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

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.


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.

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).

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). Mar. Drugs 2018, 16,  For the synthesis of the bromotyrosine carboxylic acid moiety, commercially available Omethyltyrosine (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). Scheme 2. Synthesis of the bromotyrosine carboxylic acid part (5) [17] for the first amide coupling attempts.
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 1 H 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]. Mar. Drugs 2018, 16, x FOR PEER REVIEW 4 of 25 Scheme 3. Synthesis of the tyramine derivative (6) for use in the first amide coupling approach.
The direct coupling of compounds (5) and (6) with 1-ethyl-3-(3dimethylaminopropyl)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 1 H 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]. Scheme 4. Synthesis of trifluoroacetyl protected tyramine part (17) [15] for the purpurealidin I (1) synthesis.
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 (22b-d) in the mono-brominated, mono-chlorinated and non-halogenated simplified derivatives (41-45). Reddy et al. have reported the synthesis of methyl carbamate containing bromotyrosine purpuramine K leaving the tetrahydropyranyl (THP) group to the molecule [22]. 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.
The direct coupling of compounds (5) and (6) with 1-ethyl-3-(3dimethylaminopropyl)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 1 H 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]. Scheme 4. Synthesis of trifluoroacetyl protected tyramine part (17) [15] for the purpurealidin I (1) synthesis.
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 (22b-d) in the mono-brominated, mono-chlorinated and non-halogenated simplified derivatives (41-45). Reddy et al. have reported the synthesis of methyl carbamate containing bromotyrosine purpuramine K leaving the tetrahydropyranyl (THP) group to the molecule [22]. Scheme 4. Synthesis of trifluoroacetyl protected tyramine part (17) [15] for the purpurealidin I (1) synthesis.
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 (22b-d) in the mono-brominated, mono-chlorinated and non-halogenated simplified derivatives (41-45). 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 (THPONH 2 ). 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 Et 2 O to give the free oxime (28). Trifluoroacetyl mediated deprotection of (28) using MeOH/K 2 CO 3 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).  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 (46-78) ( Table 2) have previously been reported by our group [15].  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 (46-78) ( Table 2) have previously been reported by our group [15].  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 (46-78) ( Table 2) have previously been reported by our group [15].  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 (46-78) ( Table 2) have previously been reported by our group [15].  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 (46-78) ( Table 2) have previously been reported by our group [15].  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 (46-78) ( Table 2) have previously been reported by our group [15].  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 (46-78) ( Table 2) have previously been reported by our group [15].  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 (46-78) ( Table 2) have previously been reported by our group [15].  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 (46-78) ( Table 2) have previously been reported by our group [15].  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 (46-78) ( Table 2) have previously been reported by our group [15].  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 (46-78) ( Table 2) have previously been reported by our group [15].  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 (46-78) ( Table 2) have previously been reported by our group [15].  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 (46-78) ( Table 2) have previously been reported by our group [15].  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 (46-78) ( Table 2) have previously been reported by our group [15].  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 (46-78) ( Table 2) have previously been reported by our group [15].  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 (46-78) ( Table 2) have previously been reported by our group [15]. 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). Scheme 5. Synthesis of the purpurealidin I (1) carboxylic part (22a) and a route to the simplified hydroxyimino propanamides (24a-d), R 3 substituents are given in Table 1.
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 Et 2 O 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 (30-45) 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 (46-78) ( Table 2) have previously been reported by our group [15].
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 (30-45) 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 (46-78) ( Table 2) have previously been reported by our group [15]. 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 (46-78) ( Table 2) have previously been reported by our group [15]. 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 (46-78) ( Table 2) have previously been reported by our group [15]. 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 (46-78) ( Table 2) have previously been reported by our group [15]. 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 (46-78) ( Table 2) have previously been reported by our group [15]. 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 (46-78) ( Table 2) have previously been reported by our group [15]. 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 (46-78) ( Table 2) have previously been reported by our group [15]. 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 (46-78) ( Table 2) have previously been reported by our group [15]. 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 (46-78) ( Table 2) have previously been reported by our group [15]. 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 (46-78) ( Table 2) have previously been reported by our group [15]. 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 (46-78) ( Table 2) have previously been reported by our group [15]. 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 (46-78) ( Table 2) have previously been reported by our group [15]. 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 (46-78) ( Table 2) have previously been reported by our group [15]. 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 (46-78) ( Table 2) have previously been reported by our group [15]. 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 (46-78) ( Table 2) have previously been reported by our group [15]. 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 (46-78) ( Table 2) have previously been reported by our group [15]. 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 (46-78) ( Table 2) have previously been reported by our group [15]. 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 (46-78) ( Table 2) have previously been reported by our group [15]. 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 (46-78) ( Table 2) have previously been reported by our group [15].

Stereochemistry
It has previously been reported that the configuration of the N-oxime can be determined by analysis of the 13 C 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-

Stereochemistry
It has previously been reported that the configuration of the N-oxime can be determined by analysis of the 13 C 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-

Stereochemistry
It has previously been reported that the configuration of the N-oxime can be determined by analysis of the 13 C 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-

Stereochemistry
It has previously been reported that the configuration of the N-oxime can be determined by analysis of the 13 C 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-

Stereochemistry
It has previously been reported that the configuration of the N-oxime can be determined by analysis of the 13 C 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-

Stereochemistry
It has previously been reported that the configuration of the N-oxime can be determined by analysis of the 13 C 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-

Stereochemistry
It has previously been reported that the configuration of the N-oxime can be determined by analysis of the 13 C 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 disulfidebridged psammaplin A analog supported this observation [29]. We, therefore, expect the

