Artemisinin-Derived Dimers: Potent Antimalarial and Anticancer

Mar 24, 2016 - S. B. Tsogoeva at the University of Erlangen-Nuremberg. His research focuses on artemisinin-derived natural product hybrids as potent a...
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Artemisinin-Derived Dimers: Potent Antimalarial and Anti-Cancer Agents Tony Fröhlich, Aysun Çapc# Karagöz, Christoph Reiter, and Svetlana B. Tsogoeva J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01380 • Publication Date (Web): 24 Mar 2016 Downloaded from http://pubs.acs.org on March 25, 2016

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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Medicinal Chemistry

Artemisinin-Derived Dimers: Potent Antimalarial and Anti-Cancer Agents Tony Fröhlich, Aysun Çapcı Karagöz, Christoph Reiter and Svetlana B. Tsogoeva* Department of Chemistry and Pharmacy, Organic Chemistry Chair I and Interdisciplinary Center for Molecular Materials (ICMM), University of Erlangen-Nürnberg, Henkestrasse 42, 91054 Erlangen, Germany. Dedicated to Professor Gary H. Posner on the occasion of his 75th birthday

ABSTRACT: The development of new efficient therapeutics for the treatment of malaria and cancer is an important endeavor. Over the past 15 years, much attention has been paid to the synthesis of dimeric structures, which combine two units of artemisinin, as lead compounds of interest. A wide variety of atemisinin-derived dimers containing different linkers demonstrate improved properties compared to their parent compounds (e.g. circumventing multidrug resistance), making the dimerization concept highly compelling for development of efficient antimalarial and anti-cancer drugs. The present Perspective highlights recent developments on different types of artemisinin-derived dimers and their structural and functional features. Particular emphasis is put on the respective in vitro and in vivo studies, exploring the role of the length and nature of linkers on the activities of the dimers,

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and considering the future prospects of the dimerization concept for drug discovery.

1. Introduction Artemisinin was first extracted from the Chinese medicinal plant Artemisia annua L. (sweet wormwood) in 1972 by Youyou Tu (Nobel Prize 2015).1 It was used for centuries to treat fever, including malaria-related fever.2-5 It is an enantiomerically pure sesquiterpene containing a 1,2,4-trioxane ring, which can be regarded as a combination of hemiketal, hemiacetal and lactone functions (Figure 1).6-13 In order to improve the pharmacological properties of artemisinin, several semisynthetic derivatives were developed: dihydroartemisinin, artesunic acid and artemether. All three artemisinin derivatives proved to be highly active against malaria parasites and different cancer cell lines like leukemia, colon cancer, and others.8,1419

Artemisinin and sodium artesunate, which is the water-soluble salt of artesunic

acid, were recommended by the World Health Organization (WHO) to be administered in combination with classical antimalarial drugs for reliable chemotherapy of humans (Artemisinin-based Combination Therapy - ACT), while WHO strongly discourages use of artemisinin monotherapy due to resistance development issues.20,21 Although the mechanism of action of artemisinin is still

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not completely understood, it is generally accepted that the endoperoxide linkage within the 1,2,4-trioxane system is essential for its antimalarial activity.22,23

Figure 1. Structures of artemisinin and its semi-synthetic derivatives

The majority of fatal malaria infections in humans are caused by the protozoan parasite Plasmodium falciparum, which infests erythrocytes of its hosts.11,12,24 The parasite uses the hemoglobin of erythrocytes as a source of amino acids. The enzymatic degradation of the hemoglobin takes place in the food vacuoles of the Plasmodium parasite. During this digestion process, heme-iron is released, which can react with the endoperoxide moiety of artemisinin and consequently induces its cleavage via Fe(II) Fenton reaction.25 Generated reactive oxygen species (ROS) and carbon-centered radicals are widely accepted as key intermediates responsible for the antimalarial activity of artemisinin and its derivatives.26-28

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It is proposed that the activity of artemisinin towards tumor cells is based on a mechanism

similar

to

the

one

proposed

for

antimalarial

activity

(Scheme 1).13,22,24,25,29-33

Scheme 1. Proposed pathway for activation of artemisinin by Fe(II)

The advantage of artemisinin and its semi-synthetic analogue artesunic acid as anti-cancer agents lies not only in their potency as toxic agents to cancer cells, but also in their low toxicity to normal cells.34,35 As tumor cells contain more Fe(II) ions than cells of healthy tissue, oxidative cleavage of the endoperoxide bridge of artemisinin by intracellular iron Fe(II) occurs, which leads to the formation of peroxyl free radicals and ROS and induction of oxidative stress, DNA damage, alkylation of target proteins and apoptosis takes place selectively in these cells (Scheme 1). Artemisinin’s mode of action against cancer involves the inhibition of metastasis,36 cancer-related signaling pathways,37-39 and angiogenesis.40,41 Although artemisinin and its simple derivatives have been utilized for the treatment of malaria and cancer for a long time, the emergence of resistance to this class of compounds and the fact that the factors leading to this resistance are still ACS Paragon Plus Environment

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largely unknown motivates the search for new artemisinin-based agents.21,42 Very recently, the situation has become more dire because even the therapy with artemisinin in combination with other drugs (ACT) fails in strains of malaria rampant in several Asian countries.43,44 A promising approach to circumvent multidrug resistance is the synthesis of novel artemisinin-derivatives using the technique of hybridization or dimerization,45-50 as well as coupling artemisinin to other synthetic 1,2,4-trioxane derivatives.51,52 Thus, a huge variety of 1,2,4-trioxane-derived dimers are known to be more active than the corresponding monomers.53 Despite several excellent general reviews on artemisinin and its derivatives,6-9,11-13 up to now a review focused on artemisinin-derived dimers is lacking in the literature. In this Perspective, we address the emerging and highly attractive dimerization approach, and discuss recent advancements in developing artemisinin-derived dimeric antimalarial and anti-cancer agents. Results published from 1997 until the end of 2015 are presented. Particular emphasis is put on the nature and length of the linkers for dimer design, the in vitro and in vivo activities of the most promisinig dimers, their advantages over single drugs, and their potential to address important issues relating to drug resistance. The actual and potential advantages and disadvantages of 1,2,4-trioxane-derived dimers in relation to established drugs are also discussed.

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The different contributions on artemisinin derived dimers have been organized into sections according to the linker types (symmetric or nonsymmetric, general methods for their preparation), and their corresponding biological activities (antimalarial or anti-cancer).

2. Different synthetic approaches to artemisinin-derived dimers 2.1 Dimers obtained via reaction between Artemisinin-derivatives and symmetric or nonsymmetric linker molecules A straightforward method in order to obtain artemisinin-derived dimers is to couple two molecules of the artemisinin-derivative, acting either as nucleophile (e.g. alcohol or amine) or electrophile (e.g. bromide or carboxylic acid), with different linker molecules, which bear functional groups exhibiting opposite reactivity that of the artemisinin-derivative. Depending on the linker molecule used (symmetric or nonsymmetric), it is possible to synthesize either symmetric (Figures 2 and 3) or nonsymmetric (Figure 4) dimers via this general method as shown in Scheme 2. Starting from 1999 until 2015 the following symmetric artemisinin-derived dimers have been synthesized utilizing the method described above (Scheme 2): C-10

non-acetal

dimers 1a-c

and

2a-d

(Figure 2),54,55

C-16

substituted

artemisinin-derived dimers 3a-f’,56 olefin dimers 457 and 5,58 C-10 non-acetal

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dimers 6a-f, 7a/b and 8a/b,59 phosphate dimers 8c-h, bipiperidine-linked dimer 9, carbonate-linked dimer 10a and carbamide-linked dimer 10b,60 dimers 11a-c, 12a-e and 13,61,62 dimers 14a-d (Figure 3) with two trioxane moieties linked to an aminoquinoline entity,63 artesunic acid homodimers 15a-d,64,65 dihydroartemisinin ether dimers 16a-e,66 C-10 non-acetal dimer 17,67 amide-linked dimer 18 containing a piperazine moiety,68 triazole-linked dimers 19a-e and 20a/b,69 C-10 non-acetal dimers 21a-d and 22a-d,70 dimers 23 and 24 containing ferrocene as linker.71

Scheme 2. General equation for the synthesis of dimers with symmetric or nonsymmetric linkers (with selected examples)

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H

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H

O O O

O O O

O

O

H

O

H

O O O

H

O

O O O

O

H

O O

O O O

O

X

X

X n

O trans, trans: 1a trans, cis: 1b cis, cis: 1c

a) X = S; n = 2-5: 3a-d b) X = CH2; n = 2, 3: 3e/f ( 3e'/f' ( )

para: 2a meta: 2b X=

O

O

O O ),

para: 2c ( ), 2c' ( ), 2c'' ( meta: 2d H

H

H

O O

O O O

O

O

O

H

H

O

O

O

O

O

O O O

O

O O

H

O O O

O

O

O

O O n

5

H

H

O O O

O O O

O

O O

O H

O n

O

O X

H

O O O

O n

O

H

H

O O O

O O O

O

O N

n = 2; X = OMe: 8a, X = OPh: 8b, X = Me: 8c n = 1; X = OMe: 8d, X = OPh: 8e n = 3: X = OMe: 8f, X = OPh: 8g, X = Me: 8h

O

H

O

O P O O n X

O

n = 2: 7a n = 3: 7b

O O O

O

O

O O

O

H

H

O

O O O

X

O

n ortho; n = 1: 6a, n = 0: 6d meta; n = 1: 6b, n = 0: 6e para; n = 1: 6c, n = 0: 6f

4

O O

N 9

H

H

O O

O

X

O O

X = O: 10a X = NH: 10b

O O

O

O

O

O

O O O

13 O 11b

11a

cis: 12a; trans: 12b

X= OH 11c

n n = 1; , : 12c; , : 12c' n = 2; , : 12d n = 3; , : 12e

Figure 2. Structures of 1,2,4-trioxane dimers 1-13 connected via symmetric linkers

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H

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H O O

H

O O O

O

H

O

O O O

O

O

O O

H O O O O

X

O

N H

NH O

X

H

O

O

O

N H

H

O O O

O

O O O

O

O O

Cl

N N

X = -(CH2)nN-; n = 2: 14a n = 3: 14b n = 4: 14c

n

X=

X = -O(CH2)2O-; , : 16a; , : 16b; , : 16c -(CH2)4-; , : 16d -S-S-; , : 16e

X=

N

OH

Br

15b

14d

H

15c

H

O O O

O

O O O

O

O

O

O

O

H

O

O N H

O OO

N N

O O

H

O

H N O

O 17

18

H

H O O O

O O O

O

O O n

N N N

X

N N N

m X=

n = 1, m = 3: 19a; n = 1, m=6: 19b n = 1, m = 7: 19c n = 2, m = 6: 19d; n = 2, m = 7: 19e n = 1: 20a n = 2: 20b

O n

H

S

H

O O O

O O O

O

O O

n = 1: 21a; n = 2: 22a n = 1: 21b; n = 2: 22b X= O

n

n X

n = 1: 21c; n = 2: 22c

n = 1: 21d; n = 2: 22d

H

H

O O O

O O

O

X

n = 1, 6: 15a/d

O

O

O O

O O

Fe

O

O O

O O

H

O O Fe

O O

23

24

O

O O O H

Figure 3. Structures of 1,2,4-trioxane dimers 14-24 connected via symmetric linkers

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Besides dimers connected by symmetric linkers, the same method was applied to prepare the following nonsymmetric dimers with nonsymmetric linkers: dihydroartemisinin acetal dimers 25-30 (Figure 4)61,62 and artesunic acid dimer 31 containing betulin as linker, which is a natural product and is known for its anti-cancer activity toward human lung cancer cells.64 The biological activities of these dimers will be discussed in the following sections: section 3.1 (antimalarial) and section 4.1 (anti-cancer).

Figure 4. Structures of dimers 25-31 connected via nonsymmetric linkers

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2.2 Dimers accessible via direct reaction of two artemisinin derivatives Another route for the synthesis of 1,2,4-trioxane-derived dimers is direct reaction (Friedel-Crafts condensation, esterification, four-component aza-Wittig reaction, copper-mediated Huisgen 1,3-dipolar cycloaddition, click chemistry, Ugi reaction etc.) of two artemisinin-derivatives without the use of a separate linker molecule. The linker, which is present in the final dimer product, is derived from the functional groups of the employed artemisinin-derivatives (Scheme 3).

Scheme 3. General equation for the synthesis of dimers accessible via direct reaction of two artemisinin-derivatives

These linkers can be either symmetric or nonsymmetric depending on the applied reaction type and the type of functional groups present in the starting materials. Further

distinction

can

be

made

between

reactions,

where

one

artemisinin-derivative is reacting with itself (e.g. dihydroartemisinin) or where two different artemisinin-derivatives (e.g. amine and acid chloride) are reacting with ACS Paragon Plus Environment

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each other forming the dimer (Scheme 3). This method allows a great variety of dimers to be synthesized, which would not be accessible by directly reacting linker molecules with artemisinin derivatives, Using direct reaction of two different artemisinin-derivatives, the groups of Posner, Thebtaranonth, O’Neill, N’Da, Woerdenbag, Pras, Jung, Gong, Barua, Chancharunee, Sasaki were able to synthesize the following dimers (Figure 5): dimer 32 containing an aromatic methoxy unit as linker, furan-linked dimer 33,54 dimers linked on C-16 position 34a-g,56 amide-linked dimer 35,60 amine-linked triazine dimers 36a-f,72 ether dimers 37a/b,73 amide dimers 38a/b and 39 containing a free amine unit, thioether dimer 40,74 artemisinin-guanidine dimers 41a-h,75 dimers 42a/b containing a triazole unit as linker,69 N-protected amino acid ester dimers 43a-e,76 amine-linked dimers 44a-f.77 The antimalarial and anti-cancer activity of these dimers will be discussed in sections 3.2 and 4.2.

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Figure 5. Structures of dimers 32-44

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2.3.1 Dimers obtained via post-modification of olefin 4 Another method for obtaining novel artemisinin-based dimers is to post-modify already synthesized dimers. For example in 2003 Posner’s group published a very important synthetic route, where artemisinin was converted in two steps into olefin dimer 4, which was used as starting material for the synthesis of many different dimers (Scheme 4/Figure 6).57 The modification steps leading to these dimers range from different types of oxidation reactions including epoxidation and etherification reactions to various esterification reactions (Scheme 4). One advantage of this method is that the properties of already very active dimers can be easily altered and improved. Since 2003, the following artemisinin-derived dimers have been synthesized using olefin dimer 4 (Figure 2) as precursor: dimers 45a-i (Figure 6),57 carboxylic acid dimer 46 and isonicotinate N-oxide dimer 47,78 dimers 48a-f,79 hydrazine dimer 49a, ketal dimer 49b-i, ether dimer 49k, ester dimers 49j, l and m, amide dimers 49n-s and oxadiazoles 49t/u,80 silylamide trioxane dimer 49v,81 fluorescent dansyl trioxane dimer 50b and coumarin trioxane dimer 50a,82 sulfone trioxane dimers 51a-f,83 phosphate and thiophosphate ester dimers 52a-j,84,85 dimers containing a pyridine moiety 53a and 53b (Figure 6),86 dimer 54 (Figure 6), which is very similar in structure to dimer 53b,87 dimer 55,88 piperazine dimers 56a-d89 and ferrocene containing dimer 57.71 The biological activity of these dimers (Figure

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6) will be discussed in the following paragraphs: section 3.3.1 (antimalarial) and section 4.3.1 (anti-cancer).

Scheme 4. Selected examples for post-modifications starting from olefin dimer 4

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Figure 6. Structures of dimers 45-57 derived from olefin 4

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2.3.2 Dimers obtained via post-modification of miscellaneous other artemisinin-derived dimers Besides olefin 4, other dimers were also post-modified using different reaction types such as oxidation, epoxidation, Bingel-Hirsch reaction, fluorination and oxime formation, just to name a few examples (Scheme 5). O

A

A

O

A

O 60c .

A S O

S O

O 66b

OH

A

O O

A OH

O epoxydation of dimer 11a

oxidation of thioether

60a oxidation of dimer 11a

H O O = O O

A

A

Linker

oxime formation using dimer 11c

fluorination of dimer 2c Bingel-Hirsch reaction using dimer 17

A

A F

F

F 58a/b

A

A

A O

F

A

A

O

O

O

HO

O N 60f

O C60 67 .

