Structural Modifications of Nature-Inspired Indoloquinolines: A Mini Review of Their Potential Antiproliferative Activity

Cryptolepine, neocryptolepine and isocryptolepine are naturally occurring indoloquinoline alkaloids with various spectrum of biological properties. Structural modification is an extremely effective means to improve their bioactivities. This review enumerates several neocryptolepine and isocryptolepine analogues with potent antiproliferative activity against MV4-11 (leukemia), A549 (lung cancer), HCT116 (colon cancer) cell lines in vitro. Its activity towards normal mouse fibroblasts BALB/3T3 was also evaluated. Furthermore, structure activity relationships (SAR) are briefly discussed. The anticancer screening of neocryptolepine derivatives was performed in order to determine their cytotoxic and growth inhibitory activities across the JFCR39 cancer cell line panel.

These alkaloids are composed of the tetracyclic indoloquinoline ring system, which only differ with respect to the orientation and site of their indole and quinoline ring junctures ( Figure 1). By introducing the appropriate motif at the certain positions, their derivatives can increase better biological activity than the mother core [3]. Many researchers have achieved the structural modification of cryptolepine scaffold for the purpose of improving antitumor activity, while the pharmacological properties of the analogues are being studied deeply.
The bioactivities of many antitumoral agents are related to their interactions with the DNA molecule, which is regarded as a classical target for these drugs in clinical use. The basic mechanism of antitumoral activity of these drugs is to affect the replication, expression, transcription and other physiological functions of the DNA, which causes the tumor cell death [4].
In 1990, Yamato et al. synthesized the indoloquinoline derivatives 4 (Figure 2), and screened its biological properties in vitro and in vivo. The compounds 4 showed potential antitumor activity (P388 leukemia in mice), DNA intercalative property, and ability to induce topoisomerase II dependent DNA cleavage [5]. In 1997, Deady et al. studied a series of cryptolepine derivatives 5 and evaluated their antitumoral activity in a series of murine and human tumor cell lines such as the mice lung cancer cells (LLC), mice leukemia cells (P388), human leukocyte cells (JL). These compounds appear to be mixed topoisomerase I/II inhibitors in the human leukemia cell lines studied [6].
In 1998, Bonjean et al. verified the cryptolepine alkaloids bound tightly to DNA as a typical intercalating agent by various means of absorption, such as fluorescence, circular, and linear dichroism, as well as by a relaxation assay using DNA topoisomerases. At the same time, they provided direct evidence that DNA is the primary target of cryptolepine. The mechanism of the compounds inhibiting tumor cell proliferation is mainly based on the synthesis of DNA inhibition, not the inhibition of proteins and RNA [7].
In 2002, Lisgarten, John N., reported that cryptolepine interacts with the DNA fragment d(CCTAGG)2 in a base-stacking intercalation mode by using X-ray crystallography. It was found that cryptolepine intercalated between pyrimidine bases of the fragment in the form of π-π accumulation. This is the first single crystal structure of DNA intercalator complex, which is the small molecule to bind a non-alternating (pyrimidine-pyrimidine) DNA sequence [8]. Many researchers have achieved the structural modification of cryptolepine scaffold for the purpose of improving antitumor activity, while the pharmacological properties of the analogues are being studied deeply.
The bioactivities of many antitumoral agents are related to their interactions with the DNA molecule, which is regarded as a classical target for these drugs in clinical use. The basic mechanism of antitumoral activity of these drugs is to affect the replication, expression, transcription and other physiological functions of the DNA, which causes the tumor cell death [4].
In 1990, Yamato et al. synthesized the indoloquinoline derivatives 4 (Figure 2), and screened its biological properties in vitro and in vivo. The compounds 4 showed potential antitumor activity (P388 leukemia in mice), DNA intercalative property, and ability to induce topoisomerase II dependent DNA cleavage [5]. Many researchers have achieved the structural modification of cryptolepine scaffold for the purpose of improving antitumor activity, while the pharmacological properties of the analogues are being studied deeply.
