Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Rui Filipe Jesus Pereira

Mestrado em Química Departamento de Química e Bioquímica 2019/2020

Orientador Maria Luísa Cardoso do Vale, Professora Auxiliar, FCUP Coorientador Maria de La Salette Reis, Professora Catedrática, FFUP

Todas as correções determinadas pelo júri, e só essas, foram efetuadas.

O Presidente do Júri,

Porto, ______/______/______

FCUP III Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Agradecimentos

Tem sido um privilégio trabalhar neste último ano no laboratório 2.24. do DQB e ter a possibilidade de completar a minha tese de mestrado, apesar das adversidades que este ano trouxe a todos nós. Gostaria de agradecer a todas as pessoas que tornaram isto possível. Primeiro, gostaria de agradecer à minha orientadora Profª. Luísa do Vale, pela oportunidade de trabalhar num projeto inteiramente novo dentro do seu grupo de investigação, pela confiança, apoio e orientação ao longo do ano. Uma palavra de apreço e gratidão para a Dr. Cidália Pereira pois foi ela que propôs o projeto no grupo e ajudou-me nos meus primeiros passos no projeto. Sem ela não teria conseguido chegar onde cheguei. Agradeço também à Dr. Sandra Silva pelo apoio contínuo ao longo do ano, pela sua experiência e conhecimento que me ajudaram, especialmente no estudo das propriedades físico-químicas dos compostos. Agradeço ao Prof. Eduardo Marques por ter disponibilizado o seu laboratório para a realização desses mesmos estudos, e pela orientação e contribuição na análise dos resultados. Um especial agradecimento para a minha coorientadora Profª. Salette Reis (FFUP) e à Dr. Marina Pinheiro (FFUP), pela colaboração estabelecida entre os dois grupos que me deu condições para realizar testes que de outra forma não seriam possíveis. E, apesar da minha presença física no laboratório do seu grupo ter sido comprometida, devido ao Covid-19, sempre me apoiaram em orientaram de todas as maneiras possíveis. Gostaria de agradecer também a todos os meus colegas do laboratório, especialmente ao Dr. Xiao Loureiro, Dr. Ivo Dias, Sara e todos que partilharam comigo esta longa jornada de trabalho, aprendizagem e amizade. Finalmente, palavras não são suficientes para agradecer à minha família, amigos, e à Rafaela. O apoio deles e incentivo foi fundamental para completar esta etapa. Nunca poderia ter feito isto sem vocês.

IV FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Resumo

A pele é o maior órgão do corpo humano e por isso um importante e acessível meio de administração de fármacos. A entrega transdérmica de fármacos oferece inúmeras vantagens sobre métodos de administração convencionais, tais como efeito de ação prolongado, aplicação controlável e indolor, biodisponibilidade melhorada, etc. No entanto, a pele é uma barreira efetiva contra agentes externos, principalmente devido a estrutura da camada mais externa denominada estrato córneo, que se apresenta como o principal obstáculo a permeação/penetração de fármacos. Por consequência, apenas alguns fármacos podem ser administrados pela pele, o que torna este método um desafio, merecedor de investimento.

De forma a ultrapassar a barreira do estrato córneo, potenciadores químicos de permeação têm sido usados para perturbar a organização lipídica e/ou promover a solubilidade dos fármacos nas membranas, entre outros mecanismos. No entanto, os novos potenciadores ainda necessitam de provar o seu valor como potenciais substitutos dos já usados em formulações dérmicas. Certas qualidades são necessárias para a próxima geração de potenciadores tais como: atividade melhorada e universal para a entrega de fármacos tanto lipofílicos como hidrofílicos, biodisponibilidade, baixa citotoxicidade e irritação para a pele, etc.

Nesta perspetiva, este projeto teve como objetivo a síntese, caracterização físico-química e avaliação da atividade de derivados anfifílicos de aminoácidos cíclicos, com elevada capacidade de estabelecer pontes de hidrogénio, como promotores de permeação dérmica para a entrega de fármacos. Assim, foram sintetizadas três famílias de compostos: os derivados da 4-hidroxiprolina, os derivados da 3,4-dihidroxiprolina e os derivados de um β-aminoácido pentacíclico não natural (mimético da prolina), num total de 18 compostos finais. Foram ainda determinados os valores de concentração micelar crítica para alguns compostos selecionados por tensiometria. Os compostos apresentaram valores de cmc na ordem dos 0.05 mmol∙Kg-1, e mostraram possuir elevada atividade superficial.

Palavras-chave: administração transdérmica de fármacos, potenciador químico de permeação, aminoácido, β-aminoácido cíclico, molécula anfifílica, síntese.

FCUP V Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Abstract

Skin is the largest organ in the human body and thus an interesting and readily available means to deliver drugs on and into it. Transdermal Drug Delivery (TDD) offers numerous advantages over conventional drug administrations, such as prolonged duration of action, painless and controllable application, improved bioavailability, etc. However, the skin is an effective barrier against external agents, mainly due to the Stratum Corneum (SC) layer structure that provides the main obstacle to the permeation/penetration of drugs. Therefore, there are very few drugs that can be delivered through the skin, making TDD a challenge, worthy of investment. To overcome the SC barrier, chemical permeation enhancers (CPE) have been used to affect the lipid organization and/or promote solubility of drugs through the membranes, besides other mechanisms. Though, novel CPEs have yet to prove their value as potential substitutes for the enhancers already used in dermal formulations. Certain qualities are necessary for the next generation of CPEs such as: Increased and universal activity for both lipophilic and hydrophilic drugs, bioavailability, low cytotoxicity and irritation to skin cells, amongst others. In this regard, this project aims at the synthesis, physicochemical characterization, and evaluation of cyclic amino acid amphiphiles derivatives with enhanced donor/acceptor hydrogen bonding sites, as enhancers for the transdermal delivery of drugs. Herein, three families of compounds were synthesized, hydroxyproline derivatives, dihydroxyproline derivatives and pentacyclic unnatural -amino acid derivatives (proline mimetics), in a total of 18 final compounds. Furthermore, the critical micellar concentration values of a few selected compounds were also determined by tensiometry. The compounds present cmc values in the order of 0.05 mmol·Kg-1 and showed to be highly surface active.

Keywords: Transdermal drug delivery, chemical permeation enhancer, amino acid, cyclic β-amino acid, amphiphile, synthesis VI FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Table of contents

Agradecimentos ...... III Resumo ...... IV Abstract ...... V Table of contents ...... VI List of Figures ...... IX List of Schemes ...... X List of Tables ...... XI List of Abbreviations and Acronyms ...... XII List of Symbols ...... XIV Chapter 1: Introduction ...... 1 1.1. Scope ...... 2 1.2. Objectives and Work plan ...... 3 1.3. Introduction ...... 5 1.3.1. Skin structure ...... 5 1.3.2. Permeation routes ...... 6 1.3.3. Penetration of drugs through the skin ...... 8 1.3.4. Drug flux evaluation methods ...... 10 1.3.5. Permeation enhancement strategies and mechanisms ...... 14 1.3.6. Chemical Penetration Enhancers ...... 17 1.3.7. Amino acid-based enhancers ...... 19 1.3.8. Synthesis of amino acid amphiphiles ...... 30 1.4.9. Cyclic β-amino acids, synthesis and applications ...... 33 Chapter 2: Results and Discussion ...... 37 2.1. Synthesis ...... 38 2.1.1. Synthesis of the 4-hidroxyproline derivatives ...... 41 A) Introduction of the lipophilic chain at the hydroxyl group...... 42 B) Introduction of the alkyl chain into the amino group by reductive amination 43 C) Introduction of the alkyl chain into the carboxylic acid group with TMSCl 44 D) Removal of the Cbz group by hydrogenation ...... 45 E) N-methylation by reductive amination ...... 46 F) N-acetylation of the derivatives using acetyl chloride ...... 46 2.1.2. Synthesis of the 3,4-dehydro-L-proline derivatives ...... 48 FCUP VII Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

2.1.2.1. Synthesis of derivatives 16/18 ...... 50 A) N-Boc protection with di-tert-butylcarbonate ...... 50 B) Introduction of the alkyl chain into carboxylic acid group with PyBop . 51 C) Boc removal with TFA ...... 52 D) N-alkylation/acylation of 14 ...... 52

E) Dihydroxylation with OsO4 ...... 53 2.1.2.2. Synthesis of derivative 20 ...... 54 2.1.3. Synthesis of the proline mimetic derivatives ...... 55 2.1.3.1. Preparation of the unnatural β-amino acids ...... 58 2.1.3.2. Preparation of the β-unnatural amino acid derivatives ...... 62 2.1.3.2.1. Synthesis of 29a-b ...... 62 2.1.3.2.2. Synthesis of 32a-b ...... 65 2.1.3.2.3. Synthesis of 35 ...... 65 2.2. Physicochemical properties ...... 66 2.2.1. Critical micellar concentration ...... 66 2.2.2. Melting Points ...... 70 Chapter 3: Conclusions ...... 73 Chapter 4: Materials and Methods ...... 77 4.1. Chemicals ...... 78 4.2. Synthesis and characterization ...... 79 4.3. Surface Tension ...... 80 Chapter 5: Synthesis...... 83 5.1. Synthesis of the 4-hydroxyproline derivatives ...... 84 5.1.1. Synthesis of methyl (2S,4R)-4-(O-dodecanoyl)hydroxyprolinate (2a) / (2S,4R) -4-(O-dodecanoyl)hydroxyproline (2b) ...... 84 5.1.2. Synthesis of methyl (2S,4R)-4-(O-dodecanoyl)-N-methyl-hydroxyprolinate (3a) / (2S,4R)-4-(O-dodecanoyl)-N-methyl-hydroxyproline (3b) ...... 85 5.1.3. Synthesis of methyl (2S,4R)-1-acetyl-4-(O-dodecanoyl)hydroxyprolinate (5a) / (2S,4R)-1-acetyl-4-( O-dodecanoyl)hydroxyproline (5b) ...... 86 5.1.4. Synthesis of methyl (2S,4R)-N-dodecyl-4-hydroxyprolinate (10) ...... 88 5.1.5. Synthesis of dodecyl (2S,4R)-N-Cbz-4-hydroxyprolinate (6) ...... 89 5.1.6. Synthesis of dodecyl (2S,4R)-4-hydroxyprolinate (7) ...... 90 5.1.7. Synthesis of dodecyl (2S,4R)-N-methyl-4-hydroxyprolinate (8) ...... 91 5.1.8. Synthesis of dodecyl (2S,4R)-N-acetyl-4-hydroxyprolinate (9) ...... 92 5.1.9. Synthesis of (2S,4R)-N,N-dimethyl-4-(O-dodecanoyl)-2-(O-methyl)- hydroxyprolinium iodide (4a) ...... 93 5.2. Synthesis of 3,4-dehydro-proline derivatives ...... 94 5.2.1. Synthesis of (2S)-N-tert-butyloxycarbonyl-3,4-dehydroproline (12) ...... 94

VIII FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

5.2.2. Synthesis of dodecyl (2S)-N-tert-butyloxycarbonyl -3,4-dehydro-prolinate (13) ...... 95 5.2.3. Synthesis of dodecyl (2S)-3,4-dehydro-prolinate trifluoroacetate (14) ...... 96 5.2.4. Synthesis of dodecyl (2S)-N-acetyl-3,4-dehydroprolinate (15) ...... 97 5.2.5. Synthesis of dodecyl (2S,3R,4S)-N-acetyl-3,4-dihydroxyprolinate (16) ...... 97 5.2.6. Synthesis of dodecyl N-methyl-3,4-dehydro- prolinate (17) ...... 98 5.2.7. Synthesis of methyl 3,4-dehydro-N-dodecyl-prolinate (19) ...... 99 5.2.8. Synthesis of methyl (2S,3R,4S)-N-dodecyl-3,4-dihydroxyprolinate (20) ... 100 5.3. Synthesis of β-unnatural amino acid derivatives ...... 101 5.3.1. Synthesis of (1R,5S)-6-azabicyclohept-3-ene-7-one (23a-b)...... 101 5.3.2. Synthesis of (1R,2S)-2-aminocyclopent-3-ene-1-carboxylic acid (24a) .... 102 5.3.3. Synthesis of (1S,2R)-2-aminocyclopent-3-ene-1-carboxylic acid hydrochloride (24b) ...... 103 5.3.4. Synthesis of 2-[(tert-butoxycarbonyl)amino]cyclopent-3-ene-1-carboxylic acid (25) ...... 104 5.3.5. Synthesis of dodecyl 2-[(tert-butoxycarbonyl)amino]cyclopent-3-ene-1- carboxylate (26) ...... 105 5.3.6. Synthesis of 5-[(dodecyloxy)carbonyl]cyclopent-2-en-1-ammonium trifluoroacetate (27) ...... 106 5.3.7. Synthesis of dodecyl 2-(dimethylamino)cyclopent-3-ene-1-carboxylate (28) ...... 107 5.3.8. Synthesis of dodecyl 2-(dimethylamino)-3,4-dihydroxycyclopentane-1- carboxylate (29) ...... 108 5.3.9. Synthesis of dodecyl 2-[(tert-butoxycarbonyl)amino]-3,4- dihydroxycyclopentane-1-carboxylate (30) ...... 109 5.3.10. Synthesis of 5-[(dodecyloxy)carbonyl]-2,3-dihydroxycyclopentan-1- ammonium trifluroacetate (31) ...... 110 5.3.11. Synthesis of dodecyl 2-acetamido-3,4-dihydroxycyclopentane-1- carboxylate (32a) ...... 110 5.3.12. Synthesis of methyl (1R,2S)-2-aminocyclopent-3-ene-1-carboxylate hydrochloride (33) ...... 111 5.3.13. Synthesis of methyl (1R,2S)-2-dodecanamidocyclopent-3-ene-1- carboxylate (34) ...... 112 5.3.14. Synthesis of methyl (1R,2R,3S,4R)-2-dodecanamido-3,4- dihydroxycyclopentane-1-carboxylate (35) ...... 113 References ...... 115 Supplementary material ...... 121

FCUP IX Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

List of Figures

Figure 1: Families of compounds synthetized ...... 3 Figure 2: Skin structure, adapted from servier medical art ...... 5 Figure 3: Drug permeation pathways through the skin [15] ...... 7 Figure 4: Quantitative and qualitative techniques for skin penetration monitorization , adapted from [19] ...... 10 Figure 5: Open and closed vertical Franz diffusion cells [19] ...... 11 Figure 6: Schematic representation of the two possible ways of enhancers to interact with the SC intercellular lipid envelop.[24] ...... 15 Figure 7: Amino Acid-Based Transdermal Penetration Enhancers, adapted from [36] ...... 19 Figure 8: NMR spectrum of 5b containing evidence of rotamers...... 47 Figure 9: Surface tension curves...... 67 Figure 10: DSC curves for all compounds analyzed: A) O-acylated; B) N-alkylated/acylated, C) O-alkylated derivatives ...... 70 Figure 11: DSC curve of compound 31 recorded with Thermal analysis program...... 71 1 Figure 12: H NMR spectrum of compound 2a (400 MHz, CDCl3)...... 122 13 Figure 13: C NMR and DEPT spectra of compound 2a (101 MHz, CDCl3)...... 122 1 Figure 14: H NMR spectrum of compound 2b (400 MHz, CDCl3)...... 123 Figure 15:13C NMR spectrum of compound 2b (101 MHz, MeOD)...... 123 1 Figure 16: H NMR spectrum of compound 3a (400 MHz, CDCl3)...... 124 13 Figure 17: C NMR and DEPT spectra of compound 3a (101 MHz, CDCl3)...... 124 1 Figure 18: H NMR spectrum of compound 3b (400 MHz, CDCl3)...... 125 13 Figure 19: C NMR and DEPT spectra of compound 3b (101 MHz, CDCl3)...... 125 1 Figure 20: H NMR spectrum of compound 4a (400 MHz, CDCl3)...... 126 13 Figure 21: C NMR and DEPT spectra of compound 4a (101 MHz, CDCl3)...... 126 1 Figure 22: H NMR spectrum of compound 5a (400 MHz, CDCl3)...... 127 13 Figure 23: C NMR and DEPT spectra of compound 5a (101 MHz, CDCl3)...... 127 1 Figure 24: H NMR spectrum of compound 5b (400 MHz, CDCl3)...... 128 13 Figure 25: C NMR and DEPT spectra of compound 5b (101 MHz, CDCl3)...... 128 1 Figure 26: H NMR spectrum of compound 7 (400 MHz, CDCl3)...... 129 13 Figure 27: C NMR and DEPT spectra of compound 7 (101 MHz, CDCl3)...... 129 1 Figure 28: H NMR spectrum of compound 8 (400 MHz, CDCl3)...... 130 13 Figure 29: C NMR and DEPT spectra of compound 8 (101 MHz, CDCl3)...... 130 1 Figure 30: H NMR spectrum of compound 9 (400 MHz, CDCl3)...... 131 13 Figure 31: C NMR and DEPT spectra of compound 9 (101 MHz, CDCl3)...... 131 1 Figure 32: H NMR spectrum of compound 10 (400 MHz, CDCl3)...... 132 13 Figure 33: C NMR and DEPT spectra of compound 10 (101 MHz, CDCl3)...... 132 1 Figure 34: H NMR spectrum of compound 16 (400 MHz, CDCl3)...... 133 13 Figure 35: C NMR and DEPT spectra of compound 16 (101 MHz, CDCl3)...... 133 1 Figure 36: H NMR spectrum of compound 20 (400 MHz, CDCl3)...... 134 13 Figure 37: C NMR and DEPT spectra of compound 20 (101 MHz, CDCl3)...... 134 1 Figure 38: H NMR spectrum of compound 29 (400 MHz, CDCl3)...... 135 1 Figure 39: H NMR spectrum of compound 32 (400 MHz, CDCl3)...... 136 13 Figure 40: C NMR and DEPT spectra of compound 32 (101 MHz, CDCl3)...... 136 1 Figure 41: H NMR spectrum of compound 35 (400 MHz, CDCl3)...... 137 13 Figure 42: C NMR and DEPT spectra of compound 35 (101 MHz, CDCl3 ...... 137

X FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

List of Schemes

Scheme 1: Paths for synthesizing amphiphilic amino acid derivatives, adapted from [60]...... 30 Scheme 2: Chemoenzymatic separation of diesters ...... 34 Scheme 3: Desymmetrization of anhydrides with chiral amines...... 34 Scheme 4: Dipolar Cycloadditions of Diazoalkanes...... 35 Scheme 5: 2,2-dipolar cycloaddition of chlorosulfonyl isocyanate followed by acid hydrolysis. . 36 Scheme 6: Synthetic route for the synthesis of all 4-hydroxiproline derivatives ...... 41 Scheme 7: Acylation reaction mechanism in acidic conditions, exemplified for 1b ...... 42 Scheme 8: Reductive amination reaction mechanism, exemplified for 1a-10 ...... 43 Scheme 9: Esterification reaction mechanism with TMSCl as coupling agent ...... 44 Scheme 10: N-Cbz removal reaction mechanism ...... 45 Scheme 11: Acylation reaction mechanism in basic conditions, exemplified for 2b-5b ...... 47 Scheme 12: Synthetic route for the synthesis of 16, 18 and 20 ...... 48 Scheme 13: N-Boc protection reaction mechanism ...... 50 Scheme 14: Esterification reaction mechanism with Pybop as coupling agent ...... 51 Scheme 15: Boc removal reaction mechanism with TFA ...... 52 Scheme 16: Dihydroxylation reaction mechanism with OsO4 ...... 53 Scheme 17: Synthetic route for the synthesis of the β-amino acids 24a-b ...... 55 Scheme 18: Synthetic route for the synthesis of the compounds 29a-b, 32a-b and 35 ...... 56 Scheme 19: [2+2] Cycloaddition reaction mechanism with CSI...... 58 Scheme 20: Enzymatic resolution reaction mechanism...... 60 Scheme 21: Lactam ring opening reaction mechanism ...... 61 Scheme 22: Dimethylation reaction mechanism by reductive amination ...... 63 Scheme 23: Hofmann elimination like reaction mechanism ...... 63 Scheme 24: Compounds used in the tensiometry experiments...... 66 Scheme 25: Atom numeration ...... 79

FCUP XI Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

List of Tables

Table 1: Examples of chemical permeation enhancers [22] ...... 18 Table 2: Examples of Amino acid-based enhancers ...... 22 Table 3: Protecting groups ...... 31 Table 4: Coupling reagents for peptide synthesis...... 32 Table 5: Compounds synthetized ...... 38 Table 6: Interfacial parameters for the amino acid amphiphile derivatives ...... 68 Table 7: Melting points of the derivatives analyzed by DSC...... 71 Table 8: Reagents used ...... 78 Table 9: Synthesis of methyl (2S,4R)-4-(O-dodecanoyl)hydroxyprolinate ((2a) / (2S,4R) -4-(O- dodecanoyl)hydroxyproline (2b) ...... 84 Table 10: Synthesis of methyl (2S,4R)-4-(O-dodecanoyl)-N-methyl-hydroxyprolinate (3a) / (2S,4R)-4-(O-dodecanoyl)-N-methyl-hydroxyproline (3b) ...... 85 Table 11: Synthesis of methyl (2S,4R)-1-acetyl-4-(O-dodecanoyl)hydroxyprolinate (5a) / (2S,4R)-1-acetyl-4-( O-dodecanoyl)hydroxyproline (5b) ...... 87 Table 12: Synthesis of methyl (2S,4R)-N-dodecyl-4-hydroxyprolinate (10) ...... 88 Table 13: Synthesis of dodecyl (2S,4R)-N-Cbz-4-hydroxyprolinate (6) ...... 89 Table 14: Synthesis of dodecyl (2S,4R)-4-hydroxyprolinate (7) ...... 90 Table 15: Synthesis of dodecyl (2S,4R-N-methyl-4-hydroxyprolinate (8) ...... 91 Table 16: Synthesis of Dodecyl (2S,4R)-N-acetyl-4-hydroxyprolinate (9) ...... 92 Table 17: Synthesis of N-tert-butyloxycarbonyl-3,4-dehydroproline (12) ...... 94 Table 18: Synthesis of dodecyl-N-tert-butyloxycarbonyl -3,4-dehydro-prolinate (13) ...... 95 Table 19: Synthesis of dodecyl 3,4-dehydro-prolinate trifluoroacetate (14) ...... 96 Table 20: Synthesis of methyl-3,4-dehydro-N-dodecyl-prolinate (19)...... 99 Table 21: Synthesis of methyl (2S,3R,4S)-N-dodecyl-3,4-dihydroxyprolinate (20) ...... 100 Table 22: Synthesis of (1R,5S)-6-azabicyclohept-3-ene-7-one (23) ...... 101 Table 23: Synthesis of (1R,2S)-2-aminocyclopent-3-ene-1-carboxylic acid (24a) ...... 102 Table 24: Synthesis of (1S,2R)-2-aminocyclopent-3-ene-1-carboxylic acid hydrochloride (24b) ...... 103 Table 25: Synthesis of 2-[(tert-butoxycarbonyl)amino]cyclopent-3-ene-1-carboxylic acid (25) 104 Table 26: Synthesis of dodecyl 2-[(tert-butoxycarbonyl)amino]cyclopent-3-ene-1-carboxylate (26) ...... 105 Table 27: Synthesis of 5-[(dodecyloxy)carbonyl]cyclopent-2-en-1-amonium trifluoroacetate (27) ...... 106 Table 28: Synthesis of dodecyl 2-(dimethylamino)cyclopent-3-ene-1-carboxylate (28) ...... 107 Table 29: Synthesis of dodecyl 2-(dimethylamino)-3,4-dihydroxycyclopentane-1-carboxylate (29) ...... 108 Table 30: Synthesis of dodecyl 2-((tert-butoxycarbonyl)amino)-3,4-dihydroxycyclopentane-1- carboxylate (30) ...... 109 Table 31: Synthesis of 5[(dodecyloxy)carbonyl]-2,3-dihydroxycyclopentan-1-ammonium trifluroacetate (31) ...... 110 Table 32: Synthesis of dodecyl 2-acetamido-3,4-dihydroxycyclopentane-1-carboxylate (32) .. 111

XII FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

List of Abbreviations and Acronyms

AAG l-ascorbic acid 2-glucoside Boc tert-Butyloxycarbonyl group Benzotriazol-1-yloxytris(dimethylamino)phosphonium BoP hexafluorophosphate Cbz Benzyloxycarbonyl group CLE Corneocyte lipid envelope cmc Critical micelar concentration [(1-Cyano-1-ethyloxycarbonylmethylideneamino-oxy) COMU (dimethylamino) (morpholin-4-yl) carbenium hexafluorophosphate] CPE Chemical permeation enhancer CPD Cyclopentadiene CSI Chlorosulfonyl isocyanate DBU 1,8-diazabicyclo[5.4.0]undec-7-ene DCC N,N’-dicyclohexylcarbodiimide DDAA Dodecyl N, N-dimethylamino acetate DDAIP Dodecyl-2-(dimethylamino)propionate DDAK Dodecyl 6-(dimethylamino) hexanoate DDEAC Dodecyl-6-aminohexanoate DIC N,N'-Diisopropylcarbodiimide DLGL Dilauramidoglutamide lysine DPPC Dipalmitoylphosphatidylcholine DSC Differential scanning calorimetry ESI-Ms Electrospray ionization mass spectrometry FITC Fluorescein-isothiocyanate

Fmoc 9-Fluorenylmethyloxycarbonyl group

HPMC Hydroxypropyl methylcellulose HP-β-CD (2-hydroxypropyl)-β-cyclodextrin HOAt 1-hydroxy-7-azabenzotriazole HOBt 1-hydroxybenzotriazole IC50 Half maximal inhibitory concentration IL Ionic liquid

FCUP XIII Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

LPP Lipid-protein-partitioning

NEt3 Triethylamine

NMF Natural moisturizing factor Nuclear magnetic resonance; Proton (1H-NMR) and Carbon NMR (13C-NMR) NMO N-methylmorpholine N-oxide PB Phosphate buffer PG Propylene glycol; Benzotriazol-1-yloxytris(pyrrolidino)phosphonium PyBoP hexafluorophosphate SB Stratum Basale SC Stratum corneum SG Stratum granulosum SPT Solubility-Physicochemical-Thermodynamic SS Stratum spinosum TBDPS tert-butyldiphenylsilyl group O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium TBTU tetrafluoroborate TDS Transdermal delivery systems TFA Trifluoracetic acid TLC Thin-layer chromatography TMSCl Trimethylsilyl chloride

XIV FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

List of Symbols

m Mass, molal concentration t time

C0 Initial concentration D Diffusion coefficient K Partition coefficient h Thickness of the diffusional pathlenght F Force δ Chemical shift γ Surface tension Amount of substance in mol, number of carbon atoms in the alkyl n chain, R Real gas constant

NA Avogadro's number T Absolute temperature

훤푚푎푥 Maximum surface excess

as Minimal superficial area per molecule

Chapter 1: Introduction

2 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

1.1. Scope

The act of administering a drug, as simple it may seem, is one of the most important steps in medical practices, and the administration method influences greatly their success. Some examples of the most common administration routes are the oral, intravenous, transdermal, ocular, nasal, etc. Transdermal drug delivery systems (TDDS) allow the administration of a therapeutic agent through intact skin. TDDS have been a topic of rising interest for the last years since they represent a viable substitute for the most common administration routes. Topical applications for medical purposes are used for centuries, mostly as gels or creams to treat local conditions like inflammations and wounds. [1] Though, the focus of TDDS is the delivery of a drug to the systemic blood circulation at a controlled rate. Transdermal administration has several advantages over the oral and the parenteral routes. The direct delivery to the blood circulation avoids gastrointestinal and liver first-pass effects, allowing the use of drugs that are not suitable for oral medicines due to metabolism or common side effects. [2, 3] Also, TDDS are painless, unlike hypodermic injections, that are often associated with pain and stress during application, and do not generate as much hazardous medical waste, are non-invasive with easy administration, inexpensive and generally with patient approval. At last, they can provide a sustained effect during long periods, something only transdermal delivery can achieve. Both the oral and parenteral route lack control over drug release on blood circulation, with high levels of drug concentration in the plasma shortly after administration, followed by a gradual decline without sustained effect. [2, 4] Despite the advantages, TDDS has not yet achieved its full potential. Its application is limited to a small number of drugs with low molecular masses and favored lipophilic behavior and drug doses in dermal patches are small with only a few milligrams. [1] The main obstacle to the performance of TDDS is the skin, that prevents the flux of toxins and other physical/chemical species into the body and minimizes water loss and thus it has a very low permeability for the penetration of foreign molecules. [4] The Stratum corneum (SC), the outermost layer of the skin constitutes the main barrier to drug penetration. Consequently, several approaches have been made to develop novel TDDS with increased drug permeation across the SC.

