Kinetic and X-ray crystallographic investigations of substituted 2-thio-6-oxo-1,6-dihydropyrimidine–benzenesulfonamides acting as carbonic anhydrase inhibitors

Accepted Manuscript Kinetic and X-ray crystallographic investigations of substituted 2-thio-6oxo-1,6-dihydropyrimidine-benzenesulfonamides acting as carbonic anhydrase inhibitors Daniela Vullo, Claudiu T. Supuran, Andrea Scozzafava, Giuseppina De Simone, Simona Maria Monti, Vincenzo Alterio, Fabrizio Carta PII: DOI: Reference:

S0968-0896(16)30419-9 http://dx.doi.org/10.1016/j.bmc.2016.06.005 BMC 13061

To appear in:

Bioorganic & Medicinal Chemistry

Received Date: Revised Date: Accepted Date:

13 April 2016 31 May 2016 2 June 2016

Please cite this article as: Vullo, D., Supuran, C.T., Scozzafava, A., Simone, G.D., Monti, S.M., Alterio, V., Carta, F., Kinetic and X-ray crystallographic investigations of substituted 2-thio-6-oxo-1,6-dihydropyrimidinebenzenesulfonamides acting as carbonic anhydrase inhibitors, Bioorganic & Medicinal Chemistry (2016), doi: http:// dx.doi.org/10.1016/j.bmc.2016.06.005

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Kinetic and X-ray crystallographic investigations of substituted 2-thio-6-oxo-1,6dihydropyrimidine-benzenesulfonamides acting as carbonic anhydrase inhibitors

Daniela Vullo,1 Claudiu T. Supuran1,2 Andrea Scozzafava,1 Giuseppina De Simone,3 Simona Maria Monti,3 Vincenzo Alterio3* and Fabrizio Carta1*

1

Università degli Studi di Firenze, Dipartimento di Chimica, Laboratorio di Chimica Bioinorganica,

Rm. 188, Via della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy. 2

Università degli Studi di Firenze, Dipartimento Neurofarba, Sezione di Scienze Farmaceutiche e

Nutraceutiche, Polo Scientifico, Via U. Schiff 6, 50019 Sesto Fiorentino, Florence, Italy. 3

Istituto di Biostrutture e Bioimmagini-CNR, via Mezzocannone 16, 80134 Naples, Italy.

Abstract: Herein we report an in vitro kinetic evaluation against the most relevant human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms (I, II, IX and XII) of a small series of lactate dehydrogenase (LDH, EC 1.1.1.27) inhibitors. All compounds contain a primary sulfonamide zinc-binding group (ZBG) substituted with the 2-thio-6-oxo-1,6-dihydropyrimidine scaffold. By means of X-ray crystallographic experiments we explored the ligand-enzyme binding modes, thus highlighting the contribution of the 2-thio-6-oxo-1,6-dihydropyrimidine moiety to the stabilization of the complex.

Keywords: Carbonic Anhydrase (CA); X-Ray crystallography, Sulfonamides, 2-Thio-6-oxo-1,6dihydropyrimidines.

_____ *Corresponding authors. (FC) Phone: +39-055-4573005; Fax: +39-055-4573385; e-mail: [email protected]; [email protected].

(VA)

Phone:

+39-081-2534579;

Fax:

+39-081-2536642;

e-mail:

