Development of Ribonucleotide Reductase Inhibitor

Send Orders for Reprints to [email protected] Mini-Reviews in Medicinal Chemistry, 2013, 13, 000-000

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Development of Ribonucleotide Reductase Inhibitor: A Review on Structure Activity Relationships Narayana S.H.N. Moorthy*, Nuno M.F.S.A. Cerqueira, Maria J. Ramos and Pedro A. Fernandes REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, s/n, Rua do Campo Alegre, 4169-007 Porto, Portugal Abstract: Ribonucleotide reductase (RNR, E.C. 1.17.4.1), which is composed of two dissimilar proteins (subunits), often referred as R1 (containing polythiols) and R2 (containing non-heme iron and a free tyrosyl radical), which contribute to the role played by the enzyme. RNRs are one of the important targets in anticancer and antiviral drug development and many RNR inhibitors have been discovered at the end of the 20th century; many of them are already in clinical use. Triapine (3-AP) is one of the important RNR inhibitors belonging to the class of thiosemicarbazone derivatives, used in the treatment of various cancers. The structure activity relationship (SAR) studies on the investigated RNR inhibitors showed that the nitrogen atom in the pyridine (or other heterocycles) forms coordination complexes with the metal ions along with the imine, oxo and thio atoms of the thiosemicarbazone or semicarbazone pharmacophores. The computational analyses results in the adenine and purine derivatives suggest that the nitrogen atoms in the adenine rings make several hydrogen bonds with the water molecules present in the active site, as well as Gly249 and Glu288 residues. The OH group in third position of the sugar moiety interacts with the Ser217 (C=O) and the water molecules through hydrogen bonds. The aromatic rings in the molecules interact with the tyrosine residues. The thiosemicarbazone or semicarbazone derivatives explain that the flexibility and polar properties in the thiosemicarbazone or semicarbazone pharmacophoric regions allow the molecules to coordinate with the metal ion (especially iron) present in the RNR enzymes. This review concluded that RNR inhibitors composed of different fragments such as aryl, heteroaryl, sugar moiety, polar groups, flexible bonds, etc which are required for the binding of the molecules to the RNR enzymes. Further, the fragmental analysis of the RNR inhibitors on different toxicological and metabolic targets can provide significant novel molecules with acceptable pharmacokinetic properties.

Keywords: Ribonucleotide reductase inhibitors, cancer, thiosemicarbazone, triapine. INTRODUCTION Cancer is one of the leading causes of death worldwide and accounts for 7.9 million deaths (around 13% of all deaths) in 2007 and with an estimated 13 million deaths in 2030 [1-3]. Over the last 50 years, many molecules have been developed as anticancer agents against various types of cancers. The majority of anticancer molecules in the clinical world exert their activity by inhibiting cellular processes in normal or cancerous cells. It is important to have significant anticancer molecules with less effect on normal cells and considerable effect on the cancer cells (and targets), which is a major hurdle nowadays. However, a high percentage of them fail clinically due to their lack of selectivity and adverse effects (including toxicity) [3-6]. Most of the currently used anticancer drugs directly make covalent linkages with the DNA or RNA, or indirectly by interacting and inhibiting several enzymes that are required for their (DNA or RNA) synthesis, maintenance or repair *Address correspondence to this author at the REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, s/n, Rua do Campo Alegre, 4169-007 Porto, Portugal; Tel/Fax: +351-220 402 506; E-mails: [email protected], [email protected], [email protected] 1389-5575/13 $58.00+.00

