Targeting DNA and Topoisomerase I with Indolocarbazole Antitumor Agents

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Christian Bailly Introduction

In the cell, DNA primarily exists in a supercoiled form and therefore unwinding of DNA is required prior to using it for transcription, replication, or recombination. These functions require the services of enzymes called topoisomerases, which cleave and religate one or two strands of DNA, and hence allow the DNA to unwind [1]. For more than 15 years, these enzymes have attracted considerable attention as targets for cancer therapeutics since many drugs inhibiting them, such as topotecan, adriamycin, and etoposide, are used extensively in the clinic to combat different forms of cancers, from colorectal cancer, to brain tumors and hematological malignancies.

There are three classes of topoisomerase enzymes in human cells [2]. Topoisomerase I introduces breaks in one strand of DNA and mediates DNA relaxation in a process that does not require any cofactor. In contrast, topoisomerase II cuts the two strands of the double helix and changes the topology of the DNA by catalyzing the passage of one double-stranded segment of DNA through a double-strand break produced in a second DNA segment. Topoisomerase III, which was identified more recently [3], cleaves single-strand DNA and binds covalently to the 5' end of the cleaved DNA [4]. There are, as yet, no specific inhibitors for topoisomerase III, whereas there are many for the two other enzymes. Both topoisomerases I and II have been shown to constitute critical cellular loci for a number of clinically important antitumor agents [5-7].

The plant alkaloid camptothecin (CPT) represents the archetype of the topoisomerase I poisons. The mechanism by which this natural product kills cancer cells involves the stabilization of an intermediate binary complex in which one DNA strand has undergone strand scission and is covalently attached to the enzyme. By binding to this complex and preventing religation, the topoisomerase poison precludes DNA replication and transcription, and thereby leads to the death of cells attempting to undergo theses processes [8]. CPT shows little or no affinity for DNA or topoisomerase I alone but interacts strongly and specifically with the enzyme-DNA complex. It is worth remembering that for almost a quarter of a

Small Molecule DNA and RNA Binders: From Synthesis to Nucleic Acid Complexes. Volume 2.

Edited by M. Demeunynclc, C. Bailly, W. D. Wilson

Copyright © 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

ISBN: 3-527-30595-5

century, CPT was an orphan drug in the sense that its primary molecular site of action was unknown. The discovery in 1985 that topoisomerase I was a specific molecular target for CPT has rekindled interest in the design of analogs. Once the target was identified and characterized, it took not less than 10 years to develop safe analogs such as the drugs topotecan (TPT, Hycamtin) and irinotecan (IRT, Camptosar) (Fig. 20.1), which are now in clinical use [9]. A variety of second- and third-generations CPT derivatives, such as lurtotecan, exatecan, rubitecan, and di-flomotecan (Fig. 20.1), are currently in various stages of clinical development [10, 11].

Over the past 10 years, the number of topoisomerase I poisons has greatly expanded and now includes more than 60 members with very diverse structures and origins [12, 13]. However, only a very few have revealed significant antitumor ac-

Topoisomerase Poisons

9-aminoCPT

topotecan rubitecan diflomotecan

BN80915

exatecan

DX-8951f iurtotecan

G7147211

karetinecin

BNP-1350

OH O

Fig. 20.1. Camptothecin (20-S-CPT) and various tumor-active analogues.

karetinecin

BNP-1350

OH O

HaC-gj-CHs tBu

HaC-gj-CHs

snatecan

DB-67

OH O

snatecan

DB-67

OH O

Fig. 20.1. Camptothecin (20-S-CPT) and various tumor-active analogues.

tivities in vivo. Apart from the CPTs, the only class of tumor-active topoisomerase I inhibitors is arguably that of the indolocarbazoles (IND) reviewed here.

Naturally Occurring Indolocarbazoles

IND is the generic name for a group of compounds possessing a planar 6-ring nucleus. The group includes several microbial metabolites, in particular K252a, AT2433-B1, and rebeccamycin. But the chief among the indolocarbozoles is the antibiotic staurosporine, a fascinating natural product, the discovery, chemical structure, and structure-activities of which have been presented elsewhere [14,15].