The Stratum Corneum, the outermost layer of the skin, prevents the loss of moisture from internal tissues and it has a selective interaction with permeants. In order to take full benefit of the advantages of transdermal delivery, investigation in novel TDDS is required. FCUP 3 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

1.2. Objectives and Work plan

One of the most widely used approaches in TDDS is the use of chemical permeation enhancers. CPEs are a promising response to mitigate the ineffectiveness of transdermal drug delivery. This project has the ambition of making a contribution to this field, through the synthesis of novel families of amino acid-based enhancers. Following previous studies where the potential of proline derivates as potent permeation enhancers is reported [5, 6], the design of novel compounds based on proline and proline mimetic derivatives is proposed. The aim is to establish relationships between the structures of the compounds and their physicochemical properties, biological activity and permeation enhancement capacity.

The workplan proposed involved the following tasks:

• Design and synthesis of three novel families of amphiphiles using different amino acids as headgroups and a 12-carbon alkyl chain as non-polar tail introduced in different functional groups: 4-hydroxiproline derivatives; 3,4-dihydroxiproline derivatives); and proline mimetic amphiphilic compounds (Fig.1).

Figure 1: Families of compounds synthetized

4 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

• Evaluation of the physicochemical properties of the synthetized amphiphiles, specifically the determination of the critical micellar concentration by tensiometry. • Evaluation of the toxicities of all compounds synthesized in HaCaT(keratinocyte) and 3T3 (fibroblasts) cell lines. The best performing compounds would be selected for further tests. • Evaluation of the permeation enhancement capacities of the selected compounds, through diffusion cells (Franz cells), using two different drugs to represent different characteristics (size and polarity).

The current project was carried out at the Department of Chemistry and Biochemistry of FCUP, in the research group ORCHIDS “Organic Chemistry-Inspired Design and Synthesis of Bioactive Molecules” part of LAQV REQUIMTE, under supervision of Professor Maria Luísa Cardoso do Vale and co-supervision of Professor Maria de la Salette Reis (Cathedratic professor at FFUP). The project was performed in collaboration with Professor Maria de la

Salette Reis and Doctor Marina Pinheiro, members of the research group MB2 “Molecular Biophysics and Biotechnology” part of LAQV REQUIMTE, at FFUP.

FCUP 5 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

1.3. Introduction

1.3.1. Skin structure

The skin is the largest human organ, responsible for about 15 % of the total body weight, covering 2 m2 of surface area in adults. [2, 7] It is a multilayered structure composed of 3 larger layers: epidermis, dermis and hypodermis. The epidermis, the uppermost layer of the skin is organized into five Strata: the stratum corneum (SC), the outermost layer and also known as the non-viable epidermis which is 10-20 μm thick, and the stratum lucidum (SL), stratum granulosum (SG), stratum spinosum (SS) and stratum Basale (SB) which together represent the viable epidermis, measure 50 to 100 μm and are avascular. [7] Deeper into the skin is the dermis, with a thickness of about 1–2 mm thick. The dermis is composed of fibroblasts that compose the extracellular matrix, alongside high concentrations of collagen, elastin and glycosaminoglycans. It is rich in vascular circulation, necessary for drug absorption.

Figure 2: Skin structure, adapted from servier medical art

The SC constitutes the main barrier to drug permeation. It is mostly composed of corneocytes, separated by an intercellular lipid domain. The corneocytes are non-living keratin- filled cells. [8] These cells are structurally stabilized by corneodesmosomes, proteinaceous complexes responsible for ensuring the integrity of the stratum corneum. Also, between the cells, there are constricted junctions composed of claudin and blocking proteins that enhance the paracellular barrier functions of the skin.

6 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

The organization of the SC if often described in terms of the “brick and mortar” model, where the keratin filled corneocytes represent the bricks and the intercellular lipids, the mortar. [9] The composition of the intercellular lipid mixture differs significantly from other cellular membranes, its particularly rich in ceramides (40-50 wt%), fatty acids (7-13%) and cholesterol (20-33%) with minor quantities of cholesterol sulphate (2-5 % wt%) and is organized in bilayer arrays, forming an impermeable barrier to drug diffusion. [10] At physiological temperatures, these lipids remain in a solid crystalline or gel phase. The molecular arrangement of the lipid of the extracellular matrix has been extensively studied and multiple molecular models have been proposed to explain their organization. [9] In the “domain-mosaic” model [11], the lipid mixture is organized in two regions, the gel domains known as grains (a crystalline phase) enclosed by a fluid phase known as the grain boundaries. The tri-lamellar sandwich model was proposed by Bouwstra et al. [12] and describes alternating crystalline and fluid phases. The repeating units comprise the central region, composed of unsaturated linoleic acid and cholesterol in a fluid or sublattice liquid phase, and two adjacent regions, where the head groups and the alkyl chains have decreased mobility, resulting in a crystalline phase. At last, in the single gel phase model [13] the entire lipid matrix behaves as s single phase in a closely packed state with reduced mobility. Such a compact phase explains the low permeability to both hydrophilic and hydrophobic molecules of this lipid matrix. The lipid membrane that surrounds and anchors corneocytes in the SC, known as the corneocyte lipid envelop (CLE), has also a very distinct lipidic composition from other cells, being primarily a monolayer of ω-OH-ceramides (CER). The CLE can also function as a semi- permeable membrane that can allow the passage of water, while restricting the loss of larger hygroscopic molecules, such as filaggrin (filament aggregation protein) breakdown products (smaller peptides and free amino acids), out of the corneocytes. [7]

1.3.2. Permeation routes

A drug or molecule applied to the skin surface has three potential pathways to penetrate across the SC: Intercellular route, Transappendageal route (via hair follicles and associated sebaceous glands) and Transcellular route, across the continuous epidermis. [14]

FCUP 7 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Figure 3: Drug permeation pathways through the skin [15]

The transappendageal approach encompasses penetration through sweat ducts, hair follicles. Its contribution to percutaneous transport if often considered secondary since appendages account for only 0.1% of the total surface of the skin. However, different studies have suggested that these routes can be significant positive contributors to drug permeation of both lipophilic and hydrophilic molecules. [15] Sweat glands are full of aqueous solution, a possible route for polar drugs. The follicular glands, in turn, are filled with lipoidal sebum, the ideal environment for nonpolar molecules with less resistance than the SC bulk. Also, these routes may be a quicker pathway to drug permeation than inter- and intracellular alternatives, mostly because penetration through shunt routes allows bypassing the rigid “brick and mortar” cellular structure of the SC. [16] However, this behavior is crucially dependent on the conditions of the experiment, the application site and the drug characteristics. For example, water can interfere in this process, since hydration of the tissue results in skin volume enlargement, and most likely the shunt routes begin to close. The transcellular or intracellular route is identified as the polar path though the SC. [17] The intracellular keratin matrix is highly hydrated and polar and so, ideal for polar molecules. However, after the protein matrix inside each cell, the molecules need to pass through the lipid bilayers of the CLE. So, permeation through this route is a complex process of repeated partitioning between the keratinocyte polar environment and the extracellular matrix. Also, to pass the lipid bilayers, there are between 4 and 20 layers of hydrophobic chains and hydrophilic head groups between each cell. Despite these difficulties, this approach seems to be the main contributor in the permeation of polar drugs. The intercellular route is a continuous pathway through the lipid bilayers between the keratinocytes. [16] It starts with partitioning between the donor solution (environment in contact with the skin) and the SC. The molecules then diffuse through the lipid domain until they reach the viable epidermis, where the drug is partitioned into a more aqueous environment. Although

8 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems the lipid bilayer represents a small part of SC, the literature reports its importance for many years as a key pathway for drug penetration. [17] The complex lipid matrix structure offers a penetration route for different kinds of molecules, despite being the main responsible to the barrier properties of the skin. It allows molecular transport through the hydrophobic chains for non-polar drugs or through the polar head groups for polar drugs. The relative importance of the different pathways may vary depending on the physico- chemical properties of formulation and permeant and may also vary with time.

1.3.3. Penetration of drugs through the skin

The process of drug penetration includes all moments from when the molecule is released from the dosage form until it is absorbed into the blood circulation. The physicochemical characteristics of the permeant and the specific characteristics of the skin on the application site are the factors that control these processes. A simple way to consider the factors affecting drug permeation is via the equation for steady state flux, or Fick´s law. (Eq. 1)

푑푚 퐷퐶0퐾 = (Eq. 1) 푑푡 ℎ

This law plots the cumulative mass of drug, m, passing per unit of area through the membrane (flux of drug ), where 퐶0 is the constant concentration of drug on the initial application dose, K is the partition coefficient, D the diffusion coefficient and h is the thickness of the diffusional pathlength. [4]

The partition coefficient, K, affects the first step of drug transport, the partitioning of the molecule from the applied vehicle to the lipid matrix of the SC, and then the partitioning between the SC lipophilic domain and the viable epidermis, a more hydrophilic domain. K sets the equilibrium of affinity between a hydrophilic environment and a hydrophobic one for a certain molecule. Molecules with intermediate partition coefficients (logK 2-3) will be soluble in the lipid matrix of the SC and will be also sufficiently hydrophilic to partition into the more aqueous viable epidermis. [14, 18] The diffusion coefficient, D, affects the speed and effectiveness of the permeation of the molecule along the intercellular route of the SC. It depends of the molecular size of the drug and its solubility. According to literature, there exists an inverse relationship between FCUP 9 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

molecular size and permeation, so the smaller the molecule, the higher the permeation. For a successful topical and transdermal delivery, molecules should be less than 500 Da in size. [14] Larger molecules, such as proteins or peptides, cannot naturally overcome the skin barrier unless the transport is facilitated. At the same time, the drug must have lipid solubility, to potentiate a higher diffusion coefficient in the intercellular environment of the SC, but also moderate aqueous solubility, to be soluble in the donor solution, maximizing the initial concentration and thus maximizing the flux of permeation. There are many other physicochemical parameters that affect diffusion, namely melting point (that affects solubility), ionization, binding potential, and environment characteristics like viscosity and tortuosity. [14, 18] From Fick’s law it is evident that maximum drug permeation is achieved when the h- value is low. However, this factor cannot be controlled. The typical thickness of the SC is about 10-15 µm and in the intercellular pathway the h-value may exceed 150 µm. due to its non- linearity. [14, 18] When a drug does not possess the above mentioned ideal physicochemical properties necessary for transdermal delivery, it has either to be manipulated or permeation enhancement methods have to be used. [14, 18]

10 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

1.3.4. Drug flux evaluation methods

Drug permeation/flux into the systemic circulation is the goal of TDDS. The effect of delivering the active substance through transdermal delivery systems is influenced by partitioning and diffusion, as stated in chapter 1.3.3. These parameters not only define the quantity of drug that penetrates the skin, but also the time the drug requires to enter and to overcome the SC, the time required to reach systemic circulation, the mass of drug that reaches circulation over time, the reversibility of the delivery and the saturation limit of permeation speed, among others. All these factors are essential for the clinical purpose of TDD .[19] Any topical formulation validation requires monitorization of drug permeation, which can only be done in models of human skin, where drug flux is controllable. Only after obtaining this information, the in vivo skin samples are used. In general, the goal is to follow the accumulation of drugs on the receptor solution, as well as the quantity of drug in different skin layers. The techniques for analyzing drug penetration effects can be subdivided into quantitative and qualitative methods. [19] Quantitative “in vitro” methods are used to monitor the drug flux through the model membrane used over time. The drug flux is related to the diffusion area and the amount of drug accumulated at the receptor cavity under the membrane. There are several quantitative techniques that can be combined to achieve better results. The most used ones are diffusion cells, skin-PAMPA and tape stripping with HPLC (Figure 4).

Techinques

Qualitative or Quantitative semi- quantitative

Confocal Tape stripping Microscopic Diffusion cells Skin-PAMPA RAMAN with HPLC techniques spectroscopy

Figure 4: Quantitative and qualitative techniques for skin penetration monitorization , adapted from [19] FCUP 11 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Diffusion cells are the most common used model for permeation assessment especially since the development of the “Franz cell” in 1970. Due to this, this technique will be explored in more detail than other methods. Diffusion cells consist of 2 chambers, the upper chamber or donor chamber for drug application, the lower or acceptor chamber to analyze the amount of drug flux, and between the two chambers a membrane through which the drug must pass. They can operate in static or flow-through state. In static cells, the chambers and the membrane maybe placed either vertically, like in the “Franz cells”, or horizontally. Flow-through state diffusional cells have a similar physical displacement of the chambers as static cells, with the particularity that in the acceptor chamber a continuous supply of fresh solvent flows and that the permeant is being constantly removed from the cell. Franz cells with an open top work at atmospheric pressure but most of the cells used are closed, resulting in higher pressure, which in turn leads to higher drug permeation flux. [19] Nowadays, most systems use an autosampler attached, which besides facilitating the work also leads to more consistent results.

Figure 5: Open and closed vertical Franz diffusion cells [19]

Diffusion cells can be used for two types of tests, release tests or skin permeation tests. For release tests, synthetic membrane models should be used, and the detected masses are in µg to mg range. For skin penetration testing, human skin is the best model, and the detected active substance mass is in the range of pg to ng. Ideally, release testes should be used first as a preliminary study, and when the preparation is of good quality, skin penetration studies can be conducted.

12 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Concerning Skin PAMPA, parallel artificial membrane permeability assay, it is performed in a 96-well plate and allows the fast determination of the passive membrane permeability of molecules with the advantage of its low cost and high throughput. Each cell contains a donor phase at the bottom and a hydrated skin model on top. Skin-PAMPA membranes are specifically composed of cholesterol, free fatty acid, and a ceramide-analogue compound that imitates the features of the lipid matrix of the SC. Tape striping is a commonly used, minimally invasive method for testing the penetration of topically applied formulations through the stratum corneum. Several layers of the SC are removed by an adhesive tape and analyzed by HPLC for example. This method allows the quantification of drug accumulated at the SC, as well as the drug distribution along the skin layer.

To obtain reproducible and reliable data of dermal permeability in quantitative methods, several parameters, such as the sink condition, the incubation time and temperature, the mixing, the hydration of the membrane and the drug dose amount must be considered. [19, 20] The sink condition is the ability of the acceptor solution to dissolve at least 3 times the amount of drug that is in the dosage form. Thus, the acceptor solution must have high dissolution capacity for the drug under study and, at the end of the experiment, the concentration of acceptor solution should not exceed 20 % of the drug’s solubility in the releasing matrix. This requirement is necessary to avoid interferences of drug flux due to concentration gradients. The incubation time should reflect in-use conditions and may vary from a few minutes to a maximum of 24h. It is important to ensure that the model membrane remains undamaged until the end of the experiment, and thus skin integrity tests are required prior to the dermal permeation evaluation. The incubation temperature should be physiologically relevant (32±1°C) and skin hydration should be optimal and constant. Depending on the sample amount two types of experiments can be defined: finite or infinite dose measurements. In infinite dose tests, the donor solution never runs out of permeant and a continuous flux of drug is observed. However, finite doses resemble more the conditions of dermal formulations used in patients. One of the most important elements in permeation studies is the membrane model used. The ideal model is isolated human skin, but it is often difficult to obtain many samples under good conditions for permeability testing. [21] Many laboratories use human cadaver skin FCUP 13 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

obtained from banks authorized to perform such operations. In alternative, animal skin can be used, and a wide range of animal models has proven suitable for this purpose (primate, porcine, rat and mouse models). In particular, porcine skin presents biochemical properties similar to human skin, giving results which are in good agreement with human skin models. Also, pig ear skin presents superior results compared to rat skin, especially for lipophilic compounds. [21] For simpler tests, there are other options that can give results good enough to detect potential candidates for permeation. Polymeric membranes of polysulfone, cellulose, acetate/nitrate mixed esters, and others represent some of the synthetic models used. They have the advantage of being highly tunable, with manipulable properties such as hydrophilicity and pore size, and can be also produced on a large scale. [21] Tissue cultured-derived skin equivalents can also be used for permeation assessment as alternatives to animal skin. These models are comprised of cultured human skin cells and a 3D tissue structure composed of extracellular lipid components mixed into the cell culture. Despite being representative of the SC layer, the permeability of this tissue is usually higher than in vivo skin samples. However, their use is highly supported for evaluation of skin irritation. In fact, these models were found to present an architecture, homeostasis and lipid composition similar to native human tissue, and constitute good models for toxicological studies.

The analytical data, however, are not enough to evaluate drug permeation. The damage performed on the skin model by the permeant is not evaluated with quantitative techniques, but with qualitative methods, such as microscopic and spectroscopic methods. [19] Microscopic techniques can give important information about the distribution of drugs on the different skin layers or explain permeation mechanisms, without sample destruction or, in some cases, low sample interferences. These techniques are applicable to almost all types of model membranes, in vitro, ex vivo, and even sometimes in vivo. Fluorescence microscopy is commonly used for mechanism comprehension or permeant localization, with the use of fluorescent labeled material that can be easily localized in the SC or lower layers. [19] Other techniques can provide information about molecular structure and changes after the experiment, such as confocal Raman spectroscopy. Through the observation of vibrational energy levels in site, it is possible to obtain elucidating information about modifications in the structure of skin components and to follow the penetration of active substances.

14 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

1.3.5. Permeation enhancement strategies and mechanisms

The main challenge of TDD is to design a system that can successfully deliver any drug through the skin. Much research has been conducted in this area over the past few decades, creating new powerful methods for enhancing dermal drug delivery. These methods can be mainly divided into passive enhancement and active enhancement. [22] Passive or chemical methods are designed to increase the natural driving force of molecules through the skin, the diffusion coefficient, and the solubility in the SC by applying chemicals. This can be accomplished by: (1) improving the permeation capacity of the formulation, using drug vehicles like vesicular systems, microemulsions, nanoparticles, among others. (2) improving the permeation capacity of the drug, which can be achieved through an increase in drug concentration to favour drug partitioning. To this end, strategies to solubilize more drug in the donor solution are applied like ion pairs, eutectic mixtures, supersaturated systems, among others. (3) changing the SC natural barrier properties by using chemical permeation enhancers, that modify the SC and reduce the natural skin's resistance to penetration. [4, 22] Regarding chemical penetration enhancers (CPEs), they can have 3 different mechanisms of action, according to the LPP theory presented by Barry and Co. (1989). [17, 23] They can interact with the intercellular lipid phase, the intracellular protein domains of the SC or affect the partitioning of drugs into the skin. In the intercellular lipid phase, enhancers may break down lipid packing and/or fluidize the lipid chains, creating a more permeable domain. This can be achieved by interacting at three different regions associated with the lipid bilayers: CPE can interact with the head groups, altering hydration spheres and thus disturbing lipid packing. This interaction results in a more fluid lipid domain, and thus enhanced flux of hydrophilic/lipophilic penetrants. They may also extract lipids from the bilayer creating holes on the lipid phase. Interaction at the aqueous domain of the lipid bilayer may provoke swelling, increasing the domain volume and changing the bilayer thickness. CPE are also able to insert between the hydrophobic FCUP 15 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

chains, creating stereochemical impediment, disturbing their packing and thus increasing lipid fluidity.

Figure 6: Schematic representation of the two possible ways of enhancers to interact with the SC intercellular lipid envelop.[24]

In the intracellular protein domain, permeation enhancers interact with keratin, changing its conformation and opening water channels in the cells, facilitating the transport of hydrophilic molecules. [23] They also interact with corneodesmosomes, altering the interconnections between corneocytes. At last, solvents may enter the SC and alter its chemical environment, thus changing its solution properties, increasing partitioning of the drug into the skin. [23] Recently, another theory on the action of enhancers which can complement the LPP theory has emerged. The LPP theory does not explain why a given enhancer cannot increase permeability for all drugs, or why the activity of an enhancer is concentration dependent but some enhancers have higher activity at lower concentrations and others at high concentrations. According to the Solubility-Physicochemical-Thermodynamic (SPT) theory proposed by Michniak-Kohn et al., [25] drug flux depends primarily on thermodynamic activity of the enhancer and not on concentration. Moreover, it assumes that drug flux is mostly affected by two parameters: the solubility of the drug/enhancer in the formulation and the SC environment, and the physicochemical interactions between the drug and enhancer. For both these parameters there are optimum values, and the action of an enhancer is always drug dependent. Overall, the most safe and potent enhancers are mostly related to lipid interaction because the intercellular pathway provides the best chances for drug permeation. A good

16 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

chemical enhancer should fulfill the following requirements: specificity to drug permeation without any other bioactivity; non-harmful in any way, nontoxic, non-irritating and non- allergenic; rapid onset of effect, foreseeable and reproducible; reversibility when removed from the application site; unidirectional activity, enabling drug permeation without water loss from inner tissues; compatible with drug and excipients; cosmetic acceptance from consumers. [22]

Concerning active or physical methods, they use external forces to facilitate the permeation of drugs into the SC or to bypass the SC directly. These methods are divided in electrical assisted methods (like iontophoresis, electroporation and radiofrequency), mechanical methods (like microneedles, abrasion, perforation and skin stretching,), and other physical methods like ultrasounds, laser radiation or thermophoresis. Many comparisons have been made between the two strategies. Physical methods surpass chemical methods on efficacy and safety. [26-29] Although many new permeation enhancers in the last 10 year have been developed, almost only the first-generation chemical enhancers are on the market, as e.g. ethanol, oleic acid or propylene glycol. This happens for two reasons: The new candidates do not improve the drug flux significantly more than the current ones used or the new enhancers that factually improve drastically drug permeation have safety problems. [26, 27] Concerning physical techniques, the microneedles and electric based techniques have achieved a great improvement on potency with also high safety standards. [27] On the other hand, chemical permeation enhancers may be more suitable for the market due to their many advantages over physical technologies. Transdermal formulations of drugs with chemical enhancers are generally cheaper than physical equipment. They can be adjusted in detail with chemical modifications, different sizes and forms of application, like creams, gels, and patches. Also, producing patches or creams in large scale is easier and quicker than producing the physical enhancement devices. From a market standpoint and consumer opinion, chemical enhancer-based strategies are attractive for being easy to use, non-invasive and self- administrable. [22, 30] Finally, due to recent progress in the understanding of their mechanism of action, chemical enhancers may have an important role in future high impact applications of TDDS, such as macromolecular delivery. More recently, the two approaches have been merged to create a hybrid approach in the same formulation, chemically and physically, to enable new high-impact applications. [31]

FCUP 17 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

1.3.6. Chemical Penetration Enhancers

The vast diversity of molecules known as CPE calls for a systematic classification to allow some organization within this group of compounds (table 1). However, there are several parameters that can be considered when attempting to categorize them. Classifications based on their origin (natural, synthetic, or semi-synthetic), on their mechanism of action ( solvents, amphiphilic molecules and peptides [22]) and their functional class ( hydrocarbons, alcohols, amines, carboxylic acids, amino acids, esters, amides, sulfoxides) are the most common. [32- 35] In this text we will use the classification based on functional group because it better fits our purposes. As we are mainly interested in amino acid derivatives, we will center the following discussion on this class of compounds.

18 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Table 1: Examples of chemical permeation enhancers [22]

Ethanol Glycerol N-methylpyrrolidone Propylene glycol DMSO (rac.)

Oleic acid

Dodecanol

N-dodecylazenpan-2-one (Azone) N-dodecylpyrrolidone

Limonene Menthol Farnesol

Sorbitan monooleate

Ascorbyl palmitate

Isopropyl myristate

Ethyl oleate

Glycerol monolaurate (rac.)

Dodecyl 2-dimethylaminopropionate (DDAIP) (rac.)

Dodecyl N-acetylproline (Pro2)

Transkarbam 12

FCUP 19 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

1.3.7. Amino acid-based enhancers

Amino acid amphiphiles are a new type of chemical enhancers with several advantages that justify investment and exploitation. These compounds possess an amino acid as polar head (amino acid residue) linked to a hydrophobic chain. According to the LPP theory, this structure should lead to strong interactions with the lipid bilayers of the SC. Thus, the compounds can interact with the polar head and alkyl chains at once, creating disruptions in the lipid organization, reducing chain order and increasing fluidity. [36]

Figure 7: Amino Acid-Based Transdermal Penetration Enhancers, adapted from [36]

As stated in literature, the length of the alkyl chain has a significant importance on the effect on the lipid barrier. Alkyl chains with 10 to 12 carbon atoms present the best profile. These chains are shorter than those of the fatty acids found in the lipid matrix of the SC (18 carbons or more), free or attached to ceramides. So, the shorter amphiphiles will be incorporated into the lipid packing, forming pores under the shorter chains, thereby reducing the interaction between ceramides and increasing fluidity. [37, 38] The effect of the polar head structure is less pronounced than the hydrophobic counterpart. However, since the head group is located in the hydrophilic region of the lipid bilayers, it should be small enough to be incorporated in the bilayer, but different from the ceramide head group to provoke packing irregularities.[22, 39] Moreover, polar groups having the ability to establish hydrogen bonds are vital for several reasons. First, they increase the hydration sphere of the head groups, and more water molecules mean a bigger aqueous phase between each lipid bilayer, which improves drug transport for hydrophilic molecules. Also, the increase of water in the SC can lead to additional permeation enhancement. Water is a

20 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

recognized powerful natural CPE, and, even though the mechanism of action is not fully understood, it helps permeation of both lipophilic and hydrophilic molecules. [32] Also, hydrogen bonding is directly responsible for lipid extraction from the lipid domain, another way of causing holes in the packing. Another structural property that may have a significant role in the potency of enhancers that has not been much explored yet is the chirality of the head group. The importance of stereochemistry in drug research and development is well acknowledged. In fact, most biological membranes have a chiral environment, and that feature can affect many important aspects of drug penetration, such as melting point, solubility, and stability, which may vary with stereochemical purity. Similarly, many topical formulations use excipients with known chirality, such as cellulose. Despite much evidence that drug chirality may have consequences on drug permeation, there is not much research on the influence of chirality of enhancers and their interaction with chiral drugs, enantiomeric pure or not. [39]

Amino acid-based enhancers have at least one chiral center (except glycine). They are usually small and contain several polar groups capable of hydrogen bonding. The hydrogen bonding ability is important not only for some penetration enhancement mechanisms, but also for water injection into the SC. Free amino acids and peptides are known humectants and moisturizers responsible for the natural moisturizing factor (NMF) of the SC. So, the use of amino acid-based CPE can indirectly bring a higher amount of water into the SC, increasing permeation even further. Until now, only a few permeation enhancers have been approved for clinical use due to common toxicity or skin irritation issues. The balance between potency and safety is probably the biggest challenge, although there seems to be no close relationship between these two factors. [35, 40-42] Certain mechanisms of action tend to be safer than others. Compounds that usually extract lipids from the bilayer are associated with keratin denaturation, leading to skin irritation. Non-ionic derivatives are usually less toxic than ionic ones. Furthermore, the presence of labile bonds which confer enhanced biodegradability also contributes to reduce toxicity. Since most of the metabolic activity in the skin occurs only in the viable epidermis, the enhancer can be active at the SC, and when it reaches the lower layer, it breaks down into non-toxic compounds. Amino acid-based amphiphiles consisting of amino acids linked to fatty alcohols by ester bonds should comply with this requirement. Ester bonds are easily broken on the viable epidermis, reducing the toxicity that the molecules may have, because the breakdown products are natural compounds present on the skin.[22]

FCUP 21 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Table 2 summarizes the most relevant information about some of the best amino acid- based enhancers described in the past 12 years, such as the chemical structure, the permeation enhancement data, the type of drug used as permeant and the toxicity of the compound.