[7-10]. Ribonucleotide reductase (RNR, E.C. 1.17.4.1), also known as ribonucleoside diphosphate reductase is a key enzyme involved in the de novo synthesis by converting the ribonucleotides into the corresponding deoxyribonucleotides, which are the building blocks for DNA replication. This enzyme is currently one of the main targets for DNA inhibition and the drugs that are available in the market have anticancer, antiviral and antibacterial properties. RNRs are mechanistically fascinating because of their free radical chemistry, unusual metallocofactors and complex regulatory mechanisms. The reaction catalyzed by RNRs involves the replacement of the C2'-hydroxyl group on the ribose moiety by a hydrogen atom. In spite of apparently simple, the reaction requires radical catalysis and follows an uncommon chemistry. The products of such reaction and after a phosphorylation steps are the precursors needed for both synthesis and repair of the deoxyribonucleic acid (DNA). All RNRs share a complex and exquisitely controlled radical-dependent redox chemistry and require the generation of a radical for substrate activation. In addition, all RNRs share a unique allosteric regulation system that allows an appropriate balance of each of the four deoxynucleotides (dNTPs) needed for DNA replication and repair. One might © 2013 Bentham Science Publishers

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Mini-Reviews in Medicinal Chemistry, 2013, Vol. 13, No. 13

expected that an enzyme performing such an essential and common function would have been conserved within all organisms. The evolutionary origin and relationships between these enzymes remain elusive, instead, these enzymes may exhibit different protein primary sequence and substrate preference or even metallocofactor usage. In order to differentiate them, the diversity of their metallocofactors has provided the basis for their subdivision into three main classes. Class I enzymes produce a stable tyrosyl radical on one protein subunit in a reaction of a dinuclear iron center with molecular oxygen. Class II enzymes use the cofactor cobalamin for radical generation. Class III enzymes are strictly anaerobic and form a stable glycyl radical with the help of an iron–sulfur protein and S-adenosyl methionine. Class I RNRs are the topic of this paper and are found in practically all eukaryotic organisms, from yeast and algae to plants and mammals. Some prokaryotes and viruses also express this type of RNRs. The enzymes of this class of RNRs are composed of two subunits, called R1 and R2. The substrate turnover reaction and the allosteric control of the

Moorthy et al.

enzyme take place in the monomers of subunit R1. Subunit R2 is composed of two monomers that house a diferric tyrosyl radical cofactor, which is required for starting the catalytic reaction on the monomers of subunit R1. For a successful enzymatic reduction to take place, both subunits have to interact with each other and form a holo-complex [11-16] (Fig. (1)). The expression of RNR enzymes (elevated level of RNR R2) in the cancer cells correlates with chemoresistance, cellular invasion and malignant progression. In colorectal cells, this enzyme (RNR R2) silencing by siRNA, inhibits hyperplasia and invasiveness, which cause infiltration and metastasis in these cells [17]. Over 80% of the human tumors are found to contain mutations in p53. p53R2 (mutated form of p53), an analog of RNR R2 in mammalian cells, correlated with a survival of colorectal cancer with advanced grade III and grade IV tumors, rather than earlier grade I and grade II tumors. Mammalian RNR R2 and p53R2 are located in the cytoplasm and nucleus, respectively. The function of the former one is regulated by cell cycle and the p53R2

Fig. (1). X-Ray structure of proteins R1 and R2 of RNR class I. RNR is a holoenzyme (2 2 or R12R22), subunits, R1 composed by two identical monomers with 761 residues. The active site constituted by five conserved residues, Cys439, Cys225, Cys462, Glu441 and Asn437 in each monomer of R1. The other dimer 2 named R2 has 375 residues in each monomer with a stable neutral tyrosyl free radical at position 122, coupled to a binuclear iron (Fe2O2) cluster in each monomer required for generation and stabilization of the radical (by an oxobridged binuclear Fe(III) complex).

