The drug likeness analysis of anti-inflammatory clerodane diterpenoids

Inflammation is an active defense response of the body against external stimuli. Long term low-grade inflammation has been considered as a deteriorated factor for aging, cancer, neurodegeneration and metabolic disorders. The clinically used glucocorticoids and non-steroidal anti-inflammatory drugs are not suitable for chronic inflammation. Therefore, it’s urgent to discover and develop new effective and safe drugs to attenuate inflammation. Clerodane diterpenoids, a class of bicyclic diterpenoids, are widely distributed in plants of the Labiatae, Euphorbiaceae and Verbenaceae families, as well as fungi, bacteria, and marine sponges. Dozens of anti-inflammatory clerodane diterpenoids have been identified on different assays, both in vitro and in vivo. In the current review, the up-to-date research progresses of anti-inflammatory clerodane diterpenoids were summarized, and their druglikeness was analyzed, which provided the possibility for further development of anti-inflammatory drugs.


Background
Inflammatory diseases include a vast array of disorders and defense reaction of organisms against external stimulations that are characterized by inflammation symptoms, such as allergy, autoimmune diseases, asthma, glomerulonephritis, hepatitis, inflammatory bowel disease (IBD), reperfusion injury and transplant rejection [1][2][3]. Long term low-grade inflammation has been considered to play a deteriorated role in many diseases, such as aging, cancer, metabolic disorders, and neurodegeneration [4][5][6][7]. The occurrence of chronic diseases has triggered prolonged inflammation that induces the expression of robust pro-inflammatory mediators and cytokines [8,9], which lead to the pathogenesis of inflammation-associated chronic diseases. Tumor necrosis factor (TNF)-α is one of the most potent pro-inflammatory cytokines and signals [10,11]. Through binding to its receptors, TNFR1 and TNFR2, TNF-α plays critical roles in apoptosis, cell proliferation and immune responses [11,12]. Interleukin-1β (IL-1β) is one of the inflammatory markers belonging to the IL-1 family of cytokines [13,14]. IL-6 and IL-12 display a pro-inflammatory action via stimulating IL-1 secretion [15]. IL-10, as the most important anti-inflammatory cytokine, represses pro-inflammatory responses and limits inflammation-induced tissue disruptions [16,17]. Prostaglandin E2 (PGE2) is derived from arachidonic acid produced by cyclooxygenase (COX)-1 and/or COX-2 [18], which is a principal mediator of inflammation in diseases such as rheumatoid arthritis and osteoarthritis [19]. Nitric oxide (NO) is free radical acting as a cellular signaling molecule in inflammation process [20,21]. NO is synthesized from I-arginine through the action of nitric oxide synthase (NOS) family [22], which has been associated with the pathogenesis and progression of inflammatory-related diseases [20,23]. Macrophages exit throughout the body, which play important roles in tissue development, inflammation and anti-pathogenic defense [24][25][26]. A variety of biologically active molecules related to the beneficial and harmful consequences of inflammation are produced by macrophages. Therefore, therapeutic intervention for

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Chinese Medicine macrophages and their related products has attracted widespread attention in the treatment of inflammatory diseases. [24,27].
Currently, the clinically used inflammation-treating drugs include non-steroidal anti-inflammatory drugs (NSAID) and glucocorticoids (SAID). Both classes of anti-inflammatory drugs could induce unpleasant side effects, and not suitable for chronic use [28][29][30]. Thus, it's urgent to discover and develop new effective and safe drugs to alleviate inflammation. Tons of scientific evidence has indicated that natural medicines represent a big treasure to develop potential therapeutic agents for inflammation-related diseases [31,32]. The clerodane diterpenoids are a large class of naturally occurring bicyclic diterpenoids (Fig. 1), found in lots of plant species, especially the families of Labiatae, Euphorbiaceae and Verbenaceae [33]. More and more research interests have been attracted in recent years for their wide and potent biological activities [34]. Salvinorin A, a clerodane diterpenoid isolated from Salvia divinorum, was found as an agonist of the κ-opioid receptor. It is the first opioid receptor agonist without nitrogen and non-alkaloid hallucinogen. Many clinical trials results support the physiological effects of salvinorin A is mediated through κ-opioid receptor-serotonin-2A pathway [35][36][37][38][39][40]. A lot of studies have disclosed the potential anti-inflammatory activity of clerodane diterpenoids. Herein, an up-to-date and comprehensive review of clerodane diterpenoids with anti-inflammatory property was provided, and their drug-likeness was analyzed through SwissADME, which enable further development of clerodane diterpenoids for the treatment of inflammation related diseases.