The indolocarbazoles can be classified in two groups. The closed series refers to compounds for which the glycosyl residue in linked to the IND chromophore via the two indole nitrogens, and the open series refers to the compounds with one free indole NH group and the other linked to the carbohydrate. The following section describes how these naturally occurring IND were exploited for the development of novel anticancer agents targeting topoisomerase I and/or DNA.

Staurosporine and Analogs with a Pyranose Sugar Moiety

Staurosporine was first isolated in 1977 from Streptomyces staurosporeus [16] and has since been isolated from various actinomycete strains (e.g. S. actuosus) as well as from some marine organisms [17]. This IND alkaloid, possessing antifungal and hypotensive activities, was initially described as an inhibitor of protein kinase C (PKC) [18] by competing with the ATP binding in the catalytic domain of PKC. In fact, PKC defines a family of serine/threonine-specific protein kinases, which consists of at least 12 isoforms [19]. The conventional PKC isoenzymes, which are calcium-dependent and activated by diacylglycerol/phorbol ester (e.g. PKCa, and Pn) are particularly sensitive to staurosporine but the drug also affects other protein kinases such as PKA, PKG, and protein tyrosine kinases [20, 21] as well as non-kinase proteins. For example, staurosporine potently stimulates the expression of the vasoprotective and anti-atherosclerotic enzyme NOS III and this activity seems to be unrelated to protein kinases inhibition [22].

Many synthetic derivatives of staurosporine have been designed [14] and a few of them display antitumor activities such as 7-hydroxystaurosporine (UCN-01) and 4'-N-benzoylstaurosporine (PKC412 or CGP41251) (Fig. 20.2). UCN-01 exhibits different cellular effects (cell cycle checkpoint abrogation, Gi delay, induction of apoptosis) depending on the dose and cell type used but has no effect on topoisomerase I [23]. PKC412 is interesting because in addition to being a potent and specific PKC inhibitor, it presents the additional advantage of modulating the multidrug resistance phenotype in cancer cells [24, 25]. However, despite their low toxicity profile, both PKC412 and UCN-01 display high binding to human plasma proteins, a problem which may restrict their clinical development [26].

Y^OCHg NHCHg

Y^OCHg NHCHg staurosporine

Smallmolecule Drugs

Fig. 20.2. Staurosporine and two synthetic derivatives currently undergoing clinically trials: N-benzoyl-staurosporine (PKC412) and 7-hydroxy-staurosporine (UCN-01).

H^CH3

y^och3 NHCHg

H^CH3

y^och3 NHCHg

UCN-01

Fig. 20.2. Staurosporine and two synthetic derivatives currently undergoing clinically trials: N-benzoyl-staurosporine (PKC412) and 7-hydroxy-staurosporine (UCN-01).

Thus far, no poisoning activity against topoisomerase I has been reported with staurosporine. PKC412. or UCN-01. Topoisomerase I poisons generally induce cell cycle arrest at the G2 phase and the G2 checkpoint implicates the correct functioning of different kinases, in particular human checkpoint kinase 1 (Chkl). In inhibiting this enzyme, UCN-01 is capable of overriding the G2 arrest induced by topotecan, therefore potentiating the cytotoxicity of this topoisomerase I inhibitor [27]. Similar effects have been recently reported with SB-218078 (Fig. 20.3), another IND derivative analogous to K252a [28]. UCN-01 sensitizes the cytotoxic action of drugs such as mitomycin C, cisplatin, 5-fluorouracil, and methotrexate

HO0^

COOCH3

K252a

N N'

HO7'

CEP-751 (KT-6587)

SB-218078

SB-218078

COOCH3

cep-1347

[23, 29] and phase II clinical trials associating UCN-01 with DNA-damaging agents and antimetabolites are currently ongoing.

