**2. Discussion**

#### *2.1. New Kinase Inhibitors Discovered from Marine Organisms*

Marine-derived natural products include a wide range of molecular classes such as alkaloids, macrolides, polypeptides, and terpenoids, and have different and interesting structures that play an important role in biological activities and clinical drug applications. As it pertains to the kinase research, a number of marine-derived kinase inhibitors have come from many different sources and target many different protein kinases (Figure 2). In the last five years covered in this review, from 2014–2019, there have been many results reported in the kinase research, and accordingly new biologically active kinase inhibitors that were discovered from various ocean life forms that include bacteria and cyanobacteria, fungi, animals, algae, soft corals and sponges. The initial reports of these natural product small molecules most typically include some rudimentary bioactivity test data, and further research can or does go on to explore more deeply the pharmacological potential of each. The new natural products reported during the period of this review are described systematically in the following sub-sections, divided by the source organisms reported for each.

**Figure 2.** Availability of some marine-derived kinase inhibitors with activity on the phylogenetic tree of the human protein kinase family. Color codes indicate the producing or source organisms. Yellow: Marine bacteria. Green: Marine fungi. Gray: Marine soft coral. Light blue: Marine animals. Dark blue: Marine algae. Red: Marine sponges. Adapted from refs [31] and [32] with permission, the original illustration reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com), 2019.

#### 2.1.1. Kinase Inhibitors from Marine Bacteria

In the time period covered in this review, 22 new natural product kinase inhibitors (**1–22**) were reported after being isolated from marine bacteria. These are listed in Table 1 and shown by structure in Figures 3–9. The specific kinases reportedly involved in the biological activities of these compounds are described in the following paragraphs. Since staurosporine and its derivatives have been isolated and reported from marine bacteria, these are considered for the purpose of this review as being marine-derived natural products even though staurosporine itself and other analogues are also produced by terrestrial organisms. These are accordingly summarized in this section, for basic discoveries, and also in Section 2.2, for the preclinical and clinical candidates.


**Table 1.** A summary of kinase inhibitors isolated from marine bacteria, 2014–2019.

In 2015, Goutam Dey et al. reported the anticancer activity of a lipopeptide, iturin A (**1**, Figure 3), which was isolated from the marine bacterium *Bacillus megaterium* [33]. In the same report, compound **1** was shown to inhibit p-Akt kinase, p-MAPK and p-GSK3β [33]. In other investigations, iturin A has been found to induce antiproliferative and apoptotic effects in breast cancer cells in vitro (IC50 values of MDA-MB-231, MCF-7,MDA-MB-468 and T47D cells were 7.98 ± 0.19, 12.16 ± 0.24, 13.30 ± 0.97 and 26.29 ± 0.78 μM, respectively) and in vivo [33]. These results sugges<sup>t</sup> that iturin A, with its mechanism of Akt inhibition, may be useful for the development of a drug for this or other types of cancers. In 2018, Biao Zhou et al. reported the isolation of three new indolocarbazole alkaloids (**2**–**4**) along with nine known compounds (including **5**) from the marine bacterium *Streptomyces* sp. A65 [34]. Compounds **3**–**5** (Figure 4) inhibited PKC and BTK (IC50 between 0.25 μM and 1.91 μM), and compound **2** was not active [34]. The structural similarities of **2**–**5** along with the observed biological activities that include IC50 values across two or more orders of magnitude show the beginnings of a natural structural activity relationship (SAR). It would be interesting if this could be expanded by accessing further analogues through targeted isolation studies or synthetic chemistry.

In 2018, Le-Le Qin et al. reported two new indolocabazole analogues, compounds **6** and **7** (Figure 4) along with a related compound (**8**, Figure 5), isolated from a marine bacterium *Streptomyces* sp. A68 [35]. These compounds inhibited PKC-<sup>α</sup>, ROCK II and BTK with IC50 values ranging from 0.17 to 3.24 μM, and had cytotoxic activity in vitro against PC3 human prostate cancer cells [35]. The indolocarbazole alkaloid scaffold represented in these molecules has also been popular for the development of kinase inhibitory drugs. Comparing compounds **2**–**7**, it can be considered that the position of the oxygen atom on the tetrahydrofuran subunit and its para-hydroxy and carbamic acid moieties may have important effects on the kinase inhibitor activity of each molecule.

