Design, synthesis and in-vitro biological evaluation of antofine and tylophorine prodrugs as hypoxia-targeted anticancer agents

Article abstract of DOI:10.3390/molecules26113327

Phenanthroindolizidines, such as antofine and tylophorine, are a family of natural alkaloids isolated from different species of Asclepiadaceas. They are characterized by interesting biological activities, such as pronounced cytotoxicity against different human cancerous cell lines, including multidrug-resistant examples. Nonetheless, these derivatives are associated with severe neurotoxicity and loss of in vivo activity due to the highly lipophilic nature of the alkaloids. Here, we describe the development of highly polar prodrugs of antofine and tylophorine as hypoxia-targeted prodrugs. The developed quaternary ammonium salts of phenanthroindolizidines showed high chemical and metabolic stability and are predicted to have no penetration through the blood–brain barrier. The designed prodrugs displayed decreased cytotoxicity when tested under normoxic conditions. However, their cytotoxic activity considerably increased when tested under hypoxic conditions.

Full text of DOI:10.3390/molecules26113327

molecules
Article
Design, Synthesis and In-Vitro Biological Evaluation of
Antofine and Tylophorine Prodrugs as Hypoxia-Targeted
Anticancer Agents
Ziad Omran 1,
* ,
, Chris P. Guise 2, Linwei Chen 3, Cyril Rauch 4, Ashraf N. Abdalla 5 , Omeima Abdullah 5
Ikhlas A. Sindi 6, Peter M. Fischer 7 , Jeff B. Smaill 2, Adam V. Patterson 2 , Yuxiu Liu 3 and Qingmin Wang 3
1
Department of Pharmaceutical Sciences, Pharmacy Department, Batterjee Medical College,
Jeddah 21442, Saudi Arabia
Auckland Cancer Society Research Centre, School of Medical Sciences, The University of Auckland,
2
Private Bag 92019, Auckland 1142, New Zealand; chrispguise@hotmail.com (C.P.G.);
j.smaill@auckland.ac.nz (J.B.S.); a.patterson@auckland.ac.nz (A.V.P.)
3
State Key Laboratory of Elemento-Organic Chemistry, Research Institute of Elemento-Organic Chemistry,
College of Chemistry, Nankai University, Tianjin 300071, China; chenlinwei@mail.nankai.edu.cn (L.C.);
liuyuxiu@nankai.edu.cn (Y.L.); wangqm@nankai.edu.cn (Q.W.)
4
5
6
School of Veterinary Medicine and Science, University of Nottingham, College Road,
Sutton Bonington LE12 5RD, UK; Cyril.Rauch@nottingham.ac.uk
College of Pharmacy, Umm Al-Qura University, Makkah 21955, Saudi Arabia;
anabdrabo@uqu.edu.sa (A.N.A.); oaabdullah@uqu.edu.sa (O.A.)
Department of Biology, Faculty of Sciences, King Abdulaziz University, Jeddah 21589, Saudi Arabia;
easindi@kau.edu.sa
School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, UK; Peter.Fischer@nottingham.ac.uk
Correspondence: ziad.omran@bmc.edu.sa or ziadomran@hotmail.com
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Omran, Z.; Guise, C.P.;
Chen, L.; Rauch, C.; Abdalla, A.N.;
Abdullah, O.; Sindi, I.A.; Fischer, P.M.;
Smaill, J.B.; Patterson, A.V.; et al.
Design, Synthesis and In-Vitro
7
*
Abstract: Phenanthroindolizidines, such as antofine and tylophorine, are a family of natural alkaloids
isolated from different species of Asclepiadaceas. They are characterized by interesting biological
activities, such as pronounced cytotoxicity against different human cancerous cell lines, including
multidrug-resistant examples. Nonetheless, these derivatives are associated with severe neurotoxicity
and loss of in vivo activity due to the highly lipophilic nature of the alkaloids. Here, we describe the
development of highly polar prodrugs of antofine and tylophorine as hypoxia-targeted prodrugs.
