Effective Synthesis and Antifouling Activity of Dolastatin 16 Derivatives

Some derivatives of dolastatin 16, a depsipeptide natural product first obtained from the sea hare Dolabella auricularia, were synthesized through second-generation synthesis of two unusual amino acids, dolaphenvaline and dolamethylleuine. The second-generation synthesis enabled derivatizations such as functionalization of the aromatic ring in dolaphenvaline. The derivatives of fragments and whole structures were evaluated for antifouling activity against the cypris larvae of Amphibalanus amphitrite. Small fragments inhibited the settlement of the cypris larvae at potent to moderate concentrations (EC50 = 0.60-4.62 μg/mL), although dolastatin 16 with a substituent on the aromatic ring (24) was much less potent than dolastatin 16.

In 2017, we reported the total synthesis and antifouling activity of 1 and two intermediates, northern carboxylic acid fragment 2 and southern amine fragment 3, to reveal highly potent activity of 1 (EC 50 < 0.03 µg/mL) and moderate to low activities of 2 and 3 (EC 50 > 10 and 1.17 µg/mL, respectively) [38]. With these results in hand, we envisioned that additional compounds related to 1 would show further potential toward the development of a green antifouling material. For quick access to these compounds, secondgeneration synthesis of the two unusual amino acids was required because derivatization of the amino acids was difficult with the previous methodology [37]. In this paper, we describe our efforts to synthesize derivatives of dolaphenvaline and dolamethylleuine as well as some derivatives of 1. Evaluations of the antifouling activity toward the cypris larvae of the barnacle A. amphitrite were also conducted.

Results and Discussions
For the preparation of dolaphenvaline derivatives, the C-H activation reaction focused on amide 4, which was obtained from L-valine in 3 steps according to known method [39]. The synthetic details to obtain dolaphenvaline derivatives 7 and 8 are shown in Scheme 1. The installment of the aromatic ring on 4 was accomplished in a regio-and diastereoselective manner, confirmed by 1 H NMR, in the presence of a palladium catalyst and silver salt without solvent to give amide 5 [39] (see Supplementary Material Figures S1-S14 for NMR spectra). This high diastereoselectivity was rationalized by steric repulsion between the methyl group and the phthaloyl group in the transition state shown in the brackets. Acidic hydrolysis, followed by protection with a Boc group, afforded Boc-dolaphenvaline 7, previously reported by us [37]. First synthesis of 8, having a p-hydroxy group on the benzene ring, was also possible through the same pathway when p-siloxyiodobenzene was employed in the C-H activation step. In 2017, we reported the total synthesis and antifouling activity of 1 and two intermediates, northern carboxylic acid fragment 2 and southern amine fragment 3, to reveal highly potent activity of 1 (EC50 < 0.03 μg/mL) and moderate to low activities of 2 and 3 (EC50 > 10 and 1.17 μg/mL, respectively) [38]. With these results in hand, we envisioned that additional compounds related to 1 would show further potential toward the development of a green antifouling material. For quick access to these compounds, secondgeneration synthesis of the two unusual amino acids was required because derivatization of the amino acids was difficult with the previous methodology [37]. In this paper, we describe our efforts to synthesize derivatives of dolaphenvaline and dolamethylleuine as well as some derivatives of 1. Evaluations of the antifouling activity toward the cypris larvae of the barnacle A. amphitrite were also conducted.