**Figure 3.** Structure of the lipopeptide iturin A (**1**).

**Figure 4.** Structures of indolocarbazole alkaloids **2**–**7**.

**Figure 5.** Structure of compound **8**.

In 2018, Yong-Jun Jiang et al. reported eight new and one known cyclizidine-type alkaloids including **9–12** (Figure 6) from the marine-derived actinomycete *Streptomyces* sp. HNA39 [36]. This class of molecules was first discovered only in 1982 from *Streptomyces* sp. NCIB 11649 [37–39]. From this group, compound **9** inhibited the in vitro survival of PC3 human prostate (IC50 = 0.52 ± 0.03 μM) and HCT116 human colorectal (IC50 = 8.3 ± 0.1 μM) cancer cells [36]. Compound **9** has four hydroxy groups on the indolizine core subunit, which may be important for producing the selectivity observed, since the mechanism of action is likely related to the Michael acceptor on the side chain. Furthermore, compounds **9**–**12** had some moderate inhibition against the ROCK2 (IC50 7.0 ± 0.8 to 42 ± 3 μM) [36].

In 2018, Jia-Nan Wang et al. reported nine new indolocarbazole alkaloids from the marine bacterium *Streptomyces* sp. DT-A61 [40]. Among these, compound **13** (Figure 7) was shown to inhibit ROCK2 (IC50 = 5.7 nM) [40]. The results of biological activity tests showed compound **14** (Figure 7) to be cytotoxic to PC3 human prostate cancer cells (IC50 = 0.16 μM) [40]. In 2017, Xiang-Wei Cheng et al. isolated eight known and one new indolocarbazole alkaloid, 12-*N*-methyl-k252c (**15**, Figure 7), from the marine bacterium *Streptomyces* sp. A22 [41]. From this sample set, only **15** reportedly inhibited protein kinases (IC50 = 0.91–1.84 μM against various targets) [41].

**Figure 6.** Structures of cyclizidine-type alkaloids **9**–**12**.

**Figure 7.** Structure of indolocarbazole alkaloids **13**–**15**.

In 2018, Biao Zhou et al. 15 staurosporine derivatives from the marine bacterium *Streptomyces* sp. NB-A13 [42]. Among these, six were new (**16**–**21**; Figure 8), and each one except for **16** inhibited PKC-θ (IC50 0.06 to 9.43 μM) [42]. The most active of them was compound **21**, which also inhibited SW-620 cells in vitro (IC50 = 9.99 nM) at even greater potency than staurosporine (IC50 = 25.10 nM) [42]. Structurally, compound **21** bears an additional hydroxy group in the bis-indole ring system that is shared with staurosporine, indicating one possible avenue for medicinal chemistry optimization of other molecules in this chemical class.

**Figure 8.** Structures of staurosporine derivatives **16**–**21**.

From the marine bacterium *Streptomyces* sp. XMA39, Yong-Jun Jiang et al. in 2018 isolated the known compound medermycin, and four new structurally related naphthoquinones, strepoxepinmycins A–D [43]. All of these marine natural products were growth inhibitors for different human pathogenic bacteria, and compound **22** (Figure 9) showed ROCK2 inhibition (IC50 > 20 μM) and cytotoxic activity against HCT-116 and PC3 cancer cells in vitro with IC50 > 40 mM [43].

**Figure 9.** Structure of compound **22**.

#### 2.1.2. Kinase Inhibitors from Marine Fungi

In the time period covered in this review, 16 new natural product kinase inhibitors (**23–38**) were reported after being isolated from marine fungi. These are listed in Table 2 and shown by structure in Figures 10–12. The specific kinases reportedly involved in the biological activities of these compounds are described in the following paragraphs.

In 2017, Dong-Ni Chen et al. reported the structure of sclerotiorin (**23**, Figure 10) isolated from the marine fungus *Penicillium* sp. Strain ZJ27 [44]. At the same time, sclerotiorin was shown to very weakly inhibit PknG (IC50 = 76.5 μM) [44]. In 2014, Dong-Cheol Kim et al. reported that methylpenicinoline (compound **24**, Figure 10) was isolated from marine fungus *Penicillium* sp. and inhibited p-p38 MAPK pathways, although no specific activity value was noted [45]. The authors proposed that **24** might be useful for further development toward the treatment of inflammatory and neuroinflammatory diseases, so the results of ongoing work may demonstrate their follow-up. Mycoepoxydiene (**25**, Figure 10) was reported in 2014 by Wen-Jiao Li et al., after it was isolated from the marine fungal strain *Diaporthe* sp. HLY-1 [46]. Compound **25** was shown to induce apoptosis and inhibit phosphorylation of IKK, and it was suggested that the inhibition of IKK activity and human cholangiocarcinoma (CCA) cells such as SK-ChA-1 (IC50 =12.5 ± 0.2 μM) and Mz-ChA-1 (IC50 = 35.6 ± 0.2 μM) may fully or partially explain the observed apoptosis [46]. These results sugges<sup>t</sup> that compound **25** could be used as a lead molecule for the design and development of potent and selective new inhibitors of IKK with a potential for therapy of human cholangiocarcinoma disease [15,46]. In 2014, Kunlai Sun et al. reported a new indole-diterpenoid (**26**, Figure 10) that was isolated from the marine fungus *Aspergillus flavus* OUCMDZ-2205 [47]. This compound was found to inhibit PKC-β (IC50 = 15.6 μM), at 10 μM arrested A549 cells in the cell cycle S phase, and showed antibacterial activity against *Staphylococcus aureus* (MIC = 20.5 μM) [47]. In 2015, Wen-Liang Chen et al. isolated xyloketal B (compound **27**, Figure 10) from the fungus *Xylaria* sp. (No. 2508) that was collected in a mangrove [48]. Compound **27** was shown at 300 μM to inhibit p-Akt (inhibition rate ~ 34%) and p-ERK1/<sup>2</sup> (inhibition rate ~ 40%) protein expression in vitro, and the determinants of anti-proliferation and migration effects against glioblastoma U251 cells (IC50 = 287.1 ± 1.0 μM) in vitro included TRPM7-regulated PI3K/Akt and MEK/ERK signaling [48].