The developed quaternary ammonium salts of phenanthroindolizidines showed high chemical
and metabolic stability and are predicted to have no penetration through the blood–brain barrier.
The designed prodrugs displayed decreased cytotoxicity when tested under normoxic conditions.
However, their cytotoxic activity considerably increased when tested under hypoxic conditions.
Biological Evaluation of Antofine and
Tylophorine Prodrugs as Hypoxia-
Targeted Anticancer Agents.
Molecules 2021, 26, 3327. https://
doi.org/10.3390/molecules26113327
Academic Editors: Jacinta Serpa and
Sofia C. Nunes E
Received: 9 May 2021
Accepted: 29 May 2021
Published: 1 June 2021
Keywords: phenanthroindolizidine; antofine; tylophorine; hypoxia; prodrugs; solid tumors
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
Cancer is a complex disease characterized by the loss of control of cell division. It is
the second cause of mortality in the world after cardiovascular diseases. Chemotherapy has
shown reasonable success in treating cancer; however, the lack of selectivity of chemother-
apy drugs, which causes severe side effects, and the emergence of resistance are two major
drawbacks preventing the effective clinical use of chemotherapeutic agents [1–4].
Natural products have been the major source of the currently available drugs. About
50% of the FDA-approved small molecule anticancer drugs introduced from the early 1940s
Copyright:
© 2021 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 (https://
creativecommons.org/licenses/by/
4.0/).
to the end of 2014 were either natural products or directly derived from them [
example is the phenanthroindolizidine family, a group of alkaloids with general structure
) isolated from different species of Asclepiadaceas (Figure 1). These alkaloids display
5]. One
(
1
very interesting biological activities, such as pronounced cytotoxicity against different
human cancerous cell lines. Prototype compounds, such as tylocrebrine, tylophorine, and
Molecules 2021, 26, 3327. https://doi.org/10.3390/molecules26113327
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 3327
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antofine (Figure 1), exhibit low nanomolar to picomolar half-maximal growth inhibition
(GI50) values, as well as effectiveness against multidrug-resistant human cancerous cell
lines [
diverse mechanisms [
inhibition of DNA, RNA and protein synthesis [
thase [13 15], the inhibition of dihydrofolate reductase [13
Protein-1, and NF-κB, and the down-regulation of cyclin D1 [16,17].
6
]. Phenanthroindolizidines exert their anticancer activity through a combination of
] such as the inhibition of hypoxia-inducible factor-1 (HIF-1) [ ], the
12], the inhibition of thymidylate syn-
15], the inhibition of Activator
7
8
9–
–
–
Figure 1. Chemical structure of some phenanthroindolizidine alkaloids.
Notwithstanding their therapeutic potential, no compound in this class has yet suc-
cessfully passed clinical trials. The main drawbacks are their severe central nervous system
toxicity and a significant loss of anti-cancer activity when administered in vivo
These drawbacks are attributed to the highly lipophilic nature of these alkaloids [
[
18
6
,
,
19].
19
]
that enables them to cross the blood–brain barrier (BBB) and to undergo high nonspecific
protein binding and rapid metabolism.
One proposed solution has been the use of more polar analogues that cannot cross the
BBB and therefore could have less toxic side effects [19]. Nonetheless, efforts to increase
the polarity of this type of alkaloid (e.g., by opening the indolizidine ring or introducing
hydroxyl groups on the E ring) have led to significant losses of cytotoxicity [20,21].
The aim of the present work was to develop polar prodrugs of phenanthroindolizidine
alkaloids. The rationale was that the polarity of the new derivatives would prevent them
from penetrating the BBB, thereby attenuating their neurotoxicity. The prodrugs were de-
signed to be activated only within the cancerous microenvironment by targeting the unique
biochemical alterations in cancer cells, i.e., the low concentrations of molecular oxygen
(hypoxia). This, in turn, would further increase the selectivity of the proposed molecules.
