Synthesis of novel shikonin derivatives and pharmacological effects of cyclopropylacetylshikonin on melanoma cells

molecules

Article

Synthesis of Novel Shikonin Derivatives

and Pharmacological Effects of

Cyclopropylacetylshikonin on Melanoma Cells

1

2

3

Christin Durchschein , Antje Hufner

, Beate Rinner , Alexander Stallinger ,

Alexander Deutsch , Birgit Lohberger , Rudolf Bauer * and Nadine Kretschmer ¹

4

5

1,

1

Institute of Pharmaceutical Sciences, Department of Pharmacognosy, University of Graz,

Universitaetsplatz 4, 8010 Graz, Austria; christin.durchschein@uni-graz.at (C.D.);

nadine.kretschmer@uni-graz.at (N.K.)

Institute of Pharmaceutical Sciences, Department of Pharmaceutical Chemistry, University of Graz,

2

Universitaetsplatz 1, 8010 Graz, Austria; antje.huefner@uni-graz.at

Division of Biomedical Research, Medical University of Graz, Roseggerweg 48, 8036 Graz, Austria;

3

beate.rinner@medunigraz.at (B.R.); alexander.stallinger@medunigraz.at (A.S.)

Division of Hematology, Medical University of Graz, Auenbruggerplatz 15, 8036 Graz, Austria;

4

alexander.deutsch@medunigraz.at

Department of Orthopedics and Trauma, Medical University Graz, Auenbruggerplatz 5, 8036 Graz, Austria;

5

birgit.lohberger@medunigraz.at

*

Correspondence: rudolf.bauer@uni-graz.at; Tel.: +43-316-380-8700

Received: 9 October 2018; Accepted: 20 October 2018; Published: 30 October 2018

Abstract: Despite much research in the last centuries, treatment of malignant melanoma is still

challenging because of its mostly unnoticeable metastatic spreading and aggressive growth rate.

Therefore, the discovery of novel drug leads is an important goal. In a previous study, we have isolated

several shikonin derivatives from the roots of Onosma paniculata Bureau & Franchet (Boraginaceae)

which evolved as promising anticancer candidates.

β,β-Dimethylacrylshikonin (1) was the most

cytotoxic derivative and exhibited strong tumor growth inhibitory activity, in particular, towards

melanoma cells. In this study, we synthesized eighteen novel shikonin derivatives in order to

obtain compounds which exhibit a higher cytotoxicity than 1. We investigated their cytotoxic

potential against various melanoma cell lines and juvenile skin ﬁbroblasts. The most active

compound was (R)-1-(1,4-dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl

cyclopropylacetate (cyclopropylacetylshikonin) (

inhibitory activity towards two melanoma cell lines derived from metastatic lesions (WM164 and

MUG-Mel2). Further investigations have shown that induced apoptosis caspase-dependently,

6). It revealed signiﬁcant stronger tumor growth

6

increased the protein levels of cleaved PARP, and led to double-stranded DNA breaks as shown by

phosphorylation of H2AX. Cell membrane damage and cell cycle arrest were not observed.

Keywords: shikonin derivatives; cyclopropylacetylshikonin; apoptosis; melanoma

1

. Introduction

Skin cancer is a major public health concern all over the world. According to the WHO, one of

three diagnosed cancers is a skin cancer. Melanoma is the most dangerous type of skin cancer

because of its often unnoticeable metastatic spreading and aggressive growth rate. Only approx.

4

% of human skin cancers are melanoma but cause around 80% of all skin cancer deaths and

mortality rates are increasing worldwide [ ]. In early stages, skin-localized melanomas are complete

removed by local surgery. However, advanced metastatic melanomas remain a challenge in treatment.

1

Molecules 2018, 23, 2820; doi:10.3390/molecules23112820

www.mdpi.com/journal/molecules

Molecules 2018, 23, 2820

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Although progress in elucidating the genetic and molecular aberrations of this disease has been

accomplished in recent years and novel drugs, such as vemurafenib (BRAF inhibitor), ipilimumab

(

anti-CTLA-4 monoclonal antibody), and nivolumab (anti-PD-1 antibody), have been approved,

we are far from understanding the pathogenesis of this disease precisely [ ]. In addition, it has

to be remembered that medicinal treatments still have limited efﬁcacy due to resistances of tumors, low

response rates as well as adverse side effects [ ]. In the development of new anticancer drugs, natural

products have always played an important role. Approximately 87% of all approved small molecules

anticancer drugs are natural products or are derived from them [ ]. Famous natural products in

2

3

4

this area are vinca alkaloids isolated from Catharantus roseus G. Don, like vinblastine and vincristine,

anthracyclines from Streptomyces bacteria such as doxorubicin, and taxanes from different Taxus species,

like paclitaxel and docetaxel. During the last decades, many studies have demonstrated the broad

pharmacological spectrum of shikonin and derivatives, natural naphthoquinones derived from the

roots of Chinese herbs such as Arnebia euchroma (Royle) I. M. Johnston, Lithospermum erythrorhizon

Siebold & Zuccarini, or Onosma paniculata Bureau & Franchet. Traditionally, these roots are used to

treat several diseases including cancer. In previous studies, roots of O. paniculata were phytochemically

and pharmacologically investigated and emerged as promising research objects [

5–8]. We were able to

isolate several shikonin derivatives and investigated their effects on various tumor cell lines including

leukemia, medullary thyroid carcinoma, glioblastoma, colon cancer, breast cancer, and melanoma [

Overall, -dimethylacrylshikonin (

Interestingly, it exhibited the strongest cytotoxic activity against several melanoma cell lines [

5

–

8

].

β

,

β

1

) was the most cytotoxic compound of all isolated derivatives.

8

Therefore, we decided to concentrate our studies on the effects in melanoma cells. We could show

that it led to apoptosis induction by caspase activation as well as cell cycle arrest [ ]. In general,

the anticancer effects of shikonin derivatives are largely attributed to the induction of cell cycle arrest,

apoptosis, and/or necroptosis and the inhibition of migration and invasion [ 11]. In this study, we are

addressing the question of whether the structure of can be modiﬁed to ﬁnd derivatives which are

8

8–

1

more cytotoxic. In previous investigations, we conﬁrmed that the naphtoquinone scaffold is more

crucial for the entire activity than the side chain [12]. Moreover, the presence of phenolic hydroxyl

groups increases the activity [13,14] and the side chain seems to modify the entire activity [15–17].

We now have synthesized eighteen novel shikonin derivatives and evaluated their cytotoxic effects

against various melanoma cell lines and skin ﬁbroblasts. Finally, the pharmacological effects of the

most active derivative were investigated in more detail.

2

. Results and Discussion

2

.1. Synthesis of Novel Shikonin Derivatives

Shikonin derivatives possess strong growth inhibitory activity towards various cancer cell

lines [18

crucial for the entire activity than the side chain [12]. However, there are indications that the side

chain inﬂuences the entire activity [12 15 17]. The hydroxyl groups at the naphthazarin scaffold are

–20]. In a previous investigation, we conﬁrmed that the naphtoquinone scaffold is more

,

–

necessary for maintaining its drug-like property. Also all known anthracycline antitumor antibiotics

and mitoxantrone include a quinizarin skeleton with free hydroxyl groups. These might be important

for DNA binding and cell or tissue bioavailability [21]. Therefore, we decided to modify the side

chain of

compound

groups resulting in the corresponding cycloalkylideneacetates

was investigated by preparing cycloalkylacetates to and cyclohexenylacetate 10. To study the effect

1

which was the most cytotoxic derivative in a previous study [

, we formally connected the two methyls of dimethylacylate with one to four methylene

to . The inﬂuence of the double bond

8]. To keep close to our lead

1

2

5

6

9

of the spacing CH resp. CH group, cyclopropyl to cyclohexylcarboxylates 11 to 14 together with the

2

cyclohexenylcarboxylates 15 and 16 were synthesized. 3-methylcyclopropylcarboxylate 17 and bicyclic

derivatives 18

Table 1).

, 19, and 20 were produced to analyze the space ﬁlling of an additional methylene moiety

(

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Table 1. List of investigated shikonin esters.

OH

O

OR

5

'

8

1

4

'

1

'

6

'

5

OH

O

Cpd.

R

Cpd.

R

O

1

2

11

4

''

O

12

1''

2''

α

O

3

4

13

14

O

5

6

15

16

5

''

O

1

''

3

''

O

7

8

17

18

rel.

1

''

O

rel.

5

''

7''

2

''

3

''

O

9

19

20

rel.

5

''

3

''

O

7

''

1

0

1

''

α

Since shikonin is easier and in bigger amounts acquirable than 1, we used shikonin as

starting material for our synthesis. Shikonin (100% R-isomer, seeSupplementary Material)

was acylated as usual via Steglich esteriﬁcation with the corresponding carboxylic acid,

dicyclohexylcarbodiimide (DCC) as a coupling reagent and 4-dimethylaminopyridine (DMAP) as

catalyst in dichloromethane (Scheme 1) [16,21–25]. As outlined in Scheme 2, cyclohexylideneacetic

acid (4a) and cycloheptylideneacetic acid (5a) resulted from condensation of the corresponding

cycloketone with chloroacetic acid mediated by sodium hydride and diethylphosphite [26].

Cyclobutylideneacetic acid (2a) and cyclopentylideneacetic acid (3a) were synthesized via

Horner–Wadsworth–Emmons reaction [27] followed by hydrolysis with lithium hydroxide

monohydrate in aqueous THF/MeOH [28]. The C=C double bond of ethyl cyclopentylideneacetate

was partially isomerized resulting in a 1:3 mixture of cyclopentylideneacetic acid (3a) and

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2

-cyclopentenylacetic acid (3b). 7a was prepared analogously to 3-ethylpentanoic acid via Grignard

reaction using (bromomethyl)cyclobutane and carbon dioxide (Scheme 3) [29].

Scheme 1. Acylation of shikonin.

Scheme 2. Syntheses of cycloalkylideneacetic acids 2a to 5a. (

i) (EtO) P(O)-CH -COOEt, NaH, Et O,

2 2 2

cyclobutanone or cyclopentanone (ii) LiOH H O, THF, H O, MeOH, (iii) 1. HP(OMe) , NaH,

×

2

ClCH COOH, cyclohexanone or cycloheptanone, glyme 2. HCl, H O.

2

Scheme 3. Synthesis of cyclobutylacetic acid (3a).

2

.2. Cytotoxicity of Investigated Shikonin Derivatives

Since all synthesized derivatives were 100% R-isomers, the ﬁrst question was if the chiral center

inﬂuences the overall pharmacological activity. Therefore, different samples of with varying

enantiomeric ratios were prepared. Melanoma cells were treated with these compounds up to

0.0 M and for 72 h and the effect was compared to (isolated from roots). We did not detect

1

µ

1

signiﬁcant differences in the cytotoxicity indicating that the chiral center has no inﬂuence on the

activity. In addition, it made no difference if the compound was synthesized (70/30 mixture) or

isolated (see Figure S1, Supplementary Material).

Subsequently, all derivatives were investigated regarding their cytotoxicity against various

melanoma cell lines (SBcl2, WM9, WM164, and MUG-Mel2) and juvenile skin ﬁbroblasts. Cells were

treated with different concentrations of

Supplementary Material) and their effects compared to

cancer progression (cutaneous melanoma, radial growth phase) and are very sensitive to

2

to 20 (Figure 1 and, additionally, Figures S2 and S3,

. SBcl2 cells represent an early state of

and

1

many other compounds. WM9, WM164, and MUG-Mel2 are derived from melanoma metastases

located at the left axillary node (BRAF mutated), right upper arm with stage IV superﬁcial spreading

melanoma (BRAF mutated), and from very fast growing metastasis of the left shoulder (NRAS

mutated), respectively [30]. Especially WM164 and MUG-Mel2 cells are less susceptible to many

cytotoxic compounds and, therefore, difﬁcult to treat. This is also reﬂected by our results. For example,

compounds 2, 4, and 7 were considerably less cytotoxic towards WM164 and MUG-Mel2 cells than

towards the other cell lines. However, this does not apply to all cycloalkylidenes and cycloalkylacetates.