These enhancers have been used to promote a higher absorption through skin in different classes of drugs, including anti-inflammatory steroids (Hydrocortisone), nonsteroidal anti-inflammatory drugs (Indomethacin) anti-virals (Adefovir, Tenofovir) antineoplastics (Fluorouracil), antibiotics (Metronidazole) and local anesthetics (Tetracaine, Ropivacaine). To refer that hydrophobic (Tetracaine, Hydrocortisone, Indomethacin) and hydrophilic drugs (Theophylline, Adefovir, Tenofovir, Fluorouracil, Metronidazole, Ropivacaine) are represented

22 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Table 2: Examples of Amino acid-based enhancers

Polar Flux Rate head Enhancer structure ER /µg. cm−2 Drug Donor conditions Cytotoxicity Ref amino .h−1 acid 6- 17.8 42.2 ± 14.3 Theophylline 5% drug in 60% PG 75.6 ± 12.7 (Dimethyl 175.2 ± 43.2 4.78 Hydrocortisone 2% drug in 60% PG µM amino)he 27.6 µM [43] 13.6 19.0 Adefovir 2% drug in PB pH 4.8 IC50 - xanoic IC50 - 3T3 HaCaT acid - 8.7 Indomethacin 2% drug in 60% PG 68.2 ± 11.5 40.0 70.3 ± 7.7 Theophylline 5 % drug in 60% PG 182.6 ± 6.7 µM L-Proline µM [5] IC50 - 47.0 6.54 ± 0.87 Hydrocortisone 2 % drug in 60% PG IC50 - 3T3 HaCaT) 80 % viability for 240.55 ± Hep G2, MCF 7 and A549 β-alanine 5.87 Tenofovir 2 % drug in 4 % HPMC [44] 21.06 cells, at 100 µg/ml 2.5 % drug in 1 % HPMC 1.87 37.08 ± 1.85 Tetracaine and HP-β-CD* 100 % HEK cells viability at Serine [45] 0.14 µM 2.96 5.74 ± 0.74 Ropivacaine 2.5 % drug in 1 % HPMC*

Glycine- 1 % drug in gel formed 7.2 17.7 ± 1.3 Metronidazole ------[46] Histidine with 5 % enhancer

851.47 ± 177 ± 14 7.14 Fluorouracil 0.5 % drug in water 387 ± 19 69.49 µM L-Leucine µM [6] TC50 - 2.28 29.92 ± 5.33 Hydrocortisone 0.1 % drug in water TC50 - 3T3 HaCaT

FCUP 23 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Amino acid derivatives have a long history in application in dermal delivery. The first reports of amino acid derivatives used as absorption enhancers go back to 1985, when Fix and Pogany [47] patented lysine esters of fatty alcohols as permeation enhancers; however they were not applied in dermal delivery, but in rectal or gastrointestinal absorption. In 1989, Wong et al. [48] designed biodegradable enhancers to mitigate the proved side effects of the first established skin permeation enhancer azone (skin irritation after long exposure). They synthetized, among other molecules, amino acid esters, which upon cleavage of the ester bond would result in alcohols and substituted amino acids, bioavailable compounds. From this work, dodecyl N, N-dimethylamino acetate (DDAA), a glycine derivative, showed the best permeation improvement in comparison to Azone. These results lead to the development of new enhancers, designed to improve the capabilities of DDAA and in 1993, Buyuktimkin et al. [49] described the synthesis and permeation enhancement effects of a novel amino acid derivative, dodecyl-2- (dimethylamino)propionate (DDAIP), the first amino acid-based enhancer (alanine derivative) to be patented and commercialized. Later, a new series of ω-amino acid-based derivatives was synthetized and tested [50]. These derivatives were designed as open azone analogous to replace the rigid ring structure by a flexible chain (a 5 or 6-carbon linking group between the nitrogen and carboxylic acid). and the amide bond by an ester bond, to guarantee biodegradability. Among them, DDEAC, dodecyl-6-aminohexanoate, was found as the most promising derivative with higher enhancement capacity and lower toxicity than azone. Later it was discovered that this enhancer is only active after the capture of CO2, yielding the active structure named Transkarbam 12. [51] The unnatural ω-amino acid-based derivative known as DDAK (Dodecyl 6- (dimethylamino) hexanoate), described in table 2, was synthetized and tested by Novotný et al. in 2008. [43] This enhancer was designed as an upgraded version of previously reported CPE because the polar head is comparable to the dimethylalanine head group present in the enhancer DDAIP (table 1), but its also a ω-amino acid, like the 6-aminohexanoates (Transkarbam 12 being the most effective). Compared with existing amino acid derivatives, DDAK showed to be a better and more effective enhancer, with reversible activity, capable of enhancing penetration of highly hydrophilic and lipophilic molecules. In terms of safety, this CPE shows good results, with a high IC50 value for human dermal cell lines and low oral toxicity tests in mice. This enhancer is considered until today on of the most promising amino acid-based enhancers to future clinical and cosmetic applications. [48]

24 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Several other amino acid derivatives were synthesized and tested for permeation enhancement, using different amino acid moieties, such as, proline [5, 6, 52-55], serine [38, 45, 52, 56], lysine [47, 57, 58], sarcosine [5, 59] among others. Vávrova et al. (2003) [56], conducted a study involving glycine and serine derivatives, to yield analogs of ceramide, the most prevalent lipid in the CLE. However, the researchers concluded that if an enhancer possesses a similar structure to the ceramides (serine head group with 2 alkyl chains of 14 and 24 carbons each), it does not have permeation enhancement capabilities. By altering the length of the hydrophobic chains to 12 carbons, the molecule acts as a moderate enhancer. The same group (Janůšová et al.) [5] tested a large number of natural amino acid-based enhancers, using the information previously gathered to yield the best possible enhancer. The authors synthetized several amino acid-based CPE and performed a systematic study on the influence of the following parameters on the enhancement potential: (1) alkyl chain length (2) monomeric or dimeric structure (3) the nature of the headgroup (glycine, L- and D-alanine, β-alanine, sarcosine, and L/D-proline), in what concerns its hydrogen bond donor or acceptor capacity and chirality. The authors confirmed that the optimal length for alkyl chains is twelve carbon atoms, consistent with the literature, and demonstrated that single-chained enhancers are better that double-chained enhancers. For the different head groups, enhancement activity of H-bond donor amino acids is lower than that of H-bond acceptor amino acids. In fact, L-proline and sarcosine disubstituted amide derivatives were the best enhancers of the list of amino acids used. Also, a derivative of proline with a tertiary N-methyl group instead of acetyl was synthetized, to mimic DDAK and DDAIP. This change resulted in a decrease of activity, which was expected due to the reduction of the number of H-bond acceptor sites. As for the stereochemistry, the action of proline and alanine-based enhancers is not stereoselective, since no differences between L- and D- enantiomers were observed. The authors concluded that the best enhancer synthetized was the L-proline derivative with an ester bond to an alkyl chain of 12 carbons and an acetyl group. This

enhancer presented reversible action, positive synergy with propylene glycol, IC50 values to human dermal cell lines are lower than commercially used enhancers, and proved activity and safety in in vivo models, specifically rats. FCUP 25 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

The use of different alkyl chain lengths in amphiphilic derivatives has been vastly reported in literature [37-39] However, only a few on amphiphilic derivatives with unsaturated chains are available. The β-alanine derivative with oleic acid as the alkyl chain (table 2) was first reported by Rambharose et al. in 2018. [44] In this work the focus was to study the influence of nature of the alkyl chain as well as the number of alkyl chains present at the head group level. Therefore, different fatty acids with different instauration degrees (oleic acid, linoleic acid and linolenic acid) were used. Fatty acids are a well-known category of CPE, especially the unsaturated ones, due to their cis double bonds disrupt the packed structure of the intercellular lipids, thus effectively decreasing either resistance or the length of the diffusion path. The results obtained showed that esters of fatty acids with β-alanine headgroups have in fact higher potency than free fatty acids as CPE, and monooleate ester derivative (in table 2) displayed the best result for permeation of the drug tenofovir, without any toxic effects. This result confirms that single chained enhancers work better than double chained or more.

Amino acid based amphiphiles possessing + or – charge (surfactants [60]), although generally more toxic and irritant than their neutral counterparts, can be can also be used for transdermal delivery. Anionic surfactants have strong lipid solubilizing abilities and protein denaturing capacities, and thus are generally associated with cytotoxic effects to skin cells. However, when derived from natural molecules such as amino acids, anionic surfactants present reduced cytotoxicity. Glutamic acid-based amphiphilic derivatives are known anionic surfactants due to the presence of the 2 carboxylic acid groups, with acceptable biocompatibility. [61] Teixeira et al 2013 [57] used lysine-based double chained anionic and nonionic surfactants and tested them as CPE, obtaining promising results. The anionic derivatives with 16 carbon alkyl chains were the most active for the delivery of the drug ropivacaine, (used in cationic form), because the negative charge ensured favorable electrostatic interactions with the drug. Both anionic and nonionic surfactants did not affect the HEK cell line viability. Cationic surfactants are also known for their high cytotoxicity. They interact with intercellular keratin via polar head interactions, causing protein denaturation, and thus can damage human skin. Here again, the introduction of the amino acid as polar head group lead to low cytotoxicity and biocompatibility. [60]

26 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Examples of cationic surfactants applied in TDDS refer essentially to Gemini cationic surfactants. This class of surfactants has received much attention in recent years due to their enhanced physicochemical properties, which made them suitable for biomedical applications. Gemini surfactants are known to have enhanced interfacial properties when compared to their monomeric analogues. They present lower cmc values and autoagregation in solution to yield micelles or liposomes, which lead to lower concentrations being sufficient to achieve the desired structures used in their biological applications. Amino acid-based Gemini surfactants used in transdermal delivery systems can be used as skin permeation enhancers or drug vehiculation strategies through the skin. [45, 58]

The serine-based Gemini surfactant mentioned in table 2 was evaluated as a potent CPE by Teixeira et al. [45] In this work, the efficiency of a series of serine-based Gemini surfactants as CPE, with different alkyl chain lengths and headgroup charges, was tested. The authors also adopted a different experimental strategy to test the permeation enhancement of the novel surfactants. The skin model was pretreated with serine-based surfactant enhancer solution for one hour prior to the application of the drug formulation. This method generally allows higher enhancement results, and it does not take into account drug/enhancer interactions at the donor formulation.

According to the work, the serine-based cationic gemini surfants can act as potent CPE in lower concentration solutions due to their strong interaction with the lipid layer of the SC. Also, the most efficient derivatives were the cationic surfactants, despite being associated with higher skin irritation than nonionic surfactants. Due to the low concentration needed for these gemini surfactants to be active, they didn´t show significant damage to cell lines and skin samples at the concentration studied, 16µM.

Through a computational study using an established DPPC (Dipalmitoyl phosphatidylcholine) membrane model, they observed, after insertion of surfactant in the lipid bilayers, an increase of the fluidity of the lipid domain due to polar head interactions, creating large restrictions to lipid packing. Although DPPC membrane models are well established and optimized, it would be more realistic to see the mechanism of action in models using lipids from the SC, such as ceramides and cholesterol, as presented in [62].

Muzzalupo et al also worked with amino acid-based Gemini surfactants, specifically cationic lysine derivatives [58], They entrapped the drug inside a vesicle, formed by the surfactant self-assembly, in order to facilitate the drug transport through FCUP 27 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems the skin. Thus, the loaded liposomes can enhance transdermal drug delivery through different mechanisms:

(1) They can desegregate and release the drug before coming in contact with the SC and the resulting surfactant monomers act as chemical permeation enhancers. (2) They can be absorbed by the SC and mix the surfactant molecules within the lipid bilayers of the SC, liberating the drug. This behavior provokes skin permeation enhancement through lipid fluidization, but it also improves drug partitioning. (3) They can deform and go intact through the SC, reaching the systemic system where the drug is delivered.

The authors proved, in their case, the cationic liposomes behave as drug penetration permeation enhancers after topical application, but also allow sustained and controlled drug delivery in the case of parenteral administration.

Peptides have also been studied concerning the potential for dermal delivery. They can be chemically manipulated into different derivatives, including through the introduction of alkyl chains to provide them amphiphilic properties. Amphiphilic peptides can be used in transdermal drug delivery systems, but this possibility is still underexplored. The first case of dipeptide derivatives tested as CPEs is reported by Vávrova et al. [38] Although the peptides were designed as ceramide analogous, the compounds synthetized (with 2 alkyl chain of 12 carbons and residues of glycine/serine + serine/glycine) were inactive, however. Yet, their inactivity may have been due to the presence of two alkyl chains (later proved as a disadvantage in CPEs), and not necessarily due to the dipeptide headgroup. The peptide derivative mentioned in table 1 was described by Sugibayashi et al. in 2018. [46] They developed a new peptide with 2 residues of amino acids (glycine + histidine) and one alkyl chain to be used as a gel for the dermal formulation and verified that it also was active as CPE. They prepared the formulations of this peptide in concentrations to achieve a gel phase (elongated micellar aggregates) and performed tests of incorporation and dermal delivery of metronidazole, a hydrophilic drug, in combination with other CPE, like co-solvents. They also tested the incorporation of ivermectin, a hydrophobic drug, in a gel spray formulation for dermal delivery. The authors demonstrated that this new compound could act as a chemical permeation enhancer with aggregation properties, allowing a higher concentration of drug in the

28 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

donor formulation leading to higher partitioning of the drug into the skin. This peptide derivative may be able to fluidize the SC to increase diffusion, but this mechanism of action needs further investigation. Also, synergistic activity with other CPE´s, mostly first- generation enhancers like propylene glycol and isopropyl myristate was observed Hikima et al 2013 [63], used a novel peptide Gemini amphiphilic molecule (dilauramidoglutamide lysine, DLGL), and tested it as a CPE for the delivery of l-ascorbic acid 2-glucoside (AAG), a hydrophilic compound of interest for cosmetic applications. DLGL is a tripeptide (glutamic acid-lysine-glutamic acid) with two alkyl chains of 12 carbons. This compound allowed high skin accumulation of AAG through a combination of multiple mechanisms such as protein denaturation, SC lipid removal (polar head interactions) , lipid fluidization ( alkyl chain interactions) and also SC hydration, due to the presence of multiple H-Bonding groups and negative charges.

In 2020 a new family of CPEs was synthesized using fatty acids and amino acids. [54] The alkyl chain and the polar head group were combined through an ionic bond, thus creating an ionic liquid, a new emerging class of chemical permeation enhancers. Ionic liquids are a new class of solvents interesting for biorelated applications, including skin permeation enhancement. Ionic liquids (IL) can either open the tight packing of the SC lipids and promote fluidization, if the IL is hydrophobic, or promote partitioning into the cells by opening channels of transcellular permeation, if the IL is hydrophilic [51]. IL can also enhance permeation through lipid extraction.

In this work, two different fatty acids, oleic or linoleic acids were combined with different amino acid ethyl esters (β-alanine, leucine, and proline), to create different IL. Their physicochemical properties were determined, and they were then used as solvents in the drug donor phase (to increase solubility) and as CPE at the same time. The L- proline ethyl ester linoleate exhibited the best results among all derivatives and was capable to significantly increase the permeation of a small drug, ibuprofen, and a macromolecule, Fluorescein-isothiocyanate (FITC). The capacity of delivering a macromolecule through the skin is an important achievement for CPE because it has been one of the most difficult obstacles to overcome. Zheng et al. [6] have also recently (2020) synthetized and tested new amino acid- based ionic liquids, including the L-leucine ionic liquid derivative described in table 1. [46] The authors performed a study intended to combine the qualities of IL’s with the already optimized amino acid derivatives, creating new and improved chemical permeation enhancers. FCUP 29 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

In this work, IL with multiple amino acid derivatives were prepared, specifically glycine, L-proline, and L-Leucine-based amphiphiles, and the latter two were the most active ones. These amino acid-based IL interact with the intercellular lipid domain by lipid fluidization and lipid extraction, improving greatly the permeation of both hydrophilic and hydrophobic drugs.

30 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

1.3.8. Synthesis of amino acid amphiphiles

Due to the presence of various chemical functional groups in their structure, (carboxylic acid, amino, and side chain functional groups) amino acids can be transformed into a large variety of derivatives. [60, 64]

Scheme 1: Paths for synthesizing amphiphilic amino acid derivatives, adapted from [60]

The possible derivatizations of amino acids are described in scheme 1. For the obtention of amphiphilic structures, the long alkyl chain can be either introduced at the carboxylic acid or at the amino group. The presence of functional groups in the side chain offers yet another possibility for the introduction of the alkyl chain.

The carboxylic acid may be derivatized by reaction with a fatty alcohol, obtaining O-alkyl esters, or with long-chain amines, yielding N-alkyl amides. Both these reactions can only be accomplished by using N-protected amino acids. To be effective, protecting groups, must fulfill some requirements:

(1) They need to be of easy introduction into the correct functional group, (2) They have to be stable to a broad range of reaction conditions, (3) They must confer solubility in the most common solvents, (4) They must prevent or minimize epimerization during coupling (5) They need to be safely removed at the end of the synthetic process or when the functional group requires manipulation. [65] FCUP 31 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Removal of the protecting groups should be fast, efficient, and render easy to eliminate byproducts. Wherever two or more protective groups coexist in the same molecule, they must be orthogonally deprotected. [65] Orthogonal deprotection means that two different protection groups can be removed selectively under specific reaction conditions. [65]

The following table resumes the most used protection group for each functional group in amino acids and conditions for deprotection

Table 3: Protecting groups

Function Protecting groups Removal conditions

Boc 25-50% CF3COOH-DCM

Amine Cbz H2 / Pd-C Fmoc Piperidine/DMF

tert-butyl ester CF3COOH Carboxylic acid methyl ester Basic conditions

benzyl ester H2 / Pd-C

tert-butyl ether CF3COOH

+ - Hydroxyl (R-group) TBDPS ether Bu4N F

benzyl ether H2 / Ni

For the formation of the ester or amide bonds, the carboxylic acid group needs first to be activated as it is not very reactive. Coupling agents are commonly used with this purpose. Their action involves the formation of a more reactive carboxylic acid derivative due to the presence of a better leaving group. [53]

Some examples of commonly used coupling agents are presented in the following table (table 4). [54,55].

32 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Table 4: Coupling reagents for peptide synthesis.

Concerning the introduction of the alkyl chain at the amino group, this can be achieved by amidation with a fatty acid or acyl chloride, giving rise to a N-alkanoyl derivative. The amino group may also be alkylated to yield long-chain N-alkyl derivatives by two different methods: by reaction with a long-chain alkyl halide, yielding a tertiary amine or an ammonium salt, or by reductive amination of a fatty aldehyde/ketone yielding a secondary amine. [66] The reducing agents commonly used in these reactions are

sodium triacetoxyborohydride (NaBH(OAc)3), or sodium borohydride (NaBH4).

Combination of all these reactions is the general toolkit to yield most amino acid derivatives, and the possibilities are endless. These reactions can be used to derivatize any type of amino acids (α or β, natural or unnatural).

In the next chapter we will discuss the synthesis of cyclic β-amino acids, which will be further used to obtain amphiphilic derivatives using the reactions just described.

FCUP 33 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

1.4.9. Cyclic β-amino acids, synthesis and applications

β-Amino acids, with their amino group bonded to the β carbon rather than the α carbon, are non-natural amino acids (except for β-alanine). Although not as popular as α-amino acids, β-amino acids also play an important structural role in peptides and different heterocycles, and their free forms and derivatives exhibit interesting pharmacological effects. [67, 68] These amino acids can be classified as open chained or cyclic. Open chained chiral β-amino acids can be divided in three categories according to the position of the substitution (chiral carbon): α-substituted; β-substituted or α,β-disubstituted.

Cyclic β-amino acids may exist as a carbocyclic ring having both an amino and a carboxyl group as substituents, or as a heterocyclic ring with the nitrogen atom being part of it. These compounds can be found in some natural products. In 1989-90 two Japanese research groups independently isolated the β-amino acid cispentacin [(1R,2S) ‐2‐aminocyclopentanecarboxylic acid], which was found to exhibit antifungal activity, from the culture broth of a Bacillus cereus strains. [69, 70] Other experiments proved that bacteria, cyanobacteria, fungi, and plants incorporate β-amino acids into secondary metabolites that serve as tools to secure their survival in competition with other organisms [59]. When incorporated into peptides, β-amino acids increase stability against degradation by enzymatic activity. This enhanced stability is due to the lack of enzymes capable of cleaving the peptide bond between α-amino acids and β-amino acids, especially in mammals.

Carbocyclic β-amino acids are useful intermediates (building blocks) for the enantioselective synthesis of alkaloids and other derivatives. The most common methods to synthesize enantiopure carbocyclic β-amino acids involve [59]:

(1) desymmetrization of cyclic meso compounds used as precursors. (2) stereoselective cycloaddition reactions. (3) non-stereoselective cycloadditions followed by separation of the resulting racemic mixture Desymmetrization of cyclic meso compounds (diesters or anhydrides) can be performed with two procedures, involving the regioselective reaction with an enzyme, or a chiral reagent.

For example, chemoenzymatic hydrolysis of diesters allows to form selectively a ester, and the resulting free carboxylic acid group can be transformed into an amino

34 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

group through the Curtius reaction (ethyl chloroformate addition followed by reaction with sodium azide) and subsequent heating in the presence of excess benzyl alcohol.

Scheme 2: Chemoenzymatic separation of diesters

The diesters required for this reaction can be obtained through the opening of cyclic anhydrides with methanol under reflux.

On the other hand, when chiral amines are used to open the anhydride, one major diastereomer with only one amide bond will form. Here again the carboxylic acid group can be transformed into an amino group through the Curtius reaction, resulting in the final β-amino amide North has published the desymmetrization of anhydrides by reaction with (S)-prolinates affording chiral amides. [72]

Scheme 3: Desymmetrization of anhydrides with chiral amines.

Another way to synthetize alicyclic β-amino acids effectively is through cycloaddition reactions. These reactions can be stereoselective or not and are used either to create the carbocyclic skeleton of the target molecule or to produce a carbocyclic scaffold to be elaborated into the final product. 1,3-Dipolar cycloaddition of diazoalkanes is one of the methods used to obtain cyclopropane derivatives. In the example presented in scheme 4, addition of diazomethane to a chiral enoate (to obtain stereoselectivity) and photolysis of the resulting pyrazoline, followed by acid hydrolysis of the acetal function and oxidation of the hydroxyl groups yields cyclopropane β-amino acids. [71] FCUP 35 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Scheme 4: Dipolar Cycloadditions of Diazoalkanes

Many additional methods of cycloadditions can be used, such as addition of carbenes and sulfur ylides, stereoselective Diels–Alder cycloadditions, among others. [71]

However, all these methods aim at the synthesis of enantiomeric pure β-amino acids. A different approach is to synthetize the racemic mixture of the β-amino acid and then resolve it, using either diastereomeric salt formation or enzymatic resolution. The synthesis of the racemic mixtures can be achieved through the 2,2-dipolar cycloaddition of chlorosulfonyl isocyanate to different cycloalkenes. This method has become a well-known route for the synthesis of cycloalkane-fused β-lactams, which yield alicyclic β-amino acids, after hydrochloric acid treatment. [70, 72]

36 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Scheme 5: 2,2-dipolar cycloaddition of chlorosulfonyl isocyanate followed by acid hydrolysis.

The resolution of the racemic mixture formed can be performed at different stages of the synthetic route. Either the β-lactam ring can be stereospecifically opened through enzymatic resolution giving rise to only one of the enantiomeric pure amino acids. In this case, to obtain the other enantiomer, hydrochloric acid treatment of the remaining lactam is required. On the other side, the racemic mixture of final β-amino acids can be separated with other techniques like diastereomeric salt formation as mentioned before.

Chapter 2: Results and Discussion

38 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

2.1. Synthesis

As mentioned before, this project is aimed at the design and synthesis of novel cyclic amino acid-based amphiphiles to be applied as chemical permeation enhancers. Therefore, several amphiphiles have been synthetized to perform a systematic evaluation of the effect of structural properties on their aggregation behavior, biocompatibility, and skin permeation enhancement capacities. For such, the following issues were addressed in this work.

• Proline and proline mimetics as headgroups – influence of stereochemistry, chirality • Number of hydroxyl groups at the headgroup moiety – influence of hydrophilicity • Position of the alkyl chain – influence of hydrophilicity / lipophilicity and biodegradability. • Presence of hydrogen bond acceptor or donor groups – influence of hydrogen bonding.

In this chapter, the results of the synthesis of all compounds (table 5) will be described and discussed.

Table 5: Compounds synthetized

Acronyms Generic Name Molecular structure

methyl (2S,4R)-4-(O- LHyPOMe dodecanoyl)-hydroxyprolinate

methyl (2S,4R)-4-(O- LHyPNOMe2 dodecanoyl)-hydroxyprolinate

methyl (2S,4R)-N-acetyl-4-(O- LHyPNAcOMe dodecanoyl)-hydroxyprolinate

(2S,4R)-4-(O- LHyP dodecanoyl)hydroxyproline

FCUP 39 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

(2S,4R)-4-(O-dodecanoyl)-N- LHyPNMe methylhydroxyproline

(2S,4R)-N-acetyl-4-(O- LHyPNAc dodecanoyl)hydroxyproline

dodecyl (2S,4R)-4- HypO12 hydroxyprolinate

dodecyl (2S,4R)-N-methyl-4- HypNMeO12 hydroxyprolinate

dodecyl (2S,4R)-N-acetyl-4- HypNAcO12 hydroxyprolinate

methyl (2S,4R)-N-dodecyl-4- HypN12OMe hydroxyprolinate

(2S,4R)-N,N-dimethyl-4-(O-

LHyPNMe2OMe, I dodecanoyl)-2-(O-methyl)- hydroxyprolinium iodide

dodecyl (2S,3R,4S) -N-acetyl- Hy2PNAcO12 3,4-dihydroxyprolinate (16)

methyl (2S,3R,4S)-N-dodecyl- Hy2PN12OMe 3,4-dihydroxyprolinate (20)

40 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

dodecyl (1R,2R,3S,4R)-2- acetamido-3,4- (-) Hy2CpNAcO12 dihydroxycyclopentane-1- carboxylate

dodecyl (1S,2S,3R,4S)-2- acetamido-3,4- (+) Hy2CpNAcO12 dihydroxycyclopentane-1- carboxylate

dodecyl (1R,2R,3S,4R)-2- (dimethylamino)-3,4- (-) Hy2CpNMe2O12 dihydroxycyclopentane-1- carboxylate

dodecyl (1S,2S,3R,4S)-2- (dimethylamino)-3,4- (+) Hy2CpNMe2O12 dihydroxycyclopentane-1- carboxylate

methyl (1R,2R,3S,4R)-2- dodecanamido-3,4- Hy2CpNLOMe dihydroxycyclopentane-1- carboxylate

FCUP 41 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

2.1.1. Synthesis of the 4-hidroxyproline derivatives

A total of 11 hydroxyproline-derived amphiphiles have been synthetized. The synthetic pathway for the obtention of these compounds is present is scheme 6. Throughout the whole project, efforts were made to optimize the reaction conditions. For the obtention of 4-hydroxiproline amphiphiles 3 different 4-hydroxiproline derivatives (1a- c) have been used as starting materials.

