HPLC purity: 99.8% (effector 6: not detected).
2-((1-Methyl-2-nitro-1H-imidazol-5-yl)methyl)phthalazin-1(2H)-one (10). To phthalazinone
9
(0.205 g, 1.40 mmol) in DMF (2 mL) was added NaH (60% dispersion in mineral oil) (0.084 g, 2.10 mmol)
and the resulting solution stirred for 30 min at room temperature. Nitroimidazole 19 (0.27 g, 1.54 mmol)
was added and the solution was stirred for 30 min, then quenched on ice. The resulting suspension
was filtered, the collected solid washed with water (5 mL), X4 (5 mL) and dried in vacuo to yield 10
(0.04 g, 10%) as a yellow solid, mp 207–209 ◦C. δH ((CD3)2SO) 8.49 (1H, s, H-4), 8.29 (1H, dd, J = 7.84,
0.7 Hz, H-8), 7.98 (2H, d, J = 3.7 Hz, H-7, H-6), 7.93–7.87 (1H, m, H-5), 7.19 (1H, s, H-40), 5.47 (2H, s,
CH2), 4.00 (3H, s, CH3). δC ((CD3)2SO) 158.4 (C = O), 145.6 (C), 138.9 (C), 134.1 (C), 133.9 (C), 132.4

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(CH), 129.3 (C), 128.4 (CH), 127.1 (CH), 127.9 (C), 125.9 (CH), 43.6 (CH2), 34.4(CH3). HRMS calcd for
C13H12N5O3 (M + H) m/z 286.0935, found 286.0941. LRMS m/z 286.1 (100%, M + H). HPLC purity:
94.4% (effector 10: 0.1%).
2-((1-Methyl-2-nitro-1H-imidazol-5-yl)methyl)isoquinolin-1(2H)-one (11). To isoquinolinone
2a (0.10 g, 0.69 mmol) in DMF (2 mL) was added a 1.0 M THF solution of LiHMDS (0.90 mL, 0.90 mmol)
and the mixture was stirred for 2 h. Nitroimidazole 19 (0.12 g, 0.69 mmol) in DMF (2 mL) was added
dropwise and the reaction was stirred overnight. Water (2 mL) was added and the resulting mixture
extracted with EtOAc (2 × 5 mL). The organic fractions were washed with water (5 mL), brine (5 mL)
and dried over Na2SO4. Solvent was removed in vacuo and the crude product was purified by column
chromatography (2:1, DCM, EtOAc) to yield 11 (0.11 g, 55%) as a yellow solid, mp 207–210 ◦C. δH
((CD3)2SO) 8.25 (1H, dt, J = 8.1, 0.6 Hz, H-8), 7.74 (1H, ddd, J = 9.2, 7.0, 1.3 Hz, H-6), 7.68 (1H, dd,
J = 7.9, 0.7 Hz, H-7) 7.55 (2H, d, J = 7.6 Hz, H-5, H-3), 7.16 (1H, s, H-40), 6.71 (1H, d, J = 7.4 Hz, H-4),
5.33 (2H, s, CH2), 3.99 (3H, s, CH3). δC ((CD3)2SO) 161.0 (C = O), 145.7 (C), 136.9 (C), 134.6 (C), 132.7
(CH), 132.0 (CH), 127.8 (CH), 127.1 (CH), 127.0 (CH), 126.3 (CH), 125.2 (C), 106.0 (CH), 40.9 (CH2), 34.3
(CH3). HRMS calcd for C14H13N4O3 (M + H) m/z 285.0982, found 285.0978. LRMS 2 m/z 85.1 (100%, M
+ H). HPLC purity: 96.7% (effector 2a: not detected).
8-Hydroxy-2-methylquinazolin-4(3H)-one (12). To a stirred solution of quinazolinone 17 (400 mg,
2.10 mmol) in dry DCM (10 mL) was added a 1.0 M DCM solution of BBr3 (4.8 mL, 4.8 mmol) and
the mixture stirred at 30 ◦C for 72 h. Solvent was removed in vacuo, the residue cooled to 5 ◦C and
aqueous NaOH (2.5 M, 30 mL) was added. The mixture was stirred at 20 ◦C for 15 min, filtered and
the pH of the filtrate adjusted to 5 with aqueous HCl (6 M). The mixture was chilled at 5 ◦C for 1 h.