The assumption that an increase of steric bulkiness results in decreased cytotoxic activity [16] does

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not apply to the cytotoxicity of our cycloalkylideneacetates series

2

to

5

with

5

as the most potent one.

This regularity was also not strictly observed for cycloalkylacetates

1 and 14.

Cyclopropylacetate

WM164 and MUG-Mel2 than

major clinical problems and respond poor to most treatment options.

6

to

9 and cycloalkane carboxylates

1

6

turned out to be signiﬁcantly more active against the metastatic cell lines

(Table 2). This is of special interest because these kinds of cells cause

was also more cytotoxic against

1

6

the melanoma cell lines used than 11, which was the most active derivative in a previous study [20].

However, it also exhibited cytotoxicity against juvenile skin ﬁbroblasts (IC₅₀= 1.6 ± 0.4 µM). To better

assess its cytotoxicity against nontumorigenic cells, 6 was also tested on two other healthy cell types.

On the one hand, human embryonic epithelial cells (HEK-293), a well-established nontumorigenic

cell line, was used. On the other hand, we used isolated human adult ﬁbroblasts to study the

cytotoxicity against another type of ﬁbroblasts. Fibroblasts have been shown to display distinct

transcriptional patterns depending on their origin [31]. Compared to juvenile ﬁbroblasts, IC₅₀

values of

against adult ﬁbroblasts (IC₅₀= 6.4

nontumorigenic types of cells. Nevertheless, toxicity of chemotherapeutics to healthy cells is a

well-known problem in cancer therapy and leads to undesirable side effects in patients. For example,

vinblastine, a commonly used chemotherapeutic, exhibited IC₅₀values towards melanoma cells and

6

were 3.4 fold higher towards HEK-293 cells (IC₅₀= 5.4

±

0.7 µM) and 4.0 fold higher

±

0.7 M). This shows that the cytotoxicity varies in different

µ

lung ﬁbroblasts within the same concentration range [

commonly used chemotherapeutic—which showed the same or even a higher cytotoxicity against

HEK-293 cells than against breast cancer and leukemia cells [32 33]. However, quinones and derivatives

8]. Another example is doxorubicin—again a

,

are also members of the PAINS group. PAINS (Pan-Assay Interference Compounds) possess common

structural motifs that lead to strong activities in biological assays. PAINS structures occur in natural

products (e.g., vitamin K2 and thymoquinone) as well as synthetic drugs. Even some approved

chemotherapeutics such as mitoxantrone and doxorubicin contain a PAINS motif. PAINS structures

lead, for example, to reactions with nucleophiles such as thiols or amines and cause redox cycling.

Quinones including shikonin derivatives possess strong redox activity. Therefore, they can react with

nucleophiles, for example, in the side chains of proteins [34]. This, in turn, can lead to adverse

side effects. To overcome or reduce these adverse effects, one might be tempted to use smart

6

-loaded targeted nanoparticles. It has been reported that blood vessels of tumors are leaky allowing

nanoparticles to penetrate speciﬁcally into the tumor tissue. In addition, lymphatic drainage in tumors

is poor retaining the accumulated nanoparticles and allowing the drug to be released [35]. Moreover,

shikonin-loaded nanoparticles improved the antitumor effects of shikonin in glioma cells in vitro and

the particles accumulated in the brain of rats [36]. For melanoma, it has been demonstrated recently

that self-assembled nanomicelles of clotrimazole improve drug delivery and apoptosis and, at the

same time, inhibit tumor progression [37]. Therefore, we assume that this might be a promising way

for further development of 6. However, development, characterization as well as in vitro and in vivo

testing of such nanoparticles goes beyond the scope of the current work.

Returning to the structure activity relationship of our synthesized compounds, methylation of

the cyclopropane moiety of 11 further reduced the activity (see compound 17). An additional double

bond in the carbocyclus, as present in compounds 10, 15 and 16, did not enhance cytotoxicity and,

compared to the corresponding monocyclic derivative, an additional methylene bridge as presented in

the bicyclic derivatives 18 to 20 provides no regularity concerning activity.

In summary, (R)-1-(1,4-dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl

cyclopropylacetate (cyclopropylacetylshikonin,

values towards the metastatic cell lines WM164 and MUG-Mel2 were 1.7 fold (WM164, p = 0.05) and

.3 fold (MUG-Mel2, p 0.001) lower than those of , while the effectiveness against the other

melanoma cell lines was similar compared to 1 (Table 2).

6) was the most active derivative of this series. The IC₅₀

2

≤

1

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Figure 1. Results of the XTTassay. Melanoma cells (SBcl2, WM9, WM164, and MUG-Mel2) and juvenile

skin ﬁbroblasts were treated with 5.0 M of to 20 for 72 h (mean SEM, n = 4). Compound serves as

reference compound. Results of all tested concentrations can be found in the Supplementary Material.

µ

2

±

1

Table 2. IC₅₀values ( M) after 72 h treatment with or (mean SEM, n = 4). IC₅₀

µ

1

6

±

values were determined using the four-parameter logistic curve and individual values of all

independent experiments.

Compound

SBcl2

WM9

WM164

MUG-Mel2

1

6

1.1 ± 0.1

0.9 ± 0.7

2.7 ± 0.3

3.2 ± 0.8

8.3 ± 0.3

4.9 ± 1.7

7.2 ± 0.5

3.2 ± 0.3

2

.3. Pharmacological Effects of Cyclopropylacetylshikonin (6)

Since revealed the strongest growth inhibitory activity towards the melanoma cell lines used,

its pharmacological effects were investigated in more detail. Figure 2 summarizes the results of the

ApoToxGlo assay. This assay determines cell viability, compound cytotoxicity, and the activity of

caspase 3/7, indicative for apoptosis induction, simultaneously in the same well. Cell viability of WM9

and MUG-Mel2 was slightly reduced after 24 h at 7.5 M (Figure 2A). However, this change was not

6

™

µ

statistically signiﬁcant. WM164 cells showed no reduction of cell viability but a slight, however, again

not statistically signiﬁcant increase. Cytotoxicity was minimally decreased in all cell lines after 48 h

(

Figure 2B). Caspase 3/7 activity peaked after 24 h with the maximum at 5.0

The strongest activation was found for WM9 cells. In WM164, there was just a marginal increase

in caspase 3/7 activity. In previous investigations, we demonstrated that also induced apoptosis

caspase 3 dependently after 24 h [ ]. The induction of apoptosis by shikonin derivatives was also

reported in other types of cancer such as medullary thyroid carcinoma cells [ ], human gastric cancer

cells, and human breast cancer cells [38 39]. To conﬁrm apoptosis induction, immunoblotting and ﬂow

µM and 7.5 µM (Figure 2C).

1

8

6

,

cytometric experiments were performed. Caspases are produced as inactive precursors and cleaved

into active enzymes when needed. Thereby, many factors, e.g., the induction of double-stranded

DNA breaks, can lead to caspase activation. When DNA double-stranded breaks occur, H2AX is

phosphorylated as consequence and, subsequently, induces apoptosis. Activated caspases, in turn,

cleave other proteins, for example poly (ADP-ribose) polymerase (PARP) [40]. Therefore, protein

expression of cleaved PARP and phosphorylated H2AX was investigated. The levels of both proteins

were increased by

6 as shown in Figure 3A–C. Our results indicate that DNA damage caused by 6

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might be one reason for apoptosis induction. Also, by using FACS experiments, caspase 3 cleavage

was seen in MUG-Mel2 and, particularly, in WM9 cells (Figure 3D), conﬁrming the previous results.

Furthermore, the combination of

6 and the caspase inhibitor Z-VAD-FMK led to a declineof caspase 3

activation again conﬁrming the involvement of caspases.

Since a joint decline of viability and cytotoxicityin the ApoToxGlo

necrosis induction, the CytoTox 96 Non-Radioactive Cytotoxicity Assay (LDH assay) was performed

™ assay can be a hint of

®

(

Figure 4A). The results show that

incubation. This indicates that necrosis is not involved in the observed cell death.

Previous investigations have also demonstrated that 1 affected the amount of melanoma cells in

the S- (SBcl2 and WM35 cells) or G2/M-phase (WM9 and WM164 cells) [ ]. Also shikonin led to a cell

cycle arrest in different cancer cell lines and immortalized human keratinocytes [41 45]. Therefore,

the effects of on cell cycle distribution were investigated (Figure 4B–D). However, did not lead to a

signiﬁcant cell cycle arrest, suggesting a different mode of action in these cell lines.

6 did not signiﬁcantly damage cell membrane up to 5.0 µM and 48 h

8

–

6

Figure 2. Results of the ApoTox-Glo

and caspase 3/7 activity ( ) in different melanoma cells after 6 h, 24 h, and 48 h treatment with

(mean SEM, n = 6). Staurosporine (10.0 M) served as positive control for apoptosis induction

caspase activity after 24 h: WM9: 724.8 3.8%; WM164: 898.6 18.4%, and MUG-Mel2: 637.7 64.8%

and after 48 h: WM9: 181.1 ± 4.1%, WM164: 226.6 ± 22.5%, and MUG-Mel2: 314.1 ± 41.3%).

™Triplex assay showing cell viability (A), cytotoxicity (B),

C

6

(

±

µ

±

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Figure 3. Western blot (

different melanoma cells. (

DNA damage marker phosphorylated H2AX. The effect of

evaluated by immunoblotting under control conditions and in the presence of 5.0

for 24 h. Fold change is normalized to controls (mean SD, n = 3). ( ) One representative immunoblot

out of three is shown. -Actin was used as loading control. ( ) Results of cleaved caspase 3 FACS

measurements after treatment with (5.0 and 7.5 M) and 24 h incubation with or without caspase 3

inhibitor (INH), which conﬁrmed the results above (mean ± SEM, n = 6).

A

–

A

C

) and ﬂow cytometric experiments (

) Densitometric analysis of the apoptotic marker cleaved PARP and (

on three different melanoma cell lines was

M and 7.5 M of

D

) concerning apoptosis induction in

B) the

6

µ

6

±

C

β

D

6

µ

Figure 4.

damage to the cell membrane in different melanoma cells (mean

control was used to determine maximal LDH release. ( ) Treatment of melanoma cells with 6

(A

) Results of lactate dehydrogenase (LDH) assay showing that

SEM, n = 6). Maximum LDH release

did

6 caused no signiﬁcant

±

B

–D

not lead to signiﬁcant changes in the cell cycle distribution (B: WM9, C: MUG-Mel2, and D: WM164

cells, 24 h of incubation, mean ± SEM, n = 3).

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3

. Material and Methods

.1. Chemicals and Synthesis of Derivatives

.1.1. Chemicals

Shikonin was obtained from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China).

Reagents and solvents were obtained from commercial suppliers. Solvents were dried and puriﬁed

using standard techniques.

3

.1.2. Synthesis

Compounds were synthesized as described below and in the Supplementary Material. Derivatives

with additional chiral centers in the acyl side chain were not separated into diastereomers. Solvents

were of p.a. quality, if not stated otherwise. Analytical thin layer chromatography (TLC) was

performed using aluminium foil plates coated with silica 60 F₂₅₄(Merck, Darmstadt, Germany).

