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Design and Synthesis of Novel Non Steroidal AntiInflammatory Drugs and Carbonic Anhydrase Inhibitors Hybrids (NSAIDs-CAIs) for the Treatment of Rheumatoid Arthritis Silvia Bua, Lorenzo Di Cesare Mannelli, Daniela Vullo, Carla Ghelardini, Gian Luca Bartolucci, Andrea Scozzafava, Claudiu T Supuran, and Fabrizio Carta J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01607 • Publication Date (Web): 11 Jan 2017 Downloaded from http://pubs.acs.org on January 11, 2017
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Design and Synthesis of Novel Non Steroidal Anti-Inflammatory Drugs and Carbonic Anhydrase Inhibitors Hybrids (NSAIDs-CAIs) for the Treatment of Rheumatoid Arthritis
Silvia Bua,1 Lorenzo Di Cesare Mannelli,2 Daniela Vullo,3 Carla Ghelardini,2 Gianluca Bartolucci,1 Andrea Scozzafava,3 Claudiu T. Supuran1* and Fabrizio Carta1*
1
University of Florence, NEUROFARBA Dept., Sezione di Scienze Farmaceutiche, Via Ugo Schiff
6, 50019 Sesto Fiorentino (Florence), Italy 2
Dept. of Neuroscience, Psychology, Drug Research and Child Health - NEUROFARBA -
Pharmacology and Toxicology Section, University of Florence, Florence, Italy. 3
University of Florence, Polo Scientifico, Laboratorio di Chimica Bioinorganica, Rm. 188, Via
della Lastruccia 3, 50019 Sesto Fiorentino (Florence), Italy.
Keywords: Carbonic Anhydrase Inhibitors (CAIs), Non Steroidal Anti-Inflammatory Drugs (NSAIDs), Metalloenzymes, Rheumatoid Arthritis (RA), Hybrids.
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Abstract.
We report the synthesis of a series of hybrid compounds incorporating 6- and 7-substituted coumarins (carbonic anhydrase, CA inhibitors) and clinically used NSAIDs (indomethacin, sulindac, ketoprofen, ibuprofen, diclofenac, ketorolac, etc., cyclooxygenase inhibitors) as agents for the management of rheumatoid arthritis (RA). Most compounds were effective in inhibiting the RA over-expressed hCA IX and XII, with KIs in the low nanomolar-subnanomolar ranges. The antihyperalgesic activity of such compounds was assessed by means of the paw-pressure and incapacitance tests using an in vivo RA model. Among all tested compounds, the 7-coumarine hybrid with ibuprofen showed potent and persistent antihyperalgesic effect, up to 60 min after administration.
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Introduction. Rheumatoid arthritis (RA) is a chronic and systemic inflammatory disease caused by a faulty autoimmune response, which primary affects the lining of the joints, thus causing erosion of the cartilage, bone damage and joints deformity at the latter stages. The dimension as well as the impact of RA in the society are well reported within the Rochester Epidemiology Project.1 Data showed that in the period 1995 – 2007, each year about 4.1 % of the American population was diagnosed with RA. The incidence of the disease was strictly age dependent been present in 0.9 % among patients aged 18-34 years, 5.4 % among those aged over 85 years, with the peak (8.9 %) comprised in the population aged between 65-74 years. Data analysis of the same period among gender showed that the incidence rates increased up to 2.5 % each year in women, conversely a decrease of about 0.5 % was observed in men.1 RA symptoms have high impacts on the life quality of the affected patients, which are progressively unable to carry out activities in every domain of their lives such as work, social relations and leisure.2 State-of-the art on RA pharmacological treatments include two main classes: i) drugs acting to slow or stop the course of the disease and to inhibit the joints tissutal damages and ii) those acting to ease the symptoms.3,4 The first include the Disease-Modifying-Antirheumatic-Drugs (DMARDs), biologics, JAK-inhibitors and corticosteroids, the latter are represented by the Nonsteroidal-AntiInflammatory-Drugs (NSAIDs). Usually a therapeutic RA protocol accounts for the combination of both drug classes along with the recommendation to conduct proper physical activities, with the intent to tackle the symptoms as well as the progression of the pathology.3,4 Despite recent progresses in RA treatment, there is still no effective cure. Many research efforts are constantly reported in the literature, thus offering innovative diagnostic tools, new biological targets and a multitude of potential drug lead compounds.5,6 Recently, we reported that human (h) carbonic anhydrases (CAs, EC 4.2.1.1) isoforms IX and XII are over-expressed in inflamed synovium of patients affected from juvenile idiopathic arthritis ACS Paragon Plus Environment
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(JIA), which is the most common RA disease in pediatric age.7 Previous studies also showed abnormal expressions of the hCAs I, III and IV in specimens of RA affected patients, but only antibodies against these isoforms have been detected, withour measurements of the enzymatic activity in the synovium of the affected patients.
