betulin-derived dimers and hybrids with potent antimalarial and antiviral activities

Accepted Manuscript Synthesis of New Betulinic acid/Betulin-derived Dimers and Hybrids with Potent Antimalarial and Antiviral Activities Aysun Çapcı Karagöz, Maria Leidenberger, Friedrich Hahn, Frank Hampel, Oliver Friedrich, Manfred Marschall, Barbara Kappes, Svetlana B. Tsogoeva PII: DOI: Reference:

S0968-0896(18)31441-X https://doi.org/10.1016/j.bmc.2018.11.018 BMC 14622

To appear in:

Bioorganic & Medicinal Chemistry

Received Date: Revised Date: Accepted Date:

9 August 2018 31 October 2018 13 November 2018

Please cite this article as: Karagöz, A.C., Leidenberger, M., Hahn, F., Hampel, F., Friedrich, O., Marschall, M., Kappes, B., Tsogoeva, S.B., Synthesis of New Betulinic acid/Betulin-derived Dimers and Hybrids with Potent Antimalarial and Antiviral Activities, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc. 2018.11.018

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Synthesis of New Betulinic acid/Betulinderived Dimers and Hybrids with Potent Antimalarial and Antiviral Activities

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Aysun Çapcı Karagöz,[a] Maria Leidenberger,[b] Friedrich Hahn,[c] Frank Hampel,[a] Oliver Friedrich,[b] Manfred Marschall,[c] Barbara Kappes,[b] and Svetlana B. Tsogoeva*[a] [a] Organic

Chemistry Chair I and Interdisciplinary Center for Molecular Materials (ICMM), FriedrichAlexander University of Erlangen-Nürnberg, Nikolaus-Fiebiger-Straße 10, 91058 Erlangen, Germany [b] Institute of Medical Biotechnology, University of Erlangen-Nürnberg, Paul-Gordon-Straße 3, 91052 Erlangen, Germany [c] Institute for Clinical and Molecular Virology, University of Erlangen-Nürnberg, Schlossgarten 4, 91054 Erlangen, Germany Betulin/Betulinic acid-Artesunic acid Hybrids

R2 R1 EC50 (P. falciparum) down to 0.09 µM EC50 (HCMV) down to 0.24 µM

antimalarial antiviral

H

R1 =

O O O O

-Art ; -OH

O O

O O

R2 = -COOH; -CH2Art

Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com

Synthesis of New Betulinic acid/Betulin-derived Dimers and Hybrids with Potent Antimalarial and Antiviral Activities Aysun Çapcı Karagöz,[a] Maria Leidenberger,[b] Friedrich Hahn,[c] Frank Hampel,[a] Oliver Friedrich,[b] Manfred Marschall,[c] Barbara Kappes,[b] and Svetlana B. Tsogoeva[a] Organic Chemistry Chair I and Interdisciplinary Center for Molecular Materials (ICMM), Friedrich-Alexander University of Erlangen-Nürnberg, NikolausFiebiger-Straße 10, 91058 Erlangen, Germany b Institute of Medical Biotechnology, University of Erlangen-Nürnberg, Paul-Gordon-Straße 3, 91052 Erlangen, Germany c Institute for Clinical and Molecular Virology, University of Erlangen-Nürnberg, Schlossgarten 4, 91054 Erlangen, Germany a

———

ARTICLE INFO

ABSTRACT

Article history: Received Received in revised form Accepted Available online

Severe malaria and viral infections cause life-threatening diseases in millions of people worldwide every year. In search for effective bioactive hybrid molecules, which may possess improved properties compared to their parent compounds, a series of betulinic acid/betulin based dimer and hybrid compounds carrying ferrocene and/or artesunic acid moieties, was designed and, synthesized de novo. Furthermore, they were analyzed in vitro against malaria parasites (growth inhibition of 3D7-strain P. falciparum-infected erythrocytes) and human cytomegalovirus (HCMV). From this series of hybrids/dimers, the betulinic acid/betulin and artesunic acid hybrids 11 and 12 showed the most potent activities against P. falciparum and HCMV. On the strength of results, additive and/or synergistic effects between the natural or semisynthetic products, such as betulinic acid-/betulin- and artesunic acid-derived compounds, are suggested on the basis of putatively complex modes of antimicrobial action. This advantage may be taken into account in future drug development.