1. Introduction

Recently some of us reported a series of substituted 2-thio-6-oxo-1,6-dihydropyrimidines as efficient inhibitors of the human Lactate Dehydrogenase (LDH; EC 1.1.1.27), which were shown to constitute interesting lead molecules for new anticancer agents.1 The inhibition of LDH, and in particular of the A-isoform, was demonstrated to be a novel therapeutic approach for the treatment of tumors.2 This enzyme plays a key role in sustaining the aerobic glycolytic metabolism in hypoxic tumors. This condition, also referred as the Warburg’s effect, constitutes a hallmark of this tumor type.3 Specifically, LDH-A stereoselectively converts pyruvate into lactate through a NADH oxidation process.1,2 Low oxygen tension, which as mentioned above is a characteristic feature of many tumors, triggers the expression of LDH-A (and many other proteins) through the hypoxiainducible-factor-1α (HIF-1α) cascade,4 which is also involved in many other signaling pathways.5 Of particular interest is the over-expression of isoforms IX and XII of the zinc enzyme α-carbonic anhydrase (CA, EC 4.2.1.1), which is also dependent on the HIF-1α cascade.6 hCA IX and XII, in analogy to the other 10 catalytically active α-CAs reported in humans, are quite efficient catalysts in promoting the hydration of carbon dioxide as reported in eq.1. 6a, 7

CO2

CA H2O

HCO3

H

eq.1

Such a reaction is essential for the achievement of many biological processes, which include pH homeostasis, respiration, electrolyte secretion, bone resorption, biosynthesis of lipids, nucleic acids and glucose.7-12 pH control is of particular interest in cell biology as slight variations of the intra-/ extracellular pH ratio (pHi/pHe) of up to 0.2 units may affect vital biochemical pathways, thus resulting in normal cells being switched to cancerogenic ones. 13 The role of hCAs in cancer biology, which may be considered as the result of an evolutionary achievement, has been understood in deep details in the last 15 years, after the discovery of the HIF-1α pathway which regulates the overexpression of these two proteins in many hypoxic tumors.14 Specifically the isoforms IX and XII cooperate with many other proteins through complex mechanisms yet to be clarified, and regulate the tumor pH.14 Such events not only guarantee the tumor cells survival in the harsh environment of the hypoxic tumor, but are also essential for cancer sustainment and progression events, by a reprogrammed metabolism in which glycolysis plays a pivotal role.14a, 15 The use of selective hCA inhibitors (CAIs) against the tumor associated isoforms IX and XII was recently validated as an antitumor strategy, both for the management and diagnosis of hypoxic tumors.14a To date the hCA IX monoclonal chimeric antibody Girentuximab (Rencarex ®), and its

iodo radiolabeled derivative (Redectane ®) are present in the market for both the treatment of renal cell carcinoma (RCC) and for imaging purposes. We also reported that small molecule tyrosine kinase inhibitors such as Imatinib (Gleevec®), Nilotinib (Tasigna®) and Pazopanib (Votrient ®) possess in vitro nanomolar (nM) affinities (KIs) against the tumor associated isoforms hCA IX and XII, thus supporting the concept that therapeutic effects of these drugs might be also due to the inhibition on these hCA isoforms in addition to their main protein kinase target. 16, 17 Our research interests towards the identification of novel chemical classes which possess selective inhibition profiles against various CAs, drove us to the discovery in recent years of the coumarins and their congeners,18 the sulfocoumarins,19 the phenols,20 the polyamines,21 the dithiocarbamates (DTCs),22 the xanthates (XATs) and thioxanthates (TXTs) as novel classes of CAIs.23 They were demonstrated to possess different inhibition mechanisms, by means of kinetic as well as X-ray crystallographic experiments on adducts with various hCA isoforms.18-23 Simultaneously, we performed synthetic derivatizations on known chemical classes acting as CAIs, with the aim to better address hCA selectivity issues.24 Our efforts led to advance of the sulfonamide derivative SLC-0111 into phase I clinical trials for the treatment of metastatic solid tumors in patients showing positive response to hCA IX and XII markers. 25 On pursuing our investigations on selective small molecule CAIs bearing the sulfonamide as zinc binding group (ZBG), we turned our attention on the LDH inhibitors of the type A-E, recently reported (Figure 1). 1b

Figure 1. Chemical structures of SLC-0111 and sulfanilamide substituted 2-thio-6-oxo-1,6dihydropyrimidines A-E.1b