The bioactivities of many antitumoral agents are related to their interactions with the DNA molecule, which is regarded as a classical target for these drugs in clinical use. The basic mechanism of antitumoral activity of these drugs is to affect the replication, expression, transcription and other physiological functions of the DNA, which causes the tumor cell death [4].
In 1990, Yamato et al. synthesized the indoloquinoline derivatives 4 ( Figure 2), and screened its biological properties in vitro and in vivo. The compounds 4 showed potential antitumor activity (P388 leukemia in mice), DNA intercalative property, and ability to induce topoisomerase II dependent DNA cleavage [5]. In 1997, Deady et al. studied a series of cryptolepine derivatives 5 and evaluated their antitumoral activity in a series of murine and human tumor cell lines such as the mice lung cancer cells (LLC), mice leukemia cells (P388), human leukocyte cells (JL). These compounds appear to be mixed topoisomerase I/II inhibitors in the human leukemia cell lines studied [6].
In 1998, Bonjean et al. verified the cryptolepine alkaloids bound tightly to DNA as a typical intercalating agent by various means of absorption, such as fluorescence, circular, and linear dichroism, as well as by a relaxation assay using DNA topoisomerases. At the same time, they provided direct evidence that DNA is the primary target of cryptolepine. The mechanism of the compounds inhibiting tumor cell proliferation is mainly based on the synthesis of DNA inhibition, not the inhibition of proteins and RNA [7].
In 2002, Lisgarten, John N., reported that cryptolepine interacts with the DNA fragment d(CCTAGG)2 in a base-stacking intercalation mode by using X-ray crystallography. It was found that cryptolepine intercalated between pyrimidine bases of the fragment in the form of π-π accumulation. This is the first single crystal structure of DNA intercalator complex, which is the small molecule to bind a non-alternating (pyrimidine-pyrimidine) DNA sequence [8]. In 1997, Deady et al. studied a series of cryptolepine derivatives 5 and evaluated their antitumoral activity in a series of murine and human tumor cell lines such as the mice lung cancer cells (LLC), mice leukemia cells (P388), human leukocyte cells (JL). These compounds appear to be mixed topoisomerase I/II inhibitors in the human leukemia cell lines studied [6].
In 1998, Bonjean et al. verified the cryptolepine alkaloids bound tightly to DNA as a typical intercalating agent by various means of absorption, such as fluorescence, circular, and linear dichroism, as well as by a relaxation assay using DNA topoisomerases. At the same time, they provided direct evidence that DNA is the primary target of cryptolepine. The mechanism of the compounds inhibiting tumor cell proliferation is mainly based on the synthesis of DNA inhibition, not the inhibition of proteins and RNA [7].
In 2002, Lisgarten, John N., reported that cryptolepine interacts with the DNA fragment d(CCTAGG) 2 in a base-stacking intercalation mode by using X-ray crystallography. It was found that cryptolepine intercalated between pyrimidine bases of the fragment in the form of π-π accumulation. This is the first single crystal structure of DNA intercalator complex, which is the small molecule to bind a non-alternating (pyrimidine-pyrimidine) DNA sequence [8].
In 2012, Boddupally et al. have synthesized a series of 11-substituted cryptolepine derivatives 6 ( Figure 2). The compound 6 showed the most potent anticancer activity with IC 50 = 0.97 µM against HCT-116 colon cancer cell line and IC 50 = 2.33 µM against Raji lymphoma cells in further cytotoxic test in vitro. At the same time, this compound showed a strong inhibition of c-MYC expression [9].
Gu and Lu demonstrated the binding of aniline-substituted cryptolepine derivatives with calf-thymus DNA presumably via an intercalation mechanism and studied the binding mode of these derivatives with duplex DNA by Surflex-dock software. They reported that these derivatives intercalated into the base-pairs, and reacted with DNA via mainly π-π interaction with medium, moreover the functional groups substituted on aniline ring affected the binding abilities [10,11].