Shikonin derivatives were detected in VIS. Some intermediates were additionally visualized by UV

2

54 nm and spraying with molybdatophosphoric acid and subsequent heating. Preparative thin layer

chromatography (PTLC) was done on analytical aluminium or plastic foil plates coated with silica 60

F₂₅₄(Merck, Darmstadt, Germany) and developed until the solvent front reached 8 cm. For column

chromatography (CC), silica gel 60 (63–200

µm, Merck, Darmstadt, Germany) was used. Infrared

spectra were recorded on a Bruker Alpha Platinum ATR spectrometer (Bruker, Kennewick, WA, USA).

1

13

H and C-NMR spectra were recorded on Varian 400 MHz UnityINOVA spectrometer (400 and

00 MHz, respectively, Varian, Palo Alto, CA, USA) using deuterated chloroform (CDCl ) as the

1

3

1

13

solvent and TMS as internal standard. H- and C-resonances are indicated as given in the Table 1.

Assignments marked with an asterisk are interchangeable. The purity of all synthesized compounds

was veriﬁed using NMR and analytical HPLC. The purity exceeded 95%. Liquid chromatography

coupled with electrospray ionization-tandem mass spectrometry (LC-ESI-MS) analyses were carried

out on a Dionex Ultimate 3000 UHPLC (Thermo, San José, CA, USA) coupled with a Thermo LTQ

XL linear ion trap mass spectrometer equipped with an H-ESI II probe in the negative mode using

◦

5

00 nm as acquisition wavelength (source heater temperature, 250 C, capillary temperature, 200 C,

source voltage, 3.5 kV, capillary voltage, 14 V, sheath gas ﬂow, 50 arbitrary units, auxiliary gas ﬂow,

0 arbitrary units). As stationary phase, a Kinetex C18 column (2.6 m, 100 2.10 mm, Phenomenex,

−

1

µ

×

Torrance, FL, USA) was used. The mobile phase consisted of water (A) and acetonitrile (B). The gradient

was set as follows 0–45 min: 55–100% B. Flow rate was 0.2 mL/min. Column temperature was set at

◦

3

0 C.

3

.1.3. General Procedure for the Acylation of Shikonin

◦

A solution of shikonin in abs. CH Cl (0.1 mmol/5 mL) was cooled to 0 C under argon

2

atmosphere and DCC was added. DMAP was added after stirring for 15 min. After further stirring for

5 min, the corresponding acid was added and stirring was continued for 14 h to 5 days with slowly

1

warming up to room temperature. After addition of 1 mL cyclohexane/0.1 mmol shikonin, the mixture

was concentrated under reduced pressure at room temperature to approx. 0.5 mL/0.1 mmol shikonin.

®

The mixture was ﬁltered over 3 mm silica and 2 mm celite (eluent: petroleum ether/CH Cl = 1:0 to

2

1:2). Product containing fractions were evaporated and submitted to ﬂash CC and/or repeated PTLC

(

cyclohexane/CH Cl mixtures). Due to the rapid decomposition of raw acylshikonin mixtures on

2

evaporation to higher concentrations (c > approx. 0.2 M acylshikonin) and degradation of acylshikonins

on prolonged contact with silica, intermediate solutions were not to be concentrated to dryness and all

isolation and puriﬁcation steps had be performed at a good pace.

(

0

R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 2-cyclobutylideneacetate (

.1 mmol Shikonin, 0.20 mmol DCC, 30 mol DMAP, and 0.1 mmol 2-cyclobutylideneacetic acid (2a);

reaction time 5 days; PTLC on silica (developed twice with cyclohexane/CH Cl = 2:1 and twice

2):

µ

2

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with cyclohexane/CH Cl = 1:2) and PTLC on silica with cyclohexane/CH Cl = 2:1 (three times

2

developed),

2

, yield: 3%. 2: R = 0.31 (silica, cyclohexane/CH Cl = 1:4); IR (ATR): 2957 (w), 2919 (m),

f

2 2

≈

2950 (br), 2851 (w), 1716 (m), 1668 (m), 1609 (m), 1568 (m), 1454 (m), 1263 (s), 1202 (s), 1170 (s),

− 0 0

108 (m), 703 (w) cm ; H-NMR (CDCl ): δ 1.58 (s, 3H, H-6 ), 1.69 (s, 3H, H-5 ), 2.12 (quint, J = 7.9 Hz,

3

1

2

0

H, H-3 ), 2.49 (dtm, J = 14.9, 7.4 Hz, 1H, H-2 ), 2.62 (dtm, J = 14.9, 5.5 Hz, 1H, H-2 ), 2.88 (t, J = 7.6 Hz,

00

H, H-2 , H-4 ), 3.14 (qm, J = 7.7 Hz, 2H, H-2 , H-4 ), 5.14 (tm, J = 7.1 Hz, 1H, H-3’), 5.68 (quint,

H, J = 2.1 Hz, H- ), 6.02 (dd, J = 7.1, 4.4 Hz, 1H, H-1’), 6.96 (d, J = 0.9 Hz, 1H, H-3), 7.18 (s, 2H, H-6,

H-7), 12.44 (s, 1H, C5-OH), 12.59 (s, 1H, C8-OH); C-NMR (CDCl₃):

1

α

13

00

δ

17.6 (C-3 ), 18.0 (C-6’), 25.8

00 00

C-5’), 32.4 (C-2 *), 32.9 (C-2’), 34.1 C-4 *), 69.0 (C-1’), 111.6 (C-4a, C-α), 111.8 (C-8a), 118.1 (C-3’), 131.6

(

0

(

C-3), 132.5 (C-6, C-7), 135.9 (C-4’), 148.9 (C-2), 165.1 (COO), 166.2 (C-5), 166.8 (C-8), 169.8 (C-1 ), 177.4

−

(

C-1), 179.0 (C-4); MS (ESI ) m/z (%): 819 (16) [2(M

−

H) + Cl + Na] , 785 (37) [2(M

−

H) + Na] , 417

H] , 137 (44); [M] calculated for

−

and 415 (14) [M

C H O : 382.1416.

2

−

2H + Cl] , 382.11 (100) [M] ; 381.21 (53) [M

−

2

22

6

(

0

R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 2-cyclopentylideneacetate (

.1 mmol Shikonin, 0.20 mmol DCC, 25 mol DMAP, and 0.11 mmol 2-cyclopentylideneacetic acid (3a);

reaction time 16 h; PTLC on silica (developed twice with cyclohexane/CH Cl = 2:1 and twice with

3):

µ

2

cyclohexane/CH Cl = 1:2) and PTLC on silica with cyclohexane/CH Cl = 2:1 (four times developed),

2

3

, yield: 5%.

3

: R = 0.37 (silica, CH Cl ); IR (ATR): 2959 (w), 2918 (w),

≈

2950 (br), 2850 (w), 1717 (m),

f

2

−

1 1

1648 (m), 1609 (vs), 1569 (m), 1453 (m), 1230 (m), 1188 (vs), 1114 (s) cm ; H-NMR (CDCl ): 1.58 (s,

3

0

00

3

H, H-6’), 1.68 (m, 2H, H-4 ), 1.69 (s, 3H, H-5’), 1.75 (m, 2H, H-3 ), 2.48 (m, 3H, H-2’ and H-2 ), 2.63

0

(

dtm, J = 15.3, 5.4 Hz, 1H, H-2’), 2.75 (m, 2H, H-5 ), 5.15 (tm, J = 7.2 Hz, 1H, H-3’), 5.90 (quint, 1H,

J = 1.9 Hz, H- ), 6.02 (dd, J = 7.4, 4.7 Hz, 1H, H-1’), 6.97 (d, J = 0.8 Hz, 1H, H-3), 7.18 (s, 2H, H-6 and

H-7), 12.43 (s, 1H, C5-OH), 12.60 (s, 1H, C8-OH); C-NMR (CDCl₃):

α

13

00

δ

18.0 (C-6’), 25.5 (C-4 ), 25.8

), 111.7 (C-4a), 111.8 (C-8a),

18.1 (C-3’), 131.6 (C-3), 132.4 (C-7), 133.5 (C-6), 135.8 (C-4’), 149.2 (C-2), 165.5 (COO), 166.1 (C-5), 166.7

00 00 00

(

C-5’), 26.4 (C-3 ), 32.9, 33.0 (C-2’, C-2 ), 36.2 (C-5 ), 68.7 (C-1’), 110.9 (C-α

1

0

−

H)+Na] , 395.30 (100)

(C-8), 171.5 (C-1 ), 177.7 (C-1), 179.1 (C-4); MS (ESI ) m/z (%): 813 (10) [2(M

−

[M − H] ; [M − H] calculated for C H O : 395.1495.

23

24

6

(

R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 2-cyclohexylideneacetate (

4):

0.1 mmol Shikonin, 0.20 mmol DCC, 25 µmol DMAP, and 0.11 mmol 2-cyclohexylideneacetic acid (4a);

reaction time 16 h; Flash-CC on silica (3 g) with petroleum ether/CH Cl = 0:1 to 1:0) and PTLC on

2

silica each developed twice with cyclohexane/CH Cl = 2:1 and twice with cyclohexane/CH Cl = 1:2);

2

4

, yield: 13%.

4

: R = 0.31 (silica, CH Cl ); IR (ATR): 2919 (m),

≈

2950 (br) (OH), 2851 (w), 1720 (m)

f

2

−

1

(

C=O), 1643 (m), 1610 (s), 1570 (m), 1451 (m), 1266 (m), 1233 (m), 1201 (s) (COC), 1147 (s), 777 (w) cm

H-NMR (CDCl ): 1.58 (s, 3H, H-6’), 1.59-1.70 (m, 6H, H-3 , H-4 , H-5 ), 1.69 (s, 3H, H-5’), 1.75 (m,

;

1

00

3

00 00

2H, H-3 ), 2.23 (t, J = 6.5 Hz, 2H, H-2 ), 2.48 (dt, J = 15.0, 7.3 Hz, 1H, H-2’), 2.62 (dtm, J = 15.2, 5.4 Hz,

0

1

H, H-2’), 2.75 (t, J = 5.5 Hz, 2H, H-6 ), 5.15 (tm, J = 7.2 Hz, 1H, H-3’), 5.70 (s, 1H, H-

α), 6.02 (dd, J = 7.4,

4

.7 Hz, 1H, H-1’), 6.98 (d, J = 0.8 Hz, 1H, H-3), 7.18 (s, 2H, H-6 and H-7), 12.44 (s, 1H, C5-OH), 12.60

1

3

00 00 00

s, 1H, C8-OH); C-NMR (CDCl ): δ 18.0 (C-6’), 25.8 (C-5’), 26.2 (C-4 ), 27.8 (C-5 ), 28.6 (C-3 ), 30.0

3

(

0

00

C-6 ), 32.9 (C-2’), 38.1 (C-2 ), 68.6 (C-1’), 111.6 (C-4a), 111.9 (C-8a), 112.1 (C-

C-3), 132.4 (C-7), 132.6 (C-6), 135.9 (C-4’), 149.1 (C-2), 165.4 (COO), 166.0 (C-1 ), 166.3 (C-5), 166.8

C-8), 177.6 (C-1), 179.1 (C-4); MS (ESI ) m/z (%): 841 (11) [2(M

100) [M − H] ; [M] calculated for C H O : 410.1729.

α

), 118.0 (C-3’), 131.7

0

−

− −

−

H) + Na] , 410.23 (47) [M] , 409.23

−

2

4

26

6

(

0

R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 2-cycloheptylideneacetate (

.1 mmol Shikonin, 0.20 mmol DCC, 25 mol DMAP, and 0.11 mmol 2-cycloheptylideneacetic acid (5a);

reaction time 16 h; PTLC on silica (developed twice with cyclohexane/CH Cl = 2:1 and twice with

5):

µ

2

cyclohexane/CH Cl = 1:2) and PTLC on silica with cyclohexane/CH Cl = 2:1 (four times developed),

2

5

, yield: 10%.