All these compounds share with SLC-0111 the N-(4-sulfamoylphenyl)acetamide portion (depicted in blue color in Figure 1), which contains the primary sulfonamide ZBG essential for CA inhibition and the N-acetamide moiety as fragment of the ureido moiety. Our recent investigations on CAIs containing the ureido moiety, including SLC-0111, clearly reported that such a group ensures the inhibitors tails with enough flexibility to adopt the most favorable conformations within the enzyme active site. Such an aspect resulted particularly significant on stabilizing the enzyme-ligand adducts, demonstrating the importance in targeting the hydrophobic/hydrophilic regions of the active site to solve selectivity issues.26 The aim of this study was to explore whether the introduction of the 2-thio-6-oxo-1,6dihydropyrimidine scaffold, a moiety which can be involved in both hydrophobic and hydrophilic interactions, as well as the simple substitution of a nitrogen atom of the ureido group with an sp3 carbon may affect the preferential orientation of the inhibitor tail, thus leading to selective inhibition of the physiologically relevant hCAs. We evaluated in vitro the inhibitory activity of compounds A-E against the physiological relevant isoforms hCA I, II, IX and XII and we investigated their binding modes to hCA II by means of X-ray crystallography.

2. Results and Discussion

2.1. Chemistry SLC-0111 was obtained according to synthetic procedures earlier reported by this group. 27 Compounds A-E were provided by Genentech Inc. (USA) and were either commercially available (A and B) or obtained according to the literature (C-E).1b, 28, 29 Compounds B, C, D and E, which contain a chiral center in the thiopropanamide/thiobutanamide moiety, were used in the subsequent experiments as racemic mixtures.

2.2. Carbonic anhydrase inhibition

All compounds A-E were tested in vitro for their inhibition activity against the physiologically relevant hCA isoforms I, II, IX and XII, by means of the stopped-flow carbon dioxide hydration assay30 and their activities were compared to the standard CAI acetazolamide (AAZ) and the phase I clinical anticancer drug SLC-0111 (Table 1).

Table 1. Inhibition data of human CA isoforms I, II, IX and XII with compounds A-E, AAZ and SLC-0111 by a stopped flow CO2 hydrase assay.30 KI (nM) * Compounds

hCA I

hCA II

hCA IX**

hCA XII

A

59

7.6

8.5

8.3

B

32

6.4

24

24

C

48

5.1

19

7.8

D

83

5.0

9.7

9.2

E

34

6.1

37

9.1

SLC-0111

5080***

960***

45***

4.5***

AAZ

250

12

25

5.7

All data were obtained from 3 different assays, errors  5-10 % of the reported value, CO2 hydrase,

*

stopped-flow assay. **Catalytic domain. *** From ref. 31

As reported above all compounds were good inhibitors of the tested hCA isoforms, with KIs spanning between 83 and 5.0 nM. hCA I resulted the least inhibited one with this series of compounds, although B and E were rather active with KIs of 32 and 34 nM, respectively. Substitution of the fluoro atom in E with a chlorine, to afford C, resulted in a 1.41 fold decrease of the inhibition potency, which was further reduced when no substitution at the same position occurred, as in compound D, with a KI of 83 nM. Interestingly the simple substitution in D of the αthiopropanamide moiety with the α-acetyl one, as in compound A, resulted in a sensible increase of the inhibition potency against hCA I (KI 59 nM). All the compounds tested resulted particularly active in inhibiting hCA II, having KIs lower than the standard AAZ and comprised within a 2.6 nM unit range. Among the tested compounds, those bearing the α-thiopropanamide and the bis/mono-chlorophenyl moieties at position 4 of the 1,6dihydropyrimidine ring (C and D) resulted the most active ones, with KIs of 5.1 and 5.0 nM. Conversely to what observed for hCA I, removal of the methyl pendant in D to afford A, resulted in a slight worsening of the hCA II inhibition activity (KI 7.6 nM). Finally, compounds B and E showed comparable inhibition potencies against hCA II with KIs of 6.4 and 6.1 nM, respectively.