**Table 2.** A summary of kinase inhibitors isolated from marine fungi, 2014–2019.

**Figure 10.** Structure of compounds **23**–**27**.

In 2015, Dong-Cheol Kim et al. [49] reported a new dihydroisocoumarin derivative (compound **28**, Figure 11) from the marine fungus *Aspergillus* sp. SF-5976, and this molecule inhibits p-p38 MAPK [49]. In 2016, Jong Won Kim et al. reported the discovery of stachybotrysin (**29**, Figure 11), which was isolated from the marine fungus *Stachybotrys* sp. KCB13F013 and found to inhibit the RANKL-induced activation of p-ERK, p-JNK and p-p38 MAPK [50]. The kinase inhibition activities of compound **28** and **29** were determined by Western blot analyses. In 2016, Jutta Wiese et al. reported pannorin (**30**), alternariol (**31**), and alternariol-9-methylether (**32**), which were isolated from the marine fungus, *Botryotinia fuckeliana* [51]. Compounds **30**–**32** (Figure 11) showed potent inhibition of GSK-3 in vitro

with IC50 = 0.35 ± 0.04 μM, 0.13 ± 0.04 and 0.20 ± 0.04 μM, respectively [51]. The highly oxygenated benzocoumarin scaffolds represented in these molecules are apparently effective groups for GSK-3β inhibition, and these could merit further development through medicinal chemistry approaches although the current leads show very weak antibacterial and cytotoxic effects. In 2015, Wonmin Ko et al. reported alternaramide (**33**, Figure 11), which was isolated from the marine fungus *Alternaria* sp. SF-5016 [52]. This compound inhibited the formation of p-JNK and p-p38 MAPK, as determined by Western blotting, suggesting that it could be useful in the treatment of various acute, systemic, and neurological inflammatory diseases [52]. In 2015, Kwang-Ho Cho et al. reported citreohybridonol (**34**, Figure 11) after it had been isolated from the extract of the marine-derived fungus *Toxicocladosporium* sp. SF-5699 [53]. Compound **34** was pursuantly shown to inhibit the activation of p38 MAPK pathways by the Western blot analysis [53].

**Figure 11.** Structure of compounds **28**–**34**.

In 2016, M García-Caballero et al. reported that toluquinol (**35**, Figure 12), isolated from the marine fungus *Penicillium* sp. HL-85-ALS5-R004, inhibited the phosphorylation of p-Akt and p-ERK1/<sup>2</sup> in vitro and VEGF-C-induced lymphatic vessel formation and corneal neovascularization in mice [54]. Compound **35** plays a demonstrated role in inhibiting angiogenesis and lymphangiogenesis, which is meaningful in connection with the implications of tumour-induced lymphangiogenesis and lymphatic metastasis. In 2016, Bin Wu et al. reported on biscogniauxone (**36**, 12), a new isopyrrolonaphthoquinone isolated from the deep-sea (2800 m) fungus *Biscogniauxia mediterranea* [55]. Compound **36** moderately inhibited GSK-3β (IC50 = 8.04 μM) and had almost negligible antibacterial activity against *Staphylococcus epidermidis* and methicillin-resistant *S. aureus* (IC50 ~ 100 μM) [55]. Marine-derived fungi from the deep sea are promising but severely under-studied biological resources, and these possess abundant novel and bioactive secondary metabolites that urgently need to be mined if adequate access to the

resource can be obtained [57]. In 2017, Nguyen Thi Thanh Ngan et al. reported that citrinin H1 (compound **37**, Figure 12), isolated from the marine fungal strain *Penicillium* sp. SF-5629, inhibited p-p38 MAPK expression in vitro [56]. Additionally in 2017, Marion Navarri et al. reported that dihydrosecofuscin (compound **38**, Figure 12), isolated from a deep-sea (765 m) fungus *Oidiodendron griseum* UBOCC-A-114129, inhibited CLK1 (IC50 = 15.6 μg/mL) [58]. Compound **38** showed weak antibacterial activities (MIC ~ 100 μg/mL) against Gram-positive bacteria, and the authors noted a potential use of this CLK1 inhibitor for the treatment of Alzheimer's due to the connection between the particular kinase and disease [58].

