- Email: [email protected]
Synthesis and evaluation of 4-(1,3,4-oxadiazol-2-yl)-benzenesulfonamides as potent carbonic anhydrase inhibitors
Journal Pre-proofs Synthesis and evaluation of 4-(1,3,4-oxadiazol-2-yl)- benzenesulfonamides as potent carbonic anhydrase inhibitors Chaofu Yang, Yan Feng, Xu Yang, Mingxia Sun, Zhenwang Li, Xuan Liu, Liang Lu, Xianyu Sun, Jiwen Zhang, Xinhua He PII: DOI: Reference:
S0960-894X(19)30852-2 https://doi.org/10.1016/j.bmcl.2019.126874 BMCL 126874
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
15 November 2019 23 November 2019 27 November 2019
Please cite this article as: Yang, C., Feng, Y., Yang, X., Sun, M., Li, Z., Liu, X., Lu, L., Sun, X., Zhang, J., He, X., Synthesis and evaluation of 4-(1,3,4-oxadiazol-2-yl)- benzenesulfonamides as potent carbonic anhydrase inhibitors, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.126874
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier Ltd.
Synthesis
and
evaluation
benzenesulfonamides
as
of
4-(1,3,4-oxadiazol-2-yl)-
potent
carbonic
anhydrase
inhibitors Chaofu Yang
a,b,
Yan Fengd, Xu Yangb, Mingxia Suna, Zhenwang Lic, Xuan Liub, Liang Lub,
Xianyu Sunc, Jiwen Zhanga, Xinhua Heb* a
College of Chemistry & Pharmacy, Northwest A&F University, Yangling 712100, Shaanxi,
China; b
State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of
Pharmacology and Toxicology, 27 Taiping Road, Haidian district, Beijing 100850, China; c
Department of Pharmacy, College of Animal Science and Technique, Heilongjiang Bayi
Agricultural University, Daqing, China; d College of Pharmacy, Hebei University, Baoding 071002, Hebei, China. Abstract: Human Carbonic anhydrase (hCA) I and II are crucial targets for anti-acute mountain sickness. Twenty-one 4-(1,3,4-oxadiazol-2-yl) benzenesulfonamides were synthesized and screened against these two isoforms. The results illustrated that 5c, 5g, 5h, 5k were more potent against both hCA I and II than clinical drug AAZ. In particular, the value of compound 5c with hCA I (18.08nM) was over 84-fold more than of AAZ with hCA I. The data of docking simulations were also in accord with the tendency of inhibitive activities. Furthermore, compound 6h, the methanesulfonate of 5h, showed better anti-hypoxia activity than AAZ in vivo, making it interesting lead compound. Keywords: benzenesulfonamide derivatives; hCA inhibitors; cytotoxicity; anti-hypoxia; docking simulations
Carbonic anhydrases (CAs) are essential zinc-containing enzymes, which can catalyze the interconversion of CO2 / H2O and HCO3- / H+ ions ion. There are 7 families CAs in nature, but only α-CA exists in humans
1-2.
Up to now, it is found that 16 isoforms of human Carbonic
anhydrases(hCAs) have been found which are distributed in different cells, organelles and tissues and which possess individual kinetic properties
3-5.
The hCA I and II are cytosolic and the
drug targets for the glaucoma, epilepsy and acute mountain sickness 6-9. With the world development and social progress，many people for career or travelling reach
Corresponding author. Email: [email protected] (J. Zhang); [email protected] (X. He)
high altitude area. However, Acute Mountain Sickness (AMS) emerged as a significant medical issue with rapid ascent. The incidence of AMS was over 75% at 4,500 m in reported studies 10. The clinical drugs preventing AMS are Acetazolamide, Methazolamide, Ibuprofen and Dexamethasone (Fig. 1)
11.
However, acetazolamide, a carbonic anhydrase inhibitor (CAI), is the
only FDA approved pharmaceutical prophylaxis for AMS. But its side-effects, such as headache, drowsiness, nausea or vomiting, limited its clinical application12. It’s significant to synthesize potent CA I and CA II inhibitors with few side-effects for AMS preventing and treatment owing to their key roles in AMS pathogenesis 13. Benzsulfonamide group is an ideal building-block for CAs inhibitors design, recently a lot of benzsulfonamide derivatives were reported as CA I, II, IX and XII inhibitors14-17, And it is revealed that the tails of benzsulfonamide analogs derivatives are essential for CAs inhibition and selectivity. Herein, we design benzenesulfonamide derivatives contained 1,3,4-oxadiazole in their hydrophobic tails. Totally, 21 compounds were then synthesized and evaluated of their CA inhibitory activities against hCA I and hCA II, which are relevant to AMS and in the hope of reducing side effects.
O
N N N H
S
O O S NH 2 O
N N N
O S NH 2 O
S
Methazolamide
Acetazolamide
O OH OH
HO H
HO F
O
H
O Ibuprofen
Dexamethasone
Fig. 1. Structure of clinical drugs for the treatment of AMS
O H 2N S O
O
R
N N
Fig. 2. The Molecular Structure of benzenesulfonamide derivatives bearing 1,3,4-oxadiazole The design of benzenesulfonamide derivatives bearing 1,3,4-oxadiazole was divided into three segments (Fig. 2). The first one was benzenesulfonamide head which was recognized as linking zinc and vital amino acids of hCA active center18. The second segment was 1,3,4-oxadiazole.