Clerodane diterpenoids with anti-inflammatory property
Data was collected from Web of Science, Google Scholar, Scopus, and Pubmed via using the keywords clerodane diterpenoid and inflammation, and a total of 65 clerodane diterpenoids were found with anti-inflammatory property (Fig. 2). Either in vitro or in vivo bioassays have been used to determine the antiinflammatory activity of clerodane diterpenoids. To better organize the review, the clerodane diterpenoids were introduced based on the anti-inflammatory bioassays (Table 1).
Dextran-induced edema induces an anaphylactoid reaction, which is widely used as an acute experimental model of inflammation [86]. Trans-crotonina showed inhibitory effect at the rate of 31.9% in dextran-induced edema [80]. Histamine is known to increase vascular permeability [87]. Histamine-induced edema is widely used to screen anti-inflammatory drugs [88]. Trans-crotonina showed inhibitory effect in histamine-induced edema [80].
Arachidonic acid-derived LTs are highly related to inflammatory diseases such as asthma, allergic rhinitis and cardiovascular disease [89]. Calcium ionophore A23187-stimulated peritoneal macrophages (PM) were used as an in vitro model to evaluate SA-indcued production of LT [90]. SA concentration-dependently inhibited LTB4 biosynthesis in isolated PM, and suppressed myeloperoxidase activity, cell infiltration, LTC4 production, and vascular permeability, in the peritoneal cavity, but not the production of PGE2 [79].
In summary, a total of 65 clerodane diterpenoids were reported with potential anti-inflammatory property. While, their anti-inflammatory activity was only evaluated on either in vitro cellular models or in vivo animal models, no clinical study was carried out till now. In the future, clinical trials must be performed to authenticate the therapeutic effect of clerodane diterpenoids in alleviating inflammatory responses.

Comparison of drug-likeness of anti-inflammatory clerodane diterpenoids with marketed drugs
SwissADME is a tool provided by the Swiss institute of Bioinformatics [93], which was used to predict molecular descriptors of the above summarized clerodane diterpenoids. To better summarize the drug-likeness properties of all these compounds, the following descriptors were described, inluding molecular weight (MW), number of hydrogen bond acceptors (HBA) and donors (HBD), number of stereogenic centers, and number of rotatable bonds (RB). The fraction of sp3 carbon (Fsp 3 ) is defined as the ratio of sp3 hybrid carbon to the total carbon number. The fraction of carbon and aromatic heavy atoms (Far) is the ratio of the number of aromatic heavy atoms to the total number of heavy atoms [94].
The predicted results were shown in Additional file 1: Table S1, which were grouped according to the previously described categories. Absorption, distribution, metabolism, and excretion (ADME) are critical factors in drug development [95], drug similarity and medicinal chemistry friendliness. Additional file 1: Table S2 showed the physicochemical properties, molecular polar surface area (PSA), pharmacokinetics, LogS and iLOGP, as well as bioavailability characteristics of clerodane diterpenoids (Supplementary Material). Especially for LogP [96] and LogS [97], more than one algorithm was used.
To better compare those anti-inflammatory clerodane diterpenoid derivatives with marketed drugs, the MW, HBA, HBD [98], Log P, PSA, RB, and stereogenic centers were evaluated; and the marketed drugs were divided into natural products, natural products derivatives, natural product type macrocycles, polycyclic compounds, synthetic compounds, and assumed synthetic compounds [99,100] (Fig. 3).

Size: molecular weight
Lopinski's rule of five defined the suitable traditional therapeutic agents [101], including less than 500 Da molecular mass, no more than 10 HBA, no more than 5 HBD, and an octanol-water partition coefficient LogP not great than 5. According to the SwissADME results, the mean MW for the anti-inflammatory clerodane diterpenoids was 452.4 Da (Fig. 3a). Most NSAIDs [102], and 61% of clerodane diterpenoids are with the MW less than 500 Da, which match to Lopinski's rule of five.

