© 2015 Nurcan Berber*, Mustafa Arslan*, gdem Bilen**, Zubeyde Sackes**, Nahit Gender**, #, and Oktay Arslan**

*Department of Chemistry, Faculty of Art and Sciences, Sakarya University, Sakarya, 54147 Turkey **Department of Chemistry, Faculty of Art and Sciences, Balikesir University, Balikesir, 10145 Turkey Received June 6, 2014; in final form February 12, 2015

A new series of phthalazine substituted P-lactam derivatives were synthesized and their inhibitory effects on the activity of purified human carbonic anhydrase (hCA I and II) were evaluated. 2#-Indazolo[2,1-b]phthala-zine-trione derivative was prepared with 4-nitrobenzaldehyde, dimedone, and phthalhydrazide in the presence of TFA in DMF, and the nitro group was reduced to 13-(4-aminophenyl)-3,3-dimethyl-3,4-dihydro-2#-indazolo[1,2-b]phthalazine-1,6,11(13#)-trione with SnCl2 ■ 2H2O. The reduced compound was reacted with different aromatic aldehydes, and phthalazine substituted imines were synthesized. The imine compounds undergo (2+2) cycloaddition reactions with ketenes to produce 2#-indazolo[2,1-b]phthala-zine-trione substituted P-lactam derivatives. The P-lactam compounds were tested as inhibitors of the CA isoenzyme activity. The results showed that all the synthesized compounds inhibited the CA isoenzyme activity. 1-(4-(3,3-dimethyl-1,6,11-trioxo-2,3,4,6,11,13-hexahydro-1#-indazolo[1,2-b]phthalazin-13-yl)phenyl)-2-oxo-4-p -tolylazetidin-3-yl acetate (IC50 = 6.97 ^M for hCA I and 8.48 ^M for hCA II) had the most inhibitory effect.

Keywords: P-lactam, imine, carbonic anhydrase, enzyme inhibitor.

DOI: 10.7868/S0132342315040119


2-Azetidinones usually named as P-lactams are well-known heterocyclic compounds among organic and medicinal chemists [1]. Their ring system has a common structural feature of a number of broad spectrum P-lactam antibiotics, including penicillins, carbapenems, cephalosporins, monobactams, sul-baktams, nocardicins, and tazobactams, which are used as chemotherapeutic agents to threat microbial diseases [2—5]. The compounds are very important due to their wide range of biological activities, such as antimicrobial [6], antiviral [7], antihyperlipidemic [8], anti-inflammatory [9], and antibiotic activity[10]. P-Lactames are also known as potent inhibitors of some enzymes [11]. The compounds form covalent adducts with the membrane-bound bacterial transpeptidases known as penicillin-binding proteins [12].

Carbonic anhydrases (CAs, EC are Zn2+ containing metalloenzymes that catalyse the reversible hydration of CO2 to bicarbonate ion [13, 14]. All known CAs can be grouped in five evolutionarily unrelated CA families designated a-, P-, y-, 8-, and s-CA [15—19]. Several isoforms of a-class are available in all

# Corresponding author (phone: +90 266-612-1278; fax: +90 266612-1215; e-mail: ngencer@balikesir.edu.tr).

vertebrate tissues. Apart from this, P-class isoforms are common in higher plants and algae, including cyano-bacteria. The 6-CAs are primarily found in marine diatoms [20]. Higher vertebrates, including humans, in many different cellular layouts and tissue distributions, have 16 different a-CA or CA-related proteins (CARP) isoenzymes [21, 22]. There are sixteen isozymes which are characterized and many of them are involved in critical physiological processes [23]. This metalloenzyme family is involved in many physiologic or pathologic processes, such as respiration and transport of CO2/bicarbonate between metabolizing tissues and lungs, pH and CO2 homeostasis, and tum-origenicity [24, 25].

Most of the drugs used in human medicine are heterocyclic compounds containing nitro group, they are widely spread in nature, and their utilization is becoming progressively important as biologically active pharmaceuticals, agrochemicals, and functional materials [26]. Phthalazine derivatives have been greatly used as therapeutic agents owing to their anticonvulsant, car-diotonic, vasorelaxant, and anti-inflammatory properties [27—30] and antimicrobial activity [31].

In this study, a new series of phthalazine substituted P-lactam derivatives were synthesized and their inhib-

itory effects on the activity of purified human carbonic anhydrase (hCA I and II) were evaluated.


For assessment of physiologically suitable human CA isozyme (hCA I and II) inhibitory activity, new P-lactam derivatives synthesized were subjected to CA inhibition assay with CO2 as a substrate. The results showed that phthalazine substituted P-lactam derivatives (IVa—i) inhibited the CA enzyme activity. Inhibition values (IC50) of the synthesized compounds


against CAs were shown in the table. We determined the IC50 values of 6.97-20.51 |M and 8.48-24.84 |M for hCA I and hCA II, respectively.

2#-Indazolo[2,1-&]phthalazine-trione (I) was synthesised with 4-nitrobenzaldehyde, dimedone, and phthalhydrazide in the presence of TFA in DMF. The nitro containing phthalhydrazide was reduced with tin(II) chloride in ethanol. The amino phthalhydrazide was reacted with dichloro acetyl chloride or acetoxy acetyl chloride to get the final products (IVa—i) shown in Scheme 1.