Scheme 6: Synthetic route for the synthesis of all 4-hydroxiproline derivatives

The first step in any of the reaction sequences consists in the introduction of the alkyl chain into the hydroxyproline derivative at either of the three functional groups present.

The introduction of the hydrocarbon chain into the hydroxyl group of compound 1a-b was performed by acylation with dodecyl chloride yielding compounds 2a-b.

Compound 1a was further transformed into the N-alkylated derivative 10 by reductive amination of dodecyl aldehyde.

For the introduction of the alkyl chain at the carboxylic acid group, the N-Cbz protected amino acid (1c) was used. Thus, condensation of 1c with dodecyl alcohol in the presence of TMSCl (trimethylsilyl chloride, coupling agent) afforded 6 which, upon removal of the protective group Cbz by hydrogenation, lead to the obtention of 7.

42 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

The transformation of compounds 2a-b and 7 into the corresponding N-methyl or N-acetyl derivatives was performed either by reductive amination of formaldehyde (3a-b and 8) or by reaction with acetyl chloride (5a-b,9)

Compound 3a was quaternized by methylation with CH3I yielding 4a. Each of these reactions will be discussed in detail in the next subsections

A) Introduction of the lipophilic chain at the hydroxyl group.

The introduction of the hydrophobic chain at the hydroxyl group of methyl-4- hydroxiprolinate hydrochloride salt 1a or 4-hydroxyproline 1b was achieved by condensation with dodecyl chloride. This reaction was performed in TFA (trifluoroacetic acid) at 0°C, according to literature [73]. The reaction mechanism is presented in the following scheme 7.

Scheme 7: Acylation reaction mechanism in acidic conditions, exemplified for 1b

This method allows for the insertion of the alkyl chain only at the desired position since it avoids interfere2nce of the amine as a competitive nucleophile because in acidic conditions the amino group (pk’2 = 9.65) is protonated and thus it is not nucleophilic. After one hour the formation of a new product was observed, however due to the presence of starting reagent the reaction was left under stirring overnight.

After 15 hours, compounds 2a-b were treated with diethyl ether and precipitated as white solids. Both compounds were obtained in high yields. (2a: η = 95 %; 2b: η = 84 %). FCUP 43 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

B) Introduction of the alkyl chain into the amino group by reductive amination

The introduction of the alkyl chain into the amino group of 4-hydroxiproline derivative 1a was achieved through reductive amination of dodecyl aldehyde. This reaction was performed in basic conditions to neutralize the ammonium chloride salt. Thus, dodecyl aldehyde was added to 1a in DCE and left stirring for 30 minutes to form the iminium ion, which was then reduced by reaction with NaBH(OAc)3. This reagent is a mild reductive agent, that selectively reduces imines and enamines in the presence of aldehydes. The reaction mechanism is presented in scheme 8.

Scheme 8: Reductive amination reaction mechanism, exemplified for 1a-10

Compound 10 was isolated by column chromatography and obtained as a white solid in relatively low yields (35% and 38 %). Due to the starting reagent not being visible by TLC, the monitorization of the evolution of the reaction was probably not done properly. One explanation for the low yields would be that the reactions were not complete. Optimization in further experiments will be required, by for example testing different reactions times.

44 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

C) Introduction of the alkyl chain into the carboxylic acid group with TMSCl

The starting amino acid used initially was N-Boc-hydroxyproline, a N-protected amino acid intended to avoid intermolecular coupling resulting in peptide formation

Condensation with dodecan-1-ol in the presence of TMSCl should yield the desired dodecyl ester derivative. However, the yields obtained were very low, possibly due to unwanted removal of Boc during the condensation reaction, result of the increasing acidity of the medium due to the release of HCl in the course of the reaction. The deprotected amino group thus formed may react with other amino acid molecules in solution that did not yet form the ester bond, yielding a peptide. Also, this amino acid derivative presented some problems of solubility, which may also have contributed to the low yields observed.

To overcome these problems, N-Cbz derivative 1c was used. 1c showed to be more soluble than the Boc protected amino acid, most probalby due to the larger size and higher hydrophobicity of this protective group. Furthermore, Cbz is more stable against harsh acidic conditions, allowing the use TMSCl as the coupling agent for the esterification. Removal of the Cbz group is performed under neutral conditions (hydrogenation) to yield the free amine.

The mechanism of esterification of 1c with dodecan-1-ol using TMSCl as coupling agent is described below (scheme 9)

Scheme 9: Esterification reaction mechanism with TMSCl as coupling agent FCUP 45 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

TMSCl was added to a suspension of hydroxyproline derivative 1c in DCM, and the mixture was left stirring for 30 minutes, to activate the carboxylic acid group. Only then the alcohol was added, to avoid O-silylation of the alcohol that would reduce its nucleophilic character.

Desired product 6 was obtained as an oil after isolation by column chromatography (71 % yield).

D) Removal of the Cbz group by hydrogenation

The deprotection of 6 was performed in an hydrogenator reactor using palladium on carbon as catalyst. This reaction to obtain 7 follows the mechanism presented in scheme 10.

Scheme 10: N-Cbz removal reaction mechanism

46 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

According to this mechanism, this reaction should exclusively yield the desired product and toluene, (which is removed by evaporation). However, another product was observed by TLC, and so column chromatography was necessary to isolate the target compound. Final compound 7 was obtained as a white solid in yields of 64 % and 34 % for the two experiences performed. The formation of the secondary product referred explains the low yields obtained in the reaction that usually yields the target compound almost quantitatively. The lower yield of the second experiment may be associated with the longer reaction time (15 hours) that probably lead to more degradation.

E) N-methylation by reductive amination

The synthesis of compounds 3a-b/8 was achieved through reductive amination using formaldehyde. The reaction mechanism is analogous to the one explained in scheme 8.

The resulting products 3a-b and 8 were purified and obtained as white solids. The methyl ester derivative 3a was obtained in slightly higher yields (75 %) compared to the free carboxylic acid 3b (60%), mainly due to its lower hydrophilic character that allows for an easier isolation. Product 8 was also obtained with comparable good yields (84%)

Compound 4a was synthetized though N-alkylation of 3a, using iodomethane as

electrophile and a few drops of DMF. This reaction follows a SN2 type mechanism. After 12 hours, DCM was added and the mixture was washed with water to remove the DMF. Quaternary ammonium salt 4a was obtained as a yellow oil. (65%)

F) N-acetylation of the derivatives using acetyl chloride

Acetylation of the amino group of 2a-b and 7 was performed by reaction with

acetyl chloride in the presence of NEt3. The acetamide was selected because in literature it is reported [5] that the presence of this group imports a higher potential for biological applications compared to other protecting groups. The reaction mechanism is presented in scheme 11 for the acetylation of 2b. Since the amino group has higher nucleophilicity when compared to the hydroxyl group, it easily attacks the acyl chloride.

FCUP 47 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Scheme 11: Acylation reaction mechanism in basic conditions, exemplified for 2b-5b

After 4h, the mixture was treated with water and the desired compounds 5a-b/9 (from 2a, 2b and 7 respectively) were purified by column chromatography (η 5a= 49 %; η 5b= 29 %; η 8 = 75 %). The reaction yield for the obtention of derivative 5b was lower than the others due to the presence of secondary products than harmed the purification by column chromatography. NMR analysis of all the products revealed the presence of rotamers, due to the acetamide formation. This is in accordance with reports in the literature [74] and is due to the rigidness of the newly formed amide bond. This was confirmed by an NMR 1H analysis of compound 5b in DMSO at 100ºC, where instead of two signals for the three protons from the acetyl group (figure 8), only one signal was observed.

Figure 8: NMR spectrum of 5b containing evidence of rotamers.

48 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

2.1.2. Synthesis of the 3,4-dehydro-L-proline derivatives

Amino acid 3,4-dehydro-L-proline or its methyl ester were used as starting materials for the obtention of the dihydroxyproline derivatives. Comparison of the performance of these compounds with the analogues of the 4-hydroxyproline just described should allow to infer about the influence of the presence of one more hydroxyl group in their physicochemical as well as biological properties. Two different positions were chosen for the introduction of the alkyl chain, namely the carboxylic acid group and the amine group.

Two 3,4-dehydro-L-proline derivatives were successfully synthetized (16 and 20), while the synthesis of the derivative 18 is still in course.

The synthetic route to the obtention of these compounds is outlined in scheme 12.

Scheme 12: Synthetic route for the synthesis of 16, 18 and 20

Amino acid 3,4-dehydro-L-proline 11a was used as starting material for the synthesis of derivatives with the alkyl chain at the carboxylic acid group (16/18). The FCUP 49 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems amino acid was initially protected with di-tert-butylcarbonate to form the N-Boc derivative 12. Then, the alkyl chain was introduced into the carboxylic acid group, through esterification with dodecyl alcohol yielding 13. Removal of the Boc group afforded compound 14 which was transformed into 17 by methylation, via reductive amination, or into compound 15 by acylation. Dihydroxylation of 15 yielded final product 16, and dihydroxylation of 17 should yield 18, which, however, has not yet been synthetized.

To obtain compound 20, 3,4-dehydro-L-proline methyl ester 11b was used as starting material. This transformation is straightforward with no need for protection/deprotection strategies. Thus, the first step was the introduction of the alkyl chain into the amino group by reductive amination with dodecanal, yielding 19 followed by dihydroxylation to afford the final product 20.

In the following subsections, these reactions will be described in detail.

50 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

2.1.2.1. Synthesis of derivatives 16/18

A) N-Boc protection with di-tert-butylcarbonate

Before introduction of the alkyl chain by esterification, it was necessary to protect the amino group, to avoid intermolecular coupling between the amino group and the carboxylic acid group in the condensation reaction. The selected protecting group was Boc (t-Butyl carbamate), vastly used in peptide synthesis and known to promote solubility of the amino acid in organic solvents.

Treating 11a with NaOH and di-tert-butyl dicarbonate (Boc2O) yielded compound 12 according to the mechanism shown in scheme 13.

Scheme 13: N-Boc protection reaction mechanism

The lone pair in the nitrogen attacks one of the carbonyl groups of Boc2O

t displacing CO2 and BuOH. The protonated carbamate is deprotonated in alkaline conditions and the final N-protected product is formed.

After 4 hours, the compound was isolated from the reaction mixture by extraction. Acidification to pH 3-4 (HCl) was necessary to guarantee that the compound was in its carboxylic acid form and thus minimize loss due to solubility in the aqueous phase. Care was taken not to lower the pH too much, since this may cause removal of the Boc protecting group. In all the experiences performed, the desired product was the only present after extraction, so no further purification was required. The yields were high in all cases.

FCUP 51 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

B) Introduction of the alkyl chain into carboxylic acid group with PyBop

Introduction of the alkyl chain was performed by condensation of the N-Boc amino acid 12 in the presence of PyBoP as coupling agent, according to the mechanism explained in the following scheme 14.

Scheme 14: Esterification reaction mechanism with Pybop as coupling agent

The organic base in solution abstracts a proton from 12 to yield the carboxylate anion that is nucleophilic enough to attack PyBop. This coupling agent activates the carboxylic acid group by transforming the -OH into a better leaving group. After activation, the alcohol is added, and attacks intermediary B at the carboxylic acid group through an addition elimination reaction, yielding final product 13.

52 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

The product formed is less polar than the fatty alcohol used, simplifying the separation through column chromatography. However, the reaction showed some efficiency problems (32%). Attempts were made to improve the yield of the reaction, by changing the coupling agent (TBTU, O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate, 37% yield ), and using a stronger organic base (DBU, 1,8- diazabicyclo[5.4.0]undec-7-ene, 21% yield), without success. This problem was not yet resolved, and optimization is necessary.

C) Boc removal with TFA

Deprotection of compound 13 to 14 was performed using trifluoroacetic acid, according to the following mechanism (scheme 15).

Scheme 15: Boc removal reaction mechanism with TFA

After 12 hours, excess TFA was removed by several treatments with diethyl ether followed by evaporation. The resulting unprotected compound (14) was obtained as a brown oil, in good yields (90%).

D) N-alkylation/acylation of 14

N-methylation of 14 was performed by reductive amination (17, 22 %) as described in section 2.1.1. E) . Concerning acetylation, yields were much higher and FCUP 53 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems again NMR analysis showed some peak spiting caused by rotamers (15, 81 %) which had already been observed for the 4-hydroxyproline derivatives.

E) Dihydroxylation with OsO4

The syn insertion of 2 hydroxyl groups was achieved by oxidation with OsO4 as catalyst, and N-methylmorpholine N-oxide (NMO) as co-oxidant. Both reagents react according to the following catalytic cycle (scheme 16)

Scheme 16: Dihydroxylation reaction mechanism with OsO4

After 4 hours of stirring, the mixture was filtered using a filtering plate with silica and celite, treated with saturated solution of Na2S2O3 and then isolated by column chromatography. The final compound 16 was obtained as a white solid (48 % yield).

54 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

2.1.2.2. Synthesis of derivative 20

The N-alkyl derivative 20 was synthesized in two steps using 3,4-dehydro-L- proline methyl ester 11b as starting reagent.

The first reaction was the reductive amination with dodecanal. This reaction follows the same mechanism of action explained in scheme 8. Since the starting material was not very soluble in DCE, a higher reaction time was required (48 hours instead of 24) and even so, the best yield achieved was 49 % to obtain 19. Dihydroxylation of derivative 19 occurred without major issues, giving rise to 20 as a light orange oil (31 % yield) which was solid at 0ºC. This step had low yield and thus optimization will be required.

FCUP 55 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

2.1.3. Synthesis of the proline mimetic derivatives

After the obtention of all proline derivatives, the next objective was to synthetize derivatives with similar ring structure and amino acid behavior (proline mimetics) but with new features in their structure. Therefore, unnatural pentacyclic β-amino acids were chosen to serve as precursors to a new family of di-hydroxylated derivatives, with the lipophilic chain introduced in two different functional groups (carboxylic acid or amine) in analogy to the derivatives described in section 2.1.2. The new compounds synthetized (29a-b, 32a-b, 35) are listed in table 5.

The synthesis of these compounds was challenging because the synthetic routes initially designed were not always successful. While sometimes just a change in the order of the steps performed lead to a positive outcome, in some cases new methods had to be implemented.

The unnatural β amino acids 24a-b necessary for the synthesis of the proposed derivatives are not commercially available. Thus, they were synthesized from cyclopentadiene (CPD) through 1,2-dipolar cycloaddition of chlorosulfonyl isocyanate, followed by resolution and hydrolysis of the enantiomeric cyclic lactam formed. The procedure followed is already described in literature [72, 75] (scheme 17)

Scheme 17: Synthetic route for the synthesis of the β-amino acids 24a-b

56 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Scheme 18: Synthetic route for the synthesis of the compounds 29a-b, 32a-b and 35

Once prepared, these β-amino acids were transformed into the β-amino acid derivatives 29a-b, 32a-b and 35 according to the synthetic route present in scheme 18.

The synthesis of compound 29a/b started with the N-Boc protection of amino acid 24a/b with di-tert-butylcarbonate yielding compound 25a/b. Introduction of the alkyl chain into the carboxylic acid group, through condensation with dodecyl alcohol, gave rise to 26a/b, and subsequent removal of the Boc group lead to compound 27a/b. This compound was transformed into 28a/b by dimethylation, via reductive amination. Dihydroxylation yielded final product 29a/b. The last compounds 28a/b and 29a/b were FCUP 57 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems contaminated with colored impurities, and so the reaction sequence was changed in an attempt to obtain the pure target compounds (29a/b).

This time, dihydroxylation was carried out on 26a/b, yielding compound 30a/b. Then, removal of Boc originated the corresponding ammonium trifluroacetate 31a/b. However, attempted methylation of this salt by reductive amination to yield 29a/b was not successful. Alternatively, 31a/b was used to prepare the N-acetylated derivative 32a/b, through reaction with acetyl chloride. For the synthesis of target compound 35, the first step was methylation of amino acid 24a with MeOH/TMSCl. The product formed (33) was acylated with dodecyl chloride to form compound 34, which, upon dihydroxylation, gave rise to compound 35 in good yields. Attempts to introduce the alkyl chain at the amino group by N-alkylation through reductive amination of dodecyl aldehyde with 33 were also performed, but the reaction did not yield the target monoalkylated derivative, but the dialkylated one 36

In the following subsections all the reactions will be discussed in detail.

58 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

2.1.3.1. Preparation of the unnatural β-amino acids

The key amino acids 24a-b (Scheme 18) were obtained via a cycloaddition followed by enzymatic resolution of lactam 23a-b and subsequent acid hydrolysis of the remaining enantiomerically pure lactam.

The [2+2] cycloaddition reaction is the most crucial step in obtaining good quantities of amino acid 24a-b. It is performed by adding chlorosulfonyl isocyanate (CSI, 22) to CPD (cyclopentadiene). This reaction is highly sensitive, and both the yield and the products obtained may vary according to minor differences in temperature, solvent, and reaction time. The optimized conditions described in literature [75] allow a maximum yield of 95 %. Fresh doubly distilled CPD, controlled temperature (-20ºC, MeOH bath cooled with a cryocooler), use of an amber round bottomed flask, dropwise addition of 22 and 40 minutes of stirring were the best reaction conditions found.

Scheme 19: [2+2] Cycloaddition reaction mechanism with CSI

A constant and homogenous temperature of -20ºC during the addition of CSI leads to a [2+2] cycloaddition, resulting in the formation of a four membered ring. Two new σ bonds are formed at the expense of the two π bonds, one from CPD and another from CSI according to the mechanism illustrated in scheme 19. Even with this optimized conditions, two products are formed, the desired compound 23 and its constitutional isomer compound 23.1. Both compounds behave very similarly, with close retention factors in TLC, however, their separation is easily achievable by column chromatography. Theoretically, there is a 50% chance for both products to form but this does not happen. In fact, 98% of the product formed is compound 23.

However, the experiences were not as successful as the ones reported in literature, mostly because the conditions could not be fully reproduced in our lab. Most FCUP 59 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems experiences were performed on a static ice/ salt bath. The first attempts to perform this reaction showed low efficiency with yields lower than 20 %. To improve these yields, several adjustments were tested: use of distillated diethyl ether (did not result in any improvement of yield); use of an Dewar vessel for the static ice/salt bath (allowed for constant temperatures at -20ºC but no drastic improvements in yield) ; cooling the CSI to 0ºC before dropwise addition (no pronounced effect on yield). However, one adjustment was crucial to reach the values of 50 % yield, the use of doubly distilled CPD.

CPD has the natural tendency to form dimers at room temperature, so the double distillation serves two purposes, the first one provides the energy to break the dimers and the second one is to purify the CPD. Initially, the distillation was not being carried out correctly. After the first distillation, the heating mantle was turned off and the distillate was added to the destination flask, still hot, to perform distillation of the CPD a second time. As the temperature in the flask was still higher than 40 ºC (boiling point of CPD), the second distillate obtained was probably contaminated with impurities, explaining the constant low yields of 23 achieved initially.

Thus, to fix this issue, the first distillate was transferred into the distillation flask only after the temperature inside the distillation system was below 40 ºC, and the second distillate was collected only up to the temperature of 40 ºC. This adjustment allowed to synthetize 23 in yields around 50 %. However, the CPD distillation occurred always at slightly higher temperatures than the boiling point described for CPD in literature (44°C instead of 40°C), showing the possibility of some impurities in the distillate.

Using the doubly distilled CPD, the best yield achieved was 60 %, for the reaction performed at temperatures below -20 ºC (cryocooler), using a MeOH bath in constant stirring, which assured a more homogeneous cooling of the reaction mixture.

The reaction conditions for the reduction step, essentially pH and temperature, have to be strictly controlled in order to avoid degradation of product 23. According to theoretical studies [76], ring opening of the lactam, my occur during reduction but is less favored in alkaline conditions. So, the reduction step with sodium sulfite solution was carried out at pH 8-9 adjusted with KOH 20%.

Extraction of compound 23 with ethyl acetate and purification by silica gel column chromatography using Hex/AcOEt 1:3 as eluent was performed as stated in literature [75] without issues. The product was isolated as a racemic mixture (23a, 23b)

60 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

To obtain the enantiomeric pure β amino acids, the enzymatic resolution of the racemic mixture of (±) 23 was performed. Thus, candida antarctica was used to “digest” one of the enantiomers of the lactam, using methyl tert-butyl ether (MTBE) as solvent. A catalytic trio of amino acid residues is responsible for the activity of candida antarctica. The trio consists of aspartate (Asp), histidine (His) and serine (Ser) [77] and the mechanism of action is outlined in scheme 20.

24a

23a Scheme 20: Enzymatic resolution reaction mechanism

The enzyme preferably digests the enantiomer 23a leaving enantiomerically pure lactam 23b in solution. The reaction mixture was filtered and compound 23b was

recovered from the filtrate after removal of the solvent and recrystallization (Et2O, 99%). Compound 24a, in turn, was recovered from the remaining filtered solid, upon washing with hot water and recrystallization (water/acetone, 98%). The washed enzyme can be reused up to 3 times, although It loses activity each time. According to literature, in the first cycle of use, the enzyme takes around 7 days to fully resolve the racemic mixture. In subsequent uses longer time periods are required.

Compound 24b was obtained by acid hydrolysis of the enantiomerically pure 23b according to the mechanism shown in scheme 21 and isolated by precipitation from

Et2O/EtOH (99%). FCUP 61 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Scheme 21: Lactam ring opening reaction mechanism

HCl at 10% is acidic enough to open the lactam ring and the reaction is cleaner than when higher concentrated solutions of HCl are used. The yields of this reaction were very high.

62 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

2.1.3.2. Preparation of the β-unnatural amino acid derivatives

The following reactions were performed in order to obtain a family of amphiphilic compounds containing unnatural proline mimetic β-amino acids as head groups. The lipophilic chain was introduced in the same positions as in the dehydroproline derivatives (section 2.1.2.) and using the same N-protection groups in the final products.

2.1.3.2.1. Synthesis of 29a-b

The synthetic route outlined for the obtention of the final products 29 was similar to the one outlined in scheme 5 for the synthesis of compound 18.

Initially, compound 24a/b was N-protected with Boc. The reaction was performed in the same conditions explained in section 2.1.2. A) and the reaction mechanism is described in scheme 6. This reaction was performed several times with variable yields. The low yields obtained in some of the experiments (≈30 %) can be associated with mistakes during the acidification of the mixture that led to the unwanted removal of the Boc group. In most cases, the yields obtained were around 60 to 70 %, but higher yields (25a - 93%) were achieved with some adjustments. These were a slower addition of the

Boc2O (30 minutes) and a rigorous control of pH, which has to be maintained as values above 9, during the addition, to avoid protonation of the amino acid.

The product formed 25a/b were recrystallized from AcOEt/hexane to yield white crystals. However, the recrystallization was difficult to perform and in most cases compound 25a/b was used in the subsequent reaction as dense colorless oils.

The introduction of the alkyl chain into the carboxylic acid group of 25a/b was performed according to the conditions presented in section 2.1.2. (B) using PyBop as coupling agent, according to the mechanism there described (scheme 14). In this case, compound 26a/b was obtained with yields between 40 and 60 % with one exception (29 %) due to loss of compound during treatment. Since the yields were satisfactory, no other coupling agents were tested.

Deprotection of compound 26a/b to yield 27a/b was performed using trifluoroacetic acid (for mechanism see scheme 15). The resulting unprotected compound was obtained as a white solid in quantitative yield.

Dimethylation of 27a/b by reductive amination of formaldehyde was the most problematic step, against what was expected. The first problem was that the reducing agent was difficult to dissolve in DCE, more than usual, resulting in the deposition of a FCUP 63 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

solid residue at the bottom of the flask. Also, after a few hours, a red color started to appear in the mixture, present in TLC as a narrow shadow along the plate (signs of degradation). Along with the color, a strong smell characteristic of organic amines evolved. This unexpected behavior resulted in difficulties to isolate the desired product

28a/b. Double methylation by reductive amination using NaBH (OAc)3 as reducing agent follows the mechanism presented in scheme 22:

27

28

Scheme 22: Dimethylation reaction mechanism by reductive amination

However, looking at this mechanism one realized that after addition of formaldehyde, dehydration forms an unstable iminium ion, that can´t be stabilized via enamine formation because it lacks carbons adjacent to the carbon of the newly formed N-C bond. This group, in basic conditions, can be a good leaving group, in a reaction similar to the Hofmann elimination [78], resulting in the possible degradation of the amino acid. This problem was not present in the former derivatives (proline derivatives) due to the amino group being present as part of the ring, restricting the possibility for elimination.

Scheme 23: Hofmann elimination like reaction mechanism

64 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Due to this degradation, isolation of the dimethylated derivatives was difficult and the yields obtained were low. Compounds 28a-b were obtained as light red oil (signs of colored impurities) (28a – 26%; 28b – 39 %).

Dihydroxylation of 28a/b to yield 29a/b follows the mechanism already explained in section 2.1.2.1. E). The reaction performed resulted in extra secondary products that hindered the purification. Final compound 29a/b was confirmed as obtainable by NMR and MS.

As this reaction sequence gave low yields of the target compounds29a/b, a new approach was tested, from compound 26a/b onwards, differing from the previous one in the order the reactions were carried out.

This time, dihydroxylation was performed on 26a/b prior to Boc-removal. Derivative 30a/b was obtained as a pale orange solid, easily isolated through precipitation with diethyl ether in good yields compared to the dihydroxylation of compound 28a/b (30a- 85%; 30b – 63%). Deprotection of 30a/b with TFA yielded 31a/b as a white solid (31a - 67 %; 31b – 54%). Until this point, the new approach was better than the previous one, giving rise to products in higher yields and easier to purify.

Finally, reductive amination of formaldehyde with 31a/b should yield compound 29a/b. But this time, despite no visible degradation, the product formed was not 29a/b. It is possible that the two hydroxyl groups present could compete with the amino group as nucleophiles and attack the aldehyde in solution, forming a stable cyclic acetal. However, further analysis is required to prove this hypothesis

FCUP 65 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

2.1.3.2.2. Synthesis of 32a-b

Precursors 31a-b were also used to synthetize the N-acetyl derivatives 32a-b, analogous of proline mimetic 16, by reaction with acetyl chloride. The reaction mechanism was already explained in scheme 10. However, unlike the previous proline derivatives, no rotamers were observed in the NMR spectra of the products (32a – 85%, 32b – 70 %)

2.1.3.2.3. Synthesis of 35

Concerning the synthesis of N-alkylated/acylated derivatives, initially compound 24a was transformed into the corresponding methyl ester hydrochloride salt by reaction with TMSCl in MeOH [79] This reaction follows the mechanism presented in scheme 9. Evaporation of the crude of the reaction lead to desired compound 33 with no need for further purification (97%). To introduce the alkyl chain at the amino group of 33, reductive amination of dodecanal was performed. However, by NMR the insertion of 2 alkyl chains was observed, even when the reaction was repeated with less than 1 equivalent of dodecyl aldehyde. This behavior was not expected because, in other reported syntheses of amino acid derivatives with primary amines [80], the insertion of a long alkyl chain causes enough stereochemical hindrance to prevent the introduction of a second alkyl chain. However, in this case, the dialkylated product was the main product formed (36). Other methods for N-alkylation were not tested due to lack of time.

Transformation of 33 into acylated derivative through reaction with dodecyl chloride yielded 34, which was isolated by column chromatography and recystalized to yield a white solid (57%). Dihydroxylation of this derivative with OsO4 yielded the target compound 35 in 66 % yield. The mechanisms of these reactions are analogous to those described in section 2.1.2.1 D) and E).