8-11
On the other hand, in the above-mentioned
study,7 we have demonstrated overexpression of CA IX/XII (by using RT-PCR) as well as an increased enzymatic activity due to these isoforms in the samples from JIA patients. In order to better clarify the physio-/pathological role of CAs in RA, we kept turning our attention towards the carbon dioxide hydration reaction (CO2 + H2O ↔ HCO3– + H+), which is the main transformation catalyzed by these enzymes.12 According to the equilibrium reaction, CAs over-expression increases the ionic species concentrations. Since bicarbonate ions are vital building blocks in cells life and are quickly recycled, a local extracellular acidosis is established. The dependence between low-pH values with the intensity of inflammation processes as well as their related pain symptoms, were demonstrated in RA affected patients.13,14 In addition tissue acidosis was found to be detrimental for both the humoral and cellular immunity processes.15 Considering thus the interplay between various CA isoforms and the arthritis-like diseases, we attempt here to target several of the enzymes presumably involved in this condition by dual inhibitors incorporating both a CA-binding moiety (of the coumarin type) and a cyclooxygenase inhibitor of the NSAID type, the most widely used pain-relief medication to date.16 Herein we report the design, synthesis of low molecular weight hybrid compounds containing a selective hCA IX and/or XII inhibitor head, such as the coumarin one,17 linked through a physiological cleavable linker to a commercially available, clinically used NSAID tail, of the carboxylic acid type cyclooxygenase inhibitor (indomethacin, sulindac, ketoprofen, ibuprofen, diclofenac, ketorolac, etc.). The ability of such compounds to inhibit the relevant hCAs activity as well as to reduce the pain symptoms were assessed by mean of in vitro kinetics and in vivo RA model respectively. Our scope was not to investigate cyclooxygenase inhibition role in RA with the hybrid compounds, which is well documented for
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decades, but to show that CA IX/XII inhibition may represent an additional anti-inflammatory mechanism, poorly investigated at this moment for the management of RA.
Results and Discussion.
Compounds Design and Synthesis. The drug design rationale of this project is based upon the synthesis of small molecular hybrids containing a selective hCA IX (XII) inhibitor head linked through a physiological cleavable linker to a carboxylic acid NSAID (COX inhibitor) tail in a 1:1 ratio (Figure 1). As mentioned above, we do not expect COX inhibitory activity of our compounds once that the COOH moiety essential for this COX inhibition has been derivatized.17d
Figure 1. Rational design of the hybrids herein reported.
Since the 6- and 7-substituted coumarins are usually reported selective inhibitors of the hCA IX/XII over the other isoforms,
18
we planned our hybrids up on these moieties and thus ensuring they are
committed with our recent findings on hCA IX and XII over-expression in synovium specimen of JIA affected patients.
7
For the scope of this work we considered a simple 2 carbon linear unit
spacer connecting the hCA IX (XII) inhibitor head and the NSAID terminal section. In particular,
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we focused on the ethanolamine fragment, which allows us: i) to insert a physiologically cleavable moiety, such as the amide, by making use of the NSAID carboxylic function; ii) to install the biologically compatible and not cleavable ether moiety, thus virtually ensuring a single break point on the hybrid; iii) all needed synthetic intermediates are easily accessible by means of efficient procedures. As reported in scheme 1, the 6- and 7-coumarin substituted hybrids 4a-h and 5a-h respectively, were obtained by coupling the commercially available NSAIDs 1a-h, by means of their Nhydroxysuccinamide (NHS) activated esters 2a-h, with the in situ liberated amines 3a and b (Scheme 1).
