ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2014

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Pharmacy 182

Development of HIV-1 ProteaseInhibitors and Palladium-Catalyzed Synthesis of ArylKetones and N-Allylbenzamides

LINDA AXELSSON

ISSN 1651-6192ISBN 978-91-554-8818-5urn:nbn:se:uu:diva-211672

Dissertation presented at Uppsala University to be publicly examined in B41, BMC,Husargatan 3, Uppsala, Friday, 17 January 2014 at 09:15 for the degree of Doctor ofPhilosophy (Faculty of Pharmacy). The examination will be conducted in Swedish. Facultyexaminer: Professor Morten Grötli (Göteborgs Universitet).

AbstractAxelsson, L. 2014. Development of HIV-1 Protease Inhibitors and Palladium-CatalyzedSynthesis of Aryl Ketones and N-Allylbenzamides. Digital Comprehensive Summaries ofUppsala Dissertations from the Faculty of Pharmacy 182. 84 pp. Uppsala: Acta UniversitatisUpsaliensis. ISBN 978-91-554-8818-5.

The use of palladium-catalyzed reactions to introduce new carbon-carbon bonds is afundamental synthetic strategy that has been widely embraced due to its high chemo-and regioselectivity and functional group tolerance. In this context, Pd(0)-catalyzedaminocarbonylations using Mo(CO)6 instead of toxic and gaseous CO and with allylamine as thenucleophile were investigated. The aminocarbonylated product dominated over the Mizoroki-Heck product, and (hetero)aryl iodides, bromides and chlorides gave N-allylbenzamides in goodyields.

In this thesis improvements to an existing protocol for the Pd(II)-catalyzed synthesis of arylketones from five benzoic acids and a variety of nitriles are also presented. Addition of TFAimproved the yields and employing THF as solvent enabled the use of solid nitriles, and the arylketones were isolated in good yields.

The pandemic of HIV infection is one of the greatest public health issues of our time andapproximately 35.3 million people worldwide are living with HIV. There are currently manydrugs on the market targeting various parts of the viral reproduction cycle, but the problemsof resistance warrant the search for new drugs. HIV-1 protease makes the virus mature intoinfectious particles. In this thesis a new type of HIV-1 protease inhibitor (PI) is presented, basedon two of the PIs on the market, atazanavir and indinavir, but it has a tertiary alcohol, as wellas a two-carbon tether between the quaternary carbon and the hydrazide β-nitrogen. A total of25 new inhibitors were designed, synthesized and biologically evaluated, the best compoundhad an EC50 value of 3 nM.

Based on this series a project aimed at synthesizing macrocycles spanning the P1-P3 areawas initiated. Macrocycles often tend to have an improved affinity and metabolic profilecompared to their linear analogs. Introduction of a handle in the para position of the P1benzyl group proved difficult, despite efforts to synthesize intermediates containing either abromo-, hydroxy-, methoxy-, silyl-group protected hydroxy- or an alkyne-group. The lactoneintermediate was abandoned in favor of an alternative synthetic route and initial studies werefound to be promising. This new approach requires further investigation before the targetmacrocycles can be synthesized.

Keywords: palladium, aminocarbonylation, aryl ketones, decarboxylation, HIV, proteaseinhibitor, tertiary alcohol, macrocycles

Linda Axelsson, Department of Medicinal Chemistry, Box 574, Uppsala University, SE-75123Uppsala, Sweden.

© Linda Axelsson 2014

ISSN 1651-6192ISBN 978-91-554-8818-5urn:nbn:se:uu:diva-211672 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-211672)

Till min pappa Kent

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Appukkuttan, P., Axelsson, L., Van der Eycken, E., Larhed, M.

Microwave-Assisted Mo(CO)6-mediated, Palladium-Catalyzed Amino-Carbonylation of Aryl Halides using Allylamine: from Exploration to Scale-up. Tetrahedron Lett. 2008, 49 (39), 5625-5628.

II Axelsson, L., Veron, J-B., Sävmarker, J., Lindh, J., Odell, L., Larhed, M. An Improved Palladium(II)-Catalyzed Method for the Synthesis of Aryl Ketones from Aryl Carboxylic Acids and Organonitriles. Submitted

III Mahalingam, A. K., Axelsson, L., Ekegren, J. K., Wannberg, J.,

Kihlström, J., Unge, T., Wallberg, H., Samuelsson, B., Larhed, M., Hallberg, A. HIV-1 Protease Inhibitors with a Transition-State Mimic Comprising a Tertiary Alcohol: Improved Antivir-al Activity in Cells. J. Med. Chem. 2009, 53, 607-615. A fourth manuscript, which will not be published is also in-cluded in the thesis:

IV Axelsson, L., Mane, R., Holmgren, S., Odell, L., Larhed, M.

HIV-1 Protease Inhibitors with a Tertiary Alcohol as a Transi-tion-State Mimic: Investigating Macrocyclic Inhibitors with P1 Side Chain Links to the P3 Position. Manuscript

Reprints were made with permission from the respective publishers.

Contents

1. Introduction ............................................................................................... 13 1.1 Palladium chemistry ........................................................................... 13 

1.1.1 Introduction to Palladium Chemistry .......................................... 13 1.1.2 Aminocarbonylations .................................................................. 15 1.1.3 Palladium(II)-Catalyzed Decarboxylative Reactions ................. 16 

1.2 Human Immunodeficiency Virus and Acquired Immune Deficiency Syndrome (HIV/AIDS) .......................................................... 19 

1.2.1 Introduction to HIV/AIDS .......................................................... 19 1.2.2 The Life Cycle of HIV ................................................................ 20 1.2.3 Antiviral Drug Targets and Antiretroviral Therapies ................. 22 1.2.4 HIV-1 Protease ........................................................................... 23 1.2.5 HIV Protease Inhibitors .............................................................. 24 1.2.6 Antiretroviral Therapy (ART) .................................................... 27 

2. Aims of the Present Study ......................................................................... 29 

3. Development of a Pd(0)-Catalyzed Aminocarbonylation Protocol to Synthesize N-Allylbenzamides (Paper I) ...................................................... 30 

3.1 Overview and Adjustments of Conditions ..................................... 30 3.2 Scope of Aryl Iodides .................................................................... 31 3.3 Scope of Aryl Bromides and Chlorides ......................................... 32 3.4 Scale up .......................................................................................... 34 

4. Pd(II)-catalyzed Method for Synthesizing Aryl Ketones from Benzoic Acids and Nitriles (Paper II) ....................................................................... 36 

4.1 Solvent Screen and Adjustments of Conditions ............................ 36 4.2 Scope of Nitriles ............................................................................ 39 4.3 Scope of Aryl Carboxylic Acids .................................................... 41 

5. HIV-1 Protease Inhibitors: Improved Antiviral Activity in Cells (Paper III) ...................................................................................................... 43 

5.1 Chemistry ....................................................................................... 43 5.2 Biological Evaluation .................................................................... 47 5.3 X-ray Crystallography ................................................................... 54 

6. HIV-1 Protease Inhibitors with a Tertiary Alcohol as a Transition-State Mimic: Investigating Macrocyclic Inhibitors with P1 Side Chain Links to the P3 Position .............................................................................................. 58 

6.1 Chemistry ....................................................................................... 58 Appendix - Experimental data for 45-77 ............................................. 64 

7. Concluding remarks .................................................................................. 70 

Acknowledgements ....................................................................................... 71 

References ..................................................................................................... 73 

Abbreviations

AIBN 2,2'-azobis(2-methylpropionitrile) AIDS acquired immunodeficiency syndrome aq. aqueous Ar aryl Arg L-arginine Asp L-aspartic acid ART antiretroviral therapy ATV atazanavir Bn benzyl Bu butyl CA capsid protein CC50 inhibitor concentration that decreases the cell proli-

feration by 50% CF-MAOS continuous flow microwave-assisted organic syn-

thesis Cmpd compound CYP cytochrome P-450 DBU 1,8-diazabicyclo[5.4.0]undec-7-ene DCE 1,2-dichloroethane DCM dichloromethane DFT density functional theory DLV delavirdine DIPEA diisopropylethylamine DME 1,2-dimethoxyethane DMF dimethylformamide DMSO dimethyl sulfoxide DNA deoxyribonucleic acid DRV darunavir EC50 inhibitor concentration that reduces the cytopathic

effect of the virus by 50% EDC N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide

hydrochloride EFV efavirenz equiv. equivalent(s) Et ethyl FDA US Food and Drug Administration

FTC emtricitabine GC gas chromatography Gly glycine HAART highly active antiretroviral therapy HATU O-(7-azabenzotriazole-1-yl)-N,N,N',N'-

tetramethyluronium hexafluorophosphate HIV human immunodeficiency virus HOMO highest occupied molecular orbital HTS high-throughput screening IDV indinavir IN integrase INSTI integrase strand transfer inhibitor Ki inhibition constant, Ki = [E][I]/[EI] L ligand LC liquid chromatography LPV lopinavir LUMO lowest unoccupied molecular orbital MA matrix protein mCPBA 3-chloroperoxybenzoic acid Me methyl MS mass spectrometry MW microwaves NC nucleocapsid protein NFV nelfinavir NMO N-methylmorpholine N-oxide NMP N-methyl-2-pyrrolidone NMR nuclear magnetic resonance NNRTI non-nucleoside reverse transcriptase inhibitor NRTI nucleoside reverse transcriptase inhibitor NtRTI nucleotide reverse transcriptase inhibitor NVP nevirapine PCC pyridinium chlorochromate PDC pyridinium dichromate PDB Protein Data Bank Ph phenyl PI protease inhibitor Pr propyl PR protease RNA ribonucleic acid room temp room temperature RP reverse-phase RT reverse transcriptase RTV ritonavir SIV simian immunodeficiency virus

SQV saquinavir SU surface glycoprotein TBAF tetra-N-butylammonium fluoride TBDMS tert-butyldimethylsilyl TBDPS tert-butyldiphenylsilyl TDF tenofovir disoproxil fumarate temp temperature THF tetrahydrofuran Thr L-threonine TPV tipranavir UNAIDS The Joint United Nations Programme on

HIV/AIDS UPAM universal protease associated mutations WHO World Health Organization

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1. Introduction

1.1 Palladium chemistry

1.1.1 Introduction to Palladium Chemistry The late transition metal palladium is one of the most versatile, useful and significant metals in organic chemistry. Many well known carbon-carbon bond coupling reactions1 (summarized in Figure 1) have been developed since the Wacker process in the late 1950s,2 which was the starting point for the development of palladium-catalyzed reactions.

There are four reasons why palladium is involved in so many useful reac-tions.3 Firstly it is a moderately large metal from the second row in the periodic table, which contributes to both its inherent stability and a controlled but versatile reactivity pattern. Secondly, as Pd prefers the 0 and II oxidation states, it will not be involved in radical or one-electron processes. Thirdly, palladium favors the tetrahedral d10(0) and square planar d8(II) complexes, rendering it a “soft” status, it is therefore reactive towards many of the soft π and non bonding electron donors (n-donors). Palladium tends to react in many concerted processes of low activation energy because of the small gap between its HOMO and LUMO energies; thus, it has a high affinity for nonpolar π-compounds, such as alkynes, alkenes and arenes or n-donors such as amines, imines, nitriles, phosphines, etc. The fourth factor is the high electronegativity of Pd (2.2 on the Pauling scale); thus the Pd-C bond will be relatively nonpolar, making it unreactive towards polar func-tional groups such as ketones, esters, alkyl halides, etc. In this respect it behaves in a manner completely opposite to Grignard and lithium reagents and complements those reactions.3

14

Cross-couplings:

Suzuki-Miyaura

Stille-Migita Negishi Hiyama-Hatanaka

Kumada-Corriu

Sonogashira-Hagihara

Mizoroki-Heck

Figure 1. Palladium(0)-catalyzed coupling reactions.1

The ligands fine-tune the catalytic properties of palladium through their ster-ic and electronic properties. Palladium often binds to four ligands, each of which contributes two electrons to form stable 16- or 18-electron complexes, associated with the Pd(II) or Pd(0) oxidation state. Stable 16 e- Pd(II) com-plexes or Pd(II) salts are often used as pre-catalysts in Pd0 chemistry.1,3 Some of the ligands, bases and Pd-sources used in this thesis are summarized in Figure 2.

Figure 2. Important ligands, Pd-catalysts and bases used in this thesis.

Although some of the earliest processes were Pd(II)-catalyzed, e.g. the Wacker process, the generation of a Pd(0) complex that needs to be re-oxidized to Pd(II) at the end of many Pd(II) reactions has hampered the de-velopment of new Pd(II)-catalyzed processes. Common re-oxidants are for example O2,

4 p-benzoquinone5 and hydrogen peroxide.4 The most environ-

15

mentally benign of these is oxygen but its use is limited because of the high pressures often required during small scale synthesis.6

In Pd(0) catalysis the arylpalladium species is formed through oxidative addition, which changes the oxidation state from 0 to II; the aryl source, the aryl halide or an aryl halide surrogate, is also an oxidant. In Pd(II)-catalyzed reactions the arylpalladium complex is commonly formed through a trans-metallation of an arylmetaloid species, such as an arylborane,7 through C-H activation of an arene8 or through decarboxylation of a benzoic acid.9,10

1.1.2 Aminocarbonylations The insertion of CO into aryl-, vinyl- or benzylpalladium complexes, fol-lowed by a reaction with different nucleophiles is known as a carbonylation reaction. The products are carboxylic or keto acids, esters, amides or alde-hydes.11,12 When an amine is used as the nucleophile, the reaction is called aminocarbonylation and the amide product is formed without the need for coupling reagents in the stoichiometric amounts normally associated with the creation of an amide bond.

The mechanism for Pd(0)-catalyzed aminocarbonylation is outlined in Figure 3.11,12 The palladium(II) salt or stable 16 e- complex working as a pre-catalyst is reduced to the catalytically active 14 e- Pd0L2 at the same time as the phosphine ligands, amines, solvents or other reagents are oxidized.3 In the oxidative addition that takes place in step 1, the oxidation state of Pd changes from 0 to II as two new bonds to R and X are formed. Step 2 is a CO co-ordination step and step 3 is a 1,1-insertion, where the acylpalladium complex is formed. Finally the direct attack of the amine on this complex forms the product and Pd0L2 is regenerated. With some other nucleophiles this last step is thought to occur via a reductive elimination but with amines it is believed to occur via a direct attack on the carbonyl group.11

Figure 3. The proposed mechanism for aminocarbonylations.11,12 Pd has oxidation state II unless otherwise indicated.

