Probing cytochrome P450 (CYP) bioactivation with chloromethylindoline bioprecursors derived from the duocarmycin family of compounds

Article abstract of DOI:10.1016/j.bmc.2021.116167

The duocarmycins belong to a class of agent which has great potential for use in cancer therapy. Their exquisite potency means they are too toxic for systemic use, and targeted approaches are required to unlock their clinical potential. In this study, we

Full text of DOI:10.1016/j.bmc.2021.116167

Bioorg. Med. Chem. 40 (2021) 116167
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Probing cytochrome P450 (CYP) bioactivation with chloromethylindoline
bioprecursors derived from the duocarmycin family of compounds
Natalia Ortuzara, Kersti Karua, Daniela Presab, Goreti R. Moraisb, Helen M. Sheldrakeb,
Steve D. Shnyderb, Francis M. Barniehb, Paul M. Loadmanb, Laurence H. Pattersonb,
,
,*
Klaus Porsb *, Mark Searceyc
a Department of Pharmaceutical and Biological Chemistry, UCL School of Pharmacy, 29-39 Brunswick Square, London WC1N 1AX, UK
b Institute of Cancer Therapeutics, School of Pharmacy and Medical Sciences, Faculty of Life Sciences, University of Bradford, BD7 1DP, UK
c School of Pharmacy, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK
A R T I C L E I N F O
A B S T R A C T
Keywords:
The duocarmycins belong to a class of agent which has great potential for use in cancer therapy. Their exquisite
potency means they are too toxic for systemic use, and targeted approaches are required to unlock their clinical
potential. In this study, we have explored seco-OH-chloromethylindoline (CI) duocarmycin-based bioprecursors
for their potential for cytochrome P450 (CYP)-mediated cancer cell kill. We report on synthetic and biological
explorations of racemic seco-CI-MI, where MI is a 5-methoxy indole motif, and dehydroxylated analogues. We
show up to a 10-fold bioactivation of de-OH CI-MI and a fluoro bioprecursor analogue in CYP1A1-transfected
cells. Using CYP bactosomes, we also demonstrate that CYP1A2 but not CYP1B1 or CYP3A4 has propensity
for potentiating these compounds, indicating preference for CYP1A bioactivation.
Cytochrome P450
Duocarmycin
Bioactivation
Bioprecursor
Cytotoxicity
1. Introduction
however the lack of tumour selectivity and lack of therapeutic index
have prevented further progression and regulatory approval.11–13 In
The cytochrome P450 enzymes (CYP) are a family of constitutive and
inducible oxidases that play central roles in the metabolism of xenobi-
otics and endogenous compounds. Members belonging to the CYP1
subfamily participate in the metabolism of carcinogens originating from
chemical pollutants,1 including polycyclic aromatic hydrocarbons
(PAHs), nitroaromatics, and arylamines.2 Exposure to such xenobiotics
could have a long-term effect on human health, including increased risk
of developing cancer. The CYP1A1 isoform often generates more reac-
tive intermediates through its catalytic activity of PAHs that are capable
of binding with DNA and causing genetic mutations.1 However, due to
frequent intratumoral expression and innate capacity to metabolise xe-
nobiotics, some CYPs including CYP1A1 could be a target for locore-
gionally activated cancer therapeutics.3–7
recent years, several efforts have been pursued that are focused on the
development of prodrugs9 and antibody–drug conjugates,14–16 as stra-
tegies to selectively deliver these ultrapotent duocarmycin chemotoxins
to tumour tissue. Many of these approaches have focussed on modifi-
cation of the CI trigger unit via the pendant phenolic OH (or NH2) to
deactivate these agents and prevent the spirocyclization mechanism
necessary for DNA alkylation and cell killing.17
Instead of synthetic manipulation of the phenolic group, our strategy
has been focused on the inactivation of the duocarmycins by complete
removal of the key OH group. This deactivated pharmacophore, has
been suggested to be evolved from ancestral precursors18 although a
recent report19 on the biosynthetic pathway of CC-1065 provides evi-
dence that the installment of the key phenolic OH is generated from a
tyrosine building block by 4-hydroxyphenylacetate 3-hydroxylase.
Regardless of origin, the de-hydroxylated compound is highly lipophilic
and lends itself towards being a substrate for phase 1 CYP metabolism.
In previous reports, we successfully demonstrated that duocarmycins
lacking the phenolic OH, such as the cyclopropapyrroloindole (CPI)-
derived bioprecursors 2 (ICT2700) and 3 (ICT2706) (Fig. 1), are capable
The phenol-containing duocarmycins, e.g. duocarmycin SA (1,
Fig. 1) are a family of natural products recognized as ultrapotent cyto-
toxins.8,9 Their mechanism of action involves spirocyclization of the
deep-embedded seco-OH-chloromethylindoline (CI) fragment to trigger
production of an N3-adenine covalent adduct upon binding of the minor
groove of DNA.10 Four duocarmycins have entered clinical evaluation,
* Corresponding authors.
E-mail addresses: k.pors1@bradford.ac.uk (K. Pors), M.Searcey@uea.ac.uk (M. Searcey).
https://doi.org/10.1016/j.bmc.2021.116167
Received 20 February 2021; Received in revised form 16 April 2021; Accepted 19 April 2021
Available online 21 April 2021
0968-0896/Crown Copyright © 2021 Published by Elsevier Ltd. All rights reserved.

N. Ortuzar et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116167
mechanistic understanding and comparison, we also synthesised
analogue 17 bearing a 5-positioned fluoro group and ortho to the point
of hydroxylation to generate the classical CI pharmacophore.
2. Results and discussion
2.1. Synthesis
The synthesis of the dehydroxy alkylation subunit of ICT2700 (2)
and ICT2706 (3)20,21 utilised a 5-exo-trig-radical cyclization onto a vinyl
chloride,26 and this was the initial approach taken to the more simple CI
analogues (Scheme 1). Starting from commercially available 2-bromoa-
niline or 2-bromo-4-fluoroaniline, di-Boc-protection of the amino group
with subsequent removal of one protecting group was more efficient
than attempts to mono-Boc protect directly. Alkylation with 1,3-dichlor-
opropene and subsequent cyclization using tris(trimethylsilyl)silane
(TTMSS) followed by Boc deprotection and conjugation with 5-
methoxy-indole 2-carboxylic acid gave target compounds 16 and 17 in
reasonable yield (Scheme 1, strategy A).
Figure 1. Duocarmycin SA, bioprecursors ICT2700 and ICT2706 and CI-based
analogues; TMI = trimethoxyindole and 5′-MI = 5-methoxyindole.
In order to investigate a potentially stereoselective synthesis, the
lengthier synthetic strategy based on the method established by War-
pehoski to prepare pyrrole chloroindoline duocarmycin analogues was
also studied and was utilised for the synthesis of the active analogue
seco-CI-MI (Scheme S1).27 Both fluoro-2-nitrobenzene and 1,3-difluoro-
5-nitro-benzene were treated under basic conditions with dimethyl
malonate to generate compounds 20 and 21. Reduction and conversion
to the mesylates 24 and 25 was followed by a one-pot reduction/pro-
tection/intramolecular alkylation to generate target subunits 26 and 27.
While diol 22 was efficiently transformed into respective 24, synthesis
of fluoro derivative 25 was accomplished in poor yield (34%), and
further attempts to improve the yield of this reaction were unsuccessful.
Equally, the one-pot ring closure to afford fluoro derivative protected
indoline 24 (yield = 34%) gave poor conversions. The presence of the
fluoro substituent on the aromatic ring impacted negatively on the
generation of target compounds and hence this approach was shown to
be only suitable for the preparation of unsubstituted dehydroxy-seco-CI-
MI 16 (Scheme 2, strategy B).
of undergoing regioselective aryl oxidation by CYP1A120-22 and
CYP2W121, 23 to generate cytotoxins via seco-duocarmycin OH metab-
olites in a tumour-selective manner (bioactivation outlined in Fig. 2).
