**3. Influence on arachidonic acid metabolites and the reninangiotensin system**

HBO should be viewed as a factor for increased availability of oxygen as an active molecule in changing vascular function. HBO, CYP450 activity alternations, and arachidonic acid (AA) metabolism are connected in many different pathways. Besides vascular reactivity changes due to epoxidation reactions, Hjelde et al. showed that anti-inflammatory effect of HBO is mediated by reducing expression of cyclooxygenase-2 and reducing the number of intercellular adhesion molecules and therefore reducing adhesion and infiltration of leucocytes [24].

In various aspects of metabolic diseases, evidence from different studies suggests a role for enzymes involved in arachidonic acid (AA) metabolism, including cytochrome P450 (CYP) epoxygenases and soluble epoxide hydrolase (sEH), and their eicosanoid metabolites (epoxyeicosatrienoic acids (EETs)) [25–27]. EETs have been shown to exert beneficial effects on diabetes-related endothelial dysfunction, enhanced cardio protection, and alleviation of diabetic nephropathy. In contrast, CYP4A proteins were upregulated in the livers of mice with genetically induced and diet-induced diabetes [28].

Arachidonic acid in endothelial cell can be metabolized in three different pathways: CYP450 enzymes (omega-hydroxylase and epoxygenase), cyclooxygenase and lipoxygenase, and nonenzymatic degradation of arachidonic acid in the presence of free radicals to isoprostane [29]. Epoxygenase is a cytochrome P450 family of enzymes (primarily CYP2C and CYP2J families), which in the endothelial cell produces 4 epoxyeicosatrienoic acid (EETs) isomers (5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET), of which 14,15-EETs and 11,12-EETs are the most active metabolites [30]. In most cell types and organs, EETs can be present as dihydroxyeicosatrienoic acids (DHETs) [31], which are more stable and less bioactive than EETs. DHETs are produced by sEH hydrolysis of EETs [32]. There is no evidence of EET production in a smooth muscle cell. In a smooth muscle cell, cytochrome P450 ω-hydroxylase promotes the production of 20-hydroxy-eicosatrinoic acid (20-HETE), which is a vasoconstrictor. Cyclooxygenase (COX) is an enzyme existing in two isoformes, COX-1 and COX-2, involved in the synthesis of prostanoid from arachidonic acid (AA). The resulting prostanoids act in contradiction, causing vasodilation (prostaglandin D2, prostaglandin E2, and prostacyclin I2) and vasoconstriction (prostaglandin F2α and thromboxane A2). Hypoxia activates the COX pathway, where mostly prostacyclin, PGI2, is generated. It diffuses into the smooth muscle cell in which it activates the enzyme adenylate cyclase and increases the amount of cyclic adenosine monophosphate (cAMP). cAMP promotes the opening of several types of potassium channels, resulting in hyperpolarization of the smooth muscle membrane with consequent vasodilation [33]. Lipoxygenase is an enzyme that from AA generates 12- and 15-hydroxy eicosatrienoic acids (HETEs) as the major active metabolites in the endothelial cell [29, 34].

synthesis inhibitor. There was no effect of HBO on ANGII reactivity of these aortic ring preparations nor was there a difference in serum concentrations of ANG-(1–7) [3]. mRNA and protein expression of several CYP isoforms that are involved in EET synthesis were also shown to be upregulated in aortic samples of animals, where DM was caused by streptozocin [3]. Both HBO as a treatment and in vitro hyperbaric oxygenation have been shown to change reactivity of rat thoracic aortic ring preparations to certain compounds [20, 48]. It is well known that changes in oxygen availability are crucial in the control of vascular tone, leading to changes in production of, or vessel sensitivity to, vasoconstrictor and vasodilator metabolites of arachidonic acid and nitric oxide (NO) [40, 49, 50]. The production of EETs is known to