**Figure 12.** Structure of compounds **35**–**38**.

#### 2.1.3. Kinase Inhibitors from Marine Soft Coral

In the time period covered in this review, two new natural product kinase inhibitors (**39** and **40**) were reported after being isolated from marine soft coral (see Table 3). Dihydroaustrasulfone alcohol (**39**, Figure 13), is a molecule that was reported in 2015 by Pei-Chuan Li et al. after being isolated from soft corals [59]. This compound inhibited ERK/MAPK and PI3K/AKT, and was suggested as a lead that could be pursued for the prevention and treatment of arterial restenosis [59]. In 2017, pachycladin A (**40**, Figure 13) was re-isolated from the soft coral *Cladiella pachyclados* found in the Red Sea, and a newly shown activity (absent from the previous report of this compound in 2010 [61]) was that it inhibited two members of the EGFR family when screened at 10 μM [60].

**Table 3.** A summary of kinase inhibitors isolated from marine soft coral, 2014–2019.


**Figure 13.** Structure of compounds **39**–**40**.

#### 2.1.4. Kinase Inhibitors from Marine Animals

In the time period covered in this review, 14 new natural product kinase inhibitors (**41–54**) were reported after being isolated from marine animals. These are listed in Table 4 and shown by structure in Figures 14–16. The specific kinases reportedly involved in the biological activities of these compounds are described in the following paragraphs.

In 2014, didemnaketals D and E (compounds **41** and **42**, Figure 14) were reportedly discovered from marine tunicates of the genus *Didemnum*, and these compounds inhibited several kinases (CDK5, CK1, DyrK1A, and GSK3) at 10 μg/mL and were moderately antimicrobial in vitro against *S. aureus* and *Bacillus subtilis* [62]. In 2015, Youssef [63] et al. reported the three new purine alkaloids **43**–**46** (Figure 15), together with seven known compounds (**45**–**52**, Figure 15) from the marine tunicate *Symplegma rubra*, collected from the Saudi Red Sea coast (5–7 m depth). The authors reported that these compounds were found to be moderate inhibitors of di fferent kinases (CDK5, CK1, DyrK1A, and GSK-3), with some activity at 10 μg/mL, but noted that the observed IC50 values (>10 μM) were "not su fficient to pursue with these compounds into the in vivo studies" [63]. This report was somewhat vague and did not specify the individual activity of each molecule.


**Table 4.** A summary of kinase inhibitors isolated from marine animals, 2014–2019.

In 2017, frondoside A (compound **53**, Figure 16) was isolated from *Cucumaria frondosa*, the Atlantic sea cucumber, and reported as being an e ffective inhibitor of PAK1 (IC50 ~ 1.2 μM), LIMK (IC50 ~ 60 μM), AKT (IC50 ~ 59 μM) and A549 lung cancer cells (IC50 ~ 1–3 μM) in vitro, indicating that it may be useful in the treatment of malignancies [64,65]. Additionally in 2017, the molecule 1-deoxyrhodoptilometrin (compound **54**, Figure 16), which was first discovered by Wright et al. from the Echinoderm *Colobometra perspinosa* [67], was re-isolated from the marine echinoderm *Comanthus* sp. [66]. This compound was subsequently reported to inhibit the IGF1-R kinase, focal adhesion kinase, and EGF receptor kinase with IC50 = 5, 8.4 and 4 μM, respectively [66].

**Figure 14.** Structures of didemnaketals D (**41**) and E (**42**).

**Figure 15.** Structures of purine and pyridine alkaloids (**43**–**52**).

**Figure 16.** Structure of compounds **53**–**54**.

#### 2.1.5. Kinase Inhibitors from Marine Algae

In the time period covered in this review, 10 new natural product kinase inhibitors (**55–64**) were reported after being isolated from marine algae. These are listed in Table 5 and shown by structure in Figure 17, Figure 18 and Scheme 1. The specific kinases reportedly involved in the biological activities of these compounds are described in the following paragraphs.