Studies conducted on the CI pharmacophore, which is structurally
the simplest member of the duocarmycins, indicated that while the Boc-
protected analogue 4 and trimethoxyindole (TMI) 5 (Fig. 1), for
example, retain potent DNA alkylation, only the latter retains low-nM
antiproliferative activity against L2110 cells.24,25 Since CI is the mini-
mum potent pharmacophore of all of these alkylating natural products,
and to complement previous data, we were interested in exploring
whether dehydroxy analogues of 4 were also suitable for targeting
tumour-expressed CYPs. Our previous studies20, 21 have indicated that
the 5-methoxy indole (MI) motif on the “right-hand” segment of the
pharmacophore is best tolerated for CYP1A1 bioactivation, and here we
have utilised the same motif linked to the deactivated CI pharmacophore
to probe potential for CYP bioactivation. Herein, we report on the syn-
thesis of seco-CI-MI 7 (synthesis reported in Scheme S1) and its dehy-
droxy analogue 16 and investigate their potential for bioactivation by
CYP1A1, 1A2, 1B1 and 3A4, and also report on the metabolic profile. For
2.2. Chemosensitivity
seco-CI-MI (7) and seco-CI-NHBoc (6, Scheme S1) and bioprecursors
Scheme 1. Strategy A: Synthesis of dehydroxy-seco-CI-MI derivatives. Reagent
and conditions: i. Boc2O, cat. DMAP, THF, reflux, 16 h, ii. K2CO3, MeOH, reflux,
3 h; 10: 83%, 11: 76%; iii. NaH, DMF, 0 ◦C, 30 min, then 1,3-dichloropropene,
RT, 3 h; 12: 87%, 13: 80%; iv. TTMSS, AIBN, toluene, 3 h, 90 ◦C; 14: 74%, 15:
77%; v. HCl in EtOAc (2.7 M soln), RT, 3 h; vi. EDC, 5-methoxyindole-2-carbox-
ylic acid, DMF, RT, 16 h; 16: 62%, 17: 64%.
Figure 2. Oxidative bioactivation by CYP isoforms is affected by A ring
configuration in the alkylating subunit and the presence of R fragments on the
DNA minor groove binding motif.
2

N. Ortuzar et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116167
Scheme 2. Strategy B: Synthesis of dehydroxy-seco-CI-MI derivatives. Reagent and conditions: i. NaH, (CO2CH3)2|CH, THF, 0 ◦C, then reflux, 16 h.; 20: 88%, 21: 86%;
ii. DIBAL-H, THF, 0 ◦C, 1 h.; 22: 43%, 23: 28%; iii. MsCl, Et3N, CH2Cl2, 0 ◦C, 1 h; 24: 71%, 25: 35%; iv. H2 , Pd/C, Et3N, Boc2O, THF, RT, 16 h; 26: 70%, 27: 34%; v.
TBAC, DMF, 90 ◦C, 5-16 h, 14: 86%; vi & viii. See Scheme 1 for conditions.
14, 16 and 17 (Scheme 1) were evaluated for their ability to inhibit the
growth of cell lines deficient in CYPs (EJ138 and CHOwt) and proficient
in CYP1A1 (RT112 and CHO1A1). IC50 values for compound 6 were in
CYP3A4, was also shown to potentiate the antiproliferative activity of
compounds 16 and 17. CYP1A1 and 1A2 belong to the same CYP1
family member with over 70% homology, and the smaller size of the CI
pharmacophore when compared with previously reported CBI and CPI
pharmacophores21 suggests that truncated CI duocarmycins can be
accommodated by the active site of CYP1A2, perhaps explaining why
bioprecursor 16 showed extensive metabolism when incubated with
human CYP1A1 bactosomes, however we were not able to identify the
authentic hydroxylated metabolite 7. Similar extensive metabolism and
lack of compound 7 identification was observed with CYP1A2 bacto-
somes (Fig. S1). Incubation of 16 in the presence of glutathione as a way
to detect a putative spirocyclic active species by CYP1A2 also failed to
identify a recognisable conjugate between the highly reactive gluta-
thione with the electrophilic spirocyclopropane (Fig. S2).
the range of 6–11
μ
M and are akin to results reported previously,25 while
seco-CI-MI was significantly more potent with IC50 values in the range
30–55 nM (Table 1). Amongst the seco-CI analogues, it was expected that
replacement of tert-butyloxy group CI-Boc (Scheme S1) with the planar
5-MI motif in CI-MI would favor association of this compound in the
DNA minor groove leading to an increase in antiproliferative activity;
the data here are in accordance with observations made between com-
pound CI-Boc and the seco-CI-TMI analogue.24 Bioprecursors 16 and 17
displayed a notable enhanced antiproliferative activity in the CYP1A1-
22
expressing RT11222 and CHO1A120,
cell lines, with 16 approxi-
mately 10-fold more potent in CHO1A1 compared with CHOwt cells.
Fluorinated analogue 17 appeared to be slightly less potent than 16
suggesting introduction of a fluoro group is detrimental to bioactivation
by CYP1A1. Although this differential is relatively small, the data sug-
gest that antiproliferative activity of bioprecursors 16 and 17 may be
potentiated in the presence of CYP1A1.
In an attempt to identify the active metabolite(s), we decided to use
human recombinant CYP1A1 and assay conditions previously
described.28 Bioprecursor 16 (50
μM) was incubated for 1 h with a
reconstituted protein system (RPS; consisting of human NADPH cyto-
chrome P450 reductase, freshly prepared 1,2-dilauroyl-sn-glycero-3-
phosphocholine, 100 mM potassium phosphate, pH 7.4) and subsequent
analysis revealed not only the presence of compound 7 but also higher
amounts of metabolites M2-M6 (Fig. 3A). The results indicate the use of
RPS provides a superior assay when attempting to identify very small
amounts of metabolites; 61.4% of bioprecursor 16 was metabolised after
1 h of which 0.9% was identified as metabolite 7 (Fig. S5). Furthermore,
incubation of authentic metabolite 7 with CYP1A1 led to the generation
of a major metabolite M1 (M4 in Fig. 3B) with m/z = 327.1. This is a
mass unit loss of 30, indicating the possibility of OCH3 loss from the DNA
minor groove binding motif and perhaps indicates preferred site of
metabolism.
2.3. CYP metabolism of the CI pharmacophore
To further elucidate involvement of CYPs in the bioactivation of
bioprecursors 16 and 17, and to establish the CYP isoforms involved,
compounds 16 and 17 were evaluated against a panel of recombinant
CYP enzymes. This was accomplished by incubating 16 and 17 with
several bactosomes (CYP null as control, 1A1, 1A2, 1B1, and 3A4) for 1 h
at 37 ◦C, and any metabolites produced following incubation with spe-
cific CYP bactosomes were extracted and added to the EJ138 bladder
cancer cells for antiproliferative activity evaluation using the previously
reported methodology.21
The antiproliferative activity of bioprecursor 17 indicated that bio-
activation would also occur (Tables 1 and 2). Using RPS/CYP1A1 system
we performed a time-dependent experiment and monitored metabolite
generation over a period of 60 min (Fig. 4). Two mono-hydroxylated
metabolites were generated with intact chloroethyl fragment, however
as with compound 16 we were not able to identify spirocyclized product.
The latter can be generated in position 6 (natural product configuration)
The CYP1A1-extracted metabolite fractions were shown to produce a
7-fold potentiation for bioprecursor 16 and 5-fold for bioprecursor 17
(Table 2), which is largely in accordance with the CHO/CHO1A1 cell
data for these compounds. Furthermore, CYP1A2, but not CYP1B1 or
Table 1
or in position 4, which is theoretically
a possible route for
Growth inhibition (IC50 = µM) of chloromethylindolines against human bladder
carcinoma cell lines, EJ138 and RT112, parental CHO cell line and the CYP1A1-
transfected variant.
bioactivation.29
Next, we performed studies with liver endoplasmic reticulum sam-
ples to reflect the major site of drug metabolism to fully address the
multiple CYP interactions with the most promising bioprecursor 16.