Chirality: number of stereogenic centers
The number of stereocenters has an important impact on the chiral structure and self-assembly of molecular aggregates. The number of stereogenic centers in clerodane diterpenoids was 6, which was comparable with those of natural products, natural products derivatives,natural product type macrocycles, polycyclic compounds, synthetic compounds, and assumed synthetic compounds (Fig. 3b). The natural product type macrocycle have the highest value of the stereogenic center, 12. For the synthesis of chemical compounds, more chiral centers causes more difficulty and higher cost of synthesis [99]. The number of stereogenic centers in the anti-inflammatory clerodane diterpenoid derivatives meets the standards for new drug development.

Polarity: PSA and HBD/HBA
A major challenge in drug discovery is the prediction of permeability, which, along with solubility, governs the skill of drugs and candidates transport across the gastrointestinal membrane and thus contributes to the overall exposure in systemic circulation and brain penetration. PSA, a descriptor defined as the sum of surfaces of nitrogen or oxygen atoms, plus hydrogen atoms attached to oxygen or nitrogen atoms in a molecule, has been widely used with a discrete success to model permeability and other ADME-related properties [103]. The topological PSA (TPSA) is often implemented in the drug-discovery pipeline [104]. However, the limitation of PSA should be considered. PSA does not take into account the contribution of polarity arising from electronegative atoms in drugs besides nitrogen and oxygen. And the different electronegativity of the atoms of the molecule produces a redistribution of the electron density that in principle involves the entire molecule. PSA is highly correlated with hydrogen bonding (HB. In principle, quantum mechanics calculations could also improve the description of HBA and HBD properties and the polarity of the molecules [105]. The PSA mean values were 96.1 Å 2 for clerodane diterpenoids, 86.9 Å 2 for polycyclic drugs, and 105.3 Å 2 for natural products. Additionally, the HBA/HBD and PSA values of anti-inflammatory clerodane diterpenoids are positively correlated with their MW (Figs. 3d-f and 4). According to the rule of 5, most of the anti-inflammatory clerodane diterpene derivatives might have good oral absorption.

Molecular flexibility: rotatable bonds and aromatic character
RBs are the number of bonds that allow free rotation, excluding those adjacent to triple bonds, connect hydrogen or halogen atoms, or in rings containing less than five single bonds [106]. The amide C-N bond is excluded due to its high rotational energy barrier. Reduced molecular flexibility (less RBs), and low PSA or total HB imply good oral bioavailability [107]. The number and percentage of RBs are descriptors for the biological effect of molecules. Compounds with less than 7 RBs exhibited oral bioavailability of more than 20% [108]. The increased RBs number interrupts the permeation rate to reduce oral bioavailability.
The mean numbers of RBs for the summarized clerodane diterpenoids, the polycyclic compounds, natural products, natural product derivatives, and synthetic drugs were 5. 92, 7.4, 9.4, 7.4, and 5.4, respectively (Fig. 3g). The less RBs for the clerodane diterpenoids indicated a good permeation rate. The summarized clerodane diterpenoids have a mean Fsp 3 of 0.64 because they possess an aromatic character.

Lipophilicity: LogP
LogP is normally determined in the hit to lead stage of drug discovery [109]. Lipophilicity of a drug is the ratio between its concentrations at the equilibrium in 1-octanol and water, commonly described as LogD [110]. The partition coefficient (P) is the specific distribution coefficient (D) at any given pH (typically neutral) [111].
The logP values of the anti-inflammatory clerodane diterpenoids are different by using different predicted method on Swiss ADME. Among all logP index, MLOGP is the most discrepant one (Fig. 5). Compare to the synthetic compounds, assumed synthetic compounds, natural products, natural derivatives, natural product macrocycles, and natural products polycyclic, the logP value of the clerodane diterpenoids (3.0) is higher, indicating lower oral bioavailability.