The aim of this review is to present an overview of the potential of neocryptolepine and isocryptolepine as scaffolds for the design and development of new anticancer drugs. Both compounds have also the linearly arranged tetracyclic plane as same as cryptolepine, so they can be expected as candidates of antitumoral agent, although they have a slightly weaker capability to intercalate into DNA and inhibit human topoisomerase II [12].
Molecules 2019, 24 FOR PEER REVIEW 3 In 2012, Boddupally et al. have synthesized a series of 11-substituted cryptolepine derivatives 6 ( Figure 2). The compound 6 showed the most potent anticancer activity with IC50 = 0.97 μM against HCT-116 colon cancer cell line and IC50 = 2.33 μM against Raji lymphoma cells in further cytotoxic test in vitro. At the same time, this compound showed a strong inhibition of c-MYC expression [9].
Gu and Lu demonstrated the binding of aniline-substituted cryptolepine derivatives with calfthymus DNA presumably via an intercalation mechanism and studied the binding mode of these derivatives with duplex DNA by Surflex-dock software. They reported that these derivatives intercalated into the base-pairs, and reacted with DNA via mainly π-π interaction with medium, moreover the functional groups substituted on aniline ring affected the binding abilities [10,11].
The aim of this review is to present an overview of the potential of neocryptolepine and isocryptolepine as scaffolds for the design and development of new anticancer drugs. Both compounds have also the linearly arranged tetracyclic plane as same as cryptolepine, so they can be expected as candidates of antitumoral agent, although they have a slightly weaker capability to intercalate into DNA and inhibit human topoisomerase II [12].
In addition, we have also engaged in modifying isocryptolepine derivatives in three ways: the amino substituent effect at C6, and N11 methyl localization effect, and the substituent group effect at C2 of quinoline moiety [19].
In addition, we have also engaged in modifying isocryptolepine derivatives in three ways: the amino substituent effect at C6, and N11 methyl localization effect, and the substituent group effect at C2 of quinoline moiety [19].
Some compounds with higher antiproliferative activity are listed in Tables 1 and 2. The SAR (structure activity relationships) studies reveal that the most necessary strategy is introducing ω-aminoalkylamino group in the side chain. Among the tested 84-neocryptolepine derivatives and 44-isocryptolepine derivatives, the most potent compounds with higher activity contain the -NH(CH 2 ) 3 NH 2 side chain.  Among them, 11-(3-amino-2-hydroxy)propylamino derivatives 7e and 7f were the most cytotoxic with a mean IC 50 value of 0.042 µM and 0.057 µM against the MV4-11 cell line, 0.197 µM and 0.190 µM against the A549 cell line, and 0.138 µM and 0.117 µM against the HCT116 cell line.
We propose the reason that the amino terminus domain in the side chain would react with negative charged phosphate groups in DNA, which increases the insertion ability of the complex to DNA.
The methyl localization effect is an important additional contributor to increase the activity. The activity is greatly reduced (10~30 fold lower) by changing the Me group from N5 to N6 (i.e., 8) of neocryptolepine derivatives. The Me group can increase the activity of isocryptolepine derivatives, as found by a comparison of the compounds 10e and 10c.
Replacing H atom at the C2 with MeO, Br, Cl, Me, or replacing H at C9 by COOMe can improve the antiproliferactive activity of neocryptolepine derivatives. Each group contributes differently to the antiproliferactive activity of different tumor cells. For example, 7c with Br at C2 shows the best antiproliferactive activity against MV4-11 leukemia cells, and this modification did not increase antiproliferactive activity against normal BALB/3T3 cells, but unexpectedly it decreases 2-fold the antiproliferactive activity against A549 lung cancer cells. It can be suggested that this modification increases the selectivity of the tested compound, improving their effect on leukemic cells, without increasing possible adverse effects on normal cells. The substituent effect at C2 of isocryptolepine core also can change the activity; NO 2 is the effective group to increase the activity against cancer but unfortunately also normal cells (10c and 10e). Therefore, we must examine our best to find an effective substituent for further improving the activity.
We boldly tried to replace the 5-nirtogen atom with oxygen atom, forming 9, which significantly drop the antiproliferactive activity [16].