In 2015, Shuai-Yu Wang et al. reported compound **55** (Figure 17) as a multi-target RTKs inhibitor such as FGFR2, FGFR3, VEGFR2 and PDGFR-α (with in vitro inhibition rates of 57.7%, 78.6%, 78.5% and 71.1%, respectively), which was isolated from *Rhodomela confervoides*, a red alga, and this molecule also inhibits p-PKB/Akt [68,69]. Compound **55** may accordingly present a scaffold from which to develop new multi-target RTKs inhibitors. In 2017, Sung-Hwan Eom et al. reported that the polyphenolic molecule eckol (**56**, Figure 17), purified from the edible brown seaweed *Eisenia bicyclis*, inhibited *Propionibacterium acnes* mediated phosphorylation of Akt [70]. Eckol is also proposed as an anti-inflammatory agen<sup>t</sup> and antioxidant. In 2017, fucoxanthin (**57**, Figure 17), a well-known marine carotenoid, was shown to inhibit Akt/NF-κB and involved MAPK pathways in vitro, suggesting that it could be a potential therapeutic agen<sup>t</sup> acting by this mechanism for the therapy of neurodegenerative diseases [71–73]. Further validation studies are obviously necessary, and likely ongoing. In 2018, Youn Kyung Choi et al. reported the isolation of bis (3-bromo-4,5-dihydroxybenzyl) ether (**58**, Figure 17) from *Polysiphonia morrowii*, a red alga [74]. Compound **58** selectively inhibited p-ERK, demonstrating that it could have potential in the treatment of inflammatory diseases [74]. In 2014, Paudel et al. postulated that since the polyphenolics 6–6 bieckol and pholorofucofuroeckol A (**59** and **60**, Figure 17) inhibited p-ERK1/<sup>2</sup> and p-JNK activation, these hold potential value for treating pulpitis and oral diseases [75]. These two compounds were isolated from *Eisenia bicyclis*, a brown alga [75].


**Table 5.** A summary of kinase inhibitors isolated from marine algae, 2014–2019.

**a** This molecule is synthetic but designed based on a marine natural product. **b** Hoiamide A is JNK activator.

**Figure 17.** Structure of compounds **55**–**60**.

Apratoxin A is a cyanobacterial, or "blue-green algal", natural product macrocycle that was first reported by Luesch et al. in 2001 as a product of a hybrid polyketide synthase-nonribosomal peptide synthetase (PKS/NRPS) biosynthetic pathway [83]. In 2017, Wei-Jing Cai et al. later designed and synthesized a series of cyclic depsipeptides based on apratoxin A, including apratoxin S10 (**61**, Scheme 1) [76]. This analogue of apratoxin A was selected as a desirable drug lead with cancer cell growth (IC50 = 5.97 nM against HCT116) and RTKs inhibitory activity [76]. The synthetic route of generating apratoxin S10 is depicted in Scheme 1. The synthesis of apratoxin S10 followed the reported strategy used to produce the lead compound, apratoxin A, with some modification to introduce new functionality [76].

**Scheme 1.** Synthesis and structure of apratoxin S10 (**61**).

In 2015, Mahesh Sapkota [78] et al. reported bromo-honaucin A (**62**, Figure 18), which was first isolated from marine cyanobacterium *Leptolyngbya crossbyana* and chemical synthesis was completed by Hyukjae Choi and William H. Gerwick et al. [77]. Compound **62** inhibited AKt and ERK, and the authors noted a potential use of this AKt and ERK inhibitor for the treatment of bone lysis due to the connection between the particular kinase and disease. In 2015, Zhengyu Cao et al. [81] reported the discovery of hoiamide A (**63**, Figure 18), which was first isolated from marine cyanobacterium *Moorea producens* by Alban Pereira et al. [79] and found to stimulate and increase the hoiamide A-induced activation of JNK. Compound **63** showed the inhibition of neurite outgrowth (IC50 = 4.89 nM). In 2016, Jeffrey D. Serrill et al. [82] reported coibamide A (**64**, Figure 18) (first isolated from marine cyanobacterium *Leptolyngbya* sp. [81]) inhibited VEGFA/VEGFR2 expression in mice and showed antitumor activities against glioblastoma xenografts.

**Figure 18.** Structure of compounds **62**–**64**.

#### 2.1.6. Kinase Inhibitors from Marine Sponges

In the time period covered in this review, 42 new natural product kinase inhibitors (**65–106**) were reported after being isolated from marine sponges. These are listed in Table 6, shown by structure in Figures 19–26 and Schemes 2–5, and described in detail in the following paragraphs.