Hypoxia, or oxygen deficiency, is a common feature of most solid tumors and is
considered a negative prognostic factor [22]. Hypoxia results from the disorganized tumor
vasculature, where the intercapillary distances usually exceed the oxygen diffusion range
(around 200
and chemotherapy [24]. Additionally, hypoxia induces metastasis [25], promotes angio-
genesis and vasculogenesis [26 27], and enhances invasiveness [28]. Given the important
role of hypoxia in tumor development and progression, and given its relative absence in
healthy tissues [29], hypoxia represents an attractive therapeutic target [22 30 32]. The
main approach to targeting hypoxia consists of the development of bioreductive prodrugs
that can be activated in hypoxic tissues by enzymatic reduction [33 36]. Designing drug
µm) [23]. Hypoxia is responsible, at least in part, for tumor resistance to radio-
,
,
–
–
delivery systems targeting tumor-hypoxia is another promising approach [37–39].
Nitro(hetero)cylic derivatives are now well established as prodrugs that can be acti-
vated by reductive metabolism mediated by the oxidoreductase enzymes present in human
cells [40]. This reduction is repressed by molecular oxygen, making this type of prodrug
specific to the hypoxic tumor microenvironment [40]. Some of these prodrugs, such as
PR-104 and evofosfamide, also known as TH-302, (Figure 2), have reached Phase II and
III clinical trials, either as monotherapy or in combination therapy to treat various types
of solid tumors [33,40–42]. The permanent positive charge of the tarloxotinib bromide
(Figure 2) renders the molecule less permeable to cells than the parent drug, thereby de-
creasing the toxicity. This attenuation of toxicity permits the administration of such type

Molecules 2021, 26, 3327
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of quaternary ammonium prodrugs at higher doses than can be achieved with the parent
drugs. Additionally, the relative positioning of positive charge and lipophilic head group of
this prodrug class leads to sustained tumor residence over time [43]. Tarloxotinib bromide
is currently in phase II clinical trials to treat non-small cell lung cancer with HER2 activating
mutations [44].
Figure 2. Chemical structure of some hypoxia-targeted nitro(hetero)aromatic-based prodrugs.
During this work, we successfully applied the approach employed to synthesize
tarloxotinib bromide to develop quaternary ammonium salts of antofine and tylophorine
alkaloids as hypoxia-targeted prodrugs.
2. Results and Discussion
2.1. Chemistry
Both enantiomers of tylophorine and (R)-antofine were obtained as previously de-
scribed [45] with slight modification (Figure 3). Briefly, dibromides
the corresponding N,N-diethylpyrrolidine-2-carboxamide. Amides
2
3
were alkylated by
were then treated
with nBuLi and TMEDA, followed by sodium borohydride, to yield alcohols
finally reduced using triethylsilane and trifluoroacetic acid to give the desired alkaloids.
The target quaternary ammonium salts 5a were successfully synthesized by reacting ap-
propriate precursor alkaloids with 5-(bromomethyl)-1-methyl-4-nitro-1H-imidazole [43 46].
The prodrugs 5a obtained with reasonable yields (52–68%) after purification by silica gel.
4, which were
–c
,
–
c
Figure 3. Chemical synthesis of prodrugs 5a–c.

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2.2. Physicochemical Properties
The physicochemical properties of the new compounds were evaluated (Table 1). The
water solubility of the alkaloids was considerably increased, up to 80-fold, when they
were transformed to their corresponding quaternary ammonium salts 5a–b. Consequently,
these derivatives lost their BBB penetrability, as predicted by the BBB-Parallel Artificial
Membrane Permeability Assay (BBB-PAMPA), whereas their parent alkaloids had free
passage through the BBB. Additionally, the developed salts had significantly less affinity
for plasma proteins than did their parent alkaloids.
