Design, Synthesis, and Biological Evaluation of Artemisinin-Indoloquinoline Hybrids as Potent Antiproliferative Agents

A series of artemisinin-indoloquinoline hybrids were designed and synthesized in an attempt to develop potent and selective anti-tumor agents. Compounds 7a–7f, 8 and 9 were prepared and characterized. Their antiproliferative activities against MV4-11, HCT-116, A549, and BALB/3T3 cell lines in vitro were tested. Nearly all of the tested compounds (7–9, except for compounds 7d and 7e against HCT-116) showed an increased antitumor activity against HCT-116 and A549 cell lines when compared to the dihydroartemisinin control. Especially for the artemisinin-indoloquinoline hybrid 8, with an 11-aminopropylamino-10H-indolo[3,2-b]quinoline substituent, the antiproliferative activity against the A549 cell line had improved more than ten times. The IC50 value of hybrid 8 against A549 cell lines was decreased to 1.328 ± 0.586 μM, while dihydroartemisin showed IC50 value of >20 µM in the same cell line. Thus, these results have proven that the strategy of introducing a planar basic fused aromatic moiety, such as the indoloquinoline skeleton, could improve the antiproliferative activity and selectivity towards cancer cell lines.


Introduction
Artemisinin (1), a sesquiterpene lactone from Artemisia annua, was isolated as a result of an extensive survey for antimalarial agents in Chinese traditional herb medicines by Chinese scientists since the early 1970s [1].Today, 1 and its derivatives, dihydroarteminisin (DHA, 2) and artesunate (3), are used in the first-line treatment for multidrug-resistant malaria [2,3].Besides the antimalarial activity, artemisinin and its semisynthetic analogs are endowed with the potential of anti-tumor [4][5][6][7], antiangiogenic [8,9], anti-inflammatory [10], anti-metastasis [11], and growth inhibition effects [12].The most unique and important structural feature installed in artemisinin is a peroxide group in the 1,2,4-trioxane moiety, which can react with an iron complex to produce cytotoxic free radicals and selectively induces apoptosis in many high free iron level cell lines, such as cancer cells [13].This biological sequence makes artemisinin and its analogs potent anticancer lead compounds.However, compared with many traditional cancer chemotherapeutic medicines of natural origin, such as camptothecin [14], doxorubicin [15], etc., simple artemisinin analogs are still less potent [4,16].High dosage and frequent administration would be required in order to achieve the same effectiveness in the anticancer treatment due to their short half-lives.
Many more potent artemisinin-derived antitumor agents are being developed [17][18][19].One of the important strategies for an improved action is the generation of hybrid molecules, which involves the covalent linking of the artemisinin or its analogs with some more potent and target-selective moieties [20].For instance, the 9-aminoacridine moiety, which is a key structure in Amsacrine, an antineoplastic agent for acute lymphoblastic leukemia, was linked with dihydroartemisinin (2) to form the hybrid.The antiproliferative activity of the hybrid compounds has been increased [21].This multiple target strategy led to the design of various hybrids like artemisinin-chalcone, in which the synergistic effect of chalcone on artemisinin was demonstrated [7,22], as shown in Figure 1.
The indoloquinoline skeleton has been widely found in many alkaloids [23][24][25].In particular, alkaloids from plants are promising candidates of new lead compounds in the search for new drugs against infectious diseases and cancers [26,27].Anticancer potential of indoloquinoline alkaloids are comprehensively documented in the recent review articles [28][29][30].Indeed, the indoloquinoline alkaloids like neocryptolepine (i), cryptolepine (ii), and isocryptolepine (iii), isolated from Cryptolepis sanguinolenta (Lindl.)Schltr.have been used as scaffolds for drug discovery, since this plant is used as a traditional herbal medicine in West and Central Africa (Figure 2) [31,32].Its planar fused ring system can intercalate into the DNA of tumor cells [33,34].Besides double-helical DNA, indoloquinoline derivatives have also been found to bind DNA triplexes as well as G-quadruplexes with high affinity [35].Thus, many indoloquinoline-featured compounds have been studied as antitumor agents [36][37][38][39][40][41][42][43].Neocryptolepine i can intercalate into DNA and inhibit topoisomerase II [36].On the other hand, cryptolepine ii, a regioisomer of i, is also a DNA intercalating agent and inhibits topoisomerase II, showing a high level of cytotoxicity [42].A series of 5,11-dimethylindolo [2,3b]quinolines or 6,11-dimethylindolo [2,3-b]quinolines derivatives have been tested for their chemotherapeutic activities, showing a cytotoxicity against several human cancer cell lines, with IC50 value ranging from 0.6 to 9 μM [44][45][46].In these studies, the substituents, such as the Br, MeO, or Me groups, attached on the indoloquinoline core were shown to have highly infectious activities.Metal ions, such as ruthenium(II), osmium(II) or copper(II), have been introduced to the indoloquinolines scaffold for assaying their antiproliferavitve activity.Anticancer activity in vitro and in vivo have shown that the osmium(II) or copper(II) complex displayed significant growth-inhibitory activity [47][48][49].5H-indolo[2,3-b]quinoline (DiMIQ) derivatives containing an amino acid or a dipeptide at the C-9 position also shown impressive antitumor activity.The attachment of the hydrophilic amino acid or the peptide has increased its hydrophilic properties and made the modified DiMIQ less in vivo toxic and promising anticancer agent [36,43].In our previous study, we also developed a series of indoloquinoline-derived antitumor agents like 4 and 6, by modification at the C-11 position of i and at the C-6 position of iii using diverse ω-aminoalkylamino substituents (Figure 2) [43][44][45].The target selectivity of the indoloquinoline moiety towards cancer cell lines has been well documented in these studies.Based on these facts, we are considering whether the potency of artemisinin analogues against cancer cell lines could be improved by linking to the indoloquinoline moiety.Thus, in this work, a series of dihydroartemisinin-indoloquinoline hybrids 7-9 were designed and synthesized.Their potencies as an antitumor agent were evaluated by antiproliferative screening.