5

: R = 0.40 (silica, CH Cl ); IR (ATR): 2918 (m),

≈

2950 (br), 2850 (w), 1717 (m), 1609 (s),

f

2

−

1

1569 (m), 1560 (m), 1453 (m), 1201 (m), 1136 (s), 779 (w) cm ; H-NMR (CDCl ): 1.52–1.55 (m, 2H,

3

0

00

0

00

0

00

H-4 , H-5 ), 1.55 (s, 3H, H-6 ), 1.62-1.69 (m, 2H, H-3 , H-6 ), 1.69 (s, 3H, H-5 ), 2.42 (m, 2H, H-2 ),

Molecules 2018, 23, 2820

11 of 21

0 0 00

.49 (dtm, J = 14.8, 7.3 Hz, 1H, H-2 ), 2.61 (dtm, J = 14.9, 5.0 Hz, 1H, H-2 ), 2.85 (m, 2H, H-7 ), 5.15

2

(

1

(

6

(

0

α), 6.01 (ddd, J = 7.2, 4.6, 0.8 Hz, 1H, H-1 ), 6.98 (d, J = 0.8 Hz,

tm, J = 7.4 Hz, 1H, H-3 ), 5.77 (s, 1H, H-

H, H-3), 7.18 (s, 2H, H-6, H-7), 12.44 (s, 1H, C5-OH), 12.60 (s, 1H, C8-OH); C-NMR (CDCl ): δ 18.0

1

3

0

00

0

00

C-6 ), 25.8 (C-5 ), 26.6 (C-6 ), 27.9 (C-3 ), 29.0 (C-4 ), 29.8 (C-5 ), 32.3 (C-7 ), 33.0 (C-2 ), 39.2 (C-2 ),

0

8.5 (C-1 ), 111.6 (C-4a), 111.9 (C-8a), 114.7 (C-

α

), 118.1 (C-3 ), 131.7 (C-3), 132.4 (C-7), 133.5 (C-6), 135.8

0

00

−

C-4 ), 149.2 (C-2), 165.2 (COO), 166.1 (C-5), 166.7 (C-8), 169.2 (C-1 ), 177.7 (C-1), 179.2 (C-4); MS (ESI )

−

m/z (%): 869 (15) [2(M

−

H) + Na] , 424.25 (45) [M] , 423.22 (100) [M

−

H] 159 (31), 137 (39); [M]

calculated for C H O : 424.1886.

25

28

6

(

0

R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl cyclopropylacetate (

.2 mmol Shikonin, 0.4 mmol DCC, 80 mol DMAP, and 0.25 mmol cyclopropylacetic acid (6a);

reaction time 16 h; CC on silica (7 g) with cyclohexane/CH Cl = 2:1 to 1:4; , yield 26%. : R = 0.29

2950 (br), 2916 (w), 2857 (w), 1742

6):

µ

6

2

f

(

silica, cyclohexane/CH Cl = 1:4); IR (ATR): 3080 (w), 2972 (w),

≈

2

−

1 1

s), 1610 (s), 1570 (m), 1454 (m), 1231 (m), 1204 (s), 784 (m) cm ; H-NMR (CDCl ): 0.17–0.24 (m, 2H,

3

0

00

0

H-2 , H-3 ), 0.56–0.63 (m, 2H, H-2 , H-3 ), 1.02–1.13 (m, 1H, H-1 ), 1.58 (s, 3H, H-6 ), 1.69 (s, 3H,

H-5 ), 2.23–2.37 (m, 2H, H-α), 2.49 (dtm, J = 15.0, 7.5 Hz, 1H, H-2 ), 2.63 (dtm, J = 15.0, 5.5 Hz, 1H, H-2 ),

5

7

0

.14 (tm, J = 7.1 Hz, 1H, H-3 ), 6.05 (ddd, J = 7.3, 4.5, 1.0 Hz, 1H, H-1 ), 7.01 (d, J = 1.0 Hz, 1H, H-3),

13

00

.19 (s, 2H, H-6, H-7), 12.43 (s, 1H, C5-OH), 12.59 (s, 1H, C8-OH); C-NMR (CDCl₃):

δ

4.4, 4.5 (C-2 ,

0

00

C-3 ), 7.0 (C-1 ), 18.0 (C-6’), 25.8 (C-5’), 32.9 (C-2’), 39.5 (C-

α), 69.4 (C-1’), 111.6 (C-4a), 111.8 (C-8a),

1

17.8 (C-3’), 131.5 (C-3), 132.7 (C-7), 132.8 (C-6), 136.0 (C-4’), 148.4 (C-2), 166.8 (C-5), 167.4 (C-8), 172.0

−

(

COO), 176.8 (C-1), 178.3 (C-4); MS (ESI ) m/z (%):1165 (5) [3M

−

3H + Na + K] , 1147 (7) [3M

−

2H +

−

K] , 1131 (11) [3M

−

2H + Na] , 761 (1) [2(M

−

H) + Na] , 637 (2) [2M

−

3H

−

RCOOH] , 370.22

−

(32) [M] , 369.25 (100) [M − H] ; [M] calculated for C H O : 370.1416.

21

22

6

(

5

R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl cyclobutylacetate

mol Shikonin, 0.15 mmol DCC, 30 mol DMAP, and 53 mol cyclobutylacetic acid (7a);

reaction time 16 h; PTLC on silica (developed twice with cyclohexane/CH Cl = 2:1 and one

(7):

0

µ

2

time with cyclohexane/CH Cl = 1:2) and an additional PTLC on silica (developed twice with

2

cyclohexane/CH Cl = 2:1);

7

, yield: 21%. 7: R = 0.21 (silica, cyclohexane/CH Cl = 1:4); IR (ATR):

2

f

2 2

2

959 (w), 2918 (m),

≈

2950 (br), 2851 (w), 1737 (m), 1609 (s), 1568 (m), 1453 (m), 1264 (m), 1229 (m),

−

1

0

1

202 (s), 1158 (m), 777 (w) cm ; H-NMR (CDCl ): 1.58 (s, 3H, H-6 ), 1.69 (s, 3H, H-5 ), 1.70–1.78 (m,

3

0

00

2H, trans H-2 , trans H-4 ), 1.81–1.97 (m, 2H, H-3 ), 2.09–2.20 (m, 2H, cis H-2 , cis H-4 ), 2.46 (dtm,

0

J = 15.0, 7.5 Hz, 1H, H-2 ), 2.49 (d, J = 7.5 Hz, 2H, H-

J = 7.8 Hz, 1H, H-1 ), 5.11 (tm, J = 7.3 Hz, 1H, H-3’), 6.00 (ddd, J = 7.7, 4.2, 0.9 Hz, 1H, H-1’), 6.96 (d,

J = 1.0 Hz, 1H, H-3), 7.18 (s, 2H, H-6, H-7), 12.42 (s, 1H, C5-OH), 12.57 (s, 1H, C8-OH); C-NMR

α

), 2.60 (dtm, J = 14.7, 5.9 Hz, 1H, H-2 ), 2.71 (quint,

0

13

00 00 00 00

δ 18.0 (C-6’), 18.5 (C-3 ), 25.8 (C-5’), 28.2, 28.3 (C-2 , C-4 ), 32.2 (C-1 ), 32.9 (C-2’), 41.2 (C-α),

(CDCl₃):

6

(

−

9.2 (C-1’), 111.6 (C-4a), 111.8 (C-8a), 117.8 (C-3’), 131.4 (C-3), 132.7 (C-7), 132.9 (C-6), 136.0 (C-4’), 148.5

−

C-2), 166.9 (C-5), 167.4 (C-8), 171.5 (COO), 176.8 (C-1), 178.3 (C-4); MS (ESI ) m/z (%): 789.37 (8) [2(M

−

H) + Na] , 384.21 [M] (52), 383.26 (100) [M − H] ; [M] calculated for C H O : 384.1573.

22

24

6

(

0

R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl cyclopentylacetate (

.1 mmol Shikonin, 0.15 mmol DCC, 25 mol DMAP, and 0.1 mmol cyclopentylacetic acid (8a);

reaction time 17 h; CC on silica (7 g) with cyclohexane/CH Cl₂= 2:5 and PTLC on silica

8):

µ

2

with cyclohexane/CH Cl = 2:5 (three times developed);

8

, yield 10%.

8: R = 0.26 (silica,

f

2

cyclohexane/CH Cl = 1:4); IR (ATR): 2951 (m, br), 2866 (w), 1738 (s), 1608 (s), 1569 (m), 1452 (m), 1230

2

− 00 00 0

s), 1200 (s), 1111 (s), 777 (m) cm ; H-NMR (CDCl ): 1.12–1.23 (m, 2H, H-2 , H-5 ), 1.58 (s, 3H, H-6 ),

3

1

(

00 00 0 00 00

.52–1.68 (m, 4H, H-3 , H-4 ), 1.69 (s, 3H, H-5 ), 1.78–1.88 (m, 2H, H-2 , H-5 ), 2.25 (sept, J = 7.7 Hz,

H, H-1 ), 2.37–2.43 (m, 2H, H-

H, H-2 ), 5.13 (tm, J = 7.3 Hz, 1H, H-3 ), 6.03 (ddd, J = 7.3, 4.4, 0.8 Hz, 1H, H-1 ), 6.99 (d, J = 1.0 Hz,

H, H-3), 7.19 (s, 2H, H-6, H-7), 12.43 (s, 1H, C5-OH), 12.59 (s, 1H, C8-OH); C-NMR (CDCl ): δ 18.0

0 00 00 00 00 0 00

C-6 ), 25.0 (C-3 , C-4 ), 25.8 (C-5’), 32.4, 32.5 (C-2 , C-5 ), 33.0 (C-2 ), 36.5 (C-1 ), 40.4 (C-α), 69.2

C-1 ), 111.6 (C-4a), 111.8 (C-8a), 117.9 (C-3 ), 131.5 (C-3), 132.7 (C-7), 132.8 (C-6), 136.0 (C-4 ), 148.5

1

0

α

), 2.47 (dtm, J = 15.1, 7.5 Hz, 1H, H-2 ), 2.61 (dtm, J = 15.0, 5.7 Hz,

0

13

3

(

0

Molecules 2018, 23, 2820

12 of 21

−

(

2

C-2), 166.8 (C-5), 167.4 (C-8), 172.1 (COO), 176.8 (C-1), 178.3 (C-4); MS (ESI ) m/z (%): 1231 (9) [3M

−

− −

H] ; [M] calculated for

H + K] , 1215 (8) [3M

−

2H + Na] , 398.23 (34) [M] , 397.30 (100) [M

−

C H O : 398.1729.

23

26

6

(

5

R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl cyclohexylacetate

mol Shikonin, 0.15 mmol DCC, 30 mol DMAP, and 53 mol cyclohexylacetic acid (9a); reaction

time 19 h; ﬂash CC on silica (8 g) with cyclohexane/CH Cl = 2:1 to cyclohexane/CH Cl = 0:1) and

(9):

0

µ

2

an additional PTLC on silica (developed three times with cyclohexane/CH Cl = 2:1 and one time

2

with cyclohexane/CH Cl = 1:2);

9, yield: 36%.