 Corresponding author: e-mail: [email protected]

Keywords: Betulin Betulinic acid Artesunic acid Ferrocene Natural product hybrid Antimalarial activity Antiviral activity

1. Introduction There is urgent need for new effective drugs against lifethreatening diseases, such as malaria and virus infections. Human cytomegalovirus (HCMV) is an opportunistic viral pathogen of the Herpesviridae family which infects newborns and immunosuppressed individuals with high virulence, leading to severe morbidity and mortality. Ganciclovir and its pro-drug valganciclovir have been commonly used to treat and prevent HCMV infection or reactivation, respectively. Moreover, very recently, a novel anti-HCMV drug, letermovir (LMV)/Prevymis, was approved. However, the development of HCMV resistance to ganciclovir and valganciclovir has been reported in patients[1], and the impact of putative resistance formation to LMV prophylaxis still has to be determined. Plasmodium falciparum is the most fatal parasite and causes malaria, transmitted by the bite of the Anopheles mosquito. According to the World Malaria Report 2017, 216 million malaria cases (which increased comparing to 2015) and 445 thousand malaria deaths were reported in 2016 worldwide.[2] Moreover, a concerning problem successively emerged over the period that drug has been applied in malaria therapy, i.e. multidrug resistance against artemisinin and its derivatives.[3-5] Artemisinin and its semisynthetic derivatives, which are currently among the most effective antimalarial drugs, are used in regimens of combination therapy (ACTs). The combination of two or more drugs, which act via

2009 Elsevier Ltd. All rights reserved.

different modes of action is recommended by the WHO to prevent or delay the development of resistance.

OH

OH O

HO

O O

HO

Betulin (1)

O

O

Betulinic acid (2)

O Fe

O O O

HN

O

O O

OH O

N Fe

Artesunic acid (3)

Cl

N

O

Artemisininferrocene hybrid (5)

Ferroquine (4)

Fig. 1. Bioactive natural products and synthetic derivatives: betulin (1), betulinic acid (2), artesunic acid (3), ferroquine (4) and artemisinin-ferrocene hybrid (5).

H

Fe Fe

O O O

O

O

O

O

O

O

O

HO

Fe O O O

Fe

O O

7

6

O

8 OH O O

O HO

O O

Fe

OH O

O

Fe

O

O

9

10

HO

O

H O O

O O

HO

O O O

O

O O

O

O O

11

OH

O

O O

O

12

H

Fig. 2. Hybrids 6 - 12 investigated in vitro against P. falciparum 3D7 parasites and human cytomegaloviruses (HCMV) replication. Due to the malaria parasite’s resistance towards existing drugs, alternative experimental compounds are under investigation. Drug discovery studies play a crucial role in developing new lead compounds in order to overcome consequences of those diseases and drug resistance.[6] Natural products have been an important source of hit and lead compounds for drug discovery. Most currently available drugs are either natural products or are inspired by them.[7-9] The natural products (specialized metabolites) are unique components with utilizable complex molecular structures by their very nature (e.g. paclitaxel, artemisinin). Facing this potential advantage, efficient hybrid molecules between these secondary metabolites and additional, heterologous moieties, have been repeatedly synthesized in a bioactive form.[10-17] The hybrid supposedly not only has the properties of its components, but may also have new or enhanced properties generated by the components’ synergistic coaction. In our previous studies, we have shown that the natural products are more efficient when they are hybridized.[18-28] In the present study, the natural products betulin (1), betulinic acid (2) and artesunic acid (3) were chosen due to their efficient therapeutic properties (Fig. 1). Betulin was discovered in the 18th century from birch cork and it is regarded as the first bioactive compound isolated from a plant.[29] Betulin is a natural triterpene occurring abundantly in the bark of birch trees.[30] It can be obtained in vast quantities as byproduct, generated every year by the forest industry.[31] Betulin and its derivatives have a wide spectrum of biological and pharmacological properties, such as antiviral and anti-HIV,[32, 33] anti-inflammatory,[34] anti-cancer[35-38] and antimalaria.[39] Artemisinin, a highly effective natural product, is an enantiopure sesquiterpene lactone and occurs in Artemisia annua L.[40, 41] Its discoverer Youyou Tu was awarded the 2015 Nobel Prize in physiology or medicine.[42] Artemisinin has an endoperoxide bridge which is proposed to be responsible for the mode of activation via its cleavage by a source of Fe2+ or

heme.[43] Following the reductive activation of this endo peroxide bridge, carbon-centered free radicals alkylate the parasite proteins.[44, 45] Artemisinins are a very important class of antimalarial agents for the treatment of malaria. Not only that, artemisinin and its derivatives show high efficiency against different viruses and cancer cells.[46-50] Inspired by the highly active antimalarial compound ferroquine (4)[51, 52] which is under clinical trials,[53] and by our previously reported artemisinin-ferrocene hybrid (5) with high antileukemia and antiviral activities,[21, 22] we have also included the ferrocene structure into the new molecules as a linker or a subunit. Furthermore, we have analyzed the activity of all new compounds against Plasmodium falciparum 3D7 strain and human cytomegalovirus (HCMV).

