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Article

Synthesis and Antiproliferative Activity of Marine Bromotyrosine Purpurealidin I and Its Derivatives

1
Drug Research Program, Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, Viikinkaari 5 E (P.O. Box 56), University of Helsinki, FI-00014 Helsinki, Finland
2
Drug Research Program, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, Viikinkaari 5 E (P.O. Box 56), University of Helsinki, FI-00014 Helsinki, Finland
3
Institute of Chemistry, Technische Universität Chemnitz, 09107 Chemnitz, Germany
*
Author to whom correspondence should be addressed.
Current address: Government First Grade College, Chamarajanagar 571313 (Affiliated to University of Mysore), India.
Mar. Drugs 2018, 16(12), 481; https://doi.org/10.3390/md16120481
Received: 6 November 2018 / Revised: 20 November 2018 / Accepted: 27 November 2018 / Published: 3 December 2018
(This article belongs to the Special Issue Marine Bioactive Natural Product Studies in Europe)

Abstract

:
The first total synthesis of the marine bromotyrosine purpurealidin I (1) using trifluoroacetoxy protection group and its dimethylated analog (29) is reported along with 16 simplified bromotyrosine derivatives lacking the tyramine moiety. Their cytotoxicity was evaluated against the human malignant melanoma cell line (A-375) and normal skin fibroblast cells (Hs27) together with 33 purpurealidin-inspired simplified amides, and the structure–activity relationships were investigated. The synthesized simplified analogs without the tyramine part retained the cytotoxic activity. Purpurealidin I (1) showed no selectivity but its simplified pyridin-2-yl derivative (36) had the best improvement in selectivity (Selectivity index 4.1). This shows that the marine bromotyrosines are promising scaffolds for developing cytotoxic agents and the full understanding of the elements of their SAR and improving the selectivity requires further optimization of simplified bromotyrosine derivatives.

1. Introduction

Globally, cancer is the second leading cause of death and in 2018 it is estimated to lead to 9.6 million deaths [1] and malignant melanoma is one of the most life-threatening cancers due to resistance to most therapies [2]. While prevention is important, there is a continuous need for novel treatments. The marine environment provides a potential source for discovering new drug lead molecules, especially against cancer. So far, four medicinal products originating from marine ecosystems have been registered for the treatment of different kinds of cancer such as leukemia, metastatic breast cancer, and ovarian cancer [3,4].
Bromotyrosines are a large and structurally diverse group of bromine-containing marine alkaloids which have shown a variety of biological functions including antimicrobial, antiviral, antifungal and in particular, anticancer activity [5,6,7,8] Bromotyrosines are mainly isolated from the marine sponges of the order Verongida. For representative articles about secondary metabolites of Verongida order, see, for example, Fattorusso group’s work [9,10].
Purpurealidin I (1; Figure 1) together with several other bromotyrosines have been isolated from the Indian sea sponge Pseudoceratina (Psammaplysilla) purpurea [11]. Structurally similar aplysamine 2 (2) was isolated from the Australian marine sponge, Aplysina sp. [12]. Purpurealidin I (1) has been found to be cytotoxic when tested against various cancer cell lines (ovarian cancer A2780S and cisplatin-resistant variant A2780CP (SCP5), non-small cell lung cancer A549, human breast cancer MCF7 and glioma U251MG cells), as well as non-cancer cell line NIH3T3 (normal mouse fibroblasts) [6]. Two other bromotyrosines aplysamine 4 (3) [13] and JBIR-44 (4) [14] were isolated from P. purpurea and have been tested against human cervical carcinoma HeLa cells [5]. A comparably strong cytotoxic effect was observed and there was no difference between the compounds with a longer or shorter alkyl chain attached to the tyramine part. This presents the opportunity for the design of simplified analogs of marine bromotyrosines as the long alkyl chain does not seem to be essential for cytotoxicity. In our previous studies, simplified amide-linked bromotyrosines inspired by purpurealidin I (1) displayed good Kv10.1 channel inhibition [15]. Kv10.1 potassium channel regulates many fundamental functions in a cell, for example cell cycle progression and cellular proliferation [16]. We report here the total synthesis of the marine natural product purpurealidin I (1) and a related tetrabrominated analog of aplysamine 2 (2; also, a dimethyl analog of 1). Medicinal chemistry strategies to simplify their structures are also outlined. Furthermore, we have evaluated these compounds for selective cytotoxic effects to skin cancer cells and discussed their structure-activity relationships.

2. Results

2.1. Chemistry

The purpurealidin I (1) skeleton can be viewed as a secondary amide. The retrosynthetic pathway (Scheme 1) illustrated that the synthesis of the bromotyrosine carboxylic acid part could be initiated from O-methyltyrosine (7) and the corresponding amine part from tyramine (8).
For the synthesis of the bromotyrosine carboxylic acid moiety, commercially available O-methyltyrosine (7) was brominated [17] and subjected to an esterification reaction with SOCl2 in MeOH. The ester (10) was converted to oxime (11) using sodium tungstate and hydrogen peroxide, following a literature procedure (Scheme 2) [17,18,19,20]. The corresponding carboxylic acid subunit (5) was then synthesized via the LiOH-mediated hydrolysis of methyl ester (11) in 90% yield (See the Supplementary Information for the experimental details).
The first attempts of amide coupling were made using the free primary amine (6) (Scheme 3). Tyramine (8) underwent bromination followed by N-Boc protection to give (13) [19,20,21,22,23] which was then subjected to O-alkylation with Boc-protected 3-chloro-N-methylpropan-1-amine (15) [24] to give (14) in 83% yield. The trifluoroacetic acid (TFA)-mediated Boc deprotection of (14) gave the diamine (6) in quantitative yield.
The direct coupling of compounds (5) and (6) with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was unsuccessful. This was likely due to the interfering free hydroxy group within (5). A condensation reaction of ester (11) with compound (6) also proved unsuccessful. Several alternative coupling conditions were tried (Supplementary Information, Table S1) Unfortunately, each resulted in the formation of inseparable mixtures with poor yields of the desired product, purpurealidin I (1), as determined by 1H NMR and LC-MS analyses. After several additional approaches proved unsuccessful (data not shown), trifluoroacetyl was found to be a suitable protecting group for the secondary amine (Scheme 4). The Boc-protected bromo tyramine (13) was O-alkylated with (18) to produce (16). Treatment of (16) with TFA led to the selective Boc deprotection and led to the formation of the desired target amine (17) in 77% overall yield [15].
An initial attempt to synthesize the immediate precursor of purpurealidin I (1) by direct condensation of (17) with hydroxylamine ester (11) was unsuccessful. This was likely due to the presence of the hydroxylamino moiety. We then decided to introduce the free hydroxylamino group after the amide coupling. The bromotyrosine carboxylic acid fragment (22a) was prepared via the Erlenmeyer–Plöchl azlactone method (Scheme 5) in 84% overall yield [25,26]. This procedure was also used for the preparation of the carboxylic acid fragments (22bd) in the mono-brominated, mono-chlorinated and non-halogenated simplified derivatives (4145). Reddy et al. have reported the synthesis of methyl carbamate containing bromotyrosine purpuramine K leaving the tetrahydropyranyl (THP) group to the molecule [22].
The synthesis of purpurealidin I (1), began with the bromination of p-hydroxybenzaldehyde, followed by methylation to give (19a). The requisite azlactone (20a) was then prepared by the condensation of (19a) with N-acetylglycine (Scheme 5) and subjecting the resulting product to hydrolysis using a 10% aqueous solution of HCl to give pyruvic acid (21a). Compound (21a) was then converted into the THP-protected oxime (22a) by reaction with O-(tetrahydropyran-2-yl)-hydroxylamine (THPONH2). The crude oxime was then subjected to EDC coupling with (17) under microwave conditions to produce (26) in a moderate yield (56%; Scheme 6). The THP group was removed with a 2 M solution of HCl in Et2O to give the free oxime (28). Trifluoroacetyl mediated deprotection of (28) using MeOH/K2CO3 resulted in purpurealidin I (1) in an overall yield of 12% (11 steps). The aplysamine 2 tetrabromo derivative (29), with a dimethylamino moiety at the tyramine fragment, was synthesized using purpurealidin E (25) [15,27] in the coupling with (22a) (Scheme 6).
The simplified derivatives of purpurealidin I (1) were prepared in an analogous manner (Scheme 5) with the appropriate anilines and benzylamines (Table 1) followed by THP deprotection. The yields of the amide couplings ranged from 19–87%. The THP deprotection was achieved using TFA for the various simplified derivatives of (1), as heating with 2 M HCl in Et2O proved sluggish. Several different conditions were attempted in the synthesis of compound (31) (see Experimental Section 4.1.2). After purification on silica gel, the yields of the final hydroxyimino propanamides (3045) ranged from 13–50%.
Before finalizing the purpurealidin I (1) synthesis, several synthetically simpler amide analogs containing the tyramine fragment and a series of compounds with substituted phenyl rings (Ar in Table 2) instead of the bromotyrosine part were synthesized and screened. The synthesis of these simplified amide derivatives (4678) (Table 2) have previously been reported by our group [15].

2.2. Stereochemistry

It has previously been reported that the configuration of the N-oxime can be determined by analysis of the 13C NMR C-8 carbon shifts, which are known to be approximately 26–30 ppm (E configuration) or over 35 ppm (Z configuration) [28]. The reported X-ray structure of a disulfide-bridged psammaplin A analog supported this observation [29]. We, therefore, expect the stereochemistry of all bromotyrosines synthesized herein to be E, since the benzylic C-8 shifts in 13C NMR were around 28 ppm. This was supported by the crystal structure of pyridin-2-yl analog (36), which was determined by the single crystal X-ray diffraction (Figure 2; see Supplementary Information for experimental details and crystallographic data). An intramolecular hydrogen bond between the secondary amide hydrogen atom H2 and the lone electron pair of oxime nitrogen atom N1 could explain the observed E geometry of the oxime.

2.3. Biological Activity

The cytotoxicity of the synthetic purpurealidin I (1) and compounds (2978) against cancer cells was primarily evaluated in human malignant melanoma A-375 cell line at the single concentration of 50 µM (Table 3). The compounds demonstrating over 80% cytotoxicity were selected for confirmatory dose-response experiments in the same cell line, and CC50 (cytotoxic concentration that caused death of 50% cells) was calculated (Table 3). We furthermore aimed to evaluate the potential of the compounds to selectively perturb the growth of cancer cells. Therefore, the compounds with the highest cytotoxic activities (CC50 below 15 µM) were studied for cytotoxicity in normal human fibroblast cell line Hs27 (Table 3). The degree of selectivity towards cancer cells can be expressed by selectivity index (SI). High values show selectivity towards cancer cells, while values <2 suggest general cytotoxicity of the compound [30]. Camptothecin, a naturally occurring alkaloid with known high selectivity to cancer cells (SI 92.3, Table 3) was used as a positive control. Most of the compounds demonstrated general cytotoxicity (SI < 2, Table 3). The highest selectivity to cancer cells (SI 4.1, Table 3) was shown for the compound (36).