66 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

2.2. Physicochemical properties

The evaluation of the physicochemical properties of the synthetized amphiphiles had the purpose of providing the information necessary to the execution of the subsequent experiments on permeation and toxicity. Specifically, the determination of the critical micellar concentration (cmc) is essential for understanding the concentration range at which these compounds can be tested in upcoming experiments in order to successfully evaluate the influence of their organization in solution (monomer or aggregate) in their biological/biophysical properties.

2.2.1. Critical micellar concentration

The cmc was determined by surface tension measurement. The solubility of the compounds in buffer solution was a major concern, as only the water-soluble compounds, at the concentrations used for the preparation of the stock solutions (2-4 mmol∙Kg-1), were eligible for cmc determination.

The derivatives chosen for the study are presented in scheme 24:

10 20

3a 8 9

Scheme 24: Compounds used in the tensiometry experiments.

These compounds were selected in order to allow a systematic assessment of the influence of the differences in structure on their aggregation behavior. Thus, compounds 10 and 20 were selected to evaluate the influence of the hydroxyl groups present in the proline ring; comparison between 3a, 8 and 10 should allow to assess the FCUP 67 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

70

N12HyPOMe 65 10 N12DiOHHyPOMe 20 N(CH3)HyPOC12COOH 60 3a N(CH3)HyPCOO12 8 55 N(COCH3)HyPCOO12 9

50

1 -

45

/mN·m γ 40

35

30

25

20 -8 -7 -6 -5 -4 -3 -2 -1 0 1 log ( c /mmmol·kg-1)

Figure 9: Surface tension curves influence of the position of the alkyl chain, and comparison between 8 and 9 should allow to conclude about the influence of the N-acyl group as a hydrogen bond acceptor.

Figure 9 shows the surface tension versus log(concentration) curves obtained for the five derivatives. All the amphiphiles were soluble at rt.

The critical micellar concentration was determined as the” break points” in the γ vs. ln (m/mº) curves as illustrated in figure 9. From the surface tension curves, other interfacial parameters can be obtained. The maximum surface excess (훤푚푎푥) can be calculated according to the following equation:

1 휕훾 훤푚푎푥 = − ( ) (Eq. 2) 푛푅푇 훿ln (푚/푚°) 푃,푇 where R is the ideal gas constant, T is the absolute temperature, [∂γ /(∂ ln(m/mº)] is the slope of the surface tension plot just below the cmc, m is the surfactant molal concentration, and n is the Gibbs prefactor corresponding to the number of free chemical species at the interface. For these compounds, it was considered n = 2. Through the

68 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

maximum surface excess, the minimal surface area per molecule as can be obtained, according to equation 3.

1 푎푠 = (Eq.3) 푁퐴훤푚푎푥

Where NA is the Avogadro constant. The surface tension at cmc (γcmc) was obtained directly form the surface tension curves.

Table 6: Interfacial parameters for the amino acid amphiphile derivatives

-1 -1 2 Compound cmc /mmol·Kg γcmc /mN·m as /nm 10 0.039 27.49 0.86 20 0.061 25.62 0.98 3a 0.242 27.07 1.52 8 0.052 26.07 0.82 9 0.034 26.08 1.86

First, all compounds studied were surface active, as seen by the reduction of the surface tension during addition.

Considering compounds 10 and 20, the difference in their structure is the presence of an extra hydroxyl group in compound 20. The surface tension curves of this two amphiphiles are almost identical and their cmc, surface area per molecule and minimum surface tension values are only slightly different. Compound 20 presents a higher cmc value, a result that is in good agreement with what was expected as the presence of one more group in the proline ring increases its polarity and lead to higher affinity with water.

Compound 3a is the only one with a free carboxylic acid group. At pH 5.5, the carboxylic acid is deprotonated and bears a negative charge, thereby promoting its solubility in an aqueous environment. For this reason, compound 3a presents the highest cmc value of all the compounds studied.

Compounds 8 and 9 have alkyl chains linked to the carboxylic acid group through an ester bond. The only difference between them is the substituent on the nitrogen atom. Compound 9 has an acetyl attached to the nitrogen, which confers slightly higher polarity than a methyl group as present in 8 and may also increase the size of the head group. In fact, the surface area per molecule of compound 9 is higher than 8 as expected. The

higher as may be responsible for the slightly lower cmc values observed for compound 9. FCUP 69 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Comparing 10 and 8 which differ only in the position of the alkyl chain, small differences can be seen in the surface tension at cmc, as well in the cmc values, with 8 presenting a slightly higher cmc. This is in agreement with what was expected, as the polarities of the compounds are very similar, as both have the amino group partially charged at pH 5.5 and have a free hydroxyl group.

70 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

2.2.2. Melting Points

In order to assess the melting points of all derivatives that were obtained as solids, differential scanning calorimetry (DSC) was performed.

The following figures describe the analysis of 9 compounds. Solid derivatives with melting points lower than 30ºC were not subjected to DSC analysis. The DSC curves are grouped according to the chemical structures of the compounds. Melting points are presented in table 7. The most energetic phase transitions were the ones recognized for the assessment of the melting temperatures.

1 400 A 1200 B 2b 3b 3a 5b

1000 A B 900 800

10 400 600

2000 DSC DSC uW / DSC DSC uW / 35 0 400 -100 -2000 -4000 200 -6000 30 50 70 90 -600 0 25 50 75 100 125 150 175 200 20 70 120 170 220 T / °C T / °C

1400

C 31 32 16 1200

1000 C 800 600

400

DSC uW / 200

0

-200

-400 -600 25 50 75 100 125 150 175 200 T / °C

Figure 10: DSC curves for all compounds analyzed: A) O-acylated; B) N-alkylated/acylated, C) O-alkylated derivatives FCUP 71 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

The melting temperature is defined by the extrapolated beginning of the curve and is interpreted as point of intersection of the tangent with the point of maximum slope, on the principal side of the peak, with the base line extrapolated. [81] The base line was extrapolated at least 3 times and the mean of the intersection points obtained gave rise to the final value for the melting point. Figure 11 illustrates this procedure.

Table 7: Melting points of the derivatives analyzed by DSC.

Graph Group Compound Melting Point / ºC 2b 169.5 3b 98.5 A O-acylated 3a 37.0 5b 70.8 N-alkylated 10 36.4 B N-acylated 35 90.4 31 104.0 C O-alkylated 32 118.1 16 66.2

1,200 1,100 1,000 0,900 0,800

0,700

0,600 104.1 ºC 103.9 ºC

0,500 103.9 ºC DSC/ mW DSC/ 0,400 103.9 ºC 0,300 0,200 0,100 0,000 -0,100 -0,200

30 40 50 60 70 80 90 100 110 120 130 140

T / ºC

Figure 11: DSC curve of compound 31 recorded with Thermal analysis program.

Chapter 3: Conclusions

74 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Concerning the synthesis of the amphiphilic derivatives, 18 final compounds were successfully synthetized. However, some reaction steps were performed in low yields. To overcome this issue, optimization will be required. Also, three compounds that were initially designed were not yet obtained successfully. In other to synthetize the desired compounds, new methodologies must be established. To obtain compound 18, the planned synthetic route can be followed, but the methylation by reductive amination will require higher quantities of reductive agent and a stronger organic base (with pKa higher than 11, the pKa of the pyrrolidine ring) to achieve better yields. To optimize the yield of compounds 29a-b, the problems with the dimethylation reaction must be dealt with. One possibility is to perform the reductive amination in slightly acidic conditions (acetic acid). This way, the theorized degradation reaction will not occur for lack of base in solution. To obtain the unnatural β-amino acid N-alkyl derivative, methyl (1R,2R,3S,4R)-2- dodecyl-3,4-dihydroxycyclopentane-1-carboxylate from compound 33, the introduction of the alkyl chain by reductive amination must be performed in a secondary amine, to avoid dialkylation. The primary amine can be selectively monomethylated before the introduction of the alkyl chain, by preparing, for example, an arylsulfonamide derivative from the primary amine that reacts with methyl iodide giving rise to a methylamido derivative. Then, the protective group can be removed to yield the secondary amine.

Regarding the physicochemical studies, all the studied compounds have amphiphilic behavior and are surface active, important characteristics to the application in dermal delivery. Moreover, their cmc values are low, so these compounds are likely to be biologically active as aggregates (concentrations higher than cmc) in solution. The evaluation of the physicochemical behavior of all the β-amino acid derivatives would have been of great interest because the influence of this kind of head group in the aggregation properties was never studied before, but this was not possible due to solubility problems.

For future studies, two approaches are possible:

First, the same aqueous conditions can be used but, since only a few derivatives are in fact water soluble, the remaining ones would need chemical modifications to improve water solubility. This could be achieved by imparting charge to the derivatives,

FCUP 75 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems either through quaternization of the amino group or deprotonation of the carboxylic acid group, for example.

The second approach would be to use all compounds as synthetized (non-ionic) and change the conditions to solubilize them. To achieve this, other solvents may have to be used, like dimethyl sulfoxide, propylene glycol, ethanol, among others. This could be beneficial because many of these solvents have skin permeation enhancement capacities and can act synergistically with the enhancers. However, the conditions for determination of the cmc values would be different, so these tests would have to be repeated for all the compounds.

.

Chapter 4: Materials and Methods

78 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

4.1. Chemicals

All chemicals used in this work are listed in table 8. All solvents used were pro- analysis quality.

Table 8: Reagents used

Name Formula/Acronym M / g·mol-1 Density /g·mL-1

Dodecyl aldehyde CH3(CH2)10CHO 184.32

Triethylamine NEt3 101.19 0.726

Sodium triacetoxyborohydride NaBH(OAc)3 211.94 Formaldehyde 37 % HCHO 30.03 1.083

Dodecyl chloride CH3(CH2)10COCl 218.77 0.946

Dodecyl alcohol CH3(CH2)10CH2OH 183.34 N.N-Diisopropylethylamine DIEA 129.25 0.755 1.8-diazabicyclo[5.4.0]undec-7- DBU 152.24 1.02 ene 2-(1H-Benzotriazole-1-yl)- 1.1.3.3-tetramethylaminium TBTU 321.09 tetrafluoroborate Benzotriazol-1-yl- oxytripyrrolidinophosphonium PyBoP 520.40 hexafluorophosphate Trimethylchlorosilane TMSCl 108.64 0.856

Iodomethane CH3I 141.94 2.28

Trifluoroacetic acid TFA/CF3COOH 114.02 1.48

Acetyl chloride CH3COCl 78.49 1.104

Di-tert-butyl dicarbonate Boc2O 218.25 N-methylmorpholine N-oxide NMO 117.15 2.5 wt% in Osmium tetroxide OsO4 254.23 tBuOH Chlorosulfonyl isocyanate CSI 141.53 1.626 Cyclopentadiene CPD 66.103 0.786

FCUP 79 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

4.2. Synthesis and characterization

The syntheses were performed according to procedures already described in literature [60, 64, 75]. Optimizations performed are described in detail in chapters 2 and 5. All reactions and purifications were monitored by thin layer chromatography

(TLC) on silica gel 60 F254 pre-coated aluminum sheets of 0.25 mm thickness, from Merck. Phosphomolybdic acid was used as stain to follow the reactions by TLC. In column chromatographies SDS silica gel was used (60 A, 30-70 µm, surface area of 550 m2/g). All column chromatographies were performed under pressure, with collection of eluent fractions of 10 to 25 mL. The eluents use in each case are referenced in the experimental section, chapter 4. The characterization of the compounds was performed by NMR, at CEMUP (UP) and MS, at DQB/FCUP. 1H and 13C NMR spectra were specifically obtained on a Bruker Avance III 400 spectrometer. Chemical shifts (δ) are presented in ppm and coupling constants (J) are quoted in Hz. The sign multiplicity is represented as follows: s (singlet), bs (broad singlet), d (doublet), dd (doublet of doublets), ddd (doublet of doublet of doublets), t (triplet), dt (doublet of triplets), ddt (doublet of doublet of triplets), q (quartet); m (multiplet).

The three families of derivatives synthetized were numbered following IUPAC recommendations according to the following scheme and the same notation was used for NMR purposes.

Scheme 25: Atom numeration

Mass spectra were recorded on a Finnigan Surveyor instrument, fitted with a Finnigan LCQ DECA XP MX mass detector (Finningan Corp. San José, Calif. USA) and Atmospheric Pressure Ionization (API) with Eletrospray ionization (ESI) interface. The samples were analysed by direct injection and the spetra were obtained in positive mode (m/z 50-1500) Specific optical rotations were obtained using a Perkin Elmer Polarimeter 341 with reference to the sodium D line (λ =589 nm) in chloroform. The results are presented

-1 -1 1 as [훼]퐷 푇 in deg·dm · g · mL , given by equation 2.

80 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

100 × 훼 [훼]푇 = (Eq. 2) 퐷 푙 ×푐

Where, α is the measured rotation, l is the path length in dm, 푇 is the temperature in ºC and c is the concentration in g/100mL. Melting points were obtained at FCUP | DQB - Lab&Services, with a Hitachi DSC7020, using a heating scale from 20 to 200ºC, a heating rate of 2ºC/min and

recorded in a dynamic N2 atmosphere flowing at 50 mL/min. The apparatus was calibrated with indium, tin and benzoic acid. All samples were prepared in aluminum crucibles hermetically sealed.

4.3. Surface Tension

The cmc can be determined by measuring the surface tension (γ) of a solution with increasing surfactant concentration. Addition of surfactant increases the number of monomers in solution and due to their tendency to accumulate at the surface, the surface tension decreases until the critical concentration is reached. After the cmc, the surface tension stabilizes because at this point, the concentration of free monomer is roughly constant and represents the cmc value.

In this work, the surface tension was measured using the wilhelmy plate method. The platinum plate used is made of platinum-iridium alloy and present roughened surfaces (to minimize the contact angle, θ) and has the following geometric dimensions: length 10 mm, width 19.9 mm and thickness 0.2 mm. This method measures the force (F) with which the platinum plate is puled downward by an air/water interface, and it can be expressed according to the following equation (Eq.4).

퐹 = 2훾푙 (Eq.4)

where 훾 is the surface tension and 푙 is the known plate width. Before each experiment the plate is thoroughly cleaned and flamed. The solutions used for the interfacial characterization were prepared on the same day of the experiment or the day before, in concentrations between 2-4 mM. The measurements were performed in a DCATT 11 surface tensiometer and temperature was kept constant at the desired value (± 0.1ºC) using a thermostatted Julabo water bath.

This method and equipment were used to measure the surface tension of the aqueous solution (phosphate buffer pH 5.5; 0.8 % NaCl). Successive aliquots of the FCUP 81 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems amphiphiles stock solution, freshly prepared in buffer solution, were added to the thermostated vessel with 25 mL under constant stiring. The surface tension value in each individual measurment was allowed to vary only 0.005 mN·m-1 Between the additions, the solution was stirred for 1.5 min and given a 5 min equilibrium time. These experiments were performed by Dr. Sandra Silva at lab 3.35 from DQB (CIQUP), in collaboration with Prof. Eduardo Marques.

Chapter 5: Synthesis

84 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

5.1. Synthesis of the 4-hydroxyproline derivatives

5.1.1. Synthesis of methyl (2S,4R)-4-(O-dodecanoyl)hydroxyprolinate (2a) / (2S,4R) - 4-(O-dodecanoyl)hydroxyproline (2b)

The amino acids 1a/1b were dissolved at 0ºC in TFA (≈ 5 ml) and lauroyl chloride (1.5 eq) was added drop by drop with continuous stirring. The mixture was left at rt. for 15h. After this time, the mixture was treated with diethyl ether followed by evaporation until total removal of TFA. Compounds 2a and 2b were precipitated from diethyl ether and collected by suction filtration as hydrocloride satls (white solids). In table 9 are listed the reactions performed.

Table 9: Synthesis of methyl (2S,4R)-4-(O-dodecanoyl)hydroxyprolinate ((2a) / (2S,4R) -4-(O- dodecanoyl)hydroxyproline (2b)

2a/2b 1a/b (M1a=181.62g (M2a=363.22 g Starting /mol and M1b = TFA CH3(CH2)10COCl η Exp /mol and M2b = 131.131 g/mol) V/mL V/mL (n/mmol) material 349.20 g/mol) % m/g (n/mmol) m/g (n/mmol) 1 2.860 1a 1.505 (8.289) 5.00 2.865 (12.39) 95 (7.875) 2 0.8921 1a 0.5011 (2.759) 5.00 0.9570 (4.139) 89 (2.456) 3 2.977 1b 1.153 (11.68) 5.00 4.050 (17.51) 73 (8.526) 4 2.254 1b 1.004 (7.656) 5.00 2.656 (11.48) 84 (6.455)

The spectroscopic data (1H and 13C-NMR) of compounds 2a/2b are presented below.

1 2a H NMR (400 MHz, CDCl3) δ 5.46 – 5.33 (m, 1H , C(4)H), 4.57 (dd, J = 10.9,

7.3 Hz, 1H, C(2)H), 3.84 (s, 1H, COO-CH3), 3.81 (dd, 1H, C(5)HH), 3.57 – 3.48 (m, 1H,

C(5)HH), 2.56 (ddt, J = 14.4, 7.3, 1.54 Hz, 1H, C(2)HH), 2.44 (ddd, J = 14.3, 10.9, 5.1 Hz,

1H, C(2)HH), 2.34 (t, J = 8.2 Hz, 2H,-CH2-COOR-), 1.66 – 1.55 (m, 2H, -CH2-CH2-COOR-

),), 1.38 – 1.18 (m, 16H, CH3-(CH2)8-CH2-CH2-), 0.90 (t, J = 6.9 Hz, 1H, CH3-(CH2)8- 13 CH2-CH2-). C NMR (101 MHz, CDCl3) δ 172.85 (COOR), 168.18 (COO-), 71.55 (C(4)), FCUP 85 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

58.03 (C(2)), 53.40 (-COO-CH3), 50.86(C(5)), 34.69 (C(3)), 33.73-22.50 (CH3-(CH2)10-

COOR), 13.92(CH3-(CH2)10-COOR). Rf = 0.25 (DCM/MeOH 20:1)

1 2b H NMR (400 MHz, MeOD) δ 5.64 – 5.57 (m, 1H, C(4)H ), 4.68 (dd, J = 10.3,

7.7 Hz, 1H, C(2)H), 3.87 (dd, J = 13.2, 4.9 Hz, 1H, C(5)HH), 3.65 (dt, J = 13.2, 1.5 Hz, 1H,

C(5)HH), 2.76 (ddt, J = 14.5, 7.7, 1.6 Hz, 1H, C(3)HH), 2.65 – 2.58 (m, 1H, C(3)HH), 2.56

(t, 2H, -CH2-COOR- ), 1.87 – 1.73 (m, 2H, -CH2-CH2-COOR-), 1.55 – 1.39 (m, 16H, CH3- 13 (CH2)8-CH2-CH2-), 1.05 (t, J = 6.9 Hz, 3H, CH3-(CH2)8-CH2-CH2-). C NMR (101 MHz,

MeOD) δ 172.87(COOR), 168.67 (COO-), 71.71 (C(4)), 57.91 (C(2)), 50.59(C(5)), 34.38

(C(3) ), 33.39-22.14 CH3-(CH2)10-COOR, 13.27 (CH3-(CH2)10-COOR). MS (ESI,MeOH): calc. 314.44 [M+H]+; found 314.47. Rf = 0.25 (DCM/MeOH 10:1)

5.1.2. Synthesis of methyl (2S,4R)-4-(O-dodecanoyl)-N-methyl-hydroxyprolinate (3a) / (2S,4R)-4-(O-dodecanoyl)-N-methyl-hydroxyproline (3b)

Compounds 2a/2b were dissolved in DCE (10 mL), triethylamine (2 eq.) was added and the mixture was stirred for 15 min at rt. Then, an aqueous solution of formaldehyde 37% (2 eq.) was added and, after 15 minutes of stirring, sodium triacetoxyborohydride (2 eq.) was added. The mixture was stirred at rt for 12 h, quenched with HCl 1M and the product was extracted with DCM (3 x 20 mL). The DCM extracts were washed with water and dried (Na2SO4). The solvent was removed, and the crude product was subjected to column chromatography on silica gel using DCM/MeOH (10:1) as eluent. Compounds 3a/3b were recrystallized from DCM/diethyl ether as white solids. In table 10 are listed the reactions performed.

Table 10: Synthesis of methyl (2S,4R)-4-(O-dodecanoyl)-N-methyl-hydroxyprolinate (3a) / (2S,4R)-4-(O- dodecanoyl)-N-methyl-hydroxyproline (3b)

2a/2b 3a/3b NEt3 (M3a=341.26 (M2a=363.22 g HCHO Starting V/mL NaBH(OAc)3 g /mol and η Exp /mol and M2b = V/mL (n/m m/g (n/mmol) M3b = 327.47 material 349.20 g/mol) (n/mmol) % mol) g/mol) m/g (n/mmol) m/g (n/mmol) 1 2a 1.400 1.30 0.30 2.017 0.9887 75 (3.854) (9.3) (3.7) (9.517) (2.897) 2 2b 0.9705 0.70 0.40 1.221 0.5443 60 (2.779) (5.0) (4.5) (5.726) (1.662)

86 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Spectroscopic data of compound 3a/3b are presented below:

1 3a: H NMR (400 MHz, CDCl3) δ 5.30 – 5.16 (m, 1H, C(4)H), 3.76 (s, 3H, COO-

CH3), 3.59 (dd, J = 10.8, 6.3 Hz, 1H, - C(5)HH), 3.31 (dd, 1H, J = 8.4, 7.6 Hz, -C(2)H-),

2.46 (dd, 1H, C(5)HH), 2.45 (s, 3H, NCH3), 2.40 – 2.33 (m, 1H, C(3)HH), 2.30 (t, J = 7.5

Hz, 2H, -CH2-CH2-COOR-), 2.14 (ddd, J = 13.9, 7.3, 2.7 Hz, 1H, C(2)HH), 1.67 – 1.56 (m,

2H, -CH2-CH2-COOR-), 1.39 – 1.18 (m, 16H, CH3-(CH2)8-CH2-CH2-), 0.89 (t, J = 6.9 Hz, 13 3H, CH3-(CH2)8-CH2-CH2-). C NMR (101 MHz, CDCl3) δ 173.54 (-CH2-COOPro),

173.08 (-COOCH3), 72.72 (C(4)) ,66.09 (C(2)) , 61.53 (C(5)) 52.14 (-COO-CH3) , 40.60

(NCH3) 37.27 (C(2)), 34.46-22.80 (CH3-(CH2)10-COOR), 14.23 ( CH3-(CH2)8-CH2-CH2-). MS (ESI,MeOH): calc. 342.47 [M+H] +; found 343.00. Rf = 0.73 (DCM/MeOH 20:1)

1 3b: H NMR (400 MHz, CDCl3) δ 5.34 – 5.26 (m, 1H, C(4)H), 4.37 (dd, J = 13.0,

5.6 Hz, 1H, -C(5)HH ), 3.84 (t, J = 9.0 Hz, 1H, C(2)H), 2.97 (s, 3H, -NCH3), 2.91 (d, J =

12.9 Hz, 1H, C(5)HH), 2.48 (dd, J = 9.1, 3.6 Hz, 2H, -C(3)H2), 2.32 (t, J = 7.6 Hz, 2H, (-

CH2-COOR-), 1.61 (dd, J = 14.2, 7.1 Hz, 2H, -CH2-CH2-COOR-), 1.35 – 1.19 (m, 16H, 13 CH3-(CH2)8-CH2-), 0.89 (t, J = 6.8 Hz, 3H, CH3-(CH2)8-CH2-). C NMR (101 MHz, CDCl3)

δ 172.86 (-CH2-COOPro), 170.56 (-COOH), 72.22 (C(4)), 69.73 (C(2)), 61.48 (C(5)) , 42.59

(-NCH3), 36.54 (C(3)), 34.30 -22.80 (CH3-(CH2)10-COOR-) 14.23 ( CH3-(CH2)8-CH2-CH2- ). MS (ESI,MeOH): calc. 328.47 [M+H]+; found 328.80. Rf = 0.44 (DCM/MeOH 10:1)

5.1.3. Synthesis of methyl (2S,4R)-1-acetyl-4-(O-dodecanoyl)hydroxyprolinate (5a) / (2S,4R)-1-acetyl-4-( O-dodecanoyl)hydroxyproline (5b)

Compounds 2a/2b were dissolved in DCM and 2 eq. of NEt3 were added. The mixture was placed in an ice bath and acetyl chloride was added drop by drop for one hour. After 4 hours, the mixture was washed with water. The organic extract was dried and, after solvent evaporation, the crude was purified by column chromatography using as eluent DCM/MeOH 10:1. Compounds 5a/5b were obtained as white solids. In table 11 are listed the reactions performed. FCUP 87 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Table 11: Synthesis of methyl (2S,4R)-1-acetyl-4-(O-dodecanoyl)hydroxyprolinate (5a) / (2S,4R)-1-acetyl- 4-( O-dodecanoyl)hydroxyproline (5b)

5a/5b 2a/2b (M2a=363.22 NEt3 (M5a=369.25 Starting g /mol and M2b = CH3COCl Exp V/mL g/mol and M5b η % 349.20 g/mol) V/mL (n/mmol) material (n/mmol) = 355.25 g/mol) m/g (n/mmol) m/g (n/mmol) 1 2a 2.011 2.00 0.40 1.010 49 (5.537) (14.4) (5.60) (2.735) 2 2b 0.4823 1.00 0.10 0.1412 29 (1.381) (7.17) (1.40) (0.3975)

1 5a: H NMR (400 MHz, CDCl3) δ 5.36 – 5.31(m, 1H, C(4)H), 4.54 (t, J = 8.0 Hz,

1H, C(2)H), 3.91 (dd, J = 11.5, 4.7 Hz, 1H, C(5)HH), 3.78+3.74 (2x s, 3H, COO-CH3, rot ),

3.57 (dt, 1H, J = 11.6, 1.6 Hz, C(5)HH), 2.41 – 2.33 (m, 1H, C(3)HH ), 2.29 (t, J = 7.5 Hz,

2H, CH2-COOR- ), 2.21 (ddd, J = 13.7, 10.2, 6.2 Hz, 1H, C(3)HH ), 2.07+1.99 (2x s, 3H,

-NCOCH3, rot), 1.66 – 1.54 (m, 1H, -CH2-CH2-COOR-), 1.37 – 1.18 (m, 16H, CH3-(CH2)8- 13 CH2-), 0.87 (t, J = 6.8 Hz, 3H, CH3-(CH2)8-CH2- ). C NMR (101 MHz, CDCl3) δ 173.42

(-CH2-COOR-),172.48 (COO-), 169.54 (NCOCH3), 72.69 (C(4)), 57.49 (C(2)), 53.44(C(5)),

52.57(-COO-CH3), 35.21(C(3)), 34.36-22.81 (CH3-(CH2)10-COOR), 22.39, (NCOCH3), + 14.24 ( CH3-(CH2)8-CH2-CH2-). MS (ESI,MeOH): calc. 370.50 [M+H] ; found 370.20. Rf = 0.58 (Hex/ EtOAc 1:3)

1 5b: H NMR (400 MHz, CDCl3) δ 5.36 – 5.25 (m, 1H, C(4)H), 4.63 (t, J = 7.9 Hz,

1H, C(2)H), 3.86 (dd, J = 11.8, 4.6 Hz, 1H, C(5)HH), 3.60 (dt, J = 11.8, 1,6 Hz, 1H, C(5)HH),

2.59 – 2.44 (m, 1H, C(2)HH), 2.42 – 2.32 (m, 1H, C(2)HH), 2.29 (t, J = 7.5 Hz, 2H, -CH2-

COOR- ), 2.10 (2x s, 3H, -NCOCH3, rot), 1.68 – 1.52 (m, 2H, -CH2-CH2-COOR- ), 1.25 13 (m, 16H, CH3-(CH2)8-CH2-), 0.87 (t, J = 6.9 Hz, 3H, CH3-(CH2)8-CH2-). C NMR (101

MHz, CDCl3) δ 173.42 (-CH2-COOR-), 172.84 (COO-), 171.95 (NCOCH3), 72.13 (C(4)),

58.15 (C(2)), 53.83 (C(5)), 34.32 (C(3)), 34.20-22.81 (CH3-(CH2)10-COOR), 22.41 + (NCOCH3), 14.24 ( CH3-(CH2)8-CH2-CH2-) . MS (ESI,MeOH): calc. 356.48 [M+H] ; found 356.47. Rf = 0.84 (DCM/MeOH 10:1)

88 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

5.1.4. Synthesis of methyl (2S,4R)-N-dodecyl-4-hydroxyprolinate (10)

To a solution of 1a in DCE (10 mL) triethylamine (1.5 eq.) was added. After 15 min, dodecanal (1.1eq.) was added and the mixture was stirred for another 30 minutes, followed by addition of sodium triacetoxyborohydride (1.1 eq.). The mixture was stirred at rt for 24 h, quenched with HCl 0.5 M and the product was extracted with DCM (3 x 20

mL). The DCM extracts were washed with water and dried with Na2SO4. The solvent was removed, and the crude product was subjected to column chromatography on silica gel using DCM/methanol (10:1) as eluent. Compound 10 was obtained as a white solid after 3 hours in the high vacuum bomb. In table 12 are listed the reactions performed.