9: R = 0.21 (silica, cyclohexane/CH Cl = 1:4); IR

f

2 2

2

(

1

ATR): 2920 (s),

≈

2950 (br), 2850 (m), 1738 (s), 1610 (s), 1570 (m), 1450 (s), 1230 (s), 1204 (s), 1160 (m),

−

1

00

111 (m), 782 (w) cm ; H-NMR (CDCl ): 0.92–1.05 (m, 2H, H-2 ax, H-6 ax), 1.09–1.22 (m, 1H,

3

0

00

0

00

H-4 ax), 1.21-1.33 (m, 2H, H-3 ax, H-5 ax), 1.59 (s, 3H, H-6 ), 1.62-1.76 (m, 5H, H-2 eq, H-3 eq,

H-4 eq, H-5 eq, H-6 eq), 1.76–1.86 (m, 1H, H-1 ), 2.24 (dd, 12.4, 4.7 Hz, 1H, H-

5

J = 7.2 Hz, 1H, H-3 ), 6.04 (ddd, J = 7.3, 4.3, 0.8 Hz, 1H, H-1 ), 6.99 (d, J = 0.8 Hz, 1H, H-3), 7.18 (s, 2H,

H-6, H-7), 12.42 (s, 1H, C5-OH), 12.58 (s, 1H, C8-OH); C-NMR (CDCl ):

00

α

), 2.29 (dd, 12.4,

0

.0 Hz, 1H, H-

α

), 2.47 (dtm, J = 14.8, 7.5 Hz, 1H, H-2 ), 2.61 (dtm, J = 15.1, 5.7 Hz, 1H, H-2 ), 5.12 (tm,

0

13

0

δ

18.0 (C-6 ), 25.7 (C-5 ), 2

3

0

00

0

00

0

×

26.0, 26.1 (C-3 , C-4 , C-5 ), 3

C-4a), 111.8 (C-8a), 117.9 (C-3 ), 131.5 (C-3), 132.7 (C-7), 132.8 (C-6), 136.0 (C-4 ), 148.5 (C-2), 166.9

×

33.0 (C-2 , C-2 , C-6 ), 35.0 (C-1 ), 42.1 (C-

α

0

), 69.2 (C-1 ), 111.6

0

(

−

H)+Na] ,

(

C-5), 167.4 (C-8), 171.8 (COO), 176.8 (C-1), 178.3 (C-4); MS (ESI ) m/z (%): 845 (12) [2(M

−

4

12.29 (75) [M] , 411.12 (100) [M − H] ; [M] calculated for C H O : 412.1886.

24

28

6

(

5

R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl 1-cyclohexen-1-ylacetate (10):

mol Shikonin, 0.12 mmol DCC, 15 mol DMAP, and 53 mol 1-cyclohexen-1-ylacetic acid (11a);

reaction time 13 h; PTLC on silica with cyclohexane/CH Cl = 1:1 (three times developed); 10, yield

0

µ

2

5

%. 10: R = 0.48 (silica, cyclohexane/CH Cl = 1:4); IR (ATR): 2926 (m), 2857 (w), 2837 (w), 1738 (m),

f

2 2

−

1

608 (s), 1569 (m), 1436 (m), 1409 (m), 1335 (m), 1229 (s), 1202 (s), 1147 (m), 1112 (m), 777 (m) cm

;

1

00

0

H-NMR (CDCl ): 1.51–1.68 (m, 4H, H-4 , H-5 ), 1.55 (s, 3H, H-6 ), 1.69 (s, 3H, H-5 ), 1.97–2.03 (m,

3

00 00 0

2

H, H-6 ), 2.03–2.09 (m, 2H, H-3 ), 2.47 (dtm, J = 15.1, 7.6 Hz, 1H, H-2 ), 2.62 (dtm, J = 15.0, 5.7 Hz,

0

0 00

α), 5.13 (tm, J = 7.2 Hz, 1H, H-3 ), 5.62 (s br, 1H, H-2 ), 6.01 (ddd, J = 6.5, 3.5,

1

0

H, H-2 ), 3.01 (s, 2H, H-

.9 Hz, 1H, H-1’), 6.97 (d, J = 1.0 Hz, 1H, H-3), 7.18 (s, 2H, H-6, H-7), 12.42 (s, 1H, C5-OH), 12.57 (s, 1H,

13

00 00 00

δ 18.0 (C-6’), 21.9 (C-5 ), 22.7 (C-4 ), 25.3 (C-3 ), 25.8 (C-5’), 28.6 (C-6’),

C8-OH); C-NMR (CDCl₃):

2.9 (C-2’), 43.6 (C- ), 69.5 (C-1’), 111.6 (C-4a), 111.9 (C-8a), 117.9 (C-3’), 126.5 (C-2 ), 130.6 (C-1 ),

31.4 (C-3), 132.7 (C-7), 132.9 (C-6), 136.0 (C-4’), 148.4 (C-2), 167.0 (C-5), 167.5 (C-8), 170.7 (COO), 176.7

0

00

3

1

α

−

(

C-1), 178.2 (C-4); MS (ESI ) m/z (%): 841 (5) [2(M

−

H) + Na] , 410.23 (35) [M] , 409.33 (100) [M

−

H] ; [M] calculated for C H O : 410.1729.

2

4

26

6

(

0

R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl cyclopropanecarboxylate (11):

.1 mmol Shikonin, 0.15 mmol DCC, 25 mol DMAP, and 0.1 mmol cyclopropanecarboxylic acid (11a);

reaction time 22 h; CC on silica (7 g) with cyclohexane/CH Cl = 1:1 to 1:4; 11, yield 20%. 11: R = 0.28

µ

2

f

(

silica, cyclohexane/CH Cl = 1:4); NMR data ﬁt with literature [20].

2 2

(

0

R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl cyclobutanecarboxylate (12):

.1 mmol Shikonin, 0.15 mmol DCC, 25 mol DMAP, and 0.1 mmol cyclobutane carboxylic acid (12a);

reaction time 22 h; double PTLC on silica with cyclohexane/CH Cl = 1:4; 12, yield 40%. 12: R = 0.35

µ

2

f

(

silica, cyclohexane/CH Cl = 1:4); IR (ATR): 2980 (w),

≈

2950 (vbr), 2947 (m), 2866 (w), 1736 (s), 1611

2

−

1

0

vs), 1570 (m), 1455 (s), 1204 (s), 1162 (s), 787 (w) cm ; H-NMR (CDCl ): 1.58 (s, 3H, H-6 ), 1.69 (s, 3H,

3

0

00

0

H-5 ), 1.87–2.07 (m, 2H, H-3 ), 2.19–2.36 (m, 4H, H-2 , H-4 ), 2.47 (dtm, J = 14.7, 7.7 Hz, 1H, H-2 ), 2.61

0

00

0

(dtm, J = 14.9, 5.7 Hz, 1H, H-2 ), 3.22 (quintd, J = 8.5, 0.7 Hz, 1H, H-1 ), 5.12 (tm, J = 7.3 Hz, 1H, H-3 ),

0

6

.02 (ddd, J = 7.4, 4.5, 0.9 Hz, 1H, H-1 ), 6.96 (d, J = 1.0 Hz, 1H, H-3), 7.18 (s, 2H, H-6, H-7), 12.43 (s, 1H,

13

0

00

C5-OH), 12.59 (s, 1H, C8-OH); C-NMR (CDCl₃):

δ

17.9 (C-6 ), 18.4 (C-3 ), 25.1, 25.2 (C-2 , C-4 ), 25.8

C-5’), 32.9 (C-2’), 38.0 (C-1 ), 69.0 (C-1’), 111.6 (C-4a), 111.8 (C-8a), 117.8 (C-3’), 131.4 (C-3), 132.6 (C-7),

32.8 (C-6), 136.0 (C-4’), 148.5 (C-2), 166.7 (C-5), 167.2 (C-8), 174.1 (COO), 176.9 (C-1), 178.4 (C-4); MS

0

(

1

Molecules 2018, 23, 2820

13 of 21

−

(

3

ESI ) m/z (%): 1165 (3) [3M

−

3H + Na + K] , 1147 (6) [3M

−

2H + K] , 1231 (8) [3M

−

2H + Na] ,

−

70.11 (42) [M] , 369.17 (100) [M − H] ; [M] calculated for C H O : 370.1416.

21

22

6

(

0

R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl cyclopentanecarboxylate (13):

.1 mmol Shikonin, 0.15 mmol DCC, 25 mol DMAP, and 0.1 mmol cyclopentanecarboxylic acid (13a);

reaction time 17 h; double PTLC on silica with cyclohexane/CH Cl = 1:4; 13, yield 41%. 13: R = 0.32

µ

2

f

(

1

(

1

silica, cyclohexane/CH Cl = 1:4); IR (ATR): 3050 (w), 2954 (m), 2919 (w), 2870 (w), 1737 (s), 1608 (s),

2 2

− 0

570 (m), 1452 (m), 1205 (s), 1141 (s), 767 (m) cm ; H-NMR (CDCl ): 1.58 (s, 3H, H-6 ), 1.57–1.66

3

1

0

00

0

00

m, 2H, H-3 , H-4 ), 1.66–1.75 (m, 2H, H-3 , H-4 ), 1.69 (s, 3H, H-5 ), 1.75–1.87 (m, 2H, H-2 , H-5 ),

00 00 0

.87–1.99 (m, 2H, H-2 , H-5 ), 2.47 (dtm, J = 14.7, 7.5 Hz, 1H, H-2 ), 2.62 (dtm, J = 14.8, 5.7 Hz, 1H,

0

00

0

H-2 ), 2.82 (tt, J = 7.3, 8.4 Hz, 1H, H-1 ), 5.12 (tm, J = 7.3 Hz, 1H, H-3 ), 6.02 (ddd, J = 7.3, 4.4, 0.8 Hz, 1H,

0

H-1 ), 6.98 (d, J = 0.9 Hz, 1H, H-3), 7.19 (s, 2H, H-6, H-7), 12.43 (s, 1H, C5-OH), 12.59 (s, 1H, C8-OH);

1

3

0

00

0

C-NMR (CDCl ):

δ

18.0 (C-6 ), 2

×

25.8 (C-5 , C-3 , C-4 ), 29.9, 30.0 (C-2 , C-5 ), 33.0 (C-2 ), 43.8

3

0

(

1

3

C-1 ), 69.0 (C-1 ), 111.6 (C-4a), 111.8 (C-8a), 117.8 (C-3 ), 131.4 (C-3), 132.6 (C-7), 132.8 (C-6), 135.9

0

−

C-4 ), 148.7 (C-2), 166.7 (C-5), 167.2 (C-8), 175.4 (COO), 176.9 (C-1), 178.5 (C-4); MS (ESI ) m/z (%):

−

− −

H] ; [M] calculated for C H O :

22 24 6

189 (7) [3M

84.1573.