The resulting precipitate was purified by column chromatography (1:1–1:0, EtOAc, X4) to yield 12
(0.23 g, 63%) as a tan powder, mp 261–263 ◦C (lit. [47] mp 253–258 ◦C). δH ((CD3)2SO) 12.14 (1H, br s,
NH), 9.36 (1H, s, OH), 7.49 (1H, dd, J = 7.9, 1.4 Hz, H-5), 7.25 (1H, t, J = 7.9 Hz, H-6), 7.14 (1H, dd,
J = 7.8, 1.4 Hz, H-7), 2.37 (3H, s, 2-CH3). δC ((CD3)2SO) 162.2 (C = O), 153.0 (C), 152.7 (C), 138.4 (C),
126.7 (CH), 121.9 (C), 118.6 (CH), 115.9 (CH), 21.9 (CH3). LRMS m/z 177.2 (100%, M + H). These data
are in good agreement with literature values [47,60].
2-Methyl-8-((1-methyl-2-nitro-1H-imidazol-5-yl)methoxy)quinazolin-4(3H)-one (13).
To
phenol 12 (0.069 g, 0.39 mmol) in DMF (2 mL) was added K2CO3 (0.16 g, 1.16 mmol) followed
by nitroimidazole 19 (0.069 g, 0.57 mmol). The mixture was stirred for 72 h, diluted with water and
collected by filtration. The crude compound was purified by column chromatography (40:1, DCM,
MeOH) to yield 13 (0.057 g, 47%) as a white solid, mp 253– 256 ◦C. δH ((CD3)2SO) 12.24 (1H, br s, NH),
7.72 (1H, dd, J = 8.0, 1.1 Hz, H-5), 7.54 (1H, dd, J = 8.0, 1.1 Hz, H-7), 7.39 (1H, t, J = 8.0 Hz, H-6), 7.39
(1H, s, H-40), 5.38 (2H, s, CH2), 4.06 (3H, s, NCH3), 2.34 (3H, s, CH3). δC ((CD3)2SO) 161.5 (C = O),
153.4 (C), 152.1 (C), 140.5 (C), 133.5 (C), 128.6 (CH), 125.9 (CH), 122.0 (C), 119.2 (CH), 118.7 (CH), 61.0
(CH2), 34.3 (CH3), 21.6 (CH3); C-NO2 was not observed. HRMS calcd for C14H14N5O4 (M + H) m/z
316.1040, found 316.1039. LRMS m/z 316.1 (100%, M + H). HPLC purity: 95.6% (effector 12: 0.4%).
8-Methoxy-2-methylquinazolin-4(3H)-one (17). A mixture of acid 16 (8.37 g, 42.5 mmol) and
Pd/C (5%, 0.10 g) in EtOH (100 mL) was stirred under H2 (60 psi) for 16 h. The mixture was filtered
through diatomaceous earth, the pad washed with EtOH (50 mL) and solvent removed in vacuo to give
2-amino-3-methoxybenzoic acid (7.05 g, 99%) as a tan powder which was used directly. CDI (6.70 g,
41.3 mmol) was added to a stirred solution of 2-amino-3-methoxybenzoic acid (7.05 g, 42.2 mmol)
in dry DMF (100 mL) at 70 ◦C and the mixture stirred for 10 min. A ◦solution of aqueous NH3 (108
mL, 1.05 mol◦) was added slowly and the mixture was stirred at 70 C for 16 h. The mixture was
cooled to 20 C, diluted with EtOAc (300 mL), washed with water (3
×
100 mL), brine (100 mL),
dried (MgSO4) and solvent removed in vacuo to give crude 2-amino-3-methoxybenzamide (5.21 g,
74%) as a tan solid which was used directly. Acetyl chloride (1.43 mL, 20.2 mmol) in dry THF (2 mL)
was added dropwise to a stirred solution of 2-amino-3-methoxybenzamide (1.66 g, 10.0 mmol) and
pyridine (1.06 mL, 13.0 mmol) in dry THF (50 mL) at 20 ◦C and the resulting mixture stirred for 16 h.