Scheme 5. Selected examples for post-modifications of miscellaneous artemisinin-derived dimers

Between 2002 and 2009, using the synthesis method described above (Scheme 5) the groups of Posner, Elsohly and Jung prepared the following post-modified ACS Paragon Plus Environment

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dimers: 58a/b,55 59a-e,58 60a-f,61 61a-c,90 62b-e,91 63a-k,92 64a-p,93 65a-o,94 sulfurlinked dimers 66a/b74 and fullerene dimer 67 (precursor: dimer 17)67 (Figure 7). The in vitro and/or in vivo antimalarial activity of these post-modified artemisinin-derived dimers, as well as their anti-cancer activity are discussed in the following paragraphs: Antimalarial (section 3.3.2) and Anti-Cancer (section 4.3.2).

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H

H

H O O O

O O O

O

O

H

O

O O

O O O

O

O

O O

O

O

H

H

O

O O

X

O

O

O HO

F

F

F

F

O

OH R

, : 58a , : 58b

HO

R

O

O R = -CO2Me: 59a R= -CO2H: 59b -CH2OH: 59c -OP(O)(OEt)2: 59d O

H O O O

O

OH

O O

O

X=

O

60a

P O

HO O 60e

60b

O

OPh 59e

O S O O 60d

H 60c

O

HO

N

60f

O O

H

O

O O O

O O

O

R1 R1 = NO2, R2 = H: 61a R1/R2 = NO2: 61b R1 = SO2Me, R2 = H: 61c

Art

OH H HO

Art

H

O

O

R2

H

O O

R

H O

R = H: 62b R = Ph: 62c R = (CH2)3Br: 62d R = (CH2)3S(O2)Ph: 62e

H OH 62a

O O O

H O O O

H O

O O

H

O

O O

O

H

O O

O X

X = OH: 64a (R); 64b (S) X X = O 63l X=

R = H: 63a R = Me: 63b/b' (Z/E-isomer) R = CH2CH=CH2: 63c R= R = CH2-c-Pr: 63d R = i-Bu: 63e R = t-Bu: 63f

HN Ar

63g NMe2

OMe 63j P OMe O OEt P OEt 63k O

63h

O 63i O H

H

H

O O

O O O

O

O

Y = SO2: 66a,

O

Y S O

O

S O

66b

Ar = Ph: 64c Ar = Ph-3-F: 64d Ar = Ph-4-F: 64e Ar = Ph-3-Me-4-F: 64f Ar = Ph-3,4-diF: 64g Ar = Ph-3,5-diF: 64h Ar = Ph-3-CF3: 64i

H

O O O

O O O

O

O

O

O

O O

O

X= O

N OR (Z-isomer)

O C60 67

X=

Ar = Ph-4-CF3: 64j Ar = PH-3-Cl-4-F: 64k Ar = Ph-3-Cl: 64l Ar = Ph-4-Cl: 64m Ar = Ph-4-Br: 64n Ar = Ph-4-CN: 64o Ar = Ph-4-NO2: 64p

O O Y R

Y = O: R = -CCH2CCH: 65a R = -CH2CHCH2: 65b R = -CH2CH2F: 65c R = -CH2Cl: 65d R = -CH2CH2Cl: 65e R = -CH2Ph: 65f R = -CH2Ph-4NO2: 65g R = -Ph: 65h R = -Ph-4-Me: 65i

Y = O: R = -Ph-4-F: 65j R = -Ph-2-Cl: 65k R = -Ph-4-Cl: 65l Y = S: R = -CH2CH3: 65m R = -C(CH3)3: 65n R = -Ph: 65o

Figure 7. Structures of dimers 58-67 derived from miscellaneous artemisinin derivatives

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3. Antimalarial activities 3.1.1 Antimalarial activity of dimers with symmetric linkers In the following, antimalarial activities of dimers 1-15, 23 and 24 connected by symmetrical linkers prepared by the first method (Figures 2 and 3) are discussed. In general, it can be said, that all dimers 1-15, 23 and 24 exhibit high antimalarial activities against both chloroquine-resistant (K1, Dd2, W2) and chloroquine-sensitive (3D7, NF54, HB3, D6) strains of Plasmodium falciparum (P. f.). This is important as chloroquine, which is one of the most widely used antimalarial drug, in many cases is no longer effective due to upcoming drug resistance. For all synthesized compounds the IC50 values are within the nM range (0.04-205.42 nM) and are comparable or in many cases even superior to the IC50 values of either established drug chloroquine (9.8-744 nM) or artemisinin monomer (6.6-42.5 nM) and their derivatives: dihydroartemisinin (2.09-14.8 nM) and artesunic acid (0.82 nM) (Table 1). Especially, phosphate-linked dimers 8 a-c and 8 g/h, which show IC50 values up to 0.04 nM against the chloroquine-resistant K1 strain and up to 0.09/0.21 nM against the chloroquine-sensitive HB3/3D7 strains of P. f., were up to 4700-times more potent than chloroquine and up to 300-times more effective than the parent drug artemisinin.59,60 Therefore, these compounds are among the most active artemisinin dimers synthesized so far, and hence, a phosphate ester linkage should be considered as a worthwhile option in future 20 ACS Paragon Plus Environment

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artemisinin dimer synthesis. Other linkers such as dibenzoic acid esters/dibenzyl ethers (6a-f), dicarboxylic acid esters (7a/b), diamines (9), carbonate (10a) and urea (10b) considerably decreased antimalarial activity (0.46 nM to > 60 nM) compared to the aforementioned phosphate dimers.59,60 In particular, urea-linked dimer 10b, which was one of the least active compounds, had an IC50 > 60 nM against the 3D7 strain, perhaps due to its high polarity.60 Another interesting observation can be made by looking at Table 1. With only a few exceptions, C-10 non-acetals (dimers 5, 6a/b-f, 7a/b, 8a-f, 9, 10a; IC50 < 6.0 nM) seem to be more effective against malaria parasites than C-10 acetals (dimers 11a, 12a/b/c’-e, 13, 14a-d, 23, 24; IC50 > 5.0 nM), which could be explained by their higher stability. The in vivo experiments with dimers 14b and 14d performed by Lombard’s group on P. vinckei infected mice support this assumption.95 Even though both dimers 14b and 14d were demonstrated to have a strong in vivo antimalarial effect at even very low doses and were able to decrease parasitemia to extremely low levels, recrudescence took place for both dimers after 7 to 18 days. This effect may be explained by metabolic instability of the synthesized hybrid-dimers due to presence of the C-10 acetal functionality present in both molecules. One might assume that this problem could have been solved by replacing the oxygen at the C-10 position with a carbon atom. However, this was not further investigated. 21 ACS Paragon Plus Environment

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Table 1. In vitro antimalarial activity of dimers 1-15, 23 and 24 with symmetric linkers, sorted from highly active (blue), active (green) to less active (red) Activity (IC50 in nM) 9.8 – 744 TC50 > 14 881 6.6 – 42.5 TC50 > 1686 2.09 – 14.8 TC50 = 1407 0.82

Reference compounds chloroquine59,61-63,71,95 artemisinin54-62 dihydroartemisinin56,61,63,71 artesunic acid96 Activity Comp (IC50 in nM) 8c60 0.04 ± 0.02 8a59 0.09 ± 0.10 8b59 0.18 ± 0.20 8a59,60 0.20 ± 0.30 8g60 0.21 ± 0.07 15d96 0.3 11b62 0.3 11c62 0.3 96 15b 0.32 960 0.46 ± 0.08 8h60 0.50 ± 0.30 8b59,60 0.50 ± 0.10 3f/3f’56 0.91/1.1 11b62 1.1 7b59 1.1 ± 0.8 11c62 1.2 12c62 1.2 6d59 1.3 ± 1.1 7a59 1.4 ± 1.1 6a59 1.6 ± 0.4 2c’55 1.7 12c62 1.7 7a59 1.8 ± 1.4 2c54 1.9 54 2d 1.9 3e’/3e56 1.4/10.5 6e59 2.1 ± 1.3 Toxicity Comp (TC50 in µM) 3e’/3e56 10/10 3f/3f’56 2.8/4.9 11a61 0.709 11b61 0.257 11c61 0.384 11b62 > 7.644

P. f. Strain K1 HB3 HB3 K1 3D7 3D7 W2 D6 3D7 3D7 3D7 K1 K1 D6 HB3 W2 W2 HB3 HB3 HB3 NF54 D6 K1 NF54 NF54 K1 HB3

Comp 6f59 7b59 6a59 6e59 6d59 558 11c61 2c’’55 12a62 6c59 10a60 8d60 8f60 1362 11b61 14b63 12b62 3c56 11b61 8e60 11a61 11a61 2371 14a63 3a56 12a62 12d62

Cell line

Comp

Vero Vero Vero Vero Vero Vero

11c62 12a62 12b62 12c62 12c’62 12d62

Activity (IC50 in nM) 2.4 ± 0.8 2.4 ± 0.8 2.6 ± 0.9 2.7 ± 1.6 2.9 ± 1.9 2.9 3.8 3.9 4.6 4.6 ± 1.4 4.60 ± 0.40 4.8 ± 0.4 4.80 ± 0.02 5.1 5.3 5.31 ± 0.67 5.4 5.7 5.8 6.0 ± 0.3 6.1 6.3 7.2 ± 1.2 7.37 ± 0.47 7.4 8.0 8.5 Toxicity (TC50 in µM) > 7.619 > 7.336 > 7.336 > 0.080 > 0.800 0.296

P. f. Strain/Cell line

P. f. Strain K1 K1 K1 K1 K1 NF 54 W2 NF 54 D6 K1 3D7 3D7 3D7 W2 D6 D10 D6 K1 W2 3D7 W2 D6 3D7 D10 K1 W2 W2

3D7, W2, HB3, K1, D6, D10, Dd2 Vero NF54, K1, HB3, 3D7, D6, W2 Vero 3D7, Dd2, D10, K1, D6, W2 Vero 3D7 P. f. Activity Comp Strain (IC50 in nM) 14b95 8.7 ± 2.3 3D7 11c61 11.2 D6 3d56 12.0 K1 3b56 13.0 K1 12b62 13.3 W2 12d62 14.0 D6 12e62 14.1 W2 12e62 17.7 D6 54 1c 18 NF54 1362 18.6 D6 14d63 19.62 ± 1.76 D10 12c’62 21.9 W2 457 24 NF54 14b63 28.43 ± 1.17 Dd2 14d95 29.5 ± 8.5 3D7 2471 29.6 ± 4.2 3D7 2a54 30 NF54 12c’62 31.9 D6 1b54 35 NF54 2b54 36 NF54 6b59 42.2 ± 1.9 K1 14a63 46.08 ± 0.57 Dd2 14d63 55.68 ± 15.20 Dd2 10b60 > 60 3D7 54 1a 77 NF54 14c63 89.00 ± 5.71 D10 14c63 205.42 Dd2

Cell line

Comp

Toxicity (TC50 in µM)

Cell line

Vero Vero Vero Vero Vero Vero

12e62 1362 14a63 14b63 14c63 14d63

> 0.764 > 0.806 10.00 ± 6.77 0.68 ± 0.65 5.43 ± 0.62 74.82 ± 18.06

Vero Vero CHO CHO CHO CHO

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Rigid molecular substructures such as aromatic subunits or double/triple bonds seem to be not beneficial for antimalarial activity, as can be seen for dimers 1a-c (IC50 (NF54) = 18-77 nM), 2a/b (IC50 (NF54) = 30/36 nM), 4 (IC50 (NF54) = 24 nM) and 6b (IC50 (K1) = 42.2 nM) (Table 1). The most active dimers contain saturated aliphatic linkers, although the optimal linker-length is dependent on the synthesized dimer and cannot be strictly defined: Sometimes a shorter linker might yield higher activity (e.g. dimers 12c vs. 12d/e), but sometimes the opposite effect can be observed (e.g. dimer 3e vs. 3f or dimer 8e vs. 8g). Another interesting result can be seen in the case of stereochemistry. Sometimes it seems to make a difference, which diastereomer is applied in biological investigations, but other times there is almost no difference in antimalarial efficacy. For instance the diastereomers of 2c (β,β; 2c’: α,β; 2c’’: α,α) were tested for their antimalarial activity against P. falciparum NF54, but surprisingly exhibit almost the same IC50 values.55 On the other hand, dimer 3e’, possessing an α/αconfiguration at the C-9/C-9’-position, was 8-fold more active than its corresponding diastereomer 3e (α/β-configuration), indicating that in some cases stereochemistry might play an important role concerning the biological activity of artemisinin-derived dimers.56

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3.1.2 Antimalarial activity of dimers with nonsymmetric linkers In vitro antimalarial activity of dimers 25-30, containing non-symmetric linkers and prepared by the first method (Figure 4), was evaluated against the chloroquinesensitive D6 clone and the chloroquine-resistant W2 clone of P. falciparum.61,62 In addition, their cytotoxicity and selectivity index (SI = TC50/IC50 antimalarial activity) were determined against mammalian Vero cells. A SI ≥ 10 is generally considered to be sufficient to indicate that antimalarial activity is not due to cytotoxicity,97 which for all tested compounds was the case. Moreover, all dimers exhibited strong in vitro antimalarial activities, with IC50 values in the nM range (3.0-43.8 nM for D6 clone and 4.2-45.4 nM for W2 clone). They were more potent than reference compounds chloroquine and artemisinin, but less active than dihydroartemisinin (Table 2).61,62 Table 2. In vitro antimalarial activity of dimers 25-30 with nonsymmetric linkers, sorted from active (green) to less active (red) Reference compound

Activity (IC50 in nM)

P. f. Strain

chloroquine61,62

29.7 – 744.1

D6, W2

Comp 61

25 3062 2561 2962 3062 2661 Comp 26

61

Activity (IC50 in nM) 3.0 3.8 4.2 4.7 5.1 5.8 Toxicity (TC50 in nM) 7417

P. f. Strain

Comp 61

D6 D6 W2 W2 W2 D6

26 2962 2761 2761 2861 2861

Cell line

Comp

Vero

29

62

Activity (IC50 in nM) 5.9 17.2 24.2 25.8 43.8 45.4 Toxicity (TC50 in nM) > 745

P. f. Strain W2 D6 D6 W2 D6 W2 Cell line Vero

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2761

773

Vero

3062

> 78

Vero

Dimers 25/26 and 30 displayed excellent activity against both clones (IC50 < 10 nM). However, none of these dimers could reach the extraordinary efficacies of dimers presented in section 3.1.1 (e.g. phosphate dimers 8 IC50 values below 1 nM), which could be explained by the fact that dimers 25-30 all are C-10 acetals. Surprisingly, phenol containing dimer 25 was the most active compound with an IC50 value of 3.0 nM against P. falciparum D6 and was 15-times more active than alcohol dimer 28, even though this dimer is less rigid.61 Thus structure activity relationships (SAR) seem to be different for non-symmetric and symmetric dimers. However, it is difficult to make a definite statement, because the majority of synthesized dimers are symmetric and consequently further investigations are needed.