Table 1. Physiochemical properties prodrugs 5a,b and their parent alkaloids.
PBS (pH 7.4)
Solubility
µM Mean ± SE
Permeability (Papp)
Log [10 cm/s]
PPB (% of Bound
Compound)
LogD
Mean ± SE
−6
Compound
Mean ± SE
Mean ± SE
5a
154 ± 0
162 ± 3
27 ± 2
2 ± 0
0.69 ± 0.01
0.38 ± 0.04
3.68 ± 0.03
3.14 ± 0.02
<−7
<−7
−5.6 ± 0.58
−5.4 ± 0.17
87.0 ± 15
58.9 ± 0.9
98.1 ± 0.15
93.9 ± 0.7
5b
(R)-Antofine
(S)-Tylophorine
Ondansetron
(Reference)
96 ± 3
-
-
-
Mebendazole
(Reference)
-
-
-
-
-
3.26 ± 0.01
-
-
Chlorpromazine
(Reference)
-
-
-
-
−5.4 ± 0.13
−5.1 ± 0.12
<−7
-
Clozapine
(Reference)
-
Ranitidine
(Reference)
-
Verapamil
(Reference)
-
89.4 ± 0.4
The chemical and metabolic stability of quaternary ammonium salts was also evalu-
ated to ensure that our prodrugs would not be transformed into the parent alkaloids before
arriving at their site of action, the hypoxic tumor. The developed prodrugs displayed
high plasma stability when incubated with either mouse or human plasma (Figure 4A,B),
attested by t1/2 values ranging between 63 and 145 h, Table 2. Interestingly, after 24 h of
incubation in either mouse or human plasma, no more than 1% of compounds 5a or 5b
were transformed into their parent drugs.
Figure 4. Stability of prodrugs 5a,b in mouse (A), and in human (B) plasma.

Molecules 2021, 26, 3327
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Table 2. Mouse and human plasm stability for prodrugs 5a,b, t1/2: half-life.
t1/2, h
(Mouse Plasma)
t1/2, h
(Human Plasma)
Compound
5a
65.1 ± 0.7
144.7 ± 4.7
5b
73.0 ± 1.7
63.3 ± 0.1
Verapamil
(Reference)
13.0 ± 1.0
12.2 ± 0.4
Propantheline
(Reference)
<3
<3
Similarly, consistent with their reduced lipophilicity the developed quaternary am-
monium salts showed significantly higher metabolic stability than was observed for their
parent alkaloids following incubation with mouse liver microsomes (Figure 5). The alkaloid
half-lives were increased by three- to four-fold upon their transformation to the correspond-
ing ammonium salts (Table 3). It is also noteworthy that less than 1% of compounds 5a or
5b was metabolized into the parent drug under these conditions.
Figure 5. Mouse hepatic microsomal stability for prodrugs 5a,b and their parent alkaloids.
Table 3. Mouse hepatic microsomal stability for prodrugs 5a,b and their parent alkaloids. Kel:
elimination rate constant, t1/2: half-life.
−1
Compound
5a
Kel, min
t1/2, min
286.9 ± 6.5
404.0 ± 3.2
67.5 ± 2.1
127.5 ± 3.0
0.010 ± 0
0.002 ± 0
0.002 ± 0
0.005 ± 0
5b
(R)-Antofine
(S)-Tylophorine
Imipramine
(Reference)
0.083 ± 0
8.3 ± 0.4
Propranolol
(Reference)
0.021 ± 0
33.5 ± 1.0
2.3. Cytotoxic Activity
The cytotoxicity of the compounds 5a–c and their parent alkaloids was evaluated by
the 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay against
5 cancerous and 2 non-cancerous cell lines (Table 4). All three quaternary ammonium
salts 5a–c showed significantly decreased cytotoxicity (up to 1000-fold) against cancerous

Molecules 2021, 26, 3327
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and noncancerous cell lines alike under normoxic conditions when compared with the
parent drugs.