Accordingly, 16 was incubated in the presence of human and rat liver
microsomes, both representing a rich source of drug-metabolising CYP
enzymes. In both sets of microsomes, a time-dependent disappearance of
compound 16 accompanied by concomitant formation of metabolites
M16, M22 and M23 was observed, though not in similar proportion
Compd ID
EJ138
RT112
CHOwt
CHO1A1
6
8.1 ± 2.0
0.038 ± 0.008
>25.0
11.1 ± 2.8
0.055 ± 0.004
>25.0
7.3 ± 1.1
0.030 ± 0.004
>25.0
6.5 ± 0.8
0.035 ± 0.004
>25.0
7
14
16
17
15.0 ± 1.9
8.04 ± 0.9
11.4 ± 2.2
1.2 ± 0.5
18.6 ± 1.2
5.04 ± 0.9
14.8 ± 3.8
6.2 ± 1.3
aAll IC50 values are in
μM and the mean ± SD of at least three independent assays.
3

N. Ortuzar et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116167
Table 2
Growth inhibition of bioprecursors 16 and 17 after incubation with CYP bactosomes against EJ138 bladder cancer cell lines.
EJ138
ICb50
EJ138 + Ca
EJ138 + CYP1A1
EJ138 + CYP1A2
EJ138 + CYP1B1
EJ138 + CYP3A4
Compd ID
IC50
IC50
PFc
IC50
PF
IC50
PF
IC50
PF
16
17
15.0 ± 1.9
18.6 ± 1.2
17.2 ± 4.1
15.8 ± 3.2
2.5 ± 0.9
3.1 ± 1.0
6.9
5.1
1.9 ± 0.9
9.1
3.1
13.5 ± 1.2
11.9 ± 3.0
1.3
1.3
19.8 ± 6.8
16.8 ± 5.1
0.9
0.9
5.11 ± 0.5
aC = null bactosomes; bIC50
=
μ
M; cPotentiation factor (IC50 in EJ-138 + control bactosomes/IC50 in EJ-138 + CYP isoform metabolites).
Figure 4. Time-dependent incubation of compound 17 with human CYP1A1
recombinant enzyme. M = unidentified metabolites.
expression in human liver. Growth inhibition data substantiate that this
enzyme made no contribution to the bioactivation of 16 (Table 2),
which is a desirable feature in the further development of these tumour-
activated duocarmycins bioprecursors.
3. Conclusion
In this study, we have explored CI duocarmycin-based bioprecursors
for their potential for CYP bioactivation. Collectively, the data indicate
extensive metabolism of these compounds by CYPs; however, of those
CYPs investigated, only CYP1A family members appear to be linked to
potentiation of bioprecursors 16 and 17 in cancer cells. No metabolite
from incubation of 16 with bactosomes, rat or human liver microsomes
was shown to co-elute with the authentic CI-MI metabolite, however it
was detected using human recombinant CYP1A1.
Although CYP1A2 was able to potentiate bioprecursors, it is possible
that no significant toxicity will be seen in the liver as it is a detoxification
organ and generally copes well with the metabolism of drugs such as
cyclophosphamide, which is known to be activated in the liver to a DNA
interstrand crosslinking agent.30 Accordingly, targeting overexpressed
CYP1A1 in tumours for therapeutic intervention remains a viable route
given the liver is a robust organ capable of tolerating high concentration
of chemicals without suffering severe damage in the short term. This
work provides chemical exploration to target compound synthesis and
enzymatic explorations of the simplest member of the duocarmycin
family of compounds. Importantly, the data add further support to the
concept of designing duocarmycin bioprecursors.
Figure 3. CYP1A1 metabolic studies of compounds 7 and 16. Top chromato-
gram: comparison of metabolism profiles of 16 using purified human CYP1A1
recombinant enzyme and CYP1A1 bactosomes. Bottom chromatogram: compar-
ison of metabolism profiles of compounds 7 and 16 using CYP1A1 recombinant
enzyme. In both cases, incubation time was 1 h. Apart from M1, M = uniden-
tified metabolites.
(Supporting Information). Metabolites M16 and M22 are the predomi-
nant metabolites produced by human liver microsomes (Fig. S4), while
microsomes from rat liver produced mainly M23 (Fig. S3). Human
CYP3A4 is one of the most abundant drug-metabolising CYP isoforms in
human liver and, on average, accounts for half of the total CYP
4

N. Ortuzar et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116167
4. Materials and methods
tert-Butyl
N-(2-bromo-phenyl)-N-(3-chloro-allyl)carbamate
(12):
Compound 10 (6.5 g, 24 mmol) was dissolved in DMF (60 mL), cooled to
0 ◦C and treated with NaH (60%, 2.87 g, 71.7 mmol) portionwise over
15 min. The resulting solution was stirred at 0 ◦C for a further 15 min. E/
Z-1,3-Dichloropropene (7.4 mL, 71.7 mmol) was then added and the
reaction mixture warmed to RT. After 3 h, the reaction was quenched
with sat. NaCl and the aqueous phase extracted with EtOAc (3 × 25 mL).
The combined organic extracts were then dried (MgSO4), filtered and
concentrated in vacuo. Column chromatography (2% EtOAc in hexane)
gave 12 (7.36 g, 87%) as a pale golden oil; Rf 0.40 (10% EtOAc in
hexane); IR (neat) υmax 3020, 2976, 2929, 1697, 1365, 1159, 754, 724
cmꢀ 1; 1H NMR (CDCl3, 400 MHz) δ 7.61 (d, 1H, J = 7.3 Hz, ArH), 7.30
(d, 1H, J = 6.8 Hz, ArH), 7.17 (m, 2H, ArH), 6.04 (m, 2H, =CH), 4.51
(dd, 1H, J = 15.7, 5.9 Hz, CH2), 4.41 (dd, 1H, J = 12.4, 5.2 Hz, CH2),
4.30 (dd, 1H, J = 15.8, 6.3 Hz, CH2), 3.86 (dd, 1H, J = 12.0, 6.0 Hz,
CH2), 1.54 (s, 2H, (CH3)3), 1.35 (s, 7H, (CH3)3); 13C NMR (CDCl3, 100
4.1. Chemistry
All chemicals were reagent grade. All anhydrous solvents used were
bought as such and presumed to conform to manufacturer’s standards.
NMR spectra were recorded on a Bruker Advance AM spectrometer at
the frequencies of 400 MHz (1H), 100 MHz (13C). Chemical shifts (δ) are
reported in parts per million (ppm). H and 13C chemical shifts were
1
referenced to the residual solvent peak. Elemental analysis was carried
out using a Carlo Erba CHN1108 Elemental Analyser. Melting points
(mp) were recorded using a Bibby Stuart Scientific SMP3 Melting Point
Apparatus. Infrared spectra were recorded as neat samples using a
Nicolet Smart Golden Gate Spectrometer (Avatar 360 FT-IR E.S.P). Mass
spectra (MS) were recorded using a ThermoQuest Navigator Mass
Spectrometer operated under Electrospray Ionization in positive (ES+)
or negative (ESꢀ ) modes. High-resolution mass spectra (HRMS) were
recorded using a Micromass Q-TTOF Global Tandem Mass Spectrometer,
and data were manipulated using the MassLab 3.2 software system.
Chromatographic separations were performed on silica gel for flash
chromatography (particle size 40–63 µm) Analytical-TLC was performed
on Merck precoated silica gel 60 F254 TLC plates. The TLC plates were
visualised using a variety of techniques: visualisation under UV light,
phosphomolybdic acid (10% soln. in EtOH), ninhydrin (10% soln. in
EtOH) followed by heating.