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The drawbacks of making a carbonylation reaction with gaseous reagents and high pressure reaction conditions are considerable. Carbon monoxide is a toxic and flammable gas. In 2002 Kaiser et al. reported the use of a solid CO source, Mo(CO)6, as a gas-free alternative in a palladium-catalyzed ami-nocarbonylation reaction.13 Compared to some of the alternatives, Cr(CO)6, W(CO)6, Fe(CO)5, Fe3(CO)12, Co2(CO)8, investigated by Wannberg and Larhed in 2003,14 it gives a better yield, can be bought for a reasonable price, is not highly toxic and does not undergo sublimation (as Cr(CO)6 does). The addition of the base DBU was also beneficial for the reaction outcome as it induced liberation of carbon monoxide. At higher temperatures this happens very quickly, and this is thought to occur via formation of a Mo(DBU)2(CO)4 complex (Figure 4).14

Figure 4. The role of DBU in the aminocarbonylation reactions.14

The carbonylation reaction using Mo(CO)6 has been well explored at the Organic Pharmaceutical Chemistry Division of Uppsala University; numer-ous nucleophiles, conditions and aryl halides have been evaluated.15-21

A reaction setup based on the two-chamber system developed by Skyd-strup et al.22 and using Mo(CO)6 as an ex situ CO-source23 has also been developed. This setup can, for instance, prevent the reduction of nitro-groups during the aminocarbonylation reaction.24

1.1.3 Palladium(II)-Catalyzed Decarboxylative Reactions The use of benzoic acids as arylpalladium precursors has received a lot of attention during the last decade.10,25 Benzoic acids have many benefits com-pared to other aryl sources; they are available in a wide variety at a reasona-ble price; in addition they are non toxic and inert. Furthermore, gaseous CO2 is the only byproduct produced when the aryl palladium species is formed. Since 2002, Myers9,26,27 and Gooβen10,25,28-30 and their co-workers have used Pd(II)-catalyzed decarboxylations in numerous Suzuki- and Heck- type cross-coupling reactions (Figure 5). These reactions are often endothermic and difficult, but can work with certain ortho-substituted benzoic acids. Gooβen expanded the scope so that the ortho-substituent was no longer ne-cessary by using bimetallic Cu-Pd or Ag-Pd systems.10,28-30

Ketones are a fundamental organic functional group. Many natural prod-ucts and pharmaceuticals contain an aryl ketone, often with a substituent in one or both ortho positions.31,32 Previously, synthesis of aryl ketones from benzoic acids was limited to harsh methods using organic lithium reactants in stoichiometric amounts,33 or Friedel Craft acylations34 that were often moisture-sensitive and could suffer from a lack of chemo- and regio-control.

17

A more recent protocol is to use toxic arylstannanes to synthesize sterically hindered aryl ketones.35

The first example of an arylpalladium species being added to a nitrile triple bond was reported in 1970.36 In the early twenty-first century the pio-neering work by Larock et al. received more attention and enabled synthesis of aryl ketones from arenes or arylboronic acids and nitriles (Figure 5).37,38 Nitriles are excellent coupling partners because of their low cost and diversi-ty, while arylboronic acids have limitations associated with their stability, costs and eco-friendliness. The initial product is the ketimine which needs to be hydrolyzed to the corresponding aryl ketone.

Figure 5. Examples of the decarboxylative Heck and Suzuki reactions and the syn-thesis of aryl ketones from arenes/aryl boronic acids and nitriles.

In 2010 Lindh et al.39 published a paper on the synthesis of aryl ketones in which an initial Pd(II)-catalyzed decarboxylation of a benzoic acid to gener-ate the arylpalladium species was followed by carbopalladation of a nitrile. After protonation and release of the ketimine intermediate a hydrolysis gives the aryl ketone product. Importantly, sterically congested aryl ketones could be produced as the aryl carboxylic acid needs to have an activating ortho substituent to facilitate the decarboxylation process.10,40,41

Figure 6 depicts a plausible mechanism based on an ESI-MS study, in which cationic palladium intermediates were detected. The following key steps are involved: 1) the carboxylic acid is co-ordinated to the Pd(II) center to form complex A; 2) the benzoic acid is decarboxylated to give Ar-Pd complex B; 3) the nitrile is coordinated to produce complex C; 4) 1,2-carbopalladation of the nitrile gives complex D; and 5) protonation of the ketimine by the benzoic acid results in the free ketimine. To better under-stand the mechanism, DFT calculations have been carried out on sterically congested aromatic substrates. The suggested mechanism based on DFT calculations by Svensson et al.42 is depicted in Figure 7, in which acetate replaces TFA and the calculations are based on acetonitrile. The neutral complexes in a mechanism can also be important and the formation of catio-nic complex D (when the fourth ligand is a nitrile) is less likely than K

18

(when the fourth ligand is a benzoic acid) in the study presented in section 4, since then the nitrile is no longer the solvent.

Figure 6. Suggested catalytic cycle based on the cationic palladium(II) complexes found in an ESI-MS study.39

Figure 7. Suggested mechanism based on DFT calculations.42

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1.2 Human Immunodeficiency Virus and Acquired Immune Deficiency Syndrome (HIV/AIDS)

1.2.1 Introduction to HIV/AIDS The human immunodeficiency virus (HIV) has caused a pandemic and HIV/AIDS is considered one of the world's most serious health issues. Since the first reported cases of rare diseases caused by an unknown virus among homosexual men in the US in 1981, such as pneumonia caused by Pneumo-cystis carinii43 and Kaposi's sarcoma (a rare cancer of the skin due to a virus infection),44,45 approximately 35 million people have died from AIDS ac-cording to WHO reports.46 UNAIDS reports that approximately 35.3 (32.2-38.8) million adults and children were living with HIV at the end of 2012.47 On a global scale 0.8% of the adult population (aged 15-49 years) has HIV/AIDS. The area with the highest prevalence is sub-Saharan Africa where 25.0 million people were infected by HIV in 2012. The second most affected area is south and south-east Asia, with 3.9 million (Figure 8).47

Figure 8. The number of people infected with HIV in the world at the end of 2012.47

In 2001 29.4 million people were living with the disease, but the growth in numbers seen over this 11-year period is partly due to an increase in life expectancy as antiretroviral therapy reaches more people infected with HIV each year. In 2012, 1.6 million people died from AIDS-related causes. This was a decline from 2005, which was the peak year when most people died (2.3 million). By the end of 2012, 9.7 million people eligible for treatment in low- and middle-income countries (10.6 million worldwide) were receiving antiretroviral therapy.48 Of the people who qualified for treatment according to the WHO 2010 guidelines (<350 CD4 cells/mm³)49 in low- and middle-income countries in 2012, 64% were receiving it.50

20

Between the years 1995 and 2011, 14 million life years have been saved in low- and middle-income countries.51 Gratifyingly, the number of newly infected persons per year is decreasing; 2.3 million were newly infected in 2012, compared with 3.2 million in 2001 (Figure 9).

a) b)

c)

Figure 9. a) Number of people living with HIV on a global scale, 2001-2012 b) Number of people newly infected with HIV on a global scale, 2001-2012. c) AIDS deaths on a global scale, 2001-2012.52 (Reprinted with kind permission from UNAIDS.)

There are two subtypes of HIV: HIV-1 and HIV-2. Both are thought to have originated by transmissions from simian immunodeficiency virus (SIV)-infected non-human primates; HIV-1 from chimpanzees/gorillas and HIV-2 from sooty mangabeys. HIV-2 is rare outside of West Africa and is less common and pathogenic than HIV-1.53

1.2.2 The Life Cycle of HIV Figure 10 depicts the replication cycle of HIV. HIV infects cells containing CD4 receptors, which are present to a high extent in CD4+-T-lymphocytes, an important part of the immune system.54 Macrophages and some cells in the brain also contain CD4 receptors.55 The virus attaches itself to the host cell by an interaction between viral glycoprotein gp120 (also called SU) and the host cell CD4 receptor.56

Once bound to the cell, additional binding to chemokine co-receptors CXCR4 and/or CCR5 occurs. Conformational changes in the transmembrane protein gp41 (also called TM) promote fusion and entry. Once the virus has

21

been uncoated, the virion is decapsidated, which releases the viral RNA into the host cytosol.57 The viral single-stranded RNA chain contains three major genes: gag, pol and env, and six minor genes: vif, vpr, vpu, rev, nef and tat (the minor genes code for regulatory and accessory proteins).58 Reverse tran-scriptase makes a double-stranded DNA copy of the viral RNA. Integrase incorporates the viral DNA into the host chromosomes and they start to make new viral RNA.59

Viral proteins are manufactured through the regular cell machinery: Env in the endoplasmatic reticulum and Gag and Gag-Pol in the ribosomes. Env codes to protein g160, which is cleaved by a human protease called furin in the host cell to g41 and gp120.60 Env, viral RNA, Gag and Gag-Pol then leave the cell as an immature virion and HIV-1 protease (PR) cleaves the Gag and Gag-Pol proteins to generate a mature virus particle.58

Gag is cleaved to form the structural proteins MA (matrix protein, p17), CA (capsid protein, p24), SP1 (spacer peptide 1, p2), NC (nucleocapsid pro-tein, p7), SP2 (spacer peptide 2, p1) and P6. During the maturation process, Gag-Pol is cleaved to form the functional proteins RT (reverse transcriptase), IN (integrase), PR (protease) and RNase H.61,62

Figure 10. Replication cycle of HIV. (Illustration from Ensoli,63 reprinted with kind permission from Nature reviews cancer.)

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1.2.3 Antiviral Drug Targets and Antiretroviral Therapies Since the FDA approved zidovudine (ZDV also known as azidothymidine, AZT) in 1987 a total of 26 substances against HIV are currently on the mar-ket (as of November 2013).64 In total, 39 antiretroviral drug combinations have been marketed; of these, three are multiclass combination products and three have been withdrawn. There are more drugs available for the treatment of HIV than for all other viral infections put together.56

Nucleoside Reverse Transcriptase Inhibitors (NRTIs) The FDA approved six NRTIs for clinical use between the years 1987 and 2004: zidovudine (originally developed for use against leukemia), didano-sine, stavudine, lamivudine, abacavir and emtricitabine (FTC).65

These drugs are phosphorylated in vivo to the 5' triphosphate form, and "faulty" building blocks for DNA synthesis are generated. They can be mod-ified in either the nucleic base moiety, the sugar moiety or both. Reverse transcriptase inhibition occurs by chain termination; the NRTIs lack the 3'-hydroxyl group, so the next nucleotide is not incorporated.56

Nucleotide Reverse Transcriptase Inhibitors (NtRTIs) One NtRTI has been approved by the FDA; tenofovir disoproxil fumarate (TDF).65 NtRTIs are very similar to NRTIs but because a phosphonate group is attached, this group cannot be cleaved off by esterases. TDF needs two phosphorylation steps to be useful as a substrate/chain terminator for RT. However, the presence of two negative charges from the phosphonate group limits their transport into cells, thus tenofovir is taken as a prodrug.65

Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) Between 1996 and 2011, five NNRTIs entered the market: rilpivirine, etravi-rine, delavirdine (DLV), efavirenz (EFV) and nevirapine (NVP).65 These drugs function by binding non-competitively to an allosteric site, situated close to the active site (15 Å), on the reverse transcriptase enzyme and pre-venting the conformational changes needed for the enzymatic process.56

HIV integrase strand transfer inhibitors (INSTIs) The FDA has approved three INSTIs: raltegravir (2007), elvitegravir (2012, only available as a combination drug elvitegravir/cobicistat/FTC/TDF called Stribild®) and dolutegravir (2013).59,66 They inhibit a crucial step in the HIV life cycle when integrase integrates viral DNA into the host cell chromosom-al DNA. Integrase first forms a pre-integration complex, as it binds to the viral DNA and is reverse-transcribed, and it then trims the 3' ends of the viral DNA by cutting off two terminal nucleotides. The pre-integration complex is transported to the nucleus, where a pair of transesterification reactions cova-lently bind the viral DNA into the cellular DNA, through a process called

23

strand transfer.67 Regular cellular repair mechanisms seal the viral DNA in the chromosome. Raltegravir prevents the covalent bonds forming between divalent cations in the catalytic core of the integrase and the phosphodiester backbone of the DNA.59

Fusion inhibitors (FIs) Enfuvirtide is the first, and currently the only (as of November 2013), fusion inhibitor to be approved by the FDA (2003).68 It is a linear 36-amino acid synthetic peptide that inhibits gp41, preventing the conformational changes needed for fusion of the viral and cellular membranes. This drug is only available as a sub-cutaneous injection56 and has rather limited use; it is main-ly used when other drug alternatives have failed.

Entry inhibitors: CCR5 co-receptor antagonists Maraviroc (2007) targets the CCR5 receptor found on some human cells. Viral gp120 subsequently cannot associate with the receptor and viral entry is thus prohibited. Some types of HIV use the CXCR4 receptor instead of CCR5, in which case maraviroc is in-effective.69

Protease inhibitors (PIs) There have been 11 PI drugs approved for clinical use against HIV (although two are no longer marketed). The subject of PIs will be discussed in more detail in section 1.2.5.

1.2.4 HIV-1 Protease HIV-1 protease is a dimeric enzyme, with 99 amino acids in each dimer.70-74 It cleaves Gag and Gag-Pol polyproteins to create a mature, infectious virus. Inhibition of HIV-1 PR prevents HIV-virions from developing into infec-tious particles. The active dimers, which are responsible for the activity of the enzyme, only form when the concentration of monomers is high, which happens just after budding has occurred or in the forming bud.75

The protease has two flexible flaps which, in the opened form, help the substrate/inhibitor to enter the active site. When closed the flaps facilitate the cleavage of substrate/inhibitor. For example, Ile50 and Ile150, which are situated in the flap region, keep the substrate/inhibitor in place in the active site, via hydrogen bonds formed with a structural water molecule which in turn forms hydrogen bonds with carbonyl groups from the sub-strate/inhibitor.70,76

The catalytic triad in the active site, Asp-Thr-Gly, indicates that HIV-1 PR is a member of the aspartic acid protease family and the mechanism for the cleavage is thus the same as for all aspartic proteases (Figure 11).70,77 One of the two aspartic acids 25 and 125, one on each dimer, is deprotonated and functions as a base and deprotonates a water molecule, forming the hy-

24

droxyl group which makes a nucleophilic attack on the carbonyl to form the intermediate diol. This unstable tetrahedral intermediate breaks down to the hydrolyzed carboxylic acid and amine.

Figure 11. The mechanism of the peptide bond cleavage.70,77

Figure 12 shows the nomenclature for different parts of both the endogenous substrate and the inhibitors.78 The group at the α-carbon of the amino acid closest to the scissile bond is called P1; this fills the S1 pocket on the PR on the N-terminal side. On the C-terminal side it is called P1' and S1' and so on.

Figure 12. The nomenclature used to describe the amino acids and their correspond-ing pockets in the protease.78 A dashed line shows where the scissile bond is si-tuated.