3. Discussion

SAR

The observed melanoma cell line activity for the compounds was in the range of 4–43 µM (CC50 in A-375 cells, Table 3). Both Purpurealidin I (1) and its dimethylated analog (29) showed cytotoxicity to melanoma cells (CC50 4.3 and 6.3 μM, respectively). Maintaining the hydroxylamine linker but replacing the longer tyramine fragment with the aniline moiety (e.g., (36) 4.7 μM) or benzyl amine with a one-carbon chain (e.g., (31) 12.4 μM) retained the activity. This indicates that the tyramine part is not essential for the activity. The cytotoxic activity of the pyridine derivatives with a hydroxyimino amide was found to be in the order of pyridin-2-yl (36) > pyridin-3-yl (37) > pyridin-4-yl (38). Furthermore, pyridin-3-yl methyl amide (39) retained the activity. Two bromine atoms in the tyrosine part seem to be essential for the cytotoxicity since the non-halogenated (44) or mono-halogenated (42 and 45) pyridin-2-yls were inactive. However, compound (41) with two mono-brominated p-methoxyphenyl rings showed activity even though the mono-brominated (42), with pyridin-2-yl group, was inactive. This may imply a different binding mode exists for this bis mono-brominated p-methoxyphenyl compound. However, both structural data of the binding sites and the mechanism of action are currently unknown, and cytotoxicity of these compounds cannot be accounted for.
The synthesis of the simplified amides with the tyramine end allowed for the more feasibly exploration of the aromatic substituents at the tyrosine part of the molecule. The CC50 (A-375 cells) values were retained with the best compounds, monomethylamino m-dichloro-p-methoxy (65) (6.4 μM) and m-iodo-p-methoxy (74) (6.2 μM). Non-halogenated amides in the tyrosine part (59) and (61) were also inactive, and o-bromo (46) or o-fluoro (57) substitution resulted in low activity. The CC50 values did not show a significant difference when compared the monomethylated amines at the end of the tyramine part to the dimethylated ones (e.g., CC50 in A-375 cells 6.2 μM for (74) and 8.4 μM for (73)). Replacement of the amino group in the tyramine end to isopropyl in compounds (49) and (52), as well as the addition of the morpholine moiety in (55) resulted in the loss of the activity.
Purpurealidin I (1) and its dimethylated analog (29) showed no selectivity in cytotoxicity between melanoma A-375 cell line and normal human fibroblast cell line Hs27 (SI 1.2 and 0.7, respectively, Table 3). Changing the linker from hydroxyimino amide to amide did not improve the selectivity, and different aromatic substitution on the tyrosine fragment also had no effect (SI’s varied between 0.5–1.3) However, when the longer tyramine part was replaced with directly attached aniline, some improvement in the selectivity was observed (1 SI 1.2 compared to 33 SI 2.0 or 36 SI 4.1). The pyridin-2-yl compound 36 displayed the best, albeit only moderate, selectivity (SI 4.1, Table 3).

4. Materials and Methods

4.1. Synthesis Experimental

4.1.1. General

All reactions were carried out using commercially available starting materials unless otherwise stated. The melting points were measured using a Stuart SMP40 automated melting point apparatus and are uncorrected. 1H NMR (300 MHz) and 13C NMR (75 MHz) spectra were measured in CDCl3, d6-DMSO, CD3OD, or d6-acetone at room temperature and were recorded on a Varian Mercury Plus 300 spectrometer or Bruker AV400 MHz NMR with smart probe. Chemical shifts (δ) are given in parts per million (ppm) relative to the 1H and 13C NMR reference solvent signals (CDCl3: 7.26 and 77.16 ppm; CD3OD: 3.31 and 49.00 ppm; d6-DMSO: 2.50 ppm and 39.52; d6-acetone: 2.05 and 29.84 ppm). Multiplicities are indicated by s (singlet), br s (broad singlet), d (doublet), dd (doublet of doublet), ddd (doublet of doublet of doublets), t (triplet), dt (doublet of triplets), q (quartet) and m (multiplet). The coupling constants J are quoted in Hertz (Hz). LC-MS and HRMS-spectra were recorded using a Waters Acquity UPLC®-system (Milford MA, USA) with Acquity UPLC® BEH C18 column (1.7 µm, 50 × 2.1 mm, Waters, Wexford, Ireland) with Waters Synapt G2 HDMS (Milford MA, USA) with the ESI (+), high resolution mode. The mobile phase consisted of H2O (A) and acetonitrile (B) both containing 0.1% HCOOH. Microwave synthesis were performed in sealed tubes using Biotage Initiator+ instrument equipped with an external IR sensor. The flash chromatography was performed with Biotage SP1 flash chromatography purification system with 254 nm UV-detector or Biotage Isolera™ Spektra Systems with 200–800 nm UV-detector using SNAP 10, 25, 50 or 100 g cartridges (Uppsala, Sweden). The TLC-plates were provided by Merck (Darmstadt, Germany, Silica gel 60-F254) and visualization of the amine compounds was done using ninhydrin staining and THP ethers with vanillin staining.

4.1.2. Experimental Procedures

General Procedure for the Formation of Azlactones

Aldehyde 19ad, acetylglycine (1.5 equiv.) and anhyd. NaOAc (1.5 equiv.) were dissolved in Ac2O (10–15 mL) and the reaction mixture was stirred at 80 °C overnight. Afterwards, the reaction mixture was cooled to room temperature and poured into water (50 mL). The formed precipitate was filtered, washed with water (4 × 20 mL) and dried in vacuo. The obtained crude product 20ad was used in the subsequent step without further purification.
Marinedrugs 16 00481 i001
4-(3,5-Dibromo-4-methoxybenzylidene)-2-methyloxazol-5(4H)-one (20a).
3,5-Dibromo-4-methoxybenzaldehyde 19a (1.57 g, 5.33 mmol) gave 20a as a grey solid (1.94 g, 97%). 1H NMR (400 MHz, CDCl3) δ 8.26 (s, 2H), 6.92 (s, 1H), 3.93 (s, 3H), 2.43 (s, 3H). 1H NMR is in accordance with the literature [26].
Marinedrugs 16 00481 i002
4-(3-Bromo-4-methoxybenzylidene)-2-methyloxazol-5(4H)-one (20b).
3-Bromo-4-methoxybenzaldehyde 19b (2.00 g, 9.30 mmol) gave 20b as a yellow solid (2.70 g, 98%) 1H NMR (400 MHz, CDCl3) δ 8.43 (d, J = 2.1 Hz, 1H), 7.98–7.94 (m, 1H), 7.02 (s, 1H), 6.95 (d, J = 8.6 Hz, 1H), 3.96 (s, 3H), 2.41 (d, J = 0.7 Hz, 3H). 1H NMR is in accordance with the literature [25].
Marinedrugs 16 00481 i003
4-(3-Chloro-4-methoxybenzylidene)-2-methyloxazol-5(4H)-one (20c).
3-Chloro-4-methoxybenzaldehyde 19c (2.43 g, 14.3 mmol) gave 20c as a yellow solid (2.66 g, 74%). 1H NMR (400 MHz, CDCl3) δ 8.28 (d, J = 2.1 Hz, 1H), 7.89 (dd, J = 8.6, 2.1 Hz, 1H), 7.02 (s, 1H), 6.98 (d, J = 8.6 Hz, 1H), 3.97 (s, 3H), 2.41 (s, 3H).
Marinedrugs 16 00481 i004
4-(4-Methoxybenzylidene)-2-methyloxazol-5(4H)-one (20d).
4-Methoxybenzaldehyde 19d (3.03 g, 22.3 mmol) gave 20d as a yellow solid (2.21 g, 46%). 1H NMR (400 MHz, CDCl3) δ 8.06 (d, J = 8.5 Hz, 2H), 7.11 (s, 1H), 6.96 (d, J = 8.9 Hz, 2H), 3.87 (s, 3H), 2.39 (s, 3H).

General Method for the Hydrolysis of Azlactones

Azlactone 20ad was dissolved in a 10% solution of HCl in H2O (30 mL). A capillary tube was introduced in the flask to allow the reflux despite a solid layer forming while heating. The reaction mixture was refluxed overnight. The reaction mixture was cooled to room temperature and poured into cold water (2 × 10 mL). The resulting precipitate was filtered, washed with water (4 × 20 mL) and dried in vacuo. The obtained crude product 21ad was used in the subsequent step without further purification.
Marinedrugs 16 00481 i005
3-(3,5-Dibromo-4-methoxyphenyl)-2-oxopropanoic acid (21a)
Azlactone 20a (1.74 g, 4.63 mmol) gave acid 21a as a yellowish solid (1.46 g, 89%). 1H NMR (400 MHz, d6-DMSO) δ 8.06 (s, 2H), 3.80 (s, 3H), 3.32 (s, 2H); 13C NMR (101 MHz, d6-DMSO) δ 165.7, 151.8, 143.3, 134.5, 132.9, 117.3, 106.1, 60.5. NMR showed the enol tautomer. 1H NMR is in accordance with the literature [25].
Marinedrugs 16 00481 i006
3-(3-Bromo-4-methoxyphenyl)-2-oxopropanoic acid (21b)
Azlactone 20b (3.33 g, 9.11 mmol) gave 21b as a dark red solid (1.89 g, 79%). 1H NMR (400 MHz, d6-DMSO) δ 9.26 (bs, 1H), 8.10 (d, J = 2.1 Hz, 1H), 7.69–7.64 (m, 1H), 7.10 (d, J = 8.7 Hz, 1H), 6.35 (s, 1H), 3.85 (s, 3H). NMR showed the enol tautomer. 1H NMR is in accordance with the literature [25].
Marinedrugs 16 00481 i007
3-(3-Chloro-4-methoxyphenyl)-2-oxopropanoic acid (21c)
Azlactone 20c (2.66 g, 10.6 mmol) gave 21c as a brown solid (1.92 g, 79%). 1H NMR (400 MHz, CD3OD) δ 7.91 (d, J = 2.1 Hz, 1H), 7.59 (ddd, J = 8.6, 2.1, 0.5 Hz, 1H), 7.03 (d, J = 8.7 Hz, 1H), 6.40 (s, 1H), 3.89 (s, 3H). NMR showed the enol tautomer.
Marinedrugs 16 00481 i008
3-(4-Methoxyphenyl)-2-oxopropanoic acid (21d)
Azlactone 20d (2.21 g, 10.2 mmol) gave product 21d as a brown solid (1.78 g, 90%). 1H NMR (400 MHz, CDCl3) δ 7.75 (d, J = 8.7 Hz, 2H), 6.92 (d, J = 8.9 Hz, 2H), 6.64 (s, 1H), 3.84 (s, 3H). NMR showed the enol tautomer. 1H NMR is in accordance with the literature [31].