Stereochemistry
It has previously been reported that the configuration of the N-oxime can be determined by analysis of the 13 C 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-

Stereochemistry
It has previously been reported that the configuration of the N-oxime can be determined by analysis of the 13 C 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-

Stereochemistry
It has previously been reported that the configuration of the N-oxime can be determined by analysis of the 13 C 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 disulfidebridged psammaplin A analog supported this observation [29]. We, therefore, expect the

Stereochemistry
It has previously been reported that the configuration of the N-oxime can be determined by analysis of the 13 C 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 disulfidebridged psammaplin A analog supported this observation [29]. We, therefore, expect the

Stereochemistry
It has previously been reported that the configuration of the N-oxime can be determined by analysis of the 13 C 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 disulfidebridged psammaplin A analog supported this observation [29]. We, therefore, expect the

Stereochemistry
It has previously been reported that the configuration of the N-oxime can be determined by analysis of the 13 C 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 disulfidebridged psammaplin A analog supported this observation [29]. We, therefore, expect the

Stereochemistry
It has previously been reported that the configuration of the N-oxime can be determined by analysis of the 13 C 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 disulfidebridged psammaplin A analog supported this observation [29]. We, therefore, expect the

Stereochemistry
It has previously been reported that the configuration of the N-oxime can be determined by analysis of the 13 C 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 disulfidebridged psammaplin A analog supported this observation [29]. We, therefore, expect the

Stereochemistry
It has previously been reported that the configuration of the N-oxime can be determined by analysis of the 13 C 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 disulfidebridged psammaplin A analog supported this observation [29]. We, therefore, expect the Br N(CH 3 ) 2

Stereochemistry
It has previously been reported that the configuration of the N-oxime can be determined by analysis of the 13 C 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 13 C 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.

Stereochemistry
It has previously been reported that the configuration of the N-oxime can be determined by analysis of the 13 C 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 disulfidebridged 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 13 C 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. ORTEP representation (50% probability ellipsoids) of the molecular structure of (36). The CHCl3 molecule as a packing solvent has been omitted for clarity.

Biological Activity
The cytotoxicity of the synthetic purpurealidin I (1) and compounds (29-78) 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 Figure 2. ORTEP representation (50% probability ellipsoids) of the molecular structure of (36). The CHCl 3 molecule as a packing solvent has been omitted for clarity.

Biological Activity
The cytotoxicity of the synthetic purpurealidin I (1) and compounds (29-78) 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 CC 50 (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 (CC 50 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).

SAR
The observed melanoma cell line activity for the compounds was in the range of 4-43 µM (CC 50 in A-375 cells, Table 3). Both Purpurealidin I (1) and its dimethylated analog (29) showed cytotoxicity to melanoma cells (CC 50 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 CC 50 (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 CC 50 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., CC 50 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).

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. 1 H NMR (300 MHz) and 13 C NMR (75 MHz) spectra were measured in CDCl 3 , d 6 -DMSO, CD 3 OD, or d 6 -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 1 H and 13  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.

Experimental Procedures
General Procedure for the Formation of Azlactones Aldehyde 19a-d, acetylglycine (1.5 equiv.) and anhyd. NaOAc (1.5 equiv.) were dissolved in Ac 2 O (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 20a-d was used in the subsequent step without further purification. 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.

Experimental Procedures
General Procedure for the Formation of Azlactones Aldehyde 19a-d, 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 20a-d was used in the subsequent step without further purification.     300 spectrometer or Bruker AV400 MHz NMR with smart probe. Chemical shifts (δ) are given in parts per million (ppm) relative to the 1 H and 13 C 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 µ m, 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.

Experimental Procedures
General Procedure for the Formation of Azlactones Aldehyde 19a-d, 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 20a-d was used in the subsequent step without further purification.  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.

Experimental Procedures
General Procedure for the Formation of Azlactones Aldehyde 19a-d, 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 20a-d was used in the subsequent step without further purification.    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.

Experimental Procedures
General Procedure for the Formation of Azlactones Aldehyde 19a-d, 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 20a-d was used in the subsequent step without further purification.

General Method for the Hydrolysis of Azlactones
Azlactone 20a-d was dissolved in a 10% solution of HCl in H 2 O (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 21a-d was used in the subsequent step without further purification. General Method for the Hydrolysis of Azlactones Azlactone 20a-d 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 21a-d was used in the subsequent step without further purification.

General Procedure for THP-Protection
Carboxylic acid 21a-d 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 General Method for the Hydrolysis of Azlactones Azlactone 20a-d 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 21a-d was used in the subsequent step without further purification.

General Procedure for THP-Protection
Carboxylic acid 21a-d 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 General Method for the Hydrolysis of Azlactones Azlactone 20a-d 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 21a-d was used in the subsequent step without further purification.

General Procedure for THP-Protection
Carboxylic acid 21a-d 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 General Method for the Hydrolysis of Azlactones Azlactone 20a-d 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 21a-d was used in the subsequent step without further purification.

General Procedure for THP-Protection
Carboxylic acid 21a-d and THPONH 2 (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 H 2 O (2 × 20 mL). The aqueous layer was extracted back with EtOAc (2 × 10 mL). The combined organic layers were dried over Na 2 SO 4 , filtered and concentrated in vacuo. The obtained crude product 22a-d was used in the subsequent step without further purification.
Origin Graphing and Analysis, version 8.6 (OriginLab, Northampton, MA, USA) was used for determination of CC 50 values. The cancer cell selectivity index (SI) was calculated as a ratio of CC50 values between Hs27 fibroblasts and A-375 melanoma cells.

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.