Table 12: Synthesis of methyl (2S,4R)-N-dodecyl-4-hydroxyprolinate (10)

10 1a (M1a= NEt3 CH (CH ) CHO NaBH(OAc) (M10=313.48 η Exp 181.62 g /mol V/mL 3 2 10 3 g /mol) m/g (n/mmol) m/g (n/mmol) m/g (n/mmol) (n/mmol) m/g % (n/mmol) 1.30 1.715 2.698 0.9015 1 1.511 (8.320) 35 (9.33) (9.304) (12.73) (2.876) 1.30 1.550 2.662 0.9915 2 1.523 (8.327) 38 (9.33) (8.409) (12.56) (3.162)

1 H NMR (400 MHz, CDCl3) δ 4.52 – 4.42 (m, 1H, C(4)H), 3.70 (s, 3H, COOCH3),

3.51 (t, J = 7.7 Hz, 1H, C(2)H), 3.42 (dd, J = 10.1, 5.6 Hz, 1H, C(5)HH), 2.65 – 2.54 (m,

1H, C(5)HH), 2.50 – 2.41 (m, 2H, -N-CH2-CH2-), 2.23 (bs, 1H, OH) 2.21 (dt, J = 14.2, 7.2

Hz, 1H, C(3)HH), 2.05 (ddd, J = 13.4, 7.9, 3.1 Hz, 1H, C(3)HH), 1.45 (dd, J = 19.1, 12.3

Hz, 2H, CH3-(CH2)9-CH2-), 1.35 – 1.15 (m, 18H), 0.87 (t, J = 6.9 Hz, 3H, CH3-(CH2)9-CH2- 13 ). C NMR (101 MHz, CDCl3) δ 174.28 (COO-), 70.50 (C(4)), 64.51 (C(2)), 61.54 (C(5)),

54.67 (-N-CH2-CH2-) 51.93 (COOCH3), 39.70 (C(3)), 32.04- 22.81 (CH3-(CH2)10-NR), + 14.23 (CH3-(CH2)10CH2-). MS (ESI,MeOH): calc. 314.48 [M+H] ; found 314.80 . Rf = 0.64 (Hex/ EtOAc 1:3)

FCUP 89 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

5.1.5. Synthesis of dodecyl (2S,4R)-N-Cbz-4-hydroxyprolinate (6)

The amino acid 1c was suspended in DCM and 1.1 eq of trimethylsilyl chloride (TMSCl) was added. The mixture was left stirring for 30 minutes at 0ºC. Then 1.1 eq of dodecanol (CH3(CH2)10CH2OH) was added. After 2h the reagents were completely dissolved, and the mixture was allowed to react for 12 hours. The solvent was then removed and the crude residue submitted to column chromatography on silica gel, using Hex/ EtOAc 3:1 as eluent. Compound 6 was obtained as a colorless oil in the two reactions performed, described in table 13.

Table 13: Synthesis of dodecyl (2S,4R)-N-Cbz-4-hydroxyprolinate (6)

1c (M1c= 6 (M6=433.29 TMSCl V/mL CH (CH ) CH OH η Exp 256.26 g /mol 3 2 10 2 g /mol) (n/mmol) m/g (n/mmol) m/g (n/mmol) m/g (n/mmol) % 1.555 0.80 1.273 1 1.573 (3.766) 62 (6.068) (6.3) (6.832) 1.535 0.80 1.226 2 1.789 (4.287) 71 (5.990) (6.3) (6.579)

The characterization (NMR and MS) of compound 6 is described below.

1 H NMR (400 MHz, CDCl3) δ 7.41 – 7.22 (m, 5H, -CH2-Ph), 5.26 – 4.95 (m, 2H, -

CH2-Ph), 4.56 – 4.41 (m, 2H, C(2)H + C(4)H), 4.21 – 4.06 (m, 1H, C(5)HH), 3.95 (t, J = 6.8

Hz, 1H, C(5)HH), 3.74 – 3.49 (m, 1H, CH2-OCOR-), 2.39 – 2.22 (m, 1H, C(3)HH), 2.16 –

2.01 (m, 1H, C(3)HH), 1.91 (bs, 1H, C(4)OH), 1.70 – 1.55 (m,1H, -CHH-CH2-OCOR-), 1.54

– 1.40 (m, 1H, CHH-CH2-OOCR-), 1.38 – 1.13 (m, 18H, CH3-(CH2)9-CH2-), 0.88 (t, J = 13 6.8 Hz, 1H, CH3-(CH2)9-CH2-). C NMR (101 MHz, CDCl3) δ 172.92+172.71 (COO-, rot),

155.12+154.73 (N-COO-CH2-, rot), 136.64+136.44 (NCOOCH2-CH-, rot), 128.60-127.92

(-CH2-Ph), 70.33+69.59 (C(4), rot), 67.38+67.34 (-CH2-Ph, rot), 65.62+65.53 (C(5), rot),

58.18+57.94 (C(2), rot), 55.38+54.77 (CH2-OCOR-, rot), 39.42+38.62 (C(3), rot), 32.04-

22.82 (CH3-(CH2)10-CH2-) , 14.24 (CH3-(CH2)10CH2-) MS (ESI,MeOH): calc. 434.59 [M+H]+; found 434.47. Rf = 0.37 (Hex/ EtOAc 1:3)

90 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

5.1.6. Synthesis of dodecyl (2S,4R)-4-hydroxyprolinate (7)

Compound 6 was dissolved in MeOH (30 mL) and Pd-C was added (5% wt). The mixture was stirred for 4h at rt under high pressure hydrogen atmosphere (50 bar) using an hydrogenator. The mixture was then filtered through celite under vacuum to remove the catalyst. The crude was subjected to column chromatography using Hex/AcOEt 1:3 and DCM/MeOH 10:1 as eluents. Compound 7 was obtained as a white solid after complete removal of the solvent in the high vacuum bomb. The reactions performed are listed in table 14.

Table 14: Synthesis of dodecyl (2S,4R)-4-hydroxyprolinate (7)

Exp. 6 (M6=417.29 g /mol) 7 (M7=299.25 g /mol) η % m/g (n/mmol) m/g (n/mmol)

1 2.190 (5.248) 1.006 (3.361) 64

2 1.189 (2.849) 0.3012 (1.006) 35

The detailed description of NMR spectra for compound 10 is presented below:

1 H NMR (400 MHz, CDCl3) δ 4.46 – 4.40 (m, 1H, C(4)H ), 4.13 (t, J = 6.7 Hz, 2H,

CH2-OCOR-), 4.03 (t, J = 7.9 Hz, 1H, C(2)H), 3.17 (dd, J = 11.4, 4.2 Hz, 1H, C(5)HH), 3.00

(dd, J = 11.4, 1.6 Hz, 1H, C(5)HH), 2.42 (bs, 1H, OH), 2.55 – 2.34 (m, 1H, NH), 2.28 –

2.15 (m, 1H, C(3)HH), 2.07 – 1.98 (m, 1H, C(3)HH), 1.64 (m, 2H, -CH2-CH2-OCOR), 1.30 13 (m, 18H, CH3-(CH2)9-CH2-), 0.90 (t, J = 6.8 Hz, 3H, CH3-(CH2)9-CH2-). C NMR (101

MHz, CDCl3) δ 174.95 (COO-), 72.25 (C(4)), 65.35 (C(5)), 58.20 (C(2)), 55.20 (RCOO-CH2-

CH2-), 39.57(C(3)) , 31.91-22.69 (CH3-(CH2)10-OOCR), 14.11 (CH3-(CH2)10CH2-) MS (ESI,MeOH): calc. 300.46 [M+H]+; found 300.40. Rf = 0.29 (DCM/MeOH 10:1)

FCUP 91 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

5.1.7. Synthesis of dodecyl (2S,4R)-N-methyl-4-hydroxyprolinate (8)

The synthesis of 7 was performed according to the procedure described in 5.1.2. After treatment, the mixture was subjected to column chromatography on silica gel using DCM/MeOH 10:1 as eluent. Compound 8 was obtained as a white solid. Table 15 summarized the reactions performed.

Table 15: Synthesis of dodecyl (2S,4R-N-methyl-4-hydroxyprolinate (8)

7 (M7=299.25 g NEt3 HCHO NaBH(OAc) 8 (M8=313.26 g Exp. /mol) V/mL V/mL 3 /mol) η % m/g (n/mmol) m/g (n/mmol) (n/mmol) (n/mmol) m/g (n/mmol)

1 0.2013 0.20 0.20 0.5220 0.1127 54 (0.6727) (1.4) (2.4) (2.463) (0.3598) 2 0.2503 0.30 0.30 0.7172 0.2200 84 (0.8364) (2.2) (3.7) (3.384) (0.7025)

The detailed description of NMR spectra for compound 10 is presented below:

1 H NMR (400 MHz, CDCl3) δ 4.52 – 4.45 (m, 1H, C(4)H), 4.14 (t, J = 6.8 Hz, 2H,

CH2-OCOR-), 3.49 (dd, J = 10.1, 5.8 Hz, 1H, C(5)HH), 3.41 (t, J = 8.0 Hz, 1H, C(2)H), 2.48

(s, 3H, NCH3), 2.45 (dd, J = 10.2, 4.1 Hz, 1H, C(5)HH), 2.33 – 2.22 (m, 1H, C(2)HH), 2.33

– 2.22 (bs, 1H, OH), 2.10 (ddd, J = 13.5, 7.8, 2.9 Hz, 1H, C(2)HH ), 1.70 – 1.61 (m, 2H,

CH2-CH2-OCOR), 1.39 – 1.21 (m, 18H, CH3-(CH2)9-CH2-), 0.90 (t, J = 6.8 Hz, 3H, CH3- 13 (CH2)9-CH2- ). C NMR (101 MHz, CDCl3) δ 173.02 (COO-), 70.37(C(4)) , 65.92 (C(1)),

65.18 (C(4)), 64.30 (RCOO-CH2-CH2-), 40.61 (NCH3), 40.15 (C(2)), 32.04-22.81 (CH3- + (CH2)10-OOCR), 14.24 (CH3-(CH2)10CH2-). MS (ESI,MeOH): calc. 314.48 [M+H] ; found 314.93 , Rf = 0.55 (DCM/MeOH 10:1)

92 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

5.1.8. Synthesis of dodecyl (2S,4R)-N-acetyl-4-hydroxyprolinate (9)

7 9

Compound 7 was dissolved in DCM and 1.1 eq. of NEt3 was added. The mixture 7 7 was placed in an ice bath and acetyl chloride was added, drop by drop, for one hour. After 4 hours, the mixture was washed with water. The organic extract was dried, and, after solvent evaporation, the crude was purified by column chromatography using as eluent DCM/MeOH 10:1. Compound 9 was obtained as a colorless oil at rt. The reactions performed are listed in table 16.

Table 16: Synthesis of Dodecyl (2S,4R)-N-acetyl-4-hydroxyprolinate (9)

7 (M7= 299.25 g 9 (M9= 341.26 g NEt V/mL CH COCl Exp /mol) 3 3 /mol) η % (n/mmol) V/mL (n/mmol) m/g (n/mmol) m/g (n/mmol) 1 0.5848 (1.954) 0.55 (3.9) 0.14 (2.0) 0.5005 (1.467) 75 2 0.2260 (0.7552) 0.20 (1.4) 0.05 (0.8) 0.1350 (0.3956) 52

1 H NMR (400 MHz, CDCl3) δ 4.61 – 4.48 (m, 1H, C(4)H ), 4.61 – 4.48 (m, 1H,

C(2)H), 4.13 (t, J = 6.8 Hz, 2H, CH2-OCOR-), 3.80 (dd, J = 10.9, 4.6 Hz, 1H, C(5)HH),

3.55 – 3.49 (m, 1H, C(5)HH), 2.35 – 2.17 (bs, 1H, OH), 2.35 – 2.17 (m, 1H, C(3)HH ), 2.13

(dd, J = 7.7, 5.1 Hz, 1H, C(3)HH), 2.07 + 1.97 (2x s, 3H, -NCOCH3, rot), 1.71 – 1.59 (m,

2H, CH2-CH2-OCOR ), 1.30 (d, J = 18.1 Hz, 18H, CH3-(CH2)9-CH2- ), 0.90 (t, J = 6.9 Hz, 13 3H, CH3-(CH2)9-CH2-). C NMR (101 MHz, CDCl3) δ 172.41 (COO-), 169.77 (NCOCH3),

70.32 (C(4)), 65.42 (C(5)), 57.57 (C(2)), 55.82 (RCOO-CH2-CH2-), 38.07 (C(3)), 31.91-22.68

(CH3-(CH2)10-OCOR), 22.23 (NCOCH3), 14.11 (CH3-(CH2)10CH2-). MS (ESI,MeOH): calc. 342.49 [M+H]+; found 342.60, Rf = 0.57 (DCM/MeOH 10:1)

FCUP 93 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

5.1.9. Synthesis of (2S,4R)-N,N-dimethyl-4-(O-dodecanoyl)-2-(O-methyl)- hydroxyprolinium iodide (4a)

Compound 3a (0.4735 g, 1.388 mmol) was dissolved in 10 eq. of CH3I and one drop of DMF was added. The mixture was stirred at r.t. until total consumption of 3a (TLC: 24h). After evaporation of the iodomethane, the mixture was dissolved in DCM and washed with water to remove the DMF. The organic phase was dried and after solvent removal compound 4a was obtained as a yellow oil. (0.4342 g, 0.8986 mmol, 65 % yield) The NMR characterization of the product obtained is described below.

1 H NMR (400 MHz, CDCl3) δ 5.54 – 5.43 (m, 1H, (C(4))H), 5.21 (dd, J = 10.1, 7.9

Hz, 1H, C(2)H), 4.66 (dd, J = 13.2, 6.8 Hz, 1H, C(5)HH), 4.28 (dd, J = 13.2, 4.5 Hz, 1H,

C(5)HH ), 3.95 – 3.79 (m, 6H, -N(CH3)2), 3.48 + 3.37 (2x s, 3H, COOCH3, rot), 2.96 (ddd,

J = 15.6, 10.2, 7.9 Hz, 1H, C(3)HH), 2.74 – 2.61 (m, 1H, C(3)HH), 2.45 – 2.27 (m, 2H, CH2-

COOR- ), 1.66 – 1.51 (m, 2H, CH2-CH2-COOR-), 1.35 – 1.17 (m, 16H, CH3-(CH2)8-CH2- 13 ), 0.86 (t, J = 6.8 Hz, 3H CH3-(CH2)8-CH2-). C NMR (101 MHz, CDCl3) δ 173.27 (-CH2-

COOR-), 165.59 (COO-), 72.98 (C(2)), 72.03 (C(5)) , 68.66 (C(4)), 54.57-54.21 (N(CH3)2),

50.14 (COOCH3), 34.08 (CH2-COOR-), 33.24 (C(3)), 32.01- 22.79 (CH3-(CH2)10-COOR), + 14.22 (CH3-(CH2)8-CH2-). MS (ESI,MeOH): calc. 356.53 [M] ; found 356.53 Rf = 0.57 (DCM/MeOH 10:1)

94 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

5.2. Synthesis of 3,4-dehydro-proline derivatives

5.2.1. Synthesis of (2S)-N-tert-butyloxycarbonyl-3,4-dehydroproline (12)

To a solution of 11a in dioxane/water (2:1, 18 mL), NaOH 1M (6,00 mL) was

added at 0ºC. Then di-tert-butyl dicarbonate (Boc2O) (1.1 eq) was added in small portions followed by addition of a 1M NaOH solution until pH 8-9. The mixture was stirred at room temperature for 4 hours. The reaction mixture was diluted in EtOAc (30 mL) and the layers separated. The aqueous layer was acidified with 10% HCl until the pH dropped

to 3-4 and then extracted with EtOAc. The organic layers were dried with Na2SO4. After filtration and removal of the solvent compound 12 was obtained as a colorless oil, homogeneous by TLC. All the reactions performed are listed in table 17.

Table 17: Synthesis of N-tert-butyloxycarbonyl-3,4-dehydroproline (12)

11a (M11a=113.12 g Boc O m/g 12 (M12= 213.23 g /mol) Exp. /mol) 2 η % (n/mmol) m/g (n/mmol) m/g (n/mmol)

1 0.2478 0.5356 0.3738 80 (2.191) (2.454) (1.753) 2 0.2114 0.4598 0.2718 68 (1.869) (2.107) (1.275) 3 0.2048 0.5213 0.3164 82 (1.810) (2.389) (1.484)

The detailed description of NMR spectra for the compound 12 is presented below:

1 H NMR (400 MHz, CDCl3) δ 6.08 – 5.70 (m, 2H, C(3)H + C(4)H), 5.05 (d, J = 42.9 13 Hz, 1H, C(2)H ), 4.35 – 4.14 (m, 2H, C(5)H2), 1.48 (d, 9H, -NCOC(CH3)3 ). C NMR (101

MHz, CDCl3) δ 129.93+128.71 (C (4), rot), 124.86+124.47 (C(3), rot), 81.78+80.85

(NCOC(CH3)3, rot) 66.46+66.36 (C(2), rot), 53.92+53.48 (C(5)), 28.52-28.40 + (NCOC(CH3)3). MS (ESI, MeOH): calc. 214.23 [M+H] ; found 214.13, Rf = 0.58 (Hex/ EtOAc 1:3)

FCUP 95 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

5.2.2. Synthesis of dodecyl (2S)-N-tert-butyloxycarbonyl -3,4-dehydro-prolinate (13)

To a solution of 12 in DCM triethylamine (NEt3) (2 eq) was added, followed by (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBoP) (1.1 eq). After 15 minutes of stirring at room temperature, dodecanol was added (1.1 eq). The mixture was stirred for 24 hours, quenched with HCl 1M and the product extracted with

DCM. The organic layer was washed with saturated NaHCO3, H2O and 1M HCl for several times until elimination of almost all 1-hydroxybenzotriazole (HOBt). The organic layer was dried with anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude was then submitted to column chromatography using Hexane:EtOAc (6:1) as eluent. Compound 13 was obtained as a colorless oil. In subsequent experiments, some changes have been introduced on the reaction conditions, namely the use of another organic base and a different coupling agent. All experiments are described in table 18.

Table 18: Synthesis of dodecyl-N-tert-butyloxycarbonyl -3,4-dehydro-prolinate (13)

12 (M12= NEt3 PyBop Dodecanol 13 (M13= 381.29 Exp. 213.23 g /mol) V/mL m/g g /mol) η % m/g (n/mmol) m/g (n/mmol) (n/mmol) (n/mmol) m/g (n/mmol) 1 0.3164 0.40 1.423 0.5211 0.1804 32 (1.484) (2.870) (2.734) (2.797) (0.4731) DBU PyBop V/mL m/g (n/mmol) (n/mmol) 2 0.2718 0.40 0.6566 0.2728 0.1014 21 (1.275) (2.674) (1.262) (1.464) (0.2659) NEt3 TBTU V/mL m/g (n/mmol) (n/mmol) 3 0.3738 0.30 0.6292 0.3593 0.2471 37 (1.753) (2.152) (1.960) (1.928) (0.6481)

1 H NMR (400 MHz, CDCl3) δ 6.01 – 5.84 (m, 1H, C(3)H), 5.77 – 5.64 (m, 1H,

C(4)H), 5.06 – 4.87 (m, 1H, C(2)H), 4.31 – 4.01 (m, 4H, C(5)H2 + -CH2-CH2-OOCR), 1.67 –

1.55 (m, 2H, -CH2-CH2-OOCR), 1.46 + 1,42 (2x s, J = 19.0 Hz, 9H, NCOC(CH3)3 ), 1.40 13 – 1.14 (m, 18H, CH3-(CH2)9-CH2-), 0.86 (t, J = 6.8 Hz, 3H, CH3-(CH2)9-CH2-). C NMR

(101 MHz, CDCl3) δ 170.79 (COO-) , 153.56 (NCOC(CH3)3), 129.38 (C(3)), 124.96 (C(4)),

80.26 (NCOC(CH3)3), 66.79 (C(2)) , 65.49 (CH2-CH2-OOCR), 53.42 (C(5)) ,32.02-22.79

(CH3-(CH2)10-CH2-) , 28.53-28.42 (NCOC(CH3)3), 14.21 (CH3-(CH2)10-CH2-). Rf = 0.59 (Hex/ EtOAc 6:1)

96 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

5.2.3. Synthesis of dodecyl (2S)-3,4-dehydro-prolinate trifluoroacetate (14)

To a solution of 13 in DCM at (0ºC) trifluoroacetic acid (TFA) (10.0 eq) was added and the mixture was left stirring at room temperature for 8h. Then, the mixture was

treated with Et2O followed by evaporation until total removal of TFA. Compound 14

precipitated from Et2O and was collected by suction filtration as a white solid. All the experiments performed are listed in table 19.

Table 19: Synthesis of dodecyl 3,4-dehydro-prolinate trifluoroacetate (14)

13 (M13= 381.29 g TFA 14 (M14 = 395.23 g /mol) Exp /mol) η % V/mL m/g (n/mmol) m/g (n/mmol)

1 0.2471 1.00 0.2280 89 (0.6481) (0.5769) 2 0.2800 1.00 0.2605 90 (0.7343) (0.6591)

Spectroscopic data of compound 14 is described below.

1 H NMR (400 MHz, CDCl3) δ 5.99 – 5.90 (m, 1H, C(3)H), 5.90 – 5.79 (m, 1H,

C(4)H), 5.18 (d, J = 2.2 Hz, 1H, C(2)H), 4.34 – 4.00 (m, 4H, C(5)H2 + -CH2-CH2-OOCR),

1.73 – 1.53 (m, 1H, -CH2-CH2-OOCR), 1.40 – 1.14 (m, 18H, CH3-(CH2)9-CH2-), 0.84 (t, J 13 = 6.9 Hz, 1H, CH3-(CH2)9-CH2-). C NMR (101 MHz, CDCl3) δ 167.69 (COO-), 127.73

(C(3)), 124.05 (C(4)), 67.76 (CH2-CH2-OOCR) 66.05 (C(2)), 52.70 (C(5)), 32.05-22.82 (CH3- + (CH2)10-CH2-), 14.24 (CH3-(CH2)10-CH2-) MS (ESI,MeOH): calc. 282.45 [M] ; found 282.53, Rf= 0.35 (DCM/MeOH 10:1)

FCUP 97 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

5.2.4. Synthesis of dodecyl (2S)-N-acetyl-3,4-dehydroprolinate (15)

The synthetic methodology followed in the synthesis of 15 has been already described in section 5.1.3. The desired product was obtained as a colorless oil (0.1506 g, 0.4656 mmol, 81% yield). The NMR data for the compound 15 is presented below:

1 H NMR (400 MHz, CDCl3) δ 6.07 – 5.94 (m, 1H, C(3)H), 5.88 – 5.72 (m, 1H,

C(4)H), 5.21 – 5.00 (m, 1H, C(2)H), 4.48 – 4.36 (m, 1H C(5)H), 4.34 – 4.20 (m, 1H, C(5)H),

4.20 – 4.04 (m, 2H, -CH2-CH2-OOCR), 2.11 + 1.99 (2x s, 3H, -NCOCH3, rot), 1.72 – 1.55

(m, 2H, -CH2-CH2-OOCR), 1.40 – 1.14 (m, 18H, CH3-(CH2)9-CH2-), 0.88 (t, J = 6.9 Hz,

3H, CH3-(CH2)9-CH2-). Rf = 0.42 (DCM/MeOH 20:1)

5.2.5. Synthesis of dodecyl (2S,3R,4S)-N-acetyl-3,4-dihydroxyprolinate (16)

. Compound 15 (0,1506 g, 0,4656 mmol) was dissolved in acetone (10 mL), N- methylmorpholine N-oxide (NMO, 3 eq) and OsO4 (0.1 mL, 2.5 wt% in tBuOH) were added. The mixture was stirred until total consumption of reagent 15 (TLC: 2h). The mixture was filtered over celite and silica and washed with acetone. After total removal of the solvent, DCM was added to the resulting crude and the solution was washed with

Na2S2O3 and dried. Then, the crude was submitted to column chromatography using DCM/MeOH 10:1. Compound 16 was obtained as a white solid (0,080 g, 0,2234 mmol, 48 % yield) The detailed description of NMR spectra for compound 16 is presented below:

98 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

1 H NMR (400 MHz, CDCl3) δ 4.44 – 4.06 (m, 5H, C(2)H + C(3)H + C(4)H + -CH2-

CH2-OOCR), 3.89 – 3.46 (m, 2H, C(5)H2 ), 2.71 (bs, 2H, OH), 2.07 + 1.95 (2x s, 3H, -

NCOCH3, rot), 1.78 – 1.50 (m, 2H, -CH2-CH2-OOCR), 1.40 – 1.14 (m, 18H, CH3-(CH2)9- 13 CH2-), 0.88 (t, J = 6.6 Hz, 1H, CH3-(CH2)9-CH2-). C NMR (101 MHz, CDCl3) δ 170.58

(COO-), 74.19 (C(3)) , 70.76 (C(4)), 65.87 (-CH2-CH2-OOCR), 64.68 (C(2)), 52.01 (C(5)),

31.91-22.68 (CH3-(CH2)10-CH2-), 21.74 (-NCOCH3), 14.11 (CH3-(CH2)10-CH2-). MS (ESI, MeOH): calc. 358.49 [M+H]+; found 358.33; 314.60 [M - COCH3], Rf = 0,34 (DCM/MeOH 10:1)

5.2.6. Synthesis of dodecyl N-methyl-3,4-dehydro- prolinate (17)

The synthetic method followed for the obtention of 17 has been already described in section 5.1.2. Compound 17 was obtained as a colorless oil (0.040 g, 0.13 mmol, 22 % yield) and its structure confirmed by NMR analysis.