K252a and Analogs with a Furanose Sugar Moiety

The antibiotic K252a, produced by Nocardiopsis species [30, 31] is also a potent protein kinase inhibitor acting through competitive inhibition of ATP-binding domains. It modulates the activity of many different kinases including PKC but it is particularly interesting for its inhibitory action on the tyrosine kinase of the neurotrophin-specific Trk receptor [32-35]. This activity has been exploited to develop Trk-specific inhibitors, leading to the discovery of potent compounds such as CEP-701 (KT-5555) and CEP-751 (KT-6587), which preferentially inhibit autophos-phorylation and signaling of Trk family receptors at nanomolar concentrations in vitro and display potent antitumor activity in vivo [36-39]. Combined with androgen ablation, these IND derivatives may be particularly useful for the treatment of prostate cancers [40-42] and have also revealed significant activity against neuroblastoma and medulloblastoma [43, 44]. These tumors of the neuroectoderm, which are among the most common tumors in childhood, generally express one or more of the tyrosine kinase receptors TrkA, TrkB, and/or TrkC targeted by CEP-751. However, in addition to their anti-Trk activity, these analogs of K252a also interfere with other proteins such as the platelet-derived growth factor, epidermal growth factor receptor, and probably other as yet unidentified tyrosine, serine, or threonine kinases [37].

CEP-751 and CEP-701 are candidates for cancer treatment and a phase I clinical trial for solid tumors in adults has been reported with CEP-701 [45]. But further modifications of the K252 structure can lead to compounds with a totally different spectrum of activity. For example, the K252a analog CEP-1347 displays a broad neuroprotective profile and is currently in clinical trials for Parkinson's disease. This 3,9-bis-ethyl-thiomethyl derivative (Fig. 20.3) has no effect on PKC and TrkA but is a very potent inhibitor of the c-jun N-terminal kinases (JNK) signaling cascade acting via an inhibition of the mixed lineage kinases (MLK), a family of serine/threonine kinases [46-48]. Targeting MLK with K252a-derived IND may be an effective strategy for blocking neurodegeneration associated with Parkinson's disease and very recently, various analogs of CEP-1347 possessing different alkyl-thiomethyl groups at positions 3,9 have been described [49].

Unlike staurosporine and rebeccamycin, K252a exhibits taxol-like functional activity. K252a and K252b both support the growth of taxol-dependent Tax 2-4 cells and synergize with taxol (paclitaxel), suggesting that they could specifically inhibit kinases that contribute to the destabilization of microtubules [50].

K252a shows no effect on DNA topoisomerases and has no significant interaction with DNA. However, in the course of a screening program Yamashita and co-workers found two semisynthetic derivatives named KT6006 and KT6528 (Fig. 20.4) which can stimulate DNA cleavage by topoisomerase I [51]. KT6006 is a 3,9-bis-hydroxy derivative of K252a and, as will be discussed below, it is now well

20.2 Naturally Occurring tndolocarbazoles | 543 H OH H

KT-6528 KT-6661 KT-6006

Fig. 20.4. Topoisomerase I inhibitors derived from K252a.

KT-6528 KT-6661 KT-6006

Fig. 20.4. Topoisomerase I inhibitors derived from K252a.

established that the presence of OH groups on the IND framework markedly changes the capacity of the drug to interact with DNA and topoisomerase I. KT6528 bears a CH2OH group on the furanose ring instead of a COOCH3 group common to K252a and KT6006 as well as the analog KT6661. In addition, the drugs differ by their functionality of the upper ring. K252a and KT6006 have an amide function as for staurosporine whereas KT6528 bears an imide, as with re-beccamycin and the bis-glycosyl antibiotic AT2433. DNA relaxation experiments indicated that KT6528 strongly unwinds supercoiled DNA with a potential comparable to that of drugs like adriamycin (a classical anthracycline intercalator) whereas its analog KT6006 showed little effect in this type of relaxation assay. These observations lead to the conclusion that KT6528 bearing the N-hydroxy maleimide ring was a strong DNA intercalator in contrast to KT6006 considered as a weak intercalator. These two compounds exhibit different binding affinities for DNA but are equally potent at stabilizing topoisomerase I-DNA covalent complexes. Thus this pioneer work revealed that there is no direct relationship between the strength of DNA interaction and the capacity of these IND derivatives to inhibit topoisomerase I [51].