Biological Evaluation
The results of the antiproliferative activity screening for compounds 7-8 are summarized in Tables 1 and 2. Biological activities of the compound 9 obtained from the isocryptolepine derivative 6 and artesunate 3 are cited in Reference [54] due to its low purity.
First, their antiproliferative activities against human leukemia cell line MV4-11 were evaluated (Table 1).All tested compounds have shown an antitumor activity with an IC50 in the microM range.Compound 7b turned out to be the most active hybrids (IC50 about 0.072 μM).Compared to the corresponding amino-modified indoloquinoline intermediate 4a, the antiproliferative activity of the artesunate-indoloquinoline hybrid 7a was weakened, with an increased IC50 value from 0.066 ± 0.023 μM to 0.286 ± 0.079 μM.We also observed the same trend for the intermediate 4d and the hybrid 7d.Agent 9, which is a hybrid from artesunate and 6-(3-aminopropylamino)-11Hindolo[3,2-c]quinoline (6), showed a better potency than agent 7a, which reflects the difference of the counterpart in the artesunate hybrids.Compounds 7b, 7d, 7e, 7f, 8 and 9 were then selected for the next evaluation.In order to test the selectivity of these hybrids between the normal cells and cancer cells, the normal mice fibroblast BALB/3T3 cells, human lung cancer cells A549 and human colon cancer cells HCT116 were employed in the evaluation of the second step.The clinical chemotherapy drug cisplatin and antimalarial drug dihydroartemisinin were used as positive control.Generally, these tested compounds inhibited cell proliferation of these three cell lines, though the selectivity towards the HCT116 cells was better than the others.As shown in Table 2, all the tested agents (7-9) have an enhanced antiproliferative activity against the lung cancer cells A549 with IC50 values ranging from 1.33 to 5.06 µM, which are more active in comparison to DHA (2) due to the introduced planar fused ring system.Especially compound 8, with an IC50 value of 1.328 ± 0.586 μM better than the both positive control, produced the best result in the screening.On the contrary, DHA (2) showed a poor activity against A549, with an IC50 of more than 20 µM.All the tested agents exhibited an antiproliferative activity superior to the positive control cisplatin.When against the HCT116 cell lines, the best result was provided by compound 8.It inhibited the HCT116 cells proliferation with an IC50 of 0.557 ± 0.085 μM.

General Methods
The commercially obtained reagents were directly used without further purification.The 1 H-NMR and 13 C-NMR spectra were measured by a Varian INOVA-600 spectrometer with CDCl3 as the solvent unless otherwise indicated.High-resolution mass spectra were obtained on a Bruker micrOTOF II-SKA spectrometer.The intermediate 4 and 6 were synthesized according to the method we previously reported [50][51][52].

Figure 1 .
Figure 1.The structure of artemisinin and its analogues, and subsequent hybrid compounds.