In 2014, Fabien Plisson et al. reported some new bromopyrrole alkaloids including hymenialdisine (**65**, Figure 19) that were isolated from *Callyspongia* sp. [84]. This interesting class of molecules has been studied for several decades, owing to their peculiar structural skeleton and associated pharmacological effects [85,86]. Compound **65** inhibited CK1, CDK5, GSK3β and SW620 (3.1 μM), KB-3-1 (2.0 μM), which are targets for treating neurodegenerative diseases [84]. In 2014, the β-carboline alkaloids **66**–**68** (Figure 19), including hainanerectamines B and C, were isolated from the marine sponge *Hyrtios erecta* after it was collected in Hainan, China [87]. These molecules have been shown to inhibit the dual kinase Aurora A with relatively high IC50 values of 10 μg/mL–25 μg/mL [88]. In 2014, Mohamed R. Akl et al. reported the isolation of sipholenol A from the sponge *Callyspongia siphonella*, collected in the Red Sea [89]. A semisynthetic analogue of sipholenol A, sipholenol A-4-*O*-3-,4--dichlorobenzoate (**69**, Figure 19) was associated with the suppression of the Brk and FAK signaling pathway in vitro and in vivo, also inhibited MDA-MB-231, MCF-7, BT-474 and T-47D breast cancer cells with IC50 values of 7.5 μM, 15.2 μM, 20.1 μM and 25.1 μM, respectively, making it a potentially interesting pro-drug for sipholenol A [89].


**Table 6.** A summary of kinase inhibitors isolated from marine sponges, 2014–2019.

a This molecule is synthetic but designed based on a marine natural product.

**Figure 19.** Structure of compounds **65**–**69**.

In 2015, Karianne F. Lind et al. reported that barettin (**70**, Figure 20), which was first isolated and described in 1986 [90], inhibited RIPK2 (IC50 = 8.0 μM), CAMK1a (IC50 = 5.7 μM), SIK2 (IC50 = 6.1 μM) and produced anti-inflammatory effects in vitro [91,92]. In 2015, Rebecca Horbert et al. reported the synthetic compound **71** (Figure 20) with a 3,5-diarylpyrazin-2-one core that was based on hamacanthins A and B, which are sponge natural products of the bis-indole alkaloid class [93]. Compound **71** inhibited PDGF-Rβ with good potency (IC50 = 0.02 μM), and was cytotoxic in vitro against PDGF-R dependent cancer cells [93]. The accompanying structure-activity relationship indicated that the added 3--methoxy and 4--hydroxyphenyl substituents led to an increase in the inhibitory activity against PDGF-Rβ, helping to direct future related research studies [93]. Fascaplysin (**72**, Figure 20) previously was isolated from the marine sponge *Fascaplysinopsis* species [94] and shown to inhibit CDK-4/D1 with moderate potency (IC50 = 0.35 μM) and some selectivity amongs<sup>t</sup> kinases [95]. This compound is known to have multiple other mechanisms of action, including DNA binding. More recently, in 2015, S. Mahale et al. synthesized a non-planar analogue of fascaplysin, *N*-(biphenyl-2-yl) tryptoline (**73**, Figure 20), and reported that this retained some weak but selective inhibition of CDK-4/D1 (IC50 ~10 μM) [96]. Although this represents a marked drop in potency, compound **73** was successfully designed to overcome DNA intercalation that had been considered a liability of the more active **72** [96]. Compound **73** was validated as being a preferable anticancer agen<sup>t</sup> than **72**, including the results from PK studies in mice [96]. It has been further suggested that compound **73** constitutes a lead scaffold from which drug candidates could be designed for the treatment of certain cancers [96–98]. The synthetic route of production for **73** is depicted in Scheme 2 [96], which includes commercially available starting materials (biphenyl 2-carboxylic acid and tetrahydro-β-carboline), commonly used reagents [hexafluorophosphate benzotriazole tetramethyl uronium (HBTU) and *<sup>N</sup>*,*<sup>N</sup>*-diisopropylethylamine (DIEA)], and runs in good yield (~70%). The further development of fascaplysin analogues and derivatives through medicinal chemistry will accordingly help overcome the common "supply problem" of advancement of natural product drug candidates. In 2017, Sonia Sharma et al. synthesized 4-chlorofascaplysin (**74**, Scheme 3; a marine sponge alkaloid fascaplysin analogue) and found it to be cytotoxic to breast cancer cells in vitro (IC50 = 0.3 μM) and to inhibit PI3K/AKt/mTOR in vitro and in vivo [99]. The synthetic route of 4-chloro fascaplysin is depicted in Scheme 3, where the two-step synthesis includes low cost materials and high yield reactions [99].