Lastly, the third fragment was the variable tail that was used to control the hydrophobicity and associated to the activity. General synthesis method of new target compounds(5a-u) is outlined in Scheme 1. For the first step, methyl 4-sulfamoylbenzoate (2) was obtained from 4-sulfamoylbenzoic acid (1) in methanol under refluxing. Then 4-(hydrazinecarbonyl)benzenesulfonamide(3) was achieved by the reaction of methyl 4-sulfamoylbenzoate(2) and hydrazine hydrate. Thirdly, 4-(hydrazinecarbonyl)benzenesulfonamide (3) and different aldehydes were refluxing in ethanol to produce the Schiff Bases (4a-u). The last one, 1,3,4-oxadiazoles was synthesized through the reaction of Schiff Bases with the dehydrating agents (POCl3) in literature19. However, this reaction was intense and may affect the stability of other groups. After many attempts, 4a-u were converted to the corresponding benzenesulfonamide derivatives linking 1,3,4-oxadiazole(5a-u) under the action of NaClO and green light conditions at room temperature in a good yield. The synthesized compounds were characterized by melting point detection, NMR and HRMS analyses (Supplementary data). Scheme 1. Synthesis of benzenesulfonamide derivatives a O H 2N S O
COOH
O H 2N S O
i
O
O H 2N S O
ii
O
1
O HN NH2 3
2 O H 2N S O
iii
O
O H 2N S O
iv
HN N
R
4a-u O H 2N S O
O
v
N
O H 2N S O
O
6h
R
CH3SO3H
N
N N
5h
5a
No. 5h
R
No. N
5o
R O
N
5b
5i
5p
O
O
N
O
5c
5j
5q O
O
O O
5d
R
N N 5a-u
N N
No.
O
5k
5r
O
5e
5l
Cl
5s
5f
N
Cl
5g
5m
5n
Cl
5u
Cl
5t
N
N
Br
aReagents
and conditions: (i) CH3OH, refluxing; (ii) NH2NH2, ethanol, 50 °C; (iii) R-CHO, ethanol, refluxing; (iv) NaClO, green light, r. t.; (v)CH3SO3H, Methanol. The titled compounds 5a-u were screened for their inhibition efficacy of hCA I and hCA II, compared with approved drug acetazolamide (AAZ). The hCA inhibition activity was tested the change in absorbance at 405nm of 4-NPA to 4-nitrophenolate ion20. The results are summarized in Table 1. a) All the evaluated compounds had better inhibitive potential of the hCA I with Ki from 18.08 nM to 232.18 nM than the standard drug AAZ (Ki=1532.01 nM). Among them, 5q had the weakest affinity with Ki=232.18 nM. Four compounds 5c, 5g, 5k and 5r displayed the best inhibitory potential (Ki ranging between 18.08 and 26.01) that 58-fold of AAZ. b) The cytosolic isoform hCA II was moderately inhibited by all the synthesized compounds with Ki in the range of 3.02-31.41nM. It was found that seven compounds 5b, 5l, 5m, 5n, 5o, 5q, 5s were less effective inhibitory potency (Ki>16.68nM) than the reference drug AAZ(Ki=13.52), but the others behaved as better inhibitors than AAZ. In particular, 5c was the best inhibitor with Ki value 3.02 nM. c) From the above results, benzenesulfonamide derivatives containing 1,3,4-oxadiazole were effective inhibitors of hCA I and hCA II. In the same time, the tail fragment significantly affected the inhibitory activity to hCA. When the tail fragment contained benzene or naphthalene moiety, the compounds generally have better biological activity than the others. Furthermore, benzene moiety having the hydrophobic groups methyl, methoxy or chloro group were found to the best inhibitors, such as 5c and 5g. Changing benzene group with a cycloalkane moiety, the inhibitory activity of 5s, 5t and 5u displayed slightly decrease. Interestingly, introduction of polar groups pyridine moiety, it was observed that the compounds 5l-5o demonstrated worse biological activity. Finally, with a simple carboxylate tail, 5q exhibited the worst activity. Table 1 Inhibitory potency data for compounds 5a-u and AAZ against isozymes hCA I and hCA Ⅱ Compound NO.
Ki(nM) Ⅰ
5a 5b 5c 5d
92.20 103.74 18.08 40.28
Ki(nM) b
Ⅱ
Compound NO.
Ⅰ
Ⅱ
7.48 18.85 3.02 10.69
5l 5m 5n 5o
210.44 NT NT 167.23
18.34 19.46 25.05 29.00
5e 5f 5g 5h 5i 5j 5k
110.39 48.15 20.59 45.66 87.30 38.02 26.01
8.65 6.57 5.03 7.03 5.17 7.00 5.15
5p 5q 5r 5s 5t 5u AAZ
100.66 232.18 22.85 90.53 57.09 33.67 1532.01
7.96 31.41 7.50 16.68 9.80 6.84 13.52
NT: Not Tested; b Mean from 3 different assays (errors were in the range of ± 5-10% of the reported values) Moreover, in support of the structure activity relationship (SAR), the molecular docking was performed in Sybyl 2.1.1 by Surflex-Dock model. The representative compounds 5c, 5h, 5i, 5j, 5o, 5q and AAZ were docked with hCA I(PDB entry: 2FW4) and hCA Ⅱ(PDB entry: 3HS4) (Table 2). The scores are positive correlation to the attachment of ligands with the best conformation to receptors. It integrated the calculation results of hydrogen bond, van der Waals force, polar and hydrophobic interaction. The docking scores for the newly synthesized compounds 5c, 5h, 5i, 5j, 5o, 5q and AAZ hCA I (4.3164-6.0242) and hCA II (3.4666-6.9408) were consistent with the sequence of biological activities. In addition, The results indicated that the compounds 5c, 5h and 5j had excelent combination ability to hCA I and hCA Ⅱ . For instance, Fig.3 suggested the compound 5c was capable to form hydrogen bond, arene-arene conjugation and hydrophobic interaction with hCAⅡ. Table 2 The total scores of molecular docking of compounds 5c, 5h, 5i, 5j, 5o, 5q and AAZ within the hCA I and hCA II active site Total score c
Compound NO. 5c 5g 5h 5k 5o 5q AAZ c
hCA I
hCA II
6.0242 6.9534 6.0047 5.5235 5.3042 4.4252 4.3164
6.9408 6.6256 6.5644 6.3969 5.7670 5.7020 3.4666
Total score=-log Kd.