3.2 Antimalarial activity of dimers accessible via direct reaction of two artemisinin derivatives By comparing the antimalarial activities (P. f. K1, Dd2, 3D7 and NF54) of dimers 32-36 (Figure 5), which were prepared via direct reaction of two artemisinin derivatives, observations similar to those discussed in section 3.1.1 can be made. 25 ACS Paragon Plus Environment

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The most active compounds were again C-10 non-acetals, i.e., dimers 32, 33 and 35.54,60 These compounds exhibited IC50 values below 4 nM; all C-10 non-acetals (dimers 36a-f) were considerably less active (IC50 = 5.5-35.9 nM) but were still very potent antimalarial compounds (Table 3).72 Furthermore, the most active compound, amide dimer 35 (IC50 (3D7) = 0.03 nM), which was up to 400-fold more potent than artemisinin (IC50 of 12.3 ± 1.40 nM) and artemether (IC50 of 2.11 ± 0.61 nM), contained an aliphatic linkage.60 In contrast, dimers 32 and 33, which were less active (IC50 (NF54) =1.3/3.2 nM), had aromatic moieties (phenyl and furan) as linkers. Table 3. In vitro antimalarial activity of dimers 32-36 prepared via direct reaction of two artemisinin derivatives, sorted from highly active (blue), active (green) to less active (red) Activity (IC50 in nM) 13.8/300 7.81 – 12.3

Reference compounds chloroquine diphosphate salt artemisinin54,56,60

P. f. Strain/Cell Line NF54, Dd2 K1, NF54, 3D7

dihydroartemisinin56,72

0.81 – 8.8 TC50 = 147.7 ± 50.0 µM

K1, NF54, Dd2 CHO

artesunate72

< 5.2/8.1

NF54, Dd2

2.11 ± 0.61

3D7

artemether Comp 60

35 3254 34g56 34f56 3354 34e56 34c60 34b56 34a56 34d56 36d*72 Comp 34a

72

56

60

Activity (IC50 in nM) 0.03 ± 0.01 1.3 2.1 2.3 3.2 3.4 3.9 4.3 4.4 5.0 5.5 ± 0.4 Toxicity (TC50 in µM) > 130

P. f. Strain

Comp 72

3D7 NF54 K1 K1 NF54 K1 K1 K1 K1 K1 NF54

36f* 36f*72 36e*72 36d*72 36a*72 36e*72 36a*72 36b*72 36c*72 36c*72 36b*72

Cell line

Comp

Vero

36a*

72

Activity (IC50 in nM) 7.9 ± 0.5 10.25 ± 1.7 10.3 ± 1.9 10.6 ± 1.2 11.6 ± 2.7 12.9 ± 1.9 13.6 ± 2.1 25.8 ± 2.4 31.8 ± 5.7 33.1 ± 2.7 35.9 ± 0.8 Toxicity (TC50 in µM) 17.0 ± 9.5

P. f. Strain NF54 Dd2 Dd2 Dd2 NF54 NF54 Dd2 NF54 NF54 Dd2 Dd2 Cell line CHO

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34b56 34c60 34d56 34e56 34f56

> 130 63 2.5 2.4 1.1

Vero Vero Vero Vero Vero

34g56

5.5

Vero

36b*72 0.9 ± 0.0 CHO 36c*72 >100 CHO 36d*72 >100 CHO 36e*72 0.7 ± 0.3 CHO 36f*72 >100 CHO * all of these compounds were converted into oxalate salts for reasons related to stability

Dimer 35 is even comparable in activity to the previously mentioned organophosphate dimers 8a-h (IC50 (K1) up to 0.04 nM; IC50 (3D7) up to 0.21 nM), although the connection between the two artemisinin subunits is completely different, i.e., amide vs. organophosphate ester. Dimers 34a-g (Figure 5) linked via the C-16 position of the trioxane moiety exhibit similar antimalarial activities as dimers 33 and 35.56 The dimers were tested against P. falciparum K1. To compare the malarial activity of the derivatives to their cytotoxicity against healthy cells, the IC50 value for African green monkey kidney fibroblast (Vero cells) was additionally determined. All seven dimers 34a-g (Figure 5) proved to be more active (IC50 of ≤ 5 nM) than artemisinin (IC50 of 12.1 and 7.81 nM, respectively) against K1 parasites. Furthermore, the antimalarial activity of dimers 34a-c is much higher than their cytotoxicity against Vero cells; consequently, the compounds can be concluded to be selective. 3.3.1 Antimalarial activity of dimers obtained via post-modification of olefin 4 This section discusses the antimalarial activity of dimers 45-57 (Figure 6). These dimers were prepared via the post-modification of the previously mentioned 27 ACS Paragon Plus Environment

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dimer 4, which contains a symmetric olefin linker (Figure 6). The present section is divided into two parts: in vitro (Table 4) and in vivo (Table 5) experiments. Regarding in vitro antimalarial activity, all of the dimers derived from dimer 4 are highly active against malaria parasites. Dimers 45-47 were considerably more active against the chloroquine-sensitive NF 54 strain of P. f. than precursor 4 (IC50 = 24 nM), with IC50 values ranging from 0.53 to 3.0 nM.57,78 Isonicotinate N-oxide dimer 47, alcohols 45a/b and ketone 45g were the most active compounds, exhibiting IC50 values below 1 nM. These dimers were up to 18-fold more active than artemisinin (IC50 = 8.8 nM) and up to 3-fold more potent than sodium artesunate (IC50 = 1.5 nM). Dimers 50a/b showed high efficacy in chloroquineresistant Dd2 strains of P. f., with IC50 values of 2.4 nM and 3.9 nM, respectively.82 All of these dimers were prepared from the olefin dimer 4 through reactions in which only the C-C double-bond was utilized for chemical transformations. Therefore, these dimers belong to the group of C-10 non-acetals, explaining their consistently high antimalarial activity and confirming the assumption regarding the role of dimer stability, which was discussed in section 3.1.1. Only the ferrocene-containing dimer 57 exhibited an IC50 value above 4 nM (IC50 (3D7) = 30.2 nM) and was less active than both its alcohol precursor 45a (IC50 (3D7) =

1.1 nM)

and

reference

compounds

chloroquine

and

dihydroartemisinin (IC50 (3D7) = 9.8/2.4 nM).71,98 Therefore, the free alcohol group 28 ACS Paragon Plus Environment

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of dimer 45a appears to be important for its antimalarial activity. A similar result can be seen by comparing the activities of alcohol dimers 45a/b with carboxylic acid dimers 45c/d. The succinylation of dimers 45a/b decreased its activity by nearly a factor of 6, a considerable effect.57 Interestingly, all dimers containing a carboxylic acid function (compounds 45c/d/h/i and 46) exhibited nearly the same activity (IC50 (NF54) = 2.0-3.0 nM). Therefore, it appears that a free carboxylic acid has less of a positive effect on antimalarial efficacy than a free alcohol group, perhaps due to the former’s higher polarity. However, because of their better solubility in water, the increased polarity could be beneficial in vivo Table 4. In vitro antimalarial activity of dimers 4, 45-47, 50 and 57, sorted from highly active (blue), active (green) to less active (red)

chloroquine71

Activity (IC50 in nM) 9.8 ± 2.8

artemisinin57,78,82

6.9 – 9.0

Dd2, NF54

2.4 ± 0.4

3D7

1.5

NF54

2730 ± 96.0

Dd2

507.0 ± 35.0

Dd2

Reference compounds

dihydroartemisinin71 sodium artesunate

78 82

deoxydihydroartemisinin deoxy derivative of 50a Comp 4778 45b57 45a57 45g57 45a98 45e57 45c57 45h57

82

Activity (IC50 in nM) 0.53 0.59 0.87 0.91 1.1 ± 0.5 1.7 2.0 2.1

P. f. Strain

Comp

NF 54 NF 54 NF 54 NF 54 3D7 NF 54 NF 54 NF 54

45i57 4678 50a82 45f57 45d57 50b82 457 5771

P. f. Strain 3D7

Activity (IC50 in nM) 2.4 2.4 2.4 ± 0.15 2.8 3.0 3.9 ± 0.3 24 30.2 ± 4.2

P. f. Strain NF 54 NF 54 Dd2 NF 54 NF 54 Dd2 NF 54 3D7

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For this potentially positive aspect of increased polarity, the carboxylic acid dimers 45c/d and 45h were investigated in vivo in P. berghei-infected mice.57 These dimers indeed showed higher efficacy than the drug candidate artelinic acid administered intravenously (iv) and the clinically used drug sodium artesunate administered orally (po). The best effect was achieved by intravenous injection of 10 mg/kg of dimers 45c/d or 45h, resulting in more than 80% parasite suppression. Furthermore, it must be noted neither 45c/d nor 45h had toxic side effects or caused behavioral modification in mice due to drug administration. Motivated by these promising results, carboxylic acid dimer 46 and isonicotinate N-oxide dimer 47 were also examined in vivo for their antimalarial efficacies on P. berghei-infected mice.78 Both compounds, which were administered either intravenously or orally, were considerably more effective than the clinically used sodium artesunate. In addition, a small preliminary toxicity study in mice revealed that carboxylic acid dimer 46 is safer than dimer 47 and sodium artesunate. Specifically, there were fewer deaths among the animals that were administered carboxylic acid dimer 46 and an absence of negative effects on animal weight gain. Four additional in vivo studies were published regarding compounds 48, 49 and 51 by the Posner group. In these experiments, the antimalarial efficacy of these dimers was determined in P. berghei-infected mice (Table 5).79,80,81,83 Depending on oral or subcutaneous dosing regimens (1 x 10-144 mg/kg or 3 x 10-30 mg/kg) 30 ACS Paragon Plus Environment

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dimers 48a-f, 49b-e, 49g-i, 49m/n and 51c were able to cure 100% of the malaria-infected mice; no parasites were detected in the blood 30 days post-infection. In addition, neither toxic side effects nor changes in behavior were observed in any of the cured animals.79,80,83 All of these dimers outperformed the reference compounds sodium artesunate, artemether, both of which are already used in the clinic. These dimers also outperformed the fully synthetic trioxane peroxide drug development candidate arterolane (OZ277) maleate, which is in phase II clinical trials. None of the reference compounds significantly prolonged the average survival time (6.5-12.4 days) compared to the untreated control group (survival time: 6.4-6.8 days), even at the highest doses administered in these studies. Table 5. In vivo antimalarial activity of dimers 48, 49 and 51 tested on P. berghei-infected mice. Activities are highlighted in different colors according to survival number of days (>30: blue; 20.7-27.7: green; 6.0-17.7: red) Comp 48e

48b

48d

48a 48c 48f

Activity* > 30.0a) 25.3b) 14.7c) 27.0a) 10.0b) > 30.0c) > 30.0a) 15.0b) 12.7 15.0a) 13.3b) > 30.0c) > 30.0a) 12.3b) > 30.0a) 10.0b) 7.0c)

Comp 49m

48a

49g 49h 49b 49i 48b

Activity* > 30a) 14.0b) 11.0c) > 30a) 14.7b) 10.0c) > 30a) 14.0b) 8.7c) > 30a) 13.3b) > 30a) 11.0b) > 30a) 8.3b) > 30a) 8.0b)

Comp 49p 49r 49q 49u 49k sodium artesunate arterolane (OZ277) maleate

Activity* 7.0b) 10.3c) 10.0b) 10.0c) 6.3c) 7.2a) 6.5b) 8.2c)

a) three oral doses of 30 mg/kg, b) three oral doses of 10 mg/kg, c) single oral dose of 30 mg/kg

Ref.80 > 57a) 23a)

49v artemether

a) single oral dose of 8 mg/kg combined with 24 mg/kg mefloquine hydrochloride

Ref.81

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untreated control

6.8

49n

sodium artesunate

9.1 ± 3.7a) 8.6 ± 3.5b) 7.5 ± 0.4c)

49o

a) single subcutaneous dose of 30 mg/kg, b) single subcutaneous dose of 10 mg/kg, c) three oral doses of 30 mg/kg

Ref. 49d

49e

49l 49j

79

49a > 30a) 16.3b) 49f 8.3c) > 30a) 49t 15.7b) 49s 9.0c) 49p *Survival time (in days)

> 30a) 7.0b) 27.7a) 6.0b) 24.7a) 7.7b) 20.7a) 6.0b) 17.7b) 10.0c) 15.7b) 10.0c) 13.7c) 12.7c) 11.3a)

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22.8a) 30.0 (all cured) 20.4a) c) 30.0 (3/5 cured) 16.4a) 14.8a) 14.6a) 11.0a) 11a) 12.4c)

51c

c)

51b 51d 51e 51a 51f artemether sodium artesunate untreated control

7.6b) 6.4

a) single oral dose of 54 mg/kg, b) single oral dose of 72 mg/kg, c) single oral dose of 144 mg/kg

Ref.83

Outstanding results were achieved with silylamide trioxane dimer 49v (Figure 6) (similar in structure to amides 49n-s) in conjunction with ACT, which is considered the best available treatment for uncomplicated forms of malaria caused by P. falciparum. In these analyses, mefloquine hydrochloride was used as a second antimalarial agent.81 The administration of a single oral dose of only 8 mg/kg of silylamide 49v combined with 24 mg/kg mefloquine hydrochloride was sufficient to cure all malaria-infected mice, whereas mefloquine hydrochloride alone at a single oral dose of 22 mg/kg was not curative. The experiment was terminated on day 57, and at that time, the blood of the surviving mice was free of malaria parasites. In addition, neither toxic side effects nor behavioral change were observed in any of the malaria-infected mice cured by trioxane 49v in combination with mefloquine hydrochloride. In contrast, the well-known ACT trioxane drug

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artemether combined with mefloquine hydrochloride did not cure the infected mice and prolonged survival only until day 23. Another example in which ACT yielded very successful results is the case of sulfone trioxane dimers 51c/d.83 Combining either dimer 51b or 51c at a significantly lower dose of 54 mg/kg (dose needed for monotherapy: 144 mg/kg) with 13 mg/kg mefloquine hydrochloride resulted in > 99.9% parasitemia suppression on day 3 after infection and completely cured all mice. Mefloquine hydrochloride (13 mg/kg) alone was not curative and prolonged the average survival to only day 11. By analyzing the molecular structure of the most active dimers, 48e and 49v, it becomes apparent that an amide side chain with a lipophilic substituent (i.e., t-butyl (48e) or trimethylsilyl (49v)) has a positive antimalarial effect, whereas a side chain containing an aromatic subunit (e.g.: 48d, 48f, 49j, 49r, 49u, 49k etc.) does not contribute in a positive way to antimalarial activity. Interesting mechanistic studies have been carried out for the dansyl dimer 50b.82 The deoxygenated forms of the artemisinin and dansyl dimer 50b were investigated in vitro for their antimalarial efficacy towards P. f. (Dd2) parasites. As expected, these forms were completely inactive, exhibiting IC50 values > 500 nM. This experiment proves the importance of the endoperoxide bridge for the biological activity of artemisinin derivatives. Furthermore, the distribution of a fluorescent 33 ACS Paragon Plus Environment

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dansyl dimer 50b was examined in living, intraerythrocytic-stage P. falciparum parasites using microscopic imaging. The rapid accumulation of fluorescent trioxanes was observed in parasitized erythrocytes. The trioxanes were localized within digestive vacuole-associated neutral lipid bodies of trophozoites and schizonts and surrounded the developing merozoite membranes. Interestingly, an increase in non-specific cytoplasmic localization was detected with respect to iron chelation, demonstrating that iron plays an important role in terms of the antimalarial activity promoted by artemisinin-derived compounds. In addition, the presence of artemisinin-induced peroxyl radicals was verified in parasite membranes by utilizing an oxidative-sensitive BODIPY lipid probe. As expected, these results were specific to artemisinin derivatives containing an endoperoxide moiety given that the deoxygenated counterparts cannot label neutral lipid bodies or induce oxidative membrane damage. 3.3.2 Antimalarial activity of dimers obtained via post-modification of miscellaneous artemisinin-derived dimers Post-modified dimers 58-66 (Figure 7) were investigated either in vitro (Table 6) and/or in vivo (Table 7) for their antimalarial activity. With respect to in vitro activity, fluorinated C-10 non-acetal dimers 58a/b (Figure 7) were 2-4 times less potent against chloroquine-sensitive P. f. (NF54) parasites (28 nM and 15 nM) than artemisinin. In contrast, the non-fluorinated 34 ACS Paragon Plus Environment

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precursors 2c-c’’ (Figure 2) were 2-5 times more potent (Table 1) than artemisinin.55 These data indicate that incorporating fluorine into aliphatic linkers is not beneficial for antimalarial activity. Surprisingly, bis-benzyl alcohol dimer 59c was the most active compound (IC50 (NF54) = 0.77 nM), despite bearing an aromatic subunit.58 59c was even more active than its phosphorylated counterparts (dimers 59d/e), which is in contrast to the observations made in section 3.1.1. Therefore, the position and type of the phosphate linkage within the molecule seems to be highly important. For watersoluble phthalic acid dimer 59b, which was the least active compound, a decrease in activity by a factor of 460 was observed compared to dimer 59c, an effect that was likely due to the increased polarity of this dimer. The promising in vitro results of dimers 59a/c encouraged Posner’s group to perform in vivo experiments for these compounds.58 These compounds were tested according to a published protocol using either oral (po) or subcutaneous (sc) single administrations at doses of 3.10 or 30 mg/kg. Both of the dimers were more active than clinically used sodium artesunate, with ED50 values as high as 0.06 mg/kg (sc) and 2.6 mg/kg (po). Neither toxicity nor behavioral modifications were observed in the mice due to drug administration. Table 6. In vitro antimalarial activity of dimers 58-60, sorted from highly active (blue), active (green) to less active (red) Reference compounds