2. Results and Discussion Chemistry Pursuing our interest in natural product hybrids and dimers, we have designed and synthesized new hybrid compounds consisting of betulin (1), betulinic acid (2), artesunic acid (3) and ferrocene subunit (Fig. 2). In total, five new hybrids (6-10) were synthesized via various esterification reactions which are depicted in Scheme 1. Betulin, betulinic acid and artesunic acid were supplied commercially and used without pre-modification. The derivatives of ferrocene were synthesized according to published protocols.[54]

OH

OH O

HO

1

HO OH

1

Fe

6

7

i

i

13 H O O O

i

O

O O

OH O

8

3

Fig. 3. X-Ray crystal structure of hybrid 6.

OH

OH

O

9

O

2

HO

1

Cl HO Fe

ii

Cl

ii O

10

14

Scheme 1. Synthetic routes of betulinic acid-/betulin-derived dimers and hybrids 6 – 12; i) DCC, DMAP, CH2Cl2, 0 oC  rt, ii) DMAP, CH2Cl2, 0 oC  rt. Furthermore, here we additionally, report antiviral and antimalarial activity of the previously published hybrids 11[55] and 12[56], for the first time. Taken together, we have evaluated the structure activity relations of these new compounds according to their behavior in in vitro analysis against Plasmodium falciparum 3D7 strains and human cytomegalovirus compared to a reference drug. Mono- and di-esterified betulin-ferrocene hybrids 6 and 7 were obtained in 55% and 78 % yields, respectively. For the di-esterification of betulin, the reaction mixture needed to be stirred for 5-6 days whereas monoesterification was completed after overnight stirring. The crystals of hybrid 6 were obtained by vapour diffusion crystallization in CH2Cl2 (solvent) and hexane (antisolvent)[57], afterwards the crystal structure was unambiguously determined via X-ray crystallography (Fig. 3).[58] Hybrid 6 was further used for a hybridization reaction to obtain artesunic acid-betulin-ferrocene hybrid 8. The ester bond was formed between the free alcohol group of betulin-ferrocene hybrid 6 and artesunic acid in 60% yield. Besides hybridization, dimerization of natural products by nature[59] or synthetically has been also reported as highly potent approach towards bioactive agents.[10] Thus, in the present study, synthesis and biological evaluation of new dimers of betulin and betulinic acid were carried out as well. Ferrocene dicarboxylic chloride (14) was employed as linker compound to synthesize betulinic acid dimer 9 and betulin dimer 10. The reactions were performed in the presence of DMAP in dry CH2Cl2 under N2 and resulted in desired homodimers 9 and 10. Dimers were isolated in 28% and 40 % yields, respectively. Artesunic acid-betulin and artesunic acid-betulinic acid hybrids (11 and 12) were synthesized according to our previously reported protocols. All products were precipitated in CH2Cl2/hexane mixture, dried under high vacuum and their purity confirmed via elemental analysis.

3. Biological activities In vitro antiviral activity against HCMV in primary human fibroblasts The antiviral activity of the compounds was analyzed against HCMV, strain AD169-GFP which, by the expression of green fluorescent protein (GFP), allows the quantitation of viral replication. In vitro infection experiments were performed with cultures of primary human foreskin fibroblasts (HFFs) and measurements of antiviral activity were performed according to a previously established protocol.[60-62] As reference compounds, anti-HCMV drug ganciclovir and hybrid precursors, artesunic acid, betulin and betulinic acid were included. Replication studies revealed varying degrees of antiviral activity, even reaching submicromolar concentrations in case of two hybrids (Table 1). Artesunic acid-betulinic acid hybrid 12 exerts the highest activity among all compounds analyzed here, with an EC50 value of 0.24 µM, while the parent compounds artesunic acid and betulinic acid had an EC50 of 5.41 µM and 3.62 µM, respectively (Table 1). Thus, the hybrid was 15 fold more active than betulinic acid and 23 fold more active than artesunic acid. Moreover, it is superior to the clinically used drug ganciclovir. As an analogue of 12, the artesunic acid-betulin hybrid 11 also revealed enhanced activity when compared to the parent compounds with an EC50 of 0.88 µM. Moreover, EC50 values were markedly lower compared to the previously reported in vitro EC50 of the clinically used drug ganciclovir. Hybrids 11 and 12 revealed a reinforcing effect of their components as a result of hybridization of these natural products. Hybrids 7, 8 and 9 possess moderate activity. However, EC50 values for 6, 8 and 10 could not be determined due to the induction of cytotoxicity. There is no obvious structural correlation to the toxicity. The ferrocene moiety might not be the reason for toxicity because hybrid 6 was toxic, whereas hybrid 7 was not.