Results and Discussions
For the preparation of dolaphenvaline derivatives, the C-H activation reaction focused on amide 4, which was obtained from L-valine in 3 steps according to known method [39]. The synthetic details to obtain dolaphenvaline derivatives 7 and 8 are shown in Scheme 1. The installment of the aromatic ring on 4 was accomplished in a regio-and diastereoselective manner, confirmed by 1 H NMR, in the presence of a palladium catalyst and silver salt without solvent to give amide 5 [39] (see Supplementary material for NMR spectra). This high diastereoselectivity was rationalized by steric repulsion between the methyl group and the phthaloyl group in the transition state shown in the brackets. Acidic hydrolysis, followed by protection with a Boc group, afforded Boc-dolaphenvaline 7, previously reported by us [37]. First synthesis of 8, having a p-hydroxy group on the benzene ring, was also possible through the same pathway when p-siloxyiodobenzene was employed in the C-H activation step. Boc-dolamethylleuine 15 was accessed through a [2+2] addition reaction in the presence of organocatalyst 11 as the key step to construct two contiguous asymmetric carbon centers, as shown in Scheme 2 [40]. The reaction between isovaleraldehyde (9) and propionyl chloride (10) at −40 • C provided volatile lactone 12 in a highly stereoselective manner (minor diastereomer could not be observed in crude 1 H NMR), which was next converted into carboxylic acid 13 by the treatment with NaN 3 and NH 4 Cl in DMSO. Before the Staudinger reaction, i.e., the conversion of the azide group to an amino group, benzyl ester formation was necessary since the Staudinger reaction of 13 resulted in low yield (<20%). The Staudinger reaction with benzyl ester 14 and subsequent protection with Boc 2 O proceeded smoothly to give 15 in 59% yield, previously reported by us (specific rotation of the current compound was completely identical with that of the previous one). Boc-dolamethylleuine 15 was accessed through a [2+2] addition reaction in the presence of organocatalyst 11 as the key step to construct two contiguous asymmetric carbon centers, as shown in Scheme 2 [40]. The reaction between isovaleraldehyde (9) and propionyl chloride (10) at −40 °C provided volatile lactone 12 in a highly stereoselective manner (minor diastereomer could not be observed in crude 1 H NMR), which was next converted into carboxylic acid 13 by the treatment with NaN3 and NH4Cl in DMSO. Before the Staudinger reaction, i.e., the conversion of the azide group to an amino group, benzyl ester formation was necessary since the Staudinger reaction of 13 resulted in low yield (<20%). The Staudinger reaction with benzyl ester 14 and subsequent protection with Boc2O proceeded smoothly to give 15 in 59% yield, previously reported by us (specific rotation of the current compound was completely identical with that of the previous one).

Scheme 2. Synthesis of dolamethylleuine derivatives.
With the effective synthetic route to the two unusual amino acids established, we launched the preparation of dolastatin 16 derivatives according to the previous report [38]. Condensation between 8 and proline benzyl ester gave amide 16 in which the hydroxy group was then acetylated under standard conditions (Scheme 3). After hydrogenolysis of peptide 17, coupling of resulting 18 with dolamethylleuine benzyl ester 19 gave peptide 20. In order to proceed with structure-activity relationship studies, 20 was converted into benzyl ether 22 in two steps through methanolysis of the acetate, followed by treatment of the resultant peptide 21 with BnBr, K2CO3 and KI.  With the effective synthetic route to the two unusual amino acids established, we launched the preparation of dolastatin 16 derivatives according to the previous report [38]. Condensation between 8 and proline benzyl ester gave amide 16 in which the hydroxy group was then acetylated under standard conditions (Scheme 3). After hydrogenolysis of peptide 17, coupling of resulting 18 with dolamethylleuine benzyl ester 19 gave peptide 20. In order to proceed with structure-activity relationship studies, 20 was converted into benzyl ether 22 in two steps through methanolysis of the acetate, followed by treatment of the resultant peptide 21 with BnBr, K 2 CO 3 and KI. Boc-dolamethylleuine 15 was accessed through a [2+2] addition reaction in the presence of organocatalyst 11 as the key step to construct two contiguous asymmetric carbon centers, as shown in Scheme 2 [40]. The reaction between isovaleraldehyde (9) and propionyl chloride (10) at −40 °C provided volatile lactone 12 in a highly stereoselective manner (minor diastereomer could not be observed in crude 1 H NMR), which was next converted into carboxylic acid 13 by the treatment with NaN3 and NH4Cl in DMSO. Before the Staudinger reaction, i.e., the conversion of the azide group to an amino group, benzyl ester formation was necessary since the Staudinger reaction of 13 resulted in low yield (<20%). The Staudinger reaction with benzyl ester 14 and subsequent protection with Boc2O proceeded smoothly to give 15 in 59% yield, previously reported by us (specific rotation of the current compound was completely identical with that of the previous one).