Activity (IC50 in nM)

P. f. Strain/ Cell line

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

chloroquine61,62 artemisinin55,58,61,62 Comp 59c58 60e62 59a58 60e62 59e58 59d58 Comp 61

60a 60c61

Activity P. f. (IC50 in nM) Strain NF 54 0.77 1.4 D6 NF 54 1.6 2.1 W2 NF 54 3.7 NF 54 3.0 Toxicity (TC50 in nM) > 7270 > 7475

29.7– 744 TC50 > 14 881 6.6 – 42.5 TC50 > 1686 Activity Comp (IC50 in nM) 5.2 60a61 6.7 60b61 11.7 11.8 60d61 11.8 60b61 14.0 Cell line Vero Vero

Comp 61

60b 60d61

P. f. Strain W2 D6 D6 D6 W2 W2

W2, D6 Vero NF54, 3D7, D6, W2 Vero Activity Comp (IC50 in nM) 58b55 15 58a55 28 60c61 45.5 60c61 47.1 59b58 360 Toxicity (TC50 in nM) 825 6403

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P. f. Strain NF 54 NF 54 D6 W2 NF 54

Cell line Vero Vero

The post-modified dimers 60a-f (Figure 7) showed strong activities against chloroquine-sensitive (D6) and chloroquine-resistant (W2) clones of P. f., with IC50 values in the nM range (1.4-45.5 nM for D6 clones, and 2.1-47.1 nM for W2 clones) (Table 6).61 Dimer 60e was found to be the most active hybrid in this study, being approximately 7-fold more active than artemisinin and 21 to 354-fold more active than chloroquine. However, dimer 60e was less active than its alcohol precursor 11c (Figure 1), perhaps due to the increased hydrophilicity evoked by the introduced succinic acid moiety. The 5-carbon-linked C-10 non-acetal trioxane ester dimers 61a-c (Figure 7) were tested in vivo for their antimalarial activity against P. berghei.90 After treatment of infected mice using a single oral dose (7.1 mg/kg) of either trioxane dimers 61a-c or artemether combined with mefloquine hydrochloride (21 mg/kg), parasitemia on day 30 was determined. Nitrobenzoate ester dimer 61a exhibited a similar efficacy 36 ACS Paragon Plus Environment

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as artemether (4 of 5 mice were free of parasites after 30 days). In contrast, dinitrobenzoate ester hybrid 61b and sulfonylbenzoate ester hybrid 61c were potent at curing all malaria-infected mice (no parasites were detected in the blood of any of the 5 mice on day 30 post-infection). A single oral dose of 21 mg/kg of mefloquine hydrochloride alone cured 1 mouse out of 5 and therefore was considerably less effective. Artemether and all of the benzoate ester dimers 61a-c result in at least 99.5% suppression of parasitemia on day 3 post-infection. Furthermore, none of the mice exhibited signs of toxicity or behavioral changes. Table 7. In vivo antimalarial activity of dimers 62-65 tested on P. berghei-infected mice. The activities are highlighted in different colors according to survival number of days (>30: blue; 20.0-27.8: green; 6.5-19.5: red) Comp

Activity*

62e 62c 62d 62b 62a artemether mefloquine artemether alone lumefantrine vehicle (no drug)

a)

Activity*

Activity*

Comp

Activity*

a)

63i

64a 64k 64p 64h 64d 64e 64m

24.8 24.8a) 22.0a) 21.8a) 20.3a) 18.8a) 18.3a)

65l 65c 65j 65o 65d 65i 65b

26.3a) 25.5a) 24.5a) 24.5a) 24.3a) 24.3a) 24.0a)

14.3b)

63f

23.8a)

64i

18.0a)

65g

23.8a)

11.7c)

63k vehicle (no drug)

21.5a)

64o

17.8a)

65h

23.5a)

7.3

64c

17.0a)

65m

23.3a)

artemether

40.0a)/19.0

64f

13.5a)

65k

21.8a)

mefloquine

21.0d)

64n

12.5a)

64b

19.5a)

lumefantrine

14.0e)

64j infected mice (no drug)

10.8a)

65a

18.5a)

6.5

65f

17.5a)

artemether

20.3a)

infected mice

7.0

mefloquine alone

20.3

artemether

21.5a)

63a 63h 63l 63d

7.3

Ref.

60.0a) 60.0b) 46.8c)

a)

Comp

52.5 51.8a) 48.3a) 41.0a) 60.0a) 26.3b) 27.3a)

30.0 23.7a) 20.7a) 19.0a) 18.6a) 24.0a) 20.0c)

a) single dose of 6 mg/kg trioxane combined with 18 mg/kg mefloquine, b) single dose of 6 mg/kg trioxane combined with 18 mg/kg lumefantrine, c) compound tested 91 alone: 18 mg/kg

63g

Comp

63b

a) 1x 6 mg/kg combined with 18 mg/kg mefloquine, b) 1x 6 mg/kg combined with 18 mg/kg lumefantrine, c) only trioxane 150 mg/kg, d) only mefloquine 18 mg/kg, e) only lumefantrine 18 mg/kg

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56.0a)

63j

63c

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a) 1x 5 mg/kg trioxane combined with 15 mg/kg mefloquine hydrochloride

Ref.92

56.0a)

64l93

26.8a)

41.3b) 46.8c)

64g93 64b93

25.5a) 25.0a)

17.8

a) 1x 6 mg/kg trioxane combined with 18 mg/kg mefloquine hydrochloride

Ref.93 65n94 65e94

mefloquine alone

30.0a) 27.8a)

Ref.94 *Survival number of days

After these promising results, in the following year, Posner’s group studied the in vivo antimalarial efficacies of 5-carbon-linked trioxane orthoester dimers 62b-e and its precursor 62a (Figure 7) in P. berghei-infected mice (Table 7).91 All of the malaria-infected mice receiving either the trioxane drug artemether alone or lumefantrine alone died before day 15. A combination of both prolonged survival up to day 24 but was still not curative. Mefloquine hydrochloride alone at a single oral dose of 18 mg/kg prolonged survival of the infected mice up to 20 days. Orthoester sulfone hybrid 62e plus mefloquine was completely curative and was much higher in efficacy than the antimalarial drug artemether. Furthermore, the compound was non-toxic and did not affect behavior. Posner’s group carried out further in vivo-studies in P. berghei-infected mice using

the

two-carbon-linked

alcohol/carbamates 64a-p93

and

artemisinin-derived

dimer

(thio)carbonates 65a-o

oximes 63a-k,92

(Figure 7).94

Oxime

dimers 63a-c and 63g, 63h and 63j in combination with mefloquine were more efficacious than the combination of artemether and mefloquine, achieving animal survival times longer than 40 days (Table 7). As a second experiment, oxime dimers alone or in combination with lumefantrine were administered to mice, and 38 ACS Paragon Plus Environment

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survival time was examined. Achieving survival times of more than 41 days for these combinations, both oxime hybrids 63c and 63g surpassed all reference drugs or their combinations. The alcohol dimer 64a/b as well as dimers 64g (3,4-difluorophenyl NH-aryl carbamate), 64k (3-chloro-4-fluorophenyl NH-aryl carbamate) and 64l (3-chlorophenyl NH-aryl carbamate) prolonged the survival time of malaria-infected mice beyond day 24. These results were compared to those for artemether plus mefloquine or mefloquine alone, which exhibited 20-day survival times. It was observed that the substitution at the 3-position has a significant effect on antimalarial potential. With the exception of dimers 65a/f, all of the other artemisinin-derived thiocarbonates displayed longer survival times than artemether with mefloquine. Dimers 65c (fluoroethyl carbonate), 65e (chloroethyl carbonate), 65l (chlorophenyl carbonate) and 65n (thiocarbonate) were the most effective compounds of the last-mentioned investigation, with the achieved average survival times being longer than 25 days.

4. Anti-cancer activities 4.1.1 Anti-cancer activity of dimers with symmetric linkers The anti-cancer activities of dimers 1-3 and 6-24, which are connected by symmetrical linkers (Figures 2 and 3) are discussed below. The previously mentioned aminoquinoline-linked dimers 14b/d (Figure 3), which exhibited relatively poor antimalarial activity in comparison to the other 39 ACS Paragon Plus Environment

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dimers, were tested for their in vitro anti-cancer activity against renal (TK-10), melanoma (UACC62) and breast (MCF-7) cancer cell lines (Table 8). These dimers were among the most active compounds against cancer cells.95 Both of the hybrids surpassed the growth inhibitory effect of etoposide, a well-known anti-cancer drug used as reference standard, with GI50 values ranging from 0.03 µM (MCF-7) to 0.08 µM (TK-10), respectively. Thus, both compounds can be regarded as highly active against the tested cancer cell lines, being up to 74‑fold more active than etoposide. Consequently, the required properties or structural characteristics for a compound to have a high anti-cancer activity appear to differ from those required for antimalarial activity. This difference can also be seen in the case of the ferrocene-containing dimers 23 and 24, which exhibited only moderate antimalarial activity but are highly active against leukemia.71 Specifically, these dimers were 6‑fold more effective against leukemia CCRF-CEM cells (IC50 0.07 µM and 0.08 µM) than their parent compound dihydroartemisinin (IC50 of 0.48 µM). Looking closely at Table 8 and comparing the IC50 values of C-10 acetals (dimers 11-16 and 19-23) and C-10 non-acetals (dimers 1, 2, 6-10, 17, 18 and 24), it becomes apparent that members of both groups exhibit in vitro anti-cancer activity. Although ferrocene-containing dimer 23 is a C-10 acetal and dimer 24 is a C-10 non-acetal, they share the same anti-leukemia activity (CCRF-CEM cells).71

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Therefore, the specific structure of the linker as a whole appears to be of greater importance. However, as there are many differences between cancer types, e.g., with respect to cell structure and gene expression, a given dimer may be highly active against one cancer cell line but totally inactive against another. This fact makes it difficult to identify a general correlation between molecular structure and activity. Take for instance the ether-linked dimers 11a-c (Figure 2), which were tested in vitro for their anti-cancer activity against four human solid tumor cell lines (SK-MEL, KB, BT-549 and SK-OV-3) and for their cytotoxicity towards noncancerous mammalian cell lines (Vero and LLC-PK11).61 The IC50 values demonstrate that hybrids 11a-c were significantly more potent against SK-MEL, KB and BT-549 cells (up to 0.24 µM) than the control drug doxorubicin (1.73-2.04 µM). In contrast, these dimers were completely inactive towards SK-OV-3 cells (Table 8). Therefore, given that different cancer cell lines are often used by different groups, it is very difficult, or in many cases impossible, to compare the results obtained by different research groups. The most efficient way to investigate the anti-cancer efficacy of newly synthesized compounds is to perform a drug screen against a large number of different cancer cell lines. Then, further investigations should be performed with the most active compounds, and similar compounds to these should be synthesized. 41 ACS Paragon Plus Environment

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This method was applied for dimers 6a-f, 7a/b and 8a/b. These dimers were screened by the National Cancer Institute (NCI) for their anti-cancer effects using 60 human cancer cell lines. Phosphate-linked dimers 8a/b proved to be the most potent compounds exhibiting promising activity against leukemia, colon and certain melanoma and breast cancer cell lines.59 However, both dimers demonstrated little activity towards lung, central nervous system (CNS) and renal cancer cell lines. Unlike the phosphate ester dimers, the other tested artemisininderived hybrids 6a-f and 7a/b demonstrated poor anti-cancer activity in the NCI assays. Therefore, the anti-tumor activity of both phosphate-linked dimers 8a/b was further

investigated

in

HL-60

leukemia

and

Jurkat

cell

lines

using

dihydroartemisinin and doxorubicin as reference compounds. Dimers 8a/b showed excellent activity towards the HL-60 cells, with IC50 values (0.14 µM/0.24 µM) superior to that of doxorubicin (0.51 µM) and dihydroartemisinin (1.21 µM) (Table 8). Regarding the results of the Jurkat cell line, doxorubicin proved to be more active than the phosphate ester hybrids, which were still 10‑fold more active than dihydroartemisinin. In addition, both phosphate dimers were tested for general toxicity to normal cells and were not toxic towards lymphocytes even at doses above 100 µM. Motivated by these promising results, the cytotoxic activity of additional phosphate-linked dimers 8c-h, as well as bipiperidin-linked dimer 9, carbonate42 ACS Paragon Plus Environment

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linked dimer 10a and urea-linked dimer 10b, was evaluated against HL-60 cells.60 The IC50 values of phosphate-linked dimers 8a-h and bipiperidin dimer 9 were comparable to that of doxorubicin, ranging from 0.05 to 1.04 µM. Hybrid 8f was the most potent among the tested dimers, showing a nearly 50-fold higher activity (IC50 = 0.05 µM) than dihydroartemisinin (IC50 = 2.41 µM). In addition to their excellent anti-cancer activity, the synthesized dimers were not toxic to normal cells. Specifically, compounds 8a/b did not affect the growth of peripheral blood mononuclear cells (PBMCs), even at concentrations above 250 µM. The carbonateand urea-linked dimers 10a/b exhibited no anti-cancer effect at all against the tested HL-60 cells. These results are very interesting as they are similar to those observed in the case of tests for antimalarial activity. Specifically, it was again observed that a urea subunit (dimer 10b) does not seem beneficial, whereas a phosphate ester linkage (dimers 8a-h) drastically improves biological activity. Dimers 1a-c, 2a-d (Figure 2)54,55 12a-e, 13 (Figure 3)62 and 16b66 were also investigated in vitro for their growth inhibitory activities against a diverse panel of 60 human cancer cell lines at the NCI. The results of this drug screening indicated that all dimers are particularly active against leukemia cells, especially dimer 12a. This dimer showed a GI50 (50% growth inhibition) value below 0.005 µM and was one of the most active compounds.54,55,62,66 Furthermore, most of the dimers were also very effective against one or more cell lines in each of the human cancer cell 43 ACS Paragon Plus Environment

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line panels. For example, compound 11b inhibited the growth of 24 of the 60 cell lines, even at concentrations below 0.01 µM.62 Furthermore, preliminary in vivo anti-cancer experiments using the NCI mouse hollow fiber assay were performed for dimers 2c,54 12a.62 and 16b.66 Generally, this assay is used to obtain initial qualitative indications of in vivo drug efficacy for compounds that have reproducible activity in in vitro anti-cancer drug screening assays. Dimer 2c showed considerable anti-cancer effect via both intraperitoneally (ip) as well as subcutaneous (sc) injections and diminished the viable cancer cell mass. These data suggest that dimer 2c is potent and stable in this in vivo assay. Dimer 12a performed similarly in regard to anti-cancer activity (total score: 36) to the positive control, paclitaxel (total score: 32 ± 4), which can be regarded as sufficient to apply the dimer in xenograft studies. Therefore, the anti-tumor efficacy of diether-linked dimer 12a was further investigated using a subcutaneous (sc) xenograft model of the HL-60 cell line. Treating xenografts with a single daily dose of 50 mg/kg (sc) for 10 days resulted in an optimal% T/C of less than 40%. Consequently, compound 12a can be considered active in this experiment. Ether-linked dimer 16b was selectively cytotoxic to leukemia cells and showed no acute toxicity in vivo even at a maximum tolerated dose of 400 mg/kg. These experiments corroborate the previous assumption regarding the role of stability (of C-10 acetals vs. non-acetals) in terms of anti-cancer activity. Although dimers 12a 44 ACS Paragon Plus Environment

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and 16b are both C-10 acetals, they still appear to be active and even stable in in vivo experiments. Regarding stereochemistry, a very interesting observation was made when examining dimers 16a-e (Figure 3).66 Only dimers 16b, 16d and 16e, which all have β,β-stereochemistry at C-10 position, exhibited high in vitro anti-proliferative effects in murine keratinocytes, indicating that stereochemistry might be important for antitumor activity. Table 8. In vitro anti-cancer activity of dimers with symmetric linkers, sorted from highly active (blue), active (green) to less active (red) Reference compounds