–
–
–
–
MHz) δ 154.1 (E/Z C O), 153.9 (E/Z C O), 140.9 (E/Z ArCN), 140.5
(E/Z ArCN), 133.2 (E/Z ArCH), 133.0 (E/Z ArCH), 130.5 (E/Z ArCH),
130.0 (E/Z ArCH), 128.8 (E/Z ArCH), 128.7 (E/Z ArCH), 128.4 (E/Z =
CH), 128.0 (E/Z = CH), 127.5 (ArCH), 121.5 (E/Z = CHCl), 120.5 (E/Z
= CHCl), 119.9 (ArCBr), 80.6 (C(CH3)3), 48.9 (E CH2), 45.9 (Z CH2),
28.4 (minor rotamer C(CH3)3), 28.1 (major rotamer C(CH3)3); MS (ES +
) m/z calcd for C14H17BrClNO2 [M] 347.0, 345.0. Found [M + 1] 348.0,
346.0; Analysis calcd for C14H17BrClNO2: C, 48.51; H, 4.94; N, 4.04.
Found: C, 48.66; H, 5.06; N, 4.07.
tert-Butyl N-(2-bromophenyl)carbamate (1031): 2-Bromoaniline (5 g,
29.1 mmol) was dissolved in anhyd⋅THF (50 mL) and treated with Boc2O
(14.3 g, 65.7 mmol) and DMAP (0.71 g, 5.81 mmol). The reaction
mixture was then heated under reflux for 16 h. The solution was sub-
sequently cooled and partitioned between HCl (0.5 M, 50 mL) and EtOAc
(50 mL). The aqueous phase was further extracted with EtOAc (3 × 25
mL), and the combined organic extracts washed with sat. NaCl solution,
dried (MgSO4), filtered and concentrated in vacuo. The crude oil was
redissolved in CH3OH (50 mL) and treated with K2CO3 (12 g, 87.3
mmol). This heterogenous reaction mixture was then heated under
reflux for 3 h. The solution was subsequently cooled, filtered and par-
titioned between HCl (0.5 M, 50 mL) and EtOAc (50 mL). The aqueous
phase was further extracted with EtOAc (3 × 25 mL), and the combined
organic extracts washed with sat. NaCl solution. These were then dried
(MgSO4), filtered and concentrated in vacuo. Column chromatography
(100% hexane) gave 10 (6.55 g, 83%) as a very pale golden oil; Rf 0.57
(10% EtOAc in hexane); IR (neat) υmax 3413, 2977, 2930, 1731, 1513,
1431, 1148, 745 cmꢀ 1; 1H NMR (CDCl3, 400 MHz) δ 8.06 (d, 1H, J = 8.2
Hz, ArH), 7.41 (dd, 1H, J = 8.0, 1.3 Hz, ArH), 7.19 (ddd, 1H, J = 8.2, 7.4,
1.2 Hz, ArH), 6.91 (br s, 1H, NH), 6.81 (ddd, 1H, J = 7.8, 7.6, 1.5 Hz,
tert-Butyl
N-(2-bromo-4-fluorophenyl)-N-(3-chloroallyl)carba-mate
(13): Compound 13 was synthesized and purified as described above for
12, starting from compound 11 (3.76 g, 14.2 mmol). Yield: 80% (oil); Rf
0.40 (10% EtOAc in hexane); IR (neat) υmax 2977, 2928, 1699, 1488,
1379, 1366, 1295, 1254, 1193, 1159, 880, 859, 763, 672 cmꢀ 1; 1H NMR
(CDCl3, 400 MHz) δ 7.34 (m, 1H, ArH), 7.14 (m, 1H, ArH), 7.02 (m, 1H,
ArH), 6.04 (m, 2H, =CH), 4.48 (dd, 1H, J = 15.4, 6.1 Hz, CH2), 4.39 (dd,
1H, J = 14.7, 4.0 Hz, CH2), 4.24 (dd, 1H, J = 15.8, 6.9 Hz, CH2), 3.81
(dd, 1H, J = 14.9, 4.6 Hz, CH2), 1.52 (s, 2.3H, (CH3)3), 1.35 (s, 6.7H,
(CH3)3); 13C NMR (CDCl3, 100 MHz) δ 161.1 (d, JCF = 251.0 Hz, E/Z
–
–
ArCF), 161.0 (d, JCF = 251.2 Hz, E/Z ArCF), 154.0 (E/Z C O), 153.8 (E/
–
Z C O), 137.2 (E/Z ArCN), 136.8 (E/Z ArCN), 131.3 (d, JCF = 8.6 Hz, E/
–
Z ArCH), 130.9 (d, J = 9.0 Hz, E/Z ArCH), 128.5 (E/Z = CH), 127.2 (E/Z
= CH), 124.3 (E/Z ArCBr), 124.2 (E/Z ArCBr), 121.8 (E/Z = CHCl),
120.9 (E/Z = CHCl), 120.4 (d, J = 25.3 Hz, E/Z ArCH), 120.2 (d, J =
25.4 Hz, E/Z ArCH), 115.2 (E/Z ArCH), 115.0 (E/Z ArCH), 80.8 (C
(CH3)3), 48.9 (E CH2), 45.8 (Z CH2), 28.3 (minor rotamer, C(CH3)3),
28.1 (major rotamer, C(CH3)3); MS (ES
+ ) m/z calcd for
C14H16BrClFNO2 [M] 365.0, 363.0. Found [M + 1] 366.0, 364.0;
Analysis calcd for C14H16BrClFNO2: C, 46.11; H, 4.42; N, 3.82. Found: C,
46.11; H, 4.60; N, 3.65.
ArH), 1.45 (s, 9H, (CH3)3); 13C NMR (CDCl , 100 MHz) δ 152.4 (C O),
–
–
136.3 (ArCNH), 132.2 (ArCH), 128.3 (A3rCH), 123.8 (ArCH), 120.1
(ArCH), 112.4 (ArCBr), 81.1 (C(CH)3)3, 28.3 (3C, C(CH)3)3; MS (ES + )
m/z calcd for C11H14BrNO2 [M] 273.0, 271.0. Found [M + 1]+ 274.0/
272.0; Anal calcd for C11H14BrNO2: C, 48.55; H, 5.19; N, 5.15. Found: C,
48.30; H, 5.04; N, 5.20.