Fig. 3. 3D and 2D interaction diagram of 5c and hCA Ⅱ.
In the process of developing pharmaceutical products, many candidate molecules are abandoned at the stage of subsequent studies. they often relate to poor harmacokinetic and/or toxicological properties. In order to reduce the high risk, ADME/T PREDICTOR 9.5 was used to predict the properties of newly synthesized compounds at PH7.4. As can be seen in Table 3, the titled compounds had high BBB Filter and no Absn Rick, Cyp Rick, ADMET Risk, problems for “rule of five”, Tox Risk except for 5a, 5k, 5l, 5m,5s. But there was some trouble with 5c, 5g, 5h and 5k with high biological activity that the solubility of them was poor. Thus 6h was prepared from 5h and methanesulfonic acid for the purpose of increasing water solubility. Table 3 The ADME/T prediction properties of titled compounds d
d
Compound NO.
S+Sw
BBB Filter
Absn Rick
Cyp Rick
Tox Risk
Rule Of 5
ADMET Risk
5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m 5n 5o 5p 5q 5r 5s 5t 5u AAZ
0.012 0.008 0.007 0.0002 0.008 0.005 0.003 0.009 0.062 0.046 0.001 0.054 0.056 0.032 0.052 0.095 0.242 0.012 0.096 0.039 0.023 3.957
High High High High High High High High High High High High High Low High High High High High High High Low
0.797 1.096 1.397 2.185 0.223 0.761 1.000 0.212 1.000 1.693 1.768 0.046 0.413 0.000 0.83 0.819 1.154 1.000 0.365 0.885 0.881 1.000
0.631 0.406 0.315 1.045 0.712 0.540 0.740 0.498 0.414 0.277 0.930 0.743 0.151 0.721 0.000 0.317 0.000 1.000 0.084 0.365 0.186 0.000
2.722 2.000 2.000 2.000 1.000 1.000 1.000 1.822 1.000 1.000 3.000 3.000 3.000 2.409 1.760 1.433 2.000 2.000 2.775 2.064 2.000 1.000
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
4.150 3.502 3.712 5.230 1.935 2.301 2.740 3.532 2.414 2.971 5.699 3.790 3.564 3.130 2.590 2.570 3.154 4.000 3.224 3.314 3.068 2.000
at PH=7.4, S+Sw value >0.01, the solubility is good in water; When the BBB Filter is high, the compound is easy to pass through the blood-brain barrier; When Absn Rick ≤3.500, Cyp Rick ≤2.500, Tox Risk≤2.000 and ADMET Risk ≤7.500, the compound is acceptable as a candidate drug; Rule of 5 means the problems are against Lipinski rules.
The cytotoxicity of the compounds three selected compounds (5c, 5g, 6h) and AAZ was assayed toward HEK293T healthy embryonic kidney cell line by MTS assay21. All cells were incubated for 48 h with different test compound concentrations (5, 25, 50, 100, 200 uM). The results showed that the cytotoxicity of 5c, 5g, 6h was very small and similar to AAZ. In addition, the cytotoxicity did not increase with concentration (Fig. 4). Next, anti-hypoxia effect of 5c, 5g, 6h and AAZ were test in male mice. At the normobaric hypoxia test, the mice were fed for 3 days with different compound at 400 mg/kg each time, once a day. After the last time for 1.5 hours, the mice were quickly put into 280-285ml wide-mouth bottles that were sealed by the rubber plug with vaseline, in which 10.0 g sodium lime was placed. The survival time of mice was recorded and processed by SPSS 24(Fig. 5). The data displayed that the treatment with 6h and AAZ significantly extended the survival time of Hypoxia mice. It also infered the poor solubility of 5c and 5g in water reduced absorption in vivo.
5c
5g
6h
Fig.4. Cytotoxicity studies of 5c, 5g, 6h and AAZ
AAZ
*
P≤0.05 versus CK; ** P≤0.001 versus CK
Fig. 5. The survival time of mice with selective compounds at 400mg/Kg In summary, 21 benzenesulfonamide derivatives bearing 1,3,4-oxadiazole were designed, synthesized and evaluated of inhibitory activities against hCAⅠand hCAⅡ. They exihibited high potency against both hCA I and II. Additionally, the selective compounds 5c, 5g, 6h and AAZ were tested the cytotoxicity and anti-hypoxia effect. The results showed that the cytotoxicity of 5c, 5g, 6h was very low, and anti-hypoxia ability of 6h was better than AAZ. From the above data, it can be concluded that the benzenesulfonamide derivatives having 1,3,4-oxadiazole are promising lead compounds for development of anti-AMS drugs and need to optimize water solubility. References 1. Krishnamurthy VM, Kaufman GK, Urbach AR, et al. Carbonic anhydrase as a model for biophysical and physical-organic studies of proteins and Protein Ligand binding. Chem. Rev. 2008; 108: 946-1051. 2. Vats L, Sharma V, Angeli A, et al. Synthesis of novel 4-functionalized 1,5-diaryl-1,2,3-triazoles containing benzenesulfonamide moiety as carbonic anhydrase I, II, IV and IX inhibitors. European Journal of Medicinal Chemistry, 2018; 150: 678-686. 3. Falsini M, Squarcialupi L, Catarzi D, et al. 3-Hydroxy-1 H-quinazoline-2, 4-dione as a new scaffold to develop potent and selective inhibitors of the tumor-associated carbonic anhydrases IX and XII, J. Med. Chem. 2017; 60: 6428-6439. 4. Luca LD, Mancuso F, Ferro S, et al. Inhibitory effects and structural insights for a novel series of coumarin-based compounds that selectively target human CA IX and CA XII carbonic anhydrases. Eur. J. Med. Chem. 2018;143: 276-282. 5. Slawinski J, Brzozowski Z, Zolnowska B, et al. Synthesis of a new series of N 4-substituted 4-(2-aminoethyl) benzenesulfonamides and their inhibitory effect on human carbonic anhydrase cytosolic isozymes I and II and transmembrane tumorassociated isozymes IX and XII. Eur. J. Med. Chem. 2014; 84: 59-67.
