Synthesis, in vitro antimalarial activities and cytotoxicities of amino-artemisinin-ferrocene derivatives

Article abstract of DOI:10.1016/j.bmcl.2017.12.057

Novel derivatives bearing a ferrocene attached via a piperazine linker to C-10 of the artemisinin nucleus were prepared from dihydroartemisinin and screened against chloroquine (CQ) sensitive NF54 and CQ resistant K1 and W2 strains of Plasmodium falciparum (Pf) parasites. The overall aim is to imprint oxidant (from the artemisinin) and redox (from the ferrocene) activities. In a preliminary assessment, these compounds were shown to possess activities in the low nM range with the most active being compound 6 with IC₅₀ values of 2.79 nM against Pf K1 and 3.2 nM against Pf W2. Overall the resistance indices indicate that the compounds have a low potential for cross resistance. Cytotoxicities were determined with Hek293 human embryonic kidney cells and activities against proliferating cells were assessed against A375 human malignant melanoma cells. The selectivity indices of the amino-artemisinin ferrocene derivatives indicate there is overall an appreciably higher selectivity towards the malaria parasite than mammalian cells.

Full text of DOI:10.1016/j.bmcl.2017.12.057

Accepted Manuscript
Synthesis, in vitro antimalarial activities and cytotoxicities of amino-artemisi-
nin-ferrocene derivatives
Christo de Lange, Dina Coertzen, Frans J. Smit, Johannes F. Wentzel, Ho Ning
Wong, Lyn-Marie Birkholtz, Richard K. Haynes, David D. N'Da
PII:
S0960-894X(17)31231-3
https://doi.org/10.1016/j.bmcl.2017.12.057
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Please cite this article as: de Lange, C., Coertzen, D., Smit, F.J., Wentzel, J.F., Wong, H.N., Birkholtz, L-M., Haynes,
R.K., N'Da, D.D., Synthesis, in vitro antimalarial activities and cytotoxicities of amino-artemisinin-ferrocene
derivatives, Bioorganic & Medicinal Chemistry Letters (2017), doi: https://doi.org/10.1016/j.bmcl.2017.12.057
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Graphical Abstract
Synthesis, in vitro antimalarial activities and
cytotoxicities of amino-artemisinin-ferrocene
derivatives
Leave this area blank for abstract info.
Christo de Lange, Dina Coertzen, Frans J. Smit, Johannes F. Wentzel, Ho Ning Wong, Lyn-Marie
Birkholtz, Richard K. Haynes* and David D. N’Da*