Scheme 2. Synthesis of dolamethylleuine derivatives.
With the effective synthetic route to the two unusual amino acids established, we launched the preparation of dolastatin 16 derivatives according to the previous report [38]. Condensation between 8 and proline benzyl ester gave amide 16 in which the hydroxy group was then acetylated under standard conditions (Scheme 3). After hydrogenolysis of peptide 17, coupling of resulting 18 with dolamethylleuine benzyl ester 19 gave peptide 20. In order to proceed with structure-activity relationship studies, 20 was converted into benzyl ether 22 in two steps through methanolysis of the acetate, followed by treatment of the resultant peptide 21 with BnBr, K2CO3 and KI. Functionalized southern fragment 20 was further coupled with the northern fragment 2 to give peptide 23 for the macrolactonization reaction (Scheme 4). In the previous studies by Pettit and us, Shiina's conditions by 2-methyl-6-nitrobenzoic anhydride (MNBA) [41][42][43] provided a low yield of 1 (22% by Pettit, 31% by us). In order to improve the reaction yield, extensive optimizations were performed, eventually finding that Mukaiyama's conditions using 2-chloro-1-methylpyridimium iodide (CMPI) [44] gave the target compound 24 in 64% yield over 2 steps (deprotection of benzyl groups and macrolactonization).
Functionalized southern fragment 20 was further coupled with the northern fragment 2 to give peptide 23 for the macrolactonization reaction (Scheme 4). In the previous studies by Pettit and us, Shiina's conditions by 2-methyl-6-nitrobenzoic anhydride (MNBA) [41][42][43] provided a low yield of 1 (22% by Pettit, 31% by us). In order to improve the reaction yield, extensive optimizations were performed, eventually finding that Mukaiyama's conditions using 2-chloro-1-methylpyridimium iodide (CMPI) [44] gave the target compound 24 in 64% yield over 2 steps (deprotection of benzyl groups and macrolactonization). Additional syntheses of northern fragments, benzyl ester 25 and benzyl ether 29, were carried out as shown in Scheme 5. Benzyl ester 25 was prepared by esterification reaction of 2 in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI). For the synthesis of 29, installation of a benzyl group to the prolinol moiety at the stage of 26 was essential because direct etherification into 29 from the corresponding alcohol resulted in a complex mixture. The subsequent condensation reaction between carboxylic acid 28 and the amine obtained by removal of the Boc group of 27 [45] proceeded cleanly to give 29 in good yield.  Additional syntheses of northern fragments, benzyl ester 25 and benzyl ether 29, were carried out as shown in Scheme 5. Benzyl ester 25 was prepared by esterification reaction of 2 in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI). For the synthesis of 29, installation of a benzyl group to the prolinol moiety at the stage of 26 was essential because direct etherification into 29 from the corresponding alcohol resulted in a complex mixture. The subsequent condensation reaction between carboxylic acid 28 and the amine obtained by removal of the Boc group of 27 [45] proceeded cleanly to give 29 in good yield. Functionalized southern fragment 20 was further coupled with the northern fragment 2 to give peptide 23 for the macrolactonization reaction (Scheme 4). In the previous studies by Pettit and us, Shiina's conditions by 2-methyl-6-nitrobenzoic anhydride (MNBA) [41][42][43] provided a low yield of 1 (22% by Pettit, 31% by us). In order to improve the reaction yield, extensive optimizations were performed, eventually finding that Mukaiyama's conditions using 2-chloro-1-methylpyridimium iodide (CMPI) [44] gave the target compound 24 in 64% yield over 2 steps (deprotection of benzyl groups and macrolactonization). Additional syntheses of northern fragments, benzyl ester 25 and benzyl ether 29, were carried out as shown in Scheme 5. Benzyl ester 25 was prepared by esterification reaction of 2 in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI). For the synthesis of 29, installation of a benzyl group to the prolinol moiety at the stage of 26 was essential because direct etherification into 29 from the corresponding alcohol resulted in a complex mixture. The subsequent condensation reaction between carboxylic acid 28 and the amine obtained by removal of the Boc group of 27 [45] proceeded cleanly to give 29 in good yield. The antifouling activities of synthetic samples were evaluated as EC 50 values against the cypris larvae of A. amphitrite by exposure of each compound for 48 h (Table 1, Figure 2). For comparison, EC 50 values for the previous compounds 1-3 are also shown in the table.
Installation of a functional group on the aromatic ring of 1 decreased the antifouling activity to moderate (24, EC 50 = 1.74 µg/mL). We next investigated the biological activity of the fragments. All samples showed antifouling profiles with low toxicity against cypris larvae of the barnacle A. amphitrite. Among the fragments examined, compounds Boc-3, 21, and 25 were more active with EC 50 values below 1 µg/mL. Protection of the southern fragment with a Boc group improved the EC 50 value (Boc-3, EC 50 = 0.79 µg/mL). We believe this improvement is due its lower polarity than 3 (EC 50 = 1.17 µg/mL) by protection of the amino group. It was revealed that functional groups at the p-position of the aromatic ring affected the antifouling activity of the southern fragment: a hydroxy group (21, EC 50 = 0.60 µg/mL) had a slightly decreased EC 50 value compared to Boc-3, but a benzyloxy group (22, EC 50 = 4.62 µg/mL) dramatically reduced the antifouling activities to 4.62 µg/mL. These results indicate that steric bulkiness at this position affected the activity. A benzyl ester of the northern fragment (25, EC 50 = 0.90 µg/mL) showed much higher potency than 2 (EC 50 > 10 µg/mL). Again, the less polar fragment was more active than the corresponding more polar one. Interestingly, a benzyl ether (29, EC 50 = 3.27 µg/mL) weakened the antifouling activity, suggesting the importance of the lactate moiety or the presence of a carbonyl group for the northern fragment. The antifouling activities of synthetic samples were evaluated as EC50 values against the cypris larvae of A. amphitrite by exposure of each compound for 48 h (Table 1, Figure  2). For comparison, EC50 values for the previous compounds 1-3 are also shown in the table. Installation of a functional group on the aromatic ring of 1 decreased the antifouling activity to moderate (24, EC50 = 1.74 μg/mL). We next investigated the biological activity of the fragments. All samples showed antifouling profiles with low toxicity against cypris larvae of the barnacle A. amphitrite. Among the fragments examined, compounds Boc-3, 21, and 25 were more active with EC50 values below 1 μg/mL. Protection of the southern fragment with a Boc group improved the EC50 value (Boc-3, EC50 = 0.79 μg/mL). We believe this improvement is due its lower polarity than 3 (EC50 = 1.17 μg/mL) by protection of the amino group. It was revealed that functional groups at the p-position of the aromatic ring affected the antifouling activity of the southern fragment: a hydroxy group (21, EC50 = 0.60 μg/mL) had a slightly decreased EC50 value compared to Boc-3, but a benzyloxy group (22, EC50 = 4.62 μg/mL) dramatically reduced the antifouling activities to 4.62 μg/mL. These results indicate that steric bulkiness at this position affected the activity. A benzyl ester of the northern fragment (25, EC50 = 0.90 μg/mL) showed much higher potency than 2 (EC50 > 10 μg/mL). Again, the less polar fragment was more active than the corresponding more polar one. Interestingly, a benzyl ether (29, EC50 = 3.27 μg/mL) weakened the antifouling activity, suggesting the importance of the lactate moiety or the presence of a carbonyl group for the northern fragment. 0.10 0.63 >10 1 EC50 (50% effective concentration), 2 LC50 (50% leathal concentration), 3 according to [38].