Development of Ribonucleotide Reductase Inhibitor

provides the links between RNRs and cancer. Through a p53 dependent mechanism, the expression levels of RNR subunits R1, R2, and p53R2 were reduced by chlorophyllin treatment in HCT116 (p531/1) and HCT116 (p532/2) cells [17-19]. The overexpression of RNR R1 cancer cells (especially lung cancer cells) leads to gemcitabine-resistant cancer cells. However, an administration of RNR R1-specific siRNA causes downregulation of RNR R1 activity and is an effective strategy to overcome RNR R1 overexpression in drug resistant tumor cells (pancreatic, non-small cell lung cancer, human cervical and other cancer cells) [20,21]. RNRs have low activity in resting cells, pronounced activity in rapidly growing normal cells and very high activity in cancer cells. Previous studies have clearly indicated that RNRs play a fundamental role in the critical early events involved in tumor promotion and its activity is tightly linked to the neoplastic expression state [22-24]. Several potentially useful classes of RNR inhibitors were reported, which include: (1) Free-radical scavengers: hydroxyurea, trimidox, didox, etc., (2) Iron chelators: triapine, PIH, 311, etc., and (3) Substrate analogs (gemcitabine) [12, 25-27]. Currently many RNR inhibitors are in clinical trials for cancer treatment and these molecules act on DNA synthesis, providing a good target for the anticancer drug development program. Hence, in this review, we focus on the structural requirements (structure activity relationships (SAR)) of the radical scavenger and iron chelating RNR inhibitors using various computational analyses, and the results are summarized here to help in the development of novel RNR inhibitors. RNR INHIBITORS DEVELOPMENT RNR inhibitors elicit their inhibitory activities in different mechanism pathways such as the radical scavengers that destroy the tyrosyl radical required for the reaction (e.g. -(N)-heterocyclic carboxaldehydes, thiosemicarbazones and hydroxyurea), iron chelators destroy the metal complex that produces the radical required for the catalytic reaction (e.g. desferal and parbactin), peptidomimetic inhibitors that preclude the dimerization of active holoenzyme structure or substrate analogues that preclude the binding of the natural substrate in the active site (e.g. gemcitabine) (Table 1) [12,28-34]. Structures of various RNR inhibitors are provided in (Fig. 2). In this review special attention will be given to the iron chelators and radical scavenging inhibitors. Iron chelators and radical scavengers are inhibitors of subunit R2 of RNRs, that either destroy the radicals that are settled on the tyrosine residue (radical scavengers) or inactivate the radical, preventing its generation by chelating the iron of the binuclear cluster (iron chelators). As the radical is buried c.a. 10Å from the surface of the protein, these kinds of inhibitors become generally small and act as planar molecules in order to facilitate their approach to the free radical. The first inhibitor of this kind able to preclude RNR activity was hydroxyurea (HU). HU was the first RNR inhibitor investigated as an antineoplastic agent in humans, in 1960s. Its synthesis involved many complex crystallization steps [35], but today it has a simple synthesis reaction involving hydroxylamine, HCl and KCN. HU is a wellknown RNR inhibitor that is used in myeloproliferative disorders, specifically polycythemia vera and essential


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thrombocytopenia. It is also used to reduce the rate of painful attacks in sickle-cell disease and has antiretroviral properties in diseases such as AIDS [35-37]. The simplest explanation for the successful HU inhibition of DNA synthesis is that it starves the DNA polymerase at the replication forks for dNTPs. Its main mechanism of action is elicited through the inhibition of the RNR enzymes by scavenging the tyrosyl free radical that is required for the catalytic process. In spite of the success observed in HU, it presents serious side effects such as the decrease of blood cells (anemia), platelets (thrombocytopenia), and white blood cell counts (leucopenia and neutropenia), which can decrease patient resistance to infections. Nevertheless, HU is still the one that has been predominantly used in cancer therapy [38,39]. Based on the success obtained with HU, similar compounds were synthesized aiming to achieve similar or better results. In this process, it has found that guanazole is another interesting compound since it is chemically distinct from HU but it also possesses high inhibitory activity against RNR. In some cases, as in the case of HeLa cells, it has been found that this compound is not as effective as HU. However, this seems to be overcome by the presence of iron chelating agents that potentiate the activity of this compound [40]. Another interesting compound is carbamoyloxyurea, an oxidation product of HU, that has RNRs as a primary target but also a second site of action which also appears to be in the pathway of DNA synthesis. The inhibition results were quite appealing in several in vitro studies, but the observed toxicity precluded the development of this drug for further studies. However, this molecule served as a base for the development of novel hydrazones and other semicarbazone derivatives [37,40,41]. These compounds have become very popular because the combinations of these compounds with different RNR inhibitors have shown antagonistic activity on the CDP reductase enzyme, a factor that potentiates the global action of these inhibitors. In these particular cases, the pyrazoloimidazole (IMPY), 4-methyl-5-aminoisoquinoline thiosemicarbazone (MAIQ), inox and 5`deoxy inox have shown very interesting properties. In addition, recent studies have shown that IMPY can be a useful agent in combination with other drugs to provide synergic inhibition of RNRs [28,42-45]. Some compounds such as deoxyadenosine, pyridine-2-carboxaldehyde, thiosemicarbazones, IMPY, 3,5-diamino-1,2,4-triazole (quanazole), 3,4,5-trihydroxy benzohydroxamic acid and 3,4-dihydroxy benzohydroxamic acid have also been shown to possess DNA excision repair activity in addition to RNR inhibitory activity (Table 1) [28,46,47]. Thiosemicarbazone and semicarbazone are important pharmacophores for the development of RNR inhibitors. The semicarbazone derivatives were developed for antiviral and anticancer activity since the mid of the 20th century. The antiviral activity of thiosemicarbazones was reported first in 1950 [48]. Subsequently, purine-6-carboxaldehyde thiosemicarbazone (1969) and 2-acetylpyridine thiosemicarbazone (1979) were reported, possessing antiviral activity against cytomegalovirus and antimalarial activity against P. berghei respectively [48-52].