−

2H + K] , 384.13 (62) [M ], 383.28 (100) [M

−

(

5

R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl cyclohexanecarboxylate (14):

mol Shikonin, 0.12 mmol DCC, 15 mol DMAP, and 53 mol cyclohexanecarboxylic acid (14a);

reaction time 16 h; PTLC on silica with cyclohexane/CH Cl = 1:4 (twice developed) and an additional

0

µ

2

PTLC on silica (developed twice with cyclohexane/CH Cl = 1:1 and twice with cyclohexane/CH Cl

2

=

1:2); 14, yield 10%. 14: R = 0.24 (silica, cyclohexane/CH Cl = 1:4); IR (ATR): 2927 (m),

≈

2950 (br),

f

2

−

852 (w), 1736 (s), 1609 (s), 1570 (m), 1451 (m), 1230 (m), 1203 (m), 1162 (m), 1159 (s) cm ; H-NMR

1 1

2

(

00 00 00 00 00

CDCl ): 1.18–1.38 (m, 3H, H-3 ax, H-4 ax, H-5 ax), 1.40-1.54 (m, 2H, H-2 ax, H-6 ax), 1.58 (s, 3H,

3

0

00

0

00

H-6 ), 1.62–1.69 (m, 1H, H-4 eq), 1.69 (s, 3H, H-5 ), 1.74–1.81 (m, 2H, H-3 eq, H-5 eq), 1.94 (dm,

J = 11.9 Hz, 2H, H-2 eq, H-6 eq), 2.38 (tt, J = 11.2, 3.7 Hz, 1H, H-1 ), 2.47 (dtm, J = 15.0, 7.3 Hz, 1H,

H-2 ), 2.61 (dtm, J = 14.8, 5.8 Hz, 1H, H-2 ), 5.12 (tm, J = 7.3 Hz, 1H, H-3 ), 6.03 (ddd, J = 7.3, 4.5, 0.9 Hz,

0

00

0

1

H, H-1 ), 6.97 (d, J = 1.0 Hz, 1H, H-3), 7.18 (s, 2H, H-6, H-7), 12.43 (s, 1H, C5-OH), 12.58 (s, 1H, C8-OH);

1

3

0

00 00 00 0 00 00

C-NMR (CDCl₃):

δ

18.0 (C-6 ), 2

×

25.4 (C-3 , C-5 ), 25.7 (C-4 ), 26.9 (C-5 ), 28.9, 29.0 (C-2 , C-6 ),

0

3

3.0 (C-2’), 43.3 (C-1 ), 68.9 (C-1’), 111.6 (C-4a), 111.9 (C-8a), 117.8 (C-3’), 131.4 (C-3), 132.6 (C-7), 132.8

−

(C-6), 135.9 (C-4’), 148.7 (C-2), 166.7 (C-5), 167.3 (C-8), 174.7 (COO), 177.0 (C-1), 178.5 (C-4); MS (ESI )

−

− −

m/z (%): 817 (5) [2(M

C H O : 398.1729.

−

H) + Na] , 398.16 (51) [M] , 397.28 (100) [M

−

H] ; [M] calculated for

23

26

6

(

R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl cyclohex-1-enecarboxylate

15): 0.1 mmol Shikonin, 0.17 mmol DCC, 25 mol DMAP, and 0.1 mmol cyclohex-1-enecarboxylic

acid (15a); reaction time 16 h; PTLC on silica (developed twice with cyclohexane/CH Cl = 2:1 and

µ

2

one time with cyclohexane/CH Cl = 1:2) and an additional PTLC on silica (developed twice with

2

cyclohexane / CH Cl = 2:1); 15, yield 13%. 15: R = 0.37 (silica, cyclohexane / CH Cl = 1:4); IR (ATR):

2

f

2

−

1

2

917 (m),

≈

2950 (br), 2890 (w), 1712 (m), 1609 (s), 1569 (m), 1452 (m), 1229 (s), 1203 (s), 1077 (m) cm

;

1

0

00

0

H-NMR (CDCl ): 1.57 (s, 3H, H-6 ), 1.60–1.69 (m, 4H, H-4 , H-5 ), 1.69 (s, 3H, H-5 ), 2.20–2.32 (m,

3

0 00 0 0

4

H, H-3 , H-6 ), 2.52 (dtm, J = 15.0, 7.2 Hz, 1H, H-2 ), 2.64 (dtm, J = 15.1, 5.4 Hz, 1H, H-2 ), 5.14 (tm,

0

J = 7.2 Hz, 1H, H-3 ), 6.03 (ddd, J = 7.1, 4.6, 0.8 Hz, 1H, H-1 ), 6.96 (d, J = 1.0 Hz, 1H, H-3), 7.09 (m, 1H,

0

13

H-2 ), 7.19 (s, 2H, H-6, H-7), 12.43 (s, 1H, C5-OH), 12.59 (s, 1H, C8-OH); C-NMR (CDCl ):

C-6 ), 21.4, 22.0 (C-5 , C-4 ), 24.1 (C-3 *), 25.8 (C-5 ), 25.9 (C-6 *), 32.9 (C-2 ), 69.2 (C-1 ), 111.6 (C-4a),

11.9 (C-8a), 117.8 (C-3 ), 129.9 (C-1 ), 131.5 (C-3), 132.5 (C-7), 132.6 (C-6), 135.9 (C-4 ), 141.1 (C-2 ),

δ

18.0

3

0

00

0

00

0

(

1

0

00

0

00

−

48.9 (C-2), 166.1 (COO), 166.3 (C-5), 166.9 (C-8), 177.4 (C-1), 178.9 (C-4); MS (ESI ) m/z (%): 813 (11)

−

[

2(M − H) + Na] , 396.24 (44) [M] , 395.29 (100) [M − H] ; [M] calculated for C H O : 396.1573.

2

3

24

6

(

R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl cyclohex-3-enecarboxylate

16): 0.1 mmol Shikonin, 0.20 mmol DCC, 30 mol DMAP, and 0.1 mmol cyclohex-3-enecarboxylic

µ

Molecules 2018, 23, 2820

14 of 21

acid (16a); reaction time 16 h; two subsequent PTLC on silica each one developed twice with

cyclohexane/CH Cl = 2:1 and twice with cyclohexane/CH Cl = 1:2); 16, yield: 29%. 16: 1:1 mixture

2

of diastereomers; R = 0.28 (silica, CH Cl ); IR (ATR): 3025 (vw), 2917 (m),

≈

2950 (br), 2849 (w), 1735 (s),

f

2

−

1

0

1

608 (s), 1568 (m), 1452 (m), 1220 (s), 1201 (s), 1155 (s) cm ; H-NMR (CDCl ): 1.59 (s, 3H, H-6 ), 1.70

3

0 00 00 00

(

s, 3H, H-5 ), 1.68–1.81 (m, 1H, H-6 ), 2.00–2.08 (m, 1H, H-6 ), 2.10–2.17 (m, 2H, H-5 ), 2.26–2.32 (m,

00 0 0 00

H, H-2 ), 2.49 (dtm, J = 15.0, 7.3 Hz, 1H, H-2 ), 2.59–2.71 (m, 2H, H-2 and H-1 ), 5.13 (tm, J = 6.6 Hz,

2

1

0

00

0

1

H, H-3 ), 5.66–5.76 (m, 2H, H-3 , H-4 ), 6.05 (m, 1H, H-1 ), 6.98 (2 d, 1H, J

≈

1 Hz, H-3), 7.19 (s, 2H,

3

0

H-6, H-7), 12.42 (s, 1H, C5-OH), 12.58 (2 s, 1H, C8-OH); C-NMR (CDCl ):

2

δ

18.0 (C-6 ), 24.2 and

3

0

00

0

00

0

00

4.3 (C-5 ), 24.9 and 25.0 (C-6 ), 25.8 (C-5 ), 27.3, 27.4 (C-2 ), 33.0 (C-2 ), 2

×

39.3 (C-1 ), 69.1, 69.2

0

00

(

C-1 ), 111.6 (C-4a), 111.8 (C-8a), 117.7, 117.8 (C-3 ), 124.9, 125.0 (C-3 ), 2

×

126.8 (C-4 ), 131.3 (C-3), 2

0

×

132.7 (C-7), 2

×

132.9 (C-6), 136.0 (C-4 ), 2

×

148.5 (C-2), 2

×

167.0 (C-5), 167.5, 167.6 (C-8), 174.4,

−

1

74.5 (COO), 176.6, 176.7 (C-1), 178.1, 178.2 (C-4); MS (ESI ) m/z (%): 813.41 (7) [2(M

−

H) + Na] ,

−

3

95.16 [M − H] ; [M − H] calculated for C H O : 395.1495.

23

24

6

(

R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl trans 2-methylcyclopropanecarboxylate

17): 0.1 mmol Shikonin, 0.2 mmol DCC, 82 mol DMAP, and 0.11 mmol 2-methyl-cyclopropane

carboxylic acid (cis/trans mixture ca. 1:3,5) (17a); reaction time 15 h; CC on silica (7 g) with

cyclohexane/CH Cl = 2:1 to 0:1 and PTLC on silica with cyclohexane/CH Cl = 2:1 (ﬁve times

µ

2

developed); two diastereomers of 17, yield 3%. 17: R = 0.26 (silica, CH Cl ); IR (ATR): 2960 (w),

f

2

919 (w),

≈

2950 (br), 2856 (w), 1732 (s), 1609 (s), 1569 (m), 1559 (m), 1454 (m), 1406 (m), 1205 (m),

−

1

00

1

177 (m), 1159 (s), 781 (m) cm ; H-NMR (CDCl ): 0.70–0.79 (m, 2H, 2

×

H-3 ), 1.12–1.20 (m, 7H, 2

×

3

0

00

cyclopropyl-CH , 1

H-6 ), 1.70 (s, 6H, H-5 ), 2.42-2.53 (m, 2H, 2

H-3 ), 6.00 (tm, J = 5.9 Hz, 2H, H-1 ), 6.99 (d, J = 0.9 Hz, 1H, H-3), 7.00 (d, J = 0.9 Hz, 1H, H-3), 7.18

×

H-3 ), 1.20–1.26 (m, 1H, H-3 ), 1.36–1.48 (m, 4H, 2 H-1 , 2

×

H-2 ), 1.58 (s, 6H,

3

0

×

H-2 ), 2.57–2.66 (m, 2H, 2

×

H-2 ), 5.08–5.16 (m, 2H, 2

×

0

1

3

00

(s, 4H, H-6, H-7), 12.44 (2s, 2H, C5-OH), 12.58 (s, 2H, C8-OH); C-NMR (CDCl ):

δ

2

×

17.2 (C-3 ),

3

0

00

0

2

×

17.7 (C-2 ), 3

×

17.9 (cyclopropyl-CH , C-6 ), 2

×

21.2 (C-1 ), 25.8 (C-5 ), 32.9 (C-2 ), 69.2, 69.3

131.6 (C-3), 132.6 (C-7), 132.7 (C-6), 136.0 (C-4 ), 148.6

3

0

(C-1 ), 111.6 (C-4a), 111.8 (C-8a), 117.7 (C-3 ), 2

×

−

(

7

3

C-2), 166.5 (C-5), 167.0 (C-8), 2 × 173.3 (COO), 2 × 177.2 (C-1), 178.7, 178.8 (C-4); MS (ESI ) m/z (%):

−

61.32 (12) [2(M − H)+Na] , 370.14 (46) [M] , 369.26 (100) [M − H] ; [M] calculated for C H O :

21

22

6

70.1416.