Solvent was removed in vacuo, the residue dissolved in aqueous NaOH (0.5 M, 25 mL) and stirred

Molecules 2019, 24, 1559
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for 1 h. The solution was carefully neutralized with aqueous HCl (0.5 M) and stirred at 5 ◦C for 1 h.
The precipitate was filtered and recrystallized from MeOH/water to yield 17 (0.91 g, 48%) as a white
solid, mp 263–266 ◦C (lit. [61] mp 262–263 ◦C). δH ((CD3)2SO) 10.79 (1H, br s, NH), 7.61 (1H, dd, J = 7.8,
1.4 Hz, H-5), 7.36 (1H, t, J = 7.9 Hz, H-6), 7.30 (1H, dd, J = 8.0, 1.4 Hz, H-7), 3.87 (3H, s, OCH3), 2.34 (3H,
s, CH3). δC ((CD3)2SO) 161.7 (C = O), 154.0 (C), 152.9 (C), 139.4 (C), 126.0 (CH), 121.6 (C), 116.7 (CH),
114.7 (CH), 55.8 (CH3), 21.5 (CH3). LRMS m/z 191.1 (100%, M + H). These data are in good agreement
with literature values [60,61].
5-(Chloromethyl)-1-methyl-2-nitro-1H-imidazole (19). To a solution of alcohol 18 (0.50 g,
◦
3.2 mmol) in THF (10 mL) at 0 C was added diisopropylethylamine (0.67 mL, 3.8 mmol) and
methanesulfonyl chloride (0.3 mL, 0.38 mmol) and the mixture was stirred for 30 min. EtOAc (20 mL)
was added and the solution washed with 1M aqueous HCl (20 mL), dried over MgSO4 and solvent
removed in vacuo. The crude material was purified by column chromatography (2:1, X4, EtOAc) to
yield 19 (0.51 g, 91%) as a yellow solid, mp 99–100 ◦C (lit. [43] mp 94–96 ◦C). δH (CDCl3) 7.19 (1H, s,
4-H), 4.63 (2H, s, CH2), 4.08 (3H, s, CH3). δC (CDCl3) 146.4 (C-NO2), 132.9 (C), 128.6 (CH), 34.3 (CH3),
33.9 (CH2). LRMS m/z 176.1 (100%, M35 + H), 178.1 (36%, M37 + H). These data are in good agreement
with literature values [43].
1-Methyl-2-nitro-1H-imidazole-5-carbaldehyde (20). To alcohol 18 (1.0 g, 6.36 mmol) in
chloroform (70 mL) was added MnO2 (2.76 g, 31.8 mmol) and the mixture was heated to reflux
overnight. After cooling, the resulting slurry was filtered through diatomaceous earth and solvent
removed in vacuo to yield 19 (1.5 g, 76%) as a pale yellow solid, mp 112–114 ◦C (lit. [62] mp 114–115 ◦C).
δH (CDCl3) 9.93 (1H, s, CHO), 7.81 (1H, s, H-4), 4.36 (3H, s, CH3). δC (CDCl3) 180.4 (C = O), 148.3
(C-NO2), 139.4 (CH), 132.4 (CH), 35.6 (CH3). LRMS 188.1 (100%, M + CH3OH). These data are in good
agreement with literature values [35].
1-(1-Methyl-2-nitro-1H-imidazol-5-yl)ethan-1-ol (22). A 3 M solution of MeMgBr in Et2O
(0.32 mL, 0.97 mmoL) was added to a solution of TiCl4 (0.11 mL, 0.97 mmol) in DCM (4 mL) at
–78 ◦C. The mixture was allowed to warm to –30 ◦C and added to a solution of aldehyde 20 (0.15 g,
◦
◦
0.97 mmol) in DCM (4 mL) at –30 C. This mixture was maintained at –30 to –20 C for 3 h, then
quenched by addition of saturated aqueous NH4Cl (5 mL). The aqueous fraction was extracted
with DCM (3 × 10 mL) and solvent removed in vacuo. The crude mixture was purified by column
chromatography (50:1, DCM, MeOH) to yield 22 (0.09 g, 53%) as an orange solid, mp 101–103 ◦C. δH
(Acetone-d6) 7.06 (1H, s, H-4), 5.05–4.94 (1H, m, CH), 4.59 (1H, br s, OH), 4.08 (3H, s, NCH3), 1.61 (3H,
d, J = 6.6 Hz, CH3). δC (Acetone-d6) 147.3 (C-NO2), 141.6 (C), 124.7 (CH), 60.3 (CH), 33.9 (CH3), 21.1
(CH3). LRMS m/z 172.2 (100%, M + H). These data are in good agreement with literature values [49].