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Table 1.

Moorthy et al.

Ribonucleotide Reductase Inhibitors. Comp No

Name of the Inhibitors

Activity Category

Pyrimidine derivatives 1

Gemcitabine

R1 protein inactivation

2

Tezacitabine


3

DMDC (MDL101731)


4

Cytarabine


1

Fludrabine


2

Cladribine


3

ADP-S-HBES-S-dGTP


Adenine derivatives

Urea, hydroxyureas and hydrazones 1

Hydroxyurea


2

Caracemide

Inactivator of sulfhydryl group

3

Trimodox

Radical scavenger

4

PIH

Iron chelators

5

311

Iron chelators

6

N-Carbamoyloxy urea


Semicarbazones and thiosemicarbazones 1

3-AP (Triapine)

Iron chelators

2

3-AMP

Iron chelators

3

5-AP

Iron chelators

4

5-AMP

Iron chelators

5

Methisazone

Iron chelators

1

Clofarabine


2

Cisplatin


3

Didox

Radical scavenger

4

Nitric oxide

5

Alkoxyphenols

Radical scavenger

6

SNAP

Iron chelator

7

DFO

Iron chelators

8

Guanazole

9

Pyrazoloimidazole

10

Resveratrol

11

3-Methyl-4-nitrophenol

12

Motexafin gadolinium (MGd)

Miscellaneous



5

OH HO

O

OH

H N

O N

O

O HN

OH

O H2 N

Caracemide

HON

N H

NH 2

Trimidox

Hydroxyurea

NH2 R R

H3 C

N N

N NH 2

OH HO

HO

N

N

N

N

N

N

H 2N

H 2N

NH

NH

NH

NH

S

S

PIH

5-AP = R= H; 5-AMP = R= CH3

3-AP (Triapine) = R= H; 3-AMP = R= CH3

O

O

311

N

S

N

NH 2

N H

NH

H N

H 2N

N

N

S

N

N O

Purine-6-carboxaldehyde thiosemicarbazone

H 3C

Methisazone

O

S

N

O

R

O

N N H

N H

OH H 2N

CH3

N H

Hydroxyurea

2-Acetylpyridine thiosemicarbazone derivatives

H2 N

NH2

N H O

N-Carbamoyloxy urea (Reactive intermediate)

Fig. (2). Structure of the some important hydroxyurea and thiosemicarbazone based RNR inhibitors.

Subsequent studies showed that these compounds possessed anticancer activities against different cancer cell lines. In the sixties, several thiosemicarbazones with anticancer (1956) and antiviral (1973) [53,54] effects were discovered and a huge amount of research was carried out that eventually led to the commercialization of methisazone (Nmethylisatin-thiosemicarbazone), a potent inhibitor of RNR. This compound is effective as prophylaxis against smallpox and vaccinia viruses [55-57].