Table 1. EC50 values of betulin (1), betulinic acid (2), artesunic acid (3), dihydroartemisinin (DHA), chloroquine, ganciclovir, hybrids 6-12 and ferrocene monocarboxylic acid (13) against Plasmodium falciparum and HCMV. P. falciparum

HCMV

Compound

MW (g/mol)

EC50 (µM)

EC50 (µM)

Betulin (1)

442.73

3.938

1.99 ± 0.45

Betulinic acid (2)

456.71

1.419

3.62 ± 1.21

Artesunic acid (3)

398.45

0.0097

5.41 ± 0.61

DHA

284.35

0.0026

>10

Chloroquine Phosphate

515.86

0.0083

n.d.

Ganciclovir

255.23

n.d.

2.6 ± 0.5

6

657.36

27.854

ctx/nda

7

866.79

63.669

5.29 ± 1.32

8

1021.17

1.491

10.75 ± 0.51

9

1151.45

11.752

2.46 ± 0.85

10

1123.48

54.024

ctx/nda

11

809.14

0.255

0.88 ± 0.06

12

823.12

0.085

0.24 ± 0.00

13

230.04

91b

>10b

1+3c

-

0.0125

nd

ctx/nd, microscopically detectable cytotoxicity approximately 20 % at 3.3 µM, no antiviral activity detectable. n.d: not determined. b Previously reported.[22] c Combination of betulin and artesunic acid in a 1:1 ratio. a

In vitro antimalarial activity against Plasmodium falciparum 3D7 strains The antimalarial activity of the hybrids, betulin, betulinic acid, artesunic acid and chloroquine was tested according to in vitro growth inhibition of 3D7-strain P. falciparum-infected erythrocytes. The EC50 values were determined and evaluated (Table 1). The most active compounds are 12 and 11 with EC50 values of 0.09 µM and 0.23 µM, respectively. Even though these hybrids are active in the nanomolar range, they did not surpass the activity of artesunic acid and chloroquine. However, artesunic acid-betulinic acid hybrid 12 is 16 fold more active than betulinic acid, and artesunic acid-betulin hybrid 11 is 20-fold more active than betulin. The hybrid 8, which bear a 1,2,4-trioxane moiety, is moderately active, showing 1.47 µM inhibition concentration. When we compare the betulin-ferrocene hybrid 6 with artemisinin-based hybrid 8, where betulin is the common unit in those two hybrids, it could be stated that the 1,2,4-trioxane moiety significantly enhanced the activity for 8. Furthermore, the hybrids 6, 7, 9 and 10, which do not contain 1,2,4-trioxane unit, did not show notable activity against P. falciparum 3D7 strains. Although, the hybrid compounds 6, 7, 9, and 10 which bear a ferrocene moiety, are more active than ferrocene monocarboxylic acid, they did not outperform activity of parent compounds betulin, betulinic acid and artesunic acid. Furthermore, artesunic

acid alone is more active than hybrids 6, 7, 9, and 10 against P. falciparum. Thus, we assume that these hybrids do not hydrolyze to artesunate or artesunic acid. Otherwise we would have reached at least the activity of artesunic acid. For example, a 1:1 combination of betulin and artesunic acid (1+3) shows an EC50 value of 0.012 µM, similar to artesunic acid that is 0.009 µM (Table 1). Nonetheless, for further investigations, hybrids with more stable linkers are under preparation in our laboratory and will be published elsewhere.