R
O
EDCI.HCl NHS
O OH
O
O R
DMF dry
CF3COO O
N O
1a-h
O
N DMF dry
H3N
O
O O NH R
3a=6-O-alchil 3b=7-O-alchil
2a-h
a= Indometacin b= Sulindac c= Ketoprofen d= Ibuprofen
O
O
4a-h=6-O-alchil 5a-h=7-O-alchil
e= Dichlofenac f= Flurbiprofen g= Ketorolac h= Naproxen
Scheme 1. General synthetic scheme of hybrid compounds 4a-h and 5a-h.
The key intermediates 3a,b were obtained by means of Williamson’s etherification of the commercially available 6-and 7-hydroxycoumarins with the Boc-protected 1-bromoethylamine 6, followed by deprotection with trifluoroacetic acid (TFA) (Scheme 2). O
O
HO
Br
H N
O O
O
K2CO3/DMF 60°C
O
O N H
O
O
TFA
O DCM 0°C
H3N
O
CF3COO 6-OH/7-OH Coumarin
6
7a,b
Scheme 2. Synthesis of the key intermediates 3a,b.
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3a,b
O
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All final compounds (Figure 2) as well as their intermediates were characterized by means of 1H, 13
C,
19
F-NMR spectroscopy, and HRMS and were >95% pure, as determined by HPLC (see the
Experimental Protocols for details).
O
O
O
NH N
O
HN
O
NH
O
4c-Ketoprofen
O
S O
4a-Indometacin
4b-Sulindac
O
O
O
O
O
O
4d-Ibuprofen
O
O O
O O
HN
O
F
NH O
NH Cl
Cl
O
O
O
O
O
O
O
NH
O
Cl
O
O
F O
O
O
N O
4f-Flurbiprofen
NH
NH
4g-Ketorolac
4e-Dichlofenac
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(S) O
O 4h-Naproxen
O
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O
O
O
O
F O
NH N
O
O HN
O
NH
O
5c-Ketoprofe n
O
O
O
S O
5a-Indometacin
O
O
O
O
O
O
NH
O
Cl
O
O
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5b-Sulindac
O
O
O
O
O
O
5d-Ibuprofen
O
O
O
O
O HN
O
F
N
NH O
NH Cl
O
5f-Flurbiprofen
Cl
NH
NH
(S) O
O
5g-Ketorolac
5h-Naproxen
5e-Dichlofenac
Figure 2. Structures of the compounds 4a-h and 5a-h.
Carbonic anhydrase inhibition.
All synthesized compounds, 4a-h and 5a-h, were tested in vitro for their inhibitory properties against the physiological relevant hCA isoforms (I, II, IV, VII; IX and XII) by means of the stopped-flow carbon dioxide hydration assay,19 and their activities were compared to the standard CAI acetazolamide (AAZ) (Table 1).
Table 1. hCA I, II, IV, VII, IX, and XII inhibition data with compounds 4a-h and 5a-h by a Stopped-Flow, CO2 Hydrase Assay. 19 KI (nM)*
4a
hCA I
hCA II
hCA IV
hCA VII
hCA IX**
hCA XII
>100
>100
2.6
>100
31.3
59.1
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4b
>100
>100
7.5
>100
30.8
81.7
4c
>100
>100
4.4
>100
54.7
>100
4d
>100
>100
0.44
>100
>100
>100
4e
>100
>100
5.6
>100
28.9
92.6
4f
>100
>100
0.81
>100
23.5
5.9
4g
>100
>100
0.79
>100
31.8
>100
4h
>100
>100
0.73
>100
>100
>100
5a
>100
>100
8.3
>100
20.1
6.5
5b
>100
>100
9.0
>100
27.9
7.7
5c
>100
85.4
9.5
>100
>100
57.8
5d
>100
>100
9.1
>100
>100
39.0
5e
>100
>100
9.1
>100
89.7
80.8
5f
>100
>100
8.8
>100
>100
>100
5g
>100
>100
9.4
>100
>100
>100
5h
>100
>100
9.8
>100
>100
80.7
12
74.2
2.5
25.1
5.7
AAZ 250
*
Means from three different assays. Errors were within ±5−10% of the reported values (data not
shown).**Catalytic domain.