6. Supuran CT. Carbonic anhydrases: Novel therapeutic applications for inhibitors and activators. Nat. Rev. Drug Discov. 2008, 7, 168-181. 7. Arslan T, Türkoglu E A, Sentürk M, Supuran CT. Synthesis and carbonic anhydrase inhibitory properties of novel chalcone substituted benzenesulfonamides. Bioorganic & Medicinal Chemistry Letters, 2016; 26: 5867-5870. 8. Supuran CT, Scozzafava A. Carbonic anhydrase inhibitors and their therapeutic potential. Expert Opinion on Therapeutic Patents, 2005;10(5): 575-600. 9. Ekinci D, Cavdar H, Talaz O, Sentürk, et al. NO-releasing esters show carbonic anhydrase inhibitory action against human isoforms I and Ⅱ. Bioorganic & Medicinal Chemistry, 201018,3559–3563. 10. Leaf DE.; Goldfarb DS. Mechanisms of action of acetazolamide in the prophylaxis and treatment of acute mountain sickness. J Appl Physiol. 2007; 102: 1313–1322. 11. Supuran CT; Scozzafava A. Carbonic anhydrase inhibitors and their therapeutic potential. Expert Opinion on Therapeutic Patents.2005; 10(5):575-600. 12. Harrison MF, Anderson PJ, Johnson JB, et al. Acute Mountain Sickness Symptom Severity at the South Pole: The Influence of Self-Selected Prophylaxis with Acetazolamide. PLoS One. 2016; 11(2): e0148206. 13. Bozdag M, Altamimi ASA, Vullo D, et al. State of the Art on Carbonic Anhydrase Modulators for Biomedical Purposes. Curr Med Chem. 2019; 26(15): 2558-2573. 14. Zaksauskas A, Capkauskait E, Jezepcikas L, Design of two-tail compounds with rotationally fixed benzenesulfonamide ring as inhibitors of carbonic anhydrases. European Journal of Medicinal Chemistry, 2018; 156: 61-78. 15. Swain B, Digwal CS, Angeli A, et al. Synthesis and exploration of 2-morpholino-4-phenylthiazol-5-yl acrylamide derivatives for their effects against carbonic anhydrase I, II, IX and XII isoforms as a non-sulfonamide class of inhibitors. Bioorganic & Medicinal Chemistry, 2019; 27: 115090. 16.Vats L, Sharma V, Angeli A, et al. Synthesis of novel 4-functionalized 1,5-diaryl-1,2,3-triazoles containing benzenesulfonamide moiety as carbonic anhydrase I, II, IV and IX inhibitors. European Journal of Medicinal Chemistry, 2018, 150: 678-686. 17. Akocak S, Lolak N, Bua S, et al. Synthesis and biological evaluation of novel N,N’-diaryl cyanoguanidines acting as potent and selective carbonic anhydrase II inhibitors. Bioorganic Chemistry, 2018; 77: 245-251. 18. Imran S, Taha M, Ismail NH, et al. Synthesis, biological evaluation, and docking studies of novel thiourea derivatives of bisindolylmethane as carbonic anhydrase II inhibitor. Bioorganic Chemistry, 2015; 62: 83–93. 19. Conole D, Beck TM, Smith MJ, et al. Synthesis and methemoglobinemia-inducing properties of benzocaine isosteres designed as humane rodenticides. Bioorganic & Medicinal Chemistry, 2014; 22:2220-2235. 20. Arslan M; Şentürk M, Fidan İ, et al. Synthesis of 3,4-dihydroxypyrrolidine-2,5-dione and 3,5-dihydroxybenzoic acid derivatives and evaluation of the carbonic anhydrase I and Ⅱ inhibition. J Enzyme Inhib Med Chem, 2015; 30(6): 896–900. 21. Idrees D, Hadianawala M, Mahapatra AD, et al. Implication of sulfonylurea derivatives as prospective inhibitors of human carbonic anhydrase II. International Journal of Biological
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Highlights
A series of benzenesulfonamide derivatives have been synthesized. The titled compounds were screened for hCA I and II in vitro. The selected compounds were evaluated anti-hypoxia activity in vivo. In Silico Docking Simulations were in support of SAR. Declaration of Interest Statement
The authors declare that they have no known competing fnancial interests or personal relationships that could have appeared to influence the work reported in this paper.
Graphical Abstract
Synthesis
and
evaluation
of
Leave this area blank for abstract info.