Some thiosemicarbazones also have anti-leukemic effects. 2-Formylpyridine thiosemicarbazone reported by Brockman et al. [53] in 1956 was the first compound with such type of features. Almost ten years later, in 1965 [58], -(N)heterocyclic thiosemicarbazones were reported having iron chelator characteristics, and one was found to inhibit RNRs [59]. Recently, Triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone) (3-AP) an efficient inhibitor of RNRs was developed as an anticancer drug, which has also reached

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clinical phase II for the treatment of several cancer types [60,61]. Triapine is developed by Vion Pharmaceuticals, Inc., and has been demonstrated to possess a broad spectrum of activity in animal tumor models, showing efficacy in both in vivo and in vitro tests and an ability to inhibit tumor cell growth in the murine L1210 leukemia, murine 109 lung carcinoma and human A2780 ovarian carcinoma models. Clinical tests indicate that it is 65 to 1000 times more potent than FDA approved HU. Further developments of these types of compounds, such as 3-amino-4-methylpyridine-2carboxaldehyde thiosemicarbazone (3-AMP), an analog of triapine, showed much better therapeutic effects against L1210 leukemia, M-109 lung carcinoma and A2780 human ovarian carcinoma as triapine [62]. RECENT PROGRESS IN RNR INHIBITORS In succession to HU, thiosemicarbazone and semicarbazone derivatives were developed, and the heteroaryl carboxaldehyde thiosemicarbazones derivatives (HCTs) were found to be strong RNR inhibitors [63-66]. Several HCT inhibitors inhibit RNRs at concentrations >1000-fold lower than those required for HU to achieve the same effectiveness [63,67]. Among HCTs, triapine is an effective RNR inhibitor with its iron chelating property and has been used for the treatment of various tumors. Several phase-I clinical trials showed that triapine produced no cardiovascular, central nervous system (CNS) or other major adverse effects at effective therapeutic concentrations. Triapine is currently manufactured under Good Manufacturing Practice (GMP) and undergoing phase II clinical trials as a cancer chemotherapeutic agent [43, 68-72]. Based on these promising results, triapine has also been used in combination with other drugs. For instance, a phase I trial of triapine followed by the administration of adenosine analog fludarabine in adults with refractory acute leukemias and aggressive myeloproliferative disorders (MPD) showed that triapine 105 mg/m2 followed by fludarabine 30 mg/m2 daily for five days is active in refractory myeloid malignancies and studies are continuing for patients with aggressive MPD [73]. Another phase I study on the combination of triapine and gemcitabine has provided tolerability and efficacy (initial level) of these drugs. The phase II study of the same combination (gemcitabine plus triapine) in advanced pancreatic carcinoma was reported, which revealed that this combination is associated with moderate toxicity in patients with advanced pancreatic cancer. Two-stage trial of this combination was stopped after stage I due to lack of antitumor activity [74]. A study of the same combination (triapine plus gemcitabine) in advanced biliary tract adenocarcinoma suggested that the associated response rate was not exceeding 30% in patients with adenocarcinoma of the biliary tract. Also triapine enhances the cellular uptake and DNA incorporation of gemcitabine in tumor cell lines. Additional clinical trials are ongoing to evaluate the role of triapine in combination with fludarabine for patients with hematologic malignancies (NCT00381550), and in combination with radiation for pancreatic carcinoma (NCT00288093), or radiation and cisplatin for cervical and vaginal carcinoma (NCT00941070) [75-77].

Moorthy et al.