Exo-(R)-1-(1,4-dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-3-yl)-4-methylpent-3-enyl bicyclo[2 .2.1]hept-5-

ene-2-carboxylate (18) and endo-(R)-1-(1,4-dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-3-yl)-4-methylpent-3-

enyl bicyclo[2.2.1]hept-5-ene-2-carboxylate (19): 0.1 mmol Shikonin, 0.20 mmol DCC, 25

and 0.11 mmol bicyclo[2.2.1]hept-5-ene-2-carboxylic acid (Exo (18a)/Endo (19a)/ = 1.0:4.1); reaction

time 16 h; PTLC on silica (developed three times with cyclohexane/CH Cl = 2:1 and one time with

µmol DMAP,

2

cyclohexane/CH Cl = 1:2) and PTLC on silica (developed twice with cyclohexane/CH Cl = 2:1

2

and three times with cyclohexane/CH Cl = 1:2), yield: 18, 6% and 19 10%. 18: 1:1 mixture of

2

diastereomers, R = 0.31 (silica, cyclohexane/CH Cl = 1:4); IR (ATR): 2972 (w), 2917 (m),

≈

2950 (br),

f

2

−

1

2

(

850 (w), 1734 (m), 1609 (s), 1568 (m), 1453 (m), 1230 (m), 1203 (m), 1163 (s), 1147 (m) cm ; H-NMR

00 00 00 0 0

CDCl ): δ 1.37–1.51 (m, 4H, H-3 , H-7 a, H-7 b), 1.58 (s, 3H, H-6 ), 1.60 (s, 3H, H-6 ), 1.69 (s, 3H,

3

0

00

H-5 ), 1.70 (s, 3H, H-5 ), 1.88–1.95 (m, 2H, H-3 ), 2.28-2.34 (m, 2H, H-2 ), 2.48 (dt, J = 14.8, 2H, 7.7 Hz,

H-2 ), 2.62 (dt, J = 15.0, 2H, 5.7 Hz, H-2 ); 2.94 (s, br, 2H, H-4 ), 3.06 (s, br, 1H, H-1 ), 3.08 (s, br, 1H,

H-1 ), 5.13 (tm, J = 7.3 Hz, 1H, H-3’), 5.15 (tm, J = 7.3 Hz, 1H, H-3’), 6.04 (td, J = 4.3, 0.9 Hz, 1H, H-1’),

6

0

00

0

00 00

.06 (td, J = 4.4, 0.9 Hz, 1H, H-1’), 6.12–6.20 (m, 4H, H-5 , H-6 ), 7.00 (d, J = 1.0 Hz, 1H, H-3), 7.01 (d,

0

00

J = 1.0 Hz, 1H, H-3), 6.12–6.19 (m, 4H, H-5 , H-6 ), 7.18, 7.19 (2s, 4H, H-6, H-7), 12.43 (2s, 2H, C5-OH),

1

3

00

18.0 (C-6’), 25.8 (C-5’), 30.3, 30.5 (C-3 ), 33.0, 33.1

12.59 (2s, 2H, C8-OH); C-NMR (CDCl ):

δ

2

×

3

0

00

00 00

(C-2’), 41.6, 41.7 (C-4 ), 43.1, 43.3 (C-2 ), 2

×

46.4 (C-7 ), 46.5, 46.7 (C-1 ), 69.2 (C-1’), 111.6 (C-4a),

0

111.9 (C-8a), 117.8, 117.9 (C-3’), 2 131.4 (C-3), 2

×

132.7 (C-7), 2

×

132.9 (C-6), 135.6 (C-6 ), 2

×

136.0

0

(C-4’), 138.2, 138.3 (C-5 ), 148.6 (C-2), 2

×

166.9, 167.0 (C-5), 2

×

167.5 (C-8), 174.9, 175.0 (COO), 176.7,

−

− −

H) + Na] , 408.18 (42) [M] , 407.22

176.8 (C-1), 2

×

178.3 (C-4); MS (ESI ) m/z (%): 837 (5) [2(M

−

Molecules 2018, 23, 2820

15 of 21

−

(

100) [M

−

H] ; [M] calculated for C H O : 408.1573. 19: 1:1 mixture of diastereomers, R = 0.26

24

6

f

silica, cyclohexane/CH Cl = 1:4); IR (ATR): 2973 (w), 2917 (m),

s), 1569 (m), 1452 (m), 1231 (m), 1202 (m), 1165 (s), 1108 (m), 709 (w) cm ; H-NMR (CDCl ): 1.32

d, J = 8.3 Hz, 2H, H-7 a), 1.39–1.51 (m, 4H, H-3 , H-7 b), 1.58 (s, 3H, H-6 ), 1.61 (s, 3H, H-6 ), 1.69

s, 3H, H-5 ), 1.72 (s, 3H, H-5 ), 1.88–2.00 m, 2H, H-3 ), 2.46 (dt, J = 14.9, 7.5 Hz, 2H, H-2 ), 2.59 (dt,

J = 15.0, 5.3 Hz, 2H, H-2 ), 2.93 (s, br, 2H, H-4 ), 3.04 (dtd, J = 8.9, 4.0, 1.1 Hz, 2H, H-2 ), 3.28 (s, br,

H, H-1 ), 5.11 (tm, J = 7.1 Hz, 1H, H-3 ), 5.16 (tm, J = 7.3 Hz, 1H, H-3 ), 5.88 (dd, J = 5.7, 2.8 Hz, 1H,

H-6 ), 5.90 (dd, J = 5.7, 2.8 Hz, 1H, H-6 ), 5.94 (ddd, J =

.6, 0.6 Hz, 2H, H-1 ), 6.19 (dd, J = 5.7, 3.0 Hz, 1H, H-5 ), 6.22 (dd, J = 5.7, 3.1 Hz, 1H, H-5 ), 6.96 (d,

≈

2950 (br), 2850 (w), 1733 (m), 1609

2

−

1 1

3

0

00

0

00

0

00

0

2

0

00

0

≈

7.5, 4.2, 0.8 Hz, 1H, H-1 ), 5.96 (ddd, J =

≈

7.5,

0

00

4

J = 0.9 Hz, 1H, H-3), 6.99 (d, J = 0.9 Hz, 1H, H-3), 7.18 (s, 4H, H-6, H-7), 12.42 (s, 2H, C5-OH), 12.57 (2s,

1

3

0

0 00 0

1

H, C8-OH); C-NMR (CDCl ):

δ

2

×

18.0 (C-6 ), 2

×

25.8 (C-5 ), 29.0), 29.3 (C-3 ), 32.9, 33.0 (C-2 ), 2

3

0

00

0

00

0

×

42.6 (C-4 ), 2

×

43.4 (C-2 ), 46.0 (C-1 ), 49.7, 49.9 (C-7 ), 69.2, 69. 4 (C-1 ), 2

×

111.6 (C-4a), 111.9

0

00

(

C-8a), 117.9, 118.1 (C-3 ), 131.3, 131.4 (C-3), 132.0, 132.2 (C-6 ), 132.6, 2

×

132.8, 132.9 (C-6, C-7), 135.8,

0 00

1

[

35.9 (C-4 ), 138.0, 138.1 (C-5 ), 148.6 (C-2), 167.0 (C-5), 167.7 (C-8), 173.6 (COO), 176.5 (COO, C-1),

−

76.7 (C-1), 178.2 (C-4); more polar diastereomer: MS (ESI ) m/z (%): 408.25 (33) [M] , 407.31 (100)

−

M − H] ; less polar diastereomer: MS (ESI ) m/z (%): 408.17 (43) [M] , 407.21 (100) [M

−

H] ; [M]

calculated for C H O : 408.1573.

24

6

(

R)-1-(1,4-Dihydro-5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl bicyclo[2.2.1]heptane-2-

ylacetate (20): 50 mol Shikonin, 0.12 mmol DCC, 15 mol DMAP, and 53 mol bicyclo[2.2.1]heptane-2-

ylacetic acid (20a); reaction time 16 h; PTLC on silica with cyclohexane/CH Cl = 1:4 (twice

µ

2

developed) and an additional PTLC on silica (developed twice with cyclohexane/CH Cl = 1 : 1

2

and twice with cyclohexane/CH Cl = 1:2); 20, yield 19% (sum of isomers). 20: R = 0.24 (silica,

2

f

cyclohexane/CH Cl = 1:4); IR (ATR): 2948 (m), 2917 (m),

≈

2950 (br), 2868 (w), 1738 (s), 1609 (s),

2

− 00

570 (m), 1453 (m), 1204 (s), 1173 (m), 1159 (s), 783 (m) cm ; H-NMR (CDCl ): 1.06 (m, 1H, H-3 ),

3

1

0

00

.13 (m, 1H, H-7 ), 1.15 (m, 1H, H-5 ), 1.24 (m, 1H, H-6 ), 1.32 (dquint, J = 9.9, 1.5 Hz, 1H, H-7 ),

0

.48 (m, 1H, H-5 ), 1.51 (m, 1H, H-6 ), 1.53 (m, 1H, H-3 ), 1.58 (s, 3H, H-6 ), 1.69 (s, 3H, H-5 ), 1.91

0

00

(quint, J = 6.5 Hz, 1H, H-2 ), 1.98 (s, br, 1H, H-1 ), 2.20 (m, 1H, H-

α

), 2.23 (m, 1H, H-4 ), 2.34 (dd,

0

J = 15.2, 7.9 Hz, 1H, H-

α

), 2.46 (dt, J = 15.0, 7.5 Hz, 1H, H-2 ), 2.60 (dt, J = 15.5, 5.2 Hz, 1H, H-2 ), 5.12 (t,

0

J = 7.2 Hz, 1H, H-3 ), 6.02 (dd, J = 7.4, 4.7 Hz, 1H, H-1 ), 6.98 (s, 1H, H-3), 7.18 (s, 2H, H-6, H-7), 12.43 (s,

13

0 0 00 00

H, C5-OH), 12.58 (s, 1H, C8-OH); C-NMR (CDCl ): δ 18.0 (C-6 ), 25.8 (C-5 ), 28.5 (C-5 ), 29.8 (C-6 ),

3

1

2

4

0

00

×

33.0 (C-2 ), 2

×

35.2 (C-7 ), 36.8 (C-4 ), 37.8, 37.9 (C-3 ), 38.5, 38.6 (C-2 ), 41.1, 41.3 (C-1 ), 2

×

1.3 (C- ), 69.2, 69.3 (C-1’), 111.6 (C-4a), 111.9 (C-8a), 117.9 (C-3’), 131.4, 131.5 (C-3), 132.7 (C-7), 132.8

α

(

C-6), 136.0 (C-4’), 148.5 (C-2), 2

C-4); MS (ESI ) m/z (%): 869.37 (9) [2(M

×

166.9 (C-5), 2

×

167.4 (C-8), 171.9 (COO), 2

×

176.8 (C-1), 2

×

178.3

−

H) + Na] , 424.23 (49) [M] , 423.25 (100) [M

−

H] , [M]

calculated for C H O : 424.1886.

25

28

6

Synthesis of carboxylic acids are described in the Supplementary Material.

3

.2. Isolation of β,β-dimethylacrylshikonin (1)

Dried roots of Onosma paniculata Bureau & Franchet (Boraginaceae) were acquired in Kunming,

China at a medicinal market in October 2003. The identity of the plant material was determined by

genomic analysis [46]. Compound was isolated as described previously [ ]. In brief, freshly ground

roots were extracted with petroleum ether by exhaustive Soxhlet extraction and was isolated by

1

8

1

preparative HPLC consisting of a Varian R PrepStar SD-1 (Agilent Technologies, Santa Clara, CA, USA)

with Dynamax R solvent delivery system and an absorbance detector model UV-1. A VDSpher 100

RP18 column (250

was used as stationary phase. The mobile phase consisted of (A) water and (B) acetonitrile and the

following gradient was used. 0 45 min: 70 100% B; 45 60 min: 100% B. was identiﬁed by NMR

Varian 400 MHz UnityINOVA spectrometer, Varian, Palo Alto, CA, USA) and CD measurements

×

25 mm, 10 µm) (VDS Optilab Chromatographie Technik GmbH, Berlin, Germany)

−

1

(

Molecules 2018, 23, 2820

16 of 21

(Jasco J-715 Spectropolarimeter, Eurisotop, Saint-Aubin CEDEX, France); the purity was determined

by HPLC and was 95%.