Table 4. Cytotoxicity of compounds 5a–c and their parent alkaloids under normoxia (nM, IC50).
Compound
HEK293
5080 ± 519
950 ± 174
-
CHO-K1
1680 ± 542
3490 ± 843
-
MCF7
1530 ± 144
1498 ± 128
8050 ± 0343
2 ± 0
62 ± 13
101 ± 88
HCT116
1038 ± 21
3304 ± 1562
9019 ± 1128
2 ± 0
85 ± 6
440 ± 36
RKO
2034 ± 267
4056 ± 181
8821 ± 3081
6 ± 1
202 ± 3
338 ± 061
SW480
1350 ± 0.126
7245 ± 490
9862 ± 1693
9 ± 02
69 ± 20
1148 ± 0244
MRC5
327 ± 147
3313 ± 163
3943 ± 127
6 ± 2
27 ± 2
95 ± 6
5a
5b
5c
(R)-Antofine
(S)-Tylophorine
(R)-Tylophorine
30 ± 43
16 ± 26
-
33 ± 50
35 ± 42
-
We also checked if the designed prodrugs were able to liberate the active alkaloids in
hypoxic tumors by testing them under hypoxic conditions using an anaerobic chamber. The
three prodrugs 5a and their parent alkaloids as well as the reference compounds PR-104A,
–
c
the parent alcohol form of the phosphate pre-prodrug PR-104, and evofosfamide were
tested using the HCT116 and H460 cell lines under both normoxic and hypoxic conditions
(Table 5). The prodrugs were much less cytotoxic than the parent drugs, in agreement with
our observations using the other cell lines described above (Figure 6).
Table 5. Cytotoxicity of compounds 5a–c and their parent alkaloids under normoxia and hypoxia (IC50, nM).
Cell Line HCT116 H460
DR DR
IC50
(Normoxia)
IC50
(Hypoxia)
IC50
(Normoxia)
IC50
(Hypoxia)
HCR
HCR
Compound
5a
16,471.5 ± 11,571
23.9 ± 7
4489.0 ± 698
35.1 ± 15
9055.0 ± 7592
1711.0 ± 965
16,471.5 ± 11,571
23.9 ± 7
424.7 ± 51
16.7 ± 9
1463.3 ± 411
50.1 ± 18
3024.7 ± 656
56.4 ± 35
860.7 ± 122
20.1 ± 3
8.8
0.4
11.3
0.5
1.5
0.6
10.5
85
15,847.0 ± 6339
29.2 ± 12
77,598.3 ± 22,402
107.9 ± 28
51,024.7 ± 22,402
174.9 ± 78
618.0 ± 44
25.4 ± 9
26
0.6
24
543
689
128
543
719
293
(R)-Antofine
5b
3220.0 ± 1071
101.9 ± 32
6500.0 ± 1812
160.9 ± 77
533.0 ± 47
17.2 ± 1
(S)-Tylophorine
5c
1.1
7.8
1.1
2.5
251
(R)-Tylophorine
PR-104A
-
-
1337 ± 386
4322.7 ± 1931
-
-
Evofosfamide
Figure 6. Cytotoxicity of compounds 5a–c and their parent alkaloids under normoxia and hypoxia against (A) HCT116 and
(B) H460 (IC50, nM).

Molecules 2021, 26, 3327
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The extent to which a prodrug is deactivated relative to its parent drug under normoxic
conditions is defined as the Deactivation Ratio (DR). The DR can be simply calculated as
DR = IC50 (prodrug)/IC50 (parent drug). The developed prodrugs 5a–c displayed excellent
DRs ranging between 128 and 719. When tested under hypoxic conditions, the parent
antofine and tylophorine drugs showed similar cytotoxicities to those seen under normoxic
conditions. By contrast, prodrugs 5a
–c showed significantly higher cytotoxicity under
hypoxia than under normoxia (Table 4).