This observation can now be considered as a general rule within the IND series; DNA binding and poisoning of topoisomerase I are clearly two distinct aspects, not necessarily correlated. Interestingly, the two topoisomerase I poisons KT6006 and KT6528 showed antitumor activity in the murine P388 leukemia model in vivo, whereas K252a and the compound KT6661 (Fig. 20.4), both inactive against topoisomerase I showed no significant antitumor activity in vivo. Therefore, the possibility that the anticancer activity was mediated by topoisomerase I was raised and this observation has obviously greatly encouraged the search for tumor-active IND derivatives targeting topoisomerase I. But thus far, in the closed series no other potent topoisomerase I has been reported.

Rebecca mycin

Rebeccamycin is another glycosylated IND which has profoundly inspired the development of antitumor agents, especially those targeting topoisomerase I, although this natural product is relatively weakly active against this enzyme

[52]. Rebeccamycin was originally isolated from the actinomycete strain C-38,383 (ATCC 39243) which is considered to be a member of the species Saccharotrix aerocolonigenespreviously known as Nocardia aerocolonigenes [53]. The structure of the drug was solved by NMR and X-ray analysis [54] and the absolute configuration was determined by a total synthesis [55]. Unlike the aforementioned IND such as staurosporine and K252a, its glycosyl moiety is linked to only one of the two indole nitrogens and this is the central point for its mechanism of action. The N-glycoside is a /?-D-4-methoxyglucose residue which is orientated at right angles to the plane of the planar aromatic ring. In addition, the symmetric heterocyclic ring system of rebeccamycin is substituted with chlorine atoms at positions 1,11. These chlorines originate from sodium chloride present in the fermentation broth because the replacement of NaCl by NaBr produced the l,ll-deschloro-l,ll-dibromo-rebeccamycin [56]. The drug has no effect on PKC and DNA topoisomerase II. Its antitumor activity seems to be rather correlated to its inhibitory potency against topoisomerase I. Rebeccamycin is highly toxic to tumor cells and its anticancer activity can be further enhanced by introducing fluoro substituents on the IND moiety. Recently, three new fluoroindolocarbazoles (compounds A-C in Fig. 20.5), obtained biosynthetically by feeding fluorotryptophan to culture of S. aerocolonigenes ATCC 39243, were found to be more potent than rebeccamycin against P388 leukemia in mice [57, 58].

Rebeccamycin is essentially insoluble in water (<1 pg mL-1) and, therefore, soon after its discovery, chemists from Bristol-Myers Squibb initiated the design of analogs with an improved water solubility profile. A diethylaminoalkyl side chain was introduced on the N6 imide and the water-soluble analog BMS 181176 (NSC655649) was shown to possess antitumor activity similar to that of rebeccamycin [59]. This first designed rebeccamycin analog was shown to bind to duplex DNA and to unwind supercoiled plasmid DNA, providing evidence for an intercalative mode of binding [60]. Later, additional spectroscopic measurements fully confirmed that in general (but there are a few notable exceptions) rebeccamycin and its analogs behave as typical DNA intercalating agents [61].

h3co rebeccamycin h3co rebeccamycin

h3co

nsc655649 (bms181176)

fluoroindolocarbazoles

A: r-| = f r2 = h r3 = ch3 B: r1 = f r2 = h r3 = h c:ri = h r2 = f r3 = h

Fig. 20.5. Rebeccamycin, NSC655649 and fluoro analogs.

NSC655649 exhibits a broad antitumor activity in vitro against pediatric solid tumors [62|. This compound has been described as a catalytic inhibitor of topoisomerase II (without inducing enzyme-mediated DNA double-strand breaks) [58] and it has been evaluated in phase I clinical trials in both adult and pedria-tric patients [63, 64]. A N-de-ethylated metabolite, presumably produced by the cytochrome P450 3A4 isoenzyme, has been isolated in patients treated with NSC655649 [65]. This compound is currently undergoing phase II clinical trials for the treatment of hepatobiliary and gall bladder cancers [58].