4-(1,3,4-oxadiazol-2-yl)benzenesulpotent
fonamides
carbonic
as
anhydrase
inhibitors Chaofu Yang a,b, Yan Fengd, Xu Yangb, Mingxia Suna, Zhenwang Lic, Xuan Liub, Liang Lub, Xianyu Sunc, Jiwen Zhang a, Xinhua Heb*
O H 2N S O
O N N 5c
O H 2N S O
O
N
N N 5h Compound 5c with hCA II
O H 2N S O
O N N
N
CH3SO3H
3
S0960-894X(19)30852-2 https://doi.org/10.1016/j.bmcl.2019.126874 BMCL 126874
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
15 November 2019 23 November 2019 27 November 2019
Please cite this article as: Yang, C., Feng, Y., Yang, X., Sun, M., Li, Z., Liu, X., Lu, L., Sun, X., Zhang, J., He, X., Synthesis and evaluation of 4-(1,3,4-oxadiazol-2-yl)- benzenesulfonamides as potent carbonic anhydrase inhibitors, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.126874
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier Ltd.
Synthesis
and
evaluation
benzenesulfonamides
as
of
4-(1,3,4-oxadiazol-2-yl)-
potent
carbonic
anhydrase
inhibitors Chaofu Yang
a,b,
Yan Fengd, Xu Yangb, Mingxia Suna, Zhenwang Lic, Xuan Liub, Liang Lub,
Xianyu Sunc, Jiwen Zhanga, Xinhua Heb* a
College of Chemistry & Pharmacy, Northwest A&F University, Yangling 712100, Shaanxi,
China; b
State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of
Pharmacology and Toxicology, 27 Taiping Road, Haidian district, Beijing 100850, China; c
Department of Pharmacy, College of Animal Science and Technique, Heilongjiang Bayi
Agricultural University, Daqing, China; d College of Pharmacy, Hebei University, Baoding 071002, Hebei, China. Abstract: Human Carbonic anhydrase (hCA) I and II are crucial targets for anti-acute mountain sickness. Twenty-one 4-(1,3,4-oxadiazol-2-yl) benzenesulfonamides were synthesized and screened against these two isoforms. The results illustrated that 5c, 5g, 5h, 5k were more potent against both hCA I and II than clinical drug AAZ. In particular, the value of compound 5c with hCA I (18.08nM) was over 84-fold more than of AAZ with hCA I. The data of docking simulations were also in accord with the tendency of inhibitive activities. Furthermore, compound 6h, the methanesulfonate of 5h, showed better anti-hypoxia activity than AAZ in vivo, making it interesting lead compound. Keywords: benzenesulfonamide derivatives; hCA inhibitors; cytotoxicity; anti-hypoxia; docking simulations
Carbonic anhydrases (CAs) are essential zinc-containing enzymes, which can catalyze the interconversion of CO2 / H2O and HCO3- / H+ ions ion. There are 7 families CAs in nature, but only α-CA exists in humans
1-2.
Up to now, it is found that 16 isoforms of human Carbonic
anhydrases(hCAs) have been found which are distributed in different cells, organelles and tissues and which possess individual kinetic properties
3-5.
The hCA I and II are cytosolic and the
drug targets for the glaucoma, epilepsy and acute mountain sickness 6-9. With the world development and social progress，many people for career or travelling reach
Corresponding author. Email: [email protected] (J. Zhang); [email protected] (X. He)
high altitude area. However, Acute Mountain Sickness (AMS) emerged as a significant medical issue with rapid ascent. The incidence of AMS was over 75% at 4,500 m in reported studies 10. The clinical drugs preventing AMS are Acetazolamide, Methazolamide, Ibuprofen and Dexamethasone (Fig. 1)
11.
However, acetazolamide, a carbonic anhydrase inhibitor (CAI), is the
only FDA approved pharmaceutical prophylaxis for AMS. But its side-effects, such as headache, drowsiness, nausea or vomiting, limited its clinical application12. It’s significant to synthesize potent CA I and CA II inhibitors with few side-effects for AMS preventing and treatment owing to their key roles in AMS pathogenesis 13. Benzsulfonamide group is an ideal building-block for CAs inhibitors design, recently a lot of benzsulfonamide derivatives were reported as CA I, II, IX and XII inhibitors14-17, And it is revealed that the tails of benzsulfonamide analogs derivatives are essential for CAs inhibition and selectivity. Herein, we design benzenesulfonamide derivatives contained 1,3,4-oxadiazole in their hydrophobic tails. Totally, 21 compounds were then synthesized and evaluated of their CA inhibitory activities against hCA I and hCA II, which are relevant to AMS and in the hope of reducing side effects.
O
N N N H
S
O O S NH 2 O
N N N
O S NH 2 O
S
Methazolamide
Acetazolamide
O OH OH
HO H
HO F
O
H
O Ibuprofen
Dexamethasone
Fig. 1. Structure of clinical drugs for the treatment of AMS
O H 2N S O
O
R
N N
Fig. 2. The Molecular Structure of benzenesulfonamide derivatives bearing 1,3,4-oxadiazole The design of benzenesulfonamide derivatives bearing 1,3,4-oxadiazole was divided into three segments (Fig. 2). The first one was benzenesulfonamide head which was recognized as linking zinc and vital amino acids of hCA active center18. The second segment was 1,3,4-oxadiazole.