3

.3. Cell Culture

Human melanoma cell lines (SBcl2, WM9, WM164, and MUG-Mel2) were cultured in RPMI 1640

®

medium (Gibco , Thermo Fisher Scientiﬁc, Waltham, MA, USA), 2 mM L-glutamine (Gibco ), 10% fetal

®

bovine serum (FBS, Gibco ), and 1% penicillin-streptomycin (Pen/Strep, Gibco ). Human juvenile

ﬁbroblasts were isolated from donated foreskin tissue. Pieces were excised and treated with Dispase

II (Roche, Vienna, Austria). After enzymatic digestion, cells were grown in high-glucose Dulbecco’s

®

modiﬁed Eagle’s medium (DMEM, Gibco ) supplemented with 2 mM L-glutamine, 10% FBS, and 1%

Pen/Strep. Human epithelial cells (HEK-293) were kept in Dulbecco’s Modiﬁed Eagle’s Medium:

®

Nutrient Mixture F-12 (DMEM/F12, Gibco ), 2 mM L-glutamine, 10% FBS, and 1% Pen/Strep. Human

adult ﬁbroblasts were kindly provided by Ass. Prof. Dr. Beate Rinner (Medical University of Graz)

and culture in DMEM, 2mM L-glutamine, and 10% FBS. All cells were kept in a humidiﬁed 5% CO

2

◦

atmosphere at 37 C and passaged at 90% conﬂuence by trypsinization with 0.25% trypsin–EDTA

®

solution (Gibco ).

3

.4. Sample Preparation for In-Vitro Experiments

◦

20 C and diluted to the respective

concentrations in fresh medium before each experiment. Final ethanol concentration was 0.5% in each

well. Therefore, control cells represent vehicle-treated cells (0.5% ethanol). This concentration did not

affect the cells as shown in benchmark experiments (without ethanol).

All stock solutions were prepared in ethanol, stored at

−

3

.5. Cell Viability Assay (XTT)

Cell viability was analyzed in accordance with the manufacturer’s protocol (Roche Diagnostics,

Mannheim, Germany; cell proliferation kit II (XTT), cat. no. 11 465 015 001). This assay is

0

based on the cleavage of the yellow tetrazolium salt XTT (sodium 3 -[1-(phenylaminocarbonyl)-

3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate) into an orange formazan dye

by metabolic active cells. The color changes only in viable cells and can be directly quantiﬁed using

a scanning multiwell spectrophotometer. In brief, 5000 cells/well were seeded into 96-well plates

◦

(

100

µ

L, ﬂat bottom) and grown for 24 h in a 37 C, 5% CO atmosphere before various concentrations

2

of test compounds were added. Control cells were treated with 0.5% ethanol. After 72 h, 50

freshly prepared XTT solution (5 mL of XTT plus 100 L of electron coupling reagent) was added,

incubated for 2 h or 4 h, and analyzed using a Hidex Sense Microplate Reader (Hidex, Turku, Finland).

Compound served as reference (5.0 M). The assay was performed at least two times, with two or

three replicates each.

µL of a

µ

1

µ

3

.6. ApoTox-Glo™ Triplex Assay

This assay was conducted following the manufacturer’s instructions (Promega, Fitchburg, WI,

USA, cat. no. G6320). In short, 10,000 cells/well of WM9, WM164, and MUG-Mel2 cells were seeded

in 96-well plates (100 L, white, ﬂat bottom) and treated with various concentrations of for 6 h, 24 h,

and 48 h. Staurosporine (10.0 M) served as positive control for apoptosis induction. Afterwards,

µ

6

µ

2

3

0

µ

L of a freshly prepared viability/cytotoxicity reagent was added to each well, brieﬂy mixed for

◦

0 sec by orbital shaking (300–500 rpm), and incubated for 30 min at 37 C. Fluorescence was measured

at 400_E_X/505_E_m(viability) and 485_E_X/520_E_m(cytotoxicity) by using a Hidex Sense Microplate Reader.

®

Subsequently, 100

µ

L of a freshly prepared Caspase-Glo 3/7 reagent was added to each well, brieﬂy

mixed by orbital shaking (300–500 rpm, 30 s), and incubated at room temperature for another 30 min.

Luminescence was measured using a Hidex Sense Microplate Reader. Each assay was performed at

least two times, with three replicates each.

Molecules 2018, 23, 2820

17 of 21

3

.7. Western Blot Analysis

Cells were seeded at a density of 200,000 c/mL and grown over night before the test compound

was added for 24 h. Whole cell protein extracts were prepared with lysis buffer (50 mM Tris-HCl

pH 7.4, 150 mM NaCl, 50 mM NaF, 1 mM EDTA, 10% NP-40, 1% Triton-X, and protease inhibitors),

subjected to SDS-PAGE (10 or 12%), and blotted onto PVDF membranes (Roth, Karlsruhe, Germany).

Primary antibodies against PARP, phosphoH2AX (Ser139), and β-actin were purchased from Cell

Signaling Technology (Danvers, MA, USA). Blots were developed using corresponding horseradish

peroxidase-conjugated secondary antibodies (Dako, Jena, Germany) at room temperature for 1 h and

the Amersham

™ ECL™ prime Western blotting detection reagent (GE Healthcare, in accordance with

the manufacturer’s protocol). Chemiluminescence signals were detected by the ChemiDocTouch

Imaging System (BioRad Laboratories Inc., Herkules, CA, USA). Images were processed using

ImageLab 5.2 Software (BioRad Laboratories Inc.) and normalized to their loading controls.

3

.8. Caspase 3 Cleavage Measured by FACS

Cells were seeded at a density of 200,000 c/mL and grown over night before the test compound

was added for 24 h. After incubation with 6 in presence or absence of a caspase inhibitor (Z-VAD-FMK),

cells were harvested by trypsinization and ﬁxed and permeabilized with Cytoﬁx/CytopermTM

TM

solution (BD Biosciences, Frankling Lakes, NJ, USA). The pellet was resuspended in Perm/Wash

buffer and stained with FITC-conjugated monoclonal active caspase 3 antibody (BD Biosciences).

Finally, cells were resuspended in 1 mL PBS and analyzed with a LSRII

™ (BD Biosciences) ﬂow

cytometer equipped with a 488 nm argon ion laser and a 635 nm red diode laser (BD Biosciences).

Untreated cells were used as a negative control.

®

3

.9. CytoTox 96 Non-Radioactive Cytotoxicity Assay (LDH Assay)

®

Cell membrane integrity was analyzed using the CytoTox 96 Non-Radioactive Cytotoxicity

Assay (G1780, Promega, Fitchburg, WI, USA) according to the manufacturer’s instructions. Aliquots

100 L) of 100,000 c/mL were seeded into 96-well plates (ﬂat bottom), grown for 24 h, and treated

with various concentrations of (2.5 M, 5.0 M, 7.5 M, and 10.0 M) for 6 h, 24 h, and 48 h.

L of 10X Lysis Solution were added to control wells 45 min

(

µ

6

µ

To measure maximal LDH release, 10

before adding the CytoTox96 Reagent. To measure the amount of released LDH, 50

were transferred to a fresh 96-well plate and 50

µ

®

µ

L of each well

®

µ

L of CytoTox96 Reagent were added followed by a

30 min incubation period. Finally, 50

µL of Stop Solution was added and absorbance was recorded at

4

90 nm (Hidex Sense Microplate Reader). Absorbance values were corrected by background values

and the percentage of LDH release was calculated using the following formula. 100

LDH release (OD490)/maximum LDH release (OD490).

×

experimental

3

.10. Cell Cycle Analysis

Cells (200,000 c/mL) were treated with the respective IC₅₀concentration for 24 h and harvested

◦

by trypsinization. Afterwards, they were ﬁxed with 70% ice cold ethanol for 10 min and at 4 C.

After washing with PBS, the cell pellet was resuspended in PI-staining buffer (50

Beckman Coulter, Brea, CA, USA) and incubated for 15 min at 37 C. Cell cycle distribution was

analyzed using a LSRII™ (BD Biosciences) ﬂow cytometer.

µ

L/mL PI, RNAse,

◦

3

.11. Statistical Analysis

Statistical analysis was carried out using SigmaPlot 13.0 (Systat Software Inc., Chicago, IL, USA).

IC₅₀values were determined using the four-parameter logistic curve, at least ﬁve concentrations of the

test compound, and two different cell passages each tested in two or three independent wells. Results

are expressed as mean ± SEM. P values < 0.05 were considered as statistically signiﬁcant.

Molecules 2018, 23, 2820

18 of 21

4

. Conclusions

In this study, eighteen novel shikonin derivatives and one known derivative were synthesized.

Afterwards, their cytotoxic effects against several melanoma and skin ﬁbroblast cell lines were

investigated. Compound was chosen as reference because it was the most active isolated derivative

so far. To keep close to this lead compound, the two methyl groups of the side chain of were formally

connected with different numbers of methylene groups and the inﬂuence of the double bond and

1

the spacing CH resp. CH group was investigated as well. In addition, three cycloalkenyl and three

2

bicyclic derivatives were prepared to examine the effect of unsaturation and space ﬁlling with an

additional methylene moiety.

In general, all derivatives exhibited cytotoxic activity, but most of them were less active than

For example, the prepared cycloalkylideneacetates to and cycloalkylacetates to 10 were less

active against the metastatic cell lines than . Furthermore, neither the removal of the spacing CH

resp. CH group nor bicyclic derivatives led to an overall increase of cytotoxicity compared to

1.

2

5

7

1

1.

2

However, the cyclopropane derivative

WM164 and MUG-Mel2 than . The effect was also stronger than the effect of the already known

derivative 11. Therefore, was pharmacologically investigated in more detail. We could show that

led to apoptosis induction after 24 h while the cell membrane was not damaged within this time and

at the concentration used. Apoptosis was induced caspase dependently as shown by the ApoToxGlo

assay, FACS experiments in combination with a caspase inhibitor, and Western blot experiments.

Moreover, led to double-stranded DNA breaks as shown by phosphorylation of H2AX which can

lead to subsequent apoptosis induction. However, did not lead to cell cycle arrest as often caused

6 was considerably more active against the metastatic cell lines

1

6

™

6

by H2AX phosphorylation. In summary, the results demonstrate that

compound to develop antimelanoma compounds.

6 could represent a novel lead

Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/23/11/2820/

1

13

s1 . Studies on the enantiomeric purity and associated pharmacological effects of 1; NMR-spectra ( H and C or

HMBC) of the new shikonin derivatives; synthesis of cycloalkylidenacetic acids 2a to 5a and cyclobutylacetic acid

1

(

7a); H-NMR spectra of synthesized acids; HPLC runs of synthesized compounds; results of the XTT assay.

Author Contributions: C.D. performed the XTT and ApoTox-Glo^T^MTriplex assay, prepared cells for FACS and

Western blot measurements, analyzed liquid chromatography–mass spectroscopy (LC–MS) data and drafted

the manuscript; A.H. synthesized all compounds (except for shikonin and 1), performed and analyzed NMR

measurements and drafted the manuscript; B.R. and A.S. did the FACS measurements; B.L. conducted Western

blotting; A.D. and R.B. analyzed and interpreted the data and contributed to drafting the manuscript; N.K. isolated

compound 1, performed the LDH assay, analyzed and interpreted the data, and drafted the manuscript.

Funding: The authors acknowledge ﬁnancial support by the Austrian Science Fund (FWF, P27505).

Acknowledgments: We thank Guenther Heubl (Ludwig-Maximilian-University, Munich) for the identiﬁcation

of the plant material using genomic analysis; Eva-Maria Pferschy-Wenzig (University of Graz, Graz) for her

help with LC–MS measurements; Martin Schmid and Mag. Magdalena Taschwer (University of Graz, Graz)

for performing chiral HPLC measurements; and Ing. Andrea Fleck (University of Graz, Graz) for recording the

IR spectra.

Conﬂicts of Interest: The authors declare no conﬂicts of interest.

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Article Doi

Synthesis of novel shikonin derivatives and pharmacological effects of cyclopropylacetylshikonin on melanoma cells

DOI: 10.3390/molecules23112820

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