1.2.5 HIV Protease Inhibitors There are eight cleavage sites in the endogenous substrates for the PR (Gag or Gag-Pol); three of them are Phe-Pro or Tyr-Pro.72,79 Many of the nine inhibitors on the market are peptidomimetic (with the exception of tiprana-vir) and they are mimicking this very selective cleavage site. The tetrahedral intermediate formed during the cleavage of the scissile bond is replaced by a non-hydrolysable transition state mimic in the inhibitors; the most common-ly used is the hydroxyethylene moiety.61 Figure 13 shows some of the transi-tion state mimics that have been used in inhibitors, most of them are second-ary alcohols.70

25

Figure 13. Transition state mimics.70

The first X-ray structure of HIV-PR was obtained in 1989,76 and the first protease inhibitor, saquinavir (SQV), was approved by the FDA in 1995. This drug was developed during work on a renin inhibitor, which explains the relatively short development time. In the following years, ritonavir (RTV, 1996), indinavir (IDV, 1996) and nelfinavir (NFV, 1997) were ap-proved for clinical use against HIV (Figure 14).80

The more peptide-like a compound is, the more likely it is to have poor bioavailability, due to its poor solubility, high molecular weight, rapid meta-bolism, high protein binding and low membrane permeability etc.81 Especial-ly the first generation of HIV-PIs suffered from low bioavailability and a high pill burden, partly due to rapid degradation by cytochrome P-450 en-zymes.82 Many of the PIs are now given together with ritonavir, as a phar-macokinetic "booster"; ritonavir is a CYP3A4 inhibitor and the other PIs will thus be less extensively metabolized by this enzyme.71 Another problem with the first generation of PIs involves the severe side effects, which are partly the result of high doses; these include metabolic disorders (for example hyperlipidaemia, lipodystrophy and insulin resistence), as well as nausea, vomiting and diarrhea.73,79,81

Amprenavir, which is no longer marketed but has been replaced by its prodrug fosamprenavir, was the first PI in the second generation (1999). It was soon followed by lopinavir (LPV, 2000), atazanavir (ATV, 2003), fo-samprenavir (FPV, 2003), tipranavir (TPV, 2005) and darunavir (DRV, 2006) (Figure 12).80

The second generation of PIs has a better pharmacokinet-ic/pharmacodynamic profile: atazanavir was the first PI that could be given once daily. Also, the toxicity was lessened and the side effects were less severe.71,74,79,82

Resistance is another major issue for this drug class, partly because of high HIV mutation rate;83,84 and PIs from both first and second generations have this problem. In an HIV-infected (untreated) human, approximately 10.3 billion virions are produced each day. The half-life for the virus in plasma is 6 h, the mutation rate is 3×10-5/base pair/replication cycle, and the genome includes 10,000 base pairs. These facts together indicate that one mutation could occur in each nucleotide of HIV each day.85 ATV has a resis-tence profile that is distinct from other HIV PIs.56 TPV, on the basis of being

26

developed from an HTS hit, is more adaptable to alterations in the binding site and is used as a last resort when the disease fail to respond to other anti-viral drug regimens. Because of concerns about the safety profile it is not an early choice for drug-naive HIV-patients.79 The bis-THF ligand in DRV is involved in extensive hydrogen binding to the backbone of the S2 pocket of the PR and it is therefore less susceptible to mutations and alternations of the amino acids R-groups.86

Saquinavir (SQV)

Ritonavir (RTV)

Indinavir (IDV)

Nelfinavir (NFV)

Lopinavir (LPV)

Atazanavir (ATV)

Fosamprenavir (FPV)

Tipranavir (TPV)

Darunavir (DRV)

Figure 14. The nine protease inhibitors presently approved for clinical use by the FDA.80

27

1.2.6 Antiretroviral Therapy (ART) Highly active antiretroviral therapy (HAART)87,88 or as it has become more commonly known in the last couple of years antiretroviral therapy (ART), is a combination of three or more anti-HIV drugs taken simultaneously to tar-get many different aspects of the viral lifecycle at the same time, with the aim of preventing the emergence of resistance. It is now very uncommon to choose monotherapy as a treatment option, and the morbidity and mortality of treated patients has decreased considerably since the start of HAART/ART. No less than eight diffent combinations of HIV drugs in one pill are now available, all aiming to increase patient adherence/compliance and decrease the pill burden.64

Many factors will determine the appropriate combination of drugs for a person taking HIV-medicines for the first time. The United States Depart-ment of Health and Human Services lists three main choices (Figure 15):89 1) an NNRTI-based regimen with Atripla®, a combination pill of efavirenz (an NNRTI), emtricitabine/FTC (an NRTI) and tenofovir disoproxil fuma-rate/TDF (an NtRTI); 2) a PI-based regimen of ATV or DRV boosted with RTV and Truvada® (a combination pill containing FTC and TDF); 3) INSTI-based regimen that consists of raltegravir or dolutegravir and Truvada®. Do-lutegravir can also be combined with lamivudine and abacavir (both NRTIs) The combination drug Stribild® consisting of elvitegravir, cobicistat (a cy-tochrome P450 inhibitor), TDF and FTC is also recommended.

Fusion, entry and integrase inhibitors are the newest and most costly anti-HIV drugs; they are available to a higher extent in developed countries. WHO recommendations for the low- and middle-income countries are TDF, efavirenz and FTC or lamivudine as a combination pill. In a resource-limited setting, it is not recommended that patients start treatment with PIs (but they are in the guidelines as a second line ART) because of the pill burden, cost and associated side effects.90

28

(1)

NNRTI-

based

efavirenz

FTC

TDF

(2)

PI-based

atazanavir/ritonavir FTC TDF

darunavir/ritonavir FTC TDF

(3)

INSTI-

based

raltegravir

FTC TDF

INSTI-

based

dolutegravir

FTC TDF

INSTI-

based dolutegravir

lamuvidine

abacavir

INSTI-

based

elvitegravir/cobicistat

FTC TDF

Figure 15. Recommendations for first-line treatment of drug-naive patients from the United States Department of Health and Human Services.89

29

2. Aims of the Present Study

The specific objectives of this thesis are presented below.

To develop a protocol for the Pd(0)-catalyzed aminocarbonyla-tion reaction with Mo(CO)6 as the CO source and allylamine as the nucleophile, and to investigate if the reaction could be chemo-selective without formation of the Mizoroki-Heck byproduct.

To develop an improved protocol for the decarboxylative Pd(II)-catalyzed addition of nitriles to ortho-substituted benzoic acids to produce aryl ketones and to decrease the excess of nitrile used in the reaction.

To design and synthesize atazanavir- and indinavir-inspired HIV-1 protease inhibitors with a tertiary alcohol and a two-carbon tether between the quaternary carbon and the hydrazide β-nitrogen.

To design and synthesize P1-P3 macrocyclic HIV-1 protease in-hibitors with a tertiary alcohol and a two-carbon tether between the quaternary carbon and the hydrazide β-nitrogen.

30

3. Development of a Pd(0)-Catalyzed Aminocarbonylation Protocol to Synthesize N-Allylbenzamides (Paper I)

3.1 Overview and Adjustments of Conditions When allylamine is a substrate in a Mo(CO)6-mediated Pd(0)-catalyzed ami-nocarbonylation of an aryl halide (section 1.1.2 in the introduction) will the product be a styrene via a Mizoroki-Heck reaction91,92 or the aminocarbony-lation product, the N-allylbenzamide (Figure 16)? If there is competition between the 1,1-insertion process with CO and the 1,2-insertion-β-elimination sequence of the Mizoroki-Heck reaction could the reaction be controlled and chemoselective? The allyl moiety can for example be used as a handle in macrocyclizations.

Figure 16. Aminocarbonylation or Mizoroki-Heck reaction.

The aminocarbonylation reaction between 2-iodotoluene (1a) and allylamine (2) was chosen as a model reaction (Table 1). The procedure was based on an earlier protocol by Wannberg and Larhed.14 Initial test reactions were performed with different irradiation times and temperatures and 1.5 equiv. of 2. Suitable conditions were found to be Mo(CO)6, Pd(OAc)2 as precatalyst and DBU as base in 1,4-dioxane, in a sealed vial, in the microwave reactor for 10 min at 125 ºC. It was not necessary to run the reaction in an inert at-mosphere or to use a phospine ligand, even without a dedicated ligand full conversion was observed.

It was obvious early on that the carbonylation reaction was dominating over the Mizoroki-Heck reaction; no Mizoroki-Heck product was detected according to GC-MS. The slower 1,2-alkene insertion in a Mizoroki-Heck reaction could not compete with the rapid CO insertion followed by attack of 2 in the aminocarbonylation reaction.93 Further, in an experiment in which Mo(CO)6 was not added, no Mizoroki-Heck product was formed, suggesting that these conditions are not suitable for Mizoroki-Heck reactions.

31

3.2 Scope of Aryl Iodides To investigate the scope of the reaction, six different aryl iodides were tested. As Table 1 shows, satisfactory yields of 72-81% of N-allyl benza-mides 3a-f were obtained. Electronic effects did not have an influence on the yield as both electron-rich (Table 2, entries 2-4) and electron-poor (entry 5) aryl iodides gave similar yields. The aminocarbonylation also worked with heterocyclic iodides; 3-iodofuran (1e) and 2-iodothiophene (1f) provided 3e and 3f in good yields (72% and 76% respectively, entries 6 and 7). The Mi-zoroki-Heck product or dehalogenated products were detected in no more than trace amounts in all experiments, according to LC-MS and NMR.

Table 1. Aminocarbonylation of aryl iodides with allylamine.

Entry Ar-I Product Yield (%)

1

1a

3a

73a

2

1b

3b

76a

3 4

1c

3c

81a 78b

5

1d

3d

76c

6

1e

3e

72a

7

1f

3f

76a

Isolated yields >95% pure according to GC-MS and 1H-NMR. Reagents and conditions: a Aryl iodide 1a-1c, 1e, 1f (0.4 mmol) was dissolved in 3 mL of 1,4-dioxane in a 5 mL micro-wave transparent vial. Next, 1.5 equiv. of allylamine 2, 1.0 equiv. Mo(CO)6, 3 equiv. DBU and Pd(OAc)2 (7 mol%) were added, the vial was sealed, and the reaction was exposed to MW heating for 10 min at 125 °C. bA 20 mL microwave transparent vial was charged with 2.0 mmol 1-iodo-4-methoxybenzene, 10 mL 1,4-dioxane and the same amounts of Pd-catalyst, base and Mo(CO)6 as in a and exposed to MW heating for 15 min at 130 °C. cLike a but using 3 equiv. 2, 2 equiv. Mo(CO)6, 6 equiv. DBU and 14 mol% Pd(OAc)2.

32

3.3 Scope of Aryl Bromides and Chlorides The protocol was then expanded to include the synthesis of N-allylbenzamides from hetero(aryl) bromides. A more reactive catalytic sys-tem was deemed necessary based on previous experience with aminocarbo-nylation reactions with aryl bromides.21 Hermann's palladacycle94 and the additive [(t-Bu)3PH]BF4

95,96 were chosen as a more productive system. The time was increased to 15 min and the temperature was set at 140 ºC. Dibro-moaryls 1g-1i furnished di-N-allylbenzamides 3g-3i in good yields, 73-81% (Table 2, entries 1-3), with no trace of mono-carbonylated or dehalogenated byproducts. A lower yield of 43% was obtained with benzophenone 1j (entry 4). Product 3k was isolated in 56% yield and the ester functionality intact (entry 5). Heterocyclic aryl bromides were well tolerated, and 2-bromofuran (1l), 3-bromofuran (1m) and 2-bromothiophene (1n) produced their respec-tive N-allylbenzamides, 3l, 3e and 3f, in good yields of 66-76% (entries 6-8).

Encouraged by the satisfactory results with aryl iodides and aryl bromides an attempt to carry out the reaction with aryl chlorides was made. In earlier aminocarbonylation protocols using aryl chlorides the reaction benefited from an increased reaction time and elevated temperature and incomplete conversion occurred when the experiments were run at the same time and temperature as the arylbromides. Thus, the appropriate conditions for aryl chlorides were 20 min at 160 ºC.16,97-101 Both furoyl- (3e) and thienyl- (3f) derivates were isolated in good yields (73 and 69% respectively; entries 10 and 11, Table 2) as was benzamide 3a (67%, entry 9).

A few aryl halides did not give the desired product (Figure 17). For ex-ample when the aryl halide had an amine as an ortho-substituent, as was the case with 2-iodoaniline, no trace of the product was detected according to LC-MS analysis, probably due to poisoning of the Pd-catalyst.102 The nitro-containing compound 1-bromo-4-nitrobenzene was reduced to its corres-ponding aniline under these conditions, according to LC-MS analysis.103

33

Table 2. Aminocarbonylation of aryl bromides and chlorides with allylamine.

Entry Ar-halide Product Yield (%)

1

1g

3g

73a

2

1h

3h

76a

3

1i

3i

81a

4

1j

3j

43b

5

1k

3k

56b

6

1l

3l

76b

7

1m

3e

66b

8

1n

3f

72b

9

1o

3a

67c

34

Table 2. continued

10

1p

3e

73b

11

1q

3f

69b

Isolated yields >95% pure according to GC-MS and 1H-NMR. Reagents and conditions: aAryl halide 1g-q (0.4 mmol) was dissolved in 3 mL of 1,4 dioxane in a 5 mL microwave transpar-ent vial. Next, 3 equiv. of allylamine 2, 2.0 equiv. of Mo(CO)6, 6 equiv. of DBU, Hermann’s palladacycle (5 mol%) and [(t-Bu)3PH]BF4 (14 mol%) were added, the vial sealed, and the reaction was exposed to MW heating for 15 min at 140 °C. bLike a but using 1.5 equiv. 2, 1.0 equiv. Mo(CO)6, 3 equiv. DBU, 2.5 mol% Hermann’s palladacycle and 7 mol% [(t-Bu)3PH]BF4.

cLike b but using 3.5 mol% Hermann’s palladacycle and 9 mol% [(t-Bu)3PH]BF4 and increasing the reaction time to 20 min and the temperature to 160 °C.

Figure 17. Some aryl halides did not give the desired product. The conditions used were the same as in Table 1 for the 2-iodoaniline and as in Table 2 for the 1-bromo-4-nitrobenzene.

3.4 Scale up The gaseous CO released during carbonylation reactions has previously pre-vented the development of protocols for large scale microwave reactors (>20 mL).104 The aminocarbonylation of 1-iodo-4-methoxybenzene (1c) was cho-sen as a model reaction to investigate this. Initial upscaling to a 2 mmol scale in a 20 mL sealed MW transparent borosilicate glass vial, with a slightly elevated temperature of 130 °C and increased time to 15 min, furnished 3c in an excellent 78% yield (Table 1, entry 4).