Lastly, the third fragment was the variable tail that was used to control the hydrophobicity and associated to the activity. General synthesis method of new target compounds(5a-u) is outlined in Scheme 1. For the first step, methyl 4-sulfamoylbenzoate (2) was obtained from 4-sulfamoylbenzoic acid (1) in methanol under refluxing. Then 4-(hydrazinecarbonyl)benzenesulfonamide(3) was achieved by the reaction of methyl 4-sulfamoylbenzoate(2) and hydrazine hydrate. Thirdly, 4-(hydrazinecarbonyl)benzenesulfonamide (3) and different aldehydes were refluxing in ethanol to produce the Schiff Bases (4a-u). The last one, 1,3,4-oxadiazoles was synthesized through the reaction of Schiff Bases with the dehydrating agents (POCl3) in literature19. However, this reaction was intense and may affect the stability of other groups. After many attempts, 4a-u were converted to the corresponding benzenesulfonamide derivatives linking 1,3,4-oxadiazole(5a-u) under the action of NaClO and green light conditions at room temperature in a good yield. The synthesized compounds were characterized by melting point detection, NMR and HRMS analyses (Supplementary data). Scheme 1. Synthesis of benzenesulfonamide derivatives a O H 2N S O
COOH
O H 2N S O
i
O
O H 2N S O
ii
O
1
O HN NH2 3
2 O H 2N S O
iii
O
O H 2N S O
iv
HN N
R
4a-u O H 2N S O
O
v
N
O H 2N S O
O
6h
R
CH3SO3H
N
N N
5h
5a
No. 5h
R
No. N
5o
R O
N
5b
5i
5p
O
O
N
O
5c
5j
5q O
O
O O
5d
R
N N 5a-u
N N
No.
O
5k
5r
O
5e
5l
Cl
5s
5f
N
Cl
5g
5m
5n
Cl
5u
Cl
5t
N
N
Br
aReagents
and conditions: (i) CH3OH, refluxing; (ii) NH2NH2, ethanol, 50 °C; (iii) R-CHO, ethanol, refluxing; (iv) NaClO, green light, r. t.; (v)CH3SO3H, Methanol. The titled compounds 5a-u were screened for their inhibition efficacy of hCA I and hCA II, compared with approved drug acetazolamide (AAZ). The hCA inhibition activity was tested the change in absorbance at 405nm of 4-NPA to 4-nitrophenolate ion20. The results are summarized in Table 1. a) All the evaluated compounds had better inhibitive potential of the hCA I with Ki from 18.08 nM to 232.18 nM than the standard drug AAZ (Ki=1532.01 nM). Among them, 5q had the weakest affinity with Ki=232.18 nM. Four compounds 5c, 5g, 5k and 5r displayed the best inhibitory potential (Ki ranging between 18.08 and 26.01) that 58-fold of AAZ. b) The cytosolic isoform hCA II was moderately inhibited by all the synthesized compounds with Ki in the range of 3.02-31.41nM. It was found that seven compounds 5b, 5l, 5m, 5n, 5o, 5q, 5s were less effective inhibitory potency (Ki>16.68nM) than the reference drug AAZ(Ki=13.52), but the others behaved as better inhibitors than AAZ. In particular, 5c was the best inhibitor with Ki value 3.02 nM. c) From the above results, benzenesulfonamide derivatives containing 1,3,4-oxadiazole were effective inhibitors of hCA I and hCA II. In the same time, the tail fragment significantly affected the inhibitory activity to hCA. When the tail fragment contained benzene or naphthalene moiety, the compounds generally have better biological activity than the others. Furthermore, benzene moiety having the hydrophobic groups methyl, methoxy or chloro group were found to the best inhibitors, such as 5c and 5g. Changing benzene group with a cycloalkane moiety, the inhibitory activity of 5s, 5t and 5u displayed slightly decrease. Interestingly, introduction of polar groups pyridine moiety, it was observed that the compounds 5l-5o demonstrated worse biological activity. Finally, with a simple carboxylate tail, 5q exhibited the worst activity. Table 1 Inhibitory potency data for compounds 5a-u and AAZ against isozymes hCA I and hCA Ⅱ Compound NO.
Ki(nM) Ⅰ
5a 5b 5c 5d
92.20 103.74 18.08 40.28
Ki(nM) b
Ⅱ
Compound NO.
Ⅰ
Ⅱ
7.48 18.85 3.02 10.69
5l 5m 5n 5o
210.44 NT NT 167.23
18.34 19.46 25.05 29.00
5e 5f 5g 5h 5i 5j 5k
110.39 48.15 20.59 45.66 87.30 38.02 26.01
8.65 6.57 5.03 7.03 5.17 7.00 5.15
5p 5q 5r 5s 5t 5u AAZ
100.66 232.18 22.85 90.53 57.09 33.67 1532.01
7.96 31.41 7.50 16.68 9.80 6.84 13.52
NT: Not Tested; b Mean from 3 different assays (errors were in the range of ± 5-10% of the reported values) Moreover, in support of the structure activity relationship (SAR), the molecular docking was performed in Sybyl 2.1.1 by Surflex-Dock model. The representative compounds 5c, 5h, 5i, 5j, 5o, 5q and AAZ were docked with hCA I(PDB entry: 2FW4) and hCA Ⅱ(PDB entry: 3HS4) (Table 2). The scores are positive correlation to the attachment of ligands with the best conformation to receptors. It integrated the calculation results of hydrogen bond, van der Waals force, polar and hydrophobic interaction. The docking scores for the newly synthesized compounds 5c, 5h, 5i, 5j, 5o, 5q and AAZ hCA I (4.3164-6.0242) and hCA II (3.4666-6.9408) were consistent with the sequence of biological activities. In addition, The results indicated that the compounds 5c, 5h and 5j had excelent combination ability to hCA I and hCA Ⅱ . For instance, Fig.3 suggested the compound 5c was capable to form hydrogen bond, arene-arene conjugation and hydrophobic interaction with hCAⅡ. Table 2 The total scores of molecular docking of compounds 5c, 5h, 5i, 5j, 5o, 5q and AAZ within the hCA I and hCA II active site Total score c
Compound NO. 5c 5g 5h 5k 5o 5q AAZ c
hCA I
hCA II
6.0242 6.9534 6.0047 5.5235 5.3042 4.4252 4.3164
6.9408 6.6256 6.5644 6.3969 5.7670 5.7020 3.4666
Total score=-log Kd.