4. Conclusions In summary, we have reported the synthesis and biological activities of a number of new betulin and betulinic acid hybrids/dimers. The in vitro activity analysis of these hybrids against P. falciparum and HCMV was observed in the nanomolar to micromolar range. Notably, amongst the others, the artesunic acid-betulinic acid hybrid 12 and artesunic acid-betulin hybrid 11 exhibited the lowest EC50 value within the series. Moreover, those hybrids with EC50 in the range of 0.24 - 0.88 µM against HCMV were more effective than their parent compounds and were also active at lower concentrations when compared to the reference drug ganciclovir. In conclusion, hybridization of betulin/betulinic acid and artesunic acid can be suggested for new directions in drug discovery. 5. Experimental Section Chemistry: General Information. All reactions were performed in flame-dried glassware under a nitrogen atmosphere. After column chromatography, all hybrids were precipitated in CH2Cl2/Hexane mixture to yield a pure compound for elemental analysis and biological tests. CH2Cl2 was dried initially over CaCl2 and then distilled from P2O5. All other solvents were purified by distillation or were purchased in HPLC-quality. Reagents obtained from commercial sources were used without further purification. TLC chromatography was performed on pre-coated aluminium silica gel SIL G/UV254 plates (Macherey-Nagel & Co.). The detection occurred via fluorescence quenching or development in a phosphomolybdic acid solution (10 % in EtOH). All products were dried in highvacuum (10-3 mbar). 1H NMR and 13C NMR spectra were recorded at room temperature on a Bruker Avance spectrometer operating at 300 MHz and 400 MHz. ESI Mass spectra were recorded on a Bruker micrOTOF II focus TOF MS-spectrometer. Elemental analysis (C, H, N), carried out with an Euro EA 3000 (Euro Vector) machine and an Elementar vario MICRO cube machine, is within ± 0.43% of the calculated values confirming a purity of > 95%. Betulin (1) was obtained from Carl Roth, Germany. Artesunic acid (3) and betulinic acid (2) were obtained from ABCR (Karlsruhe, Germany). General Procedure for the Synthesis of Hybrids 6, 8, and 7 In a flame-dried flask under a nitrogen atmosphere, betulin and appropriate monocarboxylic acid were dissolved in anhydrous CH2Cl2. At 0 oC, DMAP and DCC were added and the reaction mixture left to be continually stirred at room temperature overnight. The reaction was controlled via TLC, UV light and phosphomolybdic acid as indicator. The precipitated urea was filtered and solvent was removed under reduced pressure. The crude product was purified by column chromatography using Hexanes/EtOAc as eluent to yield hybrids. Hybrid 6: Betulin (1) (150 mg, 0.34 mmol, 1.0 equiv.), ferrocene monocarboxylic acid (13) (78 mg, 0.34 mmol, 1.0