N 2 -Dml-OH 13
Lithium perchlorate (2.12 g, 20.0 mmol), was dissolved in 10 mL anhydrous Et 2 O. TMS-quinine 11 (400 mg, 1.00 mmol) and CH 2 Cl 2 (20 mL) were added to this solution which was then cooled to −40 • C. DIEA (4.36 mL, 25.0 mmol) and isobutyraldehyde (0.920 mL, 10.0 mmol) were then added to the solution. Propionyl chloride (1.74 mL, 20.0 mmol) was dissolved in CH 2 Cl 2 (5.0 mL). The solution of propionyl chloride was then added dropwise to the reaction over the course of 3 h. Upon completion of the addition, the reaction was allowed to stir at −40 • C for 16 h. After this time, Et 2 O was added to the solution. The resulting mixture was filtered through a pad of celite and washed with Et 2 O. The solution was concentrated at a light vacuum and diluted with CH 2 Cl 2 . The solution was washed with sat. NH 4 Cl and brine, dried over Na 2 SO 4 and concentrated at a light vacuum to give crude lactone, which was used in the next step without further purification.
To a solution of the crude lactone in DMSO (30 mL) were added NaN 3 (1.30 g, 20.0 mmol) and NH 4 Cl (535 mg, 10.0 mmol) at room temperature. The mixture was heated at 50 • C, diluted with aqueous HCl, extracted with EtOAc, washed with brine, dried over Na 2 SO 4 , and concentrated in vacuo. The crude product was purified using