Fig. 3. 3D and 2D interaction diagram of 5c and hCA Ⅱ.
In the process of developing pharmaceutical products, many candidate molecules are abandoned at the stage of subsequent studies. they often relate to poor harmacokinetic and/or toxicological properties. In order to reduce the high risk, ADME/T PREDICTOR 9.5 was used to predict the properties of newly synthesized compounds at PH7.4. As can be seen in Table 3, the titled compounds had high BBB Filter and no Absn Rick, Cyp Rick, ADMET Risk, problems for “rule of five”, Tox Risk except for 5a, 5k, 5l, 5m,5s. But there was some trouble with 5c, 5g, 5h and 5k with high biological activity that the solubility of them was poor. Thus 6h was prepared from 5h and methanesulfonic acid for the purpose of increasing water solubility. Table 3 The ADME/T prediction properties of titled compounds d
d
Compound NO.
S+Sw
BBB Filter
Absn Rick
Cyp Rick
Tox Risk
Rule Of 5
ADMET Risk
5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m 5n 5o 5p 5q 5r 5s 5t 5u AAZ
0.012 0.008 0.007 0.0002 0.008 0.005 0.003 0.009 0.062 0.046 0.001 0.054 0.056 0.032 0.052 0.095 0.242 0.012 0.096 0.039 0.023 3.957
High High High High High High High High High High High High High Low High High High High High High High Low
0.797 1.096 1.397 2.185 0.223 0.761 1.000 0.212 1.000 1.693 1.768 0.046 0.413 0.000 0.83 0.819 1.154 1.000 0.365 0.885 0.881 1.000
0.631 0.406 0.315 1.045 0.712 0.540 0.740 0.498 0.414 0.277 0.930 0.743 0.151 0.721 0.000 0.317 0.000 1.000 0.084 0.365 0.186 0.000
2.722 2.000 2.000 2.000 1.000 1.000 1.000 1.822 1.000 1.000 3.000 3.000 3.000 2.409 1.760 1.433 2.000 2.000 2.775 2.064 2.000 1.000
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
4.150 3.502 3.712 5.230 1.935 2.301 2.740 3.532 2.414 2.971 5.699 3.790 3.564 3.130 2.590 2.570 3.154 4.000 3.224 3.314 3.068 2.000
at PH=7.4, S+Sw value >0.01, the solubility is good in water; When the BBB Filter is high, the compound is easy to pass through the blood-brain barrier; When Absn Rick ≤3.500, Cyp Rick ≤2.500, Tox Risk≤2.000 and ADMET Risk ≤7.500, the compound is acceptable as a candidate drug; Rule of 5 means the problems are against Lipinski rules.
The cytotoxicity of the compounds three selected compounds (5c, 5g, 6h) and AAZ was assayed toward HEK293T healthy embryonic kidney cell line by MTS assay21. All cells were incubated for 48 h with different test compound concentrations (5, 25, 50, 100, 200 uM). The results showed that the cytotoxicity of 5c, 5g, 6h was very small and similar to AAZ. In addition, the cytotoxicity did not increase with concentration (Fig. 4). Next, anti-hypoxia effect of 5c, 5g, 6h and AAZ were test in male mice. At the normobaric hypoxia test, the mice were fed for 3 days with different compound at 400 mg/kg each time, once a day. After the last time for 1.5 hours, the mice were quickly put into 280-285ml wide-mouth bottles that were sealed by the rubber plug with vaseline, in which 10.0 g sodium lime was placed. The survival time of mice was recorded and processed by SPSS 24(Fig. 5). The data displayed that the treatment with 6h and AAZ significantly extended the survival time of Hypoxia mice. It also infered the poor solubility of 5c and 5g in water reduced absorption in vivo.
5c
5g
6h
Fig.4. Cytotoxicity studies of 5c, 5g, 6h and AAZ
AAZ
*
P≤0.05 versus CK; ** P≤0.001 versus CK
Fig. 5. The survival time of mice with selective compounds at 400mg/Kg In summary, 21 benzenesulfonamide derivatives bearing 1,3,4-oxadiazole were designed, synthesized and evaluated of inhibitory activities against hCAⅠand hCAⅡ. They exihibited high potency against both hCA I and II. Additionally, the selective compounds 5c, 5g, 6h and AAZ were tested the cytotoxicity and anti-hypoxia effect. The results showed that the cytotoxicity of 5c, 5g, 6h was very low, and anti-hypoxia ability of 6h was better than AAZ. From the above data, it can be concluded that the benzenesulfonamide derivatives having 1,3,4-oxadiazole are promising lead compounds for development of anti-AMS drugs and need to optimize water solubility. References 1. Krishnamurthy VM, Kaufman GK, Urbach AR, et al. Carbonic anhydrase as a model for biophysical and physical-organic studies of proteins and Protein Ligand binding. Chem. Rev. 2008; 108: 946-1051. 2. Vats L, Sharma V, Angeli A, et al. Synthesis of novel 4-functionalized 1,5-diaryl-1,2,3-triazoles containing benzenesulfonamide moiety as carbonic anhydrase I, II, IV and IX inhibitors. European Journal of Medicinal Chemistry, 2018; 150: 678-686. 3. Falsini M, Squarcialupi L, Catarzi D, et al. 3-Hydroxy-1 H-quinazoline-2, 4-dione as a new scaffold to develop potent and selective inhibitors of the tumor-associated carbonic anhydrases IX and XII, J. Med. Chem. 2017; 60: 6428-6439. 4. Luca LD, Mancuso F, Ferro S, et al. Inhibitory effects and structural insights for a novel series of coumarin-based compounds that selectively target human CA IX and CA XII carbonic anhydrases. Eur. J. Med. Chem. 2018;143: 276-282. 5. Slawinski J, Brzozowski Z, Zolnowska B, et al. Synthesis of a new series of N 4-substituted 4-(2-aminoethyl) benzenesulfonamides and their inhibitory effect on human carbonic anhydrase cytosolic isozymes I and II and transmembrane tumorassociated isozymes IX and XII. Eur. J. Med. Chem. 2014; 84: 59-67.