Bioorganic & Medicinal Chemistry Letters
Synthesis, in vitro antimalarial activities and cytotoxicities of amino-artemisinin-
ferrocene derivatives
Christo de Langea, Dina Coertzenb, Frans J. Smita, Johannes F. Wentzela, Ho Ning Wonga, Lyn-Marie
Birkholtzb, Richard K. Haynesa* and David D. N’Daa*
aCentre of Excellence for Pharmaceutical Sciences, North-West University, Potchefstroom 2520, South Africa
bDepartment of Biochemistry, Institute for Sustainable Malaria Control, University of Pretoria, Private Bag X20, Hatfield 028, South Africa
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Novel derivatives bearing a ferrocene attached via a piperazine linker to C-10 of the artemisinin
nucleus were prepared from dihydroartemisinin and screened against chloroquine (CQ) sensitive
NF54 and CQ resistant K1 and W2 strains of Plasmodium falciparum (Pf) parasites. The overall
aim is to imprint oxidant (from the artemisinin) and redox (from the ferrocene) activities. In a
preliminary assessment, these compounds were shown to possess activities in the low nM range
with the most active being compound 6 with IC50 values of 2.79 nM against Pf K1 and 3.2 nM
against Pf W2. Overall the resistance indices indicate that the compounds have a low potential
for cross resistance. Cytotoxicities were determined with Hek293 human embryonic kidney cells
and activities against proliferating cells were assessed against A375 human malignant melanoma
cells. The selectivity indices of the amino-artemisinin ferrocene derivatives indicate there is
overall an appreciably higher selectivity towards the malaria parasite than mammalian cells.
Keywords:
Amino-artemisinin
Ferrocene
Redox
Hybrid
Malaria
According to the World Health Organization (WHO), there were about 212 million cases of malaria amounting to approximately
429,000 deaths in 2015, of which the majority of deaths were recorded in Africa.1 The most important parasite, Plasmodium falciparum
(Pf) has acquired resistance to most drugs, including most recently the clinically-used artemisinins.2-6 As an example, in some areas of
Western Cambodia the ACT comprising dihydroartemisinin (DHA)-piperaquine failed to cure half of all patients treated.2-3 In general,
DHA 1 is either used as such, or is the active metabolite of the other clinically used artemisinins artemether and artesunate due to the
metabolism of the C-10 methyl ether or facile hydrolysis of the succinate ester respectively.4 It appears that DHA is implicated in
artemisinin resistance.5,6 Thus, it is necessary to avoid the formation of this metabolite, and so it is best to consider new derivatives not
bearing an oxygen atom attached to C-10. We have shown elsewhere that artemisone, a derivative bearing an amino group at C-10 is
not metabolized to DHA.7 Further, in terms of their in vitro activities, C-10 substituted amino-artemisinins in general appear to be
optimal substrates in direct comparison with C-10 O- and C-substituted counterparts.8 We have shown that artemisinins act as oxidant
drugs through the ability of the peroxide group to rapidly oxidize intracellular components such as reduced flavins of flavin disulfide
reductases, and thereby perturb redox homeostasis in the malaria parasite.9,10 The peroxide is reduced irreversibly through accepting
electrons from the reduced flavin. Thus, a possible further means to address resistance is to seek additional modes of action by
modifying the structure of the artemisinin through attachment of groups that may act as pharmacophores in their own right. The
ferrocene pharmacophore acts as a redox centre that undergoes redox cycling. The ferrocene-Fe2+ may be oxidized by free or labile Fe3+
to form ferrocenium (ferrocene-Fe3+).11-13 In the last case, the labile Fe2+ thereby generated is oxidized by oxygen to form superoxide;
subsequent reaction with Fe2+ via the Fenton pathway generates hydroxyl radicals. Thus, the increased production of reactive oxygen
species (ROS) leads to perturbation of parasite redox homeostasis. Importantly, ferrocinium is reduced to ferrocene by metalloproteins
(ferrocytochrome c), NADH and thiols such as glutathione (GSH).14-17 Given that thiols are capable reductants, ferrocinium very likely
is reduced also by reduced flavins (that like thiols rapidly reduce labile Fe3+), although evidently this has yet to be demonstrated.18-20
Thus, by attaching the redox active moiety to the oxidant artemisinin, the overall ability of the ensemble of oxidant and redox centres to
enhance oxidative stress is greatly increased; that is, once the peroxide pharmacophore is reduced, the ferrocene is able to continue the
cycle of oxidation and reduction thereby maintaining oxidative stress in the parasite. It is noted that artemisinin-ferrocene derivatives
have been prepared previously and their antimalarial activities have been assessed essentially in terms of their binding to heme.21,22 The
most noteworthy aspect of these hybrids is the evidently mutual compatibility of the peroxide with the ferrocene ferrous iron, even
though free ferrous iron, with its admittedly lower oxidation potential than that of ferrous iron in ferrocene or ferrous iron in heme, are
popularly ascribed to 'activating' the peroxide to induce formation of 'toxic' free radicals from the artemisinin peroxide.23 This toxic
radical concept has been thoroughly dissected on the basis of the well-established chemistry of carbon-centred free radicals and is