General Procedure for THP-Protection

Carboxylic acid 21ad and THPONH2 (2 equiv.) were dissolved in dry ethanol (15–20 mL). The reaction mixture was stirred at room temperature for 18–48 h under argon atmosphere. The reaction mixture was concentrated under reduced pressure and then EtOAc (20 mL) was added to the residue. The organic layer was washed with a 2 M solution of HCl in H2O (2 × 20 mL). The aqueous layer was extracted back with EtOAc (2 × 10 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo. The obtained crude product 22ad was used in the subsequent step without further purification.
Marinedrugs 16 00481 i009
(E)-3-(3,5-Dibromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanoic acid (22a)
Carboxylic acid 21a (1.16 g, 3.30 mmol) and THPONH2 (0.97 g, 8.3 mmol, 2 equiv.) were used. The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, gradient elution: (DCM/MeOH, 0→10%) to give 22a as a yellow oil (1.44 g, 97%). 1H NMR (400 MHz, CDCl3) δ 7.48 (s, 2H), 5.50–5.41 (m, 1H), 3.95–3.88 (m, 2H), 3.84 (s, 3H), 3.69–3.54 (m, 2H), 1.93–1.83 (m, 2H), 1.77–1.65 (m, 2H), 1.64–1.58 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 163.7, 163.3, 153.2, 149.9, 133.6, 118.2, 102.2, 62.4, 60.8, 29.6, 28.1, 24.9, 18.6.
Marinedrugs 16 00481 i010
(E)-3-(3-Bromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanoic acid (22b)
Carboxylic acid 21b (2.00 g, 7.32 mmol) and THPONH2 (1.71 g, 14.6 mmol, 2 equiv.) were used. 22b was obtained as an oil (2.74 g, quant.). 1H NMR (400 MHz, CDCl3) δ 7.54 (d, J = 2.2 Hz, 1H), 7.23 (dd, J = 2.2, 8.5 Hz, 1H), 6.81 (d, J = 8.5 Hz, 1H), 5.46 (d, J = 3.1 Hz, 1H), 3.94–3.87 (m, 2H), 3.86 (s, 3H), 3.69–3.65 (m, 2H), 1.91–1.81 (m, 2H), 1.76–1.66 (m, 2H), 1.65–1.56 (m, 2H).
Marinedrugs 16 00481 i011
(E)-3-(3-Chloro-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanoic acid (22c)
Carboxylic acid 21c (1.86 g, 8.13 mmol) and THPONH2 (1.90 g, 16.3 mmol, 2 equiv.) were used. 22c was obtained as a yellow oil (3.05 g, quant). 1H NMR (400 MHz, CDCl3) δ 7.36 (d, J = 2.2 Hz, 1H), 7.18 (dd, J = 8.4, 2.2 Hz, 1H), 6.84 (d, J = 8.4 Hz, 1H), 5.47 (d, J = 3.1 Hz, 1H), 3.87 (s, 3H), 3.86 (s, 2H), 3.63 (dd, J = 7.6, 3.4 Hz, 2H), 1.91–1.82 (m, 2H), 1.77–1.66 (m, 2H), 1.65–1.52 (m, 2H).
Marinedrugs 16 00481 i012
(E)-3-(4-Methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanoic acid (22d)
Carboxylic acid 21d (1.73 g, 7.98 mmol) and THPONH2 (1.87 g, 16.0 mmol, 2 equiv.) were used. 22d was obtained as a yellow oil (2.78 g, quant). 1H NMR (400 MHz, CDCl3) δ 7.23 (d, J = 8.7 Hz, 2H), 6.81 (d, J = 8.7 Hz, 2H), 5.47–5.43 (t, 1H), 3.87 (s, 2H), 3.76 (s, 3H), 3.67–3.59 (m, 2H), 1.93–1.80 (m, 2H), 1.72–1.65 (m, 2H), 1.60–1.51 (m, 2H).
Marinedrugs 16 00481 i013
(E)-N-[3,5-Dibromo-4-[3-(2,2,2-trifluoro-N-methylacetamido)propoxy]phenethyl]-3-(3,5-dibromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanamide (26)
A 20 mL Biotage MW tube was charged with carboxylic acid 22a (0.24 g, 0.53 mmol), amine 17 [15] (0.25 g, 0.53 mmol), EDC·HCl (0.15 g, 0.79 mmol, 1.5 equiv.), HOBt (0.11 g, 0.79 mmol, 1.5 equiv.), DIPEA (0.10 g, 0.79 mmol, 1.5 equiv.) and dry DCM (15 mL). The reaction was MW irradiated for 2 h at 60 °C (5 bar). The reaction mixture was diluted with DCM (25 mL), washed with water (2 × 15 mL) and a 1 M solution of HCl in H2O (15 mL). The organic layer was dried over Na2SO4 and concentrated in vacuo. The crude product was purified by flash chromatography twice, 25 g, gradient elution: (heptane/EtOAc, 0→40%) to 23 as a pale yellow viscous liquid (0.16 g, 34%). 1H NMR (400 MHz, CDCl3) δ 7.50 (s, 2H), 7.34 and 7.33 (2 s, 2H, rotamers ratio 2:1), 6.92 (t, J = 6.2 Hz, 1H), 5.38 (t, J = 2.8 Hz, 1H), 4.06–4.00 (m, 2H), 3.93 (d, J = 13.1 Hz, 1H), 3.84 (s, 3H), 3.83 (d, J = 13.1 Hz, 1H), 3.80–3.72 (m, 2H), 3.65–3.54 (m, 3H), 3.43 (ddt, J = 12.9, 8.2, 6.6 Hz, 1H), 3.23 (m) and 3.11 (m) N-CH3 rotamers 2:1, 2.77 (td, J = 7.3, 1.7 Hz, 2H), 2.23–2.09 (m, 2H), 1.89–1.54 (m, 6H).
Marinedrugs 16 00481 i014
(E)-N-[3,5-Dibromo-4-[3-(2,2,2-trifluoro-N-methylacetamido)propoxy]phenethyl]-3-(3,5-dibromo-4-methoxyphenyl)-2-(hydroxyimino)propanamide (28)
THP ether 26 (0.14 g, 0.16 mmol), a 2 M solution of HCl in Et2O (4 mL), dry DCM (4 mL) and dry MeOH (0.3 mL) were added to a 20-mL sealed tube and heated in an oil bath at 70 °C for 3 h. The reaction mixture was then concentrated in vacuo. The crude product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 10 g, gradient elution: (heptane/EtOAc, 0→30%) to give 28 as a pale yellow viscous liquid (0.068 g, 54%). 1H NMR (400 MHz, CD3OD) δ 7.48 (s, 2H), 7.43 and 7.42 (2 s, 2H, rotamers ratio 2:1), 4.00 (t, J = 6.0 Hz, 2H), 3.82 (m, 5H), 3.78–3.70 (m, 2H), 3.44 (t, J = 7.1 Hz, 2H), 3.24 (q) and 3.10 (m) N-CH3 rotamers 2:1, 2.76 (t, J = 7.1 Hz, 2H), 2.25–2.08 (m, 2H).
Marinedrugs 16 00481 i015
(E)-N-[3,5-Dibromo-4-[3-(methylamino)propoxy]phenethyl]-3-(3,5-dibromo-4-methoxyphenyl)-2-(hydroxyimino)propenamide, purpurealidin I, (1)
Compound 28 (0.050 g, 0.062 mmol) and K2CO3 (0.018 g, 0.12 mmol, 2.0 equiv.) in MeOH (5 mL) and H2O (0.5 mL) were refluxed for 2.5 h. The reaction mixture was concentrated in vacuo and partitioned between EtOAc (10 mL) and water (4 mL). The aqueous layer was back-extracted with EtOAc (10 mL). The combined organic layers ware dried over Na2SO4, filtered and the solvent was removed in vacuo to give 1, as a pale yellow viscous liquid (0.040 -g, 91%). 1H NMR (300 MHz, CDCl3) δ 7.49 (s, 2H), 7.26 (s, 2H), 4.16–4.11 (m, 2H), 3.84–3.83 (m, 5H), 3.42–3.35 (m, 2H), 2.93 (t, J = 7.2 Hz, 2H), 2.71 (t, J = 6.9 Hz, 2H), 2.53 (s, 3H), 2.09–2.00 (m, 2H). 13C NMR (75 MHz, CDCl3) δ 163.6, 152.5, 151.3, 150.4, 138.2, 135.9, 133.5, 133.0, 118.3, 117.8, 71.8, 60.7, 49.0, 40.0, 35.5, 34.3, 29.0, 28.0. HRMS (ESI+): calcd. for C22H26N3O4Br4 [M + H]+, 711.8657; found, 711.8660.
Marinedrugs 16 00481 i016
(E)-N-[3,5-Dibromo-4-[3-(dimethylamino)propoxy]phenethyl]-3-(3,5-dibromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanamide (27)
A 20-mL Biotage MW tube was charged with purpurealidin E 25 [15,27] (0.17 g, 0.44 mmol), carboxylic acid 22a (0.20 g, 0.44 mmol), EDC·HCl (0.13 g, 0.66 mmol, 1.5 equiv.), HOBt (0.10 g, 0.66 mmol, 1.2 equiv.), DIPEA (0.12 mL, 0.66 mmol, 1.2 equiv.) and dry DCM (15 mL). The tube was sealed and microwave irradiated at 60 °C for 5 h. The reaction mixture was diluted with DCM (10 mL) and washed with water (2 × 15 mL), a 1 M solution of HCl in H2O (15 mL) and brine (15 mL). The organic phase was dried over Na2SO4 (anhyd.), filtered and volatiles were removed in vacuo. The crude product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, isocratic elution: (DCM/MeOH, 7:3) to give 27 as a yellow oil (0.30 g, 83%). 1H NMR (400 MHz, CDCl3) δ 7.48 (s, 2H), 7.31 (s, 2H), 6.94 (t, J = 6.2 Hz, 1H), 5.36 (t, J = 2.8 Hz, 1H), 4.03 (t, J = 5.7 Hz, 2H), 3.89 (s, 1H), 3.82 (s, 3H), 3.58 (dd, J = 4.9, 8.1 Hz, 3H), 3.42 (m, 1H), 3.23–3.10 (m, 2H), 2.75 (t, J = 7.3 Hz, 2H), 2.69 (s, 6H), 2.29 (m, 2H), 1.89–1.59 (m, 6H).
Marinedrugs 16 00481 i017
(E)-N-[3,5-Dibromo-4-[3-(dimethylamino)propoxy]phenethyl]-3-(3,5-dibromo-4-methoxyphenyl)-2-(hydroxyimino)propanamide (29)
THP ether 27 (0.30 g, 0.36 mmol) was dissolved to dry DCM (7 mL), and TFA (3 mL) was added under argon atmosphere. The reaction mixture was stirred at room temperature for 17 h. It was then diluted by adding DCM (10 mL) and washed with a 2 M solution of NaOH in H2O (15 mL) and water (15 mL) until pH was neutral. The aqueous phase was back extracted with DCM (10 mL) and the combined organic phase was dried over Na2SO4, filtered and concentrated in vacuo to give crude product (0.21 g, 80%). The crude product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, gradient elution: (DCM/MeOH, 2→20%) to give 29 as a yellow oil (0.074 g, 28%). 1H NMR (400 MHz, CD3OD) δ 7.48 (s, 2H), 7.43 (s, 2H), 4.02 (t, J = 5.9 Hz, 2H), 3.82 (s, 3H), 3.44 (dd, J = 6.7, 7.6 Hz, 2H), 3.35 (s, 2H), 3.15–2.98 (m, 2H), 2.85–2.69 (m, 2H), 2.64 (s, 6H), 2.15 (dq, J = 5.8, 7.7 Hz, 2H). 13C NMR (101 MHz, CD3OD) δ 165.4, 153.8, 152.5, 152.1, 140.0, 137.4, 134.5, 134.4, 118.8, 118.6, 71.8, 61.0, 57.3, 44.6, 41.4, 35.2, 28.8, 27.7. HRMS (ESI+): calcd. for C23H27Br4N3O4 [M+H]+ 725.8813, found: 725.8809.