equiv.), CH2Cl2 (11.1 mL), DMAP (124 mg, 1.02 mmol, 3.0 equiv.), DCC (88.6 mg, 0.43 mmol, 1.3 equiv.). Column conditions: Hexanes/EtOAc 4:1, Rf = 0.3, (UV and phosphomolybdic acid). Yield: 55%. 1H NMR (300 MHz, CDCl3): δ = 4.84 (s, 2H), 4.72 (s, 1H), 4.61 (s, 1H), 4.41 (s, 3H), 4.22 (s, 5H), 3.91 - 3.94 (d, J= 10.9 Hz, 1H), 3.16 - 3.19 (m, 1H), 2.47 - 2.57 (m, 1H), 1.88 - 2.08 (m, 3H), 1.40 - 1.76 (m, 20H), 1.18 - 1.32 (m, 6H), 1.07 (s, 3H), 1.01 (s, 3H), 0.96 (s, 3H), 0.83 (s, 3H), 0.76 (s, 3H) 0.67 - 0.70 (d, J= 9.2, 1H) ppm. 13C NMR (75 MHz, CDCl3): δ = 172.25, 150.30, 149.06, 148.86, 110.03, 99.72, 97.60, 79.11, 71.59, 71.35, 70.40, 70.23, 70.19, 69.83, 62.74, 55.43, 50.53, 48.91, 47.96, 46.71, 42.89, 41.02, 39.00, 38.85, 37.77, 37.28, 34.97, 34.33, 30.13, 29.83, 28.12, 27.55, 27.27, 25.35, 20.94, 19.31, 18.41, 16.25, 16.22, 15.50, 14.96. Anal. Calcd. for [4xC41H58FeO3+1xCH2Cl2]: C, 73.29; H, 8.72; Found: C, 73.04; H, 8.87. [M+Na]; HRMS (ESI): m/z calcd. for [C41H58FeNaO3] +: 677.3628, found: 677.3628. Hybrid 7: Betulin (1) (100 mg, 0.226 mmol, 1.0 equiv.), ferrocene monocarboxylic acid (13) (104 mg, 0.452 mmol, 2.2 equiv.), CH2Cl2 (10 mL), DMAP (55 mg, 0.452 mmol, 2.2 equiv.), DCC (93 mg, 0.452 mmol, 2.2 equiv.). Column conditions: Hexanes/EtOAc 4:1. Yield: 78 %. Rf = 0.7 (UV and phosphomolybdic acid). 1H NMR (300 MHz, CDCl3): δ = 4.80 4.80 (d, J= 8.0 Hz, 4H), 4.73 (s, 1H), 4.62 (s, 1H), 4.45 - 4.57 (m, 1H), 4.38 - 4.40 (d, J= 6.6 Hz, 4H), 4.20 (s, 10H), 3.92 - 3.95 (d, J= 11.1 Hz, 1H), 2.49 - 2.57 (m, 1H), 1.89 - 2.12 (m, 3H), 1.61 1.77 (m, 12H), 1.43 (m, 6H), 1.34 (s, 3H), 1.09 (s, 6H), 1.03 (s, 4H), 0.95 (s, 6H), 0.90 (s, 3H) ppm. 13C NMR (100 MHz, CDCl3): δ = 14.85, 16.13, 16.21, 16.81, 18.21, 20.87, 23.98, 27.16, 28.13, 29.71, 30.02, 34.17, 37.14, 37.67, 37.94, 38.36, 40.96, 42.80, 46.40, 47.88, 48.80, 50.33, 55.44, 62.65, 69.80, 69.83, 70.05, 70.10, 70.20, 70.33, 71.26, 71.34, 71.57, 72.30, 80.55, 109.95, 150.17, 171.40, 172.14 ppm. Anal. Calcd. for C52H66Fe2O4: C, 72.06; H, 7.68; Found: C, 72.02; H, 7.70. [M]; HRMS (ESI): m/z calcd. for [C52H66Fe2O4] +: 866.3656, found: 866.3672. Hybrid 8: Hybrid 6 (54 mg, 0.082 mmol, 1.0 equiv.), artesunic acid (37.8 mg, 0.098 mmol, 1.2 equiv.), CH2Cl2 (7 mL), DMAP (20.03 mg, 0.164 mmol, 2.0 equiv.), DCC (20.2 mg, 0.098 mmol, 1.2 equiv.). Column conditions: CH2Cl2/MeOH 99:1. Yield: 60 %. Rf = 0.7, (UV and phosphomolybdic acid). 1H NMR (300 MHz, CDCl3): δ = 5.77 - 5.81 (d, J= 9.9 Hz, 1H), 5.43 (s, 1H), 4.81 - 4.82 (t, J= 3.9 Hz, 2H), 4.72 (d, J= 1.9 Hz, 1H), 4.60 - 4.61 (m, 1H), 4.43 - 4.51 (m, 2H), 4.39 - 4.40 (t, J= 3.9 Hz, 3H), 4.23 (s, 2H), 4.19 (s, 5H), 3.91 - 3.94 (d, J= 11.1, 1H), 2.71 - 2.76 (m, 2H), 2.48 - 2.67 (m, 4H), 2.32 - 2.42 (m, 1H), 2.17 (s, 1H), 1.89 - 2.06 (m, 4H), 1.58 - 1.74 (m, 13H), 1.43 (s, 6H), 1.39 (s, 3H), 1.25 - 1.33 (m, 10H), 1.06 (s, 3H), 1.00 (s, 3H), 0.97 (s, 3H), 0.95 (s, 3H), 0.82 - 0.86 (m, 13H) ppm. 13C NMR (75 MHz, CDCl3) δ: 172.25, 171.95, 171.31, 150.30, 110.06, 104.57, 92.20, 91.61, 81.48, 80.25, 71.59, 71.36, 70.23, 70.19, 69.83, 62.74, 55.50, 51.68, 50.40, 48.88, 47.98, 46.70, 45.35, 42.88, 41.02, 38.46, 37.96, 37.75, 37.39, 37.18, 36.34, 34.96, 34.21, 32.69, 31.91, 31.72, 31.18, 30.10, 29.80, 29.46, 29.41, 28.12, 27.24, 26.39, 26.10, 24.75, 24.70, 23.76, 22.79, 22.12, 20.94, 20.36, 19.28, 18.26, 16.70, 16.29, 16.21, 14.93, 14.28, 12.26. Anal. Calcd. for C52H84FeO10: C, 70.57; H, 8.29; Found: C, 70.70; H, 8.51. [M] (MALDI-TOF): m/z calcd. for [C60H84FeO10]: 1020, found: 1020. General Procedure for the Synthesis of Hybrids 9 and 10. In a flame-dried flask under a nitrogen atmosphere, betulin or betulinic acid was dissolved in anhydrous CH2Cl2. At 0 oC, DMAP and ferrocene dicarboxylic acid chloride were added and the reaction mixture left to be continually stirred at room