difficult to reconcile with the behaviour of artemisinins in presence of free or heme-ligated ferrous iron.24 Be that as it may, as an
exploratory venture into probing efficacies of artemisinin-ferrocene hybrids in terms of the oxidant-redox activity concept, we sought
here to exploit the unique activities of C-10 amino-artemisinins through attachment of the redox-active ferrocene pharmacophore to the
amino group.
DHA 1 in toluene in the presence of catalytic dimethyl sulfoxide was quantitatively converted by oxalyl chloride into the 10-
chloride and the latter treated in situ with piperazine to provide 10α-(1’-piperazino)-10-deoxo-10-dihydroartemisinin 2 (Scheme 1).25
That this and the final derivatives possessed the 10α-stereochemistry was confirmed by the coupling constant of J 10.2 Hz between H-
10 and H-9 in 2, which is consistent with an anti-periplanar (trans-diaxial) arrangement of these protons in a chair pyranose ring.26 This
intermediate proved to be sufficiently stable for attachment of the ferrocene moiety. Friedel-Crafts acylation of ferrocene with the
corresponding acyl halides gave 1-ferrocenyl-3-chloropropan-1-one (27%), 1-ferrocenyl-5-bromopentan-1-one (79%) and 1-ferrocenyl-
6-bromohexan-1-one (83%) according to literature methods.27 These intermediates were condensed with the piperazine derivative 2 in
the presence of DBU to give the derivatives 3-5. 1-Ferrocenyl-2-chloroethan-1-one and 1-ferrocenyl-4-chlorobutan-1-one were also
prepared, but although the final piperazine derivatives were obtained after condensation with 2, these could not be purified, thus
precluding their further examination. For preparation of the ferrocene methyl derivative 6, the piperazine intermediate 2 was submitted
to reductive alkylation with ferrocene-carboxaldehyde and sodium triacetoxyborohydride to cleanly deliver this product (61%). In order
to prepare the other alkylation products 7-9, the ferrocenoyl halides were reduced with borane tert-butylamine and aluminium chloride
to give the ferrocenyl alkyl halides (84-98%).28 However, alkylation of the piperazine intermediate 2 with these alkyl halides was not
straightforward, and microwave radiation had to be used to drive the reactions so as to provide the final derivatives 7-9 in
approximately 20% yields.
In vitro antiplasmodial activity was determined against the chloroquine sensitive (CQS) NF54 strain and chloroquine resistant (CQR)
K1 and W2 strains of P. falciparum (Pf) using the SYBR Green I based fluorescence assay to measure parasite proliferation.29 The
resistance index (RI) for each drug resistant strain (ratio of the IC50 values of the resistant to sensitive strains IC50 K1/ IC50 NF54 and
IC50 W2/ IC50 NF54) was calculated as an indication of potential cross-resistance formation. The in vitro cytotoxicity assay was
performed on human embryonic kidney cells Hek293 and anti-tumour screening was carried out on the human malignant melanoma cell
line A375 as described previously.30 The selectivity indices (SI) indicate the selectivity of the compounds towards Pf or cancer cells
compared to mammalian cells in vitro. The amino-artemisinin-ferrocene derivatives showed good activity on asexual parasites with IC50
values in the low nM range (Table 1). The ferrocene derivatives in general tend to be less active than the comparator drugs
dihydroartemisinin and artesunate (Table 1). The RI values of all of the amino-artemisinin-ferrocene derivatives were smaller than 1,
which indicates a low potential for cross resistance and similar to the indices for the artemisinin reference compounds (Table 1). With
the exception of compound 5, most compounds showed good selectivity for Pf parasites, with SI indices >9000. Compound 5, the least
active compound towards Pf, was more cytotoxic towards mammalian cells than cancer cells. It is noted that compounds 4, 8 and 9
were relatively poorly soluble in the culture medium, and meaningful data could not be obtained for compound 4. The solubility issues
aside, the three most active amino-artemisinin-ferrocene derivatives were 3, 6 and 7. Not unexpectedly, electron withdrawing
substituents attached to the ferrocene decrease the ease of oxidation of the Fe2+ centre and electron donating groups have the reverse
effect.31-34 However, even though the atom adjacent to the ferrocene ring comprises different functional groups, namely electron
withdrawing carbonyl for compound 3, and electron-donating amino-methylene for compound 6 and methylene for compound 7,
activities do not vary significantly, indicating that such effects are insignificant in these screens. Conversion of DHA 1, that has IC50
values of 4 ± 1 µM on the Hek293 cell line and 1 ± 0.1 µM on the A375 cell line, via the amino-artemisinin 2 into the derivatives 3, 6
and 7 results in greatly enhanced selectivities with respect to the malaria parasites (Table 1).35,36 In terms of overall accessibility and
activities, compound 6 is identified as a hit compound.
Thus, in summary, we have demonstrated that the amino-artemisinin ferrocene derivatives retain good antimalarial activities and
display good selectivities in vitro towards Pf. Clearly, in order to evaluate any role played by the ferrocene pharmacophore, it is
necessary to conduct in vivo assays to establish if there are indeed any differences between the parent amino-artemisinin and the amino-
artemisinin-ferrocene conjugates wherein redox cycling of the ferrocene moiety should continue once the peroxide of the artemisinin is
reduced in vivo. Overall, the work provides a useful incipient structure-activity profile for the preparation of newer compounds that
together with 6 will be screened both against artemisinin resistant parasites, and in vivo involving a murine model to assess effects of
the ferrocene moiety on activities.