The efficacy of prodrug activation under hypoxic conditions can be expressed by
the Hypoxia Cytotoxicity Ratio (HCR), which can be calculated for a given prodrug as:
HCR = normoxic IC50/hypoxic IC50. Prodrugs 5a–c displayed interesting HCRs ranging
between 1.5 and 26, thereby confirming the selectivity of these new derivatives for hypoxic
tumors (Table 5).
3. Materials and Methods
3.1. Chemistry
(13aR)-2,3,6-trimethoxy-10-((1-methyl-4-nitro-1H-imidazol-5-yl)methyl)-10,11,12,13,13a,
14-hexahydro-9H-dibenzo[f,h]pyrrolo[1,2-b]isoquinolin-10-ium bromide (5a). To a solution
of (R)-antofine (0.17 g, 0.48 mmol) in CHCl3 (30 mL) was added 5-(bromomethyl)-1-methyl-
4-nitro-1H-imidazole (0.16 g, 0.73 mmol), and the reaction mixture was heated at reflux for
20 h under an atmosphere of argon. After the mixture was evaporated under vacuum, the
residue was purified by column chromatography on silica gel (CH2Cl2:CH3OH = 10:1) to
give the quaternary ammonium salt as a yellow solid (yield 52%). Mp:177–179 ◦C; purity:
>99%; 1H-NMR (400 MHz, DMSO-d6)
δ 8.24 (s, 2H), 8.14 (s, 1H), 7.87 (d, J = 9.0 Hz, 1H),
7.53 (s, 1H), 7.33 (d, J = 9.0 Hz, 1H), 5.21 (s, 2H), 5.01 (d, J = 17.1 Hz, 1H), 4.87–4.70 (m, 2H),
4.12 (s, 3H), 4.08 (s, 3H), 4.06 (s, 3H), 3.90–3.45 (m, 7H), 2.50–2.37 (m, 1H), 2.32–2.20 (m, 2H),
1.98–1.80 (m, 1H); 13C-NMR (100 MHz, DMSO-d6)
δ 158.0, 149.7, 149.3, 147.4, 139.8 (2C),
130.5, 124.9, 124.5, 123.9, 122.1, 120.7, 118.3, 115.8, 105.1, 104.8, 104.6, 67.9, 62.6, 56.0, 55.6,
51.5, 50.7, 33.3, 26.8, 24.0, 18.5. HRMS (ESI) calcd for C29H33N3O5 (M-Br)+ 503.2415, found
503.2301. The spectra are joined in the Supplementary Materials.
(13aS)-2,3,6,7-tetramethoxy-10-((1-methyl-4-nitro-1H-imidazol-5-yl)methyl)-10,11,12,13,13a,
14-hexahydro-9H-dibenzo[f,h]pyrrolo[1,2-b]isoquinolin-10-ium bromide (5b). The syn-
thetic procedure was similar to that of compound 5a. Compound 5b was obtained as
yellow solid (0.17 g, 58%). Mp:165–167 ◦C; purity: >99%;1H-NMR (400 MHz, DMSO-d6)
δ
8.11 (s, 1H), 8.10 (s, 1H), 8.07 (s, 1H), 7.45 (s, 1H), 7.21 (s, 1H), 5.13 (s, 2H), 4.99 (d,
J = 17.5 Hz, 1H), 4.76–4.66 (m, 2H), 4.07 (s, 3H), 4.06 (s, 3H), 3.99 (s, 3H), 3.96–3.85 (m,
5H), 3.81–3.69 (m, 2H), 3.69–3.49 (m, 2H), 2.46–2.15 (m, 3H), 1.94–1.80 (m, 1H).13C-NMR
(100 MHz, DMSO-d6) δ 149.9, 149.5, 149.4, 149.3, 147.9, 140.2, 124.5, 124.2, 124.1, 122.9, 121.2,
105.0, 104.93, 104.85, 104.4, 68.2, 63.1, 56.51, 56.48, 56.4, 56.1, 51.9, 51.0, 31.2, 27.2, 24.5, 18.8.