**Figure 20.** Structure of compounds **70**–**73**.

**Scheme 2.** Synthetic preparation of the fascaplysin derivative, compound **73**.

**Scheme 3.** Synthesis of 4-chloro fascaplysin (**74**).

In 2015, Clinton G. L. Veale et al. created a series of synthetic bisindole compounds including **75** and **76** (Figure 21), based on deoxytopsentin that was isolated from marine sponge *Topsentia pachastrelloides*[100]. Compounds **75** and **76** inhibit PK from methicillin resistant *S. aureus*(IC50 = 60 and 16 nM, respectively), and had good in vitro antibacterial activity (MIC ~ 10 μg/mL) [100]. Compound **77** (Figure 21) is among the series of synthetic analogues based on **75** and **76**, and it had enhanced inhibitory activity against MRSA PK (IC50 = 1.4 nM) [100]. Additionally in 2015, Mohamed R. Akl et al. reported an RTK inhibitor, araguspongine C (compound **78**, Figure 22), was isolated from the marine sponge *Xestospongia* sp. [101]. Sherif S. Ebada et al. reported the productive discovery of 25 bromopyrrole alkaloids in 2015, from the Indonesian marine sponges *Stylissa massa* and *Stylissa flabelliformis* [102]. This sample set included dispacamide E (compound **79**) [103], the brominated aldisines **80**–**82** [103,104], (–)-mukanadin C (**83**) [105,106] and (–)-longamide (**84**) [107], these compounds, shown in Figure 22, were moderate inhibitors of GSK-3, DYRK1A and CK-1 (IC50 0.6 ~ 6.4 μM), and may have future development potential for treating various diseases [102]. In 2015, Fei He et al. reported seven new derivatives of adociaquinone that were isolated from a marine sponge, *Xestospongia* sp., collected in Indonesia [108]. Among these, compounds **85** and **86** (Figure 22) moderately but selectively inhibited CDK9/cyclin T and CDK5/p25 at 3–6 μM IC50, while **87** and **88** (also Figure 22) moderately inhibited

most protein kinases without selectivity (IC50 = 0.5 ~ 7.5 μM) and compound **88** showed marginal activity against DYRK1A [108].

**Figure 21.** Synthetic analogues (**75**–**<sup>77</sup>**) of the marine bisindole alkaloid deoxytopsentin.

**Figure 22.** Structure of compounds **78**–**88**.

In 2015, Esther A. Guzmán et al. [109] reported microsclerodermin A (**89**, Figure 23), from the marine sponge *Amphibleptula cf. madrepora*, and this compound inhibited GSK3β and pancreatic cancer cell viability in vitro (IC50 = 2.4 μM). In 2015, Shou-Ping Shih et al. isolated a marine polycyclic quinone called halenaquinone (compound **90**, Figure 23) from the marine sponge *Petrosia* sp. [110]. This molecule

is a broad spectrum tyrosine kinase inhibitor, as well as in vitro cytotoxin against multiple cell types (IC50 = 0.18 ~ 8.0 μg/mL) [110]. In 2016, Germana Esposito et al. reported chloromethylhalicyclamine B (**91**, Figure 23) after it was isolated from the sponge *Acanthostrongylophora* sp., and suggested that it "can efficiently interact with the ATP-binding site of CK1δ (IC50 = 6 μM) in spite of its globular structure, very different from the planar structure of known inhibitors of CK1δ [111]. As the authors also suggested, this molecule can be a lead molecule for the creation of new CK1δ/ε inhibitors. In 2016, Ran Wang et al. discovered stellettin B (**92**, Figure 23) from the marine sponge *Jaspis stellifera*, and it inhibited phosphorylation of PI3K and Akt [112]. Moreover, compound **92** exhibited potent activitivity against human glioblastoma cancer SF295 cells (IC50 = 0.01 μM).

**Figure 23.** Structure of compounds **89**–**92**.

In 2016, María Roel et al. reported that crambescidins 816, 830, and 800 (compounds **93**–**95**, Figure 24) were isolated from the marine sponge *Smenospongia* sp. [113]. These compounds inhibited tumor cell proliferation by suppressing CDK 2/6 expression and activated the cell CDK inhibitors -2A, -2D and -1A, suggesting some potential as anticancer agents [113]. In 2017, Nadège Loaëc et al. reported polyandrocarpamines A and B (**96** and **97**, Figure 24) as synthetic analogues of the 2-aminoimidazolin-4-one scaffold previously isolated from marine calcareous sponges *Leucetta* and *Clathrina* [114]. Compounds **96** and **97** were found to be nM potency inhibitors of DYRKs (IC50 = 0.17 ~ 0.88 μM) and CLKs (IC50 = 0.32 ~ 8.6 μM), and the 2-aminoimidazolone scaffold was accordingly proposed as having promise for developing new kinase inhibitors [114].