tert-Butyl 3-chloromethyl-2,3-dihydroindole-1-carboxylate (14): A so-
lution of 12 (116.0 mg, 0.33 mmol) in anhyd. toluene (10 mL) was
degassed with N2 for 15 min and then treated with AIBN (12.6 mg,
0.077 mmol) and TTMSS (114 μ
l, 0.37 mmol) before heating at 90 ◦C for
3 h. The reaction was then cooled and concentrated in vacuo. Column
chromatography (5% EtOAc in hexane) gave 14 (66.3 mg, 74%) as a
clear colourless oil; Rf 0.83 (15% EtOAc in hexane); IR (neat) υmax 2975,
tert-Butyl N-(2-bromo-4-fluorophenyl)carbamate (1132): Compound 11
was synthesized and purified as described above for 10, starting from 2-
bromo-4-fluoroaniline (5 g, 26.5 mmol). Yield: 76% (white solid). Rf
0.60 (10% EtOAc in hexane); mp = 35.5–37.7 ◦C; IR (neat) υmax 3343,
3077, 2992, 1694, 1514, 1479, 1365, 1275, 1238, 1155, 1057, 1022,
850, 811, 775, 731 cmꢀ 1; 1H NMR (CDCl3, 400 MHz) δ 8.02–7.90 (dd,
1H, J = 9.1, 5.6 Hz, ArH), 7.18–7.16 (ddd, 1H, J = 9.1, 7.8, 2.9 Hz, ArH),
6.96–6.91 (dd, 1H, J = 7.8, 2.9 Hz, ArH), 6.77 (br s, 1H, NH), 1.45 (s,
1697, 1484, 1388, 1163, 1140, 1014, 856, 748, 708 cmꢀ 1
;
1H NMR
(CDCl3, 400 MHz) δ 7.88–7.49 (br d, 1H, ArH), 7.23 (m, 2H, ArH), 6.97
(ddd, 1H, J = 7.5, 7.5, 0.9 Hz, ArH), 4.11 (dd, 1H, J = 11.0, 9.7 Hz,
CH2N), 3.94 (m, 1H, CH2N), 3.77 (dd, 1H, J = 10.6, 4.4 Hz, CH2Cl), 3.69
(m, 1H, CH), 3.55 (dd, 1H, J = 10.4, 4.4 Hz, CH2Cl), 1.59 (s, 9H, (CH3)3);
13
–
–
C NMR (CDCl , 100 MHz) δ 151.3 (C O), 142.1 (ArC), 129.4 (ArC),
3
9H, C(CH3)3); 13C NMR (CDCl3, 100 MHz) δ 157.8 (d, JCF = 246.1 Hz,
127.9 (ArCH), 123.3 (ArCH), 121.2 (ArCH), 114.0 (ArCH), 80.0 (C
(CH3)3), 51.0 (CH2Cl), 46.2 (CH), 41.3 (CH2N), 27.3 (3C, C(CH3)3);
HRMS (ES + ) calcd for C14H18ClNO2 [M + 1] 268.1104. Found [M + 1]
268.1112.
–
ArCF), 152.5 (C O), 132.8 (d, JCF = 3.0 Hz, ArCN), 121.3 (d, JCF = 7.8
–
Hz, ArCH), 119.2 (d, JCF = 25.6 Hz, ArCH), 115.1 (d, JCF = 21.6 Hz,
ArCH), 112.5 (d, JCF = 9.9 Hz, ArCBr), 81.2 (C(CH3)3), 28.3 (3C, C
(CH3)3); MS (ES + ) m/z calcd for C11H13BrFNO2 [M] 291.0, 289.0.
Found [M + 1] 292.0, 290.1; Anal calcd for C11H13BrFNO2: C, 45.54; H,
4.52; N, 4.83. Found: C, 45.92; H, 4.54; N, 4.71.
Alternatively, a solution of 26 (0.47 g, 1.45 mmol) in DMF (4.7 mL)
was treated with TBAC (1.01 g, 3.6 mmol) and heated at 90 ◦C for 5 h.
The reaction mixture was concentrated in vacuo. Column
5

N. Ortuzar et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116167
chromatography (5% EtOAc in hexane) gave 14 (335 mg 86%) as a clear
colourless oil that matched the compound obtained by the alternative
route.
and concentrated in vacuo. Column chromatography (7% EtOAc in
hexane) gave 20 (15.86 g, 88%) as a pale yellow solid; Rf 0.77 (50%
EtOAc in hexane); mp = 59.7–63.9 ◦C; IR (neat) υmax 3012, 2958, 2866,
tert-Butyl 3-chloromethyl-5-fluoro-2, 3-dihydroindole-1-carbox-ylate
(15): Compound 15 was synthesized and purified as described above for
14, starting from compound 13 (113.8 mg, 0.31 mmol). Yield: 77% (pale
golden oil); Rf 0.32 (10% EtOAc in hexane); IR (neat) υmax 2975, 2871,
1751, 1613, 1578, 1518, 1431, 1341, 1270, 1199, 1031, 794, 718 cmꢀ 1
;
1H NMR (CDCl3, 400 MHz) δ 8.07, (dd, 1H, J = 8.2, 0.9 Hz, ArH), 7.66
(ddd, 1H, J = 7.6, 7.6, 0.9 Hz, ArH), 7.56–7.51 (m, 2H, ArH), 5.34 (s, 1H,
3
CH), 3.81 (s, 6H, OCH3); 13C NMR (CDCl , 100 MHz) δ 167.6 (2C, C O),
–
–
148.8 (ArCNO2), 133.6 (ArCH), 131.4 (ArCH), 129.3 (ArCH), 127.9
(ArC), 125.3 (ArCH), 54.1 (CH), 53.1 (2C, OCH3); MS (ES + ) m/z calcd
for C11H11NO6 [M] 253.1. Found [M + Na]+ 276.0; Anal. calcd for
C11H11NO6: C, 52.18; H, 4.38; N, 5.53. Found: C, 52.03; H, 4.46; N, 5.58.
Dimethyl 2-(5-fluoro-2-nitrophenyl)malonate (2133): Compound 21
was synthesized and purified as described above for 20, starting from
compound 19 (10.4 mL, 0.09 mmol). Yield: 86%; Rf 0.18 (10% EtOAc in
1
1699, 1488, 1390, 1366, 1254, 1161, 1143, 880, 859, 763 cmꢀ 1; H
NMR (CDCl3, 400 MHz) δ 7.82–7.37 (br d, 1H, ArCH), 6.93 (m, 2H,
ArCH), 4.14 (m, 1H, CH2), 3.94 (m, 1H, CH2), 3.73 (dd, 1H, J = 10.4,
4.7 Hz, CH2), 3.67 (m, 1H, CH2), 3.57 (dd, 1H, J = 10.2, 8.2 Hz, CH2),
1.57 (br s, 9H, C(CH3)3); 13C NMR (CDCl , 100 MHz) δ 160.3 (C O),
–
–
3
152.8 (ArCF), 142.4 (ArC), 138.5 (ArC), 116.3 (ArCH), 115.9 (ArCH),
115.7 (ArCH), 77.8 (C(CH3)3), 52.9 (CH2Cl), 47.4 (CH2N), 29.0 (3C, C
(CH3)3), 14.7 (CH); HRMS (ES + ) calcd for C14H17ClFNNaO2 [M + Na]+
308.0829. Found [M + Na]+ 308.0815.
◦
hexane); mp = 78.8–79.7 C; IR (neat) υmax 3089, 2965, 2355, 1751,
1720, 1588, 1522, 1435, 1353, 1255, 1157, 1006, 834, 742 cmꢀ 1; H
1
(3-Chloromethyl-2,3-dihydroindol-1-yl)-(5-methoxy-1H-indol-2-yl)
methanone (16): Compound 14 (0.15 g, 0.56 mmol) was added to a so-
lution of HCl in EtOAc (1.5 mL, 2.7 M) and left stiirred at RT for 3 h. After
this time, the reaction mixture was concentrated in vacuo and subse-
quently redissolved in DMF (1.5 mL). This solution was then treated
with EDC (0.32 g, 1.68 mmol) and 5-methoxyindole-2-carboxylic acid
(118.4 mg, 0.62 mmol), and stirred at RT for 16 h. After this time the
reaction mixture was concentrated in vacuo. Column chromatography
(15% EtOAc in hexane) gave 16 (118.5 mg, 62%) as an off-white solid.
NMR (CDCl3, 400 MHz) δ 8.19 (dd, 1H, J = 9.0, 5.2 Hz, ArH), 7.26 (m,
2H, ArH), 5.43 (s, 1H, CH), 3.86 (s, 6H, CH3); 13C NMR (CDCl3, 100
–
–
MHz) δ 167.1 (2C, C O), 164.75 (d, J = 258 Hz, ArCF), 144.8
CF
(ArCNO2), 131.3 (d, JCF = 9 Hz, ArC), 128.2/128.1 (J = 10 Hz, ArCH),
118.6 (d, JCF = 25 Hz, ArCH), 116.4 (d, JCF = 24 Hz, ArCH), 54.0 (CH),
53.3 (2C, OCH3); MS (ES ꢀ ) m/z calcd for C11H10FNO6 [M] 271.1.