CYP P450 3A13 was found to be involved in oxygen sensing, mediating ductus arteriosus constriction to oxygen, together with endothelin-1 [55]. Considering this, along with the interaction of arachidonic acid pathways with nitric oxide pathways in oxygen sensitivity [49], regional differences of arachidonic acid metabolite roles, and various conflicting evidence [49], it is clear that role of CYP450 enzymes in oxygen homeostasis is very complex and may be

In the literature, there are a lot of studies on animal models of diabetes mellitus that confirmed impaired mechanisms of vasodilation and vasoconstriction. Streptozotocin-induced diabetes mellitus in rats demonstrates attenuated vasodilation response to acetylcholine [56, 57]. Experiments on healthy mouse coronary arteries demonstrate that vasodilation to acetylcholine is accomplished 50% by NO and 50% by EDHF. In spontaneously diabetic mouse type II

Unfirer et al. [13] first investigated mechanisms of vasorelaxation in diabetic animal models after HBO exposure. Thoracic aortal rings from SD rats were used to evaluate vasorelaxation responses to acetylcholine after preconstruction with noradrenalin. With NG-nitro-L-arginine methyl ester (L-NAME)-(NOS inhibitor), indomethacin-(COX inhibitor), and N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide (MS-PPOH)-(CYP 450-epoxygenase inhibitor), they investigated which pathway is involved in enhanced vasorelaxation responses in diabetic and healthy rats after HBO exposure. HBO exposure protocol was performed in therapeutic range [58]. DM duration of 6 weeks did not change vasorelaxation response in diabetic group, and after application of inhibitors, results showed that the NO pathway is dominant in macrocirculation. In the diabetic and healthy groups, after HBO exposure, there was partial inhibition of vasorelaxation after NOS inhibition, which indicates that other pathways were included in vasorelaxation mechanisms. MS-PPOH partially blocked vasorelaxation in both HBO groups, which indicates that HBO changes vasorelaxation mechanisms to alternative pathways—enhanced production or sensitivity to EETs. Indomethacin did not inhibit vasorelaxation in any group, so COX pathway did not have influence. These findings were verified with upregulation of eNOS and COX-1 enzymes in the diabetic HBO

[42]. EETs have been recognized to induce vasorelaxation

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current in smooth muscle cells, in addition to others (including pro-angio-

be reduced with a decrease in PO2

genic, anti-inflammatory, and pro-fibrinolytic effects) [51–54].

significant factor mediating the responses to HBO.

**4. Changes in acetylcholine pathways**

(db/db), that ratio is 81% to production of EDHF [12].