Activity (IC50 in µM) 0.009 – 23.27

doxorubicin59-61,65,71,68 etoposide95 taxol61

nc (not cytotoxic) TC50 = 1.29 0.83 – 5.89 0.02 – 4.10 TC50 = 0.53 TC50 = 4.39

artemisinin65,68,71

12.9 – 36.9

dihydroartemisinin59,60,68,71

0.46 – 101

artesunic acid65 Activity Comp (IC50 in µM) 14b95 GI50 = 0.03 14d95 GI50 = 0.03 14d95 GI50 = 0.04 14b95 GI50 = 0.05 8f60 0.05 ± 0.02 8a60 0.07 ± 0.02 2371 0.07 ± 0.06 2471 0.08 ± 0.03 14b95 GI50 = 0.08 14d95 GI50 = 0.08 8h60 0.10 ± 0.04 8a59 0.143 ± 0.02 8c60 0.16 ± 0.06 64 15a 0.2 ± 0.03

1.20/1.80 Cell line MCF-7 MCF-7 UACC62 UACC62 HL-60 HL-60 CCRF-CEM CCRF-CEM TK-10 TK-10 HL-60 HL-60 HL-60 CEM/ADR5000

Cell line CCRF-CEM, CEM/ADR5000, HL-60, Jurkat, SK-MEL, KB, BT-549, SK-OV-3, A-549, SK-MEL-2, XF498, HCT-15, SK-V3 Vero LLC-PK11 MCF-7, UACC62, TK-10 SK-OV-3, BT-549, KB, SK-MEL Vero LLC-PK11 A-549, SK-V3, SK-MEL-2, XF498, HCT-15, CCRF-CEM, CEM/ADR5000 HL-60, Jurkat, HCT-15, A-549, SK-V3, SK-MEL-2, XF498, CCRF-CEM, CEM/ADR5000 CEM/ADR5000, CCRF-CEM Activity Comp Cell line (IC50 in µM) 1868 0.63 SK-V3 3f/3f’56 0.76/1.1 KB 8d60 0.77 ± 0.18 HL-60 1868 0.84 HCT-15 1868 0.85 A-549 8e60 1.04 ± 0.28 HL-60 15a64 1.2 ± 0.1 CCRF-CEM 15b65 1.21 ± 0.15 CEM/ADR5000 15c65 1.51 ± 0.11 CEM/ADR5000 11a61 1.53 BT-549 3e/3e’56 1.6/1.6 BC 15c65 1.60 ± 0.70 CCRF-CEM 2371 1.80 ± 0.46 CEM/ADR5000 56 3e/3e’ 1.8/2.3 KB

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11b61 11b61 8b59 8g60 11c61 8b60 11a61 3f/3f’56 960 1868 1868 11c61 11b61 Comp 3e’/3e56 3f/3f’56 11a61 11a61

0.24 0.24 0.241 ± 0.08 0.25 ± 0.05 0.26 0.27 ± 0.11 0.32 0.36/1.1 0.40 ± 0.17 0.45 0.46 0.48 0.56 Toxicity (TC50 in µM) 10/10 2.8/4.9 TC50 = 0.18 nc (not cytotoxic)

KB BT-549 HL-60 HL-60 KB HL-60 KB BC HL-60 SK-MEL-2 XF498 BT-549 SK-MEL

15b65 15d65 15d65 8a59 11c61 2471 8b59 10a60 10b60 11c61 11a61 11a61 11b61

Cell line

Comp

Vero Vero Vero LLC-PK11

11b61 11b61 11c61 11c61

2.16 ± 0.99 2.94 ± 0.33 3.04 ± 0.44 7.7 ± 0.02 8.00 8.20 ± 4.43 13.2 ± 3.1 > 100 > 100 na (not active) na na na Toxicity (TC50 in µM) > 8.03 TC50 = 0.10 TC50 = 5.60 TC50 = 0.15

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CCRF-CEM CCRF-CEM CEM/ADR5000 Jurkat SK-MEL CEM/ADR5000 Jurkat HL-60 HL-60 SK-OV-3 SK-MEL SK-OV-3 SK-OV-3 Cell line Vero LLC-PK11 Vero LLC-PK11

The activity of artesunic acid dimers 15a-d (Figure 3) has been analyzed in CCRF-CEM

human

leukemia

cells

and

its

multidrug-resistant

subline,

CEM/ADR5000.64,65 Dimer 15a showed encouraging cell growth inhibitory activity in both cell lines (IC50 (CCRF-CEM) = 1.2 ± 0.1 µM and IC50 (CEM/ADR5000) = 0.2 ± 0.03 µM) relative to artemisinin and artesunic acid itself (Table 8). In addition, it was observed that multidrug-resistant cells were not cross-resistant to any of the prepared artesunic acid dimers. This result indicates that artemisininderived dimers could be utilized to successfully combat multidrug-resistance, which is an ever-growing problem in cancer therapy. No IC50 values are presented in Table 8 for triazole-linked dimers 19a-e and 20a/b69 and dimers 21/22 (Figure 3)70, which contain aromatic subunits. Rather, their biological activity was determined by measuring the in vitro growth inhibitory effect at fixed concentrations (10, 20 and 50 µM) on the following cancer cell lines: 46 ACS Paragon Plus Environment

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HEP-2 (liver cancer), THP-1 (leukemia), HCT-15 (colon cancer) and A-549 (lung cancer). PC (prostate cancer), IMR-32 (neuroblastoma), Hela (cervical cancer) and MCF-7 (breast cancer). In these studies, it was possible to demonstrate that artemisinin-derived dimers can indeed compete with standard anti-cancer drugs, such as 5-fluorouracil, mitomycin and taxol. For example, triazole-linked dimer 20a, which was the most potent dimer against the HCT-15 colon cancer cell line, exhibited a growth inhibition of 75% at a concentration of 10 µM, which was higher than that of 5-fluorouracil (65% GI at 20 µM).69 Dimers 19e, 20a/b exhibited remarkable growth inhibition against the THP-1 leukemia cell line, with values ranging from 82 to 97% at a concentration of 50 µM. These values were again comparable with 5-fluorouracil (73% GI at 20 µM). Dimer 19e demonstrated very promising results (71% GI at 50 µM) against the HEP-2 human liver cancer cell line, exhibiting efficacy comparable to that of mitomycin (58% GI at 10 µM). Finally, dimers 21a-d and 22a-d showed remarkable growth inhibitory activities against the A-549 human cancer cell line, with values in the range of 56-77% at a concentration of 10 µM. These values were superior to that of artemisinin (26% GI at 10 µM) and comparable with that of taxol (56% GI at 10 µM).70 These results indicate that in contrast to antimalarial activity, anti-cancer activity is not impaired when the dimer contains rigid linkers, such as aromatic subunits (dimers 20-22) or unsaturated C-C bonds (alkyne; dimers 21/22). 47 ACS Paragon Plus Environment

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4.1.2 Anti-cancer activity of dimers with nonsymmetric linkers Dimers 25-28 (Figure 3) were tested against four human solid tumors (SK-MEL, KB, BT-549 and SK-OV-3) and two noncancerous mammalian cell lines (Vero and LLC-PK11) (Table 9).61 Although dimers 25 and 26 are inactive, dimers 27 and 28 exhibited moderate to very good activity (IC50 values up to 0.08 µM (BT-549)). Against melanoma (SK-MEL) and oral cancer (KB), alcohol 28 was considerably more active than olefin 27. Therefore, as in the case of antimalarial activity, a free hydroxyl group appears to be important for biological activity. Furthermore, hybrid 28 is 17-fold more active than paclitaxel against SK-MEL cell lines. Dimer 31 (Figure 4), in which betulin was applied as a natural product linker, was tested in vitro against leukemia cells (CCRF-CEM) and its multidrug resistant subline (CEM/ADR5000).64 Dimer 31 possessed an IC50 of approximately 43 µM against both cell lines and was more active than its parent compound betulin. However, this dimer was significantly less active than artesunic acid. Table 9. In vitro anti-cancer activity of dimers 27, 28 and 31 with nonsymmetric linkers, sorted from highly active (blue), active (green) to less active (red) Reference compounds doxorubicin61,64

Activity (IC50 in µM) 0.037 – > 5.2 nc (not cytotoxic)

Cell Line CCRF-CEM, CEM/ADR5000, SK-MEL, KB, BT-549, SK-OV-3 Vero

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TC50 = 1.29 0.02 – 4.10 TC50 = 0.53

LLC-PK11 SK-OV-3, BT-549, KB, SK-MEL Vero

betulin artemisinin64

TC50 = 4.39 50.8 ± 16.0 26.9/36.9

LLC-PK11 CCRF-CEM, CCRF-CEM CEM/ADR5000

artesunic acid64

1.2/1.8

CEM/ADR5000, CCRF-CEM

taxol61 64

Comp 2861 2861 2861 2761 2761 Comp 2761 2761

Activity (IC50 in µM) 0.08 0.16 0.24 0.27 2.58 Toxicity (TC50 in µM) 0.43 > 16.11

Cell Line

Comp

BT-549 KB SK-MEL KB BT-549

2761 3164 3164 2761 2861

Cell line

Comp

Vero LLC-PK11

2861

Activity (IC50 in µM) 12.93 42.1 ± 5.9 42.9 ± 6.9 na (not active) na Toxicity (TC50 in µM) > 1.57 0.10

Cell Line SK-MEL CEM/ADR5000 CCRF-CEM SK-OV-3 SK-OV-3 Cell line Vero LLC-PK11

4.2 Anti-cancer activity of dimers accessible via direct reaction of two artemisinin derivatives In this section, the activities of symmetrical and/or non-symmetrical dimers 3244 (Figure 5) against various cancer cell lines are discussed (Table 10). Many of these dimers are very active, with IC50 values below 1 µM. One of the most active compounds was amide dimer 38a, which exhibited extraordinary activity against breast cancer (IC50 (MCF-7) = 0.008 µM).74 It is worth mentioning that this compound is very similar in structure to amide dimer 35 (Figure 5), which was the most active compound against malaria parasites (Table 3). Beyond the antimalarial activity of amide-linked dimer 35, its anti-cancer potential was also investigated.

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With an IC50 value of 0.10 µM towards HL-60 cells, it was significantly more active than dihydroartemisinin (IC50 of 2.41 µM).60 Interestingly, dimer 38b, which differs from dimer 38a only in terms of linker length, has a very low activity (IC50 (MCF-7) > 30.2 µM). It appears that an increase in linker length reduces the cytotoxicity potential. In a second study, hybrid 38a was investigated against five different cancer cell lines (non-small cell lung carcinoma (A-549), adenocarcinoma (SK-V-3), malignant melanoma (SK-MEL-2), central nerve system tumor (XF498) and colon adenocarcinoma (HCT-15)) to further examine the biological activity of this dimer.68 Dimer 38a was more active than artemisinin against all five cell lines but was less active than dihydroartemisinin and doxorubicin. As one of 21 trioxane derivatives, dimer 38a was additionally tested for its in vivo activity in a chorioallantoic membrane (CAM) assay.67 In this assay, it is determined how the derivatives inhibit angiogenesis in a chicken embryo membrane, which is used as a measure for activity against tumor invasion and metastasis. Compared to artemisinin, dihydroartemisinin (25% inhibition each), (-)-fumagillin (57%) and (-)-thalidomide (50%), dimer 38a (43%) showed moderate activity. Sulfur-linked dimer 40 (Figure 5) was examined for its anti-cancer potential and showed high activity against the MCF-7 cell line (IC50 of 0.03 µM) as well as P388, EL4 and HT-29, with IC50s lower than 1 µM.74 Moreover, dimer 40, with an IC50 of 50 ACS Paragon Plus Environment

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9.3 µM, had a higher antitumor potential than taxol (IC50 of 13.1 µM), 5-fluorouracil (17.3 µM) and cisplatin (14.7 µM) against the oral cancer cell line YD-10B.99 In a third assay, dimer 40 showed similar efficiency as compound 38a in terms of its effect on A-549, SK-V3, SK-MEL-2 and XF498 cells (IC50 values between 4.9 and 7.6 µM).68 Tested in vivo, dimer 40 displayed harmful cytotoxicity, and most of the embryos of the CAM assay died at the given concentration.67 This example illustrates that activity is not the only important factor when searching for new drug candidates for cancer treatment. Although dimer 40 was one of the most active compounds described in this section, its toxicity is problematic for future application. Concerning the anti-cancer potential of the dihydroartemisinin dimers 37a/b (Figure 5), studies have demonstrated that stereochemistry plays a crucial role for dimer activity (Table 10).73,100This conclusion was reached by applying the MTT assay and comparing the activity of α,β-dimer 37a (IC50 of 0.35 µM), β,β-dimer 37b and the reference compound artemisinin against EN2 tumor cells. Specifically, dimer 37a is > 20-fold more effective than dimer 37b (7.2 µM). Both of the dimers were more active than artemisinin (IC50 of 11.5 µM).73 Introducing a guanidine moiety into artemisinin dimers was intended to avoid the solubility problems of artemisinin-derived hybrids that generally occur during in vivo experiments.75 In this study, the in vitro activity of the artemisinin51 ACS Paragon Plus Environment

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guanidine dimers 41a-h (Figure 5) was examined against three cancer cell lines: human non-small cell lung cancer (A-549), human colorectal cancer (HT-29) and human breast cancer (MDA-MB-231). All eight hybrids possessed higher activity than the positive control dihydroartemisinin in all three cell lines. Dimers 41a-h were generally highly active against HT-29 cells and effective against A-549 cells. However, these dimers possessed only moderate activity against MDA-MB-231 cells. Regarding all three cancer types, dimer 41f was the most promising candidate among the investigated compounds, with IC50 values of 0.24 µM for A-549, 0.02 µM for HT-29 and 1.42 µM for MDA-MB-231 cells. The anti-cancer potential of trioxane dimers 42a/b (Figure 5) against HCT-15 cells (colon cancer), A-549 cells (lung cancer) and THP-1 cells (leukemia) was determined, and both hybrids inhibited the growth of colon HCT-15 cells 2-fold more (63% for dimer 42a and 70% for dimer 42b) than artemisinin (31% growth inhibition).69 Dimer 42b also surpassed artemisinin regarding the activity against lung A-549 cells (48% compared to 29%) and shows similar activity as 5-fluorouracil for leukemia THP-1 cells (79% for 50 µM of dimer 42b and 73% for 20 µM of 5-fluorouracil). The four dimers 43b-e (Figure 5) were examined for the activity against the colon cancer cell line Caco-2. As their IC50 values ranged between 2.04 and 8.03 µM, the hybrids were at least 3-fold more active than dihydroartemisinin (IC50 52 ACS Paragon Plus Environment

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of 26.87 µM) (Table 10).76 The results of this study indicated that the application of a

(2-aminoethyl)glycine

backbone

connected

to

a

C-10

non-acetal

deoxoartemisinin yielded hybrids that were active against Caco-2 cells (IC50 value of 2.87 µM for dimer 43b). This effect can subsequently be improved upon by the introduction of other amino acids, such as lysine, to further increase the hybrid’s activity (IC50 of 2.04 µM for hybrid 43d). Dimers 44a-f (Figure 5) were tested against the two human breast cancer cell lines MDA-MB-231 and BT-474, as well as against the nontumorigenic breast cell line MCF-10A (Table 10).77 Regarding both cancer cell lines, all of the hybrids 44a-f were more active than artemisinin. Additionally, dimer 44f showed similar activity towards BT-474 cells as artemisinin dimer succinate (IC50 of 0.096 and

0.10 µM,

respectively),

with

a

selectivity

of

490

(Selectivity =

IC50 (MCF-10A)/IC50 (BT-474)). Table 10. In vitro anti-cancer activity of dimers 37-41, sorted from highly active (blue), active (green) to less active (red) Reference compounds

Activity (IC50 in µM)