temperature overnight. The reaction was controlled via TLC, UV light and phosphomolybdic acid as indicator. The solvent was removed under reduced pressure. The crude product was purified by column chromatography using Hexane/EtOAc as eluent to yield hybrids. Hybrid 9: Betulinic cid (2) (100 mg, 0.22 mmol, 1.0 equiv.), ferrocene dicarboxylic acid chloride (34 mg, 0.11 mmol, 0.5 mmol), CH2Cl2 (10 mL), DMAP (27 mg, 0.22 mmol, 1.0 equiv.). Column conditions: Hexanes/EtOAc 7:3. Yield: 28 %. Rf = 0.47, (UV and phosphomolybdic acid). 1H NMR (300 MHz, CDCl3): δ = 4.90 - 4.96 (d, J= 17.7 Hz, 4H), 4.77 (s, 2H), 4.72 (s, 4H), 4.64 (s, 2H), 3.78 (s, 2H), 3.02 - 3.22 (m, 4H), 2.30 - 2.39 (m, 4H), 2.08 - 2.10 (m, 4H), 1.87 (s, 2H), 1.71 (s, 6H), 1.25 - 1.57 (m, 35H), 1.01 - 1.03 (d, J= 5.7 Hz, 12H), 0.97 (s, 6H), 0.83 (s, 6H), 0.76 (s, 6H), 0.68 - 0.71 (d, J= 8.8, 2H) ppm. 13C NMR (75 MHz, CDCl ) δ: 171.54, 166.86, 150.07, 110.17, 3 79.15, 74.83, 74.76, 72.60, 72.24, 71.57, 58.13, 55.58, 50.82, 49.45, 46.79, 42.75, 41.02, 39.05, 38.94, 38.30, 37.42, 36.34, 34.57, 31.92, 30.53, 30.03, 28.17, 27.60, 25.72, 21.08, 19.58, 18.48, 16.33, 16.29, 15.52, 14.89.Anal. Calcd. for C72H102FeO8: C, 75.10; H, 8.93; Found: C, 74.63; H, 8.73. [M] (MALDI-TOF): m/z calcd. for [C72H102FeNaO8]+: 1174, found: 1174. Hybrid 10: Betulin (1) (100 mg, 0.23 mmol, 2.0 equiv.), ferrocene dicarboxylic acid chloride (34 mg, 0.11 mmol, 1.0 equiv), CH2Cl2 (10 mL), DMAP (55 mg, 0.452 mmol, 4.1 equiv.). Column conditions: Hexanes/EtOAc 7:3. Yield: 40 %. Rf = 0.25, (UV and phosphomolybdic acid). 1H NMR (300 MHz, CDCl3): δ = 4.84 - 4.85 (d, J= 5.5 Hz, 4H), 4.72 (s, 2H), 4.61 (s, 2H), 4.41 - 4.44 (d, J= 11.2 Hz, 2H), 4.38 (s, 4H), 3.94 3.98 (d, J= 10.8 Hz, 2H), 3.16 - 3.22 (m, 2H), 2.47 - 2.56 (m, 2H), 1.79 - 2.08 (m, 12H), 1.71 (s, 9H), 1.50 - 1.60 (m, 12H), 1.37 - 1.45 (m, 12H), 1.26 - 1.29 (m, 6H), 1.10 (s, 6H), 1.01 (s, 6H), 0.97 (s, 6H), 0.83 (s, 6H), 0.76 (s, 6H), 0.67 - 0.71 (d, J= 9.7, 2H) ppm. 13C NMR (75 MHz, CDCl3) δ: 170.88, 150.30, 110.03, 99.73, 98.04, 79.12, 73.36, 73.07, 71.48, 62.97, 55.42, 50.51, 48.94, 47.97, 46.72, 42.91, 41.07, 39.02, 38.84, 37.79, 37.30, 34.92, 34.34, 30.14, 29.82, 28.14, 27.55, 27.24, 25.36, 20.99, 19.33, 18.45, 16.28, 16.24, 15.50, 14.97. Anal. Calcd. for C41H58FeO3: C, 76.97; H, 9.51; Found: C, 76.55; H, 9.61. [M+Na]; HRMS (ESI): m/z calcd. for [C72H106FeNaO6]+: 1145.7233, found: 1145.7238. Biological experiments: General Information HCMV GFP-based replication assay HCMV GFP-based multi-round replication assays were carried out over a duration of seven days by infecting of primary human foreskin fibroblasts (HFFs) with a GFP-expressing recombinant human cytomegalovirus strain AD169 (HCMV AD169-GFP) as described before.[46, 62] All data represent mean values of determination in quadruplicate (HCMV infections performed in duplicate, GFP quantitation of total cell lysates performed in duplicate). Processing and evaluation of data was performed by the use of Excel. Values represent means and standard deviations. Growth inhibition studies in Plasmodium falciparum 3D7 strains Plasmodium falciparum culture: P. falciparum 3D7 parasites were cultured in type A-positive human erythrocytes at a hematocrit of 5 % in RPMI 1640 supplemented with 25 mM HEPES, 0.1 mM hypoxanthine, 50 µg/ml gentamycin and 0.5 % albumax I. Cultures were incubated at 37 oC under controlled atmospheric conditions of 5 % O2, 3 % CO2, and 92 % N2 at 95 % relative humidity.