Boc-Dpv(OAc)-Pro-Dml-OBn 20
To 15 (1.09 g, 3.40 mmol) was added TFA/CH 2 Cl 2 (1:4 v/v, 25 mL). After 1 h of stirring at room temperature, the solution was concentrated in vacuo to afford crude 19, which was used in the next step without further purification.
To a solution of 17 (550 mg, 1.05 mmol) in CH 3 OH (5.25 mL) was carefully added Pd(OH) 2 /C (110 mg, 20 wt%) under Ar atmosphere. The solution was purged with H 2 gas and stirring was continued under H 2 atmosphere at room temperature for 16 h. The solution was filtered through celite and concentrated in vacuo to afford crude 18, which was used in the next step without further purification.
To a solution of the crude 19 (610 mg, 2.60 mmol) and crude 18 (1.25 g, 2.60 mmol) in CH 3 CN (26 mL) were added DMTMM (740 mg, 2.60 mmol) and Et 3 N (2.17 mL, 15.6 mmol) under Ar atmosphere. After 24 h of stirring at room temperature, the mixture was concentrated in vacuo. The residue was purified using silica gel column chromatography To a solution of 21 (12.1 mg, 0.0194 mmol) in CH 3 CN (0.16 mL) were added K 2 CO 3 (8.90 mg, mmol), BnBr (8.00 µL, 0.0669 mmol) and KI (1.10 mg, 6.63 µmol) under Ar atmosphere at room temperature. The mixture was stirred for 24 h, quenched with sat. NaHCO 3 , extracted with EtOAc, washed with brine, dried over Na 2 SO 4 , filtered, and concentrated in vacuo. The crude product was purified using silica gel column chromatography (Hexane:Acetone = 80:20) to afford 22 (  To 20 (380 mg, 0.570 mmol) was added TFA/CH 2 Cl 2 (1:4 v/v, 19 mL). After 1 h of stirring at room temperature, the solution was concentrated in vacuo to afford crude amine, which was used in the next step without further purification.
To a solution of the crude amine and 2 (340 mg, 0.570 mmol) in CH 3

Dolastatin 16 Acetate 24
To a solution of 23 (110 mg, 0.100 mmol) in CH 3 OH (1.00 mL) was carefully added Pd(OH) 2 /C (22.0 mg, 20 wt%) under Ar atmosphere. The solution was purged with H 2 gas and stirring was continued under H 2 atmosphere at room temperature for 16 h. The solution was filtered through celite and concentrated in vacuo to afford crude carboxylic acid, which was used in the next step without further purification.
A solution of the crude carboxylic acid 10a (9.50 mg, 0.0100 mmol) in CH 3 CN (6.3 mL) was dropwised to a refluxing solution of 2-chloro-1-methylpyridinium iodide (13.0 mg, 0.0130 mmol) and Et 3 N (0.0150 mL, 0.110 mmol) in CH 3 CN (3.1 mL) over a 3 h period via addition funnel. The addition funnel was rinsed with a total of 0.6 mL of CH 3 CN. The mixture was refluxed for overnight. After being cooled to ambient temperature, the mixture was concentrated in vacuo. The residue was purified by silica gel column chromatography on silica gel (Hexane:EtOAc = 20:80) to afford 24 as a colorless oil (