More interesting data were obtained for the tumor associated isoform hCA IX. Compound B was the least potent among the series, with a KI of 24 nM and comparable to the standard AAZ (KI 25 nM). The introduction of both the α-thiobutanamide and the 3’,4’-dichlorophenyl amide moieties in position 4 of the 1,6-dihydropyrimidine scaffold (compound C), resulted in a slight enhancement of the inhibition potency (KI 19 nM). However, the substitution of the 3’-chloro atom with a fluoro, to afford compound E, spoiled the inhibition activity as demonstrated by the higher KI value among the series (37 nM). Better results were obtained for the monochloro substituted derivative of C, i.e., compound D (KI 9.7 nM), and even better for the α-thioacetylderivative A (KI 8.5 nM), which resulted the most potent in the tested series. In analogy to what observed for hCA IX, compound B was the less effective within the compound series in inhibiting the hCA XII isoform (KI 24 nM). Conversely, the 3’,4’-dichloro substituted derivative C was more potent than its monochloro analog D (KIs 7.8 and 9.2 nM, respectively). Interestingly, substitution of the 3’-chloro atom in C with a fluorine, as in compound E, did not significantly affect the inhibition potency against hCA XII (KI 9.1 nM). Finally, removal of the methyl pendant in D to afford A resulted in a 0.9 fold enhancement of the inhibition potency.

2.3 X-Ray crystallography. To identify the molecular basis responsible for the high binding affinity of the investigated compounds against hCAs, we determined the X-ray crystal structure of the hCA II/D adduct at 1.70 Å resolution. Co-crystallization experiments were performed on the racemate of the inhibitor D, and the electron density maps clearly revealed both enantiomers (R) and (S) within the enzyme active site, with a 50% occupancy for each of them (Figure 2).

Figure 2. Active site region of the hCA II/D adduct, showing the A-weighted |2Fo-Fc| map (contoured at 1.0 ) relative to the inhibitor molecule. Enantiomers of compound D are shown in cyan (R) and magenta (S), respectively. The helical region 131-135 is highlighted in blue.

As for all primary sulfonamide CAIs investigated to date,32 in both enantiomers of compound D the SO2NH2 moiety was observed anchored to the zinc ion, in a tetrahedral geometry, through coordination of the nitrogen, in its deprotonated form, to zinc ion. Furthermore, this nitrogen is also involved in a hydrogen bond interaction with the Thr199OG atom. Additionally, the NH backbone of Thr199 established another hydrogen bond with a sulfonamide oxygen atom (Figure 3).

Figure 3. LIGPLOT diagram33 of A) hCA II/D(R) and B) hCA II/D(S) adducts. Inhibitor bonds are shown in violet, protein residue bonds in brown. Hydrogen bonds are green dashed lines. Atoms involved in van der Waals contacts are represented by red semicircles with radiating spokes.

The organic scaffold of the inhibitor did not establish polar interactions within the enzyme cavity, but was involved in a number of van der Waals contacts (Figure 3). The presence of the chiral center within the thiopropanamide moiety did not affect the spatial orientation of the 4-phenyl-1,6dihydropyrimidine tails, as in both enantiomers the terminal ends were perfectly superimposable, and located on the upper border of the active site. This finding can be ascribed to the strong van der Waals contacts established by the tails with residues belonging to the helical region 131-135, namely Phe131 and Gly132 (see Figure 3 for details). Several studies aimed at developing selective CAIs, already identified this region as a hot spot to be targeted in the drug design process, being one

of the most variable sections among the different hCA isoforms (Figure 4).32, 34 Therefore we can reasonably assume that the lower binding affinities showed by compound D against the tumor associated hCA IX and XII may be ascribed to the single point “mutations” at positions 131 and 132. Primary sequence alignments showed the Phe131Val and Gly132Asp mutations for hCA IX, and Phe131Ala and Gly132Ser ones for hCA XII respectively. Further reduction of the binding affinity was observed for hCA I, and again two further residue substitutions involved in inhibitor recognition, namely Thr200His and Val121Ala, may contribute to the overall destabilizing effect (Figure 4).