Cell line

HL-60, P388, EL4, Bewo, HT-29, MCF-7, A-549, SK-V3, SK-MEL-2, XF498, HCT-15 taxol74,99 0.0001 – 13.1 MCF-7, HT-29, EL4, P388, Bewo, YD-10B mitomycin74 0.06 – 11.8 HT-29, Bewo, MCF-7, P388, EL4 artemisinin68,73,100 11.5 – 723 EN2 tumor cells, SK-MEL-2, A-549, SK-V3, XF498, HCT-15, murine EAT HCT-15, A-549, SK-V3, SK-MEL-2, XF498, HL-60, HT-29, MDA-MB-231, dihydroartemisinin60,68,75,76 0.46 – 26.87 Caco-2 77 artesunate 7.8 – 46 BT-474, MCF-10A, MDA-MB 231 Activity Activity Activity Comp (IC50 in Cell line Comp (IC50 in Cell line Comp (IC50 in Cell line µM) µM) µM) MDA-MB38a74 0.008 MCF-7 41h75 1.26 7.7 KB 34f56 231 doxorubicin60,68,74

0.018 – 10.8

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

41f75

HT-29

34d56

0.03

MCF-7

41g

75

1.33

41b75

0.05

HT-29

41f75

1.42

41d75

0.06

HT-29

41e75

1.57

40

0.02

74

75

77

1.3

41g 44f77

0.09 0.096

HT-29 BT-474

44e 43d76

1.7 2.04 ± 0.31

3560

0.10 ± 0.07

HL-60

41b75

2.08

76

75

41a 41h75 41c75 41c75

0.10 0.10 0.17 0.24

HT-29 HT-29 A-549 HT-29

43b 34g56 4068 43e76

2.87 ± 0.2 3.5 3.53 3.58 ± 0.98

41f75

0.24

A-549

37a100

3.6 ± 0.35

37a73 41e75 4074 4074 41e75 4074 41b75 41d75 41a75 44b77 44d77 41h75 38a74

0.35 ± 0.07 0.36 0.37 0.39 0.40 0.64 0.64 0.68 0.72 0.79 0.88 0.98 1.1

EN2 tumor cells A-549 EL4 HT-29 HT-29 P388 A-549 A-549 A-549 BT-474 BT-474 A-549 HT-29

34d56 34g56 38a68 44a77 4068 44b77 4068 4068 34e56 44c77 38a68 44c77 44a77

4.0 4.0 4.36 4.6 4.86 5.0 5.44 5.63 5.7 5.7 5.76 5.8 6.6

41a75

1.17

MDA-MB-231

37b73

7.2 ± 1.7

41d75

1.18

MDA-MB-231

34f56

7.4

34e56 44f77

1.2 1.2

BC MDA-MB-231

4068 38a68

7.56 7.63

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38a68

BC MDA-MB231 MDA-MB231 MDA-MB231 BT-474 Caco-2 MDA-MB231 Caco-2 BC HCT-15 Caco-2 murine EAT cells KB KB SK-V3 MCF-10A SK-V3 MCF-10A XF498 A-549 KB MCF-10A XF498 BT-474 BT-474 EN2 tumor cells

7.83

HCT-15

8.03 ± 0.42

Caco-2

3974

8.9

HT-29

4099

9.3

YD-10B

38a 38a74

9.94 10.1

SK-MEL-2 EL4

44b77

43c

76

68

11

MDA-MB-231

77

44c 44d77 38a74 44d77

12 12 12.1 15

MDA-MB-231 MDA-MB-231 Bewo MCF-10A

38a74

16.8

P388

44a77 3974 3974 3974 4074 34c56 44e77 44e77 3974 38b74 38b74 38b74 38b74

19 20.9 21.3 22.5 22.8 23 25 26 > 27.3 > 30.2 > 30.2 > 30.2 > 30.2

MDA-MB-231 MCF-7 P388 EL4 Bewo KB MCF-10A MDA-MB-231 Bewo P388 EL4 Bewo MCF-7

38b74

30.5

HT-29

BC

37b100

31.2 ± 3.8

SK-MEL-2 A-549

34c56 44f77

36 47

murine EAT cells BC MCF-10A

Table 10. (continued) Comp 34c56 34d56

Toxicity (TC50 in µM) 63 2.5

Cell line Vero Vero

Comp 34e56 34f56

Toxicity (TC50 in µM) 2.4 1.1

Cell line Vero Vero

Comp 34g56

Toxicity (TC50 in µM) 5.5

Cell line Vero

4.3.1 Anti-cancer activity of dimers obtained via post-modification of olefin 4 In addition to being highly active against malaria, dimers derived from olefin 4 also proved to be very promising anti-cancer agents. As in the case of malaria, 54 ACS Paragon Plus Environment

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alcohol dimer 45a (Figure 6) was among the most potent compounds, with IC50 values of 9.2-23.3 nM against prostate cancer (C2H, C2G, C1A and C2D),78 20/50 nM towards leukemia cells (CCRF-CEM and CEM/ADR5000),98 43 nM against breast cancer (MTLn3)87 and 0.1 µM towards colon cancer (HCT-116) (Table 11)101,102. These activities are only a subset of the observed beneficial effects of 45a. Alcohol dimer 45a was even more active than doxorubicin (IC50 = 28.7-75.9 nM; C2H, C2G, C1A and C2D), a well-known chemotherapy, and therefore can be considered a potentially useful anti-cancer drug candidate.78 Interestingly, ferrocene-containing dimer 57, which was less active than the other dimers against malaria, was one of the most active anti-cancer compounds, with an IC50 of 10 nM against CCRF-CEM leukemia cells.71 A similar result was observed for the ferrocene dimers 23 and 24 (Figure 3). Consequently, in contrast to antimalarial efficacy, a ferrocene moiety appears to have a positive effect on anti-cancer activity and should be considered in future investigations. As discussed in sections 3.1.1 and 4.1.1, organophosphate dimers were among the most active compounds. Therefore, a series of other phosphate dimers (52a-i) were synthesized (Figure 6). All of these dimers were derived from olefin 4 and were tested in vitro against the following various cancer types: leukemia (Jurkat T-ALL),85

prostate

(HCT-116),101,102

cancer

cervical

(LNCaP,

C1A

adenocarcinoma

and (HeLa)

C2H),84 101,102

colon and

cancer

melanoma 55

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(1205Lu).102 Furthermore, the toxicity of 52a and 52c-j was determined via their application to either non-malignant peripheral blood mononuclear cells (PBMC)85 or HFF cells.101,102 All of the phosphate dimers, with the exception of 52j, were highly active against the tested cancer cell lines, with IC50 values below 1 µM (0.0319-0.511 µM). Only dimers 52f, 52i exhibited slightly lower anti-leukemia activities (Jurkat T-ALL), with IC50 values of 1.75 µM and 1.933 µM, values that can still be considered potent. In addition, only dimers 52a/g/h were slightly toxic against PHA-stimulated PBMC cells (IC50 = 1.477-2.281 µM), but dimer 52a showed no toxicity towards HFF cells (IC50 >50 µM). All other phosphate dimers showed no toxicity whatsoever (IC50 (PBMC) > 50 µM). These studies provide useful information regarding the relationship between chemical structure and biological activity (SAR). For instance, the monophenylphosphate ester dimer 52j was up to 100-times less potent against Jurkat, HeLa and HCT-116 cells than the diphenylphosphate ester dimer 52a. This effect might be explained by the increased hydrophilic character of hybrid 52j, resulting in a diminished ability to penetrate these cancer cells. Other changes, such as replacement of the phosphoryl oxygen (52f/i) with sulfur or altering the substituents on the phenyl subunits (52a and 52c-e), also had very large impacts on biological activity. For example, dimers 52f and 52i were approximately 9- to 18-fold less active against Jurkat cells than their oxygenated counterparts. In the case of leukemia cells, the most 56 ACS Paragon Plus Environment

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promising results were achieved with 4-chloro-phenyl-substituted dimer 52d.This dimer was not only remarkably active against leukemia cells (IC50 = 152 nM) but also inactive against PHA-stimulated PBMCs (IC50 > 40000 nM). Therefore, this dimer exhibits a therapeutic index of > 263 (IC50 (PBMC) / IC50 (Jurkat cell)), approximately 10-fold higher than that of doxorubicin. Therefore, again, and in contrast to antimalarial efficacy, an aromatic subunit with the proper substitution pattern appears to be beneficial for anti-cancer activity. This fact can also be seen in the case of the secondary amine dimer 48c (Figure 6), which contains a phenyl substituent. This dimer was among the most effective compounds and inhibited LNCaP cell growth more potently (GI50 = 17.9 nM) than the standard cytotoxic agent doxorubicin (GI50 = 45.3 nM) (Table 11).84 Dimer 52a proved to be the most potent phosphate dimer, exhibiting outstanding IC50 values of 0.04 µM against HCT-116 cells and 0.19 µM against HeLa cells, being up to approximately 300-fold more active than artesunate and up to 6‑fold more effective than the primary alcohol dimer 45a.84 Furthermore, dimer 52a was not only nontoxic to primary HFFs (IC50 = 56.69 µM) (unlike its monomeric counterpart (IC50 = 4.88 µM)) but also more potent, exhibiting up to 100-fold higher activity than its monomeric analogue. Overall, phosphate ester dimers can be regarded as remarkable anti-cancer and antimalarial agents, demonstrating the great potential of dimerization. 57 ACS Paragon Plus Environment

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Encouraged by the promising in vivo antimalarial results achieved with sulfone trioxane dimers 51b/c (Figure 6), Posner and co-workers examined both dimers in vitro for their cytotoxic activity against five different cancer cell lines: U-937 (lymphoma), HL-60 (leukemia), SK-MEL-5 (melanoma), UACC-62 (melanoma) and HeLa (cervical) (Table 11).103 Both of the dimers were very effective against all tested cancer cell lines, with IC50 values ranging from 0.03-1.1 µM. Thus, these dimers are comparable in terms of anti-cancer potency to the clinically used anti-cancer drug doxorubicin. However, the sulfone dimers 51b/51c were more favorable as they did not significantly affect the noncancerous fibroblast cell lines (WT-MEF and Hs888Lu). In contrast, doxorubicin was found to be toxic against these cells, with IC50 values of 3.4 µM (WT-MEF) and 1.4 µM (Hs888Lu). The two trioxane dimers containing a pyridine moiety (53a and 53b) (Figure 6) were investigated in vitro for their effects on growth inhibition and apoptosis in four

different

PCa

cell

lines:

C4-2,

DU145,

LNCaP

and

PC-3.86

Dihydroartemisinin, tested as a control, had very little effect on cell proliferation in PCa cells under the conditions used in this study. In contrast, the synthesized dimers 53a and 53b at both tested concentrations (10 and 25 µM) significantly reduced cell number after 72 h in all three tested cell lines (C4-2, LNCaP and PC-3). The best result was achieved by dimer 53b at a concentration of 25 µM. In

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this condition, the cell number in all three cell lines (C4-2, LNCaP and PC-3) decreased to approximately 15% of the control cell number after 72 h. Artemisinin-derived dimer 54 (Figure 6), which is very similar in structure to dimer 53b (the most active compound in the aforementioned study) was investigated for its anti-cancer efficacy on rat mammary adenocarcinoma cells (MTLn3) in vitro and in vivo.87 The IC50 values of dimer 54 (43 nM), its precursor 45a (52 nM) and the monomer dihydroartemisinin (360 nM) against MTLn3 cells clearly demonstrate the great potential of artemisinin-derived dimers. Both dimers 54 and 45a, which are similar in activity, exhibit a 7-fold higher potency in vitro than dihydroartemisinin. In addition, both dimers (p < 0.01) significantly retarded the growth of MTLn3 tumors in rats and were more potent than dihydroartemisinin (p < 0.05). Surprisingly, the effect of bis-trioxane primary alcohol dimer 45a on tumor size appeared to be longer lasting.

Table 11. In vitro anti-cancer activity of dimers 45-47, 48, 51, 52, 56, 57, sorted from highly active (blue), active (green) to less active (red) Reference compounds

Activity (IC50 in µM) 0.0096 – 23.27

doxorubicin71,77,78,83,84 gemcitabine78 artemisinin71 dihydroartemisinin71,87 artesunate102

TC50 = 1.4 ± 0.7 TC50 = 3.4 ± 1.3 0.0037 – 0.0119 26.90/36.90 0.36 – 0.68 7.1 – 42 TC50 = 71.7 ± 4

Cell Line Jurkat T-ALL, PBMC*, C1A, C2D, C2G, C2H, LNCaP, CCRF-CEM, CEM/ADR5000 Hs888Lu WT-MEF C1A, C2D, C2G, C2H CEM/ADR5000, CCRF-CEM MTLn3, CEM/ADR5000, CCRF-CEM HeLa, HCT-116, 1205Lu HFF

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45a78

Activity (IC50 in µM) 0.0092

5771

0.01 ± 0.01

Comp

Cell Line

Comp 5487

Activity (IC50 in µM) 0.052 ± 0.001

51c83

0.06 ± 0.02

55 45a78 48c84 48c84

0.013

C2H CCRFCEM BT-474

0.0154 GI50 = 0.0170 GI50 = 0.0179

48c84

GI50 = 0.0180

88

45a

78

45a

98

0.0185

0.27 ± 0.03 0.28 ± 0.15 0.28 ± 0.13 0.287 ± 0.01 0.296 ± 0.037 0.39 ± 0.27 0.511 ± 0.06 3 0.56 ± 0.18 1.06 ± 0.2 1.75 ± 0.132 1.933 ± 0.278 1.96 ± 0.66 3.3 ± 0.7 4.9 ± 0.04 5±2 6.67 ± 0.7 7.436 ± 0.54 8±3 10 ± 3 18.56 ± 1.2 32 ± 4

HeLa U-937 U-937 Jurkat T-ALL

C1A

51b102

0.07 ± 0.005

HCT-116

52e85

HCT-116

45c

89

52c

85

C1A

0.05 ± 0.02

C2D

102

C2G C2H LNCaP

GI50 = 0.0319

45a98

0.2314

HL-60

51c 51b83 51c83 52g85

52a84

GI50 = 0.0344 GI50 = 0.0363 0.0361 GI50 = 0.0394 0.04 ± 0.005 0.04 ± 0.02 GI50 = 0.0422 0.043 ± 0.001 GI50 = 0.0448 0.0474

4678

MTLn3

SK-MEL-5 1205Lu BT-474 C2D

0.022 ± 0.009 0.0233 0.03 ± 0.01

52a 52b84 4778 52b84 52a101,102 51c83 52b84 45a87 52a84 4778

Jurkat T-ALL

Comp

0.06 ± 0.03 0.06 ± 0.002 0.06 ± 0.03 0.0621

56b89 45a78 51b83

84

52h85

Activity (IC50 in µM) 0.22 ± 0.016

Cell Line

51c 52a102 56a89 4778

C2D CCRFCEM BT-474 C1A UACC-62

0.02

83

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LNCaP LNCaP C2H C2H HCT-116 UACC-62 C1A MTLn3 C2H C2G CEM/ADR 5000

51c

102 89

56d

0.07 ± 0.006 0.07 ± 0.01

BT-474

4778 45a101,102 52a85

0.0846 0.1 ± 0.001 0.1 ± 0.009

C1A HCT-116 Jurkat T-ALL

51b102 45a101,102 52f85

51b102

0.1 ± 0.006

1205Lu

52i85

83

51b 51c83 56c89 4678 51c102 51b83 4678 51b83 52d85 4678

1.1 ± 0.95 0.11 ± 0.06 0.11 ± 0.02 0.1160 0.13 ± 0.007 0.13 ± 0.09 0.1340 0.14 ± 0.09 0.152 ± 0.007 0.1587

HeLa HeLa BT-474 C2H 1205Lu HL-60 C2G SK-MEL-5 Jurkat T-ALL C1A

52a101,102

0.19 ± 0.08

HeLa

71

57 56c89 45a102 56b89 52j101 52j85 56a89 56d89 52j101 45c89

Cell Line

Jurkat T-ALL BT-474 Jurkat T-ALL HeLa HeLa Jurkat T-ALL Jurkat T-ALL CEM/ADR5000 MDA-MB231 1205Lu MDA-MB231 HCT-116 Jurkat T-ALL MDA-MB231 MDA-MB231 HeLa MDA-MB231

Table 11. (continued) Comp

Toxicity (TC50 in µM)