From all the assay data, isocryptolepine analogues are more potential as the anticancer drug candidates in comparison with the neocrytolepine analogues, compound 10e (or 10a) shows the higher antiproliferactive activity against A549 and HCT116 cancer cell, and lower antiproliferactive activity against normal cells comparing 7a, when amino-substituent is NH(CH 2 ) 3 NH 2 and substituent group is H at C2.
The mode of neocryptolepine and isocryptolepine binding to DNA was studied using UV-VIS absorption spectroscopy with salmon fish sperm DNA. From the DNA binding studies, it can be proven that the methyl localization effect and the substituent group effect at C2 of quinoline moiety influence the capability to intercalate into DNA. Two effects improve the activity of isocryptolepine to interact with DNA, the binding constant of 10f-DNA was 1.05 × 10 6 L/mol and 10g-DNA was 4.84 × 10 6 L/mol [19]. The activity of neocryptolepine to interact with DNA varied; the binding constant of 7c-DNA was 2.93 × 10 5 and 8-DNA was 3.28 × 10 5 L/mol [13].

Compare Study
The COMPARE analysis assesses the correlation coefficient between the fingerprints of the test compounds and those of the various reference compounds [23]. This system provides an information

Compare Study
The COMPARE analysis assesses the correlation coefficient between the fingerprints of the test compounds and those of the various reference compounds [23]. This system provides an information intensive approach to identify the molecular targets of new compounds. The JFCR39 COMPARE analysis-guided assay is a successful means to find new anticancer drug candidates. The COMPARE analysis is carried out by calculation of the Pearson correlation coefficient (r value) between the fingerprints of compounds X and Y. The r value is then used to determine the degree of similarity, that is, the higher the r value, the greater the similarity of X to Y. Generally, an r value of 0.5 < r < 0.75 between two agents suggests they might have a similar mechanism of action ( Figure 6).

Compare Study
The COMPARE analysis assesses the correlation coefficient between the fingerprints of the test compounds and those of the various reference compounds [23]. This system provides an information intensive approach to identify the molecular targets of new compounds. The JFCR39 COMPARE analysis-guided assay is a successful means to find new anticancer drug candidates. The COMPARE analysis is carried out by calculation of the Pearson correlation coefficient (r value) between the fingerprints of compounds X and Y. The r value is then used to determine the degree of similarity, that is, the higher the r value, the greater the similarity of X to Y. Generally, an r value of 0.5 < r < 0.75 between two agents suggests they might have a similar mechanism of action ( Figure 6).
The COMPARE analysis revealed that compounds 7d and 7k have a very good match to actinomycin D (r = 0.7 for both). Similarly, compound 7k has a slight similarity to paclitaxel (r = 0.64). Compound 7q shows some resemblance to vindesine sulfate (r = 0.58) and aclarubicinHCl (r = 0.57).
(A)  The COMPARE analysis revealed that compounds 7d and 7k have a very good match to actinomycin D (r = 0.7 for both). Similarly, compound 7k has a slight similarity to paclitaxel (r = 0.64). Compound 7q shows some resemblance to vindesine sulfate (r = 0.58) and aclarubicinHCl (r = 0.57).

Conclusions
Indoloquinoline alkaloids are important scaffolds for antitumoral drug development. This review discussed the SAR of neocryptolepine and isocryptolepine, and presents the useful methods for improving the antitumoral activity of neocryptolepine and isocryptolepine analogues. The amino substituent effect and methyl localization effect are now available strategies, but the substituent group effect at the benzene ring of the quinoline moiety has no effect on regular SAR. Thus, the antitumoral activity is highly related to the activity of interacting with DNA. The computer-assisted database analysis, COMPARE, suggested that 7d and 7k have a mode of action similar to actinomycin D. It also suggested that 7l has a mode of action similar to vindesine sulfate or aclarubicin HCl. However, the new compounds may have other unique modes of action since the correlation coefficients (r) were at relatively low levels, which present an interesting possibility to examine in further studies.