In vitro antimalarial activity assay Cultures used in cell proliferation assays were synchronized by sorbitol treatment.[63] Concentrations to inhibit parasite growth by 50% (EC50) were determined using the SYBR Green I malaria drug sensitivity assay.[64] 100 µl aliquots of a cell suspension containing ring stages at a parasitemia of 0.2% and a hematocrit of 2% were added to the wells of 96-well microtiter plates. Plates were incubated for 72 h in the presence of different drug concentrations. Subsequently, cells of each well were lysed with 100 µl lysis buffer (40 mM Tris, pH 7.5, 10 mM EDTA, 0.02% saponin, 0.08 % Triton X-100) containing 8.3 µM SYBR green. Plates were incubated for 1 h in the dark at room temperature under constant mixing before fluorescence (excitation wavelength 485 nm; emission wavelength >520 nm) was determined using a microtiter plate fluorescence reader (Victor X4; Perkin Elmer). Drugs were serially diluted 1:3, with initial drug concentrations being 243 nM for chloroquine, artesunate, betulin, betulinic acid and their hybrids. Each drug concentration was examined in triplicate and repeated at least three times. Uninfected erythrocytes (hematocrit 2%) and infected erythrocytes without drug served as controls and were investigated in parallel. Percent growth was calculated as described by Beez.[65] Data were analyzed using the SigmaPlot (version 12.0; Hill function, three parameters) and Sigma Stat programs. Abbreviations DHA, dihydroartemisinin; DMAP, 4-(dimethylamino)-pyridine; DCC, N,N’-dicyclohexylcarbodiimide; EC50, half maximal effective concentration; EtOAc, ethylacetate; HCMV, human cytomegalovirus; TMSOTf, trimethylsilyl trifluoromethanesulfonate; TLC, thin layer chromatography; P. falciparum, Plasmodium falciparum; UV, ultraviolet.

Acknowledgments S.B.T. is grateful to the Deutsche Forschungsgemeinschaft (DFG) for generous funding by grant TS 87/16-3 and to the Interdisciplinary Center for Molecular Materials (ICMM), the Graduate School Molecular Science (GSMS), as well as Emerging Fields Initiative (EFI) “Chemistry in Live Cells” supported by Friedrich-Alexander-Universität ErlangenNürnberg for research funding. M.M. greatly acknowledges funding support through the Deutsche Forschungsgemeinschaft (grant MA 1289/7-3). Financial support by the German Academic Exchange Service (DAAD) for doctoral research fellowship for Aysun Çapcı Karagöz is gratefully acknowledged. Supplementary Material Supplementary data (1H NMR, 13C NMR and mass spectra) associated with this manuscript can be found, in the online version. References and notes [1] T.E. Komatsu, A. Pikis, L.K. Naeger, P.R. Harrington, Resistance of human cytomegalovirus to ganciclovir/valganciclovir: a comprehensive review of putative resistance pathways, Antivir. Res., 101 (2014) 12-25. [2] World malaria report 2017. Geneva: World Health Organization; 2017. Licence: CC BY-NC-SA 3.0 IGO. [3] A.M. Dondorp, F. Nosten, P. Yi, D. Das, A.P. Phyo, J. Tarning, K.M. Lwin, F. Ariey, W. Hanpithakpong, S.J. Lee, N. Eng. J. Med., 361 (2009) 455-467.

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