Reassured by these promising results, the reaction was increased to a 25 mmol scale. The reactants, with intact stoichiometry, were dissolved in 125 mL 1,4-dioxane and irradiated for 15 min at 125 ºC in a Biotage Advancer batch reactor.105 The reactor had a reaction vessel designed to contain vo-lumes between 50 and 350 mL, as well as a mechanical stirrer and an adia-batic cooling system. During the reaction the pressure was at the maximum 3.0 bar, decreasing to 1.0 bar as the reaction proceeded and the CO gas was

35

consumed. Gratifyingly, N-allylbenzamide 3c was isolated in 80% yield using the upscaling equipment.

Scheme 1 Aminocarbonylation on a 25 mmol scale.

Reagents and conditions: (a) Aryl iodide 1c (25 mmol) was dissolved in 125 mL of 1,4-dioxane. Next, 1.5 equiv. of allylamine 2, 1.0 equiv. Mo(CO)6, 3 equiv. DBU and Pd(OAc)2 (7 mol%) were added, the vial sealed, and the reaction was exposed to MW heating for 15 min at 125 °C in a Biotage Advancer batch reactor, 80%.

36

4. Pd(II)-catalyzed Method for Synthesizing Aryl Ketones from Benzoic Acids and Nitriles (Paper II)

In the protocol by Lindh et al.39 used to synthesize aryl ketones from aryl carboxylic acids and organonitriles, the latter functioned as both reactant and solvent in the reaction. The protocol was therefore limited to liquid nitriles. To enable usage of solid nitriles and decrease the amount of nitrile needed for a productive process the reaction conditions were further investigated.

4.1 Solvent Screen and Adjustments of Conditions As they were the highest yielding substrates from the previous study, 2,6-dimethoxybenzoic acid (4a, 0.5 mmol) and phenylacetonitrile (5d) were chosen as a model reactants for a solvent screen. Pd(O2CCF3)2 (8 mol%) and 6-methyl-2,2'-bipyridine (9.6 mol%) were used as the catalytic system. The solvents tested were acetic acid, 10% acetic acid in dioxane, 10% acetic acid in THF, 10% acetic acid in water, dioxane, DMF, DMSO, EtOH, NMP, THF and water. The results with THF/water (10/1) looked the most promising and aryl ketone 6d was isolated in 68% yield (Table 3, entry 4), after microwave heating106 at 130º C for 4 hours, and with an additional 15 min at 130 ºC after addition of formic acid to facilitate hydrolysis of the ketimine.

Adding TFA to the reaction mixture is beneficial in at least two ways. TFA protonates and helps to release the ketimine from the intermediate Pd-complex (Figure 6, going from complex D to the ketimine; if no TFA is present the benzoic acid fills this role). TFA also helps in hydrolyzing the ketimine to form the ketone.39,42 In a previously published protocol, the au-thors found it beneficial for the reaction if TFA was added during the palla-dium-catalyzed synthesis of aryl ketones from nitriles and aryl sulfinates.107 Satisfyingly, with the addition of 1 equiv. of TFA the isolated yield of 6d was increased (89%), the reaction time was shortened to 30 min and it was no longer necessary to have a separate hydrolysis step (Table 3, entry 5). When this new protocol was used, 6a-c was isolated in yields similar to those achieved in the previous study39 or better (6c).

The compromise in decreasing the amount of nitrile used and still having a productive, high yielding protocol required further investigations. In some

37

cases reducing the nitrile concentration from 5 to 2 equivalents did not have a big impact on the reaction outcome; 6d was isolated in 89% yield when 5 equiv. of 5d were used and the yield decreased to 86% with 2 equiv. of the nitrile (Table 3, entry 6). With other nitriles the drop in yield was bigger; 5 equiv. of 5e provided 6e in 94% yield while 2 equiv. only gave a 78% yield of 6e (Table 3, entries 9 and 10). Difficulties with the purification process with large amounts of excess nitrile made it easier to produce most of the aryl ketones in Table 3 with 2 equiv. of nitrile.

Attempts were also made to reduce the catalyst loading. When 4 mol% Pd(O2CCF3)2 and 4.8 mol% ligand were used in the synthesis of 6d, the yield was slightly reduced from 86% to 84% (Table 3, entries 6 and 7). Aryl ke-tone 6d was provided in 76% yield when the catalyst loading was decreased even further to 2% (Table 3, entry 8). Reducing the amount of Pd-catalyst to 4% did not affect the synthesis of 6k (Table, entries 18 and 19). A consider-able difference was noticed (according to LC-MS analysis) when more slug-gish reactants were used (Table 4). In order to develop a robust protocol, the aryl ketones in Table 3 were synthesized using 8 mol% Pd(O2CCF3)2.

38

Table 3. Scope of nitriles.

Entry Nitrile Product Yield (%)

Entry Nitrile Product Yield (%)

1 MeCN

5a

6a

71a,b 15

5h

6h

50c

2 PrCN

5b

6b

64c,d 16

5i

6i

38c

3 PhCN

5c

6c

82a,b 17

5j

6j

68c

4 5 6 7 8

BnCN 5d

6d

68d

89a,b

86c

84e

76f

18 19

5k

6k

82c

83f

9 10 11 12 5e 6e

94a

78c

60g

45h

20

5l

6l

42c

13

5f 6f

72c 21 22 23

5m

6m

47i

37j 53k

14

5g

6g

73c

Isolated yields >95% pure according to LC-MS and 1H-NMR. Reagents and conditions: a A 5 mL microwave transparent vial was charged with Pd(O2CCF3)2 (8 mol%), 6-methyl-2,2’-bipyridine (9.6 mol%) and THF (2 mL). After stirring for 5 min 2,6-dimethoxybenzoic acid (1 mmol), nitrile (5 equiv.), TFA (1 equiv.) and water (200 µL) were added, the vial was sealed, and the mixture was heated in the microwave reactor for 30 min at 130 °C. b The highest yields obtained by Lindh et al. were 94% for 3a, 73% for 3b, 20% for 3c and 73% for 3d. c Like a but with 2 equiv. nitrile. d Like a but with no TFA, conducted on a 0.5 mmol scale, heated in the microwave reactor for 4 h at 130 °C, with 1 mL formic acid added and the mixture was heated again for 15 min at 130 °C.

39

Table 3. Continuede Like a but with 2 equiv. nitrile, 4 mol% Pd and 4.8 mol% ligand. f Like a but with 2 mol% Pd and 4.8 mol% ligand. g Like a but with 1 equiv. nitrile and 2 equiv. 2,6-dimethoxybenzoic acid. h Continous-flow scale-out; the flow was 0.5 mL/min, corresponding to 2 min in the heated zone and the temperature was 210 °C. A solution of yield determining 1a (0.71 M in THF and 10% water), 2e (5 equiv.), TFA (25 equiv.), Pd(O2CCF3)2 (4 mol%) and 6-methyl-2,2’-bipyridine (9.6 mol%) was heated in the CF-MAOS. The yield was based on work-up of an aliquot of 14 mL of the mixture, with a theoretical yield of 1 mmol of 3e. i Like a but with 1 equiv. nitrile, 2 equiv. 2,6-dimethoxybenzoic acid and 10 equiv. TFA; 1 h at 130 °C. j Like a but with 2 equiv. nitrile, 10 equiv. TFA; 1 h at 130 °C. k Like a but with 2 equiv. nitrile, 3 equiv. TFA, no water addition, dry THF and 1 h at 130 °C.

4.2 Scope of Nitriles The scope and limitations of the protocol were investigated next. Electronic effects did not seem to affect the reaction outcome; 6f was isolated in 72% yield using electron-rich nitrile 5f. The yield obtained with electron-poor nitrile 5g was 73% (Table 3, entries 13 and 14). Pd(II)-catalyzed insertion of the aldehyde108,109 competed with product formation during the synthesis of 6h (Table 3, entry 15) and only a modest yield of 50% was isolated (trace amounts of the alcohol were detected by LC-MS analysis).

Notably, heterocyclic nitriles also worked well with this protocol. The electron-poor nicotinnitrile 5k furnished 6k in 82% yield whereas the yield obtained with electron-rich furan-2-carbonitrile (5j) was lower, 68% (Table 2, entries 17 and 18). Surprisingly, sterically hindered 6i was isolated in 38% yield even though the benzoic acid has two ortho-methoxy groups and the benzonitrile has an ortho-bromine (Table 3, entry 16). No Pd(0)-catalyzed activation of the aryl bromide functionality was detected according to LC-MS, full chemoselectivity was also obtained with 6e.

Dicyanide 5l did not give the diarylated compound; only trace amounts of this product were detected according to LC-MS analysis, despite that the reaction furnished 6l in moderate 42% yield (Table 3 entry 20).

According to an article by Wang et al.110 benzofurans can be synthesized from 2-(2-hydroxyphenyl)acetonitrile (5m). During initial attempts to adjust that synthesis to fit this protocol it was deemed necessary to have a more acidic reaction mixture, with 10 equiv. of TFA, for complete in situ cycliza-tion of the intermediate. When the benzoic acid was in excess, this protocol furnished 6m in 47% yield. However, if 2 equiv. of nitrile were used, the yield dropped to 37% (Table 3, entries 21 and 22). Formation of a byproduct of benzofuran-2(3H)-one was observed (detected by LC-MS analysis and confirmed by co-elution when spiked with the authentic byproduct). The byproduct was generated via hydrolysis of the nitrile and subsequent lactoni-sation, and was competing with the desired reaction (Figure 18). In an effort to prevent the hydrolysis and byproduct formation, an experiment was car-

40

ried out with no water addition, dry THF and only three equiv. of TFA; the obtained yield of 6m was 53% (Table 3, entry 23).

Figure 18. Synthesis of benzofuran 6m. For conditions and yields see Table 3 en-tries 21-23.

Sometimes the nitrile can be more expensive or precious than the carboxylic acid and it is then necessary to reverse the stoichiometry and have the ben-zoic acid in excess. Unfortunately, aryl ketone 6e was obtained in 78% yield when benzoic acid 4a was the limiting reagent, but in only 60% when the stoichiometry was reversed (Table 3, entries 10 and 11). There are two fac-tors that are important for the reaction outcome. If the nitrile is in excess it is favorable for the co-ordination and insertion of the nitrile into the Pd-complex, while a competing background decarboxylation is less of a prob-lem with the benzoic acid in excess.

Continuous-flow microwave-assisted organic synthesis (CF-MAOS) is a fast and energy-efficient way of synthesizing large quantities of product, without the safety considerations and need for the space-taking equipment normally associated with upscaling procedures.111-113 The synthesis of 6e was chosen as a model reaction for a scale-out using a borosilicate glass, tubular reactor (3 mm inner diameter, 200 mm long). Disappointingly, local super-heating and rupture of the reactor were caused by Pd(0) precipitation on the reactor inner wall.114,115 Decreasing the palladium loading and increasing the ligand concentration were not sufficient to prevent the reactors from break-ing down. An attempt to add palladium reoxidant p-benzoquinone reduced the amount of palladium precipitation but also affected the conversion (ac-cording to GC-MS). Using 25 equiv. of TFA a scale-out was possible at 0.5 mL/min and 210 ºC, and 6e was isolated in moderate 45% yield (Table 3, entry 12). If CF-MAOS is to be used as a way of producing large quantities of aryl ketones with this methodology further optimization is required.

41

4.3 Scope of Aryl Carboxylic Acids To expand the scope of the batch reaction even further the reaction was run with several different benzoic acids and a diverse set of four nitriles: 5c, 5e, 5j and 5k. A number of (hetero)benzoic acids were scanned. The best results were achieved with 3-ethoxythiophene-2-carboxylic acid (4b), acetyl-3,5-dimethyl-1H-pyrrole-2-carboxylic acid (4c), 2,6-difluoro-4-methoxybenzoic acid (4d) and 3-bromo-2,6-dimethoxybenzoic acid (4e) (Table 4).

A decrease in reactivity using the optimized conditions from Table 3 with the less electron-rich benzoic acids meant it was necessary to increase the nitrile concentration to 5 equiv. and the processing time to 1 h (instead of 30 min) at 130 ºC to achieve full conversion. Under these conditions the reac-tion worked well with various heterocyclic acids and nitriles. Moderate to good yields of 7a, 7c, 7d, 8a, 8c and 8d (48-73%) were ob-tained using benzoic acids 4b or 4c and nitriles 5c, 5j or 5k (Table 4, entries 1, 3-5, 7 and 8).

Problems with removing excess nitrile during the purification were en-countered with all the reactions involving 2-(4-bromophenyl)acetonitrile (5e). To remedy this, the reactions were conducted with either 2 equiv. of nitrile (yielding 7b in 41% and 8b in 70%, entries 2 and 6) or with a stoichi-ometry switch (2:1 of the benzoic acid:nitrile), as was done during the syn-thesis of 9b and 10b (76% and 73% respectively, entries 10 and 14).

Carboxylic acid 4d and 4e are less electron-rich and were less reactive; according to LC-MS full conversion was not obtained. The amount of sol-vent was therefore decreased 4-fold to improve the reaction outcome and good yields of 68-75% of 9a, 9c and 10a were obtained (entries 9, 11 and 13). Nitrogen co-ordination to palladium might explain the disappointing yields of 26% of 9d and 9% of 10d (entries 12 and 17). Having the benzoic acid in excess during the synthesis of 10d increased the yield slightly to 19% (entry 18). The same stoichiometry switch provided 10c in 50% yield; in contrast a yield of 36% was obtained when the nitrile was in excess (entries 15 and 16).

42

Table 4. Matrix variation of 4 benzoic acids with 4 nitriles.

Entry Product Yield (%) Entry Product Yield (%)

1

7a

61a 9

9a

68c

2

7b

41b 10

9b

76d

3 7c

59a 11

9c

75c

4

7d

48a 12

9d

26c

5

8a

73a 13

10a

68c

6

8b

70b 14

10b

73d

7

8c

64a 15 16

10c

36c

50d

8 8d

66a 17 18

10d

9c

19d

Isolated yields >95% pure according to LC-MS and 1H-NMR. Reagents and conditions: a A 5 mL microwave transparent vial was charged with Pd(O2CCF3)2 (8 mol%), 6-methyl-2,2'-bipyridine (9.6 mol%) and THF (2 mL). After 5 min of stirring 4b, 4c, 4d or 4e (1 mmol), 5c, 5e, 5j or 5k (5 equiv.), TFA (1 equiv.) and water (200 µL) were added, the vial was sealed, and the mixture was heated in the microwave reactor for 1 h at 130 ºC. b Like a but with 2 equiv. of the nitrile.c Like a but with 500 µL THF and 50 µL water. d Like a but with 500 µL THF, 50 µL water, 2 equiv.of benzoic acid 4b, 4c, 4d or 4e and 1 equiv. nitrile.