Found [M ꢀ 1]ꢀ 270.2; Analysis calcd for C11H10FNO6: C, 48.72; H,
3.72; N, 5.16. Found: C, 48.84; H, 3.77; N, 5.18.
2-(2-Nitrophenyl)propane-1,3-diol (2234): A solution of 20 (5.14 g,
20.3 mmol) in anhyd⋅THF (56.4 mL) was added dropwise over 30 min to
DIBAL-H (84.6 mL, 0.10 mol), under N2 at 0 ◦C and stirred for 1 h. A cold
solution of HCl (2 M, 100 mL) was added at 0 ◦C to quench the reaction
and the mixture was subsequently extracted with EtOAc (3 × 50 mL).
The combined organic extracts were dried (MgSO4), filtered and
concentrated in vacuo. Column chromatography (50% EtOAc in hexane)
gave 22 (1.54 g, 43%) as a dark golden oil; Rf 0.13 (50% EtOAc in
hexane); IR (neat) υmax 3238, 2946, 2889, 2355, 1609, 1513, 1481,
◦
mp = 197.6–198.2 C; IR (neat) υmax 3266, 3067, 2932, 1623, 1521,
1482, 1404, 1199, 1167, 752, 730 cmꢀ 1; 1H NMR (CDCl3, 400 MHz) δ
9.53 (br s, 1H, NH), 8.34 (d, 1H, J = 8.1 Hz, ArH), 7.38–7.30 (m, 3H,
ArH), 7.14 (m, 2H, ArH), 7.02 (m, 2H, ArH), 4.69 (dd, 1H, J = 10.6, 9.5
Hz, CH2Cl), 4.51 (dd, 1H, J = 10.7, 4.5 Hz, CH2Cl), 3.92–3.85 (m, 5H,
OCH3, CH, CH2N), 3.63 (dd, 1H, J = 10.8, 9.2 Hz, CH2N); 13C NMR
–
(CDCl , 100 MHz) δ 160.4 (C O), 154.7 (ArCOCH ), 143.7 (ArC), 131.3
–
3
(ArC),3 131.2 (ArC), 130.6 (ArC), 129.1 (ArCH), 128.3 (ArC), 124.5
(ArCH), 124.2 (ArCH), 118.2 (ArCH), 116.8 (ArCH), 112.7 (ArCH),
105.9 (ArCH), 102.5 (ArCH), 55.7 (OCH3), 54.2 (CH2Cl), 46.9 (CH2N),
43.8 (CH); MS (ES ꢀ ) m/z calcd for C19H17ClN2O2 [M] 340.1. Found [M
ꢀ 1]ꢀ 339.2; Analysis calcd for C19H17ClN2O2: C, 66.96; H, 5.03; N,
8.22. Found: C, 66.93; H, 5.25; N, 8.29.
1352, 1229, 1057, 979, 853, 744 cmꢀ 1; H NMR (CDCl3, 400 MHz) δ
1
7.79 (d, 1H, J = 8.0 Hz, ArH), 7.59–7.52 (m, 2H, ArH), 7.39 (ddd, 1H, J
= 8.3, 6.8, 1.5 Hz, ArH), 4.0 (m, 4H, CH2), 3.57 (quin., 1H, J = 6.3, 5.9
Hz, CH), 2.85 (br s, 2H, OH); 13C NMR (CDCl3, 100 MHz) δ 150.6
(ArCNO2), 134.2 (ArC), 132.7 (ArCH), 129.1 (ArCH), 127.7 (ArCH),
124.4 (ArCH), 64.7 (2C, CH2OH), 43.8 (CH); MS (ES ꢀ ) m/z calcd for
C9H11NO4 [M] 197.1. Found [Mꢀ 1 + 2Na]+ 242.1; Anal. calcd for
C9H11NO4: C, 54.82; H, 5.62; N, 7.10. Found: C, 54.64; H, 5.46; N, 7.18.
2-(5-Fluoro-2-nitrophenyl)propane-1,3-diol (23): Compound 23 was
synthesized and purified as described above for 22, starting from com-
pound 21 (2.0 g, 7.38 mmol). Yield: 28%; Rf 0.32 (50% EtOAc in hex-
ane); mp = 79.9–82.2 ◦C; IR (neat) υmax 3251 (br), 3087, 2954, 2874,
1618, 1586, 1522, 1479, 1345, 1245, 1055, 1003, 926, 878, 837, 699
cmꢀ 1; 1H NMR (CD3OD, 400 MHz) δ 7.95 (dd, 1H, J = 9.0, 5.2 Hz, ArH),
7.42 (dd, 1H, J = 10.1, 2.8 Hz, ArH), 7.21 (ddd, 1H, J = 9.1, 7.6, 2.8 Hz,
(3-Chloromethyl-5-fluoro-2,3-dihydroindol-1-yl)-(5-methoxy-1H-indol-
2yl)methanone (17): Compound 17 was synthesized and purified as
described above for 16, starting from compound 15 (384.5 mg, 1.35
mmol). Yield: 64% (white powder); Rf 0.42 (30% EtOAc in hexane); mp
= 186.4–187.9 ◦C; IR (neat) υmax 3323, 3008, 2954, 2479, 1619, 1581,
1
1480, 1402, 1261, 1232, 1200, 1163, 1030, 810, 726, 708 cmꢀ 1; H
NMR (CDCl3, 400 MHz) δ 9.49 (br s, 1H, NH), 8.32 (dd, 1H, J = 8.6, 4.8
Hz, ArH), 7.36 (d, 1H, J = 8.9 Hz, ArH), 7.13 (d, 1H, J = 1.7 Hz, ArH),
7.02 (m, 4H, ArH), 4.71 (dd, 1H, J = 10.3, 10.3 Hz, CH2), 4.52 (dd, 1H, J
= 10.7, 4.7 Hz, CH2), 3.87 (m, 5H, OCH3, CH, CH2), 3.63 (dd, 1H, J =
10.6, 8.7 Hz, CH2); 13C NMR (CDCl , 100 MHz) δ 161.1 (C O), 160.5/
ArH), 4.93 (br s, 2H, OH), 3.91 (m, 4H, CH2OH), 3.61 (m, 1H, CH); 13
C
–
–
158.7 (J = 180 Hz, ArCF), 155.1 (A3rCOCH3), 140.1 (ArC), 133.4, 133.3
(E/Z ArC), 131.6, 131.5 (E/Z ArC), 130.6, 130.5 (E/Z ArC), 128.6 (ArC),
119.5, 119.4 (E/Z ArCH), 117.2 (ArCH), 116.0, 115.8 (E/Z ArCH), 113.0
(ArCH), 112.0, 111.8 (E/Z ArCH), 106.3 (ArCH), 102.8 (ArCH), 56.1
(OCH3), 54.7 (CH2Cl), 46.8 (CH2), 43.8 (CH); MS (ES + ) m/z calcd for
NMR (CD3OD, 100 MHz) δ 165.7 (d, JCF = 252 Hz, ArCF), 149.0
(ArCNO2), 140.1 (d, JCF = 9 Hz, ArC), 128.1 (d, JCF = 10 Hz, ArCH),
117.6 (d, JCF = 24 Hz, ArCH), 115.5 (d, JCF = 24 Hz, ArCH), 64.2 (2C,
OCH2), 46.1 (CH); MS (ES ꢀ ) m/z calcd for C9H10FNO4 [M] 215.1.
Found [M + HCOO] 260.1; Analysis calcd for C9H10FNO4: C, 50.24; H,
4.68; N, 6.51. Found: C, 50.07; H, 4.68; N, 4.52.
C
19H16ClFN2O2 [M] 358.1. Found [M + 1]+ 359.0; Analysis calcd for
C19H16ClFN2O2: C, 63.60; H, 4.49; N, 7.81. Found: C, 63.12; H, 4.11; N,
7.50.