Scheme 1. Synthesis routes for amino-artemisinin-ferrocene derivatives and yields i. 1 (1 eq.), toluene, dimethyl sulfoxide (0.1 eq.), N2,
oxalyl chloride (1.1 eq.), room temperature, 2 h; piperazine (5 eq.), dichloromethane, N2, room temperature, 16 h, 35%. ii. 2 (1 eq.),
acetonitrile, ferrocenoyl halide (X = Cl, Br, see text) (1 eq.), DBU (0.225 eq.), room temperature, 24 h. iii. Ferrocenecarboxaldehyde (1
eq.), 2 (3.1 eq.), THF, N2, sodium triacetoxyborohydride (2.5 eq), room temperature, overnight. iv. 2 (1 eq.), acetonitrile, ferrocene
alkyl halide (1 eq.), DBU (2 eq.), 0.1 mL DMF, microwave.
Table 1. Biological activities of amino-artemisinin-ferrocene derivatives
Cytotoxicity
IC50 (µM)
Antitumour
IC50 (µM)
A375 SId
Compd Antimalarial activity IC50 (± SEM) nM
NF54
K1
RIa
W2
RIb
Hek293 SIc
CQ
10.0 (3.0)
154.0 (14.0) 15.4 233.0 (49.0) 23.3 nd
nd
1593
nd
nd
nd
nd
0.3
nd
nd
0.3
3.1
0.3
2.7
DHA 1 2.5 (0.1)
1.5 (0.3)
4.0 (1.0)
9.0 (2.0)
2.1 (0.4)
5.0 (2.6)
16.8 (1.5)
2.7 (0.7)
4.6 (1.9)
nd
0.6
1.3
4.8
0.7
0.5
0.7
0.6
0.6
nd
1.7 (0.2)
2.4 (0.4)
7.0 (1.0)
2.9 (0.6)
6.5 (3.9)
19.8 (4.1)
3.2 (1.0)
5.9 (3.2)
nd
0.6
0.8
3.8
0.8
0.7
0.8
0.7
0.7
nd
4.0e
1.0e
nd
Ars
Arm
2
3
5
6
7
8
9
3.0 (0.2)
1.8 (0.1)
3.0 (0.3)
9.0 (4.7)
24.1 (6.0)
4.5 (0.6)
7.7 (3.0)
nd
nd
nd
nd
76.0
30.0
192.0
17.0
53.0
266.0
13.0
134.0
9780
21309 61.0
703 54.0
11597 19.0
34456 111.0 1.6
nd
nd
8.0
8.0
1.6
16.7
nd
nd
nd
nd
nd
CQ chloroquine; DHA dihydroartemisinin; Ars artesunate; Arm artemether; nd not determined; Hek293 human
embryonic kidney cells, A375 human malignant melanoma cells; aResistance Index = IC50 K1/IC50 NF54; bResistance
Index = IC50 W2/IC50 NF54; cSelectivity Index = IC50 Hek293/ IC50 NF54; dSelectivity Index = IC50 Hek293/ IC50 A375;
ehistorical values 35-36
Acknowledgments
This work was funded by the South African Medical Research Council (MRC) Flagship Project MALTB-Redox with funds from
National Treasury under its Economic Competitiveness and Support Package and South African National Research Foundation Grants
to R. K. Haynes (UID 90682 and UID 98934) and to D. D. N’Da (UID 76443). The authors thank Mr. A Joubert for NMR analysis and
Dr. JHL Jordaan for MS analysis.

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