1-(1-Methyl-2-nitro-1H-imidazol-5-yl)ethan-1-one (23). To a solution of alcohol 22 (0.06 g,
0.35 mmol) in CHCl3 (10 mL) was added MnO2 (0.20 g, 2.3 mmol) and the resulting solution was stirred
at reflux overnight. The resulting slurry was filtered through diatomaceous earth and solvent was
removed in vacuo to yield 23 (0.048 g, 81%) as a yellow solid, mp 71–72 ◦C (lit. [62] mp 81–83 ◦C). δH
(CDCl3) 7.77 (1H, s, H-4), 4.32 (3H, s, NCH3), 2.59 (3H, s, CH3). δC (CDCl3) 189.2 (C = O), 148.2 (C-NO2),
135.7 (CH), 132.3 (C), 35.8 (CH3), 28.6 (CH3). HRMS calcd for C6H7N3O3 (M + H) m/z 170.0560, found
170.0556. LRMS m/z 170.1 (100%, M + H).
2-(Benzyloxy)benzoic acid (26). To ester 25 (1.60 mL, 12.04 mmol) in MeCN (50 mL) was added
K2CO3 (3.30 g, 24.08 mmol) followed by benzyl bromide (1.50 mL, 12.88 mmol) and the resulting
mixture was stirred at reflux overnight. After cooling the reaction was diluted with water (25 mL) and
the aqueous fraction extracted with DCM (3
×
20 mL). The combined organic extracts were washed
with water (25 mL), dried over Na2SO4 and solvent removed in vacuo. The crude residue was dissolved
in 1,4-dioxane (50 mL) and aqueous NaOH (1.5 M, 12 mL) was added. The solution was stirred at
reflux for 2 h, cooled to room temperature and the organic residues removed in vacuo. The remaining
solution was acidified with 1M aqueous HCl, extracted with EtOAc (3
dried over Na2SO4 and solvent removed in vacuo to yield 26 (2.4 g, 82%) as a colourless solid, mp
70–71 ◦C (lit. [63] mp 73–75 ◦C). δH (CDCl3) 10.72 (1H, br s, OH), 8.21 (1H, dd, J = 7.8, 1.8 Hz, H-6),
×
25 mL), the organic extracts

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7.56 (1H, ddd, J = 9.2, 7.4, 1.8 Hz, H-4), 7.46–7.38 (5H, m, Ar-H), 7.18–7.11 (2H, m, H-5, H-3) 5.30 (2H, s,
CH2). δC (CDCl3) 165.6 (C = O), 157.6 (C), 135.2 (CH), 134.5 (C), 134.0 (CH), 129.34 (CH), 129.32 (2
CH), 128.1 (2 CH), 122.6 (CH), 118.2 (C), 113.3 (CH), 72.4 (CH2). LRMS m/z 229.2 (100%, M + H).
×
×
These data are in good agreement with literature values [64].
3.2. Assays
3.2.1. Radiolytic Reduction
DMSO stock solutions (10 mM) were transferred to a Pd/C catalyzed anaerobic chamber with
an atmosphere of 5% H2, 5% CO2, 90% N2 (Shellab Bactron, Sheldon Manufacturing Inc. Cornelius,
OR), and diluted to make 10
µM solutions in 100 mM sodium formate/5 mM sodium phosphate, pH
7.0, using buffer that had been equilibrated in the chamber for
≥
3 days to ensure deoxygenation.
Aliquots (1 mL) were then transferred to HPLC vials, sealed, removed from the chamber and irradiated
using an Eldorado 78 teletherapy cobalt-60 unit. The dose rate was ~3 Gy/min, with exact values
determined by Fricke dosimetry [65]. Samples were analyzed immediately by LC/MS, or after
incubating for 5 h at 37 ◦C.