General Method for the Amide Coupling

Carboxylic acid (0.20 g), aniline or amine (1–1.5 equiv.), EDC⋅HCl (1.5 equiv.), HOBt (1.5 equiv.), and DIPEA (1.5 equiv.) were dissolved in dry DCM (5 mL). The mixture was irradiated by microwaves for 2 h at 60 °C. The TLC indicated the completion of the reaction using vanillin as a visualization reagent. The reaction mixture was diluted with DCM (20 mL) and washed with water (2 × 15 mL) and a 2 M solution of HCl in H2O (2 × 15 mL). The organic layer was dried over anhyd. Na2SO4, filtered and concentrated in vacuo. The products were purified with flash column chromatography.
Marinedrugs 16 00481 i018
(E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(3,4-dichlorophenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]-imino]propanamide (30-THP)
Carboxylic acid 22a (0.20 g, 0.44 mmol) and 3,4-dichloroaniline (0.11 g, 0.67 mmol, 1.5 equiv.) were reacted according to the general procedure for amide coupling. The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g and Ultra 25 g, gradient elution: (heptane/EtOAc, 12→100%) to give 30-THP as a crude product (0.19 g, 73%). 1H NMR (400 MHz, CDCl3) δ 8.64 (s, 1H), 7.87 (d, J = 1.9 Hz, 1H), 7.52 (s, 2H), 7.41–7.38 (m, 2H), 5.49 (t, J = 2.6 Hz, 1H), 3.99 (d, J = 13.1 Hz, 1H), 3.89 (d, J = 13.1 Hz, 1H), 3.84 (s, 3H), 3.65–3.59 (m, 2H), 1.93–1.59 (m, 6H).
Marinedrugs 16 00481 i019
(E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(3,4-dichlorobenzyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]-imino]propanamide (31-THP)
Carboxylic acid 22a (0.20 g, 0.44 mmol) and 3,4-dichlorobenzylamine (0.090 mL, 0.67 mmol, 1.5 equiv.) were reacted according to the general procedure for amide coupling. The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, gradient elution: (heptane/EtOAc, 7→60%) to give 31-THP as a light yellow solid (0.23 g, 84%). 1H NMR (400 MHz, CDCl3) δ 7.50 (s, 2H), 7.39 (d, J = 8.2 Hz, 1H), 7.37 (d, J = 2.0 Hz, 1H), 7.19 (t, J = 5.9 Hz, 1H), 7.12 (dd, J = 2.1, 8.2 Hz, 1H), 5.38 (s, 1H), 4.54 (dd, J = 6.7, 15.2 Hz, 1H), 4.35 (dd, J = 5.8, 15.2 Hz, 1H), 3.95 (d, J = 13.1 Hz, 1H), 3.85 (d, 13.1 Hz, 1H), 3.84 (s, 1H), 3.61–3.55 (m, 2H), 1.87–1.56 (m, 6H).
Marinedrugs 16 00481 i020
(E)-N-[4-Chloro-3-(trifluoromethyl)phenyl]-3-(3,5-dibromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanamide (32-THP)
Carboxylic acid 22a (0.19 g, 0.42 mmol) and 4-chloro-3-(trifluoromethyl)aniline (0.12 g, 0.63 mmol, 1.5 equiv.) were reacted according to the general procedure for amide coupling. The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, gradient elution: (heptane/EtOAc, 7→60%). to give 32-THP as an oil (0.19 g, 72%). 1H NMR (400 MHz, CDCl3) δ 8.73 (s, 1H), 7.95 (d, J = 2.6 Hz, 1H), 7.79 (dd, J = 2.6, 8.7 Hz, 1H), 7.52 (s, 2H), 7.46 (d, J = 8.8 Hz, 1H), 5.50 (t, J = 2.7 Hz, 1H), 4.00 (d, J = 13.2 Hz, 1H), 3.90 (d, J = 13.2 Hz, 1H), 3.84 (s, 3H), 3.66–3.59 (m, 2H), 1.95–1.66 (m, 6H).
Marinedrugs 16 00481 i021
(E)-N-(3-Chloro-4-methoxyphenyl)-3-(3,5-dibromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanamide (33-THP)
Carboxylic acid 22a (0.20 g, 0.44 mmol) and 3-chloro-4-methoxyaniline (0.11 g, 0.67 mmol, 1.5 equiv.) were reacted according to the general procedure for amide coupling. The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, gradient elution: (hexane/EtOAc, 7→60%) to give 33-THP as a light yellow solid (0.20 g, 77%). 1H NMR (400 MHz, CDCl3) δ 8.51 (s, 1H), 7.70 (d, J = 2.6 Hz, 1H), 7.53 (s, 2H), 7.44 (dd, J = 2.6, 8.9 Hz, 1H), 6.89 (d, J = 8.9 Hz, 1H), 5.50–5.46 (m, 1H), 3.99 (d, J = 13.2 Hz, 1H), 3.89 (d, J = 13.2 Hz, 1H), 3.89 (s, 3H), 3.84 (s, 3H), 3.65–3.60 (m, 2H), 1.92–1.66 (m, 6H).
Marinedrugs 16 00481 i022
(E)-N-(3-Bromo-4-methoxyphenyl)-3-(3,5-dibromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanamide (34-THP)
Carboxylic acid 22a (0.20 g, 0.44 mmol) and 3-bromo-4-methoxyaniline (0.13 g, 0.67 mmol, 1.5 equiv.) were reacted according to the general procedure for amide coupling. The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, gradient elution: (heptane/EtOAc, 7→60%) to give 34-THP as a light yellow solid (0.20 g, 71%). 1H NMR (400 MHz, CDCl3) δ 8.51 (s, 1H), 7.85 (d, J = 2.6 Hz, 1H), 7.53 (s, 2H), 7.51 (dd, J = 2.6, 8.9 Hz, 1H), 6.86 (d, J = 8.9 Hz, 1H), 5.47 (d, J = 2.9 Hz, 1H), 3.99 (d, J = 13.1 Hz, 1H), 3.92–3.87 (m, 4H), 3.84 (s, 3H), 3.65–3.60 (m, 2H), 1.91–1.66 (m, 6H).
Marinedrugs 16 00481 i023
(E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(3,5-dichlorophenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]-imino]propenamide (35-THP)
Carboxylic acid 22a (0.20 g, 0.44 mmol) and 3,5-dichloroaniline (0.11 g, 0.67 mmol, 1.5 equiv.) were reacted according to the general procedure for amide coupling. The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g and SNAP KP-NH 11g, gradient elution: (heptane/EtOAc, 7→60%). to give 35-THP as a yellow oil (0.18 g, 69%). 1H NMR (400 MHz, CDCl3) δ 8.64 (s, 1H), 7.58 (d, J = 1.8 Hz, 2H), 7.52 (s, 2H), 7.12 (t, J = 1.8 Hz, 1H), 5.48 (t, J = 2.7 Hz, 1H), 3.98 (d, J = 13.2 Hz, 1H), 3.88 (d, J = 13.2 Hz, 1H) 3.84 (s, 3H), 3.65–3.59 (m, 2H), 1.92–1.66 (m, 6H).
Marinedrugs 16 00481 i024
(E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(pyridin-2-yl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino] propanamide (36-THP)
Carboxylic acid 22a (0.20 g, 0.44 mmol) and 2-aminopyridine (0.063 g, 0.67 mmol, 1.5 equiv.) were reacted according to the general procedure for amide coupling. The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, gradient elution: (hexane/EtOAc, 12→60%) to give 36-THP as a light yellow solid (0.11 g, 45%). 1H NMR (400 MHz, CDCl3) δ 9.26 (s, 1H), 8.32–8.28 (m, 1H), 8.24 (d, J = 8.4 Hz, 1H), 7.76–7.70 (m, 1H), 7.53 (s, 2H), 7.06 (ddd, J = 0.8, 4.9, 7.3 Hz, 1H), 5.47 (t, J = 2.9 Hz, 1H), 3.99 (d, J = 13.2 Hz, 1H), 3.89 (d, J = 13.2 Hz, 1H), 3.83 (s, 3H), 3.64–3,61 (m, 2H), 1.91–1.64 (m, 6H). 13C NMR (100 MHz, CDCl3) δ 160.5, 153.0, 151.8, 150.7, 148.1, 138.6, 134.6, 133.7, 120.2, 118.0, 114.2, 102.3, 62.5, 60.7, 28.8, 28.5, 25.1, 19.0. HRMS (ESI+): calculated 525.9977 (C20H22Br2N3O4), found 525.9972.
Marinedrugs 16 00481 i025
(E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(pyridin-3-yl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino] propanamide (37-THP)
Carboxylic acid 22a (0.42 g, 0.94 mmol), 3-aminopyridine (0.11 g, 1.12 mmol, 1.2 equiv.) were reacted according to the general procedure for amide coupling. The product was purified by column chromatography Biotage SNAP Cartridge KP-Sil 25 g eluent (DCM/MeOH (0→10%) gradient to give the product 37-THP as an oil (0.34 g, 68%). 1H NMR (400 MHz, CDCl3) δ 8.71 (d, J = 10.1 Hz, 1H), 8.38 (s, 1H), 8.20 (ddd, J = 1.4, 2.7, 8.4 Hz, 1H), 7.53 (s, 2H), 7.30 (dd, J = 4.6, 8.4 Hz, 1H), 5.51 (t, J = 2.9 Hz, 1H), 4.06–3.87 (m, 2H), 3.84 (s, 3H), 3.67–3.59 (m, 2H), 1.90–1.59 (m, 6H).
Marinedrugs 16 00481 i026
(E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(pyridin-4-yl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino] propanamide (38-THP)
Carboxylic acid 22a (0.20 g, 0.45 mmol) and 4-aminopyridine (0.040 g, 0.45 mmol) were reacted according to the general procedure for amide coupling. The product was purified by column chromatography Biotage SNAP Cartridge KP-Sil 10 g, eluent (DCM/MeOH, 0→10%) gradient to give the product 38-THP as an oil (0.049 g, 21%). 1H NMR (400 MHz, CDCl3) δ 8.55 (d, J = 5.6 Hz, 2H), 7.64 (d, J = 5.8 Hz, 2H), 7.52 (s, 2H), 5.51 (t, 1H), 4.04–3.86 (m, 2H), 3.84 (s, 3H), 3.66–3.59 (m, 2H), 1.85–1.60 (m, 6H).
Marinedrugs 16 00481 i027
(E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(pyridin-3-ylmethyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino) propanamide (39-THP)
Carboxylic acid 22a (0.20 g, 0.44 mmol) and 3-picolylamine (0.068 mL, 0.67 mmol, 1.5 equiv.) were reacted according to the general procedure for amide coupling. The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, gradient elution: (DCM/MeOH, 0→10%) to give 39-THP as an oil (0.20 g, 82%). 1H NMR (400 MHz, CDCl3) δ 8.55 (br, 2H), 7.63 (d, J = 7.8 Hz, 1H), 7.51 (s, 2H), 7.30–7.25 (m, 1H), 7.22 (t, J = 6.1 Hz, 1H), 5.36 (t, J = 2.4 Hz, 1H), 4.61 (dd, J = 6.6, 15.1 Hz, 1H), 4.44 (dd, J = 5.8, 15.1 Hz, 1H), 3.94 (d, J = 13.2 Hz, 1H), 3.88–3.81 (m, 4H), 3.60–3.55 (m, 2H), 1.88–1.62 (m, 6H).
Marinedrugs 16 00481 i028
(E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(4-(dimethylamino)phenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanamide (40-THP)
Carboxylic acid 22a (0.22 g, 0.49 mmol and 4-(dimethylamino)aniline (0.060 g, 0.49 mmol) were reacted according to the general procedure for amide coupling. The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, gradient elution: (EtOAc/MeOH, 0→20%) to give 40-THP as an oil (0.16 g, 56%). 1H NMR (400 MHz, CDCl3) δ 7.55 (s, 2H), 7.45 (d, J = 9.0 Hz, 2H), 6.72 (d, J = 8.5 Hz, 2H), 5.47 (t, 1H), 4.03-3.89 (m, 2H), 3.83 (s, 3H), 3.65–3.60 (m, 2H), 2.93 (s, 6H), 1.91–1.57 (m, 6H)
Marinedrugs 16 00481 i029
(E)-N,3-Bis(3-bromo-4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propenamide (41-THP)
Carboxylic acid 22b (0.40 g, 1.07 mmol) and 3-bromo-4-methoxyaniline (0.12 g, 1.29 mmol, 1.2 equiv.) were reacted according to the general procedure for amide coupling. The product was purified by column chromatography Biotage SNAP Cartridge KP-Sil 25 g eluent (DCM/MeOH (0→10%) gradient to give 41-THP as an oil (0.43 g, 72%). 1H NMR (400 MHz, CDCl3) δ 8.54 (s, 1H), 7.86 (d, J = 2.6 Hz, 1H), 7.58 (d, J = 2.2 Hz, 1H), 7.49 (dd, J = 2.6, 8.9 Hz, 1H), 7.29 (dd, J = 2.2, 8.4 Hz, 1H), 6.85 (d, J = 8.9 Hz, 1H), 6.80 (d, J = 8.5 Hz, 1H), 5.46 (t, J = 2.8 Hz, 1H), 3.99 (d, J = 13.0 Hz, 1H), 3.91 (d, J = 13.0 Hz, 1H), 3.87 (s, 3H), 3.85 (s, 3H), 3.71–3.58 (m, 2H), 1.92–1.60 (m, 6H).
Marinedrugs 16 00481 i030
(E)-3-(3-Bromo-4-methoxyphenyl)-N-(pyridin-2-yl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanamide (42-THP)
Carboxylic acid 22b (0.40 g, 1.07 mmol) and 2-aminopyridine (0.12 g, 1.29 mmol, 1.2 equiv.) were reacted according to the general procedure for amide coupling. The product was purified by column chromatography Biotage SNAP Cartridge KP-Sil 25 g eluent (DCM/MeOH (0→10%) gradient to give 42-THP as an oil (0.31 g, 64%). 1H NMR (400 MHz, CDCl3) δ 9.27 (s, 1H), 8.29 (ddd, J = 0.9, 2.0, 4.9 Hz, 1H), 8.23 (dt, J = 1.0, 8.4 Hz, 1H), 7.70 (ddd, J = 1.9, 7.4, 8.5 Hz, 1H), 7.57 (d, J = 2.2 Hz, 1H), 7.07–7.01 (m, 1H), 6.80 (d, J = 8.4 Hz, 1H), 5.47–5.44 (m, 1H), 3.99 (d, J = 13.1 Hz, 1H), 3.91 (d, J = 13.1 Hz, 1H), 3.84 (s, 3H), 3.68–3.60 (m, 2H), 1.90–-1.61 (m, 6H).
Marinedrugs 16 00481 i031
(E)-N-(3,4-Dichlorobenzyl)-3-(4-methoxyphenyl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanamide (43-THP)
Carboxylic acid 22d (0.40 g, 1.36 mmol) and 3,4-dichlorobenzylamine (0.11 g, 1.12 mmol, 1.2 equiv.) were reacted according to the general procedure for amide coupling. 43-THP was obtained as light brown solid (0.54 g, 87%). 1H NMR (400 MHz CDCl3) δ 7.37 (d, J = 8.2 Hz, 1H), 7.34 (d, J = 2.1 Hz, 1H), 7.24 (s, 1H), 7.11–7.06 (m, 1H), 6.83–6.79 (m, 2H), 5.38–5.35 (m, 1H), 4.52 (dd, J = 6.7, 15.3 Hz, 1H), 4.34 (dd, J = 5.8, 15.2 Hz, 1H), 3.95 (d, J = 13.1 Hz, 1H), 3.85 (d, J = 13.1 Hz, 1H), 3.77 (s, 3H), 3.69–3.55 (m, 2H), 1.88–1.54 (m, 6H).
Marinedrugs 16 00481 i032
(E)-3-(4-Methoxyphenyl)-N-(pyridin-2-yl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanamide (44-THP)
Carboxylic acid 22d (0.40 g, 1.37 mmol), 2-aminopyridine (0.13 g, 1.37 mmol) were reacted according to the general procedure for amide coupling. The product was purified by column chromatography Biotage SNAP Cartridge KP-Sil 25 g and 10 g, eluent (EtOAc/MeOH, 0→10%) gradient to give 44-THP as an oil (0.095 g, 19%). 1H NMR (400 MHz, CD3OD) δ 8.28 (ddd, J = 5.0, 1.9, 0.9 Hz, 1H), 8.18 (dt, J = 8.4, 1.0 Hz, 1H), 7.81 (ddd, J = 8.4, 7.4, 1.9 Hz, 1H), 7.25 (d, J = 8.8 Hz, 2H), 7.13 (ddd, J = 7.4, 5.0, 1.0 Hz, 1H), 6.83 (d, J = 8.8 Hz, 2H), 3.96-3.79 (m, 2H), 3.73 (s, 3H), 3.66–3.48 (m, 2H), 1.91–1.46 (m, 6H).
Marinedrugs 16 00481 i033
(E)-3-(3-Chloro-4-methoxyphenyl)-N-(pyridin-2-yl)-2-[[(tetrahydro-2H-pyran-2-yl)oxy]imino]propanamide (45-THP)
Carboxylic acid 22c (0.32 g, 0.98 mmol) and 2-aminopyridine (0.093 g, 0.98 mmol) were reacted according to the general procedure for amide coupling. The product was purified by column chromatography Biotage SNAP Cartridge KP-Sil 10 g, eluent (DCM/MeOH, 0→10%) gradient to give 45-THP as an oil (0.29 g, 74%). 1H NMR (400 MHz, CDCl3) δ 8.28 (ddd, J = 4.9, 1.9, 0.9 Hz, 1H), 8.24 (dt, J = 8.4, 1.0 Hz, 1H), 7.71 (ddd, J = 8.4, 7.4, 1.9 Hz, 1H), 7.40 (d, J = 2.2 Hz, 1H), 7.22 (dd, J = 8.4, 2.2 Hz, 1H), 7.04 (ddd, J = 7.4, 4.9, 1.0 Hz, 1H), 6.82 (d, J = 8.5 Hz, 1H), 5.46 (t, 1H), 3.99 (d, J = 13.1 Hz, 1H), 3.91 (d, J = 13.1 Hz, 1H), 3.85 (s, 3H), 3.68–3.56 (m, 2H), 1.90–1.57 (m, 6H).