Figure 4. Multiple sequence alignment of hCA isoforms object of this study. The three catalytic histidines are highlighted in green, residues which in hCA II have been identified as involved in inhibitor recognition are boxed, whereas residues 131 and 132 are highlighted in yellow. The alignment has been performed using Clustal Omega server. 35

Below is reported the structural superposition of compound D with SLC-0111 bound to the hCA II active site (Figure 5). It is evident that the 4-phenyl-1,6-dihydropyrimidine moiety of D is located in a different region of the active site with respect to that occupied by the 4-fluoro-phenyl substituent of SLC-0111. In this region, the bulkier tail of D establishes a higher number of van der Waals contacts with the enzyme, thus explaining the higher affinity for hCA II of this compound with respect to SLC-0111.

Figure 5. Superposition of hCA II/inhibitor adducts: SLC-0111 is reported in green (PDB code 3N4B), while the two enantiomers of D in orange and red. The enzyme is represented as a surfacemodel and residues Phe131 and Gly132 are highlighted in blue.

3. Conclusions

Herein we reported an in vitro inhibition study against several relevant hCAs (isoforms I, II, IX and XII) of a small series of LDH inhibitors which contain the sulfanilamide scaffold substituted with 2thio-6-oxo-1,6-dihydropyrimidine moieties. Interestingly, all the compounds A-E showed strong binding affinities and poor selectivity profiles, with KIs comprised in the low nanomolar (nM) range against the four CA isoforms. The only exception was represented by the catalytically less efficient hCA I which was the least inhibited among the four CAs investigated here. We explored the ligandenzyme binding modes by means of X-ray crystallographic experiments of the hCA II/D adduct at 1.70 Å resolution. As expected, the primary sulfonamide group coordinated the zinc ion, which assumed the classical tetrahedral geometry. More importantly, our experiments proved that the 2thio-6-oxo-1,6-dihydropyrimidine moiety strongly interacts with the hCA II hydrophobic region, and in particular with residues Phe131 and Gly132, thus giving a great contribution to the stabilization of the enzyme-inhibitor complex. Such an observation was also supported by the change in KIs measured against hCA I, IX and XII. Indeed, in the latter cases single point mutations located within this region reduced the enzyme/inhibitor affinity. In conclusion, crystallographic results here reported suggest that sulfanilamides substituted with the 2-thio-6-oxo-1,6dihydropyrimidine moiety could represent ideal scaffolds to generate more selective CA inhibitors.

Indeed, proper substitutions of the 1,6-dihydropyrimidine ring can be utilized to efficiently discriminate the regions 131-135 of different isoforms. These findings can be applied to other classes of CAIs possessing appropriate tails, thus giving the possibility to design selective inhibitors for future development in medicinal chemistry.

4. Experimental protocols

4.1. CA inhibition An Applied Photophysics stopped-flow instrument has been used for assaying the CA catalyzed CO2 hydration activity.30 Phenol red (at a concentration of 0.2 mM) has been used as indicator, working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH 7.5) as buffer, and 20 mM Na2SO4 (for maintaining constant the ionic strength), following the initial rates of the CA-catalyzed CO2 hydration reaction for a period of 10-100 s. The CO2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor at least six traces of the initial 5-10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled-deionized water and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min – 6 h at room temperature (15 min) or 4 °C (6 h) prior to assay, in order to allow for the formation of the E-I complex. Data from Table 1 were obtained after 6 hours incubation of enzyme and inhibitor, as for the sulfocoumarins and coumarins reported earlier. 18-23 The inhibition constants were obtained by non-linear least-squares methods using PRISM 3 and the Cheng-Prusoff equation, as reported earlier, 18-23and represent the mean from at least three different determinations. All CA isofoms were recombinant ones obtained in-house as reported earlier.18-23

4.2.