and enhance K+

Streptozocin-induced diabetes in rats (a model for type 1 diabetes mellitus) reduces the levels of protective EETs, and the reduced EET levels lead to exacerbation of stroke [35]. Tsai et al. showed impaired endothelium-dependent vasodilation of coronary arterioles caused by reduced CYP activity and EET production due to increased glucose-induced superoxide levels in coronary endothelial cells [36]. EETs might constitute a key link between insulin resistance and endothelial dysfunction [37]. Endothelial dysfunction in diabetes could also be related to the release of vasoconstrictor mediators, e.g., increased production of 20-HETE leading to activation of ROS through an NAD(P)H-dependent pathway. Diabetes alters CYP expression and 20-HETE formation, leading to upregulation of CYP4A isoforms and to elevated levels of 20-HETE [37]. Li et al. also suggested contribution of 20-HETE to endothelial dysfunction in diabetes and other insulin-resistant conditions showing the attenuation of diabetes-induced vascular dysfunction by using the 20-HETE inhibitor HET0016 [38]. Insulin-stimulated vasodilation mediated by the IRS-1/PI3K/AKT/eNOS pathway can be impaired by 20-HETE [39]. Issan et al. associated dysfunction of circulating endothelial progenitor cells and angiogenic capacity with increased levels of CYP-derived 20-HETE in diabetic patients with cardiac ischemia [39]. P450 4A metabolite 20-HETE by vascular tissue is directly dependent on the concentration of oxygen within the normal physiological range of blood and tissue PO2 [40]. It is known that various arachidonic acid metabolites (prostaglandins, EETs, HETEs) and NO are of utmost importance in the mediation of vascular reactions to vasodilators and vasoconstrictors [41–46], including hypoxia and hyperoxia stimuli [46]. In conditions of reduced blood flow, the use of HBO can significantly increase tissue oxygenation. Although all P450 enzymes require molecular oxygen, the majority of them (such as those found in the liver) require only very low PO2 levels for normal activity. Results from our previous study suggest that hyperbaric oxygen increases vascular sensitivity to EETs, instead of significantly increasing EET synthesis [3]. Our studies also show that HBO is a highly effective treatment for stroke even in the presence of long-term untreated diabetes, by inhibition of 20-HETE production [47]. Unfirer et al.'s study showed changes in the dilatation mechanisms in diabetic rats under the influence of hyperbaric oxygenation. It has been shown that hyperbaric oxygenation causes activation of the CYP450 epoxygenase pathway and increased EET production in diabetic animals exposed to HBO [13]. Furthermore, Kibel et al. showed a changed relaxation response to ANG-(1–7) influenced by HBO in healthy and diabetic animals, where they also linked to a changed mechanism and improved relaxation after HBO with CYP450 activation and EET synthesis [3, 11]. HBO was shown to increase relaxation responses to ANG-(1–7) in rat aortic rings of diabetic animals, and this effect was eliminated with the addition of an EET synthesis inhibitor. There was no effect of HBO on ANGII reactivity of these aortic ring preparations nor was there a difference in serum concentrations of ANG-(1–7) [3]. mRNA and protein expression of several CYP isoforms that are involved in EET synthesis were also shown to be upregulated in aortic samples of animals, where DM was caused by streptozocin [3].

Both HBO as a treatment and in vitro hyperbaric oxygenation have been shown to change reactivity of rat thoracic aortic ring preparations to certain compounds [20, 48]. It is well known that changes in oxygen availability are crucial in the control of vascular tone, leading to changes in production of, or vessel sensitivity to, vasoconstrictor and vasodilator metabolites of arachidonic acid and nitric oxide (NO) [40, 49, 50]. The production of EETs is known to be reduced with a decrease in PO2 [42]. EETs have been recognized to induce vasorelaxation and enhance K+ current in smooth muscle cells, in addition to others (including pro-angiogenic, anti-inflammatory, and pro-fibrinolytic effects) [51–54].

CYP P450 3A13 was found to be involved in oxygen sensing, mediating ductus arteriosus constriction to oxygen, together with endothelin-1 [55]. Considering this, along with the interaction of arachidonic acid pathways with nitric oxide pathways in oxygen sensitivity [49], regional differences of arachidonic acid metabolite roles, and various conflicting evidence [49], it is clear that role of CYP450 enzymes in oxygen homeostasis is very complex and may be significant factor mediating the responses to HBO.


**11. MDR/XDR-TB treatment-pipeline agents or compounds in clinical trials and related innovative researches**

Currently, drugs in phase III clinical trials are moxifloxacin, gatifloxacin, and meropenem [72]. Heteronemin, nephalsterol, litosterol, and kahalalides are other interesting compounds which are in pre-clinical stage [72]. Okada M *et al*. conducted a study on granulysin and a new DNA vaccine against MDR/XDR-TB and revealed that agglutinating virus of Japan/Heat-Shock-Protein65DNA+Interleukin-12-12DNA vaccine provided strong therapeutic efficacy in killing MDR/XDR-TB bacilli in mice and monkey models [73]. A recent experiment using MDR-TB monkey models which received normal and genetically altered Bacilli Calmette Gue′rin (BCG) vaccines demonstrated that these 2 groups of monkeys survived well compared to the control group [74]. Another study in XDR-TB mice model showed ability of interleukin-7 to kill the bacilli [74].