General Method for THP Deprotection

THP ethers 30-THP45-THP (0.16 g, 0.27 mmol) and TFA (3 mL) were dissolved in dry DCM (7 mL). The reaction mixture was stirred under argon atmosphere for 3 d. Subsequently, the reaction mixture was quenched with a 2 M solution of NaOH in H2O (15 mL) and extracted with DCM (2 × 15 mL). The combined organic layers were dried over anhyd. Na2SO4, filtered and concentrated in vacuo. The crude products were purified using flash column chromatography.
Marinedrugs 16 00481 i034
(E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(3,4-dichlorophenyl)-2-(hydroxyimino)propanamide (30)
The general procedure for THP deprotection was used, starting from ether 30 (0.16 g, 0.27 mmol). The worked-up reaction mixture was attempted to be purified using column chromatography Biotage SNAP Cartridge KP-Sil 25 g and SNAP Ultra 10 g without success. A successful purification was achieved by using Biotage SNAP Cartridge KP-NH 11 g, gradient elution: (heptane/EtOAc, 12→100%). The obtained oil was recrystallized from CHCl3 to give 30 as a white solid (0.052 g, 36%). Mp: 198 °C (decomposed).1H NMR (400 MHz, d6-DMSO) δ 12.38 (s, 1H), 10.30 (s, 1H), 8.08 (d, J = 2.4 Hz, 1H), 7.71 (dd, J = 2.5, 8.9 Hz, 1H), 7.57 (d, J = 8.8 Hz, 1H), 7.50 (s, 2H), 3.86 (s, 2H), 3.76 (s, 3H). 13C NMR (101 MHz, d6-DMSO) δ 162.2, 151.9, 151.3, 138.4, 136.0, 132.9, 130.8, 130.5, 125.3, 121.4, 120.3, 117.2, 60.4, 27.9. HRMS (ESI+): calculated 508.8670 (C16H13Br2Cl2N2O3), found 508.8670.
Marinedrugs 16 00481 i035
(E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-(3,4-dichlorobenzyl)-2-(hydroxyimino)propanamide (31)
Unlike in the general procedure, compound 31-THP (0.20 g, 0.33 mmol) was deprotected using a 2 M solution of HCl in Et2O (5 mL) in dry DCM (5 mL) under various conditions (sealed tube, 2 h, 60 °C, MW; sealed tube, 2 h, 70 °C, oil bath; sealed tube, 12 h, 30 °C, oil bath; reflux under argon, 60 h). The worked-up reaction mixture was purified twice by column chromatography, using Biotage SNAP KP-Sil 25 g, gradient elution: (heptane/EtOAc, 12→100%) to give 31 as a light yellow solid (0.038 g, 22%). 1H NMR (400 MHz, CDCl3) δ 7.79 (s, 1H), 7.49 (s, 2H), 7.39 (d, J = 8.2 Hz, 1H), 7.35 (d, J = 2.0 Hz, 1H), 7.10 (dd, J = 2.0, 8.2 Hz, 1H), 7.02 (t, J = 5.5 Hz, 1H), 4.45 (d, J = 6.2 Hz, 2H), 3.90 (s, 2H), 3.85 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 162.5, 153.0, 152.6, 138.1, 134.6, 133.6, 132.9, 131.9, 130.9, 129.7, 127.1, 118.1, 60.7, 42.6, 28.1. HRMS (ESI+): calculated 522.8825 (C17H15Br2Cl2N2O3), found 522.8827.
Marinedrugs 16 00481 i036
(E)-N-[4-Chloro-3-(trifluoromethyl)phenyl]-3-(3,5-dibromo-4-methoxyphenyl)-2-(hydroxyimino) propanamide (32)
The general procedure for THP deprotection was employed to deprotect 32-THP (0.19 g, 0.30 mmol). The worked-up reaction mixture was purified by column chromatography, Biotage SNAP Cartridge KP-NH 11 g, gradient elution: (heptane/EtOAc, 12→100%) to give 32 as a light yellow solid (0.055 g, 33%). 1H NMR (400 MHz, CDCl3) δ 8.63 (s, 1H), 8.13 (s, 1H), 7.90 (d, J = 2.6 Hz, 1H), 7.77 (dd, J = 2.6, 8.7 Hz, 1H), 7.51 (s, 2H), 7.46 (d, J = 8.7 Hz, 1H), 3.95 (s, 2H), 3.84 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 160.2, 153.0, 152.4, 135.9, 134.1, 133.5, 132.1, 129.1, 128.8, 127.2 (m), 123.6, 118.7 (q, JCF = 5.7), 118.0, 60.6, 27.7. HRMS (ESI+): calculated 542.8934 (C17H13Br2ClF3N2O3), found 542.8937.
Marinedrugs 16 00481 i037
(E)-N-(3-Chloro-4-methoxyphenyl)-3-(3,5-dibromo-4-methoxyphenyl)-2-(hydroxyimino)propanamide (33)
The general procedure for THP deprotection was used, starting from ether 33-THP (0.20 g, 0.34 mmol). The product was purified by column chromatography, Biotage SNAP Cartridge KP-NH 11 g, gradient elution: (cyclohexane/EtOAc, 12→100%) to give 33 as a light orange solid (0.086 g, 50%). 1H NMR (400 MHz, CDCl3) δ 8.41 (s, 1H), 8.07 (s, 1H), 7.64 (d, J = 2.6 Hz, 1H), 7.52 (s, 2H), 7.42 (dd, J = 2.6, 8.9 Hz, 1H), 6.88 (d, J = 8.9 Hz, 1H), 3.94 (s, 2H), 3.88 (s, 3H), 3.84 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 160.0, 153.0, 152.7, 152.2, 134.5, 133.6, 130.8, 122.8, 122.4, 119.6, 118.1, 112.4, 60.7, 56.6, 27.9. HRMS (ESI+): calculated 504.9165 (C17H16Br2ClN2O4), found 504.9164.
Marinedrugs 16 00481 i038
(E)-N-(3-Bromo-4-methoxyphenyl)-3-(3,5-dibromo-4-methoxyphenyl)-2-(hydroxyimino)propanamide (34)
The general procedure for THP deprotection was used, starting from ether 34-THP (0.20 g, 0.31 mmol). The worked-up reaction mixture was attempted to be purified using Biotage SNAP Cartridge KP-Sil 25 g and SNAP Ultra 10 g. A successful purification was achieved by column chromatography using Biotage SNAP Cartridge KP-NH 11 g, gradient elution: (heptane/EtOAc, 12→100%) to give 34 as a yellow oily solid (0.087 g, 50%). 1H NMR (400 MHz, CDCl3) δ 8.45 (s, 1H), 8.42 (s, 1H), 7.78 (d, J = 2.6 Hz, 1H), 7.52 (s, 2H), 7.48 (dd, J = 2.6, 8.9 Hz, 1H), 6.85 (d, J = 8.9 Hz, 1H), 3.94 (s, 2H), 3.86 (s, 3H), 3.83 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 160.1, 153.2, 152.9, 152.6, 134.6, 133.6, 131.1, 125.4, 120.4, 118.1, 112.2, 111.8, 60.7, 56.6, 27.9. HRMS (ESI+): calculated 548.8660 (C17H16Br3N2O4), found 548.8660.
Marinedrugs 16 00481 i039
(E)-3-(3,5-Dibromo-4-methxyphenyl)-N-(3,5-dichlorophenyl)-2-(hydroxyimino)propanamide (35)
The general procedure for THP deprotection was used, starting from ether 35-THP (0.18 g, 0.30 mmol). The worked-up reaction mixture was purified by column chromatography, Biotage SNAP Cartridge KP-NH 11 g, gradient elution: (heptane/EtOAc, 12→100%) to give 35 as a white solid (0.021 mg, 14%). 1H NMR (400 MHz, CD3OD) δ 7.71 (d, J = 1.8 Hz, 2H), 7.53 (s, 2H), 7.14 (t, J = 1.9 Hz, 1H), 3.90 (s, 2H), 3.81 (s, 3H). 13C NMR (101 MHz, CD3OD) δ 162.2, 152.6, 150.9, 140.2, 135.7, 134.7, 133.2, 123.2, 118.0, 117.3, 59.6, 27.3. HRMS (ESI+): calculated 508.8670 (C16H13Br2Cl2N2O3), found 508.8659.
Marinedrugs 16 00481 i040
(E)-3-(3,5-Dibromo-4-methoxyphenyl)-2-(hydroxyimino)-N-(pyridin-2-yl)propanamide (36)
The general procedure for THP deprotection was used, starting from ether 36-THP (0.087 g, 0.17 mmol). The product was purified by column chromatography, Biotage SNAP Cartridge KP-NH 11 g, gradient elution: (hexane/EtOAc, 12→100%). The obtained 36 as a white powder was further re-crystallized from CHCl3 for the X-ray to give transparent crystals (0.037 g, 51%). M.p.: 192–196 °C (decomposed). 1H NMR (400 MHz, CDCl3) δ 13.85 (s, 1H), 9.41 (s, 1H), 8.39 (d, J = 8.5 Hz, 1H), 8.32–8.27 (m, 1H), 7.89–7.84 (m, 1H), 7.57 (s, 2H), 7.19 (ddd, J = 0.8, 5.2, 7.3 Hz, 1H), 4.02 (s, 2H), 3.84 (s, 3H. 13C NMR (101 MHz, CDCl3) δ 161.7, 152.8, 151.7, 150.1, 146.4, 140.2, 135.2, 133.7, 120.4, 118.0, 115.7, 60.7, 28.2. HRMS (ESI+): calculated 441.9402 (C15H14Br2N3O3), found 441.9401. LC-MS: [M + H]+ m/z 442 (tR = 5.39 min), >99%.
Marinedrugs 16 00481 i041
(E)-3-(3,5-Dibromo-4-methoxyphenyl)-2-(hydroxyimino)-N-(pyridin-3-yl)propanamide (37)