43

5. HIV-1 Protease Inhibitors: Improved Antiviral Activity in Cells (Paper III)

The use of a tertiary alcohol as part of the transition-state mimic in protease inhibitors has often resulted in less potent inhibitors than their secondary counterparts.116-120 In 2005, Ekegren et al. combined the shielded tertiary alcohol-containing core with the ethylhydrazide group from atazanavir and the indanolamine moiety from indinavir.121 Several series of potent inhibitors were synthesized with Ki values down to 2.1 nM and good membrane per-meability profiles in a Caco-2 assay (n=0, Figure 19). However, there was room for improvement concerning metabolism and cellular antiviral activity; the best EC50 value was 170 nM.122,123

Increasing the length of the tether between the quaternary carbon and the hydrazide β-nitrogen could address these issues, a three-carbon tether (n=2, Figure 19) did not show sufficient improvement.124,125 Computational studies and X-rays of inhibitors previously synthesized suggested that compounds with a two-carbon spacer would position the hydroxyl group closer to the Asp25, with associated predicted improvements in potency (n=1, Figure 19). The P1’ part of the inhibitors was also investigated in order to improve the EC50 values. Because the indanolamine moiety in indinavir is metabolically unstable due to 3’-hydroxylation of the indan,126,127 the P2 portion of the inhibitors was also varied.

Figure 19. General structure for the new class of inhibitors, with n=1.

5.1 Chemistry Scheme 2 depicts the synthetic route for the preparation of inhibitors 20-28. An aldol condensation between commercially available γ-butyrolactone and

44

benzaldehyde furnished 12. Epoxidation using mCPBA yielded 13 which was reduced with Pd/C, HCOOH and Et3N to give tertiary alcohol 14. The lactone was opened with (1S,2R)-1-amino-2-indanol and stoichiometric amounts of 2-hydroxypyridine, and the two diastereoisomers (S)-15 and (R)-15 obtained were separated by flash column chromatography (the absolute configuration was confirmed by X-ray crystallography, section 5.3). To avoid oxidation of the secondary alcohol of the indan moiety, a protective group was needed. First the primary alcohol was protected with TBDPS-Cl, and then the secondary alcohol was protected using 2-methoxypropene. Next the silyl group was deprotected with TBAF, to give (S)-17. Oxidation with Dess-Martin periodinane afforded the corresponding aldehyde which was immediately reacted with hydrazide 18 according to a reductive amination protocol121 that yielded (S)-19 with a bromine in the P1’ position. The same route was followed to synthesize (R)-19 from (R)-15.

Inhibitors with different P1’ variations were synthesized using Suzuki-Miyaura cross-couplings with boronic acids/esters at 120 °C under micro-wave irradiation for 30 min.128-130 Purification by RPLC-MS furnished 20-26 in 20-62% yield. Stille-Migita cross-couplings gave inhibitors 27 and 28 with a pyridine in the P1’ position and yields of 40% and 31%, respectively, after preparative RPLC-MS. Scheme 2

45

Reagents and conditions: (a) I. Benzaldehyde, KOtBu, benzene, room temp, 6 h; II. H2SO4, 56%; (b) mCPBA, AIBN, DCE, reflux, 6 h, 60%; (c) Pd/C, HCOOH, Et3N, EtOAc, 80 °C, 3 h, 96%; (d) (1S,2R)-1-amino-2-indanol, 2-hydroxypyridine, DCM, sealed tube, 60 °C, over-night, 66%; (e) TBDPSCl, imidazole, DCM, room temp, overnight, 80%; (f) I. 2-methoxypropene, pyridinium p-toluenesulphonic acid, DCM, 0 °C, 6 h; II. 1 M TBAF in THF, THF, room temp, 3 h, 69%; (g) I. Dess-Martin periodinane, DCM, room temp, 30 min; II. 18, NaBH(OAc)3, AcOH, THF, room temp, overnight, 40%; (h) 20-26: R–boronic acid/ester, Pd(PPh3)2Cl2, Na2CO3 (aq.), EtOH, DME, MW, 120 C, 30 min, 20–62%. 27, 28: RSn(nBu)3, Pd(PPh3)2Cl2, CuO, DMF, MW, 120 C, 50 min, 40% and 31%, respectively. Reductive amination between (S)-17 and the benzyl or fenethyl analog121,131 of the prime side 18 gave inhibitors 31 and 32 (Scheme 3). Scheme 3

Reagents and conditions: (a) I. Dess-Martin periodinane, DCM, room temp 30 min; II. 29 or 30, NaBH(OAc)3, AcOH, THF, room temp, overnight, 21% and 28%, respectively.

Inhibitors (S)-36 and (S)-37 had a core amide instead of a hydrazide, making them shorter than the inhibitors with a one-carbon tether, previously synthe-sized by Ekegren et al.121 Benzylamine or 4-bromobenzylamine attacked epoxide 33,121 and an amide coupling with (S)-2-(methoxycarbonylamino)-3,3-dimethylbutanoic acid gave (S)-36 and (S)-37 (Scheme 4). Scheme 4

Reagents and conditions: (a) Benzylamine or 4-bromobenzylamine, Ti(OiPr)4, dry THF, 40 ºC, overnight, 41% and 57% for (S)-34 and (S)-35 respectively; (b) (S)-2-(methoxycarbonylamino)-3,3-dimethylbutanoic acid, HATU, dry DMF, DIPEA, room tem-perature, 5 days ((S)-36), 60 ºC, overnight ((S)-37), 17% and 10% for (S)-36 and (S)-37 re-spectively. (S)-36 and 37 were isolated as epimeric mixtures 3:1.

46

Several inhibitors with variation at the P2 position were synthesized by lac-tone opening of 14 with (S)-2-amino-N,3,3-trimethylbutanamide, (S)-2-amino-N,3-dimethylbutanamide or (S)-2-amino-N-(2-methoxyethyl)-3-methylbutanamide (I, II or III, Scheme 5). For inhibitors (S)-40 and (S)-41 the two diastereoisomers were separated after the reductive amination step, while the separation was made after the lactone opening for intermediate (R)- and (S)-38. Reductive amination with 18 yielded (R)- and (S)-39. The three different pyridines in the P1' position were synthesized from inhibitor (S)-39 using Stille-Migita and Suzuki-Miyaura cross-couplings (Scheme 6).

Scheme 5

Reagents and conditions: (a) (S)-2-amino-N,3,3-trimethylbutanamide or (S)-2-amino-N,3-dimethylbutanamide or (S)-2-amino-N-(2-methoxyethyl)-3-methylbutanamide, 2-hydroxypyridine, DCE, 80 ºC, overnight, 63-75%; (b) I. IBX, DCE, 80 ºC, 2 h. II. 18, DCE, AcOH, Na BH(OAc)3, room temp, overnight, 2-28%. Scheme 6

Reagents and conditions: (a) 42: 2-PyridylSn(nBu)3, Pd(PPh3)2Cl2, CuO, DMF, 120 C, 50 min, 17%. 43, 44: R'-B(OH)2, Pd(OAc)2, [(t-Bu)3PH]BF4, K2CO3, water, DME, MW, 80 ºC, 20 min, 44% and 10% respectively.

47

5.2 Biological Evaluation The results of an enzyme inhibition and cell-based assay of the 25 final compounds are shown as Ki and EC50 values in Table 5, together with toxici-ty measurements in MT4 cells, expressed as CC50 values. The one-carbon tether inhibitors A, B and C with a bromine, 2- and 3-pyridine moiety in P1' are included for comparison.121,123 Inhibitors D and E, with the three-carbon spacer, are also entered,124 along with saquinavir (SQV), ritonavir (RTV), indinavir (IDV), lopinavir (LPV), atazanavir (ATV) and darunavir (DRV), with both literature and in house antiviral activities provided. Inhibitors with the (S)-configuration 19-28, 30 and 32 were excellent enzyme inhibitors with Ki values ranging from 1.2-12 nM. Changing the core of the inhibitors from a one- to a two-carbon chain gave a 27-fold decrease in the cell-based assay; A and (S)-19 had EC50 values of 1100 and 40 nM respectively. This could possibly have been the result of an improved masking of the tertiary alcohol.

With EC50 values ranging from 3-13 nM, most of the nine inhibitors with P1' variations at the para position of the aryl, 20-28, exhibited excellent antiviral activity (except for 26). The EC50 values for six of the inhibitors were in the single digit nM range. This constitutes a 10-fold decrease com-pared to the bromo analogue (S)-19. When inhibitor 21, with an EC50 value of 7 nM, was compared with some of the PIs on the market it is substantially more potent than RTV and IDV (EC50 = 50 nM for both in an in-house as-say), in the same range as SQV, LPV and ATV (EC50 values of 10, 10 and 8 nM in the in-house assay respectively), and half as potent as DRV (EC50 = 4 nM in the in-house assay).

The benzyl and fenethyl analogs (31 and 32) were less potent; 32 was al-most inactive in the cell-based assay (EC50 = 6800 nM). Inhibitors (S)-36 and 37, which have an amide instead of the central hydrazide, were also inactive. An increase in the EC50 values was seen in the inhibitors where the indanolamine had been replaced with N-alkylated amino acids at the P2 posi-tion. Inhibitors (S)-39-44 had Ki values ranging from 1.0-3.2 nM and EC50 values of 37-170 nM. The known metabolic problems with indanolamine are an issue126,127 and further exploration of this type of inhibitor is justified. Three of the inhibitors, (S)-19, 22 and 25, showed cell toxicity properties, with CC50 values below 10 µM.

48

Table 5. HIV-1 PR enzyme inhibition, antiviral activity in a cell-based assay and cytotoxicity of inhibitors 19-44a and A-E.121,123,124

Cmpd Yield Kib

(nM)

EC50c

(nM)

CC50

(nM)

A - 2.4 1100 -

B - 12.0 900 >10

C - 5.0 180 >10

D - 3.3 850 >10

E - 2.8 170 >10

(S)-19 40 2.9 40 7.9

(R)-19 36 >5000 >10 000 >10

49

Table 5. Continued

20 35 12 8 >10

21 40 1.7 7 >10

22 31 1.2 9 4.9

23 35 2.3 3 >10

24 50 11 13 >10

25 62 8.0 3 >10

50

Table 5. Continued

26 62 1.8 30 >10

27 20 2.8 5 >10

28 51 1.7 10 >10

31 21 2.3 190 >10

32 28 8.2 6800 >10

(S)-36 10 >5000 >10 000 -

(R)-36 17 >5000 >10 000 -

51

Table 5. Continued

37 17 >5000 >10 000 -

(S)-39 28 1.7 72 >10

(R)-39 26 2200 >10 000 >10

(S)-40 3 2.9 160 >10

(R)-40 5 1240 >10 000 >10

(S)-41 2 3.2 170 >10

(R)-41 7 2755 >10 000 >10

52

Table 5. Continued

42 17 1.1 47 >10

43 44 1.0 37 >10

44 10 2.0 40 >10

SQV - 0.2

(1.4132)

10

(13132) -

RTV - 0.8

(4.2132)

50

(70132) -

IDV - 0.3

(0.5133)

50

(4181) -

LPV - 1.4

(0.0013134)

10

(17134) -

ATV - 0.5

(2.7132)

8

(3.9132) -

DRV - 1.1

(0.01686)

4

(3.7135) -

aConditions: See Scheme 2 (19-28), Scheme 3 (31, 32), Scheme 4 (36, 37), Scheme 5 (39-41) and Scheme 6 (42-44). bThe Ki values for the drugs on the market are from an in-house assay. Ki values from the literature appear in parentheses. cThe EC50 values for the drugs on the market are from an in-house assay. EC50 values from the literature appear in parentheses.

Based on their favourable Ki/EC50/CC50 profile three of the inhibitors, (S)-19, 21 and 22, were further evaluated. Intrinsic clearances and rates of mem-brane permeation in a Caco-2 assay are shown for the three inhibitors in Table 6. A and D are also included for comparison. Unfortunately inhibitors (S)-19 and 22 were extensively degraded by metabolic enzymes, in a liver microsome homogenate, and exhibited intrinsic clearance values above 300 µL/min/mg. With an intrinsic clearance value of 20 µL/min/mg inhibitor 21

53

was considered reasonably stable. Many HIV-1 PIs suffer from rapid degra-dation by cytochrome P-450 enzymes, which is part of the reason they have low oral bioavailability,82 thus it is of vital importance to have good intrinsic clearance.

Inhibitor 21 was slower to permeate the Caco-2 membranes in an assay than (S)-19 and 22 (Papp (Caco-2) = 3.5 × 10-6 cm/s for 21, 42 × 10-6 cm/s and 26 × 10-6 cm/s for (S)-19 and 22, respectively). However, the excellent EC50 value for inhibitor 21 means that permeability is unlikely to be as problemat-ic as the Papp (Caco-2) value suggests, as the membranes of HIV-infected MT4 cells are different from those of Caco-2 cells (or the discrepancy indi-cates active transportation).

Table 6. Stability of and Caco-2 permeability to selected inhibitors.

Cmpd Clint

(µL/min/mg)

Papp (Caco-2)

(× 10-6 cm/s)

C 154 33

E 94a 3-20

(S)-19 > 300 42

21 20 3.5

22 > 300 26 aParent compound remaining (%) PCR.

Since resistance is such a substantial problem for the HIV-1 PIs on the mar-ket it was important to test the same three inhibitors ((S)-19, 21 and 22) against selected and clinically relevant PI resistant isolates of HIV-1 (Table 7). Cell-free HIV was passed in media containing stepwise increasing con-centrations of the inhibitors (SQV, RTV or a symmetric diol-based inhibitor136,137) presented in Table 7 to obtain the resistant isolates.

Inhibitors (S)-19, 21 and 22 had EC50 values better than or in the same range as the wild type protease and mutated isolate G48V, L90M, often seen in patients treated with saquinavir138 (entry 2, Table 7). Compound 21 was as potent as the wild type against the mutated isolate V32I, M46I, A71V, V82A ((S)-19 and 22 retained much of their potency) (entry 4, Table 7). Amino acid V82A is located in the S1' subsite and M46I in the flap region of the protease, and the mutation causes resistance in many of the inhibitors on the market.139,140

Universal protease associated mutations (UPAMs) are four mutations at amino acids 33, 82, 84 and 90; these are often seen in patients failing HIV therapy.138 When one UPAM was present (entries 2, 4 and 5, Table 7) much of the potency was preserved, as was also the case when two UPAMs were present (entry 3); however, this was not the case when the mutation occurred in both residues 82 and 84 (entry 6). Inhibitor 21 exhibited a good profile against mutated isolates with the exception of mutations in both positions 82

54

and 84. As will be described in section 5.3, from X-ray crystal analysis of the mutated isolate L63P, V82T, I84V co-crystallized with inhibitor 21 (Fig-ure 22), it looks like this is because of a loss of hydrophobic interactions.

Table 7. Antiviral activity against some HIV-1 protease inhibitor-resistant isolates.