Solubility: LogS
If the solubility and rate of dissolution are low, an enterally administered drug will mostly be excreted [112]. Solubility of a compound is commonly expressed as logS, where S is the concentration of the compound in a saturated aqueous solution (mol/L). 85% of drugs have logS values between −1 and −5 and virtually none has value below -6 [113].
The aqueous solubility of clerodane diterpenoids is negatively correlated with MW (Fig. 6). Most of the anti-inflammatory clerodane diterpenoids have poor water solubility. Only a few examples showed the Log S values greater than -4, such as casearin B (65) and isocajucarinolide (32), which might have great potential to be developed as drugs.

Compliance of clerodane diterpenoids with the rules of drug-likeness
The "5 Rules" has been widely used as the first filter to eliminate potential drug candidates with poor oral bioavailability [107]. According to different compliance rules, the biophysical and chemical properties and molecular descriptors of the anti-inflammatory clerodane diterpenoids were evaluated. Most of them possess good drug-likeness (green in Additional file 1: Table S3).

Trends on the PK behavior of clerodane diterpenoids
Besides efficacy and toxicity, poor pharmacokinetics and bioavailability always cause drug development failures. The two pharmacokinetic behaviors, gastrointestinal absorption and brain entry, are critical at different stages of the drug discovery process. BOILED Egg, an accurate predictive model, was developed by calculating the lipophilicity and polarity of small molecules [114]. It is widely used in the filtering of chemical libraries and the evaluation of drug candidates [115].
Based on the predicted results, about 80% of antiinflammatory clerodane diterpenoids are more likely to be absorbed in the gastrointestinal (GI), mainly due to their lower MW (Fig. 7a). Among them, 53 clerodane diterpenoids have higher GI absorption, and 34 ones have a high probability as P-gp substrates (Fig. 7a).
The brain capillary endothelium for the blood-brain barrier (BBB) to exclude 100% of large-molecule neurotherapeutics and more than 98% of all small-molecule drug. BBB is the key issue in brain drug development [116]. Most of the anti-inflammatory clerodane diterpenoids have a low probability to cross the BBB (Fig. 7b). Among them, ten compounds are with potential ability to be P-gp substrates (Additional file 1: Table S5).

Conclusions
Clerodane diterpenoids are secondary metabolites widely distributed in plants, fungi, bacteria, and marine sponges. In this review, 65 anti-inflammatory clerodane diterpenoids were reviewed based on their chemical structures  and pharmacological models. The knowledge system of chemistry and bioactivity of clerodane diterpenoids continues to grow rapidly in recent years. Based on the current review, ent-clerodane diterpenoids always possess better anti-inflammatory activity than clerodane diterpenoids. And a lactone ring between C-18 and C-19 is essential for the anti-inflammation activity of clerodane diterpenoids. Although lots of evidence suggested the anti-inflammatory property of clerodane diterpenoids, the clinical application of these molecules as anti-inflammatory therapy is still far away. Till now, only evidence from in vitro cellular models or in vivo animal models was available. Clinical trials are necessary to authenticate the therapeutic effect of clerodane diterpenoids in alleviating inflammatory responses. The efficacy of these clerodane diterpenoids is not potent enough; thus, further phytochemical isolation and structural optimization are needed to enhance activity, improve specificity and reduce toxicity. Moreover, virtual docking and/or chemical biology studies should be carried out to identify the potential targets for the anti-inflammatory clerodane diterpenoids. The underlying mechanisms involved in the anti-inflammatory activity should be disclosed by pharmacological investigation.
Online bioinformatics tool SwissADME provides a lot of predicted data of a compound, such as molecular descriptors, the biophysiochemical properties, and the PK parameters. A serious of drug-likeness analyses could be performed to better understand its properties, such as MW, logP, logS, PSA, HBA, HBD, RBs, and number of stereogenic centers. For natural medicine investigation, the combination of bench research and bioinformatics prediction tools could be an effective and economical approach to discover new anti-inflammatory drugs. Regarding to the anti-inflammatory clerodane diterpenoids, future chemical and biological researches will be very bright and challenging, and they have a huge therapeutic application prospect.
Additional file 1: Table S1.Molecular descriptors of anti-inflammation clerodane diterpenoids. Table S2. Biophysiochemical properties of anti-inflammation clerodane diterpenoids. Table S3, Violations of druglikeness rules by the anti-inflammation clerodane diterpenoids. For each compound, the type of violations of each rule was described. Table S4 .Absorption and Metabolism parameters of anti-inflammation clerodane diterpenoids.