**Figure 24.** Structures of compounds **92**–**97**.

In 2018, the manzamine alkaloids were isolated from Indo-Pacific marine sponges *Acanthostrongylophora* sp., including manzamine A (**98**), 8-hydroxymanzamine A (**99**), manzamine E (**100**), manzamine F (**101**), 6-deoxymanzamine X (**102**), and the synthetic analogue 6- cyclohexamidomanzamine A (**103**) was also generated [115,116]. These compounds (Figure 25) showed mixed noncompetitive inhibition of MtSK, and the synthetic compound **103** takes advantage of the structural features to pursue applications of anti-inflammatory, antiparasitic, insecticidal, and antibacterial activities with potential for development against malaria and tuberculosis [115,116]. In 2014, Nag S. Kumar et al. reported the discovery of a series of bis-indole alkaloids based on the structure of a marine alkaloid isolated from the marine sponge *Topsentia pachastrelloides*, of which compound **104** (Figure 26) is a promising PK inhibitor [117].

**Figure 25.** Structures of compounds **98**–**103**.

**Figure 26.** Structure of the bis-indole compound, **104**.

In 2015, Yongseok Kwon et al. synthesized compound **105** (Scheme 4) as a synthetic analogue of pachastrissamine (first reported in 2002 from the marine sponge *Pachastrissa* sp.), and it has in vitro cytotoxicity along with sphingosine kinase inhibitory activity that was deemed superior to the natural lead compound [118,119]. The IC50 of compound **105** and pachastrissamine in the inhibition of SphK1 and SphK2 were 7.5 μM and 12.0 μM, 20.1 μM and 41.8 μM, respectively, suggesting that compound **105** could have preferable usage in related pharmaceutical applications [118,119]. The synthetic route of the pachastrissamine carbocyclic analogue **105** is depicted in Scheme 4. The compound (+)-liphagal (**106**, Scheme 5) was first isolated in 2006 from a marine sponge by Frederick and Andersen et al. [120] and in 2015 the chemical synthesis was completed by Markwell-Heys et al. [121]. This compound inhibited PIK-α (IC50 = 100 nM), and was also found to be cytotoxic to several tumor cell lines with IC50 ≈ 1 μM. It has been suggested that **106** has potential application as a new type of kinase inhibitor or even cancer drug [122]. The synthetic route of (+)-liphagal is depicted in Scheme 5, which initiates with (+)-sclareolide (commercially available) and generates the final product in ~10% yield after 13 reaction steps [122].

**Scheme 4.** Synthetic route and structure of pachastrissamine carbocyclic analogue **105**.

**Scheme 5.** Synthesis and structure of (+)-liphagal (**106**).

#### *2.2. Preclinical and Clinical Candidates*

The US FDA has already approved more than 34 small molecule kinase inhibitors for human use [123]. Many of these have been used for the treatment of cancers, although toxicity is of grea<sup>t</sup> concern. A number of marine-derived kinase inhibitors have entered clinic trials, and some are already in later pre-approval stages or have been recently approved (Figures 27 and 28 and Table 7). For example, plitidepsin (aplidin) is a marine natural product that was developed by the Spanish company PharmaMar as an inhibitor of EGPR, Src, JNK and p38 MAPK and, although there were dose-limiting toxicities in some cases, it was not fully withdrawn from clinical trials during phase II [124–126]. Later, it was approved for clinical use in Australia in 2018 for the treatment of relapsed/refractory multiple myeloma patients. Midostaurin is among the important drugs for aggressive systemic mastocytosis (ASM) treatment as a multi-target protein kinase inhibitor, and it was FDA-approved in 2017 [124,127–129]. Protein kinase inhibitors from the marine origin that have entered clinical trials include three kinase inhibitors (lestaurtinib [130], enzastaurin [131], CEP-1347 [14,124,127]) in phase III, three kinase inhibitors (kahalalide F [132,133], 7-hydroxystaurosporine [134,135], staurosporine [136]) in phase II and two kinase inhibitors (CEP-2563 [14,124,127], isokahalalide F [14,124,127]) in phase I clinical studies. Somewhat related, bryostatin 1 entered phase II clinical trials for testing in the treatment of Alzheimer's Disease as a PKC activator, after it was also experimentally used in phase I trials for various forms of cancer [137–139]. It will be interesting to see the future developments of this marine natural product drug candidate and others.

**Figure 27.** Structures of marine-derived kinase inhibitors in clinical use (**107**–**108**).

**Figure 28.** Structures of marine-derived kinase inhibitors, **109**–**117**, currently in clinical trials.


**Table 7.** Preclinical and clinical status of some kinase inhibitors, and bryostatin 1, with marine-derived natural product origins a,b.

Kinase C (PKC) activator.