with DST of several anti-TB agents, the lack of standardized DST methods for several present and new investigational drugs, and insufficient evidence to link such DST results to patients' treatment outcomes [78]. No DST methods for group 5 and new investigational agents currently exist [78]. Molecular DST for the second-line drugs cannot yet replace phenotypic methods [78]. Collaboration between the national TB control programme, Ministries of Public Health, and the pharmaceutical companies will be required to resolve the limitations of treatment options by the compassionate use of new anti-TB agents [78]. The WHO will be the lead in ensuring that better patient data provide a more robust

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As countries are purchasing and using second-line drugs, the likelihood of misuse and developing of TB-resistant strains increases. Currently, WHO and its partners have reached the phase of expanding MDR-TB control as a component of a comprehensive TB control programme. Launching in 2002, the Global Fund to Fight AIDS, Tuberculosis and Malaria (GFATM) expected that requests for second-line drugs for MDR-TB management should go through the Green Light Committee to prevent their misuse. The number of Green Light Committee-approved MDR-TB control programme is increasing rapidly as a result of main streaming of MDR-TB management into general TB control efforts. Expanding projects and accelerating evidence gathering are essential to further develop international policies. The TBendemic countries themselves and the ability of the technical agencies, as well as the donor

community are the factors of future success to expand MDR-TB control programmes.

10th Zonal Tuberculosis and Chest Disease Centre, Chiang Mai, 10th Office of Disease Pre‐ vention and Control, Chiang Mai, Department of Disease Control, Ministry of Public Health,

[1] The WHO/IUATLD Global Project on Anti-tuberculosis Drug Resistance Surveillance. Anti-tuberculosis drug resistance in the world 1999-2002. Third Global Report.

Communicable Diseases. Geneva : World Health Organization ; 2003.

information for future policy decision [78].

**13. Conclusion**

**Author details**

Thailand, Thailand

**References**

Attapon Cheepsattayakorn\*

Address all correspondence to<EMAIL_ADDRESS>


**Ke-Jie Xiong and Feng-Pei Du \***

Department of Applied Chemistry, College of Science, China Agricultural University, Beijing 100193, China<EMAIL_ADDRESS>
**\*** Correspondence<EMAIL_ADDRESS>Tel.: +86-10-62732507

Received: 21 June 2020; Accepted: 4 July 2020; Published: 7 July 2020

**Abstract:** Two fenclorim derivatives (compounds **6** and **7**) were synthesized by linking active sub-structures using fenclorim as the lead compound. The chemical structures of the two compounds were confirmed by NMR spectroscopy, high resolution mass spectrometry, and X-ray diffraction analysis. Their fungicidal activity against six plant fungal strains was tested. Compounds **6** and **7** both crystallized in the monoclinic system, with a P21/c space group (a = 8.4842(6) Å, b = 24.457(2) Å, c = 8.9940(6) Å, V = 1855.0(2) Å3 , Z = 4) and Cc space group (a = 10.2347(7) Å, b = 18.3224(10) Å, c = 7.2447(4) Å, V = 1357.50(14) Å3 , Z = 4), respectively. The crystal structure of compound **6** was stabilized by C–H···N and C–H···O hydrogen bonding interactions and N–H···N hydrogen bonds linked the neighboring molecules of compound **7** to form a three-dimensional framework. Compound 6 displayed the most excellent activity, which is much better than that of pyrimethanil against *Botrytis cinerea* in vivo. Additionally, compound 6 exhibited greater in vitro activity against *Pseudoperonospora cubensis* compared to that of pyrimethanil. Moreover, compound **7** exhibited strong fungicidal activity against *Erysiphe cichoracearum* at 50 mg/L in vitro, while pyrimethanil did not. Compounds **6** and **7** could be used as new pyrimidine fungicides in the future.

**Keywords:** synthesis; crystal structure; fenclorim; antifungal activity; pyrimidine


**3. Applications of cyclodextrins**

The exact potential of the CDs in the sector of pharmaceutical applications is because of their capability to affect several properties influencing the behavior and therapeutic outcomes of drugs. Cyclodextrins are typically employed to improve the solubility, permeability, stability as well as adverse effects including irritation. Generally, most of these applications are associated to their capability to form inclusion complexes.