O R2 R,

Vf '/=



X: -H, -CH3, -NO2, -OMe, -F




(IVa) (IVb) ( IVc) ( IVd) ( IVe) (IVf) (IVg) (IVh) (IVi) (IVj)

X -H -H -CH3 -CH3 -NO2 -NO2 -OMe -OMe -F -F

R1 -Cl -OCOCH3 -Cl -OCOCH3 -Cl -OCOCH3 -Cl -OCOCH3 -Cl -OCOCH3

R2 -Cl -OCOCH3 -Cl -OCOCH3 -Cl -OCOCH3 -Cl -OCOCH3 -Cl -OCOCH3

Scheme 1. Synthesis of phthalazine substituted P-lactam derivatives.

All compounds obtained were elucidated by XH NMR, 13C NMR, IR, and mass spectrometry. In the infrared spectra of compounds (IVi-i), it was possible to observe the absorption bands between 3058 and 3088 cm-1 relating to NH stretch and absorptions at 1770-1748 cm-1 from P-lactam carbonyl moiety stretching. The 1H NMR

spectra for all the synthesized P-lactam compounds show signals as singlet between 5.28 and 6.38 ppm relating to hydrogens attached to P-lactam ring, as well as the signals for aromatic hydrogens between 6.86 and 8.38 ppm. In the 13C NMR data, there is a signal of P-lactam carbonyl at 150.0-165.0 ppm.

IC50 (p.M) values of the phthalazine substituted P-lactam derivatives

Compound hCA I hCA II Compound hCA I hCA II

(IVa) 18.73 ± 0.07 24.84 ± 0.05 (IVf) 10.76 ± 0.03 11.92 ± 0.03

(IVb) 6.97 ± 0.01 8.48 ± 0.02 (IVg) 15.08 ± 0.05 19.00 ± 0.06

(IVc) 20.51 ± 0.05 24.35 ± 0.06 (IVh) 13.83 ± 0.01 15.94 ± 0.02

(IVd) 15.11 ± 0.04 17.84 ± 0.03 (IVi) 13.28 ± 0.03 12.51 ± 0.01

(IVe) 13.18 ± 0.02 17.48 ± 0.03 (IVj) 9.72 ± 0.02 14.70 ± 0.03

CA inhibitors decrease intraocular pressure by reducing bicarbonate formation in the ciliary process, thus lowering Na+ transport and flow of aqueous humour: this is the basis for their use in glaucoma treatment. Unluckily, systemic therapy with parenteral sul-phonamides results in important side effects, many of them being probably due to inhibition of CA isoforms in other tissues. Acetazolamide, which is 20 times less active against hCA I than against hCA II in erythrocytes, is the most extensively used inhibitor. However, the inhibition of various CA isoforms, which are present in tissues other than eye, results in an entire range of side effects, the most prominent being numbness and tingling of extremities, metallic taste, depression, fatigue, malaise, weight loss, decreased libido, gastrointestinal irritation, metabolic acidosis, renal calculi, and transient myopia [32—34].

Sulfonamide compounds are coordinated to the zinc(II) ion within the hCAs active site, whereas its organic scaffold fills the entire enzyme cavity, making an extensive series ofvan der Waals and polar interactions with amino acid residues at the bottom, middle, and entrance of the active site cavity [35]. Coumarin derivatives may possess various tautomeric structures, which may bind within the CA active site similarly to phenols, i.e. by anchoring to the zinc-bound water molecule/hydroxide ion [36]. Coumarins cannot connect enzyme effectively in the restricted space near

Zn2+ ion since they have bulky group and exhibit unusual binding mode not interacting with the metal ion of the enzyme [37]. We suppose that the synthesized compounds have very big pendant group to be able to bind near the zinc ion. Hence, they are much more probable to bind as the coumarin derivatives.

The results show that all the compounds (IVa—i) inhibited the enzyme activity. The following structure—activity relationship (SAR) observations can be drawn from the data. The slow cytosolic isoform hCA I and the second off target isoform hCA II were inhibited by the synthesized compounds with inhibition values in the range of 6.97-24.84 |M. The best hCA I and hCA II inhibitoramong the newly synthesized and investigated compounds was (IVb). P-Lactams have different inhibition effects. Bulky phthalazine group affects inhibition by the compounds. They probably bind as the coumarin derivatives [37, 38]. Also, the interaction occurs between enzyme active side and the beta-lactam ring. Reaction mechanism shown in Scheme 2 is similar to the earlier proposed in the literature [39]. In the proposed mechanism, the transition state, which develops anionic character after the attack of OH-, is stabilized by the coordination of adjacent Zn2+ ion to O-. Then, C-N bond cleavage occurs easily due to the strain of the P-lactam ring.

Scheme 2. Proposed mechanism of the enzyme interaction with the compounds.

In summary, enzyme inhibition is extremely important issue for drug design and biochemical applications [40—43]. The results show that new phthalazine substituted P-lactam derivatives inhibit the hCA I and II enzyme activity. Therefore, our results suggest that the compounds are likely to be adopted as candidates to treat glaucoma and may be taken for further evaluation in vivo studies.

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