Cell line

Comp

Toxicity (TC50 in µM)

Cell line

Comp

Toxicity (TC50 in µM)

Cell line

51b102 45a102 52a102 52a101 52j101 51c102 51b83

4.9 ± 0.2 48.1 ± 2.6 55.8 ± 2.8 56.69 ± 4.69 56.49 ± 3.24 57.5 ± 2.9 > 50

HFF HFF HFF HFF HFF HFF WT-MEF

51b83 51c83 51c83 52a85 52g85 52h85 52c85

> 50 > 50 > 50 1.477 ± 0.639 2.031 ± 2.192 2.281 ± 0.710 > 40

Hs888Lu WT-MEF Hs888Lu PBMC* PBMC* PBMC* PBMC*

52d85 52e85 52f85 52i85 52j85

> 40 > 40 > 40 > 40 > 40

PBMC* PBMC* PBMC* PBMC* PBMC*

*PHA-stimulated

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Interesting results were achieved with a transferrin (Tf) conjugate of hydrazide dimer 55, which was analyzed for its anti-cancer potential on different breast cancer cells.88 Treating the BT-474 breast cancer cell line with both Tf conjugates resulted in the down-regulation of survivin, c-MYC and mutated human epidermal growth factor receptor-2 (ERBB2 or HER2). Furthermore, the Tf conjugate of dimer 55 significantly inhibited the growth of BT-474 cells, with an IC50 value of 13 nm. This dimer exhibited no toxicity towards the normal breast cell line MCF-10A and was approximately 30-fold more effective than a corresponding artemisinin-derived hydrazide monomer (IC50 = 440 nM). In addition, almost 100% of the BT-474 cells were killed by applying the Tf conjugate of dimer 55 at a concentration of 0.1 µM. This concentration was still non-toxic to healthy MCF-10A cells. Therefore, the synthesized Tf dimer conjugate derived from hydrazide 55 can be considered a promising anti-cancer drug candidate. Sasaki and co-workers reported the anti-cancer potential of artemisinin-derived piperazine dimers 56a-d, which possessed a pH-dependent solubility, in two human breast tumor cell lines: BT-474 and MDA-MB231.89 Dimer 56b was the most potent compound against the BT-474 cell line, with an IC50 value of 0.022 µM, whereas piperazine dimer 56c exhibited the highest efficacy towards MDA-MB231 cells, with an IC50 of 3.3 µM. In general, all of the synthesized piperazine dimers 56a-d can be considered highly active against the tested human breast 61 ACS Paragon Plus Environment

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cancer cell line BT-474, with IC50 values ranging from 0.022 to 0.11 µM. An artemisinin-derived monomer resembling the structure of dimer 56d and the carboxylic acid dimer 45c, which served as precursor for the synthesis of dimers 56a-d, were tested as reference compounds. These compounds were shown to be considerably less effective in the inhibition of cell growth of both breast cancer cell lines, with IC50 values in the range of 0.39 to 1.3 µM against BT-474 cells. Surprisingly, the monomer control was completely inactive towards the tested MDA-MB231 cell line, with an IC50 value higher than 100 µM, exceeding the maximum concentration of the assay. 4.3.2 Anti-cancer activity of dimers obtained via post-modification of miscellaneous artemisinin-derived dimers Beyond the antimalarial activity of fluorinated C-10 non-acetal dimers 58a/b (Figure 7), Posner and co-workers also examined their in vitro anti-proliferative efficacy against murine keratinocytes.55 However, the modified dimers 58a/b (IC50 > 15 nM) were less active than their non-fluorinated counterparts 2c-c’’ (IC50 < 4 nM), which is similar to the antimalarial results. Trioxane phthalate hybrid 59a (IC50 (HeLa) = 500 nM) was 10 to 20-fold more potent than the trioxane monomer dihydroartemisinin. The trioxane diol dimer 59c (IC50 (HeLa) = 46.5 nM) was 110 to 220-fold more potent than this monomer, and both dimers were non-toxic to primary normal cervical cells.58 Therefore, diol 59c 62 ACS Paragon Plus Environment

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was again among the most active compounds, similar to what was observed in the antimalarial experiments. The in vitro cytotoxicity of post-modified dimers 60a-e (Figure 7) obtained from dimeric precursors 11a-c (Figure 2) was examined against four human solid tumor cell lines (SK-MEL, KB, BT-549 and SK-OV-3) and two noncancerous mammalian cell lines (Vero and LLC-PK11).61 Among these dimers, the anti-cell proliferative activities of hybrids 60c/d were significantly enhanced compared to the control drug doxorubicin, with dimer 60d showing selectivity towards epidermal carcinoma (KB). In contrary, dimers 60a/b were inactive even at the highest concentration applied in these experiments. Consequently, in this case, hydroxylation did not result in a positive effect on biological activity. Furthermore, succinyl acid dimer 60e and the newly synthesized oxime dimer 60f were investigated for their anti-cancer activity against the following cancer cell lines: HL-60, K562, LOX IMVI, M14, OVCAR-3, SKOV3, A498, Caki, PC-3, DU145, T-47D and MCF-7.104 Both of the dimers proved to be more active than the tested reference compound artemisinin against all 12 human cancer cell types and were up to 1000-fold more potent. Dimer 60f was the most active dimer, with IC50 values up to 0.02 µM (Table 12). These excellent results motivated ElSohly and co-workers to examine the in vivo anti-cancer efficacy of dimer 60e using the hollow fiber assay against a standard 63 ACS Paragon Plus Environment

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panel of twelve human cancer cell lines.62 Based upon the positive results found in the hollow fiber assay, dimer 60e was also further evaluated in a subcutaneous (s.c) xenograft model of the human leukemia cell line HL-60 and was considered active, with optimal% T/C less than 40%. Therefore, a succinyl moiety appears beneficial for biological activity due to its ability to improve water-solubility. Jung and co-workers examined the anti-cancer activity of sulfur-linked dimers 66a/b (Figure 7) against P388, EL4, Bewo, HT-29 and MCF-7 cancer cells (Table 12).74 Regarding EL4 cells, dimer 66a possessed a similar IC50 as doxorubicin. Additionally, sulfur-linked dimer 66a was highly active against MCF-7 cells (IC50 = 0.04 µM). The activity of this dimer is comparable to the compound before it was oxidized (dimer 40 exhibited an IC50 = 0.03 µM (section 3.1)). Dimer 66b showed reduced anti-cancer activity for four of the five cancer cell lines compared to dimer 66a. Therefore, a shorter linkage again appears to be more effective. In addition, dimer 66a was tested in vivo using the CAM assay.67 However, this dimer was toxic, and most of the chicken embryos died. The same assay was performed for fullerene dimer 67 (Figure 7) and its precursor dimer 17.67 Surprisingly, despite its large size and molecular weight of > 1400 g/mol, dimer 67 has similar activity as (-)-fumagillin (57%) and (-)-thalidomide (50%), with an inhibitory effect of 50% on CAM angiogenesis. In contrast, its precursor 17 exhibited only weak activity. 64 ACS Paragon Plus Environment

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Table 12. In vitro anti-cancer activity of dimers 60 and 66, sorted from highly active (blue), active (green) to less active/inactive (red) Activity (IC50 in µM)

Reference compounds

0.17 – 10.76

doxorubicin61,74

TC50 = 1.29 0.0001 – 8.65 taxol61,74 mitomycin74

TC50 = 4.39 TC50 = 0.53 0.06 – 11.78

artemisinin104

40 – > 60

Cell Line

Comp

Activity (IC50 in µM)

60f104 60f104 60e104 60f104 66a74 60f104 60e104

Activity (IC50 in µM) 0.02 0.03 0.04 0.04 0.04 0.055 0.06

PC-3 T-47D PC-3 HL-60 MCF-7 LOX IMVI HL-60

60c61 60c61 66b74 66a74 66a74 66a74 60f104

0.30 0.47 0.50 0.58 0.82 1.59 6.5

60f104

0.06

MCF-7

66b74

7.36

Comp

104

60e 60e104 60f104 60e104 60f104 60e104 60c61 60e104 60d61

0.075 0.08 0.15 0.2 0.2 0.2 0.22 0.25 0.28

T-47D LOX IMVI K562 MCF-7 OVCAR-3 K562 SK-MEL OVCAR-3 KB

74

66a 66b74 66b74 60f104 66b74 60f104 60f104 60f104 60e104

8.55 11.04 11.17 13 13.14 15 15 15 20

Cell Line SK-MEL, KB, BT-549, SK-OV-3, P388, EL4, Bewo, HT-29, MCF-7 LLC-PK11 SK-MEL, KB, BT-549, SK-OV-3, LLC-PK11, P388, EL4, Bewo, HT-29, MCF-7 LLC-PK11 Vero P388, EL4, Bewo, HT-29, MCF-7 HL-60, K562, LOX IMVI, M14, OVCAR-3, SKOV3, A498, Caki, PC-3, DU145, T-47D, MCF-7 Activity Cell Line Comp (IC50 in Cell Line µM) 60e104 BT-549 20 DU145 KB 25 SKOV3 60e104 HT-29 30 M14 60e104 104 60e HT-29 30 Caki 60a61 EL4 na SK-MEL 60a61 Bewo na KB 60a61 A498 na BT-549 MCF-7

60a61

na

SK-OV-3

P388 P388 Bewo Caki EL4 M14 SKOV3 DU145 A498

61

60b 60b61 60b61 60b61 60c61 60d61 60d61 60d61

na na na na na na na na

SK-MEL KB BT-549 SK-OV-3 SK-OV-3 SK-MEL BT-549 SK-OV-3

Comp

Toxicity (TC50 in µM)

Cell line

Comp

Toxicity (TC50 in µM)

Cell line

Comp

60c61 60d61 60a61

> 1.57 2.13 nc

Vero Vero Vero

60b61 60c61 60a61

nc 0.68 nc

Vero LLC-PK11 LLC-PK11

60b61 60d61

Toxicity (TC50 in µM) nc nc

Cell line LLC-PK11 LLC-PK11

5. Conclusion and Outlook

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Every year, malaria causes the death of nearly half of a million people worldwide and is responsible for more than 200 million new infections each year, primarily in the tropics.105 In addition, cancer is one of the leading causes of mortality worldwide, and caused the death of 8.2 million people and 14 million new cases in 2012.106 Drug resistance is a common issue to both diseases. Hence, to overcome drug resistance in cancer and P. falciparum strains, it is important to develop new therapeutics with novel lead structures or with different action spectrums that act selectively with high (or even significantly increased) therapeutic width (to allow higher dosage). One of the approaches to achieve these challenging goals in the fight against malaria is the combined administration of different drug molecules (i.e., combination therapy). Another very promising approach is the dimerization of known bioactive molecules. Since 1997, much attention has been paid to the development of homo-dimers of artemisinin (or other 1,2,4-trioxanes) because of its long-standing use in traditional Chinese medicine.1-5 These dimers appear to exploit already known cell death pathways (Scheme 1). Artemisinin dimers could be more stable under metabolic conditions than monomers. Hence, the drug may remain active in the blood stream for a longer time span and reduce parasitemia to crucially lower levels. This hypothesized pharmacokinetic effect is evidenced by the correlation of stability and activity in C-10 non-acetal (more stable against 66 ACS Paragon Plus Environment

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hydrolysis and more active) vs. C-10 acetal (less stable and less active) dimers. Additional attention has been paid in the present review on the role that the linker itself plays in dimer activity. Linker units containing phosphate esters (e.g., 8a-c) were found to be particularly active against malaria strains. These dimers have significantly increased potency compared to monomers. Much more recently, artemisinin has been recognized to inhibit tumor growth.13 Although the mechanism of the anti-tumor activity of artemisinin monomers themselves is not yet fully understood, the resulting dimers were occasionally found to have a tumor growth inhibiting capability that was several orders of magnitude higher than monomers.107 This remarkable behavior was attributed to multivalent binding to receptors (i.e., polyvalency) or to the DNA (itself dimeric) sequence-specific transcription factor NF-kappaB. This transcription factor plays a major role in cellular signaling pathways and in the immune system response. In addition, NF-kappaB was very recently determined to be a major player in several types of cancer.108 As in the case of dimers that are active against malaria the structure of the linker moiety proved to be essential for anti-cancer efficacy. The artemisinin homodimers 28 and 45a, which carry a free hydroxyl group in their linkers, are more active than similar compounds lacking an OH group (e.g., 27 and 46, respectively).

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Another interesting feature of dimers – in contrast respect to monomers - is their occasionally reduced toxicity, which can be significant. This characteristic may imply that the metabolic pathways in healthy cells and which are affected by the monomers, are less affected by the dimers. The active centers of enzymes, which may be inhibited by monomers, might not be blocked by the usually much larger dimers. It remains to be determined whether this assumption holds true in clinical trials, considering that hepatotoxic effects are observed in many known drugs. A further interesting observation is that the stability of the linker (C-10 nonacetal vs. acetal) does not appear to be of comparable importance for anti-tumor activity. This effect may be explained by the different point of attack for antimalarial and anti-cancer agents. Although the former act on parasites in the blood, the latter interferes with intracellular metabolism, where hydrolysis does not appear to be a major issue. In summary, considerable progress has been made with the rather recently developed dimer approach, which has already found applications in the development of new effective anti-cancer and antimalarial agents. However, several challenges must be overcome before these novel drugs can be applied in the clinical. The pharmacokinetics and pharmacodynamics (e.g., potential side effects) of the dimers under physiological conditions must to investigated. In addition, little is known regarding the factors that control stability in metabolic and detoxification 68 ACS Paragon Plus Environment

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pathways. Furthermore, the mode of action of artemisinin as an anti-cancer agent should be explored, whereas this issue appears to be sufficiently resolved with respect to its activity against malaria. One salient observation common to both potential applications is the occasionally dramatic (i.e., much more than double) improvement in activity of dimers with respect to monomers against parasites or cancer cells. Furthermore, the toxic doses (TC50) in many cases the same or even increased. As mentioned above, this effect might be due to the different metabolism of monomers and dimers. This characteristic of dimers results in a higher therapeutic width, allowing physicians to administer higher, and more effective, drug doses if necessary. Drug resistance may lead to the failure of a therapy, and resistance is a known characteristic of malignant cells, organisms, and viruses due to adaptation through mutation or inherently reduced sensitivity. As a consequence, resistant species could thrive and proliferate, whereas non-resistant species perish (i.e., the selection principle). Drug efficacy might be more prone to be impaired by resistance when the drug acts in a very specific way, e.g., by a single point of attack or in a single mode of action. Such mechanisms of action include blocking a single enzyme or the production of a single protein. Dimers could therefore offer at least a temporary relief from resistance issues (e.g., parasitemia) before the population of organisms have "learned" and re-acquired resistance. Resistance in tumor cells is more 69 ACS Paragon Plus Environment

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complicated and apparently involves the detoxification pathways of the tumor itself.109 Tumor cell populations are not fully homogenous, and drug sensitivity might differ in a population of tumor cells, allowing less sensitive cells a higher chance of surviving chemotherapy. The detoxification effects of tumor cells can be mediated by trans-membrane transport proteins, such as P-glycoprotein (P-gp) or MRP (multidrug resistance-associated protein). It is known that dimers of bioactive molecules might block the P-gp transport.107 A similar mechanism could also conceivably be true for artemisinin dimers. More research is necessary to elucidate the exact mechanistic details of the anti-cancer activity of artemisinin dimers. The full potential of the dimerization concept is far from being exploited. Almost all dimers (except, e.g., 3) are modified at the C-10 position of artemisinin. Further potential might be found in dimers formed by attaching the linker to other positions in the monomers. The linker itself has been shown to play an active role in bioactivity. More detailed investigations to elucidate the role of linker moieties are called for, especially regarding the influence of dimer stereochemistry on bioactivity. Indeed, some studies have indicated that not only the length and nature of the linker but also the stereochemistry at the C-10 position of artemisinin derivatives play significant roles in dimer activity. The dimer approach might also be further extended to other types of dimers (with different monomers, i.e., artemisinin heterodimers). 70 ACS Paragon Plus Environment

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As it is difficult to predict the activity of dimers a priori, structure-activity relationship analysis, such as those presented here, could be a useful in informing the design of effective and selective drugs that possess improved anti-cancer or antimalarial activity (or even both). Compounds could also be identified that are not toxic to normal cells and which have fewer or less harmful side effects. The dimer approach might also be beneficial for the treatment of other forms of cancer, or e.g., DNA-virus diseases (possibly because of the involvement of interference with the NF kappaB factor).71,85,98,101,102,108,110 Despite tremendous progress in the development of artemisinin derived synthetic dimers as candidates for drugs to cure malaria or certain forms of cancer, some challenges remain before clinical applications can be considered: i) elucidation of the underlying principles by which the linker structure and the dimerization itself influence drug activity and toxicity (especially with respect to the monomers); ii) investigation of the pharmacokinetics and metabolic pathways in which dimers (vs. monomers) are involved; and iii) investigation of other attributes (e.g., therapeutic width, side effects, drug resistance, administration, shelf life, formulation) that will be necessary to understand for the clinical trials that may bring the above-described compounds into the clinic. As this review has shown, due to their expected superior performance and reduced toxicity, some artemisinin dimers possess the potential to complement or even replace standard drugs in the treatment of malaria or cancer. 71 ACS Paragon Plus Environment

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Biographies Tony Fröhlich was born in Fürth (Germany) in 1989. He received his M.Sc. degree in Molecular Science from the University of Erlangen-Nuremberg in 2013. The same year he began his graduate studies under the supervision of Prof. S. B. Tsogoeva. His area of research interest includes the synthesis of novel artemisinin-based hybrids as potent anti-cancer and antimalarial agents.