X-Ray crystallography

The hCA II/D adduct was obtained as previously described.

34b

In detail, a 50-fold excess of the

inhibitor was added to a 0.1 mg/mL enzyme solution in 20 mM Tris-HCl pH 8.0. After incubation overnight at 4 °C, the complex was concentrated to 10 mg/mL. Crystals were obtained at 20 °C using the hanging drop vapor diffusion technique by equilibrating drops containing 1 µL of complex solution and an equal volume of precipitant solution consisting of 1.3 M sodium citrate, 0.1 M TRIS-HCl, pH 8.5, over a reservoirs containing 1 mL of precipitant solution. Crystals appeared after 3 days.

X-ray diffraction data were collected at 100 K, using a copper rotating anode generator developed by Rigaku and equipped with Rigaku Saturn CCD detector. Prior to cryogenic freezing, the crystals were transferred to the respective precipitant solution with the addition of 10 % (v/v) glycerol. Data were indexed, integrated, and scaled using HKL2000.36 Crystal parameters and data collection statistics are reported in Table 2. The phases were determined with CNS37 using as starting model the native structure of hCA II (PDB ID: 1CA2).38 The graphic program O38 was used to view the electron density maps, and the structures were adjusted based on the calculated electron density. Composite simulated-annealing omit maps were used regularly during the building process to verify and correct the models.37 Topology files of the inhibitor were generated using the PRODRG2 server.40 The geometric restraints of the final model were analyzed using PROCHECK.41 The refinement statistics of final model are summarized in Table 2. Coordinates and structure factors were deposited in the Protein Data Bank (accession code 5J8Z).

Acknowledgments: Dr. Peter S. Dragovich at Genentech, Inc., South San Francisco (USA) is gratefully acknowledged for providing compounds A-E. This research was financed by two EU grants of the 7th framework program (Metoxia and Dynano projects to CTS).

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Table 2. Crystal parameters, data collection and refinement statistics. Values in parentheses refer to the highest resolution shell (1.73-1.70 Å). Crystal parameters Space group a (Å) b (Å) c (Å)  (°) Number of independent molecules

P21 42.4 41.4 71.8 104.2 1

Data collection statistics Resolution (Å) Wavelength (Å) Temperature (K) Rmerge (%)a Mean I/(I) Total reflections Unique reflections Redundancy (%) Completeness (%)

50.0-1.70 1.54178 100 6.5 (29.6) 16.1 (2.7) 104026 26271 (1006) 4.0 (1.9) 97.7 (75.1)

Refinement statistics Rfactor (%)b Rfree (%)b RMSD from ideal geometry: Bond lengths (Å) Bond angles (°) Number of protein atoms Number of water molecules Number of inhibitor atoms Average B factor (Å2): All atoms Protein atoms Inhibitor atoms Water molecules Ramachandran plot Residues in most favored regions (%) Residues in additional allowed regions (%) Residues in generously allowed regions (%)

16.2 19.5 0.008 1.6 2104 234 64 12.8 11.3 22.6 22.9 88.2 11.4 0.5

= Σℎ Σ | i(ℎ )−< (ℎ )>|/Σℎ Σ (ℎ ), where (ℎ ) is the intensity of an observation and < (h )> is the mean value for its unique reflection; summations are over all reflections. a

merge

= Σℎkl|| o(ℎ )|−| (ℎ )||/Σℎkl| (ℎ )|, where and are the observed and calculated structure-factor amplitudes, respectively. free is calculated in same manner as factor, except that it uses 5% of the data omitted from refinement. b

factor

Graphical abstract