20.2.4 AT2433

The antitumor antibiotic AT2433-B1 (Fig. 20.6) was originally isolated from culture broth of Actinomadura melliaura [66, 67]. It contains a unique disaccharide consisting of a methoxyglucose and an amino sugar subunit, 2,4-dideoxy-4-methylamino-L-xylose [68]. This bis-glycosyl IND derivative does not inhibit topoisomerase I but it binds strongly to DNA and interacts preferentially with GC-rich sequences. DNase I footprinting and surface plasmon resonance studies, as illustrated in Fig. 20.6, revealed that the sequence 5'-AACGCCAG provides a privileged binding site for AT2433-B1, whereas interestingly the related indolo[2,3-a]carba-zole diglycoside iso-AT2433-Bl interacts 15 times more weakly with this sequence than the parent compound [691. Iso-AT2433-Bl is a diastereoisomer of the natural aminodisaccharide and corresponds to the incorrect structure originally proposed for AT2433-B1. The absolute stereochemistry of the amino sugar is 3S,4S in AT2433-B1 and 3R.4R in iso-AT2433-Bl [70]. Therefore, a subtle modification of the amino sugar moiety has a profound impact on the drug-DNA recognition process. The amino sugar subunit (2,4-dideoxy-4-methylamino-L-xylose) of AT2433-Bl, which is reminiscent of minor groove binding oligosaccharide portions found in the enediyne antibiotics calicheamicin yli and esperamicin Ai [71, 72], represents the primary structural determinant for binding to DNA and the basic element for GC-rich sequence selectivity [69].

AT2433-A1 is the 11-chloro analog of AT2433-B1. The presence of a chlorine atom on the IND chromophore. as in rebeccamycin. is detrimental to DNA binding but nevertheless the chlorinated compound AT2433-A1 is more cytotoxic to P388 murine leukemia cells than the AT2433-B1. The presence of a chlorine atom on the IND chromophore apparently alters the distribution profile of the drug in the cell, i.e. the amount of drug molecules found in the cytoplasm versus the nucleus, as judged from fluorescence microscopy studies (unpublished data).

Synthetic Indolocarbazole Derivatives Targeting DNA and Topoisomerase I

Over the past 10 years a large diversity of open-form IND derivatives have been synthesized. Two groups can be distinguished: analogs derived from rebeccamycin and those issued from the aglycone antibiotic BE 13793C.

CH3 CH3

AT2433-B1 HN^LJ iso-AT2433-B1

AT2433-B1 HN^LJ iso-AT2433-B1

Fig. 20.6. Surface plasmon resonance (SPR) sensorgrams for binding of AT2433-B1 and iso-AT2433-Bl to the sequence 5'-AACCCCAC. The unbound ligand concentrations in the flow solution range from 10 nM in the lowest curve to 4 jaM in the top curve [69].

Fig. 20.6. Surface plasmon resonance (SPR) sensorgrams for binding of AT2433-B1 and iso-AT2433-Bl to the sequence 5'-AACCCCAC. The unbound ligand concentrations in the flow solution range from 10 nM in the lowest curve to 4 jaM in the top curve [69].

Influence of Chloro and Bromo Substituents on the IND Chromophore

As mentioned above, rebeccamycin is characterized by the presence of chlorine atoms at positions 1,11 on the IND chromophore. Therefore, one of the first studies in this area was to evaluate the influence of these chloro substituents. Dechlorinated rebeccamycin, obtained upon hydrogenolysis of the natural product with Raney nickel in aqueous sodium hydroxide [73], remains inactive against protein kinases A and C, and its inhibitory activity against topoisomerase I is only slightly superior to that of rebeccamycin. The removal of the chlorine atoms significantly decreases the cytotoxicity of the drug. Dechlorinated rebeccamycin was found to be 10 and 36 times less toxic to murine P388 leukemia cells and B16 melanoma cells, respectively, compared with rebeccamycin [51]. The chloro groups of rebeccamycin reinforce the chemical stability of the F-ring imide heterocycle

20.3 Synthetic Indolocarbazole Derivatives Targeting DNA and Topoisomerase I | 547 -O- kinase ~-#-MIC

20.3 Synthetic Indolocarbazole Derivatives Targeting DNA and Topoisomerase I | 547 -O- kinase ~-#-MIC

Minimum Inhibitory Concentration Mic
and minimum inhibitory concentration (MIC, jiM) required to inhibit DNA cleavage by topoisomerase I by rebeccamycin and three analogs [75].