The general procedure for THP deprotection was used, starting from ether 37-THP (0.34 g, 0.64 mmol). The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, gradient elution: (hexane/acetone, 0→100%) to give 37 as a pale yellow solid (0.052 g, 18%). 1H NMR (400 MHz, CD3OD) δ 8.84 (dd, J = 2.5, 19.4 Hz, 1H), 8.30–8.16 (m, 2H), 7.53 (d, J = 8.4 Hz, 2H), 7.44–7.35 (m, 1H), 3.92 (s, 2H), 3.80 (s, 3H). 13C NMR (101 MHz, CD3OD) δ 163.8, 154.4, 153.9, 152.3, 145.3, 142.3, 137.1, 134.44, 129.41, 125.2, 118.7, 61.0, 28.7. HRMS (ESI+): calculated 441.9402 (C15H14N3O3Br2), found 441.9402.
Marinedrugs 16 00481 i042
(E)-3-(3,5-Dibromo-4-methoxyphenyl)2-(hydroxyimino)-N-(pyridin-4-yl)propanamide (38)
The general procedure for THP deprotection was used, starting from ether 38-THP (0.49 g, 0.093 mmol). The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 10 g, gradient elution: (DCM/MeOH, 0→10%) to give 38 as a brownish solid (0.0099 g, 24%). 1H NMR (400 MHz, d6-acetone) δ 8.47 (s, 2H), 7.73 (d, J = 5.5 Hz, 2H), 7.59 (s, 2H), 3.98 (s, 2H), 3.82 (s, 3H). 13C NMR (101 MHz, d6-acetone) δ 162.9, 153.5, 152.2, 151.3, 145.8, 136.7, 134.3, 118.3, 114.5, 60.9, 28.4. HRMS (ESI+): calculated 441.9402 (C15H14N3O3Br2), found 441.9400.
Marinedrugs 16 00481 i043
(E)-3-(3,5-Dibromo-4-methoxyphenyl)-2-(hydroxyimino)-N-(pyridin-3-ylmethyl)propanamide (39)
The general procedure for THP deprotection was used, starting from ether 39-THP (0.17 g, 0.32 mmol). The product was purified by column chromatography, Biotage SNAP Cartridge KP-NH 11 g, gradient elution: (DCM/MeOH, 0→10%) to give 39 as a yellowish solid (0.039 g, 26%). 1H NMR (400 MHz, CDCl3) δ 8.52 (br s, 2H), 7.75 (d, J = 7.8 Hz, 1H), 7.52 (s, 2H), 7.39–7.33 (m, 1H), 7.16 (t, J = 6.1 Hz, 1H), 4.54 (d, J = 6.1 Hz, 2H), 3.91 (s, 2H), 3.84 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 163.3, 152.7, 151.1, 148.0, 147.8, 137.5, 135.4, 134.7, 133.6, 124.4, 118.0, 60.7, 41.0, 28.2. HRMS (ESI+): calculated 455.9558 (C16H16Br2N3O3), found 455.9563.
Marinedrugs 16 00481 i044
(E)-3-(3,5-Dibromo-4-methoxyphenyl)-N-[4-(dimethylamino)phenyl]-2-(hydroxyimino)propanamide (40)
The general procedure for THP deprotection was used, starting from ether 40-THP (0.16 g, 0.27 mmol). The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, gradient elution: (hexane/EtOAc, 7→60%). The obtained product was further re-crystallized from hexane and acetone to give 40 as a yellow solid (0.07 g, 50%). 1H NMR (400 MHz, d6-acetone) δ 7.61 (s, 2H), 7.56 (d, J = 9.1 Hz, 2H), 6.72 (d, J = 9.2 Hz, 2H), 3.96 (s, 2H), 3.82 (s, 3H), 2.90 (s, 6H). 13C NMR (101 MHz, d6-acetone) δ 161.2, 153.4, 153.0, 148.8, 137.2, 134.4, 129.1, 122.0, 118.2, 113.5, 60.9, 40.9, 28.5. HRMS (ESI+): calculated 483.9871 (C18H20N3O3Br2), found 483.9876.
Marinedrugs 16 00481 i045
(E)-N,3-Bis(3-bromo-4-methoxyphenyl)-2-(hydroxyimino)propanamide (41)
The general procedure for THP deprotection was used, starting from ether 41-THP (0.43 g, 0.78 mmol). The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, gradient elution: (hexane/EtOAc, 0→60%) to give 41 as a yellow solid (0.087 g, 24%). 1H NMR (400 MHz, CD3OD) δ 7.85 (d, J = 2.5 Hz, 1H), 7.51–7.45 (m, 2H), 7.24 (dd, J = 2.2, 8.5 Hz, 1H), 6.92 (d, J = 8.9 Hz, 1H), 6.86 (d, J = 8.5 Hz, 1H), 3.88 (s, 2H), 3.81 (s, 3H), 3.78 (s, 3H). 13C NMR (101 MHz, CD3OD) δ 163.4, 155.9, 154.1, 153.3, 134.8, 133.0, 131.6, 130.5, 126.6, 121.9, 113.11, 113.09, 112.2, 112.1, 56.8, 56.6, 28.7. HRMS (ESI+): calculated 470.9555 (C17H17N2O4Br2), found 470.9554.
Marinedrugs 16 00481 i046
(E)-3-(3-Bromo-4-methoxyphenyl)-2-(hydroxyimino)-N-(pyridin-2-yl)propanamide (42)
The general procedure for THP deprotection was used, starting from ether 42-THP (0.31 g, 0.68 mmol). The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, gradient elution: (DCM/MeOH, 0→10%). The obtained solid was further re-crystallized from hexane to give 42 as a white solid (0.062 g, 25%). M.p.: 214–7 °C (decomposed). 1H NMR (400 MHz, d6-DMSO) δ 12.47 (s, 1H), 9.55 (s, 1H), 8.33 (ddd, J = 4.9, 1.9, 0.9 Hz, 1H), 8.07 (dt, J = 8.3, 1.0 Hz, 1H), 7.83 (ddd, J = 8.5, 7.3, 1.9 Hz, 1H), 7.45 (d, J = 2.1 Hz, 1H), 7.23 (dd, J = 8.5, 2.2 Hz, 1H), 7.16 (ddd, J = 7.3, 4.9, 1.0 Hz, 1H), 7.03 (d, J = 8.5 Hz, 1H), 3.84 (s, 2H), 3.80 (s, 3H). 13C NMR (101 MHz, d6-DMSO) δ 161.6, 153.9, 151.1, 150.6, 148.3, 138.5, 133.0, 130.0, 129.3, 120.0, 113.4, 112.7, 110.3, 56.2, 27.4. HRMS (ESI+): calculated 364.0297 (C15H15N3O3Br), found 364.0299.
Marinedrugs 16 00481 i047
(E)-N-(3,4-Dichlorobenzyl)-2-(hydroxyimino)-3-(4-methoxyphenyl)propanamide (43)
The general procedure for THP deprotection was used, starting from ether 43-THP (0.53 g, 1.18 mmol). The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 25 g, gradient elution: (heptane/EtOAc, 0→100%) to give 43 as a yellow solid (0.057 g, 13%). 1H NMR (400 MHz, CD3OD) δ 7.39 (d, J = 8.3 Hz, 1H), 7.35 (d, J = 2.0 Hz, 1H), 7.22–7.15 (m, 2H), 7.13–7.07 (m, 1H), 6.82–6.72 (m, 2H), 4.36 (s, 2H), 3.87 (s, 2H), 3.74 (s, 3H). 13C NMR (101 MHz, CD3OD) δ 166.3, 159.7, 153.9, 141.1, 133.2, 131.7, 131.5, 131.1, 130.3, 129.9, 128.1, 114.8, 55.6, 42.7, 29.2. HRMS (ESI+): calculated 367.0616 (C17H17N2O3Cl2), found 367.0615.
Marinedrugs 16 00481 i048
(E)-2-(Hydroxyimino)-3-(4-methoxyphenyl)-N-(pyridin-2-yl)propanamide (44)
The general procedure for THP deprotection was used, starting from ether 44-THP (0.095 g, 0.26 mmol). The product was purified by column chromatography, Biotage SNAP Cartridge KP-Sil 10 g, gradient elution: (DCM/MeOH, 2→10%). The obtained product was further re-crystallized from hexane and acetone to give 44 as a white solid (0.02 g, 28%). 1H NMR (400 MHz, CD3OD) δ 8.26 (ddd, J = 5.0, 1.9, 0.9 Hz, 1H), 8.19 (dt, J = 8.4, 1.0 Hz, 1H), 7.81 (ddd, J = 8.4, 7.4, 1.9 Hz, 1H), 7.30–7.21 (m, 2H), 7.12 (ddd, J = 7.4, 5.0, 1.1 Hz, 1H), 6.85–6.76 (m, 2H), 3.92 (s, 2H), 3.74 (s, 3H). 13C NMR (101 MHz, CD3OD) δ 163.4, 159.7, 153.2, 152.3, 149.0, 140.0, 131.2, 129.9, 121.1, 115.1, 114.8, 55.6, 28.6. HRMS (ESI+): calculated 286.1192 (C15H16N3O3), found 286.1195.
Marinedrugs 16 00481 i049
(E)-3-(3-Chloro-4-methoxyphenyl)-2-(hydroxyimino)-N-(pyridin-2-yl)propanamide (45)
The general procedure for THP deprotection was used, starting from ether 45-THP (0.29 g, 0.72 mmol). The obtained product was further re-crystallized from acetone to give 45 as a white solid (0.05 g, 20%). M.p.: 218–219 °C (decomposed) 1H NMR (400 MHz, d6-acetone) δ 8.29 (ddd, J = 4.9, 1.9, 0.9 Hz, 1H), 8.20 (d, J = 8.3 Hz, 1H), 7.84–7.78 (m, 1H), 7.42–7.40 (m, 1H), 7.29 (ddt, J = 8.4, 2.2, 0.6 Hz, 1H), 7.12 (ddd, J = 7.4, 4.9, 1.0 Hz, 1H), 7.02 (d, J = 8.5 Hz, 1H), 3.96 (s, 2H), 3.85 (s, 3H). 13C NMR (101 MHz, d6-acetone) δ 162.0, 154.7, 152.8, 151.9, 149.2, 139.1, 131.5, 130.6, 129.7, 122.4, 120.7, 114.0, 113.3, 56.4, 28.2. HRMS (ESI+): calculated 320.0802 (C15H15N3O3Cl), found 320.0802.