1 H NMR (400 MHz, CDCl3) δ 5.89 (dq, J = 6.3, 2.1 Hz, 1H, C(3)H), 5.73 (dq, J =

6.1, 1.9 Hz, 1H, C(4)H), 4.19 – 4.07 (m, 2H, CH2-OOCR), 4.06 – 3.99 (m, 1H, C(2)H), 3.92

(ddt, J = 13.9, 5.5, 2.0 Hz, 1H, C(5)H), 3.32 (dddd, J = 13.9, 5.5, 2.5, 1.9 Hz, 1H, C(5)H),

2.56 (s, 3H, N-CH3), 1.69 – 1.58 (m, 2H, -CH2-CH2-OOCR), 1.39 – 1.14 (m, 18H, CH3- 13 (CH2)9-CH2-), 0.87 (t, J = 6.9 Hz, 3H, CH3-(CH2)10-CH2-). C NMR (101 MHz, CDCl3) δ

172.24 (COO-), 130.13 (C(3)), 126.71 (C(4)), 74.12(C(2)), 65.08 (-CH2-CH2-OOCR) , 62.01

(C(5)), 41.33 (-NCH3), 31.90-22.67 (CH3-(CH2)10-CH2-) , 14.10 (CH3-(CH2)10-CH2-). Rf = 0.50 (DCM/MeOH 10:1)

FCUP 99 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

5.2.7. Synthesis of methyl 3,4-dehydro-N-dodecyl-prolinate (19)

Compound 19 was obtained from 11b according to the procedure described in section 5.1.4. After purification process, compound 19 was obtained as a colorless oil. All the reactions performed are listed in table 20.

Table 20: Synthesis of methyl-3,4-dehydro-N-dodecyl-prolinate (19)

11b (M11b= 19 (M19= 163.60 g NEt3 CH (CH ) CHO NaBH(OAc) 295.25 g η Exp /mol) V/mL 3 2 10 3 /mol) m/g (n/mmol) m/g (n/mmol) m/g (n/mmol) m/g % (n/mmol) (n/mmol) 1 0.4794 0.50 0.5965 0.6973 0.1146 13 (2.936) (3.6) (3.236) (3.290) (0.3881) 0.2826 2 0.2489 0.35 0.3200 0.2221 49 (1.521) (2.5) (1.533) (1.509) (0.7522) 3 0.2728 0.50 0.5120 0.4214 0.2195 45 (1.667) (3.6) (2.778) (1.988) (0.7434)

The detailed description of NMR spectra for compound 19 is presented below:

1 H NMR (400 MHz, CDCl3) δ 5.92 (dq, J = 6.1, 2.0 Hz, 1H, C(3)H), 5.77 – 5.65

(dq, J =, 6.11, 1.99, 1H, C(4)H), 4.18 (ddd, J = 7.5, 5.2, 2.2 Hz, 1H, C(2)H), 3.92 (ddt, J =

14.0, 5.6, 1.9 Hz, 1H C(5)H), 3.73 (s, 3H, -OCH3), 3.42 – 3.29 (m, 1H, C(5)H), 2.87 – 2.71

(m, 1H, -N-CHH-CH2-), 2.68 – 2.54 (m, 1H, -N-CHH-CH2-), 1.54 – 1.42 (m, 2H, N-CH2-

CH2-), 1.36 – 1.17 (m, 18H, - CH2-(CH2)9-CH3), 0.87 (t, J = 6.8 Hz, 3H, -CH2-(CH2)9-CH3 13 ). C NMR (101 MHz, CDCl3) δ 173.22 (COO-), 130.26 (C(3)), 126.38 (C(4)), 73.03 (C(2)),

60.07 (C(5)), 55.33 (N-CH2-CH2-), 52.18 (-OCH3), 32.04-22.81 (CH3-(CH2)10-CH2-), 14.23 + (CH3-(CH2)10-CH2-). MS (ESI, MeOH): calc. 296,47 [M+H] ; found 296,67 Rf = 0,27 (Hex/ EtOAc 6:1) Rf = 0.71 (Hex/ EtOAc 1:3)

100 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

5.2.8. Synthesis of methyl (2S,3R,4S)-N-dodecyl-3,4-dihydroxyprolinate (20)

The reaction and purification procedures followed for the synthesis of 20 were the same as used in synthesis of 16 (section 5.2.5). After column chromatography using DCM/MeOH 10:1 as eluent, compound 20 was obtained as an orange oil. The synthesis was performed 2 times as described in table 21.

Table 21: Synthesis of methyl (2S,3R,4S)-N-dodecyl-3,4-dihydroxyprolinate (20)

Exp 19 (M19=295.25 g /mol) NMO m/g 20 (M20= 329.26 g /mol) η % m/g (n/mmol) (n/mmol) m/g (n/mmol)

1 0.1146 0.1191 0.0302 32 (0.2899) (0.1177) (0.0917) 2 0.2221 0.2299 0.0750 30 (0.7522) (2.272) (0.2278)

The detailed description of NMR spectra for compound 20 is presented below.

1 H NMR (400 MHz, CDCl3) δ 4.36 (dd, J = 8.0, 5.2 Hz, 1H, C(3)H), 4.17 – 4.10 (m,

1H, C(4)H), 3.75 (s, 3H, OCH3), 3.43 (d, J = 8.0 Hz, 1H, C(2)H), 3.12 (dd, J = 10.6, 1.4 Hz,

1H, C(5)H), 2.67 (dd, J = 10.6, 4.4 Hz, 1H, C(5)H), 2.56 (m, J = 8.0, 5.2 Hz 1H, -N-CHH-),

2.42 (dt, J = 11.8, 7.4 Hz, 1H, -N-CHH-CH2-), 1.41 (m, 2H, N-CH2-CH2- ), 1.35 – 1.14 13 (m, 18H, - CH2-(CH2)9-CH3), 0.87 (t, J = 6.8 Hz, 3H, CH2-(CH2)9-CH3). C NMR (101

MHz, CDCl3) δ 172.42 (COO-), 72.42 (C(3)), 70.60 (C(4)), 69.42 (C(2)), 58.52 (C(5)), 54.63

(N-CH2-CH2-), 51.95 (-OCH3), 31.70-22.47 (CH3-(CH2)10-CH2-), 13.89. MS (ESI, MeOH): calc. 330.48 [M+H]+; found 330.80, Rf = 0.27 (Hex/ EtOAc 1:3) FCUP 101 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

5.3. Synthesis of β-unnatural amino acid derivatives

5.3.1. Synthesis of (1R,5S)-6-azabicyclohept-3-ene-7-one (23a-b)

To a solution of 1,3-cyclopentadiene (CPD) (6 mL, bi-distilled) in diethyl ether

(Et2O) (150 mL) was added, drop by drop, a solution of chlorosulfonyl isocyanate (CSI,

22) (0,8 eq) in Et2O at - 20ºC (MeOH bath cooled with a cryocooler). After complete addition, the mixture was left stirring for 40 min. The mixture was then slowly poured into a cold solution (0ºC, ice bath) of Na2SO3 0,3 M and 20% KOH in water (200 mL). After 45 min stirring at 0ºC, the organic layer was separated, and the aqueous layer extracted with ethyl acetate (3×150 mL). The organic layers were dried with Na2SO4. After solvent removal, the crude was purified by column chromatography using Hex:EtOAc 1:3 as eluent. Compound 23 was obtained as a light-yellow oil, which turned into a white waxy solid when frozen. All the reactions performed are described in table 22.

Table 22: Synthesis of (1R,5S)-6-azabicyclohept-3-ene-7-one (23)

Exp 21 (M21= 66.1 g /mol 22 V/mL 23 (M23=109.05 g /mol) η % V/mL (n/mmol) (n/mmol) m/g (n/mmol)

1 3.00 (35.7) 2.20 (25.3) 0.5314 (4.873) 19

2 3.00 (35.7) 2.20 (25.3) 0.5058 (4.665) 19

3 3.00 (35.7) 2.20 (25.3) 0.4900 (4.493) 18

4 3.00 (35.7) 2.20 (25.3) 0.3782 (3.468) 14

5 6.00 (71.3) 4.40 (50.6) 0.6530 (4.873) 12

6 3.00 (35.7) 2.20 (25.3) 1.026 (9.409) 37

7 3.00 (35.7) 2.20 (25.3) 1.391 (12.76) 50

8 3.00 (35.7) 2.20 (25.3) 1.645 (15.09) 60

The NMR obtained for compound 22 is in accordance with that described in the literature [75]

102 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

5.3.2. Synthesis of (1R,2S)-2-aminocyclopent-3-ene-1-carboxylic acid (24a)

Racemic lactam 23 was dissolved in methyl tert-butyl ether (MTBE, 200 mL) and

H2O (1.1 eq). Lipase (2g lipase /g of substrate 23) was added and the mixture was left stirring at room temperature. After 72 hours, the mixture was filtered and washed with

MTBE. Removal of the solvent yielded 23b, which was recrystallized from Et2O yielding a needle-like crystalline yellowish solid. The solid previously filtered was thoroughly washed with hot water to liberate the free β amino acid 24a from the enzyme. The water was eliminated under reduced pressure and the solid obtained was recrystallized from water and acetone to afford a light creamy solid, 24a. All the reactions performed are listed in table 23.

Table 23: Synthesis of (1R,2S)-2-aminocyclopent-3-ene-1-carboxylic acid (24a) 23 24a 23b H2O (M15=109.05 Lipase (M24a=127.14 (M23=109.05 η % η % Exp V/mL g/mol) m/g g/mol) g/mol) (n/mmol) (24a) (23b) m/g (n/mmol) m/g (n/mmol) m/g (n/mmol) 1 1.970 0.215 4.023 0.9516 0.8507 83 86 (18.07) (11.94) (7.485) (7.779) 2 4.373 0.70 8.544 1.750 1.481 69 68 (40.10) (38.88) (13.76) (13.58) 3 4.804 1.00 9.631 2.170 1.968 77 82 (44.05) (55.56) (17.91) (18.05) 4 5.513 1.00 11.12 2.783 2.755 98 99 (50.55) (55.56) (21.89) (25.26)

NMR spectra of compound 23b and 24a are according to the literature. [75]

FCUP 103 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

5.3.3. Synthesis of (1S,2R)-2-aminocyclopent-3-ene-1-carboxylic acid hydrochloride (24b)

A solution of 10% HCl (20.0 mL) was added to 23 and the mixture stirred at room temperature for 2 hours. After removal of the water the crude was dissolved in the minimum amount of hot ethanol (EtOH) and the product precipitated through addition of

Et2O. The suspension was filtered and compound 24b was obtained as a white shiny powder. The NMR spectra for compound 24b are according to the described in literature. [75] All reactions performed are described in table 24.

Table 24: Synthesis of (1S,2R)-2-aminocyclopent-3-ene-1-carboxylic acid hydrochloride (24b)

Exp. 23 (M23=109.05 g /mol) 24b (M24b=163.04 g /mol) η % m/g (n/mmol) m/g (n/mmol)

1 0.7430 0.8421 76 (6.813) (5.147) 2 1.109 1.311 79 (10.17) (8.041) 3 0.6733 1.003 99 (6.174) (6.152) 4 0.9437 1.278 91 (8.654) (7.839)

In the following syntheses, and unless otherwise stated, enantiomerically pure AA 24a and 24b were used for the obtention of all the derivatives. In order to simplify and as the experimental procedure was the same in both cases, the description does not specify the isomers used.

104 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

5.3.4. Synthesis of 2-[(tert-butoxycarbonyl)amino]cyclopent-3-ene-1-carboxylic acid (25)

The synthetic methodology followed for the synthesis of 25 was similar to the one described in section 5.2.1. The desired product 25 was obtained as white crystals after recrystallization from EtOAc/Hexane. The reactions performed are listed in table 25.

Table 25: Synthesis of 2-[(tert-butoxycarbonyl)amino]cyclopent-3-ene-1-carboxylic acid (25)

Boc2O Starting 24 (M24=127.14 g/mol) 25 (M25= 227.12 g/mol) Exp. m/g η % m/g (n/mmol) m/g (n/mmol) material (n/mmol) 1 24a 0.4741 0.9114 0.6653 79 (3.729) (4.176) (2.929) 2 24a 0.4950 0.9271 0.5531 62 (3.893) (4.248) (2.435) 3 24a 0.9597 1.821 1.107 65 (7.548) (8.344) (4.874) 4 24b 0.7998 1.566 1.003 70 (6.291) (7.175) (4.416) 5 24b 0.9565 1.417 0.6293 37 (7.523) (6.492) (2.771) 6 24a 0.9213 2.032 1.232 75 (7.246) (9.310) (5.424) 7 24a 0.3948 0.7576 0.6761 96 (3.105) (3.471) (2.976) 8 24b 0.4895 0.7830 0.2685 31 (3.850) (3.588) (1.182) 9 24a 0.5146 0.9914 0.8165 89 (4.048) (4.542) (3.595) 10 24b 0.6332 0.9104 0.3187 29 (4.980) (4.542) (1.403) 11 24a 0.3955 0.9078 0.6603 93 (3.111) (4.159) (2.907)

The detailed description of NMR spectra for compound 25 is presented below:

1 H NMR (400 MHz, CDCl3) δ 5.92 – 5.87 (m, 1H, C(3)H), 5.61 (dq, 1H, J = 5.6, 2.4

Hz C(4)H), 5.16 (t, J = 8.7 Hz, 1H, C(2)H), 4.64 (d, J = 9.9 Hz, 1H, NH), 3.45 (q, J = 8.4

Hz, 1H, C(1)H ), 2.86 (ddq, J = 17.3, 7.1, 2.4 Hz, 1H, C(5)H), 2.50 – 2.39 (m, 1H, C(5)H), 13 1.39 (s, 9H, N-CO-C(CH3)3). C NMR (101 MHz, CDCl3) δ 167.57 (C(6)), 154.82

(NCOC(CH3)3), 133.27 (C(3)), 129.57 (C(4)), 79.68 (NCOC(CH3)3), 57.46 (C(2)), 47.31

(C(1)), 32.89 (C(5)), 28.25 (NCOC(CH3)3). Rf = 0.60 (Hex/ EtOAc 1:3) FCUP 105 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

5.3.5. Synthesis of dodecyl 2-[(tert-butoxycarbonyl)amino]cyclopent-3-ene-1- carboxylate (26)

The synthesis and purification of 26 was performed according to the procedure described in 5.2.2. using PyBoP as coupling agent and NEt3 as base. Compounds 26 was obtained as a white solid. All experiments performed are described in table 26.

Table 26: Synthesis of dodecyl 2-[(tert-butoxycarbonyl)amino]cyclopent-3-ene-1-carboxylate (26)

Starting 25 (M25= NEt3 PyBop Dodecanol 26 (M26=395.30 η Exp 227.12 g/mol) V/mL m/g g/mol) m/g (n/mmol) material m/g (n/mmol) (n/mmol) (n/mmol) m/g (n/mmol) % 1 25a 0.8140 1.00 1.867 0.6843 0.5530 39 (3.584) (7.17) (3.587) (3.672) (1.399) 2 25b 1.107 1.50 2.562 0.9147 1.149 60 (4.874) (10.8) (4.923) (4.909) (2.906) 4 25b 0.6293 0.50 1.724 0.5432 0.4108 38 (2.771) (3.59) (3.313) (2.915) (1.039) 5 25a 0.7972 0.60 1.947 0.6833 0.5689 41 (3.510) (4.30) (3.741) (3.667) (1.439) 6 25a 0.3840 0.30 0.9215 0.3562 0.3474 52 (1.691) (2.15) (1.771) (1.912) (0.8788) 7 25b 0.6761 0.50 1.524 0.5231 0.3306 28 (2.976) (3.59) (2.926) (2.807) (0.8363) 8 25a 0.8956 1.20 2.163 0.7431 0.7618 49 (3.943) (7.17) (4.156) (3.672) (1.927) 9 25a 0.8125 0.60 1.864 0.6832 0.5781 41 (3.577) (4.30) (3.582) (3.666) (1.462) 10 25a 0.7274 1.00 1.613 0.6210 0.6506 51 (3.203) (7.17) (3.010) (3.333) (1.646)

Characterization data of compound 26 is presented below.

1 H NMR (400 MHz, CDCl3) δ 5.91 – 5.86 (m, 1H, C(3)H), 5.63 – 5.59 (m, 1H,

C(4)H), 5.06 (t, J = 8.4 Hz, 1H, C(2)H,), 4.68 (d, J = 9.2 Hz, 1H,NH), 4.11 (dt, 1H, J = 10.7,

7.1 Hz, COO-CHH-CH2-), 4.00 (dt, 1H, J = 10.7, 6.9 Hz, COO-CHH-CH2-) 3.35 (q, J =

8.5 Hz, 1H, C(1)H), 2.80 (ddq, J = 17.1, 6.5, 2.4 Hz, 1H, C(5)H), 2.60 – 2.42 (m, 1H, C(5)H),

1.75 – 1.55 (m, 2H, -CH2-CH2-OOCR ), 1.42 (s, 9H, N-CO-C(CH3)3), 1.37 – 1.17 (m, J = 13 17.5 Hz, 18H, CH3-(CH2)9-CH2 0.87 (t, J = 6.9 Hz, 3H, CH3-(CH2)9-CH2). C NMR (101

MHz, CDCl3) δ 173.68 (C(6)), 155.32 (NCOC(CH3)3), 133.44 (C(3)), 130.46 (C(4)), 65.45,

(-CH2-CH2-OOCR), 58.02 (C(2)), 46.55 (C(1)), 34.94 (C(5)), 32.37- 23.14 (CH3-(CH2)10-

CH2-), 29.97 (NCOC(CH3)3), 14.56 (CH3-(CH2)11-OOCR) . MS (ESI,MeOH): calc. 396.58 [M+H]+; found 396.53 Rf = 0.62 (Hex/ EtOAc 6:1)

106 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

5.3.6. Synthesis of 5-[(dodecyloxy)carbonyl]cyclopent-2-en-1-ammonium trifluoroacetate (27)

Compound 27 was obtained according to the procedure described in section

5.2.3. Compound 27 precipitated from Et2O and was collected by suction filtration as a white solid. All the experiments performed are listed in table 27.

Table 27: Synthesis of 5-[(dodecyloxy)carbonyl]cyclopent-2-en-1-amonium trifluoroacetate (27)

Starting Exp 26 (M26=395.30 g /mol) TFA 27 (M27=409.24 g /mol) η % Material m/g (n/mmol) V/mL m/g (n/mmol) 1 26a 0.5531 2.00 0.5663 99 (1.399) (1.384) 2 26a 1.149 3.00 1.352 >100* (2.907) (3.304) 3 26b 0.4108 2.00 0.4203 99 (1.039) (1.027) 4 26a 0. 5689 2.00 0.6727 >100* (1.439) (1.644) 5 26b 0. 3474 1.00 0.4093 >100* (0.8788) (1.000) 6 26a 0. 3306 1.00 0.3210 94 (0.8363) (0.7844) *- excess of solvent

The spectroscopic data of compound 27 is described below.

1 H NMR (400 MHz, CDCl3) δ 6.27 – 6.12 (m, 1H, C(3)H), 5.91 – 5.85 (m, 1H,

C(4)H), 4.45 (d, J = 7.2 Hz, 1H, C(2)H), 4.21 – 4.02 (m, 2H, CH2-CH2-OOCR), 3.33 (q, J =

8.3 Hz, 1H, C(1)H), 2.91 – 2.81 (m, 1H, C(5)HH) 2.80 – 2.69 (m, 1H, C(5)HH), 1.74 – 1.54

(m, 2H, CH2-CH2-OOCR), 1.40 – 1.12 (m, 18H, CH3-(CH2)9-CH2-), 0.88 (t, J = 6.9 Hz, 13 3H, CH3-(CH2)9-CH2-) C NMR (101 MHz, CDCl3) δ 173.05 (C(6)), 137.82 (C(3)), 127.19

(C(4)), 66.07 (-CH2-CH2-OOCR), 56.20 (C(2)), 43.38 (C(1)), 34.99 (C(5)), 32.05-22.82 (CH3- + (CH2)10-CH2-OOCR) , 14.23 (CH3-(CH2)11-OOCR) MS (ESI,MeOH): calc. 296.47 [M] ; found 296.27 Rf = 0.40 (DCM/MeOH 10:1)

FCUP 107 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

5.3.7. Synthesis of dodecyl 2-(dimethylamino)cyclopent-3-ene-1-carboxylate (28)

To a solution of 27 in DCE (10 mL) triethylamine (2 eq.) was added and the mixture was stirred for 15 min. Then formaldehyde 37 % wt (2 eq.) was added followed by sodium triacetoxyborohydride (3 eq.). The reaction was allowed to proceed at r.t. for 12 h. The solution was washed with water (20 ml, 3x) and after separation the organic layer was dried with Na2SO4. The solvent was removed, and the crude product was subjected to column chromatography on silica gel using hexane/ethyl acetate 1:3 as eluent to yield compound 28 as a light red oil. All experiments are described in table 28.

Table 28: Synthesis of dodecyl 2-(dimethylamino)cyclopent-3-ene-1-carboxylate (28)

27 28 NEt3 HCHO Starting (M27=409.24 NaBH(OAc) (M28=323.28 η Exp. V/mL V/mL 3 g /mol) m/g (n/mmol) g /mol) Material (n/mmol) (n/mmol) % m/g (n/mmol) m/g (n/mmol) 1 27a 0.5604 0.20 0.30 0.6946 0.1151 26 (1.369) (1.4) (3.4) (3.277) (0.3560) 2 27b 0.4200 0.30 0.30 0.5286 0.1056 32 (1.026) (2.2) (3.4) (2.494) (0.3267) 3 27b 0.1184 0.10 0.10 0.1345 0.036 39 (0.2893) (0.72) (1.1) (0.6346) (0.1136) 4 27a 0.6727 0.45 0.30 0.7104 0.1201 23 (1.644) (3.2) (3.4) (3.352) (0.3715)

The NMR data for compound 28 is presented below.

1 H NMR (400 MHz, CDCl3) δ 6.01 – 5.96 (m, 1H, C(3)H ), 5.75 – 5.69 (m, 1H, C(4)H), 4.22

– 4.01 (m, 2H, CH2-CH2-OOCR), 4.00 – 3.94 (m , 1H, C(2)H ), 3.17 (q, J = 8.7 Hz, 1H,

C(1)H), 2.84 (ddq, J = 16.9, 8.2, 2.5 Hz, 1H, C(5)H), 2.36 (dddd, J = 16.9, 8.7, 2.6, 1.9 Hz,

1H, C(5)H), 2.22 (s, 6H, N(CH3)2), 1.69 – 1.53 (m, 2H, CH2-CH2-OOCR), 1.45 – 1.08 (m,

18H, CH3-(CH2)9-CH2-), 0.88 (t, J = 6.8 Hz, 3H, CH3-(CH2)9-CH2-). Rf = 0.51 (Hex/ EtOAc 1:3)

108 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

5.3.8. Synthesis of dodecyl 2-(dimethylamino)-3,4-dihydroxycyclopentane-1-carboxylate

(29)

The procedure followed for the obtention of 29 is the same as described in section 5.2.5. The product was submitted to column chromatography using DCM/MeOH 10:1 as eluent. An orange oil was obtained and identified (NMR) as the target compound 29. All the reactions performed as listed in table 29.

Table 29: Synthesis of dodecyl 2-(dimethylamino)-3,4-dihydroxycyclopentane-1-carboxylate (29)

28 (M28=323.28 29 (M29= 357.39 Starting NMO m/g Exp. g/mol) g/mol) η % (n/mmol) Material m/g (n/mmol) m/g (n/mmol)

1 28a 0.1151 0.1446 0.030 24 (0.3560) (1.429) (0.084) 2 28b 0.1293 0.1304 0.045 31 (0.4000) (1.289) (0.13) 3 28a 0.1201 0.1245 0.060 45 (0.3715) (1.429) (0.17)

1 H NMR (400 MHz, CDCl3) δ 4.42 (dt, J = 6.9, 4.7 Hz, 1H, C(4)H), 4.30 (dd, J = 8.7, 6.8

Hz, 1H, C(3)H), 4.13 – 3.99 (m, 2H, CH2-CH2-OOCR), 3.11 (td, J = 8.0, 2.2 Hz, 1H, C(2)H),

2.80 – 2.68 (bs, 2H, OH), 2.73 (dd, J = 8.5, 7.8 Hz, 1H, C(1)H), 2.40 (s, 6H, N(CH3)2),

2.37 – 2.29 (m, 1H, C(5)H ), 1.80 (ddd, J = 14.4, 8.3, 4.6 Hz, 1H, C(5)H), 1.66 – 1.57 (m,

2H, CH2-CH2-OOCR), 1.39 – 1.20 (m, 18H, CH3-(CH2)9-CH2-), 0.88 (t, J = 6.8 Hz, 3H, + CH3-(CH2)9-CH2-). MS (ESI,MeOH): calc. 358.54 [M+H] ; found 358.39. Rf = 0.25 (Hex/ EtOAc 1:3)

FCUP 109 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

5.3.9. Synthesis of dodecyl 2-[(tert-butoxycarbonyl)amino]-3,4-dihydroxycyclopentane- 1-carboxylate (30)

The procedure followed for the obtention of 30 is the same as described in section

5.2.5. The resulting product (30) was recrystallized from Et2O to obtain a pale orange solid. All experiments are described in table 30.

Table 30: Synthesis of dodecyl 2-((tert-butoxycarbonyl)amino)-3,4-dihydroxycyclopentane-1-carboxylate (30)

26 (M26=395.30 30 (M30= 427.50 Starting NMO m/g Exp. g/mol) g/mol) η % (n/mmol) Material m/g (n/mmol) m/g (n/mmol)

1 26a 0.7618 0.5925 0.4603 56 (1.927) (5.858) (1.077) 2 26b 0.4606 0.3753 0.3148 63 (1.165) (3.710) (0.7364) 3 26a 0.6506 0.5122 0.6001 85 (1.646) (5.064) (1.404)

The detailed description of NMR spectra for compound 30 is presented below

1 H NMR (400 MHz, CDCl3) δ 5.58 (s, 1H, NH), 4.17 (d, J = 5.8 Hz, 2H, C(4)H + C(3)H),

4.09 (t, J = 6.7 Hz, 2H, CH2-CH2-OOCR), 4.00 (dd, J = 8.2, 4.6 Hz, 1H, C(2)H ), 3.29 (q,

9.0 Hz, 1H, C(1)H), 2.22 – 2.18 (m, 1H, C(5)H), 2.14 – 2.03 (m, 1H, C(5)H), 1.68 – 1.56 (m,

2H, CH2-CH2-OOCR ), 1.44 (s, 9H, N-CO-C(CH3)3 ), 1.37 – 1.17 (m, 18H, CH3-(CH2)9- 13 CH2-), 0.88 (t, J = 6.8 Hz, 3H, CH3-(CH2)9-CH2-). C NMR (101 MHz, CDCl3) δ 174.53

(C(6)), 157.53(NCOC(CH3)3), 79,00 (C(3)), 70.31 (C(4)), 65.51 (CH2-CH2-OOCR), 56.92

(C(2)), 42.09 (C(1)), 33.94 (C(5)), 32.05-22.82 ( CH3-(CH2)10-CH2-OOCR-) , 26.01 + (NCOC(CH3)3), 14.25 (CH3-(CH2)11-OOCR-). MS (ESI,MeOH): calc. 430.60 [M+H] ; found 430.53. Rf = 0.57 (Hex/ EtOAc 1:3)

110 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

5.3.10. Synthesis of 5-[(dodecyloxy)carbonyl]-2,3-dihydroxycyclopentan-1-ammonium trifluroacetate (31)

The synthetic procedure followed for the obtention of 31 was the same as described in section 5.2.3. Compound 31 was obtained as a white solid. All the reactions performed are listed in table 31.

Table 31: Synthesis of 5[(dodecyloxy)carbonyl]-2,3-dihydroxycyclopentan-1-ammonium trifluroacetate (31)

Starting 30 (M30= 427.50 TFA 31 (M31= 443.25 Exp g/mol) g/mol) η % V/mL Material m/g (n/mmol) m/g (n/mmol)

1 30a 0.4602 2.00 0.3180 67 (1.076) (0.7174) 2 30b 0.3418 2.00 0.1922 54 (0.7995) (0.4336) 3 30a 0.6001 2.00 0. 3541 57 (1.404) (0.7989)

The detailed description of NMR spectra is presented below.