[74] and also play a role in the cellular distribution of the compounds, but they are detrimental to the interaction with DNA. Dechlorinated rebeccamycin has significant interaction with DNA and behaves as a typical intercalating agent, whereas rebeccamycin shows little affinity for DNA. As in the AT2433 series mentioned above, the presence of the chloro groups prevents the drug from inserting its IND chromophore between two consecutive DNA base pairs [61].

Brominated rebeccamycin analogs have been synthesized and, interestingly, a 3,9-dibromo derivative was found to potently inhibit both the DNA relaxation and the kinase activities of topoisomerase I [75]. Tazi and collaborators have established that topoisomerase [ can phosphorylate splicing factors of the SR protein family, such as SF2/ASF (see Section 20.4.6) [76]. The dibrominated imide compound represented in Fig. 20.7 strongly prevents the phosphorylation of SF2/ASF by topoisomerase I even in the absence of DNA, whereas rebeccamycin itself shows little effect. A marked inhibition of protein phosphorylation was also observed with dechlorinated rebeccamycin but not with its N-methyl imide derivative or the anhydride derivative. It is worth noting at this point that inhibition of the kinase activity of topoisomerase I has also been observed with camptothecin but only in the presence of DNA [76]. The kinase activity of topoisomerase I requires ATP and it is conceivable that the IND inhibitors bind directly to the ATP-binding site of the enzyme. However, the kinase site of topoisomerase I must be different from the ATP-binding site of protein kinase C which is not inhibited by these compounds

[75]. The capacity of the four compounds shown in Fig. 20.7 to inhibit the kinase activity of topoisomerase I is directly comparable to their capacity to inhibit the DNA cleavage activity of the enzyme. The dibrominated compound is a potent inhibitor of both the kinase activity and the DNA cleavage activity of topoisomerase I. The search for IND compounds capable of interfering selectively with the kinase activity of the enzyme is actively pursued.

Modification of the Imide Heterocycle

A series of N-methyl imide, N-methyl amide, and anhydride derivatives have been synthesized to evaluate the role of the imide nitrogen of rebeccamycin in the inhibition of topoisomerase I and cytotoxicity [74]. A good correlation between topoisomerase I inhibition and cytotoxicity was observed with the imide and anhydride compounds but not with the amide derivatives (Fig. 20.8). Incorporation of a methyl group on the indole nitrogen reduces considerably the capacity of the drug to inhibit topoisomerase I as well as the cytotoxicity (compare activities of compounds 1, 3, and 5 to compounds 2, 4, and 6 in Fig. 20.8). In contrast, no correlation was observed with the amide compounds 8,8' and 9,9', which are potent

ICcn ES3 MIC

ICcn ES3 MIC

Fig. 20.8. Correlation between cytotoxicity against P388 leukemia cells and minimum inhibitory concentration (MIC. nM) required to inhibit DNA cleavage by topoisomerase I for a series of imide, anhydride and amide

derivatives of rebeccamycin. IC50 values correspond to the drug concentration that inhibits murine P388 cell growth by 50% after incubation in liquid medium for 96 hours [74].

cytotoxic agents but they inhibit neither topoisomerase I nor the PKC enzymes. The anhydride derivative 5 is significantly less cytotoxic (but still active against topoisomerase I) compared with its imide derivative 3. This is an important observation because the anhydride is one of the main metabolic forms of these glycosylated rebeccamycin derivatives, as discussed below with the antitumor drug NB-506 (see Section 20.4.7).