Methanesulfonic acid 3-methanesulfonyloxy-2-(2-nitrophenyl)-propyl
ester (2435): A solution of 22 (796.6 mg, 3.9 mmol) in anhyd. CH2Cl2
(8 mL) was treated with Et3N (1.62 mL, 11.7 mmol) and cooled to 0 ◦C. A
solution of MsCl (0.9 mL, 11.7 mmol) in anhyd. CH2Cl2 (7.5 mL) was
prepared and slowly added to the reaction mixture and subsequently
stirred for 1 h at 0 ◦C. The reaction was quenched with H2O (25 mL) and
extracted with EtOAc (3 × 25 mL). The combined organic extracts were
dried (MgSO4), filtered and concentrated in vacuo. Column chromatog-
raphy (35% EtOAc in hexane) gave 41 (0.98 g, 71%) as an off-white
Dimethyl 2-(2-nitrophenyl)malonate (2033): A solution of dimethyl
malonate (24.8 mL, 0.21 mol) in anhyd⋅THF (150 mL) was cooled to 0 ◦C
and treated with NaH (8.51 g, 0.21 mol). The resulting suspension was
then stirred at 0 ◦C for 15 min and subsequently treated with a solution
of 18 (7.55 mL, 0.07 mol) in anhyd⋅THF (50 mL). The reaction mixture
was then heated to reflux for 16 h. Upon completion, the crude reaction
mixture was poured into acetic acid (100 mL, 10% aq. soln. v/v) then
extracted with EtOAc (3 × 25 mL). The organic phase was subsequently
washed with sat. NaHCO3 solution (2 × 25 mL), dried (MgSO4), filtered
◦
crystalline solid; Rf 0.30 (50% EtOAc in hexane); mp = 95.9–97.2 C;
IR (neat) υmax 3076, 3033, 2937, 1608, 1519, 1337, 1171, 982, 941,
6

N. Ortuzar et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116167
832, 788 cmꢀ 1; 1H NMR (CDCl3, 400 MHz) δ 7.93 (dd, 1H, J = 8.6, 1.5
Hz, ArH), 7.67 (ddd, 1H, J = 7.7, 7.6, 1.3 Hz, ArH), 7.54–7.50 (m, 2H,
ArH), 4.59 (m, 4H, CH2), 4.09 (quin., 1H, J = 5.8, 5.7 Hz, CH), 3.02 (s,
6H, CH3); 13C NMR (CDCl3, 100 MHz) δ 150.0 (ArCNO2), 133.4 (ArCH),
130.2, 129.5 (ArCH), 129.2 (ArCH), 125.3 (ArCH), 67.8 (2C, CH2), 39.0
(CH), 37.5 (2C, CH3); MS (ES + ) m/z calcd for C11H15NO8S1 [M] 353.6.
Found [M + Na]+ 376.0; Analysis calcd for C11H15NO8S1: C, 37.39; H,
4.28; N, 3.96. Found: C, 37.16; H, 4.20; N, 3.76.
in triplicate.
4.3. Role of CYP in chemosensitivity of CI-based bioprecursors
4.3.1. CYP bactosomes studies
Involvement of specific CYP isoforms in the activation of 16 and 17
was determined by evaluating the chemosensitivity of CYP-generated
metabolites of 16 and 17. Metabolites were created via incubation of
Methanesulfonic acid 2-(5-fluoro-2-nitrophenyl)-3-methane-sulfony-
loxy-propyl ester (25): Compound 25 was synthesized and purified as
described above for 24, starting from compound 23 (390 mg, 1.81
16 and 17 (50 μM) in the reaction mixture (2 mM NADPH, 1 mM MgCl2,
50 mM Tris-HCl (pH 7.4), 20 pmol of CYP1A1, 1A2, 1B1, or 3A4 bac-
tosomes. The latter are human CYP isoforms co-expressed with CYP-
reductase in Escherichia coli. Concentrations in pmol CYP/mg protein
varies depending on isoform used and specific information can be ob-
tained from the manufacturer’s protocols (Cypex). Control reactions
were carried out using CYP-null bactosomes. Following 1 h incubation at
37 ◦C, metabolites were extracted using acetonitrile and centrifugation
at 10000 g for 10 min. The resultant supernatant was removed, dried
using vacuum evaporation (Genevac), and the resultant pellet resus-
pended in DMSO, and the antiproliferative activity was assessed by the
MTT assay following 96 h exposure to EJ138 cells as described above.
mmol). Yield: 35%; Rf 0.30 (50% EtOAc in hexane); mp
=
99.4–102.1 ◦C; IR (neat) υmax 3093, 3023, 2938, 1525, 1347, 1330,
1
1172, 978, 949, 833, 846, 751, 702 cmꢀ 1; H NMR (CD3COCD3, 400
MHz) δ 8.01 (dd, 1H, J = 9.1, 5.2 Hz, ArH), 7.48 (dd, 1H, J = 9.8, 2.8 Hz,
ArH), 7.28 (ddd, 1H, J = 9.1, 7.5, 2.8 Hz, ArH), 4.57 (d, 4H, J = 6.1 Hz,
CH2), 4.12 (m, 1H, CH), 3.01 (s, 6H, CH3); 13C NMR (CD3COCD3, 100
MHz) δ 165.4 (d, JCF = 254 Hz, ArCF), 147.8 (ArCNO2), 135.6 (d, JCF
=
9 Hz, ArC), 129.0 (d, JCF = 10 Hz, ArCH), 117.9 (d, JCF = 25 Hz, ArCH),
116.9 (d, JCF = 24 Hz, ArCH), 69.2 (2C, CH2), 40.2 (CH), 37.2 (2C, CH3);
MS (ES + ) m/z calcd for C11H14FNO8S1 [M] 371.0. Found [M + Na]+
393.9; Analysis calcd for C11H14FNO8S1: C, 35.58; H, 3.80; N, 3.77.
Found: C, 35.77; H, 3.72; N, 3.95.
4.3.2. Human recombinant CYP1A1 studies
A reconstituted protein system (RPS) was created by mixing 200
pmol P450 with 400 pmol hPORG3H6-delta27 and incubated for
tert-Butyl 3-methanesulfonyloxymethyl-2,3-dihydroindole-1-carboxylate
(26): Et3N (0.05 mL, 0.38 mmol), Boc2O (85.5 mg, 0.38 mmol) and Pd/C
(6.3 mg, 9.4% w/w) were added to a solution of 24 (66.9 mg, 0.19
mmol) in anhyd⋅THF (3.4 mL). The mixture was stirred for 16 h at RT
under a positive pressure of H2. The reaction mixture was filtered
through Celite and concentrated in vacuo. Column chromatography
(15% EtOAc in hexane) gave 26 (43.4 mg, 70%) as a clear, colourless,
viscous oil; Rf 0.55 (35% EtOAc in hexane). Characterisation consistent
with reference.36 similar in all respects to the previously reported
compound.