Entry Mutations

Inhibitors

used to obtain the

resistant isolate

EC50 (S)-19

(µM)

EC50 21

(µM)

EC50 22

(µM)

1 wild-type (wt) - 0.040 0.007 0.009

2 G48V, L90M SQV 0.008 0.008 0.010

3 A71V, I84V,

L90M SQV 0.044 0.007 0.019

4 V32I, M46I,

A71V, V82A

Symmetric diol-

based

inhibitor136,137

0.070 0.006 0.014

5 V32I, M46I, V82A RTV 0.074 0.024 0.027

6 M46I, V82F, I84V RTV 0.24 0.13 0.60

Ki (S)-19

(nM)

Ki 21

(nM)

Ki 22

(nM)

7a L63P, V82T, I84V 30 16 16 a Ki atazanavir 6.5 nM.

5.3 X-ray Crystallography The 1.7 Å X-ray structure (PDB code 2wkz) of wild type HIV-1 PR co-crystallized with inhibitor 21 is shown in Figure 20. The arrangements of compound B123 (PDB code 2cem) and D124 (PDB code 2uxz) with a one- and three-carbon spacer, respectively, are shown for comparison. Similar to inhi-bitor 21, compound B has a 2-pyridine in the P1' position whereas structure D has a bromine. Inhibitor 21 is rotated 180º compared to B and D. This means that, for example, Asp125 from one monomer in inhibitor 21 is Asp25 from the other monomer in B and D.141

As expected the crystal structure of 21 confirmed the (S)-absolute confi-guration of the quaternary carbon. Inhibitor 21 has seven direct hydrogen bonds to the protease and five bonds via water molecules, while B has a six/five ratio, and D has a five/three ratio. A water molecule is similarly arranged between the NH terminal of Ile50/Ile150 residues in the backbone and the two carbonyl groups of all three inhibitors. A difference in binding mode among the three inhibitors is caused by the indanolamine hydroxyl

55

group which forms one hydrogen bond to the backbone NH-group of Asp29 of the enzyme (3.0 Å) and two hydrogen bonds via a water molecule (2.8 and 3.1 Å) for 21, and two hydrogen bond to Gly127 and Asp129 for B and D.

The tertiary alcohol in inhibitor 21 forms a hydrogen bond not only with Asp25 (with a binding distance of 2.7 Å; the distance for B was also 2.7 Å, while that for D was 2.9 Å), it also forms another hydrogen bond with the backbone carbonyl of Gly27 (distance 2.6 Å; 3.3 Å for B, too far away to form a bond). This additional hydrogen bond is probably part of the reason that the two-carbon tether inhibitors have better antiviral activity than the one- and three-carbon tether inhibitors.

B

21

D

Figure 20. X-ray crystal structure of 21, B and D123,124 co-crystallized with HIV-1 protease.

56

The superimposition of inhibitors 21 and B reveals a significant difference in how well the P1 benzylic group of inhibitor 21 fits in the S1 and S3 pockets (Figure 21). In inhibitor B there is only a hydrophobic interaction between the para position of the benzyl group and Val82. The same group in inhibi-tor 21 is positioned more closely and forms three hydrophobic interactions with one of the methyl groups in Val182.142 The close vicinity also causes one of the methyl groups in the valine to bend upwards, further increasing the hydrophobic interactions.

Since the guanidine aminogroup of Arg108 is 4.0 Å away from the meta position of the benzylic group in 21 it can form edge-on cation-π interac-tions.143 This is augmented by the fact that the guanidine amino group is slightly tilted towards the inhibitor. Similar interactions cannot occur with inhibitor B since the distance in this case is 7.3 Å. However, inhibitor 21 can only interact with Ile184 via the benzylic carbon (3.4 Å) while B is situated more closely to Ile84 and can interact hydrophobically via both its ipso car-bon (3.5 Å) and the two ortho positions (3.6 and 3.9 Å). Overall, the P1 moiety of inhibitor 21 fits better in the S1 and S3 pockets, which partly ex-plains the improved inhibiting potency of the two-carbon tether series.

Figure 21. Superimposition of inhibitors 21 in grey and B in green.

The X-ray complexes of mutant L63P, V82T, I84V/wild type PR and 21 are depicted in Figure 22 (PDB code 2w10). The Ki value for 21 was 10 times higher for the mutant than for the wild type (Table 7). In position 182, one of the two methyl groups of the valine is changed to a hydroxyl group in threonine, which cannot interact hydrophobically with the inhibitor.142,144,145 In position 184 the ethyl group of the wild type isoleucine turns into a valine methyl group with less steric bulk, which decreases the propensity for hydrophobic interactions with the P1 benzylic group. The distance between the groups changes from 3.4 Å in the wild type to 4.2 Å in the mutated I84V.

57

Figure 22. The nonprime side of the wild type of the protease and inhibitor 21 in gray together with the mutant L63P, V82T, I84V and 21 in green.

58

6. HIV-1 Protease Inhibitors with a Tertiary Alcohol as a Transition-State Mimic: Investigating Macrocyclic Inhibitors with P1 Side Chain Links to the P3 Position

The benefits of forming a macrocycle in medicinal chemistry projects are often substantial.146,147 Structural pre-organization of macrocycles offers a compromise between an entropy advantage from having the bioactive con-formation before binding and enough flexibility to maximize binding inte-ractions. Peptide-based macrocycles tend to have an improved affinity for their targets, if they fit in the binding pockets. They also tend to have an improved metabolic profile since macrocycles are not easily recognized and therefore not always degraded by proteases. Some examples indicate that HIV-1 protease inhibitors can benefit from having their P1 and P3 areas linked in a macrocycle.148,149 Joshi et al.150 has also synthesized a series of 14- and 15-membered P1-P3 macrocyclic HIV-1 PIs related to the inhibitors in paper III with a three-carbon tether between the quaternary carbon and the hydrazide β-nitrogen. They were slightly more potent than their linear ana-logs.

6.1 Chemistry The synthesized acyclic inhibitors in paper III showed excellent antiviral activities and were chosen as starting points for the creation of macrocyclic analogs. Because of the metabolic instability of the indanolamine126,127 the preferred P2 group was synthesized from tert-leucine (like 39 in paper III). Computational studies indicated that 14- and 15-membered rings, linking the P1 to the P3 site, would be an appropriate size to fit into the S1-S3 pockets of the HIV-1 protease. However, a handle in the P1 position was needed in order to form the macrocyclic ring via a ring closing metathesis reaction. The initial idea, depicted in Scheme 7, was to proceed via an intermediate with a bromine in the para position of the P1 benzylic group, to later be able to perform a cross-coupling reaction for introducing a vinyl functionality via a Suzuki-Miyaura reaction (for 14-membered rings) or an allyl moiety via a Stille-Migita reaction (for 15-membered rings), that can cyclize with 48.

59

N-Boc protected tert-leucine was amide coupled with allylamine to fur-nish 54 in 65% yield and the following deprotection gave 48 in quantitative yield (Scheme 8). During the first attempts to make alkene 45 an aldol con-densation between γ-butyrolactone and 4-bromobenzaldehyde furnished the byproducts 4-bromobenzoic acid and (4-bromophenyl)methanol via a Can-nizzaro reaction151 and inconsistent yields of 45 ranging from 11% to 33% were obtained. However, switching to a more reliable Wittig reaction where a commercially available α-bromo-γ-butyrolactone was reacted with PPh3, followed by a basic workup gave the intermediate ylide, which was reacted with 4-bromobenzaldehyde, furnished 45 in 65% yield (Scheme 7). Epoxida-tion of 45 using mCPBA gave 46 in 72% yield. Since the reduction of the epoxide could not be performed using the reduction conditions mentioned in paper III (Pd/C, formic acid and triethylamine)152 without affecting and re-moving the bromine, an alternative reduction protocol was evaluated using NaBH4 as the reducing agent. Unfortunately, this route produced a diol via a Payne rearrangement153,154 and the racemic mixture of anti-isomers 55a and 55b could not be used in the synthetic route (Figure 23).

An NMR investigation excluded the formation of diol 56 (which would have been formed if both the carbonyl group and the epoxide had been re-duced) and the fact that none of the oxidative conditions tested (PCC, PDC, Pinnick, Parikh-Doering of the Swern type or Dess Martin) afforded the desired product 47 provided further proof that the diol 56 was not formed. Attempts to decrease the amount of NaBH4 to 0.2 equivalents gave the same byproducts 55a and 55b formed via the Payne rearrangement, probably as a result of reduction of the carbonyl group occurring before reduction of the epoxide. NaBH3CN was not effective as a reducing agent and only starting material was detected according to LC-MS analysis.

60

Scheme 7

Reagents and conditions: (a) I. PPh3, THF, reflux, overnight. II. water, NaOH. 3. 4-bromobenzaldehyde, toluene, reflux, overnight, 65%. (b) mCPBA, AIBN, DCE, dark, 80 ºC, overnight, 72% (c) NaBH4, dry THF, rt, 3 h. Scheme 8

Reagents and conditions: (a) allylamine, Et3N, EDC, DCM, room temp, overnight, 65% (b) TFA, room temp, 3 h, quantitative yield.

61

Figure 23 The Payne rearrangement.

With a hydroxyl group in the para position of the benzyl group, catalytic hydrogenation could be used to reduce the epoxide, and the Payne rear-rangement problem could be avoided. Importantly, formation of the corres-ponding aryl triflate could enable the Suzuki-Miyaura and Stille-Migita reac-tions to synthesize 52a and 52b. When the Wittig procedure was carried out with 4-hydroxybenzaldehyde, 57 was isolated in 43% yield, and epoxidation using mCPBA gave 58 in 55% yield (Scheme 9). Unfortunately, the reduc-tion gave very low yields and was unreliable; sometimes no product was formed under conditions that had previously provided product. The reduced product 59 was insoluble in many organic solvents, but was soluble in water and ended up in the water phase during extractions. Although compound 59 was isolated in 30% yield, and triflation155 gave 60 in 25% yield, opening the lactone using amine 48 only resulted in trace amounts of 61.

Scheme 9

Reagents and conditions: (a) I. PPh3, THF, reflux, 2 days. II. water, NaOH. 3. 4-hydroxybenzaldehyde, toluene, reflux, overnight, 43%. (b) mCPBA, AIBN, DCE, dark, 80 ºC, overnight, 55% (c) Pd/C, HCOOH, Et3N, EtOAc, 80 °C, overnight, 30% (d) N-phenyl-bis(trifluoromethanesulfonimide), K2CO3, THF, MW, 25 min, 100 °C, 25%. (e) 48, 2-hydroxypyridine, DCM, sealed tube, 60 ºC, overnight, trace amounts.

62

In an effort to improve the solubility and achieve a reliable and robust reduc-tion protocol the alcohol was silylated using TBDMS-Cl, providing 62 in 80% yield. Thereafter, epoxidation furnished 63 in 56% yield. The silyl group was removed during the reduction, both in the hydrogenation appara-tus (with Pd/C) and with two transfer hydrogenation methods: Pd/C, formic acid and triethylamine156 or ammonium formate. The insoluble intermediate 58 was obtained again, without formation of the reduced product, with or without the protecting group, according to LC-MS analysis in all cases (Scheme 10).

Scheme 10

Reagents and conditions: (a) TBDMS-Cl, imidazole, DMF, room temp, overnight, 80% (b) mCPBA, AIBN, DCE, dark, 80 ºC, overnight, 56% (c) Pd/C, HCOOH, Et3N, EtOAc, 80°C, overnight, no reduced product; Pd/C, HCOOH, Et3N, EtOAc, MW, 100 °C, 1 h, no reduced product; Pd/C, ammonium formate, DMF, 100 °C, overnight, no reduced product; Pd/C, H2

(hydrogenation apparatus), overnight, no reduced product. When the alcohol was masked as a 4-methoxy group, the Wittig route gave 64 in 29% yield followed by 80% yield of 65 in the epoxidation step. Reduc-tions performed either in the hydrogenation apparatus or with the Pd/C, for-mic acid and triethylamine protocol failed to give the tertiary alcohol 66 (Scheme 11).

Scheme 11

Reagents and conditions: (a) I. PPh3, THF, reflux, overnight. II. water, NaOH. 3. 4-metoxybenzaldehyde, toluene, reflux, 29%. (b) mCPBA, AIBN, DCE, dark, 80 ºC, overnight, 80%. (c) Pd/C, HCOOH, Et3N, EtOAc, MW, 110°C, 1 h, no product; PtO2, EtOAc, H2 (Parr apparatus), room temp, overnight, 10 psi, no product. A Sonogashira reaction gave 67 in 22% yield, mostly due to a difficult puri-fication. Unfortunately the reduction step failed to deliver 68 (Scheme 12).

63

Scheme 12

Reagents and conditions: (a) Ethynyltriisopropylsilane, PdCl2(PPh3)2, CuI, DIPEA, DMF, MW, 120 ºC, 1 h, 22%. (b) Pd/C, HCOOH, Et3N, EtOAc, 80°C, overnight, no product.

A new route was evaluated using a precursor which did not contain a lactone group. Starting from 4-bromobenzylbromide and diethyloxalate it was envi-sioned that a Grignard reaction could give 69 (Scheme. 13). An additional Grignard with allylmagnesiumbromide would be used to synthesize 70 and hydroboration-oxidation would give the key intermediate 71. However, reac-tion of commercially available (4-bromobenzyl)magnesium bromide 1 M in THF and diethyloxalate failed to provide 69. Attempts were made to syn-thesize the benzyl Grignard reagent with an antracen-THF-Mg complex157 or iPrMgCl×LiCl (turbogrignard)158 but they were both unsuccessful.

Scheme 13

Reagents and conditions: (a) (4-bromobenzyl)magnesium bromide 1 M in THF, N2-atmosphere, room temp, overnight, no product; I. Mg, antracen, bromoethane, dry THF, N2-atmosphere, room temp, 48 h to form the complex II. 1-bromo-4-(bromomethyl)benzene, room temp, 30 min, 3. diethyloxalate, room temp, 1 h, no product; I. iPrMgCl×LiCl 1.3 M in THF, N2-atmosphere, 0 °C, 10 min II. 1-bromo-4-(bromomethyl)benzene, 0 °C, 2 h, III. di-ethyloxalate (addition of the Grignard solution to the diethyloxalate), -15 °C to room temp, overnight, no product.

Inspired by the route depicted in Scheme 13 a new protocol was envisioned which is outlined in Scheme 14. A Grignard reaction between 1,4-dibromobenzene and allyl bromide furnished monosubstituted 72 in 50% yield. Oxidation using K2OsO4 × 2 H2O and catalytic amounts of NMO gave diol 73 in 87% yield, which was selectively protected with TBDMS-Cl and 74 was isolated in 93% yield. Oxidation to ketone 75 was done using Dess-Martin periodinane in 80% yield. It was predicted that a Grignard reaction with allylmagnesiumbromide would give 76 which might be turned into the

64

key intermediate 77 by oxidation to the diol, using K2OsO4 × 2 H2O, fol-lowed by cleavage with NaIO4. Although the route depicted in Scheme 14 looked promising, lack of time left for this project meant that intermediate 75 was the last compound synthesized using this route.