Halogenoacetyl Derivatives

Some of the aforementioned compounds, in particular the imide derivative 3 and the two amide derivatives 8,8' and 9,9' were found to potently inhibit the reverse transcriptase activity associated with virus particles released from CEM-SS cells infected with the HIV-1 virus, but the molecular basis of this antiviral activity is not yet known [74]. Significant anti-HIV-1 effects were also observed with a series of rebeccamycin derivatives bearing a halogenoacetyl substituent either on the sugar moiety or on the nitrogen imide. Compounds such as 4 and 6 shown in Fig. 20.9 are weak inhibitors of topoisomerase I and weakly active against HIV-1. In contrast, the potent topoisomerase I poisons such as 2, 5, and 8 strongly inhibit the HIV-1 reverse transcriptase activity. On the basis of these observations, it has been postulated that topoisomerase I contributes, at least partially, to the anti-HIV-1 properties of these compounds. The literature data showing that topoisomerase I activates HIV-1 reverse transcriptase activity [77] and that this activation can be inhibited by a CPT derivative like topotecan [78] also support this hypothesis. The discovery that such glycosylated IND derivatives inhibit HIV-1 proliferation provides novel opportunities to design anti-AIDS drugs.

Glucose and Galactose Derivatives: Stereospecific DNA Recognition

The carbohydrate moiety of rebeccamycin derivatives is absolutely essential to both DNA recognition and topoisomerase I inhibition. Attempts have been made to replace the methoxy-glucose residue of rebeccamycin with another sugar such as a glucose, a galactose, or a fucose. Little differences were observed between the glucose and galactose derivatives in terms of DNA recognition and topoisomerase I inhibition. The orientation of the 4'-hydroxyl group on the sugar residue can be inverted without altering the drug-DNA recognition process. In contrast, the removal of the 6'-OH group (which is supposed to engage a hydrogen bond with the indole NH) reduces considerably the strength of the interaction between the drug and DNA. The fucose analog of rebeccamycin (lacking the 6'-OH group) was found to be less active than the glucose analog [79].

In the naturally occurring IND such as rebeccamycin and AT2433, the carbohydrate moiety is linked to the chromophore via an equatorial /?-N-glycosidic bond which orients the sugar in a suitable position for DNA recognition. The inversion of the configuration at the C-l' of the carbohydrate residue has a major impact on

h3co

1 : R1 = CI R2 = H 2:R1=H R2 = H 3: R-) = CI R2 = COCH2Br

H3CO

4: R1 = CI R2 = COCH2Br 5: R, = H R2 = COCH2Br 6^=01 R2 = COCH3

h3co

1 : R1 = CI R2 = H 2:R1=H R2 = H 3: R-) = CI R2 = COCH2Br

H3CO

4: R1 = CI R2 = COCH2Br 5: R, = H R2 = COCH2Br 6^=01 R2 = COCH3

H3CO °COCH2Br

CH2COCH2CI ^ -O

CH2COCH2CI ^ -O

H3CO °COCH2Br

H3CO

Fig. 20.9. Correlation between anti-HIV-1 activity and minimum inhibitory concentration (MIC, jiM) required to inhibit DNA cleavage by topoisomerase I for a series of imide, anhydride and amide derivatives of

H3CO

Fig. 20.9. Correlation between anti-HIV-1 activity and minimum inhibitory concentration (MIC, jiM) required to inhibit DNA cleavage by topoisomerase I for a series of imide, anhydride and amide derivatives of

H3CO

rebeccamycin. The anti-HIV-1 activity was measured by quantification of the reverse transcriptase activity associated with virus particles released from HIV-1 Lai-infected CEM-SS cells [74].

H3CO

rebeccamycin. The anti-HIV-1 activity was measured by quantification of the reverse transcriptase activity associated with virus particles released from HIV-1 Lai-infected CEM-SS cells [74].

the capacity of the drugs to bind to the target. The comparison of the DNA-binding properties of the a- and /^-glucose, galactose, and fucose derivatives of rebeccamycin showed that only the compounds having the sugar in the /^-conformation inhibit the relaxation of supercoiled DNA by topoisomerase I and intercalate in the DNA double helix, whereas the a-isomers have no effect on topoisomerase I. The /i-N-glycosidic linkage is a key element for topoisomerase I inhibition and recognition of GC-rich sequences in DNA. But, the cytotoxicity measurements showed little differences between the a and p isomers, suggesting therefore that in this series, DNA binding and topoisomerase I poisoning contribute only partially to the cytotoxic activity [79].

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