10mins at room temperature. A freshly prepared DLPC (240 μM) in assay
buffer (100 mM KPi pH 7.4) was added and incubation continued for
additional 10 mins at room temperature. The RPS was then added to an
eppendorf tube containing assay buffer and substrate (50 μM), at a total
volume of 480
incubated at 37 ⁰C for 3 mins in a block heater (Grant Block Heater
QBD4, UK). NADPH (20 l of 25 mM stock in assay buffer) was added to
the incubating mixture at a final concentration 1 mM. Reaction aliquots
(100 l) were then removed over a 60-minute period into labelled
eppendorf tubes containing dichloromethane (200 l), gently mixed and
l of the
μl prior to the addition of NADPH. The mixture was
μ
μ
tert-Butyl 5-fluoro-3-methanesulfonyloxymethyl-2,3-dihydro-indole-1-
carboxylate (27): Compound 27 was synthesized and purified as
described above for 26, starting from compound 25 (183.0 mg, 0.49
mmol). Yield: 34% (oil); Rf 0.46 (35% EtOAc in hexane); IR (neat) υmax
3020, 2979, 2938, 1690, 1490, 1394, 1354, 1175, 1143, 961, 846, 814,
783, 736 cmꢀ 1; 1H NMR (CDCl3, 400 MHz) δ 7.57 (br d, 1H, ArH), 6.92
(m, 2H, ArH), 4.34 (dd, 1H, J = 9.9, 5.9 Hz, CH2), 4.24 (dd, 1H, J = 9.7,
7.7 Hz, CH2), 4.11 (dd, 1H, J = 11.2, 9.6 Hz, CH2), 3.87 (dd, 1H, J =
11.0, 3.6 Hz, CH2), 3.70 (m, 1H, CH) 2.99 (s, 3H, CH3), 1.55 (br s, 9H,
μ
placed on ice. Tubes were centrifuged (4500 g, 2 mins) with 200
μ
bottom organic layer carefully removed into separate tubes, and dried
using SP Genevac EZ-PLUS evaporator for 30 mins. The dried reaction
was dissolved in 50 μl of 90% acetonitrile, 10% H2O, 0.1% formic acid
and transferred into HPLC vial for LC-MS analysis.
LC-MS was carried out using a gradient method with solvent A (90%
H2O, 10% MeOH, 0.1% FA) and solvent B (90% MeOH, 10% H2O, 0.1%
formic acid) and flow rate = 0.30 mL/min; t = 0 (60% A, 40% B), t = 10
min (30% A, 70% B), t = 20 min (10% A, 70% B), t = 25 min (0% A,
100% B) and t = 26 min (60% A, 40% B). Metabolites were run using a
HiChrom RPB column (25 cm × 2.1 mm id; HIRPB-250AM; R6125) and
a Waters Alliance 2695 HPLC (Micromass, Manchester, UK) with a
photodiode array detector and connected in series with Waters Micro-
mass ZQ quadrupole mass spectrometer in ESI+ mode. Bioprecursors
and their respective metabolites were analysed using UV absorbance at
315 nm with their associated masses identified as singularly charged
ions on the MS.
(CH3)3); 13C NMR (CDCl3, 100 MHz) δ 158.5 (d, JCF = 241 Hz, ArCF),
–
152.2 (C O), 115.8 (d, JCF = 8 Hz, ArCH), 115.4 (d, JCF = 22 Hz, ArCH),
–
112.1 (ArCH), 81.3 (C(CH3)3), 70.4 (CH2OMs), 50.9 (CH2), 39.3 (CH),
37.6 (CH3), 28.4 (3C, C(CH3)3); MS (ES + ) m/z calcd for C15H20FNO5S
[M] 345.1. Found [M + Na]+ 368.1; Analysis calcd for C15H20FNO5S: C,
52.16; H, 5.84; N, 4.06. Found: C, 52.09; H, 5.53; N, 4.25.
4.2. Growth inhibition assays
MS ESI + source parameters used: Desolvation gas; 650 l/hr, cone
gas; 50 l/hr, capillary voltage; 3 kV, extraction voltage; 5 V, cone
voltage; 20 V, Rf voltage; 0.2 V, source block temperature; 120 ◦C and
desolvation temperature; 350 ◦C.
The human bladder carcinoma cell lines, RT112 and EJ-138, were
obtained from the European Collection of Cell Cultures (Salisbury, UK)
and were authenticated morphologically. CHO lines were a gift from the
late Dr. T. Friedberg, University of Dundee. Cell lines were grown as
monolayers in either RPMI 1640 (RT112 and EJ-138) supplemented
with 10% (v/v) fetal bovine serum, 1 mM sodium pyruvate, and 2 mM of
L-glutamine or DMEM (CHO and CHO-1A1) at 37 ◦C in 5% CO2. Com-
pounds were dissolved in DMSO and then diluted in complete cell cul-
4.4. Preparation of microsomes
4.4.1. Human liver
ture medium to give a broad range of concentrations (0.001–100
μM),
Approximately 5 g of liver was snap-frozen in liquid nitrogen and
ground in a percussion mortar and pestle. Powdered tissue was trans-
ferred into an ice-cold glass homogeniser along with 1 mL of homoge-
nisation buffer (50 mM Tris-HCl, pH 7.4, containing 0.1 M NaCl, 0.25 M
sucrose and two tablets of Complete Protease inhibitor cocktail) for
every 0.1 g of ground tissue. The resulting mixture was homogenised
with 30 S using a Teflon pestle with the first five strokes at greater
such that the final DMSO concentration was not greater than 0.1%.
Medium was removed from each well and replaced with compound or
control solutions, and the well plates were then incubated for a further
96 h before the MTT assay was performed as previously described.16
Results were expressed in terms of IC50 values (concentration of com-
pound required to kill 50% of cells), and all experiments were performed
7

N. Ortuzar et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116167
pressure than the rest. The tubes were kept ice-cold during the ho-
mogenisation process. The homogenate was diluted to 5 volumes of liver
weight (approximately 5 mL) and centrifuged at 2,400 g in a Sigma 6
K10 centrifuge for 10 min which sediments the cell debris, nuclei and
unbroken cells. The supernatant from the first centrifugation was
transferred into 70 mL Beckman centrifuge tubes, which were filled to
the top with homogenisation buffer. The supernatants were centrifuged
at 9,000 g in a Beckman L8-60 M centrifuge with a Beckman 45 TI rotor
for 20 min to sediment the mitochondrial fraction and any broken
fragments. After centrifugation, the supernatant was transferred to ul-
tracentrifuge tubes and centrifuged at 180,000 g for 60 min in the
Beckman L8-60 M centrifuge with a Beckman 70.1-TI rotor. The upper
lipid layer and the cytosolic supernatant were removed and the micro-
somal pellets were resuspended in microsomal buffer (0.1M Tris-HCl,
pH 7.4 containing 15% glycerol, one tablet of Complete Protease in-
hibitor cocktail) and brought to a final volume of 1 mL. Samples were
stored at ꢀ 80 ◦C.
Royal Free Hospital and University College School of Medicine.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
The authors would like to thank Yorkshire Cancer Research (Pro-
gram grant B381PA) for supporting our work focused on exploring CYPs
as targets for prodrug development. The human recombinant CYP1A1
was a gift from Prof Emily E. Scott, University of Michigan; the enzyme
was produced via NIH funded grant (R37 GM076343).
Appendix A. Supplementary material
4.4.2. Rat liver
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2021.116167.
Fresh liver (10 g) from (7–8 week old) female ACI rats was washed
◦
with cold isotonic saline (0.9% NaCl, 4 C) to remove blood and con-
nective tissue was excised. The liver was homogenised using an Ultra
Turax T25 (Janke and Kunkel, IKA Labortecnik, Staufen, Germany) in
0.01 M Tris-HCl buffer, pH 7.4 containing 0.25 M sucrose, 15% glycerol
and 0.67 mM phenylmethanesulphonyl fluoride (PMSF). Having ob-
tained a tissue homogenate, it was centrifuged at 4 ◦C isolate sub-
cellular fractions. The tissue homogenate was centrifuged at 2,400 g
for 10 min to pellet intact cells, cell debris, nuclei and unbroken cells.
The resultant supernatant was transferred to tubes and centrifuged at
12,000 g for 20 min at 4 ◦C. After centrifugation, the supernatant was
transferred to ultracentrifuge tubes and centrifuged at 180,000 g for 1 h
at 4 ◦C. The supernatant was then discarded and the microsomal pellet
was re-suspended in 0.1 M Tris-HCl buffer, containing 15% glycerol and
1 mM ethylenediaminetetraacetic acid (EDTA) at pH 7.4 and stored at ꢀ
80 ◦C. Protein concentration of the resulting rat liver microsomes was
determined using the Bradford reagent with bovine serum albumin as
the standard.
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8