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Overall, the data reported in Table 1 clearly showed that all compounds were ineffective in inhibiting the highly abundant cytosolic hCA I and II as well as the central nervous system (CNS) expressed hCA VII (KIs >100 nM). The only exception was represented from the coumarine substituted at 7-position with Ketoprofen (5c), which was a high nanomolar hCA II inhibitor (KI 85.4 nM). The following Structure-Activity-Relationships (SARs) for the remaining hCA isoforms are reported:
i)
The membrane bound hCA IV, which is of interest since it seems to be over-expressed in
RA (at least antibodies against this isoform were reported in the RA patients samples), 11 was highly inhibited by the compounds included in this study. All compounds showed KIs in the low nanomolar range and comprised between 0.44-9.8 nM, thus significantly more potent when compared to the standard CAI Acetazolamide (KI 74.2 nM). The 6-substituted coumarins (4a-h) were 1.2 to 20.7 fold more potent than the 7-substituted ones (5a-h) in inhibiting the hCA IV isoform.
ii)
Interesting data were obtained for the tumor associated 12 and RA over-expressed 7 hCA IX
isoform, as the 6-substituted compounds 4a, b and 4e-g showed KIs of 23.5 and 31.8, thus comparable to the standard CAI AAZ (KI 25.1 nM). The introduction at the 6-position of the coumarine scaffold of either Ibuprofen (4d) or Naproxen (4h) determined loss of the inhibitory activity (KIs > 100 nM). Within the 7-substituted series, only the compounds 5a and b were effective inhibitors of hCA IX (KIs 20.1 and 27.9 nM respectively), whereas the Dichlofenac counterpart (5e) was a high nanomolar inhibitor (KI 89.7 nM). All other compounds resulted ineffective, thus demonstrating that the substitution position at the coumarine ring resulted in potent effects on the inhibition potencies of these compounds.
iii)
The second RA relevant hCA isoform (i.e. XII) 12 was potently inhibited by the 6-coumarin
substituted Flurbiprofen derivative 4f (KI 5.9 nM), with a potency comparable to the standard CAI
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AAZ (5.7 nM). The introduction at the same position of the coumarin scaffold of all the NSAIDs herein considered, determined either decrease (4a,b,e) or loss (4c,d,g and h) of the hCA XII inhibition potency (see data in Table 1). Swap of the NSAID moieties from the 6- to the 7-position of the coumarin ring determined a significant increase of the inhibition potency of the derivatives 5a-d with KIs comprised in the low-medium nM range (Table 1). Noteworthy the Indomethacin and the Sulindac derivatives (5a, b) were particularly active in inhibiting the hCA XII, with KIs of 6.5 and 7.7 nM respectively, thus comparable to the standard CAI AAZ (5.7 nM). Conversely, introduction of the Dichlofenac and Naproxen moieties at the 7-position of the coumarin scaffold (5e and h) determined a slight increase of the inhibition potencies with KIs of 80.8 and 92.6 nM respectively. Finally the inhibition activities of the Ketorolac 5g and Flurbiprofen 5f derivatives resulted unvaried and worsened compared to their 6-substituted counterparts (Table 1).
Pain relieve efficacy tests
The pain relieve efficacy of compounds 4a, 4f and 5a-g (10 mg kg-1 p.o.) was evaluated in a rat model of rheumatoid arthiritis induced by intra-articular (i.a.) Complete Freund’s Adjuvant (CFA) injection. The effects of compounds herein reported were compared with 100 mg kg-1 ibuprofen, the classical NSAID administered at the active dosage usually employed in this model. hypersensitivity to a mechanical noxious stimulus (Paw pressure test) is reported in figure 3.
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Figure 3. Acute pain relieving effect of 4a, 5b, 5d, 5f, 5g and ibuprofen in a rat model of rheumatoid arthritis induced by CFA i.a. injection. Compounds were administered p.o. at the dose of 10 mg kg-1 while ibuprofen (p.o.) at the dose of 100 mg kg-1. The Paw pressure test was performed to evaluate the hypersensitivity to noxious mechanical stimuli. Measurements were performed on day 14 after CFA intra-articular injection. Compounds were suspended in 1% CMC and orally administered, evaluating the pain threshold over time. Control animals were treated with vehicle. The values represent the mean of 8 rats performed in 2 different experimental sets. ^^P