Antifouling Assay
Antifouling assay against larvae of the barnacle Amphibalanus amphitrite was conducted according to the previous literature [18,19,38]. The adult barnacles, A. amphitrite, obtained from oyster farms in Lake Hamana and a pier of Shimizu bay, Shizuoka, were kept in an aquarium at 20 • C and were fed on Artemia salina nauplii. Broods were released as I-II stage nauplii upon immersion in seawater after drying overnight. The nauplii (1.0~3.0 indiv./mL) thus obtained were cultured in 2.0 L filtered (0.2 µm) natural seawater (diluted by DW: salinity 28) containing penicillin G (20 µg/mL) and streptomycin sulfate (30 µg/mL) at 25 • C and were fed on the diatom Chaetoceros gracillis at concentrations of 40 × 10 4 cells/mL. Larvae reached the cyprid stage in 5 days. The cyprids were collected, then stored at 4 • C until use (0-day-old).
The test compounds were dissolved in ethanol and aliquots of the solution (20 µL) were transferred to wells of a 24-well polystyrene culture plates and then air-dried for 3 h at room temperature and CuSO 4 was used as positive compound. Four wells were used for each concentration (0.03, 0.1, 0.3, 1.0, 3.0, 10.0 µg/mL). To each well were added filtered (0.2 mm) natural seawater (2.0 mL, salinity 28) and six 2-day-old cyprids. The plates were kept in the dark at 25 • C for 48 h. The numbers of cyprids that attached, metamorphosed, died, or did not settle were counted under a microscope. Three or four trials were carried out for each concentration. Antifouling activity (EC 50 ) indicates the concentration reducing the larval settlement to 50% of the control (non-treatment) by Probit analysis. Toxicity of compounds were expressed as LC 50 value, which indicates the concentration showing 50% mortality estimated by Probit analysis. If mortality rate did not show over 50% at most hagh concentration (10.0 µg/mL), then LC 50 value was indicated as over 10.0 µg/mL.

Conclusions
In summary, we have developed new methodologies for the derivatives of the two unusual amino acids found in dolastatin 16 through a C-H activation reaction for dolaphenvaline and an enantio-and diastereoselective [2+2] addition reaction for dolamethylleuine. These synthetic routes enabled effective access to especially the southern fragment of dolastatin 16. Many trends of the derivatives towards antifouling activity were exhibited. Specifically, less polar small fragments showed strong antifouling activity against the cypris larvae of A. amphitrite without detectable toxicity, although the whole structure was required for extremely potent activity. These results will be useful toward the development of green antifouling materials, and further studies are in progress in our laboratory.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/md20020124/s1, NMR spectra of synthetic samples. Figure S1: 1 H and 13 C NMR spectra of compound 5. Figure S2: 1 H and 13 C NMR spectra of compound 6. Figure S3: 1 H and 13 C NMR spectra of compound 8. Figure S4: 1 H and 13 C NMR spectra of compound 13. Figure S5: 1 H and 13 C NMR spectra of compound 14. Figure S6: 1 H and 13 C NMR spectra of compound 16. Figure S7: 1 H and 13 C NMR spectra of compound 17. Figure S8: 1 H and 13 C NMR spectra of compound 20. Figure S9: 1 H and 13 C NMR spectra of compound 21. Figure S10: 1 H and 13 C NMR spectra of compound 22. Figure S11: 1 H and 13 C NMR spectra of compound 23. Figure S12: 1 H and 13 C NMR spectra of compound 24. Figure S13: 1 H and 13 C NMR spectra of compound 25. Figure S14

Conflicts of Interest:
The authors declare no conflict of interest.