4.2. Cell Lines

Human malignant melanoma cell line A-375 was kindly provided by Prof. Marikki Laiho (University of Helsinki, Finland) and Hs27 human skin fibroblast cell line was kindly provided by Dr. Carmen Escobedo-Lucea (University of Helsinki, Finland). The cells were maintained in Glutamax high glucose Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco), supplemented with 10% fetal bovine serum (FBS, Gibco), at 37 °C, 5% CO2.

4.3. Analysis of Selectivity to Cancer Cells

The cells were seeded to white frame and clear bottom 96-well plates (Perkin Elmer) at the density of 10,000 cells/well for A-375 cell line and 7500 cells/well for Hs27 cell line. The cells were grown at 37 °C, 5% CO2 until they reached 70–80% confluence (approximately 24 h). Stock solutions of test compounds and a positive control (camptothecin, Sigma-Aldrich, Saint Louis, MO, USA) were prepared in DMSO and diluted into assay medium (growth medium with 5% FBS) to the final concentration. Final DMSO concentration was 0.5% in all samples. The culture medium was removed from the plate and compounds added, 200 µL/well. After 48-h incubation, the amount of ATP, which is directly proportional to the number of cells present in culture, was quantified using CellTiter-Glo® Luminescent Cell Viability kit (Promega, Madison, WI, USA), according to manufacturer’s instructions.
Origin Graphing and Analysis, version 8.6 (OriginLab, Northampton, MA, USA) was used for determination of CC50 values. The cancer cell selectivity index (SI) was calculated as a ratio of CC50 values between Hs27 fibroblasts and A-375 melanoma cells.

5. Conclusions

Several syntheses of bromotyrosines have been reported but the synthesis of bromotyrosines with monomethylated tyramine part have not been reported before. The selective removal of the protective groups from the tyramine fragment before the coupling reaction is a challenging step in the total synthesis of purpurealidin I (1). We succeeded at this by using trifluoroacetyl protection. This route can be utilized further for the synthesis of additional bromotyrosine derivatives possessing the monomethylated tyramine fragment. The synthesized simplified analogs without the tyramine fragment retained the cytotoxic activity. The selectivity towards melanoma cell line was generally low. The highest selectivity (SI 4.1) was demonstrated in the case of pyridin-2-yl compound (36). This shows that the marine cytotoxic bromotyrosines are promising scaffolds for developing cytotoxic agents and the full understanding of the elements of their SAR is still in very early stage. Further optimization of simplified bromotyrosine derivatives is needed to attain high selectivity.

Supplementary Materials

The following are available online at https://www.mdpi.com/1660-3397/16/12/481/s1, Appendix.pdf (synthesis, NMR spectra of compounds 1 and 36, and single crystal X-ray diffraction measurements), Compound36.cif and checkcif-Compound36.pdf

Author Contributions

Synthesis, C.B., I.T., M.V., V.B., N.H., T.B. and E.M.-L.; X-ray analysis, T.R. and H.L.; Conceptualization, P.K., and P.T.; Biological screening, P.I., I.T., K.-E.L., T.B.; Writing—original draft preparation, P.K., C.B., I.T., M.V., P.I.; Writing—review and editing, P.K., J.Y.-K. and P.T.; Supervision, P.K., C.B., P.I. and P.T.; Resources, J.Y.-K., P.T. and H.L.; Funding acquisition J.Y.-K. and P.T.