Scheme 14

Reagents and conditions: (a) Mg, allylbromide, THF, room temp, overnight, 50% (b) K2OsO4 × 2 H2O, NMO, Acetone/water, room temp, overnight, 87% (c) TBDMS-Cl, imidazole, DCM, room temperature, overnight, 93% (d) Dess-Martin periodinane, dry DCM, room temp, 2 h, 80%. Appendix - Experimental data for 45-77 Compound 72 is commercially available.

(E)-3-(4-Bromobenzylidene)dihydrofuran-2(3H)-one (45). α-bromo-γ-butyrolactone (7 g, 42.43 mmol) was added to a solution of PPh3 (11.13 g, 42.43 mmol) in THF (16.8 mL) and the mixture was heated to reflux over-night. The precipitate was filtered off and dissolved in water (70 mL) and aqueous NaOH (10%, 210 mL) was added slowly to the slurry. The solution was then extracted with CHCl3 (3 × 400 mL) and the organic layers were dried (MgSO4) and evaporated (14.56 g). The crude product was added to a solution of 4-bromobenzaldehyde (7.85 g, 42.44 mmol) in 280 mL of tolu-ene. The mixture was heated to reflux overnight and left to cool while stir-ring. The solution was split and each half (1 and 2) was treated separately. 1. A precipitate of crude product (1.66 g) was formed and washed with EtOH. A second (2.75 g) and third (0.14 g) crop was yielded by recrystallization in EtOH. 2. Purification by flash column chromatography (silica gel, EtOAc/isohexane, 1:5) followed by recrystallization (first crop 3.27, second crop 0.15 g) of the product-containing fractions yielded 45. The total mass from 1 and 2 (7.97 g) was purified by dry flash column chromatography (silica gel, EtOAc/isohexane, fractions of 100 mL, starting with 0% EtOAc,

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which was increased by 0.5% each 4th fraction) to yield 45 (7.70 g, 72%). 1H NMR (CDCl3, 400 MHz): 7.51 (m, 2H) 7.44 (t, J = 3.0 Hz, 1H), 7.30 (m, 2H), 4.41 (t, J = 7.2 Hz, 2H), 3,15 (td, J = 7.2, 3.0 Hz, 2H); 13C NMR (CDCl3, 100 MHz): 172.4, 135.6, 133.8, 132.4, 131.5, 124.6, 124.5, 65.6, 27.6; MS m/z: 253 (M+H+), 255 (M+2+H+).

6, 2-(4-Bromophenyl)-1,5-dioxaspiro[2,4]heptan-4-one (46). A solution of 45 (3 g, 11.85 mmol) and mCPBA (4.91 g, 28.45 mmol) in DCE (72 mL) was heated to 80°C and AIBN (47.4 mg, 0.29 mmol) was added. The reac-tion mixture was stirred in the dark, at 80°C, overnight. After cooling to room temperature EtOAc (300 mL) was added and the mixture was washed with Na2S2O3 (300 mL), NaHCO3 (300 mL) and brine (300 mL). The organ-ic layer was dried with MgSO4 and evaporated. Recrystallization of the crude compound in EtOH resulted in 46 (1.72 g) as an off-white solid. A second recrystallization of the mother liquor gave additional 46 (0.5658 g). The total yield was 72%. 1H NMR (CDCl3, 400 MHz): m, 2H), 7.16 (m, 2H), 4.56 (td, J = 9.5, 3.8 Hz,1H), 4.38 (s, 1H), 4.32 (m, 1H), 2,48 (m, 1H), 2.06 (m, 1H); MS (ESI+): m/z: 269 (M+H+), 271 (M+2+H+).

(S)-N-Allyl-2-amino-3,3-dimethylbutanamide (48). Trifluoroacetic acid (4.5 mL) was added to a solution of 54 (1.45 g, 5.37 mmol) in DCM (30 mL) and the solution was stirred at room temperature for 3 h. The mixture was diluted with EtOAc (20 mL) and washed with aqueous NaOH (1 M, 20 mL). The water layer was re-extracted with EtOAc (2 × 30 mL) and the combined organic layers were dried and concentrated. Purification by silica flash col-umn chromatography (1% MeOH, 1% Et3N in DCM) yielded 48 (1.71 g, quantitative yield). 1H NMR (CD3OD, 400 MHz): 5.87 (m, 1H), 5.26 (m, 1H), 5.16 (m, 1H), 3.88 (m, 2H), 3.53 (s, 1H), 1.09 (s, 9H); MS (ESI+): m/z: 171 (M+H+).

(S)-tert-Butyl 1-(allylamino)-3,3-dimethyl-1-oxobutan-2-ylcarbamate (54). A solution of (S)-2-(tert-butoxycarbonylamino)-3,3-dimethylbutanoic acid (1.91 g, 8.25 mmol), allylamine (0.47 g, 8.25 mmol), Et3N (0.92 g, 9.07 mmol) and EDC (1.58 g, 8.25 mmol) in DCM (40 mL) was stirred at room temperature overnight. The solution was diluted with EtOAc (100 mL) and washed with NaHCO3 (2 × 50 mL) and brine (100 mL), and thereafter dried (Na2SO4), filtered and evaporated. The residue was purified by silica flash column chromatography (0.5% MeOH in DCM) yielding 54 (1.457 g, 65%) as a white solid. 1H NMR (CD3OD, 400 MHz): 5.84 (m, 1H), 5.21 (m, 1H), 5.10 (m,1H), 3.87 (m, 1H), 3.80 (m, 2H), 1.45 (s, 9H), 0.98 (s, 9H); MS (ESI+): m/z: 271 (M+H+).

3-[(4-Bromophenyl)(hydroxyl)methyl]tetrahydrofuran-3-ol (55a+55b). NaBH4 (0.31 g, 8.13 mmol) and 46 (1.46 g, 5.42 mmol) were stirred in dry

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THF (75 mL) at room temperature for 2.5 h. Water (100 mL) was added and the mixture was extracted with EtOAc (100 mL). The organic layer was washed with brine (100 mL), dried with Na2SO4 and evaporated. Purification by silica flash column chromatography (EtOAc, isohexane, 1:1) yielded 55a+55b (1.46 g, quantitative yield). 1H NMR (CD3OD, 400 MHz): 7.51 (m, 2H), 7.24 (m, 2H), 4.07 (s, 1H), 3.78 (d, J = 12.3 Hz, 1H), 3.72 (d, J = 12.3 Hz, 1H), 3.56 (t, J = 6.9 Hz, 2H), 1.62 (t, J = 6.9 Hz, 2H); 13C NMR (CD3OD): 136.6, 132.3, 129.4, 122.4, 66.0, 65.5, 61.4, 59.2, 31.5; MS (ESI+): m/z: 273 (M+H+), 275 (M+2+H+).

(E)-3-(4-Hydroxybenzyliden)dihydrofuran-2(3H)-one (57). PPh3 (11.13 g, 42.43 mmol) was dissolved in THF (20 mL). α-Bromo-γ-butyrolactone (7 g, 42.43 mmol) was added to the solution which was heated to reflux for two days. The precipitate was filtered off and rinsed with THF. Aqueous NaOH (10%, 210 mL) was added and the mixture was extracted with DCM (3 × 200 mL). The organic layer was dried with MgSO4 and evaporated. The crude ylide was dissolved in toluene (280 mL) and 4-hydroxybenzaldehyde (5.18 g, 42.43 mmol) was added. The mixture was heated to reflux overnight and then left to cool to room temperature. The solvents were evaporated and the crude product recrystallized in EtOH, which gave a first crop (2.16 g). The mother liquor was evaporated and the solids were redissolved in acetone (60 mL). Ether (100 mL) and isohexane (400 mL) were added to this solu-tion, resulting in formation of a precipitate, which was filtered off and re-crystallized from EtOH. The second crop was purified by flash column chromatography (EtOAc/isohexane, starting with 1:3 then 1:2). The total yield of 57 was 3.49 g, 43%. 1H NMR (CD3OD, 400 MHz): mm, 1H), 6.87 (m, 2H), 4.47 (m, 2H),m, 2H); 13C NMR (CD3OD): 174.4, 136.5, 132.2, 126.4, 120.3, 115.8, 66.10, 27.2; MS (ESI+): m/z: 191 (M+H+).

2-(4-Hydroxyphenol)-1,5-dioxaspiro[2,4]heptan-4-one (58). mCPBA (4.72 g, 27.33 mmol) and AIBN (35 mg, 0.20 mmol) were added to a solu-tion of 57 (2.17 g, 11.39 mmol) in DCE (50 mL) and the mixture was heated to reflux, in the dark, overnight. The solution was cooled to room tempera-ture, whereupon a precipitate of m-chlorobenzoic acid was formed, and re-moved by filtration. The liquid was diluted with EtOAc (175 mL) and washed with Na2S2O3 (150 mL), NaHCO3 (150 mL) and brine (150 ml). The organic layer was dried with MgSO4 and concentrated. Purification by dry flash column chromatography (silica gel, EtOAc/isohexane) yielded 58 (1.28 g, 55%). MS (ESI+): m/z: 207 (M+H+).

3-Hydroxy-3-(4-hydroxybenzyl)dihydrofuran-2(3H)-one (59). A solution of 58 (0.5 g, 2.42 mmol), Pd/C (10%, 0.065 g, 0.061 mmol), HCOOH (0.15 mL, 3.88 mmol) and Et3N (0.51 mL, 3.64 mmol) in EtOAc (4.6 mL) was

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stirred overnight at 80°C. The solution was filtered through a pad of celite and the solvent was evaporated. The solid was purified by silica flash col-umn chromatography (EtOAc/isohexane, 1:1) and washed with NaHCO3 and brine and the organic layer was dried with MgSO4 (the extra extraction was due to m-chlorobenzoic acid left from the previous step) and evaporated to yield 59 (0.162 g, 30%). 1H NMR (CD3OD, 400 MHz): 7.09 (m, 2H), 6.71 (m, 2H), 4.20 (m, 1H), 3.37 (m, 1H), 2.92 (dd, J = 23.2, 13.6 Hz, 2H), 2.35 (m, 1H), 2.10 (m, 1H); 13C NMR (CD3OD): 181.0, 157.7, 132.3, 127.1, 116.1, 76.5, 66.4, 42.9, 35.0; MS (ESI+): m/z: 209 (M+H+).

4-[(3-Hydroxy-2-oxotetrahydrofuran-3-yl)methyl]phenyl trifluorome-thansulfonate (60). N-phenyl-bis(trifluoromethanesulfonimide) (77.2 mg, 0.22 mmol), 59 (50.0 mg, 0.24 mmol), and K2CO3 (33.2 mg, 0.24 mmol) were dissolved in dry THF (500 µL) in a sealed, 2.0 mL Smith microwave transparent vial and heated to 80 ºC in the microwave reactor for 20 min. The solution was filtered and the liquid was thereafter purified by flash col-umn chromatography (silica gel, EtOAc/isohexane, 3:7) to yield 60 (20.3 mg, 25%). 1H NMR (CD3OD, 400 MHz): 7.47 (m, 2H), 7.31 (m, 2H), 4.30 (m, 1H), 4.07 (m, 1H), 3.06 (dd, J = 40.4, 13.7 Hz, 2H), 2.32 (m, 1H), 2.06 (m, 1H); MS (ESI+): m/z: 373 (M+MeOH+H+)

(E)-3-(4-((tert-Butyldimethylsilyl)oxy)benzylidene)dihydrofuran-2(3H)-one (62). Alkene 7 (700 mg, 3.68 mmol), tert-butyldimethyl chlorosilane (665.7 mg, 4.42 mmol) and imidazole (601.1 mg, 8.83 mmol) were dissolved in 10 mL DMF and stirred at room temperature overnight. The mixture was diluted with DCM (50 mL) and washed with 10% citric acid (50 mL). The water layer was re-extracted with DCM (50 mL) and the combined organic layers were washed with brine, dried with MgSO4 and concentrated. Flash chromatography (silica gel, 1% MeOH in DCM) gave 62 as white crystals (883 mg, 80% yield). 1H NMR (400 MHz, CD3OD): δ 7.72 – 7.69 (m, 1H), 7.47 (d, J = 8.6 Hz, 2H), 6.92 (d, J = 8.6 Hz, 2H), 4.48 (t, J = 7.3 Hz, 2H), 3.26 (m, 2H), 0.99 (s, 3H), 0.23 (s, 2H). MS (ESI+): m/z: 305 (M+H+)

2-(4-((tert-Butyldimethylsilyl)oxy)phenyl)-1,5-dioxaspiro[2.4]heptan-4-one (63). A solution of 62 (883 mg, 2.90 mmol) and mCPBA (1.2 g, 6.97 mmol) in DCE (20 mL) was heated to 80°C and AIBN (14 mg, 0.08 mmol) was added. The reaction mixture was stirred in the dark, at 80°C, overnight. After cooling to room temperature EtOAc (100 mL) was added and the mix-ture was washed with Na2S2O3 (100 mL), NaHCO3 (2 × 100 mL) and brine (100 mL). The organic layer was dried with MgSO4 and evaporated. Flash chromatography (silica gel, EtOAc/isohexane, 1:9) provided 63 (526 mg, 56% yield). MS (ESI+): m/z: 321 (M+H+)

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(E)-3-(4-Methoxybenzylidene)dihydrofuran-2(3H)-one (64). PPh3 (7.95 g, 30.3 mmol) was dissolved in THF (14 mL). α-bromo-γ-butyrolactone (5 g, 30.3 mmol) was then added to the solution which was heated to reflux over-night. The precipitate was filtered off and rinsed with THF (3 × 10 mL THF). Aqueous NaOH (10%, 150 mL) was added and the mixture extracted with chloroform (3 × 100 mL). The organic layer was dried with MgSO4 and evaporated. The crude ylene was dissolved in toluene (200 mL) and 4-methoxybenzaldehyde (4.13 g, 30.3 mmol) was added. The mixture was heated to reflux overnight and then left to cool to room temperature. The solvents were evaporated and the crude product was recrystallized in EtOH yielding 64 (1.78 g, 29%). 1H NMR (400 MHz, CD3OD): δ 7.51 (d, J = 8.6 Hz, 2H), 7.46 (t, J = 2.9 Hz, 1H), 6.99 (d, J = 8.6 Hz, 2H), 4.48 (t, J = 7.3 Hz, 2H), 3.84 (s, 3H), 3.25 (td, J = 7.3, 2.9 Hz, 2H). MS (ESI+): m/z: 205 (M+H+)