HRMS (ESI) calcd for C30H35N3O6 (M-Br)+ 533.2520, found 533.2403.
(13aR)-2,3,6,7-tetramethoxy-10-((1-methyl-4-nitro-1H-imidazol-5-yl)methyl)-10,11,12,13,
13a,14-hexahydro-9H-dibenzo[f,h]pyrrolo[1,2-b]isoquinolin-10-ium bromide (5c). The syn-
thetic procedure was similar to that of compound 5a. Compound 5c was obtained as yellow
solid (yield 55%). Mp:185-187 ◦C; purity: >99%;1H-NMR (400 MHz, DMSO-d6)
δ 8.11 (s,
1H), 8.10 (s, 1H), 8.08 (s, 1H), 7.45 (s, 1H), 7.20 (s, 1H), 5.13 (s, 2H), 4.98 (d, J = 17.4 Hz, 1H),
4.80–4.62 (m, 2H), 4.07 (s, 6H), 4.00 (s, 3H), 3.96–3.84 (m, 5H), 3.82–3.68 (m, 2H), 3.67–3.42
(m, 2H), 2.46–2.14 (m, 3H), 1.94–1.81 (m, 1H).13C-NMR (100 MHz, DMSO-d6)
δ 149.9, 149.5,
149.4, 149.3, 147.9, 140.2, 124.5, 124.2, 124.1, 122.9, 121.2, 117.9, 104.92, 104.90, 104.8, 104.4,
68.2, 63.1, 56.50, 56.45, 56.4, 56.0, 51.8, 50.9, 49.1, 27.2, 24.4, 18.8. HRMS (ESI) calcd for
C30H35N3O6 (M-Br)+ 533.2520, found 533.2400.
3.2. Physiochemical Properties
Water solubility, logD, and PAMPA-BBB were evaluated using the protocols we previ-
ously described [47].

Molecules 2021, 26, 3327
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3.3. Mouse/Human Plasma Stability Test
Incubations were carried out in 6 aliquots of 70
µ
L each in duplicates. The test
compounds (1
at 100 rpm. Six time points over 24 h (0, 3, 6, 9, 12 and 24 h) have been analyzed. The
reactions were stopped by adding 350 L of acetonitrile with subsequent plasma proteins
µ
M, final DMSO concentration 0.5%) were incubated at 37 ◦C with shaking
µ
sedimentation by centrifuging at 5500 rpm for 5 min. Supernatants were analyzed by
LC-MS using Shimadzu VP HPLC system including vacuum degasser, gradient pumps,
reverse phase column, column oven and autosampler. The HPLC system was coupled with
tandem mass spectrometer API 3000 (PE Sciex, Foster City, CA, USA). Both the positive
and negative ion modes of the TurboIonSpray ion source were used. The acquisition and
analysis of the data were performed using Analyst 1.5.2 software (PE Sciex, API 3000 mass
spectrometer, Foster City, CA, USA). The percentage of the test compounds remaining after
incubation in plasma and their half-lives (T1/2) were calculated.