Funding

This research was funded by European Union Seventh Framework Programme grant agreement No. FP7-KBBE-2009-3-245137 (MAREX) Exploring Marine Resources for Bioactive Compounds: From Discovery to Sustainable Production and Industrial Applications 2010–2014. J.Y.-K. thanks Academy of Finland for the project No. 285103 and P.K. thanks Academy of Finland for the project No. 315937.

Acknowledgments

We thank Paul Flemmich for the synthesis assistance, Heidi Mäkkylä for her excellent technical assistance in the biological experiments and Andrew Neal for checking the language. We also wish to thank the DDCB core facility supported by the University of Helsinki and Biocenter Finland.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Bromotyrosines purpurealidin I (1), aplysamine 2 (2), aplysamine 4 (3) and JBIR-44 (4).
Figure 1. Bromotyrosines purpurealidin I (1), aplysamine 2 (2), aplysamine 4 (3) and JBIR-44 (4).
Marinedrugs 16 00481 g001
Scheme 1. Retrosynthetic route to purpurealidin I (1).
Scheme 1. Retrosynthetic route to purpurealidin I (1).
Marinedrugs 16 00481 sch001
Scheme 2. Synthesis of the bromotyrosine carboxylic acid part (5) [17] for the first amide coupling attempts.
Scheme 2. Synthesis of the bromotyrosine carboxylic acid part (5) [17] for the first amide coupling attempts.
Marinedrugs 16 00481 sch002
Scheme 3. Synthesis of the tyramine derivative (6) for use in the first amide coupling approach.
Scheme 3. Synthesis of the tyramine derivative (6) for use in the first amide coupling approach.
Marinedrugs 16 00481 sch003
Scheme 4. Synthesis of trifluoroacetyl protected tyramine part (17) [15] for the purpurealidin I (1) synthesis.
Scheme 4. Synthesis of trifluoroacetyl protected tyramine part (17) [15] for the purpurealidin I (1) synthesis.
Marinedrugs 16 00481 sch004
Scheme 5. Synthesis of the purpurealidin I (1) carboxylic part (22a) and a route to the simplified hydroxyimino propanamides (24ad), R3 substituents are given in Table 1.
Scheme 5. Synthesis of the purpurealidin I (1) carboxylic part (22a) and a route to the simplified hydroxyimino propanamides (24ad), R3 substituents are given in Table 1.
Marinedrugs 16 00481 sch005
Scheme 6. Synthesis of purpurealidin I (1) and aplysamine 2 analog (29).
Scheme 6. Synthesis of purpurealidin I (1) and aplysamine 2 analog (29).
Marinedrugs 16 00481 sch006
Figure 2. ORTEP representation (50% probability ellipsoids) of the molecular structure of (36). The CHCl3 molecule as a packing solvent has been omitted for clarity.
Figure 2. ORTEP representation (50% probability ellipsoids) of the molecular structure of (36). The CHCl3 molecule as a packing solvent has been omitted for clarity.
Marinedrugs 16 00481 g002
Table 1. Structures of final hydroxyimino propanamides (3045) from the Scheme 5. The corresponding THP-ethers (23ad) are found in the Experimental part (Section 4.1.2) numbered as 30-THP45-THP.
Table 1. Structures of final hydroxyimino propanamides (3045) from the Scheme 5. The corresponding THP-ethers (23ad) are found in the Experimental part (Section 4.1.2) numbered as 30-THP45-THP.
Marinedrugs 16 00481 i050
CmpdR1R2R3CmpdR1R2R3CmpdR1R2R3
30BrBr Marinedrugs 16 00481 i05135BrBr Marinedrugs 16 00481 i05240BrBr Marinedrugs 16 00481 i053
31BrBr Marinedrugs 16 00481 i05436BrBr Marinedrugs 16 00481 i05541BrH Marinedrugs 16 00481 i056
32BrBr Marinedrugs 16 00481 i05737BrBr Marinedrugs 16 00481 i05842BrH Marinedrugs 16 00481 i059
33BrBr Marinedrugs 16 00481 i06038BrBr Marinedrugs 16 00481 i06143HH Marinedrugs 16 00481 i062
34BrBr Marinedrugs 16 00481 i06339BrBr Marinedrugs 16 00481 i06444HH Marinedrugs 16 00481 i065
45ClH Marinedrugs 16 00481 i066
Table 2. Structures of final amide compounds (4678) [15].
Table 2. Structures of final amide compounds (4678) [15].
Marinedrugs 16 00481 i067
CmpdArR1R2CmpdArR1R2CmpdArR1R2
46 Marinedrugs 16 00481 i068BrN(CH3)257 Marinedrugs 16 00481 i069BrN(CH3)268 Marinedrugs 16 00481 i070BrN(CH3)2
47 Marinedrugs 16 00481 i071BrN(CH3)258 Marinedrugs 16 00481 i072BrN(CH3)269 Marinedrugs 16 00481 i073BrN(CH3)2
48 Marinedrugs 16 00481 i074BrN(CH3)259 Marinedrugs 16 00481 i075BrN(CH3)270 Marinedrugs 16 00481 i076BrN(CH3)2
49 Marinedrugs 16 00481 i077BrC(CH3)260 Marinedrugs 16 00481 i078BrNH(CH3)71 Marinedrugs 16 00481 i079BrN(CH3)2
50 Marinedrugs 16 00481 i080BrN(CH3)261 Marinedrugs 16 00481 i081HNH(CH3)72 Marinedrugs 16 00481 i082BrNH(CH3)
51 Marinedrugs 16 00481 i083BrN(CH3)262 Marinedrugs 16 00481 i084BrN(CH3)273 Marinedrugs 16 00481 i085BrN(CH3)2
52 Marinedrugs 16 00481 i086BrC(CH3)263 Marinedrugs 16 00481 i087BrNH(CH3)74 Marinedrugs 16 00481 i088BrNH(CH3)
53 Marinedrugs 16 00481 i089HN(CH3)264 Marinedrugs 16 00481 i090BrN(CH3)275 Marinedrugs 16 00481 i091HN(CH3)2
54 Marinedrugs 16 00481 i092HNH(CH3)65 Marinedrugs 16 00481 i093BrNH(CH3)76 Marinedrugs 16 00481 i094HNH(CH3)
55 Marinedrugs 16 00481 i095Br Marinedrugs 16 00481 i09666 Marinedrugs 16 00481 i097HN(CH3)77 Marinedrugs 16 00481 i098BrN(CH3)2
56 Marinedrugs 16 00481 i099BrN(CH3)267 Marinedrugs 16 00481 i100BrN(CH3)278 Marinedrugs 16 00481 i101BrN(CH3)2
Table 3. Cytotoxicity of purpurealidin I (1) and compounds (2978) against human malignant melanoma cell line (A-375) and normal skin fibroblast cell line (Hs27). Camptothecin, a compound with high selectivity to cancer cells, was used as a positive control. The selectivity index of individual compounds was calculated as a ratio of CC50 in normal fibroblasts over CC50 in melanoma cells. CC50 = cytotoxic concentration that caused death of 50% cells. ND = not determined.
Table 3. Cytotoxicity of purpurealidin I (1) and compounds (2978) against human malignant melanoma cell line (A-375) and normal skin fibroblast cell line (Hs27). Camptothecin, a compound with high selectivity to cancer cells, was used as a positive control. The selectivity index of individual compounds was calculated as a ratio of CC50 in normal fibroblasts over CC50 in melanoma cells. CC50 = cytotoxic concentration that caused death of 50% cells. ND = not determined.
CompoundPrimary Test Result
(Cytotoxicity % at 50 µM in A-375 Cells)
CC50 (µM) in A-375 CellsCC50 (µM) in Hs27 CellsSelectivity Index
197.94.35.21.2
2999.96.34.50.7
30101.413.115.21.2
3199.712.422.31.8
3299.810.312.01.2
3398.913.526.42.0
34100.216.222.51.4
35101.49.612.21.3
3684.74.719.44.1
3783.526.4NDND
3871.1NDNDND
3993.222.1NDND
4099.127.8NDND
4199.219.8NDND
422.7NDNDND
4321.5NDNDND
440.4NDNDND
454.0NDNDND
4659.2NDNDND
4791.331.6NDND
4897.613.111.30.9
4938.3NDNDND
5065.0NDNDND
5193.614.811.80.8
5238.8NDNDND
5390.027.7NDND
54101.420.4NDND
5531.3NDNDND
5686.733.5NDND
5726.0NDNDND
5897.413.26.80.5
5939.6NDNDND
6093.834.135.7 1.0
610.0NDNDND
6296.517.7NDND
6399.911.011.71.1
6498.412.311.81.0
6599.76.57.21.1
6698.615.4NDND
6797.07.77.20.9
6896.814.814.91.0
6997.913.47.50.6
7045.3NDNDND
7198.627.1NDND
7299.915.3NDND
7399.38.411.11.3
7496.76.210.01.6
75101.042.9NDND
7696.619.6NDND
7794.322.1NDND
7831.7NDNDND
Camptothecin94.70.065.5492.3

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Bhat, C.; Ilina, P.; Tilli, I.; Voráčová, M.; Bruun, T.; Barba, V.; Hribernik, N.; Lillsunde, K.-E.; Mäki-Lohiluoma, E.; Rüffer, T.; Lang, H.; Yli-Kauhaluoma, J.; Kiuru, P.; Tammela, P. Synthesis and Antiproliferative Activity of Marine Bromotyrosine Purpurealidin I and Its Derivatives. Mar. Drugs 2018, 16, 481. https://doi.org/10.3390/md16120481

AMA Style

Bhat C, Ilina P, Tilli I, Voráčová M, Bruun T, Barba V, Hribernik N, Lillsunde K-E, Mäki-Lohiluoma E, Rüffer T, Lang H, Yli-Kauhaluoma J, Kiuru P, Tammela P. Synthesis and Antiproliferative Activity of Marine Bromotyrosine Purpurealidin I and Its Derivatives. Marine Drugs. 2018; 16(12):481. https://doi.org/10.3390/md16120481

Chicago/Turabian Style

Bhat, Chinmay, Polina Ilina, Irene Tilli, Manuela Voráčová, Tanja Bruun, Victoria Barba, Nives Hribernik, Katja-Emilia Lillsunde, Eero Mäki-Lohiluoma, Tobias Rüffer, Heinrich Lang, Jari Yli-Kauhaluoma, Paula Kiuru, and Päivi Tammela. 2018. "Synthesis and Antiproliferative Activity of Marine Bromotyrosine Purpurealidin I and Its Derivatives" Marine Drugs 16, no. 12: 481. https://doi.org/10.3390/md16120481

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