An investigation on the induction of replicational stress by the HU combined with histone deacetylase inhibitors (HDACi) exerts antitumor activity. HU has treated head and neck squamous cell carcinoma (HNSCC) cell lines and freshly isolated cancer cells in combination with HDACi (valporic acid (VPA), a clinically approved anti-epileptic drug). These results demonstrated that at clinically achievable levels, VPA/HU combination efficiently blocks proliferation as well as clonogenic survival, and triggers apoptosis of HNSCC cells [78-81]. Didox induces caspase dependent multiple myeloma (MM) cells apoptosis by inhibiting DNA synthesis and repair, when administrated with melphalan. The in vitro synergism studies provide the preclinical rationale that the drug alone and in combination with DNA-damaging agents can be used to improve patient outcome in MM [82]. On account of their interesting properties, the thiosemicarbazone and the semicarbazone derivatives were used as RNR inhibitors in the advanced stage of the clinical trials. Several studies were conducted in order to understand the chemical properties that justify the unique chemical features that these compounds possessed. In this regard, the SAR of RNR inhibitors can provide important information regarding the effect of scaffold and the functional groups (fragments) for the inhibitory activity. Advanced computer analyses such as quantitative structure activity relationship (QSAR) analysis, pharmacophore analysis and docking studies can provide these types of information. Here, we report some studies of this kind that have provided some specific features of the structurally different thiosemicarbazones that are responsible for the observed RNR inhibitory activity and anticancer properties. 3D-QSAR analyses (using comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) approaches) were reported on a set of 39 -(N)-heterocyclic carboxaldehyde thiosemicarbazones with RNR inhibitory activity against H.Ep.-2 cells (human epidermoid carcinoma) [30,31]. The results of the CoMFA and CoMSIA analyses suggested that the hydrogen bond acceptor groups near the nitrogen of the pyridine ring (small, electron rich and hydrophobic groups at position 5) can enhance the inhibitory activity values (the lone pairs of electrons in the nitrogen atom of the pyridine ring can form a complex with the iron ion). SAR of the compounds also showed that compounds substituted on the meta position with I, Cl and F atoms, possessed better activity and compounds have steric bulky groups such as meta -OC2H4N(CH3)2 and meta-OOCCH2OC6H5, exhibited low potency. Additionally, the presence of small bulky groups at para and meta positions of the pyridine ring are favorable to RNR inhibitory activity ((para-CH3) and (meta-CH3)). The substituents present in the less potent compounds (meta-OC2H4N(CH3)2 and meta-OOCCH2OC6H5), are oriented into steric unfavorable regions. They concluded that the presence of less steric bulky groups and electron rich substituents provides significant inhibitory activity [34]. Other QSAR and pharmacophore studies addressed to a series of -N-heterocyclic carboxaldehyde thiosemicarbazones derivatives exhibit anticancer activity by inhibiting RNR enzymes [83], and revealed that the SMR_VSA5 descriptor,


which measures the steric parameters and bulkiness of a given molecule, is the main contributor for the activity prediction in all selected models. The QSAR results showed that the van der Waals (vdW) surface area properties such as the surface polarity, hydrogen bond donor properties and hydrophilic contact surface of the molecules are important for the RNR inhibitory activity. The results obtained from the pharmacophore analysis revealed that the H-bond acceptor and the H-bond donor along with aromatic or planar ring in the molecules and the concentrated hydrophobicity in particular regions are also responsible for the inhibitory activity. The distance between the aromatic/ hydrophobicity to the H-bond donor and the acceptor groups should be connected with almost the same distance [83-85]. Ansari and co-workers have also performed similar QSAR studies addressed to some thiosemicarbazone RNR inhibitors, such as 1-formylisoquinoline thiosemicarbazone and 2-formylpyridine thiosemicarbazone derivatives [86-88]. The statistically significant results obtained from the MLR analysis pointed out that the descriptors such as heat of formation; total energy and highest occupied molecular orbital (HOMO) energy have contributed in the activity prediction of the analyzed compounds. Several QSAR studies were also performed in some hydrazine derivatives. For example, the QSAR studies on a series of 2-benzoxazolyl hydrazone derivatives against various cancer cell lines were reported by Moorthy et al [89]. The hydrophilic and hydrophobic integy moments of the molecules (vsurf_IW6 and vsurf_ID8) (measure the unbalance between the centre of mass of the position of a molecules and its hydrophilic or hydrophobic region around it) reduced the interaction energy between the molecule and the water, which improved the antitumor activity. The potential energy descriptors indicated that the flexibility of the freely rotatable bonds played a significant role in the interaction of the chemotherapeutic agents to the barriers and to reach the target. The reported results have shown that the antitumor activity could be improved with the presence of specific hydrophobic substituents and electron donating groups near the hydrazone moiety. Moreover, the formation of an intramolecular hydrogen bond has a high impact on the pharmacological activity of the compounds. Those compounds that have a 2-pyridine substituent possessed better antitumor activity; the pyrimidine and the pyrazine substitutions have less pronounced antitumor activity and the worst ones are obtained with the pyridazine derivatives. The values derived from this study indicate that the distance between the most reactive hydrophilic region and the hydrophobic region should be very small and this key feature is preponderant for the observed antitumor activity [90]. 3D-QSAR studies on Schiff bases of hydroxysemicarbazide analogs (antitumor activities against L1210 cells) using CoMFA and CoMSIA methods can also be found in the literature [91]. The predictive ability of the resulting model was evaluated against a test set (four molecules). The results explained that the presence of a hydrophobic field in both models (CoMFA and CoMSIA) justifies the lipophilic requirement of molecules for better activity. The contour maps of both methods (CoMFA and CoMSIA) described that substitution by electron-rich functional groups (hydrogen