6. Supuran CT. Carbonic anhydrases: Novel therapeutic applications for inhibitors and activators. Nat. Rev. Drug Discov. 2008, 7, 168-181. 7. Arslan T, Türkoglu E A, Sentürk M, Supuran CT. Synthesis and carbonic anhydrase inhibitory properties of novel chalcone substituted benzenesulfonamides. Bioorganic & Medicinal Chemistry Letters, 2016; 26: 5867-5870. 8. Supuran CT, Scozzafava A. Carbonic anhydrase inhibitors and their therapeutic potential. Expert Opinion on Therapeutic Patents, 2005;10(5): 575-600. 9. Ekinci D, Cavdar H, Talaz O, Sentürk, et al. NO-releasing esters show carbonic anhydrase inhibitory action against human isoforms I and Ⅱ. Bioorganic & Medicinal Chemistry, 201018,3559–3563. 10. Leaf DE.; Goldfarb DS. Mechanisms of action of acetazolamide in the prophylaxis and treatment of acute mountain sickness. J Appl Physiol. 2007; 102: 1313–1322. 11. Supuran CT; Scozzafava A. Carbonic anhydrase inhibitors and their therapeutic potential. Expert Opinion on Therapeutic Patents.2005; 10(5):575-600. 12. Harrison MF, Anderson PJ, Johnson JB, et al. Acute Mountain Sickness Symptom Severity at the South Pole: The Influence of Self-Selected Prophylaxis with Acetazolamide. PLoS One. 2016; 11(2): e0148206. 13. Bozdag M, Altamimi ASA, Vullo D, et al. State of the Art on Carbonic Anhydrase Modulators for Biomedical Purposes. Curr Med Chem. 2019; 26(15): 2558-2573. 14. Zaksauskas A, Capkauskait E, Jezepcikas L, Design of two-tail compounds with rotationally fixed benzenesulfonamide ring as inhibitors of carbonic anhydrases. European Journal of Medicinal Chemistry, 2018; 156: 61-78. 15. Swain B, Digwal CS, Angeli A, et al. Synthesis and exploration of 2-morpholino-4-phenylthiazol-5-yl acrylamide derivatives for their effects against carbonic anhydrase I, II, IX and XII isoforms as a non-sulfonamide class of inhibitors. Bioorganic & Medicinal Chemistry, 2019; 27: 115090. 16.Vats L, Sharma V, Angeli A, et al. Synthesis of novel 4-functionalized 1,5-diaryl-1,2,3-triazoles containing benzenesulfonamide moiety as carbonic anhydrase I, II, IV and IX inhibitors. European Journal of Medicinal Chemistry, 2018, 150: 678-686. 17. Akocak S, Lolak N, Bua S, et al. Synthesis and biological evaluation of novel N,N’-diaryl cyanoguanidines acting as potent and selective carbonic anhydrase II inhibitors. Bioorganic Chemistry, 2018; 77: 245-251. 18. Imran S, Taha M, Ismail NH, et al. Synthesis, biological evaluation, and docking studies of novel thiourea derivatives of bisindolylmethane as carbonic anhydrase II inhibitor. Bioorganic Chemistry, 2015; 62: 83–93. 19. Conole D, Beck TM, Smith MJ, et al. Synthesis and methemoglobinemia-inducing properties of benzocaine isosteres designed as humane rodenticides. Bioorganic & Medicinal Chemistry, 2014; 22:2220-2235. 20. Arslan M; Şentürk M, Fidan İ, et al. Synthesis of 3,4-dihydroxypyrrolidine-2,5-dione and 3,5-dihydroxybenzoic acid derivatives and evaluation of the carbonic anhydrase I and Ⅱ inhibition. J Enzyme Inhib Med Chem, 2015; 30(6): 896–900. 21. Idrees D, Hadianawala M, Mahapatra AD, et al. Implication of sulfonylurea derivatives as prospective inhibitors of human carbonic anhydrase II. International Journal of Biological
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Highlights
A series of benzenesulfonamide derivatives have been synthesized. The titled compounds were screened for hCA I and II in vitro. The selected compounds were evaluated anti-hypoxia activity in vivo. In Silico Docking Simulations were in support of SAR. Declaration of Interest Statement
The authors declare that they have no known competing fnancial interests or personal relationships that could have appeared to influence the work reported in this paper.
Graphical Abstract
Synthesis
and
evaluation
of
Leave this area blank for abstract info.
4-(1,3,4-oxadiazol-2-yl)benzenesulpotent
fonamides
carbonic
as
anhydrase
inhibitors Chaofu Yang a,b, Yan Fengd, Xu Yangb, Mingxia Suna, Zhenwang Lic, Xuan Liub, Liang Lub, Xianyu Sunc, Jiwen Zhang a, Xinhua Heb*
O H 2N S O
O N N 5c
O H 2N S O
O
N
N N 5h Compound 5c with hCA II
O H 2N S O
O N N
N
CH3SO3H
3