3.4. Mouse Microsomal Liver Stability Assay
Mouse hepatic microsomes were isolated from pooled, perfused livers of Balb/c male
mice according to the standard protocol [48]. The batch of microsomes was tested for
quality control using imipramine and propranolol as reference compounds. Microsomal
incubations were carried out in 96-well plates in 5 aliquots of 40 µL each. Liver microsomal
incubation medium contained PBS (100 mM, pH 7.4), MgCl2 (3.3 mM), NADPH (3 mM),
glucose- 6-phosphate (5.3 mM), glucose-6-phosphate dehydrogenase (0.67 units/mL) with
0.42 mg of liver microsomal protein per ml. Control incubations were performed by
replacing the NADPH-cofactor system with PBS. The additional control incubations in
PBS (with 3.3 mM MgCl2) without added microsomes were carried out in this study. Test
compound (2
µ
M, final solvent concentration 1.6%) was incubated with microsomes at
◦
37 C, shaking at 100 rpm. The incubations were performed in duplicates. Five time
points over 40 min had been analyzed. The reactions were stopped by adding 12 volumes
of 90% acetonitrile-water to incubation aliquots, followed by protein sedimentation by
centrifuging at 5500 rpm for 3 min. The supernatants were analyzed using LC-MS as
mentioned above.
3.5. Cytotoxicity Assay
The eight cell lines HEK293 (Human embryonic kidney 293), CHO-K1 (Chinese
hamster ovary K1), MCF-7 cells (Human breast adenocarcinoma), HCT116 (human col-
orectal carcinoma), RKO (Human rectal carcinoma), SW480 (human colorectal carcinoma),
H460 (Human lung cancer), and MRC5 (Normal human foetal lung fibroblast), were ob-
tained from the ATCC. The cell lines were routinely grown in alpha minimum essential
medium (
α-MEM) containing 5% foetal calf serum (FCS). The appropriate number of cells
(
HCT116 = 400 cells/well, H460 cells = 600 cells/well) were seeded into 96-well plates
under normoxic or hypoxic conditions. Cells were plated in 100 µL α-MEM containing
10% FCS, 10mM D-Glucose and 0.2mM 20-deoxycytidine. After incubation for 2 h, the
compounds were added at the appropriate concentration and incubated for further 4 h.
The cells were then washed three times with drug-free α-MEM (containing 5% FCS and
pen/strep). The plates were then incubated for 5 days under normoxic conditions. The cells
were stained with sulphorhodamine B to measure total cells [49]. The IC50 was determined
by interpolation as the compound concentration reducing staining to 50% of controls on
the same plate [50].
4. Conclusions
In conclusion, we successfully developed 1-methyl-4-nitro-1H-imidazol-5-yl qua-
ternary ammonium salts of phenanthroindolizidine alkaloids as polar hypoxia-selective
prodrugs. These new derivatives showed no predicted BBB penetration. Thus, these novel
prodrugs should be devoid of neurotoxicity, the main reason for the failure of their parent
alkaloids in clinical trials. The developed polar prodrugs 5a–c showed high chemical

Molecules 2021, 26, 3327
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and metabolic stability and displayed decreased cytotoxicity when tested under normoxic
conditions. However, their cytotoxic activity considerably increased when tested under
hypoxic conditions. These new derivatives represent promising lead compounds that can
be considered for further preclinical evaluation.
Supplementary Materials: Spectroscopic data (1H-NMR, 13C-NMR and HRMS) for compounds
5a–c are available online.
Author Contributions: Conceptualization, Z.O.; methodology, Y.L., Q.W. and O.A.; validation, C.R.,
P.M.F., J.B.S. and A.V.P.; formal analysis, I.A.S.; data curation, C.P.G., L.C. and A.N.A.; writing—original
draft preparation, Z.O. and J.B.S.; funding acquisition, Z.O. All authors have read and agreed to the
published version of the manuscript.
Funding: The authors would like to acknowledge the financial support provided by King Abdulaziz
City for Science and Technology (KACST), Grant No. 13-MED2515-10, and Batterjee Medical College,
Grant No. B-RES-2020-0028.
Institutional Review Board Statement: The study was conducted according to the guidelines of
the Declaration of Helsinki, and approved by the Institutional Ethics Review Board of the College
of Pharmacy at Umm AlQura University, (protocol code UQU-COP-EA # 143915 and date: 15
October 2017).
Data Availability Statement: The data presented in this study are available in this article.
Conflicts of Interest: The authors declare no conflict of interest.
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