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donor/electron-rich) at the ortho and meta position(s) have favorable pharmacological effects on the compounds, while para position is detrimental for the activity. The most active compound in the data set has an anthracene nucleus and the central pharmacophore region has accommodated within the sterically favored region. Meta and ortho positions are also sterically favorable regions (ortho-substitution, preferably OH, and a sterically bulky electronegative functional group at other meta-position may lead to more active compounds). The acetyl group present in the compound has oriented into a sterically unfavorable, which may lose important interactions at the active site, which is detrimental to activity. The authors concluded on the mechanism of action of these derivatives caused by the presence of the pharmacophore (RNC (=O) NHOH), which is also present in HU (RNR inhibitor) [90]. The selected models (both CoMFA and CoMSIA models) predicted the biological activities of 4.078 and 4.040 by CoMFA and CoMSIA, respectively which are near to the experimental value of HU (4.086). The pharmacophores of the derivatives are same as the established pharmacophore of RNR inhibitors. Hence these compounds have antitumor activity, which may be due to the inhibition of RNR enzymes [92]. Other QSAR studies regarding similar Schiff bases of hydroxysemicarbazides (SB-HSC), showed that the essential pharmacophore (-NHCONHOH), hydrophobicity (SlogP), molecular size/polarizability (calculated molar refractivity) and the presence of an oxygen-containing group at the ortho position are important determinants for the antitumor activities. Similar to what is observed with the thiosemicarbazone derivatives, these compounds exert an RNR inhibitory effect through chelation of the non-heme iron of the R2 subunit because they have no (N)-OH groups in the pharmacophore. The reported compounds possessed the essential pharmacophore (-NHCONHOH), which has both iron chelating and free radical scavenging properties as shown in the studies on HU. From the chemical structure point of view, the SB-HSC is the most likely target of the same enzyme as HU as mentioned earlier by Raichurkar et al [91-93]. The molecular docking results showed that the ligands can only interact with the irons of the R2 subunit in the oxidized form of the protein, i.e., after the activation of the enzyme. In the reduced form, the metallic center is protected by a cluster of aspartates and glutamate residues, which hinder the access of the inhibitors to the metallic center. These results also show that the iron interacts very closely with the nitrogen atoms of the thiosemicarbazone derivatives. The QSAR results have shown however that this might not be fundamental for the chelation activity of these compounds [83-85,94-96]. The nitrogen atoms in the pyridine molecules coordinate with the metal ions along with the imine, oxo and thio atoms of the thiosemicarbazone or semicarbazone pharmacophores. The computational analyses results on the thiosemicarbazone and the semicarbazone derivatives show that the flexibility of the bonds and the distance between the aryl groups are responsible for the RNR inhibitory activities. The analyses results on the adenine and purine derivatives suggest that the nitrogen atoms in the adenine rings make hydrogen bond

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Moorthy et al.