1 H NMR (400 MHz, CDCl3) δ 4.27 – 3.93 (m, 4H, C(4)H + C(3)H + CH2-CH2-OOCR),

3.67 (t, J = 8.7 Hz, 1H, C(2)H), 3.32 (q, J = 8.8 Hz, 1H, C(1)H), 2.24 – 2.06 (m, 1H, C(5)H2),

1.68 – 1.48 (m, 1H, -CH2-CH2-OOCR), 1.40 – 1.03 (m, 18H, CH3-(CH2)9-CH2-), 0.84 (t, J 13 = 6.8 Hz, 3H, CH3-(CH2)9-CH2-). C NMR (101 MHz, CDCl3) δ 173.64 (C(6)), 75.16 (C(3)),

69.23 (C(4)), 65.81(CH2-CH2-OOCR), 55.20 (C(2)), 39.00 (C(1)), 34.07 (C(5)) , 31.75-22.52

82 ( CH3-(CH2)10-CH2-OOCR-), 13.89 (CH3-(CH2)11-OOCR-) MS (ESI,MeOH): calc. 330.49 [M]+; found 330.80. Rf = 0.20 (DCM/MeOH 10:1)

5.3.11. Synthesis of dodecyl 2-acetamido-3,4-dihydroxycyclopentane-1-carboxylate (32a)

FCUP 111 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Compound 32 was obtained according to the procedure described in section 5.1.3. After the work up, the crude was subjected to column chromatography using DCM/MeOH 10:1 as eluent. Compound 32 was obtained as white solid. All the reactios performed as listed in table 32.

Table 32: Synthesis of dodecyl 2-acetamido-3,4-dihydroxycyclopentane-1-carboxylate (32)

Starting 31 (M31= 443.25 NEt3 CH COCl 32 (M32= η Exp g /mol) V/mL 3 371.27 g /mol) Material V/mL (n/mmol) % m/g (n/mmol) (n/mmol) m/g (n/mmol) 0.20 0.1363 1 31a 0.1922 (0.4336) 0.04 (0.6) 85 (1.4) (0.3671) 0.10 0.0740 2 31b 0.1260 (0.2842) 0.04 (0.6) 70 (0.72) (0.199)

The detailed description of NMR spectra of 32 is presented below.

1 H NMR (400 MHz, CDCl3) δ 7.11 (d, J = 5.6 Hz, 1H, NH), 4.44 – 4.35 (m, 1H,

C(3)H ), 4.17 – 4.12 (m, 1H, C(4)H), 4.15 – 4.08 (m, 2H, -CH2-CH2-OOCR), 4.01 (dd, J =

8.3, 4.6 Hz, 1H, C(2)H), 3.36 (q, J = 8.9 Hz, 1H, C(1)H), 3.28 (bs, 2H, OH), 2.27 – 2.13 (m,

2H, C(5)H2), 2.05 (s, 3H, -NCOCH3), 1.72 – 1.57 (m, 2H, -CH2-CH2-OOCR), 1.42 – 1.19 13 (m, 18H, CH3-(CH2)9-CH2-), 0.90 (t, J = 6.9 Hz, 3H, CH3-(CH2)9-CH2-). C NMR (101

MHz, CDCl3) δ 175.01 (C(6)), 172.67 (NCOCH3) , 79.81 (C(3)), 70.35 (C(4)), 65.64 (CH2-

CH2-OOCR), 56.48 (C(2)), 41.23 (C(1)), 34.61 (C(5)), 32.04-26.01+22.82 (CH3-(CH2)10-

CH2-OOCR-), 23.35 (-NCOCH3), 14.25 (CH3-(CH2)11-OOCR-). MS (ESI,MeOH): calc. 372.52 [M+H] +; found 372.60 Rf = 0.60 (DCM/MeOH 10:1)

5.3.12. Synthesis of methyl (1R,2S)-2-aminocyclopent-3-ene-1-carboxylate hydrochloride (33)

β-Amino acid 24a (0.4411 g, 3.472 mmol) was mixed in TMSCl (2 eq) and left to stirr for 15 minutes. Methanol was added to the suspension and the mixture was allowed to react for 12 hours, at rt. Then, the solvent was removed on a rotary evaporator to yield the methyl ester derivative 33 (0.6025 g, 3.391 mmol, 97 %) as HCl salt. Compound 33 was characterized by MS-ESI.

MS (ESI,MeOH): calc. 142.18 [M] +; found 142.13

112 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

5.3.13. Synthesis of methyl (1R,2S)-2-dodecanamidocyclopent-3-ene-1-carboxylate (34)

Compound 33 (0.6025 g, 3.403 mmol) was dissolved in DCM and 2 eq of NEt3 were added. The solution was placed in an ice bath at 0ºC and dodecyl chloride was added dropwise for one hour. The mixture was stirred for 12 hours until no visible change in the reaction was observed (TLC). The reaction mixture was quenched with HCl 1M and the product extracted with DCM. The organic extract was dried and, after solvent evaporation, the resulting crude was subjected to column chromatography using Hex/AcoEt 3:1 as eluent. Compound 34 was obtained as a white solid. (0.6329 g, 1.957 mmol, 57% yield) The detailed description of NMR spectra for compound 34 is presented below:

1 H NMR (400 MHz, CDCl3) δ 5.96 – 5.87 (m, J = 5.8, 3.9, 2.3 Hz, 1H, C(3)H), 5.65

(d, J = 9.1 Hz, 1H, NH), 5.62 – 5.56 (m, 1H, C(4)H), 5.42 – 5.32 (m, J = 7.2, 2.8, 1.9 Hz,

1H, C(2)H), 3.65 (s, 3H, C(6)OOCH3), 3.39 (m, 1H, C(1)H), 2.85 – 2.75 (m, 1H, C(5)H), 2.61

– 2.46 (m, 1H, C(5)H), 2.09 (td, J = 7.5, 3.5 Hz, 1H, -NH-CO-CH2-CH2-), 1.61 – 1.50 (m,

1H, -CH2-CH2-CONR), 1.32 – 1.17 (m, 16H, CH3-(CH2)8-CH2- ), 0.87 (t, J = 6.9 Hz, 3H, 13 CH3-(CH2)9-CH2-). C NMR (101 MHz, CDCl3) δ 174.05 (C(6)), 172.56 (NCOCH2-),

133.46 (C(3)), 130.00 (C(4)), 55.81 (C(2)), 51.94 (COOCH3), 45.82 (C(1)), 37.04 (C(5)),

35.04-22.80 (CH3-(CH2)10-CONHR-), 14.23 (CH3-(CH2)10-CONHR-) . MS (ESI,MeOH): calc. 324.48 [M+H] +; found 324.09. Rf = 0.23 (Hex/ EtOAc 3:1) FCUP 113 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

5.3.14. Synthesis of methyl (1R,2R,3S,4R)-2-dodecanamido-3,4- dihydroxycyclopentane-1-carboxylate (35)

The synthetic procedure followed for the obtention of 35 has been already described in section 5.2.5. The crude obtained was purified by column chromatography using Hex/AceOt 1:3 as eluent and 35 was obtained as a white solid (0.4658 g, 1.303 mmol, 66 % yield). The characterization of 35 by NMR is described below.

1 H NMR (400 MHz, CDCl3) δ 6.95 (d, J = 6.2 Hz, 1H, NH), 4.47 – 4.30 (m, 1H,

C(3)H ), 4.14 (td, J = 4.5, 2.5 Hz, 1H, C(4)H), 3.97 (dd, J = 8.2, 4.6 Hz, 1H, C(2)H), 3.71 (s,

3H, COOCH3), 3.37 (q, J = 8.8 Hz, 1H, C(1)H ), 2.26 – 2.07 (m, 4H, C(5)H2 + -CH2-CONHR-

), 1.61 – 1.50 (m, 2H, CH2-CH2-CONHR), 1.35 – 1.13 (m, 16H, CH3-(CH2)8- CH2-), 0.87 13 (t, J = 6.9 Hz, 3H, CH3-(CH2)9-CH2-). C NMR (101 MHz, CDCl3) δ 176.00 (C(6)), 175.77

(NCOCH2-), 80.04 (C(3)), 70.72 (C(4)), 56.53 (C(2)), 52.67 (COOCH3), 41.52 (C(1)), 37.09

(C(5)), 34.84-23.13 (CH3-(CH2)10-CONHR-), 14.55 (CH3-(CH2)10-CONHR-). MS (ESI,MeOH): calc. 358.49 [M+H] +; found 358.80 Rf = 0.29 (Hex/ EtOAc 1:3)

FCUP 115 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

References

1. Prausnitz, M.R. and R. Langer, Transdermal drug delivery. Nat Biotechnol, 2008. 26(11): p. 1261-8. 2. Alexander, A., et al., Approaches for breaking the barriers of drug permeation through transdermal drug delivery. Journal of Controlled Release, 2012. 164(1): p. 26-40. 3. Prausnitz, M.R., Mitragotri, S., and Langer, R., Current status and future potential of transdermal drug delivery. Nature Reviews Drug Discovery, 2004. 3(2): p. 115-124. 4. Barry, B.W., Novel mechanisms and devices to enable successful transdermal drug delivery. European Journal of Pharmaceutical Sciences, 2001. 14(2): p. 101-114. 5. Janusova, B., et al., Amino acid derivatives as transdermal permeation enhancers. Journal of Controlled Release, 2013. 165(2): p. 91-100. 6. Zheng, L., et al., Novel skin permeation enhancers based on amino acid ester ionic liquid: Design and permeation mechanism. International Journal of Pharmaceutics, 2020. 576: p. 119031. 7. Menon, G.K., New insights into skin structure: scratching the surface. Advanced Drug Delivery Reviews, 2002. 54: p. S3-S17. 8. Hansen, S., Lehr, C.M., and Schaefer, U.F., Modeling the human skin barrier- Towards a better understanding of dermal absorption Preface. Advanced Drug Delivery Reviews, 2013. 65(2): p. 149-151. 9. Kapoor, M.S., GuhaSarkar, S., and Banerjee, R., Stratum corneum modulation by chemical enhancers and lipid nanostructures: implications for transdermal drug delivery. Therapeutic Delivery, 2017. 8(8): p. 701-718. 10. Schmitt, T. and Neubert, R.H.H., State of the art in Stratum Corneum research: The biophysical properties of ceramides. Chemistry and Physics of Lipids, 2018. 216: p. 91-103. 11. Forslind, B., A domain mosaic model of the skin barrier. Acta Derm Venereol, 1994. 74(1): p. 1-6. 12. Bouwstra, J.A., et al., Structure of the skin barrier and its modulation by vesicular formulations. Prog Lipid Res, 2003. 42(1): p. 1-36. 13. Norlén, L., Skin Barrier Formation: The Membrane Folding Model. Journal of Investigative Dermatology, 2001. 117(4): p. 823-829. 14. Benson, H.A.E., Skin Structure, Function, and Permeation. Topical and Transdermal Drug Delivery, 2011: p. 1-22. 15. Lemos, C.N., et al., Chapter 6 Nanoparticles influence in skin penetration of drugs In vitro and in vivo characterization, in Nanostructures for the Engineering of Cells, Tissues and Organs. 2018. p. 187-248. 16. Barry, B.W., Drug delivery routes in skin: a novel approach. Advanced Drug Delivery Reviews, 2002. 54: p. S31-S40. 17. Barry, B.W., Mode of action of penetration enhancers in human skin. Journal of Controlled Release, 1987. 6(1): p. 85-97. 18. Marwah, H., et al., Permeation enhancer strategies in transdermal drug delivery. Drug Delivery, 2016. 23(2): p. 564-578. 19. Zsikó, S., et al., Methods to Evaluate Skin Penetration In Vitro. Scientia Pharmaceutica, 2019. 87: p. 19. 20. Bartosova, L. and Bajgar, J., Transdermal Drug Delivery In Vitro Using Diffusion Cells. Current Medicinal Chemistry, 2012. 19(27): p. 4671-4677. 21. Godin, B. and Touitou, E., Transdermal skin delivery: Predictions for humans from in vivo, ex vivo and animal models. Advanced Drug Delivery Reviews, 2007. 59(11): p. 1152-1161.

116 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

22. Kovacik, A., Kopecna, M., and Vavrova, K., Permeation enhancers in transdermal drug delivery: benefits and limitations. Expert opinion on drug delivery, 2020: p. 1-12. 23. Barry, B.W., Lipid-Protein-Partitioning theory of skin penetration enhancement. Journal of Controlled Release, 1991. 15(3): p. 237-248. 24. Schroeter, A., et al., Penetration Enhancers and Their Mechanism Studied on a Molecular Level, in Percutaneous Penetration Enhancers Chemical Methods in Penetration Enhancement: Modification of the Stratum Corneum, N. Dragicevic and H.I. Maibach, Editors. 2015, Springer Berlin Heidelberg: Berlin, Heidelberg. p. 29-37. 25. Haq, A., Chandler, M., and Michniak-Kohn, B., Solubility-physicochemical- thermodynamic theory of penetration enhancer mechanism of action. Int J Pharm, 2020. 575: p. 118920. 26. Tuan-Mahmood, T.M., et al., Microneedles for intradermal and transdermal drug delivery. Eur J Pharm Sci, 2013. 50(5): p. 623-37. 27. Schoellhammer, C.M., Blankschtein, D., and Langer, R., Skin permeabilization for transdermal drug delivery: recent advances and future prospects. Expert Opinion on Drug Delivery, 2014. 11(3): p. 393-407. 28. Maibach, H., Dermatological formulations: Percutaneous absorption. By Brian W. Barry. Marcel Dekker, 270 Madison Avenue, New York, NY 10016. 1983. 479 pp. 16 × 23.5 cm. Price $55.00 (2070 higher outside the US. and Canada). Journal of Pharmaceutical Sciences, 1984. 73(4): p. 573-573. 29. Amjadi, M., Mostaghaci, B., and Sitti, M., Recent Advances in Skin Penetration Enhancers for Transdermal Gene and Drug Delivery. Curr Gene Ther, 2017. 17(2): p. 139-146. 30. Dragicevic, N. and Maibach, H.I., Percutaneous Penetration Enhancers Chemical Methods in Penetration Enhancement, Nanocarriers. 2016. 1-384. 31. Monti, D., et al., Influence of a Combination of Chemical Enhancers and Iontophoresis on In Vitro Transungual Permeation of Nystatin. AAPS PharmSciTech, 2018. 19(4): p. 1574-1581. 32. Lane, M.E., Skin penetration enhancers. International Journal of Pharmaceutics, 2013. 447(1-2): p. 12-21. 33. Williams, A.C. and Barry, B.W., Penetration enhancers. Adv Drug Deliv Rev, 2004. 56(5): p. 603-18. 34. Dragicevic, N. and Maibach, H.I., Percutaneous penetration enhancers chemical methods in penetration enhancement: Drug manipulation strategies and vehicle effects. 2015. 1-341. 35. Karande, P., et al., Design principles of chemical penetration enhancers for transdermal drug delivery. Proceedings of the National Academy of Sciences of the United States of America, 2005. 102(13): p. 4688-4693. 36. Vávrová, K. and Hrabálek, A., Amino Acid-Based Transdermal Penetration Enhancers. 2016. p. 325-336. 37. Silva, S.M.C., et al., Structure Activity Relationships in Alkylammonium C12- Gemini Surfactants Used as Dermal Permeation Enhancers. The AAPS Journal, 2013. 15(4): p. 1119-1127. 38. Vávrová, K., et al., Synthetic ceramide analogues as skin permeation enhancers: structure-activity relationships. Bioorg Med Chem, 2003. 11(24): p. 5381-90. 39. Vávrová, K., Zbytovská, J., and Hrabálek, A., Amphiphilic transdermal permeation enhancers: structure-activity relationships. Curr Med Chem, 2005. 12(19): p. 2273-91. 40. Karande, P., Jain, A., and Mitragotri, S., Discovery of transdermal penetration enhancers by high-throughput screening. Nat Biotechnol, 2004. 22(2): p. 192-7. FCUP 117 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

41. Karande, P. and Mitragotri, S., High throughput screening of transdermal formulations. Pharm Res, 2002. 19(5): p. 655-60. 42. Arora, A., et al., Multicomponent chemical enhancer formulations for transdermal drug delivery: More is not always better. Journal of Controlled Release, 2010. 144(2): p. 175-180. 43. Novotny, J., et al., Dimethylamino acid esters as biodegradable and reversible transdermal permeation enhancers: effects of linking chain length, chirality and polyfluorination. Pharm Res, 2009. 26(4): p. 811-21. 44. Rambharose, S., et al., Novel mono, di and tri-fatty acid esters bearing secondary amino acid ester head groups as transdermal permeation enhancers. New Journal of Chemistry, 2018. 42(3): p. 2232-2242. 45. Teixeira, R.S., et al., Novel serine-based gemini surfactants as chemical permeation enhancers of local anesthetics: A comprehensive study on structure–activity relationships, molecular dynamics and dermal delivery. European Journal of Pharmaceutics and Biopharmaceutics, 2015. 93: p. 205- 213. 46. Dahlizar, S., et al., Combined Use of N-Palmitoyl-Glycine-Histidine Gel and Several Penetration Enhancers on the Skin Permeation and Concentration of Metronidazole. Pharmaceutics, 2018. 10(4). 47. Fix JA, P.S., Lysine esters used as absorption enhancing agents EP-0162747. 1985. 48. Wong, O., et al., New Alkyl N,N-Dialkyl-Substituted Amino Acetates as Transdermal Penetration Enhancers. Pharmaceutical Research, 1989. 6(4): p. 286-295. 49. Büyüktimkin, S., Büyüktimkin, N., and Rytting, J.H., Synthesis and enhancing effect of dodecyl 2-(N,N-dimethylamino)propionate on the transepidermal delivery of indomethacin, clonidine, and hydrocortisone. Pharmaceutical research, 1993. 10(11): p. 1632-1637. 50. Doležal, P., Hrabálek, A., and Semecký, V., ε-Aminocaproic Acid Esters as Transdermal Penetration Enhancing Agents. Pharmaceutical Research, 1993. 10(7): p. 1015-1019. 51. Hrabálek, A., et al., Synthesis and Enhancing Effect of Transkarbam 12 on the Transdermal Delivery of Theophylline, Clotrimazole, Flobufen, and Griseofulvin. Pharmaceutical Research, 2006. 23(5): p. 912-919. 52. Fincher, T.K., et al., In Vitro Evaluation of a Series of N-Dodecanoyl-l-Amino Acid Methyl Esters as Dermal Penetration Enhancers. Journal of Pharmaceutical Sciences, 1996. 85(9): p. 920-923. 53. Tenjarla, S.N., et al., Synthesis and evaluation of N-acetylprolinate esters — novel skin penetration enhancers. International Journal of Pharmaceutics, 1999. 192(2): p. 147-158. 54. Md Moshikur, R., et al., Design and Characterization of Fatty Acid-Based Amino Acid Ester as a New “Green” Hydrophobic Ionic Liquid for Drug Delivery. ACS Sustainable Chemistry & Engineering, 2020. 8(36): p. 13660-13671. 55. Harris, W.T., et al., n‐PentylN‐Acetylprolinate. A New Skin Penetration Enhancer. Journal of Pharmaceutical Sciences, 1995. 84(5): p. 640-642. 56. Vávrová, K., et al., l-Serine and glycine based ceramide analogues as transdermal permeation enhancers: polar head size and hydrogen bonding. Bioorganic & Medicinal Chemistry Letters, 2003. 13(14): p. 2351-2353. 57. Teixeira, R.S., et al., Lysine-based surfactants as chemical permeation enhancers for dermal delivery of local anesthetics. International Journal of Pharmaceutics, 2014. 474(1): p. 212-222. 58. Muzzalupo, R., et al., Pharmaceutical versatility of cationic niosomes derived from amino acid-based surfactants: Skin penetration behavior and controlled

118 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

drug release. International Journal of Pharmaceutics, 2017. 529(1-2): p. 245- 252. 59. Dolezal, P., Hrabálek, A., and Semecký, V., epsilon-Aminocaproic acid esters as transdermal penetration enhancing agents. Pharm Res, 1993. 10(7): p. 1015-9. 60. Infante, M.R., et al., Amino acid-based surfactants. Comptes Rendus Chimie, 2004. 7(6): p. 583-592. 61. Okasaka, M., et al., Evaluation of anionic surfactants effects on the skin barrier function based on skin permeability. Pharmaceutical Development and Technology, 2019. 24(1): p. 99-104. 62. Lundborg, M., et al., Predicting drug permeability through skin using molecular dynamics simulation. Journal of Controlled Release, 2018. 283: p. 269-279. 63. Hikima, T., et al., Skin accumulation and penetration of a hydrophilic compound by a novel gemini surfactant, sodium dilauramidoglutamide lysine. International Journal of Pharmaceutics, 2013. 443(1): p. 288-292. 64. Bordes, R. and Holmberg, K., Amino acid-based surfactants – do they deserve more attention? Advances in Colloid and Interface Science, 2015. 222: p. 79- 91. 65. Isidro-Llobet, A., Álvarez, M., and Albericio, F., Amino Acid-Protecting Groups. Chemical Reviews, 2009. 109(6): p. 2455-2504. 66. Abdel-Magid, A.F., et al., Reductive Amination of Aldehydes and Ketones with Sodium Triacetoxyborohydride. Studies on Direct and Indirect Reductive Amination Procedures1. The Journal of Organic Chemistry, 1996. 61(11): p. 3849-3862. 67. Juaristi, E., Structural Types of Relevant β‐Amino Acid Targets. 2005. p. 1-17. 68. Kuhl, A., et al., Alicyclic β-amino acids in Medicinal Chemistry. Amino acids, 2005. 29: p. 89-100. 69. Spiteller, P. and von Nussbaum, F., β‐Amino Acids in Natural Products. Cheminform, 2005. 36: p. 19-91. 70. FÜLÖP, F., The Chemistry of 2-Aminocyclopentanecarboxylic Acid, in Studies in Natural Products Chemistry, R. Atta ur, Editor. 2000, Elsevier. p. 273-306. 71. Miller, J. and S. Nguyen, The Enantioselective Synthesis of Conformationally Constrained Cyclic - Amino Acids. Mini-Reviews in Organic Chemistry, 2005. 2: p. 39-45. 72. Benedek, G., et al., Efficient Synthesis of Hydroxy-Substituted Cispentacin Derivatives. European Journal of Organic Chemistry, 2008. 2008(21): p. 3724- 3730. 73. Kristensen, T.E., The Selective O-Acylation of Hydroxyproline as a Convenient Method for the Large-Scale Preparation of Novel Proline Polymers and Amphiphiles. European Journal of Organic Chemistry, 2009. 2009(3): p. 387- 395. 74. Hu, D.X., Grice, P., and Ley, S.V., Rotamers or Diastereomers? An Overlooked NMR Solution. The Journal of Organic Chemistry, 2012. 77(11): p. 5198-5202. 75. Silva Pereira, E.C., Exploring chirality of unnatural amino acids and peptides on water sorption and stratum corneum uptake. 2019, University of Reading. 76. Coll, M., et al., Theoretical Study of the Alkaline Hydrolysis of a Bicyclic Aza-β- lactam. The Journal of Physical Chemistry B, 2000. 104(47): p. 11389-11394. 77. Li, C., et al., Analysis of the conformational stability and activity of Candida antarctica lipase B in organic solvents: insight from molecular dynamics and quantum mechanics/simulations. J Biol Chem, 2010. 285(37): p. 28434-41. 78. Hofmann, A.W.V. and Clark, J., XIV Researches into the molecular constitution of the organic bases. Philosophical Transactions of the Royal Society of London, 1851. 141: p. 357-398. FCUP 119 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

79. Li, J. and Sha, Y., A convenient synthesis of amino acid methyl esters. Molecules (Basel, Switzerland), 2008. 13(5): p. 1111-1119. 80. Silva, S.G., et al., Towards novel efficient monomeric surfactants based on serine, tyrosine and 4-hydroxyproline: synthesis and micellization properties. Tetrahedron, 2009. 65(21): p. 4156-4164. 81. Araújo, A., et al., Determination of the melting temperature, heat of fusion, and purity analysis of different samples of zidovudine (AZT) using DSC. Brazilian Journal of Pharmaceutical Sciences - BRAZ J PHARM SCI, 2010. 46.

Supplementary material

• 1H NMR spectra • 13C NMR spectra • Reaction schemes: total overview

122 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

• Characterization of compound 2a

1 Figure 12: H NMR spectrum of compound 2a (400 MHz, CDCl3).

13 Figure 13: C NMR and DEPT spectra of compound 2a (101 MHz, CDCl3). FCUP 123 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Characterization of compound 2b

1 Figure 14: H NMR spectrum of compound 2b (400 MHz, CDCl3).

Figure 15:13C NMR spectrum of compound 2b (101 MHz, MeOD).

124 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Characterization of compound 3a

1 Figure 16: H NMR spectrum of compound 3a (400 MHz, CDCl3).

13 Figure 17: C NMR and DEPT spectra of compound 3a (101 MHz, CDCl3). FCUP 125 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Characterization of compound 3b

1 Figure 18: H NMR spectrum of compound 3b (400 MHz, CDCl3).

13 Figure 19: C NMR and DEPT spectra of compound 3b (101 MHz, CDCl3).

126 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Characterization of compound 4a

1 Figure 20: H NMR spectrum of compound 4a (400 MHz, CDCl3).

13 Figure 21: C NMR and DEPT spectra of compound 4a (101 MHz, CDCl3). FCUP 127 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Characterization of compound 5a

1 Figure 22: H NMR spectrum of compound 5a (400 MHz, CDCl3).

13 Figure 23: C NMR and DEPT spectra of compound 5a (101 MHz, CDCl3).

128 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Characterization of compound 5b

1 Figure 24: H NMR spectrum of compound 5b (400 MHz, CDCl3).

13 Figure 25: C NMR and DEPT spectra of compound 5b (101 MHz, CDCl3).

FCUP 129 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Characterization of compound 7

1 Figure 26: H NMR spectrum of compound 7 (400 MHz, CDCl3).

13 Figure 27: C NMR and DEPT spectra of compound 7 (101 MHz, CDCl3).

130 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Characterization of compound 8

1 Figure 28: H NMR spectrum of compound 8 (400 MHz, CDCl3).

13 Figure 29: C NMR and DEPT spectra of compound 8 (101 MHz, CDCl3).

FCUP 131 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Characterization of compound 9

1 Figure 30: H NMR spectrum of compound 9 (400 MHz, CDCl3).

13 Figure 31: C NMR and DEPT spectra of compound 9 (101 MHz, CDCl3).

132 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Characterization of compound 10

1 Figure 32: H NMR spectrum of compound 10 (400 MHz, CDCl3).

13 Figure 33: C NMR and DEPT spectra of compound 10 (101 MHz, CDCl3).

FCUP 133 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Characterization of compound 16

1 Figure 34: H NMR spectrum of compound 16 (400 MHz, CDCl3).

13 Figure 35: C NMR and DEPT spectra of compound 16 (101 MHz, CDCl3).

134 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Characterization of compound 20

1 Figure 36: H NMR spectrum of compound 20 (400 MHz, CDCl3).

13 Figure 37: C NMR and DEPT spectra of compound 20 (101 MHz, CDCl3).

Characterization of compound 29

1 Figure 38: H NMR spectrum of compound 29 (400 MHz, CDCl3). 136 FCUP Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Characterization of compound 32

1 Figure 39: H NMR spectrum of compound 32 (400 MHz, CDCl3).

13 Figure 40: C NMR and DEPT spectra of compound 32 (101 MHz, CDCl3). FCUP 137 Unnatural β-amino acid derivatives as potential transdermal drug delivery systems

Characterization of compound 35

1 Figure 41: H NMR spectrum of compound 35 (400 MHz, CDCl3).

Figure 42: 13C NMR and DEPT spectra of compound 35 (101 MHz, CDCl

Reaction schemes: total overview

Reaction schemes: total overview