Aysun Çapcı Karagöz was born in 1984 in Zonguldak (Turkey). She received her B.Sc. and M.Sc. degrees in Chemistry from the Ege University. In 2013, she began her doctoral studies under the supervision of Prof. S. B. Tsogoeva at the University of Erlangen-Nuremberg. Her research is focused on the development of novel natural product hybrids as potent antimalarial and anti-cancer therapeutics. Christoph Reiter was born in Stuttgart (Germany) in 1985. He studied chemistry at the University of Stuttgart where he received his Diploma degree in 2010. In 2011, he joined the group of Prof. S. B. Tsogoeva at the University of ErlangenNuremberg. His research focuses on artemisinin-derived natural product hybrids as potent agents against viruses, malaria and cancer as well as on asymmetric organocatalysis.

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Svetlana B. Tsogoeva was born in 1973, studied chemistry at St. Petersburg State University, where she completed her doctoral thesis in 1998. Then, she moved to the Johann Wolfgang Goethe-University, Frankfurt am Main, for a postdoctoral research. In July 2000 she joined the Degussa AG Fine Chemicals Division as a research scientist. In January 2002 she was appointed a first junior professor in Germany at the Georg-August-University of Göttingen. Since February 2007, she is professor of organic chemistry at the Friedrich-Alexander-University of ErlangenNuremberg. Her research is currently focused on organocatalytic multi-component domino reactions, asymmetric oxidations with non-heme iron complexes and synthesis of natural product hybrids with anti-cancer, antiviral and antimalarial activities.

Acknowledgment S.B.T. is grateful to the Wilhelm Sander-Stiftung (Grant Nr. 2014.019.1), Interdisciplinary Center for Molecular Materials (ICMM) and „Dr. Hertha & Helmut Schmauser-Stiftung“ for generous research support. Financial support by the German Academic Exchange Service DAAD (doctoral research fellowship for Aysun Çapci Karagöz) is also gratefully acknowledged. Abbreviations

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1205Lu, melanoma cell line; A-549, non-small cell lung carcinoma cell line; ACHN, renal cancer cell line; ACT, artemisinin-based combination therapy; Bewo, placental chloriocarcinoma cell line; BC, breast cancer cell line; BT-474, BT-549, breast cancer cell line; C1A, C2D, prostate nonturmorigenic cell line; C2G, C2H, prostate tumorigenic and metastastic cell line; C4-2, prostate cancer cell line; CAM, Chorioallantoic membrane; CCRF-CEM, human leukemia cell line; CEM/ADR5000, multidrug-resistant human leukemia cell line; CHO, Chinese Hamster Ovarian cell line; c-MYC, proto-oncogene; COLO 205, colon cancer cell line; CNS, central nervous system; DNA, deoxyribonucleic acid; DU145, prostate cancer cell line; EL4, mouse thymoma cancer cell line; EKVX, non-small cell lung cancer cell line; FG/fg, Functional group; GI50, concentration for 50% of maximal inhibition of cell proliferation; HCT-15, colon cancer cell line; HCT-116, colon cancer cell line; HeLa, cervical cancer cell line; HEP-2, liver cancer cell line; HFF, primary human foreskin fibroblasts cell line; HER2, human epidermal growth factor receptor 2; HL-60, Human promyelocytic leukemia cell line; HOP-92, nonsmall cell lung cancer cell line; ip, intraperitoneally; HT-29, human colorectal adenomocarcinoma cell line; IC50, half maximal inhibitory concentration; iv, intravenously; IMR-32, neuroblastoma cell line; KB, human epidermoid carcinoma cell line; KM-12, colon cancer cell line; LLC-PK11, pig kidney epithelial cell line; LNCaP, prostate cancer cell line; MCF-7, breast cancer cell line; MCF-10, 74 ACS Paragon Plus Environment

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nontumorigenic breast cell line; MDA-MB-231, human breast cancer cell line; NCI, National Cancer Institute; NCI-H226, non-small cell lunger cancer cell line; OVCAR-3, OVCAR-4, ovarian cancer cell line; P388, mouse fibroblast leukemia cancer cell line; PBMC, peripheral blood mononuclear cell line; PC, PC-3, prostate cancer cell line; P. f., Plasmodium falciparum; po, oral; ROS, reactive oxygen species; SAR, structure activity relationship; SF-295, central nervous system cancer cell line; SI, selectivity index; SK-MEL-2, SK-MEL-5, melanoma cell line; SK-OV-3, human ovarian carcinoma cell line; sc, subcutaneous; TC50, toxic concentration; THP-1, leukemia cancer cell line; TK-10, renal cancer cell line; UACC62, melanoma cell line; Vero, African green monkey kidney fibroblast cell line; WHO, World Health Organization; YD-10B, oral cancer cell line. AUTHOR INFORMATION

Corresponding Author * Tel.: (+49)-(0)9131-85-22541; E-mail: [email protected]

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3. Klayman, D. Qinghaosu (artemisinin): an antimalarial drug from China. Science 1985, 228, 1049-1055. 4. Li, Y.; Wu, Y. L. An over four millennium story behind Qinghaosu (artemisinin): a fantastic antimalarial drug from a traditional Chinese herb. Curr. Med. Chem. 2003, 10, 2197-2230. 5. Miller, L. H.; Su, X. Artemisinin: discovery from the Chinese herbal garden. Cell 2011, 146, 855-858. 6. Posner, G. H. Antimalarial peroxides in the qinghaosu (artemisinin) and yingzhaosu families. Expert Opin. Ther. Patents 1998, 8, 1487-93. 7. Dong, Y.; Vennerstrom, J. L. Peroxidic antimalarials. Expert Opin. Ther. Patents 2001, 11, 1753-1760. 8. Jung, M.; Lee, K.; Kim, H.; Park, M. Recent advances in artemisinin and its derivatives as antimalarial and antitumor Agents. Curr. Med. Chem. 2004, 11, 1265-1284. 9. Ploypradith, P. Development of artemisinin and its structurally simplified trioxane derivatives as antimalarial drugs. Acta Tropica 2004, 89, 329-342. 10. Dewick, P. M. Medicinal Natural Products: A Biosynthetic Approach, 3rd ed.; John Wiley & Sons: Chicester, U.K., 2009.

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58. Paik, I.-H.; Xie, S.; Shapiro, T. A.; Labonte, T.; Narducci Sarjeant, A. A.; Baege, A. C.; Posner, G. H. Second generation, orally active, antimalarial, artemisinin-derived trioxane dimers with high stability, efficacy, and anticancer activity. J. Med. Chem. 2006, 49, 2731-2734. 59. Jeyadevan, J. P.; Bray, P. G.; Chadwick, J.; Mercer, A. E.; Byrne, A.; Ward, S. A.; Park, B. K.; Williams, D. P.; Cosstick, R.; Davies, J.; Higson, A. P.; Irving, E.; Posner, G. H.; O'Neill, P. M. Antimalarial and antitumor evaluation of novel C-10 non-acetal dimers of 10beta-(2-hydroxyethyl)deoxoartemisinin. J. Med. Chem. 2004, 47, 1290-1298. 60. Chadwick, J.; Mercer, A. E.; Park, B. K.; Cosstick, R.; O'Neill, P. M. Synthesis and biological evaluation of extraordinarily potent C-10 carba artemisinin dimers against P. falciparum malaria parasites and HL-60 cancer cells. Bioorg. Med. Chem. 2009, 17, 1325-1338. 61. Slade, D.; Galal, A. M.; Gul, W.; Radwan, M. M.; Ahmed, S. A.; Khan, S. I.; Tekwani, B. L.; Jacob, M. R.; Ross, S. A.; ElSohly, M. A. Antiprotozoal, anticancer and antimicrobial activities of dihydroartemisinin acetal dimers and monomers. Bioorg. Med. Chem. 2009, 17, 7949-7957. 62. Galal, A. M.; Gul, W.; Slade, D.; Ross, S. A.; Feng, S.; Hollingshead, M. G.; Alley, M. C.; Kaur, G.; ElSohly, M. A. Synthesis and evaluation of

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68. Jung, M.; Park, N.; Moon, H.-I.; Lee, Y.; Chung, W.-Y.; Park, K.-K. Synthesis and anticancer activity of novel amide derivatives of non-acetal deoxoartemisinin. Bioorg. Med. Chem. Lett. 2009, 19, 6303-6306. 69. Saikia, B.; Pratim Saikia, P.; Goswami, A.; Barua, N. C.; Saxena, A. K.; Suri, N. Synthesis of a novel series of 1,2,3-triazole-containing artemisinin dimers with potent anticancer activity involving huisgen 1,3-dipolar cycloaddition reaction. Synthesis 2011, 19, 3173-3179. 70. Buragohain, P.; Saikia, B.; Surineni, N.; Barua, N. C.; Saxena, A. K.; Suri, N. Synthesis of a novel series of artemisinin dimers with potent anticancer activity involving Sonogashira cross-coupling reaction. Bioorg. Med. Chem. Lett. 2014, 24, 237-239. 71. Reiter, C.; Fröhlich, T.; Zeino, M.; Marschall, M.; Bahsi, H.; Leidenberger, M.; Friedrich, O.; Kappes, B.; Hampel, F.; Efferth, T.; Tsogoeva, S. B. New efficient artemisinin derived agents against human leukemia cells, human cytomegalovirus and Plasmodium falciparum: 2nd generation 1,2,4-trioxaneferrocene hybrids. Eur. J. Med. Chem. 2015, 97, 164-172. 72. Cloete, T. T.; de Kock, C.; Smith, P. J.; N'Da, D. D. Synthesis, in vitro antiplasmodial activity and cytotoxicity of a series of artemisinin-triazine hybrids and hybrid-dimers. Eur. J. Med. Chem. 2014, 76, 470-481.

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79. Posner, G. H.; Paik, I.-H.; Chang, W.; Borstnik, K.; Sinishtaj, S.; Rosenthal, A. S.; Shapiro, T. A. Malaria-infected mice are cured by a single dose of novel artemisinin derivatives. J. Med. Chem. 2007, 50, 2516-2519. 80. Posner, G. H.; Chang, W.; Hess, L.; Woodard, L.; Sinishtaj, S.; Usera, A. R.; Maio, W.; Rosenthal, A. S.; Kalinda, A. S.; D'Angelo, J. G.; Petersen, K. S.; Stohler, R.; Chollet, J.; Santo-Tomas, J.; Snyder, C.; Rottmann, M.; Wittlin, S.; Brun, R.; Shapiro, T. A. Malaria-infected mice are cured by oral administration of new artemisinin derivatives. J. Med. Chem. 2008, 51, 1035-1042. 81. Woodard, L. E.; Mott, B. T.; Singhal, V.; Kumar, N.; Shapiro, T. A.; Posner, G. H. Malaria-infected mice are cured by a single low dose of a new silylamide trioxane plus mefloquine. Pharmaceuticals 2009, 2, 228-235. 82. Hartwig, C. L.; Rosenthal, A. S.; D'Angelo, J.; Griffin, C. E.; Posner, G. H.; Cooper, R. A. Accumulation of artemisinin trioxane derivatives within neutral lipids of Plasmodium falciparum malaria parasites is endoperoxide-dependent. Biochem. Pharmacol. 2009, 77, 322-336. 83. Rosenthal, A. S.; Chen, X.; Liu, J. O.; West, D. C.; Hergenrother, P. J.; Shapiro, T. A.; Posner, G. H. Malaria-infected mice are cured by a single oral dose of new dimeric trioxane sulfones which are also selectively and powerfully cytotoxic to cancer cells. J. Med. Chem. 2009, 52, 1198-1203.

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growth of breast cancer cells in vitro and induce down-regulation of HER family members. PLoS One 2013, 8, e59086. 90. Kyu Moon, D.; Singhal, V.; Kumar, N.; Shapiro, T. A.; Posner, G. H. Antimalarial preclinical drug development: a single oral dose of a 5-carbon-linked trioxane dimer plus mefloquine cures malaria-infected mice. Drug Dev. Res. 2010, 71, 76-81. 91. Kyu Moon, D.; Tripathi, A.; Sullivan, D.; Siegler, M. A.; Parkin, S.; Posner, G. H. A single, low, oral dose of a 5-carbon-linked trioxane dimer orthoester plus mefloquine cures malaria-infected mice. Bioorg. Med. Chem. Lett. 2011, 21, 2773-2775. 92. Mott, B. T.; Tripathi, A.; Siegler, M. A.; Moore, C. D.; Sullivan, D. J.; Posner, G. H. Synthesis and antimalarial efficacy of two-carbon-linked, artemisinin-derived trioxane dimers in combination with known antimalarial drugs. J. Med. Chem. 2013, 56, 2630-2641. 93. Conyers, R. C.; Mazzone, J. R.; Siegler, M. A.; Tripathi, A. K.; Sullivan, D. J.; Mott, B. T.; Posner, G. H. The survival times of malaria-infected mice are prolonged more by several new two-carbon-linked artemisinin-derived dimer carbamates than by the trioxane antimalarial drug artemether. Bioorg. Med. Chem. Lett. 2014, 24, 1285-1289.

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99. Nam, W.; Tak, J.; Ryu, J.-K.; Jung, M.; Yook, J.-I.; Kim, H.-J.; Cha, I.-H. Effects of artemisinin and its derivatives on growth inhibition and apoptosis of oral cancer cells. Head and Neck 2007, 29, 335-340. 100. Beekman, A. C.; Wierenga, P. K.; Woerdenbag, H. J.; Van Uden, W.; Pras, N.; Konings, A. W.; El-Feraly, F. S.; Galal, A. M.; Wikström, H. V. Artemisininderived sesquiterpene lactones as potential antitumour compounds: cytotoxic action against bone marrow and tumour cells. Planta Med. 1998, 64, 615-619. 101. He, R.; Forman, M.; Mott, B. T.; Venkatadri, R.; Posner, G. H.; AravBoger, R. Unique and highly selective anticytomegalovirus activities of artemisinin-derived dimer diphenyl phosphate stem from combination of dimer unit and a diphenyl phosphate moiety. Antimicrob. Agents Chemother. 2013, 57, 42084214. 102. He, R.; Mott, B. T.; Rosenthal, A. S.; Genna, D. T.; Posner, G. H.; AravBoger, R. An artemisinin-derived dimer has highly potent anti-cytomegalovirus (CMV) and anti-cancer activities. PLoS ONE 2011, 6, e24334. 103. Rosenthal, A. S.; Chen, X.; Liu, J. O.; West, D. C.; Hergenrother, P. J.; Shapiro, T. A.; Posner, G. H. Malaria-infected mice are cured by a single oral dose of new dimeric trioxane sulfones which are also selectively and powerfully cytotoxic to cancer cells. J. Med. Chem. 2009, 52, 1198-1203.

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