with the water molecules in the active site (Gly249 and Glu288 residues). The sugar moieties in the molecules containing OH in the third position interact with Ser217 (C=O) and with water molecules through hydrogen bonds. The aromatic rings in the molecules interact with the tyrosine residues [97-99]. CONCLUSION Over the last two decades, it has been observed a great increase in the development of potent RNR inhibitors with anticancer, antiviral and antibacterial properties using different strategies. At the same time, important SAR studies pointed out crucial information that is being used in order to get more efficient substitutions in the general structures of the RNR inhibitors. Currently, the thiosemicarbazone and semicarbazone derivatives present very interesting properties, as have been emphasized by the advanced stage of the clinical trials where they are being used. The QSAR, pharmacophore and molecular docking results have provided crucial information, which revealed the chemical features that are responsible for

the interesting activity of these compounds in several types of tumors. These features are schematically represented in “Fig. (3)”. The structural analyses of the thiosemicarbazone or semicarbazone compounds revealed that the flexibility and polar properties allow these molecules to coordinate with metal ions that are present in the enzymes. The active site of the RNRs exhibits aromatic residues (tyrosine) and an iron ion, hence flexible aromatic rings (geometry also) with polar bridges (thiosemicarbazones) are necessary for the interactions. The presence of bulky substituents in the aromatic ring also makes the region of the molecule hydrophobic and interacts with the hydrophobic part or aromatic residues. These results concluded that RNR inhibitors composed of different fragments such as aryl, heteroaryl, sugar moiety, polar groups, flexible bonds, etc are required for the binding of the molecules to the RNR enzymes and to be good ironchelators. Further fragmental analysis of the RNR inhibitors on different toxicological and metabolic targets can provide significant novel molecules with acceptable pharmacokinetic properties. The combined results of computational and

Steric bulkiness: Unfavourable Hydrophobicity: Unfavourable Electronegativity: Favourable Hydrogen bond acceptor: Unfavourable

Hydrogn bond acceptor: Favourable Hydrogen bond donor : Unf avourable Coordination bonding with metal ions

Hydrophobicity: Favourable N

S

R N C H

H N H

Hydrogen bond donor: Favourable

N H

Steric bulkiness: Favourable

Hydrogen bond donor and acceptors: Favourable Geometric f lexibility: Favourable Coordination bonding with metal ions (chelation)

Steric bulkiness: Favourable Positive charge: Favourable

Hydrogn bond acceptor: Favourable Hydrogen bond donor : Unf avourable Coordination bonding with metal ions

Steric bulkiness: Unf avourable O

Ar N

OH N H

Negative charged group: Favourable Increased OH groups (2 more): Favourable

N H

Hydrogen bond donor: Favourable

Steric bulkiness: Favourable Hydrophobicity: Favourable Big aromatic structures: Unf avourable

Fig. (3). Graphical representation of SAR of compounds derived from the computational analyses.


experimental works are a guide for the development of novel RNR inhibitors with fragmental based drug design and other molecular modeling analysis. We believe that these conclusions can provide important insights about the key features of the currently available compounds that can now be used in the development of new compounds addressed to inhibit RNRs with anticancer, antiviral and antibacterial activity. It must be mentioned however that although a great success is being observed with radical scavengers and iron chelators as RNR inhibitors, in particular with the thiosemicarbazone and semicarbazone derivatives, there are two problems that researchers have to face: i) the first is the toxicity of the compounds, which requires a careful balance between the toxicity of normal tissues vs. cancer cells in order to acquire the desired effects; ii) the second is the resistance of the host organisms to RNR inhibitors that has been observed in several treatments during therapy. This means that the challenge of RNR inhibition by scavenging the radical that is essential for catalysis or by destroying or inactivating the metal that generates the radical, now resides in the development of safer and more efficacious inhibitory compounds, which will surely be available soon, taking into account the high potency of the enzymes and the available data.


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CONFLICT OF INTEREST The authors confirm that this article content has no conflicts of interest.

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ACKNOWLEDGEMENTS One of the Authors (N.S.H.N. Moorthy) gratefully acknowledges the Foundation for Science and Technology (FCT), Portugal for a Postdoctoral Grant (SFRH/BPD/ 44469/2008).

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Received: ??????????, 2012

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Revised: ??????????, 2012

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Accepted: ??????????, 2012