2-(4-Methoxyphenyl)-1,5-dioxaspiro[2.4]heptan-4-one (65). A solution of 64 (1 g, 4.89 mmol) and mCPBA (2.03 g, 11.75 mmol) in DCE (15 mL) was heated to 80°C and AIBN (17 mg, 0.10 mmol) was added. The reaction mix-ture was stirred in the dark, at 80°C, overnight. After cooling to room tem-perature EtOAc (100 mL) was added and the mixture was washed with Na2S2O3 (100 mL), NaHCO3 (2 × 100 mL), saturated KI solution (100 mL), Na2S2O3 (100 mL), NaHCO3 (100 mL) and brine (100 mL). The organic layer was dried with MgSO4 and evaporated. Flash chromatography (silica gel, EtOAc/isohexane, 1:1) provided 65 (867 mg, 80% yield) MS (ESI+): m/z: 221 (M+H+)

2-(4-((Triisopropylsilyl)ethynyl)phenyl)-1,5-dioxaspiro[2.4]heptan-4-one (67). Compound 45 (50 mg, 0.19 mmol), ethynyltriisopropylsilane (165 µl, 0.76 mmol), PdCl2(PPh3)2 (13 mg, 10 mol%), CuI (8 mg, 22 mol%) and DI-PEA (323 µl, 1.9 mmol) were dissolved in 2 mL DMF and heated in the microwave reactor for 1 h at 120 °C. The reaction mixture was filtered through celite and rinsed with EtOAc. EtOAc (10 mL) was added and and the mixture was extracted with water (10 mL). The water phase was re-extracted with 10 mL EtOAc, the combined organic phases were washed with brine (2 × 10 mL) and dried with MgSO4 and the solvents were evapo-rated. The crude product was dissolved in 2.2 mL MeCN and purified with RPLC-MS (60-100% MeCN in 0.05% aqueous formic acid) affording 67 (15.4 mg, 22% yield). 1H NMR (400 MHz, CD3OD): δ 7.49 (d, J = 8.4 Hz, 2H), 7.32 (m, 2H), 4.53 (ddd, J = 9.8, 9.1, 3.5 Hz, 1H), 4.36 (s, 1H), 4.32 (dt, J = 9.1, 8.2 Hz, 1H), 2.49 (ddd, J = 14.4, 9.8, 8.2 Hz, 1H), 2.04 (ddd, J = 14.4, 8.2 3.5 Hz, 1H), 1.15 (m, 21H). MS (ESI+): m/z: 371 (M+H+)

3-(4-Bromophenyl)propane-1,2-diol (73). NMO (3.51 g, 29.96 mmol) and potassium osmate (0.03 g, 5%) were added to a solution of 72 (2.95 g, 14.98

69

mmol) in acetone:water (40 mL, 8:1). The reaction mixture was stirred at room temperature overnight. Sodium sulphite (2.2 g) was added and the reaction mixture was stirred for 1 h. The acetone was removed under re-duced pressure and extracted with EtOAc (4 × 20 mL), dried with MgSO4 and concentrated. Flash chromatography yielded 73 as a yellow solid (3.01 g, 87%). 1H NMR (400 MHz, CDCl3): δ 7.37 (d, J = 8.3 Hz, 2H), 7.02 (d, J = 8.3 Hz, 2H), 4.10-3.98 (m, 2H), 3.78 - 3.71 (m, 1H), 3.51 - 3.43 (m, 1H), 3.35 - 3.27 (m, 1H), 2.60 - 2.56 (m, 2H). 13C NMR (101 MHz, CDCl3): δ 137.0, 131.5, 131.0, 120.3, 72.8, 65.7, 38.9. MS (ESI+): m/z: 230 (M+H+), 232 (M+2+H+)

1-(4-Bromophenyl)-3-((tert-butyldimethylsilyl)oxy)propan-2-ol (74). TBDMS-Cl (2.06 g, 13.68 mmol) was added to a stirred solution of 73 (3.01 g, 13.03 mmol) and imidazole (1.33 g, 19.55 mmol) in dry DCM (150 mL) and the mixture was stirred overnight. The reaction mixture was washed with water, dried (MgSO4), evaporated, and purified by flash column chro-matography to yield 74 as a white solid (4.19 g, 12.12 mmol). 1H NMR (400 MHz, CDCl3): δ 7.44 – 7.39 (m, 2H), 7.13 – 7.08 (m, 2H), 3.84 (ddd, J = 6.6, 4.4, 3.7 Hz, 1H), 3.61 (dd, J = 10.0, 3.7 Hz, 1H), 3.45 (dd, J = 10.0, 6.6 Hz, 1H), 2.72 (d, J = 6.6 Hz, 2H), 2.41 (d, J = 4.4 Hz, 1H), 0.91 (s, 9H), 0.07 (d, J = 1.6 Hz, 6H).13C NMR (101 MHz, CDCl3): δ 137.4, 131.6, 131.2, 120.3, 72.6, 66.3, 39.0, 26.0, 18.4, -5.24, -5.21. MS (ESI+): m/z: 344 (M+H+), 346 (M+2+H+)

1-(4-Bromophenyl)-3-((tert-butyldimethylsilyl)oxy)propan-2-one (75). Dess-Martin periodinane (6.15 g, 14.5 mmol) dissolved in 20 mL dry DCM was added to a solution of 74 (4.18 g, 12.12 mmol) in 10 mL dry DCM. After 2 h the mixture was poured into saturated NaHCO3 (50 mL). The or-ganic layer was extracted, washed with brine, dried with MgSO4 and concen-trated. Flash chromatography yielded 75 as a yellow solid (3.33 g, 80%). 1H NMR (400 MHz, CDCl3): δ 7.46 – 7.42 (m, 2H), 7.11 – 7.05 (m, 2H), 4.23 (s, 2H), 3.78 (s, 2H), 0.93 (s, 9H), 0.08 (s, 6H). 13C NMR (101 MHz, CDCl3): δ 207.9, 132.8, 131.8, 131.4, 121.1, 69.0, 44.7, 25.9, 18.4, -5.4. MS (ESI+): m/z: 342 (M+H+), 344 (M+2+H+)

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7. Concluding remarks

This thesis describes the palladium-catalyzed synthesis of N-allylbenzamides and aryl ketones and the development of new HIV protease inhibitors. The specific results and conclusions are the following:

A new Pd(0)-catalyzed aminocarbonylation protocol was devel-oped using 17 different (hetero)aryl iodides, bromides and chlo-rides, with allylamine as the nucleophile and Mo(CO)6 as the sol-id in situ CO source. The N-allylbenzamides were isolated in good yields ranging from 43-81%, and one reaction was scaled up to 25 mmol scale. The reaction was chemoselective without a competing Mizoroki-Heck reaction.

Improvements were made to an existing protocol for a decarbox-ylative Pd(II)-catalyzed addition reaction to synthesize aryl ke-tones from aryl carboxylic acids and organonitriles. Significant increases in yield were achieved when TFA was added and THF was employed as solvent enabling the use of solid nitriles with a 2- to 5-fold excess. Using this protocol 29 (hetero)aryl ketones were synthesized in yields of up to 94% from five different ben-zoic acids with an ortho substituent and a diverse set of nitriles.

A series of 25 new HIV protease inhibitors was synthesized and biologically evaluated. These compounds were related to ataza-navir and indinavir but had a tertiary alcohol as well as a two-carbon tether between the quaternary carbon and the hydrazide β-nitrogen. The compounds obtained were up to 56 times more po-tent than the one- or three-carbon tether analogs, with EC50 values down to 3 nM. X-ray crystallography data showed that the P1-part of inhibitor 21 fitted in the S1 and S3 pockets better than an earlier series, and that there was an additional hydrogen bond to Gly27.

Attempts were made to make macrocyclic HIV-1 protease inhibi-tors in which the P1 and P3 sides were bound together. Despite synthetic efforts using a lactone-based route with bromo-, hy-droxy-, methoxy-, silyl-protected hydroxy- and alkyne-based handles, it was not possible to obtain the tertiary alcohol interme-diate. However, a route that was not based on the lactone looked promising.

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Acknowledgements

I would like to express my sincere gratitude to the following people. My main supervisor Professor Mats Larhed: you have always found the time in your very tight schedule for discussions and reviewing my work. You also create a great atmosphere in the department. Dr. Luke Odell, my co-supervisor: you have contributed so much to my projects and you’re such a skilful chemist; are there any name-reactions you don’t know? Gunilla Eriksson: you are such a genuinely caring person; without you org-farm would not be the same place. Sorin Srbu thank you for answering my computer related questions. Stefan Holmgren, my examworker: you did a great job. My mentor Dr. Annika Jenmalm Jensen: time flies when we discuss chemi-stry, teaching, personality tests etc. You have been an excellent mentor. All my co-authors: thank you! Dr. Jonas Sävmarker and Dr. Jonas Lindh: thanks for your contributions to the aryl ketone project. Dr. Maria de Rosa, Dr. Jonas Sävmarker, Dr. Per Öhrngren and Antona Wagstaff: thanks for proofreading the thesis. My past lab and room-mates: Dr. Per Öhrngren, for those endless HIV-discussions, Dr. Riina Arvela and Dr. Stefanie Sclummer for always being so cheerful, Dr. Alejandro Trejos and Olaf van der Veen for all the fun talks in that very crowded lab space over the past year, Linda Åkerbladh for clos-ing the door to our writing room for some gossip. My travelling companions to ACS in San Francisco: Dr. Anneli Nordqvist, Dr. Rebecca Fransson and Dr. Anna Lampa. Thinking of the cartwheeling, almost-thrown-out-of-the-hotel experience makes me giggle. Patrik Norde-man and Dr. Alejandro Trejos: thanks for the good time in Copenhagen.

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So many memorable colleagues have made this period in my life fun. Anna-Karin Belfrage, Dr. Fredrik Wångsell, Dr. Hanna Andersson: nice that so many people from my undergraduate studies in Linköping ended up in Upp-sala. Dr. Kristina Orrling: it was very boring to listen to all the cat discus-sions in the coffee room I think, but we had fun. Dr. Pernilla Örtqvist: I miss hearing your strong opinions about so much. Dr. Charlotta Wallinder, Dr. Anja Sandström, Dr. Ulrika Rosenström: you have a lot of great ideas about teaching, keep up the good work. Dr. Johan Gising: thanks for answering so many questions about the thesis writing process. All the ”new” PhD-students Fredrik, Ashkan, Martin, Anna, Karin, Bobo, Jonas R, Sara, Hiba and Marc: you have great years ahead of you. Alla goa vänner; Johanna och Jessica med familjer, Karin, Miche, Maria, och Anna med era små och karlar, synd att ni bor så långt bort. I sommar ska vi komma iväg på den där tjejresan tycker jag. Anneli med familj vad kul att Linnéa och Elin är sådana polare och stort tack för hjälpen med bilden. Fred-rik, Eva, Olle och Millan, vårt nyårsfirande kan jag riktigt längta till under övriga året. Alla mina kusiner och morbröder för att ni gör somrarna på Gotland lite extra trevliga. Laila och Ingela, ni är som mina extramammor. Geo med familj, vad roligt det är att ni bor i Stockholm nu. Birgitta och Christer, bättre svärföräldrar kunde man inte önska sig. Ann-Mari tack för all hjälp med barnen. Pernilla, Peter och Sam, är så härligt att ni bor såpass nära oss nu. Kusinerna och vi har så kul ihop. Min mamma Agneta, du är en klippa när det gäller att ta hand om barnbar-nen, stort tack för all hjälp med dem. Min pappa Kent, är så ledsen över att du inte fick vara med på den här resan mot en doktorstitel, tror att det hade varit jättekul att diskutera all kemi med dig. Saknar dig! Neo, Ramses och Pralin, får man tacka sina katter? Neo, du vet verkligen hur du ska hitta tillfällen när det inte passar att ligga i mitt knä. Mina älskade barn, Elin och Meja. Ni gör livet mycket jobbigare, roligare, gladare och mysigare. Jimmy, du är verkligen 7.2 du. Älskar dig för att du är en sådan person som alltid ställer upp.

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134. Sham, H.L., Kempf, D.J., Molla, A., Marsh, K.C., Kumar, G.N., Chen, C.M., Kati, W., Stewart, K., Lal, R., Hsu, A., Betebenner, D., Korneyeva, M., Vasavanonda, S., McDonald, E., Saldivar, A., Wideburg, N., Chen, X., Niu, P., Park, C., Jayanti, V., Grabowski, B., Granneman, G.R., Sun, E., Japour, A.J., Leonard, J.M., Plattner, J.J. & Norbeck, D.W. ABT-378, a Highly Potent Inhibitor of the Human Immunodeficiency Virus Protease. Antimicrob. Agents Chemother. 42, 3218-3224 (1998).

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135. De Meyer, S., Azijn, H., Surleraux, D., Jochmans, D., Tahri, A., Pauwels, R., Wigerinck, P. & de Bethune, M.-P. TMC114, a Novel Human Immunodeficiency Virus Type 1 Protease Inhibitor Active Against Protease Inhibitor-Resistant Viruses, Including a Broad Range of Clinical Isolates. Antimicrob. Agents Chemother. 49, 2314-2321 (2005).

136. N1,N6-Di[(1S)-2-methyl-1-(methylcarbamoyl)propyl]-(2R,3R,4R,5R)-2,5-di(benzyloxy)-3,4-dihydroxyhexanediamide.

137. Alterman, M., Björsne, M., Muehlman, A., Classon, B., Kvarnström, I., Danielson, H., Markgren, P.-O., Nillroth, U., Unge, T., Hallberg, A. & Samuelsson, B. Design and Synthesis of New Potent C2-Symmetric HIV-1 Protease Inhibitors. Use of L-Mannaric Acid as a Peptidomimetic Scaffold. J. Med. Chem. 41, 3782-3792 (1998).

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142. The alpha-methyl group of Val 182 has a distance of 3.8 Å to one of the ortho carbons, 3.8 Å to the meta and 3.9 Å to the para carbon of the inhibitor benzyl group. .

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144. The beta-methyl group of Val182 has a distance of 5.1 Å to the ortho, 4.3 and 4.4 Å to the meta positions, and 3.9 Å to the para position of the inhibitor benzyl group. .

145. The methyl group of mutant Thr182 has a distance of 4.0 Å to the ipso, 3.8 Å to the ortho, 3.7 Å to the meta, and 3.8 Å to the para carbon. The distance to the hydroxy oxygen is 5.7 Å for the ortho, 5.1 Å for the meta, and 5.0 Å for the para carbon of the inhibitor benzyl group.

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Acta Universitatis UpsaliensisDigital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Pharmacy 182

Editor: The Dean of the Faculty of Pharmacy

A doctoral dissertation from the Faculty of Pharmacy, UppsalaUniversity, is usually a summary of a number of papers. A fewcopies of the complete dissertation are kept at major Swedishresearch libraries, while the summary alone is distributedinternationally through the series Digital ComprehensiveSummaries of Uppsala Dissertations from the Faculty ofPharmacy.

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