3.2.2. Chemical Reduction
Compound 3c (2.14 mg, 5.9 µmol), nitrogen purged acetonitrile, ammonium chloride, and zinc
powder were transferred to a Pd/C catalyzed anaerobic chamber with an atmosphere of 5% H2, 5%
CO2, 90% N2 (Shellab Bactron, Sheldon Manufacturing Inc. Cornelius, OR). 3c was dissolved in
acetonitrile (1 mL), followed by addition of NH4Cl (32.1 mg, 0.60 mmol) and zinc powder (32.6 mg,
0.50 mmol). The resulting slurry was mixed and left for 1 h, then filtered through 0.2 µm acrodisc
filters. Filtrate samples were diluted in mobile phase (1:100) and analyzed immediately by LC/MS,
or after 3 h incubation at room temperature.
3.2.3. LC/MS Analysis
Solutions were analyzed with an Agilent 1260 series HPLC coupled with an Agilent single
stage quadrupole mass spectrometer, equipped with a Jet StreamTM electrospray ionization source
(Agilent Technologies, USA). Chromatographic separation was achieved on an Agilent Zorbax C18,
3.0
×
150 mm column with 5 µ
m particle size, maintained at 30 ◦C. The mobile phase consisted of
45 mM ammonium formate buffer, pH 3.5 (A) and 80% acetonitrile with 0.01% formic acid (B) with
a linear gradients (40–100% B over 8 min, 100–40% B over 1 min, held at 40% B for 3 min) at a flow
rate of 0.5 mL/min. The sample volume injected was 100 µ
L and the auto sampler was set at 4 ◦C.
The mass spectrometer was run in positive and negative ion ionization modes, with dual polarity
scans from 100–600 m/z. Instrument parameters of the mass spectrometer were: fragmentation voltage
70 V, gas flow 12 L/min, gas temperature 250 ◦C, sheath gas flow 10 L/min, sheath gas temperature
325 ◦C, nebuliser 35 psi, capillary voltage 2500 V, and nozzle voltage 500V. Absorbance detection was
at 276 nm (bandwidth 16 nm) with quantitation by integration of peak areas with Agilent Open Lab
CDS Chemstation software. Unirradiated samples were used for single-point calibration.
3.2.4. Biochemical PARP Inhibition Assay
Inhibition of PARP-1 activity was evaluated in a radioisotope-based filter binding assay by
Reaction Biology Corporation (Malvern PA) using human recombinant PARP-1 (residues 1-1014,
Genbank accession # NM_001618.3, MW = 114.8 kDa) expressed with a C-terminal His-tag in Sf9 insect
cells, >80% pure by SDS-PAGE. Polymerization of 32P-NAD+ (10
µM) on core histones (0.01 mg/mL)
in buffer (50 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2, 0.02% Brij35, 1 mM DTT, 1% DMSO and
20 µg/mL activated DNA (Sigma-Aldrich D4522) was measured after 1 h incubation and washout
of remaining free NAD+. Each compound was included in a 10-point, 3-fold dilution dose response
experiment and an IC50 value derived.

Molecules 2019, 24, 1559
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Author Contributions: Conceptualization, M.P.H. and W.R.W.; investigation, B.D.D. and W.-W.W.; resources,
M.P.H. and W.R.W.; writing—original draft preparation, B.D.D.; visualization, W.-W.W.; writing—review and
editing, B.D.D., M.P.H. and W.R.W.; supervision, M.P.H. and W.R.W.; funding acquisition, M.P.H. and W.R.W.
Funding: This research was funded by the Health Research Council of New Zealand grant number 14/538 and the
Cancer Research Trust New Zealand grant numbers GOT-1606 and GOT-1349.
Acknowledgments: The authors thank Adrian Blaser for assistance with NMR spectroscopy and Sisira Kumara
for HPLC analyses.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; and in the decision to
publish the results.
Abbreviations
•
•
•
•
•
•
•
•
•
•
•
•
•
•
AcCl acetyl chloride;
BnCl benzyl chloride;
CDI 1,10-carbonyldiimidazole;
DCM dichloromethane;
DIPEA diisopropylethylamine;
DMF N,N-dimethylformamide;
EtOAc ethyl acetate;
Et2O diethyl ether;
EtOH ethanol;
h hour;
MeOH methanol;
rt room temperature;
THF tetrahydrofuran;
X4 petroleum ether boiling range 40–60 ◦C.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).